Liquid Chromatography of Natural Pigments and Synthetic Dyes

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JOURNAL OF CHROMATOGRAPHY LIBRARY — volume 71

liquid chromatography of natural pigments and synthetic dyes

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JOURNAL OF CHROMATOGRAPHY LIBRARY — volume 71

liquid chromatography of natural pigments and synthetic dyes Tibor Cserháti Institute of Material and Environmental Chemistry, Chemical Research Centre, Hungarian Academy of Sciences, P.O. Box 17, 1525 Budapest, Hungary

Amsterdam ● Boston ● Heidelberg ● London ● New York ● Oxford Paris ● San Diego ● San Francisco ● Singapore ● Sydney ● Tokyo

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Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK First edition 2007 Copyright © 2007 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN-13: 978-0-444-52222-1 ISBN-10: 0-444-52222-0 ISSN: 0301-4770

For information on all Elsevier publications visit our website at books.elsevier.com

Printed and bound in The Netherlands 07 08 09 10 11 10 9 8 7 6 5 4 3 2 1

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CONTENTS Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX 1

2

Theory and practice of liquid chromatography (LC) . . . . . . . . . . . . . . . . . . . . . . 1.1 Sample preparation strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Thin-layer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1 Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.2 Practical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 High-performance liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1 Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.2 New theoretical advances in high-performance liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.3 Practical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Supercritical fluid chromatography (SFC) . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 Various electrophoretic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.1 Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5.2 New advances in the theory of electrophoretic techniques . . . . . . . 1.5.3 Practical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 3 4 7 12 13 21 42 43 43 44 45 54 55

Liquid chromatography of natural pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 2.1 Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 2.1.1 Chemistry and biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 2.1.2 Thin-layer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 2.1.3 High-performance liquid chromatography . . . . . . . . . . . . . . . . . . . 71 2.1.3.1 HPLC determination of carotenoid pigments in samples of plant origin . . . . . . . . . . . . . . . . . . . . . . . . . . 71 2.1.3.2 HPLC determination of carotenoid pigments in human and animal tissues . . . . . . . . . . . . . . . . . . . . . . . . 104 2.1.3.3 HPLC determination of carotenoid pigments in miscellaneous organic matrices . . . . . . . . . . . . . . . . . . 122 2.2 Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 2.2.1 Chemistry and biochemistry of flavonoids . . . . . . . . . . . . . . . . . . 133 2.2.2 Thin-layer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 2.2.2.1 TLC separation of flavonoids in the extracts of medicinal plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 2.2.2.2 TLC separation of flavonoids in model systems and in miscellaneous matrices . . . . . . . . . . . . . . . . . . . . 151

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2.2.3

High-performance liquid chromatography . . . . . . . . . . . . . . . . . . . 161 2.2.3.1 HPLC determination of flavonoids in plant extracts . . . . 162 2.2.3.2 HPLC determination of flavonoids in food and food products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 2.2.3.3 HPLC determination of flavonoids in beverages . . . . . . . 189 2.2.3.4 HPLC determination of flavonoids in other matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 2.2.4 Electrophoretic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 2.3 Anthocyanins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 2.3.1 Chemistry and biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 2.3.2 Thin-layer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 2.3.3 High-performance liquid chromatography . . . . . . . . . . . . . . . . . . 244 2.3.3.1 Determination of anthocyanins in wine . . . . . . . . . . . . . . 244 2.3.3.2 Determination of anthocyanins in fruits and other beverages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 2.3.3.3 Determination of anthocyanins in miscellaneous matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 2.3.4 Electrophoretic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 2.4 Chlorophylls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 2.4.1 Chemistry and biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 2.4.2 High-performance liquid chromatography . . . . . . . . . . . . . . . . . . . 286 2.4.2.1 Determination of chlorophylls in the marine environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 2.4.2.2 Determination of chlorophylls in miscellaneous matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 2.5 Miscellaneous pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 2.5.1 Thin-layer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 2.5.2 High-performance liquid chromatography . . . . . . . . . . . . . . . . . . . 326 2.5.2.1 Miscellaneous pigments in living organisms . . . . . . . . . . 326 2.5.2.2 Miscellaneous pigments in foods and food products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 2.5.2.3 Miscellaneous pigments in other matrices . . . . . . . . . . . . 337 2.5.3 Electrophoretic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 3

Liquid chromatography of synthetic dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 3.1 Application of synthetic dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 3.2 Toxicology of synthetic dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 3.3 Environmental impact of synthetic dyes . . . . . . . . . . . . . . . . . . . . . . . . . . 371 3.3.1 Physical and physicochemical methods used for the adsorption and degradation of dyes . . . . . . . . . . . . . . . . . . . . . . . . 372 3.3.2 Microbiological methods used for the degradation of dyes . . . . . . 373 3.4 Thin-layer chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374 3.4.1 Application of model systems in TLC . . . . . . . . . . . . . . . . . . . . . . 374 3.4.2 TLC determination of dyes in various matrices . . . . . . . . . . . . . . . 386

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3.5 High-performance liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . 402 3.5.1 HPLC determination of synthetic dyes in animal tissues . . . . . . . . 403 3.5.2 HPLC determination of synthetic dyes in foods, food products and waters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412 3.5.3 Degradation of synthetic dyes by physicochemical methods followed by HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432 3.5.4 Degradation of synthetic dyes by microbiological methods followed by HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469 3.5.5 HPLC separation of synthetic dyes in model mixtures . . . . . . . . . 480 3.5.6 Miscellaneous applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511 3.6 Electrophoretic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516 3.6.1 Determination of dyes in foods and food products . . . . . . . . . . . . 516 3.6.2 Environmental and toxicological application of electrophoretic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528 3.6.3 Separation of model mixtures of dyes by electrophoretic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561 Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573

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Preface

For decades, both natural pigments and synthetic dyes have been extensively used in various fields of everyday life such as food production, the textile industry, paper production, agricultural practice and research, water science and technology, and so on. Natural pigments not only have the capacity to increase the marketability of products, they also display advantageous biological activities as antioxidants and anticancer agents. Synthetic pigments, on the other hand, cause considerable environmental pollution and adverse toxicological side effects. Both classes of pigment exhibit considerable structural diversity. As the stability of the pigments against hydrolysis, oxidation and other environmental and technological conditions is markedly different, the exact determination of their composition may help when predicting the shelf-life of products and assessing the influence of technological steps on the pigment fractions, resulting in more consumer-friendly methods of processing. Furthermore, the qualitative determination and identification of the pigments may contribute towards establishing the provenance of the product. The unique separation capacity of the liquid chromatographic (LC) technique makes it the method of choice for analysing pigments in any complicated accompanying matrices. The objectives of this monograph are to provide a compilation of the newest results in this field of research, a critical evaluation of the results, a comparison of the various LC separation techniques, and the prediction of future trends in the LC analysis of pigments. The book is meant to be self-sufficient in terms of the needs of the average analytical chemist who intends to work in this fascinating field. We are confident that it will be a valuable reference book for researchers and serious students engaged in the chromatography of pigments. The author is grateful to Ms Eva Tarlós, Ms Esther Gyulassy and Mr Lajos Gál for their valuable technical assistance.

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Chapter 1

Theory and practice of liquid chromatography (LC)

The term liquid chromatography (LC) includes each chromatographic technique using a liquid mobile phase and an organic or inorganic stationary phase. According to the configuration of the stationary phase, LC techniques can be separated into planer (thin-layer) and column chromatography. Independently of the shape of the stationary phase, LC methods can be divided into normal (direct or adsorption) and reversed-phase (RP) separation methods. Adsorption LC uses a nonpolar mobile phase and a polar stationary phase, while the RP separation mode is characterized by the application of a polar mobile phase and a nonpolar stationary phase. 1.1 SAMPLE PREPARATION STRATEGIES Sample preparation is a crucial step in any chromatographic analysis. The objectives of sample preparation are the preconcentration and prepurification of solutes to be measured, with the maximum possible decrease of the disturbing components of complicated matrices such as soil, sludge, human and animal issues, blood, fuel oils and food. Undesired accompanying compounds can considerably decrease the efficacy of determination, reducing theoretical plate number, separation factor, peak symmetry, repeatability and reproducibility. Traditional separation procedures, such as shake flash extraction, are often the most time-consuming step in the chromatographic analysis. Liquid and liquid–liquid extraction (LLE) is the conventional method of treating samples prior to chromatographic separation. Because of the extremely high number of solutes to be extracted from the even higher number of matrices, the exact theory of choice of the optimal solvent or solvent mixture has not until now been developed. Although the efficacy of LLE techniques is generally high, their application is not proposed for many reasons. The LLE procedure requires considerable time and the clean-up process requires highly purified organic solvents in considerable amounts, enhancing considerably the cost of analysis. Moreover, some of these solvents are suspected of endangering the health of laboratory workers and may increase environmental pollution. A special case of liquid extraction is the Soxhlet extraction extensively used in recent decades, however, because of new preconcentration methods its importance is decreasing. It has been established that the efficacy of Soxhlet

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extraction, pressurized liquid extraction, supercritical fluid extraction and subcritical water extraction is highly similar and the application of the Soxhlet method does not enhance the efficacy of extraction [1]. A considerable number of alternative methods reducing or entirely eliminating the use of more or less toxic organic solvents have been developed and successfully applied to preconcentrate samples before chromatographic analysis. These techniques include microwave assisted extraction [2,3], and its optimized variant [4], pressurized liquid extraction [5,6] its modification using water–organic modifier mixtures instead of pure organic components [7], simultaneous steam distillation extraction [8], static and dynamic headspace for volatile compounds, continuous flow liquid membrane extraction [9,10], class-selective immunoextraction applying various immunosorbents [11,12], solid-phase extraction (SPE) [13–15] of various bioactive compounds, solid-phase microextraction (SPME) [16], and supercritical fluid extraction (SFE) [17–19]. The application of membrane-based techniques such as supported liquid membrane extraction, microporous membrane liquid–liquid extraction, polymeric membrane extraction and membrane extraction with a sorbent interface has also been reported [20]. As neither natural pigments nor synthetic dyes are volatile, head-space analysis and SPME cannot be generally applied for the prepurification of these types of compounds. The majority of the techniques listed above are only in the experimental stage and are not extensively used. However, their advantageous characteristics make probable their rapid and wide-range acceptance. Solid-phase extraction (SPE), a well established and simple preconcentration technique, can be used for liquid samples or the liquid extract of solid matrices. The sample is passed through an inert cartridge filled with solid sorbent, or it is embedded in a disc. Because of the strong adsorption capacity of the sorbent, the analytes remain bonded to the sorbent surface. After the adsorption step the sorbent is washed to remove coadsorbed components. Finally, the analytes are removed from the sorbent with a strong solvent. The SPE procedure results in a concentrated solution of analytes with the minimum quantity of undesirable compounds. For the successful SPE preconcentration a number of factors have to be taken into consideration. The sorbent has to be as selective as possible. Sorbent– analyte interactions can be divided into three classes: nonpolar, polar and ionic. Nonpolar or slightly polar analytes may be dissolved in water, which is a highly polar solvent. For these cases the application of nonpolar sorbents can be proposed. Analytes with polar functional groups are generally retained on polar sorbents. When the retention is governed by ionic interactions, an anionic sorbent should be used to retain cations and a cationic sorbent to retain anions. Sorbent capacity is also an important factor in SPE. Low adsorption capacity results in the inadequate retaining of solutes, whereas high adsorption capacity increases solvent consumption without enhancing the performance of the process. The volume of the original sample and the final volume of the purified sample also have to be taken into consideration. In general the mass of the solutes and the accompanying compounds retained by the sorbing should be less than 5 per cent of the mass of the sorbing. SFE cartridges are commercialized in a wide range of sizes, the effective sorbing volumes ranging from less than 1 ml to over 50 ml. There are numerous reports in the literature of SPE clean-up of a wide variety of analyses prior to chromatographic analysis. Thus, the application of SPE for the preconcentration of the metabolites of peptide–doxorubicin conjugate in human plasma [21], for the

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preparation of samples for determining the cyclic depsipeptide in whole blood [22], and for the prepurification of abamectin and doramectin in sheep faeces [23] have been recently reported. Supercritical fluid extraction (SFE) has been recognized as an important, solvent-free alternative to convetional liquid–solvent methods, such as Soxhlet extraction, for the removal of organic compounds from solid matrices. SFE techniques provide many advantages: reducing the use of organic solvents in analytical laboratories, shorter extraction times and the ability for on-line introduction of the extracted material to chromatographic instrumentation. The supercritical fluid extraction process can be coupled to a wide range of chromatographic techniques, including gas (GC) and supercritical fluid chromatography (SFC), as well as high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). The supercritical state of the mobile phase indicates that its physicochemical characteristics are between those of a liquid and a gas. As the viscosity of these mobile phases are near to that found in common gaseous phases, the mass transfer is very rapid in these systems, the coefficient of diffusion being about 10 times higher then in a liquid. As the pressure increases at a constant temperature, the viscosity and diffusivity of the mobile phase approach those of a liquid. The increase of temperature at a fixed pressure results in improved diffusivity while the enhancement of temperature causes reduced viscosity. These changes are more explicit near the critical point. The viscosity and diffusivity are one to two orders of magnitude higher in the supercritical than in the liquid state, even at elevated pressures. The liquidlike density, gas-like viscosity and diffusivity, and pressure-dependent solvatation capacity highly promoted the application of SFE. Because of its advantageous physicochemical characteristics, CO2 is generally applied as the unique or main component of mobile phases. The primary benefits of SFE are that the method is relatively inexpensive, applies environmental friendly mobile phases and has approximately the same solvatation power as the organic solvents used in LLE. The extraction capacity of SFE can be easily regulated by pressure, temperature or both of them. The addition of a low concentration of organic modifier, such as methanol or acetonitrile, considerably enhances the extraction power of SFE. Moreover, SFE reduces of the quantity of organic solvents required for the extraction, and decreases markedly the extraction time. SFE coupled with LC has found application in the separation and quantitative determination of numerous analyses, such as polycyclic hydrocarbons in vegetable oil [24], essential oils of Carum copticum [25] and Mentha pulegium [26], fluoroquinolones from chicken breast muscles [27], etc. The online coupling of SFE to LC for the measurement of solutes in aqueous matrices has also been described recently [28]. 1.2 THIN-LAYER CHROMATOGRAPHY Thin-layer chromatography (TLC) as an easy to carry out analytical technique was evolved more than 30 years ago. The method has found wide-ranging applications in the separation and semiqantitative determination of both organic and inorganic compounds present in very low quantities in complicated accompanying matrices. In recent years the use of different TLC techniques has been markedly enhanced. This development may be

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due to the increased automation and instrumentation of each step of TLC separation (precise spotting devices, high-performance thin-layer chromatography (HPTLC), circular rotation planar chromatography, centrifugal, gradient and forced-flow developments, densitometric evaluation with up-to-date software, etc.). Hyphenated techniques, such as TLC combined with ultraviolet and visible spectrophotometry (TLC-UV-VIS), mass spectrometry (TLC-MS), and Fourier infrared spectrometry (TLC-FTIR), have also found application in TLC analyses. These new advances markedly increased the reproducibility and repeatability of TLC measurements. Thus, TLC has recently been used in clinical medicine [29], forensic analysis [30,31], biochemistry [32], for the evaluation of pharmaceuticals [33], plant extracts [34], human toxicology [35], environmental protection studies [36,37], cosmetology [38], food analysis [39], and plant drugs [40], among others. The simplicity, rapidity and flexibility of TLC analytical systems has also contributed to the widespread use of the method. The theoretical aspects and practical employment of various TLC methodologies have been discussed in detail in excellent books [41,42], biennial reviews published in the Fundamental Reviews issue of the ACS journal Analytical Chemistry [43] and other reviews dealing with special aspects of theory and practical application of TLC [44,45]. 1.2.1 Fundamentals The basis of any TLC separations is the interaction of the compounds to be analysed with both the stationary and the mobile phase. The differences among the strengths of interaction result in the separation of compounds (solutes). Both inorganic and organic materials with a well-defined chemical structure, high specific surface areas and marked (possibly uniform) porosity have been used as stationary phases. TLC plates are prepared from these stationary phases by equally distributing them on a solid inactive support such as glass, aluminium, plastic, etc. Inorganic (gypsum) and organic compounds (polyvinylalcohols of various molecular masses) equally can be applied to bind the stationary phase to the support, increasing in this manner the mechanical stability of the stationary phase. Generally, the addition of binders exerts a negligible effect on the chromatographic characteristics (retention capacity, selectivity) of the stationary phase. Besides the chemical properties, the efficacy of sorbing depends markedly on the particle size, particle size distribution, specific surface area, pore volume, mean pore diameter and pore size distribution. Using stationary phases of smaller particle size and narrower particle size distribution results in increasing separation efficacy, better resolution, reduction of analysis time and decreases the detection limit. Traditional TLC sorbents have an average particle size of 10 – 50 µm, the particle size distribution being fairly wide. The particle size of HPTLC sorbing is about 5 µm and the particle size distribution is markedly narrower than in the case of common TLC sorbants. The separation power of HPTLC plates is generally higher than that of TLC. The overwhelming majority of TLC separations are carried out on silica or silica-based stationary phases. The retention characteristics of the silica surface can be modified by covalently binding various organic ligands such as C1-, C2-, C8-, C18- and C30 alkyl chains [46], amino, diol, cyano groups (CN) [47,48], and different chiral selectors. Ligands modify the polarity of the original silica support, the approximate polarity order being silica⬎amino

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silica⬎cyano silica and octadecyl silica. It has to be emphasized that the polarity order may considerably depend on the composition of the mobile phase and the physicochemical properties of the solutes to be separated. Because of their medium polarity, amino, diol and cyano stationary phases can be equally employed both in direct and reversed-phase separation mode using mobile phases more or less polar than the stationary phase [49]. Surprisingly, these stationary phases are not frequently used in TLC, although they demonstrate good separation capacity. Numerous TLC and HPTLC ready-made plates are commercially available facilitating the successful solution of a high number of separation problems. Besides silica, other inorganic stationary phases (neutral and acidic alumina, alumina chemically modified with C8-alkyl chain [50], magnesium silicate, diatomaceous earth and diatomaceous earth modified with τ-aminopropyltrimethoxysilane [51] have also found application in TLC. Organic compounds, mainly cellulose [52], various cellulose derivatives [53] and polyamides have also been employed as stationary phases. Although their separation characteristics differ markedly from those of silica and silica-based supports they have now been extensively used in TLC analyses. It has been reported many times that the efficacy of TLC separation can be enhanced by mixing two different stationary phases, however, these methods have not been widely applied in TLC practice [54]. Impregnation of the support has also been frequently employed for the enhancement of the efficacy of TLC separation or for the study of the strength of the interaction between the solutes and the molecules of the impregnation agent. These solid phase additives can be adsorbed on the surface of the support by immersing the plate in the solution of the impregnating agent, by predevelopment of the plates in the solution of the additive, and by mixing the additives and the support and preparing plates from this mixture [55–58]. Besides the correct selection of the stationary phase, the composition of the mobile phase also exerts a considerable influence on the success of the separation process. One of the main advantages of TLC over HPLC is the markedly higher number of solvents and solvent mixtures which can be employed as a mobile phase. Detection in HPLC is generally carried out in the presence of the mobile phase, consequently, the common UV detection of analyses cannot be performed in mobile phases with UV absorption capacity. In routine TLC practice, the mobile phase is evaporated before detection, therefore, it does not interfere with the determination of analyte spots. The solvent strength (elution power or elution capacity) of a mobile phase is related to its ability to move one or more analytes on a given stationary phase. The solvent strength is higher when the retention of analyses is lower (higher mobility). However, it has to be borne in mind that the elution strength markedly depends on the physicochemical properties of the solutes and those of the stationary phase. It is generally accepted that the elution order of pure solvents on silica is n-pentane⬎ n-hexane⬎ n-heptane⬎ cyclohexane⬎ carbontetrachloride⬎ toluene⬎ dichloromethane⬎ diisopropylether⬎ terc.-butanol ⬎ diethylether⬎ nitromethane⬎ acetonitrile⬎ 1-butanol⬎ 2-propanol⬎ ethylacetate⬎ 1-propanol⬎ acetone⬎ ethanol⬎ dioxane⬎ tetrahydrofurane>methanol>pyridine>water. The elution strength of solvents is approximately reversed for reversed-phase TLC. Mobile phases containing solvents with low boiling point, low viscosity and low toxicity are preferable. High viscosity enhances the developing time, and the removal of mobile phase components with high boiling point is sometimes cumbersome.

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The position of an analyses on the TLC plate is characterized by the retention factor Rf. It is determined by dividing the distance between the centre of the spots and the start line (zs) with the distance of the eluent front of the start line (zf): Rf ⫽

zs zf

(1.1)

The Rf value varies between 0 (analyte remains on the start) and 1 (analyses move with the front of the mobile phase). The reproducibility of the measurement of Rf value can be enhanced by relating it to the Rf value of a standard analyte. As the correlation between the Rf value of the analyte and the concentration of the stronger component in the mobile phase (C) is not linear, the RM value was introduced to linearize the relationship between the mobility of solutes and the concentration of the stronger component in the mobile phase.  1  RM ⫽ log   Rf ⫺1

(1.2)

RM ⫽ RM0 ⫹ b⭈C

(1.3)

where RM0 is the theoretical RM value of an analyte extrapolated to zero concentration of the stronger component in the mobile phase, and b is change in the RM value caused by a unit change of the concentration of the stronger component in the mobile phase. The velocity of the movement of the front of the mobile phase can be described by the rate coefficient (k): k ⫽ ( zf2 ) Ⲑt

(1.4)

where t is the time of developing. Developing time is influenced by many physical and physicochemical factors of both the stationary and mobile phases, such as the capillary forces between the pores of the support particles and the molecules of the mobile phase, and the viscosity and surface tension of the components of the mobile phase. The traditional method of determining the position of an analyte spot on the plates is a visual evaluation. However, this technique is highly subjective and depends considerably on the expertise of the analytical chemist. TLC scanners, developed for exact determination not only pinpoint position but also the area, intensity and symmetry of the spot, overcome the uncertainty of the visual evaluation. Moreover, TLC scanners make possible more accurate determination of the quantity of analyte in the spot by converting spot characteristics into peak characteristics. Peak height is the distance between the peak maximum and the baseline, whereas peak area is the area of the peak between the beginning and end of the peak and the baseline.

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The efficacy of the separation of two neighbouring spots is defined by the resolution Rs. It can be calculated by Rs ⫽ 2

( z1 ⫺ z2 ) (w1 ⫺ w2 )

(1.5)

where z1 and z2 are the distances of the spot maxima from the start, and w1 and w2 are the spot widths at the baseline. Separation is considered acceptable when the resolution is equal to or higher than 1. Eqn. 1.5 shows that the efficacy of separation can be enhanced either by increasing the distance between the spot centres (higher selectivity) or by reducing peak widths (higher efficiency). The impact of various hydrophobic and hydrophilic (electrostatic) interactive forces on the TLC separation of solutes has been extensively investigated. It has been established that adsorption, partition and ion exchange exert the highest influence on the separation process, modifying retention strengh and selectivity. The polar substructures of solutes can be adsorbed by the hydrophilic centres on the surface of the particles of the stationary phase. Higher strength of adsorption results in enhanced retention, whereas lower adsorption strength promotes the mobility of the solute. The molecular basis of the adsorption processes outlined above is not entirely elucidated. The role of permanent and inducible dipole moments of analyte and their ability to form hydrogen bonds may play a considerable role in the adsorption mechanism. Partition of solutes occurs between the stationary and mobile phases. Analyses with higher preference for the stationary phase are more strongly retained and are later eluted. 1.2.2 Practical considerations Sample application is a decisive step in TLC measurements especially in quantitative analyses. The preparative or analytical character of the separation and the volume and physicochemical properties of the sample solution influence equally the mode of sample application. The concentration of the analyte(s) of interest in the sample frequently determines the volume to be applied on the TLC plate; a relatively low concentration of analyses requires a high sample volume. Samples containing analyses liable to oxidation have to be applied in a nitrogen atmosphere. Samples can be applied onto the plates either in spots or in bands. It has been proven that the application of narrow bands results in the best separation. The small spot diameter also improves the performance of TLC analysis. The spot diameter has to be lower than 3 mm and 1 mm for classical TLC and HPTLC, respectively. It has been further established that the distance between the spot of the analyte and the entry of the mobile phase also exerts a marked impact on the efficiency of the separation process, the optimal distance being 10 and 6 mm for TLC and HPTLC plates, respectively. Loops and capillaries were employed earlier for the application of samples onto the plates. This method does not allow the exact determination of the sample volume, consequently it was not suitable for reliable quantitative work. Syringes have been developed and commercialized for the accurate application of microlitre and nanolitre volumes. A wide variety of automated application devices have been developed and are available on

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the market. They can apply different sample volumes in an predetermined order without direct contact between the plate and the solid parts of the application device. The successful analysis of samples containing the analyte(s) at very low concentration requires high sample volume, which may deteriorate the performance of the separation process. Precoated plates with concentrating (preadsorbant) zones overcome this difficulty. A zone of stationary phase with relatively low retention strength (i.e., diatomaceous earth) is applied to the bottom of the layer performing the separation. A fairly high sample volume can be applied onto the concentration zone without deteriorating separation. Because of the low adsorption capacity of the concentrating zone the analyses move together with the front of the mobile phase and are concentrated in a very narrow band at the beginning of the stationary phase carrying out the separation. Another method for the reduction of spot dimensions uses a very strong eluent for predevelopment. Analyses move together with the eluent front forming a very narrow band. Also, this procedure can be employed for the increase in performance of separation, however, it is time consuming and the strong solvent has to be entirely removed before analysis. The appropriate choice of solvent for the dissolution of sample may also reduce spot diameter. Apolar (i.e., n-hexane) and polar (water, methanol) solvents can be preferably used for adsorption and reversed-phase TLC separations, respectively. Sample volume can also be increased by the tedious method of portionwise application with intermediate removing of the solvent. The optimal distance between the spots is 10 – 15 mm for TLC and 5 mm for HPTLC plates, the distance of development varying between 10 – 15 cm for TLC and about 7 cm for HPTLC. The reliability of the quantitative analysis can be increased by applying it to parallel samples from both standards and samples on the two halves of the plate to decrease the possible systematic error caused by the nonuniformity of the stationary phase (data pair method). Numerous techniques can be employed for the development of plates, such as linear ascending, linear horizontal, circular and anticircular. Linear ascending development is the method used most frequently in practical TLC. Eluent is poured into a tank, and the lower part of the plate is immersed in the eluent. The part of the plate containing the samples has to be always over the level of the mobile phase. The mobile phase ascends by capillary forces. Reaching the developing distance the plate is taken out of the tank, and the position of the front of the mobile phase is marked for the further calculation of retention characteristics (one-dimensional development). Residues of the mobile phase can be removed at ambient temperature or any elevated temperature which does not influence the sensitivity of the following detection procedure. Separation performance of one-dimensional development can be increased by employing multiple development using various mobile phase systems. Plates have to be dried between the developments. Two-dimensional development employes two (identical or different) eluents. The sample is spotted at the corner of the plate then developed in one direction. Reaching the developing distance the plate is taken out, dried, turned 90o, and developed again in the second mobile phase. The benefit of the technique is the markedly higher separation capacity, its disadvantage is the impossibility to run the standard on the same plate, which reduces the reliability of quantitative analysis. The gas phase present in the traditional wide developing chambers may influence the separation process by adsorbing the surface of the stationary phase. The beneficial effect of the gas phase in equilibrium with the eluent can be intentionally used for the modification of the retention of solutes or it can be excluded by using very thin chambers (sandwich chambers)

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or other special TLC techniques. The temperature of the TLC chamber has to be constant in order to avoid the fluctuation of the composition of the gas and liquid phases. The differences between solute retentions determined in saturated and unsaturated chambers are considerably lower in reversed-phase TLC because the retention is governed by the partition of solutes between the polar eluent and apolar stationary phase and the impact of the adsorption centres of the original support on the retention is negligible. Moreover, mobile phases generally used in RP-TLC contain a considerable amount of water, and are less volatile. Various automated developing chambers have been designed and produced at a commercial scale. These devices enhance the reliability and reproducibility of the separation process by controlling plate preconditioning, chamber dimensions, developing time and distance, and mobile phase mobility. Some instruments make possible the application of the stepwise gradient technique, which is specially suitable for the separation of solutes with highly different retention properties in one plate. In the case of the application of the stepwise gradient technique the plate is firstly developed for a short distance with the strongest eluent separating the analyses with the highest retention capacity. Finishing the first step the eluent is removed in vacuum and the procedure is repeated with a mobile phase which has a lower elution strengh. Up to 25 steps can be applied to reach the optimal separation of any class of analyses. The advantage of the use of linear horizontal development is the reduced developing time. In this case the gravitation does not decrease the mobility of the mobile phase more than in the traditional linear ascending development. Plates are placed horizontally in the chamber and the transport of the eluent is assured by a glass frit strip, a capillary split or any other method. Circular development techniques employ circular TLC plates, the mobile phase enters the centre of the plate and the development occurs out of the centre of the plate. The sample can be applied either onto the dry layer or onto the layer under the flow of the mobile phase. Forced flow techniques exploiting centrifugal force markedly reduce developing time resulting in less lateral diffusion, and more compact spots. Furthermore, the reliability of the quantitative analysis also increases. Samples can be spotted onto the outer sphere of the circular plate and the mobile phase moves toward the centre of the plate (anticircular developing method). The developing time is short because the velocity of the eluent increases during the separation process. After completing development the plates are taken out of the developing device and the volatile components of the mobile phase are removed. Drying can be carried out by ambient or hot air flow, under vacuum or in the case of oxidable analyses under nitrogen flow. Spots of natural pigments and synthetic dyes can be visually investigated. The majority of organic and inorganic compounds do not absorb in the visible part of the spectrum, they have to be determined by other techniques. Fortunately, numerous organic molecules contain conjugated electron systems absorbing in the ultraviolet (UV) part of the spectrum. These analytes can be accurately detected on plates containing one or two fluorescent indicators mixed on the support. These fluorescent compounds can be excited by UV light (most frequently at 254 and 365 nm wavelengths) and they discharge visible radiation. UV absorbing analytes absorb this radiation and they appear as a dark spot on a green or pale blue background (fluorescent quenching). A small number of analyses can emit fluorescent light when excited with UV light. They can be detected on

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nonfluorescent plates and the presence of any nonfluorescent accompanying material does not interfere with the measurement. Analyses without absorbing visible or UV light have to be modified after development for visual or instrumental evaluation (postchromatographic derivatization). One of the principal advantages of TLC is that a high number of reactions can be employed to promote the detection, identification and quantitative determination of unknown analyses because the colour of the spot may include information about the chemical structure of the analyte. Prechromatographic derivatization offers another possibility for the detection of analyses. The objective of prechromatographic derivatization is to introduce a chromophore or UV active substructure into the analyte. Derivatization not only can increase the sensibility of detection but also may enhance the stability of the original analyte during the separation process. The prerequisite of an effective detection method is the even distribution of the reagent spray on the surface of the stationary phase in drops as fine as possible. This step of the TLC measurements has also been automated and commercial devices are available for uniform spraying of the plates. A more homogeneous distribution of the reagent can be achieved by dipping the plate in the solution of the reagent. Dipping increases the reliability of the determination and reduces the influence of error inherent in the spraying technique. Plates are generally saturated by the reagent in five seconds. It is also possible to add the reagent to the eluent when the retention capacity of the reagent is low and it moves together with the eluent front. However, it has to be taken into consideration that the presence of reagent in the mobile phase can influence the retention characteristics of the TLC system. Many derivatization procedures need elevated temperature (generally 5 – 10 minutes at 100–120° C). The temperature of the plate has to be uniform all over the surface of the stationary phase because the efficacy of the derivatiation considerably depends on the reaction temperature. Uneven distribution of heat can cause unreliable quantitative results. The main disadvantage of traditional TLC is the inherent lower reproducibility than that of the corresponding HPLC. However, the modern automated devices (spotting apparatus, developing systems, TLC scanners) combined with adequate software, markedly enhance the reproducibility of any TLC measurement making it comparable with that of HPLC. Analyses can be tentatively identified by the retention characteristics, it is commonly accepted that two compounds are identical when their Rf values are identical, taking into consideration the standard deviation of the determination. When the Rf value of an unknown molecule and that of the standard is equal, the identity of the two compounds is highly probable. The reliability of this type of identification can be considerably increased when the Rf value of the unknown analytes and the standard are determined in parallel in a minimum of four different TLC systems. When the Rf values are identical in each system, the identity of the standard and the unknown molecules can be rightly assumed. This approximation is practically accepted but is not scientifically correct. However, TLC separation coupled with the results of other physicochemical methods, such as the determination of the UV, IR and fluorescent spectra, and MS characteristics, can unambiguously identify the structure of the unknown analyte. Common TLC techniques are not suitable for exact quantitative measurements. Semiquantitative measurement of the quantity of analyses in a spot can be performed visually. Each spot of sample is applied between two spots of standard, and after development

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the size and the intensity of spots are compared. This semiquantitative technique is rapid, furthermore it needs no expensive instrumentation. However, it requires trained personnel and the reproducibility is about 10 per cent. A more advanced method is the extraction of the analyte from the stationary phase and the measurement of its concentration by spectrometry. The extraction process is time consuming and the reproducibility is always lower than that of HPLC. The advent of sensitive chromatogram spectrophotometers (TLC scanners) has revolutionized TLC methodology. In order to achieve maximal sensitivity, scanning is performed at the maximum absorption wavelength of the analyte. As the maximal wavelength may be different in the adsorbed state, in situ determination of the adsorption spectra is recommended. Scanning is commonly carried out in reflectance mode: a monochromatic light is directed onto the plate, and the intensity of the reflected diffuse light is determined with a detector system. Calibration curves have to be employed for accurate quantitative evaluation. The amount of the unknown compound and the standard has to be as similar as possible because the extrapolation outside of the highest or lowest points of the calibration curve may lead to distorted results. A calibration curve can be constructed from both the peak height or peak area. Both methods give similar results when the peaks are narrow and symmetric. The application of peak area for the calibration curve is more frequently employed. Up-to-date TLC apparatus increases not only the reproducibility but also the sensitivity of the technique; the detection of analyte in the nanogram range has often been reported. Modern instrumental TLC complies with the requirements of good laboratory practice (GLP) such as the stability of the stationary phase (surface homogeneity, standard specific surface area and pore volume, invariable retention strength and separation capcity, etc). Validation processes can also be applied for any up-to-date TLC analytical procedure. These procedures are easy to carry out, reliable, and the cost per analysis is relatively low because of the high sample throughput. As the majority of the impurities remain on the start, not interfering with the separation, the sample preparation is not complicated. Because of the basic similarity of TLC and HPLC, TLC can be used as the pilot method for HPLC analyses. The benefits of using TLC as a pilot method for HPLC are the simultaneous measurement of the retention of a high number of solutes, the rapid testing of mobile phase systems, and easier detection of analyses or impurities moving together with the eluent front, remaining at the start or showing very low mobility. Materials eluting with the eluent front do not cause problems in HPLC. However, impurities remaining at the start or showing very low mobility can adsorb on the surface of the stationary phase decreasing the theoretical plate number and the efficacy of separation. Their removal from the column is generally tedious or practically impossible. Both preventive maintenance and troubleshooting are the basic requirements for continuous and accurate chromatographic analysis not only in TLC but also in each field of chromatographic work. After opening the sealed packing of ready-made plates they are exposed to humidity and laboratory vapours. Because of the high adsorption capacity of the stationary phases they can bind volatile compounds to their surface. Unfortunately, this clandestine adsorption causes no visual change of the plate, the deteriorating effect can be detected only after the unsuccessful separation process. The adsorbed molecules may uncontrollably modify the retention characteristics of the stationary phase resulting in lower reproducibility and/or irregular spot shape. Plates can be protected by storing them

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Chapter 1

in well-closed plastic or metal (inoxidable steel) containers. Heating of the plates under strictly controlled conditions (constant temperature and heating time) generally eliminates the problem. However, it has to be borne in mind that pyridine is moderately volatile and can strongly adsorb on the slightly acidic adsorption centres of silica. The temperature and length of heating considerably depend on the physicochemical character of the adsorbed compounds and on the strength of their interaction with the stationary phase. The application of analyses onto the plates has to be carried out very carefully. Damaged parts influence the mobility of the eluent, and the changed ratio of stationary and mobile phases leads to modified retention and distorted spot symmetry. The plates cannot be damaged when loops, capillaries and syringes are employed for the application of samples onto the plates. Moreover, the application device has to be very carefully washed between spotting the various samples and standards to prevent cross-contamination. The reproducibility and repeatability of the quantitative analysis may be strongly reduced by cross-contamination of samples and standards. The partial or total inmiscibility of the various components of the eluent can produce serious problems because the unmixed components of the mobile phase can migrate independently of each other making the separation impossible. The miscibility of the components has to be verified very thoroughly. In the case of binary systems the miscibility can be controlled by using miscibility tables. However, it has to be emphasized that the addition of the third or fourth solvent or solid compounds (i.e., acids, bases, salts) to the binary mobile phase may considerably modify the mixing properties of the system. Adequate mixing tables for such a system do not exist. TLC separation has to be performed at constant temperature and without any air flow around the chamber. The composition of both the vapour and the mobile phase changes with changing temperature, which increases the variation in the retention behaviour of solutes. 1.3 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY High-performance liquid chromatography (HPLC) is one of the most frequently used chromatographic techniques. As gas chromatography can be employed for the analysis of about 20 per cent of all compounds without derivatization, HPLC can be used for the separation of each compound which is soluble in any mobile phase system. HPLC separation is carried out with a liquid mobile phase and a solid stationary phase filled in columns. The great variety of HPLC methods may be due to the fact that both stationary and mobile phases can be modified according to the requirements of the separation process. Similarly to TLC, various modes of separation can also be chosen in HPLC by combining various stationary and mobile phases of different polarities. Besides changing polarities, diverse mechanisms of separation can be employed using other physicochemical principles such as ion exchange and size exclusion. These procedures make possible the successful separation and quantitative analysis of compounds which cannot be analysed with traditional normal or reversed-phase HPLC techniques. The various theoretical and practical aspects of the use of HPLC methods have been recently discussed in exquisite books, such as the application of HPLC-MS in drug analysis [59], the theory of chromatography [60], the fundamentals of chromatography [61, 62], the practice and theory of ion-chromatography [63], problem solving in HPLC [64], the

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analysis of food [65], macromolecules [66] and peptides [67], etc. In recent years a considerable number of reviews have also been published on the more important developments in HPLC instrumentation and practical applications, such as the monitoring of exposure to neoplastic agents [68], the comparison of analytical methods for the determination of statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) [69], the analysis of chiral drugs [70], speciation of arsenics [71], monitoring haloacetic acids in drinking water [72], separation of humic substances [73], etc. HPLC techniques can be employed not only for the separation and quantitative determination of extremely small quantities of analyses but also can be used for the purification of larger amounts of compounds by preparative HPLC. The theory and practice of preparative liquid chromatography has been reviewed earlier [74]. 1.3.1 Fundamentals The theoretical plate height (H) related to the separation capacity of the system can be described by B H ⫽ A ⫹ ⫹ Cs ⭈
⫹ Cm


(1.6)

or by H ⫽ H p ⫹ H d ⫹ Hs ⫹ H m

(1.7)

where µ is the linear velocity of the eluent, Hp or A is the theoretical plate height caused by various diffusion processes, and Hd is the contribution of the molecular diffusion to the theoretical plate height. Hp depends on the type of the stationary phase, on its particle diameter and the mode of packing. Hd can be described as Hd ⫽

b ⫽ 2⭈Dm


(1.8)

where Dm is the diffusion coefficient of the eluent. The term Hd is generally negligible because of the very low coefficient of diffusion in liquid mobile phases. Hs is the theoretical plate height related to the mass transfer in the stationary phase and shows the peak broadening caused by the resistance of mass transfer to the support. It can be described by

Hs ⫽ Cs ⭈
⫽

2⭈ds2 ⭈k⬘⭈
3⭈Ds (1⫹ k⬘)2

(1.9)

where ds is the thickness of the liquid on the surface of the stationary phase, k⬘ is the capacity factor, and Ds is the diffusion coefficient of the analyte. The equations above indicate

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that thin liquid film on the stationary phase with a high Ds value on the support and a low velocity of the mobile phase enhances the separation efficacy. The term Hm, the theoretical plate height based on the mass transfer in the eluent, can be described by H m ⫽ Cm ⭈
⫽

w⭈dp2 ⭈


(1.10)

Dm

where w is a constant and dp is the mean particle diameter. Substitution of Eqns. 1.8 – 1.10 in Eqn. 1.7 results in H ⫽ H p ⫹ H d ⫹ H s ⫹ H m ⫽ 2 dp ⫹

2 k⬘ds 2u 3(1⫹ k⬘)2 Ds

⫹

dp2 u Dm

(1.11)

The theoretical plate height can also be described by H ⫽ 1Ⲑ (1ⲐH p ⫹1ⲐH pm ) ⫹ H d Hs H m

(1.12)

where Hpm is the Hp value corrected for the multipath effect. A more simple equation has also been proposed for the calculation of theoretical plate height: h ⫽ A.
1Ⲑ 3 ⫹ B Ⲑ
⫹ C Ⲑ


(1.13)

where h is the reduced plate height (H/dp), and µ is the reduced velocity (µ.dp/Dm). The value of A generally varies between 0.2 –1.7 and decreases with increasing homogeneity of the packing. The value of B is between 1.6 and 1.8, whereas C is 0.05 – 0.03. The minimum value of h is 2–3 when µ is 2–3. Thus, H ⫽ 2–3.dp is optimal in HPLC. According to the equations above a theoretical plate number of 40.000 can be achieved in a 25 cm column packed with a stationary phase of 3 µm particle size. Peak capacity is an important parameter in practical HPLC. It determines the possible number of peaks present in the chromatogram. A packed column with 5 000 theoretical plates can yield a peak capacity of 17 (k’ varying from 0.2 to 2) or a peak capacity of about 50 k’ value being between 0.5 and 20. Retention in HPLC is measured by the capacity factor: k⬘⫽ (t R ⫺ t0 ) Ⲑt0

(1.14)

where tR is the retention time of solute retained in the given HPLC system (time passed from the injection to the appearance of the peak maximum of the analyte), and t0 is the dead time. The exact measurement of dead time is of paramount importance for theoretical works and it is necessary for the accurate determination of capacity factor. Dead time can be defined as the retention time (or retention volume when it is multiplied by the flow rate) of the unretained analyte. Dead time can equal the elution volume of the solvent disturbance peak

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caused by injecting one component of the mobile phase or the elution volume of a molecule showing the lowest possible retention time. Isotopically labelled components of the mobile phase can also be employed for the determination of dead time. The method most frequently used in RP-HPLC is the injection of the solution of UV active salt (potassium iodide or sodium nitrate). However, it has to be taken into consideration that salts may interact with the analyte and/or with the components or additives of the mobile phase. The dependence of the ratio of capacity factors of an analyte on the polarity (P⬘) of the mobile phase can be described by ⬘ ⬘ k2⬘ ⫽ 10(P1⫺P2 )2 ⬘ k1

(1.15)

where k⬘2 and k⬘1 are the capacity factors of the analyte in the second and first mobile phases, and P⬘1 and P⬘2 are the polarities of the first and second mobile phases. In the case of a binary eluent system the dependence of the capacity factor on the volume fraction of the component with the higher elution strength (C) can be calculated by logk⬘⫽ logk0⬘ ⫹ bC

(1.16)

where log k⬘ is the capacity factor determined at a given concentration of the stronger component in the eluent, log k⬘0 is the logarithm of the capacity factor extrapolated to 100 per cent concentration of the weaker component of the mobile phase, and b indicates the change of the log k’ value caused by unit change of C in the mobile phase. Similarly to TLC, HPLC separations are achieved with a liquid mobile phase and a solid stationary phase reversibly adsorbing analyses. Adsorption (direct or normal phase) techniques apply a polar stationary phase such as silica, alumina, zirconia and porous glass, and a relatively nonpolar organic solvent or solvent mixture as mobile phase. Normal phase separation is suitable for the analysis of molecules with one or more functional groups and for the differentiation among isomers. The adsorption of one solute molecule on the surface of the stationary phase requires the displacement of one adsorbed eluent molecule. As polar substructures of analyte readily interact with the polar adsorption on the support surface, the difference of the strength of these interactions results in the different retention and consequent separation of the analyses. The components of the mobile phase in normal phase HPLC have to comply with the following requirements: low viscosity, relatively low boiling point, detector compatibility (low cut-off in the UV region), miscibility with a high number of other mobile phase components, negligible toxicity and environmental pollution capacity. The overwhelming majority of both normal and reversed phase HPLC separations employ silica or silica-based supports. Silica applied in HPLC is porous and noncrystalline with the general formula of SiO2.xH2O. The amount of water chemically bonded to the silica is not stoichiometric; the water molecules form silanol groups (Si-OH) with silica. The polar character of silica supports considerably depends on the number and character of silanol groups on the silica surface and they make possible the chemical modification of silica with various organic ligands. Silanols may occur in geminal (two hydroxyl groups

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on the same silica atom), vicinal (two hydroxyl groups on two neighbouring silica atoms) and isolated position. Silica stationary phases applied in HPLC generally contain isolated silanol groups. Because of the paramount importance of silanol groups in the determination of the separation characteristics of silica many methods have been developed and successfully employed for their analysis, such as traditional chemical procedures, isotope exchange, spectroscopy, etc. Silicic acid is a weak acid and it dissociates in the following manner: Si(OH)4 ⫽ Si(OH)3 O⫺ ⫹ H⫹

(1.17)

The dissociation constant is 1.6 ⫻ 10⫺10 corresponding to a pKa value of 9.8. The pKa value of the second dissociation step is 11.7: Si(OH)3 O⫺ ⫽ Si(OH)2 O22⫺ ⫹ H⫹

(1.18)

The silanol groups on the silica surface also show acidic character, the pKa value being 6.8: SiOH ⫽ SiO⫺ ⫹ H⫹

(1.19)

Silica stationary phases display some ion exchange properties, which may also influence the separation characteristics of silica. One of the main disadvantages of the use of silica and silica-based stationary phases is their instability even at slightly alkaline pH, such as 8.0. HPLC stationary phases can be characterized with the average particle diameter and the distribution of particle size. Smaller average diameter and narrow particle size distribution generally enhances the efficacy of separation. The average particle diameter can be calculated with different methods: dn ⫽ ∑ ni di ⲐN ⫽ ∑ ni di Ⲑ ∑ ni

(1.20)

where dn is the average particle diameter calculated according to the number of particles, ni is the number of particles with diameter d i , and N is the total number of particles in the sample. ds ⫽ (1ⲐS )∑ ni d 3

Ⲑ 4 ⫽ ∑ ni di3 Ⲑ ∑ ni d 2

(1.21)

where ds is the average particle diameter calculated according to the surface of particles, and S is the surface area of the sample. dm ⫽ (4p Ⲑ 3 M )冢 ∑ ni d 4 冣 ⫽ ∑ ni d 4 Ⲑ ∑ ni d 4 ⲐSni d 3

(1.22)

where dm is the average particle diameter calculated according to the mass of particles, M is the sample mass, and p is the density of the particles.

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Although smaller particle size generally increases the separation capacity of the stationary phase it also influences the permeability (P): P ⫽ (dp2 ⲐY )冤( 20 Ⲑ1⫺  0 )2 冥

(1.23)

where Y is a dimensionless shape factor (for spherical particles Y equals 180) and ε 0 denotes the external porosity (interstitial volume) in cm3. Permeability is further influenced by the linear mobile phase velocity u (cm/s), mobile phase viscosity g (g/cms) and the length of the HPLC column (cm): P ⫽ uvL ⲐP

(1.24)

The specific surface area of the stationary phase also plays a considerable role in the efficacy of separation. It is defined as the sum of the internal and external surface areas. The external surface area of spherical and uniform particles (A) is A ⫽ 6 Ⲑd 

(1.25)

where σ is the particle density (the density of nonporous silica equals 2.2 g/cm3). The specific surface areas of silica supports employed in HPLC show high variations, they are generally between 10 and 500 m2/g. The size and shape of pores, and their distribution also contribute to the separation process. It is obvious that the pore size and pore volume influence markedly the specific surface area. Silica-based stationary phases with a chemically bonded ligand on the surface can be characterized by the carbon content (grams of carbon per 100 g of packing) and by the bonding density (micromols of ligand bonded/square meter of initial silica surface area). High bonding density is preferable for the majority of separations. The number of silanol groups not covered by the ligand is lower, exerting less influence on the separation, moreover, the hydrophobic ligand enhances the stability of the stationary phase at alkaline pH. Normal-phase separation techniques can be preferably employed for the separation of nonionic molecules and positional isomers. Because of the acidic character of the surface silanol, groups of silica basic analyses are strongly bonded, sometimes exhibiting broad, tailing peaks which deteriorate separation efficacy, repeatability and reproducibility. Furthermore, the adsorption strength of the surface silica groups is not uniform, resulting also in irregular peak shape and decreasing separation performance. Reversed-phase HPLC stationary phases can be classified as ‘brush type’, ‘bulk type’ or ‘oligomeric type’. The brush phase is prepared by reacting monofunctional silanes (i.e., dimethyloctylchlorosilane) with the surface silanol groups with the elimination of hydrogen chloride formed during the reaction. The surface of the original silica support is covered with the organic ligands like bristles on a brush. The bulk phase can be produced by reacting trifunctional silanes (i.e., octyltrichlorosilane) with silica support. Trifunctional silanes react not only with the surface silanol groups but also with each other, forming a polymer coating on the silica surface. When bifunctional silanes are reacted with the silica support

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they form an oligomeric cover on the surface. Because of the different structure of bonded phases their retention properties show marked differences depending on the type of analytes and on the composition of the mobile phase. The mode of interaction between analytes and the surface of the stationary phase has also been extensively studied in RP-HPLC. It is generally accepted that hydrophobic interactions play a decisive role in the solute–stationary phase interaction. An intermolecular binding between the apolar substructure of the analyte and the hydrophobic ligand occurs and influences retention. More hydrophobic analyses are more strongly retained on stationary phases with more or longer hydrophobic ligands. The length of the alkyl chain of the ligand molecule and its surface density also exert a considerable influence on the strength and selectivity of retention. Non-silica-based RP-HPLC stationary phases have also been developed and their separation capacity has been compared with those of silica-based ones. The porous structure of crosslinked polymer gels may be responsible for the markedly different selectivity and retention characteristics. Up till now, the mode of separation on polymer stationary phases is not entirely understood at the molecular level. It has been established that the size-exclusion effect may influence the retention of analyses on polymer gels. Much effort has been devoted to the reliable prediction of the retention of analyses in any RP-HPLC system. A considerable number of quantitative structure–retention relationship (QSRR) studies have been carried out using various solutes, mobile and stationary phases. The objectives of QSRRs are the prediction of the retention of a new analyte, the elucidation of structural descriptors governing retention, the determination of the molecular mechanism of separation, the assessment of the physicochemical parameters of solutes (other than chromatographic), and the estimation of the efficacy of bioactive compounds on the basis of their chromatographic behaviour. Calculations indicate that the following intermolecular interactions influence retention: dipole–dipole, dipole-induced dipole, instantaneous dipole-induced dipole, hydrogen bonding, electron pair donor–electron pair acceptor and solvophobic interactions. The energy of dipole–dipole interactions (E) can be described by E ⫽⫺W 2 ⫺1r⫺6 冤2
12
22 Ⲑ 3kT ⫹ 2
12 ⫹ 1
22 ⫹ 3I1 I 2 12 Ⲑ 2( I1 ⫹ I 2 )冥

(1.26)

where W and k are constants, ε is the relative electric permittivity of the medium, r is the distance between the interacting molecules, T is the absolute temperature, and µ , α, and I are the dipole moment, polarizability and ionization potential of the interacting molecules, respectively. A wide variety of molecular parameters has been included in QSRR calculations, such as bulkiness-related parameters (molecular mass, refractivity, molecular volume, total energy, solvent-accessible area, Taft constant), geometry-related parameters (moments of inertia, length-to-breadth ratio, Sterimol width parameters, angle strain energy), physicochemical parameters (hydrophobic constant, Hammett constant, solubility parameters, boiling point, solvatochromic parameters), polarity-related (electronic) parameters (Swain–Lupton’s constant, dipole moments, atomic excess charges, orbital energies, superdelocalizabilities, partially charged surfaces), and molecular graph-derived (topological) parameters (adjacency matrix indices, distance matrix indices, information content

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indices). When QSRR calculation is carried out on a set of solutes with similar chemical structure (the same basic structure with different substituents) the correlation between the molecular parameters and retention is generally good. However, the physicochemical parameters influencing the retention strongly depend on the properties of the analytes, and on the character of the mobile and stationary phases. To the best of our knowledge the relationship describing the retention of any solutes in any RP-HPLC system is missing and it is improbable that this relationship will be found in the near future. The correlation between molecular characteristics and retention is usually poor in the case of analytes with basically different molecular structures (polar and apolar molecules, basic and acidic compounds, linear and ring structures, etc). The same is valid for the calculation of the relationship between bioactivity and retention of solutes. Various biological activities (pharmaceutical effect, toxicity, environmental polluting capacity, etc.) can be well correlated with retention in the case of homologues series (congeneric) and it is generally invalid for molecules of inhomogeneous chemical structures. As the logarithm of 1-octanol-water partition coefficient (log P) describes the hydrophobicity of molecules and the retention of solutes in RP-HPLC depends on the hydrophobicity, a strong correlation can be expected between the log P value and the retention of solutes in RP-HPLC. Besides log P, a considerable number of physicochemical parameters have been tested for their capacity to predict retention in RP-HPLC. Thus, Snyder’s polarity index, fraction of positively and negatively charged surface area, molecular bulkiness, nonpolar surface area, electron donor and acceptor capacity, various sterical parameters, and the energy of highest occupied molecular orbit have all been included in QSRR calculations. Only the silica-based stationary phases with covalently bonded alkyl chain, cyano and propylamino ligands have found practical applications in HPLC. Besides these common ligands, the experimental use of naphthalene, pyrene and nitroaromatic as ligands has also been reported. Silica-based stationary phases with covalently bonded cyclodextrins or cyclodextrin derivatives have been frequently employed in the separation and quantitative determination of isomer pairs. Besides silica, silica-based and polymeric stationary phases, porous graphitized carbon (PGC), zirconium oxide and its derivatives, alumina and its derivatives have been used for the solution of special separation problems which cannot be easily solved by using traditional HPLC stationary phases. PGC support possesses a rigid planar surface and it is capable of dispersion and charge transfer interactions. PGC demonstrates special retention characteristics deviating from those of silica-based stationary phases. The retention order of analyses does not follow their order of lipophilicity, proving that interactions other than hydrophobic influence the retention. Electrostatic interactions between the planar ring structures of solutes and the hexagonal graphite molecules on the PHC surface exert a marked impact on the retention. Because of its high stability at extreme pH, and the neutrality of the surface, PGC is especially suitable for the separation of a high number of polar and apolar compounds. Positional isomers with one or more polar substructures are also well separated on PGC. PGC support can be characterized by sufficient hardness to withstand high pressures, stability during the chromatographic separation process, specific surface area of 150 – 200 m2/g, mean pore diameter of⭓10 nm, absence of micropores, and uniform surface energy.

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The eluotropic strength of mobile phase constituents also differs from that determined on other RP (silica-based) stationary phases. The eluotropic strength of methanol and acetonitrile (ACN) is relatively low, while dioxane and tetrahydrofuran (THF) show high eluotropic strength. Interestingly, the eluotropic strength depends to a greater extent on the chemical character of the analyses than on silica-based RP stationary phases. It has been established that retention on PGC depends on the electron–donor and acceptor capacity of the analyses, and on the steric correspondence of the solute substructures with the hexagonal graphite units of the PGC surface. Zirconium dioxide shows both Lewis basicity and acidity, the oxygen atom bearing a negative the zirconium atom a positive charge. Zirconia exists in crystallographic and amorphous forms, demonstrating different retention characteristics. The characteristics of zirconia surface and the pore size can be easily modified by heat treatment from 200 to 2 680oC. The special stability of zirconia at extreme pH has often been demonstrated, which offers a distinct advantage over silica and alumina. Moreover, zirconia is amphoteric and shows anion exchange properties in neutral and acidic environments, and cation-exchange properties in the alkaline region. The surface nature zirconia has been modified with silanization, polybutadiene, polystyrene, polyethylene, octadecyl and carbon coating and the retention behaviour of these zirconia-based phases has been measured. Because of its higher pH stability, alumina and alumina-based stationary phases offer an alternative to silica and silica-based stationary phases. Although the overwhelming majority of separations in HPLC are carried out with the supports described above, separations using other basic principles have also found application in HPLC. Ion-pair chromatography separates ionic compounds using traditional RP stationary phases. A so-called counter-ion of opposite charge is added to the mobile phase. It forms a neutral ion-pair which can be easily separated under RP conditions. The mobile phase generally consists of water or buffer mixed with an organic modifier such as methanol or ACN. Separation in ion-exchange HPLC is based on the interaction of the charged solute with the oppositely charged surface of the stationary phase. Cation-exchange chromatography is employed for the separation of positive ions using a negatively charged stationary phase. In the case of anion-exchange chromatography, anions are retained on a positively charged stationary phase. The retention order of cations and anions is Ba2⫹
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phases the method is named gel filtration chromatography (GFC). The stationary phases applied in these separation procedures are porous particles with strictly controlled pore size. Theoretically, the analyses do not interact with the surface of the stationary phase in SEC. The molecules of solute can enter the pores of the particles of the stationary phase depending on their effective size and shape. Large solute molecules cannot enter the pores of the stationary phase; they are not retained and are eluted rapidly from the separation column. Small molecules are also not retained because they diffuse through the porous network. Only molecules with bulkiness corresponding more or less to the dimensions of pores are retained and separated during the chromatographic process. The selection of an eluent for this chromatographic technique is simpler than for other HPLC methods because generally one solvent is required. Affinity chromatography is based on specific interactions between the analyte and the corresponding molecules bonded to the stationary phase. This method is highly specific and can separate only one or several molecules in the sample according to their affinity to the components of the stationary phase (i.e., enzyme–substrate interaction). Separation is generally carried out in two steps. In the first step the interacting species (target molecules) in the sample can be adsorbed on the stationary phase and the non-interacting components are eluted. The target molecules can be eluted from the affinity column by changing either ion strength and pH and/or both of them simultaneously. 1.3.2 New theoretical advances in high-performance liquid chromatography Chromatography has been developed as an empirical method for the separation of compounds which are not or not easily separated by other analytical techniques. However, the rapid increase of practical separation procedures necessitated the development of the theory of chromatography. Theory not only explains the physicochemical procedures underlying existing chromatographic separations but also creates a solid base for the development of new technologies. Theoretical advances also promote the rapid progress of the practice in various fields of chromatographic separation such as the liquid chromatography of natural pigments and synthetic dyes. Beds of porous spherical particles are generally applied in chromatography. However, when the sample contains particles such as cell debris, etc. the sample has to be pretreated before injection, making the separation process longer. Expanded beds using beads of different sizes and densities overcome this difficulty. In order to evaluate the efficacy of the expanded bed technique the plate height (HETP), plate number (N ), resolution (Rs ), Bodenstein number (Bo), particle Peclet number (Pep ) and axial dispersion coefficient (Dax) have been calculated and compared with the corresponding values of a traditional HPLC column. N can be expressed by N ⫽ ( t r  t )2 or

N ⫽ ( L Ⲑ L )2

(1.27) (1.28)

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where σt and σL are the standard deviations in time and length units, L is the column height and t is the residence time. The correlation between the standard deviation and Dax can be described by  2t Ⲑtr2 ⫽ 2 Dax Ⲑ (Ui L )

(1.29)

where Ui is the interstitial flow rate. The Bodenstein number can be defined by Bo ⫽ Ui L ⲐDAX

(1.30)

In the case of higher dispersion  2t Ⲑtr2 ⫽ 2 ⲐBo ⫺ 2 ⲐBo2 (1⫺ e⫺Bo )

(1.31)

Dispersion can be characterized by the Pep: Pe p = Ui dp ⲐDAX

(1.32)

where dp is the particle diameter. NEB, the expanded bed plate number, can be expressed by N EB ⫽ ( L0 Ⲑ L )2 ⫽ ( L0 Ⲑ L )2 Ⲑexp2 ⫽ N Ⲑexp2

(1.33)

where L0 is the bed height before expansion and exp is the degree of expansion (L/L0). The plate height equivalent for expanded beds is HETPEB ⫽ L0 ⲐN EB ⫽ HETP⭈exp

(1.34)

The chromatographic parameters discussed above were calculated for packed beds (increased dispersion and bed length), and for expanded beds with moderate, higher and lower dispersion. The results indicated that the corresponding separation parameters of packed beds and expanded beds are commensurable, therefore, expanded beds can be successfully employed in liquid chromatography even in the case of trace analysis of synthetic dyes in waste water and sludge [75]. Novel general expressions were developed for the description of the behaviour of the height equivalent of a theoretical plate in various chromatographic columns such as unpacked (open capillary), packed with spherical nonporous particles and packed with spherical porous adsorbent particles. Particles may have unimodal or bimodal pore size distribution. The expression describing the mass balance in open capillaries is Cd C 2Cd C  2  ⫹Vx d ⫺ DL ⫹ K f  Cd ⫺ s  ⫽ 0 2  t x Rc K x

(1.35)

Cs C  2  ⫽ K f  Cd ⫺ s   t Rc K

(1.36)

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where Cd is the concentration of the solute in the liquid stream of the capillary, Cs denotes the concentration of solute in the adsorbed phase, Vx is the linear velocity of the mobile phase along the axis x of the capillary, K represents the equilibrium adsorption constant, Rc is the radius of the capillary, t denotes time, x is the axial coordinate of the capillary, L is the column length, and δ (t) is the Dirac delta function. The initial and boundary conditions for eqns 1.35 and 1.36 are at t ⫽ 0, Cd ⫽ 0, for 0 ⭐ x ⭐ L at t ⫽ 0, Cs ⫽ 0

at

at

x ⫽ 0, Vx Cd

x ⫽ L,

x⫽0 ⫺DL

C d x

⫽ Vx (t ), for t 典0

(1.37)

(1.38)

x⫽0

Cd 冨x⫽L ⫽ 0, for t 典0 x

(1.39)

The height equivalent of a theoretical plate H for open capillaries can be described by H⫽

dc K 2 2 DL ⫹ Vx Vx 2 K f (1⫹ K )2

(1.40)

where DL is the axial dispersion coefficient, dc represents the diameter of the open cylindrical capillary (dc ⫽ 2.Rc ), and Kf denotes the film mass transfer coefficient. In the case of chromatographic columns packed with spherical nonporous adsorbent particles the differential mass transfer balance can be described by C  Cd C 2Cd (1⫺  b ) 3  K f Cd ⫺ s  ⫽ 0 ⫹Vx d ⫺ DL ⫹ K t x  b R  x 2

(1.41)

Cs C  2  ⫽ K f  Cd ⫺ s   t Rc K

(1.42)

where Cd is the concentration of solute in the interstitial channels of the packed bed, Cs denotes the concentration of solute in the adsorbed phase, Vx represents the linear velocity of the mobile phase in the interstitial channels along axis x of the packed column, Rp is the radius of the nonporous particles, and ε b denotes the void fraction of the packed bed. Plate height can be calculated by H⫽

 b (1⫺  b )d p K 2 2 DL ⫹ Vx Vx 3K f ( b ⫹ (1⫺  b )K )2

where dp is the diameter of the nonporous particles (dp ⫽ 2.Rp).

(1.43)

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In the case of a chromatographic column packed with porous adsorbent particles showing a unimodal pore size distribution, the differential mass balance of the solute in a porous adsorbent particle can be described by

R ⫽ R p , K f 冢Cd ⫺ C p冨 R⫽R 冣 ⫺  p D p p

C p R

冷R⫽Rp ⫹ p
pR C p 冷R⫽Rp ⫽ 0

(1.44)

for t ⬎ 0,0 ⭐  ⭐  where Cd is the concentration of the solute in the interstitial channels for bulk flow, Cp denotes the concentration of solute in the pores of the porous particle, R is the radial direction in the porous particle, θ denotes the angular direction in the porous particle, Rp is the radius of the porous adsorbent particle, Kf is the film mass transfer coefficient, ε p denotes the porosity of the porous particle, vpR is the radial component of the intraparticle connective velocity, and Dp represents the pore diffusion coefficient. The differential mass balance of the solute in the interstitial channels of the packed column can be described by Cd C 2 Dd (1⫺  b ) C p ⫹Vx d ⫺ DL ⫹ ⭈ ⫽0 t x b t x 2

(1.45)

where Cd represents the concentration of solute in the interstitial channels of the packed bed, Cp denotes the average concentration of solute in the adsorbent particle, Vx is the linear velocity of the mobile phase in the interstitial channels along the axis x of the packed column, Rp is the radius of the porous pariles, and εb denotes the void fraction of packed bed. In the case of columns packed with spherical porous absorbent particles showing unimodal pore-size distribution the theoretical plate is

H⫽

2 DL x ⬙ L2 ⫺ ⭈ Vx Vx ( x⬘)2 DL

(1.46)

The equations are slightly modified in the case of porous adsorbent particles having bimodal pore-size distribution. The differential mass balance of solute in the micropores of the porous adsorbent particles can only be described with a complicated equation including the porosity of the macroporous and microporous region of the adsorbent particles, the concentration of the solute in the microporous region of particles, the radial component of velocity in the pores of the microporous region, the angular component of the velocity in the microporous region of particles, the pore diffusion coefficient of the solute in the microporous region, the radial coordinate of the microporous region, the angular coordinate of the microporous region, the radial coordinate of the macroporous region and the angular coordinate of the macroporous region. The results of calculations have been

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compared with experimental data and a good agreement was found between the theoretical equations and the concrete measurements. It has been stated that the new equations can be successfully employed for the description of the dependence of the plate height on the linear velocity of the mobile phase both in HPLC and capillary electrochromatography (CEC) and they promote better understanding of the physicochemical processes governing chromatographic separation of a wide variety of compounds, including natural pigments and synthetic dyes [76]. The final objective of each chromatographic analysis is the separation and quantitative determination of the maximal number of sample components in a minimal time. The performance of packed columns and monolithic columns using high mobile phase velocities has been compared. Interestingly, the pressure limitation was the decisive factor of packed columns for fast separations. Monolithic columns can be operated at higher flow rates, decreasing considerably the analysis time. However, it has been observed that the separation of some compounds markedly deteriorated at high mobile-phase velocities. This effect was tentatively explained by the slow mass transfer of the solutes under investigation [77]. As plants usually contain a high amount of pigment fractions (even up to more than 70 pigments isolated by RP-HPLC) the whole separation of the pigments is generally time consuming. The rapid RP-HPLC technique using small spherical particles or monolithic columns may enhance the data output of control laboratories increasing efficacy and decreasing expenses. As the overwhelming majority of HPLC separation is carried out in the reversed-phase mode, it is understandable that theoretical work is preferably concentrated on the various aspects of RP-HPLC systems. Thus, theory has been employed on chromatographic fingerprints frequently used in the quality control of complex natural mixtures. They can facilitate the determination of the authenticity and traceability of the natural product under investigation. Chromatographic fingerprints are chromatographic patterns of complicated multicomponent samples without the exact identification of each sample component. The application of hyphenated techniques highly increases their information content. The practical value of a chromatographic fingerprint increases with increasing separation capacity of the RP-HPLC system and the concentration distribution of each absolute molecule. Some evaluation methods use only the retention time, peak area and/or peak height of the individual components separated. A fingerprint can be regarded as a continuous signal composed of the sum of the concentration of each component. The information content of a fingerprint (Φ ) can be calculated after normalizing with its overall peak area equal to one. Each real chromatographic response is divided by the sum of each real chromatographic response. The information content is calculated by the summation of the ratios mentioned above. The advantages of this calculation method are that each characteristic of the chromatogram is taken into consideration, the peaks need not to be identified, and the noise exerts a negligible influence on the calculation. The evaluation of simulated and real chromatograms reveals that the information content of the chromatogram is the highest in the case of baseline separation and identical concentration of the components while overlapping peaks with varying intensity markedly reduce the information content. It was further established that the method facilitates the selection of chromatographic conditions producing chromatographic profiles with the highest possible information content, consequently, it can be successfully employed in quality control of mixtures of natural origin

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[78]. According to the data, the procedure can also be applied to the determination of the similarities and dissimilarities of natural pigment mixtures. Much effort has been devoted to the development of reliable calculation methods for the prediction of the retention behaviour of analyses with well-known chemical structure and physicochemical parameters. Calculations can facilitate the rapid optimization of the separation process, reducing the number of preliminary experiments required for optimization. It has been earlier recognized that only one physicochemical parameter is not sufficient for the prediction of the retention of analyte in any RP-HPLC system. One of the most popular multivariate models for the calculation of the retention parameters of analyte is the linear solvation energy relationship (LSER): log k ⫽ log k0 ⫹
V2 ⫹ sⴱ2 ⫹ a ∑ 2H ⫹ b∑ 2H ⫹ rR2

(1.47)

where k denotes the analyte retention, the subscript 2 represent solute descriptors such as molar volume (V2 ), dipolarity/polarizability (π2* ), overall hydrogen-bond acidity (Σα 2H ), overall hydrogen-bond basicity (Σβ2H), and excess molar refraction R2. The regression coefficients of descriptors denote the system (combination of mobile and stationary phases) response to these interactions. These coefficients can be measured, however the procedure is time consuming and inappropriate for practical purposes. According to the linear solvent strength theory (LSST) the retention of the analyte depends on the volume fraction (ϕ) of the organic modifier in binary mobile phase systems: log k ⫽ log kw ⫺ S

(1.48)

where log kw is the hypothetical retention of the analyte in pure water as mobile phase. Eqn. 1.48 can be used for the prediction of the retention of analyte at any volume fraction of organic modifier, however, the determination of log kw and S has to be carried out for each component of the sample. Retention of an analyte is related to the equilibrium constant (K)of its distribution between the stationary and mobile phases. Moreover, it is related to the free energy of analyte transfer from the mobile phase to the stationary phase G o: log k ⫽ log  ⫹ log K ⫽ log  ⫺⌬G o Ⲑ 2.3 RT

(1.49)

where ϕ is the ratio of the volume of the stationary phase to the volume of the mobile phase in the column. Log kw can be related by the free energy of analyte transfer from water to the stationary phase ( Gwo): ⌬Gwo ⫽⫺2.3RT log

kw 

(1.50)

The regression coefficient of eqn.1.48 can be calculated by S ⫽ log kw ⫺ log korg

(1.51)

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where korg is the retention of the analyte in a pure organic mobile phase. S can be related to the free energy ‘org’ analyte transfer from water to pure organic eluent GS ⌬Gso ⫽⫺2.3 RTS

(1.52)

According to the LSER model: log kw ⫽ log k0,w ⫹
wV2 ⫹ Sw ⴱ2 ⫹ aw ∑ 2H ⫹ bw ∑ 2H ⫹ rw R2

(1.53)

S ⫽ log k0,s ⫹
sV2 ⫹ Ss ⴱ2 ⫹ as ∑ H2 ⫹ bs ∑ H2 ⫹ rs R2

(1.54)

where subscript w denotes LSER coefficients for logkw, and subscript S represent LSER coefficients for S. Global LSER can be expressed by log k ⫽ (log k0,w ⫺ log k0,s) ⫹ (
w ⫺
s)V2 ⫹ (sw ⫺ ss)ⴱ2 +(aw ⫺ as)∑ H2 ⫹ (bw ⫺ bs)∑ H2 ⫹ (rw ⫺ rs) R2

(1.55)

It has been stated that the global LSER equation (eq. 1.55) takes into consideration simultaneously the descriptors of the analyte and the composition of the binary mobile phase and it can be more easily employed than the traditional local LSER model [79]. The prerequisite of the application of LSER calculations is the exact knowledge of the chemical structure and physicochemical characteristics of the analyses to be separated. Synthetic dyes as pollutants in waste water and sludge comply with these requirements, therefore in these cases LSER calculations can be used for the facilitation of the development of optimal separation strategy. Unfortunately, mixtures of natural pigments generally contain one or more unidentified pigment components making impossible the prediction of their retention behaviour in any RP-HPLC system. Global LSER calculations have also been applied to the study of the retention of ionizable analyses in RP-HPLC. While the retention of neutral analyses does not depend on the pH of the mobile phase the retention of analyses with one or more ionizable substructures considerably depends on the pH even at the same concentration of organic modifier in the eluent. The relationship between the retention and pH of the mobile phase and pK value of the analyte can be described by k ⫽ 冢 k HA ⫹ k A 10 pH⫺ pK 冣 Ⲑ 冢1⫹10 pH⫺ pK 冣

(1.56)

where kHA and kA are the retention of the acidic and basic forms of the analyte, and pK is the dissociation constant of the analyte. The retention factors of neutral (k0) and ionized forms (k1) can be related by the parameter f: log f ⫽ log k1 ⫺ log k0

(1.57)

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Eqn. 1.57 can be rewritten as log k ⫽ log k0 ⫹ log[1⫺ D(1⫺ f )]

(1.58)

where D is a descriptor for the degree of ionization of the analyte at the given pH of the eluent. D can be expressed by D ⫽ 10 pH⫺ pK Ⲑ 冢1⫹10 pH⫺ pK 冣

(1.59)

The retention of ionizable analyte can be calculated by log k ⫽ log k0 ⫹ .V2 ⫹ s.ⴱ2 ⫹ .H2 ⫹ b. H2 ⫹ r. R2 ⫹ d.log[1⫺ D(1⫺ f )]

(1.60)

or including the parameters taking into consideration the composition of the binary mobile phase: log k ⫽ (log k0,w ⫺ log k0,S .) ⫹ ( w ⫺ S .).V2 ⫹ (Sw ⫺ SS .).ⴱ2 ⫹ ( w ⫺  S .).H2 ⫹ (bw ⫺ bS .). H2 ⫹(rw ⫺ rS .).R2 ⫹ d.log[1⫺ D(1⫺ f )]

(1.61)

The dielectric constant of the mobile phase influences the pK value of the dissociable substructure of the analyte, it decreases with decreasing dielectric constant. This relationship can be approximately described by s w

pK ⫽ww pK ⫺ m pk 

(1.62)

where ws pK denotes the pK value determined in a given mixture of water and organic modifier, and ww pK represents the pK value determined in water. The validity of the theoretical calculations was controlled by concrete practical measurements. The calculated and experimental data show excellent agreement with the correlation coefficient showing highly significant correlation [80]. As the pH of the mobile phase markedly influences the retention of ionizable compounds, it can be assumed that the separation capacity of RP-HPLC for ionizable analyses can be modified by changing the pH of the mobile phase. The theory of effect of pH gradient on the performance of RP-HPLC systems has been recently elaborated. The basic equation describing the dependence of the retention of the solute on the gradient of pH or organic modifier is: VR⬘

∫0

1 dV ⫽1 V0 ki

(1.63)

where V denotes the cumulative volume of the mobile phase flowing through the column since the beginning of the gradient, V0 is the column void (‘dead’) volume and ki the

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retention factor of the solute corresponding to the composition of the mobile phase at the column inlet. The fractional band migration (dx) is dx ⫽ dV Ⲑ (V0 .ki )

(1.64)

and the reduced retention volume (V⬘R) is VR⬘ ⫽ VR ⫺ V0

(1.65)

The reduced retention time (t⬘R) is dt Ⲑ (t0 .ki ) ⫽ 1

(1.66)

The dependence of the retention factor on the pH of the mobile phase at constant concentration of organic modifier can be described for monoprotic acids as k ⫽ f[HA] k[HA] ⫹ f[A⫺ ] k[A⫺ ]

(1.67)

and monoprotic basis k ⫽ f[BH⫹ ] k[BH⫹ ] ⫹ f[B] k[B]

(1.68)

where the subscript is related to the dissociated or nondissociated forms of acid or base, and f is the mole fraction of the individual forms, which can be calculated by f[HA] ⫽

1 K a Ⲑ[H⫹ ]⫹1

(1.69)

f[A⫺ ]1⫺ f[HA]

(1.70)

where Ka is the dissociation constant of the ionizable group of the analyte, and [H+] is the hydrogen ion concentration. The dependence of the retention of ionizable analyses on the pH can be calculated for acids by k⫽

k[HA] ⫹ k[A⫺ ]10 pH⫺ pK a 1⫹10 pH⫺ pK a

⫽

k[A⫺ ] ⫹ k[HA]10 pK a ⫺ pH 1⫹10 pK a ⫺ pH

(1.71)

and for bases k⫽

k[BH⫹ ] ⫹ k[B]10 pH⫺ pK a 1⫹10 pH⫺ pK a

⫽

k[B] ⫹ k[BH⫹ ]10 pK a ⫺ pH 1⫹10 pK a ⫺ pH

(1.72)

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When the pH of the mobile phase changes linearly in time the pH change can be described by pH ⫽ pH 0 ⫹ at

(1.73)

where pH0 denotes the starting pH and a represents the programmed rate of pH change. Accordingly, the retention factor ki is for acids ki ⫽

k[A⫺ ] ⫹ k[HA]10 pK a ⫺( pH0 ⫹at ) 1⫹10 pK a ⫺( pH0 ⫹at )

(1.74)

and bases ki ⫽

k[BH⫹ ] ⫹ k[B]10( pH0 ⫹at )⫺ pK a 1⫹10( pH0 ⫹at ) ⫺ pK a

(1.75)

Also in this case the calculated (predicted) retention values showed good agreement with the experimental results. It has been concluded that pH gradient elution may enhance the separation efficacy of RP-HPLC systems when one or more analyses contain dissociable molecular parts [81]. As numerous natural pigments and synthetic dyes contain ionizable groups, the calculations and theories presented in [80] and [81] and discussed above may facilitate the prediction of the effect of mobile phase pH on their retention, and consequently may promote the rapid selection of optimal chromatographic conditions for their separation. Because of its advantages (high sensitivity and selectivity, low cost and miniaturization) amperometric detection has been frequently used in flow injection analysis (FIA) and RPHPLC. However, it has been established that the peak area (detector response) considerably depends on the flow rate. A general approach has been proposed to predict the effect of flow rate on the peak area in FIA and RP-HPLC. The general form of the correlation describing the flow in a parallel plate cell with short rectangular electrodes is

1Ⲑ 3

1Ⲑ 3

Sh ⫽ 1.85Re Sc

 dc   L  el

1Ⲑ 3

(1.76)

where Sh ⫽ km.dc /D is the Sherwood number, Re ⫽ ((U/S).dc /v is the Reynolds number, Sc ⫽ v/D is the Schmidt number, km is the electroactive species mass transport coefficient, D denotes its diffusion coefficient, v represents the solution kinematic viscosity, U is the solution volumetric flow rate, S is the channel cross-section, Lel is the electrode length and dc denotes the cell hydraulic diameter (dc ⫽ 4.S/P), P is the perimeter of the cross-section. Complicated theoretical considerations have resulted in the establishment of a logarithmic relationship between peak area and flow rate. Experimental data showed good correspondence between the predicted and measured values of peak area [82]. Amperometric detection has not been frequently applied in the RP-HPLC analysis of natural pigments and

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synthetic dyes, but because of its advantageous characteristics its acceptance and application in pigment and dye analysis can be expected. Surface diffusion considerably influences the mass transfer inside the intraparticle space of a porus adsorbent. The surface diffusion coefficient Ds can be expressed by  ⫺Es  Ds ⫽ Ds0 exp   RT 

(1.77)

where Ds0 is the frequency factor, Es denotes the activation energy of surface diffusion, R is the universal gas constant, and T is the absolute temperature. Es can be considered as the fraction of the isosteric heat of adsorption (Qst): Es ⫽ ⬘(⫺Qst )

(1.78)

where α⬘ is the ratio of Es to -Qst. Replacing Es with eqn.1.77, eqn.1.78 becomes ⫺⬘(⫺Qst )  Ds ⫽ Ds0 exp    RT 

(1.79)

Molecular diffusivity (Dm) can be expressed by

Dm ⫽ 7.4⭈10⫺8

(asv Msv )1Ⲑ 2 T 0.6 svVb.a

(1.80)

where α denotes the association coefficient, M is the molecular mass, v represents viscosity, and Vb is the molar volume at the normal boiling point. Subscripts ‘a’ and ‘sv’ represent the analyte and solvent, respectively. Intraparticle diffusivity (De) can be related to pore diffusivity (Dp) and surface diffusivity by De ⫽ Dp ⫹ p KDs

(1.81)

where K is the adsorption equilibrium constant. It has been supposed that the surface diffusion is a form of molecular diffusion, restricted by adsorption interaction. Consequently: ⫺ (⫺Qst )  Ds ⫽ Dm exp    RT 

(1.82)

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where β is the ratio of adsorptive interaction between adsorbate molecules and the surface of the stationary phase to -Qst. It has to be observed that the ratio of Ds /Dm depends on the temperature of the system. Molecular diffusivity can be expressed by  ⫺Em  Dm ⫽ Dm0 exp   RT 

(1.83)

where Dm0 and Em are the frequency factor and the activation energy of molecular diffusion, respectively. Em is related to the vaporization energy of the solvent corresponding to the energy needed for making a hole having the size of the solvent molecule. Finally Ds can be described by ⫺E ⫺ (⫺Qst )  Ds ⫽ Ds0 exp  h  RT  

(1.84)

where Eh denotes the activation energy of the hole-making process (kj mol⫺1). It has been concluded from the theoretical considerations and experimental measurements that surface and molecular diffusion are intercorrelated. It was further stated that this novel model makes possible the exact interpretation of the mass transfer mechanism and of the intrinsic characteristics of surface diffusion in RP-HPLC [83]. Natural extracts generally contain molecules with highly different retention characteristics which cannot be separated under isocratic conditions. The application of gradient elution is a necessity for these types of natural samples. However, the optimization of gradient elution on the base of isocratic data is cumbersome and the prediction of retention in gradient elution from isocratic data is difficult. Retention in an isocratic system can be described by a polynomial function: log k ⫽ c0 ⫹ c1 ⫹ c22

(1.85)

where c are regression coefficients. The quadratic member in eqn.1.85 can be negligible in a narrow concentration range of an organic modifier, resulting in the well-known eqn.1.48. The volume fraction of the stronger component of the mobile phase can be substituted by the normalized polarity parameter PNm: log k ⫽ c0 ⫹ c1 PmN

(1.86)

The polarity parameter for ACN – water mixtures is PmN ⫽ 1⫺

1.33 1⫹ 0.47

(1.87)

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The fundamental equation of gradient elution is tg ⫺t0

t0 ⫽

∫ 0

dt k ((t ))

(1.88)

where t0 is the dead time, tg denotes the retention time of the analyte in the gradient system, and k.(ϕ (t)) represents the descriptor of the analyte retention factor at the column inlet as a function of time. The retention time tg is related to tg ⫽

t0 log(2.3k0 b ⫹1) ⫹ t0 ⫹ t D b

(1.89)

where b is related to the solvent strength by b ⫽ S⬘t0

(1.90)

where ϕ ⬘ is the slope of the gradient program, tD is related to the time delay till the gradient reaches the column inlet (dwell time), and k0 is the retention factor at the beginning of the gradient: k0 ⫽ 10(logkw ⫺S 0)

(1.91)

where ϕ 0 denotes the mobile phase composition at the start of the gradient (ϕ ⫽ ϕ0 ⫽ ϕ⬘t). The method has been proposed for the prediction of retention data in isocratic systems from data measured in gradient elution and vice versa [84]. Similar calculation methods may be very important in the analysis of natural extracts containing pigments with highly different chemical structure and retention characteristics. The calculations make possible the rational design of optimal separation conditions with a minimal number of experimental runs. The theoretical considerations discussed briefly above have been further enlarged and the enhanced calculation of optimal gradient programmes was achieved involving three factors: gradient slope, initial eluent composition and gradient curvature. In the case of an ACN organic modifier the retention of an analyte can be described by  1.33  log k ⫽ c0 ⫹ c1 PmN ⫽ c0 ⫹ c1  1⫺  1⫹ 0.47 

(1.92)

In the case of assymetric peak shape the efficacy of a chromatographic system (N) can be expressed by N⫽

41.7(t R ⲐW 2 ) ( B ⲐA) ⫹1.25

(1.93)

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where W and B/A denote peak width and asymmetry factor measured at 10 per cent of peak height. Eqn.1.92 supposes that the efficacy is independent of the ratio of organic modifier. It is only partially true, because efficacy is not entirely independent of the composition of the mobile phase. The authors stated that the application of their computation method based on the peak purity criteria facilitates the development of optimal gradient elution programmes [85]. This optimization method can be also employed in the RP-HPLC analysis of natural pigments. The linear solvent strength (LSS) model combined with QSRR calculations has been applied for the prediction of retention in gradient RP-HPLC. It was established that total dipole moment (µ), electron excess charge of the most negatively charged atom (δ Min) and water-accessible molecular surface area (AWAS) exert the highest impact on the retention: retention parameter ⫽ k1 ⫹ k2
⫹ k3Min ⫹ k4 AWAS

(1.94)

where ki are regression coefficients. The method was proposed for the approximate prediction of the retention of analytes in gradient elution and for the facilitation of the development of optimal gradient elution strategy [86]. This prediction and optimization procedure is similar to those discussed above, consequently, its application in the field of RP-HPLC analysis of natural pigment may be similar. Theoretical equations have been developed for the description of the evolution of analyte zones in time- and space-dependent fields. It was established that the evolution of analyte zones can be described by the iterative summation of the local density of analyte zones with the mesoscopic approach involving Lagrangian description, the theory of continuity and the assumption of local equilibrium. Equations relating local retention factor and local diffusion coefficient to local mobile phase concentration can be employed for the prediction of migration and spreading. Moreover, the equations can predict the bandwidth under gradient elution [87]. Although the good correlation between the results of theoretical calculations and experimental values demonstrates the applicability of the method, the equations are fairly complicated for practical applications in the analysis of natural pigments and synthetic dyes. RP-HPLC can be used not only as an analytical tool for the separation and quantitative determination of various molecular species at low concentrations but also as a convenient method for preparative scale purification of expensive chemicals. The objectives of preparative chromatography markedly differ from those of analytical HPLC. The aim of preparative HPLC is the yield of the highest amount of material at a given degree of purity under minimal time and cost. For the optimization of preparative HPLC processes exact knowledge of the competitive isotherm models of the interacting species and their mass transfer kinetics is required. For the calculation of single component band profiles the general rate (GR) model was employed, while the transport-dispersive (TR) model was used for modelling multicomponent band profiles. The GR model was applied by ignoring the influence of the external mass transfer resistance and the influence of surface diffusion. The differential mass balance of species in the mobile phase can be described by:

e

C p C C 2C ⫹u ⫽  e DL 2 ⫺ (1⫺  e ) t x t x

(1.95)

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where C and Cp are the analyte concentrations in the mobile and the stagnant liquid phase, respectively, x denotes the distance along the column, t represents time, ε c is the external porosity of the bed, µ denotes the superficial velocity of the mobile phase, and DL is the axial dispersion coefficient. Similarly, the mass balance for the analyte in the stagnant liquid phase (within the pore of particles) can be described by: p

C p t

⫹ (1⫺  p )

C p  q 1   ⫽ 2 ⭈  Deff r 2  t r r  r 

(1.96)

where ε p is the mesopore porosity of the particles, r denotes the distance from the particle centre, and Deff represents the pore diffusion coefficient. Deff can be calculated by Deff ⫽ Dp  p ⫽

p 

Dm ⫽

 2p (2 ⫺  p )2

(1.97)

Dm

where Dp denotes the pore diffusivity, Dm is the molecular diffusivity, and θ is the pore tortuosity expressed by

⫽

(2 ⫺  p )2

(1.98)

p

The set of initial and boundary conditions were defined as C (0, x ) ⫽ 0 for 0具 x 具 L

(1.99)

Cp (0, x, r ) ⫽ 0 and q(0, x, r ) ⫽ 0 for 0具 x 具 L and 0具r 具 Rp

(1.100)

where q denotes the concentration of analyte in the solid phase, and Rp is the particle radius. Boundary conditions for eqn.1.94 are for t⬎0, at x ⫽ 0
f 冤C f (t ) - C f (t ,0)冥 ⫽  e DL

C (t ,0) x

(1.101)

and for t ⬎ 0,at x ⫽ L

C ⫽0 x

(1.102)

Boundary conditions for eqn.1.95 are for t⬎0, at r ⫽ Rp Cp 冢t , r ⫽ Rp 冣 ⫽ C

for t 典0, at r ⫽ 0,

Cp (t , r ) r

⫽0

(1.103)

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The transport-dispersive model consists of one differential mass balance equation for each component, i, in the mobile phase: Ci q C  2C ⫹ F i ⫹
i ⫽ e DL 2i t t x T x

(1.104)

where F denotes the phase ratio and ε T is the total porosity of the bed. The results of theoretical calculation using both general rate and transport-dispersive models were in good agreement with the overloaded band profiles determined experimentally, therefore, the method has been found to be suitable for the prediction of band profiles [88]. Natural pigments were generally used as a complicated mixture of various compounds with chromophore substructure. Their separation by preparative RP-HPLC is not necessary, and the application of preparative RP-HPLC for the purification of one or more pigment fractions is not expected in the near future. The retention of analyses in RP-HPLC markedly depends on the adsorption of the organic constituent of the mobile phase on the surface of the stationary phase. The excess adsorption isotherms of ACN, THF and methanol were measured on silica support modified with C1, C6, C8, C10, C12 and C18 monomeric phase and a model was developed for the description of the retention of solutes from the binary mobile phase. The dependence of the retention factor on the partition coefficient can be described by k ⫽ K

(1.105)

where K is the solute partition coefficient, and ϕ is the phase ratio, and [k⫽(VR⫺V0)/V0]. According to these relationships the exact determination of both the volume of the mobile phase and the volume of the stationary phase in the column are required for the calculation of the retention factor. The relationship between solute retention volume, VR(c), and its adsorption, Γ (c) can be expressed by VR (c) ⫽ V0 ⫹ S

d ( c ) dc

(1.106)

where V0 is the void volume of the column, and S denotes the adsorbent surface area. The solute distribution between the two liquid phases (eluent and adsorbed phase in equilibrium) can be expressed by cs ⫽ K p ce

(1.107)

where cs is the solute concentration in that phase at the equilibrium with the eluent, ce denotes the solute equilibrium concentration in the eluent, and Kp represents the solute distribution constant between the mobile phase and adsorbed phase. The adsorption process can be modelled by (cs ) ⫽ K H⭈cs ⫽ K H K p ce

(1.108)

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where KH is the Henry adsorption constant. The final equation describing the solute retention in the binary mobile is VR (cel ) ⫽ V0 ⫺ Vs ⫹ K p (cel )冤Vs ⫹ SK H 冥

(1.109)

where VR(cel ) is the solute retention as a function of eluent concentration, V0 is the total volume of the liquid phase in the column, Vs denotes the volume of the adsorbed layer, Kp(cel) represents the distribution coefficient of the solute between the mobile phase and adsorbed phase, S is the adsorbent surface area, and KH is the Henry constant for the solute adsorption from the pure organic component (adsorbed layer) on the surface of the bonded phase. The Henry constant can be calculated from KH ⫽

VR (100) ⫺ V0 S

(1.110)

Similarly, Kp(cel) can also be calculated by K p (cel ) ⫽

K 2 corganic ⫽ K1 celuent

(1.111)

It has been experimentally and theoretically verified that the organic component of the mobile phase adsorbs on the surface of the stationary phase forming a mono- or multimolecular layer. The volume of the adsorbed layer is not dependent on the length of the alkyl chain [89]. The precise measurement of competitive adsorption isotherms not only of theoretical importance but may help the optimization of chromatographic processes in both analytical and preparative separation modes. The methods applied for the experimental determination of such isotherms have been recently reviewed [90]. Frontal analysis using various flow rates can be successfully applied for the determination of competitive adsorption isotherms [91]. The mass balance in frontal analysis for a component i and for a volume element in a fixed bed can be depicted by ci 1⫺  qi (c ) c ⫹ ⭈ ⫹
i ⫽ 0 i ⫽ 1, N t  t x

(1.112)

where c denotes the concentration in the liquid phase, q represents the concentration in the solid phase, ε is the column porosity, u is the linear velocity, and t and x are the time and space coordinates, respectively. The initial and boundary conditions for frontal analysis are ci (t ⫽ 0) ⫽ ciInit

i ⫽ 1, N

and ci (t , x ⫽ 0) ⫽ ciFeed

(1.113) i ⫽ 1, N

(1.114)

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According to the equilibrium dispersive model and adsorption isotherm models the equilibrium data and isotherm model parameters can be calculated and compared with experimental data. It was found that frontal analysis is an effective technique for the study of multicomponent adsorption equilibria [92]. As has been previously mentioned, pure pigments and dyes are generally not necessary, therefore, frontal analysis and preparative RP-HPLC techniques have not been frequently applied in their analysis. Although the performance of liquid chromatographic systems has increased considerably in recent decades, the complete separation of each component in a complex sample (i.e., the extract of pigments from any plant, flower or fruit) cannot be achieved by using a single chromatographic column. The development of multidimensional separation techniques overcomes or decreases this difficulty by coupling two or more columns with highly different modes of retention. Theoretically, the plate number of such systems is the product of the plate number of the columns coupled together. A wide variety of single systems have been coupled, such as HPLC-capillary zone electrophoresis (CZE), HPLC-GC, and HPLC-HPLC using RP, normal phase, and/or SEC separation modes. In order to faci-litate the comparison of the performance of the individual separation units, retention data have to be normalized. The scaled retention factor Xa can be expressed by

Xa ⫽

Rti ⫺ Rt0 Rt f ⫺ Rt0

(1.115)

where Rti is the retention time of any analyte i, Rtf is the retention time of the last eluting analyte, and R0 is the retention time of the unretained analyte. The efficacy of the coupling of an octadecylsilica (C18) and carbon-clad zirconia has been demonstrated in the analysis of oligostyrene olgomers and isomers [93,94]. The results published in [93] and [94] clearly demonstrate the advantageous separation characteristics of the two-dimensional separation system. Its application for the analysis of complicated pigment mixtures may considerably enhance our knowledge of their composition. A new criterion has been proposed for the evaluation of the performance of two-dimensional hyphenated chromatography. It was stated that the so-called overlap index is a reliable descriptor of the efficacy of any two-dimensional chromatographic system [95]. Although the overwhelming majority of theoretical papers in liquid chromatography are dealing with the various aspects of RP-HPLC separation, theoretical advances have also been achieved in some other separation modes. Thus, a theoretical study on the relation between the kinetic and equilibrium quantities in size-exclusion chromatography has been published. In adsorption chromatography the probability of adsorbing an analyte molecule in the mobile phase exactly r-times is described by

W (r ) =

(kt 0 )r exp(⫺kt 0 ) r!

(1.116)

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where k is the rate constant representing the probability per unit time that a molecule in the mobile phase adsorbs on the solid phase, and t0 is the time it takes the eluent to flow completely trough the column. The excluded volume V0 is V0 ⫽ t 0 rf

(1.117)

where rf denotes the flow rate. It was established that the equations developed are suitable for the description of SEC and adsorption separation mechanism in the framework of a single unified theory [96]. The theory of chromatography of linear and cyclic polymers with functional groups has also been developed. A linear in-pore partition function Q(1) was created for polymeras with one specific functional group a. Q(1) can be expressed by Q (1) ⫽ Q (1) ⫽ Q (0) ⫹ 2.wa ..Pa

(1.118)

where Q(0) is the partition function of a corresponding non-functionalized polymer, and the second term accounts for the specific interaction of the functional group. Pa(z) denotes the sub-partition function of a chain having one point a at distance z from the wall, and δ represents the characteristic length of interaction. It was assumed that δ is of the order of chain unit size. Parameter wa is positive when the functionalized unit adsorbs more strongly that the other ones. For polymers with two specific groups, a and b, the in-pore partition function can be described by: Q (2) ⫽ Q (0) ⫹ 2(wa Pa ⫹ wb Pb ) ⫹ 22 wa wb Pab

(1.119)

where Pa denotes the sub-partition function of a non-functional chain having one point near the pore walls, Pb represents as for Pa a sub-partition function of a non-functional chain contacting the pore walls by its point b, and Pab is the sub-partition function of a chain with both points a and b at the pore walls. The parameters wa and wb account for the additional adsorptive interaction of the specific chain units a and b. The distribution coefficients of a polymer with one or two functional groups (K(1) and (2) K ) can be expressed by K (1) ⫽ K (0) ⫹ qa pa

(1.120)

and K (2) ⫽ K (0) ⫹ qa pa ⫹ qb pb ⫹ qa qb pab

(1.121)

where K(0) denotes the distribution coefficient of a non-functional polymer, qa and qb are the reduced functional group interaction parameters, and Pab represents the reduced one- and two-point contact possibilities. The equations above can also be used for the description of distribution coefficient of cyclic polymers after slight modifications. It has been found that the calculations may facilitate the separation of polymers according to the functional group, moreover, the method is suitable for the analysis of linear and cyclic polymers, as well as

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comb-like and semicyclic ones [97]. The majority of natural pigments are in monomeric form in their original state. However, after extraction or other purification steps, pigments are liable to oxidation and form polymers with more or less unknown chemical structure. For the separation and structure elucidation of oxidated pigment polymers, such methods can be successfully applied. Because of its capacity to separate effectively cations and anions ion chromatography has also found application in chromatographic practice. A nonlinear model has been developed for the prediction of the retention of polyvalent weak acid anions in anion chromatography. In the case of strong acid anions containing only one acidic group eluted by a mobile phase with monoanionic additive, the retention can be described by log k ⫽ const ⫺ ( y Ⲑx )log C

(1.122)

where y represents the charge of the solute, x is the charge of the ion in the mobile phase, and the constant depends on the column and the composition of the stationary and mobile phases. The average charge (av) of the weak acid solute anions is m

av ⫽ ∑ i i

(1.123)

i⫽I

where αi is the fraction of species of anions carrying i negative charges. The dependence of the retention factor on weak anions from the concentration of the monoanionic mobile phase can be described by: m

log k ⫽ a ∑ i ilog C ⫹ b1⭈log  h ⫹ b2 log sh ⫹ d

(1.124)

i⫽1

where α h denotes the fraction of species of anions with highest charge, αsh represents the fraction of species of anions with the second highest charge, and a, b1, b2 and d are constants depending on the characteristics of the column, mobile phase and solute. When the absolute value of the highest charge of the solute is 2, αh can be expressed by h ⫽

K a1 K a 2 [H⫹ ]2 ⫹ K a1[H⫹ ]⫹ K a1 K a 2

sh ⫽

K a1[H⫹ ] ⫹ 2

[H ] ⫹ K a1[H⫹ ]⫹ K a1 K a 2

(1.125)

(1.126)

where [H⫹] denotes the function of concentration of the mobile phase indicating that the fraction of species of solute is also a function of the concentration of the mobile phase. For computational purposes eqn. 1.123 can be rewritten as y ⫽ ax1 ⫹ b1x2 ⫹ b2 x3 ⫹ d where x1 ⫽ average log C, x2 ⫽ log α h, x3 ⫽ log α sh, and y ⫽ log k.

(1.127)

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When the effect of the change of the average charge is neglected, eqn. 1.112 can be reduced to log k ⫽ alog C ⫹ b1log  h ⫹ b2 log sh ⫹ d

(1.128)

Experimental values correlated well with the predicted ones proving that the model is a valuable addition to the theory of ion chromatography [98]. Many synthetic dyes are sulphonated derivatives of complicate organic molecules. Because of their strong adsorption capacity their separation under traditional RP-HPLC conditions is sometimes difficult. Anion chromatography offers an advantageous alternative for the analysis of this type of synthetic dyes. A thermodynamic approach has also been employed on ion interaction chromatography (IIC) to predict the retention of neutral and ionic analyte species. The basic equations describing retention are

k⫽

k⫽

c1 冦a[H]b f ⫹ 冤冢 a[H]b f 冣2 ⫹1冥1Ⲑ 2 冧

(⫾ zE )

⫹ c2 [H]

(1+ c3 [H])⭈冦1⫹ c4 [H] 冤 a[H] f ⫹ 冦冢 a[H] f 冣 ⫹1冧1Ⲑ 2 冥 b

b

⫾2 zE

c1 冦a[H]b f ⫹ 冤([LH] f )2 ⫹1冥1Ⲑ 2 冧

冢1⫹ d3 [LH]1Ⲑb 冣

2

⫹ d2 [LH]1s Ⲑb

(⫺2 z E )

冧

(1.129)

(1.130)

where a, b and f are constants depending on the chromatographic system, zE and zH denote the charges of the solute E and the IIR H, [H] and [LH] represent the concentration of IIR in the mobile and stationary phases, respectively, and c1–c4 and d1–d4 are the fitting para-meters. c1 and d1 denote the capacity factor without IIR in the mobile phase, they are related to the electrostatic interaction with the charged surface (KEL ), c2 and d2 are related to the thermodynamic equilibrium constant for ion-pair formation in the stationary phase (KEHL ), c3 and d3 are related to the thermodynamic equilibrium constant for ion-pair formation in the mobile phase (KEH), c4 denotes the thermodynamic equilibrium constant for adsorption of IIR onto the stationary phase (KHL ) and d4 is the total ligand surface concentration. The validity of this theoretical model has been controlled by real experiments and good correlation was found between the experimental and calculated values [99]. Although the theoretical approximation and the mechanism of separation described and discussed in [97] and [98] are different, the practical objectives of both methods are similar, to predict the retention of charged analyte species. As has been previously mentioned these chromatographic procedures can find application in the analysis of charged dye molecules. The performance of HPLC separations can be increased not only by the application of two- or multidimensional techniques but also by the use of simulated moving bed (SMB) [100–102] or true moving bed (TMB) techniques [103,104]. SMB is a multicolumn separation technique allowing the continuous separation of analyses with higher productivity and smaller eluent consumption than the traditional single-column procedures. TMB

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exploits the advantages of the addition of a counter-current motion of the solid phase in comparison with the liquid phase. These methods have been extensively used for separations of isomers and fine chemicals and to the best of our knowledge they have not been employed in the analysis of natural pigments and synthetic dyes. 1.3.3 Practical considerations The traditional HPLC instrument is composed of two different parts: the first part separates the components of the sample and the other part accomplishes the detection of the components separated. The part of the HPLC carrying out the separation contains a column, an injection device and the eluent delivery system (pump with filters, degasser and transfer tubing, eventually a mixer for gradient elution). One or more detectors, a signal output device coupled with appropriate software, are responsible for detection and primary data evaluation. Pumps deliver the eluent or the different components of the eluent into the column with a precise, constant and reproducible flow rate. Injectors introduce the sample into the mobile phase under high pressure. There are several approaches to injection in HPLC, such as syringe injection via septum, a combination of a septum and syringe or a valve injection. Valve injection is the method of preference in up-to-date HPLC instrumentation. Detectors are responsible for monitoring the analytes in the mobile phase, leaving the column after the separation process. The ideal HPLC detector has to comply with the following requirements: high sensitivity, universal or specific response, wide linear dynamic range, no or negligible extra-column band broadening and stable response independent of column temperatures and flow rates. Unfortunately, detectors may approximate but do not reach the strict conditions of an ideal detector. Numerous detectors have been developed based on the physicochemical characteristics of the analyte to be detected. Thus, ultraviolet–visible (UV-vis), infrared, fluorescence, atomic absorption, coulometry, amperometry, refractive index detectors (RI), and mass spectrometric detectors have been developed and applied depending on the type of the solutes to be analysed. The UV-vis detector is one of the most popular and general detectors because many organic molecules adsorb in the UV and/or visible region. The sensitivity of detection can be enhanced by the derivatization of analyses showing no absorbance in the UV-vis region, however, this procedure is sometimes time consuming, cost enhancing and causes band broadening, deteriorating the performance of the analysis. The diode-array detector makes possible the measurement of the UV-vis spectra of analyses facilitating identification and enhancing the sensitivity of detection for compounds with different UV-vis absorption maxima. Some molecules can absorb UV energy and emit light at a higher wavelength during the shift of energy state. The advantages of fluorescence detection are high selectivity and high sensitivity. Analyses without flourescence capacity can be derivatized. The refractive index (RI) detector measures the difference in RI between the eluent and the analyte in the eluent. The RI detector can detect each compound (universal detector), however its sensitivity is lower than that of other detectors. Radioactive detection can selectively measure radioactive components in the sample. It is very specific and can be used only for this special purpose. IR and Fourier-transform (FTIR) can also be employed as detector.

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IR spectra are suitable for the identification of a wide variety of organic compounds, however the signal of the eluent generally overshadows the weak sign of the analyte, reducing detector sensitivity. In recent decades the hyphenated technique, HPLC-MS, also become the method of preference in HPLC practice. Various techniques have been developed and applied for MS detection such as thermo-spray interface, atmospheric ionization (API), electrospray or ionspray ionization (ESI or ISI), and particle beam ionization. The operating life of an HPLC column highly depends on the maintenance and care devoted to the column. Samples and mobile phases have to be clear and without solid particles. They have to be filtered before application. The application of a precolumn is proposed to increase the life of the analytical column. The inlet and outlet filters of the analytical column can assemble particles from the eluent and sample which were not previously removed during the preparation of the sample and the mobile phase. In the case of gradient elution, the components of the buffer can precipitate at a higher concentration of organic modifier, the solubility of the buffer has to be verified at the highest concentration of organic modifier before the analytical procedure. Even in the case of maximal care, columns can accumulate impurities resulting in reduced performance. Washing of the column with a series of solvents or solvent mixtures can restore the separation capacity. Other processes, such as dissolving or compression of the column inlet bed, can also deteriorate column efficacy. Partial or entire refilling of the column is the best method to overcome this difficulty. Columns have to be stored in the solvent proposed by the manufacturer, never use buffers, salts, acids or bases during storage, even for a night. Appropriate filtering and degassing of the mobile phase is a prerequisite of successful analytical work. Besides the purity control of the components of the mobile phase, the stability of the eluent flow has also to be verified. The modification of the retention time of standards and/or sample components with well-known retention time can be caused by pump disfunction, variation of column temperature and the composition of the mobile phase. 1.4 SUPERCRITICAL FLUID CHROMATOGRAPHY (SFC) Supercritical fluid chromatography is the name for all chromatographic methods in which the mobile phase is supercritical under the conditions of analysis and the solvating properties of the fluid have a measurable effect on the separation. SFC has some advantages over GC and HPLC: it extends the molecular weight range of GC, thermally labile compounds can be separated at lower temperatures, compounds without chromophores can be sensitively detected, and the use of open-tubular and packed columns is feasible. SFC can be employed in both the analysis of natural pigments and synthetic dyes, however it has not been frequently applied in up-to-date analytical practice. 1.5 VARIOUS ELECTROPHORETIC TECHNIQUES Capillary electrophoresis (CE) separates sample components within a capillary tube under the effect of an electric field employed across the two ends of the capillary. The different

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magnitudes and directions of migration velocities of analyses influence their separation. The migration parameters can be easily modified by changing the electrophoretic media, the concentration and pH of the buffer. The partial charge of the analyte, the degree of its ionization, the interaction between the analyte and the additives in the electrophoretic buffer, and the characteristics of the capillary surface may also influence the separation. Common CE techniques employ a narrow bore capillary (10 – 100 µ m i.d.) and a high potential, up to 30 kV. The use of capillaries with small internal diameter permits efficient heat dissipation, which allows the application of high voltages without solvent boiling. The theoretical plate number of CE techniques is higher than any other traditional analytical procedure. Because of its high efficacy CE has found application in many fields of analytical chemistry, such as biochemistry, pharmaceutical research, environmental analysis, etc. In the last five years a considerable number of books and reviews have been published on the various aspects of CE analysis. Thus, excellent books have been published on clinical and forensic applications [105], on the use of CE in pharmaceutical analysis [106], and on the theoretical and practical bases of CE separations [107]. The analysis of small molecules [108], natural organic matter [109], metabolites [110] and biomolecules [111] have also been reviewed. The application field of micellar eletrokinetic chromatography has also been evaluated [112]. 1.5.1 Fundamentals The instrumentation of CE is relatively simple. The capillary carrying out the separation is filled with a buffer, and both its ends are immersed into two buffer reservoirs that are kept at the same level. A high voltage is applied across the capillary. Generally, the sample is introduced at the anodic end and the detector is placed at the opposite (cathodic) end. Various CE techniques have been developed and applied to the analysis of both charged and neutral analyses. In capillary zone electrophoresis (CZE) the sample is placed in the capillary between two similar buffer solutions, and a potential is applied across the capillary. The migration of charged species in the sample depends on their charge-to-mass ratios. Analyses with higher charge are retained in the capillary. Capillary gel electrophoresis (CGE) employs a gel-filled capillary. The pores of the gel act as sieves, and the analyses are eluted according to their charge and size. Instead of gels, polymer solutions can be also applied in CGE. This technique is mainly used for the separation of oligonucleotides, peptides and proteins. Micellar electrokinetic chromatography (MEKC) has been developed for the separation of neutral analytes. An anionic surfactant over its critical micellar concentration is added to the background electrolyte. Analyses are partitioned between the apolar core of the surfactant micelles and the polar aqueous buffer according to their hydrophobicity. Capillary isotachophoresis (CITP) uses two different electrolytes, a leading and a terminating one. After sample injection a potential is applied across the two different buffers. Because of the two different buffers the electric field along the length of the column is not constant. The bands of the individual sample components are sharpened under the effect of the changing electric field, and they migrate at the same velocity. The method can be applied to the preconcentration of dilute samples. Capillary isoelectric focusing (CIEF) is suitable for the separation of amphoteric analyses in a pH gradient. A continuous pH gradient is built up in the column by using ampholytes under a potential field. Amphoteric analyses migrate to the point where their net charge equal to zero and they form stationary and sharply focused zones.

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Electroosmotic flow (EOF) is an important parameter in CE techniques. It is defined as the bulk flow of solvent in the capillary under the effect of a potential field. The unreacted hydroxyl groups on the surface of the fused silica capillaries dissociate when the pH of the buffer is higher than 2.5. The negatively charged surface attracts the cations of the buffer, and the layer of positive charges form a double layer which creates a potential difference near to the capillary wall (zeta potential). The solvated cations migrate toward the cathode, transporting the solvated solvent molecules with them. Electrophoretic flow (µ ep) is the flow of ions. Under the potential applied, cations and anions move to the cathode and anode, respectively. Electroosmotic flow is larger than the electrophoretic flow. Because of the dominating role of EOF each analyte (cationic, anionic and neutral) has a tendency to move towards the cathode. The cation migration velocity is composed of the electrophoretic migration and the electroosmotic flow. Neutral analyses are not separated under common CE conditions, they move together with the electroosmotic flow. The migration velocity of anions is the difference between electroosmotic flow and electrophoretic migration, consequently their migration velocity is lower than that of neutral compounds and cationic ones. One of the most important factors influencing CE separation is the composition of the buffer. A change in the pH of the buffer can modify the separation of charged analyses with various charge-to-size ratios in CZE. The separation of neutral compounds by MEKC can be achieved by the addition of anionic, cationic or neutral surfactants, i.e., sodium dodecylsulphate, ethoxylated long-chain alcohols and cetyltetrabutylammonium bromide. The efficacy of CE separation techniques can be further enhanced by adding modifiers to the buffer such as methanol, ACN, bile salts, cyclodextrins and cyclodextrin derivatives, hydroxypropylcellulose, hydroxypropylmethylcellulose and tetraalkylammonium salts. Traditional CE techniques apply aqueous buffers for separation which are unable to dissolve strongly hydrophobic analyte components. These molecules can be precipitated in the aqueous media during the separation process. The addition of organic modifiers can overcome this difficulty, however they lengthen the analysis time and reduce the velocity of the electroosmotic flow. 1.5.2 New advances in the theory of electrophoretic techniques The objectives of each theoretical approach are not only the explanation of the experimental results or failures of practice but also the prediction of new possibilities to increase the sensitivity, separation capacity and velocity of the chromatographic procedure under investigation. Numerous theoretical reviews deal with the problems of the CE separation technique. In recent years the methods to enhance the precision in CE by the modification of operational parameters [113], the theory and methodological improvements of sample stacking of cationic and anionic solutes in CE [114–116], and the results and difficulties of the application of conductivity detection in CE technologies [117] have been reviewed. The peak distortion in CZE electrophoregrams by electromigration dispersion has been modelled and the peak centre was related by a1 ⫽

lL
app A V

(1.131)

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where a1 denotes the peak centre, l, L, µAapp and V are the length from injection to detection, the total length of capillary, the apparent mobility of the solute and the voltage applied, respectively. The standard deviation of the Gaussian part (a22) can be expressed by a22 ⫽ 2 DA t D ⫹

l0⬘2 12

(1.132)

where DA denotes the diffusion coefficient of solute, tD is the time from injection to detection, and l⬘0 is the effective injection length. The peak distortion in distance units can be described by a3 ⫽⫺2 z A AA c1A l0

(1.133)

where zA is the charge of the solute, clA denotes the concentration of the solute in the injection zone, and l0 is the length of injection. AA is a constant depending on the starting conditions: AA ⫽⫺

(
A ⫺
co )(
A ⫺
counter ) B cco
A (
co ⫺
counter )

(1.134)

where µA denotes the mobility of the solute, µco and µ counter are the mobilities of the co-ion of the solute and the counter-ion, respectively, and ccoB is the co-ion concentration in the background electrolyte. To facilitate calculations, eqns 1.31–1.33 were converted from time to distance dimensions. It was established that the total variance of the peak includes the variance due to diffusion and due to electromigration diffusion: M2 ⫽ 6.115 ⫺1.523 ⫻10⫺1 G ⫹1.524 ⫻10⫺3 G 2 ⫺ 5.12 ⫻10⫺6 G 3 a22

(1.135)

where M2 is the second moment of the peak (total peak variance), and G denotes the percentage of the Gaussian variance defined by G⫽

a22 x100 1 a22 ⫹ a1 a3 9

(1.136)

The results of theoretical calculations were compared with the concrete measurements using paraquat and 4-aminopyridine as model compounds. In the case of paraquat the fitness of the calculated values to the theoretical ones (RES) was expressed by RES ⫽

Aexp ⫺ AHVL max Aexp

x100

(1.137)

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where Aexp denotes the experimental absorbance data point, AHVL the theoretical max absorbance calculated, and Aexp represents the absorbance at the peak maximum. The injection length (l0) was calculated by l0 ⫽

Anorm l

(1.138)

I cAP RAP

where Anorm denotes the normalized area (AU), cIAP is the injected aminopyridine concentration and l is the length from injection to detection. The normalized area was computed by Anorm ⫽

a0 M1

(1.139)

where M1 denotes the first moment of the peak. The efficacy (N) was calculated by N⫽

M12 M2

(1.140)

The diffusion coefficient (DA0 ) for small ions can be expressed by DA0 ⫽


0A kT zAe

(1.141)

where µA0 is the mobility at zero ionic strength at the temperature of the measurement, T denotes the absolute temperature, zA represents the solute charge number, e is the electronic charge, and k is the Boltzmann constant. The mobility at different capillary temperatures can be calculated by
0 (306K) ⫽

298 0
298K 306

(1.142)

It has been found that the equations describe correctly the peak shape determined experimentally, and can be applied for the prediction of peak distortion [118]. Organic modifiers have been frequently employed in CE to increase the solubility of hydrophobic solutes in the aqueous buffer system. Unfortunately, many organic modifiers are UV absorbent and cannot be used without considerable loss of sensitivity of detection. A contactless conductivity detection system has been developed which extends the application range of UV-absorbing solvents [119]. As both natural pigments and synthetic dyes absorb in the visible part of the spectra, the application of UV-absorbing organic modifiers in their CE analysis does not cause detection problems.

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Another study investigated the occurrence of system peaks in background electrolytes containing multivalent weak ionic species, such as phosphoric acid and phtalic acid. It has been experimentally proven that system peaks can disturb the determination of solute species migrated near to the system peaks and this phenomenon on has to be taken into consideration at the design stage of the composition of the background buffer [120]. The electrophoretic mobilities of cationic analytes in water, in non-aqueous methanol and acetonitrile were measured and the influence of ionic strength and ion-pair formation on the mobility was determined. It was assumed that the mobility µ of the ion at infinite dilution (µ 0,i) and the viscosity of the pure solvent are constant:
act,i f  ⫽
0,i ⫽ const.

(1.143)

where µ act,i denotes the mobility of a fully charged ion at a given ionic strength, and f is the correction factor that depends on the ionic strength of the background electrolyte. The Stokes radius (rStokes ) of solutes was calculated by rStrokes,i ⫽

zi e0 6
0,i 

(1.144)

where zi denotes the charge number of the ion, and e0 is the elementary charge. It was established that ion–solvent interactions are responsible for the high mobility in water, and ion–ion interactions and ion-pair formation also play a considerable role in mobility. The application of an organic solvent in CE offers a new possibility to change the selectivity of CE systems. Moreover, the various ion–solvent interactions and ion-pair formations can be exploited for increased separation efficacy. Its application for CE analysis of ionic synthetic dyes may be expected in the future [121]. The CE behaviour of inorganic anions in water–methanol mixtures has also been extensively studied. The specific conductivity σ of electrolytes was converted into equivalent conductivity by ⫽

 zc

(1.145)

where z denotes the number of elementary charges and c is the concentration of perchloric acid. The correlation between the equivalent conductivity of a strong 1:1 electrolyte on its concentration can be described by  ⫽  ⬁ ⫺ ( A ⬁ ⫹ B)c1Ⲑ 2

(1.146)

A⫽

0.82 ⫻106 (T )3 Ⲑ 2

(1.147)

B⫽

82 (T )1Ⲑ 2

(1.148)

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where Λ∞ denotes the electrolyte equivalent conductivity at infinite dilution, T is the absolute temperature, and ε represents the relative dielectric constant. The actual mobility of an ion in a univalent electrolyte depends on the ionic strength according to  8.20 ⫻105
i⬁   I 42.75  
i0 ⫽
i⬁ ⫺    ⫹ (T )1Ⲑ 2  ⫻  ⫺1 Ⲑ 2 I )    (1⫹ 50.29a(T ) 

(1.149)

where µ ∞i denotes the absolute mobility, a is the distance of closest approach between ion i and its counter-ion, and l represents ionic strength. Although the model CE system was different from that used in [118] the conclusions were similar on the decisive role of ion–solvent interaction and ion-pair formation [122]. CE techniques have been applied not only to the separation of organic and inorganic analytes but also to the determination of complex formation constants (K). K is defined by

K⫽

[ AX ] [ A][ X ]

(1.150)

where [AX], [A] and [X] are the equilibrium concentrations of the formed complex, the analyte and the ligand, respectively. The effective mobility (µeff) of the solute is the weighted average of the mobilities of all forms of solutes in the CE system:
eff ⫽

[ A] [ AX ] 1
A ⫹
AX ⫽ 冢[ A]
A ⫹[ AX ]
AX 冣 ⫽ ∑ xi
i [ A]⫹[ AX ] [ A][ AX ] ct i

(1.151)

where ct is the total additive concentration. The interaction constant can be expressed by K⫽

1uA ⫺ ueff ct ueff ⫺ uAX


eff,1 ⫽

1 
A ⫺
eff,1   ⫹
AX K  ct ,i 

(1.152)

(1.153)

Eqn.1.152 can be linearized and the formation constant can be calculated from the slope of the plot µeff versus (µA-µeff)/ct. This method was employed for the measurement of the interaction of pyridinium and benzylaminopyridinium with dimethyl-β -cyclodextrine.

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It is well known that complex forming with cyclodextrins and cyclodextrin derivatives can markedly increase the stability of the complexed guest molecule, enhancing the shelflife of the product. The beneficial effect depends considerably on the complex stability. The method outlined above can be used for the selection of an optimal complexing agent for increasing the food products containing light and oxygen-sensitive natural pigments [123]. Although CE separations can be reasonable well described by the classical theoretical relationships for electrophoretic migration, slight deviations from the theory occur in the case of many classes of solutes. Thus, it has been reported that the CE separation of oligosaccharides follow the general rule [124], while the description of the separation of DNA in polymer solutions necessitated a new mathematical model. The drag forces were expressed by Fe ⫽ FDNA ⫹ Fp

(1.154)

where Fe represents the electrophoretic force, FDNA is the drag force on the DNA without contact with polymer molecules and Fp denotes the drag force imparted by the polymer obstacle during transient entanglement coupling with the DNA. The electrophoretic force is Fe ⫽ qeff N DNA E

(1.155)

where qeff denotes the effective charge per base pair, NDNA is the number of base pairs of the DNA molecule, and E is the electric field strength. The drag force on DNA is FDNA ⫽ fDNA N DNA


(1.156)

where v is the velocity of the DNA and fDNA is its translational friction coefficient per base pair in polymer solution. The drag force of polymer molecules can be described by Fp ⫽ fp
p

(1.157)

where p is the number of polymer molecules dragged by the DNA and fp denotes the friction coefficient of the polymer, which depends on the viscosity and on the radius of gyration of the polymer, Rp:  KM t1⫹d  Rp ⬵    6.2 N A 

1Ⲑ 3

(1.158)

where K and d are constants characterizing the polymer–solvent system, Mr is the molecular mass of polymer and NA is Avogadro’s number. The effective mobility (µ p) of a DNA molecule is
p ⫽

qeff N DNA E fDNA N DNA ⫹ fpP

(1.159)

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The average number of polymers dragged by one DNA molecule (X) can be calculated by X⫽

eff N c
% E Ld

(1.160)

where NC is the number of polymer–DNA collisions, µ is the average electrophoretic mobility, and τeff denotes the effective time constant of polymer–DNA contact, which depends on the size of the DNA: eff ⫽ xeff N DNA

(1.161)

where Xeff represents the effective time constant per base pair. The effective time constant depends on the polymer concentration by X eff (C ) ⫽ a ⫹ bC

(1.162)

where C is the polymer concentration. The effective time constant can be expressed by eff ⫽ (a ⫹ bC ) N DNA

(1.163)

Combining eqns 1.159 and 1.162: X⫽

(a ⫹ bC ) N DNA N C
% E Ld

Nc ⫽

(1.164)

CN A ( RDNA ⫹ Rp )2 Ld Mr

(1.165)

where π.(RDNA⫹Rp)2 is the collision cross-sectional area. The radius of gyration of the DNA molecule RDNA is related to 1Ⲑ 2

1 2 L3p 2 L4p  L  2 RDNA ⫽  LC Lp ⫺ Lp ⫹ ⫺ 2  1⫺ e C   LC Lp   LC   3

(1.166)

where LC denoted the contour length of the DNA and Lp represents the persistence length of the DNA. The average effective mobility of DNA was given by
% ⫽ ( X ⫺ P )
p⫹1 ⫹ ( P ⫹1⫺ X )
p

(1.167)

where P is an integer value P⬍X⬍P⫹1. It was found that the calculated values of electrophoretic mobility agreed well with the experimental data, proving the validity of this theoretical approximation [125].

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A different theory has also been proposed for the CE separation of DNA. The relationship between the free flow electric mobility (µ 0) and the diffusion coefficient of DNA (D0) can be described by D0 kBT ⫽
0 Q

(1.168)

where Q is the charge. However, it was established that eqn. 1.167 does not correctly give the behaviour of DNA in free-flow electrophoresis. Another equation describes the migration of small DNA solutes in a gel concentration:   R 2

⫺  
(C ) ⫽ (C ) ⫽ e⫺KC ⫽ e 4  a 
0

(1.169)

where K ≈ R2 denotes the retardation factor, and a ≈ C⫺1/2. As the application of eqn.1.168 is difficult because ϕ cannot be easily measured, the following equation was proposed for random gels:
(C ) ⬵
0

1 1 ⫽ 2 1⫹ KC 1⫹ (1⫺ ) 3

(1.170)

The migration of longer DNA molecules in gels can be described by ⫽

a 2
0 E k BT

(1.171)

where v is the solvent viscosity. The ratio of migration can be expressed by 1Ⲑ 2

  1  2  2  2 
 ⬇   ⫹
0  3 N   5 ⫹ 2     

(1.172)

where 1/β is the ratio of µ/µ0 in very strong fields. The total mobility of DNA fragments (µtot) on the polymer solution is
tot ⫽
rep ⫹
CR

(1.173)

where µ rep is due to DNA reputation and µ CR denotes the constant release. It was admitted that the separation mechanism of CE is not entirely elucidated and further theoretical works are needed for the exact interpretation of the molecular base of CE separation [126].

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The classical theory of electrophoretic migration has also been successfully applied to the description of the behaviour of human growth hormone peptides. According to the classical theory, the mobility (µ) of a peptide can be described by u⫽

Kz M 2Ⲑ3

(1.174)

where z is the net charge of the peptide, K denotes the proportionality constant, and M represents the molecular mass of the peptide. The electrophoretic mobility can also be expressed by

u⫽

冤(l Ⲑt p ) ⫺ (l Ⲑt M )冥

(1.175)

E

where l is the separation distance (the distance between the injection point and the detector), tp is the retention time, tM is the retention time of a neutral marker (messily oxide), and E is the electrical field strength (the applied voltage divided by the total length of the capillary across which the voltage is applied). The relationship between the electrophoretic mobility and the radius (r) and the net charge of the peptide is

u⫽

1.6⭈10⫺12 zf ( kr )(1⫹ krb ) 6r (1⫹ kr ⫹ krb )

(1.176)

where k denotes the inverse screening length, f(kr) is the Henry function, v is the buffer viscosity, and rb is the radius of the buffer ions. The radius of the peptide molecule can be calculated from the molecular mass, Avogadro number, and the partial specific volume. The dependence of the inverse screening length on the characteristics of the buffer is expressed by 1Ⲑ 2

 8Ne2 I  k⫽  1000 DkT 

(1.177)

where e is the electronic charge, I denotes the molar ionic strength, D is the dielectric constant, k represents the Boltzmann constant, and T is the absolute temperature. Henry’s function can be calculated by f (kr ) ⫽ 1⫹

0.5 1⫹ exp冤2.8冦1⫺ log(kr )冧冥

kr 具10

(1.178)

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The final equation used for the calculation of the elctrophoretic mobility of peptides was u⫽

0.0258冢 z ⲐM 2 Ⲑ 3 冣 1⫹ 冢19.8 ⲐM 1Ⲑ 3 冣

(1.179)

A good agreement between the measured and calculated values of electrophoretic mobilities was established [127]. The capacity factor of neutral analyses in MEKC can be expressed by k ⫽ (t r ⫺ t0 ) Ⲑ 冤 t 0 (1⫺ t r Ⲑtm )冥 ⫽ K (Vs ⲐVm )

(1.180)

where tr is the retention time of the analyte, t0 denotes the retention time of an unretained solute moving at µ EOF, tm represents the micelle retention time, K is the partition coefficient, Vs is the volume of the micellar phase, and Vm is the volume of the mobile phase. The CE theories briefly discussed above can be applied to the prediction of the electrophoretic mobility of natural pigments and synthetic dyes too, and for the design of a CE system for their optimal separation. 1.5.3 Practical considerations The efficacy of CE separation depends considerably on the type of capillary. Fusedsilica capillaries without pretreatment are used most frequently. Its outside is coated with a polymer layer to make it flexible and to lessen the occurrence of breakage. The polymer coating has to be dissolved with acid or burned away at the detection point. Capillaries with an optically transparent outer coating have also found application in CE. The objectives of the development of chemically modified capillary walls were the elimination of electroosmotic flow and the prevention of adsorption on the inner wall of the capillary. Another method to prevent the adsorption of cationic analyses and proteins is the use of mobile phase additives. The modification of the pH of the buffer, the addition of salts, amines and polymers have all been successfully employed for the improvement of separation. The design of an effective CE separation system is highly facilitated by previous knowledge of some physicochemical parameters of analyses, such as ionic charge, molecular mass, solubility and stability. Generally, the best separation of small charged species can be achieved by CZE, while MEKC can be employed for the separation of small uncharged analyses. The washing of capillaries with dilute alkaline solution is advisable before analysis. The alkaline solution can be followed by deionized water and buffer. Capillaries can be washed between runs too. Samples can be introduced into the capillary by hydrodynamic and electrokinetic methods. The hydrodynamic method applies a pressure difference (5 – 10 sec) between the two ends of the capillary. The pressure difference can be achieved by overpressure, vacuum or by creating a height difference between the levels of the buffer and sample reservoirs. In the case of electrokinetic injection, the injection end of the capillary is dipped into the sample for a few seconds and a voltage of some thousand volts is applied.

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The disadvantage of this injection method is that the concentration of analyses with higher migration velocity will be higher in the injected sample than in the bulk of the sample. Similarly to HPLC, UV-vis detection is used the most frequently. For the detection of analyses without UV-vis absorbance a chromophore has to be added to the buffer. In these systems the analyses produce a negative peak. Fluorescence, conductivity, electrochemistry and mass spectrometry have also found application in the detection systems of CE technologies. When peaks are not observed, the set of detector and integrator, the correct polarity of voltage, the exact wavelength of the detector, the blockade of capillary, the inefficacy of injection, etc. have to be controlled. When too many peaks appear, the purity of samples and/or standards, and the degradation of the buffer or sample have to be controlled. Peak irregularity, such as tailing and distortion, can be caused by too concentrated a sample, too large an injection volume, unsuitable buffer composition, or adsorption of analytes on the inner wall of the capillary. Uncontrollable adsorption, unstable light source, changed viscosity, inadequate temperature control, unstable current, height difference between reservoirs, etc. may also result in poor reproducibility. Inaccurate detector and integrator set, adsorption on the capillary inner wall, too small a sample volume or too dilute a sample, and low absorbance and/or high absorbance of the buffer may cause decreased sensitivity.

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109 P. Schmitt-Koplin and J. Junkers, Capillary zone electrophoresis of natural organic matter. J. Chromatogr.A 998 (2003) 1–20. 110 P. Britz-McKibbin and S. Terabe, On-line preconcentration strategies for trace analysis of metabolites by capillary electrophoresis. J. Chromatogr.A 1000 (2003) 917–934. 111 A.M. Garcia-Campana, L. Gamiz-Gracia, W.R.G. Baeyens and F.A. Barrero, Derivatization of biomolecules for chemiluminescent detection in capillary electrophoresis. J. Chromatogr.B 793 (2003) 49–74. 112 T. Welsch and D. Michalke, (Micellar) electrokinetic chromatography: an interesting solution for the liquid phase separation dilemma. J. Chromatogr.A 1000 (2003) 935–952. 113 B.X. Mayer, How to increase precision in capillary electrophoresis. J. Chromatogr.A 907 (2001) 21–37. 114 J.P. Quirino and S. Terabe, Sample stacking of cationic and anionic analytes in capillary electrophoresis. J. Chromatogr.A 902 (2000) 119–135. 115 J.P. Quirino and S. Terabe, Sample stacking of fast-moving anions in capillary zone electrophoresis with pH-suppressed electroosmotic flow. J. Chromatogr.A 850 (1999) 339–344. 116 Z.K. Shihabi, Stacking in capillary zone electrophoresis. J. Chromatogr.A 902 (2000) 107–117. 117 A.J. Zemann, Conductivity detection in capillary electrophoresis. TrAC Trends in Analytical Chemistry 20 (2001) 346–354. 118 G.L. Erny, E.T. Bergström and D.M. Goodall, Electromigration dispersion in capillary zone electrophoresis. Experimental validation of use of the Haarhoff–Van der Linde function. J. Chromatogr.A 959 (2002) 229–239. 119 J. Muzikar, T. van de Goor, B. Gas and E. Kenndler, Extension of the application range of UVabsorbing organic solvents in capillary electrophoresis by the use of a contactless conductivity detector. J. Chromatogr.A 924 (2001) 147–154. 120 J.L. Beckers, P. Gebauer and P. Bocek, Why robust electrolytes containing multivalent ionic species can fail in capillary zone electrophoresis. J. Chromatogr.A 916 (2001) 41–49. 121 S.P. Porras, M.-L. Riekkola and E. Kenndler, Electrophoretic mobilities of cationic analytes in non-aqueous methanol, acetonitrile and their mixtures. Influence of ionic strength and ion-pair formation. J. Chromatogr.A 924 (2001) 31–42. 122 S. Descroix, A. Varenne, C. Adamo and P. Gareil, Capillary electrophoresis of inorganic anions in hydro-organic media. Influence of ion-pairing and solvation phenomena. J. Chromatogr.A 1032 (2004) 149–158. 123 P. Bartak, P. Bednar, L. Kubacek and Z. Stransky, Advanced statistical evaluation of complex formation constant from electrophoretic data. Anal. Chim. Acta 407 (2000) 327–336. 124 B. Lagane, M. Treilhou and F. Couderc, Capillary electrophoresis: theory, teaching approach and separation of oligosaccharides using indirect UV detection. Biochem. Mol. Biol. Educ. 28 (2000) 251–255. 125 H.J. Jung and Y.C. Bae, Theory for the capillary electrophoretic separation of DNA in polymer solution. J. Chromatogr.A 967 (2002) 279–287. 126 G.W. Slater, M. Kenward, L.C. McCormick and M.G. Gauthier, The theory of DNA separation by capillary electrophoresis. Curr. Opin. Biotechnol. 14 (2003) 58–64. 127 D.J. Winzor, Classical approach to interpretation of the charge-dependence of peptide mobilities obtained by capillary zone electrophoresis. J. Chromatogr.A 1015 (2003) 199–204.

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Natural pigments are abundant in the plant and animal kingdoms. The chemical structures of pigments show high variation including linear, cyclic and oligocyclic substructures. Besides of their capacity to colour foods and food products they show marked health effects too. As has been previously mentioned, the low volatility and low thermal stability of the majority of natural pigments make impossible the application of traditional gas-chromatoraphic methods for their analysis. However, pyrolysis gas chromatography has been successfully employed in special cases for pigment analysis. The classification of liquid chromatographic methods according to the chemical structure of the pigments is sometimes misleading. Various pigment classes such as carotenoids and chlorophylls, can occur together and can be determined in one chromatographic process. These chromatographic techniques are discussed only in one subsection. Chromatographic methods employed for the separation and quantitative determination of natural pigments have been previously reviewed. 2.1 CAROTENOIDS The basic structural characteristic of carotenoids is a symmetrical tetraterpene skeleton. The end groups of the tetraterpene structure can be modified into six membered rings. The main groups of these pigments are the carotenes without oxygen in the molecule and xanthophylls containing one or more oxygen. Carotenoids are generally highly lipophilic and insoluble in water. Derivatives esterified by strongly polar substituents such as polysaccharides show better water solublity. Carotenoids and carotenoid derivatives are one of the most widespread natural pigment classes. Besides their colouring ability they show marked biological activities too. Their antioxidant capacity has already been established [1]. It has been found that carotenoid pigments can demonstrate chain-breaking antioxidant effects [2] and these effects considerably depend on the chemical structure of the pigment and on the environmental conditions [3]. They play an important role in several other biochemical processes [4]. Thus, they enhance the bright colour of the marine environment [5] and they are responsible for the brightest colours in the indument of birds [6, 7]. Carotenoids play a considerable role in numerous sexual signals, increasing mating and/or breeding success [8,9]. It has been further demonstrated that á-carotene, lutein and â-cryptoxanthin show antitumour initiating effects [10].

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2.1.1 Chemistry and biochemistry The basic structure of carotenoids is a conjugated polyprenoid chain. Carotenoids exert a positive impact on human welfare. They show marked singlet molecular oxygen quenching capacity [11] and they protect against photosensitized peroxidation of lipids [12]. 2.1.2 Thin-layer chromatography Because of their versatility and simplicity, TLC methods have been frequently applied to the separation and semi-quantitative determination of carotenoid pigments in synthetic mixtures and various biological matrices. The retention of pure carotenoid standards has been measured in different TLC systems. Separations have been carried out on silica plates using three mobile phases: (1) petroleum ether–acetone, 6:4 v/v; (2) petroleum ether-tert–butanol 8:2 v/v, and (3) methanol–benzene–ethyl acetate 5:75:20 v/v. Carotenoids were dissolved in benzene and applied to the plates. Developments were performed in presaturated normal chambers. The chemical structure and the RF values of the analytes measured in the three mobile phases are listed in Table 2.1. It was concluded from the retention data that mobile phase 3 is the most suitable for the separation of this set of carotenoids [13]. The efficacy of various normal and RP-TLC systems for the separation of the colour pigments of Capsicum annuum was compared. Neutral aluminium oxide, silica gel, diatomaceous earth, silica gel–diatomaceous earth 1:1, cellulose, cyano, diol- and amino modified silicas were employed as stationary phases for adsorption TLC. Polyamide and modified silica layers were used for RP-TLC as received, the other stationary phases were impregnated by overnight predevelopment in n-hexane – paraffin oil, 95:5 v/v. Cabon tetrachloride, n-hexane, chloroform, ACN, acetone, THF, pyridine, acetic acid, and their various mixtures were applied as mobile phases for adsorption TLC. Methanol, 1-propanol, ACN, acetone, THF, pyridine and dioxane served as organic modifiers for RPTLC. Distilled water, buffers at various pH (solutions of and dipotassium hydrogen phosphate or potassium dihydrogen phosphate) and solutions of lithium chloride formed the aqueous phase. Carotenoids were extracted from a commercial paprika sample by acetone (1g paprika shaken with 3 ml of acetone for 30 min), the solution was spotted onto the plates. Development was carried out in a sandwich chamber in the dark and at ambient temperature. After development (15 cm for normal and 7cm for HPTLC plates) the plates were evaluated by a TLC scanner. The best separations were realized on impregnated diatomaceous earth stationary phases using water–acetone and water–THF–acetone mixtures as mobile phases. Some densitograms are shown in Fig.2.1. Calculations indicated that the selectivity of acetone and THF as organic modifiers in RP-TLC is different [14]. A slightly different set of TLC and RP-TLC methods has been applied for the separation of the colour pigment of chilli powders. The pigment profile has been used for their classification, applying principal component analysis (PCA), a multivariate mathematical statistical method. Pigments were extracted from six chilli powders of different origin (Thailand, India, China, Malaysia, Bali and Pakistan) by shaking 3 g of chilli powder with 10 ml of acetone for 30 min. The suspension was centrifuged and the supernatant was collected. The solid rest was extracted as long as it was nearly colourless. The combined extracts were evaporated to 1 ml final volume. Developments were performed in sandwich

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TABLE 2.1 THE RF VALUES OBTAINED FOR CAROTENOIDS BY USE OF DIFFERENT MOBILE PHASES

No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 a

Carotenoid


-carotene -cryptoxanthin
-cryptoxanthin Zeaxanthin Lutein Nigroxanthin -carotene monoepoxide
-carotene monoepoxide
-carotene diepoxide Lutein epoxide Antheraxanthin Violaxanthin Cycloviolaxanthin Cucurbitaxanthin A Capsanthin 3,6-epoxide Capsanthin Capsanthin 5,6-epoxide Capsorubin Capsanthol (6
R) Capsanthol (6
S) 5,6-diepikarpoxanthin 6-epikarpoxanthin 5,6-diepilatoxanthin 5,6-diepicapsokarpoxanthin

See structures above Mobile phase composition see in the text Reprinted with permission from J. Deli [13]. b

RF value in mobile phaseb

End groupa R

Q

1

2

3

a d d d d d c c c f f f g g g d f k d d h i h h

a b a d e j b a c e d f g d k k k k l l d d f k

0.93 0.76 0.75 0.46 0.46 0.52 0.92 0.92 0.91 0.46 0.45 0.41 0.58 0.50 0.43 0.38 0.35 0.32 0.25 0.38 0.35 0.26 0.36 0.28

0.92 0.83 0.83 0.72 0.73 0.78 0.92 0.90 0.90 0.58 0.55 0.46 0.74 0.72 0.61 0.60 0.47 0.42 0.26 0.68 0.36 0.38 0.45 0.30

0.93 0.80 0.80 0.51 0.52 0.63 0.91 0.91 0.91 0.47 0.47 0.44 0.77 0.63 0.55 0.44 0.39 0.38 0.23 0.49 0.39 0.24 0.33 0.30

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2.00 90% Acetone

85% Acetone

0.00 Start (a)

Front

Start

Front (b) 0

95% Acetone

00 (c)

Start

Front

Fig. 2.1. Separations of the pigments of Capsicum annuum on impregnated diatomaceous earth using acetone–water 85:15 (a), 90:10 (b) and 95:5 (c) mixtures as mobile phases. Reprinted with permission from T. Cserháti et al. [14].

chambers at ambient temperature in the dark. Chromatographic profiles were recorded at 340 and 440 nm to measure both yellow and red pigments. The intra-day and inter-day reproducibilities of the RF values and peak areas were measured by the analysis of the same sample four times a day during five consecutive days, and the relative standard deviation was calculated. PCA was performed on the parameters of the seven largest pigment fractions separated on impregnated diatomaceous earth as the stationary phase and acetone–water 17:3 v/v as the mobile phase. The chromatographic profiles at 340 and 440 nm of the chilli powder originating from Bali are shown in Fig. 2.2. The results established that the best separation in adsorption TLC can be achieved on silica layers using hexane–THF mixtures as mobile phases. The relative standard deviations were 3.4 and 5.3 per cent and 6.1 and 8.6 per cent for the RF values and peak areas, respectively, proving the moderate reproducibility of TLC results. PCA found marked differences between the chilli powders proving that this TLC separation method of pigments combined with PCA can be successfully applied for the classification of chilli powders [15].

