Recent Advances in Environmentally Compatible Polymers: Cellucon '99 Proceedings

RECENT ADVANCES IN ENVIRONMENTALLY COMPATIBLE POLYMERS RECENT ADVANCES IN ENVIRONMENTALLY COMPATIBLE POLYMERS Editors...

Author: John F Kennedy | Glyn O Phillips | Peter A Williams

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RECENT ADVANCES IN ENVIRONMENTALLY COMPATIBLE POLYMERS

RECENT ADVANCES IN ENVIRONMENTALLY COMPATIBLE POLYMERS Editors: JOHN F KENNEDY Director of the Birmingham Carbohydrate and Protein Technology Group, School of Chemistry, The University of Birmingham, Birmingham B 15 2TT, England UK. and Director of ChembiotechLaboratories, University of Birmingham Research Park. Birmingham B15 2SQ, England, UK and Professor of Applied Chemistry, The North East Wales Institute of Higher Education, Wrexham, Clwyd LLll 2AW. Wales, UK

GLYN 0 PHILLIPS Chairman of Research Transfer Ltd (Newtech Innovation Centre), and Professorial Fellow of The North East Wales Institute of Higher Education, Wrexham, Clwyd, LLll 2AW, Wales, UK and Professor of Chemistry, The University of Salford, England, UK PETER A WILLIAMS Head of the Multidisciplinary Research and Innovation Centre, and the Centre of Expertise in Water Soluble Polymers, and Professor of Polymer and Colloid Chemistry. The North East Wales Institute of Higher Education, Wrexham, Clwyd, LL11 2AW, Wales, UK Guest Editor: HYOE HATAKEYAMA Professor of Applied Physics and Chemistry, Department of Applied Physics and Chemistry, Faculty of Engineering and Graduate School of Engineering, Fukui University of Technology, 3-6-1 Gakuen, Fukui, Fukui 910-8505, Japan

W O O D H E A D PUBLISHING LIMITED

Published by Woodhead Publishing Ltd Abington Hall, Abington, Cambridge CB 1 6AH, England www.woodheadpublishing.com First published 2001 Reprinted 2006 0 2001, Woodhead Publishing Limited The authors have asserted their moral rights

Conditions of sale This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publishers cannot assume responsibility for the validity of all materials. Neither the authors nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from the publisher. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN-13: 978-1-85573-545-3 ISBN-I 0: 1-85573-545-8

Printed in Great Britain by Antony Rowe Ltd, Chippenham. Wiltshire

CONTENTS Preface .............................................................................................................................

xv

PART 1: AN OVERVIEW OF THE DEGRADATION OF POLYMER MATERIALS 1.

-

Degradation of important polymer materials an overview of basic reactions B R h b y .....................................................................................................................

3

PART 2: SYNTHESIS AND DERIVATISATION OF BIOCOMPATIBLE POLYMERS 2.

3.

4.

5.

6.

7.

8. 9.

Conjugated oligomers bearing furan and thiophene heterocycles: synthesis, characterization and properties related to electronic conduction and luminescence C Coutterez and A Gandini .....................................................................................

17

Polyamides incorporating furan moieties. Novel structures and synthetic procedures M Abid, S Gharbi, R El Gharbi and A Gandini ......................................................

27

Saccharide- and lignin-based polycaprolactones and polyurethanes H Hatakeyama, Y Izuta, T Yoshida, S Hirose and T Hatakeyama .........................

33

Cellulose as a raw material for levoglucosenone production by catalytic pyrolysis G Dobele, G Rossinskaja, T Dizhbite, G Telysheva, S Radtke, D Meier and 0 Faix ...............................................................................................................

47

New ionic polymers by subsequent functionalization of cellulose derivatives M Vieira, T Liebert and Th Heinze .........................................................................

53

Preparation and characterization of carbamoylethylated and carboxyethylated konjac mannan S Takigami, Y Suzuki, A Igarashi and K Miyashita ...............................................

61

Plastification of cellulosic wastes M Durhn, M Moya, E Umaiia and G JimCnez .........................................................

67

Synthesis and thermal properties of epoxy resins derived from lignin S Hirose, M Kobayashi, H Kimura and H Hatakeyama .........................................

73

10. Effect of modification on the functional properties of rice starch M A M Noor and M N Islam ..................................................................................

79

vi

Contents

11. Succinylation of chemically modified wool keratin - the effect on hygroscopicity and water absorption N Kohara, M Kanei and T Nakajima ......................................................................

91

12. Natural polymers for healing wounds J F Kennedy, C J Knill and M Thorley ...................................................................

