Zhaolun Fang
Flow Injection Separation and Preconcentration
Weinheim · New York · Basel · Cambridge · Tokyo
Professor Zhaolun Fang Institute of Applied Ecology Academia Sinica P.O. Box417 110015 Shenyang. China
This book was carefully produced. Nevertheless. author and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements. data. illustrations. procedural details or other items may inadvertently be inaccurate.
Published jointly-by VCH Verlagsgesellsehaft mbH. Weinheim (Federal Republic of Germany) VCH Publishers. Inc .. New York. NY (USA)
Editorial Director: Dr. Christina Dyllick. Karin Sora
Library of Congress Card No. applied for
A CIP catalogue record for this book is available from the British Library
Die Deutsche Bibliothek - CIP-Einheitsaufnahme Faag, Zlutolua: Flow injection separation and preconcentration I Zhaolun Fang. - Weinheim ; New York ; Basel ; Cambridge ; Tokyo : VCH.1993 ISBN 3-527-28308-0
Cl VCH Verlagsgesellsehaft mbH. D-6940 Weinheim (Federal Republic of Germany). 1993 Printed on acid-free and low-chlorine paper All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form -by photoprinting. microfilm. or any other means- nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book. even when not specifically marked as such, are not to be considered unprotected by law. Printing: Strauss Offsetdruck GmbH. D-6945 Hirschberg 2 Bookbinding: Verlagsbuchbinderei C. Kriinkl, D-6148 Heppenheim Printed in the Federal Republic of Germany
Foreword
Ever changin~. science transfonns itself through new concepts and tools. which render older ones obscure. Thus authoring a monograph on a field in rapid development may seem to be a futile exercise. since the state of the an is changing even as the author writes. There are. however. qualities of an everlasting value: thoroughness. attention to detail and an ability to identify the key concepts. This. in tum. requires a profound knowledge and experience. which can be obtained only through a lifetime in the field. deep interest in the subject and personal panicipation in the an of experimentation. The author has shown to he in possession of all these qualities. while keenly focusing on one of the most imponant features of flow injection: the ability to handle chemical separations. He,thus defined the scope of the book. which begins with the right blend of concepts and definitions. deals with relevant flow methodologies and emphasizes important experimental details in a way never done before. The body of the work is a> valuable as it is unique. since it is aimed at the chemical aspects of separations for which flow-injection is a vehicle. One realize~ once again that this book could only be written hy someone who was not blinded by the trappings of flow manipulations and/or computerization. but who has kept sight of the most important feature of flow-injection: it~ ability to manipulate and monitor chemical reactions reliably so that reproducible results are obtained even under non-equilibrium conditions. Therefore as flow-injection becomes funher transformed into sequential injection, flow-injection microproce.ssing and perhaps even flow injection synthesis. this book will retain its value. It is said that a travel book tells more about its author than about the traveled territory. Zhaolun Fang has achieved a rare balance while mapping the landscape of flow-injection based separations. by critically presenting contributions of many. including his Chinese colleagues. His book gives us an opponunity to view the outstanding contributions of Chinese analytical chemists in the context of international science. Only someone who has worked extensively in such different countries as Denmark, Germany and China could succeed in this endeavor.
Seattle, December 1992
Jaromir Ruzicka
Preface
Despite the rremendous progress made in analytical instrumentation in the past few decades. the development of basic analytical techniques in the chemical laboratory has been rather slow and incompatible with requirements of the computer age. The sample pretreatment stage. which often involves manual separations such as extraction, sorption. distillation, precipitation and dialysis. quite frequently forms the bouleneck and weakest link of the entire analytical procedure. not only in terms of efficiency but also in reliability and sample/reagent consumption. It is therefore mandatory that more efforts be directed towards the development of efficient automated on-line sample pretreatment techniques which could remove the bouleneck. The flow injection technique has. proved to be an effective tool for achieving such goals. Originally considered to be merely a technique for automation of serial assays. flow injection analysis has gradually evolved into a powerful technique for substituting tedious manual separation procedures by producing stronger contacts between chemistry and the analytical instrument: in fact. this has now become one of the most active research fields in automated continuous flow analysis. persistently stimulating further interests in revolutionizing conventional operations in the analytical laboratory. This book is the result of a prolonged excitement which I experienced together with my colleagues both in China and in Europe through the past decade on the previously mentioned quest for better sample pretreatment methods with extensive implementation of the flow injection technique. The reader may already be aware of several good monographs on flow injection analysis mentioned in Chapter I. which give general treatments of the technique. A monograph on non-chromatographic continuous separation techniques ha-; also been published recently by Valcarcel and Luque de Castro (Chapter 3. ref. [13]). However. separation and preconcentration methods based on flow injection principles and techniques warrant a more dedicated and detailed treatment owing to the fast development and strong interest in this panicular field. The book is intended to be used both as a source reference book for the research analytical chemist and as a laboratory handbook for the routine analyst who wish to automate or/and improve the performance of their instrumental analytical procedures. particularly when dealing with samples with complex matrices. such as those frequently encountered in environmental. agricultural and clinical applications. The book is composed of nine chapters with a brief introduction on the basic principles (Chapter I) and instrumentation (Chapter 2), followed by separate chapters on liquid-liquid extraction (Chapter 3). sorption (Chapter4), gas-liquid separation (Chapter 5), dialysis (Chapter 6) and precipitation (Chapter 7). Finally, two separate chapters are devoted to environmental and agricultural applications (Chapter 8) and clinical and pharmaceutical applications (Chapter 9). Electro-deposition and dissolution is not
VIII
Preface
included in the book because the available bibliography is still too little to warrant the inclusion of a separate chapter. but some related material may be found in the book by · Valcarcel and Luque de Castro. The bibliography given in the book is not intended to be exhaustive. but attempts were made to include all imponant contributions before the end of 1991 and some from 1992: also. whenever possible. tabulated comparison of the performance for different techniques was made. so that the reader can procure a general picture of the state of the an with minimum reading. Special care has been taken to provide sufficient practical details for the techniques and procedures in order that the book may serve as a guide and assist the practising analyst in constructing their own flow injection separation/preconcentration systems. I wish to express my gratitude to all my colleagues in the Flow Injection Analysis Research Centre of the Institute of Applied Ecology who have arduously contributed to the accumulation of a substantial amount of material included in this book. Panicularly, thanks are due to Mr. Guanhong Tao who has created most of the an work used in this book. I wish to extend my sincere thanks to Professor Jaromir Ruzicka and ~ofessor Elo Hansen. in whose laboratory in Chemistry Depanment A of the Technical University of Denmark we conducted our earliest cooperative studies on on-line column preconcentrations. and to whom I am indebted for many inspirations in the succeeding years of my research career in flow injection analysis. I also wish to express my gratitude to Dr. Bernhard Welz not only because of the many fruitful research cooperations we have had in his laboratories in the Bodenseewerk of Perkin-Elmer but also because without his encouragement and suppon this book would never have materialized. The financial support from the Natural Science Foundations of China and Bodenseewerk. Perkin-Elmer GmbH for many research projects. the achievements of which are included in this book. are highly appreciated. Finally. I wish to acknowledge my appreciation for the most pleasant editorial cooperation I have had with Dr. Hans Ebel and Mrs. Karin Sora and Mr. Peter Biel from VCH Publishers.
Oberlingen. September 1992
Zhaolun Fang
Contents
1
Introduction 1.1 Historical Perspectives 1.2 Basic Principles of FIA 4 1.2.1 WhatisFIA? 4 1.2.2 Dispersion of Sample and Reagent Zones 5 1.2.3 The Evaluation of Dispersion 6 1.2.4 Factors Influencing Dispersion 8 1.3 General Characteristics of Fl Methods for Separation and Preconcentration I 0 1.4 Fundamental Aspects of Fl Separation and Preconcentration II 1.4.1 Classification of A Separation Techniques II 1.4.2 Enrichment Factor (EF) 12 1.4.3 Enhancement Factor (N) 12 1.4.4 Concentration Efficiency (CE) 13 1.4.5 Consumptive Index (CI) 14 1.4.6-Phase Transfer Factor (P) 14 1.4.7 Time-based and Volume-based Sample Loading in Fl Preconcentration Systems 15 1.4.8 The Evaluation of FI Preconcentration System Efficiency 15 1.5 Liquid Chromatographic and FI Separation Methods · 18
•
2
General Instrumentation 21 2.1 Liquid Delivery Devices 21 2.1.1 General 21 2.1.2 Peristaltic Pumps 22 2.1.3 Reciprocating Piston Pumps 26 2.1.4 Syringe Pumps 27 2.2 Injection and Multi-functional Valves 29 2.2.1 General 29 2.2.2 Six-port Rotary Valve 29 2.2.3 Eight-channel Sixteen-port Multifunctional Valve 2.2.4 Three-layer Commutator Valve 33 2.2.5 Bypasses in Valve Designs 33 2.3 Transport Conduits and Mixing Reactors 35 2.3. I Transport Conduits 35 2.3.2 Mixing Reactors 36 2.3.3 Manifolds for Connection of Conduits 37
30
X
comems 2.4 Detectors 38 2.4. 1 Spectrophotometers 38 2.4.2 Atomic Absorption Spectrometers 40 2.4.3 Induction Coupled Plasma (ICP) Emission Spectrometers 42 2.4.4 Electrochemical Detectors 42 2.4.5 Other Detectors 44
3
Liquid-liquid Extraction 47 3.1 General 47 3.2 Instrumentation 48 3.2.1 Phase Segmentors 48 3.2.2 Extraction Coils 52 3.2.3 Phase Separators 52 3.2.4 Integrated Liquid-liquid Extractor 59 3.3 Theoretical Aspects of Fl Liquid-liquid Extraction 59 3.3.1 Mechanism of Phase Transfer in FI Liquid-liquid Extraction 59 3.3.2 Dispersion in FI Liquid-liquid Extraction 61 3.4 FI Manifolds for Liquid-liquid Extraction 63 3.4.1 General 63 3.4.2 Sample Introduction Modes 64 3.4.3 Segmentation and Extraction Modes 65 3.4.4 Phase Separation Modes 67 3.4.5 Flow Exit Modes 68 3.4.6 Modes of Delivery of Separated Phase to Detector 69 3.4.7 Modes of Derivatization 70 3.4.8 Fllterative Flow Reversal Liquid-liquid Extraction System without Phase Separation 71 3.4.9 Multiple-stage Fl Liquid-liquid Extraction Systems 73 3.5 Coupling of Fl Liquid-liquid Extraction Systems to Various Detectors 74 3.5. I Spectrophotometers 74 3.5.2 Flame Atomic Absorption Spectrometers 76 3.5.3 Electrothermal Atomic Absorption Spectrophotometers (ETAAS) 80 3.5.4 ICP Spectrometers 80 3.5.5 Coupling of Fl Liquid-liquid Extraction Systems to Gas and Liquid Chromatographs 81
4
Sorption 85 4.1 Introduction 85 4.2 Classification of FI Column Techniques 86 4.3 Dispersion in FI Column Preconcentration Systems 87 4.3.1 Dispersion in Sample Loading 87 4.3.2 Dispersion in Sorption and Elution 88 4.3.3 Dispersion in Eluate Transport and Post Column Reactions 89 4.4 Practical Considerations in the Design and Operation of FI Column Preconcentration Systems 90 4.4.1 Column Designs 90
Coment.~
4.4.2 Column Loading 93 4.4.3 Column Washing and Equilibration 95 4.4.4 Elution 96 4.S Column Packings 98 4.5.1 General Requirements for On-line Column Packings 98 4.5.~ Chelating Jon-exchangers 98 4.5.3 C 1x Bonded Silica Gel 100 4.5.4 Polymer Sorbents 100 4.5.5 Strongly Basic Anion Exchangers 101 4.5.6 Strong!~ Acidic Cation Exchangers 101 4.5.7 Activated Alumina 102 4.5.8 Water Adsorbents 102 4.6 FJ On-line Column Separation and Preconcentration Systems 103 4.6.1 General I 03 4.6.2 Systems for On-line Separation of Interferents 103 4.6.3 Column Preconcentration Systems for Flame AA and ICP Emission Spectrometry 105 4.6.4 Column Preconcentration Systems for Hydride Generation and Cold Vapor AAS 112 4.6.5 Column Preconcentration Systems for Graphite Furnace AAS 114 4.6.6 Column Separation and Preconcentration Systems for Spectrophotometry 120 4.6.7 Column Preconcentration Systems for Chemiluminescence Detenninations 123 4.6.8 Column Preconcentration Systems for Ion-selective Electrode Detectors 123 4.7 Sorption Preconcentration for Solid Phase Optosensing 124 4.7.1 General 124 4.7..:! Practical Considerations in the Design of Solid Phase FI Optosensing Systems 125 4. 7.3 Solid Phase Absorptiometry 126 4.7.4 Solid Phase Fluorimetry 128 5
Gas-liquid Separation 129 5.1 Introduction 129 5. 1.1 General 129 5.1.2 Classification of FI Gas-liquid Separation Systems 130 5.2 Gas-liquid Separators for FIA 131 5.2.1 General 131 5 .2.2 Gas-diffusion Separators 131 5.2.3 Gas-diffusion Membranes 134 5.2.4 Gas-expansion Separators for Vapour Generation Atomic Spectrometric Systems 135 5.3 A Gas-diffusion Separation Systems 138 5.3.1 Basic Gas-diffusion Separation Systems 138 5.3.2 Gas-diffusion Preconcentration Systems 138 5.3.3 Factors Influencing Mass Transfer in FI Gas-diffusion Separation Systems 140
XI
XII
Coment.1·
5.4 Coupling of Fl Gas-diffusion Separation Systems to Various Detectors 14::! 5.4.1 Spectrophotometric Detectors 142 5.4.2 Optosensing Using Optical Fibers 144 5.4.3 Chemiluminescence Detectors 145 5.4.4 Electrochemical Detectors 146 5.4.5 Mass Spectrometric Detectors 147 5.5 FI Vapour-generation Systems 148 5.5.1 Hydride-generation Systems 148 5.5.::! Cold Vapour Generation Systems 156 6
Dialysis 159 6.1 General 159 6.2 Fundamental Aspects of Fl On-line Dialysis 160 6.3 Dialyzers 162 6.4 On-line Dialysis Membranes 163 6.5 Fl On-line Dialysis Manifolds 164 6.5.1 Basic manifold Configurations 164 6.5.2 Manifolds with Dialyzer as Sample Loops 164 6.5.3 Donnan Dialysis Preconcentration System 165 6.6 Coupling of FI On-line Dialysis to Various Detectors 166 6.6.1 Spectrophotometers 166 6.6.2 Electrochemical Detectors 167 6.6.3 Atomic Absorption Spectrometers 167
7
Precipitation 169 7.I Introduction 169 7.'2 On-line Precipitate Collectors 170 7 .2.1 General 170 7.2.2 Stainless Steel Filters 171 7.2.3 Disposable Membrane Filters 172 7 .2.4 Packed-bed Filters 172 7 .2.5 Knotted Reactors 173 7.2.6 Choice of Precipitate Collectors 174 7.3 FI Manifolds for On-line Precipitation-dissolution 175 7.3.1 On-line Filtration Systems Without Precipitate Dissolution 175 7.3.2 On-line Filtration Systems with Precipitate Dissolution 177 7.3.3 Filterless System without Precipitate Dissolution 178 7.3.4 Filterless Systems with Precipitate Dissolution 179 7.4 Some Fundamental Aspects of On-line Precipitation-dissolution 183 7.4.1 Kinetic Effects in On-line Precipitation and Coprecipitation 183 7 .4.2 Kinetic Effects in Precipitate Dissolution 184 7 .4.3 Precipitate Forms in Continuous Precipitate Collection 185 7.5 FI Variables for On-line Precipitation-dissolution Systems 186 7.5.1 Sample or Precipitant Volume 186 7.5.2 Sample Flow-rate 186 7.5.3 Reaction Coil Dimensions 187 7.5.4 Flow-rate of Dissolution Solvent 188
Contem.,
XIII
7.6 A Method' With On-line Continuous Precipitation 188 7.6.1 Indirect Methods Involving On-line Continuous Precipitation 18!1 7.6.2 FI On-line Preconcentration Methods With Continuous Precipitationdissolution 191 7.6.3 Reduction oflnterference Effects in Flame AA Using Continuou~ Precipitation 193 8
Environmental and A~ricultural Applications 197 8.1 General !97 8.2 Waters 197 8.2.1 Determination of Trace Elements 197 8.2.2 Determination of Trace Anions 202 8.2.3 Determination of Surfactants 202 8.2.4 Determination of Nitrogen and Nitrogen Compounds 203 8.2.5 Other Determinations 204 8.3 Plant and Animal Tissues 204 8.3.1 Determination of Trace Elements 204 8.3.2 Determination of Anionic Constituents 206 8.4 Beverages 206 8.5 Milk 20X 8.6 Soils and Sediment~ 209 8. 7 Other Agricultural Samples 210 8.8 Selected Analytical Procedures 211 8.8.1 Spectrophotometric Determination of Total Nitrogen in Soils with On-line Gas-diffusion Separation 211 8.8.2 Spectrophotometric Determination of Anionic Surfactants in Water with On-line Solvent Extraction 213 8.8.3 Electrothermal Atomic Absorption Spectrometric Determination of Trace Metals in Sea Water with On-line Sorbent Extraction Separation and Preconcentration 215 8.8.4 Hydride Generation Atomic Absorption Spectrometric Determination of Selenium and Arsenic in Soils and Biological Materials 218
9
Clinical and Pharmaceutical Applications 9.1 General 221 9.2 Blood and Serum 221 9.2.1 Determination of Trace Elements 9.2.2 Determination of Urea 223 9.2.3 Perchlorate 223 9.2.4 Gaseous Constituents 224 9.3 Urine 224 9.3.1 Determination of Trace Elements 9.3.2 Galactose 225 9.3.3 Amines 226 9.4 Pharmaceuticals 226 9.4.1 Carboxylic Acid Drugs 226 9.4.2 Sulphonamides 226 9.4.3 Codeine 227
221
221
224
XIV
Contents
9.4.4 Local Anaesthetics 227 9.4.5 Vitamins 227 9.4.6 Other Pharmaceuticals 227 9.5 Selected Procedures 229 9.5.1 Spectrophotometric Determination of Codeine in Pharmaceutical Preparations by Fl Solvent Extraction 229 9.5.2 Spectrophot
10 References 239 II
Index
253
1
Introduction
II is not within the scope of this book to provide the reader with a comprehensive treatment on the most basic aspects of flow-injection analysis (FIA). However. the main principles of FIA will be discussed briefly in this chapter, with special reference to separation arid preconcentration techniques. to help the reader gain a deeper insight into the related techniques. For a more comprehensive treatment of the basics of FlA. the reader may consult the second edition of "Flow Injection Analysis". authored by Ruzicka and Hansen II], and other monographs on the topic listed in the references [::?..3 ].
1.1
Historical Perspectives
The development of Fl separation and preconcentration techniques is a logical extension following the advent of FlA. The inception of FIA in tum is the result of a long search for better laboratory techniques in solution manipulation which could match the efficiency of the computer age. The- important stages of development in this quest for efficiency and autorruuion in the chemical laboratory are shown in Figure 1.1.. which also shows the relation between the various techniques for automated solution analysis and the scheme for their classification. The first attempts for automation of solution handling which made up the bulk of labour ih an analytical laboratory was simply mechanizing and simulating the traditional manual batch operations under a conveyer-belt concept. This was an approach which ~ proved to be cost effective and efficient enough to gain widespread acceptance. Batdunalyzers which were more successful include the Dupont "sample bag" analyzers and the parallel centrifugal analyzers. They are. however, expensive devices. and their use is rather limited. moreover, they are not designed to perform separations. An important breakthrough in labOratory automation was the introduction of continuous flow analysis by Skeggs in 1957[4]. With this system analytical chemistry was for the first time performed in conduits instead of discrete vessels, which greatly improved the efficiency of serial assays. After being marketed by Technicon under the trade name AutoAnalyzer. the system became the most widely accepted equipment for automated solution analysis before the advent of FlA. Yet the conventional concept of performing
2
I Introduction
I
AUTOMATED SOLUTION ANALYSIS
I
I
I
I Coutiuuous flow aualysers j
Batch ADalysers j Sequential diacrete (coni/flyer belt) aiUIIys•r•
~
-
Alr-•e11mented contlnuou• flow eiUIIyser•
-
Flow-Injection analyser•
Centrlluflal analyaers
Fig.l.l: Stages of development and classification of automated solution analysis. chemistry under equilibrium conditions was strictly adhered to in this system. as for discontinuous batch procedures. To achieve this. air bubbles were introduced into the flow to ensure homogeneous mixing, which constituted the most important feature of Skeggs "s system. i.e .. air-segmented continuous flow. This approach, although effective in serving its purpose of steady state reading under flow conditions. later confined the further development of continuous flow analysis. The requirement of achieving equilibrated conditions seriously restricted the efficiency of the technique. so that sampling frequencies were typically only in the range of 20-40 h- 1• A new stage of important development was initiated in the mid-seventies by substituting the segmented flow principle by the non-segmented principle of FIA, and abandoning the principle of steady state readout. Although non-segmented flow analysis systems were reponed even before the introduction of FlA. the breakthrough in renovation of concept was achieved mainly through the effons of Ruzicka and Hansen (I ,6 J through a series of publications initiated in 1975. At the end of 1990. after sixteen years of development. FIA has emerged as a new concept on solution manipulation in the analytical laboratory. with more than 3200 publications. According to the estimation of Ruzicka and Hansen [ 1]. during its first ten years of development. the total number of publications on the subject has achieved an average doubling time of about 1.3 years. which is a very remarkable rate of development for a specific analytical technique. In the last three or four years, with an increase in the total accumulated number of publications, the doubling time has gradual1y increased to approximately 4.8 years (Fig. 1.2) [5], but still is definitely one of the fastest developing techniques in modem analytical chemistry. FIA was soon found to be a powerful technique, not only for performing serial analysis but also for separation operations. In fact, on-line separation processes were incorporated into FI systems from the early days of development of FlA. The first
1.I Historical Perspectives
3
CUMUL. NO.
FIA PUBL 1QDOO
To •4.5 Y
1000
10
75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90
YEAR F"q:.l.l: Cumulative number of publications on AA since 1975. TD. doubling time of publications.
separation techniques to be used were based on dialysis and solvent extraction. followed by gas diffusion, ion-exchange and electro-deposition. The use of FI for preconcentration purposes and the use of on-line precipitation were introduced much later. but most of tbe separation techniques have made considerable progress since their introduction. FI separation and preconcentration have become a field which is attracting extensive interest, and is shown to be one which best demonstrates the dramatic effects of renovation of tr3ditional practice in the analytical laboratory. This is because traditional separation processes are usually tedious and time consuming. involving multiple stages of careful operation, with frequent transfers of solutions from one vessel to another, and thus ~t:posing the sample to serious contamination risks, particularly in trace analysis. The ~on sequence often forms the bottleneck of an analytical procedure. and therefore .::; coe which most urgently requires renovation.
4
I Introduction
1.2
Basic Principles of FIA
1.2.1
What Is FIA?
This question may appear superfluous for a book dedicated to a specific domain of
FlA. such as this one. The reason why a discussion on the definition of FIA is given here is due to the fact that recent developments in the technique seem to exceed the perimeters set by previous definitions; and it also appears that a better characterization of the essential features of FIA is necessary for full exploitation of its merits in on-line separation applications. · In the first edition of their well-known monograph. "Flow-Injection Analysis'', published in 1981, Ruzicka and Hansen [6] defined FIA as "A method based on injection of a liquid sample into a moving unsegmented continuous stream of a suitable liquid. The injected sample forms a zone, which is then transported toward a detector that continuously records the absorbance, electJl?de potential, or any other physical parameter, as it continuously changes as a result of the passage of sample material through the ftow cell", and the technique "is based on a combination of sample injection, controlled dispersion, and exact timing''- later often referred to as the three basic principles or cornerstones of FlA. The basic components of an FIA system described by this definition may be illustrated by a very simple schematic diagram (Fig. 1.3).
s
c
W·
R F'~g.l.3:
Scbematic diagram of buic PIA
system showing the various components. P. pmnp: S. sample injector; C. carrier; R.
p
reagent: M. mixing (reaction) coil: D. flowthrough detector: and W. waste.
Seven years later. in the second edition of their book, as the result of a better understanding of its great potentials, the definition has been revised to read: "Infonnationgathering from a concentration gradient formed from an injected, well-defined zone of a fluid, dispersed into a continuous unsegmented stream of a carrier", but the three cornerstones have remained unchanged [ 1]. Despite the broad scope covered by the revised definition, it still seems inadequate to incorporate all the variations of the technique appearing in related publications. Occasionally. one may notice violations of the definition by techniques rightfully classified as FIA by their authors.
12 Basic Principles of FIA
5
Tile at.rwr mair segmentation. and the injection of sample solution into a contin-
-ay ....... aream. resulting in a transient output signal. was once considered to be lk _ . • z 2
- live feature of FIA, however, a brief scan through the most recent FIA
-n.., .-ne
AA systems are segmented by gas segments (see hydride generation sys-
5
"*• reveals that its development has somewhat invalidated these distinctions.
lmiS in Chapter 5). some are discontinuous (see, for example, preconcentration systems for graphite furnace AAS in Chapter 4), and fluid zones containing the analyte are not
always well-defined in the sense that they may not be encompassed by clear boundaries wben Introduced (an example is the concentrate zone formed during elution of an adsorbed analyte from an on-line column). although the process may be perfectly repro....-;t+ IB spile of these dilemmas, two basic features of FIA remain unchanged, i.e.:
• •
Reproducible manipulation of sample and reagent zones through precise ummg. Quantitative evaluation of analyte concentration under thermodynamically nonequilibrated conditions.
These formed the basis for a revised definition of FlA. recently proposed by the Author r7J. i.e.: A non-chromatographic flow analysis technique for quantitative analysis, performed b)· rrproducihly manipulating sample and reagent zones in a flow stream under thermo~cal/y non-equilibrated conditions. 11lis definition differentiates FIA from segmented continuous flow analysis in the mode of manipulation of samples and/or reagents in fluid zones (the latter applies continuous sample introduction), but more importantly, in the non-equilibrium conditions of operation and measurement for FIA (the latter applies steady state equilibrium conditions for measurements). 1be definition also differentiates Fl techniques which implement packed columns for oo-line separations from chromatographic techniques by explicitly mentioning the nondlromatographic principle and purpose of AA. This will be discussed in more detail in Sec. 1..5. Despite all the efforts in precisely defining FlA. the matter still appears to be a source of considerable controversy. However, the development of AA will only be !'wtber stimulated by research activities guided under different ideologies .
. ... .,
Dispersion of Sample and Reagent Zones
The most imponant physical phenomenon in the manipulation of sample or reagent ._. zcoes in a non-segmented continuous flow, which is characteristic of at least a , _ fll at A systems, is the dispersion of the zones into a continuous stream, called a.t mrrwr. Wbcrl a sample zone is injected or introduced by other reproducible means ae IIIJe c::ln"Jer_ tbe latter may be either a non-reagent fluid, which simply transports the
6
I lntroduclioll
sample zone to a flow-through detector, or a reagent which, in addition to the transport function, later reacts with the sample to form a detectable or separable species. Less frequently, the reagent zone is injected into the carrier stream of a sample, which then takes up the function of transportation of the reacted zones (reversed FlA or rFIA). In all cases, the carrier acts as a diluent of the injected zone when they disperse iino each other during the transport. Dispersion between sample and reagent or diluent is not an exclusiVe process for FlA. since this will always happen in any batch analytical procedure or other flow analysis method when one attempts to homogenize a mixture of sample. reagent and diluent. The explicit difference in the FI dispersion process is that it is reproducible and controllable. The difference of the two dispersion processes is illustrated in Fig. 1.4 a and b. Obviously, the dispersion of a randomly introduced defined volume of reagent into a defined volume of sample in a container, as shown in Fig. 1.4 a is neither reproducible nor controllable. In order to obtain reproducible readout, one is obliged to homogenize the mixture of sample and reagent by shaking or stirring, and. wait for the reaction to achieve equilibrium. This latter measure is mandatory because the point of initiation of reaction is not well-defined and any attempt to reproducibly monitor the reaction before the achievement of thermodynamic equilibrium will inevitably fail. In the FI dispersion process the situation is completely different. As shown in Fig. 1.4 b the extent of dispersion of the injected zone into the carrier is determined by molecular diffusion. but mainly by convection due to differences in the flow velocity of fluid elements located at different points along the radial axis. With the thin conduits of 0.31.5 mm i.d. usually used in FIA systems. and in the absence of air segments. no random turbulence is produced. The mixing through convection can be made so reproducible that the longitudinal distribution profiles of analyte or reagent concentration in the conduit. monitored at the flow-cell of the detector against the time following injection I residence time) for a series of injections of the same solution may be precisely overlapped. Under such conditions. neither physical nor chemical equilibration is required for achieving reproducible readout at any preselected residence time.
1.2.3
The Evaluation of Dispersion
In FI systems the sample or reagent zones may be manipulated to produce a required degree of dispersion. The dispersion is therefore controllable, and may be designed to satisfy different analytical objectives. This calls for a quantitative evaluation of the dispersion. The dispersion coefficient D, proposed by Ruzicka and Hansen, is used most often for such evaluation [1,6]. and is simply the ratio of the concentration of the constituent of interest in a fluid element of the injected zone before and after the dispersion has taken place, expressed by:
1.2 Basi,· Principles
of FIA
7
6 F L
0
w
Fig.IA: Comparison of mixing and dispersion conditions in a. batch mode and b. flow-injection mode of operation.
co
D=C
( 1.1)
Where co is the original concentration of the constituent of interest in the solution to be injected , and C the concentration of that fluid element of the dispersed solution zone which is under consideration. When the fluid element with the highest concentration is concerned (i.e. readout at FI peak maximum), equation 1.1 is expressed as:
co cmax
D=--
(1.2)
8
I Introduction
Where emu is the concentration of the constituent at peak maximum of the dispersed zone. When fluid elements on the rising or falling slopes of the concentration gradient of the dispersed zone are concerned, the equation js expressed as: (1.3)
Where ('Vcld is the concentration of the constituent in that fluid element on the gradient of the FI peak which is under consideration. D is therefore dimensionless, and is a value greater than unity. the value reflecting the dilution factor of the fluid element being studied. FI systems with dispersion coefficients above 10 are classified as high dispersion systems. those between 2 and I 0, medium dispersion systems. while those below 2 are categorized as low dispersion systems. Although D values less than unity are sometimes used to describe preconcenttation systems where final concenttations of a constituent are higher than the original, this approach is not adopted in this book. The mechanisms of concentration increase in preconcenttation systems are quite different from those of sample dispersi~ and it may not seem appropriate to use the dispersion coefficient to describe the extent of preconcenttation. FI systems with medium and large dispersion coefficients are used to achieve sample dilutions, usually to bring the analyte concenttation into an appropriate range for readout. but medium dispersion systems are also used in single channel FI systems, where reagents are used as carrier streams, to attain adequate mixing of sample and reagent. Low dispersion systems are used whenever one intends to prevent the original concenttation of the analyte in the injected fluid zone from being diluted by the carrier. Such systems are therefore most important for preconcenttation purposes. where highest sensitivity enhancement effects are always pursued.
1.2.4
Factors Influencing Dispersion
The FI experimental parameters or factors which may influence dispersion include: • • • • • •
sample volume carrier flow-rate flow-rate ratio between sample carrier and meJging reagent geomebical dimensions and configurations of manifold components viscosity of the fluids temperature
Under normal conditions, the last two factors have very limited effect on the dispersion, and in most cases may be neglected.
1.2 Basic Principles of FIA
9
Although it has been stated as a rule by Ruzicka and Hansen that dispersion diminishes with a decrease in Aow-rate in a narrow tube [ 1,6], this statement holds true only at extremely low flow-rates where molecular diffusion rates approach the rate of convection craled by the flow. Karlberg and Pacey have shown experimentally that with a fixed manifold, the flow-rate have very little influence on the dispersion within a broad range of flow-rates (1.6-4.0 ml min- 1), used most frequently in FIA [2]. As expected, the effect of carrier/reagent (C / R) flow ratio on dispersion is much more pronounced, and the dilution effect from this facror may be predicted fairly well from the flow ratio itself. Thus a flow ratio of 1 implies a 1:1 dilution of the injected zone after the merging point of carrier and reagent. Since the ratio of the total flow to carrier flow (C +R)/C in this case equals to 2, the dispersion coefficient is increased by a factor of 2 .. The influence of the volume of the injected zone (often the sample volume) on dispersion is one which has been studied most frequently. and the decrease of dispersion by increasin_g tbe ipjected sample volume is well documented. However. this statement is only valid below a certain volume, mainly determined by the geometrical features of the manifold. With most FI systems, the upper limit of sample volume which may be effectively used for manipulating dispersion is in the range 100-200 Jll. Nevertheless, larger sample volumes may be used with some sacrifice in sampling frequency, when the smallest dispersion or highest sensitivity is desired. The lower applicable limit of sample volume is determined by the construction of the injector, but usually will be within the range of 10-30 JJL Varying the sample volume is a very convenient way of manipulating the dispersion. However. the dispersion coefficients cannot be expected to be varied by more than a factor of 3-4. The effects of geometrical features of the FI manifold on dispersion are more complicated. and deserve a more detailed treatment. The influence of the length of the conduit (distance travelled) on dispersion has also been expressed as a rule by Ruzicka and Hansen, stating that '"the dispersion of the sample zone increases with the square root of the distance travelled through an open narrow tube" [ 1.6]. However, this is valid only when the conduits are straight. In the case of coiled conduits. which are often used in FI to save space as well as to ensure tidiness and system stability. the contribution of coil length to dispersion is less pronounced. This is due to the development of secondary flows which promote radial dispersion and limit the axial dispersion. When the tubes are knotted or knitted to produce a three-dimensionally disoriented flow, the radial secondary flows are further enhanced (cf. Sec. 2.3.2), and fluid zones may travel for long distances without noticeably increasing dispersion. Knotted or knitted reactors are therefore very useful in prolonging the residence time of an injected zone without appreciably increasing the dilution from the carrier. In a study on the influence of the inner diameter of the conduits on dispersion. Karlberg and Pacey [2] concluded that within a practical range of 0.35--0.9 mm i.d. tbe dispersion contribution is nof critical, the experiment producing only a variation within 3.0-3.8 in dispersion coefficient. However, the manifold used in the study was
10
1 IntroductiOn
a medium dispersion system; when working with a low dispersion FI system connected to an AAS detector, the Author and co-workers [8] have shown that the inner diameter of conduits may contribute more to the dispersion. With a straight conduit of 15 emlong, D increased from 1.02, using a 0.35 mm i.d. conduit, to 1.41, using a 1.05 mm i.d. conduit, when injecting 65 J.Ll of sample. This implies a 30 % decrease in the analyte signal. Therefore, the contribution of tube inner diameter to dispersion should not be overlooked, particularly in preconcentration systems, which are always, at least partially, low dispersion systems. A precaution which should be taken in this context is that one should avoid unintentional local broadening of the conduits through carelessly made tube connections (cf. Fig. 2.9). The broad sections between non-flushed ends act as small mixing chambers which may significantly increase dispersion.
1.3
General Characteristics of FI Methods for Separation and Preconcentration
FI methods of separation and preconcentration exhibit some extremely favourable features over their batch or even continuous flow counterparts. The general merits sum~ marized below are-valid, irrespective of the separation principles and equipment involved: •
• •
• • • • • •
High sample throughput, 1-2 of:ders of magnitude higher than batch procedures, with shon operation times typically in the range of 10-200 s per determination (including separations). High enrichment efficiencies for preconcentration systems, typically a factor of 5-50 higher than batch procedures. Low sample consumption, 1-2 orders of magnitude lower than batch procedures. This is a feature particularly imponant for valuable samples. such as blood. or for samples which have to be transponed to the laboratory from distant collection sites. Low reagent consumption, 1-2 orders of magnitude lower than batch procedures. This is a feature particularly imponant when expensive reagents are used. High reproducibility, typically in the range 1-3% r.s.d .. Simple automated operation which allows implementation with continuous monitoring systems and use in process control. Low contamination risks owing to closed and inen separation systems used. a feature particularly imponant for trace analysis. Possible enhancement in selectivity by applying kinetic discrimination. Very limited laboratory bench space and utensils required. Excluding the detector, sometimes a Fl on-line separation/preconcentration system occupies no more space than an electronic typewriter.
1.4
Fundom~ntal
Aspects of FJ Separation and
Preconc~ntrmion
II
A feature which may be considered as a drawback of Fl separation prqcedures is the incompleteness of mass transfer between phases, which is usually unacceptable in batch separation methods. However, the incompleteness of physical and chemical processes is not a feature which solely belongs to FI separation systems, but is common to all FI systems, except those which apply instantaneous reactions. Since FI is basically ·a technique for the reproducible monitoring of analytical signals under thermodynamically non-c:quilibrated conditions (cf. Sec.l.2). this does not deteriorate the precision or sensitivity of a properly designed and calibrated FI system. including those used for separations. On the contrary, the highly reproducible non-equilibrium conditions are the very basis for fast operations and selectivity enhancements through kinetic discrimination. Nevertheless, this feature has to be kept in mind in designing and optimizing an FI separatiort sys1em, and may become an important source of enor in the analysis of real samples when identical equilibrium conditions cannot be reached for standards and samples.
1.4
Fundamental Aspects of FI Separation and Preconcentration
1.4.1
Classification of FI Separation Techniques
FI separation techniques may be classified according to the type of interface across which mass transfer takes place. Hitherto, the t)1'C of separations reported include: liquid-liquid solvent extraction dialysis gas diffusion liquid-gas hydride generation cold vapour generatton liquid-solid ion-exchange adsorption sorbent extraction precipitation-dissolution coprecipitation-dissolution electrodeposition-stripping The chapters in this book are divided mainly according to this classification. Most techniques may also be classified according to the medium used for separation. e.g. membrane techniques, colunm techniques and filtration techniques. Dialysis, gas diffusion and some solvent extraction and hydride generation methods belong to the first category, while sorption methods based on adsorption, ion-exchange and sorbent extraction belong to the second. Techniques using similar separation media share some common principles in the design of separators and operations. The reader is therefore advised to refer to the related chapters for more information when reading a specific chapter with materials presented according to the first scheme of classification.
12
1 I niJ"OdMction
Separation methods and systems may also be classified according to their objectives for separation. Separations may be effected to overcome interferences to improve the selectivity, or to enhance the sensitivity through a preconcentration process, or both. In this book those belonging to the first category will be referred to as separation methods and systems, and those belonging to the other two, preconcentration methods and systems.
1.4.2
Enrichment Factor (EF)
The enrichment factor, abbreviated EF, is a criterion used most often for evaluating preconcentration systems [9]. Despite its frequent usage, the precise meaning of the term is not well-defined. Theoretically, the term is meant to be the ratio between the analyte concentration in the concentrate, C,, and in the original sample, Cs. i.e.,
EF= Ce Cs
(1.4)
In practice, the evaluation of EF is not as simple and straightforward as it seems, because the actual analyte concentration in the concentrate, C,, is usually unknown. Therefore, an approximation of EF is usually accepted by defining EF as the ratio of the slopes of the linear section of the calibration curves before and after the preconcentration, all based on the detector responses to Cs· The evaluation is therefore based on response enhancements, and not on the actual concentration increases. Nevertheless, the EF values deduced will be close to the true value if the analytical conditions, including detector response characteristics. remain generally unchanged for the two calibrations. In order to achieve high sensitivities, in FI preconcentration systems the readout is almost always made at the FI peak maximum, i.e., by evaluating the peak height. Enrichment factors are sometimes deduced simply by comparing the peak heights before and after the preconcentration. A disadvantage of such direct comparisons is that usually the analyte signals before preconcentration are extremely low (otherwise a preconcentration would not be necessary). This mode of evaluation is subject to large errors. and therefore is not recommended.
1.4.3
Enhancement Factor (N)
In FI preconcentration systems, sometimes the analyte signals are enhanced during the introduction of the concentrate into the detector through mechanisms other than increase in concentration of analyte, such as enhancement through organic solvent effect in flame AAS when organic solvents are used as the concentrate media. The analyte signals may also be influenced by variations in detector response characteristics of the
I .4 Fundamental Aspects of Fl Separation and Preconceruration
13
Fl preconcentration system compared to one without preconcentration. For example. the response of a flame AAS detector may be influenced by a change in the solution introduction rate (cf. Sec. 2.4.2, equation 2.1 ). These effects should be differentiated from enrichment effects in order to obtain a valid evaluation of the preconcentration performance. This may be realized by separately detennining the enhancement factor under similar operational conditions but without preconcentration. In this book the two factors will be differentiated whenever possible, otherwise the total enhancement factor. expressed as N, will be used instead of EF. Fang et ai.[IO] have shown that when sensitivity enhancement factors exist, other than an increase in concentration of the analyte in solution, the enhancement effects will be multiplicative on EF. Provided that different factors have independent enhancement mechanisms, the total enhancement factor N, will· be the product of the individual enhancement factors, N~o N2, .. .N,, and the enrichment factor EF. i.e.,
N, =N1 ·N2 · ... ·N, ·EF
(1.5)
Note that the N values may not always be larger than unity, since some variations in analytical conditions may have negative effects on the sensitivity.
1.4.4
Concentration Efficiency (C£)
One of the main reasons for the implementation of Fl preconcentration systems is their high efficiencies compared to batch systems. Although EF is indispensable for the evaluation of preconcentration systems, when used exclusively, it does not provide adequale information on their efficiencies. High enrichment factors are not necessarily associated with high efficiencies, as these may be achieved at the expense of long preconcentration periods of hours or even days, consuming liters of sample. In a study on ion-exchange column preconcentration for flame AAS, Fang et al.[9) have suggested the use of concentratioa efficiency (C£) for the evaluation and comparison of the efficiency of various systems. CE is defined as the product of the enrichment factor EF and the sampling frequency in number of samples analyzed per minute, expressed in min -•. lberefore, iff is the sampling frequency expressed in samples analyzed per hour, CE = EF ·if/fJJ)
(1.6)
1be value dictates the factor of enrichment of an analyte achieved by the system in one minute. In this book the use of this concept is extended to all preconcentration systems, irrespective of the principle of separation. This makes it possible to use CE values not only as a criterion for refining the designs of preconcentration manifold systems but also for comparison of the efficiencies of preconcentration procedures based on different separation principles.
14
1.4.5
I lmroduction
Consumptive Index (C/)
The consumptive index. C/ reflects another aspect of preconcentration system efficiency. i.e.• efficiency of sample utilization. This concept. recently proposed by the Author [11], is defined as the volume of sample, in milliliters, consumed to achieve a unit EF. and is expressed by the equation:
Cl = .!::!_
EF
(1.7)
where Vs is the volume of sample consumed to achieve the EF value. Efficiency in this sense may be very important when the analysis is sample limited, as for body fluid analysis, or when large numbers of samples have to be collected and transported to distant laboratories.
1.4.6
Phase Transfer Factor (P)
In FI preconcentration methods, quite often the analyte in a sample may not be completely transferred into the concentrate due to insufficient equilibrium time, and sometimes because of inadequate capacity of the collection medium (e.g. sorption column capacity). In the first category, the loss of analyte does not always deteriorate the enrichment factor. since the loss might be compensated for by an increase in sample loading rate. It does not imply a serious loss in precision either, since the losses may be very reproducible for both sample and standard. However, under such conditions, as well as when breaking-through of analyte occurs due to insufficient capacity of the collection medium, the results are more likely to be affected by matrix effects and interferences from competing species. Therefore, this is an important aspect which should also be evaluated together with the other criteria. The completeness of analyte transfer from the sample phase into the concentrate phase. either directly. as in solvent extraction or gas diffusion methods, or indirectly through a collection medium, as with sorption columns or coprecipitation, may be quantified by the phase transfer factor P [ 10], defined as the ratio between the analyte mass in the original sample, ms. and that in the concentrate m~. P= me
m.,
( 1.8)
In column preconcentration systems P is sometimes referred to as retention efficiency (%E), defined by Hartenstein et al.[l2] as the analyte percentage in a sample recovered from a column following sorption and elution. In this book, onJy P will be used to describe the completeness of analyte transfer. since it may be used in a more general sense.
1..J Fundamental Aspects of Fl Separation and Preconcenrration
1.4.7
15
Time-based and Volume-based Sample Loading in FJ Preconcentration Systems
In Fl preconcentration systems the sample volume being processed may be determined either by fixing the time interval for sample introduction (loading) into the preconcentration system. under a defined sample flow-rate. or by using a carrier stream to displace a fixed sample volume. defined by the volume of a sample loop. The former approach is referred to as time-based sampling. and the latter. volume-based sampling. Schematic diagrams of systems adopting the two different approaches are shown in Fig. 2.5 a and b. Although both approaches have been used successfully in a number of publications. time-based sampling systems are more straightforward to operate. since they obviate the need for preliminary loading of samples into a loop, and the subsequent displacement of the sample by a carrier stream. Consequently, they are also generally more efficient than volume-based systems. They are. of course. more dependent on flow stabilities of the pump systems. High quality pumps are therefore required, panicularly for systems incorporating packed columns or filters which create large impedances in the flow. In order to ensure flow stability. the samples always have to be pumped into. and not drawn into the preconcentration system in this mode of operation. In this context. volume-based sampling is much less dependent on flow stability. provided one makes cenain that the sample loops are completely filled with the sample solution before introduction into the preconcentration system. The sample may be drawn or sucked into the loop by the pump located downstream. which has the advantage of avoiding risks of cross contamination in the pump.
1.4.8
The Evaluation of FI Preconcentration System Efficiency
It has been shown recently that a reasonably close prediction on the achievable limit of sensitivity enhancement effects and efficiencies of FI preconcentration systems may be derived from known experimental data and parameters based on a mathematical model used for Fl-flame AAS systems [ 10]. Such predictions are shown to be very useful in setting up a target for funher improvement. and may be used to locate the weaker links in the design of a preconcentration system. Here the model is subjected to a more general treatment to include FI preconcentration systems using other flow-through detectors. In batch preconcentration procedures, the concentrates are almost always homogeneous. Assuming a complete transfer of the analyte mass ms from sample phase to the concentrate phase, the analyte concentration before the preconcentration (in the original sample), C5 , and after the preconcentration (in the concentrate), Cr. will be msfVs and msfVr. respectively. The ratio of the two volumes. V.IV... which is tenned the phase ratio, will be equivalent to the enrichment factor EF. expressed as C.,/C,.
16
1 introduction
When the analyte transfer from sample to concentrate is incomplete, i.e .• the phase transfer factor P is less than unity.
EF= Cr Cs
= PVs V,
(1.9)
This equation cannot be applied to most FI preconcentration systems because the distribution of analyte in different fractions of the concentrate is usually not uniform as in batch procedures. where fractions containing different concentrations of the analyte are almost always homogenized and made up to a predetermined volume before obtaining a steady-state readout in the final assay. In Fl. the concentration gradient is reproducibly preserved in on-line introduction of the concentrate, and monitored as such by the detector in the form of a transient signal. This feature of FI preconcentration systems is an important benefit of the approach. as higher EF may be achieved by monitoring the fraction of concentrate which contains the highest concentration of the analyte, using peak evaluation. However. it also complicates the prediction of EF in a FI preconcentration system, since the peak shape reflecting the distribution of the analyte in the concentrate depends on many factors. including: • • • •
the geometric design and dimensions of components of the FI preconcentration system the kinetic characteristics of the chemical and/or physical processes involved the phase transfer mode the flow-rate used for formation and introduction of the concentrate
Since quantifying such factors would be extremely difficult, an estimation of the peak concentration was attempted based on the volume of solvent used to complete the phase transfer and analyte transport into the detector [ 10]. a parameter which of course is closely related with all the previously-mentioned factors. The width at the peak base of a Gaussian peak is 4 u, u being the variance of the peak. Considering that FI peaks in preconcentration systems are almost never symmetrical, but rather often include a long tailing, the width at peak base was taken as 5 u or 2.1W. W being the half peak width (W 2.435 u). The volume of ·concentrate was estimated, based on the time between peak rise and falling to baseline, defined by 2.1 W, and symbolized by r,, expressed in minutes or seconds. The volume of concentrate V, was then derived from its flow-rate q, as V, = qjr,. Assuming a homogeneous solution, in a graphical presentation describing C, as a function of V (volume of concentrate processed), C, is constant, the total analyte mass in the concentrate which is introduced into the detector therefore may be presented by the area within the rectangle formed with V, and C, (Fig. 1.5). The introduction of the concentrate which conunns a continuously changing concentration gradient of the analyte. produces a peak with V, as base. A close simulation of the peak form was made using a triangle with V, as base and peak maximum as the apex. If C,. is the average concentration of the analyte in the concentrate, i.e., the concentration in a homogeneous solution, then a triangle with a height of 2C,
=
1.4 Fundamental Aspcrts of Fl Separation and Preconcentration
I7
v Fig.l.S: Schematic diagram showing relationship between concentrate volume V,. and pea}; concentration of analyte in the concentrate. volume: concentration: W, half pea}; width. Shaded area refers to analyte mass in homogeneous concentrate with volume V,. and concentration C,. Solid line describes the profile of a typical elution or dissolution pea}; with half peak width W. Broken line is the triangle formed with V,. as base and having the same area as shaded rectangle [ 10]. (Reproduced by permission of Royal Society of Chemistry)
v,
c,
covers the same area (analyte mass) as the rectangle. A reasonably close estimation of the analyte concentration and EF at the peak therefore was made by multiplying the values oblained under a homogeneous assumption by a factor of two. resulting in the
following equalion: EF= 2PV,
V,.
(1.10)
Since for time based sampling it is more convenient to express the sample and concentrate volumes in terms of time and flow-rates. i.e., V,. = qt,. and v. = Q,t,. Q., being the sample introduction rate and 1., introduction time in seconds, equation L 10 may be expressed as
EF= 2PQs. t, qt,.
(1.111
This equation is valid only when the amount of sample processed is directly related the sample loading time, which occurs when the system is completely chemically equilibrated to conditions optimized for the transfer before. the initiation of the preconcentration process. When an equilibration step is omitted to improve the efficiency, a to
18
I Introduction
correction factor z•. is deducted from the sample loading time. t... corresponds to the time used by the sample solution for equilibration, during which very low amounts of analyte, if any, are collected. t•. is evaluated by extrapolating an analyte response-~, graph to z.ero. Equation 1.11 is then modified to give: EF
= 2PQs (Is- lw) qt,
(1.12)
When other sensitivity enhancement factors with independent mechanisms are taken into consideration, it follows from equations 1.5 and 1.12 that:
N = 2N1N2 ... NnPQs · (Is - lw) qt,
(1.13)
When EF in equation 1.4 is substituted by the relationship in equation 1.7 , and V,, by Q,t,
Cl = t,q 2P
1.5
(1.14)
Liquid Chromatographic and FI Separation Methods
Many similarities in equipment and methodology exist between HPLC and FI separation methods, particularly those involving column separation techniques, and occasionally, some confusion in classification does occur in published literature. However, the objectives, underlying principles and technical requirements in hardware for HPLC and FIA are quite different. Although the two techniques have much to borrow from each other, they should rightfully belong to two different disciplines. When FI techniques are used for separation. the objective is to separate a single analyte or group of analytes from interfering sample components or matrices. often simultaneously achieving some degree of preconcentration, whereas HPLC is used for the separation between multi-components in a sample. The FI separation process is quite similar to batch filtration or solvent extraction procedures. and no chromatographic processes are involved. This is also true for FI separation systems incorporating on-line columns. The analytes are sorbed on the columns, analogous to collection of precipitates on a filter, and during this process the interfering sample components are allowed to pass to waste. Later on. the collected analytes are completely eluted and determined on-line with a suitable detector. with no concern in the chromatographic separation properties
I .5 Liquid Chromatographic and FJ Separation Methods
19
of the column. Often the strongest applicable eluent is chosen for the elution to obtain shon elution times and high enrichment factors. 'lllenoodynamic equilibration is an imponant factor for achieving highly efficient chromatographic separations, whereas Fl separations are almost always performed under non-equilibrated conditions in order to achieve optimum performance. Thus, in Fl separation procedures, completeness of the separation reaction. and therefore of analyte recovery are not pursued. This will not deteriorate the precision or accuracy of the results as long as the losses are reproducible and the systems are properly calibrated. The differences in objective and principle between HPLC and Fl separations bring major dissimilarities in the related equipments. The Fl flow systems produce much less flow impedance during operation. even when packed columns are implemented. Columns are tnuch shoner. and packing materials are much coarser when they are used to serve Fl separation purposes. This in tum substantially lowers the requirement to withstand high pressures for the valves and pumps of a Fl system, so that high pressure pumps such as those used in HPLC are not necessary. On the other hand, the high versatility of Fl often demands multi-functional valves, and multi-channel pumps. These differences may be better understood after comparing the related instrumentation described in Chapter 2 to standard HPLC equipment.
2
General Instrumentation
Only general purpose equipment used in FI systems is described in this chapter, together with its proper usage, particularly in separation and preconcentration applications. Included are solution delivery (propulsion) devices, injection and multi-functional valves, transpon and ·mixing systems, and the most frequently used detectors. Devices used for specific separation purposes are described in the corresponding chapters on individual separation techniques.
2.1
Liquid Delivery Devices
2.1.1
Genera]
The liquid delivery or propulsion device is a basic piece of equipment in all flow analysis systems. However, certain characteristics of FIA call for specific requirements in its propulsion systems. FI systems are in essence low pressure systems. with seldom more than a few bars pressure developed in the conduits. Therefore, expensive high pressure liquid delivery devices such as those used for HPLC are not necessary. On the other hand. FIA is a technique based on highly reproducible timing,- a feature which demands pulseless and reproducible flow-rates in liquid delivery. The high versatility of FIA also demands easily manageable propulsion devices which will not depreciate the flexibility of the technique. HPLC pumps, expensive as they are, do not always meet all these requirements. The main features of an ideal liquid delivery device for FIA may perhaps be summarized as follows: • • • •
Reproducible flow-rates both on a shon tenn (hours), and a long tenn (days) basis. Multi-<:hannel capability, providing at least four parallel pumping channels to eoswe high versatility. Pulse-free fluid delivery. Resistance to strong reagents and solvents.
22
2 General lnstrumenlation
• •
Flow-rates readily adjustable. Low initial invesbnent and running cost.
Unfonunately, none of the presently available liquid delivery devices can meet all these requirements. Those described in this chapter are the ones whose performance matches closest with the requirements of FI systems, particularly for separation purposes, and are used most broadly in such applications.
2.1.2
Peristaltic Pumps
General Characteristic.f of the Peristaltic Pump Owing to its high versatility. the peristaltic pump is undoubtedly the most often used liquid propulsion device in FlA. The main disao\tal'n4ges of such devices seem to be their flow pulsation. a lack in long term flow-rate :stability.'tlnd low resistance (of the pump tubes) to organic solvents and high concentrauons of strong acids. However. when used properly. most of the drawbacks of the penstaJUc pumv t:an often. though not always, be overcome. Pulsation seldom poses a problem in FI separation and/or preconcentration systems in which packed columns or long reactors are arranged on-line. or where gas-liquid separators are used. On-line separators often function as an effective pulse-damping system, so that pulsations are noticeable only when pump rotation speeds are extremely low, a condition which in any case should be avoided in FI operations. Long-term drifts in the flow-rate of peristaltic pumps are unavoidable, owing to the gradual aging of plastic pump tubes. Nevenheless, good precision can be ensured with frequent calibration. which is usually readily accomplished in FI procedures. It is also good practice to regularly check the more critical flow-rates (such as the sampling flowrate in time-based sampling preconcentration systems) every 2-3 days, or bener still, everyday. When noticed, the flow-rate drifts arising from variations in the propenies of the pump tubes may be readily compensated by changing the pump rotation speed accordingly. When fixed rotation speed pumps are used, the effects caused by the drift often may be compensated for by adjusting other experimental parameters. Thus, variations in sample volume caused by a drift in sampling flow-rate may be compensated by an increase in sampling time for time-based preconcentration systems.
Delivery of Organic Solvents Using Peristaltic Pumps Organic solvents are used quite frequently in FI separation systems, but most of them cannot be pumped through the tygon pump tubes used most often with peristaltic pumps, at least not for extended periods. Several choices of solvent resistant pump tubes, such as the Solvaflex, Verdoprene, Neoprene, Marprene and Viton pump tubes, are available, and
2.1 Liquid Delivery Devices
23
AQ
OR OR
AQ
a
b
Fig.2.1: Displacement bottle for organic solvent delivery. a, organic solvent density higher than water. b, organic solvent densicy.lower than water. B, glass bottle; C, bottle cap furnished with liquid flow inlet and outlet and gas outlet (not shown); OR. organic phase; AQ. aqueous phase; PTFE tubing extending to bottom of bottle.
may be used to pump a certain range of solvents; but none of the materials are usable for all common st>lvents. A list of the applicable solvents for each pump tube species may be obtained from the manufacturers. Solvent-proof pump tubes are. however, usually much more expensive, although some claim a longer tube lifetime. This would appear to be a serious drawback of the peristaltic pump in many separation applications. However. a cheap and practical solution for the propulsion of organic solvents which are immiscible with water is to use a solvent displacement bottle. A schematic diagram of a typical displacement bottle is shown in Fig. 2.1. This is simply a thick-walled glass reagent bottle, sealed with a threaded cap made of solvent resistant plastic. The cap is furnished with an inlet and outlet. through which PTFE tubes are fixed via threaded connectors. and also an outlet to facilitate the release of air bubbles. The bottle is first filled with the organic solvent. and securely capped. with the air release outlet open. Water is pumped through the inlet using normal pump tubes. with the inlettube reaching to the bottom of the bottle and the outlet tube for the solvent extending shortly into the bottle when the water forms the lower phase. and vice versa when the solvent forms the lower phase. Pumping is continued until ·all the air in the bottle is expelled from an air release outlet. The outlet is then blocked with a blind threaded fining. When water is pumped further into the bottle. the organic solvent is displaced at identical flow-rate through the outlet Any accumulation of air bubbles in the bottle due to accidental introduction of air or to the use of undegassed solvents may lead to an enhancement in flow pulsation and a deterioration in flow stability. It is therefore a wise policy to often check the upper part of the displacement bottle for air bubbles, and release them before a degradation of performance occurs. For trouble-free operation, it is also good practice to degas beforehand lhe solvent and water used for the displacement.