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100

67

900

440 nm sample V

340 nm sample V

14

7 1

6 8

5 3 2

−100

5

1213 91011

4

195 mm

−100

5

195 mm

Fig. 2.2. Separation of the colour pigments of chilli powder on impregnated diatomaceous earth eluted with acetone–water (17:3 v/v). Reprinted with permission from T. Cserháti et al. [15].

Allenic and acetylenic carotenoids in marine algae serve as chemosystematic markers and as food chain indicators. Because of their importance, much effort has been devoted to the separation and identification of new carotenoid species. Gyroxanthin (3S,5R,6S,3
S,5
R,6
R)5,6-epoxy-6,7,7
,8
-tetradehydro-5,6,5
,6
-á,á-carotene-3,19,3
, 5
-tetrol was extracted from the dinoflagellate Gymnodinium galatheanum. Carotenoids were separated in the first step on silica gel–CaCO3 1:1 (w/w) with petroleum ether–Me2CO–benzene–i–propanol 75:20:4:1 (v/v) and on silica gel Ca(OH)2–MgO–CaSO4 10:4:3:1 stationary phase in the second step. For further purification of the samples the following TLC systems were employed: silica gel–CaCo3 4:1 w/w mobile phase being n-hexane–Me2CO–i-propanol 68.5:30:1.5 v/v and silica gel–diatomaceous earth–Ca(OH)2– MgO 14:16:9:9 w/w using the same mobile phase. It was established that gyroxanthin occurs as a 19-dodecanoate-3
-acetate diester [16]. The carotenoid composition of the tissues of white storks (Ciconia ciconia) feeding on introduced crayfish (Procambarus clarkii) was studied by TLC. Blood samples of nestling storks were centrifuged within 24 h of collection and the blood plasma, body fat and skin samples were stored at 20oC. Blood plasma was mixed with acetone at the ratio of 1:3 v/v then centrifuged at 13 000g for 10min. The supernatant was employed for TLC analysis. Skin and fat samples were ground by hand, mixed with 40 ml of diethyl ether, sonicated for 1min and left to stay for 2h. The organic phase was evaporated and the pigment rest was redissolved in acetone for TLC. Crayfish samples were similarly treated as fat and skin, only they were ground more strongly. Separations were carried out on silica gel plates with a fluorescence indicator using three mobile phases: (1). petroleum ether– acetone–diethylamine 10:4:1 v/v; (2). hexane–acetone 3:1 v/v, and (3). benzene–ethyl acetate 1:1 v/v. The TLC systems separated well astaxanthin and canthaxanthin. Astaxanthin was identified not only by the retention behavior in TLC but also by chemical reaction. After saponification (KOH in methanol) the formation of astacene was observed. Reduction (NaBH4 in ethanol) yielded crustaxanthin.

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The results indicated that astaxanthin is the dominant carotenoid in crayfish-eating stork while only lutein occurred in the plasma of the control stork population [17]. The quantity of á-carotene in Brazilian fortified pasta has also been measured by TLC. The content of total carotenoids was determined by extracting the ground paste with the mixture of hexane–acetone–ethanol 10:7:6 v/v followed by cold saponification for 1h with a 40 per cent solution of KOH. After saponification carotenoids were transferred to petroleum ether. á-carotene was purified in a column consisting of MgO–HyfloSupercel 1:2. It was eluted with 4 per cent diethyl ether in petroleum ether. The other pigment fractions were eluted with acetone, transferred to petroleum ether, and applied to a silica gel layer. The plates were developed with 3 per cent methanol in benzene. The presence of epoxides was determined by exposing the plates to HCl vapours. The colour of epoxides changed from yellow or orange to blue or green. The method has been proposed for the determination of the â-carotene content of fortified pastas [18]. Because of their complementary character, TLC and HPLC can be used simultaneously for the easier solution of complicated separation problems. Thus, the determination of capsaicinoids in fruit of hot pepper Capsicum annuum L. by spectrophotometry, TLC and HPLC has been reported. Samples were homogenized with acetone followed by a homogenization with acetone–petroleum ether 1:1 v/v until the tissue was nearly white. The extract was filtered and the acetone was washed out by small amounts (0.01 ml) of water. The ether phase was dried with anhydrous Na2SO4 and concentrated in vacuum at 30°C. The extract was separated on silica TLC plates using a petroleum ether–acetate–methanol (75:20:5) mobile phase. The capsaicinoids were scraped off the layer and further analysed by HPLC. The RF values of carotenoids and capsaicinoids are listed in Table 2.2. It was stated that the method can be employed for the measurement of carotenoids in hot peppers [19]. TABLE 2.2 THE RF VALUES OF CAROTENOIDS AND CAPSAICINOIDS FORM CAPSICUM ANNUUM

Carotenoids

RF


-carotene Cryptoxanthin Zeaxanthin Antheraxanthin Violaxanthin Capsanthin Capsorubin Neoxanthin Capsaicinoids

0.98 0.64 0.58 0.42 0.35 0.29 0.23 0.18 0.31

Reprinted with permission from I. Perucka et al. [19].

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Another study employed both TLC and HPLC for the analysis of carotenoids of Calendula officinalis L. TLC separation of all E(trans)-á,á-carotene, cryptoxanthin, zeaxanthin and lutein was performed on a silica layer using petroleum ether– i–propanol–CHCl3 (90:10:70 v/v). The same carotenoid pigments were separated by RP-HPLC using an ODS column (2504 mm, i.d.). The organic modifiers were methanol, THF and ethyl octane. The flow rate was 1 ml/min, pigments were detected at 440 nm [20]. The dependence of the quantity of
-carotene and lutein in Biomphalaria glabrata and Helisoma trivolvis (Colorado and Pennsylvania strains) snails on the diet and parasitism were measured by high performance RP-TLC. Five whole snail bodies and five digestive gland–gonad complexes (DGG) were washed with artificial spring water and then homogenized with 2 ml of acetone. The supernatant was filtered, the rest was washed with 2  200 ml of acetone. The combined organic fraction was evaporated under nitrogen atmosphere and redissolved in 200 – 1000 ml of heptane. Standards were prepared in dichloromethane. C-18 HPTLC plates with pre-absorbent zones were prewashed by predevelopment in dichloro methane–methanol (1:1, v/v). The developing chamber was pre-equilibrated for 15min. The mobile phase consisted of petroleum ether (37.8–53.5 °C)–ACN–methanol (1:1:2, v/v). The developing distance was approximately 7cm and developing time was about 19min. Pigment degradation was prevented by carrying out the experiments in subdued light. Plates were evaluated by a TLC scanner at the maximum wavelenth of lutein (448 nm) and á-carotene (455 nm). The results are compiled in Table 2.3. The results indicated that yolk diet significantly increased the concentration of pigments while parasitism has no significant effect on the amount of pigments. The data demonstrate that the RP-HPTLC method can be used for the determination of pigments in snails [21]. TABLE 2.3 WEIGHT PERCENTAGE OF
-CAROTENE AND LUTEIN IN THE WHOLE BODY AND DGG OF H.TRIVOLVIS (CO) SNAILS MAINTAINED ON A YOLK (Y) DIET OR LETTUCE (L) DIET

Pigment

Sample

Ya

La


-carotene

Whole body DDG Whole body DGG

0.0008620.00025b 0.001900.00052b,c 0.002190.00038b 0.005200.00097b

0.002200.00031 0.008910.00059 0.008460.0010 0.02100.0020

Lutein

Mean (wt %)S.E.; n  5 individual snails for each sample. n  4 individual snails after elimination of one value with the statistical Q-test. c Concentrations significantly reduced (Student’s t-test, P0.05) compared with snails on L diet. a

b

(Continued on next page)

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TABLE 2.3 (continued) Pigment

Sample

Y*

L*


-carotene Lutein

DGG DGG

0.003700.00061 0.003840.00058**

0.01390.0021 0.01060.0010

Snail DGGs; mean (wt%)S.E.; n  5 individual snails for each sample. Concentrations significantly reduced (Student’s t-test, P0.05) compared with snails on L diet.

*

**

WEIGHT PERCENTAGE OF
-CAROTENE AND LUTEIN IN THE WHOLE BODY AND DGG OF ECHINOSTOMA TRIVOLVIS INFECTED (I) AND UNINFECTED (U) H. TRIVOLVIS (PA) SNAILS

Pigment

Sample

U*

I*


-carotene

Whole body DDG Whole body DGG

0.005640.0011 0.01420.0029 0.005420.00084 0.006020.0013

0.007420.00082 0.01220.0026 0.003890.00085 0.005510.0014

Lutein

Mean (wt %) S.E.; n  5 individual snails for each sample.

*

WEIGHT PERCENTAGE OF
-CAROTENE AND LUTEIN IN THE WHOLE BODY AND DGG OF ECHINOSTOMA CAPRONI INFECTED (I) AND UNINFECTED (U) B. GLABRATA SNAILS

Pigment

Sample

U*

I*


-carotene

Whole body DDG Whole body DGG

0.02200.0025 0.006940.00062 0.01010.0018 0.006020.0013

0.01950.0022 0.007840.00092 0.01110.00092 0.005510.0014

Lutein

Mean (wt %) S.E.; n  5 individual snails for each sample. Reprinted with permission from R. T. Evans et al. [21].

*

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2.1.3 High-performance liquid chromatography RP-HPLC is the method of preference for the separation and quantitative determination of carotenoid pigments on both plant and animal tissues and in algae, insects and bacteria. The popularity of RP-HPLC is mainly due to the relative simplicity, to the decreasing environmental loading caused by the organic components of the mobile phase, the low wavelength cut and the rapidity of the analysis. The overwhelming majority of techniques apply octadecylsilica (ODS or C18) analytical columns of 4 – 4.6 mm internal diameter (i.d.) with a particle size of 5m. Because of slightly different separation characteristics, a C30 stationary phase has also found application in the RP-HPLC analysis of carotenoids. 2.1.3.1 HPLC determination of carotenoid pigments in samples of plant origin Surprisingly, traditional column chromatography has also been employed for the separation and quantitative determination of carotenoid composition of tea leaves. Pigments were extracted by homogenizing 50g of tea leaves (or 10g of tea dust) with 100 ml of methanol. The suspension was filtered and the solid residue was extracted with 100 ml of acetone followed with 100 ml of peroxide-free diethyl ether. The combined extract was washed with water and evaporated in vacuum. Chlorophylls and fats were removed by refluxing the residue with 50 ml of 10 per cent potassium hydroxide for 20min at 60°C under nitrogen atmosphere and stored overnight at ambient temperature in the dark. Pigments were extracted with peroxide-free diethyl ether, washed free of potassium hydroxide, concentrated and stored in light petroleum ether (40–60°C). Carotenoids were separated on an alumina column using various mobile phases such as mixtures of peroxide-free diethyl ether and petroleum ether (40–60°C), and acetone and petroleum ether. Pigments were tentatively identifed by their UV–visible spectra and by comparing their retention behaviour with those of authentic standards. The pigment composition of Assam, Cambod and China teas are compiled in Table 2.4. The data in Table 2.4 clearly show that the pigment composition of teas depends considerably on the type of tea cultivars while the effect of the manufacture on the quantity of pigments is negligible (illustrated in Table 2.5). The results demonstrate that traditional column chromatography may facilitate the measurement of the amount of pigments in teas and can be used for the following of pigment degradation during manufacturing [22]. Traditional column chromatography has also been employed for the extraction of carotenoids from palm oil. Separations were carried out on silica columns, carotenoids were eluted with n-hexane while the free fatty acids of the oil were removed from the stationary phase with ethyl acetate. The recovery of the method was 45 per cent and the purity of the cartotenoid fraction about 20 per cent w/w [23]. Because of their commercial importance, the pigment composition of paprika (Capsicum annuum) and chilli powder (Capsicum frutescens) have been intensively investigated by various HPLC methods [24–26]. An ODS column (250  4.6 mm i.d.; particle size 6 m) was applied for the determination of the carotenoid composition of paprika paste. Pigments were extracted three times with methanol and twice with diethyl ether. The combined extract was saponified with 30

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TABLE 2.4 CAROTENOID AND CHLOROPHYLL COMPOSITION OF TEA LEAVES AND DUST (MG/100 G DRY WT)a

Carotenoids

Pytoene Antheraxanthin Lycopene Pytofluene  -carotene
-carotene
-zeacarotene Aurochrome Mutatochrome
-cryptoxanthin Cryptoflavin Cryptoxanthin-5,8-di-epoxide Lutein Zeaxanthin Lutein-5,6-epoxide Violaxanthin Neoxanthin Chlorophyll-a Chlorophyll-b Toral chlorphyll Total carotenoid

Assam

China

Leaf

Dust

Leaf

1.07 1.65 3.25 0.09 0.31 8.14 2.69 0.16 0.17 0.89 0.48 Trace 10.0 11.2 0.46 1.49 0.33 782 314 1 095 36.7

0.39 0.33 0.56 0.04 0.21 3.96 0.22 Trace 0.21 0.03 0.50 0.05 6.13 5.91 3.17 Trace 0.23 183 69.5 255 6.10

2.18 1.77 2.81 0.08 2.31 32.1 0.97 0.23 0.31 3.25 0.08 0.46 211 17.71 0.06 0.25 1.97 1034 437 1 471 73.1

Dust

Cambod Leaf

0.62 1.70 0.41 1.70 0.53 2.83 0.06 0.05 0.81 1.87 11.67 16.0 0.04 1.37 Trace 0.19 0.59 0.23 0.07 2.67 0.36 0.22 0.07 0.46 7.09 16.7 7.01 15.91 1.67 0.12 0.11 0.71 0.06 1.58 271 719 101 255 376 1 002 24.7 49.3

Dust 0.46 0.37 0.53 0.03 0.44 6.11 0.09 0.03 0.58 0.05 0.41 0.05 6.56 6.88 2.27 0.09 0.12 281 70.9 249 11.5

a Data are average of three trials in triplicate as mean with standard deviation less than 0.9 per cent. Trace  less than 0.01. Reprinted with permission from R. Ravichandran [22].

per cent KOH in methanol. Separation of pigments was performed with gradient elution consisting of water–methanol and dichloromethane mixtures. The flow rate was 1.2 ml/min and pigments were detected at 450 nm. A typical chromatogram illustrating the good separation capacity of the HPLC system is shown in Fig. 2.3. The quantitative results are compiled in Table 2.6. For peak identification see Fig. 2.3. Storage experiments proved that the carotenoid composition of products did not change markedly in one year, the brownish colouration may be due to the oxidation of flavonoid compounds [27]. A microbore ODS column was employed for the separation of the paprika pigments and the performance of the column was compared with that of the normal HPLC column.

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TABLE 2.5 PIGMENT DEGRADATION DURING TEA MANUFACTUREa

Total Total Total Total Poly Catechins carotenoid Chlorophyll Chlorophyll-a Chlorophyll-b phenols (a:b) Green leaf Withered leaf Fermented dhool Dried tea Brew Tea residue

73.1 70.3 61.6 24.7 9.9 16.4

1 471 1 441 1 202 376 247 129

1 034 1 014 830 271 186 84.8

437 412 372 101 58.5 42.3

31.7 29.1 13.4 10.5 5.8 7.0

24.6 22.6 11.7 9.6 4.2 6.3

a

Chlorophyll expressed as mg/g dry weight. Carotenoid expressed as mg/100 g dry weight. Data are average of three trials in triplicate as mean with the standard deviation less then 0.8 per cent. Reprinted with permission from R. Ravichandran [22].

Pigments were extracted by shaking 1g of paprika powder with 3 ml of acetone for 30min. After finishing the extraction, the suspension was centrifuged and the supernatant collected. The procedure was repeated four times. The collected organic phases were diluted to 100 ml with acetone. Pigments were separated on a normal (150  4 mm i.d., particle size 5 m) and on a microbore ODS column (150  2 mm i.d., particle size 4 m) using gradient elution. The steps of gradient elution are shown in Table 2.7. Carotenoids were detected at 440 nm. Columns were not thermostated and separations were performed at room temperature (202°C). The mean and the relative standard deviation of retention time and peak area were computed from three parallel measurements. The carotenoids capsanthin, zeaxanthin and
-carotenein and the extracts were tentatively identified comparing their retention time with those of authentic standards. The chromatographic profiles of pigments separated on normal and microbore columns are displayed in Fig. 2.4. High differences were found between the separation capacity of normal and microbore columns. Using the normal column 34 pigment fractions were separated while 75 carotenoids were separated in the microbore column. The relative standard deviation of retention times was less than 1.5 per cent demonstrating the stability and reproducibility of the RP-HPLC system. However, the reproducibility of the peak area was considerably lower (3 – 5 per cent). This finding can be tentatively explained by the fact that the baseline separation of carotenoids was not achieved in each case and the integration of partially separated fractions was subjected to higher uncertainty. The results illustrated that a microbore column can be successfully used for the separation of various pigment fractions of paprika powder having higher separation capacity than the traditional normal RP-HPLC columns [28].

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8

22

14 20 11 13 12 1

0

5

10

2

15

3

23

10

6 4 5 7

9

19 15

25 20 Time (min)

17 16

30

21

18

35

40

Fig. 2.3. Characteristic chromatogram of paprika paste. Detection at 450 nm. Peak identification: 1  Capsorubin; 2  5,6-Diepikarpoxanthin; 3  Capsanthin-5,6-epoxide; 4  Capsanthin-3,6-epoxide; 5  Violaxanthin; 6  Luteoxanthin 2; 7  Luteoxanthin 1; 8  Capsanthin; 9  Antheraxanthin; 10  Mutatoxanthin; 11  Cucurbitaxanthin A; 12  (9/9
Z)-Capsanthins; 13  (13/13
Z)Capsanthins; 14  Zeaxanthin; 15  Nigroxanthin; 16  (9Z)-Zeaxanthin; 17  (13Z)-Zeaxanthin; 18  Cryptocapsin; 19  -Cryptoxanthin; 20 
-Cryptoxanthin; 21  (Z)-Cryptoxanthin; 22 
-Carotene; 23  (Z)-
-Carotene. Reprinted with permission from J. Deli et al. [27].

The efficacy of various RP- and normal HPLC columns for the separation of colour pigments of chilli powder has also been compared. Pigments were extracted with acetone as previously described. Columns included in the experiments were: (1) ODS (150  4 mm i.d., particle size, 5m; carbon loading, 9.7 per cent); (2) ODS (150  2 mm i.d., particle size 4m, carbon loading, 9.5 per cent); (3) silica (150  2 mm i.d., particle size 4 m); (4) ODS (125  2 mm i.d., particle size 5 m, carbon loading, 4.9 per cent); (5) RP-Alu (150  2 mm i.d., particle size 5m, carbon loading 4.0 per cent). Mobile phases for RP-HPLC columns consisted of methanol–ACN (80:20, v/v) (solvent A) and water (solvent B). Gradient elution began with 15 per cent A to 40 per cent A in 10min, to 80 per cent A in 15 min, 10 min hold, to 90 per cent A in 10min, to 97 per cent A in 3 min, final hold. Pigments were eluted from the silica column with the following gradient: from 100 per cent n-hexane to 75 per cent THF in 25 min, final hold. Flow rates for columns 1–5 were 0.80, 0.17, 0.22, 0.50 and 0.17 ml/min, respectively. Pigments were detected at 340 and 440 nm. The chromatographic profiles of pigments measured on different columns are shown in Fig. 2.5.

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TABLE 2.6 CAROTENOID COMPOSITION IN RED TOMATO-SHAPED PAPRIKA (1), DIFFERENT PAPRIKA PASTE (2–5) AND GROUND PAPRIKA (6 AND 7) (%)

Peak

1

2

3

4

5

6

7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Total carotenoid content mg/g dry weight

2.1 0.7 1.8 2.0 1.3 0.7 0.7 33.1 2.2 2.4 5.4 0.9 3.2 8.8 0.9 0.1 0.3 0.8 0.1 6.8 1.9 13.7 — —

2.2 1.0 1.1 2.0 0.7 0.7 0.5 33.2 0.2 4.2 6.4 1.6 6.6 9.0 1.0 0.2 0.7 1.0 0.3 5.9 1.4 11.3 3.0 2.85

2.1 1.3 1.9 2.0 — 0.6 0.3 40.8 0.6 3.7 7.8 1.8 6.3 10.7 1.2 0.1 0.4 0.5 0.3 4.8 1.1 10.4 — 3.62

2.5 1.0 1.6 1.4 — 0.2 0.2 36.5 0.6 2.4 6.8 3.2 8.6 11.5 — 0.3 2.0 0.9 0.2 4.6 2.0 8.2 2.7 2.66

2.6 — 1.5 2.0 1.2 0.6 0.5 38.2 — 2.5 6.8 3.4 6.2 12.3 — 1.41 2.3 1.0 0.2 4.2 1.6 6.2 1.8 1.47

2.1 0.6 1.7 1.4 — 0.5 0.2 46.0 1.0 4.0 7.8 1.8 3.7 12.1 1.3 0.1 0.2 0.3 0.1 5.8 0.9 8.6 — 4.18

3.1 1.3 2.3 2.1 — 0.7 — 32.1 1.0 2.6 6.3 3.6 6.8 8.7 1.1 0.7 1.6 0.3 0.3 4.1 1.2 8.8 1.6 2.88

Reprinted with permission from J. Deli et al. [27].

It was established that the best separations can be achieved in ODS columns, however the separation capacity depends considerably on the type of stationary phase and on the dimensions of the column [29]. RP-HPLC has been also employed for the study of the efficacy of microwave-assisted extraction (MAE) of carotenoid pigments from paprika powders. The study was motivated by the fact that the traditional extraction methods are time consuming, and they require a relatively great amount of organic solvents increasing environmental pollution. Moreover, pigments are strongly bonded to the insoluble cell components making difficult their extraction. Each extraction process was carried out on suspensions consisting of 40 mg of paprika powder and 2 ml of extracting agent. Extracting agents were water mixed with acetone,

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TABLE 2.7 COMPOSITION OF GRADIENT ELUTION FOR THE SEPARATION OF COLOUR PIGMENTS OF PAPRIKA (CAPSICUM ANNUUM) POWDERS IN NORMAL (150  4 MM I.D.) AND MICROBORE (150  2 MM I.D.) OCTADECYLSILICA COLUMNS. A  ACN – METHANOL (1:4, V/V); B  BIDISTILLED WATER. EACH GRADIENT STEP WAS LINEAR. FLOWRATES FOR NORMAL AND MICROBORE COLUMNS WERE 2 ML/MIN AND 0.6 ML/MIN, RESPECTIVELY

Time (min)

Normal column A vol per cent

0 10 15 17 60

80 90 95 97 97

Time (min)

Microbore column B vol per cent

0 5 6 9 60

15 15 95 97 97

Reprinted with permission from T. Cserháti et al. [28].

dioxane, ethanol, methanol and THF in volume ratios of 15, 30, 45, 60, 75 and 90 per cent vol. After extraction the samples were centrifuged and the UV-vis absorption at 395, 410, 425, 455, 470 and 485 nm was measured and the chromatographic profiles of the supernatants were determined. The separation of pigments was performed on an ODS column (125  3 mm i.d., particle size 5 m) using gradient elution. The steps of gradient elution are compiled in Table 2.8. The flow rate was 0.5 ml/min and pigments were detected at 440 nm. Measurements were performed at ambient temperature. The data were evaluated by the spectral mapping technique and by multivariate linear regression analysis. It was found that optimal conditions for the microwave-assisted extracting of carotenoids from paprika powder were 120 s extraction time and 50 W electric power. Calculations proved that the efficacy of extraction increases with increasing concentration of the organic component in the extracting media and the selectivity differences between the organic modifiers are relatively low. Both the extraction strength and the extraction selectivity significantly depended on the physicochemical parameters of the organic component: o

Extraction strength  0.153  (1.42  0.22).103. a R  0.8075, F5%  52.47, sxy  0.015, P  0.0001

(1) o

First coordinate of the selectivity map  96.03  (1.48  0.26). a R  0.7351, F5%  32.92, sxy  21.65, P  0.0001

(2) o

Second coordinate of the selectivity map  147.69  (1.37  0.22). a R  0.7646, F5%  39.41, sxy = 18.31, P  0.0001

(3)

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Capsanthin

Zeaxanthin β-Carotene

0

Minutes

60

(a)

Absorbance 440 nm

Capsanthin

(b) 0

Zeaxanthin

β-Carotene

Minutes

60

Fig. 2.4. Separation of the colour pigments of Capsicum annuum on a normal (a) and microbore column (b). Reprinted with permission from T. Cserháti et al. [28].

where  is the calculated dielectric contant of the extracting agents using the additivity rule, R is the regression coefficient, F5% is the test of the null hypothesis, and sxy is the standard error of the estimate (n  30). The chromatograms of pigments extracted with water–acetone (10:90, v/v) and water–THF (75:35, v/v) are shown in Fig. 2.6. It was concluded from the data that MAE can be applied for the efficient extraction of carotenoid pigments from paprika powder, however the amount and composition of the extracted pigment depends considerably on the composition of the extracting agent [30]. The stability of the pigments of paprika powder under various storage conditions has also been investigated by RP-HPLC. Samples of paprika powders were vacuum packed

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Absorption 340 nm

Absorbance 440 nm

78

0

(b)

130 Minutes

Absorption 340 nm

Absorption 440 nm

Minutes

0

0

180 Minutes

(d)

200 Minutes

Absorption 440 nm

Absorbance 340 nm

(c)

0 (e)

0

130

(a)

140 Minutes

0 (f)

140 Minutes

Fig. 2.5. Separation of colour pigments of chilli powder on column 1 (a,b), on column 2 (cd) and on column 5 (e,f). For conditions see text. Reprinted with permission from T. Cserháti et al. [29].

and normal atmosphere packed in plastic bags and were stored in the dark and under an 18 W daylight tube at 221oC. Parallel samples for the analysis of the quantity and composition of pigments were taken from each treatment at 4, 8, 12, 16, 20, 24 and 28 days of storage time. Extraction of pigment was carried out by homogenizing 2.0g of paprika powder with 15.0 ml of acetone for 3min. After extraction the suspension was centrifuged, the supernatant collected and the procedure was repeated until the rest was quasi colourless. The combined extracts were dried with anhydrous Na2SO4 and evaporated to dryness at 35°C. RP-HPLC separation of pigments was performed in an ODS column (250  4 mm i.d., particle size 10 m) using an ODS precolumn (4  4 mm i.d., particle size 5 m). Pigments were detected at 450 nm. The components of the gradient elution were acetone–water (75:25, v/v) (eluent A) and acetone–methanol (75:25, v/v) (eluent B). Elution was iniciated at 0 per cent B, increased to 65 per cent B in 10min, to 100 per cent

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TABLE 2.8 GRADIENT ELUTION FOR THE REVERSED-PHASE HIGHPERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION OF COLOUR PIGMENTS IN THE EXTRACTS OF PAPRIKA (CAPSICUM ANNUUM). ELUENT A, METHANOL–ACETONITRILE (80:20, V/V); ELUENT B, BIDISTILLED WATER

Time (min)

A (%)

B (%)

0 10 25 35 45 55 58 120

15 40 80 80 90 90 97 97

85 60 20 20 10 10 3 3

Reprinted with permission from G. A. Csiktusnádi-Kiss et al. [30].

B in 60 min. The flow rate was 1 ml/min. The photodegradation of pigments was prevented by carrying out the measurements under subdued light. The effect of storage conditions, such as storage time, presence or absence of light and oxygen on the decomposition rate of carotenoids was evaluated by stepwise regression analysis and principal component analysis. Typical chromatograms showing the decrease of the amount of pigments in paprika powders are shown in Fig. 2.7. Calculations proved that the overall decomposition rate of pigments significantly depends on the storage conditions: Total peak area  4.92.104  (3.940.85).102.storage time in days  (5.751.37).103.presence of light  (3.901.37).103.presence of oxygen

(4)

The presence of light and oxygen and the length of storage equally decreased the amount of pigments of paprika powder. Interestingly, the relative impact of the three storage conditions was highly similar. It has been concluded from the data that the RP-HPLC technique combined with multivariate mathematical statistical methods is suitable for the evaluation of the effect of storage conditions on the overall velocity and selectivity of pigment decomposition in paprika powders [31]. A similar RP-HPLC method has been employed for the determination of the effect of reduced glutathion (GLT) and hydroxypropyl-
-cyclodextrin (HP-
-CD) on the stability of the colour pigments of paprika. Samples were prepared by mixing 5 per cent GLT (w/w) and 5 per cent HP-
-CD (w/w) with paprika powder and stored at room temperature (221°C) in diffuse light in Petri dishes covered by glass plates. Samples without additives served as a control. 1g of paprika was taken after 14, 28, 42 and 56 days of storage

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Absorption 440 nm

80

0

50 Retention time, min

0

50 Retention time, min

100

Absorption 440 nm

(a)

(b)

100

Fig. 2.6. Separation of colour pigments of paprika powders extracted with water–acetone (10:90, v/v; chromatogram a) and water–THF (75:35, v/v, chromatogram b). Reprinted with permission from G. A. Csiktusnádi-Kiss et al. [30].

time. Carotenoid pigments were exhaustively extracted with acetone and the absorption of the extracts was measured at 395, 410, 425, 440, 455, 470, 485 and 500 nm. RP-HPLC separation of pigments was performed on an ODS column (150  4 mm i.d., particle size 5 m). Solvents A and B were methanol–ACN (80:20, v/v) and bidistilled water, respectively. Initial eluent composition was 80 per cent A changing to 90 per cent A in 10min, to 97 per cent A in 2 min final hold to 60 min. The flow rate was 2 ml/min and the detection wavelength 450 nm. Capsanthin, zeaxanthin and
-carotene served for the tentative identification of these pigment fractions. Some typical chromatograms are shown in Fig. 2.8.

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Absorbance (AU)

0.08 0.06 0.04 0.02 0.00 0

5

10

15

20

0

5

10

15

20

(a)

25 30 35 40 Retention time (min)

45

50

55

60

45

50

55

60

Absorbance (AU)

0.04 0.03 0.02 0.01 0.00

(b)

25

30

35

40

Retention time (min)

Fig. 2.7. Separation of pigments of Capsicum annuum on a LiChrocart RP-18 column after 28 days of storage, using water–acetone–methanol gradient elution, detection wavelengh 450 nm, flowrate; 1ml/min. Storage conditions: a: darkness, absence of oxygen, b: light, absence of oxygen. Reprinted with permission from H. Morais et al. [31].

It was established that neither GLT nor HP-
-CD are suitable for the enhancement of the stability of carotenoid pigments in paprika powders [32]. Not only RP-HPLC but also normal-phase HPLC found application in the analysis of the carotenoid composition of Capsicum annuum and the results achieved in the different HPLC systems have been compared. Pigments were homogenized and extracted exhaustively with acetone. The combined extracts were mixed with 100 ml of ethyl ether and washed with 100 ml of 10 per cent aqueous NaCl solution. The organic fraction was washed with saturated Na2SO4 solution and dried over anhydrous Na2SO4. Saponification of pigments was carried out by adding to the carotenoid solution 100 ml of 20 per cent NaOH and 20 per cent methanol. After 1h incubation time the phases were separated and the organic fraction was washed again with water and evaporated to dryness at 35°C. Normal-phase HPLC separation of pigments was performed in a silica column (150  3.5 mm i.d., particle size 10 m). Gradient elution was carried out with hexane-acetone mixtures. RP-HPLC separations were performed in an ODS column of the same dimensions. Pigments were detected at 450 nm. Authentic standards were

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Absorption 430 nm

82

3 2 1

0

Absorption 430 nm

(a)

60 Time, min

Absorption 430 nm

2

3

2

3

1

1

0 (b)

0

60 Time, min

(c)

60 Time, min

Fig. 2.8. Reversed-phase HPLC chromatograms (430 nm) of colour pigments of paprika powder after 56 days of storage time. a  control, b  5 per cent GLT, c  5 per cent HP-
-CD. Peak indentification: 1  capsanthin; 2  zeaxanthin; 3 
-carotene. Reprinted with permission from T. Cserhati et al. [32].

employed for the identification of carotenoids. Some typical chromatograms are shown in Fig. 2.9. The chromatograms show marked differences indicating that the quantity and composition of pigments depends considerably on the type of varieties under investigation. The carotenoid composition of the three varieties determined by RP and normal-phase HPLC are compiled in Table 2.9. The data demonstrated that these varieties have high provitamin A activity [33]. RP-HPLC was further used for the measurement of the chromatographic profile of the pigments of chilli powder (Capsicum frutescens). Pigments were extracted by shaking 1g of chilli powder with 30 ml of acetone for 30min. After the extraction step the mixture was centrifuged and the supernatant was collected. This procedure was repeated as the residue became nearly colourless. Supernatants were collected and concentrated to 1 ml by evaporation. An aliquot of the sample was tenfold diluted and injected into the column through a 20 l loop. The

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Reverse phase HPLC 40 I. 35 A

Relative area

30 25 20

B

15 10 5

C

0 10

13

16

20 24 Retention times, min.

28

32

36

Normal phase HPLC 60 II. A

50

Relative area

40 B

30

20 C

10

0 2

7

17 Retention times, min.

26

34

Fig. 2.9. Separation of carotenoid pigments by reversed-phase (I) and normal-phase (II) HPLC. The relative areas of carotenoids (retention time) are shown for latoxanthin (9.7), capsorubin (11.4), neoxanthin (13.1), capsanthin 5,6-epoxide (13.25), violaxanthin (14.4), capsanthin 3,6-epoxide (15.3), luteoxanthin (16.1), curcurbitaxanthin B (17.3), capsanthin (17.8), cyclo-violaxanthin (19.3), antheraxanthin (19.3), mutatoxanthin (20.9), 9-cis-capsanthin (23.0), lutein (24.0), zeaxanthin (24.3), 9-cis-zeaxanthin (27.2), 15-cis-zeaxanthin (28.4), canthaxanthin (29.6), cryptocapsin (31.5), -cryptoxanthin (33.9),
-cryptoxanthin (34.4),
-apo-8
-carotenal (34.9), -carotene (40.8) in RP-HPLC separation mode. Retention times of pigments separated in the normal phase mode are:
-carotene (1.85), cis-
-carotene (2.35), carotene (2.68), cyprocapsin (3.32), cryptoflavin (4.05),
-cryptoxanthin (7.38), -cryptoxanthin (7.63), antera-xanthin (9.33), lutein (11.55), cucurbitaxanthin A (13.47), luteoxanthin (17.27), zeaxanthin (19.56), cis-zeaxanthin (20.25), mutatoxanthin (23.67), cycloviolaxanthin (24.60), 9-cis-capsanthin (26.08), capsanthin (27.50), capsanthin 2,6-epoxide (28.75), violaxanthin (30.82), capsorubin (33.12), neoxanthin (33.63). A: Mulato, B: Guajillo, C: Ancho varieties. Reprinted with permission from O. Collera et al. [33].

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TABLE 2.9 CAROTENOID COMPOSITION IN THE THREE VARIETIES OF C. ANNUUM IN REVERSEDPHASE AND NORMAL PHASE HPLC

No.

Pigment (order of elution)

Ancho

Guajillo

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Latoxanthin Capsorubina Neoxanthina Capsanthin 5,6-epoxide Violaxanthin Capsanthin 3,6-epoxide Luteoxanthin Curcurbitaxanthin B Capsanthin Cycloviolaxanthin Antheraxanthina Mutatoxanthin 2 Mutatoxanthin 1 9-cis-capsanthin Luteina Zeaxanthinb 9-cis-zeaxanthin 15-cis-zeaxanthin Canthaxanthin Cryptocapsin -cryptoxanthin
-cryptoxanthinb
-Apo-8
-carotenala,b -caroteneb
-carotenea,b Non-identified compounds Weight (%) Total carotenoids (mg/100 g dw)
-caroteneb cis-
-carotene -carotene Cryptocapsin Cryptoflavinec
-cryptoxanthinb -cryptoxanthin Anteraxanthinb Luteinb Cucurbitaxanthin A Luteoxanthin

5.55 2.17 — — 14.5 — — 10.5 9.69 — Trace 0.30 0.33 10.1 — 4.29 — — — 0.24 5.28 9.70 Ref. 2.1 20.9 4.39 100 7.52 19.7 — 13.8 — 8.06 — — Trace Trace — 2.67

1.56 0.35 0.28 — 13.2 — — — 12.6 0.51 7.07 0.31 0.30 10.0 — 1.88 0.50 4.79 — 10.0 2.24 9.53 Ref. 1.27 17.9 5.74 100 100% 16.2 4.29 7.65 6.87 4.13 4.43 10.8 Trace Trace — —

1 2 3 4 5 6 7 8 9 10 11

Mulato 1.53 4.20 2.20 1.43 22.0 11.6 2.10 — 11.2 — Trace 1.23 — 9.93 0.88 3.56 — — 0.78 0.62 0.72 6.10 Ref. 2.98 14.9 2.00 100 7.24 11.0 8.80 8.50 5.63 3.50 0.33 7.08 Trace 0.02 2.75 2.75

(Continued on next page)

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TABLE 2.9 (continued)

No.

Pigment (order of elution)

Ancho

12 13 14 15 16 17 18 19 20 21 22

Zeaxanthin cis-zeaxanthin Mutatoxanthin Cycloviolaxanthin 9-cis-capsanthinb Capsanthina,b Capsanthin 2,6-epoxide Violaxanthin Capsorubinb Neoxanthinb Latoxanthin Non-identified compounds Weight (%)

8.63 8.40 Trace — 17.7 5.82 Trace Trace 1.40 Trace 0.71 15.7 100

Guajillo 4.41 — 3.42 1.15 7.88 Trace 0.85 Trace 0.30 6.00 0.88 20.8 100

Mulato 0.03 1.79 7.30 0.29 0.12 0.05 1.16 11.1 0.67 5.93 2.96 20.3 100

Most carotenoids were assigned only by their retention time in HPLC and UV-Vis spectra. a Compounds isolated and identified by UV, visible, infrared and mp. b Compared with reference standard. c Carotenoids which were assigned only by normal phase HPLC. Reprinted with permission from O. Collera et al. [33].

analytical column was of 1504 mm i.d. filled with ODS support (particle size 5 m). Gradient elution was applied for the separation of pigments. The organic component of the mobile phase consisted of methanol–ACN (8:2, v/v), the linear gradient was from 15 per cent to 80 per cent organic component in 50min, isocratic hold for 30min, to 7 per cent organic modifier in 10 min, and a final isocratic hold for 10 min. The flow rate was 1 ml/min. Diode-array detection (DAD) was set to 300 – 600 nm including the absorption maximum of both yellow (340 nm) and red pigments (440 nm). The column was not thermostated, separations were performed at ambient temperature. The RP-HPLC profile of chilli powder is shown in Fig. 2.10. The chromatograms demonstrate the complexity of the pigment composition of chilli powder. They further illustrate that a higher amount of pigments are detected at 340 nm than at 440 nm, that is, the chilli powder contains more yellow than red pigments. Red pigments are more strongly retained in the column suggesting that they are more hydrophobic than the yellow ones. The method was proposed for the analysis of chilli powders, facilitating their identification [34]. A similar study has been carried out in order to test the capacity of RP-HPLC for the authenticity test of chilli powders on the basis of pigment composition. Carotenoid pigments were extracted by shaking 3 g of chilli powder with 10 ml of acetone for 30 min. The supernatant was decanted and the procedure was repeated as the solid rest was nearly colourless. The collected organic phases were evaporated and redissolved in the mobile phase. Separations were performed on a narrow-bore ODS column (150  2 mm i.d., carbon loading, 9.5 per cent). Eluents A and B were methanol–ACN (80:20, v/v) and bidistilled water, respectively. Gradient elution was initiated by 15 per cent A increased to 80 per cent A in 25 min, held for 10 min, increased to 90 per cent A in 10 min, held for 10 min, increased to 97 per cent A in 3 min and held for 62 min. Each step of gradient elution was linear. Measurements were

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Absorbance 440 nm

Absorbance 340 nm

86

0 (a)

0

80 Time, minutes

(b)

80 Time, minutes

Fig. 2.10. Separation of the colour pigments of chilli powder on an octadecylsilica column. Detection wavelengths 340 (a) and 440 (b) nm. Reprinted with permission from T. Cserháti et al. [34].

carried out at ambient temperature (211°C). Yellow and red pigments were detected at 340 and 440 nm, respectively. The flow rate was 0.17 ml/min. Principal component analysis was employed for the elucidation of the similarities and dissimilarities among the chromatographic profile of chilli powders. Some typical chromatograms are shown in Fig. 2.11. The chromatographic profile of chilli powders differs according to both the detection wavelength and the origin of the chilli powder. This finding indicates that both yellow and red pigments are present and their ratio depends on the origin of the chilli powder. Principal component analysis of chromatographic data illustrated that the chilli powders can be readily separated according to their origin using RP-HPLC combined with multivariate mathematical statistical methods [35]. Lycopene, a carotenoid occurring mainly in tomatoes, is extensively applied as a natural colouring agent in food and food products. Unfortunately, lycopene is insoluble in water but soluble in highly toxic hydrophobic organic solvents such as benzene, chloroform, methylene chloride, etc. Moreover, lycopene decomposes easily during the extraction process. In order to increase the yield of lycopene from tomatoes supercritical fluid extraction (SFE) was employed for the preconcentration of lycopene and
-carotene from the pulp and skin of ripe tomatoes and their purity was checked by RP-HPLC. Whole fresh tomatoes were ground and filtered in vacuum. The remaining moisture was adsorbed by mixing the sample with silica gel. Tomato skin and seed were dried for 24h in an air drier at 35°C and stored at 5°C. Extraction was carried out at pressures between 2 500–4 000 psi and at different temperatures (40–80°C) for 30min. RP-HPLC measurements were performed in an ODS column (250  4.6 mm i.d.; particle size 5m). The injected volume was 2l and the mobile phase contained methanol-THF-water (67:27:6 v/v). Carotenoids were detected at 446 nm. A typical chromatogram of the extract is shown in Fig. 2.12. The chromatogram clearly shows that the selectivity of the supercritical extraction procedure is fairly high and it can be used for the extraction of lycopene from tomatoes. The yields achieved in the presence or absence of an entrainer are listed in Table 2.10. It was found that chloroform as an entrainer increases considerably the yield while n-hexane is ineffective. Yield also increased with increasing temperature [36].

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Absorbance 340 nm

Absorbance 340 nm

Liquid chromatography of natural pigments

120

0 (a)

Absorbance 440 nm 0 (b)

0 (c)

120 Minutes

Absorbance 440 nm

Minutes

120 Minutes

0 (d)

120 Minutes

Fig. 2.11. Separation of colour pigments of chilli powders. a: origin Malaysia, detection wavelength 340 nm; b: origin Malaysia, detection wavelength 440 nm; c: origin Thailand, detection wavelength 340 nm; d: origin Thailand, detection wavelength 440 nm. Reprinted with permission from a. Kósa et al. [35].

Fig. 2.12. HPLC of the extract at 40°C and 4 000 psi with the entrainer. The identified components are in the order: first peak (retention time 2.60min)  reference; second peak (retention time 22.69min)  trans-lycopene; third peak (retention time 25.22min 
-carotene. Reprinted with permission from E. Cadoni et al. [36].

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TABLE 2.10 PERCENTAGE YIELDS OF LYCOPENE AND
-CAROTENE OBTAINED FROM CAMONE TOMATOES AND FIELD TOMATOES AT VARIOUS TEMPERATURES AND 4 000 PSI

Temperature (°C)

Tomatoes

Entrainer

Lycopene (mg/100 g)


-carotene (mg/100 g)

40 40 40 40 50 60 70 80 Soxhlet

Camone Camone Field Skin Skin Skin Skin Skin Skin

Chloroform n-hexane Chloroform

14.920.12 2.350.07 3.710.05 3.800.05 12.420.30 18.760.52 48.100.07 64.410.12 77.080.07

1.940.06 0.310.04 0.530.05 15.450.55 24.610.16 32.420.68 33.501.52 34.880.42 37.760.37

Reprinted with permission from E. Cadoni et al. [36].

Another supercritical CO2 extraction method employed vegetable oil as co-solvent. Sun-dried tomatoes (50 per cent moisture content) were further dried at 70oC and 20mbar for 70–80h. The final product, containing about 6 per cent moisture, was ground to a particle size of 1 mm. Pressure and temperature of extraction varied between 335–450 bar and 45–70°C, respectively. Hazelnut, almond, peanut and sunflower seed oils were included in the experiments, their concentration being between 1 and 20 per cent. RP-HPLC analysis of the extracts was performed in an ODS column (250  4.6 mm i.d.; particle size 5m). Lycopene and
-carotene were separated by isocratic elution, the mobile phase being methanol–THF–water (67:27:6, v/v). The flow rate was set to 1.5 ml/min and analytes were detected at 475 nm. The good separation of lycopene and
-carotene is illustrated in Fig. 2.13. It has been concluded from the data that the use of hazelnut oil resulted in higher extraction efficacy. The extraction yield increased with increasing extraction time and decreasing particle size of the raw material. Higher pressure, temperature and flow rate also enhanced the amount of lycopene in the extract. It was found that the optimal extraction conditions are the following: pressure 450 bar, temperature 65 – 70°C, CO2 flow rate 18 – 20kg CO2/h, particle size of the raw material 1 mm, and about 10 per cent of co-solvent. It was stated that the method can extract about 60 per cent of the total lycopene present in dried tomatoes [37]. Analytical and preparative high-speed counter-current chromatography (HSCCC) has also found application for the separation of lycopene from crude extract of tomato paste. An aliquot of 2g of crude extract of tomato paste was percolated five times with 50 ml of chloroform–methanol (2:1) at ambient temperature. The combined organic phase was evaporated to dryness under a nitrogen stream. The nonaqueous two-phase system

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Liquid chromatography of natural pigments

22

Lycopene

20 18 16 14 β-carotene

12 10 8 Lycope-

6 4

Time (min)

Fig. 2.13. HPLC analysis of the extract in supercritical CO2 at 66°C and 450 bar with vegetable oil as co-solvent. Reprinted with permission from G. Vasopollo et al. [37].

consisted of n-hexane, dichloromethane and ACN (10:3.5:6.5, v/v). The sample was dissolved in the lower phase. Extraction efficacy was controlled by RP-HPLC. Separation was carried out in an ODS column (250  4.6 mm i.d.) at 30°C. The isocratic mobile phase was ACN–methanol–chloroform (47:47:6, v/v). The flow rate was 1.0 ml/min and solutes were detected at 472 nm. Analytical HSCCC separated well the carotenoid pigments of tomato paste as demonstrated in Fig. 2.14. It was found that the method is suitable for the extraction of 8.6mg of lycopene (RPHPLC purity 98.5 per cent) from 100mg of crude extract of tomato paste containing about 9 per cent of lycopene. The technique has been proposed for the preparative separation of lycopene from tomato paste [38]. The effect of temperature on the RP-HPLC behaviour of
-carotene isomers has been extensively investigated and the results were employed for the separation of carotenoids of tomato juice extract. Carotenoids were extracted from food samples of 2g by adding magnesium carbonate to the sample and then extracted with methanol–THF (1:1, v/v) in a homogenizer for 5min. The extraction step was repeated twice. The collected supernatants were evaporated to dryness (30°C) and redissolved in methanol–THF (1:1, v/v). Separations were performed on a polymeric ODS column (250  4.6 mm i.d.; particle size 5m). The isocratic mobile phase consisted of methanol–ACN–isopropanol (54:44: 2, m/m). The flow-rate was 0.8 or 2.0 ml/min. The effect of temperature on the retention times of lycopene and four
-carotene isomers is shown in Table 2.11. The data indicated that the temperature exerts a considerable influence on the retention time and separation of
-carotene isomers. Low temperature enhances the efficacy of separation. The chromatoraphic profile of a tomato juice extract at a column temperature of 7°C, flow rate 2.0 ml/min and further chromatographic conditions (see caption) is shown in

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CH3

1.0 Absorbance (254 nm)

CH3 CH3

1

CH3

CH3

CH3

H3C CH3

CH3

CH3

0.8 5

0.6 0.4

6

2 3

0.2

4

0.0 0

1

2

3

4

5

Time (h)

Fig. 2.14. Chromatogram of the crude lycopene by analytical HSCCC. Solvent system: n-hexanedichloromethane–acetonitrile (10:3.5:6.5/v:v:v) ; stationary phase: upper phase; mobile phase: lower phase; flowrate: 1.0ml/min; revolution speed: 1 800 rpm: sample: 15 mg dissolved in 1ml lower phase; retention of the stationary phase: about 50 per cent; peak 5: lycopene. Reprinted with permission from Y. Wei et al. [38].

Fig. 2.15. The figure illustrates again that better separations can be achieved at lower column temperatures [39]. The carotenoid content of thermally processed tomato-based food products has also been investigated by RP-HPLC. Extraction of tomato-based food products was performed at 0°C under golf fluorescence light. The internal standard
-apo-8
-carotenal, and the filter aids magnesium carbonate and Celite at 10 per cent of the weight of the sample were added to the samples. The amounts of the samples depended on the expected carotenoid content and was 300 g for soups, 200 g for tomato and vegetable juice, 100 g for whole tomatoes, ketchup, spaghetti sauce and tomato puree, and 50 g for tomato paste and tomato sauce. The mixture was blended for 20min with THF then filtered thorugh a Whatman No. 1 filter paper. The solid rest was extracted two or three times until it was almost colourless. The combined extracts were concentrated to about two-thirds at 35°C and were partitioned into methylene chloride (250 ml) and salt water (150 ml). The organic phase was dried over anhydrous sodium sulphate, and concentrated to 10 ml. Pigment solutions were diluted to 50 ml with methylene chloride and diluted for RP-HPLC analysis. Separation of pigments was carried out in an ODS column (250  4.6 mm i.d.; particle size 5 m). The mobile phase consisted of ACN (40 per cent), methanol (20 per cent), methylene chloride 20 per cent and n-hexane (20 per cent). Detection wavelength was 450 nm, except for lycopene (470 nm),
-cryptoxanthin (445 nm), phytofluene (350 nm), phytoene (290 nm) and
-carotene (400 nm). A characteristic chromatogram of carotenoids in the extract of vegetable soup is shown in Fig. 2.16. The good separation of carotenoids indicates that this RP-HPLC method can be successfully employed for the separation and quantitative determination of carotenoid pigments in tomato-based food products. Some data are compiled in Table 2.12.

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TABLE 2.11 RETENTION TIMES OF LYCOPENE AND FOUR
-CAROTENE ISOMERS, USING DIFFERENT COLUMN TEMPERATURES BETWEEN 0°C AND 30°C, FLOWRATE 2.0 ML/MIN (FOR FURTHER CHROMATOGRAPHIC CONDITIONS SEE TEXT)

Temperature

Retention time (min)

(°C)

30 25 20 15 10 5 0

Lycopene

(E)-
-carotene

9(Z)-
-carotene

13(Z)-
-carotene 15(Z)-
-carotene

12.77 16.21 26.64 41.47 80.14 159.04 293.73

16.00 19.19 26.64 36.03 55.45 86.99 133.25

17.43 21.28 30.71 41.47 65.72 105.44 164.16

18.43 22.13 30.71 41.47 60.74 92.13 133.25

18.43 22.13 30.71 41.47 60.74 86.99 124.27

Reprinted with permission from V. Böhm [39]. 7 5.000

1.41

mV

4

9 0.000 −0.556 0.00

5.00

10.00

11 10

15.00 Time (min)

7a

20.00

7b

25.00

32.00

Fig. 2.15. Chromatographic profile of a tomato juice extract at a column temperature of 7°C. Peak identification: 4 
-apo-8
-carotenal; 9  (E)-
-carotene; 11  13(Z)-
-carotene; 10  9(Z)-
carotene; 7  lycopene; 7a  9(Z)-lycopene; 7b  15(Z)-lycopene. Reprinted with permission from V. Böhm [39].

The importance of these types of investigations was emphasized because the consumption of foods with high carotenoid content may reduced the risk of lung and other epithelial cancers [40]. The carotenoid content of orange juices has also been investigated by RP-HPLC. Thus, a non-endcapped C30 column was employed for the analysis of saponified carotenoids in

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10

20

31220

30

40

50 0.5

0.5

Wulare 0

35

5

5

30

A 16 0.4 8 3 0.5

3

10 12

15

AU

2

2 0.01

9

0.3

AU

15 0.3

16

0.5

0.4

4

4

14

1

1

16 0.2 0.01

30

(mla)

35

9

3

10

0.1

1

18

0.1

0

0

0.2

11

16' 11 12 14

1' 4

17

5 6 7

18' 0

0

2

17'

0

10

20

30

40

50

(min)

Fig. 2.16. HPLC profile of carotenoids in an extract of vegetable soup. An expansion of the profile from 30 to 39 is shown in the inset (A). Monitored wavelengths were 436, 440, 464, and 409 nm for peaks 9, 10, 11, 12, and 14, respectively, in the inset (A). Peak identification: 1  1  all-trans-lutein and cis-lutein; 2  5,6-dihydroxy-5,6-dihydrolycopene (lycopene-5,6-diol); 3 
-apo-8
-carotenal (internal standard); 4  lycopene 1,2-epoxide; 5  lycopene 5,6-epoxide; 6  1,2-dimethoxyprolycopene (tentative identification); 7  5,6-dimethoxy-5,6-dihydrolycopene; 8  lycopene; 9  pheophytin b; 10  neurosporene; 11 
-carotene; 12  pheophytin a; 13 
-carotene; 14  pheophytin a isomer and
-carotene; 15  -carotene; 16 and 16  all-trans-
-carotene, cis
-carotene; 17 and 17  all-trans- or cis-phytofluene; 18 and 18  all-trans- or cis-phytoene. Reprinted with permisson from L. H. Tonucci et al. [40].

orange (Citrus sinensis) juice. Solid components of orange juices were precipitated by mixing 2 ml of ZnSO4.H2O solution with 25 ml of juice. After mixing, 2 ml of K4[Fe(CN)6].H2O solution was added and the suspension was mixed again. After 10min of precipitation time the suspension was centrifuged. Carotenoids in the solid rest were dissolved

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in acetone and the organic phase was shaken with petroleum ether and water. The petroleum ether fraction was evaporated to dryness, and the solid rest was redissolved in 6 ml of diethyl ether and 6 ml of methanolic KOH. Samples were stored overnight in the dark at ambient temperature, then extracted with 20 ml of diethyl ether. The ether fraction was washed alkaline free with distilled water, dried with sodium sulphate, and evaporated to TABLE 2.12 CAROTENOIDS (MILLIGRAMS PER 100 G) IN TOMATO-BASED SOUPS, TOMATO JUICE, VEGETABLE JUICE AND QUALITY CONTROL SAMPLE

Sample Tomato soup MeanSDa CV,c % Nd Vegetable beef soup MeanSD CV,c % N Minestrone soup MeanSD CV,c % N Vegetarian vegetable soup MeanSD CV,c % N Tomato juice MeanSD CV,c % N Vegetable juice MeanSD CV,c % N Quality control (vegetable juice) MeanSD CV,c % N Tomato soup MeanSDa 0.090.02 CV,c % 21 Nd 6

-carotene


-carotene

-carotene

-carotene

ndb

0.230.047 1.950.41

0.170.016

6

6

4

6

0.460.05 11 4

1.510.29 19 4

Ce

C

4

4

0.210.12 56 5

0.920.39 43 5

C

C

5

5

0.410.18 44 5

1.500.37 25 5

C

C

5

5

nd 6

0.270.04 13 6

1.740.20 11 6

0.180.03 19 6

0.210.03 14 3

0.830.14 17 3

C

C

3

3

0.250.01 5 9

0.910.11 12 9

C

C

9

9

10.922.92 27 6

1.530.20 13 4

1.720.172 10 6

0.720.176 0.110.03 25 29 6 6 (Continued on next page)

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TABLE 2.12 (continued)

Sample

Vegetable beef soup MeanSD CV,c % N Minestrone soup MeanSD CV,c % N Vegetarian vegetable soup MeanSD CV,c % N Tomato juice MeanSD CV,c % N Vegetable juice MeanSD CV,c % N Quality control (vegetable juice) MeanSD CV,c % N

Lutein

Lycopene

0.110.03 0.310.034 28 11 4 4

Neuro sporene

Phytoene

C

0.350.06 0.190.02 16 10 4 4

trf

0.280.11 0.170.09 38 52 4 4

nd

4

0.150.08 1.480.83 53 56 5 5

C

0.160.04 1.930.51 26 26 5 5

C

5

Phytofluene Lycopene5,6-diol

4

5

tr

5

0.600.07 0.310.04 12 13 4 5

0.060.02 10.771.07 24 10 6 6

1.230.27 22 6

1.900.19 0.830.14 10 17 6 6

0.110.03 29 6

0.080.02 9.660.12 21 1 3 3

C

1.710.16 0.690.07 9 10 3 3

0.050.01 10 3

0.090.01 8.560.53 9 6 9 9

C

3

9

5

1.690.07 0.730.037 0.080.01 4 5 12 9 9 9

a

SD, standard deviation. nd, not detected. c CV, coefficient of variation. d N, number of values used to calculate the mean and SD. e C, co-eluted with pheophytins. f tr, trace, below 0.005 mg/100 g. Reprinted with permission from L. H. Tonucci et al. [40]. b

dryness in vacuum. Pigments were dissolved in 0.5 ml of acetone or methyl-tert-butyl ether (MTBE) and diluted in 1.0 ml of methanol. Measurements were performed in a C30 column (250  4.6 mm i.d.; particle size 5m). The initial mobile phase composition was methanol–water–MTBE (90:5:5, v/v), it was changed to methanol–MTBE 95:5, v/v) at 12 min, then to methanol–MTBE (86:14, v/v) at 8 min, and to methanol–MTBE (74:25, v/v)

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at 30 min. Final composition consisted of methanol–MTBE (50:50, v/v) reached at 50min. The flow rate was 1 ml/min and carotenoids were detected at 290, 350, 430 and 486 nm. Pigments were well separated by the method and the chromatographic profile depended considerably on the detection wavelength as demonstrated in Fig. 2.17. The spectral characteristics of orange juice carotenoids are compiled in Table 2.13. It has been stated that the RP-HPLC method developed for the analysis of carotenoids in orange juice separates 39 pigment fractions in a reasonable running time of 40 min [41]. Another study employed an ODS column and different mobile phase composition for the measurement of carotenoids in orange juice. Citrus fruits were hand-squeezed and the juice was filtered. Aliquots of 5 ml of juice were extracted with ethyl acetate (3  50 ml) containing 0.004 per cent butyl hydroxytoluene (BHT). The organic phase was dried with 50 g of anhydrous sodium sulphate and the aqueous phase was mixed with 50 ml of mehanol and 100 ml of 1 M NaCl, extracted with 75 and 25 ml of ethyl acetate. The ethyl acetate fractions were combined, evaporated to dryness at 40°C and redissolved in the mobile phase. Extracts were analysed in an ODS column (250  4.6 mm i.d.; particle size 5 m). The mobile phase consisted of ACN–methanol–1,2-dichloroethane (60:35:5, v/v) containing 0.1 per cent BHT, 0.1 per cent triethylamine and 0.05 M of ammonium acetate. The column was not thermostated and the flow rate was 1 ml/min. Pigments were detected

24

25

23 80 18

22 20

Absorbance (mAU)

60 40 20

28 27

11 9 12 19

7 6 8 15

100 80

33 26

29 31

36 35 37

430 nm

60 40 20

486 nm

1 34 5

34

350 nm

0 10

20 30 Time (min)

40

Fig. 2.17. Saponified carotenoids in orange juice. Chromatographic conditions are given in text. Chromatograms from absorbance monitoring at 430, 486 and 350 nm, respectively, are shown, all at identical attenuation. Peak identification: 1, 3, 5, 8, 26 and 29  unidentified peaks; 4  valenciaxanthin; 6  neochrome; 7  trollichrome; 9  antherxanthin; 11  cis-anthexanthin; 12  neoxanthin; 19  auoxanthin B; 20  cis-violaxanthin; 22  leutoxanthin; 23  mutatoxanthin A; 24  mutatoxanthin B; 25  lutein; 27  zeaxanthin; 28  isolutein; 31  -cryptoxanthin; 33 
-cryptoxanthin; 34  phytofluene; 35  -carotene; 36  æ-carotene; 37 
-carotene. Reprinted with permission from R. Rouseff et al. [41].

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TABLE 2.13 SPECTRAL CHARACTERISTICS OF ORANGE JUICE CAROTENOIDS

Peak no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

Carotenoid

Valenciaxanthin Neochrome

Antherxanthin cis-anthexanthin Neoxanthin

Auroxanthin A Auroxanthin B cis-violaxanthin Leutoxanthin Mutatoxanthin A Mutatoxanthin B Lutein Zeaxanthin Isolutein

-cryptoxanthin
-cryptoxanthin Phytofluene -carotene -carotene
-carotene

RTa(min)

4.87 5.07 5.3 5.53 5.99 7.68 8.83 9.98 10.25 10.49 10.79 11.25 11.68 11.95 12.56 13.25 13.56 14.25 15.1 16.06 16.41 17.17 18.21 19.1 19.39 20.77 21.93 22.31 25.15 25.88 26.87 28.84 30.01 31.36 35.66 37.93 38.77 39.54 40.12

a RT  retention time. s  spectral shoulder. Reprinted with permission from R. Rouseff et al. [41].

Observed (nm) Peak I

Peak II

S355.5 332.5 349.5 351.5 374.5 399.5 397.5 S400.5 S421.5

371.5 347.5 369.5 369.5 391.5 421.5 421.5 419.5 443.5 431.5 441.5 434.5 397.5 419.5 420.5 421.5 421.5 401.5 401.5 435.5 417.5 419.5 427.5 427.5 445.5 424.5 449.5 439.5 443.5 445.5 445.5 347.5 450.5 347.5 445.5 399.5 450.5 400.5 449.5

418.5 414.5 396.5 397.5 400.5 379.5 379.5 411.5 395.5 399.5 S406.5 S406.5 S424.5 405.5 S427.5 S418.5

S421.5 332.5 S429.5 333.5 S424.5 379.5 379.5

Peak III

367.5 393.5 389.5 413.5 447.5 447.5 443.5 471.5 459.5 471.5 481.5 445.5 447.5 442.5 425.5 424.5 463.5 441.5 441.5 451.5 451.5 471.5 450.5 476.5 467.5

473.5 365.5 477.5 367.5 473.5 425.5 478.5 425.5

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III

0.035 I 0.030 0.025 II Au

0.020 0.015 IV 0.010 V

VI

0.005 0.000 0.00

2.00

4.00

6.00

8.00

10.00 12.00 14.00 16.00 18.00 20.00

Fig. 2.18. HPLC chromatogram of carotenoids in an authentic sample of orange juice: I Sudan I, II lutein, III zeaxanthin, IV
-cryptoxanthin, V -carotene, VI
-carotene. For chromatographic conditions see text. Reprinted with permission from A. M. Pupin et al. [42].

at 450 nm. Identification and quantification of carotenoids was achieved by using authentic standards; the synthetic dye Sudan I served as internal standard. The good separation capacity of the method is illustrated in Fig. 2.18. Recoveries varied between (61.313.3) per cent for
-carotene and (78.111.2) per cent for lutein (see Table 2.14). Because of the acceptable recovery and good separation capacity, the method was proposed for the determination of adulteration of retail samples of orange juice [42]. The carotenoid composition of leafy vegetables has also been measured by various RPHPLC techniques. Thus, the
-carotene content of some green leafy vegetables of Western India has been determined. Total carotenes were determined after extracting the samples with acetone followed by n-hexane. After overnight saponification with 5 per cent methanolic KOH the samples were extracted with petroleum ether (40 – 60°C) and the absorption of the organic phase was measured at 450 nm. The amount of
-carotene was measured by RP-HPLC using an ODS column (250  4.6 mm i.d.; particle size 5m). The isocratic mobile phase consisted of ACN–methanol–dichloromethane (90:8:2, v/v). The flow-rate was 1 ml/min and pigments were detected at 450 nm. The total and
-carotene contents of green leafy vegetables are compiled in Table 2.15. It has been stated that the results provide meaningful information for dietary planning, taking into consideration vitamin A intake [43]. Another liquid chromatographic method has been developed for obtaining standards and RP-HPLC quantification of leafy vegetable carotenoids. Carotenoids were extracted

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TABLE 2.14 LEVELS OF CAROTENOIDS (MG/LITRE) IN AUTHENTIC (HAND-SQUEEZED ORANGE JUICE, FROZEN CONCENTRATED ORANGE JUICE (FCOJ) AND IN FROZEN CONCENTRATED ORANGE PULP WASH (FCOPW), BOTH DILUTED TO 12° BRIX. (N  5 FOR PERA RIO, N  4 FOR NATAL, N  3 FOR VALENCIA AND HAMLIN VARIETIES, AND N  2 FOR FCOPW)

Variety

Lutein

Zeaxanthin


-cryptoxanthin

-carotene


-carotene

Total

Pera Rio Mean Natal Mean Valencia Mean Hamlin Mean Baia Lima Pera Coroa

0.14–0.23 0.18 0.04–0.06 0.05 0.06–0.07 0.07 0.03–0.08 0.05 0.05 0.08 0.08

0.30–0.53 0.39 0.06–0.12 0.09 0.04–0.10 0.08 0.06–0.27 0.16 0.04 0.10 0.15

0.10–0.19 0.13 0.01–0.03 0.02 0.02 0.02 0.02–0.06 0.04 0.08 0.01 0.05

0.04–0.18 0.09 0.03–0.04 0.03 0.03–0.07 0.05 nd–0.02 0.01 nd 0.02 0.05

0.05–0.08 0.07 0.02–0.03 0.03 0.02–0.05 0.04 nd–0.02 0.01 0.02 nd 0.04

0.63–1.21 0.86 0.18–0.28 0.22 0.17–0.31 0.25 0.11–0.45 0.27 0.19 0.21 0.37

FCOJ 1a 2b 3c Mean SD FCOPW

0.06 0.10 0.09 0.08 0.02 0.01–0.03

0.09 0.22 0.14 0.15 0.07 0.02–0.04

0.05 0.08 0.08 0.07 0.02 0.01

0.02 0.03 0.02 0.02 0.01 nd

0.04 0.05 0.04 0.04 0.01 nd

0.26 0.48 0.37 0.37 0.11 0.04–0.08

nd  not detected (0.02 mg/); n  number of analysed samples. Results not corrected for recovery. a Variety Hamlin. b Variety Pera Rio. c Mixture of several varieties. Reprinted with permission from A. M. Pupin et al. [42].

from 50 – 60g of curly lettuce by cold acetone, then partitioned into petroleum ether, concentrated in vacuum and separated in an open column (MgO:Hyflosupercel, 1:1, activated for 2h at 110oC). Fractions were eluted with ethyl ether, acetone and petroluem ether. The purity of carotenoid pigments was verified by RP-HPLC. Separations were carried out in an ODS column (150  4.6 mm i.d.; particle size 3m). The components of the mobile phase were acetonitrile, methanol and ethyl acetate containing 0.05 per cent triethylamine. A concave gradient curve was employed from 95:5:0 to 60:20:20 in 20min. The detection wavelength for carotenoids was 438 (neoxanthin), 441 (violaxanthin), 439 (lactucaxanthin), 447 (lutein), and 454 (
-carotene). The carotenoid concentrations found in leafy vegetables and calculated by one-poiny calibration are compiled in Table 2.16.

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TABLE 2.15 TOTAL AND
-CAROTENE CONTENTS OF GREEN LEAFY VEGETABLES ANALYSED IN THE PRESENT STUDY (FW  FRESH WEIGHT)


-carotene (mg/100 g FW)

Name

Botanical name

Total carotenes (mg/100 g FW)

Amaranth Bengalgram leaves Bathua Colocasia leaves — Fenugreek leaves Amaranthus sp — — — — Onion stalks Mayalu Radish leaves Shepu Spinach

Amaranthus gangeticus Cicer arietinum

22 610 15 870

2 430 9 700

Chenopodium album Colocasia antiquorum

6 939 28 942

6 492 2 359

Pergularia daemia Trigonella foenum graecum

4 766 24 352

3 616 10 226

Am,aranthus sp Digera arvensis Trianthama monogyna Lucas aspera Potulaca quadrifida Allium cepa Basella rubra Raphanus sativus Peucedaruum graveolens Spinacia oleracea

10 970 17 150 7 490 6 248 3 088 5 470 5 433 11 930 21 565 12 896

8 875 14 390 3 990 3 066 1 195 2 970 4 345 6 540 5 500 2 851

Reprinted with permission from V. S. Nambiar et al. [43].

It was stated that the method is relatively inexpensive, suitable for the preparation of carotenoid standards and provides a high sample throughput [44]. The addition of plant extracts to food products has also been investigated by RP-HPLC. Methods have been developed for the analysis of carotenoids, xanthophylls, lutein extracted from tagete, bixin and norbixin extracted from annatto, safflower, curcuma, santal and yellow gardenia extracts, as well as from carminic acid and carmine. Separations were carried out in an ODS column (150  4.6 mm i.d.; particle size 5 m). The composition of the mobile phase and the detection wavelength depended on the pigment to be separated. Thus, lutein, canthaxanthin,
-cryptoxanthin, lycopene and
-carotene were eluted using gradient elution. Eluents A and B were methanol–ACN (6:11, v/v) and THF, respectively. Separation began with 95 per cent A for 0 – 5 min, then to 20 per cent A in 20 min. A chromatogram showing the baseline separation of carotenoids is shown in Fig. 2.19. Norbixin and bixine were separated by a different gradient: eluent A water–ACN (3:2, v/v), eluent B water–ACN (1:1, v/v); 0 – 15 min 100 per cent A to 0 per cent A, 15 – 25 min, isocratic conditions, the flow rate was 1 ml/min and pigments were detected at 450 nm. Curcumins were

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TABLE 2.16 COMPARISON OF CAROTENOID COMPOSITION (G/G) OF LEAFY VEGETABLES OBTAINED BY ONE-POINT CALIBRATION, STRAIGHT LINE EQUATION AND RESPONSE FACTORS

Carotenoid

Sample number

One-point calibration

Straight line equation

Neoxanthin

1 2 3

7.0 10.4 11.5 24.3 17.7 17.7 21.8 12.3 11.7 10.8 12.6 7.9 21.0 19.5 23.0 8.2 22.9 21.7 24.5 6.2

6.7 10.3 11.1 23.9 16.8 17.2 21.4 14.0 12.0 11.5 13.7 9.4 19.5 18.7 22.7 10.4 22.6 21.6 25.2 8.0

5.6 6.7 9.0 24.7 15.2 14.8 16.7 6.2 8.7 7.6 9.5 11.0 15.6 15.4 17.9

5.4 6.6 8.7 24.0 14.2 14.4 16.4 8.1 9.0 8.1 10.3 12.0 15.0 14.8 17.7

CV between samples Violaxanthin

CV between samples Lactucaxanthin

CV between samples Lutein

CV between samples
-carotene

1 2 3 1 2 3 1 2 3 1 2 3

CV between samples Curly lettuce Neoxanthin

CV between samples Violaxanthin

CV between samples Lactucaxanthin

CV between samples Lutein

1 2 3 1 2 3 1 2 3 1 2 3

(Continued on next page)

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TABLE 2.16 (continued)

Carotenoid

CV between samples
-carotene

Sample number

One-point calibration

Straight line equation

1 2 3

8.4 16.9 18.2 19.4 7.0

10.2 17.0 18.1 20.0 8.1

9.5 8.1 13.8 28.5 20.9 12.0 28.3 40.1 49.7 33.0 67.4 34.3 32.7 19.2 47.3 42.5

9.2 7.7 13.3 28.5 19.4 12.2 27.9 39.4 47.7 31.9 66.6 35.6 32.9 19.9 48.6 42.5

14.4 13.1 20.1 23.6 20.9 23.6 27.7 14.3 61.4 77.8 80.7 14.2 28.4 40.5 39.4 18.5

13.9 12.9 19.3 22.4 19.4 22.9 27.3 16.9 59.0 74.6 79.8 15.2 28.6 40.4 40.5 18.7

CV between samples Roquette Neoxanthin

CV between samples Violaxanthin

CV between samples Lutein

CV between samples
-carotene

1 2 3 1 2 3 1 2 3 1 2 3

CV between samples Cress Neoxanthin

CV between samples Violaxanthin

CV between samples Lutein

CV between samples
-carotene

CV between samples

1 2 3 1 2 3 1 2 3 1 2 3

(Continued on next page)

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TABLE 2.16 (continued)

Carotenoid

Sample number

One-point calibration

Straight line equation

Neoxanthin

1 2 3

9.1 18.2 18.4 35.3 15.4 23.4 24.9 23.9 41.1 69.0 61.6 24.9 24.9 43.1 40.9 27.3

8.9 18.0 17.7 35.0 14.4 23.8 24.5 27.1 39.8 66.7 60.9 25.3 25.1 44.8 42.0 28.6

CV between samples Violaxanthin

CV between samples Lutein

CV between samples
-carotene

1 2 3 1 2 3 1 2 3

CV between samples CV  coefficient of variation (%). Reprinted with permission from M. Kimura et al. [44].

separated by the same method as norbixine and bixine. Carminic acid and carmin were determined in the same RP-HPLC column. The mobile phase components were aqueous solutions of sodium hydrogen carbonate (eluent A 8g/l); eluent B 5g/l in water–ACN 1:1. The gradient was initiated with 100 per cent A and it was decreased to 40 per cent A in 30min. The flow rate was again 1 ml/min. Dyes were detected at 520 nm. The methods outlined above were proposed for the identification of natural colourants added to food products [45]. RP-HPLC-UV has also been employed for the separation and quantification of the biologically active ingredients of saffron (Crocus sativus L) such as crocin 1–4 and crocetin. The chemical structures of crocetin, a carotenoid derivative, and those of its glycosides are shown in Fig. 2.20. Crocetins were extracted from ground saffron (20mg) and from Zhizi (dried fruit of Gardenia jasminoides Ellis, 100mg) by adding 5 ml of 80 per cent methanol and by mixing the suspension in the dark at room temperature for 2h. After extraction the samples were filtered and the filtrates were used for HPLC analysis. Analytes were separated in an ODS column (150  3.9 mm i.d.; particle size 4m) at room temperature. Solvents A and B were methanol and 1 per cent aqueous acetic acid, respectively. The gradient was: 0–1min 40 per cent A; to 55 per cent A in 1–6min; to 75 per cent A in 6–23min; to 90 per cent A in 23–25; final hold, 5min. The flow rate was 1 ml/min and UV-vis spectra were detected between 200–600 nm. Typical chromatograms

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1 3

Absorbance

5

2

4

10

20 Temps (min)

Fig. 2.19. Chromatogram of carotenoid solution in THF. 1: lutein, 2: canthaxanthin, 3:
-cryptoxanthin, 4: lycopene, 5:
-carotene. ODS column, 5m, 150mm  4.6mm. Mobile phase: eluent A (methanol-acetonitrile 6:11), eluent B (THF); gradient profile: 0–5min, isocratic conditions 95 per cent A; 5–20min, gradient to 20 per cent A. Flow rate 1ml/min. Detection 450 nm. Reprinted with permission from C. Tricard et al. [45]. CH3

O

CH3 OR"

'RO CH3

HOH2C HO HO

O

CH3

O

HOH2C HO HO

O OH

CH2

OH

O

HO HO

O

OH X

Y Crocin 1: Crocin 2: Crocin 3: Crocin 4: Crocetin:

R' = R" = X R' = X, R" = Y R' = X, R" = H R' = Y, R" = H R' = R" = H

Fig. 2.20. Structures of crocetin and crocins 1–4. Reprinted with permission from N. Li et al. [46].

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illustrating the differences between the crocin content of samples are shown in Fig. 2.21. The contents of crocins and crocetin in various samples are compiled in Table 2.17. Calibration curves were linear in the range of 5–300g/ml and the RSD values of intraday and inter-day variabilities were between 0.3–10.5 per cent. It has been concluded from the results that the method can be applied for the quality control of crocin-containing medicinal herbs [46]. The compositions of commercial saffron products have been determined by a different RP-HPLC technique. The optimization of the extraction process was carried out by adding 200 ml of solvent to 400mg of saffron and stirring the suspension in light and in the dark at 25, 30, 40 and 50°C. The highest yields were obtained by a methanol–water (50:50, v/v) extracting agent at 25°C in the dark with 1h extraction time. Extracts were centrifuged and filtered before analysis. Separation was performed in an ODS column (150  3.9 mm i.d.; particle size 4 m) at 30oC. Gradient conditions were: 20–70 per cent methanol at 1 per cent methanol/min increase. The flow rate was 1 ml/min and analytes were detected at their maximum absorbance. The good separation performance of the system is illustrated in the chromatograms in Fig. 2.22. The results of quantitative analyses are compiled in Table 2.18. It was stated that the low R.S.D. values (lower than 2 per cent) makes the method suitable for saffron analysis [47]. The effect of various extraction conditions on the yield of saffranal has also been investigated by RP-HPLC. Separations were performed in an ODS column (250  4.9 mm i.d.; particle size 5m) using 75 per cent ACN as isocratic mobile phase. It was found that the heating of stigmas at 80oC for 30min before extraction increased the concentration of safranal compared with freeze-dried stigmas [48]. 2.1.3.2 HPLC determination of carotenoid pigments in human and animal tissues Because of the numerous beneficial effects of carotenoids in living organisms many efforts have been devoted to the development of chromatographic techniques for their measurement in human and animal tissues. Ion exchange chromatography and gelfiltration chromatography have been employed for the ligand-binding characterization of xanthophyll carotenoids to solubilized membrane proteins from human retina. The study was motivated by the fact that the macula of retina contains a considerable amount of lutein and zeaxanthin which can protect against age-related macular degeneration cauisng blindness. It has been established that macula or peripherial retina is rich in proteins binding endogenous xanthophyll carotenoids [49]. Another study emphasized that lutein can also protect against age-related macular degeneration, various tumours and cardiovascular diseases. In order to elucidate the effect of age on the lutein uptake and storage the lutein concentration in adipose tissue and buccal mucosa cells (BMC) in 12 young (26.90.8 yr) and 17 older subjects (67.31.1 yr) was measured by HPLC before and after supplementation. Lutein concentration increased in BMC after supplementation but it did not change in adipose tissue. No significant difference was found between the age groups. As it has been suggested that epoxy-
-carotenes may reduce the incidence of lung cancer the intestinal absorption of 5,6-epoxy-
-carotene and 5,8-epoxy-
-carotene in humans after oral administration has been investigated by RP-HPLC. Capillary blood samples

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250

mAU

200 2 150

6

3

1

100 4

50

5

0 (a)

0

5

10

15

20

25

1

250 200 mAU

6 150 2

100 50

7 0 (b)

0

5

3 8 4

10

15

5 20

25 6

250

mAU

200 1

150 100 50

2 73 8

0 0 (c)

5

10

15 Time (min)

20

25

Fig. 2.21. Representative HPLC chromatograms of the extracts of (a) Spanish saffron spiked with standards, (b) Iranian saffron and (c) Zhizi purchased from Nanjing. Peaks: 1  crocin 1; 2  crocin 2; 3  crocin 3; 4  crocin 4; 5  crocetin; 6  13-cis-retinoic acid (internal standard); 7 and 8  cis-crocins. Reprinted with permission from N. Li et al. [46].

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TABLE 2.17 QUANTITIES OF CROCIN 1–4 AND CROCETIN IN HERBS OBTAINED FROM DIFFERENT SOURCES

Saffron (%)a

Compound

Crocin 1 Crocin 2 Crocin 3 Crocin 4 Crocetin

Zhizi (%)a

Spain

Iran

Tibet

Hong Kong

Nanjing

9.000.71 4.600.35 1.300.12 0.360.06 0.060.01

2.900.21 1.900.12 0.870.05 0.230.01nd 0.050.01

2.800.06 1.700.06 0.710.05 nd nd

0.200.01 0.060.003 0.050.003 nd nd

0.370.02 0.150.004 0.090.01 nd nd

a

Data are presented as percentage of the dried herbs, and expressed as meanSD of triplicate analyses for each herb. nd  not detected. Reprinted with permission from N. Li et al. [46].

were collected before and 3h after of oral administration, and centrifuged immediately. Plasma was analysed instantly or stored at 20°C. Venous blood samples were taken at 0, 2, 4, 6, 8 and 24h after administration and were treated similarly. Ethanol and ethyl acetate were added to the plasma in the volume ratio of 1:2:1. The internal standard retinyl acetate, BHT was also added and the suspension was vortexed then centrifuged. The supernatant was separated and the solid rest was vortexed again with 2 per cent vol. of hexane and 2 per cent vol. of ethyl acetate. The collected supernatants were mixed with 1 per cent vol. of water, vortexet and centrifuged again. The organic fraction was evaporated to dryness and redissolved in propan-2-oldichloromethane (2:1, v/v). RP-HPLC separation was performed in an ODS column (100  3.6 mm i.d.; particle size 3m) with a 20min linear gradient from methanol–water (3:1, v/v) with 10 mM ammonium acetate to methanol–dichloromethane (4:1, v/v), final hold 20min. The flow rate was 0.8 ml/min. Analytes were detected at various wavelengths (445 nm for 5,6-epoxy-
-carotene,
-carotene and other plasma carotenoids; 400 and 425 nm for other carotenoids; 315, 325 and 340 for retinoids). The chromatographic profiles of carotenoids in human plasma are displayed in Fig. 2.23. The chromatograms clearly show that that the method is suitable for the separation of carotenoids in human plasma and 5,6-epoxy-
-carotene is absorbed by humans after oral administration [50]. A C30 RP column has been applied for the separation of carotenoids present in various matrices such as human serum, raw and thermally processed carrots, a Duniella algae-derived preparation and a poultry feed. Samples were mixed with deionized water and CaCO3, homogenized and extracted with acetone–hexane (1:1, v/v).

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0.3 1 0.2

5

4

440 nm

310 nm

440 nm

250 nm

Is

0.0 9 2 3

6

7 8

10

0.0

Absorbance (O.D.)

(a)

(b)

0.3 4

Is 0.2

5

1

0.0 6 7 8

23

9 10

0.0 0.3 4

Is

5

0.2

0.0

9

1 3

6

8

10

0.0 (c) Time (min)

Fig. 2.22. Chromatograms of methanol:water (50 per cent, v/v) saffron extracts from Mancha (a), Rio (b) and Sierra (c) types, simultaneously recorded at 250, 310 and 440 nm, including 4-nitroaniline as internal standard. The mobile and stationary phases were a linear gradient of methanol–water from 20 to 70 per cent in 50min, and an ODS column, respectively. The flowrate was 1ml/min, temperature 30°C and sample size 50l. The following compounds are present: picrocrocin (1), HTCC (2), 3-entiobiosile-kaempferol (3), -crocin (4), crocin 2 (5), crocin 3 (6), safranal (7), crocin 4 (8), crocin 5 (9), crocin 6 (10) and internal standard (I S.). Reprinted with permission from P. Lozano et al. [47].

Extracts were further purified on neutral alumina cartridges conditioned by passing through 5 ml of hexane. Extracts were loaded in hexane and washed by 5 ml of hexane. The - and
-carotenes were removed by 3.5 ml of acetone–hexane (10:90, v/v), other carotenoids were eluted with acetone–hexane 30:70 and 70:30 v/v. Prepurification of pigments was performed in subdued light under a stream of nitrogen. Analyses were carried out in a C30 column (250  4.6 mm i.d.,; particle size 5m) using isocratic mobile phase composed of methyl-tert-butyl ether (MTBE)–methanol (3:97 and 38:62, v/v) at a flow rate of 1 ml/min. The column was not thermostated; separations were achieved at room temperature (about 23oC). Carotenoids were detected at 453 and 460 nm (lutein). The

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TABLE 2.18 SAFFRON METABOLITE CONCENTRATIONS AND RATIOS BETWEEN THEM IN THE THREE SPANISH SAFFRON QUALTITY SAMPLES (MANCHA, RIO AND SIERRA), AND A MANCHA COMMERCIAL SAMPLE, ANALYSED BY HPLC UNDER THE CONDITIONS DESCRIBED IN THE TEXT.

Wavelength (nm)

Compound (AComp/AI.S.)

Mancha (AComp/AI.S.)

Rio (AComp/AI.S.)

Sierra (AComp/AI.S.)

Commercial sample (AComp/AI.S.)

250

Picrocrocin HTCC Kaempferol Safranal -crocin Crocin 2 Crocin 3 Crocin 4 Crocin 5 Crocin 6

1.23 0.05 0.08 0.06 3.85 1.81 0.07 0.40 0.32 0.14

0.75 0.03 0.06 nd 2.30 1.01 0.02 0.25 0.21 0.07

0.22 nd 0.05 nd 2.10 1.15 0.04 0.26 0.32 0.16

0.51 0.04 0.04 0.03 3.43 1.48 0.08 0.32 0.39 0.15

310 440

nd, not detected. HTCC, 2,6,6-trimethyl-4-hydroxy-1-carboxaldehyde-1-cyclohexene. Reprinted with permission from P. Lozano et al. [47].

chromatographic profiles of carotenoids extracted from human serum are shown in Fig. 2.24. The method has been proposed for the determination of geometrical carotenoid isomers in various matrices [51]. As lutein and zeaxanthin play an important role in the prevention of makula degeneration and the biological activity depends considerably on the type of isomer, the corresponding carotenoid stereo-isomers have been measured in retina and spinach. Separation of stereoisomers was carried out in a C30 column (250  4.6 mm i.d.; particle size 3m) using isocratic elution for retina and gradient elution for spinach samples, as illustrated in Fig. 2.25. Fig. 2.25 demonstrates that the stereoisomers can be well separated in the C30 column. The concentration of carotenoid stereoisomers are compiled in Table 2.19. The data in Table 2.19 indicate that the carotenoid content increases with increasing growing time. Boiling exerts a negligible effect on both the amount and composition of carotenoids. Commercial samples contain markedly lower quantities of carotenoids [52]. The effect of vitamin E supplementation on -tocopherol and
-carotene concentrations in tissues from pasture- and grain-fed cattle was also elucidated with HPLC analysis. The investigation was motivated by the fact that -tocopherol influences beneficially meat colour and stability [53], and the presence of
-carotene can modify the amount of -tocopherol in tissues [54]. Samples were extracted with hexane and the concentration of
-carotene was assessed by HPLC. Some data are listed in Table 2.20. It was concluded from the data that

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2

0.08

AU

4

0.04 7 6 1 3 0.00

5

(a)

15

25

35

2

0.08

AU

4

5

0.04

7 6 1 3

0.00

5 (b)

15

25 Time (min)

35

Fig. 2.23. Reversed-phase gradient HPLC profiles of carotenoids in human plasma. A human volunteer was given an oral dose of 5,6-epoxy-
-carotene (9.1 mol). Plasma was analysed for carotenoids before (a) and 6h after (b) the oral dose. Peak identification: 1, bilirubin; 2, lutein; 3, zeaxanthin; 4,
-cryptoxanthin; 5, 5,6-epoxy-
-carotene; 6, lycopene; 7,
-carotene. The detection wavelength was 445 nm. AU, absorbance unit. Reprinted with permission from A. B. Barua [50].

vitamin E supplementation decreases the concentration of
-carotene in tissues, resulting in reduced carcase fat yellowness which influences marketability of meat [55]. The effect of saponification on the concentration of carotenoids in fatty foods has also been investigated by RP-HPLC. Sausages containing 5.6 per cent powdered paprika were extracted exhaustively with chloroform–methanol (2:1, v/v). The extracting solvent contained 0.01 per cent butylated hydroxyanisole (BHA). An aliquot of the combined extracts was evaporated to dryness and saponified at 50°C for 5min with 10 per cent KOH in methanol in the presence of 0.01 per cent BHA. Free carotenoid pigments were extracted with diethyl ether, washed with water, dried over anhydrous Na2SO4 and evaporated under

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Absorbance, 453 nm

3

1

(a) 0

5

8

2 5 6 7

4

10

15

20

25

30

35

Absorbance, 453 nm

6

5 1 2 3

0

10

20

4

30

7

40

50

60

(b)

Absorbance, 453 nm

12

3−6 7−11

0 (c)

13

1−2

10

20

30

40

50

60

70

Retention time, min

Fig. 2.24. C30 chromatograms of carotenoids extracted from human serum: (a) xanthophylls fraction, 7:93 (v/v) MTBE–methanol mobile phase; (b) - and
-carotenes fraction, 11:89 (v/v) MTBE–methanol mobile phase; (c) lycopene fraction, 38:62 (v/v) MTBE–methanol mobile phase. Tentative peak identifications: (a) 1, 13-cis-lu- lutein; 2, 13
-cis-lutein; 3, all-trans-lutein; 4, zeaanthin; 5–7, unidentified
,-carotenoids; and 8,
-cyrptoanthin; (b) 1–2, unidentified æ-carotene isomers; 3, 15-cis-
-carotene; 4, 13-cis-
-carotene; 5, all-trans--carotene; 6, all-trans-
-carotene; and 7, 9-cis-
-carotene; and (c) 1–11 and 13, cis-lycopene isomers; and 12, all-trans-lycopene. Reprinted with permission from C. Emenhiser et al. [51].

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0-21 min.: aceton/waser 86:14 (v/v) 21-25 min.: linearer gradient 25-40 min.: aceton/wasser 97:3 (v/v)

50

Chlorophyll b All-E β -carotin

00 Chlorophyll a

50 Au

All-E lutein

9, 13-ZZ β-carotin

00 50 13' -z lutein 13 -z lutein

00 50

β-apo-8'-carotenal standard All-E zexanthin 9-Z lutein 9'-Z lutein

13, 15-ZZ β-carotin 9-Z β -carotin 13-Z

0 0

5

10

15

(a)

20 min

25

30

35

0-30 min.: aceton/waser 86:1 (v/v) All-E lutein

4

Au

3 13 -z zeaxanthin 13' -z lutein

2

All-E zexanthin 9'-Z lutein

4

9-Z lutein

13 -z lutein

9 -z zeaxanthin

1 0

0

5

10

(b)

20

25

0-30 min.: aceton/waser 86:1 (v/v) All-E lutein

10 8

Au

15 min

All-E zexanthin

6 13 -z zeaxanthin 4

9'-Z lutein

13' -z lutein

9-Z lutein

9 -z zeaxanthin

13 -z lutein

2 0 0 (c)

5

10

15 min

20

25

Fig. 2.25. (a–c) Comparison of HPLC separation of carotenoid isomers with a C30 column. Column temperature 25°C, flow rate 1ml/min, detection 450 nm. Mobile phase was acetone and water mixtures. Reprinted with permission from T. Glaser et al. [52].

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TABLE 2.19 QUANTITATIVE EVALUATION OF CAROTENOID-STEREOISOMERS IN VARIOUS SPINACH SAMPLES (G/G SPINACH)

13-Z-lutein 13
-Z-lutein all-E-lutein 9-Z-lutein 9
-Z-lutein All lutein All E-zeaxanthin 13,15-ZZ
-carotene 13-Z
-carotene 9,13
-ZZ
-carotene AllE
-carotene 9-Z
-carotene All
-carotene All carotenoids

1

2

3

4

5

6

1.7 1.7 76.5 3.6 5.2 88.7 3.9 1.4 3.1 3.6 61.5 9.2 78.8 171.4

2.5 3.1 75.9 3.8 6.3 91.6 3.6 1.6 3.3 3.1 63.5 10.2 81.7 176.9

2.1 2.4 93.3 7.1 10.2 115.1 6.3 1.7 3.3 5.5 70.5 10.2 91.7 213.1

2.8 2.5 98.1 7.4 10.4 121.2 6.5 1.6 3.7 3.9 74.0 7.2 90.4 218.1

1.2 1.3 57.1 2.8 2.0 64.4 1.1 1.2 1.5 4.8 37.5 5.1 50.1 115.6

1.3 1.4 48.5 2.2 2.4 57.1 1.3 1.0 1.2 4.3 36.0 4.7 47.2 105.6

1  spinach after five weeks of growing time (raw); 2  spinach after five weeks of growing time (boiled); 3  spinach after eight weeks of growing time (raw); 4  spinach after eight weeks of growing time (boiled); 5  commercial spinach (raw); 6  commercial spinach (boiled). Reprinted with permission from T. Glaser et al. [52].

nitrogen flow. Saponified and non-saponified samples were dissolved in acetone. Carotenoid analyses were performed in an ODS column (250  3.9 mm i.d.; particle size 5m). Components of gradient elution were acetone-water (100:50, v/v) (A) and acetone–water (100:5, v/v). Elution began with 100 per cent A for 5min, to 50 per cent A in 5min (curve convex), to 100 per cent B in 30min (curve concave) and 10min hold. The flow rate was 1 ml/min and pigments were detected in the wavelength range of 350 – 550 nm. Characteristic chromatographic profiles of saponified and non-saponified sausage extract are shown in Fig. 2.26. The chromatograms demonstrate that the resolution capacity of the method is high and a considerable number of carotenoids can be separated. Furthermore, they indicate that saponification markedly decreases the number of pigment fractions. The quantitative results are compiled in Table 2.21. The data indicated that saponification causes a loss of some pigments such as capsanthoin and
-carotene [56]. The selective absorption and carotenoid concentration in eggs, avian and animal tissues have been extensively studied. These investigations were motivated by the findings that carotenoids can function as ultraviolet filters in eye lenses [57], can accumulate in eggs [58], and can influence the colour that is used as signals of quality to attract males [59,60]. However, the number of investigations concerning analysis of carotenoids in avian taxa is surprisingly low [61,62]. Eggs of common moorhen (Gallinula chloropus), the American

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TABLE 2.20 MEAN CONCENTRATION (WITH POOLED S.E.0 OF -TOCOPHEROL AND
-CAROTENE IN LIVER, FAT AND MUSCLES (g/g TISSUE) FROM PASTURE- AND GRAIN-FED CATTLE WITH OR WITHOUT VITAMIN E SUPPLEMENT (N  8)

Pasture

-tocopherol Liver Fat Muscle
-carotene Liver Fat Muscle

Grain

Control

Supplemented

Control

Supplemented

Pooled SE

LD SM GM

20.9ba 40.2a 4.5a 4.4a 5.8a

27.7a 37.0a 4.6a 4.3a 6.1a

5.9c 5.2c 1.8b 2.0b 2.4b

24.1ab 17.7b 4.3a 5.3a 6.0a

1.30 2.35 0.21 0.21 0.21

LD SM GM

12.1a 0.99a 0.16a 0.09a 0.22a

0.8c 0.10c 0.01c 0.01b 0.03c

1.0c 0.09c 0.03c 0.03b 0.05c

0.43 0.058 0.008 0.008 0.008

8.1b 0.67b 0.10b 0.05b 0.16b

a

Means within the same row with the same letter are not statistically different (P 0.05). LD  m. longissimus dorsi; SM  m. semimembranosus; GM  m. gluteus medius. Reprinted with permission from A. Yang et al. [55].

coot (Fulica americana) and the lesser black-backed gull (Larus fuscus) were collected and used for the determination of carotenoids or were artificially incubated. On the day of hatching the chicks were killed and the tissues analysed. Extraction of pigments was achieved by homogenizing 0.2 – 0.5g of egg yolk and/or tissues with 2 ml of (1:1, v/v) mixture of 5 per cent aqueous NaCl and ethanol. An aliquot of 3 ml of hexane was added to the suspension and homogenized again. The hexane phase was separated by centrifugation, and the procedure was repeated twice. The collected organic phase was evaporated to dryness, and redissolved in 1 ml of methanol–dichloromethane (1:1, v/v). The supernatant was injected into an ODS column (250  4.6 mm i.d.; particle size 5m). Carotenoids were separated with a gradient consisting of acetonitrile–methanol (85:15, v/v) and acetonitrile–dichloromethane–methanol (70:20:10, v/v). The carotenoid concentrations measured in egg yolks and tissues of newly hatched moorhen, coot and gull are compiled in Table 2.22. It was concluded from the data that the RP-HPLC technique can separate the carotenoid pigments present in egg yolk and tissues of newly hatched wild birds. The results further indicated that avian embryos can discriminate between various carotenoids [63]. An HPLC method was also employed for the study of the effect of dietary supplementation of zeaxanthin on the photoreceptor death in light-damaged Japanese quail. It was established that zeaxanthin protects the photoreceptors from light-induced death [64].

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Chapter 2 39

27

0.32 Internal standard 18

41

9

37

Au

0.24

32

23

0.16

30 29 31 26 28 25 24

13

0.08 7

21

34 36 38

22

15 14

3

35 40

12 11 10

43 42 44

20

0.00 20.0

40.0 Minutes

(a)

60.0

9

0.40

Au

0.32

0.24

0.16

12 13

0.08

11 10

3 6 57 12 4 8

16 15 14

Internal standard 18

32 33

21 19

0.00 10.0 (b)

20.0

30.0 Minutes

40.0

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TABLE 2.21 CAROTENOID COMPOSITION OF NON-SAPONIFIED AND SAPONIFIED SOBRASADA SAUSAGE EXTRACT AND RETENTION TIME FOR EACH PIGMENT RESOLVED. FOR PEAK IDENTIFICATION SEE FIG. 2.26

Peak No.

tRa

Carotenoid composition (g/g dry weight)b Non-saponilied extract

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

17.1 17.4 18.1 18.4 18.6 18.9 19.2 19.6 19.9 20.4 20.8 21.2 22.0 22.5 22.7 22.9 23.5 25.8 28.4 30.8 31.5 32.6 33.0 33.9

Saponified extract

nd nd 0.29  0.04 nd nd nd 0.41  0.09 nd 6.8  0.3 0.13  0.02 1.1  0.1 0.74  0.06 1.7  0.1 0.12  0.03 0.12  0.03 nd nd

0.26  0.02 0.25  0.02 7.4  0.4 0.49  0.06 0.65  0.04 0.67  0.03 2.2  0.1 0.36  0.03 60  3 1.5  0.1 7.7  0.5 5.7  0.3 5.0  0.3 0.44  0.04 0.48  0.05 0.18  0.07 1.1  0.1

nd 0.15  0.07 1.3  0.1 0.44  0.12 10  1 0.24  0.10

0.65 0.02 nd 1.8 0.1 nd nd nd (Continued on next page)

Fig. 2.26. Reversed-phase HPLC separation of (a) Sobrasada sausage extract and (b) saponified Sobrasade sausage extact in an ODS column at maximum absorbances at each point in time. Peak identification: 1 – 2, 4 – 6, 8, 12, 14 – 17  unidentified free; 3  capsorubin; 7  violaxanthin; 9  capsanthin; 10  anteraxanthin; 11  cis-capsanthin; 13  lutein and zeaxanthin; 18  cantaxanthin, internal standard; 19  cryptoxanthin; 20, 24, 25, 28  unidentified monoester; 21 
-cryptoxanthin; 22  capsorubin monoester; 23, 26, 27, 29  capsanthin monoester; 30, 31  lutein-zeaxanthin monoester; 32 
-carotene; 33  cis-
-carotene; 34, 37, 39, 41, 43  capsanthin diester; 35  capsorubin diester; 36, 38, 40, 42, 44  unidentified diester. Reprinted with permission from J. Oliver et al. [56].

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TABLE 2.21 (continued)

Peak No.

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

tRa

34.3 34.6 35.5 36.5 37.1 37.7 40.3 42.8 43.4 45.0 47.4 49.5 50.2 51.5 53.3 54.8 56.7 58.4 60.2 64.6

Carotenoid composition (g/g dry weight)b Non-saponilied extract

Saponified extract

0.33  0.10 4.9  0.4 22  1 0.30 0.13 8.1  0.4 4.1 0.3 2.3  0.1 5.1  0.2 nd 2.2  0.1 3.6  0.1 0.72  0.11 15  0.3 0.74  0.33 30  1 2.7  0.2 20  0.3 1.4  0.1 4.6  0.2 0.54  0.06

nd nd nd nd nd nd nd 2.3  0.1 0.31  0.003 nd nd nd nd nd nd nd nd nd nd nd

a

Retention time of each peak. Data are given as meanstandard error of the mean (n  5, nd: not detected). Reprinted with permission from J. Oliver et al. [56].

b

The selective absorption of carotenoids in the common green iguana (Iguana iguana) has also been invstigated by RP-HPLC. Iguanas received a basal diet supplemented with various carotenoids for 5 – 28 days and then the concentration of carotenoids, vitamin A and -tocopherol was determined in the blood plasma. Plasma samples of 0.1 ml were mixed with 0.2 ml of ethanol then diluted with 0.1 ml of water. The suspension was vortexed and extracted twice with n-hexane. The combined extracts were evaporated to dryness, redissolved in 0.2 ml of 2-propanol and used for RP-HPLC analysis. Measurements were carried out in a C30 column (250  4.6 mm i.d.; particle size 5m). The components of the gradient elution were methanol–0.4g/l aqueous ammonium acetate (90:10, v/v) (eluent A) and methanol–methyl-tert-butyl-ether–water (8:90:2, v/v with 0.1g/l ammonium acetate in water) (eluent B). Retinol, carotenoids and -tocopherol were detected at 325, 450 and 290 nm, respectively. Chromatograms of plasma carotenoids are shown in fig. 2.27. The differences between the chromatograms illustrate that the composition of

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TABLE 2.22 CAROTENOID CONCENTRATIONS IN EGG YOLK FROM GULL, MOORHEN AND COOT AND IN TISSUES OF NEWLY HATCHED MOORHEN, COOT AND GULL

Carotenoids in egg yolk (g/g)*

Gull (n  20)

Moorhen (n  10)

Coot (n  10)

Total Lutein Zeaxanthin Canthaxanthin B-cryptoxanthin Echinenone
-carotene

71.66.5a 14.21.2a 5.11.2a 120 1.3 2.4 0.2a 9.2 0.7a 18.81.9a

47.56.90b 14.90.9a 8.10.7b nd 5.20.5b 1.20.1b 13.81.4b

131.06.0c 45.73.9b 17.41.6c nd 26.22.1c 0.40.0c 33.42.7c

*Values are means  S. E. M., numbers with different superscripts are significantly (P0.05) different with respect to row. CAROTENOID COMPOSITION (PERCENTAGE OF TOTAL) IN TISSUES OF NEWLY HATCHED MOORHEN

Carotenoids

Yolk

Lutein (L) Zeaxanthin(Z) B-cryptoxanthin Echinenone
-carotene L/
-carotene L-Z/
-carotene Lutein (L) Zeaxathin (Z) B-cryptxanthin
-carotene L/-
carotene L-Z/
-carotene

31.41.0 17.00.9

Liver

Kidney

Lung

Heart

Breast muscle

Leg muscle

Skin

Bile

3.30.2 22.01.4 5.30.3 18.61.1

32.42.2 30.62.7 22.21.5 29.51.4 28.42.4 10.10.5 23.01.7 16.51.3 17.51.2 20.62.0 17.61.2 5.30.2

11.00.5 18.51.2 15.41.0

17.91.1 19.81.2 16.81.1 13.51.0 14.01.3 16.51.3

2.60.2 10.41.0

6.100.3

4.10.2

5.900.3 9.30.5

5.40.3 3.700.3

4.30.3

29.01.1 57.73.6 25.901.7 12.10.8 14.91.0 19.91.3 18.71.3 14.61.1 60.04.3 1.08 0.85 0.06 2.68 2.05 1.12 1.58 1.95 0.17 1.67 32.92.1 18.31.2

0.15

1.57

8.40.4 15.81.2 7.80.3 13.41.3

4.58

3.16

1.99

2.68

3.15

15.41.1 17.01.4 15.81.3 18.81.2 12.21.0 12.40.8 12.70.7 10.70.8 14.20.9 13.00.9

0.26 8.60.5 7.20.5

16.01.1 34.12.7 35.92.2

39.02.1 33.72.2 38.72.1 35.62.0 38.82.5 30.72.7

22.51.4 37.72.6 22.31.2 1.46 0.22 0.71

21.01.6 24.51.3 23.41.1 22.11.3 23.41.7 42.23.3 0.73 0.69 0.68 0.85 0.52 0.20

2.27

0.43

1.31

1.32

1.21

1.13

1.49

1.08

0.37

(Continued on next page)

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TABLE 2.22 (continued) CAROTENOID COMPOSITION (PERCENTAGE OF TOTAL) IN TISSUES OF NEWLY HATCHED GULL

Carotenoids

Egg yolk

Liver

Heart

Leg muscle

Lutein (L) Zeaxanthin (Z) Canthaxanthin B-cryptoxanthin Echinenone
-carotene L/
-carotene LZ/
-carotene

19.01.1 7.10.4 16.81.3 3.40.2 12.91.0 26.32..40.1 0.72 0.99

7.10.4 2.10.1 21.21.3 5.80.3 20.81.7 36.72.1 0.19 0.25

51.13.3 19.11.3 5.70.3 4.50.2 5.30.3 2.10.1 24.33 33.42

55.34.2 19.51.1 4.70.3 3.10.21 3.50.2 2.1 39.5 53.43

Absorbance

Absorbance

Absorbance Absorbance

Reprinted with permission from P. F. Surai et al. [63].

(a)

1

(b)

1

0.01 2 0 0.02 2 0

4

(c) 3

0.1 1 0

5 (d)

0.05

1 2

0 10

12

14

16

18

20

22

24

26

28

30

32

34

Elution time (min)

Fig. 2.27. Representative HPLC chromatograms of carotenoids found in the plasma of green iguanas after being fed with a carotene-deficient diet (a) or a diet supplemented with
-carotene (b), canthaxanthin (c) and
-apo-8
-carotenoic acid ethyl ester (d) recorded at 450 nm. Enumerated peaks are: (1) lutein (21.3min); (2) zeaxanthin (22.2min); (3) undefined peak co-eluted with zeaxanthin (22.2min); (4) canthaxanthin (23.1min); and (5) apo-8
-carotenoic acid ethyl ester (26.7min). Retention times in parentheses. Reprinted with permission from J. Raila et al. [65].

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TABLE 2.23 CONCENTRATIONS OF PLASMA RETINOL (1, NG/ML), LUTEIN (2, NG/ML), ZEAXANTHIN (3, NG/ ML), CANTHAXANTHIN (4, NG/ML) AND -TOCOPHEROL (5, G/ML) IN CONTROL AND EXPERIMENTAL GREEN IGUANAS AFTER INGESTING DIETS SUPPLEMENTED WITH DIFFERENT CAROTENOIDS (80 MG/KG DIET) FOR 28 DAYS (MEANS.D., N  5)

Feeding group

n

1

2

3

4

5

Control
-carotene Canthaxanthin
-apo-8
carotinoic acid ethyl ester

5 5 5 5

5212.2 5419.3 5520.5 757.2

18353.6c 19155.8b 535202.9a 316141.5b

132.8b 254.8a nc 2211.4a

21.6d 31.6d 1949302.3a 74.0d

3.481.578 4.071.516 3.630.819 3.620.745

Values with different superscripts in the same column are significantly different at P0.05 (a,b), P0.01 (a,c) and P0.001 (a,d) determined by one-way ANOVA and Tukey test. nc  not calculated because the HPLC chromatogram revealed an undefined peak co-eluted with zeaxanthin that was also detectable in the canthaxanthin diet. Reprinted with permission J. Raila et al. [65].

diet exerts a considerable influence on the carotenoid content and composition in plasma. The quantitative results are listed in Table 2.23. The results illustrate that iguanas absorb selectively the polar carotenoids and this process can be followed by RP-HPLC measurement of separate carotenoids in plasma [65]. The potential dietary and biochemical bases of the sexually based dichromatism has been studied by using RP-HPPLC for the measurement of the quantity and composition of carotenoids in the plumage of male and female American goldfinches supplemented with various carotenoids. The pigment concentration in feathers was measured by trimming pigmented barbules suspended in 1 ml of acidified pyridine and held at 85°C for 3h. After cooling the mixture 1 ml of water and 5 ml of hexane was added. The suspension was shaken for 2min, centrifuged, and the hexane phase separated. The organic phase was evaporated to dryness and dissolved in the HPLC mobile phase (methanol–acetonitrile, 50:50, v/v  0.05 per cent triethylamine). Pigments were separated on a C30 column (250  4.6 mm i.d.; particle size 5m). The flow rate was 1.2 ml/min and pigments were detected at 450 nm. The retention times of pigments were: canary xanthophyll B, 9.7; canary xanthophyll A, 10.7; lutein, 15.7; zeaxanthin, 18.3; and canthaxanthin, 24.8min. The quantitative results are compiled in Table 2.24. The results demonstrated that male finches incorporate a significantly higher amount of pigments in their feathers than females do [66]. Another RP-HPLC technology has been developed for the investigation of the carotenoid concentration in the diet, blood and tissues of zebra finch (Taeniopygia guttata). Diet mix (0.5) was pulverized and extracted three times with 2 ml of THF. The

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TABLE 2.24 COMPARISON OF PLUMAGE PIGMENT CONCENTRATIONS AND COMPOSITIONS PRESENT IN MALE AND FEMALE AMERICAN GOLDFINCHES FED A SEED DIET DURING MOULT IN CAPTIVITY WITHOUT AND WITH CANTHAXANTHIN SUPPLEMENTATION

Without canthaxanthin supplementation Concentration (g/g feather) Canary xanthophyll B Canary xanthophyll A Lutein Percentage composition (of total plumage pigments) Canary xanthophyll B Canary xanthophyll A Lutein With canthaxanthin supplementation Concentration (g/g feather) Canary xanthophyll B Canary xanthophyll A Lutein Canthaxanthin Percentage composition (of total plumage pigments) Canary xanthophyll B Canary xanthophyll A Lutein Canthaxanthin

Males (n  5) 53.530.8 44.721.1 12.96.87

45.16.1 42.33.9 12.72.1 Males (n  7) 29.214.8 27.013.4 6.24.9 369.0366.6

8.34.6 7.83.5 2.11.5 81.88.5

Females (n  8) 25.88.7 24.88.9 22.6

Z 2.20 1.76 1.17

P 0.03 0.08 0.24

44.93.7 42.72.6 12.41.2

0 0 0.41

0.99 0.99 0.68

Females (n  3) 14.84.1 24.513.6 7.35.4 42.047.9

Z 1.57 0.28 0.28 2.5

P 0.12 0.78 0.78 0.01

18.16.9 32.020.1 9.57.0 40.533.8

1.94 1.94 1.39 1.94

0.05 0.05 0.16 0.05

Mann–Whitneys U test (Z reported) was used to examine sex differences in carotenoid pigmentation. MeansS.D. are provided for each group. Reprinted with permission from K. J. McGraw et al. [66].

suspension was centrifuged and the supernatant was evaporated to dryness and redissolved in the HPLC mobile phase. An aliquot of samples was saponified with 5 per cent KOH in methanol. Blood samples were centrifuged (3 000 rpm for 10min) and carotenoids were extracted from the plasma fraction by mixing 200 ml of ethanol with 25 ml of plasma. After vortexing the suspension 100 ml of tert-butyl methyl ether was added and the mixture was vortexed again. After centrifugation 200 ml of supernatant was evaporated to dryness and dissolved in the mobile phase. An aliquot of plasma samples was also saponified. Tissue samples (liver and adipose tissues) and eggs were treated as described above. Carotenes and xanthophylls were analysed in a C30 column (250  4.6 mm i.d.). Separation of carotenoids was achieved by the isocratic mobile phase methanol–methylene chloride (50:50, v/v)  0.05 per cent triethylamine while

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121

a

Lutein Zeaxanthin

70

Anhydrolutein

% of total carotenoids

60

β-cryptoxanthin

50 a

b

b

30 20

a

a

40

b

b

a a

a.b

a

c

b d

c

10

b

c

c d

0 Diet

Serum

Liver

Fat

Yolk

Fig. 2.28. Relative abundance of carotenoid pigments in zebra finch diet, plasma and tissue. The carotenoid profile of each sample was determined by conventional reversed-phase HPLC. Letters denote significant differences in carotenoid composition (within tissues only) as determined by post hoc paired comparisons. Reprinted with permission from K. J. McGraw et al. [67].

xanthophylls were separated by methanol–ACN (50:50, v/v)  0.05 per cent triethylamine. The flow rate and the detection wavelength were the same in both cases (1.2ml/min and 450 nm, respectively). The composition of carotenoid pigments in zebra finch diet, plasma, liver, fat and yolk are shown in Fig. 2.28. The data clearly show that 2
,3
-anhydrolutein was not present in the diet but occurred in the plasma, liver, adipose tissue and egg of zebra finch. It has been concluded that zebra finch metabolizes anhydrolutein from lutein present in the diet [67]. A similar study has been carried out to determine the selective pigment incorporation into the feathers of songbirds. The carotenoid content of the feathers of yellow warbles (Dendroica petechia), common yellowthroat (Geothylpis trichas) and evening grosbeak (Coccothraustes vespertinus) has been measured by RP-HPLC. Surface lipids were removed from feathers by washing them with ethanol and hexane and pigments were extracted with acidified pyridine at 95oC for 3h. Separations were performed in a C30 column (250  4.6mm i.d.) using isocratic elution with methanol–ACN (1:1, v/v). Flow rate was 1 ml/min and pigments were detected between 250 – 600 nm. A typical chromatogram is shown in Fig. 2.29. The concentrations of lutein and zeaxanthin in the samples are compiled in Table 2.25. The data demonstrate that these birds selectively incorporate lutein in the plumage, however, the underlying biochemical process is as yet unknown [68].

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0.055 OH HO

0.050

all-E lutein

0.045 0.040 0.035

Au

0.030 0.025 0.020 0.015 9-Z lutein

9'-Z lutein

13-Z lutein

13'-Z lutein

0.010 0.005

450.00 375.00 16.00

18.00

20.00

22.00 24.00 Minutes

26.00

28.00

λ (nm)

30.00

Fig. 2.29. Representative HPLC chromatogram of yellow feathers from an adult male common yellowthroat. All-trans-lutein was found in these feathers, plus cis isomers that were formed during the thermochemical extraction process. Chromatograms for the other two yellow-coloured bird species studied here were identical to this one. Reprinted with permission from K. J. McGraw et al. [68].

2.1.3.3 HPLC determination of carotenoid pigments in miscellaneous organic matrices Because of their considerable role in human wellfare, carotenoids have been measured not only in plants as primary sources and in human and animal tissues but also in a wide variety of other matrices to find new and economical sources for carotenoids. Thus, the carotenoid accumulation capacity of algae and microalgae have been vigorously investigated. A validated liquid chromatography–electrospray mass spectrometry method have been developed and employed for the separation and quantitative determination of

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TABLE 2.25 CAROTENOID COMPOSITION (PERCENTAGE OF TOTALS.E.) OF FULLY FORMED FEATHERS, BLOOD AND UNKERATINIZED LIPID FRACTIONS FROM MATURING FEATHER FOLLICLES IN FOUR PASSERINES THAT DISPLAY YELLOW PLUMAGE COLOURATION

Species Common Yellowthroat

Yellow warbler

Evening grosbeak

Site

n

Lutein (%)

Zeaxanthin (%)

Feather Blood Follicle Feather Blood Follicle Feather

5 4 1 7 1 1 10

100 892 85 100 85 86 100

0 92 4 0 12 13 0

Reprinted with permission from K. J. McGraw et al. [68].


-carotene,
-cryptoxanthin, lutein, zeaxanthin, canthaxanthin and astaxanthin in the powder of Spirulina Pacifica (Spirulina platensis) microalgae. RP-HPLC measurements were carried out in two ODS columns (200  2.1 mm i.d.; particle size 5m and 100  2.1 mm i.d.; particle size 5 m) connected in series. Pigments were isocratically separated by the mobile phase consisting of ACN-methanol (0.1M ammonium acetate)–dichloromethane (71:22:7, v/v). The flow rate was 300l/min. UV detection was carried out at 449 nm. MS detection was performed under both postitive-ion and negative-ion modes. The chromatographic profiles of the extract of Spirulina platensis under various conditions is shown in Fig. 2.30. The chromatogram illustrates that the method is suitable for the separation and detection of zeaxanthin,
-cryptoxanthin and
-carotene in the extract of the microalgae Spirulina platensis. Some validation parameters of the method are listed in Table 2.26. It was further established that the sensitivity is higher under positive-ion than under negative conditions [69]. Not only RP-HPLC but also other chromatographic techniques have found application in the analysis of carotenoids. Thus, lutein has been isolated and purified from the microalgae Chlorella vulgaris using high-speed counter-current chromatography (HSCCC). Lutein was extracted from liophilized algae by adding 250 ml of 10.0M aqueous KOH containing 2.5 per cent ascorbic acid to 100g of algae. The mixture was heated to 60oC for 10min then lutein was extracted at room temperature with 50 ml of dichloromethane. The suspension was centrifuged and the supernatant was separated. The procedure was repeated until the solid rest was almost colourless. The combined extract was washed with 100 ml of water, and evaporated to dryness. HSCCC isolation of lutein was carried out with a twophase solvent system consisting of n-hexane–ethanol–water in various ratios. The efficacy of the isolation process was controlled with RP-HPLC. The chromatograms of crude lutein

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Chapter 2 1.18e7 cps 3

Intensity, cps

1.5e6 1.0e6 1 2

5.0e5

5

10

15

Intensity, cps

2.5e5

20 Time, min

25

30

35

2.66e5 cps 1

568

2.0e5 1.5e5 1.0e5 5.0e4

520

540

560

580 m /z

600

620

7.62e4 cps 2

552 Intensity, cps

640

60000 40000 20000

520

560

580 m /z

600

620

640

3.54e5 cps 3

536

3e5 Intensity, cps

540

2e5 1e5

520

540

560

580 m /z

600

620

640

Fig. 2.30. Total ion current chromatogram of alga Spirulina Platensis algae sample by LC–TurbolSP–MS and positive–ion mass spectra of the carotenoids identified: 1  zeaxanthin; 2 
-cryptoxanthin; 3 
-carotene. Reprinted with permission from M. Careri et al. [69].

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TABLE 2.26 INTRA-DAY AND INTER-DAY REPEATABILITY OF THE LC-TURBOL SP-MS TECHNIQUEa

Analyte

Lutein Zeaxanthin Canthaxanthin


-cryptoxanthin
-carotene

n

50 100 50 100 0 20 10 50 50 100

RSD (%) Intra-dayb

Inter-dayc

0.8 4.5 6.4 3.8 3.5 4.1 2.6 4.3 2.2 1.0

6.8 5.9 6.4 5.8 1.3 3.6 12 3 5.4 10

a

Mobile phase: CH3CN–MeOH with 0.1 M CH3COONH4–CH2Cl2(71:22:7, v/v). b Calculated from mean values (n  5). c Calculated from mean values (n  15). Reprinted with permission from M. Careri et al. [69].

and the lutein fraction separated by HSCCC are shown in Fig. 2.31. It was suggested that HSCCC can be applied not only for the preparation of lutein but also for the isolation and purification of other compounds of biological activity [70]. The effect of various concentrations of additives on the astaxanthin production of the green microalgae (Haematococcus pluvialis) has also been investigated by RP-HPLC. NaNO3 (0, 0.15, 0.25, 0.5, 0.75 and 1 g/l), acetate and malonate (0, 0.25, 0.5, 1 and 2 per cent, w/v) were added to the liquid culture of the microalgae. Separation of pigments was performed in an ODS column (250  4 mm i.d.; particle size 5 m). Eluents of gradient elution were: water (eluent A), methanol (B) and acetone (C). Elution was initiated at 9 per cent A, 76 per cent B, 15 per cent C; 9 min 5 per cent A, 45 per cent B, 50 per cent C; 15 min 4 per cent A, 38 per cent B, 58 per cent C; 17min 3 per cent A, 27 per cent B, 70 per cent C; 22min 3 per cent A, 27 per cent B, 70 per cent C; 25 min 100 per cent C; 26 min 100 per cent C. The flowrate was 1 ml/min and pigments were detected with a diode array detector at 444 and 476 nm. The results of HPLC analyses are compiled in Table 2.27. It was stated that the new low toxicity RP-HPLC method is suitable for the separation of chlorophylls a and b, carotenes and xanthophylls and can be used for the investigation of the effect of additives on the pigment production of Haematococcus pluvialis [71]. New spectrophotometric and RP-HPLC methods were developed for the detection of adulteration in cochineals. The insects were cleaned over as sieve and dried at 60oC. The dried

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Chapter 2

2500 A

Absorbance

2000 1500 1000 500 0

0

5

(a)

10 Time (min)

15

20

60

A

Absorbance

40

20

0 (b)

0

30

60 Time (min)

90

120

Fig. 2.31. Chromatogram of crude lutein from the microalga Chlorella vulgaris by HPLC analysis, A  lutein. Conditions: column: reversed-phase C18 column (250  4.6 mm i.d., 5 m); mobile phase: methanol–dichloromethane–acetonitrile–water (67.5:22.5:9.5:0.5, v/v); flow rate: 1.0ml/min; detection at 450 nm (a). Chromatogram of crude lutein from the microalga Chlorella vulgaris by preparative HSCCC separation, A  lutein. Conditions: column: multilayer coil of 1.6mm i.d. PTFE tube with a total capacity of 230ml; rotary speed: 800rpm; solvent system: n-hexane–ethanol–water (4:3:1, v/v); mobile phase: lower phase (ethanol–water); flow rate: 1ml/min; detection at 254 nm; sample size: 200 mg; retention of the stationary phase: 58 per cent (b). Reprinted with permission from H.-B. Li et al. [70].

insects were ground and about 0.125103 kg was homogenized with 10 ml of methanol–water (65:35, v/v). Pigments were extracted at 80oC, the suspension was centrifuged and the supernatant was used for spectrophotometry and RP-HPLC. Chromatographic separation was carried out in an ODS column (250  4.6 mm i.d.; particle size 5 m) The constituents of the mobile phase were water (A), methanol (B) and 5 per cent orthophosphoric acid in water (C). Gradient elution was: 0–11min 50 per cent A, 40 per cent B, 10 per cent C;

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TABLE 2.27. QUANTITATIVE CAROTENOID COMPOSITION (PG/CELL), OBTAINED BY HPLC ANALYSIS, OF H. PLUVIALIS CELLS IN THE STATIONARY PHASE IN CULTURES WITH DIFFERENT CONCENTRATIONS OF NITRATE (G/L), ACETATE AND MALONATE (% W/V)

Nitrate 0 0.15 0.25 0.5 0.75 1 Acetate 0 0.25 0.5 1 2 Malonate 0 0.25 0.5 1 2

Astaxanthin free

Astaxanthin Cantaxanthin ester

Echinenone


-carotene

Other carotene

0.400.00 0.110.00 0.190.00 nd nd nd

24.110.38 5.410.46 0.400.01 nd nd nd

0.620.04 0.140.00 0.070.00 nd nd nd

0.500.01 0.130.00 0.190.00 nd nd nd

0.530.00 0.650.18 0.690.01 1.160.00 1.030.00 1.060.01

3.080.07 3.300.04 4.920.04 5.280.00 5.480.00 5.090.01

0.380.00 0.330.00 0.430.01 0.300.01 0.800.00

1.480.00 17.590.7 28.920.00 30.610.03 48.500.00

0.200.00 30.240.0 0.360.00 0.270.00 0.310.00

0.250.00 0.640.02 1.190.02 0.820.00 0.900.01

2.250.01 1.520.00 2.340.14 1.780.01 2.570.02

10.80.01 3.350.91 2.960.09 1.550.01 2.460.00

0.270.00 0.160.00 0.210.00 0.130.00 1.310.02

0.590.00 8.620.11 9.990.23 11.550.01 73.160.51

0.080.00 0.120.00 0.130.00 0.120.00 0.520.06

0.100.00 0.250.02 0.320.01 0.310.00 1.000.04

0.900.00 0.800.00 0.930.01 0.710.00 3.180.41

4.650.00 2.020.17 1.740.35 0.760.01 4.850.26

Reprinted with permission from M. Orosa et al. [71].

12–25min to 90 per cent B, 10 per cent C, final hold 6 min. The flow rate was 1.2 ml/min and pigments were detected at 420 and 500 nm. The chromatographic profiles of pure and adulterated cochineal pigments are shown in Fig. 2.32. The chromatograms demonstrate that the method is suitable for the separation of colourants added to cochineal, that is, it can be used for the detection of adulteration of this pigment mixture. The quantitative data obtained by RP-HPLC are compiled in Table 2.28. The data indicated that both the spectrophotometric and RP-HPLC methods can be successfully employed for the elucidation of adulteration of cochineal [72]. The extraction of carotenoids produced during methanol waste biodegradation was also followed by RP-HPLC. It has been previously established that chromatoraphic solvents after distillation can be used for the bacterial production of carotenoids [73,74]. The strain Methylobacterium organophilum has been used for the biodegradation of methanol. Methanol was biodegraded in three days, then the suspension was centrifuged (5 000g), washed twice with fresh culture medium and centrifuged. Pellets were dissolved in

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Chapter 2 22 420 nm 18 ca

14

dc VII

dcII

6

ca

dcIV

10 Absorbance . 103

420 nm

420 nm

dcIII dcIII,

dc VII

dcII

−2 22

500 nm

18 ca

dcIII

dcII

fk ka

fk ka

2

ca

dcIV

dcIV, dcVII, fk, ka,

500 nm

dcIII dcIV

ca

dcIII

500 nm dcIV

ca

dcVII, fk,

dcIII dcIV

14 10

dc VII

6

−2

(a)

fk ka

dcII

2 0

dc VII

10

20

fk ka

dcII

30 0

10

20

Retention time (min) (b)

ka

dcII

30 0

10

20

30

(c)

Fig. 2.32. Chromatograms obtained at 420 and 500 nm for pure cochineal (a) and for cochineal adulterated by erythrosin (b), at a concentration of 0.35103 kg per kg cochineal, and by trans-
-carotene (c) at a concentration of 6103 kg per kg cochineal. Cochineal pigments: dcII, dcIII, dcIV and dcVII, unknown pigments of D. coccus Costa; ca, carminic acid; fl, flavokermesic acid; and ka, kermesic acid. * marks the peak of the added colourant. Reprinted with permission from M. González et al. [72].

methanol, centrifuged again, and 2 ml of 0.01 M NaOH in methanol was added to 5 ml of supernatant. The mixture was incibated for 8 h under nitrogen atmosphere at ambient temperature in darkness. Separation of carotenoids was carried out in an ODS column (250  4 mm i.d.; particle size 10 m). The isocratic mobile phase consisted of ACN–methanol (0.1 M ammonium formate): dichloromethane (71:22:7, v/v). The flow rate was 0.7 ml/min and solutes were detected at 450 and 480 nm. Electrospray ionization–mass spectrometry was in positive-ion mode, N2 gas flow 10l/min, nebulizer pressure 50 psi, drying gas temperature 350°C, and capillary voltage 3 500 V. Mass spectra were obtained between m/z 500–600. A typical chromatogram of the saponified extract of M. organophylum is shown in Fig. 2.33. Because of the good separation capacity, the RP-HPLC method has been proposed for the measurement of carotenoids produced by M. organophylum from methanol waste [75]. The composition and amount of pigments in marine environments have also been vigorously investigated. Thus, on RP-HPLC method has been developed for the study of the effect of variable irradiance on the xanthophyll cycle of the seagrass Zostera marina. Extraction of pigments from seagrass was carried out by grinding the samples with acidwashed sand in the presence of 1 ml of 90 per cent acetone and the liquid phase was

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TABLE 2.28 DETECTION OF COLOUR ENHANCEMENT AND DETECTION OF FRAUD

Adulteranta

C420(103)b

C500(103)b

C420/C500

1536

1507

1.020.03

2038 27411 34314

1496 1506 1527

1.370.05 1.830.07 2.270.09

1596 1888 2139

1877 219 25011

0.850.03 0.850.04 0.850.04

25312 29115 33513

1496 14810 1659

1.700.07 1.690.09 2.030.08

23411 28214 32119

1526 1689 19211

1.540.09 1.680.10 1.670.07

Cochineal Cochineal–tartrazine 0.30103 0.40103 0.50103 Cochineal–erythrosin 0.30103 0.35103 0.40103 Cochineal–riboflavin 0.60103 0.70103 0.80103 Cochineal–
-carotene 5.0103 6.0103 7.0103 a

kg adulterant per kg cochineal. kg pigments per kg of cochineal. (meanstandard deviation, n  3). Reprinted with permission from M. González et al. [72].

b

employed for RP-HPLC analyses. The chromatographic profile of the extract of lightadapted Z. marina is shown in Fig. 2.34. The chromatogram proves the good separation capacity of the RP-HPLC system used for the analysis of pigments in seagrass. Some data concerning the pigment composition of seagrass are collected in Table 2.29. It has been concluded from the data that the position of leaves does not influence the carotenoid content and composition [76]. RP-HPLC has found application in the study of the feeding activities of the dominant copepod species of the Belgian coastal zone. The gut pigments of adult copepods were extracted by grinding them with 90 per cent cold acetone, the extract was filtered and used for RP-HPLC measurement. Separations were performed in an ODS column (250  4.6 mm i.d.; particle size 5m). Components of gradient elution were methanol–0.5 M aqueous ammonium acetate (pH 7.2, 80:20, v/v, eluent A), eluent B was ACN–water (90:10, v/v), and eluent C ethyl acetate. The flowrate was 1 ml/min. The carotenoid pigments found in Acartia clausi, Centropages hamatus, and Temora longicornis before, during and after the Phaecystis globosa peak in 1998 are compiled in Table 2.30.

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0

Chapter 2

2

4

6

8

10

12

14

Fig. 2.33. Chromatogram of the saponified extract of M. organophylum. Retention times (min) of analytes were: astaxanthin (4.34), extracted carotenoid (4.65), Lutein (5.02), canthaxanthin (6.55) and
-carotene (10.15). Reprinted with permission from P. Stepnowski et al. [75].

0.10 0.08 7

AU

0.06 0.04 0.02

4 1 2

3

6 8

5

0.00 2.00

4.00

6.00

8.00

10.00 12.00 Minutes

14.00

16.00

18.00

20.00

Fig. 2.34. Sample chromatogram of light-adapted Z. marina leaf sample. Peak identification: 1  neoxanthin, 2  violaxanthin, 3  antheraxanthin, 4  lutein, 5  zeaxanthin; 6  chlorophyll b, 7  chlorophyll a, 8 
-carotene. Reprinted with permisson from P. J. Ralph et al. [76].

The results illustrate that RP-HPLC can contribute to the elucidation of the feeding activity of copepod species [77]. The pigment composition of Phaeocyctis globosa and Imantonia rotunda in Belgian coastal waters has been measured by RP-HPLC. Samples for RP-HPLC pigment analyses

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TABLE 2.29 DIFFERENCE IN XANTHOPHYLL PIGMENT CONCENTRATIONS ALONG A SERIES OF NUMBER 2 LEAF BLADES (MEANS.E.M., n  5).

Position (cm)

Neoxanthin

Violaxanthin

Antheraxanthin

Lutein

0–3 3–6 6–9 9 – 12 12 – 15 ANOVA

163.72.5 165.42.3 172.65.9 177.29.5 166.517.7 0.839

101.411.6 81.89.5 88.61.7 76.74.9 79.82.9 0.322

29.213.3 57.15.4 58.612.7 49.50.9 38.99.9 0.324

309.28.5 340.627.3 344.829.2 51.83.4 306.317.9 0.377

Position (cm)


-carotene

Z-A-V

EPS

0–3 3–6 6–9 9 – 12 12 – 15 ANOVA

50.313.5 50.017.4 48.41992 38.91652 46.17.3 0.987

130.624.9 141.31.7 147.714.9 126.25.8 118.812,9 0.753

0.890.03 0.780.03 0.800.02 0.830.01 0.830.02 0.157

Leaves were dark-adapted; therefore, there is no detectable level of zeaxanthin. Concentrations are nmol pigment (mol chl a  b), the P value from one factor ANOVA is displayed below each column. V-A-Z  xanthophyll pool (violaxanthin, antheraxanthin, zeaxanthin); EPS  epoxidation state. Reprinted with permission from P. J. Ralph et al. [76].

were sonificated with 2 ml of acetone. Then they were centrifuged, and the supernatant was injected into the column. The details of HPLC measurement are described in ref. [77]. Pigments were detected at 436 nm. Typical chromatograms of Phaeocyctis globosa and Imantonia rotunda are shown in Fig. 2.35. The chromatograms indicate that the pigment composition of the two cultures are similar but not identical. The numerical comparison of the cultures is demonstrated in Table 2.31. It was proposed that the results of HPLC analysis have to be completed with microscopic studies for the safe identification of phytoplankton taxa [78]. The effect of microcapsulation and storage on the isomerization and chemical decomposition of astaxanthin was investigated by RP-HPLC. Microencapsulation of astaxanthin in a chitosan matrix cross-linked with glutaraldehyde was performed by the method of multiple emulsion/solvent evaporation. Astaxanthin was extracted from microcapsules four times with dichloromethane–methanol (50:50, v/v). The suspension was centrifuged after each extraction step, then the collected supernatants were evaporated to dryness and used for HPLC measurements. Separation was performed in an ODS column (250  4.6 mm i.d.; particle size 5 m). The column was not thermostated. The isocratic mobile phase consisted of 85 per cent of methanol, 5 per cent dichloromethane, 5 per cent ACN, and 5 per cent water.

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Chapter 2

TABLE 2.30 CAROTENOID PIGMENTS DETECTED IN ACARTIA CLAUSI (A), CENTROPAGES HAMATUS (C), AND TEMORA LONGICORNIS (T) BEFORE, DURING AND AFTER THE PHAECYSTIS GLOBOSA PEAK IN 1998 AND AFTER 90 MIN OF STARVATION IN FILTERED SEAWATER. CROSSES IN BRACKETS CORRESPOND TO DETECTABLE BUT VERY LOW AMOUNTS OF PIGMENT. TAXONOMIC GROUPS CORRESPONDING TO EACH PIGMENT ARE ALSO PRESENTED

Carotenoid pigment

Peridin Fucoxanthin

Taxonomic group

Dinoflagellates Diatoms and prymnesiophytes 19
-Hexanoyl- Prymnesiophytes fucoxanthin Astaxanthin Animals Alloxanthin Cryptophytes Lutein Green algae

Before Phaecyctis peak

During Phaecyctis peak

After Phaecyctis peak

After starvation

A X X

C X X

T X X

A

C

A

C

T

X

X

X

T X X

A C

X

T X X

X

X

X

X

X

X

X

X

X

X X

X

Reprinted with permission from S. Gasparini et al. [77].

Astaxanthin was detected at 480 nm. The chromatographic profiles of microencapsulated and free astaxanthin after eight weeks of storage are shown in Fig. 2.36. It was concluded from the results that the method is suitable for the study of the stability of miroencapsulated astaxanthin. The HPLC data proved that microencapculated astaxanhtin did not suffer isomerization or chemical decomposition under the experimental conditions [79]. Because of the high theoretical and practical importance of the analysis of caronetoid pigments, the chromatographic techniques applied have been frequently reviewed [80–83]. The analysis of the pigment (carotenoid and chlrophyll) content of vegetable oils has been specially reviewed [84]. A simple and rapid RP-HPLC method was developed for the determination of retinoid in galenicals. Commercial preparations were diluted, filered and used for separation. Measurements were carried out in an ODS column (150  4.6 mm i.d.: particle size 3 m). Solvents A and B were methanol–10 mM ammonium acetate (75:25, v/v) and methanol–THF (84:16, v/v), respectively. The flow rate was 0.8 ml/min. Gradient conditions were: 0 – 25 min, 0 per cent B; 35 min, 100 per cent B, isocratic for 10 min. Typical chromatograms are shown in Fig. 2.37. The repeatability of peak area ranged between 0.48 –3.2 per cent for UV-DAD and 0.57 – 3.1 per cent for fluorescence detection. The reproducibility varied between 0.26 – 4.6 per cent. It was found that the method is precise, selective, sensitive and linear, therefore, it can be employed for the routine quality control of this class of drugs [85].

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133

Phaeocystic globase colonies

0.16 0.14 0.12

5

AU

0.10 0.08 0.06

2

0.04 1

0.02

7 8

910

13

0.00 2.00

4.00

6.00

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 Minutes 5

Imantonia rotunda

11

1.60 1.40 7 1.20

AU

1.00 6 0.80 8

0.60 0.40

1

2

0.20

9 10

13

0.00 2.00

4.00

6.00

8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 Minutes

Fig. 2.35. HPLC absorbance chromatograms of cultures of Phaeocyctis globosa colonies and Imantonia rotunda made at 436 nm. Retention time is given in minutes. AU  absorption units. Pigments: 1, chlorophyll-c; 2, chlorophyll-c1c2; 3, cis-flucoxanthin; 4, 19
-butanoxyloxyfucoxanthin; 5, fucoxanthin; 6, 19
-hexanoxyloxyfucoxanthin; 7, diadinoxanthin; 8, diatoxanthin; 9, phytilated chlorophyll-clike; 10, chlorophyll-a allomer; 11, chlorophyll-a; 12, chlorophyll-a epimer; 13,
,
-carotene. Reprinted with permission from E. Antajan et al. [78].

2.2 FLAVONOIDS 2.2.1 Chemistry and biochemistry of flavonoids Flavonoids are secondary metabolites generally occurring in various plants as glycosides. The chemical structure of flavonoids shows high variety. The basic structure of flavons and flavonols is the 2-phenylbenzo-gamma-pyrone. Flavonoids generally contain two phenol rings linked with a linear three-carbon chain (chalcones) or with three carbon

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Chapter 2 TABLE 2.31 HAPTOPHYTE BIOMARKER PIGMENTS OF THE TWO STRAINS ISOLATED FROM BELGIAN COASTAL WATERS EXPRESSED AS RATIOS TO CHLOROPHYLL-A

Strain

Ratio to Chl-a Chl-c3

Phaeocyctis globosa Imantonia rotunda

0.208 0.269

But-fuco fuco 0.000 0.060

Hex-fuco

0.350 0.746

0.000 0.296

50

50

40

40

30 20 10

45°C 35°C 25°C Initial

0 0

(a)

2

4

6 8 10 Time (min)

12

Absorbance (mAU)

Absorbance (mAU)

Reprinted with permission from E. Antajan et al. [78].

30 20 10

45°C 35°C 25°C

0

14

0

(b)

2

4

6 8 10 Time (min)

12

14

Fig. 2.36. Representative HPLC profiles of the extract of microcapsules, initially and after eight weeks of storage (a) and pure astaxanthin after eight weeks of storage (b). Chromatographic conditions are described in the text. Reprinted with permission from I. Higuera-Ciapara et al. [79].

atoms forming a five-membered ring strucutre (aurones). The basic structural formulae are listed in Fig. 2.38. Similarly to carotenoids, flavonoids also show considerable biological activity. Their beneficial effect in cancer and heart diseases has been proven many times [86–89]. It has further been established that flavonoids improve cardiovascular remodelling and vascular function in NO-deficient hypertension [90]. Moreover, flavonoid intake reduces the risk of chronic diseases [91], and beneficially influences inflammations [92], and ulcer formation [93,94]. It has been established that the free radical scavenger activity of flavonoids is responsible for the antioxidant effect [95,96]. The beneficial antitumour effect of quercetin has also been demonstrated [97,98]. Isoflavones belonging to the phytoestrogens also expose marked biological activity [99]. Their effect and fate of isoflavones in organisms have been extensively investigated

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50

40

30

4

mRU

3 2 1

20

5

10

0 10

20

(a)

30 Time (min)

40

50

4 5.0E+05

4.0E+05

µV

b 3.0E+05 5 2.0E+05 a 3

1

1.0E+05

2

0.0E+00 10.00 (b)

20.00

30.00 Time (min)

40.00

50.00

Fig. 2.37. Gradient LC separation of the ‘retinoid solution’ components and retinoic acid isomers by (A) UV-DAD detection (350 nm) and (B) fluorescence detection with on-line photoreactor switched (a) off and (b) on with irradiation at 366 nm. Peak identification: 1  13-cis retinoic acid; 2  9-cis retinoic acid; 3  all-trans retinoic acid; 4  vitamin A palmitate; 5 
-carotene. Reprinted with permission from R. Gatti et al. [85].

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Chapter 2

O A

B

B O

A O

O

O

Fig. 2.38. Basic chemical structure of flavones, flavonols and flavonoids (chalcones and aurones).

