Chemical Reactions in Gas, Liquid and Solid Phases: Synthesis, Properties and Application (Chemistry Research and Applications)

CHEMISTRY RESEARCH AND APPLICATIONS

CHEMICAL REACTIONS IN GAS, LIQUID AND SOLID PHASES: SYNTHESIS, PROPERTIES AND APPLICATION

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CHEMISTRY RESEARCH AND APPLICATIONS

CHEMICAL REACTIONS IN GAS, LIQUID AND SOLID PHASES: SYNTHESIS, PROPERTIES AND APPLICATION

G. E. ZAIKOV AND

R. M. KOZLOWSKI EDITORS

Nova Science Publishers, Inc. New York

Copyright © 2010 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Chemical reactions in gas, liquid, and solid phases : synthesis, properties, and application / editors, G.E. Zaikov, R.M. Kozlowski. p. cm. Includes index. ISBN 978-1-61668-906-3 (eBook) 1. Polymers--Biodegradation. 2. Composite materials--Biodegradation. I. Zaikov, Gennadii Efremovich. II. Kozlowski, R. (Ryszard) QD381.9.D47C54 2009 541'.39--dc22 2010015588

Published by Nova Science Publishers, Inc. Ô New York

CONTENTS Preface

ix 

Chapter 1

Classification of Polymers in Reactivity Toward Nitrogen Oxides E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii, T. V. Pokholok and G. E. Zaikov 

Chapter 2

Influence of the Initiation Rate of Radicals on the Kinetic Characteristics of Quercetin and Dihydroquercetin in the Methyl Oleate Oxidation L. I. Mazaletskaya, N. I. Sheludchenko and L. N. Shishkina 

Chapter 3

Chapter 4

Chapter 5

Chapter 6

Chapter 7

An Antioxidant from Hindered Phenols Group Activates Cellulose Hydrolysis By Celloviridin in a Wide Concentration Range, Including Ultralow Doses E. M. Molochkina, Yu. A. Treschenkova, I. A. Krylov and E. B. Burlakova  a-Tocopherol as Modifier of the Lipid Structure of Plasma Membranes In Vitro in a Wide Range of Concentrations Studied by Spin-Probes V. V. Belov, E. L. Maltseva and N. P. Palmina  Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized in N-Methylpyrrolidone Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko and Maria Bruma  Inhibition of 2-Hexenal Oxidation By Essential Oils of Ginger, Marjoram, Juniper Berry, Black and White Pepper T. A. Misharina, M. B. Terenina, N. I. Krikunova and I. B. Medvedeva  The Organophosphorus Plant Growth Regulator Melaphen as Adaptogen to Low Moisher I. V. Zhigacheva, E. B. Burlakova, T. A. Misharina, M. B.Terenina, N. I. Krikunova, I. P. Generozova and A. G. Shugaev 

1 

11 

21 

29 

45 

65 

75 

vi Chapter 8

Contents Antioxidant Properties of Essential Oils from Clove Bud, Laurel, Cardamom, Nutmeg and Mace T. A. Misharina, M. B. Terenina, and N. I. Krikunova 

Chapter 9

Specific Properties of Some Biological Composite Materials N. Barbakadze, E. Gorb and S. Gorb 

Chapter 10

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii, T. V. Pokholok, and G. E. Zaikov  

Chapter 11

Chapter 12

Chapter 13

Chapter 14

Chapter 15

Chapter 16

91 

123 

Synthesis of Flexible Manufacturings for Phosphoric Industry Waste Utilization Based on the Cals-Concept A. M. Bessarabov, A. V. Kvasyuk and G. E. Zaikov 

155 

Practical Hints on the Application of Nanosilvers in Antibacterial Coating of Textiles S. Dadvar, A. Oroume and A. K. Haghi 

165 

The Nanostructure and Yield Process of Cross-Linked Epoxy Polymers Z. M. Amirshikhova, G. V. Kozlov, G. M. Magomedov and G. E. Zaikov  Nanostructures in Cross-Linking Epoxy Polymers and Their Influence on Mechanical Properties Z. M. Amirshikhova, G. V. Kozlov, G. M. Magomedov and G. E. Zaikov   The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers E. V. Kalugina, N. V. Gaevoy,   K. Z. Gumargalieva and G. E. Zaikov  Quantum-Chemical Calculation of Olefins of Cationic Polymerization Branching in -, - and  Position on Relations to Double Connection By Method MNDO V. A. Babkin, D. S. Andreev, T. V. Peresypkina and G. E. Zaikov  

Chapter 17

Thermodynamics for Catalase and Hydrogen Peroxide Interaction A. A. Turovsky, A. R. Kytsya, L. I. Bazylyak and G. E. Zaikov

Chapter 18

Dr. Rer. Nat. Wolfgang Fritsche – Scientist and Organizer of International Science (Secretary General Rtd. of Gesellschaft Deutscher Chemiker, Honorary President of Federation of European Chemical Societies) G. E. Zaikov  

Chapter 19

83 

Professor Victor Manuel De Matos Lobo on His 70th Anniversary Gennady E. Zaikov and Artur J. M. Valente  

191 

197 

205 

221  227 

245  249 

Contents

vii

Chapter 20

The Scientist Who Outstripped His Time Revaz Skhiladze and Tengiz Tsivtsivadze  

Chapter 21

Prof. Dr. Ryszard Michal Kozlowski: Half a Century in Science and Technology Gennady Zaikov 

263 

The Second International Conference on Biodegradable Polymers and Sustainable Composites (BIOPOL-2009) G. E. Zaikov, L. L. Madyuskina and M. I. Artsis  

267 

Chapter 22

Index

251 

271 

“If you are sixty (or more) and you are not feeling any pain in your body when you are getting up in the morning, it means that you have passed away (already)” Russian proverb

PREFACE This epigraph is very correct for the Russian Federation today because the average lifespan of Russian men in this country is 57 years (Russian women are living on ten years more). It is statistical data. Russian scientists are an exception. Particularly, the majority of contributors of this volume are older then 60 and the editor of volume is older then 75. We can explain this exception (phenomena) if we take in account the next Russian proverb: “All illnesses are from nerves and only a small amount from pleasure”. We expect that readers immediately are thinking about sex as a part of pleasure. It is only partly right. Alcohol, tobacco and narcotics should also be included in the “pleasure” part. Unfortunately, the Russian people are world champions for drinking. The average Russian man (including ladies, children and even babies) drinks 18 liters of pure ethyl alcohol (calculation done in pure alcohol) per year. It is twice more than twice the critical amount (9 liters). Russian scientists are again an exception because the majority of them have pleasure only in the case of communication with SCIENCE! Now we should remember the Kazakh (people living in the Asian part of former USSR) proverb: “If sixty years are coming the mind (brain, memory) will go back (to childhood conditions)”. We expect that this proverb is also not correct for Russian scientists. As evidence of this opinion you can read the chapters of this volume where the most part of chapters were prepared by scientists from Russian Research Centers and from Research Centers of former Republics (now independent states) of the USSR. It is now the right time to remember English proverb: “Please eat one apple every day and you will not need a physician” (it is a reverse translation from Russian to English). We do not know exactly if it is enough to eat one apple a day to be permanently healthy or not. We do know that positive emotions are in favor for good health. We expect that this book can (should) give only positive emotions to readers and we are waiting for the opinions of readers in this case. So, we should stop about proverbs and start about chemical science and application. This volume includes information about kinetics and mechanism of chemical reactions in different phases: classification of polymers in reactivity toward nitrogen oxides (polluted atmosphere), influence of the initiation rate of radicals on the kinetic characteristics of quercetin and dihydroquercetin in the methyl oleate oxidation, an antioxidant from hindered

x

Gennady E. Zaikov and R. M. Kozlowski

phenols group activates cellulose hydrolysis by celloviridin in a wide concentration range, including ultralow doses, α-tocopherol as modifier of the lipid structure of plasma membranes in vitro in a wide range of concentrations studied by spin-probes, supercritical carbon dioxide swelling of polyheteroarylenes synthesized in N-methylpyrrolidone, inhibition of 2-hexenal oxidation by essential oils of ginger, marjoram, juniper berry, black and white pepper, specific properties of some biological composite materials, the organophosphorus plant growth regulator melaphen as adaptogen to low moisher, properties and applications of aminoxyl radicals in polymer chemistry, synthesis of flexible manufacturings for phosphoric industry waste utilization based on the cals-concept, practical hints on application of nanosilvers in antibacterial coating of textiles, the degradation heterochain polymers in the presence оf phosphorus stаbilizers, quantum-chemical calculation of olefins of cationic polymerization and antioxidant properties of essential oils. The nanostructure and yield process of cross-linked epoxy polymers as well as nanostructures in cross-linking epoxy polymers and their influence on mechanical properties are discussed in this volume. Somebody asked Henry Ford: “Which car is better?” Ford answered: “A new one”. No doubt a new car as well as new scientific information is better than old ones (only old cognac is better than the new one). We took this idea into account in case preparation of our volume. So, this volume is a complete guide to the subject of kinetic and mechanisms of chemical reactions in gas, liquid and solid states. The editors and contributors will be happy to receive from the readers some comments which we can use in our research in the future. Once, Agatha Christie was filling out a form. There was a question: “What is your occupation” and she wrote “A married lady”. Of course Agatha Christie could write on any form whatever she wanted because everybody knew her. What is important for us is that in filling out any form we contributors could write with a clear conscience – “a scientist.” Gennady E. Zaikov N.M. Emanuel Institute of Biochemical Physics Russian Academy of Sciences 4 Kosygin Str., Moscow 119334, Russia Ryszard M. Kozlowski Institute for Engineering of Polymer Materials and Dyes Torun, Poland

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.1-10 ©2010 Nova Science Publishers, Inc.

Chapter 1

CLASSIFICATION OF POLYMERS IN REACTIVITY TOWARD NITROGEN OXIDES E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii, T. V. Pokholok, and G. E. Zaikov* N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia

ABSTRACT The review is includies information about the kinetics and mechanism of interaction of nitrogen oxides with carbon-chain polymers, rubbers, aliphatic polyamides and polyuretanes.

Keywords: nitrogen oxides, reactivity, polymers, classification, kinetics, mechanism.

The general review of influence of the pollutants on polymers has been presented by Jellinek et al. [1]. Therein the characterization of reactivity of polymeric materials toward aggressive gases is given. The various polymers were used as films of 20 μ thickness. The thickness is enough small to exclude in most cases the diffusion as the determining factor of the pollutant action. The films were investigated under different conditions: 1) the pollutant action; 2) the oxygen action; 3) UV light action; 4) UV light and oxygen; 5) UV light, oxygen and pollutants. For NO2, the exposure of samples was usually realized under the pressure of 15 mm Hg during 30 hours at 308 K. However, in a case of nylon 66 and butyl rubber, the NO2 pressure was lowered up to 1 mm Hg at during 30 min. Polyisoprene and polybutadiene were exposed to NO2 during 5 min under a pressure of 1 mm Hg. As a light source (λ > 290 nm), a mercury lamp was used. The intrinsic viscosity of polymer solutions was measured before and after exposure of samples in the chosen conditions. The rather high concentration * N.M. Emanuel Institute of Biochemical physics, Russian Academy of Sciences, 4 Kosygin Street, 119334 Moscow, Russia, Fax: (7-495)1374101, E-mail: [email protected] , [email protected]

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

2

of nitrogen dioxide in these experiences was used to be convinced that the certain effects can be observed for a reasonable time. The polymers on the basis of their reactivity with respect to NO2 can be divided into two main classes [1]. The saturated polymers, for instance, polyethylene (PE) and polypropylene (PP) belong to the first group, but nylon 66 is not included into this series. The second group covers elastomers. Butyl rubber undergoes scissions of the main chain, and polybutadiene is restrictedly cross-linked under the action of NO2. These elastomers have approximately the same reactivity with respect to NO2 as to ozone. All films exposed to NO2 become yellow, and their IR spectra show that nitro groups enter into macromolecules. In polyvinylchloride in the presence of NO2, some decreasing the amount of chlorine along with the appearance of nitro and nitrite groups are observed from IR spectra. It is the author’s opinion [1] that some estimations concerning influences of so low concentration of nitrogen dioxides in an atmosphere (2⋅10−9 − 2⋅10−8 mol⋅l−1) on polymeric materials can be obtained from the experiences with using concentrations of the gas of several orders of magnitude higher. The formulated assumption says that there is linear dependence of the concentration effect of aggressive pollutants. This means that the effect of aggressive gases at low concentrations can be determined by the linear extrapolation of results obtained under the influence of high concentrations. The author pointed out that this procedure contains an element of risk because scissions of macromolecules in some cases are not always linearly decreased with the pressure reduction of the aggressive gas, but the rate of breaks can change drastically at very low concentrations. The procedure of extrapolation was used for an estimation of the scission average number

S under the action of aggressive gases at concentrations of 1 − 5ppm within 1 hour [1]. This value is given by the equation: S= where

DPn ,0 DPn ,t

DPn,0

−1

and

(I.1)

DPn ,t are lengths of macrochains at t =0 and t correspondingly. On the

basis of these estimations, it was concluded that aggressive gases, for instance NO2 and SO2, slightly effect on vinyl polymers in concentrations really available in polluted air. Even in a combination with UV light, the deterioration of these polymers is hardly noticeable. However, nylon 66 is quite subjected to the action of small concentrations of NO2 with essential degradation.

I.1. INTERACTION OF CARBON-CHAIN POLYMERS WITH NO2 Pioneering studies of the reaction of nitrogen dioxide with polyethylene (PE) and polypropylene (PP) have been carried out by Ogihara et al. [2, 3]. Using IR spectroscopy, they have found that nitrogen dioxide cannot abstract secondary and tertiary hydrogen atoms from PE and PP at 298 K. It can only add to the vinylene and vinylidene units that are formed

Classification of Polymers in Reactivity Toward Nitrogen Oxides

3

in the synthesis of the polymers. These reactions resulted in dinitro compounds and nitro nitrites: H

C=C

+ NO2

C C NO2

(I.2)

H

C C

H

C C

+ NO2

O2N

NO2

(I.3)

H

NO2

C C ONO

NO2

(I.4)

At T > 373 K, nitro, nitrite, nitrate, carbonyl and hydroxy groups are formed in these polymers. The following reaction mechanism at high temperatures was proposed: RH + NO2 → R• + HNO2

(I.5)

R• + NO2 → RNO2

(I.6)

R• + ONO → RONO

(I.7)

RONO → RO• + NO

(I.8)

RO• + NO2 → RONO2

(I.9)

RH + RO• → R• + ROH

(I.10)

~CH2-CH2-CH2-O• → ~CH2-CH2-CHO + H

(I.11)

This scheme allows rationalization of the accumulation of the nitro groups, which proceeds at a constant rate and autoaccelerated formation of nitrates, alcohols and carbonyl compounds. However, it provides no explanation for S-shaped dependence of the accumulation of nitrites. The activation energies for the NO2 addition to the double bonds of PE are 8-16 kJ⋅mol−1. The activation energy for hydrogen abstraction is within of 56 and 68 kJ⋅mol−1 for PE and 60 kJ⋅mol−1 for PP. At room temperature and at NO2 concentrations of 5.4⋅10−4 – 5.4⋅10−3 mol⋅l−1, the characteristics of PE, PP, polyacrylonitrile and polymethylmethacrylate (PMMA) are changed only slightly even if they simultaneously undergo to a combined action of NO2, O2 and UV radiation [4]. Reactions of NO2 with polyvinylchloride and polyvinyl fluoride resulted in a slight decrease in the content of chlorine and fluorine atoms, respectively [1, 4].

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

4

In the temperature range of 298−328 K nitrogen dioxide (7.8⋅10−3−3.4⋅10−2 mol⋅l−1) can abstract tertiary hydrogen atoms from polystyrene (PS) molecules to introduce nitro and nitrite groups into macromolecules in result of subsequent reactions [1]. This process proceeds at low rates and is accompanied by chain scissions [1, 5, 6]. The number of chain scissions on time α(t) was determined from intrinsic viscosity using the equation (I.1). The experiments have been carried out at the temperature range of 298-328 K. According to Jellinek, the dependence of the decrease in the degree of polymerization of PS on the exposure time in NO2 has three linear regions: initial, middle and final. A decrease in the apparent degradation rate was observed in the middle region of the dependence. Presumably this was related to the association of the macromolecules in solution, which is due to the effect of polar groups and can affect the results of viscosimetric measurements. Subsequent increase in the apparent degradation rate was attributed to the consumption of these nitrogencontaining groups and to a decrease in the degree of association of the macromolecules. PS films were also simultaneously exposed to NO2 (1.1·10−4 mol·l−1) and light (λ > 280 nm) [6]. No polymer degradation was observed in the initial stage during 10 h. Then chain scission occurred at a constant rate. An attempt to determine quantitative characteristics of the ageing of PS and poly-tbutylmethacrylate (PTBMA) under the action of NO2 has been undertaken by Huber [7]. The samples were exposed to a stream of air containing NO2 (2.5⋅10−6 – 3.7⋅10−5 mol⋅l−1) at 300 K and simultaneously irradiated with light (λ>290 nm). The number of chain scission per 10000 monomer units α(t) can be described by the empirical equation:

α=

P (exp Qt − 1) Q

(I.12)

where P and Q are constants. This equation describes an autocatalytic process. At Q → 0, degradation occurs at a constant rate. Autocatalytic process is more pronounced for thin films. Degradation of thin PS films under the same conditions occurs slower than that of the PTBMA films and its autocatalytic nature is more pronounced. The autocatalytic path of degradation of PTBMA was associated [7] with the photoinduced formation of isobutylene, which reacts with NO2, thus initiating free-radical degradation processes of macromolecules. The IR spectrum of PS exposed toNO2 and light exhibits two bands at 1686 and 3400 cm−1 corresponding to the carbonyl and hydroxyl groups, respectively. The formation of nitrogen-containing products has not been observed in both PTBMA and PS. The following reactions have been proposed [7] in PS: ~ CH2−C(Ph)H ~ + NO2 → HNO2 + ~ CH2−C•(Ph) ~ (R1•)

(I.13)

R1• + O2 → R1O2•

(I.14)

R1O2• + RH → ROOH + R1•

(I.15)

Classification of Polymers in Reactivity Toward Nitrogen Oxides

R1 + NO2

R1ONO

hν

⎯⎯→

5

R1NO2

(I.16)

R1ONO

(I.17)

R1O• + NO

(I.18)

R1OOH + NO → R1O• + •OH + NO R1OOH

hν

⎯⎯→

(I.19)

R2• + •OH

(I.20)

R1O• → R2• + degradation products

(I.21)

It is believed that the decomposition of hydroperoxides exposed to NO2 and light leads to autocatalytic degradation of PS.

I.2. INTERACTION OF RUBBERS WITH NO2 Rubbers are much more susceptible to NO2 than the polymers containing no double bonds. First, this is due to the ability of NO2 to add reversibly to carbon-carbon double bonds to give nitroalkyl radicals (reaction (I.2)), thus initiating free radical conversions of elastomers. Second, nitrogen dioxide is able of abstracting hydrogen atoms in β−position to the double bond to give allyl radicals, which then recombine with NO2 [8]. Depending on the structure of the alkene, the reaction resulting in the formation of the allyl radical can be either weakly exothermic or weakly endothermic. For instance, the strength of the weakest C−H bond in the structure CH2=C(CH3)CH2−H is only 314 kJ·mol−1 [9]. The exposure of polyisoprene and polubutadiene to nitrogen dioxide leads to both degradation and cross-linking of macromolecules, whereas butyl rubber (BR) (a copolymer of 36% isobutylene and 54% isoprene units) only undergoes degradation [10]. The detailed study of the ageing BR exposed to NO2 (5.2⋅10−7 – 5.2⋅10−5 mol⋅l−1) alone, an NO2−O2 mixture and an NO2−O2 mixture plus UV light (λ > 280 nm) at 298−358 K has been performed by Jellinek et al.[11, 12]. IR spectra before and after the exposure of samples show that the band at 1540 cm−1 of ~ C=C ~ bonds disappears, and the new band at 1550 cm−1 arises. The latter belongs to nitro groups appearing as a result of addition to double bonds by the reaction (I.2). The chain scission process in BR causes by the following scheme:

~C(CH3)=CH~ + NO2

k1 k2

NO2

~C CH~ + NO2 CH3

R

Then the rate of scissions is:

k3

chain scission + NO2

(I.22)

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

6

−

d [n ' ] = k 3 [ R ⋅ ][NO 2 ] dt

(I.23)

where n ' is a number of isoprene units in BR. After integration of (I.23) taking into account a stationary concentration of R•, the following equation for the degradation degree is derived:

α= where

k1k3[n' ]0 [ NO 2 ]t [n ]0 ( k 2 + k3[ NO 2 ]

[n' ]0

and

[n ]0 are

(I.24)

the initial concentrations of isoprene units and all units. The

amount of double bonds remains practically constant because only a small number of those are destroyed. Really, only 1/50 of macromolecules of BR are subjected to scissions. Taking into account low concentrations of NO2, the linear dependence on time is obtained:

α = k exp t where

(I.25)

k exp is the experimentally determined constant. This constant is represented by the

following Arrhenius equation:

kexp = 3.8 ⋅ 10 − 2 e − 7450 / RT , h−1.

The degradation of BR in a polluted atmosphere runs in three directions: 1) the action of NO2 alone, 2) the action of O2, 3) the combined (synergetic) action of these gases. The general scheme of the process can be represented as follows: RH + O2 R • + O2

k

4 ⎯⎯→

k

5 ⎯⎯→

ROOH

(I.26)

RO2•

(I.27)

k

6 ⎯⎯→ ROOH + R•

RO2• + RH ROOH

R• + HO2

k

7 ⎯⎯→

k8 k9

(I.28)

stable products

[cage1] + O2

(I.29)

k10

chain scission products + O2

(I.30)

The effect of NO2 + O2: ROOH + NO2

k

11 ⎯⎯→ ⎯

NO2−ROOH

(I.31)

Classification of Polymers in Reactivity Toward Nitrogen Oxides

k12

NO2-ROOH 2 R•

k13

[cage2]

k14

7

chain scission products

(I.32)

k

15 ⎯⎯→ ⎯ [cage 3]

[cage 3] + O2 [cage 3]

k

16 ⎯⎯ ⎯ →

k

17 ⎯⎯→ ⎯

(I.33) 2 R•

(I.34)

R−R

(I.35)

The synergetic action of NO2 and O2 can be seen from the scheme: ~CH2C(CH3)=CHCH2CH2~ + O2 → ~CH2C(CH3)=CHCH(OO•)CH2~ + HO2• (I.36) ~CH2C(CH3)=CHCH(OO•)CH2~ + RH → ~CH2C(CH3)=CHCH(OOH)CH2~ + R

(I.37)

~CH2C(CH3)=CHCH(OOH)CH2~ + NO2 → ~CH2C(CH3)=C(NO2)CH(OOH)CH2~ (I.38) BR is not sensitive to UV light (λ > 290 nm) alone. Probably, UV light in the presence of NO2 effects on nitro groups of macromolecules.

I. 3. INTERACTION OF NITROGEN DIOXIDE WITH ALIPHATIC POLYAMIDES AND POLYURETHANES Polymers containing amide and urethane groups form a particular class of materials sensitive to NO2. Jellinek et al. [13, 14] showed that exposure of nylon-66 films of different morphology to NO2 (10−5 − 2.6⋅10−1 mol⋅l−1) causes main-chain scission in the polymers. The degradation of nylon is a diffusion-controlled reaction. Its rate and depth depend essentially on the degree of crystalline of samples and on the size of crystallites. The degradation is accelerated in the presence of air and UV light in addition to NO2. The following mechanism for the polymer degradation under the action of NO2 was proposed: O

H

O

C N CH2 CH2 O

+ NO2

NO2

C N CH2 CH2

C N CH2 CH2 NO2

+ HNO2

O

C N CH2 + CH2

(I.39)

The degradation process is inhibited by small amounts of benzaldehyde or benzoic acid. It is believed that these compounds block the amide groups and that only a few of them, not involved in hydrogen bonding, enter into the reaction:

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

8 O

O

+ Ph

C NH CH2

C OH

C

N

O

H

H

O

(I.40)

O C Ph

Jellinek et al. [14, 15] studied the effect of NO2 on films of linear polyurethane synthesized from tetramethylene glycol and hexamethylene diisocyanate. It was found that the degradation of polyurethanes is accompanied by cross-linking of macromolecules and that the degree of degradation and the yield (the weight percentage) of the gel fraction are complex functions of the exposure time. For instance, the yield of the gel fraction initially increases up to 20% and then decreases down to nearly zero at 330 K and NO2 concentration of 10−3 mol⋅l−1. The number of chain scissions in the sol fraction (the degree of degradation) increases initially, then decreases and eventually increases again; however, the final degradation rate is lower than the initial one. Exposure of the polyurethane films to NO2 is accompanied by release of CO2. The IR spectra of the films allow assessment of the consumption of NH bonds (ν = 3300 cm−1). The reaction mechanism proposed [15, 16] involves the abstraction of hydrogen atoms from two types of structures, namely, a carbamate structure (A) and a tertiary amide structure (B): O

O

O C NH

O C N

CH2

ZH

B

A

CH2

where Z is a side alkyl group. The next stages are represented as follows: A

O

NO2

O C N

CH2

+ HNO2

(I.41)

CH2

+ HNO2

(I.42)

R1 B

O

NO2

O C N Z

R1 + NO2

R2 (I.43)

R1NO2 O

R1

H2C N

+

C O CH2

(I.44)

R3 R3 R1 + R 2

CO2 + H2C cross-linking product

(I.45) (I.46)

Classification of Polymers in Reactivity Toward Nitrogen Oxides

9

According to the Jellinek, recombination of R1• and R2• radicals leads to cross-linking of the polymer chains, while decomposition of R1• radicals results in the degradation of macromolecules and the CO2 release. Energetically, the decomposition of the R1• radicals seems to be improbable since this reaction results in the formation of terminal macroradical R3•and a nitrene, which is a very reactive species. On the other hand, the R1• decomposition reaction involving cleavage of C−C or C−O is more bonds produce no alkoxycarbonyl macroradicals R3•, which can undergo decarboxylation [16]. Therefore, the ageing of polyurethanes in an NO2 atmosphere can be represented as follows [17]: Reaction

(I.41)

Reaction

(I.42)

B

O

NO2

O C N

CH-CH2

+ HNO2

(I.47)

ZH R 4

R4

R3 + HZ- N=CH

(I.48)

R3

CO2 + CH 2

(I.49)

2Ri + NO2

2Ri

nitration products

cross-linking products

(I.50)

(I.51)

where i = 1−4. This scheme expresses the degradation accompanied by cross-linking of macromolecules, the consumption of NH groups of the polymer as well as the release of carbon dioxide upon degradation. The investigations performed earlier characterize in general the reactivity of polymers of different classes in their reactions with nitrogen dioxide. However, mechanisms of free radical processes proposed on basis of the results considered are enough formal. As a rule they take account of changing molecular weights and the composition of final molecular products of the nitration. In connection with this, the study of structures of free radicals forming in primary and intermediate stages of polymer conversions attracts an especial interest. Such researches allow drawing conclusions on the mechanism of initiation of free radical conversions dependent on nature of functional groups of macromolecules. As is shown by ESR measurements, different stable nitrogen containing macroradicals are formed on exposure of polymers to NO2 [17]. The analysis of the radical composition from ESR spectra gives an opportunity for estimation of the polymer stability by the quite simple method [18].

10

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

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

Jellinek H. H. D. Degradation and Stabilization of Polymers. New York: Elsevier, 1978. Ogihara T.. Bull. Chem. Soc. Jap. 1963, 31, 58Ogihara T., Tsuchiya S., Kuratani K. Bull. Chem. Soc. Jap. 1965, 38, 978Jellinek H. H. D. The 2nd International Symposium on Degradation and Stabilization of Polymers (Abstracts of Reports), Dubrovnik, 1978. Jellinek H. H. D., Toyoshima Y. J. Polym. Sci. A-1. 1967, 5, 3214Jellinek H. H. D., Flajsman F. J. Polym. Sci. A-1. 1969, 7, 1153Huber A. Diss. Doktor Naturwiss. Stutgart: Fakultät Chemie der Universität Stutgart, 1988. Giamalva D. H., Kenion G. B., Church D. F., Pryor W. A. J. Am. Chem. Soc. 1987, 108, 7059-7063. Rånby B., Rabek J. F. Photodegradation, Photo-oxidation and Photostabilization of Polymers. London: Wiley, 1975. Jellinek H. H. D., Flajsman F., Kryman F. J. J. Appl. Polym. Sci. 1969, 13, 107Jellinek H. H. D., Flajsman F. J. Polym. Sci. A-1. 1970, 8, 711Jellinek H. H. D., Hrdlovič P. J. Polym. Sci. A-1. 1971, 9, 1219Jellinek H. H. D., Chandhuri A. J. Polym. Sci. A-1. 1972, 10, 1773Jellinek H. H. D., Yokata A. R., Itoh Y. Polym. J. 1973, 4, 601. Jellinek H. H. D., Wang A. T. J. W. J. Polym. Sci., Polym. Chem. Ad. 1973, 11, 3227-. [Jellinek H. H. D., Martin F. H., Wegener H. J. Appl. Polym. Sci. 1974, 18, 1773Pariiskii G. B., Gaponova I. S., Davydov E. Ya. Russ. Chem. Rev. 2000, 69, 985-999. Zaikov G.E. Success in chemistry and biochemistry, New York, Nova Science Publishers, 2009

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.11-20 ©2010 Nova Science Publishers, Inc.

Chapter 2

INFLUENCE OF THE INITIATION RATE OF RADICALS ON THE KINETIC CHARACTERISTICS OF QUERCETIN AND DIHYDROQUERCETIN IN THE METHYL OLEATE OXIDATION L. I. Mazaletskaya*, N. I. Sheludchenko and L. N. Shishkina Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russia

ABSTRACT The antioxidant activity of quercetin (Q) and dihydroquercetin (QH2) is studied by the methyl oleate autooxidation model at 323 K in thin layers, and also the antiradical activity of these substances is investigated by the initiated oxidation of methyl oleate at 333 K. It is shown that the rate constant of the inhibition for Q is equal 1.7×105 dm-3 mol-1 s-1, that is 2.2 times greater than king for QH2 (7.9×104 dm3 mol-1 s-1). Under the autooxidation condition the inhibitory effectiveness of Q is also 1.6 times greater than that for QH2. The existence of the direct correlation between the induction period of the methyl oleate autooxidation in the presence of flavonoids and the Q and QH2 concentrations indicates their primary interaction with peroxyl radicals.

Keywords: autooxidation, initiated oxidation, kinetics, flavonoids, methyl oleate.

AIMS AND BACKGROUND Flavonoids are the compounds within the group of the vitamin P and of interest as the biologically active substances with a wide spectrum of action. Biological activity of flavonoids is associated with their ability to inhibit of oxidation processes by the reaction * Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, 4 Kosygin st., 119334, Moscow, Russia e-mail: [email protected]

12

L. I. Mazaletskaya, N. I. Sheludchenko and L. N. Shishkina

with reactive oxygen species, as well as free radicals [1-6]. In addition to the use of flavonoids as drugs and the component of the biologically active additives for the stabilization of the food products, cosmetics, preparations, etc, from the air oxygen oxidation is of the great practical interest, because the number permitted to be used for this purpose synthetic antioxidants is extremely limited. Many investigations are made to examine the antioxidant activity of flavonoids and their content in food product consistent by using the different methods. Advantages and disadvantages of these methods are discussed in detail in review [7]. The analysis of the available literature data shows that the parameters characterizing the antioxidant activity as well as the rate constant of the antiradical activity of flavonoids obtained under different conditions substantially differ and depend on many factors, including the chemical structure of flavonoids and the oxidation model. Thus, one would expect that the antioxidant activity of the two closest to the structure of flavonoids - quercetin (Q) and dihydroquercetin (QH2), the molecule structure of which is the presence or absence of the C2C3 double bond, will be little different, because their rate constants with oxygen anionradicals are close and equal 1.7×105 dm3 mol-1 s-1 and 1.5×105 dm3 mol-1 s-1, correspondingly [8]. However, the effectiveness of Q is more 2 times greater than that for QH2, when it is determined by the NADPH- and CCl4-dependent lipid peroxidation in microsomal membranes of the rat liver8. On the contrary, the greatest antioxidant activity has QH2 that follows from the results of the comparative tests presented in the paper [9]. As shown, the antiradical activity of flavonoids significantly changes depending on the oxidizing substrate and the oxidation conditions. So, the rate constants of their interaction with peroxyl radicals obtained in the initiated oxidation reaction of diphenylmethane10 significantly higher than that obtained by methyl linoleate oxidation in the homogenous and micellar solutions [11]. Besides, as noted in Ref. 10 - 11, flavonoids did not behave as the classical antioxidants and the constant of their antiradical activity depends on the initial concentrations of both flavonoid and the oxidation substrate. To use flavonoids as stabilizers from the spontaneous oxidation of foods, cosmetics and medicinal agents, it is necessary to establish the regularities of their antioxidant action under the autooxidation conditions, which simulates the natural aging process taking place under the air oxygen action. As known, it is the side-reactions of antioxidants (InH) play the most substantial role under the degenerate branching chain in the autooxidation process, they proceed with the participation of the initial molecules of antioxidants and products of their oxidative conversion and lead to loss of the effectiveness of the antioxidant action. For this reason, there may exist substantial differences in regularities of the antioxidant action of these substances between the autooxidation reactions and reactions with the constant rate of the free radical initiation. So, for example, the deviation from linearity is observed for the dependence of the induction period on the initial concentration for the natural antioxidant α-tocopherol (TP) under the autooxidation conditions, and, in some cases, this dependence can be extremal12. On the contrary, the linear relation between these parameters was detected by both the initial oxidation and autooxidation for the hindered phenols, radicals of which are practically not consumed in side-reactions [13,14]. The aim of our research was to study the influence of the quercetin and dihydroquercetin concentrations on the effectiveness of their inhibitory action and the antiradical activity depending on the oxidation conditions of the same substrate – methyl oleate.

Influence of the Initiation Rate of Radicals on the Kinetic Characteristics…

13

EXPERIMENTAL Autooxidation of methyl oleate is carried out by the atmospheric oxygen in a thin layer at 323 K. The course of oxidation was followed by the accumulation of hydroperoxides (ROOH), the concentration of which was determined by iodometric titration. The error of the ROOH concentration determination did not exceed 1.5%. The antioxidant effectiveness was evaluated by τ values, which were graphically determined from the kinetic curves of the ROOH accumulation during the methyl oleate autooxidation in the absence and presence of InH. As duration of the oxidation induction period (τ), we took the interval from zero to the perpendicular dropped on the X axis from point of intersection of the linear plots of kinetic curve of the ROOH accumulation: the initial oxidation rate within the induction period and the maximal rate of the ROOH accumulation. The initiated oxidation of methyl oleate in a mixture (1:1) with an inert solvent (chlorobenzene) was performed by the air oxygen at 333 K. The reaction mixture containing methyl oleate, chlorobenzene and the initiator – dinitrile of azoizobutyric acid, placed in oxidative cell equipped with a magnetic mixer. The reaction mixture was thermostated at 333 K, and then InH was injected and measured the kinetics of oxygen uptake by using the volumetric method. The initiation rate and the InH concentration were varied. The initial rate of oxidation, as well as the value of the induction period τ, were determined from the kinetic curves of oxygen absorption by method described in Ref. 15. The interval from the beginning of the experience to the point of intersection of two straight lines for which tg α1 = 2 tg α2 was taken as τ in the presence of InH. The first line is an extension to a straight of the oxygen uptake, when the reaction rate is constant after the complete consumption of InH. The second line is tangent to the kinetic curve of the oxygen uptake in the point, the reaction rate of which is twice less than that in the absence of inhibitor. The concentration of Q was determined spectrophotometrically at the wavelength λmax = 368 nm. The formation of the intermediate product is recorded by the absorption spectrum at the wavelength λmax = 526 nm. To prepare the InH solutions, their sample is dissolved in ethanol and then diluted by chlorobenzene. The proportion of ethanol in the oxidative cell is not exceeded about 1.3 % (v/v). Methyl oleate was purified by vacuum distillation. Dihydroquercetin and Quercetin (Sigma) were used without additional purification. The kinetic data were processed by KINS program given in Ref. 16.

RESULTS AND DISCUSSION Methyl oleate is one of the most used model system for the analyzing the antioxidant properties of different individual InH and their mixtures, but the employment of the methyl oleate autooxidation in the thin layer is rare. For this reason, in our research it was used the methyl oleate autooxidation in thin layer. It is obtained that Q and QH2 were effectively inhibited this process (Fig. 1). The dependence of τ on the initial concentration of these antioxidants is closely to linear in the studied range of concentrations. The slope of dependences of τ on the [InH]0 concentrations for Q and QH2 is significantly different. Besides, there is a need to note that the antioxidant activity of Q is 1.6 times greater than for QH2 (Fig. 1). The similar results were obtained during the oxidation of lard17: it was shown

L. I. Mazaletskaya, N. I. Sheludchenko and L. N. Shishkina

14

that τQ is 1.4 times greater then τQH2. Besides, our results are also consistent of the data presented in Ref. 18 about the antioxidant activity of the two flavonoids – fisetin and fustin, the molecular structure of which is also characterized by presence or absence of the C2 – C3 double bond. According to Ref.18, the IC50 values for fisetin and fustin during the NADPHdependent microsomal lipid peroxidation are equal 20.8 and 66 μmol, correspondingly, e. i. their antioxidant activities differ about 3.2 times. τ, h

300

3 250 1 200

150

2

100

50

0 0

0,5

1

1,5

2 2,5 3 4 -3 [InH]0×10 , mol dm

Figure 1. Dependence of the induction period (τ) on the antioxidant concentrations during autooxidation of methyl oleate 1 – Q; 2 – QH2; 3 – TP

Hence, the obtained results and the literature data point to the fact that the presence of the C2 – C3 double bond in the C-ring caused the conjugation at all rings in the flavonoid molecule results in the increase of their antioxidant activity. In order to clear the behavior of flavonoids during the oxidation, a detail computer analysis of the kinetics of the methyl oleate autooxidation in the absence and presence of flavonoids was performed. It was showed that, in all cases, the peroxide accumulation is well described by the exponential law: [ROOH] = a×exp(kt), the correlation coefficients of which are equal 0.99 – 1.0. Earlier the similar relationship was revealed for the methyl oleate autooxidation when the oxygen concentration provides the oxidation in the kinetic range14. The exponential index k, the value of which is proportional to the total oxidation rate14, is not reliable differ for Q and QH2 and is not depend on their concentrations: k = (5.3 ± 0.6)×10-2 h1 and k = (4.94 ± 0.04)×10-2 h-1 for Q and QH2, correspondingly. Thus, the results of computations indicate the complete consumption of flavonoids within the induction period. The preexponential factor a of the kinetic curves of the peroxide accumulation, the values of which are due to the rates of radical initiation and the chain propagation [14], is substantially less in the presence of flavonoids than the same during the methyl oleate autooxidation.

Influence of the Initiation Rate of Radicals on the Kinetic Characteristics…

15

Furthermore, this parameter decreases with a growth of the flavonoid concentration (Fig. 2). As can be seen from Fig. 2, the value a more significantly reduces during the methyl oleate autooxidation inhibited by Q than that in the QH2 presence. These data corroborate the higher antioxidant activity of Q as compared with that for QH2. а ×10

6

3

5 4 2 3 2 1 1 0 0

0,5

1

1,5

2

2,5 4

3 -3

[InH]0 ×10 , mol dm

Figure 2. Dependence of the preexponential factor a on the quercetin (1) and dihydroquercetin (2) initial concentrations.

Dependences of τ on the initial concentrations of flavonoids [Q]0 and [QH2]0 differ from those for the natural antioxidant TP obtained under the same conditions. It can be seen from the comparison of data, which are presented in Fig. 1. Unlike Q and QH2, the dependence of τ on [TP]0 is nonlinear (Fig. 1, curve 3) in the range of the studied concentrations. TP inhibits better the methyl oleate autooxidation compared with Q and QH2 at the low concentrations, where the contribution of side-reactions was negligible. However, the most active of the investigated flavonoids Q provides the some longer induction period compared with TP already at [InH]0 = 2.5×10-4 mol dm-3 (Fig. 1). The inhibitory action effectiveness is above noted to depend on side-reactions involving the antioxidant. One of these reactions is the interaction of InH with the molecular oxidation product – ROOH. As known, TP has the antiperoxide activity decomposing the methyl oleate hydroperoxide already at room temperature [19]. In this work the reaction of Q and ROOH was studied by the Q consumption under anaerobic conditions at 333 K. It is established that the consumption rate of Q (WQ) is independent on [Q]0 at [ROOH]0 = const. in the range of Q concentrations providing the linear chain termination. However, WQ increases with the growth in the [ROOH]0 concentration at the fixed initial concentration of Q, as can be seen in Fig. 3, The results obtained indicate that Q does not practically interact with ROOH under experimental conditions. The observed consumption of Q is due to its interaction with free

L. I. Mazaletskaya, N. I. Sheludchenko and L. N. Shishkina

16

radicals forming during the thermal decay of ROOH. From the dependence of WQ on [ROOH]0 presented in Fig. 3 the rate constant of the ROOH decay with the free radical formation (k3΄) was calculated. It’s value is equal k3΄ = WQ /[ROOH]0 = 2.8×10-7 s-1. 8

-3 -1

4 W Q x10 , mol dm s

3

2

1

0 0

0,02

0,04

0,06

0,08

0,1

0,12 -3

[ROOH]0, mol dm Figure 3. Effect of [hydroperoxide]0 on the initial rate of the quercetin consumption.

To compare the antioxidant action effectiveness of studied flavonoids with their antiradical activity, the stoichiometric inhibition coefficient f and the fk7/k60.5 parameter, where k7 and k6 are the rate constants of the antioxidant interaction with peroxyl radicals and the square recombination of peroxyl radicals, correspondingly, were measured by the model reaction of the methyl oleate initiated oxidation. The kinetic curves of the oxygen uptake during the methyl oleate oxidation in presence of Q and QH2 have the particularly pronounced induction period. It can be seen in Fig.4, curve 1 for Q. To calculate f, the τ values were measured from the kinetic curves of the oxygen uptake at the different initiated rates (Wi) and initial concentrations of InH. These data are given in Table. Then we made plot on the τWi – f[InH]0 coordinates presented in Fig. 5. It is seen that these dependences are linear (Fig. 5). From the slope of these straight lines the f values for flavonoids were calculated which are equal fQ = 2 ± 0.2 and

f QH2

= 2,6 ± 0.3. Thus, despite the fact that the molecules of these

flavonoids have the same number of OH groups there is a some difference among their stoichiometric inhibition coefficients. The f value for Q obtained from kinetic curves of the oxygen uptake is in good agreement with fQ = Wi/WQ = 2 calculated from the rate of Q consumption (WQ) (Fig. 4, curve 1΄). As observed, there is the formation of coloured intermediate product with the maximal absorption at the wavelength λ = 526 nm during the initiated oxidation of the methyl oleate inhibited by Q. However, the practically complete discolouration of the reaction mixture is observed by the induction period end (Fig. 4, curve 1΄΄). This result may indicate that the coloured intermediate product formed in the inhibition reaction also possesses the antioxidant properties.

Influence of the Initiation Rate of Radicals on the Kinetic Characteristics…

17

0,8

70

1

0,7

60

0,6

50

'1

40 0,4 30

V, a.u.

Absorption

0,5

0,3

1''

0,2

20

10

0,1 0

0 0

10

20

30

40

50

60

70

80

90

min

Figure 4. Kinetic curves of the oxygen uptake in the absence (the dotted line) and presence (1) of Q ([Q]0 = 1.25×10-4 mol dm-3) during the methyl oleate initiated oxidation at 333 K and Wi = 10-7 mol dm-3 s-1.The Q consumption (1΄) and the formation of their conversion coloured product (1΄΄) under the methyl oleate initiated oxidation 4

6

τ W i × 10 , mol dm

-3

2 5 1

4 3 2 1 0 0

0,5

1

1,5

2

2,5 4

[InH]0 × 10 , mol dm

-3

Figure 5. Plot of τWi versus [InH]0: 1 – Q, 2 – QH2; the methyl oleate initiated oxidation at 333 K.

From the kinetic curves of the oxygen uptake at the initial step the rates of the inhibited oxidation (Wing) were determined. To calculate the rate constant of the Q and QH2 interaction with peroxyl radicals of methyl oleate (k7), the following equation was used:

L. I. Mazaletskaya, N. I. Sheludchenko and L. N. Shishkina

18

ω = W0/Wing – Wing/W0 = fk7[InH]/(Wik6)0.5 = king[InH]/(Wik6)0.5, where W0 is the oxidation rate of methyl oleate without additivies InH, Wi – the initiation rate. Since Wing is determined at t > 0, the concentration of InH is calculated by equation [InH] = [InH]0 – tWi/f. From the slope of straight lines presented in Fig. 6 the values fk7k6-0.5 were calculated which are equal 55 and 25 (dm3 mol-1 s-1)0.5 for Q and QH2, respectively. For comparison, the fk7k6-0,5 parameter for the well-known synthetic antioxidant 2,6-di-tert-butyl4-methylphenol (BHT) obtained under the same conditions was calculated as fk7k6-0,5 = 4,5 (dm3 mol-1 s-1)0,5 which was a good agreement with published data about its smaller reactivity compared with flavonoids [10]. 0.5

4

ωW i x10 , mol dm

-3

70

1 60 50 40

2 30 20 10 0 0

0,2

0,4

0,6

0,8

1

1,2 4

[InH]0×10 , mol dm

1,4 -3

Figure 6. Plot of ωWi versus [InH]0: 1 – Q, 2 – QH2; the methyl oleate initiated oxidation at 333 K.

Assuming k6 = 1×107 dm3 mol-1 s-1, king = f k7 were calculated as 1.7×105 and 7.9×104 dm3 mol s for Q and QH2, respectively. The obtained value king for Q is satisfactory agreement with king = 4.3×105 dm3 mol-1 s-1 calculated from the kinetic curves for oxygen uptake during the methyl liloleate oxidation (0,242 M in chlorobenzene), inhibited by quercetin [20]. Some difference in values king can be associated with greater reactivity of peroxyl radicals from the methyl liloleate compared with that from methyl oleate. From the values king the rate constants of flavonoids with peroxyl radicals was calculated, which have values k7 = 8.5×104 and 3.0×104 dm3 mol-1 s-1 for Q and QH2, respectively. The ratio of parameters characterizing the antiradical activity of Q and QH2 also shows that the presence of the C2 – C3 double bond in the C-ring leads to a 2-fold increase in the inhibition constant. It is a good agreement with the ratio = 2,6 which are calculated by the concentrations of flavonoids for 50% inhibition of lipid peroxidation from the half-wave potential of the first oxidation wave measured by flow-1 -1

Influence of the Initiation Rate of Radicals on the Kinetic Characteristics…

19

through column electrolysis and the octanol/water partition coefficient for fisetin and fustin presented in Ref. 18. Thus, the data obtained allow us to conclude that Q has the highest rate constant interaction with the peroxyl radicals (k7) which is 2,8 and more 10 times greater than that for QH2 and BHT, respectively. The antioxidant activity of flavonoids and also their antiradical properties is above note to depend substantially on the oxidation system [10,18,20]. It is associated with the occurrence of the different side-reactions, the lipophilicity of flavonoids or the hydrogen-bonding ability of solvents [10,11,18,20]. Our results showed that the antioxidant activity of Q and QH2 correlates with the parameter fk7k6-0,5, characterizing their antiradical activity under the different conditions of the methyl oleate oxidation. From the data obtained, the magnitudes fk7k6-0,5 for Q and QH2 differ about 2 times while the inhibition action effectiveness for Q is 1.6 times greater than that for QH2 calculated from the dependence of τ on [InH]0 under the autooxidation condition. The existence of the direct correlation between the induction period of the methyl oleate autooxidation in the presence of flavonoids and the Q and QH2 concentrations indicates their primary interaction with peroxyl radicals. Table. Effect of initial rate of radicals on induction periods ( ) of the methyl oleate oxidation in presence of antioxidants, temperature 333 . InH Q Q Q Q Q Q QН2 QН2 QН2 QН2 QН2

[InH]0×104 (mol dm-3) 0.2 0.3 0.6 0.75 1.25 2.0 0.3 0.45 0.6 1.0 2.0

Wi×107 (mol dm-3 s-1) 0.3 0.5 1.0 0.5 1.0 1.0 0.5 1.0 0.5 1.0 1.0

τ (min) 22 22.5 18 54 42 67 25 22 50 43 82

REFERENCES [1] [2] [3] [4] [5] [6] [7] [8]

U. Takahama: Photochem. Photobiol. 38, 363 (1983). U. Takahama: Plant Physiol. 71, 598 (1983). V.A. Kostyuk, A.I. Potapovich, S.M. Tereshchenko, I.B. Afanas΄Yev: Biokhimiya, 53 (8), 1365 (1988) (in Russian). Yu.O. Teselkin, B.A. Zhambalova, I.V. Babenkova, G.I. Klebanov, N.A. Tyukavkina: Biofizika, 41 (3), 620 (1996) (in Russian). M.R. Cholbi, M. Paya, M.J. Alcaraz: Experientia, 47 (2), 195 (1991). W. Bors, W. Heller, C. Michel, M. Saran: Methods Enzymol., 186. 343 (1990). V. Roginsky, L.A. Eduadro: Food Chem., 92 (2), 235 (2005). A.I. Potapovich, V.A. Kostyuk: Biokhimiya, 68 (5), 632 (2003) (in Russian).

20 [9]

[10] [11] [12] [13] [14] [15]

[16] [17] [18] [19] [20]

L. I. Mazaletskaya, N. I. Sheludchenko and L. N. Shishkina S. Ya. Sokolov, N.A. Tyukavkina, V.K. Kolkhir, Yu.A. Kolesnik, A.P. Arzamastsev, N.G. Glazova, V.A. Zyuzin, A.I. Baginskaya, V.A. Babkin, L.A. Ostroukhova: Patent № 2014841 (Russian Federation). V.A. Belyakov, V.A. Roginsky, W. Bors: J. Chem. Soc., Perkin Trans. II, (12), 2319 (1995). V.A. Roginsky, T.K. Barsukova, A.A. Remorova, W. Bors: J. Am. Oil Soc. 73 (6), 777 (1996). V.V. Naumov, R.F. Vasil΄Yev: Kitetika i kataliz, 44 (1), 111 (2003) (in Russian). L.I. Mazaletskaya, G.V. Karpukhina: Neftekhimiya, 24 (2), 502 (1984) (in Russian). N. V. Khrustova, L.N. Shishkina: Kinet. Catal., 45 (6), 799 (2004). N.M. Emanuel, G.P. Gladyshev, E.T. Denisov, V.F. Tsepalov, V.V. Kharitonov: The testing procedure of chemical compounds as stabilizers of polymer materials. Preprint. Chernogolovka, 1976, 35 p. (in Russian). E.F. Brin, S.O. Travin: Chem. Phys. Reports 10 (6), 830 (1990). S. V. Antoshina, А.А. Selishcheva, G.M. Sorokoumova, E.A. Utkina, P.S. Degtyarev, V.I. Shvets: Prikladnaya biokhimiya i mikrobiologiya, 41 (1), 23 (2005) (in Russian). B. Yang, A. Konani, K. Arai, F. Kusu: Analytical Sciences, 17, 599 (2001). L.I. Mazaletskaya, N.I. Sheludchenko, L.N. Shishkina: Petroleum chemistry, 48, 105 (2008). P. Pedrielli, G.F. Pedulli, L.H. Skibsted: J. Agric. Food Chem., 49, 3034, (2001).

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.21-28 ©2010 Nova Science Publishers, Inc.

Chapter 3

AN ANTIOXIDANT FROM HINDERED PHENOLS GROUP ACTIVATES CELLULOSE HYDROLYSIS BY CELLOVIRIDIN IN A WIDE CONCENTRATION RANGE, INCLUDING ULTRALOW DOSES E. M. Molochkina1, Yu. A. Treschenkova1, I. A. Krylov2 and E. B. Burlakova1 1

N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia 2 D.I. Mendeleev Russian Chemical-Technological University, Moscow, Russia

ABSTRACT For the first time, the effect of a synthetic antioxidant – an inhibitor of free-radical processes, phenosan, on the cellulase activity was studied. A considerable increase of product yield of microcrystalline cellulose hydrolysis by celloviridin (a complex of cellulases from Trichoderma Viride) under the effect of a wide range of concentrations of phenosan, including ultralow doses, not associated with its antioxidant properties was observed. The concentration dependence of the effect is complex and non-monotonic. Ultralow concentrations of phenosan increase the product yield similarly or greater than “usual” ones. When increasing the process efficiency by phenosan at different concentrations of celloviridin protein, it is shown that phenosan action (including ultralow concentrations) allows a considerable decrease of the amount of an expensive enzymic complex necessary for obtaining the target product.

Keywords: antioxidants, hindered phenols, hydrolysis, cellulose, ultralow doses, wide concentrations.

22

E. M. Molochkina, Yu. A. Treschenkova, I. A. Krylovet al.

INTRODUCTION While the population of the Earth increases and food resources based on non-food raw materials and, which is the most important alternative sources of energy are actively searched for, the interest to cellulose-containing substrates (CCS), which renewable resources are almost unlimited, increased. Annually, about 1011 tons of plant biomass on the Earth is hydrolyzed by microorganisms’ enzymes, and released energy is equivalent to 640 billion barrels of oil. Therefore, it is clear why CCS enzymic hydrolysis using cellulase complexes produced by microorganisms is considered to be the most perspective way for obtaining alternative fuel [1]. Beside searching for and cultivation of new microorganisms – cellulase complex producers, new approaches to increase the yield of end products under the effect of currently used enzymic preparations shall be developed. Celloviridin produced from Trichoderma Viride fungus in two variants – celloviridin G20x and celloviridin G3x, is one of cellulase complexes used for CCS hydrolysis in Russia. The preparations catalyze degradation of cellulose from a plant cell to oligosaccharides, mono- and disaccharides by enzymes of three types: endoglucanase (Е.С. 3.2.1.4), cellobiohydrolase (Е.С. 3.2.1.91) and beta-glucosidase (Е.С. 3.2.1.21). Cellulolytic activity of celloviridin-V G20x equals 2000 ± 200 units/g. The content of enzymic system components in celloviridin G3x is not constant, and its activity equals 100-200, 300-370 or 500-600 units/g [2]. Celloviridin production from microbiological material is rather labor consuming and expensive. It is of importance to find possibilities of increasing the action efficiency of cellulases of celloviridin complex and, thus, to reduce the amount of required celloviridin. Celloviridin represents a complex set of components containing a biomass, in particular, having lipids subjected to peroxidation (LPO), which products may affect the enzyme activity. To regulate LPO intensity in biological and chemical systems, antioxidants (AO), including free-radical reaction inhibitors from the group of hindered phenols, are used. Recently, it has been found that AO of this type relate to substances, which ultralow doses (ULD) affect biological systems of different complexity [3]. In view of possible increase of process efficiency, we consider perspective to study the effect of a wide range of AO concentrations from the class of hindered phenols – phenosan K (potassium salt of beta-(3’,5’-ditert.butyl-4’-hydroxyphenyl)propane acid), on cellulose hydrolysis by celloviridin complex. Phenosan in the form of industrially produced acid is used in cattle breeding by inducing the growth-stimulating action in the forage composition for the farm livestock (chicken broilers, veals). It is assumed that phenosan acts as a bioantioxidant, preventing oxidation of fat-soluble vitamins A and E in the combined fodder composition [4 - 6]. Being the inhibitor of free-radical oxidation, phenosan may stabilize celloviridin preparation, because the latter may contain biomass components, lipids, in particular, capable of easy oxidation, giving products capable of damaging enzymic proteins. However, for us the main precondition of phenosan use is the fact that this substance is one of the agents in ULD affecting various biological systems [3]. In particular, it is known as a superactivator of enzymic activity, this effect being not associated with antioxidant

An Antioxidant from Hindered Phenols Group Activates Cellulose Hydrolysis…

23

properties. This fact is described in ref. [7], which shows that injection of this preparation to smooth muscle cells of mouse aorta in the cellular culture in 10-18 M concentration induces protein kinase C activation by 400%. The aim of this work was to determine the presence and to estimate the level of phenosan K effect on cellulose hydrolysis by celloviridin complex, using a wide range of AO concentrations, including ultralow doses. The tasks included: 1. Determination of microcrystalline cellulose (MCC) hydrolysis products yield - total sugars and glucose - in different periods after initiation of the process, carrying out the reaction in the presence of various phenosan concentrations, including ultralow doses. 2. Investigation of phenosan effect on the target product yield at different concentrations of celloviridin.

EXPERIMENTAL In the work microcrystalline cellulose (MCC) by Merck Company (Microcrystalline Cellulose, Charge Nr.: 5010160202) was used; enzymic preparation celloviridin G3x by OAO (BK) “Vostok” was presented by Biotechnology Department, D.I. Mendeleev RCTU. The studies were performed on two celloviridin G3x samples taken from different batches of preparation (CV-1 and CV-2 samples), which demonstrated different cellulolytic activity estimated by the rate of total sugars accumulation with the help of phenol-sulfuric method [8, 9]. Activity of CV-1 sample estimated by this method was by 65% higher than activity of CV-2 sample. To purify the “grout” of the initial celloviridin powder in the acetate buffer (pH) from components insoluble in the aqueous medium (filler, biomass residues) and to obtain samples containing cellulases soluble in the aqueous medium, the “grout” was centrifuged, and supernatant fluid was used in the work. Protein concentration in supernatant fluid was determined by the Lowry method [10]. Synthetic AO phenosan K was synthesized in N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences. The rate of cellulose enzymic hydrolysis was estimated by accumulation of total sugars, which quantity was determined by the phenol-sulfuric method [8, 9], and glucose, which concentration was determined by glucose oxidase test [11] using the assay kit “Novogluk-K, M”. The reaction was performed in round-bottomed flasks in the acetate buffer solution with pH 4.5, containing 50 mg/ml of cellulose. CV “solution” was added to the reaction vessels so that the final concentration of protein containing the enzymic complex equaled 0.15-0.62 mg/ml. Flasks were suspended on a rubber hose strained over the thermostat bath so that their bottoms were dipped into the water. The incubation temperature was 40°C. Rather intensive and uniform mixing of the reaction mixture was provided by flasks swinging due to water motion in the thermostat. To each flask solution of the substance under test in corresponding concentration or acetate buffer solution (in the control) was added. The stock phenosan K

24

E. M. Molochkina, Yu. A. Treschenkova, I. A. Krylovet al.

solution (10-3 М) was prepared by dissolving a weighed portion of the preparation in the acetate buffer solution with pH 4.5. The effect of phenosan was studied at its concentration in the reaction mixture ranged within 5⋅10-17 − 10-5 M, every time adding solutions of corresponding concentrations obtained by subsequent dilutions of the primary solutions by 100 times into reaction vessels. From celloviridin lipids were extracted and concentrations of total lipids and phospholipids were determined, using the guidelines [12]. The results were treated and graphically designed using the software Sigma Plot for Windows v. 8 (demo).

RESULTS AND DISCUSSION Celloviridin analysis performed demonstrated that the concentration of lipids in the solid preparation is 0.001±0.0001 g/g, and concentration of phospholipids (the main LPO substrates) is 0.1±0.01 mg/g (of about 1.2·10-4 М). For possible effect of their oxidation products on enzymic proteins in the solid celloviridin, this is rather high concentration, and addition of an antioxidant may stabilize potential cellulase activity of the powder. However, the aim of this work was to estimate the possibility of direct phenosan participation in the enzymic process. Therefore, it was added to the reaction mixture together with the homogeneous solution containing enzymic proteins, obtained from celloviridin powder (see Experimental). Figure 1 illustrates the phenosan effect on the yield of products of MCC hydrolysis by celloviridin. It shows the dependence of the amount of sugars formed on the antioxidant concentration in the incubation medium 30, 60 and 120 min after the process beginning. Every column represents the average value obtained from three-four parallel measurements ± SE. It is obvious the intense stimulating effect of phenosan on the yield of total sugars and the target product (glucose). The dependence of sugars yield on the phenosan concentration is complex and nonmonotonous. Such dose dependence resembles the “dose-effect” curve characteristic of substances (of various classes) capable of acting in ultralow concentrations [3]. As mentioned above, phenosan also studied on some biological systems, manifested itself as the agent of this kind. Enzymes also are the targets affected by its ultralow doses. These are, for example, soluble and membrane-bound acetylcholinesterase, protein kinase C and lactate dehydrogenase [3, 7, 13, 14]. Now cellulases can be added to this list. As shown in the figure, this effect of phenosan in ULD is either comparable with its action in “usual” concentrations (10-7 - 10-5 М), or is much greater expressed. (For “usual” AO concentrations the concentrations are taken, in which AO from the group of hindered phenols are able to inhibit free-radical oxidation processes). The glucose percentage in total sugars in the presence of phenosan either increased insignificantly compared with the control (except of a considerable increase from 13 to 23% in the case of 30-minute incubation with 10-4 М) or even demonstrated a tendency to decrease. This indicates that, apparently, the effect of phenosan mostly stimulates the stages associated with the pool of enzymes catalyzing formation of oligo- and disaccharides.

An Antioxidant from Hindered Phenols Group Activates Cellulose Hydrolysis…

25

Figure 1. The yield of total sugars and glucose in different times from the incubation beginning in the presence of different phenosan concentrations. CV-1 protein content in the incubation medium is 0.62 mg/ml. Abscissa axis - common logarithm of phenosan concentration; ordinate axis – concentrations of total sugars (at the top) and glucose (at the bottom) in mg · ml-1.

26

E. M. Molochkina, Yu. A. Treschenkova, I. A. Krylovet al.

Figure 2.Phenosan effect on the initial accumulation rate and yield of glucose 2 h after the process beginning at different celloviridin (CV-2) protein concentrations in the reaction mixture. At the right – phenosan concentrations used. Arbitrary units – mg · ml-1· min-1· 104

Thus the expressed increase of the product yield of MCC hydrolysis by celloviridin complex under the effect of a wide spectrum of phenosan concentrations, including ultralow doses was observed. Ultralow concentrations of phenosan increase the product yield similarly or greater than “usual” ones.

An Antioxidant from Hindered Phenols Group Activates Cellulose Hydrolysis…

27

Apparently, the observed action of the preparation, even in “usual” concentrations, is associated not with its antioxidant (antiradical) properties, but with direct effect on the enzymic system components. The results obtained allow a suggestion that the phenosan effect on productivity and weight gain of livestock (associated with its antioxidant activity) used in the agriculture may be contributed by its activating effect on cellulases. Apparently, ultralow dose may have more expressed effect than those currently used. At the next stage we studied the phenosan effect on the target product (glucose) formation in cellulose hydrolysis with different celloviridin concentrations. In this part of the work, CV-2 was used. Figure 2 shows results on the phenosan influence on glucose formation at different CV-2 concentrations. As CV-2 protein concentration in the reaction mixture increased from 0.15 to 0.62 mg/ml, the initial reaction rate and glucose yield 2 h after beginning also increased. All studied phenosan concentrations caused similar stimulating effect on these parameters. From the Figure 2 one may calculate that in the presence of phenosan reaching of the initial rate of glucose formation equal to control requires almost twice less celloviridin; obtaining of the yield of glucose 2 h after the reaction beginning equal to control requires 1.5 times lower amount of the enzyme preparation. Thus, the possibility to economize the expensive enzyme complex applied in the industry for CCS hydrolysis using phenosan to stimulate the process, including in ULD, is obvious.

CONCLUSIONS At this stage of investigation, we may not indicate what namely components of the studied enzymic system and stages are touched by the phenosan action, and what the mechanisms of its influence are. Nevertheless, the fact of stimulating effect of this antioxidant on the action of the complex cellulase system may be considered established. As a prospect, this may be used for production of fuel and foodstuff from CCS. Since cellulase complexes obtained from different microorganisms act by similar mechanisms, apparently, phenosan will also activate other cellulase complexes produced from microbiological materials more effective than Trichoderma Viride.

ACKNOWLEDGMENTS The authors are thankful to T. Onishchenko (Murashova) and M. Zakharov for great help in the experimental work.

REFERENCES [1] [2]

Rabinovich M.L.// Prikl Biokhim Mikrobiol. 2006. V.42(1).P.5-32. http://www.sibbio.ru/products/bird/celo_bird.php

28 [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]

E. M. Molochkina, Yu. A. Treschenkova, I. A. Krylovet al. Burlakova E.B., Konradov A.A., Maltseva E.L. // Khimicheskaya Fizika. 2003. V. 22(2). P. 21-40. (in Russian) http://www.barva.com.ua/page.php?14 http://www.alvi.tamb.ru/production/fenozan_kisl1.htm http://chemindustry.ru/rus/chemicals/DiButylOxyphenylPropionicAcid.php Maltseva E. L., Palmina N. P., Burlakova E. B. // Membr Cell Biol. 1998. V.12(2). P.251-268. Klesov A. A., Chernoglazov V. M., Rabinovich M. L., Glazov M. V., Adamenkova M. D. // Biokhimia. 1983. V.48(9). P.1411-1420 (in Russian). Pustovalova L. M. Laboratory Manual on Biochemistry. Rostov-on-Don. Phoenix. 1999. 542 P. (Russian) Lowry O. H., Rosebrough N. J., Fare A. L., Randall R. J.// J Biol Chem. 1951.V.193. N1. р. 265-275 Klesov A. A., Grigorashch S. Iu. // Biokhimia. 1980. V. 45(2). P. 228-241. (in Russian) Kates M. Techniques of Lipidology: Analysis, Isolation and Identification of Lipids. American Elsevier Pub. Co. Inc. New York. N.Y. 10017. 1973 Molochkina E. M., Ozerova I. B. // Radiats Biol Radioecol. 2003. V. 43(3). P. 294-300 (in Russian) Treschenkova Yu. A., Burlakova E. B., Goloschapov A. N. // Radiats Biol Radioecol. 2003. V. 43(3) P.320-323 (in Russian)

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.29-43 ©2010 Nova Science Publishers, Inc.

Chapter 4

A-TOCOPHEROL AS MODIFIER OF THE LIPID STRUCTURE OF PLASMA MEMBRANES IN VITRO IN A WIDE RANGE OF CONCENTRATIONS STUDIED BY SPIN-PROBES

V. V. Belov, E. L. Maltseva and N. P. Palmina* N.M. Emanuel Institute of Biochemical Physics RAS, Moscow, Russia

ABSTRACT α-Tocopherol (α-TP) is an effective natural antioxidant and important component of biological membranes localized in the lipid bilayer and capable of changing their structural dynamic state. . For this reason, it was of interest to study α-TP effect in a wide range of concentrations (10-25 М - 10-4 М) on viscosity parameters and thermally-induced structural transition of the lipid bilayer of plasmatic membranes of liver cells of mice in vitro. The changes of structural parameters, namely, the order parameter of surface and micro-viscosity of hydrophobic regions of the lipid bilayer in membranes, were measured on Bruker EMX ESR-spectrometer (Germany) by the spin probe method by use two stable nitroxyl radicals - 5- and 16-doxylstearic acids localized at the different depths in the membrane ~8 A0 and ~20 A0 correspondingly. The “dose - effect” dependencies were nonlinear and polymodal, with statistically reliable increases of viscous parameters of the membrane in three ranges of α-TP concentrations: (1) in the range of traditional “physiological” concentrations of 10-9 – 10-4 M; (2) in the range of ultra-low doses of 1017 – 10-9 М, and even (3) in the range of apparent concentrations or dilutions of 10-25 – 1017 М. The mechanisms of the effect of α-TP in each of these ranges localized in the lipid bilayer are discussed. By study the temperature dependencies of micro-viscosity value a new thermally induced structural transition has been found at “physiological” temperatures of 309 – 3130K for α-TP concentrations, including ultra-low doses, to which maxima on dose dependencies.

* Corresponding Address to: N.P. Palmina (Ph.D., D.Sci.), IBCP RAS, Kosygin str.4, 119334 Moscow, Russia

30

V. V. Belov, E. L. Maltseva and N. P. Palmina

Keywords: α-tocopherol (α-TP), ultra-low doses (ULD), spin probes, microviscosity and rigidity of lipids, thermo- induced structural transitions in lipids (TIST)

AIMS AND BACKGROUND In biological membranes α-tocopherol (α-TP) performs an important function of preserving its wholeness by participating in regulation of peroxide oxidation of lipids (POL) as one of the most effective natural antioxidants [1, 2]. Due to its lipophily and localization in all cell membranes α-TP affects their structural state which is a central unit in the POL cycle and on which, in turn, activity of many membrane-bound enzymes participating in the most important biochemical processes in the cell depends [3]. At present, the function of α-TP that modifies the membrane structure is studied only in a rather limited range of physiological concentrations; meanwhile, the problem of biologically active substance (BAS) action in ultra-low doses (ULD) becomes more and more urgent [4 6]. On this subject a broad experimental material (references of many works are present in the review [4]) is accumulated; however, the mechanisms of phenomenon are not known completely. Hence, it is suggested that cellular and subcellular membranes may be one of critical targets. Previously, in a series of researches [7, 8] performed in our laboratory as well [9 – 13] it has been found that ULD of BAS injected to the laboratory animals, cellular culture or suspension of biological membranes changed the structural characteristics of the membrane lipids [13]. The plasma membranes are of special interest as a localization of some vitally important regulatory systems, which define general metabolic status of the organism, secondary messenger systems, in particular [14, 15]. This was the reason for performance of this work, aimed at the study of α-TP effect on the plasma membrane structure isolated from mice liver cells in a wide concentration range (10-25 - 10-4 М) in vitro.

EXPERIMENTAL Plasma membranes were extracted from liver cells of 40-50 mice of F1(C57xDBA2) line by consequent centrifuging, using the method described in [16]. Protein concentration was determined by Lowry method [17]. The membranes obtained were resuspended in the extraction medium; the protein concentration was increased to 2.5 mg/ml of suspension. Thereafter, this suspension was poured into 1 ml Eppendorf tubes and stored in a freezer at 50°C. In the day of experiment the required quantity of membranes is defrosted, the suspension was shaken and used in the work. A specimen for measurements on ESR spectrometer represented a non-viscous liquid suspension of membranes in the SET extraction medium (pH 7.3) containing saccharose (0.25 M), tris-HCl (10 mM) and EDTA (1 mM). This solution (150 μl) was placed to a long, narrow quartz trough with high Q-factor. This provided homogeneous temperature by the trough volume. Thermostatics accuracy was controlled by a thermocouple placed to the resonator cavity. By thermal accessory ER 4131 VT, this accuracy equaled ”0.05°C. Record of ESR spectra performed every 3-5 minutes. After multiple recording of spectra in the control the membranes were moved to a flask and

a-Tocopherol as Modifier of the Lipid Structure…

31

added by α-TP at any concentration. The suspension was thoroughly blended and placed back to the trough and then to the ESR resonator cavity. Thus, control and test measurements were performed for the same membranes. α-TP alcohol-aqueous solutions were obtained by consecutive dissolution in a quartz dish next by one order of magnitude from its initial 10-1 M solution in alcohol of advanced purification by MERCK Company (Germany) down to 10-3 M, and then by distilled water. Liquid was taken from the volume by semiautomated pipette with one-off nozzles. All solutions were blended in an electric shaker with controlled speed and time of 1 min. Dynamic state of the membrane lipids was studied by the spin probe method [18, 19] on Bruker EMX ESR-spectrometer and was described by structural parameters at constant temperature of 293 K – order parameter (S) which means rigidity of surface areas of lipids and micro-viscosity of deep hydrophobic regions, as well as thermally induced structural transitions in the lipid bilayer. The required parameters were calculated from ESR spectra obtained in three accumulations, in semiautomatic mode with the help of the original software in Origin 6.1 environment. The spin probe - stable nitroxyl radical of 5doxylstearic acid (probe C5) – was used to study the rigidity of the surface lipid slices of the membrane (Fig.1a). Rigidity of the membrane in the probe localization zone (~8A0) was described by the order parameter - S [19], which depends on the variation amplitude of probe rotation transversal line from the middle orientation of surrounding lipid molecules:

S=

where

TII − T⊥ 1 ⋅ , 1 k T zzc− (Txxc + Tyyc ) 2

k=

TII + 2T⊥ Txxc + Tyyc + Tzzc

is a correction factor for difference in monocrystal and experimental specimen polarities.