~
24
Grnerallnstrummtaticm
Propa Usa~e of Peristaltic PumfJ.\'
Despite the basic nature of its function in most Fl systems. users of peristaltic pumps often do not operate them properly. including people who have used them for years. Since its proper usage is of primary importance. particularly in many on-line separation systems where flow impedances are often significantly higher than normal FIA systems, it may be necessary to stress the important points here. and provide some useful hints for their correct manipulation. a
The performance of the pump depends strongly on the pressure of the compression cam or band which presses the pump tubes against the rollers. This pressure is usually controlled by a spring loaded screw which can be adjusted by the operator. A continuous delivery of liquid do not always indicate stable and reproducible flow. and some experience is needed to make the correct adjustment. A recommended procedure of setting the pressure is as follows: (i)
(ii)
b
Connect the pump tubes to the FI manifold to be used, with the valve turned to a position where downstream flow resistance is highest. and engage the pressure band or cam. Stan the pump and release the pressure of the cam or band until the flows in all the pump tubes stop. Gradually increase the pressure until continuously flowing streams are obtained for all the channels, and further tighten the pressure·setting screw one extra tum.
The pressure on the pump tubes should be readjusted when the type or number of pump tubes or the flow manifolds are changed. Periodical readjustments should also be made when pump tubes gradually age under usage. Note that pressure adjustments optimized for open ended pump tubes (i.e. without load) may have to be readj~sted when the pump tubes are later connected to a manifold, particularly when the flo·w impedance of the manifold is high. as when packed columns are used in the system. Some operators tend to overtighten the screws to make sure that the flows will not be influenced by a change in impedance. However. this may significantly shorten the lifetime of the pump tubes as well as deteriorate the reproducibility of the flow due to partial or complete adhesion of the inner walls of the pump tubes under excessive pressures. In order to ellttend the pump tube lifetime, as well ao; to obtain best performance of the pump. the pump tubes should always be lubricated usin~ a small amount of silicone oil. Moreover. the pressure on the pump tubes should be released by disengaging the cam or band whenever the pwnp is not in use for relatively loog periods. e.g.. during coffee or lunch breaks. Pump tubes should be rinsed with water at the end of a working day.
2.1 Liquid Delivery Devices
c
d
e
f
25
New and used pump tubes. or pump tubes of different materials or brands should not be used together when a common cam or band is used to exen the pressure. It is rarely possible to optimize the pressure for all the pump tubes simultaneously. Such precautions are not necessary when separate cams are used for each individual pump tube. The flow-rate of a pump channel should only be varied by changing the pump tube or the rotation speed of the pump. One should never attempt to vary the flow-rate by adjusting the pressure screw. Although releasing the screw with a deviation from optimum pressure may lower the flow-rate, such flows are extremely sensitive to impedance variations in the flow conduits. and are neither stable nor reproducible. Commercial pump tubes are always colour-coded on their collars (used to keep the ·tubes in position during propulsion) to facilitate identification of flowrates. However, owing to difference:- in the quality of the plastic materials. the same colour-coding may produce substantially different flow-rates with different brands of a specific species (e.g. PVC) of pump tube, even when the same pump and rotation speeds are used. The actual flow-rate may also differ from the nominal flow-rate specified by the manufacturer due to the relatively large flow impedance created in Fl systems with packed columns or long. thin reaction Jines, as already mentioned in (a). It is therefore a wise policy to take the colour-coding only as a rough guide. and make actual measurements of the flow-rate for each channel. Pulsation is a common demerit for all peristaltic pumps. However, pulsations may be damped to a negligible degree by lubrication of the pump tubes, by correct adjustment of the compression cams or bands, and by using sufficiently high rotation speeds. For systems with high flow impedance, shortening the pump tubes (particularly with larger bores) may also help to decrease pulsations through suppression of the "bellows effect". Although air-filled reservoirs are sometimes used as pulse-dampeners. and may be effective in absorbing the pulsation to some extent, such devices cannot be generally recommended. When the flow impedances are different in the two valve positions (as in systems using packed columns as a special form of sample loop) the pulse dampener act!. as a reservoir to partially store the flow stream in the position with higher impedance, and decreases the flo\\. In the second position, with a decrease in impedance, a momentary surge in the flow-rate will occur when the stored fluid is released from the reservoir. Such major flow fluctuations may be the source of a deterioration in precision.
2 G~n~rallnstrutMmarion
26
2.1.3
Reciprocating Piston Pumps
Reciprocating piston pumps similar to those used in HPLC, but with much lower maximum working pressures of 30-:60 bars, have also been used successfully for Fl separation purposes, mainly by Japanese workers. Such pumps have several favorable features compared to peristaltic pumps, particularly for on-line separation purposes. These include: • • •
Better flow stability. both on a short term and-particularly on a long term basis. Capability of pumping against high impedance created by, e.g.• packed columns or on-line filters without disturbing the flow stability. Strong resistance to organic solvents, often used in on-line separation systems.
However, an important demerit of piston pumps, which limits their versatility, is the small number of available channels (usually two). A piston pump with an equal number of channels as an eight-channel peristaltic pump would be much more expensive and bulkier than the latter. Piston pumps are therefore often supplemented by peristaltic pumps. using the latter for purposes where flow-rate stabilities are less critical, such as filling samples into a sample loop. Another shortcoming of the piston pump, specific for atomic spectrometric detection systems which are equipped with pneumatic nebulizers, is its disability in working against suction. When the pump rates are lower than the uptake rates set for the nebulizer, the suction from the nebulizer deactivates the check valves of the pump. and the pump will lose control of the flow. The pump then behaves as if it were an open tube, the flowrate of the liquid passing through it being determined by the intensity of the nebulizer suction. The piston pump is therefore limited only to applications where the pumping rates are higher than the free aspiration rates of the nebulizer. A typical example of a piston pump suitable for Fl applications is the Japanese Sanuki DMX-2300-T all solvent reciprocating pump which has a plunger diameter of 2.5 mm and a stroke length of only lmm (stroke volume 4.9 J.tl). The two channels of the pump work intermittently. compensating each other to give a low pulsation stable flow. When one channel propels the carrier stream, and the other, a reagent, the two solutions are introduced alternately in small increments of 4.9 JJ.l, which are readily and reproducibly mixed during transport to the detector. High precisions of 0.07% r.s.d. on a shon term basis. and 0.18% r.s.d. on a long term basis have been obtained in our laboratory. using such a pump to inject a dye solution detected by a photometric system.
2.1 Liquid Delivery Devices
2.1.4
27
Syringe Pumps
Hitherto syringe pumps have not been used extensively in FI systems; however, a recently developed sinusoidal flow syringe pump designed by Ruzicka et al. [I] has shown promise in a number of applications. and will be described here briefly. A schematic diagram of the pump is shown in Fig.2.2. The movement of the piston of a disposable plastic syringe is controlled by the rotation of a cam, resulting in a sinusoidal flow-rate - time pattern for the fluid delivered from the syringe. Such flow-rate patterns, though non-linear, are completely pulseless. and can be made highly reproducible within each cycle, when the sector of cam movement is fixed by means of micro-switches and computer control. An important feature of the pump is the substitution of check valves by computer controlled multi-channel Fl valves, which improves the reliability and versatility of the system. Synchronous action of the syringe piston and the valve allows the carrier and sample to be loaded into the syringe in one half cycle, and delivered into the reaction and detection system in the second half. The advantages of such a liquid delivery system are: • • • •
Pulseless flow delivery over a wide flow-rate range. highly reproducible both on short and long tenn basis. Strong resistance to concentrated acids and bases, and organic solvents. Flow-rates unaffected by relatively large impedance variat; ..ms in the flow system. Low running cost compared to peristaltic pumps, because no pump tubes are consumed.
The shortcomings are:
•
Decreased sampling frequencies due to the requirement of a carrier loading stage.
•
Disability in perfonning time-based sampling in various preconcentration systems Chapter 4 ). Reduced versatility compared to peristaltic pumps, owing to the small number of available channels, and restrictions in flow-rate ratios of the channels, which can only be changed by varying the barrel size of one of the syringes. Possible deterioration of flow-rate reproducibility when used with atomic spectrometric systems with pneumatic nebulizers [2). The flow pattern may be noticeably disturbed by the suction of the nebulizer. particularly during the flow reversal stage at each half cycle. 1see
•
•
28
2 General lnstrumelllation Lood .,.,.
I
I I
------.---------------1 I
' I
Reagent ayrlnge
( llooour••••• .,.,.)
1 I
Sample ayrlnge
------.-----.----------.
Reagent ayrlnge
Sample reaervolr
Fig.2.2: Schematic diagram of the operation principle of a sinusoidal flow pump using an 8-pon valve. L. reaction coil; W, waste; C. computer; and D, detector [1].
2.2 Injection and
Multi~functional
2.2
Injection and Multi-functional Valves
2.2.1
General
\-all'l'J
29
A large number of different designs of valves for performing sample injection and/or other flow functions have been described in the Fl bibliography. some borrowed from related fields such as liquid chromatography. However, the requirements of Fl valves are quite different from valves used in HPLC. It is not necessary for Fl valves to withstand extremely high pressures as for HPLC, but Fl valves are often required to perform simultaneously other functions in addition to sample injection, particularly in separation system!.. and they are actuated much more frequently than HPLC valve~. Therefore. Fl valves should be more versatile, and should be able to provide a large number of leak-proof operations using a wide range of solvents. The author will not refer exhaustively to the various valve designs reponed in the literature. since some of the earlier designs, such as the membrane type and sandwich type rotary valves. described in some other monographs [3.4]. are now seldom used. Only those designs which are used most frequently in on-line separation systems will be described in this section .
....,..,., ·-·-
Six-pon Rotary Valve
The principle of operation of a two-layer six-pon rotary valve, with its design borrowed from HPLC valves. is shown in Fig. 2.3. The valve has three channels engraved on the contact surface of the rotor and six pons on the stator. or \'icr versa. The valve is simple in construction. but is not sufficiently versatile to perform the more complicated functions often encountered in on-line separations, in which case a group of such valves may be used to achieve the desired perfonnance.
w NS
PILL ~:
INJBCJ'
Schematic dia~ram of a 6-pon injector valve. S. sample; NS. new sample: C. carrier. D. reaction coil and detector: L. sample loop: and W. wBSle.
30
2 General Instrumentation
~ss
w
Fig.2.4: 8-channel 16-pon multifunctional valve as used in dual column ion-exchange preconcentration for flame AAS. S~> S2. samples; E1> E2. eluent, Ct. C2. ion-exchange columns; AAS. AA spectrometer: and W, waste (cf. Fig. 2.5 d) ref. (5] by permission of Elsvier scientific Publishing Co..
2.2.3
Eight-channel Sixteen-port Multifunctional Valve
A design which has shown great versatility in FJ separation systems, and which will be extensively referred to in the examples presented in later chapters is the eightchannel multi-functional valve first reported by Fang et al. in 1984. This valve was used as a principle component in a dual column on-line ion-exchange preconcentration system for flame AAS [5]. Commercial versions with such a design are available from Chinese (Zhaofa, Syntone). Swedish (Tecator), and Japanese (Hitachi) FJ instrument manufacturers. The original design of the 8-channel valve, used in the dual column ion-exchange preconcentration system is shown in Fig. 2.4, as applied in dual ion-exchange column preconcentration for flame AAS. A schematic presenwion of this application is shown in Fig. 2.5 d. Despite its high versatility, the valve features a very simple construction of a two-layer rotBJ)' design, with eight channels on both the rotor and the stator, and the channels unifonnly positioned around the axis of the rotor, separated 45° from each other. When the valve is actuated, the rotor turns through an angle of 45°, and all the
2.2 Injection and Multi-functional Valves (a)
31
INJBCT
PILL
w LS
LS
v
v (b)
INJBCI'
FILL
v
w
c~-+
El
s~-+
+--•D +--•w
v
Flg.l.S. (a)(b): Applications of the 8-c:bannel multifunctional valve. A, volume-based sample injection. V. multifunctional valve: X. blocked channel; S, sample; C. carrier: LS. sample loop: W. waste line: D. detector.
channels on the rotor are shifted one channel position in relation to the valve stator. In the original design. the valve was turned manually. the channel port connections were push-fined, and the rotor was made from PTFE, while the stator was made from PVC. In more recent applications. and in the commercial versions, the valves are made automatic. either electronically or computer controlled. The sections which come into contact with solutions are usually fabricated from fluorocarbon plastic materials and threaded fittings are used for line connections. A few of the many functions which could be performed by this valve are shown in Fig. 2.5, and more will be presented in applications in the later chapters. Not all the channels are required in every application; the unused channels are blocked using a blind connector if necessary, or simply left open, if not.
32
2 General Instrumentation
(c)
FILL
INJECI'
W1
W1 LR
M
M
R
c
D
R
LR
c
D
W2
W2 LS
8
s
c
c
LS
W1'
v
v
ELUTE
(d)
LOAD
v.
v.
s. B E
p
+v..
Fig.2.5 (c)(d): Applications of the 8-channel multifunctional valve. c. merging zones application in which injected sample and reagent zones merge at point M. d. application for dual sorption column preconcentration with which a second valve is required to multifunctional valves: V11 , 2-way valve; elute the two columns sequentially. V, S. S 1• S2; samples: E. eluent: R. reagent; B. buffer: C. carrier: LS. LR. sample and reagent loops; W, W', WI. WI'. W2. waste lines; note that Wand W', WI and WI' are the same waste lines; D. detector.
v,.
A six-channel valve, the various functions of which have been described in detail in Ruzicka and Hansen's book had a design similar to, but even simpler than the eightchannel valve [3]. All its functions could be performed equally well using the 8-channel valve. but not vice versa.
2.2 Injection and Multi-functional ValveJ
v
v
~
~
~
s c
w D
..._
33
s
c
L v
w D
...__ FILL
INIBCI'
Fll-2-6: Schematic diagram of a 3-layer commutatator injector. L. sample loop: S. sample: C. carrier: W, waste: and D, detector.
2.2.4
Three-layer Commutator Valve
A special type of injection valve. developed and most frequently used by the Brazilian group in Piracicaba. is called a "commutator" by its inventors, and features a three-layer sandwich design, with the middle layer sliding between the two outer layers (Fig. 2.6). This valve is also capable of performing very many complicated functions, described in detail by Krug et al.[6)
2.2.5
Bypasses in Valve Designs
In some injection valve designs. a bypass, usually made of a relatively long piece of thin plastic tubing is connected to the inlet and valve, parallel to the sample loop. but having a much larger hydrodynamic impedance than the latter (Fig. 2.7). This bypass is meant to avoid an interruption of carrier flow during the turning of the valve when all valve channels are momentarily blocked, or when the valve is in the loading position. This may have some favorable effect in improving the reproducibility of flow, particularly when the valves are operated manually by inexperienced personnel. However with automatically controlled valves, the turning of the valves usually takes only a fraction of a second; the blockage of the channels of a valve resembling the design in Fig. 2.4 is very shon and reproducible. The small and momentary surge in the flow following the valve tum has little effect, if any, on the flow stability when the rea4outs are made on the peak. which occurs at least a few seconds after the actuation of the valve. This is evident from the high reproducibilities of less than 1% r.s.d. often reponed for such valves. On the other hand, the adverse effects of the bypass often much outweigh its benefits in many on-line separation systems. Therefore, such designs are not recommended, and special precautions are required if used. Some reasons are given below:
34
:? Genera/Instrumentation
FILL BYPASS
INJECT BYPASS
Fig.2.7: Schematic presentation of the principle of a bypass for an injector. S, sample.
The dimensions of the bypass always have to be balanced against those of the sample loop, otherwise a substantial fraction of the carrier stream will bypass the sample loop during sample injection. This not only prolongs the time for discharging the sample, but the bypassing stream also dilutes the injected sample when it merges again with the main stream. Moreover. the approach is only applicable when relatively short sample loops are used. When a long and thin conduit, such as a knotted reactor, or a packed column is used as a sample loop, as is often the case in FI separation systems, the impedance in the main stream becomes so large that tens of meters of bypass tubing may be required to limit the flow through it. during sample injection or elution. This will create an even higher impedance in the flow during the sample loading stage, when the bypass serves as the only open conduit. This may be sufficient to render the procedure unpractical.
23 Transport Conduits and Mixing Reactors
2.3
Transport Conduits and Mixing Reactors
2.3.1
Transpon Conduits
35
Transport conduits are an integral component of any flow analysis system. The function of transport conduits ~~ to provide connections between the different components
ot the flo" system. In FlA. PTFE tubings of 0.35-1.0 mm i.d. are used most often for ~uch purposes. Although the outer diameter is not critical, tht" tube walls should not be thmner than 05 mm to ensure adequate mechanical strength. PTFE tubing of 0.35 mm i.d. is used when the lowest dead volume or dispersion is required in a length of connection. e.g. the connection between a valve and nebulizer of an atomic spectrometer. Although tubings with smaller diameter than that WOIJ)d produce even lower dispersion. they are not recommended due to increased risks of blockage by accidentally introduced suspended particles in the flow stream. 0.5-0.7 mm i.d. tubings are suitable for most purposes. the dimensions providing a reasonable compromise between low dead volume and low hydrodynamic resistance. The larger diameter tubings of I mm and above are used in systems with high flow-rates. sometimes with the evolution of gases, as in hydride generation systems. Large bore tubings are used in such cases to decrease the impedance of the flow. The larger diameter will not mcrease the sample dispersion appreciably owing to the gas segmentation. Connection tubes may be connected to the different components or prolonged either by push-fitting using different diameter tubings or by means of threaded fitting connectors. Push-tit connections are convenient and easy to make. and may be used wherever h1gh internal pressures in the conduits arc not expected. e.g., sampling probes and waste lines. However. they are relatively unreliable. and are a common source of leakages. particularly in column separation systems where flow impedance is high. Therefore. threaded fitting connectors. or push-fit connectors with a slip-proof design such as those shown in Fig. 2.8 are strongly recommended for most on-line separation systems.
Fig.2.8: A connector with a slip-proof push-fit connection (left) and a threaded fining connection. P. plastic tubing: E. male end with slip-proof design: B. connector body: 0. rubber 0-ring: F. threaded fining; and PTFE tubing with flanged end.
Special care should be taken in making push-tit connections between two tubing!with identical o.d. using an outer sleeve. The two tubings should be pushed full wa~ down the sleeve until they meet each other with the ends flush, so that no dead volume ~~
36
2 Genera/Instrumentation
left between the ends (Fig. 2.9). Such dead volumes may function as miniature mixing chambers which, when positioned at critical pans of the manifold, may increase the dispersion and degrade the precision of the determination. In order to make a reliable connection with a threaded fitting connector. the ends of the PTFE tubing have to be flanged and fitted with rubber o-ring washers as shown in Fig. 2.8 or fitted with specially produced ferrules (sometimes without flanging). Both the latter and connection lines with J'e4ldy flanged ends may be rather expensive, but in order to make a reliable flanged end in the laboratory, a special flanging tool will be necessary.
Fig.2.9: Push-fit connection of tubings by an outer sleeve. Note that the tube ends are slanted to facilitate pushing in. Right, correct connection. Left. faulty connection.
2.3.2
Mixing Reactors
The main function of mixing reactors is to promote the reproducible radial mixing of two or more components merged through a T-piece. The reactor is usually made of PTFE tubing with the same range of dimensions as for the transport conduits. The tubes are coiled. knotted. or knitted to produce a secondary flow in the radial plane by varying the flow direction. This enhances radial mixing, and decreases the axial dispersion of an injected sample plug. Although coiled reactors of about JO mm coil diameter are most often used. the most dramatic effects are obtained by using knitted or knotted reactors (Fig.2. 10). These reactors were first introduced by Engelhardt and Neue for applications in HPLC [7). and referred to in some references as three-dimensionally disoriented reactors (or 3-D reactors). While in coiled reactors the flow directions are changed mainly on a two-dimensional plane. with a knotted reactor the flow directions are changed on a three-dimensional basis. and hence its name. Owing to its strong capability ~n limiting dispersion, it has been recommended not only for mixing but also for transport conduits and. sample loops (8).
Fang et al.[9) recently also reponed on a special function of such reactors in using them for on-line precipitate collection. Precipitates are formed in the reactor and collected almost quantitatively on the reactor tube walls presumably through centrifugal force created from the secondary flow. With the aid of such collectors no filters are necessary to separate the precipitate. Details on the technique are given in Chapter 7.
2.3 Transport Conduits and Mixing Reactors
F"~g.l.IO:
37
A knotted reactor
The so-called ••single bead pearl string reactor"first described by Reijn [I 0], and other packed reactors using inert packing materials are also shown to be effective as mixing reactors [4). However they are more difficult to produce and less flexible than the knitted or knotted reactors. and, perhaps for this reason, are not used as broadly.
2.3.3
Manifolds for Connection of Conduits
FI manifolds are essentially made up of a network of T-connections, connecting the different components of the system. The many T-connectors in a system may be integrated into a single compact block, which then serves as a convenient base for building the manifold. One such part commercialized by Tecator (Hoganas, Sweden) is termed a ..Chemifold", and is shown in Fig. 2.11. Instead of a T-configuration, Y or W-configurations (as in figure) for the merging conduits may also be used for producing better mixing effects.
Fig.l.ll: A Chemifold
38
~ Gt'nt'ra/lnstrumt'ntatwn
2.4
Detectors
In principle, any detection system which could be adapted for flow-through detection may be used as detectors for FlA. However, some detectors are inherently more suitable than others in the interfacing, and therefore are used more frequently in Fl systems. These include the spectrophotometer (visible and UV). atomic absorption and ICP spectrometer. chemiluminescence and various electrochemical detectors. and will be discussed here in more detail.
2.4. 1
Spectrophotometers
General Visible and UV spectrophotometers are by far the most frequently used type of detectors in FI systems. This is also true for FI separation systems. Provided the light source intensity is strong enough, a conventional batch spectrophotometer can easily be converted into a flow-through spectrophotometer by substituting the conventional cuvette with a flow-through cell. FloM·-ce/1.~
A most often used flow-cell configuration, which may be furnished with either glass or quartz windows. is shown in Fig. 2.12. Flow cells of 18 J.LI capacity in the light path (1.:5 mm diameter. I0 mm long) are commonly used. The inlet and outlet conduits of the cell are so arranged that the stream is fed in from the bottom of the cell and leaves from the top to facilitate the release of accidentally introduced air bubbles. Care should be taken not to reverse the flow directions through the cell. in order to avoid the trapping of air bubbles in it. In some applications. separation and detection systems are integrated by packing the flow cell with partially transparent sorbent material. The analyte may be first collected on the sorbent, transformed in situ into a detectable species, and detected in the cell. This technique, termed sorbent absorptiometry. will be discussed in Chapter 4. Schlieren Effects in Spectrophotometry A frequent trouble encountered in spectrophotometric readouts is the generation of spurious peaks due to differences in refractive properties of the sample and carrier/reagent. In case of a substantial difference in the refractive index. the parabolic interfaces at the two ends of the sample zone create convex and concave lens effects on
2.4
IN
OUT
END VIEW
END VIEW
(INLET)
IN
Detector.~
39
OUT
SIDE VIEW
(OUTLET)
F"tg.l.l2: A spectrophotometrir: flow-cell. Arrow shows light path.
the leading and tailing pans of the dispersed zone, respectively. producing a displacement of the baseline in these section~ (Fig. 2.13). Such phenomenon. called the "Schlieren effect", will not be observed in batch procedures or in air-segmented continuous flo\\ systems where the samples and reagents are always homogenized before the final measurements in the cuvette or flow-cell. and therefore is a matrix effect quite characteristic of FI spectrophotometric procedures. When the response of the analyte peak is high. the spurious pe8ks usually can be easily differentiated from the main peaks_ However. when the analyte peak response is low. it may be seriously distoned by the interfering peaks. and measures should be taken to eliminate them. One way of overcoming such interferences is to match the refractive indices of the sample and carrier/reagent. This approach. though sometimes effective, is inconvenient. especially when it is the refractive index of the sample which has to be adjusted in order to match the carrier/reagent. A more convenient approach is to disturb the parabolic profile at the sample/carrier interface by promoting radial mixing using knitted or knotted reactors. Such a solution is found to be most effective when the reactor is closely connected to the flow-cell. Any straight section of conduit between the reactor and cell will provide an opponunity for the restoration of the paraboJic interface. Further information on the elimination of Schlieren effects are given in the chapters on individual separation techniques.
40
2 General Instrumentation
Flg.2.13: Recording trace of typical baseline displacement from Schlieren effects in spectrophotometry. Right. injection of 10% (w/v) sodium chloride into distilled w*r carrier. Left. injection of distilled water into 10% (w/v) sodium chloride carrier. Arrows on recording are injection points. Horizontal arrow, direction of chart scan.
2.4.2
Atomic Absorption Spectrometers
The flame atomic absorption spectrometer is inherently a flow-through detector, with which the sample solutions are continuously fed into the nebulizer-burner system through suction. Despite the relatively large volume of the spiay chamber (usually about 100 ml) in comparison to the spectrometric flow-cell, the detector was shown to have very little contribution to the dispersion of the injected sample in comparison to other components of the Fl system [ 11]. With careful optimization, as little as 50-80 pi sample may be injected to achieve 80-95% of the steady state signal obtained by conventional sample introduction (see Fig. 2. 14). The performance of the flame atomic absorption spectrometric detector is enhanced substantially by the Fl mode of sample introduction. Besides the decrease in sample volume already mentioned. additional contributions which may be of interest in separation and preconcentration systems include: •
• • • •
Strong tolerance to high concentrations of dissolved solids in the sample. 30% m/v NaCl and saturated lithium metaborate solutions have been successfully introduced into an AA spectrometer for hundreds of-determinations without interruption [12]. Matrix effects due to differences in viscosity are minimized, if a pump is used (as usually is, in Fl) to deliver the sample carrier [13]. Organic solvents may be used as sample carriers to enhance the sensitivity [14]. Dynamic range of calibration may be expanded by multistage automated on-line dilutions through sequential injections (zone penetration), zone sampling, etc [15]. When slotted tube atom traps are used to enhance the sensitivity for some analytes, the lifetime of the quartz tubes may be prolonged 8-10 fold by FI sample introduction [16].
For more details on the above, the reader may consult the related monographs and reviews [ 15,17].
2.4 De1ec10rs
41
... 0.12 0.10
§G.III -e• !G.III CI.IM
O.CI2
0
Fig.l.l4: Response and dispersion characteristics of a Perkin-Elmer flame AA spectrometer under low dispersion Fl conditions of operation. I0 mg 1- 1 Pb test solution under 7.2 ml min- 1 nebulizer free uptake rate. Shaded areas A. conditions under which >90% steady state signals are achieved; B, conditions under which 80-90% steady state signals are achieved. (From reference II. Reproduced with permission from Royal Society of Chemistry.)
An important feature of FI-ftame AAS systems, sometimes overlooked, is that the sample introduction flow-rate can be controlled independently from that adjusted for the pneumatic nebulizer. In earlier publications. when the carrier flow-rates were lower than the free uptake, attempts were made to compensate for the difference in flow-rate between the sample carrier and the uptake rate of the nebulizer by introducing water or air to improve the precision. Later. these were shown not to be necessary when the carriers were de-gassed, and all fc:ms of flow compensation were found tu degrade the sensitivity to some extent [ 18j. On the other hand, it was reponed in quite an early stage of development that the nebulizing efficiency could be significantly improved by starving the nebulizer to some extent. The decrease in signal is therefore not proportional to the decrease in sample/carrier How-rate, and this feature can be made use of to improve the absolute sensitivity of the method. In the study of a Perkin-Elmer AA spectrometer. it was shown that the anaiyte response and the sample introduction rate follow an empirical relationship described by the equation [ 19]: (q
(2 II
where A is the absorbance at the lower flow-rate: A', the steady state absorbance obtained with conventional sample introduction; Q, the minimum sample/carrier flowrate to reach A'; and q, the sample/carrier introduction rate used, expressed in ml min- 1• Q is termed the saturation uptake rate. AAS determinations based on hydride and cold vapour generation, electrothermal atomization with graphite furnaces have also been used successfully with FJ systems to improve the overall performance of these techniques. Special requirements on the atomization-detection systems for hydride and cold vapour generation will be discussed in Chapter 5, and for the graphite furnace, in Chapter 4.
42
2.4.3
2
Gt>n~rallnstrumt'ntation
Induction Coupled Plasma (ICP) Emission Spectrometers
The ICP spectrometer has much in common with the flame AA spectrometer in its pneumatic sample introduction system. However, usually less compromise is required in the flow-rate during sample introduction, because optimum uptake rates of ICP spectrometers are much lower than flame AA systems. Nevertheless, various attempts were made to further improve the sensitivity by other FI techniques, including direct injection into the torch [20], and thermospray interfacing [21 ]. A great advantage of ICP spectrometry over AAS is its multi-element capability, which. combined with the versatility of FI on-line dilution and separation could create a powerful combination with great potentials. Hitherto, the majority of publications, however, deal with the determination of a single analyte, using peak integration rather than peak height evaluation, and some even with photographic recording. Therefore, the benefits of the technique is far from being fully exploited. The reason for this situation is that currently most commercial ICP spectrometers are not designed to monitor and record transient signals, which are characteristic of FlA. and the FI-ICP combination may not enter a major phase of development before ICP spectrometers which are able to do so ·are available.
2.4.4
Electrochemical Detectors
A large variety of electrochemical detectors have been used in FlA. including those based on potentiometry. amperometry. conductimetry, voltametry, and chronopotentiometry. There appears to be no generally accepted detector design for each species. and the type of detectors are so diverse that even a brief discussion in this book will prove to be too lengthy. Only one of the species which has been applied most broadly. the ion-selective electrode. will be discussed here in some detail. The earlier setup of using ion-selective electrodes as m:t FI detector employed an electrode tilted at about 45°. with the sample/carrier flowing continuously over its sensing surface. The electrode was adjusted so that the surface film thus formed kept contact with the effluent solution. the latter being maintained at constant level in the out-flow reservoir. in which the reference electrode was submerged. This cascade design, seemingly simple. requires some skill, experience, and patience from the operator, and appears to lack the robustness of a routinely applicable detector. Wall-embedded ion-selective sensors are more suitable for routine operations. The sensing surface can be made to be either a section of a glass or plastic tubular electrode or a pan of a conduit through which the sample/carrier solution flows. The reference electrodes are easily located somewhere downstream. Two designs. suitable for FI applications. are shown in Figs. 2.1 5 and 2.16.
2.4 Detectors
43
T
M Fig~15:
A tubular How-through hquid membrane ion-selective electrode. T. PVC tubing: M. membrane containing elec~active component: H. housing containing internal reference solution: R. Ag/AgCI electrode 122].
/------L A
:··.• B
M
S1
D
S2
0 Y~g~16:
Solid membrane flow-through ion-selective electrode. A, threaded cylinder for fixing solid membrane: B. main block with inlet and outlet: Sl, silicone rubber disk with hole in centre: S2. silicone rubber disk with slot which forms the flow path: M. solid membrane: D. copper contact disk with lead. L soldered to disk [23].
44
2 Genera/Instrumentation
The first design is suitable for liquid membrane electrodes, and was first reported by Meyerhoff and Kovach for student experiments in the determination of potassium [::!2]. On one side of the walls of a thin PVC tubing, a piece was cut off to fonn a small hole, a stainless steel needle of suitable diameter was inserted to block the hole, and a tetrahydrofuran solution containing PVC, plasticizer, electro-active material (e.g. valinomycin for potassium electrode), etc. was applied several times on the hole. After drying, the applied material fonned a membrane, making up a pan of the tubing wall, the needle was removed, and the tubing was enclosed in a chamber which contained a suitable internal reference solution and the inner reference electrode (e.g. Ag/AgCI). The second design reponed by Van der Linden et al. is suitable for solid membrane electrodes [23]. A small area of the sensing membrane disk was exposed to the stream flowing through the cavity, the volume of which was defined by the slot cut in the 0.3 mm thick spacer. The FI mode of sample introduction for ion-selective electrodes shows several advantages over conventional practice: a
b
c
2.4.5
The difficulties in manual operation in deciding the appropriate readout time are avoided. The exposure time of the electrode to the sample is fixed in FI operation. thereby improving the precision of measurements. Selectivity of the electrodes are often improved through kinetic discrimination. Interfering ions usually respond slower than the analyte. The precise timing of FIA can be utilized to control the readout time before the interfering effects become noticeable, while in batch operations one always waits until not only the analyte response, but also the response from the interferent reaches steady state. The sensing surface of selective electrodes are easily affected or damaged by hazardous materials in real samples. The short exposure time of the FI mode of operation decreases such risks and prolongs the lifetime of the electrodes. If necessary. an on-line pre-separation could also be more readily incorporated in an Fl system than in batch systems. On-line gas diffusion, dialysis, and column separation systems may all be used for such purposes without drastically affecting the sample throughpuL
Other Detectors
Chemiluminescence and Bioluminescence Detectors The chemiluminescence and bioluminescence detectors have much to benefit from FI techniques. Chemiluminescence and bioluminescence reactions are often rapid, and the duration of the signals extremely short; therefore only the signals from slower reactions can be reproducibly monitored by batch procedures. whereas reactions in the millisecond range may be readily monitored in an FI system. Such detectors are often constructed in a spiral fonn to increase the light flux.
2.4 Detector.t
45
Fluorimetric detectors The perfonnance of fluorimetric detectors can also be enhanced using FI techniques. The flow-cells of such detectors used for HPLC may readily be adapted for use in FI systems. The reproducibility of fluorescence measurements are reponedly improved by better control of the reaction conditions in the FI systems, and selectivity may be enhanced by on-line removal of potential quenching interferents using Fl separation techniques.
3
Liquid-liquid Extraction
3.1
General
Liquid-liquid exuaction is one of the most frequently used separation techniques in the analytical laboratory. Despite its indisputable effectiveness in the removal of interfering matrices. and the preconcentration of trace analytes, its popularity has been somewhat impaired under batch operation conditions due to the tediousness of the operation, which may also induce complications in trace analysis through contaminations from the utensils and laboratory environment. A further nuisance created by batch extractions is the often annoying odor and toxicity of organic solvent vapours released into the laboratory atmosphere. Such undesirable consequences may be largely avoided using FI liquid-liquid extraction. and may be considered as another merit which adds to the general advantages of FI on-line separation methods mentioned in Sec. 1.3. FI liquid-liquid extraction systems are readily automated, while the closed extraction system greatly minimizes contamination risks as well as release of solvent vapours. A further advantage of FI liquid-liquid extraction is that. in contrast to most other Fl separation techniques, particularly on-line dialysis and gas-diffusion. much higher phase transfer factors can be achieved by FI liquid-liquid extraction (P values of 0.80 - 0.99 are typical). This may be important for achieving higher sensitivities. lower sample consumptions or/and bener precisions. . The first reports on implementing liquid-liquid extraction with FI systems was made by Karlberg and Thelander (I], and Bergamin et al.(2] in 1978. Since then. the technique aroused such broad interest that the number of related publications has attained the largest number in individual FI separation and preconcentration techniques. Irrespective of manual batch procedures or automated FI methods, liquid-liquid extractions normally go through three successive sequences, i.e.: • • •
Dispensing of defined volumetric ratios of immiscible organic and aqueous phases into a container. Bringing of the two phases into intensive contact with each other for effecting mass transfer between the two phases. Physical separation of the two phases.
In FI liquid-liquid extraction, usually these three sequences are executed respectively through the use of:
48
3 Liquid-liq11id Extraction
•
• •
A phase segmentor (with appropriate liquid propulsion devices) by which the immiscible organic and aqueous inflows are brought into contact with regular alternate segments in a defined ratio and delivered through a single outlet channel. An extraction coil in which the analyte is transferred from one phase to the other. A phase separator which continuously separates the segmented stream into two parts, with the one composed of a single phase used for determination.
Although most Fl determinations involving liquid-liquid extractions are made after separation of the two phases, in some of the more recent contributions. determinations are also made without phase separation, therefore excluding phase separators from the system.
3.2
Instrumentation
3.2.1
Phase Segmentors
Merging-tube Segmentors Phase segmentors with a T-design , modified from Technicon A-4 connectors, were the first to be used for phase segmentation in FI liquid-liquid extraction systems [1]. The segmentor, shown schematically in Fig. 3.1, is composed of a glass capillary inlet for the aqueous phase and a glass tube outlet for the segmented stream. The organic phase is introduced through a platinum capillary which merges with the inlet and outlet tubes in a perpendicular direction. A PTFE tube is inserted in the outlet tube to vary the volume and glass surface area of the outlet cavity by adjusting the longitudinal position of the PTFE tubing. By doing so. the segment sizes could be changed conveniently. Later, various designs of merging segmentors have been reported. After a comparison of different designs. including the T, Y and W configurations for the merging tubes. Kawase [3] found no significant differences in the segmentation patterns. However. the smoothness of the bores of the segmentor was found to affect the regularity of the segmentation as did also the pulsations from the liquid propulsion system, and the volume of the cavity influences the size of the segments. With such segmentors the segment of organic phase formed at the juncture of the merging tubes is dislodged when the hydrodynamic force imposed on it by the aqueous stream is equal to the interfacial force which holds it in place. Since the hydrodynamic force increases with an increase in flow-rate, the organic phase segments will be small when the flow-rate of the aqueous phase is high. Excessively small segments tend to
3.2 Instrumentation
49
OR
t
AQ . .
Fig.3.1: Schematic diagram of a merging-tube segmentor. OR, organic phase; AQ, aqueous phase: Pt. platinum capillary [ 1].
disappear due to adherence to the extraction tube walls and rejoin to form irregular larger segments. One way to reduce the hydrodynamic force of the aqueous flow force is to use an outflow channel with larger diameters for the merging tube separator. However, this does not solve the problem because under high flow-rates the two phases tend to flow in parallel in a wide-bored tube, and segmentation actually occurs in the extraction coil under il~ controlled conditions. Therefore, the merging-tube designs appear to have some inherent weaknesses in obtaining 1-tighly repeatable or controllable phase segmentation over large aqueous flow-rate ranges.
Coaxial Segmentors Recently, phase-segmentors with a coaxial design have been introduced by Swedish workers [4.5]. Such segmentors are based on droplet formation of one phase in another, and are reponed to _produce more reproducible and controllable segmentation than the merging-tube designs. The design seems to have overcome the principal shortcomings of merging-tube segmentors, and might be a good substitute for the latter, particularly when large phase ratios with high aqueous flows are used to produce more significant preconcentration effects. The "falling drop" phase segmentor designed by Backstrom and Danielsson [4). which appears to be the first coaxial segmentor reponed, was shown tQ provide regular segmentation at high phase ratios and/or total flow-rates. A more detailed investigation of coaxial segmentors was undertaken by Kuban et al.[5). A significant improvement in performance over previous designs was reponed with a 2-3-fold enhancement in segmentation repeatability. Seginentation behavior is largely predictable, and segment lengths can be varied over a very wide range from 2 to 50 mm for the organic phase and from 3 to 300 mm for the aqueous phase. The coaxial segmentor shown schematically
50
3 Liquid-ilqu1d Exrracrion
, . _ AQ
Fig.3.2: Schematic diagram of a coaxial falling-drop segmentor. OR. organic phase; AQ. phase: SP. segmented pha...e (4.5].
aqueou~
in Fig. 3.2 is composed of two pans. a small Perspex or glass chamber with an inlet and a conical outlet. and a glass capillary which is insened into the conical pan of the chamber. The organic solvent is introduced from the capillary (0.05-0.35 mm i.d.' forming droplet-; at its end. Following release. the droplets move into the outflow channel of the chamber which is filled by the aqueous sample stream flowing through it, and are transponed by the stream through the outlet. where phase segmentation occurs. The segmented stream is then funher delivered into the extraction coil. The volume of the droplet formed V. is governed by the interfacial tension Tofo· the inner diameter of the capillary d;. the acceleration due to gravity g. and the densit) difference between the two phases :jp. and may be calculated from the equation: Trd;Tofo
l'= - - g:jp
(3.1)
It appears that the length of a segment in the extraction coil might be fairly well predicted when the inner diameter of the coil tubing is defined, however, the actual segment length is not easily calculated because some transpon of the organic phase takes place in the film at the tubing wall, and the ends of the segments are not flat but parabolic. which also affects the length, particularly for shon segments.
32 Jnstrumrntation
51
Various factors which influence the segmentation perfonnance of coaxial segmentors using Freon-113. CHCIJ. IBMK and CCG as test solvents are summarized as follows: a
b
c
d
e
f
Of the different materials used for the construction of the segmentor chamber (including Perspex. PVDF. glass and a combination of glass and PVDF). all-glass chambers produce the lowest relative standard deviations in the segment lengths. and work properly over a very wide range of flow-rates for both phases. Small drops of organic solvents are occasionally trapped on the surface of hydrophobic material. and the use of such materials for the chamber housing should better be avoided. The length of the organic segment increases linearly with the inner diameter of inlet capillary at constant flow-rates of the two phases. The best repeatability ( <5fk r.s.d.) of the segment length are obtained with 0.10-0.35 mm i.d. capillaries for all organic solvents tested. The length of the OJltanic segment is affected by the distance from the end of the glass capillary to the outflow tube (located at the end of the conical section of the chamben below 3 mm. Under such conditions the droplets are drawn off before their full development. owing to the limited space available. The repeatability is then significantly degraded. Above 4 mm the droplets are released freely into the -conical section of the chamber. and the segment length is almost unaffected by the distance. In contrast to merging-tube segmentors which are strongly influenced by hydrodynamic conditions. the length of organic phase segments of coaxial segmentors is prctctically unaffected hy the aqueous phase flow-rate within large flow-rate ranges studied (0.~5-10.0 ml min- 1). This implies that the length of the aqueous segments. and hence the phase ratio. increases linearly with the aqueous phase flow-rate when that of the organic phase is kept constant. The length of the organic phase segment is not affected by the flow-rate of the OJltanic phase from 0.1 ml min- 1 to below a certain upper limit. which depends on the inner diameter of the capillary and the solvent species. The limiting flow-rates for Freon-113. chlorofonn and carbon tetrachloride were found to be > 1.6. 1.1 and 0.5 ml min- 1 for capillaries with 0.3.0.2 and 0.1 mm i.d. respectively. while the corresponding values for IBMK were 1.6. 0.6 and 0.3 ml min -I respectively. The segmentation repeatability is affected by the cleanliness of the chamber walls and the flat end of the glass inlet capillary. Regular washing with ethanol and occasional grinding of the capillary tip are recommended for avoiding deterioration in performance.
More recently Kuban and Ingman [6] reponed a dual channel dropping segmentor which allows the simultaneous mixing of aqueous solutions of sample and reagent and segmentation of the resulting homogeneous mixture by an immiscible organic solvent.
52
3 Liquid-liquid £'Ctraction
also based on the principle of the coaxial segmentor. The design differs from the previous coaxial designs in that the aqueous phases are introduced through the capillary, the inner diameter of which determines the segment size. The applicability of the method was demonstrated in the determination of copper using APDC and extraction into chloroform. However, it is doubtful that such an approach can be used under large Aglorg. phase ratios, since extremely small organic phase segments will be formed, which may combine in a non-controllable manner on their passage through the extraction coil.
3.2.2
Extraction Coils
Extraction coils in which phase transfer of the analyte occurs in the segm~nted stream are usually helically coiled PTFE tubing with 0.5 - 1.0 mm i.d.. Such coils are suitable for transfer of analyte from an aqueous sample into an organic solvent, but tubings of glass or other hydrophilic materials are required for reversed transfers. These are discussed further in Sec. 3.3.1. With fixed inner diameters for the tubing, the extraction time is determined by the length of the coil and the flow-rate_ of the segmented stream. The extraction time in tum determines the completeness of mass transfer. As mentioned previously. with adequate extraction time. the phase transfer factor P typically falls in the range 0.80- 0.99. Kawase 131 studied the extraction efficiency and band broadening (dispersion) of extraction coils of different lengths and tube ~!Jner di~eters from 03 to 2.0 mm. The results seem to favour the use of ~o:s mm 1.d. tubin~ 'for better extraction efficiencies and mammum band broadening. Nord et al.f71 stipulated the importance of the interfacial area-to-volume ratio on kinetics of the extraction process which also lead to the preference of small diameter tubing for the extraction coil. Their studies also indicate that the phase transfer as decreased with an mcrease in t~e flow-rate Of the segmented stream, and. particularly with short extraction coils. {>base transfer factors are enhanced 6). decreasing the flo\\--rate of the se!!Jrierited stream. However. the beneficial effects of using lo\\· flow-rates to 1mprove mass transfer have to be carefully balanced against sacrifices m sample throughput.
3.2.3
Phase Separators
Whenever implemented, the phase separator is usually the most important component which determines the performance of FI liquid-liquid extraction systems, although this component is totally excluded in some extraction systems (cf. Sec. 3.4.8). Phase separators are expected to function efficiently. and to achieve good separation of the two phases in the smallest feasible dead volume in order to reduce the dispersion.
32 Instrumentation
53
w
t 2
SP-+ 1
~
OR Fig.3.3: A T-tube gravitational phase separator for liquid-liquid extraction. I. PTFE tube; 2. glass T-tube; 3. PTFE strip: SP. segmented phase: OR. organic phase: W. waste for aqueous phase containing residual organic phase [ 1].
Phase separators may be classified according to the different mechanisms of phase separation. The earlier separator designs were all based on differences in densities of the immiscible phases. These are designated here as gravitational separators. Later. separator designs were introduced which were based on different affinities of the immiscible phases to a separation membrane. hence the name. membrane phase separators. The latter come closer to the requirements of an ideal phase separator. and are used almost exclusively nowadays under a variety of different designs. including sandwich and tubular membrane separators. Gravitational Phase Separators
A typical gravitational separator is shown in Fig. 3.3. The separator is usually made of glass with a T-configuration, the segmented flow entering the separator from the horizontal side arm, and the two separated phases leaving in opposite directions through the two vertical arms. The outflow direction of the two immiscible phases will depend on their relative densities. the phase with the higher density leaving from the lower arm and vice versa. A small PTFE strip is inserted into the segmented flow inlet arm of the separator, extending into the organic phase outlet arm to achieve better separation of the phases. The hydrophobic PTFE strip is wettable by most organic solvents but not by water, and therefore acts as a guide for directing the organic flow into the appropriate
54
3 Liquid-liquid Extraction SP
AQ
~
t
1
3
2
•
OR
Fig.3.4: Schematic figure of a sandwich-type gravitational phase separator for liquid-liquid extraction. 1. grooved stainless steel upper block; 2. stainless steel lower block: 3. grooved PTFE disk: SP. segmented phase: AQ. aqueous phase; OR. organic phase (8].
outlet ann [ 1]. In order to achieve better separation effects. the aqueous phase which nonnally carries the injected sample is drawn off from the outflow ann of the separator at a slightly higher flow-rate than its inflow rate. By doing so. a small fraction of the organic phase is forced to leave the separator from the aqueous outflow ann. This sacrifice reduces the chances of entraining aqueous phase into the separated organic fraction which is funher transponed to the detector for signal evaluation. The accidental entrainment of aqueous phase into a photometric flow-cell may cause contaminations which can only be removed by interrupting the operations and washing the cell with ethanol. An obvious advantage of gravitational phase separators is their simple construction. the separator often being transfonned from standard T-connectors for flow analysis with little additional effon. However. when using such designs considerable care and experience are required to avoid contamination of the flow-cell with aqueous phase. particularly at the beginning of the operation. It is also difficult to separate a segmented stream com~ posed of two phases with similar densities or more complicated extraction systems such as water-methanol-chlorofonn using these separators (9]. De Ruiter et al.[8) described a rather non-conventional gravitational phase separator (Fig. 3.4) which resembles a sandwich-type membrane phase separator (cf. next section). but without a membrane. A grooved PTFE disk was sandwiched between two stainless steel blocks, with one of the blocks grooved to fonn a cavity of 8 - 43 J.tl volume together with the groove on the disk. An inlet and outlet was provided on the grooved upper block for introduction of the segmented flow and leading the aqueous phase to waste. A second outlet was furnished on the un-grooved lower block which was connected with the cavity through an aperture in the groove of the PTFE disk. The organic phase
3.2 Jnstrumentalion
55
with higher density was recovered from this outlet both by gravity and by its affinity to the lower half of the cavity made of PTFE material. Similar to other gravitational phase separators, this separator has the advantage of no need for regular change of membranes. However. hitheno its application hao; been rather limited. and it is still doubtful that its overall performance could compete with membrane phase separators.
Sandwich-type Membrane Phase Separators The first sandwich-type membrane separator used in FI liquid-liquid extraction was described by Kawase et al.[9] in 1979. Owing to its better performance compared to gravitational separators, it soon became the most frequently used type of phase separators. As its name implies. sandwich separators are usually composed of two separate organic solvent resistant plastic blocks, furnished with grooves or cavities. with microporous membrane sandwiched in between. Sometimes the membranes are supponed by PTFE coated metal filter screens to improve the durability of the membranes [ 10]. Factors which are reponed to influence the phase separation efficiency of sandwichtype membrane separators include:
a
• • • • •
The.membrane area exposed to phase transfer. The porosity of the membrane The volume and geometrical fonn of the mini-chambers of the separator. The impinging angle of the segmented flow on the membrane. The pressure drop across the membrane.
Although the phase separation efficiency will no doubt be enhanced by larger membrane areas, the mechanical stability of the membrane is reduced by an increase in area, while analyte dispersion may also be increased. Screen supponed and polyethylenebacked membranes are more durable (cf. Sec. 3.2.3. Separation membranes) so that larger exposed membrane areas are applicable. However. the screens panially obstruct the membranes. and their use is not always justified when overall performance, including analyte dispersion is considered. The effects of geometrical aspects of the mini-chambers of sandwich-type separators, panicularly those chambers through which the segmented stream flows. were studied by some workers. Parallelepiped and spiral groove-type chambers and cylindrical cavity-type chambers of different volumes have been suggested for various applications. In principle, provided that the exposed membrane area is sufficient, smaller chambers should favour a reduction in dispersion. This principle should generally hold true for the outlet cavity of the "clean"separated phase. However, the volume of the chamber for the segmented flow should be at least four to five times larger than the volume of an individual segment of the aqueous phase in order to avoid, at any moment, filling of the chamber with aqueous phase alone. This is essential for smooth operation.
56
3 Liquid-liquid Extraction
There seems little to choose between the different geometrical forms of the cavity, except that cylindrical cavity-type chambers may not be suitable for some solvents. Motomizu and Korechica [ 11 ) indicated that the working principles of the groove-type and cylindrical cavity-type separators may be different. Assuming the use of a hydrophobic membrane, in the groove-type separator, the organic and aqueous phase segments move alternately over a thin film covering the membrane surface, while in a cylindrical cavity the surface is always covered by a relatively thick layer of organic solvent. The latter type of separator will not work effectively in an extraction system when the density difference of the two phases are small (e.g. water/benzene). Some workers stress the importance of the impinging angle of the segmented flow on the membrane, indicating that slant-wise impinging of the flow on the hydrophobic membrane facilitates the penetration of the organic phase [10.12). However, other workers found little difference in various modes of segmented flow entry [13]. This incongruity might be due to the different groove or cavity dimensions and flow-rates, as well as the resistance of the impedance coil used in separate studies. Backstrom et al.[ 10] studied the effect of variations in pressure drop across the membrane on the separation efficiency by adjusting the length of a narrow-bore tubing connected to the outlet of the phase separator. The narrow tubes are designated as flow restrictors, or impedance coils. which conform with its function of increasing the pressure drop by increasing the flow impedance. Their studies show that separation efficiencies are lowered with insufficient pressure drop across the membranes; but excessive pressure drops induce intrusion of aqueous phase through the membrane. With a 0.2 J.tm pore size membrane. a 50 em length of 0.7 mm i.d. tubing producing 0.05 ann. was sufficient to reduce organic phase losses to 0.4 %. A design used successfully in the Author's laboratory [14]. which combined important features from groove-type [15] and cylindrical cavity-type [12) designs is shown in Fig. 3.5. The separator was made of PTFE. An inlet and outlet were furnished on the lower block which accommodated the groove for the segmented flow. The upper block had only one outlet reserved for the organic phase. A relatively small membrane area (.18 mm2 ) was used for the separator both to increase the membrane durability and to decrease the analyte dispersion. No support screens were used in order to increase the effective area of the membrane. and the bottom of the groove for the segmented flow was slightly slanted to create an impinging angle without excessively increasing the groove volume, which was made to be 27 J.d. The groove for the organic phase was made shallow (approximately 1 mm ) to avoid intensive dispersion and collapse of the unsupported membrane into the low pressure side. The blocks were joined tightly with two metal plates which are screwed together. An impedance coil was used with the separator to adjust the pressure drop across the membrane. Although better than 99% separation of the organic phase from the segmented flow may often be achieved using membrane separators, the small amount of residual organic phase in the aqueous fraction renders the latter useless for determination purposes. When determinations in both phases are desired, a dual-membrane sandwich phase sepanttor,
.. : ......
3.2 lnstmmematioll
57
1cm Fig.3.5: Schematic diagram of a sandwich-type membrane phase separator for liquid-liquid e>.traction. A. B. PTFE blocks: M. mirroporous PTFE membrane: G. slanted groove for segmented phase: P. metal plate for fastening A. B block' hy four screw~ (not shown 1.
with three cavities may be used 162]. The segmented phase is introduced into the middle cavity. separated by a hydrophobic and
High phase separation efficiencies of 90 - I OO'il- feasible. Low analyte dispersion. owing to smaller dead volume of the separator. High phase ratios applicable. resulting in higher enrichment factors (£FJ. High total (aqueous+ organic phase) flow-rates pennissible. leading to increased sample throughputs and concentration efficiencies (C£). Applicable to a large range of organic solvents, irrespective of density differences between the two phases. Easier manipulation.
The main drawback of the sandwich-type membrane separator is associated with the limited lifetime of the membrane. Operations are interrupted periodically due to breakdown of the membrane. which is particularly objectionable for applications in process control or continuous monitoring. In such cases. it is advisable to use a supporting grid for the membrane to prolong its lifetime, possibly with some sacrifice in the phase separation efficiency.
58
3 Liquid-liquid £rtraction
Separation Membranes
Separation membranes for liquid-liquid extraction may be classified into hydrophobic and hydrophilic types. based on their affinity to non-polar or polar solvents respectively. The choice of a particular type depends on whether the organic phase or the aqueous phase is collected for the final determination. Hydrophobic microporous membranes are used most often since the organic phase is most frequently collected for determination. Such membranes may again be classified into three different forms. i.e. sheeh or tapes with reinforced plastic backing, simple sheets or tapes without backing and tubular membranes. The PTFE membrane is by far the most frequently used hydrophobic membrane, owing to its strong resistance to organic solvents, and its availability in various pore sizes and forms. Nord and Karlberg [15] conducted a detailed study on the durability of different hydrophobic membrane· materials and recommended the use of PTFE membranes with polyethylene backing. However, whether this should be used will depend on other factors on performance discussed in the previous section. Although PTFE membranes of 0.2 - 20 11m pore size have been used by different workers, most prefer the use of pore sizes in the vicinity of 1.0 11m. Larger membrane pore size facilitates mass transfer through the membrane, however, the water intrusion pressure of the membrane. at which penetration of aqueous phase occurs, is lowered compared to the smaller pore size membranes, and the optimization of pressure drop across the membrane becomes more critical. PTFE membranes with about 0.1 mm thickness are often used in membrane separators. but thicker or multiple layer membranes may be used to improve the durability. There are only a few reports on the use of hydrophilic membranes for separation of a ''clean" aqueous phase from the segmented phase [16.17]. It appears that. hitheno. only filter papers (e.g. Whatman No. J or 5) were used for such purposes. Column Phase Separator
Toei [ J8] recently reponed on a phase separator for liquid-liquid extraction which is in the form of a packed column. The column, produced from a 20 mm length of 8 mm i.d. tubing. was packed with highly absorbent paper from babies' diapers. A small aqueous sample of about I 0 Jtl was injected into a continuous flow of organic solvent to form a single aqueous segment. A segmentor was therefore not required. After transfer of the analyte from the aqueous into the organic phase in an extraction coil, the aqueous segment was transponed through the separation column, in which the water from the sample was absorbed by the dry hydrophilic packing. The "clean" organic phase was then delivered to the flow-cell of the detector. The main advantage of this interesting approach is its extreme simplicity. However, the limitations are quite obvious: the system cannot be used for preconcentration purposes, owing to restriction in the sample volume; columns may have to be changed much more frequently than the membranes in membrane separators because of the limited capacity of the column; and enhanced analyte dispersion in the organic phase after
33 Theoretical Aspects of Fl Liquid-liquid Extraction
59
separation. owing to the small sample volume. Nevertheless, the method may be useful in cases where higher sensitivities are not required, and separation from interferents in the aqueous phase is the main objective for separation.