[100–102]. Isoflavonoid has been used as dietary biomarkers [103,104]. It has been further established that isoflavonoids influence receptor expression [105] and induce immune changes [106,107]. The individual variation in metabolism of isoflavones has also been extansively studied [108,109]. It has been further demonstrated that flavonoids show antiinflammatory effects on adjuvant arthritis [110], and protect mice from two types of lethal shock caused by endotoxin [111]. 2.2.2 Thin-layer chromatography 2.2.2.1 TLC separation of flavonoids in the extracts of medicinal plants TLC as a rapid and easy-to-carry-out method has been frequently applied for the separation of the active ingredients in various medicinal plants. Its widespread application in this field can be partially explained by the fact that TLC requires a less cumbersome prepurification procedure of plant extracts than HPLC. The components of callus cultures of different plant species have been investigated by TLC. The investigations were motivated by the finding that the in vitro cultures of higher plants can produce compounds of considerable medicinal and industrial importance. Besides other secondary metabolites, the experiments included the determination of flavonoids in Cucumis sativus L.), the anthocyanins in Rudbecka hirta L., etc. Flavonoids were successively extracted from the pulverized callus (15g) with chloroform and methanol, and the methanol extract was concentrated and employed for TLC analysis. Anthocyanins were extracted from dry and pulverised callus (5g) with methanol–HCl (100:8.6, v/v). The extracts were evaporated and dissolved in methanol. Silica gel, cellulose and C18 stationary phases were employed for the investigations using various mobile phases. Flavonoids (vitexin, isovitexin and isoorientin) were separated on RP-TLC plates using the mixture of tetrahydrofuran–water–phosphoric acid (60:60:1, v/v/) as a mobile phase. Anthocyanins (cyanidin-3-malonylglucoside, cyanidin-3-monoglucoside, petunidin3-monoglucoside, and malvidin-3-monoglucoside) were separated by two dimensional TLC mobile phases being n-amylalcohol–acetic acid–water (2:1.1:1, v/v) and formic acid–hydrochloric acid–water (10:1:3, v/v) for the first and second development, respectively. It was concluded from the data that TLC can be applied for the preliminary measurement of flavonoid and anthocyanin pigments in complex accompanying matrices without using cumbersome prepurification techniques necessitated by HPLC [112].

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Synthetic mixtures of flavonoids (catechin, ellagic acid, gallic acid monohydrate, methyl gallate, kampherol, rutin), coumarins (herniarin, scopoletin, umbelliferone) and the ethyl acetate-soluble fraction of the methanolic extract of Phyllanthusemblica L. (Euphorbiaceae) leaves were separated by both normal and RP-TLC and the effect of densitometer and video-documentation settings on the quantitative results has been investigated. Normal-phase separations were performed on silica layers using toluene– ethanol–formic acid–n-hexane (60:18:4:18, v/v) and THF–2-propanol–n-hexane (23:2:75, v/v) for flavonoid and coumarins, respectively. RP-TPLC separations were carried out on C18 stationary phases using methanol–water (1:1, v/v)  1 per cent o-phosphoric acid and THF–2-propanol–CAN– water (37:5:5:53, v/v) for flavonoids and coumarins. It was concluded from the comparison of quantitative data that both video camara and TLC densitometers can be used for the measurement of flavonoids and coumarins in the extract of Phyllanthus emblica L. [113]. HPTLC was employed for the detection of the adulteration of ‘Espinheira Santa’ (Maytenus ilicifolia and Maytenus aquifolium, Celastraceae) with the extract of Sorocea bomplandii (Moraceae). Plants were dried at 40°C, ground and for the extraction of flavonoids an aliquot of 3g was boiled with 30 ml of methanol for 30 min under agitation. The extract was diluted with the same volume of water and the mixture was concentrated until the chlorophylls were precipitated. The supernatant was fractionated in an ionexchange column (60 x 1cm i.d.) using water followed by methanol for the elution of flavonoids. Flavonoids were separated on silica HP-TLC plates with ethyl acetate–formic acid–water (6:1:1, v/v) as the mobile phase and they were detected by immersing the plate in a solution of diphenylboric acid–2-aminoethylester (100 mg) and PEG 400 (500 mg) in 100 ml of methanol. It was supposed that the overwhelming majority of Maytenus spp. flavonoids are triglycosilated derivatives while the flavonoids of S. bomplandii were less strongly retained on the silica plate [114]. Normal-phase TLC has been employed for the separation of two new flavans in the extract of the undergorund tubers of Cyperus conglomeratus Rottb. (family Cyperaceae). The underground tubes were dried, ground and were extracted with with petroleum ether: diethyl ether–methanol (1:1:1, v/v) for 24 h at ambient temperature. The extract was defatted with cold methanol. The components of the extract were preliminarily separated by traditional column chromatography followed by GC/MS and TLC. New flavans (5-hydroxy-7,3
,5
-trimethoxyflavan and 5,7-hydroxy-3
,5
-dimethoxyflavan) were separated on silica TLC layers using petroleum ether–diethyl ether (1:3, v/v) with the RF values of 0.50 and 0.37 for dimethoxy and trimethoxy derivatives, respectively [115]. Because of the diaphoretic and laxative effects, the composition of Sambuci flos (Sambucus negra L., black elder) has been extensively investigated by TLC. Samples for TLC analysis were preparated by refluxing 1.0 g of air-dried, powdered flowers of Sambucus negra with 10 ml of methanol for 30 min. The suspension was filtered, and the filtrate was concentrated and redissolved in 5 ml of methanol. Separation was performed on silica layers using 10 different mobile phases: 1  ethyl acetate–formic acid–acetic acid–water (100:11:11:27, v/v); 2  ethyl acetate–formic acid–water (8:1:1, v/v); 3  ethyl acetate–formic acid–water (88:6:6, v/v); 4  ethyl acetate–methyl–ethyl ketone– formic acid–water (50:30:10:10, v/v); 5  ethyl acetate–methyl–ethyl ketone–formic acid–water (60:15:3:2, v/v); 6  ethyl acetate–formic acid–acetic acid–methyl–ethyl

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ketone–water (50:7:3:30:10,v/v); 7  1-butanol–acetic acid–water (12:3:5, v/v); 8  1-butanol–acetic acid–water (66:17:17, v/v); 9  ethyl acetate–methanol–water (100: 13.5:10, v/v); 10  ethyl acetate–methanol–formic acid–water (100:13.5:2.5:10, v/v). Detection was carried out by spraying the plates with 1 per cent methanolic diphenylboryloxyethylamine followed by 5 per cent ethanolic polyethylene glycol 4 000. The RF values measured in the 10 mobile phases are compiled in Table 2.32. The application of various mathematical statistical methods such as the information content derived from Shanon’s equation, calculation of discriminative power and formation of cluster dendograms indicated that the best separation can be achieved by mobile phases 2 and 10 [116]. Normal-phase TLC using a silica stationary phase was employed for the optimization of the separation of flavonoid content of Matricariae flos (Chamomilla recutita L. Rauschert). Air-dried and powdered plant material was extracted by refluxing for 10 min with methanol. The suspension was filtered, evaporated and the residue was redissolved in methanol. The mobile phases included in the experiments were: 1  ethyl acetate– methylethylketone–formic acid–water (50:30:10:10, v/v); 2  ethyl acetate–methanol– water (75:15:10 v/v); 3  ethyl acetate–formic acid–water (80:10: 10, v/v); 4  ethyl acetate–formic acid–water (100:20:30, v/v); 5  ethyl acetate–formic acid–acetic acid–water (100:11:11:27, v/v); 6  n-butanol–acetic acid–water (66:17:17, v/v); 7  ethyl acetate–methanol–formic acid–water (75:10:5:10, v/v); 8  ethyl acetate–acetic acid–water (80:10:10, v/v). Development was carried out in the linear ascending mode at

TABLE 2.32 INPUT DATA: RF VALUES OF FLAVONOIDS AND PHENOLIC ACIDS OF SAMBUCI FLOS, AND DEVELOPMENT TIME

Mobile phase t(min) Compound

1 40

2 27

3 36

4 38

5 6 30 38 RF value

7 95

8 100

9 39

10 41

Phenolic acid A Phenolic acid B Phenolic acid C Flavonoid A Flavonoid B Phenolic acid D Phenolic acid E Flavonoid C Flavonoid D

0.98 0.94 0.76 0.67 0.62 0.57 0.51 0.44 0.35

0.92 0.87 0.71 0.54 0.51 0.46 0.41 0.27 0.17

0.81 0.73 0.51 0.34 0.31 0.28 0.20 0.11 0.03

0.96 0.91 0.83 0.74 0.70 0.64 0.58 0.49 0.38

0.88 0.82 0.74 0.32 0.28 0.26 0.21 0.08 0.04

0.76 0.71 0.65 0.59 0.58 0.48 0.41 0.50 0.30

0.80 0.75 0.72 0.67 0.63 0.52 0.48 0.56 0.42

0.85 0.80 0.71 0.58 0.53 0.41 0.36 0.31 0.14

0.89 0.84 0.65 0.57 0.52 0.42 0.37 0.27 0.16

0.91 0.86 0.79 0.73 0.63 0.61 0.56 0.46 0.33

Phenolic acid A  ferulic acid; phenolic acid B  caffeic acid; flavonoid A  isoquercitrin; flavonoid B  hyperoside; phenolic acid E  chlorogenic acid; flavonoid C  rutin. Reprinted with permission from Z. Males et al. [116].

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TABLE 2.33 INPUT DATA — RF VALUES OF CHAMOMILLE FLAVONOIDS AND RELATED COMPOUNDS SEPARATED IN MOBILE PHASES TESTED (1 – 8)

Component

Herniarin Flavonoid 1 Apigenin-7-diacetylglucoside Apigenin-7-acetylglucoside Flavonoid 2 Apigenin-7-glucoside Luteolin-7-glucoside Chlorogenic acid Flavonoid 3 Rutin Flavonoid 4 Flavonoid 5

Mobile phase 1

2

3

4

5

6

7

8

0.94 0.89 0.85

0.85 0.71 0.72

0.93 0.83 0.77

0.94 0.90 0.87

0.94 0.88 0.83

0.90 0.82 0.80

0.91 0.84 0.81

0.92 0.69 0.66

0.85

0.67

0.77

0.87

0.83

0.80

0.81

0.63

0.79 0.67 0.60 0.47 0.44 0.42 0.36 0.38

0.62 0.58 0.55 0.48 0.40 0.36 0.32 0.24

0.69 0.56 0.48 0.35 0.32 0.28 0.23 0.17

0.87 0.72 0.64 0.52 0.49 0.46 0.41 0.34

0.83 0.68 0.59 0.48 0.46 0.43 0.37 0.39

0.80 0.74 0.69 0.64 0.60 0.56 0.48 0.41

0.81 0.66 0.58 0.47 0.46 0.42 0.36 0.29

0.49 0.42 0.34 0.26 0.24 0.20 0.16 0.18

Reprinted with permission from M. Medic-Saric et al. [117].

ambient temperature. The RF values determined in the eight mobile-phase systems are compiled in Table 2.33. The data were evaluated by information theory and numerical taxonomy. It was found that the best separation of flavonoids can be achieved in mobile phases 2, 3 and 5 [117]. Because of the carminative, astringent, aphrodisiac and diuretic effects of the members of the genus Wrightia (family: Apocyanaceae) the indole and flavanoid content of Wrightia tinctoria, W. Tomentosa and W. Coccinea has been investigated by HP-TLC, HPLC, spectrophotometry and infrared methods. The scheme of the separation of the components of Wrightia tinctoria is shown in Fig. 2.39. Extract of leaves was concentrated and purified with traditional column chromatography using a silica stationary phase. Elution was successively performed with benzene, benzene – ethyl acetate (90:19; 80:20, 50:50, v/v), ethyl acetate, ethyl acetate – methanol (50:50, v/v) and methanol. Silica HP-TLC was carried out with the mobile phases of chloroform–methanol (95:5, and 98:2, v/v; solvents I and II) and ethyl acetate–formic acid–water (100:11: 11:27, v/v). RP-HPLC separations were made in an ODS column using isocratic elution mode (methanol–water, 80:20, v/v). The various analytical techniques proved that W. tinctoria and W. tomentosa contains indigotin, indirubin, isatin, tryptanthrin, indoxyl-yielding glycosides, anthranillate and rutin, while W. coocinae contains only anthranillate and rutin. The possible formation of indigotin, indirubin and tryptanthrin is shown in Fig. 2.40 [118].

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Chapter 2 0.8

Indigotin(1) Indirubin(2) Tryptanthrin(3) Isatin(4) Anthranillate(5) Rutin(6)

0.7 0.6

% W/W

0.5 0.4 0.3 0.2 0.1 0 Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

Sep

Oct

Nov

Dec

Sep

Oct

Nov

Dec

−0.1 Months 0.8

Indigotin(1) Indirubin(2) Tryptanthrin(3) Isatin(4) Anthranillate(5) Rutin(6)

0.7 0.6

% W/W

0.5 0.4 0.3 0.2 0.1 0 Jan

Feb

Mar

Apr

May

Jun

Jul

Aug

−0.1 Months

Fig. 2.39. Isolation of chemical constituents of Wrightia tinctoria. A similar scheme was followed for the isolation of chemical constituents from W. tomentosa and W. cocinea. Reprinted with permission from A. V. Muruganandam et al. [118].

The sesquiterpene lactone and flavonoid composition of the Arnica angustofolia Vahl subsp. attenuata (Greene) Maguire in the flowerheads was determined by TLC and GC/MS technologies. The objectives of the investigations were the elucidation of the presence of methylated flavonoids in the flowerheads, and the assessment of the possibility of the use of flavonoid profiles for the identification of subspecies. Flavonoid aglycones (FA) were exhaustively extracted from flowerheads with dichloromethane, then macerated with 10 ml of methanol. The methanol soluble fraction was firstly separated in a Sephadex LH20 column with methanol mobile phase, and FA fraction was employed for TLC, GC and GC/MS analyses. The flavonoid aglycones identified in flowerheads of Arnica angustifolia are

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141

R H

O2

N H

RR H

+1a

N H

N H

H

N H

1 (R = beta-D-glucosyl) HOH

RR

OO

O N H

N H

N H Scheme I - Palusible sequence of formation of indigotin from indican OH O

HOH H+

O

air

N H

N H

O

O

O HO

N H

N H

-H2O

H

N H

O

O

Scheme II - Plausible sequence of formation of indirubin from indoxyl ans isatin O

O

O

OH NH2

N NH2 O

N O O Scheme III - Plausible sequence of formation of tryptanthrin from anthranillic acid and isatin

Fig. 2.40. Plausible sequence of formation of indigotin from indican (I), of indirubin from indoxyl and isatin (II), and of tryptanthrin from anthranillic acid and isatin (III). Reprinted with permission from A. V. Muruganandam et al. [118].

listed in Fig. 2.41. The measurements revealed that the taxa differ considerably in their chemical composition and the differences between the flavanons, flavones, and flavonols can help their precise classification [119]. The diuretic and cholagog effect of Ononis arvensis motivated the development of a new two-dimensional paper chromatographic and TLC method for the measurement of onion in its roots and aerial parts. Samples were dried, ground and extracted with methanol (70 per cent, w/v) for 2 h. The supernatant was diluted and used for TLC separation on a cellulose stationary phase. The first eluent was 3 per cent formic acid, and the second was n-butanol–acetic acid–water (4:1:5, v/v) for both TLC and paper chromatography. Spots were scraped off, extracted with methanol and the absorption was measued at 260 nm. It was found that the average ononin content in roots and aerial parts was 0.1530.0278 (per cent) and 0.4980.045 (per cent), respectively. Because of the simplicity, the method was

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Chapter 2 R2 O

R1

OH

1 2 3 4 5

R2

O

R1 OH OCH3 OCH3 OCH3 OCH3

OH R2 OH OCH3 OH OCH3 OH

R3 Ha Ha,b,c Ha,b,c OHa,b,c OCH3 b R2

6 7 8 9

R2

R3

OH OCH3 OCH3 OCH3

OCH3 OCH3 OCH3 OCH3

Ha Ha,b,c OHa OCH3b,c R2

O

R3 OH

OH

OH

O

R1

R2

R3

OH OH OCH3 OH OH OCH3

OH OCH3 OCH3 OH OCH3 OCH3

Ha Ha Ha OHa OCH3a OHa R2

O

R1

16 17 18 19 20 21 22

O

R1

R2

R3

OH OH OCH3 OCH3 OH OH OCH3

OH CH3 OH OCH3 OH OH OH

Ha Ha Ha Ha OHa OCH3a OCH3a

R3 OH

CH3O OH

a

O

R3

CH3O

23 24 25 26 27 28

R3

R1

R1

O

R1

10 11 12 13 14 15

O

R1

R3

O

R1

R2

R3

OH OH OH OCH3 OH OCH3

OH OCH3 OH OH OCH3 OH

Ha Ha OCH3a OCH3a OCH3a Ha

identified in ssp. attenuata, bidentified in ssp.angustifolia, cidentified in ssp. tomentosa

Fig. 2.41. Flavonoid aglycones identified in flowerheads of Arnica angustifolia. Reprinted with permission from T. J. Schmidt et al. [119].

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proposed for the detrmination of ononin in the extracts of roots and aerial parts of Ononis arvensis L. [120]. A new HP-TLC method has been applied for the quantitative analysis of flavonoids in Passiflora coerulea L. The objective of the experiments was the separation and identification of the compound(s) responsible for the anxiolytic effect of the plant. Samples were extracted with 60 per cent ethanol or refluxed three times with aqueous methanol, and the supernatants were employed for HPTLC analysis. Separation was performed on a silica layer prewashed with methanol and pretreated with 0.1 M K2HPO4, the optimal mobile phase composition being ethyl acetate–formic acid–water (9:1:1,v/v). It was established that the best extraction efficacy can be achieved with 60 – 80 per cent aqueous methanol. The HPTLC technique separates 10 different flavonoids, which can be used for the authenticity test of this medicinal plant [121]. Another HPTLC method has been developed for the separation of kaempferol and quercetin in the extract of Ginkgo biloba leaves showing beneficial effect in brain diseases. Leaves were refluxed with methanol for 30 min then filtered. The filtrate was refluxed with 25 per cent HCl for 60 min then neutralized with ammonia and the clear supernatant was applied for HPTLC. Silica plates were predeveloped in chloroform–methanol (1:1, v/v). Separation was performed with toluene–acetone–methanol–formic acid (46:8:5:1, v/v) as the mobile phase using incremental multiple development. A densitogram illustrating the good separation charactersitics of the system is shown in Fig. 2.42. The relative standard deviation (RSD) of the method was low (1.37 and 1.40 for kaempferol and quercetin,

Analysis a: c 100

80 5

[mV]

60

40

3

1

4

20 2

0 10 Wavelength: 254 nm

20

30

40

50

60

[nm]

Fig. 2.42. HPTLC densitogram showing the separation of kaempferol (3) and quercetin (5) from other matrix components (1,2,4). Reprinted with permission from A. Jamshidi et al. [122].

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respectively), the recovery values varied between 94.11 – 97.06 per cent. Because of the good validation parameters this simple and selective technique has been proposed for the separation and quantitative determination of kaempferol and quercetin in the extract of Ginkgo biloba leaves [122]. HPTLC has also been applied for the separation and quantitative determination of flavonoids in the extracts of Vaccinium myrtillus L. and Vaccinium vitis-idaea L. They can be employed for the treatment of bacterial infection of the urinary tract, the inflammation of pancrease and for the reduction of blood glucose level. Extracts were prepared by boiling three times 10 g of air-dried and powdered leaves with 80 per cent methanol for 1 h. The collected extracts were concentrated, dissolved in hot water, filtered, washed with petroleum ether, and extracted with 3  10 ml of ethyl acetate. The organic phase was evaporated to dryness and redissolved in methanol. Separation was performed on silica layers using a gradient programme. The steps of the gradient programme are listed in Table 2.34. The amounts of flavonoids measured in the leaves of Vaccinium myrtillus L. and Vaccinium vitis-idaea L. are compiled in Table 2.35 [123]. A simple TLC method has been developed for the separation and identification of flavons and flavon glycosides in the extract of Phillyrea latifolia L. The leaves (100 g) were defatted in 1 l of chloroform for 24 h and then extracted with 2  1 l of ethanol–water (80:20, v/v). The collected extracts were concentrated and extracted again with n-hexane to remove chlorophylls and other apolar constituents. Analytes were extracted with ethyl acetate. Both normal phase and RP-TLC have been used for the separation of flavonoids. The results are compiled in Table 2.36. It was concluded from the data that TLC can be successfully applied for the quality control of plant extracts containing various flavone derivatives [124]. The leaf flavonoids of the cruciferous species such as Camelina sativa, Crambe abyssinica, Crambe hispanica, Thlaspi arvense, Brassica napus and Sinapis alba were separated and identified with the combination of HPLC, TLC and paper chromatography. Flavonoid aglycones were extracted by cutting fresh three-week-old leaves in tiny pieces and boiled in 50 ml of 2 M HCl for 45 min. After cooling the suspension was extracted with 3  50 ml of ethyl acetate. The combined extracts were evaporated to dryness and the residue was redissolved in 95 per cent of ethanol. TLC separation was performed on cellulose plates using n-butanol–acetic acid–water (4:1:5, v/v), HCl–acetic acid–water (3:30:10, v/v) and acetic acid–water (50:50, v/v) as mobile phases. RP-HPLC was carried out in ODS columns (250  9.4 mm i.d.; particle size 5 m and 150  4 mm i.d., particle size 5 m). Flavonoid aglycones were separated using different gradients consisting of water–ACN mixtures. Analytes were detected at 220, 254 and 300 nm. Flavonoid glycosides were extracted by boiling 100 g of fresh leaves in methanol for 10 min. The extract was evaporated to dryness and dewaxed with hexane. The solid residue was dissolved in water, filtered and washed three times with n-butanol. The butanol fractions were evaporated and the rest was dissolved in water. RPHPLC separation was performed similarly as in the case of flavonoid aglycones. HPLC fractions showing UV spectra characteristics for flavonoids were collected and separated by semi-preparative TLC as described above. Fractions were scraped, eluted and chromatographed again using two-dimensional TLC on a cellulose stationary phase (eluent 1: n-butanol–acetic acid–water, 4:1:5, v/v); eluent 2: 15 per cent acetic acid). The flavonoid composition in a range of Brassicaceae determined by TLC are compiled in Table 2.37.

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TABLE 2.34 THE TWO- AND THREE-STEP GRADIENT PROGRAMMES USED FOR THE ANALYSIS OF EXTRACTS FROM VACCINIUM MYRTILLUS L. AND VACCINIUM VITIS-IDAEA L. MIXTURE OF TOLUENE, HEXANE, ETHYL ACETATE, AND METHANOL WERE USED AS MOBILE PHASES

Step 1

Step 2

Step 3

60

50

50

30 10 3

25 25 3

25 25 3

55

50

60

25 20 9

40 10 9

30 10 9

60

50

30 10 9

25 25 9

Three-step gradient elution for quercetin (Stepwise gradient elution: hRF  52) Toluene–hexane 7:3 (% v/v)  0.1 ml formic acid Ethyl acetate (% v/v) Methanol (% v/v) Development distance (cm) Multiple gradient elution for hyperin (Plate dried after each step; final hRF  41) Toluene–hexane 7:3 (% v/v)  0.1 ml formic acid Ethyl acetate (% v/v) 2-propanol (% v/v) Development distance (cm) Multiple gradient elution for isoquercetin and avicularin (Plate dried after each step; final hRF  64) Toluene–hexane 7:3 (% v/v)  0.1 ml formic acid Ethyl acetate (% v/v) 2-propanol (% v/v) Development distance (cm) Reprinted with permission from H. D. Smolarz et al. [123].

It was stated that the chromatographic analysis of the flavonoid profile may help the determination of taxonomic relationships between these species [125]. The optimization of the separation of flavonoid glycosides of Mentha piperita (Laminaceae) was carried out on silica, amino, cyano and C18 HPTLC statinoary phases. The investigation was motivated by the spasmolytic, carminative and cholagogue characteristics of the plant. Air-dried and powdered leaves of peppermint (300 g) were extracted with methanol–water 1:1 v/v at ambient temperature. The suspension was filtered, concentrated to 200 ml acidified to pH 3 with formic acid and separated in an ODS column (400  40 mm i.d.; particle size 40 m).

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TABLE 2.35 COMPARISON OF THE HYPERIN, ISOQUERCETRIN AND AVICULARIN CONTENT, DETERMINED BY VIDEOSCANNING, OF LEAVES OF VACCINIUM MYRTILLUS L. AND VACCINIUM VITIS-IDAEA L.

Flavonoid

Species

Amount in dry leaves (g/g1)

Limit of detection (ng)

Hyperin

Vaccinium myrtillus L. Vaccinium vitis idaea L. Vaccinium myrtillus L. Vaccinium vitis idaea L. Vaccinium vitis-idaea L.

450 380 134 460 290

130 130 170 70 80

Isoquercetrin Avicularin Quercetin

Reprinted with permission from H. D. Smolarz et al. [123].

Polyphenols were eluted with various mixtures of water–methanol (8:2, 7:3, 6:4 and 5:5 v/v). Further purification was achieved on a cellulose layer, and on an ODS SPE cartridge. The TLC conditions used for the measurements are listed in Table 2.38. The RF values measured on the HPTLC plates are compiled in Table 2.39. Because of the good separation capacity chemically modified stationary phases were proposed for the rapid and economical analysis of peppermint polyphenols [126]. Because rutin and its aglycone show marked beneficial activities, such as antioxidant, antiinflammatory, antiallergic, antiviral and anticancer effects its measurement in various medicinal plants has been extensively studied. Leaves of Tephrosia purpurea (Fabaceae), Leptadenia reticulata (Asclepiadaceae) and Ruta graveolens (Rutaceae) were airdried, powdered and an aliquot of 1g was refluxed with 3  25 ml of methanol for 15min. The combined extracts were filtered and the volume was adjusted to 100 ml with methanol. Rutin was separated on silica HPTLC plates using ethyl acetate–n-butanol–formic acid–water (5:3:1:1, v/v) as the mobile phase. After development the plates were dried and scanned at 366 nm. Some densitograms are shown in Fig. 2.43. The densitograms show the good separation capacity of the method. The validation parameters were: instrumental precision 0.10 per cent, repeatability 0.937, limit of detection 40 ng/spot, limit of quantification 150 ng/spot, linearity 0.999 (correlation coefficient), range 150–750 ng/spot, intra-day precision 0.31 – 2.62, inter-day precision 0.78 – 5.60. The rutin content of medicinal plants (per cent, w/w) were: Tephrosia purpurea 4.650.225, Leptadenia reticulata 5.320.082, Ruta graveolens 2.610.0057. Because of the appropriate validation parameters, the application of this HPTLC technique was suggested for the sensitive and specific measurement of rutin in these medicinal plants [127]. Flavonol truxinix esters with possible myorelaxant activity were determined in Pseudotsuga menziesii using various TLC stationary and mobile phases. Because of chemotaxonomical interest the same investigations were carried out on 34 species from the family of Pinaceae. Dried and pulverized needles were exhaustively extracted with chloroform followed with methanol. The chloroform fraction was evaporated to 5 ml and

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TABLE 2.36 TLC DATA OF THE ISOLATED COMPOUNDS AND OF FLAVONE, FLAVONE GLYCOSIDE AND BIFLAVONE REFERENCE STANDARDS

Compound or reference

RF value for TLC system

standard

Compounsd 1 Luteolin-4-glucoside Compound 2 Apigenin-7-O-glucoside Compound 3 Luteolin-7-O-glucoside Compound 4 Apigenin Compound 5 Luteolin Amentoflavone (3
-8-biapigenin) Bartramiaflavone (biluteolin) Philonostiflavone (2
-8-biluteolin)

S1S1

S1S2

S1S3

S1S4

S1S5

S1S6

0.49 0.48 0.51 0.50 0.49 0.49 0.95 0.95 0.96 0.94 0.95

0.05 0.05 0.06 0.06 0.06 0.05 0.41 0.40 0.31 0.33 0.36

0.31 0.32 0.39 0.38 0.44 0.43 0.13 0.12 0.19 0.18 0.20

0.07 0.08 0.06 0.07 0.08 0.08 0.47 0.46 0.21 0.22 0.10

0.76 0.75 0.74 0.75 0.73 0.75 0.48 0.46 0.30 0.31 0.74

0.55 0.56 0.68 0.68 0.78 0.77 0.47 0.49 0.60 0.61 0.29

0.91 0.94

0.08 0.15

0.19 0.27

0.00 0.00

0.80 0.76

0.58 0.86

S1S1: silica stationary phase, eluent ethyl acetate–formic acid–water (6:1:1, v/v); S1S2: silica stationary phase, eluent toluene–ethyl formate–formic acid (4:4:1, v/v); S1S3: C18 stationary phase, eluent methanol–water–acetic acid (25:25:3, v/v); S1S4: silica stationary phase, eluent toluene–pyridine–formic acid (100:20:7, v/v); S1S5: cellulose stationary phase, eluent acetic acid–water (1:1, v/v); S1S6: C18 stationary phase, eluent methanol–water–acetic acid (78:25:3, v/v). Reprinted with permission from A. Pieroni et al. [124].

methanolic extract was evaporated to dryness, the residue was dissolved in 5 ml of hot water, and stored under refrigeration for 24 h, then it was filtered, evaporated again to dryness and redissolved in 5 ml of methanol. Separations were performed on diol and RP-18 plates using multiple gradient development. Some densitograms are shown in Fig. 2.44. The best separation was achieved by using 2D TLC on coupled silica and RP-18 stationary phases illustrating the advantages of multiple development strategies for the analysis of microcomponents in complicated plant matrices [128]. The separation characteristics of unmodified silica, diol, cyano, cellulose and RP-18 stationary phases were compared for the analysis of ellagitannins, gallotannins and flavonoid glycosides in the aerial parts of Erodium cicutarum (Geraniaceae) (stork’s bill). Its decoction has been used in cases of dysentery, fever, wounds and worm infections. The air-dried and powdered plant material (300 g) was extracted three times with water–acetone (1:1, v/v) at ambient temperature. The collected extract was filtered and concentrated. The aqueous phase was acidified to pH 3 with formic acid and separated in an ODS column. Polphenols

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TABLE 2.37 FLAVONOID COMPOSITION IN A RANGE OF BRASSICACEAE USING TLC

Plant species

Flavonolsb

Varieties,

Flavonesb

accessions or cultivarsa Isorhamnetin Kaempferol Quercetin Luteolin Crambe hispanica var. hispanica Cambe abyssinica Cambe hyspanica var. glabrata Camelina sativa (false flax) Brassica napus (oilseed canola) Brasica oleracea Brassica oleracea

Apigenin

2 acc. 1 acc. 2 acc. 2 acc.

   

   

   

   

   

22 acc.











Excel Westar Quantum (red glaze, kale) (broc 3, green glaze broccoli) 1 acc. Ochre

   

   

   

   

   











 

 

 

 

 









































Sinapis arvense Sinapis alba (white mustard) Eruca vesicaria subsp. sativa (garden rocket) Raphamus sativus Rauola (radish) Capsella bursa pastoris (shepherd’s burse) Descurainia sofia (flixweed)

, present; , not detectable. a Saskatoon Research Centre collection. b Indicated flavonoid aglycones found in acid-hydrolysed extracts. Reprinted with permission from J. Onyilagha et al. [125].

were eluted with water–methanol (9:1, 8:2; 7:3, 6:4 and 5:5, v/v). Further purification was obtained in an Sephadex LH-20 column eluted with methanol or acetone–methanol mixtures (9:1, 8:2, 7:3, v/v). The mobile phases employed for the HPTLC separation of flavonoids and tannins were n-hexane–acetone 1:1, v/v (cyano stationary phase); diisopropyl ether–acetone–water–98 per cent formic acid 60:30:5:5, v/v (diol); diisopropyl

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TABLE 2.38 CONDITIONS USED FOR CHROMATOGRAPHY

Stationary phase

Mobile phase composition

Proportions (v/v)

Compounds analyseda

HPTLC NH2 HPTLC RP-18W HPTLC CN

Acetone–glacial acetic Water–methanol n-hexane– tetrahydrofuran Diisopropyl ether– acetone–water–98% formic acid Diisopropylether– acetone–water–98% formic acid Ethylacetate–formic acid–glacial acetic acid Toluene–ethyl formate– formic acid–water

8515 6040 6040

Flavonoids Ca, Ra Flavonoids Ca, Ra Ca, Ra

50301010

Caffeetannins

50301010

Caffeetannins

100111126

Flavonoids Ca, Ra

51001010

Caffeetannins

HPTLC Si 60

TLC Si 60

TLC Si 60 TLC Si 60

a

Ca, caffeic acid; Ra, rosmarinic acid. Reprinted with permission from I. Fecka et al. [126].

TABLE 2.39 COMPARISON OF THE RF VALUES OF PEPPERMINT POLYPHENOLS ON HPTLC PLATES

RF on:a

Compound

Caffeic acid Rosmarinic acid Eriocitrin Hesperidin Luteolin-7-O-rutinoside Diosmin a

NH2, I

RP-18W, II

CN, III

Si 60, IV

0.77 0.09 0.29 0.48 0.23 0.42

0.49 0.37 0.42 0.26 0.19 0.26

0.40 0.19 0.03 0.03 0.03 0.03

0.76 0.68 0.15 0.17 0.14 0.16

Stationary phases (all HPTLC plates and mobile phases (I,II,III, or IV). Reprinted with permission from I. Fecka et al. [126].

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d

1000 900 800 700 600 500 400 300 200 100 0

c

b

a 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Fig. 2.43. Densitometric thin-layer chromatograms of (a) rutin standard, and resolution of rutin in (b) Tephrosia purpurea, (c) Leptadenia reticulata, and (d) Ruta graveolens; scanning was performed at 366 nm. Reprinted with permission from V. P. Kumar et al. [127].

6,7,8 10,5 9

4 1

5 1 0

−0.000 0 [mV]

4

−0.025

3 2

10

−0.060 3 −0.110

−0.075

(a)

(b)

3

6,2

6,8

−0.100 10.16

4 10

−0.050

20.00 40.00 60.00 80.00 100.00 mm Migration distance

2

2

20.00 40.00 60.00 80.00 100.00 mm Migration distance

−0.160 3.40 (c)

20.00 40.00 60.00 80.00 100.00 mm Migration distance 1

1

0.31

[mV]

0.21 0

1

1

1

2 3

0.21

4 7 10 5

2 0

9 6

8

0

0

7

0.21

4 10

0.11

3 6

8

2 3

4

10 6

8

0.010 -0.050

(d)

20.00 40.00 60.00 80.00100.00 mm Migration distance

(e)

20.00 40.00 60.00 80.00 100.00 mm Migration distance

(f)

20.00 40.00 60.00 80.00 100.00 mm Migration distance

Fig. 2.44. Densitograms obtained from flavonoids in extracts from: a, d, Pseudotsuga menziesii, B, E, Cedrus atlantica, c. f, Cedrus atlantica var. glauca. 1, hyperoside; 2, astragalin; 3, transtiliroside; 4, trans-ditiliroside; 5, daglesioside I; 6, daglesioside II; 7, daglesioside III; 8, daglesioside IV; 9, unknown flavonoid 2, 10, unknown flavonoid 1. Separations a, b and c were performed on RP-18 plates developed with mobile phase methanol–water–formic acid (70:30:6, v/v). Densitograms d, e and f were obtained on diol stationary phase using multiple gradient development (first step ethyl acetate–chloroform, 10:90, v/v, second step ethyl acetate–chloroform, 70:30, v/v). Detection at 366 nm. Reprinted with permission from M. Krauze-Baranowska et al. [128].

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TABLE 2.40 RESULTS FROM TLC ANALYSIS OF AQUEOUS (AQ) AND METHANOL (ME) EXTRACTS FROM THE EROIUM GENERA SPECIES INVESTIGATED

Compound

Geranin Dehydrogeranin Corilagin 3-galloylshikimik acid Methyl gallate 3-Oglucoside Rutin Hyperin Hyperin-6-gallate Isoquercitrin Luteolin-7-Oglucoside

E.ciconium

E.cicutarium

E.manescavi

E.pelargoniiflorum

Aq

Me

Aq

Me

Aq

Me

Aq

Me

   

   

   

   

   

   

   

   

















    

    

    

    

    

    

    

    

, absent; , present. Reprinted with permission from I. Fecka et al. [129].

ether–acetone–98 per cent formic acid 5:4:1, v/v (silica); water–methanol–98 per cent formic acid 49:50:1 or 69:30:1, v/v (RP-18). The results are compiled in Table 2.40. The data indicated that tannins, phenolic acids and flavonoids present in this class of medicinal plants are compounds with potentially promising biological activity [129]. 2.2.2.2 TLC separation of flavonoids in model systems and in miscellaneous matrices The study of the separation characteristics of model solutions of flavons and flavonoids and the elucidation of the quantitative relationships between the molecular structure and physicochemical parameters of solutes and their retention behaviour may promote the rational design of separation systems showing the highest possible efficacy. The RPHPTLC behaviour of coumarins and flavonoids has been studied in detail using methanol, 2-propanol, ACN, dioxane and THF as organic modifiers. Separation of model compounds was obtained on RP-18 plates and the relationship between the RM values and the concentration of the organic modifier was calculated by RM  RM0  m.C per cent where RM is the RM value of a solute in the presence of C concentration of organic modifier and RM0 is the RM value of the solute extrapolated to zero concentration of organic modifier related to the lipophilicity of the solute. The parameters of the equation achieved by methanol and 2-propanol organic modifiers are compiled in Table 2.41.

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TABLE 2.41 CONSTANTS FOR THE EQUATION RM  RM0  M.C%: R4°M FOR C  0 AND M THE ABSOLUTE VALUE OF THE SLOPE. THE CORRELATION COEFFICIENTS FOR THE PLOTS WERE BETWEEN 0.988 AND 0.999. SELECTIVITIES AS äRM0 RELATIVE TO 4-HYDROXYCOUMARIN

50–70% methanol and 0.01 M phosphate buffer (pH 4)

20–70% 2-propanol and 0.2 M phosphate buffer (pH 4)

No.

RM0

m

äRM0

RM0

m

äRM0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1.97 1.65 1.34 2.60 0.78 4.49 — 3.31 2.97 2.28 3.04 1.93 2.37 2.01 1.98 2.42 3.45 3.47 3.94 1.26

2.91 2.72 2.34 3.20 2.18 4.70 — 3.94 4.18 3.67 3.29 3.33 3.52 3.18 3.30 3.27 3.82 3.82 5.33 3.45

— 0.32 0.63 0.63 1.19 2.54 — 1.34 1.00 0.31 1.07 0.04 0.40 0.04 0.01 0.45 1.48 1.50 1.97 0.71

1.50 1.25 1.17 1.19 0.39 2.47 2.59 2.04 1.28 1.43 2.20 1.24 0.69 1.23 1.00 2.04 2.91 2.31 2.72 2.41

1.53 3.21 3.36 2.17 2.14 3.25 3.38 3.80 3.08 4.11 3.70 4.29 2.02 3.42 3.34 3.49 6.70 4.41 6.28 3.06

— 0.25 0.33 0.31 1.11 0.97 1.09 0.59 0.22 0.07 0.70 0.26 0.81 0.27 0.50 0.54 1.41 0.81 1.22 0.91

Compounds: 1  4-hydroxycoumarin; 2  umbelliferon; 3  4-methyl-esculetin; 4  isopimpinellin; 5  esculin; 6  flavone; 7  -naphtoflavone; 8  kaempferol; 9  quercetin; 10  isoquercitrin; 11  robinetin; 12  robinin; 13  myricetin; 14  luteolin 7-O-glucoside; 15  rutin; 16  hesperetin; 17  hespiridin 18  naringin; 19  pelargonin chloride; 20  polargonin chloride; 21  malvin chloride. Reprinted with permission from M. L. Bieganowska et al. [130].

The retention data and calculations demonstrated that RP-HPTLC can be applied for the efficient separation of coumarins and flavonoids. Furthermore, it was stated that the results make possible the rapid optimization of any separation problem [130]. The retention behaviour of the same set of coumarins and flavonoids was also investigated on polyamide and alumina layers using an aqueous mobile phase with various organic modifiers. The retention of solutes showed high variations on polyamide plates according

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TABLE 2.42 EQUATION CONSTANTS FOR RM  RMW  MC, RMW (INTERCEPT) AT C5  0 AND ABSOLUTE VALUES OF SLOPE (M) FOR ALUMINA AS ADSORBENT

30–70% acetonitrile No.

m

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

1.75 0.98 0.85 3.86 1.43 5.04 2.87 1.52 1.33 2.79 1.62 0.27 1.33 0.54 0.35 1.73 1.22 1.69 3.49 0.61 2.29

RMw 0.58 0.31 1.68 2.37 0.46 1.47 1.10 1.89 1.38 2.66 1.02 0.61 0.83 1.36 1.15 0.76 0.81 0.75 1.77 1.46 1.66

50–90% methanol

60–85% doixane

m

RMw

m

RMw

3.04 1.01 0.32 3.66 1.26 2.71 4 074 2 048 1 053 5.04 3.17 2.92 3.13 2.65 1.59 1.69 1.87 1.68 3.22 1.80 2.41

3.25 0.37 0.36 3.46 0.38 1.89 3.49 2.70 1.16 4.81 2.89 2.57 3.30 2.92 1.01 0.84 2.10 0.81 2.98 2.53 2.55

4.04 4.39 2.06 3.89 5.15 2.49 1.94 6.88 2.82 4.25 3.99 — — — 8.39 3.03 3.25 1.61 6.97 5.89 —

2.77 2.74 1.79 1.94 4.77 0.84 0.79 5.98 2.34 3.73 2.30 — — — 6.56 1.80 2.80 1.25 4.66 3.84 —

For identification of compounds see Table 2.41. Reprinted with permission from M. L. Bieganowska et al. [131].

to the type of organic modifier (methanol, 2-propanol, ACN, THF and dioxane) emphasizing the importance of the correct selection of organic modifier. Best separations were obtained with methanol and 2-propanol, THF showed the poorest separation capacity. The parameters of equations describing the dependence of the retention on the concentration of the organic modifier on an alumina stationary phase are compiled in Table 2.42. The coefficient of corrleation varied between 0.816 and 0.999. It was established that the selectivity of the alumina layer is higher than that of polyamide, therefore, its application for the separation of coumarins and flavonoids was proposed [131]. The adsorption characteristics of Florisil and silica were studied using a similar set of coumarins and flavonoids as in references [130] and [131]. The parameters of the equation describing the dependence of the RM value on the concentration of the stronger component in the binary mobile phase are listed in Table 2.43.

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TABLE 2.43 PARAMETERS OF THE EQUATION RM  RM0  m.LOG S FOR TLC ON FLORISIL WITH 70–90 PERCENT METHANOL, 75–95 PERCENT ISOPROPANOL, OR 75–95 PERCENT DIOXANE IN DICHLOROMETHANE AS MOBILE PHASE FOR TLC ON SILICA WITH 30–70 PERCENT ISOPROPANOL OR 40–80 PERCENT DIOXANE IN DICHLOROMETHANE AS MOBILE PHASE

No. of compound

Florisil RM0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Slope

MeOH

i-PrOH

DX

MeOH

i-PrOH

5.319 11.428 9.307 15.898 7.76 12.526 13.018 15.398 11.356 16.536 20.482 — — 12.869 13.219 9.655 17.51 — 17.47

10.375 8.086 — 13.608 11.373 13.08 11.016 — 11.062 18.215 18.23 — — 8.115 14.662 12.98 — — —

11.242 11.409 — 13.507 11.203 13.032 11.446 — 12.303 19.586 20.554 — — 5.041 9.143 10.198 — — —

2.951 6.337 4.227 7.114 4.201 5.498 7.131 — 5.635 9.566 8.193 — — 6.611 6.404 4.576 7.557 — 6.886

5.526 4.414 — 10.79 5.947 4.187 5.888 — 5.645 5.894 6.817 — — 4.066 6.981 6.148 — — —

No of compound

5.881 6.153 — 6.794 5.843 5.531 6.098 — 5.833 8.305 6.946 — — 2.417 4.532 4.683 — — —

Silica RM0

1 2 3 4 5

DX

Slope

i-PrOH

DX

i-PrOH

DX

0.716 0.802 3.593 2.298 2.186

1.58 1.89 5.287 3.251 3.719

0.525 0.716 1.376 1.031 1.344

0.980 1.288 2.239 1.468 2.144

(Continued on next page)

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TABLE 2.43 (continued)

No of compound

Silica RM0 i-PrOH

6 7 8 9 10 11 12 13 14 15 16 17 18 19

0.419 0.731 — 3.871 5.363 3.593 5.973 — 1.804 3.756 3.994 6.3 7.972 5.275

Slope DX

i-PrOH

DX

0.793 2.013 7.927 3.728 6.374 5.666 4.88 7.927 2.713 5.975 4.227 5.133 — 4.792

0.428 0.530 — 1.834 2.670 1.376 2.613 — 0.813 1.601 1.543 3.142 3.806 2.545

0.647 1.215 3.622 1.693 2.948 2.811 1.981 3.622 1.413 2.710 1.690 2.368 — 2.212

Reprinted with permission from A. Petrucznyk et al. [132].

It has been concluded from the data that both Florisil and silica can be applied for the TLC separation of coumarins and flavonoids. The best separations were obtained on Florisil stationary phase and methanol and 2-propanol as mobile phase additives [132]. The retention behaviour of 23 flavonoids was studied using different stationary and mobile phases. The compounds included in the experiments were: flavonols kaempferol, astragalin, robinin, tiliroside, quercetin, isoquercitrin, hyperoside, avicularin, rutin, myricetin, acacetin, apigenin, 7-glucoside of apigenin, luteolin, 7-glucoside of luteolin, iso-orientin, rhoifolin, naringenin, naringin, hesperitin, hesperidin. The RF values of the model compounds were determined on silica and diol stationary phases (mobile phases: ethyl acetate and methanol mixtures), on cyanopropyl silica (ethyl acetate and dichloromethane mixtures), aminopropyl silica (ethyl acetate, methanol and water mixtures), and ODS (methanol and water mixtures). It was established that the selectivity difference between diol and silica is relatively low, while the retention characteristics of silica and aminopropyl phases are highly different. It was further stated that these results can be used for the optimization of the separation of these classes of solutes by TLC [133]. The retention behaviour of flavonoids has also been extensively studied on silica stationary phases using heptane, benzene or dichloromethane as weaker components of the binary mobile phase and ethyl acetate and methyl ethyl ketone as modifier. Flavones (3-hydroxy, 5-hydroxy and 7-hydroxyflavone, tectochrysin, chrysin, apigenin, genkwanin, baicalein), flavonols (galangin, pilloin, kaempferol, rhamnetin, quercetin, robinetin,

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morin), flavanons (pinocembrin, naringenin) and pinostrobin chalcone were included in the experiments. It was found that the RM value generally decreases linearly with increasing concentration of organic modifier in the mobile phase. It has been stated that the results can be used for the rapid optimization of the separation of a given set of flavonoids [134]. The separation of an other set of flavonoids (apigenin, luteolin, kaemferol, quercetin, myricetin, rhamnetin, isorhamnetin, 3,8-biapigenin, gentisein, isogentisin and norathyriol) has been achieved by using silica plates with chloroform–acetone–formic acid (76:16.5:2.5, v/v) and toluene–ethyl acetate–formic acid (5:4:1, v/v) mobile phases [135]. A combined method was developed for the analysis of flavonoids using both RP-HPLC and normal-phase TLC. RP-HPLC analyses were carried out in an ODS column (150  4.6 mm i.d.; particle size 10 m). Isocratic mobile phases contained 60 or 50 per cent methanol and 0.1 per cent acetic acid. Gradient elution also consisted of a mixture of methanol and water. The flow rate was 1 ml/min and solutes were detected at 254 nm. The fractions were collected, evaporated to dryness and redissolved in methanol and applied on to silica HPTLC plates. The three step elution of plates was performed with 5, 10 and 15 per cent methanol in ethyl acetate containing 0.1 per cent of formic acid. A typical RPHPLC chromatogram is shown in Fig. 2.45. The measurements illustrated that neither NPTLC nor RP-HPLC can separate each of the solutes while their combined application resulted in the baseline separation of each component [136]. Microcrystallin cellulose triacetate (MCTA) has also been employed in the TLC analysis of flavanones. Plates were home-made by mixing 3 g of silica with 9 g of MCTA in 35 ml of ethanol and the suspension was spread on to 20  10 cm plates. A considerable number 50.0

10 9

40.0

6 7

[mV]

30.0

20.0 1 2 10.0

3,4

8 5

0.0

10.0 0.00

15.00

30.00

45.00

60.00

Time (min)

Fig. 2.45. Gradient elution chromatogram of flavonoids investigated. Peak identification: 1  naringin; 2  hesperidin; 3  quercitrin; 4  myricetin; 5  naringenin; 6  hesperetin; 7  luteolin; 8  apigenin; 9  flavone; 10  acacetin. Reprinted with permission from M. A. Hawryt et al. [136].

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Fig. 2.46. Densitogram of racemic naringenin on MCTA layers eluted with water–methanol (80:20, v/v). The development distance was 15cm; the separation time 2 h. 0.5 l (full line) and 1l (dashed line) of ()-naringenin solution (4 mg/ml). Reprinted with permission from L. Lepri et al. [137].

of compounds was investigated using aqueous solutions of ethanol, 2-propanol, and methanol–ethanol as mobile phases. The densitogram of naringenin is shown in Fig. 2.46. The densitogram clearly shows that the MCTA stationary phase is suitable for the separation of enantiomers. The retention data of the compounds investigated are complied in Table 2.44. The data indicated that MCTA can be successfully applied for the enantiomeric separation of a wide variety of racemic pairs using aqueous organic mobile phases [137]. The TLC analysis of flavonoids was performed not only in the extract of medicinal plants and model mixtures but also in various other matrices. Thus, phenolic compounds in red wines have also been determined by TLC. Wine samples were acidified to pH 2.0 with 0.1 M HCl and 25 ml of acidified wine was extracted with 2  25 ml of diethyl ether. The organic phase was evaporated to dryness and redissolved in 5.0 ml of methanol. Separation of phenolic compounds was performed on silica layers using 11 different mobile phases. In order to find the best separation system, information theory and cluster analysis was applied. The RF values determined in 11 mobile phases are compiled in Table 2.45. The measurements prove that the wines contain gallic acid, caffeic acid, apigenin, kaempferol, p-coumaric acid and naringenin. According to the mathematical statistical calculations the best mobile phase consisted of benzene–ethyl acetate–formic acid (30:15;5, v/v) [138]. TLC and HPLC have been employed for the investigation of the flavonoid composition of three genotypes of dry bean (Phaseolis vulgaris) differing in seedcoat colour. Extraction of pigments was carried out by soaking 100 g of beans in distilled water then separating the seedcoats from the cotiledon. The dried and ground seedcoats were extracted sequentially with hexane, ethyl acetate, methanol and methanol–water (1:1, v/v). TLC separations were performed on cellulose layers using butanol–acetic acid–water (4:1:5, v/v) as a mobile phase. Extracts were also separated with preparative RP-HPLC on in ODS column (250  10 mm i.d.; particle size 5 m). The composition of the isocratic mobile phase was ACN–water (3:7, v/v) at the flow rate of 1.0 ml/min. Analytical RP-HPLC was carried out with ACN–water (1:3, v/v) for quantification and with a gradient from ACN–water (1:9, v/v)

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TABLE 2.44 RETENTION (hRF1, hRF2)a AND RESOLUTION (, RS)b DATA FOR ENANTIOMERIC COMPOUNDS ON MICROCRYSTALLINE CELLULOSE TRIACETATE PLATES WITH SILICA GEL 60 GF254 AS BINDER (TEMPERATURE 25°C)

Compound

Eluent

hRF1

hRF2



RS

Alfametrin Fenpropathrin Fenoxaprop-ethyl Taxifolin Hesperetin Naringenin Flavanone 6-methoxyflavanone 6-hydroxyflavanone (4S,5R)/(4R,5S)-4-methyl-5phenyl-2-oxazolidone (4R,5S)/(4S5R)-1,5-dimethyl-4phenyl-2-imidazolidone trans-4-chlorostilbene oxide 2-phenylcycloheptanone 1-(9-fluorenyl)ethanol N-benzylproline ethyl ester gamma-(trityloximethyl)gamma-butyrolactone 2,3-O-isoprpylidene1-1-4-4-tetra-henylthreitol 2-methyl-1-indanone 3-methyl-1-indanone Troger’s base N-tBOC-3-(2-naphtyl)-Ala

80:20 c 80:20 c 80:20 d 80:20 c 80:20 e 80:20 e 80:20 c 80:20 c 80:20 c 80:20 e

23 30 36 44 23 23 22 24 36 62

29 34 46 48 27 28 24 27 39 72

1.37 1.20 1.52 1.17 1.24 1.30 1.12 1.17 1.14 1.58

1.7 1.2 2.2 1.3 1.5 1.6 0.4 0.8 0.8 2.6

80:20 e

75

86

2.06

2.5

80:20 d 60:40 d 80:20 d 40:60 d 70:30 d

25 17 26 19 48

40 31 44 22 50

2.00 2.20 2.24 1.20 1.08

4.3 3.5 3.0 1.0 0.4

50:50 d

18

20

1.14

0.7

80:20 e 80:20 e 80:20 c 80:20 c

50 52 23 69

57 58 44 72

1.33 1.28 2.64 1.16

1.8 1.6 3.6 0.8

RF  100.   (1/RF1  1)/(1/RF2  1). RS  2  (distance between the centres of two adjacent spots)/(sum of the width of the two spots in the direction of development). c Ethanol–water; migration distance 12 cm; separation time 3 h. d 2-propanol–water; migration distance 14 cm; separation time 6 h. e Methanol–water. migration distance 16 cm. separation time 2.5 h. Reprinted with permission from L. Lepri et al. [137]. a

b

to ACN–water (9:1, v/v) in 20 min, the total analysis time being 40 min. TLC investigations indicated the presence of flavonoid polymers (condensed tannins) while RP-HPLC measurements proved that astragalin is the most important pigment component in the seedcoats of dry beans [139].

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TABLE 2.45 RF VALUES OF THE PHENOLIC COMPOUNDS

Phenolic compound

Phenolic compound I Phenolic compound II Phenolic compound III Phenolic compound IV Gallic acid Phenolic compound V Caffeic acid Kaempferol Apigenin p-coumaric acid Naringenin

RF for mobile phase 1

2

3

4

5

6

7

8

9

10

11

0.04 0.07 0.13 0.23 0.27 0.43 0.39 0.31 0.44 0.53 0.52

0.11 0.17 0.25 0.32 0.39 0.43 0.48 0.41 0.52 0.58 0.57

0.11 0.15 0.28 0.48 0.51 0.52 0.66 0.70 0.72 0.73 0.75

0.05 0.08 0.11 0.23 0.36 0.53 0.59 0.73 0.69 0.77 0.83

0.05 0.07 0.12 0.18 0.25 0.49 0.60 0.70 0.77 0.78 0.82

0.08 0.09 0.11 0.22 0.30 0.54 0.64 0.74 0.76 0.80 0.84

0.06 0.07 0.09 0.20 0.25 0.36 0.41 0.30 0.40 0.48 0.46

0.06 0.08 0.11 0.22 0.28 0.61 0.68 0.76 0.81 0.84 0.88

0.09 0.11 0.13 0.25 0.35 0.71 0.76 0.81 0.86 0.85 0.89

0.11 0.20 0.27 0.43 0.51 0.74 0.71 0.83 0.80 0.83 0.90

0.08 0.12 0.22 0.47 0.54 0.72 0.76 0.84 0.86 0.85 0.89

Mobile phase composition: 1  toluene–ethyl acetate–formic acid (30:10:10, v/v); 2  toluene–ethyl acetate–formic acid (55:20:25, v/v); 3  toluene–acetone–formic acid (7:6:1, v/v); 4  benzene–ethyl acetate–formic acid (30:15;5, v/v); 5  chloroform–methanol–formic acid (15:3:2, v/v); 6  chloroform–methanol–formic acid (147:30:23, v/v); 7  Toluene–chloroform–acetone–formic acid (8:4:3:3, v/v); 8  chloroform–methanol–formic acid (37:8:5, v/v); 9  chloroform–methanol–formic acid (36:9:5, v/v); 10  ethyl acetate–chloroform–formic acid (24:21:5, v/v); 11  ethyl acetate–chloroform–formic acid (23:21:6, v/v). Reprinted with permission from V. Rastija et al. [138].

The differences between the pigment composition among white carnation (Danthus caryophyllus L.) cultivars have been investigated by two-dimensional thin-layer chromatography. Pigments were extracted from the petals of fully opened flowers of 13 cultivars by mixing 10 g of petals (fresh weight) with 200 ml of methanol at ambient temperature. The extract was filtered, evapoated to dryness and redissolved in 50 ml of water. The aqueous phase was extracted with petroleum ether, followed by ethyl acetate. The organic and aqueous phases were separately evaporated to dryness, and dissolved in 3 ml of methanol. Two-dimensional TLC separation was performed on cellulose layers using n-butanol–acetic acid–water (6:1:2, v/v) and 2 per cent acetic acid as first and second mobile phases, respectively. Flavonoids separated by two-dimensioal TLC were isolated, hydrolyzed in 2 N HCl at 100°C for 1 h, then extracted with diethyl ether. The organic phase was evaporated to dryness and dissolved in 99 per cent of methanol. The extracts were separated with TLC using n-butanol–acetic acid–water (4:1:5, v/v; upper phase) and 30 per cent acetic acid. Two-dimensional TLC revealed the presence of 17 separate compounds. The results of the two-dimensional TLC measurements are compiled in Table 2.46.

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TABLE 2. 46 ORGANIC ACIDS. FLAVANONES AND FLAVANOLS IN THE PETALS OF 13 WHITE CARNATION CULTIVARS

Cultivar

California White Sonneto Silky U Conn Sim Swan Albivette Florence Delphi Tobia Bagatel White Lucena Kaly White Barbara White Mind

Flower colour

White White White White White White White White White White Pure white Pure white Pure white

Pigment constituents Organic acid

Flavanones (naringenin)

Flavonol (kaempferol)

            

            

          ± ± 

, present; , absent; , a very small amount present. Reprinted with permission from T. Onozaki et al. [140].

It was concluded from the data that cultivars form three groups according to the composition of pigments, which reflects the differences between the flavonoid biosyntheses [140]. Flavonoids in propolis have also been investigated by TLC using a silica stationary phase and various mobile phases. Samples of 14 propolis with different geographical origin were extracted with 80 per cent ethanol and analysed with normal-phase TLC. The retention data are compiled in Table 2.47. Using numerical taxonomy it was found that the best separations were obtained by chloroform–methanol–(98–100 per cent) formic acid (44.1:3:2.35) and n-hexane–ethyl acetate-glacial acetic acid (31:14:5, v/v) as mobile phases. As the flavonoid profile of the propolis samples showed considerable differences, the method has been proposed for the authenticity test and traceability of various propolis products [141]. TLC has been applied for the control of the synthesis of new 8-C-glucosylflavones such as orientin, parkinsonin A, isoswertia-japonin, parkinsonin B, 5-methyl orientin, 7-methyl orientin and 5,7-dimethylorientin. The purity of the products were checked on a silica stationary phase using hexane–ethyl acetate (5:1 and 3:1, v/v), and acetone–ethyl acetate–water–acetic acid (25:35:5:1, v/v) as mobile phases [142].

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TABLE 2.47 RF VALUES OF PHENOLIC ACID AND FLAVONOID STANDARDS

Standard

Phenolic acids o-coumaric acid p-coumaric acid Caffeic acid Ferulic acid Flavonoids Flavanone Naringenin Flavone 3-hydroxyflavone 6-hydroxyflavone 6
-hydroxyflavone 7-hydroxyflavone 3,6-dihydroxyflavone 3,7-dihydroxyflavone Morin Chrysin Quercetin Galangin Apigenin Kaempferol

Mobile phase 1

2

3

4

5

6

7

8

9

10

11

0.55 0.55 0.38 0.56

0.38 0.36 0.26 0.32

0.51 0.51 0.30 0.49

0.51 0.49 0.33 0.49

0.37 0.34 0.22 0.28

0.48 0.47 0.34 0.45

0.73 0.69 0.43 0.63

0.75 0.75 0.62 0.70

0.28 0.22 0.23 0.28

0.47 0.42 0.25 0.41

0.34 0.34 0.22 0.51

0.67 0.54 0.88 0.77 0.67 0.52 0.46 0.54 0.54 0.23 0.62 0.39 0.65 0.44 0.51

0.40 0.37 0.62 0.51 0.39 0.32 0.30 0.36 0.36 0.16 0.38 0.27 0.44 0.33 0.37

0.62 0.58 0.92 0.80 0.61 0.46 0.42 0.51 0.50 0.14 0.60 0.27 0.64 0.47 0.50

0.65 0.44 0.86 0.76 0.62 0.51 0.42 0.52 0.48 0.14 0.53 0.28 0.57 0.33 0.39

0.38 0.24 0.66 0.56 0.36 0.28 0.26 0.34 0.33 0.13 0.36 0.22 0.37 0.21 0.23

0.62 0.44 0.85 0.66 0.56 0.48 0.46 0.46 0.47 0.23 0.56 0.35 0.60 0.37 0.40

0.75 0.52 0.91 0.82 0.75 0.56 0.47 0.56 0.54 0.13 0.68 0.30 0.72 0.39 0.47

0.76 0.73 0.92 0.83 0.80 0.73 0.70 0.72 0.70 0.32 0.74 0.60 0.85 0.67 0.77

0.85 0.35 0.69 0.70 0.51 0.38 0.36 0.37 0.44 0.14 0.41 0.20 0.59 0.31 0.36

0.80 0.38 0.55 0.55 0.56 0.34 0.29 0.43 0.39 0.00 0.51 0.00 0.53 0.26 0.29

0.91 0.16 0.81 0.85 0.46 0.47 0.28 0.49 0.32 0.04 0.58 0.14 0.45 0.11 0.10

Composition of mobile phases: 1  toluene–ethyl acetate–(98–100%) formic acid (36:12:5. v/v); 2  cyclohexane–ethyl acetate–(98–100%) formic acid; 3  toluene–ethyl acetate–glacial acetic acid (36:12:5, v/v); 4  cyclohexane–ethyl acetate–glacial acetic acid (31:14:5, v/v); 5  nhexane–ethyl acetate–(98–100%) formic acid (31:14:5, v/v); 6  toluene–acetone–(98–100%) formic acid (38:10:5, v/v); 7  n-hexane–ethyl acetate–glacial acetic acid (31:14:5,v/v); 8  petroleum ether(40–70°C)–ethyl acetate–(98–100%) formic acid (30:15:5, v/v); 9  carbon tetrachloride–acetone–(98–100%) formic acid (35:10:5, v/v); 10  n-hexane–ethyl acetate–glacial acetic acid (30:20:1.5. v/v); 11  chloroform–methanol–(98–100%) formic acid (44.1:3:2.35, v/v). Reprinted with permission from I. Jasprica et al. [141].

Because of its importance, the application of planar chromatography for the analysis of various secondary metabolites in plants such as heterocyclic oxygen compounds (coumarins, flavonoids, anthocyanins, etc.) has been reviewed many times [143,144]. 2.2.3 High-performance liquid chromatography The analytical chemical methods including both TLC and HPLC employed for the determination of bioflavins have also been previously reviewed [145,146].

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2.2.3.1 HPLC determination of flavonoids in plant extracts Similarly to TLC a high number of HPLC technologies have been developed and employed for the separation and quantitative determination of the biologically active components in medicinal plants and in extracts of medicinal plants. Thus, the components of the extracts of medicinal herbs such as Rhizoma chuanxiong and Gingko biloba have been separated by RP-HPLC and information theory has been applied to the chromatographic fingerprint. Samples of R. chuanxiong were dried, ground and an aliquot of 0.5 g was homogenized with 30 ml of methanol for 2 min. The suspension was centrifuged, the liquid phase was filtered, concentrated to 1 ml, and diluted to 5 ml with methanol. G. biloba products were extracted with 20 ml of methanol for 15 min, filtered, evaporated to 1 ml and diluted to 5 ml with methanol. RP-HPLC analyses of R. chuanxiong samples were performed in an ODS column (200  4.6 mm i.d.) using two gradients, eluents A and B being methanol and water–K2HPO4–H3PO4, pH 3, respectively. Gradient 1 started with 40 per cent eluent A and 60 per cent eluent B, then it was changed to 80 per cent A 20 per cent B after 50min. Gradient 2 started with 10 per cent eluent A and 90 per cent eluent B, then it was changed to 100 per cent A after 60min. Flow rates were set to 0.7 and 1 ml/min, respectively. The column was thermostated at 25oC. The components of G. biloba products were separated in an ODS column (250  4 mm i.d.). The starting mobile phase was water–ACN–isopropanol–citric acid (1000:200:30:4.92, w/w) and then it was linearly changed to water–ACN–isopropanol–citric acid (1000:470:50:6.08, w/w) after 25 min. The flow rate was 1.0 ml/min. Typical chromatograms of extracts are shown in Fig. 2.47. The chromatograms show the good separation capcacity of the RP-HPLC methods. It was stated that information theory employed for the chromatographic profile can be used for the quality control of medicinal herbs and extracts [147]. The well-known beneficial effect of Cynara scolymus (Asteraceae) (bile and liver diseases, dyspepsic syndrome, reduction of blood cholesterol) motivated the exact measurement of its bioactive components. The dried extracts (80 mg) were dissolved in 2 ml of 15 per cent (v/v) aqueous methanol, sonificated for 5min then filtered before injection. RP-HPLC was carried out in an ODS column (250  2 mm i.d.; particle size 5 m). Components of the gradient elution were 0.03 per cent aqueous TFA solution (A) and ACN (B). The gradient started with 90A – 10B and changed to 64A – 36 B in 35 min. The column temperature was 25oC, the flow rate 0.2 ml/in. MS detection was performed in ion-negative ESI mode, with spray voltage 3.5 kV, capillary temperature 200°C. Data were recorded between 350 – 1000 m/z. The chromatographic profile of an extract at 330 and 210 nm is shown in Fig. 2.48. The compounds were well separated under the RP-HPLC conditions proving the advantage of the use of a narrow-bore column. The quantity of monocaffeoylquinic acids, dicaffeoylquinic acids, and flavonide found in 12 extracts are compiled in Table 2.48. The validation parameters of the method such as linearity, precision, accuracy, specificity and robustness were good, therefore, the method has been proposed for commercial control laboratories [148]. The active components of the herbaceaous perennial plant Hypericum perforatum are antiinflammatory, antidepressive and healing agents, therefore, their analysis is of considerable importance for health care. Samples were prepared by extracting the dried flowering tops by hot methanol. RP-HPLC separations were performed in an ODS column (250  4.6 mm i.d.; particle size 5 m) thermostated at 30°C. The steps of gradient elution are listed in Table 2.49.

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400 2 mAU

300 200 100 0

1 0

10

20

(a)

30 40 50 Retention time (min)

400

60

70

60

70

2'

mAU

300 200 1'

100 0

0

10

20

(b)

30 40 50 Retention time (min)

Au

0.1 0.05

(c)

0

0

5

10

15

20

25

0

5

10

15

20

25

0

5

20

25

Au

0.1 0.05 0

(d)

Au

0.1 0.05 0

(e)

10 15 Retention time (min)

Fig. 2.47. Chromatographic fingerprints from Rhizoma chuanxiong using gradient elution 1 (a) and 2 (b). Chromatographic fingerprints of a standard extract (c), one extract (d) and one product (e) of Gingko biloba at 360 nm. Peak 1 represents luteolin. Reprinted with permission from F. Gong et al. [147].

A typical chromatogram representing the separation of Hypericum perforatum extract with the HPLC-MS attributions of the components detected is shown in Fig. 2.49. The validation parameters of the method (linearity, stability, reproducibility of the injection integration and repeatability, and robustness) were acceptable, therefore, it was found suitable for routine analysis of the composaition of the extracts of Hypericum perforatum [149]. The flavonoid content of the tinctures of Calendula officinalis L., Passiflora incarnata L., and Silybum marianum (L.) Gaertn. was investigated by HPLC-DAD and HPLC-MS. The anti-inflammatory effect, and the beneficial influence to treat hepatic injuries, tension

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3

700

330 nm

600

mAU

500 400 300

4

200

9

2

100

5

6 7

20

25

1

8 10

0 (a)

5

10

15

30

35

40

210 nm 800

mAU

600 400 200 11

0 10

(b)

5

10

15

20

25

30

35

40

Time (min)

Fig. 2.48. Separation of an extract by HPLC under optimized conditions in a 250 mm  2 mm i.d., 5 m particle, C18 column. The mobile phase was a gradient prepared from 0.03 per cent TFA in water (a) and acetonitrile (b): mobile phase composition (%) was changed from 90a:10b to 64a:36b in 35 min. The flow rate was 0.2 ml/min, the temperature 25°C, and detection was performed at 210 and 330 nm. Peak assignments: 1  pseudochlorogenic acid; 2  neochlorogenic acid; 3  chlorogenic acid; 4  cryptochlorogenic acid; 5  cynarin; 6  cynaroside; 7  scolymoside; 8  3,4-di-Ocaffe- oylquinic acid; 9  1,3-di-O-caffeoylquinic acid; 10  4,5-di-O-caffeoylquinic acid; 11  cynaropikrin. Reprinted with permission from M. Hausler et al. [148].

and difficulty of falling asleep motivated the investigation. Samples were prepared by extracting 200 g of dried flowers of calendula, dried flowering tops of passion flower, and dried fruits of milk-thistle with 1 000 ml of 40 and 60 per cent (v/v) ethanol. TLC analyses were carried out on silica plates using ethyl acetate–formic acid–acetic acid–water (100:11:11:26) as the mobile phase for calendula and passion flower extracts, and chloroform–acetone–formic acid (75:16.5:8.5, v/v) for milk-thistle tincture. RP-HPLC-DAD was performed in an ODS column (250  4 mm i.d.; particle size 5 m) thermostated at 26°C. The steps of gradient elution are listed in Table 2.50. The pH of the mobile phase (pH 3) was held constant with H3PO4.

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TABLE 2.48 AMOUNTS OF MONOCAFFEOYLQUINIC ACIDS (MNOCQA), DICAFFEOYLQUINIC ACIDS (DICQA), AND FLAVONIDES (FLAV) IN DRIED ARTICHOKE EXTRACTS. RESULTS ARE EXPRESSED AS PER CENT (%)SD; ALL SAMPLES WERE INJECTED IN TRIPLICATE

Extract 1 Extract 2 Extract 3 Extract 4 Extract 5 Extract 6 Extract 7 Extract 8 Extract 9 Extract 10 Extract 11 Extract 12

MNoCQA

DiCQA

Flav

1.280.02 1.660.06 1.360.01 1.090.02 0.750.00 0.910.00 0.480.00 0.660.01 2.550.01 4.240.04 3.600.02 2.060.02

0.430.01 0.030.0 0.410.00 0.440.00 0.320.01 0.450.00 0.200.00 0.420.01 0.200.00 0.260.00 0.100.00 0.100.00

0.070.00 0.130.00 0.160.00 0.130.00 0.090.00 0.110.00 0.060.00 0.090.01 0.260.00 0.520.0 0.270.00 0.270.00

Reprinted with permission from M. Hausleret et al. [148] TABLE 2.49 LINEAR GRADIENT PROGRAMME FOR HPLC ANALYSIS

Time (min)

Solvent A (%)a

Solvent B (%)b

Solvent C (%)c

Initial 10 30 40 55 56 65

100 85 70 10 5 100 100

0 15 20 75 80 0 0

0 0 10 15 15 0 0

Solvent A  water–85% phosphoric acid (99.7:0.3, v/v). Solvent B  acetonitrile. c Solvent C  methanol. Reprinted with permission from M. Brolis et al. [149]. a

b

Solutes were tentatively identified by atmospheric pressure ionization (API)–electrospray– mass selective detector (gas temperature 350°C, flow rate 10 l/min, nebulizer pressure 30 psi, quadrupole temperature 30°C, capillary voltage 3 500 V).

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4

5

0.025 1

AU

0.020

10 0.015

6 2 15

3 0.010

7

11 8

13 14 12

9

0.005

16

0

20

40

60

Min

Fig. 2.49. Profile of Hypericum perforatum extract with the HPLC-MS attributions of the components detected. 1  chlorogenic acid isomer; 2  3-O-p-coumaroylquinic acid; 3  chlorogenic acid; 4  rutin; 5  hyperoside; 6  isoquercitrin; 7  3,3
,4
,5,7-pentahydroxyflavanone 7-Orhamnopyranoside; 8  quercitrin; 9  quercetin; 10  I3,II8 biapigenin; 11  pseudohypericin; 12  hypericin; 13  hyperforin analogue; 14  hyperforin analogue; 15  hyperforin; 16  adhyperforin. Reprinted with permission from M. Brolis et al. [149]. TABLE 2.50 COMPOSITION OF THE MOBILE PHASE USED FOR THE HPLC-DAD ANALYSIS

Time (min)

Solvent A (%)a

%ACN

Flow (ml/min)

0.10 10.00 15.00 30.00 35.00 42.00 50.00

88.0 82.0 82.0 55.0 0.0 0.0 88.0

12.0 18.0 18.0 45.0 100.0 100.0 12.0

1.30 1.30 1.30 1.30 1.30 1.30 1.30

a

H2O Reprinted with permission from A. R. Bilia et al. [150].

Chromatographic conditions were the same as for HPLC-DAD, only orthophosphoric acid was replaced by formic acid. Chromatographic profiles of calendula, milk-thistle and passion flower tinctures (each 60 per cent v/v, ethanol) are shown in Fig. 2.50. Analytes were well separated under the

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3m 600

500

500

400

400

300

3c

3p 600 7p

500 5m−6m 7m 8m

300

5p

400 mAU

600

mAU

mAU

6c

300 4m

200

4p

200

200

2m 100

5c 2c 4c 1c

7c

6p

100

100

0 10

20

2p

1m

0

(a)

1p

30

Time (min)

40

0 10

(b)

20

30

Time (min)

10

40 (c)

20

30

40

Time (min)

Fig. 2.50. Profiles of calendula (a), milk-thistle (b) and passion flower (c) tinctures (each 60 per cent v/v, ethanol) with the HPLC-MS attributions of the components detected. 1c  quercetin-3-O-rutinosylrhamnoside; 2c  rutin; 3c  isorhamnetin-3-O-ruti-nosylrhamnoside; 4c  isoquercitrin; 5c  isorhamnetin-3-O-gluco-sylglucoside; 6c  narcissin; 7c  isorhamnetin-3-O-glucoside; 1m  taxifolin; 2m  siliydianin; 3m  silychristin; 4m  oxy derivative of silybin/isosilybin isomers; 5m and 6m  2,3-dehydro derivatives of silybin/isosilybin isomers; 7m  silybin; 8m  isosilybin; 1p  6,8-diC-glucosylapigenin; 2p  isoschaftoside; 3p  shaftoside; 4p  homoorientin; 5p  isovetexin-2-O-glucoside; 6p  vitexin. Reprinted with permission from A. R. Bilia et al. [150].

RP-HPLC conditions proving the good application parameters of the technique. The quantitative data are compiled in Table 2.51. Because of the acceptable accuracy, specificity and reproducibility, the method was proposed for the analysis of other herbal drugs too [150]. The high sensitivity of hyphenated techniques such as HPLC-MS has also been exploited in the identification and structural studies of flavonoid glucosides [151] and the application of other hyphenated techniques such as LC-MS-MS and LC-NMR for the analysis of plant constituents has been discussed earlier [152]. A new poly(7-oxobornene-5,6-dicarboxylic acid-block-norbornene)-coated silica has been synthesized and applied for the separation of flavonoids in model systems and in the extracts of onion, elder flower blossom, lime blossom, St. John’s Wort and red wine. Separation was performed in a (150  4 mm i.d.; particle size 7 m) column at room temperature. Flavonoids (quercitrin, myricetin, quercetin, kaempferol and acacetin) were separated with gradient elution: water–ACN (20 mmol TFA) from 78:22 to 70:30 v/v in 3min. The flow rate was 2 ml/min. The separation of the standard mixture is shown in Fig. 2.51. It has been stated that the method is rapid, accurate and the MS detection makes possible the reliable identification of flavonoids [153]. RP-HPLC found application in the separation and identification of the main flavonoids in weld (Reseda luteola L.). The aerial parts of the weld were dried, ground and extracted with various solvents and solvent mixtures such as methanol, ethanol, water, methanol–water

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TABLE 2.51 LIST AND AMOUNTS (MG/ML; MEANSSD OF RESULTS FROM THREE REPLICATE ANALYSES) OF FLAVONOLS PRESENT IN CALENDULA. FLAVOLIGNANS IN MILK-THISTLE AND FLAVONS PASSION FLOWER TINCTURES

Calendula tinctures Quercetin-3-rutglc Rutin Isorhamnetin-3-rutglc Isoquercitrin Isorhamnetin-3-glcglc Narcissin Isorhamnetin-3-glc Total flavonols

60% v/v tincture 2.10.08 4.70.23 16.60.52 2.00.06 7.10.60 21.40.72 3.20.11 57.1

40% v/v tincture 1.80.09 4.00.29 18.60.80 1.60.10 9.40.64 20.40.86 2.60.13 58.4

Milk-thistle tinctures

60% v/v tincture

40% v/v tincture

Taxifolin Silydianin Silychristin Oxy derivative 2.3-dehydro derivatives Silybin Isosilybin Total flavanolignans

5.50.21 10.30.33 52.51.06 18.80.84 19.90.65 39.40.92 29.40.79 170.3

Passion flower tinctures

60% v/v tincture

40% v/v tincture

Passion flower tinctures Vicinin-2 Schaftoside/isoschaftoside Homoorientin Isovitexin-2-glc Vitexin Isovitexin Total C-glycosylflavones

17.00.58 59.61.48 25.60.89 36.11.14 18.30.89 58.91.45 215.5

14.21.01 47.51.56 21.71.21 26.51.02 15.00.97 39.81.23 164.7

2.90.17 4.80.28 31.50.99 9.60.60 13.80.55 13.50.46 15.40.70 88.6

Reprinted with permission from A. R. Bilia et al. [150].

(8:2, v/v) and ethanol–water (8:2, v/v). Extractions were performed at room temperature and at the boiling point of the solvent; the extraction time varied between 5 and 240 min. Flavonoids were separated in an ODS column (250  4.6 mm i.d.; particle size 5 m) at 25oC. Gradient elution started with methanol–1 per cent aqueous acetic acid (5:95, v/v). The concentration of methanol was increased to 40 per cent in 25 min, to 60 per cent in 40 min, and to 90 per cent in 50min (final hold, 5min). The flow rate was 1 ml/min and analytes were detected at 350 nm. The chromatographic profile of some extracts are presented in Fig. 2.52. The chromatograms

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169

6 1 2

4

3

mAU, 254 nm

5

0

0.5

1.5

2.5

3.5

4.5

5.5

[Min]

Fig. 2.51. LC of a flavone standard mixture. For chromatographic conditions see text. Peak identification: 1  quercitrin; 2  myricetin; 3  quercetin; 4  kaempferol; 5  acacetin. Reprinted with permission from C. W. Huck et al. [153].

demonstrate that both the quantity and composition of the extracted flavonoids markedly depend on the conditions of extraction. The quantitative results are compiled in Table 2.52. It has been concluded from the data that the optimal extraction conditions are methanol– water (8:2, v/v) as extracting agent, boiling temperature and 15min extraction time [154]. Besides their importance in health care, the analysis of flavonoids can contribute to the solution of taxonomical problems too. Thus, leaf flavonoids have been used as systematic characters in the genera Lavandula and Sabaudia. Fresh and dry leaves were mixed with 100 per cent and 70 per cent methanol, respectively, and heated at 70°C for 5min. Extraction time varied between 8h and overnight. Chlorophyll was removed with petroleum ether (40 – 60°C) and the rest was evaporated to dryness. The extracts were redissolved in 80 per cent metanol and investigated by two-dimensional paper chromatography, paper electrophoresis, preparative paper chromatography and RP-HPLC. Separation was carried out in an ODS column (250  4mm i.d.; particle size 5m). Analytes were separated by two different gradient elution programmes using mixtures of water, methanol and acetic acid. The Column was thermostated at 25°C and the flow rate was 1 ml/min. These combined chromatographic methods allowed the unambiguous or tentative identification of the following flavonoids: luteolin 7-O-glucoside (1), luteolin 7-O-glucuronide (2), chrysoeriol 7-O-glycoside (3), apigenin 7-O-glucoside (4), apigenin 7-O-glucuronide (5), derivative of 4 or 5 (6), luteolin 7,4
di-O-glucuronide (7), luteolin 7-O-glucoside-4
-O-glucuronide (8), 6-OH-luteolin 7-O-glycoside (9), scutellarein 7-O-glycoside (10), vitexin (11), hypoaletin 8-O-glucuronide (12), derivative of 12 (13), hypoaletin 4
-methyl ether 8-O-glucuronide (14), isoscutellarein

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Absorbance / mAU

170

Chapter 2 C

980 780 580

B

380 A

180 −20 0

5

10

15

20

25

Absorbance / mAU

(a) 980

35

40

45

50

55

60

C

Rutin

350 nm

780 580 B

380 A

180 −20 0

Absorbance / mAU

30 Time / min

5

10

15

20

25

(b)

30 35 Time / min

980

45

40

50

55

60

350 nm

Rutin

780 580

C

380 180

A B

−20 0

(c)

350 nm

Rutin

5

10

15

20

25

30

35

40

45

50

55

60

Time / min

Fig. 2.52. HPLC analysis of (a) a methanol extract, (b) a methanol–water extract and (c) a water extract. (A) Luteolin-3
7-diglucoside; (B) luteolin-7-glucoside; (C) luteolin. Reprinted with permission from D. Cristea et al. [154].

8-O-glucuronide (15), hypoaletin 7-O-glucoside (16), isoscutellarein 7-O-glycoside (17), apigenin (18), genkwanin (19), xanthomicrol (20), and salvigenin (21). The presence of flavonoids in species, subspecies and varieties of Lavandula and Sabaudia based on HPLC, PC and electrophoresis results is compiled in Table 2.53. It was concluded from the data that flavonoid patterns are systematically informative at the infrageneric level and may facilitate the taxonomic classification of these types of plant species [155]. Negative atmospheric pressure chemical ionization (APC) low-energy collision activation mss spectrometry has also been employed for the characterization of flavonoids in extracts of fresh herbs. Besides the separation, quantitative determination and identification of flavonoids, the objective of the study was the comparison of the efficacy of the various detection systems in the analysis of flavonoids in herb extracts. Freeze-dried herbs (0.5g of chives, cress, dill, lovage, mint, oregano, parsley, rosemary, tarragon and thyme) were ground and extracted with 20 ml of 62.5 per cent aqueous methanol. After sedimentation the suspension was filtered and used for HPLC analyses. Separations were carried out in an

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TABLE 2.52 YIELDS (G/100 G DRY PLANT) OF THE THREE WELD FLAVONOIDS AS A FUNCTION OF EXTRACTION TIME. AT ROOM TEMPERATURE AND BOILING TEMPERATURE

Extraction time (min)

5 15 30 60 120 240

Room temperature

Boiling temperature

A

B

C

A

B

C

0.194 0.202 0.238 0.346 0.370 0.396

0.151 0.165 0.183 0.226 0.292 0.271

0.044 0.079 0.086 0.124 0.167 0.161

0.364 0.448 0.456 0.443 0.429 0.422

0.294 0.357 0.363 0.366 0.359 0.358

0.171 0.233 0.239 0.241 0.246 0.242

A  luteolin; B  luteolin-7-glucoside; C  luteolin-3
,7-diglucoside. Reprinted with permission from D. Cristea et al. [154]. TABLE 2.53 PRESENCE OF FLAVONOIDS IN SPECIES. SUBSPECIES AND VARIETIES OF LAVANDULA AND SABAUDIA BASED ON HPLC. PC AND ELECTROPHORESIS RESULTSe. NUMBERS REFER TO FLAVONOIDS IN THE TEXT

Compound Texton

Section Stoechase L.Stoechas subsp. stoechase L. stoechas subsp. luiseiri L. viridis Section Dentata L.dentata var.dentatae Section Lavandula L. angustifolia subsp. angustifolia L. angustifolia subsp. pyrenaica L. angustifolia subsp. delphinensis

Flavone glycosides

6-OH Flavone, glycosides

Flavone, C-glyco sides

1/2

3

4/5

6

7/8

9

10

11

  

  

  

  

  

  

  

  

























































 (Continued on next page)

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TABLE 2.53 (continued)

Compound Texton

Flavone glycosides

L. lanata L. latifolia Section Pterostoechas L. canariensis L. coronopifolia L. maroccana L. mairei var. mairei L. minutolii var. minutolii L. multifida L. rotundifolia Section Subnuda L. aristibracteata L. dhofarensis L. subnuda Compound texton

Section Chaetostachys L. bipinnata Sabaudia grou S. atriplicifolia Section Stoechase L.Stoechas subsp. stoechase L. stoechas subsp. luiseiri L. viridis Section Dentata L.dentata var.dentatae Section Lavandula L. angustifolia subsp. angustifolia L. angustifolia subsp. pyrenaica

6-OH flavone, glycosides

Flavone, C-glyco sides

1/2

3

4/5

6

7/8

9

10

11

 

 

 

 

 

 

 

 

      

      

      

      

      

      

      

      

  

  

  

  

  

  

  

  

8-OH flavone 8-glycosides

8-OH flavone 7glycosides

Flavone aglycones (external)

12

13

14

15

16

17

18

19

20

21





















































































































































(Continued on next page)

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TABLE 2.53 (continued)

Compound texton

L. angustifolia subsp. delphinensis L. lanata L. latifolia Section Pterostoechas L. canariensis L. coronopifolia L. maroccana L. mairei var. mairei L. minutolii var. minutolii L. multifida L. rotundifolia Section Subnuda L. aristibracteata L. dhofarensis L. subnuda Section Chaetostachys L. bipinnata Sabaudia group S. atriplicifolia

8-OH flavone 8-glycosides

8-OH flavone 7 glycosides

Flavone aglycones (external)

12

13

14

15

16

17

18

19

20

21





















 

 

 

 

 

 

 

 

 

 

      

      

      

      

      

      

      

      

      

      

  

  

  

  

  

  

  

  

  

  









































Extract subjected to electrophoresis; , flavonoid not detected by HPLC; , ,  relative amounts of flavonoids present (low, medium, or high UV absorbance of the flavonoid peak. respectively. Reprinted with permission from T. M. Upson et al. [155].

e

ODS column (250  4.6mm i.d.; particle size 5m). Components of the linear gradient elution were 30 per cent methanol in water (1 per cent formic acid) (solvent A) and 100 per cent methanol (solvent B). The gradient was 25–40 per cent B in 10min, 40–43 per cent B in 14min, and 43–84 per cent B in 6min. The flow rate was 1 ml/min. Spectra were recorded between 220 and 450 nm. The parameters of single-stage MS were: source temperature 80°C, probe temperature 450°C, cone voltage 30 V and corona discharge 2.7kV. The characterstics of the triple-stage MS were: corona current, cone 30–40 V, source temperature 120°C, probe temperature 400°C, cone gas nitrogen (120l/h), desolvation gas N2 (600l/h). The scan was in both cases in the range of 120 – 1000 u. Some ion cromatograms are shown in Fig. 2.53. The chromatograms demonstrate that the flavonoids separated under RP-HPLC conditions with gradient elution can be effectively identified by MS techniques. The retention time and name of the flavonoids measured in various herb extracts are compiled in Table 2.54. The data demonstrated that negative APCI-MS-MS can be successfully employed for the determination of flavonoid profiles in complicated herb extracts [156].

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100

6

%

2.00 3.00

4 5

4.00 1+2

3

8 7

0 2.00

4.00

6.00

(a)

8.00 Time

10.00

100

12.00

14.00

3

16.00

m/z 269

6 % 2

2.00 0

(b) 100

3

m/z 299 % 4

2.00 0 1.00

(c)

2.00

3.00

4.00

5.00

6.00

7.00 Time

8.00

9.00

10.00

11.00

12.00

13.00

Fig. 2.53. HPLC-MS total ion chromatogram of mint extract (a). Flavonoid components Nos 1–8 were identified: 1  m/z 593 luteolin-7-rhamnoglucoside; 2  m/z 579 naringenin-7-rhamnoglucoside; 3  m/z 609 hesperetin-7-rhamnolucoside; 4  m/z 577 apigenin-7-rhamnoluoside; 5  m/z 607 diosmein-3-rhamnoglucoside; 6  m/z 359 rosmarinic acid; 7  m/z 795 acacetin-acetyl-glucosyl-rhamnoglucoside; 8  m/z acacetin-rhamnoglucoside. HPLC-MS ion chromatogram of m/z 269 (b) and m/z 299 (c) in parsley extract. Flavonoid components nos 1–4 were identified: 1  m/z 563 apigenin-7-apiosylglucoside; 2  m/z 605 apigenin-acetyl-apiosylglucoside; 3  m/z 593 diosmetin-apiosylglucoside; 4  m/z diosmetin-acetyl-apiosylglucoside. Reprinted with permission from U. Justesen [156].

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TABLE 2.54 RETENTON TIME (TR) AND NAME OF THE FLAVONOIDS FOUND IN VARIOUS HERB EXTRACTS

Herb Chives (Allium schoenoprasum) Cress (Lepidium sativum) Dill (Anethum graveolens) Isorhamnetin-glucuronide Lovage (Levisticum officiale) Mint (Mentha var.)

tR (min)

Tentative name

3.1 3.5 3.6 3.2 3.5 3.6 5.3 9.4

Quercetin-glucoside Isorhamnetin glucoside Kaempferol glucoside Quercetin-glucoside Isorhamnetin glucoside Kaempferol glucoside Quercetin-ramnoglucoside Quercetin-glucuronide Kaempferl-glucuronide

5.3 6.1 6.1 6.2 6.9 7.9 8.9

Quercetin-ramnoglucoside Luteolin-rhamnoglucoside Luteolin-7-rhamnoglucoside Naringenin-rhamnoglucoside Hesperetin-rhamnoglucoside Apigenin-rhamnoglucoside Diosmetin-rhamnoglucoside

(Diosmin). rosmarinic acid

(Origanum vulgare)

Parsley (Petroselinum crispum)

Rosemary (Rosmarinus officialis) Tarragon (Artemisia dranunculus) Thyme (Thymus vulgaris)

9.1 14.1 14.2 5.5 8.8 9.2 15.2 8.5 8.6 10.1 10.1 8.8 9.2 8.5

Acacetin-acetyl-glucoside-rhamnoglucoside Acacetin-rhamnoglucoside Oregano Luteolin-glucoside Rosmarinic acid Apigenin-acetyl-diglucoside Diosmetin-acetyl-apiosy glucoside Apigenin-7-apiosylglucoside Diosmetin-apiosylglucoside Apigenin-acetyl-apiosylglucoside Diosmetin-acetyl-apiosylglucoside Rosmarinic acid Luteolin-acetyl-glucuronide Diosmetin-ramnosylglucoside

5.9 6.8 7.7 8.8 10.0 10.0 12.5

Eriodictyol-glucuronide Luteolin-glucoside Luteolin-glucuronide Rosmarinic acid Luteolin-acetyl-glucoside Apigenin-glucuronide Luteolin-diglucoside

Reprinted with permission from U. Justesen et al. [156]

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2.2.3.2 HPLC determination of flavonoids in food and food products Because of the advantageous dietary effects of flavonoids they have been vigorously investigated in food and food products. The objectives of these measurements were the separation and quantitation of well-known flavonoids in foods and the identification of new flavonoids. An HPLC-ESI MS method has been developed for the isolation and identification of new quercetin derivatives in the leaves of Eruca sativa (Mill). Fresh leaves (500g) were homogenized with 1 200 ml of methanol–water (7:3, v/v), the suspension was macerated for 24h at ambient temperature, then it was filtered, concentrated to 50 ml and diluted with water to 500 ml. The extract was applied to an Amberlite XAD-2 column (75  8cm i.d.) and was washed subsequently with 1l of water and 1l of diethyl ether. The glucoside fraction was eluted with 1.5l of methanol and the eluate was concentrated in vacuum and liophilized. Further fractionation was performed in an ODS column (200  2.5 mm i.d.) using methanol–water mixtures as mobile phases. Elution started with 100 ml of water and continued by increasing the ratio of methanol in steps of 10 per cent (100 ml each). Fractions of 7 ml were collected and used for further separation by HPLC-ELSD (evaporative light scattering detector), HPLC-DAD and HPLC-ESI MS (electospray ionization). HPLCDAD analysis was carried out in an ODS column (250  4.6mm i.d.; particle size 5m) using a linear gradient of water (0.05 per cent TFA) and ACN at a flow rate of 1 ml/min. The gradient started at 5 per cent ACN and finished at 100 per cent ACN in 30min. HPLC-ELSD was performed under the same chromatographic conditions as HPLC-DAD using an ELSD detector (temperature 40°C, pressure of compressed air 240 kPa). HPLCESI MS measurements were made in an ODS column (100  2 mm i.d.; particle size 5m) using the same linear gradient elution as before. The flow rate was 0.2 ml/min. The spray capillary voltage for ESI in positive-ion mode was 4kV, the temperature of the heated inlet capillary was 230°C. Nitrogen was the sheath gas (482 MPa) and also the auxiliary gas. The chromatogaphic results were completed with IR spectroscopy, 1 H NMR and 13C NMR measurements. The new quercetin derivaties were identified as quercetin 3,3
4
-tri-O--D-glucopyranoside, quercetin 3
-(6-sinapoyl-O--D-glucopyranosil)-3,4
-di-O-á-D-gluco-pyranoside, and quercetin 3-(2-sinapoyl-O--D-glucopyranosil)-3
-(6-sinapoyl-O--D-glucopyranosil)-4
-O--D-glucopyranoside [157]. Flavonoids in peppermint leaves (Mentha piperita folium) have also been separated by RP-HPLC using gradient elution. In order to select the best extracting solvent, peppermint leaves were extracted with petroleum ether, chloroform, ethyl ether, ethyl acetate, acetone, methanol, ethanol, 80 per cent and 30 per cent ethanol at room temperature and boiling water. As ethanol extracted the highest amount of flavonoids it was used for further investigations: 3g of leaves were ground and agitated with 50 ml of ethanol for 15min then with 50 ml for 10min and finally with 25 ml for 5min. The combined extracts were evaporated to dryness at 30°C redissolved in 5 ml of methanol and injected into the ODS column (250  4.6 mm i.d.; particle size 5 m). The constituents of gradient 1 were water–formic acid (19:1, v/v) (A) and methanol (B). The gradient started with 30 per cent B, 30 per cent B, 15min, 40 per cent B, 20min, 30min, 45 per cent B, 50min, 60 per cent B, 52min, 80 per cent B. Solvents for gradient 2 were water–phosphoric acid (999:1, v/v, A) and ACN (B):0min 17 per cent B, 35min 23 per cent B, 37min, 36 per cent B, 57min, 56 per cent B, 59min, 100 per cent B, 63min, 100 per cent B. The flow rate was in both cases 1 ml/min and chromatograms were evaluated

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at 280, 320 and 350 nm. The effect of the extraction method on the chromatographic profile of peppermint extract is illustrated in Fig. 2.54. The retention times of analytes determined in gradients 1 and 2 are listed in Table 2.55. 1.688 8

9

1.125 AU

10

0.583 7

a

b

−.0.000 Om

c

20m40.0s

(a)

41m20.0s

61 m

Time 2.244

7

1.495 AU

9 b

0.747

8

a c −0.002 Om

10

20m40.0s

(b)

41m20.0s

61 m

Time 1.848

7

1.231 AU

1 3 0.615

−0.002 Om (c)

6

2

4

9 8

5

21m20.0s

42m40.0s

10

63 m

Time

Fig. 2.54. High-performance liquid chromatography profile of a peppermint sample (gradient no.1), extracted with ethyl ether (a) and ethanol (b). Chromatographic profile of a sample extracted with ethanol (gradient no.2) (c). Detection at 320 nm. For peak identification see Table 2.55. Reprinted with permission from F. M. Areias et al. [158].

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TABLE 2.55 PEPPERMINT PHENOLIC COMPOUNDS SEPARATED BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (RT1 AND RT2  RETENTION TIMES IN MIN MEASURED IN GRADIENTS 1 AND 2)

No

Compound

RT1

RT2

a 1 2 b 3 4 5 c 6 7 8 9 10

Eriodictyol 7-O-heteroside Eriodictyol 7-O-rutinoside Eriodictyol 7-O-glucoside Luteolin 7-O-heteroside Luteolin 7-O-rutinoside Luteolin 7-O-glucoside Hesperetin 7-O-rutinoside Apigenin 7-O-heteroside Apigenin 7-O-rutinoside Rosmarinic acid 5,6-OH-7,8,3
,4
-OMe-flavone Pebrellin (5,6-dihydroxy-7,8,4
-trimethoxyflavone) Gardenin B (5-hydroxy-6,7,8,4
-tetramethoxyflavone)

9.2 — — 22.43 — — — 26.48 — 24.53 54.38 56.14 60.29

— 12.05 14.19 — 18.02 20.08 23.46 — 25.22 28.10 52.29 55.55 62.19

Reprinted with permission from F. M. Areias et al. [158].

The results indicated that the separation and quantitative determination of this class of compounds make possible the application of the method for the quality control and authenticity test of peppermint leaves [158]. The performance of various ODS columns for the separation of flavonoids in onion and celery has been compared. Flavonoids were extracted from 0.25g of lyophilized onion with 20 ml of 60 per cent aqueous methanol. The extracting agent contained 125g kaempferol as internal standard and 20 mM sodium diethyldithiocarbamate as antioxidant. Extract was mixed with 6 M of HCl to give a solution of 1.2 M HCL in water–methanol 1:1 and refluxed at 90°C for 2 h. After hydrolysis the samples were filtered, diluted and the pH was adjusted to 2.5 with TFA. Samples of celery were treated similarly but isorhamnetin was used as an internal standard, and the hydrolysis was carried out in 2 M HCl for 4h at 90°C. Both isocratic and gradient elutions were employed for the measurements using water–methanol–ACN mixed in various ratios. Typical chromatograms illustrating the effect of acid hydrolysis on the flavonoid composition of onion and celery are shown in Fig. 2.55. The mesurements indicated that the separation capacity of ODS columns for flavonoids shows marked differences; the selection of the column is of predominant importance for the success of the analysis. It was further established that the method allows the separation of rutin, quercetin-3-glucoside, quercetrin, myricetin, luteolin, quercetin, apigenin, kaempferol and isorhamnetin and can be used for the analysis of flavonoids in onion and celery [159]. Flavonoids have also been analysed in the peel and pulp of different apple varieties by RP-HPLC. The varieties Golden, Red Delicious, Granny Smith and Reineta Green were inluded in the experiments. Methanol containing 1 per cent 2,6-di-tert-butyl-4-methylphenol

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IS

Absorbance (365 nm)

Absorbance (365 nm)

IS

Lt IS

Qc

Ap

0 (a)

5

10

15

IS

20

Retention time (min)

0

25 (b)

5

10

15

20

25

Retention time (min)

Fig. 2.55. Gradient reversed-phase HPLC analysis of flavonoids in white onions (a) and celery (b). ODS column of 150  3.9mm i.d; particle size 5m. Mobile phase: 20min gradient of 15–35 per cent acetonitrile in water adjusted to pH 2.5 with TFA. Fowrate: 1ml/min. Upper and lower traces represent samples before and after hydrolysis, respectively. Detection wavelength 365 nm. IS  internal standard; Qc  quercetin; Ap  apigenin; Lt  luteolin. Reprinted with permission from A. Crozier et al. [159].

was used for the extraction. Samples of 5g of peel or 10g of pulp were sonificated with 10 ml of methanol for 1 h, followed by 10 ml for 30 min, and 5 ml for 30 min. The combined exracts were diluted to 25 ml and filtered before HPLC analysis. Analytes were separated in an ODS column (250  4.6mm i.d.; particle size 5m). Solvents for gradient elution were aqueous 0.01 M phosphoric acid (A) and methanol (B). Elution started with 5 per cent B, 50 per cent B for 10min, 70 per cent B for 5min, 80 per cent B for 5min, and 100 per cent B for 5min. The column was not thermostated and the flow rate was set to 1 ml/min. The method successfully separated the solutes as illustrated in Fig. 2.56. The amounts of phenolic compounds in peels and pulps from the apple varieties are compiled in Table 2.56. The data demonstrate that the amount and composition of phenolic compounds depend considerably on the variety of apple. Because of the high recovery values (95 – 105 per cent), the wide linearity range (0.7 – 120 g/ml), and the low detection (0.21 – 0.63 g/ml) and determination limit (0.71 – 2.00g/ml) the method was proposed for the analysis of phenolic compounds in apples [160]. RP-HPLC combined with DAD and ESI-MS has been employed for the determination of flavonol aglycones and glucosides in berries. Cultivated blackcurrants (Ribes nigrum ‘Ojebyn’), strawberries (Fragaria x ananassa ‘Senga Sengana’), red raspberries (Rubus idaeous ‘Ottawa’), wild lingonberries (Vaccinium vitisidea), bilberries (Vaccinium myrtillus), rowanberries (Sorbus aucuparia), chokeberries (Aronia mitchurinii ‘Viking’), sea buckhorn berries (Hypophae rhamnoides), crowberries (Empetrum hermaphroditum), whortleberries (Vaccinium uliginosum) and cranberries (Vaccinium oxycoccos) were included in the experiments. Flavonoid glycosides were extracted with aqueous methanol and hydrolized to the

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Chapter 2 9 10

Gallic acid 3 4

0.01 Au

6 8 12

11

13

2 1

0

5

7

5

10

15

20

Time (min)

(a)

5

Gallic acid 0.01 Au 7 3

1

0 (b)

5

6 9

4

8

2

10

15

20

Time (min)

Fig. 2.56. HPLC chromatogram of (a) Golden peel and (b) Golden pulp extracts at 280 nm. Peaks: 1  procyanidin B3; 2  procyanidin B1; 3  ()-catechin; 4  procyanin B2; 5  chlorogenic acid; 6  ()-epicatechin; 7  caffeic acid; 8  phloretin derivative; 9  phloridzin; 10  rutin; 11, 12 and 13  flavonol glucosides. Reprinted with permission from A. Escarpa et al. [160].

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TABLE 2.56 CONTENTS OF PHENOLIC COMPOUNDS IN PEELS AND PULPS FROM GOLDEN. REINETA. RED AND GRANNY SMITH APPLES Peak no.

Contenta (mg/kg fresh sample)

Analyte Golden

Reineta

Red

Granny

8 9 10 11 12 13

Procyanidin B3b Procyanidin B1b ()-catechin Procyanin B2b Chlorogenic acid ()-epicatechin Caffeic acid Anthocynidinb Phloretin derivative Phloridzin Rutin Flavonol glucosidec Flavonol glucosidec Flavonol glucosidec

25–66 32–53 66–164 69–166 17–37 82–168 nd nd 58–71 31–71 136–237 35–57 67–180 nd

125–158 103–242 229–460 388–581 100–440 238–439 8–38 nd 53–100 83–418 327–671 19–146 37–403 nd

11–14 127–172 297–445 433–659 113–157 248–481 nd 585–1037 61–122 104–159 136–504 67–146 190–369 nd

70–124 173–241 374–486 558–574 6–60 246–312 10–14 nd 29–31 12 390–414 164–179 nd nd

Pulps 1 2 3 4 5 6 7 8 9

Procyanidin B3b Procyanidin B1b ()-catechin Procyanin B2b Chlorogenic acid ()-epicatechin Caffeic acid Phloretin derivative Phloridzin

21–27 10–11 28–49 23–32 29–57 19–34 6–9 5–8 4–8

33–41 57–67 113–136 82–94 266–357 91–111 9–11 19–21 16–20

20–28 11–21 44–70 34–54 63–106 36–59 2.0 2–4 13–14

61–100 62–84 136–182 97–105 28–71 71–97 4–5 7.0 5.0

1 2 3 4 5 6 7

a

Mean values for six apples. Each apple was analysed in triplicate. Quantified as catechin. c Quantified as rutin. nd, not determined. Reprinted with permission from A. Escarpa et al.[160]. b

corresponding aglycones by HCl. RP-HPLC separations were performed in an ODS column (125  3 mm i.d; particle size m). Solvents for gradient elution were 1 per cent aqueous formic acid (A) and methanol (B). The gradient was: 0 – 10 min, 10 – 13 per cent of B in A; 10 – 25 min, 13 – 70 per cent of B in A; 25 – 29 min, 70 per cent B in A; 29 – 30min, 70 – 100 per cent of B in A; 30 – 35 min, 10 per cent of B in A. The flow rate was 0.5 ml/min. The ESI-MS conditions were: temperature of heated capillary, 225°C; voltage 4.5kV. Scan

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mass spectra were m/z 200 – 1 000. Helium was the collision gas. DAD spectra were recorded between 220 and 450 nm. Sugar moieties were identified by GC-MS after derivatization. The identification process is visualized in Fig. 2.57. The retention times and tentative structures of flavonol glycosides are compiled in Table 2.57. 100 14,42 13,60 14,17

50

Base peak ion chromatogram, full scan M/Z 275-1000 source induced dissocaition off 15,50

19,33

17,01 17,55

0 Relative abundance %

(a) 13,59

100

14,41 14,16

0 10

11

12

13

14

15

Reative abundance %

18

19

20

Full scan ms, peak at 14.1 min

80 60 612,0

40 20

306.2

0 300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

m/z -Deoxyhexose -hexose 303,3

100

550

Full scan ms-ms, M/Z 611> peak at 14.1 min

80

-Deoxyhexose

60

465,0

40 20 0 200

250

300

350

400 m/z

(d)

Reative abundance %

17

(M+H)+ 611,0

100

(c)

450

500

550

600

Full scan MS3,M/Z 611 > 303> peak at 14.1 min 257,1

100 80 60

229,3

40 20

128,9

153,3

190,9 203,8

165,1

0 80

(e)

16

Time(min)

(b)

Reative abundance %

Extracted ion chromatogram of M/Z 303, source induced dissociation on

50

100

120

140

160

180

200

213,3 228,6 220

233,4

247,1

240

257,9 260

284,9 275,4 280

300

m/z

Fig. 2.57. Identification of a flavonoid diglycoside from unhydrolized chokeberry sample. (a) Positive total ion chromatogram, (b) ion source collision-induced dissociation chromatogram for the ion m/z 303 (quercetin), (c) full scan MS spectrum of the peak at 14.1min, (d) full scan MS-MS spectrum of the [MH]* ion, (e) fragmentation spectrum (MS3) of the m/z 303.3 ion. Reprinted with permission from S. Hakkinen et al. [161].

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TABLE 2.57 RETENTION TIMES AND TENTATIVE STRUCTURES OF FLAVONOL GLYCOSIDES IN THE UNHYDROLYSED BERRY EXTRACTS

Berry

Retention time

Tentative structure

Bog whortleberry

5.2 14.4 15.0 17.9 14.1 14.4 15.5 10.5 14.1 14.4 14.9 14.3 15.0 15.4 17.8 14.9 16.6 14.7 14.5 14.9 15.4 15.8 13.3 13.8 14.5–15.0 15.2 15.7 12.3 14.3 15.0

Q-pentose Q-hexose Q-pentose Q Q-deoxyhexose-hexose Q-hexose Q-hexose-pentose Q-hexose-hexose Q-deoxyhexose-hexose Q-hexose K-hexose-pentose Q-hexose Q-pentose Q-deoxyhexose Q-pentose-pentose Q-hexose M Q-hexose-deoxyhexose Q-hexose-pentose Q-hexose Q-pentose Q-deoxyhexose M-deoxyhexose-hexose M-hexose Q-deoxyhexose-hexosea Q-deoxyhexose-hexosea K-deoxyhexose-hexose Q-deoxyhexose-hexoseb Q-deoxyhexose-hexoseb Q-hexose-CH3

Chokeberry

Rowanberry

Cranberry

Crowberry Lingonberry

Blackcurrant

Sea buckthornberry Bilberry

Q, quercetin; K, kaempferol; M, myricetin. a There are two diglycosides with m/z 611 in blackcurrant; they have different retention times in HPLC and different MS-MS spectra. In the faster-eluting diglycoside, the sugars are fragmented separately. In the more slowly eluting glycoside, the fragmented sugars are linked together. b There are two diglycosides with m/z 611 in sea buckthorn berry; they have different retention times in HPLC and different MS-MS spectra. In the faster-eluting diglycoside, 449 is the strongest ion in MS-MS, indicating the initial loss of a hexose sugar (162). In the more slowly eluting diglycoside. 464 is the strongest ion in the MS-MS. indicating the initial loss of deoxyhexose sugar (146). Reprinted with permission from S. Hakkinen et al. [161].

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It was stated that this combined technique (HPLC-DAD, HPLC-ESI-MS, GC-MS) allows the safe identification of flavonol aglycones and glycosides and can be used for the analysis of these compounds in berries [161]. The flavonoid content of honey has also been frequently investigated by HPLC. Thus, 15 flavonoids were found in the Australian jelly bush honey (Leptospremum polygalifolium), myricetin, luteolin and tricetin being the main constituents. The flavonoid composition of the New Zealand manuka (Leptospermum scoparium) honey differed markedly from the Australian one containing mainly quercetin, isorhamnetin, chrysin and luteolin. The method was proposed for the authenticity test of honey floral origin [162]. Another study used RP-HPLC for the determination of quercetin, luteolin, apigenin and kaempferol in honey and various other food products. The amounts of flavonoids found are compiled in Table 2.58. The data demonstrate again that the flavonoid content and profile are highly different in various foods and food products which has to be taken into consideration in the design of special diets [163]. Because of the importance of soybean and soybean products in both human and animal nutrition their flavonoid content has been investigated many times. Thus, HPLC-UV and HPLC-MS have been applied for the determination of flavonoids and other phytochemicals in soybean extracts and in onion with and without hydrolysis. 1g of onion was homogenized and mixed with 8 ml of methanol–water (8:2, v/v). After 2h the suspension was centrifuged at 4°C and the supernatant injected. Powdered soybean (500 mg) was defatted by 2  10 ml of hexane and further treated as the onion sample. Flavonoids were hydrolyzed by mixing 2 ml of extract with 2 ml of 2 M HCl and heated to 130°C for 2 h. The solution was neutralized with 4 M of NaOH. Separation was performed in an ODS column (125  4.6 mm TABLE 2.58 FLAVONOID CONTENT OF SOME FOODS

Black tea Linden flower Sage Rosehip Violet carrot juice Grape molasses Tarhanad Honeyd Urica sp.d

Quercetina

Luteolin

Apigenin

Kaempferol

34.89b 21.78 27.20.8 16.70.2 83.70.5 169228 5.920.12 —b 0.870.02

—b — 110.7 — — — — — —

—c — — — — — — 29.34 14355

1109 1132 — — — —

Amounts are expressed in g/l. Meanstandard deviation. c Not detected. d Amounts are expressed in mg/100 g. Reprinted with permission from S. Karakaya et al. [163]. a

b

2.420.1 —

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1.987 4.358

(d)

5.712 7.178 Daidzin 8.415 Genistin 11.800 Daidzein

2.335

Time [min]

16.00 Genistein

Time [min]

(b)

Relative absorbance

17.586 Genistein

12.867 Daidzein

Relative absorbance

(c)

12.540 Quercetin

Relative absorbance

8.885 Naringin (ISTD)

Time [min] 7.472 Daidzin 8.905 Genistin 9.337 Naringin (ISTD)

(a)

12.617 Quercetin

Relative absorbance

i.d.; particle size 3 m). Solvents for gradient elution were: ACN–water–formic acid (10:90:5, v/v, A) and ACN-water-formic acid (90:10:5, v/v, B). The gradient conditions were: 0–3min 0 per cent B; 3–4 min 0–17 per cent B; 4–22 min 17–28 per cent B; 22–23 min 28–50 per cent B; 23–29.5 min 50 per cent B. The Column was not thermostated and separations were carried out at room temperature. UV detection was carried out at 270 nm. MS detection was carried out as follows: source temperature, 120°C; cone voltage, 40eV; accelerator lens potential 0.5kV, multiplier voltage 650 V, and m/z range 100–800. Chromatographic profiles of onion and soybean extracts with and without acid hydrolysis are represented in Fig. 2.58. The chromatograms demonstrated the good separation capacity of the method, the differences between the retention times may be due to the changing temperature of the analytical

Time [min]

Fig. 2.58. HPLC analysis of an onion extract without (a) and with acid hydrolysis (b five-fold diluted), and soybean extract wihout (c three-fold diluted) and with acid hydrolysis (d, five-fold diluted). Reprinted with permission from W. Andlauer et al. [164].

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column. Some unidentified peaks with low retention times were found on the chromatogram of unhydrolysed onion extract. MS measurement proved that peaks are quercetin glucoside, quercetin diglucoside and isorhamnetin 4
-glucoside. The main isoflavones found in the soyabean extract were daidzin (670, 692 mg/kg), genistin (602, 622 mg/kg), daidzein (100, 103 mg/kg) and genistein (144, 148 mg/kg). The first and second values were calculated with external and internal standards, respectively. The retention times, limit of detection and repeatabilities of the analytes are compiled in Table 2.59. It has been concluded from the results that this easy-to-carry-out method can be used even in routine laboratories for the quantitative analysis (UV detection) and for the identification (ESI-MS) of this class of phenolic compounds in various complicated matrices [164]. A similar RP-HPLC method has been developed for the determination of isoflavones in soybean powder. Ground soybean powder (100 g) was extracted four times with 300 ml of hexane, then the defatted powder was extracted with six solvents or solvent mixtures such as acetone, methanol–water (4:1, v/v), methanol–ethyl acetate–acetone (1:1:1, v/v), acetone–0.1 M HCl (5:1, v/v), methanol–water (4:1, v/v) plus 0.1 M HCl (5:1, v/v), and methanol–ethyl acetate–acetone (1:1:1, v/v) plus 0.1 M HCl (5:1, v/v). An aliquot of 0.5 g defatted or non-defatted soybean powder was shaken for 2h or sonificated for 1h with 5 ml TABLE 2.59 RETENTION TIMES (tR). LIMITS OF DETECTION (LODS; S/N  3) AND REPEATABILITIES (RSD) OF PHYTOCHEMICALS

Catechin Epicatechin Malvin chloride Daidzin Rutin Genistin Naringin Myricetin Resveratrol Morin Daidzein Quercetin Naringenin Genistein Apigenin Kaempferol Formononetin Biochanin A Flavone

tRSD (min)

LOD (pmol)

RSD

3.280.08 5.570.16 7.030.06 7.280.06 8.080.15 8.530.15 8.880.21 9.870.13 10.810.13 11.190.12 12.100.12 13.140.12 15.250.14 16.460.14 17.550.16 17.870.16 22.500.18 27.540.08 28.180.09

40 40 40 6 8 6 8 13 16 42 6 16 8 8 16 37 8 8 16

5.4 11.5 4.7 5.7 4.5 5.1 3.1 7.2 4.2 5.1 4.4 4.4 4.8 4.1 6.2 8.2 6.0 5.8 3.9

Reprinted with permission from W. Andlauer et al. [164].

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of solvent at ambient temperature. After extraction the suspensions were centrifuged and the supernatant evaporated to dryness and redissolved in 5 ml of methanol. Measurements were performed in two different ODS columns (250  4.6 mm i.d. and 50  2.1 mm i.d.). Particle size was in both cases 5 m. Isocratic and gradient elution techniques have been applied using various mixtures of methanol–water and ACN–water. The best separation was achieved in the column of 250 mm length using water (A) and ACN (B) gradient elution. The gradient started at 5 per cent B; to 10 per cent B in 5 min; to 25 per cent B in 15 min; to 32 per cent B in 27 min. Detection wavelength was 262 nm and flow rate was set to 2.0 ml/min. The column was thermostated at 25°C. The separation of isoflavones under optimal chromatographic conditions is illustrated in Fig. 2.59. The isoflavone contents in defatted soybean powders extracted with various techniques are compiled in Table 60. mAU

6

1.2E+06

1.0E+05

4 8.0E+04

3

6.0E+04 13 1

12

4.0E+04 9 5

11

2.0E+04 7

2 0.0E+00

5.00

8

10.00

15.00

20.00

25.00

Time (min)

Fig. 2.59. HPLC chromatogram of isoflavones in soybean extract after addition of acetylglycitin standard by employing an acetonitrile:water gradient solvent system. Conditions described in text. Peaks: 1  6-O-malonylaidzin; 2  6-O-malonylglycitin; 3  6-O-malonylgenistin; 4  daidzin; 5  glycitin; 6  genistin; 7  6-O-acetyldaidzin; 8  6-O-acetylglycitin; 9  daidzein; 10  glycitein; 11  6-O-acetylgenistin; 12  genistein; 13  fluorescein (IS). Reprinted with permission from T. H. Kao et al. [165].

ISOFLANONE CONTENTS (G/G) IN DEFATTED SOYBEAN POWDER AS AFFECTED BY VARIOUS EXTRACTION METHODS.

Glein

Gein

Din

Glin

Gin

Mdin

Mglin

Mgin

Adin

Agin

Total

A1 B2 A B A B A B A B A B

Acetone Acetone Acetone –acid3 Acetone –acid Mix4 Mix Mix–acid– A Mix–acid 80% Methanol 80% Methanol 80% Methanol–acid 80% Methanol–acid

nd 606 30.5 37.1 11.3 9.8 5.8 30.9 15.6 25.7 34.7 22.7

6.9 4.0 5.6 6.5 4.3 4.0 5.4 6.0 4.8 5.4 6.5 5.0

7.4 8.3 31.5 37.6 13.7 11.0 31.2 31.2 16.7 27.0 34.5 23.4

7.0 15.6 312.1 396.6 36.1 28.6 277.5 340.3 156.6 263.5 207.1 186.6

nd 1.6 34.7 25.8 6.5 5.5 38.2 45.5 17.5 43.4 75.0 29.5

7.1 19.1 276.4 311.8 29.1 25.2 248.8 274.4 128.9 202.7 178.4 166.5

3.6 11.4 415.0 611.7 13.9 11.4 451.8 512.8 145.3 379.7 363.9 326.3

1.3 4.1 51.5 75.1 2.4 2.3 50.8 54.8 22.8 21.1 90.5 40.4

3.5 17.4 489.8 603.8 19.7 11.6 454.9 512.9 186.6 339.9 323.0 307.3

nd 3.0 6.5 8.3 3.2 3.1 6.2 5.7 3.8 5.2 6.8 5.6

3.5 3.5 8.4 9.8 3.6 3.5 8.1 8.4 5.4 7.0 8.3 6.8

39.9 95.6 1 660.8 2 121.9 143.6 117.7 1 579.5 1 821.5 703.6 1 319.8 1 327.5 1 119.5

A  supersonic extraction 1 h. B  shaking extraction 2 h. 3 acetone–0.1 MHCl (5:1. v/v). 4 Mix  methanol–ethyl acetate–acetone (1:1:1. v/v). nd  not detected. Dein  daidzein; Glein  glycitein; Gein  genistein; Din  daidzin; Glin  glycitin; Gin  genistin; Mdin  6-O-malonyldaidzin; Mglin  6-O-malonylglycitin; Mgin  6-O-malonylgenistin; Adin  6-O-acetyldaidzin; Agin  6-O-acetylgenistin. Reprinted with permission from T. H. Kao et al. [165].

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1 2

Chapter 2

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The highest recovery (over 82 per cent for the majority of isoflavones) was obtained by using defatted soybean powder as raw material. It was further concluded from the data that acetone–0.1 M HCl (5:1, v/v) was the most effective extracting agent and the shaking the suspension for 2h yielded a higher amount of isoflavones than sonification did. Moreover, it was observed that the addition of HCl and defatting the sample generally increased the recovery [165]. Tandem mass spectrometry (MS-MS and MS-MS-MS) has also been employed for the identification of flavone aglycones and glucosides in soybean pods. Soybean pods (Glycine max ‘Tomahare’) (0.3 – 0.6g) were homogenized in 1.5 ml of 80 per cent aqueous ethanol, then the suspension was heated at 50°C for 1h. After cooling, the mixture was centrifuged and the supernatant was used for the analysis. Each separation was performed in an ODS column (250  4.6mm i.d.; particle size 5m). Solvents for gradient elution were acetic acid–water (pH  3, A) and ACN (B); from 5 per cent B to 65 per cent B in 90min, then to 100 per cent B in 10min, final hold 5min. UV-vis spectra of the analytes were recorded. MS conditions in positive-ion mode were: source temperature, 500oC; sprayer needle voltage, 4kV; capillary temperature 210oC, m/z range 100–1 000. MS-MS was carried out at a collision energy of 23 per cent. The chromatographic profiles of soybean pod extract determined by APCI-MS and UV are shown in Fig. 2.60. It was stated that the combination of UV-vis detection with MS detection techniques is a powerful tool for the identification of flavone aglycones and glucosides in soybean pods [166]. Supercritical fluid chromatography (SFC) has also found application in the separation of polyphenolic compounds. Supercritical fluid extraction (SFE) of grape seed was performed with CO2 (465atm, 2h, mass flow rate 50g/min) followed with CO2 containing 15 per cent methanol for 2 h at a flow rate of 50g/min. The separation of phenolic compounds was carried out in different bonded silica columns (250  4.6 mm i.d.; particle size 523m) and the performance of the columns was compared. Analytes were detected at 280 nm. The impact of column temperature, CO2 pressure, and the type and concentration of additives and modifiers was studied in detail. The optimal conditions for the separation of polyphenolic analytes were: two diol columns coupled sequentially; flow rate 2 ml/min liquid; CO2 pressure 125atm; column temperature 40°C; modifier methanol containing 0.25 per cent citric acid; and modifier programme: 93/ 7 per cent hold for 1min; to 83/17 per cent at 1.7 per cent/min; to 55/45 per cent CO2/methanol at 4 per cent/min; final hold, 10min. Chromatograms illustrating the baseline separation of analytes in grape seed extract are shown in Fig. 2.61. It has been concluded from the data that the SFC separation of polyphenolic compounds can be obtained in a relatively short analysis time and the efficacy of the separation is acceptable, therefore, the method can be considered as an alternative to existing HPLC techniques [167]. 2.2.3.3 HPLC determination of flavonoids in beverages Tea (Camellia sinensis) is one of the most frequently consumed beverages in the world and, consequently, an important agricultural product [168]. It has been proved many times that tea may reduce cholesterol level, hypertension, and shows antioxidant and anti-microbial effects [169]. Because of its importance, a considerable number of analytical methods have been developed for the separation and quantitative determination of the constituents of tea [170,171]. Thus, the application of high-speed counter-current chromatography [172,173], and HPLC-APCI-MS [174] have been reported.

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100 90

3 HPLC-APCI-MS chromatogram (TIC, abundance)

Relative abundance

80 70 60

4,5

1

50 40 2

30

6

20

7

10 0 20

25

30

35

40

(a)

45

50

55

60

65

70

65

70

Time (min) 1.0 HPLC-UV chromatogram (260 nm, absorbance)

7

3

0.75

Au

4,5

0.5

0.25

6 1 2

0.0 20 (b)

25

30

35

40

45

50

55

60

Time (min)

Fig. 2.60. Simultaneous HPLC-APCI-MS (a) and HPLC-UV (b) chromatograms of the soybean pod extract. Chromatographic conditions see in text. Peak identification; 1  7,4
-dihydroxyflavone-7O-
-D-glucoside; 2  luteolin-7-O-
-D-glucoside; 3  apigenin-7-O-
-D-glucoside; 4  7,4
dihydroxyflavone; 5  apigenin-7-O-
-D-glucoside-6-O-malonate; 6  luteolin; 7  apigenin. Reprinted with permission from S. M. Boué et al. [166].

The various chemical processes influencing the colour formation in tea have been vigorously investigated. The self-association of black tea polyphenol theaflavin and its complexation with caffeine [175], and the role of epicatechin quinone in the synthesis and degradation of theaflavin [176] have been studied in detail.

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4.939

19.932

28.466

Liquid chromatography of natural pigments

6 200

1

23.875

7

150

15.620 16.224

50

13.845

100

3.756

mAU

8

5

0

5

10

15

25

20

4

5

6

20.194

7

23.806

3

2

200

15.986

12.702

7.198

6.008

1

19.797

Time (min)

(a)

8

50

15.993

13.778

100

3.773

mAU

150

0

5 (b)

10

15

20

25

Time (min)

Fig. 2.61. Separation of grape seed extract (a) and grape seed extract spiked with mixture of eight compounds (b) using optimized conditions. Chromatographic conditions are discussed in the text. Peak identification: 1  2-phenylethanol; 2  vanillin; 3  ferulic acid; 4  protocatechoic acid; 5  caffeic acid; 6  gallic acid; 7  catechin; 8  epicatechin. Reprinted with permission from A. Kamangerpour et al. [167].

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RP-HPLC has also been used for the analysis of flavan-3-ols and theaflavins during the study of the oxidation of flavan-3-ols in an immobilized enzyme system. Powdered tea leaves (200mg) were extracted with 3  5 ml of 70 per cent aqueous methanol at 70°C for 10min. The combined supernatants were filtered and used for HPLC analysis. Flavan-3ols were separated in a phenyl hexyl column (250  4.6 mm i.d.; particle size 5 m) at 30°C. Solvents A and B were 2 per cent acetic acid in ACN and 2 per cent acetic acid in water, respectively. Gradient elution was: 0–10min, 95 per cent B; 10–40min, to 82 per cent B; to 40–50min 82 per cent B. The flow rate was 1 ml/min. Theaflavins were determined in an ODS column (100  4.6 mm i.d.; particle size 3m) at 30°C. The flow rate was 1.8 ml/min and solvent B was the isocratic mobile phase. The data demonstrated that flavan-3-ols disappear during the oxidation process while the amount of theaflavins with different chemical structures increases [177]. A different RP-HPLC method was developed and applied for the separation of four major catechin, gallic acid and caffeine in green, Oolong, black and puerh teas. Ground tea samples (1.9–3.8g) were extracted with 3  20 ml of 80 per cent aqueous methanol for 3h then 2  20 ml of 80 per cent methanol containing 0.15 per cent HCl for 3h. The combined extract was filtered, diluted and used for HPLC measurements. HPLC separation was carried out in an ODS column (250  4.5 mm i.d.; particle size 5m). Solvents A and B were water–acetic acid (97:3, v/v) and methanol, respectively. Gradient elution started with 100 per cent A for 1min and to 63 per cent B in 27min. The flow rate was 1 ml/min. Some chromatograms of tea extracts are collected in Fig. 2.62. The chromatographic profiles of teas are basically similar, containing the same components, however the quantity of the individual fractions shows considerable differences. The quantitative results are compiled in Table 2.61. As the method is rapid and selective it was proposed for quality control during tea manufacturing [178]. Another simple and reliable RP-HPLC method was developed for the determination of catechins in tea leaves and tea extracts. Tea leaves (0.25g) were sonicated with 20 ml of ethanol–water (10:90) for 20min. The suspension was filtered and centrifuged before HPLC analysis. Separation was performed in an ODS column (150  4.6mm i.d.) at 30°C using gradient elution. Solvents A and B were 0.1 per cent aqueous orthophosphoric acid and 0.1 per cent orthophosphoric acid in methanol, respectively. The Linear gradient started with 20 per cent B, 0–5min; to 24 per cent B, 5–7 min; 24 per cent B, 70–10 min; to 40 per cent B, 10–20min; to 50 per cent B, 20–25 min. The Flow rate was 1 ml/min and chromatograms were recorded at 210 and 280 nm. A characteristic chromatogram illustrating the good separation capacity of the method is shown in Fig. 2.63. The amounts of catechins found in teas are compiled in Table 2.62. The recovery was 88.5 – 106.9 per cent, the limit of detection 0.19 – 0.58 g/ml, the limit of quantitation 0.64 – 3.87  g/ml, and the linearity range 0.22 – 102.23 g/ml. Because of the rapidity, simplicity and robustness, the method was proposed for routine laboratories dealing with the analysis of catechins in teas [179]. A slightly different RP-HPLC technology has been developed and applied for the determination of catechins in tea infusions. Tea infusions were prepared by the traditional method, — filtered and used for HPLC analysis. Separation was carried out in an ODS column

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6

mAU

50

5

25

4

2

1 0 0

5

(a)

10 20 15 Minutes - 280 nm band = 10 nm

25

3 25

mAU

20 6

15

5

10 5

1 2

0

0

5

mAU

(b)

10 20 15 Minutes - 280 nm band = 10 nm

25

6

40 35 30 25 20 15 10 5 0

1

2 0

5

(c) 45 40 35 30 m 25 A 20 U 15 10 5 0

3 4

5

10 20 15 Minutes - 280 nm band = 10 nm

25

6

5

1 2

0 (d)

7

4

5

10

3 4

15

20

25

Minutes - 280 nm band = 10 nm

Fig. 2.62. HPLC chromatogram of (a) jasmin (green) tea, (b) Fujian Oolong tea, (c) pu-erh tea and (d) black tea at 280 nm. Peak identification: 1  gallic acid (GA); 2  ()-epigallocatechin (EGC); 3  ()-epigallocatechin gallate (EGCG); 4  epicatechin (EC); 5  ()-epicatechin gallate (ECG); 6  caffeine (CA); 7  ()-catechin gallate (CG). Reprinted with permission from Y. Zuo et al. [178].

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TABLE 2.61 THE CONTENTS OF INDIVIDUAL CATECHINS. GALLIC ACID AND CAFFEINE IN TEAS.

Content (mg/g tea)

GA

EGC

EGCG

EC

ECG

CA

CG

Pu-erh Meifoo green tea Shanghai green tea Hangzou lung ching Jasmine Fujian Oolong Jiangxi Oolong Fujian black

5.53 0.74

6.23 27.7

1.99 52.7

3.24 10.3

1.32 21.8

22.4 26.8

— —

0.37

30.8

51.1

7.25

11.3

23.0

—

1.84

37.6

62.4

6.60

16.3

28.5

0.81

1.13 1.42

27.6 10.0

54.2 22.2

6.90 2.63

15.8 6.06

29.6 7.44

— 0.27

1.67

15.9

28.2

2.96

6.45

18.7

—

1.36

4.45

21.6

—

2.06

5.71

3.79

mAU

For abbreviations see Fig. 2.61. Reprinted with permission from Y. Zuo et al. [178].

7

700 600 500 400 300 200 100 0

6

2 1

0

10

4 3

8

5 5

10

9

11

15

20

25

Min

Fig. 2.63. Chromatogram of green tea extract. For chromatographic conditions see text. Peak identification: 1  gallic acid; 2  ()-GC (gallocatechin); 3  theobromin; 4  ()-EGC (epigallocatechin); 5  ()-C (catechin); 6  caffeine; 7  ()-EGCG epigallocatechin gallate; 8  ()-EC (epicatechin); 9  ()-GCG (gallocatechin gallate); 10  ()-ECG epicatechin gallate; 11  ()-CG (catechin gallate). Reprinted with permission from H. Wang et al. [179].

(250  4.6 mm i.d.; particle size 5m) at room temperature. Gradient 1 consisted of aqueous acetate buffer (1.0 mM acetic acid, 1.0 mM sodium acetate in water, pH 4.5) and ACN. The gradient programme was as follows: from 12 to 21 per cent ACN in 0–18min; then 21–65 per cent ACN in 18–40min at a flow rate of 0.7 ml/min. Gradient 2 used the same aqueous

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TABLE 2.62 CONTENT OF CATECHINS IN TEA LEAVES AND GREEN TEA DRY EXTRACTS (% W/W)

Compound

()-GC ()-EGC ()-C ()-EGCG ()-EC ()-GCG ()-ECG ()-CG

catechins

Black teas

Green teas

Dry extracts

Ceylon

Keemum

Sencha

Longjing

RGT

Gunpower

A

B

0.03 0.12 0.02 0.71 0.13 nd 0.53 nd 1.54

nd 0.10 nd 0.43 0.04 nd 0.32 nd 0.89

0.25 4.96 0.12 6.13 0.93 0.09 1.09 nd 13.57

0.17 1.22 0.18 7.74 0.55 0.24 1.95 0.03 12.08

0.24 3.37 0.12 6.53 0.67 0.19 1.16 0.03 12.31

0.22 3.03 0.07 6.11 0.68 0.17 1.38 0.02 11.68

2.01 8.04 0.74 19.46 2.69 1.70 3.74 0.23 38.61

0.9 7.55 0.31 14.5 1.71 0.5 3.3 0.15 28.92

For symbols see Fig. 2.62. Reprinted with permission from H. Wang et al. [179].

solvent but ACN was replaced by methanol: from 30 to 50 per cent methanol in 0–40min at a flow rate of 0.5 ml/min. Gradient 3 applied ACN and aqueous ascorbate buffer (1.0mM acetic acid, 1.0mM sodium acetate, 0.10mM ascorbic acid in water). ACN concentration changed from 15 to 19 per cent in 0–16min; then 19–31 per cent (16–40min) at a flow rate of 0.7 ml/min. Spectra were detected between 210–400 nm. The retention times of analytes varied according to the gradient employed and they were between 8.7 and 28.4min. A typical chromatogram showing the good separation of catechins is shown in Fig. 2.64. The amounts of catechins found in tea infusions are listed in Table 2.63. Interestingly, the catechin level was the highest in black tea, indicating that the catechin loss is relatively low during the production process of black tea. It was concluded from the results that all three tea types are good sources of these potentially beneficial catechins [180]. An isocratic elution system has also been developed for the determination of catechins, caffeine and gallic acid in green tea. Extraction of analytes was carried out by infusing 3g of tea with 150 ml of boiling water for 5min. The infusion was filtered and used for HPLC analysis. The effect of various concentrations of ethanol on the efficacy of the extraction has also been investigated. Separation was performed in an ODS column (150  4.6mm i.d.) using methanol–water–orthophosphoric acid (20:79.9:0.1, v/v) as the isocratic mobile phase. The Column was thermostated at 30°C and the flow rate was 1.0 ml/min. Analytes were detected at 210 nm. The effect of ethanol concentration on the chromatographic profile of analytes is shown in Fig. 2.65. The chromatograms indicate that a higher concentration of ethanol markedly deteriorates the separation capacity of the RP-HPLC system; its concentration has to be held under 15 per cent in the samples. The amounts of analytes in the various tea samples are compiled in Table 2.64.

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2

4

Absorbance

Green tea 1 6

3 5

0

10

20

30

40

Time in minutes

Fig. 2.64. Representative chromatogram of tea infusions. Conditions: C18 column; acetonitrile–aqueous acetate buffer gradient; absorbance at 210 nm. Peak identities: 1  epigallocatechin (EGC); 2  caffeine; 3  epicatechin (EC); 4  epigallocatechin gallate EGCG); 5  epicatechin gallate (ECG); 6  internal standard (naringenin). Reprinted with permission from W. E. Bronner et al. [180]. TABLE 2.63 CATECHIN LEVELS IN TEA INFUSIONS. MG/DL

Black tea Green tea

Jasmin tea

Infusion time

EGC

EC

EGCG

ECG

3 min Literature 3 min 20 min Literature 3 min 20 min Literature RSD(%)

6 1–39 2 4 5–87 5 7 1–27 5

4 1–44 1 2 2–65 2 3 1–12 4

12 3–48 6 9 5–190 5 8 2–47 12

11 4–218 3 5 4–377 3 5 2–34 7

For symbols see Fig. 2.64. Reprinted with permission from W. E. Bronner et al. [180].

The correlation coefficients of the linear correlations were over 0.988, and the precision varied between 1.50 and 8.88 per cent. Because of its simplicity, reliability and precision the method was proposed for the routine analysis of these compounds in all kinds of tea and tea products [181].

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60% ethanol

mAU

2000 1500 1000 500 0 0

10

20

(a)

30 Min

40

50

1400

mAU

1200

30% ethanol

1000 800 600 400 200 0 0

10

20

mAU

(b)

30 Min

1400 1200 1000 800 600 400 200 0

40

50

15% ethanol

0

10

20

(c)

30

40

50

Min 1400 1200

Water

mAU

1000 800 600 400 200 0 (d)

0

10

20

30 Min

40

50

Fig. 2.65. Effect of solvent for sample preparation on the separation of analytes. The chromatographic conditions are described in the text. 1  gallic acid; 2  ()-GC; 3  ()-EGC; 4  ()-C; 5  caffeine; 6  ()-EGCG; 7  ()-EC; 8  ()-GCG; 9  ()-ECG. Reprinted with permission from H. Wang et al. [181].

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TABLE 2.64 CATECHINS, CAFFEINE AND GALLIC ACID IN DIFFERENT TEA SAMPLES (MG/100 ML)

Components

Gunpowder

RGT

Sencha

Keemun

Sri Lanka

Gallic acid ()-GC ()-EGC ()-C Caffeine ()-EGCG ()-EC ()-GCG ()-ECG

catechins

0.78 2.57 29.7 0.69 23.9 32.6 5.58 0.51 4.26 76.0

1.65 3.05 30.1 1.22 30.3 31.4 6.48 0.91 5.03 78.2

0.74 2.81 36.2 1.41 28.9 28.8 9.54 1.02 4.92 84.6

3.33 0.40 0.90 nd 38.2 0.95 nd nd 1.19 3.44

2.79 1.57 1.84 0.50 22.9 1.16 1.45 nd 2.92 9.43

nd, not detected. For details and symbols see text. Reprinted with permission from H. Wang et al. [181].

R2 OH O

HO

R3 R1

OH

O

Myricetin:R1=R2=R3=OH Quercetin:R1=R2=OH, R3=H Kaempferol:R1=OH, R2=R3=H

Fig. 2.66. Chemical structures of flavonols in tea. Reprinted with permission from H. Wang et al. [182].

Another isocratic elution method was applied for the determination of flavonols in green and black tea leaves and green tea infusions by RP-HPLC. The chemical structures of the flavonols studied are shown in Fig. 2.66. Infusions of teas were prepared by mixing 1g of tea leaves with 100 ml of boiling water for 5min, then they have filtered and used for HPLC analysis. The infusion step was repeated three times. Flavonoids were hydrolysed by mixing 1g of tea leaves with 40 ml of 60 per cent aqueous ethanol and 5 ml of 6 M HCl. The suspension was heated at 95°C for 2 h, then filtered and the volume was adjusted to 50 ml with 60 per cent aqueous ethanol. Separation was performed in an ODS column (150  4.6mm i.d.) operated at 30°C. The isocratic mobile phase consisted of 30 per cent aqueous ACN in 0.025 M KH2PO4, and the pH was adjusted to 2.5 with 6 M HCl. The

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250

mAU

200

1

150 100 3 50 0 0

2

4

6

8

10

12

14

16

18

Min

Fig. 2.67. A typical chromatogram of flavonols in tea leaves. The chromatographic conditions are described in the text. 1  myricetin; 2  quercetin; 3  kaempferol. Reprinted with permission from. H. Wang et al. [182].

flowrate was 1 ml/min. The good separation capacity of the method is illustrated by the chromatogram in Fig. 2.67. The amounts of flavonols in different tea leaves and in different infusions of gunpowder tea are compiled in Table 2.65. It has been established that the flavonoid content in the ground samples was considerably higher than in the unground ones. It was further stated that the results can be employed for the determination of the daily intake of these compounds [182]. HPLC has also been applied for the determination of flavanols and phenolic acids in the fresh shoots of tea. A considerable number of extraction procedures were tested and the recoveries were compared. Tea shoots (15g) were mixed with 100 ml of boiling water and the suspension was filered. The solid residue was washed with 3  10 ml of distilled water and the combined extracts were evaporated to dryness. The dry extract was dissolved in chloroform, ethyl acetate, methanol and water and the solutions were used for RP-HPLC analysis (first method). Method 2: fresh shoots (15g) were mixed with 180 ml of methanol for 4 min at 20°C and the further steps were the same as in method 1. Method 3 was the same as method 2 but the extraction times were 3, 4, 5, 6 and 7 min. Maximal yield was obtained by method 3 using 5min blending time. The components of the tea solution were separated in an ODS column (250  4.6 mm i.d.; particle size 5m) at 35°C. The detection range was between 220 and 600 nm and the flow rate was 1.2 ml/min. Gradient elution consisted of a linear gradient from 8 to 31 per cent ACN in 2 per cent aqueous acetic acid in 50 min then to 100 per cent ACN in 2 min, final hold 3min. A typical chromatogram of the tea extract is represented in Fig. 2.68. It has been established that methanol extracted a higher amount of tea components than chloroform, ethyl acetate or cold and boiling water did. The repeatability of the method was high (CV 3–9 per cent) and the recoveries varied between 88–116 per cent [183]. An HPLC method was employed for the study of the effect of pH on cream particle formation and solid extraction yield of black tea. Broken black tea (60g) was brewed with 2l of boiling distilled water for 10min then filtered. After cooling down, pH of aliquots of 45 ml were adjusted to 1.2, 2, 3, 7, 9, 11 and 13 with 3 M HCl and NaOH, respectively.