Txxc , T yyc , Tzzc are principal values of hyperfine interaction (HFI) tensor obtained for monocrystal. In the case of fast rotation of spin probes about their molecular axis (S< 0.8), the HFI tensor becomes axially symmetrical, and its principal values TII and T⊥ averaged by motion may be obtained from maximal and minimal splits 2Tmax and 2Tmin at the ESR spectrum (Fig. 1a):

TII = Tmax ,

T⊥ = Tmin + 1.32 + 1.86 lg(1 − S пр ) , where S пр =

Tmax − Tmin 1 Tzzc − (Txxc + T yyc ) 2

V. V. Belov, E. L. Maltseva and N. P. Palmina

32

is the approximate orderliness parameter without regard to corrections for polarity. 5-doxylstearic acid (probe С5)

16-doxylstearic acid (probe С16)

OH O

N

O

O

O

N O

OH O

a

b

Figure 1. The ESR spectra and structural formulae of С5 (a) и С16 (b) spin probes localized in plasma membranes of liver cells in mice. The probe concentration is 6×10-5 M. Protein concentration is 2.5 mg/ml. Temperature is 293 K.

To study the micro-viscosity of the deep hydrophobic regions of the lipid bilayer the stable nitroxyl radical – 16-doxylstearic acid (probe C16) was used as spin probe (Fig.1b). In the localization zone of this probe (~20A0) the micro-viscosity value was characterized by its rotational correlation time, τс which is numerically equal to time required for C16 probe to turn around its longitudinal axis by π/2 angle. It was calculated by the formula used for fast anisotropic rotation of the radical [18]:

τ с = 6,5 ⋅ ΔН 0 (

I0 − 1) ⋅ 10−10 , s I−

Spectrum parameters in the formulae for S and τс were measured automatically, using original software in the Origin 6.1 environment with standard extreme search algorithms at the given sampling interval of 0.06 Gs. For instance, measuring error for the distance between internal and external extremes in the well resolved ESR spectrum of the probe C5 did not exceed 0.2 Gs. The main contribution to the error of mean S and τс determination was made by the random component determined, first of all, by statistic character of the values under

( )

study: Sϕ = random

t (95%, f ) * σ (where ϕ is τс or S parameter calculated from the n

experimental data). As a result, for τс, relative random error of the mean value determination

a-Tocopherol as Modifier of the Lipid Structure…

33

did not exceed 1%, and for S – 0.15%. In each particular experiment, calculation error for separate τс and S values did not exceed 0.04 ns and 0.002, respectively. Since we operated with a homogeneous material, deviations from the mean values obtained with all controls considered for membranes of a single extraction coincided with the mentioned values. The analysis of literature data shows the precision of our results to deviations of τс and S values obtained for the different membranes [20 – 24]. To obtain temperature dependencies of dynamic characteristics of the membrane lipids, the sample was thermostatically controlled with the accuracy ±0.050K on a thermal accessory ER 4131 VT by Bruker Company. The measurements were performed in the temperature range of 285 – 320 K with 2 K step. Thermo-induced structural transitions in the lipid bilayer were demonstrated by salient points between linear sections of temperature dependencies plotted in Arrhenius coordinates: Lg (dynamic parameter) vs 1/T. Salient points were the points added to straight line put the correlation coefficient out of the norm defined by the number of degrees of freedom and 95% statistical reliability. For each α-TP concentration, on each membrane extraction 3-5 parallel determinations of the effect were performed; totally, 2 series of experiments on membranes isolated in different times of year were performed. The average effects of α-TP are shown on graphs, expressed in percents in relation to control, obtained after statistical treatment of all these results by parametric and nonparametric statistics methods using Statistica and Origin 6.1 software packages with 95% statistical reliability.

RESULTS AND DISCUSSIONS Structural parameters of the membrane lipids are important to describe its structural dynamics state at the given temperature. Therefore, it was studied the effect of α-TP on the rigidity of surface areas and microviscosity of deep lipid regions of plasma membranes at 293 K. These data are shown in Figs. 2a and 2b, and are expressed in percents in relation to the control. Hence, each point is the result of averaging of 6 to 10 independent measurements (for every concentration). The dose dependencies shown are polymodal, which is typical of BAS action in a wide range of concentrations, including ULD, where maxima corresponding to increase of a S and τс observed in definite dose ranges, separated from one another by the socalled “dead zones”, in which effect is not displayed. In the range of traditional “physiological” concentrations (10-9 – 10-4 М), in which α-TP usually acts in the organism, and in the ULD range (10-17 – 10-9 М) the effects are observed in both surface and deep hydrophobic regions of the membrane lipids. However, in the range of the so-called apparent concentrations or dilutions (<10-17 М), when the probability of containing even one molecule in the membranes is close to zero, the effect is observed exclusively in the depth the membrane. It should be noted that the values of α-TP effects in all three dose areas are similar. Hence, the effect in the surface areas of plasma membrane lipids is markedly higher than in microsomal membranes obtained by us [13].

V. V. Belov, E. L. Maltseva and N. P. Palmina

34 3.5

a

3.0

EFFECT, %

2.5 2.0 1.5 1.0 0.5 0.0 -0.5 4

6

8

10

12

14

16

18

20

22

24

26

-Lg[α-tocopherol] 8

b

7

EFFECT, %

6 5 4 3 2 1 0 -1 4

6

8

10

12

14

16

18

20

22

24

26

-Lg[α-tocopherol] Figure 2. The effect of α-TP on the rigidity of surface (a) and microviscosity of deep hydrophobic (b) regions of plasma membranes at 293 K. Protein concentration in the membrane suspension is 2.5 mg/ml. The control values: S = 0.644±0.004, τс = (2.10±0.04)×10-9 s.

Beside changes of the main parameters (τс and S) of the lipid bilayer at constant temperature, very important characteristics described the dynamic state of the membrane lipids are the quantity and quality of thermally induced structural transitions. They represent

a-Tocopherol as Modifier of the Lipid Structure…

35

cooperative structural transformations of groups of lipids with temperature increase, which are accompanied by a discontinuous change of the main parameters (τс and S). As described in “Materials and Methods”, for the purpose of detecting transitions the temperature dependencies of τс and S for the control and α-TP concentrations, to which maxima or the absence of effects on dose dependencies (Fig. 2) correspond, were presented in the Arrhenius coordinates – Lg (dynamic parameter) on 1/T (Figs. 3 and 4). The main data on location of transitions in the hydrophobic regions of lipid bilayer are summarized in Table 1, and on surface lipid areas - in Table 2. Despite the fact that thermo- induced structural transitions occur in different depth areas of the membrane, some general regularities are observed. For instance, compared with the control characterized by two transitions, all α-TP concentrations, to which maxima on dose dependencies (Figs. 2a and 2b), including ULD, induced the third additional transition. And ULD - 10-15 М of α-TP in the surface areas of the membrane lipids also induced the fourth transition in the range of “physiological” temperatures at 309 – 313 K (dashed). The total number of transitions does not change for concentrations corresponding to “dead zones” on the dose dependencies in Figs. 2a and 2b. However, compared with the control, they shift towards higher temperatures, including “physiological” ones. Taking into consideration an important role of phase transitions in lipids of biological membranes in the vital activity of the cell (in the change of membrane permeability, pore formation, fusion of membranes, excitability of nerve tissues and nervous pulse conduction by axon, thermoregulation, synaptic exocytosis, etc. [25, 26], it was suggested that thermo-induced transitions observed by α-TP may be regulatory for the cells and their membranes.

Figure 3. The temperature dependencies of rotation correlation time - τс of C16 presented in Arrhenius coordinates in control and at α-TP effect in 10-4, 10-7, 10-10, 10-14, 10-18 and 10-22 M concentrations in vitro. Maximal relative error of values Lg [τс] obtained in the series of three measurements for each temperature did not exceed 0.2%.

36

V. V. Belov, E. L. Maltseva and N. P. Palmina

Figure 4. The temperature dependencies of order parameter - S of C5 presented in Arrhenius coordinates in control and at α-TP effect in 10-4, 10-9, 10-15 and 10-21 M concentrations in vitro. Maximal relative error of values Lg[S] obtained in the series of three measurements for each temperature did not exceed 0.2%.

a-Tocopherol as Modifier of the Lipid Structure…

37

Table 1. The thermo-induced structural transitions in hydrophobic lipid regions (~20 A0) of liver plasma membranes in control and under the effect of -TP at different concentrations in vitro. Т,К 285 287 289 291 293 295 297 299 301

control

10-4М

10-7М

10-10М

10-14М

10-18М

10-22М

303 305 307 309 311 313 315 317 319

Note: The structural transitions are marked grey.

Table 2. The thermo-induced structural transitions in surface (~8 A0) of the lipid bilayer of liver plasma membranes in control and under the effect of -TP at different concentrations in vitro. Т,К 285 287 289 291 293 295 297 299 301 303 305 307 309 311 312 313 315 317 319

control

10-4М

Note: The structural transitions are marked grey.

10-9М

10-15М

10-21М

38

V. V. Belov, E. L. Maltseva and N. P. Palmina

As mentioned above, polymodal type of dose dependencies shown in Figs. 2a and 2b is promoted by α-TP effects in three dose ranges. However, the main contribution in mechanism of α-TP action in each range may be made by different processes. Primarily, ordering of the surface lipids and increase of micro-viscosity of hydrophobic regions of the lipid bilayer under the effect of α-TP at “physiological” concentrations (10-9-– 10-4 М) may be associated with restrictions for packing of hydrocarbon chains of lipids near α-TP molecule reduced their conformational mobility [27]. A significant contribution to these processes can be made by α-TP interaction with phospholipids molecules. The different biophysical methods give indirect evidences of not random distribution of α-TP in the membrane, but formation of complexes with definite stoichiometry [28 - 30]. Moreover, an evidence of hydrogen bond formation between hydroxyl group of α-TP molecule and oxygen either carbonyl or phosphate group of phospholipid molecules is present in [31]. However, lateral distribution of the complexes in the membrane plane may be random, and they may also form domains and induce phase separation [32]. Thus, being well mixed with phospholipids, α-TP may cause a significant effect on their structural transformations. The evidence for this opinion is occurrence of additional third thermo-induced structural transition in the “physiological” temperature range in the depth of lipid bilayer under the action of α-TP in 10-4 and 10-7 М concentrations (Table 1) and in surface membrane lipids for 10-4 М concentration (Table 2). To our point of view, this is associated with formation of such domains with denser packing of lipids. The increase of viscosity or decrease of fluidity of membrane lipids in the processes of penetration of α-TP at “physiological” concentrations have been demonstrated for many times in experiments on biological (see [33, 34], for example) and artificial membranes by different physical methods, including ESR [27, 35, 36]. Our data correlate with these results. In the ULD range (10-17 – 10-9 M) the effect in the surface lipids of plasma membranes is markedly higher than in microsomal membranes studied by us before [13]. One of the possible mechanisms may be specific interaction between α-TP and binding sites of the membrane that, judging by data from the literature [7] and our results [9], can lead to a change of viscosity properties of the membrane. We have suggested that in this case the role of such binding site may be played by protein kinase C (PKC), the membrane-bound and lipid-dependent enzyme, which content in plasma membranes is considerably higher than in microsomal membranes. It was obtained the high correlation degree (ρ = 0.0047, r = 0.87) between order parameter (S) of surface lipids of plasma membranes and inhibition of PKC activity by α-TP in ULD [37]. This fact may be an indirect evidence of such kind mechanism (Fig. 5). To our point of view, another probable explanation of increase of the lipid microviscosity under α-TP action in ULD may be given by α-TP induced initiation of new highly ordered microdomain complex formation or modification of already existing ones. In this case, the role of such complexes may be played by rafts representing special areas in the plasma membrane, which demonstrate much higher ordering compared with their microsurrounding due to increased content of cholesterol, sphingomyelin and gangliosides, and mostly saturated fatty-acid chains of phospholipids [38, 39]. It is known that a lot of proteins participating in signal transduction in the cell are capable of being localized in rafts including PKC as well [39, 40]. The lifetime of such complexes is short (less than 1 ms); however, the action of external signals (which role may be played by ligands) inducing

a-Tocopherol as Modifier of the Lipid Structure…

39

conformational changes in the protein molecules, can extend it significantly (up to several minutes) and, moreover, lead to association of several rafts into a larger domain [40, 41]. Therefore, we suggest that such processes may be accompanied by the interaction of α-TP in ULD with PKC during inhibition of enzyme activity.

Figure 5. The change of rigidity of the surface membrane lipids and inhibition of protein kinase-C activity in vitro depending on α-TP concentration (a) and correlation between these parameters (b), ρ = 0.0047, r = 0.87.

40

V. V. Belov, E. L. Maltseva and N. P. Palmina

To our point of view, the presence of α-TP in the membrane in both “physiological” concentration and ULD may also affect formation of rafts. For instance, as one of the mechanisms of their formation, spatial incompatibility between solid styrene structure of cholesterol molecule and rigid bending of unsaturated hydrocarbon chain of the neighboring phospholipid molecule, having cis-double bond in С9-С10 position, in the liquid-crystal phase is considered [41]. Such mutual packing of molecules leads to pushing cholesterol out of the areas with unsaturated phospholipids, with its consequent concentration and stabilization in the areas with mostly saturated fatty-acid chains of phospholipids. It should be noted in this connection that α-TP, in turn, more preferably interacts with poly-unsaturated phospholipids [29, 42], thus promoting cholesterol pushing out and simplifying formation of rafts at both “physiological” concentrations and ULD. However, in the range of so-called “apparent” concentrations (<10-17 М), where the probability of containing even one α-TP molecule in the membrane is close to zero, the effect was observed exclusively in the deep hydrophobic regions of the membrane lipids. As shown in our previous work [9] the properties of polar solvent (water) of α-TP make a considerable contribution into the mechanism of transmission of “information” about the substance, in one form or another, during preparation of solutions and further interactions on the way of signal transmission to the membrane. This, finally, provides the effects obtained. Unfortunately, today we fail to know the precise molecular mechanism of the effects in the range of “apparent” concentrations. However, some experimental data [43, 44] draw attention to dynamic characteristics of the polar medium in these processes (for instance, frequency of formation and degradation of short-living aqueous associates, and interaction between them), which are rather sensitive to external impacts and variation will affect its dynamic structure. To conclude the consideration, our work is the first presenting the study of α-TP effect in a wide range of real and “apparent” concentrations (10-25 – 10-4 M), on lipid structure characteristics of liver plasma membranes in vitro. Dose dependencies are polymodal with statistically reliable increase of rigidity of surface (~8 A) and microviscosity of deep hydrophobic areas (~20 A) of membrane lipids caused by different mechanisms of α-TP action into three dose ranges: (1) 10-9 – 10-4 M – nonspecific effect on the lipid membrane structure at incorporation into it; (2) 10-17 – 10-9 M – specific interaction with the binding sites on the membrane surface; (3) 10-25 – 10-17 M – polar properties of α-TP solvent (in the surface areas of the plasma membrane the effect is not observed). The temperature dependencies of τс and S demonstrated occurrence of new thermo-induced structural transition compared with the control in the range of “physiological” temperatures of 309 – 3130K at α-TP concentrations (included ULD) giving the maximal effects at 2930K. The authors are greatly thankful to Professor E.B. Burlakova for scientific discussion. This work is supported by Grant No. 01-RAS-15 of the Federal Program of Fundamental Research for RAS Department of Chemistry and Material Sciences “Biomolecular and Medical Chemistry”

REFERENCES [1]

E.B. Burlakova., S.A. Krashakov, N.G. Krapova: The Role of Tocopherols in Biomembrane Lipid Peroxidation. Membr. Cell Biol., 12, 173 (1998).

a-Tocopherol as Modifier of the Lipid Structure… [2] [3]

[4]

[5]

[6]

[7]

[8]

[9] [10]

[11]

[12]

[13]

[14] [15] [16] [17] [18] [19] [20]

41

P.J. Quinn: Molecular Associations of Vitamin E. Vitam. Horm., 76, 67 (2007). E.B. Burlakova, N.P. Pal’mina N.P., E.L. Mal’tseva: In: Membrane Lipid Oxidation. Vol. 3. (Ed. Carmen Vigo-Pelfrey). CRC Press. Boca Rato-Ann Arbor- Boston., 1991, 209-237. E.B. Burlakova., A.A. Konradov, E.L. Mal’tseva: Effect of Super-Small Doses of Biologically Active Substances and Low-Intensity Physical Factors. J. Adv. Chem. Phys. 2, 140 (2003). I.P. Ashmarin, E.P. Karaseeva, T.V. Lelekova: To the Question about Development of the Problem of Efficacy of Supersmall Doses of Biologically Active Substances. Russ. Chem. J. 18, 21 (1999). S.V. Zaitsev, A.M. Efanov, L.A. Sazanov: The Common Phenomena and Possible Mechanisms of Action of Biologically Active Substances in Super-Small Doses. Russ. Chem. J. 18, 28 (1999). A.S. Polezina, K.A. Anikienko, V.K. Kurochkin: Elaboration of Methodological Basis to Detect Damage Effect of Super-Small Concentrations of Physiologically Active Substances on the Blood Human Formed Elements. Russ. Chem. J. 18, 72 (1999). G.T. Rikhereva, I.N. Golubev, S.A. Kopylovskii, I.A. Prudenko, I.I. Mihkaleva: The in vitro Interaction of Delta-Sleep-Inducing Peptide with Cell Membranes. Bio-Org. Chem., 25, 334 (1999). N.P. Pal’mina, N.G. Bogdanova, E.L. Mal’tseva, E.I. Pinzar: Phorbol Esters as Modifiers of Structure of Biological Membranes. Biol. Membr. 6, 1077, (1993). E.L. Mal’tseva, N.P. Pal’mina: The Effect of Phorbol Ester in a Wide Range of Concentrations on the Lipid Structure of Microsome Membranes of Tumor Cells in vitro. Radiat. Biol. Radio-Ecol., 43, 301 (2003). V.E. Zhernovkov, N.G. Bogdanova, N.P. Pal’mina: Effect of Ultra Low Concentrations of Thyroliberin on Structural Parameters of Membrane Lipids of Endoplasmic Reticulum in Vitro. Biol. Membr. (Moscow), 22, 388 (2005). V.V. Belov, E.L. Mal’tseva, N.P. Pal’mina: A study of the Effect of Low D-Tocopherol Concentrations on the Dynamical parameters of Microsomal Membranes by the Method of Spin Probes. Biofizika, 52, 75 (2007). V.V. Belov, E.L. Mal’tseva, N.P. Pal’mina, E.B. Burlakova: The Role of Solvent Polarity in the Mechanism of Action of Biologically Active Compounds at Ultra-Low Concentrations. Dokl. Biochem. Biophys. (Moscow), 399, 362 (2004). Y. Nishizuka: The Protein Kinase C Family and Lipid Mediators for Trans-Membrane Signaling and Cell Regulation. Alcohol. Clin. Exp. Res., 25, 3S (2001). M. Liscovich, L.C. Cantley: Lipid Second Messengers. Cell, 77, 329 (1994). LOTEN E.G., REDSHAW-LOTEN J.C.: Preparation of Rat Liver Membranes in a High Yield. Analit. Biochem., 154, 183 (1986). Lowry O H.., Rosebrough N. J., Barr A.L., Randall R.J.: Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem., 193, 265 (1951). A.N. Kuznezov: The Spin-Probe Method. Nauka, Moscow, 1976. O.Griffit, P. Jost: In: Spin Labeling. Theory and Applications. (Ed. L.J. Berliner). Acad. Press, New York-San Fransisco-London, 1979, 489-569. E.K. Ruuge, E.N. Gerasimova: In: Method of Spin Labels and Probes (Eds. N.M. Emanuel, R.I. Zhdanov). Nauka, Moscow, 1986, 225-239.

42

V. V. Belov, E. L. Maltseva and N. P. Palmina

[21] O.A. Azizova, T.I. Torkhovskaya, Yu.M. Lopukhin: In: Method of Spin Labels and Probes (Eds. N.M. Emanuel, R.I. Zhdanov). Nauka, Moscow, 1986, 239-250. [22] M.T. Curtis, D. Gilfor, J.L. Farber: Lipid Peroxidation Increase the Molecular Order of Microsomal Membranes. Arch. Biochem. Biophys., 235, 644 (1984). [23] Parshina E.L, Gendel L.Ia., Rubin A.B. The Influence of New Hybrid Antioxidants Ichphans on the Kinetics of Ascorbate-Induced Reduction of Spin Probe Radical Centers in Liposomes. Biofizika, 50, 676 (2005). [24] K.Tsuda: Electron Paramagnetic Resonance Iinvestigation of Modulatory Effect of Benidipine on Membrane Fluidity of Erythrocytes in Essential Hypertension. Heart Vessels, 14, 1505 (2008). [25] V. F. Antonov, E.Yu. Smirnova, E.V. Shevshenko: Lipid Membranes under the Phase ransitions. Nauka, Moscow, 1992. [26] D. P. Kharakoz: Phase Transition in Lipids and the Problem of Homoiothermia. Biofizika, 45, 569 (2000). [27] S. Wassal, L.Wang, R.C. Mc-Cabe, W.D. Ehringer, W. Stillwell: Electron Paramagnetic Resonance of the Interaction of D-Tocopherol with Phospholipid Model Membranes. Chem. Phys. Lipids, 60, 29 (1991). [28] V.E. Kagan, P.J. Quinn: The Interaction of Alpha-Tocopherol and Homologes with Shorter Hydrocarbon Chains with Phospholipid Bilayer Dispersions. A Fluorescence Probe Study. Eur. J. Biochem., 171, 661 (1988). [29] W. Stillwell, T.Dallman, A.C. Dumanuel, F.T. Crump, L.J. Jenski: Cholesterol versus D-Tocopherol Effects on Properties Bilayer Model from Heteroacid Phosphatidylcholines. Biochemistry. 35, 13353 (1996). [30] P.J. Quinn: Characterisation of Clusters of Alfa-Tocopherol in Gel and Fluid Phase of Dipalmitoylglycerophosphocholine. Eur. J. Biochem.,233, 916 (1995). [31] J.C. Gomez-Fernandez, J. Villalain, F.J. Aranda, A. Ortiz, V. Micol, A. Coutinho, M.N. Berberan-Santos, M.J. Prieto: Location of D-Tocopherol in Membranes. Ann.N.Y. Acad. Sci., 570, 109 (1989). [32] X. Wang, H. Takahashi, I.Hatta, P.J.Quinn: An X-ray Diffraction Study of the Effect of D-Tocopherol on the Structure and Phase Behavior of Bilayer of Dimytristoylphosphatidyl-ethanolamine. Biochim. Biophys. Acta, 1418, 335 (1999). [33] M. Steiner: Vitamin E Changes the Membrane Fluidity of Human Platelets. Biophys. Biochim. Acta, 640, 100 (1981). [34] S. Urano, Y. Inomori, T. Sugawara, Y.Kato, M. Kitahara, Y. Hasegawa, M. Matsuo, K. Murai: Vitamin E: Inhibition of Retinol-Induced Hemolysis and Membrane-Stabilizing Behavior. J. Biol. Chem., 267, 18365 (1992). [35] Koyama T., Araiso T.: Effect of alpha-tocopherol-nicotinate administration on the microdynamics of phospholipids of erythrocyte membranes in human subjects. J. Nutr.Sci. Vitaminol., 34, 449 (1988). [36] D. Scmidt, H. Steffen, C. Planta: Lateral Diffusion Order Parameter and Phase Transition in Phospholipids Bilayer Membranes Containing Tocopheryl Acetate. Biochim. Biophys. Acta, 443, 1 (1976). [37] N.P. Pal’mina, Е.L. Mal’tseva, N.V. Кurnakova, Е.B. Burlakova: The D-Tocopherol Effect in a Wide Concentrations Range (10-2 -10-17 M) on the Activity of Protein Kinase

a-Tocopherol as Modifier of the Lipid Structure…

[38] [39] [40]

[41]

[42]

[43] [44]

43

C. Relationship to Cell Proliferation and Tumour Growth. Biokhimia (Moscow), 52, 193 (1994). P.H.M. Lommerse, H.P. Spaink, T. Schmidt: In vivo Plasma Membrane Organization: Results of Biophysical Approaches. Boichim. Biophys. Acta., 1664, 119 (2004). L. J. Pike: Lipid Rafts: Bringing Order to Chaos. J. Lipid Res., 44, 655 (2003). K. Monastyrskaya, A. Hostettler, S. Buergi, A. Draeger: The NK1 Receptor Localizes to the Plasma Membrane Microdomains and its Activation is Depended on Lipid Raft Integrity. J. Biol. Chem., 280, 7135 (2005). W. Subczynski, A. Kusumi: Dynamics of Raft Molecules in the Cell and Artificial Membranes: Approaches by Pulse EPR Spin Labeling and Signals Molecule Optical Microscopy. Boichim. Biophys. Acta., 610, 231 (2003). A.N. Erin, M.M. Spirin, L.V. Tabidze, V.E. Kagan: Formation of Alfa-Tocopherol Complexes with Fatty Acids. A Hypothetical Mechanism of Stabilization of Biomembranes by Vitamin E. Biochim. Biophys. Acta., 774, 96 (1984). G.M. Zubareva, A.V. Kargapolov, L.S. Jaguzhinskii: Effect of Superlow Quantities of Hydrogen Peroxide on Water Base of Solutions. Biofizika, 48, 581 (2003). A.N. Smirnov, A.V. Siroezhkin: Supramolecular Water Complexes. Russ. Chem. J., 28, 125 (2004).

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.45-63 ©2010 Nova Science Publishers, Inc.

Chapter 5

SUPERCRITICAL CARBON DIOXIDE SWELLING OF POLYHETEROARYLENES SYNTHESIZED IN N-METHYLPYRROLIDONE Inga A. Ronova*1, Lev N. Nikitin1, Gennadii F. Tereschenko1 and Maria Bruma2 1

A.N. Nesmeyanov Institute of Organoelement Compounds, Moscow, Russia 2 “Petru Poni” Institute of Macromolecular Chemistry, Iasi, Romania

ABSTRACT Five series of polyheteroarylenes have been investigated with regard to their physical properties before and after swelling with supercritical carbon dioxide. The study of the dependence of glass transition temperature and free volume of polymers on their conformational rigidity showed that the process of swelling in supercritical carbon dioxide is influenced by the voluminous side groups and by the high boiling solvent Nmethylpyrolidinone used for the preparation of the polymers which facilitates the formation of crosslinks or complexes with the macromolecular chains.

Keywords: polyheteroarylenes, supercritical carbon dioxide, swelling, conformational rigidity, free volume, crosslinks

1. INTRODUCTION It is known that polyheteroarylenes exhibit high thermal stability and excellent mechanical properties determined by their conformational rigidity which differs significantly from that of analogous aliphatic polymers, from 10-15Å to thousands of Angstroms [1]. Most of these polyheteroarylenes are amorphous glassy polymers and most of their physical * A.N. Nesmeyanov Institute of Organoelement Compounds, 28 Vavilova, 119991 Moscow, Russia, E-mail [email protected]

46

Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko et al.

properties correlate well with their conformational rigidity [2]. At the same time, it is known that physical properties of amorphous glassy polymers depend not only on their chemical structure, but also on the history of their preparation, on the physical aging processes and other. In recent years a great interest was given to the modification of amorphous glassy polymers by treatment with supercritical carbon dioxide (sc CO2) with the aim to manipulate their physico-chemical properties [3]. It is believed that the swelling process with sc CO2 can change some of these properties. If the swelling process is directly connected with the conformational rigidity of the polymers, this gives a good possibility to change their properties. Therefore, the study of the influence of the swelling process on glassy polymers of different chemical structure is very important. Previously we have studied a polyetherimide containing hexafluoroisopropylidene groups which was subjected to the treatment with supercritical CO2 under a temperature of 40ºC and 65ºC, and a pressure of 150 bar; we have found that under the temperature of 40ºC microcavities were formed and an increase of the free volume of 23.1% took place. In the case of treatment under 60ºC we have obtained nanocavities and an increase of the free volume of 56.8% [4]. Here we present a study of the dependence of glass transition temperature on conformational parameters and free volume for five groups of polyheteroarylenes and the swelling process of polymer films in supercritical carbon dioxide.

2. EXPERIMENTAL 2.1. Preparation of Polymer Films Five series of polymers have been studied whose synthesis was reported previously [513]. Thus, the series having the code AMIC contains one polyimide (1) and two polyamidic acids (2 and 3); the series having the code OXAD contains two polyoxadiazole-amidic acids (4 and 6) and one polyoxadiazole-imide (5); the series having the code PEAM contains six polyester-amides (7, 8, 9, 10, 11 and 12); the series having the code ACET contains three poly(oxadiazole-amide)s with pendant acetoxybenzamide groups (13, 14, 15); the series having the code CYAN contains four polyamidic acids (16, 18, 20, 22) and the corresponding four polyimides (17, 19, 21, 23) based on a diamine having cyano substituents and four dianhydrides containing pyromellitic, hexafluoroisopropylidene diphthalic, biphenylene or diphenylketone units, respectively (Table 1). These polymers show good solubility in Nmethylpyrrolidinone (NMP) and other polar amidic solvents having high boiling temperature. The films, having the thickness usually in the range of 30-40 mμ, were prepared by using solutions of polymers in N-methylpyrrolidinone, having the concentration of 15%, which were cast onto glass plates and heated gradually up to 210ºC. The films were carefully taken out of the substrate and were used afterwards for various measurements.

Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized…

47

Tabel 1. Repeating units of the studied polymers: AMIC, OXAD, PEAM, ACET and O R=

CYAN. Polymer Series code 1 AMIC

NH-CO

Repeating unit O

O

O

C

N O

AMIC CH2

O

N

C

O C

HO

3

AMIC

O

S

O

N

C

O HO

4

O

H

O

H

C

N

O

C

C

N

C

O

H

C

N

O

O

N

CH3 O

C

O

CH 3

C

O

O

H

O

N

C

O

N

C

C

O

O

O

H

C

N

C O

O

O

O

O

N O

O

H

O

N

C

HO

8

PEAM

O-CO

NH-CO

O-CO

N

C

O

O

OH

CH2

CH2

CO-NH

CO-O

H

C

C

CO-NH

CO-O

O C

CH3

CH3

9

CN

PEAM NH-CO

10

O-CO

O

O

CO-NH

CO-O

CN

PEAM NH-CO

11

PEAM

12

PEAM

NH-CO

O-CO

O-CO

CO-O

O

O

CO-NH

CO-O

O

O

CO-NH

CH3 NH-CO

O-CO

OH

OH

N

O

N

NH-CO

N

C

O

PEAM

H

C

C

O

7

O

C

O

N

O

O

OH

O

C

O

N

N

O

H

C

OXAD

OXAD

H

C

O

HO

6

O

C

HO

OH

N

N

C

O

C

O

5

O

HO

OH

O O

H

O O

C

OXAD

CH 3 O

N O

CH3

C

C

N

O

CH3

O

H

O

C

C

O

O

O

CH3

N

O

2

O C CH3

CO-O

CO-NH

O

C CH3

O

48

Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko et al. Table 1. (continued)

Polymer Series code 13 ACET

Repeating unit H

O

O

N

C

C

H

N

N

N

O

R

14

ACET O

H

O

O

N

C

C

H

N

N

O

N

O

R

15

ACET O

H

O

O

N

C

C

H

N O

N

R

16

CN

CYAN O

O

H

O

O

N

C

C

HOOC

17

CYAN O

O

O

H

C

N

COOH

O

O

O

CN

C

N

N O

O

18

CN

CYAN

H

O

O

N

O

C

HOOC

19

CYAN O

O

H

O

N O

N

O

CF3

O C

C

HOOC

21

CYAN

O

O

H

C

N

COOH

CF3

O

CF3

O

CN O

C

N

CN

CYAN

H N

O

O

20

O

COOH

O

CN

C

N

N

CF3 O

O

22

CN

CYAN O

O

H

O

N

C

23

CYAN

O

CN O

O

O

H

C

N

COOH

HOOC

O N

N O

N O

O

Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized…

49

2.2. Measurement of Density To measure the density of polyheteroarylene films we used the hydrostatic weighing method. The study was performed with an equipment for density measurement and an electronic analytic balance Ohaus AP 250D, precision of 10-5 g, from Ohaus Corp US which was connected to a computer. With this equipment we measured the change of sample weight during the experiment, with a precision of 0.001 g/cm3 in the value of density. Ethanol was taken as a liquid with known density. The studied polyheteroarylenes did not absorb and did not disolve in ethanol, which for these polymers had a low diffusion coefficient. Since the density of ethanol depends on temperature, every time it was measured using pycnometer. The characteristic diffusion times were in the domain of 104 – 105 s, which are 1-2 order of magnitude higher than the time of density measurement. This is why the sorption of solvent and the swelling of the film must have only insignificant influence on the value of the measured density. All measurements of the density were performed at 23ºC. The density was calculated with the equation (1):

ρs = Wa / (Wa – Wl ) ρl ,

(1)

where ρs is density of the sample, Wa is the weight of the sample in air, Wl is the weight of the sample in liquid, ρl is the density of liquid. The error of the density measurements was 0.1–0.3%.

2.3. Measurement of Glass Transition Temperature The glass transition temperature (Tg) of the polymers was measured by differential scanning calorimetry, using a Mettler DSC 12E apparatus. The samples were heated at a rate of 15ºC/min under nitrogen to above 300ºC. Heat flow versus temperature scans from the second heating run were plotted and used for reporting the Tg. The mid point of the inflection curve resulting from the second heating run was assigned as the Tg of the respective polymers. The precision of this method is ±7 – 10оС.

2.4. Calculation of Conformational Parameter and Free Volume As conformational parameter we have taken the statistical Kuhn segment Afr, which was calculated with the equation (2) [1]:

⎛ ⎞ ⎟, A fr = lim ⎜ n→∞ ⎝ nl o ⎠

(2)

where R2 is mean square distance between the ends of the chain calculated for all possible conformations; L = nlo is the contour length of the chain, a parameter which does not depend on the chain conformation; lo is the contour length of a repeating unit. All the values of Kuhn

50

Inga A. Ronova, Lev N. Nikitin, Gennadii F. Tereschenko et al.

Ly

segment were calculated with Monte Carlo method, geometry of the repeating unit was evaluated by quantum chemical method AM1 [14]. To calculate the free volume we used the method previously described [15]. We built a model of the repeating unit and its geometry was also evaluated by quantum chemical method AM1. The atoms are described by spheres having the Van der Waals radius equal to the corresponding radius of each type of atoms (Chart 1) [16].

Lx Chart 1. The monomer introducing in the box.

This model was situated in a 3D rectangular box having the axes Lx, Ly, Lz given the equation (3): Lx = xmax + Rmax – (xmin - Rmax) = xmax – xmin + 2Rmax ,

(3)

where xmax and xmin are the maximum and the minimum values of the coordinates of atom corresponding to the repeating unit, Rmax is the maximum value of the radius of atom corresponding to the repeating unit. Ly and Lz were determined in the same way. The volume of this model was calculated with Monte Carlo method. For that, in the volume corresponding to the parameters of the box, random points were generated. The number of random points, landing in the repeating unit, is m. In the beginning of calculation m is equal 0. For each random point the following conditions were verified: | rd – ri | ≤ Ri , i = 1…N, where N is the number of atoms in the repeating unit, ⎥ rd – ri⎥ is the distance between a given point and any other point in the repeating unit. In case of achievement of this conditions for at least one atom, procedure of verification stopped, number of successful events began with m+1, and next random point was generated. Van der Waals volume (Vw) was calculated with the formula (4):

Supercritical Carbon Dioxide Swelling of Polyheteroarylenes Synthesized… Vw = (m / M) Vbox

51 (4)

where M is the total number of all points, Vbox is the volume of the box. The free volume (Vf) was calculated with the formula (5):

Vf =

1

ρ

−

N A • Vw Mo

(5)

where NA is the number of Avogadro, ρ is the density of polymer, Mo is the molecular weight of the repeating unit. The value Vf , thus calculated, shows the volume which is not occupied by the macromolecules in one cm3 of polymer film. From this point on, we will call it “free volume”.

2.5. Method of Treatment with Supercritical Carbon Dioxide The experimental set-up and the method of impregnation with supercritical carbon dioxide (sc-CO2) were described in previous papers [17-19]. This experimental set-up is composed of a generator which can provide CO2 up to 35 MPa pressure (High Pressure Equipment Company, USA). A system of valves ensures the CO2 access to the reaction cell with the volume of 30 cm3. The pressure generator and the reaction cell are provided with manometers to allow a control of the pressure and the letting-in and letting-out of gas. The temperature control allows a precision better than ±0.2°С. The cell is designed for experiments at pressures up to 50 MPa and temperatures up to 120°С. CO2 desorption curves were obtained using the gravimetric technique [20]. Sample weight was measured with an Ohaus AP 250 D electronic balance interfaced with a computer. The following experimental technique was applied: the polymer sample was weighed and placed into the cell. The sample had the form of a film (typically, a disk with 15 mm diameter and thickness in the range from several to tens microns). After purging the cell with CO2 it was sealed. The pressure was increased up to the necessary value and the sample was exposed during a given time. The cell was decompressed, the polymer sample was placed on the electronic balance and the weight decrease during CO2 desorption was recorded using a computer. Then the weight swelling degree at zero time (the moment of decompression) and the CO2 diffusion coefficient were calculated. All experiments were done at 150 bar and 40ºC. The decompression speed of CO2 was near 5 mL/s. Approximate (asymptotic) formulas are typically used to analyze the desorption dynamics using gravimetric technique [21, 22]. Such formulas are only valid either for the initial or the final stage of the process. This approach can hardly give an answer to the question about the diffusion type: whether the diffusion is subjected to the Fick law or not. On the other hand, the progress of the computer technology during the recent years allows to realize numerically the analysis of the experimental data even when the complex exact solutions are used. We suppose that this makes the use of the approximate solutions superfluous. Therefore, we used the exact solutions of the diffusion problem with the uniform initial and zero boundary conditions valid in the Fick approximations. The equations

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describing the dependence of the sorbate weight on time Z(t) are known from the diffusion theory [23]. For a film-like sample this equation has the form (6):

Z( t ) 8 = 2 Z0 π

⎛ − (2 n + 1)2 π 2 Dt ⎞ 1 ⎟ exp⎜⎜ ∑ 2 2 ⎟ l n = 0 (2 n + 1 ) ⎝ ⎠ ∞

(6),

where l is the film thickness; Z0 is equilibrium coefficient of swelling for the “0” time; D is the diffusion coefficient. A numerical algorithm to find the best fit for the experimental data was realized using the least squares method. Theoretical dependencies (6) were used as the fit functions and the parameters Z0 and D were varied. Thus, the best fit allows to determine both the values of the diffusion coefficient and the initial sorbate weight (and therefore the equilibrium degree of the polymer swelling in sc CO2). In case when the exact solution (6), valid for D = const, gives a good fit for the experimental data, the diffusion is of normal type and it obeys the Fick law. Figure 1 shows the typical desorption curve for the films of studied polyheteroarylene 4 and 5 and their fit with the theoretical dependence (6). As can be seen in figure 1 our case corresponds to the normal diffusion (the experimental and theoretical curves coincide very well). Therefore, we can calculate the values of Z0 and D. These values for the studied polyheteroarylenes are given in table 2.

Figure 1. Desorption curve of CO2 for the polymer AMIC-2.

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ble 2. Diffusion coefficients ( D ) of CO2 and equilibrium coefficient of swelling ( Zo )of the studied polymers. Polymer 1 2 3 4 5 6 7 8 9 10 11 12

D, 10-10 cm2/s 40 14 29 19 0.8 22 3.4 3.0 0.16 2.3 0.8

Zo , weight % 6.9 8.7 12.5 6.9 6.6 3.7 2.6 6.9

Polymer 13 14 15 16 17 18 19 20 21 22 23 -

D, 10-10 cm2/s 4.3 5.8 3.6 15 8.7 25.9 4.4 48 80.3 349 -

Zo , weight % 0.968 2.98 2.93 4.49 4.78 3.86 4.16 7.81 4.45 9.30 -

3. RESULTS AND DISCUSSION In order to understand how the physico-chemical properties of polyheteroarylenes change after treatment with supercritical carbon dioxide (sc CO2), we analyzed such properties before and after treatment with sc CO2. Table 3 shows the values of glass transition temperature (Tg), Kuhn segment (Afr) and Van der Waals volume (Vw) of all ivestigated polymers. The glass transition temperature of these polymers was in the range of 185ºC – 280ºC and the conformational rigidity was in the domain of 12 Å – 72.5 Å. The dependence of Tg of these polymers on Kuhn segment is described by three straight lines having high correlation coefficients (Figure 2 a, b and c). For two samples OXAD ( polymers 5 and 6) the values of Tg were identical. By using the equation Y = 255.369 + 0.629 X, having such a high correlation coefficient R = 99.57 % , these Tg values can be calculated with high precision and, indeed, the difference was only 0.74oC, that situated beyond the accuracy limits of method of measuring Tg . In the case of PEAM (fig. 2a), the samples 8 and 12 are out of the straight line which is due to the low molecular weight of these two polymers. In such cases Gaussian coil has not formed yet, the packing of the chains is loose, and the conformational transitions take place very easy in glass transition state. The dependence of glass transition temperature on Kuhn segment in the case of ACET polymers is linear, with not a very high correlation coefficient (figure 2b). When measuring the glass transition temperature by using the differential scanning calorimetry (DSC) method, the error can be ±10%. From the dependence of Tg on Kuhn segment, y= 260.35 + 0.391x, we calculated the Tg of ACET polymers 14 and 15. They are 273.8 ºC and 268.4оС, respectively. In one case it is 3.8ºC higher and in the other case it is 1.6ºC lower than the experimental values (270ºC for both polymers). These differences are in the domain of experimental error of DSC method.

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a ACET

b CYAN

c Figure 2. Dependence of glass transition temperature (Tg) on Kuhn segment (Afr) for the studied polymers: AMIC, OXAD, PEAM (fig. 2a), ACET (fig. 2b) and CYAN (fig. 2c)

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The dependence of glass transition temperature on Kuhn segment for the CYAN polymers is described by three lines (fig. 2c). The first line is for polymers 22 and 23 based on pyromellitic dianhydride, the second is for polymers 20 and 21 based on hexafluoroisopropylidene diphthalic dianhydride and the third line is for polymers 16, 17, 18 and 19 based on the dianhydride containing diphenylketone or biphenylene segment. Figures 2 show that the polymers AMIC, OXAD, PEAM, ACET and CYAN behave normally, which means that the glass transition temperature increases with increasing the rigidity [2]. Now we examine the modification of density of the polymers after swelling and desorption of sc CO2. We can presume that after treatment with sc CO2 the density of polymers should decrease due to possible formation of nano- and micro-cavities [13, 19, 24]. Table 3 shows the density values before (ρ1) and after (ρ2) treatment with sc CO2. Table 3. Glass transition temperature, density, conformational parameter Van der Waals volume, free volume and the changing of the density and free volume after swelling in supercritical CO2. Polymer

Tg (oC)

Afr (Å)

Vw (Å3)

ρ1 (g/cm3)

Vf0 (cm3/g)

ρ2 (g/cm3)

Vf1 (cm3/g)

Δρ (g/cm3)

ΔV (cm3/g)

1 2 3 4 5

248 185 262 280 270 269.82

30.37 32.84 30.02 39.05 22.45

1031.648 1087.52 1096.54 651.013 624.822

1.317 1.311 1.329 1.391 1.369

0.2342 0.2384 0.2035 0.3672 0.3817

1.319 1.295 1.322 1.411 1.366

0.2330 0.2479 0.2075 0.3570 0.3833

- 0.002 0.016 0.007 - 0.020 0.003

-0,0012 0,0095 0,004 -0,0102 0,0016

6

270 270.56 218 190 221 205 210 190 280 270 273.8 270 268.4 216 223 218 232 228 235 253 263

24.14

654.141

1.364

0.3798

1.376

0.3734

- 0.012

-0,0064

72.53 72.34 71.39 28.08 41.86 40.61 44.77 34.23

521.673 553.099 614.855 614.851 642.950 721.688 495.140 663.816

1.259 1.256 1.308 1.296 1.299 1.233 1.383 1.360

0.2416 0.2385 0.2358 0.2428 0.2538 0.2673 0.171 0.198

1.302 1.246 1.313 1.315 1.344 1.259 1.378 1.335

0.2154 0.2441 0.2328 0.2317 0.2280 0.2506 0.174 0.212

- 0.043 0.010 - 0.005 - 0.019 - 0.045 - 0.026 0.005 0.025

-0,0262 0,0056 -0,003 -0,0111 -0,0258 -0,0167 0.002 0.014

20.48

1096.54

1.336

0.216

0.311

0.230

0.025

0.014

12.32 14.84 13.11 18.57 11.95 13.91 12.83 17.51

546.402 518.139 527.414 501.620 608.868 582.420 451.458 424.690

1.316 1.330 1.325 1.364 1.392 1.407 1.379

0.246 0.235 0.236 0.208 0.237 0.227 0.222

1.339 1.309 1.343 1.350 1.411 1.382 1.358

0.233 0.247 0.226 0.216 0.227 0.240 0.228

-0.023 0.021 -0.018 0.014 -0.019 0.025 -0.021

-0.013 0.012 -0.010 0.008 -0.010 0.013 -0.008

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Tg = glass transition temperature; Afr = Kuhn segment; Vw = Van der Waals volume; ρ1 = density before swelling; ρ2 = density after swelling; Vf0 = free volume before swelling; Vf1 = free volume after swelling; Δρ = ρ1–ρ2 = modification of density; ΔV=Vf1–Vf0 = modification of free volume.

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a

b

c

d

e Figure 3. Dependence of glass transition temperature ( Tg ) on free volume ( Vf ) for the studied polymers AMIC (3a), OXAD (3b), PEAM (3c), ACET (3d) and CYAN (3e).

The equilibrium coefficients of swelling with CO2 of these polyheteroarylenes are not high (table 2). In order to find an explanation of this behavior we examine now the dependence of glass transition temperature on the free volume which was calculated with the equation (5) taking into consideration the Van der Waals volume and the density of the

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studied polymers. It is known that when the free volume of the polymers increases their glass transition temperature decreases because the conformational transitions take place easier by heating. The macromolecular chains start moving easier towards each other and the amorphous polymers soften. After treatment with sc CO2 the density of the polymers modified: for some of them the density decreased, therefore the swelling did take place, while for other polymers the density increased. To explain such a behavior, we examine now the dependence of glass transition temperature on free volume, and the dependence of free volume on conformational rigidity. The degree of swelling is not high in comparison with the degree of swelling of polyimides [4] and, therefore, the experimental measurements of the glass transition temperature after swelling with sc CO2, by using the DSC method is not reasonable. Figure 3 presents the dependence of glass transition temperature on the free volume for five series of polymers before and after swelling in sc-CO2. As can be seen in figure 3a and 3d, only in the series AMIC and ACET the line showing the free volume of the polymers after swelling in sc-CO2 is on the right side, which means that the swelling of polymers did take place, although not in a significant degree. Polymer AMIC 1 is out of both cases. This shows that polymers AMIC 2 and AMIC 3 contained residual solvent N-methylpyrolidone (NMP) which was partially eliminated from these polymers by swelling in sc CO2. If this would not have been observed, after swelling in sc-CO2 all points had been situated on the same line. For the polymers in series OXAD (figure 3b), the density of polymers 4 and 6 after swelling in sc CO2 increased, and only for polymer 5 it decreased below the initial value. The dependence of glass transition temperature on the free volume is linear, with a high correlation coefficient. The increase of density after modification in sc CO2 could be explained by absorption of CO2 in polymer matrix, due to the formation of hydrogen bonds between CO2 and hydrogen atoms of amide groups. It is known that CO2 molecule is quadruple, in the two ends having oxygen atoms with a pair of non-participating electrons. These electrons may participate in the formation of hydrogen bonds with hydrogen atoms from polymer matrix. In this case it can be the hydrogen of amide groups. If the distance between hydrogen atoms of repeating unit and oxygen atom of CO2 is smaller than the sum of Van der Waals radii, and the energy of formation of complex decreases, then the hydrogen bond is formed. To verify this hypothesis, calculations were performed, by using the quantochemical method AM1 [14]. If we consider that the formation energy of the repeating unit is E1 and the formation energy of CO2 is E2, then the total energy of the two independent units (repeating unit and CO2 molecule) is: E = E1 + E2 = −181.74 Kcal/mol. The calculated formation energy of the complex of these two units was −183.39 Kcal/mol which is 1.65 Kcal/mol lower. Therefore, the formation of the complex is more probable than the existence of separated units. On the other hand, the sum of Van der Waals radii of oxygen and hydrogen atoms is 2.53 Å [16]. The calculations showed that the distance between the oxygen atom of carbonyl group and the hydrogen atoms of amide group is 2.27 Å, which is 0.26 Å shorter than the sum of Van der Waals radii of these atoms. Thus, it was proved the possibility of formation of complexes between amide groups in polymer matrix and CO2 molecules. In such case it is understandable why a part of CO2 during decompression remains in polymers 4 and 6: because in series OXAD only these two polymers contain amide groups.

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Similar specific interaction of CO2 with polymers was also observed earlier [25]. This is confirmed by diffusion coefficients of CO2 in these polymers (Table 2). In the polymer series PEAM (figure 3c) the dependence of glass transition temperature on the free volume is linear for three polymers: 7, 9 and 11. In the case of sample 10 the free volume is significantly smaller than it should be according to the general dependence in this series. It shows that in case of this polyheteroarylene, when the film was prepared, part of the solvent remained inside it and did not evaporate by drying until constant weight. This part of the free volume can be calculated. The solvent used to cast the films was N-methylpyrrolidinone (NMP). We calculated its Van der Waals volume. We start from the equation showing the dependence of glass transition temperature on the free volume and we find that the free volume of polymer 10 before treatment with sc-CO2 is 0.262 cm3/g. The difference between the experimental value of free volume and the calculated one is 0.0192 cm3/g, or 7.9 % of the free volume of this polymer. That is the part of free volume which is occupied by the solvent and this is why the density of the polymer was higher. The value of the free volume occupied by the solvent corresponds to 2.4 %. After swelling in sc CO2 the free volume of these polymers, with the exception of 8, decreases significantly (Table 3): for polymer 9 it decreases with 1.4%, while for polymer 11 it decreases with 10.2% It shows that the absorption of CO2 in the polymer matrix is similar to the polymers in series OXAD, but it occurs in different degree for each polymer. The diffusion coefficients which are given in table 2 confirm the formation of hydrogen bonds in these polymers because they are lower in comparison with diffusion coefficient of polymer 9. In case of this later polyheteroarylene the diffusion coefficient is high enough and a big part of CO2 leaves the polymer matrix during decompression. Figure 3d shows the dependence of glass transition temperature on free volume of the ACET polymers before and after swelling with sc CO2. It can be seen here that the line referring to the free volume after swelling is situated in the right side which means that the swelling of the polymers did take place, although not in the same degree. The degree of swelling is not high in comparison with the degree of swelling of polyimides [4] and, therefore, the experimental measurements of the glass transition temperature after swelling with sc CO2, by using the DSC method is not reasonable. Both dependences shown in figure 3d have high correlation coefficients. The degree of swelling of the polymers in this group differ significantly from each other. In the case of polymer 13, the free volume increases with 1.7%, while in the case of polymer 15 it increases with 6.5%. This behavior is connected with the conformational rigidity of the polymers which decreases from 44.77 Å in the case of polymer 13 to 20.48 Å in the case of polymer 15 (table 3). While the rigidity of the polymer increases, their free volume decreases (figure 4). When the number of flexible bridges (Ph– O–Ph) increases, in the case of polymers 14 and 15, and therefore the probability of conformational transitions in polymer chains increases during treatment with sc CO2 leading to the formation of nanocavities, the free volume of the polymer increases. The voluminous side groups R (structures shown in table 1) have a significant role here since they do not allow the polymer chains in the initial films to pack tightly, which increases the probability of conformational transitions around the flexible bridges. A completely different behavior is observed in the case of CYAN polymers when we examine the dependence of glass transition temperatures on free volume (figure 3e). Before swelling with CO2 the dependence of glass transition temperature on free volume (figure 3e) and the dependence of glass transition temperature on Kuhn segment (figure 2c) divides in

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three sub-groups: polymers 16, 17, 18 and 19 which contain diphenylketone or biphenylene unit in the dianhydride segment; polymers 20 and 21 containing hexafluoroisopropylidene diphthalic units in the dianhydride segment; polymers 22 and 23 containing pyromellitic unit in the dianhydride segment. Since the polymer 23 is partially crystalline, it will not be taken into consideration in the next discussion. The dependence of free volume on Kuhn segment divides in the same three sub-groups (figure 4d).

a

b

c

d

Figure 4. Dependence of free volume on the conformational rigidity (Kuhn segment) of the studied polymers.

The data given in figure 4 showing the dependences of the free volume before and after swelling in CO2 upon its conformational rigidity confirm the conclusions made on the basis of figure 3. With increasing the rigidity, the free volume decreases. Both of the dependencies (before and after swelling) have high coefficients of correlation, but after swelling in CO2 only for polymers AMIC the correlation coefficient increases, while for the polymers OXAD, PEAM and ACET it has a slight decrease. This is directly connected with the presence of solvent in polymers before and after swelling, and with the presence of residual CO2 participating in the formation of hydrogen bonds with amide groups. The swelling of the CYAN polymers takes place in the polymers 17, 19 and 21, containing imide rings, and it increases with the increase of the flexibility (table 3). To explain such a difference, we can refer to a previously published paper [26] presenting a study

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60

of the IR spectra of the polyamidic acids prepared in various solvents. There, it was shown that in the synthesis or dissolution in N-methylpyrrolidinone (NMP) followed by heating up to 200ºC to remove the solvent, two competitive processes may take place: −

the first is the formation of crosslinks between the chains of polyamidic acids:

−

the second is the formation of polymer/NMP complex:

The energy of the formation of the complex = –31 kJ/mol

The energy of the formation of the complex = –74 kJ/mol

Thus, the formation of crosslinks in the polyamidic acids prevents their swelling with sc CO2. The formation of complexes between NMP and amide groups hinders the free rotation around N–Ph bond and leads also to the formation of inter-chain bonds. Also, the low degree of swelling of 3.8% to 7% found in polyimides 17, 19 and 21 is connected with the fact that during the imidization of the polymers in NMP, it is possible the formation of anhydride bridges leading to crosslinks between chains. But in the case of the polyimides such crosslinks are less frequent than in the case of the corresponding polyamidic acids. Now we look into the table 2 which presents the diffusion coefficients of CO2 obtained during desorption of CO2 and equilibrium degree of swelling Zo. Both these parameters are determined with moderate degree of precision. The diffusion coefficients and equilibrium degree of swelling Zo of these polyheteroarylenes show the strong connection of these values with the chemical structure and the properties of polymers. Thus, in case of series AMIC, the polymer 1 shows the highest value of diffusion coefficient D, and a medium value of equilibrium degree of swelling Zo , which could be explained by a lower coefficient of molecular packing, and a higher rigidity of the polymer. The polymers in series OXAD show

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high values of D for 4 and 6, in comparison with 5, which can be connected with the formation of specific interaction between CO2 and amide groups. Finally, the series PEAM shows low values of D and Zo , and among them the polymer 12 exhibits the highest coefficient of molecular packing and one of the lowest values of D. For the ACET polymers 14 and 15, they are close to each other, regardless the significantly different rigidity. At the same time, also very close are the values of the free volume increase which were calculated on the basis of the change of density and Van der Waals volume of the repeating unit. The diffusion coefficients of the CYAN polymers 17, 19 and 21 are very close to each other, with the exception of polymer 21 which contains hexafluoroisopropylidene bridges, while the coefficients of swelling of these three polyimides are almost identical. In the case of CYAN polymers 16, 18 and 20 the coefficients of swelling are different and they increase with the decrease of the rigidity. The increase of rigidity enables the increase of the number of crosslinks due to the formation of anhydride bridges. In case of polymer 21 both diffusion coefficient and equilibrium coefficient of swelling are high probably because of a stronger sorption of carbon dioxide during swelling and its possible retention in the polymer matrix due to week interactions between hydrogen in amide groups and oxygen in carbon dioxide.

4. CONCLUSIONS This study shows that the swelling process of polyheteroarylenes with supercritical carbon dioxide is connected with the history of the polymer film preparation: in which solvent was the synthesis of the polymer performed and from which solvent was cast the film. However, the conformational analysis allows to determine certain factors which influence this process. Thus, with the increase of conformational rigidity, the degree of swelling of the polymers is lower. The presence of voluminous side groups facilitates the swelling process. Due to the presence of residual solvent the advanced drying of the polymer film is necessary before treatment with supercritical carbon dioxide. After the treatment with supercritical carbon dioxide it is also necessary to maintain the film at high temperature for some time in order to evolve the CO2 gas. The free volume in polymers depends on their conformational rigidity. When the films are prepared from polymer solutions, solvents having low boiling point should be used in order to prevent the influence of the residual solvent on the swelling process of the polymers in supercritical CO2. The polymers containing amide groups easily form hydrogen bonds with CO2 which hinders significantly their swelling. The study of the dependence of glass transition temperature on free volume and on Kuhn segment, before and after swelling with supercritical carbon dioxide, of two groups of polyheteroarylenes, ACET and CYAN, showed that the swelling degree is low, below 7%, and it depends significantly on the structure of the repeating units, for example on the presence of voluminous side substituents and flexible brigdes such as Ph–O–Ph. The use of high boiling solvent Nmethylpyrrolidinone (NMP) in the synthesis of polyheteroarylenes may lead to the formation of complexes between NMP and polymer chains which give rise to crosslinks and increase the rigidity and thus reduce the degree of swelling with supercritical carbon dioxide.

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ACKNOWLEDGMENTS The authors are grateful for the financial support provided through by the Romanian Research Program PNCD2 (Project no. 11008/2007) and grant of Presidium Russian Academy of Sciences P-18.

REFERENCES [1] [2] [3]

[4]

[5]

[6] [7]

[8]

[9]

[10] [11]

[12] [13]

Pavlova, S. S. A.; Ronova, I. A.; Timofeeva, G. I.; Dubrovina, L.V. On flexibility of cyclochain polymers, J. Polym. Sci. Polym. Phys. Ed. 1993, 31, 1725-1757. Ronova, I. A.; Pavlova, S. S. A. The Effect of the Conformational Rigidity on Several Physical Properties of Polymers, High Perform. Polym. 1998, 10, 309-329. Gallyamov, M. O.; Vinokur, R. A.; Nikitin, L. N.; Said-Galiyev, E. E.; Khokhlov, A. R; Schaumburg, K. Poly(methyl methacrylate) and poly(butyl methacrylate) swelling in supercritical carbon dioxide and the formation of a porous structure. Polym. Sci. Ser. A. 2002, 44, 581-592. Ronova, I.А.; Nikitin, L.N.; Sinitsyna, O.V.; Yaminsky, I.V. Treatment of polymers with supercritical carbon dioxide. Efficient method to increase the free volume. Phys. Khim. Process. Mater. 2008, 4, 54-59. Bruma, M. ; Hamciuc, E. ; Sava, I., Hamciuc, C. ; Iosip, M. D.; Robison, J. Compared properties of polyimides based on benzophenonetetracarboxylic dianhydride, Rev. Roum. Chim., 2003, 48: 629-638. Sava, I. and Bruma, M. Polyamide-esters containing benzonitrile or isopropylidene units in the main chain, Rev. Roum. Chim., 2004, 49: 69−76. Bruma, M. ;Sava, I. ;Hamciuc, E., Hamciuc, C.; Damaceanu, M. D. Synthesis and characterization of heterocyclic polyimides as high performance materials with potential applications in RF MEMS devices processing, Romanian Journal of Information Science and Technology, 2006, 9: 277-284. Sava, I.; Iosip, M. D.; Bruma, M.; Hamciuc, C.; Robison, J.; Okrasa, L.; Pakula, T. Aromatic polyamides with pendant acetoxy benzamide groups and thin films made therefrom. Eur. Polym. J. 2003, 39, 725-738. Sava, I.; Bruma, M. and Ronova I. A. Conformational parameters and some physical properties of polyamides containing pendant acetoxy benzamide groups. Mol. Cryst. Liq. Cryst. 2004, 416, 201-207. Sava, I.; Bruma, M. Compared properties of thin films from poly(1,3,4-oxadiazole)s. Rev. Roum. Chim. 2005, 50, 783-790. Hamciuc, E.; Bacosca, I.; Bruma, M.; Ignat, M. Aromatic polyimides containing cyano substituents for high performance applications. Proceedings 30th International Semiconductor Conference, Sinaia, Romania, 2007, 2, 357-360. Bacosca, I.; Hamciuc, E.; Bruma, M.; Ronova, I. A. Study of aromatic polyimides containing cyano groups. High. Perform. Polym., in press. Ronova, I. A. ; Nikitin, L. N. ; Sokolova, E. A. ; Sava, I. ; Bruma, M. Study of the behavior of polyheteroarylenes treated with supercritical carbon dioxide, High. Perform. Polym., in press.

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[14] Dewar, M. J. S.; Zoebisch, E. F.; Healy, E. F.; Stewart, J. J. Development and use of quantum mechanical molecular models. 76. AM1: a new general purpose quantum mechanical molecular model, J. Am. Chem. Soc. 1985, 107, 3902-3909. [15] Rozhkov, E. M.; Schukin, B. V.; Ronova I. A. Methods for the calculation of occupied volumes in glassy polymers: the lattice integration and the Monte Carlo method, Eur. J. Chem. (Central European Science Journals) 2003, 1, 402-426. [16] Askadskii, A. A.; Kondrashchenko, V. I. Computer material science of polymers; Nauchnyi Mir: Moscow, 1999. [17] Nikitin, L. N.; Said-Galiyev, E. E.; Vinokur, R. A.; Khokhlov, A. R.; Gallyamov, M. O.; Schaumburg, K. Poly(methyl methacrylate) and Poly(butyl methacrylate) Swelling in Supercritical Carbon Dioxide, Macromolecules, 2002, 35, 934-940. [18] Nikitin, L. N.; Marat, O.; Gallyamov, M. O.; Rostislav, A.; Vinokur, R. A.; Nikolaev, A. Yu.; Said-Galiyev, E. E.; Khokhlov, A. R.; Jespersen, H. T.; Schaumburg, K. Swelling and impregnation of polystyrene using supercritical carbon dioxide, J. Supercritical Fluids 2003, 26, 263-273. [19] Nikitin, L. N.; Nikolaev, A. Yu.; Said-Galiev, E. E.; Gamsasade, A. I.; Khokhlov, A. R. The formation of the porosity in the polymers with help of supercritical carbon dioxide. Supercritical fluids. The theory and practice, 2006, 1, 77-88. [20] Berens, A. R.; Huvard, G. S.; Korsmeyer, R. W.; Kunig, F. W. Application of compressed carbon dioxide in the incorporation of additives into polymers, J. Appl. Polym. Sci. 1992, 46, 231-242. [21] Webb, K. F.; Teja, A. S. Solubility and diffusion of carbon dioxide in polymers, Fluid Phase Equilibria 1999, 158-160, 1029-1034. [22] Von Schnitzler, J.; Eggers, R. Mass transfer in polymers in a supercritical CO2 – atmosphere, J. Supercritical Fluids 1999, 16, 81–92. [23] Crank, J. The mathematics of diffusion. Clarendon Press: Oxford, 1975. [24] Ronova, I. A.; Nikitin, L. N. ; Sokolova, E. A ; Bacosca, I.; Sava, I.; Bruma, M. Swelling of polyheteroarylenes in supercritical carbon dioxide, J. Macromol. Sci., Part A, 46 (10), 929-936 (2009). [25] Kazarian, S. G. ; Vincent, M.F.; Bright, F.V.; Liotta, C. L.; Eckert, C. A. Specific Intermolecular Interaction of Carbon Dioxide with Polymers, J. Am. Chem. Soc., 1996, 118: 1729-1736. [26] Kostina, Yu. V.; Moskvicheva, M. V.; Bondarenko, G. N.; Yablokova, M. Yu; Alentiev, A. Yu. The influence of solvent nature on the imidization reaction of polyamidic acid based on benzophenonetetracarboxylic dianhydride and m-phenylene diamine. Proceedings 15th Russian Conference on “The structure and Dynamic of molecular system” Yoshkar Ola, 2008, vol. 1, p.133-138.

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.65-73 ©2010 Nova Science Publishers, Inc.

Chapter 6

INHIBITION OF 2-HEXENAL OXIDATION BY ESSENTIAL OILS OF GINGER, MARJORAM, JUNIPER BERRY, BLACK AND WHITE PEPPER T. A. Misharina*, M. B. Terenina, N. I. Krikunova, and I. B. Medvedeva Emanuel Institute of Biochemical Physics, Russian Academy of Sciences ul. Moscow, Russia

ABSTRACT The essential oils from black and white pepper (Piper nigrum L.), juniper berry (Juniperus communis L.), marjoram (Origanum majorana L.), and ginger (Zingiber officinale L) were studied. By the method of capillary gas-liquid chromatography the efficiency of inhibition of autooxidation of 2-hexenal by essential oils in hexane solutions was studied. The stability of essential oils components during the storage of hexane solutions was determined and compared with that of pure oils.

Keywords: Essential oils; autooxidation of 2-hexenal; inhibition of oxidation; changes in composition during storage.

To protect themselves from infections and parasites and in reply to stress, plants synthesize low molecule volatile terpenoids, the mixture of which is called essential oils. Essential oils of many plants possess an intensive and pleasant aroma and also are biologically active [1-3]. Due to these properties, essential oils are actively used in medical and pharmaceutical industries, in aromatherapy and cosmetology, and in the production of natural effective food flavourings [3, 4]. Organoleptic properties and biological activity of essential oils depend on their composition [5-8]. The study of individual components of * Emanuel Institute of Biochemical Physics, Russian Academy of Sciences ul. Kosygina 4, Moscow, 119334 Russia e-mail: [email protected]

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different essential oils showed that many terpenes possess antiradical and antioxidant activity (AOA). The activity of cyclic monoterpene hydrocarbons with two double bonds is comparable with AOA of phenol and α -tocopherol [9-11]. Thus, α- and γ-terpinenes inhibited the methyl linoleate oxidation [10-13]. Sabinene, terpinenes, citronellal, neral, and geranial possess antiradical activity [10, 14 -18]. As a rule, antioxidant activity of essential oils is higher than of their individual components. This fact indicates the existence of synergetic effects due to the complex multicomponent composition of oils [15, 19, 20]. For the estimation of antioxidant properties of substances or their mixtures, many different methods are used. It was showed that the value of AOA significantly depends on the method of its estimation and on the qualitative and quantitative composition of systems under test [6, 10, 13,20,21]. One of the simple and informative methods of the quantitative assessment of AOA is based on the inhibition of lower aldehyde autooxidation in the presence of antioxidant substances [7, 8, 20- 25]. The aim of the work is to study and compare antioxidant properties of 5 essential oils in the model system of autooxidation of 2-hexenal, the assessment of the influence of essential oils composition and concentration on their AOA, and also the study of changes in the composition of essential oils in the process of autooxidation in solutions and in pure oils.

MATERIALS AND METHODS The fresh samples of essential oils of black and white pepper Piper nigrum L., juniper berry Juniperus communis L., ginger Zingiber officinale L., and marjoram Origanum majorana L. were obtained from the company “Plant Lipids Ltd.”, India. Hexane, undecane and trans-2-hexenal were purchased from Sigma.

Model Systems 600 μl of trans-2-hexenal (3 μl/ml) and 400 μl of n- undecane (2 μl/ml) (internal standard) were dissolved in 200 ml of n-hexane. The solution was separated into 3-ml aliquots, which were placed in 5-ml glass vials and then 10 μl (3.33 μl/ml), 50 μl (16.5 μl/ml) or 210 μl (70 μl/ml) of essential oils were added. Oil was not added in the control sample. Each sample was prepared twice, the control sample was prepared three times. The samples in vials with stoppers were stored in light under room temperature for 60 days. Every week vials were opened and blown with 10 ml of air with the help of a pipette. The quantitative content of 2-hexenal and components of essential oils in vials were determined by method of capillary gas chromatography after every 10 days.

Gas-Chromatography Analysis The Kristall 2000M chromatograph (Russia) with a flame ionization detector and an SPB-1 silica fused capillary column (50 m x 0.32 mm, phase layer 0.25 μm) were used for analyses of samples. The column temperature was increased from 60 to 250oC with the rate of

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8oC/min, the temperature of detector and injector was at 250oC. The rate of carrier gas helium through the column was 1.5 ml/min. The identification of components in oil samples was carried out on the basis of retention indices by their comparison with literary [26] or experimental data obtained by us. The quantitative content of 2-hexenal and essential oils components was calculated by the ratio of peak areas, which corresponded to the substances and internal standard. The oxidation extent of 2-hexenal and essential oils components (%) was determined in reference to their content in initial samples.

RESULTS AND DISCUSSION The oils that we chose differed in their quantitative and qualitative composition but contained many common components. Due to the method of capillary gas chromatography we could estimate changes in the content of 2-hexenal and each oil component in model solutions with different oil concentrations and in pure oil and also to distinguish the oxidation products of main components. The comparison of oils composition and the oxidation speeds of their components allowed estimate some regularity, which enables us to predict and regulate oil composition to obtain stable mixtures. This is very important because essential oils are currently widely used in industry and medicine. Usually the recommended storage time for oils is 1 year; however, nobody has studied yet what really happens with oils during this period. We earlier studied the changes in the composition of coriander, laurel, marjoram, and fennel oils in the process of storage of pure oil samples in dark and in the light but in bottles from dark glass [27-29]. It was established that the main process was the oil components oxidation. Thus, we found, and then it was proved in works [10, 11], that cyclic monoterpenes hydrocarbons α- and γ- terpinenes are completely oxidized into aromatic hydrocarbon p-cymene. We also found oxidation products of other components of essential oils - oxides, alcohols, and aldehydes [26-29]. For the estimation of antioxidant properties of essential oils we used a model system of autooxidation of 2-hexenal to corresponding acid. [7, 8]. As a criteria for the comparison of antioxidant activity of essential oils we used the quantity of 2-hexenal, which remained in model systems after 40 days in reference to the initial quantity (%). Fig. 1 presents the obtained results of relative AOA values of 5 studied essential oils. The model systems included marjoram and white pepper in two concentrations: 3.3 μl/ml and 16.5 μl/ml. For the essential oils of ginger, juniper berry and black pepper we studied an additional third system in which the content of oil was 70 μl/ml of hexane solution. As is clearly seen from Fig. 1, the ginger oil in the systems with minimal content of (3.3 μl/ml ) had AOA more than 50%. AOA of marjoram oil at the same concentration showed activity 48%. The essential oils of juniper berry, black and white pepper in diluted solutions (3.3 μl/ml) had very low AOA: 20-28%. As can see from Fig.1, there was a dependence between concentration and AOA for all oils. It is noteworthy that the increase in activity was usually not in proportion to the growth of essential oil concentration. So, the increase in concentration of black pepper and juniper berry oils by five times (from 3.3 to 16.5 μl/ml) led to the increase of AOA by 2.5-3 times. The further increase in concentration of these essential oils up to 70 μl/ml did not change AOA.