3.2.4
Integrated Liquid-liquid Extractor
In order to simplify the connections and conduit arrangements of Fl liquid-liquid extraction systems. Sahlestrom and Karlberg [19] proposed a self-contained system which integrated all the necesslll)' components on a compact block, including engraved conduits for sample and reagent mixing. an engraved segmentor, a detachable extraction coil. a sandwich membrane separator with spiral shaped grooves, and a rinsing system for the flow-cell. The integrated system, later commercialized by Tecator, was designed to facilitate routine use. The system could be operated with a start-up time of 3-5 min. and has shown good reliability over extended working periods. However, the engraved conduits are sealed with a high density rubber gasket which is only resistant to a specific range of organic solvents such as chloroform and carbon tetrachloride. Some solvents, such as isobutyl methyl ketone (IBMK) cannot be used with the system, because prolonged contact of the solvent with the rubber caused excessive swelling which may ultimately block the conduits.
3.3
Theoretical Aspects of FI Liquid-liquid Extraction
3.3.1
Mechanism of Phase Transfer in FJ Liquid-liquid Extraction
The transfer of analyte from one phase to another in liquid-liquid extraction is achieved at the interface between the two immiscible phases. With appropriate agitation of the phases, mass transport within the two phases is of minor importance for the transfer efficiency. Maximum exposure of the phase interface is therefore pursued in batch extractions by vigorous shaking, resulting in the formation of highly dispersed droplets of one phase in the other. However, such conditions are not achievable in continuous flow systems such as FlA. Nevertheless, in most cases phase transfer factors of 0.8-0.99 can be achieved in extraction coils usually in no more than a minute. This high extraction efficiency cannot be fully explained by merely considering the mass transfer at the interface on the two ends of the segments, the area of which is indeed very
60
3 Liquic/-/i,,uid Extraction
PTFE TUBE
a
b
Fi~.3.6:
Schematil' diagram showing film formation in a liquid-liquid extraction coil tubing. a. static conditions: b. flow conditions. AQ. aqueous phase: OR. organic phase: F. organic film 1201.
limited. This induced further investigations into the mechanism of phase transfer in Fl liquid-liquid extrdction systems. With the aid of an ingenious photographk technique. Nord and Karlber~ [20[ were able to provide evidence on the fonnation of a thin film of 0.01-0.05 mm thick on the extraction coil tube walls by the phase which wetted the tube material. In most applications the analyte is extracted from the aqueou~ phase into an organic phase in order to increase the organic phase interfacial area relative to its volume. PTFE tube:- wettable by the solvent are used so that organic solvent film~ are fonned on the tube walls (Fig. 3.6). The film thickness increases with an increase in flow-rate. and disappears when the flow is stopped. The film largely increases the phase transfer area. particular!~ in relatively narrow tube~. and i:-. responsible for the high efficiency in phase transfer. Nord and Karlberg [20] derived an equation relating the film thickness td,. em) to the inner diameter of the tube (R. cm1. the viscosity (TJ. poise) of the film fonning phase. the flow-rate (u.cm ,-I land the surface tension 17. dyne em-:):
d.
=COliS( "
R
(IIIJ17l"
where K is an empirical constant equal to 1/2 or 2/3. From equation 2. it follows that the film thickness increases with an increase in viscosity of the film fonning phase and a decrease in the interfacial surface tension. The film thicknes~ may therefore vary considerably for different solvents. The solvent rna~ be characterized in respect to film formation by the ratio between its viscosity and interfacial tension. TJ '.-: low ratios being associated with the formation of thin films.
33 Thenr-etica/ Aspects of Fl Liquid-liquid Extraction
61
There is no evidence that the film thickness contributes significantly to the phase transfer efficiency, however, thicker film~ may induce the break-up of small segments of the film forming phase to form larger segments during the extraction and cause the development of irregular segmentation. Thick films can also enhance analyte dispersion (cf. next section).
3.3.2
Dispersion in FI Liquid-liquid Extraction
In Fl liquid-liquid extraction systems analyte dispersion may occur in four stages of operation: • • • •
dispersion dispersion dispersion dispersion detector
before phase segmentation during extraction in the phase separator after phase separation, during transport to the detector, and in the
In principle. analyte dispersion before segmentation and after phase separation (i.e. stages I and 4) may be controlled as in normal FIA since the flowing stream is composed of a single phase. When high sensitivities are required, the dispersion should be minimized by injecting sufficiently large sample volumes, using short, thin conduits or knotted reactors for transportation to the segmentor and detector. The sample volume should be so chosen that dispersion is minimized not only before the segmentation but also after phase separation. This is particularly important when a large phase ratio is used to achieve preconcentration. Ignoring dispersion effects in the extraction and separation, and assuming that no reagents are merged, 2()0.4()() J£1 sample volumes are normally sufficient to achieve dispersion coefficients close to unity for relatively short conduits of about 30 em (0.5-().7 mm i.d.). However. with a phase ratio of ten, even assuming 100% separation effiCiency, the concentrate phase containing the separated analyte will only amount to a volume of 20 - 40 Jll. Such small volumes may result in large dispersion coefficients during their transport to the detector even in relatively short conduits. In order to minimize dispersion effects in the last stage. the sample volume has to be increased further so that t!Je final concentrate volume will reach a minimum of I 00-200 J£1, the specific volume dej>ending on the length and quality of the transport conduit. To achieve this, instead of increasing the sample loop volume to impractical extents, time-based sampling (cf. Sec. 1.4.7) may be applied. The merging of reagents into the sample before segmentation should also be carefuJJy designed to minimize dilution effects. In order to achieve high phase transfer factors, the extraction coil lengths are usually substantially longer than normal Fl reaction coils. This feature would have been a principle source of analyte dispersion in the extraction system; however, the phase
62
3 Liquid-liquid Extraction
FILM
CONCENTRATION
------2
(
8
•
)
•• •• • •
.(
.
• ·••• •• I•
4
I• I•
---0----- [J --.
m
--- w ---
I
FLOW
--+
Fig.3.7: Schematic diagram showing the analyte dispersion mechanism in a ftow of segmented phases (20).
segmentation hinders the mass transport between segments of the same phase, making dispersion effects less serious. On the other hand, the film formation on the extraction tube walls which is responsible for the high extraction efficiencies also provides a "bridge'" in between neighbouring segments of the tube-wetting phase. This instigates mass transfer from the leading segments backward to the succee~ing segments, causing dispersion of the analyte. The phenomenon has been studied in detail by Nord and Karl berg [20] and Lucy and Cantwell [21 ]. Under the prevailing laminar flow conditions in a straight extraction conduit. the film on the tube walls may be regarded as static. Assuming extraction of the analyte from the aqueous into an organic phase which wets the tube walls, the analyte in a flowing aqueous plug is partially transferred into the organic film surrounding it (Fig. 3.7). On subsequent substitution of the aqueous sample plug by an organic segment, the analyte in the organic film disperses into the bulk of the segment through molecular diffusion and convection by secondary flows generated in the forward movement of the flow. When the next aqueous plug is introduced, mass transfer occurs between the plug and an organic film which contains analyte material from the previous plug. This material is transferred into the next organic segment, causing axial dispersion of the analyte.
3.4 Fl Manif(Jids for Liquid-/1quid l:.xlraclion
63
When the analyte is initially in the film-forming organic phase. axial dispersion will cease when the analyte has been completely transferred into the aqueous phase, since the aqueous plugs will be effectively isolated from each other by the surrounding organic phase. However. before this stage analyte dispersion will proceed in the usual manner. The axial dispersion increases with an increase in the film thickness. This is due to the longer migration distances in the thicker film. and to the larger fraction of organic solvent (containing the analyte) being retained to mix with later segments. Factors which decrease the film thickness are therefore beneficial in reducing the axial dispersion 1cf. Sec. 3.3.1 ). With coiled extraction conduits. where secondary flows in the radial direction develop. the films on the extraction tuhe walls are no more stagnant and axial dispersion is reduced. Nevertheless. unless the film gains th~ same flow velocity as the organic segments. which is extremely unlikely owing to the affinity of the solvent to the tube material, axial dispersion will exist. However. axial dispersion need not be associated with a decrease in sensitivity provided the sample volume is large enough. The phase separator may be an important source of dispersion if the dead volume of the separator is not carefully minimized. An important benefit of membrane phase separators is that the associated chambers or cavities. particularly for outflow of the "clean" separated phase. can be made extremely small.
3.4
FI Manifolds for Liquid-liquid Extraction
3.4. 1
General
The number of variations in the design of Fl manifolds for liquid-liquid extraction hac; surpassed that of any other individual FI separation techniq1.1es. This may be due both to the broad interest in Fl liquid-liquid separation and to the larger number of individual components in the manifold. While this may be beneficial for the user in choosing the best manifold design for a particular application. it poses difficulties for a comprehensive description of the different variations. In this section an attempt is made to characterize the different modes for each of the five basic parts of a FI liquidliquid extraction manifold (excluding the propulsion and detection systems which are described in Chapter 2). Then the user can select the proper mode of each part for a specific application, and integrate the parts to form an appropriate manifold design. A typical FI liquid-liquid extraction separation systems may be divided into the following parts:
64
3 Liquid-liquid Extraction
• • • • •
Sample introduction Segmentation and extraction Phase separation Flow exit Delivery of separated phase to detector
The different operation modes and designs for each part will be discussed separately.
3.4.2
Sample Introduction Modes
Either volume-based or time-based sampling (cf. Sec. 1.4.7) may be used for sample introduction in a FI liquid-liquid extraction system. Volume-based sampling is preferred for separations where high sensitivities are not required, and therefore only small sample volumes are injected. By using a fixed volume sample loop or cavity, low sample volumes down to I 0 Jll may be injected precisely into a carrier stream, whereas the precision of introducing small sample volumes by a time-based approach is relatively low, owing to timing errors and pump pulsations. On the other hand, in liquid-liquid extraction systems where preconcentration effects are pursued, large sample volumes are required both to achieve high phase ratios and to reduce analyte dispersion. The loading and subsequent injection of the large sample loop then substantially lower the sample throughput. Since under such conditions equally good precision may be obtained using time-based sampling. this mode is recommended for liquid-liquid extraction preconcentration systems. It may be necessary to point out here that the function of time-based sampling in FI liquid-liquid extraction differs from other Fl separation-preconcentrations in not being directly related with enrichment effects. When the analyte is collected on/in a preconcentration medium (e.g., sorption column, filter or acceptor solution) before being presented to the detection system, provided the collection capacity of the medium is not exceeded, the enrichment factor will increase linearly with the sampling time. In liquidliquid extraction, the enrichment factor EF is determined mainly by the phase ratio and phase transfer factor. EF will be influenced by the sampling time only when the time is so short that dispersion effects become significant due to the small sample volume. When the sample volume is sufficient for achieving a D value approaching unity, the effect of sample volume on EF will disappear, and a longer sampling period will only be justified when the volume of the separated concentrate is of concern (e.g., for the filling of a second sample loop in parallel determinations). Volume-based sample injections are usually made by injecting samples into a nonsegmented carrier stream which is subsequently segmented by the extractant downstream. Dispersion of the injected sample before segmentation is therefore a matter of concern if loss of sensitivity through dispersion should be avoided. Toei [22] suggested segmentation of the carrier stream prior to the sample injection to reduce the dispersion of
3.4 Fl Manifolds for Liquid-liquid Extraction
65.
small samples. The sensitivity was increased by 50% compared to a conventional system with segmentation following injection. with improvements also in precision and sample throughputs. However. the sample volume used in the study was rather small (20 Ill). and not in the usually injected volume range where high sensitivities are pursued. On the other hand, if necessary, there are also other ways of reducing the sample dispersion during transpon of the sample from the injector to the segmentor. such as using a knotted reactor. Therefore it seems still too early to generalize the advantages of injecting into a segmented flow.
3.4.3
Segmentation and Extraction Modes
General Hitheno. most FI liquid-liquid extractions are performed in a multi-segmented flow comprised of the two immiscible phases. Since the extraction coil is merely an extension of the outlet of the segmentor. the 1wo components may be viewed upon as a single part in the Fl manifold. This pan of the manifold is closely connected with the propulsion system which should meet the demands for the delivery of organic solvents. Although these may be satisfactorily propelled by solvent resistant piston pumps, peristaltic pumps are used more frequently. often making the displacement bottle (cf. Sec. 2.1.1) an indispensable component of this pan of the manifold. For a description on the various modes of segmentation. the reader may refer to Sec. 3.:!.1. However. liquid-liquid extractions may also be achieved with a single segment or without phase segmentation. The single segment method reponed by Toei [18] using a column phase separator has been described in Sec. 3.2.3. Another single segment system reponed by Valcarcel's group (23.24.:!5] requires none of the conventional components for Flliquid-liquid extraction and phase separation. Because of its uniqueness. the system will be treated separatel) in a later section (cf. Sec. 3.4.8).
Extraction by Solvent Circulation In the normal FI extraction mode the enrichment factor is limited by the phase ratio which, in tum. is restricted by practical factors. A relatively complex extraction system was described by Atallah et al.[26] in which the continuously pumped sample is extracted by a small volume of organic phase trapped in a closed loop which incorporates the segmentor, extraction coil, phase separator and detector flow-cell. A simplified schematic diagram of the circulated extraction pan of the manifold in shown in Fig. 3.8 to demonstrate the principles. It differs from other extraction systems in that the separated extractant is not directed to waste after passing through the flow-cell. but is circulated to the segmentor to initiate a new round of extraction with freshly entered ponion of the same sample. The enrichment factor depends on the number of circulation
66
3 Liquid-liquid Extraction
s
Fig.3.8: A manifold for liquid-liquid extraction with solvent circulation. S. aqueous sample: SG. phase segmentor: SP. phase separator: R. restrictor or impedance coil: P. pump: V. v·. valve (two positions): OS. organic solvent: D. detector (26).
cycles achievable in a given time. at the end of which the concentrate is expelled to waste. while new solvent is introduced into the system (valve turned to bottom right position in Fig. 3.8). The system has been applied to the determination of trace amounts of uranium in a synthetic nuclear waste reprocessing solution. achieving an EF value of 4 with 3 ml sample [27].
Non-segmented £rtracrion Using Membranes Extractions from one phase to another phase may be achieved without segmentation of the phases by using a sandwich-type membrane module similar to a gas-diffusion or dialysis membrane separator [28]. The sample and extractant streams flow independently in the two corresponding channels separated by a membrane (Fig. 3.9). Different membranes have been used for various purposes. With a microporous PTFE membrane wettable by the organic phase. the analyte in an aqueous sample is transferred through the membrane into the organic phase flowing on the opposite side of the membrane. The main drawback of this approach is its low phase transfer efficiency. which usually amounlc; to only 8-18 'k of that achievable with segmented extractions. However, the technique may be applied to the analysis of concentrated samples. where its defect may become an advantage in avoiding a dilution step. Such systems can be used for extractions involving two aqueous phases when the membrane is loaded with a suitable organic solvent.
3 ..J Fl M
67
r---+W
AQ -- ----------------- -M OR
'----+
D
FiJ!.3.9: Schematic diagram oi a sandwich-type membrane extracuon module used for non-segmented liquid-liquid extraction. AQ. aqueou~ phase: OR. organic phase: M. membrane: D. detector: W. waste !2R].
Audunsson [29) reponed on a sandwich-type extraction module equipped with liquid membranes. prepared by immersing hydrophobic microporous membranes (e.g. PTFE membranes) in organic solvents for about 15 min. The inert merrt'branes then act as suppons for the immobilized solvent. When an aqueous sample passes by the membrane. non-ionic components in the sample are extracted into the hydrophobic liquid film and transferred into an appropriate acceptor solution on the other side of the membrane. When the acceptor remains stagnant while the sample flows continuously for a defined period, a preconcentration is effected in the acceptor solution, which is subsequently transferred to the detector. The procedure is equivalent to extraction and back-extraction in a single step. More details on such a system used for sample cleanup in gas-liquid chromatograph~ is presented in Sec. 3.7. A non-segmented extraction system_ also based on membrane transfer. but using a silicone rubber tube as the membrane. was described by Melcher [30]. Aqueous samples are injected into a tubular cell which houses the membrane tube. Organic compounds in the sample penneate the silicone rubber membrane and arc collected in an extractant flowing through the bore of the membrane tube. The extractant is then delivered to the detector.
3.4.4
Phase Separation Modes
The different modes of separation applied in FI liquid-liquid extraction manifolds may be characterized by the various separator designs described in Sec. 3.2.3. and will not be elaborated further here. However. liquid-liquid extractions can also be achieved without phase separations. At least two such modes have been reported. As mentioned in the previous section, the one developed by Valcarcel's group [23.24.25) is described in Sec. 3.4.8. In the system reponed by Thommen et al.[31J extraction is performed in a segmented stream in the conventional FI mode. They avoided phase separation by using a specially constructed capillary flow-cell coupled with optical fibers, and by using a specially designed computer software for soning the signals from individual segments. Following extraction, the segmented flow was delivered directly into the capillary flowcell which used optical fibers to transmit light from and to a spectrophotometer. The
68
3 Liquid-liquid Extraction
R
R
w
SP w
a
b
p
c Fag.3.10: Schematic presentation of the flow exit modes of A liquid-liquid extraction systems. For details. see text. SP; phase separator. R. restrictor or impedance coil; D. detector. W, waste: P. pump.
optical fiber was made to illuminate less than J Jll of the cell. the light being transmitted perpendicular to the flow direction. Such an arrangement significantly reduced the interfering effects from refractive index differences in the two phases, making it possible to son out the signals from the alternate segments and achieve "digital phase separation"using a simple soning program. The analyte concentrations in both phases may be monitored simultaneously with this system. While the obvious advantage of the system is its simplicity in hardware and its reliability in operation, an equally obvious demerit is the loss in sensitivity associated with the use of a capillary flow-cell. However, this shoncoming may be disregarded when high sensitivities are not of concern.
3.4.5
Flow Exit Modes
The mode of exit of the residual segmented phase after leaving the separator and the exit of the separated "clean" phase after passing through the detector flow-cell is important for achieving smooth separation, high separation efficiencies and, when membrane separators are used, for prolonging the membrane lifetimes. The different modes of operation and arrangement of the exit conduits are shown in Fig. 3. JO a - c. The exit system in Fig. 3. JO c functions under forced outflow effected through the suction of a
3.-1 Fl Man(fold.r.for Liquid-liquid Extraction
69
pump. which may be the same one used for sample and solvent introduction. This mode of exit is used mostly for gravitational separators with which control of the flow-rate of the separated phase through the flow-cell can improve the reliability of the separation system. When the analyte is extracted from an aqueous sample into an organic solvent. the flow-rate of the latter through the flow-cell (sucked by the pump) is made slightly lower than its inflow to ensure that no aqueous phase is entrained into the flow-cell (cf. Sec. 3.2.3. Gravitational phase separators). The common features for the exit modes in Fig. 3.10 a. b are the incorporation of a • restrictor or impedance coil in the exit conduit of the residual segmented phase and the absence of pump suction. Such armngements are suitable for membrane phase separators. with which the separation efficiencies are readily cont~!,l~d by adjusting the length of the impedance coil. An improved balance of pressure on the two sides of the membrane may be achieved by merging the two exit flows as shown in Fig. 3.10 b. This is reponed to be beneficial for preventing evaporation of the solvent in the separator [ 19). However, it appears that such effects may be valid only if the merged exit conduit downstream of the merging point is sufficiently thin and long.
3.4.6
Modes of Delivery of Separated Phase to Detector
The separated extrdcts containing the analyte are presented to the detector through different modes of delivery. depending on specific features of the detection systems. The modes may be categorized as: • • •
on-line direct delivery on-line collection-delivery off-line collection-delivery
The different modes are shown schematically in Fig. 3.1 I a, b. c. respectively. with an impedance coil as the exit mode. Mode a. which is characterized by continuous delivery of the separated phase to the detector immediately following separation. is used mainly for spectrophotometric. fluorimetric and chemiluminescence detectors, although it is also occasionally used with atomic spectrometric detectors when optimum sensitivities are not required. Mode b is used mainly for flame AA and ICP spectrometric systems. which require specific sample uptake rates for achieving optimum sensitivities. The exit flow-rates of the separated phase are normally too low to meet the demands of the detector, panicularly when large phase ratios are used to achieve high EF values. For this reason a supplementary interface. composed of an additional sampling valve, is used to collect a defined volume of the separated phase (concentrate) under the low flow-rates optimized for the extraction and separation. The collected concentrate is then injected into a carrier and delivered to the detector at the required flow-rates for obtaining optimum detection signals.
70
3 Liquid-liquid E:ctraC'tinn
R
R
w SP
w SP
w a
w b
R
w
SP
+@rw sv
c Fig..3.11: Schematic presentation of the modes of delivery of separated phase to the detector. For details, see text. SP. phase separator: R. restrictor or impedance coil: V. extract collection valve: CR. gas or liquid carrier: SV. extract collection vial: W. waste: D. detector.
Mode c is reserved for detection systems such as electrothermal AAS. which by nature are not suitable for continuous operations. The liquid-liquid separations are therefore completed off-line ao; a sample work-up procedure. The concentrates or extracts are collected and stored in small vessels before presenting to the detector. Therefore the extraction system can be physically separated from the detection system.
3.4.7-
Modes of Derivatization
In Fl liquid-liquid extraction systems, derivatizing reactions may be implemented at almost any part of the manifold to enhance the selectivity or/and sensitivity of the determination ( 13]. Chemical reagents may be merged with the sample immediately after injection, or during the segmentation (as practiced with the dual channel dropping segmentor described in Sec. 3.2.1 ), or in the extraction coil, or merged with the separated phase containing the analyte after extraction. The first three modes may be associated with extraction or determinative chemistries or both, however, the merging of reagents into a segmented stream in the extraction coil (third mode) leaves some doubt upon the regularity of the segmentation after merging. The last mode can be used only for determinative purposes: in spectrophotometric methods it has the disadvantage of limiting the reagent solvent to that of the analyte concentrate in order to avoid interferences from refractive index differences, thus often requiring the use of extra displacement bottles.
3.4 Fl Maniji1ld.1 for Liquid-liquid Extraction
3.4.8
71
FI Iterative Flow Reversal Liquid-liquid Extraction System without Phase Separation
Valcarcel's group [23.24.25) reponed on a Flliquid-liquid extraction system in which none of the typical components for on-line extraction are incorporated (Fig 3.12 a. b). This is achieved by injecting a single segment of sample into a continuously flowing immiscible extractant, using a conventional sample injection valve (or rice \'Crsa). and propelling the injected segment back and fonh with reproducible iterative action in an extraction coil, using the immiscible phase as a carrier stream. Assuming the injection of a hydrophobic extractant segment into an aqueous sample stream flowing in a PTFE extraction coil, the hydrophobic solvent continuously forms a film on the extraction tube wall at the tailing pan of the segment, where analyte transfer from the aqueous phase to the organic phase occurs. The transferred mass disperses into the bulk of the segment when the flow direction reverses. Simultaneously, a new tailing section of the segment is created at its opposite end. thus forming a new site of mass transfer. The highest transfer efficiency naturally occurs at the two ends of the segment. In order to detect the analyte in the extractant without separation. two detection modes are used. The first [23,24] involved the incorporation of the detector flow-cell in the central section of the loop of the injector valve, and is used for extracting the analyte from aqueous to organic phase. The loop and flow-cell is filled with the hydrophobic extractant at the beginning of an extraction cycle. During the extraction, the solvent segment is only allowed to flow half-way through the loop before reversal of the flow direction, so that at no point during the extraction does the aqueous phase enter the flowcell. From Fig. 3.12 bone could see that only half of the extractant segment is monitored. The loop has to be rinsed by a flow of methanol (introduced on the turning of a second v.alve) after each determination cycle to avoid contamination of the flow-cell by traces of the aqueous phase. The peak summits in the recordings in Fig. 3.12 a represent the signals obtained at the tailing pan of the segment. while the valleys represent those from the middle section of the segment. The group of peaks may demonstrate the potential of the technique in studying the kinetic features of the extraction process. The second detection mode was used for transfer of an analyte from the organic to the aqueous phase [25]. The organic sample is injected into a aqueous extractant containing a reagent and carried through an extraction coil to the flow-cell located downstream. The iterative change of the flow direction is synchronized with the injection in such a way that the organic segment approaches, but never reaches the flow-cell. Thus, the analyte transferred into the interfacing section of the aqueous phase is being monitored. resulting in a similar recording as that in Fig. 3.12 a. The method was used to determine total polyphenols in olive oil. A prerequisite for successful operation of an iterative extraction system is the implementation of a precise timing unit, preferably a microcomputer, with a pump capable of rotating in both directions.
72
3 Liquid-liq11id £ttroction
(b)
Fig.3.12: FI iterative flow reversal liquid-liquid extraction system without phase separation. a, schematic diagram showing the conduit connections of the system at two positions of the valve. Left. filling of organic solvent into loop: right. iterative extraction of sample with recorded signal. OR. organic solvent: S+R, sample and reagent: W, waste: D. spectrophotometer detector. b. schematic presentation of the iterative extraction principle. D. spectrophotometer How-cell (23].
3.4 FJ Manifolds for Liquid-liquid Extraction
13
Despite its advantages. the proficiency of the iterative extraction technique in achieving high enrichment effects seems rather limited. owing to restrictions in applying large phase ratios. Although that summation of the peak maximum signals of multiple peaks can improve the sensitivity, it is doubtful that the detection limit can be enhanced through such measures. Another limitation of the technique is that it cannot be applied to detectors which consume the extractant. such as flame AA and ICP spectrometers.
3.4.9
Multiple-stage FI Liquid-liquid Extraction Systems
By directing the separated phase of an extraction unit into another extraction unit instead of into the detector flow-cell. two or more extraction units may be connected in tandem. Thus. highly specific determinations may be achieved after multiple stage extractions, including complicated back extractions to remove interferents in complex matrices. Bengtsson and Johansson [32] reponed a two-stage extraction system used as sample work-up system for the determination of trace heavy metals with ETAAS. Shelly et al.[33 J described an automated three-stage extraction procedure for the isolation of polycyclic aromatic compounds from complicated sample matrices. The system was used successfully as a sample preparation equipment for determinations with HPLC using a video fluorimetric detector. The performance of the multi-stage extraction system was funher optimized by Rossi et al.[34) to improve the analyte reco'very. reproducibility and sample throughput. These authors stressed the importance of optimizing the performance of the entire system, asserting that merely linking several individual successful simple systems does not necessarily produce a working complex system. One of the problems encountered in non-optimized multi-stage extractions. which do not occur in single-stage operations, is back -flushing.
74
3 Liq11id-liq11id £wraction
3.5
Coupling of FI Liquid-liquid Extraction Systems to Various Detectors
3.5.1
Spectrophotometers
General
The spectrophotometer is the most frequently used detector in FI liquid-liquid extraction applications. This may be due to the large number of reagents available which often not only form extractable species with the analyte but also function as chromogenic reagents. A funher reason might be found in the suitability of the spectrophotometric flow-cell"for such applications. The advantages may be summarized as follows: • • • •
no limitations to the flow-rate and flow direction through the flow-cell no limitations to the position of the flow-cell in the FI manifold. such as locating the cell in the sample loop and upstream of the pump the glass or quanz flow-cell is not attacked by organic solvents interference effects due to refractive index differences normally do not occur after the phase separation.
Not all these favourable propenies exist for other detectors such as flame AA spectrometers. However. a main disadvantage of the spectrophotometer flow-cell is the hydrophilic nature of the cell walls and windows. This propeny makes the cell vulnerable to contaminations by accidentally entrained aqueous phase which is difficult to remove. When such contaminations do happen. the difference in refractive index of the two phases will deteriorate the precision of the measurements. However. the cell and extrdction unit may be cleaned by rinsing with a water miscible organic solvent such a'> ethanol. methanol or acetone.
Typical Manifolds
A cunently used typical FI manifold design for liquid-liquid extraction with spectrophotometric detection, integrated from the various functional pans described in Sec. 3.4. is shown schematically in Fig. 3. I 3. The manifold features volume-based sampliag. merging of a reagent before segmentation, use of a merging tube segmentor, delivery of the organic solvent using a displacement bottle, a PTFE extraction coil connected to a sandwich-type membrane separator, an impedance coil connected to the outflow of die sample waste, direct delivery of the separated phase to the flow-cell and merging of die exit conduits from the two phases.
3.5 Coup/in~: ofF/ Liquid-liquid Extruc1wn Svs1ems to Various Detectors
15
c w
R
---+ Water
p
DB Fig.3.13: s,hemati' diagram of a typical Fl manifold for liquid-liquid extra(;lion spectrophotometry. P, pump: DB. displacement boule: C. carrier, R. reagent: S. sample: SG, phase segmentor: EC. extraction coil: SP. phase separator: R. restrictor or impedan'e coil: D. dete(;lor: W. waste.
Other combinations are of course feasible. such as using time-based sampling to introduce larger sample volumes or using dropping segmemors to achieve more reproducible segmentations. This will depend on the individual objectives of the application.
Performance of Fl Liquid-liquid Extraction Spectrophotometric Systems The performance of some selected applications using Fl liquid-liquid extraction spectrophotometric systems are shown in Table 3.1. The examples show that generally a sample throughput of 30-60 h- 1 and a precision of 1-2% r.s.d. may be achieved. The sample volume depends on whether preconcentrations are intended in the determinations. Without such considerations. I 0(}..500 ~I injections are typical. while preconcentrations often involve the introduction of a few milliliters of sample. An interesting extension of the liquid-liquid extraction system for the determination of lead (cf. Table 3.1) reponed by Novikov et al.l60], warrants funher discussion. In order to determine lead at lower concentration levels in. natural waters. the extraction system was combined with on-line ion-exchange column (packed with Chelex-100) preconcentration to improve the sensitivity. In spite of the low precision (9% r.s.d.) of the combined system. which is rather disappointing, the attempt does show the general feasibility of combining different on-line separation techniques in tandem for spectrophotometric determinations. The coupling of Fl liquid-liquid extraction systems to fluorimetric and chemiluminescence detectors are similar to, and no more difficult than the interfacing with spectrophotometers.
3 Liquid-liquid Extraction
76
Table 3.1
Perfonnance of typical FI liquid-liquid extraction spectrophotometric procedures Sample
Analyte
Reagent/solvent
Sample volume (Jll)
I
R.s.d.
(h-1)
(%)
Ref.
Cationic surfactants
water
Orange II/CHCI3
20
60
1.5
3
Anionic surfactants
water
500
60
1.2
9
Molybdenum
plants
2200
30
Manganese
steels
250
24
Lithium
blood urine alloys, soilleachate. sea water beers
methylene blue/ CHCI3 KSCN/Sn(II)/ isoamyl alcohol tetraphenylphosphonium/CHCI3 14-crown-4 deriv. /CHCh DC 18C6* /CHCh/ dithizone
50
>100
600
45
0.5
60
100
60
3.3
61
Lead
Bittering compounds
iso-octane
2 0.97
57 58,59
• dicyclohexyl-18-crown-6
3.5.2
Aame Atomic Absorption Spectrometers
General The majority of applications in atomic spectrometry involve the use of the flame AAS detector. A detailed review of the field is given by VaJcarcel and GaJiego [35] and Tyson [36]. FI liquid-liquid extractions are implemented for different objectives including preconcentration, interference removal and indirect detennination of anions and organic anaJytes. The coupling of FI liquid-liquid extraction systems to a flame AA spectrometer pose no major difficulties, occasional entrainment of traces of aqueous phase into the detector usuaJiy will not produce noticeable effects, while the presentation of the anaJyte to the detector in an organic solvent extract may create 2-3 fold extra sensitivity enhancements for many elements compared to sample introduction in the aqueous phase. However, a few points discussed below are to be noted to produce an optimized interfacing.
3.5 Couplmg of Fl Liquid-liquid Ertractimr Systems to Variou.f Detectors
77
Practical Consideration.\ A specific feature of Fl liquid-liquid extraction systems for flame AAS is the need for adapting the flow-rate of the separated concentrate (almost always the organic phase) to that of the spectrometer\ nebulizing system. which is normally about an order of magnitude higher than the former. This is particularly important when large enrichment factors are pursued by using high sample/extractant ratios. Since it is impractical to apply sample flow-rates higher than approximately 15 ml min- 1• a contemplated enrichment factor of 20 would limit the extractant to less than 0.8 ml min- 1• Excessively depleted sample introduction rates create starvation conditions in the nebulizing system which deteriorate both the sensitivity and precision of the measurement. When higher sensitivitjes are not required, this obstacle may be overcome by compensating the flow-rate difference with an additional flow of the same solvent as the concentrate. This is readily achieved by connecting aT-piece between the injector and the nebulizer, and aspirating by suction the compensating solvent flow through an introduction tube on the side arm. This approach has been used by Sweileh and Cantwell [37] to eliminate interferences from an iron matrix in the determination of zinc following extraction of the Zn(SCNJ: complex from an aqueous sample by IBMK. lron(III) was reduced to the divalent state by ascorbic acid before the extraction, which kept it in uncomplexed form in the aqueous phase in the presence of thiocyanate. However. in most reported FI-AAS systems using on-line liquid-liquid extraction, the delivery mode b described in Sec. 3.4.6. involving a preliminary collection of the separated extractant in the loop of an injector valve is preferred. The collected fraction may then be presented to the nebulizer under optimum flow conditions either using a pumped aqueous carrier stream or simply by freely aspirated air-flow. The latter produced higher sensitivitiel>. [14) probably owing to a further enhancement in the aspiration rate. In liquid-liquid extraction the organic phast: is introduced into the nebulizer almost always in a discontinuous mode. Periodical changes in the flame conditions are therefore inevitable. In order to optimize the flame conditions while making readings for the organic concentrate. the flames should purposely be made leaner in preliminary adjustments while aspirating aqueous solutions. A background corrector is also desirable. though not indispensable. for eliminating background shifts due to changes in flame conditions. When choosing an extraction system one should also be aware that not all organic solvents suitable for spectrophotometric determinations can be used favourably with a flame AA nebulizer-burner system. IBMK can provide excellent performance, and therefore is used most frequently in liquid-liquid extractions for flame AAS. On the other hand, carbon tetrachloride and chloroform, which are often used with spectrophotometric detectors, may pose problems because of their very poor combustion properties.
Typical Manifold Designs A typical manifold for Flliquid-liquid extraction with flame AA spectrometric detection, using time- based sampling, which is suitable for preconcentration and general use,
78
3 Liquid-liquid Extraction
v
• mllmln
RG
1.8
s
16
Water
0.8
AAS
w
--------w aoo ••
D p
Fig.3.14: Schemal.> diagram of a A manifold for liquid-liquid extraction flame atomic absorption specnometry. P, pump; RG. reagent; S, aqueous sample; SG, phase segmentor, D. displacement bottle: E. extraction coil: PS, membrane phase separator: R, restrictor or impedance coil: W, waste: V, injector valve: AAS, flame atomic absorption spectrometer (14].
is shown in Fig. 3.14. The carrier for the final introduction of the collected extract may be either pump delivered water or freely aspirated water or air. However, our group has observed a doubling of the sensitivity for lead, following a KI/IBMK extraction, when using air, compared to water [14]. Sample change may be achieved during injection of the collected extract into the spectrometer. During this stage the extract is directed to waste. The duration of this sequence should be long enough to avoid carryover between samples within the extraction system. During the extraction sequence, the loop filling time should also be sufficient to produce an extract volume at least 30% larger than the loop volume (usually 1~200 ttl) in order to prevent carryover between samples in the loop. The implementation of a knoned reactor loop may be helpful in reducing dispersion and hence the minimum washing volume required for avoiding carryover. The loop filling time directly affects the sample throughput, and therefore the concentration efficiency of a preconcentration procedure. It appears that the filling time could be shortened by using higher total flow-rates for the sample and extractant. However, Nord and Karlberg [38] have observed a significant reduction in the phase transfer efficiency above a total flow-rate of approximately 7 ml min- 1, which cannot be improved by prolonging the residence time. They ascribed the phenomenon to the disruption of small
3.5 CtJupling of Fl Liquid-liquid Extraction Systems to VoritJU.t DeteciOrs
79
organic segments under high flow.:rates. which decreased the extraction efficiencies. Yet the evidence for the explanation seems not to be sufficient. and high phase transfer factors have been achieved in the author's laboratory at high total flow-rates of 16.8 ml min- 1 [14].
Preconcentration Systems Despite the great potentials of using FI liquid-liquid extraction for achieving analyte preconcentrations for flame AAS. the number of applications are surprisingly few compared to sorption column preconcentration procedures. The performance of some published procedures ( 14,38.39] are shown in Table 3.2. Despite the additional enhancement effects (rom the organic solvents. the concentration efficiencies and consumptive indices generally tend to be inferior to those achievable by sorption procedures (cf. Sec. 4.6.3). However. the technique may be more tolerant to interferences from complex matrices, and certainly warrants further exploitation in its application to trace analysis of complex samples.
Table 3.2 Performance of Fl liquid-liquid extraction preconcentration procedures for flame AAS Analyte Reagent/ solvent
Sample. volume
Phase ratio
EF*
CE*
(min- 1) (ml)
(mil
CJ
f
R.s.d
Ref.
1.0
(h-1) (%)
APDC!IBMK APDC!IBMK
4.0
13
19
13
0.21
40
Zn
4.0
10
16
II
0.25
40
6
38 38
Pb
APDC!IBMK
4.0
12
23
15
0.17
40
4
38
Ni
APDC!IBMK HDEHP/IBMK
4.0
10
18
12
0.22
40
4
In
32
10
>10
KI/IBMK
12
20
60
1.5 1.4
38 39
Pb
60 60
Cu
>10 <0.2 60
0.20
14
EF* and C£". enrichment factors and concentration efficiencies including organic solvent enhancement effects.
Indirect Methods Indirect determinations using flame AAS based on the formation of ion-pairs have been used extensively by Valcarcel's group [40,41,42,43.44] for the determination of perchlorate, nitrate and nitrite and surfactants. The extraction system used were similar to that shown in Fig. 3.14, except that the outflow of extractant from the injector loop was controlled by a pump instead of using an impedance coil. The determinations are based on extraction of ion-pairs formed between the analyte and a reagent containing the metal
80
3 Liquid-liquid Extrac·tion
species (often copper) which is determined by AAS. Further details on the methods are given in Sec. 8.2.3 and 8.3.2. The aqueous/organic phase ratios were relatively low in these applications, since preconcentration was not the main objective of the determinations.
3.5.3
Electrothermal Atomic Absorption Spectrophotometers (ETAAS)
Direct introduction of extracts into a graphite furnace atomizer, following a liquidliquid extraction, is not feasible, owing to the discontinuous nature of ETAAS operations. Earlier systems on combining Fl liquid-liquid extraction with ETAAS were off-line sample work-up systems [45,46]. The systems all involve the use of a double extraction method based on the same chemistry. The dithiocarbamate complexes of trace metals are extracted into Freon 113. After phase separation, the extract is segmented by an acid solution of mercury(ll). which forms stronger complexes with dithiocarbamates, thus displacing the analyte metals. Following a second separation. the organic phase containing the mer,ury- dithiocarbamate is discarded, and the aqueous phase containing the back-extracted metals is collected and determined by ETAAS. More recently. the method of Backstrom et al.[45] was further refined to produce an on-line method which is programmed to operate ih parallel with an ETAAS system [4]. The final extract is collected in a 23 ttl sample loop of an injector, and subsequently delivered into the graphite furnace using a low air flow provided by a peristaltic pump. The system has been used for the determination of Cd, Co. Cu, Fe, Ni and Pb. achieving 50-I 00 fold enrichments at sampling frequencies of 30 h- 1 with precisions of 1.5-2.7% r.s.d., and yielding detection limits lower than 5 ng 1- 1•
3.5.4
ICP Spectrometers
The number of applications of FI liquid-liquid extraction separation to ICP emission spectrometry (ICPES) is rather few [47,48,49.50]. The coupling of extraction systems to the ICPES detec!or pose less difficulties in terms of differences in flow-rate requirements, since the normal uptake rates of the ICP nebulizers are much lower than flame AAS nebulizers. However, the plasma is more sensitive to changes in solvent conditions, sometimes requiring stringent control of the completeness of aqueous phase exclusion from the introduced extract. Manzoori and Miyazaki [49] reported on an indirect method for the determination of fluoride. The method involves the formation of lanthanum/alizarin complexone/fluoride complex and its extraction into hexanol containing N,N- diethylaniline. Fluoride is determined by introducing the separated organic phase into the plasma and measuring the intensity of the La II 333.75-nm line. These workers found it necessary to connect two
3.5 Coupling of Fl Liquid-liquid Extraction Systems to Various Detectors
81
membrane separators in series in order to completely remove the aqueous phase from the separated extractant. Entrainment of traces of water into the plasma caused severe noise. Menendez Garcia et al.[50] combined on-line liquid-liquid extraction separation with hydride generation gas-liquid separation for the determination of arsenic with ICPES. Arsenic in the aqueous sample is extracted as Asl3 into xylene which is continuously mixed on-line with sodium borohydride in dimethylformarnide and acetic acid solutions. Arsine is generated in the organic phase and separated in a gas-liquid separator which prevents most of the xylene vapour from entering the plasma. The method was used to improve the sensitivity and to remove interferences from transition metals in the determination of low levels of arsenic in white metal, cast iron, cupro-nickel etc ..
3.5.5
Coupling of FI Liquid-liquid Extraction Systems to Gas and Liquid Chromatographs
General Fl liquid-liquid extraction systems may be coupled to gas and liquid chromatographs in all the three modes presented in Sec. 3.4.6. The objectives for the separation may involve sample cleanup or/and preconcentration for gas and liquid chromatography, but also post-column derivatization for the latter. in fact most applications in the latter field belong to this category.
Coupling to Gas Chromatographs (GC) The earliest application in this field was an off-line procedure involving collection of the extract in vials before the gas chromatographic determination [51]. The method was used for the determination of terodiline in blood serum. The first on-line coupling of a liquid-liquid extraction system to GC was reported by Roeraade [52] in I 985 in the determination of traces of aliphatic and aromatic hydrocarbons in waste water. achieving high sensitivity and precision, but involving rather complex instrumentation. A simpler system was reported by Fogelquist et al.[53], which was used for the determination of halocarbons in sea water. However, the coupling involved relatively large modifications in the GC instrument, rendering it unsuitable for normal routine sample injections. A more recent design, reported by Ballesteros et al. [54], and shown schematically in Fig. 3.15, appears to be free of the deficiencies mentioned above. A small sample volume (4 Jl.l) HPLC injection valve (V) was used to collect the separated extract after removing the last traces of aqueous phase using an on-line desiccating minicolumn (DC). The valve was connected to the chromatograph through a piece of 0.5 mm i.d. stainless steel tubing by inserting its needle end into the injection port (IP) of the chromatograph.
82
3 Liquid-liquid Enraction
OR
s
D
p
GC Fig..3.15: Schematic diagrdi'Tl of an on-line Fl sample cleanup system for a gas chromatograph. OR. organic solvent: S. aqueous sample: SG. phase segmentor; EX. extraction coil; SP membrane phase separator: W. waste: DC. desiccator; V. 6-pon injector valve; B, heater: SC. stopcock: I. injection pon of gas chromatograph; D. detector: GC. gas chromatograph [54].
The tube was heated electrically to 25-175°C. On actuation of the valve, the collected extract was injected into the chromatograph introduced by the GC carrier gas. Using ethyl acetate as extractant. four phenols were determined in water samples with a precision of 0.8-3.0tf( r.s.d .. The sample throughput capacity of the extraction system is 30 h- 1• but actual sampling rates are determined by the GC programme. When n-hexane is used as extractant. acetic anhydride may be incorporated in it as a derivatization reagent for the determination of mixtures of phenols. cresols and chlorophenols using the above system. Audunsson [29.55] reponed on the coupling of an on-line liquid membrane extraction system to GC. The combined system was used for sample clean-up and preconcentration of p.g 1- 1 levels of amines in urine. The liquid membrane exiraction system is described in Sec. 3.4.3 which features a stagnant acceptor solution to enrich the separated analyte during the extraction. After the preconcentration period, the aqueous extract in the collection groove of the extraction unit was displaced by fresh acceptor stream and carried to the GC instrument which was equipped with an automatic sampling interface. A 1.510 p.l heancut of the displaced extract was made when the injector of the interface was activated, introducing the injected volume into the heated injection pon of the GC. The' extract flow was interrupted for about 5 s during the injection, inducing large pressure fluctuations in the system. Such disturbances were minimized when the acceptor stream was stopped during the injection. Using this system, it was possible to decrease the analyte concentration a thousandfold without alteration of the precision which was 3.5-4% r.s.d. for amine concentrations down to I JJ.g 1- 1 in urine.
3.5 Coupling of Fl Liquid-liquid Ertraction Systl'm.1· to Various Detectors
83
Coupling to High Performance Liquid Chromatographs (HPLCJ The post-column connection of an HPLC to Fl liquid-liquid extraction appears to present no major difficulties. and effluent from the chromatograph column may be connected to the solvent segmentor of the extraction system or between this point and the extraction coil. The pre-column connection of an on-line liquid-liquid extraction system with an HPLC involves filling the separated extract into the sample loop of an HPLC injection valve. The sequence of liquid-liquid separation and the actuation of the valve should be synchronized to ensure reproducible injection of an appropriate section of the extract. An example of such a system is that reponed by Farran et al.l56) for the determination of pesticides using n-heptane as extractant.
4
4.1
Sorption
Introduction
Non-chromatographic separations based on ion-exchange and adsorption have been used extensively for enhancing the selectivity and sensitivity of analytical methods. Although most procedures involve some kind of continuous flow operation, nevertheless they are mostly off-line batch procedures which require considerable operational efforts. Automation of sorption separation procedures is therefore a topic which has attracted much interest. Fl on-line separation and preconcentration by sorption is an area which has shown great promise in this respect, and in fact, is an area which has become one of the most active research fields in automated solution analysis in recent years. In FlA. on-line separations by sorption are always connected with the use of columns. The first attempt in implementing an ion-exchange micro-column in a Fl system was that made by Bergamin et al.ll] for the preconcentration and determination of ammonium ions reponed in 1980. This was followed by a contribution by Burguera et a1.[2] who reponed on the successive elution of zinc and cadmium from an ion-exchange column with subsequent determination of the metals by chemiluminescence. However, the development of the technique was rather slow before the application of Fl column preconcentration to flame AAS. the first contribution of which was made by Olsen et al. in 1983 (3j. The work opened a major period of development not only for Fl column techniques. but also for AAS. with an exponential growth of related publications in the following years. Hitherto. the majority of publications on Fl column separation and preconcentration are connected with atomic spectrometric methods, particularly flame AAS. This is not at all surprising. considering the dramatic effects demonstrated by such combinations. However. the Fl bibliography also shows a steady growth of the number of publications on using on-line column separation and preconcentration for other detection systems, including spectrophotometry. ion-selective electrode, contluctimetry. ICP-MS, fluorescence, etc .. The general merits of FI separation and preconcentration summarized in Chapter I are all well demonstrated by Fl sorption column techniques. The technique is inherently easier to operate than other separation methods, and the equipment generally more robust. An additional benefit over other separation techniques is its extremely high versatility, owing to the availability of a broad range of choice for different sorbents, complexing systems, and eluents. However, there are some restrictive features which are characteristic
86
4 Sorption
of such techniques which may have to be kept in mind for the proper design of FI column separation systems : a
b
c
4.2
The on-line packed columns create different degrees of extra flow impedance. the extent depending on the geometrical dimensions of the column and packing material, as well as the liquid flow-rate through the column. The requirements on the pump quality is therefore more stringent than other separation systems. Preconditioning of analyte collection medium (i.e. the packed column) necessary following each elution, a sequence which is not required in other separation methods. A sample loading and elution stage is always included in the separation procedure, between which drastic changes in the composition of flow occur. This feature may create difficulties with some detectors (cf. Sec. 4.6.6)
Classification of FI Column Techniques
In the classification scheme in Sec. 1.4.1, the first three entries under liquid-solid separation methods. i.e., ion-exchange, adsorption, and sorbent extraction, all belong to column separation techniques. While in the batch approach, separations based on these principles may be performed either by static equilibration or by a column technique. online columns are invariably used in FI separations, both for convenience and efficiency. FI column separation systems based on different sorptive mechanisms do not differ strongly in the principles of system design and optimization of operational parameters. Therefore. the principles discussed in the following sections are genenilly applicable to the different approaches. Differentiated by the purpose of separation, FI column separation systems may be classified into: • • •
Systems used exclusively for separation. i.e. for selectivity enhancement. Systems used for separation and preconcentration, i.e. for sensitivity enhancement (often achieved with an improvement in selectivity). Integrated systems used both for preconcentration and in situ detection on the column (solid phase optosensing).
Those methods belonging to the first category are generally less demanding in system design, and in the simplest case a separation system may be constructed by merely connecting a column to the sample loading line. The discussions in Sec. 4.3 and Sec. 4.4 refer mainly to separation systems belonging to the second category, which also forms the
-13 Di.tprrsion in Fl Column Pn·com·cllfi'Otion System.t
87
bulk of publications on Fl sorptive separation methods. However. the principles outlined in these sections will no doubt also be useful for the design of systems belonging to the other categories.
4.3
Dispersion Systems
In
FI Column Preconcentration
The importance of dispersion control in FIA has been stressed in Chapter I. This is a vital factor in the optimization of FI column systems designed for sensitivity enhancements, and should be taken into consideration at all phases of method development. The fundamental and practical aspects for minimizing dispersion in the different sequences of operation are discussed separately in the following sections.
4.3.1
Dispersion in Sample Loading
One of the characteristic features of FI column preconcentration procedures is that the analyte has to be sorbed on a column before elution into the detector. This collection process is often termed ··sample loading .. in preconcentration procedures, and should be differentiated from filling samples into a sample loop, sometimes also referred to as sample loading. The Iauer is a sequence for all FIA procedures excepting those which introduce samples based on timing. and should better be designated as '"sample filling··. Either volume-based or time-based sample loading may be applied for column preconcentration. The merits and demerits of the two approaches have been compared in Sec. 1.4.7, with general preference towards time-based sample loading systems. With time-based loading, the sample dispersion in this stage can often be neglected. since it precludes the use of sample loops - often the main source of dispersion. In order to obtain high enrichment factors in preconcentration systems, sample volumes are usually much larger than those used for normal FlA. Large sample loops measuring a few milliliters capacity are quite common in volume-based preconcentration systems. In such loops sample dispersion into the carrier stream during transport to the column may be quite significant, particularly at the tailing edge of the sample bolus where the sample zone has to travel the entire length of the loop before reaching the column. The result is a prolongation of the loading period if the sample is expected to be completely transferred. The dispersion is most serious when large diameter sample loops made of > 1.2 mm i.d. tubing are used to decrease the loop length when 5-10 ml (or even larger) sample volumes are loaded. Although dispersion may be effectively decreased by using
88
4 Sorption
narrow bore tubing of 0.5 mm i.d., especially in a knotted reactor configuration, this will produce excessive back pressure due to the long loop length of, occasionally, more than ten meters. This is one reason why usually time-based sample loading are preferred over volume-based sample loading. Practical measures for decreasing the dispersion in volume-based sample loading will be discussed in Sec. 4.4.2.
4.3.2
Dispersion in Sorption and Elution
Here the term ''dispersion" is used in a more general sense, not only referring to the spatial distribution of the analyte in solution, but also in the column. The dispersion of the analyte during sorption and elution is influenced by at least the following factors:
• • • • •
the the the the the
geometrical dimensions of the column propenies of the sorbent material (in relation to the analyte) propenies of the eluent eluent flow-rate ~esign of flow system in relation to the column.
On-line columns packed with inen packing materials usually have very limited effect on the dispersion of an injected sample. When an active sorptive material is substitured in the column. the sorbent contributes significantly to the dispersion characteristics of lhe sorbed analyte. Despite the differences between Fl column preconcentration and HPLC discussed in Chapter 1. the sorption and release of the analyte in Fl on-line column is in principle a chromatographic process. and it may be used to predict and explain the dispersive behavior of the analyte within this sequence. In HPLC the injected sample volumes are usually small: before elution. the loaded sample is found as a thin band located at the end of the column where the sample is applied. In Fl column preconcentration the sorbed analyte band is acted upon by the large volume of sample solvent even before the elution stage. The sample band is dispersed downstream in different degrees, depending on the distribution coefficient of the analyte between the stationary phase (column packing) and the mobile phase (the sample solvent). Thus, breakthrough of analyte may occur before the saturation of lhe column capacity. It is not sufficiently clear how the dispersion characteristics of this "pre-elution" during sample loading will influence the dispersion in the final elurion, even without breakthrough. A mathematical model of the elution process for on-line column preconcentration proposed by Li and Tsalev [4] shows that the elution peak form is independent of the analyte distribution on the column. However, the system they used for modeling was a rather idealized one, which composed of a micro-column packed with silanized glass beads impregnated with a very thin layer of sorbent. 1be situation might be somewhat different when the sorbed analytes reach into the depth of the sorptive materials.
4.3 DiJpcr.ficm ;, Fl Column Prcco11cc'lllraticm Sy.~tcm.\
89
The on-line monitoring of the elution signal i:,. an important feature for FI on-line preconcentration which endows high efficiency to the method. In batch ope~ations. column preconcentrat1on!> are almost always made by collecting the entire eluate fraction containing the con~entrated analyte. The readout is then based on a homogeneous solution with an averaged concentration of the different eluate fractions. In Fl column preconcentration. the peak height of the elution peak is almost invariably used. i.e., the measurements are made on that eluate mini-fraction which contains the maximum analyte concentration. Because of this peculiarity of the Fl on-line approach. the elution peak form and dispersion characteristics are much more important than in batch methods. The importance of the peak width on the enrichment factor is explained in Sec. 1.4.8. Experimental parameters should therefore be carefully optimized to produce sharp elution peaks. The dispersive characteristics· in this process is different from those of an open tube FI system. and "dispersion" should preferably be used as a chromatographic term, involving not only molecular <.liffusion and flow convection, but also mass transfer between the stationary and mobile phases.
4.3.3
Dispersion in Eluate Transport and Post Column Reactions
After elution from the column, the eluate carrying the concentrate zone may either be ttansported directly to the detector or be further processed to produce a detectable species before being transported to the detector. The former case refer to systems where the analyte can be determined without transformation, e.g. in AAS. ICPES or selective electrode potentiometric measurements. The latter category include most applicatiom. in spectrophotometry. fluorimetry and chemiluminescence. which usually require a post column reaction. In both cases. minimization of dispersion in the conduits between the column and detector is essential for obtaining better enrichment factors. For a direct transportation. the shortest and thinnest conduits that are practical should be used in order to avoid unnecessary dispersion. The dispersion contribution of various transport conduits in FI-AAS systems have been studied in detail. and the importance of tube diameter. length and configuration have been stressed [5]. The dispersion contribution of this seemingly unimportant system component may often be overlooked. However. to obtain optimum performance. careful minimization of dispersion is required. especially when the columns are small. and the concentrate zones in the eluate well-focused. A shortest possible length. not exceeding 20 em of 0.35 mm i.d tubing has been recommended for the transport conduits in AAS applications, and should be also applicable to other detection systems which do not require post column reactions. In systems where post column reactions are required, reagent streams are usually merged with the eluate stream via a T-piece. Often the pH condition of the eluate may not be suitable for the reaction. in which case an appropriate buffer has to be introduced either with the reagent or before hand as a separate merging stream. Then, a certain length of reaction coil is necessary to allow sufficient development of the reaction. All these
90
4 Sorption
factors contribute to the dispersion of the eluted analyte zone, and should be optimized carefully so that the preconcentration effects will not be lost significantly in post column reactions. Practical measures to decrease dispersion in this stage are discussed in a later section.
4.4
Practical Considerations in the Design and Operation of FI Column Preconcentration Systems
4.4.1
Column Designs
The column design strongly influences the performance of preconcentration systems. The capacities of the columns used in FI preconcentration range from 15 ~tl to over 400 ~tl. depending on the specific purpose, but are generally much smaller than those used in batch procedures. In different reports, column diameters may be as small as 1.5 mm. or as large as 7.5 mm. The optimum column design for achieving high efficiency and low consumptive index depends on several factors, including: the sample loadi• rate and volume, the breakthrough capacity of the sorbent for the analyte, the specificity and particle size of the packing material, and the extent of interference. Column performance will also depend considerably on the specific properties of the column pacJcinl. Nevertheless, some general guidelines on the design of columns for FI preconcentration have been suggested for atomic spectrometric applications [6]. These should be generally applicable to other detection systems as well: a
b
With columns of identical capacities, and provided that the breakthrough caJ*ities are not exceeded, large aspect ratios give higher enrichment factors and show stronger tolerance to interferences. The aspect ratio is defined by Marshall and Mottola as the ratio between the column length L, and average column diameter d, i.e. L/d [7]. However, the upper limit of EF values achievable ua.g columns with large aspect ratios may be limited by the extensive back pressURS generated at high sample loading rates. With columns of similar aspect ratios, the larger the column capacity, the strongu the tolerance to interferences, and the lower the EF value. For flame AAS ..t ICP spectrometry, columns of 100 ~tl with aspect ratios of 1~15 seem to be a reasonable compromise for achieving favorable performances both in semitivities and recoveries. Admittedly, such columns are relatively small and may
4.4 Pratical Considerations
91
E
\
(a) E
(b)
B
(c) E
Y~g.4.1:
On-line packed columns. a, uniform-bored column with push-fit connections; T. Tygon tubing; F, plastic foam or screen; R, sorbent packing; E. PTFE tubing. b, uniform-bored column with threaded fitting connections; A, plexiglas or PTFE column body, B. threaded fitting; C. 0-ring; D, conical insen; F, plastic foam or screen; R. sorbent packing; E, PTFE tubing. c, conical micro-column made from a pipet tip with push-fit connections; P. section of Eppendorf pipet tip; S, thick-walled silicon rubber tubing; F, plastic foam; R, sorbent packing.
c
not pennit large sample volumes with extended loading periods, nevertheless, long preconcentration periods are not recommended when optimum efficiency and consumptive features are pursued, and this should not be taken as a serious demerit. Finer particle size of the column packing improves the breakthrough capacity and enhances the EF values by producing sharper elution peaks. However, the increased back pressure will limit the sample loading to lower flow-rates and restrict the highest achievable EF. For packings having favourable kinetic properties, a reasonable compromise might be a particle size of 80-100 mesh, at a loading rate of 8-9 ml min- 1•
92
4 Sorption
d
Smaller column-capacities of 15-20 Ill (with decreased loading rates) are required for some applications which have strict limitations on the concentrate volume. such as for graphite furnace AAS, whereas columns used for hydride generation AAS require larger capacities of more than 250 Ill. owing to the large dead volume of the FI manifold used for hydride generation AAS.