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TABLE 2.65 CONTENT OF FLAVONOLS IN DIFFERENT TEA LEAVES (G/KG DRY LEAVES)A AND IN DIFFERENT INFUSIONS OF GUNPOWDER TEA (MG/L)a

Tea leaves Green tea Gunpowder Zhejiang RGT Sencha Longjing Black tea Qimen Ceylon Infusions of gunpowder tea First Second Third Total

Myricetin

Quercetin

Kaempferol

1.590.04(22.1)b,c 0.930.04(15.1)

4.050.02(56.3) 2.840.03(46.2)

1.560.04(21.6) 2.380.03(38.7)

1.320.03(15.8) 0.830.02(16.5)

3.750.04(44.7) 1.790.02(35.6)

3.310.04(39.5) 2.410.01(47.9)

0.240.02(6.7) 0.520.04(9.9)

1.040.02(29.0) 3.030.02(57.5)

2.310.01(64.3) 1.720.02(32.6)

6.400.02 4.200.03 2.700.02 13.3

23.870.06 15.900.10 10.010.03 49.78

9.010.02 6.500.03 3.490.02 19.00

a

All the data for content of flavonols are average values of triplicate analyses. and are given with standard deviations. b The tea infusions were made according to the method described in the text. c Data in parentheses are expressed as percentage of the compound in three flavonoids. Reprinted with permission from H. Wang et al. [182].

Another experiment was carried out by adjusting the pH of the water and then they were employed for the preparation of the tea infusion. HPLC resulted in the separation of more than 50 components in the broken black tea infusion as demonstrated in Fig. 2.69. Unfortunately, the majority of peaks have not been identified and the chromatographic profiles only indicate the presence of many compounds in the infusions. Some results are presented in Table 2.66. The measurement indicated that theaflavins and tea catechins play a decisive role in the formation of cream particles and tea colour [184]. RP-HPLC with gradient elution was employed for the study of the influence of theaflavins and thearubigins on the adsorption of black tea on calcium carbonate. Separation of tea constituents was performed in an ODS column (250  4.9mm i.d.; particle size 5 ìm). Aqueous solvent was 1 per cent citric acid, pH adjusted to 2.8 with sodium hydroxide and the organic solvent was ACN. The gradient initiated at 8 per cent ACN, was increased to 31 per cent in 50min. Theaflavins and thearubigins were detected at 460 nm, while total polyphenolics were detected at 280 nm. The flow rate was 1.5 ml/min. The results demonstrated the involvement of theaflavins and thearubigins in the adsorption process [185].

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11 10

1

1000 18

2 500 12 13

3 9 6 4 5 78

1920

15 16 14 17

21

22 23

24

0 0

5

10

15

20

25

30

35

40

Fig. 2.68. HPLC trace of an extract of Australian-grown fresh tea shoots monitored at 280 nm: linear gradient 8–31 per cent acetonitrile in aqueous 2 per cent acetic acid over 50min. Peak identification: 1  theogallin; 2  gallic acid; 3  theobromine; 4  isochlorogenic acid; 5  gallocatechin; 6  epigallocatechin; 7  catechin; 8  p-coumarylquinic acid; 9  chlorogenic acid; 10  caffeine; 11  epigallocatechin gallate; 12  epicatechin; 13  p-coumaric acid; 14  3-(p-hydroxyphenyl) propionic acid; 15  gallocatechin gallate; 16  quercetin 3-rhamnosylglucoside; 17  epigallocatechin 3,5-digallate; 18  epicatechin gallate; 19  catechin gallate; 20  quercetin 3-glucoside; 21  quercetin glycoside; 22  kaempferol 3-rhamnosylglucoside; 23  kaempferol glycoside; 24  epicatechin 3,5-digallate. Reprinted with permission from L. Yao et al. [183].

The separation characteristics of RP-HPLC and capillary electrophoresis were compared for the analysis of tea catechins and theaflavins. Tea samples (0.1g) were extracted with 10 ml of boiling water and incubated at 90°C for 30 min. Tea extract was mixed with 20 per cent ACN in the ratio of 1:2 v/v, centrifuged and used for HPLC and CE analyses. RP-HPLC separations were performed in an ODS column (110  4.6 mm i.d.; particle size 5 m) at 32°C. Solvents for gradient elution were 5 per cent aqueous ACN containing 0.035 per cent (v/v) TFA (A) and 50 per cent ACN containing 0.025 per cent TFA (B). The Gradient began with 10 per cent B, it was increased to 20 per cent at 10 min, to 40 per cent at 16 min, to 50 per cent at 20min and back to 40 per cent from 25 to 27 min. CE investigations were performed in a 40cm  50 m i.d. capillary. The buffer solution was prepared by mixing 800 l of 500 mM boric acid (pH 7.2), 200l of 100mM KH2PO4 (pH 4.5), 450 l of 20 mM -cyclodextrin ( CD) and 550  l of ACN. The sample was injected by pressurized injection of buffer, sample, buffer, each at 2.5kPa for 3s. Separation was performed for 10min at 25kV at 30°C. The detection wavelength for both HPLC and CE was 205 nm. Chromatograms (A, B) and electropherograms (C, D) of a green tea and a black tea sample are shown in Fig. 2.70. The chromatograms and electrophoregrams indicate that CE is a more rapid method than HPLC because it separates the 16 analytes in 10 min. However, the detection limit was considerably higher than that of HPLC. The validation parameters (linearity, relative standard deviation of retention times, the reproducibility of

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Chapter 2 LCR 280, 4

550, 100

of TEA06 . D

24

15

1

120 100

mAU

80 27

60

18 7

20

12 35

40

14

21

16

22

2 10 3 4 5 8 13 6 11 9

17 20

23 30 32 33 34 25 26 29 31

10

36 3738 4041 43

20

(a)

44 45

47 48 49

30

50

40

Time (min) LCR 280, 4 1

10 12

550, 100

of TEA05 . D

24

120

29 15

100 27

mAU

80 60

26 25 18

35

40 17

20

7 2 3 11 1314 4 6 89

28

30 31 33 34 32 36 3738394041 43 42

16

10 (b)

19 21 22 20

20

30

47 44 45

48

50 49

46

40

Time (min)

Fig. 2.69. HPLC diagram of tea infusion monitored at 280 nm. (a) Infusion with pH 9.45; (b) infusion with pH 4.9. Reprinted with permission from Y. Liang et al. [184].

within-day and between-day analyses) of both methods were acceptable, suggesting that both techniques can be applied for the separation and quantitative determination of tea catechins and theaflavins. The amount of major tea polyphenols are compiled in Table 2.67. The coefficients of correlation again demonstrate the good agreement between the results determined by HPLC and CE. Because of the shorter analysis time and lower solvent consumption, the CE method has been proposed for this type of measurement [186].

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TABLE 2.66 HPLC RESULTS OF INFUSIONS WITH DIFFERENT PHS AT 280 NM

Peak

Compounds

Retention

Peak area (mm2)

time (min) pH 4.90 15 21 22 24 29 35 47 48 49 50 Total of 50 peaks

Gallic acid ()EGC ()EC Caffeine ()EGCG ()ECG TF TF3G TF3
G TF3.3
DG 24.43

9.392 14.184 14.834 15.562 19.277 24.408 38.961 40.801 41.913 42.294 7.65

965.41 240.69 128.42 9 957 1 281 556.23 235.31 223.31 46.44 261.23 2.88

pH 9.45 1 151 233.40 130.27 12 964 38.94 303.20 None None 24.52 68.83

TF  theaflavin; TF3G  theaflavin-3-gallate; TF3
G  theaflavin-3
-gallate; TF3,3
G  theaflavin3.3
-digallate. For other symbols see text. Reprinted with permission from Y. Liang et al. [184].

HPLC-DAD and HPLC-MS were employed for the determination of flavonols, flavones and flavanones in tea and other food products. For the analysis of aglycones freeze-dried samples were extracted by adding 40 ml of 62.5 per cent aqueous methanol to 0.500g of sample then the total volume was adjusted to 50 ml with 6 M HCl. The suspension was refluxed for 2h and after cooling down it was diluted with 100 ml of methanol and sinicated for 5 min. Liquid samples were treated similarly but the original sample volume was 15 ml and red wines were hydrolysed for 4h. For the determination of flavonoid glycosides the samples were extracted but the hydrolysis step was omitted. Separations were performed in ODS columns (250  4.6mm i.d.; particle size 5m). The components of acid hydrolysate were separated by solvents methanol–water (30:70, v/v) containing 1 per cent formic acid (A) and methanol (B). The linear gradient was 25–86 per cent B in 50min. Solvents for glycoside separation were 1 per cent aqueous formic acid (A) and ACN (B). The gradient was 5–60 per cent B in 60min. The Flow rate was in both cases 1 ml/min and analytes were detected in the range of 220–450 nm. MS parameters were: source temperature 150°C, probe temperature 450°C, cone voltage 30eV, corona discharge 1.6–1.9kV. Negative-ion mass spectra for aglycones and glycosides were taken between m/z 120–450 and 120–650, respectively. The quantitative results are compiled in Table 2.68. It has been stated that the extraction, hydrolysis, and RP-HPLC separation method is specific and sensitive for the analysis of flavonols, flavones and flavanons. The data can be used for the estimation of the daily intake of these compounds [187].

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204

TF3 TF4

TF1

TF2

CG

CA

T

A

C

EC

ECG

Caffeine

EGC

200 mV

0

TF3 TF4

20

EGCG

Minutes

TF1

CG

10

(b)

TF2

T

CA

C

EC

EGC

GA

EGCG

200 mV

ECG

Caffeine

(a)

ECG

GA

TFd

TFs C

CGTFc

EC

Caffeine

EGC

20 mAU

Caffeine

(c)

2 (d)

4

GA

TFd

TFs EGC EC TFb TFc

EGCG EGCG

20 mAU

6 Minutes

8

10

Fig. 2.70. Chromatograms (a, b) and electropherograms (c, d) of a green tea and a black tea sample. Reprinted with permission from B.-L. Lee et al. [186].

EC

6.06 7.27 5.27 4.90 6.06 6.93 8.59 9.91 0.49 0.33 4.27 4.85 1.75 1.88 1.41 2.19 y0.120.899x 0.988

HPLC

CE

5.34 6.13 9.97 8.98 12.66 13.79 12.58 13.21 0.07 0.11 3.35 3.28 3.58 3.07 6.82 8.92 y0.220.915x 0.981

HPLC

EGCG CE

36.53 35.59 28.07 21.06 23.46 24.56 18.62 19.92 0.60 0.12 30.61 29.41 7.70 3.57 2.84 3.23 y1.530.99x 0.977

HPLC

CE

18.10 23.12 35.46 32.93 29.83 30.96 16.85 19.11 0.30 0.12 11.82 12.24 8.99 7.36 5.52 9.18 y1.011.00x 0.977

TFs HPLC

CE

0.88 1.79 1.50 5.56 1.81 3.65 1.03 4.32 1.03 0.68 0.66 2.02 0.66 3.63 10.70 17.28 y0.780.629x 0.965

Page 205

Japanese green tea Long-jing Jasmine Chrysanthemum Pu-erh Iron Buddha Oolong tea Ceylon tea y-HPLC:x-CE r-coefficient of correlation

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Liquid chromatography of natural pigments

TABLE 2.67

Reprinted with permission from B.-L. Lee et al. [186].

205

Apple Apricot Bean, green Blackcurrant Blueberry Broccoli Brussels sprouts Celery, leaf Celery, stalk Cherry Cowberry Cranberry Grapefruit, pulp Grapes, blue Grapes, green Kale Leek Lemon, pulp Lime, pulp

18 1 2 3 1 5 2 3 4 1 1 1 2 2 1 4 2 1 1

2.00.4 2.6 1.60.6 3.70.1 7.3 3.72.5 0.60.1

1.0 21 16 0.50.1 3.73.0 0.2 122

0.4

Kaempferol

Hesperetin

Naringenin

Myricetin

Apigenin

Luteolin

7528 1.60.9

208 0.50.3

0.10.1

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6.03.4 0.90.1

0.5 0.40.1

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536

473 3.11.3 17 43

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TABLE 2.68

4521 1812 347

0.60.6 312 9.01.0

112 0.8

1.1

1855

1.00.6 1.5

0.50.1

1.40.5 1.40.8

1.60.5

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Liquid chromatography of natural pigments

Onion, red Onion, spring Onion, yellow Orange, pulp Orange, juice Parsley Pear, peel Plum, blue Red currant Red raspberry Red wine Rosebud Saladsb Strawberry Sweet pepper green Sweet pepper. red Sweet pepper yellow Tea Tomato

0.40.2 150.4

a

Number of samples analysed. Five different salads (cabbage) were analysed, cabbage lettuce, China cabbage, oxheart cabbage, iceberg salad, savoy, Duplicate analyses of two individual samples of each type of salad were performed, each with a content below 1.0 mg/100 g fresh weight. Reprinted with permission from U. Justesen et al. [187]. b

207

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RP-HPLC has been employed for the determination of flavonoids and other phenolic compounds in cranberry juice. The neutral and acidic analytes were preconcentrated octadecyl silica SPE cartridges conditioned with distilled water (neutral analytes) or with 0.01 M HCl (acidic compounds). Hydrolysis of samples was carried out in aqueous methanol solution acidified with 6 M HCl at 35°C for 16h. Chromatographic separation was performed in an ODS column (150  4.6mm i.d.; particle size 5m). Solvents A and B were water–acetic acid (97:3, v/v) and methanol, respectively. The gradient started with 0 per cent B (flow rate, 0.9 ml/min), reached 10 per cent B in 10min (flowrate, 1.0 ml/min) and increased to 70 per cent B in 40min (flowrate, 1.0 ml/min). Analytes were detected at 280 and 360 nm. Some typical chromatograms are presented in Fig. 2.71. The concentrations of flavonoids and phenolic acids are compiled in Table 2.69. It was stated that the SPE-HPLC procedure makes possible the simultaneous determination of phenolic compounds and flavonoids, therefore, it can be employed for the measurement of these classes of analytes in other fruit juices [188]. The content of organic acids and phenolic compounds in fruit juices and drinks has also been investigated by RP-HPLC-DAD. Fruit juices were centrifuged then filtered before injection, fruit drinks were filtered and injected without any other prepurification step. Separation was performed in an ODS column (250  4.6mm i.d.) at 40°C. Components of linear gradient were aqueous sulphuric acid (pH 2.5, solvent A) and methanol (solvent B). The initial flow rate of solvent A was 0.35 ml/min. The flowrate of solvent B was increased from 0 to 0.45 ml/min from 15 to 75min, and held at 0.45ml/min for 5min. The Spectra of analytes were recorded at 200–600 nm. It has been stated that the method separates 10 non-phenolic and 21 phenolic solutes in 80min, therefore, it can be employed for the authenticity test of these products and for the detection of spoilage [189]. Diosmin, hesperidin and naringin have also been determined in different citrus fruit juices and pharmaceutical formulations by reversed-phase high-performance liquid chromatography. Raw materials were sonicated at ambient temperature for 15min, filtered, mixed with internal standard solution, centrifuged and injected in to the RP-HPLC system. Tablets containing flavonoid glucosides were sonicated with methanol–dimethyl sulphoxide (DMSO) 1:1 v/v, the suspension was filtered and the liquid phase was injected. Separation was carried out in an octylsilica column (250  4.6 mm i.d.; particle size 5m) at 34°C. The isocratic mobile phase consisted of THF–water–acetic acid (21:77:2 v/v). The flow rate was 0.85 ml/min and analytes were detected at 280 nm. Chromatograms illustrating the separation of a standard mixture, commercial orange juice sample and a handsqueezed grapefruit juice sample are shown in Fig. 2.72. The peaks of analytes were symmetrical, sharp and well separated, and the retention times were 12.0, 14.0, 16.6, and 20.1 for diosmin, hesperidin, naringin, and internal standard, respectively. The concentrations of analytes in commercial and hand-sqeezed citrus fruit samples and in tablets are compiled in Table 2.70. The linearity range was 0.25–20g/ml, and the relative standard deviation of the method varied between 0.7 and 3.6 per cent. This simple, specific and accurate technique has been proposed for the separation and quantitative determination of these compounds in citrus fruit juices and pharmaceutical formulations [190]. The concentration of xanthohumol and other prenylflavonoids in hops and beer has been investigated by HPLC-MS. Beer samples were degassed, sonicated, and diluted with

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209

3

30 25

mAU

20 15 10 2

5

1

0

0

5

10

(a)

15

20

25

30

35

1

45

40

45

40

45

4

10

mAU

40

Minutes - 280 nm band = 10 nm

2

5

3

0

0

5

10

(b)

15

20

25

30

35

Minutes - 280 nm band = 10 nm 8

mAU

50

7

4 25 5 6

2 3 1 0

0 (c)

5

10

15

20

25

30

35

Minutes - 280 nm band = 10 nm

Fig. 2.71. HPLC chromatogram of the neutral (a) and acidic fractions (b) and the acid-catalysed hydrolysed product of freshly squeezed cranberry juice (c) at 280 nnm. Peaks in a: 1  ()-catechin; 2  myicetin; 3  quercetin (added as internal standard). Peaks in b: 1  anthocyanin derivative I; 2  benzoic acid; 3  p-anisic acid; 4  quercetin (added as internal standard). Peaks in c: 1  ()-catechin; 2  anthocyanin derivative I; 3  anthocyanin derivative II; 4  benzoic acid; 5  anthocyanin derivative III; 6  p-anisic acid; 7  myricetin; 8  quercetin. Reprinted with permission from H. Chen et al. [188].

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TABLE 2.69 CONCENTRATIONS OF FLAVONOIDS AND PHENOLIC ACIDS IN CRANBERRY JUICE SAMPLES

Component

Concentration (mg/l) Canned cranberry juice

()-catechin Chlorogenic acid p-coumaric acid Benzoic acid p-anisic acid Myricetin Quercetin

1.0 5.1 5.2 34 nd 1.7 nd

Hydrolysed products of the canned juice 2.0 nd 6.1 28 nd 2.9 12

Fresh cranberry juice 8.1 nd nd 41 2.2 8.3 nd

Hydrolysed product of freshly squeezed juice 9.8 nd nd 178 3.2 47 175

nd, Not detected. Reprinted with permission from H. Chen et al. [189].

ethanol–water (5:95 v/v). Herb teas containing hops were extracted with boiling water. Ground hops (400mg) were sonicated with 100 ml of methanol then filtrated and diluted with methanol–formic acid (99:1, v/v) and used for HPLC analysis. Separations were carried out in an ODS column (250  4.6mm i.d.; particle size 5m). The Linear gradient started with 40 per cent ACN in 1 per cent aqueous formic acid. ACN concentration was increased to 100 per cent in 15min and held for 5min. The flow rate was 0.8 ml/min. The conditions of APCI in the positive-ion mode were: temperature of nebulizer interface 500°C, corona discharge needle 8kV, discharge current 3A, orifice plate voltage 55 V. The results of HPLC-MS-MS analysis of a methanolic extract of hops and those of a commercial beer are presented in Figs. 2.73, 2.74. The prenylflavonoid contents are listed in Table 2.71. The coefficient of variation of the within-day precision of the analysis varied between 3.8 and 11.4 per cent. The correlation coefficient of linearity was in each case over 0.998. The data presented indicate that the method can be applied for the separation, identification and quantitation of prenylflavonoids in hops and beer [191]. HPLC with fluorescence detection was employed for the analysis of riboflavin (RF), flavin mononucleotide (FMN) and flavin–adenin dinucleotide (FAD) in beer, wine and other beverages. The investigation was motivated by the finding that these compounds are responsible for the so-called ‘taste of light’ which develops in beverages exposed to light. Samples were filtered and injected in to the analytical column without any other pretreatment. Separations were carried out in an ODS column (200  2.1mm i.d.; particle size 5m). Solvents A and B were 0.05 M phosphate buffer (pH 3) and ACN, respectively. The

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211 n

h

c IS

IS

IS h

d

Absorbance 280nm

Absorbance 280nm

Absorbance 280nm

d n

d

0 20 (a) Time (min)

20 (b) Time (min)

20 (c) Time (min)

Fig. 2.72. Examples of HPLC chromatograms: (a) calibration standards; (b) commercial orange fruit juice sample; (c) hand-sqeezed grapefruit juice sample. Peaks: (d) diosmin; (h) hesperidin; (n) naringin; (IS) rhoifolin (internal standard). Reprinted with permission from. F. I. Kanaze et al. [190].

gradient started with 95 per cent A, and changed to 75 per cent A in 8 min and the flow rate was 0.6 ml/min. Excitation and emission wavelengths for the fluorescence detection were 265 and 525 nm using a 500 nm cut-off filter. The good separaton of the analytes under the optimal conditions is demonstrated in Fig. 2.75. The quantities of analytes in the samples and the sensorial values of control and samples spiked with RF are compiled in Table 72.

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TABLE 2.70 CONCENTRATIONS OF DIOSMIN. HESPERIDIN. AND NARINGIN IN COMMERCIAL AND HAND-SQUEEZED CITRUS FRUIT JUICE SAMPLES (G/ML). AND IN TABLETS

Citrus fruit juice samples Orange 1 2 3 4 5 6 7 8 9 10 MeanS.D. Range Tangerine 1 2 3 4 5 6 MeanS.D. Range Lemon 1 2 3 MeanS.D. Range Grapefruit 1 2

Diosmin (g/ml)

Hesperidin (g/ml)

Naringin (g/ml)

22.9 64.4 45.9 26.4 7.9 26.4 21.9 22.7 72.3 35.7 34.7 7.9

141.8 443.0 247.0 207.1 53.8 217.2 102.9 138.5 735.3 179.5 246.6 53.8

— — — — — — — — 2.3 — — —

9.6 6.7 21.2 7.4 14.4 15.7 12.5 6.7

52.1 45.9 48.9 86.1 170.5 237.7 166.7 45.9

— — — 1.2 0.5 0.7 0.8 0.5

1.6 11.9 17.8 10.4 1.6

15.9 47.2 28.8 30.6 15.9

— 3.8 — — —

1.9 13.7

164.0 43.9

584.7 351.3

Reprinted with permission from F.I.Kanaze et al. [190]

The repeatabilities of retention time and concentration were between 0.29–0.70 per cent and 1.35–3.06 per cent, respectively. The LOD and LOQ values were 0.49–1.97 and 1.72–6.57g/l. The linearity range varied between 2.00–222.46 [192]. Another RP-HPLC method was applied for the determination of gallic acid, transveratrol, quercetin and rutin in red wines. Samples of wines were filtered and injected into

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Rel. int. (%) Rel. int. (%)

100

Rel. int. (%)

100

Rel. int. (%)

m/z 355 100

100

213

179

289,678

2

50

1

0 m/z 241

121

m/z 341

165

72,845

3

50 0

5

50

4

9870 6

0 m/z 409

165

1002

7 8

50 0 0.0

5.0

10.0

15.0

20.0

Time (min)

Fig. 2.73. HPLC-MS-MS analysis of a methanolic extract of hops. peaks: 1  isoxanthohumol; 2  xanthohumol; 3  2
,4-dihydroxy- chalcone (internal standard); 4  8-prenylnaringenin; 5  desmethylxanthohumol; 6  6-prenylnaringenin; 7  3
geranylchalconaringenin; 8  6-geranylnaringenin. Prenylflavomoids were detected in a single HPLC run by multiple-reaction ion monitoring; vertical lines in the panels indicate start of a new scanning period. For other details see text. Reprinted with permission from. J. F. Stevens, et al. [191].

the analytical column without any other pretreatment. Measurements were carried out on in ODS column (250  4mm i.d.; particle size 5m). Components of the mobile phase were acetic acid (A), methanol (B) and water (C). The Gradient started with A–B–C (5:15:80, v/v) at the flow rate of 0.4 ml/min. After 5min the mobile phase was changed to A–B–C (5:20:75, v/v) at the flow rate of 0.5 ml/min and at 30min the composition was A–B–C (5:45:50, v/v) at the same flow rate and it remained the same till 50min. The chromatographic profile of a red wine sample determined at 280 and 360 nm is shown in Fig. 2.76. The retention times for analytes were 6.7, 36.5, 37.1 and 41.5min for gallic acid, rutin, trans-veratrol and quercetin, respectively. It was found that the concentration of analytes shows high variations between the red wine samples as demonstrated by the quantitative data in Table 2.73. It was suggested that the method can be used for the measurement of phenolic compounds not only in wines but also in other alcoholic beverages such as beers and liqueurs [193]. A combination of various chromatographic techniques, such as RP-HPLC-DAD, RPHPLC-MS, solid-phase microextraction (SPME) – GC, was employed for the elucidation

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Rel. int (%) Rel. int (%)

100

Rel. int (%)

100

Rel. int (%)

m/z 355 100

100

179

245,820

1 2

50 0 m/z 241

121

m/z 341

165

52,655

3

50 0 58,066

5 4

50 0 m/z 409

165

7

3958

6

50 0 0.0

5.0

10.0

15.0

20.0

Time (min)

Fig. 2.74. HPLC-MS-MS analysis of a commercial beer. Peaks: 1  isoxanthohumol; 2  xanthohumol; 3  2
,4-dihydroxychalcone (internal standard); 4  8-prenylnaringenin; 5  6-prenylnaringenin; 6  probably 8-geranylnaringenin 7  6-geranylnaringenin. Prenylflavomoids were detected in a single HPLC run by multiple-reaction ion monitoring; vertical lines in the panels indicate start of a new scanning period. For other details see text. Reprinted with permission from J. F. Stevens et al. [191].

of the similarities and dissimilarities between the red wines from the Portuguese mainland and the Azores Islands. For the extraction of polyphenolic compounds 5 ml of wine was neutralized to pH 7 with 1 M NaOH, diluted to 10 ml with distilled water and 5 ml of solution was loaded into a neutralized ODS cartridge. The cartridge was washed with 20 ml of dihydrogenphosphate buffer (pH 7) then it was dried under nitrogen flow. Analytes were eluted with 5 ml of ethyl acetate, the solution was evaporated to dryness and redissolved in methanol. Polyphenolics were separated in an ODS column (100  4.6mm i.d.; particle size 5m) at 35°C. Solvents were 0.4 per cent (v/v) ortho-phosphoric acid (A) and ACN (B). The Gradient programme was: 0–5min 100 per cent A; 20min, 85 per cent A; 60min 37.5 per cent A. The flow rate was 1 ml/min. A characteristic chromatogram illustrating the good separation capacity of the RP-HPLC system is shown in Fig. 2.77. The amounts of analytes are compiled in Table 2.74. The results indicated that the concentration of polyphenols in red wines depends on both the grape variety and on the exogenous factors. The validation parameters of the method were good, the recoveries were in each case over 98 per cent, the coefficients of variation were between 1.3 per cent and 4.3 per cent, and the limit of detection ranged from 10g/l to 0.1mg/l. It was stated that the method is suitable for the determination of silbene compounds and quercetin in red wines [194].

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215

TABLE 2.71 PRENYLFLAVONOID CONTENTS IN BEERS AND HERB TEAS. MG/L (C.V., n2)a

No. US MAJOR BRANDS 1 2 3 4 Northwest/US microbrews 5 6 7 8 Imported beers 9 10 11 12 Other beverages 1 2 3 No. US major brands 1 2 3 4 Northwest/US microbrews 5 6 7 8 Imported beer 9 10 11 12

Beer type

Xanthohumol

Isoxanthohumol 8-Prenylnaringenin

Lager/pilsner Lager/pilsner Lager/pilsner Lager/pilsner

0.034 (4.0) 0.009 (1.8) 0.014 (4.1) —

0.50 (1.3) 0.68 (0.1) 0.40 (3.0) —

0.013 (1.7) 0.014 (4.2) 0.017 (0.3) —

Am. Porter Am.hefeweizen Strong ale India pale ale

0.69 (1.1) 0.005 (0.9) 0.24 (0.2) 0.16 (3.6)

1.33 (3.8) 0.30 (0.2) 3.44 (1.5) 0.80 (8.3)

0.24 (0.7) 0.008 (2.5) 0.11 (0.5) 0.039 (5.2)

Imported stout Imported lager Imported pilsner Imported pilsner

0.34 (2.4) 0.002 (15) 0.028 (8.8) 0.012 (3.5)

2.10 (0.4) 0.04 (3.2) 0.57 (1.2) 1.06 (0.3)

0.069 (2.2) 0.001 (141) 0.021 (1.8) 0.008 (4.0)

Non-alcohol beer Herb tea Herb tea

0.003 (14) 0.004 (18) —

0.11 (1.9) 0.009 (3.7) —

0.003 (9.2) 0.002 (2.7) —

Beer type

6-prenylnaringenin

6-geranylnaringenin

Total

Lager/pilsner Lager/pilsner Lager/pilsner Lager/pilsner

0.0034 (9.2) 0.038 (1.0) 0.031 (1.4) —

0.011 (2.3) 0.006 (0.1) 0.005 (1.4) —

0.59 0.75 0.46 —

Am. Porter Am.hefeweizen Strong ale India pale ale

0.56 (2.2) 0.011 (2.1) 0.20 (0.3) 0.146 (2.4)

0.074 (7.9) 0.001 (2.1) 0.006 (15) 0.016 (8.0)

2.90 0.33 4.00

Imported stout Imported lager Imported pilsner Imported pilsner

0.17 (0.7) 0.001 (28) 0.055 (2.0) 0.022 (7.7)

0.007 (6.8) — 0.007 (6.3) 0.001 (16)

2.68 0.04 0.68

(Continued on next page)

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TABLE 2.71 (continued)

No.

Beer type

6-prenylnaringenin

6-geranylnaringenin

Total

Other beverages 1 2 3

Non-alcohol beer Herb tea Herb tea

0.007 (8.2) 0.004 (5.4) —

— — —

0.12 0.02 —

a Trace of desmethylxanthohumol also present. Reprinted with permission from J.F. Stevens et al. [191].

2

20 %F

15

25

25

20

20 15

15

15

1

%F

20

2

%F

25

%F

25

3

3

1

2

1 3

10

10

10

10

5

5

3 5

(a)

5 5

6

7

(b)

5

6

7

(c)

5

6

7

(d)

5

6

7

Fig. 2.75. Chromatographic analysis of a standard solution (a), an orange juice (b), a white wine (c), and a wheat beer (d). 1  FAD; 2  FMN; 3  RF. Time scale in min; F  fluorescence. Reprinted with permission from C. Andrés-Lacueva et al. [192].

The efficacy of various solid-phase extraction procedures for the preconcentration of pigments from red wines before RP-HPLC analysis has been extensively studied. SPE cartridges were preconditioned with 3 ml of acetone, 3 ml of methanol and 3 ml of distilled water then red wines were loaded into the cartridges at a flow rate of 0.2 ml/min until the pigments appeared at the vent. After loading the cartridges were washed with 5 ml of distilled water and pigments were removed with ACN, methanol, THF and dioxane, each of them containing 10 per cent (v/v) conc. formic acid. The sample capacities of SPE sorbents are compiled in Table 2.75. Desorption experiments indicated that the pigments are very strongly bonded to SPE sorbents X and XI, while they can be easily eluted from the octadecylsilica SPE cartridge. Because of their high sample capacity and high preconcentration factor, ODS SPE cartridges were employed in further experiments. RP-HPLC-DAD analyses were carried out in an ODS column (250  4mm i.d.; particle size 5m) at ambient temperature. Solvents A and B were

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TABLE 2.72 FAD. FMN AND RF CONCENTRATION (G/L) FOR WINES. BEERS AND FRUIT JUICES AND THE ESTIMATE OF THE AVERAGE INTENSITY OF THE DEFECT SUNLIGHT FLAVOUR AFTER EXPOSURE TO LIGHT OF SAMPLES

Sample No.

Flavin content FAD (g/l)

Pinot Grigio wines Clone 1 nd 2 nd 3 nd 4 nd 5 nd 6 nd 7 nd 8 nd Vineyard (Vallagarina, Trentino, Italy) 9 nd 10 nd 11 nd 12 nd Producer 13 nd 14 nd 15 nd 16 nd Beers 17 45.8 18 19.3 19 23.2 20 45.4 21 65.2 22 50.2 Fruit juices 23 104.3 24 171.8 25 100.8 26 131.0

FMN (g/l)

Sensorial values RF (g/l)

Control

SampleRF

nq nq nq nq nq nq nq nq

85.5 94.8 63.2 77.2 56.4 64.6 69.1 77.7

0.38 0.38 0.43 0.43 0.43 0.71 0.29 0.29

1.88 1.88 2.00 2.00 1.86 2.00 2.00 2.00

nd nd nd nd

89.9 114.9 104.5 106.4

0.63 0.88 1.00 0.25

1.75 1.88 1.88 1.75

3.3 nq 2.9 nq

67.4 44.3 83.3 105.8

0.22 0.11 0.78 1.00

2.00 1.11 1.89 2.00

nd nd nd 8.1 nd nd

291.7 235.8 169.0 272.3 398.9 507.9

0.29 0.14 0.29 0.43 0.43 0.29

0.57 1.14 1.29 1.86 1.43 0.71

17.3 54.4 39.4 36.7

68.3 21.7 39.2 3493

0.14 0.11 0.14 2.00

1.89 1.88 1.75 2.00

nd, not detected; nq, not quantified. Sample identification: 1  SMA 505 (Istituto Agrario S. Michele a/A, Italy), 1996; 2  SMA 514 (Istituto Agrario S. Michele a/A, Italy), 1996; 3  GM 1 (Forschungsanstalt Geisenheim. Germany), 1996; 4  GM 2 (Forschungsanstalt Geisenheim, Germany), 1996; 5  R 6 (Vivai Cooperativi Rauscedo. Italy), 1996; 6  49207 Fr (Staatliches Weinbauinstitute Freiburg, Germany), 1996; 7  INRA-CV 52 (INRA, France), 1996; 8  INRA-CV 53 (INRA, France), 1996; 9  Bagolé. 1996; 10  Palazzina, 1996; 11  Acquaviva PR; 12  Sabbioni; 13  Gaierhof; 14  Girelli; 15  Mezzacorona; 16  Lagari; 17  Heineken; 18  Forst Premium; 19  Moretti; 20  Franziskaner Hefe-Weissbier; 21  Maisel’s Weisse; 22  Forst Sixtus; 23  Pineapple; 24  Orange; 25  Grapefruit; 26  Tropical (addition of RF declared). Reprinted with permission from C. Andrés-Lacueva et al. [192].

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Chapter 2 0.4 1 0.3

Abs

280 nm 0.2

0.1

0 0.06 0.05

Abs

0.04

2 360 nm

0.03 0.02 0.01 0 5

10

15

20

25

30

35

40

45

50

min

Fig. 2.76. Chromatogram for a red wine sample using gradient elution and photodiode array detection. The peaks correspond to: 1  gallic acid, measured at 280 nm; 2  rutin, measured at 360 nm. Reprinted with permission from M. López et al. [193].

bidistilled water–cc. formic acid (9:1, v/v) and bidistilled water–ACN–cc. formic acid (6:3:1, v/v), respectivly. The gradient started with 100 per cent A, decreased to 75 per cent in 20min, remained at 75 per cent till 45min, further decreased to 50 per cent in 85min and remained the same till 150min. The flow rate was 1 ml/min. Some chromatograms illustrating the effect of preconcentration on the results of RP-HPLC measurements are shown in Fig. 2.78. The chromatograms demonstrate that the SPE preconcentration step enhances the sensitivity of the measurements, therefore, its application in the analysis of the pigments of red wines is highly recommended [195]. The newly developed monolithic-type column has also found application in the HPLC determination of wine phenolics. Red wine samples were filtered and injected into the column without any other pretreatment. Separations were performed in an ODS monolithic column (100  4.6mm i.d.) at 301°C. Solvent A was methanol–double-distilled water (2.5:97.5, v/v) at pH 3 with H3PO4, and solvent B consisted of methanol–double-distilled water (50:50, v/v) at pH 3 with H3PO4. Conditions of gradient elution were as follows: 0–10min 100

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TABLE 2.73 PHENOLIC COMPOUND CONCENTRATIONS AND PHENOL DETECTED IN SEVERAL COMMERCIAL RED WINES

Wine

Phenolicsa (mg/l)

Gallic acid (g/ml)

Rutin (g/ml)

Transresveratrol (g/ml)

Quercetin (g/ml)

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

1 857 2 315 1 848 2 050 1 950

53.3 46.2 48.1 38.8 27.7

nd nd nd 4.62 nd

nd 1.34 nd nd nd

nd nd nd nd 4.66

nd, not detected. a Total phenols are expressed as gallic acid equivalents (GAEs). Reprinted with permission from M. López et al. [193].

2.5

3

35.870 36.758

Stop

4 5

41.168

2 32.217

30.158

29.017

33.400 33.633

27.746 26.125

1.0

25.195

1.5

26.988 30.008

2.0

24.292

Absorbance at 306 nm

1

0.5 Retension time (min)

Fig. 2.77. Chromatogram of a reverse-phase HPLC of Azorean (Basalto) red wine monitored at 306 nm. Peaks: 1  trans-pieceid; 2  cis-pieceid; 3  trans-resveratrol; 4  cis-resveratrol; 5  quercetin. Reprinted with permission from J. A. B. Baptista et al. [194].

per cent A; 15min, 82 per cent A; 20min, 75 per cent A; 22min, 65 per cent A; 30 min, 0 per cent A, 34 min, 0 per cent A. Flow rate was 2.1 ml/min. The good resolution of analytes is illustrated in Fig. 2.79. The numerical data are listed in Table 2.76. The results indicated that the calibration curves are significantly linear. The LODs varied between 8 and 161  g/l, the intra-day and inter-day RSDs were under 3 per cent and 6 per cent, respectively, and the

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TABLE 2.74 CONCENTRATION OF HYDROXYSTILBENE COMPOUNDS AND QUERCETIN (MG/ML) IN DIFFERENT WINES FROM PORTUGUESE MAINLAND AND AZORES ISLANDS

Wine and vintage

Stilbenes (mg/l)

Piceid

Lagos,1993c Frei Bernardo Tino Talha, 1997d J.P., 97c Uva Estremadura, 1998d Bairrada, 1995c Dao, 1996d Ouro Velho, 1998d Douro, 1995d Continental Portugal average value Basalto, 1997d Terras do Conde, 1994d S. Miguel, 1998c Azorean average value

Quercetin total (mg/l)

Resveratrol

Total (mg/l)

transa

cisb

Total

trans

cis

Total

2.16 2.21 2.88 1.11 2.81 4.95 3.10 2.05 1.96 2.58

0.11 0.35 0.37 0.55 0.63 0.09 0.28 0.26 0.12 0.31

2.27 2.56 3.25 1.66 3.44 5.04 3.38 2.31 2.08 2.89

1.04 1.93 0.84 1.13 0.63 2.87 1.84 1.73 2.02 1.56

0.31 0.25 0.72 0.30 0.20 0.21 0.23 0.26 0.24 0.30

1.35 2.18 1.56 1.43 0.83 3.08 2.07 1.99 2.26 1.86

3.62 4.74 4.81 3.09 4.27 8.12 5.45 4.30 4.34 4.75

7.62 4.48 4.46 4.69 2.79 4.26 2.39 4.73 3.96 4.38

2.07 3.62 0.19 1.96

0.30 0.10 0.15 0.18

2.37 3.72 0.34 2.14

5.21 2.35 2.53 3.36

0.50 1.70 0.28 0.83

5.71 4.05 2.81 4.19

8.08 7.77 3.15 6.33

2.96 1.81 11.95 5.57

a

Quantified as trans-resveratrol. Quantified as cis-resveratrol. c Monovarietal. d Blended variety. Reprinted with permission from J. A. B. Baptista et al. [194]. b

recoveries were 95–103 per cent. The data proved that the separation characteristics of monolithic column are better than those of traditional ODS columns, and that direct injection makes possible the rapid analysis of polyphenolics in both white and red wines [196]. The optimal conditions for MS and DAD detection of phenolic compounds in wine have been elucidated. Separation of analytes in wine was carried out in an ODS (250  4.6 mm i.d.; particle size 3m) at room temperature. Eluents A and B were 4.5 per cent formic acid in water and eluent A–ACN (90:10, v/v, B), respectively. The linear gradient started at 0 per cent B, reached 50 per cent B in 25min; 80 per cent B in 35min, and final hold for 20min. The flow rate was 0.7 ml/min. Analytes were detected at 280 nm. APCI and API-ES measurements were performed by applying various fragmentor voltages and two ionization modes (positive and negative). Nitrogen was employed as a nebulizing and

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TABLE 2.75 SPE SORBENTS USED FOR THE PRECONCENTRATION OF PIGMENTS OF RED WINES AND THEIR SAMPLE CAPACITY

No.

Sorbent Type

Sample capacity (ml red wine)

I II III IV V VI VII VIII IX X XI

Silica Octadecylsilica Cyanopropyl Alumina (acidic) Alumina (basic) Alumina (neutral) Diol Aminopropyl Florisila Accell Plus QMAb Carbon black

1 10 1 1 1 1 1 8 1 5 5

a

Polar. higly active. weakly basic adsorbent. Silica-based hydrophilic. strong anion exchanger with large pore size. Reprinted with permission from G. A. Csiktusnádi-Kiss et al. [195]. b

drying gas. API-ES conditions were: N2 pressure, 380 Pa; drying gas, 10l/min at 350°C; ion spray voltage 4 000 V. Mass range was m/z 50–500 for low-molecular mass phenols and m/z 150–1 000 for flavan-3-ol compounds. The results indicated that the sensitivity of APCI is inferior to that of the API-ES detection technique. The DAD chromatogram and selected mass chromatograms of flavan-3-ols obtained by negative- and positive-ion modes are shown in Fig. 2.80. The mass chromatograms clearly demonstrate that both negative and positive ion mode can be used for the detection of this class of analytes using the optimal fragmentor voltage of 60 V. The successful application of the HPLC-DADMS technique for the analysis of a wine sample is demonstrated in Fig. 2.81. It was concluded from the data that API-ES-MS detection offers an excellent possibility for the analysis of phenolic solutes decreasing the possible errors caused by coelution of two or more analytes [197]. Another study employed a similar RP-HPLC method for the determination of trans- and cis-resveratrol, catechin, epicatechin, quercetin and rutin in wines and musts. Wine samples were filtered and diluted when necessary and used for analysis without any other pretreatment. Separation was performed in an ODS column (150  4 mm i.d.; paricle size 5 m) at ambient temperature. The gradient began with ACN-5 per cent aqueous acetic acid (9:91, v/v) for 0–10 min; to 25:75 in 1 min; hold for 11 min; to 70:30 in 1 min, hold for 5 min. The flow rate was 1 ml/min. Analytes were detected by DAD. Fluorescence detection used 280/315 nm (excitation/emission) for catechin and epicatechin; 314/370 nm for trans-resveratrol and 260/370 nm for cis-resveratrol. Chromatograms of a red wine sample obtained at different

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0.014

Absorbance 440 nm

Absorbance 440 nm

0.014

0

0 0

(a)

150

0

(b)

Minutes

0.01

Absorbance 570 nm

Absorbance 570 nm

0.01

0

0 0

(c)

150 Minutes

150 Minutes

0

(d)

150 Minutes

Fig. 2.78. RP-HPLC chromatograms of a red wine without preconcentration (a) and with preconcentration (b) at 440nm, and without preconcentration (c) and with preconcentration (d) at 570 nm. For chromatographic conditions see text. Reprinted with permission from G. A. Csiktusnádi-Kiss et al. [195].

wavelenghts is shown in Fig. 2.82. The investigations proved that the sensitivity of fluorescence (FL) detection is higher than that of DAD but the amounts of compounds measured by both detection modes are highly similar as demonstrated in Table 2.77. Detection and determination limits ranged between 0.06–0.21g/ml and 0.22–0.67  g/ml, respectively, the RSD was 1.6–8.6 per cent and the linearity range was 0.2–25  g/ml depending on the type of analyte. It was concluded from the results that the procedure can be applied for the determination of these analytes in wines [198]. The separation and identification of flavanol–anthocyanin adducts in wine and in model solutions were performed with RP-HPLC coupled to DAD or ESI-MS. The investigation was motivated by the assumption that the formation of flavanol–anthocyanin complexes may influence the organoleptic characteristics of wine during ageing. Measurements were carried out in an ODS column (250  2 mm i.d.; particle size 5 m) at 30°C. The flow rate was 0.25 ml/min. Solvent A was water–formic acid (95:5), solvent B consisted of ACN–solvent A (80:20, v/v). The Gradient elution began with 3 per cent B for 7min; to 20 per

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256 nm 1

20

mAU

15 2

10

3 4

5

7 14 6

9

0 0

5

10

15

20

25

30

35

[min] 324 nm

A

20

mAU

15 11 10

5

B

8

10

5 0 0

5

10

15

20

25

30

35

[min] 15

20

mAU

15

365 nm

13 12

10 5 0 0

5

10

15

20

25

30

35

[min]

Fig. 2.79. Chromatograms of a white (I) and red wine sample (II). (LC-DAD signals at three different wavelenghts: 256, 324, 365 nm). Peak identification: 1  gallic acid; 2  protocatechuic acid; 3  p-hydroxybenzoic acid; 4  vanillic acid; 5  caffeic acid; 6  ()-catechin; 7  syringic acid; 8  p-coumaric acid; 9  ()-epicatechin; 10  ferulic acid; 11  trans-resveratrol; 12  rutin; 13  myricetin; 14  cis-resveratrol; 15  quercetin; A  caftaric acid; B  coutaric acid. Reprinted with permission from M. Castellari et al. [196].

cent B in 15 min; to 30 per cent B in 8min; to 40 per cent B in 10min; to 50 per cent B in 5 min, and to 80 per cent B in 5min. DAD detection was carried out between 220–600 nm. The conditions of the positive ion mode ESI-MS were: m/z 150–2 000; source voltage, 4.5kV; capillary voltage, 23.5 V; capillary temperature, 250°C; collision energy for fragmentation,

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TABLE 2.76 POLYPHENOLIC CONSTITUENTS (MG/L) IN RED AND WHITE WINE

Red wine (n  5)

Gallic acid Protocatechuic acid p-hydroxybenzoic acid Vanillic acid Caffeic acid ()-catechin Syringic acid p-coumaric acid ()-epicatechin Ferulic acid trans-resveratrol Rutin Myricetin cis-resveratrol Quercetin Caftaric acid Coutaric acid

White wine (n  9)

Max

Min

Max

Min

61 8.9 0.3 2.2 8.7 40 2.2 3.1 23 4 1.5 10 4.4 0.2 6.2 75 6.2

39 3.4 nd 1.6 2.2 23 1.3 0.5 18 nd 0.2 3.6 1.7 nd 3.5 20 2

13 1.2 0.07 0.4 7.7 13 0.98 1.4 4.4 0.47 0.32 2.7 0.26 nq 1.7 79 18

1.6 0.5 nd nd 0.6 3.1 0.41 nd 2 nd nd nd nd nd nd 12 1.2

nd, not detected, concentration lower than LOD. nq, not quantified, concentration lower than LOD  10. Reprinted with permission from M. Castellari et al. [196].

25 per cent for MS2, 30 per cent for MS3 and 35 per cent for MS4. Two flavanol–anthocyanin adducts were isolated from the wine sample as illustrated in Fig. 2.83. It was concluded from the data that the interactions between carbocations resulting from the cleavage of tannin interflavanis bonds and anthocyanins occur in wine [199]. 2.2.3.4 HPLC determination of flavonoids in other matrices Because of their beneficial biological effects, the fate of flavonoids in human tissues has been extensively investigated [200, 201]. Mass spectrometry as a reliable detection technique has been frequently used in the HPLC analysis of flavonoids [202, 203]. The content of quercetin in human plasma after consuming canned green tea has been determined by RP-HPLC and electrochemical detection. Hydrolysis of quercetin in green tea samples was carried out by mixing 1 ml of tea with 1.5 ml of mobile phase, ultrasonicated for 3 min, mixed again with 0.5 ml of 6 M HCl and heated at 90oC for 2 h. After cooling the sample was filtered, diluted 100 times and injected into the column.

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40 20 0 −20

7.202

4.522

2.302

mAU

DAD1 B , Sig=280,

2

225

4

6

N

8

2.437

3000 2000 1000 0

4.704

min MSD1 289,

Catechin

2

(M-H)Epicatechin

4

N

6

8

min MSD1 305, N 2.280

15000 10000 5000 0

Epigallocatechin

2

4

6

8

min MSD1 457, Epigallocatechin-gallate

4.548

4000 2000

N

0 2

4

6

8

min 7.246

MSD1 441, 15000 10000 5000 0

N

Epicatechin-gallate

2

4

min

6

2

P

4.595

2.349

2.909

mAU

60 40 20 0 −20

8 7.315

DAD1 B, Sig=280,

4

min

6

8

10000

P Epicatechin

4.756

2.463

MSD1 291, Catechin

5000 0 4

2

8

6 min

MSD1 307, P 2.302

40000 30000 20000 10000 0 2

Epigallocatechin

4

6

8

min MSD1 459, P

8000 6000 4000 2000 0

4.635

Epigallocatechin-gallate

2

4

6

8

min MSD1 443, P

30000

Epicatechin-gallate 7.370

20000 10000 0 2

4

6

8

min

Fig. 2.80. DAD chromatogram and selected mass chromatograms of flavan-3-ols obtained by negative (N) and positive (P) ion mode API-ES-MS coupled to HPLC-DAD. Reprinted with permission from S. Pérez-Magarino et al. [197].

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Chapter 2 DAD1 B, Sig=280,4 Ref=off Catechin

mAU

2000

Epicatechin

1000

Gallic acid

Epicatechin-gallate

0 0

10

20

30

50

60

70

50

60

70

API-ES negative 30.228

MSD1 TIC, MS file

40 min

75.570

10.287

3000000

44.132

5000000

1000000

0 0

10

20

30

40 min

Fig. 2.81. DAD and TIC (total ion current) chromatogram of a wine sample using negative-ion APIES detection. Reprinted with permission from S. Pérez-Magarino et al. [197].

Plasma samples were mixed (0.01 l) with 50 l of 2 M acetate buffer (pH  5) containing 10 mg/ml ascorbic acid and 20 l of 500 U -glucuronidase. The suspension was held at 37°C for 3h then extracted with 3  200 l of ethyl acetate. The combined organic extracts were evaporated to dryness and redissolved in 100 l of methanol–water (4:6, v/v). Separation was performed in a microbore ODS column (150  1mm i.d.; particle

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mAbs

150

227

ch2 280nm 1

100 50

2

0 200

ch2 300nm

150 100 4

150 0 200

ch3 360nm

6

150 100 150 0 0

10

20

30

min

Fig. 2.82. Chromatograms for a red wine sample using gradient elution and photodiode array detection. Flow rate, 1ml/min. Peak identification: 1  catechin; 2  epicatechin; 4  trans-resveratrol; 6  quercetin. Reprinted with permission from P. Vinas et al. [198].

size 3 m) at 40°C. The detection unit consisted of a glassy carbon working electrode, an Ag/AgCl reference electrode, and a stainless steel auxiliary electrode. The detection potential was set to 0.5 V versus Ag/AgCl. The isocratic mobile phase was methanol–water (4:6, v/v) containing 0.5 per cent phosphoric acid. The flow rate was 25 l/min. Typical chromatograms of commercial green tea and plasma samples are shown in Fig. 2.84. The chromatograms prove that both quercetin and luteolin are well separated from the impurities in both tea and plasma matrices. The method was very sensitive, the detection limit being 0.3 pg of quercetin, therefore it was proposed for the further study of the fate of quercetin in human tissues [204]. An octylsilica stationary phase was employed for the HPLC detemination of the flavone aglycone diosmetin in human plasma. Plasma samples (1 ml) spiked with diosmetin and internal standard were mixed with 2 ml of 0.1 M phosphate buffer (pH  2.4) and the suspension was extracted with 6 ml of diethyl ether by shaking the mixture for 30 min at 37°C. After centrifugation the diethyl ether phase was separated, evaporated to dryness and redissolved in 100 l of mobile phase. Diosmetin was determined in an octylsilica column (250  4.6 mm i.d.; particle size 5 m) at 43°C. The isocratic mobile phase consisted of methanol–water–acetic acid (55:43:2, v/v). The flow rate was 0.9 ml/min and analytes were detected at 344 nm. Chromatograms demonstrating the separation capacity of the RPHPLC system are shown in Fig. 2.85. The intra-day and inter-day precision ( % RSD) were between 1.6–4.6 and 2.2–5.3 per cent, respectively. Recoveries varied according to the

PHENOL CONTENT IN DIFFERENT TYPES OF WINES (G/ML)

124.3 71.4 55.3 108.3 65.6 53.7 31.6 76.0 56.0

33.1

32.0

11.6

10.9

15.0 21.9 6.79 24.4 13.6 10.8 2.18 6.15 3.69 nd 3.90

trans-resveratrol

cis-resveratrol

Quercetin

15.6 431

16.8 21.2 6.93 24.8 13.6 11.4 2.74 6.30 3.80

nda nd nd nd nd nd nd nd nd

— — — — — — — — —

nd 2.35 nd 1.64 nd nd 0.38 nd 0.50

0.25 2.36 nd 1.58 nd nd 0.48 nd 0.50

nd nd nd nd nd nd 0.16 nd nd

0.99

nd

—

nd

0.06

nd

4.10

3.90

—

nd

0.16

nd

0.039 nd nd nd nd nd 0.021 nd nd

nd

34.3 30.0 22.7 nd nd 12.6

— — — — — — — — —

nd

—

0.71

—

Page 228

124.8 71.3 55.9 108.2 65.8 52.0 30.5 77.8 55.3

Rutin

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a

nd, not detected. Reprinted with permission from P. Vinas et al. [198]. Chapter 2

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6000000

23.8 5000000

m/z = 781 epi-Mc3glc OH OH

4000000

O

HO

m/z= 781 cat-Mv3glc OH OH

Intensity

OCH3 OH OH HO

3000000

OH OCH3

OCH3 OH

OH

OGlc

OH HO

OH

2000000

O

HO

O+

O+

OCH3 OGlc

26.9

OH

1000000

0 5

10

15

20

25

30 Minutes

35

40

45

50

55

Fig. 2.83. Mass chromatogram extracted from the total ion current (TIC) of a wine. Reprinted with permission from E. Salas et al. [199].

2

Peak current height

1 nA

0.1 nA

1

0.1nA

1

(a)

(b)

Retention time (min)

2

(c)

Fig. 2.84. Chromatograms of a solution prepared from commercial canned green tea (a), blank plasma (b) and sample plasma (c) of the same subject at 1h after ingestion of 340ml commercial green tea. Peak identification: 1  quercetin; 2  luteolin (internal standard). For chromatographic conditions see text. Reprinted with permission from D. Jin et al. [204].

amount of diosmetin, being 89.7–92.0 per cent for 50 and 200 ng/ml concentrations, and the limit of quantification was about 10 ng/ml (1 ml of plasma sample). Because of the simplicity, specificity, precisity and accuracy the method was proposed for the studies of the fate of diosmin in humans [205].

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4

4

3

3

mAU

mAU

230

2

1

I

2

II

1

0

0 0

(a)

Page 230

10 20 Time (min)

0 (b)

10 20 Time (min)

Fig. 2.85. Examples of chromatograms. (a) extract of 1ml blank plasma; (b) plasma sample obtained from a female healthy subject 2h after oral administration of a Daflon tablet (diosmin/hesperidin, 450/50 mg) containing 61.2 ng/ml of diosmetin. Peaks: I  internal standard, 7-ethoxycoumarin; II  diosmetin. Reprinted with permission from F. I. Kanaze et al. [205].

The considerable biological impact of phyto-oestrogens has motivated the development of various chromatographic methods for their analysis in living organisms. Thus, an HPLCMS-MS procedure has been employed for the determination of seven phyto-oestrogens in human serum and urine [206], in rat serum [207], and in the study of rat oral bioavailability [208]. Automated on-line and off-line SPE methods were developed for the determination of isoflavones and lignans in urine. The chemical structures of analytes are shown in Fig. 2.86. 1 ml samples of urine were mixed with the internal standard (4-methylumbelliferyl glucuronide/4-methylumbelliferyl sulphate) and with -glucuronidase/sulphate solution, then it was thermostated overnight at 37°C. Off-line SPE was carried out using cartridges preconditioned with 2 ml of methanol and 2 ml of water (flow rate, 1 ml/min). Urine samples were loaded into the cartridge with the same flow rate, then the cartridge was washed with 3 ml of 0.1 per cent NH4OH-MeOH (90:10, v/v), 2 ml of water and 2 ml of 70 per cent methanol in water. Analytes were eluted with 2 ml of MeOH. The eluate was evaporated to dryness, and redissolved in the mobile phase. Separations were performed in an ODS column using gradient elution (solvents were 10 mM ammonium acetate (pH  6.5) and methanol–ACN (50:50, v/v). Negative-ion atmospheric pressure chemical ionization was

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OH

HO

231

OH

O

OH

O

O

HO

O H

O HO

Genistein

Equol

Daidzein

HO

OH

OH

O

O

OH

OH

HO

OH O-DMA

Enterodiol

HO O O

OH

Enterolactone

Fig. 2.86. Chemical structures of the iosflavones daidzein, genistein, O-desmethylangolensin, equol, and the lignans enterolactone and enterodiol. Reprinted with permission from Z. Kuklenyik et al. [209].

used for the detection of analytes. The APCI conditions were: nebulizing gas temperature, 500°C; and corona needle voltage, 3 V. It was found that the recoveries were 83–94 per cent for the on-line SPE procedure, and 65–80 per cent for the off-line SPE. Detection limits were 0.1–0.7 ng/ml for on-line SPE and 0.4–3.3 ng/ml for off-line SPE. Reproducibility varied between 4–12 per cent (online SPE) and 18–19 per cent (off-line SPE). Because of the rapidity and good validation parameters, the procedure was proposed for epidemiological studies of human exposure to the compounds [209]. Solid-phase borate complexation coupled with RP-HPLC has been employed for the measurement of polyhydroxyflavones in human blood plasma, vegetables and redwine. The chemical structures of polyhydroxyflavones included in the investigation are shown in Fig. 2.87. Vegetables were homogenized, centrifuged and the supernatant was applied for analysis. Human plasma was heparinized before analysis. The outer skins of onion were

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Chapter 2 OH

OH OH

OH

O

HO

O

HO OH

OH

OH

O

O

Quercetin

Fisetin OH OH

O

HO

OH HO

O

OH OH

O-rutinose

O

O

Kaempferol Rutin OH OH O

HO

OH

HO

O

OH OH

Myricetin

O

OH

HO

OH O

Morin

Fig. 2.87. Structures of polyhydroxyflavones. Reprinted with permission from H. Tsuchiya [210].

ground, extracted with 50 per cent aqueous ethanol, centrifuged and the supernatant was used for HPLC measurements. Phenylboric acid cartridges were preconditioned with 1 ml of 28 per cent aqueous ACN containing 1 per cent TFA, 1 ml of water and 1 ml of 0.5 M potassium phosphate buffer (pH  8.5). After loading, the cartridge it was washed with 1 ml of the same buffer. Solutes were eluted with 4  0.5 ml of 28 per cent aqueous ACN containing 1 per cent TFA. Separation was performed in an octylsilca column (250  4.6 mm i.d.; particle size 5 m) at 50°C using isocratic elution (TFA–ACN–water (1:28:71, v/v). Polyhydroxyphenols were detected at 370 nm. Characteristic chromatograms are shown in Fig. 2.88. The concentrations of quercetin were 127–144.9 g/g in the outer skin and 3.89–4.04 g/g in the edible part of the onion. Red wines contained 0.10–6.83 g/ml quercetin, 2.09–15.45 g/ml myricetin, 0.20–1.82 g/ml morin and 0.20–1.18 g/ml kaempferol. The polyhydroxyflavones were absent in broccoli, lettuce,

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6 3

5 3

3

3 5

4

5

mAbs

12 4

6 5

2 2

4

6

0 0 (a)

5

10

15

Retention time (min)

0

5

10

15

(b) Retention time (min)

0

5

10

15

(c) Retention time (min)

0

5

10

15

(d) Retention time (min)

Fig. 2.88. Chromatograms obtained from standard, onion, wine and plasma samples. (a) standard polyhydroxyflavones (5.00g/ml of each), (b) onion skin etract, (c) wine, and (d) plasma spiked with quercetin (5.00g/ml). Detection at 370 nm. Peaks: 1  rutin; 2  myricetin; 3  fisetin (internal standard); 4  morin; 5  quercetin; 6  kaempferol. Reprinted with permission from H. Tsuchiya [210].

garlic and apple. Recoveries varied between 75.7–104.6 per cent, the RSD values ranged form 0.8 per cent to 10.2 per cent. It was suggested that the technique can be applied in nutritional and pharmacokinetic investigations [210]. 2.2.4 Electrophoretic methods The advantages of CE and MEKC (small sample size, high separation efficacy and speed) have been exploited in the analysis of flavonoids too. The results obtained in the analysis of wines have been reviewed earlier [211]. CE has also been employed for the determination of polyphenols in green tea. The flow injection system extracted the tea sample (1.75 g) with 100 ml of boiling water, filtered the suspension then diluted it with water and injected it into the capillary. Separation was carried out in a fused silica capillary of 57 cm  75 m i.d.  375 m o.d using 0.15 M H3BO3 buffer at pH 8.5 and a working voltage of 20 kV. Analytes were detected at 210 nm. The effect of buffer pH and concentration on the separation efficiency is visualized in the electrophoregrams in Fig. 2.89. The concentrations of polyphenols found in the different tea varieties are compiled in Table 2.78. It was stated that the technique is suitable for the determination of flavonols in different tea varieties. The low detection limit (0.04 g/ml for flavonols and 1.2  g/ml for caffeine), the short analysis time (20 min) and the good separation efficacy make the method superior to the accepted HPLC procedures [212]. As the separation characteristics of liquid chromatographic and electrophoretic techniques markedly differ from each other, combined methods using the advantages of both procedures have been successfully used for the analysis of flavonoids. Thus, the use of CZE-UV, HPTLC-UV and GC-MS for the measurement of flavonoids in seeds and root exudates of Lotus pedunculatus has been reported. The rooting solution and seed exudate were passed through cellulose acetate filters to bind the flavonoids. After extraction,

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Chapter 2

0.006

0.1 M H3BO3 ph 8.0

3

8.000e-3

10 11

0.004

6.000e-3 AU

AU

1+2

11

4.000e-3 2.000e-3

4+5+6

0.000 3

4

5

6

7

8

5

6

7

8

0.008 0.1 M H3BO3 ph 8.5

3 0.006

10 11 12 13 14

10 AU

11

0.004

0.1 M H3BO3 pH 8.5

0.006

3

10 11

0.004 8

8 0.002

1

2

0.000

6

7

9

0.002

6

8

9

10

14

7

6

1 2

0.000

4+5

9

4+5 6

7

8

9

10

11

12

13

14

15

(e)

(b)

0.008 3

11

0.004

8

0.002

7+8

4+5 6

7

8

9

5 6 4

12

2

0.000 5

11

9

6 1

10

0.006

10

0.004 0.002

0.15 M H3BO3 pH 8.5

3

0.1 M H3BO3 pH 9.5

AU

0.006

AU

9

(d) 0.008

(c)

7+8

4+5 4

9

9

6

2

1

0.000e-3

(a)

AU

10

9

7+8

0.002

0.05 M H3BO3 pH 8.5

3

9

7

0.000 10

Time (min)

11

12

13

14

6

(f)

8

10

12

14

16

18

Time (min)

Fig. 2.89. (a, b, c) Influence of the pH of the buffer on the resolution of the peaks. (d, e, f) Influence of the buffer concentration on the resolution of the peaks. Electropherograms of a standard mixture of polyphenols. Peaks: 1  EOFcaffeine; 2  adenine; 3  theophylline; 4  EGC; 5  ()-epicatechin; 6  ()-epicatechin; 7  EGCG; 8  ECG; 9  quercetin; 10  gallic acid; 11  caffeic acid. Reprinted with permission from L. Arce et al. [212].

flavonoids were washed out with 2  5 ml of methanol, then the methanol solution was evaporated to dryness and redissolved in methanol. Silica HPTLC plates have been applied for the preseparation of analytes. Two-dimensional separation of flavonoid aglycones was performed with chloroform–methanol–formic acid (93:6:1, v/v) in the first direction and with toluene–ethyl acetate–methanol–acetic acid (70:25:4:1, v/v) in the second one. Flavonoid glycosides were separated with 2-propanol–toluene–ethyl acetate–water (50:10:25:12.5, v/v) and ethyl acetate–methyl ethyl ketone–formic acid–water (50:30:10:10, v/v) in the first and second direction, respectively. Individual spots were eluted with methanol and used for CZE-UV and GC-MS. A fused silica capillary (65 cm  75 m i.d.  375 m o.d.) was applied for the separation. The electrophoresis buffer contained 50 mM boric acid and 10 mM sodium tetraborate at pH 8.5. Injection of samples was carried out under gravity from a height of 50 mm for 30 s. The current was 21 A. UV-VIS spectra were recorded between 195–365 nm. Typical electropherograms

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TABLE 2.78 ANALYSIS OF POLYPHENOLS IN REAL SAMPLES (FIGURES CORRESPOND TO CONCENTRATIONS IN G/ML) a

Caffeine Adenine EGC Epicatechin Catechin EGCG ECG Gallic acid

Caffeine Adenine EGC Epicatechin Catechin EGCG ECG Gallic acid

Caffeine Adenine EGC Epicatechin Catechin EGCG ECG Gallic acid

Caffeine Adenine EGC

Pompadour

Hornimans

770.84.6(773.73.3) 45.91.7(44.560.7) 85.822.0(90.642.8) 97.333.0(93.322.4) 32.060.4(93.322.4) 316.43.3(327.02.5) 177.42.6(171.92.0) 88.142.2(84.281.8)

319.23.5(319.52.9) 0.640.06(0.740.08) —b 8.621.2(10.10.9) 11.21.6(11.00.4) 26.40.9(26.21.8) 19.91.5(21.40.9) 40.40.3(40.71.8)

Burco

Akfa

504.03.8(510.11.5) — 80.80.4(79.142.0) 6.80.6(5.80.1) 8.30.3(8.70.8) 9.120.5(9.41.2) 1.120.3(0.980.2) 32.41.3(30.51.1)

408.92.6(409.42.7) — 2.20.1(1.90.5) 4.70.9(5.40.4) 5.80.2(5.30.2) 13.20.9(12.11.1) 6.20.4(6.70.3) 25.41.0(25.01.2)

CayCiceg

Filiz

197.91.5(192.42.0) 19.30.9(17.61.2) — 2.020.3(2.50.4) 0.850.05(0.720.09) 3.90.1(3.50.8) 0.740.08(0.80.01) 7.00.6(6.10.2)

307.93.1(310.42.2) 23.431.6(22.81.3) — 14.640.9(15.40.6) 2.190.8(2.040.4) 7.20.5(7.70.4) 3.660.9(3.450.08) 15.01.1(15.40.5)

Pompadour

Hornimans

Demcay

Rize

216.062.3(194.22.1) 20.50.8(19.40.3) —

545.43.2(554.01.9) — — (Continued on next page)

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Chapter 2 TABLE 2.78 (continued)

Epicatechin Catechin EGCG ECG Gallic acid

Pompadour

Hornimans

Demcay

Rize

7060.8(7.10.5) 1.620.4(1.050.4) 9.460.7(10.00.4) 4.30.1(4.90.3) 18.931.2(20.21.0)

9.10.7(9.90.8) 2.20.3(2.10.4) 12.90.9(13.40.7) 6.30.4(6.90.5) 31.71.6(30.61.3)

a

In parentheses concentrations found by using the standard addition method (g/ml). b —  not detected. Reprinted with permission from L. Arce et al. [212].

are shown in Fig. 2.90. The electropherograms illustrate that the preseparation and prepurification of flavonoids can be obtained by two-dimensional HPTLC even in the case of complicated accompanying matrices [213]. Tea catechins have also been vigorously investigated with various electrophoretic techniques. Capillary electrokinetic chromatography [214] and MEKC methods have also found application in the analysis of microcomponents of teas [215,216]. The performance of HPLC and CE for the separation of tea catechins has been frequently compared [217]. The separation efficiency of borate–phosphate–SDS based MEKC and RP-HPLC for green tea catechins has also been compared. Spray-dried green tea extract (GTE) was dissolved in a water–formic acid solution (99.7:0.3, v/v) and used for HPLC separation. Samples for MEKC contained 1 per cent acetone. MEKC measurements were performed in an undeactivated fused silica capillary (total length, 47 cm; effective length, 40 cm; 50 m i.d.  375 m o.d.). The capillary was conditioned by washing with 1 M NaOH for 3 min, 0.1 M NaOH for 3 min, water for 3 min and running buffer for 3min. Running buffer consisted of 20 mM potassium hydrogen phosphate–50 mM sodium tetraborate–200 mM sodium dodecyl sulphate (SDS) (3:1:2, v/v). Buffer pH was adjusted to pH  7 with 0.1 M HCl. Samples were injected hydrodynamically at the anodic end int low-pressure mode (0.5 psi for 1 s). Separations were performed at 30kV for 5min, the capillary temperature being 29°C. Analytes were detected at 200 nm. RP-HPLC separations were carried out in an ODS column (250  3.0mm i.d.; particle size 5m). Components of the gradient elution were: water–methanol–formic acid (74.7:25:0.3, v/v, A) and ACN–formic acid (99.7:0.3, v/v, B). The gradient was as follows: 0–8 min, 100 per cent A; 100 per cent B in 24 min. Analytes were detected at 270 nm. The chromatograms and electropherograms of standard mixture and GTE are shown in Fig. 2.91. The chromatograms and electropherograms illustrate that the analysis time of high-performance capillary electrophoresis (HPCE) is markedly lower (about 4.5 min), the sensitivity is higher and the solvent consumption is considerably lower. Moreover, the repeatability of migration times was better than that of RP-HPLC. Because of the advantageous application parameters the HPCE technique was proposed for the routine analysis of tea extracts [218].

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Absorbance (mAU) at 270 nm

Liquid chromatography of natural pigments

48

237

4

2

5

36

6

1

24

3 12

08

35

Retention time (min)

(a) Absorbance (mAU) at 270 nm

48

36

4 2

24 5 RL 2

RL 1 12

3

6

1 0 8

(b)

35

Retention time (min)

Absorbance (mAU) at 270 nm

20 4 14

8

5

RT1 1

2

3

RT2 6

RT3

2

0 (c)

8

Retention time (min)

35

Fig. 2.90. Electropherograms of sterile root exudate before (a) and after incubation with Mesorhizobium loti (b) and Rhizobium leguminosarum bv. trifolii (c). From their UV spectra, peaks 1–6 were identified as resorcinol, rhamnetin, catechin, quercetin glycoside, quercetin aglycone and hesperidin. Differences in the retention times of the same compounds in different samples could be due to the variations in buffer temperature or sample composition. Reprinted with permission from H. L. Steele et al. [213].

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II

12

EGCG

ECG

I

40

ECG

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35

10

30

20 15

C

EC

2 0

GCG

EGC

10 GC

2

mAU

GCG

25

EC

EGC C

4

EGCG

6 GC+GA

mAU

8

0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min IV EGCG

0

2.5 5.0 7.5 10.0 12.5 15.0 17.5 min III C

0

0.042

0.028 0.026

0.038

0.024

0.034

0.022

0.030

0.020

2.0

2.5

3.0 min

3.5

4.0

4.5

ECG 1.5

2.0

2.5

GA

Ac 1.5

0.006

EC

0.010

0.008 0.006

EGC

0.014 GCG

GC

0.010

GC C

0.012

0.018

Ac

0.014

0.022 GA

0.016

0.026

EGC GCG EGCG ECG EC

0.018

3.0 min

3.5

4.0

4.5

Fig. 2.91. (I) HPLC chromatogram of pure standards (all standard solutions were 100 g/ml); (II) HPLC chromatogram of GTE sample (concentration: 1 000 g/ml; injection volume: 1l). UV detection was performed at 270 nm; for other details see text. (III) HPCE electropherogram of the standard mixture (C, 500 g/ml; GC, 200 g/ml; others, 1 000 g/ml); (IV) HPCE electropherogram of GTE sample. UV detection was performed at 200 nm; for other details see text. Peak identification: GC, ()-gallocatechin; EGC, ()-epigallocatechin; C, ()-catechin; EGCG, ()-epigallocatechin-3-gallate; EC, ()-epigallocatechin; GCG, ()-gallocatechingallate; ECG, ()-epicatechingallate; GA, gallic acid; Ac, acetone (EOF marker). Reprinted with permission from M. Bonoli et al. [218].

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2.3 ANTHOCYANINS 2.3.1 Chemistry and biochemistry Anthocyanidin aglycones are polyhydroxy and polymethoxy derivatives of the 2-phenylbenzopyrylium cation. Anthocyanins are the glycosides of anthocyanidins and they are generally water soluble. Glycosylation can occur at various sites such as 3-,7-3
- and 5
-glycosides. The sugar moiety can be acylated by both aromatic and aliphatic acids. Anthocyanidins are widely distributed in the plant kingdom. They are frequently responsible for the colour of flowers, fruits, roots and leaves. Anthocyanins are extensively used as natural food colourants [219,220]. Similarly to other natural pigment classes, such as carotenoids, flavonoids, etc., anthocyanins also have beneficial biological effects. They show marked protective effects against peroxynitrite-induced endothelial dysfunction and vascular failure [221], have a considerable role in disease prevention [222], influence human skin keratinocyte proliferation and differentiation [223], and modify tumour development [224]. 2.3.2 Thin-layer chromatography Simlarly to the analysis of carotenoid and flavonoid derivatives, the simple, rapid and easyto-carry-out TLC techniques have also found application in the separation of anthocyanins. Thus, the anthocyanin pigments responsible for the red colour of wine have been separated by HPTLC. The chemical stuctures of anthocyanin pigments found in the skin of grapes (Vitis vinifera L.) are listed in Fig. 2.92. Extraction of colour pigment was carried out by macerating berries with MeOH–HCl (99.9:0.1, v/v) at 25°C for 16 h. The filtered and concentrated solution was applied to an ion exchange column. The column was washed with 10 per cent aqueous formic acid solution, then anthocyanins were eluted with MeOH–water–HCl (65:35:0.1, v/v). The eluate was evaporated and freeze dried. Anthocyanins were further purified in an ODS semi-preparative column (250  10 mm i.d.; particle size 10 m) using gradient elution of 10 per cent formic acid dissolved in water (solvent A) and in methanol (solvent B) at a flow rate of 5 ml/min. The gradient was: 0 per cent B for 3 min; to 45 per cent B in 2 min; 45 per cent B for 10 min; to 60 per cent B in 15 min, to 100 per cent B in 2 min; 100 per cent B for 5 min. Peaks were identified by MS (cone potential, 60 V, capillary potential 3.5kV). Extraction of red wine pigments was performed in ODS cartridges preconditioned with methanol followed by water. Wine samples were applied to the cartridge and washed with 3  2ml of water. Anthocyanins were eluted with 4 ml of ethanol–formic acid (90:10, v/v) and the solution was used for HPTLC separation. Samples were spotted onto C18 reversed-phase plates and separated by the mobile phase methanol–water–TFA (55:45:1, v/v). The results of combined HPTLC RP-HPLC are shown in Fig. 2.93. The total integrated area minus the areas of peaks of 3-glucosides, 3-acetylglucosides and 3-p-coumaryl-3-glucosides divided by the total integrated area was considered as the polymeric pigment fraction. The amounts of polymeric pigments in wines are compiled in Table 2.79. It was concluded that the HPTLC method is suitable for the separation of the three main anthocyanin classes occurring in red wine. Moreover, it allows the determination of the relative amounts of polymeric pigments [225]. TLC and HPLC have been applied for the separation of the flower pigments of some Delphinium species. Anthocyanins were extracted with 70 per cent aqueous ACN containing

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Chapter 2 R1 OH + O

HO

R2 O OH

OH

OR3

O OH

OH

R1 = OH, R2 = H

Cyanidin

R1 = OH, R2 = OH

Delphinidin

R1 = OCH3 , R2 = H

Peonidin

R1 = OCH3 , R2 = OH

Petunidin

R1 = OCH3 , R2 = OCH3

Malvidin

R3 = H

: Glucoside

R3 = -CO-CH3

: Acetylglucoside

R3 = -CO-CH=CH-

R3 = -CO-CH3

: Acetylglukoside

R3 = -CO-CH=CH-C6H4-OH

: Coumaroylglucoside

Fig. 2.92. General structure of anthocyanins found in Vitis vinifera grapes. Reprinted with permission from M. Lambri et al. [225]. TABLE 2.79 PRECISION OBTAINED IN THE PERCENTAGE DETEMINATION OF POLYMERIC PIGMENTS (n  35).

Wine

Amount of polymeric pigment (%) (meanSD)

CV(%)

A B C D E F G H

94.42.57 88.92.04 94.42.14 92.41.19 93.22.14 88.62.43 93.91.00 84.41.40

2.7 2.3 2.3 1.3 2.3 2.7 1.1 1.7

Reprinted with permission from M. Lambri et al. [22].

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HPLC

241

5a

1600 1400

mAU

1200 5b 1000 800 4a

600 1a 400

a

200

b

3a (1-5)c

2a 1b 2b3b

4b

c

0 10

12

14

16

18 20 22 T (minutes)

b, HPTLC

a,HPTLC Rf 0.59

Glycosides

24

26

28

30

c, HPTLC Rf 0.38

Acetylglycosides

Rf 0.23

Coumaroylglycosides

Fig. 2.93. HPLC identification of the anthocyanins in the 3-glucoside, 3-acetylglucoside, and 3-pcoumaroylglucoside fractions separated by HPTLC (for which the corresponding HPTLC chromatograms are given). Peak identification: 1a  delphinidin-3-glucoside; 2a  cyanidin-3- glucoside; 3a  petunidin-3-glucoside; 4a  peonidin-3-glucoside; 5a  malvidin-3-glucoside; 1b  delphinidin-3acetylglucoside; 2b  cyanidin-3-acetylglucoside; 3b  petunidin-3-acetylglucoside; 4b  peonidin-3acetylglucoside; 5b  malvidin-3-acetylglucoside; 1c  delphinidin-3-coumaroylglucoside; 2c  cyanidin-3-coumaroylglucoside; 3c  petunidin-3-coumaroylglucoside; 4c  peonidin-3-coumaroylglucoside; 5c  malvidin-3-coumaroylglucoside. Reprinted with permission from M. Lambri et al. [225].

3 per cent TFA at 5°C. The extract was filtered and used for HPLC analysis. Extracts were hydrolyzed with 2 N HCl and employed for TLC for the separation of anthocyanidins. TLC investigations were carried out on cellulose layers using water–HCl–acetic acid (10:3:30, v/v) as the mobile phase. RP-HPLC measuremements were performed in an ODS column (150  4.5 mm i.d.). Components of the linear gradient were 25 per cent ACN, 20 per cent acetic acid and 0.5 per cent TFA in water (solvent A) and 0.5 per cent aqueous TFA (solvent B). The gradient was: from 30 per cent to 100 per cent A in 50 min. The flow rate was 1 ml/min and analytes were detected at 510 nm. TLC measurements demonstrated that the hybrids D. cardinale  D. grandiflorum and D. grandiflorum  D. nudicaule contain only delphidin originated from D. gradiflorum. D. cardinale and D. nudicaule contained only pelargonidin. The concentration of anthocyanins separated by RP-HPLC and the chemical strucutres of the main components are compiled in Table 2.80.

Pigment no. Rt (min)

2 6.71

6.85

3

4

5

6

7

8

9

10

10.83

11.09

11.55

15.87

16.07

18.47

18.72

19.49

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80.51 83.43

20.03 21.84

25.36

26.32

6.44 3.60 27.21

Chapter 2

Standard cultivars A B C D E Parents and hybrids F G H I J K L M

1

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RELATIVE ANTHOCYANIN COMPOSITION OF PETALS OF ELPHINIUM CULTIVARS. INTERSPECIFIC HYBRIDS AND THEIR PARENTS BY HPLC ANALYSIS. RELATIVE LEVEL OF ANTHOCYANIN (AREA PERCENTAGE AT 510 NM) ARE REPORTED (VALUES UNDER 2PER CENT ARE EXCLUDED)

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19.88

20.37

20.65

21.01

21.28

21.67

22.37

22.77

28.07

28.20

3.51 15.46 38.59

62.94 55.38 59.52

6.16

5.89

21.23

12.76

61.20

16.53 52.37 19.61 40.94 53.44 15.06

6.35 14.07 3.36 30.98

Page 243

Standard cultivars: A  D. hybridum ‘Galahad’; B  D. hybridum ‘Astorat’; C  D. hybridum ‘Summer Skies’; D  D. hybridum ‘Blue Bird’; E  D. belladonna ‘Belladonna Imp.’ Parents and hydrids: F  D. Cardinale Hook; G  D. grandiflorum L.; H  D. nudicaule Torr. and A. Gray; I  D. cardinale x D. grandiflorum; J  D. grandiflorum x D. nudicaule no.1; K  D. grandiflorum x D. nudicaule no.2; L  D. grandiflorum x D. nudicaule no.3; M  D. grandiflorum x D. nudicaule no.4. Peak identification: 5  delphinidin-3-rutinoside-7-glucoside; 9  delphinidin-3-rutinoside; 13  violdelphin; 22  cyanodelphin. Reprinted with permission from K. Honda et al. [226].

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7.72

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31.15

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19.86

Liquid chromatography of natural pigments

Rt (min) Standard cultivars A B C D E Parents and hybrids F G H I J K L M

243

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Chapter 2 OH

OH

OH

OH O

HO

O

Glc-O

OH

OH

O-Glc-Rha

O-Glc-Rha OH

OH delphidine 3-rutinoside-7-glucoside

delphidin 3-rutinoside OH

OH

OH

OH O

pB-Glc-pB-Glc-o

pB-Glc-pB OH

Glc Glc

O-Glc-Rha OH

O

O Glc

Glc

pB

OH O-Glc-Rha

OH

pB violdelphin

cyanodelphin

Fig. 2.94. Chemical structures of the main anthocyanins found in the petals of Delphinium cultivars. Reprinted with permission from K. Honda et al. [226].

The chemical structures of the main anthocyanins are shown in Fig. 2.94. The results illustrated that TLC can also be used for taxonomical studies [226]. Anthocyanins and flavonol glucosides were studied by various chromatographic techniques in the purple flowers of Dendrobium cv. ‘Pompadour’. Colour substances were extracted by mixing approximately 1 kg of flowers with 10 l of methanol–acetic acid–water (4:1:5, v.v). The extract was concentrated and further purified by TLC and HPLC. Preparative HPLC was carried out in an ODS column (150  19 mm i.d.) using gradient elution. Solvents A and B were 1.5 per cent aqueous H3PO4 and 1.5 per cent H3PO4-20 per cent acetic acid and 25 per cent ACN, respectively. Anthocyanins were eluted by a gradient starting with 60 per cent A to reach 15 per cent A in 40 min. The flow rate was 4 ml/min and analytes were detected at 530 nm. Analytical TLC was performed using the following mobile phases: n-butanol-2 N HCl (1:1, v/v), 1 per cent HCl and acetic acid–HCl–water (15:3:82, v/v). Analytical RP-HPLC was also performed in an ODS column (250  4.6 mm i.d.) at 40oC. The gradient began at 60 per cent A and decreased to 15 per cent A in 30 min. The flow rate was 1 ml/min and anthocyanins were detected at 530 nm. Solutes were further indentified by FAB-MS and 1HNMR. Flavonoid glucosides were also separated and identified by TLC and HPLC [227]. 2.3.3 High-performance liquid chromatography 2.3.3.1 Determination of anthocyanins in wine Because of their considerable impact on the quality of wine, anthocyanins have been intensively investigated in wines using a wide variety of HPLC techniques [228]. RP-HPLC was

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employed for separation of anthocyanins from red grapes. The extraction of anthocyanins were performed by macerating red grape skins (V. vinifera) in methanol containing 5 per cent 1 N HCl at 4°C. After repeated maceration the combined methanol phases were concentrated at 30°C, dissolved in water and washed with n-hexane. Analytes were further purified in a silica column (50  2.5 cm i.d.). Impurities were eluted with water and anthocyanins were removed with methanol containing 1 per cent HCl. Anthocyanins were isolated in an ODS column (46  2.6 cm i.d.; particle size 24–40 m) with gradient elution. Solvents were 10 per cent acetic acid (A) and methanol (B). Gradient conditions were: 0 per cent B for 40 min; 5 per cent B over 40 min; 5 per cent B over 80 min; and 10 per cent B over 60 min. The flow rate was 6 ml/min and anthocyanins were detected at 520 nm. Analytes were further purified in another ODS column (100  25 mm i.d.) using the same solvents. The gradient was 5 per cent B over 22min; 10 per cent B in 3 min; hold for 5 min; 15 per cent B over 15min. HPLCDAD was performed in an ODS column (250  4 mm i.d.; particle size 5m) at 30°C. Gradient employed 5 per cent formic acid (A) and ACN (B) starting with 10 per cent B reaching 35 per cent B over 50 min. The flow rate was 1.5 ml/min. It was established that the main components of red grape skin anthocyanins were the 3-monoglucoside derivatives of delphidin, cyanidin, petunidin, peonidin and malvidin [229]. Procyanidins in white wines were analysed using RP-HPLC and fluorescence detection. Wine samples were diluted (1/3) and injected into an ODS column (250  4 mm i.d.; particle size 5m). Solvents were 20 mm acetic acid (A) and methanol (B). Separation was carried out with the gradient: 0–5 min, 98 per cent A; 5–12 min, to 93 per cent A; 12–18 min, to 89 per cent A; 18–28 min, to 85 per cent A; 28–38 min, to 70 per cent A; 38–48 min, to 60 per cent A; 45–55 min, to 50 per cent A. Excitation and emmision wavelengths were 275 and 322 nm and the flow rate was 0.5 ml/min. The presence of procyanidin dimers B1, B2, B3, B4 and procyanidin trimers C1 and T2 was proven, as illustrated in Fig. 2.95. The results of wine analyses are compiled in Table 2.81. It was concluded from the results that the method is simple and sensitive, therefore, it can be applied for the rapid determination of procyanidins in wine [230]. Gel permeation chromatography (GPC) has also found application in the analysis of anthocyanin pigments in rosé cider and red wine. Anthocyanins were preconcentrated in an ODS column (90  25 mm i.d.). Samples were passed through the column, the impurities were removed by washing the column with distilled water then anthocyanins were eluted with methanol. GPC fractionation was carried out in a 950  25 mm i.d. column using acetone–8 M urea (6:4) as the mobile phase. The pH was adjusted to 2.0 with HCl. The flow rate was 1 ml/min. HPLC analysis was performed in an ODS column (150  4.6 mm i.d.) at 30°C. Gradient conditions were: 0–10 min; 20 per cent methanol–80 per cent 0.01 M KH2PO4; to 50 per cent methanol for 40min; final hold 15 min. The flow rate was 1ml/min and analytes were detected at 520 nm. The HPLC profiles of samples and GPC fractions are shown in Fig. 2.96. It has been stated that the GPC method is suitable for the separation of polymeric anthocyanins in both cider and red wine [231]. The optimum conditions for the MS detection of anthocyanin derivatives have also been intensively studied. Grape skin extract was prepared by macerating skins with methanol–formic acid (95:5) for three days changing the extracting agent each day. The combined extracts were evaporated and diluted with water. RP-HPLC measurements were carried out in an ODS column (150  4.6 mm i.d.; particle size 5 m) at 30°C. The solvents were water–formic acid (90:10, v/v, A) and methanol–water–formic acid (45:45:10, v/v, B). The gradient was from 35 to 95 per cent B in 20 min; to 100 per cent B in 5 min; final hold

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Chapter 2 B3 43.64 43.608 B1 (+)-Catechin 40.781 47.1st T2 48.718 B4 81.608 C1

246

100

80

B

%F

60

40

20

0 10

20

30 Time (min)

40

30

Fig. 2.95. Chromatographic analysis (HPLC/fluorimetry) of procyanidins in white wine. Reprinted with permission from S. Carando et al. [230]. TABLE 2.81 PROCYANIDINS: ANALYTICAL CHARACTERISTICS AND CONCENTRATION IN SIX FRENCH WHITE WINES

Compounds

Rt (min)

LOQ (g/l)

Ranged concentrations (mg/l)

B3 B1 T2 B4 B2 C1

43.3 43.7 47.2 48.7 51.1 51.7

12 15 13 16 16 13

0.91–2.17 0.83–1.38 0.84–1.67 0.28–0.47 nd 0.56–1.11

Reprinted with permission from S. Carando et al. [230].

5 min. The flow rate was 0.8 ml/min. DAD detection ranged from 240–600 nm. APCI and ESI were employed in both positive and negative ionization modes. ESI conditions were: nebulizer pressure, 380 Pa; drying gas flow and temperature 10 l/min and 350°C, and Vcap 4 000 V. APCI conditions were: carrier gas flow, 3 l/min; corona discharge needle, 5 000 V at 90°C. Typical chromatograms are shown in Fig. 2.97. The chromatograms illustrate the

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247 fr. 1 fr. 2

Starking delicious juice 1

fr. 3 9

1

6

7 8

5 2

fr. 4

34

1 1 Rose cider 9

5

6

9

7

6

8

7

fr. 5 5 10 15 20 25 30 35 40 45 50 55 60 65 Retention time (min)

5 10 15 20 25 30 35 40 45 50 55 60 65 Retention time (min)

Fig. 2.96. HPLC profiles of Starking Delicious juice, rosé cider, and GPC fractions from rosé cider recorded at 520 nm. Each lyophilized sample was dissolved in 10 per cent EtOH (20 mg/ml). Peak 1 with retention time of 27.5min is Cyn-3-gal, which is the main anthocyanin in apple, and peaks 5–9 are unidentified anthocyanins, which may be newly produced during the vinification process. Reprinted with permission from T. Shoji et al. [231].

good separation performance of the method. Furthermore, they demonstrate that the anthocyanin profile of young and aged wines differs considerably. The anthocyanins found in skin extracts and young and two-years old wines are listed in Table 2.82. It has been concluded from the results that the combination of DAD and MS detection techniques highly facilitate the identification of anthocyanin compounds and may help the better understanding of chemical and biochemical processes underlying wine elaboration and ageing [232]. Interestingly, an ion-pair normal-phase HPLC method was developed for the determination of high-molecular-mass grape phenolics. Seed and skin extracts were prepared with 66 per cent (v/v) aqueous acetone for 24 h. The organic phase was evaporated at 35°C and the residue was lyophilized. Pefractionation of extracts was carried out in a silica column (250  10 mm i.d.; particle size 10 m) at a flow rate of 4.5 ml/min using the same gradient as the analytical separation. Analytes were detected at 280 nm. Samples at 0–20, 20–30, 30–40, 40–50 and 50–65 min were collected, concentrated and injected into the analytical column. Samples of proanthocyanidins were subjected to acid-catalysed thiolysis too. Ethanol was removed from wines and the analytes were extracted by SPE using ODS

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9

0.100

Abs

5 1 0.050

7

2 3

11 14, 12 13 15 17 19

6

30

23 25 24 27

28

0.000 5.00

10.00

20.00

(a)

30.00

min

9

0.100

Abs

11

0.050

12

5 6

1

78 2

4 3

10 14 16 13 15 18 19 20 21

23 24 25 28

29

30

0.000 5.00

(b)

10.00

20.00

30.00

min

Fig. 2.97. (a) Chromatogram of young Tinto Fino wine. (b) Chromatogram of aged Tinto Fino wine. Peak identification: 1  delphinidin-3-glucoside; 2  Dp-gls-py derivativea; 3  cyanidin3-glucoside; 4  unk.; 5  petunidin-3-glucoside; 6  Pt-gls-py derivativea; 7  peonidin-3-glucoside; 8  unk.; 9  malvidin-3-glucoside; 10  Pn-gls-py derivativea; 11  Mv-gls-py derivative; 12  unk.; 13  unk.; 14  Dp-(6-acetyl)-3-glucoside; 15  vitisin B; 16  unk.; 17  Cy-(6-acetyl)-3 glucoside; 18  polymer Mv-cata; 19  Pt-(6-acetyl)-3 glucoside; 20  dimer Mv-cata; 21  dimer Mv-cata; 22  Pn-(6-acetyl)-3 glucoside; 23  Mv-(6-acetyl)-3 glucoside; 24  Dp-(6-coumaroil)-3 glucoside; 25  Mv-(6-caffeoil)-3 glucoside; 26  Cy-(6-coumaroil)-3 glucoside; 27  Pt-(6-coumaroil)-3 glucoside; 28  Pn-(6-coumaroil)-3 glucoside; 29  Mv condenseda; 30  Mv-(6-coumaroil)-3 glucoside. Reprinted with permission from I. Revilla et al. [232]. (aProposed structure).

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TABLE 2.82 ANTHOCYANIN COMPOUNDS DETECTED IN VARIOUS SKIN EXTRACTS AND WINESa

Peak no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Skin extract

Wines

Ext CS

Ext TF

TF (young)

TFb (aged)

CSb

Garnachab

Gracianob

*c ndd *c ndd *c *c *c ndd *c ndd *c ndd ndd *c ndd ndd *c ndd *c ndd ndd *c *c *c *c *c *c *c ndd *c

*c *c *c ndd *c ndd *c ndd *c ndd *c ndd ndd *c ndd ndd *c ndd *c ndd ndd *c *c *c *c *c *c *c ndd *c

*c *c *c ndd *c *c *c ndd *c ndd *c *c *c *c *c ndd *c ndd *c ndd ndd ndd *c *c *c ndd *c *c ndd *c

*c *c *c *c *c *c *c *c *c *c *c *c *c *c *c *c ndd *c *c *c *c ndd *c *c *c ndd ndd *c *c *c

*c *c *c *c *c *c *c *c *c *c *c *c * *c *c *c ndd *c *c *c *c *c *c *c ndd ndd *c *c *c *c

*c ndd *c ndd *c *c *c ndd *c *c *c *c *c *c *c *c ndd ndd ndd *c ndd ndd *c ndd ndd ndd *c ndd *c *c

*c *c *c *c *c *c *c ndd *c ndd *c *c *c *c *c ndd *c *c *c *c *c ndd *c *c *c ndd *c *c *c *c

a

CS. Cabernet Sauvignon; TF. Tinto Fino. Two-year aged wines. c Detected. d Not detected. Numbers refer to anthocynins in Fig. 2.97. Reprinted with permission from I. Revilla et al. [232]. b

support. Separation was performed in a silica column (250  4 mm i.d.; particle size 5 m). Solvents for gradient elution were methylene chloride–methanol–formic acid–water (0:97:2:1, v/v, A) and (83:14:2:1, B) both containing 20 mM heptanesulphonic acid. The gradient started from 0 to 60 per cent A in 50 min; to 100 per cent A in 5 min; final hold

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Chapter 2

600 520 nm

mAU

400 300 200 100 0 10

20

30

(a)

40 min

50

60

600

70

520 nm

mAU

400 300 200 100 0 (b)

10

20

30

40 min

50

60

70

Fig. 2.98. Separation at 520 nm of red wine (Cabernet Sauvignon) extract before (a), and after (b) addition of 20 mm heptanesulphonic acid to the mobile phase. In (a), the two broad peaks at approximately 40 and 55 min are anthocyanins before heptanesulfonic acid addition; and in (b), the two peaks at approximately 12 and 15 min are the same anthocyanins after heptanesulphonic acid addition. Reprinted with permission from J. A. Kennedy et al. [233].

10 min. The effect of the ion-pairing agent on the retention of wine pigments is illustrated in Fig. 2.98. The chromatograms indicate that the ion-pairing agent exerts a marked effect on the retention of anthocyanins in the silica stationary phase [233]. A simple and rapid RP-HPLC procedure was developed for the determination of anthocyanins in red wines. The basic structures and equilibria of anthocyanins present in wine are shown in Fig. 2.99. Separations were performed in an ODS column (250  4 mm i.d.; particle size 5 m) at 50°C. The buffer applied was 10 mM KH2PO4H3PO4 at pH 1.6. Solvents A and B were ACN–buffer (5:95, v/v) and ACN–buffer (50:50, v/v). Gradient conditions were from 10 per cent B to 45 per cent B in 30 min; to 100 per cent B in 1 min, 100 per cent B for 3 min. Analytes were detected at 518 nm. The retention times of anthocyanins are compiled in Table 2.83. The chromatogram of Cabernet Sauvignon wine is shown in Fig. 2.100. It was concluded from the results that the baseline separation of each analyte under investigation, and the good linearity and ruggedness of the method allow its application for the routine analysis

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251

R1

R1 OH

HO

O

O

HO

R2

H

HO

OH

O

HO

OH

OH

H R2

Gl O

OH

OH

H

Gl

H

OH

Chalcon nearly colourless

H

O

Carbonilbase colourless

CH2-OR3

-Gl

+H2O, -H+ R1

O

O

OH + O

HO

R2 O

OH O

HO O

-H+

+H2SO3, -H+

Gl

O OH H SO3H

OH

Quinoidal base

Flavylium ion

violet

R2

R2

Gl

OH

R1

R1

OH

Gl

Flavene sulfonate

red

clourless

Fig. 2.99. Structure and equilibria of the anthocyanins present in species Vitis vinifera at wine pH. The groups R1, R2, R3 are listed in Table 2.83. Reprinted with permission from B. Berente et al. [234]. TABLE 2.83 ANTHOCYANINS INVESTIGATED IN THIS STUDYa

Peak

tR (min)

Anthocyanin

R1

R2

1 2 3 4 5 6 7 8 9

5.3 7.7 9.3 11.9 13.2 22.1 23.0 29.5 30.1

Delphinidin-3-glucoside Cyanidin-3-glucoside Petunidin-3-glucoside Paeonidin-3-glucoside Malvidin-3-glucoside Paeonidin-3-acetylglucoside Malvidin-3-acetylglucoside Paeonidin-3-coumaroylglucoside Malvidin-3-coumarylglucoside

-OH -OH -OH -H -OCH3 -H -OCH3 -H -OCH3

-OH -H -OCH3 -OCH3 -OCH3 -OCH3 -OCH3 -OCH3 -OCH3

R3

-H

Acetyl -Coumaryl

a

The positions of groups R1, R2, R3 are shown in Fig. 2.97. Reprinted with permission from B. Berente et al. [234].

of red wines. Moreover, the anthocyanin composition may facilitate the authenticity test of red wines [234]. A similar RP-HPLC method was developed and employed for the separation and quantitative determination of nine anthocyanins in red grape cultivars and red wines. Analysis

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Chapter 2

140

5

120

Absorbance / mAbs

100 80 60 7 40

1 3

4

20

9

6

2

8

0 0

5

10

15

20

20

30

35

t / min

Fig. 2.100. Chromatogram of the Cabernet Sauvignon wine used for the method optimization. Numbers refer to anthocyanins in Table 2.83. Reprinted with permission from B. Berente et al. [234].

was carried out in an ODS column (150  3.9 mm i.d.; particle size 5 m) at 25°C. Solvents were composed of water–ACN (40:60, v/v) adjusted to pH 1.3 with perchloric acid (B) and water–ACN (95:5, v/v) at the same pH. Gradient conditions were: 0 min, 5 per cent B; to 10 per cent B in 5 min; to 20 per cent B in 25 min; to 25 per cent B in 38 min; to 35 per cent B in 50 min; to 100 per cent B in 53 min, 100 per cent B in 55. The flow rate was 1.5 ml/min and spectra were recorded between 250 and 600 nm. The anthocyanin profiles of two different red wines are shown in Fig. 2.101. The comparison of the anthocyanin profile of grapes and wines showed marked differences, indicating that the fermentation process modifies the composition of anthocyanins. The results concerning the change of anthocyanin concentration during ageing in oak barrels or in stainless steel tanks are compiled in Table 2.84. Surprisingly, the data in Table 2.84 indicate that neither the type of storage nor the length of ageing exert a considerable effect on the composition of anthocyanins [235]. Size-exclusion chromatography combined with RP-HPLC-MS was employed for the separation of pyranoanthocyanins from red wine. Wine samples (10 ml) were acidified with 3 M HCl to pH 1 then sodium bisulphite was added at a concentration of 400 g/l. After 15 min reaction time the treated wine was loaded into a gel column (200  15 mm i.d.). Pigments were eluted with 95 per cent ethanol followed with 100 per cent methanol. The various fractions were acidified to pH 1, concentrated and redissolved in water. HPLC-DAD was carried out in an ODS column (150  4.6 mm i.d.; particle size 5 m) at 35°C. Solvents were 0.1 per cent aqueous TFA (A) and ACN (B). The gradient started with 10 per cent B for 5 min; to 15 per cent B for 15 min; isocratic for 5 min; to 18 per cent B for 5 min; to 35 per cent B for 20 min. The flow rate was 0.5 ml/min and analytes were detected at 520 nm. MS conditions were: sheath and auxiliary gas were a mixture of nitrogen and

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0.16 0.14

Cabernet-Bauvignon

Pn-3-Gl-Ac Mv-3-Gl-Ac

0.12

Pt-3-Gl

0.04

Pn-3-Gl

0.08 Df-3-Gl

Au

0.10

0.06

253

0.02

Pn-3-Gl-Cm Mv-3-Gl-Cm

Mv-3Gl

Liquid chromatography of natural pigments

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Pn-3-Gl-Cm Mv-3-Gl-Cm

Tempranllie

Mv-3-Gl-Ac

Mv-3Gl Pt-3-Gl

Pn-3-Gl

Df-3-Gl

0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0.00

Cy-3-Gl

Au

Minutes

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Minutes

Fig. 2.101. Chromatograms of Cabernet Sauvignon and Tempranillo wines recorded at 520 nm. Peak identification: Df-3-GL  delphinidin-3-O-glucoside; Cy-3-Gl  cyanidin-3-O-glucoside; Pt-3-Gl  petunidin-3-O-glucoside; Pn-3-Gl  peonidin-3-O-glucoside; Mv-3-Gl  malvidin-3-O-glucoside; Pn-3-Gl-Ac  paeonidin-3-O-acetylglucoside; Mv-3-Gl-Ac  malvidin-3-O-acetylglucoside; Pn-3Gl-Cm  peonidin-3-coumaroylglucoside; Mv-3-Gl-Cm  malvidin-3-coumarylglucoside. Reprinted with permission from E. Revilla et al. [235].

helium, the flow rate being 1.2 l/min and 6 l/min, respectively. The capillary voltage was 4 V and the temperature was 195°C. Spectra were recorded in positive-ion mode (m/z 120 – 1 500). It was found that HPLC-DAD cannot separate all the pigment fractions without the prefractionation of the wine sample. Ethanol eluted seven fractions from the gel column. Eluates 1, 2, 3 and 4 were mixed, forming fraction A, while the other combined eluates formed fraction B. The list of anthocyanins found in fractions A and B and their retention time in RP-HPLC are shown in Tables 2.85 and 2.86. The chemical structures of pigments found in fraction A and B are shown in Figs. 2.102 and 2.103. It was stated that the method allows the separation and identification of new pigments in red wine and this combined technique can be applied for the characterization of new pigments in other red wines [236]. A similar method was employed for the investigation of anthocyanin-derived pigments in port wines. Wine samples were directly applied into an SEC column (250  16 mm i.d.) and were eluted with methanol–water (20:80, v/v) obtaining fraction A followed with methanol (fraction B). The HPLC chromatograms of fractions A and B are shown in Fig. 2.104. The results indicated that the prefractionation of pigments in low molecular mass and high molecular mass fractions may enhance the separation power of the method.

RELATIVE AMOUNT OF EIGHT ANTHOCYANINS IN FOUR TEMPRANILLO WINES (VINTAGE 1999) DURING THEIR AGEING IN OAK BARRELS (O) OR IN STAINLESS STEEL TANKS (S)

Wine

1

3 3

Cy-3-Gl

Pt-3-Gl

Pn-3-Gl

Mv-3-Gl

Mv-3-Gl-Ac

Pn-3-Gn-Cm

Mv-3-Gl-Cm

55 145 235 25

15.20 15.72 14.82 15.09

1.22 1.29 1.31 1.43

14.32 15.15 14.85 13.87

4.25 4.20 4.16 4.52

56.98 56.29 54.69 57.14

3.10 2.90 3.57 2.82

0.80 0.47 0.99 0.74

4.13 3.98 5.61 4.38

145

14.92

1.69

13.95

4.23

56.50

3.30

0.76

4.64

235

15.27

1.44

14.43

3.96

55.16

3.53

0.69

5.51

55 145 235 55 145 235 25

15.81 15.65 15.16 13.95 14.19 13.57 12.81

1.54 1.60 1.51 1.03 1.03 0.91 1.02

14.56 14.64 15.10 14.26 14.08 13.80 13.04

4.50 4.38 3.72 3.08 2.81 2.67 3.03

56.14 55.73 55.10 59.35 58.62 58.58 61.66

2.86 2.98 3.49 2.79 3.08 3.49 3.18

0.68 0.72 0.67 0.50 0.67 0.79 0.61

3.90 4.29 5.24 5.05 5.51 6.23 4.56

145

12.87

1.14

13.49

3.08

59.75

3.64

0.64

5.40

235

11.59

0.91

13.94

3.18

59.51

3.68

0.72

6.47

55 145 235

13.76 13.64 13.85

0.32 1.09 0.96

13.91 13.60 13.30

3.04 3.04 3.22

60.39 58.97 58.18

3.15 3.15 3.60

0.43 0.84 0.91

4.00 5.51 5.97

For symbols see Fig. 2.101. Reprinted with permission from E. Revilla et al. [235].

Chapter 2

4 4 4

Df-3-Dl

Page 254

2 2 2 3 3 3 3

Relative amount (%)

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Oak barrel Oak barrel Oak barrel Stainless steel tank Stainless steel tank Stainless steel tank Oak barrel Oak barrel Oak barrel Oak barrel Oak barrel Oak barrel Stainless steel tank Stainless steel tank Stainless steel tank Oak barrel Oak barrel Oak barrel

Days of ageing

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Type of ageing

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TABLE 2.84

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The procedure was proposed for the study of the change in pigment composition during ageing [237]. Another method used a semi-preparative ODS column for the prefractionation of wine anthocyanins. Alcohol from wine samples was evaporated at ambient temperature, the aquoeus rest was centrifuged, filtered and applied into an ODS column (230  15 mm i.d.). The column was washed with water, then anthocyanins were eluted with the following TABLE 2.85 CHROMATOGRAPHIC DATA OF COMPOUNDS IDENTIFIED IN FRACTION A OF THE WINE

Peak no.

Retention time (min)

Identification

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

21.0 21.5 28.3 29.3 34.0 35.3 36.0 37.3 42.3 43.6 45.0 45.2 46.6 47.3 47.8 49.5 49.8 50.0 50.3 50.7 51.4 51.7 52.2 52.5 53.0 54.1 54.4 56.2 56.5 57.1

Delphinidin-3-glucoside pyruvic derivative Delphinidin-3-glucoside Petunidin-3-glucoside Petunidin-3-glucoside pyruvic derivative Peonidin-3-glucoside Peonidin-3-glucoside pyruvic derivative Malvidin-3-glucoside Malvidin-3-glucoside pyruvic derivative Unknown Malvidin-3-glucoside-4-vynil-procyanidin dimer Peonidin-3-( p-coumaroyl)glucoside pyruvic derivative Malvidin-3-( p-coumaroyl)glucoside pyruvic derivative Unknown Peonidin-3-glucoside-4-vynilcatechin Malvidin-3-glucoside-4-vynilcatechin Peonidin-3-glucoside-4-vynilphenol Peonidin-3-glucoside-4-vynilcatechol Malvidin-3-glucoside-4-vynilepicatechin Malvidin-3-glucoside-4-vynilcatechol Malvidin-3-( p-coumaroyl)glucoside-4-vynilcatechin Malvidin-3-(p-coumaroyl)glucoside-4-vynilepicatechin Peonidin-3-glucoside-4-vynilphenol Malvidin-3-glucoside-4-vynilphenol Peonidin-3-glucoside-4-vynilguaiacol Malvidin-3-glucoside-4-vynilguaiacol Peonidin-3-( p-coumaroyl)glucoside-4-vynilcatechol Malvidin-3-( p-coumaroyl)glucoside-4-vynilcatechol Peonidin-3-( p-coumaroyl)glucoside-4-vynilphenol Malvidin-3-( p-coumaroyl)glucoside-4-vynilphenol Malvidin-3-( p-coumaroyl)glucoside-4-vynilguaiacol

The chemical structures of pigments found in fraction A are shown in Fig. 2.102. Reprinted with permission from C. Alcalde-Eon et al. [236].

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TABLE 2.86 CHROMATOGRAPHIC DATA OF COMPOUNDS IDENTIFIED IN FRACTION B OF THE WINE

Peak no.

Retention time (min)

1

9.9

2

18.7

3

20.0

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18a 18b 19a

21.8 24.5 25.7 26.6 28.4 33.6 35.6 37.8 38.3 39.4 40.1 40.4 40.7 41.1 41.5 41.6 41.9

19b 20 21a

42.2 2.5 43.1

21b 21c

43.3 43.4

22 23 24 25 26 27 28 29 30 31

43.5 44.7 45.8 46.5 47.0 47.7 48.0 48.3 48.9 49.2

Identification Direct condensation product between catechin and delphinidin-3-glucoside Direct condensation product between catechin and peonidin-3-glucoside Direct condensation product between catechin and malvidin-3-glucoside Delphinidin-3-glucoside Unknown Cyanidin-3-glucoside Unknown Petunidin-3-glucoside Peonidin-3-glucoside Malvidin-3-glucoside Unknown Unknown Malvidin-3-glucoside-8-ethyl-procyanidin dimer Petunidin-3-glucoside-8-ethyl-catechin Petunidin-3-glucoside-8-ethyl-catechin Unknown Malvidin-3-glucoside-8-ethyl-gallocatechin Malvidin-3-glucoside-8-ethyl-catechin Malvidin-3-glucoside-8-ethyl-gallocatechin Direct condensation product between catechin and peonidin-3-(p-coumaroyl) glucoside Peonidin-3-glucoside-8-ethyl-catechin Malvidin-3-glucoside-8-ethyl-catechin Direct condensation product between catechin and malvidin-3-(p-coumaroyl) glucoside Malvidin-3-glucoside-8-ethyl-epicatechin Direct condensation product between catechin and malvidin-3-(p-coumaroyl) glucoside Unknown Malvidin-3-acetylglucoside Petunidin-3-( p-coumaroyl)glucoside-8-ethylcatechin Malvidin-3-caffeoylglucoside Petunidin-3-( p-coumaroyl)glucoside Petunidin-3-( p-coumaroyl)glucoside-8-ethyl-catechin Malvidin-3-( p-coumaroyl)glucoside-8-ethyl-catechin Malvidin-3-( p-coumaroyl)glucoside-8-ethyl-catechin Peonidin-3-( p-coumaroyl)glucoside Malvidin-3-( p-coumaroyl)glucoside

Reprinted with permission from C. Alcalde-Eon et al. [236].

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Liquid chromatography of natural pigments

257 R1

R1

OH

OH +

+

O

HO

O

HO

R2

R2

O-Glucose-R3

O-Glucose O

OH

COOH Peak 2: R1 = R2 = OH Peak 3: R1 = OH, R2 = OCH3 Peak 5: R1 = OCH3, R2 = H Peak 7: R1 = R2 = OCH3

Peak 1: R1 = R2 =OH, R3 = H Peak 4: R1 = OH, R2 = OCH3, R3 = H Peak 6: R1 = OCH3, R2 = H, R3 = H Peak 8: R1 = R2 = OCH3, R3 = H Peak 11: R1 =OCH3, R2 = H, R3 = p-coumaric acid Peak 12: R1 = R2 = OCH3, R3 = p-coumaric acid (b)

(a)

R1

R1

OH

OH +

+ O

HO

R2

O

HO

R2 O-Glucose-R4

O-Glucose-R4

OH

O

O

O

HO

OH

R3 OH

OH Vinylphenol derivatives (R3 = H) Peak 16: R1 = OH, R2 = OCH3, R4 = H Peak 22: R1 = OCH3, R2 = H, R4 = H Peak 23: R1 = R2 = OCH3, R4 = H Peak 28: R1 = OCH3, R2 = H, R4 = p-coumaric acid Peak 29: R1 = R2 = OCH3, R4 = p-coumaric acid

OH

R3

Peak 10: R1 = R2 = OCH3, R3 = (epi)catechin, R4 = H Peak 14: R1 = OCH3, R2 = R3 = R4 = H Peak 15: R1 = R2 = OCH3, R3 = R4 = H(catechin) Peak 18: R1 = R2 = OCH3, R3 = R4 = H(epicatechin) Peak 20: R1 = R2 = OCH3, R3 = H, R4 = p-coumaric acid (catechin) Peak 21: R1 = R2 = OCH3, R3 = H, R4 = p-coumaric acid (epicatechin) (d)

Vinylcatechol derivatives (R3 = OH) Peak 17: R1 = OCH3, R2 = H, R4 = H Peak 19: R1 = R2 = OCH3, R4 = H Peak 26: R1 = OCH3, R2 = H, R4 = p-coumaric acid Peak 27: R1 = R2 = OCH3, R4 = p-coumaric acid Vinylguajacol derivatives (R3 = OCH3) Peak 24: R1 = OCH3, R2 = H, R4 = H Peak 25: R1 = R2 = OCH3, R4 = H Peak 30: R1 = R2 = OCH3, R4 = p-coumaric acid (c)

Fig. 2.102. Structures of all the compounds found in fraction A: (a) anthocyanins; (b) A-type vitisins; (c) pyranoanthocyanins originated by reaction between anthocyanins and vynilphenol, vynilcatechol or vynilguaiacol; (d) pyranoanthocyanins originated by reaction between anthocyanins and vynil(epi)catechin. Reprinted with permission from C. Alcalde-Eon et al. [236].

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258

Chapter 2 OH +

O

HO

R1 OH O

HO

OH OH

OH

+

R1 OH

R2 HO

O

O-Glucose-R3 OH

+

R2 O-Glucose-R3

OH Peak 4: R1-R2-OH, R3-H Peak 6: R1=OH, R2=R3=H Peak 8: R1=oh, R2=OCH3, R3=H Peak 9: R1=OCH3, R2=R3=H Peak 10: R1-R2-OCH3, R3-H Peak 23: R1=R2=OCH3, R3=aetic acid Peak 25: R1-R2-OCH3, R3-caffeic acid Peak 26: R1=OH, R2=OCH3, R37p-coumaric acid Peak 30: R1=OCH3, R2=H, R3=p-coumaric acid Peak 31: R1=R2=OCH3, R3=p-coumaric aci (a) R3

Peak 1: R1-R2-OH, R3-H Peak 2: R1=OCH3, R2=R3=H Peak 3: R1=OCH3, R2=H, R3=p-coumaric acid Peak 19a: R1=R2=OCH3, R3=p-coumaric acid Peak 21a: R1-R2-OCH3, R3-p-coumaric acid Peak 21c: R1=R2=OCH3, R3=p-coumaric acid (b)

OH

HO HO

O

OH

HO CH-CH3

R4

R1 OH

O+

HO

R2 O-Glucose-R5 OH

(c)

Peak 13: R1-R2-OCH3, R3-(epi)catechin, R4-H, R5-H Peak 14: R1-OH, R2-OCH3, R3-H, R4-H, R5-H Peak 15: R1-OH, R2-OCH3, R3=H, R4=H, R5=H Peak 17: R1=R2=OCH3, R3=H, R4=OH, R5=H Peak 18a: R1=R2=OCH3, R3=H, R4=H, R5=H Peak 18b: R1-R2-OCH3, R3-H, R4-OH, R5-H Peak 19b: R1=OCH3, R2=R3=R47R5=H Peak 20: R1-R2-OCH3, R3-R4-R5-H Peak 21b: R1-OH, R2-OCH3, R3=R4=R5=H (epicatechin) Peak 24: R1-OH, R2-OCH3, R3-H, R4-H, R5-p-coumaric acid Peak 27: R1=OCH3, R2=R3=R4=H, R5=p-coumaric acid Peak 28: R1=R2=OCH3, R3=R4=H, R5=p-coumaric acid Peak 29: R1=R2=OCH3, R3=R4=H, R5=p-coumaric acid

Fig. 2.103. Structures of all the compounds found in fraction B: (a) anthocyanins and acylated anthocyanins; direct condensation products between flavanols and anthocyanins; (c) dimers resulting from the condensation mediated by acetaldehyde between anthocyanins and flavanols. Reprinted with permission from C. Alcalde-Eon et al. [236].

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259

gradient: from 100 per cent of 2 per cent acetic acid to 100 per cent methanol–2 per cent acetic acid (1:1, v/v) in 280 min; methanol for 60 min. The flow rate was 2.8 ml/min and anthcyanins were detected at 520 nm. The three fractions eluted separately from the column were analysed in a C16 column (150  4.6mm i.d.). Gradient components were 1 per cent formic acid (A) and ACN (B). Gradient conditions were 5 per cent B to 15 per cent B over 2 min; 15 per cent B over 8 min, to 20 per cent B over 15 min; to 25 per cent B over 10 min; to 30 per cent B over 9 min; to 40 per cent B over 5 min; to 50 per cent B for 5 min; to 60 per cent B over 6 min. The flow rate was 0.7 ml/min and analytes were detected at 525 nm. The capillary temperature and voltage were 180°C and 3 V, respectively. The combined method successfully separated anthocyanins such as delphidin 3-glucoside (Dp-3g), cyanidin 3-glucoside (Cy-3g), petunidin 3-glucoside (Pt-3g), peonidin 3-glucoside (Pn-3g), malvidin 3-glucoside (Mv-3g), delphidin 3-acetylglucoside, petunidin 3-acetylglucoside, malvidin 3-acetyl-glucoside, malvidin 3-caffeoylglucoside, petunidin 3-coumaroylglucoside and malvidin 3-coumaroylglucoside. It was concluded from the data that the prefractionation of wine anthocyanins is a prerequisite of successful HPLC analysis [238]. The synthesis of two new red wine pigments by nucleophilic addition of vinylphenols to malvidin 3-glucoside has been described. The structures of the two new pigments and their formation are shown in Fig. 2.105. HPLC-DAD and HPLCMS confirmed the occurrence of these two anthocyanin derivatives in red wine [239]. 2.3.3.2 Determination of anthocyanins in fruits and other beverages The anthocyanin content of fruits and berries has also been frequently investigated. Besides traditional columns, the application of narrow-bore columns coupled with electrospray ionization detection in the analysis of anthocyanins in berries has also been reported [240]. Both isocratic and gradient elution techniques have been employed for the separation of anthocyanic pigments in red fruit juices, concentrated juices and syrups. Fruit juices were filtered and preconcentrated in ODS SPE cartridges. Syrups were diluted before filtering and SPE treament. Cartridges were activated by 5 ml of methanol followed with 5 ml of water. Analytes were eluted with methanol. Separation was performed in an ODS column (250  4.5 mm i.d.; particle size 5 m) using two isocratic mobile phases: water–ACN–formic acid (81:9:10 and 84:6:10, v/v). Gradient elution conditions were: 0–25 min 84 per cent water–6 per cent ACN; to 65 per cent water over 35 min; held for 45 min. The mobile phase always contained 10 per cent formic acid. The flow rate was 1 ml/min. Some typical chromatograms are shown in Fig. 2.106. The chromatograms demonstrate that the anthocyanin profile of red fruit juices show considerable differences which can be used for their authenticity test. Moreover, the chromatograms illustrate the good separation power of the HPLC system. The identified anthocyanins and anthocyanidins and their occurrence in various red fruit juices are compiled in Table 2.87. It was concluded from the data that the method is suitable for the differentiation between fruits and may help the detection of adulteration [241]. An HPLC separation method using acetone in the mobile phase revealed that anthocyanins of blackcurrant seeds readily react with acetone, as shown in Fig. 2.107. This finding indicates that the use of acetone in the mobile phase may lead to distorted results in the analysis of anthocyanins [242].

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Chapter 2 3

mAU

200

7 5

100

6

1

8

4

2 0 10

0

20

40

30

50

Min

(a)

8 120

mAU

4 80 3 2 40 1

13 10 12 5 67 9 16 11 15 17 14 18 19 20

0 0 (b)

10

20

30 Min

40

50

60

Fig. 2.104. HPLC chromatograms, recorded at 520 nm, of fraction a (upper lane) and b (lower lane) eluted from Toyopearl gel column with 20 per cent and 100 per cent methanol. Peak identifications: Fraction a, 1  Mv 3-gluc; 2  Mv 3-gluc-4-vinyl; 3  Mv 3-gluc-py derivative; 4  Pt 3(acetyl)gluc-py derivative; 5  Mv 3-(acetyl)gluc-py derivative; 6  Pt 3-(coumaroyl)gluc-py derivative; 7  Mv 3-(coumaroyl)gluc-py derivative; 8  Pn 3-(coumaroyl)gluc-py derivative. Fraction b, 1  Mv 3-gluc-4-ethyl-cat; 2  Mv 3-gluc-4-vinyl-PC dimer; 3  Mv 3-(acetyl)gluc-4vinyl-PC dimer; 4  Pn 3-(coumaroyl)gluc-py derivative; 5  Mv 3-gluc-4-vinyl-cat; 6  Dp 3-gluc-py derivative; 7  Mv 3-(acetyl)gluc-4-vinyl-cat; 8  Mv 3-(coumaroyl)gluc-py derivative; 9  Mv 3-(coumaroyl)gluc-4-vinyl-PC dimer; 10  Mv 3-gluc-4-vinyl-cat; 11  Mv 3(coumaroyl)gluc-4-ethyl cat; 12  Mv 3-(acetyl)gluc-4-vinyl-cat; 13  Mv 3-(coumaroyl)gluc; 14  Dp 3-(acetyl)gluc-py derivative; 15  Mv 3-(coumaroyl)gluc-4-vinyl-cat; 16  Mv 3-gluc-4vinylphenol; 17  Mv 3-(caffeoyl)gluc-4-vinylphenol; 18  Pn 3-(coumaroyl)gluc-4-vinylphenol; 19  Mv 3-(coumaroyl)gluc-4-vinylphenol; 20  Mv 3-(acetyl)gluc-4-vinylphenol. Mv, alvidin; Dp, delphinidin; Pt, petunidin; Pn, paeonidin; py, pyruvic acid; gluc, glucoside; Cat, ()-catechin or ()-epicatechin; PC, procyanidin. Reprinted with permission from N. Mateus et al. [237].

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Liquid chromatography of natural pigments

261 OMe

OMe

OH

OH O+

HO

O+

HO

OMe

OMe +

pH 1.5

Oglc OH

R1

Oglc

R2

O

OH 1: malvidin 3-glucoside 2a: R1 = OH, R2 = H 3a: R1 = R2 = OCH3

R1

R2 OH

2b: R1 = OH, R2 = H 3b: R1 = R2 = OCH3

Fig. 2.105. Formation of pyranoanthocyanins. Reprinted with permission from A. E. Hakansson et al. [239].

Normal-phase separation mode has also been employed for the HPLC-MS analysis of proanthocyanidins in foods and beverages. Polyphenol extraction from various samples was performed by SPE. The cartridge was conditioned with 3  5 ml methanol followed with 3  5 ml water. After sample loading, the cartridges were dried in vacuum and the analytes were eluted with 10 ml of acetone–water–acetic acid (70:29.5:0.5, v/v). Solid samples (cinnamon, grape seed extract, peanut skin, peanut nutmeat, apple, almond seedcoat) were extracted before SPE, liquid samples (green tea beverage, red wine, grape juice) were loaded after filtering. Separation was carried out in a silica column (250  4.6 mm i.d.; particle size 5 m) at 37°C. Solvents for gradient elution were dichloromethane (A), methanol (B) and acetic acid–water (1:1, v/v, C). Concentration of C was constant (4 per cent) during the separation. Gradient conditions were: from 14 per cent B in A to 28.4 per cent B in A in 30 min; to 50 per cent B in 30–60 min; to 86 per cent B in 60–65 min; 65–70 min isocratic. Analytes were detected at 280 nm. Fluorescence detection was carried out at 276 and 316 nm excitation and emission wavelengths. MS conditions in the negative-ion mode were: 0.75 M ammonium hydroxide as buffering agent (flow rate 0.04 ml/min), and capillary and fragmentor voltages 3 kV and 75 V, respectively. The nebulizing pressure was 25 psi and the drying gas temperature 350°C. The mass range was set to m/z 100–3 000. Some characteristic chromatograms are shown in Fig. 2.108. The chromatograms show the good separation performance of the system and illustrate the marked difference between the oligomer profiles of samples. It was stated that the method can be applied for the analysis of proanthocyanidins in a wide variety of foods and food products. Because of the higher sensitivity and selectivity, the fluorescence detection mode was proposed [243]. A combined technique has been developed for the preparative isolation of anthocyanins from blackcurrant (Ribes nigrum) fruits. Frozen berries (370 g) were extracted three times with 1l of methanol containing 0.1 per cent TFA at 4°C for 6 h. The combined exracts were concentrated, diluted to 250 ml with water and extracted with ethyl acetate. The aqueous phase was concentrated to 100 ml and applied in an ion-exchange column. The column

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Chapter 2 DP3GLU CY3GAL DP3ARA CY3GLU

MV3GLU

PT3GLU

CY3GLU

PN3GLU

DP3GAL

Absorbance (525 nm)

Absorbance (525 nm)

DP3GLU

PN3GAL MV3GAL PT3GAL CY3ARA PN3ARA PT3GLU PT3ARA PN3GLU MV3GLU MV3ARA

0.00

0

40.00

20.00

(a)

20

(b)

Minutes

40

60

min CY3-2GLU-RUT

Absorbance (525 nm)

Absorbance (525 nm)

CY3SOPHO

CY32GLU-RUT CY 3GLU PG 3SOPHO

0

(c)

20

40 min

CY3-2XYL-RUT

CY3GLU CY3-SOPHO

CY3RUT

CY3RUT

60

0

(d)

20

40

60

min

Fig. 2.106. Separation and identification of various anthocyanin red fruit juices: a, grape; b, blueberry; c, raspberry; and d, red currant. Mobile phase: water–acetonitrile–formic acid (84:6:10, v/v). Abbreviations: Dp: delphidin, Cy; cyanidin, Pt: petunidin, Pn: peonidin, Mv: malvidin, glu: glucose, gala: galactose, ara: arabinose, ruti: rutinose, sopho: sophorose, sam: sambubiose, xyl: xylose. Reprinted with permission from J.-P. Goiffon et al. [241].

was washed with water and anthocyanins were eluted with methanol containing 0.05 per cent TFA. The samples were further purified by SEC using 30–60 per cent methanol with 0.05 per cent TFA. Analytical and preparative separations were carried out in ODS columns (250  4.6 mm i.d.; particle size 5 m, and 250  10 mm i.d.; particle size 10 m) using the same solvents: formic acid–water (1:9, v/v, A) and formic acid–water–methanol (1:4:5, v/v, B). Gradient conditions for analytical HPLC were: 90 per cent A, 0–4 min; to 0 per cent A in 4–17 min; 4min isocratic. For preparative HPLC the conditions were: 90 per cent A, in 0–2 min; to 40 per cent A, in 2–5 min; to 10 per cent A, in 5–17 min; to 0 per cent A over 1 min. The flow rates were 1.0 and 4.0 ml/min. TLC control was carried out on cellulose layers using formic acid–ccHCl–water (25:24:51, v/v). SEC measurements indicated that anthocyanidin 3-rutinosides and 3-glucosides were better separated on the Toyopearl HW-40 F stationary phase than on Sephadex LH-20. The chromatographic profiles of preparative and analytical separation are shown in Fig. 2.109. The chromatograms illustrate the acceptable separation of analytes in preparative conditions. The HPLC and TLC data are compiled in Table 2.88.

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TABLE 2.87 IDENTIFIED ANTHOCYANINS AND ANTHOCYANIDINS AND THEIR OCCURRENCE IN VARIOUS RED FRUIT JUICE

Compound no.

Anthocyanin and Anthocyanidin

Occurrence in fruit

1 2 3 4 5 6 7 8 9 10 11

Dp 3-gala Cy 3,5-diglu Cy 3-sam, 5-glu Dp 3-glu Cy 3-sopho Cy 3-gala Pg 3,5-diglua Dp 3-ara Dp 3-ruti Cy 3-(2Gglu-ruti) Cy 3-glu

BB EL EL BB, G, BC RB BB

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Cy 3-sam Pg 3-sopho Pg 3-gala Cy 5-glub Pn 3,5-diglu Pt 3-gala Cy 3-ara Cy 3-(2Gxyl-ruti) Cy 3-ruti Pt 3-glu Pg 3-glu Mv 3,5-diglu Dpc Pn 3-gala Pt 3-ara Pg 5-glub Pg 3-ara Pn 3-glu Cy 3-L-rha g Mv 3-gala Pn 5-glub Cyc Pn 3-ara Mv 3-glu Mv 5-glub

BB BC RC, MC, RB RC, MC, RB, BB, G, BL, EL, S EL RB S, RB G BB BB RC RC, MC, BL, RB BB, G S G BB BB, S

BB, G BB

BB BB. G (Continued on next page)

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Chapter 2 TABLE 2.87 (continued)

Compound no.

Anthocyanin and Anthocyanidin

37 38 39 40

Ptc Pgc Pnc Mvc

Occurrence in fruit

For symbols see Fig. 2.106. BC  blackcurrant; RC  redcurrant; S  strawberry; MC  morello cherry; RB  raspberry; EL  elderberry; BB  blueberry; G  grape. a Commercial standard not found in fruits. b Obtained by partial hydrolysis of the 3.5-diglucoside. c Obtained by hydrolysis of the corresponding anthocyanins. Reprinted with permission from J.-P. Goiffon et al. [241].

R1

R1 OH

O+

HO

R2

OH O

O+

HO

OG

R2 OG

O

OH 3d, 40°C

R1 1/6 OH 2/7 OH 3/8 OH 4/9 OH 5/10 OCH3

R2 H OH H OH OCH3

OH

G rutinose rutinose glucose glucose glucose

Fig. 2.107. The formation of pyranoanthocyanins 6–10 from anthocyanins 1–5 and acetone. Reprinted with permission from Y. Lu et al. [242].

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265

P1 350

P2

300

mAU

250 200

P3

150

P8 − P12

P4

100

P5 P6 P7

50 0 0

5

10

15

20

(a)

25

30

35

40

35

40

min

P3 1000

mAU

800 600

P4

400

P5

200 0 0

5

10

15

20

(b)

25

30

min

P3 P4

mAU

400

P2

300 Mixed oligomers 200 100 0 0

(c)

5

10

15

20

25

30

35

40

min

Fig. 2.108. (a) Whole Red Delicious apple extract. (b) Cinnamon extract. Labels correspond to the predominant A-type oligomers in the extract. (c) Peanut skin extract. UV traces at 280 nm for a 10 l injection of various samples. Peak identification: P1  monomers; P2  dimers; P3  trimers; P4  tetramers; P5  pentamers; P6  hexamers; P7  heptamers; P8  octamers; P9  nonamers; P10  decamers; P11  undecamers; P12  dodecamers. Reprinted with permission from S. A. Lazarus et al. [243].

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The data prove that the retention order of anthocyanins deviates from each other in HPLC and TLC suggesting the involvement of a different retention mechanism. It was stated that the preseparation of anthocyanins by size-exclusion chromatography is a prerequisite of the successful preparative separation by RP-HPLC [244]. Novel pyranoanthocyanins have also been isolated and identified in blackcurrant (Ribes nigrum) seed using HPLC, 2D NMR and ES-MS. Blackcurrant seeds were extracted with acetone–water (70:30, v/v) and the components of the extract were separated in a polyamide column followed by HPLC-DAD. The new pigments were finally separated in an MCI-HP20 column. The chemical structures of anthocyanins 1–2 and the novel pyranoanthocyanins 3–6 with the pyrano[4,3,2-de]-1-benzopyrylium core structure are shown in Fig. 2.110. It was stated that the analytical method developed separated well the novel pyranoanthocyanins [245]. A similar combined method has been employed for the isolation and identification of new pyranoanthocyanins from blackcurrant (Ribes nigrum) seed [246].The chemical structures of the new pyrananthocyanins are shown in Fig. 2.111. As it has been demonstrated that the consumption of strawberries has health benefits [247,248] the composition of the strawberry has been vigorously investigated [249,250].Various physicochemical methods have been employed for the isolation and identification of a new anthocyanin (5-carboxypyranopelargonidin 3-O--glucopyranoside) in strawberry (Fragaria ananassa). Strawberries were extracted twice with methanol containing 0.5 per cent TFA at 4°C. The combined extracts were filtered, concentrated under reduced pressure, extracted with ethyl acetate and purified by ion-exchange and size exclusion chromatography using gradient elution. Preparative RP-HPLC was carried out in an ODS column (250  22 mm i.d.; particle size 5 m) using formic acid–water (1:19, v/v, A) and formic acid–water–methanol (1:9:10, v/v, B) as mobile phase components. The following gradient was employed: from 10 per cent B to 100 per cent B in 45min; isocratic over 13 min. The flow rate was 14 ml/min. An ODS column (250  23 mm i.d.; particle size 5 m) was employed for analytical separation. Formic acid–water (1:18, v/v, A) and formic acid–water–methanol (1:9:10, v/v, B) were used as mobile phase components. The two gradients consisted of: from 10 per cent B to 100 per cent B in 23 min; isocratic over 5 min (gradient 1); from 10 per cent B to 30 per cent B in 3 min; isocratic over 42 min; from 30 per cent to 100 per cent B in 7 min; isocratic over 5 min (gradient 2). The flow rate was 0.75 ml/min. Identification of the new pigment was performed by UV-visible spectroscopy, 2D-NMR and electrospray LC-MS [251]. Similar combined chromatographic technology was applied for the isolation and identification of flavanol–anthocyanin complexes from strawberry (Fragaria ananassa Dutch). Strawberries (1.25 kg) were extracted in 3  1.5 l of methanol containing 0.5 per cent TFA at 3°C. Extract volume was reduced, then it was extracted with ethyl acetate, and purified in an ion-exchange column and followed by SEC. SEC was performed in a Sephadex LH-20 column (100  5 cm) using methanol–TFA–water (19.8:0.2:80.0, v/v) as the mobile phase for the elution of monomeric anthocyanins. Flavanol–anthocyanin complexes were eluted by changing the compostion of the mobile phase to 49.5:0.5:50.0, v/v. Complexes were further separated by preparative HPLC using formic acid–water (0.5:9, v/v, A) and methanol–formic acid–water (5:0.5:4, v/v, B) as mobile phase components. The gradient was as follows: from 10 per cent B to 100 per cent B in 45 min; isocratic over

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3000 4

2 1 2500

mAU

2000 3

1500

1000

500 5

6

0 0

5

10 Min

(a)

15

4 400 350 2

300

mAU

250 200 150

1 3

100 50

5 6

0 0 (b)

5

10

15

20

25

Min

Fig. 2.109. Preparative (a) and analytical separations (b) of anthocyanins in fruits of the blackcurrant (Ribes nigrum) at 52010 nm. The amount of sample applied to the preparative column was 4.3 mg. Peak identification: 1  Dp 3-glu; 2  Dp 3-rut; 3  Cy 3-glu; 4  Cy 3-rut; 5  Pn 3-rut; 6  Mv 3-rut. Reprinted with permission from C. Froytog et al. [244].

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Chapter 2 TABLE 2.88 CHROMATOGRAPHIC DATA ON THE ANTHOCYANINS IN FRUITS OF THE BLACKCURRANT (RIBED NIGRUM)

Pigment

Online HPLC

Dp 3-glu Dp 3-rut Cy 3-glu Cy 3-rut Pn 3-rut Mv 3-rut

tR (min)

Area (%)

TLC hRF

11.09 11.76 12.21 12.88 13.57 14.58

12.9 30.6 9.3 43.6 1.5 2.1

15 35 25 48 60 57

Reprinted with permission from C. Froytog et al. [244].

R1

R1 OH

OH

+

+

O

HO

O

HO

OH

OH

HO

HO

O

O

OH

OH

OH

O

O

O OH

CH3

OR2

OH OR2

1 R1 = H, R2 = rhamnose

3 R1 = H, R2 = rhamnose

2 R1 = OH, R2 = H

4 R1 = OH, R 2 = rhamnose 5 R1 = H, R2 = H 6 R1 = OH, R2 = H

Fig. 2.110. Chemical structures of anthocyanins 1–2 and the novel pyranoanthocyanins 3–6. Reprinted with permission from Y. Lu et al. [245].

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269 R

R

OH

OH O+

HO

OH

O+

HO

OH OG

OG O

O CH3

C

1/5 2/6 3/7 4/8

R

G

H OH H OH

rut rut glc glc

OH

1-4

5-8

Fig. 2.111. Chemical structures of pyrananthocyanins. Reprinted with permission from Y. Lu et al. [246].

13 min. The flow rate was 14 ml/min. Analytical HPLC was carried out in an ODS column (200  5 mm i.d.; particle size 5 m) using the same solvents as for the preparative HPLC. The gradient was from 10 per cent B to 100 per cent B in 23 min; isocratic over 5 min. The 1 H-1H NOESY NMR spectra and other spectroscopic methods identified the flavanol–anthocyanin complexes [252]. Their structure is shown in Fig. 2.112. SEC has also been employed for the fractionation of apple procyanidins. Apple procyanidins (apple condensed tannins: ACTs) were extracted by homogenizing 3 kg of apple with 0.1 per cent (w/w) potassium pyrosulphite solution. After holding the mixture at 4°C for 24 h the suspension was centrifuged and the supernatant was filtered. The filtrate was applied to a Sepabeads SP-850 column (285  25 mm i.d.), then the column was washed with 300 ml of water. The crude apple polyphenol fraction was eluted with 200 ml of 80 per cent ethanol and evaporated to 65 ml. Further purificat ion of the CAP fraction was achieved by SEC columns. Samples were firstly loaded in to a column of 285  25 mm i.d., the column was washed with 200 ml of distilled water, and the phenolic compounds were eluted with 250 ml of 40 per cent ethanol and 100 ml of 60 per cent acetone. The ethanolic fraction contained the monomeric catechins and oligomeric procyanidins and it was purified in an ODS SPE cartridge. The resulting solution and the acetone eluate were mixed, obtaining ACT fraction, which was loaded into another SEC column (950 mm  25 mm i.d.). SEC fractions were analysed by RP-HPLC using an ODS column (150 mm  4.6 mm i.d.; particle size 5 m). Solvents were mixtures of methanol–0.01 M KH2PO4 in volume ratios of (2:8, v/v, A) and (5:5, v/v, B) adjusted to pH 2 with phosphoric acid. The gradient consisted of 10 per cent A for 0–10min; to 100 per cent B in 40 min, isocratic for 15min. The flow rate was 1 ml/min and analytes were detected at 280 nm. The chemical structures of monomeric flavan-3-ols and procyanidin oligomers in apples are shown in Fig. 2.113. Typical chromatograms illustrating the effect of SEC prefractionation on the chromatographic

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Chapter 2

R OH O

HO

OH OH

OH

HO HO O

OH O

OH

OH OH

Fig. 2.112. The structures of the flavan-3-ol(4 → 8)pelargonidin 3-O-
-glucopyranosides (1–4) isolated from strawberry extract. The letter A denotes the aglycone ring systems belonging to the anthocyanidin substructure, whereas the letter F denotes the aglycone ring system belonging to the flavanol substructure. Reprinted with permission from T. Fossen et al. [252].

profiles of analytes are compiled in Fig. 2.114. MALDITOF spectra of SEC fractions indicated that fraction 4 contains mainly tetramers, fraction 5 four tetra-heptamers, fraction 6 hepta- and octamers while no polymerized compound was detected in fraction 7. It has been conluded from the results that the combination of SEC with RP-HPLC allows the successful separation of procyanidin oligomers and it can be employed for the analysis of hydrolysable tannins and anthocyanins in tea and wine [253]. 2.3.3.3 Determination of anthocyanins in miscellaneous matrices The anthocyanins in other food and food products have also been investigated [254,255].Thus, the procyanidin composition of chocolate has been investigated by RPHPLC with electrospray ionization mass spectrometric and tandem mass spectrometric detection. Chocolate samples of 10 g were frozen, ground, and 1 g was homogenized twice with 10 ml of n-hexane for 5 min at 30°C. The defatted sample was air-dried and sonicated with acetone–water–acetic acid (70:29.8:0.2 v/v) for 10 min at 30°C. The organic phase was evaporated and the aqueous part was filtered and used for HPLC analysis without further purification. Separation was carried out in an ODS column (250  4.6 mm i.d.; particle size 5 m) at ambient temperature. The mobile phase components were 0.2 per cent aqueous acetic acid (A) and ACN (B). The gradient started from 6 to 25 per cent B in 0–18 min; to 60 per cent B in 18–20 min; isocratic 20–25. Optimized MS conditions were as follows: negative-ion mode; source voltage, 3.9kV; capillary voltage, 31 V, capillary temperature, 300°C; sheath and auxiliary gas was nitrogen. Spectra were recorded between m/z 250–2 000. Some typical mass chromatograms are shown in Fig. 2.115. It has been

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271 OH

OH

OH

OH O

HO

O

HO

OH

OH

R

OH

OH

OH

O

HO

R OH

R=

OH: (-)-epicatechin

R=

OH: procyanidin B2 (epi-epi)

R=

OH: (+)-catechin

R=

OH: pocyanidine B1 (epi-cat) OH

OH

OH

OH O

HO

O

HO

OH

OH

OH

OH OH

OH

OH

OH OH

OH

OH

R

O

HO

OH

OH

OH

OH

O

HO

O

HO

OH

O

HO

OH

n

OH OH

OH OH

procyanidin C1 (epi-epi-epi)

O

HO

OH OH procyanidin oligomers n = 0 - 13

Fig. 2.113. Structures of monomeric flavan-3-ols and procyanidin oligomers in apples. Reprinted with permission from A. Yanagida et al. [253].

stated that the method is rapid, does not need complicated sample preparation procedures and gives structural information for the identification of analytes [256]. The separation and identification of two novel anthocyanins from red onion, Allium cepa, have also been reported. Scales of 2.37 kg red onion were cut and extracted twice with methanol containing 0.5 per cent TFA. The extract was filtered, extracted with ethyl acetate and the aqueous phase was further purified in an ion-exchange and an SEC column. SEC separation was performed in a Sephadex LH-20 column (100  5 cm i.d.) applying methanol–water–TFA (39.6:60:0.4, v/v) as the mobile phase at a flow rate of 2.5 ml/min.