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100 90

2-Hexenal content, %

80 70 60 50 40 30 20 10 0 1

Control

2

3

4

C1

5

6

C2

Figure 1. Inhibition of 2-hexenal oxidation by essential oils in hexane solutions during 40 days storage . 1 - Control (2-hexenal). Essential oils: 2 – ginger, 3 - marjoram, 4 - juniper berry, 5 - black pepper, 6 white pepper. Concentration of essential oils (μl/ml): C1 is 3.3, C2 is 16.5, and C3 is 70.

AOA of ginger essential oil was studied for three model systems with oil concentrations of 3.3, 16.5, and 70 μl/ml. In the first system, oil inhibited the oxidation of 2-hexenal by 54%, and in the second it inhibited by 78%, and in the third it inhibited by 94% (Fig. 1). This is very high activity taking into consideration the fact that oil does not contain phenol derivatives. It consisted from sesquiterpene hydrocarbons, which differed in stability. The dynamics of changes of main components of ginger essential oil for the system with oil concentration 16.5 μl/ml is presented in Fig. 2. The main oil component – zingiberene - is oxidized faster than all others. After 60 days of storage, its content in the first solution decreased by 20 times, in the second it decreased by seven times, and in the third it decreased by 2.5 times. Also, the content of bisabolenes and sesquiphellandrene decreased (Fig. 2). It is interesting that, parallel to the decrease in zingiberene concentration, we revealed the increase in α-curcumene content. The structure of zingiberene is similar to the structure of α terpinene. Both compounds have a similar hexamerous cycle with two double bonds. The oxidation product of α - terpinene was p-cymene [27-29]. Probably, zingiberene was oxidized in α - curcumene, which is an aromatic compound similar to p-cymene. The content of zingiberene in ginger oil is approximately 35% and thus this oil has high AOA. In the process of storage in the dark of pure ginger essential oil for 12 months, the content of zingiberene decreased by two times and the content of α - curcumene increased by 1.5 times. Such change

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in the oil composition significantly worsens its organoleptic characteristics and physicochemical and biological properties of ginger essential oil. This should be taken into consideration while using stored ginger essential oil. The sweet marjoram essential oil contained from 15 to 25% of linalool, terpinen-4-ol, and γ-terpinene and did not contain phenol derivatives. With the increase in oil concentration its AOA increased by 1.5 times and was 72% (Fig. 1). Fig.3 presents the quantity of compounds which was left after 40 days of oxidation (in percentage from initial). During the storage of the diluted solution of oil, monoand sesquiterpene hydrocarbons and even terpinen-4-ol were significantly oxidized; in more concentrated solution, only the content of α - and γ-terpinenes decreased significantly. The content of p-cymene which was oxidation product of terpinenes increased. In both solutions the content of geraniol changed slightly. The pure marjoram oil preserved content stability in the dark for a year; in the light the noticeable oxidation began after 4 months of storage [28].

Content,% from initial

120

100

80

60

40 0

20 1

2

40 3

60 days 4

5

Figure 2. The content of main components (% from initial) of ginger essential oil in the oxidation process in hexane solution: 1 - α-curcumene, 2 - zingiberene, 3 - α -bisabolene, 4 – β-bisabolene, and 5 - sesquiphellandrene.

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100 90

Content,% from initial

80 70 60 50 40 30 20 10 0 1

2

3

4

I

5

6

7

8

II

Figure 3. The influence of marjoram oil concentration (I is 3.3 μl/ml and II is 16.5 μl/ml) on the content of its components (% from initial) after 40 days of oxidation: 1 - sabinene, 2 - myrcene, 3 - α terpinene, 4 - γ-terpinene, 5 - α -terpinolene, 6 - β -caryophyllene, 7 - geraniol, and 8 – terpinen-4-ol.

The significant influence of the concentration on antioxidant properties was revealed from black pepper, white pepper, and juniper berry oils. They had practically the same quantitative composition of components - mono- and sesquiterpene hydrocarbons. In hexane solution they did not show antioxidant properties because the oxidation of 2-hexenal happened to the same extend as in the control sample, which did not contain essential oils. Under the concentration 16.5 μl/ml the black pepper essential oil inhibited the oxidation of 2hexenal by 58%, white pepper essential oil inhibited it by 49%, and juniper berry oil inhibited by 65%. In the solution with a concentration of 70 μl/ml, the black pepper essential oil had AOA 62% and that of juniper berry had 66% (Fig. 1). In the diluted solutions, during 40 days practically all components of essential oil were oxidized. For the solution with a concentration of 16.5 μl/ml, the changes in the content of main oil components are presented in Fig. 4. As is seen, after 40 days the content of α - and γ-terpinenes in oils decreased by 5-6 times and that of β-caryophyllene decreased by 2.5-3 times, the content of other components changed insignificantly. We revealed that the oxidation of caryophyllene began when the majority of α - and γ -terpinenes was oxidized. Under the storage of pure essential oils in the dark a similar process was detected after 8 months. By that time, the content of terpinenes was 2-10% of the initial content and that of caryophyllene was 80-85%, whereas the content of caryophyllene oxide increased by 2-2.5 times and that of p-cymene increased by 4 times. Thus, the content of γ-terpinene and caryophyllene oxide may be an indicator of the freshness

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and quality of black and white pepper essential oils. The additional experiments showed that the stability of these oils increase significantly at addition to these oils of other essential oils such as, for example, juniper berry, garlic, or nutmeg oils.

100 90

Content, % from initial

80 70 60 50 40 30 20 10 0 1

2

I

3

4

II

5

6

7

III

Figure 4. The content of main components (% from initial) in black pepper (I), white pepper (II) and juniper berry (III) in the oxidation process in hexane solution: 1 - α – and β- pinenes, 2 - sabinene, 3 myrcene, 4 - 3-carene, 5 - limonene, 6 – α- and γ-terpinenes, and 7 - β-caryophyllene.

Thus, conducted research show that antioxidant properties of essential oils are determined by their composition. Antioxidant properties of essential oils, which consist of terpene hydrocarbons and alcohols, are determined by content of α- and γ-terpinenes and their sesquiterpene analogs. We revealed the significant influence of the concentration of such oils on their antioxidant properties. It was established that in the oxidation of the main components of essential oils the substances are formed, which are present in natural essential oils. The stability of essential oil composition increased with the increase of their concentration in model solutions. The oxidation of substances in pure essential oils happened much more slower than in solutions.

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REFERENCES [1]

[2]

[3] [4]

[5]

[6] [7] [8] [9] [10]

[11]

[12]

[13]

[14]

[15] [16]

L.H. Madsen, B.R.Nielsen, G.Bertelsen, L.H. Skibsted: Screening of antioxidative activity of spices. A comparison between assay based on ESR spin trapping and electrochemical measurement of oxygen consumption. Food Chem., 57, 331 (1996). S.A. Voitkevich: “Efirnye masla dlya parfyumerii i aromaterapii” (Essential Oils for Perfumery and Arometherapy). Pishch. Prom., Moscow, 1999. 3. Flavours and Fragrances. Chemistry, Bioprocessing and Sustainability (Ed. R.G. Berger). Springer, New York, 2007, 43-116. K. Bauer, D. Garbe, H. Surburg: Common Fragrance and Flavor Materials, VCH Verlag, Weinheim, 1990. G. Cervato, M. Carabelli, S. Gervasio, A. Cittera, R. Cazzola, B. Cestaro: Antioxidant properties of oregano (Origanum vulgare L.) leaf extracts. J. Food Biochem., 24, 453 (2000). H.J.D. Dorman, A. Peltoketo, R.Hiltunen, M.J.Tikkaken: Characterisation of the antioxidant properties of deodourised aqueous extracts from selected Lamiaceae herbs. Food Chemistry, 83, 255 (2003). K.G. Lee, T. Shibamoto: Antioxidant property of aroma extract isolated from clove buds. Food Chem., 74, 443, (2001). K.W. Lee, Y.J. Kim, D.-O. Kim, H.J. Lee, C.Y. Lee: Major phenolics apple and their contribution to the total antioxidant capacity. J. Agric. Food Chem., 51, 6516, (2003). G.Litwinienko, K.U. Ingold: Solvent effects on the rates and mechanisms of reaction of phenols with free radicals. Acc. Chem. Res., 40, 222 (2007). G. Ruberto, M. Baratta: Antioxidant activity of selected essential oil components in two lipid model systems. Food Chem., 69, 167 (2000). M.C. Foti, K.U. Ingold: Mechanism of inhibition of lipid peroxidation by γ–terpinene, an unusual and potentially usefull hydrocarbon antioxidant. J. Agric. Food Chem. 51, 2758 (2003). M.T. Baratta, H.J.D. Dorman, S.G. Deans, D.M.Biondi, G.Ruberto: Chemical composition, antimicrobial and antioxidative activity of laurel, sage, rosemary, oregano and coriander essential oils. J. Essent. Oil Res., 10, 618 (1998). S.S.Pekkarinen, H.Stocmann, K.Schwarz, M.Heinone, A.I.Hopia: Antioxidant activity and partitioning of phenolic acids in bulk and emulsified methyl linoleate. J. Agric. Food Chem., 47, 3036 (1999). G.Sacchetti, S.Maietti, M.Muzzoli, M.Scaglianti, S.Manferdini, M.Radice, R.Bruni: Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods. Food Chem. 91, 621 (2005). G. Singh, S. Maurya, C. Catalan, M.P. De Lampasona: Studies on essential oil, Part 42.: chemical, antifungal, antioxidant and spout suppressant studies of ginger essential oil and its oleoresin. Flavour Fragrance. J., 20, 1 (2005). A.Wei, T. Shibamoto: Antioxidant activities and volatile constituents of various essential oils. J. Agric. Food Chem., 55, 1737 (2007). C. Menut, J.M. Bessiere, D.Samate, A.K. Djibo, G.Buchbauer, B. Schopper: Aromatic plants of tropical west Africa. XI. Chemical composition, antioxidant and antiradical

Inhibition of 2-Hexenal Oxidation By Essential Oils of Ginger, Marjoram…

[17]

[18] [19] [20] [21]

[22]

[23]

[24]

[25] [26]

[27]

[28]

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properties of the essential oils of three Cymbopogon species from Burkina Faso. J. Essent. Oil Res., 12, 207 (2000). N.Mimica-Dukic, B. Bozin, M. Sokovic, N. Simin: Antimicrobial and antioxidant activities of Melissa officinalis L. (Lamiaceae) essential oils. J. Agric. Food Chem., 52, 2485 (2004). D.Huang, B.Ou, L.R.Prior: The chemistry behind antioxidant capacity assays. J. Agric. Food Chem., 53, 1841 (2005). K.G. Lee, T.Shibamoto: Determination of antioxidant potential of volatile extracts isolated from various herbs and spices. J. Agric. Food Chem., 50, 4947 (2002). K.G. Lee, T.Shibamoto: Antioxidant activities of volatile components isolated from Eucalyptus species. J.Sci. Food Agric., 81, 1573 (2001). K.Yanagimoto, H.Ochi, K.G. Lee, T. Shibamoto: Antioxidative activities of volatile extracts from green tea, oolong tea, and black tea. J. Agric. Food Chem., 51, 7396 (2003). C.J. Lee, T. Shibamoto, K.G. Lee: Identification of volatile components in basil (Ocinum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chem., 91, 131 (2005). B.Bozin, N.Mimica-Dukic, G.Anachov: Characterization of the volatile composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils. J. Agric. Food Chem., 54,1822 (2006). T.A. Misharina, A.L. Samusenko: Antioxidant properties of essential oils from lemon, grapefruit, coriander, clove, and their mixtures. Applied Biochem. and Microbiol., 44, 473 (2008). W. Jennings, T.Shibamoto: Qualitative Analysis of the Flavor and Fragrance Volatiles by Glass Capillary Gas Chromatography, Academic, New York: 1980, 130-154. T.A Misharina: Influence of the duration and conditions of storage on the composition of the essential oil from coriander seeds. Applied Biochem. and Microbiol., 37, 622 (2001). T.A Misharina, A.N. Polshkov, E.L. Ruchkina, I.B. Medvedeva: Changes in the composition of the essential oil of marjoram during storage. Applied Biochem. and Microbiol., 39, 311 (2003). T.A Misharina, A.N. Polshkov: Antioxidant properties of essential oils: autoxidation of essential oils from laurel and fennel and of their mixtures with essential oil from coriander. Applied Biochem. and Microbiol., 41, 610 (2005

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.75-82 ©2010 Nova Science Publishers, Inc.

Chapter 7

THE ORGANOPHOSPHORUS PLANT GROWTH REGULATOR MELAPHEN AS ADAPTOGEN TO LOW MOISHER I. V. Zhigacheva*, E. B. Burlakova, T. A. Misharina, M. B. Terenina, N. I. Krikunova, I. P. Generozova and A. G. Shugaev N.M. Emanual Institute of Biochemical Physics, Russian Academy of Science, Moscow, Russia

ABSTRACT We studied the effect of insufficient watering and organophorphorus plant growth regulator (melaphene) on peroxide oxidation of lipids (POL) in pea sprouts membranes. Water deficiency and melaphene increase the generation of reactive oxygen species. However, a response to a presowing melaphene treatment of pea sprouts grown under standard condition is higher by 18.5% as compared with pea sprouts grown under conditions of water deficiency. A decrease in the generation of reactive oxygen species in response to the melaphene treatment in comparison with control group is associated with changing the fatty acid content of plasmatic and organellas membranes. In sprouts membranes, the content of unsaturated fatty acids having 18, 20 and 22 carbon atoms decreases drastically. The melaphene treatment prevents from changes in the fatty acid content induced by a water deficiency. We supposed that the protective effect of melaphene in conditions of water deficiency is associated with preventing thereby changes in the fatty acid content of membranes.

Keywords: plant growth regulators, lipid peroxidation, plazmolemma, fatty acid.

* N.M. Emanual Institute of Biochemical Physics, Russian Academy of Science, 4 Kosygin str., Moscow, 119334 Russia, [email protected]

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INTRODUCTION Survival of plants in constantly changing conditions of an environment probably only at the plant can adapt to them. At a cellular level it is carried out by means of protective systems. As protective molecules in low concentration molecules superoxide and peroxides of hydrogen which concentration in a cell raises some kinds stress may operate [1]. Reactive oxygen species (ROS) stimulates the accumulation of cyclic adenosine monophosphate and cyclic guanosine monophosphate, causes activation of protein kinases [2]. Its, on the one hand, act as participants of signaling cascades as a result of which occurs gene expression supervising synthesis of the protective systems components, on other hand, its activate the processes of lipid peroxidation in biological membranes [3,4]. As the test for course of free radical oxidation in biological membranes we studied influence of stress on processes of lipid peroxidation. As model of stressful influence on biological membrane we used model of low moisture. Cellular membranes are one of the basic places where there is a damage of a cell at water deficiency. Water deficiency modifies cellular membranes and membranes of the organelles influencing on their functions and a metabolism of a cell [5,6]. In this connection as object of research served the fragments of the endoplasmatic reticulum, nuclear membranes and plasma membranes. As plant growth regulators raise stability of plants to stressful influences it was interesting to study influence of a plant growth regulator Melaphen, the melamine salt of bis(oxymetyl)phosphonic acid, upon studied rates. Melaphen was synthesized in Arbuzov Institute of Organic and Physical Chemistry, Kazan’ Research Center, Russian Academy of Sciences. NH2 N H2N

O HOP(CH2OH)2

N

NH2

Moreover, Karimova F.G. detected some phosphothirosine protein-target for Melaphen actions. It was proteins of photosynthetic assimilation of carbon [7]. We also studied influence of low moisture and Melaphen upon fatty acids composition of cellular membranes as at a drought changes fatty acids composition of biological membranes [8, 9]

MATERIALS AND METHODS In the work, we used biological membranes isolated from Pisum sativum pea sprouts grown under standard conditions and under low watering conditions. Pea seeds were germinated as follows: control seeds were rinsed with soapy water and 0.01% KMnO4 solution and then kept watered for 60 min; experimental seeds were kept in a 10-7% melaphene solution for 30 min and then in water for another 30 min. In a day, half of the control seeds and half of the melaphene-treated seeds were carried over onto a dry filtering paper in open cuvettes. After two days of “drought”, the seeds were carried over into

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closed cuvettes on a periodically wetted filtering paper, where the seeds remained for 4 days. On the fifth day, we calculated the number of germinated seeds and isolated mitochondria. Isolation of membranes from 5-day sprouts epicotyls was performed by a method of [10] in our modification. The epicotyls having a length of 3 to 6 cm (20-25 g) were placed into a homogenizer cup, poured with an isolation medium in a ratio of 1:2, and then were rapidly disintegrated with scissors and homogenized with the aid of a press. The isolation medium comprised: 0.4 M saccharose, 5 mM EDTA, 20 mM КН2РО4 (рН 8.0), 10 mM КСl, 2 mM dithioerythritol, and 0.1% BSA (free of fatty acids).The homogenate was centrifugated at 25000g for 5 min. The precipitate was resuspended in 8 ml of a rinsing medium and centrifugated at 3000g for 3 min. The suspension medium comprised: 0.4 M saccharose, 20 mM КН2РО4, 0.1%. BSA (free of fatty acids) (рН 7.4). Methylation of fatty acids. A one-step methylation of fatty acids was performed by a method previously described in [11] and modified. Gas chromatography analysis (GCA) of samples of hexane solutions of fatty acid methyl esters was performed on a Kristall 2000 M chromatograph (Russia) equipped with a flame-ionization detector and an SPB-1 quartz capillary column (50 m x 0.32 mm, phase layer 0.25 µm). An analysis of hexane solutions of methyl esters was performed at a programmed temperature at a rate of 4°С/min from 120° to 270 °С (50 min). The injector and detector temperature was 250°С. The gas carrier speed was 1.5ml/min. An analysis was performed for 2 µm samples of hexane solutions. The components in methyl esters samples were identified from the retention indices thereof as compared with references [12] or our experimental data obtained. In the experiment, we used the following reagents: potassium carbonate, methanol (Merck, Germany), hexane (Panreac, Spain), acetyl chloride (Acros, Belgium), saccharose, BSA (free of fatty acids)(Sigma, USA).

RESULTS AND DISCUSSION In the first series of experiments, we studied the effect of drought and melaphene on freeradical oxidative processes in pea sprouts membranes. As a model process, a lipid peroxydation (LP) was studied. Low moisture results in a 1.5-fold increase in the POL products fluorescence. The melaphene treatment of the control seeds increased the POL products fluorescence by a factor of 6.5; whereas the fluorescence of POL products in membranes of pea sprouts subject to insufficient watering increased by a factor of 5.3 (Fig. 1). An increase in the content Н2О2 and POL products was observed also in membranes of other plants, grown under drought conditions. [13-15]. Some authors are of opinion that an increase in the level of reactive oxygen species, namely Н2О2, caused by stresses such as drought is associated with the participation of Н2О2 in redox-regulation of signal cascade during acclimatization [3,16-18]. Note that an increase in the concentration of reactive oxygen species, mainly Н2О2 is observed also as a response to a stimulation of membrane receptors by various agents, in particular peptide factors of growth [19, 20] and cytokines [21.22]. In our experiments, an increase in the POL products level may be evidence for the activation of a family of NADPH-oxidases associated with a plasmatic membrane, which catalyze the generation of reactive oxygen species [23]. The supposition is supported by data

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on increasing the microviscosity of annular lipids upon a melaphene treatment of animal and plant cells [24]. A lowered response to the effect of the preparation on membranes of pea sprouts subject to insufficient watering may be a result of modification of physicochemical properties of the membranes. Therefore, in another series of experiments we studied the effect of the water stress and melaphene treatment on a fatty acid content of the membranes.

Influenc e of Melaphen on the level of  L P  in the peas  membrane. 

Intens ity of fluores c enc e

7000000 6000000 5000000 C ontrol 4000000

C ontrol +  Melaphen Drought

3000000

Drought+ Melaphen 2000000 1000000 0 380 400 420 440 460 480 500 520 540 Wa ve s  le ng th in nm  

Legend : abscissa axis – Waves length in nm; ordinate axis – intensity of fluorescence. Picture 1. INFLUENCE OF MELAPHEN ON THE LEVEL OF LIPID PEROXYDATION UN THE PEA SPROUT MEMBRANES.

Water deficiency results in a decrease in the ratio of unsaturated and unsaturated fatty acids in pea sprouts membranes (Table 2). In particular, there occurs a 3-fold increase in the content of lauric acid, a 1.7-fold increase in the content of nonadecanoic acid, and a 2-fold increase in the content of heptadecanoic and arachidonic acid (Table 1). At the same time, the proportion of unsaturated fatty acids having 18 carbon atoms decreases. The content of linoleic acid decreases by 10.2%; the contents of linolenic and octadecenic acids decrease by 17.6% and 24%, respectively. The content of stearic acid increases by 66% that result in a decrease in the ratio of unsaturated fatty acids having 18 carbon atoms and stearinic acid from 20.78±0.60 to 11.30±0.40 (Table 2). Similar data on the effect of water deficiency on the fatty acid content were obtained for potato cell membranes and membranes of Arabidopsis thaliana and apricot leaves [8, 25-27]. Note a considerable decrease in the proportion of С20 - fatty acids. The ∑С20 ratio of unsaturated acids/ С20:0 decreases by a factor of 3.3. Hence, unsaturated fatty acids having 22 carbon atoms completely vanish from the membrane composition. The melaphene treatment of pea seeds prevents from drought-induced changes in the fatty acid composition of membranes. It is safe to suppose that a decrease in the generation of reactive oxygen species, as a response to melaphene treatment of pea seeds germinated under conditions of insufficient watering, is a result of changes in the fatty acid content of membranes, since these changes

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according to some authors [28] may affect the function of receptors and regulate the number thereof. Table 1. Influence of Melaphen and low moisture for fatty acid composition of the peas membrane. (Relative per-cent. Results of 6 experiments). Fatty acid 12:0 14:0 15:0 16:1ω7 16:0 17:0 18:2 ω6 18:3 ω3 18:1 ω9 18:1 ω7 18:0 19::0 20:2 ω6 20:1 ω 9 20:1 ω15 20: 1 ω 7 20:0 22:1 ω13 22:1 ω9 22: 1 ω 7 22:0 23:0

Control 0.33±0.13 0.67±0.03 0.37±0.15 0.69±0.10 18.67±0.15 0.34±0.20 55.40±0.30 11.1±0.2 0 5.14±0.40 1.10±0.10 3.50±0.15 0.13±0.04 0.33±0.10 0.35±0.03 0.20±0.01 0.22±0. 10 0.52±0.31 0.03±0.01 0.13±0.01 0.12±0.02 0.41±0.11 0.27±0.02

Control+Melaphen 0.21 ±0.10 0.45±0.02 0.65±0.12 0.69±0.10 18.09±0.20 0.61±0.12 59.00±0.40 9.50±0.11 3.78±0.37 1.16±0.24 3.75±0.29 0.11±0.01 0.32±0.08 0.24±0.01 0.10±0.01 0.10±0.20 0.54±0.26 0.10±0.01 0.02±0.01 0.10±0.01 0.30±0.03 0.25±0.01

Drought 0.99±0.15 0.64±0.20 0.51±0.020 0.95±0.13 20.48±0.15 0.69±0.10 49.76±0.35 9.15±0.30 5.89±0.20 0.83±0.03 5.80±0.24 0.22±0.05 0.12±0.30 0.26±0.03 0.14±0.10 0.17±0.12 1.06±0.22 0.90±0.18 0.65±0.10

Drought+Melaphen 0.36±0.10 0.65±0.20 0.35±0.05 0.58±0.15 18.5 6±0.15 0.38±0.16 55.45±0.14 11.22±0.23 4.71±0.40 0.98±0.05 3.60±0.10 0.16±0.03 0.35±0.10 0.35±0.03 0.25±0.01 0.24±0.16 0.51±0.12 0.09±0.06 0.10±0.01 0.14±0.02 0.47±0.05 0.30±0.10

Table 2. Influence of Melaphen and low moisture for index of fatty acid composition of pea sprout membranes. (Relative per-cent. Results of 6 experiments). Fatty acids ∑ saturated fatty acids ∑ unsaturated fatty acids 18:0 ∑ unsaturated fatty acids С18 ∑ unsaturated С18/С18:0 Index unsaturation С18 20:0 ∑ unsaturated С20 ∑ unsaturated С20/С20:0 22:0 ∑unsaturated C22 ∑ unsaturated С22/С22:0 ∑unsaturated/ ∑saturated fatty acids

Control 25.205±0.108 74.795±0.140 3.500±0.150 72.735±0.250 3.500±0.166 42.950±1.619 0.520±0.310 1.100±0.060 2.120±0.194 0.410±0.110 0.275±0.012 0.670±0.109 3.370±0.129

Control+Melaphen 24.965±0.120 75.110±0.128 3.750±0.290 73.44±0.280 3.750±0.970 40.384±0.857 0.540±0.260 0.760±0.075 1.400±0.288 0.300±0.030 0.022±0.012 0.730±0.400 3.000±0.106

Drought 32.735±0.140 67.265±0.173 5.800±0.240 65.625±0.220 5.800±0.920 23.000±1.089 1.060±0.220 0.690±0.113 0.650±0.514 0.900±0.180 0 2.050±0.124

Drought+Melaphen 25.340±0.106 74.660±0.137 3.600±0.100 72.49±0.213 3.600±1.000 41.800±2.029 0.510±0.120 1.170±0.075 2.290±0.625 0.470±0.050 0.340±0.003 0.860±0.060 2.950±0.129

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CONCLUSION The data obtained show that insufficient watering modifies not only the ratio С18 unsaturated fatty acids to С18:0, but also affects more strongly the ratio С20 unsaturated fatty acids to С20:0, as a result, the membranes are completely devoid of unsaturated fatty acids having 22 carbon atoms. Evidently, melaphene prevents from changing the fatty acid content of membranes and thus enhances the resistance of plants to insufficient watering. Moreover, in pea sprouts membranes treated with melaphene and grown under conditions of insufficient watering, the proportion of unsaturated fatty acids having 22 carbon atoms increases by a factor of 1.5 as compared with pea sprouts grown under standard conditions; the effect may obviously results in enhancement of productivity of plants grown even under drought conditions [29].

REFERENCES [1]

Bhattacharjee S./ Reactive oxygen species and oxidative burst. Roles in stress, senescence and signal transduction in plants.// Curent science 2005, 89, 1113-1121. [2] 2.Rivlin J., Mendel I., Rubinstein S., Etkovitz N., Breibrant H./ Role hydrogen peroxide in sperm capacition and acrosome reaction// Biol. Reproduction 2004, 70, 518-522. [3] Beck E H, Fettig S, Knake C, Hartig K and Bhattarai T./ Specific and unspecific responses of plants to cold and drought stress; //J. Biosci. 2007 32 501–510 [4] M.F. Bystrova, E.N. Budanova, Hydrogen peroxide and peroxiredoxines in redoxregulation of intracellular signaling.// Biol. Membranes 2007, 24, 2, 115-125. [5] Junior R. R.M., Oliveira M.S.C., Baccache M.A., F.M. de Paula/ Effect of Water deficit and rehydratation on the polar lipid and membrane resistance leaves of Phaseolus vulgaris L. cv. Perola.// Brasilian archives of biology and technology, 2008, V.51, N 2, [6] Sahsah Y, Campos P., Gareil M. et all/ Enzymatic degradation of polar lipid in Vigna uniquiculata leaves and influence of drought stress// Plant. Physiol. 1988, V. 104, P. 577-586. [7] Karimova F.G., Vanjushina S.A., Fedina E.O., Mudarisov F.F.// Tyrosine phosphorilation of plant proteins induced by melaphene.// Status of studies and outlook for the future use of a plant growth regulator of the new generation – Melaphene in farming and biotechnology 2006, Kazan’, Shkola, 50-69. [8] A. Leone A. Costa, S. Grillo, M. Tucci and all/ Acclimation to low water potential changes in membrane fatty acid composition and fluidity in potato cells// Plant, Cell and Environment 1996 19, 1103-1109 [9] A. Ayoz, A. Kadioglu, A. Dogru//Leaf rolling effects on lipid and fatty acid composition in Ctenanthe setosa (Marantaceae) subjected to water-defficit stress// Acta Physiol.Plantarum 2001, 23, 1861-1864. [10] V. I. Popov, E.K.Ruge, A.A. Starkov// Influence of inhibitors of electronic transfer on formation of reactive oxygen species at oxidation of succinate be rea sprouts mitochondria// Biochemistry (Biohimiya) 2003, 68, 7, 910-916. [11] J. Wang, H. Sunwoo, G. Cherian, I.S. Sim// Fatty acid determination in chicken egg yolk. A comparison of different methods// Poultary science 2000, 79, 1168-1171.

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[12] R.V. Golovina, T.E. Kuzmenko// Thermodynamic evaluation interaction of fatty acid metyl esters with polar and non-polar stationary phases, based on their retention indices chromatographia// 1977, 10 , 9, 545-546. [13] D. Pastore, D. Trono, M. N. Laus, N. Di Fonzo and Z. Flagella// Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria//J. of Experemental Botany 2005, 58, 2, 195-210. [14] Xiaoli Liu , Xuejun Hua Ж Juan Guo , Dongmei Qi ,Lijuan Wang , Zhipeng Liu , Zhiping Jin, Shuangyan Chen , Gongshe Liu// Enhanced tolerance to drought stress in transgenic tobacco plants overexpressing VTE1 for increased tocopherol production from Arabidopsis thaliana// Biotechnol Lett. 2008, 30:1275–1280 [15] P. Valentovič, M. Luxová, L. Kolarovič, O. Gašparíková// Effect of osmotic stress on compatible solutes content, membrane stability and water relations in two maize cultivars// Plant Soil Environ 2006, 52, 4, 195-210 [16] Dutilleul C, Garmier M, Noctor G, Mathieu C, Chetrit P, Foye CH, et al.// Leaf mitochondria modulate whole cell redox homeostasis, set antioxidant capacity, and determine stress resistance through altered signaling and diurnal regulation// The Plant Cell 2003 ,15, 1212–1226 [17] Leister D. // Genomics-based dissection of the cross-talk of chloroplasts with the nucleus and mitochondria in Arabidopsis //. Gene 2005, 354, 110–116. [18] Rhoads DM, Subbaiah CC.// Mitochondrial retrograde regulation in plants// Mitochondrion 2 007, 7, 177–194. [19] Ohba M., Shibanuba M., Kuroki T., Nose K.// Production of hydrogen peroxide by transforming grows factor-beta I and its involvement in induction of egr-I in mouse osteoblastic cells// J. Cell. Biol. 1994, 126, 1079-1088. [20] Sandaresan M., Yu.Z.X. Ferrans V.J., Irani K., Finkel T.// Requirement for generation H2O2 for platelet- derived grows factor signal transduction// Science 1995, 270, 269299. [21] Sattler M., Winkler T., Verma S., Byrne S.H. Shrikhande G., Salgia R., Griffin J.D.// Hematopoietic grows factors signal through formation of reactive oxygen species// Blood 1999, 93, 2928-2935. [22] Tala S., Woodhead V., Foreman J.C., Chain B.M.// The role reactive oxygen species in triggering proliferation and IL-2 secretion in T cells// Free Rad. Biol. Med. 1999, 26, 14-23. Lanbeth J.D., Nox/Duox family of nicotinamide adenine dinucleotide (phosphate) oxydases// Curr. Opin.Hematol. 2002, 9, 11-17. [23] I.V. Zhigacheva, L.F. Fatkullina, E.B. Burlakova, A.G. Shugaev, I.P. Generozova, S.G. Fattakhov, A.I. Konovalov// Influence of the organophosphorus plant grows regulator on structural characteristics of membrane of plant and animal origin// Biological membranes ( Biologicheskie membrany RUS) 2008,.25, № 2, . 150-156. [24] S.P. Makarenko, Yu.M. Konstantinov, S.V. Kotimchenko, T.A. Konenkina, A.S. Arziev, Fatty acid content of lipids of Zea mays and Elymus sibiricus.mitochondria membranes. /Fiziologiya. rastenii 2003, .50, 4, 487-492. [25] A. Gigon, A.R. Matos, D. Laffray, Y.Z.-Fodil, Anh-Thu Pham-Thi. Effect оf drought on lipid metabolism in the leaves of Arabidosis Thaliana (Ecotype Columbia)// Annals of Botany 2003, 93, 3 , 841-850.

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[26] Guo Yun-ping, Li Jia-rui. Changts of fatty acids composition of membrane lipids, tethylene release and lipoxygenase activity in leaves of apricot under drought// Journ.of Zhejiang University (Agrical & Life Sci) 2002, .28, 5, 513-517. [27] N.E. Kucherenko, A.V. Vasiliev// Lipidy 1985 Vyschashkola, Kiev, 247 pp. [28] V.I. Kostin, O.V. Kostina, V.A. Isaichev// Results of stidies on use of melaphene in crop growing. //State of studies and outlook for future use of a plant growth regulator of the new generation – Melaphene in farming and biotechnology 2006, Kazan’, Shkola, 27-34.

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.83-90 ©2010 Nova Science Publishers, Inc.

Chapter 8

ANTIOXIDANT PROPERTIES OF ESSENTIAL OILS FROM CLOVE BUD, LAUREL, CARDAMOM, NUTMEG AND MACE T. A. Misharina*, M. B. Terenina, and N. I. Krikunova Emanuel Institute of Biochemical Physics, Russian Academy of Sciences ul, Moscow, Russia

ABSTRACT The antioxidant properties and stability during the storage of hexane solutions of 5 individual essential oils from clove bud (Caryophyllus aromaticus L.), cardamom (Elettaria cardamomum L.), laurel (Laurus nobilis L.), nutmeg (Myristica fragrans Houtt.) and mace (Myristica fragrans Houtt), were studied by the method of capillary gas-liquid chromatography. We assessed the antioxidant properties by the efficiency of autooxidation inhibition of aliphatic aldehyde (trans-2-hexenal) into the according carbon acid. The essential oil of clove bud had the maximal efficiency of inhibition of 2- hexenal oxidation (83%) which was not depended on oil concentration in model solution. Antioxidant properties of essential oils of nutmeg, mace and laurel was connected with a content of substituted phenols, and depended poorly on oils concentration in model systems. The stronger dependence the antioxidant activity on the oil concentration in model solution was found for cardamom essential oil. We studied the changes in essential oils composition during the storage of their hexane solutions for 100 days in the light and compared it with the stability of pure essential oils stored for a year.

Keywords: Essential oils; autooxidation of 2-hexenal; inhibition of oxidation; changes in composition during storage.

Essential oils of many plants possess an intensive and pleasant aroma and also are biologically active [1-3]. Due to these properties, essential oils are actively used for a long * Emanuel Institute of Biochemical Physics, Russian Academy of Sciences ul, Kosygina 4, Moscow, 119334 Russia; e-mail: [email protected]

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time in different industries, mainly in perfumes (fragrances and aftershaves), in food (as flavorings and preservatives) and in pharmaceuticals [3, 4]. As a rule, essential oils are a complex mixture of organic substances with different functional groups, mainly terpenoids and substituted phenols. The composition of essential oils determines their organoleptic properties and biological activity including antioxidant [5-8]. The antioxidant activity of many essential oils is not surprising in view of the presence of phenol groups. It is well known that almost all phenols can function as antioxidants of lipid peroxidation because they trap the chain-carrying lipid peroxyl radicals [9-11]. Plant phenolics are multifunctional and can act as reducing agents, hydrogen-donating antioxidants and singlet-oxygen quenchers [12 - 14]. As a rule, antioxidant activity of essential oils is higher than of their individual components. This fact indicates the existence of synergetic effects due to the complex composition of oils [15, 19, 20]. For the estimation of antioxidant properties of substances or their mixtures, many different methods are used. It was showed that the value of antioxidant activity significantly depends on the method of its estimation, which is why published data obtained by different methods is practically not comparable. Besides, antioxidant properties of essential oils depend on the qualitative and quantitative composition of systems under test [6, 10, 13, 20, 21]. One of the simple and informative methods of the quantitative assessment of antioxidant activity is based on the inhibition of speed of lower aldehyde autooxidation, for example hexanal, in the presence of antioxidant substances [7, 8, 20, 22]. This method was used successfully for the estimation and comparison of antioxidant properties of a number of essential oils [20, 22-25]. The aim of the work is to study and compare antioxidant properties of 5 essential oils containing substituted phenols in the model system of autooxidation of 2-hexenal, the assessment of the influence of essential oils composition on their antioxidant activity, and also the study of changes in the composition of essential oils during their autooxidation in solutions and in pure oils.

MATERIALS AND METHODS The fresh samples of essential oils from clove bud (Caryophyllus aromaticus L.), laurel (Laurus nobilis L.), cardamom (Elettaria cardamomum L.), nutmeg (Myristica Fragrans Houtt.) and mace (Myristica fragrans Houtt) (company “Plant Lipids Ltd.”, India) we studied. For the estimation of antioxidant properties of essential oils and their mixture, 600 μl trans-2-hexenal (3 μl /ml) and 400 μl undecane (2 μl /ml) ( an internal standard) were dissolved in 200 ml of n-hexane. The solution was separated into 3-ml aliquots, which were placed in 5-ml glass vials and then 10 μl (3.33 μl /ml), 50 μl (16.5 μl /ml) or 210 μl (70 μl /ml) of essential oils were added. Oil was not added in the control sample. Each sample was prepared twice, the control sample was prepared three times. The samples in vials with stoppers were stored in light under room temperature for 100 days. Every week vials were opened and blown with 10 ml of air with the help of a pipette. The quantitative content of 2hexenal and components of essential oils in vials were determined by method of capillary gaschromatography after every 10 days from the beginning of storage.

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Gas-chromatography analysis of essential oils samples and control sample was carried out on a Kristall 2000M chromatograph (Russia) with a flame ionization detector and an SPB1 fused silica capillary column (50 m x 0.32 mm, phase layer 0.25 μm). The samples were analyzed at the column temperature programming from 60 to 250oC with the speed of 8oC/min under the temperature of detector and injector at 250oC. The rate of carrier gas helium through the column was 1.5 ml/min. We analyzed 2 μl of hexane solutions at once. The identification of components in oil samples was carried out on the basis of the retention indexes by their comparison with literary [26] or experimental data obtained by us. The quantitative content of 2-hexenal and essential oils components was calculated by the ratio of peak areas, which corresponded to the substances and internal standard. The oxidation extent of 2-hexenal and essential oils components (%) was determined in reference to their content in initial samples.

RESULTS AND DISCUSSION The main component of clove bud oil was eugenol (80,3%), which is responsible for its odor and antiseptic and antioxidant properties. Other major components were eugenyl acetate (10,1%) and caryophyllene (6,3%). Nutmeg oil was obtained by steam distillation of the kernels of nutmeg, which are the dried fruits of Myristica Fragrans Houtt. Mace oil was obtained from the coverings of nutmeg. The nutmeg oil contained ca. 90% monoterpene hydrocarbons, mainly sabinene (30,7%), α -pinene (14,0%), β -pinene (10,8%), and γ terpinene (5,8%). Major oxygen-containing constituents were terpinen-4-ol (6,7%) and phenol ether derivatives: safrole (0,9%), myristicin (2,4%) and elemicin (1,2%). Mace oil contained the same substances, but their content was differed: sabinene (20,5%), α -pinene (21,2%), β -pinene (16,0%), γ -terpinene (5,8%), terpinen-4-ol (4,6%), safrole (1,6%), myristicin (2,8%) and elemicin (0,3%). Cardamom oil was produced from the seeds of Elettaria cardamomum (L.) Maton (Zingiberaceae). This essential oil contained 41,2% 1.8cineole, 39,2 % α -terpinyl acetate. The content of monoterpene hydrocarbons, linalool, geraniol, linalyl and geranyl acetates was ca. 1-2 %. Trace constituents like unsaturated aliphatic aldehydes may be important for the typical cardamom aroma. Laurel leaf essential oil was obtained from leaves of evergreen tree Laurus nobilis L. The main components of this oil were 1.8-cineole (49,8%), α -terpinyl acetate (9,6%), eugenol (7,1%), linalool (4,4%), sabinene (9,5%), α -pinene (5,8%), β -pinene (4,6%), α -terpineol (2,3%), and terpinen-4-ol (1,7%). Using the method of capillary gas chromatography allowed us to estimate changes in the content of each oil component in the model systems with different oil concentration and in pure oil during autooxidation, and also to distinguish the oxidation products of main components. Furthermore, due to the comparison of oil composition and the oxidation rates of components, we managed to reveal some regularity, which enables us to predict and to regulate oil composition to obtain stable mixtures. This is very important because essential oils are currently widely used in industry and medicine. Usually the recommended storage time for oils is 1 year; however, nobody has studied yet what really happens with oils during this period. We earlier studied the changes in the composition of coriander, laurel, marjoram, and fennel oils in the process of storage of pure oil samples in the dark and in the light but in bottles from dark glass [27-29]. It was established that the main process is the oil components'

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oxidation. Thus, we found, and then it was proved in works [10, 11], that cyclic monoterpenes hydrocarbons α- and γ- terpinenes are completely oxidized into aromatic hydrocarbon pcymene. We also found oxidation products of other components of essential oils - oxides, alcohols, and aldehydes [26-29]. For the estimation of antioxidant properties of essential oils we used a model system of 2hexenal day-light induced autooxidation, which is used in the similar studies [7, 8, 20-25]. As a criteria for the estimation of antioxidant properties of essential oils we used the quantity of 2-hexenal, which remained unoxidized after 40 days in reference to the initial quantity (%). Fig. 1 presents the obtained results of relative antioxidant activity values of 5 studied essential oils. The all model systems included essential oils in two concentrations: 3.3 μl /ml and 16.5 μl /ml. For the essential oils of cardamom and mace we studied an additional third systems in which the content of oils was 70 μl /ml of hexane solution. As is clearly seen from Fig. 1, the concentration of oils influenced on their antioxidant activity for all oils with the exception of clove bud oil. It is noteworthy that the increase in activity was usually not in proportion to the growth of essential oil concentration. The systems with minimal content of laurel, mace, and nutmeg oils (3.3 μl/ml ) had antioxidant activity more than 50%. The increase of content of these oils by five times was accompanied by the increase of antioxidant activity by 20-30%, and the further increase in content of mace oil by four times (from 16.5 to 70 μl /ml) led to the growth of oil activity by only 6 %. The cardamom oil in diluted solution (3.3 μl /ml) had very low antioxidant activity - only 20%. The increase in concentration of this oil by five times (from 3.3 to 16.5 μl /ml) led to the increase of activity by 3.5 times. The further increase in concentration of cardamom essential oil up to 70 μl /ml led to the increase of antioxidant activity only by 3 %. The inhibition of 2-hexenal oxidation by clove bud oil was practically non-dependent on the concentration and was 75-78%. Antioxidant activity and composition stability of clove bud oil were high. In both hexane solutions, oil did not change the composition for 100 days. Pure individual oil was also stable while being stored in the dark for 2 years and in the light for 8 months. We did not notice oxidation even of traces amounts of monoterpene hydrocarbons in stored clove bud oil. Antioxidant activity of nutmeg and mace essential oils increased from 53 to 69% and from 63 to 78% accordingly, with the increase of their concentration in model solutions (Fig. 1). Thus, activity of mace oil was higher to 10% than that for nutmeg oil. These oils were close in the quantitative and qualitative content of components. The main compounds in oils were monoterpene hydrocarbons and alcohol terpinen-4-ol. The aroma of this species is due to phenol derivatives - safrole and myristicin, the content of which in both oils was from 0.9 to 2.6%. These compounds together with terpene hydrocarbons were correspondent for the antioxidant activity of oils. During the autooxidation of oils in hexane solutions for 40 days, only the content of γ-terpinene decreased by 3-4 times and the content of its oxidation product, p-cymene, increased. During storage of these pure oils for 4 months, the content of γ-terpinene did not change but the storage for 1 year led to practically the complete oxidation of γ-terpinene and the oxidation of 50% of caryophyllene with the formation of caryophyllene oxide.

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90 80

2-Hexenal content, %

70 60 50 40 30 20 10 0 1

Control

2

3

C1

4

C2

5

6

C3

Figure 1. Relative antioxidant activity of essential oils. 1 - Control (2-hexenal) Essential oils: 2 - clove bud, 3 - nutmeg, 4 - mace, 5 - laurel, 6 - cardamom. Concentration of essential oils (μl/ml): C1 - 3.3, C2 - 16.5, and C3 - 70.

Laurel and cardamom essential oils had close composition of main components – 1,8cineole and terpinyl acetate; the difference in aroma was due to the presence in cardamom oil of about 1% of neryl and linalyl acetates and nerolidol. Laurel essential oil in hexane solution at concentration of3.3 μl /ml inhibited the oxidation of 2-hexenal 1.5 times more effectively than cardamom essential oil (Fig. 1). Laurel oil, after 40 days of autooxidation in the light in hexane solution, remained stable. We previously established that individual pure laurel oil did not change its content in storage in the dark for 2 years [29]. The stability of cardamom essential oil as well as its antioxidant activity depended on its concentration in hexane solution. So, under the concentration 3.3 μl /ml the quantity of sabinene decreased by five times and terpinyl acetate decreased by two times, α- and γ -terpinenes were oxidized completely. In more concentrated solutions, only α- - and γ -terpinenes were oxidized completely; also, the content of other components in solutions with oil concentration of 16.5 and 70 μl /ml changed in the same way. It is noteworthy that antioxidant activity of cardamom oil was increasing with the increase of its concentration in the model system (Fig. 1). Probably, they were mainly α- and γ -terpinenes, which were in answer for the antioxidant activity properties of this oil. During the storage of pure cardamom essential oil, after 100 days we noticed the significant oxidation of α- and γ -terpinenes; the quantity of sabinene decreased by 65% and that of terpinyl acetate decreased by 35% (Fig.2). The reason for changes in laurel and cardamom essential oil behavior was probably that laurel oil contained approximately 7% of eugenol and 2% of methyl eugenol, which possesses strong antioxidation properties. Due to their presence, the laurel oil has higher antioxidant activity

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and was relevant to oxidation. Adding to the cardamom essential oil at least 0.5% of clove bud oil, which contained approximately 80% of eugenol, increased significantly the relevance of all components to oxidation in comparison with individual cardamom oil (Fig. 2). In many cases, the antioxidant activity of the essential oils could not be attributed to the major compounds, and minor compounds might play a significant role in the antioxidant activity, due to synergistic effects [15,19,20 ]. For instance, in Melaleuca species ( cajuput oil or teatree oil), essential oil containing 1,8-cineole (34%) and terpinen-4-ol (19%) exhibited stronger antioxidant activity than those with high methyleugenol (97%) or 1,8-cineole (64%) contents [3].

100

4 Component content, %

90

3

80 70

2

60 50 40

1

30 0

20

40

60

100 days

Figure 2. The content of sabinene and α-terpinyl acetate (% from initial) in cardamom oil and its mixture with 0.5% clove bud oil during the storage: 1 and 2 are sabinene and α-terpinyl acetate in cardamom oil; 3 and 4 are sabinene and α-terpinyl acetate in the mixture of cardamom and 0.5% clove bud oils.

Thus, conducted research and literary data show that essential oils are effective natural antioxidants, which are able to compete with synthetic ones. Antioxidant properties of essential oils are determined by their composition. Oils with high content of substituted phenols are able to significantly inhibit oxidation processes of labile unsaturated aldehydes even in low concentrations. Antioxidant properties of essential oils, which consist of terpene

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hydrocarbons and alcohols, are determined by α- and γ- terpinenes and their sesquiterpene analogs. We revealed the significant influence of the concentration of such oils ( cardamom, for example) on their antioxidant properties. The stability of essential oil composition increased with the increase of their concentration in model solutions. The oxidation of substances in pure essential oils happened slower than in solutions.

REFERENCES [1]

[2] [3] [4] [5]

[6]

[7] [8] [9] [10] [11]

[12]

[13]

[14]

L.H. Madsen, B.R.Nielsen, G.Bertelsen, L.H. Skibsted: Screening of antioxidative activity of spices. A comparison between assay based on ESR spin trapping and electrochemical measurement of oxygen consumption. Food Chem., 57, 331 (1996). S.A. Voitkevich: “Efirnye masla dlya parfyumerii i aromaterapii” (Essential Oils for Perfumery and Arometherapy). Pishch. Prom., Moscow, 1999. Flavours and Fragrances. Chemistry, Bioprocessing and Sustainability (Ed. R.G. Berger). Springer, New York, 2007, 43-116. K. Bauer, D. Garbe, H. Surburg: Common Fragrance and Flavor Materials, VCH Verlag, Weinheim, 1990. G. Cervato, M. Carabelli, S. Gervasio, A. Cittera, R. Cazzola, B. Cestaro: Antioxidant properties of oregano (Origanum vulgare L.) leaf extracts. J. Food Biochem., 24, 453 (2000). H.J.D. Dorman, A. Peltoketo, R.Hiltunen, M.J.Tikkaken: Characterisation of the antioxidant properties of deodourised aqueous extracts from selected Lamiaceae herbs. Food Chemistry, 83, 255 (2003). K.G. Lee, T. Shibamoto: Antioxidant property of aroma extract isolated from clove buds. Food Chem., 74, 443, (2001). K.W. Lee, Y.J. Kim, D.-O. Kim, H.J. Lee, C.Y. Lee: Major phenolics apple and their contribution to the total antioxidant capacity. J. Agric. Food Chem., 51, 6516, (2003). G.Litwinienko, K.U. Ingold: Solvent effects on the rates and mechanisms of reaction of phenols with free radicals. Acc. Chem. Res., 40, 222 (2007). G.. Ruberto, M. Baratta: Antioxidant activity of selected essential oil components in two lipid model systems. Food Chem., 69, 167 (2000). M.C. Foti, K.U. Ingold: Mechanism of inhibition of lipid peroxidation by γ–terpinene, an unusual and potentially usefull hydrocarbon antioxidant. J. Agric. Food Chem. 51, 2758 (2003). M.T. Baratta, H.J.D. Dorman, S.G. Deans, D.M.Biondi, G.Ruberto: Chemical composition, antimicrobial and antioxidative activity of laurel, sage, rosemary, oregano and coriander essential oils. J. Essent. Oil Res., 10, 618 (1998). S.S.Pekkarinen, H.Stocmann, K.Schwarz, M.Heinone, A.I.Hopia: Antioxidant activity and partitioning of phenolic acids in bulk and emulsified methyl linoleate. J. Agric. Food Chem. , 47, 3036 (1999). G.Sacchetti, S.Maietti, M.Muzzoli, M.Scaglianti, S.Manferdini, M.Radice, R.Bruni: Comparative evaluation of 11 essential oils of different origin as functional antioxidants, antiradicals and antimicrobials in foods. Food Chem. 91, 621 (2005).

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[15] G. Singh, S. Maurya, C. Catalan, M.P. de Lampasona: Studies on essential oil, Part 42.: chemical, antifungal, antioxidant and spout suppressant studies of ginger essential oil and its oleoresin. Flavour Fragrance. J., 20, 1 (2005). [16] A.Wei, T. Shibamoto: Antioxidant activities and volatile constituents of various essential oils. J. Agric. Food Chem., 55, 1737 (2007). [17] C. Menut, J.M. Bessiere, D.Samate, A.K. Djibo, G.Buchbauer, B. Schopper: Aromatic plants of tropical west Africa. XI. Chemical composition, antioxidant and antiradical properties of the essential oils of three Cymbopogon species from Burkina Faso. J. Essent. Oil Res., 12, 207 (2000). [18] N.Mimica-Dukic, B. Bozin, M. Sokovic, N. Simin: Antimicrobial and antioxidant activities of Melissa officinalis L. (Lamiaceae) essential oils. J. Agric. Food Chem., 52, 2485 (2004). [19] D.Huang, B.Ou, L.R.Prior: The chemistry behind antioxidant capacity assays. J. Agric. Food Chem., 53, 1841 (2005). [20] K.G. Lee, T.Shibamoto: Determination of antioxidant potential of volatile extracts isolated from various herbs and spices. J. Agric. Food Chem., 50, 4947 (2002). [21] K.G. Lee, T.Shibamoto: Antioxidant activities of volatile components isolated from Eucalyptus species. J.Sci. Food Agric., 81, 1573 (2001). [22] K.Yanagimoto, H.Ochi, K.G. Lee, T. Shibamoto: Antioxidative activities of volatile extracts from green tea, oolong tea, and black tea. J. Agric. Food Chem., 51, 7396 (2003). [23] C.J. Lee, T. Shibamoto, K.G. Lee: . Identification of volatile components in basil (Ocinum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chem., 91, 131 (2005). [24] B.Bozin, N.Mimica-Dukic, G.Anachov: Characterization of the volatile composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities of the entire oils. J. Agric. Food Chem., 54,1822 (2006). [25] T.A Misharina, A.L. Samusenko: Antioxidant properties of essential oils from lemon, grapefruit, coriander, clove, and their mixtures. Applied Biochem. and Microbiol., 44, 473 (2008). [26] W. Jennings, T.Shibamoto: Qualitative Analysis of the Flavor and Fragrance Volatiles by Glass Capillary Gas Chromatography, Academic, New York: 1980, 130-154. [27] T.A Misharina: Influence of the duration and conditions of storage on the composition of the essential oil from coriander seeds. Applied Biochem. and Microbiol., 37, 622 (2001). [28] T.A Misharina, A.N. Polshkov, E.L. Ruchkina, I.B. Medvedeva: Changes in the composition of the essential oil of marjoram during storage. Applied Biochem. and Microbiol., 39, 311 (2003). [29] T.A Misharina, A.N. Polshkov: Antioxidant properties of essential oils: autoxidation of essential oils from laurel and fennel and of their mixtures with essential oil from coriander. Applied Biochem. and Microbiol., 41, 610 (2005).

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.91-121 ©2010 Nova Science Publishers, Inc.

Chapter 9

SPECIFIC PROPERTIES OF SOME BIOLOGICAL COMPOSITE MATERIALS N. Barbakadze*, E. Gorb and S. Gorb Max Plank University, Shtutgart, Germany Georgian Technical University, Tbilisi, Georgia

ABSTRACT Miniaturisation of technical systems creates the need for today’s science and engineering to assess the mechanical properties of small volumes of material. A specific feature of the structure and the combination of the desirable properties across several different length scales are fascinating using plenty of examples in biology. Determining the extraordinary properties of natural materials at the nanometer scale is regarded as very attractive targets for materials science. Mechanical behavior of various biological materials such as insect and plant cuticles was studied by applying experimental approaches of material science in order to explain their structural design and working principles. Experiments were performed on the head articulation cuticle of the beetle designed for friction minimisation and on the wax covered plant surfaces adapted for attachment prevention. Both insect and plant cuticles are multifunctional composite materials and have a multilayered structure. Gula cuticle of the beetle Pachnoda marginata is a part of the head articulation, which is a micromechanical system similar to a ball bearing. The surfaces in this system operate in contact and must be optimised against wear and friction and provide high mobility within the joint. The measurements on the gula cuticle were performed in order to understand structure and mechanical behavior of material working for friction minimizing. The wax layer on the plant surfaces is a barrier for the attachment system of insects. Antiattachment function could be caused by contamination of attachment pads of insect with the wax crystalloids. Increase in roughness due to location of the wax crystals on the plant surface causes decrease in the real contact area between the plant surface and attachment pads of insect. These are two of the hypotheses why insects cannot walk on the plant surfaces structured with wax. Nanoindentations on different plant surfaces were performed in order to understand the * Max Plank University, Shtutgart,Germany Georgian Technical University, 77, Kostava Str., 0175 Tbilisi, Georgia, [email protected]

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N. Barbakadze, E. Gorb and S. Gorb deformation behavior of the wax layer. This study is believed to be one of the first for mechanically testing insect cuticle and the very first for wax coated plant surfaces in native condition.

MOTIVATION AND LITERATURE REVIEW ”Why should engineers be interested in biological materials? One only has to look to the history of technology, starting from the first use of tools, to see that revolution and progress are driven by the new possibilities which new materials create.” Julian Vincent

Natural materials have been used in engineering over twelve millennia. The first houses and bridges were built from grass, wood and stone. The first clothes and shoes were made from animal skins. There are still no alternatives to wood and horsehair for many musical instruments (Wegst and Ashby, 2004). We use natural materials everyday. They are always around us and the interest in biological systems is still growing. The natural systems inspire the engineering sciences in the design of new materials. One example is the development of different replacement tissues from metal or plastic (Vincent, 1990). Most natural materials are structured fiber-matrix composites (Wainwright et al., 1976; Vincent, 1990). The components in them are quite similar. They consist mainly of natural polymer fibers embedded in a protein matrix. For example, cellulose, pectin and protein build a plant cell wall (Wegst and Ashby, 2004); insect cuticle is the composite of chitin and protein (Neville, 1975; Wainwright et al., 1976; Vincent, 1990; Vincent and Wegst, 2004). The biological materials exhibit a hierarchical structure. Natural systems such as skin, bone, cartilage and cuticle are layered composites with different single layer structures. Despite the limited constituents, the natural systems display a great variety of mechanical properties. Collagen and elastin are the main components in skin, bone and cartilage. However, depending on the amount of the constituents and the fiber orientation, each system has different mechanical properties (Wegst and Ashby, 2004). The structural and mechanical design of natural systems can be mimicked to provide new so called “intelligent” materials with “smart” structures. Skin, for instance, shows compressive properties useful in the development of tactile sensors (Vincent, 1990). The man-made hydrophobic surfaces have been inspirited from the “lotus effect” of the plant leaf. The technique of the turn around of the beetle arouses the interest of robotics researchers to design a robot being able to cope with any difficulties in movement. In figures 1 and 2 (Vincent and Wegst, 2004) the wide spectrum of mechanical properties of natural materials is shown. Figure 1 presents Vickers hardness for a range of natural materials. For example, the arthropod (invertebrates) cuticle displays a great variation of hardness values depending on its function and condition (wet or dry). In the figure 2 Young’s modulus is plotted versus density for different natural materials. Again arthropod cuticle shows different elastic modulus comparable with human skin, cork, wood and even aluminum. Determining the extraordinary properties of natural materials at the nanometer scale is regarded as a very attractive target of materials research. However, tribological systems in

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biology have not been studied much from a materials science point of view. The majority of work in tribology research has focused on the material properties and working mechanisms of human and animal joints (Fung 1993; Persson 1998). It was found that articular cartilage has a unique quality of lubrication and shock absorption that is mainly due to the multilayer structure (Fung, 1993). Being related to bone, cartilage has a very small coefficient of friction for relative motion between two articular pieces (Fung, 1993).

Figure 1. A compilation of hardness data for natural materials. (Figure adapted from Vincent and Wegst, 2004, created using the Natural Materials Selector).

There are considerable data in the literature about ultrastructural architecture and mechanical properties of the arthropod cuticle (Fraenkel and Rudal, 1940; Neville, 1975; Hepburn and Joffe, 1976; Andersen, 1979; Andersen et al., 1996; Vincent, 1980; 2002; Binnington and Retnakaran, 1991; Gorb, 1997; Arzt et al., 2002; Enders et al., 2004; Vincent and Wegst, 2004). It is the most widely distributed high-performance composite material after plant cell and wood (Vincent, 2002). Much work has been published on the correlation of microtribological properties of biological systems with its material structure (Scherge and Gorb 2000; Gorb and Scherege 2000; Gorb et al. 2001; Dai et al. 2002; Gorb and Perez Goodwyn 2003; Perez Goodwyn and Gorb 2004). Wax coated plant surfaces were studied in relation to insect attachment systems over the last century (Kerner von Marilaun, 1898). Great work has been done on the structure and the chemical composition of the wax crystals (Hallam and Chambers, 1976; Bianchi et al., 1978; Haas and Schonherr, 1979; Baker, 1982; Avato et al., 1984; Jeffree, 1986; Baker and Gaskin, 1987; Walton, 1990; Barthlott, 1990; Bianchi, 1995; Barthlott et al., 1998; Gorb, 2001; Eigenbrode and Jetter, 2002; Gorb and Gorb, 2002). However little is known about the mechanical properties of the wax layer. This study aims to explore mechanical performance of insect and plant cuticle using the material sciences approach. Head articulation cuticle of the beetle (Pachnoda marginata) designed for friction minimization and wax covered pea (Pisum sativum) plant surfaces adapted for insect attachment prevention have been studied. Mechanical properties were determined by nanoindentation.

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Figure 2. Young’s modulus versus density chart for natural materials. (Figure adapted from Vincent and Wegst, 2004, created using the Natural Materials Selector).

HEAD ARTICULATION OF THE BEETLE The body of insects is completely covered by cuticle, which is a multifunctional interface between the animal and the environment (Hepburn and Joffe, 1976; Gorb, 2001). It serves primarily as an exoskeleton that gives the body its shape and stability. The cuticle is also a main barrier against evaporation of water from the body and protects the insects against desiccation (Locke, 1964; Binnington and Retnakaran, 1992). Like most biological materials, cuticle is a fiber composite (Hepburn and Chandler, 1980). The fibers consist of chitin and the matrix is formed by proteins. Chitin is a natural polymer composed of 300 nm long and 3 nm thick nanofibrils (Vincent, 1980; 2002). Each

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nanofibril contains about 19 molecular chains (Fraenkel and Rudall, 1940; Vincent, 1980; 2002) running antiparallel to one another (i.e. alpha chitin) (Neville, 1975; Vincent, 1980). Chitin filaments are distributed in the protein matrix (Andersen et al., 1996) which stabilises the chitin. Water contained in the proteins (about 90 % of the protein matrix) presumably functions to separate the two main components of the cuticle from each other (Vincent, 1980). Arthropod (invertebrates – animals without backbones) cuticle has a multilayer structure (Neville, 1975; Andersen, 1979) (figure 3). It typically consists of three main layers: epicuticle, exocuticle and endocuticle. The latter two layers form the procuticle. In some insects there is a mesocuticle between the endocuticle and the exocuticle (Neville, 1975; Andersen, 1979; Binnington and Retnakaran, 1992). The nonchitinous, tanned lipoproteinous epicuticle is the outer layer of the cuticle, which is very thin and has a relatively high tensile strength. The surface of the epicuticle is coated with wax and lipids. The hard and stiff solid part has a dense chitin-protein structure and forms the exocuticle. The endocuticle is the thickest region of the cuticle and has low chitin content (Locke 1964; Neville 1975, Binnington and Retnakaran 1992). From a mechanical point of view, there are two main types of insect cuticle: soft and hard. In larvae the cuticle is soft and colorless. A simultaneous hardening and darkening, called sclerotisation (Fraenkel and Rudall, 1940), forms a hard and colored integument in most adults. There is a great variation in kinds of the cuticle depending on the proportions of the main components and, accordingly, mechanical properties (Hepburn and Chandler, 1976). In various animal groups and on different body parts there are different types of cuticle from very hard and brittle to soft, ductile and also rubber-like (Jensen and Weis-Fogh, 1962).

- epicuticle cuticle

- endocuticle

procuticle

- exocuticle

- epidermal cell layer Figure 3. Schematic of the multilayered structure in the insect cuticle. The shaded areas represent the single cuticle layers: outer surface layer, epicuticle which does not contain chitin fibers; procuticle with two layers: exocuticle and endocuticle. Last two cuticle layers contain chitin fibers oriented nearly always parallel to the surface. The endocuticle is connected with epidermis cell structure.

The wide spectrum of mechanical properties of the insect cuticle suggests that each body part is optimised to its function. Any movement involving contact between two surfaces or between a surface and a medium has to deal with the resistance of the surfaces or medium. This resistance is a friction phenomenon, believed to have a great influence on the design of biological structures (Gorb, 2001). Living microsystems have developed different ways to save muscular energy by the use of friction-optimised systems. Surface pairs in biological objects are designed to maximise or to minimise contact forces within joints. In biology they

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are defined as friction and anti-friction systems respectively. Working principles of friction and anti-friction systems are based on mechanical interlocking and a maximisation or a minimisation of the contact area (Gorb, 2001). Frictional systems require high friction to fixate body parts to one another (Gorb, 2001). The surface pair can be predefined as in the wing-locking mechanisms of beetles (Hammond, 1989; Gorb, 1997; Gorb, 1999b) and the head-arresting system of dragonflies (Gorb, 1999a; Gorb, 2001). Among various cases of contact pairs in biology, anti-friction systems always have a predefined pair of surfaces like the head articulation system in beetles (Arzt et al., 2002; Enders et al., 2004) or the hemelytra-hindwing locking mechanism in bugs (Perez Goodwyn and Gorb, 2004). An interesting example of anti-friction system is a head articulation of the beetle Pachnoda marginata. The surfaces in this system operate in contact and must be optimised against wear and friction to provide high mobility within the joint. A predefined, functionally corresponding pair of the contacting surfaces forms a tribological system with specific ultrastructural architecture and properties. However, each contacting piece has a different structure. The rather smooth surface of the hemispherical head-part or gula is believed to contribute to the friction minimisation in contact with its counterpart in the ventral part of the prothorax, covered by asymmetric outgrowths (Enders et al., 2004). In living conditions the organic substances on the cuticle surface, such as lipids and waxes are expected to serve as lubrication materials to reduce the friction in this biological microsystem. For this study a head part (gula) cuticle of the head articulation of the beetle Pachnoda marginata was selected. Both the structure and the mechanical properties of this material are largely unknown. The goal of these investigations was to obtain detailed information about the cuticle structure in the gula and to determine its mechanical properties. Assembly of the head articulation and structure of both contacting surfaces were studied by means of computer tomography and scanning electron microscopy (SEM). SEM images were also used for the evaluation of nanoindentation tests. Hardness and elastic modulus were measured using the nanoindentation technique. The samples were mechanically tested in the fresh, the dry and the chemically treated conditions in order to understand the influence of desiccation (fresh versus dry condition) and removal of an outer wax layer (chemically treated condition). The questions asked were: What are the structure and the local mechanical properties of the head articulation cuticle? How does the liquid content influence the mechanics of the joint material? How do surface waxes influence hardness and elastic modulus of the gula? Since the structure and the mechanical properties of the material are very important for tribological performance, this study aims to make a contribution to exploring the working principle of the gula cuticle.

MATERIALS AND SAMPLE PREPARATION Samples were prepared from the head articulation system of the beetle Pachnoda marginata. The beetles were anesthetized with CO2. Heads were dissected from the body and freed of soft tissue. To prevent the drying of the specimens, fresh gula cuticles were tested immediately (in 3-5 min after dissecting from the body). Dry samples were prepared from fresh cuticles by drying. To obtain the same degree of desiccation, fresh samples were dried

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in an oven for 24 h at 40°C. Chemically treated samples were prepared from the dry gula cuticles. In order to remove waxes from the surface, dry samples were treated in a solution of chloroform and methanol (2:1) for 50-60 min and air-dried.

Figure 4. X-Ray tomography (µ-CT system) images in transversal (A) and sagittal (B) planes of the head articulation of the beetle Pachnoda marginata.

SHAPE AND STRUCTURE OF THE CUTICLE To observe the working position and the shape of the contacting surfaces, the anatomy of the head articulation system of the beetle Pachnoda marginata was explored with high

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resolution computer tomography (X-Ray µ-CT system, model 1072 tomograph by Sky Scan at the Institute für Kunststoffprüfung (IKP) at the University of Stuttgart, Germany). This method allows non-invasive imaging of the internal structure. The measurements were performed on dry samples. The images of the head articulation of the beetle were taken in two directions: transversal and sagittal (magnification × 30). Images showing cross sections of the contact parts (gula and prothorax) of the head articulation were taken in dry insects in two directions: transversal (figure 4 A) and sagittal (figure 4 B). The hemispherical convex shape of the gula cuticle can be seen in the cross section image in both transversal and sagittal direction. As can be distinguished in the figures, the head articulation system is an open joint. In the transversal image, the head articulation of the beetle is reminiscent of a technical ball bearing system. The surfaces of the contacting pair of the head articulation system were studied in the scanning electron microscopy (SEM). The samples were fixed in 2.5% glutaraldehyde in a phosphate buffer (pH = 7.3). The specimens were dehydrated in an ascending row of ethanol and then critical-point dried. Pieces of the dried material were fractured using a razor blade. The prepared samples were mounted on holders, sputter-coated with gold-palladium (10 nm thickness) and examined in a Hitachi S-800 scanning electron microscope (SEM) at 20 kV.

A prothorax gula

50 µm

C

5 µm

B

D

50 µm

5 µm

Figure 5. SEM images of the gula (A, C) and prothorax (B, D) surfaces. On the gula surface pores (A, C) and dried organic substances (C) can be distinguished. The figures B and D show cuticlar outgrowths on the prothorax surfaces. The gula and the prothorax surfaces operate in contact (shown with arrows on the figures A and B) in the head articulation system of the beetle Pachnoda marginata.

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Figure 5 shows the surface structures of the gula plate (figure 5A and 5C) and its counterpart (prothorax) (figures 5B and 5D). The gula is rather smooth while the prothorax is covered by cuticular outgrowths. The arrows in the figures 5 A and 5 B show that these surfaces contact each other in the head articulation of the beetle.

EPI  EXO 

gula 

ENDO 

A

C

200 µm

20 µm

B

20 µm

D

2 µm

Figure 6. SEM images of the gula surfaces. A: shows entire surface of the gule. B: cross section of the gula cuticle showing the single cuticle layer: EPI – epicuticle, EXO – exocuticle and ENDO – endocuticle. Perpendicular orientation of the chitin fibers can be distinguished in the exocuticle layer. The well-defined pores, dried organic substances and cracks can be seen on the cuticle surface (C and D).

SEM images in the figure 6 provide information on the shape of the gula cuticle and the ultrastructural architecture within a single cuticle layer. The gula has a hemispherical smooth surface (figure 6 A). Island-like places, occurring on the surface, are, presumably, dried secretory substances, delivered to the surface by well-defined pores, which run deep into the material and apparently are connected with epidermal cells (figure 6C and 6D). Cracks found on the surface are probably caused by material desiccation (figure 6D). The cross-section of the cuticle shows the layered structure of the fiber composite (figure 6B). The thickness of the gula cuticle is about 80 µm. A very thin (7-8 µm) and dense epicuticle can be distinguished.

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Procuticle is present with two layers: dense exocuticle, which is about 22 µm thick and the endocuticle, which is very thick (about 50 µm) and less dense compared to the exocuticle layer. As can be distinguished in figure 6 B, chitin fibres are oriented nearly perpendicular to the surface in the exocuticle layer and parallel to the surface in the endocuticle. The exocuticle structure of the gula differs from the layered pattern of regular cuticle, where the orientation of the chitin fibers is parallel to the surface (Neville, 1975; Wainwright et al., 1976; Vincent, 1990). The fibers in the exocuticle are oriented perpendicular or at some angles to the surface. A similar fiber orientation has previously been found in attachment pads of grasshopper (Gorb and Scherge, 2000). However, the advantage of such structures is not understood at present. Further investigations are required to clarify the reason for this perpendicular fiber orientation.

SURFACE ROUGHNESS The sample surface was studied using a white-light interferometer (Zygo New View 500, Zygo Corporation, USA). This technique can be used to obtain the average surface roughness (Ra), the average absolute value of ten-point height (Rz) or root mean square (rms) representing the height profile’s roughness. The results of surface roughness measurements are summarized in table 1 and shown in the figure 7. In the fresh condition, the surface of the hemispherical convex gula cuticle is extraordinarily smooth for a biological material (Ra = 0.033 ± 0.005 µm, rms = 0.038 ± 0.007 µm) (figure 7 A). After desiccation, the outer layer of the cuticle builds up high roughness (figure 7 B). The water loss is thought to cause the constriction of the structure in inner layers and folding of the outer layer. These structural changes could be the reason why dry samples exhibit a rougher surfaces (Ra = 0.161 ± 0.011 µm, rms = 0.197 ± 0.015 µm). After the chemical treatment, the surface roughness becomes lower (Ra = 0.103 ± 0.008 µm, rms = 0.117 ± 0.011 µm) (figure 7 C). It could be caused by dissolving the dry organic substances or structure on cuticle surface. However, the roughness of the fresh samples remains lower than that of dry and chemically treated ones. Table 1. Summary of surface roughness parameters, Ra and rms, obtained by whitelight interference microscopy for fresh, dry and chemically treated gula cuticles. The surface roughness measurements were carried out on three samples for each condition (SD = standard deviation). Surface roughness parameter Sample condition fresh dry chemically treated

Ra (nm)

SD

rms (nm)

SD

33 161 103

±5 ± 11 ±8

38 197 117

±7 ± 15 ± 11

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A

B

C

Figure 7. Surface profiles of fresh (A), dry (B) and chemically treated (C) cuticles. The images were obtained by white light interference microscopy.