When properly made. both columns with push-fit or threaded fittings for connections to the valves and nebulizers are satisfactory for preconcentration purposes. Nevertheless. columns with threaded fittings are strongly recommended for long term reliability and better tolerance of. high sample loading rates. Columns with end fitting~ and threading either inside or outside the columns have been reported, and there seems little to choose between the two alternatives. Some reported column designs are shown in Fig. 4. I. A commercially available column with a conical cavity. designed for Fl column preconcentration. has proved to be both convenient and reliable over long periods of continuous o~ration. The structure of the column is shown in Fig. 4.2.
F
Fig.4.2: Schematic diagram of a Perkin-Elmer conical column used for on-line preconcentration. B.. outer column housing: C. conical column with lid. L: A. threaded fittings: 0. 0-rings; F. porou~ plasuc filters: E. PTFE tubings with flanged ends; and R. sorbent packing
.J..J Protico/ Considerations
4.4.2
93
Column Loading
Sample Loading Rates
General discussions on the benefits and limitations of time-based and volume based sampling are given in Chapter 1. For preconcentration by sorption, the sampling sequence is always associated with loading samples on a packed column, either directly, by aspirating the sample from its container (time-based sampling), or indirectly, by displacing the sample from a fixed volume loop using a carrier stream (volume-based sampling). The EF value of a preconcentration system is directly related to the amount of sample loaded on the- column, which in tum is determined by the loading time and the loading flow-rate. Volume-based sampling usually takes more time to load the same volume of sample when compared to the time-based alternative owing to stronger dispersion effects (cf. Sec. 4.3.1 ). The dispersion may be minimized by using sample loops with a knotted configuration (cf. Sec. 2.3.2), or by introducing small air segments at the two ends of the sample zone. The latter approach was proposed by Xu et al.[8] for preventing dispersion between the sample and elueni zones in a time-based sampling preconcentration system for flame AAS. but. with some modification, may equally well be used to limit the sample dispersion during loading in a volume-based system. The introduced air segments will not interfere in the final determinations even using a spectrophotometric detector, since they will have left the system when elution is initiated. Although large loading rates are desirable for achieving high EF and C£ values. the maximum loading flow-rate~ are limited by both the kinetic features of the sorptive packing material and the capability of the fluid delivery system in maintaining stable flow under high back pressures. In most cases, kinetic requirements are the limiting factors. and most good quality peristaltic pumps would perform sufficiently well even at the upper limits set by kinetic requirements. Generally, the linear flow-rate of the sample through an on-line column is much higher than in batch procedures where samples are often loaded by gravity. the contact time of sample and sorbent for FI systems being often less than a second. The kinetic requirements on the column packings are therefore much more important than in batch procedures (cf. Sec. 4.5.1). Excessively high loading rates will inevitably lead to incomplete retention of the analytes due to insufficient contact time between sample and packing, even before the breakthrough capacity has been reached. Sensitivity enhancements gained by increasing the sample volume will therefore be panially lost through a decrease in the phase transfer factor P (cf. Sec. 1.4.6). With a defined loading period, the amount of analyte retained on the column, reflected in the analytical response of the eluate, always reaches a maximum with an increase in sample loading rate in time-based sampling. beyond which an increase in sample volume leads to decrease in signal due to insufficient contact time. Although complete retention of the analyte is not a prerequisite for FI procedures, systems with excessively low P values are more vulnerable to interferences. The optimized sample "loading rate should therefore be based on an overall consideration for sensitivity and
4 Sorplion
94
selectivity. and often reflects a compromise between them. A reasonable sample loading rate (carrier flow-rate in volume-based loading) to stan with is 4-5 ml min- 1• Carry-over and Cross Comamination in Column Loading
Time-based sampling is achieved most conveniently using peristaltic pumps. the sample being transferred directly through the pump tube before loading on the column. This may produce carry-over and cross contamination problems through sorption of the analyte or matrix constituents on the tube walls. Sample loading by pump suction downstream of the column may avoid such defects: however. such operations are not recommended because the suction through a tightly packed column is significantly weakened. resulting in depressed and unstable solution flow rates which deteriorate the performance of the preconcentration. Furthermore, if samples are to be sucked into a column. they have to be de-gassed before loading, otherwise air bubbles created in the conduit and column will degrade the precision of the flow-rate. This would make the procedure very inconvenient. However. time-based sample loading by pumping through pump tubes onto the column can be achieved efficiently and precisely by ~aking some precautions and following the guidelines mentioned below 16]: a
b
c
As a warm-up period, peristaltic pumps should be allowed to run under working conditions by pumping water through the column for about 15 min. during which the flow-rate is likely to drift. The sample volumes introduced will vary, and results collected in this staning-up stage will not be reliable. When programmable pumps are used in an automated on-line preconcentration system. it is advantageous to increase the revolution rate of the sampling pump during the elution stage to speed up the changing over of samples in the pump tube in order to increase the sampling frequency. particularly when the elution period is short (e.g. 10 s). A segment of air is always introduced in between samples when the sampling probe is lifted out of the previous sample solution. and acts as a barrier: dispersion at sample interfaces will therefore be negligible even at high flow-rates. Sampling pump tubes should be restricted to the shortest possible lengths in order to change samples rapidly and decrease sample consumption. Some adherence of sample constituents on the pump tube walls will be inevitable: this may be washed off by the leading section of the next sample. which is pumped to waste before the commencement of the loading sequence. The time and sample volume required for the washing will depend on the area of pump tube walls. and therefore the length of tubing exposed to the samples. Short pump tubes are therefore desirable and may be prolonged in order to reach the sample solution and other components of the manifold by connecting pieces of 0.5 mm i.d. PTFE tubing. which act as sample probes.
4.4 Pratical Con.fidtration.f
95
Waste Effluent Discharge
Another practical consideration in sample loading is on the route for discharge of effluent from the column. It may not be of consequence for some applications whether the effluent is discharged through the flow-cell or a separate waste line. However, in many cases the effluent may exen a deleterious effect on the flow-through detector. Such examples include introducing sample effluents with high dissolved solids contents into a flame AA or ICP spectrometer nebulizer-atomization system, or the introduction of sample effluents containing constituents which may poison an ion-selective electrode flow-through detector. Simple manifold designs often allow the waste containing the sample matrix to flow into the detector during sample loading, but whenever necessary, discharge of the effluent to waste is easily accomplished using suitable valve and manifold designs (cf. Sec. 4.6.3), and such designs are recommended for long term trouble-free manipulation even if no deleterious effects are observed with simpler designs.
4.4.3
Column Washing and Equilibration
In batch procedures for column preconcentration, the operation almost always go through two column washing stages before and after sample loading. The columns are usually equilibrated by washing with a buffer solution at the pH required for sample loading before the preconcentration. and washed again with distilled water after the sample loading to remove the residual sample in the column before elution. Such procedures are simulated in Fl column preconcentration using volume-based sampling by using the buffered sample carrier as a washing solution to achieve the above mentioned goals. However. this precluded use of the more efficient time-based sampling. or require more sophisticated manifolds with separate lines for washing. which all deteriorate the efficiency. After a detailed study on the effect of such washing treatments in Fl on-line preconcentration with flame AAS. Fang et al.(9] concluded that washing and/or equilibration procedures showed no advantages in terms of sensitivity and precision over a simpler and more efficient system without such sequences. Admittedly. when an equilibratio~stage is excluded, breakthrough of the analyte cannot be avoided during the initial stage of sample loading. owing to unsuitable pH conditions created during the previous elution. However, the loss is reproducible. Since samples usually are buffered during loading. this early stage of sample loading is used as a sequence for column equilibration, with the same function as equilibrating with a buffer solution. The loss of sample is not excessive, since normally equilibration may be achieved in a few seconds. Yet, this results in a simpler and more efficient column preconcentration system. The introduction of small air segments between the sample and eluent zones, as proposed by Xu et al.[8], can further shonen the time for equilibration, and a 40% increase in EF was reponed, compared with a similar system without the introduction of air segments.
96
4 Sorption
Washing columns with distilled water after sample loading was shown to create losses of analyte due to a deviation from the optimum pH. but was reported not to produce any favourable effects with flame AAS [9]. This may not remain valid for other detection systems where the residual sample; matrix may interfere with the final determination in the eluate. Thus. washing or using a carrier to transport the sample to the column is often necessary in spectrophotometric applications to overcome matrix effects (cf. Sec. 4.6.6. Compensation of Schlieren effects). The washing step after sample loading was also found to be indispensable in graphite furnace AAS systems [ 10]. especially when the sample contains substantial amounts of dissolved solids. Although extremely small columns were used. the introduction of residual sea water sample remaining in the column created such intensive background absorption that corrections were not feasible. A washing step may also be necessary for JCP spectrometric applications where matrix elements exert strong spectral interferences on the analyte. The Fl preconcentration system for graphite furnace with a column washing sequence is shown in Fig. 4.11.
4.4.4
Elution
Eluent Requirements
a
b
c
d
e
In on-line elution. the kinetic features of the process are much more imponant than for off-line batch procedures. Weak eluents requiring long equilibrium periods may be used successfully for off-line procedures, but cannot be used for on-line applications, since slow elutions may significantly degrade the enrichment factors and/or the concentration efficiencies. The eluent should not attack the packing material noticeably for at least hundreds of elutions. This may not be a vital factor for off-line procedures. since sometimes the sorbent may even be digested to release the analytes: but an on-line system is expected to run for days and weeks without changing the columns. Highly concentrated acids and bases may be effective eluents and may not be harmful to the sorbent; however. they may create problems in some detectors such as the AAS nebulizing systems through corrosion or blockages. On-line modification or dilution of the eluates are feasible. but only at the expense of a sacrifice in sensitivity. Schlieren effects occurring at the interface between sample/carrier and eluent in spectrophotometric determinations can be a serious source of interference (cf. Sec. 4.6.6). Therefore, the refractive index of the eluent should be as close as possible to that of the interfacing sample, carrier, or wash solution in such applications. In the atomic absorption spectrometer, organic solvent eluents may create additional enhancement effects. The effect of organic solvents in flame AAS is
.J ..J Pratical Con.fidrration.1·
97
well known. and may be conveniently exploited to create higher enhancement factors in the preconcentration. This feature should therefore also be taken into consideration in the choice of an eluent.
Elution Flow-rate The elution flow-rate is an important parameter in column preconcentration which is usually optimized for maximum sensitivity. However, the speed of elution is also a crucial factor for the efficiency of on-line preconcentration systems. because in most cases the eluent flow is connected directly (some after merging with reagent streams) with the detector. This is panicularly important for detection systems which require a certain sample delivery rate for optimum response, e.g., the flame AA or ICP spectrometer, and will be discussed in more detail in section 4.6.3: Ignoring the flow-rate requirements of the detector (as may be done in spectrophotometry), the optimum flow-rate for elution will depend on how strongly the analyte is retained on the sorbent. and on the releasing strength of the eluent. When maximum EF is pursued. low elution rates are favoured. Experimentally, elution rates may be decreased until EF approaches a steady value. If optimum CE value is the objective, then a compromise has to be made between EF and sampling frequency. and higher elution rates giving somewhat lower peak signals may have to be adopted.
Direction of Eluent Flow The flow direction of the eluent is a factor which has drawn attention from the very beginning of the development of on-line column preconcentration. Reversal of the flow direction between the loading and elution stages was necessary to avoid the column . becoming more and more tightly packed. which could in tum influence the flow-rate and produce leakage problems. With strongly sorbed species. reversed flow elution is also beneficial in decreasing the dispersion of the concentrate during elution. and improving the enrichment factors. This may be readily achieved by using an 8-channel valve. such as that described in Sec. 2.2. An example of reversed flow elution used in AAS is shown in Fig. 4.8.
98
4 Sorplion
4.5
Column Packings
4.5.1
General Requirements for On-line Column Packings
Sorbents used successfully as packing materials for batch column preconcentration purposes are not always adaptable to FI column preconcentration systems. The special requirements for FI preconcentration sorbents may at least include the following: a b
c
The extent of swelling and shrinking should be negligible when being transfanned from one fonn to another, or when solvent conditions are changed. The mechanical properties should be strong enough to withstand high linear flow-rates through the column, and to maintain long column life times. Fibrous sorbents are. for example, usually mechanically unsatisfactory. The kinetic properties should be sufficiently favourable to allow easy retaining and elution of the analyte by an appropriate eluent. Sorbents which retain an analyte so strongly that they can be recovered only by complete destruction of the sorbent obviously cannot be used in FJ preconcentration systems.
Requirements for packing materials may be less stringent when the columns are used for separation purposes where the interferents are removed by the column, and no preconcentrations are involved. In such applications the column may be used for extended periods without regeneration. and in extreme cases may even be discarded when they are exhausted. In addition to the requirements mentioned above. packings used in sorbent absorptiometry should be transparent enough to transmit adequate light energy at the wavelengths where measurements are made.
4.5.2
Chelating Ion-exchangers
Hitherto the most frequently used packing materials for preconcentration columns were chelating ion-exchangers. Chelex-100 was the sorption material used in the first report on FI column preconcentration, which was applied to flame AAS [3). Lately, it has also been used for on-line column preconcentration of lead in combination with a FJ solvent extraction spectrophotometric method [11). Despite its success in batch preconcentration methods, and its favourable properties in complexing a large number of heavy metals. it did not fully meet the requirements for an ideal packing material for on-line applications. In a detailed study on the swelling properties of the resin, Fang et a!. [ 12) provided some guidelines for its usage in on-line columns, which may allow smooth running of the preconcentration system. Nevertheless, the overall perfonnance was non-ideal.
4.5 Column Packings
99
The Japanese product Muromac A-1. which has the same imminodiacetic functional group as Chelex -100, was reported to be free from the troublesome swelling properties of the latter, and has been used successfully in a number of AA and ICP spectrometric applications with column preconcentrations [ 13,14]. A prototype column packed with another imminodiacetic resin was used in a preconcentration system for ICP-MS [66]. The CPG-8Q chelating ion-exchanger, with quinolin-8-ol functional groups azoimmobilized on porous glass, is perhaps the column packing material used most often in FI preconcentration systems. mostly with flame AAS detection [6,12.1 5.16]. This is · because of its excellent mechanical kinetic properties due to immobilization of the functional groups on an easily accessible porous glass surface. However. the fine particle size and irregular shape of the particles produce considerable back pressure with the longer and thinner columns. Therefore only the larger particles, obtained by sieving. should be used for column packing. An important shortcoming of the exchanger is its relatively low exchange ~ apacity (5.5 fLEq g- 1 for Zn) [ 15], which explains its relatively low tolerance to interferents. One reported case was the unsatisfactory recovery for the preconcentration of cadmium in sea water samples [16]. The exchanger should therefore be used either for metal analytes which form very strong complexes with quinolin-8-ol. or for samples with low concentrations of competing ions, or with the introduction of suitable masking agents. Quinolin-8-ol immobilized on silica was used for on-line preconcentration purposes with an ICP-MS system [ 17]. Isotope dilution and standard addition methods were applied for the determination of trace metals in a sea water standard reference material. A similar ion-exchanger was used for on-line preconcentration of cobalt with catalytic spectrophotometric determination 118]. The Chinese origin 122 resin. with salicylic functional groups has been used successfully for a number of applications [6.12,19]. This resin is no more available. but substituted by a similar Chinese resin. 50 I. The latter was used for the preconcentration of cobalt from water samples [9). Good results have been reported for Co. Cu. Ni. Pb. and Zn in sea water samples with flame AAS detection. but the salicylic functional group did not form sufficiently strong complexes with cadmium to give good recoveries. The Amberlite IRA-743 ion-exchange resin which exhibits specificity for boron was used by Sekerka and Lechner [63] for on-line preconcentration of boron with spectrophotometric detection by the azomethine-H method. In an application of FI sorption preconcentration to ICP spectrometry. Wang and Barnes [20] made a detailed study on two chelating resins, one_ with a poly(dithiocarbamate) group (PDTC), and the other with a carboxymethylated poly(ethylenimine)isocyanate group (CPPI). Previously the PDTC resin was used as a non-reversible chelating resin for the preconcentration of 52 elements using a batch procedure. Most of the metals could be recovered only after complete digestion of the exchanger. However, in the FI preconcentration system some metals could be completely eluted. In a study on the behavior of 35 elements collected by the two resins. 22 were quantitatively recovered from one or both of the resins, using 2M HN0 3 as eluent. Unfor-
I00
4 Sorption
tunately. the alkaline earth metals which were not retained by CPPI and PDTC in batch procedures were retained by both resins in the Fl on-line preconcentration method. Recently. Yuan et al. [70] used a CPPI resin packed column for the on-line preconcentration of zinc from sea water with ICP atomic fluorescence spectrometric detection.
4.5.3
C 18 Bonded Silica Gel
Non-polar or medi!Jm polar sorbents may be used to collect metal complexes of low polarity from the aqueous phase by reversed phase adsorption. The metal complexes are subsequently eluted by a suitable solvent such as methanol or ethanol. The technique, termed sorbent extraction. or solid phase extraction, has been used in the batch mode for sample cleaning and preconcentration purposes. Karlberg et al. (47] were the first to use Cu1 octadecyl bonded silica gel as sorbent for the separation and determination of caffeine in pharmaceutical preparations by a Fl on-line column technique with spectrophotometric detection. Ruzicka and Amdal [21] have shown the great potential of this technique in on-line column preconcentration of heavy metals for flame AAS. Metal complexes of a number of well-known complexing agents were sorbed on Cu1 and eluted by ethanol. Among the complexing agents studied, sodium dimethyl dithiocarbamate (DDC) forms stable complexes with most of the important heavy metals, many even in strongly acidic medium and appears to be the mos~ promising. Interference from common matrix elements such as calcium and magnesium are also much less pronounced than most chelating ion-exchangers. Fang et al. [22] used the CwDOC system to determine lead, copper and cadmium in a sea water matrix with flame AAS and obtained good recoveries. The C 1s-DDC sorbent extraction system has also been used successfully in FI preconcentration of heavy metals in sea water and other water samples using graphite furnace AAS ( 10,23]. C 111 has also been used successfully as packing material in a flow-cell for solid phase absorptiometry [61].
4.5.4
Polymer Sorbents
Amberlite XAD polymeric adsorbents have been used for the preconcentration of trace elements in batch procedures followed by AAS or photometric measurements. The adsorbent series also shows promise in on-line preconcentration systems. Plantz et al.[24] used XAD-4 (polystyrene divinyl benzene based) to collect the bis(carboxymethyl)dithiocarbamate complexes of Co, Cu, Cr. Ni, Mo, Pt, and V by reversed phase adsorption, and eluted the complexes with 0.1 M NR.OH, with the subsequent determination made by ICP-MS. Xu et al.[25] used the polyacrylate adsorbent XAD-8 to collect the chloro-complex of gold, which was subsequently eluted by ethanol and determined by flame AAS.
4.5 Column Packings
4.5.5
101
Strongly Basic Anion Exchangers
Strongly basic anion exchange columns may be used as a clean-up medium to remove anion interferents. Although these exchangers lack the selectivity of chelating ion-exchangers. they have nevertheless been used successfully in some on-line preconcentration applications. Anion exchangers were used for collection of Se(VI) [26]. and the chloro-complex of tin [27). in FI hydride generation AAS systems. Interference~ from cations which do not form negatively charged complexes may be effectively suppressed. However, due to the low selectivity of the packing. relatively large column capacities have to be reserved for anionic interferents in the sample matrix. Thus either the sample load has to be limited to relatively small volumes or the column dimensions have to be enlarged, both leading to degraded enrichment factors. Ferreira et al. also used an anion exchanger, Dowex 1-X8. to retain the chloro-complex of zinc. followed by sodium hydroxide elution [28). This enhanced the selectivity of the final spectrophotometric determination. Reagent loaded anion exchangers may be used as selective sorbents for the preconcentration of certain analytes. Okabayashi [29) reported an anion-exchange resin loaded with alizarin fluorine blue sulphonate-lanthanum complex which was used for the preconcentration of fluoride. with subsequent determination using an ion-selective electrode. Hernandez et al.[30) used a chromoazurol modified anion exchanger to collect aluminium from serum samples. The analyte was eluted using IM NaOH and determined by flame AAS. In some applications. the complexing agent was introduced together with the sample forming an anionic complex which was collected by an anionic-exchanger. Porta et al.[31) used a macroporous anion-exchange resin (AG MP 1) to preconcentrate the metallic complexes formed with ~-hydroxy-7- iodoquinoline-5-sulphonic acid. followed by ICP spectrometric determination. Tesfalidet et a1.[32] reponed on a novel application of an on-line anion-exchanger packed bed. loaded with borohydride reductant for hydride generation AAS. The packed reactor provided the multi-function of separating cation. interferents. analyte preconcentration. and reaction environment.
4.5.6
Strongly Acidic Cation Exchangers
These are used mainly for the on-line separation of cationic interferents. Marshall and van Staden [33) reponed on a fine example of using a fully sulphonated strongly acidic resin (Bio-Rad AG50W-X8) for the on-line removal of interfering base metals in a FI hydride generation AAS system. However. non-selective cation-exchangers may also be used for preconcentration purposes, an early example is the preconcentration of ammonium ion in natural waters using Amberlite IR-120, followed by spectrophotometric determination using Nessler reagent [ 1].
102
4.5.7
4 Sorption
Activated Alumina
Activated alumina is a sorbent which is characterized by long-term stability under
both acidic and basic conditions. It has been used quite often as column packing in ICP applications, particularly in its acidic form, for the pre concentration of oxyanions [34,35]. Its basic form has been used successfully for the collection of Cr(III) [36], and cadmium [37] from urine. In a detailed study on the behavior of various oxyanions on activated alumina (acidic) Cook et al.[35] have shown that while arsenate, chromate, molybdate, phosphate, selenate, and vanadate were all well retained on the sorbent. only chromate and molybdate could be reasonably well eluted using 1M N~OH, or stronger alkali solutions. About 80% of phosphate and selenate could be eluted using IM KOH, whereas arsenate and vanadates may be de-sorbed only by using stronger eluents such as 5M KOH.
4.5.8
Water Adsorbents
Toei [38] reponed on a column packed with the inner fibers of a disposable diaper for babies, which was used as a separator to remove the aqueous phase from a segmented solvent extraction system. Only the organic phase flows into the detector. The column phase separator is simpler than other solvent extraction phase separators (cf. Sec. 3.2.3). The number of samples which could be treated before renewal of the disposable column depends on the volume of the aqueous sample injected.
4.6 Fl On-lint• Col11mn Separation and Prenmcemration Systems
4.6
FI On-line Column Separation and Preconcentration Systems
4.6.1
General
103
FI on-line column separation and/or preconcentration have been applied to various detection systems. including atomic absorption and JCP spectrometers, spectrophotometers and electrochemical detectors. The basic components of the manifolds for different detection systems are quite similar. usually consisting of the following parts: • • • • •
Single or dual multi-channel peristaltic pumps A multi-functional valve or a system consisting of 2-3 simpler valves Single or dual on-line micro-columns Transpon conduits and other reaction lines Flow-through detector
However. the coupling of the FJ manifold to different detectors calls for specific requirements which warrant separate discussions.
4.6.2
Systems for On-line Separation of Interferents
The manifold designs are usually simple and straightforward when on-line columns are used to enhance sensitivity by retaining the interferents; this procedure is ofren termed '"on-line clean-up·- of the sample. Columns which selectively retain the interferent. but allow the analyte to pass freely. are connected in the sample introduction line either before ot behind the injection valve (Fig. 4.3 a. b). Szpunar-Lobinska et al.[39] compared the two approaches in the determination of biuret in urea fenilizers by spectrophotometry, and strongly recommended the second one (Fig. 4.3 b) beeause: a
b
The precision for the manifold in Fig. 4.3 b was one order of magnitude better than that in Fig. 4.3 a. The reason for the difference might probably be that for the manifold in Fig. 4.3 a. during filling of the sample loop, the sample has to be sucked through a packed column. This may produce air bubbles in the sample and deteriorate the sampling precision. The manifold in Fig. 4.3 b required only one-third of the sample volume used for the manifold in Fig. 4.3 a, obviously owing to the larger dead volume before the sample loop for the latter.
104
4 Sorption
s a p
b
s w
p F"ag.4.3: Schematic diagrams of A manifold with on-line columns used for sePar-ation of interferences. a, pre-injection separation: b, post-injection separation: S, sample: C. packed column; D, detector. and W. waste.
c
The manifold in Fig. 4.3 a required much more frequent column regeneration because of the larger sample volume processed.
The capacity of the columns used in such applications may be larger than those used for preconcentration purposes since dispersion of sample is less important at this stage. This makes it possible to periodically regenerate or replace the columns after a large number of determinations. In the foregoing example, regeneration of the column was required only every other day [39]. The clean-up separation manifolds are quite similar for ·different detection systems. They can also be integrated with separation manifolds based on other principles such as gas-liquid, or liquid-liquid separation. A good example is that described by Marshall and van Staden [33]. These workers used an on-line ion-exchange column to remove interfering base metals in the determination of selenium and arsenic with a FI hydride generation AAS system, which also incorporated a gas-liquid separator. The system is shown schematically in Fig. 4.4. More recently, Tyson et al.[40] reponed on a column separation system which effectively removed the interference of large concentrations of copper in the determination of hydride forming elements. When the analyte is retained on the column instead of the interferents, the FI manifold resembles those used for on-line preconcentration purposes, because finally the analyte will have to be eluted in each cycle in order to be determined in the detector. The only difference would be in the amount of sample loaded. The reader may refer to the manifolds used for column preconcentration described in the sections which follow.
4.6 Fl On-line Column Separation and Preconcelllratinn Systems
CR--r-~.-~~~~
A R
p
4.6.3
105
Fig.4.4: Schematic diagram of FI hydride generation AAS system with on-line removal of metal interferems using on-line sorption column. S. sample: C. ion-exchanger column: CR. buffer carrier: A. acid: R, reductant: SP, gas-liquid separator: W. waste; Ar. argon flow: AAS. heated quartz atomizer [33].
Column Preconcentration Systems for Flame AA and ICP Emission Spectrometry
A specific feature of flame AA and ICP emission spectromettic detectors is that certain sample introduction rates are required to achieve optimum response (see Sec. 2.4.2). Therefore, when coupling Fl column preconcentration systems to flame AA or ICP spectrometers the elution process cannot be optimized independently as for off-line procedures, because the process is simultaneously a sample introduction process requiring independent optimum conditions. In the FI mode the optimum sample introduction canier flow-rate for different flame AA spectrometers varies within 4-10 ml min- 1• Usually such flow-rates are too high to provide sufficient contact time between the eluent and column packing in order to achieve equilibrium. The optimum elution flow-rate under on-line conditions is therefore a compromise between the two conditions. The optimum elution rates giving maximum sensitivity in flame AAS are almost always lower than the optimum sample introduction rate, being in the range 2-4 ml min-•. Elution rates may be higher when stronger eluents are applied, or when the analytes are more weakly sorbed. In ICP spectrometry, optimum sample introduction rates are much lower than those for flame AAS. In a FI column preconcentration system the optimum conditions for elution are closer to the required sample introduction rates. Therefore, on-line elution flow-rates are quite similar to the normal sample introduction rates of 1-2 ml min-• for ICP spectrometric systems. A schematic diagram of the simplest FI on-line column preconcentration system for flame AAS is shown in Fig. 4.5. The first of such systems was reported by Olsen et al. in 1983 in a pioneering paper in this field [3]. These simple systems are single line manifolds with one or two injection valves and a column arranged in series with the detector. Such manifolds are operated using volume based sampling. with defined volumes of sample and eluent injected through a single or two separate valves sequentially. Although simple in construction, such manifolds are not as simple to operate, and suffer from other defects such as waste discharge into the detector and uni-directional flow through the column.
I06
-1 Sorption
s
E
CR
w
p Fij!.•I.S: Schematic ligun: of" single-channel Fl manifold for on-line preconcemration with flame AAS detection. S. sample injection valve: E. eluent injection valve: C. packed sorption column: D. flam.: AAS detector: W. waste: CR. carrier (31.
Water
v
a
b
FigA.6: Schematil" diagram of a Fl manifold for high efficiency on-line column preconcentration for flame AAS with coumercurrent elution. a. sample loading sequence: b. elution sequence. P 1• P~: peristaltic pumps: E. eluent: S. sample: B. buffer/reagent: C. conical column: V injector valve: W. waste: and AAS. flame AA detector [16].
A more efficient system using time-based sample loading on a CPG-8Q ion-exchange column and a single rotary valve is shown in Fig. 4.6 116]. Almost the same manifold design has been used in a sorbent extraction preconcentration system for AAS [:22]. This manifold design incorporates all the practical features for achieving optimum performance mentioned in the previous sections. including time based sample loading on a conical micro-column with effluent discharge into waste. reversed flow elution without washing sequence. and shon transpon conduits. A dual pump system is shown in the
.J .l1 Fl On-line Column Separation and Prcnmccmrotion S.\'Jtcm.~
107
original application. but when an extra channel is provided on the rotor (or stator) for eluent discharge during sample loading. the system may be operated equally well using a single pump running through the entire analytical cycle. but at the expense of a larger consumption of eluent. Recycling of the unused eluem is not recommended owing to contamination risks. The manifold in Fig. 4.6 wa!'- modified by Xu et al.l8] by introducing small air segments at the sample/eluent interface to restrict mutual dispersion between the two zones (cf. Sec. 4.4.3). When the number of channels are increased to eight both on the rotor and stator of the multi-functional valve. the same components may be used to set up a dual column system shown in Fig. 4.7 II~). This manifold. featuring two columns operated in parallel, may be used to further improve the efficiency. particularly when long sample loading periods are used to obtain high enrichment factors. The two columns are loaded simultaneously. and eluted sequentially either using a double pump systen. as in Fig. 4.7 or using an additional two-way valve as in Fig. 2.6 d . However, the manifold may also be arranged to have two columns loaded and eluted alternately, i.e., while one sample i!-. loaded the foregoing sample is eluted 119). The alternating column system in reference 19 used a volume-based sampling approach which was not very efficient: a modification of the system using an R-channel valve. featuring more efficient time-based sampling. is shown in Fig. 4.8. The efficiencie!'o of the two systems would be similar if the loading periods double the elution period!-.. When longer elution periods or shorter loading periods are adopted. bener efficiencie~ will be obtained in the alternating mode. With extended loading sequences. where the bulk operation time is consumed in sample loading. parallel loading becomes more efficient. because then the elution time will be much shorter than the loading time. Both systems described above have limitations In the parallel mode either doublt: pumps or dual valves (as shown in Figs. 4.7, 2.6 d) 19) have to be used to control the sequential elution of the columns. This is not necessary in the alternating mode. However. the sample consumption is usually larger for the laner because the time for sample changeover is determined by the sample loading time. and not by the normally shorter elution time. On the other hand. unless separate pumps are used for loading and elution. both systems lack the possibility of independent control over the sample changeover time. Therefore. if minimum sample consumption is required. a more complicated three-pump system will be necessary. FI column preconcentration manifolds used for flame AAS can be easily adapted to work with ICP spectrometry. usually adjusting the elution flow-rate to lower values. Thus the manifold in Fig. 4.7. which was originally designed for flame AA has been readily adapted to an ICP system with minor modifications [41,42]. The performance of some selected examples in column preconcentration for flame AA and ICP spectrometry are shown in Table 4.1 and Table 4.2
108
4 Sorption
v
(a)
r:-1-~-~-------'
L_j R..------ ,' SA----~~~~~--__, ~--~~--~~---+
R.------
• w
_______ _
r:~..:-
L.:_r F\.------
sA-----Pillf--""--+----i~--~~--~~--+
R.-----s.---oflll!!l~....i--,J,-----1
v (c)
w Preconcentration
l -·
Fag.4.7a-c: a, b, schematic diagram of a dual column FI on-line preconcentration manifold for flame AAS with parallel sample loading and sequential elution using two pumps. a, loading sequence; b, elution sequence for column CA. P1. Pn, peristaltic pumps; SA, S8 , samples; EA. Ea; eluent (2M HN03); RA. Ra. ammonium acetate buffer; T, timer for pump control; V, 8-channel multifunctional valve; CA. Ca; columns packed with chelating ion-exchangers; W, WA. Wa. waste flows; W A•, same waste line as W A; and AAS, flame AA detector [12]. c, Sequence of operation for pumps and valve. T, switching point of valve position.
4.6 Fl On-line Column Separarwn and Preconcenrration Systems
5min 100
109
10 mgl-1
~r1
100
75
(d)
A o-3
5
10
0.2
Z$ 1)-1
0
B
A
scan
c
--~>
Fig.4.7d: Chan recordin!! obtained in the preconcentration of lead usin!! the dual column system. A. a standard series of 2>-100 J.lg 1- 1 Pb: B. 100 J.lg 1- 1 Pb in sea water matrix. C. recording obtained from standard series of 2.>-10 mg 1- 1 Pb using conventional direct sample introduction. Reproduced by permission of Elsevier Science Publishers
S1 B
w
E
C1
w
w w
C2
C2
S2 B
v
v
w
a
b
FigA.B: Schematic diagram of a dual column Fl on-line preconcentration manifold for flame AA or ICP emission spectrometry with alternating column loading and elution. a, load C2, elute Cl; b. load CI. elute C2. Sl, S2, samples: B, buffer; E, eluent; V, 8-channel multifunctional valve; Cl, C2, sorption columns: D, detector; W, waste.
Table 4.1
Perfnnnance of representative Fl on-line column preconcenlralion methods for flame /\1\S
Analyle and snrhed species Cd, Pb, Zn Cu,C'o,Cd,Ni,Ph,Zn Ni, Cu. Ph. C'd Pb.Cd,Cu,Zn Pb Cd,fe(III),Cr(III) Cu,Mn( II),Ph.Zn Cu, Cd, Pb Cu,Pb complexes with DDC,quinolin8-ol, PAN, PAR Cu,Cd,Pb-DDC Au-chloro-complex
----
Column packing/eluenl
EF
CF.
(min·- 1) C'helex-1 00/2M HNOJ C'PO-RQ/1 M HN01 (dual) 122 resin/2M HN0.1 (duai)C'helex-100,122 resin, CPG-HQ/2M HN03 Alumina/2M HN03 Muromac A-I/2M HN01
Cl (ml)
f
(h .. I)
13 5no 20-28 50-100
6 17 13-19 50-I()()
0.15 0.20 0.1 H-0.25 0.10-0.20
27 2 40 60
250 46-96
46
0.10 0.10-0.22
II
10-21
CPG-RQ/2M HCI C1R bonded silica/ethanol
25-31
50-62
60(EF*)
"'20 (CE*)
C:::1R bonded silica/ethanol XAD-R/ethanol
19-25(EF*) 35(EF')
38-50 35(CE')
0
R.s.d.
Ref.
(%)
-
.e..
1.5-4.1 1.2-3.2
3 15 19 12
13
1.4 0.6-1.4
64 14
0.05-0.()6 ......0.()6
120
1.2-I.H
"'20
-
16 21
OJJ6-0.07 0.14
120 60
1.0-1.4 . 1.4
22 25
~
i g·
Table 4.1 Perfonnance of representative on-line column preconcentration methods for ICP emission spectrometry Analyte and sorbed species
Column packing/eluent
Ba.Be,Cd,Co,Cu,Mn, Chelex- Hl0/2M HNO~ Ni.Pb AI.Cr(lll),Fe(lll), Muromac A- I/2M HNO-'
n.v
Sc etc .• 22 elements Bi,Ce,Co,Ni, V
PllTf'.CPPI chclnting resins/ 2M HNO-' CPG-8Q/2M BCI
EF
CE {min- 1)
(ml)
(h-1)
10-30
5-15
1-3
30
6
41.42
34-113
10-32
0.1
17
2.3-4.4
65
!Sc)
3.3
().(i
3
3
20
24-41
24-41
0.3-0.5
()0
-
tl)
C/
I
R.s.d.
Ref.
(%)
~
0..
~
c
7 2.8-7.7
44
~
~
tj ~
f
c,..
ii3
5· ::.
1::1
it ~
~
g..., ....
:::s
~
5'
:::s
,C,..
~
~
112
4 Sorption a
b
v,
CA
8
88 8 E
C8
+v,. Fig.4.9: Schematic diagram of a dual column FI on-line preconcentration manifold for vapour generation AAS with parallel column loading and sequential elution. a. elution sequence for column CA: b. loading sequence. VI, 8-channel multifunctional valve (missing channels in figure are blocked); Vu. 2-way valve for controlling column elution sequence. SA. SB, samples: 8, buffer: E. eluent; R, reductant: SP, gas-liquid seperator: A, quanz tube atomizer: Ar, argon flow: W, waste [26].
4.6.4
Column Preconcentration Systems for Hydride Generation and Cold Vapor AAS
Fig. 4.9 shows a FI column preconcentration manifold for hydride generation or cold vapor AAS determinations using dual columns [26,43]. Single column systems are of course feasible using the four channel valve design and a single pump. A direct combination of the on-line column preconcentration system used for flame AA to the Fl hydride generation manifold was readily achieved because the acid conditions and flow-rates for elution from the preconcentration columns in the determination of selenium and bismuth were not very much different from those required for the ensuing hydride generation reaction. A similar column preconcentration system has been applied successfully for cold vapor determination of mercury with minor modifications in the operational parameters [44.45]. An integrated membrane gas-liquid separator and absorption cell has also been used for cold vapor determinations with a column preconcentration system but EF values were lower by 70% [45]. The performance of column preconcentration systems for hydride generation and cold vapour AAS are shown in Table 4.3. Tesfalidet and lrgum [32] reported on a packed membrane diffusion cell for heterogeneous hydride generation with AAS detection which also achieved some extent of preconcentration of the hydride forming element. This system will be treated in detail in Chapter 5.
Table 4.3
Performance of on-line column preconcentration methods for vapour generation AAS
Analyte and sorbed Column packing/eluent species Se(VI) Bi(lll)
•
Sb(IIJ) Sn(IV)-chloro complex Hg(ll)
EF
(dual) D-201 strongly basic 15 anion exchanger/ I M HCI (dual) CPG-RQ/2M HCI 30 CPG-RQ/4M HCI 20 D-201,Dowexi-RX strong basic 4 anion exchanger/0.1 M HN0 3 (dual) CPG-8Q. 501 chelating 20 resin/2M HCI in 0.5% thiourea
CE (min- 1)
C/ (ml)
I
R.s.d. (%)
Ref.
(h-')
IR
0.7
50
1.1
26
25 20 5
0.5 0.75 0.75
50 60 70
1.0 1.0 2.2
26 71 27
20
0.75
60
1.2
43,45
..,""0..... ~ §": '"' ('") ~
1::
~
c;;,
~
s::a
i3
g· Q ;:,
., tl.
~
g ~ ~ §" ~
...
~
-YJ
114
4 Sorption
Collected "0 CD
CD
"C
u
c:
ia
Discarded
u
10
.l:l
Ill
0Ill
i5
.l:l
< Eluate volume -
Fig.4.10: Schematic diagram demonstrating the principle of eluate zone-sampling in Fl column preconcentration for ETAAS [9]. Reproduced by permission of Royal Chemical Society.
4.6.5
Column Preconcentration Systems for Graphite Furnace AAS
Cenain specific features of graphite furnace AA demand special requirements in the design and operation of the column preconcentration systems. lmponant differences from flame AA applications include the following [ 10]: a
b
c
In contrast to flame AA atomizers. which are inherently flow-through detectors, the graphite furnace atomizer is normally operated in the batch mode with discrete sampling. Therefore. the preconcentration of analyte cannot be achieved in a continuous flow mode as for flame AAS, but are operated in parallel and synchronized with the atomization process in a discontinuous mode. The sample volume which can be conveniently and reproducibly processed in a graphite furnace is much smaller than that in flame AAS. the maximum volume being no more than I 00 J.tl. When using a L'vov platform and an ethanol sample solvent, the maximum allowable volume decreases to about 70 J.tl. The eluate volume therefore has to be limited to below this volume in the case of ethanol elution. A special zone-sampling procedure has been adopted to introduce only the most concentrated fraction of the eluate into the furnace. while discarding the rest, because it was impossible to complete the elution in such small volumes, even using a· small column of 15 J.tl. This has been achieved with good reproducibility by collecting the eluate in a 0.35 mm i.d. PTFE tubing of 60-70 111 volume with a pre-determined delay time following the beginning of the elution (see Fig. 4.10) [10.23]. Graphite furnace AAS is much more sensitive to matrix interferences than flame AAS. In flame AA the small amount of residual sample in the micro-columns may be safely disposed into the nebulizer-burner system during elution. For graphite furnace applications, even introducing a small fraction of a heavy sample matrix into the furnace may create disastrous effects in the determinations, often despite the use of sophisticated background correction facilities. Therefore, for most graphite furnace applications a column washing step which removes the residual sample before the elution is indispensable.
4.6 Fl On-line Column Separation and Preconuntration Systems
115
Fig. 4.11 and Table 4.4 show the manifold design and operational sequences of a Fl sorbent extraction column preconcentration system for graphite furnace AAS proposed by the Author and co-workers which incorporated the considerations mentioned above [I 0]. The system was computer controlled, and, owing to the complication of the process, would be difficult to operate otherwise. The procedure consisted of 5 steps (7 steps in a later modification [23]). which is much longer and more complicated than those for flame AAS. but system components are almost exactly the same as for flame AA, except using a 4:5 channel multi-functional valve. The samples were loaded on an extremely small conical column, packed with CtM· with only 15 Jil capacity, at lower flow-rates than those used for flame AAS. The column was then washed with deionized water in the same direction as sample loading; but later it was reponed that countercurrent washing with 0.02% nitric acid is more efficient in eliminating the background interference (23]. The retained analyte was then very slowly eluted with ethanol into a 0.35 mm i.d. PTFE collector tube. which stored the central section of the eluate but discarded the leading section. The elution was temporarily interrupted at a predetermined time when the required central section of the eluate had entered the collector. The collected fraction was then introduced into the furnace using a stream of low air flow. and the elution was completed sending the residual eluate to waste. In later studies. anempts were made to simplify the procedure with a direct elution of the fraction of eluate into the furnace by controlling the time interval for the collection (23}. This. however, produced some deterioration in the sensitivity and precision. Further details on the operation of column preconcentration methods for graphite furnace are given in Sec. 8.8.3. The characteristic data on the performance of column preconcentration systems for graphite furnace AAS is shown in Table 4.5
116
4 Sorption
Sequence 1
(a)
P2 On
P1 Off
Sequence 2
(b)
P2 On
P1 On
w Sequence 3
(C)
P2 Ott
P1 On
w
4.6 Fl On-line Column Separation and Preconcentration
S_vstem.~
117
Sequence 4
(d)
P2 On
P1 Off
w
Sequence 5
(e)
P2 On
P1 Off
w Fig.4.11 a-e: FI manifold and operation sequence of sorbent extraction preconcentration for ETAAS. P 1• P2. peristaltic pumps; C, micro-conical column (15 11-L packed with C 18 sorbent): V, multifunctional valve; L, eluate collector (75 11-l. 0.35 mm i.d.); W, waste; and GF. graphite furnace [ 10].
'IBble 4.4 Sequence of operation of Fl on-line sorbent extraction preconcentration for ETAAS Sequence Duration (s)
Pump Medium pumped
2
10 20
3
60
I I 2 2
4
30
I
Air Ethanol Sample Sample DOC solution Water
I
Air Ethanol
5 Total
30 150
Flow-rate (ml min- 1)
Function
0.8 0.5 5.0 2.1 0.4 1.6 0.8 0.15
Dispense eluate from previous elution into furnace Elution of residual analyte from previous elution Sample change Sample loading on sorbent column
-00
....
Column rinsing Evacuation of eluate collector Sample elution and and eluate collection
f 5' ~
Table 4.5
Perfonnance of Fl on-line sorbent extraction preconcentration methods for bonded silica)
Analyte and sorbed species
Eluent
Pb-DDC complex Cd, Cu, Ni, Pb-DDC complex Cd, Pb, Cu, Ni, Co, Fe-APDC complex Co-DOC
Ethanol Ethanol
As( III), As(lll)+As(V)-D DC
Acetonitrile Ethanol Ethanol
T:F
(min- 1)
Cl (ml)
(h-1)
10.4 6.2-9.9
0.10 0.11-0. I R
24 22
20({'d) 225(Co)
-
O.OR (Co)
7(Co)
42 210 7.6
11 18 3.8
2f1 "17-27
CF.
-
0.13 0.1.~
. 0.4
I
17 5 30
~TAAS
(column packing C 18
1
Detection limit (ngl-'>
3 Cd O.R,Cu 20, Ni 40, Pb 7 Cd 0.4,Cu IO,Co I. Fe 25.Ni 9,Pb 4 6 1.7 110-150
R.s.d.
Ref.
(%)
1.9 2.0-3.3
10 23
3-5
67
5
68
:!:!
c
'l' ~
"'
(") :;)
10
55
""' 0..
~
69
:!
::
c:,
~
"'~. ~
::
§ ~
~
~
;
=:
~
~ :::. I:)
:: ,c;,
~ ~"'
-o
120
4 Sorption
4.6.6
Column Separation and Preconcentration Systems for Spectrophotometry
General Although the first contribution in FI on-line column preconcentration was one which used a spectrophotometer as detector [ 1], and despite its broad applicability to both inorganic and organic analyte species, hitherto such contributions are much fewer than applications in AAS. There might be at least two important reasons for the slower development. The first is associated with interferences in the signal readout, arising from variations in the refractive index (Schlieren effects) between sample (or carrier) and eluent, the second reason is the requirement for coordinating the chemical conditions of the elution and the ensuing chromogenic reactions, which is not always easy or even possible. Difficulties originating from refractive index differences do not exist in AAS or ICP applications, whereas coordination of elution and reaction conditions are rarely necessary. However, theSe obstacles are not always easy to overcome in spectrophotometry, and the topic will be given a more detailed ueatment here.
Compensation of Schlieren Effects Interferences in spectrophotometry due to cufferences in refractive indiCes between sample and carrier/reagent have been discussed in Sec. 2.4.1. Such mterferences are significantly enhanced in column preconcentration procedures because of the inevitable existence of a sample (or carrier) and eluent interface usually characterized by large compositional differences. Occasionally, when the sorbed species are eluted slowly, the elution peaks can be resolved from the preceding Schlieren effect peaks. in which case the interference will not be serious. Such conditions were reported by Wu and Qi [46] in the determination of uranium using arsenazo-m, following elution from a levextrel preconcentration column using DTPA. However, in order to achieve better enrichment effects, strong eluents are preferred which are capable of rapid elutions. Then the elution peak apex where readouts are made will appear at the frontiers of the eluate zone, where unfortunately one will find the most serious interferences. When the interfering peaks are relatively small and reproducible, they may simply be taken as blank values in the calibration. However, large interferences created by using. for example, ethanolic eluents for aqueous samples are much more difficult to deal with. Measures discussed in Sec. 2.4.1 for overcoming such matrix effects, i.e., matrix matching, and erihancement of mixing by using various reactors may be used to decrease the interfering effects, but complete elimination is difficult. Karlberg [47] reported the use of a special flow-cell with which the light passed through the cell perpendicular to the flow direction. A similar principle was adopted in a micro flow-cell using optical fibers to transmit light through a capillary, from and to a spectrophotometer, described by Thommen et al.[48]. Although the interfering effects
4.6 F/ On-lim· Column Separmion ami Preconcentration Systems
121
may be largely eliminated by such designs, the sensitivity of the determinations are inevitably degraded due to the shon light path. Nevenheless, if the main objective of the separation is enhancement in selectivity, the implementation of such flow-cells may be an effective solution. In a detailed study on the compensation of Schlieren effects using a diode-array spectrometer. Zagatto et al.l49j recommended the use of a dual-wavelength spectrophotometer as a best solution. Almost complete compensation of Schlieren signals as high as I absorbance has been achieved using this approach. The method has been used successfully by Ferreira et al.[28] in the spectrophotometric determination of zinc in plants. following an on-line column separation. This. however. calls for special instrumentation which is not always available in the laboratory. Lacy et al.[60) proposed the use of peak area instead of peak height to alleviate Schlieren effect interferences. This may be effective when interference signals are not very serious compared to the analytical signals. but the approach also requires special functions from the spectrophotometer which are usually not available. In most cases a reasonable degree of compensation of Schlieren effects may be achieved by taking the following measures: a
b
After sample loading. wash the columns either with the carrier (as in volumebased sample loading) or with a special wash solution whose refractive indices have been matched with the eluent by introducing "inert., solutes into one of the neighbQring phases. Use knotted or pearl-string reactors for transport or reagent mixing in between the columns and the flow-cells.
Dilution Effects in Post-column Reactions
In spectrophotometric determinations, a chromogenic reaction is almost always necessary after the elution of the analyte to transform it into a detectable species. Therefore. reagents have to be introduced into the eluate flow which inevitably dilute the eluate. Since the pH or solvent conditions for elution are often quite different from that required by the reaction. the need arises for adapting the conditions to those of the spectrophotometric determination. On-line addition of a buffer solution to the eluate is often necessary in order to achieve the required pH conditions for the reaction. The amount of buffer required may be quite substantiaL depending on the difference in elution and reaction conditions. resulting in large dilutions of the eluate. Therefore usually concentrated buffer solutions are used to decrease the dilution. Thus, for the determination of boron with azomethine-H following a column preconcentration and elution with I M phosphoric acid reponed by Sekerka and Lechner [63]. a 2M ammonium phosphate buffer (pH 6.6) was merged with the eluate to adjust the pH. Even with this relatively high buffer concentration, a buffer flow-rate twice that of the eluate was required. Long sample loading
122
4 Sorption
periods were therefore used to compensate for the loss in sensitivity through dilutions, which in tum limited the sampling frequency to 10-20 h- 1• Nevertheless, the method is still much more efficient than batch procedures, and, with a detection limit of 1 Jlg 1- 1• is one of the most sensitive methods for boron ever reponed.
Manifold Designs Fl manifolds for column separation and preconcentration in spectrophotometry are diverse, and there is hardly one which may be considered typical. However, the reader may refer to the manifold used in the detennination of boron just mentioned (63]. Another interesting contribution by Novikov et al.[l I) combined ion-exchange column preconcentration with on-line solvent extraction followed by spectrophotometric detection. The eluate from the column preconcentration was released into an on-line liquid-liquid extraction system. An advantage of this approach is that interferences from Schlieren effects are avoided, since the eluate does not flow directly to the detector. Selectivity and sensitivity are also enhanced due to the combination of two separation procedures. The system has been used successfully for the detennination of lead in alloys. soil leachates and sea water. Lei et al.(50) reponed on an ingenious manifold design for avoiding refractive index interference effects ·in spectrophotometric detection when using eluents which are immiscible with the sample. Samples were loaded on a packed column for a defined time period and eluted by an immiscible eluent. After elution, the immiscible eluate plug was not transponed directly to the detector, but was directed into the sample loop of a second valve. The inlet and outlet of the valve were furnished with stainless steel tubings between which the conductivity of the flow can be continuously monitored. The second valve was actuated when the eluate plug sandwiched between fluids of different conductivity entered the loop: the plug is then transponed to the detector by a carrier which had the same composition as the eluent. The system was used successfully for the determinaiion of mercaptans in gasoline using a column packed with Amberlite URA401-S anion exchange resin which was eluted by an aqueous alkaline solution, and was also used for the determination of aniline in benzene. General Performance
Owing to technical obstacles discussed in the previous paragraphs, the enhancement in sensitivity and selectivity in spectrophotometric applications using on-line columns are not as dramatic as for atomic spectrometric methods. EF values of 10-15, with sampling frequencies of 20 - 30 h -• are typical for preconceiltrations, yielding CE values of 3-7, and C/ values may be as high as 0.5-1 mi. Nevertheless, the approach is much more efficient than off-line batch procedures, and shows promise in the trace analysis of analyte species which are not suitable to be analysed by atomic spectrometry or by other separation techniques in combination with spectrophotometry.
o/.fl Fl 011-line Column Si!pararioll and Preconcemration S.vstems
4.6.7
I 23
Column Preconcentration Systems for Chemiluminescence Determinations
The first report on the use of ion-exchange columns with chemiluminescence detection was that by Burguera et al.f51] on the determination of zinc and cadmium through inhibition of the cobalt catalyzed chemiluminescence generation from luminol. The chloro-complexes of zinc and cadmium were retained by an anion exchanger and eluted separately using sodium hydroxide and nitric acid. Apparently, preconcentration effects were not pursued. Although applications for column separation or preconcentration systems coupled to chemiluminescence determinations are few, published reports show no panicular difficulties in such applications. except for the requirement of an adjustment of the chemical conditions of the eluate to suit the chemiluminescence reaction. Interferences due to refractive index effects are not likely to occur, owing to the ofLen used spiral shape of the chemiluminescence flow-cell. and to the fact that light emission is measured perpendicular to the direction of the flow. Therefore, column washing is usually not as important as for spectrophotometric applications. so that time-based sample loading manifolds such as those used for flame AAS may be used to advantage for improving the concentration. efficiencies. Alwanhan et al.(52] reported on a column preconcentration method for iron(ll) coupled with chemiluminescence detection through iron(Il)-enhanced oxidation of luminol in alkaline hydrogen peroxide. Time-based sampling was used with a mini-column packed with quinolin-8-ol immobilized on controlled pore glass. An extremely high sensitivity with a detection limit of 10- 1 ~ M was achieved with 3 ml of sample. More recently, Elrod et al.(53] used a different chemiluminescence reaction system , Fe(II)- brilliant sulfoflavin-hydrogen peroxide. also using time-based sampling on a quinolin-8-ol column, to determine iron(IIl in sea water. However, the sensitivity (detection limit, 0.45 nM with 4.4 ml sample) was somewhat inferior.
4.6.8
Column Preconcentration Systems for Ion-selective Electrode Detectors
Although reports on on-line column preconcentrations for ion-selective electrodes are rather few, the applications d() show considerable enhancements in selectivity and sensitivity. Interferences from Schlieren effects do not exist in ion-selective electrode systems, which obviates the need for column washing, and time-based sampling may be used conveniently. However, sample wastes and strong eluents may exert corrosive effects on the selective electrode membranes. It is therefore advisable to use a manifold which can direct the sample effluent to waste during sample loading, and the acidity and composition of the eluent should be carefully chosen or modified following elution (e.g. by merging a suitable buffer) to avoid gradual deterioration of electrode response.
124
4 Sorption
Risinger [54] reported a FI on-line ion-exchange preconcentration system for the determination of copper using a copper ion-selective electrode detector and a CPG-8Q column. The analyte was loaded on the column by time-based sampling while anions and inert sample components were directed to waste without contacting the electrode. The sorbed analyte was then eluted by acid and neutralized by merging with a buffer stream before passing the electrode. The detection limit for copper was improved more than two orders of magnitude using 5 ml sample (f 12 h- 1) compared to 50 ttl injections without requiring excessive equilibration times. Matrix effects were reduced because of the separation and because all measurements were made in the same buffer solution. Okabayashi et al.[29] reported on a dual preconcentration column system for the determination of low concentrations of fluoride using a fluoride electrode. 1 ml of sample was sorbed selectively on an anion-exchanger loaded with alizarin fluorine blue sulphonate-lanthanum complex. The columns were loaded alternately, and successively eluted with I M sodium chloride - 0.5 M sodium chloride. A detection limit of 1 JLg 1- 1 was achieved at a sampling frequency of 24 h- 1•
=
4. 7
Sorption Preconcentration for Solid Phase Optosensing
4.7.1
General
Optosensing on a solid phase which retains a detectable species in a packed flowthrough detector is a relatively new technique, developed in recent years following the pioneering work of Yoshimura et al.[55,56] Hitherto most applications are based on absorptiometry. but at least three applications on fluorimetry have been reported [57 ,58,59]. A special feature of the technique is the integration of the separation and detection components in the flow system, with the sorption columns located in the flow-cells. The methods are characterized by direct sensing of the detectable species on the sorbent (solid phase) instead of releasing them into a flow stream which is then transported to the detector. This feature renders extremely high sensitivities to the technique, owing to the absence of unavoidable dilutions from the eluent. The following options are available for retention of the detectable species on the column, which is simultaneously the flow-cell:
4.7 Sorption Preconuntration for Solid Phase Optosensing
a b c d
125
Direct retention of an analyte which can be detected without derivatization. Retention of a detectable derivative of the analyte following on-line or off-line derivatization. On-column derivatization of a retained analyte or analyte derivative into a detectable species, by introducing reagents into the column following retention. On-column derivatization of the retained analyte or analyte derivative by reagents immobilized on the column prior to retention.
The sorbed species may either be eluted after each determination or after a set of determinations. The latter approach increases the sample throughput, but is only applicable when the response is linear over a wide range.