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Chapter 2 5

2

1

5

4

3

10

15

20

25 30 35 40 Retention time (min)

45

50

55

60

65

5

fr. 1

2

fr. 4

3 fr. 5 fr. 2

1 fr. 6

4 AU fr. 3

AU

5

10

15 20 25 30 Retention time (min)

35

fr. 7

5

10 15 20 25 Retention time (min)

30

35

Fig. 2.114. RP-HPLC profiles of ACTs and SEC fractions (fr.) of ACTs. Each lyophilized sample was dissolved in water (1 mg/ml), and analysed by RP-HPLC. Upper chromatogram: RP-HPLC profile of ACTs. Lower chromatograms with fraction numbers: RP-HPLC profiles of SEC fractions of ACTs. The numbers of identified peaks in each chromatogram are (1) procyanidin B1 (PB1), (2) ()-catechin, (3) procyanidin B2 (PB2), (4) procyanidin C1 (PC1), 5 ()-epicatechin (EC). AU means relative absorbance units (at 280 nm). For details on the RP-HPLC conditions see text. Reprinted with permission from A. Yanagida et al. [253].

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EC m/z 289 (I) C PB2

Relative abundance

m/z 577 (II)

m/z 865 (III)

m/z 1153 (IV)

m/z 1441 (V)

m/z 1729 (VI)

0

2

4

6

8

12

14 16 18 Time (min)

20

22

24

26

28

Fig. 2.115. Mass chromatograms of catechin monomers (m/z 289) and procyanidin oligomers (dimer through hexamers m/z 577 to 1729). C, ()-catechin, EC, ()-epicatechin, and PB2, procyanidin B2 as identified by the retention times of authentic standard. Reprinted with permission from J. Wollgast et al. [256].

Preparative and analytical HPLC were carried out in an ODS column using gradient elution. The gradient was composed of methanol, water and formic acid. The chemical structures of the new pigments were elucidated by UV-VIS, 2D NMR and LC-MS. MS conditions were: capillary 3 kV, cone 30 and 60 V, extractor 7 V, sources block temperature 120°C, desolvation temperature 150°C [257].The chromatographic profile of the SEC fraction containing the new pigments is shown in Fig. 2.116. The chemical structures of the new derivatives identified by various spectroscopic techniques are shown in Fig. 2.117. New anthocyanin pigments have also been isolated and identified from the cell cultures of carrot, Nentes scarlet-104 local variety. Overnight extraction of pigments was carried out by acidified methanol at 4°C. The concentrated methanolic solution was concentrated and washed with chloroform and diethyl ether to remove lipids and chlorophyll. Similarly to the methods discussed above, the eluate was prepurified by ion-exchange and SEC methods. Anthocyanins were hydrolysed by 1 N HCl for 1 h, the acyl goups were extracted with diethyl ether, and the aglucones were extracted with amyl alcohol. Anthocyanidin aglycones were separated in an ODS column (250  4.6 mm i.d.; particle size 10 m) using methanol–acetic acid–water (7:1:2, v/v) as the isocratic mobile phase. Carbohydrate moieties

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Chapter 2 2

1 3 4 5

10 min

15

20

Fig. 2.116. The HPLC profile of the Sephadex LH-20 fraction containing the carboxypyranocyanidins 1–4. Reprinted with permission from T. Fossen et al. [257].

OH OH + O

HO

HO O O O O

OH

OH OH O O

1 O OH 2

Fig. 2.117. The structures of 5-carboxypyranocyanidin 3-O-
-glucopyranoside (1) and 5-carboxypyranocyanidin 3-O-(6-O-malonyl-
-glucopyranoside (2) isolated from red onions. Reprinted with permission from T. Fossen et al. [257].

were separated by paper chromatography. Anthocyanins were identified by MS and NMR. Anthocyanins found in the callus cultues were cyanidin-3-lathyroside [cyanidine-3-O{-Dxylopyranosyl (1 → 2) -D-galacto pyranoside}] and cyanidine-3--D-glucopyranoside [258].

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Anthocyanins were separated and identified even in the tubers of pinta boca (Solanum stenotomum). Tubers were boiled for 15 min then the coloured zones were separated, ground and freeze-dried. An aliquot of 40 mg was dissolved in water (pH adjusted to 0.5 with HCl) and applied on a C18 SPE cartridge previously activated by acidified methanol and water. The loaded cartridge was washed with water and analytes were eluted with methanol containing 0.1 per cent HCl. De-acylated anthocyanins were prepared by alkaline hydrolysis. Separation was carried out in an ODS column (150  4.6 mm i.d.; particle size 5 m) at 35°C. Solvents were 0.1 per cent aqueous TFA (A) and ACN (B). Gradient conditions were isocratic 10 per cent B, for 5 min; from 10 per cent to 15 per cent B over 15 min; isocratic 15 per cent B for 5 min; from 15 per cent B to 18 per cent B over 5 min; from 18 per cent B to 35 per cent B over 20 The flow rate was 0.5 ml/min and analytes were detected at 520 nm. MS conditions were: sheath (1.2 l/min) and auxiliary gas (6 l/min) were mixtures of nitrogen and helium; capillary voltage, 4 V; capillary temperature, 195°C. Spectra were taken in the positive-ion mode ranging from m/z 120–1 500. The chromatogram of pinta boca tuber concentrate is shown in Fig. 2.118. The chromatogram illustrates that the pinta boca tuber concentrate contains a considerable number of pigment fractions which are not entirely separated under the chromatographic conditions employed. The retention time and proposed identity of the pigment fractions are compiled in Table 2.89. It was established that each pigment was acylated by hydroxycinnamic acid and the acylation increased the stability of the pigments. Furthermore, the data demonstrated that the tubers of pinta boca can be employed as a natural colourant in the food industry [259]. Because of their marked importance in human nutrition, the analytical techniques employed for the separation of biologically active compounds in potatoes (Solanum tuberosum), tomatoes (Lycopersicum esculentum) and jimson weed (Datura stramonium) seeds have been reviewed before [260]. 100

6a−6b

4

mAU

80 400

60 5 40

1

2a 7a−7b

300

20

mAU

2b

3

8

0 200

38

40

42

44 min

46

48

100

0

0

30

20

30

40

50

min

Fig. 2.118. Chromatogram recorded at 520 nm corresponding to pinta boca tuber concentrate. Reprinted with permission from C. Alcalde-Eon et al. [259].

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Chapter 2

TABLE 2.89 RETENTION TIME AND PROPOSED IDENTITY FOR ALL THE COMPOUNDS FOUND IN THE CONCENTRATE OF PINTA BOCA TUBERS

Peak no.

Proposed identity

Retention time

1 2a 2b 3 4 5 6a 6b 7a 7b 8

Petunidin 3-O-p-coumaroylrutinoside-7-O-glucoside Petunidin 3-O-caffeoylrutinoside-5-O-glucoside Delphinidin 3-O-p-coumaroylrutinoside-7-O-glucoside Peonidin 3-O-caffeoylrutinoside-5-O-glucoside Petunidin 3-O-(cis-p-coumaroyl)rutinoside-5-O-glucoside Petunidin 3-O-feruloylrutinoside-5-O-glucoside Peonidin 3-O-(cis-p-coumaroyl)rutinoside-5-O-glucoside Malvidin 3-O-p-coumaroylrutinoside-5-O-glucoside Peonidin 3-O-feruloylrutinoside-5-O-glucoside Malvidin 3-O-feruloylrutinoside-5-O-glucoside Petunidin 3-O-p-coumaroylrutinoside

36.2 37.3 37.5 39.3 40.1 40.9 42.0 42.4 42.7 43.2 44.4

Reprinted with permission from C. Alcalde-Eon et al. [259].

The anthocyanin composition of flowers has also been frequently investigated by chromatographic methods. Acylated anthocyanins and flavonols have been extracted from the flowers of Dendrobium cv. ‘Pompadour’ and separated by TLC and RP-HPLC techniques. Fresh flowers (approximately. 1 kg) were extracted with 10 l of methanol–acetic acid–water (4:1:5, v/v), and the extract was concentrated and analysed by preparative and analytical RP-HPLC using ODS columns (150  19 mm i.d.; and 250  4.6 mm i.d.) at 40°C. The gradient consisted of methanol–water–acetic acid in various volume ratios. Flow rates were 4 and 1 ml/min for preparative and analytical separations, respectively. Cyanidin 3 (6-malonylglucoside)-7,3
-di(6-synapilglucoside) and the demalonyl derivative were detected in the flowers [261]. The anthocyanin profile of the flowers of Vanda (Orchidaceae) was investigated with a similar technique. Flowers (2 kg) were extracted with 10 l of methanol–acetic acid–water (9:1:10,v/v) at ambient temperature for 24 h. The extract was purified by column chromatography, paper chromatography, TLC and preparative RP-HPLC. Analytical HPLC was carried out in an ODS column (250  4.6 mm, i.d.) at 40°C. Gradient conditions were from 40 per cent to 85 per cent B in 30 min (solvent A: 1.5 per cent H3PO4 in water; solvent B: 1.5 per cent H3PO4, 20 per cent acetic acid and 25 per cent ACN in water). The flow rate was 1 ml/min and analytes were detected at 530 nm. The chemical structures of acylated anthocyanins present in the flowers are compiled in Table 2.90. The relative concentrations of anthocyanins in the flower extracts are listed in Table 2.91. It can be concluded from the results that the complex separation and identification methods (TLC, HPLC, UV-vis and 1H NMR spectroscopy, FAB-MS) allow the separation, quantitative determination and identification of anthocyanins in orchid flowers [262].

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TABLE 2.90 ACYLATED ANTHOCYANINS FROM THE FLOWERS OF VANDA

R1 O

O O

HO

O

OH HO

+ O

OH

O

HO HO

OH

O

O OMe

R3

OH

OH

OH O

OH

O

O

OH OR2

CH3O

R3 OH

No. of anthocyanins

R1

R2

R3

1 2 3 4 5 6 7 8

OH H OH OH OH H H H

H H Malonic acid Malonic acid Malonic acid Malonic acid Malonic acid Malonic acid

OCH3 OCH3 H or OCH3 OCH3 H H or OCH3 OCH3 H

Reprinted with permission from F. Tatsuzawa et al. [262].

A novel pigment has been isolated from the petals of Rosa hybrida with complex chromatographic techniques and the structure was elucidated with spectroscopic methods and high resolution fast-atom bombardement mass spectrometry, 1H NMR, and FTIR. Anthocyanins were extracted from 7.9 kg of petals of Rosa hybrida cv. ‘M’me Violet’ with 80 per cent aqueous ACN containing 0.1 per cent TFA. The extract was purified in a Sephadex LH-20 column, and the fraction eluted with 80 per cent ACN was further fractionated in a HP-20 column using water, 15, 20 and 30 per cent ACN as mobile phases. The last fraction was lyophilized and separated by preparative RP-HPLC using an ODS column (50  5 cm i.d.). Solvents were 0.5 per cent aqueous TFA (A) and water–methanol

DISTRIBUTION OF ANTHOCYANINS IN THE FLOWER EXTRACT OF VANDAa

Anthocyanin (as %)c

Flower

4.5  3.7 4.1 14.0 8.1 8.6  5.1 3.9 3.6 6.5 10.0 6.9 5.5 3.6 4.4

C

2b

11.1 13.7 12.6 12.0 5.9 4.4 6.0 14.7 13.1

7.7  14.2 10.0 11.6 4.6 8.3 9.6 9.0 6.3  7.3 12.4 13.1 11.0 9.2 7.7

4.0 5.6 6.2 8.3 8.3 14.2 11.9 6.4 7.9 6.3  4.9 3.1   3.3 10.4

3

4

5

6

7

8

3.9 4.6 7.7 4.9 9.1 8.9 6.6  13.8

10.3 9.0  3.4 3.5  3.3 11.1   6.0 4.6   4.7  4.4

30.4 72.6 40.8 34.3 29.9 32.8 54.6 9.0 11.9 13.7 30.2 9.3 14.8 18.5 13.4 13.4 4.8

  14.4 21.7 9.9 10.4 3.3 49.2 3.5   11.6 3.7  10.6 5.3 3.1

—

 4.6 3.3     3.4 

31.0 36.5 21.8 19.6 15.0 12.6 24.6 25.0 21.0

56.9 92.0 79.3 81.8 84.5 75.7 90.0 85.3 91.4 92.6 89.7 87.8 86.2 74.5 88.2 83.4 89.9

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 4.8   7.3 5.6   8.0 3.0 4.5 7.1 12.2 10.1 5.8 5.5 7.2

1

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B

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I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII

A

Total

a

Chapter 2

Percentage of total absorbance of all detected anthocyanins at 530 nm in HPLC analysis. and —, under 3per cent. The anthocyanin number was the same as in Table 2.90. Rt(min); 1 (14.4), 2 (15.9), 3 (16.3), 4 (16.4), 5 (16.5), 6 (17.5), 7 (17.9) and 8 (18.1); unidentified anthocyanins A (12.1), B (13.2), C 915.2). b 2. Phalaenopsis anthocyanin 1; 6. Phalaenopsis anthocyanin 2; 7. Phalaenopsis anthocyanin 3; 8. Phalaenopsis anthocyanin 4. c I  Vanda ustii Golamco. Claustro. and de Mesa (one more unidentified peak at Rt 18.7 min was observed as 28.7per cent; II  Vanda tricolor Lindl. var. suais (Rchb.f.) Lindl.; III  Vanda Rovert’s Delight; IV  Vanda Taweewan x Vanda Chindavat; V  Vanda Thananchai x Vanda Ratirat; VI  Vanda Bud Mellot x Vanda Gordon Dillon; VII  Vanda Lenavat Rose x Vanda Gordon Dillon; VIII  Vanda Fuchs Delight; IX  Vanda coerulae Griff. ex Lindl.; X  Vanda coerulescens Griff.; XI  Vanda Varabth ‘Y-54’; XII  Vanda Wirat; XIII  Vanda Manuvadee ‘Chiaki’; XIV  Vanda Gordon Dillon x Vanda Bangkok Blue ‘Kamiya’; XV  Vanda Manuvadee; XVI  Vanda Kultana Blue; XVII  Vanda Manuel Torres. Reprinted with permission from F. Tatsuzawa et al. [262].

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TABLE 2.91

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279

OH OH O+

HO

O O O HO OH

Fig. 2.119. Chemical structure of the novel pigment rosacyanin B. Reprinted with permission from Y. Fukui et al. [263].

1:1 v/v, containing 0.5 per cent TFA (B). The linear gradient started at 20 per cent B and reached 100 per cent B in 80 min. The flow rate was 32 ml/min and analytes were detected at 520 nm. Further purification was performed in another ODS column (30  2 cm i.d.) employing 0.1 per cent aqueous HCl (A) and 50 per cent ACN and 0.1 per cent HCl in water (B). Separation began with 70 per cent B for 30 min, then changed to 100 per cent B over 20 min. The chemical structure of the novel pigment, rosacyanin B is shown in Fig. 2.119. It was established that rosacyanin B is the first C-4 substituted anthocyanidin isolated from intact plants [263]. Because of their considerable commercial importance, the composition and stability of anthocyanin-rich extracts have also been investigated [264]. The SEC and HPLC techniques described above were employed for the analysis of four solid and four liquid commercial anthocyanin-rich extracts. The results of SEC and HPLC measurements are compiled in Table 2.92 and Table 2.93, respectively. The measurements indicated that various chromatographic techniques provide useful information on the quality of commercial anthocyanin-rich extracts and they can be successfully applied for the quality control of this class of products [265]. The formation of new anthocyanin-alkyl/aryl-flavanol pigments was studied with HPLC-DAD and HPLC-MS. Model solutions containing mv3gl, the procyanidin dimers B4 (catechin-epicatechin), B2-3
-O-gallate (epicatechin-epicatechin-3
-O-gallate), formaldehyde, acetaldehyde, propionaldehyde, benzaldehyde, iso-butyraldehyde and iso-valeraldehyde have been prepared and the formation of new components during the incubation was followed with HPLC-DAD and HPLC-MS. HPLC-DAD was performed in an ODS column (250  4.6 mm i.d.) at 25°C. Solvents consisted of water–formic acid (95:5, v/v, A) and ACN (B). The gradient started with 10 per cent B and linearly increased to 35 per cent B in 50 min. The flow rate was 1.5 ml/min. HPLC-MS applied a shorter ODS column (150  4.6 mm i.d.; particle size 5 m) at 35°C. The gradient was composed of water–TF and ACN. MS conditions were: capillary voltage and temperature 3 V and 190°C, respectively.

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Chapter 2 TABLE 2.92 TOTAL MONOMER (MAF). RED (RPAF) AND YELLOW-BROWN POLYMER (YBPAF) ANTHOCYANIN FRACTIONS OF COMMERCIAL ANTHOCYANIN-RICH EXTRACTS

Sample

MAFa (%)

RPAFa (%)

MAF-RPAF (%)

YBPAFa (%)

Solid 1 Solid 2 Solid 3 Solid 4 Liquid 1 Liquid 2 Liquid 3 Liquid 4

76.02.5 42.12.6 54.00.4 55.21.5 31.20.5 41.10.8 46.32.0 39.40.4

22.42.4 42.53.5 31.31.2 32.21.6 39.50.3 36.70.6 51.01.7 22.80.5

98.4 84.6 85.3 87.4 70.7 77.8 97.3 62.0

1.60.2 15.41.0 14.71.6 12.60.4 29.30.8 22.20.8 2.70.3 37.80.5

a Mean value of at least three determinationsSD. Reprinted with permission from M. P. Prodanov et al. [265].

The spectra were recorded in the positive-ion mode in the range of m/z 120–1 500. Some chromatograms illustrating the effect of aldehydes on the interaction of mv3gl and B2-3
gallate are shown in Fig. 2.120. The chromatograms demonstrate that different aldehydes influence differently the formation of anthocyanin–flavanol pigments. The results of HPLC-MS measurements are compiled in Table 2.94. Because free aldehydes display an unpleasant aroma in Port wine these reactions may improve the quality of wines and contribute to the colour formation [266]. 2.3.4 Electrophoretic techniques Because of their high separation capacity, short analysis time, low reagent consumption and simplicity, various electrophoretic methods have found application in the separation and quantitative determination of anthocyanins in various complex matrices [267].The different techniques used for the measurement of anthocyanins in beverages [268], the application of capillary electrophoresis (CE) for the analysis of natural food pigments [269], the use of CE for the determination of anthocyanins in foods [270] and in medicinal plants [271] have been previously reviewed. The migration order of wine anthocyanins in CE has been studied in detail and the results have been compared with those obtained by RP-HPLC-MS. Wines were filtered and used for the analyses without any other pretreatment. Wine samples of 10 ml were freeze-dried, redissolved in methanol and applied for semi-preparative fractionation. CZE measurements were carried out in a fused-silica capillary (46 cm effective length, 75 m i.d.). The capillary was conditioned with 0.1 M NaOH (2 min), water (2 min) and running buffer (5 min). The buffer consisted of 50 mM sodium teraborate (pH  8.4) containing 15 per cent (v/v)

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TABLE 2.93 INDIVIDUAL TOTAL MONOMER (TMA) AND TOTAL ACYLATED MONOMER (TAcMA) ANTHOCYANIN CONTENTS OF COMERCIAL ANTHOCYANIN-RICH EXTRACTS

Sample

Dp-3-Gl

Cy-3-Gl

Pt-3-Gl

Pn-3-Gl

Mv-3-Gl

Dp-3Gl-AC

Solid 1 Solid 2 Solid 3 Solid 4 Liquid 1 Liquid 2 Liquid 3 Liquid 4

— 82.25.7 42128.9 7.10.4 58.90.4 95.53.8 36.60.4 29.30.8

— 15.01.7 57.51.3 nd 8.40.2 14.30.2 4.80.3 6.80.6

— 87.77.2 30315.3 4.50.2 63.11.2 103.92.7 24.85.9 33.83.4

— 57.12.4 1362.9 1 3.30.1 14.10.7 55.20.5 2.50.1 51.50.3

— 35028.4 10158.2 21.71.1 77.37.0 3711.6 15.41.5 1000.3

— 24.80.8 22.70.4 nd 4.70.4 16.70.7 3.50.1 9.01.2

Cy-3-Gl-Ac

Pt-3-Gl-Ac

Pn-3-Gl-Ac

Mv-3-Gl-Ac

TMA

TAcMA

— 7.00.2 19.43.2 nd nd nd nd nd

— nd 21.61.5 nd nd nd nd nd

— nd 16.83.7 nd nd nd nd nd

— nd 5851.8 2.40.1 nd nd nd nd

— 624 2 151 39 220 657 87.6 230.4

— 31.8 139 2.4 4.7 17.6 3.5 9.0

Solid 1 Solid 2 Solid 3 Solid 4 Liquid 1 Liquid 2 Liquid 3 Liquid 4

TMA and TAcMA are the sum of all monomer and all acylated monomer anthrocyanins. respectively. expressed as mg Mv-3-Gl/100 g dry matter. nd, not detected amounts. Mean value of at least three determinationsSD. Reprinted with permission from M. P. Prodanov et al. [265].

methanol. Separations were performed at 25 kV and at 10°C. Samples (30nl) were hydrodinamically injected (50 mbar  6 s) at the anode and analytes were detected at the cathode. RP-HPLC was performed in an ODS column (200  4.6 mm i.d.; particle size 5m). Solvents were 5 per cent (v/v) aqueous formic acid (A) and ACN (B). The gradient started with 10 per cent B, to 30 per cent B in 65min; to 50 per cent B in 10 min; to 100 per cent B in 2 min. The flow rate was 1 ml/min and anthocyanins were detected at 520 nm. MS conditions were: nitrogen flow, 30l/min; electrospray ionization interface, 150°C; cone voltage, between 40–80 V. The chromatogram and electrophoregram of a wine sample are shown in Fig. 2.121. The chromatogram and electropherogram illustrate that both separation techniques are suitable for the analysis of anthocyanins in wine. It can be further established that the elution order of anthocyanins is different in HPLC and CE. This finding indicates that the mechanism of retention is entirely different in these techniques. The retention (tR) and migration times (tm) of wine anthocyanins are compiled in Table 2.95.

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Chapter 2 Isovaleraldehyde

Acetaldehyde

Mv3gl

Mv3gl Abs 520 nm

Abs 520 nm

18 17 2

3

Pn3gl

Pn3gl 16

0

10

20 30 Rt (min)

40

0

50

17

10

20 30 Rt (min)

Pn3gl

21 22

16

20 30 Rt (min)

Pn3gl

40

0

50

10

20 30 Rt (min)

40

50

Mv3gl

Abs 520 nm

26

Abs 520 nm

24 18 23

Isobutyraldehyde

Mv3gl

25 Pn3gl 16

Pn3gl 17 16

10

17

16

Formaldehyde

0

50

Mv3gl Abs 520 nm

Abs 520 nm

Mv3gl

10

40

Propionaldehyde

Benzaldehyde

0

19 20

18

16

20 30 Rt (min)

40

50

0

10

28

17 18

20 30 Rt (min)

40

50

Fig. 2.120. Chromatograms recorded at 520 nm of model solutions (six days reaction time at 35ºC) containing mv3gl and B2-3
-gallate mediated by different aldehydes (mv3gl), malidin-3-glucoside; Pn3gl, peonidin-3-glucoside. Numbers refer to pigments in Table 2.94. Reprinted with permission from J. Pissarra et al. [266].

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283 Isovaleraldehyde

Acetaldehyde Mv3gl

3

Mv3gl

4 Abs 520 nm

Abs 520 nm

2

5 7

Pn3gl Pn3gl

1

0

10

6

2 3 20 30 Rt (min)

40

0

50

10

20 30 Rt (min)

40

50

Propionaldehyde 14

Benzaldehyde Mv3gl

8 11

3 Pn3gl

2

Mv3gl

Abs 520 nm

Abs 520 nm

9

12

10

Pn3gl

1

0

10

1

20 30 Rt (min)

40

50

0

10

2 3

13 15

20 30 Rt (min)

40

50

Fig. 2.120. (Continued)

The retention data prove that the CZE method separates the pigments in a considerably shorter analysis time than HPLC does. It was supposed that the good separation capacity and low reagent consumption make CZE a valuable alternative to RP-HPLC [272]. 2.4 CHLOROPHYLLS 2.4.1 Chemistry and biochemistry Chlorophylls are macrocyclic tetrapyrrole derivatives containing in their natural form a chelated magnesium ion. The basic structure of chlorophyll pigments is shown in Fig. 2.122. Chlorophylls occur very frequently in the plant kingdom, they are responsible for the colour of vegetables and some fruits. They also occur in algae and several bacteria. Chlorophylls in plants are photoreceptors and in photosynthesis the presence of a closed circuit of conjugated double bonds allows them to absorb light. Because of their predominant importance as photoreceptors a considerable number of analytical methods have been developed for the separation and quantitative determination. The analytical methods applied for the measurement of chlorophylls and carotenoids in food products have been reviewed previously [273].

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Chapter 2

TABLE 2.94 NEW PIGMENTS DETECTED IN THE MODEL SOLUTIONS BY LC-MS ANALYSIS RESULTING FROM THE REACTION BETWEEN MV3GL AND DIMER B4 AND BETWEEN MV3GL AND B2-3
-O-GALLATE MEDIATED BY DIFFERENT ALDEHYDESa

Aldehyde

Peak no.

Peak identity

Acetaldehyde

16 2 and 3 4 1 2 and 3 5 and 7 6 1 2 and 3 8 and 11 9 and 10 1 2 and 3 12 and 14 13 and 15 16 17 and 18 2 and 3 ndb 16 2 and 3 17 and 18 19 and 20 nd 16 21 22 16 17 and 18 23 and 24 nd 16 25 26 16 17 and 18 27 and 28 nd

mv3gl-epicat mv3gl-ethyl-epicat mv3gl-ethyl-B4 mv3gl-cat mv3gl-ethyl-epicat mv3gl-3methylbutyl-epicat mv3gl-3methylbutyl-B4 mv3gl-cat mv3gl-ethyl-epicat mv3gl-benzyl-epicat mv3gl-benzyl-B4 mv3gl-cat mv3gl-ethyl-epicat mv3gl-propyl-epicat mv3gl-propyl-B4 mv3gl-epicat mv3gl-ethyl-epigal mv3gl-ethyl-epicat mv3gl-ethyl-B2gal mv3gl-cat mv3gl-ethyl-epicat mv3gl-ethyl-epigal mv3gl-3methylbutyl-epigal mv3gl-3methylbutyl-B2gal mv3gl-epicat mv3gl-benzyl-B2gal mv3gl-benzyl-epigal mv3gl-epicat mv3gl-ethyl-epigal mv3gl-propyl-epigal mv3gl-propyl-B2gal mv3gl-epicat mv3gl-methyl-epigal mv3gl-methyl-B2gal mv3gl-cat mv3gl-ethyl-epigal mv3gl-isobutyl-epigal mv3gl-isobutyl-B2gal

Isovaleraldehyde

Benzaldehyde

Propionaldehyde

Acetaldehyde

Isovaleraldehyde

Benzaldehyde

Propionaldehyde

Formaldehyde

Isobutyraldehyde

a

cat, ()-catechin; epicat, ()-epicatechin, epigal, ()-epicatechin-3-O-gallate. Not detected in HPLC-DAD chromatogram. Reprinted with permission from J. Pissarra et al. [266]. b

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6

0.4

AU

0.3

10

0.2 9

3 1

0.1

13

7

5 2

12

11

8

4

14

0.0 0

10

20

30 Time (min)

40

50

60

8 4

6

mAU

4 9 2

2 1

5 3

6

8 7

11 12 13

10

0 7

8

9

10 Time (min)

11

12

13

Fig. 2.121. Liquid chromatogram at 520 nm of a 2002 vintage Tannat red wine (upper lane). Electrophoregram of the same wine at 599 nm (lower lane). For RP-HPLC and CZE details see text. Peak identification on the chromatogram: 1  delphinidin-3-O-glucoside; 2  cyanidin-3-O-glucoside; 3  petunidin-3-O-glucoside; 4  derivative of petunidin-3-O-glucoside and pyruvic acid; 5  peonidin-3-O-glucoside; 6  malvidin-3-O-glucoside; 7  derivative of malvidin-3-O-glucoside and pyruvic acid; 8  delphinidin-3-O-(6-O-acetyl)glucoside; 9  dimer of malvidin-3-O-glucoside and catechin; 10  petunidin-3-O-(6-O-acetyl)glucoside; 11  dimer of malvidin-3-O-glucoside and catechin; 12  peonidin-3-O-(6-O-acetyl)glucoside; 13  malvidin-3-O-(6-O-acetyl)glucoside; 14  malvidin-3-O-(6-O-p-coumaroyl)glucoside. Peak identification on the electrophoregram: 1  malvidin-3-O-(6-O-p-coumaroyl)glucoside; 2  malvidin-3-O-(6-O-acetyl)glucoside; 3  peonidin-3-O-(6-O-acetyl)glucoside; 4  malvidin-3-O-glucoside; 5  peonidin-3-O-glucoside; 6 and 7  dimer of malvidin-3-O-glucoside and catechin; 8  petunidin-3-O-(6-acetyl)glucoside; 9  derivative of malvidin-3-O-glucoside and pyruvic acid; 10  derivative of petunidin-3-O-glucoside and pyruvic acid; 11  petunidin-3-O-glucoside; 12  delphinidin-3-O-glucoside; 2  cyanidin-3O-glucoside. Reprinted with permission from D. Calvo et al. [272].

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TABLE 2.95 RETENTION (TR) AND MIGRATION TIMES (TM) OF WINE ANTHOCYANINS

Compound

tR (min)

tm (min)

Delphinidin-3-O-glucoside Cyanidin-3-O-glucoside Petunidin-3-O-glucoside Derivative of petunidin-3-O-glucoside and pyruvic acid Peonidin-3-O-glucoside Malvidin-3-O-glucoside Derivative of malvidin-3-O-glucoside and pyruvic acid Delphinidin-3-O-(6-O-acetyl)glucoside Dimer of malvidin-3-O-glucoside and catechin Petunidin-3-O-(6-O-acetyl)glucoside Dimer of malvidin-3-O-glucoside and catechin Peonidin-3-O-(6-O-acetyl)glucoside Malvidin-3-O-(6-O-acetyl)glucoside Malvidin-3-O-(6-O-p-coumaroyl)glucoside

13.32 17.12 20.3 22.95 24.12 26.42 29.64 30.72 34.92 37.07 38.65 41.95 43.77 56.87

12.42 12.64 12.22 11.6 10.63 10.34 11.5 — 10.9 11.35 11.25 9.95 9.8 9.47

Reprinted with permission from D. Calvo et al. [272].

R N N H

CH3

Mg N

N

H COOR1 H3COOC

H

O

Phytyl =

Fig. 2.122. Basic structure of chlorophyll pigments.

2.4.2 High-performance liquid chromatography Because of their low separation efficacy, TLC techniques have been recently replaced by various HPLC methods which are more precise and reliable.

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2.4.2.1 Determination of chlorophylls in the marine environment Chlorophylls and other pigments have been frequently investigated in marine samples. As the amount of chlorophyll may be used as a marker for phytoplankton production its determination is of paramount importance in sea research [274]. HPLC data have been used for the study of phytoplankton community structure [275] and for the indentification of phytoplankton groups [276]. Earlier advances in HPLC pigment analysis have been reviewed [277]. The effect of column type, column temperature and conditions of gradient elution have been investigated in detail and the optimal methods have been applied for analysis of phytoplankton pigments. Ten RP-HPLC columns (three C8, five C18, one C14 and one C30, column length between 100 – 250 mm, column diameter 3.2–4.6 mm, particle size 3, 3.5 and 5) were included in the optimization experiments. Column temperatures were 40, 45 or 60°C and the flow rate ranged 0.6–1.0 ml/min. Solvents for gradient elutions were methanol–water 70:30, 28 mM tetrabutyl ammonium acetate, pH  6.5 (A) and methanol (B). Methanol was replaced with ethanol for the C30 column. Gradients started at a specified percentage of B and reached 100 per cent B at the end. Algal monocultures and field samples were chilled while disrupted by sonification, then they were extracted with acetone or ethanol, filtered and used for RP-HPLC analysis. The chromatographic profiles of various algal monocultures are shown in Fig. 2.123. It has been found that the method is simple, shows good separation capacity and can be easily employed for the analysis of phytoplankton pigments [278]. Normal-phase HPLC has also found application in the analysis of pigments in marine sediments and water-column particulate matter. Sediments were extracted twice with methanol and twice with dichloromethane. The combined extracts were washed with water, concentrated under vacuum and redissolved in acetone. Nomal-phase separation was performed with gradient elution solvents A and B being hexane–N,N-disopropylethylamine (99.5:0.5, v/v) and hexane–2-propanol (60:40, v/v), respectively. Gradient conditions were: 100 per cent A, in 0 min; 50 per cent A, in 10 min; 0 per cent A in 15 min; isocratic, 20 min. Preparative RP-HPLC was carried out in an ODS column (100  4.6 mm i.d.; particle size 3 m). Solvent A was methanol–aqueous 0.5 N ammonium acetate (75:25, v/v), solvent B methanol–acetone (20:80, v/v). The gradient was as follows: 0 min, 60 per cent A; 40 per cent A over 2 min; 0 per cent A over 28 min; isocratic, 30 min. The same column and mobile phase components were applied for the analytical separation of solutes. The chemical structure and retention time of the major pigments are compiled in Table 2.96. It was found that the separation and identification of carotenol chlorin esters by various chromatographic techniques may help the better understanding of the pathway of carbonmineralization in the marine environment [279]. Another RP-HPLC method using DAD and fluorescence detection was also employed for the analysis of pigments in sediment. Freeze-dried sediment samples (1–2g) were extracted with 3 ml of acetone, centrifuged, filtered, dried under nitrogen flow and redissolved in acetone before RP-HPLC analysis. Separation was carried out in an ODS column (250  4.6 mm i.d.; particle size 5 m). Solvents were methanol–0.5 M aqueous ammonium acetate, pH  7.2 (80:20, v/v, A), ACN–water (90:10, v/v, B) and ethyl acetate (C). The gradient started with 100 per cent A, enhanced to 100 per cent B in 4 min; then to 25 per cent B and 75 per cent C over 34 min. Excitation and emission wavelengths for fluorescence detection were 440 and 660 nm, respectively. Pigments were identified by MS-APCI. The chemical structure and retention time of pigments are compiled in Table 2.97. It was

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Chapter 2

Dunaliella tertiolecta CCMP 1320

Gyrodinium galatheanum

26

23

15

39 3 31

26 27

11 21 14

6

(a)

34 41

(d)

10 19

1

25

28 29

41 43

Synechococcus of. elongatus CCMP 1829 27 25

Pycnococcus provasolil CCMP 1203 31

mAU (450nm)

39 9

43

43

39 18

14 11 16

4 (b)

a

26 26

42,43 (e) 34 41

Micromonas pusitte CCMP 1545 13

a

b

39

11 a 25

5

10

39

3

(c) 0

22

Guillardia theta CCMP 327

31 27

14

4

39

h 27

15

n 42,43

34 20

25

42

24 (f)

27

6

0

5

10

15

33

41

20

25

Retention time (min)

Nannochloropsis sp. 1 CCMP 531

Amphidinium carterae CCMP 1314 3

27

19

39

7

14

27

9

41 43

25

(g)

(j)

8

mAU (450nm)

Pelgococcus subvinidis CCMP 1429 9

43

Emillania hudeyi 15 CCMP 373

27

3

1 19

1

3

39

39

10 6

(h)

39

17 2023

41 43

1

(k)

12 19

2

Chrysochromutina polylepsis 15 CCMP 1757

1

37

42,43

19

1 10 10

39 37, k,L

39

3 d

(i) 0

5

10

19

27

42,43

15

20

3 5

m

25

(l)

0

5

Retention time (min)

23

10

15

27

20

41 43

25

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concluded from the data that pigments spearated by RP-HPLC can be applied as biomarkers of hypoxic/anoxic waters [280]. HPLC chromatographic profiles have also been employed for the investigation of phytoplankton dynamics. Seawater samples (10–20l) were filtered, the filter freeze-dried and extracted with N,N-dimethylformamide. The extract was centrifuged, mixed with the ionpairing solution (0.5 M ammonium acetate), filtered and submitted to HPLC analysis. Spring samples were analysed in a C8 column using two solvents: methanol–0.5 M ammonium acetate (80:20, v/v, A) and methanol (B). Gradient conditions were: 100 per cent A at 0–5 min; 35 per cent A in 25min; 0 per cent A, 40–55 min. Separation of pigments in summer samples was carried out in an ODS column. Solvent A was the same as before, solvent B was methanol–acetone (70:30, v/v). The gradient started at 0 per cent B and reached 100 per cent B in 20 min followed with isocratic hold for 15 min. The flow rate was 1 ml/min. The pigment ratios to the total chlorophyll for eight taxonomic groups based on CHEMTAX analysis are compiled in Table 2.98. It can be concluded from the data that HPLC is an adequate tool for the investigation of phytoplankton dynamics [281]. Another RP-HPLC technique has been developed and employed for the analysis of pigments in marine phytoplankton. Phytoplankton were concentrated from seawater by filtering, and the frozen filters were ground with acetone, diluted with water to 90 per cent acetone, extracted overnight and centrifuged. Separation was performed in an ODS column (250  4.6 mm i.d.; particle size 5 m). Mobile phase components were methanol–0.5 M ammonium acetate, pH  2 (80:20, v/v, A), ACN–water (90:10, v/v, B) and ethyl acetate (C). Gradient conditions were; 0 min, 100 per cent A; 4 min, 100 per cent B; 18 min, 20 per cent B and 80 per cent C; 21min, 100 per cent B; 23.5min, 100 per cent A; 29min, 100 per cent A. The flow rate was 1 ml/min and pigments were detected by DAD and by fluorescence detector (excitation and emission wavelengths were 425 and 660 nm, respectively). Some chromatograms illustrating the change of pigment concentration and composition in time are shown in Fig. 2.124. It was established that RP-HPLC allows the rapid and reliable determination of a wide variety of phytoplankton pigments, therefore it can help with the identification of phytoplankton taxa and can be employed for the study of algal groups too [282].

Fig. 2.123. Chromatograms of algal monocultures from various algal classes analysed in the Exlipse XDB C8 column showing the elution position of additional pigments not previously shown with the same method. Peak identification: 1  chloropyhll c3; 2  monovynil chl c3; 3  chlorophyll c2; 4  Mg 3,8-divynil pheoporphyrin a5 monomethyl ester; a, b, c, d, e, f, g, h, i, j, k, l, m, n  unknown; 5  chlorophyll c1; 6  chlorophyllid a; 7  peridinin; 8  peridinin isomer; 9  19
-butanoyloxy fucoxanthin; 10  fucoxanthin; 11  neoxanthin; 12  4-Keto-19
-hexanoyloxyfucoxanthin; 13  prasinoxanthin; 14  violaxanthin; 15  19
-hexanoyloxy fucoxanthin; 16  asaxanthin; 17  dianidochrom; 18  unknown (myxo-like spectra); 19  dianidoxanthin; 20  dinoxanthin; 21  antheraxanthin; 22  alloxanthin; 23  diatoxanthin; 24  monadoxanthin; 25  zeaxanthin; 26  lutein; 27  canthaxantin; 28  gyroxanthin diester-like 1; 29  gyroxanthin diester-like 2; 30  divinyl chlorophyll b; 31  monovinyl chlorophyll b; 32  divinyl chlorophyll b
; 33  crocoxanthin; 34  monovinyl chlorophyll b
; 35  chlorophyll  allomer 1; 36  chlorophyll  allomer 2; 37  phytylated chlorophyll c-like; 38  divinyl chlorophyll a; 39  monovinyl chlorophyll a; 40  divinyl chlorophyll a
; 41  monovinyl chlorophyll a
; 42 
,-carotene (-carotene); 43 
,
-carotene (
-carotene). Reprinted with permission from L. van Heukelem et al. [278].

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Chapter 2 TABLE 2.96 CHEMICAL STRUCTURE AND RETENTION TIME IN RP-HPLC OF THE MAJOR PIGMENTS

Pigment

Retention time (min)

Chlorophyll-a Pheophytin-a Pyropheophitin-a Pheophorbide-a Pyropheophorobide-a Isofucoxanthin-dehydrate Fucoxanthin dehydrate Fucoxanthin-hemiketal Isofucoxanthin dehydrate pheophorbide a ester Isofucoxanthin dehydrate pheophorbide a ester Isofucoxanthin dehydrate pyropheophorbide a ester

23.5 26.4 28.1 5.0 6.9 10.7 12.0 6.4 24.4 22.9 25.4

Reprinted with permission from R. Goericke et al. [279].

TABLE 2.97 SUMMARY OF PIGMENT CHARACTERISTICS FROM HPLC-DAD AND HPLC-MS ANALYSIS

Pigment

Retention time (min)

Chlorophyll-a Pheophityn-a Chlorophyll-b Pheophytin-b Chlorophyllone-a Bacteriochlorophyll-e1 Bacteriochlorophyll-e2 Bacteriochlorophyll-e3 Bacteriopheophytin-e1 Bacteriopheophytin-e2 Bacteriopheophytin-e3 Fucoxanthin Zeaxanthin
,
-carotene

31.6 37.0 40.0 34.7 18.0 8.5 9.5 20.5 25.5 26.5 27.0 15.0 23.5 39.5

Reprinted with permission from N. Chen et al. [280].

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291

3 4 1200 1000

mAU

800 600

8

400

7 6 5

200

1

10 9 11

2

12

0 0

5

10

(a)

15 Min

20

25

34 200 8 7 9 10 11 56

mAU

100

1 12

2

0 −100 −200 −300 0

5

10

15 Min

20

25

10

15 Min

20

25

(b)

12.5 10

3

mAU

7.5 5 2.5 0 −2.5 −5 0

(c)

5

Fig. 2.124. HPLC absorbance chromatograms of phytoplankton samples collected from the study site on 24 November 2000 (a), 25 November 2000 (b) and 1 December 2000 (c). Peak identification: 1  chlorophyll-a; 2  chlorophyll-b; 3  chlorophyll-c2; 4  peridin; 5  fucoxanthin; 6  neoxanthin; 7  19
-hexanoylfucoxanthin; 8  diadinoxanthin; 9  alloxanthin; 10  lutein; 11  zeaxanthin; 12 
-carotene. Reprinted with permission from C. K. Wong et al. [282].

9/15/2006

PIGMENT RATIOS TO THE TOTAL CHLOROPHYLL-A FOR EIGHT TAXONOMIC GROUPS BASED ON CHEMTAX ANALYSIS

Cluster

Fuco

Hex-Fuco

Neo

Viol

Allo

Zea

Chl b

DV Chl a

Total Ch l a

I

Dinoflagellates Diatoms Chrysophytes Prymnesiophytes Chlorophytes Cryptophytes Prochlorophytes Cyanobacteria

1.06 0 0 0 0 0 0 0

0 0 0.37 0 0 0 0 0

0 0.47 0.11 0 0 0 0 0

0 0 0 1.71 0 0 0 0

0 0 0 0 0.08 0 0 0

0 0 0 0 0.06 0 0 0

0 0 0 0 0 0.23 0 0

0 0 0 0 0.01 0 0.27 0.35

0 0 0 0 0.46 0 0.68 0

0 0 0 0 0 0 1 0

11 1 1 1 1 1 0 1

II

Dinoflagellates Diatoms Chrysophytes Prymnesiophytes Chlorophytes Cryptophytes Prochlorophytes Cyanobacteria

1.06 0 0 0 0 0 0 0

0 0 0.37 0 0 0 0 0

0 0.47 0.15 0 0 0 0 0

0 0 0 1.71 0 0 0 0

0 0 0 0 0.08 0 0 0

0 0 0 0 0.05 0 0 0

0 0 0 0 0 0.23 0 0

0 0 0 0 0.01 0 0.37 0.35

0 0 0 0 0.46 0 1.22 0

0 0 0 0 0 0 1 0

1 1 1 1 1 1 0 1

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Taxa

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TABLE 2.98

0 0.75 0.15 0 0 0 0 0

0 0 0 1.71 0 0 0 0

0 0 0 0 0.05 0 0 0

0 0 0 0 0 0.29 0 0

0 0 0 0 0.01 0 0.27 2.06

0 0 0 0 0.29 0 0.52 0

1 1 1 1 1 1 1 1

B

Dinoflagellates Diatoms Chrysophytes Prymnesiophytes Chlorophytes Cryptophytes Prochlorophytes Cyanobacteria

1.06 0 0 0 0 0 0 0

0 0 0.62 0 0 0 0 0

0 0.86 0.13 0 0 0 0 0

0 0 0 1.71 0 0 0 0

0 0 0 0 0.25 0 0 0

0 0 0 0 0 0.26 0 0

0 0 0 0 0.02 0 0.21 0.42

0 0 0 0 1.02 0 2.06 0

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A

Page 293

Per  peridinin; But-fuco  19
-butanoyloxyfucoxanthin; fuco  fucoxanthin; Hex-fuco  19
-hexanoyloxyfucoxanthin; Neo  neoxanthin; Viol  violaxanthin; Allo  alloxanthin; Zea  zeaxanthin; Chl b  chlorophyll-b; DV chl a  divinyl chlorophyll-a. Reprinted with permission from K. Furuya et al. [281].

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A similar RP-HPLC method has been applied for the investigation of pigments in sediments and numerous source biota. Extraction of pigments was carried out with acetone, methanol, THF and their mixtures. Typical chromatograms showing the good separation performance of the RP-HPLC system are shown in Fig. 2.125. Percentage compositions of chlorophyll-a and bacteriochlorophyll-a derivatives are compiled in Table 2.99. It was established that the extraction capacity of THF is higher than that of traditional extracting agents such as methanol and acetone. It was further stated that RP-HPLC can be successfully employed for the analysis of tetrapyrrole pigments in sediments and source biota [283]. RP-HPLC has also been applied for the study of the effect of salinity on the diversity of prokaryotic and eukariotic phytoplankton. Seawater samples were filtered and the filters were sonificated with acetone and left for 24 h at 4°C. Separation was performed in an ODS column (250  4.6 mm i.d.; particle size 5 m). The concentrations of pigments are compiled in Table 2.100.




z

0.007

0.05




F



C01

(d)


0.015

z




0.05

Absorbance (AUFS)

(a)

z

c1/c1

M z

E



 c1/c1

F

(e)

(b) F




0.015

0.05

z c1/c2 C01 C D

0 (c)

10



20

30 Time, min.

40

50

F c1/c1 P

 C01

10

60 (f)

E

20

30

40

Time, min.

Fig. 2.125. HPLC chromatograms generated at 440 nm. Pure cultures: (a) Synechococcus elongatus, (b) ‘2 micron picosphere’, (c) Cyclotella choctawatcheena. Phytoplankton field communities (d) Twin Key Basin, (e) Rabbit Key Basin, (f) Sandy Key Basin.

-carotene, Z  zeaxanthin, a  chlorophyll-a, N  myxoxanthophyll, E  echinenone, c1/c2  chlorophyllsc1/c2, F  fucoxanthin, Col  unknown carotenol, Cone  unknown carotenone, D  diadinoxanthin, P  peridin. Reprinted with permission from J. W. Louda et al. [283].

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It can be concluded from the data that RP-HPLC, together with other techniques such as microscopy, flow cytometry and DNA analysis, can contribute to the determination of the effect of salinity on the diversity of phytoplankton [284]. Some RP-HPLC technics used for the pigment analysis of phytoplankton have been compared. Octyl and ODS columns were included in the experiments. The column length varied between 100–250mm i.d., the width ranged between 3.0–4.6 mm, and the particle size was 3.0, 3.5 and 5 m. Separations were carried out with gradient elution composed of two or three solvents. Acetone and methanol were used as extracting agents. RP-HPLC separated 24 pigments but the results were somewhat different depending on the method applied [285]. A similar extraction and RP-HPLC method was employed for the study of the spatial variability of phytoplankton composition and biomass. Seawater samples were filtered and the filters were frozen, ground and sonicated with acetone–water (90:10, v/v). The extract was mixed with tetrabutylammonium acetate (500:165, v/v) and injected into an ODS column (150  4.6 mm i.d.; particle size 3 m). Alloxanthin, 19
-butanoylfucoxanthin, fucoxanthin, peridinin, prasinoxanthin, zeaxanthin, 19
hexanoylfucoxanthin and chlorophyll-b were separated and used as taxonomic markers. However, the results indicated that the pigment profile itself is not specific to particulate taxa and can be used only with a combination of other analytical techniques [286]. HPLC has been further employed for the mesurement of the spatial and temporal variabilities of phytoplankton community structure [287], for the investigation of the seasonal change of algal pigments [288], for the study of the seasonal and interannual change of phytoplankton communities [289] and for the assessment of the tidal and diurnal periodicities of pigment profiles [290]. The separation capacities of binary and ternary gradient systems for the measurement of pigments in sediment, microbial mat and a microbial culture (Chlorobium phaebacteroides) have been compared. Samples were sonicated by acetone, the suspension was centrifuged and the supernatant was filtered. The extraction step was repeated and the combined extracts were dried and redissolved for RP-HPLC analysis. Separation was carried out in ODS columns (150  4.6 mm i.d.; particle size 3 m). Gradient components were 0.01 M aqueous ammonium acetate, methanol, ACN and ethyl acetate mixed in various volume ratios. The flow rate was 0.7 ml/min. MS conditions were: APCI source operated in the positive-ion mode; capillary temperature, 150°C; APCI vaporizer temperature, 450°C; discharge current, 5m. It was established that the best separation can be obtained using 300 mm column length and 3 m particle size. The optimal gradient composition varied according to the sample to be analysed. Chromatographic profiles of sediment extract and microbial mat extract are shown in Fig. 2.126. Gradient conditions for these chromatograms were: 0–5 min, 5 per cent 0.01 M aqueous ammonium acetate (A) – 80 per cent methanol (B) – 15 per cent ACN (C); 100 min, 20 per cent B – 15 per cent C – 65 per cent ethyl acetate (D); 105 min, 1 per cent B – 1 per cent C – 98 per cent D; isocratic, 5 min. It was found that this new RP-HPLC-DAD-MS method separated the pigments well, therefore, its application in geochemistry, limnology and oceanography was proposed [291]. The same chromatographic technique was employed for the separation and identification of chlorophyll and bacteriochlorophyll pigments in a calcite/gypsum microbial mat. Some chromatograms showing the pigment distribution in various samples are depicted in Fig. 2.127. The chromatographic and structural characteristics of the pigments are compiled in Table 2.101.

PERCENTAGE COMPOSITION OF CHLOROPHYLL-A AND BACTERIOCHLOROPHYLL-A DERIVATIVES IN TWO SURFICIAL FLOCS AND A 1.2 M CORE FROM WHIPRAY BASIN, SITE NO. 2

Surfacial floc 0–2

4–6

12–14

16–18

18–20

20–22

48.70 5.00 0.01 15.70

60.53 20.21

3.17 27.36 22.51

8.01

12.07 2.78

27.19

7.46

2.80

6.14 1.23 4.08

40.91

7.86 1.55 29.38

53.02

0.09 0.70 3.02 1.81

15.24 0.30 12.21 0.76 0.86 0.18 0.54 1.22 1.27

0.00 0.00

1.28

0.08 0.19 0.30 0.32

1.13

2.52 0.00 0.00 0.00 1.09

2.69 6.3

2.34 6.35

0.51 6.18

1.38 6.02

0.47 5.78

0.44 4.98

24–26

30–32

13.80

14.60 3.00 8.00 0.80

13.10 0.90

0.26 0.81 17.11 2.20

0.22 1.86 9.74 0.21

naa na

na na

31.92

0.37 0.37

35.25

14.71

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12.43 41.31

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3.90 46.20 2.70

47.00

13.49 18.97 29.77

41.56

44.40

58.40

40.88

0.00 0.00 0.86

0.00 0.00 0.00 0.37

0.28 3.96

0.38 6.4

Chapter 2

Chlorophyll-a derivativesb CHLide-a PCHLide-a PBIDa PPBIDa CYCLO CHLa-Allo ChLa ChLa-Epi PTINa-Allo PTINa PTINa-Epi pPTINa PBIDa-SEs pPBIDa-SEs CHLs/SUM Pheos CHls/SUM Ptins pBIDa/CYCLO PTINa/pPTINa Totals and Org.c ‘Chl-a’. nmol/g-C Corg. %-dry wt.

Barren

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Depth below sea floor (cm)

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TABLE 2.99

0.34

0.64

0.59

0.56

0.17

0.38

0.31

69.1 20.9 4.6 5.5

45.0 40.0 8.2 6.9

48.7 51.3

8.2

65.4 20.9 4.4 9.3

26.5 36.1 6.3 6.7

46.0 54.0

32.0 57.0 10.5

84.0

100.0

48.90 25.60 0.80

53.70 15.90 0.90

80.54 11.46 0.00

49.78 27.75 1.62

0.00 72.83 0.00

53.74 38.80 0.00

0.00 100.02 0.00

35.25 62.23 2.52

0.00 99.96 0.00

14.71 85.29 0.00

na na na

na na na

2.17 0.31 0.00

1.17 0.65 0.04

0.00 0.37 0.00

0.74 0.54 0.00

0.00 0.47 0.00

0.16 0.28 0.01

0.00 0.28 0.00

0.06 0.32 0.00

16.0

8.77 4.71

7.93 6.38

3.63 1.81

0.86 0.00

3.11 1.67

0.83 0.00

2.66 0.94

0.73 0.00

1.22 0.18

3.89

0.36

0.91

1.01

0.63

1.21

0.83

1.65

0.73

1.04

61.46

71.39

36.91

6.27 57.43

3.70 6.10

7.08 45.36

81.50

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0.45

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Liquid chromatography of natural pigments

‘BCHLa’. nmol/g-C Bacteriochlorophyll-a derivativesb BCHLab BPTNa BPTINa-Epi pBTINa Percentages pPBIDaCYCLO SUMPTINsa PBID & pPBIDa-SEs nmol/g-Org-C PPBIDaCYCLO PTINsa PBID & pPBIDa-SEs Ratio: nmol/nmol ‘CHLa’/‘BCHLa’ PPBIDaCYCLO/BCHLaBTINa PTINsa/BCHLaBP TINsa Chlorophyll-a derivativesb CHLide-a PCHLide-a PBIDa pPBIDa CYCLO CHLa-Allo CHLa CHLa-Epi PTINa-Allo

60.82

4.84 2.19 (Continued on next page) 297

Surfacial floc 34–36

42–44

54–56

56–58

64–66

82–84

90–92

100–102

13.63 1.38 17.38

26.98

12.30

19.44

12.15

73.00

13.33

80.60

23.50

8.57 2.55 19.97

5.35 1.22 19.34

0.61

17.66 0.11 0.27 0.00 0.56

5.74 1.45 15.49 2.51 8.90 0.00 0.00 0.11 0.61

11.63 0.05 0.16 0.16 0.56

1.93 0.00 0.00 0.00 0.34

0.00 0.00 0.00 0.52

0.68 3.05 0.53

0.66 2.98 0.48

0.57 4.08 0.33

0.74 5.36 0.35

0.76 2.7 0.14

0.15 1.72 0.03

24.8 56.4 8.8 10.0

0.48 4.75 0.36

0.40 3.2 0.27

80.4 19.6

25.3 53.7 6.1 14.9

0.00 0.00

85.1 14.9

17.7 60.6 8.6 13.1

76.1 11.8 12.2

30.4 48.6 9.2 11.7

68.5 12.3 19.3

25.81

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0.53 4.02 0.34

0.24

2.98 0.00 0.00 0.00 0.52

0.00 0.00

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PTINa PTINa-Epi pPTINa PBIDa-SEs pPBIDa-SEs CHLs/SUM Pheos CHls/SUM Ptins pBIDa/CYCLO PTINa/pPTINa Totals and Org.c ‘Chl-a’. nmol/g-C Corg. %-dry wt. ‘BCHLa’. nmol/g-C Bacteriochlorophyll-a derivativesb BCHLab BPTNa BPTINa-Epi pBTINa

Depth below sea floor (cm)

100.0

na  not available. organic carbon not determined on surficial flocs. CHLa and BCHLa equate to the sum of the respective parents and all derivatives; CHL  chlorophyll. CHLide  chlorophylide. PBID  pheophorbide. PTIN  pheophitin. ‘B’  bacterio. ‘P’  pyro. Allo  allomer (13/2-hydroxy). Epi  epimer. CYCLO  cyclo(pyro)pheophorbide-a-enol. ‘SEs’ steryl esters. Reprinted with permission from J. W. Louda et al. [283].

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TABLE 2.99 (continued)

a

b

Chapter 2

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TABLE 2.100 PIGMENT COMPOSITION. IN ìg/L. ALONG THE SALINITY GRADIENT. AS DETERMINED FROM HPLC ANALYSES

Survey date

Salinity (%)

Chl c

Per

Fuc

Neo

Pra

Vio

18/05/03

4 5.4 8 11 15 22.4 31.6 37

0.763 2.052 1.155 0.058 0.200 0 0 0

0.121 0.980 0.412 0 0 0 0 0

0.224 0.234 0.852 0.283 0.515 0 0 0

0.110 0 0.194 0.037 0 0 0 0

0.202 0 0 0 0 0 0 0

0.128 0.228 1 0 0 0 0 0

26/05/03

4 5.4 8 11 15 22.4 25 31.6 35.6

0.755 2.719 1.330 0.909 0.139 0 0 0 0

0.087 1.198 0.432 0.191 0 0 0 0 0

0.294 0.788 0.183 0.190 0.377 0.181 0 0 0

0.036 0 0.202 0.128 0 0 0 0 0

0 0 0 0 0 0 0 0 0

0.018 0.088 1.258 0.982 0 0 0 0 0

Survey date

Salinity (%)

Diadino

All

Lut

Zea

Chl b

Chl a

-carotene


-carotene

18/05/03

4 5.4 8 11 15 22.4 31.6 37

0.065 0.630 1.102 0.156 0.422 0 0 0

0.880 1.214 0.237 0 0 0 0 0

0.048 0.042 0.646 0.140 0 0 0 0

0 0 0.382 0.076 0 0 0.561 6.657

0.855 0.046 1.180 0.176 0 0 0 1.805

4.064 5.498 8.411 1.714 2.008 0.531 2.374 18.80

0.137 0.064 0 0 0 0.206 1.193 15.68

0.289 0.181 0.588 0.117 0.102 0.877 17.38 508.5

26/05/03

4 5.4 8 11 15 22.4 25 31.6 35.6

0.078 1.044 0.652 0.561 0.144 0 0 0 0

0.650 0.865 1.332 0.241 0 0 0 0 0

0.025 0.060 0.664 0.886 0 0 0 0.540 0.909

0 0 0.303 0.139 0.043 0 0 2.847 3.909

0.176 0.063 1.468 1.366 0 0 0 0 1.626

3.220 7.644 8.595 7.982 1.766 3.492 5.350 9.410 13.57

0.118 0.119 0.168 0.022 0 0.150 0.388 4.684 21.38

0.152 0.316 0.464 0.381 0.362 0.870 2.319 182.0 363.4

Chl c  chlorophyll-c; Per  peridin; Fuc  fucoxanthin;  Neo  neoxanthin; Pra  prasinoxanthin; Vio  violaxanthin; Dia  diadinoxanthin; All  alloxanthin; Lut  lutein; Zea  zeaxanthin; Chl b  chlorophyll-b; Chl a  chlorophyll-a. Reprinted with permission from M. Estrada et al. [284].

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Chapter 2

46

Response

40

6 2

8 45

1

0

10

20

3

7

42

10 9

18 11 15 20 12 17 14 13 16 19 21

30

40

43

32 29 35 36 26 27 31 37 4 34 28 30 23 38 33 25 39 44 45 22 24

50

60

70

80

59 51 55 48 52 58 60 50 53 57 61 63 62 49 64 5456 47

90

100

110

Retention time / min

Fig. 2.126. HPLC-DAD (300–800 nm) chromatograms of Priest Pot sediment extract (upper lane) and microbial mat extract (lower lane). Peak identification: a  scytonemin; b  siphonaxanthin; c  scytonemin (stereoisomer of a; d, e, f, 1, 1a, 1b, 4, 4a, 5, 6, 7a, 8, 10, 12, 12a, 13a, 18a, 20a, 20b, 21a, 24c, 30a, 46a, 46b  unidentified carotenoid; 2, 3,  chlorophyllone; 3a  diatoxanthin; 5a  lutein; 6a  zeaxanthin; 7, 9  phaephorbide-a methyl ester; 7  alloxanthin; 11  monadoxanthin; 11a  carotene diol; 13  pyropheophorbide-b methyl ester; 13b  adonirubin; 14, 16, 17, 18b, 19a, 22, 25b, 27a, 31a, 32, 32c, 36, 38, 44  unidentified chlorin; 15  cantaxanthin; 18  pyropheophorbide-a methyl ester; 18c  rhoidoxanthin; 19  bacteriophaephytin c/d; 20  crocoxanthin; 21  pyrochlorophyll b; 22a  okenone; 23  chlorophyll-a; 23a  echinenone; 24  bacteriopheophytin; 24a, 30, 31, 34a  bacteriopheophytin-a; 24b  unidentified bacteriopheophytin; 25  chlorophyll-a epimer; 25a, 27, 28, 29, 33  bacteriopheophytin c; 26  pyrochlorophyll-a; 32a  bacteriopheophytin-d; 32b  bacteriopheophytin-a epimer; 34  phaeophytin-b; 35  hydroxypheophytin-a; 37  hydroxypheophytin-a epimer; 37a  pyrobacteriophaeophytin-a; 39  purpurin-7 phytyl ester; 40  pheophytin-a; 41, 42a, 43a  carotene; 42  pheophytin-a epimer; 43  pyrophaeophytin-b; 45  purpurin-18 phytyl ester; 46  pyrophaeophytin-a; 47  phaeophorbide-a ester; pyrophaeophorbide-a ester; 48, 50  phaeophorbide-a ester; 49, 51  unidentified chlorin ester; 52, 53, 54  pyrophaeophorbide-b ester; 55, 56, 57, 58, 59, 60, 61, 62, 63, 64  pyrophaeophorbide a ester. Reprinted with permission from R. L. Airs et al. [291].

It has been stated that these new RP-HPLC and APCI-LC-MS methods separate a higher number of pigments than previous ones. Although the pigment profiles may help the safe classification of the members of bacterium communities they alone are not suitable for the classification [292]. Another RP-HPLC procedure was applied for the study of the distribution and stability of steryl chlorin esters in copepod faecal pellets from diatom grazing. Pigments were sonicated for 15 min with acetone at 0°C and the procedure was repeated until the extract became colourless. The organic phase was evaporated and the fraction containing the free alcohols was separated by TLC (silica stationary and dichloromethane mobile phases) and analysed by gas chromatography. RP-HPLC measurements were performed in an ODS

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TABLE 2.101 ASSIGNMENT OF CHLORIN COMPONENTS IN MICROBIAL MAT

Peak no.

tR (min)

Assigmenta

Esterifying alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 34 35 36 37 38

26.2 32.9 34.5 35.6 37.3 38.6 39.4 43.2 44.1 45.2 45.9 47.4 49.4 49.4 52.4 53.6 53.8 54.1 54.8 55 56.2 56.6 58.9 59.2 61.4 62 62.4 63.7 64.4 64.6 65.4 67.4 68.9 71 71.6 74.7 82.4

Bchl c Bchl d Bchl d Bchl d Bchl c Bchl d Bchl d Bchl a Bchl d Bchl c Bchl d Bchl c Bchl c Bchl d Bchl d Bchl c Bchl c Bchl d Bchl d Chl a Bph c Bph d Bph d Bph c Bph d Bph d Bph c Bph a Bph d Bph c Bph d Bph d Bph d Bph d Pbph a Ph a Pph a

Farnesol Geranyl geraniol Tetradecanol Geranyl geraniol Geranyl geraniol Tetradecanol Tetradecanol Phytol Hexadecanol Farnesol Hexadecanol Farnesol Farnesol Octadecanol Geranyl geraniol Geranyl geraniol Geranyl geraniol Geranyl geraniol Tetradecanol Phytol Geranyl geraniol Tetradecanol Tetradecanol Tetradecanol Hexadecanol Hexadecanol Hexadecanol Phytol Hexadecanol Hexadecanol Hexadecanol Octadecanol Octadecanol Octadecanol Phytol Phytol Phytol

Bchl  bacteriochlorophyll; Bph  bacteriophaeophytin; Chl  chlorophyll; Ph  pheophytin; Pbph  pyrobacteriophaeophytin; Pph  pyrophaeophytin. Reprinted with permission from R. L. Airs et al. [292].

a

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37

2.00

20 21

1.50

22 25 28 24 27 29 31

1.00 18

0.50

35 36 33 32

15

8 12

38

0.00

(a) 0.10

36 35

0.08 28

0.06

31

20

0.04

16

38

21 24 26

8

0.02 Response

33 3234 30 37

12

0.00 (b) 36 35 37

0.08

33 32 34 28 29

0.06

19 26 25 31 12 17 21 23 15

0.04 0.02

38

22 13

0.00 (c) 12

28

0.15

29

36 31 35 32 33

0.12 0.09 20 21 10

0.06

37 38

11 1

0.03

3 5 8 2 4 67 9

34

14

26 23

0.00 15

(d)

25

35

45

55 65 75 85 Retention time / min

95

105 115

Fig. 2.127. HPLC-DAD chromatograms (300 – 800 nm) plotted at the maximum absorbance of microbial mat extracts from (a) the living cyanobacterail mat, (b) 8 – 10cm depth, (c) 10–12cm depth, and (d) 12 – 14 cm depth. Numbers refer to pigments in Table 2.101. Reprinted with permission from R. L. Airs et al. [292].

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column (150  4.6 mm i.d.). The flow rate was 1 ml/min and analytes were detected by DAD (350–700 nm). Solvents were methanol (A), water (B) and acetone (C). The gradient was 80 per cent A, 20 per cent B for 0–5 min; to 60 per cent A, 10 per cent B, 30 per cent C after 15 min, isocratic 25 min; to 30 per cent A, 10 per cent B, 60 per cent C after 25 min, to 5 per cent A, 5 per cent B, 90 per cent C after 45 min; isocratic to 95 min. MS-APCI conditions were: vaporizer 450°C; capillary 300°C; corona 7 A; sheath gas pressure 20 psi. Chromatographic profiles of various samples are shown in Fig. 2.128. It was stated that the RP-HPLC-MS technique is suitable for the separation of steryl esters of the chlorophyll-a transformation products of pyrophaeophorbide and consequently can be employed for the study of such transformations [293]. A similar study has been carried out to separate the steryl esters of pyrophaephorbide-b produced by algae. Typical chromatograms are shown in Fig. 2.129. The chromatographic profiles illustrate that the method separates well the analytes under investigation and allows the detection of differences between the samples of various origins. Steryl choline esters (SCEs) found in the samples are listed in Table 2.102. It can be concluded from the data that the method is suitable for the investigation of the fate of chlorophylls in algae [294]. HPLC has found applications in a wide variety of studies concerning the marine environment [295]. It has been employed for the identification of the components of microphytobenthic communities [296], for the investigation of the change in phytoplankton communities [297] in many sampling sites such as the Mediterranean Sea [298], Equatorial Pacific [299], Mississipi River-influenced continental shelf [300], etc. 2.4.2.2 Determination of chlorophylls in miscellaneous matrices Because of their theoretical and commercial importance, chlorophylls have been measured in a wide variety of matrices such as fruits, plants, olives and various model systems. Thus, RP-HPLC was employed for the investigation of chlorophyll derivatives occurring during fruit storage. Extraction of pigments was carried out by homogenizing 2 g of peel of cherimoya fruit (Annona cherimola, Mill.) cv. ‘Fino de Jeta’ with 100 ml of 400 mm sucrose in 50 mm Tris-HCl (pH  8.0) for 120 s. The suspension was filtered then centrifuged at 4°C. The solid residue was mixed with 10 ml of 100 mM phosphate buffer (pH  6.5) containing 0.2 per cent Triton X-100. Separations were made in an ODS column (250  4 mm i.d.; particle size 5 m ). A similar column with 10 mm i.d. was employed for semipreparative separation. Gradient elution started with 100 per cent A (80 per cent methanol in 1 M ammonium acetate) and changed in 15 min to 100 per cent B (80 per cent methanol in acetone) and held isocratically as long as the pigments were eluted. Flow rates were 1 and 4 ml/min for the analytical and semipreparative columns, respectively. Analytes were detected by DAD between 400 and 670 nm and by fluorescence detector, the excitation and emission wavelengths being 440 and 660 nm. Chromatograms detected spectrophotometrically at 660 nm and fluorometrically are depicted at Fig. 2.130. The chromatograms illustrate that the sensitivity of fluorimetric detection is considerably higher than that of spectrophotometry. It was concluded from the results that the RP-HPLC method is suitable for the separation of chlorophyll derivatives and can be applied for the study of the degradative process of chlorophylls [301].

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Chapter 2

i+i'

Algal culture h

j+j'

g' g a

k

e+f

(a) o

Algal control (48 h)

SCEs

j+j'

i+i' a k

(b)

p l

r

k

Pellets (48 h)

q

n

b

s

j+j' t

i+i'

m u

(c) l Pellets aged 30 days k

c

b j+j'

d

t

m u

(d) 20

30

40

50 Time (min)

60

SCEs n-s 70

80

Fig. 2.128. HPLC-MS summed base peak mass chromatograms of total extracts of (a) T. weissflogii culture, (b) control, (c) faecal pellets immediately after grazing (48h) and (d) pellets after ageing in filtered seawater in the dark for 30 days. Peak identification: a  phaeophorbide-a; b  pyrophaeophorbide-a; c  132-chlorophyllone-a; d  132-epi-chlorophyllone-a (carotenoid); ef  132-hydroxyhlorophyll-a, 15
-hydroxylactone, chlorophyll-a; g  chlorophyll-a; g
 chlorophyll-a epimer; h  chlorophyll-a-like; i  hydroxyphaeophytin-a  unknown; i’  hydroxyphaeophytin-a epimer; j  phaeophytin-a; j’  phaeophytin-a epimer; k  purpurin-18phytyl ester; l  pyrophaeophytin-a; m  chlorine-a-like; t  132-oxopyrophaeophytin-a?; u  132-oxopyrophaeophorbide -24-methylcholesta-5,24(28)-dien-3
-yl-ester?; SCE Sterol: n  C272 d.b.a; o  C27 2 d.b. C28 2 d.b.; p  C29 2 d.b.; q  C27 1 d.b.; r  C28 1 d.b.  C29 2 d.b.; s  C29 2 d.b. Reprinted with permission from H. M. Talbot et al. [293]. (ad.b  number of double bonds. *  carotenoid.)

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TABLE 2.102 SCE ASSIGMENTS AND STRUCTURES

Peak

Assignment

a b

Pyrophaeophorbide-b colesta-5.24-dien-3
-ol ester Pyrophaeophorbide-b colestra-5.22-dien-3
-ol and 24-methylcholestra-5.24(28)dien-3
-ol esters Pyrophaeophorbide-b colest-5-en-3
-ol ester Pyrophaeophorbide-b 24-methylcolest-5-en-3
-ol and pyrophaeophorbide-a colesta5.24-dien-3
-ol ester Pyrophaeophorbide-a colesta-5.22-dien-3
-ol and 24-methylcholesta-5.24(28)-dien3
-ol esters Pyrophaeophorbide-a colest-5-en-3
-ol ester Pyrophaeophorbide-a 24-methylcolest-5-en-3
-ol esters

c d e f g

Reprinted with permission from H. M. Talbot et al. [294].

The effect of ethylene on the decomposition of chlorophyll in intact radish (Raphanus sativus L.) cotyledons was also assessed by HPLC and HPTLC. Six-day-old seedlings were exposed to 100 ppm ethylne for 12 h then exposed to fresh air for 24 h in the dark at 25°C. Cotyledons were homogenized with cold acetone, filtered, the precipitate was dried and used as a crude enzyme preparation. The reaction mixture containing chlorophyll-a and enzyme preparation was incubated then analysed with HPTLC and RP-HPLC. In order to separate chlorophyllide-a, C-132-OH-chlorophyll-a, chlorophyll a and chlorophyll-a
, ODS HPTLC plates were developed with methanol–acetone–water (15:8:1, v/v). RP-HPLC measurements were carried out in an ODS column (250  4 mm i.d.; particle size 7 m) using an isocratic mobile phase (methanol–acetone–water, 15:10:1, v/v). The flow rate was 0.8 ml/min and analytes were detected fluorimetrically (excitation 405 and emission 660 nm). The dependence of the composition of the reaction mixture on the reaction time is illustrated in Fig. 2.131. It was established that chlorophyll-a decomposes firstly to C-132-HO Chl-a and then to a colourless fluorescence derivative. It was further supposed that similar investigations may provide valuable information on how to suppress the Chl decomposition in fruits and vegetables [302]. Another RP-HPLC study used a zinc–phtalocyanine internal standard in the analysis of chlorophylls in different plant samples. Spinach, lettuce, iceberg lettuce and endive samples were frozen and 10–20 g of sample were mixed with 10 g of quartz sand and 10 g of soluble starch. An aliquot of 1–4 g was mixed with 2 ml of buffer (pH  7), 13 ml of N,N-dimethylformamide (DMF) then homogenized. Further purification of samples was performed in ODS SPE cartridges preconditioned with 2 ml methanol and 5 ml of aqueous phosphate buffer (pH  7). Cartridges were washed with buffer and methanol–buffer mixtures, and analytes were removed with 6 ml of methanol followed with 4 ml of DMF. Separation of chlorophylls was carried out in an ODS column (250  2 mm i.d.; particle size 4 m) at 31°C. The flow rate was 0.28 ml/min. Conditions of gradient elution were: 0–9.5 min, 100 per cent methanol; to methanol–acetone–DMF (80:15:5 v/v)

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Chapter 2 8 Culture (0 h.) ∗-carotenoid

12

5' 5

3

4 6 3'

(a)

∗ ∗ ∗

8'

4'

6'

10 11

∗

8 Absorbance (400+430 nm)

Control (48 h.)

12 (b)

10 11 3 3'

∗

6' 6 4' 5

8'

7

Faecal pellets (48 h.)

9 6 6' 13 8 8' ∗ (c) 5

∗ 20

10 11 ∗ ∗ ∗ ∗

12

40 Time (min)

SCFs (a-g)

14

60

80

Fig. 2.129. HPLC chromatograms (400430 nm) of tota; extracts of (a) Tetraselmis suecica, (b) control, (c) faecal pellets immediately after grazing (48h). Peak identification: 1  pyrophaeophorbide-b; 2  pyrophaeophorbide-a; 3  chlorophyll-b; 3
 chlorophyll-b epimer; 4  chlorophyll-a; 4
 chlorophyll-a epimer; 5  phaeophytin-b; 5
 phaeophytin-b epimer; 6  hydroxyphaeophytin-a; 6
 hydroxyphaeophytin-a epimer; 7  pyrophaeophytin-b; 8  phaeophytin-a; 8
 phaeophytin-a epimer; 9  pyrophaeophytin-a; 10  C-15
OH lactone chlorophyll-b; 11  C-132OH chlorophyll-b; 12  C-15
OH lactone chlorophyll; 13  C-15
OH lactone phaeophytin-a; 14  purpurin-18-phytyl ester. Reprinted with permission from H. M. Talbot et al. [294].

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Ch1 660 nm

Liquid chromatography of natural pigments

mAU

150

307

6

100

50 5 2 1

3 4

7

8

0

0

10

20

30

Min 6

3 4

5

Fluorescence intensity

2

1

0

7

8

20

10

30

Min

Fig. 2.130. Elution profile by RP-HPLC of the chlorophyll derivative pigments analysed. The pigments were detected spectrophotometrically at 660 nm and fluorimetrically using excitation and emission wavelengths at 440 and 660 nm, respectively. Peak identification (numbers in parentheses are retention times in min): 1  chlorophyllide-b (3.10); 2  chlorophyllide-a (4.98); 3  pheophorbide-b (7.44); 4  pheophorbide-a (8.85); 5  chlorophyll-b (14.74); 6  chlorophyll-a (16.40); 7  pheophytin-b (21.49); 8  pheophytin-a (23.38). Reprinted with permission from L. Almela et al. [301].

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Chapter 2

Reaction time

CH2 CH3

CH

0 min

H3C

H3C

N

N

N

N

CH2-CH3

CH3

CH2 CH2

O COOCH3 COOH (R=C20H39)

Fluorescence intensity ex/em: 405/660 nm

Chlorophyll a

CH2 CH3

CH H3C

5 min

H3C

N

N

N

N

CH2 HO CH2

CH2-CH3

CH3 O COOCH3

COOH (R=C20H39) 2 C-13 -OH-chlorophyll a

Chl a

90 min Standard

0

10

20

30

40

Retention time (min)

Fig. 2.131. Tracing of HPLC chromatograms at 0, 5 and 90 min. The experimental conditions are described in the text. Data are typical results. Reprinted with permission from M. Adachi et al. [302]

in 6.5 min. Excitation and emission wavelengths of analytes varied according to the type of analyte. Typical chromatograms of pigments are shown in Fig. 2.132. The chromatograms cearly show that chlorophylls decompose under acidic conditions to pheophytins. The chlorophyll concentrations found in leafy vegetables are compiled in Table 2.103.

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2 Fluorescence activity (arbitrary units)

Fluorescence activity (arbitrary units)

3

2

1

X 0

1 3

5

4

6

7 8

7

10

20 Time [min]

(a)

30

0 (b)

10

20 Time [min]

9

30

Fig. 2.132. Chromatogram of spinach, stored frozen until analysis by HPLC (A) and after acidifying the same pigment extract with 0.2ml M HCl per 10 ml extract and exposure to air and light for 15 h at 20°C (B). Zinc–phtalocyanine was used as an internal standard (IS). Peak identification: 1  chlorophyll-b; 2  chlorophyll-a; x  unknown degradation product; 3  IS; 4  pheophytin-b; 5  pheophytin-a; 6  chlorophyll-b
; 7  chlorophyll-a
; 8  pheophytin-b
; 9  pheophytin-a
. Reprinted with permission from T. Bohn et al. [303]. TABLE 2.103 CHLOROPHYLL CONTENT OF SOME LEAFY VEGETABLES

Vegetable

n

Chl a (g/g)

Chl b (g/g)

Total

Ratio of chlorophyll

Spinach Endive Lettuce Iceberg

5 5 3 3

69120 27326 28318 191

1937 837 705 41

88418 35633 35324 221

3.600.21 3.290.08 4.040.06 5.070.09

Chlorophyll content (S.D.) of four fresh and immediately frozen green leafy vegetables. Analyses were carried out using zinc–phtalocyanine as an internal standard. Reprinted with permission from T. Bohn et al. [303].

The main recovery of the IS was 10012 per cent and the r values of the calibration curve were always over 0.9993. It can be concluded from the data that the method allows the reliable determination of chlorophylls and related compounds in various plant materials [303]. TLC and RP-HPL were applied for the separation and identification of oxidized chlorophylls and metallochlorophillic complexes of copper in table olives. Pigments were extracted

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Chapter 2

with N,N-dimethylformamide and preconcentrated on silica TLC plates. Petroleum ether (65 to 95°C)–acetone–diethylamine (10:4:1, v/v) and petroleum ether (65 to 95°C)–acetone–pyridine (10:4:2.5, v/v) were the mobile phases. HPLC analyses were performed in an ODS column (250  4.6mm i.d.; particle size 5m). Solvent A was water–0.05

16

9

16'

14 7

5

4

20 14'15

6

11

17

12

(a) 16

16'

9

14

7

12 3

5

4

8

6

10 11

20

15 14' 12 13

18

19

17

21

(b)

0

5

10

15 Time (min)

20

25

30

Fig. 2.133. HPLC chromatogram of pigment extracts from table olives cv. Gordal: (a) healthy fruits and (b) altered fruits. Peaks: 1  15-glyoxilic acid pheophorbide-b; 2  15-glyoxilic acid pheophorbide-a; 3  Cu-15-glyoxilic acid pheophorbide-a; 4  pheophorbide-b; 5  pheophorbide-a; 6  pyropheophorbide-a; 7  15-glyoxilic acid pheophytin-b; 8  Cu-15-glyoxilic acid pheophytin-b; 9  15-glyoxilic acid pheophytin-a; 10  Cu-15-glyoxilic acid pheophytin-a; 11  15
-OH-lactonepheophytin-b; 12  15
-OH-lactone-pheophytin-a; 13  15-formylpheophytin-b; 14  pheophytin-b; 14
 pheophytin-b
; 15  15-formylpheophytin-a; 16  pheophytin-a; 16
 pheophytin-a
; 17  pyropheophytin-b; 18  Cu-pheophytin-a; 19  Cu-15-formylpheophytin-a; 20  pyropheophytin-a; 21  Cu-pyropheophytin-a. Reprinted with permission from B. Ganul-Rojas et al. [304].

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M tetrabutylammonium acetate or 1 M amonium acetate–methanol (1:1:8, v/v) and solvent B consisted of aetone–methanol (1:1, v/v). The chromatographic profiles of healthy and altered fruits are depicted in Fig. 2.133. The method allows the separation and identification of the following new chlorophyll derivatives: Cu-15-formylpheophytin-a, Cu-15-glyoxilic acid pheophytin-a, Cu-15-glyoxilic acid pheophytin-b, 15
-OH-lactone-pheophytin-b and 15
-OH-lactone-pheophytin-a. Because of its high separation capacity, the technique has been proposed for the investigation of the colour change in table olives and the detection of oxidized chlorophylls and Cu complexes in foods and food products [304]. Another RP-HPLC method was employed for the separation and quantitative determination of various pigments in olive oils. Both liquid-phase distribution (LPD) and SPE have been used for the extraction of pigments. Oils were distributed between n-hexane (containing lipids and carotenes) and DMF (other pigment fractions). The DMF fraction was treated with Na2SO4 solution and extracted again with n-hexane–diethyl ether (1:1, v/v). The organic phase was dried and redissolved in acetone. SPE used an ODS stationary phase. The samples dissolved in n-hexane were loaded into the cartridge, washed with n-hexane and the pigments were eluted with 5 ml of acetone. Separation was performed in an ODS column (250  4.6 mm i.d.; particle size 5 m) using gradient elution (mixtures of 0.05 M tetrabutylammonium and 1 M ammonium acetate, methanol and acetone). The flow rate was 1.5 ml/min and chlorophylls were detected fluorimetrically, other pigments with DAD. It was established that the efficacy of both extraction methods (LPD and SPE) was similarly high, varying between 94.44 and 105.88. It was further found that the level of maturation and the type of industrial extraction technology exerts a considerable effect on the amount and composition of pigments in the oils. The amounts of virgin oil pigments are compiled in Table 2.104. It was demonstrated that by using a multivariate mathematical statistical method (principal component analysis) the HPLC data can be employed for the classification of olive variety and for the authenticity test of oils as illustrated in Fig. 2.134 [305]. The decomposition of chlorophyll-a and
-carotene in the presence of fatty acid esters was also studied with an RP-HPLC technique in model systems and the effect of illumination on the decomposition rate was elucidated. Analytes have been extracted from the model solutions by SPE. A mixture of 0.5g of TABLE 2.104 VIRGIN OLIVE OIL PIGMENTS (MG/KG OLIVE OIL (PPM))

Olive oil pigment

Mean values

Median values

Range

Chlorophyll-b Chlorophyll-a Pheophytin-b Pheophytin-a Neoxanthin Violaxanthin Lutein

0.92 0.29 1.2 12.09 0.91 0.89 7.82

0.41 0.01 0.92 10.75 0.86 0.51 6.82

0–5.19 0–6.18 0.05–9.72 2.06–37.06 0.12–2.36 0.00–5.15 3.96–14.78

Reprinted with permission from A. Cichelli et al. [305].

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23.92 26.84

19.00

16.34

5.84

26.13

Chapter 2

Virgin olive oil

27.08

Refined olive oil

26.04 55

.21

Channel A

21.78

23.85

Inject 00/00/00 02:00:29

Channel A

Inject 00/00/00 04:03:28

Channel A

Inject 00/00/00 00:17:48

312

Adultered olive oil

Fig. 2.134. Comparison between three different olive oil samples. Reprinted with permission from A. Cichelli et al. [305].

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magnesium oxide and 0.5g of diatomaceous earth was layered on to a C18 cartridge preconditioned with 6 ml of methanol and 12 ml of n-hexane. Samples of 3 ml were loaded onto the cartridge and all-trans-
-carotene, its cis isomers, methyl stearate, methyl oleate and methyl linoleate were eluted with 24 ml of n-hexane. Chloroform and its isomers were eluted with 6 ml of acetone. Saponification of the first fraction was carried out with methanolic potassium hydroxide for 2h. Separation was performed in an ODS column (250  4.6mm i.d.; particle size 5m). Carotenes and carotene isomers were separated by methanol–ACN–THF (54:42:1, v/v) as the mobile phase, the flow rate being 1 ml/min. Analytes were detected at 450 nm. Another isocratic mobile phase (methanol–ACN–water; 94:5:1, v,v) was employed for the separation of Chl-a and its isomers at a flow rate of 0.8 ml/min. Solutes were detected at 660 nm. Chromatograms illustrating the good separation capacity of the RP-HPLC system are shown in Fig. 2.135. The percentage changes of all-trans-carotene and its cis isomers in the presence of Chl-a and fatty acid esters, and the percentage changes of Chl-a and its isomers in the presence of
-carotene and fatty acid esters during illumination for varied lengths of time are compiled in Table 2.105. The results demonstrated that Chl-a is more sensitive to isomerization and decomposition in the presence of fatty acid esters than
-carotene. The decomposition rate was in both cases of first order and the effect of fatty acid esters markedly depended on the length of the hydrophobic apolar alkyl chain [306].

3

Absorbance ( 660 nm)

Absorbance ( 450 nm)

3

5

4 1 2

1 2

0.00

8.00

4

16.00

Retention time (min)

0.00

6.00

12.00

18.00

Retention time (min)

Fig. 2.135. Upper lane: HPLC chromatogram of all-trans-
-carotene and its isomers in the presence of Chl-a and methyl stearate during illumination for 3h. Peak identification: 1  13,15-cis-
carotene; 2  15-cis-
-carotene; 3  all-trans-
-carotene; 4  9-cis-
-carotene; 5  13-cis-
carotene. Lower lane: HPLC chromatogram of Chl-a and its isomers in the presence of
-carotene and methyl linoletae during illumination for 3 h. Peak identification: 1  Chl-a isomer I; 2  Chl-a isomer II; 3  Chl-a; 4  Chl-a
. Chromatographic conditions are described in text. Reprinted with permission from B. H. Chen et al. [306].

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HPLC has been applied for the separation of synthetic C8 substituted bacteriochlorophyll-d analogues. The chemical structures of analytes are shown in Fig. 2.136. Epimers of Zn-MBPhes-d were prepurified in a silica column and separated in an ODS column (250  10 mm i.d.). The separation conditions were different for epimer pairs: Zn-2R and Zn-2S: methanol–water (5:1, v/v), retention time 51 and 55min; Zn-3R and Zn-3S: methanol–water (4:1, v/v), retention time 51 and 63min; Zn-4R and Zn-4S: methanol–water (8.5:1.5, v/v), retention time 28 and 30min; Zn-5R and Zn-5S: methanol–water (4:1, v/v), retention time 36 and 39min. The flow rate was in each instance 2 ml/min [307]. Capillary electrophoresis coupled with a laser-induced fluorescence (LIF) detector has also been applied for the analysis of copper chlorophyll in olive oils. Samples were

TABLE 2.105 PERCENTAGE CHANGES OF ALL-TRANS CAROTENE AND ITS CIS ISOMERS IN THE PRESENCE OF CHL A AND FATTY ACID ESTERS, AND THE PERCENTAGE CHANGES OF CHL A AND ITS ISOMERS IN THE PRESENCE OF
-CAROTENE AND FATTY ACID ESTERS DURING ILLUMINATION FOR VARIED LENGTHS OF TIMEa

Time (min)

Methyl stearate
-carotene 13.15-di-cis

15-cis

All-trans

9-cis

0 10 30 60 120 180

3.2 4.4 5.1 5.1 5.7 5.8

1.3 1.0 1.9 0.9 0.6 0.5

84.0 83.0 80.0 76.0 70.8 70.8

4.1 0.2 0.9 0.8 0.7 0.7

7.4 11.4 12.1 17.2 22.2 22.2

0 10 30 60 120 180

3.2 4.1 4.0 3.6 5.4 5.5

1.3 3.1 3.5 2.9 3.2 3.1

84.0 81.0 81.0 80.0 75.0 74.8

4.1 0.6 1.4 0.6 0.3 0.6

7.4 11.2 10.1 12.9 16.1 16.0

0 10 30 60 120 180

3.2 5.2 5.6 5.7 6.4 6.5

1.3 1.2 1.3 0.7 0.5 0.4

84.0 81.5 80.3 79.8 80.8 80.5

4.1 1.7 1.5 1.5 1.8 1.7

7.4 10.4 11.3 12.3 10.5 10.9

13-cis

(Continued on next page)

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TABLE 2.105 (continued)

Time (min)

Methyl stearate
-carotene Chl a

0 10 30 60 120 180

Chl a Isomer II

Chl a

Chl a


9.1 19.4 38.6 35.4 40.5 40.7

8.2 13.2 24.1 31.1 28.5 29.1

82.7 58.9 28.0 25.1 25.0 25.0

0.0 8.5 9.3 8.4 6.0 5.2

0 10 30 60 120 180

9.1 6.4 12.6 18.4 29.5 29.2

8.2 9.3 10.1 8.4 7.5 16.1

82.7 82.9 75.0 70.1 60.0 46.0

0.0 1.4 2.3 3.1 3.0 8.7

0 10 30 60 120 180

9.1 2.4 5.6 10.4 12.5 11.7

8.2 6.3 9.4 12.1 5.8 7.9

82.7 77.9 72.0 63.1 59.0 59.0

0.0 13.4 13.0 14.4 22.7 21.4

a

Average of duplicate analyses. Reprinted with permission from B. H. Chen et al. [306].

preconcentrated on silica SPE cartridges. Samples of 1 000 mg were loaded, the lipophilic components were removed by 30 ml of n-hexane and the pheophytins were eluted with 18 ml of acetone. The acetone was evaporated and the residue redissolved in 1 ml of acetone–water (80:20). Separations were performed in a capillary of 50 cm total length and 75m i.d. using borate buffer (25mM, pH  8.6). Samples were injected hydrodynamically. The voltage was 30kV, separation time 15min, the capillary was thermostated at 25°C. The electrophoregrams of fresh extra virgin olive oil and extra virgin olive oil with a long shelf life are shown in Fig. 2.137. The validation parameters of the method were: RSD per cent of areas, 1.49; RSD per cent of migration times, 0.56; recovery ranged 91.90 – 98.15 per cent; RSD of recovery was 2.67. The coefficient of correlation was 0.996. It was stated that the repeatability, reproducibility and accuracy of method is good, consequently, it is suitable for the quantitative determination of copper chlorophyll in olive oil [308].

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Chapter 2 OH

N

R8

N

Mg N

N R12

COO-farnesyl O R8 = Et, n-Pr, iso-Bu, neo-Pentyl

(a)

OH

N

R8

N

N

N

MeOOC O 1: R8 = Et

2: R8 =CH2CH2Ph

3: R8 = CH=CH2

(b)

4: R8 = trans-CH=CHPh

5: R8 = cis-CH=CHPh R1

OH

R2 N

N

R8

Zn N

MeOOC

N

O

Zn-1R: R1 = Me, R2 = H, R8 = Et

Zn-1S: R1 = H, R2 = Me, R8 = Et

Zn-2R: R1 = Me, R2 = H, R8 = CH2CH2Ph

Zn-2S: R1 = H, R2 = Me, R8 = CH2CH2Ph

Zn-3R: R1 = Me, R2 = H, R8 = CH=CH2

Zn-3S: R1 = H, R2 = Me, R8 = CH=CH2

Zn-4R: R1 = Me, R2 = H, R8 = trans-CH=CHPh Zn-4S: R1 = H, R2 = Me, R8 = trans-CH=CHPh (c) Zn-5R: R1 = Me, R2 = H, R8 = cis-CH=CHPh

Zn-5S: R1 = H, R2 = Me, R8 = cis-CH=CHPh

Fig. 2.136. Molecular structures of (a) naturally occurring magnesium chlorin, bacteriochlorophyllsd (Bchls-d), (b) their synthetic analogues, methyl bacteriopheophorbides-d (MBPhes-d, and (c) 3
epimeric zinc complexes (Zn-MBPhes-d). Reprinted with permission from S. Sasaki et al. [307].

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Page 317

3.113

0.18

6.443

7

1 5.000

4 0.10

5 6.991

2

6.850

4.730

Absorbance

3

6 0.00

0

5

10 Minutes

Fig. 2.137. Left: electrophoregram of chlorophyll pigments in fresh extra virgin olive oil, added with copper chlorophyllin: (1) chlorophyllin B; (2) pheophytin B; (3) pheophytin B
; (4) chlorophyllin A; (5) pheophytin A; (6) pheophytin A
; (7) copper chlorophyllin. Right: electrophoregram of chlorophyll pigments in extra virgin olive oil, with a long shelf-life, added with copper chlorophyllin: (1) chlorophyllin B; (2) chlorophyllin derivatives; (3) chlorophyllin A; (4) copper chlorophyllin. Reprinted with permission from L. Del Giovine et al. [308].

2.5 MISCELLANEOUS PIGMENTS Besides the great pigment classes such as carotenoids, flavonoids, anthocyanins and chlorophylls a wide variety of other pigments have been separated, quantitated and identified by different liquid chromatograpchic techniques. The chemical structures of these pigments show high diversity. Unfortunately, in the majority of cases the biological activity of these

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molecules has not been elucidated in detail and thier benifcial or toxic effects need further investigation. 2.5.1 Thin-layer chromatography Both adsorption and partition TLC have been used for the separation of tetraphenylporphyrin derivatives newly synthetized. The chemical structure of porphyrins are listed in Table 2.106. Silica plates were activated at 120°C for 30 min then cooled in a desiccator. Separations were performed with dichloromethane–methanol mixed in various volume ratios. C18 plates were not pretreated and separation was carried out with mixtures of acetone–water. It was established that both adsortion and RP-TLC can be applied for the separation of tetraphenylporphyrin pigments. Topological indexes may help the better understanding the physicochemical procedures underlying the separation [309]. Isolation of curcumin from turmeric has been obtained by TLC. It has been reported that curcumin [(E,E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-one] may have antitumour and anticancer activities. Turmeric was ground and a sample of 20g was refluxed with 50 ml of dichloromethane for 1 h. The suspension was filtered and concentrated at 50°C. The residue was triturated with 20 ml of n-hexane. The rest was analysed by TLC using a silica stationary phase and dichloromethane–methanol (97:3, v/v) as the mobile phase. It was found that TLC separated the crude extract in three fractions with RF values of 0.49, 0.22, and 0.085 [310]. A combined method using TLC-Fourier transform infrared and RP-HPLC-DAD detection was applied for the separation and tentative identification of the main pigment fraction of raisins. For the optimization of the extraction process 1g of raisins was ground with 2g of acid-washed sand, and 8.5 ml of extracting agent (water, methanol, acetone, ACN, THF, dioxane and water–organic mixtures in volume ratios of 1:3; 1:1; 3:1). After extraction the samples were centrifuged and the visible spectra of the supernatant was measured between 400–700 nm. As the best results were obtained with methanol, a column of 100  10 mm i.d. was filled with the raisin–sand mixture and it was washed with methanol. Fractions of 1 ml were collected and their visible spectra were measured as described above. Silica and alumina ready-made TLC plates were impregnated by overnight predevelopment in n-hexane–parafin oil (95:5, v/v) for RP-TLC analyses. Extracts of raisins were spotted onto the plates and were developed with aqueous solutions of methanol, acetone, THF and dioxane in the dark. The main pigment fraction was measured by TLC-FTIR on-line coupling using a diffuse reflectance accessory (resolution, 4cm1; scan 1 024). Off-line FTIR was performed by scraping off the main pigment fraction and extracting exhaustively with methanol. The extract was evaporated to dryness in vacuunm, redissolved in 20 l of methanol and the FTIR spectra were measured using a KBr pellet of 4mm diameter (resolution, 4cm1; scan 128). RP-HPLC separation of pigments was carried out in an ODS column (250  4 mm i.d.; particle size 5 m) using a THF–water gradient elution: 0–5 min, 100 per cent water; 25–45 min, 50 per cent water; 55–80 min, 5 per cent water. The flow rate was 1 ml/min and spectra were detected between 300–600 nm. Densitograms of various raisin extracts measured at different wavelengths

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TABLE 2.106 THE PORPHYRINS INVESTIGATED H3C R R O

O

CH3

O

O

O

R

O R

N H

N H N H N

N

R R O

N H N

N

O

O R

O H3C

O

O

R

CH3

Basic structural formula for molecules 1–5 and 6–10 No. Series A 1 2 3 4 5 Series B 6 7 8 9 10

R

Name

-H -CH3 -C5H11 -C10H21 -C16H33

5,10,15,20-Tetra-(4-hydroxyphenyl)porphyrin 5,10,15,20-Tetra-(4-methoxyphenyl)porphyrin 5,10,15,20-Tetra-(4-pentyloxyphenyl)porphyrin 5,10,15,20-Tetra-(4-decyloxyphenyl)porphyrin 5,10,15,20-Tetra-(4-hexadecyloxyphenyl)porphyrin

-H -CH3 -C5H11 -C10H21 -C16H33

5,10,15,20-Tetra-(3-methoxy-4-hydroxyphenyl)porphyrin 5,10,15,20-Tetra-(3.4-methoxyphenyl)porphyrin 5,10,15,20-Tetra-(3-methoxy-4-pentyloxyphenyl)porphyrin 5,10,15,20-Tetra-(3-methoxy-4-decyloxyphenyl)porphyrin 5,10,15,20-Tetra-(3-methoxy-4-hexadecyloxyphenyl)porphyrin

Reprinted with permission from M. Podgórna et al. [309].

are shown in Fig. 2.138. Unfortunately, the RP-TLC stationary phase was strongly IR absorbent and the high background adsorbance made it impossible to use the TLC-FTIR on-line coupling technique. The off-line TLC-FTIR spectra of pigment extracts were similar to those of D-erythrose, and their difference spectra were similar to those of D-fructose. It was concluded from the comparison of FTIR spectra that the main pigment fractions of

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Chapter 2 340 nm

340 nm

10 170 (a) Distance of development (mm)

−0.04 10 170 (b) Distance of development (mm)

0.19 Absorbance (AU)

Absorbance (AU)

Absorbance (AU) −0.04

670 nm

0.48

0.40

−0.04 (c)

10 170 Distance of development (mm)

Fig. 2.138. Densitograms of colour pigments of raisins. Alugram RP-18/UV254 plates; mobile phase water–THF (3:7):raisin 8, detection wavelength 340 nm (a); raisin 3, detection wavelength 340 nm (b); raisin 8, detection wavelength 670 nm (c). Reprinted with permission from T. Cserháti et al. [311].

raisins are supposedly composed of saccaheride units such as erythrose, which is the decomposition product of glucose and fructose [311]. A similar TLC-FTIR and RP-HPLC-MS technique was applied for the study of the colour pigments of Tichoderma harzianum. The investigations were motivated by the supposition that the determination of the pigment profile may help the identification of Trichoderma species. T. harzianum was fermented for 72 h then 400 ml of fermentation broth was extracted with 400 ml of ethyl acetate and the organic phase was evaporated to dryness. RP-TLC separations were carried out on RP-18W/UV254 layers using acetone–water mixtures as mobile phases and two-step gradient elution. Gradient elution always started with water–acetone (1:9, v/v; separation distance 3 cm). Mobile phases for the second gradient step contained 55, 60, 65 and 70 per cent acetone. After development the plates were dried and evaluated with a TLC scanner at 400 nm. As the in situ FTIR evaluation of plates was hindered by the strong absorbance of the stationary phase, the two main fractions were scraped off the layer and extracted exhaustively with acetone. The acetone was evaporated and the residue was redissolved in 20l of acetone, and measured by a traditional FTIR method using KBr pellets. The spectra were tentatively identified by using an FT-IR spectra library. RP-HPLC measurements were performed in an ODS column (250  4 mm i.d.; particle size 5 m) using gradient elution. Separation started with 5 per cent acetone for 0–5min; increased to 67 per cent in 25min; changed to 94 per cent in 15 min; final hold, 40 min. The flow rate was 1ml/min and pigment profiles were recorded between 320 and 450 nm. MS-APCI conditions were used in the positive-ion mode, corona discharge 5 A, orifice potential was 20 V, quartz tube temperature 200°C. Nitrogen was employed as the nebulizing and curtain gas. The best separations of pigments by RP-TLC using two-step gradient elution and by RP-HPLC are illustrated in Fig. 2.139. It was found that pigments are not baseline separated by RP-TLC even in the optimal gradient system and the number of fractions is lower than that obtained by RP-HPLC. FT-IR indicated that pigments contain oxygen and unsaturated substructures in the pigment molecules. MS indicated molecular masses between 470–500 for pigments. It was supposed

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321

Reflectance 400 nm

4 3 5 1 2

6 7

0

160 Distance of development, mm

Fig. 2.139. Upper lane: densitograms of colour pigments of Trichoderma harzianum. Alugram RP18W/UV254 plates. Gradient: water–acetone (10:90, v/v) for 3cm; water–acetone (45:55, v/v) for 13cm. Detection wavelength 400 nm. Lower lane: RP-HPLC chromatogram of colour pigments of Trichoderma harzianum. Reprinted with permission from G. Csiktusnádi Kiss et al. [312].

that the main pigment fractions are oxidation polymers originating from monomers with polar and unsaturated substructures [312]. The combination of normal and RP-TLC-FTIR was also applied for the investigation of the colour pigments of chestnut sawdust. Colour pigments were extracted by boiling 1g of chestnut sawdust with 5ml of distilled water. After cooling at ambient temperature the suspension was centrifuged and the supernatant was used for TLC analyses without any other pretreatment. The efficacy of extraction, the visible spectra of the supernatant and its titration curve were measured in separate experiments. Normal-phase TLC separations were carried out on silica, alumina and diatomaceous stationary phases. The same stationary phases were impregnated by overnight predevelopment in n-hexane–paraffin oil (95:5, v/v) for RP-HPLC. Various mixtures of n-hexane, acetone, ACN, THF, dioxane, methanol, ethanol, 1- and 2-propanol, ethylcellosolve and water were applied as mobile phases. In each case the separation distance was about 17cm. After development, the plates were evaluated by a TLC scanner at 340 nm. On-line and off-line FTIR measurements were performed as described above. The efficacy of extraction was 5 per cent indicating that the overwhelming majority of pigments are insoluble in water. This fact suggests the polymeric character of the pigments. Some typical densitograms are shown in Fig. 2.140. The densitograms demonstrate that the separation capcity of normal and RP-TLC differs considerably. It was further established that solutes were strongly retained on alumina and impregnated alumina stationary phases, suggesting the acidic character of the watersoluble pigment fractions. The off-line FTIR spectra of the two main pigment fractions are depicted in Fig. 2.141. On-line FTIR spectra of the pigment fractions suggested that each pigment contains both hydroxyl and carboxyle groups and they are tannic acid-like compounds with high molecular mass [313].

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Absorbance, AU 400 nm

0.02

9

6 8 10 2 1

3 7 45

11

0 0

Minutes

120

Fig. 2.140. Densitograms of colour pigments of chestnut sawdust on silica (a) and impregnated silica (b) layers at 340 nm. Multistep gradient development: water–THF 1:1 (v/v) for 3cm; water–THF 3:7 (v/v) for 8cm; then water–THF 1:4 (v/v) to the end of the development. Reprinted with permission from T. Cserháti et al. [313].

Ommochromes belong to the naturally occurring pigments present in many members of the animal kingdom mainly in insects. Their presence in the central nervous system of the silkworm Bombyx mori was proved by using cellulose and silica stationary phases. The RF values of standards and the isolated pigments are compiled in Tables 2.107 and 2.108. The good correspondence of the retention values of standard ommin and the red pigment from ganglia makes it possible but does not prove that the red pigment is ommin and not xanthommatin. The retention values suggested that the red pigment contains 3-hydroxykynurenin. The data illustrate that TLC combined with other physicochemical methods, such as light and electron microscopy and SDS-polyacrylamide gel electrophoresis (SDS-PAGE), can also be successfully employed in physiological studies [314]. Two-dimensional TLC, HPLC-DAD and HPLC-APCI were applied for the separation and identification of anka pigments synthesized on rice grains by Monascus purpureus DSM 1379. The chemical structures of anka pigments are shown in Fig. 2.142. Pigments were purified by extracting the anka powder with n-hexane (orange pigments) followed by methanol (red pigments). Crude pigment extracts were further purified by recrystallization. Two-dimensional TLC was performed on silica/diatomaceous earth plates using n-hexane–ethyl acetate (7:3, v/v) (first dimension) and n-hexane–ethyl acetone (7:3, v/v) (second dimension). The RF values of pigments are compiled in Table 2.109. The retention values demonstrated that the separation of anka pigments considerably depends on the composition of the mobile phase, furthermore, it was found that the TLC

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100

% Transmittance

γaCO2γa8CO2--CH3 -CH2-

γ-OH

-CH3 -CH235 4000

Wavenumber (cm−1)

500

% Transmittance

100

-CH3 -CH2-

γC=0 γBCO2-

35 4000

γ−0H

γaaCO2Wavenumber (cm−1)

500

Fig. 2.141. Upper lane: off-line FTIR spectrum of the pigment fraction in the middle of the chromatogram (silica stationary phase, multistep development); lower lane: off-line FTIR spectrum of the strongly retained pigment fraction (impregnated silica stationary phase, multistep development). Reprinted with permission from T. Cserháti et al. [313].

data can be used for the optimization of the purification method for these pigments. RPHPLC separation of anka pigments was performed in an ODS column (250  4.6mm i.d.) using an isocratic mobile phase (ACN–water, 80:20 v/v). The flow rate was 0.8 ml/min and pigments were detected at 233 nm. The conditions of HPLC-MS-MS with APCI interface were: vaporizer and capillary temperatures, 300°C and 180°C, respectively; corona voltage, 4 kV; electron multiplayer voltage, 1.6 kV; sheath and auxiliary gas was nitrogen; argon was the collision gas. The effect of detection wavelengths on the chromatographic profile of pigments is illustrated in Fig. 2.143. The variation of the reproducibility of the retention time was less than 1 per cent and the recovery varied between 100.7 and 103.9 per cent. The results indicated that the combined method can be used for the separation, identification and quantitation of anka pigments [315].

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TABLE 2.107 COMPARISON OF TLC RF VALUES OF THE RED PIGMENT EXTRACTED FROM LARVAL GANGLIA AND FROM THE EGGS OF B. MORIa

Compound

(a)

(b)

(c)

Silica gel

Cellulose

Silica gel

Cellulose

Silica gel

Cellulose

Standard ommin from eggs

0.84

0.80

0.45

Red pigment from ganglia

0.83

0.80

0.46

Chemically synthesized xanthommatin

0.90

0.29 0.09 0.28 0.08 0.36

0.61

0.55

0.10 0.00 0.10 0.00 0.06

0.20 0.00 0.20 0.00 0.29

a

Solvent systems: (a) 80per cent formic acid–metahmol–HCl (80:15:0.5); (b) collidine–lutidin–water (1:1:2); (c) collidine–water (3:1); all by volume. The pre-coated thin-layer plates were silica gel and cellulose. Reprinted with permission from H. Sawada et al. [314].

TABLE 2.108 CELLULOSE TLC RF VALUES OF HYDROLYSATES OF B. MORI LARVAL GANGLIA EXTRACTS OF XANTHORENIC ACID AND 3-HYDROXYKYNURENINEa.

Compound

(a)

(b)

(c)

(d)

Xanthurenic acid 3-hydroxykynurenine Acid hydrolysate of the standard compound from eggs Acid hydrolysate of the red pigment from ganglia

0.72 0.48 0.48 0.49

0.77 0.62 0.63 0.62

0.76 0.37 0.38 0.38

0.61 0.41 0.40 0.40

a Solvent systems: (a) butanol–acetic acid–water (4:1:2); (b) pyridine–butanol–water (1:1:1); (c) collidine–water (3:1); (d) propanol–ethyl acetate–water (7:1:2); all by volume. Reprinted with permission from H. Sawada et al. [314].

Two new 10,10
-linked dihydroanthracenones were isolated from an indigenous Australian toadstool belonging to the subgenus Dermocybe of Cortinarius by TLC and chiral HPLC, and their chemical structure was elucidated by 1HNMR and CD (circular dichroism). The chemical structures of pigments investigated are depicted in Fig. 2.144. Extraction of pigments was performed by macerating 122g of fruit bodies with 800 ml of ethanol at ambient temperature for 4 h. The liquid phase was evaporated to dryness and

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C7H15

O

325 C5H11

O O

O O

O

O

O

O

O I

II

C7H15

O

C5H11

O O

O O

O

O

O

O

O

III

IV C7H15

O

C5H11

O O

O NH

O

NH

O O

O

V

VI

Fig. 2.142. Structures of anka pigments and two red nitrogen analogues. Pigments: I  anakaflavin, II  monascin, III  monascorubrin, IV  rubropunctatin, V  monascorubramine, VI  rubropunctatamine. Reprinted with permission from S. S. Teng et al. [315].

partitioned between 100 ml of water and 3  100 ml of ethyl acetate. The combined organic fraction was dried with Na2SO4, evaporated to dryness and separated by TLC. TLC separation was carried out in a silica stationary phase using toluene–ethyl acetate–acetic acid (50:49:1) as the mobile phase. After development, two yellow spots (RF 0.14 and 0.17) and two fluorescence ones (RF 0.19 and 0.25) were detected. Fluorescence fractions were further purified in a Sephadex LH-20 column (400  35 mm i.d.) employing methanol as the mobile phase. Chiral separation was performed in a Daicel Chiralpak AD column (250 x 4.6 mm i.d.; particle size 10  m). The isocratic mobile phase consisted of ethanol at a flow rate of 0.5 ml/min. It can be concluded from the results that the combination of various chromatographic techniques such as TLC, gel permeation chromatography and chiral HPLC with other physicochemical methods (1H NRM, CD) is a valuable tool for the isolation, separation and identification of new anthracenone pigments [316].

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TABLE 2.109 RF VALUES OF PIGMENTS ELUTED WITH N-HEXANE–ETHYL ACETATE (7:3, V/V) AND N-HEXANE–ACETONE (2:1. V/V)

Pigment

First dimension

Second dimension

Ankaflavin (yellow, C7)a Monascin (yellow, C5)a Rubropunctatin (orange, C5)a Monascorubrin (orange, C7)a Red pigment 1b Red pigment 2b

0.45 0.40 0.38 0.38 0.01 0.01

0.49 0.44 0.50 0.45 0.19 0.17

a

Identified anka pigment (colour, saturated side chain on ketonic carbonyl group). Pigment not identified. Reprinted with permission from S. S. Teng et al. [315]. b

Time (min)

Time (min) 0.628

AU

0.628

3

6 4 1

5 2 100 nm 392 nm

0.6

100 nm 392 nm

0.6

Time (min)

Time (min)

Fig. 2.143. Chromatograms of pigments detected by various wavelengths. Peaks: 1  rubrupunctatamine; 2  monascorubramine; 3  monascin; 4  rubropunctatin; 5  ankaflavin; 6  monascorubrin. Reprinted with permission from S. S. Teng et al. [315].

2.5.2 High-performance liquid chromatography Because of the high number of individual pigments in an even higher number of accompanying matrices, a wide variety of extraction and HPLC separation techniques have been developed and applied for the prepurifaction, preconcentration, separation and quantitative determination of the pigments under investigation. 2.5.2.1 Miscellaneous pigments in living organisms Various chromatographic techniques have been employed for the purification and characterization of B-phycoeryhtrin (B-PE) from the unicellular red algae Porphyridium cruentum. B-PE is a biliprotein containing a chromophore. The research was motivated by the importance of B-PE in light-sensing elements in biosensors and by the possible application in food

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OR2

OH

327

1 R1 = R2 = H 2 R1 = Me, R2 = H 3 R1 = R2 = Me

O Me

R1O

OH OMe OH

OH

O

OH

O

Me Me

Me OMe

OR

Me HO

OMe

O

OH

OH

OMe Me HO

OMe

O

OMe

OH

4R=H 5 R = Ac

OH

6

OMe OH

OMe OH

O

O Me

Me OMe Me

OH

HO

OMe Me

O

OH 7

OMe

OH

HO

OMe

O

OH

OMe

8

Fig. 2.144. Chemical structures of anthracenone pigments. Reprinted with permission from K. Beattie et al. [316].

and cosmetics instead of synthetic dyes. Extraction of B-PE was achieved by sonicating 50g of wet cell mass in 50 ml of 1 M acetic acid–sodium acetate buffer (pH  5.5). After sonication the suspension was centrifuged and the solid rest extracted again. The combined extract was saturated to 65 per cent with (NH4)2SO4, held for 2h and centrifuged. The pellet was suspended in 1 ml of 50 mM acetic acid–sodium acetate buffer (pH  5.5) and dialysed overnight against 1l of the same buffer. The dialysate was loaded into a DEAE-cellulose column (170  25 mm i.d.), washed with 90 ml of the same acetate buffer and the biliproteins were eluted with 100 ml of 0.25 M acetate buffer followed with 100 ml of 0.35 M acetate buffer. The fractions containing PEs were loaded into a Sephadex G-100 column (600  25 mm i.d.) and eluted with 0.02 M phosphate buffer (pH  7.0). The pink elates were saturated overnight again to 65 per cent with (NH4)2SO4, centrifuged, resuspended in 5mM sodium phosphate

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Chapter 2

Relative absorbance

0.02

0.01

0.00

0

4

8

12 16 20 Retension time (min)

24

28

32

Fig. 2.145. Semipreparative HPLC separation of the subunits from P. cruentum B-PE. The elution profile monitored at 226 nm is shown. The identity of each peak was detemined from the visible absorption spectra, the known distribution and content of PEB (phycoeryhtrobilin) and PUB (phucourobilin) and SDA-PAGe. Dotted line symbolizes the gradient in percentage of acetonitrile (for quantitative details see text). The order of subunit elution is , ,
. The elution profile shows a partial resolution of at least three γ- species. Reprinted with permission from R. Bermejo et al. [317].

buffer (pH  7.0) and dilalysed again in the same buffer. Separation of B-PE subunits was carried out in a C4 column (150  4.6 mm i.d.). Solvents for gradient elution were 0.05 per cent TFA in ACN (A) and 0.05 per cent TFA in water (B). Gradient conditions were: 0–3 min, 5 per cent A; 8 min, 40 per cent A; 28min, 60 per cent A; 31 min, 95 per cent A. The flow rate was 0.8 ml/min and analytes were detected at 226 nm. The good separation of B-PE subunits is illustrated in Fig. 2.145. The data demonstrated that the purification and RP-HPLC method is suitable for the separation of the subunits of B-phyroerythrin from their unicellular algae P. cruentum [317]. A modification of the method described above was also employed for the purification of B-PE and R-phycocyanin from P. cruentum [318]. Besides RP-HPLC, expanded-bedadsorption combined with ion-exchange chromatography has also been employed for the isolation and purification of R-phycoerythrin from the red alga Gracilaria verrucosa. The scheme of extraction, isolation and purification of R-phycoerythrin is shown in Fig. 2.146. It has been stated that the method combining expanded-bed adsorption with ion-exchange chromatography is an effective and inexpensive procedure for the preparative isolation and purification of this pigment from G. verrucosa [319]. 2.5.2.2 Miscellaneous pigments in foods and food products RP-HPLC with DAD and MS detection has also been employed for the determination of anthraquinone pigments in the extract of Rubia tinctorum. The names and chemical structures of anthraquinone pigments found in R. tinctorum are compiled in Table 2.110.

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Best condition for extraction of alga

Suitable concentration of (NH4)SO4 for eluting expanded-bed adsorption column

3 g portions of 250 g minced alga

Different ratios of dH2O to weight of the alga

The ratio of 1:1 is best

Extraction of 550g frozen alga using method described on left

Filter extraction with nylon stockings and then centrifuge

329

Isolation and purification of Rphcoerithrin from 700 g G. verrucosa

Extraction of alga as well as isolation of R-phycoerithrin using expanded bed column described on the left

Elute with 0.1 M (NH4)2SO4 from expanded-bed adsorption column applied to the ionexchange column

Applied to expanded-bed adsorption column Elution of ion-exchange column with different concentrations of NaCl Different concentrations of (NH4)2SO4 for eluting column Measuring yield of purified Rphycoerithrin from G verrucosa 0.1 M (NH4)2SO4 best concentration for elution

Fig. 2.146. Outline of full process of extraction, isolation and purification of R-phycoerythrin from G. verrucosa. Reprinted with permission from G. Wang [319].

Pigments were extracted from the root of R. tinctorum by refluxing 2.5g of root material with 100 ml of water–ethanol (1:1, v/v). After 3h the mixture was filtered, a sample of 500 l was taken and the rest of the liquid phase was evaporated to dryness. The solid rest was mixed with 100 ml of 2 per cent (v/v) H2SO4 and refluxed for 48h. Samples of 500 l were taken after 0, 0.5, 2, 4, 12, 24 and 48h. Separation of pigments was performed in an ODS column (250  4.6 mm. i.d.; particle size 5 m). The linear gradient programme consisted of ammonium formate–formic acid (0.2 M, pH  3) EDTA (30mg/ml (solvent A) and ACN (B). The gradient started with 27 per cent B for 0–6min; to 60 per cent B in 6–20min; 3min hold; 23–25min to 70 per cent B, 10min hold. The flow rate was 1.0 ml/min and analytes were detected at 254 nm. HPLC-ESI-MS measurements employed an ODS column (250  2.1mm i.d.; particle size 5m) at the flow rate of 0.2 ml/min. ESI parameters in the negative-ionization mode were: spray voltage, 4.5 kV; sheath and auxiliary gas, nitrogen; capillary temperature, 200°C; capillary voltage, 12.00 V. Conditions of APCI interface were: vaporizer temperature, 450°C; discharge current, 10A; capillary temperature, 150°C. Some characteristic chromatographic profiles and mass spectra are shown in Fig 2.147. The UV profile of an acidic hydrolysate is depicted in Fig. 2.148. The chromatograms illustrate that the RP-HPLC

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TABLE 2.110 ANTHRAQUINONE STRUCTURES

CH2

O O

R5

O O

OH

O

R1 R2

H

OH R3

R6

HO

HO HO

H

HO

Primeverose

R4

O

Anthraquinone aglycone moiety

No.

Name

R1

R2

R3

R4

R5

1 2 3 4 5 6 7 8 9 10 11 13 14 15 16

Lucidin primeveroside Ruberythric acid Lucidin glucoside Alizarin glucoside Pseudopurpurin Munjistin Lucidin Alizarin Xanthopurpurin Purpurin Quiizarin 2-hydroxymethylanthraquinone Anthraquinone 1.8-dihydroxyanthraquinone 2.6-dihydroxyanthraquinone

OH OH OH OH OH OH OH OH OH OH OH H H OH H

CH2OH X CH2OH Y COOH COOH CH2OH OH H H H CH2OH H H OH

X H Y H OH OH OH H OH OH H H H H H

H H H H OH H H H H OH OH H H H H

H H H H H H H H H H H H H OH OH

R6 H H H H H H H H H H H H H H OH

X  primeverose; Y  Glucose. Reprinted with permission from G. C. H. Derksen et al. [320].

method separates adequately both anthraquinone glycosides and aglycones. It was further established that the sensitivity of UV detection is higher than that of MS detection. However, it was emphasized that MS is a valuable tool for the on-line identification of unknown pigments present in R. tinctorum extract [320]. The red pigments in meat and meat products have also been vigorously investigated [321,322]. Red pigments in Parma ham have been separated by HPLC and identified by ESI-MS, absorption and fluorescence spectrometry. The unknown red pigment was extracted from Parma ham by homogenizing 5 g of minced ham with 20 ml distilled water. Then the suspension was centrifuged and filtered and three volumes of ice-cold acetone were added to the eluate and it was incubated for 15min in an ice bath. After incubation

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331

4.50

100

254 nm

1

80 2

60 40

5 6

20 0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

42

44

Relative abundance

Time (min)

(a)

100

563.0

100

80 60

269.2

608.71126.9

40 20

533.1

m/z

100

80

80

60

60

60

40 239.3

1172.5 1691.7

299.1

80

40 647.0

100

20

(b)

1066.8 578.4 1600.7

m/z

20

(c)

158.8 226.9

283.1

40 255.2

m/z

20

(d)

239.3

m/z

Fig. 2.147. UV (254) trace of a crude extract of Rubia tinctorum roots and mass spectra (single MS) of the chromatographic peaks for individual anthraquinones: lucidin primeveroside (1), ruberythic acid (2), pseudopurpurin (5) and munjistin (6). Mass spectra (a-b) were obtained with NI-ESI with post-column addition of ammonia. Reprinted with permission from G. C. H. Derksen et al. [320].

the suspension was centrifuged again, an equal volume of distilled water was added to the supernatant and the mixture was loaded into an ODS cartridge (12 ml/2g) preconditioned with 15 ml of methanol and 15 ml of distilled water. The cartridge was washed with 25 ml of distilled water and the pigment was removed with 10 ml of 75 per cent acetone. Separation of pigments was carried out in an RP column of 150  4.6 mm i.d. using isocratic elution (methanol–0.01 M Na2HPO4, 76:24, v/v; pH  9). The flow rate was 0.5 ml/min at room temperature. Fluorescence detection used 415 and 590 nm for excitation and emission wavelengths, respectively. ESI-MS conditions were: needle and capillary voltage 2681 and 1230 V; chamber temperature, 105°C. The chromatographic profile of the red pigment is depicted in Fig. 2.149. On the basis of the results of absorption and fluorescence spectra, HPLC and ESI-MS, it was concluded that the red pigment in Parma ham is Zn-protoporphyrin IX, the iron in the heme replaced by Zn [323]. RP-HPLC has been applied for the determination of betalain pigments in various plants too. The researches were motivated by the commercial importance of betalain pigments as natural food colourants. An RP-HPLC method was developed for the measurement of betalain pigments in prickly pear (Opuntia ficus-indica) fruits. The chemical structures of betanin and indicaxanthin found in the prickly pear are depicted in Fig. 2.150. Pigments were extracted by homogenizing fresh fruit flesh with methanol (1:5, w/v). The suspension was filtered and the liquid phase was applied for spectrophotometry and RP-HPLC. Liquid chromatographic separation was performed in an ODS column (250  4.6 mm i.d.; particle size 5 m) at ambient temperature. Gradient elution started with 1 per cent aqueous acetic acid and changed to 12 per cent solvent B in

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Relative abundance

332 100 1 80

254 nm

60

2

3

40

4

8

6

20

10

0 0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

42

44

Relative abundance

Time (min) 476.7 431.0

100 80

269.1

80 60

60

403.1

40

40 20

401.1

100

862.8

253.2

20 0

0 200

300

400

500

600

700

800

900 1000

200

300

Relative absorbance

m/z

406

500

600

700

800

900

m/z 260

266 247

266 247

400

260 231

231

406

416

415

Wavelength (nm)

Fig. 2.148. UV (254 nm) profile of an acidic hydrolysate of an aqueous alcoholic extract of Rubia tinctorum (after 30 min). The mass spectra (sinlge MS) obtained with NI-ESI with ammonia added post-column and the UV-Vis spectra of peaks 3 and 4 are depicted, lucidin primeveroside (1), ruberythic acid (2), lucidin glucoside (3), alizarin glucoside (4), munjistin (6), alizarin (8), and purpurin (9). Reprinted with permission from G. C. H. Derksen et al. [320].

30 min. Solvent B was 1 per cent acetic acid in ACN. The flow rate was 1 ml/min and pigments were detected by DAD. Chromatograms showing the separation of pigments are depicted in Fig. 2.151. It can be conluded from the results that the method is suitable for the separation and quantitative determination of betalain pigments in prickly pear fruits [324]. The same RP-HPLC method was applied for the screening and identification of betalains in cactus pears (Opuntia stricta, Opuntia undulata, Opuntia ficus-indica). Pigments were extracted by stirring 10 g of homogenized fruits with 50 ml of ethanol–water (80:20, v/v). The suspension was centrifuged and the supernatant was filtered. LC-MS separations were carried out in another ODS column (150  4.6 mm i.d.; particle size 5 m) at ambient temperature using gradient elution (from 88 mM aqueous acetic acid to 88 mM acetic acid in ACN in 30 min). Positive-ion ESI was applied for the identification of pigments. The chromatographic profiles of cactus pear extracts are depicted in Fig. 2.152. The

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333

Absorbance 415 nm

H

Flurorescence intensity (Ex 415 nm, Em 590 nm)

Z

P

1

2 0

Z

1'

2' 5 10 15 20 25 Retention time (min)

0

5 10 15 20 25 Retention time (min)

Fig. 2.149. HPLC elution profile of the red pigment from Parma ham. Traces 1 and 1
, standard solutions. Traces 2 and 2
, Parma ham extract. Peaks are as follows: H, hemin; Z, zinc protoporphyrin IX; and P, protoporphyrin IX. Reprinted with permission from J. Wakamatsu et al. [323].

O

CH2OH

+ N

O OH

+ N

HO HO

H COO-

H COO-

HO HOOC HOOC Betanin

N H H

N H H

COOH

COOH Indigoxanthin

Fig. 2.150. Chemical structures of betanin and indicaxanthin. Reprinted with permission from J. A. Fernández-López et al. [324].

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0.020 484 nm

0.004

535 nm

0.010

2

0.002 AU

AU

0.015

1

0.005

2 0.000

0.000

−0.002

2 0.020

535 nm

1 −

0.004

484 nm

0.010

AU

AU

0.015

0.005

0.002

0.000

0.000 −0.002 0.00

5.00

10.00 15.00 20.00 25.00 30.00 Minutes

0.00

5.00

10.00 15.00 20.00 25.00 30.00 Minutes

Fig. 2.151. Left: HPLC chromatograms of betalain pigments from reddish purple prickly pear fruits. Right: HPLC chromatograms of betalain pigments from yellow purple prickly pear fruits. Peaks: 1  indicaxanthin; 2  betanin. Reprinted with permission from J. A. Fernández-López et al. [324].

chromatograms clearly show that the composition of pigments and their relative concentration depends considerably on the cactus species. It was found that the highest concentration of pigments occurs in Opuntia stricta pears [325]. The occurrence of anthraquinones in vegetables, herbs and liquors was investigated by using RP-HPLC, RP-HPLC-MS and GC-MS. The chemical structures of anthraquinone derivatives are shown in Fig. 2.153. Samples with high moisture content were homogenized, freeze-dried and extracted in a Soxhlet extractor with ACN for 4–5 h. Liquid materials were acidifed with HCl, extracted with equal volume of ethyl acetate and evaporated to dryness. Prepurification was performed in a silica column (7 g). Sample solutions were loaded into the column and it was washed with 10 ml of pentane and the analytes were removed with 20 ml of acetone–pentane (1:1, v.v). The liquid phase was evaporated to dryness and redissolved in the mobile phase. RP-HPLC was carried out in an ODS column (250  4 mm i.d.). Solvents A and B for gradient elution were ACN–methanol (95:5, v/v) and 0.1 per cent aqueous acetic acid, respectively. The gradient started with 50 per cent A and changed to 100 per cent A in 43 min. The flow rate was 0.8 ml/min and anthraquinones were detected by DAD at 435 nm. RP-HPLC results were verified by GC. LC-MS measurements were performed in another ODS column (100  2.0 mm i.d.) using a linear gradient elution from 60 per cent 10 mM aqueous ammonium acetate to 72 per cent ACN in 16 min. The flow rate was 0.2 ml/min. MS conditions were: corona, 5 A; sheath and auxiliary gas, nitrogen; capillary temperature, 200°C; collision gas, argon. A typical chromatogram showing the baseline separation of pigments and the internal standard danthron is shown in Fig. 2.154. The concentrations of emodin, chrysophanol and physcion in vegetables, herbs and herbal flavoured liquors are compiled in Table 2.111. It has been concluded from the chromatographic data that the concentration of

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335

O. undulata

O. stricta

O. ficus-indica 2

2 0.01 AU

0.01 AU

2

0.01 AU

3 3 535 nm

535 nm 1 2 184 nm

0.0

20.0

2

2 3

3

10.0

3

535 nm

484 nm

30.0

0.0

1

484 nm

10.0

20.0

30.0

0.0

3

10.0

Time (min)

Time (min)

20.0

30.0

Time (min)

Fig. 2.152. HPLC patterns of betalains from cactus pear studies. Peak 1  indicaxanthin (tR  16.8min); 2  betanin (tR  19.6min); 3  isobetanin (tR  22.8min). Reprinted with permission from J. A. Fernández-López et al. [325]. OH

OH

OH

O

OH

O

CH3

CH3

HO

O

O Emodin

OH

Chrysophanol

O

OH

OH

O

OH

CH3

OMe O Physcion

O Danthron

Fig. 2.153. Chemical formula of the investigated anthraquinones. Reprinted with permission from S. O. Mueller et al. [326].

anthraquinones shows high variation between the samples but the majority of vegetables, herbs and herbal liquors contain more than one type of anthraquinone [326]. Gel filtration chromatography carried out on a Sephadex stationary phase has also been employed for pigment analysis. The decolourization of molasses spent wash (MSW) by the white-rot fungus Flavodon flavus was followed with this technique. Untreated and treated MSW samples were diluted up 10 per cent and were loaded into a Sephadex G-50

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column preconditioned with 0.01 M phosphate buffer (pH  7.0). MSW fractions were eluted with the same buffer. Eluates of 3 ml volume were collected and the absorption was measured at 475 nm. A chromatogram illustrating the efficacy of the decolourization process is shown in Fig. 2.155. The data clearly demonstrate that gel filtration chromatography can be successfully applied for the control of the decolourization of pigments of high molecular mass and unknown composition [327].

Standard 19.2 min 60 50

Emodin 17.3 min

Chrysophanol 24.6 min

mAU

40

Physcion 24.6 min

30 20 10

10

20 t [min]

30

Fig. 2.154. RP-HPLC separation of anthraquinones of a sample obtained from watercress. Absorbance at 435 nm. Reprinted with permission from S. O. Mueller et al. [326]. TABLE 2.111 EMODIN. CHRYSOPHANOL AND PHYSCION IN VEGETABLES. HERBS AND HERBAL FLAVOURED LIQUORS (K1 – K11). ANALYSED BY RP-HPLC AND GC-MS. DATA ARE GIVEN AS RANGE. IN MG/KG (N 3) AND WERE NOT CORRECTED FOR RECOVERY

Sample

Sum of anthraquinonesa Freshb

Latuca sativa var. capitata cv. ‘Cabbage lettuce’ Phaseolus vulgaris Beans Pisum sativum Gardenpeas Brassica chinensis Chinese cabbage Latuca sativa var. capitata cv. ‘Iceberg salad’ Cichorium intibus var. folio cv. ‘Chicory’ Cichorium intibus var. folio cv. ‘Radicchio’

Dryc

Emodin

Freshb

0.06–5.9 1.0–174 0.02–0.03 0.05–36 0.4–342 0.02–0.06 0.04–3.6 0.2–21 0.02–0.03 — — — 0.04 0.74 0.01 — — — — — —

Dryc 0.5 0.2–0.70 0.1–0.2 — 0.24 — —

(Continued on next page)

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337

TABLE 2.111 (Continued) Chrysophanol Freshb Latuca sativa var. capitata cv. ‘Cabbage lettuce’ Phaseolus vulgaris Beans Pisum sativum Gardenpeas Brassica chinensis Chinese cabbage Latuca sativa var. capitata cv. ‘Iceberg salad’ Cichorium intibus var. folio cv. ‘Chicory’ Cichorium intibus var. folio cv. ‘Radicchio’

Dryc

0.01–0.03 0.5 0.02–3.5 0.12–33 — — — — 0.01 0.17 — — — —

Sum of anthraquinonesa Vitis vinifera Grape vine leaves 0.4–0.8 Rhizoma graminis Couch grass root 0.2–0.7 Plantagines lanceolatea Plantain herb 0.4–0.7 Fagopyrum esculentum Buckwheat — Artemisia annua Wornwood 0.1 K1 K2 K3 K4 K5 K6 K7 K8 K9 K10 K11

Physcion

— 0.54–0.86 0.07–0.08 1.03–1.28 — 0.05–0.07 6.08–7.60 — 0.05–0.08 — 0.05–0.06

Emodin

0.1–0.2 0.06–0.2 0.1–0.2 — 0.1 — 0.08–0.14 0.02–0.03 0.24–0.30 — 0.01–0.02 2.03–2.55 — 0.02–0.03 — 0.010–0.016

Freshb

Dryc

0.03–5.8 0.01–32 0.02–3.6 — 0.02 — —

0.5–173 0.1–308 0.1–21 — 0.34 — —

Chrysophanol

Physcion

0.1–0.2 0.05–0.2 0.1–0.2 — —

0.2–0.4 0.08–0.3 0.2–0.3 — —

— 0.36–0.58 0.03–0.04 0.64–0.79 — 0.02–0.03 2.91–3.65 — 0.02–0.03 — 0.02–0.03

— 0.09–0.14 0.016–0.020 0.15–0.18 — 0.01–0.02 1.14–1.40 — 0.010–0.017 — 0.010–0.017

—, not detectable.  above limit of detection. but not quantifiable. a Maximum sum of all anthraquinones detected. b mg/kg fresh weight. c mg/kg dry weight. Reprinted with permission from S. O. Mueller et al. [326].

2.5.2.3 Miscellaneous pigments in other matrices The composition of natural dyes in ancient textiles has also been investigated in detail. Thus, the application of RP-HPLC with DAD and MS detection for the separation and identification of natural dyes in historical Coptic textiles has been reported. The chemical

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Chapter 2 0.04

0.035

Fungus treated MSW

OD at 475 nm

0.03

Untreated MSW

0.025 0.02 0.015 0.01 0.005 0 80

90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 Fraction #

Fig. 2.155. Gel filtration chromatography of MSW in a Sephadex G-50 column. Solid line represents 10 per cent diluted MSW decolourized by the live immobilized fungus and the broken line represents fractionation of untreated, 10 per cent diluted MSW. The colour absorbance of the fractions was read at 475 nm. MSW treated with the fungus for five days was used for gel filtration. Reprinted with permission from C. Raghukumar et al. [327].

structure and common name of the natural pigments included in the investigation are listed in Fig. 2.156. Pigments were extracted from the textiles by hydrolysing 0.5–3 mg of sample in 3 M HCl–ethanol (1:1, v/v; 400 l) at 100°C for 20 min. The mixture was filtered and the liquid phase was evaporated to dryness. The residue was redissolved in water–methanol (1:1, v/v) before HPLC analysis. Separations were performed in an ODS column (150  2.1 mm i.d.; particle size 5 m) at a flow rate of 0.2 ml/min. The components of the mobile phase were 0.3 per cent (v/v) aquoues formic acid (A) and ACN (B). The gradient was: 5 per cent B for 2 min; to 60 per cent B in 30 min; to 100 per cent B in 35min; isocratic to 60 min. Column temperatures for MS and DAD detection were 30 and 20°C, respectively. ESI conditions were: capillary voltage, 3.5 kV; cone voltage, 30 V; source temperature, 120°C; desolvation temperature, 300°C; cone gas flow rate, 60l/h; desolvation gas flow rate, 500 l/h. APCI conditions were: corona current, 2 A; cone voltage, 50 V; source temperature, 130°C; desolvation temperature, 500°C; cone gas flow rate, 100l/h; desolvation gas flow rate, 300l/h. The retention times of some pigments are compiled in Table 2.112. The presence of luteolin, apigenin, rhamnetin, kaempferol, alizarin, purpurin, xanthopurpurin, monochloroalizarin and indirubin in the historic Coptic textiles was demonstrated. It was stated that various MS detection modes combined with DAD detection are a powerful tool for the identification of pigments in textiles [328]. A slightly different method using RP-HPLC-DAD has also been employed for the separation and identification of natural pigments in archeological Coptic textiles. The hydrolysis of textile fibres was carried out as described above. However, the insoluble residue was treated with 200 l of warm pyridine and filtered again. Separation was performed in an ODS column (250  4.6 mm i.d.; particle size 5 m) using two gradient programmes. Programme I was for the analysis of hydrolysate: from 100 per cent A (5 per cent ACN and

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339 OH

Carminic acid Me

Gallic acid CO2 H

HO HO

OH

OH

OH OH OH

HO

OH

OH

O

O

Ellagic acid

O

HO2 C

OH

Myricetin OH

O

O

HO

HO

OH

OH

OH OH

O

HO

OH

O

O Luteolin

O Lawsone

O

HO

OH

OH OH

O

OH

O

Quercetin

H N

Indirubin

OH OH OH

OH

HN

O Indigotin

O

Apigenin

H N

O

HO

O O

O

HO

N H

OH O

OH

O

O

6-Bromoindigotin

H N

Br

Kaempferol O

HO

N H

OH O

OH OH

6,6¢-Dibromoindigotin

O

O H N

Br

Rhamnetin O

OMe

N H

OH

O

OH OH OH Alizarin

OH

O

Br

OH Laccaic acid A

HC2C O

OH

O

OH

HC2 C O

OH

O

OH

HC2C

O

HO

CH2 -CH2 -NHAC OH

HO

OH

O Laccaic acid B

Purpurin

OH

O

HC2C

HO CH2-CH2 -OH OH

HO

OH

O Laccaic acid E

OH

OH

Laccaic acid C

HC2C O

OH

CH2-CH2(NH2)-CO2H OH O

OH

OH

O

OH

HC2C

HC2C HO

HC2C O

CH2-CH2-NH2 OH

HO

Fig. 2.156. Chemical structure and common name of the natural pigments included in the investigation. Reprinted with permission from B. Szostek et al. [328].

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Chapter 2 TABLE 2.112 RETENTION TIME OF SOME PIGMENTS

Compound

tR (min)

Compound

tR (min)

Gallic acid Ellagic acid Lawsone Quercetin Kaempferol Alizarin Indirubin Laccaic acid B Laccaic acid E

3.0 14.0 17.2 19.5 22.0 25.3 29.8 14.5 12.1

Carminic acid Myricetin Luteolin Apigenin Rhamnetin Purpurin Laccaic acid A Laccaic acid C

12.8 16.7 19.3 21.5 24.7 28.1 14.7 11.7

Reprinted with permission from B. Szostek et al. [328].