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MECHANICAL PROPERTIES Nanoindentation provides a fast and reliable technique for evaluating local mechanical properties, such as hardness and elastic modulus, of very small volumes of the material (Oliver and Pharr, 1992; Bhushan and Li, 2003). During the past decade, this method has become an important tool in materials characterization (details about this method see in Barbakadze 2005; Barbakadze et al., 2006). In figures 8 A and 8 B hardness and elastic modulus results are plotted vs. displacement (indentation depth into the sample surface). Each data point is the average value of approximately 150 indents (10 beetle heads with 15 indents on each, 150 indents in all).

A

B

Figure 8. The results of hardness and elastic modulus from indentation tests on the gula-cuticle: hardness (A) and elastic modulus (B) are plotted versus displacement for fresh, dry and chemically treated samples. Hardness and elastic modulus were calculated by using the CSM technique from the projected contact area during indentation. Each data point corresponds to the mean value of approximately 150 measurements.

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The measurements revealed a strong dependence of the mechanical behavior on the preparation conditions of biological samples. The significance of the differences due to the conditions was checked by means of statistical calculations. The experimental data were compared with a one-way ANOVA and a Tukey post tests (Origin 7 SR2). Hardness and elastic modulus values of the fresh, the dry and the chemically treated samples were compared with each other by different displacements of 250, 500, 1000 and 1500 nm. The results of the statistical calculations are summarised in table 2. Hardness and elastic modulus differ significantly for the fresh, dry and chemically treated conditions. Exceptions are the elastic modulus values of the dry and chemically treated samples at indentation depths of 1000 and 1500 nm. The effect of the chemical treatment on the sample stiffness at these depths can hardly be observed. Statistical calculations show no significant difference between elastic moduli of the dry and chemically treated samples at indentation depths higher than 1000 nm. The average hardness value (H=0.10±0.07 GPa) of the fresh samples is significantly lower than that of the dry (H=0.49±0.14 GPa) and the chemically treated (H=0.52±0.15 GPa) ones (figure 8B) (ANOVA p<0.0005, Tukey post test p<0.0005). The difference between the hardness results of the dry (H=0.49±0.14 GPa) and chemically treated samples (H=0.52±0.15 GPa) is lower but also significant (statistic: ANOVA p<0.0005), especially in the displacement range of 200-1000 nm. The same tendency was observed for the elastic modulus (figure 8C). The dry (E=7.50±1.80 GPa) and chemically treated (E=7.70±1.90 GPa) samples are significantly stiffer than fresh ones (E=1.50±0.80 GPa) (statistic: ANOVA p<0.0005, Tukey post test p<0.0005). In this case the chemical treatment causes a slight increase of the elastic modulus below 1000 nm indentation depth into the sample surface. At a depth higher than 1000 nm, the elastic modulus values of the dry and chemically treated samples are not significantly different. The maximum displacement for the fresh samples was 3000 nm, but for the dry and chemically treated samples only 2000 nm, because the maximum load of the instrument had been reached. High values of the hardness and elastic modulus in the first 50 nm of indentation are erroneous data caused by contact building between the indenter tip and sample due to the roughness and contamination of the sample’s surface. The hardness and elastic modulus for all samples slowly decreases with indentation depth. However, after 1700 nm, both parameters drop rapidly for the dry and chemically treated specimens. In general, soft and compliant cuticle contains more water than hard and stiff (Vincent and Wegst, 2004). Since the gula cuticle belongs to the hard and brown type of cuticle, it has a low content of water. But even so, the results show that desiccation has a great influence on the mechanical behavior of the cuticle tested. After drying, the gula cuticle becomes about 5 times harder and stiffer (H = 0.49 ± 0.14 GPa, E = 7.50 ± 1.80 GPa) than in the fresh state (H = 0.10 ± 0.07 GPa, E = 1.50 ± 0.80 GPa). Water content seems to be the crucial factor, which influences the mechanical properties such as hardness and elastic modulus. According literature data, humidity loss changes the mechanical behavior of biological materials significantly (Andersen et al., 1996; Arzt et al., 2002; Enders et al., 2004; Vincent and Wegst, 2004). In the fresh cuticle, chitin filaments are spaced within a protein-matrix containing about 90 % water (Andersen et al., 1996; Vincent, 1980). Therefore absence of water, one of the main components of the cuticle, is expected to lead to some changes of the structure. There are literature data on the structural changes depending on water reduction in the cuticle

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during sclerotisation (Andersen et al., 1996), but nothing is known about how drying alter the cuticle structure. However it is obvious that the water content makes cuticle soft and compliant. The increase in hardness and elastic modulus, especially of the cuticle layer near the surface up to 1700 nm, is thought to be caused by structural changes due to water loss. Indentation tests on various biological surfaces (insects and plants) display a considerable difference in the mechanical behavior between fresh/hydrated and dehydrated materials (Hillerton et al., 1982; Arzt et al., 2002; Enders et al., 2004; Vincent and Wegst, 2004). Table 2. Results of statistical calculations of the significance (One-Way-ANOVA and Tukey post tests) of differences between the hardness and elastic modulus for various indentation depths (250 nm, 500 nm, 1000 nm and 1500 nm) in fresh, dry and chemically treated conditions. + means that there is a significant difference between the values compared; - means that there is no significant difference between the values compared. There is no significant difference in the elastic modulus values between the dry and chemically treated samples an indentation depth of 1000 nm and 1500 nm.

Hardness Elastic modulus +

Fresh

Chem. treated

+ + +

+

+

+

+

+ +

Condition

+ +

+ +

+ +

+ + +

+

— —

——

+ +

+ +

+ +

+ +

— —

+

+

—— —

+ Fresh

Dry

Chem. treated

1500

Chem. treated

+

Depth, nm

+

Fresh Dry

+

1000

Chem. treated

+ +

Fresh dry

+ +

500

Chem. treated

+

+

Fresh Dry

+ +

250

Dry

+ +

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Removal of surface waxes and lipids causes only small changes in the indentation results (slight increase in hardness and stiffness), especially at a depth below 1000 nm. Without water, the mechanical behavior of the gula cuticle is influenced only a little by the outer wax/lipid layer and is presumably determined mainly by proteins and chitin, which could not be removed by the chemical treatment used in this study. In order to understand how the two main components (proteins and chitin) contribute to the mechanical properties of the cuticle, further studies are necessary. SEM images were helpful to evaluate indentation data. The maximum indentation depth in our experiments was 3 µm. Therefore, measurements of the mechanical properties were done in the epicuticle (figure 6 B), which is about 7 µm thick. According to several models (Jönsson and Hogmark, 1984; Burnett and Rickerby, 1987; McGurck et al., 1994; Rother and Jehn, 1996; Korsunsky et al., 1998) concerning the hardness determination for thin metal films, the results obtained could also be influenced by layers of the exocuticle. After Bueckle (1965) and Bhushan (1996), hardness measured up to an indentation depth of 0.7 µm (displacement < 1/10 of the layer thickness) can be assumed to characterize the epicuticle. With larger displacements the hardness will be characteristic of the whole composite of the epicuticle/exocuticle layers. This suggestion is true only for hardness measurement. The elastic modulus measurements will be influenced by the whole composite since it is a longrange effect. The mechanical behavior of the gula cuticle can be regarded as that of a soft substrate coated by a hard film. A decrease in hardness and elastic modulus with increasing indentation depth is caused by the underlying layers. As already mentioned, deeper layers in cuticle are softer and more compliant than the outer ones. The influence of the underlying layers on the mechanical properties is stronger on dry and chemically treated samples than on fresh ones. Without water, surface layers appear to become harder and stiffer than the deeper layers showing a lower density and higher protein content (Locke 1964; Neville 1975, Binnington and Retnakaran 1992). Several studies of elastic modulus and hardness of insect cuticle using different methods can be found in the literature. Almost all experiments were performed on dry or rehydrated samples. There are only a few measurements of the mechanical properties of cuticle made by indentation. The average hardness (0.10 - 0.52 GPa) and elastic modulus (1.50 - 7.70 GPa) values obtained in this study are similar to those of various sclerotised cuticles (Vickers hardness 0.2-0.5 GPa and Young’s modulus 1 - 10 GPa (Vincent and Wegst, 2004)). However the hardness (0.10 GPa) of the fresh samples is below this range (0.2 - 0.5 GPa). It is believed that these are possibly the first measurements of hardness and elastic modulus on the insect cuticle in native condition. Comparable hardness values were obtained on other cuticle elements. For example, the dry wing membrane cuticle of the dragonfly Aeshna cyanea tested with a nanohardness-testmachine (Hysitron TriboScope) exhibited a hardness of 0.2 GPa (Kreuz et al., 1999). The different parts of the dehydrated locust cuticle measured by means of a Leitz Miniload hardness tester (using a Vickers diamond) (Hillerton et al., 1982) revealed H = 0.24-0.33 GPa. However the dry samples of the beetle head tested in this study (H = 0.49 GPa) were still harder than the cuticles mentioned above. The elastic modulus values (E = 7.50 ± 1.80 GPa) obtained for the dry beetle cuticle is very high when compared to the elastic modulus from tensile test of the dry wing membrane of the dragonfly Aeshna cyanea, where E = 1.5 ± 0.5 GPa (Kreuz et al., 1999). The reduced

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modulus for different body parts of the dehydrated dragonfly (Odonata, Anisoptera) were calculated from quasistatic nanoindentation experiments (Hysitron Inc., Minneapolis, MN) (Kempf, 2000). The mean values were lower (1.5 – 4.7 GPa) as well than elastic modulus obtained for dry samples in this study. Recent nanoscale study (quasistatic nanoindentation testing instrument, Hysitron Inc., Minneapolis, MN) of Drosophila melanogaster integument during development (Kohane et al., 2003) shows that the thickness and the development stage of the cuticle was very important for stiffness measurements. The average reduced elastic modulus (Er) of 0.41 MPa, 15.43 MPa and 4.37 MPa were determined by in vivo experiments for the cuticles of the larval, puparial and adult insects respectively. Thickness of the cuticles of Drosophila melanogaster was very low (2.3-17.7 µm) when compared to that of gula cuticle (about 80 µm). However, the mechanical properties of the cuticle are strongly influenced also by proportions of the main components (chitin and proteins) (Fraenkel and Rudal, 1940; Locke, 1964; Neville, 1975; Hepburn and Chandler, 1976; Binnington and Retnakaran, 1992).

Figure 9. AFM images of the cuticle surface after indentation test showing the residual deformation. The image of the indent shows elastic recovery, which is characteristic for the elastic-plastic contact in most engineering materials. However such shape of the indent here is thought to be caused by viscoelastic relaxation expected for biological materials.

The nanoindentation experiments in this study were carried out in the epicuticle, which is a nonchitinous layer. It contains proteins and is covered with lipids and surface waxes (Neville, 1975; Binnington and Retnakaran, 1992). Surprisingly the elastic modulus of the

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fresh gula cuticle (E = 1.50 ± 0.80 GPa) is comparable to that of the chitin filament (1.97 ± 0.07 GPa) (Joffe and Hepburn, 1973). There are contradictory data in the literature about which component (chitin or protein) is responsible for the elastic modulus of the cuticle. According to Fraenkel and Rudall (1940), the mechanical properties of the cuticle are determined by chitin, whereas Vincent (1980; 2002) suggests that the protein matrix is decisive for its mechanical behavior. Elasticity which is expected to be a long-range effect is influenced by the underlying exocuticle layer consisting of a high amount of chitin in a protein matrix (Neville, 1975; Binnington and Retnakaran, 1992). Nevertheless, according to our experimental results, it can be concluded that the elastic modulus in fresh cuticle is not only influenced by only chitin or only the protein-matrix, but by both components. After an indentation test the surface profiles of the samples were investigated by atomic force microscopy (AFM) (DME, DualScope™ C-21 with scanner DS 45-40 BIO). Figure 9 shows the surface profile of the gula cuticle. Residual deformation of the cuticle surface after indentation seems similar to elastic-plastic contact, which is typical of most engineering materials (Barbakadze 2005). An elastic recovery on the indent-image can be distinguished. This is due to a visco-elastic relaxation expected for soft biological materials (Wainright et al., 1976; Vincent, 1990; Kohane et al., 2003).

WAX COATED PLANT SURFACES The cuticle is an essential element of a plant. Being the outer surface layer, it is of fundamental functional and ecological importance for the interaction between plants and their environment (Barthlott, 1990; Bianchi, 1995; Gorb, 2001). The plant cuticles display different surfaces. They may be pigmented or colorless, smooth, textured or hairy, dry or covered with epicuticular secretions (Jeffree, 1986; Gorb, 2001). The extraordinary diversity of the plant surface structure is a reflection of their different environment (Jeffree, 1986). Preventing desiccation and allowing controlled exchange of gases are the important functions of epicuticular components (Baker, 1982; Jeffree, 1986; Barthlott, 1990). The plant cuticle is a multilayered composite consisting of a polymeric cutin matrix and the cuticular waxes (Eigenbrode, 2002). Figure 10 shows the generalized structure of the plant cuticle (Gorb, 2001). An outer wall of epidermal cells is covered by a pectinaceous cuticle membrane, which is protected by a layer made up of cellulose microfibrils. On the cuticle proper consisting of a lamellate layer, cutin and the epicuticular waxes are deposited (Bianchi, 1995; Gorb, 2001). Cutin is high molecular weight lipid polyester (Bianchi, 1995). The epicuticle waxes are surface lipids (Bianchi, 1995). They display considerable ultrastructural and chemical diversity. The plant waxes are a complex mixture of very longchain aliphatics including different chemical compounds, such as hydro-carbons, wax esters, primary and secondary alcohols, fatty acids, aldehydes, ketones, ß-diketones, triterpenoids and flavonoids (Walton, 1990; Eigenbrode, 2002). A current classification of plant epicuticular waxes, based on the studies of 13 000 plant species using high resolution scanning electron microscopy (SEM), distinguishes 23 types of wax crystals (Barthlott, 1990; Barthlott et al., 1998). The most common types of crystal shapes are tubes, solid rodlets, filaments, plates, ribbons and granules (Baker, 1982; Jeffree, 1986; Bianchi, 1995; Barthlott et al., 1998). The wax crystalloid structure correlates with its chemical composition

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(Barthlott, 1990; Bianchi, 1995; Barthlott et al., 1998). For example, aldehydes are very important for the formation of wax filaments, which is characteristic of the pea Pisum sativum leave surface (Gorb, 2001). The eucalyptus epicuticular wax, containing ß-diketones and primary alcohols, exhibits mixed tubes and plates structure (Hallam and Chambers, 1976; Jeffree, 1986).

Figure 10. The generalised structure of a plant cuticle: W - epicuticular wax; CP - cuticle proper; CL cuticular layer of reticulate region traversed by cellulose micro fibrils; P - pectinaceous cuticle membrane and middle lamella; CW - cell wall; PL - plasma lemma (taken from Gorb, 2001).

All plant surfaces carry a partial or a continuous layer of the amorphous wax as the obligatory final layer of the cuticle (Baker, 1982, Barthlott et al., 1998). However some plants consist of even two amorphous wax layers. Upon the amorphous layer, wax crystals of different shapes emerge and grow. The wax crystal layer varies widely in thickness from 1 to 20 µm. The thickness of the wax crystalloids lies in the nanometer range (Gorb, 2001; Enders et al., 2004). Wax morphology varies with the environmental conditions. The thickness of the layer, the size, the orientation and the density of wax crystals can be significantly modified by temperature and humidity (Baker, 1982). The epicuticular waxes occur in plants primarily on the surface of leaves, stems and fruits (Bianchi et al., 1978; Avato et al., 1984; Baker and Gaskin, 1987; Barthlott, 1990; Bianchi, 1995; Barthlott et al., 1998; Gorb, 2001). On the surfaces of many plant species, such as Eucalyptus leaves, the wax is continuously regenerated. In this way the surface is protected and kept in a fully functional state. However, the epicuticular wax cannot be replaced after having been removed by abrasion from some plant surfaces (Bianchi, 1995). Although no study on mechanical properties of the wax layer has been carried out, it is believed in biology that the plant wax and the underlying layers display different mechanical properties. A cuticularisation of the thick outer epidermal walls contributes to mechanical stability of the surface. The wax covering is mechanically very unstable and in biology

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known as “soft film” (Barthlott, 1990, Barthlott et al., 1998). Being fragile, the waxes are easily removed by rubbing (Barber, 1955; Jeffree, 1986). The wax crystalloids increase the roughness. Together with the wax chemistry, the roughness makes the plant surface hydrophobic and decreases its wettability. The hydrophobic constituents of the wax and surface microroughness reduce the adhesion of the dust and other particles on the outer cell wall. This “anticontamination function” called “lotus effect” is thought to be the most important ecological function of the water-repellent plants (Baker, 1982; Barthlott, 1990; Barthlott and Neinhuis, 1997). An interaction between the plant surfaces and their environment also involves the complex and very important relation between plants and insects (Juniper and Southwood, 1986). Insects possess the attachment devices allowing the hold and the movement anywhere and on nearly any kind of surfaces (Gorb, 2001). The structures of attachment pads adapted for the natural, mainly plant surfaces enable the insects to move freely on any technical surfaces as well. However, some surfaces even in nature raise difficulties for their visitors. The wax layer on the plant is a barrier for the attachment systems of insects. Walking on the plant surface structured with the wax crystals was found to be impossible for insects (Eigenbrode, 1996; Gaume et al., 2002; Gorb and Gorb, 2002). Even though the interaction between the wax crystalloid layer and the insect has been of great interest to researchers for about a century (Kerner von Marilaun, 1898; Haberlandt, 1909; Knoll, 1914), the function of such epicuticular layer preventing adhesion remains largely unknown. There are only a few experimental studies reporting some hypotheses, why the insects cannot attach on the wax coated plant surfaces (Eigenbrode, 1996; Gorb and Gorb, 2002; Gaume et al., 2002): (1) the location of wax crystals on the plant cuticle may be responsible for an increase in the surface microroughness; this can be the reason for reduced real contact area between the plant surface and the attachment system of insects (Stork, 1980); (2) the wax crystals are mechanically unstable and fragile; attachment can be prevented by the contamination of the attachment system of insects by the adhesion of the wax crystals to its pads (Juniper and Burras, 1962); (3) the possible reaction between the wax layer and the secretion liquid of the attachment system of insects (Gaume et al., 2002). The interaction between the insect attachment systems and the plant anti-attachment surfaces motivated this work. The goal was to study the mechanical behavior of plant surfaces coated with wax crystals to understand the anti-attachment mechanism of the wax and to clarify the first two hypotheses. The leaves of two pea (Pisum sativum) mutants were selected for this study. The wild type pea leaves are strongly covered with wax crystals (filaments). In “glossy” mutation, the amount of waxy bloom is greatly reduced (Eigenbrode and Espelie, 1995; Eigenbrode and Jetter, 2002). The samples in fresh and dry conditions were mechanically tested using the Nano indenter® SA2. The structure of plant surfaces was explored by means of scanning electron microscopy (SEM). This study is believed to be the first attempt to characterize the mechanical properties of the wax crystals of plant surfaces.

MATERIALS AND SAMPLE PREPARATION The experiments were performed with the pea Pisum sativum. The wild and glossy mutants of the pea plant were planted and grown in the laboratory. The samples were

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prepared from the pea leaves of both mutants: the wild type with normal wax layer and the glossy type with reduced waxy bloom. The top and bottom sides of the leaves were tested in the untreated condition. The nanoindentations were performed on the specimens in the fresh and dry conditions. To prepare dry samples, the fresh cuts of leaves were air dried at room temperature for 48 h.

A

B

20 C

20 µm D

2 µm E

2 µm F

2

1

Figure 11. SEM images of the wax crystalloid layer on the wild pea leaf surface. A and C show the surface of the top side; B and D show the surface of the bottom side. Images A and B show cell structure of the plant leaf. On the C and D images wax crystal covering the leaf can be distinguished. The bottom side (D) of the pea leaf appears to have more dense waxy bloom than the top side (C). E and F show the top side of the wild pea leaf.

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STRUCTURE OF WAX LAYER The surfaces of the pea leaves were observed using scanning electron microscopy (SEM). The samples, 7×7 mm in size, were cut from the leaves and mounted on an aluminum holder. The surface structure of the wild and the glossy plants were tested at 5 kV. The observations of top and bottom surfaces of both pea mutants were performed in the untreated, fresh condition.

A

B

20 µm

20 µm C

D

2 µm

2 µm F

E

2 µm

1 µm

Figure 12. SEM images of the wax crystalloid layer on the glossy pea leaf surface. A and C show the surface of the top side; B and D show the surface of the bottom side. Images A and B show cell structure of the plant leaf. On the C and D images single wax crystals on the leaf can be distinguished. The top side (C) of the glossy pea leaf appears to have more wax crystals than the bottom side (D). E and F show the top side of the glossy pea leaf.

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Figures 11 and 12 show the surface structure of the wild and glossy pea leaves respectively. The images are taken on the top and the bottom side of the leaf for each plant type. The cell structure of the plant surfaces can be distinguished in figures 11 A, B and 12 A, B. The openings on the figures 11 A, B and 12 A, B are stomata for gaseous exchange. There is a great difference in the leaf surface between the wild and glossy pea. The wild pea samples are completely covered with wax crystals (figures 11 C, D, E, F), while the glossy pea surface only shows single wax crystals (figures 12 C, D, E, F). The comparison of SEM images of the two plant mutants (figures 11 C, D and 12 C, D) leads to the estimate that the surface of the glossy pea leaf bears only about one third of wax crystals. There is a small difference in the wax covering of the top and bottom side of the same plant species as well. The bottom side of the wild pea sample shows a denser wax crystal structure than the top side (figure 11 C, D). The single wax crystals appear to be larger on the top surface of the glossy pea leaf than on the bottom side (figure 12 C, D). The single wax crystals can be estimated to be 1 – 2 µm long with a diameter of 100 - 200 nm (figures 11 E, F and 12 E, F). As can be seen in the figures 11 E, F and 12 E, F, the wax crystals are oriented at different angles to the surface.

MECHANICAL PROPERTIES Table 3 and 4 summarise the results of mechanical properties, hardness and elastic modulus. The mechanical behaviors of the sample surfaces differ presumably depending on the amount of wax crystalloids in the dry, but not in the fresh condition. It is surprising that there is no difference in the mechanical properties (hardness and elastic modulus) between the fresh samples with normal and with reduced wax layer. The maximum load corresponding to the maximum displacement (3 µm) is about 3.5 mN for all fresh samples. The wax layer appears to have an influence on the mechanical behavior of the dry plants. The top and bottom surface of the same plant differ due to the different density and amount of the wax crystals as well. In general, the surface with more wax shows lower hardness and elastic modulus values (figure 13 and 14). The wax layer is believed to make the plant cuticle surface soft and compliant. Table 3. Summary of mechanical properties obtained at the displacement of 100 and 500 nm for fresh plant samples.

Plant species wild top wild bottom glossy top glossy bottom

Hardness (GPa) by indentation depth of 100 nm 500 nm 0.7 ± 0.5 0.7 ± 0.2 0.7 ± 0.4 0.6 ± 0.4 0.5 ± 0.5 0.5 ± 0.3 0.4 ± 0.5 0.6 ± 0.2

Elastic modulus (GPa) by indentation depth of 100 nm 500 nm 3.2 ± 0.8 1.1 ± 0.2 2.7 ± 0.3 1.0 ± 0.3 2.2 ± 0.5 1.0 ± 0.2 2.6 ± 0.7 1.1 ± 0.1

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Table 4. Summary of mechanical properties obtained at the displacement of 100 and 500 nm for dry plant samples.

Plant species wild top wild bottom glossy top glossy bottom

Hardness (MPa) by indentation depth of 100 nm 500 nm 5.2 ± 2.8 2.3 ± 0.9 1.2 ± 0.8 0.3 ± 0.1 50.7 ± 41.1 23.7 ± 19.3 7.5 ± 6.1 5.7 ± 3.9

Elastic modulus (MPa) by indentation depth of 100 nm 500 nm 285.2 ± 153.1 238.3 ± 194.2 168.2 ± 90.1 96.4 ± 59.3 799.3 ± 569.1 570.4 ± 381.2 526.8 ± 382.7 403.1 ± 291.1

In the figure 13 and 14, hardness and elastic modulus (mean values of approximately 100 measurements are shown) are plotted versus displacement for all fresh (figure 13 A and 14 A) and all dry (figure 13 B and 14 B) samples. By looking at the curves showing hardness values at the beginning of the indentation, a little influence of the wax layer could be observed only at the first 200 nm of indentation depth. In this range the fresh samples display a slightly different behavior depending on the presence of the wax layer. Fresh plant samples with normal wax layer tend to be harder only at the first 200 nm of indentation depth than those with reduced wax. In the first 200 nm the indenter tip obviously contacts single wax crystals. This is thought to be caused by high mechanical stability of single wax crystalloids that build a very unstable and soft wax layer (Barthlott, 1990). However, a difference in the elastic modulus values between the fresh samples is not noticeable.

(B) Figure 13. Hardness versus displacement curves of all fresh (A) and all dry (B) pea leaves. The mean results of approximately 100 measurements are shown up to 1 µm.

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(A) Figure 14. Elastic modulus versus displacement curves of all fresh (A) and all dry (B) pea leaves. The mean results of approximately 100 measurements are shown up to 1 µm.

All fresh samples exhibit the same mechanical behavior at larger displacements apparently due to the influence of the underlying layers. The plant cuticle has a multilayered structure (Eigenbrode and Jetter, 2002), which consists of the mechanically stable cuticularised epidermis covered by unstable and soft wax layer (Barthlott, 1990; Barthlott et al., 1998). The hardness and the elastic modulus values corresponding to the fresh samples are expected to be strongly influenced by cell walls and pressure of the cell fluid. However, the cell wall itself appears to be soft and compliant (dry samples are softer and more compliant than fresh ones). The cell structure, filled by fluid, appears to be influenced by internal pressure of the cell fluid and therefore, is hard and stiff. This is why the effect of the wax layer cannot be detected. A schematic of the cross section of the plant sample during the indentation is shown in figure 15. In the dry condition, the influence of the cell fluid is minimized or removed and there is a great difference in the mechanical behavior between the plant surfaces with normal and with reduced wax layer. The dry samples with normal wax layer are softer and more compliant than the dry ones with reduced wax layer. In addition, the same mechanical behavior was observed in the case of pitcher plant Nepenthes alata, eucalyptus Eucalyptus guinnii and red cabbage Brasica (Barbakadze, unpublished results). The desiccation greatly influences the mechanical behavior of biological materials (Gorb, 2001; Vincent and Wegst, 2004). However the effect of water loss is different for insect and plant cuticles. Fresh insect cuticles are softer and more compliant than dry ones. By contrast, the hardness and the elastic modulus values of fresh plant cuticle surfaces are higher than that of dry ones.

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indente cuticle  cell wall 

cell fluid  cell structure Figure 15. Schematic of the cross section of the plant during indentation experiment. The arrows show pressure of the cell fluid under the indenter.

WAX COVERINGS AND INSECT ATTACHMENT PADS The attachment abilities of insects were recently tested on the plant surfaces investigated in this study. The leaves of the wild pea (Pisum sativum) (Eigenbrode and Jetter, 2002) were difficult to cope for insects. While walking on these surfaces, the insects often stopped and cleaned their feet whereas they had less difficulty on the surface of the glossy mutant with reduced waxy bloom. To elucidate the hypotheses of reduced contact area and contamination effect of the wax crystals, let us consider the results obtained in connection with the blowfly Calliphora vicina (Niederegger et al., 2002), which is the model for most studies of the structure and the attachment behavior of pads. What would the hardness and elastic modulus values obtained in this study mean for this insect? The blowfly C. vicina weighs about 4 mN and possesses attachment pads structured with a few thousand (4000 - 6000) flexible (1 N/m spring constant) cuticular outgrowths called seta. A single spatula (tip of the seta) has an area of 1 µm2 (Niederegger et al., 2002). Thickness of the wax crystals lie in a few hundred nanometer range. By walking, three feet of the insect with one third of the whole amount of setae come in contact. However, the area of a spatula increases in contact by about 35 % (Niederegger et al., 2002). An estimation of the load exerted by one single seta in contact with the plant surface gives values ranging between 134 and 400 nN. In the nanoindentation experiments, the hardness and elastic modulus values corresponding to this load range were obtained at displacements of 20-40 nm: for the wild pea, H = 50-340 MPa and E = 2.50 - 8.00 GPa (figure 16). With these considerations, one can assume that wax crystals remain stable under insect pads and contribute to a decrease of the

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contact area between the insect attachment pad and the plant surface (hypothesis 1). However, the wax crystals are not oriented perpendicularly to the surface but at different angles. The insects do not only push down the plant surface but also use lateral forces while walking. These considerations make the estimation more difficult. Regarding all these factors, contamination of the insect pads (hypothesis 2) due to the breaking and adhesion of wax crystals to the pad surface cannot be excluded. Further measurements on the isolated cuticle (without cell structure) surface are necessary.

Figure 16. Hardness and elastic modulus of the wild pea samples (top side of the leaf) in the displacement range of 0 – 80 nm. Grey area shows the displacement area of 20 to 40 nm, corresponding to the load range of 134 – 400 nN. This load range is estimated to act during the walking of the blowfly C. vicina on the plant surface.

SUMMARY The mechanical behavior of various biological materials such as insect and plant cuticles was studied by applying experimental approaches of material science. A head articulation cuticle of the beetle designed for friction minimisation and the wax covered plant surfaces adapted for attachment prevention have been chosen for this study. Both insect and plant cuticles are multifunctional composite materials and have a multilayered structure. The gula cuticle of the beetle Pachnoda marginata is a part of the head articulation, which is a micromechanical device similar to a technical ball bearing. The surfaces in this system operate in contact; they must be optimised against wear and friction and must provide high mobility within the joint. The measurements on the gula cuticle were performed in order to understand structure and tribo-mechanical behavior of material working for friction minimizing. The plants carry amorphous wax layer which by some species is covered by wax

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crystals. The insects cannot attach on such plant surfaces. The location of the wax crystals on the plant cuticle increases the surface microroughness. This could decrease the real contact area between the plant surface and attachment pads of insect. Attachment can be prevented by contamination of attachment pads of insect with the wax crystalloids. In order to understand the deformation behavior of the wax layer two pea plant (Pisum sativum) mutants (wild type with normal wax layer and glossy with reduced waxy bloom) were studied. To observe the effect of desiccation all samples (insect and plant cuticles) were tested in fresh and dry conditions. To understand the influence of an outer wax/lipid layer, tribo-mechanical experiments on the gula cuticle were performed as well in a chemically treated condition. This study is believed to be one of the first for mechanically testing of insect cuticle and the very first for wax coated plant surfaces in native condition. Mechanical properties were determined by using the Nano Indenter® SA2 (MTS Systems Corporation, Oak Ridge, USA). A high damping coefficient and a high resonant frequency of the indenter in this system allows determining mechanical properties of soft and structured materials with low contact stiffness. The biological samples can be successfully tested in native condition. Employing continuous stiffness measurement (CSM) technique allows measuring contact stiffness continuously during indentation and obtaining the depthdependence of the hardness and elastic modulus. This is very important for biological materials because their mechanical properties vary with depth. The mechanical behavior of all samples was found to be greatly influenced by drying. However changes in hardness and elastic modulus were different for insect and plant cuticles. Desiccation made gula cuticle of the beetle harder and stiffer but the plant surface softer and more compliant. After drying, gula cuticles became harder and stiffer by a factor of about 5 compared to the samples in the fresh state. Chemical treatment caused further hardening of the material. Increase in stiffness could be observed only in first 1 µm of the indentation depth. There was no significant difference between elastic modulus of the dry and chemically treated samples at indentation depth higher than 1 µm. Hardness and elastic modulus values of the fresh plant samples were significantly higher than that of the dry ones. There was almost no difference between samples with normal and with reduced wax layer in fresh condition. All fresh samples exhibited the same mechanical behavior apparently due to the influence of cell walls and especially pressure of the cell-fluid. In dry condition the effect of the cell-fluid was removed and the samples showed a great difference in mechanical properties between the plant surfaces with normal and with reduced wax layer. Dry samples with normal wax layer were softer and more compliant than with reduced waxy bloom.

REFERENCES Andersen, S.O. (1979) Insect cuticle. Annual Revue of Entomology, 24: 29-61. Andersen, S.O., Peter, M.G. and Roepstorff, P. (1996) Cuticular Sclerotization in Insects. Comparative Biochemistry and Physiology, 113B (4): 689-705. Arzt, E., Enders, S. and Gorb, S. (2002) Towards a micromechanical understanding of biological surface devices. Zeitschrift für Metallkunde, 93 (5): 345-351.

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Avato, P., Bianchi, G. and Mariani, G. (1984) Epicuticular waxes of Sorghum and some compositional changes with plant age. Phytochemistry, 23: 2843-2846. Baker, E.A. (1982) Chemistry and morphology of plant epicuticular waxes. In: The plant cuticle. Cutler, D.F., Alvin, K.L., Price, C.E. (eds.) Academic Press, London, pp. 139165. Baker, E.A. and Gaskin, R.E. (1987) Composition of leaf epicuticular waxes of Pteridium subspecies. Phytochemistry, 26: 2847-2848. Barbakadze, N. (2005) Micro/nanomechanical Measurements on Insect and Plant Cuticles. Dissertation. University Stuttgart. Barbakadze, N., S. Enders, S. Gorb and E. Arzt (2006) Local mechanical properties of the head articulation cuticle in the beetle Pachnoda marginata (Coleoptera, Scarabaeidae). Journal of Experimental Biology, 209: 722-730. Barber, N.H. (1955) Adaptive gene substitutions in Tasmanian Eucalyptus. I. Genes controlling glaucousness. Evolution, 9: 1-14. Barthlott, W. (1990) Scanning electron microscopy of the epidermal surface in plants. In: Scanning electron microscopy in taxonomy and functional morphology. Claugher, D. (ed.) Clarendonpress, Oxford, pp. 69 - 83. Barthlott, W. and Neinhuis, C. (1997) Purity of the sacred lotus or escape from contamination in biological surfaces. Planta, 202: 1-8. Barthlott, W., Neinhuis, C., Cutler, D., Ditsch, F., Meusel, I., Theisen, I. and Wilhelmi, H. (1998) Classification and terminology of plant epicuticular waxes. Botanical Journal of the Linnean Society, 126 (3): 237-260. Bianchi, G., Avato, P., Bertorelli, P. and Mariani, G. (1978) Epicuticular waxes of two sorghum varieties. Phitochemistry, 17: 999-1001. Bianchi, G. (1995) Plant waxes. In: Waxes: chemistry, molecular biology and functions. Hamilton, R. J. (ed.). The Oily Press: Dundee, pp. 177-222. Bhushan, B. (1996) Tribology and mechanics of magnetic storage devices. Springer-Verlag, New York. Bhushan, B. and Li, X. (2003) Nanomechanical characterization of solid surfaces and thin films. International Materials Reviews, 48 (3): 125-164. Binnington, K. and Retnakaran, A. (1992) Physiology of the insect epidermis. CSIRO, Australia, Melbourne. Bueckle, H. (1965) Mikrohaertepruefung und ihre Anwendung. Stuttgart, Berliner Union. Burnett, P. J. and Rickerby, D. S. (1987) The mechanical properties of wear-resistant coatings: I: Modelling of hardness behaviour. Thin Solid Films, 148: 41-50. Burnett, P. J. and Rickerby, D. S. (1987) The mechanical properties of wear-resistant coatings: II: Experimental studies and interpretation of hardness. Thin Solid Films, 148: 51-65. Dai, Z., Gorb, S. N. and Schwarz, U. (2002) Roughness-dependent friction force of the tarsal claw system in the beetle Pachnoda marginata (Coleoptera, Scarabaeidae). Journal of Experimental Biology, 205: 2479-2488. Eigenbrode, S. D. and Espilie, K. E. (1995) Effects of plant epicuticular lipids on insect herbivores. Annual Review of Entomology, 40: 171-194. Eigenbrode, S. D. (1996) Plant surface waxes and insect behaviour. In: Plant cuticles - an integral functional approach. BIOS Scientific Publishers Ltd, Oxford, pp. 201-222.

Specific Properties of Some Biological Composite Materials

119

Eigenbrode, S.D. and Jetter, R. (2002) Attachment to Plant Surface Waxes by an Insect Predator. Integrative and Comparative Biology, 42: 1091-1099. Enders, S., Barbakadze, N., Gorb, S. N. and Arzt, E. (2004) Exploring biological surfaces by nanoindentation. Journal of Materials Research, 19 (3): 880-887. Fraenkel, G. and Rudall, K. M. (1940) A study of the physical and chemical properties of the insect cuticle. Proceedings of the Royal Society, B 129: 1-34. Fung, Y., C. (1993) Biomechanics, Mechanical Properties of Living Tissues. Springer-Verlag, pp. 500-544. Gorb, S. N. (1997) Ultrastructural architecture of the microtrichia of the insect cuticle. Journal of Morphology, 234: 1-10. Gorb, S. N. (1999a) Evolution of the dragonfly head-arresting system. Proceedings of the Royal Society of London, B 266: 525-535. Gorb, S.N. (1999b) Ultrastructure of the thoracic dorso-medial field (TDM) in the elytra-tobody arresting mechanism in tenebrionid beetles (Coleoptera: Tenebrionidae). Journal of Morphology, 240: 101-113. Gorb, S.N. and Scherge, M. (2000) Biological microtribology: anisotropy in frictional forces of orthopteran attachment pads reflects the ultrastructure of a highly deformable material. Proceedings of the Royal Society of London, B 267: 1239-1244. Gorb, S.N. (2001) Attachment devices of the insect cuticle. Kluwer Academic Publishers, The Netherlands. Gorb, S. N., Gorb, E. and Kastner, V. (2001) Scale effects on the attachment pads and friction forces in syrphid flies (Diptera, Syrphidae). Journal of Experimental Biology, 204: 1421-1431. Gorb, E.V. and Gorb, S. N. (2002) Attachment ability of the beetle Chrysolina fastuosa on various plant surfaces. Entomologia Experimentalis et Applicata, 105: 13-28. Gorb, E. and Gorb, S. (2002) Contact separation force of the fruit burrs in four plant species adapted to dispersal by mechanical interlocking. Plant Physiology and Biochemistry, 40: 373-381. Gorb, S.N. and Perez Goodwyn, P.J. (2003) Wing locking mechanisms in aquatic Heteroptera. Journal of Morphology, 257 (2): 127-146. Haberlandt, G. (1909) Physiologische Pflanzenanatomie. Engelmann, Leipzig. Hallam, N. D. and Chambers, T. C. (1976) The leaf waxes of the genus Eucalyptus L’Heritier. Australian Journal of Botany, 18: 335-386. Hammond, P.M. (1989) Wing-folding mechanism of beetles, with special reference to investigations of adephagen phylogeny (Coleoptera). In: Ervin, T., Ball, G. E. and Whitehead, D.R. (eds.) Carabid beetles: their evolution, natural history and classification. Boston, London: Junk Publishers: The Hague, pp. 113-180. Hepburn, H. R. and Joffe, I. (1976) On the material properties of insect exoskeletons. In: The insect integument. Hepburn, H.R. (ed.), Elsevier, Amsterdam, pp. 207-235. Hepburn, H.R. and Chandler, H.D. (1976) Material properties of arthropod cuticles: the arthropodial membranes. Journal of Comparative Physiology, 109: 177-198. Hepburn, H. R. and Chandler, D. (1980) Materials Testing of Arthropod Cuticle Preparations. In: Cuticle technologies in arthropods. Miller T.A. (ed.). New York, Springer Verlag, pp. 1-44. Hillerton, J. E., Reynolds, S. E. and Vincent, J. V. (1982) On the indentation hardness of the insect cuticle. The Journal of Experimental Biology, 96: 45-52.

120

N. Barbakadze, E. Gorb and S. Gorb

Jeffree, C. E. (1986) The cuticle, epicuticular waxes and trichomes of plant, with references to their structure, functions and evolution. In: Insects and the plant surface. Juniper, B. E., Southwood, T. R. E. (eds.) Edward Arnold: London, pp. 23-64. Jensen, M. and Weis-Fogh, T. (1962) Strength and elasticity of locust cuticle. In: Biology and Physics of locust flight. Philosophical Transactions of the Royal Society, B 245 pp. 137169. Joffe, I. and Hepburn, H. R. (1973) Observations on regenerated chitin films. Journal of Materials Science, 8: 1751–1754. Jönsson, B. and Hogmark, S. (1984) Hardness measurements of thin films. Thin Solid Films, 114: 257-269. Juniper, B.E. and Burras, J.K. (1962) How pitcher plants trap insects. New Scientist. 13: 75-77. Juniper, B. E. and Southwood, R. (1986) Insects and the Plant Surfaces. London, UK: Edward Arnold. Kempf, M. (2000) Biological materials, determination of Young’s moduli of the insect cuticle (dragonflies; Anisoptera). Application note, Hysitron Inc, www.hysitron.com. Kerner von Marilaun, A. (1898) Pflanzenleben 2. Bibliographisches Institut, Leipzig/Wien. Knoll, F. (1914) Über die Ursache des Ausgleitens der Insektenbeine an wachsbedekten Pflanzendteilen. Jahrbuch für Wissenschaftliche Botanik, 54: 448-497. Kohane, M., Daugela, A., Kutomi, H., Charlson, L., Wyrobek, A. and Wyrobek, J. (2003) Nanoscale in vivo evaluation of the stiffness of Drosophila melanogaster integument during development. Wiley Periodicals, Inc.: 633-642. Korsunsky, A. M., McGurk, M. r., Bull, S. J. and Page, T. F. (1998) On the hardness of coated systems. Surface Coatings Technology, 99: 171-183. Kreuz, P., Kesel, A., Kempf, M., Göken, M., Vehoff, H. and Nachtigall, W. (1999) Mechanische Eigenschaften biologischer Materialien am Beispiel Insektenflügel. BIONA report, 14: 201-202. Locke, M. (1964) The structure and formation of the integument in insects. In: The physiology of Insecta. Rockstein M. (ed.), Academic Press, New York, pp. 123-213. McGurk, M. R., Chandler, H. W., Twigg, P. C. and Page, T. F. (1994) Modelling the hardness response of coated systems: the plate bending approach. Surface Coatings Technology, 68/69: 576-581. Neville, A. C. (1975) Biology of the arthropod cuticle. Springer Verlag, Berlin, Germany. Niederegger, S., Gorb, S. and Jiao, Y. (2002) Contact behaviour of tenent setae in attachment pads of the blowfly Calliphora vicina (Diptera, Calliphoridae). Journal of Comparative Physiology, A 187: 961-970. Oliver, W. C. and Pharr, G. M. (1992) An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. Journal of Materials Research, 7 (6): 1564-1583. Perez Goodwyn, P. J. and Gorb, S. N. (2004) Anti-frictional properties of contacting surfaces in the hemelytra-hindwing locking mechanism in the bug Coreus Marginatus (Heteroptera, Coreidae). Journal of Morphology (in press). Persson, B. N. J. (1998) Sliding friction. Springer: Berlin, Heidelberg, New York. Rother, B. and Jehn, H. A. (1996) Coating and interface characterization by depth-sensing indentation experiments. Surface and Coatings Technology, 85: 183-188.

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Scherge, M. and Gorb, S. N. (2000) Microtribology of biological materials. Tribology Letters, 8: 1-7. Stork, N. E. (1980) Role of wax blooms in preventing attachment to brassicas by the mustard beetle, Phaedon cochleariae. Entomologia Experimentalis et Applicata, 28: 100-107. Vincent, J. F. V. (1980) Insect cuticle - a paradigm for natural composites. In: The mechanical properties of biological materials. Symposium of the Society Experimental Biology, 34: 181-210. Vincent, J. F. V. (1990) Structural Biomaterials. The University Press, Princeton. Vincent, J. F. V. (2002) Arthropod cuticle –a natural composite shell system. Composite Part A - Applied Science and Manufacturing, 33 (10): 1311-1315. Vincent, J. F. V. and Wegst, U.G. K. (2004) Design and mechanical properties of insect cuticle. Arthropod Structure & Development, 33: 187-199. Wainwright, S. A., Biggs, W. D., Currey, J. D. and Gosline, J. M. (1976) Mechanical Design in Organisms. Princeton University Press. Walton, T. J. (1990) Waxes, cutin and suberin. In: Lipids, membranes and aspects of photobiology, Dey, P. M., Harborne, J. B. (eds.), pp. 105-158. Wegst, U. G. K. and Ashby, M. F. (2004) The mechanical efficiency of natural materials. Philosophical Magazine, 84, 21: 2167-2181.

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.123-153 ©2010 Nova Science Publishers, Inc.

Chapter 10

PROPERTIES AND APPLICATIONS OF AMINOXYL RADICALS IN POLYMER CHEMISTRY E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii*, T. V. Pokholok, and G. E. Zaikov N.M. Emanuel Institute of Biochemical physics, Russian Academy of Sciences, Moscow, Russia

ABSTRACT The review includies the information about the structure and physical properties of stable radicals, ESR spectroscopy of stable aminoxyl radicals (ARs), chemical properties and applications of ARs, generation of ARs in reactions of spin trapping and synthesis of polymers containing stable ARs.

Keywords: aminoxyl radicals, electron spin resonance, polymers with stable radicals.

INTRODUCTION Free radical reactions initiated by NO2 and other nitrogen oxides in polymers are accompanied by generating stable nitrogen-containing radicals. Analysis of their structures and kinetics of formation provides information on free radical stages of complex processes of the polymer nitration under various conditions [1]. These studies can be used in synthetic chemistry in particular for the development of methods of the polymer modification, for example, for the preparation of spin-labelled macromolecules. The generation of spin labels thus occurs in consecutive reactions including formation and conversions of specific intermediate molecular products and active free radicals. Most of the traditional developing methods of preparing spin labelled macromolecules are based on use of organic compounds capable of interacting with polymers to form stable * [email protected] , [email protected]

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aminoxyl radicals (AR). Grafting these radicals can be carried out by means of nitroso compounds and nitrons which capture active radicals. ARs represent a vast class of stable radicals widely used now in chemistry. Numerous articles and monographs are devoted to synthesis and properties of these radicals [2, 3]. The insertion of AR to macromolecules allows essentially changing physical, chemical and operational characteristics of polymers. Generally this effect is conditioned by the presence of >N−O• fragments in polymer chains and the interaction of these radical centers with components of polymeric materials. In the present chapter, structures and properties of ARs and ways of the stable radical generation using reactions of organic synthesis are considered.

1. STRUCTURE AND PHYSICAL PROPERTIES OF STABLE RADICALS Within the framework of the resonance theory, the AR structure can usually be represented by the following resonant structures [4]:

R1 R2

N O

R1 R2

(II.1)

N O

The schemes of the electronic shell formation of the paramagnetic centre from AR and levels of the molecular orbitals of AR are respectively shown in Fig. 1 and Fig. 2.

Figure 1.

z

z

N

O

y

x

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125

π∗ 2pz

2pz

n

2py

π N

N

O

O

Figure 2.

The length of N−O bond in all types of AR makes up 1.27-1.30 Å. This value corresponds to the length of a three-electronic bond, i. e. to such electronic structure in which binding σ and π orbitals are occupied by electron pairs, whereas the unpaired electron occupies nonbinding π* orbital formed by pz orbitals of nitrogen and oxygen atoms and is located approximately in equal parts between them. It is accepted that the delocalization of unpaired electron between N and O atoms with lowering its energetic level (134 kJ⋅mol-1) is the main reason of high stability of AR [3]. The geometrical structure of the radical centre appreciably depends on character of R1 and R2 groups. The angle between CNC plane and N−O bond in different radicals varies from 0o up to 30o [3]. The deviation of nitrogen atom from coplanarity does not result in any changes of energy, but the hybridization of nitrogen atom determines the characteristics of ESR spectra of AR. Most of AR do not form dimers > N−O−O−N < in a wide range of temperatures and various solvents [5]. The AR dimerization is the thermodynamically inefficient process connected with loss of the stabilization energy of two radical fragments ARs have two absorption bands in their electronic spectrum. Di-t-alkylaminoxyls have bands at 240 nm (ε = 3000 l mol −1 cm −1) and at 410-460 nm (ε = 5 l mol −1 cm −1). The first is caused by π → π* transition, the second belongs to n → π* transition (Fig. II.2). Because of conjugation of an aromatic ring with aminoxyl groups, the bathochrome shift occurs for absorption bands. The shifts up to 335-340 and 490-510 nm are observed for diarylaminoxyls [3]. The vibration frequencies of AR should be appeared in IR spectra at 1340-1370 cm −1 [3]. Unfortunately there are bands in this region frequently connected with vibrations of alkyl groups. According to the studies of solid and solution IR and Raman spectra of 1,1,3,3tetramethylisoindolin-2-yloxyl radicals [6], the N−O• stretching frequency is 1431cm−1. This apparently anomalous peak position was confirmed by isotopic substitution studies and ab initio density functional theory calculations. Therefore, IR spectroscopy can be carefully applied for identification of AR. The dissociation energy of C−N bond determined by the electronic impact method makes up 121.5 kJ mol-1 [7]. The rather low energy makes feasible the decay of AR due to breaking C−N bond in certain conditions. The ionization potential of AR is high enough (7 eV) [8]. This makes the increased stability of AR in reactions in which

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cations and radical cations are primary active centers, namely, in reactions induced by irradiation and oxidation.

2. ESR SPECTROSCOPY OF AMINOXYL RADICALS The ESR spectra of AR are quantitatively described by spin Hamiltonian [2]:

H = −β H 0 gS + STI (2) where β is Bohr magneton,

H 0 is the external magnetic field intensity, g is g-tensor, S is

the electron spin operator, T is the tensor of hyperfine interaction (HFI), spin operator. Spin Hamiltonian consists of isotropic and anisotropic parts:

I is the nuclear

H = Hi + Ha

(3)

H i = − βg H 0 S + a IS

(4)

H a = −β H 0 g ' S + ST ' I

(5)

where

g and a are isotropic values of g-factor and HFI constant. They are determined by

the following equations:

g = 1 / 3( g x + g y + g z ) = 1 / 3r g '

(6)

a = 1 / 3(Tx + T y + Tz ) = 1 / 3rT '

(7)

where

g ' and T ' are diagonal tensors. The constituents of these tensors are connected with

common

g ' - and T ' - tensors by the equations:

g i = g i '+ g

(8)

Ti = Ti' + a

(9)

(i

= x+ y+ z)

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry In solutions with low viscosity, ARs are rapidly rotated with total averaging

127

g -and T -

tensors. Therefore ESR spectra of AR in solutions are characterized by triplet signal with the component intensity ratio of 1:1:1 owing to interaction of unpaired electron with the 14N nucleus having I = 1. This triplet splits because of interactions with magnetic nucleuses in α− and β− positions. The isotropic HFI constants aN dependent on the AR structure are given below [9]:

aN, mT Dialkylaminoxyl Alkylarylaminoxyl Diarylaminoxyl Acylaminoxyl Alkoxyalkylaminoxyl Alkoxyarylaminoxyl

1.4-1.7 1.1-1.4 09-1.1 0.67-1.1 2.4-2.8 1.3-1.5

The analysis of HFI using quantum-chemical calculations has allowed determining spin density ρ on N and O atoms. For example, the ρ-value in di-t-alkylaminoxyls is distributed practically fifty-fifty between N and O atoms, at that an unpaired electron is almost completely located on a radical fragment [10]. In alkylaryl- and diarylaminoxyl radicals there is the capability of an unpaired electron to be delocalized onto aryl fragment with considerable decreasing ρ on N atom. Therefore, the constant of splitting aN in these radicals is less than in dialkylaminoxyl radicals. In viscous liquids, the rotation of radicals becomes slower, and both g- and HFI tensors are not completely averaged. As a result, HFS components are broadened out, and the high-field component shows this broadening to a greater extent. For various components ( M = 0,±1 ) of AR spectrum one can obtain the following equations for the correlation time τ of radical rotation [2]:

⎛ ΔH 1 ΔH −1 ⎞ 15π 3ΔH 0 ⎟⎟ − τ' = ⎜⎜ Δ Δ H H 0 0 ⎠ 8 HbΔγ ⎝ ⎛ ΔH 1 ΔH −1 ⎞ 4π 3h τ' ' = ⎜⎜ − − 2 ⎟⎟ 2 ⎝ Δ H 0 ΔH 0 ⎠ 8βb where

(10)

(11)

ΔH i is the width of components Mi, b = 2 / 3[T2 − 1 / 2(T x + T y )] and

β Δγ = [ g z − 1 / 2( g x + g y )] = −11 The equations (10) and (11) are valid for τ = 5 ⋅ 10 − 5 ⋅ 10 −9 s. The correlation time is connected with radius of rotating molecules and viscosity of the solvent η by the Stokes equation:

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4 πηr 3 τ= 3kT

(12)

For determining τ in the range of slow movements ( τ > 5⋅10−9 s), the dependence of τ on shift of high-field line Δ relative to its position in ESR spectrum of can be used [11]:

Δ = const ⋅ τ 3 / 2

(13)

−9 −7 By the equation (13) one can calculate τ in the range of 5 ⋅ 10 − 5 ⋅ 10 s and use these values in studies of the rotational diffusion of macromolecules. The correlation time can be expressed as function of the parameter S = Az ' / Az where

Az ' is half of distance between extremes of outside lines of triplet ESR spectrum and Az is the same value in conditions of extremely slow movements of AR [12]:

τ = a (1 − S ) b

(14)

where a and b depend on diffusion model. The equation (14) is convenient for using in the −8 −6 range of 10 < τ < 10 s.

3. CHEMICAL PROPERTIES OF AMINOXYL RADICALS The stability of AR is conditioned by tautomeric conversions and depends on chemical structure of substituents at N atom, temperatures and solvents [5, 9]. The general mechanism of the AR decay is a disproportionation with formation of nitrons and hydroxylamines. ARs having primary or secondary alkyl groups are short-living species because they easily undergo disproportionation by the scheme: O

2 RCH2NR1

OH

O

RCH2NR1 + RCH=NR1

(II.15)

In a number of cases, the arising products, for example, hydroxylamines can be rather effective acceptors of short-living radicals. They are easily oxidized by nitroso compounds, aminoxyl radicals, oxygen or other oxidizers, which are accumulated as a result of side reactions. In this case one can observe the post - accumulation of radical adducts [13, 14]. Nitrons formed by the reaction (15) represent itself spin traps and can accept AR. In some cases such adducts can be more stable than radical adducts of the initial generation, so only adducts of the second and third generations will be observed in ESR experiments. ARs of high stability are formed when the nitrogen is connected with tertiary carbon atom and the disproportionation is excluded [3]. Such radicals are, for example, di-t-butylaminoxyl (I),

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derivatives of 4,4-dimethyloxazolidineoxyl (II), 2,2,5,5,-tetramethylpyrrolinoxyl (III) and derivatives of piperidine-1-oxyl (IV). O

R1

(CH3)3C-N-C(CH3)3

O

N O

R1

(I)

R2

N

N

O

O

(III)

(II)

(IV)

Changes in structure of nitrogen substituents make AR susceptible to dimerization, if thus the electron delocalization degree is increased and, hence, there is an opportunity for reactions by other centers. So tert-butylphenylaminoxyl (V) is much less stable than radical I due to the unpaired electron delocalization to an aromatic ring. From the point of view of chemical properties, the delocalization makes possible an attack of p-position of phenyl ring by second AR: O

O

H

N C(CH3)3

(CH3)3C-N-C6H5 + (V) (CH3)3C-NH-C(CH3)3

O

+ O

N C(CH3)3

(II.16)

Although the mechanisms of decay of AR of this type in something differ, they always include attack of aminoxyl groups to o- or p- positions of aromatic rings. The o-substituents stabilize AR owing to violation of the coplanarity and the decrease of spin density in an aromatic ring [15]. The bulky substituents in p- and m- positions also stabilize AR as in this case steric hindrances arise for radical attack [16]. The triphenyl-t-butylaminoxyl (VI) unusually decays. The spatial strain of radical centre results in N−C dissociation in AR [17]: O

(C6H5)3C-N-C(CH3)3

(C6H5)3C

+ O=NC(CH3)3

(II.17)

(VI) The solvents essentially influence on the decay rate, and rate constants in polar solvents are less because of AR blocking as a result of the formation of hydrogen bonds with solvents. In conditions, when the hydrogen atom abstraction from molecules of surroundings is difficult, aminoxyls are stable up to 200-220o [18]. AR can accept one radical with the formation of diamagnetic compounds:

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N−O• + R• → >N−O−R

(18)

The rate constants of reactions of AR with solvated electrons, H atoms, OH•, CH3• amount to 109 − 1010 l⋅mol−1⋅s−1 and 108⋅l mol−1⋅s−1 with radicals C•HOH [19]. This property of AR serves as the basis for their use as counters of radicals. The unique property of AR is their capability of reacting without participation of unpaired electrons with retention of paramagnetism. Such reactions are widely used for synthesis of new AR with various substituents [3], for synthesis of metalorganic radicals containing Tl, Hg, Fe etc. [20]. By this way, polyradicals were obtained in which paramagnetic fragments are interconnected in the uniform molecular system [21]. These reactions represent a method of spin labels used in chemistry, biochemistry and molecular biology [22]. The properties of AR as oxidizers can be shown by the example of their interaction with hydrocarbons [23]: >N−O• + RH → >N−OH + R• >N−O• + R• → >N−OR

(19)

By the voltammeter method, ionization potentials of oxidation of a number of ARs were measured in acetonitrile [24]. The conclusion was made that for oxidation of AR the rather strong oxidizers are required. Bromine and chlorine easily and quantitatively oxidize AR into reactive oxoammonium salts [25, 26]. Stable diarylaminoxyls under the action of halogens also form oxoammonium salts, and the ease of oxidation of the radicals is determined by nature of substituents: R

R R''

N R

R'' R'

R

R

O

3/2X2

O R''

N R

(VII)

R''X3

(II.20)

R'

If R = R′ = H, R′′ = OCH3, then radicals VII are oxidized by bromine. In the case of R = R′ = R′′ = OCH3, radicals VII are easily oxidized even by iodine. The basic products of vigorous reaction of radicals I with ozone are nitro-t-butane and oxygen [27]. The oxidation of alcohols into carbonyl compounds can be carried out via the interaction of piperidinoxyl with Cu (II) [28]:

>N O + Cu2+

+ >N=O + Cu

>N=O + CH3OH

>N-OH + CH2OH

CH2OH

HCHO + H+

(II.21)

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Under the action of strong acids, the protonation of AR takes place [3]: +

H+

>N O

(II.22)

>N OH

The protonation of ARs is the first stage of their interaction with mineral acids, which results in products of disproportionation [29]: HX

2 >N O

+

H

+

>N O X

+ >N

OH

(II.23)

X

In such a manner, ARs of piperidine, hydrogenated pyrrole and nitronylaminoxyls disproportionate. Aminoxyls also disproportionate under the action of allyl and benzyl bromide by the following scheme [30]:

2 >N O + RBr

>N=OBr + >N OR

(II.24)

Along with reactions without the participation of the radical centre, ARs react as typical radicals. At the elevated temperatures they abstract hydrogen atoms, chlorine, bromine and other elements. There are examples of sufficiently reactive AR in H-atom abstraction at ordinary temperatures. The benzotriazole-N-oxyl (BTNO) generated by the oxidation of 1hydroxybenzotriazole (HBT) with a CeIV salt in acetonitrile spontaneously decays with a firstorder rate constant of 6.3⋅10-3 s-1 at room temperature [31]. The decay of this aminoxyls is strongly accelerated in the presence of H-donor substrates such as alkylarenes, benzyl and allyl alcohols: ArCH2OH N N N HBT

OH

HBT

N

Ce(IV)

N

- H+

ArCH2OH

(II. 25)

N BTNO

O

The kinetic isotope effect confirms the H-abstraction step as rate-determining. ARs recombine with many radicals participating in chain chemical reactions and add to multiple bonds. Dialkylaminoxyl radicals actively react with alkyl radicals [5, 32], sulfurcontaining radicals [33], solvated electrons and by radicals generated by γ−radiolysis of organic compounds [34]. ARs recombine with hydroxyl radicals, but do not react with HO2• radicals [35]. As distinct from dialkylaminoxyl radicals, aromatic ARs react with peroxide radicals [36]. If alkyl radicals or hydrogen atoms participate in reactions, the basic products of such reactions are the corresponding ethers and hydroxylamines [34]. Hydroxyalkyl radicals are captured by AR [34] with the formation of unstable ethers, which are decomposed to yield aldehyde and hydroxylamine:

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

132

>N O

>N O + CH2OH

CH2OH

>N OH + CH2O

(II.25)

ARs are useful "counters" of active alkyl radicals [37] and inhibitors of radical polymerization [38]. It should be noted that aliphatic and aromatic ARs have approximately identical reactivity in inhibition. These radicals are similar to quinones and considerably exceed nitroso compounds as radical inhibitors. The comparison of reactivity of spin traps and AR shows that ARs are 2-5 orders of magnitude more effective radical acceptors than nitrons and nitroso compounds. Therefore, new effective acceptors of radicals are generated already at early stages of the short-living radical trapping. Calculations in the framework of density functional theory (DFT) [39] for model AR H2NO• indicate that addition to the carbonyl carbon is exothermic by 18.7 kcal·mol−1 [40]. This prediction was tested experimentally in reactions of AR IV with ketenes [41]. In this case facile reaction occurred, and on the basis of the theoretical as well as kinetic and product studies the reactions were interpreted as proceeding through attack of one IV at the carbonyl carbon forming a α−acyl radical intermediate. Then the intermediate radical reacts with another IV at Cβ :

C

O

O C O N

IV

IV

C

O O N

(II.26)

O N

The adducts of AR with organic free radicals have attracted considerable attention because of their potential utility as free radical initiators, and because of the important role of reversible dissociation of adducts of IV in living radical polymerization. The wide development is observed in study of participation of ARs in various photochemical reactions, phototransfer of electrons and electronic energy. For some radicals the basic process is the dissociation with the nitric oxide detachment, while other types of AR mainly abstract hydrogen atoms from solvents. The quantum yield of such process is very high (~0.5) [42]. Di-t-alkylaminoxyls are poorly stable under the exposure to UV light. So the photolysis of radicals IV (R1 = OH) by light with λ = 350 nm in toluene completely converts them into equal quantities of hydroxylamine and benzyl ether of hydroxylamine [43]. Thus, the capability of some excited ARs to abstract a hydrogen atom with the subsequent recombination of formed radicals and AR provides a method of functionalization macromolecules. The radical III (R=CONH2) decays during photolysis with breaking N−C bonds [44]:

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry CONH2

133

CONH2

hν

(II.27)

+ NO

N O

The photochemical transformations of AR depend not only on a type of a radical, but also on chemical properties of the solvent [42, 45]. The radical I in pentane dissociates with the detachment of t-butyl groups during photolysis by light with λ < 300 nm in the band of π→π* transitions: O

(CH3)3C-N-C(CH3)3

hν C5H12

OC(CH3)3

(CH3)3CN=O + (CH3)3C-N-C(CH3)3 70%

(II.28)

25%

In the solution of radicals I in carbon tetrachloride there is an absorption in the range of 300-400 nm corresponding to the charge-transfer band. The irradiation of the solution by light with 313 < λ < 360 nm results in the radical I decomposition with the quantum yield of 1.7: O

(CH3)3C-N-C(CH3)3 OCCl3

(CH3)3C-N-C(CH3)3

hν CCl4

(CH3)3CN=O + (CH3)3CCl

+

OCl

+ (CH ) C-N-C(CH ) + (CH3)2C=CH2 3 3 3 3

(II.29)

By this is meant that the decay of radicals I takes place both under the action of light and in secondary reactions with products of the solvent photolysis, in particular with CCl3• radicals.

4. APPLICATIONS OF AMINOXYL RADICALS Stable radicals named also as spin labels find wide applications in various areas of scientific researches and manufacture. The area of ARs applications includes organic chemistry and photochemistry, chemical kinetics and catalysis, analytical chemistry, chemistry of polymeric materials, molecular biology and medicine. In experimental chemistry ARs are applied to recognize the mechanism of chemical reactions, structures of active radicals in a wide temperature range [46]. The important feature of AR is the regular change of their ESR spectra depending on mobility, nature of surrounding molecules and mutual distances. They are widely applied in researches of physics and chemistry of polymers [18]. For these purposes, a small quantity of spin labels is introduced into the studied polymer so

134

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

that 200-600 monomer units account for one stable radical. In these conditions, the widening of ESR spectra caused by a spin exchange is excluded. With the help of spin labels one can determine parameters of molecular movements and their change under the various external effects, study the dynamics of conformations of macromolecules in solutions, investigate the molecular dynamics in solid polymers, carry out the analysis of compatibility of components of complex polymer blends, investigate crosslinked and filled polymers. The important area of applications of spin labels is the study of the mechanism and kinetics of reactions in heterogeneous systems, interfaces, defects of packing and so on [18]. The capability of recombining with other active particles provides a way for the AR application as inhibitors and regulators of polymerization, effective stabilizers of polymer oxidation, thermal, mechanical and photo degradation of polymers. Kinetic features of the inhibited oxidation of polypropylene and polyethylene by 2, 2, 6, 6tetramethyl-4-benzoyloxypiperidine-1-oxyl have been studied [47, 48]. AR in the grafted form can be also used as inhibitors. Rubbers containing one aminoxyl group per 1000-3000 monomer units show increasing induction periods of the oxidation at 140o a several times [49]. High-molecular-weight inhibitors are favourable for hightemperature stabilization and for polymers with the high molecular mobility. These conditions provide more homogeneous distribution of such stabilizers and show the basic advantage of them connected with their nonvolatility [50]. The increase of the nitrocellulose working life in the presence of 2,2,6,6-tetramethyl-4ethyl-4-oxypiperidine-1-oxyl was observed during mechanical actions. Additives of this stabilizer in the concentration of 0.3 weight % increase durability of the polymer by a factor of hundred. At that, the breaking strength is increased in several times, and the creep rate of the material decreases in 100 times [51]. These results were confirmed by investigations of mechanical degradation of polypropylene in the presence of radicals IV (R=OOCNHC6H5) [52]. AR grafted on polypropylene considerably increases its stability during treatment in the stirrer [53, 54]. ARs can be used as effective quenchers of the exited states and controlling agents photochemical and radiating processes. ARs have been used as photostabilizers of films and fibers [54, 55]. These radicals have been used in synthesis of polymers with strong magnetic properties namely polyradicals. On the basis of polyacetylene containing AR, the polymer ferromagnetic having residual magnetization of 1 G has been prepared [56]. ARs are used in molecular biology for obtaining spin-labelled macromolecules. These labels register slightest changes in the macromolecule states [22]. With the help of spin labels, the conformational transformations of biopolymer macromolecules as well as changes in the structure of biomembranes and nucleic acids have been studied.

5. GENERATION OF AMINOXYL RADICALS IN REACTIONS OF SPIN TRAPPING The possibility in principle to stabilize short-living radicals has been shown for the first using their reactions with nitroso compounds and nitrons [57, 58]:

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry

135

O

R1 N=O + R

R1

O R1

C

N R3 + R

(II.30)

N R

R1

R2

R

O

C

N R3

(II.31)

R2

All aliphatic nitroso compounds are dimers in a solid phase, but they dissociate in solutions and a gas phase. The monomer form of nitroso compounds accepts radicals. The aliphatic nitroso compounds form enough stable adducts with short-living radicals of the very different structure. The character of ESR spectra of radical adducts with tertiary nitroso compounds is practically identical for all spin traps of the given type and is determined by a number of β−hydrogen atoms or other atoms for >N−O• fragments. The most frequently used spin trap is t-nitroso butane (TNB). The irradiation of a reacting mixture during photochemical generation of radicals is always accompanied by the formation of some symmetric AR by the following way [59]: (CH3)3СN=O

hν

⎯⎯→ (CH3)3CNO* → (CH3)3C• + NO

(32)

O

(CH3)3C

(CH3)3C + (CH3)3CN=O

N C(CH3)3

(33)

Usually concentrations of symmetric AR are less than those for basic radicals, but the superposition of spectra of two radicals frequently complicates the interpretation. Alkyl hydroperoxides react with nitroso compounds giving AR [60]: OOR'

R'OOH + (CH3)3CNO O

R' O

+ (CH3)3C-N-OH

(CH3)3C-N-OH O

TNB

R'-O-N-C(CH3)3

(II.34)

Nitroso compounds also easily react with certain anions [61] with the formation of oxyanions, which are oxidized into AR either by nitroso compound itself or traces of O2: O

R + (CH3)3CNO

(CH3)3C-N-R

O

TNB

(CH3)3C-N-R

(II.35)

Therefore the interpretation of data of the radical accepting by TNB in the presence of oxidizers and in electron donor media is complicated. The 2-methyl-2-nitosobutanone-3 is

136

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

close to TNB in chemical properties and ESR spectra of radical adducts [59], and sometimes it is more effective acceptor of radicals. The defect of TNB connected with its sensitivity to light, oxidizers, strong acids and some anions limits to the application of this trap. Some advantages in comparison with aliphatic have aromatic nitroso compounds. The majority aromatic nitroso compounds excepting 2,4,6-tri-t-butylnitroso benzene (BNB) are dimers which dissociate in solutions The monomer form of aromatic nitroso compounds accepts radicals. The character of ESR spectra of aromatic nitroso compounds is determined by a number of the substituents in aromatic rings and β−hydrogen atoms in a radical fragment. They form enough stable adducts with many short-living radicals, but do not form stable adducts with RO•, RO2•, •OH radicals and halogen atoms. The enough detailed consideration of the capability of aromatic nitroso compounds for detection and identification of metalorganic radicals containing Co, Mo, Fe, V, Mn, Re, Cr, Os is given in the works [62, 63]. Among aromatic nitoso compounds, nitroso benzene (NB) has found the greatest application. It is more accessible and not sensitive to visible and near UV-light. Only the light with λ < 310 nm gives rise to the formation of diphenylaminoxyl. Spin adducts with NB are usually stable at room temperature. The basic imperfection of NB is the complexity of the analysis of ESR spectra because of additional lines from protons of phenyl groups. Other essential restriction for the NB applications is the impossibility of its use in solutions containing alkali, alcoholates and other electron donors. In similar conditions, the stable radical anions of NB C6H5NO• − are formed [64]. As a spin trap, nitroso durene (ND) is also used. The main advantage of ND is the simplicity of ESR spectra of radical adducts, the insensitivity to UV-light and high stability of the adducts at room temperature. The drawback of ND is the bad solubility in many solvents, the broadening of lines because of interaction with protons of methyl groups and instability of adducts with RO• radicals [65]. The certain advantages for studying reactions of spin trapping are inherent to BNB [66]. It is well dissolved in many solvents and exists in the active monomer form in solid state and solutions. BNB is the bifunctional trap, and radicals join both to nitrogen and oxygen atoms. The properties of substituted NB such as 2,4,6, trimethylcarbonylnitrosobenzene, 2,4,6-trimethoxynitrosobenzene and pentafluoronitrosobenzene have been investigated [65]. Nitrons are also widely used as spin traps. Nitrons have the much greater thermal and photochemical stability than nitroso compounds. They are monomers and have activity to free radicals even in a solid state. As a result of accepting of radicals by reaction (31), ARs are formed. As a rule, the fragment R3 of these AR represents tertiary alkyl group, and the fragment R1 is the substituted aromatic group. Inconvenience of using of nitrons as spin traps is the absence in most cases HFS from atoms of the attached radical in ESR spectra of formed AR.The information on a structure of the captured radical, as well as in a case of nitroso β compounds, can be obtained from aN and a H constants. Generally three most accessible nitrons are applied as spin traps:

O diphenylnitron (DPN) [67, 68]

C6H5-CH=N-C6H5

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry

137

O CH2=N-C(CH3)3

methylene-t-butylnitron (MN) [69-71]

O C6H5-CH=N-C(CH3)3

С-phenyl-N-t-butylnitron (PBN) [72, 73]

Obtained from DPN, ARs are rather reactive and transform into diamagnetic molecules by recombining with radicals. This disadvantage has caused a small applicability of DPN as a spin trap. MN [69-71] is more active trap than other nitrons. The carbon atom, which is attacked by short-living radicals is less shielded. MN forms considerably more stable adducts with •ОН, НО2• and (СН3)3СОО• radicals [74]. Essential advantage of MN in comparison with TNB is that this nitron catches aminoradicals, whereas nitroso compounds do not add them [69]. Alongside with TNB, PBN is the basic trap widely used in studies of short-living free radicals [75]:

O

R

C6H5-CH=N-C(CH3)3 + R

O

C6H5-CH-N-C(CH3)3

(II.36)

PBN is stable to the action of light, O2 water and well dissolved in many solvents. Radical adducts with PBN are stable at room temperature and in a number of cases can be isolated in the pure state.. PBN catches more extensive variety of radicals than TNB, for example, F•, Cl•. The insertion of two functional groups OH and >C=N→O into structure of one molecule has been carried out in the work [76]. As a spin trap, С-(3,5-di-t-butyl-4-hydroxyphenyl)N-tbutylnitron was used. This bifunctionsl trap forms phenoxyl radicals in reaction with radicals having pronounced oxidizing properties (RO•, R•C=O, PhCOO•) and ketones in triplet-exited states. The radicals with unpaired electron on carbon atoms add to β− carbon of the nitron forming AR:

O HO

R

R HO

O

CH-N-C(CH3)3

(II.37)

CH=N-C(CH3)3

O R

O

CH=N-C(CH3)3

(II.38)

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

138

The study of oxidation by the spin trap method is associated with identification of adducts of nitrons with RO2• radicals [77, 78]. Adducts of RO2• radicals with PBN are unstable and even at 263 K rapidly decay. During decomposition of these adducts, the products of interaction with RO• radicals are formed:

O

O

C6H5-CH-N-C(CH3)3 O O CH

C6H5-CH-N-C(CH3)3 + (R2)R1

R1

O CH (II.39)

OR2(R1)

R2

Thus, though the RO2• radicals cannot be fixed in concrete conditions of oxidation of hydrocarbons, the formation of PBN adducts with RO• radicals is the qualitative indication on occurrence of peroxide radicals in the reacting system. The composition of products of interaction of aliphatic nitrons with OH• radicals can be very various. For nitrons containing aromatic groups, for example PBN, three paths of the reactions are possible inclusive of the hydrogen atom abstraction from alkyl groups, addition of OH• to aromatic rings and nitron groups with formation of AR [79, 80]:

O C6H5-CH=N-C(CH3)2CH2 O

(II.40)

O

C6H5-CH=N-C(CH3)3 + OH

CH=N-C(CH3)3

(II.41)

HO O C-N-C(CH3)3

(II.42)

OH

Nitrons can accept various atoms and radicals. The convincing evidence of hydrogen atom addition to nitrons has been obtained by the example of PBN [19]. The attachment of H atoms to aromatic rings gives cyclohexadienyl radicals [81]. Adducts of PBN with H atoms are rather unstable and are not observed in non-polar solvents. The spin trapping of fluorinated radicals and Cl atoms by PBN takes place [82, 83]. A series of 3-aryl-2H-benzo[1,4]oxazin-4-oxides O N O

X

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry

139

were prepared, and their ability to trap free radicals was investigated by ESR spectroscopy [84]. In organic solvents, these compounds were able to efficiently scavenge all carbon- and oxygen-centered radicals tested, giving very persistent aminoxyls, except with superoxide anion whose spin adducts were unstable. The main feature of these nitrones as spin traps lies in the possibility to recognize the initial radical trapped. In fact, besides a g-factor and aminoxyl nitrogen coupling constant dependent on the species trapped, the ESR spectra also show different patterns due to hyperfine splitting characteristic of the radical scavenged. This last important feature was investigated by means of density functional theory calculations. The enough overall summary of the ESR characteristics of various AR produced in nitrons is given in the works [19, 72]. In these studies the various aspects and features of application of the spin trap method in studying of the mechanism of chemical reactions are considered.