4.7.2
Practical Considerations in the Design of Solid Phase FI Optosensing Systems
Techniques and manifolds used for other on-line sorption ·separation methods described in the previous sections may be used to advantage for solid phase optosensing systems. . A large number of different column packings mentioned in Sec. 4.5 may be used for this purpose: however, a specific requirement would be its transparency. Obviously, opaque sorbent materials are not suitable owing to excessive attenuation of the light intensities. Even using satisfactory packing materials, light attenuation and scattering are unavoidable. frequently demanding modification or adaptation of the spectrophotometers to meet the more stringent requirements. Because of considerations both in light attenuation and flow impedance. when standard flow-cells with I em path lengths are used, the packing material never takes up the entire length of the cell cavity. This has the disadvantage of leaving some void volume in the cell. requiring the spectrophotometer to be set up in an odd venical position in order to keep the surface of the sorbent level and stable [57]. This also obviates the possibility of applying more efficient reversed flow elution (cf. Sec. 4.4.4, direction of eluent flow). Special flow-cells/columns are therefore preferred which have shoner path lengths. A flow-celVcolumn packed with C 18 used by Ruzicka et al.[60,61] for the determination of phosphate by absorptiometry is shown in Fig. 4.12. The light was transmitted radially through the column. which had an i.d. of only 2 mm, at a levrl where the bulk of the analyte was retained. Owing to light attenuation in such systems, the light source of the spectrophotometer may have to be modified to provide higher intensities. With double-beam spectrophotometers equipped with photomultipliers, normally the detection system should be sensitive enough for such applications, however. the reference beam will have to be attenuated by a suitable means to balance the two beams.
126
4 Sorption
+--IN
w
c
p
-------B
--+ OUT Fig.4.12: Schematic diagram of microcolumn block for optosensing in sorbent absorptiometry. B. outer housing for column; C. glass column; G. rubber gasket: F. threaded fittings; W. window in housing; and P. sorbent packing (60].
4.7.3
Solid Phase Absorptiometry
A general advantage of solid phase absorptiometry over liquid phase systems is that the technique is relatively free from Schlieren effect interferences. Although change of composition in the flow during elution can still produce signal deviations, the extent is rather moderate due to the shoner path length and the more intensive mixing in a packed column .. The effect will be funher decreased when the light intensity is monitored perpendicular to the flow direction as in the flow-cell/column described in Fig. 4.12. Funhermore, usually the analytical readouts are not made at a point where interference effects are most serious, as in liquid phase measurements. These are well-illustrated by a recording in the determination of phosphate (Fig. 4.13), obtained using the flow-cell in Fig. 4.12. Phosphate was sorbed on C 1s sorbent as the 12-molybdophosphoric acid complex and reduced to molybdenum blue with ascorbic acid on the column. After measuring the absorbance, the sorbed species was eluted with methanol. This produced small spikes at the aqueous reagent/methanol interface as well as baseline shifts due to the change in refractive index, but had no deleterious effects on the readout. A 40-fold
127
4.7 Sorption Preconcrlllralwn for Solid Pha.H' OptosensinK
3
R
R
E
IL I R
E
Fig.4.13: Recording of a typical FI profile for sorbent absorptiometric optosensing of sorption of phosphomolybdate on C 18 sorbent (L), reduction of the sorbed species by ascorbic acid (R). and elution by methanol CE>. The flow is momentarily stopped during reduction . .6 A are si~l heights related to phosphate content in samples. I, 2. 3, signals corresponding to 0, 25, and 50 llg 1- 1 P; BLK, blank; SC, si~l generated by Schlieren effects [60].
increase in signal compared to the homogeneous liquid phase approach was obtained with this method, using a flow-cell path length only one-fifth as long. An excellent example of solid phase absorptiometry through analyte derivatization by on-column reaction with a immobilized reagent is that reponed by Valcarcel's group (62]. 1-(2- pyridylazo)-2-naphthol (PAN) was immobilized on Dowex 50W X4-400 cation exchanger which was packed in a I mm light path flow-cell. The analyte copper was retained in the cell forming the Cu(II)/PAN complex by time-based or volume based sample loading, and subsequently eluted by thioglycollic acid. With volume-based sampling, the thioglycollic acid was included in the carrier, so that analyte retention was immediately followed by an elution to achieve a high sampling frequency of 110 h -• when ·injecting 300 1'1 of sample. The immobilized PAN resin may be used for at least 200 injections.
128
4.7.4
4 Sorption
Solid Phase Fluorimetry
Pereiro Garcia et al. reported on a sensitive fluorimetric optosensing method for the determination of trace amounts of aluminium [57]. The analyte was preconcentrated by being sorbed in a flow-cell packed with Kelex 100 (a quinolin-8-ol derivative) immobilized on Amberlite XAD-7. The fluorescence intensity of the complex was measured on the sorbent, optical communication between the fluorimeter and the flow-cell being achieved using an optical fiber bundle arranged perpendicular to the flow direction. The sorbed species was later eluted using 2M HCl. Chen et al.[58] described a sensitive and selective fluorimetric method for the determination of fluoride by measuring the fluorescence of the ternary complex of zirconium, Calcein Blue and fluoride retained on DEAE-Sephadex anion-exchanger in a 1.1 mm i.d. flow-cell. The detection limit for fluoride was 1 Jl.g 1- 1 at a sampling frequency of 30 h- 1 and consuming only 0.5 ml of sample. An excellent precision of 1% r.s.d. was reported, and the tolerance to most interfering ions has been improved substantially, some by an order of magnitude. compared to a manual method based on the same reaction. In a third method reported by de Ia Torre et al.[59], beryllium was determined by sensing the fluorescence of its complex with morin which was immobilized on Dowex I X4-100 anion-exchanger. Again, good selectivity and sensitivity were obtained. It is worth noticing that apparently all the three reported methods did not suffer from interferences due to refractive index variations. Although this is an advantage in common with solid phase absorptiometry, the magnitude of interference, if any, appears to be even less than the latter.
5
Gas-liquid Separation
5.1
Introduction
5.1.1
General
In flow-injection analysis, volatile analytes or analyte compounds may be separated from interferents in an ill-defined sample stream and transplanted into a liquid or gaseous acceptor stream with well-defined composition. Reaction conditions for effecting the gasliquid separation and detection of the separated species may be optimized independently. often greatly enhancing the selectivity of the determinations. The gas-liquid separations are effected through ·on-line separators incorporated in the Fl manifolds. The effects of the separation process are often equivalent to batch distillation or isothermal distillation procedures, such as the Conway micro-diffusion method I J ], developed some forty years ago, which are much less efficient and consume much more sample and reagent. Like most other Fl analytical processes, the separations are also almost always performed under non-equilibrated condition!.. and the phase transfer factors P are rarely higher than 0.3. usually being in the range 0.05~.2. While this sometimes may have some unfavourable effects on sensitivity. they may be compensated for; whenever necessary. by preconcentration measures during the gas-liquid separation. On the other hand. the non-equilibrium conditions may be exploited favourably to improve selectivity through kinetic discrimination lcf. Sec. 5.5.1 Tolerance of interferences in FI hydride generation systems). In 1979, Baadenhuijsen and Seuren-Jacobs 121 were the first to report on a FJ gas diffusion separation system with a semi-permeable dimethylsilicone rubber membrane. used for the determination of carbon dioxide in plasma. In the same year. Zagatto et al.[3) introduced an isothermal distillation FI system in which ammonia diffused from a flowing donor liquid film across an air-gap and absorbed by a flowing acceptor film on the opposite side of the gap. However, later developments on gas diffusion separations mainly followed the approach of Baadenhuijsen and Seuren-Jacobs, obviously due to its simpler design and higher versatility. The first theoretical study on an Fl gas-diffusion separation system was attempted by van der Linden [4], who used a tank-in-series model for the mathematical evaluation of the separation process. The first report on using a gas expansion separator for separation of volatile hydrides from the liquid reaction mixture in a Fl system with AAS detection was made by Astrom
130
5 Ga.t-liquid Separation
in 1982 in the hydride generation determination of bismuth [5), and in 1986, Pacey et al.[6] were the first to propose the use of a dual-phase membrane gas-diffusion system for the same purpose. Despite the apparently narrow range of analytes which could be determined by effecting a gas-liquid separation. the gain in selectivity is usually so significant that the related techniques have undergone rapid development since the first contributions: and the range of analytes has expanded from inorganic to include volatile organic constituents. such as ethanol and acetone.
5.1.2
Classification of FI Gas-liquid Separation Systems
FI gas-liquid separation systems may be classified according to the chemical processes involved. In most cases the analyte is transformed into a volatile species by means of a suitable acid-base chemical reaction before the separation: such as for the release of carbon dioxide, sulphur dioxide. hydrogen cyanide, hydrogen sulfide. Sometimes the analyte is sufficiently volatile to be separated from the liquid phase under elevated temperatures without funher reaction. Among these are ethanol, ozone. and chlorine dioxide. The separated gas phase containing the analyte may be transponed, usu'ally with the help of a suitable carrier gas. directly to the detector, such as in hydride generation AAS, or it may be absorbed by a liquid acceptor stream and then transponed. with or without a derivatization reaction. to the detector. as usually performed in spectrophotometric systems. Introduction of a reagent into the acceptor stream not only enhances the selectivity but also the sensitivity by creating more favourable kinetic conditions for mass transfer. When the separated gaseous analyte species reacts with a reagent to form a different species on the acceptor side. the concentration of the gaseous analyte is maintained at a very low level at the separation interface. which favours the funher release of the analyte. Thus. the transfer of carbon dioxide is enhanced by using a basic acceptor stream, and the transfer of ammonia is enhanced by an acidic acceptor. FI gas-liquid separation systems may also be classified according to the physical means of separation. Two main approaches are available for performing on-line gasliquid separations in FIA systems, i.e.: a b
Separation by gas diffusion through a liquid impermeable micro-porous membrane. Separation by gas expansion, often assisted by a suitable carrier gas, in a chamber where the liquid phase is directed to waste, and the gas released from an upper outlet.
5.2 Gas-liquid Separators for FIA
131
Separation by gas diffusion is suitable for general use, while the latter is used almost exclusively for vapour generation applications with atomic spectrometric detection. where the separated gases are transported directly to the detector without further derivatizations. Gas-diffusion separations may be further divided into single and dual-phase separation systems. Single-phase system~ are used for spectrophotometry, electrochemistry and chemiluminescence etc., with which liquid phase is used in both donor and acceptor channels. Dual-phase systems use a suitable gas as the acceptor stream, while the donor stream is liquid; such systems are used with mass spectrometric (cf. Sec. 5.4.6) or electron capture detection [7 j, but may also be used as a substitute for gas expansion separation in vapour generation atomic spectrometric determinations (cf. Sec. 5.5.1 FI hydride generation manifolds with dual phase gas diffusion separators)
5.2 5.2.1
Gas-liquid Separators for FIA General
Gas-liquid separator designs used in FlA may be divided into two main categories, depending on whether the separation is based on gas diffusion or gas expansion. However, there can hardly be a clear margin drawn between the process of diffusion and expansion. The main difference between the two species of separators used to be that a membrane is almost always used for gas diffusion from one channel to another channel. whereas gas expansion separators are characterized by separation chambers with significantly larger capacities. but usually without a membrane. However. recently there is an increasing tendency of implementing membranes in expansion chambers. leaving the geometrical aspect as the only difference between the two species of separators.
5.2.2
Gas-diffusion Separators
There are mainly two designs of gas diffusion separators, i.e., the sandwich type and the tubular type, the schematic diagrams of which are shown in Fig. 5.1 and 5.2. A common feature of the two designs is that they have sepru:ate channels for a donor and an acceptor stream separated by a membrane which is permeable to the gaseous analyte species. The difference between the two designs is in the form of the membrane.
132
5 Gas-liquid Separation ACCEPTOR IN
G
ACCEPTOR OUT
DONOR .....,.. IN
G
A
D
b
c
F'tg.5.1: A typical sandwich-type membrane gas-diffusion separator. a, side view: A. B. plastic blocks with F. threaded fittings; and G, engraved grooves; M, microporous gas-diffusion membrane. b. top view showing position and configuration of a straight channel groove. c, a simplified schematic presentation of the sandwich-type gas-diffusion separator; D. donor stream; A, acceptor stream; M. membrane.
A typical structure of the sandwich type gas-diffusion separator is shown in Fig 5.1. As its name implies, the membrane which separates the donor and acceptor streams is sandwiched between two half blocks on which are engraved channels that are minor images of each other. An inlet and an outlet port, extending from the channels and furnished with connectors, are provided on each block. The blocks, usually made of
5.2 Gas-liquid Separa10rs for FJA
133
D
ON
--+
t ~I
L..
0
T
I
I 1
t
r
I
~w
'
A
Fig.S.2: Schematic figure of a tubular membrane gas-diffusion separator. DN, donor stream; T. inner microporous membrane tubing; W. waste; 0. outer tube of nonporous material with inlet and outlet for acceptor stream, A; D. to detection system. plexiglas PVC or PTFE, are held together tightly by screws to avoid leakages between the blocks and the inserted membrane. The configuration of the engraved channels are usually straight, but occasionally winding or spiral. with semi-circular, rectangular or triangular cross-sections. The dimensions of the channels may vary in the range 0.1-1.0 mm in depth, 1-3 mm in width and 3-10 em in length. Sometimes the channels are filled with glass beads or the membranes are lined with nylon or stainless steel gauzes to provide mechanical support for the membrane in order to prolong the membrane 1ifetime. Fig 5.2 is a schematic diagram of a tubular type gas-diffusion separator. The separator is column shaped. with two concentric channels. The tubular membrane forms the inner channel. usually reserved for the donor stream, and extends beyond the outer channel to be connected to the suppl\ and waste conduits. The terminals of the outer channel are extended to inlet and outlet ports on the column which are furnished with connectors for the acceptor stream conduits. The outer channels are made of plastic or glass tubing with 5-10 mm i.d .. and the inner channels made of membrane tubing, such as Gore-Tex microporous PTFE tubing. with dimensions of 2-4 mrn i.d .. 3-5 mm o.d. and 8-20 em long. Various designs of tubular membrane gas-diffusion separators have been reported by Aoki et al.(8J, Nagashima et al.[9] and Motomizu et al.[ 10]. Occasionally, the tubular separator is used without an outer channel. i.e .. without an acceptor stream, when the purpose of the separation is to remove non-hazardous gaseous products in the ftow stream. in which case the gases are simply released into the atmosphere. The separator is then merely a coil of microporous membrane tubing. With toxic gases, or when more complete removal of gases are desired, the porous tubing may be housed in an outer chamber which is connected to a vacuum source such as a water aspirator. Such a system was used by Nyasulu [11] for the removal of sulphur dioxide generated in the on-line destruction of thiosulfate when an indirect potentiometric FI method was used to determine silver in photographic fixing solutions. In this application the gas-diffusion tube was made into a single-bead-string configuration by inserting glass beads into the tubing in order to promote mixing and minimize dispersion.
134
5 Gas-liquid St>paration TO VACUUM
t
t Reagent In
Reagent out
Fig.5.3: A sandwich-type reagent degasser. A. B. plastic blocks; D. sintered metal disk; M. microporous membrane; and G. engraved groove (12].
A modification of the sandwich type gas-diffusion separator wac; used by Pedersen et al.[ 12] as a degasser in an FI system for monitoring and controlling of a biological waste water treatment plant with biological removal of phosphate and niuate. The degasser, used to maintain long term low stable flow-rates. was installed for every reagent and was placed between the reagent bottles and the pump intake. A schematic diagram of the degasser is shown in Fig. 5.3. The construction features a single outlet on the acceptor side which was connected to a vacuum source and the PTFE membrane was backed by a sintered metal support on the acceptor side. Another approach for on-line degassing of solutions is to use a standard sandwich type gas-diffusion separator. One of the ports of the acceptor channel is blocked and the other connected to a vacuum source or to a pump which evacuates the gas from the channel. Such an arrangement was used by Hinkamp and Schwedt ( 13] in the determination of total phosphorus in waters with amperometric detection to remove gas bubbles generated in the reaction stream after an on-line continuous digestion in a microwave oven. Gas-diffusion separators have been integrated with detection cells to produce more compact systems both in optical sensing and atomic absorption spectrometry. These will be described in more detail in the sections on the coupling to individual detectors (cf. Sec. 5.4.2, 5.5.2)
5.2.3
Gas-diffusion Membranes
The membranes used in F1 gas-diffusion separation systems may be classified according to the mechanism of mass transfer of gaseous components across them. Microporous or heterogeneous membranes, usually made of PTFE or polypropylene, allow peneua-
5.2 GaJ-liquid Separators for F/A
135
tion of gaseous components by diffusion through a gas layer trapped in the pon:_s of the membrane, whereas mass transfer in non-porous or homogeneous membranes. such as silicone rubber. depends on the difference in solubility of the gaseous components between the membrane material and the liquid streams. Since the membranes are usually hydrophobic. ionic transfers are hindered. However, occasionally large pore membranes of 3-5 pm. which may accommodate aqueous solution in the pores, are used to effect gas-diffusion and dialysis simultaneously (cf. Sec. 5.5.1 ). The required properties of membranes used in FI gas-diffusion applications may include the following: a
b c d e
Membranes should be permeable only to gases, and impermeable to other constituents in the liquid phase. Membranes should be hydrophobic to avoid mass transfer through dialysis. Membranes should exhibit good kinetic properties, i.e., allow efficient transfer of gaseous phase across the interface. The mechanical strength of membranes should be sufficiently strong to allow reasonably long lifetimes under continuous operations. Membranes should be chemically resistant to common reagents to ensure stable performance for extended periods. The surface properties of membranes should be such that they will not be easily clogged by suspended materials in the donor streams.
PTFE membranes 0.01-0.08 mm thick and 0.1-0.45 J.lm pore size come close to the above requirements except for its relatively low mechanical strength. However, with some mechanical support (cf. Sec. 5.2.2) the lifetime may be prolonged. Polypropylene membranes were reponed to have better mechanical properties [14], and higher water entrance pressures [ 15] than PTFE membranes and may be a good substitute for the latter. The lifetime of the membrane will also depend on the sample species. With relatively clean water samples a membrane could last for months under continuous operation. whereas the membrane may have to be changed everyday if the sample contains appreciable amounts of suspended materials.
5.2.4
Gas-expansion Separators for Vapour Generation Atomic Spectrometric Systems
In vapour generation methods for atomic spectrometry, the detection of the analyte is achieved in the gaseous phase; the amount of gas produced is normally much larger than separations based on collection in a liquid- acceptor stream, and a carrier gas is usually used to assist the efficient separation and transport of the gaseous analyte to the detector
136
5 Gas-liquid Seporotion
(cf. Sec. 5.5). In the earlier phase of development of Fl vapour generation methods, the Vijan-type U-tube gas-liquid separators were used most frequently to separate the, gaseous phase from the reaction mixture [ 16]. The gas-liquid separation occurs mainly in the expanded bulb of the separator, in a conventional Vijan-type separator the gas expands upwards through an outlet into the atomizer, while the liquid flows under gravity to waste from a second outlet, the U-tube bend forming a liquid seal to prevent outflow of gas from this end. Wang and Fang [ 17] commented on the importance of a miniaturization of the separator on the basis of the conventional Vijan design, owing to the much smaller sample volumes often used in FI systems. A typical glass U-tube separator is shown in Fig 5.4 a. The internal capacity of the bulb was decreased to about 50 % of a normal Vijan separator to minimize dispersion. Although effective in improving the sensitivity, this also introduced additional risks in incomplete gas-liquid separation. In order to prevent entrainment of liquid into the heated quartz atomizer, and induce deleterious effects in the readout as well as poisoning of its surface, it is strongly recommended to control the out-flow of the liquid from the separator outlet using a pump, a measure which is not necessary in conventional Vijan-type separators. The draw-out flow-rate should be made substantially higher than the liquid total in-flow to ensure that no liquid enters the atomizer. A fraction of the evolved gas is inevitably drawn out with the waste liquid; however, the amount is relatively small compared to the total gas volume, so that the effect on sensitivity is negligible. Later, it was realized that with a forced out-flow of waste the U-configuration of the outlet tube contributed nothing to the completeness or efficiency of separation, and it is quite sufficient to withdraw the waste liquid at the lowest point of the separator. This resulted in substantially simplified designs such as the commercialized version shown in Fig. 5.4 b. The separation performance was further improved by packing the separator half-full with glass beads of approximately 3 mm diameter. which decreases aerosol formation and foaming. An additional precaution for preventing migration of liquid particles into the heated quartz cell was taken by Welz and Schubert-Jacobs [ 18] by connecting a length of microporous PTFE tubing. with one end blocked, to the outlet of the gas-liquid separator, the tube protruding into a secondary outer tube with a larger diameter (Fig. 5.4 c). A cylindrical gas expansion separator was proposed by Ikeda [19]. which features an inner reservoir to accept inflow of the reaction mixture. The gas was released from the mixture in the reservoir into the separator and carried off into the atomizer by an argon flow, while the liquid phase was drawn to waste after overflowing from the reservoir into an outer ring. The reservoir was apparently intended to ensure more complete release of the hydride. however, the reported detection limit for arsenic was no better than that using U-tube separators owing to the worse precision. Furthermore, the sample throughput was substantially degraded because of the longer residence time of the reaction mixture in the separator and perhaps also because of its larger dead volume. The interference effects may also be enhanced using such designs because kinetic discrimination cannot be fully exploited owing to the delay in reaction time.
5.2 Gas-.liquid Separators for FIA
137
D
D
w
w
1----1
1cm
8
b
c
Fig.5.4: Gas-expansion gas-liquid separators. a, modified Vijan-type U-tube separator: b. PerkinElmer W-configuration separator: and c. modified W-configuration separator with PTFE tube scrubber. G-L. gas-liquid mixture: D. to detection system; W. to waste pump; B. glass beads: T. microporous PTFE tube with blocked end; 0, outer tube with gas outlet.
Recently. Marshall and van Staden [45] described a gas expansion separator used for FI hydride generation AAS which is separated into two compartments by a cotton gauze membrane. The reaction mixture is allowed to flow on the membrane from the top compartment, where a flow of argon gas is directed parallel to the membrane to sweep the hydrogen and hydrides into a quartz atomizer, while the liquid phase is pumped out from the base of the lower compartment (cf. Sec. 5.5.1).
138
5 Gas-liquid Srparation
s CA A
A
F~g.S.S:
1--_..,w Schematic diagram of a typical FI manifold with gas-diffusion separation and volumebased sampling. CR. carrier stream: S. sample; R. reagent for fonnation of volatile analyte species; SP. membrane gas-diffusion separator; A. acceptor stream: D. detector and W, waste outlets for donor and accep1or streams.
5.3
FI Gas-diffusion Separation Systems
5.3.1
Basic Gas-diffusion Separation Systems
Fig. 5.5 is a schematic diagram of a typical FI manifold for gas diffusion separations. A defined volume of sample is injected into a carrier stream and, if necessary, is merged with an reagent to transform the analyte into a volatile species. The resulting donor stream is transported through a length of mixing coil to provide adequate mixing and reaction time, and thence into the donor channel of the gas-diffusion separator where the gaseous analyte species diffuses through the membrane into the acceptor channel and is absorbed by the acceptor stream which is propelled continuously to the flowthrough detector. The acceptor streams may or may not contain reagents, depending on the analyte s~cies and the detection system. The ftow-Jates of the donor and acceptor streams typically vary in the range 0.5-1.5 ml min-•, and are usually identical for the two streams to achieve optimum transfer efficiencies (cf. Sec. 5.3.4).
5.3.2
Gas-diffusion Preconcentration Systems
When the acceptor stream is stopped for a predetermined period while the sample in the donor stream is pumped continuously through the donor channel of the gas diffusion separator, a preconcentration of the gaseous analyte may be effected in the acceptor
5.3 Fl Gas-diffusion Separation Systems
139
Rt Ra p
a Fig.5.6: A manifold with gas-diffusion separator nested in sample loop of .the injection valve. used for preconcentration of volatile species by time-based sampling (sample loading sequence). AS. autosampler. T. heating thermostat (optional); GDS, gas-diffusion separator; V. injection valve; R 1• reagent for generation of volatile species: R2 • acceptor reagent stteam; R3. derivatization reagent (optional); D , detector; W, waste; a, valve position in sample injection sequence. Crossed circles in valve represent blocked channels [20].
stream. at the expense of a lower phase transfer factor. This involves the introduction of larger sample volumes. making the volume-based sampling approach as shown in Fig. 5.5 rather inconvenient. A time-based sampling gas-diffusion preconcemration Fl system with a sandwich type separator nested in the sample loop is shown schematically in Fig 5.6. The system was reported by Zhu and Fang [20] in the determination of total cyanide. The samples were aspirated from an autosampler to facilitate sample changeover, and acidified before passing through a heated (60°C) PTFE reaction coil to liberate the hydrogen cyanide. The sample stream containing the hydrogen cyanide was transported through the donor channel of the separator while the basic acceptor stream was temporarily stopped for a defined period by switching the loop out of the flow. During this preconcentration period the hydrogen cyanide penetrating the membrane was collected in the acceptor solution. At the end of this sequence, the acceptor flow was restored on actuation of the valve, and the collected cyanide was transported to the detection system after derivatization to produce a detectable species. With a 50-s preconcentration period consuming 2 ml of sample a 3.5-fold enhancement in signal was obtained compared to using a system resembling that in Fig. 5.5. A similar preconcentration system was used by Schulze et al.[l4] for the determination of ammonia. achieving 10-fold sensitivity enhancement in a 10-min preconcentration period.
140
5 Gas-liquid Separation
A disadvantage of the above system is in the mode of sample changeover, which requires synchronization of the action of an autosampler with that of the injection valve. This not only complicates the instrumentation but also introduces air segments into the donor flow, bringing deleterious effects on the membrane due to pressure fluctuations in the separator channels. This last defect may be avoided by stopping the pump when the sample probe of the autosampler is lifted out of the liquid surface, or by including an additional valve in the system, which further complicates the system. A more versatile gas-diffusion preconcentration system using the S-channe! multifunctional valve described in Sec. 2.2 is shown in Fig. 5.7 a. b [21]. Not all the valve channels are used in this application, and the unused channels are blocked. This system do not require autosamplers for controlling the sample volume. which in this case is determined by the flow-rate and the time of actuation of the valve. The preconcentration factor depends on the length of the sample loading period. Introduction of air segments in between samples is also avoided, which is beneficial for prolonging the membrane lifetime. In the sample load (preconcentration) sequence a section of the acceptor stream is stopped in the gas-diffusion separator while the main stream bypasses the separator and flows to the detector to define the baseline of the measurement. The gaseous analyte species diffuses through the membrane, and is collected in the acceptor solution. At the end of the loading sequence the valve is switched to injection position and the acceptor concentrate is carried by the acceptor stream to the detector. The system was used for the determination of ammonium ions at ultra-trace levels. With a 3 min preconcentration period, 60-fold signal enhancement was obtained compared to a separation system without preconcentration. achieving a detection limit of l JJ.g 1- 1 •
5.3.3
Factors Influencing Mass Transfer in FI Gas-diffusion Separation Systems
Factors influencing the mass transfer and performance of FI gas-diffusion separation systems were studied in detail, and discussed by Karlberg and Pacey [22] using chlorine dioxide as the gaseous analyte. Some important observations mainly based on their results include: a
b
c
The mass transfer of the gaseous analytes increases with a decrease in sample/carrier flow-rate, particularly when the gaseous analyte is transformed into another irreversible chemical species in the acceptor solution. The highest phase transfer factor is obtained when the flow-rate ratio of donor and acceptor streams is one, and decreases with a deviation from unity to either direction. Slightly higher peak signals are obtained with the donor and acceptor streams flowing countercurrently than flowing concurrently. But the enhancement is mainly due to a change in peak form and not to an increase in the mass transfer.
53 Fl ml min-t
s
1.5
c
1.5
Ga.~-diffusion
Separation Systems
141
-
1.2
v Fig.S.7a: FI manifold of gas-diffusion preconcentration system using multifunctional valve with time-based sampling (valve in injection position). Experimental conditions are for the preconcentration of ammonia. S. sample: C. carrier (water); R 1, reagent for generation of volatile species (base); R2. acceptor reagent stream (buffered acid-base indicator):· V. 8-channel multifunctional valve (unused channe's are not shown),; W. waste flows.
3mln
A
2 M 1
N
0
Fig.S.7b:
''
-
-m
chart recordings of spectrophotometric measurements of 0.1 p.g 1- 1 ammonia using 0.5--3 min sample loading (preconcentration) periods. Arrows point to signals obtained without preconcentration. i.e., with equal donor and acceptor flow-rates [21].
5 Ga.~·liquid Separation
142
d
e f
g
Optimum mass transfer occurs when the pressures on the donor and acceptor channels of the separator are equal. both for supported and unsupported membranes. With a fixed channel length, the mass transfer increases with an increase in the width of the separator channels. Serpentine and spiral-shaped channels enhance mass transport of the gaseous phase by an increase in turbulence compared to straight channels. with spiralshaped channels giving the best efficiencies in gas transport. Gas transpon improves with a decrease in channel depth. However. the relationship is non-linear due to the increase of linear flow-rate with more shallow channels, which shortens the contact time between the donor stream and the membrane.
5.4
Coupling of FI Gas-diffusion Separation Systems to Various Detectors
5.4.1
Spectrophotometric Detectors
The most frequently used detector in FI systems with gas-diffusion separation is the spectrophotometer. Quite often the gas-diffusion process offers sufficient selectivity to allow relatively non-specific chemical reactions in the acceptor stream to detect the analyte. Thus, carbon dioxide, sulfur dioxide, hydrogen sulfide. ammonia may all be determined using suitable acid-base indicators in appropriate buffer solutions used as the acceptor streams. The concentration of the buffer solutions may be adjusted to suit a cenain concentration range for the analyte. In order to funher enhance the selectivity and/or sensitivity more specific reagents may be introduced in the acceptor streams. In the previously mentioned example on the determination of cyanide [20] a modified pyrazolone-isonicotinic acid reaction was used for such purposes. Interferences due to Schlieren effects seem not to have been reponed in gas diffusion spectrophotometric systems. This is understandable, since the matrix composition of acceptor streams is usually quite uniform, and the refractive index is little affected after absorbing the gaseous analytes. For the gas-diffusion spectrophotometric determination of carbonate, sulfide, sulfite and ammonium nitrogen, the volatile compounds generated under suitable pH conditions penetrate the membrane in a gas-diffusion separator and are collected in acceptor streams containing the appropriate chromogenic reagents. When the separation process is sufficiently selective, the determination may be based on a protolytic reaction, with the
5.4 Coupling of FJ Gas-diffusion Separation System.f to Various Detectors
143
pH variations in the acceptor stream indicated by a suitable acid-base indicator. When additional selectivity is required in the detection, more specific reagents are then used in the acceptor streams. When acid-base reagents are used, the indicator species as weJI as the composition and concentration of the buffer must be carefully chosen to achie?ve good stability of baseline. and to provide the necessary sensitivity and linear range in the calibration graph. Van der Linden [23] discussed the conditions affecting the linearity and sensitivity of such determinations on the basis of a theoretical treatment, indicating that higher sensitivities are obtained using lower total buffer concentrations. accompanied by narrower linear ranges. and highest sensitivities are achieved with no buffers in the acceptor streams except the indicators. Ammonia in whole blood and plasma is determined by adding sodium hydroxide to the sample and releasing the ammonia into an acceptor stream containing phenol red indicator [24] (cf. Sec. 9.2.4). Various specific reagents have been applied by different workers to improve the selectivity and/or sensitivity of spectrophotometric determinations in the acceptor streams. Stratka et al.[25] determined residual aqueous ozone using the redox reagents potassium indigo trisulfonate and bis(terpyridine)iron(Il) after a gas-diffusion separation which also transferred chlorine across the membrane. The reagents showed sufficient specificity for ozone to make a selective determination after the separation. Recently Sonne and Dasgupta [26] determined sulfide in the presence of sulfite and thiosulphate etc. by controlling the pH of the donor stream to a range which was effective in liberating hydrogen sulfide without releasing major amounts of sulfur dioxide. The hydrogen sulfide was coJlected in an alkaline acceptor stream containing pentacyanonitrosyl-ferrate(II) which reacted selectively with the sulfide. The reaction product is unstable. but was of no consequence in the determination due to the reproducibility of timing in the A system. Sulfite was determined in the same mixture by acidifying the donor stream to a pH as to generate sulfur dioxide significantly without significant decomposition of the thiosulfate. Sulfite was then detected selectively by the bleaching of a pararosaniline acceptor stream. Tanaka et al.[27] described an interesting application on the simultaneous determination of cyanide and thiocyanate which were transferred simultaneously from a phosphoric acid donor stream into the acceptor stream of a tubular PTFE membrane separator. The membrane featured a large pore size of 3.5 !Jm, making it possible not only for the penetration of gaseous hydrogen cyanide but also for the passage of ionic cyanide and thiocyanate in a dialysis process. The same reaction system of chloramineT/pyridine/barbituric acid was used for the two analytes by discriminating them kinetically. At pH 8.1 thiocyanate reacLo; very slowly with chloramine-T, so that cyanide may be determined without interference from thiocyanate. At pH 6.0 both analytes react very rapidly with chloramine-T so that a total concentration of the two analytes may be obtained.
144
5 Gas-liquid Separution
Pacey's group [28] reponed an extremely selective spectrophotometric method for the detennination of chlorine dioxide which exhibited 5400-fold higher selectivity for chlorine dioxide over chlorine. The high selectivity resulted from a synergistic combinalion of the removal of transition metal interferents by gas-diffusion. kinetic discrimination in gas transfer efficiency across the membrane and optimization of the acceptor stream composition by the addition of oxalic acid. Volatile organic constituents may also be detennined by gas-diffusion spectrophotometry with high selectivity and efficiency. Thus Marstorp et al.[29] detennined oxidized ketone bodies (i.e. the sum of acetoacetic acid and acetone) in milk after an off-line pretreatment at l00°C, during which the acetoacetic acid was decarboxylated to acetooe. Acetone in the donor stream diffused at room temperature through a PTFE membrane into a reagent acceptor stream containing hydroxylammonium chloride and methyl orange at pH 3.7. Acetone reacts with hydroxylamine to shift the hydroxylammoniumhydroxylamine equilibrium which creates a pH change that changes the colour of lhe indicator. A high sample throughput of 100 h- 1 was achieved. The combination of gas-diffusion separation and enzyme reactions in spectrophotometry produced methods with outstanding selectivity by combining the high selectivity of enzyme reactions with that of gas-diffusion separations. The selectivity is further enhanced through specific chemical derivatizations in the acceptor streams. Petersson ct al.[30] were the first to use such an approach for the selective detennination of urea in whole blood. An interesting recent development of this approach is the integration of the enzyme reactor and the gas-diffusion separator by Spinks and Pacey [31]. Urease was immobilized by adsorption on a PTFE membrane after addition of perfluoroakyl chains to the fn:e amine groups of the enzyme. On contact with the membrane, urea in the donor stream was convened enzymatically into ammonia which penetrated the membrane and was detennined by measuring the absorbance change of an acid-base indicator. A stopped-flow period of 1 min was used to improve the sensitivity. The enzyme consumption in Ibis application was extremely low, and the leaching of enzyme from the membrane seems not excessive, the reponed half-life of the immobilized enzyme membrane being about 14 days.
5.4.2
Optosensing Using Optical Fibers
Ruzicka and Hansen [32] introduced in 1985 the concept of optosensing ~t active sarfaces using FI integrated microconduits and optical fibers. A sandwich-type gas-diffuiion separator was fntegrated with a pan of the detection system as shown schematically ill Fig. 5.8. The gaseous analyte penetrates the PTFE membrane and is collected by a buffered acceptor stream containing an acid-base indicator. The colour change of the iadicator is monitored directly in the acceptor channel using a two-branched optical fibres. The incident light from a spectrophotometer is transmitted by one branch of the oprical
5.4 Co11plint: ofF/ Ga.t-dijfruion Separation Svstems lo Vario11s Detectors
145
A-+---...,
t
s
! 0
Fig.S.8: Schematic diagram of an integnued gas-diffusion separation optosensing detection systt:m. A. acceptor stream with reagent; S. reacted sample with generated volatile species; 0. common outlet to waste for donor and acceptor streams: F. two-branched optical fibers; M. microporous PTFE membrane [32).
fiber to a point on the acceptor channel of the gas-diffusion separator which was produced from black PVC. The light traverses through the acceptor liquid layer and is reflected by the white background of the membrane. traversing again through the liquid layer, and transmitted by the other branch of the optical fiber back to the spectrophotometer. This system was used for the measurement of urea after an enzymatic degradation into ammonia.
5.4.3
Chemiluminescence Detectors
The combination of Fl gas-diffusion separation with chemiluminescence has produced selective methods for the determination of chlorine and chlorinated species. HolloweiJ et al.[33] determined chlorine dioxide by a chemiluminescent reaction with luminol. following a gas-diffusion separation. A T-spiral flow ceU was mounted directly in front of the photomultiplier to maximize the detection of the light emission. Potential interferences from transition metals were removed by the gas-diffusion process, since they do not pass
146
5 Gas-liquid S~pararion
through the membrane. Chlorine is also transported across the membrane. but chlorine dioxide penetrates through the membrane at a preferential rate of 3.1 to I over chlorine. Since the luminol reaction may be manipulated to produce 500-fold higher signals for chlorine dioxide than for an identical concentration of chlorine. this resulted in a method which was over 1500 (3.1 x 500) times more selective for chlorine dioxide than for chlorine: and interferences from iron, manganese and other oxychlorinated compounds were eliminated. A similar system was used by Gord et al.[34] to determine free chlorine (Cb/HOCI!OCI-) in aqueous systems. based on the chemiluminescent reaction of hypochlorite with lophine (2.4.5-triphenylimidazole). In order to maximize the chlorine transfer across the membrane, low pH conditions were established in the donor stream to ensure that the majority of the chlorine species was converted to gaseous chlorine. Chlorine was dissociated to hypochlorite ions under high pH conditions in the acceptor stream which then merged with the luminescent reagent in the T-spiral flow-cell, producing an instantaneous chemiluminescent reaction. Although many of the transition metals produce chemiluminescence with lophine. interferences from transition metals a-; high ao; I M concentrations were not observed in this application owing to the effectiveness of the gas-diffusion separation.
5.4.4
Electrochemical Detectors
Potentiometric
Gas-sensing probes based on pH electrodes covered with gas permeable membranes have been used for some time for the potentiometric determination of gaseous analytes such as ammonia. The internal electrolyte of the probe. which forms a thin film between the electrode sensing surface and the membrane, is static and usually consists of a concentrated solution of the ionic species of the analyte gas in order to ensure reversibility of the diffusion process. Although effective as selective sensors. the achieving of steady state signal as well as return to baseline using such electrodes are time consuming. Such disadvantages are prevented in FI gas-diffusion systems by moving the potentiometric detector away from the membrane separator. and using a continuously flowing electrolyte as acceptor stream. In 1981. Meyerhoff and Fraticelli [35] appear to be the first to report on the coupling of a gas-diffusion separation system to a flow-through potentiometric detector. Ammonia was isolated from the donor stream containing the sample by penetrating a microporous membrane and collection in an acceptor buffer stream, and then determined selectively using a tubular nonactin polymer membrane electrode. The precision ( <7% r.s.d.) and sample throughput (30 h-I) of this early application was rather low. Coetzee and Gunaratna [36] reported on the potentiometric determination of free chlorine in water using a silver/silver chloride electrode following a gas diffusion separation using a acceptor stream buffered at pH 4.5.
5.-J C:oupiing of FJ Ga.~-diftusion Separation Srsrmu to Various Detectors
147
Figuerola et a1.[37] determined free cyanide and cyanide present in weak complexes sequentially using two silver iodide/silver sulfide electrodes with an intervening gasdiffusion separator. Fol1owing the potentiometric determination of free cyanide with the first How-through electrode. the effluent was acidified, and the evolved hydrogen cyanide from weakly complexed species was transferred through the membrane of the gas-diffusion separator. collected in an alkaline acceptor stream and determined with the second electrode. More recently. in the determination of cyanide with a metallic silver-wire electrode Frenzel et al.[38] have shown that relatively non-selective potentiometric sensors may be used to make selective determinations by enhancing the selectivity using gao;-diffusion separations. Potential interferences from sulfide, sulfite and nitrite which may form interfering gaseous species were removed by a pre-oxidation with permanganate and dichromate.
Conductimetric The combination of gas-diffusion separation with conductimetry. recently reponed by de Faria and Pasquini [39], provided a selective. precise and economic approach for the determination of ammonia. and also for nitrates and nitrites, after pre-reduction using an on-line zinc column. This approach appears to be a good substitute for the spectrophotometric methods on the determination of ammonia nitrogen.
Amperometric Kunnecke and Schmid [40] introduced a gas-diffusion separation system combined with an immobilized alcohol oxidase column used for the determination of ethanol in beverages by amperometry. Ethanol vapour from the samples diffused through a siliconemodified polypropylene membrane and was collected in a potassium phosphate buffer acceptor stream before passing through the immobilized enzyme column where the ethanol was transformed into hydrogerr peroxide. The peroxide was determined using an amperometric detector with excellent precision (cf. Sec. 8.4).
5.4.5
Mass Spectrometric Detectors
The feasibility ofFI mass spectrometry was demonstrated by Canham and Pacey [41] using a gas-diffusion separator as an interface between the FI system and a quadrupole mass spectrometer. The same workers [42] used the dual phase gas diffusion FI system with a mass spectrometer to study the formation of different species of volatile hydrides of selenium and arsenic. The combined technique offered extremely high selectivity and sensitivity through the synergistic combination of the selectivity of FI gas diffusion separation and
148
5 Gas-liquid s~partllion
mass spectrometry. A sandwich type separator with Gore-Tex microporous membrane of 0.45 pm pore size, supponed by a nylon mesh on the acceptor side, was used for the separation, and dry helium gas flowing through the acceptor channel was used as carrier to transpon the volatile hydrides to the detector.
5.5
FI Vapour-generation Systems
5.5.1
Hydride-generation Systems
G~n~ra/
Hydride generation atomic absorption specttometry is undoubtedly one of the most popular techniques for the determination of trace amounts of arsenic, selenium, bismuth, antimony and other hydride-forming elements in various sample matrices. Volatile covalent hydrides are formed with the evolution of hydrogen usually by addition of sodium or potassium borohydride. In batch procedures the reactions are carried out in a reaction chamber, and the generated hydrides and hydrogen are swept into a heated quanz atomizer by a carrier gas, usually argon or nitrogen. High sensitivities are achieved with the batch hydride generation technique compared to fl1m1e AAS, and determinations are fairly fast, but sample and reagent consumptions are relatively large. The technique also suffers from interferences, which occasionally may be quite serious, both in the liquid and gas phase reactions. Attempts were made to automate hydride generation determinations and decrease sample consumption using segmented continuous flow systems, but the improvements, ·if any, were only marginal. Asttom [5] was the first to use a FJ system for hydride generation AAS. In the determination of bismuth, high sensitivity was obtained with only 700 ~tl of sample and achieving a high sampling frequency of 180 h- 1• This work was soon followed by many other contributions. involving the determination of selenium, arsenic, lead, antimony, and tellurium, and more recently, tin and germanium, and also by combination of the technique to ICP-OES and atomic fluorimetry using both gas-expansion and dual-phase gas diffusion separators. The important contributions in this field will be dealt with in the following sections.
5.5 FJ
'v'apour-~enerarion
Systems
149
AAS
Ar 120 mllmln
mll•ln
24
NaSH..
Carrier
2
w
10
e
400 IlL
w
v Fig.5.9: Schematic diagram of a typical FI vapour generation AAS system (valve in sample fill position). V, injector valve: SP. gas-liquid separator; S, acidified sample: AAS. quanz cell atomizer; W, waste flows; a, b, reaction coils; Ar. stabilized argon flow. Note that the outflow rate from the separator is higher than the total inflow rate of sample carrier and reagent.
Basic Fl Hydride Generation Manifolds with Gas-expansion Separators
The basic Fl manifold configuration for hydride generation atomic spectrometry has varied little since the early publications, and a typical manifold using the gas-expansion separator in Fig. 5.4 b is shown in Fig. 5.9. The injected samples are usually preacidified to contain 1M HCI and transported by an IM HCl carrier stream to merge with the borohydride reductant flow at a confluence point. The reaction mixture passes through a length of reaction coil and merges with an inert carrier gas flow which carries the liquid-gas mixture into the gas expansion separator. The separation of the gas from the reaction mixture is achieved as described in Sec. 5.2.4. and the hydride is transported into the heated T-shaped quartz atomizer for atomization. The following points in the design of a FI hydride generation AAS system, based mainly on our own experiences using gas expansion sepatators, is worth mentioning [43,44]: a
b
Carrier flow-rates much higher than other Fl systems are required in Fl hydride generation AAS for achieving good precision. The total flow-rate of the reaction mixture is usually in the range 6-10 ml min-• in order to minimize the irregular pulsation effects due to the evolution of hydrogen gas in the reaction coil. Sample volumes of approximately 500 J.tl, which are much larger than those normally used in other FI systems, are often necessary to achieve sensitivities comparable to the batch hydride generation procedures. This may be due to the relatively large dead volume of the gas-expansion separator and the quartz
150
c
d
e
f
5 Gas-liquid S~paration
atomizer cell. This is supported by the fact that, with low sample volumes, the sensitivity increases with a decrease in the dimensions of the gas-liquid separator and the atomizer tube diameter. In spite of the larger sample volume in comparison to most other FIA systems, the consumption is usually less than S% of that in batch procedures. A steady flow of inert carrier gas (usually argon) is required to strip and carry the evolved hydride and hydrogen gases smoothly through the separator into the quartz atomizer. The most often used location in the manifold for introducing the carrier gas is between the separator and the reaction coil, but other points have also been used, and the location seems not to be very critical. Carrier gas flowrates of 50-200 ml min- 1 are typical. Although the flow-rate of the carrier gas is not critical, it should not fluctuate excessively when merged with the reaction mixture which is segmented by generated hydrogen gas in a thin conduit. It is mandatory that the inen gas maintain a steady flow under relatively large pressure fluctuations in the reaction coil, which is important in suppressing the pulsations generated by the discontinuous release of hydrogen gas segments into the gas-liquid separator. To achieve this, Wang and Fang [17] have recommended the incorporation of a copper disk throttle with a tiny aperture of 0.2 mm in the outlet of the pressure gauge of the inen gas tank. A back pressure of about 3 kg cm- 2 could be maintained with a gas flow as low as 150 ml min- 1, which proved to be sufficient for ensuring uniform flow. An alternative is to use a length of capillary tubing as a throttle, but flow-meters with precision needlevalve controls are available which could be controlled directly to produce the required conditions. Larger inner diameters of about 1.0- 1.2 mm for reaction coil and transpon tubing. compared to typical i.d. of 0.5 - 0.7 mm for other systems, are recommended for reducing the back pressure and fluctuations in the conduits due to hydrogen evolution. The larger diameter does not increase dispersion significantly owing to the larger sample volumes, the usually shon reaction coils, and segmentation of the flow stream following reductant addition. The importance of the withdrawal rate of solution waste from the gas-expansion separator was stressed in Sec. 5.4.2. Forced withdrawal using a pump is strongly recommended in preference over free outflow of reaction waste for ensuring optimum precision and long term trouble-free operation. This aspect should not be overlooked, at least in the design of a FI hydride generation system using gasexpansion separators. When gas-diffusion separators are used, the requirements may be different. Shon reactor lengths of approximately I 0 em (I mm i.d. tubing) are recommended for general use following the borohydride reduction. The hydride forming reactions are usually very fast, so that in most cases shon reaction times of a few seconds are sufficient for achieving optimum sensitivity while suppressing the interfering reactions (cf. next section). The smaller exposed tube-wall
5.5 Fl Vapour-generation Systems
151
areas also minimizes sorption of reduced metals which are potential interferents. Short reactor lengths also favour smoother operations, since the back pressure increases significantly with lengthening of the conduit due to excessive evolution of hydrogen gas. However, some analytes with higher valency states, e.g. As(V), show higher sensitivities under longer reaction times, sometimes requiring as long as 280 em coil lengths [ J8]. A pre-reduction to the lower valency state is then recommended, rather than using long reaction coils. Marshall and van Staden [45] described a hydride generation AAS system which incorporated a special gas expansion separator with a cotton gauze membrane described in Sec. 5.2.4. The argon carrier gas was not merged with the reaction flow before entering the separator as usually done, but was used to flush the gases from on top of the cotton gauze membrane to achieve better precision. The system was used for the determination of As. Bi, Sb, Se and Te and has proved to be reliable. However, the detection limits using 300 pi sample (e.g. 8 pg 1- 1 for As) appear to be two orders of magnitude inferior than those usually reported (cf. Table 5.1). The reason for this large difference is not explained. Liversage et al.(46] appear to be the first to attempt combination of an FI hydride generation manifold to an ICP-OES system. The reaction mixture was directed to a conventional pneumatic nebulizer where an argon gas flow was introduced. The nebulizer was connected in turn to a U-tube gas expansion separator the outlet of which was connected to the ICP torch. The outflow of waste from the separator was not controlJed by a pump; this might be one of the reasons for the relatively low reproducibility of 7.2% r.s.d. obtained for As. Another reason might be the introduction of relatively large amounts of hydrogen gas into the plasma when using this mode of separation.
F/ Hydride Generation Manifolds with Dual-phase Gas-diffusion Separators Gas-liquid separation in FI hydride generation have also been achieved using dualphase gas-diffusion separators both in a sandwich and tubular configuration. Yamamoto et al.[47] were the first to report on such a system using a tubular PTFE microporous separator (cf. Sec. 5.2.2) in the hydride generation AAS determination of arsenic, achieving a characteristic concentration of 0.06 pg 1- 1• The system is shown schematically in Fig. 5.10, and features an air-segmented carrier stream, with introduction of argon carrier gas upstream of the reductant merging point. The PTFE separation tube was 25 em long, encased in a glass outer tube having a single outlet port to release the diffused gases into the atomizer. No carrier gas was used in the outer channel. Despite the favourable results obtained by Yamamoto et al., results on using similar tubular separators reported by Wang and Fang (44] and Welz and Schubert-Jacobs [18] show inferior performance in sensitivities and reliability compared to gas-expansion separators, and the latter was preferred. The contradictory reports in the evaluation of gasdiffusion and gas-expansion separators are probably due to the difference in performance
152
5 Gas-liquid Separation
AAS
AIR
S
Ar
HCI G
BH
Fig5.10: Schematic figure of a FI hydride generation AAS system with segmented carrier stream and tubular membrane dual phase gas diffusion separator reponed in ref. 48. S. sample; Ar. argon flow; T. microporous PTFE tubing: G, dual-phase gas-diffusion separator. BH, borohydride reductant; W, waste; and AAS, quartz atomizer cell.
of the gas-expansion separators used in the comparisons rather than differences in the gas-diffusion separators. Barnes and Wang [48,49] studied the performance of both sandwich and tubular-type dual-phase membrane gas-diffusion separators in the determination of As(V) by hydride generation ICP-OES. and obtained better detection limits with a sandwich design, while both were found to be superior to using a gas-expansion separator. Their results seem to suggest that dual-phase membrane separators are better suited at least for combination to ICP-OES systems in comparison to gas expansion separators. but further research efforts may be necessary to reach decisive conclusions. A sandwich-type gas-diffusion separator was used by Chan and Hon [50] for the determination of bismuth by hydride generation AAS, achieving a detection limit of 0.17 p.g 1- 1 using 200 p.l sample. A separator similar to that in Fig. 5.1 was used with a donor groove of 1 x 3 x 50 mm and an acceptor groove of 3 x 3 x 50 mm. A 10 x 60 mm, 200-mesh stainless steel screen was used to support the microporous PTFE membrane by fixing the edges of the screen to the acceptor block using a length of adhesive tape which had a slot cut in the centre to expose the groove region. No carrier gases were used on the donor side, but nitrogen gas was introduced at 200 ml min- 1 into the acceptor channel to carry the diffused gases to the atomizer. FJ Hydride Generation with Integrated Reaction-separation Systems An interesting development of FI hydride generation systems is the use of a solid phase tetrahydroborate reagent in the reduction step reported by Tesfalidet and Irgum [51] and the subsequent integration of the reaction and separation system [52]. In the first report, a column packed with a macroporous strongly basic anion-exchange resin
5.5 Fl Vapour-generation Systems
153
was transformed into the tetrahydroborate fom• hy passing the sodium salt through the column. After being washed with water, an acidified sample was injected through the column which initiated the hydride generation reaction. The column was then regenerated. The procedure was relatively slow, requiring 4 min for each cycle, and the detection limit of 1.5 JLg 1- 1 for arsenic(III), obtained using a flame-in-tube atomizer. was only fair compared to previously reported Fl hydride generation procedures. However, the solid phase reduction approach has stimulated the conception of heterogeneous hydride generation in a packed membrane cell. in which the hydride generation and the gasliquid separation processes are combined. The membrane cell used in the study was quite similar to a standard gas-diffusion separator shown in Fig. 5.1, except that much wider channels of 5 - 10 mm were used. This necessitated the support of the PTFE microporous membrane using a polypropylene mesh on the acceptor side of the membrane. The donor cavity was packed with the anion exchanger which was transformed into the tetrahydroboratc form and washed. The injection of an acidified sample through the exchanger bed triggered the hydride generation reaction, and the hydrides formed were transferred without delay into the acceptor channel where they were flushed by a constant flow of hydrogen gas into a flame-in-tube atomizer. A schematic diagram of the system used is shown in Fig. 5.11.
N
t SIR
B
Fig.S.ll: Schematic diagram of an integraled 'reaction-separation cell for hydride generation reponed in ref. 52. A. B. plastic blocks with engraved cavities. inlets and outlets; C I· cavity packed with P, anion-exchanger: C2. cavity for separated gas; M. microporous PTFE membrane: N. suppon nening: SIR. inlet for acidified sample and borohydride regenerent; W. waste flow: H2. hydrogen carrier gas; D. flame-in-tube atomizer.
The integrated reaction-separation system was tested for arsenic, selenium and antimony and was shown to provide improved tolerance to interferences due to better conditions in kinetic discrimination, but further refinements appear to be necessary to improve the sample throughput and sensitivities.
-
Table 5.1 Perfonnance and characteristic data of representative Fl hydride generation
~
AAS methods Analyte
Bi Se
As Se As,Se,Te Sb,Bi As As,Se,Sb Te,Bi,Sn
Sample
Aqueous Wheat,orchard leaves,soils, coal fty. ash Soils,rice, waste waters Geological Wheat,orchard leaves. coal fly ash Waters Soil,sludge, sea water, blood,urine,etc.
* Characteristic concentration
Separator
U-tube U-tube
Sample volume
I
Detection limit
(ltl)
(h-')
700 400
180 250
(/18
1-'>
0.08 0.06
R.s.d.
Ref.
(%) 0.2-0.8 1.6
v.
~ .s· s::
~
s 17
E:
1"'Ia g·
U-tube
400
220
0.10
1.5
44
U-tube U-tube
200 500
so 120
0.2 0.08-0.6
-
53 59
1000
150
0.07
2.5
60
500
180
0.06-0.27
-
18•
Tubular membrane W-tube
5.5 Fl Vapour-generation Systems
155
Performance of Fl H_vdride Generation AAS 'The analytical perfonnances of some typical Fl hydride generation AAS systems using different gas-liquid separators are shown in Table 5.1. Most sampling frequencies are at least 2-3 fold higher than batch procedures, while the sample consumptions are usually less than 1/20 of the latter to achieve similar detection limits. The relatively low sampling frequency of reference 53 was probably due to the low carrier ftow or large capacity of the separator used. The characteristic concentrations of FI hydride generation AAS with 200--500 J.d sample injection are similar to batch procedures using I 0-20 ml sample, but are somewhat lower than batch methods using maximum allowable sample volumes of 50 mi. However, absolute sensitivities and detection limits of FI procedures are 1-2 orders of magnitude better. T~lerance
of Interferences in FJ Hydride Generation systems
An outstanding advantage of FJ hydride generation AAS is the significant improvement in selectivity. The tolerable concentrations of interfering metallic ions in FI procedures are often reponed to be one to two orders of magnitude higher than batch -systems. The following reasons may be responsible for improvement of tolerance to interferences in FI methods: a
b
c
'The reaction time for the hydride generation process in an FJ system can be precisely controlled by the flow-rate and line lengths to favour the main reactions which are usually fast. The slower interfering reactions are often suppressed by using shorter reaction coil lengths. Such kinetic discriminations are not possible in balch procedures. The high ftow-rate of sample and reagent through the reaction conduit and gas-liquid separator leaves little possibility of accumulating reduced metal or metal boride deposits therein. Such deposits are often considered to be important sources of interference in hydride generation AAS. The smaller sample volume introduces a smaller absolute amount of interferent into the hydride generation system. Since the absolute amount of interferent has often been shown to be more important than its relative concentration in competing with the analyte hydride for free radicals in the atomization process, the beneficial effect of the smaller sample volume in FI hydride generation AAS is obvious.
156
5 Gas-liquid S~poration
B
5.5.2
Fig.S.12: An integrated reaction-separalion-detection A system for cold vapour determination of mercury by AAS. [21,56] A. B. plexiglas blocks held together by screws (not shown): C. reaction coil; D. acidified sample and carrier; E. borohydride reductant; G. grooved channel for flow of reaction mixture; F. waste flow; M. microporous PTFE membrane; I, incident light path; J, slot for gas diffusion into light path.