0.1 per cent TFA) to 100 per cent B (0.1 per cent TFA in ACN) in 60 min. Program II was for pyridine extract: 0–9 min, 80 per cent A to 100 per cent B in 15 min. The column temperature in both cases was 40°C. The retention times and the standard deviations of the retention times of pigments are compiled in Table 2.113. The data in Table 113 illustrate that pigments are well separated in the RP-HPLC system applied. Some characteristic chromatographic profiles are shown in Fig. 2.157. The data indicated that the textiles were dyed with madder (Rubia tinctorum) and wild madder (Rubia peregrina). It was found that the historic textiles contain alizarin, purpurin, luteolin, apigenin, carminic acid, ellagic acid, gallic acid, indigotin, and laccaic acids A and B. The chromatograms illustrate that the method is suitable for the separation of pigments extracted from ancient textiles, however, the change of baseline indicates the presence of other, probably polymeric pigments in the extract [329]. A combination of analytical techniques such as RP-HPLC with DAD and fluorescence detection, GC-MS, optical microscopy, scanning electron microscopy, and FTIR was employed for the investigation of ancient Greek polychromy. Three astrogaloi (knucklebones used in popular games) painted pink, red and yellow were included in the experiments. Samples for RP-HPLC measurements (0.1–0.5 mg) were hydrolysed with 20 l of 30 per cent v/v HCL added to 600 l of methanol. Hydrolysis was carried out in an ultrasonic bath for 1h at 60°C. After hydrolysis the sample was diluted to 2 ml with water and extracted with 3  2 ml of ethyl acetate. The combined extract was evaporate to dryness and redissolved in 50 l of mobile phase. RP-HPLC analyses were performed in an ODS column (250  4.6 mm i.d.) using gradient elution. Solvents A and B were 19 per cent (v/v) ACN in water containing 0.1 per cent v/v TFA and 50 per cent aqueous ACN. The gradient profile was 100 per cent A to 5 min; to 100 per cent B in 15min; isocratic for 20 min. In order to reach maximal sensitivity, detection wavelengths for DAD and the fluorescence detector were changed during the separation. The chromatographic profiles of the extract of the superficial layer of the pink

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TABLE 2.113 CHROMATOGRAPHIC RETENTION TIMES FOR EXAMINED DYES

Chemical compound Gradient programme I Gallic acid Laccaic acid B Carminic acid Ellagic acid Laccaic acid A Lawson Luteolin Apigenin Munjistin Alizarin Purpurin Indigotin Gradient programme II Indigotin Indirubin 6-bromoindigotin 6.6’-dibromoindigotin

Retention time (min)

Standard deviation

6.1 14.7 15.8 17.4 18.9 22.0 24.6 27.6 30.3 32.1 35.5 35.9

0.07 0.16 0.51 0.12 0.09 0.10 0.13 0.18 0.12 0.12 0.10 0.10

7.5 8.5 12.7 18.3

0.04 0.05 0.05 0.06

Reprinted with permission from J. Orska-Gawris et al. [329].

astragalos obtained by DAD and fluorescence detection are depicted in Fig. 2.158. The data achieved by the various analytical techniques suggested that a red indigotin lake (tyrian purple) and madder lake were employed for painting the astragalos [330]. RP-HPLC methods have been frequently applied for the investigation of various chemical, biochemical and biophysical processes in in vitro model systems. Thus, the separation of new compounds achieved by enzymatic oxidation of phloridzin was carried out by semi-preparative RP-HPLC. Phloridzin was incubated with a polyphenol oxidase prepared from apple pulp for 6h at 30°C under air agitation. After incubation the suspension was filtered, stabilized by NaF and injected into the RP-HPLC column using diluted acetic acid–ACN gradient. The new compounds were isolated and identified by NMR and MA techniques. The proposed mechanism of the formation of new phloridzin derivatives 3 and 4 is shown in Fig. 2.159. The results illustrate that RP-HPLC can be successfully used for the study of enzymatic processes in model systems [331]. An RP-HPLC technique was employed for the analysis of bilirubin, one of the main components of pigment gallstones. The aim of the study was the determination of the inhibition of chlolesterol crytallization under bilirubin deconjugation. Bilirubin in rat bile was measured in an ODS column (250  4.5 mm. i.d.; particle size 5 m). Separation was

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Fig. 2.157. Chromatograms of acid hydrolisates from Coptic textiles: red silk fibre (sample 6) (a), red wool fibre (sample 10) (b), orange wool fibre (sample 4) (c), and green wool fibre (sample 14) (d). Chromatograms obtained with gradient programme I at 255 nm. Peak identification: 1  gallic acid; 2  laccaic acid B; 3  carminic acid; 4  ellagic acid; 5  laccaic acid A; 6  luteolin; 7  apigenin; 8  alizarin; 9  purpurin; 10  indigotin. Reprinted with permission from J. Orska-Gawris et al. [329].

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Purpurine

Alizarine HPLC-fluroresce

30

Purpurine

40 Time (min)

20

30 Time (min)

40

Fig. 2.158. HPLC chromatogram of a sample of the superficial layer of the pink astragalos. Reprinted with permission from M. P. Colombini et al. [330].

performed with a gradient 60–100 per cent (v/v) methanol in 20 min. The flowrate was 2 ml/min and analytes were detected at 450 nm. The study illustrated that results obtained by HPLC techniques can promote the better understanding of biochemical processes such as the formation of pigment gallstones [332]. The synthesis, glucuronidation and hepatic transport of new aromatic congeners of bilirubin have also been followed by an RP-HPLC method. The chemical strucutres of bilirubin derivatives are shown in Fig. 2.160. RP-PLC separation of bilirubin derivatives was performed in an ODS column (250  4.6 mm i.d.; particle size 5 m) at 34°C. Analytes were eluted isocratically with 0.1 M di-n-octylamine acetate in 8 per cent aqueous methanol. The flow rate was 0.75 ml/min and bilirubins were detected at 454 nm. Typical chromatograms are depicted in Fig. 2.161. The chromatograms demonstrate that RP-HPLC is a valuable tool for the study of the synthetic process of new bilirubin derivatives [333]. A normal-phase HPLC method was employed for the separation of 11-cis- and alltrans-retinals. Separation was performed in a silica column (150  6.0 mm i.d.; particle size 3 m) with an isocratic mobile phase (n-hexane with 15 per cent ethyl acetate and 0.15 per cent ethanol). The flow rate was 1 ml/min and retinols were detected at 360 nm. The method separated well the 11-cis- and all-trans-retinals as demonstrated in Fig. 2.162. The results emphasize again the decisive role of chromatographic methods in the elucidation of the mechanism of various biochemical processes [334]. The interaction of linear tetrapyrroles such as biliverdin and bilirubin with nitrogen-oxiderelated species has also been investigated by RP-HPLC. Analyses were performed in an ODS

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HO

OGlu

HO

O

OGlu O

O enzymatic oxidation

OH

OGlu Michael-type addition

OH

O HO HO

Phloridzin

H

PPO O

O

O

OH

O A ring aromatization via prototropic shift

OGlu

OH

OH

water addition and B ring rearomatization

HO

O

OGlu

HOOC

HO

HO

OGlu

+ HOOC

HO

OH

OH

OH

OH HO Compound 3 (2 atropisomers with unassigned 60:40 ratio)

OH

OH enzymatic oxidation HO

HO

OGlu

PPO

O

O

O

O

O HOOC

OGlu

HO

OGlu

HO

O OH

+ HOOC

O

OGlu

HOOC

+

HOOC

O

O

O

OH HOOC

O

OGlu

HO

OGlu

HO

+ HOOC

O

O O

Compound 4 (2 enantiomers with unassigned 65:35 ratio)

Fig. 2.159. Proposed mechanism for the formation of compounds 3 and 4. Reprinted with permission from C. Le Guernevé et al. [331].

column (250  4.6 mm i.d.; particle size 10 m). Solvents A and B were 100 mM ammonium acetate (pH  5.5)–2-methoxyethanol–methanol (45:5:50, v/v) and 2methoxyethanol– methanol (5:95, v/v), respectively. The gradient changed from 100 per cent A to 100 per cent B in 11min. The flow rate was 1.4 ml/min and analytes were detected at 450 nm [335]. The formation of trimeric oligomers of 5,6-dihydroxyindole-2-carboxilic acid (DHICA) a key molecule in melanin synthesis was also followed by RP-HPLC. The chemical structure of monomer units (DHICA) and 5,6-dihydroxyindole (DHI) and those of the oligomeric products arising from the biomimetic DHICA oxidation are listed in Fig. 2.163. Both analytical and semi-preparative separations of the reaction mixtures were carried out in

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exo HO2C

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O

CH2

N H

N H

N H

N H

O

1 and Bilirubin-IX alfa

CO2H

O

CH2

N H

N H

HO2C

N H

N H

O

2 and Bilirubin-XIII alfa

CO2H HO2C

O

CH2

N H

N H

N H

N H

O

3 and Blirubin-III alfa

Fig. 2.160. Linear representation of bilirubin IX, III and XII (solid lines) and their analogues (1–3) with phenyls superimposed (dashed lines). Reprinted with permission from J. O. Brower et al. [333]. 2 Absorbance (450 nm)

1 3

IXα XIIIα

IIIα 15

25 Time (min)

35

Fig. 2.161. Superimposed reverse-phase HPLC chromatograms of mixtures containing (lower) bilirubin XIII, IX and III and (upper) diphenyl bilirubins XIII (2), IX (1) and III (3). The mobile phase was 0.1 M di-n-octylamine acetate in 8 per cent water/methanol at a flow rate of 0.75 ml/min. Reprinted with permission from J. O. Brower et al. [333].

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0.04 A360 483 nm 470 nm

Absorbance

0.03

1 0.02 2 0.01

11-cls

all-trans

0.00 400

450

(a)

500 550 600 Wavelength (nm)

650

700 (b)

4 6 8 Retention time (min)

10

Fig. 2.162. Absorption spectra of Amphiop1 expressed in HEK293s cells (a) and the HPLC patterns of retinal oximes (b). Absorption spectra and the HPLC patterns were measured before (a, curve 1, and b, top trace) and after irradiation at 520 nm for 2 min (a, curve 2, and b, middle trace). The HPLC pattern of retinal oximes extracted from a mixture of irradiated and non-irradiated bovine rhodopsin in equal amounts is indicated as a reference (b, bottom trace). The absorption maxima of the original pigment and its phoroproduct are shown in panel (a). Reprinted with permission from M. Koyanagi et al. [334].

H N

HO

HO

HO COOH

COOH

DHICA HO

HO

HO

HO

NH

HO

NH

HO

NH

+ TRIMERS

COOH

COOH N H

COOH HO

N H

HO

1

2

COOH HO 3

Fig. 2.163. Oligomeric products arising from biomimetic DHICA oxidation: dimers 4,4
-biindolyl, 4,7
-biindolyl, 7,7
-biindolyl and trimers. Reprinted with permission from A. Pezzella et al. [336].

ODS columns (250  4.6 mm i.d.; 250  22 mm i.d.). The isocratic mobile phase for the analytical separation consisted of 0.05 M ammonium citrate (pH  2.5) containing 10 per cent ACN. Preparative separation was performed with 0.4 M formic acid–methanol (6:4, v.v) as the mobile phase. Enantiomers wre measured in a chiral phase column ((R)-3,5-dinitrobenzoylphenylglycinepropylsilyl) (250  4.6 mm i.d.; particle size 5 m). Mixtures of 0.5 per cent formic acid and 2-propanol were employed for the separation of the various isomers [336]. The good separation capacity of the method is illustrated in Fig. 2.164. 2.5.3 Electrophoretic techniques Because of the advantageous characteristics (low capillary and operating costs, short analysis, small amount of waste products, etc.) CE methods have found application in the

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15.00 1,acetylated derivative 10.00 AU*10−3

II

I

5.00

III

0.00 30.00 35.00 Time (min)

30.00

Fig. 2.164. HPLC elution profile of the products arising by tyrosinase-catalysed oxidation of dimer 1, at 45 min of reaction. The acetylated derivative of 1 was identified by co-injection with an authentic sample. UV detection at 280 nm. See text for details. Reprinted with permission from A. Pezzella et al. [336].

O R

HO O

O

R

OH

OH

O

O O HO

O CHO

O

CHO

R = C5H11: xanthomonasin A (a)

C7H15: xanthomonasin B

R O O

O

O O

R = C5H11: rubropuntatin (b)

C7H15: monascorubrin

Fig. 2.165. Structures of Monascus yellow pigments (a) and Monascus red pigments (b). Reprinted with permission from T. Watanabe et al. [338].

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Without xanthomonasinA

Xanthomonasin A Trp-P-2(NHOH) Trp-P-2(NHOH) + xanthomonasin A 0

2

(a)

4 6 Migration time (min)

8

Trp-P-2(NHOH)

0

(b)

2

4 6 Migration time (min)

8

Fig. 2.166. Separation of xanthomonasin-A and remaining Trp-P-2(NHOH) in the short-contact mode (a) and in the long-contact mode (b). (a) In the short-contact mode, the MEKC conditions were as follows: capillary, 50 m i.d.  36 cm; running solution, 10 mM SC solution in 25 mM phosphate buffer at pH 7; applied voltage, 15 kV; temperature, 20°C; detection, 264 nm; concentration, 0.1mM xanthomonasin A and 0.1mM Trp-P-2(NHOH). (b) In the long-contact mode, the MEKC conditions were: running solution, 10 mM SC solution in 25 mM phosphate buffer containing 0.1mM xanthomonasin A at pH 7. The negative shift of the baseline (indicated by the arrow) shows that the zone of xanthomonasin-A passed the detection window. The other conditions are the same as those in the short-contact mode. Reprinted with permission from T. Watanabe et al. [338]

analysis of natural food pigments. The CE analysis of lac, cochineal, safflower, gardenia, monascus and elderberry pigments has been previously reviewed [337]. MEKC has been applied for the study of the effect of Monascus pigments on the decomposition of the mutagenic 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (TrpP-2(NHOH)). The chemical structures of yellow and red pigments are shown in Fig. 2.165. MEKC measurements were carried out in a capillary of 36 cm total length (effective length, 31.4cm; internal diameter, 50 m). Running separation solution was 10 mM sodium cholate (SC) in 25 mM sodium phosphate buffer (pH  7.0). Measurements were carried out at 9, 12, 15 and 18 kV, and the detection wavelength was 264 nm. The mutagenic agent Trp-P-2(NHOH) and the pigment were mixed and after various interaction times they were separated by MEKC. Typical eletropherograms are depicted in Fig. 2.166. The data proved that the degradation of the mutagenic agent was accelerated in the presence of Monascus pigment, indicating that the pigment catalyses the decomposition of 3hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole. The short analysis time (8 min) makes the method suitable for the study of similar molecular interactions [338].

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280 N. Chen, T.S. Bianchi, B.A. McKee and J.M. Bland, Historical trends of hypoxia on the Louisiana shelf: application of pigments as biomarkers. Org. Geochem. 32 (2001) 543–561. 281 K. Furuya, M. Hayashi, Y. Tabushita and A. Ishikawa, Phytoplankton dynamics in the East China Sea in spring and summer as revealed by HPLC-derived pigment signatures. Deep Sea Res. 11 (2003) 367–387. 282 C.K. Wong and C.K. Wong, HPLC pigment analysis of marine phytoplankton during a red tide occurrence in Tolo Habour, Hong Kong. Chemosphere 52 (2003) 1633–1640. 283 J.W. Louda, J.W. Loitz, D.T. Rudnick and E.W. Baker, Early diagenetic alteration of chlorophyll-a and bacteriochlorophyll-a in a contemporaneous marl ecosystem; Florida Bay. Org. Geochem. 31 (2000) 1561–1580. 284 M. Estrada, P. Henriksen, J.M. Gasol, E.O. Casamayor and C. Pedrós-Alió, Diversity of planktonic photoautotrophic microorganisms along a salinity gradient as depicted by microscopy, flow cytometry, pigment analysis and DNA-based methods. FEMS Microbiol. Ecol. 49 (2004) 281–293. 285 H. Claustre, S.B. Hooker, L. Van Heukelem, J.-F. Berthon, R. Barlow, J. Ras, H. Sessions, C. Targa, C.S. Thomas, D. van der Linde and J.-C. Marty, An intercomparison of HPLC phytoplankton pigment methods using in situ samples: application to remote sensing and database activities. Marine Chem. 85 (2004) 41–61. 286 L. Lampert, B. Quéguiner, T. Labasque, A. Pichon and N. Lebreton, Spatial variability of phytoplankton composition and biomass on the eastern continental shelf of the Bay of Biscay (north-east Atlantic Ocean). Evidence for a bloom of Emiliana huxleyi (Prymnesiophyceae) in spring 1998. Cont. Shelf Res. 22 (2002) 1225–1247. 287 Y. Obayashi, E. Tanoue, K. Suzuki, N. Handa, Y. Nojiri and C.S. Wong, Spatial and temporal variabilities of phytoplankton community structure in the Northern North Pacific as determined by phytoplankton pigments. Deep Sea Res. Part I: Oceanogr. Res. Papers 48 (2001) 439–469. 288 A. Metaxatos and L. Ignatiades, Seasonality of algal pigments in the sea water and interstitial water/sediment system of an Eastern Mediterranean coastal area. Est. Coast. Shelf Sci. 55 (2002) 415–426. 289 Y. Dandonneau, P.-Y. Deschamps, J.-M Nicolas, H. Loisel, J. Blanchot, Y. Montel, F. Thieuleux and G. Bécu, Seasonal and interannual variability of ocean color and composition of phytoplankton communities in the North Atlantic, equatorial Pacific and South Pacific. Deep Sea Res. II 51 (2004) 303–318. 290 C. Brunet and F. Lizon, Tidal and diel periodicities of size-fractionated phytoplankton pigment signatures at an offshore station in the southeastern English Channel. Est. Coast. Shelf Sci. 56 (2003) 833–843. 291 R.L. Airs, J.E. Atkinson and B.J. Keely, Development and application of a high resoltuion liquid chromatographic method for the analysis of complex pigment distributions. J. Chromatogr.A 917 (2001) 167–177. 292 R.L. Airs and B.J. Keely, A high resolution study of the chlorophyll and bacteriochlorophyll pigment distributions in a calcite/gypsum microbial mat. Org. Geochem. 34 (2003) 539–551. 293 H.M. Talbot, R.N. Head, R.P. Harris and J.R. Maxwell, Distribution and stability of steryl chlorin esters in copepod faecal pellets from diatom grazing. Org. Geochem. 30 (1999) 1163–1174. 294 H.M. Talbot, R.N. Head, R.P. Harris and J.R. Maxwell, Steryl esters of pyrophaephorbide b: a sedimentary sink for chlorophyll b. Org. Geochem. 30 (1999) 1403–1410. 295 E. Buffan-Dubau and K.R. Carman, Diel feeding behavior of meiofauna and their relationship with microalgal resources. Limnol. Oceanogr. 45 (2000) 381–395. 296 T.S. Bianchi, E. Engelhaupt, P. Westman, T. André, C. Rolff and R. Elmgren, Cyanobacterial blooms in the Baltic Sea: natural or human-induced? Limnol. Oceanogr. 45 (2000) 716–726.

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297 T.S. Bianchi, C. Rolff, B. Widbom and R. Elmgren, Phytoplankton pigments in Baltic Sea seston and sediments: seasonal variability, fluxes and transformations. Estuar. Coast. Shelf Sci. 55 (2002) 369–383. 298 F. Vidussi, J.-C. Mary and J. Chiavérini, Phytoplankton pigment variations during the transition from spring bloom to oligotrophy in the northwestern Mediterranean Sea. Deep Sea Res. 47 (2000) 423–445. 299 H.W. Higgins and D.J. Mackey, Algal class abundances, estimated from chlorophyll and carotenoid pigments, in the western Equatorial Pacific under El Niño and non-El Niño conditions. Deep Sea Res. 47 (2000) 1461–1483. 300 N.N. Rabalais, N. Attila, C. Normandeau and R.E. Turner, Ecosystem history of Mississipi River-influenced continental shelf revealed through preserved phytoplankton pigments. Marine Poll. Bull. 49 (2004) 537–547. 301 L. Almela, J.A. Fernández-López and M.J. Roca, High-performance liquid chromatographic screening of chlorophyll derivatives produced during fruit storage. J. Chromatogr.A 870 (2000) 483–489. 302 M. Adachi, K. Nakabayashi, R. Azuma, H. Kurata, Y.Takahashi and K. Shimokawa, The ethylene-induced chlorophyll catabolism of radish (Raphanus sativus L.) cotyledons: production of colorless fluorescence chlorophyll catabolite (FCC) in vitro. J. Japan Soc. Hort. Sci. 68 (1999) 1139–1145. 303 T. Bohn and T. Walczyk, Determination of chlorphyll in plant samples by liquid chromatography using zinc-phtalocyanine as an internal standard. J. Chromatogr.A 1024 (2004) 123–128. 304 B. Ganul-Rojas, L. Gallardo-Guerrero and M. I.minguez-Mosquera, Identification of oxidized chlorophylls and metallochlorophillic complexes of copper in table olives (cv. Gordal) with green staining alteration. J. Food Prot. 62 (1999) 1172–1177. 305 A. Cichelli and G.P. Pertesana, High-performance liquid chromatographic analysis of chlorophylls, pheophytins and carotenoids in virgin olive oils: chemometric approach to variety classification. J. Chromatogr.A 1046 (2004) 141–146. 306 B.H. Chen and M.H. Liu, Relationship between chlorophyll a and
-carotene in a lipidcontaining model system during illumination. Food Chem. 63 (1998) 207–213. 307 S. Sasaki, M. Omoda and H. Tamiaki, Effects of C8-substituents on spectroscopic and selfaggregation properties of synthetic bacteriochlorophyll-d analogues. J. Photochem.Photobiol. A: Chem. 162 (2004) 307–315. 308 L. Del Giovine and F. Fabietti, Copper chlorophyll in olive oils: identification and determination by LIF capillary electrophoresis. Food Contr. 16 (2005) 267–272. 309 M. Podgórna and P. Kus, Separation of tetraphenylporphyrin derivatives by adsorption and partition thin-layer chromatography. J. Planar Chromatogr. — Mod. TLC 13 (2000) 166–170. 310 A.M. Anderson, M.S. Mitchell and R.S. Mohan, Isolation of curcumin from turmeric. J. Chem. Educ. 77 (2000) 359–360. 311 T. Cserháti, E. Forgács, M. Candeias, L. Vilas-Boas, R.Bronze and I. Spranger, Separation and tentative identification of the main pigment fraction of raisins by thin-layer chromatography–Fourier transform infrared and high-performance liquid chromatography–ultraviolet detection. J. Chromatogr. Sci. 38 (2000) 145–150. 312 G. Csiktusnádi Kiss, E. Forgács, T. Cserháti and J.A.Vizcaino, Colour pigments of Tichoderma harzianum. Preliminary investigations with thin-layer chromatography–Fourier transform infrared spectroscopy and high-performance liquid chromatography with diode array and mass spectrometric detection. J. Chromatogr. A 896 (2000) 61–68. 313 T. Cserháti, E. Forgács, M.H. Morais and A.C. Ramos, TLC-FTIR of color pigments of chestnut sawdust. J. Liq. Chrom.& Rel. Technol. 23 (2001) 1435–1445.

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314 H. Sawada, M. Nakagoshi, K. Mase and T. Yamamoto, Occurrence of ommochrome-containg pigment granules in the central nervous system of the silkworm Bombyx mori. Comp. Biochem. Physiol. Part B 125 (2000) 421–428. 315 S.S. Teng and W. Weldheim, Analysis of anka pigments by liquid chromatography with diode array detection and tandem mass spectrometry. Chromatographia 47 (1998) 529–536. 316 K. Beattie, C. Elsworth, M. Gill, N.M. Milanovic, D. Prima-Putra and E. Raudies, Austrocolorints A1 and B1: atropisomeric 10,10
-linked dihydroanthracenones from an Australian Dermocybe sp. Phytochemistry 65 (2004) 1033–1038. 317 R. Bermejo, E.M. Talavera and J.M. Alvarez-Pez, Chromatographic purification and characterization of B-phycoerythrin of Porphyridium cruentum. Semipreparative high-performance liquid chromatographic separation and characterization of its subunits. J. Chromatogr.A 917 (2001) 135–145. 318 R. Bermejo Román, J.M. Alvárez-Pez, F.G.A. Fernández and E.M. Grima, Recovery of pure B-phycoeryhtrin from the microalga Porphyrium cruentum. J. Biotechnol. 93 (2002) 73–85. 319 G. Wang, Isolation and purification of phycoeryhtrin from red alga Gracilaria verrucosa by expanded-bed-adsorption and ion-exchange chromatography. Chromatogaphia 56 (2002) 509–513. 320 G.C.H. Derksen, H.A.G. Niederlander and T.A. van Beek, Analysis of anthraquinones in Rubia tinctorum L. by liquid chromatography coupled with diode-array UV and mass spectrometric detection. J. Chromatogr.A 978 (2002) 119–127. 321 R. Sakata, Studies on physicochemical characteristics of red pigments in meat products. Animal Sci. J. 71 (2000) 1–16. 322 G. Parolari, L. Gabba and G. Saccani, Extraction properties and absorption spectra of dry cured ham made with and without nitrate. Meat Sci. 64 (2003) 483–490. 323 J. Wakamatsu, T. Nishimura and A. Hattori, A Zn–porphyrin complex contributes to the bright red color in Parma ham. Meat Sci. 67 (2004) 95–100. 324 J.A. Fernández-López and L. Almela, Application of high-performance liquid chromatography to the characterization of the betalain pigments in prickly pear fruits. J. Chromatogr.A 913 (2001) 415–420. 325 J.A. Fernández-López, R. Castellar, J.M. Obán and L. Almela, screening and mass-spectral confirmation of betalains in cactus pears. Chromatographia 56 (2002) 591–595. 326 S.O. Mueller, M. Schmitt, W. Decant, H. Stopper, J. Schlatter, P. Schleier and W.K. Lutz, Occurrence of emodin, chrysophanol and physcion in vegetables, herbs and liquors. Genotoxicity and anti-genotoxicity of the anthraquinones and of the whole plants. Food Chem. Toxicol. 37 (1999) 481–491. 327 C. Raghukumar, C. Mohandass, S. Kamat and M.S. Shailaja, Simultaneous detoxification and decolorization of molasses spent wash by the immobilized white-rot fungus Flavodon flavus isolated from a marine habitat. Enz. Microbiol. Technol. 35 (2004) 197–202. 328 B. Szostek, J. Orska-Gawrys, I. Surowiec and M. Trojanowicz, Investigation of natural dyes occurring in historical Coptic textiles by high-performance liquid chromatography with UVVis and mass spectrometric detection. J. Chromatogr.A 1012 (2003) 179–192. 329 J. Orska-Gawris, I. Surowiec, J. Kehl, H. Rejniak, K. Urbaniak-Walczak and M. Trojanowicz, Identification of natural dyes in archeological Coptic textiles by liquid chromatography with diode aray detection. J. Chromatogr.A 989 (2003) 239–248. 330 M.P. Colombini, A. Carmignani, F. Modugno, F. Frezzato, A. Olchini, H. Brecoulaki, V. Vassilopoulo and P. Karkanas, Integrated analytical techniques for the study of ancient Greek polychromy. Talanta 63 (2004) 839–848. 331 C. Le Guernevé, P. Sanoner, J.-F. Drilleau and S. Guyot, New compounds obtained by enzymatic oxidation of phloridzin. Tetrahedron Lett. 45 (2004) 6673–6677.

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332 K. Nakai, S. Tazuma, T. Nishioka and K. Chayama, Inhibition of cholesterol crystallization under bilirubi deconjugation: partial characterization of mechanism whereby infected bile accelerates pigment stone formation. Biochem. Biophys. Acta 1632 (2003) 48–54. 333 J.O. Brower, D.A. Lightner and A.F. McDonagh, Aromatic congeners of bilirubin: synthesis, stereochemistry, glucuronidation and hepatic transport. Tetrahedron 57 (2001) 7813–7827. 334 M. Koyanagi, A. Terakita, K. Kubokawa and Y. Shichida, Amphioxus homologs of Go-coupled rhodopsin and peropsin having 11-cis- and all-trans-retinals as their chromophores. FEBS Lett. 531 (2002) 525–528. 335 H. Kaur, M.N. Hughes, C.J. Green, P. Naughton, R. Foresti and R. Motterlini, Interaction of bilirubin and biliverdin with reactive nitrogen species. FEBS Lett. 543 (2003) 113–119. 336 A. Pezzella, D. Vogna and G. Prota, Synthesis of optically active tetrameric intermediates by oxidation of the melanogenic procursor 5,6-dihydroxyindole-2-carboxilic acid under biomimetic conditions. Tetrahedron: Assymetry 14 (2003) 1133–1140. 337 T. Watanabe and S. Terabe, Analysis of natural food pigments by capillary electrophoresis. J. Chromatogr.A 880 (2000) 311–322. 338 T. Watanabe, T.K. Mazumder, A. Yamamoto, S. Nagai, S.Arimoto-Kobayashi, H. Hayatsu and S. Terabe, A simple and rapid method for analyzing the Monascus pigment-mediated degradation of mutagenic 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole by in-capillary micellar electrokinetic chromatography. Mutat. Res. 444 (1999) 75–83.

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Liquid chromatography of synthetic dyes

Liquid chromatography of synthetic dyes is less complicated and less problematic than the analysis of natural pigments. The number of individual natural pigment fractions in any matrix may be high, sometimes over 30–40 different molecules. Natural pigments in the same matrix can be structurally similar, which makes difficult their separation, or they can be structurally highly different necessitating gradient elution techniques which increases considerably analysis time because of the re-equilibration of the chromatographic system. Synthetic dyes are commonly applied alone or in combination with a very limited number of other dyes, facilitating their separation and quantitative determination. As the number of dyes in a sample is relatively low, spectrophotometric methods can also be successfully used for the determination of their concentration in various matrices. 3.1 APPLICATION OF SYNTHETIC DYES A huge number of dyes have been synthesized and used mainly for dying textiles. According to their chemical structures they are generally classified as azo, triphenylmethane (trityl), indigoid, phtalocyanine, anthracene and diazobenzene dyes. However, the structural characteristics of dyes sometimes overlap, uniting in the molecule more than one structural element, making impossible the unambiguous classification. Besides their use in the textile industry, various dyes have found application in a wide variety of other fields of up-to-date research and industrial activity. Synthetic dyes have been employed in various chromatographic procedures not only as analytes but also as additives to the analytical system. The addition of dyes has increased the selectivity of separation and the sensitivity of detection. The effect of dye–protein interaction has been mainly exploited in affinity chromatography of bioactive macromolecules. Thus,
,
’-azoisobutyronitrile, Procion Green H-4G and Procion Brown MX 5BR have been employed in the affinity membrane chromatography of lysozyme for the enhancement of separation efficacy. It has been established that dyes considerably improve the separation efficacy dependent on the chemical strucutre of the dye [1]. Another study has investigated the interaction of L-glutamate oxidase with triazine dyes (reactive blue 2, reactive brown 10, reactive red 2, reactive yellow) to select the optimal ligand for affinity chromatography. It was established that dyes allow the 335-fold purification of glutamate oxidase in a single step [2]. The interaction of Cibacron Blue F3GA, a triazine dye with hydroxypropyl--cyclodextrin, was studied in

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detail and the results were applied in the affinity chromatographic purification of L-lactase dehydrogenase. The enzyme was bonded to the dye in the affinity matrix and eluted with a mobile phase containing cyclodextrin. It was established that the technique allows 15-fold purification in one step with an 89 per cent recovery [3]. Procion red has been applied for the purification of alcohol dehydrogenase from bovine liver crude extract by dye–ligand affinity counter-current chromatography. In order to increase the efficacy of separation, 0.05 per cent of procion red was mixed in the two-phase solvent system. The measurement of enzyme activities and the results of SDS-PAGE proved that the enzyme can be purified in one step with counter-current chromatography exploiting dye–ligand interaction [4]. Cibacron Blue F3GA has been successfully employed for the purification of wheat flour high-Mr glutenin subunits by dye–ligand chromatography. Glutenin subunits were eluted from the immobilized dye ligand by SDS and the subunits were well separated from each other. The recovery of the method was high. It was supposed from the results that electrostatic interactions occur in the glutenin subunits and the dye molecule [5]. Fluorescent DNA-intercalating dyes were employed for the separation of DNA by ion-pair RP denaturing HPLC. It was found that the addition of the asymmetrical cyanine DNA-binding dye SYBR Green 1 neither modifies the separation capacity of the HPLC system nor influences retention time, it only increases the sensitivity of fluorescence detection [6]. Various synthetic dyes such as 6-carboxyfluorescein, hexafluorofluorescein, tetrachlorofluorescein, carboxytetramethyl-rhodamine and indodicarboxycyanine have been employed for the labelling of oligonucleotides, and the labelled products were separated and purified by RPHPLC-DAD, RP-HPLC-MS and capillary gel electrophoresis. It was established that the method allows the separation of oligonucleotides and makes possible the purity control of the analytes [7]. The fluorogenic dye (naphtalene-2,3-dicarboxaldehide) has been applied for the labelling of bioactive peptides and amino acids for electrochromatography in microchips. A glass chip containing channels filled with polymer monolith has been used for RP-electrochromatography. Polymer monoliths were formed in the channels using UV light-initiated polymerization. Laser-induced fluorescence detection was applied. It was stated that the separaton is extremely fast (six analytes in 45 s), the efficacy is high (600 000 plates/m) and better than that of the corresponding CE method [8]. Rhodamine B was employed as a model substrate for the investigation of the adsorption characteristics of sol–gel-derived silicate films using various physicochemical methods such as FTIR and single-molecule fluorescence spectra. The results demonstrated that the conditions of drying of the silica film markedly change its adsorption characteristics [9]. Rhodamine has also been employed for the labelling of lysozyme and the labelled lysozyme was used for the investigation of its adsorption to the polishing marks on a silica surface. The measurements demonstrated that lysozyme adsorbs to the solution marks of nanometer depths on the silica surface and the character of the adsortion changes with the ionic strength and pH of the eluting solution [10]. A new isoelectric buffer 2,2-bis(4-morpholinyl)propanoic acid was synthesized for CE and the anionic dye Orange G was employed as an indirect detection probe. It was found that the employment of the new buffer together with Orange G allows the detection of many inorganic and organic ions at the submicromole per litre level [11]. Coomassie Brillian Blue G was applied for the study of the interaction of surfactants with dye. The results of the theoretical study were employed for the development of a CE method for the separation and determination of anionic surfactants at the nanogram/ml level with a

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recovery of 98–102 per cent [12]. A synthetic dye was employed for the labelling of mitochondrial proteins in CE [13,14]. Near infrared dyes were employed for increasing the sensitivity of the detection of deoxynucleotide conjugates by CE [15]. The fluorescence dye NanoOrange has been used for the nanomolar quantitation of human serum albumin by CE [16]. Furthermore, dyes were employed as markers of the degree of citotoxicity of mining effluent to rainbow trout cell lines and to the ciliated protozoan, Tetrahymena thermophita. Alamar blue and carboxyfluorescein diacetate were employed for the measurements of metabolic activity and membrane integrity, respectively [17]. 3.2 TOXICOLOGY OF SYNTHETIC DYES The possible toxicity of dyes has been vigorously investigated [18]. Thus, it was established that the decomposition of azo dyes can produce carcinogenic aromatic amines [19]. It was further found that the microbial toxicity of reactive azo dyes also influences their biodegradation rate [20]. The growth inhibitory effect of 30 synthetic dyes was determined on 22 bacteria. The results demonstrated that both the chemical type of the dye and the biological characterstics of the bacteria exert a similar impact on the extent of toxicity [21]. The fungicidal effect of diazobenzene dyes has also been demonstrated [22]. It was further established that textile dye waste waters are toxic to the male reproductive system of albino rats and mice [23]. The toxicity of anthraquinone dyes during thermophilic and mesophilic anaerobic treatments has also been demonstrated [24]. The toxicity of acid dye effluent during ozone treatment was also established [25]. The change of toxicity of the intermediates of C.I. Direct Red 28 dye through sequential anaerobic and aerobic treatment was also monitored and a decrease in toxicity was found [26]. The bioaccumulation of HC Orange No.1 and the antioxidant response of goldfish Carassius auratus were also observed [27]. The possible mutagenic effect of dyes has also been investigated in detail. Thus, the mutagenicity of the commercial dye CI Disperse Blue 291 containing 2-[(2-bromo-4,6dinitrophenyl)azo]-5-(diethylamino)-4-acetanilide has been determined using the Salmonella assay. The results proved unambiguously the mutageneic effect of the dye [28]. Another study demonstrated that dye plant effluents show marked mutagenic effect [29]. The mutagenicity of the effluents from textile/dye industries of Sanganer, Jaipur (India) has been assessed [30]. Dyes and dye intermediates such as dichlorobenzidine congeners show not only mutagenic but also carcinogenic activity [31]. The mutagenecity, genotoxicity and cytotoxicity of dichlorobenzenes have also been demonstrated [32]. The toxic effect of metal waste leachate and dye waste leachate was monitored by using the Allium cepa chromosome aberrations assay. The inhibition of mitotic index, induction of chromosomal aberrarion, and micronulei formation were observed. It was further established that the toxicity of dye waste leachate was lower than that of metal waste leachate [33]. 3.3 ENVIRONMENTAL IMPACT OF SYNTHETIC DYES The significant amount of dyes released into the environment enhances markedly the environmental pollution. Various methods, both physical and microbiological, have been

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employed for the degradation or removal of dye pollutants for organic and inorganic matrices, such as ground and waste water, sludge, soil, etc. [34, 35]. 3.3.1 Physical and physicochemical methods used for the adsorption and degradation of dyes The adsorption of dyes on solid organic or inorganic supports can be used for the removal of dyes from waters but it does not solve the problem: the dyes remain intact and the need for degradation is remains [36]. A wide variety of physical and physicochemical methods and their combinations have been tested for their efficacy to decompose dye residues in waste-waters, textile and dye factory effluents, ground waters, etc. Physical methods include photoxidation in aqueous dispersion of H2O2/
-FeOOH [37], ozonation [38], iron powder reduction and photooxidation [39], and catalytic combustion on hematite [40]. The efficacy of ozonation combined with sonolysis for the decomposition of pararoseaniline (C.I. Basic Red 9) monohydrochloride has been investigated and the good performance of the system was demonstrated [41]. The photocatalytic decomposition of azo dyes reactive black 5 and reactive yellow 145 over a newly deposited TiO2 was also observed [42]. The degradation of dyes with various chemical structures on nanoporous polyoxotungstate anatase composite under visible-light irradiation was investigated. Dyes included in the experiments were azo derivatives (Congo Red, Methyl Orange, Ponceau G, Orange II, Eriochrome Blue Black), anthraquinone dyes (Alizarin S), heteropolyaromatic (Methylene Blue), fluorescent (Neutral Red, Rhodamine B), and sulphonic (Fuchsin Acid) compounds. Degradation products were detected and identified by ES-MS and ion chromatography. The results allowed the elucidation of the possible pathway of photodegradation [43]. The efficacy of the various oxidation systems used for the removal of dyes from dyebath effluent has been compared. The oxidation systems included in the investigation were O3, H2O2/UV-C and TiO2/UV-A procedures. It was found that both ozonation and H2O2/UV-C treatment can be successfully used for the degradation of dyes in the effluents [44]. A photocatalytic membrane reactor was applied for the removal of azo dye Acid Red 18 from water using titanium dioxide catalysator. It was established that the dye was decomposed completely in 5 h [45]. The monitoring of oxidative degradation of the reactive azo dye Reactive Orange 4 during the UV-H2O2 process was carried out. The kinetics of decolourization followed a pseudo-first order [46]. A similar UV-H2O2 process was applied for the study of the degradation of the anionic acidic dye Acid Orange 7. Also in this case a pseudo first-order kinetics was observed [47]. The photocatalytic oxidation of the dye Acid Blue 9 adsorbed on TiO2 was also followed [48]. A mechanism of photodegradation was proposed including the excited hot electrones and hydroxyl radicals generated on the surface of TiO2. Fenton and photo-Fenton oxidation of textile effluents was also investigated. It was found that the combination of Fenton, Fenton-like and photo-Fenton reactions can be applied for the effective degradation of the organic material in textile effluents [49]. The Fenton process has been employed for the removal of the reactive dye R94H from textile waste-water. The results demonstrated that the dye can be degraded by simultaneous addition of H2O2 and Fe2
to the waste-water [50]. An electrochemical method has also been developed and applied for the treatment of textile dye waste-water. The method applied Ti/Pt as the anode

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and stainless steel as the cathode. The results indicated that this procedure can be employed for the treatment of dye waste-water [51]. A different electrochemical methodology was employed for the treatment of waste-water containing Acid Orange II. The best conditions for colour removal were: ferrous coagulation (FeII/dye 0.5 mol/mol); 30 min electrolysis (cell voltage, 20.0V; air flow 0.1 m3/h) [52]. The efficacy of another electrolysis system using an activated carbon-fibre electrode for the degradation of 29 dyes was measured. It was established that the chemical structure of dyes exerts a considerable effect of their stability [53]. The photoelectrochemical degradation of naphtol blue black diazo dye on a WO3 film electrode was investigated. It was found that the electrode system promotes the rapid decomposition of the dye [54]. The capacity of TiO2 thin-film electrode for the photoelectrocatalytic degradation of the reactive dye Remazol Brilliant Orange 3R was determined. The data proved that the dye can be readily removed with this procedure [55]. TiO2 catalyst was further applied for the photo-assisted bleaching of 15 dyes of various chemical structures in visible light. The data suggested that the the decomposition of dyes considerably depended on their chemical structure, the quality of light and the crystal form of the catalyst [56]. The photocatalytic and photochemical decomposition of the azo dye Solophenyl Green BLUE under the effect of UV light and catalysators such as TiO2 and different activated carbons was monitored and the efficacy of the system was demonstrated [57]. Similar experiments were carried out to study the solar photocatalytic degradation of eight commercial dyes in TiO2 suspensions. It was established that each dye can be removed under experimental conditions but the degradation rate of dyes depends markedly on their chemical structure [58]. The effect of adsorption on the photodegradation of five commercial dyes was studied using surface bond-conjugated TiO2/SiO2 photocatalyst. The substructures of dyes responsible for their adsorption on the surface of a catalysator were tentatively identified as a penta-heterocycle N group and a sulphonate group. It was further found that the triazine ring and its substituted version are the most stable against irradiation [59]. Photocatalytic oxidation has also been applied to enhance the decomposition rate of the azo dye Procion Red MX-5B. It was concluded from the results that the decomposition rate increases with increasing concentration of titanium dioxide and hydrogen peroxide in the dye solution. Higher UV light intesity also enhanced the degradation rate [60]. The photochemical and photocatalytic decomposition of the indigoid dye acid blue 74 has been followed by various analytical methods, such as UV-VIS spectrophotometry, FTIR spectroscopy and 1H NMR. It was established that the decomposition was accelerated under the combined effect of UV and H2O2. The first decomposition product of the dye was identified as isatinsulphonic acid [61]. 3.3.2 Microbiological methods used for the degradation of dyes Microbiological methods have been frequently employed for the biodegradation of dyes [62,63]. These technologies apply various strains such as Pseudomonas species [64]. Escherichia coli has also been applied for the decolourization of azo dyes [65]. Both anaerobic and aerobic technologies have been employed for the decolourization of dyes [66,67]. The decolouration capacity of other micro-organisms such as Pleurotus ostreatus [68], Lentinula (Lentinus) edodes [69], Irpex lacteus [70], Sclerotium rolfsii [71], Funalia trogii ATCC 200800 [72], and Pseudomonas aeruginosa [73] has also recently been

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demonstrated. The separation, purification and identification of enzymes or enzyme systems responsible for the biodegradation of synthetic dyes has also been vigorously investigated [74–76]. Thus, the involvement of lignin peroxidase in the degradation of Remazol Brilliant Blue R was reported [77]. Not only the microbiological decomposition of the intact original dye molecules but also the further microbiological degradation of their intermediates was investigated. The decomposition capacity of both aerobic and anaerobic sludge systems was measured. It was found that the oxidation of the substituents on the aromatic ring or on the side chain of test compounds occurs under aerobic conditions. Anaerobic conditions result in the formation of substituted toxic amines [78]. 3.4 THIN-LAYER CHROMATOGRAPHY Because of their simplicity, easy-to-carry-out, low requirement of prepurification, and rapidity, TLC methods have been frequently applied in the analysis of synthetic dyes. However, the majority of invetigations were performed in model systems to establish the optimal separation conditions, and to determine various physicochemical parameters of dyes. The number of studies dealing with the analysis of dyes in complicated natural accompanying matrices is relatively low. 3.4.1 Application of model systems in TLC The separation of synthetic red pigments has been optimized for HPTLC separation. The structures of the pigments are listed in Table 3.1. Separations were carried out on silica HPTLC plates in presaturated chambers. Three initial mobile-phase systems were applied for the optimization: A  n-butanol–formic acid (100
1); B  ethyl acetate; C  THF–water (9
1). The optimal ratios of mobile phases were 5.0 A, 5.0 B and 9.0 for the prisma model and 5.0 A, 7.2 B and 10.3 C for the simplex model. The parameters of equations describing the linear and nonlinear dependence of the retention on the composition of the mobile phase are compiled in Table 3.2. It was concluded from the results that both the prisma model and the simplex method are suitable for the optimization of the separation of these red pigments. Multivariate regression analysis indicated that the components of the mobile phase interact with each other [79]. The hydrophobicity of some monoazoic dyes was determined by RP-TLC. The chemical structure of analytes are depicted in Fig. 3.1. RP-TLC plates were prepared by impregnating silica plates for 24 h in a hexane–paraffin oil (90:10, v/v) mixture. Mobile phases consisted of methanol–0.5 M HCl mixed in various volume ratios. Methanol concentration varied between 30–60 per cent in steps of 6 and 3 per cent. The RM value characterizing molecular hydrophobicity was calculated by RM  log(1RF 1)

(3.1)

and the RM values determined at various methanol concentrations were linearly extrapolated to 100 per cent water. The regression parameters of the equations are listed in Table 3.3. The results indicated that the hydrophobicity values determined by RP-TLC, water–octanol

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TABLE 3.1 THE STRUCTURES OF THE PIGMENTS HO

R1

R2 N R3

N R4

Name

R1

R2

R3

R4

R-202 R-203 R-405

COOH H COOH

SO3Ca/2 SO3Na SO3Ca/2

CH3 Cl CH3

H CH3 Cl

HO

R1

N

R2 N

Name

R1

R2

R-205 R-219 R-220

H COOCa/2 COOH

S03Na H SO3Ca/2

Reprinted with permission from K. Morita et al. [79].

partition and by theoretical calclations show considerable differences. It was supposed that this discrepancy may be due to the inherent different physicochemical processes used for the measurements [80]. The RP-TLC behaviour of some common food dyes was investigated in detail. The chemical structure of dyes are listed in Fig. 3.2. Measurements were carried out on RP-18 silica plates using aqueous ammonium sulphate (0.1; 0.5; 1.0 M), ethanol and acetone in various volume ratios. Developments were performed at room temperature (222oC) in chambers previously saturated with the vapours of the mobile phase. It was found that the presence of dissociable anorganic salt modifies markedly the RP retention behaviour of dyes. The retention of dyes generally decreases with increasing concentration of the organic modifier in the mobile phase. It was further concluded that RP-TLC can be successfully used for the separation of this class of synthetic food dyes [81].

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TABLE 3.2 REGRESSION EQUATIONS AND COEFFICIENTS FOR DETERMINATION OF THE hRF VALUES OF RED PIGMENTS. FIRST ORDER MODEL: hRF  b0
b1.x1
b2.x2 + b3.x3; SECOND ORDER MODEL WITH INTERACTION: hRF  b0
b1.x1
b2.x2
b3.x3
b11.x12
b22.x22
b33.x32
b12.x1.x2
b23.x2.x3
b31.x3.x1 R-202

R-203

R-205

R-219

R-220

R-405

41.294 5.431 -23.347 — 0.908

-6.196 34.040 -19.898 20.538 0.917

-12.661 36.615 -17.738 22.354 0.914

-9.581 13.193 -11.553 43.473 0.811

66.966 — -32.776 — 0.811

60.232 — -24.709 -7.082 0.877

44.843 — -16.567 — 12.346 — 6.389 — — — 0.928

60.144 — -27.255 -5.620 — 32.279 — — — — 0.893

46.360 — -12.388 — — — — — — — 0.824

20.059 — — -19.103 — — — — -25.700 49.871 0.898

First-order model b0 b1 b2 b3 R2

Second-Order Model with Interaction b0 b11 b22 b33 b12 b23 b31 b1 b2 b3 R2

46.723 — -11.206 -2.989 — — — — — — 0.912

25.706 5.226 -8.726 — — — 12.580 — — — 0.928

Reprinted with permission from K. Morita et al. [79].

The retention behaviour of mono- and ditetrazolium salts under various normal and reversed-phase TLC conditions and the correlation between retention characteristics and physicochemical parameters of solutes has been investigated in detail. The RF values of seven monotetrazolium and nine ditetrazolium salts were determined on alumina stationary phases and alumina stationary phase impregnated by overnight predevelopment in n-hexane–paraffin oil 95:5 (v/v). The chemical structures of di- and monotetrazolium salts are depicted in Fig. 3.3. Mobile phases for adsorption TLC were mixtures of n-hexane–1propanol in volume ratios between 40–65 per cent of 1-propanol in steps of 5 per cent. RP-TLC measurements were carried out with water–1-propanol mobile phases, the ratio of 1-propanol ranging 5–27.5 per cent. RM values and the correlation between the RM values and the concentration of the organic modifier (C) in the mobile phase was calculated as described above. The regression parameters are compiled in Table 3.4. The data illustrate that the dyes can be separated under both normal and reversed-phase conditions. Calculations suggested that the ring structure of dyes lies parallel to the surface of the stationary phase in both normal and RP-TLC and the polar substructure of analytes interacts with the adsorption centres of the support [82].

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377

(a) HO3S

HO OH

N=N

HO3S

I

OH

N=N

II HO

SO3H

OH

N=N

SO3H

IV

III

HO3S

OH

N=N

(b)

HO3S

COOH

V

HO3S

N=N

COOH

VII

N(CH3)2

VI

N(C2H5)2 HO3S

N=N

N=N

H N

N=N

VIII HO

HOOC

OH

N=N

HOOC

N=N

XII

XI

HO HOOC

OH

N=N

XIII

HOOC

HOOC

N=N

XIV

COOH

NH2 HOOC

N=N

N(CH3)2

N=N

XV

HOOC

COOH

XVI

N(C2H5)2 HOOC

N=N

N =N

XVIII

XVII

HOOC

NH2

N=N

H N

XIX

Fig. 3.1. Monoazoic derivatives with solubilizing (a) sulphonic acid group, and (b) carboxylic acid group. Reprinted with permission from E. Seclaman et al. [80].

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TABLE 3.3 REGRESSION PARAMETERS DETERMINED BY TLC EQUATIONS

Compound

RMO

b

n

r

r2

F

8 8 8 8 8 8 8 8 8 8 8 6 8 8 8 8 6 8 8

0.946 0.908 0.935 0.925 0.879 0.890 0.904 0.985 0.89 0.91 0.87 0.99 0.83 0.91 0.96 0.94 0.97 0.95 0.98

0.865 0.825 0.875 0.856 0.774 0.792 0.818 0.970 0.80 0.83 0.79 0.99 0.77 0.83 0.91 0.87 0.95 0.88 0.97

51.52 28.37 42.37 35.72 20.58 22.92 27.08 199.77 23.35 30.57 19.44 226.20 13.30 28.75 67.56 46.33 90.51 54.12 255.18

RMRMO
b(% methanol) I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII XIX

0.12 -0.19 0.89 1.56 -0.06 1.02 2.16 2.71 0.64 0.26 0.18 2.79 -0.21 1.23 0.63 2.70 3.29 1.91 3.20

-0.019 -0.014 -0.028 -0.038 -0.021 -0.031 -0.044 -0.054 -0.022 -0.015 -0.017 -0.046 -0.009 -0.035 -0.025 -0.018 -0.053 -0.040 -0.041

n  number of the experimental determination for each compound. r  regression coefficient. F  fisher test. Reprinted with permission from E. Seclaman et al. [80].

Similar investigations were carried out using 2-propanol as the organic modifier instead of 1-propanol. The parameters of linear regression analyses are compiled in Table 3.5. It has been concluded from the results that the retention on both stationary phases is of mixed character; hydrophilic, electrostatic and steric molecular parameters are equally involved in the retention [83]. The same RP-TLC investigations were carried out by using ethanol, THF and dioxane as organic modifiers. The parameters of regression analyses are compiled in Table 3.6. It was established that the type of organic modifier exerts a negligible effect on the lipophilicity but influences considerably the specific hydrophobic surface area of the dyes. The role of hydrogen-donor and -acceptor capacity of the polar molecular subtructures in the retention was demonstrated [84]. The strength and selectivity of the organic modifiers mentioned above have beeen calculated by the spectral mapping technique. The potency values of organic modifiers (related to the elution strength) are depicted in Fig. 3.4. The positions of the organic modifiers in Fig. 3.4. illustrate that, except for THF,

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379 SO3Na

HO

HO

NaO3S

N

N

N

N NaO 3S

N

N CO2Na SO3Na

Tartrazine (E-102; C.I. 19140)

Sunset Yellow FCF (E-110; C.I. 15985)

O

O NaO3S

O

O

NaO3S Quinoline Yellow FCF (E-104; C.I. 47005)

N

NaO3S

NaO3S

HO

NaO3S

HO N

N

N

NaO3S

NaO3S NaO3S

NaO3S Ponceau 6R (E-126; C.I. 16290)

Amaranth (E-123; C.I. 16185)

O

O

O

NaO

O SO3Na

NaO3S I

I

N

CO2Na

H

N H

Indigo Carmine (E-132; C.I. 73015)

Erithrosine (E-127; C.I. 45430)

NaO3S

SO3 Na N

OH

NHAc

N

NaO3S SO3-

N=N

N=N SO3Na SO3Na

SO3Na

Brilliant Blue FCF (E-133; C.I. 42090) Brilliant Black BN (E-151; C.I. 28440)

Fig. 3.2. The structures of the dyes investigated. Reprinted with permission from D. M. MilojkovicOpsenica et al. [81].

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Menotetrazolium salts R1

N

N

R3

R2

R3

No

R1

1

-H -H

-H -H

-H

2 3

-H

-H

-NO2

4

-H

-H

-NO2

5

-H

-H

-H

6

-H

-H

R2

R4

-I

NO2

ClN

N +

R4

-H

General structure

CH3

7

R1-O-CH2-O-R2

-H

I

Ditetrazolium salts

N

N

R3

R4

N

N

N

N

R7

R1 N

N +

R2

R1

R2

R8

R6 General structure

R5

No

R3

R4

R5

R6

R7

R8

1

-OCH3

-OCH3

-H

-H

-OCH3

-OCH3

-OCH3

-OCH3

2

-OCH3

-H

-NO2

-NO2

-OCH3

-OCH3

-OCH3

-H

3

-H

-NO2

-H

-H

-H

-H

-H

-NO2

4

-H

-H

-H

-H

-CH3

-CH3

-H

-H

5

-H

-H

-NO2

-NO2

-OCH3

-OCH3

-H

-H

6

-H

-H

-H

-H

-H

-H

-H

-H

7

-H

-H

-H

-H

-OCH3

-OCH3

-H

-H

8

R1-O-CH2 -O-R2

-H

-H

-OCH3

-OCH3

R7-O-CH2 -O-R8

9

-H

-H

-H

-OCH3

-OCH3

-H

-NO2

2 Cl-

-NO2

Fig. 3.3. Chemical structure of tetrazolium salts. Reprinted with permission from T. Cserháti et al. [82].

their elution strength does not differ significantly, therefore, its application in the RP-TLC of tetrazolium salts was proposed. The selectivity map of organic modifiers is shown in Fig. 3.5. The distribution of the points representing organic modifiers indicates that not only the elution strength but also the selectivity of THF deviates considerably from that of other modifiers [85].

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TABLE 3.4 PARAMETERS OF LINEAR CORRELATION BETWEEN THE RM VALUE OF TERAZOLIUM SALTS AND THE 1-PROPANOL CONCENTRATION (C, VOL.%) IN THE ELUENT. RM  RM0
B.C

Parameter

1

2

3

4

5

6

7

8

9

3.02 3.29 4.04 0.9712

2.04 2.90 0.58 0.9998

2.08 3.85 5.06 0.9832

2.09 4.29 7.72 0.9152

No. of monotetrazolium salts RMO -102 x ba 103 x sbb rc

1.31 2.02 1.98 0.9860

0.89 1.19 2.65 0.9138

1.34 1.71 3.32 0.9325

1.12 1.48 2.21 0.9580

1.25 1.74 3.19 0.9387

1.39 1.93 4.27 0.9144

1.79 2.90 1.73 0.9982

2.06 3.20 1.15 0.9993

2.24 3.70 1.73 0.9989

1.43 2.06 3.97 0.9484

1.45 1.99 1.69 0.9894

2.10 2.17 3.71 0.9463

2.72 3.60 1.41 0.9999

1.66 0.69 2.14 0.8213

0.59 0.48 1.07 0.9338

1.06 1.60 3.07 0.9055

1.18 3.50 8.09 0.9504

0.55 2.01 4.75 0.8480

1.12 1.82 2.02 0.9879

2.26 2.35 5.74 4.53 11.41 10.23 0.9626 0.8924

1.43 1.64 5.66 0.7630

1.88 3.26 3.84 0.9604

1.97 3.55 2.42 0.9951

1.13 1.28 2.11 0.9382

1.94 4.37 1.07 0.9997

No. of ditetrazolium salts RMO -102 x ba 103 x sbb rc

2.89 2.95 5.30 0.9279

No. of monotetrazolium salts RMO -102 x ba 103 x sbb rc

0.33 1.67 6.24 0.7375

No. of ditetrazolium salts RMO -102 x ba 103 x sbb rc a

Decrease in the RM value caused by 1 per cent increase in 1-propanol concentration in the mobile phase. Standard deviation of b. c Coefficient of correlation. Reprinted with permission from T. Cserháti et al. [82]. b

The lipophilicity and specific hydrophobic surface area of 42 synthetic dyes were also measured on alumina-based RP-TLC layers as described above. The common and IUPAC names of the non-homologue series of synthetic dyes are compiled in Table 3.7. Methanol and water mixtures were employed as mobile phases, the methanol concentration varying between 20–95 per cent in steps of 5 per cent. Linear correlations were calculated separately for each synthetic dye between the methanol concentration in the mobile phase and the RM values. The regression parameters are compiled in Table 3.8. It was found that the regression paramters of synthetic dyes show high differences,

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TABLE 3.5 PARAMETERS OF LINEAR CORRELATION BETWEEN THE RM VALUE OF TERAZOLIUM SALTS AND THE 2-PROPANOL CONCENTRATION (C, VOL.%) IN THE ELUENT. RM  RM0
B.C

Normal phase

No. of monotetrazolium salts

TLC Parameter 1 RMO -102 x ba 103 x sbb rc

2.11 2.78 4.90 0.9563

2

3

4

5

6

7

8

9

1.81 2.30 5.55 0.9227

2.32 2.79 4.26 0.9669

1.92 2.28 4.87 0.9377

2.19 2.73 2.88 0.9837

2.32 2.90 3.41 0.9798

2.31 2.80 3.92 0.9717

2.71 3.41 8.43 0.9191

2.64 3.35 8.36 0.9178

2.50 3.35 8.61 0.9137

2.32 2.92 6.51 0.9330

1.85 0.76 2.81 0.7708

3.28 3.11 9.90 0.9995

1.76 4.45 1.58 0.7551

3.06 3.87 8.99 0.9267

8

9

No. of ditetrazolium salts RMO -102 x ba 103 x sbb rc

2.02 6.66 2.01 0.8037

Reversed-phase

No. of monotetrazolium salts

TLC Parameter 1 RMO -102 x ba 103 x sbb rc

0.46 2.29 0.99 0.9991

2

3

4

5

6

7

1.07 1.26 4.95 0.9992

0.85 2.23 1.48 0.9978

1.19 2.42 6.94 0.8678

1.08 2.76 8.92 0.8102

0.56 2.53 4.57 0.9687

1.12 2.00 6.31 0.8455

0.94 5.20 0.01 0.9999

1.49 1.74 5.43 0.8483

1.94 3.57 6.85 0.9336

1.89 3.06 6.86 0.9126

1.20 2.10 5.52 0.8856

1.87 4.01 8.47 0.9212

No. of ditetrazolium salts RMO -102 x ba 103 x sbb rc a

2.46 6.99 1.55 0.9993

2.03 2.21 4.00 4.23 2.31 12.13 0.9983 0.8955

Decrease in the RM value caused by 1 per cent increase in 2-propanol concentration in the mobile phase. b Standard deviation of b. c Coefficient of correlation. Reprinted with permission from T. Cserháti et al. [83].

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TABLE 3.6 PARAMETERS OF LINEAR CORRELATIONS BETWEEN THE RM VALUE OF TETRAZOLIUM SALTS AND THE ETHANOL, TETRAHYDROFURAN AND DIOXANE CONCENTRATION (C VOL.%) IN THE ELUENT

Ethanol organic modifier Parameter

RMO 102  ba 103  sbb rc

No. of monotetrazolium salts

1

2

3

4

5

6

7

0.43 2.10 3.06 0.9696

0.95 1.20 0.98 0.9902

0.56 1.38 0.72 0.9959

1.13 2.34 0.50 0.9993

1.18 2.90 2.52 0.9889

0.79 2.76 2.80 0.9849

1.13 2.16 1.13 0.9959

No. of ditetrazolium salts Parameter

RMO 102  ba 103  sbb rc

9

No. of ditetrazolim salts

1

2

3

4

5

6

7

8

9

2.25 4.76 1.80 0.9978

2.22 3.06 5.95 0.9476

1.40 1.88 1.65 0.9886

1.68 2.02 1.19 0.9948

1.70 2.38 2.27 0.9866

1.22 2.42 3.55 0.9692

1.66 2.54 2.478 0.9825

1.86 2.50 3.49 0.9720

1.85 2.36 6.60 0.9001

8

9

8

9

Tetrahydrofuran organic modifier Parameter 1

2

No. of monotetrazolium salts 3

4

5

6

0.82 1.45 1.03 1.52 1.43 1.06 RMO 102  ba 12.14 15.49 13.94 16.00 14.74 13.69 103  sbb 6.43 7.92 4.45 0.01 3.96 2.47 rc 0.9986 0.9987 0.9995 0.9999 0.9996 0.9998 Parameter

RMO 102  ba 103  sbb rc

8

7 0.99 2.17 3.42 0.9427

No. of ditetrazolium salts 1

2

3

4

5

6

7

1.68 4.04 9.89 0.9209

2.22 1.93 2.26 2.31 1.64 2.31 5.12 16.77 19.09 21.14 16.49 20.37 4.37 8.41 14.85 3.96 9.40 15.34 0.9858 0.9987 0.9970 0.9998 0.9984 0.9972

1.74 1.80 3.92 3.88 9.32 10.95 0.9032 0.8706

(Continued on next page)

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TABLE 3.6 (continued) Dioxane organic modifier Parameter

RMO 102  ba 103  sbb rc

No. of monotetrazolium salts

1

2

3

4

5

6

7

0.58 3.02 3.59 0.9478

1.12 2.52 4.18 0.9736

0.81 2.28 1.75 0.9941

1.32 3.48 7.37 0.9580

1.29 4.44 7.68 0.9579

0.93 3.76 9.51 0.9160

1.34 3.76 8.08 0.9372

Parameter

8

9

8

9

2.21 3.70 4.85 0.9448

2.16 3.79 5.55 0.9411

No. of ditetrazolium salts 1

2.36 RMO 102  ba 4.98 103  sbb 12.02 rc 0.8610

2

3

2.20 1.93 3.39 5.64 9.19 14.51 0.8549 0.9134

4

5

6

7

1.85 3.03 4.23 0.9382

1.91 3.17 1.48 0.9989

1.37 2.27 3.32 7.08 7.79 10.54 0.9491 0.9683

a

Decrease in the RM value caused by 1 per cent increase in dioxane concentration in the eluent. Standard deviation of b. c Coefficient of regression. Reprinted with permission from T. Cserháti et al. [84]. b

suggesting that the RP-alumina stationary phase can be applied for the separation of this class of analytes. Quantitative structure-retention calculations indicated that the polar surface area of dyes correlate negatively with the hydrophobicity parameters [86]. The lipophilicity and specific surface area of a similar set of synthetic dyes was also determined on an alumina-based RP-TLC stationary phase and the linear relationship between the two hydrophobicity parameters was calculated. The result of the calculation is depicted in Fig. 3.6. The good correlation between these physicochemical parameters indicated that from the chromatographic point of view these compounds behave as a homologous series of analytes, however, their chemical structures are markedly different [87]. TLC coupled with mass spectrometry employing desorption electrospray ionization has been used for the separation of synthetic dyes. The chemical structures of dyes included in the investigation are shown in Fig. 3.7. ODS HPTLC plates (10  10 cm) were used as the stationary phase; the mobile phase consisted of methanol-tetrahydrofuran (60:40, v/v) containing 50–100 mM ammonium acetate for the positive-ion test and of methanol–water (70:30, v/v) for the negative-ion test. Test mixtures for negative- and positive-ion mode detection consisted of methyleneblue, crystal violet, rhodamine 6G

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385

Tetrahydrofuran

Potency

2-Propanol (Reversed)

2-Propanol (Adsorption)

1-Propanol (Reversed)

1-Propanol (Adsorption)

Ethanol

Dioxane

32

7

Fig. 3.4. Potency values (related to the elution strength)2 standard deviations of solvents, simultaneously taking into consideration each tetrazolium salt. Reprinted with permission from E. Forgács et al. [85].

and fluorescein, naphtol blue black, and fast green FCF, respectively. After development, the plates were dried at room temperature or at 110°C for 30 min. The schematic illustration of the ES-MS detection system is depicted in Fig. 3.8. A typical ion chromatogram illustrating the efficacy of the ES-MS detction system is depicted in Fig. 3.9. The results demonstrated that the method is suitable for the evaluation of traditional TLC plates in both the negative- and positive-ion modes. The limit of detection was in the nanogram range, proving the applicability of the technique for residue analysis of environmental pollutants [88]. A similar TLC-ES-MS method was employed for the analysis of the chemical structures, and mass-to-charge ratios observed of the analytes are shown in Fig. 3.10. Rhodamine dyes were used as test compounds for the detection of the positive-ion mode while FD&C dyes were detected in the negative-ion mode. Stationary phases for

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Chapter 3

F1 190

1-Propanol (Adsorption)

Dioxane Tetrahydrofuran

F2

100 50

180

Fig. 3.5. Similarities and dissimilarities between the selectivity of solvents, simultaneously taking into consideration each tetrazolium salt. Two-dimensional nonlinear selectivity map. Number of iterations, 547; maximal error, 1.42  102. Reprinted with permission from E. Forgács et al. [85].

the separation of rhodamines were C8 and C2 hydrophobic supports, and the mobile phase consisted of methanol–water (80:20, v/v) containing 200 mM ammonium acetate. FD&C dyes were separated on an ODS plate in water–ACN (70:30, v/v). Characteristic chromatographic profiles illustrating the detection capacity of the system are listed in Fig. 3.11. It was supposed that the technique can be employed for the detection of a wide variety of analytes separated by TLC on different stationary phases [89]. 3.4.2 TLC determination of dyes in various matrices Because of the relatively low sensitivity of TLC methods they have not been frequently applied for the analysis of samples containing trace amounts of synthetic dyes. However, TLC techniques have been proven to be a rapid, reliable and valuable tool for the easy following of synthetic procedures for the preparation of new dyes. Thus, the synthesis of some bifunctional reactive triazine dyes has been monitored by TLC. The synthetic pathways a and b are shown in Fig. 3.12. The purity of intermediates was controlled by TLC using a silica stationary phase (RF value of 2-allylamino-4,6-chloro-1,3,5-triazine  0.48

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TABLE 3.7 COMMON AND IUPAC NAMES OF SYNTHETIC DYES 1

Acridine O

N,N,N,N-Tetramethyl-3,6-acridinediamine mono-hydrochloride

2

Amidoblack

4-Amino-5-hydroxy-3-[4-nitrophenyl)azo]-6-(phenylazo)2,7-naphtalenedisulphonic acid disodium salt

3

Azobenzene

1,2-Diphenyldiazene

4

Bengal Rose

3,4,5,7-Tetrachloro-3,6-dihydroxy-spiro[isobenzofuran-1(3H), 9-[9H]xanthen]-3-one] disodium salt

5

Brilliant Green

N-[4-[[-(Diethylamino)phenyl]phenylmethylethylene]2,5-cyclohexadien-1-ylidene]-N-ethylethanaminium sulphate

6

Bromthymol Blue 4,4-(3H-2,1-Benzoxathiol-3-ylidene)bis[2-bromo-3-methyl-6(1-methylethyl) phenol]S,S-dioxide

7

Carminic Acid

7-á-D-Glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydoxy-1-methyl9,10-dioxo-2-anthracene-carboxylic acid

8

Congo Red

3,3-[[1,1-biphenyl]-4,4-diylbis-(azo)]bis[4-amino-1-naphtalene -sulphonic acid] disodium salt

9

Coumassie R250

N-[4-[[4-[(4-ethoxyphenyl)amino]phenyl][4-[ethyl[(3-sulphophenyl) methyl]amino]phenyl]methylene]-2,5-cyclohexadien-1-ylidene]N-ethyl-3-sulphobenzenemethan-aminium monosodium salt

10

Coumassie R G250

N-[4-[[4-[(4-ethoxyphenyl)amino]phenyl[4-[ethyl[(3-sulphophenyl) methyl]amino]2-methylphenyl]methylene]-3-methyl-2,5-cyclohexadien1-ylidene]-N-ethyl-3-sulphobenzen emethanaminium monosodium salt

11

Crystal Violet, Gentian Violet

N-[4-[Bis[4-dimethylamino)-phenyl]methylene]-2,5-cyclohexadien1-ylidine]-N-methyl-methan-aminium chloride

12

Eosin Yellow

2,4,5,7-Tetrabromo3,6ihydroxyspiro[isobenzofuran 1(3H), 9-[9H]xanthen-3-one disodium salt

13

Evan’s Blue

6,6’-[3,3-Dimethyl[1,1-biphenyl]-4,4-diyl)bis(azo)bis[4-amino5hydroxy-1,3-naphtalelenedisulphonicacid]-tetrasodium salt

14

Hematoxylin

7,11b-Dihydroxybenz[b]indeno[1,2-d]pyran-3,4,6a,9,10 (6H)pentol

15

Janus Green B

3-(Diethylamino)-7-[[4-dimethylamino)phenyl[azo]-5-phenylphenazinium chloride

16

Litmus

Natural dye mixture

17

Malachit Green

N-[4-[[4-(Dimethylamino)phenyl]phenyl-methylene]-2,5-cyclo-hexadien -1-ylident]-N-methyl-methanaminium chloride

18

Methylene Blue

2,2-Methylenebis[3,4,5-trihydroxy-benzoic acid]

19

Methyl Green

4-[[4-(Dimethylamino)phenyl][4-(dime-thylimino)-2,5-cyclo-hexadien1-ylidene]methyl]-N-ethyl-N,N-dimethylbenzeneami-nium bromide chloride (Continued on next page)

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Chapter 3

TABLE 3.7 (continued)

20

Methyl Violet

N-[4-[Bis[4-dimethylamino)-phenyl]methylene]-2,5-cyclohexadien1-ylidine]-methanaminium chloride

21

Neutral Red

N8N8,3-Trimethyl-2,8-phenazinediamine monohydro chloride

22

Nile Blue

5-Amino-4-(diethylamino)benzo[a]phen-azoxonium hydrogen sulphate

23

Orange GS

4-[[4-(phenylamino)phenyl]azo]-benzene-sulphonic acid monosodium salt

24

Orcein

Oxidation product of orcein

25

p-Methoxy-azo-benzene

26

p-Dimethylamino azobenzene - N,N-Dimethyl-4-(phenylazo)benzenamine

27

Pararosaniline

4-((4-Aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene) mehtyl) benzenamine monohydrochloride

28

Yellow AB

1-Phenylazo-2-naphtalenamine

29

Phloxim B

2’,4’,5’,7’-Tetrabromo-4,5,6,7-tetra-chloro-3’,6’-dihydroxy-spiro [isobenzofuran-1] (3H),9’-[9H]xanthen-3-one-sodium salt

30

Pyronin G Pyronin Y

N-[6-(Dimethylamino)-3H-xanthene-3-ylidene- dene]-N-methylmethanaminium chloride

31

Rubin C

2-Amino-5-[(4-amino-3-sulphophenyl)(4-imino-3-sulpho-2,5cyclohexadien-1-ylidene)acid disodium salt

32

Safranin O

3,7-Diamino-2,8-dimethyl-phenylphenazinium chloride

33

Sudan Black B

2,3-Dihydro-2,2-dimethyl-6-[[4-(phenyl)azo-1H-perimidine

34

Sudan III

1-[[4-(Phenylazo)phenyl]azo]-2-naphthalenol

35

Sudan IV

1-[[2-Methyl-4-[(2-methylphenyl)-azo]phenyl]azo-2-naphtalenol

36

Sudan Red/ Scarlet Red

2,3-Dihydro-2,2-dimethyl-6-[[4-(phenyl-azo)-1-naphtyl] azo]-1H-perimidine

37

Thionine

3,7-Diaminophenothiazin-5-ium chloride

38

Trypan Blue

3,3’-[(3,3’-Dimethyl[1,1’-biphenyl]-4,4’-diyl)bis(azo)]bis[5-amino4-hydroxy-2,7-naphtalenedisulphonicacid]tetrasodium salt

39

Trypan Red

4,4’-[(3-Sulpho[1,1’-biphenyl]-4,4’-diyl) bis(azo)[3-amino-2,7naphtalenedisulphonic acid] pentasodium salt

40

Rhodamin B

N-[9-(2-Carboxyphenyl)-6-diethylamino)-3H-xanthen-3-ylidene]N-ethylethanaminium chloride

41

2,6-dichloroindo phenol sodium

2,6-Dichloro-4-[(4-hydroxyphenyl)imino] phenol -2,5-cyclohexadien1-one sodium

42

Methyl Red

2-[[4-(Dimethylamnio)phenyl]azo]-benzoic acid

Reprinted with permission from T. Cserháti et al. [86].

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TABLE 3.8 PARAMETERS OF THE LINEAR RELATIONSHIPS BETWEEN THE RM0 VALUES OF SYNTHETIC DYES AND THE CONCENTATION OF METHANOL IN THE ELUENT (C VOL.%). RM  RM0
B.C Dye number

Common name

RM0

b  102

Rcalc

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Acridine O Amidoblack Azobenzene Bengal Rose Brilliant Green Bromthymol Blue Carminic Acid Congo Red Coumassie G Red Coumassie R Crystal Violet Eosin Yellow Evan’s Blue Hematoxylin Janus Green B Litmus Malachite Green Methylene Blue Methyl Green Methyl Violet Neutral Red Nile Blue Orange GS Orcein p-Methoxy-azo-benzene p-Dimethylaminoazobenzene Pararosaniline Yellow AB Phloxim B Pyronin G Rubin C Safranin O Sudan Black B Sudan III Sudan IV Sudan Red Thionine Trypan Blue Trypan Red Trypan Red 2,6-dichloroindophenol Sodium Methyl Red

2.78 4.08 3.68 4.90 4.92 4.67

5.15 4.31 4.61 5.85 7.66 6.36

0.9970 0.9901 0.9959 0.9926 0.9947 0.9994

4.12 5.39 6.00 3.19 3.35

5.11 7.06 7.94 5.20 3.72

0.9898 0.9985 0.9901 0.9938 0.9934

4.22 3.70 3.93 2.12 4.33 3.34 2.53 3.76 2.50 3.85 3.76 3.53 2.17 3.93 5.13 2.68 -2.90 2.81 6.12 5.40 5.58 5.74 1.85

6.58 5.01 5.43 5.22 6.78 5.38 4.59 5.68 5.10 5.71 4.75 4.46 4.38 5.10 5.93 4.64 -5.66 5.27 6.52 5.59 5.39 5.57 3.66

0.9963 0.9981 0.9990 0.9944 0.9926 0.9914 0.9828 0.9980 0.9973 0.9981 0.9995 0.9979 0.9968 0.9992 0.9943 0.9952 0.9893 0.9955 0.9944 0.9966 0.9983 0.9967 0.9782

4.29 1.80 2.54 -2.78

6.12 3.53 3.28 -6.12

0.9985 0.9681 0.9949 0.9703

Reprinted with permission from T. Cserháti et al. [86]

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RMO 12

RMO = 0.19 + (0.76 ± 0.04) . b r = 0.9567

-4 -6

15 b

Fig. 3.6. Relationship between the lipophilicity (RM0) and specific hydrophobic surface area (b) of synthetic dyes (n  41). Reprinted with permission from T. Cserháti et al. [87].

in n-heptane–diethylether, 1:1, v/v; RF value of 2-allyoxy-4,6-dichloro-1,3,5-triazine  0.68 in n-heptane–diethylether, 3:1, v/v). The retention values of the new synthetic dyes a, b and c were: a, RF  0.39, mobile phase n-propanol–ammonia 3:1, v/v; b, RF  0.77, mobile phase n-propanol–ammonia 1:1, v/v; c, RF  0.82, mobile phase n-propanol–ammonia 1:1, v/v. The data proved that normal-phase TLC carried out on a silica stationary phase can be used for the control of synthesis of new triazine-based dyes [90]. The synthesis of disperse dyes derived from 1-indanylidenemalononitrile has been performed and the purity of products was checked by TLC. The synthesis scheme is shown in Fig. 3.13. The substituents of the new disperse dyes are listed in Table 3.9. The study proves again that TLC can be applied for the rapid control of synthetic pathways and the purity of compounds newly synthetized [91]. TLC has been applied for the purity control of the newly synthetized o,o-dihydroxyazo dyes and their chromium complexes. The structures of 7-hydroxy-o,o-dihydroxyazo dyes and their chromium complexes are listed in Fig. 3.14. TLC purity check of o,o-dihydroxyazo dyes and their chromium complexes was performed on silica layers using 5 per cent water/ethanol and 5 per cent water–dimethylsulphoxide as the mobile phase, respectively. The formula and RF values of 7-hydroxy-o,o-dihydroxyazo dyes and their chromium complexes are compiled in Table 3.10. The retention values indicated that the TLC technique applied is suitable for the purity control of the these new dye compounds [92]. Food pigments, sweeteners and a preservatives were quantitatively determined by TLC in sparkling and non-sparkling drinks. TLC separations were carried out on ready-made silica plates using 2-propanol–12.5 per cent aqueous ammonia (10:2, v/v) and ethanol–2propanol–12.5 per cent aqueous ammonia (10:40:1, v.v) as mobile phases for the separation of pigments and sweeteners and preservatives, respectively. Samples were used as

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O OH

N (CH3)2N

N(CH3)2

S

O

O

HO methylene blue (M)+ = m/z 284

fluorescein (M - H)- = m/z 331 HO

N(CH3)2

O

O S O

NN

O H

S HO (CH3)2N

naphtol blue black (M - 2H)2- = m/z 285

N(CH3)2 crystal violet (M)+ = m/z 372

O NN

H2N

O2N O

OR1 R4 CH3CH2N R2

O H

S

O

O

CH3CH2NHCH2

R5 NCH2CH3 R3

rhodamine 6G R1 = CH2CH3; R2, R3 = H; R4, R5 = CH3 (M)+ = m/z 443

rhodamine B R1, R4, R5 = H; R2, R3 = CH2CH3 (M)+ = m/z 443

O HO

S

OH

NCH2CH3

O fast green FCF {(M)+ - 3H)}2+= m/z 381

O

S

O

OH

Fig. 3.7. Dyes used in these experiments and the mass-to-charge ratio of the major ion from each that was observed or monitored. Reprinted with permission from G. J. Van Berkel et al. [88].

received for the measurements of pigments, and they were concentrated 20-fold for the separation of sweeteners and preservatives. The chromatographic parameters of pigments are listed in Table 3.11. The food pigments detected and quantified in beverages are compiled in Table 3.12.

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Chapter 3 TLC plate cameras

manualcontrolled x,y,z

probe/emitter

computercontrolled x,y HV

injection valve

syringe pump

(b) Sampling Detail separated component liquid microjunction (20 - 50 µm) probe/emitter 127 µm 254 330 635 µm µm µm

(a) Probe Detail stainless steel tee spray

stepping

nebulizing gas

scanning eluting solvent

plate movement 200 µm

reversed-phase C18 layer

spray flow eluting solvent

gas flow

nebulizing gas eluting solvent

Fig. 3.8. Schematic illustration showing the sampling probe and its components, and the combined manual and computer-controlled manipulator stages used to position the TLC plate relative to the sampling probe. Inset (a) shows the detail of the sampling/emitter probe. Inset (b) shows the sampling probe at the surface of a TLC with the formation of a liquid microconjunction. Reprinted with permission from G. J. Van Berkel et al. [88].

Because of the simplicity and rapidity, the method was proposed for the analysis of drinks in laboratories without other expensive chromatographic equipment [93]. A combined method including TLC, HPLC, GC-MS and FTIR was employed for the study of the mechanism of the degradation of Navitan Fast Blue S5R by Pseudomonas aeruginosa. The culture medium containing the dye and its decomposition products was centrifuged and analysed by HPLC. The lyophilized supernatant was investigated by TLC

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Liquid chromatography of synthetic dyes 145 ng

100 Relative Abundance

393

2.5

1.4 ng

1.5 blank

50

0.5 1

2

3

14 ng

1.4 ng

blank 0 0

1

2

3

4

5

6

Time (min)

Fig. 3.9. Single-ion monitoring mass chromatogram (m/z 443, dwell time 500 ms) obtained in the positive-ion mode during the sequential step sampling (30 s sampling time) of four separate bands from developed spots (0.4 l each) of differing amounts of rhodamine 6G, namely, 0 (blank solvent), 1.4, 14 and 145 ng. Reprinted with permission from G. J. Van Berkel et al. [88].

and FTIR. TLC measurements were carried out on silica layers using chloroform–ethanol (9:1, v/v) as the mobile phase. A degradation product with the RF value of 0.12 was tentatively identified as an aliphatic carboxilic acid. RP-HPLC analyses were performed in an ODS column. On the basis of the chromatographic data a decomposition pathway was proposed (Fig. 3.15.) [94]. Both normal-phase TLC and RP-HPLC were applied for the investigation of the formation of indigo by recombinant mammalian Cytochrome P450. Samples for TLC analysis were prepared by centrifuging the cultures at 1 200g/5 min. The pellets were washed with cold buffer (100 mM Tris at pH  7; 0.5 mM EDTA and 500 mM sucrose), resuspneded in the same buffer and extracted three times with CHCl3. The combined extracts were concentrated and spotted on silica plates. Plates were developed with CHCl3methanol (50:1, v/v) as the mobile phase. Colour pigments were separated in an octylsilica column (150  4.6 mm i.d.) using a methanol–buffer gradient. According to the UV-visible spectra, retention characteristics and MS results, the colour pigment synthesized by cytochrome P450 was identified as indigo [95]. Normal-phase TLC has been employed for the control of the synthesis of some new reactive azo dyes containing the tetramethylpiperidine fragment. The chemical structure of the basic molecule and the substituents of the new derivatives are shown in Fig. 3.16. The new derivatives were characterized by their RF values determined in different mobile phases. Compositions of mobile phases were n-propanol–ammonia (1:1, v/v) for dye 1.2 (RF  0.84); n-propanol–ammonia (2:1, v/v) for dyes 1.3 (RF  0.50) and 1.4 (RF  0.80); and n-heptane–diethyl ether (1:1, v/v) for dyes 1.5 (RF = 0.80) and 1.6 (RF  0.76). The results indicated that together with other physicochemical methods such as IR and 1H NMR, normal-phase TLC is a valuable tool for the purity control and identification of new synthetic dyes [96].

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Chapter 3 O C R6

R3 N R2

O

O OR1 R7

OH I

C I

N R5 R4

I

I

Rhodamine 6G (1) R1, R2, R4 = CH2CH3, R3, R5 = H,R6, R7 = CH3 1 = m/z 443

O

O

HO

FD & C Red # 3 (4) (4 - H)- = m/z 834

Rhodamine B (2) R1, R6, R7 = H, R2, R3, R4, R5 = CH2CH3 N=N Rhodamine 123 (3) R1 = CH3, R2,R3,R4,R5,R6,R7 = H 3 = m/z 345 O CH3CH2

HO3S

OH S

O S O OH

FD & C Yellow # 6 (7) (7 - H)- = m/z 407 (7 - 2H + Na)- = m/z 429

O

N CH3

O H3C

R

SO3

HN N

NCH2CH3

O

N

N

O

OH S OH OH

CH3

CH3

N

CH3 Caffeine (8) (8+H)+ = m/z 195 (8+Na)+ = m/z 217

FD & C Green # 3 (5) R = OH (5 - 3H)2- = m/z 381 (5 -2H)- = m/z 763 FD & C Blue # 1 (6) R=H (6 -3H)2- = m/z 373 (6 - 2H)- = m/z 747

OH Acetaminophen (9) (9+H)+ = m/z 152 (9+Na)+ = m/z 174

O OH O O

CH3

Aspirin (10) (10+Na+) = m/z 203 (10+2Na-H)+ = m/z 225

Fig. 3.10. Structure and mass-to-charge ratio observed for the compounds investigated. Reprinted with permission from G. J. Van Berkel et al. [89].

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Relative abundance

Liquid chromatography of synthetic dyes

Relative abundance

0.019 mm/s R6G/B = 0.65 R6G/B = 0.86

10 5 0 0

2

4

6

8

10

12

14

16

18

20 15

0.044 mm/s R6G/B = 0.66 R6G/B = 0.85

10 5 0 0

(b)

Relative abundance

3

15

(a)

2

4

6

8

10

12

14

16

18

100 0.019 mm/s R6G/B = 0.66 R6G/B = 0.83

50

0 0

(c)

2

1

20

395

2

4

6

8 10 12 Distance, mm

14

16

18

Fig. 3.11. Positive-ion SRM ion current profiles for 1 (m/z 443–415 black trace), 2 (m/z 443 – 415, red trace), and 3 (m/z 345–285, blue trace) obtained during development lane scans of replicate development lanes of the RP C2 TLC separation of a mixture (50 ng each) of rhodamines 6G (1), B (2), and 123 (3) at surface scan rates of (a) 19, (b) 44, and (c) 190 m/s using a DESI solvent (methanol) flow rate of 0.5 l/min. Dwell time was 100 ms for each transition. Signal levels were normalized to the signal in panel (c). Chromatographic resolution, R, calculated from the data is shown in each respective panel. Reprinted with permission from G. J. Van Berkel et al. [89].

TLC separation of the components of black dye commercial product (BDCP) was performed on silica layers. The chemical structures of the dye components are shown in Fig. 3.17. Dyes were extracted from the effluent of the dye processing plant, from the untreated river water and from the drinking water treatment plant. The organic extracts were further concentrated and purified using a copolymer of styrene divinyl benzene. The mobile phase for TLC separation consisted of toluene–ethyl acetate (8:1, v/v). The RF values of dye components were 0.43 (C. I. Disperse Violet 93), 0.48 (C. I. Disperse Orange 37) and 0.59 (C. I. Disperse Blue 373), respectively. A complicated chromatographic separation system was developed and applied for the fractionation of the components of sludge extract for a mutagenecity test. The steps of the

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Chapter 3 H N

N N

Cl

H3C

OCH2CH=CH2

N

N

Cl

N

N

O

CH3

N

CH3 CH3

OCH2CH=CH2

Formula 1.

Formula 2.

H3C

H N

N N

CH3 N

N

CH3 CH3H

OCH2CH=CH2 O Formula 3.

Fig. 3.12. Synthesis of the compounds by the methods (a) and (b). A  -NHC6H4SO3H (dye 1a); A  -NHCH2CHCH (dye 1b); A  -OCH2CHCH2 (dye 1c). SP  N-(4-sulpho)phenyl-3methyl-5-pyrazolone; m-PDA = 1,3-phenylendiamine-4-sulphonic acid. Reprinted with permission from T. Konstantinova et al. [90].

C(CN)2

R2

C(CN)2 R1

+

CH

+ OH2

R1 CHO

R2

Fig. 3.13. Synthesis scheme. Reprinted with permission from N. Almonasy et al. [91].

fractionation process are shown in Fig. 3.18. It was proposed that the this type of effluent has to be characterized more throroughly by both chemicl and toxicological methods [97]. Differential pulse polarography (DPP) was employed for the determination of the dyes Carmoisine, Allura red and Ponceau 4R in sweets and soft drinks, and the results were compared with those obtained by RP-HPLC. HPLC separations were performed in an ODS column using gradient elution. Solvent A was 3 mM aqueous triethylamine solution mid pH adjusted to 6.5 by orthophosphoric acid. Methanol was solvent B. The gradient started with 10 per cent B and was increased to 70 per cent B in steps of 3 per cent. The column temperature was 22°C and the flow rate 1 ml/min. Food samples were diluted and filtered when it was necessary and injected without any other pretreatment. The retention

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TABLE 3.9 LIST OF PREPARED COMPOUNDS

Compound

R1

R2

a b c d e f g h

H H H H H H H CH3

H Cl CH3 OCH3 C6 H5 N(CH3)2 N(C2H5)2 N(C6H13)2

Reprinted with permission from N. Almonasy et al. [91]. R1

R2

I

H

H

II

H

CH3

III

NO2

H

R2 HO N

R1

N OH

HO H2O

H2O

H2O

OH R2

O

Cr

R1

N

O N

N O

N

Na+.nH2O

O CH3

Cr

HO

O

O N

II' N

R1 R2

I' III' IV'

HO R1

R2

H H NO2

H Cl H

Fig. 3.14. The structures of 7-hydroxy-o,o’-dihydroxyazo dyes and their chromium complexes. Reprinted with permission from H. Kocaokutgen et al. [92].

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Chapter 3 TABLE 3.10 FORMULA AND RF VALUES OF 7-HYDROXY-O,ODIHYDROXYAZO DYES AND THEIR CHROMIUM COMPLEXES

Dye

Formula

RF

I II III IV I’ II’ III’ IV’

C16H12N2O2 C17H14N2O2 C16H11N2O2Cl C16H11N3O4 NaC32H20N4O6Cr.4.5H2O C17H12N2O3Cr.4H2O NaC32H18N4O6Cr.2H2O NaC32H18N6O10Cr.2.5H2O

0.73 0.74 0.81 0.71 0.48a 0.76a 0.57a 0.79a

a

DMSO/H2O Reprinted with permission from H. Kocaokutgen et al. [92].

TABLE 3.11 DETECTION OF FOOD PIGMENTS ON CHROMATOGRAMS

Additive

RF

Colours

Patent blue V (E-131) Quinoline yellow (E-104) Brilliant blue FCF (E-133) Tartrazine (E-102) Azorubine (E-122) Ponceau 4R (E-124) Curcumine (E-100) Indigo carmine (E-132) Cochineal (E-120) Methyl violet Mixed carotenes (E-160a) Plain caramel (E-150a) Erythrosine B (E-127) Orange yellow S (E-110)

0.49 0.6 0.76 0.5 0.48 0.26 0.7 0.5 0.05 0.85 0.26 0 0.59, 0.70 0.55

Blue Yellow Navy blue Yellow Pink Pink Yellow Nay blue Red Navy blue Detected at 366 nm Brown Pink Orange

Reprinted with permission from I. Baranowska et al. [93].

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TABLE 3.12 FOOD PIGMENTS DETECTED AND QUANTIFIED IN THE BEVERAGES ANALYSED

No.

Beverage

Pigments found

concentration (g/L)

1 2 3 4

Orangeade Orangeade Orangeade Orangeade

5 6 7 8

Orangeade Orangeade Orangeade Grape beverage

9

Grape beverage

Quinoline yellow Quinoline yellow Tartrazine Ponceau 4R Azorubin Ponceau 4R Azorubin Ponceau 4R Tartrazine Patent blue V Tartrazine Patent blue V Ponceau 4R Azorubine Unidentifieda Azorubine Orange yellow S Patent blue V Unidentifieda beta-carotene beta-carotene beta-carotene beta-carotene Plain caramel Plain caramel Plain caramel Plain caramel

0.33 0.03 0.19 0.29 0.33 0.52 0.33 0.29 0.15 0.03 0.21 1.2 0.02 0.07 — 0.33 0.05 0.25 — — — — — — — — —

10 11 12 13

Grape beverage Grape beverage Lemon-apple beverage Melon-strawberry-raspberry beverage

14 15 16 17 18 19 20 21 22 23

Pepsi blue-type DEW-type beverage Orange beverage Orange beverage Orange beverage Orange beverage Cola-type beverage Cola-type beverage Pepsi-type beverage Pepsi-type beverage

a

Yellow spot of unidentified pigment at the origin. Reprinted with permission from I. Baranowska et al. [93].

times of dyes were 6, 8 and 11 min for Ponceau 4R, Allura red and Carmoisine, respectively. The correlation coefficient of the calibration curve was always higher than 0.9998. The standard deviation of the HPLC method depended considerably on the type of sample, being between 5.7 and 0.06 per cent. The concentrations of dyes found by DPP and HPLC are compiled in Table 3.13. The results demonstrated the good agreement between the methods, indicating that DPP can also be applied to the analysis of dyes in this class of food products [98]. The concentration of Sudan I, 1-phenylazo-2-naphtalenol, added illegally to foodstuffs has been determined by atmospheric pressure chemical ionization tandem mass

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Chapter 3

NaSO3 N=N

N=N

NH O3SNa

Navitan Fast BlueS5R Azoreduction NaSO3 NH2+H2N

NH2+H2N

NH NaSO3

1,4 Diamino Naphthalene

Metanilic acid

COOH

Peri acid Oxidative decarboxylation and ring cleavage

OH

Desulfonation Salicylic acid Oxidative determination

OH

Oxidative decarboxylation

OH

NH2 Catechol

Aniline

Ortopathway reactions O

COOH COOH

Beta-Ketoadipic acid

TCA Cycle

Fig. 3.15. Proposed pathway for the degradation of Navitan fast blue S5R by P. aeruginosa. Reprinted with permission from C. V. Nachiyar et al. [94].

spectrometry and isotope dilution, and the results were compared with those obtained by HPLC. Extraction of Sudan I from foodstuffs was carried out by mixing 1 g of sample with 10 ml of ACN. The suspension was filtered, and the liquid phase was evaporated to dryness and redissolved in the mobile phase. RP-HPLC measurements were carried out in an ODS column (250  4.6 mm i.d.; particle size 5 m) thermostated at 40°C. The isocratic

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Liquid chromatography of synthetic dyes SO3H

401

A1

OH

N N

N H

HO3S

SO3H

N

N

A1

N

Fig. 3.16. Characteristic data for the dyes with formula I. Reprinted with permission from P. PetrovaMiladinova et al. [96].

Blue Component of BDCP (C.I. Disperse Blue 373)

Br

O2N

CH3CONH N

N

N(CH2CH=CH2)2

NO2

OMe

Violet Component of BDCP (C.I. Disperse Violet 93) Br

O 2N

N

N

NO2

N(C2H5)2 CH3COHN

Orange component of BDCP (C.I. Disperse Orange 37) Cl

O2N

C2H5 N

N

N

CH2CH2CN

Cl

Fig. 3.17. The chemical structures determined for the dye components of BCDP using NMR and MS. Reprinted with permission from G. A. Umbuzeiro et al. [97].

mobile phase consisted of methanol–0.55 M acetic acid (90:10, v/v). The flow rate was 1 ml/min and Sudan I was detected at 476 nm. The results demonstrated that the sensitivity of the APCI-MS method was considerably higher than that of the traditional RP-HPLC prodecure [99].

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Chapter 3

Extract Sephadex LH-20 column (20x400 mm) MeOH : CHCl3 = 1:1, 10 ml/Fr

F1 – F25 (F8-10)

1/2 1/2

1/2 Ultra pack ODS column (26x300 mm) Fr. 1-81 75% CH3CN Fr. 82-104 100% CH3CN, 6 ml/Fr.

Fr 1-104 (Fr. 101) YMC-Pack ODS-AM324 column 0-20 min 90% MeOH 20-60 min 90-100% MeOH 60 min 100% MeOH, 3 ml/Fr Fr 1-91 (Fr. 39,40) Luna 5µ Phanyl-Hexyl column (10x250 nm) 0-50 min 70% CH3CN 50-70 min 70-100% CH3CN 70 min 100% CH3CN, 3 ml/Fr

Fr 1-91 (Fr. 31,32)

Fig. 3.18. Scheme for the bioassay-directed fractionation/chemical analysis performed with the sludge extract. Reprinted with permission from G. A. Umbuzeiro et al. [97].

3.5 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY Similarly to the liquid chromatographic analysis of natural pigments, HPLC, especially RP-HPLC, plays a decisive role in the determination of synthetic dyes in a wide variety of accompanying matrices, such as human and animal tissues, food and food products, ground- and waste-waters, sludge, soil, etc. [100].

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TABLE 3.13 CONCENTRATIONS OF DYES IN (A) SOFT BEVERAGES AND (B) SWEETS DETERMINED BY DPP AND HPLC Sample

Dye

Found (mg/l)

E(%)

DPP (separate)

HPLC

Ponceau 4R E 124 Carmoisine E 122

223.1 34.2

209.8 39.1



6.0 14.3

Aromatic plants bitter Cherry bitter Blood orange soda Pink grapefruit soda

Ponceau 4R E 124 Ponceau 4R E 124 Carmoisine Allura red E 129 Allura red E 129

72.5 78.0 3.7 7.8 1.8

68.5 75.9 4.3 8.5 1.6


5.5
2.7 16.2 9.0
11.0

(b) Sweets Acid drop Gelled sweet 1

Ponceau 4R Ponceau 4R

19.6 35.2

21.0 33.1

7.1 6.0

(a) Soft beverages Pomegranate syrup 1 Pomegranate syrup 2

E  relative difference between DPP and HPLC measurements. Reprinted with permission from S. Chanlon et al. [98].

3.5.1 HPLC determination of synthetic dyes in animal tissues Triphenylmethane dyes and their leuco metabolites have been separated with an RPHPLC technique. The chemical structure of dyes and the leuco metabolites are shown in Fig. 3.19. Dyes were extracted from trout muscle by mixing 5 g of tissue with 10 ml of citrate buffer and 50 ml of ACN. The suspension was homogenized and then centrifuged (4 200 g for 5 min). The supernatant was mixed with 25 ml of dichloromethane and a small amount of salt. After shaking for 1 min, the organic phase was separated and evaporated to dryness. The rest was redissolved in 5 ml of ACN containing 5 per cent acetic acid. The solution was loaded on a ion-exchange SPE cartridge preconditioned with 5 ml of ACN containing 5 per cent of acetic acid. The cartridge was washed consecutively with 2.5 ml of acetone, 5 ml of methanol and 5 ml of ACN. The cartridge was dried and the dyes were eluted with 10 ml of ACN containing 5 per cent of 35 per cent ammonia solution. The eluate was dried and redissolved in 0.05 M ammonium acetate–ACN (50:50 v/v). RP-HPLC-vis measurements were performed in an ODS column (250  2 mm i.d.; particle size 5 m). A 10  2 mm column filled with lead(IV) oxide was employed for the post-column oxidation of the analytes. The method of post-column oxidation allowed the simultaneous detection of each dye and metabolite in the visible range. Dyes were eluted with a two-phase step gradient programme: mobile phase 1 (0.05 M aqueous ammonium

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Chapter 3 R

N+Me2

Me2N R=H Malachite Green R = NMe2 Crystal Violet R

NMe2

Me2N R=H R = NMe2

Leukomalachite Green Leukocrystal Violet

Fig. 3.19. Structures of triphenylmethane dyes and metabolites. Reprinted with permission from J. A. Tarbin et al. [101].

acetate pH  4.5–ACN, 35:65, v/v for 10 min) followed by mobile phase 2 for 20 min (0.05 M aqueous ammonium acetate pH  4.5–ACN, 20:80, v/v). The flow rate was 0.3 ml/min and analytes were monitored at 618 nm. The isocratic mobile phase for LC-MS was 0.05 M aqueous ammonium acetate pH  4.5–ACN, 25:75, v/v. MS parameters for the positive-ion ESP mode were: capillary voltage, 2.9 kV; source temperature, 150oC; con voltage, 30 – 110 V; offset, 5 V. The good separation of analytes is illustrated in some typical chromatograms shown in Fig. 3.20. Recoveries for HPLC-vis varied between 69.1–71.3 per cent (MG) with RSD values 4.4–7.0 per cent. The same values for CV were 87.0–95.5 per cent and 8.1–16.5 per cent, respectively. The validity parameters of LMG (91.2–97.3 per cent, RSD 2.3–14.3 per cent) and LCV (96.4–114.5 per cent RSD 3.0–23.6 per cent) were similar. The LOD values were in general lower than 1 g/kg. It was concluded from the measurements that the extraction method is rapid and simple and allows the reproducible and sensitive determination of the dyes by both HPLC-vis and HPLC-MS techniques [101]. Another RP-HPLC method has also been developed and applied for the determination of MG, LMG, gentian violet (GV) and leuco gentian violet (LCV) in catfish and trout tissues. The chemical structures of MG and LMG were previously presented. The chemical structure of VG and LVG are shown in Fig. 3.21. Dyes were extracted mixing 20.0 g of

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10000 9000

MG LMG

8000 7000

CV

LCV

6000 (a)

5000 0

4

8

12

16

20

24

28

32

0

4

8

12

16

20

24

28

32

Peak height

10000

(b)

9000 8000 7000 6000 5000 10000 9000 LMG

MG

8000 7000

LCV

CV

6000 5000 0 (c)

4

8

12

16 20 Time/min

24

28

32

Fig. 3.20. HPLC-vis chromatograms. (a) Mixed standard 10 g/kg equivalent; (b) blank trout muscle extract, and (c) trout muscle spiked at g/kg. Peak heights in mV. Detection wavelength 618 nm. Peak identification: MG  malachite green; CV  crystal violet; LMG  leucomalachit green; LCV  leucocrystal violet. Reprinted with permission from J. A. Tarbin et al. [101].

homogenized fish with 3 ml of aqueous 0.25 mg/ml hydroxylamine.HCl, 5 ml of aqueous 0.05 M p-toluene sulphonic acid and 20 ml of aqueous ammonium acetate (pH adjusted to 4.5 with acetic acid). The suspension was homogenized, 90 ml of ACN was added and the homogenization was repeated. Basic alumina (20 g) was added to the mixture, shaken vigorously and centrifuged. The supernatant was separated, and the pellet was extracted again with 30 ml of ACN. The combined supernatant was diluted with 100 ml of de-ionized water, 50 ml of methylene chloride and 2 ml of diethylene glycol. The samples were shaken and after equilibration the bottom layer was separated. The extraction step was repeated with 50 ml of methylene chloride. The collected organic phases were evaporated. SPE purification was carried out by using a neutral alumina cartridge bonded to a Bond Elut PRS cartridge. The SPE system was equilibrated with 5 ml of ACN. Samples were redissolved in 2 ml of methylene chloride and loaded into the SPE cartridges. The system

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Chapter 3

N(H3)2 (H3C)2N

N(H3)2 (H3C)2N

LGV (colorless)

N(H3)2

GV (violet)

N+(CH3)2Cl-

Fig. 3.21. Chemical structures of leucogentian violet (LGV) and gentian violet (GV). Reprinted with permission from L. G. Rushing et al. [102].

was washed with 5 ml of ACN, then the alumina cartridge was removed. Analytes were eluted from the PRS cartridge with ACN-0.1 M ammonium acetate buffer (50:50, v/v). HPLC separation was performed in an cyano column (250  4.6 mm i.d.; particle size 5 m) coupled with a PbO2 column (20  2.0 mm i.d.). The isocratic mobile phase consisted of ACN–buffer (60:40, v/v). The flow rate was 1 ml/min and dyes were detected at 588 nm. Separations were also carried out in a SynChropak 150  4.6 mm i.d. SCD-100 column using ACN–buffer 55:45 as the mobile phase. The linear regression between concentration and peak areas of analytes was measured in the range of 0.5–50 ng/injection, the recovery values were determined at 5–20 ng/g levels. Chromatograms illustrating the separation efficacy of the HPLC system are shown in Fig. 3.22. The correlation coefficients of the linear regressions were high (over 0.9999), the recovery values varied according to the concentration and type of the analyte from 49(2) per cent to 90(3) per cent. It can be concluded from the data that the method is reliable and can be applied for the quantitative analysis of these dyes in trout and catfish tissues [102]. The toxicity and metabolism of MG and LMG in rats and mice was also investigated with HPLC methods. The structures of MG, LMG and demethylated derivatives are shown in Fig. 3.23. Dyes and their derivatives were extracted from livers using samples of 1–10 g. They were homogenized with 2 ml of 250 mg/ml aqueous hydroxylamine–HCl, 3 ml of 50 mM toluene sulphonic acid, 10 ml of 100 mM aqueous ammonium acetate (pH  4.5) and 30 ml of ACN. After homogenization, 2 g of NaCl was added to the samples and the mixture was centrifuged at 3 000 rpm for 10 min at 25°C. The supernatant was shaken with 40 ml of water, 2 ml of diethylene glycol and 90 ml of methylene chloride. The organic phase was evaporated to 1 ml and submitted to SPE prepurifation. HPLC-DAD was performed in a CN column (250  4.6 mm i.d.) coupled with a PbO2 oxidative post-column. Solvents A and B for gradient elution were 100 mM ammonium acetate (pH  4.5) and ACN, respectively. The gradient started with 60 per cent A (0–10 min); and changed to 50 per cent A in 10–25 min. The flow rate was 1 ml/min and analytes were detected at 618 nm. APCI-MS of LMG and metabolites was carried out in an ODS column (250  4.6 mm i.d.; particle size 5 m) using gradeint elution: from buffer–ACN 50:50 v/v to 100 per cent ACN in 10 min. MG and metabolites were separated in a nitrile column (250  4.6 mm i.d.; particle size 5m) using isocratic elution (50 mM ammonium acetate–ACN, 40:60, v/v). MS conditions were: temperature of ion source, 150°C; heated nebulizer probe, 500°C; nitrogen was used as the probe and bath gas. Molecular ion chromatograms of liver extract of rat feed

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100 LGV LMG

LMG

LGV

0.005 AUFS

GV MG

GV

5 ppb splked trout MG

10 ppb splked catflsh control catflsh

0 0 (a)

5

10 Time (min)

control trout

15

0

5

(b)

10 Time (min)

15

Fig. 3.22. (a) Overlay chromatograms of 10 ng/g (ppb) LMG, LGV, MG and GV fortified catfish and control catfish, 0.8 equiv./100 l injection, SCD column, 588 nm. (b) Overlay chromatograms of 5 ng/g (ppb) LMG, LGV, MG and GV fortified trout and control trout, 0.8 equiv./100 l injection, SCD column, 588 nm. Reprinted with permission from L. G. Rushing et al. [102].

R1

R1

N

N+ R2

R2

R3 N

R3 N R4

R4

Fig. 3.23. Structures of malachite green (MG), leucomalachite green (LMG) and demethylated derivatives. Reprinted with permission from S. J. Culp et al. [103].

with leucomalachite green and malachite green are shown in Fig. 3.24. The chromatograms clearly prove that the combined HPLC-DAD and HPLC-MS techniques well separate MG, LMG and their metabolites and can be employed for the study of their fate in living organisms such as rats and mice [103]. A similar extraction and HPLC method has been employed for the investigation of the persistence of GV and LGV in channel catfish (Ictalurus punctuatus) muscle. Some chromatograms illustrating the decrease of GV and LGV concentrations in a catfish sample are

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Chapter 3

shown in Fig. 3.25. The concentrations of LGV and GV found in catfish samples are compiled in Table 3.14. It was concluded from the data that GV rapidly metabolizes to LGV, which persist, therefore, it can be used for monitoring the catfish exposure to GV [104]. Another RP-HPLC method has been developed and applied for the determination of residues of malachite green in aquatic animals. MG and LMG were extracted by 9.66

100 %

m/z 345

(a) 0 6.92

100 %

m/z 275

(b) 0 8.82

100 %

m/z 289

(c) 0 10.71

100 %

m/z 303

(d)

0 12.50

100

m/z 317

%

6.06

(e)

0 13.84 m/z 331

%

100 7.83

(f) 0 100

1.83

% (g)

2.04

0

13.94 TIC 8.82 13.10 6.08 10.71 12.60 6.927.83 3.69 4.22 Time

Fig. 3.24. Left: LC-APCI/MS molecular ion chromatograms obtained at 20 V from a liver extract of a rat fed 580 ppm leucomalachite green (LMG) for 28 days. (a) Mass chromatograms of m/z 345, malachite green N-oxide (MG N-ox); (b) m/z 275, tetradesmethyl LMG; (c) m/z 289, tridesmethyl LMG; (d) m/z 303, didesmethyl LMG; (e) m/z 317, desmethyl LMG (12.5 min) and didesmethyl MG N-ox (6.08 min); (f) m/z 331, LMG (13.94 min) and desmethyl MG N-ox (7.83 min); and (g) total ion chromatogram. Right: LC-APCI/MS molecular ion chromatograms obtained at 20 V from a liver extract of a rat fed 600 ppm malachite green (MG) for 28 days. (a) Mass chromatograms of m/z 273, tetradesmethyl MG; (b) m/z 287, tridesmethyl MG; (c) m/z 301, didesmethyl MG; (d) m/z 315, monodesmethyl MG; (e) m/z 329 MG; and (f) total ion chromatogram. Reprinted with permission from S. J. Culp et al. [103].

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homogenizing 2 g of sample with 2 ml of McIlvaine solution at pH  3.0, 100 l of 1 M p-TSA and 50 l of 1 mg/ml methanolic N,N,N’,N’-tetramethyl-1,4-phenylenediamine hydrochloride. The McIlvaine solution was prepared by mixing 18.9 ml of 0.2 M sodium hydrogen phosphate and 81.1 ml of 0.1 M citric acid. The suspension was homogenized again with 12 ml of ACN then centrifuged at 3 400 g at 15°C for 5 min. The supernatant was separated and the residue was mixed with McIlvaine solution of pH  6.0 and 12 ml of ACN and centrifuged again. The combined liquid phases were mixed with 6 ml of dichloromethane, centrifuged and the organic phase was loaded onto an aromatic sulphonic acid-bonded SPE cartridge. The cartridge was washed with 1.5 ml of methanol and dried. Analytes were eluted with 3 ml of an eluent mixture consisting of 2.5 ml of 25 per cent (m/v) ammonium hydroxide, 2.5 ml of 1.0 mg/ml methanolic ascorbic acid and 45 ml of methanol. RP-HPLC-DAD measurements were performed in an ODS column (100  3.0 mm i.d.). The mobile phase consisted of 50 mM sodium perchlorate, 25 mM of sodium acetate and 25 mM of 1-pentanesulphonic acid (pH adjusted to 4.0 with acetic acid) and ACN (2:3, v/v). The flow rate was 0.6 ml/min and analytes were detected at 620 nm. Leuco forms of the dyes were oxidized with lead(IV) oxide. HPLC-MS analyses were carried out in another ODS column (50  2 mm i.d.) using 50 mM ammonium acetate (pH  4.4) and ACN (2:3, v/v) as the mobile phase at a flow rate of 200 l/min. MS conditions were: ionization voltage,
5 500 V; source temperature, 400oC; and entrance, declustering and

100 5 ng LGV

Response at 588 nm (0.02 AUFS)

unknown

0.5 ng GV

reagent blank standard control 1-hr 7-hr 15-day 79-day

0 4

6

8 Time

10

12

Fig. 3.25. Composite overlay of HPLC chromatograms of the control reagents blank; standard containing 5 ng LGV and 0.5 ng GC; 0.5 g equivalent of control catfish sample; 1 h post-dosing sample; 7 h post-dosing sample; 15 days post-dosing sample; and 79 days post-dosing sample. Samples are mean of n  5. Reprinted with permission from H. C. Thompson Jr et al. [104].

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Chapter 3 TABLE 3.14 CONCENTRATION OF LEUCOGENTIAN VIOLET (LGV) AND GENTIAN VIOLET (GV) IN MUSCLE TISSUE OF CATFISH EXPOSED TO 100 NG/ML GV IN WATER FOR 1 H

Time post-dosing

LGV (ng/g)

GV (ng/g)

Controlsa 1h 2 h1min 4h 7h 1 day 2 days 5 days 8 days 15 days 22 days 33 days 51 days 79 days

0.00.1 11.71.80 6.82.2 15.94.3 15.53.6 15.13.1 13.53.3 9.43.3 9.72.8 5.72.2 3.30.5 2.80.9 1.50.6 3.10.5

mdlb 50.1 0.80.3 mdl mdl mdl mdl 0.30.2 mdl mdl mdl mdl mdl mdl

a

Taken prior to dosing. mdl  0.2 ng/g for GC. Mean and standard deviation of single determinations of five fish at each sampling interval. Reprinted with permission from H. C. Thompson Jr et al. [104]. b

focusing potentials were -9, 40 and 180 V, respectively. The chromatographic profiles of MG residues extracted from various samples are shown in Fig. 3.26. The correlation coefficients of the linear regressions were always higher than 0.9999 showing the good linear response of the HPLC systems. Recoveries varied according to the concentration of MG and LMG and the animal species, from 43.8 to 105 per cent. The concentrations of LMG found in various samples are compiled in Table 3.15. It was stated that the method is suitable for the analysis of MG and LMG in control laboratories [105]. A similar HPLC technique was employed for the investigation of the persistence of MG in juvenile eels (Anguilla anguilla). Extracts were separated in phenyl-hexyl and octyl silica columns (both 50  4.6 mm i.d.; particle size 3 m) in series. The mobile phase was composed of 60 per cent ACN and 40 per cent 0.05 M ammonium acetate buffer (pH  4.5). The flow rate was 0.6 ml/min and analytes were detected at 620 nm. The concentrations of MG and LMG are compiled in Table 3.16. The data prove that this HPLC method can be used for the investigation of the persistence of MG and LMG in animal tissues [106]. The methods described above were completed by tandem mass spectrometry and applied for the determination of MG in various edible aquatic animals. The baseline separation of MG and LMG extracted from different tissues is illustrated in Fig. 3.27.

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0.005

0.005

1000

LMG

Counts/s

BG

A620 (AUFS)

A620 (AUFS)

MG

BG

MG MG

(a)

0

10

20

(b)

0

10

LMG 20

BG MG (c) 0

10

20

Retention time (min)

Fig. 3.26. Typical elution HPLC profiles of MG residues extracted from (a) salmon spiked at 20 g/kg LMG and MG each; (b) residue-incurred salmon fillet (2.9 g/kg). Analysis of the residueincurred salmon was repeated using the LC-MS-MS system as shown in (c); the profile shows the monitoring of the m/z  329.5 to m/z 313.3 fragmentation. The elution positions of MG, LMG and the internal standard brilliant green (BG) are indicated. Note: BG is not detected in the m/z  329.5 to m/z 313.3 trace (c) and its position is therefore depicted as an under broken arrow. Reprinted with permission from A. A. Bergwerft et al. [105].

Concentrations of LMG found in the samples of edible aquatic animals are compiled in Table 3.17. It was stated that the method allows the detection of MG and LMG at low residue levels, therefore, it is suitable for quality control purposes [107]. A derivative of Congo Red was synthesized and an RP-HPLC method was developed for its determination in plasma and brain. The chemical structure of the new derivatives (sodium 3,4-diaminonaphtalene-1-sulphonate, RCA) is shown in Fig. 3.28. Extraction of RCA from plasma was carried out by vortexing 1 ml of plasma with 1 ml of 15 sodium disulphide in 5 mM of tetra-n-butylammoniumiodide (TEBA) and 10 g of internal standard (sodium 4-aminonaphtalene-1-sulphonate), then the suspension was centrifuged (800 g for 10 min) and used for SPE prepurification. Hamster brain (about 0.9 g) was homogenized with 4 ml of 6 per cent cystein in 0.4 M ZnSO4 in water. After homogenization the sample was mixed with 4 ml of 0.4 M Ba(OH)2, 2 ml of 5 per cent sodium disulphide in 5 mM of TEBA and centrifuged at 30 000 g for 10 min. The supernatant was loaded into an SPE cartridge filled with ENV
hydrophobic phase. Cartridges were preconditioned with one column volume of methanol, water and 1 per cent sodium disulphide in 5 mM TEBA in water. After loading, the cartridges were washed with two volumes of 1 per cent sodium disulphide in 5 mM TEBA and one volume of water–methanol (9:1, v/v). Analytes were eluted with 2  500 l of methanol. The combined extract was evaporated to dryness and redissolved in the mobile phase for RP-HPLC analysis. Measurements were performed in an ODS column ((250  4.6 mm i.d.; particle size 5 m). The mobile phase was composed of 50 mM Na2SO4 in 5 mM TEBA in water–methanol (82:18, v/v). The flow rate was 1 ml/min

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TABLE 3.15 SUMMARY OF THE RESULTS FROM THE ANALYSIS OF 18 MOST PROBABLY CULTURED TROUT, 10 EEL AND 20 SALMON PRODUCTS

Species

LMG (g/kg)

Species (/product)

LMG (g/kg)

Species (/product)

LMG (g/kg)

Trout Trout Trout Trout Trout Trout Trout Trout Trout Trout Trout Trout Trout Trout Trout Trout

nda 1.3 2.1 2.1 nd nd 1.8 nd 1.6 14.9 1.9 2.8 2.2 2.8 4.2 1.5

Trout Trout Eel Eel Eel Eel Eel Eel Eel Eel Eel Eel Fresh salmon Fresh salmon Fresh salmon Smoked salmon

nd 2.8 1.7 9.7 nd nd nd 7.0 nd 1.5 2.4 nd 2.9 0.2 (MS) 0.2 (MS) 0.2 (MS)

Smoked salmon Smoked salmon Smoked salmon Smoked salmon Smoked salmon Smoked salmon Smoked salmon Smoked salmon Canned salmon Canned salmon Canned salmon Canned salmon Canned salmon Canned salmon Canned salmon Canned salmon

nd (MS) nd (MS) 0.2 (MS) nd (MS) nd (MS) nd (MS) nd (MS) nd (MS) nd (MS) nd (MS) nd (MS) nd (MS) nd (MS) nd (MS) nd (MS) nd (MS)

The fish were purchased from local retailers and vendors. Samples were analysed by HPLC-Vis or by LC-MS/MS as indicated by ‘MS” in brackets. One LMG was detected and reported in this table. a nd  not detected. Reprinted with permission from A. A. Bergwerft et al. [105].

and analytes were detected at 210 nm. The validation parameters (accuracy, precision, sensitivity and recovery) were also measured. Typical chromatograms illustrating the successful separation of RCA and internal standard from the elements of accompanying matrices are shown in Fig. 3.29. It was found that the addition of the ion-pairing agent, TEBA, is necessary for the effective separation of RCA from the other analytes. The correlation coefficient of the linear relationship between peak area and RCA) concentration was 0.98 and 0.99 for brain and plasma. The other validation parameters are compiled in Table 3.18. Because of the good validation parameters, the RP-HPLC method combined with SPE has been proposed for the investigation of the pharmacokinetics of RCA in plasma and brain [108]. 3.5.2 HPLC determination of synthetic dyes in foods, food products and waters A considerable number of foods and food products contain allowed synthetic colourants to increase the commercial value of the merchandise. Unfortunately, the illegal use of synthetic

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TABLE 3.16 AVERAGED TISSUE CONCENTRATIONS OF RESIDUES OF MG FOUND IN GLASS EEL TREATED WITH MG

Time point

Residue

(h)

Prior to treatment 24a 23.67 23.33 23 22 21 18 15 12 0 0.33 0.67 1 2 3 6 9 12 24 48 72 96 120 240 480 720 1 152 1 487

MG

LMG

Averaged concentration (range) (g kg1)

SD (g kg1)

4 21(12–28) 95(79–125) 194(131–267) 218(167–262) 388(283–469) 381(255–456) 435(343–523) 411(281–501) 411(314–543) 206(141–275) 236(172–352) 196(109–284) 245(123–318) 208(110–263) 205(113–359) 178(148–288) 168(129–242) 142(95–173) 97(54–128) 44(32–57) 30(16–49) 21(10–38) 18(5–42) 9(4–13) 7(2–13) 2(ndb–4) 2(nd–5) 2(nd–8)

4 4 13 44 24 51 66 59 71 75 47 60 63 66 50 73 44 37 27 21 9 12 9 10 4 4 1 2 3

Averaged concentration (range) (g kg1) 7 7(3–12) 21(12–37) 68(40–141) 84(45–137) 177(154–250) 263(205–372) 446(349–635) 543(469–605) 613(448–799) 639(452–940) 675(519–893) 586(367–824) 746(647–1004) 659(480–880) 766(533–926) 699(484–919) 689(585–783) 676(613–777) 690(552–767) 761(590–949) 831(542–1 295) 714(464–913) 667(399–932) 576(419–733) 505(323–658) 420(231–669) 304(140–473) 139(75–253)

SD (g kg1)

N

5 3 7 31 25 32 49 87 44 95 176 111 139 106 141 112 134 76 65 64 125 231 150 176 93 100 143 111 57

9 10 10 9 10 10 10 10 10 10 10 10 10 10 10 10 9 10 10 10 10 9 10 10 9 9 9 10 9

(Continued on next page)

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TABLE 3.16

(continued)

Time point

Residue

(h)

MG Averaged concentration (range) (g kg1)

1 920 2 400

LMG SD (g kg1)

nd(nd–nd) nd(nd–nd)

na nac

Averaged concentration (range) (g kg1)

SD (g kg1)

N

25 12

9 10

28(nd–84) 15(nd–43)

Samples were taken at given time points from the start (-24 h) to the end of the exposure (0 h), and after treatment (from 0 to 2 400 h). The range of concentrations determined in a series per time point is given as minimum and maximum concentrations. SD  standard deviation. a These samples were taken immediately after preparation of the MG-containing bath water. b nd  not detected. c na  not applicable. Reprinted with permission from A. A. Bergwerft et al. [106]. (a)

Signal (cps)

MG

(b) LMG

MG

(c) MG

LMG

(d) LMG

MG

200

100

100

100

150

80

80

80

60

60

60

40

40

40

20

20

20

LMG

100 50 0

0 0

10

20

0 0

10

20

10 0 Time (min)

0 20

0

10

20

Fig. 3.27. Typical LC-MS (MRM from m/z 329 to m/z 313) chromatograms of (a) standard at 1 ng/ml, (b) blank salmon, (c) salmon spiked at 0.10 g/kg and (d) retailer-purchased trout containing LMG at 0.15 g/kg. Reprinted with permission from P. Scherpenisse et al. [107].

dyes not allowed in food frequently occurs. The separation and quantitative determination of the level of dyes legally permitted and the detection of illegal colourants in food is of paramount importance. Concequently, a considerable number of HPLC techniques have been developed for the analysis of synthetic dyes in foods [109]. Thus, ion-interaction

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TABLE 3.17 RESULTS OF THE DETERMINATION OF LMG IN SAMPLES OF EDIBLE AQUATIC ANIMALS PURCHASED FROM LOCAL RETAILERS Code

Species

A B C D E F G H I J K L M N O P Q R

Salmon Salmon Salmon Salmon Shrimps Shrimps Salmon Shrimps Salmon Shrimps Pangasius Trout Pangasius Pangasius Trout Salmon Salmon Trout

LMG content (gkg1)

Remark

CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
7 0.15 CC
CC
CC
CC
CC
24

Fresh Fresh Smoked Smoked Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh Canned Smoked Fresh

CC
 calculated decision limit. Reprinted with permission from P. Scherpenisse et al. [107].

NH2 NH2

SO3Na

Fig. 3.28. RCA structure formula. Reprinted with permission from R. Pirola et al. [108].

HPLC has been employed for the identification and determination of red dyes in confectionery. The chemical structures, commercial names, European Community (CE) number, colour index (CI) number and name, and food and drug (F and D) name of the red dyes included in the investigation are listed in Fig. 3.30. Dyes were extracted from confectionery by stirring 1.00 g of sugar ball with 10 ml of methanol until the balls were entirely decolourized. The extract was diluted 10-fold with water, filtered and used for

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Chapter 3 6

6

4

4 mV

mV

4

RCA

IS

RCA IS

mV

6

IS

2

2

2

0

0

0

0

10

(a)

20 min

30

0

10

(b)

20 min

30

0

20 min

30

6

6

6

10

(c)

RCA IS IS

mV 2

mV

2

2

IS

4

4 mV

4

RCA

0

0 0 (a)

10

20 min

30

0 0

(b)

10

20 min

30

0 (c)

10

20 min

30

Fig. 3.29. Upper lane: (a) plasma blank; (b) plasma standard: RCA 10 g/ml; and (c) plasma sample: RCA 8.72 g/ml, I.S. 10 g/ml. Lower lane: (a) brain tissue blank; (b) brain standard: RCA 10 g/ml; and (c) brain sample: RCA 1.04 g/ml, I.S. 10 g/ml. Reprinted with permission from R. Pirola et al. [108].

HPLC. Separation was performed in an ODS column (250 x 4.6 mm I.D., particle size 5 m). The isocratic mobile phase was composed of water–ACN (70:30, v/v) containing 5.0 mM octylamine at pH 6.4. The column was thermostated at 25°C and the flow rate was 1 ml/min. Red dyes were detected at 520 nm. Typical chromatograms of confectionery extracts are depicted in Fig. 3.31. Because of the low detection level

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TABLE 3.18 PRECISION, ACCURACY AND RECOVERY FOR THE DETERMINATION OF RCA IN PLASMA AND BRAIN TISSUE BY THE DESCRIBED PROCEDURES

Nominal Intra-assay concentration precision (g/ml or g/g) (CV%) (n10) Plasma

1 10 100

Brain tissue

1 2 5

Intra-assay precision (CV%) (n6)

Intra-assay accuracy (%) (n10)

Intra-assay Recovery accuracy (%) (%) (meanSD) (n6)

6.2 2.08 3.5

7.8 3.3 4.6

96.7 97.8 101.1

95.4 100.5 99.9

76.13.5 78.82.5 79.12.9

7.5 4.4 3.1

8.6 5.2 4.5

95.1 99.5 96.9

94.0 97.7 98.1

74.34.5 78.14.5 80.13.8

Reprinted with permission from R. Pirola et al. [108].

Molecular structure

Commercial CE name number

CI number and name

F and D name

14720 Acid Red 14

Food red 3

SO3Na N N

SO3Na Carmoisine E122

OH HO NaO3S

SO3Na

N N Amaranth

E123

16185 Acid red 27

Food red 9

Ponceau 4R E124

16255 Acid red 18

Food red 7

SO3Na

HO NaO3S

N N NaO3S SO3Na

Fig. 3.30. Molecular structures, commercial common names, European Community (CE) number, colour index (CI) number and name, food and drug name of the three red food dyes studied. Reprinted with permission from M. C. Gennaro et al. [110].

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Chapter 3

(b)

5.04

OFFS time (min)

(B)

30

25

20

15

10

5

30

25

20

15

10

5

(a)

CH . 1 C. S

2 . 50 ATT

0

7.84

3.04

0

0 OFFS 0 2 . 50 ATT CH . 1 C. S (A)

18.61

Absorbance 0.001 AUFS

(b)

6.08

Absorbance 0.001 AUFS

18.21

(12 g/l), the method allows the safe determination of these dyes in food products such as confectionerys [110]. Ion chromatography has also been used for the analysis of eight synthetic food colourants in drinks. The chemical structures of dyes (Amaranth, AMA; Brilliant Blue, BRI; Indigo Carmin, IND; New Red, NEW; Ponceau 4R, PON; Sunset Yellow, SUN; Tartrazine, TAR; Allura Red, ALL) are shown in Fig. 3.32. Separations were performed in an ODS clumn (250  4.0 mm i.d.; particle size 13 m) using ternary gradient elution (solvent A  2.0 M HCl; solvent B  ACN; solvent C  water). The gradient started at 10 per cent A
50 per cent B for 0–9.5 min; 95 per cent B + 2.5 per cent C for 9.6–20.0 min; 10 per cent A
90 per

time (min)

Fig. 3.31. (A) Chromatogram recorded for the French product: a  E123, b  E124. (B) Chromatogram recorded for the Italian product: b  E124. Reprinted with permission from M. C. Gennaro et al. [110].

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419 SO3Na NaO3S

N(C2H5)CH2

N

OH

NHCOCH3

N

N(C2H5)CH2 +

SO3-

SO3Na

NaO3S

SO3Na BRI H N NaO3S

NEW

O SO3Na N H

O

HO

NaO3S

N N

IND

NaO3S

SO3Na

HO

SO3Na

N NaO3S

PON

COONa

N

TAR HO OMe

NaO3S

N NaO3S

HO

SO3Na

N N

N

H3C SO3Na

SO3Na

ALL HO AMA

N NaO3S

N

SUN

SO3Na

Fig. 3.32. Structures of eight synthetic food colourants. Reprinted with permission from Q. Chen et al. [111].

cent B for 20.1–35.0 min; 10 per cent A
50 per cent B in 35.1 min. The flow rate was 1.5 ml/min and the detection wavelength was changed during the separation process (625, 430, 480, 525 and 625 nm). Chromatograms demonstrating the good separation capacity of the method are shown in Fig. 3.33. The results of the analysis of real samples are compiled in Table 3.19.

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Page 420

Chapter 3

0.1

AMA

ALL SUN

0.2

PON

BRI IND TAR

AU

0.3

NEW

0.4

0.0

0

5

0

5

0

5

10

15 20 Minutes

25

10

15 20 Minutes

25

30

35

10

15 20 Minutes

25

30

35

(a)

30

35

0.05

AU

0.03

PON

TAR

0.04

0.02 0.01 0.00 -0.01

(b) 0.10

TAR

0.08

0.04

SUN

AU

0.06

0.02 0.00 -0.02 (c)

Fig. 3.33. Chromatograms of standard solution where the concentrations of all analytes were 40 g/ml except the Brilliant blue concentration which was 20 g/ml (a) and the final solutions of real samples: (b) sample D; (c) sample F. For analytical conditions see text. Reprinted with permission from Q. Chen et al. [111].

The coefficients of correlation of the linear relationship between peak area and analyte concentration were high, ranging from 0.9978 to 0.9998; and recoveries varied between 94.7 and 109.0 per cent. It was established that the procedure is reliable and reproducible, therefore in can be applied for the analysis of these synthetic dyes in drinks [111]. An RP-HPLC method has also been developed for the determination of six food dyes (Sunset Yellow, E-110; Carminic acid, E-120; Carmoisine, E-122; Amaranth, E-123;

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TABLE 3.19 ANALYSIS OF REAL SAMPLES

Contenta (g/ml or g/g)

Sample BRI A B C D (wild jujube) E F

0.82  0.04

TAR

15.6  0.7 21.1  0.3 4.0  0.2 325.0  4.0

SUN

PON

42.5  1.0 67.0  1.4

176.0  4.0

13.6  0.5 11.6  0.5

Meanstandard deviation (n  4), g/g for solid sample (sample F) and g/ml for the other samples. Reprinted with permission from Q. Chen et al. [111]. a

Ponceau 4R, E-124; and Erythrosine, E-127) using a buffered mobile phase. Separation of dyes was performed in an ODS column (150  3.9 mm i.d.; particle size 3 m). Components of the mobile phase were methanol (eluent A) and 0.1 M NaH2PO4/Na2HPO4 buffer (pH  7). The gradient elution started with 20 per cent A and reached 100 per cent in 2 min, final hold 4 min. The flow rate was 2 ml/min and dyes were detected at 520 nm. The baseline separation of dyes in 6 min is illustrated in Fig. 3.34. Commercial samples were diluted and injected into the analytical column without any pretreatment. The amounts of dyes found in the samples are compiled in Table 3.20. It was concluded from the good validation parameters that the technique is specific, sensitive, accurate and rapid. Consequently, its application for the determination of these synthetic dyes in drinks was proposed [112]. Another RP-HPLC technique has been applied for the determination of synthetic food dyes in soft drinks with a minimal clean-up. Separation of dyes was obtained in an ODS column (150 x 4 mm i.d.; particle size 5 m). Solvents A and B were methanol and 40 mM aqueous ammonium acetate (pH  5), respectively. Gradient conditions were: 0–3 min, 10 per cent A; 3–5 min, to 25 per cent A; 5–8 min, 25 per cent A; 8–18 min, to 75 per cent A; 18–20 min, 75 per cent A. The flow rate was 1 ml/min and dyes were detected at 414 nm. The separation of synthetic dyes achieved by the method is shown in Fig. 3.35. The concentrations of dyes found in commercial samples are compiled in Table 3.21. The quantification limit depended markedly on the type of dye, being the highest for E-104 (4.0 mg/l) and the lowest for E-102 and E-110 (1.0 mg/l). The detection limit ranged from 0.3 mg/l (E-102 and E-110) to 1.0 mg/ml (E-104 and E-124). It was suggested that the method can be applied for the screening of food colourants in quality control laboratories [113]. Sulphonated azo dyes were separated and quantitated in various food products by ion-pair liquid chromatography with DAD and electrospray MS detection. The chemical structure of sulphonated azo dyes included in the investigation are shown in Fig. 3.36. Dyes were separated in an ODS column (125  2.0 mm i.d.; particle size 5 m) using gradient elution. An aqueous solution of 3 mM triethylamine (pH adjusted to 6.2 with acetic acid) and methanol

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Chapter 3

100 E-127

60

40

Methanol (%)

0.06

80

E-122

E-124 E-110

E-123

0.08

E-120

Absorbance (volts)

0.10

0.04 20

0.02

0 0.0

1.0

2.0

3.0

4.0

5.0

Times (minutes)

Fig. 3.34. Chromatogram of a dye mixture obtained by the proposed method and gradient profile. Reprinted with permission from J. J. Berzas-Nevado et al. [112]. TABLE 3.20 ANALYSIS OF COMMERCIAL SAMPLES

Found (mg/l) Sample

Dyes

Direct measurement

Standard addition

Bitter kas

Carmoisine (E-122) Ponceau 4R (E-124) Carmoisine (E-122) Amaranth (E-123) Sunset Yellow (E-110) Ponceau 4R(E-124) Amaranth (E-123) Amaranth (E-123) Ponceau 4R(E-124)

35.3  0.1 8.40  0.5 18.5  0.1 38.0  0.3 7.8  0.2 49.8  0.2 30.0  0.1 150.8  0.4 271.0  0.5

32.8  0.2 10.5  0.1 17.5  0.3 38.5  0.3 7.3  0.2 49.4  0.3 30.2  0.3 153.2  0.2 273.0  0.3

Bitter kalty

Grenadine Gelatine

Reprinted with permission from J. J. Berzas-Nevado et al. [112].

were the solutions A and B. The gradient started at 95 per cent A, changed to 80 per cent in 5 min; to 70 per cent in 10 min; to 50 per cent in 15 min; to 45 per cent in 30 min; and to 30 per cent in 35 min. The flow rate was 0.2 ml/min and azo dyes were detected at 430 nm. Soda samples (1 ml) were diluted with methanol–water (1:1, v/v) to 505 ml, sonicated, filtered and injected into the analytical column. Fruit jam (1.5 g), salted fish and

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500

mV

1

2 0

8

4

0

67

3 5

Minutes

20

Fig. 3.35. HPLC chromatogram of the colour additives. E-102, 7.6 min; E-124, 11.5 min; E-104, 12.9 min (first isomer); E-110, 13.1 min; E-104, 14.9 min (second isomer); E-122, 17.1 min; E-104, 18.1 min (third isomer); E-104, 18.4 min (fourth isomer). Reprinted with permission from M. S. Garcia-Falcón et al. [113].

TABLE 3.21 FOOD COLOURS CONCENTRATIONS IN ANALYSED BEVERAGES (MIN–MAX; N  3)

Samples

Soft drinks (trade name)

Colour

Concentration (mg/l)

1

With fruit juice (Radical orange)

2 3

With fruit juice (Radical apple temptation) With fruit juice (Radical wild fruits seduction)

4 5 6

With fruit juice (Radical pineapple emotion) With fruit juice (Kas lemon) With fruit juice (Kas orange)

7 8 9

With fruit juice (Fanta lemon) With flavour extracts (Casera lemon) With flavour extracts (Casera orange)

E-102 E-110 E-102 E-122 E-124 E-102 E-102 E-102 E-110 E-104 E-104 E-102 E-110

21.5–22.5 3.9–4.1 7.6–8.3 32.7–33.4 17.7–18.2 5.8–6.1 2.0–2.1 11.9–12.3 2.0–2.1 3.8–4.0 4.0–4.3 17.8–18.1 6.0–6.2

Reprinted with permission from M. S. Garcia-Falcón et al. [113].

salted vegetable (0.5 g) were extracted with 3  5 ml of methanol–water (1:1, v/v), the extracts were centrifuged, combined and filtered prior to analysis. The chromatographic profiles of standard dyes detected by DAD and MS are shown in Fig. 3.37. The concentration of azo dyes found in real samples are compiled in Table 3.22. The linear relationship

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Chapter 3 Ponceau R (C.I. 16150)

CH3

CH3

SO3Na

HO

SO3Na

HO

H3C

Ponceau 3R (C.I.16155)

N CH3

N

N

N

CH3

SO3Na

SO3Na New Coccine (C.I. 16255)

Orange I (C.I. 14600)

HO

NaO3S

OH

N

N NaO3S

N NaO3S

N

SO3Na Sunset Yellow FCF (C.I.15985)

Orange II (C.I. 15510) HO

HO N

N NaO3S

NaO3S

N

N

SO3Na Metanil Yellow (C.I. 13065) SO3Na N NH

N

Fig. 3.36. Chemical structure, name and colour index of the dyes used. C.I.  colour index. Reprinted with permission from M.-R. Fuh et al. [114].

between the concentration of analytes and the peak area was highly significant (R2 values were always 0.999), and the detection limit was 0.01 g/ml. It was established that triethylamine is an effective ion-pair agent, moreover, it does not influence ES-MS detection. The analysis of commercial samples demonstrated that the the method can be used for the detection of sulphonated azo dyes in a wide variety of food products [114]. A separation method using RP-HPLC and electrospray-tandem mass spectrometry was developed for the simultaneous determination of Sudan-azo dyes in hot chilli products. The chemical structures of the azo dyes included in the investigation are listed in

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425

1

30

10

20

8

4 10

2

5

3

9

67

0 (a) 7

10

Absorbance ( ×105)

8 6 9

6 8 3

4

12

5 4

2 0 (b)

5

10

15

20 Time (min)

25

30

35

Fig. 3.37. LC-UV-MS chromatogram of sulphonated azo dye standard solution. (a) LC-UV chromatogram (430 nm); (b) LC-ES-MS reconstructed chromatogram. (1) Tartrazine (tR  11.52 min); (2) amaranth (tR  12.25 min); (3) new coccine (tR  14.24 min); (4) sunset yellow FCF (tR  15.82 min); (5) allura red AC (tR  18.00 min); (6) ponceau R (tR  20.37 min); (7) ponceau 3R (tR  21.27 min); (8) orange I (tR = 21.96 min); (9) orange II (tR  25.65 min); (10) metanil yellow (tR  28.25 min). Reprinted with permission from M.-R. Fuh et al. [114].