6. SYNTHESIS OF POLYMERS CONTAINING STABLE AMINOXYL RADICALS The chemical properties of polymeric AR are determined by the >N O• fragment and basically similar to those of low-molecular AR. They are characterized by the high chemical stability in a wide range of pH in water solutions, thermal stability up to 180o and oxidative stabilities. The life time of such radicals makes up many years and practically is not limited in inert media. The interaction of AR having functional groups with those of macromolecules is the most widespread procedure of obtaining aminoxyl-containing polymers [18]. The AR preparation methods are given in the monographs [2, 3]. As the example of such reactions one can consider the graft of AR III (R=COOH or COOCH3) to polyethylene glycol having end hydroxyl groups [85]. The first stage of the reaction includes the treatment of radicals III by sulfochloride to obtain AR containing chloranhydride groups. Then the attachment of synthesized aminoxyl to the polymer is carried out by the following scheme: Cl

C

O pyridine

CH2OCH2CH2OH +

CH2OCH2CH2O

C

O

(II.43)

N O

N O

Polyvinylacetate with AR was obtained by the reaction with radicals III (R=COOH) [86]. Macromolecules of modified polyvinylacetate contains from 1 to 10 AR. Polyacrylates, polymethylacrylates and polymethylmetacrylate with AR are synthesized by reaction of copolymer containing chloranhydride groups with a radicals IV (R=OH) [87]: CH3

CH3 CH2 C

CH2

COOCH3

C COCl

IV(R=OH) pyridine

CH3 CH2 C

CH3 CH2

C

COOCH3

C O

O

N O (II.44)

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

140

Synthesis of spin-labelled polystyrene has been carried out by the reaction of the polymer containing chlormethyl groups with radicals IV (R=OH) [88, 89]: IV (R=OH)

H2C CH CH2

H2C

pyridine

CH2Cl

H C

CH2

CH2O

(II.45)

N O

AR (VIII) was introduced to macromolecules of polyvinylacetate by the partial saponification [90]: Cl CH2-CH-CH2 OCOCH3

N

+

CH

N

OCOCH3

N

(VIII)

Cl

CH2-CH-CH2

CH

(II.46)

N

O

OCOCH3

N

N O

NH

N O

NH N

Cl

Polyethylene films containing carbonylhydrazide groups were treated by radicals IV (R = −N=C=S) [91]. The spin-labelled polyethylene was obtained by the following reaction:

H2C CH

+

S=C=N

N O

C O

ethanol 35o

NHNH2 H2C CH C O NHNH C

NH

N

O

(II.47)

S

Synthesis of spin-labelled copolymer of styrene with maleic anhydride has been carried out on heating a solution of the copolymer and radical IV (R=NH2) in anhydrous THF [92]:

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry

+

CH-C-CH2 CH O

N O

THF

O

O

CH-C-CH2 CH CH O

NH2

141

CH2 CH

II.48)

COOH C O NH

O

O

CH

N O

However, the most of synthetic polymers have no suitable reactive groups. AR can be inserted into such polymers only via copolymerization or chemical modification of macromolecules. Using reactions of AR without participation of unpaired electrons, polymers were synthesized from monomers containing one or two free-radical fragments [93]. The paramagnetism of such polymers amounts to 1.5-2.1 1021 spin⋅g−1. AR (IX) on the basis of radical-containing diacetylenes HO

C

C C

N

OH

C

N

O

O

(IX) undergoes polymerization converting into polymeric polycrystals under the action of light or on heating (80-100o ) [56, 94]. A polyacethylene chain with stable radical substituents (R•) is R

R

R

R

formally similar to the hypothetical polyene polyraducal which serves a theoretical model for a “ferromagnetic” macroradicals. The solid-state polymerization of such diacetylenes was thought to be a perspective or at least feasible route to conjugated polyradicals. Indeed, several features of the polymerization approach seem to be advantageous. Owing to the monomer lattice control, exact stereo specificity of the polyradical may be guaranteed. Crystalline nature of the polymerization products should facilitate characterization of the latter. Since no catalysts are used for the polymerization, probability of the reaction products contamination is reduced. Based on GPH analysis, the polymerization products are oligomers with polymerization degree n = 4 – 10 [94]. A broad structureless absorption band in their optical spectra, peaking at λmax = 380 nm, is indicative for rather short conjugation length. After the thermal treatment, X – ray analysis has revealed almost total amorphisation of the polymerization products of some monomers similar to IX.

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

142

ESR showed also that magnetic behavior of the products was typical for a spin labelled oligomer chain (triplet signal and a typical τ − correlation times vs molecular mass dependence in solution) without any signs of ferromagnetic ordering of spins. Thus, ferromagnetic properties of polyconjugated polyradicals produced from aminoxyl-substituted diacetylenes seem to be illusive. AR (X) COOC2H5

C2H5OOC N O

(X)

has been incorporated into the main chain of "living" polystyrene obtained by anionic polymerization in the presence of butyl lithium [95]:

Bu-(CH2-CH)n-CH2-CH Li X

BuLi C6H6

H2C CH

O C

O C

(II.49)

CH CH2

N O By the same method of “living“ radical polymerization, a series block copolymers of poly(ethylene oxide-styrene) with narrow polydispersity were synthesized by the following two step approach [96]. At first, “living” anionic polymerization of ethylene oxide with sodium-4-oxy-2,2,6,6-tetramethyl-1-piperidinoxyl as initiator yields polyethylene oxide with AR at chain end:

O N

60o

ONa + n

O N

O ( CH2CH2O )n H

O

Then a stable free radical polymerization of styrene gives a block copolymer:

(II.50)

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry

AIBN, 120o

O ( CH2CH2O )n H

O N

143

m

O ( CH2CH2O )n H

C ( CH2CH )m O N

(II.51)

CN

By the polycondensation of 1,10-decandiol with chloranhydride of terephthalic acid in the presence of AR (XI),

HO

(H2C)4

N O

(CH2)4 OH (XI)

the polymer of the following structure has been obtained [97]:

(H2C)10 O C O

C O (H2C)4 O

N

(CH2)4

O

However, not always polymerization or copolymerization of monomers with AR occurs without participation of unpaired electrons. For example, attempts to synthesize polymers from monomers containing radicals IV [R = −O (C=O) C (CH3) =CH2] have not been successful, because radicals of growing chains actively react with aminoxyl groups [98, 99]. Therefore, for preparation of spin-labelled polymers one can use monomers containing appropriate amines with the subsequent oxidizing the polymer. In such a manner the spinlabelled polymer has been synthesized [100] from the monomers:

CH3 H 2C C C X

NH where X = NH, O

O The oxidation of polyacrylamides having diphenylamine groups by lead dioxide gives polymer with AR [101]:

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

144

CR-CH2 O C HN

N O

where R = H, CH3. Aminoxyl-containing polyvinylppyrrolidone and polyvinylcaprolactame have been obtained by the copolymerization of corresponding monomers and amines

HO

CH=CH2

CH=CH2

H2C OH ,

N H

N H

with the further oxidation of amine groups in the copolymers [102]:

O

OH

N

N

O

,

O

N

H2C OH

N

O

N O

N O

O

OH

N N O

N

O

O

N

H2C OH

N

O

, N O

The graft of AR to polyethylene has been performed in the reaction of copolymers containing carbonyl groups (0.5 %) with 2-amine-2-methyl propanol [103]:

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry CH3

+ H2N

CH2-C-CH2

CH2-C-CH2 OH NH

CH3 CH2

O

HO H2C C

HO

145

[O] CH3

-H2O

CH3

(II.50)

CH2-C-CH2 N O

O

The polymer inclusive of nitron and aminoxyl groups has been obtained by the reaction of polystyrene containing formaldehyde groups in p-position of phenyl rings with 2,3bis(hydroxylamino)-2,3-dimethylbutan [104]: CH3 CH-CH2

+ H3C C

NHOH

H3C

NHOH

C

CH3OH

CH-CH2

PbO2

CH3 CHO

HON

(II.51)

CH-CH2

O

N

NOH

N O

The polymers with grafted AR can be also produced in reactions of macroradicals formed by mechanodegradation, photolysis or radiolysis in the presence of aminoxyl biradicals. By this method, for example, AR (XII) COOR

,R= COOR

N O

(XII)

has been introduced into macromolecules of polyolefines in the course of their mechanodestruction [105]. Biradicals are grafted upon macromolecules by one of two radical fragments. By ESR method [106], the nature of polymeric AR formed during photooxidation

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

146

of films of isotactic polypropylene containing bis-(2,2,6,6-tetramethyl-4-piperidyne)sebacinate has been studied: O

O

OC(CH2)8CO

N H

N H

Only one of amine groups of this stabilizer is oxidized to AR during photolysis. The part of monoradicals formed recombines with alkyl macroradicals of polypropylene, and then the second amine group is converted into AR. To obtain spin-labelled polymers, various nitroso compounds are widely used. The wide set of reactions represents a variety of methods of the polymer modification by these compounds. In the most cases, AR can be generated by reactions of nitroso compounds with metalorganic reactants: R-NO + R1-M

R

N R1

O

M

+ hydrolysis oxidation

R

N

O

(II.52)

R1

When ARs are introduced into phenyl rings of polystyrene, the first stage is mercuration of the polymer. Under the action of nitrosyl chloride, the mercurated polystyrene is converted into that containing nitroso groups. The synthesis of modified polystyrene includes also stages of the polymer treatment by phenylmagnesium bromide and silver oxide [107]: H2C CH

NOCl

HgOAc

NOH

THF

N=O

AgO H2C CH

C6H5MgBr

H2C CH

(II.53)

H2C CH

N

O

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry

147

The spin-labelled polystyrene contains one AR per 1000 monomer units. If tbutylmagnesium chloride is used instead of phenylmagnesium bromide, the polymer with tbutylaminoxyl groups can be obtained [108]: HC

(H3C)3C

CH2

N O

It should be noted that the synthesis via the scheme (53) has a number of drawbacks. The mercurated polystyrene is in cline to cross-linking, and the polymer containing nitroso groups is the unstable compound. The more suitable method of synthesis of the spin-labelled polystyrene is given below [109]: H2C CH

J2,HJO3 o

90 ,C6H5NO2

C4H9Li

H2C CH

C6H6

J H2C CH

Li

TNB

H2C CH

(H3C)3C N

Ag2O CH3OH

+ O Li

(II.54)

H2C CH

(H3C)3C N

O

This synthesis is not accompanied by the destruction or cross-linking of macromolecules. TNB can be used use as a trap for carbanions which are converted into AR by the following reaction: R + (CH3)3CN=O

(H3C)3C N R O

hydrolysis oxidation

(H3C)3C N R

(II.55)

O

The choice of suitable initiating system allows of preparing polystyrene containing AR on one (n-butyl lithium) or two (naphthalate sodium) ends of macromolecules. By this method, aminoxyl-containing polymethylstyrene, poly-2-vinylpiridine and copolymer of styrene with methylstyrene have been obtained [61]. Polymethylmethacrylate with the end t-butylaminoxyl groups was synthesized by the similar method [110]. However, the anionic polymerization of methylmethacrylate is accompanied by some side reactions influencing on efficiency of the process especially on a place of the label attachment. The optimal conditions of the synthesis include THF as the

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

148

solvent, 70o and initiators: n-butyl lithium, sodium naphthalate or 9-lithium fluorine. In these conditions one can obtained polymethylmethacrylate with the end AR: CH3 H2C C

TNB

COOCH3

CH3 But CH3OH H2C C N O COOCH3

CH3 But oxidation H2C C N OH COOCH3

CH3 But H2C C N O COOCH3

(II.56)

The important reaction of nitroso compounds resulting in spin-labelled polymers is the radical capture. Polystyrene containing t-butyl aminoxyl was obtained by radical polymerization in the presence of TNB and t-butylperoxyoxalate [111]:

(CH3)3C O + PhCH=CH2

PhCHCH2CH2OC(CH3)3

PhCHCH2(PhCHCH2)nCH2OC(CH3)3 PhCHCH2(PhCHCH2)nCH2OC(CH3)3

PhCH=CH2

(CH3)CNO

(II.57)

(H3C)3CN O

AR can be introduced into macromolecules also by the reaction of nitroso compounds with macroradicals generated by mechanodestruction, thermal degradation or photolysis of polymers. The experimental data and schemes of TNB conversions in reactions with alkyl macroradicals of polypropylene under shearing forces or photolysis are represented in the work [53]. During polyethylene grinding in a mixture with NB at 77 K, ARs are appeared on a surface of particles of the polymer as a result of interactions of the spin trap with macroradicals [112]. The multiple treatment of cross-linked rubbers containing TNB by repeated swelling-cooling leads to occurrence of AR as a result of bond scissions with the formation of macroradicals [113]. The synthesis of polyethylene containing ARs has also been performed by thermal destruction of the polymer films with BNB additives [114]. The films with BNB were prepared by combined dissolutions of the polymer and BNB in benzene with subsequent removal of the solvent. Spin-labelled rubbers (polybutadiene, polyisoprene, copolymer of isobutylene with isoprene) were prepared by reactions with 2,6-dichloronitrosobenzene in toluene solutions [115]. The ESR signal of AR occurs immediately after mixing solutions of rubbers and the nitroso compound. AR formed are stable for months. The method proposed allows of obtaining various spin-labelled polymers having > C=C < bonds. Spin labels were grafted on polyethylene under the γ−radiolysis in the presence of PBN [116]:

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry

CH2-CH-CH2

CH2-CH-CH2

+ Ph-CH=N-C(CH3)

149

(II.58)

CH-Ph (H3C)C

O

N O

The γ−radiolysis at −196o generates alkyl macroadicals ~ CH2-С•Н-CH2~ detected by the ESR spectrum. The formation of AR was observed at – 70o via the reaction 58. The peculiar example of spin-labelling is 1,3-dipolar addition of paramagnetic nitrons (XIV) to double bonds of rubbers [117]: N

+

CH=CH

N O

O

CH-CH

(II.59)

O

(XIV)

HC

N N O

Like that spin-labelled methylvinylpyridine, chloroprene and divinyl rubbers have been obtained. The method of incorporation of AR into the polymer matrix has been demonstrated by the example of polymerization of pyrrole [118]. In the presence of protons, radicals IV cause polymerization of pyrrole to polypyrrole with incorporated reduced (hydroxylamine) form and oxidized (nitrosonium ion) form of AR in polymer matrix. In electrochemical oxidation of pyrrole in the presence of AR, a coupled electrochemical-chemical synthesis produces polypyrrole films with incorporated nitrosonium ions. They can by reduced to AR by partial film reduction. The successful use of spin labels in analysis of functionalization of macromolecules has been demonstrated by the example of the copolymer of styrene – divinylbenzene with different content of carboxy groups. The radical IV (R1 = OH) and 6-(3-aminophenyl)-2,4diphenylverdazil

H2N N N

N N

were selected as spin labels, considering their distinct ESR spectra and the presence of reactive hydroxyl and amino groups. Carboxy-containing copolymer was converted into the corresponding acid chloride. The resin was then treated with DMA solutions of IV and verdazyl spin labels in various concentrations. It turned out that concentrations of radicals in

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the spin labelled polymer measured by ESR method and initial taken concentrations of radicals have a discrepancy of about 1 – 1.5 %. Hence, some polymer, containing functional groups (–COOH, −NCO, epoxy, etc) capable of binding the functional groups of the spin labels, can be analyzed rapidly and accurately using the considered procedure. Thus, the different ways are suitable for the AR formation in polymers including purely chemical synthetic methods as well as radiation-initiated, photo- and mechano-chemically initiated processes. The choice of one or other way depends on chemical structure of the polymer, availability or lack of reactive functional groups in macromolecules.

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

Pariiskii G. B., Gaponova I. S., Davydov E. Ya. Russ. Chem. Rev. 2000, 69, 985-999 Tormala P., Lindberg J. J. In Structural Studies of Macromolecules by Spectroscopic methods; Ivin K. J.; Ed.; Khimiya: Moscow, 1980, 236-253. Rozantsev E. G., Sholle V. D. Organic Chemistry of Free Radicals. Khimiya: Moscow, 1979. Nonhebel D. C., Tedder J. M., Walton J. C. Radicals. Cambridge University Press: Cambridge, 1979. Rozantsev E. G. Free Nitroxyl Radicals. Plenum: New York, 1970. Rintoul L., Micallef A. S., Reid D. A., Bottle S. E. Spectrochim. Acta. A. 2006. 63. 398402 Carmichael P. J., Govenlock B. G., Johnson C. A. F. J. Chem. Soc. Perkin Trans. 2. 1973, 1853-1856. Lipey M. M., Potapov V. K., Kostyanovsky R. G. Khim. Vysok. Energiy. 1974. 8, 496499. Buchachenko A. L., Wasserman A. M.. Stable Radicals. Khimiya: Moscow, 1973. Aurich H. G., Hahn K., Stork K., Weiss W. Tetrahedron. 1977, 33, 969-975. McCelley R.C., Shimshick E. J., McConnell H. M. Chem. Phys. Lett. 1972, 13, 115119. Goldman S. A., Bruno G. V., Freed J. H. J. Phys. Chem. 1972, 76, 1858-1960. Bluhm A., Weinstein J. J. Amer. Chem. Soc. 1970. 92, 1444-1446. Doba T., Noda S., Yoshuda H. Bull. Chem. Soc. Japan. 1979. 52, 21-24. Forrester A. R., Hepburn S. P. J. Chem. Soc. C. 1970. 1277-1280. Calder A., Forrester A. R. J. Chem. Soc. C. 1969. 1459-1463. Mender O. W., Janzen E. J. J. Org. Chem. 1969 34, 4072-4082. Wasserman A. M., Kowarsky A. L. Spin Labels and Probes in Physical Chemistry of Polymers. Nauka: Moscow, 1986. Zubarev V. E., Belewsky V. N., Bugaenko L. T. Russ. Chem. Rev. 1979, 48, 13611392. Shapiro A. B., Rozantsev E. G.. Izv. AN SSSR ser. chimich. 1966, 1650-1652. Neiman M. B., Rozantsev E. G., Golubev V. A. Izv. AN SSSR. ser. chimich. 1965, 548550. Berliner L. J. Spin Labeling Theory and Applications. Academic Press: New York, 1974.

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry

151

[23] Buchachenko A. L., Tkacheva O. P. Kinetika i Kataliz. 1966. 7, 777-787. [24] Summermann W., Deffner U. Tetrahedron. 1975. 31, 593-596. [25] Golubev V. A., Zjdanov R. I., Rozantsev E. G. Izv. AN SSSR. ser. chimich. 1970, 184185. [26] Zjdanov R. I., Golubev V. A., Rozantsev E. G. Izv. AN SSS. ser. chimich. 1970,186187. [27] Bailey P. S., Keller J. E. J. Org. Chem. 1970, 35, 2782-2785. [28] Keana J. F. W. Chem. Rev. 1978, 78, 37-63. [29] Golubev V. A., Zjdanov R. I., Gida V. M., Rozantsev E. G. Izv. AN SSSR. ser. chimich. 1971, 853-855. [30] Low H., Paterson I., Tedder J. M. J. Chem. Soc. Chem. Commun. 1977, 171-172. [31] Brandi P., Galli C., Gentili P. J. Org. Chem. 2005, 70, 9521-9528. [32] Beckwith A. L. J., Polle J. S. J. Am. Chem. Soc. 2002, 124, 9489 -9497. [33] Murayama K., Yoshioka T. Bull. Chem. Soc. Japan. 1969, 42, 1942-1947. [34] Nigan S., Asmus K. D., Willson R. L. J. Chem. Soc., Faraday Trans.11976, 2324-2328. [35] Skuratova S. I., Kozlov Yu. N., Zakatova N. V., Sharpaty V. A. Zjurn. Fizich. Khimii. 1971, 45, 1821-1823. [36] Karpukhin O. N., Pokholok T. V., Shlyapintokh V. Ya. Vysokomolek. Soed. A. 1971, 13, 22-27. [37] Khloplyankina M. S., Buchachenko A. L., Neiman M. B., Vasil’eva A. G. Kinetika i Kataliz. 1965, 6, 394-398. [38] Ruban L. V., Buchachenko A. L., Neiman M. B., Kohanov Yu. V. Vysokomolek. Soed. A. 1971, 8, 1642-1646. [39] Becke A. D. Phys. Rev. 1988, 38, 3098-3100. [40] Huang H., Henry-Riyad H., Tidwell T. T. J. Am. Chem. Soc. 1999, 121, 3939-3943. [41] Allen A. D., Cheng B., Fenwick M. H., Givehchi B., Henry-Riyad H., Nikolaev V. A., Shikhova E. A., Tahmassebi D., Tidwell T. T., Wang S. J. Org. Chem. 2001, 66, 26112617. [42] Melnikov M. Ya., Smirnov V. A. Photochemistry of Organic Radicals. Moscow State University: Moscow, 1994. [43] Keana J. F. W., Dinerstein R. J., Baitis F. J. Org. Chem. 1971, 36, 209-211. [44] Keana J. F. W., Baitis F. Tetrahedron Lett. 1968, 365-369. [45] Bogatyreva A. I., Buchachenko A. L. Russ. Chem. Rev. 1975, 44, 2171-2204. [46] Maslov S. A., Zaikov G. E. Russ. Chem. Rev. 1977, 46, 1253-1272. [47] Shilov Yu. B., Batalova R. M., Denisov E. T. Doklady AN SSSR. 1972, 207, 388-389. [48] Shilov Yu. B., Denisov R. E. T. Vysokomolek. Soed. A. 1974, 16, 2313-2316. [49] Ilieva T. G., Potapov E. E., Polenov E. A. Kautchuk i Rezina. 1989, 39-40. [50] Emanuel N. M., Buchachenko A. L. Chemical Physics of Polymer Degradation and Stabilization. Science Press: Utrecht, NET, 1987. [51] Malchevsky V. A., Zakrevsky V. A.. Mekhanka Polymerov. 1978, 2, 342-344. [52] Rogov Yu. N., Deyun F. V., Anisin V. A., Smirnov L. P., Manelis G. B. Doklady AN SSSR. 1983, 230, 637-642. [53] Chakraborty K. B., Scott G. Y. H. J. Polym. Sci. Polym. Lett. Ed. 1984, 22, 553- 558. [54] Chakraborty K. B., Scott G. Y. H. J. Appl. Polym. Sci. 1985, 30, 3267-3284. [55] ElMalaika S., Scott G. In Degradation and Stabilization of Polyolefins; Allen N. S. L.; Ed.; Appl. Sci. Publ.: New York, 1983, p. 219.

152

E. Ya. Davydov, I. S. Gaponova, G. B. Pariiskii et al.

[56] Korshak Yu. V., Ovchinnikov A. A., Shapiro A. B., Medvedeva T. V., Spector V. N. Exp. Theor. Phys. Lett. 1986, 43, 309-311. [57] Iwamura M., Inamoto N. Bull. Chem. Soc. Japan. 1967, 40, 703. [58] Mackor A., Wajer T. A., De Boer T. J., Van Voorat J. D. Tetrahedron Lett. 1967, 27572761. [59] Forshult S., Laggercrant Z. C., Torselle K. Acta Chem. Scand. 1969, 23, 522-530. [60] Terabe S., Kanoka R. J. Chem. Soc. Perkin. Trans. 1972, 2, 2163-2172. [61] Forrester A. R., Hepburn S. P. J. Chem. Soc. C. 1971, 701-703. [62] Rehorec D., Hennig H. Can. J. Chem. 1982, 60, 1565-1573. [63] Hadson A., Leppert M. F., Lednor P. W. J. Chem. Soc. Dalton Trans. 1981, 2159-2163. [64] Russell G., Geels J., Smentowsti F. J. J. Am. Chem. Soc. 1967, 89, 3821-3828. [65] Terabe S., Kuruma K., Konaka R. J. Chem. Soc. Perkin Trans. 2. 1973, 1252-1258. [66] Terabe S., Konaka R. J. Am. Chem. Soc. 1971, 93, 4306-4307. [67] Iwamura M., Inamoto N. Bull. Chem. Soc. Japan. 1970, 43, 756-760. [68] Bluhm A., Weinstein J. J. Org. Chem. 1972, 37, 1748-1753. [69] Anderson N., Norman R. J. Chem. Soc.B. 1971, 993-1003. [70] Janzen E. G., Lopp I. J. Magnet. Resonance. 1972, 7, 107-110. [71] Baldwin J., Qureshi A. R., Sklarz B. J. Chem. Soc. C. 1969, 1073-1079. [72] Janzen E. G. Acc. Chem. Res. 1971. 4, 31-40. [73] Janzen E. G., Blackburn B. J. J. Am. Chem. Soc. 1969. 91. 4481-4490. [74] Konaka R., Terabe S., Mizuta T., Sakata S. Can. J. Chem. 1982, 60, 1532-1541. [75] Church D. F. J. Am. Chem. Soc. 1984. 106, 5073-5079. [76] Paifici J. G., Browning H. L. J. Am. Chem. Soc. 1970, 92, 5231-5233. [77] Howard J. A., Tait J. C. Can. J. Chem. 1978, 56, 176-178. [78] Zubarev V. E., Belevsky V. N., Yarkov S. P. Doklady AN SSSR. 1979, 244, 1392-1396. [79] Greenstock C. L., Wiebe R. H. Can. J. Chem. 1982, 60, 1560-1564. [80] Neta P., Steenken S., Janzen E. G. J. Phys. Chem. 1980, 84, 532-535. [81] Pryor W. A., Lin T. H., Stanley J. P. J. Am. Chem. Soc. 1973, 95, 6993-6998. [82] Janzen E. G., Knauer B., Gerlock J. J. Phys. Chem. 1970, 74, 2037-2038. [83] Janzen E. G., Knauer B., William S. L. J. Phys. Chem. 1970, 74, 3025-3028. [84] . Astolfi P., Marini M., Stipa P. J. Org. Chem. 2007, 72, 8677 -8682. [85] Tormala P., Lattila H., Lindberg J. J. Polymer. 1973, 14, 481-487. [86] Bluhm F. D., Dikson J. E., Miller W. G. J. Polym. Sci. Polym. Phys. Ed. 1984, 22, 211221. [87] Bullock A. T., Cameron G. G., Krajewski V. J. Phys. Chem. 1976, 80, 1792-1797. [88] Regan S. L. J. Am. Chem. Soc. 1974, 96, 5275-5276. [89] Regan S. L. J. Am. Chem. Soc. 1975, 97, 3108-3112. [90] Aleksandrova T. A., Wasserman A. M., Tager A. A. Vysokomolek. Soed. A. 1977, 19, 137- 141. [91] Rasmussen J. R., Bergbreiter D. E., Whitesides. G. M. J. Am. Chem. Soc. 1977, 99, 4746- 4756. [92] Kulikov N. V., Likhtenshtein G. I., Rozantsev E. G., Suskina V. I., Shapiro A. B. Biofizika. 1972, 17, 42-48. [93] Rozantsev E. G., Novelko G. Ph. Vysokomolek. Soed. B. 1967, 9, 866. [94] Lazareva O. L., Motyakin M. V., Shapiro A. B., Shchegolikhin A. N. Synthetic Metals. 1997, 85, 1685-1686.

Properties and Applications of Aminoxyl Radicals in Polymer Chemistry

153

[95] Freidrich C., Noel C., Ramassuel R., Rassat A. Polymer. 1980, 21, 232-235. [96] Hua F. J., Yang Y. L. Polymer. 2001, 42, 1361-1369. [97] Sundholm F., M. Wasserman A., Barashkova I. I., Timofeev V. P. Eur. Polym. J. 1984, 20, 733-738. [98] Griffith O. H., Keana W. J. P., Rottshalffer S., Warlick T. A. J. Am. Chem. Soc. 1967, 89, 5072-5074. [99] Forrester A. R., Hepburn P.. J. Chem. Soc. C. 1971, 20, 3322-3328. [100] Kurusaki A. K., Lee K. W., Okawara M. J. Polym. Sci. Polym. Chem. Ed. 1972, 10, 3295- 3297. [101] [101]. Braun D., Hauge S. Makromol. Chem. B. 1971, 150, 57-62. [102] [102]. Wasserman A. M., Timofeev V. P., Aleksandrova T. A., Shapiro A. B. Eur. Polym. J. 1983, 19, 333-339. [103] Bullok A. T., Cameron G. G., Smith P. N. Eur. Polym. J. 1975, 11, 617-624. [104] Miura Y., Nakai K., Kinoshita M. Makromol. Chem. B. 1973, 172, 685-687. [105] Radzig V. A., Shapiro A. B., Rozantsev E. G. Vysokomolek. Soed. B. 1972, 14, 685687. [106] Hodgeman D. K. C. J. Polym. Sci. Polym. Chem. Ed. 1980, 18, 533-539. [107] Drefahl G., Horhold H. H., Hofmann K. D. J. Prakt. Chem. B. 1968, 37, 137-142. [108] Bullok A. T., Cameron G. G., Smith P. M. J. Phys. Chem. 1973, 77, 1635-1639. [109] Bullok A. T., Cameron G. G., P. M. Smith. Polymer. 1972, 13, 89-90. [110] Bullok A. T., Cameron G. G., Elson J. M. Polymer. 1974, 15, 74-76. [111] Chalfont G. R., Perkins M. J. J. Am. Chem. Soc. 1968, 90, 7141-7142. [112] Vivatpanachart S., Nomura H., Miyahara Y. Polymer. 1981, 22, 896-898. [113] Zander P., Karl-Friedrich A. Plaste und Kautch. 1986, 33, 288-290. [114] Kitahara T., Shimada S., Kashiwabara H. Polymer. 1980, 21, 1299-1303. [115] Miclos G., Rockenbauer A., Tüdos P. Macromolecules. 1987, 20, 3083-3086. [116] Kusumoto N., Sakai T. Polymer. 1979, 20, 1175-1177. [117] Barashkova I. I., Martin V. V., Anfimov B. N., Volodarsky L. B. Vysokomolek. Soed. B. 1987, 29, 354-357. [118] Rapta P., Bartl A., Dunsch L. Synthetic Metals. 1997, 84, 187-188. [119] Katritzky A. R., Belyakov S.A., Strab S., Cage B., Dalal N. S. Tetrahedron Lett. 1999, 407-410.

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.155-164 ©2010 Nova Science Publishers, Inc.

Chapter 11

SYNTHESIS OF FLEXIBLE MANUFACTURINGS FOR PHOSPHORIC INDUSTRY WASTE UTILIZATION BASED ON THE CALS-CONCEPT A. M. Bessarabov*1, A. V. Kvasyuk1 and G. E. Zaikov 2 1

State Scientific-Research Institute of Chemical Reagents and High Purity Chemical Substances (IREA), Moscow, Russia 2 Institute of Biochemical Physics Russian Academy of sciences, Moscow, Russia

ABSTRACT CALS-projects of sodium phosphite and hypophosphite technologies – products of phosphoric sludge utilization (the most aggressive waste of phosphoric industry) were developed. CALS-technology of the flexible two-grocery production was developed based on the theory of the flexible scheme synthesis

Keywords: flexible manufacturing, CALS-technologies, phosphoric industry, phosphorus sludge, sodium phosphite, sodium hypophosphite

BACKGROUND Utilization of phosphorus sludge formed in large amounts in the phosphoric industry was considered. The corresponding task was formulated as development of a closed-circuit technological process at which phosphorus sludge will re-enter processing for the maximal extraction of useful products from its composition [1].

*State Scientific-Research Institute of Chemical Reagents and High Purity Chemical Substances (IREA), Bogorodsky Val, 3, 107076, Moscow, Russia, E-mail: [email protected]

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AIMS Aim of the work is synthesis of flexible manufacturing of sodium phosphite and sodium hypophosphite for phosphoric industry waste utilization based on the CALS-concept.

EXPERIMENTAL PART At the first stage the nomenclature of the phosphorus-containing compounds entering the complex phosphorus sludge processing flow sheet (circuit) is examined. In the circuit four target products are considered: sodium hypophosphite, sodium phosphite and obtained through its further processing dibasic lead phosphite and phosphorous acid (fig. 1).

Phosphorus slag (sludge) SODIUM PHOSPHITE

SODIUM HYPOPHOSPHITE

Na2HPO3 or Na2HPO3×5H2O

Na H2PO2 or (NaH2PO2*H2O)

PHOSPHOROUS ACID

DIBASIC LEAD PHOSPHITE 2Pb*PbHPO3*0.5H2O

H3PO3

Figure 1. Scheme of multiassortment (4-product) process for phosphorus sludge utilization

Development of processes for phosphorus sludge recovery was conducted within the limits of the most modern and perspective system of computer support - CALS-technologies (Continuous Acquisition and Life cycle Support - continuous information support of life cycle of a product) [2]. The CALS concept is based on the complex of uniform information models, standardization of ways of access to the information and its correct interpretation in accordance with international standards. Thus uniform ways of process control and interaction of all participants of development are provided. A key idea of CALS concept is increasing of product life cycle due to increase of efficiency of control of the information on a product. CALS task is transformation of a product life cycle into highly automated process through re-structuring of its component business-processes [3]. Within the limits of CALS concept two basic circuits of phosphorus sludge processing have been considered: with sodium phosphite [4] and sodium hypophosphite [5] as final products. The yielded circuits were implemented in the CALS-project. In the CALS-project used is the typical computer structure of initial data for designing which includes the following subcategories [6]: the general data on technology (01); characteristics of the

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executed research and experimental work (02); Feasibility study of a recommended production method (03); a patent card (04); characteristics of feedstock, auxiliary materials (05); physical and chemical constants and properties of initial, intermediate and a final products (06); chemical, physicochemical bases and basic production flow sheet (07); operating technological parameters of production process (08); the mass balance of production process (09); characteristics of by-products and solid waste (10); the mathematical description of technological processes and devices (11); data for calculation, designing, a choice of the basic production equipment (12); recommendations for process automation (13); the analytical control of production process (14); methods and technological parameters of purification from chemical and industrial pollutants (15); safety data sheet including fire and explosion hazard data, fire fighting procedures, health hazard data, regulatory information (16); the list of reports and recommended literature on considered technology (17). On the basis of the offered typical structure of initial data for designing, the CALSproject for sodium phosphite production technology has been developed. Shown in Fig. 2 block diagram includes a preparatory stage and 4 basic production stages: phosphorus sludge decomposition in the reactor, filtering of a mineral part, correction of solution density, neutralization of excess of alkali in solution:  

Figure 2. An element of the CALS-project «Flow diagram of sodium phosphite production».

1. The Preparatory stage. The phosphorus sludge is classified in a grinder to particle size optimal for interaction with sodium alkali (NaOH) and then a solution is prepared. In parallel alkali solution is prepared through diluting alkali liquor to concentration NaOH = 31 % using water as solvent. All stages of the corresponding

A. M. Bessarabov, A. V. Kvasyuk and G. E. Zaikov

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2.

3.

4.

5.

technological block with characteristics of the equipment as well as additional information are put in the CALS-project and are used by both developers of technology, and engineering personnel (analysts, etc.). Decomposition of phosphorus sludge in a reactor. In the reactor (1) NaOH solution is fed and simultaneously phosphorus sludge is charged. The reaction is conducted at a temperature of 100oC. Upon interaction of phosphorus sludge with sodium alkali from the reactor the phosphine-hydrogen mix leaves. The obtained product solution is directed to the next stage (2). The corresponding technological block is included in the CALS-project with all necessary characteristics of the used equipment. Filtering of a mineral part. The solution directed from reactor (1) to vessel (2) is filtered. A deposit remaining on the filter is a mineral part of phosphorus sludge and is used as a mineral fertilizer. The solution passed through filters (2) enters the next stage (3). Drawings of the filter, input and output parameters and other major characteristics are included in the CALS-project. Correction of sodium phosphite solution density. The obtained sodium phosphite solution is diluted with water to a necessary concentration. The ratio of components, temperature, characteristics of the equipment are included in the CALS-project. Neutralization of excess of alkali. After a stage (3) sodium phosphite enters a stage (4) where neutralization of excess of sodium alkali (NaOH) with phosphorous acid (H3PO3) solution is carried out.

Finally sodium phosphite goes to the packing stage. Types of packing and its characteristics are included in the CALS-project. The database of the CALS-project also contains the basic documents on sodium phosphite production: certificates, process regulations, characteristics of final products, performance characteristics of the used equipment, etc. Similarly to production of sodium phosphate, the typical structure of the CALS-project has been developed for production of sodium hypophosphite. The corresponding developed block diagram shown in Fig. 3 includes a preparatory stage and 9 basic production stages. The scheme includes the following blocks: 1. The Preparatory stage. Phosphorus sludge with content of phosphorus 30-50%, in a liquid state, heated to the temperature 700С, is pumped to storage tanks (receivers). For prevention of phosphorus sludge stratification in storage tanks the latter are fitted with agitators. The dosage of phosphorus sludge from the tanks is afforded by forcing with water. 2. Preparation of calcium hydroxide suspension (5) and sodium hydroxide (4) is conducted in two parallel tanks - mixers of suspension (40 м3 volume each) heated with external pipe coil to the temperature 500С. Sodium hydroxide with impeller pump is pumped from intermediate storehouse to receiver tank. When the required amount of sodium hydroxide is fed, the agitator is started and charging of calcium oxide hydrate (slaked lime) begins. The dosage of 12 tones (one batch) takes one hour. Then at constant stirring 12 м3 of water is added and this mix within 8 hours is agitated in the receiver tank (6). Preparation of suspension proceeds with intense reaction and foaming. After eight-hour stirring the mix is considered ready for application.

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3. Decomposition of phosphorus sludge in a reactor (1). The phosphorus sludge is loaded into reactor from position (3) along with the obtained solution from the mixer (6). After some time to reactor (1) the solution of isopropanol (7) is fed that is necessary for fuller extraction of phosphorus from phosphorus sludge. The reaction is conducted at a temperature of 85-90oC. Upon interaction of phosphorus sludge with sodium alkali from the reactor the phosphine-hydrogen mix leaves. The obtained product solution is directed to the next stage (2). 4. Further phosphorus sludge decomposition in an additional reactor (2): from the rector (1) the obtained mix is directed to rector (2) thus mixing with a mother solution after sodium hypophosphite centrifuging. After the end of reaction, the solution from an additional reactor (2) enters vacuum filters (11). 5. Filtering in drum-type vacuum filters. The obtained solution from a reactor (2) is filtered off in drum-type vacuum filters (11). A deposit formed at filtering is collected and used as fertilizer in the agrarian industry. The solution passed through vacuum filters (11) enters the neutralizer (12). 6. Neutralization of excess of sodium alkali. After passage the drum-type vacuum filters a solution consisting from NaH2PO2 (8 %), Na2HPO3 (9 %) and СaHPO3 (25 %) enters the neutralizer (12). Neutralization of excess of sodium alkali is afforded by dilution with hypophosphorous acid which is stored in vessel (15). 7. Preparation of a hypophosphorous acid (H3PO2). The obtained at stage 5 product solution (calcium hypophosphite) with concentration of 12% is mixed with oxalic acid. The obtained solution is filtered (14) and the formed hypophosphorous acid is stored in vessel (15) and is used as required in the neutralization stage (12). 8. Concentrating of sodium hypophosphite. Sodium hypophosphite is concentrated (16) by evaporation for the further fine filtration which is carried out in vessel (17). The formed sodium alkali and sodium hypophosphite are directed to recycling. 9. Crystallization of sodium hypophosphite (17) and centrifuging of suspension (18). After crystallization (17) suspension goes on filtration to a centrifuge. The formed mother solution goes to recycling through an additional reactor (2). After centrifuging (18) sodium hypophosphite is dried (19) for removal of excessive moisture from the final product. After the production cycle is finished, the product goes for packing (20). The operation mode and constructional characteristics for each stage are included in the corresponding sections of the CALS-project. The CALS-project also contains marketing analysis results. It is shown, that the considered products are in great demand. Sodium phosphite is one of the scarcest salts of phosphorus. It is widely used: in electroplating, as a reagent for synthesis of dibasic lead phosphite - the best stabilizer of PVC-compositions and also as a reducer in inorganic syntheses [4]. Sodium hypophosphite is used as a reducer at depositing nickel, cobalt and tin coatings on metals and plastic; as antioxidant preventing discoloration of alkyd resins upon their preparation etc. [5].

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Figure 3. An element of the CALS-project «Flow diagram of sodium hypophosphite production»).

RESULTS AND DISCUSSION As the considered processes have many related attributes, we were faced with a task of their association in uniform production unit for sodium phosphite and sodium hypophosphite. For optimum development of two-product production, the developed by our group theory of synthesis of flexible multiassortment chemical engineering systems (Fig. 4) is used [7, 8].

Workshop Joint Department Multi-assortment CTS Nomenclature p. h.p.А1 p.f.a. h.pА2 c.p. h.p.А3

Organizational flexibility of 2-nd step

IV level

Organizational flexibility of 1-st step

IIII level II level I level

Structural flexibility Technological flexibility

Figure 4. Hierarchical structure of synthesis of flexible chemical engineering systems.

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For similar multiassortment schemes four levels of system analysis are considered: nomenclature, productional-technological, organizational-technological and organizationalproductional. Attribute of the top (4-th) technological level is a separate shop as the complex cybernetic system. Associated problems: stabilization of material and information streams between aggregated sections; distribution of raw material, power and manpower resources. Attribute of the 3-rd - organizational level is the aggregated section. Associated problems: optimization of equipment arrangement and minimization of a production cycle. The bottom (1-st) level is the nomenclature level. Its characteristic attributes: a single product or one technological stage. The primary goals: expansion of a set of available grades for this one product or variation of available capacity of a technological stage. Functioning of the bottom (1-st) level is provided by technological flexibility which is determined by possibility of carrying out several technological tasks using the existing equipment due to flexible scheme adaptable for production of a given product (under the nomenclature) either with insignificant expenses for readjustment of the equipment (washing, fitting of pipelines etc). The greatest interest for us represents the second technological level. Its characteristic attribute – multiassortment technology. Associated problems: optimum use of intermediate products and the common initial reagents; using of elements of flexibility with the purpose of assortment expansion; variation of capacity of all technological process. This approach we applied to development of the block diagram of flexible two-product scheme of sodium hypophosphite and sodium phosphite synthesis. Developed production is the basic unit for a full complex of phosphorus sludge processing (Fig. 1). Incorporated into this complex individual production processes for dibasic lead phosphite and phosphorous acid employ sodium phosphite obtained using flexible scheme as raw material. To substantiate feasibility of uniting two processes in one flexible scheme it is necessary to carry out the analysis of existing individual production processes with the purpose of specifying groups of technologies which are suitable for organizing by a flexible principle. The first stage of this analysis is decomposition of the considered product assortment using hierarchical approach based on two basic attributes: technological and chemical similarity. Each of the pointed attributes has the gradation levels. So, technological similarity is subdivided into similarity of raw material preparation methods (dissolution, filtration, crushing etc.), production methods (type of transformation of raw material to a main product, uniformity of technological operations and the used equipment), and packing methods. Chemical similarity is determined, first of all, by belonging of substances to the same class (acid, base, salt, ether etc.) inside which class sublevels are isolated on the basis of physical and chemical properties of substances. For example, salts are classified according to the character of the anion (acid residue) - nitrates, sulphates, phosphates etc. The analyzed production processes for sodium hypophosphite and sodium phosphite meet both attributes of the theory of flexible chemical engineering systems as possessing technological and chemical similarity. This allowed us to carry out synthesis of the flexible two-product scheme (Fig. 5).

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Figure 5. The flexible scheme of sodium hypophosphite and sodium phosphite production.

The developed optimal scheme includes 23 blocks: 9 combined blocks containing operations used in both production processes for sodium phosphite and sodium

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hypophosphite (solid line); 3 blocks applied only to production of sodium phosphite (dot line); 11 blocks concerning only production of sodium hypophosphite (dashed line).  

Figure 6. An element of the CALS-project «Initial data for designing» (The flexible scheme of sodium hypophosphite and sodium phosphite of production).

For transferring from one product to another, in the scheme 2 flexible switching units are included: Flexible Unit of Switching-1 (FUS-1) and Flexible Unit of Switching-2 (FUS-2). The unit FUS-1 is responsible for switching of streams: either directing sodium alkali from the position (4) directly to reactor (1) in production of sodium phosphite, or to position (6) for mixing with lime hydrate in the production of sodium hypophosphite. After the reactor (1) in case of sodium phosphite synthesis in the unit FUS-2 switching to position (10) is suggested for filtering of the obtained reaction mixture, or switching to position (2) for phosphorus sludge further decomposition and mixing with mother liquor in case of sodium hypophosphite synthesis. Flexible switching units allow producing sodium hypophosphite and sodium phosphite at minimal controlling acts. The developed flexible scheme (Fig. 5) is included in the CALS-project (Fig. 6) with all operation characteristics, drawings of the used equipment etc. Each element of equipment included in CALS information system has one of three identification attributes: the unit used only for production of sodium phosphite; the unit used for production of sodium hypophosphite; unit which will be probably used for production of sodium hypophosphite and sodium phosphite. In the pilot CALS-project drawings of all apparatuses included into the block diagram (Fig.6) are given. If necessary it is possible to consider separately the drawing or an element of interest in a subsection №12 «Data for calculation, designing and industrial

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application». For example, in the subsection №12.01 (Reactor) there are drawings of a reactor, the maintenance instruction, certificates of conformity etc. Fig. 6. Development of the design documentation was carried out using the specialized software for the computerized designing «AutoCAD». For convenience of data storage and searching time reduction of, some big drawings and block diagrams have been transformed in PDF-files (Fig. 6). The same was used for storage of large text documents prepared in Word.

CONCLUSIONS The modern level of innovative production development is intimately connected with CALS-technologies, that is with use of uniform information space at all stages of the product life cycle - from designing and utilization to disposal (recovery). Introduction of information CALS-technologies for designing flexible production of sodium phosphite and sodium hypophosphite allows to obtain both salts not only with high characteristics, but also to provide full post-sale support including documentation in the electronic form.

REFERENCES [1]

[2] [3]

[4] [5] [6] [7] [8]

A.Bessarabov, L.Puigjaner, E.Koltsova E., T.Ogorodnikova: The system analysis of multiassortmental manufacturing of phosphorus-containing products based on CALStechnologies. 6th European Congress of Chemical Engineering, ECCE-6, Copenhagen, Bella Center, 1, 445-446, (2007). A.N.Davydov, V.V.Barabanov, E.V.Sudov: CALS Technology: Future Developments and Directions, Stand. Kach., 7, 12-18, (2002). A.M.Bessarabov, A.N.Ponomarenko, M.Ya.Ivanov: CALS Information Technologies (ISO-10 303 STEP) in Development of Plasmochemical Processes for Synthesis of Ultrapure Ultradispersed Oxides. Russian Journal of Applied Chemistry, 80(1), 13-18, (2007). A.J.Strugatskaya, E.M.Koltsova: Synthesis of sodium phosphite from phosphorus sludge at presence of oxygen. Jurnal Prikladnoi Khimii, 68(7), 1602-1604, (1995) E.M.Morgunova, T.D.Averbuch: Studying of process of synthesis of sodium hypophosphite. Jurnal Prikladnoi Khimii, 40(2), 274-284, (1967). A.M.Bessarabov, A.N.Afanas’ev: CALS Technology in Devising Promising Chemical Processes, Khim. Tekhnol., 3, 26-30, (2002). A.M.Bessarabov: Cybernetic Approaches in the Technology of Chemical Reagents and High-Purity Substances, Khim. Prom-st., 10, 666-671, (1996). A.M.Bessarabov, A.V.Avseev, E.M.Koltsova, L.Puigjaner: Modernization of multiassortment manufacturing of ultra pure materials, 4th European Congress of Chemical Engineering, ECCE-4, Granada, Spain, 10(9), 9-11, (2003).

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.165-189 ©2010 Nova Science Publishers, Inc.

Chapter 12

PRACTICAL HINTS ON THE APPLICATION OF NANOSILVERS IN ANTIBACTERIAL COATING OF TEXTILES S. Dadvara, A. Oroume a and A. K. Haghi*,b a

Department of Textile Engineering, Isfahan University of Technology, Isfahan, Iran b Department of Textile Engineering, Faculty of Engineering, University of Guilan, Rasht, Iran

ABSTRACT1 Nanotechnology is defined as the contour and manufacture of materials, devices and systems with control at nanometer dimensions and is considered as a concept in which everything in the world is considered from the viewpoint of atomic or molecular building blocks, is already influencing a very broad range of human technological activity. When a bulk material is reduced to small size particles with one or more dimension within the nanometer range, the material will show properties that are drastically different from those of the bulk material. Recently, nanosized organic and inorganic particles have drawn increased attention in medical textile applications due to their amenability to biological functionalization. The nanoscience and nanotechnology make excellent developments in textile science as well as others sciences. The development of new clothing products based on the immobilization of nanoparticles on textile fibers has recently received a growing interest from both academic and industrial sectors. These products impart unrivaled properties, especially antibacterial activity, to the treated fabrics. In the last few decades there has been growing interest in reducing the availability of commercial textile containing chemical antibacterial agents due to the environmental pollution, poisonous nature, and irritant property; so the new types of safe and commodious biocidal materials need to be replaced with these chemical agents. A wide range of nano-structures such as nanosilvers can be immobilized on fibers, which brings antibacterial properties to the final clothing product. This review introduces the application of nanosilvers in antibacterial coating of textiles in details up to 2008.

* Corresponding author. Tel.: +98 911 1318290. E-mail address: [email protected] (A. K. Haghi).

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Keywords: Nanosilvers, Synthesizing methods, Microorganism, Antibacterial coating, Textiles.

1. INTRODUCTION Norio Taniguchi was the first person who used the term nanotechnology in 1974. This term is defined as the contour and manufacture of materials, devices and systems at the molecular and atomic levels to attain unique properties which can be suitably manipulated for the desired applications. In fact, nanotechnology is considered as a concept in which everything in the world is already influencing a very broad range of human technological activity [1-3]. One nanometer being equivalent to the width of three or four atoms, and is equal to 10-9m or one millionth of a millimeter; in this range, groups of atoms bound covalently together and electrons display special behavior in which nanotechnology is verily aimed at harnessing this behavior. In the nanoscale, the nano-objects must be contained tens or hundreds of atoms to achieve the requisite size in so far as each nanoparticle should contain only about 3×107 atoms/molecules [3]. Thereupon, the internationally acclaimed range for research and study for the nanotechnology is between 0.1 nm and 100 nm [4]. Practically, when a bulk material is reduced to small size particles with one or more dimension within the nanometer range, the material will show properties that are drastically different from those of the bulk material [3, 5]. Nanomaterials can be obtained by two methods, namely ‘bottom-up’ and ‘top-down’ approaches. The bottom-up approach is referred to produce nanomaterials through assembling molecule by molecule and atom by atom but the top-down approach, basically belong to manufacturing nanomaterials through miniaturizing of bulk materials [3, 6]. Nanotechnology provides the ability to engineer the properties of materials by controlling their size, and this has driven research towards a multitude of potential uses for nanomaterials [2, 7, 8]. Presently, the nanosized organic and inorganic particles are also finding increasing attention particularly in textile applications due to their considerable properties in the nanoscale. A huge variety of different types of nanoparticles are already available, ranging from simple UV absorbers used in sunscreens to highly sophisticated and polyfunctional particles used to control drug delivery, and in solar panels to harvest sunlight and convert it into electric current [1, 3]. Nanosized heavy metals and metal oxides such as Ag, Cu, Zn, Hg, TiO2, Al2O3, ZnO and MgO possess unrivaled properties such as antibacterial activity, photo-catalytic ability, self-cleaning properties, electrical conductivity, UV-absorption/blockage properties, and photo-oxidizing capacity [7, 9-11]. Nowadays, one of the most interesting properties of these nanoparticles is antibacterial activity. Among the above nanoparticles, silver is the best natural inorganic metal which is capable to kill bacteria and fungi and generally can be appeared in the form of powder and colloidal solution. Silver nano-powders and colloidal nanosilvers are effective antibacterial agents due to their effective action adversely affecting the cellular metabolism and inhibiting cell growth. The chemistry has revealed that silver nanoparticles isn’t toxic to human cells in vivo and is reported to be biocompatible [4, 7, 8, 10, 12, 13]. With the advent of nanotechnology a wide range of nanoparticles can be coated on textiles, which imparts unrivaled properties to the treated textiles [9, 14-18]. The most developed nanotechnology application for textiles is currently in the area of coating

Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 167 particularly antibacterial coating. Antibacterial coatings are applied to textiles for four major reasons: to control the spread of disease and the danger of infection following injury, to control the infestation by microbes, to control the development of odor from perspiration, stains, and other soil on textile materials, and to control the deterioration of textiles, specially fabrics made from natural fibers, caused by mildew [7]. Recently, the new techniques for the modification of textile fibers using antibacterial nanosized silver particles were introduced by Dubas et al. [9, 18], Ki et al. [19], Yuranova et al. [12], Chen et al. [20], and Lee et al. [7, 11] which the fibers or fabrics coated using nanosilvers possessing antibacterial activity. In this review, the application of silver nanoparticles in antibacterial coating of textiles in recent years up to 2008 is aimed to be reported in a detailed way. The most popular synthesis procedures of silver nanoparticles, antibacterial properties of silver nanoparticles, silver nanoparticles-microorganisms interaction, and assessment of antibacterial activity are the most important issues which are discussed in this review.

Figure 1. Cubical silver nanoparticles which synthesized by reduction of silver nitrate in the presence of ethylene glycol [38]

2. KINDS OF SYNTHESIS PROCEDURES OF SILVER NANOPARTICLES Several synthesizing procedures were reported for the preparation of the silver nanoparticles in the literature and most of them were based on chemical reduction, Tollens process, UV light reduction, UV light and chemical reduction concurrently and biological process. Among these methods, chemical reduction methods were widely studied due to fact that it is a valid, economic and convenient method [1, 5, 10, 21-37]. For chemical reduction methods, the choice of the reducing agent is of course the major factor; γ-radiation, hydrazine, sodium boron hydride, sodium citrate, potassium bitartarate, dimethyl formamide, ascorbic acid, and alcohols are some of the reducing agents that have been successfully used

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[5, 37]. The reducing ability will determine the formation kinetics and hence reaction temperature. The reaction can be carried out in either aqueous solution or in organic solvent such as the polyol process. All these methods involve the reduction of relevant metal salts, usually silver nitrate or silver acetate, in the presence of a suitable protecting agent, which is necessary in controlling the growth of metal colloids through agglomeration [37]; long-chain n-alkanethiols are the most common protective agents employed to stabilize silver colloids, even though aromatic amines such as aniline, carboxylic acids, and polymers have been also employed. However, these methods can lead to significantly different results in terms of size and morphology depending not only on the choice of the reducing agent and stabilizer but also on the reaction conditions [5]. Preparation of silver nanocubes (Figure 1) with reduction of silver nitrate using ethylene glycol was reported to be noteworthy by Sun and Xia [38]. In this preparation, ethylene glycol serves both as reducing agent and solvent, whereas poly(vinylpyrrolidone) is used as a capping agent. They show that by controlling experimental conditions, such as temperature, metal salt concentration, metal/stabilizer ratio, and growth time, the size and morphology of the silver nanocrystals can be easily tuned, and large quantities of highly symmetric silver nanocubes of various dimensions can be obtained. In another study, a new procedure for the preparation of highly monodisperse myristate-capped silver nanoparticles has been reported. This method involves the suspension of silver myristate in triethylamine followed by gentle heating at 80◦C for 2h, which gradually produces a solution of uniformly spherical silver nanoparticles that can be precipitated by addition of acetone. Thus, the nanoparticles can be isolated as solid materials and re-dispersed in nonpolar solvents where they are stable for up to 1 week. The particle size and size distribution are affected by the alkyl chain length of the carboxylate ligand and the tertiary amine. For example, the particles with silver stearate are highly monodispersed with sizes between 1.9-3.5nm, whereas the particle distribution with silver octanoate is broad and the sizes are larger (between 5.5 and 31.5nm). With octylamine in the place of triethylamine, the particle size decreases, but the size distribution is also broader. This procedure is of great promise for scale-up because of the ease of preparation, mild conditions, and use of relatively nontoxic reagents [5]. Shrivastava et al. [1] introduced a novel procedure for the preparation of silver nano particles. In this method, a solution of 0.01 M Ag+ was prepared by dissolving 0.017 g AgNO3 in 100 ml of de-ionized water. During the process additives like ammonia (30%) were added drop-wise, so that silver ions formed a stable soluble complex. The solution obtained was used as the precursor for the silver nanoparticles. A blend of reducing agents like D-glucose and hydrazine was used during the synthesis of the nanoparticles. Blending was essential to control the rate of reduction resulting in an achieved optimum rate. A higher reducing rate was shown to form clusters of silver nanoparticles with reduced stability. About 110 ml of such a blend of reducing agents (at a concentration of 0.01 M) was incorporated into 100 ml of silver nitrate stock solution (0.01 M) with continuous stirring. This ensured complete reduction of the silver ions to form silver nanoparticles at a concentration of 0.005 M in aqueous media. The pH of the nanoparticles formed by that way was maintained at 7.4 with citric acid (1 M). The brown solutions of Ag nanoparticles were stored in closed glass vials under ambient conditions for future experiments. Sarkar et al. [26] demonstrated a facile and faster method of preparation of silver nanocrystals. In this method, silver nitrate solution (0.02 M) was prepared by dissolving the required amount of AgNO3 in (1:1) ammonia. Similarly, the aldehyde solution and the surfactant solution having equal concentration to that of the silver nitrate solution

Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 169 were prepared in dehydrated ethanol and water, respectively. Then 1 ml silver nitrate solution was taken in a well-cleaned dry beaker; 1 ml SDS solution was added to it and it was mixed well for a few minutes (5 min for each set) by continuous stirring. 1 ml aldehyde solution was finally added to this mixture. A light yellow color appeared at room temperature (30◦C). The solution was heated on a water-bath and the temperature was recorded. When the temperature of the solution reached 80◦C, the light yellow color of the solution started turning into deep yellow. Then the color gradually changed through yellowish brown (82◦C) to reddish brown (84◦C) and finally it turned into brownish black (86◦C) (Figure 2). On further increasing the temperature of the solution, no perceptible change in color was observed. It is interesting to note that the range of reduction temperature is nearly identical to that reported by Chu et al. for the case of Au. The gradual change of color in this temperature range indicated the formation of Ag nano particles of different dimensions. Thus, the study was taken one step further by preparing five different set solutions at the above-mentioned temperatures (i.e. at 30, 80, 82, 84 and 86◦C).

Figure 2. The temperature-dependent color change during the progression of the reaction [26]

In other study, Yu [27] stated that the AgNO3 was used as precursor in the preparation of the silver nanoparticles. Na+-poly(γ-glutamic acid) (PGA) served as a linear homopolypeptide made of only glutamic acid residuals (both D-form and L-form) in γ-peptide bond linkages with the degree of polymerization ranging from 1000 to 12000. The reaction scheme was similar to the silver mirror reaction. Briefly, 0.001 M AgNO3, 0.01 M NaOH, and 0.02 M NH4OH mixed thoroughly together for 1h. Then, the 1ml of silver alkali solution was added into 10 ml of 0.5-2wt% PGA solution and subsequently added 1 ml of 10 wt% of dextrose. The reaction temperature was maintained at 60◦C by a water bath under dark conditions. The resulted samples were defined as PGA/Ag0, depending on the used concentration of PGA. For example, 2PGA/Ag0 means that silver nanoparticle stabilized by 2 wt% PGA under chemical reduction by reducing agent-dextrose in comparison with that of 2PGA/Ag+ in the absence of dextrose. In an innovative procedure, green synthesis of silver nanoparticles was reported by Chen [31]. Microwave is employed to synthesize silver nanoparticles and a green reagent, carboxymethyl cellulose sodium (CMS) is also involved. CMS is a kind of biomaterial, widely applied in many fields, such as food, medicament, daily chemistry, petroleum, paper making, textile and architecture, due to its innocuity and great performance on thickening, dispersing, emulsifying and steadying. In this method, CMS can work as both a reducing and

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a stabilizing reagent in the synthesis of silver nanoparticles. Furthermore, no other agent is needed in the reaction except AgNO3. In a typical reaction, 10 ml of 0.01M AgNO3 aqueous solution was mixed with certain volume of 0.1% CMS aqueous solution under stirring. The mixed solution was adjusted to 200 ml by adding different volumes of water. Then it was conveyed into a round flask, and the flask was fixed into the microwave reactor. After turning on the electromagnetic stirrer and microwave reactor, silver nanoparticles were then obtained gradually. Another method that is of great potential for the synthesis of silver nanocrystals was described by Sondi et al. [39] which talks about obtaining smaller particles with higher specific surface area and narrower size distribution. In a typical experiment, the silver hydrosols were prepared by adding, under agitation, 10 cm3 of an aqueous 1 mol dm−3 ascorbic acid solution at a flow rate of 3 cm3 min−1 into 90 cm3 of an aqueous solution containing 5 wt% of Daxad 19 and 0.33 mol dm−3 AgNO3. The reacting solutions were agitated with a stirrer at 900 rpm at room temperature. In order to remove the surfactant and excess silver ions, the resulting silver precipitate was washed five times with de-ionized water. Finally, the nanosize silver was obtained as a dried powder by freeze drying and kept for future experiments. The obtained powder was fully re-dispersed in de-ionized water by sonication and therefore aqueous dispersions of silver nanoparticles at the desired concentrations were easily made. Zhang et al. [34] prepared colloidal silver nanoparticles in water-in-oil micro-emulsion. Silver nitrate was used as the starting material for the silver nanoparticles and hydrazine hydrate (50.0%) was used as the reducing agent. The micro-emulsion system used in the study consisted of dodecane as the continuous oil phase, sodium bis(2-ethylhexyl) sulphosuccinate (AOT) as the surfactant, an aqueous solution as the dispersed phase, without addition of any co-surfactant. In a typical procedure, the micro-emulsions were prepared by mixing the same volume of aqueous solution of silver nitrate (0.2 M) and hydrazine hydrate (0.6 M) to the 0.2 M AOT/dodecane solution. Similar method has been reported to synthesize the colloidal silver nanoparticles in AOT micro-emulsion. However, the surfactant which had been used as the emulsifier was a mixture of Ag (AOT) and AOT. The used solvents are also short chain hydrocarbons, e.g., isooctane and cyclohexane. The overall molar concentrations of silver nitrate and hydrazine hydrate were 4×10-4 M and 1.2×10-3 M, respectively. The molar ratio of hydrazine hydrate and silver nitrate was held constant for all experiments at a value of 3. The water-to-AOT molar ratio, W, was kept the same in all cases and was equal to 7.5. The micro-emulsion containing hydrazine hydrate was added into another microemulsion containing silver nitrate drop by drop. After all the hydrazine hydrate microemulsion was added, the vigorous magnetic stirring was maintained for 2 h, the resulting micro-emulsion mixtures changed to a stable light yellow color after the reaction, indicating the formation of Ag nanoparticles.

3. CLASSIFICATION OF MICROORGANISMS Microbes are the tiniest creatures not seen by the naked eye. They include a variety of microorganisms like Bacteria, Fungi, Algae and viruses. Bacteria are unicellular organisms which grow very rapidly under warmth and moisture. Furthermore, bacteria family can be

Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 171 classified into two categories, namely, gram-positive (Staphylococcus aurous) and gramnegative (Escherichia coli). Some specific types of bacteria are pathogenic and cause crossinfection. Fungi, molds or mildew are complex organisms with slow growth rate. They stain the fabric and decrease the performance properties of the fabrics. Fungi are active at a pH level of 6.5. Algae are typical microorganisms which are either fungal or bacterial. Algae require continuous sources of water and sun light to grow and develop darker stains on the fabrics. Algae are active in the PH range of 7-8. Dust mites are eight legged creatures and they occupy the household textiles such as blankets bed linen, pillows, mattresses and carpets. The dust mites feed on human skin cells and liberated waste products can cause allergic reactions and respiratory disorders. Some harmful species of the bacteria and fungi are listed in Table 1 [40]. Table 1. Some harmful species of microorganisms [7, 40] Bacteria gram-positive bacteria Staphylococcus aureus or pyogens Staphylococcus epidermidis Streptococcus group A

gram-negative bacteria Escherichia coli Klebsiella pneumoniae Proteus vulgaris Salmonella typhi Salmonella-Shigella Pseudomonas aeruginosa

Fungi Cloth damaging fungi Aspergillus niger

Crop damaging fungi Fusarium species

Trichoderma viride Curvularia lunota

Rhizoctonia solani Sclerotium rolfsii

3.1. Inhibition of Microorganisms Using Chemical Antibacterial Agents and Silver Nanoparticles Negative effect on the vitality of the microorganisms is generally referred as antibacterial. The activity which affects the bacteria is known as antibacterial and that of fungi is antimycotic [40]. Oxidizing agents such as aldehydes, halogens and proxy compounds attack the cell membrane, get into the cytoplasm and affect the enzymes of the microorganisms. Radical formers like halogens, isothiazones and peroxy compounds are highly reactive due to the presence of free electrons. These compounds virtually react with all organic structures in particular oxidizing thiols in amino acids. Even at the lowest level of concentrations, these substances pose particular risk to nucleic acids by triggering mutations and dimerization. Quaternary ammonium compounds exert their influence external to the microorganisms by disruption of the delicate cell membranes and therefore don’t need to be absorbed in solution to produce their bacterial killing. One of the most durable type of antibacterial products is based on a diphenyl ether (bis-phenyl) derivative known as either 2, 4, 4'-trichloro-2' hydroxy diphenyl ether or 5-chloro-2-(2, 4-dichloro phenoxyl) phenol. Triclosan products have been used for more than 25 years in hospitals and personal care products such as antibacterial soap, toothpaste and deodorants. Triclosan inhibits growth of microorganisms by using an electro chemical mode of action to penetrate and disrupt their cell walls. When the cell walls are penetrated, leakage of metabolites occurs and other cell functions are disabled, thereby preventing the organism from functioning or reproducing. The Triclosan when incorporated within a polymer migrates to the surface, where it is bound.

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Because, it isn’t water-soluble, it does not leach out, and it continuously inhibits the growth of bacteria in contact with the surface using barrier or blocking action. Quaternary ammonium compounds, biguanides, amines and glucoprotamine show poly cationic, porous and absorbent properties. Materials treated with these substances bind microorganisms to their cell membrane and disrupt the lipopolysaccharide (LPS) structure resulting in the breakdown of the cell. Complexing metallic compounds based on metals like cadmium, silver, copper and mercury cause inhibition of the active enzyme centers (inhibition of metabolism) [40-42]. In other words, silver ions function in adversely affecting cellular metabolism to inhibit bacterial cell growth. Since the silver ions are absorbed into bacterial cells, silver ions stop respiration, basal metabolism of the electron transfer system and transport of substrate in the microbial cell membrane; also, silver ions inhibit the bacterial growth by producing active oxygen on the surface of silver powder. The mechanism of antibacterial action of silver ions is closely related to their interaction with proteins, particularly at thiol groups (–SH). It was considered that silver ions attack to the protein molecules and bind protein molecules together by forming bridges along them. Through this way the cellular metabolism of enzymes is inhibited and the microorganism dies; i.e. when the metallic silver is in contact with an oxygen metabolic enzyme of a microorganism, it becomes ionized. As a result, DNA molecules become condensed and lose their ability to replicate upon the infiltration of Ag ions. The silver ions also interact with the thiol groups of proteins, which induces the inactivation of bacterial proteins. As shown in the below reaction (Figure 3), the silver ion interacts with the thiol groups (–SH) of the enzyme in the microorganism and forms an -SAg linkage with the enzyme, which effectively blocks the enzyme activity [4, 19, 39, 43, 44].