Cold Vapour Generation Systems
In principle. FI hydride generation AAS systems such as that shown in Fig. 5.9 may be readily transformed into cold vapour generation AAS systems for the determination of mercury using a non-heated quanz cell atomizer; however, research interests have been focused on systems based on an integrated separator - absorption cell. This ingenious integrated design. first proposed by de Andrade [54], and later modified by Fang et al.[21.55,56], is a combination of a gas-diffusion separ:ator and an absorption flow-cell. The separator-absorption cell differs from a conventional sandwich-type gas-diffusion separator in that the acceptor channel is enlarged into a light absorption path which is periodically purged with an ar:gon gas flow to clear the path from residual analyte vapour. The mercury vapour generation reaction was initiated after merging the acidified sample stream with the borohydride reductant. The reaction mixture containing hydrogen gas and mercury vapour was directed into the donor channel which was separated from the absorption path by a nylon gauze supported PTFE membrane. Mercury vapour and hydrogen diffused through the membrane directly into the absorption path to produce an absorption signal. After the valve has been switched back to sample load position, the light path was purged with argon. A characteristic concentration of 1.3 J.d- 1 and a detection limit of 0.06 Jl.g 1- 1 was achieved with 400 JJ.l sample and a sample throughput of 200 h- 1• Later the integrated system was further miniaturized and developed to include also the reaction conduits (Fig. 5.12) and used in combination with an on-line column preconcentration system [21.57]. Purging of the light-path with ar:gon was not necessary
5.5 Fl Vapour-generation Systems
157
in this system because the sampling frequency was detennined by the column loading and elution process rather than by the mercury vapour diffusion rate from the flow cell. Morita et al.[58] reponed on a FI atomic fluorescence method which used a special cylinder-type gas-liquid separator in which a string of small glass beads was hung in a vertical position. The- reaction mixture following reduction of mercury was directed to the string and was allowed to flow down to an outlet to waste. Argon gas introduced at the cylinder base carried the mercury vapour through a trap into the atomic fluorescence detector. Despite a low detection limit of 0.1 J.Lg 1- 1 was achieved with only 64 J.LI sample, the sampling frequency (35 h-I) was extremely low, probably due to the large dead volume of the separator and trap which required long washout periods.
6
Dialysis
6.1 General Dialysis separations are often used for removal of interferents in the sample matrix. The technique is based on differences in mobility of ionic or molecular constituents in a liquid phase during their transport across a semi-penneable membrane into a second liquid phase which need not be immiscible with the first. Mass transfer occurs between a donor phase and an acceptor phase separated by a membrane which selectively allows penetration of solutes by blocking the passage of macro-molecules or by differences in molecular diffusivities. The driving force of the mass transfer is the existence of a concentration gradient of the transferable solute between the two phases. The first application of on-line dialysis to a flow system seems to be that made by Skeggs [I] in his pioneering work on segmented continuous flow analysis. The first report on using on-line dialysis in a non-segmented flow system was that made by Kadish and Hall [2], whereas Hansen and Ruzicka [3] were the first to report such applications in FlA. Despite its early implementation in FIA, applications of on-line dialysis in this field have been rather few compared to other separation techniques, and mostly dedicated to the analysis of blood serum. This may be due to the fact that dialysis is a slow separation procedure compared to the speed of most Fl procedures, and the dialysis efficiencies are usually quite low. Batch procedures for separations through dialysis always require the achievement of mass transfer equilibrium. which is usually very time-consuming, whereas FIA systems with on-line dialysis are performed much more rapidly by effecting separations without reaching equilibrium. This can be realized readily by exploiting the precise-timing feature of FIA, and on-line dialysis may be achieved at high sampling frequencies of more than ]()() h-1. In FI dialysis, mass transfer is executed under reproducible flow conditions which may be interrupted sometimes. but also in a reproducible manner. FI on-line dialysis typically yield reproducibilities of 1-2% r.s.d. Although air-segmented continuous flow systems have also been used for on-line dialysis purposes, the introduction of air segments into the system defied the possibility of producing the highly reproducible conditions required for a non-equilibrated separation, and worse performances were obtained than those for non-segmented FIA systems. Owing to the short time available for solute transfer in FIA, under normal experimental parameters the solute transfer is, at best, usually less than 15%, occasionally less than 1%. This becomes a serious limitation when sensitivity of the method is of concern
160
6 Dialysis
besides the selectivity. Contrary to other FI on-line separation techniques which usually lay their stress on preconcentration procedures, FI on-line dialysis methods are seldom used for analyte enrichment purposes. Obviously. this is due to the mechanism of dialysis mass transfer which is slow and influenced not only by the analyte concentration in the donor but also, to a similar degree, by that in the acceptor stream. However, a preconcentration may be effected by changing the nature of the membrane. With an ionexchange membrane, a preconcentration could be achieved through Donnan dialysis, an example of which is given in Sec. 6.5. On the other hand, FI on-line dialysis may be used conveniently to achieve different degrees of analyte dilution.
6.2
Fundamental Aspects of FI On-line Dialysis
Bemhardsson et al.[4] proposed theoretical models to describe the mass transfer in online dialyzers. With typical dialyzer dimensions, the theoretical results based on laminar flow and plug flow models agreed reasonably well with experimental results, provided that hydrostatic pressures on two sides of the membrane are equal and excessive bulging of membrane is avoided. The phase transfer factor P (cf. Sec. 1.4.6) in on-line dialysis may be conveniently expressed by the dialysis factor, defined as the output concentration of the detector channel. CJ, normalized by division with the initial sample channel concentration, C5 [5]. The phase transfer efficiency may also be described using dialysis percentage by multiplying the dialysis factor with 100. The dialysis factor Cd/Cs depends on several experimental parameters such as permeation qualities of the membranes, membrane area, channel dimensions, analyte concentration, flow-rates of the donor and acceptor streams and their ratios, the relative flow direction of the two streams and temperature. A brief summary on the effects of various factors under FIA conditions, based mainly on results and observations obtained by van Staden and Rensburg [6] using calcium and chloride ions as model analytes are given here. Solute Concentration
Dialysis factors are little influenced by differences in analyte concentrations within relatively wide concentration ranges below 5000 mg 1- 1• However, other coexisting solutes may influence the dialysis efficiency, and interfere by changing the osmotic pressure.
6.2 Fundamental Aspects of Fl On-line Dialysis
161
Relative Flow Direction of Donor and Acceptor Streams
Within a practical flow-rate range of 1~ ml min- 1 concurrent and countercurrent ftow of donor and acceptor streams show no differences in dialysis factors. However, the reproducibility of results are better under concurrent flow conditions. Sample Volume
The sample volume has no influence on the dialysis factor. This implies that for a fixed dialyzer. the ratio of membrane surface area to sample volume will not affect the dialysis factors. Flow-rate
The dialysis factor increases with -a decrease in ftow-rate (equal How-rates for donor and acceptor streams) in the range 1.0- 3.9 ml min- 1• However, precision is better at the larger flow-rates and higher sample throughputs are feasible. Membrane Properties
Dialysis factor increases with a decrease in membrane thickness and an increase in pore size, the influence being more pronounced at higher flow-rates. Channel Length
Dialysis factor increases with an increase in channel length from 70 to 300 em (0.5 mm wide). Obviously. this also implies an enhancement in dialysis factor with an increase in the membrane area. Temperature
Dialysis factors may be noticeably influenced by temperature variations. u 1s advisable to thermostat the dialyzer. usually at 25°, for optimum results. It should be remembered that the above observations were obtained under specific experimental conditions; therefore the statements should not be over-generalized.
162
6.3
6 Dialysis
Dialyzers
Diverse designs of dialyzers have been used in continuous flow systems [7]. but the sandwich (parallel plate) type has been used almost exclusively in FlA. A typical sandwich type dialyzer is quite similar to a gas diffusion separator (cf. Fig. 5.1). The dialyzer is composed of two half blocks. usually produced from plexiglas or other more chemically resistant materials. Semi-circular. triangular or rectangular grooves 0.1-0.5 mm deep and 0.5-2 mm wide are engraved on the two blocks forming mirror images of each other. These grooves function ac; channels for the donor and acceptor streams after joining the blocks and separating the grooves by a dialysis membrane. The length of the channels may vary within a wide range from 5 em to· I 00 em. but in FIA applications channels are seldom longer than 50 em. For space saving reasons. longer channels are always arranged in a winding configuration. After inserting the membrane. the two half blocks may be clamped together by screws, or by some other more convenient means as used in some commercial designs. Sundqvist [8] reported on a commercialized microdialysis unit from Tecator which consisted of two engraved rubber plates for sandwiching the membrane. and the dialysis holder wac; designed to allow rapid exchange of membranes in 2 - 3 minutes. The channels may be filled with small glass beads of 0.1-0.3 mm diameter, the size depending on the depth of the channels. to decrease the dispersion and to serve as a support for the membrane. To facilitate filling. the channels may be wetted with a water soluble surfactant such as Triton X-100, and the open channels are filled with the beads [9]. The membrane is then placed over one of the channels and the two blocks clamped together. The beads are kept in place by inserting pieces of nylon net or plastic foam at the two ends of the channels. In order to balance the pressures on the two sides of the channels. the channel with the lower pressure (usually the donor stream) can be connected to a flow restrictor located downstream of the dialyzer. The restrictor is simply a length of thin tubing. Such measures may become necessary when one channel. usually the acceptor stream. is connected with packed reactors which significantly increase the flow impedance. On the other hand, in order to minimize clogging of the membranes, an increase in pressure on the acceptor side may be necessary when samples containing suspended sediments (e.g. urine) are analyzed. possibly with some sacrifice in the dialysis efficiency. A special tubular dialyzer for Donnan dialysis is described in Sec. 6.5. in connection with the FI manifold used for preconcentration.
6.5 On-line Dial_vsis M~mbranes
6.4
163
On-line Dialysis Membranes
The dialysis membrane is a critical factor contributing to the efficiency of the dialysis separation system. In addition to their specificity on allowing the passage of a cenain species of analyte while obstructing interfering components, which is a common requirement for all dialysis systems, on-line systems demand better mechanical and kinetic properties. Dialysis membranes of Fl systems are expected to allow the achievement of high transfer factors within very short contact times of usually less than 30 seconds, while the membranes should be able to withstand hundreds of analytical cycles without significant deterioration in their performance. Dialysis membranes usually belong to one of the following types: microporous, homogeneous and ion-exchange. of which microporous membranes with pore sizes of 1-10 pm are most frequent,,· used. Homogeneous membranes effect transfer of a species from donor to acceptor by molecular diffusion. Therefore the dialysis factor depends on the solubility and diffusivity of the species in the membrane and not on their particle sizes. Ion-exchange membranes are also microporous, but the pore walls have cations or anions attached to them. These membranes may be used to achieve preconcentration through a process termed Donnan dialysis. When an ion-exchange membrane is used to separate a high ionic strength solution from one of lower ionic strength, ions from the more concentrated solution is transported across the membrane into the more dilute solution Since the membrane does not allow penetration of ions with opposite charge, electroneutrality can only be maintained by the diffusion of ions of the same charge from the dilute solution back into the solution of higher ionic strength. When a solution of low ionic strength contains the analyte, it may be transferred across the membrane to achieve a preconcentration in the solution with higher ionic strength if the volume of the latter is smaller than the sample. Both flat and tubular ion-exchange membranes may be used for this purpose, but hitherto only Nation 811 cation-exchange tubing has been reponed in Fl applications [13] (cf. Sec. 6.5.3). Van Staden and Rensburg [6] compared the performance of eight different commercially available membrane types possibly produced from cellophane, cellulose acetate or natural cellulose with pore sizes ranging from < 1.5 to 6 pm. The best dialysis performance was obtained using Technicon type C membrane which is probably made of cellophane. This was attributed to its smaller thickness of 0.015 mm and the relatively large pore size of 4-t> pm. Some dialysis membranes are asymmetric, i.e. they allow solutes to pass through easily from one side, but much more difficult from the other side. Thus, Xie and Christian [10] reported 50% decreases in signal in the dialysis of sodium and lithium in serum when the wrong side of a Technicon type H (cellulose acetate) membrane was used, using ion-selective electrode detection. Therefore, one should be careful in distinguishing the correct side facing the acceptor stream when mounting such membranes.
164
6 Dialysis
s DONOR
ACCEPTOR
w
A
p F"~g.6.1:
Schematic diagram of a typical A manifold for on-line dialysis. C. donor sample carrier; S. sample; A. acceptor stream; DS, membrane dialyzer; R, reagent; D. detector; W. waste.
6.5
FI On-line Dialysis Manifolds
6.5.1
Basic Manifold Configurations
The basic configuration of a Fl manifold for on-line dialysis· with volume-based sampling is shown in Fig. 6.1. The manifold differs from gas-diffusion manifolds in that usually no reagents are added to the donor stream while most applications involve merging of a reagent to the acceptor stream to transform the dialysate into a detectable species.
6.5.2
Manifolds with Dialyzer as Sample Loops
Fig. 6.2 shows a schematic diagram of a manifold incorporating the dialyzer as a special form of sample loop. The system was used by Chang and Meyerhoff [11] mainly to enhance the dialysis efficiency of the salicylate analyte through a silicone rubber membrane. The diffusive penetration of salicylic acid through the membrane appears to be rather slow, therefore the donor and acceptor streams within the dialyzer were switched out of the flows using two rotary valves to allow longer dialysis periods (trapping times) of a few minutes under static conditions. A similar dialyzer sample loop approach was used by Macheras ~d Koupparis [12] in an automated FI system for drug-protein binding studies.
6.5 Fl On-line
Dialysi.~
Manifolds
165
w
Fig.6.l:
6.5.3
Schematic diagram of a Fl dialysis system with sample circulation (valve in loading position). Pl. P2: pumps: DS, membrane dialyzer nested in sample loop; A. acceptor stream: R, reagent: D. detector; W, waste Ill].
Donnan Dialysis Preconcentration System
The only application of FI Donnan dialysis appears to be that reported by Koropchak and Allen f 13j. The principle of Donnan dialysis is described in Sec. 6.4, and the FI system for preconcentration is shown in Fig. 6.3. The dialyzer, made from 1-5 meters length of 0.89 mm o.d .. 0.64 mm i.d. Nation 81 1 cation exchange tubing, formed the sample loop of a 6-port injector. and contained the acceptor solution (0.5 M strontium nitrate, 0.012 M aluminium nitrate and 0.1 M nitric acid) which was stopped in the loop during the dialysis. The loop was immersed in 400 ml of sample solution and dialyzed for 5-10 min. after which the acceptor solution in the loop wac; transported by a water carrier stream to a flame AA spectrometer. EF values of 100 were obtained · for copper and lead with 10-min dialysis periods. FI provided the possibility of using highly concentrated acceptor solutions to achieve higher EF values without blocking the nebulizer-burner system of the spectrometer.
166
6 Dialysis
c
c
• LOAD
INJECT
Fig.6.3: FI Donnan dialysis preconcentration system for flame AAS. T, ion-exchanger membrane tubing; S, sample solution; B. magnetic stir-bar; V, injector valve (crossed circles are blocked channels); C, carrier; and AAS, flame AA detector ( 13].
6.6
Coupling of FI On-line Dialysis to Various Detectors
6.6.1
Spectrophotometers
In spectrophotometric methods which incorporate an on-line dialysis system, the acceptor streams are almost always connected to the detector after merging with the appropriate reagents. Dialysis is used mostly to remove interferents, such as suspended or colloidal materials, which interfere physically with the photometric detection; but the technique is also often used to achieve different degrees of dilutions automatically. As an early example, Basson and van Staden [14] used an on-line dialyzer to achieve deproteination and dilution of serum samples in the determination of calcium by a cresolpthalein complexone FI photometric method. Owing to the dilution effects and the exclusion of potential interferents, interferences from Schlieren effects, such as those encountered in sorption separation systems (cf. Sec. 4.6.6) were not experienced in on-line dialysis spectrophotometric systems.
6.6 Coupling of Fl On-line Dialysis to l'arious Detectors
6.6.2
167
Electrochemical Detectors
On-line dialysis is a very useful technique for enhancing the selectivity, stability and lifetime of ion-selective electrodes and other electrodes for electrochemical detection FI systems. Gonon and Ogren [15] reponed on the effectiveness of on-line dialysis in preventing the clogging of an ammonia gas electrode membrane by serum proteins and urine sediments in the determination of urea following transformation to ammonia using an immobilized urease column. The effect of on-line dialysis may be further illustrated by the results obtained by van Staden [16] for the determination of chloride in milk with a chloride electrode. Casein in milk interfered in the direct determination by causing a serious positive baseline displacement in potential. An injection of 30 1-11 casein caused an almost irreversible displacement which could not be eliminated even after an hour of continuous washing with 1 M KN03 at 3.9 ml min- 1• This was due to the formation of an insoluble film of casein on the electrode surface. The incorporation of on-line dialysis in the system, however, precluded such difficulties, and resulted in a rapid and precise method achieving an r.s.d. of better than 0.5% at sampling frequencies of 120 h- 1• The implementation of on-line dialysis in electrochemical detection systems may appear to be an imperative measure in the analysis of many sample species, such as body fluids, polluted waters. soil extracts etc., which can potentially form organic films on the electro-sensing surfaces of the detectors. However, a dialysis step is not always indispensable. Masoom and Townshend [17] incorporated a membrane dialyzer to remove interferences in the enzymatic determination of glucose in ~erum, using a flow-through amperometric detector which was preceded by a glucose oxidase enzyme column. Results obtained by dialysis were compared to those using a column of copper diethyldithiocarbamate sorbed on silic:1 gel for removal of interferences. Although no deteriorations were observed on the properties of both the immobilized enzyme column and the platinum electrodes for either of the approaches, the column approach was found to be superior both in sensitivity and sample throughput, and therefore was preferred above dialysis.
6.6.3
Atomic Absorption Spectrometers
Dialysis systems are not often used with the AAS detector. This might be due to the fact that organic colloidal or suspended materials in the sample normally do not interfere in flame AA determinations so that separations are not required. If only dilutions are necessary, usually they can be achieved more conveniently and efficiently by other FI techniques. However, as shown in Sec. 6.5.3. Dorman dialysis may be used to preconcentrate trace analytes and achieve EF values exceeding 100, but at low sample throughputs of only about 6-8 h- 1• The low sampling frequency is rather objectionable for a detector which has to maintain a burning flame during the on-line dialysis preconcentration period. Therefore the use of multiple dialysis cells to achieve higher sample throughputs of 20-30 h- 1 has been proposed.
7
Precipitation
7.1
Introduction
Precipitation is one of the oldest separation techniques used in classical chemical analysis. However, its importance in modem analytical chemistry has declined due to the development of more versatile and efficient separation techniques such ao; solvent extraction and ion-exchange which are also more easily automated. Conventional operations for precipitation in the batch mode are both labour and time consuming, and require considerable operator skill. When coprecipitation methods are used to separate or preconcentrate trace constituents, the long manual procedures are particularly undesirable, as they may introduce contamination risks which are difficult to overcome. Despite the obvious drawbacks of the precipitation-dissolution manual batch procedure, little has been attempted for its automation. presumably owing to difficulties in designing efficient automated procedures for aging. quantitative transfer of precipitates on to a filter, and its subsequent dissolution or weighing [I]. Can FIA provide a solution to the automation of precipitation-dissolution and revitalize its function in modem analytical chemistry? The answer to this question came quite late, well after the application of FI techniques to solvent extraction and column separations. The reason for this delay obviously comes from the difficulties in on-line continuous manipulation of a heterogeneous system which could potentially create serious blockage problems in a standard FIA system. However, recent research efforts in this direction have been quite rewarding, and from the achievements described in this chapter the reader will see that the major difficulties in the automation of precipitation and even coprecipitation processes using Fl techniques have been overcome. The first publication dealing with on-line manipulation of precipitates using a Fl technique was in 1986 by Petersson et al. [2], on the indirect determination of sulfide by flame AAS through the formation of cadmium sulfide precipitate. However, in this work collection of precipitate was avoided by allowing the colloidal precipitate to pass through an ion-exchange column (for details see Sec. 7.6.1). The first achievements in on-line collection and dissolution of precipitates were made by Valcarcel's group in Cordoba [3,4] on indirect methods for the determination of anions, followed by a series of publications by the same group and other groups on the indirect determination of organic constituents [7,8,15,24], preconcentration methods for trace elements [5,9,11,12,18], as well as for reduction of interfering effects [I 0, 11,20], the majority of applications using flame AAS as detector. The topic has also been covered in several reviews [6,13,16].
170
7 Precipitation
Recently an efficient on-line coprecipitation-dissolution system bas been developed by Fang et al.[21] using a knotted reactor collector without filters, which has triggered a series of related publications, using flame and graphite furnace AAS as detectors [22,23]. As a result of the recent developments, separation and preconcentration procedures by precipitation, originally requiring hours of operation and a few hundred milliliters of sample and reagent in the conventional batch mode, may now be completed in less than a minute with an automated FI system, whiJe·consuming a few milliliters of sample and reagent. Risks of contamination are also drastically reduced in the FI systems, which in tum improves the reliability and precision of the determinations.
7.2
On-line Precipitate Collectors
7.2.1
General
The most important component in FI systems designed for on-line precipitationdissolution is undoubtedly the precipitate collection device. The design and geometrical dimensions of this component is a critical factor which governs the overall performance of the entire system. An ideal on-line collector should satisfy the following requirements: •
• •
• • •
Capable of collecting a few milligrams of precipitate without developing prohibitive flow impedance which could reduce a sample and reagent flow of 4-5 ml min- 1 delivered-tbrough a peristaltic pump. The geometrical design should be such that the collected precipitates are easily accessible by washing solution and dissolution reagent The geometrical design and dimensions of the collector should promote radial dispersion and limit the axial dispersion of the dissolved analyte in case of precipitate dissolution. This criterion is more important in trace analysis which requires the highest sensitivity. Produced from inert materials to accommodate different reagents and solvents, and minimize contamination from the collector itself. Robust structure which remains. stable under long term continuous use. Capable of collecting different forms of precipitates.
The performance of various precipitate collectors are discussed and compared in the following sections.
72 On-line Precipilate Collectors
8
.:::. . .
__.=:~·5:P
FLOW
b
171
==•---=___,.
--+
FLOW
Fig.7.1: Schematic diagram of stamless steel on-line filters. a, cylindrical-type; b, planer (disc) type.
7.2.2
Stainless Steel Filters
These are the type of precipitate collectors reported most often [3-9,12-14]. Such filters wert' originally used as cleaning devices in high performance liquid chromatography. The constructions of two different designs of stainless-steel filters are shown in Fig. 7.1 a and b, which all consist or an outer threaded casing, but different in the geometrical design of the filter. The cylindri::al fi Iter (Fig. 7 .l a) is characterized by a large filtration area of ca. 3 cm2 , but also has a relatively lMge dead volume of 580 J.d, whereas the planer rl; ,._ filter (Fig. 7.1 b) ha~ a much smaller area of only 7 mm 2 , and a variable inner volume of I 00-800 ; J. With f-. only filters with )X'fe sizes larger than I 11-m could be used in order to prevent decrease in flow-rate when precipitates are collected. Extremely large pore sizes (300 llm) lead to irreproducible results owing to loss of the smaller precipitate particles. Thus. the cylindrical design was recommended by Valcarcel et [7,13], who reported that the pore size of the filter had no influence on the analytical I\ signal in the range 0.5-2.0 11-m [13]. _ _. Such filters have been used successfully in a number of applications [3-9,tf-14]. However, the dispersion of the collected analyte during dissolution should be rather high, considering the large dead volume of the filter. This is of little importance when the anal:)rte concentration is high, as in the determination of certain pharmaceutical preparations, but may be an important drawback in trace analysis, where the highest sensitivities are required. Although enrichment factors of 500-700 have been reported in the preconcennation of trace analytes using such filters, these were achieved under very low sampling frequencies, and the concennation efficiencies expressed in C£ values were usually lower -. than 10 [5,9]. Adeeyinwo and Tyson [10] used a stainless-steel disc filter with 211-m pore size, 6 mm diameter, and 2 mm thickness to separate calcium from an interfering alurniniwn matrix : by oxalate precipitation. The results were inferior to those obtained using membrane filters, giving poor reproducibility. These results are consistent with the experiences o~ Valcarcel et a1.[7] using disc type stainless-steel filters mentioned above.
at:-/
1
172
7.2.3
7 P"cipilalion
Disposable Membrane Filters
-- - The report by Adeeyinwo and Tyson [10] appears to be the first one using disposable nylon membrane syringe filters for on-line precipitate collection and dissolution. 0.45 -JJm pore-s1ze-fifters Wiili3, 13, and 25 mm diameters were compared. With the 3 mm 1-L___ diameter filter, excessive back pressure occurred even at a low flow-rate of0.3 ml min-•, producing leakages at weaker connections. Much larger flow-rates could be maintained with the 25 mm filter, however, the sensitivity in the flame AAS determination of calcium using 1.5 ml min i!_o~~-~en 5~%_l~Jhan the smaJJ filter, obviously owing to dispersion within the large dead volume of such filters, which may be in the range of
[email protected] JJl for 25 mm filters. A compromise between sensitivity and trouble-free manipulation, using 13 mm filters, was recommended by these authors. The precision obtained was not very impressive, being 4% r.s.d. at 10 mg ml- 1 Ca. and worsened to 15% at 1 mg ml- 1• The life time of such filters was not mentioned, but may not be very long, judging from the thinness of the membrane material. An ev~er cellulose ac~e ~~bl~ filter of ~~__!!!.m diameter was used for the preconcentrl,ltion of coppefl)yprecipitation of the hydroxide [20]. The large dead volume !_ of the filter seems to have a strongmftuence on the perfonnanCe:Since C£ was estimated to be about 0.7 min- 1 (12-fold enrichment in about 3.5 min), with a consumptive index of about I ml. Precision data on the method was not available. The Author's own experience of using a 25 mm membrane filter for on-line coprecipitation was discouraging, the sensitivity in the determination of lead being only about 20% of that obtained using a knotted reactor (cf. Sec. 7.2.5). ·,
7.2.4
Packed-bed Filters
Packed-bed filters made from 3-4 mm i.d. PTFE [11] or tygon tubings [24], packed with polystyrene granules [11], cotton, or filter paper pulp [24] have also been used for on-line collection of precipitates. A 5 em long 3 mm i.d. filter packed with coarse polystyrene granules of 3~ mesh apparently was not sufficient to collect the finer particles, since it was used in combination with a membrane filter (3 mm i.d.) to create a graded collection [11]. This combination was reported to be effective in eliminating excessive back pressure in an on-line precipitation-dissolution system for flame AAS based on calcium oxalate reaction. The EF for Ca was 650 at a sampling frequency of 2 h-I, giving a CE of 21 min -I. Although a high EF may not be so important for Ca in the rock samples studied, the results showed a substantial improvement in sensitivity enhancement capabilities over that obtained by the same group using a larger membrane filter alone cf. Sec 7 .2.3. A packed-bed filter with filter paper pulp or cotton packing was used for the indirect determination of pharmaceuticals by spectrophotometry [24]. The filter column, 3 mm
7.2 On-line
Precipillll~
Co/l«tors
173
i.d., and JO mm long was effective in retaining the precipitate, and did not produce excessive back pressure if the precipitate was dissolved following each precipitation cycle. The capacity of the filter for collecting precipitates is rather low. and therefore is not suitable for collecting large amounts of precipitate in coprecipitation processes. Another disadvantage of such designs is the difficulty in controlling and reproducing the tightness of the packing.
7.2.5
Knotted Reactors
The basic aspects of knotted reactors are given in Sec. 2.3.2. In FIA such reactors have been used mainly to promote radial dispersion and limit the axial dispersion of sample plugs during transport to the detectors. The extension of their use to precipitate collection was quite recent. The first successful application of a knotted reactor to on-line precipitation-dissolution in a coprecipitation system. was reported in 1991 by Fang et al.[2J). The reactor was used to collect on its walls almost quantitatively the black precipitate fonned by a reaction between the coprecipitation earner, iron (II) and hexamethylene dithiocarbamate complexing agenL Flltering devices were therefore not necessary. ISO em of O.S mm i.d., J.S mrn o.d Micro-Line plastic tubing was used to make the reactor by tying interlaced knots on the tubing. but later it was found that the precipitate could be coiJected equally well using PIFE tubing with the same dimensions [23). A lcnoued reactor used for the collection of Fe(II)-HMDTC precipitate is shown in
Fig. 2.10. The mechanism of the coiiection was assumed to be the development of a sustained centrifugal force in the stream carrying the precipitate, as a result of secondary flows created in the three-dimensionaiJy disoriented configuration of the reactor r21 ]. This assumption is supported by the fact that straight, or even coiled reactors made of the very same piece of tubing were much less effective in coiJecting the precipitates. Another reason for the strong adherence of precipitate to the tube walls might be the hydrophobic nature of the tubing material and the precipitate. Therefore, it seems not likely that the collector will be effective for all kinds of precipitate. Apart from precipitate collection, the knotted reactor serves at least two other purposes in the manifold. Firstly, the three-dimensionally disoriented design ensures rapid mixing of sample and reagent solutions to minimize the inhomogenity of reaction conditions in the coprecipitation medium, therefore. no reaction coil prior to separation is necessary; and secondly, limits the dispersion of the precipitate solution after dissolution, when being transported to the detector. The last function is particularly important in achieving high enrichment factors for trace preconcentration procedures. Although the dead volume of the knotted reactor described above amounted to about 300 J.d. which was not much lower than those of the stainless-steel and disposable membrane filters mentioned previously, the three-dimensionally disoriented design is an ideal configuration for limiting the axial spreading of the dissolved species, and contribution to dispersion is negligible.
174
7 Precipitation
· The advantages of the knotted reactor precipitate collector may be summarized as follows: •
• • • • •
Relatively large capacity for precipitate collection owing to the large tube wall area. This is particularly important for coprecipitation applications where amount of precipitate tends to be larger. Low back pressures even at high flow-rates of 5-6 ml min -I owing to the filterless open tube system. Negligible loss in sensitivity from dispersion, owing to favorable three-dimensionally disoriented configuration. Made from inert material, therefore contamination-free. Easily constructed at very low cost. Long and almost permanent lifetime.
The foregoing list included almost all the required features of an ideal precipitate collector mentioned in Sec. 7.2.1, except at the moment it may not be applicable to all forms of precipitate, particularly those with a hydrophylic nature. However, much remains to be. explored in the. material of the reactor, and in the types of precipitate which are applicable with the present design. Even during the preparation of this manuscript, dithizone and APDC were added to the list of complexing agents which produced precipitates suitable to be processed in coprecipitation procedures using the knotted reactor [30].
7.2.6
Choice of Precipitate Collectors
Some general guidelines may be proposed for the choice of the proper type of precipitate collector to serve the purpose of different applications. a
b
c
When sensitivity or concentration efficiency is not an important criteria of the method, and reagents or sample solutions are not extremely corrosive, the large capacity cylindrical stainless-steel filter m~y be the best choice. For trace analyte preconcentration by precipitation, and particularly by coprecipitation, the filterless knotted reactor precipitate collector should be used whenever possible; otherwise the packed-bed column with coarse granules combined with a small diameter membrane filter appears to be the best alternative, although sample loading rates may be limited to low values by the back pressure produced by the combination. Packed-bed filters and small diameter membrane filters may be used when moderate sensitivity or preconcentration effects are required.
73 Fl Manifolds for On-line Prec:ipilalion-dissoluJion
7.3
FI Manifolds for On-line Precipitationdissolution
7.3.1
On-line Filtration Systems Without Precipitate Dissolution
175
i
------, This is the simplest type of FI manifold used for on-line precipitation. used principally with stainless-steel filters [13.16], the variations of which are shown in Fig: 7.2 a-c. In the nonnal FIA oriiFIJUnode, the sample may be either injected directly into the precipitant (Fig. 7.2 a) or injected into a water carrier and later merged with the precipitant (Fig. 7.2 b) before passing through the filter. In the reversed FIA or rFIA mode (Fig. 7.2 c), the precipitant is injected into a continuously flowing sample or blank. In each case. the precipitates fonned in the reaction coil following injection are collected on the filters -~ for a number of injections without dissolution, the filter is then washed periodically in an ultrasonic bath [4), the frequency depending on the amount and fonn of precipitate collected for eachaetenninati~n. The cylindrical stainless-steeLfiher ~ith 3 em~ filtration area was reponed to be able to ~usiain the'-collection of curdy orcg~iiillhous precipitates for 250 samples before a cleaning is needed [ 16], however, the number decreased drastically to 20 for crystalline precipitates if reproducible results are to be obtained. It is therefore questionable whether a system without precipitate dissolution should be used at all for crystalline precipitates. With this type of manifold, obviously no components can be detennined in the precipitate. They may be used either to separate an interfering species from the analyte which is transponed to the detector after the separation, or for indirect detennination of the analyte. In the latter case, the analyte forms a precipitate with a species in the reagent which could be readily detected by the detector, either directly or after a reaction. The decrease in concentration of the detectable ·species (in flame AAS often a metallic ion) was then evaluated to determine the analyte concentration. . Owing to the necessity of periodically dissolving the accumulated precipitates, large capacity filters must be used in such manifolds in order to decrease the frequency of disconnecting the filters. This of course will seriously limit their performance in achieving higher sensitivities. Such drawbacks may be at least partially compensated for by selecting a more sensitive detectable species. However, this type of manifold was designed mainJy to perfonn indirect detenninations of anions and organic constituents which are not able to be detennined by techniques such as AAS, the sensitivity factor is thus not very important. Since the analytical results are based on a difference of two measurements (i.e. a blank with high response, and the sample with lower response) (see recordings in Fig. 7 .2), and the noise level is higher with larger signals which serves as the real baseline, the errors in the measurements may be amplified, especially with low analyte concentrations.
176
7 Precipitation
s PR
w
'
a
___________
--·R
I I I I ,.,~I I
-• ~.
..
p
TIIH
s w
I
'
p
b
I I I
I
R
------------J'
--+-
.- T.[,.... c
D
•
p
Time
PR
s
' I' I
w
I
c BLK
R
--+--
I
----- -------------~
I
........ -• _!rr_ . c
D
p
TiMe
Ftg.7.l: FI on-line filuation systems without precipitate dissolution. a, nonnal FIA (nFIA) mode with sample injected into precipitant; b, nFIA mode with sample injected into water carrier before merging with precipitant; and c. reversed FlA (rFIA) mode with precipitant injected into sample or blank. S. sample; PR, precipitant; P, pump; F. filter; D. detector. W, waste; R. reagent (optional); BLK, blank [13,16].
7.3 FJ Manifolds }or On-line Precipitation-dissolution
177
P1
DS
w l--•w
PA
------.---A
Fig.7.3: Schematic diagram of a Fl on-line precipitation-dissolution system used for the indirect AAS detennination of chloride and iodide. PI, P2, pumps: S, sample injector valve; VI, V2, valves; F, filter; PR, precipitant (Ag•); WA, washing solution (HN03): DS, Jissolution solvent (6M Nf40H); R, water diluent (optional); D, detector; and W, waste [29].
7.3.2
On-line Filtration Systems with Precipitate Dissolution
An irnponant feature of such systems is that they may be used to achieve different degrees of preconcentration. When used for enrichment purposes involving the processing of larger sample volumes. the samples are always introduced using time-based sampling instead oi using sample loops. Such systems may also be used for the_sequential determination of two components when they can be precipitated together and later dissolved sequentially. Various systems for AAS with precipitate dissolution have been proposed with relatively minor modifications fS,lJ,ll,20,29]. Owing to the complications in precipitate_ washing and dissolution. they usually involve the use of three valves. and sometimes. two pumps. Fig. 7.3 is the schematic diagram of an FI on-line precipitation-dissolution \ system used for the indirect determination of mixtures of chloride and iodide by flame AAS [29]. Two pumps and three valves were used in the system. Chloride and iodide in the samples were precipitated with silver ions contained in the precipitant which also functioned as the carrier. The precipitates were collected on a stainless steel filter and --.....; a negative peak representing the total chloride and iodide content was formed due to a decrease in silver concentration in the sample zone. After a washing step, the silver chloride was selectively dissolv¢Jn__Q M _l!mmonia to produce a positive peak proportional to the chloride content alone. Although preconcentration was nol pursued in this application, it may be achieved readily using this system through time-based sampling.
l
178
7 Precipitation
s
c
E
w
Cd(ll)
c
HQ
p
Fig.7.4a: Schematic diagram showing the principle of a filterless on-line precipitation system without precipitate dissolution. C. carriers; S, sample containing sulfide; Cd(II). cadmium sulphate precipitant; E. acid eluent; HQ, quinolin-8-ol ion:Cxcbanger column; D, flame AA detector; P, pump: and W, waste.
7.3.3
Filterless System without Precipitate Dissolution
Only one such system has been reported [2], and was used for the indirect detennination of sulfides by flame AAS. using cadmium as a tag material. A schematic diagram _Jbowing the principle of the manifold is shown in Fig. 7.4 a. The system is characterized by the collection ofr~ionic ~action species (tag element) in the reagent by an online ion-exchange column, following the precipitation reaction between the analyte and the tag element. The colloidal precipi~. containing the analyte w~ atlowed io penetrate the column and transported by the effluent directly into the nebulizer of the spectrometer, and subsequently into the flame, where the precipitate suspension was decomposed and the tag element atomized and detennined. The ion-exchange column was then regenerated by eluting with an appropriate eluent. A recording of the detennination is shown in Fig 7.4 b. The system is not expected to achieve any degree of preconcentration. as is also true for other systems without precipitate dissolution. However, high sensitivity may be achieved by choosing an appropriate tag material. The advantage of the system is that peak heights are proportional to the concentration of the analyte. producing better precision at lower concentrations. compared to indirect determinations based on peak differences (see Sec. 7 .3.1 ). An important limitation of the method is that the precipitate fonned should either be colloidal or extremely fine. so that it is not retained on the column. The particle size of the sorbent may of course be optimized to allow passage of coarser particles, with some sacrifice of the adsorption of the tag ion.
7.3 Fl Man({old.1· j(Jr On-lint• Precipilation-disso/ution
179
s2-
Ill
II
"
a-o,ugmt'
,!!!..!..
1•8
A
•a
•a
., 0
••
.4
Fig.7.4b: recordings of a standard series obtained in the determination of sulfide using the system in a. The off-scale signals in between the standard peaks are those obtained by elution of column (2]. Reproduced by permission of Elsevier Science PublisheJS.
••• blank Scan-
7.3.4
Filterless Systems with Precipitate Dissolution
System for flame AAS
A schematic diagram of the simplest version of such systems used with a flame AAS detector is shown in Fig. 7.5 [21]. The system is composed of two independently controlled pumps and a 4:5 channel rotary injection valve, identical to that used for on-line column preconcentration (cf. Fig 4.11 ). The system has been used successfully for coprecipitation applications with organic precipitants. where relatively large amounts of precipitates were formed using a carrier. The core and most important characteristic of the system is the knoned reactor precipitate collector which is described in detail in Sec. 7.2.5. By using such collectors, precipitates may be collected almost quantitatively on the tube walls of the reactor, so that on-line filters are no more necessary. Some other features of the manifold also deserve mentioning. While most previous designs for on-line preconcentration have the filtering device located between the injector and detector, in this design the collector was connected to the injector as a form of sample loop, so that the effluent of the sample and reagent after the precipitation reaction was directed to waste during sample loading (precipitation) periods without using an extra valve. Another feature is the accommodation of the merging point of the sample and precipitant within the collector loop. This arrangement ensured complete dissolution of the precipitate, whereas a merging point
180
7 Precipi1111ion
PR P, ON
s
a
R .................... OS
w PR p, OFF
b
s R ...............
OS
w Fig.7.5: Schematic diagram of a Fl fiherless on-line precipitation system with precipitate dissolution using a knotted reactor (KR) as precipitate collector. a. sample loading (precipitate collection) sequence; b. precipitate dissolution sequence. P 1• P2 • peristaltiC pumps: V. 4:5 channel injector valve; PR. precipitant: S. sample; R. buffer reagent (optional): DS, dissolution solvent: KR. knoned reactor precipitate collector; and W, waste [21].
upstream of the injector. often used in previous designs, would have resulted in accumulation of precipitate in the transport conduit or reaction coil where the dissolution reagent could not reach. More details on this system, used in preconcentration by coprecipitation with Fe(II)-HMDTC is described in Sec. 7.6.2 and Sec. 9.5.3. Sys1em for ETAAS Recently the system has been adapted with minor modifications to on-line preconcentration by coprecipitation for an ETAAS system (Fig. 7.6) [23]. Owing to the discontinuous nature of the operation in ETAAS, the term "on-line" is used in the sense that
7.3 Fl Manifolds for On-line Precipitalion-dissoiUJioll
181
the FI preconcentration is conducted in parallel with the furnace program of the ETAAS system. Following precipitation and collection of the precipitate in a knotted reactor, the precipitate is dissolved in 60 ~-tl of organic solvent (IBMK) and stored in a length of thin tubing before introduction into the graphite furnace. The FI operation is rather complicated because of the incorporation of a washing sequence to remove residual sample matrix. a segmentation sequence to limit the dispersion during dissolution. and a collection sequence to store the concentrate after precipitate dissolution. This resulted in a procedure consisting of seven sequences with a total operation time of 140-160 s. Although the operation cycles of the FI preconcentration and the graphite furnace atomization program can be arranged to work synchronously with little effon. computer control of the preconcentration sequences is mandatory for reproducible results.
(a) 0.21.. HWA-HWDTC
Concentrate delivery
P1
{b) ml/min 0.2&'1t HMA•HMDTC
Preelpltat Jon
P1
ml/mln
B
182
7 Precipitation
(c) 0.21Yt
Waatllng
HMA•HMDTC
B
(d) 0.28 ..
Dlaaolutlon
HMA·HMDTC
P1
mllmln
a F'ag.7.6: Fl manifold and main operation sequences for on-line coprecipitation ETAAS determination of Cd with the Fe(ll)-HMDTC/IBMK system. a, delivery of stored concentrate (from previous sample); b, precipitation of sample with HMA-HMDTC: c. washing of precipitate with aqueous solution of HMA-HMDTC: and d, precipitate dissolution by IBMK. P 1, P2, peristaltic pumps; KR, knotted reactor, B, solvent displacement bottle; V, 4:5 channel injector valve; C, PTFE concentrate collector tube (60 1-'1); GF, graphite furnace; and W, waste [23].
7.4 Somt' Fundamental AJpt'c·ts of On-lint' Prt'dpitation-disso/ution
7.4
Some Fundamental Aspects of On-line Precipitation-dissolution
7.4.1
Kinetic Effects in On-line Precipitation and Coprecipitation
183
An imponant difference between the batch and the continuous mode of precipitation is the available reaction time. Different standing times are normally used in batch procedures to ensure the completeness of the precipitation reaction or/and the form of the precipitate to facilitate filtration and minimize contamination. Standing times of 15 min to a few hours are typical, occasionally with elevated temperatures. Such procedures are obviously not feasible in continuous on-line precipitation systems where reaction times are typically in the range of a few seconds to a few tens of seconds. Quantitative recovery of analyte through precipitate collection is therefore not likely unless the precipitation (or coprecipitation) process is extremely fast. This should not be considered as a drawback when one realizes that FIA is a technique essentially performed under physically and chemically non-equilibrium conditions, and that reproducibility of the reaction process rather than its completeness is the key issu~- of the technique. With proper calibration, good results may be obtained under nonequilibrium conditions for the precipitation. This has been pfd'J'e~ ·e~J>erimentally in the coprecipitation of cobalt and nickel with Fe(11)-HMDTC [22]. Although the anai)!IL_ collection efficiency was only approximately 50%, good sensitivity and precision were achieved . with excellent agreement of analytical results with certified values of standard reference materials, covering a large variety of different sample matrices. The optimum conditions for the precipitation may also vary when a batch procedure is adapted to work in a continuous mode. Thus the optimum pH range for the coprecipitation of lead with Fe(II)-HMDTC (pH 2-3) was found to be much narrower in the Fl procedure than in the original batch procedure (pH 1-7) [21]. This was also considered to be a kinetic effect due to insufficient time for equilibrium in the continuous mode. so that coprecipitation can only be complete under the most favorable conditions. This phenomenon could, however, be made use of to overcome certain interferences from coexisting elements by kinetic discrimination, and should not be simply regarded as a drawback of the continuous approach. In manual batch procedures one always waits for a reaction to complete, unfortunately too often allowing interfering side reactions to fully develop. The equilibrium is therefore often achieved with a significant loss in selectivity. After studying the interference effects of ten main potential interference ions in the determination of chloride by silver chloride precipitation, Valcarcel' s group has shown that the FI approach exhibited 2-12 fold greater tolerance to the interfering ions when compared to the batch manual counterpart, all performed without precipitate dissolution [3,15]. These authors concluded that the improved selectivity of continuous precipitation
184
7 Precipitation
was the result of the absence or deprivation of undesirable side reactions during the short reaction period in the FI system. The enhancement in selectivity of continuous precipitation processes is an important benefit which by far outweighs the incompleteness in precipitation. In fact, this should be an important field for future exploration.
7.4.2
Kinetic Effects in Precipitate Dissolution
The dissolution speed of the precipitate is usually not regarded as an important factor in batch procedures, as long as the dissolution could be completed in a few minutes or even somewhat longer time. For extremely slow dissolutions, the rate of dissolution may be accelerated by increasing the temperature. For on-line precipitate dissolutions, the speed of dissolution must be much faster, especially if the objective is to achieve sensitivity enhancements. Elevated temperatures are of limited use in on-line dissolution systems as this may produce undesirable gas bubbles in the system conduits and deteriorate precision. In on-line precipit~tion-dissolution systems with flame AAS detection, the volume of solvent used to dissolve the collected precipitate should be as small as possible in order to increase the concentration of the analyte in the eluent. On the other hand, the flow-rate of the dissolution solvent should be reasonably close to the free uptake rate of the nebulizer, so as not to starve the nebulizer excessively. Failing to do so will result in a decrease in sensitivity and possibly deterioration in precision due to the formation of air bubbles in the solvent. Strong solvents have to be used in order to satisfy these requirements. However, the form of the precipitate is also important for achieving fast dissolution. Gelatinous and curdy precipitates have larger surface areas than crystalline precipitates, and thus are more readily dissolved using the same solvent. In an on-line coprecipitation procedure with a sampling flow-rate of 4 ml min- 1 during the precipitation stage, 2 ml of sample is introduced in a 30 s loading period; if a preconcentration factor of 20 is pursued, the precipitate should be dissolved in approximately 0.1 ml of solvent, assuming uniform distribution of analyte in the bolus. This implies that even with a relatively low dissolution flow-rate of 2 ml min- 1 the precipitate should be completely dissolved in 3 s. This provides a rough idea of the kinetic requirements for on-line dissolution solvents used in flame AAS. Such requirements are less stringent using spectrophotometric detectors equipped with flow-cells. The optimum solvent flow through the cells will not be defined by the detector as for flame AAS, making the choice of solvents more flexible. In extreme cases, where dissolution is exceptionally slow, the solvent may even be stopped in the collector of the precipitate in order to achieve complete dissolution. This can, of course, only be accomplished at the expense of decreasing the sampling frequency and the CE of the system. Therefore, regardless of the detector used, the importance of the kinetic properties of the dissolution solvent for on-line preconcentration systems can hardly be over-emphasized.
7.4 Some Fundamental Aspects of On-line Precipitation-dissolution
185
An example of the importance of the choice of dissolution solvent is illustrated by the dissolution of the precipitate from the reaction of cobalt with 1-nitroso-2-naphthol [12]. Variations in peak. widths and heights were observed owing to the different kinetic properties of the solvents. and ethanol was chosen for its faster dissolution and cheaper cost.
7.4.3
Precipitate Fonns in Continuous Precipitate Collection
With batch precipitation procedures, the formation of crystalline precipitates are aJways preferred over gelatinous forms for ease of filtration and washing. In continuous methods, hitherto the requirements seem to be quite contrary. For continuous systems without dissolution, crystalline precipitates such as calCium oxaJate were reponed to easily clog the filter pores of a stainless-steel filter. so that an off-line dissolution in ultrasonic baths was necessary after only 20 determinations. With the same filtenngsystem more than 250"aetefminations were·reaslfife-wflen gelatinous precipitates such as ferric hydroxide were formed. Although gelatinous or curdy precipitates have smaller panicle sizes, the colloidal panicles agglomerate to form larger aggregates which are easily filterable in a continuous system; they are also less compact, allowing easier passage of solution through the precipitate bed. so that more precipitates could be collected before significant flow resistance is developed [4]. When filterless systems are used without precipitate dissolution, as described in Sec. 7.3.3, the precipitate is expected to pass freely through the column which collects the surplus precipitant ions. Obviously. only colloidal precipitates without agglomeration can be transported through a column packed with a typical sorbent panicle size of 50-I 00 mesh. Attempts in the Author's laboratory for an indirect AAS determination of sulfate. using barium as a tag material. and passing barium sulfate through a cation exchange column, were unsuccessful due to blockage of the column by the precipitate. For applications involving precipitate dissolutions, the kinetic aspects on dissolution of different forms of precipitate. also favouring the formation of curdy or gelatinous precipitates, have already been discussed in the preceding section. It thus appears that. at least based on the knowledge available. conditions for online precipitation-dissolution should generally be optimized towards the formation of gelatinous, curdy. or colloidal forms of precipitates.
....---_J
186
7 Precipitation
7.5
FI Variables for On-line Precipitation-dissolution Systems
7.5.1
Sample or Precipitant Volume
For indirect determinations without preconcentration processes, sample volumes of approximately 100 J.d for nFIA or precipitant volume for rFIA are recommended. Lower sample or reagent volumes may be used to decrease the sensitivity if necessary, but larger sample volumes are not likely to enhance the sensitivity significantly except when very long precipitation coils are used. A disadvantage of using larger sample loops, apart from the general defect of decreased sampling frequency, may be the appearance of double peaks in nFIA due to insufficient reagent in the center of the sample bolus when the reagent is contained in the carrier. The same is true for rFJA where the reagent volume is too large. In on-line precipitation-dissolution systems involving preconcentration, samples are almost always introduced on a time basis due to the larger volumes involved. The sample volume is an important parameter determining the EF, CE and CI values of the system. Within certain limits, an increase in sample volume will result in enhanced EF values. The first limitation will be the capacity for collection of precipitate of the on-line collector, which has been discussed in Sec. 7 .2. An additional limitation will be the time available for a single determination. Preconcentration (precipitation) periods longer than 2 min, resulting in a sampling frequency of approximately 25 h- 1• are considered to have very little practical significance due to reasons discussed in previous chapters (see e.g. Chapter 1). Within the time available, the amount of sample which can be loaded (processed in the precipitation) depends on the maximum applicable sample flow-rate, which in tum is determined by other factors discussed in ·the next section. Although sample volumes ranging from 2.5 [21] to 250 ml [51 have been reported for preconcentration purposes, the routinely applicable range would be in a much narrower range of about 2~10 ml.
7.5.2
Sample Flow-rate
With volume based sampling, sample flow-rates are defined by the carrier flow-rates, whereas for preconcentration systems with time-based sampling, the sample flow-rates are specified by the sample loading rates. The optimum values in both cases are, however, quite similar. The important factors influencing the choice of sample flow-rates include the following:
7.5 Fl Variables for On-line Precipitation-dissolution S_vsums
a
b
c
d
187
The impedance of the on-line precipitate coJiector. This is particui~Y, iJilpo~~ when the collection of a relatively large amount of precipitate is intended beforeD· dissolution, or when a small capacity filtering device is used to limit dispersion. Thus, with a nylon membrane filter of 0.45-JJ.m pore and 5 rom diameter the highest tolerable flow-rate was found to be only 0.3 ml min- 1 in the coiJection of a calcium oxalate precipitate l 10). The form and ~ount of the p~~ipitate fOI!Jlc~d. _Crystalline precipitates, being more compact, produce more impedance in the filters. Large amounts of precipitate formed in coprecipitation processes also create impedance which limits the upper range of the flow-rate. ~of the precipi!~tion reactio~. With slow reactions sample flow-rates have to be decreased in order to achieve a reasonable degree of equilibrium. In extreme cases. as in the on-line formation of calcium oxalate. the reaction mixture even had to be stopped in the coil to wait for the precipitation to proceed [4]. ~" ~. ·For indirect determinations without precipitate dissolution. the sample and reagent flows are transferred directly to the detector. For atomic spectrometric detectors which require an optimum uptake, the combined flows from the sample and reage~t (~,2J,oJDe~mes the d~luent). should be such that the detector will not be excess1vely starved as to detenorate 1ts performance. For photometric detectors. the flow-rates are more flexible.
The optimized sample flow-rate, which is one of the most important factors governing the efficiency of the system, is often a compromise of the above factors. and usually falls in the range of 2-6 ml min- 1.
7.5.3
Reaction Coil Dimensions
The inner diameter of the precipitation coil should be large enough to allow unimpeded passage of the precipitate suspension. However, large tubing diameters will enhance the axial dispersion of the sample (in nFIA) or reagent plug (in rFIA). The dispersion of the sample bolus in the reaction coil will not be very important if the analyte or tag material to be determined is collected as the precipitate and later dissolved, but in systems without dissolution, the dispersion of the sample plug (in nFIA) or reagent plug (rflA) in the reaction coil has to be taken into consideration if optimum sampling frequencies and sensitivities are required. Tubings with inner diameters of 0.5-0.7 mm may be used for most purposes, and the smaller diameter tubings should be used whenever possible. The coil length depends primarily on the speed of reaction and the sample plus reagent flow-rates, and varies considerably in different applications. However. extremely long ~lion coils create excessive impedance, as well as carry-over problems in the
188
7 Precipitation
system, and should be avoided. A general recommendation for the coil length might be in the range of 30-100 em. When the dispersion of the sample or reagent plug is to be limited during the transport. or when the reactor is to be used simultaneously as a precipitate collector. the reaction coil should be knotted or knitted to produce a three dimensionally disoriented flow configuration (cf. Sec. 7.2.4).
7.5.4
Flow-rate of Dissolution Solvent
In FI systems with precipitate dissolution most often the solvent flow-rates are optimizecJ'to achieve maximum sensiiivity in terlns of dissolution peak height. and to a lesser degree, highest sampling frequency. The flow-rate of a solvent for attaining these will depend on the kinetics of the dissolution. but will also be governed by the required feeding rates of the detector. When a reasonably fast dissolution solution is used. its optimum flow-rate will be approximately 2 ml min- 1 with a photometric detector, and 3-5 ml min- 1 for flame AAS detectors. using aqueous solutions. When organic solvents are used for dissolutions with flame AAS. the optimum flow-rates are substantially lower, presumably because of enhancements in nebulization efficiencies. Thus, the optimum flow- rate reported for the dissolution of Fe(II)-HMDTC precipitate using IDMK, with flame AA detection. was only 1.6 ml min- 1 [21].
7.6
FI Methods with On-line Continuous Precipitation
7 .6.1
Indirect Methods Involving On-line Continuous Precipitation
Indirect Determination of Anions by Flame AAS
Hitherto all applications using FI continuous precipitation systems to determine anions had as detector the flame AA spectrometer. The prerequisite is that a suitable cation (tag element) is required which can selectively precipitate the anion analyte. The high sensitivity of the AA detector for the determination of most metals can then be readily exploited to perform a sensitive indirect measurement of the precipitated anion via the tag element. following an on-line separation. Other methods, e.g. VIS-UV spectrophotometry, are less attractive in this respect, as an indirect method involving separation usually will not show much superiority over direct methods.
7.6 Fl Mrthod.f with On-line Continuou.J Prrcipillltion
189
The indirect methods for the determination of anions by on-line precipitation using AAS are shown in Table 7.1. It can be seen that methods with a dissolution step generally have much lower sampling frequencies than those without dissolution. One has to remember, however, that the filters have to be cleaned periodically if the precipitates are not dissolved within each cycle. Therefore, the differences in real sample throughputs will not be that significant if the time for the off-line cleaning of filters is taken into account. The indirect method for the determination of chloride in the reversed FI mode (rFIA) with reagent injected into a sample carrier have higher sensitivity, lower detection limit. and higher sampling frequency than that perfonned in the normal mode (nFIA), with the sample injected into a carrier containing the reagent [3 ). This does not appear to be a characteristic inherent in the different approaches, but seems rather to be the result of the parameters used in the study. The low sampling frequency in the nFIA mode is due to the low flow-rate of the carrier (reagent) stream, which may be readily increased by using a water carrier and merging the reagent downstream, as shown in Fig. 7.2 b. The higher sensitivity for the rFIA approach seems to be the effect of a larger sample volume available for reaction. In the nFIA mode shown in Fig. 7.2 a, the injected sample is sandwiched in between reagent zones. The volume is restricted to below 100 J.LI in order to ensure that sufficient reagent will be dispersed into the central pan of the sample bolus; whereas in the rFIA mode such restrictions for the sample (carrier) volume do not exist. The apparent constraint for nFIA in this respect can, however, also be avoided by using a carrier stream not containing the reagent, while injecting a larger sample volume. and m~,ging the precipitant downstream as in Fig. 7.2 b.
Indirect Determination of Organic Constituents When organic constituents in a sample selectively form insoluble products with certain metals. such properties can be utilized to develop sensitive indirect methods for the organics by using AAS to determine the tag metals following a separation of the precipitate, as for,anion determinations. Unfortunately. reactions selective enough to determine the organic analytes in even moderately complex matrices are few. Such methods are nonetheless quite ideal for pharmaceutical preparations where the matrices are often simple and controlled. The highest selectivity is therefore not necessary. FI on-line precipitation methods provide a much more efficient alternative compared to the manual batch methods. The various applications in this field are also shown in Table 1. Manifolds used for the determinations were quite similar to those used for the indirect determination of anions.
Table 7.1
-
Indirect detennination of anions and organics by Ff-AA on-line precipitation
Analyte
Tag Sample Element
Sulfide Chloride Chloride Ammonia
Cd Ag Ag Fe
Standards Waters Waters Standards
Oxalate
Ca
Standards
Chloride Ag Chloride/iodide Ag
Standards Foods. wines
Sulfate Sulfonamides
Pb
Anaesthetics
Co
Standards Phannaceutical preparations, urine Phannaceutical preparations Phannaceutical preparations
Cu,Ag
Chlorohexidine Cu
Manifold Figure 7.4 a 7.2 a 7.2 c 7.2 a 7.3 7.2 a 7.3 7.3 7.3 7.2 c 7.2 a 7.2 c 7.2 c 7.2 c 7.3
Conctn. range (mg 1-') O.oJ-3
I
R.s.d.