Fig. 3.38.The IUPAC names of Sudan azo dyes are as follows: Sudan I  1 [(2,4-dimethylphenyl)azo]-2-naphtalenol; Sudan II  1-(phenylazo)-2-naphtol; Sudan III  1-(4-phenylazophenylazo)-2-naphtol; Sudan IV  o-tolyazo-o-tolyazo-beta-naphtol; and Disperse Orange 13 = 4-[4-(phenylazo)-1-naphtylazo]-phenol. Azo dyes were separated in an ODS column (250 x 2.1 mm i.d.; particle size 5 m) at 35°C. The isocratic mobile phase consisted of 0.1 per cent formic acid in methanol–0.1 per cent formic acid in water (97:3, v/v). The flow rate was 200 l/min. MS conditions were: nebulizing and desolvation gas were nitrogen at the flow rates of 50 and 555 l/h, respectively; electrospray voltage, 3.0 kV; cone voltage 25 V; source temperature, 110°C; desolvation temperature, 110oC. Azo dyes were extracted from the samples by homogenizing 1 g of sample with 10 ml of acetone, then the suspension was centrifuged and an aliquot of 3 ml of supernatant was mixed with 1 ml of deionized water, filtered and used for analysis. LC-ESI-MS/Ms SRM traces of standards and spiked samples are listed in Fig. 3.39. It was found that the detection and quantitation limits depended on both the chemical structure of the dye and the character of the accompanying matrix. LOD and LOQ values in chilli tomato sauce

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Chapter 3 TABLE 3.22 CONCENTRATIONS OF AZO DYES FOUND IN SODA, FRUIT JAM, SALTED FISH AND SALTED VEGETABLES

Sulphonated azo dye found Soda-1 Soda-2 Soda-3 Soda-4 Fruit jam-1 Fruit jam-2 Fruit jam-3 Salted-fish-1 Salted fish-2 Salted-fish-3a Salted vegetable-1 Salted vegetable-2

Tartrazine Sunset yellow FCF Sunset yellow FCF Tartrazine Tartrazine Sunset yellow FCF Allura red AC Allura red AC New Coccine Tartrazine Sunset yellow FCF Tartrazine Sunset yellow FCF Tartrazine Sunset yellow FCF Tartrazine Sunset yellow FCF

Amount measured 4.9 mg l1 17.4 mg l1 15.4 mg l1 3.7 mg l1 10.0 mg l1 3.3 mg l1 33.4 mg kg1 17.9 mg kg1 19.8 mg kg1 136.0 mg kg1 118.1 mg kg1 123.7 mg kg1 49.6 mg kg1 292.5 mg kg1 132.1 mg kg1 83.0 mg kg1 105.4 mg kg1

a

The extract of this sample was diluted two-fold prior to analysis. Reprinted with permission from M.-R. Fuh et al. [114].

ranged from 3 – 11 and 5 – 17 g/kg, respectively. The same values for chilli tomato and cheese sauce varyied between 3 – 11 and 7 – 48 g/kg. The coefficient of correlation between analyte concentration and peak area was over 0.981, proving the good linearity of the analytical procedure. The precision of the intra-day repeatability also depended on the type of dye and the matrix (R.S.D. 1 – 13 per cent). Because of the high sensitivity, good validation parameters and simple sample preparation method the technique was proposed for the separation and quantitative determination of Sudan dyes in food products [115]. Another ion-interaction high-performance liquid chromatographic technique was evolved for the determination of sulphonated dyes in water. The chemical structures and common names of dyes under investigation are shown in Fig. 3.40. Dyes were extracted from spiked tap water (2.2 – 29 g/l depending on the type of dyes) acidified by 0.1 M HCl to pH  3 and loading the samples into ODS SPE cartridges (100 mg). Cartridges were preconditioned by 1 ml of methanol followed by 2 ml of phosphate buffer containing 2.4 mM of butylamine. After passing 100 ml of samples through the cartridges, the analytes were eluted with 2 ml of mobile phase (pH = 9). Separation of dyes was carried out in an ODS column (150 x 4.6 mm i.d.; particle size 5 m) at ambient temperature. The isocratic mobile phase consisted of ACN– phosphate buffer (27:73, v/v) at pH  6.7 containing 2.4 mM butylamine. The flow

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HO

HO

N

N

N

N

CH3

CH3 Sudan I (M.W. 248,28)

Sudan II (M.W. 276,34)

HO N N

N N

Sudan III (M.W. 352,40) HO N N

N CH3

N CH3 Sudan IV (M.W. 380,45) HO

N

N N

N Disperse Orange 13 (M.W. 352,40)

Fig. 3.38. Chemical structures of the azo dyes investigated and of the internal standard Disperse orange 13. Reprinted with permission from F. Calbiani et al. [115].

rate was increased during the separation process and analytes were detected at 460 nm. Chromatographic profiles of extract of spiked tap water sample and dye standards are shown in Fig. 3.41. The LOD and LOQ values showed high variations between the dyes being 4.74 – 28 g/l and 15 –56 g/l, respectively. The linearity range also depended on the type of dye varying form 15 – 498 g/l. The lowest recovery was 76±1 per cent (Acid Orange 52) the highest one 1004 per cent (Acid Red 88). It was concluded from the results that the method

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Chapter 3

(a)

6.95

m/z 249→93

1

% % 17.88 (i)

8.02 m/z 353→93 10.00 Time

m/z 353→120

m/z 381→224

(j)

17.94

8.05 m/z 353→93

2 5.00

15.00

11.30

2 100

1

9.80 m/z 277→121

2 100

%

%

m/z 381→224

m/z 249→93

2 100

(h)

5.00 (e)

11.26

1

100 %

m/z 353→120

1

100 (d)

(g)

%

% %

100 (c)

100

1

7.01

2

(f)

100 m/z 277→121 9.74 (b)

100 %

%

100

10.00 Time

15.00

Fig. 3.39. LC-ESI-MS/MS SRM traces obtained from 125 g/l standard solution of: (a) Sudan I; (b) Sudan II; (c) Sudan III; (d) Sudan IV; (e) Disperse orange 13 internal standard (100 g/l; left column) and from a blank chilli tomato and cheese sauce sample spiked with 125 g/l each (1 685 g/kg sample) of (f) Sudan I; (g) Sudan II; (h) Sudan III; (i) Sudan IV; (j) Disperse orange 13 internal standard (100 g/l; right column; injection volume 20 ). Reprinted with permission from F. Calbiani et al. [115].

applies a simple sample pretreatment and preconcentration technique, which is reliable, reproducible and can be employed for the trace analysis of these synthetic dyes in tap water [116]. LC/NMR and LC/MS have found application in the separation and identification of dyes and other xenobiotics in effluents of textile factories. Extraction of analytes was carried out by shaking 1L of waste-water with 4  20 ml of dichloromethane. The combined organic phases were dried with anhydrous sodium sulphate, filtered and evaporated to 1.0 ml. Prefractionation of the analytes was performed in an ODS column (250  4 mm i.d.; particle size 5 m) using an ACN–water gradient (from 80 to 100 per cent ACN in 30 min) at the flow rate of 0.4 ml/min. Fractions eluted at 0.1 – 10.5 min (fraction A) and 10.5 – 15.0 min (fraction B) were collected, evaporated to dryness and used for LC/NMR. LC/NMR measurements were realized using the same ODS column, gradient elution being carried out with ACN–deuterium oxide mixtures. Stopped-flow LC/NMR measurements were performed with the following gradients: for fraction A, ACN–D2O (60:40, v/v) for 5 min; to 90 per cent ACN in 25 min at the flow rate of 0.4 ml/min. The gradient for fraction B consisted of ACN–D2O (85:15, v/v) as starting composition and it was increased to 90 per cent ACN in 20 min at the flow rate of 0.8 ml/min. LC/MS analyses were carried out in the same ODS column. Gradient elution started with ACN–water (54:46, v/v) to reach 90 per cent ACN in 30 min followed by 10 min hold. APCI conditions were: vaporizer temperature, 450oC; discharge, 3 kV; capillary temperature, 150oC. Data were collected in the full-scan mode (positive and negative ions) in the range of m/z 50 – 1 000. Collision energy values ranged from 15 to 50 per cent depending

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429

Commercial Name C.I.number and name HO N

NaO3S

Acid Red 88 N

15620 Acid Red 88

HO

Acid Orange 7

15510

N

NaO3S

Acid Orange 7

N

Acid Orange 12

HO N

15970 Acid Orange 12

N

SO3Na Acid Yellow 1

OH NO2

NaO3S

10316 Acid Yellow 1

NO2 HO NaO3S

SO3Na

Acid Red 27

N

16185 Acid Red 27

N

Food Red 9

SO3Na CH3 HO N

SO3Na Xylidine Acid red 26 (2,4 Xylidine)

16150 Acid Red 26

N

SO3Na (CH3)2N

N

Acid Orange 52 N

(CH3)2N

SO3Na

HOOC N N

13025 Acid Orange 52

Acid Red 2

13020 Acid Red 2

Fig. 3.40. Structure and names of the dyes used. Reprinted with permission from M. Pérez-Urquiza et al. [116].

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0 (b)

2

R88

MO OU

MR 4

6

8 min

6

8 min

10

12

14

16

10

12

14

16

AMR

R88

MO. MR. ON.00

2

NYS. P2R

0 (a)

P2R Impurity

AMR

NY

430

4

Fig. 3.41. (a) Chromatogram of an extracted spiked tap water sample. The concentrations injected were NYS (126.1 g/l), P2R (61.2 g/l), AMR (167.1 g/l), OII (90.9 g/l), CO (116.2 g/l), R88 (245.1 g/l), MO (215.5 g/l) and MR (124.5 g/l). Chromatographic conditions: An RP-ODS stationary phase was used and the mobile phase was acetonitrile–phosphate buffer (27:73, v/v), pH 6.7 containing the ion-interaction reagent butylamine. Flow rate of 1 ml/min from 0 to 6 min and after 2 ml/min to the end. (b) Chromatogram of a dye mixture. Chromatographic conditions: mobile phase was acetonitrile–phosphate buffer (38:62, v/v), pH 6.7 containing the ion-interaction reagent butylamine. Flow rate 1 ml/min. Reprinted with permission from M. Pérez-Urquiza et al. [116].

on the type of analytes. The chemical structures of compounds identified or tentatively identified in the waste-water are listed in Fig. 3.42. The chromatographic profile of fraction A is shown in Fig. 3.43. Measurements indicate that peak 6 is 1,4-diaminoanthraquinone (disperse violet), peak 7 is disperse blue 3, and peak 10 is disperse blue 14. Peak 9 was identified as 7diethylamino-4-methylcoumarin and peak 11 as ethylene terephtalate cyclic trimer. It was concluded from the results that the combined use of hyphented techniques such as LC-NMR and LC-MS is a very efficient tool for the non-target analysis in complex environmental samples because each technique provides complementary information [117].

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Liquid chromatography of synthetic dyes

431 H

SO3H O

NCH3

O

NH2

1 8 C11H23 SO3H 2

CH3

9

C12H25

(C2H5)2N

O

O

H

SO3H

NCH3

O 3 10 C13H27 O H NCH2CH2OH

O

O

H

NCH3 O

O

O

4

O

H

NCH2CH2OH

11

O

NCH2CH2OH

O

O

O

O O

O 5 OH O

H

NCH2CH2OH

12

NH2

O

C9H19 6

OCH2CH2OH NH2

O

13 H C9H19

NCH3

O

d c b a OCH2CH2-X-OCH2CH2OH

7 O

H

NCH2CH2OH

X:(-OCH2CH2-)n

14 C9H19

Fig. 3.42. Chemical structures of compounds identified or tentatively identified in the effluent of a textile company by LC/NMR and LC/MS. Reprinted with permission from A. Preiss et al. [117].

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Chapter 3

The application of liquid chromatography–atmospheric pressure ionization–mass spectrometry in water analysis has been previously reviewed. The application of this technique for the analysis of synthetic dyes was also discussed [118]. 3.5.3 Degradation of synthetic dyes by physicochemical methods followed by HPLC The successful and possibly rapid degradation of toxic dye residues is of considerable importance form both a human and a veterinary toxicological point of view and for environmental protection. Because of the high amount of dyes loaded in the environment, much effort has been devoted to the development of physical, physicochemical and microbiological methods obtaining the degradation of dyes to non-toxic (even nutritive) derivatives. As has been previously mentioned, visible spectroscopic methods are widely applied for the measurement of the decomposition rate of dyes. However, when more than one dye molecule is simultaneously present or the primary decomposition products also absorb on

7 800 10

700

600

(mAU)

500 4 400 11 300 1,2,3 200

9

5 8

100

6

0

5

10

15 (min)

20

25

30

Fig. 3.43. UV chromatogram as obtained in the LC/NMR run of fraction A. For chromatographic conditions see text. Reprinted with permission from A. Preiss et al. [117].

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the same wavelength as the parent dye molecule, the reliability of spectrometry becomes questionable. The high separation capacity of chromatographic methods such as HPLC and CZ overcomes this difficulty. Fortunately, the character of method (physical or microbiological) applied for the degradation of dyes has a low impact on the efficacy of the chromatographic separation technique. Thus, the photocatalytic degradation of amaranth, a food azo dye, has also been followed by RP-HPLC measurements. A suspension of titanium dioxide and amaranth was subjected to strong photon flux, and the decolourization kinetics was followed by taking samples at various time intervals. The samples were filtered then injected into an ODS column (250 x 4.6 mm i.d.). The components of gradient elution were 6 mM aqueous ammonium acetate (pH adjusted to 3 with orthophosphoric acid) and ACN. The gradient started with 5 per cent ACN for 0–10 min; reached 20 per cent in 20 min; was isocratic to 25 min; and reached 80 per cent in 45 min. The flow rate was 1 ml/min and the concentration of amaranth was detected at 522 nm. Carboxilic acids and inorganic ions formed during the photodegradation were measured by ion chromatography. It was found that, during the photocatalytical process, colour intermediates were formed from the parent amaranth molecule. Their chemical structure is shown in Fig. 3.44. The results suggested that various chromatographic techniques promote the elucidation of the photocatalytical degradation process of the azo dye amaranth [119]. High-performance ion chromatoraphy has been employed for the investigation of the ozonation process of hydrolysed azo dye Reactive yellow 84 (CI). The chemical structure of the azo dye is shown in Fig. 3.45. The decolourization process was followed by visible spectrophotometry; ion chromatography was applied only for the separation of organic and inorganic ions formed during the ozonation [120]. High-performance ion chromatography has also been employed for the determination of the organic and inorganic end-products of the oxidative decomposition of Reactive yellow 84. Chromatographic separations were performed with an ion chromatograph operating in suppressed conductivity detection mode. The mobile phase consisted of an NaOH–water gradient and the flow rate was 1.5 ml/min. A typical chromatogram showing the separation of the end products of the oxidative decomposition of Reactive yellow 84 is shown in Fig. 3.46 [121]. The decolourization of the azo dye amaranth was also investigated using atomic hydrogen permeating through a Pt-modified palladized Pd sheet electrode. The decolouration products were separated by RP-HPLC in an ODS column. The isocratic mobile phase was 0.1 M aqueous orthophosphoric acid. The flow rate was 1.2  102 cm3/s and decomposition products were detected at 236 nm. The RP-HPLC system separated two analytes with retention times of 3.4 and 4.5 min, as demonstrated in Fig. 3.47. The peaks were

HO3S

HO N

HO N

N

N

Fig. 3.44. Coloured intermediates. Reprinted with permission from M. Karkmaz et al. [119].

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Chapter 3

Cl

Cl

N

N

N

N

H N

N

N H

NH

N N SO3Na

NaO3S

NaO3S SO3Na

SO3Na

NaO3S

SO3Na

N

N SO3Na

H N N

NaO3S

SO3Na

Fig. 3.45. Chemical structure of azo dye Reactive yellow 84. Reprinted with permission from M. Koch et al. [120].

Conductivity (µS)

50

3

6

40 30

7

20

2 1

10

4

5

0 2

4

6

8

10

12

Time (min)

Fig. 3.46. High-performance ion chromatographic analysis of C.I. Reactive yellow 84 after 120 min of the catalytic oxidation over Fe-Y80 catalyst. Initial conditions were 100 mg/l azo-dye, pH 5, t  50°C, catalyst concentration 1 g/l and 20 mmol H2O2. Peak identities are as follows: 1, acetate; 2, formate; 3, chloride (used for pH adjusting); 4, nitrate; 5, malonate; 6, sulphate; 7, oxalate. Reprinted with permission from M. Neamtu et al. [121].

tentatively identified as 1-naphtylamine-4-sulphonate (tR 3.4 min) and 1-amino-2-naphtol-3, 6-disulphonate (tR 4.5 min). It was concluded from the measurements that the atomic hydrogen permeating through the electrodes causes the decolourization of amaranth [122]. Nanocrystalline WO3 electrodes were also employed for the photoelectrochemical mineralization of Naphtol Blue Black (NBB) dye and the decomposition products were determined by RP-HPLC. Separations were carried out in an ODS column (250  4.6 mm i.d.; particle size 10 m) at room temperature. The mobile phase consisted of 0.5 per cent ammonium acetate
2 per cent acetic acid in 15 per cent ACN
85 per cent water. Typical chromatograms showing the decompositon of the dye and the appearance of the degradation products are shown in Figs 3.48 and 3.49. The chemical structures of NBB dye and the possible intermediate decomposition products are listed in Fig. 3.50. The chromatograms illustrate the good separation capacity of the RP-HPLC technique. It can be concluded from the results that method can be applied for the study of the photoelectrochemical degradation of the azo dye NBB [123]. An RP-HPLC method was employed for the investigation of the effect of solar light and TiO2 on decomposition of the textile dye Reactive blue 4 (RB4). The chemical structure of Reactive blue 4 is shown in Fig. 3.51. RP-HPLC measurements were performed in an ODS column. Chromatographic profiles demonstrating the effect of irradiation time and the

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Liquid chromatography of synthetic dyes

435

Decolorized by reducing agent

Decolorized by atomic hydrogen

0

5 10 Retention time / min

15

Fig. 3.47. High-performance liquid chromatograms of the amaranth solution decoloruized by atomic hydrogen and that by a reducing agent. The flow rate of eluent solution was 1.17  102 cm3/s. Reprinted with permission from Y. Yoshida et al. [122].

concentration of hydrogen peroxide are depicted in Figs 3.52 and 3.53. The chromatographic results supported the finding of other methods that solar light in the presence of TiO2 photocatalyst totally mineralysed the textile dye RB4 [124]. Another RP-HPLC method was applied for the investigation of the effect of TiO2 on the photocatalytic degradation of the leather dye Acid brown 14. The chemical structure of Acid brown 14 is shown in Fig. 3.54. Chromatographic measurements were performed in an ODS column using methanol–water (10:90, v/v) as the mobile phase. The flow rate was 1 ml/min and analytes were detected at 212 nm. The results demonstrated that Acid brown 14 is rapidly decomposed under solar light in the presence of a TiO2 photocatalyst [125]. A similar heterogeneous photocatalytic system was applied for the study of the decomposition of the anthraquinone dye, Acid blue 25 (AB25). The chemical structure of the dye and those of the first intermediates tentatively identified by HPLC-MS are shown in Fig. 3.55. RP-HPLC-DAD analysis of AB25 was carried out in a C4 column (250  4 mm i.d.; particle size 5 m) at ambient temperature. The isocratic mobile phase was composed of ACN (solvent A)–water (pH adjusted to 4.5 with acetic acid and ammonium acetate) (42:58, v/v).

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Chapter 3 6.76 7.54 12.62

Signal (mAU)

120

UV @ 280 nm

100

1

80

2 3

60 10.92 40

4 5

20

6

0 -2

0

2

4

6 8 10 Time (min)

12

14

16

Fig. 3.48. High-performance liquid chromatograms of (1) 8  105 M NBB dye, (2) 2  104M chromotrope 2B, (3) 2  104 M chromotrope 2R, (4) 1  103 M 1,2-naphtoquinone, (5) solution after photoelectrochemical degradation of NBB dye on a WO3 film electrode, at E  1.08 V versus. SCE, for 60 min, from 8  105 M NBB dye
0.5 M Na2SO4, and (6) solution after photoelectrochemical degradation of NBB dye on a WO3 film electrode, at E  1.08 V versus SCE, for 30 min, from 8 x 10-5 M NBB dye + 0.5 M NaCl. Reprinted with permission from M. Hepel et al. [123].

Naphthol blue black 100

Chromotrope 2B Chromotrope 2R

80 Signal (mAU)

1,2 Naphthoquinone

UV @ 280 nm

60

1

40 2 20 3

0 0

2

4

6 8 Time (min)

10

12

14

16

Fig. 3.49. High-performance liquid chromatograms of the NBB dye degradation solutions, recorded during the dye degradation on a WO3 electrode , at E  1.08 V versus SCE, from 2  104 M NBB
0.5 M NaCl solution with pH 4.5, for: (1) 0 min (2) 30 min, (3) 2 h. Reprinted with permission from M. Hepel et al. [123].

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437

O O

1,2-Naphtoquinone

SO3Na

NaO3S

N OH

OH

NO2

N

Chromotrope 2B

SO3Na

NaO3S

N OH

OH

N

Chromotrope 2R

Fig. 3.50. Chemical structures of NBB dye and the possible intermediate decomposition products. Reprinted with permission from M. Hepel et al. [123].

O

NH2 SO3Na Cl

O

NH

N NH

N N

SO3Na

Cl

Reactive blue 4

Fig. 3.51. Chemical structure of Reactive blue 4. Reprinted with permission from B. Neppolian et al. [124].

The flow rate was 1 ml/min and AB25 was detected at 600 nm (retention time, 10.2 min). LCMS was performed in an ODS column (150 x 3mm i.d.; particle size 3 m) using gradient elution from 0 to 100 per cent A in 25 min. MS measurements were made with ESI in negative mode. It can be concluded from the data that HPLC-DAD and HPLC-MS offer a unique possibility for the investigation of the photocatalytic degradation of Acid blue 25 and make possible the identification of coloured intermediates [126].

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Chapter 3

λ = 253 6

6 4h

8h

mV

4

mV

4

2

2

0

0

0

(a)

5

10

15

min

0

(b)

6

5

10

6 16h

24h

4 mV

mV

4

2

2

0

0

0 (c)

15

min

10

5 min

15

0 (d)

10

5

15

min

Fig. 3.52. HPLC chromatograms (RB4) as a function of irradiation time: (a) 4 h, (b) 8 h, (c) 16 h and (d) 24 h. Reprinted with permission from B. Neppolian et al. [124].

The efficacy of diamond and metal-alloy electrodes for the degradation of the textile dyes Basic yellow 28 and Reactive black 5 was also followed by RP-HPLC. The chemical structures of the textile dyes under investigation are shown in Fig. 3.56. An ODS column (150  4.6 mm i.d.; particle size 5 m) was employed for the RP-HPLC determination of

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6

439

6

λ = 253 4h

6h

4 mV

mV

4

2

2

0

0

10

5 min

0

(a)

10

5 min

0

(b)

6

6 8h

12h 4

mV

mV

4

2

2

0

0 0

(c)

10

5 min

5 min

0 (d)

10

Fig. 3.53. HPLC chromatograms (RB4) as a function of hydrogen peroxide (8.8  103 M): (a) 4 h, (b) 6 h, (c) 8 h and (d) 12 h. Reprinted with permission from B. Neppolian et al. [124].

NaSO3

N

OH N

N

SO3Na

N OH

Fig. 3.54. Chemical structure of Acid brown 14. Reprinted with permission from S. Sakthivel et al. [125].

the concentration of dyes. The mobile phase was methanol–KH2PO4.Na2HPO4 (pH  5) 20:80, v/v. The flow rate was 0.8 ml/min and the column was not thermostated. Analytes were detected at 450, 590 and 220 nm. Typical chromatograms illustrating the decomposition of Reactive black 5 are depicted in Fig. 3.57. It was established again that RP-HPLC

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Chapter 3 O

NH2 SO3Na

O

HN

Fig. 3.55. Chemical structure of Acid Blue 25 [1-amino-9,10-dihydro-9,10-dioxo-4-(phenylamino)2-anthracenesulphonic acid, monosodium salt] and the first intermediates tentatively identified by HPLC-MS. Reprinted with permission from I. Bouzaida et al. [126].

is a valuable tool for the determination of the concentration of textile dyes and their decomposition products formed during electrochemical treatments [127]. Remazol dyes such as Remazol Red RB, Remazol Golden Yellow RNL, Remazol Blue GG and Remazol Black B and their hydrolysis products were separated and identified by using RP-HPLCDAD and electrospray mass spectrometry. RP-HPLC measurements were performed in an ODS column (150  4.6 mm i.d.; particle size 5 m). The mobile phase for RP-HPLCDAD consisted of 1 per cent phosphoric acid–ACN (80:20, v/v), and the flow rate was 0.75 ml/min. Dyes were detected at 500 and 600 nm. Hydrolysis of dyes was carried out by thermostating 100 ml of 1.0 mM NaOH
0.5 g/l dye at 70°C for 15 min. After hydrolysis, 10 ml of sample were loaded into a strong anion-exchanging SPE cartridge preconditioned with 3 ml of methanol followed by 3 ml of deionized water. Analytes were eluted with 3 ml of 30 per cent (v/v) HCl in methanol. LC-MS-MS profiles of some dyes are shown in Fig. 3.58. It was found that the method is suitable for the separation and quantitative determination of these sulphonated azo dyes in effluents of textile plants [128]. The efficacy of the various methods used for the extraction of azo dyes from toy products has been determined and the results compared. The investigations were motivated by the possible toxic side-effect of the dyes. The chemical structures of azo dyes included in the study are shown in Fig. 3.59. Supercritical fluid extraction (SFE), microwave-assisted extraction (MAE) and Soxhlet extraction under various experimental conditions were applied for spiked poly(vinyl) chloride samples. Extracted dyes were separated in an ODS column (250  4.6 mm i.d.; particle size 5 m) using methanol as the mobile phase. Dyes are well separated by this method as demonstrated in Fig. 3.59. The optimal parameters of the extraction methods are compiled in Table 3.23. Recoveries depended on both the type of extraction method and the chemical structure of the dye. It was found that the highest recovery can be obtained by MAE and the extraction efficacy was the lowest for Solvent red 24 [129]. Liquid chromatographic techniques have been frequently employed for the separation and identification of the toxic decomposition products of synthetic dyes. Thus, the amount of aromatic amines formed from azo dyes in toys has been determined. The chemical structures of the dyes included in the investigation are listed in Fig. 3.60. The dyes were

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SLY C(CH3)2 OMe

N CH N

N+

CH3

CH3 CH3SO4CBWB

Na+-SO3-O-C2H4-SO2-

N N

SO3-Na+

N

SO3-Na+

HO H2N

Na+-SO3-O-C2H4-SO2-

N

Fig. 3.56. Chemical structures of textile dyes. Reprinted with permission from M. Cerón-Rivera et al. [127].

CBWB 5

-3

Detector response × 10 (au)

(a)

7b (b)

7a

0

0

5

10

15

20

Fig. 3.57. Chromatograms of CBWD (Reactive black 5) dye solution after 15 min electrolysis (b) under dynamic regime with flow at 3 ml/min in comparison with the solution before electrolysis (a). Working electrode Fe52; column OCTYL; flow rate 0.8 ml/min; mobile phase: (20:80) aqueous phosphate buffer, pH 5–methanol (2.5 min) and linear gradient buffer–methanol (20:80) to (50:50); temperature 25°C; detection wavelength 220 nm. Reprinted with permission from M. Cerón-Rivera et al. [127].

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Chapter 3 RT:4.41

Relative abundance

100

Remazol blue

80 60 40 20 0 1

2

3

4 5 6 Retention time (min)

Relative abundance

8

9

10

9

10

9

10

RT:4.45

100

Remazol black

80 60 40 20 0 1

2

3

4 5 6 Retention time (min)

7

8

RT:3.13

100 Relative abundance

7

Remazol yellow

80 60 40 20 0 1

2

3

4 5 6 Retention time (min)

7

8

Fig. 3.58. LC-MS-MS analysis of an effluent sample taken through an analytical method involving SPE. LC conditions: mobile phase acetonitrile–0.01 g/100 ml ammonium acetate in water (90:10, v/v); flow rate 0.30 ml/min; sample volume 20 l. The left-hand traces show the MS-MS ion chromatograms, while the corresponding right-hand spectra show the presence of the daughter ion indicative of each dye. RT  retention time. Reprinted with permission from W. F. Smyth et al. [128].

reduced and the decomposition products were separated by RP-HPLC using an ODS column (250  4.6 mm i.d.; particle size 5 m). Solvents A and B for gradient elution were methanol and 30 mM NaH2PO4-Na2HPO4, pH 6.9, respectively. The gradient started with 50 per cent A for 13 min, to 80 per cent A in 0.5 min, followed by 16.5 min hold. The flow rate was 1 ml/min and analytes were detected at 230 nm. The separations of aromatic amines are shown in Fig. 3.61. The results indicated that carcinogenic amines can be formed from synthetic dyes in toys and RP-HPLC is a suitable technique for the measurement of such decomposition products [130].

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B 0.30

(a)

A

CH2CH3 O2N

N=N CH2CH2OH

OH

0.25

(b)

N=N

AU

0.20 0.15 CH3

0.10 C

0.05

(c)

CH3

N=N

HO

N=N

0.00 5.00

10.00

15.00

20.00

Fig. 3.59. HPLC-UV chromatogram at 230 nm for the analysis of azo dyes. (a) Disperse red 1; (b) Solvent yellow 14; (c) Solvent red 24. Reprinted with permission from M. C. Garrigós et al. [129].

TABLE 3.23 OPTIMUM EXPERIMENTAL CONDITIONS FOR SFE, MAE AND SOXHLET EXTRACTION

Sample size (g) Extraction solvent Temperature (oC) Pressure (Mpa) Time Solvent volume (ml)

SFE

MAE

Soxhlet

0.4 CO2 120 55.2 30 min (static
dynamic) 8

0.5 MeOH 120 0.5 20 min

1 MeOH b.p. of solvent Ambient 7h

15

70

Reprinted with permission from M. C. Garrigós et al. [129].

A similar study was carried out using a different set of synthetic dyes. The chemical structures of the dyes are listed in Fig. 3.62. The decomposition products were separated in an ODS column (250  4 mm i.d.; particle size 7 m) using an isocratic mobile phase composed of methanol–water (45:55, v/v). The flow rate was 1.2 ml/min. The contents of aromatic amines determined by spectrophotometric and HPLC methods are compiled in Table 3.24. It was established that spectrophotometry can be used for the exact determination of the amines but it is inadequate for their separation. RP-HPLC proved to be a valuable method for the analysis of this class of decomposition products [131].

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Chapter 3 Dye

CAS number CI number

Disperse Red 1 O2N

N

2872-52-8

11 110

3180-81-2

11 115

CH2CH3

N

N CH2CH2OH

Disperse Red 13

Cl

O2N

N

CH2CH3

N

N CH2CH2OH

Disperse Orange 25

31 482-56-1

O2 N

N

11 227

CH2CH3

N

N CH2CH2CN

Solvent Black 3

4197-25-5

N

26150

NH CH3

N N

N

NH CH3

Solvent Orange 7

3118-97-6 12 140

CH3 HO N N

Solvent Red 24

85-83-6

26 105

HO

CH3 CH3

N

N

N

N

Solvent Yellow 2

60-11-7 N

11 020

CH3 N

N

CH3 Solvent Yellow 3

97-56-3

11 160

CH3 CH3

N

NH2

N

Solvent Yellow 14

HO

N

842-07-9

12 055

N

Fig. 3.60. Chemical structures of synthetic dyes. Reprinted with permission from M. C. Garrigós et al. [130].

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0.40 (6) (15) 0.30

(3)

AU

(7) 0.20

(11) (2) (1)

(17) (18)

(8) (5)

0.10 (4)

(10) (14) (9) (12) (13)

(16)

(20) (19) (21) (22)

0.00 5.00

10.00

15.00

20.00

25.00

30.00

Minutes

Fig. 3.61. HPLC-UV chromatogram at 230 nm for the analysis of the aromatic amines listed. (1) 1,4-Diaminobenzene; (2) 2-chloro-1,4-diaminobenzene; (3) 2,4-diaminotoluene; (4) benzidine; (5) 4,4-oxidianiline; (6) aniline and 4-nitroaniline; (7) o-toluidine; (8) 4,4-methylenedianiline; (9) 3,3-dimethoxibenzidine; (10) 3,3-dimethylbenzidine; (11) 4-chloroaniline and 2-amino-4nitrotoluene; (12) 4,4-thiodianiline; (13) p-cresidine; (14) 2,4-dimethylaniline; (15) 2-naphtylamine; (16) 4-chloro-o-toluidine; (17) 4,4-methylene-di-o-toluidine; (18) 2,4,5-trimethylaniline; (19) 4-aminobiphenyl; (20) 3,3-dichlorobenzidine; (21) 4,4-methylenbis (2-chloroaniline); and (22) o-aminoazotoluene. Reprinted with permission from M. C. Garrigós et al. [130].

High-performance ion chromatography (HPICE) was employed for the investigation of the mineralization process of reactive azo dyes under the effect of UV/H2O2 oxidation. Reactive red 120, Reactive black 5 and Reactive yellow 84 were oxidized under different experimental conditions and the organic acids (oxalic, malic, malonic, formic, acetic and succinic) formed during the decomposition process were separated in an ion-exchange column operated in suppressed conductivity detection mode. Analytes were eluted with heptafluorbutyric acid 0.4 m/l at a flow rate of 1.5 ml/min. A typical chromatogram illustrating the acid profile of the dye decomposition is shown in Fig. 3.63. [132]. A similar HPICE method was employed for the study of the oxidation of Reactive yellow 84 and Reactive red 120 by the photo-Fenton and Fenton-like processes [133]. Photocatalytic degradation has been frequently investigated by various HPLC techniques. Thus, the photodegradation of ethyl violet has been recently studied in detail [134]. The photodegradation of the aminoazobenzene acid orange 52 (AO52) was also followed by RP-HPLC. The dye was oxidized by UV/H2O2, UV/TiO2 and VIS/TiO2 systems and the primary decomposition products were separated by GC and HPLC. The chemical structure of AO52 is shown in Fig. 3.64. RP-HPLC measurements were performed in an ODS column (150  4.6 mm i.d.). The mobile phase contained 20 per cent methanol in water in 0 – 5 min, then enhanced to 100 per cent in 15 min, final hold 5 min. Decomposition products were detected at 210 nm. Compounds formed during the oxidation process carried out with UV/H2O2 are listed in Table 3.25. The results indicated that

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Chapter 3

Acid Red 85

HO O

N

N

O

S

H3C

N

N

O

NaO3S SO3Na Direct Blue 6

OH

NH2 OH N NaO3S

NH2

N N

N

SO3Na

NaO3S

SO3Na

Ponceau SS

SO3Na

HO

N

N

N

N

SO3Na Sudan III

HO N

N

N

Disperse Yellow 7

N

N N

N N

OH CH3

COOH

Mordant Orange 1 N O2N

OH

N

Disperse Orange 3 N O2 N

NH2

N

Fig. 3.62. Chemical structure of synthetic dyes. Reprinted with permission from A. Pielesz et al. [131].

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TABLE 3.24 CONTENT OF AROMATIC AMINES DETERMINED BY SPECTROPHOTOMETRIC AND HPLC METHODS

Contant of aromatic amines (g/kg dye) No. Dyea

Splitting amine

Spectrophotometry

HPLC

1 2 3 4 5 6 7

Benzidine Benzidine p-phenilenediamine, aniline p-phenilenediamine, aniline p-phenilenediamine, aniline p-PD p-PD

128 0.11 23.2 0.07 160 0.07 110.8 0.10 256 0.07 94.7 0.10 143.6 0.10

104 24.6 129.1, 31.5 73.7, 15.1 165.9, 89.2 132.8 0.088

Acid red 85 Direct blue 6 Ponceau SS Sudan III Disperse yellow 7 Mordant orange 1 Disperse orange 1

a

Dyes were analysed under more drastic conditions of reduction process. Reprinted with permission from A. Pielesz et al. [131]. 1

10 8

µs

6 4

3

2

2

4

5

0 0

5

10 Minutes

15

20

Fig. 3.63. HPICE analysis of RB5 after 45 min irradiation with 24.5 mml H2O2/l. Peak identities are as follows: 1, oxalate; 2, malonate (trace); 3, formate; 5, acetate. Reprinted with permission from M. Neamtu et al. [132].

toxic by-products can be formed during the decolouration process and RP-HPLC may help in the separation and identification of these molecules [135]. The photocatalytic decomposition products of methyl orange were separated by RPHPLC and identified by MS, MS/MS techniques. Methyl orange was irradiated in the presence of TiO2 dispersions. Samples were taken after various incubation times, filtered

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Chapter 3

N

NaO3S

CH3 N

N CH3

Fig. 3.64. Chemical structure of aminoazobenzene acid orange 52.

TABLE 3.25 ORGANIC COMPOUNDS DETECTED IN THE COURSE OF UV/H2O2 OXIDATION OF THE DYE AO52

H3C

H3C

CH3

CH3

N

N

1

2

NH2

OH

3

4

O H3C

CH3

OH

OH

N

OH

HO

OH

O

HO 6

5

HO

7

N

CH3

CH3 N

N

H 10

HOOC HO

11

N

CH3

9

CH(OH)2COOH

HO

N

N

SO3Na

8

COOH

HOOC

COOH

HOOC

COOH

HO

OH

OH

CH3COOH 12

HCOOH 13

Reprinted with permission from ref. 135. C. Galindo et al. [135].

14

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and analysed. Measurements were performed in an ODS column (250 x 4.6 mm i.d.; particle size 5 m) using ACN – 10 mM ammonium acetate pH 6.8 (24:76, v/v) as the mobile phase. Chromatographic profiles illustrating the decomposition of methyl orange are depicted in Fig. 3.65. It has been concluded from the measurements that HPLC-DAD and HPLC-MS are suitable methods for the study of the photocatalytic decomposition of methyl orange [136]. Combined analytical techniques, such as ion chromatography, GC-MS and FTIR, were employed for the investigation of the photocatalytic decomposition of triazine-containing azo dyes in the presence of TiO2 suspensions. The chemical structures of the dyes are shown in Fig. 3.66. HPLC was employed for the determination of the concentration of cyanuric acid, a stable metabolite of both dyes. The mobile phase consisted of 0.2 M orthophosphoric acid and the flow rate was 1 ml/min. The results demonstrated that the majority of metabolites were aromatic and aliphatic carboxylic acids separated and identified by FTIR, GC-MS and HPLC [137]. The effect of nanoporous TiO2 thin-film electrodes on the removal and degradation of the reactive textile dye Reactive orange 16 (R3R) was investigated by physicochemical analytical procedures including RP-HPLC. The chemical structure of the dye is shown in Fig. 3.67. Liquid chromatographic measurements were employed for the separation and detection of the decomposition products of the dye. They were realized in an ODS column

15

20

25

RT:0.00-29.99 SM:15G 263 100 302 90 80 70 60 50 40 306 30 320 276 20 10 0

322 0

5

10

15

20

25

90 80 70 60 50 40 30 20 10 0

293

320

0

216

308

5

10

15

20

218 308 292 338 322 0

5

10

320 320

15

20

276

320

306 322

5

10

15

20

25

RT:0.00-29.99 SM:15G 282 100

RT:0.00-29.99 SM:15G 100 90 80 304 70 60 50 40 30 20 10 0

200

0

25

Absorbance

10

90 80 70 60 50 40 30 20 10 0

Absorbance

Absorbance 5

Absorbance

Absorbance Absorbance

0

RT:0.00-29.99 SM:15G 304 100

RT:0.00-29.99 SM:15G 304 100

RT:0.00-29.99 SM:15G 302

100 90 80 70 60 50 40 30 20 10 0

25

90 80 70 60 50 40 30 20 10 0

304 276

308

0

5

336 320

322

10

15

20

25

Fig. 3.65. Chromatograms monitored in full-scan MS corresponding to solutions of methyl orange degraded at 0, 5, 10, 15, 22 and 27 min; each peak is characterized by its m/z value. Reprinted with permission from C. Baiocchi et al. [136].

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Chapter 3 Cl N

N

Cl

N

NH

N N SO3Na

SO3Na

Procion Red MX-5B A (MX-5B) Cl N

N N

NH

N

NH

N NaO3S SO3Na SO3Na

SO3Na Reactive Brilliant Red K-2G B (K-2G)

Fig. 3.66. The structures of triazine-containing azo dyes: (A) Procion red MX-5B (MX-5B) and (B) Reactive brilliant red K-2G (K-2G). Reprinted with permission from C. Hu et al. [137].

O NaO3SOCH2CH2

S

N

O

SO3Na

N HO

HN CO H3C

Fig. 3.67. Molecular structure of Reactive orange 16 dye. Reprinted with permission from P. A. Carneiro et al. [138].

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(250 x 4 mm i.d.; particle size 5 m) using a mobile phase consisting of methanol– water mixtures. The flow rate was 1 ml/min. The column was thermostated at 30°C. Chromatograms illustrating the degradation of the dye under different experimental conditions are depicted in Fig. 3.68. It has been established that the azo dye R3R can be decomposed under the given conditions and chromatography may help the separation and identification of the degradation products [138]. The efficacy of the solar photocatalytic decomposition of the azo dye Acid brown 14 by ZnO and TiO2 was compared and an RP-HPLC technique was applied to follow the degradation process. Chromatographic separations were carried out in an ODS column using an isocratic mobile phase of methanol–water (10:90, v/v), a flow rate of 1 ml/min, and UV detection at 212 nm. The measurements demonstrated that both ZnO and TiO2 are suitable for the promotion of the degradation of the azo dye Acid brown 14 [139]. The carcinogenic aromatic amines released from azo dyes in leather were investigated by using microwave-assisted extraction (MAE) or supercritical fluid extraction (SFE) followed by RP-HPLC. The chemical structures of dyes and aromatic amines are listed in Fig. 3.69. The flow schemes for SFE and MAE are shown in Figs 3.70. and 3.71.

100 80

5

60 40 20 0

4

3 2 1

0

1

2

3

4 5 6 Time (minutes)

7

8

9

10

Fig. 3.68. Analytical HPLC chromatograms with detection of diode array of 4.7 x 10-5mol/l of R3R dye curve (1) before and curve (2) after 180 min of photoelectrocatalysis on the TiO2 thin-film electrode biased at
1.0 V in Na2SO4 0.025 mol/l. Curve (4) before and curve (3) after photoelectrocatalysis in NaCl 0.022 mol/l and curve (5) after bleaching of 4.7  105 mol/l of R3R dye submitted to a chemical treatment by active chlorine addition. The mobile phase was methanol–water 80:20 per cent with a flow rate of 1 ml/min and controlled temperature at 30°C. The column was a Shimpack (Shimadzu) CLC-ODS, 5 m (250 mm  4.6 mm). Reprinted with permission from P. A. Carneiro et al. [138].

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Chapter 3 Azo dye

Acid Red 035

CH3

Amine o-Toluidine

O

N

CH3

OH HN N

NH2

HO3S

SO3H

Acid Orange 031

4-Chloroaniline

Cl

OH

N

Cl

N

HO3S

Acid Black 077 NH2 NH SO2

Benzidine

N N NH2 OH N

N

N

N

Acid Black 209

CH3 OH NH2

N

N HO3S

CH3

3,3'-Dimethylbenzidine CH3

HO

N

N

N

NH2

H2N

SO3H

HO3S

HO3S

NH2

N

OH

NH2

H2N CH3

SO3H

3,3'-Dimethoxybenzidine Direct Blue 015

OMe

OMe MeO

NH2 OH

N

N

N

N

OH NH2 NH2

NH2

H2N OMe

HO3S

SO3H

HO3S

SO3H

Direct Red 061

3,3'-Dichlorobenzidine Cl Cl NH2

N

N

N

Cl N

NH2

NH2

H2N Cl

SO3Na

SO3Na

Fig. 3.69. Chemical structures of the azo colourants together with the released carcinogenic amines investigated in this study. Reprinted with permission from C. S. Eskilsson et al. [140].

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Degreasing Step Ca. 0.3 of leather sample is placed in the SFE vessel. SF-CO2 at 138 bar and 40 °C for 30 min dynamic extraction

Reducing agent

CO2

Decolourizing step Addition of 1 ml of 0.1 g/ml sodium dithionite solution. Vertex mixing for 1 min. SF-CO2 at 138 bar and 40 °C for 10 min static extraction

Sequential SFE Step 1: CO2 at 138 bar and 40°C for 15 min at 4 ml/min Step 2: CO2 at 276 bar and 40°C for 15 min at 4 ml/min Step 3: CO2 with 2% of methanol at 276 bar and 40 °C for 15 min at 4 mllmin

Methanol

Collection Solid phase trapping or Solvent collection Methanol

ODS 40 or 80°C

20 ml acetone/methanol (1:1) with 100 µl of 1M HCl at 10°C

Elution volume of 1.5 ml Evaporation to 2 ml HPLC-DAD

Fig. 3.70. Flow scheme for the analytical procedure based on supercritical fluid extraction (SFE). Reprinted with permission from C. S. Eskilsson et al. [140].

Separation of amines was realized in an ODS column (250 x 3 mm i.d.; particle size 5 m) at 30oC. The flow rate was 0.3 ml/min and amines were detected at 280 nm. Solvents A and B for gradient elution were ACN and 3 mM phosphate buffer (pH  7). The gradient started with 15 per cent A for 2 min; then to 60 per cent A in 50 min. Chromatograms illustrating the separation of amines are shown in Fig. 3.72. It was established that the recoveries of both SFE and MAE were higher than those of traditional solvent extraction, therefore, their application for the analysis of carcinogenic aromatic amines in leather is highly advocated [140]. Both HPLC and GC-MS were employed for the separation, identification and quantitation of the decomposition products of indigo and indigo carmine. The chemical structures of the dyes are shown in Fig. 3.73. Carboxylic acids were preconcentrated before HPLC analysis either by ion-exchange SPE or by solid-phase microextraction. HPLC measurements were performed in a Sarasep column (300  7.8 mm i.d.) using 5 mM H2SO4 at a flow rate of 0.7 ml/min. Analytes were detected at 215 nm. The main intermediates formed during the photocatalytic decomposition are compiled in Table 3.26. The results demonstrated that

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Chapter 3

MAE vessel containing Ca. 0.3g of degreased leather sample

Addition of 20 ml buffer and 1 ml sodium dithionite solution (0.1 g/ml) MAE at 40 °C for 10 min. Filtration filtrate made up to 25 ml with methanol

Fraction 1

Extraction of the matrix with 8 ml methanol/buffer (1:1) MAE at 40°C for 10 min. Filtration filtrate made up to 10 ml with methanol

Fraction 2

Extraction of the matrix with 8 ml methanol MAE at 40°C for 10 min. Filtration filtrate made up to 10 ml with methanol

Fraction 3

Extraction of the matrix with 8 ml methanol and 200 µl of 2 MHCl. MAE at 40°C for 10 min. Filtration filtrate made up to 10 ml with methanol

Fraction 4

HPLC-DAD

Fig. 3.71. Flow scheme for the analytical procedure based on microwave-assisted extraction (MAE). Reprinted with permission from C. S. Eskilsson et al. [140].

TiO2/UV treatment results in the photocatalytic degradation of indigo and indigo carmine and chromatographic techniques may help the better understanding of the decomposition process [141]. The photo-oxidative decomposition of the mono-azo dye Acid red 27 (AC 27) in a tubular continuous-flow photoreactor was also followed by RP-HPLC. The chemical structure of the dye is depicted in Fig. 3.74. HPLC measurements were realized in an ODS column (220  4.6 mm i.d.; particle size 5 m) using an isocratic mobile phase consisting of ACN–water (30:70,v/v). The flow rate was 0.9 ml/min and the dye was detected at 254 nm. Chromatograms illustrating the effect of flow rate in the photoreactor and the influence of the length of the photoreactor on the degradation of AR27 are shown in Fig. 3.75. The chromatographic and spectrophotometric results proved that the tubular continuous-flow photoreactor is suitable for the decomposition of AR27 and HPLC is a valuable procedure to follow the degradation process [142]. HPLC-UV-MS was further applied for the monitoring of the degradation of Methyl Red and Methyl Orange by separating and identifying the decomposition products. The photocatalytic degradation was carried out in aqueous media containing oleic acid and

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500 6

Response (mV)

400

5

1

7

300 4 200

100

2

3

0 10

20

30 40 Time (min)

0

10

20

30 40 Time (min)

50

3,3-dichlorobenzidine

0 (a)

Response (mV)

80

60

40

20

4-aminobiphonyl

3,3-dimethoxybenzidine

100

0 (b)

50

Fig. 3.72. (a) Standard HPLC chromatogram. (1) Benzidine, (2) o-toluidine, (3) 4-chloroaniline, (4) 3,3-dimethoxybenzidine, (5) 3,3-dimethylbenzidine, (6) 4-aminobiphenyl; (7) 3,3-dichlorobenzidine. Detection wavelength: 280 nm; amino concentration: approximately 50 g/ml. HPLC conditions are described in the text. (b) Chromatogram of a genuine leather sample treated with the MAE procedure. Reprinted with permission from C. S. Eskilsson et al. [140].

tri-n-octylphosphine oxide-capped anatase TiO2 nanocrystal powders. The chemical structure of the dyes investigated are shown in Fig. 3.76. HPLC analyses were performed in a phenyl–hexyl column (150  3 mm i.d., particle size 3  m) employing the following gradient: from 50 mM ammonium acetate in methanol–methanol–water (5:25:70) to 5:75:20

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O

H N

N H

O

indigo chromophore group

O

H N

NaSO3 N H

NaSO3 O

indigo carmine

Fig. 3.73. Developed formulae of indigo and indigo carmine. Reprinted with permission from M. Vautier et al. [141].

HO

SO3Na

N NaO3S

N

SO3Na

Fig. 3.74. Chemical structure of AR27 (C.I.16185). Reprinted with permission from N. Danesvar et al. [142].

(a) Flow rate = 44 ml min4

(b) Flow rate = 19 ml min4

AR22

H2O2 (1)

(2)

(3)

(4)

(5)

(1)

(2)

(3)

(4)

(5)

Fig. 3.75. HPLC chromatograms of AR27, recorded during the dye degradation at different photoreactor lengths. (1) 0 cm, (2) 86 cm, (3) 172 cm, (4) 258 cm and (5) 335 cm. [AR27]0150 mg/l, [H2O2]0  650 mg/l, I058 W/m2. Reprinted with permission from N. Danesvar et al. [142].

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TABLE 3.26 MAIN INTERMEDIATE PRODUCTS DETECTED DURING PHOTOCATALYTIC DEGRADATION OF INDIGO CARMINE AND INDIGO

Compounds

Analytical techniques

2-Nitro-benzaldehyde and/or 2,3-dihydroxyindoline

GC-MS

Anthranilic acid

HPLC-UV GC-MS

21.8

137

Tartaric acid

HPLC-UV GC-MS

7.9

150

Malic acid

HPLC-UV GC-MS GC-MS

8.9

134

HPLC-UV GC-MS HPLC-UV

8.4

HPLC-UV GC-MS GC-MS

9.3

Glycolic acid

HPLC-UV GC-MS

11.3

76

Oxalic acid

HPLC-UV GC-MS HPLC-UV GC-MS HPLC-UV

6.4

90

17.9

59 121 60

Amino fumaric acid Pyruvic acid Malonaldehydic acid Malonic acid 3-Amino propenoic acid

Acrylic acid 3-Amino-2,3-dihydroxypropanoic acid Acetic acid

Retention time (min)

Molecular weight (g/mol) 151

131 88

9.3 104 87

13.8

Reprinted with permission from M. Vautier et al. [141].

in 10 min, final hold 5 min. MS conditions were for positive- and negative-ion mode: nebulizer gas (air), 1.2 l/min; curtain gas (nitrogen), 1 l/min; turbo ionspray gas (nitrogen at 350°C), 6 l/min ; needle voltage, 5 000 and 4 400 V; orifice declustering potential, 40 and 40 V, and focusing potential, 150 and 120 V. On the basis of the HPLC-MS results, the decomposition pathways proposed for Methyl Orange (MeOr) and Methyl Red (MeRed) are depicted in Fig. 3.77. The results indicated that HPLC measurements play a decisive role not only in the monitoring of the photocatalytic decomposition of dyes but also in the elucidation of the possible mechanism of degradation [143]. A similar system has been employed for the study of the photocatalytic degradation of 10 dyes. The intermediates and final decomposition products were separated and identified

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Organic dye

Chemical structure COOH

Methyl Red (MeRed)

N

CH3 N

N CH3

Methyl Orange (MeOr) -O3S

N

CH3 N

N CH3

Fig. 3.76. Chemical structures of dyes employed as target substrates in the TiO2-assisted photocatalysis. Reprinted with permission from R. Comparelli et al. [143].

by ion chromatography, XRD, NMR and IR methods. The chemical structures of the dyes included in the study are shown in Fig. 3.78. The results of HPLC analyses facilitated the elucidation of the photocatalytic degradation pathway of Methylene Blue, depicted in Fig. 3.79. [144]. A combination of physicochemical methods consisting of spectrophotometry, MS and reversed-phase ion-pair HPLC has been applied for the characterization of the textile anthraquinone dye Reactive Blue 4 (RB4) and its hydrolysis products. The chemical structure of Reactive Blue is shown in Fig. 3.80. Samples for HPLC separations were centrifuged, filtered and diluted when necessary. HPLC-DAD measurements were carried out in an ODS column (250  4.6 mm i.d.; particle size 5 m) at 40oC. The ion-pairing agent tributyl amine (1 mM) and 1 mM acetic acid were added to the components of the gradient (solvent A  water–methanol, 80:20, v/v; solvent B  water–methanol, 5:95, v/v). Elution started with 25 per cent methanol and it was increased to 95 per cent in 30 min, final hold 6 min. The flow rate was 0.5 m/min. Analytes were detected at 230, 250, 280, 485 and 598 nm. HPLC-ESI-MS analyses were realized with another ODS column (50  2 mm i.d.; particle size 3 m) at ambient temperature. The gradient was the same as described above for HPLC-DAD. The flow rate was 0.10 ml/min. The MS conditions were: capillary and cone voltages were 250 kV and 25 V, respectively. The source block and desolvation temperatures were 100 and 150oC, respectively. Nitrogen served as the desolvation gas (433 l/h) and nebulizing gas (81 l/h). The chromatographic profiles of reacted and unreacted RB4 determined by HPLC-DAD are shown in Fig. 3.81. The chromatograms demonstrate that the HPLC-DAD technique is suitable for the separation of hydrolysed and non-hydrolysed RB4 and its degradation products. The results of HPLC-MS were similar, as illustrated in Fig. 3.82. It was concluded that HPLC-DAD and HPLC-MS allow the separation and identification of RB4, its mono- and dihydrolyzed derivatives, and other degradation products such as 1-amino-4-hydroxyanthraquinone-2-sulphonic acid [145].

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HO3S

459

N

CH3 N

N

CH3 Methyl Orange

HO3S

N

N

NH2

N

2

OH

HO3S

CH3 N

N 4

HO3S

HO

N 1

HO

N

H N

N

CH3

CH3 N

N CH3

CH3

5

H N

N H N

N

COOH CH3

6

3

low molecular weight by-products

COOH

COOH

N

N

CH3

NH2

N

N

N

2

CH3

COOH methyl red N

1

OH

N

N 56

CH3 CH3

COOH

CH3

OH

OH

N

CH3

CH3 N

N

N

H N

N COOH N

OH

OH CH3

COOH

3

COOH

H N

N

N

OH CH3

N

N CH3

Fig. 3.77. Proposed degradation pathways for MeOr and MeRed during photocatalysis. The schemes illustrate that two mechanisms of degradation are independently active. Reprinted with permission from R. Comparelli et al. [143].

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Chapter 3

Dye

Chemical formula NH2

Congo Red (CR)

NH2 N=N

N=N

NaO3S

NaO3S

Methyl Orange NaO3S (MO)

N=N CH3

Ponceau G (PG)

N(CH3)2 SO3Na

HO

N=N

H2O

SO3Na OH Orange II (OH)

N=N

SO3Na

OH Eriochrome Blue Black B (EB)

O

NaO3S

OH

N=N

OH OH

Alizarin S (AS)

SO3Na O N

Methylene Blue (MB) (H3C)2N

N

Neutral Red (NR) (H3C)2N

(C2H5)2N

N(CH3)2

S+ Cr-

CH3 HCl NH2

N

N(C2H5)2-Cl

O

Rhodamine B (RB)

COOH

Fuchsin Acid (FA)

CH3 NH2 SO3Na

H2N SO3

Fig. 3.78. Chemical structures of 10 dyes photocatalytically degraded under visible-light irradiation. Reprinted with permission from Y. Yang et al. [144].

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N (H3C)2N

S+ Cr M 319

N(CH3)2

N (H3C)2N

S+

N(CH3)2

M 284 Detected M + H 285

N S+

H2N

NH2

M 258 Detected M+ H259

N

NO2

+ H2N

NH2 H2N

NH2

M 228 Detected M + H 229 NO3 +CH3COOH Detected by IC

HCOOH Detected by IC

Fig. 3.79. Photocatalytic degradation pathway of MB in the presence of H3PW12O40/TiO2. Reprinted with permission from Y. Yang et al. [144].

The adsorption of textile dyes on natural sorbents was investigated by various HPLC methods. The chemical structures and calculated molecular areas of the dyes are depicted in Fig. 3.83. Because of their different chemical structures and retention characteristics, the dyes were separated in different columns with different mobile phases. Basic blue 41 (BB41) was determined in an octylsilica column (75  4.6 mm i.d.). The mobile phase consisted of methanol–KH2PO4–Na2HPO4 buffer (pH  5) in volume ratio 1:1. Acid blue

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Chapter 3 NH2

O

SO3H

Cl N

N O

NH

NH

Cl

N

SO3H Cl N

OH or O-Cellulosate N + 2H2 O

R1

N

Cl

pH 11

N

N

T=60-80°C R1

N

+ 2H+ + 2Cl− OH or OCellulosate

HPLC Absorbance [mAU]

Fig. 3.80. Chemical structure of unreacted Reactive blue 4. Reprinted with permission from W. J. Epolito et al. [145]. 1600

500 400 300 200 100 0

(a)

Reacted RB4

a--Impurity 1 (27.4 min) b--Dihydrolyzed (27.6 min) c--Monohydrolyzed (27.8 min) d--Impurity 3 (28.2 min)

1200 1000 c

800

500 400 300 200 100 0

(b)

Unreacted RB4

600 400

a

200

d

0 0

5

10

15

20

25

30

700 HPLC Absorbance [mAU]

(d)

b

1400

DH

500

27.6 MH

UH 28.4

Impurity 1

700 (e)

h e--Dihydrolyzed (27.6 min) f--Monohydrolyzed (27.8 min) g--Impurity 2 (28.0 min) h--Unhydrolyzed (28.4 min)

1000 800

Impurity 2 & 3 27.8

600

200

0 27.0

600

1400 1200

400

100

500

1600 (c)

600

300

400

300

200

e

400 27.4

28.0 28.2 27.5 28.0 Retention time [nm]

200 28.5

f g

0 200

300

400 500 600 Wavelength [nm]

700

Fig. 3.81. HPLC analysis of reacted and unreacted RB4 (300 mg/l) total chromatogram at 598 nm (a and b), enlarged chromatogram from 27 to 28.5 min retention time (c), spectra of reacted RB4 componenets (d), and spectra of unreacted RB4 components (e) (DH, dihydrolysed; MH, monohydrolysed; UH, unhydrolysed. Reprinted with permission from W. J. Epolito et al. [145].

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5e+8

Intensity [counts]

4e+8 3e+8 2e+8 1e+8 0 (a)

0

5

10

15

20

25

30

35

40

45

50

5e+8 27.8

Intensity [counts]

4e+8 3e+8

1e+8

(b)

28.3

2e+8 26.9

28.6

0 25.0 25.5 126.0 26.5 27.0 27.5 28.0 28.5 29.0 29.5 30.0 Retention time [min]

Fig. 3.82. LC/ESI-MS analysis of reacted RB4 (500 mg/l) in negative ion mode of operation: total LC chromatogram (a) and enlarged chromatogram from 25 to 30 min retention time (b). Reprinted with permission from W. J. Epolito et al. [145].

74 (AB74) was analysed in another octylsilica column (250 x 4.6 mm i.d.). The mobile phase contained 15 per cent ACN and 85 per cent water (50 mM KCl
25 mM HCl). The concentration of Reactive black5 (RB5) was measured in an octylsilica column of 150  4.6 mm i.d. The components of the mobile phase were the same as for BB41 but the ratio of methanol was decreased to 25 per cent. The detection wavelengths were 615, 609 and 600 nm for BB41, AB74 and RB5, respectively. The amount of methylene blue in the solution used for the measurement of the relative specific area was determined in an RP-column (250  5 mm i.d.) using methanol–water 1:1 v/v as the mobile phase. It has been stated that the method using HPLC as an analytical tool can be applied for the study of the adsorption of dyes, furthermore, it can be employed for the optimization of adsorption efficacy in environmental protection studies [146]. New precursors for cyanine dyes were synthesized and the purity of the end products was checked by RP-HPLC. The chemical structures and UIPAC names of the intermediates are listed in Fig. 3.84. Purity control and the identification of the intermediates was performed in an ODS column

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OMe

Chapter 3

CH3

S N

N=N

Cr CH2CHCH2OCH3

N CH3

MeO

Basic blue 41 (BB41) 472 A2 NaO3S

O NH NH

SO3Na

O Acid blue 74 (AB74) 329 A+ Na+ SO3

O C2H4 SO2

N=N

SO3-Na+

HO H2N Na+ SO3

O C2H4 SO2

N=N

SO3-Na+

Reactive black 5 (RB5) 779 A2

Fig. 3.83. Studied dyes, their formulae, codes used in this work and calculated molecular areas. Reprinted with permission from M. M. Dávila-Jiménez et al. [146].

using water–ACN–TFA (80:19.95–0.05, v/v) as the mobile phase and MS detection mode. The results indicated that the RP-HPLC method applied can be successfully used for the analysis of this class of intermediates [147]. RP-HPLC found application in the monitoring of the alkali hydrolysis kinetics of alkali-clearable azo disperse dyes containing a fluorosulphonyl group. The chemical structures of dyes included in the experiments are shown in Fig. 3.85. Samples for RP-HPLC analysis were neutralized to pH 4.0 – 4.5 with diluted HCl mixed with five volumes of ACN and injected without any other sample preparation step. Separation was carried out in an ODS column at ambient temperature. The isocratic mobile phase consisted of ACN–water (80:20, v/v) and dyes were detected at their absorption maxima. HPLC measurements indicated that dyes are easily hydrolysed under relatively mild alkaline conditions, and the hydrolysis follows a pseudo first-order kinetics [148]. The effect of the electrochemical treatment on the degradation of some textile dyes was monitored by RP-HPLC. The chemical structures of dyes investigated are shown in Fig. 3.86.

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Fig. 3.84. Structures of products newly synthesized. Reprinted with permission from T. Deligeorgiev et al. [147].

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Chapter 3

H2O/p-Dioxane NHCOCH3 Reflux, 3 hrs FO2S

ClO2S

HCl/EtOH NHCOCH3 Reflux, 1 hr FO2S

NH2

Fig. 3.85. 4-(N,N-diethylamino)4-fluorosulphonylazobenzene dyes 1–4 used in the study. Reprinted with permission from J. Koh [148].

OH O

HO

CH

OH

CH2 O Sandocryl yellow (natural yellow 28)

O

O

Cl

Cl

SLY Sandocryl blue (vat blue 41

N H

N H

Cl

Cl

SLB Sandocryl green (basic green 4)

N(CH3)2

C

SLG Sandocryl red (basic violet 16)

N(CH3)2 :: Cl-

SLR C(CH3)2 N H3C

CH = CH + Cl-

N(C2H5)2

Sandolan orange (acid orange 7) SNO

HO NaO3S

N=N

Fig. 3.86. Structural fomulae and colour indices of the studied dyes. Reprinted with permission from M. M. Dávila et al. [149].

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467

Measurements were realized in an ODS (250 x 4.6 mm i.d.) and in an octylsilica column (150  4.6 mm i. d.) at 29°C. Particle size of both stationary phases was 5 m. Various mixtures of water–methanol and water–ACN were used as mobile phases containing phosphate buffers of different pH. Flow rate and detection wavelength also depended on the chemical character of the analytes. Chromatographic profiles illustrating the effect of electrochemical treatment on the dyes are depicted in Fig. 3.87.

0.003

0.03 SLB Org.

0

Defector response, au

0

SLY

0.015

aq. 0

0.09 SNO

0 0

10 Time, min

20

Fig. 3.87. Chromatograms of the batch solutions before (dotted lines) and after hydrolysis (continuous lines) of three dyes on a diamond electrode. Hexane extract of SLB (org.) and aqueous extract of SLY (aq.) (right axis). Column Octyl, flow rate 0.8 ml/min, temperature 29oC, detection wavelength 220 nm, mobile phase aqueous phosphate buffer (pH 5)–methanol (50:50, v/v) (SLY, SNO) and linear gradient methanol–water (40:60, v/v) to 50:50 (SLB). Reprinted with permission from M. M. Dávila et al. [149].

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Chapter 3

It was concluded from the results that RP-HPLC methods can be successfully employed for the study of the electrochemical decomposition of textile dyes [149]. The use of surface-enhanced resonance Raman spectroscopy (SERRS) as an identification tool in TLC and HPLC has been investigated in detail. The chemical structures and common names of anionic dyes employed as model compounds are depicted in Fig. 3.88. RP-HPLC separations were performed in an ODS column (100  3 mm i.d.; particla size 5 m). The flow rate was 0.7 ml/min and dyes were detected at 500 nm. A heated nitrogen flow (200oC, 3 bar) was employed for spraying the effluent and for evaporating the solvent. Silica and alumina TLC plates were applied as deposition substrates; they were moved at a speed of 2 mm/min. Solvents A and B were ammonium acetate–acetic acid buffer (pH  4.7) containing 25 mM tributylammonium nitrate (TBANO3) and methanol, respectively. The baseline separation of anionic dyes is illustrated in Fig. 3.89. It was established that the limits of identification of the deposited dyes were 10 – 20 ng corresponding to the injected concentrations of 5 – 10 g/ml. It was further stated that the combined HPLC-(TLC)-SERRS technique makes possible the safe identification of anionic dyes [150]. The effect of the composition of the mobile phase on the lateral diffusion of the dye 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (Dil) on the surface of HO N=N

NaO3S

N2H

OH N=N

SO3Na

NaO3S Food Yellow 3 CI 15985 HO

NaO3S

N=N

K

Acid Red 33 CI 17200

SO3Na

OH N=N

H3C

CH3

Acid Orange 7 CI 15510

SO3Na Food Red 1 CI 14700 NaO3S

O

HN

CH3 CH3

OH

NHO2S

CH3

N=N O

OH

Acid Violet 43 CI 60730

NaO3S

SO3Na

Acid Red 155 CI 18130

Fig. 3.88. Anionic dyes used in this study. Reprinted with permission from R. M. Seifar et al. [150].

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250

75 15985

50

14700

150 mAU

17200 15510

100

18130 25

MeOH (vol. %)

200

60730 50

0

0 0

5

10 Time (min)

15

20

Fig. 3.89. Separation of the six anionic dyes under gradient conditions in 5 m Hypersil ODS, 100  3 mm i.d. column. Eluent: (first solution) 25 mM TBANO3, 25 mM acetate buffer pH 4.7; (second solution) methanol. Flow rate, 0.7 ml/min; injection volume, 20 l; gradient indicated in the Figure; sample solution, mixture of anionic dyes, concentration of each 25 g/ml; Absorption detection at 500 nm. Reprinted with permission from R. M. Seifar et al. [150].

an ODS stationary phase was investigated by single molecular spectroscopy. The results indicated that the type of mobile phase exerts a considerable effect on the lateral diffusion of the theamphiphile dye molecule [151]. The mixed mode of sorption of the dye 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine pechlorate (Dil) at the interface of an ODS stationary phase and ACN–water mobile phase was studied by single-molecule resolution and fluorescence imaging techniques. The measurements indicated that minimally four types of adsorption sites are present on the surface of the ODS stationary phase. The desorption times of the dye are different at the different adsorption sites resulting in a deformed peak shape [152]. 3.5.4 Degradation of synthetic dyes by microbiological methods followed by HPLC The efficacy of chemical and photochemical decolourization of dyes in the effluent of textile factories has been proved many times. However, in the majority of cases these methods are relatively expensive and the solid catalysator used for the oxidation sometimes presents a new problem for environmental protection. Microbiological methods are less expensive, the dyes are decomposed to small organic and inorganic molecules which can be taken up by micro-organisms as carbon and nitrogen sources. Also, in the case of microbiological decomposition of dyes, liquid chromatographic techniques can be

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successfully employed to follow the decomposition of the original environmental pollutant for control and kinetic studies, to separate and identify the primary and secondary biodegradation products, etc. Because of the high number of synthetic dyes with entirely different chemical structures, and consequently entirely different capacity to be decomposed by micro-organisms, the ability of many species to decompose synthetic dyes has been investigated. Thus, the biodegradation of sulphonated phtalocyanin dyes by the white-rot fungus Bjerkandera adusta was followed by RP-HPLC coupled with DAD and MS. B. adusta was incubated in the presence of 200 mg/ml Reactive blue 15 (RB15) or Reactive blue 38 (RB38) and the degradation products were analysed by RP-HPLC. Preparations of lignin peroxidase (LiP) and mangane peroxidase (MnP) have also been tested for their capacity to degrade the dyes. RP-HPLC-DAD measurements were realized in an ODS column (250  4.6 mm i.d.; particle size 5 m). Eluents were water (A) and 90 per cent methanol (B) both containing 10 mM ammonium acetate. The gradient started with 100 per cent A for 0 – 8 min; to 100 per cent B in 48 min, final hold 2 min. The column was not thermostated, the flow rate was 1 ml/min and solutes were detected at 225 nm. HPLC-MS measurements were carried out in the same column at 30°C. The gradient programme was: 100 per cent A 0 – 8 min; 16 min, 25 per cent B, 18 min, 100 per cent B, 20 min, 100 per cent B. MS conditions in negative mode were: probe capillary voltage 3.25 kV at 400°C; cone voltage 43 V; source temperature 120°C. The chromatographic profiles of the decomposition products of the two dyes are shown in Fig. 3.90. The chromatograms clearly show that the decomposition products of dyes caused by the pure culture of B. adusta and MnP show a similar pattern suggesting the involvement of MnP in the decomposition process. LC-MS chromatographic profiles are shown in Fig. 3.91. The RP-HPLC measurements demonstrated that sulphophtalimide is the major metabolite formed from sulphonated phtalocyanine dyes by the white-rot fungus B. adusta [153]. Metabolites formed during the decolourization of the azo dye Reactive red 22 by Pseudomonas luteola were separated and identified by HPLC-DAD and HPLC-MS. The chemical structures of Reactive red 22 (3-amino-4-methoxyphenyl--hydroxyl-sulphone sulphonic acid ester) and its decomposition products are shown in Fig. 3.92. RP-HPLC measurements were carried out in an ODS column using an isocratic elution of 50 per cent methanol, 0.4 per cent Na2HPO4 and 49.6 per cent water. The flow rate was 0.5 ml/min, and intermediates were detected at 254 nm. The analytes of interest were collected and submitted to MS. RP-HPLC profiles of metabolites after various incubation periods are shown in Fig. 3.93. It was concluded from the chromatographic data that the decomposition process involves the breakdown of the azo bond resulting in two aromatic amines [154]. On-line HPLC methods were also developed for the continuous monitoring of the azo dye degradation processes in a bioreactor. The flow sheet diagram of the system is shown in Fig. 3.94. The dual HPLC systems allow the monitoring of the concentration of 1-amino-8-hydroxynaphtalene-3,6-disulphonic acid (H-acid), an azo dye precursor in the effluent. Separations were performed in an RP-column (125 x 4 mm i.d.; particle size 5 m) using gradient elution. Solvent A was ACN–water (10:90, v/v) containing 6 mM tetrabutylammonium hydrogensulphate and ACN was solvent B. The gradient was run from 0 to 25 min with the increase of B by 2 %/min. Then the concentration of ACN

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471 Reactive blue 15

AU

Reactive blue 15 0.10

0.20

0.08

0.15

0.06

0.10

0.04 0.05

0.02

0.00

0.00 (a)

0

10

20

30

1

0.10

0.15

0.08 AU

(d)

2

1 0.10

0.06 0.04

2

0.05

0.02 0.00

0.00 0

(b) 0.15

10

20

30

(e)

0

1

10.0

20.0

10

20

1

0.3 AU

0.10 0.2 2

0.05

2

0.1 0.00

0.0 0

(c)

10

20

Retention time (min)

30

0 (f)

30

Retention time (min)

Fig. 3.90. HPLC chromatograms (detection wavelength 225 nm) of the reaction products of RB15 (left, a–c) and RB38 (right, d–f). (a,d) Initial dye solutions (50 mg/l); (b,c) after incubation with MnP; (c,f) after incubation of 200 mg/l dye in cultures of B. adusta. The parent compounds RB15 and RB38 are not detected under these chromatographic conditions. Reprinted with permission from A. Heinfling-Weidtmann et al. [153].

was increased by 10%/min for 4 min and held at 90 per cent ACN for 4 min. The column was thermostated at 40oC. A chromatogram illustrating the separation of H-acid is shown in Fig. 3.95. It was established that the monitoring HPLC system is suitable for the control of textile dye effluents and for the detection of azo dye residues and intermediates [155]. A different HPLC system was developed for the monitoring of anaerobic azo dye degradation. The scheme of the system is shown in Fig. 3.96. The HPLC system was similar to that described in ref. [155]. A chromatogram illustrating the separation of the hydrolysed azo dye Reactive black 5 (RB5-H) and its decomposition products is shown in Fig. 3.97. This monitoring technique has also been proposed for the screening of the performance of bioreactors by measuring the composition of waste-water [156].

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O

3-SPI

O

4-SPI

N H

N H HO3S

O

SO3H O

162

182

4-SPI

3-SPI 118

162

226

226 118 80

(a)

162

182

118

162

226

226 118

50 100 150 200 250 300 m/z 0 (b)

5

10 Retention time (min)

15

50 100 150 200 250 300 m/z 20

Fig. 3.91. Chromatograms and mass spectra of (a) sulphophtalimide (SPI) and (b) reaction products of RB15 incubated with MnP of B. adusta. Chromatograms were obtained by LC-MS analysis detecting the molecular anion SPI (m/z 226  [SPI-H]) and spectra of the respective peaks were recorded by collision-induced dissociation of the molecular anion. Reprinted with permission from A. Heinfling-Weidtmann et al. [153].

The decompositon of two textile dyes in a sequencing batch reactor (SBR) was also followed by RP-HPLC. The chemical structure of dyes (Remazol brilliant violet 5R and Remazol black B) under investigation are shown in Fig. 3.98. The RP-HPLC system consisted of an ODS column (250  4 mm I.D.) using gradient elution. Components of the gradient were water and ACN containing 0.2 per cent tetrabutylammonium hydrogen sulphate. The gradient started with 25 per cent ACN and was increased to 40 per cent in 35 min. The flow rate was 1 ml/min, and dyes and decomposition products were detected at 224 nm. Some diagrams illustrating the evolution of the chromatographic peaks are shown in Fig. 3.99. The results indicated that the data obtained by RP-HPLC may promote the better understanding of the decomposition process of these textile dyes [157]. RP-HPLC has also found application in the study of the microbial decolourization of reactive azo dyes in a sequential anaerobic–aerobic system. The chemical structures of

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OMe OH (a)

N=N NaO3SOCH2CH2O2S SO3H OMe NH2

(b) NaO3SOCH2CH2O2S

OMe OH

?

N=N

OH NaO3SOCH2CH2O2S

H2N

SO3H

(c)

SO3H

(d)

Fig. 3.92. A proposed mechanism for decolourization of Reactive red 22 by Pseudomonas luteola. (a) Chemical composition of Reactive red 22, (b), (c) products resulting from complete cleavage of the azo bond of Reactive red 22, (d) the product resulting from the partial reduction of the azo bond of reactive red 22. Reprinted with permission from J.-S. Chang et al. [154].

dyes are shown in Fig. 3.100. Metabolites were extracted from anaerobic and aerobic cultures by acidifying the sample to pH 2–3 with 6 M HCl then shaken with equal volume of ethyl acetate. The organic phase was dried with anhydrous Na2SO4, evaporated to dryness and redissolved in methanol. Analyses were performed in an ODS column (150  4.6 mm i.d.). The isocratic mobile phase was 50 per cent methanol, 0.3 per cent orthophosphoric acid and 49.7 per cent water. The flow rate was 0.5 ml/min, metabolites were detected at 275 nm. The chromatographic profiles of dyes after various incubation steps are shown in Figs 3.101, 3.102, and 3.103. It was established from the results that the azo dyes are readily decomposed in the sequential anaerobic–aerobic system and the data of metabolites can be followed by RP-HPLC measurements [158]. The release of carcinogenic amines from azo dyes under the effect of a Streptomyces species was also investigated by RP-HPLC and GC-MS. The chemical structures of dyes and their reduction products are shown in Fig. 3.104. Aromatic amines were separated in an ODS column at 25°C using gradient elution. Components of the gradient were ACN (A) and water (B). The flow rate was 0.7 ml/min and metabolites were detected at 240 and 280 nm. LC-MS conditions were: nebulizer and curtain gas was nitrogen at flow rates of 10 and 8 l/h, respectively. Ion spray, orifice and ring voltages were 4 800, 40 and 170 V. Ion-spray source operated in positive-ion mode. Typical HPLC chromatograms are shown

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Chapter 3

(a)

A

CB (b)

A B C

(c) B

A

C

(d)

(e)

A

0

5

10

15

20

Elution time (min)

Fig. 3.93. The HPLC analysis on metabolites resulting from decolourization of reactive red 22 by Pseudomonas luteola (a) at the beginning of static incubation (IA  3 639 667, IB  130 140, IC 116 243), (b) after static incubation for 4.7 h (IA  2 231 542, IB = 230 559, IC = 120 563), (c) after static incubation for 23.4 h (IA  1 892 854, IB  428 414, IC  205 169), (d) 3-amino-4-methoxyphenyl -hydroxyl sulphone sulphonic acid ester (AMHSSAE), 90 per cent pure, 52 mg/l, and (e) products resulting from decolourization of Reactive red 22 by chemical reduction with SnCl2, (IA, IB, and IC represent intensities of peaks A, B, and C, respectively). Reprinted with permission from J.-S. Chang et al. [154].

in Fig. 3.105. The retention times of metabolites were as follows: 2,4-xylidine  26.41 min; 2,6-xylidine  26.93 min; 2,4,5-trimethylaniline  31 min; benzidine  15.13 min; 4-aminobiphenyl  33.64 min. It was concluded from the data that both enzymatic and chemical reductions result in the release of the same type of aromatic amines

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Waste HPLC 2

HPLC 1

Multi port value 2

Multi port value 1

Waste

P3

F4

F3

B3 F1

B4

F2

B2

B1

W1 Coloured waste water

P1

P2

Treated waste water

Fig. 3.94. Process and instrument flow sheet diagram: P1, P2 pumps; W1, heat exchanger; B1, B2, glass reactors; F1, F2, membrane cells; B3, B4 safety PTFE cells; F3, F4, HPLC filter frits. Reprinted with permission from A. Rehorek et al. [155].

which can be easily separated, identified and quantitated by RP-HPLC and GC methods [159]. The decolourization capacity of the white-rot fungus Phlebia tremellosa was investigated in detail. Dyes included in the experiments were Cibacron red, Remazol navy blue, Remazol red, Cibacron orange, Remazol golden yellow, Remazol blue, Remazol turquoise blue, and Remazol black B. The decomposition of Remazol black B was followed by RP-HPLC. Measurements were performed in an ODS column (250  4.6 mm i.d.) using ACN–water

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Chapter 3 100

0.05 0.04

80

60

0.02 0.01

40

Solvent (%)

Intensity (AU)

0.03

0.00 20 -0.01 0

-0.02 0

5

10

15 20 25 Retention time (min)

30

35

Fig. 3.95. Chromatogram of H-acid in the ultrafiltration permeate from the anaerobic reactor: injection volume 20 l, retention time 9.39 min, concentration 0.02 g/l original, 0.25 g/l after 11 h biomass treatment. Reprinted with permission from A. Rehorek et al. [155].

Waste Eluent A

Pump 4 DAD

Eluent B

Pump 3 UF-Module

BMR

Pump 2

ESIP 5441

Select B pre+analytical column

Teflon-cell Anaerobic bio-reactor

28m 1/16" PEEK BMR Valve 1 Pump 1

Valve 2 20µL loop

Fig. 3.96. Schematic figure of membrane sampling modules from bioreactor coupled to HPLC-DAD. Reprinted with permission from A. Plum et al. [156].

(60:40, v/v) containing 4.5 M CTAB as the mobile phase. The flow rate was 0.6 ml/min. The retention time of the dye was 4.57 min and the main metabolite eluted at 3.8 min [160]. Exact mass measurements were carried out on-line with RP-HPLC on a quadrupole mass spectrometer and the technique was applied for the analysis of anaerobically treated

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0.18

Absorption (-)

IN-SITU (585 nm)

ONLINE (585 nm)

0.14 0.1

RB5-H

TAHNDSDPI TAHNDS DPI

0.06 0.02 -0.02 0

Absorption (-)

(a)

1.7 1.5 1.3 1.1 0.9 0.7 0.5 0.3 0.1 0.1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

p-Base IN-SITU (270 nm)

0

1

2

3

4

5

6

(b)

7

8

9

10

11

12

13

14

15

ONLINE (270 nm)

16

17

18

19

20

Retention Time [min]

Fig. 3.97. Comparison of ion-pair chromatography separations of RB5-H and metabolites by on-line and in situ sampling coupled to HPLC-DAD from bioreactor: (A) 585 nm scanning wavelength, (B) 270 nm scanning wavelength. Peak identification: TAHNDSDP1  7-amino-8-hydroxy-1,3-naphtoquinone-3,6-disulphonate-1,2-diamine; TAHNDSDP2  dihydroxynaphtoquinone-3,6-disulphonatediimine; p-Base  2-(4-aminobenzenesulphonyl)ethanol. Reprinted with permission from A. Plum et al. [156].

Remazol brillant violet 5R Cu O Na+O3-SOCH2CH2O2S

O

NHCOCH3

N=N Na+-O3S

SO3Na

Remazol Black B OH Na+O3-SOCH2CH2O2S

NH2

N=N

N=N

NaO3S

SO3Na

SO2CH2CH2OSO3Na

Fig. 3.98. Chemical structure of the reactive dyes used in the present study. Reprinted with permission from N. D. Lourenco et al. [157].

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Chapter 3 2.5

120

2.0

100 80

1.5

60 1.0 40 0.5

Dye conc. (mg l-1)

Peak area x 10-6

478

20 0

0 0

2

4

6 Time (h)

(a)

24

2.5

160 120 100

1.5

80 1.0

60 40

0.5

Dye conc. (mg l-1)

Peak area x 10-6

140 2.0

20 0

0 0 (b)

2

4

6

8 10 Time (h)

12

24

Fig. 3.99. Examples of the evolution of the chromatographic peak areas corresponding to the dye metabolites during SBR 1 cycles in periods 2 (a) and 3 (b). Metabolites 1 (•) and 3 (ⴱ) correspond to the benzene-based and naphtalene-based amines, respectively. Metabolite 2 (o) is apparently in equilibrium with metabolite 1. The violet dye concentrations calculated form spectrophotometric analysis (ⵧ) are also presented. Reprinted with permission from N. D. Lourenco et al. [156].

textile waste-water. Different RP-HPLC methods were employed for the analytes detected in positive- and negative-ion modes. An ODS column (50  2 mm i.d.; particle size 3 m) was used for the analytes detected in the positive-ion mode. Solvents A and B for the gradient were 3 per cent ACN and 75 per cent ACN both containing 1 per cent formic acid. The gradient started with 100 per cent A and reached 100 per cent B in 8 min followed with 1 min hold. The flow rate was 0.25 ml/min. Solutes detected in the negative-ion mode were separated in a column of the same dimensions as before, filled with a phenylhexyl stationary phase. Eluents A and B were 30 per cent and 70 per cent aqueous methanol both with 1 mM of acetic acid and 1 mM of tributylamine. Gradient conditions were: 0–50 per cent B in 20 min; to 100 per cent B in 1 min, and 2 min hold. The flow rate was 0.2 ml/min. The IUPAC names of compounds investigated in both positive- and negative-ion modes are compiled in Table 3.27. It was stated that the method allows the exact determination of the molecular mass of a wide variety of compounds facilitating the analysis of textile factory effluents [161].

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A. (Monoazo-vinylsulfone structure) OH Na+O3-SOCH2CH2O2S

NHCOCH3

N=N Na+-O3S

B. (Diazo-vinylsulfone structure) Na+O3-SOCH2CH2O2S

SO3Na

N=N HO H2N

Na+O3-SOCH2CH2O2S

N=N

SO3 Na

C. (Copper complex-monoazo-vinylsulfone structure) Cu O

O

Na+O3-SOCH2CH2O2S

NHCOCH3

N=N Na+-O3S

SO3Na

Fig. 3.100. Chemical structures of: (A) Remazol brilliant orange 3R; (B) Remazol black B; and (C) Remazol brilliant violet 5R. Reprinted with permission from N. Supaka et al. [158].

5.361

A

A 3.582 6.65

11.812 1

3.767 5.35

B

2

3

Fig. 3.101. HPLC analysis on metabolites resulting from decolourization of Remazol brilliant orange 3R under anaerobic–aerobic conditions: (1) at the beginning of the anaerobic incubation; (2) after anaerobic incubation for 24 h; and (3) after aerobic incubation for 12 h. Reprinted with permission from N. Supaka et al. [158].

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Chapter 3

5.392

A

3.397 5.362

A

11

6.6

3.582

B

3

2

1

5.5

3.968 4.582

4.045

4.572

Fig. 3.102. HPLC analysis on metabolites resulting from decolourization of Remazol black B under anaerobic–aerobic conditions: (1) at the beginning of the anaerobic incubation; (2) after anaerobic incubation for 24 h; and (3) after aerobic incubation for 12 h. Reprinted with permission from N. Supaka et al. [158].

1

2

3

Fig. 3.103. HPLC analysis on metabolites resulting from decolourization of Remazol brilliant violet 5R under anaerobic–aerobic conditions: (1) at the beginning of the anaerobic incubation; (2) after anaerobic incubation for 24 h; and (3) after aerobic incubation for 12 h. Reprinted with permission from N. Supaka et al. [158].

3.5.5 HPLC separation of synthetic dyes in model mixtures There is everlasting controversy and everlasting cooperation between analytical chemists dealing with chromatography. Academic research is generally not interested in the solution of practical problems, only with the theory of separation, with the development of new separation processes and with the mathematically based explanation of retention behaviour.

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Liquid chromatography of synthetic dyes CH3 H3C

481 SO3Na

HO

N N

SO3Na Xylidine Ponceau 2 R Chemical reduction NH2

Enzymatic reduction NH2

NH2

H3 C

CH3

CH3

+

CH3

+ H3C CH3

CH3

2,6-Xyilidine

2,4,5-Trimethylaniline

2,4-Xylidine

HO

H2N

NH2 N N N

H2N

N

N

N

SO3Na

NaO3S C.I. Direct Black 38 Chemical reduction H2N

NH2

Enzymatic reduction +

NH2

Benzidine HO

4-Amino-biphenyl

N

N

N

N

N

N H2N

SO3Na

NH2

C.I. Direct Brown 1 Chemical reduction

H2N

Enzymatic reduction

NH2 Benzidine

NH2

+ 4-Amino biphenyl

Fig. 3.104. The structure of dyes and their reduction products. Reprinted with permission from M. Bhaskar et al. [159].

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15.133

0.30 0.070 0.26

1

482

0.060

30.375

0.18 A.U

3

0.040 2

0.14

26.412 25.920

1

0.030

0.10

0.010

0.06

0

0.02

2

0.020

33.783

A.U

0.22 0.050

−0.02

(a)

20 Minutes

30

40

10

20 Minutes

(b) 15.173

0.18

30

40

2

10

0.12

A.U

0.09

2

0.06

33.642

0.03

0

(c)

10

20 30 Minutes

40

Fig. 3.105. HPLC chromatograms for enzymatic reduction. (a) Xylidine ponceau-2R (1  2,4-xylidine, 2 = 2,6-xylidine, 3 = 2,4,5-trimethylaniline). (b) Direct black-38 (1  benzidine, 2  4aminophenyl). (c) Direct brown-1 (1  benzidine, 2  4-aminobiphenyl). Conditions: mobile phase, acetonitrile (A) and water (B); flow rate, 0.7 ml/min; 25°C; injection volume, 10 l; gradient elution: 0 min, A 23 per cent, B 77 per cent; 0–21 min, A 34 per cent and B 66 per cent; 21–30 min, A 60 per cent and B 40 per cent; 30–34 min, A 70 per cent and B 30 per cent; 34–37 min, A 90 per cent and B 10 per cent; and 37–40 min, A 23 per cent and B 77 per cent. Detection at 280 nm. Reprinted with permission from M. Bhaskar et al. [159].

Industrial laboratories, legislation and control offices for human welfare, food and environmental protection are mainly interested in practical methods, which are maximally rapid, easy-to-carry-out, reliable and reproducible. However, this controversy exists only on the surface of chromatographic science. Practical problems always represent a serious challenge for the theoretical chromatographers and frequently offer new directions of research. On the other hand, practical analysts cannot solve uncommon problems without adequate knowledge of the theory of separation science.

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TABLE 3.27 IUPAC NAMES OF COMPOUNDS INVESTIGATED IN BOTH POSITIVE- AND NEGATIVE-ION MODES

Positive-ion electrospray No.

IUPAC name

1 2 3 4 5 6 7 8 9 10 11 12

Sulphadiazine Sulphathiazole Sulphamerazine Sulphamethazine Sulphamethoxazole Sulphadoxine Sulphaquinoxaline Sulphaphenazole Sulphasalazine 10-Desacetylbaccatine III Baccatine III Erythromycine

Negative-ion electrospray No.

IUPAC name

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

3-Aminobenzenesulfonate 1-Aminonaphtalene-7-sulphonate 1-Aminonaphtalene-5-sulphonate Benzenesulphonate Naphtalene-1,5-disulphonate 6 Naphtalene-2,6-disulphonate 1-Hydroxynaphtalene-4-sulphonate Toluene-4-sulphonate 3-Nitrobenzenesulphonate 1-Aminonaphtalene-8-sulphonate Naphtalene-1-sulphonate Naphtalene-2-sulphonate 1-Hydroxynaphtalene-2-sulphonate Anthraquinone-2-sulphonate Methylenebis-8,8-naphtalene Reactive orange Reactive black 5 Reactive red 198

Reprinted with permission from T. Storm et al. [161].

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Thus, RP-HPLC-MS has been employed for the analysis of sulphonated dyes and intermediates. Dyes included in the investigation were Acid yellow 36, Acid blue 40, Acid violet 7, Direct yellow 28, Direct blue 106, Acid yellow 23, Direct green 28, Direct red 79, Direct blue 78 and some metal complex dyes such as Acid orange 142, Acid red 357, Acid Violet 90, Acid yellow 194 and Acid brown 355. RP-HPLC was realized in an ODS column (150  3 mm i.d.; particle size 7 m). The composition of the mobile phase varied according to the chemical structure of the analytes to be separated. For the majority of cases the mobile phase consisted of methanol–5 mM aqueous ammonium acetate (10:90, v/v). Subsituted anthraquinones were separated in similar mobile phases containing 40 per cent methanol. The flow rate was 1 ml/min for UV and 0.6 ml/min for MS detection, respectively. The chemical structure of dye intermediates investigated in this study and their retention times are compiled in Table 3.28. It was found that the method is suitable for the separation of decomposition products and intermediates of dyes but the separation of the original dye molecules was not adequate in this RP-HPLC system [162]. A similar HPLC technique was applied for the analysis of polysulphonated anionic dyes and their intermediates. Separations were carried out in two ODS columns (column I  150  3 mm i.d.; particle size 7 lm and column II  150  4.6 mm i.d.; particle size 5 lm). Aromatic sulphonic acids were eluted from column I with methanol–5 mM ammonium acetate in water (10:90, v/v). Components of gradient elutions for column II were 2.5 mM dihexylammonium acetate in water (solvent A) and 2.5 mM dihexylammonium acetate in methanol (solvent B). Naphtalenesulphonic acids were separated by gradient a (from 50 per cent B in 0 min to 80 per cent B in 35 min). Sulphonated dyes were eluted with gradient b (from 60 per cent B in min to 100 per cent B in 20 min). ESI ion source temperature was 100oC, the cone voltage was 30 V. The retention times of naphtalene sulphonic acids measured in both columns are compiled in Table 3.29. It was concluded from the data that naphtalene sulphonic acids can be successfully separated in an ODS column using gradient elution and an ion-pairing agent. The colour index, commercial names and retention times, tR (min), of dyes are compiled in Table 3.30. The retention times of the majority of dyes showed considerable differences, indicating that the method can be applied for the separation of dyes [163]. Micro high-performance liquid chromatography using mobile phases containing cyclodextrin has also been employed for the analysis of naphthalene sulphonic and aminonaphtalene sulphonic acids. Microcolumns included in the investigation were of various lengths (135–166 mm), the i.d. was always 0.32 mm, and the particle size 5–7 m. Mobile phases contained water, aqueous methanol, Na2SO4 or tetrabutylammonium hydrogensulphate, -cyclodextrin (0.01 M) and triethylamine (0.2–2.0 per cent). The flow rate ranged 3–5 l/min, the columns were not thermostated, and analytes were detected at 220–230 nm. The chemical structures of aminonaphtalene sulphonic acids are listed in Fig. 3.106. The retention factors of naphthalene sulphonic acids are compiled in Table 3.31. The retention data indicated that both columns can be employed for the separation of the dye intermediates of naphthalene suphonic acid, the retention is influenced by both the presence of a strong electrolyte and organic modifier in the mobile phase. The effect of eluent additives on the microcolumn separation and retention behaviour of aminonaphtalene sulphonic acids is illustrated in Fig. 3.107. It was

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TABLE 3.28 SULPHONATED DYE INTERMEDIATES STUDIED IN THIS INVESTIGATION

No.

Compound

tRa(min)

Naphtalene sulphonic acids 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Naphtalene-1-sulphonic acid Naphtalene-2-sulphonic acid 1-Aminonaphtalene-6-sulphonic acid 1-Aminonaphtalene-7-sulphonic acid 2-Aminonaphtalene-7-sulphonic acid 5-Aminonaphtalene-1-sulphonic acid 8-Aminonaphtalene-1-sulphonic acid 7-Amino-1-hydroxynaphtalene-3-sulphonic acid 6-Amino-1-hydroxynaphtalene-3-sulphonic acid 1,6-Dihydroxynaphtalene-3-sulphonic acid 6-Aminonaphtalene-1,3-disulphonic acid 8-Amino-8-hydroxynaphtalene-3,6-sulphonic acid 1-Amino-8-hydroxynaphtalene-3,6-sulphonic acid 1,8-Dihydroxynaphtalene-3,6-disulphonic acid 5,5-Dihydroxynaphtalene-2,2-dinaphtylamine-7,7-disulphonic acid 7-Aminonaphtalene-1,3,6-trisulphonic acid 8-Aminonaphtalene-1,3,6-trisulphonic acid

13.4 15.2 6.9 9.3 8.5 3.0 13.1 4.6 3.4 5.1 1.8 1.8 2.5 1.6 3.8 1.5 1.7

Other sulphonic acids 18 19 20

2,5-Dichloroaniline-4-sulphonic acid 1-(2,5-Dichloro-4-sulpho)-phenyl-3-methylpyrazolone 4,4-Dinitrostilbene-2,2-disulphonic acid

4.7 5.2 9.0

Anthraquinone sulphonic acids 21 22 23 24 25 26 27 28 29 a

Anthraquinone-1-sulphonic acid Anthraquinone-2-sulphonic acid 1-Aminoanthraquinone-2-sulphonic acid 1-Amino-4-hydroxyanthraquinone-2-sulphonic acid 1-Chloroanthraquinone-2-sulphonic acid 1-Amino-4-bromoanthraquinone-2-sulphonic acid Anthraquinone-1,5-disulphonic acid Anthraquinone-1,8-disulphonic acid Anthraquinone-2,6-disulphonic acid

9 13.4 8.1b 11.6b 16.8b 13.0b 1.7 3.9 2.7

Retention time, HPLC conditions: 10 per cent MeO–90 per cent 5 mM aqueous NH4OAc, flow rate 1 ml/min, other in text. b Mobile phase 40 per cent MeOH–60 per cent 5 mM aqueous NH4OAc, flow rate 1 ml/min. Reprinted with permission from M. Holcapek et al. [162].

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Chapter 3 TABLE 3.29 RETENTION TIMES, tR (MIN) IN REVERSED-PHASE HPLC OF NAPHTALENE SULPHONIC ACIDSa

No.

Compound

(a) tR (min)

(b) tR (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

7-Amino-1-hydroxynaphtalene-3-sulphonic acid 5-Aminonaphtalene-1-sulphonic acid 6-Amino-1-hydroxynaphtalene-3-sulphonic acid 2-Aminonaphtalene-7-sulphonic acid 1,6-Dihydroxynaphtalene-3-sulphonic acid 1-Aminonaphtalene-6-sulphonic acid 1-Aminonaphtalene-7-sulphonic acid 1-Amino-8-hydroxynaphtalene-3,6-disulphonic acid 6-Aminonaphtalene-1,3-disulphonic acid Naphtalene-1,5-disulphonic acid Naphtalene-1,6-disulphonic acid Naphtalene-1-sulphonic acid 8-Aminonaphtalene-1-sulphonic acid Naphtalene-2-sulphonic acid Naphtalene-1,3-disulphonic acid Naphtalene-1,7-disulphonic acid 1,8-Dihydroxynaphtalene-3,6-disulphonic acid Naphtalene-1,3,7-trisulphonic acid 8-Aminonaphtalene-1,3,6-trisulphonic acid Naphtalene-1,3,6-trisulphonic acid Naphtalene-1,3,5-trisulphonic acid Naphtalene-1,3,5,7-tetrasulphonic acid

4.2 4.6 5.4 5.4 5.6 5.7 8.1 9.2 9.8 10.2 11.0 11.4 11.5 11.7 13.0 14.0 15.8 16.7 16.9 17.4 18.0 23.8

4.6 3.0 3.4 8.5 5.1 6.9 9.3 2.5 1.8 – – 13.4 13.1 15.2 – – 1.6 1.7 – – – –

Flow rate  1 ml/min at 40°C. (a)  linear gradient, 50–80 per cent B in 35 min; A: 2.5 mM dihexylammonium acetate (DHAA) in water, B: 2.5 mM DHAA in methanol column II (150  4.6 mm i.d., V0  1.66 ml). (b)  isocratic elution, 4.5 mM ammonium acetate in 90 per cent aqueous methanol, column I (150  3.3 mm i.d., V0  0.87 ml). Reprinted with permission from M. Holcapek et al. [163]. a

found that microbore columns can be used for the separation of this class of dye intermediates when the mobile phase contains both cyclodextrin and triethylamine. The low solvent consumption and high separation capacity makes the method an attractive and possible alternative for the RP-HPLC analysis of naphthalene sulphonic acid derivatives [164]. An ion-pair liquid chromatographic method was developed for the separation of sulphothalimide (SPI) and its derivatives. The investigations were motivated by the

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TABLE 3.30 RETENTION TIMES, TR (MIN) IN GRADIENT ELUTION REVERSEDPHASE HPLC OF SULPHONATED DYESa

No.

Colour index name

Trade name

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Acid violet 7 Acid yellow 23 Acid orange 10 Acid yellow 36 Direct red 79 Acid blue 40 Acid red 118 Direct blue 106 Mordant yellow 8 Reactive green 8 Acid red 357 Direct green 26 Reactive blue 109 Direct blue 78 Direct green 28 Acid violet 90 Acid yellow 194 Acid orange 142

Egacid red 6B Egacid yellow T Egacid orange GG Egacid yellow M Saturn red L4B Egacid blue A2G Midlon red E Saturn blue LB Alizarin chrome, yellow R Ostazin olive H-G Rylan red 3G Saturn green LB Procion blue MX-26 Saturn blue L4G Saturn green L5G Rylan bordeaux B Rylan yellow 3R Rylan orange R

tR (min) 7.4 8.7 10.0 13.8 13.9 14.1 14.5 14.5 15.2 15.8 16.1 17.8 18.3 18.4 20.3 20.5 21.9 22.7

a

Linear gradient, 60–100 per cent B in 20 min. For other conditions see text. Reprinted with permission from M. Holcapek et al. [163].

finding that SPI is the main metabolite of the oxidation of sulphophtalocyanine dyes and can increase environmental pollution. The chemical structures of the compounds separated are listed in Fig. 3.108. Separation of analytes were performed in an ODS column (150  3 mm i.d.; particle size 3 m). Eluent A was water–methanol (80:20, v/v) and eluent B was water–methanol (30:70, v/v) containing 5 mM tributylamine (TrBA) and 5 mM acetic acid. The gradient started with 20 per cent B, and increased to 85 per cent B followed by 1 min isocratic hold. MS conditions were: nitrogen was employed as drying gas (550 l/h) and nebulizing gas (100 l/h). MS-MS measurements used argon as the collision gas (1.3.103 mbar); ES was applied in the negative mode; capillary voltage, 3.0 kV; temperature of probe tip 250°C; cone voltage 20 V; source temperature 120°C. The analytes were well separated by the technique as demonstrated in Fig. 3.109. The limit of detection depended on the type of analytes, ranging from 0.2 g l to 2.6 lg/l. The limit of quantitation varied between 2 and 10 g/l. It was stated that the ion-pair LCESI-MS-MS technique using TrBA as the ion-pairing agent allows the separation of

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Chapter 3 NH2 HO3S

NH2

HO3S SO3H 1-aminonaphtalene-6-sulphonic acid (Cleve-1,6-acid)

6-aminonaphtalene-1,3-disulphonic acid (amino-1-acid)

NH2

OH

HO3S

NH2 HO3S

1-aminonaphtalene-7-sulphonic acid (Cleve-1-7-acid)

7-amino-1-hydroxynaphtalene-3-sulphonic acid (gamma-acid) OH

HO3S

NH2

NH2

HO3S

SO3H

2-aminonaphtalene-7-sulphonic acid (amino-F-acid)

1-amino-8-hydroxy-naphtalene-3,6-disulphonic-acid (H-acid)

SO3H NH2

NH2

SO3H 8-aminonaphtalene-1-sulphonic-acid (Peri acid)

5-aminonaphtalene-1-sulphonic acid (Laurent acid)

SO3H NH2 HO3S HO3S

NH2

SO3H OH

8-aminonaphalene-1,3,6-trisulphonic acid (Koch acid)

6-amino-1-hydroxynaphtalene-3-sulphonic acid (1-acid)

Fig. 3.106. Structures of aminonaphtalenesulphonic acids. Reprinted with permission from P. Jandera et al. [164].

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489

TABLE 3.31 RETENTION FACTORS, K = (TR – TM)/TM OF AROMATIC SULPHONIC ACIDS ON ODS COLUMNS (A  140  0.32 MM I.D.; PARTICLE SIZE 5 M AND B  150  3.3 MM I.D.; PARTICLE SIZE 7 M). MOBILE PHASES WERE: I  0.4 M NA2SO4 IN WATER AND II  10 PER CENT (V/V) METHANOL IN 0.4 M NA2SO4 IN WATER. FOR OTHER CHROMATOGRAPHIC CONDITIONS SEE TEXT.

Sample

Naphtalene-1,3-disulphonic acid Naphtalene-1,5-disulphonic acid Naphtalene-1,6-disulphonic acid Naphtalene-1,7-disulphonic acid Naphtalene-1,3,5-trisulphonic acid Naphtalene-1,3,6-trisulphonic acid Naphtalene-1,3,7-trisulphonic acid Naphtalene-1,3,5,7-tetrasulphonic acid

Column A

Column B

I

II

I

II

2.32 1.15 1.37 4.54 0.47 0.61 0.60 0.11

2.68 1.25 1.61 5.25 1.09 0.92 0.86 0.26

5.72 0.88 2.09 8.16 0.28 0.49 0.62 0.12

0.94 0 0.22 2.21 0 0 0.24 0

Reprinted with permission from Jandera et al. [164].

sulphophtalimide and some of its derivatives, and even makes possible the separation of positional isomers [165]. Mono-ad disulphonated azo dyes were also determined by RPHPLC-atmospheric pressure ionization mass spectrometry. The chemical structure of the dyes investigated are listed in Fig. 3.110. Various columns were employed for LC-ES (ODS, 300  2.1 mm.i.d.) and LC-APCI measurements (PCN, 250  4.6 mm i.d.). The optimal conditions are listed in Table 3.32. Dyes were extracted from spiked waters by membrane extraction discs containing 500 mg of poly(styrene-divinylbenzene). Dyes were separated in the HPLC systems as illustrated in Fig. 3.111. It was established that that APCI in negative ion mode was less sensitive than ES, and APCI in the positive-ion mode showed the lowest sensitivity [166]. The influence of various structural and physicochemical parameters of the stationary and mobile phases on the tailing of a cationic dye in reversed-phase chromatography has been studied in detail. Measurements were performed in a C8 reversed-phase column (80  4.6 mm). The isocratic mobile phase was ACN–0.01 M aqueous HCl (90:10, v/v). Analyses were carried out at 20°C and the flow rate was 1–5 ml/min. The concentration of the cationic dye, 1,1-didodecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (Dil) in the model solutions varied between 0.9–309 M. The dependence of the chromatographic profile of the dye on the injected concentration is illustrated in Fig. 3.112. Calculations and mathematical modelling indicated that the peak tailing of the dye can be

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490

Chapter 3 3 8.00

mAU

6.00

6

1 2

4.00

4 5 2.00

0.00

23.00

0.00 (a)

46.00 t (min)

12.00

10.00

mAU

8.00

1

6.00

3

4.00 2

4

7 5

2.00 6 0.00

−2.00 0.00 (b)

23.00

46.00 t (min)

Fig. 3.107. Comparison of micro-HPLC separations of aromatic sulphonic acids in different mobile phases: (a) 0.005 M tetrabutylammonium hydrogensulphate (TBAS) in 15 per cent (v/v) methanol in water; (1) Laurent acid, (2) amino-F-acid, (3) Cleve-1,6- and Peri acids, (4) unidentified impurity, (5) Cleve-1,7-acid and (6) unidentified impurity. (b) 0.005 M tetrabutylammonium hydrogensulphate (TBAS) in 15 per cent (v/v) methanol in water with 0.01 M -cyclodextrin (CD): (1) Laurent acid, (2) amino-F-acid, (3) Cleve-1,6-acid, (4) Peri acids, (5) unidentified impurity, (6) Cleve-1,7-acid and (7) unidentified impurity. Column, Biosphere Si C18, 162  0.32 mm i.d.; flow rate: 5 l/min, column temperature: ambient, detection, UV, 220–230 nm. Reprinted with permission from P. Jandera et al. [164].

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Liquid chromatography of synthetic dyes SPA

SPI

491 SPAM

O

SPAA1 O

O OH NH

OH O3 S

O3 S

O

O

O3 S

SPAA2

O

O

NH2

OH

NH2

NH2

NH2

OH

O3S

O

O3S

O

O

Fig. 3.108. Structures of the compounds under investigation. Reprinted with permission from T. Reemtsma [165].

100

4-SPI

226>118

4-SPAA2

244>157

100

%

%

3

2

100 4-SPI

4-SPAA1

226>162

245>157

4-SPA

244>163

%

2 100

3-SPI

226>182

3-SPAA1

244>183

100

%

245>183

3-SPA

%

1

27 5

(a)

7

9

244>200 100

3-SPAA2

245>201

4-SPA

Retention time (min) %

100

3-SPI

226>118

1 244>227 100

4-SPAA1

%

245>227

3-SPA

%

27 3-SPAA1

100

3-SPI

226>162

%

1 8.5

10.5

(c)

Retention time (min)

12.5

13

(d)

15

17

Retention time (min)

37 100

3-SPI

226>182

%

14 5

(b)

7

9

Retention time (min)

Fig. 3.109. Expanded chromatogram sections showing the multiple reaction monitoring detection of the isomers: (a) 3-/4-SPI; (b) pure 3-SPI; (c) SPAA; and (d) SPA. Reprinted with permission from T. Reemtsma [165].

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Chapter 3

Acid Red 1 MW 509 CI 18050

NHCOCH3

HO N N

SO3Na

NaO3S Na+ COONa

Mordant Red 9 MW 518 CI 16105

HO

SO3Na

N N

SO3Na Acid Red 13 MW 502 CI 16045

HO NaO3S

N N

SO3Na

NaO3S

N

Acid Red 14 MW 502 CI 27290

OH N

SO3Na

Acid Red 73 MW 556 CI 27290

N N

HO

N N NaO3S

SO3Na Mordant Yellow 8 MW 446 CI 18821

COONa HO N N

N

SO3Na

N CH3

Acid Yellow 23 MW 534 CI 19140

HO N N

NaO3S

N

SO3Na

N COONa

SO3Na Direct Yellow 28 MW 680 CI 19555

CH3

SO3Na

S S

N N

CH3

N N

Fig. 3.110. Chemical structure of the dyes studied. MWmolecular mass. Reprinted with permission from C. Ráfols et al. [166].

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493

TABLE 3.32 OPTIMIZATION OF THE DIFFERENT OPERATING PARAMETERS

Technique

Parameter

Optimization conditions

LC-TC-MS

Flow rate Eluent Source temperature Stem temperature Tip temperature Flow rate Eluent Drying gas flow rate ESI nebulizing gas flow Electrospray voltage HV lens voltage Extraction voltage Focus voltage Source temperature Flow rate Eluent Drying gas flow rate Nebulizing gas flow APCI voltage Cone voltage Source temperature APCI probe temperature

1 ml/min CH3OH–water (50:50, v/v)
NH4ac(0.034 M) 300°C 140°C 260°C 0.3 ml/min CH3OH–water (50:50, v/v)
NH4ac(0.001 M) 200 l/h 10 l/h 2.8 kV (2–3.5 kV) 0.1 kV 20 V (20–130 V) 27.47 V 150oC 1 ml/min CH3OH–water (50:50, v/v)
NH4ac(0.03 M) 400 l/h 50 l/h 2.5 kV 15 V 180°C 500–600°C

LC-TC-MS

LC-APCI-MS

Reprinted with permission from C. Ráfols et al. [166].

described by a bi-Langmuir adsorption isotherm, which suggests a mixed retention mechanism based on the distribution of analytes between strong and weak adsorptive sites. Tailing is mainly caused by the slow desorption of the dye from the strong adsorption sites which bind the analyte by hydrogen bonding or other strong electrostatic interactive forces [167]. The pH dependence of the tailing of Dil was investigated in separate experiments. The experimental conditions were the same but the pH of the mobile phase was adjusted to different values by HCl. The effect of pH on the retention behaviour of the dye is illustrated by chromatograms in Fig. 3.113. The pH dependence of tailing was tentatively explained by the marked contribution of free silanol groups to the reversed-phase retention of the dye [168]. Single-molecule resolution and fluorescence imaging were employed for the investigation of the mixed-mode adsorption of Dil at the interface of an ODS stationary phase and ACN–water mobile phase. It has been established that the increase in the

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Chapter 3

Blue

Yellow 28

Red 13

Yellow 8

APCI mm 30 ppm

Red 14 Red 13

Sim

100

Red 1

Red 9

494

%

0 18

22

Yellow 28

26

Blue

14 rt

Red 73

Black 11

Black 17

10

APCI mm 30 ppm

Red 14 Red 13

Sim

100

6

Yellow 8

2

%

0

Yellow 28

Red 14

22

Blue 113

18

26 3 ppm

Yellow 8 Red 73

%

14 rt

Red 1

Black 17 Yellow 23

100

Sim

10

Red 13

6

Black 11

2

0 10

15

20

25

30

35

Retention time (min)

Fig. 3.111. LC-APCI-MS (PI, NI) and LC-ES-MS and time-scheduled SIM conditions of a mixture of dyes at 30 and 3 ppm, respectively. Reprinted with permission from C. Ráfols et al. [166].

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495

400 350

mAU

300 250 200 150 100 50

Residuals

0 40

(a)

20 0 −20 −40

2.0

mAU

1.5 1.0 0.5 0.0 16 (b)

17

18 Time (min)

19

Fig. 3.112. (a) Chromatograms of Dil as a function of analyte concentration over the range 0.9–309 M. This graph overlays three replicate chromatograms at each concentration. A plot of residuals between the replicate chromatograms is shown, which demonstrates high reproducibility of the experimental data. (b) Chromatograms (o) for the lowest concentration of Dil, 0.9 m, plotted on a smaller scale to show that the chromatogram is symmetric. A Gaussian curve (-) is shown for reference. Reprinted with permission from M. J. Wirth et al. [167].

concentration of the organic modifier results in the increase of the stronger specific adsorption sites [169]. A similar study was realized to study the desorption process of Dil from fused silica and silica gel. The measurements indicated that the strong adsorption sites on the surface of fused silica and chromatographic silica stationary phases are chemically identical [170].

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mAU

496

Chapter 3 200 180 160 140 120 100 80 60 40 20 0 (b)

mAU

(a) 200 180 160 140 120 100 80 60 40 20 0 45 (c)

50

55 60 65 Time (min)

70

45 (d)

50

55 60 65 Time (min)

70

Fig. 3.113. Dil chromatograms and best-fit simulation results. Mobile phase is acetonitrile–water (80:20, v/v) (a) pH 2; (b) pH 3; (c) pH 4; (d) pH 5. Reprinted with permission from E. A. Smith et al. [168].

Various liquid chromatographic techniques have been frequently employed for the purification of commercial dyes for theoretical studies or for the exact determination of their toxicity and environmental pollution capacity. Thus, several sulphonated azo dyes were purified by using reversed-phase preparative HPLC. The chemical strctures, colour index names and numbers, and molecular masses of the sulphonated azo dyes included in the experiments are listed in Fig. 3.114. In order to determine the non-sulphonated azo dyes’ impurities, commercial dye samples were extracted with hexane, chloroform and ethyl acetate. Colourization of the organic phase indicated impurities. TLC carried out on silica and ODS stationary phases was also applied to control impurities. Mobile phases were composed of methanol, chloroform, acetone, ACN, 2-propanol, water and 0.1 M sodium sulphate depending on the type of stationary phase. Two ODS columns were employed for the analytical separation of dyes. The parameters of the columns were: 150  3.9 mm i.d.; particle size 4 m and 250  4.6 mm i.d.; particle size 5 m. Mobile phases consisted of methanol and 0.05 M aqueous ammonium acetate in various volume ratios. The flow rate was 0.9 ml/min and dyes were detected at 254 nm. Preparative separations were carried out in an ODS column (250  21.2 mm i.d.) using a flow rate of 13.5 ml/min. The composition of the mobile phases employed for the analytical and preparative separation of dyes is compiled in Table 3.33. Dye fractions were separately collected, evaporated to dryness and identified by ES-MS. It was established that the separation capacity of the TLC systems applied was very low, therefore, they cannot be applied for the purity control of this class of dyes.

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497 OH

R1

N

N

R2

SO3Na

NaO3S Name MW

R1

R2

H3C-CO-NH-

Acid Red 1 (509) C.I. 18050 Acid Red 8 (450) C.I.14900

H3C

H

CH3

Chromotrope 2R (468) C.I.16570 (Acid Red 29)

HO

O Acid Red 106 (621) C.I.18110 Acid Violet 5 (678) C.I.18125

S

H3C

H N

O O H3C

S

NHCOCH3 O

Procion Red MX-5B (615) (Reactive Red 2)

Cl N NH

N N Cl Cibacron Brilliant Red 3B-A (995) C.I.18105 (Reactive Red 4)

Cl NH

O

N N N

NH

SO3Na

HN NaO3S

Reactive Orange 16 (617) C.I.17757

O

OH H3COONH

N

N

S O

O SO3Na

SO3Na

Fig. 3.114. The names, molecular masses, colour index numbers and structures of the azo dye used in this study. Reprinted with permission from M. Chen et al. [171].

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Chapter 3

TABLE 3.33 THE PERCENTAGES OF METHANOL IN THE AQUEOUS AMMONIUM ACETATE BUFFER (0.05 M) USED IN THE ANALYTICAL AND PREPARATIVE MOBILE PHASES FOR EACH DYE

Dye

Analytical HPLC

Acid red I

30% methanol

Acid red 8 Acid red 106 Acid violet 5

40% methanol 35% methanol 35% methanol

Chromotrope 2R

30% methanol

Reactive orange 16 Procion red MX-5B

30% methanol 50% methanol

Cibacron brilliant Red 3B-A

35% methanol 35% methanol

Preparative HPLC 32% methanol for 20 min and linear gradient to 50% methanol at 30 min and kept at 50% methanol 40% methanol 38% methanol 40% methanol for 20 min and linear gradient to 50% methanol at 30 min and kept at 50% methanol 35% methanol for 20 min and linear gradient to 45% methanol at 30 min and kept at 45% methanol 35% methanol 40% methanol for 10 min and linear gradient to 55% methanol at 15 min and kept at 55% methanol 38% methanol for 20 min and linear gradient To 45% methanol at 25 min and kept at 45% methanol

Reprinted with permission from M. Chen et al. [171].

It was further found that the methods separate well the dyes and impurities, as demonstrated on the preparative chromatographic profile of Reactive orange 16 in Fig. 3.115. It was concluded from the results that the method can be used in the future for the purification and analysis of a wide variety of sulphonated azo dyes [171]. Various counter-current chromatographic techniques have been frequently applied for the preparative separation and purification of different synthetic dyes. Thus, the isomeric 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione monosulphonic acids of the colour additive D&C Yellow No. 10 (Quinoline yellow) were separated by pH-zone refining counter-current chromatography (CCC). The synthesis and the chemical structures of the products are shown in Fig. 3.116. The total capacity of the tubing was about 325 ml, and was mounted on a rotating frame. The two-phase eluent system for the preparation of monosulphated components consisted of isoamyl alcohol–methyl tert-butyl ether–ACN–water (3:1:1:5, 600 ml: 200 ml: 200 ml: 1 000 ml). After equilibrating the phases the volumes of the upper organic (UP) and lower aqueous phases (LP) were 940 and 1038 ml, respectively. Ligandcontaining UP was prepared by adding 10.2 g of dodecylamine and 2.1ml of sulphuric acid to 200 ml of UP, resulting in a pH of about 1.9. The mobile phase was prepared by adding 5.62 g of ammonium hydroxide to 1000 ml of LP (pH  10.9). Analytes were dissolved and/or suspended in LP and UP. The mobile phase was pumped into the system at a flow rate of 3 ml/min, and the column was rotated at 960 rpm. The pH and UV absorption of the effluent was monitored and fractions of 6 ml were collected and controlled by analytical HPLC. RP-HPLC control of CCC separation was realized in an ODS column

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499

2.0

Absorbance

1.5

1.0

0.5

0.0

1 M 0

10

3

4 20

30

5 40

50

Time (minutes)

Fig. 3.115. Preparative HPLC chromatogram of Reactive orange 16 with a mobile phase of methanol–0.05 M ammonium acetate (35:65, v/v) at 13.5 ml/min and detection at 254 nm. Collected region M is the major component. Reprinted with permission from M. Chen et al. [171].

(250  4.6 mm i.d.; particle size 5 m) using a gradient of methanol–0.1 M aqueous ammonium acetate. Elution started with 25 per cent methanol, to 90 per cent methanol in 25 min, to 100 per cent methanol in 5 min, 10 min hold. The flow rate was 1 ml/min, and dyes and intermediates were detected at 254 and 415 nm. LC-MS was performed in an ODS column (250  2 mm i.d.; particle size 5 m) using the same gradient elution at a flow rate of 0.25 ml/min. APCI MS conditions were: vaporizer and capillary temperatures 400 and 150°C, respectively, corona discharge 5 A. The analytical chromatogram of the dye fractions and their CCC separation profile are shown in Fig. 3.117. It was stated that the method can be applied for the separation of positional isomers of highly polar compounds containing an acidic sulphonic group [172]. The same technique has been employed for the purification of Food Colour Red No. 106 (Acid red). The chemical structure of the dye is shown in Fig. 3.118. The separation process was controlled by analytical RP-HPLC carried out in an ODS column (150  4.6 mm i.d.; particle size 5 m). The mobile phase consisted of ACN–0.01 M TFA (27:73, v/v). The flow rate was 1 ml/min and analytes were detected at 254 nm. Equilibrated n-butanol and water were employed for CCC separation. OP was acidified with 40 mM of sulphuric acid, and 30 mM of aqueous ammonia was added to the LP. The coil was rotated at 800 rpm and LP was pumped at a flow rate of 1 ml/min. Fractions of 1 ml volume were taken from the effluent. The CCC profile of Food Colour Red No. 106 in CCC is shown

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500

Chapter 3

H O N O O 1.H2SO4

O

+O N N

CH3

ZnCl2

O

HO 1

2. NaOH

2 O N H O 3 NaO3S

O N O O

6SA

(-SO3Na)2

N

+

O O N NaO3S

O

8SA

Fig. 3.116. Preparation of D&C Yellow No. 10 by condensing quinaldine, 1; with phtalic anhydride, 2; and sulphonating the condensation product, 3. Reprinted with permission from A. Weisz et al. [172].

in Fig. 3.119. The chromatographic profiles of CCC fractions, the original sample and the column contents are shown in Fig. 3.120. The chromatograms demonstrated that the CCC technique is suitable for the purification of the dye, the purity of the end product being 99.9 per cent [173]. The beneficial effect of the change of the flow rate of the mobile phase has also been exploited for the improvement of CCC purification of the components of the dye Quinoline yellow (Colour Index No. 47005). The chemical structures of the components of Quinoline yellow are shown in Fig. 3.121. The two-phase system used for the purification consisted of tert-butyl methyl ether–1-butanol–ACN–0.1 M TFA (1:3:1:5 v/v). The column

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Liquid chromatography of synthetic dyes 8SA

0

5

501

6SA

10 15 20 25 30 Retention time (min)

35

415nm 40

−O S 3

O

O

O

N SO3− O 8SA

N 6SA

415nm 415nm 0 10 20 30 40 0 10 20 30 40 Retention time (min) Retention time (min)

(a)

Absorbance (415 nm)

1.45

0 0 (b)

30 Solvent front

60 90 Fraction number

120

pH

11 10 9 8 7 6 5 4 3 2

2.90

150

Fig. 3.117. Separation of 6SA and 8SA from a certified lot of D&C Yellow No. 10 by pH-zonerefining CCC. (a) HPLC analysis of colour additive and (b) HPLC analyses of the separated components. Reprinted with permission from A. Weisz et al. [172].

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502

Chapter 3 + O

(CH3CH2)2N

N(CH2CH3)2

SO3

SO3Na

Fig. 3.118. Structure of the main component (Acid red) in Food Colour Red No. 106. Molecular mass: 580. Reprinted with permission from H. Oka et al. [173].

20

12.0

18

Abs.

16

pH

10.0 Fr. 5 8.0

12 10

6.0

Fr. 4

pH

Abs.(544 nm)

14

8 Fr. 3

4.0

6 Fr. 2

4

2.0

Fr. 1

2 0

0.0 0

1.0

2.0

3.0

4.0

5.0

(hr)

Fig. 3.119. Separation of the components of Food Colour Red No. 106 by pH-zone-refining CCC. For conditions see text. Reprinted with permission from H. Oka et al. [173].

was rotated at 800 rpm, the LP was pumped at a flow rate of 0.1 ml/min, and after 20 min the flow rate was enhanced to 2 ml/min. Fractions of 1 ml volume were collected from the effluent. RP-HPLC control of the purification step was performed in an ODS column (150  4.6 mm i.d.; particle size 5 m). Gradient elution started with 5 per cent ACN in 0.05 M phosphate buffer (pH  6.0) and ACN concentration was linearly increased to 50 per cent in 30 min. The flow rate was 1 ml/min and solutes were detected at 254 nm. The CCC profile of the dye is shown in Fig. 3.122 and the RP-HPLC profiles of the fractions

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Liquid chromatography of synthetic dyes

Fr. 1

503

Fr. 2

Fr. 3

0.1 AUSF (2.54 nm)

0

5

10

15

20

0

5

(Min)

10

15

20

0

5

(Min)

Fr. 4

10

15

20

(Min)

Fr. 5 Acid red

Acid red

Column contents

Acid red

0

5

10 (Min)

15

20

0

5

10 (Min)

15

20

0

5

10

15

20

(Min)

Fig. 3.120. HPLC separation of the components of Food Colour Red No. 106. For HPLC conditions see text. Reprinted with permission from H. Oka et al. [173].

are depicted in Fig. 3.123. The technique has been proposed for the effective separation of molecules in one run which have high differences in their lipophilicity [174]. RP-HPLC was also employed for the investigation of the hydrolysis behaviour of two fluorotriazine reactive dyes Cibacron blue F-R and Cibacron yellow F-4G. Chromatographic measurements were performed in an ODS column (50  4.6 mm i.d.; particle size 5 m) at room temperature. The mobile phase was ACN–0.05 M ammonium acetate buffer containing 1 mM acetyltrimethylammonium bromide (47:53, v/v). Flow rates were 0.8 and 0.6 ml/min depending on the dyes to be separated. Dyes were detected at 275 nm. The hydrolysis of dyes was investigated both in the absence and presence of

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504

Chapter 3 Quinoline 5'

4'

1

2

7

R1

R2

6'

3'

O

2'

7'

N

8'

6 3

R3

O

4

5

Idandiode

Fig. 3.121. Structures of the isolated quinoline yellow components. Fraction 4, R1  SO3Na, R2  SO3Na, R3  H; fraction 6, R1  SO3Na, R2  H, R3  SO3Na; fraction 10, R1  H, R2  SO3Na, R3  H; fraction 13, R1  H, R2  H, R3  SO3Na. Reprinted with permission from H. Oka et al. [174].

4.0

1.0 Fr. 4 Fr. 10 Fr. 3

3.0

Fr. 5

abs (416 NM)

0.5

Fr. 6

Fr. 2

Fr. 7 Fr. 8

Fr. 1

2.0 0

0

20

Fr. 13 Fr. 11

1.0

Fr. 12 Fr. 9 0 0

20.0

24.0

28.0

30.0

(hr) 2.0 mL /min 0.1 mL/min

Flow rate

Fig. 3.122. Separation of quinoline yellow components by HSCCC. For HSCCC conditions see text. Reprinted with permission from H. Oka et al. [174].

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Liquid chromatography of synthetic dyes 10

10 Fr.2

0.5

10

Fr.3

0.5

Fr.4 (min)

Fr.1

10

0.5

Fr.5

Peak 2 (min)

10

505

0.5

0.5

Peak 1

0

0 0

5

10

15

20

25

0 0

5

(min)

10

10

15

20

25

0 0

5

(min)

20

25

0 0

5

15

20

25

0

5

10

Fr.8

15

20

25

Peak 6

Fr.9

0.5

10

(min)

10

Fr.7

0.5

10

(min)

10

Fr.6

0.5

15

(min)

10

Peak 3

10

Fr.10

0.5

0.5

Peak 4

0

0 0

5

10

15

20

25

0 0

5

(min)

10

15

20

25

10

Fr.11

15

(min)

20

25

25

5

10

15

(min)

5

20

25

10

15

20

25

0

5

10

15

20

25

(min)

10 Column contents

0.5

0 0

0 0

(min)

0.5

0 10

20

Fr.13

0.5

0

15

Peak 5

Fr.12

0.5

10

(min)

10

5

5

(min)

10

0

0 0

0 0

5

10

15

(min)

20

25

0

5

10

15

20

25

(min)

Fig. 3.123. HPLC separation of fractionated components of quinoline yellow. For HPLC conditions see text. Reprinted with permission from H. Oka et al. [174].

fabric for 120 min. The retention times and capacity factors of the dyes are compiled in Table 3.34. The retention times indicate that the hydrolysed dyes are eluted more rapidly than the non-hydrolysed ones. This phenomenon was tentatively explained by the supposition that the first step of hydrolysis is the cleavage of -F and its substitution by the more polar -OH group. Chromatographic profiles of dye mixtures obtained under different experimental conditions are depicted in Fig. 3.124. It can be concluded from the chromatographic data that the hydrolysis of reactive fluorotriazine dyes can be followed by RP-HPLC and can be used for the control of the dyeing process [175]. The effect of various experimental conditions on the alkali-hydrolysis kinetics of 4-amino-4-fluorosulphonylazo benzene disperse dyes was investigated by RP-HPLC.

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Chapter 3 TABLE 3.34 RETENTION TIMES AND CAPACITY FACTORS OF THE DYES, BLUE F-R AND YELLOW F4-G, SEPARATE AND IN MIXTURE (1:1, W/W)

Dyes

Retention time (min)

Capacity factor (k)

Blue Hydrolysed blue Yellow Hydrolysed yellow Blue in mixture (m) Hydrolysed blue (m) Yellow in mixture (m) Hydrolysed yellow (m)

4.70 3.44 3.32 2.38 7.54 5.07 4.57 3.16

1.41 0.76 0.71 0.23 1.86 0.93 0.73 0.20

0 (a)

2

4 6 8 Time (min)

10

0 (b)

2

4 6 8 Time (min)

10

0 (c)

2

5.22 6.05 7.55

4.49

2.61

7.48

5.07

2.62 2.98

2.63 3.06 3.15 3.98 5.11

7.54

3.16 4.54

3.28

4.57

Reprinted with permission from A. Zotou et al. [175].

4 6 8 Time (min)

10

Fig. 3.124. Chromatograms of a 1:1 dye mixture: (a) 40 min before addition of Na2CO3, (b) 10 min after addition of Na2CO3, and (c) 60 min after addition of Na2CO3; flow rate  0.6 ml/min, other conditions are described in the text. The peak at 4.57 min in (a) and the same peak (smaller in size) at 4.54 min in (b) are both attributed to the functional group of cibacron yellow (F); the peak at 7.54 min in (a) and the same peak (smaller in size) at 7.40 min in (b) are both attributed to the hydrolysed part of the functional group of cibacron yellow; the peaks at 5.07 min in (b) and 5.22 min in (c) are attributed to the hydrolysed part of the functional group of cibacron blue. Reprinted with permission from A. Zotou et al. [175].

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507

The chemical structures of the dyes under investigation are listed in Fig. 3.125. Hydrolyses were performed at 0.4, 1.0 and 2.0 g/l concentrations of NaOH at 80 and 90oC for 50 min. Samples were mixed with ACN in the ratio of ACN–sample 80:20 (v/v) and used for RPHPLC without any other pretreatment. Dyes were analysed in an ODS column using ACN–water 80:20 as the mobile phase. They were detected at their absorption maxima. The tendency of the alkali hydrolysis behaviour of dyes is illustrated in Fig. 3.126. The HPLC measurements indicated that this class of dyes is readily hydrolysed under mild alkaline conditions, therefore, their application in the textile industry is advocated [176]. An interesting application of dye analysis is its application for the study of the dediazoniation product formation. The scheme illustrating the coupling reaction between the 4-nitrobenzenediazonium (PNBD) ion and the sodium salt of 2-naphtol-6-sulphonic acid to yield the 6-sulphonate-2-naphtol-1-azo-p-nitrobenzene azo dye is shown in Fig. 3.127. As it was supposed that the formation of inclusion complexes with -cyclodextrin (-CD) may influence the coupling, its effect has also been investigated. RP-HPLC measurements were realized in an ODS column (250  4.6 mm i.d.; particle size 5 m) at ambient temperature. Analytes were separated by methanol–water (75:25, v/v) and ACN–water (75:25, v/v) mobile phases in the presence of -CD. The flow rate was 0.8 ml/min and the detection wavelength was set to 220 nm. The chromatograms demonstrating the baseline separation of the possible dediazoniation products are shown in Fig. 3.128. The chromatograms illustrate that the application of ACN as an organic modifier is superior to that of methanol. It was further concluded from the results that the technique can be employed for the study of X O S

F

R1

N

O Y

C2H5 N

N C2H5 R2

Dye

X

Y

R1

R2

1a 1b 1c 2a 2b 2c 3a 3b 3c 4a 4b 4c 5a 5b 5c

H H H NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2

H H H H H H Cl Cl Cl Br Br Br CN CN CN

H CH3 NHCOCH3 H CH3 NHCOCH3 H CH3 NHCOCH3 H CH3 NHCOCH3 H CH3 NHCOCH3

H H OCH3 H H OCH3 H H OCH3 H H OCH3 H H OCH3

λmax (EtOH, mm) 469 478 519 513 524 573 520 539 595 522 542 594 540 580 620

Fig. 3.125. 4-(N,N-diethylamino)-4-fluorosulphonylazobenzene dye (1–5) used in the study. Reprinted with permission from J. Koh et al. [176].

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Chapter 3 8 1a 2a 3a 4a 5a

80

6 LN (A0 / A)

Residues of parent dyes (%)

100

60 40

2 20 0

0 0

5

10 15 20 25 30 35 40 45 50

0

8 1b 2b 3b 4b 5b

80 60 40

6 LN (A0 / A)

Residues of parent dyes (%)

10 15 20 25 30 35 40 45 50 Hydrolysis time (min)

100

4

2 20 0

0 0

5

(b)

10 15 20 25 30 35 40 45 50

0

5

Hydrolysis time (min)

10 15 20 25 30 35 40 45 50 Hydrolysis time (min)

8

100 80

6 LN (A0 / A)

Residues of parent dyes (%)

5

Hydrolysis time (min)

(a)

60 40

4

2 20 0

0 0 (c)

4

5

10 15 20 25 30 35 40 45 50 Hydrolysis time (min)

0

5

10 15 20 25 30 35 40 45 50 Hydrolysis time (min)

Fig. 3.126. Alkali-hydrolysis behaviours of 1–5 at 80°C (NaOH 0.4 g/l): (a) 1a–5a; (b) 1b–5b; (c) 1c–5c. Reprinted with permission from J. Koh et al. [170].

the interaction of aromatic diazonium ions [177]. Sulphonated compounds including dyes were separated by ion-exchange HPLC-MS too. The chemical structures of the compounds included in the investigation are listed in Fig. 3.129. Separation of analytes was carried out on an aminopropyl column (125  4 mm i.d.; particle size 5 m) at room temperature. Components of ternary gradient were water (A), ACN (B) and ammonium acetate buffer (mixture of 38.5 g of ammonium acetate, 183 g of acetic acid and 980 g of water, pH  3.80) (eluent C). The gradient started at 88 per cent A 10 per cent B and 2 per cent C,

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Liquid chromatography of synthetic dyes SO3-

+

509 SO3-

N2 N

+ O+

O+

NO2

N

NO2

2.55

6.79

5.41

A (220 nm)

8.37

6.53

5.14

A (220 nm)

3.64

1

Fig. 3.127. Representative coupling reaction between PNBD and the sodium salt of 2-naphtol-6-sulphonic acid to yield the 6-sulphonate-2-naphtol-1-azo-p-nitrobenzene azo dye. Reprinted with permission from C. Bravo-Diaz et al. [177].

0 (a)

2

4

6 t / min

8

10

12

0 (b)

2

4

6 t / min

8

10

Fig. 3.128. Effect of solvent strength on the chromatograms of a standard solution prepared by dissolving -CD ([-CD]  9.5 mM) and the commercially available dediazoniation products 4-nitrophenol ([PNBOH]  2.00104 M), nitrobenzene ([NBH]  1.98104 M and 4-chloro-nitrobenhenzene ([PNBCl]  1.99104 M). (a) Mobile phase MeOH–water (75:25, v/v), (b) ACN–water (75:25, v/v). Reprinted with permission from C. Bravo-Diaz et al. [177].

reached 5 per cent A 65 per cent B and 30 per cent C in 16 min, 4 min final hold. The flow rate was 1.5 ml/min and UV detection was set to 270 nm. ESI conditions were: spray voltage 4.5 kV; capillary voltage, 46 V and capillary temperature, 250°C. APCI spectra were taken at the following parameters: vaporizer temperature, 450°C; discharge current, 5 A; capillary voltage 10 V; capillary temperature, 200°C. The chromatograms detected at 270 nm (upper lane), by APCI-MS-TIC (middle) and by ESI-MS-TIC (lower lane) demonstrating the impact of the various detection methods on the chromatographic profile of the sulphonated compounds are depicted in Fig. 3.130. It has been stated that ionexchange high-performance liquid chromatography coupled with various MS techniques

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510

Chapter 3 SO3Na SO3Na

O2N OH

Benzene sulfonic acid sodium salt

N

N

HO SO3Na

2-Naphtalene sulfonic acid sodium salt

Eriochrome Black T

O

O

SO3Na

SO3Na NaO3S O

O 2-anthraquinone sulfonic acid sodium salt

2,6-Athraquinone disulfonic acid disodium salt

SO3Na

SO3Na

SO3 SO3Na O

N

N+

Sulfochodamine B sodium salt

SO3Na

1,5-Naphtalene disulfonic acid disodium salt

SO3Na

NaO3S N

HO N

N

N

OH HN O Azophloxine

SO3K Crocein Orange G

SO3K

1,2-Benzene disulfonic acid dipotassium salt

Fig. 3.129. Structures and names of the test substances. Reprinted with permission from G. Socher et al. [178].

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Liquid chromatography of synthetic dyes 2000

511

3 5.87 min.

1750 7 10.33 min.

1500 4 6.40 min.

mAU

1250 2 5.53 min.

1000

5 6.84 min.

750

8 10.95 min.

500

6 8.51 min.

250

9 11.69 min. 10 15.00 min.

1 4.65 min.

0 0

2

4

6

8

10 Minutes

12

14

16

18

20

Fig. 3.130. HPLC chromatograms of the test mixture detected by DAD (270 nm, upper lane) by APCI-MS-TIC (middle) and by ESI-MS-TIC (lower lane). Peak identification: 1benzene sulphonic acid sodium salt; 22-naphtalene sulphonic acid sodium salt; 32-anthraquinone sulphonic acid sodium salt; 4sulphorhodamine D sodium salt; 5crocein orange G; 6eriochrome black T; 72,6-anthraquinone disulphonic acid disodium salt; 81,5-naphtalene disulphonic acid disodium salt; 9azophloxine; 101,2-benzene disulphonic acid dipotassium salt. Reprinted with permission from G. Socher et al. [178].

offer a unique possibility for the separation, quantitation and identification of sulphonated aromatic compounds differing considerably in their chemical structure. [178]. The purity and stability of three dichlorotriazine dyes applied in nonlinear optical materials was checked by RP-HPLC. The chemical structures of the dyes are shown in Fig. 3.131. Analyses were realized in an ODS column (250  4.6 mm i.d.; particle size 5 m) using gradient elution. Aqueous ammonium acetate (50 mM) and methanol were solvents A and B, respectively. The detection wavelength depended on the absorption maxima of the dye. Chromatograms illustrating the decomposition of dyes under alkaline conditions are depicted in Fig. 3.132. It was established that the application of RP-HPLC for the study of the purity and stability of dyes may facilitate their use in nonlinear optical materials. [179]. 3.5.6 Miscellaneous applications Various liquid chromatographic methods have found application in the control of the synthesis of new dye molecules. Thus, an alkali-clearable azo disperse dye with a fluorosulphonyl group was synthesized and it stability was checked by RP-HPLC. The synthesis route is depicted in Fig. 3.133. The purity control and the hydrolysis rate of the new dye was followed by RP-HPLC using an ODS column and an ACN–water (80:20, v/v)

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512

Chapter 3

(a ) Reactive Brown 23 Me Cl HO3S

N

N

N

N

N

N

N N

Me SO3H

SO3H

Cl

SO3H

(b ) Reactive orange 4 OH N N

Me Cl N

SO3H

N

N

SO3H

N Cl

(c) Reactive Red 2 HO3S

SO3H N NH

Cl

N

OH

Cl

Fig. 3.131. Structures of (a) Reactive brown 23, (b) Reactive orange 4, and (c) Reactive red 2. Reprinted with permission from K. E. Van Cott et al. [179].

isocratic mobile phase. Hydrolysis was carried out at 8.0, 9.0, 10.0, 11.0 and 12.0 pH at 60, 70, 80, 90 and 100°C. Some typical chromatograms illustrating the decomposition of the dye are shown in Fig. 3.134. The data proved again that RP-HPLC can be successfully employed for both the control of synthetic processes and for the study of hydrolysis kinetics [180]. Some new benzanthrone dyes were synthesized and applied for the one-step colouration and stabilization of polystyrene. The chemical structures of monomeric benzanthrone dye (formula 1), the stabilizer TTMP 2,2-(2,2,6,6-tetramethylpiperidine-1-yl)-4,6dichloro-1,3,5-triazine (formula 2) and the new synthetic product showing both colouration and stabillizer capacity (formula 3) are shown in Fig. 3.135. The synthesis process was controlled by TLC using a silica stationary phase and an n-heptane-acetone (1:1, v/v)

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513

Reactive brown 23

100

600

80 60 %B

mAU

400

40 200 20 0

0 0

5

10

15

20

25

(a)

30 35 Minutes

40

45

50

55

60

400 Reactive orange 4

100 80 60

200

%B

mAU

300

40 100 20 0

0 0

5

10

15

(b)

20

25

30 35 Minutes

Reactive Orange 4

40

45

50

55

60

Dichlorotriazine

800 t = 5 minutes

mAU

600 400

t = 185 minutes

200

Hydrolysis Product t = 485 minutes

0 0.0

(c)

2.5

5.0

7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 Minutes

Fig. 3.132. HPLC chromatograms of (a) Reactive brown 23 (420 nm detection), (b) Reactive red 2 (Aldrich, 538 nm), and (c) Reactive orange 4 (490 nm) during hydrolysis. Dyes were dissolved at 10 mg/ml in 50 mM Na2CO3 pH 10.5. Samples were removed for HPLC analysis at regular time intervals (t). Reactive brown was diluted to 2 mg/ml and injected; Reactive red 2 and Reactive orange 4 were diluted to 0.25 mg/ml. Column: Nucleosil C18, 5 m, 4.6250 mm; 20 l injection. Solvent programme for Reactive red 2 and Reactive brown 23: 5 min isocratic 50 mM ammonium acetate; 0–70 per cent methanol linear gradient for 35 min; 70–100 per cent methanol for 1 min; 5 min isocratic methanol. Solvent programme one for Reactive orange 4: 5 min isocratic 50 mM ammonium acetate; 0–50 per cent methanol linear gradient for 25 min; 70–100 per cent methanol for 1 min; 5 min isocratic methanol. Reprinted with permission from K. E. Van Cott et al. [79].

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514

Chapter 3 H2 O/p-Dioxane

ClO2S

NHCOCH3 Reflux, 3 hrs

HCl/EtOH

NHCOCH3

FO2S

NH2

FO2S Reflux, 1 hr

2

1

3

Scheme 1. Preparation of 4-fluorosulfanilaniline

CH3

HCl, NaNO2,0 - 5°C

N

FO2S

C2H5 N

3

N C2H5

N,N-diethyl-m-toluidine, 0 - 5°C

Scheme 2. Synthesis of 4-(4-diethylamino-2-methyl-phenylazo)benzene sulfonyl fluoride

Fig. 3.133. Preparation of 4-fluorosulphonylaniline and synthesis of 4-(4-diethylamino-2-methylphenylazo)benzene sulphonyl fluoride. Reprinted with permission from J. Koh et al. [180].

H H P P

(a)

(b)

(c)

H

H

H

P P (d)

(e)

P (f)

Fig. 3.134. Chromatogram of the synthesized hydrolysed dye and parent dye in buffer of pH 11 recorded after various times of hydrolysis at 90°C; (a) synthesized hydrolysed dye, (b) 0 min, (c) 10 min, (d) 20 min, (e) 30 min, (f) 40 min; where components P and H represent parent dye and hydrolysed dye. Reprinted with permission from J. Koh et al. [180].