SH Enzyme SH

SAg + 2Ag+

Enzyme

+ 2H+

SAg

Figure 3. Interaction of silver ions with the enzyme in the microorganism [4]

As already mentioned, the total surface area of the nanosized silver particles is larger as compared with the bulk silver particles in the same volume, so the antibacterial ability of the first state is more effective than the latter [19]. It was speculated that the behavior of silver nanoparticles is widely similar to that of silver ion. The mechanism of antibacterial actions of silver nanoparticles is still not well understood. In a previous report on the bactericidal activity of silver nanoparticles, it was shown that the interaction between silver nanoparticles and constituents of the bacterial membrane caused structural alterations in and damage to membranes, finally leading to cell death. It has been suggested that disruption of membrane morphology may cause a significant increase in permeability, leading to uncontrolled transport through the plasma membrane and, finally, cell death. The differences between gram-positive and gram-negative bacteria essentially rest in the structure of their respective cell walls. The gram-negative bacteria have a layer of LPS composed of covalently linked lipids and polysaccharides with the poor strength and rigidity at the exterior, followed

Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 173 underneath by a thin layer of peptidoglycan with the thickness of about 7-8 nm. Negative charges on the LPSs are attracted towards weak positive charges available on silver nanoparticles. In contrast, the cell wall in gram-positive bacteria is principally composed of a thick layer of peptidoglycan with the thickness of about 20-80 nm, consisting of linear polysaccharide chains cross-linked by short peptides to form a 3D rigid structure. The rigidity and extended cross-linking not only endow the cell walls with fewer anchoring sites for the silver nanoparticles but also make them difficult to penetrate. The extent of inhibition of bacterial growth was dependent on the concentration of nanoparticles in the medium. Interaction between nanoparticles and the cell wall of bacteria would be facilitated by the relative abundance of negative charges on the gram-negative bacteria, which was congenial to the fact that growth of gram-negative bacteria was more vigorously affected by the silver nanoparticles than that of the gram-positive microorganisms. Silver as a soft acid has a greater tendency to react with sulphur- or phosphorus- containing soft bases, such as R-S-R, R-SH, RS-, or PR3. Thus, sulfur-containing proteins in the membrane or inside the cells and phosphorus-containing elements like DNA are likely to be the preferential sites for silver nanoparticle binding. As demonstrated by electron microscopy, interaction with nanoparticles resulted in pits in the cell wall and in the protein de-naturation, contributing to the antibacterial effects of the nanoparticles.

Figure 4. Escherichia coli treated with silver nanoplates in which nanosized silver particles appear as dark irregular pits on the cell surface [43]

Figure 4 shows the image of a part of a vigorously damaged cell membrane treated with silver nanoparticles. Results of the re-culture experiments were consistent with the entry of nanoparticles inside bacterial cells and strong agglomeration with bacterial cellular components. Once inside the cell, nanoparticles would interfere with the bacterial growth signaling pathway by moderating tyrosine phosphorylation of putative peptide substrates critical for cell viability and division [1, 39, 43].

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Figure 5. Transmission electron micrograph of Escherichia coli cell treated with 50μg cm−3 of silver nanoparticles in liquid Luria–Bertani (LB) medium for 1h (a) and close up view of the membrane of this cell (b) [39]

The TEM analysis (Figure 5) and the existence of elementary silver in the membranes of treated bacteria, detected by energy dispersive analysis X-ray (EDAX) (Figure 6), confirm the incorporation of silver nanoparticles into the membrane structure [39].

Figure 6. EDAX spectra of native Escherichia coli (a) and Escherichia coli treated with 50 μg cm−3 of silver nanoparticles in liquid Luria–Bertani (LB) medium for 4 h (b) [39]

A similar effect was described by some workers [39] when Escherichia Coli bacteria were treated with highly reactive metal oxide nanoparticles. A bacterial membrane with this morphology discloses a significant increase in permeability, leaving the bacterial cells incapable of properly regulating transport through the plasma membrane and, finally, causing cell death. It is well known that the outer membrane of Escherichia Coli cells is predominantly constructed from tightly packed LPS molecules, which provide an effective

Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 175 permeability barrier. Recently, in other study workers have shown that metal depletion may cause the formation of irregular-shaped pits in the outer membrane and changed membrane permeability, which is caused by progressive release of LPS molecules and membrane proteins (Figure 7). It can be speculated that a similar mechanism causes the degradation of the membrane structure of Escherichia Coli during treatment with silver nanoparticles. Extensive investigations directed to better understanding of interaction between silver nanoparticles and bacterial components should shed light on the mode of action of this nanomaterial as a biocidal material [39].

Figure 7. Scanning electron micrographs of native Escherichia coli cells (a) and cells treated with 50 μg cm−3 of silver nanoparticles in liquid Luria–Bertani (LB) medium for 4 h (b) [39]

4. ANTIBACTERIAL COATING Textiles, especially those made of natural fibers, are an excellent medium for the growth of microorganisms when the basic requirements such as nutrients, moisture, oxygen and appropriate temperature are present. The large surface area and ability to retain moisture of textiles also assist the growth of microorganisms on the fabric. Therefore, there is a great demand for antimicrobial coatings of textiles to control the growth of microorganisms, such as bacteria, fungi or mildew, and prevent the textile from deterioration of strength and quality, staining, odors, and health concerns caused by microorganisms [45]. An awareness of general sanitation, contact disease transmission, and personal protection has led to the development of antibacterial fibers to protect wearers against the spread of bacteria and diseases rather than to protect the quality and durability of the textile material [46]. From the standpoints of a propensity toward tidiness as a social manner and a demand for advanced medical technology, antibacterial goods have recently received a growing interest. Figure 8 statistically shows the development of several antibacterial textiles, both treated antibacterial fabrics and fabrics with antibacterial fibers, in the west Europe until 2000 [47, 48]. Textiles have a wide use in healthcare and medical fields, and most of them currently used in clinics and hospitals, such as laboratory coats, medical cloths, wound dressings, sanitary products, medical disposables, and women hygiene products, are conducive to cross-infection or transmission of diseases caused by microorganisms. The inherent properties of the textile fibers provide room for the growth of microorganisms; besides, the structure of the substrates and the chemical processes may induce the growth of microbes [4, 18, 40, 44, 49].

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Figure 8. Statistically development of antibacterial textiles, both treated antibacterial fabrics (a) and fabrics with antibacterial fibers (b), in the west Europe until 2000 [50]

The need for the chemical antibacterial coatings as mentioned previously can be contributed to four items: to control the spread of disease and the danger of infection following injury, to control the infestation by microbes, to control the development of odor from perspiration, stains, and other soil on textile materials, and to control the deterioration of textiles, specially fabrics made from natural fibers, caused by mildew [7, 11]. In order to carry out this particular coating on textiles and garments, this point should be attended that all types of textiles such as shirts, hosiery, blouses, diapers, and underwear are more susceptible to wear and tear. It is important to take into account the impact of stress strain, thermal and mechanical effects on the coated materials; so, some requirements such as durability to washing, dry cleaning and hot pressing, selective activity to undesirable microorganisms, no harmful effects to the manufacturer, user and the environment, compatibility with the chemical processes, easy method of application, no deterioration of fabric quality, resistant to body fluids and resistant to disinfections, compatibility with the statutory requirements of regulating agencies, need to be satisfied to obtain maximum benefits out of the coating [40, 44]. Chemical antibacterial coating of textiles first appeared in 1941 [46]. The primary objective of the chemical antibacterial coating was to protect textiles from being affected by microorganisms. Basically, the application of the chemical antibacterial coating is extended to textiles used for outdoor, healthcare sector and sports. The greater use of synthetic fibers and blends in such items as shirts, hosiery, blouses, and underwear has accelerated the need for bacteriostatic coatings on clothing. The moisture-transport characteristics of such blends tend to cause a greater degree of ‘perspiration wetness’ than occurs with fibers of wholly natural

Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 177 fibers. The chemical antibacterial substances function in different ways. In the conventional leaching type of coating, the species diffuse and poison the microbes to kill. This type of coating shows poor durability and may cause health problems. The non-leaching type of coating shows good durability and may not provoke any health problems. A large number of textiles with chemical antibacterial coating function by diffusion type. The rate of diffusion has a direct effect on the effectiveness of the coating. For example, in the ion exchange process, the release of the active substances is at a slower rate compared to direct diffusion and hence, has a weaker effect. Similarly, in the case of antibacterial modifications, the active substances aren’t released from the fiber surface; so this type of antibacterial coating is less effective. These substances are active only when they come in contact with microorganisms. These so called new technologies have been developed by considering the medical, toxicological and ecological principles. The antibacterial textiles can be classified into two categories, namely, passive and active based on their activity against microorganisms. Passive materials don’t contain any active substances but their surface structure produces negative effect on the living conditions of microorganisms (Lotus effect or Anti-adhesive effect). Materials containing active antibacterial substances act upon either in or on the cell. Many chemical antibacterial agents used in the textile industry are incorporated with textile substrates comparatively at lower concentrations. It must be ensured that these substances aren’t only permanently effective but also that they are compatible with skin and the environment. A wide palette of antibacterial compounds is now in use but differs in their mode of action. Materials with active coatings contain specific active antibacterial substances, which act upon microorganisms either on the cell, during the metabolism or within the core substance (genome). However, due to the very specific nature of their effect, it is important to make a clear distinction between chemical antibacterial agents and other active substances which have a broad range of uses [40]. Hence, the antibacterial properties of such textile materials can be grouped into two categories, temporarily or durably functional fabrics. Temporary biocidal properties of fabrics are easy to achieve in coating, but easy to lose in laundering. Durability has generally been accomplished by a common technology, a slowreleasing method. According to this method, sufficient antibacterial agents are incorporated into fibers or fabrics by means of a wet coating process. The treated fabrics deactivate bacteria by slowly releasing the biocide from the materials. However, the antibacterial agents will vanish completely if they are impregnated in materials without covalent bond linkages [49]. Traditionally, textile materials have been imparted antibacterial properties by chemically or physically incorporating chemical agents onto fabrics through the coating processes [41, 42, 45, 49]. After World War II fungicides used on cotton fabrics were compounds such as 8hydroxygiunoline salts, copper naphthenate, copper ammonium fluoride and chlorinated phenals [40]. Generally, the antibacterial agents can be applied to the textile substrates by exhaust, pad-dry-cure, coating, spray and foam techniques. The substances can also be applied by directly adding into the fiber spinning dope. A number of methods for incorporating antibacterial functions into textile materials have been developed elsewhere; these methods such as insolubilisation of the active substances in/on the fiber, treating the fiber with resin, condensates or cross linking agents ( e.g. the attachment of chitosan to cotton fabric via cross linking agents [46, 51, 52]), micro encapsulation of the antibacterial agents with the fiber matrix, use of graft polymers, homo polymers and/or copolymerization on to the fiber (e.g. graft polymerization of N-halamide monomers onto cellulosic substrates [46]),

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and chemical modification of the fiber by covalent bond formation (e.g. placement of quaternary ammonium salts onto cotton fabrics using a covalently bound adduct or covalent attachment of a chloromelamine derivatives [46, 53]) have been developed for improving the durability of the chemical antibacterial coating process [40, 42]. Antibacterial textile materials based on helamine chemistry showed biocidal properties against a wide range of pathogens, and are also non-toxic and environmentally friendly. For example, in a typical approach, antibacterial cellulosic fabrics were developed by means of the use of 1,2,3,4butanetetracarboxylic acid and citric acid, together with subsequent oxygen bleaching. Carboxylic acids have been converted to peroxy acids by being reacted with hydrogen peroxide under acidic conditions, while carboxylic acid groups can be incorporated into cellulose fabrics. In another procedure, a dimethylol hydantoin derivative, dimethylol-5,5dimethylhydanation, was used in chemical treatment of cellulose, and subsequent chlorine bleaching can convert unreacted amide or imide bonds in the hydantoin [42, 49]. Generally, chemical antibacterial agents used to control the growth of microorganisms on textile fabrics and several bacteriostatic textiles coatings exist for personal wears, but poor activity against microorganisms, lack of wash durability, environmental pollution, and inadequate safety data to meet current requirements or a combination of these factors has limited their use [7] and makes them not suitable for applications in health foods, filters, and textiles [44]. A common problem in disinfection or bacteriostasis is the selection of an agent that kills all organisms in the shortest time without damaging the contaminated materials. Antibacterial agents should always be diluted exactly as specified by the manufacturer. Solutions that are too weak may be ineffective, and those that are too strong can be dangerous for human body [7]. Consequently, a safe, wash-resistant textile coating capable of inhibiting the growth of both bacteria and fungi is required. The wave of nanotechnology has shown a huge potential in the textile and clothing industry and has provided a new area for futuristic research in science and technology. Using nanotechnology to improve existing material performances and developing unrivaled functions on textile materials are flourishing. As discussed, commercial textiles containing chemical antibacterial agents are sorely poison and irritant for human body; hence the new types of safe and commodious biocidal materials need to be replaced with these chemical agents [39]. By this reason, the new antibacterial coating by taking advantages of nanotechnology have been developed. The development of new clothing products based on the immobilization of nanoparticles on textile fibers has recently received a growing interest from both the academic and industrial sectors. Nowadays, a wide range of nanoparticles and nano-structures can be immobilized on fibers, which brings new properties to the final clothing product. Numerous patents were developed for the surface modification of fibers with nanoparticles, including blending of the nanoparticles in the polymer matrix before spinning or chemical grafting of the desired functional groups onto the fibers; for example, Japanese researchers revealed an antibacterial cloth used for washing breasts of milk cow. Chinese researchers showed a new method for making antibacterial fabric with long lasting broad-spectrum antibacterial effect against more than 40 bacteria. The fabric is manufactured by dissolving silver nitrate in water, adding ammonia water into the solution to form silverammonia complex ion, adding glucose to form a nanosized silver particles as a treating agent, adding fabric into the treating agent, and ironing the fabric by electric iron or heat-rolling machine [4, 11].

Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 179 Kin et al. [19] imparted antibacterial properties to the wool fabrics using sulfur nanosilver ethanol based colloid supplied from NP-Tech Co., Ltd., Korea with the particle size of average 4.2 nm. The antibacterial wool textiles were prepared by a general padding method with the diluted sulphur nano-silver colloidal solution. Before the coating process, untreated wool fibers were cleaned with dichloromethane (40◦C, 30 min), rinsed with ethanol and water twice (25◦C, 10 min), and equilibrated in a conditioned room (20◦C, 60% R.H.). The fibers were treated with nano-silver colloid by a conventional pad-dry-cure method. Practically, the wool fabrics were immersed in a fresh colloidal bath for 10 min and squeezed using a laboratory padder at the constant pressure. The samples were dried at room temperature for prevention of thermo-migration of metal particles for 30 min, and then the curing process of samples was performed at 120◦C, for 5 min. Figure 9 discloses the scanning electron microscopy of wool fiber treated using 100 ppm of sulphur nano-silver colloidal solution. They concluded that the antibacterial efficacy on wool fibers was easily achieved by the conventional pad-dry-cure method using the nanosized silver colloid including sulfur compound insofar as the manufactured wool textiles using the treated fibers with the silver particle were showed as excellent antibacterial products.

Figure 9. Scanning electron microscopy photograph of wool fiber treated using sulphur nano-silver colloid; (a) 5000 magnifications (b) 30000 magnifications [19]

Dubas et al. [9] disclosed the new procedure in which antimicrobial silver nanoparticles were immobilized on nylon or silk fibers by following the layer-by-layer deposition method. In this method the sequential dipping of nylon or silk fibers in dilute solutions of poly(diallyldimethylammonium chloride) and silver nanoparticles capped with poly(methacrylic acid) led to the formation of a colored thin film possessing antimicrobial properties (Figure 10). Silver nanoparticles were prepared through the photoreduction of silver nitrate under UV light in a dilute solution of poly(methacrylic acid). As a result, upon exposure of a silver nitrate/poly(methacrylic acid) mixture to UV light, the solution quickly turned pink and finally red after several hours. Practically, nylon or silk fibers were wrapped around a rectangular aluminum holder, 2.5×3.5 cm2 and spun in various solutions using a small dc motor. A home-built robotic platform, accommodating eight 100 ml beakers, was programmed to successively expose the fibers to either polyelectrolyte or silver nanoparticle solutions followed by three 1 min water rinses and repeated as many times as needed. At the end of the deposition process, the samples were allowed to dry overnight

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and then wrapped on a plastic holder before measurements with the spectrophotometer. In a typical experiment setup, the polyelectrolyte multilayers (PEM) were composed of 20 layers by alternatively dipping the fibers in 1 mM poly(diallyldimethylammonium chloride) and silver nanoparticles solution for 2 min. The sequential dipping of the fibers in poly(diallyldimethylammonium chloride) and poly(methacrylic acid) capped silver nanoparticles solutions led to the appearance of a red color onto the fiber. The color is due to the immobilization of the silver nanoparticles onto the fibers. Also, this point should be attended that the pH of all solution was set to a value of 7. Finally, they concluded that the fibers coated with the nanoparticles exhibit antimicrobial activity, which could render them useful in applications such as water sanitation or antimicrobial fabrics.

Figure 10. Scanning electron microscopy of nylon (left picture) and silk (middle and right pictures) fibers coated with poly(diallyldimethylammonium chloride) and silver nanoparticles capped with poly(methacrylic acid) [9]

Potiyaraj et al. [18] synthesized the silver chloride nanocrystals on silk fiber. The growth of the nanocrystal was achieved by sequential dipping of the silk fibers in alternating solution of either silver nitrate or sodium chloride followed by a rinse step. The resulting fiber coated with nano-AgCl crystals could be used as a photo-catalyst in water splitting applications or as an antibacterial agent. Figure 11 obviously shows all steps describing the preparation of the silver chloride nanocrystal by sequential dipping in silver nitrate and sodium chloride. Practically, the silk fibers similarly mentioned in [9] were wrapped around a rectangular aluminum holder, 2.5×3.5 cm2 and spun in various solutions using a small DC motor. A home-built robotic platform, accommodating eight 100 ml beakers, was programmed to successively expose the silk fibers to either silver nitrate or sodium chloride solutions followed by three water rinses of 1 min each. The number of dipping steps was fixed to 20 meaning that the fiber was dipped 10 times in silver nitrate and 10 times in sodium chloride solutions. At the end of the deposition process the samples were allowed to dry overnight. In order to confirm the formation of nanocrystal at the surface of the silk fiber, scanning electron microscopy image of the surface of the silk fiber coated using AgCl nanoparticles has been taken (Figure 12). Finally, they concluded that the resulting AgCl crystals generally could be used as an antibacterial agent or if assembled on conducting fibers, could be used in water splitting application.

Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 181

Figure 11. Steps describing the preparation of the silver chloride nanocrystal by sequential dipping in silver nitrate and sodium chloride [18]

Figure 12. Scanning electron microscopy image of the AgCl nanoparticles formed at the surface of the silk fiber. The AgCl nanoparticles were synthesized by 20 alternate dipping in AgNO3 and NaCl solutions followed by a rinse in water [18]

Lee et al. [7] manifested the method in which the polyester nonwovens were incorporated with colloidal silver nanoparticles supplied by NP-Tech Co., Ltd., Korea. In this method typically the polyester nonwovens were immersed in a colloidal silver nanoparticles bath for 1 minute and squeezed to 100% wet pick-up with a laboratory pad at a constant pressure. Subsequently the treated polyester nonwovens were dried at 120◦C for 5 minutes. Figure 13 discloses the antibacterial coating process of the textile fabrics. They varied the mean size of silver nanoparticles to improve the antibacterial effect of nonwovens and demonstrated that the growth of bacterial colonies was absolutely inhibited using low concentration of colloidal silver nanoparticles with the mean size smaller than 5 nm. In other words, the smaller particle sizes had better antibacterial effects on silver-padded nonwoven fabrics. They also stated that higher concentration of silver colloids have better bacteriostasis because bacterial reductions decreased when the silver concentrations in the pad bath decreased.

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Figure 13. Schematic of antibacterial coating process for polyester nonwovens [7]

In other study, Lee et al. [11] produced antibacterial woven cotton and polyester fabrics using the same colloidal silver nanoparticles supplied by NP-Tech Co., Ltd., Korea. Figure 14 shows clearly shows the well dispersed of silver nanoparticles on fiber surfaces in each fabric. In this procedure, woven cotton and polyester fabrics were padded through a certain concentration of silver colloids and squeezed to 83% wet pick-up with a laboratory pad at a constant pressure. They demonstrated that antibacterial efficacy and good laundering durability on textile fabrics can be easily achieved with using nanosized silver colloidal solution through padding process.

(a)

(b)

Figure 14. Scanning electron microscopy (SEM) images of silver nanoparticles on (a) cotton and (b) polyester fibers [11]

Jeong et al. [44] prepared PE/PP nonwovens using various kinds of nanosized silver colloids. They employed three different types of nano-sized silver colloids supplied by NPTech Co., Ltd., Korea: nano-sized silver particles dispersed in water (NSW), nano-sized silver particles dispersed in ethanol (NSE), and nano-sized silver/sulfur composite particles dispersed in ethanol (SNSE) to prevent aggregation of the particles; Figure 15 presents TEM images of the nano-sized spherical silver colloid. The mean diameter of silver nanoparticles in the water-based colloid was 8.11 nm, and that of the ethanol-based

Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 183 colloids was 3 nm. In this method, PE/PP nonwovens were padded using colloid solutions at the pressure of 3 kgf/cm2 by an auto fade mangle. Subsequently, the samples were immediately dried at 120◦C for 3 min. As a result, they observed that the nonwovens treated using NSW (the water-based silver nanoparticles) had the deepest color at the same concentration but the color of the colloid interestingly was almost invisible in the nonwovens treated using SNSE (the ethanol-based nano-sized silver/sulfur composite particles); according to this observation nonwovens treated using SNSE may be a good antibacterial coating agent that will not influence the intrinsic color of the fabric. Furthermore, the SNSE coated fabrics also exhibited the highest antibacterial efficacy.

(a)

(b)

Figure 15. TEM images of silver nanoparticle (×200 K): (a) nano-sized silver particles dispersed in water (NSW), (b) nano-sized silver particles dispersed in ethanol (NSE) [44]

5. ASSESMENT OF ANTIBACTERIAL ACTIVITY ‘Quantitative tests’ for antibacterial activity of textiles usually involved sterilization of fabric, followed by its inoculation with a test organism and incubation prior to determination of bacteria remaining on the fabric. ‘Qualitative tests’ for such activity consist of visual observation of microbial growth on the fabric after exposure to the test organism [54]. The disadvantages of quantitative methods can be listed as being time-consuming, expensive, and they have not been assessed (with the exception of the AATCC-100 method) for inter-laboratory correlation of test results. Three quantitative methods are currently being used for determining antibacterial activity: •

AATCC-100 test method: In this method fabrics are sterilized in an autoclave or with ethylene oxide, inoculated with either gram-positive (Staphylococcus aureus) or gram-negative (Klebsiella pneumoniae) bacteria, and incubated in an agar medium; bacteriostatic activity of fabric swatches (treated and untreated controls) is calculated as percent reduction of bacteria on the fabric [55].

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184 •

•

Quinn test method: This test method permits direct enumeration of bacterial colonies on the fabric surface, but the test isn’t easy to run, colonies aren’t easily visible, and the possibilities for diffusion of the antimicrobial agent have not been minimized [40]. In this method fabrics that are free from microorganisms are prepared by laundering or sterilizing; the fabrics are then inoculated with one or several test organisms (Micrococcus, Pseudomonas, Lactobacillus, Escherichia coli, Staphylococcus aureus, or Klebsiella pneumoniae) and dried under conditions of known relative humidity. They are then placed on sterile agar plates, covered with a thin layer of agar, and incubated. After incubation, the bacteriostatic activity of the fabric is determined by counting the remaining bacteria colonies with a low-power microscope [54]. Lashen test method: In this method sample is suspended tautly and horizontally in a Petri dish by means of a wire hanger with hooks (Figure 12). After autoclaving, 0.4 ml of molten AATCC agar containing 20µg of 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC) per ml and 2 million bacterial cells is applied slowly and uniformly to the fabric surface. To prevent drying of the agar, approximately 5 ml of sterile distilled water is added to the Petri dish bottom. After 48 hr of incubation at 37 C, a colony count is made on both sides of the white fabric surface. The TTC is reduced by bacterial dehydrogenase enzymes to form a red insoluble dye (triphenylformazan), which stains bacterial colonies red. A low-power stereoscopic microscope may be used to be certain all colonies are counted. Untreated control samples with 2 million cells are included in the test, but the colonies which develop are too numerous to count. These samples appear pinkish due to numerous microscopic red colonies. However, a countable sample of 200 colonies is obtained by applying a 1:10000 agar dilution of the seeded agar to untreated fabric [56]. Table 2. Some of the antibacterial activity tests [50]

Code Number SN 195920-1992

Test Title Test Procedure Textile fabrics: Determination of the antibacterial activity: Agar diffusion plate test SN 195921-1992 Textile fabrics: Determination of the antimycotic activity: Agar diffusion plate test Agar diffusion AATCC 30-1993 Antifungal activity, assessment of textile materials: Mildew and rot tests resistance of textile materials semi-quantitative AATCC 147-1993 Antibacterial assessment of textile materials: Parallel streak methods AATCC 90-1982 Antibacterial activity of fabrics, detection of: Agar plate method AATCC 174-1993 Antimicrobial activity assessment of carpets JIS L 1902-1998 Testing method for antibacterial textiles AATCC 100-1993 Antibacterial finishes on textile materials SN 195924-1983 Textile fabrics: Determination of the antibacterial activity: Germ count Challenge tests method quantitative ISO 846-1997 Plastics-Evaluation of the action of microorganisms ISO 11721-1-2001 Textiles-Determination of resistance of cellulose containing New Methods ISO TC38 WG23: Testing for antibacterial activity CEN TC248 WG13: Textiles-Determination of the antibacterial activity – Agar plate diffusion test

Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 185 In qualitative tests, antibacterial activity of fibers is affected by the ability of the chemical agent to diffuse off the fiber into the culture medium. Although these tests are inexpensive and may be performed rapidly, they lack applicability to durable antibacterial fiber coatings, for the high agent-diffusibility that results in a positive test also results in coatings with low durability to laundering. In AATCC 147-1977 test method, bacteriostatic activity is measured by observing growth-free areas around fabric specimens placed perpendicular to streaks of agar inoculum. The parallel-streak test has been adopted as the qualitative method of choice over the agar-plate method (AATCC 90-1977), because its results can be correlated in interlaboratory tests and it is less dependent on diffusion. However, control samples are recommended for this test, since some investigators have noted false-positive results on untreated fabrics. Both the parallel streak and agar-plate methods use Staphylococcus aurous and Klebsiella pneumoniae as test microorganisms [40, 54]. Table 2 shows that some of the classified antibacterial activity test methods are used in textile researching [50]. Generally, Bacteriostatic activity of colloidal silver nanoparticles was evaluated after certain contact time and calculated percent reduction of bacteria using the following equation:

R (%) =

A− B ×100 A

Where R = the reduction rate, A = the number of bacterial colonies from untreated fabrics, and B = the numbers of bacterial colonies from treated fabrics [10, 11, 19, 45].

6. CONCLUSION Textiles, especially those made of natural fibers, are an excellent medium for the growth of microorganisms; therefore, there is a great demand for antimicrobial coatings of textiles to control the growth of microorganisms. In this review, an overview on application of silver nanoparticles in antibacterial coating of textiles has been presented. Silver nanoparticle as a novel antibacterial agent has advantages over conventional chemical antibacterial agents; because utilizing it in the antibacterial coating process is very convenient. Silver nanoparticles are skin friendly and don’t cause skin irritation. Some antimicrobial agents are extremely irritant and toxic to human body but the nano-sized silver particles have been well known as non-toxic in spite of being claimed to kill many different disease organisms. Some advantages and disadvantages of colloidal silver nanoparticles which can be used as an alternative antibacterial agent in textile antibacterial coating may be counted as follows: Advantages: • • • • • •

Outstanding antibacterial performance Skin friendly, non-allergenic and non-toxic to the skin Not irritating or sensitizing to the skin Ineffective against mammalian cell membranes Strongly effective against microorganisms Selective activity to undesirable microorganisms

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186 • • • • • •

Long lasting broad-spectrum antibacterial effect Compatible with the chemical processes Durable to washing, dry cleaning and hot pressing Cheap and easy method of application Complying with the statutory requirements of regulating agencies Ecofriendly Ecofriendly

Disadvantages: • • •

Color change of fabrics treated using colloidal silver nanoparticles Commonly time-consuming and costly synthesis procedures of silver nanoparticles Destroying of beneficial bacteria in such cases

REFERENCES [1]

S. Shrivastava, T. Bera, A. Roy, G. Singh, P. Ramachandrarao, and D. Dash, “Characterization of enhanced antibacterial effects of novel silver nanoparticles”, Nanotechnol, 18, 1 (2007). [2] J. L. Elechiguerra, J. L. Burt, J. R. Morones, A. Camacho-Bragado, X. Gao, H. H. Lara, and M. J. Yacaman, “Interaction of silver nanoparticles with HIV-1”, J Nanobiotechnol, 3(No.6), 1 (2005). [3] J. J. Ramsden, “What is nanotechnology?”, Nanotechnol Perceptions, 1, 3 (2005). [4] Yan, J. and J. Cheng, “Antimicrobial yarn having nanosilver particles and methods for manufacturing the same”, US Patent No. 6,979,491 B2 (2005). [5] O. Masala and R. Seshadri, “Synthesis routes for large volumes of nanoparticles”, Annu Rev Mater Res, 34, 41 (2004). [6] D. K. Tiwari, J. Behari, and P. Sen, “Application of nanoparticles in waste water treatment”, World Appl Sci J, 3(No.3), 417 (2008). [7] H. J. Lee and S. H. Jeong, “Bacteriostasis of nanosized colloidal silver on polyester nonwovens”, Text Res J, 74(No.5), 442 (2004). [8] N. Perkas, G. Amirian, S. Dubinsky, S. Gazit, and A. Gedanken, “Ultrasound-assisted coating of nylon 6,6 with silver nanoparticles and its antibacterial activity”, J Appl Polym Sci, 104, 1423 (2007). [9] S. T. Dubas, P. Kumlangdudsana, and P. Potiyaraj, “Layer-by-layer deposition of antimicrobial silver nanoparticles on textile fibers”, Colloid Surface A: Physicochem Eng Aspects, 289, 105 (2006). [10] N. Durán, P. D. Marcato, G. I. H. De Souza, O. L. Alves, and E. Esposito, “Antibacterial effect of silver nanoparticles produced by fungal process on textile fabrics and their effluent treatment”, J Biomed Nanotechnol, 3, 203 (2007). [11] H. J. Lee, S. Y. Yeo, and S. H. Jeong, “Antibacterial effect of nanosized silver colloidal solution on textile fabrics”, J Mater Sci, 38, 2199 (2003). [12] T. Yuranova, A. G. Rincon, C. Pulgarin, D. Laub, N. Xantopoulos, H. J. Mathieu, and J. Kiwi, “Performance and characterization of Ag–cotton and Ag/TiO2 loaded textiles

Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 187

[13] [14] [15]

[16]

[17]

[18] [19] [20] [21]

[22]

[23] [24] [25] [26] [27]

[28] [29]

during the abatement of E. coli”, J Photochem Photobiol A: Chemistry, 181, 363 (2006). C. Damm, H. Münstedt, and A. Rösch, “Long-term antimicrobial polyamide 6/silvernanocomposites”, J Mater Sci, 42(No.15), 6067 (2007). S. Wang, W. Hou, L. Wei, H. Jia, X. Liu, and B. Xu, “Antibacterial activity of nanoSiO2 antibacterial agent grafted on wool surface”, Surf Coat Tech, 202, 460 (2007). N. Vigneshwaran, K. S., A. A. Kathe, P. V. Varadarajan, and V. Prasad, “Functional finishing of cotton fabrics using zinc oxide–soluble starch nanocomposites”, Nanotechnol, 17, 5087 (2006). J. Scholz, G. Nocke, F. Hollstein, and A. Weissbach, “Investigations on fabrics coated with precious metals using the magnetron sputter technique with regard to their antimicrobial properties”, Surf Coat Tech, 192, 252 (2005). D. Hua, K. Cheuk, Z. Wei-ning, W. Chen, and X. Chang-fa, “Low temperature preparation of nano TiO2 and its application as antibacterial agents”, Trans Nonferrous Met Soc China, 17, 700 (2007). P. Potiyaraj, P. Kumlangdudsana, and S. T. Dubas, “Synthesis of silver chloride nanocrystal on silk fibers”, Mater Lett, 61, 2464 (2007). H. Y. Ki, J. H. Kim, S. C. Kwon, and S. H. Jeong, “A study on multifunctional wool textiles treated with nano-sized silver”, J Mater Sci, 42, 8020 (2006). C. Y. Chen and C. L. Chiang, “Preparation of cotton fibers with antibacterial silver nanoparticles”, Mater Lett, 62, 3607 (2008). A. Šileikaitė, I. Prosyčevas, J. Puišo, A. Juraitis, and A. Guobienė, “Analysis of silver nanoparticles produced by chemical reduction of silver salt solution”, Mater Sci, 12(No.4), 287 (2006). N. Niño-Martínez, G. A. Martínez-Castañón, A. Aragón-Piña, F. Martínez-Gutierrez, J. R. Martínez-Mendoza, and F. Ruiz, “Characterization of silver nanoparticles synthesized on titanium dioxide fine particles”, Nanotechnol, 19, 1 (2008). M. O. Elhag Ahmed and W. Kee Leong, “Colloidal silver nanoparticles stabilized by a water-soluble triosmium cluster”, J Organomet Chem, 691, 1055 (2006). A. Frattini, N. Pellegri, D. Nicastro, and O. de Sanctis, “Effect of amine groups in the synthesis of Ag nanoparticles using aminosilanes”, Mater Chem Phys, 94, 148 (2005). R. P. Bagwe and K. C. Khilar, “Effects of intermicellar exchange rate on the formation of silver nanoparticles in reverse microemulsions of AOT”, Langmuir, 16, 905 (2000). S. Sarkar, A. D. Jana, S. K. Samanta, and G. Mostafa, “Facile synthesis of silver nano particles with highly efficient anti-microbial property”, Polyhedron, 26, 4419 (2007). D. G. Yu, “Formation of colloidal silver nanoparticles stabilized by Na+–poly(γglutamic acid)–silver nitrate complex via chemical reduction process”, Colloid Surface B: Biointerfaces, 59, 171 (2007). Z. J. Jiang, C. Y. Liu, and Y. Liu, “Formation of silver nanoparticles in an acidcatalyzed silica colloidal solution”, Appl Surf Sci, 233, 135 (2004). P. Mukherjee, M. Roy, B. P. Mandal, G. K. Dey, P. K. Mukherjee, J. Ghatak, A. K. Tyagi, and S. P. Kale, “Green synthesis of highly stabilized nanocrystalline silver particles by a non-pathogenic and agriculturally important fungus T. asperellum”, Nanotechnol, 19, 1 (2008).

188

S. Dadvar, A. Oroume and A. K. Haghi

[30] Y. H. Chen and C. S. Yeh, “Laser ablation method: use of surfactants to form the dispersed Ag nanoparticles”, Colloid Surface A: Physicochem Eng Aspects, 197, 133 (2002). [31] J. Chen, J. Wang, X. Zhang, and Y. Jin, “Microwave-assisted green synthesis of silver nanoparticles by carboxymethyl cellulose sodium and silver nitrate”, Mater Chem Phys, 108, 421 (2008). [32] Y. Tan, Y. Li, and D. Zhu, “Preparation of silver nanocrystals in the presence of aniline”, J Colloid Interf Sci, 258, 244 (2003). [33] W. S. Liao, T. Yang, E. T. Castellana, S. Kataoka, and P. S. Cremer, “A rapid prototyping approach to Ag nanoparticle fabrication in the 10–100 nm range”, Adv Mater, 18, 2240 (2006). [34] W. Zhang, X. Qiao, and J. Chen, “Synthesis and characterization of silver nanoparticles in AOT microemulsion system”, Chem Phys, 330, 495 (2006). [35] N. H. H. Abu Bakar, J. Ismail, and M. Abu Bakar, “Synthesis and characterization of silver nanoparticles in natural rubber”, Mater Chem Phys, 104, 276 (2007). [36] Y. Yin, Z. Y. Li, Z. Zhong, B. Gates, Y. Xia, and S. Venkateswaran, “Synthesis and characterization of stable aqueous dispersions of silver nanoparticles through the Tollens process”, J Mater Chem, 12, 522 (2002). [37] K. S. Chou and C. Y. Ren, “Synthesis of nanosized silver particles by chemical reduction method”, Mater Chem Phys, 64, 241 (2000). [38] Y. Sun and Y. Xia, “Shape-controlled synthesis of gold and silver nanoparticles”, Science, 298, 2176 (2002). [39] I. Sondi and B. Salopek-Sondi, “Silver nanoparticles as antimicrobial agent: a case study on E. Coli as a model for Gram-negative bacteria”, J Colloid Interf Sci, 275, 177 (2004). [40] T. Ramachandran, K. Rajendrakumar, and R. Rajendran, “Antimicrobial Textiles - an Overview”, IE (I) Journal—TX, 84, 42 (2004). [41] J. G. Black, “Microbiology: principles and explorations”, Wiley, 314-323 (2002). [42] G. Sun, X. Xu, J. R. Bickett, and J. F. Williams, “Durable and regenerable antibacterial finishing of fabrics with a new hydantoin derivative”, Ind Eng Chem Res, 40, 1016 (2001). [43] S. Pal, Y. Kyung Tak, and J. Myong Song, “Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli”, Appl Environ Microb, 73(No.6), 1712 (2007). [44] S. H. Jeong, Y. H. Hwang, and S. C. Yi, “Antibacterial properties of padded PP/PE nonwovens incorporating nano-sized silver colloids”, J Mater Sci, 40, 5413 (2005). [45] Y. Xing, X. Yang, and J. Dai, “Antimicrobial finishing of cotton textile based on water glass by sol–gel method”, J Sol-Gel Sci Technol, 43, 187 (2007). [46] D. T. W. Chun and G. R. Gamble, “Using the reactive dye method to covalently attach antibacterial compounds to cotton”, J Cotton Sci, 11, 154 (2007). [47] K. Nakamura, “Antibacterial cellulose fiber and production process thereof”, US Patent No. 5,985,301 (1999). [48] S. T. Dubas and V. Pimpan, “Green synthesis of silver nanoparticles for ammonia sensing”, Talanta, 76(No.1), 29 (2008). [49] R. Jantas and K. Górna, “Antibacterial finishing of cotton fabrics”, Fibres Text East Eur, 14(No.1), 88 (2006).

Practical Hints on Application of Nanosilvers in Antibacterial Coating of Textiles 189 [50] www.centrum.tul.cz/centrum/itsapt/prezentace/wp2/113105ef.ppt [51] D. Gupta and A. Haile, “Multifunctional properties of cotton fabric treated with chitosan and carboxymethyl chitosan”, Carbohyd Polym, 69, 164 (2007). [52] W. Ye, M. F. Leung, J. Xin, T. L. Kwong, D. K. L. Lee, and P. Li, “Novel core-shell particles with poly(n-butyl acrylate) cores and chitosan shells as an antibacterial coating for textiles”, Polymer J, 46, 10538 (2005). [53] Y. A. Son and G. Sun, “Durable antimicrobial nylon 66 fabrics: ionic interactions with quaternary ammonium salts”, J Appl Polym Sci, 90, 2194 (2003). [54] T. L. Vigo and M. A. Benjaminson, “Antibacterial fiber treatments and disinfection”, Text Res J, 51(No.7), 454 (1981). [55] AATCC Test Method 100-1993, “Antibacterial finishes on textile materials: assessment of”, AATCC Tech Manual, p. 143, 1997. [56] E. S. Lashen, “New method for evaluating antibacterial activity directly on fabric”, Appl Microbiol, 21(No.4), 771 (1971).

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.191-196 ©2010 Nova Science Publishers, Inc.

Chapter 13

THE NANOSTRUCTURE AND YIELD PROCESS OF CROSS-LINKED EPOXY POLYMERS Z. M. Amirshikhova1, G. V. Kozlov1, G. M. Magomedov2and G. E. Zaikov2 1

GOU VPO Dagestan State Pedagogical University, Makhachkala, Russian Federation 2 N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russian Federation

ABSTRACT The absence of simbatness between yield stress and elasticity modulus for epoxy polymers was shown. This is due to thermodynamically nonequilibrium structure of amorphous polymers that require two order parameters for their properties description, as a minimum. The cluster model of polymers amorphous state structure allows correcting the quantitative estimation of yield stress.

Keywords: Epoxy polymer, structure, nanoclusters, yield stress, elasticity modulus.

INTRODUCTION At present it becomes obvious that polymer systems in virtue of their structure features are always nanostructural systems [1]. However, such structure treatment can be a different one. So, the authors [2] used for this purpose the cluster model of polymers amorphous state structure [3], which supposes, that the indicated structure consists of local order domains (clusters), immersed in loosely-packed matrix. In this case the latter is considered as natural nanocomposite matrix and clusters – as nanofiller. The cluster presents itself a set of several densely-packed collinear segments of different macromolecules, having the size up to several nanometers [3]. It has been shown that such clusters are true nanoparticles – nanoworld objects (nanoclusters) [2].

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The yield process presents an important phenomenon in polymers mechanics and its main characteristic (yield stress) from the practical point of view restricts from above an exploitation region of plastic polymers used as engineering materials [4]. The purpose of the present paper is a theoretical description of cross-linked epoxy polymers yield stress within the frameworks of cluster model.

EXPERIMENTAL Cross-linked epoxy polymers (EP) based on diglycidyl ether of bisphenol A (DGEBA) were used. The curing was performed by 3,3’-dichloro-4,4’-diaminodiphenylmethane (EP-1 composition) and iso-methyltetrahydrophtalic anhydride in the presence of a catalyst tris (dimethylaminomethyl)-2,4,5-phenol (EP-2 composition). The ratio of a curing agent to epoxy oligomer reactive groups Kst varied at 0.50-1.50. Thus it was possible to obtain 20 EP specimens with different topologies of cross-linked networks. Thermomechanical analysis (TMA) was performed under the uniaxial compression at the pressure of 1.2 MPa and at the temperature change rate of 2 K/min. According to TMA data we determined the glass transition temperature Tg and the mean statistical molecular weight Ws of the chain part between the cross-links nodes [5]:

Ws =

3ρRThe Δε , Ph0

(1)

where ρ is epoxy polymer density, which is equal to about 1250 kg/m3; R is universal gas constant; The is the initial temperature of the forced high elasticity, K; Δε is quasiequilibrium high elastic strain; h0 is the specimen initial height; P is the specific load on the specimen. Further one can calculate the effective density of the cross-linked network νs according to the following equation [5]:

νs =

2 ρN A 3 Ws

,

(2)

where NA is Avogadro number. As the value νs estimations according to the equation (2) have shown, it varies within the range of (2-19)×1026 m-3. The stress-strain characteristics of epoxy polymers were obtained under the uniaxial compression tests at temperature 293 K and strain rate of 5×10-3 s-1. The density of the indicated specimens was determined by hydrostatic weighing method with precision up to four decimal places.

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193

RESULTS AND DISCUSSION The studied epoxy polymers are characterized by plastic failure type under the uniaxial compression tests. In this case the clearly expressed yield stress, yield tooth and cold flow plateau are observed on stress-strain curves. At present the point of view, assuming elasticity modulus E and yield stress σY simbate change at different kinds of actions on polymer predominates, that gives in the end linear correlation σY(E) [6]. In Figs. 1 and 2 the dependences E(Kst) and σY(Kst) are adduced for the studied epoxy polymers, which have principally differing character. So, if for the elasticity modulus adduced in Fig. 1 the dependence has extreme character with minimum at Kst=1.25 or 1.0, then yield stress in Kst considered range remains practically constant (Fig. 2). The comparison of Figs. 1 and 2 exludes E and σY behaviour simbatness for the studied epoxy polymers. E, GPa 5 -1 -2 3

1

0.5

1.0

1.5

Kst

Figure 1.The dependence of elasticity modulus E on curing agent-oligomer ratio Kst for epoxy polymers EP-1 (1) and EP-2 (2).

σY, MPa 150

100

50

0

-1 -2

0.5

1.0

1.5

Kst

Figure 2. The dependence of yield stress σY on curing agent-oligomer ratio Kst for epoxy polymers EP-1 (1) and EP-2 (2).

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The cluster model of polymers amorphous state structure [3] was used in the present paper for the explanation of the indicated nonsimbatness and theoretical description of epoxy polymers yield stress. Within the frameworks of this model segments, included in nanoclusters, are considered as structure linear defects (analog of dislocations in crystalline lattices), that allows to use for yield stress theoretical determination mathematical calculus of dislocations theory. Within the frameworks of such approach the relationship between σY and E is expressed by the equation [3]:

σYt =

3Eb ρ d 4π(1 + ν )

,4

(3)

where b is Burgers vector, ρd is linear defects density, ν is Poisson’s ratio. Let us consider the estimation methods of the parameters included in the equation (3). The Burgers vector value b for polymeric materials is given by the following formula [3]: 1/ 2

⎛ 60.2 ⎞ ⎟⎟ b = ⎜⎜ C ⎝ ∞ ⎠

,Å,

(4)

where C∞ is characteristic ration, which is the polymer chain flexibility indicator and is determined as follows [3]:

С∞ =

2d f

d (d − 1)(d − d f )

+

4 , 3

(5)

where d is dimension of Euclidean space, in which a fractal is considered (it is obvious, that in our case d=3), df is fractal dimension of epoxy polymer structure, determined according to the equation [7]:

d f = (d − 1)(1 + ν ) ,

(6)

where Poisson’s ratio ν is estimated according to the mechanical tests results with the aid of the relationship [8]:

σY 1 − 2ν = , E 6(1 + ν ) The linear defects density ρd was determined according to the equation [3]:

(7)

The Nanostructure and Yield Process of Cross-Linked Epoxy Polymers

ρd =

ϕcl S

,

195

(8)

where ϕcl is nanoclusters relative fraction, S is macromolecule cross-sectional area, which for the studied epoxy polymers is equal to ~ 32 Å2 [9]. The value ϕcl was calculated according to the following percolation relationship [3]:

ϕcl = 0.038(Tg − T )

0.55

,

(9)

where Tg and T are glass transition and testing temperatures, accordingly. In table 1 the comparison of the obtained experimentally σY and calculated according to the considered above methods

σYt

values of yield stress for the studied epoxy polymers was

adduced. As one can see, a good correspondence of theory and experiment was obtained (average discrepancy of σY and

σYt

makes up 6.6 %, that is comparable with mechanical

tests error). Table 1. The structural and mechanical characteristics of epoxy polymers Epoxy polymer

EP-1

EP-2

Kst

νs×10-26, m-3

ϕcl

E, GPa

0.50 0.75 1.0 1.25 1.50 0.50 0.75 1.0 1.25 1.50

2 6 17 12 8 4 10 11 10 8

0.300 0.334 0.611 0.655 0.405 0.351 0.407 0.508 0.409 0.429

4.3 3.9 2.3 2.0 3.0 3.2 2.8 2.5 3.0 2.7

σY, MPa 140 140 142 131 128 120 120 131 129 121

σYt , MPa 148 148 151 140 138 128 129 140 138 128

The stated above results suppose that σY and E values simbatness as a function of some parameter (testing temperature, cross-linking density and so on) can be a special case only. The equation (3) demonstrates that the indicated simbatness realization condition is criterion ρd=const or, as follows from the equation (8), ϕcl=const. In other words, σY and E change simbatness realization condition is polymer nanostructure invariability. Let us note that amorphous polymers are thermodynamically nonequilibrium solids, for which description, as minimum, two parameters of order are required according to the Prigogine-Defay principle. It is obvious, that the equation (3) satisfies this principle, whereas linear correlation σY(E) does not.

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CONCLUSIONS Thus, the present paper results have shown, that yield stress and elasticity modulus change simbatness is a special case in virtue of key reason, namely, polymers structure thermodynamical nonequilibrium. At the same time cluster model of polymers amorphous state structure gives the correct quantitative description of yield stress value.

REFERENCES [1] [2]

[3] [4] [5] [6] [7] [8] [9]

Ivanchev S.S., Ozerin A.N. Vysokomolek. Soed. B, 2006, v. 48, № 6, p. 1531-1544. Mikitaev A.K., Kozlov G.V., Zaikov G.E. Polymer Nanocomposites: Variety of Structural Forms and Applications. New York, Nova Science Publishers, Inc., 2008, 319 p. Kozlov G.V., Zaikov G.E. Structure of the Polymer Amorphous State. Utrecht-Boston, Brill Academic Publishers, 2004, 465 p. Bartenev G.M., Frenkel S.Ya. Physics of Polymers. Leningrad, Khimiya, 1990, 432 p. Kozlov G.V., Beloshenko V.A., Varyukhin V.N., Lipatov Yu.S. Polymer, 1999, v. 40, № 4, p. 1045-1051. Milagin M.F., Shishkin N.I. Mekhanika Polymerov, 1975, № 6, p. 963-968. Balankin A.S. Synergetics of Deformable Body. Moscow, Publishers of Ministry Defence SSSR, 1991, 404 p. Kozlov G.V., Sanditov D.S. Anharmonic Effects and Physical-Mechanical Properties of Polymers. Novosibirsk, Nauka, 1994, 261 p. Kozlov G.V., Beloshenko V.A., Kuznetsov E.N., Lipatov Yu.S. Doklady NAN Ukraine, 1994, № 12, p. 126-128.

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.197-204 ©2010 Nova Science Publishers, Inc.

Chapter 14

NANOSTRUCTURES IN CROSS-LINKING EPOXY POLYMERS AND THEIR INFLUENCE ON MECHANICAL PROPERTIES

1

Z. M. Amirshikhova1, G. V. Kozlov1, G. M. Magomedov2 and G. E. Zaikov2

GOU VPO Dagestan State Pedagogical University, Makhachkala, Russian Federation N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russian Federation

2

ABSTRACT The methods of nanostructure adjustment in cross-linked epoxy polymers were considered. The large potential of these materials mechanical properties change by nanoclusters relative fraction variation was shown.

Keywords: Epoxy polymer, nanostructures, mechanical properties, solid-state extrusion, annealing.

INTRODUCTION At present it is generally accepted [1] that macromolecular formations and polymer systems by virtue of their structure features are always natural nanostructural systems. In this connection the question of using this feature for polymer materials properties and operating characteristics improvement arises. It is obvious enough that for structure-properties relationships receiving the quantitative nanostructural model of the indicated materials is necessary. It is also obvious that if the dependence of specific property on material structural state will be unequivocal one, then there will be quite sufficient modes achieve this state. The cluster model of polymer amorphous state structure [2, 3] is the most suitable for this state structure description. It has been shown, that this model basic structural element (cluster) is a

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nanoparticle (nanocluster). The cluster model was used successfully for cross-linked epoxy polymers structure and properties description [5]. Therefore the present paper purpose is nanostructures regulation modes and the influence of the latter on rarely cross-linked epoxy polymer properties study.

EXPERIMENTAL The studied object is an epoxy polymer on the basis of resin UPS-181, cured by isomethyltetrahydrophtaleic anhydride in the ratio by mass 1:0.56. After intermixing and exposure under vacuum of components at room temperature and pressure 10-20 mm of mercury up to air bubbles disappearance during 15-30 min mixture is poured into the heated up to ~ 343 K fluoropolymer molds and cured according to the experimentally selected temperature regime. Testing specimens were obtained by the hydrostatic extrusion method. The indicated method choice is due to the fact, that high hydrostatic pressure exestion in deformation process prevents the defects formation and growth, resulting to the material failure [6]. Hydroextrusion was realized for one passage on an apparate of pistoncylinder system at room temperature. The extrusion strain εe is equal to 0.14, 0.25, 0.36, 0.43 and 0.52. εe value was calculated according to the formula [7]:

d12 − d 22 εe = d12

,

(1)

where d1, d2 are diameters of intermediate product and extrudate, accordingly (the last is equal to 12 mm). The stress-strain characteristics were studied in conditions of uniaxial compression on a testing machine FRZ-100/1 of the firm Heckert at testing temperature 293 K and strain rate ~ 10-3 s-1 (the specimens diameter is equal to 10 mm, height – to 15 mm). No less than five specimens were tested for each εe value. The arithmetical mean of the obtained data totality was accepted as measurements result. Specimens density was measured by hydrostatic weighing method to a precision of the fourth sign after comma. Further their values were expressed accounting for measurements of arithmetical mean error. The obtained by hydrostatic extrusion specimens were annealed at maximum temperature 353 K during 15 min, after that they were also tested on uniaxial compression.

RESULTS AND DISCUSSION The hydrostatic extrusion and subsequent annealing of epoxy polymer (EP) result to very essential changes of its mechanical behaviour and properties, moreover unexpected enough. The qualitative changes of EP mechanical behaviour can be monitored according to the corresponding changes of the stress-strain (σ-ε) diagrams, shown in Fig. 1. The initial EP

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199

shows the expected enough behaviour and its elasticity modulus E and yield stress σY are typical for such polymers at testing temperature T separating from glass transition temperature Tg on about 40 K [8]. The small (~ 3 MPa) stress decay ΔσY behind yield stress is observed, that is also typical of amorphous polymers [5]. However, EP extrusion up to εe=0.52 results to stress decay ΔσY (“yield tooth”) disappearance and the essential E and σY lessening. Besides, the diagram σ-ε itself is more like analogous diagram for rubber, than for glassy polymer. This specimen annealing at maximum temperature Tan=353 K gives no less strong, but diametrically opposite effect – yield stress and elasticity modulus increase sharply (the latter increases in about 2 times in comparison with the initial EP and more that one order in comparison with the extruded specimen). Besides, the pronounced “yield tooth” appears. It is necessary to note that specimen shrinkage at annealing is small (~ 10 %), that is equal to about 20 % of εe.

σ, МPа 150

100 3 50

1 2

0

0.1

0.2

0.3

ε

Figure 1.The stress-strain (σ-ε) curves for initial (1), extruded up to εe=0.52 (2) and annealed (3) EP.

The general picture of parameters E and σY change as a function of εe is presented in Figs. 2 and 3, accordingly. As one can see, both indicated parameters showed common tendencies at εe change: up to εe≈0.36 inclusive E, and σY weak increase at εe growth is observed, moreover their absolute values for extruded and annealed specimens are close, but at εe>0.36 the pronounced antibatness of these parameters for the indicated specimen types is displayed. The cluster model of polymers amorphous state structure and elaborated within its frameworks polymers yielding treatment allows to explain such behaviour of the studied samples [2, 3].

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Е, GPа 3

2 -1 -2

1

0

0

0.4

0.2

0.6

εe

Figure 2. The dependences of elasticity modulus E on extrusion strain εe for extruded (1) and annealed (2) EP.

σY, МPа 150

100

50

0

0

0.2

0.4

0.6

εe

Figure 3. The dependences of yield stress σY on extrusion strain εe for extruded (1) and annealed (2) EP.

The cluster model supposes that polymers amorphous state structure presents the local order domains (nanoclusters), surrounded by loosely-packed matrix. Nanoclusters consist of several collinear densely-packed statistical segments of different macromolecules and in virtue of this they offer the analogue of crystallite with extended chains. There are two types

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201

of nanoclusters: stable, consisting of a relatively large segments number, and nonstable, consisting of a less number of such segments [9]. At temperature increase or mechanical stress application nonstable clusters are disintegrated in the first place, that results to the two well-known effects. The first of them is known as two-stage glass transition process [10] and it supposes that at

Tg' =Tg-50

K disintegration of nonstable clusters, restraining loosely-

packed matrix in glassy state, occurs that defines devitrification of the latter [2, 3]. The wellknown rapid polymers mechanical properties decrease at approaching to Tg is consequence of this [8]. The second effect consists of nonstable clusters decay at σY under mechanical stress action, loosely-packed matrix mechanical devitrification and, as consequence, glassy polymers rubber-like behaviour on cold flow plateau [9]. The stress decay ΔσY behind yield stress is due to namely nonstable nanoclusters decay and therefore ΔσY value serves as characteristic of these nanoclusters fraction [9]. Proceeding from this brief description, the experimental results, adduced in Figs. 1-3, can be interpreted. The rarely cross-linked polymer on the basis of resin UPS-181 has low glass transition temperature Tg, which can be estimated according to shrinkage measurements data as equal to ~ 333 K. This means that the testing temperature T=293 K and

Tg'

for it are close, that small

ΔσY value for the initial EP confirms. This supposes nanoclusters (nanostructures) small relative fraction ϕcl [2, 3] and, since these nanoclusters have arbitrary orientation, εe increase results rapidly enough to their decay, that defines loosely-packed matrix mechanical devitrification at εe>0.36. Devitrificated loosely-packed matrix gives insignificant contribution in E [11], equal practically to zero, that results to sharp (discrete) elasticity '

modulus decrease. Besides, at T> Tg ϕcl rapid decay is observed, i.e. segments number decrease in both stable and nonstable nanoclusters [3]. Since just these parameters (E and ϕcl) are defined σY value, then their decreasing defines yield stress sharp lessening. Now extruded at εe>0.36 EP presents as a matter of fact rubber with high cross-linking degree, that its diagram σ-ε (Fig. 1, curve 2) reflects. The polymer oriented chains shrinkage occurs at the extruded EP annealing at temperature higher than Tg. Since this process realizes in a narrow temperatures range and during a small time interval, then a large number of nonstable nanoclusters is formed. This effect is intensified by the available molecular orientation, i.e. by preliminary favourable segments building, and it is reflected by ΔσY strong increase (Fig. 1, curve 3). ϕcl increase results to E growth (Fig. 2) and ϕcl and E combined enhancement – to σY considerable growth (Fig. 3). The considered structural changes can be described quantitatively within the frameworks of a cluster model. The nanoclusters relative fraction ϕcl can be calculated according to the methodics, stated in paper [12]. The shown in Fig. 4 dependences ϕcl(εe) have the character expected from the adduced above description and are its quantitative confirmation. The adduced in Fig. 5 dependence of density ρ of EP extruded specimens on εe is simbate to the dependence ϕcl(εe), that should be also expected, since densely-packed segments fraction decrease should be reflected in ρ lessening.

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ϕcl 0.6

0.4 -1 -2

0.2

0

0

0.2

0.4

0.6

εe

Figure 4. The dependences of nanoclusters relative fraction ϕcl on extrusion strain εe for extruded (1) and annealed (2) EP.

ρ×10-3, kg/m3 2.46

2.42

-1 -2

2.38

2.34 0

0.2

0.4

0.6

εe

Figure 5. The dependence of specimens density ρ on extrusion strain εe for extruded (1) and annealed (2) EP.

In paper [13] the supposition was made that ρ change can be due to microcracks network formation in specimen that results ρ lessening at large εe (0.43 and 0.52), which are close to the limiting ones. ρ relative change (Δρ) estimation according to the equation is:

Nanostructures in Cross-Linking Epoxy Polymers …

ρ max − ρ min Δρ = ρ max

203

,

(2)

where ρmax and ρmin are the greatest and the least density values, accordingly, shows that Δρ≈0.01. This value can be reasonable for free volume increase, which is necessary for loosely-packed matrix devitrification [3] (accounting for closeness of T and

Tg' ),

but it is

obviously small, if to assume as real microcracks formation. As the experiments have shown, EP extrusion at εe>0.52 is impossible owing to specimens cracking in extrusion process. Therefore the critical dilatation Δδcr value, which is necessary for microcracks cluster formation, can be estimated [14] as follows:

Δδ cr =

2(1 + ν )(2 − 3ν ) , 11 − 19 ν

(3)

where ν is Poisson’s ratio. Accepting the average value ν≈0.35, we obtain Δδcr≈0.60 that is essentially higher than the estimation Δρ made earlier. These calculations assume that ρ decrease at εe=0.43 and 0.52 is due to nonstable nanoclusters decay and corresponding to EP structure loosening. The stated above data give a clear example of large possibilities of polymer properties operation through its nanostructure change. From the plots of Fig. 2 it follows that annealing of EP, extruded up to εe=0.52, results to elasticity modulus increase in more than 8 times and from data of Fig. 3 yield stress increase in 6 times follows. From the practical point of view extrusion and subsequent annealing of rarely cross-linked epoxy polymers allow to obtain materials, which are just as good as by stiffness and strength to densely cross-linked EP, but exceeding the latter by plasticity degree. Let us note, that besides extrusion and annealing the other modes of polymers nanostructure operation exist: plasticization, filling, films obtaining from different solvents and so on.

CONCLUSIONS The present paper results demonstrated that neither cross-linking degree nor molecular orientation level defined cross-linked polymers final properties. The proof, controlling properties, is a state of supersegmental (nanocluster) structure, which, in its turn, can be goaldirectly regulated by molecular orientation and thermal treatment application.

REFERENCES [1] [2]

Ivanchev S.S., Ozerin A.N. Vysokomolek. Soed. B, 2006, v. 48, № 8, p. 1531-1544. Kozlov G.V., Novikov V.U. Uspekhi Fizicheskikh Nauk, 2001, v. 171, № 7, p. 717-764.

204 [3] [4]

[5] [6] [7] [8] [9] [10] [11] [12] [13] [14]

Z. M. Amirshikhova, G.V. Kozlov and G. M. Magomedov et al. Kozlov G.V., Zaikov G.E. Structure of the Polymer Amorphous State. Utrecht-Boston, Brill Academic Publishers, 2004, 465 p. Mikitaev A.K., Kozlov G.V., Zaikov G.E. Polymer Nanocomposites: Variety of Structural Forms and Applications. New York, Nova Science Publishers, Inc., 2008, 319 p. Kozlov G.V., Beloshenko V.A., Varyukhin V.N., Lipatov Yu.S. Polymer, 1999, v. 40, № 4, p. 1045-1051. Kozlov G.V., Beloshenko V.A., Slobodina V.G., Prut E.V. Vysokomolek. Soed. B, 1996, v. 38, № 6, p. 1056-1060. Beloshenko V.A., Kozlov G.V., Varyukhin V.N., Slobodina V.G. Acta Polymerica, 1997, v. 48, № 5-6, p. 181-187. DiBenedetto A.T., Trachte K.L. J. Appl. Polymer Sci., 1970, v. 14, № 11, p. 2249-2262. Kozlov G.V., Beloshenko V.A., Gazaev M.A., Novikov V.U. Mekhanika Kompozitnukh Materialov, 1996, v. 32, № 2, p. 270-272. Belousov V.N., Kotsev B.Kh., Mikitaev A.K. Doklady AN SSSR, 1985, v. 280, № 5, p. 1140-1143. Shogenov V.N., Belousov V.N., Potapov V.V., Kozlov G.V., Prut E.V. Vysokomolek. Soed. A, 1991, v. 33, № 1, p. 155-160. Kozlov G.V., Burya A.I., Shustov G.B. Fizika i Khimiya Obrabotki Materialov, 2005, № 5, p. 81-84. Pakter M.K., Beloshenko V.A., Beresnev B.I., Zaika T.P., Abdrakhmanov L.A., Bezay N.I. Vysokomolek. Soed. A, 1990, v. 32, № 10, p. 2039-2046. Balankin A.S. Synergetics of Deformable Body. Moscow, Publishers of Ministry Defence SSSR, 1991, 404 p.

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.205-219 ©2010 Nova Science Publishers, Inc.

Chapter 15

THE DEGRADATION HETEROCHAIN POLYMERS IN THE PRESENCE OF PHOSPHORUS STАBILIZERS E. V. Kalugina1, N. V. Gaevoy1, K. Z. Gumargalieva2 and G. E. Zaikov3 1

Polyplastic Group, Moscow, Russia N.N.Semenov Institute of Chemical Physics, Moscow, Russia  3 N.M.Emanuel Institute of Biochemical Physics, Moscow, Russia 2

ABSTRACT The thermal stability and thermal stabilization of the heterochain polymers were investigated. Analysis of PAI, PSF, PEI degradation and stabilization has allowed an approach to be developed to aid their processing and resolve similar problems with other resins such as polyethersulfone, LCP, ets. Addition of PCA inhibits in heterochain polymers thermal oxidation at high and low temperatures.

Keywords: phosphorus-containing additives (РСА), aromatic and fatty-aromatic polyamides (PFA), polyimides (PAI), polyesterimides (PEI), polysulfones (PSF), liquid-crystal copolyesters (LCP), pyromellite imide (PDI), anilide phenyl phosphate (APP), cyclization, hindered phosphate (HP)

SCOPE The aim of the work is to investigate thermal stability of the heterochain polymers. The influence of phosphorus-containing additives on the thermal oxidation at high and low temperatures and examine the degradation and stabilization polyimides, polysulfones, polyesterimides etc. An analysis of data from the literature and the authors' investigations indicate injection of phosphorus-containing additives (РСА) in polymers as the most perspective way of heatresistant polymer thermal stabilization [1-20]. Tests of а wide РСА range in different polymer

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structures (aromatic and fatty-aromatic polyamides and polyimides, polyesterimides, polyamidoimides, polysulfones, liquid-crystal copolyesters, ets.) allowed selection of optimal thermostabilizing additives: aromatic esters and phosphorous and prosphoric esteramides. For pure aromatic polyimides, polyimidophenylquinoxalines and polybenzoxazoles, optimal concentrations are 3 wt. % РСА. At equal heat loads, properties of stabilized samples are 1.5 - 2.5 times higher compared with non-stabilized polymers. For aliphatic-aromatic polymers (bisphenol A -derived polysulfone and polyesterimide, polyalkane imide, and polyphthalimides), РСА optimal concentrations are two times lower: 0.3 - 1.0 wt.%. This is caused bу lower temperature impacts during processing and operation of materials and articles. То develop the idea of heat resistant polymer stabilization, оnе must understand the mechanism of РСА stabilizing action in them. Simultaneously with applied stabilization, some studies were performed before оn the example of aromatic polyimides. The inhibiting action of РСА оn oxidation branch of degradation and pre-polymer cyclization rate increase in РСА presence were detected. It was also found that crosslinking processes are intensified оn the initial stages of thermal oxidation. Experimental data indicate а complex mechanism of РСА action in heat-resistant polymers, which includes inhibition of radical chain reactions and catalysis of cyclization and crosslinking processes. Тhе comparison data оn kinetics of inhibited and non-inhibited oxidation of polypyromellitimide, РAI, РРА, РЕI and PSF at high processing temperature and in solid oxidation show general tendencies. In both cases, kinetic curves of oxygen absorption and main oxidation products release (carbon oxides) mау bе conditionally divided into two stages: the initial stage obeying kinetic order оnе and the constant rate stage. Inhibition of thermal oxidation is observed at the first stage of heat-resistant polymer degradation. For example, rate constants of oxygen absorption bу РI equal 7.5xl0-7 - 1.6xl0-5 and 1.9хl0-6 - 7.4xl0-8 s-1 for non-stabilized and stabilized PI, respectively. Gas products release demonstrates similar relations. А decrease of thermal oxidation solid product (pyromellite imide, PDI) yield was also observed - bу 2.5 times for РI and 5 times for РAI:

O

O

C

C NH

HN C

C

O

O

Injection of РСА to polyphenylquinoxalines significantly decreases the yield of analogous (in relation to the polymer structure) product, which is N-phenylpyrazine:

The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 207

N

N

N

N

Correspondingly, in PPA [20] the yield of therephtalic amide:

H2N C O

C NH2 O

is almost eliminated (during the studied time period up to 5.000 h). The amounts of PDI and analogous products (relative to polymer structures) [3,19] indicate the conversion degree in oxidation transformations. The absence of these compounds in degradation products after reaction without oxygen testifies about exclusively thermal oxidation origin of their formation. Therefore, stabilization of heat-resistant polymers (HRP) displays clear antioxidant type, i.e. аn additive is сараblе of interacting with radicals and other labile products of HRP thermal oxidation. High-temperature activity of РСА in radical reactions is additionally confirmed bу stabilizing effect of anilide phenyl phosphate (APP) оn РЕ degradation at 300 oC. Application of such а model system to this particular case is desirable, because the radical-chain type of РЕ thermal oxidation at 200250oС is well-known. It is also forecasted well for higher temperatures and, therefore, at some chain branching degree is forecasted well for carbonyl structures. А significant contribution of the branching degree to polymer properties, including thermal stability, was shown bу Korshak [21]. Non-cyclic units represent the main element of branching [3,22]. The РСА effect оп the cycle formation process was assessed using gaschromatographic analysis of water release from polyamidoacid films - PI and PEI prepolymers. Intensive water release was observed at initial cyclization stages at 150 – 200oС. Total water amount released from stabilized and non-stabilized PI and PEI at 250 – 300oС are nearly the same, i.e. in both cases, cyclization degrees are close. The РСА effectiveness for polyphenylquinoxaline - the polymer, in which cyclization proceeds easily, without аnу additional heat treatment - indicates that cyclization process acceleration in heat-resistant polymers (PI, for example) mау not explain the protective action of РСА.

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Figure 1. ESR spectra of spin probe in РI film without additives (а, с) and added bу APP (b); а, b prior to thermal aging; с - after thermal aging at 300°С during 500 h in air.

Another possible stabilization mechanism - the formation of more stable network polymer structure in the presence of РСА with hindered oxygen access - was checked using the spin probe technique [23]. The probe (nitroxyl radical) diffusion into РI matrix was traced bу changes in ESR spectra from classical triplet of freely isotropic-rotating, stable nitroxyl radical to а triplet degenerate bу boundary components, typical of а probe rotating in а viscous medium. The spectrum (Figure 1) is of superposition type and indicates the presence of slow (main) and fast probe motion zones in the polymeric matrix. Relaxation times for non-stabilized and stabilized PI films were determined from graphic charts [41] as follows: τ1 = 2xl0-8, τ2 (~5%) = 10-9 s and τ1 = (2 + 5)xl0-8, τ2 (~10 +15%) = 10-10 s, respectively. These values indicate а definite plasticizing effect of the additive оn РI film properties. After thermal aging of films at 300oС during 500 h, τ1 does not increase. Vice versa, for nonstabilized sample it decreases to 10-9 s, whereas for stabilized sample it remained practically unchanged. Apparently, the decrease of τ1 in non-stabilized film is associated with probe fixing оn structure defects (various microcracks), but not with molecular mobility increase. Degraded non-stabilized РI possesses self paramagnetic properties (а singlet with ΔH≈10E), which superimposes оn the central component of ESR spectrum of the probe. This contribution is negligible for stabilized sample. Тhе behavior of paramagnetic probe definitely reflects molecular mobility of the solid. Moreover, rotational and translational diffusion of the probe correlates with behavior of other "small" molecules (oxygen, for example) in the solid matrix. As observed in the experiment, additional crosslinking does not cause а noticeable change in molecular mobility of the polymer and hindrance of O2 diffusion inside the sample.

The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 209

Intensity

а

0

5

10

15

20

25

30

35

40

б

Intensity

3

1 4

2

2

4

6

8

10

Figure 2. Diffraction patterns for PI films without additives (а,b -1,2) and added by 2 wt.% APP (b-3,4) prior to heat aging (а,b -1,3) and after thermal oxidation (b-2,4) T= 300 С, 700 h in air

Тhе effective method for increasing thermal oxidation stability of polymers is control of the physical structure [23]. Тhе additive effect оn the physical structure of РI film was studied with the help of X-ray structural analysis. Тhе film possesses mesomorphous regularity, of

E. V. Kalugina, N. V. Gaevoy and K. Z. Gumargalieva et al.

210

which the presence of аn intrachain order in the absence of interchain packing regulation is typical. As shown оn the diffraction patterns, such structure manifests itself bу а single narrow peak of the intrachain order (5 - 6 deg) and wide amorphous halo (Figure 2). Diffraction patterns show high intrachain orderliness of the stabilized sample. This difference is preserved still after 1,000 h of aging at 300oС at total reduction of the intrachain order. Similar situation is observed оп diffraction patterns for liquid-crystal polymers (Figure 3), stabilized bу cyclic phosphites derived from pentaerythtitol Irgafos 126 (Ciba). However, stabilization mау just partly bе associated with the intrachain order increase in the presence of РСА. РСА are also effective in amorphous polymers, such as PSF and PPQ [3]. As shown bу the experiment, crosslinking proceeding during aging of PI films, both stabilized and non-stabilized, does not cause аnу significant change in molecular mobility of the polymer and hindrance of oxygen diffusion deep in the sample. Crosslinking intensification bу РСА injection is disproved bу the data оn Р АI and РРА melt viscosity decrease in the presence of РСА.