8 Reference
(h -•,
(%)
100 50 200
2 3 3 4 4 4 4 4 29
3-100 0.3-10 2-50 3-45 5-90 5-60 3-100 5-90(CI) 10-300(1) 2-20 2.5-35
60 100-150
1.2 2.0 3.5 2.2 6.0 5.0 8.7 5.2 6.5(CI) 4.9(1) 3.0 1.5-3
1-llxiO-~M
100
0.6
8
5-20
10
3.6
15
so 10 20 6 10 10
14 7
.,"
~ ;;·
...g·~-
7.6 Fl Methods with On-line Continuous Precipitation
7.6.2
191
FI On-line Preconcentration Methods with Continuous Precipitation-dissolution
General Preconcentration by precipitation and particularly by coprecipitation have long been used in the manual batch mode to enhance the sensitivity and se~ctivity of atomic spectrometric methods. Comprehensive treatment of such methods may be found in a number of reference books [25.26]. __M!!lual preconcentrations by direct precipitat.iimj of the trace analytes are limited by the solubility products_of the precipitates and ~~- / contamination problems. The small amount of precipitate is also difficult to deal wltliin the long manual operations. It is not surprising that the first attempts in adapting preconcentrations by precipitation to Fl systems were in this direction [5,9,12,t8i. as the small amount of P!!':ipit~ was t!lrned into an advantage for on-line filtration systems, and contamination risks were virtually eliminated. Nevertheless, the limitations in the number of selective reactions which produce precipitates with sufficiently low solubility products still exist. and perhaps even enhanced to some extent due to the kinetic features of the FJ system. Despite the preceding successes, it seems rather do.ubtful that the direct precipitation approach for trace preconcentration by FI could be applied broadly. Coprecipitation is the proc~s of transferring a substance into a precipitate of some compound if the substance r8i1; IJ form its own solid phase under the given experimental -· conditions. This transferring could involve adsorption on the surface of the collector, the I formation of isomorphic crystab. occlusion. and mechanical inclusion of other phases 1 [25j. In contrast to direct precipitation methods. preconcentrations by coprecipitation with a carrier can be used for very low concentrations of trace analytes, and a host of coprecipitation systems exists in the literature which can simultaneously handle a whole group of trace analytes. Thus once a procedure is developed, it could be used for tens of different analytes with minor modifications. Admittedly, the relatively low recoveries of some coprecipitation procedures may be further enhanced in the FI systems. This, however. will not produce a deterioration in precision as for batch procedures. in view of the high reproducibility of the FI process. This has been fully demonstrated in the example given in Sec. 7 .4.1. The real technical obstacl~ in the development of an on-line coprecipitation system is rather the amount 9f precipitate (mainly from the reaction product of the carrier) which has to be halidled. Whereas the larger amount of precipitates formed in coprecipitation methods in comparison to direct precipitations may be considered as an advantage in batch procedures,, t?i.~, h~ ~u,med into a disadvantage for continuous precipitation methods where flow impedanCe Is a major factor limiting the flow-rates and the amount of sample which can be treated in one analytical cycle. However, this impediment has been overcome, at least for some coprecipitation systems, by using knotted reactor precipitate collectors (Sec. 7.2.5).
I
j
) 92
7 Precipilalion
Performance of On-line Preconcentration Methods with Continuous Precipitationdissolution In principle. all effective FI continuous precipitation systems with dissolution may be used to achieve some degree of analyte preconcentration. The only prerequisite is that enough sample is available. Within limits defined by the capacity of the precipitate collector. the enrichment factors EF will be proportional to the amount of sample processed. The efficiencies of different preconcentration systems. however. may be quite different. A quantitative evaluation of the efficiencies of the systems may be made using the criteria originally proposed for on-line column preconcentration methods. i.e .• in tenns of CE and CJ. These are calculated for the various methods collected in Tables 7.2 and 7.3, based on the experimental data given in the individual papers. Since different enrichment factors are often reponed for the same system using different sample volumes and loading times (therefore resulting in different sample throughputs). the data are only calculated for the highest sampling frequencies. While C/ values are to a large extent independent of the sampling frequency. CE values would be somewhat higher with lower sampling frequencies. From a practical point of view. the performance of the system at higher sample throughputs are much more important. when considering a closer adaptation of the system to the high measuring. speed of the detector (in most cases, a flame AA spectrometer). In applications where organic solvents were used as dissolution agents. the resulting gain in AA signal, compared to conventional aspiration of aqueous solutions. obviously included the enhancement factors from the organic solvent effect. In cases where such enhancement effects are mentioned or obvious, ~- corresponding total enhancements are presented as N,. When the overall performance of the systems are examined. one can notice large differences in the CE and C/ values of one to two orders of magnitude. with the superiority clearly belonging to coprecipitation methods using knotted reactors. The extremely low efficiencies both in analyte enrichment and sample consumption for the system reponed in reference 18 may be principally due to the low efficiency of the packed column in collecting the precipitates. The collector was actually a large dimension pearl string reactor with 1.9 mm diameter glass beads packed in a 7 em long 2.8 mm i.d. Tygon tubing. The cavities between neighboring beads appear much too large to impede the passage of precipitate particles sufficiently. and it may be assumed that the bulk of precipitate formed was never collected on the column. As the samples were drawn through the collector after precipitation using the suction of the AA nebulizing system, the transport of the reaction mixture would not have been possible if a filtering device with better collection efficiencies, such as those described in Sec. 7.2.1. were used. It seems extremely unlikely that even a moderately efficient continuous precipitation system can ever be developed without the use of an appropriate pump. An important advantage of the Fe(II)-HMDTC coprecipitation system originally proposed as a batch procedure by Eidecker and Jackwerth [27,28] is its strong tolerance to common matrix constituents, including K. Na. Ca. Mg. Fe. and AI. for a whole series of trace metals and nonmetals. The merits of the batch procedure are fully preserved in
7.6 Fl Method.t with On-linr Continuous P"cipitatitHI
193
the FI method while the sample throughput is improved almost two orders of magnitude with full automation, and the sample consumption decreased by a factor of 40 (21 ]. The strong tolerance of the method to interferences from samples with complex matrices is well illustrated by the results for Pb. Cd, Co, and Ni in bovine liver and whole blood digests (cf. Sec. 8.3.1 and 9.5.1 ). using both flame AA [21.22] and graphite furnace AA [23].
7.6.3
Reduction of Interference Effects in Flame AA Using Continuous. Precipitation
On-line precipitation may be used to remove interfering species in the sample matrix from the analyte either by precipitating the analyte. collecting the precipitate on a filter, followed by on-line dissolution. or by precipitating the interferent. The first approach can be used simultaneously to achieve some degree of preconcentration of the analyte if necessary. In fact. all preconcentration methods are associated with complete or partial removal of the matrix which interferes in the final measurements one way or another. and in this sense may be viewed upon as methods for removing interferences. For major elements such 3!; calcium. which may suffer from various interferences in flame AAS determinations. the main interest would be in most cases the reduction of interferences. The feasibility of the on-line precipitation approach has been demonstrated by Tyson's group in the successful AAS determination of calcium in the presence of up to 1000 mg 1- 1 of aluminium by selectively precipitating the calcium as the oxalate. and later dissolving the collected precipitate using hydrochloric acid (10.13.17 .20]. To date. no publications on the second approach (i.e. precipitation of interferent) have been noted.
Table 7.2
Analyte Sample
Pb Cu Ca Co Ag Fe Ca Cu
-
Fl preconcentrarion methods for flame AAS with on-line precipitation-dissolution Precipitant
Tap water 1.5 M ammonia liquor 0.1% rubeanic Silicates acid Standard Ammoniaammonium oxalate Silicates 0.1% 1-nitroso-2naphthol Standard 0.1 M NaCI solutions 0.07 M NaOH 0.09 M Na2C03 Standard 0.1 M NaOH
"'Thermostated at 55°C
Dissolution solvent
Precipitate collector
Stainlesssteel filter dichromate Stainlesssteel filter in HN03 5% HCI Packed bed & membrane filter StainlessEthanol steel fi Iter• 50 mM sodium Packed bed thiosulfate 0.4 M H3P03 0.09 M HCI 2M HCI Membrane 2M HN03
'i
C/
I
(min- 1)
(ml)
(h-')
(pg ·-·)
(%)
6.3
0.40
IS
20
1.0
s
6.3
0.52
20
5
1.4
9
5.7
0.06
4
0.7
-
II
26 (N,)
0.26
40
6
2.5
12
1.0
2.8
12
s
3.5
18
1.3
4.6
40
-
0.90
0.8 10
3.7 2.7
3.4
12 12 17
18 18 20
CE
D.L.
R.s.d.
-
Ref.
..., ~ i.
"' "6'
i·
Table 7••l
Fl prccnnccntration methods fnr
Precipitant: Carrier ion: Precipit:ttc L'nllel·tnr: Dissolution solvent: -----·--· ---- ... Analyte Sample digests
lfMA-HMDT C Fe(ll) Knotted reactor IRMK
Cd
Cn
Ni
Bovine liver. whole hlnnd Rnvinc livt•r. whole blood. plant leaves. urine. etc. Bovine liver. Oyster tissue. urine. etc. Citrus leaves. oyster tissues. urine. etc.
N,
CE (fl'in··
Pb
name AAS with on-line coprecipilation
1)
(min
.,
Cl
Maximum frequency
D.L.
R.s.d.
Cml)
(h I)
t1•g I __ ,)
(%)
Ref.
30
66
0.05.
90
2
2.7
21
::,.
29
62
n.os
72
U.l5
1.5
22
~
'-1
~
§. ~
~ 'S
23
24
52
62
0.116
o.os
72
u
2.7
22
72
15
I.R
22
~ ~
:;:
~
r"')
~
i" ~
.,5. 3
-s·
§"
i" ~
8
Environmental and Agricultural Applications
8.1 General Despiae lht IIIIJe number of modem determinative techniques available to the environmental and agricultural analytical chemist. separation procedures involving timeconsuming manual procedures often remain an integral part of analytical methods for related samples. This is due to the inherent complexity and variability of the sample matrix of many environmental and agricultural samples. The sample throughputs are often severely hampered by tedious batch separations. which seriously limit the amount of analytical information produced within a given time. Fl separation systems have proved to be powerful techniques in resolving such problems in environmental and agricultural analysis. frequently demonstrating all the special merits of the technique stated in Sec. 1.3: It is not surprising that hitheno most applications of FI on-line separation and preconcemration are associated with such samples.
8.2
Waters
8.2.1
Determination of Trace Elements
Arsenic and Antimony Total arsenic and antimony may be determined in waters by Fl hydride generation AAS after a prereduction of arsenic(V) and antimony(V) to arsenic(III) and antimony( III). Yamamoto et al.[l] determined arsenic(IU) and (III+V). and antimony(III) and (Ili+V) selectively in thermal water using a Fl-HGAAS system. On-line reduction of arsenic(V) and antimony(V) was achieved by merging 50% and 8% KI reductant, respectively, to the strongly acidified sample stream. A detection limit of 0.2 J.Lg 1- 1 was achieved with a sampling frequency of 100 h- 1• The same group f2] determined arsenic in sea water using a tubular membrane separator. and obtained an improved detection limit of 0.07 J.Lg 1-1.
198
8 Em•ironnvfllQ/ Qnt/ Agrir:ulturQ/ App/icQtions
Wang and Fan.g-[3] determined arsenic in waste wa~ also by FI-HOAAS with good recoveries and a detection limit of 0.1 pg 1- 1• A prereduction was made by boiling the sample with 10% KI for 5 min to reduce arsenic (V)"to arse~(lll). Sperling et al.[4] determined arsenic(Ill) and total arsenic in sea and lake waters by ETAAS, following on-line reduction and preconcentration of the DOC complex using a micro-column packed with C1s sorbent, using a manifold modified on the basis of that shown in Fig. 4.11. A mixture of sodium sulphite, hydrochloric acid, sodium thio-su1phate and potassium iodide was used to achieve fast reduction of arsenic(V) to arsenic(lll), and ethanol was used to elute the sorbed arsenic-DOC complex. The detection limits for As(W) and As(ID+V) were 0.11 and 0.15 pg 1- 1 respectively. Welz and Schuben-Jacobs [5] also determined arsenic in sea water (NASS-2 and CASS-1) and riverine water (SLRS-1) by FI-HGAAS using a commercialized F1A prototype ·instrumenL designed for· combination to an AA. spectrometer, achieving a detection limit of 0.06 /JS 1- 1• The FI procedure was compared to a German standard balch procedure for the determination of arsenic, and the former was shown to achieve a 6-fold higher sampling frequency with at least 90% decrease in sample and reagent consumption.. Boron
Sekerka and Lechner [6] determined boron in natural water samples with an automated FI specttophotometric system using on-line ion- exchange preconcentration. A boron-specific ion- exchanger, Amberlite IRA-743, based on N- methylglucamine, was used to sorb the analyte in an on-line column during a sample loading period. and the column was eluted by 1 M phosphoric acid. The eluate was then meJJed with an ammonium phosphate buffer and finally with the azomethine-H reagent. A stopped-flow period for prolonging the chromogenic reaction time was required to improve the sensitivity. Detection limits of 5 /Jg 1- 1 and 1 pg 1- 1 were achieved at sample throughputs of 20 h- 1 and 10 h- 1 respectively. The method appears to be one of the most sensitive boron methods available. Good recoveries were obtained for spiked natural water samples. Cadmium
Fang et al.[7] determined cadmium in sea water by flame AAS with a dual column ion-exchange preconcentration system. Of the three different ion-exchangers investigated, only Chelex-100 gave satisfactory recovery, achieving a detection limit of 0.07 /Jg 1- 1, with a C£ value of 60 min- 1 and a sampling frequency of 60 h- 1• The same C£ value was obtained using a single column system with CPG/8-Q ion-exchanger, achieving a sampling frequency twice as high, but an EF value a factor of two lower [8). However, satisfactory recoveries for sea water were obtained only by diluting the samples 1+ 1. - Fang et al.(9] were able to obtain better recoveries (95%) for cadmium in sea water through on-line sorbent extraction preconcentration of the DOC complex on a C 18 column, also with flame AAS detection. TheN, value was 50 min-•, including enhancement
82 Wa1er.f
199
effects from the alcohol eluent, and the detection limit was 0.3 Jl.g 1- 1• The procedure was also used successfully for the determination of cadmium in tap water and NBS SRM 1643b. Despite the improved sensitivity of the ftame AAS methods with on-line column preconcentration, the detection limits achieved are still insufficient for most unpolluted water samples. Cadmium in such samples (CASS-1. CASS-2, NASS-2, SLRS-1, etc.) have been successfully determined using an ETAAS system with on-line sorbent extraction preconcentration similar to the one described in Sec. 8.8.3. A detection limit of 0.0008 1'8 1- 1 Cd was achieved with almost complete removal of the interfering matrix of sea water samples ( JO]. Pona et aJ.(JJ) also developed an on-line column preconcentration method for the determination of cadmium and other trace metals (Pb, Cu, Ni, and Fe) in Antarctic sea water using ETAAS. A detection limit of 0.0004 J18 1- 1 was achieved for cadmium. The APDC complexes of the trace metals were collected or• a micro column packed with C 18 , XAD-7 or XAD-2 sorbent and acetonitrile was used for elution. According to the authors, the main drawback of the method is at least two successive 80 111 elutions (with an intermediate evaporation in the furnace) are necessary to recover all the analytes from the column.
Chromium Chromium(VJ) was determined in fresh water samples using solid phase absorptiometty. The complex of chromium(VI) with 1,5-diphenylcarbazide was sorbed on a cation exchanger packed in a flow-cell. The detection limit was as low as 33 ng 1- 1 [12].
Cobalt Yamane et al.[ 13] determined cobalt in sea water and river waters with a catalytic spectrophotometric method after on-line preconcentration using a column packed with quinolin-8-ol immobilized on silica gel. The tartrate eluate from the preconcentration had to pass through a second column packed with a strongly acidic cation exchanger to remove iron(III) and manganese(ll) which interfere in the catalytic reaction of oxidation of protocatechuic acid by hydrogen peroxide. An extremely low detection limit of 0.005 11g 1- 1 was obtained. However, the sampling frequency was less than 2 h- 1• A much more efficient, but less sensitive method for the determination of cobalt in sea water, tap water and waste waters by flame AAS with on-line column preconcentration was proposed by Fang et al.[14). The column was packed with quinolin-8-ol immobilized on controlled pore glass. With dual columns, 48-fold sensitivity enhancement was achieved at a sampling frequency of 60 h- 1, achieving a detection limit of 0.2 J.Lg 1- 1• Cobalt is determined by Sperling et al.[ 10] in sea water with a detection limit as low as 0.004 J.Lg 1- 1 using C1s column preconcentration Fl-ETAAS. A still lower detection limit of 0.001 Jl.g 1- 1 was achieved by Porta et al.[ll] using a similar approach, making it possible to determine cobalt in Antarctic sea water.
200
8 Environmrntal and Agricultural Application.f
Coprwr The dual-column and single-column ion-exchange preconcentration flame AAS systems as well as the DDC-C 18 sorbent extraction flame AAS and ETAAS systems used for the determinalion of cadmium were also used for the determination of copper in water samples. 1be detection limit for the dual-column system was 0.07~.09 J.l8 1- 1 at 60 samples h- 1 [7]; for the sorbent extraction preconcentration flame AAS system, 0.2 J.l8 1- 1 at 120 samples h- 1 [9]; for the sorbent extraction ETAAS system, 0.02 J.l8 1- 1 at approximately 20 samples h- 1 [10]. The sensitivity of sorption column preconcentration methods with flame AAS detection should be sufficient for the determination of copper in most natural water samples. Yuan et al.[15] used an on-line dual column preconcentration system with FAAS detection to determine copper in sea water. The quinolin-8-ol copper complex was sorbed on columns packed with C 18 sorbent and eluted by methanol
Lead Fang et al.[7] determined lead in sea water with detection limits of 0.~.8 J,lg 1- 1 using a dual column on-line preconcentration Fl-AAS system with different chelating ion-exchangers. 100-fold enrichment was achieved at a sampling frequency of 60 h- 1• In a further improved system involving a single conical ion-exchange column, even higher sampling frequency of 120 h- 1 was reponed with about 30-fold enrichment, and with sample consumption approaching that of conventional flame AAS without preconcentration. Lead was also determined in sea water and other water samples by sorbent extraction preconcentration AAS with a detection limit of 3 J.lg 1- 1 [9]. The DOC complex of lead was sorbed on a column packed with C 18 sorbent and eluted with ethanol or methanol, achieving a sampling frequency of 120 h- 1 [8]. The sorbent extraction preconcentration system was miniaturized to be adapted to an ETAAS system achieving an enrichment factor of 26, and a detection limit of 0.003 JJg 1- 1, without lowering the original sampling frequency of the ETAAS procedure [16]. The interfering matrix of sea water samples was removed in the separation, so that no chemical modifiers were necessary in the ETAAS determination. Results obtained for standard reference sea water CASS-1, CASS-2, NASS-1 and riverine water SLRS-1 agreed well with certified values. The procedure is described in detail in Sec. 8.8.3. Bysouth et al.[17] improved the selectivity of immobilized quinolin-8-ol ionexchangers in the Fl-AAS determination of lead in tap water samples by adding masking agents. Interferences from 200-fold excess of iron, copper, aluminium and zinc were suppressed by using a buffer-masking solution consisting of 0.2 M boric acid, 2% triethanolamine, 2% thiourea, and 2% acetylacetone. Recoveries from tap water samples ranged from 94 to 108%.
8.2 ltatcrs
20 I
Mercury
Zhang eta!.[ 18] determined mercury at ultra-trace levels in sea water. tap water and waste waters using on-line ion-exchange preconcentration cold vapour AAS. Of three different ion-exchangers tested, CPG-8HQ produced the best sensitivity and selectivity. The sorbed analyte was eluted by 2 M hydrochloric acid in 0.5~ thiourea. An EF of 40 and detection limit of 0.002 Jl£ 1- 1 with sampling frequency of 60 h- 1 was reponed. Nickel
Sperling et aJ.( 10] determined nickel in CASS-1. CASS-2, NASS-2 standard reference sea water samples by ETAAS after on-line separation and preconcentration of its DOC complex on C 1s column, achieving a detection limit of 0.04 ll8 1- 1• The method is sufficiently sensitive to determine nickel in de-ionized water. Almost the same detection limit was obtained by Pona et al.(ll] in the determination of nickel in Antarctic sea water, using a similar approach which involved sorbing the APDC complex on a C 1x column. Multi-element Determinations with ICP-MS
ICP-MS is a powerful technique for multi-element trace determinations in water samples. However, its application to the analysis of saline waters is limited to those with dissolved solid contents below 0.2%. in order to avoid instrumental drifts caused by solid deposition on the orifice. The analysis of sea water therefore demand~ a separation of the salt matrix prior to determination by JCP-MS. Beauchmin and Berman [ 19] used an on-line column packed with silica immobilized quinolin-8-ol ion-exchanger to separate the matrix and determine Mn. Co. Ni. Cu. Pb and U in the standard reference open ocean water NASS-2 using an isotope dilution technique and a standard addition method. Plantz et al.[20) proposed an on-line sorption column system to eliminate interferences from alkali and alkaline eanh elements and anions in the ICP-MS determination of various trace metals in sea water samples. The his( carboxymethyl l- dithiocarbamate reagent was used to complex V, Cr, Ni. Co. Cu, Mo. Pt. Hg. and Bi. Under acidic conditions, the complexes were sorbed on a column packed with XAD-4. while the interferents passed freely through. The retained analytes were then eluted with 0.1 M Nf40H into the ICP-MS. The detection limits were in the range 8-80 ng 1- 1 using a 0.5 ml sample loop. The precision was in the range 3 to 5 % r.s.d.. The system was also used for the determination of trace metals in urine.
202
8 Em•ironmt'ntal and Agricultural Applications
8.2.2
Detennination of Trace Anions
Cyanide Cyanide was determined by Zhu and Fang [21] in natural and waste waters after on-line. gas diffusion separation and spectrophotometric determination of a non-stable intermediate reaction product in the pyrazolone/isonicotinic acid method. Phosphate
Phosphate was determined in rain water, sea water and ground water at JLg 1- 1 levels by Yoshimura et al.(22] using solid phase absorptiometry. The ion-associate formed by the reaction of molybdophosphate with Malachite Green was sorbed on a Sephadex dextran gel packed in a flow-cell, and the increase in absorbance was monitored to perform the determination of phosphate, resulting in a very sensitive method.
Fluoride Fluoride was determined indirectly with ICPES by Manzoori and Miyazaki [23] in waste water, tap water and lake water samples after FI extraction of the lanthanum/alizarin complexone/ftuoride complex into hexanol containing N,Ndiethylaniline. The determination range was 0.03-1.3 p.g ml- 1 fluoride.
8.2.3
Detennination of Surfactants
Anionic Kawase et. al.[24] determined anionic surfactants using liquid-liquid extraction based on the methylene blue method at an early stage of development. Sahlestrom and Karlberg [25] used the same reaction to determine anionic surfactants using a microconduit extraction system. The methods were not intended for very low concentration levels. Anionic surfactants were determined in river waters and treatment plant waters in the concentration range 0.04-3.5 p.g ml- 1 based on an ion-pair extraction reaction with methylene blue in chloroform using a FI spectrophotometric system. Time-based continuous sampling was used to improve the sensitivity for meeting the requirements of environmental monitoring [26]. After an extensive comparison of different cationic dyes and extraction solvents for the FI liquid-liquid extraction spectrophotometric determination of anionic surfactants, Motomitzu et a1.[27] recommended the use of Methylene Blue and 1,2-dichlorobenzene. A detection limit of 5 JLg 1- 1 was achieved with a sampling frequency of 20 h- 1, and a precision of 0.9% r.s.d. in the analysis of river waters (for details cf. Sec. 8.8.2).
8.2 Waters
203
Gallego et al.[28] developed an indirect AAS method for the detennination of anionic surfactants in waste waters by FI liquid-liquid extraction. The method involves the fonnation of the detergent-1,10-phenanthroline-copper(II) ion pair and extraction into IBMK. Concentration of anionic surfactants in the range 0.1 to 5.0 /-tg ml- 1 was detennined by measuring the copper in the separated organic layer using a PTFE membrane phase separator. The method was reported to be highly selective, being free from interference of non-ionic surfactant&, and a high precision of 0.8% r.s.d. was achieved.
Cationic Kawase [29] detennined cationic surfactants by FI liquid-liquid extraction with chlorofonn after ion-pair fonnation with Orange II. The method features a high precision of 0.4-0.9% r.s.d. with a sampling frequency of 60 h- 1• Martinez-Jimenez et al.[30] detennined cationic surfactants in natural, tap and waste waters indirectly by flame AAS. The method is based on the on-line extraction of the detergent-tetrathiocyanatocobaltate(II) ion-pair into ffiMK. The surfactants were determined by measurement of cobalt in the separated organic phase. A sampling frequency of 35 ± 5 h- 1 was achieved with a precision of 1.2% r.s.d.
Non-ionic Leon-Gonzalez et a1.[31] proposed an FI spectrophotometric method for the detennination ot Triton-type non-ionic surfactants based on their reaction with alizarin fluorine blue. An on-line ion-exchange column was incorporated in the system to eliminate interferences from ionic and amphoteric surfactants. In case of interferences from non-ionic surfactants, an on-line Amberlite XAD-4 adsorption column was used to retain selectively the Triton-type surfactant, which was subsequently eluted by ethanol. However, no infonnation was given regarding interferences from refractive index effects at the ethanol/aqueous interface and their elimination.
8.2.4
Detennination of Nitrogen and Nitrogen Compounds
Total ammoniacal nitrogen was detennined spectrophotometrically in a canal water sample by Son et al.[32], following gas-diffusion separation, achieving a detection limit of about 20 /-tg 1- 1• Willason and Johnson [33] detennined ammonia in sea water using a gas-diffusion spectrophotometric method. Hara et al.[34] used an on-line gas-diffusion preconcentration system for the determination of ammonium ions in distilled and natural water samples with an ion-selective electrode detector. The preconcentration was achieved by maintaining a time-based large sample flow of about 10 ml min- 1 against a low acceptor buffer flow of only 0.3 ml min- 1• A detection limit of 30--3 Jlg 1- 1 was reported.
204
8 Environmental and Agricultural Applications
The combination of gas-diffusion separation and conductimetry by Pasquini and de Faria [35] produced an extremely sensitive and selective method for the determination of ammonia in Kjeldahl digests. Ammonia from an alkalinized (NaOHIEDTA) sample digest is transferred through a PTFE gas-diffusion membrane into a de-ionized water acceptor stream and the change in conductance measured. A sampling frequency of 100 h- 1 was achieved with a precision of 1% r.s.d .. Results for total nitrogen in vegetables, animal feeds and fertilizers were in good agreement with those obtained by a conventional distillation/titration procedure.
8.2.5
Other Detenninations
Phenol The 4-aminoantipyrine standard manual method for the determination of phenol in water using a chloroform extraction was automated achieving a detection limit of 0.005 mg 1-•. and a sampling frequency of 50 h- 1 [36].
8.3
Plant and Animal Tissues
8.3.1
Detennination of Trace Elements
Arsenic and Antimony
Yamamoto et al.[ I] determined arsenic in wheat flour (NBS SRM 1567), rice flour (NBS SRM 1568) and also antimony in orchard leaves (NBS SRM 1571) and coal fly ash (NBS SRM !633a) using a FI-HGAAS system which was also used for water analysis, and obtained good agreement with certified values. On- line reduction of arsenic(V) and antimony(V) was achieved by merging 50% and 8% KI reductant, respectively, to the strongly acidified sample stream. Detection limits of 0.2 and 0.04 11g 1- 1 were achieved for arsenic and antimony, respectively, with a sampling frequency of 100 h - 1• Wang and Fang [3] successfully determined arsenic in rice flour (NBS SRM 1568) and wine by FI-HGAAS, and obtained a better detection limit of 0.1 11gl- 1 and sampling frequency of 220 h- 1, but a prereduction, involving boiling of the sample with 10% KI reductant, was necessary. Liversage et al.[37] determined arsenic in a variety of standard reference environmental samples including orchard leaves and coal fly ash using a FI hydride generation ICP spectrometric method and obtained good agreement with certified values. However, the reported precision (7.2 % r.s.d.) was rather low.
83 Plant and Animal Tissu~s
205
Cadmium, Cobalt and Nickel
Welz et al.[38] used a modified on-line coprecipitation method based on that described in Sec. 9.5.3 with Fe(II)-HMDC as carrier to preconcentrate cadmium, cobalt and nickel in a variety of standard reference plant and animal tissue samples, including citrus and tomato leaves, bovine liver, oyster and lobster tissues etc. with FAAS detection. The method showed high tolerance to interferences from transition metals such as iron and copper, and good agreement with certified values was obtained for all the samples studied. Bismuth
Yamamoto et al.( 1] determined bismuth at 0.1 mg kg- 1 levels in orchard leaves (NBS SRM 1571) by Fl-HGAAS, with a detection limit of 0.05 p.g 1- 1 in the digest and a sampling frequency of 120 h- 1• Molybdenum
One of the pioneering works on F1 liquid-liquid extraction preconcentration was on the determination of molybdenum in plant ash solutions [39]. An aqueous/organic phase ratio of 10 was used to extract the molybdenum thiocyanate complex with a sample throughput of 30 h- 1• but the sensitivity achieved in this early attempt appears to be somewhat insufficient for samples with low molybdenum contents. A more recent attempt by the same group produced a more sensitive method for molybdenum. Pessenda et a1.[40] from this group implemented an on-line cation-exchange column, packed with Dowex 50W-X8, to remove metal interferents in the determination of molybdenum in plant materials using a sensitive F1 catalytic spectrophotometric method originally developed by Fang and Xu [41] for the determination of molybdenum in waters. The sample was continuously pumped through the column, and a defined volume of the effluent was collected in a sample loop, after discarding the leading section of the flow. The collected effluent was then injected into the reaction system using 0.15 M ammonium chloride. The sorbed interferents were partially eluted from the column in reversed flow direction following each measurement, using 2 M ammonium chloride as eluent, and was fully regenerated after about 40 measurements using the same eluent. The incorporation of the separation system produced a highly selective and sensitive method for the determination of molybdenum in plants, achieving a sample throughput of 40 h- 1 and a precision of <2% r.s.d. Selenium
Wang and Fang [42] determined selenium in wheat flour (NBS SRM 1567), orchard leaves (SRM 1571) and coal fly ash (NBS SRM 1633a) by Fl-HGAAS, achieving a detection limit of 0.06 p.g 1- 1 in the digest and an extremely high sampling frequency of 25(}..300 h- 1• The method is described in more detail in Sec. 8.8.4.
206
8 Environmental and Agricultural Application.t
Zinc Ferreira et al.(43) developed an Fl spectrophotometric method for the determination of zinc in plant ash extracts using zincon. The zinc chloro-complex was retained on an anion-exchange column and subsequently eluted by dilute sodium hydroxide. Schlieren effects were overcome using dual-wavelength spectrophotometry. A sampling frequency of 45 h- 1 was achieved with an r.s.d. better than 2%.
8.3.2
Detennination of Anionic Constituents
Nitrate and Nitrite Silva et al.(44) developed an indirect Fl-AAS method for the determination of nitrate and nitrite in meats and vegetables following liquid-liquid extraction. Nitrate forms an ion pair with bis(2,9- dimethyl-1,1 0-phenanthrolinato)copper(l) which is extracted into IBMK. Copper is then determined in the separated organic phase. When the nitrite is oxidized by cerium(V)) into nitrate before the extraction, total nitrate is determined.· When nitrite is converted to nitrogen with sulfamic acid, only the original nitrate is determined.
Sulfite Sullivan et al.(45] used an Fl gas-diffusion spectrophotometric system to determine sulfite in food products including fruits. vegetables, shrimps and wines. Sulphur dioxide is separated from the sample using a gas-diffusion membrane separator. and determined by decolorization of Malachite Green. The method shows good selectivity and sensitivity, and sulfite may be determined with a detection limit of 0.1 mg 1- 1 in the food extracts, corresponding to 1-10 mg kg- 1 in the food product, and with a precision of 1-2% r.s.d ..
8.4
Beverages
Ethanol An efficient FI gas diffusion amperometric system for the determination of ethanol in beverages was developed by Kunnecke and Schmid [46]. The same authors also used the system to achieve on-line monitoring of ethanol content of cultivation media, with amperometric as well as photometric detection [47]. The system combined the selec-
8.4 Bevera[le.f
207
tivity of a gas-diffusion separation membrane with the selectivity of alCohol oxidase (immobilized on porous glass packed in an on-line column reactor) which transfonned the separated ethanol (collected in a potassium phosphate buffer acceptor stream) into hydrogen peroxide. The peroxide was then determined amperometrically or photometrically with a reagent consisting of 2.2 ·-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid) and horse radish peroxidase in potassium phosphate buffer. Silicone modified polypropylene gas-diffusion membranes with different thickness were prepared by·casting a mixture of silicone E43 and toluene (1+1 w/w), onto a polypropylene membrane and dried overnight. Different dilution rates of the separated ethanol were achieved by using membranes with different thickness of 20-60 ~tm. and undiluted samples may be analyzed within a wide range of ethanol concentration from 0.0006 to 60% with an excellent precision of 0.23-0.65% r.s.d .. The operational halflife of the immobilized enzyme was 8000 injections within 44 h and the sampling frequency of the method was I 20-180 h- 1 • The method has been used successfully in the determination of ethanol in beer. wine, spirits and medicine.
Billering Compounds in Beers The manual standard method for detennining bitterness in beer is based on an isooctane extraction of the degassed and acidified sample, and measuring at 275 nm the absorbance of the extract against pure iso-octane. This method was adapted by Sablestrom et al.[48] into an FI liquid-liquid extraction system, achieving a sampling frequency of 6()h- .
Carbon Dioxide and Sulphur Dioxide Linares et al.[49] proposed a FI system with on-line gas-diffusion for the simultaneous detennination of carbon dioxide and sulphur dioxide in wines. The two gaseous constituents were separated from the acidified sample in a sandwich-type membrane gas diffusion separator, and collected in an acceptor stream. Two detectors, one potentiometric, responsive to both analytes, and the other photometric, responsive only to sulphur dioxide (after reaction with a p-rosaniline-formaldehyde solution) were connected in series to determine the two constituents in the acceptor. The method was applied to the determination of carbon dioxide and sulphur dioxide in different types of fruity wines and the analytical results were in good agreement with those obtained by standard methods. Sulphur dioxide in wines has also been determined spectrophotometrically based on decolorization of Malachite Green following gas-diffusion separation (cf. Sec. 8.3.2).
208
8
8.5
Environm~ntal
and Agricultural Applications
Milk
Chloride Van Staden (SO] determined chloride in milk using a coaled tubular solid state chloride-selective membrane electrode after separating the interfering matrix by on-line dialysis. A sampling frequency of 120 h- 1 was achieved with a precision of 0.5% r.s.d.. With 30 1'1 sample injection. the working range covered was 250-SOOO mg 1- 1 chloride (cf. Sec. 6.6.2). Fan (51] determined chloride in milk in the range ~3000 mg 1- 1 by the Hg(SCN)l/Fe(IJ) spectrophOiometric merhod following on-line dialysis. achieving a salpling frequency of 90 h- 1 and a precision of 1.3% r.s.d.
T0111/ and free calcium Van Staden and van Rensburg f52] developed a procedure for the simultaneous determination of total and free calcium in milk using an Fl on-line dialysis system. Total calcium was detennined directly by AAS. using a dinitrogen oxide-acetylene flame to overcome interferences from phosphate (with ionization suppression using potassium ions). while free calcium was detennined by spectrophotometry, using cresolphthalein complexone. after separating it from the remaining fraction of total calcium and from interferents in the determination through an on-line dialyzer. Total and free calcium were determined simultaneously at a sampling frequency of 60 h- 1• with precisions of 1.3% and 0.85% r.s.d. respectively. This procedure and the previously described procedure for the determination of chloride in milk were combined to produce a method for the simultaneous determination of chloride, total and free calcium in milk, giving a sampling frequency of 60 h- 1[53). Marstorp et al.[54) determined oxidized ketone bodies (i.e. the sum of acetoacetic acid and acetone) in milk after an off-line pretreatment at 100°C, during which the acetoacetic acid was decarboxylated to acetone. Acetone in the donor stream diffused at room temperature through a P1FE membrane into a reagent acceptor stream containing hydroxylammonium chloride and methyl orange at pH 3. 7. Acetone reacts with hydroxylamine to shift the hydroxylammonium-hydroxylamine equilibrium which creates a pH change that changes the colour of the indicator. A high sample throughput of 100 h- 1 was achieved. Lactose
Lundback and Olsson [55] determined lactose in cow milk using a FI amperometric system incorporating an on-line dialyzer and an immobilized galactose oxidase reactor used mainly for the determination of galactose in urine (cf Sec. 9.3.2 ). The response factor of lactose (including dialysis) is 16 times lower than galactose, but the latter is not present in cow milk. This makes it possible to determine lactose using the same system.
8.6 Soil.v and Sediments
209
After on-line dialysis of the milk sample, lactose is oxidized in the galactose oxidase reactor with release of hydrogen peroxide. The latter is detected by amperometric reduction of a mediator. oxidized by hydrogen peroxide in a peroxidase catalyzed reaction. The linear range of the method with 20 J.Ll injection is in the range 0.05-300 mM.
Drug
dissol~~otion
in milk
Macheras et aJ.[56J used an automated FI on-line dialysis spectrophotometric system. incorporating the dialyzer in a sample loop, to monitor the kinetic process of drug dissolution in low fat milk. The system is similar to that shown in Fig. 6.3, and the dissolution medium· is circulated as donor stream through the dialyzer, pumping it from and back to the dissolution tank. Commercial formulations of salicylamide, propantheline bromide, nitrofurantoin and acetaminophen were used in the study. The resuhs show that the dissolution rate of drugs in milk are lower than in an aqueous buffer.
8.6
Soils and Sediments
Arsenic Wang and Fang (3] determined arsenic in soils by FI-HGAAS following digestion of the sample with a mixture of nitric, perchloric and hydrofluoric acids and reduction of arsenic(V) to (III) by Kl. Recoveries were satisfactory (cf. Sec. 8.8.4). Welz and Schubert-Jacobs (5] determined arsenic in standard reference soil (NBS 142), sludge (L 291). sediments (MESS-I and BCSS-1), etc. by FI- HGAAS, using the same prototype Fl system as for water analysis (cf. Sec. 8.2.1 ). The samples were digested with aqua regia. and a prereduction of arsenic(V) was made with potassium iodide and ascorbic acid in 5 M HCL The analyte concentrations in the sample digests were high enough to allow 100-fold dilutions of the digest, and no interferences were observed in the determinations. Good agreement with certified values was obtained.
Total Nitrogen Sun et al.[57] determined total nitrogen in soil samples by spectrophotometry with an on-line gas diffusion system after a Kjeldahl digestion (for details cf. Sec 8.8.1.).
Selenium The same method used by Wang and Fang [42] for the determination of selenium in biological materials (cf. Sec. 8.8.4) was applied to the determination of selenium in soil digests with good recoveries in the analysis of spiked samples.
210
8. 7
8 Em•ironmenta/ and A~:ricultura/ Applications
Other Agricultural Samples
Biuret Content in Fertilizers Biuret in urea fenilizers is produced through thermal decomposition of ·urea during its synthesis. Owing to its hannful effect to crops. detennination of its content in commercial fenilizers is mandator)'. Szpunar-Lobinska and Trojanowicz (58) developed a FJ spectrophotometric method for such determinations. based on the selective forma&ion of copper(IJ)-biuret complex. The only interferent in the detennination was ammonia. and this was separated from the sample by passing it through an on-line cation exchange column before the complexation reaction. with the column located either before or after the sample injection valve. The latter approach is recommended for bener performance (cf. Sec. 4.6.2). A sampling frequency of 50 h- 1 was achieved with a pmsion of 1.2-2.5% r.s.d ..
Polyphenol.,· in 0/iw• Oils Mesa et al.[.59] proposed an automatic FJ liquid-liquid extraction spectrophotometric system for the determination of polyphenols in olive oils without incorporating any of the usual components for phase separation. The system, featured by iterative change of flow direction. is described in more detail in Sec. 3.4.8. The polyphenols in olive oil samples are extracted into n-hexane after merging with a Folin-Ciocalteu reagent stream, and determined at 750 nm without phase separation. Excellent agreement was obtained between results obtained by the FIA method and the much more time-consuming and reagent-consuming manual reference method.
8.8 Selected Analytical Procedures
8.8
Selected Analytical Procedures
8.8.1
Spectrophotometric Detennination of Total Nitrogen in Soils with On-line Gas-diffusion Separation [57]
211
General Total nittogen is one of the most important determinations in soil analysis. The routine procedures based on· the Kjeldahl digestion and distillation procedures has changed little since their introduction. Despite recent attempts in automating the procedure. the efficiency still remains rather low. The FI procedure described here significantly improves the efficiency in substituting the time-consuming distillation process of batch procedures by an on-line gas diffusion separation. Although a digestion cannot be dispensed with, the micro-sampling feature of FI may be exploited to increase the number of samples processed in a single batch, and to shorten the digestion time.
Equipment Spectrophotometer equipped with a flow-cell of 10 mm light path, 18 JLI volume. Wavelength 590 nm. Chart recorder or printer readout. • Sample injector as shown in Fig. 2.5 a. e, 4-channel peristaltic pump with tygon pump tubes. • Plexiglas sandwich-type gas-diffusion separator with PTFE microporous membrane (cf. Fig. 5.1 ). • All connections made with 0.5 mm i.d. PTFE tubing.
•
Reagents and standard solutions •
Carrier: de-mineralized water
•
5 M sodium hydroxide
• •
0.1 M sodium dihydrogen phosphate buffer solution Mixed acid-base indicator: 0.02 g cresoi red, 0.04 g bromothymol blue, and 0.08 g bromocresol purple dissolved in 3 ml 0.1 M NaOH, and diluted to 100 ml with 0.1 M NaCl. Acceptor solution: 10 ml buffer solution, 4 ml mixed indicator diluted to 500 ml with distilled water and adjusted to wine red colour by adding drop-wise dilute acid or base. The absorbance of the solution at 590 nm should be within 0.25-0.3 with water as reference.
•
212
B Environmental and Agricultural Applications
ml/mln
H.O
1.5
R
1.5
A
1.5
100 ••
s
510 nm
F"tg.&.l: A manifold for the determination of total nitrogen in soil digests by spectrophotometry with gas-diffusion separation. S. sample: R. 5 M sodium hydroxide: SP. membrane gasdiffusion separator. A. buffer acceptor stream with acid-base indicator; D. detector, and W, waste outlets for donor and acceptor streams [57].
•
•
Standard solutions: ammonium-nitrogen standard series of 5, 10, 20, 30, 40, 50 mg 1- 1, prepared by dilution of 1000 mg l- 1 N~-N stock solution with water in .50 ml volumetric flasks containing 6 ml of concentrated sulfuric acid. Catalyst mixture for sample digestion: K2S04/CuS04/Se powder mixed in proportion I 00: I 0: 1.
Sample Treatment
0.5 g of air-dried soil (passed through 0.25 mm screen) is digested at boiling temperature with 6 ml concentrated sulphuric acid in a 50 ml graduated digestion tube after adding I g catalyst mixture. The digestion is continued with gentle boiling in a block digestor until a white residue is obtained (ca 40 min). After cooling. the digest is diluted to volume with de-mineralized water and left standing for some time to let the solid residue settle. The supernatant clear digest solution is used for determination without filtration. Fl Manifold and Operation
The FI manifold and operational parameters are shown in Fig. 8.1. A 10-s loading period with 30-s injection is recommended. I 00 J.ll sample is injected into a water carrier after being filled into the sample loop, and merged downstream with the sodium hydroxide to generate ammonia which is transferred through the gas diffusion membrane into the acceptor stream. The absorbance change of the acceptor is recorded and peak heights taken for N~-N concentration evaluation.
8.8 Selected Analytical Procedurrs
213
Performance of Method.
• • • • • •
Sample consumption: 200 JAI (with sample loop wash out) Reagent consumption: 5 M NaOH, 1 ml: buffer solution, I ml Sampling frequency: 90 h- 1 Precision: 1% r.s.d. at medium concentration level Analytical range: 5-50 mg J- 1 Note: The analytical concentration range may be adjusted by varying the buffer concentration in the acceptor stream. Thus. when the buffer is decreased to 4 ml/500 ml acceptor solution. and I ml/500 ml acceptor solution, the sensitivity is enhanced and the analytical ranges are lowered to 3-30 and 2-15 mg 1- 1 respectively.
Validation of the Procedure
The results from this procedure agreed well with those of a standard Kjeldahl distillation-titration procedure for nitrogen determination.
8.8.2
Spectrophotometric Determination of Anionic Surfactants in Water with On-line Soh'ent Extraction
General
Spectrophotometric batch procedures based on the solvent extraction of an ion associate formed between an anionic surfactant and a cationic dye are often used for the determination of anionic surfactants. Such procedures are usually tedious to operate. The method described here. based on an optimized on-line solvent extraction procedure for the determination of anionic surfactants reponed by Motomizu et al.[27). could demonstrate the efficiency and sensitivity which can be achieved by a FIA procedure. Equipment
• • • • •
Spectrophotometer equipped with a flow-cell of 10 mm light path. 18 t-LI volume. Wavelength: 658 nm. Chart recorder or integrator readout. 6-pon sample injector as shown in Fig. 2.4. Sample loop volume, 100 or 300 J.ll. Two double-plunger pumps or a single 4-channel peristaltic pump equipped with a displacement bottle for organic solvent delivery. T-tube phase segmentor and sandwich-type membrane phase separator. 300 em long. 0.5 mm i.d. PTFE tubing extraction coil. All connections made from 0.5 mm i.d. PTFE tubing.
214
8 Em•iro,ental and Agricultural Applications
mllmln
c
0.8
R
0.8
OS
0.8
S
w
p F'ag.8.2: Fl manifold and operational parameters for liquid-liquid extraction spectrophotometric determination of anionic surfactants. P, pumps: C. water carrier: OS. 1.2-dichlorobenzene; R. 5xl0-~ M Methylene Blue; S, sample; SG, phase segmentor; EC, extraction coil; SP, phase separator: R. restrictor or impedance coil; D. detector: W, waste [27].
Reagent.f and Standard Solutions • • • • •
Carrier: de-mineralized water Cationic dye stock solution: Sxi0- 3 M Methylene Blue (MB) aqueous solution Chromogenic reagent solution: 1 m1 MB stock solution in 100 ml 0.1 M sodium sulphate and 0.1 M acetate buffer (pH 5) Extractant: 1.2-dichlorobenzene Anionic surfactant standard solutions: Sodium dodecylsulphate (SDS) is dried at 50°C under reduced pressure (3 mm Hg) and dissolved in de-mineralized water to give a 3.5x10- 3 M stock solution. Working solutions in the ranges ~3.0xl0- 5 M (100 Jll sample) and ~7.0xl0-~ M SDS (300 Jll sample) are prepared by dilution of the stock standard.
Sample Treatment River and tap waters are analyzed without treatment
Fl Manifold and Operation The Fl manifold and operational parameters are shown in Fig. 8.2 . Either I 00 or 300 Jll sample loops are used, corresponding to the two different analytical ranges. The samples are injected into a water carrier and merged with the MB dye solution before being segmented by dichlorobenzene solvent. The extraction takes place in the 3 m extraction coil and the separated organic phase is directed to the spectrophotometer flow-cell to record the peak absorbance. The .peak height is used for evaluation.
H.H Selected Analyticu/ Pron•du"s
215
Performance of Method • • • •
Linear range: 0-3.0xlo-~ M (JOO Jll sample) .. 0-7.0xl0-s M (300 Jll sample) Detection limit (3u) (300 Jll sample): 1.8x 10-11 M SDS Sampling frequency: 20 h- 1 Precision (300 Jll samples): 0.9% r.s.d. (2.1xlo- 6 M SDS): 4.4% r.s.d. (J.4xl0- 7 M SDS)
Validation
of M~thod
Results showed good apeemen1 with those obtained by a balch Methylene Blue method
8.8.3
Electrothennal Atomic Absorption Spectrometric Determination of Trace Metals in Sea Water with On-line Sorbent" Extraction Separation and Preconcentration [ 10, 16]
General Despite the high sensitivity of ETAAS, a considerable number of imponant heavy metals cannot be directly determined in unpolluted sea water even with sophisticated background correction and matrix modification. This is due to the low concentrations and strong interference from the sample matrix. ETAAS with on-line sorbent extraction separation and preconcentration provides an ideal solution to such determinations (cf. Sec. 4.6.5). The on-line separation significantly improves the selectivity and sensitivity of ETAAS determinations without affecting its normal sample throughput. The separation is achieved by means of a 15 J.ll micro conical column packed with C1s bonded silica sorbent. The column selectively sorbs the DDC complex of a number of trace heavy metals while allowing the interfering matrix to flow to waste. The sorbed analytes are then eluted with ethanol into a 40-70 1t1 collector tube using an eluate zone-sampling technique (cf. Sec. 4.6.5) before introduction into the graphite furnace by a stream of air. The procedure described here is for the determination of lead in sea water, but may be extended to the determination of Ni, Co, Cd, As etc. in sea water with minor modifications.
Equipment •
Atomic absorption spectrometer with deuterium arc background corrector, equipped with lead hollow cathode lamp. Wavelength, 283.3 nm with 0.7 nm bandpass.
216 •
•
• •
8 Environmemal and Agricultural Applications
Graphite furnace atomizer with pyrolytic graphite coated graphite tubes and uncoated electrographite platfonns. The graphite furnace temperature program is shown in Table 8.1. The atomization pulses are recorded by high resolution graphics, and peak area is used for evaluation of results. Computerized automated FI solution processing system with two variable speed peristaltic pumps and a 4:5 channel injector valve, all separately programmable in seven steps. Tygon pump tubes are used for all solutions except ethanol. which must be delivered by a solvent resistant pump tube (e.g. Verdoprene or Marprene). Conical sorbent column (15 Jll capacity) made as shown in Fig. 4.1 c, packed with 40-63 Jlm C 18 bonded silica sorbent with octadecyl functional groups. Thin-bored 0.35 mm i.d. PTFE tubing is used for all connections and for the eluate collector to reduce dispersion. Table 8.1
Step
Graphite furnace temperature program for the determination of lead in sorbent extraction eluate Temperature/
oc
Pre-heat 1
Gas flow/ ml min- 1
lime/s Ramp
Hold
70
90
1
50
300
150 500
20 10
10 20
I
5
20 1900
5 3
300 300 300
6
2500
2
3 4
0
5
0 300
Reagents and Standard Solutions • •
• • •
All reagents should be of the highest purity available, and doubly de-mineralized water is used for preparing the reagents. Complexing reagent: 0.02% diethylammonium diethyldithiocarbamate (DOC), prepared in 0.05 M ammonia solution adjusted to pH 9 with acetic acid. Ultratrace amounts of lead in the reagent may be removed by on-line purification of the reagent immediately before merging with the sample using a 500 Jll column packed with C 1s sorbent. Washing solution: 0.02% nitric acid Eluent: ethanol or methanol Working standards: 0.2-2.0 Jlg 1- 1 Pb standards prepared by stepwise dilution of
H.X Selected Anal,vtit:al Procedures
217
1000 mg 1- 1 Pb stock solution and acidified to pH 2 with nitric acid. Note: in the report by Fang et al.[16] matrix matched working standards were required. Sperling .et al.[ 10] found this not to be necessary if a 0.02% nitric acid is used as washing solution.
Sample Treatmt'nt Sea water samples (acidified on collection) are analysed without treatment
Fl manifold and orwration
The Fl manifold. operational par.uneters and sequence are identical to those shown in Fig. 4.1J and Table 4.4, exceprlhat water washing Should be substituted by 0.02% nitric acid. Performanct> of Method • • • • • • • •
Enrichment factor: 26 (compared with 50 Jll direct injection) Characteristic concentration: 0.015 Jlg 1- 1 Pb Detection limit (3cr): 0.003 118 1- 1 Pb Precision: 1.9% r.s.d. at 0.1 Jlg 1- 1 Pb level Sample throughput: 24 h- 1 Sample consumption: 2.5 ml Reagent consumption: ethanol (methanol) 0.35 ml, 0.02% DOC \1.3 ml Suppression of interfering effects: almost complete elimination of background interference (cf. Fig. 8.3)
Validation of Method
Good agreement of analytical results with certified values of standard reference materials has been reported for CASS-1. CASS-2 coastal sea waters, NASS-2 open ocean seawater and SLRS-1 riverine water.
:~ '--l··_ _ _--JI f·~~ I f'l\_
::r. . ...-.=:~_...,._.____.1 r,, •
1
0
2 0 Time/a
I I I
2
YfiJLl: Recordings of ETAAS aaomization sipals for lead in waaer samples using on-line sorbent extraction separation and preconc:emration. (a) Blank; (b) 0.100 1'81- 1 Pb standard: (c) de-mineralized warer; (d) NASS-2 (0.041 pg 1- 1); (e) CASS-1 (0.245 pg r" 1) ; and (f) CASS-2 (0.022 1'8 1- 1) [10). (Rc:produc:cd by permission from Royal Society of Chemisay.)
8.8.4
Hydride Generation Atomic Absmption Spectrometric Detennination of Selenium and Arsenic in Soils and Biological Materials
General The advantages of FI systems with on-line gas- liquid separators over batch procedures have been discussed in detail in Sec. 5.5.1. A recommended procedure for the determination.of selenium and arsenic, mainly based on the methods proposed by Wang and Fang [3,42], is described here. The methods may be readily extended to other hydride forming elements and mercury with minor modifications.
Equipment • •
Atomic absorption spectrometer with selenium and arsenic hollow cathode lamps. Wavelengths: Se, 196.0 nm; As, 193.7 nm. Chart recorder readout. Electrically heated T~shaped quartz atomizer constructed of 7 nun i.d., 160 nun long quartz tube with an 15 nun i.d., 40 nun long side arm as inlet in the centre of the ~be. Optimum temperature for Se, 700 °C; for As, 950 °C.
8.8
•
•
S~l~ct~d
AI'IDlytical Pmcedllns
219
FI system consisting of a 5-channel peristaltic pump equipped with tygon pump tubes; a 4:4 channel injector valve; 1 mm i.d. PTFE reaction coils and connection lines connected with a Clemifold; and a gas-liquid separator of one of the types shown in Fig. 5.4 a and b. Argon carrier gas supplied from gas cylinders equipped with a orifice-type flowrestrictor capable of providing a back-up pressure of >3 kg cm-2 at 100 ml min- 1 flow-rate. Gas flow-rates are measured with a flow meter in off-line mode. both for accuracy and to prevent entrainment of aqueous constituents into the flow-meter.
Reagents anti S111ndturl Sollllions • • •
0.4% (m/Y) sodium borohydride in 0.1 M sodium hydroxide prepared freshly before the detenninatioo. lO'll (m/v) potassium iodide reductant for As(V). Se(IV) and As(lll) standards of 2, 4. 6. 8, 10 pg 1- 1 prepared in I M hydrochloric acid by 2-stase dilutions of 100 mg 1- 1 stoclt solutions.
Sample Treatment Soils and coal fly ash: 0.2-0.5 g ground samples are treated with 7 m1 nibic-perchloric acid (3: 1) mixture and 3 m1 hydrofluoric acid in a covered P1FE crucible and left standing ovemighL 1be samples are then digested on a hot plate at low boiling temperature until a clear digest is obtained. 1be perchloric acid is fwned off to 1-2 mi. For the determination of selenium, 5 ml 4 M hydrochloric acid are added and boiled for 5 min to reduce Se(VI) to Se(IV). while for arsenic. 5 ml of 10% KI are added and boiled for 5 min to reduce As(V) to As (Ill). After cooling, the digests are diluted to 25 ml with I M HCI. Plants: 0.2-1.5 g dried powdered samples are treated with 5'-10 m1 of concentrated nibic acid in a covered glass beaker and left standing overnighL The mixture is heated on a hot plate till the generation of intensive dark brown fumes, 5 ml nibic~perchloric acid (3:1) mixture are added. The mixture is further digested to produce a clear solution, and then boiled down to 1-2 ml until the appearance of large amount of perchloric acid fumes. For the determination of arsenic, 5 m1 of 10% KI are added and boiled for 5 min for the reduction of As(V). After cooling, the digest is diluted to 10 ml with 1 M HCI. Reduction of Se(VI) by boiling with 4 M HCl, as done for soil samples, is not necessary, presumably because a large fraction of the perchloric acid may have been transformed into hydrochloric acid during the oxidation of organic matter in the plant samples.
Fl Manifold and Operation The FI manifold and operational parameters" are shown in Fig. 8.4. A 5-s sample loading period with 10-s injection period is recommended.
220
8 EnvironmetUal and Agricultural Applications
Performance of method • • • • • •
Sample volume: 400 ~-&1 (total consumption ca. 800 ~-&1 including sample loop wash-out). Reagent consumption: HCI (concenttated), 0.3 ml; NaBH.t, 3 mg. Sampling frequency: 240 h- 1• Characteristic concentration: Se, 0.26; As, O.IS lAB 1- 1• Delec:tion limit (3a): Se, 0.06; As 0.10 ~-&8 1- 1• Precision: Se. 1.6%; As, 1.5% r.s.d. (2~-&gl- 1 ).
Validation of method: good agreement of analytical results with cenified values of NBS saandards has been reponed for SRM 1567 wheal flour (Se), SRM 1571 orchard leaves (Se); SRM 1633 coal fty ash (Se), SRM 1563 rice ftour (As).