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515

CH3

H N

N

CL

N

N

N

Cl N

H 3C

N

N

CH3 CH3

OCH2CH=CH2 OCH2CH=CH2 O Formula 2

Formula 1 H3C CH3 H N

N N

N

N CH3 CH3

OCH2CH=CH2 O Formula 3

Fig. 3.135. Chemical structures of compounds under investigation. Reprinted with permission from V. Bojinov et al. [181].

mobile phase. The characteristics of polystyrene prepared by the addition of compounds 1, 2 and 3 were investigated by gel permeation chromatography using tetrahydrofurane as the mobile phase at 30°C. It was stated that the new compound (formula 3) is suitable for the one-step colouration and stabilization of polystyrene. [81]. An octylsilica stationary phase coupled with DAD and chemiluminescence (CL) detection was employed for the analysis of intermediates in oxidative hair dyes and waste-water of shampooing after hair dyeing. Investigations included p-phenylenediamine (PPDA), o-phenylenediamine (OPDA), p-aminophenol (PAP), o-aminophenol (OAP), resorcinol (RE) and hydroquinone (HQ). Separation of analytes was performed in an octylsilica column (150  4.6 mm i.d.; particle size 5 m) and the composition of the mobile phase depended on the type of analytes to be detected. The column was thermostated at 25oC and the flow rate was 1 ml/min. The CL detection system is shown in Fig. 3.136. Some chromatograms illustrating the separation of analytes and the effect of the different modes of detection on the chromatographic profile are depicted in Fig. 3.137. The retention times were: PPDA  1.67 min; PAP  2.57 min; HQ  4.91 min; OPDA  5.50 min; OAP  7.82 min and RE  12.22 min. The chromatograms demonstrate that the method separates well the analytes under investigation. It was further found that except for OPDA, CL detection is more sensitive than that obtained by DAD. The linearity range varied according to the compound to be detected being between 1–10.000 ng/ml and the coefficient of correlation was always over 0.993. Detection limits ranged 0.21–32.35 ng/ml, and the standard deviation was 0.9–2.9 per cent. The recoveries of analytes from oxidative hair dyes and waste- waters of shampooing after hair dyeing are compiled in Tables 3.35 and 3.36. It was concluded from the results that chemiluminescence can be

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516

Chapter 3 ml min−1 2.2

DMSO 2.1 NaOH

Sample 1.8

H2O

Flow cell Waste

2.0 Luminol Peristaltic pump

Fig. 3.136. Schematic diagram of the FIA-CL detection. Reprinted with permission from J. Zhou et al. [182].

used for the detection of aromatic compounds not only in model solutions but also in oxidative hair dyes [182].

3.6 ELECTROPHORETIC METHODS The good separation capacity, low solvent consumption, rapidity and relative simplicity of various electrophoretic techniques have also been exploited in the separation and quantitative determination of synthetic dyes. 3.6.1 Determination of dyes in foods and food products The legal or illegal application of synthetic dyes in foods and food products increases consumer acceptance, and consequently, the profit of the producer. As a considerable quantity of foods and food products contain dyes, their determination is of considerable importance. Electrophoretic techniques have been frequently employed for dye analysis. Thus, micellar electrokinetic capillary chromatography has also been employed for the determination of synthetic colours in soft drinks and confectioneries [183]. An electrophoretic method was developed for the simultaneous determination of artificial sweeteners, preservatives and colours in soft drinks. The samples were degassed by sonication, filtered and used for analysis without any other pretreatment. Measurements were realized in uncoated fused-silica capillaries, the internal diameter being 50 m. Capillary lengths were 48.5 cm (40 cm to the detector) and 65.4 cm (56 cm to the detector). Capillaries were conditioned by washing them with (1 M sodium hydroxide (10 min), followed by 0.1 M sodium hydroxide (5 min) and water (5 min). Samples were injected hydrodinamically (250 mbar) at the anodic end. Analyses were performed at a voltage of 20 kV and the capillary temperature was 25oC. Analytes having ionizable substructure

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350

4 Net CL intensity (a.u.)

Absorbance (a.u.)

1.5 1.2 6

0.9 0.6

1

0.3

3

2

5

0.0

300 250 5

200 1 2

150 0

2

4

(a)

6 8 10 Time (min)

12

6

4

14

0

2

3 4

(b)

6 8 10 Time (min)

12

14

350

1 Net CL intensity (a.u.)

Absorbance (a.u.)

8 6 4 2

4

6

300 4

250

200

0

1

150 0 (c)

2

4

6 8 10 Time (min)

12

0

14 (d)

6

2

4

6 8 10 Time (min)

12

14

Fig. 3.137. Chromatograms of tested compounds with DASD at 275 nm and CL detection. CL reaction conditions: luminol, 1104 mol/l; pH 7.4; DMSO, 1104 mol/l; NaOH, 0.1 mol/l; flow rate, 2.8 ml/min. Separation conditions: mobile phase, A:B  10:90 (A: methanol; B: 0.1 per cent triethylmine
0.025 mol/l ammonium acetate
5x103 mol/l tetrabutyl ammonium bromide; pH 6.0; flow rate, 1.0 ml/min. Peaks: 1  PPDA; 2  PAP; 3  HQ; 4  OPDA; 5  OAP; 6  RE. (a) Chromatogram of standard solution by DAD detection (compound, 100 ng/ml); (b) chromatograms of standard solution by CL detection (compound, 100 ng/ml); (c) chromatogram of Xiandai hair dyes by DAD etection; (d) chromatogram of Xiandi hair dyes (diluted 10 times) by CL detection. Reprinted with permission from J. Zhou et al. [182].

were separated by CZE. Seven synthetic dyes (Quinoline yellow, Sunset yellow FCF, Carmoisine, Ponceau 4R, Brilliant blue FCF, Green S and Black PN), three artificial sweeteners (acesulfame K, aspartame and saccharin) and two preservatives (benzoic acid and sorbic acid) were included in the CZE experiments. CZE measurements were carried out in carbonate and borate buffers at pH  9.5. The concentrations of the buffers were 5, 10, 20 and 50 mM. Analytes were detected at 200 nm. Electropherograms illustrating the influence of SDS addition on the retention of analytes are shown in Figs 3.138 and 3.139. The results indicated that the best separation of each analyte can be obtained using 20 mM of carbonate buffer at pH 9.5, and 62 mM SDS. It was further found that the limit of quantification of the MEKC method is 0.01 mg/ml [184].

RECOVERY OF TESTED COMPOUNDS IN OXIDATIVE HAIR DYES

HPLC-CL (g/ml)

Compound

HPLC-DAD (g/ml)

Originala

Added

Founda

Recovery (%)

Originala

Added

Founda

Recovery (%)

1.27 0.03 1.47 0.02 0.29 0.01

1.00 1.00 0.50

2.18 0.03 2.39 0.07 0.76 0.02

91.3 94.7 94.4

1.39 0.02 1.35 0.01 0.30 0.01

1.00 1.00 0.50

2.46 0.05 2.26 0.05 0.80 0.01

106.3 91.4 100.8

Zhanghua

PPDA OPDA RE

0.99 0.02 1.27 0.03 0.25 0.01

1.00 1.00 0.50

1.91 0.05 2.20 0.01 0.70 0.01

91.6 92.5 90.6

1.03 0.03 1.20 0.02 0.26 0.01

1.00 1.00 0.50

2.11 0.04 2.23  0.06 0.75 0.01

107.8 103.3 98.6

Xiandai

PPDA OPDA RE

1.92 0.03 1.57 0.03 0.45 0.01

2.00 1.00 0.50

3.74 0.05 2.57 0.05 0.91 0.02

91.2 100.0 92.6

2.06 0.04 1.59 0.05 0.46 0.02

2.00 1.00 0.50

4.13  0.03 2.54  0.05 0.95  0.02

106.3 94.7 98.2

12.66 0.24 8.46 0.16

10.00 5.00

22.020.42 13.720.39

93.6 105.2

14.48 0.04 8.21 0.03

10.00 10.00

24.84  0.55 17.99  0.18

103.6 97.9

Guangming PPDA RE

Mean value  SD (n  3). Reprinted with permission from J. Zhou et al. [182].

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TABLE 3.35

a

Chapter 3

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TABLE 3.36 RECOVERY OF TESTED COMPOUNDS IN WASTE-WATERS OF SHAMPOOING AFTER HAIR DYEING

Brand

Compound

HPLC-CL (g/ml) Originala

Added

HPLC-DAD (g/ml)

Founda

Recovery (%)

Shengshida PAP

5.93

 0.01

5.00

11.23  0.02

106.0

nd

Yuanhua

PPDA PAP

106.12  2.12 3.67  0.11

50.00 5.00

157.33  1.58 8.78  0.15

102.4 102.2

nd nd

Lambia

PPDA PAP

62.62  1.25 3.30  0.08

50.00 50.00

115.03  0.12 50.93  1.48

104.8 95.3

nd nd

Youngrace

PPDA PAP

47.34  0.95 2.14  0.04

50.00 50.00

95.26  1.98 53.14  1.06

95.8 102.0

nd nd

Weicaili

PPDA PAP

73.14  1.05 10.92  0.10

50.00 50.00

123.10  1.48 61.13  1.73

99.9 100.4

nd nd

PPDA PAP

36.23  0.87 2.70  0.07

50.00 50.00

83.14  2.18 48.73  0.57

93.8 92.1

nd nd

Mean value  SD(n3). ND  not detected. Reprinted with permission from J. Zhou et al. [182]. a

Another electrophoretic method has been developed for the determination and separation of dyes in various foodstuffs. The dyes included in the investigation were Carminic acid (E120), Erythrosine (E-127), Sunset yellow (E-110), Amaranth (E-123), Ponceau 4R (E-124), Carmoisine (E-122) and Red 2G (E-128). The chemical structures of the dyes are shown in Fig. 3.140. Dyes were separated in a fused-silica capillary of 57 cm length (50 cm to the detector) and 75 m i.d. The capillary was thermostated at 25°C and the separation voltage was 20 kV (electrophoretic current, 60 A). The optimal conditions for the CZE analysis of dyes were: 15 mM Na2B4O7 buffer, pH  10.5; hydrodynamic injection for 5 s; detection wavelength 216 nm. The RSD value of migration time was 0.5 per cent while the same value for peak areas was between 0.17 and 1.32 per cent. The linearity range was 8 – 200 mg/l, and the LOD and LOQ values varied between 0.38 – 2.12 mg/l and 1.16 – 7.06 mg/l, respectively. The recovery of the method was good (95 – 111.3 per cent). Samples of beverages and molten ice were injected directly, and syrups were diluted and filtered before analysis. The results were compared with those of RP-HPLC performed in an ODS column (150 x 3.9 mm i.d.; particle size 3 m). Eluent A was methanol and eluent B was

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Chapter 3 7

150

6

11 12

100 1 50 2

(a)

3 45

8

9 10

13

0

Absorbance (mAU)

4,7 150

6

1

3a

11

100

12

5

50

9

8

2

(b)

3b 10

13

0

7 150

6

1

3a

100 50

11

4,8 5 (c)

9

2

12

10

3b

13

0 2

4

6

8 Miration time (min)

10

12

14

Fig. 3.138. Electropherogams showing the separation of caffeine (1), aspartame (2), brilliant blue FCF (3), green S (4), sorbic acid (5), benzoic acid (6), saccharin (7), acesulfame K (8), sunset yellow FCF (9), quinoline yellow (10), carmoisine (11), ponceau 4R (12), black PN (13), using 20 mM carbonate buffer, pH 9.5; containing (a) no SDS, (b) 50 mM SDS, (c) 75 mM SDS. A 48.5  50 m I.D. fused-silica capillary was used and absorbance was measured at 200 nm. Reprinted with permission from R. A. Frazier et al. [184].

NaH2PO4/Na2HPO4. Gradient elution started at 20 per cent A, increased to 100 per cent A in 2 min, final hold 4 min. Electropherograms of a standard mixture of the seven dyes (A), a sample of grenadine (B) and ice lolly with orange flavor (C) are shown in Fig. 3.141. The electropherograms demonstrate that the dyes are well separated from each other and from the impurities present in the commercial samples. The amounts of dyes found in the sample by CZE and HPLC are compiled in Table 3.37. The data prove that the numerical results of both methods are similar, therefore, CZE can also be applied for the separation and quantitative determinaton of dyes in foods [185]. CZE has been employed for the analysis of another set of dyes in foodstuffs. The chemical structures, numbers and names of the dyes included in the investigation are listed in Fig. 3.142. A fused-silica capillary column of 57 cm length (50 cm effective length; 75 m i.d.) was employed for the separations. The capillary was conditioned by 1.0 M NaOH for 20 min followed by 10 min wash with water and 10 min wash with the running buffer. The buffer was prepared by adding NaOH to 10 mM phosphoric acid to reach pH 11.0. The capillary was thermostated at 25oC and the separation voltage was 20 kV. A hydrodynamic injection mode was applied (0.5 psi, 4 s, 21 nl) and spectra of

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521 5

200 150

4

1

10a

100

Absorbance (mAU)

8

7

2

(a)

200

10b

9

13

5

150

9,10a

4

1

100 50

12

3

50 0

11

6

11

6 3

12 10b

8

7

2

(b)

13

0 5 200 4

1

150 100 50

11

6

3

7

2

(c)

10a

8

9

12

10b

13

0 2

4

6

8 Migration time (min)

10

12

14

Fig. 3.139. Electropherogams showing the separation of caffeine (1), aspartame (2), brilliant blue FCF (3), green S (4), sorbic acid (5), benzoic acid (6), saccharin (7), acesulfame K (8), sunset yellow FCF (9), quinoline yellow (10), carmoisine (11), ponceau 4R (12), black PN (13), using 20 mM carbonate buffer, pH 9.5; containing (a) 60 mM SDS, (b) 62 mM SDS, (c) 65 mM SDS. A 48.5  50 m i.d. fused-silica capillary was used and absorbance was measured at 200 nm. Reprinted with permission from R. A. Frazier et al. [184].

analytes were taken between 280 and 600 nm. Liquid samples were diluted, filtered and used for CZE without any other purification step. Jellies were extracted with methanol and centrifuged. The supernatant was evaporated to dryness and redissolved in water. The electrophoregrams of a commercial sample and the standard mixture are shown in Fig. 3.143 The electropherogams demonstrate the baseline separation of dyes even in the presence of complicated accompanying matrices such as mint syrup. The LOD and LOQ values were between 1.0 – 1.7 and 3.2 – 5.5 g/ml, respectively, and the RSD of migration time and peak areas ranged 1.3 – 1.8 per cent and 2.0 – 3.1 per cent. The migration times of the dyes were: Tartrazine, 9.97 min; Sunset yellow FCF 6.73 min; Amaranth, 8.41 min; New coccine, 7.49 min; Allura red AC, 5.43 min; Patent blue V calcium salt, 4.23 min. The amounts of synthetic dyes found in commercial samples are compiled in Table 3.38. It has been stated that the analysis time is rapid and the method makes possible the separation and determination of these dyes at ppm levels in various foodstuffs [186].

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522

Chapter 3 OH

HO NaO3S

N

NaO3S

N

N

N

SO3Na SO3Na

E-110 HO

E-112

SO3Na

HO

N

NaO3S

N

NaO3S

N

N NaO3S

SO3Na

E-123

H

SO3Na E-124

CH2 OH

O H

H

OH H

OH H

OH

O

CH3

OH

NaO

OH

HO OH

I

I COOH

I

O

O

O +

Na I

COONa

E-127

E-120

OH N

NH-CO-CH3

N SO3Na

NaO3S E-128

Fig. 3.140. Structures of dyes studied. Reprinted with permission from J. J. B. Nevado et al. [185].

CZE using cyclodextrins (CD) as buffer additives has also been employed for the determination of synthetic dyes in various food products. The synthetic dyes New coccine, Erythrosine, Allura red AC, Tartrazine, Sunset yellow FCF, Brilliant blue FCF, Indigo carmine and Fast green FCF were included in the experiments. Measurements were carried out in a used-silica capillary (47 cm length, 40 cm to the detector, 50 m i.d.). Capillary temperature was 25°C and separation voltage was 20 kV. Pressure injection was performed

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30

mAU

20

E−122 E−124

10

E−120

E−127

15

15 10

5

5

0

(a)

E−122

E−125

25 E−110

20

E−124

E−126

25

mAU

30

0 0

4

8

12 Minutes

16

20

(b)

0

2

4

6

8 10 Minutes

12

14

16

25

E−110

20

E−122

10

5

E−123

mAU

15

0 0

(c)

4

8

12 Minutes

16

20

Fig. 3.141. Electropherograms of a standard mixture of the seven dyes (a), a sample of grenadine (b), and ice lolly with orange flavour (c). Operating conditions: 15 mM borate (pH  10.5); 20 kV and 25°C. Reprinted with permission from J. J. B. Nevado et al. [185].

at 0.034 bar (0.5 psi, 3 s). Analytes were detected at 200 nm. Ice cream samples were injected after melting without any other pretreatment, fruit soda drinks were degassed before injection. The capillary was conditioned with 0.1 M NaOH, water and buffer before analysis. Running buffer consisted of 0.025 M borax – NaOH (pH  9.5); it contained various amounts of CDs. Electrophoregrams illustrating the separation of synthetic dyes from various foodstuffs are shown in Fig. 3.144. The migration times of dyes were: New coccine, 9.17 min; Erythrosine, 5.06 min; Allura red AC, 6.06 min; Tartrazine, 6.57 min; Sunset yellow FCF, 4.83 min; Brilliant blue FCF, 3.94 min; Indigo carmine, 5.37 min; fast green FCF, 4.52 min. The concentrations of dyes in the samples are compiled in Table 3.39. It has been stated that this method is rapid, simple, it does not require complicated sample preparation. It has been proposed for the determination of food colourants in real samples [187]. Synthetic dyes were determined in milk beverages by CZE too. Colourants included in the investigations were the same as in ref. [187]. Milk samples were mixed with equal volumes of ethanol, stirred for 10 min then the pH was adjusted to 2.0. The suspension was centrifuged and the supernatant was loaded into a polyamide SPE cartridge preconditioned with 2 ml of methanol followed with 2 ml of water. After loading the supernatant, the cartridge

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Chapter 3

N

NaO3S

HO

COONa N

N

NaO3S

HO

N

N

N

SO3Na SO3Na H SO3Na

H NaO3S

NaO3S

N

N

N N NaO3S SO3Na

SO3Na OMe

SO3Ca2

HO

HO N

NaO3S

N

SO3-

CH3

CH3

CH3 N+

N CH3

SO3Na

CH3

Fig. 3.142. Structures, numbers and names of the dyes used. Reprinted with permission from M. Pérez-Urquiza et al. [186].

was washed with 1 ml of water and 1 ml of methanol. Dyes were eluted with 1 ml of 0.5 per cent ammonia–methanol (1:1, v/v). Running buffer was a 15 mM disodium tetraborate and the pH of the solution was adjusted by NaOH. To influence migration time -CD was added to the running buffer in various concentrations. Separations were realized in an uncoated fused-silica capillary (50.2 cm length, 40 cm to the detector, 50 m i.d.) preconditioned with 1 M NaOH (3 min), water (5 min) and running buffer (5 min). Samples were pressure injected (0.5 psi, 5 s). Separation voltage was 25 kV and the capillary was thermostated at 25oC. Dyes were detected at 200 nm. The migration times of dyes were: New coccine, 7.91 min; Carminic acid, 5.18 min; Allur red AC, 4.72 min; Tartrazine, 5.74 min; Sunset yellow FCF, 4.11 min; Brilliant blue FCF, 3.39 min; Indigo carmine, 4.36 min; Fast green FCF, 3.88 min. Some typical electropherograms of real samples are shown in Fig. 3.145. The type and concentration of dyes found in commercial milk beverages are compiled in Table 3.40. It was found that the prepurification of dyes by SPE is simple, the analysis time is relatively short,

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E−126

30

525

E−110 E−120

10

E−122 E−124

15 E−127

mAU

20

E−123

25

5

0 0

4

8

12

16

20

Minutes

Fig. 3.143. Electropherogram of the dyes studied under optimized conditions. Electrophoretic buffer: buffer phosphates 10 mM, pH 11.0; applied voltage, 20 kV, and detection wavelength, 280 nm. Hydrodynamic injection at 0.5 psi for 4 s. All analytes have around 4.5 g/ml, and the sample has 9 g/ml of E-131 and 14.8 g/ml of E-102. Reprinted with permission from M. Pérez-Urquiza et al. [186].

and the compounds of the accompanying matrix do not interfere with the separation and quantitation of the dyes [188]. A very similar method has been employed for the determination of the same set of food colourants using a large-volume sample stacking (LVSS) technique. Carbonated beverages were degassed and submitted to SPE purification when required. Jelly samples were mixed with 10 volumes of 50 per cent aqueous ethanol, stirred at 65oC for 4 h and the aqueous phase was treated by SPE using polyamide cartridges. Milk samples were treated as described before. Samples were injected in to the capillary by pressure (2 psi) for 55 or 25 s. After injection, a -5 kV voltage was employed to stack the analytes; it takes about 3 min. Electropherograms of some real samples are shown in Fig. 3.146. The concentrations of dyes found in commercial food samples and the average migration times are compiled in Table 3.41. It was established that the LVSS technique considerably enhances the sensitivity of the determination. Detection limits for CE varied between 0.18 – 1.76 g/ml and the same values for LVSS ranged 0.002 – 0.021 g/ml. Because of the low detection limit, the method was proposed for the analysis of trace amounts of dyes in foods [189].

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TABLE 3.37 RESULTS OF THE QUANTITATIVE ANALYSIS OF DIFFERENT PRODUCTS STUDIED (CONCENTRATIONS IN MG/L). SAMPLES DID NOT CONTAIN E-120 AND E-128

Product

Method E-127

Bitter

CZE LC Grenadine tropic CZE LC Ice lolly strawberry CZE LC Syrup strawberry CZE LC Grenadine CZE LC Ice lolly orange CZE LC Syrup raspberry CZE LC

E-110

— — — — — — — — — — — — 33.4  0.1 32.8  0.2

E-122

— — — — — — — — — — 11.0  0.2 10.7  0.2 — —

E-124

E-123

37.5  0.2 9.0  0.1 35.0  0.2 8.7  0.2 119.3  0.4 130.6  0.3 121.3  0.3 128.7  0.5 34.5  0.1 17.8  0.2 33.1  0.3 16.0  0.2 141.9  0.4a 114.9  0.3a 137.9  0.3a 11.4  0.2a — 14.0  0.1 — 13.7  0.2 6.5  0.1 — 6.8  0.3 — — 39.9  0.1 — 40.7  0.2

a

Concentration in mg/kg. Reprinted with permission from J. J. B. Nevado et al. [185].

TABLE 3.38 CONCENTRATION (G/G) OBTAINED FOR THE SAMPLES ANALYSED. DYES E-123 AND E-129 WERE NOT DETECTED

Product Melon beverage Strawberry syrup Currant syrup Mint syrup Orange jelly Pineapple jelly

E-102

E-110

E-124

E-131

101 128 — 118 463 —

210 — — — 262 13

— — 326 — — —

— — — 55 — —

Reprinted with permission from M. Pérez-Urquiza et al. [186].

— — — — — — — — 8.8  0.2 8.5  0.3 2.3  0.1 2.8  0.2 — —

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0.002 AU Fast green FCF

EOF

0

2

Tartrazine

4 6 Time (min)

(a)

Absorbance (A)

Absorbance (A)

0.01 AU

8

10

EOF

0

New coccine

4 6 Time (min)

8

Absorbance (A)

Absorbance (A)

2

8

10

8

10

0.002 AU Sunset yellow FCF

EOF

0

4 6 Time (min)

(c)

0.005 AU

(b)

2

Allura red Ac

10

Tartrazine EOF

0 (d)

2

4 6 Time (min)

Fig. 3.144. Electropherograms of synthetic food colourants in two different ice cream bars (a,b), grape soda (c) and mango soda (d). Separation solution, 0.025 M borax–NaOH buffer containing 5 mM -cyclodextrin, pH 9.5; detection wavelength, 200 nm. Reprinted with permission from K.-L. Kuo et al. [187].

A different CE procedure was developed and applied for the determintion of Sunset yellow (E-110), Carmoisine (E-122) and Ponceau 4R (E-124) in ice cream. Dyes were extracted by mixing 10 g of ice cream with 30 ml of ethanol–cc.ammonia (95:5, v/v). The liquid phase was separated, filtered and the extraction step was repeated twice with 15 ml of extracting agent. Analytes were separated in a fused-silica capillary column (50 cm x 75 m i.d.). Hydrostatic injection was applied with positive nitrogen pressure (15 min). The separation voltage was 30 kV and the capillary was thermostated at 30oC. Running buffer was 25 mM sodium phosphate (pH 8) – 25 mM sodium borate (pH 8) (1:1, v/v). Electropherograms of ice creams containing synthetic dyes are shown in Fig. 3.147. Good linear correlations were found between the concentration of dyes and the corresponding peak areas, the coefficients of correlation being 0.9988 – 0.9994. Recovery values for dyes were 98.301.56 per cent, 93.411.73 per cent, 95.092.80 per cent for E-110, E-122 and E-124, respectively. It was concluded from the results that the method is rapid and inexpensive, its good reproducibility, sensitivity and linearity are comparable with those of HPLC procedures [190].

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Chapter 3 TABLE 3.39 CONTENT OF SYNTHETIC FOOD COLOURANTS IN SELECTED FOOD PRODUCTSa

Sample

Colourant

Concentration (g ml1) and (RSD, %)

Ice cream bar A Ice cream bar B Grape soda drink

Tartrazine New coccine Fast green FCF Allura red AC Sunset yellow FCF Tartrazine Tartrazine

27.88(5.37) 21.42(3.54) 9.38(7.38) 25.32(4.95) 9.34(3.81) 9.66(4.85) 11.94(3.29)

Mango soda drink Pineapple soda drink a

Values are means of triplicate determinations. Reprinted with permission from K.-L. Kuo et al. [187].

3.6.2 Environmental and toxicological application of electrophoretic methods Because of the good separation and validation parameters, various electrophoretic techniques have found application in environmental analysis too. Xanthene dyes (phloxine B and uranine) were determined in guava fruits by both CZE and RP-HPLC and the results were compared. CZE measurements were performed in a fused-silica capillary (50 cm x 75 m i.d.) washed with 0.1 M NaOH for 1 min, with water for 3 min and finally with running buffer for 5 min. The buffer was 10 mM Na2B4O7 and 50 mM H3BO3 (pH  8.5). The separation voltage was 20 kV, and samples were injected by the gravity mode. Phloxine B and uranine were detected at 546 and 493 nm, respectively. RP-HPLC analyses were carried out in an ODS column using gradient elution. Solvents were ACN and 0.5 M NH4OAc buffer. The linear gradient started with 20 per cent ACN and increased to 80 per cent ACN in 15 min. A characteristic separation of dyes is shown in Fig. 3.148. The figure illustrates the baseline separation of the dyes. Guava fruit were mixed with dry ice in 1:1 w/w ratio and stored at 25°C. The efficacy of various mixtures of methanol, acetone and acetonitrile for the extraction of dyes was screened at different pH and temperature. The influence of the presence of salts such as Na4EDTA, (NaPO3)6, MgO and Pb(OAc)2 on the recovery was also determined. Samples were further purified by SPE. Extracts of 50 – 100 ml were loaded into amino SPE cartridges containing 0.5 – 1 g of sorbent. Cartridges were activated by methanol followed by methanol acidified by HCl. After loading, the SPE columns were washed with hexane, acetone and methanol. Dyes were eluted with basic methanol (0.5 – 1.0 ml of 1 M NaOH) mixed with

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16000 Strawberry-flavored milk

Carminic acid

(c) 12000

Intensity (AU)

New coccine Strawberry-flavored yogurt

(b) 8000

New coccine

Brilliant blue FCF (a)

4000

0

0

1

2

3

4

5 Time (min)

6

7

Blueberry-flavored milk

8

9

10

Fig. 3.145. The electrophoregrams of commercially available blueberry-flavoured and strawberry flavoured milk beverages. (a) Blueberry-flavoured milk, (b) strawberry-flavoured yoghurt, and (c) strawberry-flavoured milk. New coccine, Brilliant blue FCF, or carminic acid were found in the products. The samples that contained new coccine were directly centrifuged without a pH change, while the sample that contained carminic acid was adjusted to pH 2.0 prior to centrifugation. Separating conditions: borax–NaOH buffer (pH 10) containing 7.0 mM -CD was used as a running buffer. A voltage of 25 kV was applied to a fused-silica capillary tube of 50.2 cm  50 m i.d. (40 cm from the inlet end to the detection window). Reprinted with permission from H.-Y. Huang et al. [188].

5–7 ml of methanol. It was establshed that the highest recovery can be obtained by using a methanol–ACN mixture (1:1, v/v) containing 2.5 g of MgO for 300 ml of extracting agent.The LOD values were smaller for RP-HPLC (phloxine B: 0.020 g/ml; uranine 0.054 /ml) than for CZE (phloxine B: 0.138 g/ml; uranine 0.081 /ml). Recoveries were similar for both techniques and depended highly on the concentration of dyes (about 100 per cent at 1 g/g and 68 – 79 per cent at 0.05 g/g). Phloxine B content found in real samples by CZE are compiled in Table 3.42. The optimized extraction, SPE purification and CZE separation procedure allowed the measurement of the concentration of phloxine B and uranine in guava fruits [191]. Much effort has been devoted to the applicaton of electrophoretic techniques for the analysis of dyes and dye decomposition products in industrial waste-waters. CZE with DAD and MS detection was employed for the determination of reactive dyes in spent dyebaths and waste-waters. The chemical structure of the dyes included in the experiments are shown in Fig. 3.149. Liquid samples were purified and preconcentrated by SPE. ODS cartridges were conditioned by washing with two bed volumes of methanol, four bed volumes of water and two bed volumes of 5 mM tetrabutylammonium phosphate. Aliquots of 10 – 30 ml were loaded and the cartridges were washed with 2 ml of water. Analytes were eluted with 2 ml of methanol–water (70:30, v/v). Untreated fused-silica capillaries (110 cm and 57 cm  50 m i.d.) were coupled to MS and DAD. The running

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TABLE 3.40 CONTENTS OF COLOURANTS DETERMINED IN COMMERCIAL MILK BEVERAGES

Milk sample

Mixed fruit-flavoured milk A Mixed fruit-flavoured milk B Apple-flavoured milk A Apple-flavoured milk B Apple-flavoured milk C Mango-flavoured milk Blueberry-flavoured milk A Blueberry-flavoured milk B Strawberry-flavoured milk Strawberry-flavoured yoghurt A Strawberry-flavoured yoghurt B Strawberry-flavoured yoghurt C

Colourant

Tartrazine Tartrazine Tartrazine Sunset yellow FCF Tartrazine Sunset yellow FCF Tartrazine 12 Sunset yellow FCF Tartrazine Sunset yellow FCF New coccine Brilliant blue FCF New coccine Brilliant blue FCF Carminic acid New coccine Carminic acid Carminic acid

Concentrationa

g/ml

RSD (%)

47.1 23.3 13.4 6.5 17.3 6.3 12.6 7.6 27.7 10.6 27.7 10.2 2 042 4.17 25.2 6.5 15.9 11.35

0.90 0.91 0.89 0.11 1.23 2.24 1.41 3.08 0.51 1.33 1.28 4.87 3.67 2.71 1.12 4.88 1.33 0.62

a

Values are means of tiplicate determinations. Reprinted with permission from H.-Y. Huang et al. [188].

buffer for CE-DAD was 30 per cent ACN and 30 mM ammonium acetate. CE-MS measurements were carried out with a running buffer of 40 per cent ACN and 5 mM acetate. Samples were injected by pressure at 0.5 psi for 2 s (CE-DAD) and for 15 s (CE-MS). MS conditions were: sheath liquid, 2 propanol–water, 80:20, v/v; flow rate, 2 l/min; sheath and drying gas was nitrogen at flow rates of 20 and 50 l/h, respectively. The electrospray voltage was 3 and 3.5 kV; the cone voltage was 20 V. Recovery of dyes varied between 81 – 121 per cent, the detection limit depended on the character of the accompanying matrix (23 – 42 g/l). The method was proposed for the analysis of sulphonated dyes in dye waste-waters [192]. CZE was also employed for the analysis of sulphonated azo dyes in river samples. The chemical structures of dyes are shown in Fig. 3.150. Separations were performed in a fused-silica capillary (total length 57 cm; effective length, 50 cm; 75 m i.d.). Activation of the capillary was carried out by washing it with 1.0 M NaOH for 15 min, followed by water (5 min) and the running buffer (5 min). The buffer was prepared from 10 mM

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13000 Brilliant blue FCF Allura red AC

(a) Grape-flavored jelly

8000

Intensity (AU)

Sunset yellow FCF

3000

(b) Peach-flavored jelly

Sunset yellow FCF

−2000

Taratrazine

−7000

(c) Apple-flavored milk

−12000 0

1

2

3

4

5 Time (min)

6

7

8

9

10

Fig. 3.146. Electropherograms of commercially available jelly and milk products determined by LVSS. (a) Grape-flavoured jelly, (b) peach-flavoured jelly, and (c) apple-flavoured milk. LVSS conditions were: sample injection time 25 s (2 psi); sample stacking time 2.9 min (5 kV), after which the current reached 95 per cent of its maximum value. Samples were treated with a polyamide column SPE and then separated by LVSS-CE. Reprinted with permission from H.-Y. Huang et al. [189].

phosphoric acid and tetrabutylammonium hydroxide and 25 mM triethylamine diluted to 1:5 volume ratio (pH  11.55). The separation voltage was 15 kV and the capillary was held at 25°C. Hydrodynamic injection mode was aplied for 4 s. Dyes were detected at 300 – 500 nm. Samples of river water were filtered, spiked, and the pH was adjusted to pH  3 by 0.1 M HCl. Samples of 100 ml were passed trough the SPE cartridges preconditioned with 1 ml of methanol and 2 ml of water. After loading, the analytes were eluted with 2 ml of methanol, evaporated to dryness and redissolved in 250 l of running buffer. Electropherograms showing the good separation capacity of the CZE method are shown in Fig. 3.151. The migration times of dyes under optimal conditions, the detection and quantitation limits, and the recoveries are compiled in Table 3.43. It was concluded from the results that the preconcentration step is simple, the CZE method is rapid, the separation capacity is good, and it does not require an organic solvent [193]. The microbiological decomposition of some phtalocyanine dyes was also followed by CZE and HPLC. The dyes included in the experiments were Remazol turquoise blue G133, Everzol turquoise blue and Heligon blue S4. The white-rot fungi Phanerochaete chrysosporium PC671 was grown in the presence of dyes and samples were taken from the culture media at different times during the fermentation process. They were centrifuged and the supernatant was employed for CZE and HPLC analyses. CZE measurements were realized in an untreated fused-silica capillary column (78 cm length; 58 cm effective length, 75 m i.d.). The capillary was activated by rinsing with 1 M sodium hydroxide for 0.4 min at 30oC then the same washing step was repeated at 60oC followed by rinsing for 0.4 min at 60 and 30oC. Finally, capillary was conditioned with the running

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TABLE 3.41 AVERAGE MIGRATION TIMES AND CONTENTS OF COLOURANTS DETERMINED IN COMMERCIAL FOOD SAMPLES

Food sample

Colourant

Concentrationa

Migration timeb

g/ml

RSD(%)

Min

RSD(%)

Grape-flavoured

Fast green FCF

0.90

0.79

4.38

2.60

carbonated beverage

Allura red AC

4.31

2.29

6.01

3.79

Orange-flavoured

Sunset yellow FCF 0.97

7.69

4.58

3.49

carbonated beverage

Tartrazine

0.38

3.74

6.69

1.10

Apple-flavoured

Brilliant blue FCF

0.05

0.23

4.68

0.40

carbonated beverage

Tartrazine

0.81

3.36

6.20

0.61

Grape-flavoured jelly

Brilliant blue FCF

0.07

2.34

3.86

0.44

Allure red AC

0.48

0.03

5.52

1.05

Sunset yellow FCF 0.16

6.45

4.88

0.52

0.30

6.83

8.16

0.84

Sunset yellow FCF 1.67

3.45

5.15

2.71

Tartrazine

1.70

7.49

0.47

Peach-flavoured jelly

Pineapple-flavoured jelly Tartrazine Apple-flavoured milk

3.46

a

Quantitive analysis was based on the standard addition method; colourant standards were added to each food sample in the range 1 to 5 g/ml. Values are the mean of nine measurements. b Values are the mean of nine measurements. Reprinted with permission from H.-Y. Huang et al. [189].

buffer (30°C; 4.0 min, 100 psi). The running buffer consisted of 0.025 M cetyltrimethylammonium bromide (CTAB) and 0.0385 M sodium tetraborate. The separation voltage was -12.5 kV and the capillary was thermostated at 30°C. Analytes were detected at 254 and 666 nm. Vacuum-controlled hydrodynamic injection was employed for 30 s. RPHPLC determinations employed an ODS column (250 mm x 4.6 mm i.d.). The isocratic mobile these was composed of 50 per cent ACN and 50 per cent 20 mM ammonium acetate. The flow rate was set to 0.55 ml/min. Detection wavelengths were the same as for CZE. The LOD values of dyes in RP-HPLC ranged between 0.27 and 0.37 g/ml; they varied for CZE from 0.24 to 0.35 g/ml. The samples were also investigated by atomic absorption spectrometry and differential pulse polarography. Some electropherograms illustrating the degradation of a dye are shown in Fig. 3.152. Combining the results of the

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OH N

NaO3S

E110

10

N

SO3Na

4.908

5.500 5.735

2.196

5

2.313

Absorbance mAU

533

4.471

Liquid chromatography of synthetic dyes

0 0

2

3

4

5 6 7 Minutes 200 nm Band = 10 nm

8

9

10

4.900

1

E122

NaO3S

N

N

OH

10

5

3.706 3.875

2.220

Absorbance mAU

15

SO3Na

0 1

2

3

4

5 6 7 Minutes 200 nm Band= 10 nm

40 E124

35

9

10

OH N

NaO3S

N

25 20 SO3Na

NaO3S

15 5

3.675

0.237

10

2.225

Absorbance mAU

30

8

5.600

0

0 0

1

2

3

4

5 6 7 Minutes 200 nm Band = 10 nm

8

9

10

Fig. 3.147. Electropherograms of ice cream with E-110; fruit ice cream with E-122 and fruit ice cream with E-124. Reprinted with permission from L. D. Giovine et al. [190].

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TABLE 3.42 DETERMINATION OF PHLOXINE B IN GUAVA SAMPLES FROM THE DYE-SPRAYED ORCHARD

Phloxine B found, (ng/ga)

Treatment row 5 days after 7th spraying B C F I L O MDb Overall meanc CV (%)

4 hours after 11th spraying 21(1) 250(25) 74(24)

36(10) 24(10) 7(2)

100(22) 49(4) 426(94) 111(18) 17

29(7) 26

5 days after 14th spraying

44(18) 5.1(1) 10(3) 29(19) 4.9(2) 19(8) 40

a

Each value is the mean of 3 – 5 replications. Values in parentheses are standard deviations. No dyes were detected in any control samples. b MD  maximum dye, from fruit samples with high deposits of phloxine B. c Overall mean of phloxine B concentrations (and of SDs) in guava fruits from each sampling time (MD sample excluded. Reprinted with permission from J. P. Alcantara-Licudine et al. [191]. Phloxine B Cl Cl

Cl O

Cl

O

Br

Uranine

O O

ONa

O

O

H2O Br

ONa Br

Br

H2O

0

2

4

6 8 10 12 Retention time, min

14

16

18

Fig. 3.148. Liquid chromatogram of uranine and phloxine B. Reprinted with permission from J. P. Alcantara-Licudine et al. [191].

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535 OSO3S

OH N

O

N

SO3-

N H

1 O

NH2 SO3-

O

NH

S

SO3-N

-O3S

N

N N

OSO3-

OSO3-

O

2

O

O

NH2 N

OH

S

S

O

O

OSO3-

O

3 Cl N

N -O3S

N H

N

OSO3-

O NH

S

OH N

O

N

SO3-

-O3S 4 O

SO3-

-O3S

O

O N N

Cu

N

S

N

O

O

OSO3-

5

Fig. 3.149. Reactive dyes investigated in this study. Reprinted with permission from T. Poiger et al. [192].

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Chapter 3

Structure

pKa OH

10316 Acid Yellow 1

(NYS) NO2

NaO3S

CIa number and name

NO2 (OII)

HO

10,62

15510 Acid Orange 7

(CO)

10,43

15970 Acid Orange 12

(MO)

3,20

13025 Acid Orange 52

N

NaO3S

N

HO N N

SO3Na

N

(CH3)2N

HO

CH3 CH3

SO3Na

N

SO3Na (P2R)

N

11,59

16150 Acid Red 26

10,36

16185 Acid Red 27 Food Red 9

11,06

15620 Acid Red 88

N

SO3Na HO NaO3SH3

SO3Na (AMR)

N N

SO3Na

HO NaO3SH3

(R88)

N N

a

CI = Colour Index

Fig. 3.150. Structures, numbers and names of the dyes used. Reprinted with permission from M. Pérez-Urquiza et al. [193].

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537

MO

Liquid chromatography of synthetic dyes

0.010

NYS

AMR

P2R

OII CO

0.006

R88

Absorbance (Relative units)

0.008

0.004 Spiked water sample

0.002 Standard 0.000

0

2

4 Time (minutes)

6

8

Fig. 3.151. Electropherogram of a dye mixture obtained under optimized conditions; voltage 15 kV and detection 460 nm. Injection was made in hydrodynamic mode by 4 s. The dye concentrations in the standard were 15 g/ml except methyl orange (30 g/ml); in the spiked river water they were 20 g/ml except methyl orange (40 g/ml). Reprinted with permission from M. Pérez-Urquiza et al. [193].

different analytical methods used in this study the decomposition pathway shown in Fig. 153 was proposed [194]. Because of their carcinogenic and other toxic effects, the decomposition products of dyes such as aromatic sulphonates, aryl amines, benzenediamines, benzedediols, aminophenols, 1,2-dihydroxynaphtalene have been frequently investigated by various electrophoretic techniques. The formation of carcinogenic aryl amines in azo dyes and the synthetic pathways of some dyes were also studied by CE. Measurements were performed in a fused-silica capillary coated by polyimide (60 cm total length, 52 cm effective length, 75 m i.d.) at room temperature. The capillary was activated by rinsing 1 M sodium hydroxide for 15 min, followed by water for 15 min. The buffer employed for the separation and determination of aromatic amines and the control of the formation of diazonium salts consisted of 50 mM phosphate buffer (pH 3.1 adjusted with 0.1 M NaOH) containing 10 per cent methanol.separaton conditions were: 22 kV working potential; hydrostatic injection, 10 cm for 20 sec; detection wavelength, 214 nm. The running buffer of the second method employed for the separation of H acid, Koch’s acid and Chromotropic acid

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Chapter 3 TABLE 3.43 MIGRATION TIME OF THE DYES UNDER OPTIMIZED CONDITIONS, DETECTION AND QUANTITATION LIMITS, AND ACHIEVED RECOVERIES AFTER PRECONCENTRATION OF THE SPIKED RIVER WATER ANALYSED

Dye

Migration time (min)

LOD (g/ml)

LOQ (g/ml)

Recoverya (%)

Acid yellow 1 Acid orange 7 Acid orange 12 Acid orange 52 Acid red 26 Acid red 27 Acid red 88

7.73 5.40 5.65 4.74 5.89 6.86 5.01

0.1 0.90 1.82 0.43 4.53 2.87 1.99

0.34 3.01 6.08 1.42 15.09 9.58 6.63

89 12 92 13 99 8 92 13 82 11 65 11 96 13

Mean recoveries obtained at 25, 37.5, 50, 100 and 150 g/l. Reprinted with permission from M. Pérez-Urquiza et al. [193]. a

consisted of 20 mM citrate buffer (pH adjusted to 4.5 with 0.1 M NaOH). The separation voltage was 20 kV the other conditions were the same as in the case of the previous method. An electropherogram demonstrating the good separation capacity of the CZE method is shown in Fig. 3.154. The applicability of the second CZE method for the quality control of the different starting materials used for the synthesis of dyes is illustrated in Fig. 3.155. This method allowed the detection of the carcinogenic 4-aminodiphenyl as a by-product of dye synthesis [195]. CE combined with MS and DAD detections was employed for the separation of the decomposition products of a dye in waste-water under oxidative conditions. The structure of the original dye molecule Orange II and those of its possible decomposition products (p-phenolsulfonate, o-phtalate, p-sulphobenzoate and 1,2-dihydroxynaphtalene) are shown in Fig. 3.156. Untreated capillaries or capillaries coated with linear polyacrylamide were used for the measurements. The total and effective lengths of the capillaries were 62 and 50 cm, respectively (75 m i.d.). Running buffers for CE-MS consisted of 40 mM ammonium acetate (pH  6.0) and 50 mM ammonium carconate (pH  8.5). Samples were injected at 5 kPa for 8 s. When the acetate buffer and coated capillary was used the separation voltage was -25 kV. Ion-spray voltage was set to -4.5 kV. In the case of untreated fused-silica capillaries and carbonate buffer the separation voltage was 25 kV. The running buffer for CE-UV/DAD was 50 mM ammonium carbonate buffer (pH  9.5). Samples were injected hydrodynamically (20 mm in 30 s). Separation voltage and capillary temperature were 20 kV and 30oC, respectively. The baseline separation of Orange II and its possible decomposition products is shown in Fig. 3.157.

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539

Day 0

Day 3 remazoITO.Inj1. UV3000 A 666nm

12

12

10

10

8 6

2

0

0

−2

−2

−4

−4 10

20

(a)

50

60

2

−2

10

20

30 40 Minutes

10

20

30 40 Minutes

50

60

20

30 40 Minutes

50

60

Day 7

0

0

0 (b)

25.79 30.15

Day 5

30 40 Minutes

mV or mAU

0

mV or mAU

6 8.434

2

(c)

8

4 8.434

4

25.79

14

mV or mAU

mV or mAU

14

remazoITO.Inj1. UV3000 A 666nm

16

25.79

16

50

60

2 0 −2

0 (d)

10

Fig. 3.152. Electropherograms (a–d) of Remazol TB and metabolite M1R detected at 666 nm at different time points in the kinetic study. Reprinted with permission from A. Conneely et al. [194].

It was stated that these CE-MS and CE-UV/DAD methods allow the separation and identification of the degradation products of Orange II and can be employed for the study of the decomposition process in more detail [196]. A combination of chomatographic techniques such as CZE-UV, ion-pair LC-DAD and ion-pair LC-electrospray mass spectrometry was employed for the separation of polar hydrophilic aromatic sulphonates. SPE preconcentration of aromatic sulphonates was studied in detail. Sorbents LiChrolut EN, Isolute ENV
, HR-P and Oasis HLB were included in the experiments. They were activated by rinsing 7 ml of methanol followed with 3 ml of water acidified to pH 2.5 with sulphuric acid. Water samples were passed

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Chapter 3 SO3N

SO3-

N

N

N

Cu N

N

N N

R

SO3manganese peroxidase SO3-

Cu2+

+

N

SO3-

N

N

N N

N

N N

R

SO3metabolite M1 breakup of phtalocyanine ring sysem

O electroactive metabolite M2 and other metabolites NH

+

O ptalimide, metabolite M3

Fig. 3.153. The proposed degradation pathway for copper phtalocyanine dyes using P. chrysosporum (white-rot fungi). Reprinted with permission from A. Conneely et al. [194].

through the cartridges at the flow rate of 1 ml/min. Analytes were eluted with 1 ml of 5 mM aqueous triethylamine–acetic acid followed by 6 ml of methanol–acetone (1:1, v/v). CZE was performed in various untreated fused capillary columns of 75 m i.d. The total lengths of the capillaries were 47 cm (40 cm effective length) and 64.5 cm (56 cm

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541

0.039 11

0.037

14

Volts

0.035

0.033 8 1 2

0.031

3 0.029

6 7 4

9 10

13 12

15

5

0.027 4

5

6

7 Time (min)

8

9

10

Fig. 3.154. Electropherogram for the working solution of aromatic amines. Peaks: 1  4,4-diaminodiphenylmethane; 2  4,4-oxidianiline; 3  benzidine; 4  aniline; 5  2,4-diaminoanisole; 6  2,4-toluilendiamine; 7  o-toluidine; 8  3,3-dimethylbenzidine; 9  3,3-dimethoxybenzidine; 10  p-cresidine; 11  2-naphtylamine; 12  p-chloroaniline; 13  4-aminodiphenyl; 14  1-naphtylamine; 15 = 4-chlorotoluidine; all at 10 ng/l. Conditions: buffer  50 mM phosphate 10 per cent methanol; pH  3.1; fused-silica capillary recovered with polyamide, 52 cm  75 m i.d.; applied potential 
22 kV; UV detection at 214 nm. Reprinted with permission from S. Borrós et al. [195].

effective length). Analytes were pressure injected at 0.5 psi for 5 s and were separated at 20 – 25 kV voltage. Running buffers were 12 mM ammonium acetate (pH adjusted to 10 with ammonia) and 12 mM sodium borate (pH 9.3). Capillaries were thermostated at 25°C and aromatic sulphonates were detected at 214 nm. HPLC separations were realized in an ODS column (75  4mm i.d.; particle size 4 m) using gradient elution. Solvents A and B were methanol and water (pH 6.5) both with 5 mM triethylamine and 5 mM of acetic acid. The gradient started with 100 per cent B (flow rate, 0.8 ml/min; in 6 min 100 per cent B and 1.0 ml/min; in 10 min 95 per cent B; in 15 min, 80 per cent B; in 20 min 40 per cent B; in 25 min, 30 per cent B; in 30 min, 25 per cent B). An electropherogram illustrating the separation of some aromatic sulphonates is shown in Fig. 3.158. It was further established that aromatic sulphonates can also be separated by the running buffer ammonium acetate allowing the use of MS detection. Chromatograms illustrating the separation of analytes by ion-pair HPLC are shown in Fig. 3.159. The results demonstrated that in this special case the separation capacity of ion-pair HPLC is higher than that of CZE. SPE investigations indicated that the highest recoveries can be obtained on LiChrolut EN and HR-P adsorbents. The practical applicaton of the method for the determination of aromatic sulphonates in waste-water has also been presented [197].

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Chapter 3

OH OH

NH3

OH

0.030

HO3S SO3H NH3

HO3S

SO3H

SO3H

H acid

0.029 SO3H

Volts

HO3S

Chromotropic acid

0.028 Koch acid 0.027

0.026 3

4

5

6

Time (min)

Fig. 3.155. Electropherogram from the analysis of a 1 000 ng/l sample of H-acid (81.5 per cent), using 20 mM citrate buffer, pH 4.5; fused-silica capillary recovered with polyimide, 52cm  75 m i.d.; -20 kV potential, UV detection at 254 nm. Reprinted with permission from S. Borrs et al. [195].

Micellar electrokinetic chromatography (MEKC) has been applied for the determination of benzenediamines, benzenediols and aminophenols in oxidative hair dyes. The investigations were motivated by the findings that these compounds may cause allergic dermatitis, are nefrotoxic and carcinogenic. Analyses were performed in a capillary of 67 cm  50 m at 25°C. UV detection was carried out 7 cm from the anodic end. Injections were made in hydrodynamic mode for 1 s. The capillary was conditioned by rinsing with water (25°C, 5 min), followed with 1.0 and 0.1 M of NaOH at 60°C for 10 min, then washing with water at 25oC for 10 min. A modified phosphate buffer of 50 mM was applied. Tetradecyl- and hexadecyl-trimethylammonium bromides (TTAB and CTAB) were employed as buffer additives in the concentration range of 0.0 – 30.0 mM. The separation voltage was – 20 kV. The measurements indicated that the concentration of the cationic surfactant in the running buffer exerts a considerable effect on the electrophoretic mobility of the analytes. The pH, the concentration of the buffer and the organic modifiers equally influenced the migration behaviour. The effect of organic modifiers on the electrophoretic behaviour of dye intermediates is illustrated in Fig. 3.160. The chromatopraphic parameters of analytes are compiled in Table 3.45. It was established that the method can be applied in practice for the analysis of dye intermediates in real samples [198]. An interesting application of CZE was the study of the interaction of human serum transferrin and fluorescein isothiocyanate (FITC). Measurements were carried out in an uncoated fused-silica capillary (total length 59 cm; 75 m i.d.; effective lengths for

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543 1,2-dihydroxynaphtalene m/z = 159.2

OH N

O-

N

OH Fe3+ (Fenton oxidation) +

H2O2, H

OH

SO3-

Orange II m/z 7 327,3

p-phenolsulfonate mz = 173,2

SO3-

COOH

COOH

COO-

SO3o-phthalate m/z = 165,1

p-sulfobenzoate m/z = 201,2

Fig. 3.156. The structure and molecular mass of Orange II and estimated degradation products. Reprinted with permission from S. Takeda et al. [196].

fluorescence and UV detection were 41 and 50.5 cm, respectively). FITC was mixed with the protein in various ratios, and the samples were analysed by CE to monitor the conjugation reaction. The CZE patterns of the FITC-labelled transferrin samples are shown in Fig. 3.161. The data indicated that the conjugation reaction follows a saturation curve and the amount of conjugates increases with increasing ratios of FITC in the reaction mixture as demonstrated in Table 3.46. The method using simultaneous fluorescene and UV detections was proposed for the investigations of interactions between various molecules [199]. Another study employed CE for the determination of the stoichiometry of the conjugation reaction between immonuglobulin and Lissamine rhodamine-B sulphonyl chloride (LRSC). The chemical structure of the dye is shown in Fig. 3.162. Separation of the unconjugated dye from the conjugated end product was performed by CE using an uncoated fused-silica capillary column (60 cm  75 m i.d.). The running buffer consisted of 10 mM borate and 0.5 mM sodium dodecyl sulphate. The separation voltage was 20 kV and analytes were detected by a fluorescence detector. It was concluded from the results that the CE method combined with

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Chapter 3 0.013

2

4

3

0.008 AU

1

0.003

−0.002

0

5

10

15

Time/min

Fig. 3.157. Pherogram of standard solution by CE-UV/DAD. Conditions: sample injection, 20 mm x 30 s; capillary, 62 cm  75 m i.d. untreated fused silica (50 cm to the detector); running solution 50 mM ammonium carbonate buffer (pH  9.5); CE voltage, 20 kV; detection wavelength, 240 nm; temperature, 30°C. Peaks: I, Orange II; II, p-phenolsulphonate; III, o-phtalate; IV, p-sulphobenzoate (100 mg/l of each). Reprinted with permission from S. Takeda et al. [196].

1200

21 18

Absorption [Relative intensity]

1000

15 4

800

6

8 600 22 19 11

1

400

200

0

EOF

1

3

5

7

9 Time [min]

11

13

15

Fig. 3.158. Electropherogram of a 10-compound aromatic sulphonate mixture containing 2 mg/l of each compound. Conditions: running electrolyte 12 mM sodium borate, pH 9.3, capillary 47 cm (40 cm to detectiom window)  75 m i.d., voltage 25 kV, temperature 25oC, pressure injection 0.5 psi. for 5 s, UV detection at 214 nm. For peak identification see Table 3.44. Reprinted with permission from R. Loos et al. [197].

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545

21

(a) UV-DAD

20

13 4 Relative intensity [%]

18

14+15 9

5

1 2+3

7+8

6

17

11 10

19 22

16

12

19

(b) TIC-ESI-MS

12

14+15 13

7+8 1

2

16 17

5

4

10 11

6

6

22

18

9

2+3 4

21 20

8

10 12 Time [min]

14

16

18

Fig. 3.159. IP-UV-DAD (a) and total-ion current (TIC)-ESI-MS (b) chromatogram of a 22compound aromatic sulphonate standard mixture of 4 mf/l in the SIM-NI mode. Injection volume 50 l, fragmentation voltage 80 V. Superspher RP-18 ‘fast and short’ column (75 x 4 mm i.d., 4 m particle diameter). For peak identification see Table 3.44. Reprinted wih permission from R. Loos et al. [197].

other physicochemical techniques is suitable for the study of the decomposition of the dye and for the study of its interaction with immunoglobulin [200]. 3.6.3 Separation of model mixtures of dyes by electrophoretic methods The electrophoretic behaviour of a wide variety of dyes has been achieved in model systems and the influence of various experimental conditions such as the composition,

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Chapter 3 TABLE 3.44 AROMATIC SULPHONATES NUMBERED IN THE ORDER OF THEIR LC RETENTION TIMES (UV-DAD) IN FIG. 3.159

No.

Name

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

2-Amino-1,5-DNS 1,3,6-NTS 1,3-BDS 1,5-NDS 5 2,6-NDS 1-OH-3,6-NDS 1-Amino-5-NS BS 9 1-Amino-4-NS 2-OH-3,6-NDS 1-OH-6-amino-3-NS 3-Nitro-BS 1-Amino-6-NS 4-Methyl-BS 1-OH-4-NS 4-Chloro-BS 2-Amnio-1-NS 1-Amino-7-NS 4-Chloro-3-nitro-BS 1-NS 2-NS Diphenylamine-4-sulphonate

T 2.5 2.7 2.9 3.4 4.8 7.3 8.5 8.6 9.4 11.1 11.9 12.7 13.5 13.9 13.9 15.6 16.0 16.6 17.3 17.7 18.2 18.7

Mw

Mm (m/z)

302 365 236 286 286 302 223 157 223 302 239 203 223 171 223 191/193 223 223 237/239 207 207 249

302 367 237 287 287 303 222 157 222 303 238 202 222 171 223 191 222 222 236 207 207 248

Abbreviations: NS  naphtalenesulphonate; BS  benzenesulphonate; NDS  naphtalenedisulphonate; BDS = benzenedisulphonate; NTS  naphtalenetrisulphonate; OH  hydroxy. Reprinted wih permission from R. Loos et al. [197].

concentration and pH of the running buffer, etc. on the efficacy of the separation has been investigated in detail. The synthesis of phtalocyanine dyes was followed by CE and the purity and composition of the end product was investigated by the same method. The chemical structure of the newly synthetized zinc phtalocyanine tetrasulphonic acid is shown in Fig. 3.163. An uncoated fused silica capillary (65cm  75 m i.d.) was employed for the separation of dye components. Running buffers were 10 mM ammonium acetate (pH adjusted to 9.4 with ammonia) and 10 mM potassium dihydrogenphosphate (KH2PO4) (pH  9.0). Samples were injected hydrodynamically (50 mm for 10 s). Separation was realized in the constant current mode at 30 A, which required about 28 kV separation voltage. Analytes

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547

2 (a) 1 3

Absorbance at 220 nm (arb. unit)

4

(b)

7 6 5

8 10 9

13

11 12

2 3 1

7 4 6 5

8 10 11 12

13

9

10

15

Fig. 3.160. Electropherograms of dye intermediates obtained in a phosphate buffer (50 mM) containing 18 mM TTABat pH 5.0 with addition of organic modifiers: (a) methanol (15 per cent, v/v); (b) acetonitrile (7 per cent, v/v). Other operating conditions: 20 kV, 25°C. For peak idetification see Table 3.45. Reprinted with permission from C.-E. Lin et al. [198].

were detected at 630 nm. An electropherogram showing the separation profile of zinc phtalocyanine tetrasulphonic acid is depicted in Fig. 3.164. It was stated that the CE method is suitable for the separation of phtalocyanine derivatives and can be employed for the control of synthesis pathways and monitoring the concentration of this type of dyes in textile waste-waters [201]. The synthesis of a new near-infrared cyanine dye was monitored by CE and fluorescence detection. The chemicals structure of the dye and its synthetic precursor are depicted in Fig. 3.165. The analysis of the dye was realized in fused-silica capillaries of 75 and 100 m i.d. The total and effective lengths of capillaries were 75 and 60 cm, respectively. The separation voltage was 30 kV and separations were carried out at ambient temperature. The running buffer was 2.5 mM Na2B4O7 (pH  9.2). A near-infrared laser-induced fluorescence detector was applied. Electropherograms illustrating the separation of the dye are shown in Fig. 3.166.

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Chapter 3

TABLE 3.45 PARTITION COEFFICIENTS (PMW), RETENTION FACTOR (K) AND MOBILITY DATA OF DYE INTERMEDIATES MEASURED WITH A PHOSPHATE–(TTAB) BUFFER AT PH 5.0a

Peak no.

Dye intermediates

epb

0c

k

Pmw

1 2 3 4 5 6 7 8 9 10 11 12 13

1,4-benzenediol 3-aminophenol 1,2-benzenediamine 2-aminophenol 1,3-benzenediamine 1,2-benzenediol 1,3-benzenediol 6-methyl-3-aminophenol 8 4-methoxy-1,3-benzenediamine 4-aminophenol 4-methylaminophenol 2-methyl-1,4-benzenediamine 1,4-benzenediamine

0.88 1.14 1.22 1.45 1.63 1.76 1.80 1.99 2.06 2.20 2.32 2.46 2.72

0.08 0.46 0.79 0.88 1.34 0.20 0.20 1.01 1.91 2.06 2.23 2.43 2.68

0.35 0.33 0.22 0.33 0.19 1.09 1.15 0.82 0.14 0.15 0.11 0.05 0.08

66 62 39 59 33 198 210 152 23 28 22 10 24

Mobility in units of 104 cm2 V1 s1; mc  3.19  104 cm2 V1 s1. TTAB at concentration 18 mM. c TTAB at concentration 1.6 mM. Reprinted with permission from C.-E. Lin et al. [198]. a

b

It was further found that 4.7 l of 1.1013 mol/l dye in methanol can also be detected using enriched-injection and methanol – 40 mmol/l aqueous sodium borate (98:2, v/v). Because of the low limit of detection the dye was proposed for labelling purposes [202]. Another CE method was developed and employed for the separation of the components of FD&C Red No. 3 (erythrosine). The separations were also carried out by RP-HPLC and the efficacy of the methods was compared. The chemical structures of the main components of the dye are shown in Fig. 3.167. The components of erythrosine were separated in a fused-silica capillary (43 cm effective length  75 m i.d.). The running buffer was 50 mM sodium tetraborate, 25 mM SDS (pH 9.3). Analytes were detected at 516 nm. HPLC measurements were realized in an octylsilica column (150  4.6 mm i.d.; particle size 5 m) at 35°C. Solvent A was 0.1 M aqueous ammonium acetate and solvent B consisted of methanol. The gradient programme was: 0 min, 55 per cent A; 20 min 35 per cent A; 21 min, 100 per cent B, final hold, 4 min. The flow rate was 1 ml/min. The separations of the components of the standard mixture (left) and those of a real sample (right) by CE are shown in Fig. 3.168. The electropherograms clearly illustrate that the method allows the baseline separation of the dye components even in real commercial samples. The main

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FITC c

Fluorescence (mV)

150

Conjugated transferrin

100

b

50

0 2

4 6 Migration time (min)

8

10

140

26

Fluorescence (mV)

120 21

100 80

16

60

11

40

6

20 1

0

−4

−20 0 (B)

Absorbance at 200 nm (mAU)

0 (A)

2

4 6 Migration time (min)

8

10

Fig. 3.161. (A) Zone electrophoresis patterns of FITC-labelled transferrin samples by fluorescence detection. The unbound dye (providing a main peak and several minor ones) was not removed from the samples. Experimental conditions: background electrolyte, 100 mM borate buffer, pH 8.3; voltage, 20 kV; capillary 59 cm (effective length 41 cm)  75 m i.d.; injection of samples 100 mbar x s; 20°C; detection with fluorescence detector (240 – 400 nm, broadband excitation filter and a 495 nm cut-off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 13 m (1 mg/ml) Tf and (a) 0.01 mM FITC, (b) 0.1 mM FITC, and 1 mM FITC. (B) Zone electrophoresis patterns of an FITC-labelled transferrin sample by simultaneous fluorescence (upper trace, left axis) and UV detection (lower trace, right axis). The unbound dye shows several peaks with both detections. Experimental conditions: background electrolyte, 100 mM borate buffer, pH 8.3; voltage, 20 kV; capillary 59 cm (effective length: fluorescence 41 cm, UV 50.5 cm)  75 m i.d.; injection of samples 100 mbar  s; 20°C; detection with fluorescence detector (240 – 400 nm, broadband excitation filter and a 495 nm cut off emmision filter). The reaction was left to continue for 20 h, and the reaction mixtures contained 6.5 m (0.5 mg/ml) Tf and 0.1 mM FITC. Reprinted with permission from T. Konecsni et al. [199].

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Chapter 3

TABLE 3.46 FITC TO TRANSFERRIN RATIO IN THE CONJUGATED TRANSFERRIN SAMPLES DETERMINED BY CZE

Transferrin concentration

1 mM FITC

0.1 mM FITC

0.01 mM FITC

0.13 mM (10 mg/ml) 0.066 mM (5 mg/ml) 13 µM (1 mg/ml) 6.6 µM (0.5 mg/ml) 1.3 µM (0.1 mg/ml)

2.50.5 30.5 3.50.5 nsa nsa

0.80.1 0.90.1 1.10.1 1.20.1 2.20.1

0.110.05 0.130.05 0.250.05 0.40.05 1.40.05

n (number of experiments) = 5. ns = no baseline separation of the conjugated protein molecules from unbound FITC in CZE of the reaction mixture. Reprinted with permission from T. Konecsni et al. [199]. a

SO2Cl

SO3-

(Et)2N

O

N+(Et)2

Fig. 3.162. Structure of LRSC; 9-[(4-chlorosulphonyl)-2-sulphonylphenyl]-3,6-bis(diethyl)aminoxanthylium hydroxide inner salt. Reprinted with permission from S. M. Smith et al. [200].

validation parameters of the CE procedure were: RSD, 2.03 – 5.11 per cent; recoveries, 92.7 – 105 per cent; linearity ranges, 0.06 – 20 per cent; limits of detection 0.06 – 0.05 per cent. The quantitative results obtained by CE and RP-HPLC are compiled in Table 3.47. The data demonsrate that the results of both methods are very similar. Because of the good validation parameters, lower solvent consumption and better cost efficiency, the CE method was proposed for the separation of the components of this dye [203]. Another interesting application of CE in the investigation of synthetic dyes was the determination of the dissociation constants (pKa) of sulphonated azo dyes. The dissociation constants were also determined by the traditional stpectrophotometric method and the results were compared. The chemical structures, numbers and names of the food and textile dyes included in the investigations are listed in Fig. 3.169. CE measurements were carried out in an untreated fused-silica capillary of 57 cm (50 cm effective length)  75 m i.d. Running buffers in the pH range of 5.6 and 12 were prepared by mixing phosphoric acid and sodium hydroxide, the final buffer concentration being 0.02 M. The solution of dyes

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551

SO3Na

N NaO3S

N N

N

N Zn

N

N

N

SO3Na

SO3Na

Fig. 3.163. Structure of zinc phtalocyanine tetrasulphonic acid. Reprinted with permission from J. Schoefield et al. [201].

0.010

0.008

0.006

0.004

0.002

0.000 0

5

10

15 Minutes

20

25

30

Fig. 3.164. Electropherogram of zinc phtalocyanine tetrasulphonic acid. Reprinted with permission from J. Schoefield et al. [201].

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Chapter 3

R N

Cl-+

N

NaO3S

SO3Na

Dye 1

R =

Dye 2

Cl

COOH

R = S

Fig. 3.165. Structures of dye 1 (synthetic precursor) and dye 2. Reprinted with permission from C. Yang et al. [202].

COOH

S +N

N (c)

−

− SO3

O3S

2

3 (min)

(a)

(b)

0

1

2

3

4

5

Time (min)

Fig. 3.166. Near-IR LIF electropherograms: (a) injection (50 mbar, 16 s) of 0.5 l of dye 2 as a 1.1012 mol/l solution in methanol; (b) injection of running buffer (50 mbar, 5 s); (c) injection of dye 2 as a 1.109 mol/l solution in methanol (50 mbar, 5 s). CE conditions: 2.5 mmol/l aqueous sodium borate running buffer (measured pH 9.2); 75 cm (60 cm to the detector)  100 m capillary; 30kV. Response axes: arbitrary fluorescence units. Reprinted with permission from C. Yang et al. [202].

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553 I

I NaO

O

O

I

I Co2Na

2',4',5',7'-Tetraiodofluorescein

I

I NaO

O

I O

O

NaO

I

O

I

I

Co2Na

Co2Na

2',4',5'-Triiodofluorescein

2',4',7'-Triiodofluorescein

Fig. 3.167. Structures of 2,4,5-triiodofluorescein, 2,4,7-triiodofluorescein, and 2,4,5,7tetraiodofluorescein (major components FD&C Red No. 3). Reprinted with permission from L. Evans III [203]. 30

1 40

2

25 20 mAU

mAU

30 2 20

34

15 10

5

10

0

0

10

20 30 Time (min)

6

40

1 5 0

0

10

20 30 Time (min)

40

Fig. 3.168. Left: electropherogram of a standard mixture of (1) 2,7-diiodofluorescein, (2) 2monoiodofluorescein, (3) 4,5-diiodofluorescein, (4) 2,4,7-triiodofluorescein, (5) 2,4,5-triiodofluorescein, and 2,4,5,7-tetraiodofluorescein. Right: electropherogram of an FD&C Red No. 3 sample submitted for certification; (1) 2,4,7-triiodofluorescein, (2) 2,4,5-triiodofluorescein, and (3) 2,4,5,7-tetraiodofluorescein. CE run conditions:
15 kV, 50 mM sodium borate, 25 mM SDS, pH 9.3. Reprinted with permission from L. Evans III [203].

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Chapter 3 TABLE 3.47 TRIIODOFLUORESCEINS FOUND IN CERTIFIED BATCHES OF FD&C RED NO. 3 BY HPLC AND CE

Sample

A1 A2 A3 B1 B2 B3 B4 C1 D1 E1

2,4,5-Triiodofluorescein found (%)

2,4,7-Triiodofluorescein found (%)

HPLC

CE

HPLC

CE

0.65 3.03 4.69 5.46 6.01 6.72 4.26 4.30 0.52 4.28

0.61 3.13 4.68 5.31 5.91 6.56 4.06 4.41 0.48 4.18

0.02 0.14 0.30 NPO NPO NPO NPO NPO 0.21 0.01

NPO 0.14 0.28 NPO NPO NPO NPO NPO 0.21 NPO

NPO  no peak observed. Reprinted with permission from L. Evans III [203].

contained 3 per cent (v/v) acetone as EOF marker. The capillary was thermostated at 25°C and the separation voltage was 15 kV. Samples were injected hydrodynamically for 4 s. Spectra were detected between 275 and 550 nm by DAD. Spectrophotometric determination of pKa values was carried out by measuring the spectra of the dye solutions between 270 and 600 nm at various pH (ionic strength 0.1 M) at 250.2°C. The concentration of the dye was 2  105 M and the pH dependence of the spectra was used for the calculation of pKa values. The electrophoretic mobility (me) of dyes in CE is related to me  ( Lc .Ld V ) .(1t 1teof )

(3.2)

where Lc is the length of the capillary, Ld the effective length of the capillary, t is the migration time of the analyte and teof is the migration time of a neutral marker. Dissociation constants can be calculated by pK a log[ H
] log(me  ma )  (mb  me )

(3.3)

where me is the electrophoretic mobility of the analyte at the given pH, ma is the electrophoretic mobility of the fully ionized acid, and mb is the electrophoretic mobility of the protonated species. A typical electropherogram used for the determination of pKa values is

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555 CI1 number and name

Structure NaO3S

N

(TAR) N

COONa

19140 CI Food Yellow 4 CE E-102 tartrazine FD C Yellow No. 5

HO

SO3Na HO NaO3S

N N

(SY-FCF)

15958 CI Food Yellow 3 CE E-110 Sunset Yellow FCF FD C Yellow No.6

(AMR)

16185 C.I. Food Red 9 CE E-123 Amaranth FD C Red No.2

SO3Na HO NaO3S

SO3Na

N N

SO3Na HO NaO3S

(NC)

16255 CI Food Red 7 CE E-124 New Coccine

(PBV)

42051 CI Food Blue 5 CE E-131 Patent Blue 5

N N NaO3S SO3Na SO3Ca/2 HO SO3CH3

CH3 N+

N CH3

CH3

Fig. 3.169a. Structures, numbers and names of the food and textile dyes studied. Reprinted with permission from M. Pérez-Urquiza et al. [204].

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Chapter 3 (AR-AC)

OMe HO NaO3S

N

16035 CI Food Red 17 CE E-129 Allura Red AC

N CH3

SO Na 3

(OII)

HO NaO3S

N

15510 Acid Orange 7 D C Orange 4

N

HO

(CO)

15970 Acid Orange 12

(P2R)

16150 Acid Red 26

(R88)

15620 Acid Red 88

N N

SO3Na CH3 HO CH3

SO3Na

N N

SO3Na HO NaO3S

N N

Fig. 3.169b. Structures, numbers and names of the food and textile dyes studied. Reprinted with permission from M. Pérez-Urquiza et al. [204].

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557

2

1

EOF

0

5

10 15 Time (min)

20

Fig. 3.170. Electropherogram of P2R at pH 11.9. Reprinted with permission from M. Pérez-Urquiza et al. [204]. TABLE 3.48 pKa VALUES OBTAINED BY USING CAPILLARY ZONE ELECTROPHORESIS AND SPECTROPHOTOMETRY METHODS

pKa valuea

Analyte CE

OII CO P2R P2R-1 P2R-2 AMR R88 TAR SY-FCF NC PBV AR-AC a

Spectrophotometry

1b

2c

10.65(0.08) 10.46(0.04)

10.68(0.12) 10.43(0.07)

11.42(0.13) 11.53(0.11) 10.47(0.08) 10.70(0.06) 9.49(0.08) 10.44(0.08) 11.04(0.13) 7.63(0.03) 11.45(0.14)

11.26(0.14) 11.61(0.21) 10.49(0.10) 11.06(0.08) 9.43(0.10) 10.36(0.09) 11.19(0.13) 7.58(0.09) 11.38(0.12)

10.83(0.03) 10.43(0.01) 11.59(0.02)

10.36(0.02) 11.0(0.03) 9.40(0.01) 10.36(0.01) 11.24(0.01) 7.67(0.02) 11.35(0.01)

At 0.1 M ionic strength, the stimate standard deviation is placed in parentheses. By using migration times. c By using DAD spectra. Reprinted with permission from M. Pérez-Urquiza et al. [204]. b

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Chapter 3

(1) 20 pA (3)

0

200

(u)

(2)

(u)

400 Time [s]

600

800

50 pA (1) (3)

(2)

(a) (S)

(b) 0

200

400

600

800

1000

Time [s]

Fig. 3.171. Upper trace: non-aqueous CE-ED of a malachite green sample. Signals (1) and (3) correspond to 4.1 g/ml malachite green and 7.7  106 M ferrocene (internal standard), respectively. Peak 2 has been identified as crystal violet (0.14 g/ml), the other signals resulted from unknown components (u). Lower trace: successive electropherograms of a dye mixture containing de mixure (1) 2.5 g/ml methylene blue, (2) 1.1 g/ml rhodamine B, (3) 4.2 g/morin For repetitive recording (b) 9.1 x 10-6 [FcMTMA]ClO4 was added as internal standard. Experimental conditions: capillary dimensions, 95 cm  75 m i.d.; running electrolyte, acetonitrile containing 1 M HAc and 10 mM NaAc; electrokinetic injection, 20 s 5 kV; separation voltage 20 kV; applied detection potential, 1.55 V. Reprinted with permission from F.-M. Matysik [205].

shown in Fig. 3.170. The dissociation constants detrmined by CE and spectrophotometry are compiled in Table 3.48. It was established that the results of the two methods are very similar to each other, that is, CE can be applied for the determination of the dissociation constants of azo dyes. The advantages of the CE method are that the measurements can be carried out with a very small amount of sample and the sample need not to be chemically pure [204]. Although the overwhelming majority of CE analyses were performed in aqueous environments, non-aqueous CE has also been employed for the determination of synthetic dyes. The possibility to use electrochemical detection (ED) combined with non-aqueous

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CE was investigated in detail. Various drugs and dyes, such as Malachite green, Crystal violet, Methylene blue and Rhodamine B were included in the experiments. Capillaries of 95 cm  75 m i.d. were used in the study. Detector cells consisted of a cylindrical body containing the electrodes (voltammetric microdisc electrode, platinum coil, and a silver/silver chloride reference electrode). Running electrolyte was ACN containing 1 M HAc and 10 mM NaAc. Samples were injected electrokinetically (20 s 5 kV). Working potential was 20 kV; detection potential, 1.55 V. Electropherograms illustrating the separation and detection capacity of non-aqueous CE combined with ED are shown in Fig. 3.171. It was stated that the non-aqueous system offers some marked advantages, such as the use of a wider range of working electrode potentials and enhanced stability [205]. A very similar CE-ED system was employed for the separation of a dye mixture. The effective separations of dyes are demonstrated in Fig. 3.172. The good stability of the platinum electrode response was observed again [206]. Another non-aqueous CE (NACE) system was applied for the separation of five dyes. The chemical structures of the dyes are shown in Fig. 3.173. Investigations were carried out in an uncoated capillary (total length, 77 cm; effective length, 51 cm; 50 m i.d.). A hydrodynamic injection mode was employed (5 s). The Running buffer was ACN containing various 1-butyl-3-methyl imidazolium salts such as fluoroacetate, acetate and hexafluorophosphate. The separation voltage was
18 kV and analytes were detected at 256 nm. An electropherogram showing the separation of dyes is depictedin Fig. 3.174. It was stated that the main advantages of NACE are the modification of selectivity, the enhanced solubility of lipophilic analytes, and the reduced electrophoretic current [207].

(d) 50 pA

(a) (b) (c) (1)

(2)

(3) 0

200

400

600

800

1000

Time [s]

Fig. 3.172. Non-aqueous capillary electrophoresis with electrochemical detection of a dye mixture containing (a) 1.7 g/ml malachite green, (b) 0.70 g/ml crystal violet, (c) 4.3 g/ml rhodamine B, and (d) 9.1  106 M ferrocene. Experimental conditions: capillary dimensions, 95 cm  75 m i.d.; running electrolyte, acetonitrile containing 1 M HAc and 10 mM NaAc; electrokinetic injection, 20 s 5 kV; separation voltage 20 kV; applied detection potential, 1.55 V. Reprinted with permission from F.-M. Matysik [206].

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560 Name 1 Janus Green

Chapter 3 Abrev.

Structure

MW N

JGN

N+

N

511,07 N

Cl-

N

N 2 Brilliant Cresyl Blue BCB

332,8

NH2 N N

O

NH2

N

N+

3 Sudan Black

Cl-

456,55

SBL HN

N

N HN

4 Thymolphtalein

N

N

456,55

TPH O

OH

HO

5 Phenolphtalein

PPH

HO

318,3

O

OH

Fig. 3.173. Dyes used in this study. Reprinted with permission from M. Vaher et al. [207].

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18000

561

3

5

4

16000 Absorbance (arb. units)

14000

1

12000 2

10000 8000 6000 4000 2000 0 −2000

0

2

4

6

8 10 Time (min)

12

14

16

18

Fig. 3.174. Example of NACE electropherogram of mixture of dyes (1, Janus Green; 2, Brilliant Cresyl Blue; 3, Sudan Black; 4, Thymolphtalein; 5, Phenolphtalein) in acetonitrile. Added ionic liquid was 1-butyl-3-methyl fluoroacetate (3.3 mg/ml). Applied voltage
18kV, current 8 A. Reprinted with permission from M. Vaher et al. [207].

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A acacetin 155, 156, 167 acacetin-acetyl-glucoside-rhamnoglucoside 175 acacetin-rhamnoglucoside 175 Acartia clausi 129, 131 acetic acid 457 6⬘⬘-O-acetyldaidzin 187 6⬘⬘-O-acetylgenistin 187 6⬘⬘-O-acetylglycitin 187 acid black 077, 452 acid black 209, 452 acid blue25, 435–436, 440 acid blue40, 484, 487 acid blue74, 464 acid blue9, 372 acid brown 14, 435, 439, 451 acid brown 355, 484 acid orange031, 452 acid orange10, 487 acid orange12, 429, 536, 538, 556 acid orange142, 484, 487 acid orange52, 427, 429, 536, 538 acid orange7, 372, 429, 466, 468, 536, 538, 556 acid orang II 373 acid red 502 acid red035, 452 acid red 1, 492, 497–498 acid red 106, 497–498 acid red118, 487 acid red13, 492 acid red14, 492 acid red155, 468 acid red2, 429 acid red 26, 536, 538, 556 acid red 27, 429, 454, 536, 538 acid red33, 468

acid red357, 487 acid red73, 492 acid red8, 497–498 acid red85, 446–447 acid red88, 427, 429, 536, 538, 556 acid violet43, 468 acid violet5, 497–498 acid violet7, 487 acid violet90, 484, 487 acid yellow1, 429, 536, 538 acid yellow194, 484, 487 acid yellow23, 484, 487, 492 acid yellow36, 484, 487 acridinO, 387, 389 acrilic acid 457 acylated monomer anthocyanins 281 adenine 234, 235 adhyperforin 166 alfametrin 158 algae 326 alizarin 330, 332, 339, 340, 341, 342 alizarin glucoside 330, 332 alizarin S 372, 460 Allium cepa 99, 271, 371 Allium schoenoprasum 175 alloxanthin 132, 289, 291, 293, 295, 299 all-trans-α-carotene 110 all-trans-β-carotene 110 all-trans-lutein 110, 122, all-trans-lycopene 110 all-trans-retinal 343 all-trans-retinoic acid 135 allura red 396, 399, 403, 425, 426, 521–522, 524, 527, 531, 556 almond seedcoat 261 amaranth 99, 379, 417, 419, 422, 425, 433, 519, 521, 555 Amaranthus gangeticus 99 Amaranthus sp. 99

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574 amentoflavone 147 American goldfinches 119 amidoblack 387, 389, aminoazobenzene acid orange 52, 448 o-aminoazotuluene 445 2-(4-aminobenzenesulphonyl)ethanol 477 4-aminobiphenyl 445 3-amino-2, 3-dihydroxypropanoic acid 457 4-amino-4⬘-fluorosulphonylazo benzene 505 amino fumaric acid 457 1-amino-8-hydroxynaphtalene-3, 6disulphonic acid 470 7-amino-8-hydroxy-1, 3-naphtoquinone-3, 6-disulphonate-1, 2-diamine 477 aminonaphtalene sulphonic acids 484, 486 1-amino-2-naphtol-3, 6-disulphonate 434 2-amino-4-nitrotoluene 434 3-amino propenoic acid 457 Anethum graveolens 175 Anguilla anguilla 410 2⬘, 3⬘-anhydrolutein 121 anhydrolutein 121 ankaflavin 326 annatto 99 Annona cherimola, Mill. 303 antheraxanthin 65, 68, 72, 74, 83, 84, 95, 96, 115, 130, 131, 289 anthocyanidin 239 anthocyanidin aglycone 239, 273 anthocyanin derivative 209 anthocyanin-alkyl/aryl-flavanol pigment 279 anthocyanins 254 anthranillate 139 anthranillic acid 141, 457 anthraquinone 330 anthracenone pigment 327 apigenin 147, 155, 156, 157, 159, 161, 169, 179, 184, 186, 190, 206, 339, 340, 341, 342 apigenin-acetyl-apiosylglucoside 175 apigenin-acetyl-diglucoside 175 apigenin-7-acetyl-glucoside 139 apigenin-7-apiosylglucoside 175 apigenin-7-diacetyl-glucoside 139 apigenin-7-glucoside 139, 155 apigenin 7-O-glucoside 169 apigenin-7-O-β-D-glucoside 190

apigenin-7-O-β-D-glucoside-6⬘⬘-O-malonate 190 apigenin 7-O-glucuronide 147, 169 apigenin 7-O-heteroside 178 apigenin-rhamnoglucoside 175 apigenin 7-O-rutinoside 178 apo-8⬘-carotenoic acid ethyl ester 118 Apocyanaceae 139 apple 206, 261 apricot 206 arabinose 262 Arnica angustofolia 140, 142 aromatic sulphonates 544–546 Aronia mitchurinii 179 Artemisia dranunculus 175 artichoke 165 artificial sweeteners 516 asaxanthin 289 astaxanthin 67, 123, 130, 131, 132 astaxanthin ester 127 astaxanthin free 127 astragalin 150, 155, 158 α-tocopherol 113, 116 aurochrome 72 aurones 134 auroxanthin A 96 auroxanthin B 95, 96 avicularin 145, 146, 155 azobenzene 387, 389 α, α⬘-azoisobutyronitrile 369 Azorubin 398, 399 B bacteriochlorophyll 316 bacteriochlorophyll e1, 290 bacteriochlorophyll e2, 290 bacteriochlorophyll e3, 290 bacteriopheophytin 300 bacteriopheophytin-a epimer 300 bacteriopheophytin-c 300 bacteriopheophytin-d 300 bacteriopheophytin-c/d 300 bacteriopheophytin-e1, 290 bacteriopheophytin-e2, 290 bacteriopheophytin-e3, 290 baicalein 155 β-Apo-8⬘-carotenal 83, 84, 90, 92

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575 β-apo-8⬘-carotenoic acid ethyl ester 118 bartramiaflavone 147 Basella rubra 99 basic blue 41, 461, 464 basic green 466 basic red 372 basic violet16, 466 Bathua 99 bean, green 206 Bengalgram 99 bengal rose 387, 389 bensoic acid 209 benzidine 452 beverages 452 betalain pigments 331, 332 betanin 331, 333, 334, 335 13, II8 biapigenin, 166 bilberries 179 bilirubin 109, 343 biliverdin 343 biluteolin 147 2⬘-8⬘⬘-biluteolin 147 3⬘-8⬘⬘-bipigenin 147, 156 biochanin A 186 (E, E)-1, 7-bis(4-hydroxy-3-methoxyphenyl)1, 6-heptadiene-3, 5-one 318 bixin 99, 102 Bjerkandera adusta 470 black elder 137 blackcurrant 179, 183, 206, 261 Black PN 516, 520–521 blueberry 206, 262 bog wortleberry 183 19⬘-butanoyloxyfucoxanthin 289, 293 Brassica chinesis 336 Brassica napus 144, 148 Brassica oleracea 148 Brilliant Black BN 379 Brilliant Blue FCF 379, 379, 398, 516, 520–522, 524, 529, 530, 532 Brilliant Blue BRI 417 Brilliant Cresyl Blue 560–561 Brilliant Green 387, 389, 411 broccoli 206 6-bromoindigotin 339, 341 Bromthymol Blue 387, 389 brusselssprouts 206 β-zeacarotene 72

C caffeetannins 149 caffeic acid 138, 149, 157, 159, 161, 180, 181, 191, 223, 224, 234 caffeine 193, 196, 201, 235 caftaric acid 224 C-132-OH chlorophyll-b 306 Calendula officinalis 69, 163 Camelina sativa 144, 148 Camellia sinensis 189 canary xanthophyll A 119, 120 canary xanthophyll B 119, 120 canthaxanthin 67, 83, 84, 103, 117, 118, 119, 120, 123, 124, 125, 127, 130, 289, 300 capsaicionids 68 capsanthin 65, 68, 74, 81, 82, 83, 84, 85, 115 capsanthin 2, 6-epoxide 83, 85 capsanthin 3, 6-epoxide 65, 74, 83, 84 capsanthin 5, 6-epoxide 65, 74, 83, 84 capsanthin diester 115 (9/9⬘Z)-capsanthins 74 (13/13⬘Z)-capsanthins 74 capsanthoin 112 Capsanthol (6⬘R) 65 Capsella bursa pastoria 148 Capsicum annuum 64, 66, 68, 71, 77, 78, 80 Capsicum frutescens 71, 81 capsorubin 65, 68, 74, 83, 84, 85, 115 capsorubin diester 115 capsorubin monoester 115 6-carboxyfluorescein 370 5-carboxypyranocyanidin 3-O-βglucopyranoside, 274 5-carboxypyranopelargonidin 266 carboxytetramethyl-rhodamin 370 Carassius auratus 371 carmine 99, 102 Carminic Acid 99, 102, 339, 340, 341, 342, 387, 389, 419, 519, 524, 529–530 Carmoisine 396, 399, 403, 417, 419, 422, 516, 519–521, 527 carnation 159 α-carotene 68, 72, 83, 84, 92, 93, 95, 96, 97, 98 α-carotene monoepoxide 65

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576 β-carotene 65, 68, 70, 72, 74, 81, 82, 83, 84, 86, 87, 88, 90, 93, 95, 96, 97, 98, 99, 100, 101, 102, 103, 108, 109, 110, 112, 113, 115, 117, 118, 123, 124, 125, 127, 130, 131, 135, 291, 294, 399 β,β-carotene 133, 289, 290 9(Z)-β-carotene 91 β-carotene diepoxide 65 β,ε-carotene (α-carotene) 289 β-carotene monoepoxide 65 (E)-β-carotene 91 (Z)-β-carotene 74 13(Z)-β-carotene 91 13-cis-β-carotene 110 15(Z)-β-carotene 91 15-cis-β-carotene 110 ζ-carotene 110 carotenes 63, 125 carotenoid 63, 64, 132 carrots 106 catechin 191, 192, 201, 221, 223, 226, 228, 235, 236, 237 (⫺)-catechin 224 (⫹)-catechin 180, 181, 209, 223, 238, 272, 273 catechin gallate 193, 201 catechins 73, 137, 186 catfish 404, 406–408, 410 Cedrus atlantica 150 Cedrus atlantica var. glauca 150 celery 178, 179, 206 cell cultures 273 Centropages hamatus 129 Chamomilla recutita L.Rauschert 138 cheese sauce 425 Chenopodium album 99 cherry 206 chilli 78, 86, 87, 424 chives 170, 175 4-chloroaniline 445, 452 2-chloro-1, 4-diaminobenzene 445 4-chloro-o-toluidine 445 Chlorobium phaebacteroides 295 chlorogenic acid 138, 139, 164, 166, 180, 181, 201 chlorogenic acid isomer 166 chlorophyll-a epimer 300 132-chlorophyllone-a 304

chlorophyllid a 289, 307 chlorophyll 125, 132, 137, 283 chlorophyll α allomer 2, 289 chlorophyll b 72, 130, 290, 291, 293, 295, 299, 306, 309, 311 chlorophyll c 133, 299 chlorophyll c1, 289 chlorophyll c1/c2, 294 chlorophyll c2, 289, 291 chlorophyll c3, 289 chlorophyll epimer 306 chlorophyll-a 72, 130, 133, 289, 290, 291, 294, 299, 300, 306, 309, 311 chlorophyll-a-allomer 133, 287 chlorophyll-a-epimer 133 chlorophyll-b epimer 306 chlorophyllide b 307 chlorophyllin A 317 chlorophyllin B 317 chlorophyllin derivatives 317 chlorophyllone 300 chlorophyllone-a 290 chlorophytes 292 Chlorrela vulgaris 123, 126 chokeberry 179, 183 chrysophanol 335, 336 Chromotrope 2B 437 Chromotrope 2R 437, 497–498 Chromotropic acid 542 chrysin 155, 161, 184 chrysoeriol 7-O-glycoside 169 chrysophytes 292 Cibacron Blue F-R 503 Cibacron Blue F3GA 369, 370 Cibacron Brilliant Red 3B-A 497, 498 Cibacron Orange 475 Cibacron Red 475 Cibacron Yellow F-4G 503 Cicer arietinum 99 Cichorium intibus var. 336 Ciconia ciconia 67 CI Direct Black38, 481 CI Disperse Blue291, 371 CI Direct Brown1, 481 CI Disperse Orange37, 401 CI Disperse Blue373, 401 CI Disperse Violet 93, 401 cinnamon 261 cis-antherxanthin 95, 96

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577 cis-β-carotene 83, 84, 115 9-cis-β-carotene 110 cis-capsanthin 115 9-cis-capsanthin 83, 84, 85 cis-crocin 105 cis-flucoxanthin 133 13⬘-cis-lutein 110 cis-lycopene isomers 110 cis-pieceid 219 11-cis-retinal 343 cis-resveratrol 219, 221, 222, 223, 228 cis-retinoic acid 105 3-cis retionic acid 135 9-cis-retinoic acid, 135 cis-violaxanthin 95, 96 cis-zeaxanthin 83, 85 9-cis-zeaxanthin 83, 84 15-cis-zeaxanthin 83, 84 Citrus sinensis 92 Clastranceae 137 Coccothraustes vespertinus 121 Cochineal 398 Colocasia 99 Colocasia antiquorum 99 Confectioneries 516 Congo Red 372, 387, 389, 460 common 123 common yellowthroat 121, 122 Coomassie Brilliant Blue G 370 copper chlorophyllin 317 Coumassie G250 387 Coumassie R250 387 corilagin 151 coumarins 137, 151 3-O-p-coumaroylquinic acid 166 coutaric acid 224 p-cresidine 445 Crambe abyssinica 144, 148 Crambe hispanica 144, 148 cranberry 183, 206, 208 cress 170, 175 Crocein Orange G 510 crocetin 102, 103, 105, 106 crocin 104, 107 crocin1, 105, 106 crocin 2, 105, 106, 107, 108 crocin 3, 105, 106, 107, 108 crocin 4, 105, 106, 107, 108 crocin5, 107, 108

crocin 6 107, 108 α-crocin 107, 108 crocoxanthin 289, 300 Crocus sativus L 102 crowberry 183, 206 Crystal Violet 384, 387, 389, 391, 404, 558–559 cryptocapsin 74, 83, 84 cryptochlorogenic acid 164 cryptoflavin 72, 83, 84 cryptophytes 292 cryptoxanthin 68, 69, 115 α-cryptoxanthin 65, 74, 83, 84, 95, 96, 98 β-cryptoxanthin 65, 72, 74, 83, 84, 95, 96, 97, 99, 103, 109, 110, 115, 117, 121, 123, 124, 125 cryptoxanthin-5, 8-di-epoxide 72 (Z)-cryptoxanthin 74 Cu-15-glyoxilic acid pheophytin-a 310 Cu-15-glyoxilic acid pheophytin-b 310 Cu-15-glyoxilic acid pheophorbide-a 310 Cu-15-pheophytin-a 310 Cucumis sativus L. 136 cucurbitaxanthin A 65, 74 Cu-pheophytin-a 310 Cu-pyropheophytin-a 310 curcuma 99, 102 Curcumine 398 curcurbitaxanthin A 83, 84 curcurbitaxanthin B 83, 84 curly lettuce 98 Cy-(6-acetyl)-3 glucoside 248 Cy-(6-coumaroil)-3-glucoside 248 cyanide-3-α-D-glucopyranoside 274 cyanidin 240, 262 Cyanidin 3- (6-malonylglucoside)-7, 3⬘-di(6synapilglucoside) 276 cyanidin-3-acetylglucoside 241 cyanidin-3-coumaroylglucoside 241 cyanidin-3-glucoside 241, 248, 251, 256, 259 cyanidin-3-lathyroside(cyanidine-3-O(α-Dxylopyranosil (1-2) α-D-galacto pyranoside 274 cyanidin-3-malonylglucosige 136 cyanidin-3-monoglucoside 136 cyanidin-3-O-glucoside 253, 286 cyanobacteria 292 cyanodelphin 243, 244

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578 Cyclotella choctawatcheena 294 cycloviolaxanthin 65, 83, 84, 85 Cynara scolymus 162 cynarin 164 cynaropikrin 164 cynaroside 164 Cyperaceae 137 Cyperus conglomeratus Rottb. 137 cyprocapsin 83 Cytochrom P450, 393 D D. belladonna ‘Belladonna Imp.’ 243 D&C Yellow No. 10, 501 D. Cardinale Hook 243 D. cardinale x D. grandiforum 243 D. grandiflorum L 243 D. grandiforum x D. nudicale no.1. 243 D. grandiforum x D. nudicale no.2. 243 D. grandiforum x D. nudicale no.3. 243 D. grandiforum x D. nudicale no.4. 243 D. hybridum ‘Astorat’ 243 D. hybridum ‘Blue Bird’ 243 D. hybridum ‘Galahad’ 243 D. hybridum ‘Summer Skies’ 243 D. nudicaule Torr. 243 daglesioside I. 150 daglesioside II. 150 daglesioside III. 150 daglesioside IV. 150 daidzein 231 danthron 335 Danthus caryophyllus L 159 Daturu stramonium 275 dehydrogeranin 151 delphinidin 240 delphinidin-3-acetylglucoside 241 delphinidin-3-coumaroylglucoside 241 delphinidin-3-glucoside 241, 248, 251, 255, 256 delphinidin-3-O-(6-O-acetyl)glucoside 285 delphinidin-3-O-glucoside 286 delphinidin-3-rutinoside-7-glucoside 243, 244 delphinidin-3-rutoside 243, 244 Delphinium cardinale 241 Delphinium grandiflorum X 243 Delphinium grandiflorum 241

Delphinium nudicaule 241 Delphinium species 240 delphynidin 3-O-p-coumaroylrutinoside-7-Oglucoside 276 delphynidin-3-glucoside pyruvic derivative 255 Dendrobium cv. Pompadour 244, 276 Dendroica petenchia 121 derivative of malvidin-3-O-glucoside and pyruvic acid 286 derivative of petunidin-3-O-glucoside and pyruvic acid 286 Descurainia sofia 148 desmethylxanthohumol 213 diadinoxanthin 133, 291, 294 diadzein 185, 186, 187 diadzin 185, 186, 187 1, 4-diaminobenzene 445 3, 4-diaminonaphtalene-1-sulphonate 411 2, 4-diaminotoluene 445 dianidochrom 289 dianidoxanthin 289 diatoms 132, 292, 293 diatoxanthin 133, 287, 300 6, 6-dibromoindigotin 339, 341 dicaffeoylquinic acids 162, 165 6, 8-diC-glucosylapigenin 167 3, 3⬘-dichlorobenzidine 445, 452 2, 6-dichloroindophenol sodium 388 dichlorotriazine dyes 511 dichromatism 119 4-(N, N-diethylamino)-4⬘-fluorosulphonylazobenzene 466, 507 7-diethylamino-4-methylcoumarin 430 4-(4-diethylamino-2-methylphenylazo)benzene sulfonyl fluoride 514 2, 3-dihydroxyindoline 457 dihydroxynaphtoquinone-3, 6-disulphonatediimine 477 3, 3⬘-dimethoxybenzidine 445, 452 2, 4-dimethylaniline 445 3, 3⬘-dimethylbenzidine 445, 452 1, 3-di-O-caffeoylquinic acid 164 1, 8-dihydroxyanthraquinone 330 2⬘, 4-dihydroxy-chalcone 213, 214 2, 6-dihydroxyanthraquinone 330 3, 4-di-O-caffeoylquinic acid 164

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579 1, 1⬘-dioctadecyl-3, 3, 3⬘, 3⬘tetramethylindocarbocyanine perchlorate 468, 489 3, 5-digallate 201 3, 6-dihydroxyflavone 161 3, 7-dihydroxyflavone 161 7, 4⬘-dihydroxyflavone 190 7, 4⬘-dihydroxyflavone-7-O-β-D-glucoside 190 5, 6-diepicapsokarpoxanthin 65 5, 6-diepikarpoxanthin 65, 74 5, 6-diepilatoxanthin 65 Digera avensis 99 5, 6-dihydrixyindole-2-carboxile acid 344 5, 6-dihydroxy-5, 6-dihydrolycopene 92 dill 170, 175 dimer of malvidin-3-O-glucoside and catechin 286 5, 6-dimethoxy-5, 6-dihydrolycopene 92 (4R, 5S)/(4S5R)-1, 5-dimethyl-4-phenyl-2imidazolidine 158 5, 7-dimethylorientin 160 dinoflagellates 132, 292 dinoxanthin 289 diosmetin 227, 229, 230 diosmetin-acetyl-apiosy glucoside 175 diosmetin-apiosylglucoside 175 diosmetin-rhamnoglucoside 175 diosmin 149, 208, 211 Direct Black 38, 482 Direct Blue015, 452 Direct Blue106, 484, 487 Direct Blue6, 446–447 Direct Blue78, 484, 487 Direct Brown 1, 482 direct condensation product between catechin and delphinidin-3-glucoside 256 direct condensation product between catechin and malvidin-3-(p-coumaroyl) glucoside 256 direct condensation product between catechin and malvidin-3-glucoside 256 direct condensation product between catechin and peonidin-3-( p-coumaroyl) glucoside 256 Direct Green 26, 487 Direct Green 28, 487 Direct Red 061, 484, 487 Direct Red 79, 452

Direct Yellow28, 484 Disperse Blue3, 430 Disperse Blue14, 430 Disperse Orange1, 447 Disperse Orange3, 446 Disperse Orange13, 424, 427, 428 Disperse Orange25, 444 Disperse Red1, 444 Disperse Red13, 444 Disperse Violet 430 Disperse Yellow7, 446–447 ditetrazolium salts 376, 381–384 divinyl chlorophyll a 492 divinyl chlorophyll b 289 Dp 3-(acetyl)gluc-py derivative 260 Dp 3-gluc-py derivative 260 Dp-(6-acetyl)-3-glucoside 248 Dp-(6-coumaroil)-3-glucoside 248 Dp-gls-py-derivative 248 E E(trans)-α,α-carotene 69 ε-carotene 93, 95, 96, 110 echinenone 117, 127, 294, 300 Echinostoma caproni 70 Echinostoma trivolis 70 eel 410, 412, 413 elder flower blossom 167 ellagic acid 137, 339, 340, 341, 342 ellagitannins 147 emodin 335, 336 Émpetrum hermaphroditum 179 enterodiol 231 enterolactone 231 3-entiobiosile-kaempferol 107 Eosin Yellow 387, 389 6⬘-epikarpoxanthin 65 epicatechin 186, 191, 194, 196, 201, 221, 223, 226, 228, 235, 236 (⫺)epicatechin 180, 181, 223, 224, 234, 272, 273 epicatechin gallate 193, 196, 201, 226 132-epichlorophyllone-a 304 epigallo catechin 193, 194, 196, 201 (⫺)epigallocatechin 238 (⫺)epigallocatechin-3-gallate 238 epigallocatechin 3, 5-digallate 201 epigallocatechin gallate 193, 194, 196, 201

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580 epoxy-β-carotenes 106 5, 6-epoxy-β-carotenes 106, 108, 109 Eriochrome Blue Black 372 Eriochrome Blue Black B 460 Eriochrome Black T 510 eriodictiyol 7-O-heteroside 178 eriodictiyol 7-O-rutinoside 178 eriodictyol-glucoronide 175 Erodium cicotarum 147 Eroium genera 151 Eruca vesicaria 148 erythrosin 128, 379, 419, 519, 522, 548 Erythrosine B 398 Escherichia coli 373 esculin 152 ethylene terephtalate cyclic trimer 430 Euchsin acid 460 Euphorbiaceae 137 Evan’s Blue 387, 389 evening grosbeak 121, 123 Everzol Turquoise Blue 531

florisil 154, 155 fluorescein 187, 385, 390 1-(9-fluorenyl)ethanol 158 food dyes 375 Food Colour Red No. 106, 499, 502–503 Food Red1, 468 Food Yellow3, 468 formononetin 186 15-formylphenophytin-a 310 15-formylphenophytin-b 310 Fragaria ananassa 266 Fragaria ananassa Dutch 266 Frugaria xananassa 179 fruit jam 421, 426 fruit juice 423 Fuchsin Acid 372 fucoxanthin 132, 133, 289, 290, 291, 293, 294, 295, 299 fucoxanthindehydrate 290 fucoxanthin-hemiketal 290 Fulica americana 113 Funalia trogii ATCC 200800, 373

F Fast Green FCF 385, 391, 522, 524, 528, 532 fenoxaprop-ethyl 158 fenpropathrin 158 Fenugreek 99 ferrocene 559 ferulic acid 138, 161, 191, 223, 224 fisetin 232, 233 flavan-3-ol 270, 271 flavan-3-ol(4 α−8)pelargonidin 3-O-βgucopyranosides 270 flavanol-anthocyanin adducts 222 flavans 137 flavin mononucleotide 210 flavin-adenindinucleotide 210 Flavodon flavus 335 flavone 136, 141, 152, 161, 186 flavonoid aglycones 140, 142, 144 flavonoid glycosides 144, 147 flavonoids 134, 136, 137, 143, 151, 161 flavonol aglycones 184 flavonol glucosides 180, 181 flavonol truxinix esters 146 flavonols 136, 141, 155 flavonone 158, 161 flavonons 141

G galactose 262 galangin 155, 161 gallic 157 gallic acid 191, 193, 194, 201, 223, 224, 226, 234–236, 238, 339, 340, 341, 342 gallic acid monohydrate 137 Gallinula chloropus 112 gallocatechin 201 (⫺)gallocatechin 238 (⫺)gallocatechingallate 238 gallocatechin gallate 193, 201 gallotannins 147 3-galloylshikimik acid 151 γ-(trityloximethyl)-γ-butyrolactone 158 Gardenia jasminodies Ellis 102 gardenin B (5-hydroxy-6, 7, 8, 4⬘tetramethoxyflavone) 178 γ-crotene 93 genistein 185–187, 231 genistin 185–187 genkwanin 155, 169 Gentian Violet 387, 406 gentizein 156 genus 139

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581 Geothylpis trichas 121 Geraniaceae 147 geranin 151 3⬘-geranylchalconaringenin 213 6-geranylnaringenin 213, 214, 216 Gingko biloba 162, 163 glicitein 187 glicolic acid 457 glucose 262 3-O-α-glucopyranoside 266 glucoside 152 7-glucoside-luteolin 155 8-C-glucosilflavones 160 glutathion 81 glycitin 187 15-glyoxilic acid pheophorbide-a 310 15-glyoxilic acid pheophorbide-b 310 15-glyoxilic acid pheophytin-b 310 Gracilaria verrucosa 328 grape 262 grape seed 191 grape skin extract 245 grapefruit, pulp 206, 212 grapes, green 206 grapes, blue 206 green pepper 207 Green S 516, 520–521 green tea 224 grenadine 520, 526 guava fruit 528, 534 Gyroxanthin 67 gyroxanthin diester-like1, 2, 289 H H acid 542 Hameatococcus pluvialis 125 hazelnut seed oils 88 HC orange No.1 371 Heligon Blue S4 531 Helisoma trivolvis 69 hematoxylin 387 389 hemiarin 137 139 hemin 333 hesperetin 152, 158 hesperetin 7-O-rutinoside 178 hesperetin-rhamnoglucoside 175 hesperidin 149, 152, 155, 156, 206, 208, 211, 237

hexafluorofluorescein 370 19⬘-hexanoyl-fucoxanthin 132 19⬘-hexanoyloxy fucoxanthin 133, 289, 291, 293, 295 high-speed counter current chromatography 123 homoorientin 167, 168 HSCCC 88, 123 human serum 106 3-hydroxyamino-1-methyl-5H-pyrido(4, 3blindole(Trp-P-2(NHOH)) 348 2-hydroxyanthraquinone 330 132-hydroxychlorophyll-a 304 4-hydroxycoumarin 152 5, 7-hydroxy-3⬘, 5⬘-dimethoxyflavan 137 5-hydroxy-7, 3⬘, 5⬘-trymethoxyflavan 137 3-hydroxy-flavone 155, 161 6⬘-hydroxyflavone 161 6-hydroxyflavonone 158, 161 7-hydroxy-flavone 155, 161 15⬘-hydroxylactone, chlorophyll-a 304 7-hydroxy-o, o⬘-dihydroxyazo dyes 390, 397 3-(p-hydroxyphenyl)propionic acid 201 hydroxypheophytin-a 300, 304, 306 hydroxypheophytin-a epimer 300, 304, 306 hydroxypropyl-β-cyclodextrin 81 hydroxystilbene 220 hyperforin 166 hyperforin analogue 166 hypericin 166 Hypericum perforatum 162, 163, 166 hyperin 145, 146, 151 hyperin-6⬘⬘-gallate 151 hyperoside 138, 155, 166 hypoaletin 4⬘-methyl ether 8-O-glucuronide 169 hypoaletin 7-O-glucoside 170 hypoaletin 8-O-glucuronide 169 Hypophae rhamnoides 179 I ice cream 527–528, 533 ice lolly 520, 526 Ictalurus punctuatus 407 Iguana iguana 116 Imantonia rotunda 130, 131, 133, 134 1-indanylidenemalononitrile 390 indicaxanthin 331, 334

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582 indigo 453–454, 456–457 indigo carmine 379, 398, 417, 453–454, 456–457, 522, 524 indigotin 139, 141, 339, 341, 342 indigoxanthin 333 indirubin 139, 141, 339, 340, 341 indodicarboxycyanine 370 indoxyl 141 indoxyl-yielding glycosides 139 Irpex lacteus 373 isatin, 139 141 isatin sulphonic acid 373 isobetanin 335 isochlorogenic acid, 201 isoflavones 134, 230 isoflavonoid 136 isofucoxanthin-dehydrate 290 isofukoxanthin dehydrate pheophorbide a ester 290 isogentisin 156 isohamnetin 184 isolutein 95, 96 isoorientin 136, 155 isopimpinellin 152 2, 3-O-isopropylidene-1, 1, 4, 4-tetrahenylthreitol 158 isoquercetin 138, 145, 146, 151, 152, 155, 166, 168 isorhamnetin 156 isorhamnetin-3-O-glucoside 167 isorhamnetin-3-O-glucosylglucoside 167 isorhamnetin-3-O-rutinosylrhamnoside 167 isorhamnetin-3-rutglc 168 isorhamnetin 4⬘-glucoside 186 Isorhamnetin-glucuronide 175 isoschaftoside 167, 168 isoscutellarein 7-O-glycoside 170 isoscutellarein 8-O-glucuronide 169–170 isosilybin 168 isoswertia-japonin 160 isovetexin-2⬘⬘-O-glucoside 167, 168 isovitexin 136, 168 isoxanthohumol 213, 214, 215 J Janus Green B, 387, 389, 560–561 jelly, 532

K kaempferol 137, 143, 152, 155, 156, 157, 159, 161, 167, 184, 186, 199, 200, 206, 232, 339, 340 kaempferol 3-rhamnosylglucoside 201 kaempferol glucoside 175, 201 kaempferol-glucoronide 175 kale 206 ketchup 90 Koch acid 542 L L. angustifolia subsp.angustifolia 171, 172 L. angustifolia subsp.delphinensis 171, 173 L. angustifolia subsp.pyrenaica 171, 172 L. aristibracteata 172, 173 L. bipinnata 172, 173 L. canariensis 172, 173 L. caronopifolia 172, 173 L. dhofarensis 172, 173 L. lanata 172, 173 L. latifolia 172, 173 L. mairei var. mairei 172, 173 L. maroccana 172, 173 L. minutolii var. minutolii 172, 173 L. multifida 172, 173 L. rotundifolia 172, 173 L. stoechas subs.luiseiri 171, 172 L. subnuda 172, 173 L. viridis 171, 172 laccacic acid A 339, 340, 341, 342 laccacic acid B 339, 340, 341, 342 laccacic acid C 339, 340 laccacic acid E 339, 340 C-15⬘OH lactone chlorophyll 306 C-15⬘OH lactone chlorophyll-b 306 C-15⬘OH lactone phaeophytin-a 306 15⬘-OH-lactone-pheophytin-a 310 15⬘-OH-lactone-pheophytin-b 310 lactucaxanthin 99, 100 Laminaceae 145 Larus fuscus 113 latoxanthin 83, 84, 85 Latuca sativa var. 336 Lavandula 169 lawsone 339, 340, 341 leafy vegetables 97, 98

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583 leek 206 lemon 206, 212 Lentinula edodes 373 Lepidium sativum 175 Leptadenia reticulata 146, 150 Leptospremum scoparium 184 Leptospremum polygalifolium 184 leucogentian violet 406 Leukomalachite Green 404, 405, 407 Leukocrystal Violet 404, 405 leutoxanthin 95, 96 Levisticum officiale 175 lignans 230 lime blossom 167 lime pulp 206 lingonberry 183 Lissamine rhodamine-B sulphonyl chloride 543 Litmus 387, 389 Lotus pedunculatus 233 lovage 170, 175 Lucas aspera 99 lucidin 330 lucidin primeveroside 330 ludicin glucoside 330, 332 lutcolin glucoside 175 luteolin-7-rhamnoglucoside 175 luteolin-acety-glucoside 175 luteolin-glucuronide 175 luteolin-rhanoglucoside 175 lutein 65, 69, 70, 72, 83, 84, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 108, 109, 115, 117, 118, 119, 120, 121, 123, 124, 125, 126, 130, 131, 132, 289, 291, 299, 300, 311 lutein epoxide 65 lutein-5, 6-epoxide 72 lutenoil 156 lutenoil 7-O-glucuronide 169 lutenoil 7-O-gucoside-4⬘-O-glucuronide 169 luteolin 339, 340, 341, 342 luteolin 7, 4⬘-di-O-glucuronide 169 luteolin 7-O-glucoside 178 luteolin 7-O-heteroside 178 luteolin 7-O-rutinoside 178 luteolin-3⬘7-diglucoside 170, 171 luteolin-4-glucoside 147 luteolin-7-glucoside 139, 170, 171 luteolin-7-O-β-D-glucoside 190

luteolin-7-O-glucoside 147, 152, 169 6-OH-luteolin 7-O-glucoside 169 luteolin-7-O-rutinoside 149 luteoxanthin 74, 83, 84 lycopene 72, 86, 88, 91, 92, 94, 103, 109 lycopene 1, 2-epoxide 92 lycopene 5, 6-epoxide 92 lycopene-5, 6-diol 92, 94 Lycopersicum esculentum 275 M Malachit Green 387, 389, 404, 407, 558–559 M. organophylum 130 macula 104, 108 magnesium chlorin 316 malic acid 457 malidin-3-glucoside 282 malonaldehidic acid 457 malonic acid 457 6⬘⬘-O-malonylaidzin 187 6⬘⬘-O-malonylgenistin 187 6⬘⬘-O-malonylglycitin 187 malvidin 240, 262 malvidin 3-O-feriloylrutinoside-5-Oglucoside 276 malvidin 3-O-p-coumaroylrutinoside -5-Oglucoside 276 malvidin-3-(p-coumaroyl)glucoside 256 malvidin-3-(p-coumaroyl)glucoside pyruvic derivative 255 malvidin-3-(p-coumaroyl)glucoside-4vinilcatechin 255 malvidin-3-(p-coumaroyl)glucoside-4vynilcatechol 255 malvidin-3-(p-coumaroyl)glucoside-4vynilguaiacol 255 malvidin-3-(p-coumaroyl)glucoside-4vynilphenol 255 malvidin-3-(p-coumaroyl)glucoside-8-ethylcatechin 256 malvidin-3-acetylglucoside 241, 251, 256, 259 malvidin-3-caffeoylglucoside 256, 259 malvidin-3-coumaroylglucoside 241, 251, 259, 260 malvidin-3-glucoside 241, 248, 251, 255, 256, 259, 286

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584 malvidin-3-glucoside pyruvic derivative 255 malvidin-3-glucoside-4-vynilcatechin 255 malvidin-3-glucoside-4-vynilepicatechin 255 malvidin-3-glucoside-4-vynilepicatechol 255 malvidin-3-glucoside-4-vynilguaiacol 255 malvidin-3-glucoside-4-vynilphenol 255 malvidin-3-glucoside-4-vynil-procyanidindimer 255 malvidin-3-glucoside-8-ethyl-catechin 256 malvidin-3-glucoside-8-ethyl-epicatechin 256 malvidin-3-glucoside-8-ethyl-gallocatechin 256 malvidin-3-glucoside-8-ethyl-procyanidin dimer 256 malvidin-3-monoglucoside 136 malvidin-3-O-(6-O-acetyl)glucoside 286 malvidin-3-O-(6-O-p-coumarin)glucoside 286 malvidin-3-O-acetylglucoside 253 malvidin-3-O-coumaroylglucoside 253 malvin chloride 152, 186 malvinidin-3-O-glucoside 253 maringin 211 Matricariae flos 138 Maytenus aquifolium 137 Maytenus ilicifolia 137 Mesorhizobium loti 237 methanol yellow 424, 425 Mentha piperita 145 Mentha piperta folium 176 Mentha var. 175 methylene blue 372, 384, 387, 389, 391, 458, 460, 559 4-methyl-esculetin 152 methyl green 387, 389 methyl orange 372, 449, 454, 457–460, 537 methyl red 388, 389, 454, 457–459 methyl violet 388, 389, 398 4, 4⬘-methylenedianiline 445 4, 4⬘-methylene-di-o-toluidine 445 2-methyl-1-indanone 158 3-methyl-1-indanone 158 (4S, 5R)/(4R, 5S)-4 methyl-5-phenyl-2oxazolidone 158 methyl gallate 137 methyl gallate 3-O-glucoside 151 Methylobacterium organophilum 127 5-methyl orientin 160 7-methyl orientin 160 6-metoxyflavonone 158

Mg 3, 8-divynil pheophorphyrin a5 monomethyl ester 289 mice 406 microalgae 123 milk 529-530, 532 milk beverages 529 milk-thistle 164, 166 minestrone soup 93, 94 mint 170, 175 monadoxanthin 289 monascin 325, 326 monascorubin 325, 326 monascorubramine 325 monoazoic dyes 374 monocaffeoylquinic acids 165 monotetrazolium salts 376, 381–384 monovinyl chl c3 289 monovinyl chlorophyll a 289 monovinyl chlorophyll b 289 mordant Orange1, 446–447 mordant red9, 492 mordant yellow8, 487, 492 morin 156, 161, 186, 232 munjistin 330–332, 341 mutatochrome 72 mutatoxanthin 74, 83, 85 mutatoxanthin 1, 84 mutatoxanthin 2, 84 mutatoxanthin A, 95, 96 mutatoxanthin B, 95, 96 mv 3-(acetyl)gluc-4-vinyl-cat 260 mv 3-(acetyl)gluc-4-vinyl-PC dimer 260 mv 3-(acetyl)gluc-4-vinylphenol 260 mv 3-(acetyl)gluc-py derivative 260 mv 3-(coumaroyl)gluc-4-ethyl cat 260 mv 3-(coumaroyl)gluc-4-vinyl cat 260 mv 3-(coumaroyl)gluc-4-vinyl-PC dimer 260 mv 3-(coumaroyl)gluc-py derivative 260 mv 3-(cumaroyl)gluc-4-vinylphenol 260 mv 3-gluc 260 mv 3-gluc-4-ethyl-cat 260 mv 3-gluc-4-vinyl 260 mv 3-gluc-4-vinyl cat 260 mv 3-gluc-4-vinyl-PC dimer 260 mv 3-gluc-4-vinylphenol 260 mv 3-gluc-py derivative 260 Mv-(6-acetyl)-3 glucoside 248 Mv-(6-caffeoil)-3 glucoside 248 Mv-(6-coumaroil)-3-glucoside 248

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585 Mv-gls-py derivative 248 myricathin 339, 340 myricetin 152, 155, 156, 167, 184, 186, 199, 200, 206, 209, 223, 232, 233 myxoxanthophyll 294 N nano orange 371 naphtalene-2, 3-dicarboxaldehide 370 naphtalene sulphonic acids 484–486 naphtoflavone 152 Naphtol Blue Black 373, 385, 391, 434, 436 2-naphtol-6-sulphonic acid 507, 509 2-naphtylamine 445 1-naphtylamine-4-sulphonate 434 narcissin 167, 168 naringenin 155, 156, 157, 158, 161, 186, 196, 206 naringenin-rhamniglucoside 175 naringin 152, 155, 186, 208 Navitan Fast Blue S5R 392, 400 N-benzylproline ethyl ester 158 neochlorogenic acid 164 neochrome 95, 96 neoxanthin 68, 72, 83, 84, 85, 95, 96, 99, 100, 101, 102, 130, 131, 289, 291, 293, 299, 311 Neutral Red 372, 388, 389, 460 neuro saporene 94 New Coccine 424–426, 521–522, 524, 528–530, 555 New Red 417 nigroxanthin 65, 74 4-nitroaniline 107, 445 4-nitrobenenediazonium ion 507 2-nitro-benzaldehide 457 norathyrol 156 norbixin 99 N-tBOC-3-(2-naphtyl)-Ala 158 O o-coumaric acid 161 okenone 300 5, 6-OH-7, 8, 3⬘, 4⬘-OMe-flavone 178 onion 167, 178, 184 onion stalks 99 onion red 206 onion soring 207

onion yellow 207 Ononis avensis 141, 143 Opuntia ficus-indica 332 Opuntia stirica 332 Opuntia undulata 332 orange 212 Orange G 370 Orange GS 388, 389 Orange I 424, 425 Orange II 372, 424, 425, 460, 539, 543 orange juices 92, 207 Orange Yellow 398, 399 orcein 388, 389 oregano 170, 175 organophylum 128 orientin 160 Origanum vulgare 175 oxalic acid 457 4, 4⬘-oxidianiline 445 132-oxopyrophaeophytin-a 304 132-oxopyrophaeophhorbide a-24methylcholesta-5, 24(28)-dien-3byl-ester 304 oxy derivative 167, 168 P paeonidin-3-acetylglucoside 251 paeonidin-3-coumaroylglucoside 251 paeonidin-3-O-acetylglucoside 253 paeonidin-3-O-coumaroylglucoside 253 p-anisic acid 209 pangasius 415 paprika 75, 109 paprika powders 79 pararoseaniline 372, 388, 389 parkinsonin A, B 160 parma ham 333 parsley 170, 175 Passiflora coerulea L. 143 Passiflora incarnata 163 passion flower tinctures 164, 166 Patent Blue V 398, 399, 521, 555 p-coumaric acid 157, 159, 161, 201, 223, 224 p-coumarylquinic acid 201 peanut nutmeat 261 peanut seed oils 88 peanut skin 261 pear, peel 207

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586 pebrellin (5, 6-dihydroxy-7, 8, 4⬘trimethoxyflavone) 178 pelargonin chloride 152 penoidin 3-O-(cis-p-coumaroyl)rutinoside-5O-glucoside 276 3, 3⬘, 4⬘, 5, 7-pentahydroxyflavone 7-Orhamnopyranoside 166 peonidin 240, 262 peonidin 3-O-caffeoylrutinoside-5-Oglucoside 276 peonidin 3-O-feriloylrutinoside-5-O-glucoside 276 peonidin-3-(p-coumaroy)glucoside 256 peonidin-3-(p-coumaroy)glucoside pyruvic gerivative 255 peonidin-3-(p-coumaroy)glucoside-4vynilcatechol 255 peonidin-3-(p-coumaroy)glucoside-4vynilphenol 255 peonidin-3-acetylglucoside 241 peonidin-3-coumaroylglucoside 241 peonidin-3-glucoside 241, 248, 251, 255, 256, 259, 282, 286 peonidin-3-glucoside pyruvic derivative 255 peonidin-3-glucoside-4-vynilcatechin 255 peonidin-3-glucoside-4-vynilcatechol 255 peonidin-3-glucoside-4-vynilguaiacol 255 peonidin-3-glucoside-4-vynilphenol 255 peonidin-3-glucoside-8-ethyl-catechin 256 peonidin-3-O-(6-O-acetyl)glucoside 286 peonidin-3-O-glucoside 253 peppermint 146 Pergularia daemia 99 peridin 132, 291, 294, 299 peridinin 289, 293, 295 peridinin isomer 289 persley 207 Petroselinum crispum 175 petunidin 262 petunidin-3-glucoside 256, 259 petunidin-3-coumaroylglucoside 259 petunidin 3-O-(cis-p-coumaroyl)rutinoside-5O-glucoside 276 petunidin 3-O-caffeoylrutinoside-5-Oglucoside 276 petunidin 3-O-feruloyrutinoside-5-Oglucoside 276 petunidin 3-O-p-coumaroylrutinoside 276

petunidin 3-O-p-coumaroylrutinoside-7-Oglucoside 276 petunidin-3-(p-coumaroyl)glucoside 256 petunidin-3-(p-coumaroyl)glucoside-8-ethylcatechin 256 petunidin-3-acetylglucoside 241, 259 petunidin-3-coumaroylglucoside 241 petunidin-3-glucoside 241, 248, 251 petunidin-3-glucoside pyruvie derivative 255 petunidin-3-glucoside-8-ethyl-catechin 256 petunidin-3-monoglucoside 136, 245 petunidin-3-O-(6-O-acetyl)glucoside 286 petunidin-3-O-glucoside 253, 286 Peucedaruum graveolens 99 Phaeocystis globosa 129, 130, 131, 133, 134 phaeohorbide-a 304 phaeophorbide-a ester 300 phaeophytin-a epimer 304 Phanerochaete chrysosporium PC671, 536, 541 Phenolphtalein 560–561 Phaseolis vulgaris 157, 336 pheaphorbide-a methyl ester 300 phenophytin 92 2-phenylcycloheptanone 158 2-phenylethanol 191 pheophorbide-b 307, 310 pheophytin-a 290, 300, 307, 309, 310, 311, 317 pheophytin-a epimer 300, 306 pheophytin-b 290, 300, 306, 309, 310, 311, 317 Phillyrea latifolia L. 144 Philonostiflavone 147 Phizobium leguminosarum bv. Trifolii 237 Phlebia tremellosa 475 phloretin derivative 180, 181 phloridzin 180, 181 Phloxim B 388, 389, 528, 534 phtalocyanine dyes 470, 540 p-hydroxybenzoic acid 223, 224 Phyllanthus emblica L. 137 phyrobacteriophaephytin-a 300 physcion 335, 336 phytilated chlorophyll-c-like 133 phytoene 90, 94 phytofluene 90, 94, 95, 96 phyto-oestrogens 230 phytylated chlorophyll c-like 289 picrocracin 107, 108

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587 pieceid 219 pilloin 155 Pinaceae 146 pinocembrin 156 pinostrobin chalcone 156 pinta boca 275 Pisum sativum 336 Pleurotus ostreatus 373 plum, blue 207 pluvialis 127 pn 3-(coumaroy)gluc-py derivative 260 pn 3-(cumaroyl)gluc-4-vinylphenol 260 Pn-(6-acetyl)-3 glucoside 248 Pn-(6-coumaroil)-3-glucoside 248 Pn-gls-py-derivative 248 polargonin chloride 152 polyhydroxyflavones 231 polyhydroxyphenols 232 polymer Mv-cat 248 polyphenols 73 Ponceau 3R 424, 425 Ponceau 4R 396, 398, 399, 403, 417, 419, 519, 422, 516, 520-521, 527 Ponceau 6R 379 Ponceau G 372, 460 Ponceau R 424, 425 Ponceau SS 446–447 Porphyridium cruentum 326 Potulacada quadrifida 99 poultry feed 107 prasinoxanthin 289, 295, 299 6-prenylnaringenin 213, 214, 216 8-prenylnaringenin 213, 214, 215 prenylflavonoids 208 preparative high speed counter-current chromatography 88 proanthocyanidins 247 261 probably 8-geranylnaringenin 214 Procambarus clarkii 67 Procion Brown MX 5BR 369, 497–498 Procion Green H-4G 369 Procion Red 370 Procion Red MX-5B 373, 450, 497 procyanidin 245, 271 procyanidin B1, 180, 181 procyanidin B2, 180, 181, 273 procyanidin B3, 180, 181 procyanidin C1, 272 procyanidin oligomers 271, 273

profile 177 propolis 160 protocatecholic acid 191 protocatechuic acid 223, 224 prymnesiophytes 292 pseudochlorogenic acid 164 pseudohypericin 166 Pseudomonas aeruginosa 373, 392 Pseudomonas luteola 470, 473–474 Pseudomonas species 373 pseudopurpurin 330, 331 Pseudotsuga menziesii 146, 15 pt 3-(acetyl)gluc-py derivative 260 pt 3-(cumaroyl)gluc-py derivative 260 Pt-(6-acetyl)-3-glucoside 248 Pt-(6-coumaroil)-3-glucoside 248 Pt-gls-py-derivative 248 purpurin 330, 332, 339, 340, 341, 342 purpurin-18 phytyl ester 300, 304, 306 purpurin-7 phytyl ester 300 pyrananthocyanins, 266 269 pyrochlorophyll a 300 pyrochlorophyll b 300 Pyronin G 388, 389 Pyronin Y 388 pyrophaeohorbide-a 305, 306 pyrophaeophorbide-a colesta-5, 22-dien-3β-ol ester and 24methylcolestra-5, 24(28)-dien-3b-ol esters 305 pyrophaeophorbide-a colesta-5-en-3β-ol ester 305 pyrophaeophorbide-a ester 300 pyrophaeophorbide-b 24-methylcolest-5-en3β-pl and pyrophaeophorbide-a colesta-5, 24-dien-3β-ol-ester 305 pyrophaeophorbide-b colesta-5, 22-dien-3β-ol ester and 24-methylcolestra-5, 24(28)-dien-3b-ol esters 305 pyrophaeophorbide-b colesta-5, 24-dien-3β-ol ester 305 pyrophaeophorbide-b colesta-5-en-3β-ol ester 305 pyrophaeophytin-a 290, 300, 304, 306, 310 pyrophaeophytin-b 300, 306 pyrophaeophorbide-b 306 pyropheophorbide-a 289, 310 pyropheophorbide-a methyl ester 300 pyruvic acid 457

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588 pytoene 72 pytofluene 72 Q quinoline yellow 379, 398, 399, 500, 505, 516, 520–521 quercetin 134, 143, 145, 146, 152, 155, 156, 161, 166, 167, 179, 184, 199, 200, 206, 209, 232–234, 237, 339, 340 quercetin 3-(2-sinapoly-O-α-Dglucopyranosil)-3⬘-(6-sinapoly-Oα-D-glucopyranosil)-O-α-D-glucopyranoside 176 quercetin 3⬘-(6-sinapoly-O-α-Dglucopyranosil)-3, 4⬘-di-O-α−Dgluco-pyranoside 176 quercetin 3-glucoside 201 quercetin 3-rhamnosylglucoside 201 quercetin aglycone 237 quercetin diglucoside 186 quercetin glucoside 175, 186 quercetin ramnogucoside 175 quercetin-3-O-rutinosylrhamnoside 167 quercetrin-3-rutglc 168 quiizarin 330 R Radish leaves, 99 raisin, 318, 320 Raphamus sativus, 99, 148, 305 raspberry, 262 rat, 406 Reactive Black 5, 372, 439, 441, 445, 463–464, 483 Reactive Blue2, 369 Reactive Blue4, 434, 437, 458, 462 Reactive Blue15, 470 Reactive Blue38, 470 Reactive Blue109, 487 Reactive Brilliant Red K-2G, 450 Reactive Brown 10, 369 Reactive Brown 23, 512–513 Reactive Green 8, 487 Reactive Orange 483 Reactive Orange4, 512 Reactive Orange16, 449, 450, 497–499 Reactive Red 2, 369, 512–513 Reactive Red 22, 470, 473–474

Reactive Red120, 445 Reactive Red198, 483 Reactive Yellow 369 Reactive Yellow 84 (CI) 433, 434, 445 Reactive Yellow 145, 372 red currant 207, 262 Red G2, 519 red onion 271, 274 red pigment 326 red raspberry 179, 207 red wine 157, 167, 207, 212 Remazol Black 442 Remazol Black B 440, 475, 477, 479–480 Remazol Blue GG, 440 Remazol Blue 442, 475 Remazol Brilliant Blue R 373 Remazol Brilliant Orange 3R 373, 479 Remazol Brilliant Violet 5R 477, 479–480 Remazol Golden Yellow 475 Remazol Golden Yellow RNL 440 Remazol Navy Blue 475 Remazol Red 475 Remazol Red RB 440 Remazol TB 539 Remazol Turquoise Blue 475, 531 Remazol Yellow 442 Reseda luteola 167 resorcinol 237 resveratrol 186, 220 retina 104, 108 retinoid 106, 132 retinol 116, 119 retinyl acetate 106 rhamnetin 155, 156, 237, 339, 340 Rhizoma chuanxiong 163 Rhodamine 123, 394 Rhodamine B, 370, 372, 388, 391, 394, 460, 558–559 Rhodamine 6G 384, 391, 393–395 rhoidoxanthin 300 rhoifolin 211 Ribes nigrum 179, 266, 267 riboflavin 210 robinetin 152, 155 robinin 152, 155 Rosa hybrida 277 rosacyanin 279 rosebud 207 rosemary 170, 175

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589 rosmarinic acid 149, 175, 178 Rosmarinus officialis 175 rowanberries 179, 183 ruberythic acid 330, 331, 332 Rubia tinctorum 328, 331, 332 Rubin C 388, 389 rubropunctatin 325, 326 rubropunctatarmine 325 Rubus idaeous 179 Rudbecka hirta L. 136 Ruta graveolens 146, 150 rutin 137, 138, 139, 146, 151, 152, 155, 166, 167, 168, 180, 212, 213, 218, 221, 223, 228, 232, 233 rutinose 262 S S. atriplicifolia 172, 173 safflower 99 saffranal 104, 107, 108 saffron 102, 104, 105, 106, 107 Safranin O 388, 389 salads 207 salmon 411, 412, 414, 415 salted fish 421, 426 salted vegetables 422, 426 salvigenin 170 sambubiose 262 Sambucus negra L. 137 Sandocryl Blue 466 Sandocryl Green 466 Sandocryl Red 466 Sandocryl Yellow 466 Sandolan Orange 466 Scarlet Red 388 Sclerotium rolfsii 373 santal 99 schaftoside 167, 168 scolymoside 164 scopoletin 137 scutellarein 7-Oglycoside 169 scytonemin 300 sea buckhorn berries 179, 183 seagrass 128 serum 230 Shepu 99 shrimps 415 silybin 168 Silybum marianum 163

silychristin 167, 168 silydianin 168 Sinapis alba 144, 148 Sinapis arvense 148 siphonaxanthin 300 soda 421, 426, 527–528 soft drinks 516 Solophenyl Green Blue 373 Solvent Black3, 444 Solvent Orange7, 444 Solvent Red24, 443–444 Solvent Yellow2, 444 Solvent Yellow3, 444 Solvent Yellow14, 443–444 Solanum stenotomum 275 Solanum tuberosum 275 sophorose 262 Sorbus aucuparia 179 Sorocea bomplandii (Moraceae) 137 soybean 184, 190 spaghetti sauce 90 spinach 99, 108 Spinacia oleracea 99 Spirulina pacifica 123 Spirulina platensis 123, 124 St. John’s Wort 167 steryl chlorin ester 300 strawberry 179, 207, 266 Sudan Black B 388, 389, 560–561 Sudan I 97, 399, 424, 427, 428 Sudan II 424, 427, 428 Sudan III 388, 389, 424, 427, 428, 446–447 Sudan IV 388, 389, 424, 427, 428 Sudan Red 388, 389 sulphonated azo dyes 530 6-sulphonate-2-naphtol-1-azo-p-nitrobenzene 507, 509 sunflower seed oils 88 Sunset Yellow 379, 417, 419, 422, 424, 519, 527 Sunset Yellow FCF 425, 426, 516, 520–522, 524, 528, 530, 532, 555 sweet pepper 207 sweet pepper red 207 sweet pepper yellow 207 SYBR Green1, 370 Synechococcus elongatus 294 syringic acid 223, 224 syrup 526

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590 T Taeniopygia guttata 119 tangerine 212 tartaric acid 457 Tartrazine 379, 398, 399, 425, 426, 521–522, 524, 528, 530, 532, 555 tarragon 170, 175 taxifolin 158, 167, 168 tea, 207 227 tectochrysin 155 Temora longicornis 129, 131 Tephorosia purpurea (Fabaceae) 146, 150 tetrachlorofluorescein 370 Tetrahymena thermophita 371 2⬘, 4⬘, 5⬘, 7⬘-tetraiodofluorescein -, 553 tetramethylpiperidine 393 2, 2-(2, 2, 6, 6-tetramethylpiperidine-1-yl)-4, 6-dichloro-1, 3, 5-triazine 512 5, 10, 15, 20-tetra-(3, 4methoxyphenyl)porphyrin 319 5, 10, 15, 20-tetra-(3-methoxy-4decyloxyphenyl) porphyrin 319 5, 10, 15, 20-tetra-(3-methoxy-4hexadecyloxyphenyl) porphyrin 319 5, 10, 15, 20-tetra-(3-methoxy-4hydroxyphenyl) porphyrin 319 5, 10, 15, 20-tetra-(3-methoxy-4pentyloxyphenyl) porphyrin 319 5, 10, 15, 20-tetra-(4decyloxyphenyl)porphyrin 319 5, 10, 15, 20-tetra-(4hexadecyphenyl)porphyrin 319 5, 10, 15, 20-tetra-(4hydroxyphenyl)porphyrin, 319 5, 10, 15, 20-tetra-(4methoxyphenyl)porphyrin 319 5, 10, 15, 20-tetra-(4pentyloxyphenyl)porphyrin 319 tetraphenylporphyrin derivatives 318 Tetraselmis suecica 306 theaflavin 203 theaflavin 3⬘3-digallate 203 theaflavin-3-gallate 203 theobromin 193, 201 theogallin 201 theophylline 234 4, 4⬘-thiodianiline 445 thionine 388, 389

Thlaspi arvense 144 thyme 170, 175 Thymolphtalein 560–561 Thymus vulgaris 175 tiliroside 155 tocopherol 119 tomato 86, 207 tomato juice 93, 94 tomato puree 90 tomato sauce 90 tomato soup 93 tomato-based 90 o-toluidine 445, 452 total carotenoid 72 total chlorophyll 72 total chlorophyll-a 73 total chlorophyll-b 73 toys 442 trans lycopene 87 trans-4-chlorostilbene oxide 158 trans-β-carotene 128 trans-ditiliroside 150 trans-pieceid 219 trans-resveratrol 219, 221, 223, 228 trans-tiliroside 150 trans-veratrol 212 Triathama monogyma 99 tricetin 184 Trichoderma harzianum 320 Trigonella foenumgraecum 99 2⬘, 4⬘, 5⬘-triiodofluorescein 553–554 2⬘, 4⬘, 7⬘-triiodofluorescein 553–554 2, 4, 5-trimethylaniline 445 Troger’s base 158 trout 403, 404, 406, 412, 415 Trypan Blue 388, 389 Trypan Red 388, 389 trypllichrome 95 tryptanthrin 139, 141 turmeric 318 U umbelliferone 137, 152 uranine 528, 534 urine 230 V V. vinifera 245 Vaccinium myrtillus L. 144, 145, 146, 179

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591 Vaccinium oxycoccos 179 Vaccinium uliginosum 179 Vaccinium vitis-idaea 144, 145, 146, 179 valenciaxanthin 95, 96 Vanda (Orchidaceae) 276 Vanda Bud Mellot x Vanda Gordon Dillon 278 Vanda coerulae Griff 278 Vanda coerulescens Griff 278 Vanda Fuchs Delight 278 Vanda Gordon Dillon x Vanda Bankok Blue ‘Kamiya’ 278 Vanda Kultana Blue 278 Vanda Lenavat Rose x Vanda Gordon Dillon 278 Vanda Manuel Torres 278 Vanda Manuvadee 278 Vanda Manuvadee ‘Chiaki’ 278 Vanda Rovert’s Delight 278 Vanda Taweewan x Vanda Chindavat 278 Vanda Thananchai x Vanda Ratirat 278 Vanda tricolor Lindl. 278 Vanda ustii Golamco 278 Vanda Varabth ‘Y-54’ 278 Vanda Wirat 278 vanillic acid 223, 224 vanillin 191 vat blue 41, 430 vegetable beef soup 93, 94 vegetable juice 93, 94 vegetables 231 vegetarian vegetable soup 93, 94 vicitin-2 168 violaxanthin 65, 68, 72, 74, 83, 84, 85, 99, 100, 101, 102, 115, 130, 131, 289, 293, 299, 311 violdelphin 243, 244 vitamin A palmitate 135 vitexin 136, 167, 168, 169 Vitis vinifera 239 vitisin B 248 W W. coccinea 139, 140 W. tomentosa 139, 140

weld 167 white onions 179 white rot fungi 470, 475, 531, 540 whortleberries 179 wild lingonberries 179 wine 157, 244, 252 Wrightia tinctoria 139, 140 X xanthene dyes 528 xanthohumol 208, 213, 214, 215 xanthomicrol 170 xanthophylls 63, 125 xanthopurpurin 330 xanthurenic acid 324 Xylidine Acid Red 429 Xylidine Ponceau 2 R 481–482 xylose 262 Y Yellow AB 388, 389 yellow gardenia 99 yellow throat 123 yellow warbler 121, 123 yoghurt 529–530 Z (13Z)-zeaxanthin 74 (9Z)-zeaxanthin 74 zeaxanthin 65, 68, 69, 72, 74, 81, 82, 83, 84, 85, 95, 96, 97, 98, 104, 108, 109, 110, 113, 115, 117, 118, 119, 121, 123, 124, 125, 130, 289, 290, 291, 293, 294, 295, 299, 300 zebra finch 119, 121 zinc phtalocyanine tetrasulphonic acid 551 zinc protoporphyrin 333 Z. marina 129 Z. marina leaf 130 Zostera marina 128

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