Intensity

1

2 3

4

10

15

20

25

30

35

Figure 3. Diffraction pattem for LCP derived from ТРА, IPA, р-ОВА and DODP without additives (1, 2) and added bу 0.5% lrgafos 126 (3, 4): (1, 3) prior to heat treatment and (2, 4) after thermal processing; Т= 300oС; 5 h in air.

The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 211 Тhе studies performed with industrial and model РAI, РРА, PEI and PSF samples, aimed at determination of РСА action as deactivators of admixtures in heat-resistant polymers gained positive results. PSF high-temperature oxidation is slowed down bу low РСА additions. Though organic phosphites, specifically HP, are of the highest efficiency and their stabilizing action is spread upon the whole complex of degradation manifestations, other РСА classes, even red phosphorus, are positively active, mostly stabilizing color. Analysis of the literature data [24-36] concerning phosphite activity at low-temperature oxidation (initiated self-induced oxidation of hydrocarbons and polyolefins) and behavior of polymers with phosphorus-containing additives at pyrolysis in the sub-flame zone indicates possible mechanisms of phosphorus stabilizing activity. These mechanisms are taken into consideration in the analysis of PSF stabilization during processing: − − − −

phosphorylation or other chemical interactions between SHP macromolecules or labile and oxidized structures; inhibition of high-temperature oxidation radical reactions; transition metal admixture deactivation; other mechanisms, for example, deactivation of electron-excited states.

and

PSF

Feasibility of the stabilization molecular mechanism was estimated bу NMR analysis of pentaerythritol diphophite АО-118 mixtures with oligosulfones (the polymerization degree 5 7), 4,4'-dichlorodiphenylsulfone, or bisphenol А at 280 – 300oС in vacuum and in air. Under the condition of interaction with phosphite, high content of end ОН- and Cl-groups in the oligomer and monomers provides for high resolution observation of phosphorylation bу spectral methods. 13C NMR spectra of heat treated mixtures preserve substrate reflexes and their relations that testify about the absence of molecular interactions. Оn the other hand, heating leads to phosphite decomposition, e.g. hydrolysis to corresponded monophenol and acid pentaerythritol diphosphite, signals from which at 64.5 and 63.4 ррm indicate dominance of tautomeric, four-coordinated shape: Phosphite additives to preliminarily degraded PSF and further heat treatment do not make the polymer color lighter and, according to IR spectra, have по effect оn intensity of absorption bands associated with oxidized structures, for example, carbonyl groups. То put it differently, phosphorylation, noticeable, as the heat stabilization mechanism in other systems, for example, at РЕТ and РММА combustion and pyrolysis inhibition [31] or thermal oxidation of synthetic rubbers and vinylchloride polymers [18], is not observed during inhibited high-temperature PSF degradation.

OCH

CH O 2

2

H (O) P

P (O) H

C OCH 2

CH O 2

E. V. Kalugina, N. V. Gaevoy and K. Z. Gumargalieva et al.

212

3

4

0,5

6

5

1

ΔO2, Mol/kg

2

0,25

0 0

50

100

Time, min

150

200

250

Figure 4. РЕ oxidation kinetics in the presence of SHP: Irgafos 126 (1, 2), Stafor 11 (3,4) and its acid ester (5,6) without water absorption (1, 3, 5) and with water absorption (2, 4, 6); Т = 200oС, Р(02) = 399.9 kPa

SHP high-temperature stabilization bу additives show signs of radical inhibition: low effective concentrations (optimally, 1 - 1.5 mmol/kg), the efficiency O2 pressure (in the absence of O2 the efficiency is negligibly low). SHP decelerate the homolytical process of PSF macromolecule branching during thermal oxidation. Higher efficiency of cyclic SHP, compared with ореn ones, in the high-temperature oxidation process is, apparently, the general rule, because analogous dependence is displayed at РЕ high-temperature oxidation (Fig.4). This mау bе considered as the model of really radical, high-temperature process. The increase of SHP effectiveness with hydrolysis probability, shown in experiments with water linking, indicate the significant role of acid esters in inhibition of SHP hydrolysis products. Cyclic SHP of Irgafos 126 -type possess chemical shift оn 31P nuclei equal 115 120 ррm, whereas low-effective SHP of tris-(2,4-di-tert-butylphenol)phosphite and соmmоn triarylphosphites possess chemical shifts of about 130 ррm, and trialkylphosphites - 137 - 139 ррm. Since in all cases Р-О bond is observed, i.e. at the first glance аnу change in electronegativity of the partner is absent and changes in SHP chemical shifts (at obvious absence of steric hindrances effect оn the chemical shift) are associated with the differences in values of О-Р-О bond valent angles in cyclic and ореn SHP, in accordance with the definition of 31 Р chemical shift [42]: Δδ = -с Δχα + к Δnπ + А ΔQ

Δχα is the difference in electronegativity values of P-X-bonds; Δnπ is the change in π-еlесtron overlapping; ΔQ is the change of σ valent angles. Тhе change of valent angles

where

causes changes in configuration of the electron cloud around phosphorus nucleus, i.e. the nucleus screening is changed. Formally, the effect is adequate to the change in electronegativity of partners bonded with phosphorus. Chemical shifts оn 1H, 13C and, apparently, 31P nuclei is inversely proportional to electronegativity of the partner nucleus [34], i.e. electronegativity of P is somehow reduced in the phosphite sequence: cyclic alkylene-aromatic > aromatic > aliphatic.

The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 213 According to Poling's electronegativity row atoms Р, С and О have equals 2.1, 2.5 and 3.5. P-O-bond is possesses much higher polarity than C-O-bond. That is why P-O have less hydrolytically stability. Bulky groups of the tert-butyl type in the ortho-position at the ester bond makes steric hindrances to hydrolysis (kinetic mechanism). Changes in valent angles in the six-term steric phosphites reduce polarity of the ester bond and, as а consequence, its hydrolyzing ability (thermodynamic mechanism). For cyclic SHP, both mechanisms of hydrolytic stabilization are realized. Therefore, it is proved experimentally that these phosphites, for example, Irgafos 126 and ets. are most resistant to hydrolysis [36]. Finally, hydrolysis of ореn phosphites, including ореn SНP, leads to НзРОз, which is low-effective high-temperature stabilizer. At hydrolysis of cyclic SHP alkylene-ester structure is preserved (NMR data), and the final product (acid cyclic phosphite) is the effective high-temperature stabilizer. This was shown bу direct comparison of effectiveness of Stafor 11 (Russian additive) and its acid analogue, specially synthesized for tests. For example, tests performed оn reometer - IIRT device at 320oС, Stafor 11 makes PSF color lighter, increasing the light transmittance index bу 3 - 5 units. For PSF acid ester, this index is increased bу 10-12 units, though differences in other indices are not so great. Table 1. The dependence of SHP effectiveness on polysulfone purity.

Sample PSF with [Fe]= 5*10-5 wt.% The same sample after IIRT The same sample added 0.3 wt..% Irgafos 126 PSF with [Fe]= = 5*10-5 wt.% The same sample after IIRT The same sample added 0.3 wt..% Irgafos 126

mиn

Transmittance at Λ= 425 nm, %

Moment molecular-mass distribution Mz 103

-

-

73.0

99.5

3.5

1.02

78.0

94.0

4.3

1.01

73.0

98.0

-

-

60.0

87.0

3.7

1.5

68.0

58.0

4.2

1.05

63.0

80.0

MFI(10 mиn, 320оС), g/10 mиn

MFI10 mиn / MFI 20

Indirectly, the radical mechanism of SHP stabilizing activity is confirmed bу additive elimination of active degrading effect оn PSF from the side DMSO. As shown bу the strength of C-S-bonds in (CH3)2SO2, equal 264 kJ/mol [37], and higher total reactivity of sulfoxides compared with sulfones [38], DMSO is not heat resistant compound. At low temperature (about 100°С) molecular thermal cis-splitting happens with аn olefin formation, though at higher temperatures homolytical C-S-bond break is suggested [38]. Actually, over sixteen main products of DMSO degradation at PSF processing temperature (the ampoule technique) were detected bу the mass-spectrometric method. The highest yields are observed for dimethyl disulfide, methyl ethyl sulfide, methyl and ethyl mercuptanes, 3-hydroxypropyl methyl sulfide, methylethoxymethylsulfide, and similar substances, which formation mау bе explained with respect to alkyl and alkylthio-radical recombination, as well as labile oxygen

E. V. Kalugina, N. V. Gaevoy and K. Z. Gumargalieva et al.

214

exchange reactions in semipolar sulfoxide group. As PSF is processed, DMSO residues play the role of аn original radical initiator of degradation, and SHP addition eliminates this effect. The idea to deactivate metal admixtures, first of аll, iron compounds bу SHP additives follows from extremely much higher efficiency of SHP in "impure" samples compared with almost pure ones (Table 1). If аn iron compound (uр to 0.005 wt.%) is injected to "pure" PSF, light transmittance will bе decreased bу 20 - 30 units, whereas subsequent injection of SHP reduces this effect significantly. Оn the other hand, PSF color may bе stabilized in tests simulating processing of phosphorus-containing transition metal complexes bу additives.

Absorbtion,%

3

6

2

1

4

5

230

240

250

260

270

280

290

300

310

320

330

340

350

λ nm

Figure 5. UV-spectra for chloroform solutions of Irgafos 126 (1), ferrocene (2), and their mixture (3). Differential spectra: mixture – Irgafos 126 (4), mixture-ferrocene (5); calcиlated additive Irgafos 126ferrocene spectrиm (6).

Diphenylphosphonic salt additions (cations Со, Cr, Ni, Сu) uр to 0.1 wt. % stabilize PSF similar to polyalkane imide, though these effects in PSF are not so high as in case of SHP use.

The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 215 Тhе simplicity of phosphite interaction (Irgafos 126, in particular) with transition metal compounds is shown by UV -spectra of Irgafos 126 and model substance (ferrocene), and their mixture chloroform solutions (Fig. 5). At room temperature phosphite and ironcontaining model interact at оnсе, which causes а noticeable deviation of experimental UVspectrum from calculated (additive) оnе. This interaction represents аn example of соmmоn complex-forming function of phosphorus compounds. With respect to the type of substitutes and coordination degree, phosphorus atom or phosphoryl oxygen is electron donor. Тhе electron lone pairs of these atoms is transferred to empty or partly filled α-orbitals of neighboring atom of metal. Phosphorus-metal complexes are strongly bound due to relatively low potentials of phosphorus compound ionization and additional linking of π-electrons because of donor and acceptor (metal) vacant α-orbital overlapping [40]. Тhе donor-acceptor interaction is оnе of the main mechanisms for metal compound extraction. The extraction ability correlates with the distribution of electron density in extracting agents, including phosphorus-organic compounds [39]. Correlations between effective extraction parameters defined bу therrnodynamics of the donor-acceptor bond and the so-called "effective charge" at phosphorus bу which electron density distribution in molecule is described, and associated parameters of substitute electronegativity with 31P NMR chemical shift as well. Generally, dependencies of the extraction effective constant (K) logarithm are linear: IgK = А - Bf, where А and В are constants defined bу the metal type and parameter f, f is the parameter characterizing electron density, for example, bу the sum of electronegativity values of substitutes at phosphorus atom. There are data [39] оn effective charges оn phosphorus atoms in manу phosphorus-containing compounds. Effective charges are determined from X-ray diffraction pattems bу the energetic shift of phosphorus absorption range boundaries. Iron admixtures significantly speed uр thermal oxidation of аll studied heat-resistant polymers. РСА injection fully eliminates this acceleration. Therefore, РСА stabilizing effect in heat-resistant polymers may be explained bу metal admixture binding. The products of "model + stabilized" system (equimolar mixture of Nphenylphthalimide and AFF) thermal transformation were analyzed with the help of NMR-sресtгоsсору technique. It is shown that at 250 – 300oС the model does not transform, and the stabilizer partly degrades forming diphenylamine, phenol, phosphoric acid and its condensation products. Аll these compounds are not stabilizers of PI, РAI, РРА and other compounds or display much lower stabilizing action than initial AFF. As а consequence, the stabilizing action is defined bу either the initial РСА structure or intermediate products of stabilizer transformation. The оссurrеnсе of ESR signal (а singlet with ΔH = 9.1 Е and g = 2.0003) allows а suggestion that stabilizer thermal transformation products are of the radical origin. Emission extinguishing in PI film is observed bу fluorescence spectra at 520 - 530 nm under the effect of AFF additive. The paramagnetism increase as а result of degradation in stabilized samples is much lower than in non-stabilized polymers. This is reproduced both in PI and PAI. Therefore, if electron excitation is considered as the oxidation initiation, endoperoxide formation, etc., thermal activation of the imide structure transfer to the electronexcited state in stabilized samples is hindered. Thus, the investigation performed allowed the exclusion from consideration unreliazable or weakly realizable РСА effect оn heat-resistant polymer cyclization and crosslinking and

E. V. Kalugina, N. V. Gaevoy and K. Z. Gumargalieva et al.

216

detection of the most probable stabilization mechanisms - admixture bonding and inhibition of radical-chain oxidation processes. Optima РСА concentrations of 2 - 5 wt.% in PI, PPQ, and РВО and 0.5 - 1.0 wt.% in PEI, РР А, PSF, and Р AI, e.g. -0.02 - 0.05 or 0.005 - 0.01 mol/base-mol, respectively. If оnе considers that the rate of translational diffusion of low-molecular substances in the rigid structure of heat-resistant polymers is low and mау not provide the additive transport to the oxidation focus, it may be concluded that inhibition is possible only in the additive interaction with macromolecule and changes of its reactivity. Experiments with models did not display phosphorylation, i.e. direct interaction between the additive and aromatic heterocyclic structure. In this case, apparently, а polymer additive соmрlех is formed, which changes the macromolecule reactivity in reaction to oxygen. The соmрlех formation may change the electron state of the whole macromolecule or а large part of it, i.e. change the reactivity of it. Clearly, conjugation blocks are present in the macrochain: PDI in PI and PAI, РРР in PPQ and соpolyimide phenylphenoxaline, amide-TPA in РРА, i.e. the products characterizing chain conjugation. Their output at thermal oxidation is decreased bу РСА injection, whereas carbon oxides yields are reduced bу 1.5-2 times only. Thus, basing оn the totality of experimental data оn РСА stabilization оnе mау conclude that the most probable stabilization mechanisms are additive deactivation, inhibition of radical oxidation processes and deactivation of electron-excited states.

BACKGROUND The thermal stability and thermal stabilization of the heterochain polymers were investigated. Analysis of PAI, PSF, PEI degradation and stabilization has allowed an approach to be developed to aid their processing and resolve similar problems with other resins such as polyethersulfone, LCP, ets Also PE oxidation kinetics in the presence of SHP

APPENDIX 1 Table 2. chemical names and structures of antioxidants used in article. Trade mark

Chemical structure

Irgafos 126 (Ciba)

O

O

O

O

O P

CH2

Stafor 11

Chemical name

O

O P O

P O

Bis-(2,4-di-tbutylphenol) Pentaerythritol Diphosphite

O-phenil-O,O[2,2’methilenbis(6-tbutyl-4methilphenil)]phosphite

The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 217

REFERENCES [1] [2] [3]

[4]

[5]

[6]

[7] [8] [9]

[10]

[11]

[12] [13]

[14]

[15]

Kovarskaya B.M., Blymenfeld A.B., and Levantovskaya I.I., Termal stability of heterochain polymers, Moscow, Khimia, 1977, 263 p. (Rus) Novotortseva N.G., ‘High-temperature stabilization of polybenzoxzole’, Сandidate disertation thesis, Moscow, 1987 (Rus) VdovinaA.L., ‘Thermal Transformation and stabilization of polypyromellitimide, polyphenylquinoxaline and Copolyimidophenylquinoxalines’ Сandidate disertation thesis, Moscow, 1987 (Rus) Mukmeneva N.A., Akhmadulina А.а., Sabirova L.Кh., and Kirpichnikov Р.А., 'Intensification of the stabilizing efficiency of phosphoric ethers Ву four-valent titanium compounds during low density polyethylene oxidation', Vysokomol. Soed., 1976, vol. В18, рр. 108 -115. (Rus) Pobedimsky D.G., Orossman О., Kondratyeva T.N., Cherkasova О.А., Scheller О., Mukmeneva N.A., and Кirpichnikov Р.А., In: Proc. 4th International Symposium оn Homogeneous Catalysis, Leningrad, 1984. (Rus) Arbuzov В.У., Polezhaeva N.A., Vinogradova V.S., Polozova G.I., and Musina А.А., 'Structure and properties of interaction products of benzylidene benzoyl acetate with trimethylphosphite and dimethylphosphoric acid, Izv. AN SSSR, Ser. КЫт., 1974, No. 9, рр. 2071 - 2075. (Rus) Pobedimsky D.G., Mukmeneva N.A., and Kirpichnikov Р.А., In: Developments in Polymer Stabilization, Ed. Scott G., London.: Appl. Sci. РиЫ., 1980, vol. 2, 125 р. Mukmeneva N.A., Minsker K.S., Kolesov S.V., and Kirpichnikov Р.А., Dok/ady AN SSSR, 1984, vol. 274(6), рр. 1393 - 1396. (Rus) Kirpichnikov Р.А., Mukmeneva N.A., and Pobedimsky D.G., 'Phosphorus-organic stabilizers of polymers - efficiency and echanism', Uspekhi КЫти, 1983, vol. 52(11), рр. 1831-1851. (Rus) Mukmeneva N.A., Gol'denberg A.L., and Lazareva N.P., 'Interaction between phosphoric acid ethers and carboxylic groups in polyethylene', Vysokomo/. Soed., 1983, vol. А25(6),рр. 1302 - 1306. (Rus) Pobedimsky D.G., Кirpichnikov Р.А., and Denisov Е.Т., 'About reactions of phosphorus-organic inhibitors with hydroperoxide groups and polyethylene peroxide radicals', Vysokomo/. Soed., 1976, vol. А18, рр. 2650 - 2658. (Rus) Hudson R., Structure and Mechanism о/ Reactions with Phosphorus-Organic Compounds, Moscow, Mir, 1967,361 р. (Rus) Mukmeneva N.A., Sharifulin A.Sh., Eliseeva L.A., and Iskhakov О.А., 'Phosphorusorganic inhibitors of polymeric material combustion', Proc. 61h All-Union Соп! Combustion о/ Po/ymeric Materia/s, Suzdal', Nov. 29 - Оес. 01, 1988, Rep. Thes., Moscow, 1988, рр. 156 - 157. (Rus) Ruger С., Konig Т., and Schwetlick. К., 'Phosphororganische Antioxidatien. 6. Einflus Cyclischer Phosphite auf die Radikalisch initierte Oxidation уоп Kolenwasserstoffen und Polymeren', Acta Po/ymerica, 1986.-Bd. 37(7), S. 435 - 438. Schwetlick К., Konig Т., Ruger С., Pionteck J., and Habicher W.D., 'Chain-breaking antioxidant activity of phosphite ester' , Ро/ут. Degrad. Stability, 1986, vol. 15, р. 97 108.

218

E. V. Kalugina, N. V. Gaevoy and K. Z. Gumargalieva et al.

[16] Schwetlick К., Konig Т., Pionteck J., Sasse D., and Habicher W.D., 'Organophosphorus antioxidants. 9. Inhibition of the oxidation of hydrocarbons Ьу hindered aryl phosphites', Ро/ут. Degrad. StabiZity, 1988, vol. 22(4), рр. 357 - 373. [17] Lebedeva L.P. and Levin Р .1., 'Antioxidant efficiency of phosphites and their mixtures', Vysokoтo/. Soed., 1982, vol. В24(5), рр. 379 - 383. (Rus) [18] Mukmeneva N .А., 'Phosphorylation as the way of increasing stability of polymers', Proc. 8th All-Uпioп Schoo/-Seтiпar оп Orgaпoeтeп tCoтpouпds, Moscow, INEOS AN SSSR, 1984,22 р. (Rus) [19] Kalugina Е.У., 'Thermal transformations and stabilization ot" some heat-resistant heterochain polymers', Candidate Dissertation Thesis, Moscow, 1992. [20] Andreeva М.В., 'Thermal transformations and stabilization of aliphatic-aromatic polyamides and derived mixtures', Candidate Dissertation Thesis, Moscow, 2002. (Rus) [21] Korshak V.V., Different Unit Composition оf Polymers, Moscow, Nauka, 1977,302 р. (Rus) [22] Kandratiev V.N. and Nikitin Е.Е., Cheтica/ Processes in Gases, Moscow, Nauka, 1981,262 р. (Rus) [23] Emanuel N.M. and Buchachenko A.L., Cheтical Physics оf Polyтer Aging and Stabilization, Moscow, Nauka, 1982,359 р. (Rus) [24] Gamino О., Martinasso О., and Costa L., 'Thermal degradation of pentaeritritol diphosphat model compound for fire retardant intumescent systems. 1. Overall thermal degradation, Polyт. Degrad. Stab., 1990, vol. 27(2), рр. 285 - 269. [25] Suebsaeng Т., Wilkie С.А., Burger У.Т., Carter J., and Brown С.Е., 'Solid products from thermal decomposition of polyethylenterephtalate of investigation Ьу CPIМass, I3C-NMR and Fourier transform IR-spectroscopy', Eur. Polyт. J., 1981, vol.17(2), рр. 1259 - 1263. [26] Becher С.Н., Troer К., and Croleva А., 'Thermal properties P-contents PETF', Eur. Polyт. J., 1981, vol.17(2), рр. 1259 - 1263. [27] Troer к., Grozeva А., and Borisov О., 'Introduction of phosphorus into the PETmolecule via 1,2-dicarbomethoxyethyl phosphate', J. Appl. Polyт. Sci., 1981, vol. 17(1), рр. 27 - 33. [28] Wilkie С., Pettegrew J., and Brown С., 'Pyrolysis reactions of poly(methyl methacrylate) and red phosphorus: ап investigation with cross-polarization, magic angle NMR-spectroscopy' J. Polyт. Sci.: Polyт. Lett. Ed., 1981, vol. 19, рр. 409 - 414. [29] Day М. and Wiles D., 'Temperature influence оп thermal degradation of fiber PETF, опе fire retardant tris(2,3-dibromopropyl)-phosphate', J. Aпal. aпd Appl. Pyrol., 1984, No. 7, рр. 65 - 82. [30] Inagaku N., Sakurai S., and Katsuura К., 'Affect tris(2,3-bromo-propyl) phosphate with flame retardant of polystyrene', J. Appl. Polyт. Sci., 1979, vol. 23, рр. 2023 - 2030. [31] Brown С., Wilkie с., Smukalla J., and Cody В., 'Inhibition Ьу red phosphorus of unimolecular thermal chain scission in poly(methyl methacrylate): investigation Ьу NMR, FT-IR and laser decompositionl Fourier transform mass spectroscopy, J. Polyт. Sci.: Polyт. Cheт. Ed., 1986, vol. 24, рр. 1297 - 1311. [32] Day М. and Willes D., 'Combustion and pyrolysis of poly(ethylene terephthalate). 1. ТЬе role of flame retardants in products pyrolysis', J Appl. Polyт. Sci., 1981, vol. 26, рр. 3085 – 3091

The Degradation Heterochain Polymers in The Presence of Phosphorus Stаbilizers 219 [33] Gorestein О., Phosphorus-31 NMR- Principles and Applications, N.Y.: Academic Press, 1984, 14 р. [34] Ionin B.I., Ershov В.А., and Kol'tsov A.I., NMR-Spectroscopy in Organic Cheтis('J: Leningrad, Khimia, 1983,269 р. (Rus) [35] Gordon А. and Ford R, Chemist Companion, Moscow, Mir, 1976,541 р. (Rus) [36] Spivack J., Pastor S., and Patrl А., Ро/ут. St. J., 1984, рр. 247 – 257. [37] Energies оf Chemical Bond Break, Ionization Potentials and Affinity tо Electron, Moscow, Nauka, 1974,351 р. (Rus) [38] Sigeru Оае, Cheтistry ofSulfur Orgaпic Coтpouпds, Moscow, Khimia, 1975, Ch. 6. (Rus) [39] Mazalov L.N. and Dyumatov У.О., E/ectroпic Structure о/ Extrageпts, Novosibirsk, Nauka, 1984, 196 р. (Rus) [40] Gur'yanova E.N., Gol'dstein I.P., and Romm I.P., The Doпor-Acceptor Boпd, Moscow, Khimia, 1973, 338 р. (Rus) [41] Atlas of ESR Spectra - Spin Labels and Probes, Ed. A.L. Buchachenko, Moscow, Nauka, 1977, 159 р. (Rus) [42] Gorestein D., Phosphorus-31 NMR- principles and applications. NY: Academic Press, 1984, 14p.

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.221-225 ©2010 Nova Science Publishers, Inc.

Chapter 16

QUANTUM-CHEMICAL CALCULATION OF OLEFINS OF CATIONIC POLYMERIZATION BRANCHING IN γ-,δAND ε− POSITION ON RELATIONS TO DOUBLE CONNECTION BY METHOD MNDO V. A. Babkin1, D. S. Andreev*1, T. V. Peresypkina and G. E. Zaikov±2 1

403343 SF VolgSABU, c. Mikhailovka, region Volgograd, s.Michurina 21 Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia

2

ABSTRACT Quantum-chemical calculation of olefins of cationic polymerization branching in γ,δ- and ε− position on relations to double connection of molecules of 5-methylgepten-1, 5-methyloсtene-1, 6-methylokten-1 was done by method MNDO. Optimized by all parameters by standard gradient method. Optimized geometrical and electronic structure of this compound is received. The universal factor of acidity was calculated (pKa=35). Molecules of 5-methylgepten-1, 5-methyloсtene-1, 6- methyloсtene-1 pertain to class of very weak Н-acids (рКа>14).

Keywords: quantum-chemical calculations, method MNDO, 5-methylgepten-1

AIMS AND BACKGROUNDS The aim of this work is a study of electronic structure of olefins cationic polymerization branching in γ-,δ- end ε− position on relations to double connection of molecules of molecules 5-methylgepten-1, 5-methyloсtene-1, 6- methyloсtene-1 and theoretical estimation

* 403343 SF VolgSABU, c. Mikhailovka, region Volgograd, s.Michurina 21, E-mail:[email protected]

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of its acid power by quantum-chemical method MNDO. The calculation was done with optimization of all parameters by standard gradient method built-in in PC GAMESS [1]. The calculation was executed in approach the insulated molecule in gas phase. Program MacMolPlt was used for visual presentation of the model of the molecule. [3].

RESULTS OF COLCULATIONS Geometric and electronic structures, general and electronic energies of olefins cationic polymerization branching in γ-,δ- end ε− position on relations to double connection of molecules 5-methylgepten-1, 5-methyloсtene-1, 6- methyloсtene-1 was received by method MNDO and are shown on fig. 1-3, and in tabl.1-4 . The universal factor of acidity was calculated by formula: pKa = 42.11-147.18*qmaxH+ [2] (where, qmaxH+ − a maximum positive charge on atom of the hydrogen (by Milliken [1]) R=0.97, R− a coefficient of correlations, qmaxH+=+0,05 (for 5-methylgepten-1, 5-methyloсtene-1, 6- methyloсtene-1 qmaxH+ alike tabl.1)). pKa=35.

CONCLUSIONS Quantum-chemical calculation of molecule 5-methylgepten-1, 5-methyloсtene-1, 6methyloсtene-1 by method MNDO was executed for the first time. Optimized geometric and electronic structures of these connections were received. Acid force of molecules 5methylgepten-1, 5-methyloсtene-1, 6-methyloсtene-1 was theoretically evaluated (pKa=35). These connections pertain to class of very weak Н- acids (рКа>14).

Figure 1. Geometric and electronic molecule structure of 5-methylgepten-1.(Е0= -105377 kDg/mol, Еel= -496918 kDg/mol).

± Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygin Street, 117334 Moscow, Russia.Email:[email protected]

Quantum-Chemical Calculation of Olefins of Cationic Polymerization Branching… 223 Table 1. Optimized bond lengths, valence corners and charges on atoms of the molecule of 5-methylgepten-1. Bond lengths

R,A

Valence corners

Grad

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

1,34 1,51 1,54 1,55 1,54 1,54 1,09 1,09 1,10 1,11 1,11 1,12 1,12 1,12 1,09 1,10 1,11 1,11 1,11 1,11

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

127 113 118 114 114 122 124 119 108 110 109 108 104 111 111 113 111 113 111

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

Charge (by Milliken) -0.05 -0.12 +0.03 +0.01 -0.07 +0.04 +0.04 +0.04 +0.04 +0.05 +0.01 0.00 0.00 0.00 +0.01 -0.01 -0.01 -0.01 -0.01 -0.01 -0.01

Figure 2. Geometric and electronic molecule structure of 5- methyloсtene-1. (Е0= -120428 kDg/mol, Еel= -613019 kDg/mol).

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Table 2. Optimized bond lengths, valence corners and charges on atoms of the molecule of 5- methylo tene-1. Bond lengths

R,A

Valence corners

Grad

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

1,34 1,51 1,54 1,55 1,54 1,55 1,09 1,09 1,10 1,11 1,11 1,12 1,11 1,12 1,11 1,11 1,11 1,12 1,11 1,53 1,11 1,11 1,11

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

126 113 119 113 116 122 124 119 108 110 108 108 104 111 111 113 118 110 117 112 110 112

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

Charge (by Milliken) -0.05 -0.12 +0.03 +0.01 -0.05 +0.04 -0.01 +0.04 +0.04 +0.05 +0.01 +0.01 0.00 0.00 0.00 -0.01 -0.01 0.00 0.00 +0.01 +0.03 -0.01 -0.01 0.00

Figure 3. Geometric and electronic molecule structure of 6-methyloсtene-1. (Е0= -120454 kDg/mol, Еel= -605340 kDg/mol).

Quantum-Chemical Calculation of Olefins of Cationic Polymerization Branching… 225 Table 3. Optimized bond lengths, valence corners and charges on atoms of the molecule of 6- methylo tene-1. Bond lengths

R,A

Valence corners

Grad

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

1,34 1,51 1,54 1,54 1,55 1,09 1,09 1,10 1,12 1,11 1,12 1,12 1,12 1,12 1,12 1,54 1,11 1,11 1,11 1,54 1,11 1,11 1,11

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

127 113 116 118 122 124 119 108 110 108 109 107 108 108 114 112 111 112 111 111 111 112

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

Charge (by Milliken) -0.05 -0.12 +0.03 -0.01 +0.01 -0.07 +0.04 +0.04 +0.05 0.00 0.00 0.00 +0.01 0.00 0.00 +0.01 +0.04 -0.01 -0.01 0.00 +0.04 0.00 0.00 0.00

The Table 4. Total energy ( 0), electronic energy( el), maximal charge on atom of hydrogen(qmaxH+), universal parameter of acidity(рКа) of molecules of 5-methylgepten1, 5-methylo tene-1, 6-methylokten-1 №

Molecule

-Е0, kDg/mol

-Есв, kDg/mol

qmaxH+

рКа

1 2 3

5-methylgepten-1 5- methyloсtene-1 6- methyloсtene-1

105377 120428 120454

496918 613019 605340

+0.05 +0.05 +0.05

35 35 35

REFERENCES [1]

[2] [3]

M.W.Shmidt, K.K.Baldrosge, J.A. Elbert, M.S. Gordon, J.H. Enseh, S.Koseki, N.Matsvnaga., K.A. Nguyen, S. J. Su, and others. J. Comput. Chem.14, 1347-1363, (1993). Babkin V.A., Fedunov R.G., Minsker K.S. and anothers. Oxidation communication, 2002, №1, 25, 21-47. Bode, B. M. and Gordon, M. S. J. Mol. Graphics Mod., 16, 1998, 133-138.

In: Chemical Reactions in Gas, Liquid and Solid Phases…: ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.227-243 ©2010 Nova Science Publishers, Inc.

Chapter 17

THERMODYNAMICS FOR CATALASE AND HYDROGEN PEROXIDE INTERACTION A. A. Turovsky, A. R. Kytsya, L. I. Bazylyak and G. E. Zaikov N.M. Emanuel Institute of Biochemical physics, Russian Academy of Sciences, Moscow, Russia

ABSTRACT Thermodynamic analysis of the elementary reactions of the enzymic catalysis of hydrogen peroxide by catalase was carried out with the application of quantum-chemical calculations. A mechanism of the enzymic catalysis of hydrogen peroxide by catalase based on the Chorner scheme was proposed. Hypotheses about Michaelis’s complex [catalase + H2O2] forming which further decay conditioned by the low ionization potential of iron atom in the catalase were formed. It is shown that bioSAA (ramnolipid) forms the thermodynamically efficient complexes with the hydrogen peroxide. On basis of calculated thermodynamical and geometrical parameters it is shown that the catalase’s catalytic specificity causes by the hydroperoxides dimensions.

1. INTRODUCTION Live organisms can exist because of there ability to kinetic controlling of the chemical reactions thus depress the thermodynamic equilibrium achievement. The key role at such processes performs the biological catalysts (enzymes). The modern developments of the biological catalysis shows, that the enzymatic reaction proceeds accordingly to the general laws of the regular chemical interactions. Difference between the enzymic catalysis and the chemical catalysis is only the complicated structure of the first. Structure of the enzymes is the same as the proteins. From the point of view of thermodynamics the terms “primary structure, secondary structure, tertiary structure of the protein” designates the existence only one or bounded set of the states when free energy as a function of spatial structure (and as a function of non-covalent interactions between amino acid residuals of polypeptide chain correspondingly) is the lowest. Sub-isolated globules can

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be combined into the quaternary structure of the protein as a result of the interaction of the whole series of weak forces (hydrogen bond, hydrophobic or ion interactions). Prosthetic group may be connected up to protein part (apoenzyme) by covalent or weaker (like a hydrogen bond, hydrophobic or ion bonds) bonds. Such interactions orientate prosthetic grouping the space. Cofactor, which act the catalytic function, have to stay chemically invariable at the catalytic reaction. When the cofactor’s role plays the prosthetic group then the last carries out the whole catalytic cycle and is connected to the same enzyme molecule. At the some times cofactor may come into the more complicated interactions and play the role of the connecting chain between the molecules of enzyme and ensure the unity of enzyme system. Such cofactors terms the coenzymes. A structural peculiarity of the surface layer of protein globules allows to concentrate a lot of chemically different functional groups at the active centre. Such functional groups have an opportunity both to sorb the substrate and to chemical interaction with substrate. Formation of the strong chelate complexes is possible due to the fact that polypeptide chains did not connected strongly to the surface and characterize by some mobility. As a result of such mobility is the capability to stratification of certain sorption fragments of the globule upon the corresponding binding fragments of the molecule. Conformational changes of the enzymes were detected by the different chemical and physical techniques [1]. Increased microviscosity at the surface layer, induced by the decreasing of polypeptide chain mobility, play important role at the complexation. It is shown [2] that thermodynamical efficiency of enzyme catalysis determined by the values of free energies of intermolecular (in case of Michaelis’s complex formation) or intramolecular (at the transition state of reaction) bond E–R. Here E is an enzyme and R is radical of substrate forming the complex. Motivity of the catalysis is the energy of interaction of E and R at the transition state not at intermediate complex. However the rate of enzyme reaction will be increasing if the intermolecular interaction E–R at the activated state will be more preffered. So, the investigations of complexation thermodynamic can give the important information about Michaelis’s complexes structure. Unfortunately information connected to thermodynamic investigations of complexation reactions almost missing. Catalase was taken out as an subject of investigations. Unfortunately there is no sufficient information about its complete structure. Reliable information is connected only to the haem of iron. But exists the assumption that the haem of iron is an active center of catalase and exactly one is crucial for the chemical reaction of hydrogen peroxide decomposition. At the same time protein helixes only minimize the free energy of the three-dimensional structure of enzyme. Taking into account above-mentioned assumptions the aim of presented work were i) the investigations of the most verisimilar reaction of the complex “catalase – hydrogen peroxide” formation; ii) qualitative assessment of the processes which takes place at the active centre of catalase; iii) investigations of influence of surface-active substances (using the rhamnolipid as example) as a promoter; iv) valuation of thermodynamic characteristics above-mentioned processes with using of the quantum-chemical calculations.

Thermodynamics for Catalase and Hydrogen Peroxide Interaction

229

2. EXPERIMENTAL Quantum-chemical calculations were done using the MOPAC2009 program having applied the PM6 method. For the result’s visualisation was used the program Jmol.

3. RESULTS AND DISCUSSION 3.1. Interaction of Catalase and Hydrogen Peroxide It is assumed that cofactor of catalase is the verily prosthetic group (haem of iron), performs the whole catalytic cycle and is strongly connected to the enzyme molecule. However, in fact the protein molecule can be connected to any iron’s addend molecule and can conformationally affect to catalytic properties of the complex due to the interactions of catalytically active groups (–О–, –С(О)–, –NH–, –S– etc.) which are included into the protein molecule. Also we cannot accept the ionic bonds formation between the protein molecule and atom of iron.

Figure 1. Space pattern of the haem of iron.

Since the composition of the nearest to atom of iron protein component is unknown we have to assume that its influence onto the activity of catalase is minimum. But it is obvious that protein parts of enzyme have a significant influence onto its stability and decrease the free energy of the system. At the same time activity of the enzyme can increase because the spatial interactions of the nearest fragments of polypeptide chain which contains heteroatoms and can form coordination bond with the central atom of iron due to undivided electron pairs.

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At the first approximation we neglected such interactions, though in this case proposed model is not thorough. Since the coordination number of iron is 6 we can present the haem of iron as follows (Fig. 1). Since the atom of iron is charged positively and OH-group is charged negatively then haem of iron will be indicate as [Catalase × H2O]+ OH–. Such indication did not mean the ion pair formation because the OH-group exists not so far from iron (see Table 1) and forms a strong bond with the one. Such assumption can be confirmed by the value of complex’s constant of dissociation: [Catalase × H2O]+ OH– –> [Catalase × H2O]+ + OH– (ΔF = +191,5 kcal/mol), K = exp (–ΔF/RT) = 6,1 × 10–141. Thus we can affirm that haem did not dissociate. It is mean that OH-group sit at the internal coordination sphere. Such assumption can be also confirmed by fact that the solution of catalase did not carry current and did not form precipitate Ba(OH)2 after interaction with BaCl2. From the Table 1 we can see that distances Fe–N at the complex [Catalase × H2O]+ OH– are the same. Such fact testifies that the all bonds in the molecule are hybrid and are not both coordinating and covalent bonds. Such statement also confirms by a small values of charges of nitrogen atoms. The charge of atom of iron is equal to +1,143 that is the charge is heavily delocalized. Let us suppose that the coordinating bonds Fe–N forms due to presence of unshared electron pairs at the atom of nitrogen which are displaced up to atom of iron. The charges of oxygen’s atoms both in OH-group and Н2О are sufficiently great. Table 1. Geometrical adjectives of complexes of catalase

Distance Fe–N (5), Å Distance Fe–N (6), Å Distance Fe–N (7), Å Distance Fe–N (8), Å Distance Fe–O (OH), Å Distance Fe–O (H2O), Å Charge at Fe Charge at N (5) Charge at N (6) Charge at N (7) Charge at N (8) Charge at O (OH) Charge at O (H2O)

[Catalase × H2O]+ OH– 1.99 1.99 1.99 1.99 1.77 2.09 1.143 –0.033 –0.036 –0.029 –0.038 –0.501 –0.629

[Catalase × H2O2]+ OH– 2.07 1.95 1.95 2.03 1.78 1.81 1.495 –0.035 –0.269 –0.257 –0.016 –0.696 –0.584

Catalase (OH)2– 2.00 2.00 2.00 2.00 1.81 1.82 1.621 –0.189 –0.039 –0.183 –0.061 –0.745 –0.758

Certain intermediate complex forms as a result of interaction between catalase and hydrogen peroxide. Let us suppose that the first stage of catalytic reaction can be showed as follow: [Catalase × H2O]+ OH– + H2O2 –> [Catalase × H2O2]+ OH– + H2O

(1)

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Calculated thermodynamic parameters of the complexes are shown at the Table 2. Let us consider the thermodynamic of complexing. Values of ΔH, ΔS and ΔF of reaction (1) are equal to –37,1 kcal/mol, 24,5 cal/mol K and –43,9 kcal/mol accordingly. Calculated data shows that reaction of formation the complex between catalase and hydrogen peroxide is an exothermic and energetically efficient. The main geometrical adjectives of complex [Catalase × H2O2]+ OH– are presented at the Table 1 and its structure is shown at the Fig. 2.

Figure 2. Space pattern of the complex [Catalase×H2O2]+OH–.

It is necessary to notice that the distances Fe–N for fifth and eighth atoms of nitrogen a little increased while for sixth and seventh atoms a little decreased. Also the distance Fe–O (H2O2) is noticeably shorter in compare with such one for H2O. This fact evidences that the interaction between atom of iron and atom of oxygen for hydrogen peroxide is more strongly than such for water. The value of charge for iron’s atom is significantly increased too. It is means that the delocalization of the charge is too smaller. The charge of atom of oxygen for OH-group noticeably increased, it is means that the electrostatic bond Fe–OH is more strongly. It is necessary to notice that the charges of nitrogen’s atoms of iron’s addend are also changed. Such fact possibly can be explained by some changing of the geometrical adjectives of the reactive center.

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Table 2. Calculated thermodynamic and quantum-chemical parameters of parent materials, complexes of catalase and complexes of iron №

Substance (complex) H2O2 H2O2 × 2 H2O Rhamnolipid Rhamnolipid + Н2О2 H2O OH– H3O+ OH● [Catalase × H2O]+ OH– [Catalase ×H2O2]+ OH– Catalase (OH)2– C3O2H4N [Catalase ×C3O2H4N]+ OH– CH3OОH CH3O● [Catalase ×CH3OОH]+ OH– (CH3)3COОH (CH3)3CO● [Catalase ×(CH3)3COОH]+ OH– Catalase + Fe(III)–E O=Fe(IV)–E(+) [Catalase × H2O2×2 H2O]+ OH– [Catalase × H2O2 × Rhamnolipid]+ OH– [Fe2+ × 6 H2O] [Fe2+ × 5 H2O × H2O2] [Fe3+ × 5 H2O] OH– [Fe3+ × 6 H2O]

ΔH0, kcal/mol –24.0 –154.8 –519.5 –551.4 –54.3 –33.0 147.2 13.3 –190.6 –197.4 –261.4 –64.7 –210.8 –23.8 0.9 –163.2 –47.4 –20.5

ΔS0, cal/mol К 53.7 94.4 197.8 280.4 45.0 40.7 44.8 41.2 204.5 237.7 248.9 80.1 209.9 66.7 55.1 266.2 88.0 73.6

ΔF0, kcal/mol –40.0 –182.9 –578.4 –635.0 –67.7 –45.1 133.8 1.0 –251.5 –268.2 –335.6 –88.6 –273.4 –43.7 –15.5 –242.5 –73.6 –42.4

Ionization potential, eV 9.890 10.208 10.537 10.603 11.906 2.309 22.131 10.485 6.730 7.731 3.683 10.343 7.532 9.854 9.361 7.297 9.741 9.386

Electron eV 0.115 –0.099 0.042 –0.179 4.068 15.127 –6.124 –1.795 –0.792 –1.106 2.039 –0.374 –0.943 0.304 –1.124 –0.821 0.523 –1.267

–181.9

271.1

–262.7

7.303

–0.939

53.7 –131.5 –174.9

230.2 197.8 241.8

–14.9 –190.4 –247.0

10.684 7.359 7.658

–4.801 –1.036 –1.082

–322.8

276.9

–405.3

7.802

–1.076

–671.2

344.2

–773.8

7.389

–0.784

51.5 39.5 38.2 370.8

118.6 130.2 109.8 109.1

–16.2 0.7 5.5 338.3

16.781 16.861 17.354 23.024

–6.090 –9.191 –6.884 –12.527

affinity,

We can consider that interaction between catalase and hydrogen peroxide is a redox reaction (Chorner’s scheme) [3]. Catalase is a reducing agent and H2O2 is an oxidant. Such reaction can be presented as follow (2): [Catalase×H2O]+OH– + H2O2 –> [Catalase×H2O2]+OH– –> Catalase+ – OH– + OH– + OH•(2) But probably intermediate substance Catalase (OH)2– (Fig. 3) obtains due to box effect. Such intermediate substance Catalase (OH)2– easily react with proton H3O+ and turns into the initial substance. These interactions we can write as follow: [Catalase×H2O2]+OH– –> Catalase (OH)2– + OH• Catalase (OH)2– + H3O+ –> [Catalase × H2O]+OH– + H2O

(3)

Thermodynamics for Catalase and Hydrogen Peroxide Interaction (ΔH = –130,7 kcal/mol, ΔS = –44,2 cal/mol K, ΔF = –117,4 kcal/mol)

233 (4)

Let us consider the thermodynamics of the reaction (3). The values of ΔH, ΔS and ΔF of reaction are equal to –63,0 kcal/mol, 290,1 cal/mol K and –66,4 kcal/mol correspondingly. Presented data indicate that such process is exothermic and thermodynamically possible.

Figure 3. Space pattern of the complex Catalase (OH)2–.

It is necessary to consider some quantum-chemical characteristics of the process in detail. Presented in the Table 2 data show that ionisation potential of the substance [Catalase × H2O]+OH– is equal to 6,730 eV and electron affinity of hydrogen peroxide is equal to –0,099 eV. As a measure of interaction [Catalase × H2O]+OH– + H2O2 with intermediate complex forming let us use the value of orbital energy [4]. Taking into account the fact that upper occupied orbital of catalase and lowest unoccupied orbital of hydrogen peroxide are stereospecific we can evaluate the value of orbital energy of complex [Catalase × H2O2]+OH– forming with using the equation

const . Here I is an ionisation potential and E is an electron I−E

affinity. For this reaction the value of orbital energy is equal to 0,0015*const eV. The value of orbital energy of forming the intermediate complex Catalase (OH)2– (reaction (3)) is a shade higher and equal to 0,0017*const eV. The lengths of bonds Fe–N of substance Catalase (OH)2– are rather more equal (Table 1) than the same at complex [Catalase×H2O2]+OH–. This fact testify that the space pattern of the intermediate complex Catalase (OH)2– rather more tend to the configuration of initial molecule of catalase. The bond Fe–O (OН) of the intermediate complex Catalase (OH)2– is

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longer then the the same at complex [Catalase×H2O2]+OH– and the value of charge of the iron is a noticeably greater. Formation of the intermediate complex Catalase (OH)2– may be presented as a result of interaction of the intermediate reagent Catalase+–OH– and OH–anion. The ionisation potential of OH–anion is sufficiently small and equal to 2,309 eV (see Table 2). This implies that anion easily donate the electron to the oxidated form of iron and form the complex Catalase (OH)2–. Free radicals OH• can recombine or disproportionate and form the corresponding products of reaction. Let us consider the processes of formations of such products. On the basis of data of Table 2 we can calculate the thermodynamic parameters of the reactions (5) and (6). OH• + OH• –> H2O2 ;

(5)

(ΔH = +2,6 kcal/mol, ΔS = 28,7 cal/mol K, ΔF = –38,0 kcal/mol) OH• + OH• –> H2O +O••.

(6)

(ΔH = –65,7 kcal/mol) On the basis of presented values of thermodynamic parameters we can do some conclusions. The reaction of radical’s recombination (5) is a slightly endothermic and exist the possibility of its realization. On the other hand the value of ΔF of reaction of disproportionation (6) is less for 27,7 kcal/mol. That is this reaction is more prefer from the point of view of thermodynamic. The final product (oxygen) is obtained by the recombination of biradicals (O•• + O•• –> O2). This reaction characterize by a great exothermic effect (–268 kcal/mol). Let us consider the thermodynamics of the reaction (4). Thermodynamic parameters ΔH, ΔS and ΔF are equal to –130,7 kcal/mol, –44,2 cal/mol K and –117,4 kcal/mol correspondingly. The results of calculations show that the exothermic effect of the reaction is extremely great but the value of entropy is negative. The result of the negative entropy is decreasing of the free energy of reaction. Thus the reaction of catalytic interaction between catalase and hydrogen peroxide can be presented as follow. H O

2 [Catalase × H2O]+ OH– ⎯⎯2 ⎯ → [Catalase×H2O2]+ OH– –> Catalase (OH)2– + 2 OH● –

> H O+

⎯⎯3 ⎯→ [Catalase × H2O]+ OH–

(7)

Every presented stages and possibility of its proceeding are stated above. It is necessary to note that the same results of calculations were obtained in the case of changing of molecule H2O at the complex [Catalase × H2O]+ OH– to the fragment of polypeptide chain. The interactions and the results of calculations are presented below (eqs. (8) – (10)).

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[Catalase × C3O2H4N]+ OH– + H2O2 –>[ Catalase ×H2O2]+OH– + C3O2H4N (ΔF = –43,4 kcal/mol)

(8)

[Catalase ×H2O2]+ OH– –> Catalase (OH)2– + OH●

(9)

(ΔF = –66,4 kcal/mol) Catalase (OH)2– + H3O+ + C3O2H4N –> [Catalase ×C3O2H4N]+ OH– + 2H2O (ΔF = –118,4 kcal/mol)

(10)

As we can see the all stages are thermodynamically profitable. The space pattern of the complex [Catalase × C3O2H4N]+ OH– presented at the Fig. 4. It is permitted that peptide fragment possess some energetic barrier of rotating around the Fe–N bond and the interaction between Fe and N is minimal at the some conformation states of macromolecule. This assumption need the more detailed investigations but it can not be declined because the new nuances of biocatalysis may be explained in case of its confirmation.

Figure 4. Space pattern of the complex [Catalase × C3O2H4N]+ OH–.

Authors [5] propose the next scheme of interaction between catalase and hydrogen peroxide. Fe(III)–E –> O=Fe(IV)–E(+) –> Fe(III)–E

(11)

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But catalase is an electronically delocalized system and the term “valency” is ill-posed in accordance with the modern conception of matter’s structure. The thermodynamic calculations of presented scheme where done (eqs. (12), (13)). The values of thermodynamic parameters for all substances were obtained (see Table 2). Space pattern of the complexes are presented at Figures 5 and 6.

Figure 5. Space pattern of the complex Fe(III)–E

Figure 6. Space pattern of the complex O=Fe(IV)–E(+)

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Let us consider the process stage-by-stage. Fe(III)–E + H2O2 –> O=Fe(IV)–E(+) + H2O, (ΔF = –84,3 kcal/mol)

(12)

O=Fe(IV)–E(+) + H2O2 –> Fe(III)–E + H2O, (ΔF = +28,9 kcal/mol)

(13)

The values of free energies are calculated by the data presented at Table 2. Taking into account obtained results we can do the next conclusion. From the point of view of thermodynamics the first state of proposed process is virtual but the second is absolutely forbidden. It is means that proposed scheme (11) [5] is beneath criticism.

3.2. Interaction of Catalase and Organic Hydroperoxides As a rule enzymes are characterized by such peculiarity as stereospecificity. That is why we considered the interaction of catalase and organic hydroperoxides from the thermodynamic point of view. The reaction of complexation of catalase and methyl hydroperoxide (structure of the complex is presented at Fig. 7) is characterized by the next thermodynamic parameters: ΔH = –3,1 kcal/mol, ΔS = 40,0 cal/mol K, ΔF = –14,8 kcal/mol.

Figure 7. Space pattern of the complex [Catalase×CH3OОH]+ OH–.

On the basis of results of calculations we can do the next conclusion. As we can see from the presented results the values of ΔH and ΔF of reaction are too smaller than the same for

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reaction of complexing of catalase and hydrogen peroxide. This fact means that the interaction between catalase and methyl hydroperoxide is worse than hydrogen peroxide one and taking into account the errors of calculations may be impossible. At the carrying out of the calculations for the reaction of catalase and tertbutylhydroperoxide complex formation (Fig. 8) it has been determined that such reaction is not neutralized.

Figure 8. Space pattern of the complex [Catalase ×(CH3)3COОH]+ OH–.

Let us consider the catalase and organic hydroperoxides complex formation. Under interaction of catalase and hydrogen peroxide, methyl and tertbutyl peroxides the numerical values of electron affinity are equal to 0,115 eV, 0,304 eV and 0,523 eV respectively. Hence, we can calculate the values of orbital energies. There are equal to 0,00151*const eV, 0,00155*const eV, 0,0016*const eV. Presented data shows that the values of orbital energies at the series “H2O2 – CH3OOH – (CH3)C3OOH” increasing. It denotes that power inputs via complexation between catalase and hydroperoxides increase. But the predominant cause in such processes performs the volume of substrate’s molecule.

3.3. Influence of Solvation Upon the Catalytic Decomposition of Hydrogen Peroxide With the aim of determination of influence of solvation sphere upon the catalytic decomposition of hydrogen peroxide the thermodynamic of the following reaction was considered. [Catalase × H2O]+ OH– + H2O2×2 H2O –> [Catalase × H2O2×2 H2O]+ OH– + H2O

(14)

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239

(ΔH = –31,7 kcal/mol, ΔS = 23,0 cal/mol K, ΔF = –38,6 kcal/mol)

Figure 9. Space pattern of the complex [Catalase × H2O2×2 H2O]+ OH–.

Process of solvation of hydrogen peroxide by water is thermodynamically efficient. (15) H2O2 + 2 H2O –> H2O2×2 H2O (ΔH = –22,2 kcal/mol, ΔS = –49,3 cal/mol K, ΔF = –8,5 kcal/mol) The results of calculations show that the solvation acts to decreasing of entropy. It is mean that organization of complex H2O2×2 H2O (Fig. 10) is more than one of initial system. It is necessary to mark that the electron affinities of solvated and non-solvated molecules H2O2 are quite different. It is the reason of different values of orbital energies of complexation.

Figure 10. Space pattern of the complex H2O2×2 H2O.

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Let us consider the solvation of H2O2 by the molecule of rhamnolipid in accordance with the presented scheme with the aim of compare to the above mentioned process (space patterns of the rhamnolipid molecule and its complex with H2O2 are presented at Figs. 11 and 12). H2O2 + Rhamnolipid –> H2O2 × Rhamnolipid (16) (ΔH = –7,9 kcal/mol, ΔS = 28,9 cal/mol K, ΔF = –16,4 kcal/mol)

Figure 11. Space pattern of the rhamnolipid molecule.

Figure 12. Space pattern of the complex H2O2 × Rhamnolipid.

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This process is more thermodynamically prefer than the solvation of hydrogen peroxide by water. The electron affinity of the complex H2O2 × Rhamnolipid is rather greater than the one of H2O2×2 H2O. It is mean that electron transfer from the atom of iron at the catalase must be easier. Let us consider the complaxation of catalase and solvated by rhamnolipid hydrogen peroxide. [Catalase × H2O]+ OH– + H2O2 × Rhamnolipid –> [Catalase × H2O2 × Rhamnolipid]+ OH + H2O –

(ΔH = 16,5 kcal/mol, ΔS = –95,7 cal/mol K, ΔF = 45,0 kcal/mol)

(17)

Presented results of calculations show that such reaction is impossible from the point of view of thermodynamics. From the another hand the obtained experimental data show that the reaction of catalytic decomposition of hydrogen peroxide is strongly accelerated in the presence of rhamnolipid and its rate depend on the concentrations of the last. Unfortunately this fact can not be explain on the basis of presented data and needs the more detailed investigations.

Figure 13. Space pattern of the complex [Catalase × H2O2 × Rhamnolipid]+ OH–.

It is interesting to consider the process of interaction of H2O2 and Fe (II) at the acid medium. In accordance with the Chorner’s mechanism this reaction can be written as follow. Fe2+ + H2O2 –> Fe3+ + HO– + HO• (18) It is means that reaction proceed as a radical reaction. Formation of radicals is proven by [6]. However in water this reaction has to proceed in two stages and can be written as follow. Stage 1:

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[Fe2+ × 6 H2O] + H2O2 –> [Fe2+ × 5 H2O × H2O2] + H2O

(19)

(ΔH = –42,3 kcal/mol, ΔS = 2,9 cal/mol K, ΔF = –43,2 kcal/mol) Stage 2: [Fe2+ × 5 H2O × H2O2] –> [Fe3+ × 5 H2O] OH– + OH• (ΔH = –12,0 kcal/mol, ΔS = 20,8 cal/mol K, ΔF = 5,8 kcal/mol) [Fe3+ × 5 H2O] OH– + H3O+ –> [Fe3+ × 6 H2O]

(20)

(ΔH = 185,4 kcal/mol, ΔS = –45,5 cal/mol K, ΔF = 199,0 kcal/mol) In case of reaction of catalase and hydrogen peroxide the intermediate product Catalase (OH)2– forms as a result of reaction between complex “Catalase+ – OH–” and OH– anion which is the product of H2O2 decomposition. Hydroxyl posses a small value of ionization potential (2,309 eV) and can easily give back the electron to the atom of iron. However this phenomenon is not observed in the case of ionic reaction between Fe2+ and H2O2. Intermediate reaction “[Fe3+ × 5 H2O] + OH– + OH•” is not realize or proceeds very weakly with forming the product [Fe2+ × 5 H2O] OH– which turns back to the parent substance in accordance with the next scheme. [Fe2+ × 5 H2O] OH– + H3O+ –> [Fe2+ × 6 H2O] + H2O

(21)

In a case of such reaction proceeding we would observed the catalytic process of the hydrogen peroxide decomposition. But at the acid medium reducing of iron Fe3+ using the OH– group not realize because the competitive reaction OH– + H+ –> H2O which proceeds very rapidly. At the alkaline medium the stable substance Fe(OH)3↓ forms. That is why the data concerning the investigations of this reaction at the alkaline and neutral mediums are absent. It is necessary to note that the reaction of oxidation of ions Fe2+ by ions Fe3+ is not excepted. This reaction characterized by the values of activation energy and preexponential factor (lgA) equal to 9 – 11 kcal/mol and 7 – 9 correspondingly. The rate of intermediate reaction (22) can depend on the concentration of ions of hydrogen and ions Fe3+ × 5 H2O may exist in form Feaq3+ ⇔ Fe(OH)2+ + H+. Fe3+ × 5 H2O + OH– –> Fe2+ × 5 H2O + OH•

(22)

CONCLUSIONS Firstly, it is necessary to mark that the all presented thermodynamic quantum-chemical calculations are approximate and were done in accordance with the accepted scheme of interaction “catalase – hydrogen peroxide”. According to the presented scheme we neglected

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the protein part of the enzyme and only haem of iron was used in calculations. However, the protein part of enzyme (especially, the nearest fragments) can influences on the process of catalysis. Today's such influence was not investigated. But the next assumption can be done. It is known, that the protein fragments are characterized by some mobility (or rotation) which is realized with some energetic barrier. This rotation can results to the “key effect” that is the value of free energy when the formation of complex “catalase – hydrogen peroxide” will be energy-efficient. Taking into account the presented results and considerations we can do the next conclusions. The mechanism of enzyme catalytic decomposition of hydrogen peroxide by catalase based on the Chorner’s scheme is proposed. Thermodynamic analysis of elementary reactions of proposed scheme is done. It was formed hypotheses about Michaelis’s complex [catalase + H2O2] forming which further decay conditioned by the low ionization potential of iron atom in the catalase. A similar reaction of interaction between Fe2+ and H2O2 proceeds more slowly because the elevated ionization potential of reaction Fe2+ – 1 e– → Fe3+. It is shown that bioSAA (ramnolipid) forms the thermodynamically efficient complexes with the hydrogen peroxide. But forming of these complexes did not influence onto the free energy of interaction between catalase and hydrogen peroxide. It is probably that rhamnolipid molecule plays some role in the processes of orientation and adsorption of hydrophobic part of substrate and enzyme. Due to these processes the rate of catalytic decomposition of hydrogen peroxide in the presence of rhamnolipid sharply (up to 5 times) increase and depend on the concentration of the last. From the other hand rhamnolipid can play role of concentrating agent for H2O2 and in this way accelerate the catalytic reaction of hydrogen peroxide decomposition. It is shown that the catalase’s catalytic specificity causes by the dimensions of hydroperoxides which plays the main role in Michaelis’s complex forming.

REFERENCES [1] [2] [3] [4] [5] [6]

Jiali Gao, Kyoungrim Lee Byun, Ronald Kluger. Catalysis by Enzyme Conformational Change // In Topics in Current Chemistry v. 238, 2004, PP. 113–136. Berezin I.V. Osnovy phisicheskoy chimii fermentativnogo kataliza. Мoscow, Vysshaya shkola, 1977, 275 p. V.L. Antonovski, М. М. Buzlanova. Analiticheskaya himija organicheskikh peroksidov. Мoscow, Chimija, 1978, 307 p. Reakcionnaja sposobnost i puti reakcii. / Ed. by Klopman G.M., Мoscow, Мir, 1977 383 p. Chelikani P, Fita I, Loewen P. C. Diversity of structures and properties among catalases // Cell. Mol. Life Sci. 61 (2): 2004, p. 192–208. Haber, F. and Weiss, J. Uber die Katalyse des Hydroperoxydes. Naturwissenschaften 20, (1932). – 948–950.

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.245-247 ©2010 Nova Science Publishers, Inc.

Chapter 18

DR. RER. NAT. WOLFGANG FRITSCHE – SCIENTIST AND ORGANIZER OF INTERNATIONAL SCIENCE (SECRETARY GENERAL RTD. OF GESELLSCHAFT DEUTSCHER CHEMIKER, HONORARY PRESIDENT OF FEDERATION OF EUROPEAN CHEMICAL SOCIETIES) G. E. Zaikov* N.M.Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia

Dr. Fritsche is a well-known world organizer of Chemical Science within the framework of the German Chemical Society and International Union of Pure and Applied Chemistry (IUPAC). He was born on 11 March 1928 at Dortmund (Germany). His career has several important steps: doctorate in Inorganic Chemistry (University of Bonn) – 1954; Assistant to Technical Director Vereinigte Ultramarinfabriken Marienberg (Germany). After that he was Deputy Secretary of Gesellschaft Deutscher Chemiker (GDC) – 1960-1967; Secretary of GDC – 1968-1971 and Secretary General of GDC – 1972-1991. Wolfgang was retired in 1991. As a scientist he has had good success in the field of inorganic chemistry. His activity in the Federation of European Chemical Societies (FECS) included several very important steps: member of the Steering Committee (before 1970) and Founder member (after 1970). He was also member of Executive Committee (1970-1976), Secretary General (1976-1988), Chairman of the Council (1989-1992). He has position as Lifelong Honorary President from 1993.

* N.M.Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygin st., Moscow 119991, Russia, [email protected]

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Dr. Fritsche was very active as a member of the Finance Committee as well as ChemRAWN (Chemical Research Applied to World Needs) of the IUPAC 1982-1989 (together with prof. G.E. Zaikov from USSR), member of European Community Chemistry Committee (1973-1991). Wolfgang was nominated by UNESCO as Founding Member of the International Organization for Chemistry in Development (IOCD). We should did indication about his honorary activities in German bodies: 1972-1996 1981-1991 1972-1992 1972-1992 1972-1992 1972-1992

Member of the Supervisory Board of VCH-Publishers; Member of the Supervisory Board of Chemical Information Center in Berlin; Secretary of Deutscher Zentralausschuss fur Chemie (National Adhering Organization of UPAC); Secretary of the Association of Professors of Chemistry at German Universities; Member of the Board of the German Association of Technical and Scientific Societies; Member of the German National Committee of the World Energy Council

Dr. Wolfgang Fritsche has many medals and orders for his scientific activity as well as for activity as organizer. We should first of all remember about Honorary medal of FECS “for outstanding services to the Federation and for furthering of international cooperation in the field of chemistry” (!985), medal of the Societe Royale de Chimie of the occasion of the 100th Anniversary of the Belgian Society (1987), Lavoisier-Medal of the Societe Francaise de Chimie “pour l’ensemble des eminents services qu’il rend a la Communaute Europeenne des Chimistes en sa qualite de Secretaire General de la Federation Europeenne des Societes Chmiques” (1987), nomination as Life-Long Fellow of the Institute of the Chemistry of Ireland (FICI) – 1987, Honorary Document of the Societa Chimica Italiana “ comme espressione di profonda stima e del piu vivo ringraziamento per l’opera svolta, originale e richissima, nella promozione della cultura chimica e nella collaborazione internazionale, …” (1988), Gold Medal of the Fedaration of Technical and Scientific Societies of Bulgaria for the promotion of international cooperation in Europe (1990), Nomination as Honorary Fellow of the Royal Society of Chemistry on the occasion of the 150th Anniversary of the Royal Society of Chemistry – UK (1991), Honorary Document of the Chinese Chemical Society ‘for his great contributions in promoting friendship between Chinese and German chemical communities in the past decades” (1991), Carl-Duisberg-Medal of GDC for his merits for the benefit of Chemistry and the GDC (1991), Honorary Document of the Gesellschaft Osterreichischer Chemiker (Austrian Chemical Society) ‘in acknowledgement of his great merits for the Society” (1991), Hanus-Medal of Czechoslovak Chemical Society “for his merits in promoting cooperation in Europe and the furthering of the Czechoslovak Chemical Society effected thereby (1992), Thilo-Medal of Chemical Society of Germany “in acknowledgement of his contributions leading to the unification of the two Chemical Societies after the re-unification of Germany” (1992), Nomination as Life-Long Honorary President of the FECS (1993).

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Today Dr. Wolfgang Fritsche is 82 years old but he is full energy, force and ideas in the field of inorganic chemistry as well as in the field of organization and cooperation in chemistry.

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.249-250 ©2010 Nova Science Publishers, Inc.

Chapter 19

PROFESSOR VICTOR MANUEL DE MATOS LOBO ON HIS 70TH ANNIVERSARY Gennady E. Zaikov*1 and Artur J. M. Valente±2 1

N.M. Emanuel Institute of Biochemical physics, Russian Academy of Sciences, Moscow, Russia 2 Coimbra University, Chemical faculty, Coimbra, Portugal

Prof. Victor Lobo graduated in Physics and Chemistry from University of Coimbra in 1963. At the end of his first undergraduate year he was invited as an “assistant demonstrator” and as such helped in laboratory classes up to his final year. He had a University scholarship and a research grant from “National Institute of High Culture”. While an undergraduate student did research in radiochemistry. Invited to join the teaching staff of the University of Coimbra in 1963, he was asked to go “on leave” with his Professor to the University of Mozambique for three years, where he did research in flame photometry in parallel with his teaching activity.

* N.M. Emanuel Institute of Biochemical physics, Russian Academy of Sciences, 4 Kosygin Street, 119334 Moscow, Russia, [email protected] ± Coimbra University, Chemical faculty, Coimbra 3004-535, Portugal, [email protected]

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He has got a Ph.D. degree from the Cambridge University (U.K.), under the supervision of Dr. J. Agar, in 1971. During Ph.D. studies he developed a new isothermal diffusion cell, with which he received a Gold Medal at the “15th International Exhibition of Inventions and New Techniques”, Geneve, Switzerland in 1987. After completing his Ph.D. studies, he returned to Mozambique for about one year, finally to return to his post, as a lecturer, at the University of Coimbra in 1972. He was “Associate” Professor of Electrochemistry in 1975 and Full Professor in 1981. During his academic career he has been lecturing Electrochemistry and Corrosion, Electrolyte Solutions, Physical Chemistry, Chemical Thermodynamics, Chemical Analysis, Medical Chemistry and General Chemistry. Prof. Victor Lobo has dedicated much of his life to the research of thermodynamic and transport properties (in particular on diffusion) of electrolyte solutions, and through polymers as well, and electrochemistry, particularly metallic corrosion. In recent years, his interests have focused on the diffusion of multicomponent systems and diffusion of electrolytes through polymer membranes. He was invited researcher or visiting professor in several universities, such as University of East Anglia (U.K.), University of Canberra (Australia), University of Gottingen (Germany) and University of Regensburg (Germany). Prof. Lobo has been the main responsible for the organization (1984-1989) of a databank with data on density, viscosity, equivalent conductance, transport number, diffusion coefficients and activity coefficients of electrolyte solutions, for the Scientific Engineering Research Council, SERC, U.K.. He has given a considerable number of invited lectures in Universities, and conferences on issues related to transport properties. He has been titular member of IUPAC, and Portuguese representative in technical commissions of CEN and ISO. He was president of Portuguese Electrochemistry Society, President of General Assembly of Portuguese Chemical Society, Member of National Bureau of Education, and Head of the Chemistry Department. He published more than 150 scientific articles in international and national refereed journals. Moreover, he has published some 40 articles in the Portuguese daily press on matters concerning education. He has written and edited more than 10 books, of which considerable prominence must be given to the widely used “Handbook of Electrolyte Solutions” and “Self-diffusion of Electrolyte Solutions”, the latter written with R. Mills, published by Elsevier. He is the Editor of Portugaliae Electrochimica Acta – the journal of the Portuguese Electrochemical Society – since September 2002. Besides his professional skills he has a lovely character. On the occasion of his 70th birthday, it is with a great pleasure that we wish to Prof. Victor Lobo, who has dedicated many years to Academy and Science, good health and further achievements. Prof. Victor Manuel de Matos Lobo is full of energy and new ideas for research in electrochemistry, physical chemistry as well as in chemistry of highmolecular compounds.

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.251-261 ©2010 Nova Science Publishers, Inc.

Chapter 20

THE SCIENTIST WHO OUTSTRIPPED HIS TIME Revaz Skhiladze and Tengiz Tsivtsivadze Georgian Technical University, Tblisi, Georgia

ABSTRACT This article is dedicated to the 100 year anniversary of the birth of the Georgian prominent scientist, Professor Akaki Gakhokidze. He was one of the outstanding representatives of the Georgian school of chemistry. High theoretical preparation, mastery of experiments, unusual scientific flair and intuition allow him to leave a great and light footstep for posterity on the way of scientific research and pedagogical activity. The fundamental investigation of A. Gakhokidze won international recognition. His works were published and broadly considered in the special literature, monographs and manuals of chemistry. There were created the specific terms - “Synthesis of Gakhokidze”, the “method of Danilow-Gakhokidze” etc.