Ar 120 ..., .. In
..1/.in
2C
0.4f.NaBH4 1M HCI
F"~g.8.4:
2 10
8
w
Fl manifold and operational parameters of hydride generation AAS system (valve in sample fill position) for Se and As. V, injector valve; L. sample loop; SP, gas-liquid separator; S, acidified sample; C, acid carrier. AAS. quanz cell atomizer; W, waste flows; a, b, reaction coils; Ar. stabilized argon flow [3].
9
Clinical and Pharmaceutical Applications
9.1
General
Owing to the complexity of the matrices of clinical and pharmaceutical samples, separation procedures often constitute an integral part of the related analytical methods. Since these are fields where frequently large sample workloads are involved. automation and improvements in the efficiencies of the corresponding separation procedures are highly desirable. Some clinical samples or pharmaceutical constituents (particularly in body fluids) may also be very valuable or only available in small quantities. thus requiring micro-analytical techniques. A growing trend in clinical and pharmaceutical analysis is the on-line monitoring of certain constituents in the body or in industrial processes. FI separation techniques have the potential of simultaneously providing solutions to aU these requirements in many applications. The imponant ones are summarized in this chapler.
9.2
Blood and Serum
9.2.1
Determination of Trace Elements
Cadmium
Welz et al.[l] determined cadmium in whole blood digests by flame AAS following on-line coprecipitation using a modified procedure of that used for the determination of lead. Cadmium is coprecipitated with the carrier Fe(II)-HMDTC which is collected in a knotted reactor and dissolved by IBMK. A 52-fold signal enhancement was obtained with ·a sampling frequency of 72 h- 1 (for details cf. Sec. 9.5.3).
222
9 Clinical and Pharmaceutical Applications
Fang and Dong [3] determined cadmium in whole blood digests by ETAAS with on-line coprecipitation-dissolution also using the Fe(II)-HMDTC system, achieving a detection limit of 0.003 J.tg J- 1•
Lead Fang et al.[2) detennined lead in whole blood digests by flame AAS using an on-line coprecipiwion system based on coprecipitation with Fe(II)-HMDTC. The precipitate is collected with a knoned reactor in a filterless Fl system (for details d. Sec 9.5.3). A 44-fold signal enhancement was achieved with a sampling frequency of 90 h- 1•
Uthium Lithiwn in blood was determined by Kimura et a1.(4.S] by FJ liquid-liquid extraction with proton-dissociable chromogenic 14-crown-4 derivatives as the extractionspectrophotometric reagent. Lithiwn may be determined selectively in blood under a high sodium background of I 30-1 60 mM, after extraction of the lithium complex into chloroform. The method also features a low sample conswnption of 20 pi and high sample throughput of 100 h- 1• A 14-crown-4 derivative has also been incorporated in a PVC membrane to construct a coated-wire lithium ion-selective electrode used for the determination of lithium in blood sera, after on-line dialysis separation in a FIA system [6]. An estimation of the overall selectivity of the method over the sodium content, including that of the dialysis membrane and the electrode, resulted in a selectivity coefficient k of 1/50. This selectivity is not sufficient to overcome interferences from relatively large fluctuations in sodium contents, and corrections based on a simultaneous evaluation of the sodium content are required under such circumstances. Nickel
Fang and Dong [3] determined nickel in whole blood digest by ETAAS following on-line coprecipitation-dissolution using the same fiherless system as for cadmium. A detection limit of 0.02 f..Lg 1- 1 was reached, making it possible to determine nickel in the blood of unexposed persons. Selenium
Welz and Schubert-Jacobs [7) determined selenium in standard reference whole blood samples (Seronorm 904, 905, 906) by Fl-HGAAS using the prototype of a commercialized Fl system, FIAS-200, and obtained good agreement with certified values. The performance of the Fl procedure was compared to a standard IUPAC batch procedure to show the advantages of the former.
9.2 Blood and Smun
223
McLaughlin et al.[8] also detennined selenium in blood plasma and serum by FIHGAAS, following digestion with nitric, sulphuric and perchloric acids. Although good agreement was obtained between the proposed method and an ETAAS procedure, both the detection limit (1.2 1'8 1- 1), within run precision (5.8% r.s.d. at 20 1'8 1- 1). and sampling frequency (90 h- 1) were significantly worse than those reponed by Welz [7] and those obtained in our laboratory. The inferior perfonnance appear to be mainly due to the gas-liquid separation system, which seems to have a fairly large dead volume and which used free out-flow instead of forced withdrawal of waste.
9.2.2
Detennination of Urea
Petersson et al.[9] developed a miniaturized optosensing FIA system for the detennination of urea in undiluted whole blood, based on the optical detennination of ammonia generated from the sample under the action of urease. A description of the optosensing system is given in Sec. 5.4.2 with a schematic diagram (Fig. 5.8). A special feature of the gas-diffusion separation system is that the separating barrier between the donor and . acceptor streams consists of a hydrophobic microporous membrane and a hydrophilic membrane between which is sandwiched a gel of covalently immobilized urease. The blood sample was injected using an alkaline carrier, and the dispersion was minimized to achieve a dispersion coefficient D close to 1. The reaction and sensing then proceed through three stages. In the first, the injected sample zone gets into contact with the enzyme layer, and sample urea is degraded to ammonium ions. In the second stage the alkaline carrier succeeding the ~ample zone transforms the ammonium ions into ammonia which penet:ates the hydrophobic membrane, and in the third. the ammonia is transferred into the acidic buffer acceptor stream containing the acid-base indicator, producing a change in absorbance. The activity of the membrane sandwich remained constant for at least a week under continuous daily operation.
9.2.3
Perchlorate
Gallego and Valcarcel [ 10] determined perchlorate in human serum and urine by an indirect flame AAS method based on the fonnation of an ion-pair between copper(l)-6methylpicolinealdehyde azine and perchlorate which was extracted into ffiMK. Perchlorate was determined in the range 0.1 to 5 IJg 1- 1 by measuring the copper signal with a sampling frequency of 45 h- 1•
224
9 Clinical and Pharmaceutical Applications
9.2.4
Gaseous Constituents
Carbon dioxide The first application of FI gas-diffusion separation reported by Baadenhuijsen [II] was on the determination of carbon dioxide in plasma by spectrophotometry. resulting in a fast automated procedure. A dimethylsilicone rubber membrane was used for separating the carbon dioxide from the acidified sample. Fan et al.[ 12] described a similar procedure using a PTFE microporous membrane. The procedure is described in detail in Sec. 9.5.2.
Ammonia FI gas-diffusion separation systems with membrane separators have been used successfully for the detennination of ammonia in whole blood and plasma both with a potentiometric [13] and a spectrophotometric detector (14].
9.3
Urine
9.3.1
Determination of Trace Elements
Arsenic Welz and Schubert-Jacobs [7] determined arsenic in standard reference urine (Seronorm 108) using FJ-HGAAS. Owing to the presence of organic forms of arsenic in urine, the determination of total arsenic requires a nitric-sulfuric-perchloric acid digestion of the sample followed by reduction of arsenic(V) with KI. In spite of a high dilution factor, the residual perchloric acid in the digest caused an interference, resulting in low values when calibrations were made with aqueous standards. Matching of the perchloric acid content in the standard and sample was unsuccessful, and satisfactory results can be obtained only by a standard addition method. This interference effect has not been observed in the determination of arsenic in other biological samples also involving perchloric acid digestion [ 15.].
Cadmium Burguera and Burguera [16] developed a FJ liquid-liquid extraction spectrophotometric method for the determination of cadmium in urine using dithizone. Original urine samples were directly injected into a buffered earner (pH 10.5) containing tartrate, and
9.4 Urine
22S
hydroxylamine and hexacyanoferrate as masking agents. Cadmium was extracted by dithizone in chlorofonn, and after phase separation, absorbance of the organic phase was measured at 518 nm. The method was used to determine cadmium in urine at levels higher than 0.2 J.l.8 1- 1• with a precision of better than 3% r.s.d .. Welz et al.[l] determined cadmium in the digest of several standard reference urine samples using the same on-line coprecipitation procedure for serum analysis described in Sec. 9.2.1, and obtained good agreement with certified values. Dong and Fang [17] recently developed a FI-AAS method to determine cadmium in undigested urine by flame AAS following on-line ion-exchange preconcentration. The methOd is described in detail in Sec. 9.5.4.
Cobalt and Nickel The same on-line coprecipitation procedure used by Welz et al.[ I] for the determination of cadmium in urine digests was also used successfully to determine cobalt and nickel in standard reference urine samples by FAAS.
Multi-element Determinations hy ICP-MS V, Cr. Ni. Co. Cu. Mo. Pt. Hg and Bi in urine have been determined by ICP-MS following an on-line column retention/elution of the bis(carboxymethyl)- dithiocarbamate complex, achieving detection limits in the range 8-80 ng 1- 1 [18j. Cr. Ni, and Pt were determined in a urine standard reference material NBS-SRM 2670, and the results agreed well with the recommended values.
9.3.2
Galactose
Lundback and Olsson [ 19] determined galactose in urine using a FI amperometric system incorporating an on-line dialyzer and an immobilized galactose oxidase reactor (Fig. 9.1 ). Following on-line dialysis of the urine sample. and the removal of ascorbic acid using a copper(IJ) saturated Bond-Eiut column, D-galactose in the sample is oxidized in the galactose oxidase reactor with release of hydrogen peroxide. The latter is detected by amperometric reduction of a mediator, oxidized by hydrogen peroxide in a peroxidase catalyzed reaction.
9.3.3
l\T!nnnes
Audunsson [20] developed an on-line liquid membrane separation technique for sample cleanup in the determination of p,g 1- 1 levels of amines in urine by gas-liquid chromatography (GLC). The technique is described in detail in Sees. 3.4.3 and 3.5.5. The technique improved the detection limit more than two orders of magnitude compared to that obtained by normal GLC with pure aqueous standards.
226
9 Clinical and Pharmaceutical Applications
s
H.O B
w
R
Fig.9.1: Manifold of a A amperometric system for the determination of galactose in urine. S, sam-
ple; DS, on-line dialyzer: GOR. immobilized galactose oxidase reactor; Cu(U), copper(U) saturated Bond-Eiut column for removal of ascorbic acid, POD, peroxidase reactor: B. phosphate buffer. pH 7; R. 5 mM potassium hexacyanoferrate and 0.1 M sodium phosphate, pH 7; D. amperometric detector: and W. waste.
9.4
Pharmaceuticals
9.4.1
Carboxylic Acid Drugs
Stewart et. al.[21] have shown the usefulness of F1 on-line ion-pair extraction in the determination of carboxylic acid drugs. using salicylic acid, valproic acid, and ibuprofen as model drugs. After a comparison of different chromophoric and ftuorophoric cationic dyes in chloroform extractant, Gentian Violet was recommended as counterion for spectrophotometric determination and Acridine Orange was recommended for ftuorimetric determination. The system was used for post column detection in HPLC.
9.4.2
Sulphonamides
Sulphonamides in pharmaceutical preparations and urine were determined indirectly using flame AAS by continuous precipitation with copper or silver, as proposed by Montero et al.[22]. The copper method exhibited a better selectivity, and only this was used for determinations in urine. The precipitate, formed by injecting one of the cations into a carrier containing the sample, was collected on an on-line filter, and the peak absorbance of the residual metal in the stream passing through the filter was measured. The decrease in peak height compared to a blank was then related to the sulphonamide
9.4 Pharmoc:eulicals
227
concentration. Sulphonamide was determined in the range 2.5 - 3.5 p.g ml- 1 with a sampling frequency of 1~150 h- 1 and a precision of 1.5-3Ci- r.s.d .. The equivalent manual batch procedure was limited to a sample throughput of 5 h- 1• and the selectivity and sensitivity were also worse.
9.4.3
Codeine
As one of the earliest applications of Fl liquid-liquid extraction. Karlberg et a1.!23] determined codeine in acetylsalicylic acid tablets. Codeine is extracted as its picrate ion pair into chloroform, and determined by spectrophotometry following phase separation. A sampling frequency of 60 h- 1 was achieved with a precision of about I'*- r.s.d.(cf.
Sec. 9.5.}).
9.4.4
Local Anaesthetics
Montero et al.[24] developed an indirect AAS method for the determination of local anaesthetics (lidocaine. tetracaine and procaine hydrochlorides) in pharmaceutical preparations using Fl on-line precipitation-dissolution. A cobalt solution is injected into a carrier stream containing the sample. and the precipitate formed is retained on an online stainless steel filter. The determinations were made by measuring the residual cobalt concentration in a similar way as for the indirect determination of sulphonamides (cf. Sec. 9.4.2). A sampling frequency of JOO h- 1 was achieved with a precision of 0.6% r.s.d •.
9.4.5
Vitamins
Karlberg and Thelander [25] determined vitamin 8 1 in pharmaceutical preparations with an FI liquid-liquid extraction fluorimetric system using the thiochrome method. A sampling frequency of 70 h- 1 may be achieved with a precision of about 1% r.s.d ..
9.4.6
Other Pharmaceuticals
Berberine and Benzethonium Sakai [26] developed an FI liquid-liquid extraction spectrophotometric method for the determination of berberine and/or benzethonium in pharmaceutical preparations based on ion-association with tetrabromophenolphthalein ethyl ester (TBPE-H) and extraction of
228
9 Clinical and Pharmaceutic'al Applications
the blue ion-associate into I ,2-dichioroethane. The sampling frequencies achieved were 45 h- 1 for berberine and 30 h- 1 for benzethonium. The precisions were about I% r.s.d..
Procyclidine Procyclidine hydrochloride was detennined in pharmaceutical preparations by Fossey and Cantwell [27] using a FI liquid-liquid extraction system. based on ion-pair extraction of the drug with picrate into chloroform. A high sampling frequency of 240 h- 1 was shown to be feasible with a precision of I% r.s.d..
EM/april Kato [28] reponed on the spectrophotometric determination of enalapril in pharmaceutical preparations using FI liquid-liquid extraction based on ion-pair formation with Bromothymol Blue, and subsequent extraction into dichloromethane. The sample throughput wa." 80 h-I. and the precision was about I % r.s.d..
Scopolamine Hydrobromide Chen et al.[29] determined scopolamine hydrobromide in tablets by FI solvent extraction using a membrane phase separator, based on ion-pair formation with Bromocresol Green and extraction into chloroform. A sampling frequency of 60 h- 1 was achieved with a precision of 0.8% r.s.d.. The results were in ·good agreement with a standard batch procedure. 1be method was used for examination of content uniformity in scopolamine hydrobromide tablets.
Atropine Sulphate
Qu et aJ.[30] detennined atropine sulphate by FI solvent extraction based on ion-pair formation with Bromocresol Green and extraction into chloroform. A sampling frequency of 60 h- 1 and a precision of 0.7% r.s.d. were reported.
9.5 Se/ecu:J Fu~c·edures
:.29
9.5
Selected ProcedurLS
9.5.1
Spectrophotometric Determination of Codeine in Pharmaceutical Preparations by FI Solvent extraction [23,31]
General The method is based on the fonnation of codeine-picrate ion pair and extraction into chlorofonn at pH 6.5. This pH is important for minimizing interferences. The procedure was developed for deten mnation of codeine in acetylsalicylic acid tablets but may be adapted to other preparations.
Equipment • • • • • •
Spectrophotometer equipped with a flow-cell of I 0 mm light path, 18 .Jll volume. Wavelength 405 nm. Chart recorder or printer readout Sample injector as shown in Fig. 2.5. with 40 JLI sample loop. 4-channel peristaltic pump with tygon pump tubes. Chlorofonn is delivered with a lhsplacement boule (cf. Fig. 2. 1) PTFE T-tube segmentor PTFE sandwich-type membrane phase separator with PTFE microporous membrane. Extraction coil and connections are made from 0.5 mm i.d. PTFE tubing. Extraction coil length. 2.0 m.
Reagents and Standard Solutions • • • •
Carrier: 0.065 M phosphate buffer, pH 6.5 Picrate reagent: 0.025 M picrate in 0.065 M phosphate buffer, pH 6.5. Neutralize picric acid with sodium hydroxide, add buffer and adjust to pH 6.5. Extractant: Chlorofonn Standard solutions: prepare standards in the range 2-5x w- 4 M from phannacopeia quality codeine phosphate. Match the composition as close as possible to that of the sample when necessary.
Sample Treatment Tablets are homogenized and dissolved in 0.065 M phosphate buffer (pH 6.5) to make up a sample solution of approximately 3.5XI0-4 M codeine. Filter when necessary.
9 Clinical and Pharmoceutical Applications
230
ml/mln
c
2.0
PR
0.8
Water
1 .6
40 Ill
s
EC
w
p
D F"~g.9.2:
Fl manifold for the spectrophotometric detennination of codeine by solvent extraction. P, pump; DB. displacement bottle for delivery of chloroform; C. carrier, 0.065 M phosphate buffer, pH 6.5: PR, picrate reagent: S. sample; SG. phase segmentor; EC. extraction coil; SP. phase separator; R. restrictor or impedance coil: D. detector; W, waste (23,31].
FJ Manifold and Operation The FI manifold and operational parameters are shown in Fig. 9.2. 40 Jll of sample is injected into the phosphate buffer carrier and merged with the picrate reagent. Segmentation of the flow with chlorofonn is effected through the T-segmentor, the solvent being delivered by pumping water into the displacement bottle. After extraction in the 2 m extraction coil the two phases are separated in the membrane separator, and the organic phase is delivered to the flow cell. Peak heights are used for evaluation.
Peiformance of Method • • • •
Sampling frequency: 60 h- 1 Precision: 1.5'ii r.s.d. at 3.5xl0-4 M level Sample consumption: 100 Jll (including washout) Organic solvent consumption: 1.5 ml
9.5 Se/ecled
9.5.2
Procedure.~
:!31
Spectrophotometric Determination of Carbon Dioxide in Blood with Gas Diffusion Separation [11, 12]
Principle
Carbon dioxide in acidified blood samples is transferred through a hydrophobic microporous membrane and dissolved in an acceptor stream of basic buffer solution containing an acid-base indicator. The absorbance change is measured by spectrophotometry. Equipment
•
• • • •
Spectrophotometer equipped with a flow-cell of 10 mm light path, 18 l.tl volume. Wavelength 410 nm (normal peaks) or 580 nm (negative peaks). Chan recorder or printer readout. Sample injector as shown in Fig. 2.5. 4-channel peristaltic pump with tygon pump tubes. Plexiglas sandwich-type gas-diffusion separator with PTFE microporous membrane. All connections made with 0.5 mm i.d. PTFE tubing.
Reagents and Standard Solutions
• • • • • •
•
Carrier: de-mineralized water 0.2 M sulphuric acid 0.2 M sodium bicarbonate 0.2 M sodium carbonate Acid-base indicator: I g Cresol Red in I 00 ml 0.1 M sodium hydroxide Acceptor solution: I 0 ml 0.2 M sodi urn bicarbonate. 5 ml 0.2 M sodium carbonate and 4 ml Cresol Red indicator solution diluted to 1000 ml with de-mineralized water. When measurements are made at 410 nm, the absorbance of the solution is adjusted to 0.2--{).3 with water as reference by adding drop-wise dilute acid or base. At 580 nm the absorbance of the solution should be adjusted to within 0.8-0.95 with water as reference. Standard working solutions: 0, 8, 16, 24, 32,40 mM sodium bicarbonate solutions prepared by dilution from I 000 mM stock solution .
FI Manifold and Operation
The manifold and operational parameters are shown in Fig. 9.3. 35 ~.tl of blood sample is injected into the water carrier and merged with 0.2 M sulphuric acid, on passing through the gas-diffusion separator the evolved carbon dioxide is transported through the membrane into the acceptor stream producing a change in absorbance, the peak height of which is used for evaluation of results.
232
9 Clinical and Pllarmaceutical Applications mllmln 35 11
s
CR
1.0
R
1.8
A
1.8 410 nm or 680 nm
Fig.9.3: FI manifold for the determination of carbon dioxide in blood Y>ith gas-diffusion separation. CR. water carrier: S. sample; R. 0.2 M sulphuric acid: SP. membrane gas-diffusion separator: A. buffered acceptor with acid-base indicator; D, detector and W, waste outlets for donor and acceptor streams (12].
Peiformanc:e of Method • • • • •
Sample consumption: about 100 J.tl (with wash-out) Sampling frequency: 90 h- 1 Precision: 0.9% r.s.d. Analytical range: 0-40 mM C02 Recoveries: 94-106%
9.5.3
Flame Atomic Absorption Spectrometric Determination of Lead and Cadmium in Whole Blood and Urine with On-line Preconcentration by Coprecipitation [1,2]
Principle Lead, cadmium and other trace metals are coprecipitated from blood and urine matrices and collected on-line on the tube walls of a knotted reactor. lron(D), formed by reduction of iron(ill) using ascorbic acid. is used as a coprecipitation carrier to form a black precipitate with HMDTC. Therefore. 200 mg 1- 1 iron are added to all sample digests to provide a minimum iron concentration. The collected precipitate is subsequently dissolved by mMK and transponed to the flame atomizer of an AAS system. The method features high sensitivity and sample throughput with high tolerance to the principle matrix interferents, including iron and copper.
9.5 Selected Pr(l(;edures
233
Equipment
•
•
•
•
Perkin-Elmer M 2100 atomic absorption spectrometer with deuterium lamp background corrector. Cadmium and lead hollow cathode lamps operated at 4 rnA and 7 rnA. and 228.8 nm and 283.3 nm wavelength respectively. Spray chamber used without impact system. Acetylene flow-rate 1.0 I min- 1, air flow-rate 10 I min- 1 (extra-lean flame). Peak height evaluation with a time constant of 0.5 s. Fl peaks recorded by a printer or ch: 1rt recorder. FlAS-200 flow injection s;. .Lem with two peristaltic pumps and one 4:5 channel injector valve which all are separately programmable. All solutions are pumped through tygon pump tubes except IBMK, which is delivered through a solvent displacement bottle (cf. Fig. :? 1). Knotted reactor precipitate collector: ma(.h: by tying 5-mm-diameter interlaced knots in 150 em long. 0.5 mm i.d., 1.8 mm o.d. Micro-Line (Thermoplastics Scientifics Inc.) ur PTFE tuhi11g (cf. Fig. 2.9). All connections made of 0.5 rum i.d. PTFE tubing except connection between injector valve and the spectrometer nebulizer which is 0.35 mm i.d ..
Reagents and Standard Solutions
•
•
• • •
0.25% hexamethylene ammonium hexamethylene dithiocarbamate (HMAHMDTC) precipitant solution: prepared fresh each day by dissolving 125 mg reagent in 50 ml water containing 0.25 m1 5% m/v lithium hydroxide. Ascorbic acid reductant: 1% m/v solution for cadmium and 2% m/v for lead, prepared by dissolving 0.5 or 1.0 g ascorbic acid in 50 ml 0.2 M aqueous potassium chloride solution containing 4 ml of 0.2 M hydrochloric acid. 10 g 1- 1 iron(III) solution: prepared by dissolving iron(III) nitrate in water. Nitric and hydrochloric acids for digestion: purified by sub-boiling distillation. Standard working solutions: 0-10 pg 1- 1 Cd, and 0-200 pg 1- 1 Pb are prepared by diluting 1000 Jlg 1- 1 stock solutions with 0.1 M nitric acid.
Sample Treatment
Blood samples: 1.0 ml whole blood is digested with 5 ml nitric acid in a sealed PTFE digestion vessel using a microwave digestion system MDS-81 D (Kumer Analysentechnik, Rosenheim, Germany), and treated according to the manufacturer's recommendations. Transfer the clear digests into quartz digestion tubes, and add 0.1 ml perchloric acid and 0.1 ml sulphuric acid. Heat the digest to 150-170 oc in a digestion block and evaporate to near dryness. Dissolve the residue in a few milliliters of water by gentle heating; add 500 J.Ll 10 g 1- 1 iron(III) solution and dilute to 25 ml with de-mineralized water.
234
9 Clinical and Pharmaceutical Applications
Urine samples: Digest 10 ml of urine with 5 ml nitric acid in a quanz digestion tube, heating to 130 °C for two hours in a digestion block. After cooling, add 0.1 ml perchloric acid and 0.1 ml sulphuric acid. Heat the digest to 15~170 oc in a digestion block and evaporate to near dJ)'IIess. Dissolve the residue in a few milliliters of water by gentle heating; add 500 J.tl 10 g 1- 1 iron(ID) solution and dilute to 25 ml with de-mineralized water.
Fl Manifold and Operation The FI manifold for the on-line coprecipitation preconcentration is shown in Fig. 9.4 with the operational parameters. The recommended sample loading period is 30 s, and the dissolution period, 10 s. Longer periods yield higher EF values, but may induce larger losses of precipitate during the collection.
Performance of Method • • • • • • • • •
Sampling frequency: 90 h- 1 Enhancement factor: 44 (Pb), 40 (Cd) Enrichment factor (EF): 20 Concentration efficiency (CE): 30 min-• Detection limit (30'): 2 J.tg 1- 1 Pb, 0.2 J.t8 1- 1 Cd . Precision: 2.7% r.s.d. (Pb), 1.5 % r.s.d. (Cd) Sample consumption: 2.5 ml (including washout) IBMK consumption: 1.0 ml Tolerance of iron content: 250 mg 1-• in digest
Validation of Method Good agreement of results with certified values of standard reference whole blood samples. Seronorm 901, 902 (Pb); Seronorm 905 and 906 (Cd); and standard reference urine samples, Lyphochek 2, Lanonorm 2 and 3 (Cd).
95 Se/ec1ed ProcedurrJ
:!35
ml/mln
a Aa
w HMDTC
P, OFF
b
w Fig.9.4: Fl manifold for the FAAS detennination of lead and cadmium in blood and urine by on-line coprecipitation. a. sample loading (precipitate collection) sequence: b. precipitate dissolution sequence. P 1, P2 , peristaltic pumps: V. 4:5 channel injector valve: S. sample: KR. knotted reactor precipitate collector; and W. waste [ 1.2].
236
9 Clinical and PhorfTI/lceutica/ Applications
9.5.4
Aame Atomic Absorption Spectrometric Determination of Cadmium in Undigested Urine with On-line Ion-exchange Preconcentration [17]
General
The sensitivity of flame AAS is insufficient for the determination of cadmium in urine at normal levels. ETAAS methods require sample digestion and/or chemical modification of the sample matrix. resulting in complicated procedures. In this procedure, cadmium is determined efficiently by flame AAS in undigested urine following an on-line separation of the matrix and preconcentration of the analyte using a micro-column packed with CPG- 8HQ ion-exchanger (quinolin-8-ol immobilized on porous glass). Eq11ipment
•
•
Perkin-Elmer M 2100 atomic absorption spectrometer with deuterium lamp background corrector and FIAS-200 flow injection system. Cadmium hollow cathode lamp operated at 5 rnA and 228.8 nm wavelength. Flame conditions as recommended by manufacturer. Micro ion-exchange column: 200 ~tl volume, produced from an Eppendorf pipette tip as described in reference [31] and shown in Fig. 4.1 c, packed with 125-177 ~tm particle size CPG-8HQ ion-exchanger (Pierce Chemicals).
Reagents and Standard Solutions
• • •
0.5 M ammonium acetate buffer solution adjusted to pH 9 with ammonia liquor. 2 M hydrochloric acid eluent Cadmium standard series of 0. 10. 20, 30. 40, 50 llg 1- 1• made by diluting I 00 mg 1- 1 standard stock solution with 0.001 M nitric acid. ·
Sample Treatment
Urine samples are acidified on collection to contain I% (v/v) nitric acid. The dilution effect is compensated for in the final result by a correction factor. Before the determination, the samples (approximately 10 ml) are adjusted to pH 3 by adding a few drops of concentrated ammonia liquor. The small dilution effect from variation in the sample volume due to the addition of base is neglected. Fl Manifold and Operation
The FI ion-exchange preconceniration manifold is shown in Fig 9.5 together with the experimental parameters. 0.5 mm i.d. PTFE tubes are used for all connections except the connection between the valve and nebulizer which is 0.35 mm i.d..
95 Selecled Proudurr.r
237
Water ml/min
E
a
w
c
[:1:
s B
p,
v
Water
ml/mln E
P:t o. Pa
b
w
c
s B
p,
v
F"ag.9.5: FI manifold for the FAAS detennination of cadmium in urine with on-line ion exchange column preconcentration. a. sample loading sequence: b. elution sequence. PI· P2: peristaltic pumps; E. 2 M HCI eluent; S, sample: B, 0.5 M ammonium acetate buffer, pH 9; C, conical ion-exchange column packed with CPG-8HQ: V, injector valve; W, waste; and AAS. flame AA detector (17].
238
9 Clinical and Phormaceutica/ Applications
During the 40 s sample loading period, the standard or urine sample is pumped through the conical micro-column, entering from the thinner end, after merging with the ammonium acetate buffer. Simultaneously. de-mineralized water is being aspirated from a container by the nebulizer suction to establish the baseline of the readout. The eluent pump (P 1) is stopped during this sequence. At the beginning of the 20 s elution stage. the valve position is changed and the eluent pump PI is actuated to effect a countercurrent elution into the spectrometer. Simultaneously, sample change is achieved via pump P2 (the flow-rate is lowered in this stage to save sample). Performance of Method
• • • • • • •
Sample volume: 2.5 ml Sampling frequency: 60 h- 1 Enrichment factor (EF): 30 Concentration efficiency (CE): 30 min-• Detection limit C3a): 0.3 JJg 1- 1 Precision: l.6'if r.s.d. (20 JJg 1- 1 level) Column life time: >300 samples
Validation of the Method
Certified value of standard reference urine sample GBW 09103: 0.053±0.003 mg 1- 1 Cd: found value: 0.051±0.002 mg 1- 1 Cd (n=3).
Note The capacity of the ion-exchange column is important for achieving good recoveries and sensitivity in the analysis of urine samples. Low capacities may result in low recoveries (e.g. 78% recovery for 100 JJl column) while large capacitic: ~:;;~ · :..1"e the EF value (e.g. EF = 21 for 400 pi column). 200 Jll columns give optimum overall performance.
References
Chapter 1 [1) J. Ruzicka and E. H. Hansen. FloM·-Injection Analy.fis, Second Edition, John Wiley & Sons. New York, 1988. (2) B. Karlbe~ and G. E. Pacey, FloM·-Injection Analysis - A Practical Guide. Elsevier. Amsterdam. 1989. (3) M. Valcarcel and M. D. Luque de Castro, Flow-Injection Anal)·sis: Applications. Ellis Horwood. Chichester, 1981.
-
Principle.~
and
(4] L. Skeus. Am. J. Clin. Path .. 28 (1957) 311. (5] Z-L. Fang. L-J. Sun and S-K. Xu. Anal. Chim. Acta. 26/ (19921 557. (6] J. Ruzicka and E. H. Hansen.
Flot~·
Injection Analysis. first edition. John Wiley and Sons.
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Anal. At. Spectrom., 7 (1992) 293.
(II] z-L. Fanr,. Spectrocllim. Acta Re,·.. 14 (1991) 235. (12) S.D. Hanstein. J. Ruzicka and G.D. Christian, Anal. Chem., 57 (19851 21.
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(25) J.A.G. Mesa. P.Linares, M.D. Luque de Castro and M. Valcarcel, Anal. Chim. Acta, 235 (1990) 441. 126] R.H. A&allah. J. Ruzicka and G.D. Christian, Anal. Chem .. 59 (1987) 2909. (27) R.H. Atallah. G.D. Christian and S.D. Hartenstein, Analyst, J/3 (1988) 463. 128) Y. Sahlestrom and B. Karlberg, Anal. Chim. Acto. 179 (1986) 31.5. 129) G~ Audunsson .. Anal. Chem .• 58 (1986) 2714.
(30) R.G. Melcher. Anal. Chim. Acto. 214 (19H8) 299. (31) C. Thommen. A Fromageat. P. Obergfell and H.M. Widmer. Anal. Chim. Acta. 234 (1990) 141. (32) M. Bengasson and G. Johansson, Anal. Chim. Acta, 158 (1984) 147. (33) D. C. Shelly. T. M. Rossi and l.M. Warner, Anal. Chem .. 54 (1982) 87. (34) T. M. Rossi. D.C. Shelly and l.M. Warner, Anal. Chem .. 54 (1982) 2061: (35) M. Valcan:el and M. Gallego, Chapter 5 in FIDM• Injection Atomic Spectroscopy, editor, J.L Burpera. Marcel Dekker, New York. 1989.
(36) J.F. Tyson, Sp«trochim. Acta Rn·., 14 (1991) 169. (37) J.A. Swcileh llld F.F. Cantwell, AMI. Chem., 57 (1985) 420. (38) L Nord and B.
KarlberJ, Altai. Chim. Acta, 145 (1983)
151.
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250
Rifer~nces
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z. Anal. Chem .. 329 (1988) 728.
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[42] X. Wang and Z-L. Fang, Fenxi Huaxue, 14 (1986) 738. [43] J.R. Ferreira, E.A.G. Zagatto, M.A.Z. Arruda and S.M.B. Brienza, Analyst,Jl5 (1990) 779. [44] M. Silva, M. Gallego and M. Valcarcel, Anal. Chim. Acta, 179 (1986) 341. [45] JJ. Sullivan, T.A. Hollingworth, M.M. Wekell, R.T. Newton and J.E, Larose. J. Assoc. Off. Anal. Chem., 69 (1986) 542. (46] W. Kunnecke and R.D. Schmid, Anal. Chim. Acta, 234 (1990) 213. (47] W. Kunnecke and R.D. Schmid. J. Biotechnol.. 14 (1990) 127. [48] Y. Sah1estrom, S. Twengstrom and B. Karlberg. Anal. Chim. Acta. 187 (1986) 339. [49] P. Linares, M.D. Luque de Castro and M. Valcarcel, Anal. Chim. Acta, 225 (1989) 443. [50) J.F. van Staden. Anal. Lells ... /9 (1986) 1407. [51] S-H. Fan, Shipin yu Fajiao Gongye. 5 (1990) 76. [52] J.F. van Staden and A. van Rensburg, Analyst. Jl5 (1990) 605. [53] J.F. van Staden and A. van Rensburg. Fresenius J. Anal.
Ch~m.,
337 (1990) 393.
[54] P. Marstorp, T. Anfalt and L. Andersson, Anal. Chim. Acta. 149 (1983) 281. [55] H. Lundback and B: Olsson, Anal. Le11s. 18 (1985) 871. (56] P. Macheras, M. Koupparis and C. Tsaprounis,Jnter. J. Plwrm., 33 (1986) 125. [57] L-J. Sun. L. Li and Z-L. Fang, Turang Tongbao, 17 (1986) 37. (58] J. Szpunar-Lobinska and M. Trojanowicz, Analyst, 115 (1990) 319. [59) J.A.G. Mesa, P. Linares, M.D. Luque de Castro and M. Valcarcel, Anal. Chim. Acta, 235 (1990) 441.
Chapte1 9 [l] B. ¥.:etz, S-K. Xu ~,ad M. Sperling, Appl. Speorosc. 45 ( 1991) 1433.
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.";jh
[4] K. Ktmura. S. Iketani, H. Sakamoto and T. Shono, Anal. Sciences. 4 (1988\ 221. (5) K. Kimura. S. Iketani. H. Sakamoto anc: T. Snono, Analyst. J/5 (1990) 125i. [6] R.Y. Xie and G.D Christian, Ana' r:.,"n. 58 (l986) 1806. [7) B. Welz and M. Schubert-Jacobs.
Atr"IL
[8] K. f\1-:Laughlin, D. Dadgar. M.R.
~myth
Spcrtmsc .. 12 (1991) 91. a;.d D. ~-1cMaster, Analyst, JJ5 (191)0\ '275.
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Index
acetoae. determination in milk, by gas-diffusion. spectrophotometry 144,208 alumina. activated 102 - preconcentration of lead 110 aluminium, determination - by solid phase absorptiometry I 28 amines, determination - in urine, by GLC. with on-line liquid-liquid exttaction clean-up 225 ammonia, determination - in blood, by gas-diffusion. spectrophotometty 224 - by ion-selective electrode." with gas-diffusion separation 146 - indirect, by FAAS with on-line precipitation 190 ammonium nitr~en, determination - in waters. by ion-selective electrode, with gasdiffusion preconcemratioil 203 - in waters; by spectrophotometry. with gas-diffusion preconcentration 140 - in waters. by spectrophotometry. with ga.~-dif fusion separation 203 - in Kjeldahl digests. by conductimetr)'. with gas-diffusion separation 204 amperometry - gas-diffusion separation for 147 anaesthetics, local, determination in pharmaceutical preparations, by liquid-liquid extraction spectrophotometry 227 - in pharmaceutical preparations: by indireCt FAAS, with on-line precipitation 190 antimony, determination - by HGAAS, with column preconcentration 113 - in plant tissues, by HGAAS 204 - in thermal water, by HGAAS I 97 arsenic, determination - by hydride generation ICPES 151 f - by liquid-liquid exttaction hydride generation ICPES 81 - in plant tissues, by HGAAS 204,218 ff - in plant tissues, by ICPES 204 - in sea waters, by HGAAS 154, 197, 198
- in sea waters, by ETAAS with sorption column separation 198 - in soils, by HGAAS 209,218 ff - in thermal water, by HGAAS 197 - in urine. by HGAAS 224 in waste waters, by HGAAS 198 atomic absorption spectrometers - dispersion in 41 - effects of sample introduction rate 41 atomic absorption spectrometry, name - column preconcentration systems for 105 ff - -performance of 110 - coprecipitation preconcentration system for 179 f - Donnan dialysis preconcentration for 167 - enhancement of performance by FIA 40 - indirect determinations with on-line precipitation 188 ff - interference reduction by on-line precipitation 193 - liquid-liquid extraction preconcentration systems for 76 ff - - performance of 79 atomic absorption spectrometr)'. electrothermal - colurim preconcentration systems for 114 f - coprecipitation preconcentration systems for 180 ff - liquid-liquid extraction preconcentration for 80 - - performance of 119 atomic absorption spectrometry, hydride (vapour) generation see hydride generation AutoAnalyser 1 automated solution analysis 2 - classification of 2 bismuth, determination - by HGAAS with column preconceniratiorl 112f. - in plant tissues, by HGAAS 154, 205 - in urine. by ICP-MS with column preconcentration 225 - in waters, by HGAAS 154
254
Index
beryllium, detl.'rmination - by solid phase fluorimetry 128 boron, determination - in natural water, by spectrophotometry, with column preconcentration 121 f. 198 cadmium, determination - in blood. by FAAS with on-line coprecipitation 195.221,232 ff - in bovine liver, by FAAS with on-line coprecipitation 195 - in plant tissues. by FAAS with on-line coprecipitation 195. 205 - in sea water. by ETAAS with column preconcentration 199 - in sea water, by FAAS with column preconcentration 198 - in urine. by FAAS with column preconcentration 225. 236 ff - in urine. by FAAS with on-line coprecipitation 195. 225. 232 ff - in urine, by liquid-liquid extraction spectrophotometry 224 calcium. determination - by FAAS. with on-line precipitation 194 - inmilk 208 carbon dioxide, determination - in blood. by gas-diffusion spectrophotometry 231 ff - in human plasma. by gas-diffusion spectrophotometry 224 - in wines. by gas diffusion spectrophotometry 207 .carboxylic acid druKS- determination - by liquid-liquid extraction spectrophotometry 226 carrier stream, definition 5 C 18 bonded Silica gel COlumn SU also ion• exchangers - preconcentration of heavy metals 100. 110 - preconcentration of heavy metals in sea water 198 ff.215 ff - in solid phase absorptiometry 125 f Cbelex-100 column see also ion-exchangers - in A preconcentration 110 f - preeoncentration of heavy metals in sea water 198 Chemlfolds 37 chemiluminescence detecton 44 - column preconcentration systems for 123 - gas-diffusion separation with 145 f chloride. determtaatioa - indirect. by FAAS. with on-line precipitation 177. 190 - in milk. by ion-selective elcc:trode, with online dialysis 167.208 chloriae diuide, - by gas diffusion chemilumineseenee 144 ff
detenliut._
chlorine, free, determination - in water. by gas-diffusion chemiluminescence 146 - in water. by ion-selective electrode, with gasdiffusion separation 146 chlorohexidine, determination - in pharmaceutical preparations, indirect. by FAAS. with on-line precipitation 190 chromium, determination - in fresh water. by solid phase absorptiometry 199 - in urine, by ICP-MS with column preconcentration 225 cobalt. determination - in bovine liver, by FAAS with on-line coprecipitation 205 - by FAAS, with on-line precipitation 194 - in oyster tissues, by FAAS with on-line coprecipitation 195 - in plant tissues. by FAAS with on-line coprecipitation 205 - in sea water. by ETAAS with column preconcentration 199 - in sea water, by FAAS with column preconcentration 199 - in sea water, by catalytic spectrophotometry with column preconcentralion 199 - in urine. by FAAS with on-line coprecipitation 195,225 - in urine. by JCP-MS with column preconcentration 225 codeine, determination - by liquid-liquid extraction spectrophotometry 227,229 ff column separation and preconeentration systems see sorption column systems columns, sorption - conical 91 f - designs of 90 ff - packings for 98 ff - -requirements of 98 - with push-fit coMections 91 f - with threaded fittings connections 91 f
concentration efliciency (CE) -
definition 13 of liquid-liquid extraction FAAS system 79 of precipitation FAAS systems 192, 194, 195 of sorption column ETAAS systems I 19 of sorption column FAAS systems I 10 of sorption column ICPES systems Ill of sorption column spectropho&omettic systems 122 - of sorption column vapour pneration AAS systems 113
COIIdlldimetry - ps-diffusion sepualion for 147 COIIIIIden 35 f - threaded fminp 35 f
Index
- push-fit 35 f consumptive index (Cf) - definition 14 - equation for 18 - of liquid-liquid extraction FAAS systems 79 - of precipitation FAAS systems 192. 194, 195 - of sorption column ET AAS systems 119 - of sorption column FAAS systems 110 - of sorption column ICPES systems Ill - of sorption column spectrophotometric systems 122 - of sorption column vapour generation AAS systems 113 continuous no"· analysis - air-segmented systemo I f - non-segmented. development of 2 copper, determination - by FAAS. with on-line precipitation 194 - by ion-selective electrode. with column preconcentration 124 - in plant tissues. by FAAS with on-line coprecipitation 205 - in sea water. by FAAS and ET AAS with column preconcentration 200 - by solid phase absorptiometry 127 - in urine. by JCP-MS with column preconcentration 225 coprecipilation systems - filterless. with precipitate dissolution 179 f - - determination of lead and cadmium in blood and urine by FAAS 233 ff - - ET AAS detection 180 ff - - FAAS detection 179 f - kinetic effects in 183 f - preconcentration methods 191, 195 CPG/8Q ion-exchange column see also ionexchangers - preconcentration of heavy metals I 06. II 0 f, 113. 124 - -heavy metals in sea water 198 ff cyanide, determination - simultaneous with thiocyanate, by spectrophotometry. with gas-diffusion separation 143 - in natural and waste waters. by spectrophotometry with gas-diffusion separation 139,
202
- as sample loop 164 f dispersion - in FAAS detectors 41 - conduit inner diameter influence on 9 - conduit length influence on 9 - in Fl systems 6 - flow-rate influence on 8 f - - comparison to batch systems 6 f - in knotted reactors 173 - m liquid-liquid extraction Fl systems 61 ff - sample volume influence on 9 - in sorption column systems 87 ff dispersion coefficient - definition 7 - evaluation of 7 - classification 8 displacement bottle for organic soh·ent delh·ery 23 distillation, isothermal 130 drugs, dissolution of · - in milk, by spectrophotometry. with on-line dialysis 209 elution in column systems 88 f - direction of. 97 - dispersion in, 88 - eluent requirements 96 f - now-rate requirements for 97 enhancement factor (N) - definition 13 - equation for 18 - evaluation of 16 f - in liquid-liquid extraction FAAS 79 enrichment factor (EF) - definition 12 - equation for 18 - of Donnan dialysis FAAS system~ 165 - relationship with enhancement factor 13 - of sorption column ETAAS systems 119 - of sorption column FAAS systems I 10 - of sorption column ICPES systems Ill - of sorption column spectrophotometric systems 122 - of sorption column vapour generation AAS systems 113 ethanol, determinatloa - in beverages, by gas-diffusion amperornetry
206
dialysis sylleiDS - deve~entof JS9
- Donnan 165 f - manifold. basic 164 - manifold. with dialyzer as sample loop 164 f - nws transfer in JS9 f - for spectrophotometry 166 f dialysis factor I 60 - effecu of experimenlal factors on 160 f ....,.,. 162f - membranes for 163 f
255
filters " ' precipitate collectors
now-lnjectioD aulysls basic system 4 definition 4 f development of I ff
-
preconcentration systems see separation systems - principles 4 ff - publications, cumulative number of 2 f
Index
- separation systems 10 ff - - c:haracteristic:s of 10 - - classification of 11 f - - efficiency evaluation of 15 ff - - mass transfer in 11 f - - mathematic:al model for 15 ff ftuorlde, determination , - indirect ICPES method, with liquid-liquid extraction 80, 202 - by ion-selective electrode, with column preconcentration 124 - by solid phase fluorimetry 128 nuorimetry 45 - solid phase 128 galactose. determination - in urine, by amperometry with on-line dialysis and column separation 225 gas-diffusion separation systems - basic manifold 138 - for determination of carbon dioxide in blood 231 ff - for determination of total nitrogen in soils 212 ff - development of 130 f - with dual-phase membrane separators 152 - for electroc:hemic:al detectors 146 ff - for prec:onc:entration 138 ff - for spectrophotometry 142 ff gas-diffusion separators 131 f - dual phase 151 f - integrated reaction-separation system 153 - integrated reaction-separation-detection system 156 - mic:roporous membranes for 134 f - sandwich type 131 f - - as degasser 134 - tubular type 133 gas-expansion separators 135 ff gas-liquid separation systems - classification of 130 f gas-liquid chromatography - coupling of FI liquid-liquid extraction to 81 f high performance liquid chromatography - comparison with FIA 18 f - coupling of FI liquid-liquid extraction to 83 hydride generation systems see also vapour generation systems - basic manifolds for, in AS 149 - column preconcentration systems for 112 f - design of 149 ff - determination of As and Se in soils and biological materials 218 - development 148 - integrated reaction-separation system for 152 - interferent removal for, by ion-exchange . 104f
- interferent, tolerance to 135 - performance of, with AAS detec:tion 154 f indirect methods - by liquid-liquid extraction FAAS 79 f - by precipitation FAAS 188 ff induction coupled plasma emission spectrom· etry - ion-exchange c:Oiumn prec:onc:entration system for 105 f. Ill - liquid-liquid extraction system 80 f induction coupled plasma mass spectrometry - with column prec:onc:entration 201 - combination with Fl. potentials of 42 injection valves 29 ff - bypasses in 33 f - commutator 33 - rotary, 8-c:hannel I ~pon 30 f - -column (dual) prec:onc:entration with 32, 108f - -gas-diffusion prec:onc:entration with 140 f - - merging zones operation with 32 - -time-based sampling with 31 - - volume-based sampling with 31 - rotary, six-pon 29 ion-exc:bangen 98 ff - chelating 98 f - - 122 salicylic: functional resin 99 - - Amberlite IRA-743 99 - - Chelex-100, propenies of 98 - - CPG-8Q, propenies of 99 - - Muromac A-I 99 iodide, determination - by indirect FAAS, with on-line precipitation 167. 190 ion-selective electrodes - advantages of combination with FIA 44 - column prec:oncentration systems for 123 f - dialysis separation for 167 - with gas-diffusion separation 146 f - solid membrane, flow-through 43 f - wall-embedded, tubular 42 f knotted reactors 36 f - decrease of dispersion in 173 - precipitate collection with 36, 173 f. 179 ff - Schlieren effect elimination with 39 lactose, determination - in milk, by amperometry, with on-line dialysis 208 lead determination - in blood, by FAAS with on-line coprecipitation 195, 222 - in bovine liver, by FAAS with on-line .coprecipitation 205 - in sea water, by ETAAS with column precon" centration 200, 215 ff
ltulex
- in sea water, by FAAS with column preconcenuaaion 200 - in tap water, by FAAS with on-line ~cipilll· tion 194 liquld·liquid extraction systems 47 ff - components of 63 f - determination of anionic surfactants in waters 213 ff - determination of codeine in pharmaceutical preparations
229 ff
-
delivery of separated phase 10 detectors 69 f derivatization ill 70 r detectors. coupling to 74 ff dispersion mechanism in 61 ff ellil modes for 68 r elltraction coils for ~2 - iftleJralcd 59 - iterative flow-~versal in 71 f - merit~ of A ~ysaem 47 - manifolds for 63 - - in speclrophotomeny 74 --in FAAS 76ff - multiple-slaJe 73 - non-segmented. using membranes 66 r - perforrnancr of, in spectrophotometry 75 f - phase separators stt phale aeparators - phase transfer factors in Fl systems 48 - phase transfer mechanism 59 ff - sample introduction in Flsysaems 64 f - segmentation modes for 65 r - sel!mentors stc phase segmenturs - with solvent circulation 65 f - without phase separation 67 r lithium. delerMillation - in blood. by liquid-liquid extraction with.ionsel«aive electrode 222 - in blood. ~· liquid-liquid extraction with spectrophotometry 222 IUSI spectrallldry - Jas-diffusion separator as interface for FIA 147 -annsfer - in A dialysis 159 f - in Fl separation 10. 14 - in sorption columns 93 - in JIS diffusion separation 140 f - in liquid-liquid extraction 48, 59
..........
- for gas-diffusion separators 134 f - -properties RqUiRd 135 - for liquid-liquid extraction separators 58
.-rcu.,, •ea~....._
- by cold vapour AAS 156 f - by cold vapour AAS with column ~oncentration 112 f - in sea water. by cold vapour AAS with column preconcentraaioo 201
257
- in urine, by JCP-MS with column prec:oncenlration 225 mixiiiJ reactors see olso tnnsport conduhs 35ff - knoned stt also knotted reactors 36 f - dimensions of, in precipitation systems 187 r - sinJie bead pearl strinl! 37 IDOiybdenum. delermiutialt - in plant tissues, by catalytic spectrophotome· try, with ion-exchan,e separaaion 205 - in plant tissues. by liquid-liquid extraction spectrophotometry 205 - in urine, by ICP-MS with column preconcenaralion 225 Muroaw: A·l iall-adluie C'OIII- su Glso ioDoeXdlaiiJen - in ~oncentration of llavy metals 110 r
nickel. dele..-llllliaa - in blood. by ETAAS. with on-line coprecipitatioa 222 - in plant tissues. by FAAS with on-line c:oprecipitation 195, 205 - in sea water, by ETAAS with c:olumn preconcentration 201 - in urine. by FAAS with on-line c:oprecipitatioa 19S. 225 - in urine, by JCP-MS with column preconc:entration 225 nitrate, deteratinalloa - in meat (with nitrite) by liquid-liquid extrac· aion spectrophotometry 206 Ditrogea, total, determilllllloll - in soils. by ga.'i·diffusion spectrophotometry 209.211 ff optical fibers, - in opcosensing J44 optoseesing systems - l!BS-diffusion )44 f - solid phase 124 r - - desip of 125 f - - sorption preconcemratioa for 124 f oxalate, dettnn....._ - by FAAS, with on-line pncipilalion 190 perdllorate, ~ - in serum and urine, by iDdircct FAAS with liquid-liquid extraction 223 peristaltic ....... - characteristics of 22 - flow-rate adjustments for 25 - proper usa,e of 24 - pulsations, elimination of 25 - pump tubes for 22 ff - - colour coding of 25 - organic solvent delivery with 22 f
258
Index
phase segmentors, for !iquid-liquid extraction 4Kff - coaxial 49 ff - -factors influencing performance of. 51 - falling drop see coa.xial - - dual channel 51 f - merging tube (T-tube) 48 f pbase separators, for liquid-liquid separation - column type 58 - gravitational, sandwich type 54 - gravitational, T-tube type 53 - membrane-sandwich type 55 ff - -geometrical aspects of 55 ff - - merits of 57 pbase transfer - in liquid-liquid extraction - - mechanism 59 ff pbase transfer factor (P) - definition 14 - in dialysis Fl systems 160 - in liquid-liquid extraction Fl systems 47 pbenol, determination - poly-, in olive oils. by liquid-liquid extraction spectrophotometry 210 - in water, by liquid-liquid extraction spectrophotometry 204 pbospbate, determination - by solid phase absorptiometty 126 - in sea, ground and rain waters, by solid phase absorptiometr)· 202 platinum, determination - in urine, by ICP-MS with column preconcentration 225 polymer sorbents - Amberlite XAD-4, preconcentration of heavy metals with I 00 - Amberlite XAD- 7. in solid phase fluorimetry 128 - Amberlite XAD-8, preconcentration of gold with 100 precipitate - dissolution flow-rate. for FAAS 188 - dissolution of, kinetic effects in 184 - forms, in on-line precipitate collection 185, 187 precipitate collectors, on-line 170 ff - choice of 174 - disposable membrane filters 172 - general requirements 170 - impedance of 187 - knotted reactors 173 - packed bed filters 172 - stainless steel filters 171 precipitation systems see also copredpitatlon systems - development of 169 - FJ variables for 186 - filterless, without precipitate dissolution 178
- kinetic effects in precipitation 183 f - kinetic effects in precipitate dissolution 184 - on-line filtration without dissolution 175 ff - on-line filtration with dissolution 177 f - preconcentration methods 191 ff, 194 preconcentratioo - by coprecipitation 179 ff, 191 ff - by Donnan dialysis I 65 f - by gas-diffusion I 38 f - by liquid-liquid extraction, for FAAS 79 - by precipitation 177. 191 ff - by sorption columns 87 ff pumps - technical requirements of. in FlA 2 I f - peristaltic sec peristaltic pumps - reciprocating see reciprocating piston pumps - syringe see syringe pumps, sinusoidal reciprocating piston pumps - advantages and disadvantages of 26 restrictors for FI liquid-liquid extraction 69 reversed FIA (rFIA) 6 - comparison of sensitivity with nFlA 189 - with on-line precipitation 175 f, 186, 189 sample loading - dispersion in. for sorption column systems 87 r - in precipitation systems 186 f - in sorption column preconcentration systems 93 ff - - carry-over in 94 - time-based 15 - -with 8-channel 16-port valve 31 - - in gas-diffusion preconcentration systems l39f - - in precipitation preconcentration systems 177 - - in sorption column preconcentration systems 94 - volume-based 15 - -with 8-channel 16-port valve 31 Schlieren effects 38 ff - compensation of 38 - - in column systems for spectrophotometry 120f - generation of 38 f - in sorption column systems 96 segmentors see phase segmentors selenium, determination - in blood, by HGAAS 222 f - by HGAAS, with column preconcentration 112 f - in plant tissues, by HGAAS 20S, 218 ff - in soils. by HGAAS 209,218 ff silver, determination - by FAAS, with on-line precipitation 194 solid phase absorptiometry 126 f - immobilized reagents in 127
lnde:r
solid phase nuorimetry 128 solvent extraction see liquid-liquid extraction sorbent extraction see also sorption column systems and C11 bonded silica gel - with C IX bonded silica gel and XAD polymer sorbents I00 - determination of traee metals in sea water by ETAAS 215 ff sorption column systems 85 ff - for chemiluminescence 123 - classification of 86 - components of I03 - determination of cadmium in urine by FAAS 236ff - determination of trace metals in sea water by ETAAS 215 ff - development of 85 - dispersion in 87 rr - elution in 96 f - forETAAS 119 - for FAAS 105 ff, I 10 - -manifolds for preconcentration I 06 ff - for hydride generation AAS 113 - for JCPES 105 ff, I II - for interferent removal 103 rr - -manifolds 103 ff - for ion-selective electrodes 123 r - with on-line precipitation 178 - performance of - - sample loading in 93 rr - special requirements for 86 - for spectrophotometry 120 ff - -manifolds 122 - washing columns in, 95 f - waste discharge in, for FAAS 95 spectrophotometers - flow-cells for 38 r spectrophotometry - gas-diffusion separation systems for 142 ff - liquid-liquid extraction systems for 74 ff - sorption column systems for 120 ff sulfate, determination - by indirect FAAS, with on-line precipitation 190 sulfide, determiaatioa - by gas-diffusion spectrophotometry 143 - by indirect FAAS, with on-line precipitation 169, 178, 190 sulfite, detenDiutioa - in food. by gas-diffusion spec:trophotometry 206 ...plloaamides, determiaatloa - in pharmaceutical preparations, by liquid-Jiq-
259
uid extraction spectrophotometry 226 - in pharmaceutical preparations and urine, by indirect FAAS, with on-line precipitation 190
sulphur dioxide, determination - in wines, by gas-diffusion spectrophotometry 207 surfactants, determination - anionic. in river and waste waters. by liquidliquid extraction spectrophotometry 202 f. 213 ff - cationic, in natural. tap and waste waters by liquid-liquid extraction spectrophotometry 203 - non-ionic, by spectrophotometry, with column separation 203 syringe pumps. sinusoidal - advantages and disadvantages of 27 - principles of operation 27 f tin, determination - in foods, by HGAAS with column preconcentration 113 time-based sampling see sample loading transport conduits see al.fo mixing reactors 35 ff - dimensions of, in FIA 35 uranium, determination - by spectrophotometry, with column preconcentration 120 urea, determination - ·in blood, by gas-diffusion spectrophotometry 223 U-tube gas-liquid separator 136 f vanadium, determination - in urine. by ICP-MS with column preconcentration 225 vapour generation systems set also hydride generation systems - gas expansion separators for 135 ff - integrated reaction-separation-detection system for 156 vitamin a•• determination - in pharmaceutical preparations, by liquid-liquid extraction spectrophotometry 227 volume-based sampliDI see sample loading zinc, determiaatioa - in plant ash extracts, by spectrophotometry. with ion-exchanse separation 121,206 - in sea water, by FAAS, with ion-e:xchanp preconcentration 110