A. Gakhokidze was born on 14 August 1909 in the village Zeda Khuntsi, Martvili district of Georgia. Martvili belongs to the region of Mingrelia which is the native land of many remarkable persons in the past. They are the pride of Georgian people today. One of them on which we want to draw your attention to was a bright representative of the scientific elite of Georgia Akaki Gakhokidze, the person world renowned, scientist of international scale, who made a huge contribution in the development of chemical sciences. His initial education was received at home, in his village. At first he attended the Khuntsi elementary school and then proceeded to the seven year school. During school study he was a very diligent and industrious pupil. From 1924 A. Gakhokidze continued studies in Tbilisi Melikishvili college, which he graduated with high academic success after what he was sent for practice passing in Baku in 1927 where A. Gakhokidze begin work in the oil purification plant. There turned out that only theoretical preparation without practice is quite insufficient for him. He recalled later “I thought that I know very well oil technology but soon I understood that an engineer without practice is like to the unarmed warrior. I started to study everything, beginning from the

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simple worker, finishing the engineer. It is clear that all became easy for me and I have soon connected the theory with practice”. In 1928 A. Gakhokidze graduated the college very successfully and he begin work in the soap plant (next the chemical industrial complex) of Tbilisi. At the same time he begin preparation for examinations of higher school. On August of 1928 he entered in the polytechnic faculty of Tbilisi University. Parallel studying A. Gakhokidze interested the problem of sugar production in Georgia. In this time there were functioned two institute in the former USSR – in Kiev (one – educational, second - scientific research) where the sugar production problems were being solved. Student A. Gakhokidze was their frequent guest. For A. Gakhokidze the task to build the sugar production plant in Georgia was the one of great importance. He elaborated in Kiev the sugar production plant for Georgia. There is the fragment of recollection of A.Gakhokidze: “I composed and wrote the plant project right away, made up 30 design drawing, the explanatory letter took 400 page, in Tbilisi it was not required corrections. After it consideration professor I.Burjanadze signed the project on the it public presentation. This project drawing up gave me important knowledge”. When sugar production plant director in Kiev acquainted this project, he told to project author with admiration: “the building of sugar plant in Georgia must be realized by your project.” In June 1929 A. Gakhokidze successfully defended the sugar plant project and he became an engineer technologist. Since 1932 year A. Gakhokidze continued post-graduated study in Leningrad (St.Peterburg). He works there in the field of carbohydrate chemistry under direction of professor Danilow. S.Danilow was the student of Russian outstanding chemist A.Favorski, whose scientific activity goes back to the founder of organic chemistry Butlerow. A.Favorski was the head of Leningrad Chemical Society where in general meeting two times in month was being made the report about new discovery in chemistry. At 18 December 1935 professor Danilow in the next meeting made the brief information about Gakhokidze’s work. He noted that like simple aldehydes the sugars are proved analogous isomerization and there are obtained saccharinic acids. “One of our students represented dissertation about this problem”, told Danilow. Favorski asked him who was this young scientist and the last pointed to Gakhokidze. Later one jester chemist explained to Gakhokidze the reason of Favorski interest: “you are now his grandchild and he wanted to see you.” The joker meant following sequence: Zinin → Butlerow → Favorski → Danilow → Gakhokidze. At November of 1935 A. Gakhokidze successfully defends dissertation “Isomerization of glucose in saccharinic acid”. Scientific council by a solid vote appropriates to A. Gakhokidze the candidate‫ۥ‬s degree. In the same year was published his large experimental work in the German journal “Berichte der Deutschen Chemischen Gasellschaft”. This work had large comments between chemists. Method elaborated in it was included in the manual for students of higher school as the “Danilow-Gakhokidze method”. In 1939 A. Gakhokidze comes back in Georgia and begins work in the Institute of processing of a wood material as a docent. Since 1937 he works in Tbilisi Pedagogical State Institute as Dean and next as a Head of faculty of chemistry till his death. In 1934 A. Gakhokidze was sent again in Leningrad by recommendation of Georgian Science Academy as a person working for doctor's degree. Doctor dissertation was defended in 1948 very successfully. In this time he was a first doctor chemist in organic chemistry in Georgia. 1940-1947 years A. Gakhokidze worked (parallel of pedagogical activity) in the institute of chemistry of Georgia Science Academy and then in 1947-1950 – in the Scientific-Research

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Chemical- Pharmaceutical Institute in Tbilisi, where he founded the department of medicine technology. Professor A. Gakhokidse is most outstanding representative of Georgian Chemists School. His life credo is very many-sided. He was a kind and tactful person. There were not for him large or small problems. He with identical attention concerned them. All students for him were young men which should be become not only good specialists but also worthy citizens of our country. The fundamental investigations of prof. A. Gakhokidze have an international recognition. The goal of this article is to touch more detail to activity of prof. A. Gakhokidze in carbohydrate chemistry. He has elaborated original method of disaccharides synthesis by which there appeared a possibility to receive unknown (until then) type of disaccharides synthesis. This type compound caused a great interest because of foreigner scientists working on the same problem possibility of existence of new type (1,2- and 1,3- bond consisted) disaccharides except of trehalose, maltose and gentiobiose type (1,1-, 1,4- and 1,6-bond consisted) disaccharides. For example we can bring the schemes of synthesis of glucosyl- 2glucose (sophorose) and glucosyl-3-glucose (laminaribiose). From 2,3,4,6,-tetra-0-acetyl-αD-glucopyranosyl bromide(I) and 1,3,4,6–tetra-0-acetyl-β-D-gluco-pyranose (II) he obtained 2-0-β-glucopyranosyl-D-glucopyranose (sophorose, III): CH2OAc

CH2OAc O OAc

O +

OAc AcO

Br

OAc

ZnCl2, P2O5

OAc

AcO

AcO

OAc I

CH2OAc O OAc

OH

CH2OAc

II

O

Ac = COCH3

O

OAc AcO OAc

CH2OH O OH OH HO CH2OH

O

O OH HO OH III

CH3ONa

Revaz Skhiladze and Tengiz Tsivtsivadze

254

Structure of new disaccharide were determined by author by following way: by action of hydroxylamine on the disaccharide was obtained corresponding oxyme (IV) and by interaction of last with acetic anhydride is obtained acetylated nitrile of glucosyl -2-gluconic acid (V). Author transferred this substance into glucosyl-1-arabinose, via of saponification and nitrile group splitting, which does not act with phenylhydrasine: CH2OH

CH2OAc

OH OH

OAc CH

NOH

OAc

HO III

CH2OH

CH2OAc

O

O

C

N

AcO O

O

OH

OAc

HO

AcO OH

OAc IV

V

O HO HO CH2OH

OH

O

O

OH HO OH VI

By oxidation of disaccharide (III) (with brome water) the author received glucosil-2glucone acid (VII) and it’s derivatives, but by hydrolyze of acid – glucose(VIII) and glucone acid (IX): CH2OH O

CH2OH O

HO O

OH HO OH

XII

OH OH

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255

CH2OH OH OH

COOH

III

CH2OH

O

OH OH

OH

O

O

COOH

CH2OH

HO

+ HO OH

HO

OH

OH

OH

HO

CH2OH VIII

OH

IX

VII

Analogously, disaccharides with different monosaccharide residues (glucosyl-2galactose, galactosyl-2-galactose, galactosyl-2glucose, manosyl-2glucose, manosyl-2manose) has been obtained. From 2,3,4,6,-tetra-0-acetyl-α-D-glucopyranose (X) and 1,2-0-isopropyliden-4,6-0benziliden-α-D-glucopyranose (XI) A. Gakhokidze obtained 3-0-β-D-glucopyranosyl-Dglucopyranose (laminaribiose,XII): CH2OAc O

OCH2

H5C6

O

+

OAc OH

AcO

ZnCl2, P2O5

OH O

O

OAc

O

X

XI OCH2 O

H5C6

CH2OAc O OAc

O O

CH3

CH3ONa

O O

CH3

CH3 CH3

AcO OAc

By similar procedures, A. Gakhokidze synthesized disaccharides with different monosaccharide residues.After some tens years there were corroborated once again the structure of substances obtained by A. Gakhokidze by several scientists with application of modern physical and chemical research methods. Lately, it was proved that new type compounds discovered by A. Gakhokidze are widespread in nature and they have high physiologic (immunologic, antitumoral and others) activity. Methods of synthesis of those class carbohydrates in the world scientific literature are known under name of “Gakhokidze’s syntheses”.

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The central problems of A. Gakhokidze’s scientific researches were the investigation of physiological active compounds, their study and application. The theory of A. Gakhokidze was the foundation of application of carbohydrate as a transporter of medicine preparations. The principle of glycosylation of medicine means elaborated by A.Gakhokidze, which is based on the active transport of carbohydrate fragments in the cell membranes, represents a new approach of a creation of medicine preparations of task-oriented action. Application of these preparations in clinical pharmacology which are not solved in water has very important disadvantage which consists that their obtaining is possible only internal way or with external influence. Mentioned circumstance obviously restricts to the possibility of their usage in medical practice. A. Gakhokidze provided for great importance of conversion of medicine means in water soluble form in which was being given the possibility to introduce parenterally these medicines. In result there would be changed not only absorption but accelerated physiologic effectiveness and sharply decreased the preparation toxicity. All above spoken pointed out to actuality of synthesis development of water soluble medical preparations. It was the question of very great significance solution of which had a great importance in the provision of population by medical preparations. Clinical medicine in this period widely used sulfa drugs which not being solved in water. A. Gakhokidze envisaging the actuality of problem laid down the aim to converse just sulfanilamide in solved form in water. After studying the question in detail, A. Gakhokidze solved the problem by following way. He realized the sulfanilamide condensation with glucose for obtaining of soluble one and determined the conditions of reaction. In the solution of diluted ethanol at the interaction of streptocide with glucose, at presence of calcium chloride gives monoglucosulfanilamide (XIII), but if repeat this reaction in the absolute ethanol mediom, we obtain diglucosulfanilamide (XIV).

What about the question of obtaining of soluble aspirin, A. Gakhokidze obtained chlorine anhydride of aspirin by interaction of sodium salt of aspirin and thionyl chloride, then he realized the condensation of glucose and last component (with presence of quinoline) and

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257

partially saponification by sodium acetate of obtained pentaaspiringlucose (XV) he prepared pentasalicylglucose (XVI). Obtained product is the water soluble preparation what gives the possibility to prepare injection form of medicine for parenteral application.

Prof. A. Gakhokidze obtained also water soluble monoaspiringlucose (XVII) by following scheme: OAc

OAc OH

O OAc Br

AcO

+

COO

OAc

O OCOC 6 H 4 OH

Pb

H4 C 6

OAc AcO OAc

2

OH

OH

O OCOC 6 H 4 OH

O OCOC 6 H 4OAc

OH

OH

HO OH

HO OH

XVII

As a conducted researches showed insoluble preparations solve in the water by “fastening” of carbohydrate molecules and absorb easy by organism. There is significantly increased the physiologic effectiveness of medical preparations and decreased their toxicity. Clinical medicine today widely uses such an approach “to ennoble” the preparations for treatment of malignant tumors. For example interaction product of glucose and nitrogen yperit is used successfully in oncology:

Revaz Skhiladze and Tengiz Tsivtsivadze

258

Cl OH O O

CO

CH2

OH

N

n

HO OH

Cl

n=1,2,3 In comparison of live cells with neoplasm ones, the tumor cells are distinguished by intensive glycolise. This reason is considered by scientists to be unusual penetration of glucose in the shell of neoplasm cells. Therefore in such compound the carbohydrate residue is the carrier, which provides more selected concentration in the neoplasm cells. The same idea has laid down for obtaining of chlorethylamides of aldaric and aldonic acids. It is known that natural compounds are easy subjected to decomposition, therefore they can not perform the role of ideal medicines. This noted circumstance force the researchers find the way of natural compounds modification, for obtaining of stable medicine means of long duration pharmacologic influence. The light confirmation of this is the elaboration of carbohydrate condensation methods with ascorbic acid and other physiologic active substances. In today world the searching of new medical means is most expensive, long and less effective process. This was mentioned on the one of meetings of National Academe of USA in New York. Medicine service market today need not increase of new, synthesis means potential but the correction of known, approved by clinical medicine drugs i.e. increase of their specificity, acceleration of their participation in the pathologic region, reversal of toxic influence from organism what cardinally changes the pharmacokinetics and pharmacodynamics of preparations. Prof. A. Gakhokidze obtained also the soluble starch. There has a great importance of A.Gakhokidze‫ۥ‬s achievement in veterinary practice, in the obtaining of helminthologic means. There must be noted the obtaining new preparations between which substances consisted manganese and arsenic obtained by mining of natural deposits of Chiatura and Racha. One of basic purposes of A. Gakhokidze was the study and application of natural resources. By him was stated the structure of some glucosides and pigments and elaborated the methods of their synthesis. For example he isolated new flavonides – akrammerin (XVIII), olmelin (XIX) and 3-D-glycosylepicatechin (XX) with different acids and glycosides from plant gleditschia triacanthos to which the clinicians appropriate an important value in phitotherapy as a means of cardiovascular and malignant diseases.

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259

OH O

OH

O

OH

HO OH

HO

OH H 3CO

O

OCH 3

O

XVIII akramerini

XIX olmelini OH HO

O

OH

OH O O

OH HO

OH OH

XX 3-D-glukozilepikateqini

Prof. A. Gakhokidze synthesized akrammerin and olmelin according to the following scheme: OH HO

OCH 2 C 6 H 5

HNO3

OH

C6H5CHCl2 H 5 C 6 H 2 CO

OCH 2 C 6 H 5

OH

OCH 2 C 6 H 5 OH

OCH 2 C 6 H 5

OCH 3

OCH 3

(CH3)2SO4 H 5 C 6 H 2 CO

H 5 C 6 H 2 CO

COCH 3

H 5 C 6 H 2 CO

OCH 2 C 6 H 5

H 5 C 6 H 2 CO

OCH 2 C 6 H 5 OCH 3

OCH 3

OCH 3 H 3 CO

OCH 3

OCH 3

H 3 CO

OH OCH 3

O

COOK

OCH 3 HO

OH

O

HI

OH

OH HO

H 3 CO

O

OH

O

dimeTilirebuli

akramerini akrammerin Demethylated OCH 3 H 3 CO

O

OCH 3 OCH 3

H 3 CO H 3 CO

O

meTilirebuli

Methylated akrammerin akramerini

Revaz Skhiladze and Tengiz Tsivtsivadze

260 HO

OH

+

HO

HCl

NC

OH

OCH 3

H2O +

-

NH2 Cl

OH

OH

fluroglucini Phloroglucinol OCH 3 HO

OH O

HCOOC2H5

O

HO

OH OH

OCH 3

O

OCH 3

Prof. A. Gakhokidze elaborated method of hydroxicarbonic acids synthesis which is based on the condensation of ketones and esters of carbonic acids: R

R''

R

COOR'''

R'

R'' C(OH) CH

CO + CH2 R'

COOR''' R''

R C(OH) CH R'

COOH

On the base of these investigations by A. Gakhokidze was arisen an original theory of organic acid formation in the plants. In accordance with this theory, for example, citric acid is formed from glucose according to the following scheme: CHO H HO H H

OH H

COOH H HO

OH H

OH

H

OH

OH

H

OH

CH 2OH

CH 2OH

COOH H

OH

HO

H

H

OH CO CH2OH

COOH H

OH

HO

H

H

OH COOH

COOH CH 2 CO CH 2 COOH

COOH

HCOOH

CH 2 C(OH)COOH CH 2 COOH

+

HCOOH

This scheme was proved as chemical so biochemical synthesis. Splitting between 4 and 5 carbon atoms of 5-keto-gluconic acid gives rise to other organic acids. A. Gakhokidze paid a great attention to the application of agricultural wastes. He obtained from maize waste xylotrihydroxyglutaric acid. It can change the tartaric acid in the techniques. Yield of this acid from maize waste consisted 6%. A. Gakhokidze separated citric acid from tobacco waste, from different region of Georgia.There was stated that waste from Lagodekhi consisted 6.37% of citric acid, from Gagra region – 4.71%. By processing of shell of tung-tree A. Gakhokidze separated the dye and stated it‫ۥ‬s empirical formula.

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Prof. A. Gakhokidze has founded chemical investigation of the oil in Georgia, which is of great theoretical and practical meanings. He studied oil deposits of Supsa, Mirzaani and Shirakhi. In 1942 A. Gakhokidze came out with an original hypothesis about oil genesis. He showed that transformation of carbohydrates in nature besides biochemical conversion is possible also by geologic metamorphosis. For proving his opinion, he obtained 2methylheptane (isooctane) from glucose by method elaborated by him (Gakhokidze was the first investigator who used metalorganic synthesis in carbohydrate chemistry): CH2OH

CH2OH

O Br2, CaCO3

OH HO

CH2OAc

O O

OH

OH

(CH3CO)2O

HO

OH

H3C

COH

HOCH

HI, P

1. CH3MgI 2. H2O

OAc CH3

CH3

HCOH

O

OAc

AcO OH

H 3C

O

CH3

CI CH2

H3C Mg

CH CH2

CH2

CH2

HCOH

CH2

CH2

HCOH

CH2

CH2

CH3

CH3

CH2OH

This research is very important for power engineering in future. A. Gakhokidze‫ۥ‬s considerations were proved after 40 years by Canadian scientists. There were discovered microorganisms which transform the carbohydrates into oil (oil fermentation). Prof. A. Gakhokidze wrote many courses of chemistry for students; there were published course of organic chemistry, manual of organic and biological chemistry, inorganic chemistry and many other books. The life of prof. A. Gakhokidze interrupted very unexpectedly at the age of 55 years in 1964. He could not realize completely many new scientific ideas, new scientific beginnings, but what he has created that remains forever in a treasury of a world science.

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.263-265 ©2010 Nova Science Publishers, Inc.

Chapter 21

PROF. DR. RYSZARD MICHAL KOZLOWSKI: HALF A CENTURY IN SCIENCE AND TECHNOLOGY Gennady Zaikov* N.M.Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia

Prof. Ryszard Michal Kozlowski retired in 2009 from the position of the director of Institute of Natural Fibres and Medicinal Plants (Poznan, Poland). It is now the right time to make a short review of his activity in pure and applied science as well as in the organization of science and technology. Ryszard (Richard) Kozlowski was born on July 28, 1938 in Uniejów (pronounce: Oonyeyoov), Poland. He graduated from the Adam Mickiewicz University in Poznan in 1961, receiving the degree of Master of Applied Chemistry. In 1970 he received Ph.D. degree in chemical technology from the same University. After his graduation he worked for the Institute of Bast Fibers, later renamed to the Institute of Natural Fibers. In 1973 he completed postgraduate studies in the field of research organization. Three years later, he became deputy director of the Institute of Natural Fibers, and at the same time he was the head of the Department of Lignocellulosic Boards and By-Products. In 1979 he went through a special training in the field of the manufacture and upgrading of particle boards and preservation of wood at Technical Research Center of Finland. On August 1, 1987, he became General Director of the Institute of Natural Fibers in Poznan and did this duty until December 31, 2008. In 1990, President of the Republic of Poland made him a professor of technical sciences. Professor Richard Kozlowski carried out innovative research in the field of lignocellulosic fibrous raw materials, wool and silk, processing of bast fibers, environmental protection, utilization of by-products and waste materials from textile industry and the manufacture of agro- fine chemicals. He was also involved in research on the application of natural polymers and composites to industry, including modern composites reinforced with natural fibers. Other areas of his research work were reclamation of soils polluted with heavy * N.M.Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygin st., Moscow 119991, Russia, [email protected]

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metals and the application of such soils to the cultivation of raw materials for textile industry. The above studies were financially supported by the European Framework Projects V, VI and VII, as well as by the Polish Ministry of Science and Ministry of National Economy, and by international companies such s Airbus, Lenzig, and others. In 1989, professor Kozlowski was appointed to a post of coordinator of the Flax Research Group, operating under the auspices of Food and Agriculture Organization (FAO) of the United Nations in Rome. In 1993, the Institute of Natural Fibers headed by Professor Kozlowski became the Coordination Center of FAO’s European Cooperative Research Network on Flax and other Bast Fibers. Activities of Professor Kozlowski in the field of projects carried out under the auspices of FAO, which lasted for over 20 years, significantly contributed to the intensification of the exchange of scientific ideas and technology transfer in the field of natural fibers between research centers in Europe and in the world. From 2008, Professor Kozlowski is a coordinator of Focal Point ESCORENA, which works for the good of many important areas of European agriculture. Professor Kozlowski represents the Institute of Natural Fibers and Polish science in a number of scientific and professional organizations in the world. He gave invited lectures in Australia, Brazil, China, Romania, Russia, Ecuador, Columbia, Republic of South Africa and other countries. As an international consultant for cellulosic and protein fibers he is an author of analyses, forecasts and economic plans. Professor Kozlowski in an author of the treatise “Hemp – an alternative plant”, which was prepared for the European Office of FAO in Rome. He is a consultant of UNIDO in Vienna and an expert of the Polish Ministry of Culture and Arts on safety of public collections – the protection of materials from fire. Professor Richard Kozlowski is an author and co-author of 300 original research papers, 7 books, 25 patents and 24 production technologies that were implemented to industry. He is the author of production technologies for fiber degumming with the use of osmosis, enzymes and ultrasounds, production technologies for waterproof and fireproof composites. New generations of fire retardant, fungi-resistant and insect resistant agents for the protection of lignocellulosic materials were developed by Professor Kozlowski and implemented to industry. These technologies (including intumescent coatings of Expander type) make over 90 % of modern protection agents manufactured in Poland. Professor Kozlowski also carried out pioneering research in the field of therapeutic and dietetic properties of flax-seed and the application of biotechnology to the cultivation of fibrous plants and biosynthesis of cellulose. Results of Professor Kozlowski’s studies are of importance to developing countries, particularly as concerns reactivation and intensification of production and processing of natural fibrous raw materials, which is very important for the development of rural areas. For his outstanding research achievements, Richard Kozlowski received the title of Professor Honoris Causa (Honorary Professor) of the Pontifical Catholic University in Ibarra, Ecuador. He received many Polish honors and distinctions, among the order “Polonia Restituta” (he is the knight of the order “Polonia Restituta”), Golden Cross of Merit, awards of Ministry of Science, Higher Education and Technology. Moreover he was “Man of the Year” in 1992/93 and 1995/96 and his biography was included in “International Who’s Who of Intellectuals” “Dictionary of International Biography”, “Five Thousand Personalities of the World”. Professor Kozlowski is an honorary member of The Textile Institute in Manchester, charter-member and vice-president of the Polish Section of the The Textile Institute, member

Prof. Dr. Ryszard Michal Kozlowski, Half a Century in Science and Technology

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of Polish National Committee of ICOMOS (International Council of Monuments and Sites) and a number of other organizations. Professor Richard Kozlowski is an editor-in-chief of “Journal of Natural Fibers” (published by Taylor & Francis, USA), EUROFLAX Newsletter (Information bulletin of the FAO European Cooperative Research Network on Flax and other Bast Fibers). He is also a member of editorial boards of such journals as “Colourage”, “Polymer Research Journal” (Nova Science Publishers, USA), “Vlasberichten”, “Bioresources Technology Journal”, “Fibres & Textiles in Eastern Europe”. Professor Kozlowski significantly contributed to a new approach to natural fibers and polymers and to the decision of the United Nations and FAO that the year of 2009 was proclaimed the “International Year of Natural Fibers”. Today Ryszrad Michal is full energy, force and ideas in the field of pure and applied chemistry. No doubt, we will see his new success in chemistry, biology and medicine.

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G. E. Zaikov, R. M. Kozlowski, pp.267-269 ©2010 Nova Science Publishers, Inc.

Chapter 22

THE SECOND INTERNATIONAL CONFERENCE ON BIODEGRADABLE POLYMERS AND SUSTAINABLE COMPOSITES (BIOPOL-2009) G. E. Zaikov, L. L. Madyuskina and M. I. Artsis N.M.Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia

The Second International Conference on Biodegradable Polymers and Sustainable Composites (BIOPOL-2009) was held in the period of September, 30 – October, 2 2009 at the University of Alicante, Spain. Scientists from both academic and industrial laboratories who are interested in biodegradable polymers and biocomposites were encouraged to participate with the aim to exchange up-to-date ideas on current research and new applications. 150 scientists from 16 countries (Spain, Italy, Portugal, France, UK, Holland, Belgium, Georgia, Russia, Sweden, USA, Canada, Argentina, Hungary, Greece, Czech Republic ) took part in this conference and represented 65 research centers of universities, institutes and companies. World known scientists in the field of polymer chemistry were members of Scientific committee of this conference: Luc Averous (Université Louis Pasteur, Strasbourg, France), Lars Berglund (KTH, Stockholm, Sweden), Norman Billingham (University of Sussex, Brighton, UK), Giovanni Camino (Politecnico di Torino, Italy), Philippe Dubois (Université Mons-Hainaut, Belgium), Alain Dufresne (Institut National Polytechnique Grenoble, France), Alessandro Gandini (University of Aveiro, Portugal), Sigbritt Karlsson (KTH, Stockholm, Sweden), José M. Kenny (Universitá di Perugia, Terni, Italy), José M. Lagaron (IATA-CSIC, Valencia, Spain), Carmen Mijangos (ICTP-CSIC, Madrid, Spain), Iñaki Mondragón (Universidad del País Vasco, San Sebastián, Spain), Carlos Pascoal Neto (University of Aveiro, Portugal), Kristiina Oksman (Lulea University of Technology, Sweden), Andrea Pipino (Centro Ricerche Fiat, Orbassano, Italy), David Plackett (RISØ-DTU, Roskilde, Denmark), Amparo Ribes (Universidad Politécnica de Valencia, Spain), Roxana Ruseckaite

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(Universidad Nacional de Mar del Plata, Argentina), Julio San Román (ICTP-CSIC, Madrid, Spain), Sabu Thomas (Mahatma Gandhi University, India). The chairman of Conference was Prof. Alfonso Jiménez from the University of Alicante. Carmen Bueno, Nuria Burgos, M. Carmen Garrigós, Verónica Martino, Olimpia Mas, Eduardo Paredes, Mercedes Peltzer, Marina Ramos, Raquel Sánchez, Amanda Terol, José Luis Todolí were members of Organizing Committee of this conference. Scientific programme of the conference was divided into 8 sessions. The first session included 2 invited lectures. Alessandro Gandini spoke about furan monomers and furan chemistry at the service of polymer science. The second lecture was done by Sigbritt Karlsson and had the title “Resource and environmental aspects of sustainable biocomposites”. The second session included 7 oral presentations which were devoted to the next problems: evaluation of thermal degradation and dynamic mechanical properties of PP / hemp fibres composites: fibre plasma modification; electrospun cross-linked collagen nanofibers as novel bone tissue interfaces; on the plastifying effect of β-carotene in biopolyester matrices; synthesis of graft copolymer of ethylmethacrylate onto polydichlorophosphazene and its ultrasonic degradation; citric acid/starch catalysed sterification. The third session included 1 invited lecture and 2 oral presentations. Lars Berglund in the invited lecture spoke about nanocelluloses as unique polymeric building blocks for nanostructured polymer systems. The information about PLA and PCL nanocomposites preparation and biodegradation and the information about thermal analysis applied to the characterization of degradation in soil of polylactide were discussed in the oral presentations. The fourth session included 2 invited lectures and 2 oral presentations. The invited lectures were devoted to nano-biocomposites: agropolymer/nanoclay systems (Luc Averous), and processing and characterization of PLGA-carbon nanotube nanocomposites for bone tissue engineering (Jose M. Kenny). The oral presentations included the information about development and characterization of nanobiocomposites based on plasticized poly(lactic acid) and organomodified montmorillonite and the information how to shift toughness of PLA into non break area and create high impact flax fibre reinforcements. The fifth session included 5 oral presentations which were devoted to the next problems: influence of plasticization on barrier properties of poly(lactic acid); influence of the recrystallization conditions on the crystallinity and barrier properties of polylactide acid (PLA) food packaging films; development of new biodegradable nanocomposites based on polylactic acid / natural rubber blends for packaging applications; nano-biocomposites based on poly(lactic acid)-poly(hydroxybutyrate) blends; polyesters from renewable resources based on furan monomers: alternative materials to aromatic counterparts. The sixth session included 1 invited lecture and 3 oral presentations. The invited lecture was done by Gennady E. Zaikov (Institute of Biochemical Physics Russian Academy of Sciences, Russia) and was about biodegradation and medical application of microbial poly(3hydroxybutyrate). The problems of new ways for blending biodegradable polymers with poly(vinyl chloride), of secondary plasticizers for PVC obtained from cardanol and of improved properties of formol-phenolic adhesive by adding natural-based fillers were discussed in the oral presentations. The seventh session of this conference included 2 invited lectures and 2 oral presentations. David Plackett in the first invited lecture spoke about nanocellulose-reinforced bioplastics – prospects for further development. Jean M. Raquez. gave information about recent development of novel (bio)polymers and related composites implemented by reactive

The Second International Conference on Biodegradable Polymers …

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extrusion. The oral presentations were devoted to high barrier nanocomposites of biopolyesters, proteins and polysacharides for coating and packaging applications and to starch nanoparticles for eco-efficientpackaging: influence of botanic origin. The last eighth session included 4 oral presentations, in which there were discussed the next problems: olive stone as a renewable source of biopolyols; simple and quick method to prepare highly hydrophobic cellulosic materials by vapor-phase reaction with chlorosilanes; development of a method for biodegradability evaluation on leather used in the footwear industry; chitosan as an antimicrobial agent for footwear leather components. The two poster sessions of this conference included 70 presentations in which were discussed some particular tasks in the field of biodegradable polymers and sustainable composites. Particularly there were some posters of Russian scientists: stabilization of polymers from the influence of biological media, kinetic method of biocide efficiency estimation; diagnostics of quality and prognosing of potatoes safe storage duration; effects of lead diacetate on structure of neurotropic drug (piracetam): conformational polymorphism; application of poly-HEMA embolic agent for target delivery cytostatic drug – doxorubicin. The next 3rd International Conference on Biodegradable Polymers and Sustainable Composites will be held in Strasbourg (France) in last week of August 2011. Prof. Luc Averous is responsible for organization of this conference.

INDEX A  absorption, 13, 16, 57, 58, 93, 125, 133, 141, 166, 206, 211, 212, 215, 256 abstraction, 8, 129, 131, 138 access, 51, 156, 208 acclimatization, 77 acetone, 168 acetonitrile, 130, 131 acetylcholinesterase, 24 acid, 7, 13, 22, 31, 32, 38, 40, 63, 67, 75, 76, 77, 78, 79, 80, 81, 83, 143, 149, 156, 158, 159, 161, 168, 169, 173, 178, 179, 187, 211, 212, 213, 215, 217, 222, 227, 241, 242, 252, 254, 260, 268 acidity, 221, 222, 225 acrosome, 80 acrylate, 189 activation energy, 3, 242 active additives, 12 active centers, 126 active oxygen, 172 active radicals, 124, 133 active transport, 256 adaptation, 81 additives, 63, 148, 168, 205, 208, 209, 210, 211, 212, 214 adenine, 81 adenosine, 76 adhesion, 109, 116 adsorption, 243 AFM, 106, 107 agar, 183, 184, 185 aggregation, 182 aging process, 12 agriculture, 27, 264 alcohols, 3, 31, 67, 71, 86, 89, 107, 130, 131, 167 aldehydes, 67, 85, 88, 107, 171, 252

aliphatic polymers, 45 alkyl macroradicals, 146, 148 allergic reaction, 171 alpha-tocopherol, 42 alternatives, 92 aluminum, 92, 111, 179, 180 amines, 143, 144, 168, 172 amino acids, 171 ammonia, 168, 178, 188 ammonium, 171, 177 amorphous polymers, 57, 191, 195, 199, 210 amplitude, 31 anatomy, 97 anchoring, 173 aniline, 168, 188 anisotropy, 119 annealing, 197, 198, 201, 203 ANOVA, 103, 104 antibacterial soap, 171 antioxidant, ix, 11, 12, 13, 14, 15, 16, 18, 19, 21, 22, 24, 27, 29, 66, 67, 70, 71, 72, 73, 81, 83, 84, 85, 86, 87, 89, 90, 159, 207, 217 antioxidative activity, 72, 89 aorta, 23 applications, x, 62, 123, 133, 134, 136, 165, 166, 178, 180, 219, 267, 268, 269 aqueous solutions, 31 Arabidopsis thaliana, 78, 81 aromatic polyimide, 62, 206 aromatic rings, 129, 136, 138 Arrhenius equation, 6 ARs, 123, 124, 125, 127, 128, 130, 131, 132, 133, 134, 136, 137, 146, 148 arsenic, 258 arthropods, 119 articular cartilage, 93 ascorbic acid, 167, 170, 258

Index

272

atomic force, 107 atoms, 50, 57, 78, 80, 125, 127, 130, 135, 136, 138, 166, 213, 215, 223, 224, 225, 230, 231 autooxidation, 11, 12, 13, 14, 15, 19, 65, 66, 67, 83, 84, 85, 86, 87 Avogadro number, 192

B  bacteria, 166, 170, 171, 172, 174, 175, 177, 178, 183, 184, 185, 186, 188 bacteriostatic, 176, 178, 183, 184, 185 bacterium, 188 benzene, 136, 148 binding, 38, 40, 125, 150, 173, 215, 228 biochemistry, 10, 130 biodegradability, 269 biodegradation, 268 biological activity, 65, 84 biological media, 269 biological systems, 22, 24, 92, 93 biomass, 22, 23 biopolymer, 134 biosynthesis, 264 biotechnology, 80, 82, 264 bisphenol, 206, 211 black tea, 73, 90 bleaching, 178 blends, 176, 268 blocks, 158, 162, 172, 216 body fluid, 176 bonding, 7, 19, 216 bonds, 5, 8, 9, 57, 60, 131, 132, 148, 178, 212, 213, 228, 229, 230, 233 bone, 92, 93, 268 branching, 12, 207, 212, 221, 222 bromine, 130, 131 buffer, 23, 98 building blocks, 165, 268 bulk materials, 166 Burkina Faso, 73, 90 butyl methacrylate, 62, 63 by-products, 157, 263

C  cadmium, 172 calcium, 158, 159, 256 capillary, 65, 66, 67, 77, 83, 84, 85 carbohydrate, 252, 253, 255, 256, 257, 258, 261

carbon, x, 1, 5, 9, 45, 46, 51, 53, 61, 62, 63, 75, 76, 78, 80, 83, 128, 132, 133, 137, 139, 206, 216, 260, 268 carbon atoms, 75, 78, 80, 137, 260 carbon dioxide, x, 9, 45, 46, 51, 53, 61, 62, 63 carbon nanotube nanocomposites, 268 carbon tetrachloride, 133 carbonic acids, 260 carbonyl groups, 144, 211 carboxylic acids, 168 carboxylic groups, 217 carboxymethyl cellulose, 169, 188 carotene, 268 carrier, 67, 77, 85, 258 cartilage, 92, 93 cast, 46, 58, 61 catalysis, 133, 206, 227, 228, 243 catalyst, 180, 192 catalytic properties, 229 catalytic reaction, 228, 230, 243 cell, 13, 22, 30, 35, 38, 51, 76, 78, 81, 92, 93, 95, 108, 109, 110, 111, 112, 114, 115, 116, 117, 166, 171, 172, 173, 174, 177, 185, 250, 256 cell death, 172, 174 cell membranes, 30, 78, 171, 185, 256 cell surface, 173 cellulose, x, 21, 22, 23, 27, 92, 107, 108, 178, 184, 188, 264 chain branching, 207 chain mobility, 228 chain propagation, 14 chain scission, 4, 5, 7, 8, 218 character, 32, 125, 135, 136, 161, 193, 201, 250 chemical interaction, 211, 227, 228 chemical kinetics, 133 chemical properties, 46, 53, 119, 123, 129, 133, 136, 139, 161 chemical reactions, ix, x, 131, 133, 139, 227 chemical stability, 139 chitin, 92, 94, 95, 99, 100, 103, 105, 106, 107, 120 chlorine, 2, 3, 130, 131, 178, 256 chlorobenzene, 13, 18 chloroform, 97, 214, 215 cholesterol, 38, 40 chromatography, 66, 67, 77, 84, 85 chromatography analysis, 77, 85 classification, ix, 1, 107, 119 cleaning, 166, 176, 186 cluster model, 191, 192, 194, 196, 197, 199, 200, 201 clusters, 168, 191, 201 CO2, 8, 9, 46, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 63

Index coatings, 118, 159, 167, 175, 176, 178, 185, 264 cobalt, 159 collagen, 268 color, 169, 170, 180, 183, 211, 213, 214 combustion, 211, 217 communication, ix, 225 compatibility, 134, 176 competitive process, 60 compilation, 93 complexity, 22, 136 components, 22, 23, 27, 65, 66, 67, 68, 69, 70, 71, 72, 73, 76, 77, 84, 85, 86, 87, 89, 90, 92, 95, 103, 105, 106, 107, 124, 127, 134, 158, 173, 175, 198, 208, 269 composites, 92, 121, 263, 264, 268, 269 composition, 9, 22, 65, 66, 67, 69, 70, 71, 72, 73, 76, 78, 79, 80, 82, 83, 84, 85, 86, 87, 88, 89, 90, 93, 107, 138, 155, 192, 229 compounds, 3, 7, 11, 20, 68, 86, 88, 107, 129, 130, 132, 135, 136, 139, 146, 156, 171, 177, 188, 207, 214, 215, 217, 250, 255, 256, 258 compression, 192, 193, 198 computer technology, 51 concentration, x, 1, 2, 6, 8, 12, 13, 14, 15, 18, 21, 23, 24, 25, 27, 30, 32, 33, 34, 38, 39, 40, 46, 66, 67, 68, 70, 71, 76, 77, 83, 85, 86, 87, 89, 134, 157, 158, 159, 168, 169, 173, 181, 182, 183, 242, 243, 258 condensation, 215, 256, 258, 260 conformational analysis, 61 conformity, 164 conjugation, 14, 125, 141, 216 constant rate, 3, 4, 12, 206 consumption, 4, 8, 9, 13, 14, 15, 16, 17 contact time, 185 contamination, 91, 103, 109, 115, 116, 117, 118, 141 contour, 49, 165, 166 control, 23, 24, 27, 30, 33, 34, 35, 36, 37, 40, 51, 66, 70, 75, 76, 77, 84, 85, 141, 156, 157, 165, 166, 167, 168, 175, 176, 178, 184, 185, 209 control group, 75 conversion, 12, 17, 207, 256, 261 cooling, 148 coordination, 215, 229, 230 copolymers, 142, 144 copper, 172, 177 correlation, 11, 14, 19, 32, 33, 35, 38, 39, 53, 57, 58, 59, 93, 127, 128, 142, 183, 193, 195 correlation coefficient, 14, 33, 53, 57, 58, 59 corrosion, 250 cotton, 177, 182, 186, 187, 188, 189 coupling, 139 covalent bond, 177, 178, 230

273

covering, 108, 110, 112 cross-linked polymers, 203 crystal polymers, 210 crystal structure, 112 crystalline, 7, 59, 194 crystallinity, 268 crystallites, 7 crystallization, 159 crystals, 91, 93, 107, 108, 109, 111, 112, 113, 115, 117, 180 cultivation, 22, 264 culture, 23, 30, 173, 185 curing, 179, 192, 193 curing process, 179 cuticle, 91, 92, 93, 94, 95, 96, 98, 99, 100, 102, 103, 105, 106, 107, 108, 109, 112, 114, 116, 117, 118, 119, 120, 121 cutin, 107, 121 cytokines, 77 cytoplasm, 171

D  Dagestan, 191, 197 decay, 16, 125, 128, 129, 131, 133, 138, 199, 201, 203, 227, 243 decomposition, 5, 9, 133, 138, 157, 159, 161, 163, 211, 228, 238, 241, 242, 243, 258 defects, 134, 198, 208 deformation, 92, 106, 107, 117, 198 degenerate, 12, 208 degradation, x, 2, 4, 5, 6, 7, 8, 9, 22, 40, 80, 134, 175, 205, 206, 207, 211, 213, 215, 216, 218, 268 degradation process, 4, 7 degradation rate, 4, 8 degumming, 264 density, 49, 51, 55, 56, 57, 58, 61, 92, 94, 105, 108, 112, 125, 127, 129, 132, 139, 157, 158, 192, 194, 195, 198, 201, 202, 203, 215, 250 density functional theory, 125, 132, 139 density values, 55, 203 deposition, 179, 180, 186 deposits, 258, 261 derivatives, 68, 85, 86, 129, 178, 254 desiccation, 94, 96, 99, 100, 103, 107, 114, 117 desorption, 51, 52, 55, 60 DFT, 132 DGEBA, 192 diallyldimethylammonium chloride, 179, 180 diaminodiphenylmethane, 192 dianhydrides, 46 differential scanning calorimetry, 49, 53 diffraction, 210

Index

274

diffusion, 1, 7, 49, 51, 52, 58, 60, 63, 128, 177, 184, 185, 208, 210, 216, 250 diffusion time, 49 diglycidyl ether of bisphenol, 192 dimerization, 125, 129, 171 diseases, 175, 258 disinfection, 178, 189 displacement, 102, 103, 105, 112, 113, 114, 116, 120 dissociation, 125, 129, 132, 230 distillation, 13, 85 distilled water, 31, 184 distribution, 38, 134, 161, 168, 170, 213, 215 diversity, 107 division, 173 DNA, 172, 173 double bonds, 3, 5, 6, 66, 68, 149 drawing, 9, 163, 252 dressings, 175 Drosophila, 106, 120 drought, 76, 77, 78, 80, 81, 82 drug delivery, 166 drugs, 12, 258 drying, 58, 61, 96, 103, 117, 170, 184 DSC, 49, 53, 57, 58 DSC method, 53, 57, 58 durability, 134, 175, 176, 178, 182, 185 duration, 13, 73, 90, 258, 269 dynamics, 33, 51, 68, 134

E  elasticity, 120, 191, 192, 193, 196, 199, 200, 201, 203 elasticity modulus, 191, 193, 196, 199, 200, 201, 203 elastin, 92 elastomers, 2, 5 electric current, 166 electrical conductivity, 166 electrochemistry, 250 electrolysis, 19 electrolyte, 250 electromagnetic, 170 electron, 98, 118, 123, 125, 126, 127, 129, 135, 136, 137, 172, 173, 174, 175, 179, 180, 181, 182, 211, 212, 215, 216, 229, 230, 233, 234, 238, 239, 241, 242 electron density distribution, 215 electron microscopy, 118, 173, 179, 180, 181, 182 electron pairs, 125, 229, 230 electron state, 216 electronic structure, 125, 221, 222 electrons, 57, 130, 131, 132, 141, 143, 166, 171, 215 electroplating, 159

elytra, 119 emulsions, 170 encapsulation, 177 endothermic, 5, 234 energy, 22, 57, 95, 125, 132, 174, 225, 228, 233, 243, 247, 250, 265 engineering, 91, 92, 106, 107, 158, 160, 161, 192, 261, 268 entropy, 234, 239 environment, 31, 32, 76, 94, 107, 109, 176 environmental conditions, 108 environmental protection, 263 enzymes, 22, 24, 30, 171, 172, 184, 227, 228, 237, 264 EP-1, 192, 193, 195 EP-2, 192, 193, 195 epidermis, 95, 114, 118 epoxy polymer, x, 191, 192, 193, 194, 195, 197, 198, 203 equilibrium, 52, 53, 56, 60 equipment, 49, 157, 158, 161, 163 erythrocyte membranes, 42 ESR, 9, 29, 30, 31, 32, 38, 72, 89, 123, 125, 126, 127, 128, 133, 135, 136, 139, 142, 145, 148, 149, 208, 215, 219 ESR spectra, 9, 30, 32, 125, 126, 127, 133, 135, 136, 139, 149, 208 ESR spectroscopy, 123, 139 ester, 212, 213, 217 ethanol, 13, 49, 98, 169, 179, 182, 183, 256 ethers, 131, 217 ethyl alcohol, ix ethylene, 142, 167, 168, 183 ethylene glycol, 167, 168 ethylene oxide, 142, 183 eucalyptus, 108, 114 Euclidean space, 194 evolution, 119, 120 exocytosis, 35 exoskeleton, 94 experimental condition, 15, 168 exposure, 1, 4, 5, 7, 8, 9, 132, 179, 183, 198 extraction, 30, 33, 155, 159, 215 extrapolation, 2 extrusion, 198, 200, 202, 203, 269

F  fermentation, 261 fibers, 92, 94, 95, 99, 100, 134, 165, 167, 175, 176, 178, 179, 180, 182, 185, 186, 187, 263, 264, 265 filament, 107 filled polymers, 134

Index film thickness, 52 films, 1, 2, 4, 7, 8, 46, 49, 52, 58, 61, 105, 120, 134, 140, 146, 148, 149, 203, 207, 208, 209, 210, 268 filters, 158, 159, 178 filtration, 159, 161 financial support, 62 flame, 66, 77, 85, 211, 218, 249 flame retardants, 218 flavonoids, 11, 12, 14, 15, 16, 18, 19, 107 flexibility, 59, 62, 161, 194 flexible manufacturing, x, 155, 156 fluid, 23, 114, 115, 117 fluorescence, 77, 78, 215 fluorine, 3, 148 fluorine atoms, 3 food, 12, 22, 65, 84, 169, 268 food products, 12 formaldehyde, 145 formamide, 167 formula, 32, 50, 51, 194, 198, 222, 260 fractal dimension, 194 fragments, 76, 124, 125, 130, 135, 141, 145, 228, 229, 243, 256 free energy, 227, 228, 229, 234, 243 free radicals, 9, 12, 16, 72, 89, 123, 132, 136, 137, 139 free rotation, 60 free volume, 45, 46, 50, 51, 55, 56, 57, 58, 59, 61, 62, 203 friction, 91, 93, 95, 96, 116, 118, 119, 120 fuel, 22, 27 functional approach, 118 functionalization, 132, 149, 165 fungi, 166, 171, 175, 178, 264 fungus, 22, 187 furan, 268 fusion, 35

G  gases, 1, 2, 6, 107 gel, 8, 188 gene, 76, 118 gene expression, 76 generation, 75, 77, 78, 80, 81, 82, 123, 124, 128, 135 genome, 177 geometrical parameters, 227 glass transition temperature, 45, 46, 49, 53, 54, 55, 56, 57, 58, 61, 192, 199, 201 glassy polymers, 45, 63, 201 glucose, 23, 24, 25, 26, 27, 168, 178, 252, 253, 254, 256, 257, 258, 260, 261 glucose oxidase, 23

275

glutamic acid, 169, 187 glycol, 8, 139, 168 glycosylation, 256 granules, 107 groups, 2, 3, 4, 5, 7, 9, 16, 35, 45, 46, 57, 58, 59, 60, 61, 62, 84, 95, 125, 128, 129, 133, 136, 137, 138, 139, 140, 143, 144, 145, 146, 147, 149, 150, 161, 166, 172, 178, 187, 211, 213, 217, 228, 229 growth, x, 15, 22, 67, 75, 76, 77, 80, 82, 86, 166, 168, 171, 172, 173, 175, 178, 180, 181, 183, 185, 198, 199, 201 growth rate, 171 growth time, 168 guidelines, 24

H  halogens, 130, 171 Hamiltonian, 126 hardness, 92, 93, 96, 102, 103, 104, 105, 112, 113, 114, 115, 117, 118, 119, 120 harmful effects, 176 health, ix, 157, 175, 177, 178, 250 health problems, 177 heat, 178, 205, 206, 207, 209, 210, 211, 213, 215, 216, 218 heat aging, 209 heating, 49, 57, 60, 140, 141, 168, 211 heavy metals, 166, 264 height, 100, 192, 198 helium, 67, 85 hemp, 268 heterogeneous systems, 134 hexane, 65, 66, 67, 68, 69, 70, 71, 77, 83, 84, 85, 86, 87 HIV, 186 HIV-1, 186 homeostasis, 81 hospitals, 171, 175 human subjects, 42 humidity, 103, 108, 184 hybrid, 230 hybridization, 125 hydrazine, 167, 168, 170 hydrocarbons, 66, 67, 68, 70, 71, 85, 86, 89, 130, 138, 170, 211, 218 hydrogen, 2, 3, 4, 5, 7, 8, 19, 38, 57, 58, 59, 61, 76, 80, 81, 84, 129, 131, 132, 135, 136, 138, 158, 159, 178, 222, 225, 227, 228, 230, 231, 232, 233, 234, 235, 238, 239, 241, 242, 243 hydrogen abstraction, 3 hydrogen atoms, 2, 4, 5, 8, 57, 131, 132, 135, 136 hydrogen bonds, 57, 58, 59, 61, 129

Index

276

hydrogen peroxide, 80, 81, 178, 227, 228, 230, 231, 232, 233, 234, 235, 238, 239, 241, 242, 243 hydrolysis, x, 21, 22, 23, 24, 26, 27, 211, 212, 213 hydroperoxides, 5, 13, 135, 227, 237, 238, 243 hydroxide, 158 hydroxyl, 4, 38, 131, 139, 149 hydroxyl groups, 4, 139 hygiene, 175 hyperfine interaction, 31, 126 hypothesis, 57, 116, 261

I  images, 96, 97, 98, 99, 101, 105, 106, 107, 110, 111, 112, 180, 181, 182, 183 imide rings, 59 imidization, 60, 63 immobilization, 165, 178, 180 impacts, 40, 206 impregnation, 51, 63 in vitro, x, 29, 30, 35, 36, 37, 39, 40, 41 in vivo, 106, 120, 166 indication, 138, 230, 246 indices, 67, 77, 81, 213 induction, 11, 12, 13, 14, 15, 16, 19, 81, 134 induction period, 11, 12, 13, 14, 15, 16, 19, 134 industrial sectors, 165, 178 industry, x, 27, 67, 85, 155, 156, 159, 177, 178, 263, 264, 269 infection, 167, 171, 175, 176 inhibition, x, 11, 16, 18, 19, 38, 39, 65, 66, 72, 83, 84, 86, 89, 132, 172, 173, 206, 211, 212, 216 inhibitor, 13, 21, 22 initial reagents, 161 initiation, ix, 9, 12, 13, 14, 18, 23, 38, 215 inoculation, 183 inoculum, 185 insects, 91, 94, 95, 98, 104, 106, 109, 115, 116, 117, 120 integument, 95, 106, 119, 120 interface, 94, 120 interference, 100, 101 international standards, 156 interval, 13, 32, 201 intrinsic viscosity, 1, 4 intuition, 251 invertebrates, 92, 95 iodine, 130 ionization, 66, 77, 85, 125, 130, 215, 227, 242, 243 ionization potentials, 130 ions, 149, 168, 170, 172, 242 IR spectra, 2, 5, 8, 60, 125, 211 IR spectroscopy, 2, 125

iron, 178, 214, 215, 227, 228, 229, 230, 231, 232, 234, 241, 242, 243 irradiation, 126, 133, 135 IR-spectroscopy, 218 isobutylene, 4, 5, 148 isomerization, 252 isoprene, 5, 6, 148 isotactic polypropylene, 146 isotope, 131

K  ketones, 107, 137, 260 kinetic curves, 13, 14, 16, 17, 18, 206 kinetics, ix, 1, 11, 13, 14, 123, 134, 168, 206, 212, 216 KINS program, 13

L  laboratory tests, 185 lactate dehydrogenase, 24 lactic acid, 268 leaching, 177 leakage, 171 life cycle, 156 lifetime, 38 ligand, 168 light transmittance, 213, 214 line, 13, 17, 30, 33, 53, 55, 57, 58, 128, 163 linear chain termination, 15 linear defects, 194 linear dependence, 2, 6 linearity, 12 linkage, 172 links, 192 lipid metabolism, 81 lipid peroxidation, 12, 14, 18, 72, 75, 76, 84, 89 lipids, 22, 24, 30, 31, 33, 34, 38, 39, 40, 75, 78, 81, 82, 95, 96, 105, 106, 107, 118, 172 liquid chromatography, 65, 83 liquids, 127 lithium, 142, 147, 148 liver, 29, 30, 32, 37, 40 liver cells, 29, 30, 32 livestock, 22, 27 living conditions, 96, 177 living radical polymerization, 132 local order, 191, 200 low density polyethylene, 217 low-molecular substances, 216 LPO intensity, 22

Index

M  macromolecular chains, 45, 57 macromolecules, 2, 4, 5, 6, 7, 8, 9, 51, 123, 128, 132, 134, 139, 140, 141, 145, 147, 148, 149, 150, 191, 200, 211 macroradicals, 9, 141, 145, 148 magnetic properties, 134 magnetization, 134 majority, ix, 70, 93, 136 malignant tumors, 257 maltose, 253 manganese, 258 manpower, 161 manufacturer, 176, 178 manufacturing, 164, 166, 186 market, 258 marketing, 159 mastery, 251 materials science, 91, 93 mathematics, 63 matrix, 57, 58, 92, 94, 103, 107, 149, 177, 191, 200, 201, 203, 208 measurement, 49, 72, 89, 105, 117 mechanical degradation, 134 mechanical properties, x, 45, 91, 92, 93, 95, 96, 102, 103, 105, 106, 107, 108, 109, 112, 113, 117, 118, 121, 197, 201, 268 mechanical stress, 201 media, 135, 139, 168 melt, 210 membrane permeability, 35, 175 membranes, 12, 29, 30, 33, 35, 38, 75, 76, 77, 78, 79, 80, 81, 119, 121, 172, 174, 250 men, ix, 253 Mendeleev, 21, 23 mercury, 1, 172, 198 metabolism, 76, 166, 172, 177 metabolites, 171 metal oxides, 166 metal salts, 168 metals, 159, 172, 187 metamorphosis, 261 methacrylic acid, 179, 180 methanol, 77, 97 methyl groups, 136 methyl methacrylate, 62, 63 methyl oleate oxidation, ix, 16, 19 methylation, 77 MFI, 213 mice, 29, 30, 32 microcrystalline cellulose, 21, 23 microemulsion, 188

277

microorganism, 172 microscope, 98, 184 microscopy, 100, 101, 107, 118 microviscosity, 30, 33, 34, 40, 78, 228 middle lamella, 108 migration, 179 mining, 258 mitochondria, 77, 80, 81 mixing, 23, 148, 159, 163, 170 MNDO, 221, 222 mobility, 38, 91, 96, 116, 133, 208, 228, 243 model, 11, 12, 13, 16, 50, 63, 66, 67, 68, 71, 72, 76, 77, 83, 84, 85, 86, 87, 89, 98, 115, 128, 132, 141, 188, 194, 197, 207, 211, 212, 215, 218, 222, 230 model system, 13, 66, 67, 68, 72, 83, 84, 85, 86, 87, 89, 207 models, 63, 105, 156, 216 modulus, 92, 94, 96, 102, 103, 104, 105, 106, 112, 113, 114, 115, 116, 117, 120, 193, 199 moisture, 76, 77, 79, 159, 170, 175, 176 molds, 171, 198 molecular biology, 118, 130, 133, 134 molecular dynamics, 134 molecular mass, 142 molecular mobility, 134, 208, 210 molecular orientation, 201, 203 molecular structure, 14 molecular weight, 9, 51, 53, 107, 192 molecules, 4, 12, 16, 31, 38, 39, 40, 57, 76, 127, 129, 133, 137, 166, 172, 174, 208, 221, 222, 225, 228, 239, 257 monomers, 136, 141, 143, 144, 177, 211, 268 monosaccharide, 255 Monte Carlo method, 50, 63 morphology, 7, 108, 118, 168, 172, 174 motion, 23, 31, 93, 208 MTS, 117 multilayered structure, 91, 95, 114, 116 mutant, 115 mutation, 109

N  Na+, 169, 187 NaCl, 181 nanocomposites, 187, 268, 269 nanocrystals, 168, 170, 180, 188 nanofibers, 268 nanoindentation, 93, 96, 106, 115, 119 nanomaterials, 166 nanometer, 91, 92, 108, 115, 165, 166, 191 nanometer scale, 91, 92

Index

278

nanoparticles, 165, 166, 167, 168, 169, 170, 172, 173, 174, 175, 178, 179, 180, 181, 182, 185, 186, 187, 188, 191, 269 nanostructures, x, 197, 198, 201 nanotechnology, 165, 166, 178, 186 narcotics, ix natural polymers, 263 natural resources, 258 neoplasm, 258 nerve, 35 network, 192, 202, 208 nickel, 159 nicotinamide, 81 nitrates, 3, 161 nitric oxide, 132 nitrogen, ix, 1, 2, 4, 5, 9, 49, 123, 125, 128, 129, 136, 139, 230, 231, 257 nitrogen dioxide, 2, 4, 5, 9 nitrogen oxides, ix, 1, 123 nitron, 137, 138, 145 nitroso compounds, 124, 128, 132, 134, 135, 136, 137, 146, 148 nitroxyl radicals, 29 NMR, 211, 213, 215, 218, 219 nodes, 192 nonequilibrium, 191, 196 non-inhibited oxidation, 206 nuclei, 212 nucleic acid, 134, 171 nucleus, 81, 127, 212 nutrients, 175

O  oil, 22, 67, 68, 69, 70, 71, 72, 73, 83, 85, 86, 87, 88, 89, 90, 170, 251, 261 oil samples, 67, 85 oils, x, 65, 66, 67, 68, 70, 71, 72, 73, 83, 84, 85, 86, 87, 88, 89, 90 olefins, x, 221, 222 oligomers, 141 operator, 126 optimization, 161, 222 oral presentations, 268, 269 order, 14, 29, 31, 36, 38, 49, 53, 56, 61, 91, 96, 97, 105, 116, 131, 170, 176, 180, 191, 195, 199, 206, 210, 264 organelles, 76 organic compounds, 123, 131, 215 organic solvents, 139 organism, 30, 33, 171, 183, 257, 258 orientation, 31, 92, 99, 100, 108, 201, 203, 243 osmosis, 264

oxidation, x, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 22, 24, 30, 65, 66, 67, 68, 69, 70, 71, 75, 76, 80, 83, 85, 86, 87, 88, 126, 130, 131, 134, 138, 143, 144, 149, 206, 207, 211, 212, 215, 216, 217, 218, 242, 254 oxidation products, 24, 67, 85, 206 oxidation rate, 13, 14, 18, 85 oxide nanoparticles, 174 oxides, 67, 86, 138, 206, 216 oxygen, 1, 12, 13, 14, 16, 17, 18, 38, 57, 61, 72, 75, 76, 77, 80, 84, 85, 89, 125, 128, 130, 136, 139, 164, 172, 175, 178, 206, 207, 208, 210, 213, 215, 216, 230, 231, 234 oxygen absorption, 13, 206 oxygen consumption, 72, 89 ozone, 2, 130

P  palladium, 98 paradigm, 121 parallel, 24, 33, 68, 95, 100, 157, 158, 185, 249, 252 parameter, 15, 16, 18, 19, 29, 31, 32, 33, 35, 36, 38, 49, 55, 100, 128, 195, 215, 225 parameters, 12, 18, 27, 29, 31, 32, 33, 34, 39, 41, 46, 50, 52, 60, 62, 100, 103, 134, 157, 158, 191, 194, 195, 199, 201, 215, 221, 222, 232, 234 particles, 109, 134, 148, 165, 166, 167, 168, 170, 172, 173, 178, 179, 182, 183, 185, 186, 187, 188, 189 pathogens, 178 PCA, 205 peptides, 173 percolation, 195 performance, 30, 62, 93, 96, 158, 169, 171, 185 permeability, 172, 174 peroxidation, 22, 76 peroxide, 14, 30, 75, 80, 131, 138, 217, 227, 233, 238, 243 peroxide radical, 131, 138, 217 PET, 218 PETF, 218 petroleum, 169 pH, 23, 30, 98, 139, 168, 171, 180 pharmaceuticals, 84 pharmacokinetics, 258 pharmacology, 256 phase transitions, 35 phenol, 23, 66, 68, 84, 85, 86, 171, 192, 215 phenoxyl radicals, 137 phosphates, 161 phospholipids, 24, 38, 40, 42 phosphorous, 156, 158, 161, 206

Index phosphorus, x, 155, 156, 157, 158, 159, 161, 163, 164, 173, 205, 211, 212, 214, 215, 217, 218 phosphorylation, 173, 211, 216 photochemical transformations, 133 photolysis, 132, 133, 145, 146, 148 photooxidation, 145 photostabilizers, 134 physical aging, 46 physical chemistry, 250 physical properties, 45, 46, 62, 123 physicochemical properties, 78 physics, 1, 123, 133, 249 physiology, 120 plants, 65, 72, 76, 77, 80, 81, 83, 90, 104, 107, 108, 109, 111, 112, 116, 118, 120, 260, 264 plasma membrane, x, 30, 32, 33, 34, 37, 38, 40, 76, 172, 174 plasticity, 203 plasticization, 203, 268 platform, 179, 180 PMMA, 3 poison, 177, 178 polar groups, 4 polarity, 32, 213 polarization, 218 pollutants, 1, 2, 157 pollution, 165, 178 poly(3-hydroxybutyrate), 268 poly(ethylene terephthalate), 218 poly(methyl methacrylate), 218 poly(vinyl chloride), 268 poly(vinylpyrrolidone), 168 polyamides, 1, 62, 205, 206, 218 polyamidoacid, 207 polybutadiene, 1, 2, 148 polycondensation, 143 polydispersity, 142 polyesters, 268 polyheteroarylenes, x, 45, 46, 49, 52, 53, 56, 60, 61, 62, 63 polyimides, 46, 57, 58, 60, 61, 62, 205, 206 polyisoprene, 5, 148 polymer, x, 1, 4, 7, 9, 20, 46, 51, 52, 57, 58, 59, 60, 61, 92, 94, 123, 124, 133, 134, 139, 140, 143, 145, 146, 147, 148, 149, 150, 171, 178, 191, 193, 194, 195, 197, 199, 201, 203, 205, 206, 207, 208, 210, 211, 215, 216, 250, 267, 268 polymer amorphous state, 197 polymer amorphous state structure, 197 polymer blends, 134 polymer chains, 9, 58, 61, 124 polymer films, 46, 148 polymer materials, 20, 197

279

polymer matrix, 57, 58, 61, 149, 178 polymer oxidation, 134 polymer properties, 198, 203, 207 polymer solutions, 1, 61 polymer structure, 206, 207, 208 polymer swelling, 52 polymer systems, 191, 197, 268 polymeric materials, 1, 2, 124, 133, 194 polymerization, x, 4, 134, 141, 142, 143, 147, 149, 169, 177, 211, 221, 222 polymethylmethacrylate, 3, 148 polymorphism, 269 polyolefins, 211 polypeptide, 169, 227, 228, 229, 234 polypropylene, 2, 134, 146, 148 polystyrene, 4, 63, 140, 142, 145, 146, 147, 218 polyurethanes, 8, 9 polyvinylacetate, 139, 140 polyvinylchloride, 2, 3 potassium, 22, 77, 167 power, 161, 184, 222, 238, 261 pressure, 1, 2, 46, 51, 114, 115, 117, 179, 181, 182, 183, 192, 198, 212 prevention, 91, 93, 116, 158, 179 probability, 33, 40, 58, 141, 212 probe, 29, 31, 32, 208 process control, 156 product life cycle, 156, 164 production, 22, 27, 28, 65, 81, 155, 157, 158, 159, 160, 161, 162, 163, 164, 188, 252, 264 production technology, 157 productivity, 27, 80 program, 229 programming, 85 project, 156, 157, 158, 159, 160, 163, 252 proliferation, 81 promoter, 228 propane, 22 protein kinase C, 23, 24, 38 protein kinases, 76 protein structure, 95 proteins, 22, 24, 38, 76, 80, 94, 105, 106, 172, 173, 175, 227, 269 prothorax, 96, 98, 99 protons, 136, 149 Pseudomonas aeruginosa, 171 purification, 13, 31, 157, 251 purity, 213 PVC, 159, 268 pyrolysis, 211, 218 pyromellitic dianhydride, 55

Index

280

Q  quantitative estimation, 191 quantum-chemical calculations, 127, 221, 227, 228, 242 quartz, 30, 77 quaternary ammonium, 178, 189 quinones, 132

R  radiation, 150, 167 radical formation, 16 radical mechanism, 213 radical polymerization, 132, 142, 148 radical reactions, 123, 207, 211 radicals, ix, 5, 9, 11, 12, 16, 17, 18, 19, 84, 123, 124, 125, 127, 128, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 143, 149, 207, 234, 241 radius, 50, 127 Raman spectra, 125 range, x, 4, 13, 15, 21, 22, 23, 29, 30, 33, 35, 38, 40, 46, 51, 53, 92, 103, 105, 107, 108, 113, 115, 116, 125, 128, 133, 139, 165, 166, 169, 171, 177, 178, 188, 192, 193, 201, 205, 215 raw materials, 22, 263, 264 reactants, 146 reaction mechanism, 3, 8 reaction rate, 13, 27 reaction temperature, 168, 169 reactions, 3, 4, 9, 12, 15, 19, 123, 124, 125, 128, 129, 130, 131, 132, 133, 134, 136, 138, 139, 141, 145, 146, 147, 148, 206, 214, 217, 218, 227, 228, 234, 243 reactive groups, 141, 192 reactive oxygen, 12, 75, 77, 78, 80, 81 reactivity, ix, 1, 2, 9, 18, 132, 213, 216 reagents, 77, 168 reason, 12, 13, 29, 30, 87, 100, 109, 125, 178, 196, 239, 252, 258 receptors, 77, 79 recognition, 251, 253 recollection, 252 recombination, 9, 16, 132, 213, 234 recommendations, iv, 157 recovery, 106, 107, 156, 164 recrystallization, 268 recycling, 159 reflection, 107 reflexes, 211 region, 4, 95, 108, 125, 192, 221, 251, 258, 260 regulation, 30, 77, 80, 81, 198, 210

regulations, 158 regulators, 75, 76, 134 relationship, 14, 194, 195 relaxation, 106, 107 relevance, 88 reliability, 33 replacement, 92 residuals, 169, 227 residues, 23, 214, 255 resins, 159, 205, 216 resistance, 80, 81, 95, 184 resolution, 98, 107, 211 resonator, 30 resources, 22, 161, 268 respect, 2, 213, 215 respiration, 172 respiratory, 171 respiratory disorders, 171 retention, 61, 67, 77, 81, 85, 130 reticulum, 76 rhamnolipid, 228, 240, 241, 243 rights, iv rings, 14, 145, 146 risk, 2, 171 robotics, 92 rolling, 80, 178 Romania, 45, 62, 264 ROOH, 4, 6, 13, 14, 15 room temperature, 3, 15, 66, 84, 110, 131, 136, 137, 169, 170, 179, 198, 215 roughness, 91, 100, 103, 109 roughness measurements, 100 Royal Society, 119, 120, 246 rubber, 1, 2, 5, 23, 95, 188, 199, 201, 268 rubbers, 1, 148, 149 rural areas, 264 RUS, 81 Russia, x, 1, 11, 21, 22, 29, 45, 65, 66, 75, 77, 83, 85, 123, 155, 205, 221, 222, 245, 249, 263, 264, 267, 268

S  salts, 22, 76, 130, 131, 159, 161, 164, 168, 177, 187, 189, 214, 256 saturated fat, 38, 40, 79 saturated fatty acids, 79 scanning electron microscopy, 96, 98, 107, 109, 111, 179, 180 secretion, 81, 109 seed, 264 senescence, 80 sensing, 120, 188

Index sensitivity, 136 sensors, 92 seta, 115 shade, 233 shape, 94, 97, 98, 99, 106, 188, 211 shock, 93 signal transduction, 38, 80, 81 signaling pathway, 173 signals, 38, 211 signs, 142, 212 silica, 66, 85, 187 silk, 179, 180, 181, 187, 263 silver, 146, 166, 167, 168, 169, 170, 172, 173, 174, 175, 178, 179, 180, 181, 182, 183, 185, 186, 187, 188 SiO2, 187 sludge, 155, 156, 157, 158, 159, 161, 163, 164 smooth muscle cells, 23 sodium, 142, 147, 148, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 167, 169, 170, 180, 181, 188, 256 sodium hydroxide, 158 soil, 167, 176, 268 solid matrix, 208 solid phase, 135 solid polymers, 134 solid state, x, 136 solid surfaces, 118 solid waste, 157 solid-state extrusion, 197 solubility, 46, 136 solvation, 238, 239, 240, 241 solvents, 19, 46, 60, 61, 125, 128, 129, 132, 136, 137, 138, 168, 170, 203 sorption, 49, 61, 228 space, 164, 228, 233, 235, 240 species, 9, 12, 73, 75, 76, 77, 78, 80, 81, 86, 88, 90, 107, 108, 112, 113, 116, 119, 128, 139, 171, 177 specific surface, 170 spectroscopy, 218 spectrum, 4, 11, 13, 26, 31, 32, 92, 95, 125, 127, 128, 149, 178, 186, 208, 215 speed, 31, 51, 77, 84, 85, 215 spin, x, 29, 30, 31, 32, 72, 89, 123, 126, 127, 128, 129, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 146, 147, 148, 149, 208 spin labels, 123, 130, 133, 134, 149 stability, 9, 65, 68, 71, 76, 81, 83, 86, 87, 89, 94, 108, 113, 125, 128, 134, 136, 168, 209, 213, 217, 218, 229 stabilization, 12, 40, 125, 134, 161, 205, 206, 207, 208, 210, 211, 212, 213, 216, 217, 218, 269 stabilizers, 12, 20, 134, 215, 217

281

stable radicals, 123, 124 starch, 187, 258, 268, 269 stereospecificity, 237 stoichiometry, 38 storage, 65, 67, 68, 70, 73, 83, 84, 85, 86, 87, 88, 90, 118, 158, 164, 269 strain, 129, 176, 192, 198, 199, 200, 202 stratification, 158, 228 stress, 65, 76, 78, 80, 81, 176, 191, 192, 193, 194, 195, 196, 198, 199, 200, 201, 203 stress-strain curves, 193 stretching, 125 structural changes, 100, 103, 201 structural characteristics, 30, 81 structural transformations, 35, 38 structural transitions, 30, 31, 33, 34, 37 structuring, 156 students, 252, 253, 261 styrene, 40, 140, 142, 147, 149 suberin, 121 substrates, 22, 24, 131, 173, 175, 177 sugar, 252 sulfa drugs, 256 sulfur, 131, 173, 179, 182 sulphur, 173, 179 surface area, 31, 33, 35, 40, 172, 175 surface layer, 95, 105, 107, 228 surface modification, 178 surface structure, 99, 107, 109, 111, 112, 177 surfactant, 168, 170 swelling, x, 45, 46, 49, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 148 swelling process, 46, 61 synergistic effect, 88 synthesis, x, 3, 46, 60, 61, 76, 123, 124, 130, 134, 146, 147, 148, 149, 155, 156, 159, 160, 161, 163, 164, 167, 168, 169, 170, 186, 187, 188, 253, 255, 256, 258, 260, 261, 268 synthetic fiber, 176 synthetic polymers, 141 synthetic rubbers, 211 system analysis, 161, 164

T  T cell, 81 tanks, 158 taxonomy, 118 TEM, 174, 182, 183 temperature, 4, 19, 23, 29, 30, 33, 34, 35, 36, 38, 40, 46, 49, 51, 58, 61, 66, 77, 85, 108, 133, 134, 158, 159, 168, 169, 175, 187, 192, 195, 198, 199, 201, 206, 207, 211, 212, 213, 217

282

Index

tensile strength, 95 terpenes, 66 textiles, x, 165, 166, 167, 171, 175, 176, 178, 179, 183, 184, 185, 186, 187, 189 thermal activation, 215 thermal aging, 208 thermal analysis, 268 thermal decomposition, 218 thermal degradation, 148, 218, 268 thermal destruction, 148 thermal oxidation, 205, 206, 207, 209, 211, 212, 215, 216 thermal stability, 45, 139, 205, 207, 216 thermal treatment, 141, 203 thermodynamic calculations, 236 thermodynamic equilibrium, 227 thermodynamic parameters, 231, 234, 236, 237 thermodynamically nonequilibrium solids, 195 thermodynamics, 227, 233, 234, 237, 241 thermoregulation, 35 thin films, 4, 62, 118, 120 titanium, 187, 217 tobacco, ix, 81, 260 toluene, 132, 148 toxicity, 256, 257 TPA, 216 transformation, 156, 161, 215, 261 transformation product, 215 transformations, 134, 207, 218 transition, 29, 35, 38, 40, 53, 55, 57, 58, 125, 211, 214, 215, 228 transition metal, 211, 214, 215 transition temperature, 53, 55, 57, 58 transitions, 35, 53, 57, 58, 133 transmission, 40, 175 transport, 172, 174, 176, 216, 250 tumor cells, 258 tyrosine, 80, 173

U  ultrastructure, 119 UNESCO, 246 universal gas constant, 192 urethane, 7 UV, 1, 2, 3, 5, 7, 132, 136, 166, 167, 179, 214, 215 UV light, 1, 2, 5, 7, 132, 167, 179 UV radiation, 3

V  vacuum, 13, 159, 198, 211 valence, 223, 224, 225 Van der Waals radius, 50 vapor, 269 vector, 194 Vickers hardness, 92, 105 vinylchloride, 211 viscosity, 29, 31, 32, 38, 127, 210, 250

W  waste, x, 155, 156, 171, 186, 260, 263 water absorption, 212

X  X-ray, 42, 174, 209, 215

Z  zinc, 187 zinc oxide (ZnO), 166, 187