97

PART 3: PRODUCTION AND USE OF BIOCOMPATIBLE MATERIALS 13. Improvement of alginate fiber mixing with phosphoryl polysaccharides S Tokura, H Tamura, Y Tsuruta, C Nagaei and K Itoyama ..................................

107

14. Preparation of cellulose viscose for various matrices B Lonnberg, S Ciovica, T Strandberg, T Hultholm and K Lonnqvist .................. 1 13 15. Synthesis and properties of novel polyelectrolyte on the basis of wood polymer G Shulga, G Zakis, B Neiberte and J Gravitis ......................................................

123

16. Utilising the potential of wood fibre L Salmtn and U-B Mohlin ....................................................................................

129

17. Composites from banana tree rachis fibers (Musa Giant Cavendishii AAA) M Sibaja, P Alvarado, R Pereira and M Moya .....................................................

139

18. Temperature and concentration dependency on equilibration in polysaccharide electrolyte hydrosol M Takahashi, M Mishima, T Yamanaka, T Hatakeyama and H Hatakeyama ..... 145 19. Hydrolysed lignin. Structure and perspectives of transformation into low molecular products M Ja Zarubin, S R Alekseev and S M Krutov .......................................................

155

20. Products of lignin modification: promising adsorbents of toxic substances T Dizhbite, A Kizima, G Rossinskaya, V Jurkjane and G Telysheva .................. 161 21. Characterisation and adsorption of lignosulphonates and their hydrophobized derivatives on cellulose fibre and inorganic fillers G Telysheva, T Dizhbite, A Kizima, A Volperts and E Lazareva ........................

167

PART 4: BIODEGRADABLE POLYURETHANE-BASEDPOLYMERS 22. Biodegradable and highly resilient polyurethane foams from bark and starch J-J Ge, W Zhong, Z-R Guo, W-J Li and K Sakai .................................................

175

Contents

vii

23. Biodegradable polyurethanes derived from waste in the production of bean curd and beer K Nakamura, M Iijima, E Kinoshita and H Hatakeyama ......................................

18 1

24. Biodegradable polyurethane composites containing coffee bean parchments H Hatakeyama, D Kamakura, H Kasahara, S Hirose and T Hatakeyama ............ 19I 25. Biodegradable polyurethane sheet derived from waste cooking oil S Srikumlaithong, C Kuwaranancharoen and N Asa ............................................

197

26. Biodegradable polyesters prepared with dimethyl succinate, butanediol and monoglyceride Y Taguchi, A Oishi, K Fujita, Y Ikeda and T Masuda .........................................

205

Preparation and thermal properties of polyurethane composites containing fertilizer N Yamauchi, S Hirose and H Hatakeyama ...........................................................

21 I

28. Biodegradable polymers derived from lactide and lactic acid S H Kim and Y H Kim ..........................................................................................

217

29. Biodegradable polyurethane foams from molasses Y Hazutani ............................................................................................................

227

27.

30. Biodegradable polyurethane foams derived from molasses K Kobashigawa, T Tokashiki, H Naka, S Hirose and H Hatakeyama .................. 229 31. Polyurethane from pineapple wastes M Moya. J Vega, M Sibaja and M Durin .............................................................

235

32. Preparation and physical properties of saccharide-based polyurethane foams Y Asano, H Hatakeyama, S Hirose and T Hatakeyama .......................................

.24 1

33. Biodegradable polymer in seed protein from corn J Magoshi and S Nakamura ...................................................................................

247

PART 5: ANALYSIS AND CHARACTERISATION OF NEW POLYMERS AND MATERIALS 34.

The complete assignment of the "C CPMAS NMR spectra of native cellulose by using 13Clabelled glucose T Erata, T Shikano, M Fujiwara, S Yunoki and M Takai .....................................

261

...

Vlll

Contents

35. 13CCPMAS NMR and X-ray studies of cellooligosaccharideacetates as a model for cellulose triacetate H Kono, Y Numata, N Nagai, M Fujiwara, T Erata and M Takai ........................

269

36. Thermal and mechanical properties of cellulose acetates with various degrees of acetylation in dry and wet states T Asai, H Taniguchi, E Kinoshita and K Nakamura .............................................

275

37. DSC and TG studies on cellulose-based polycaprolactones H Hatakeyama, H Katsurada, N Takahashi, S Hirose and T Hatakeyama ........... 28 1

38. TG-FTIR studies on cellulose acetate-based polycaprolactones T Yoshida, H Hatakeyama, S Hirose and T Hatakeyama .....................................

289

Thermal analysis of functional paper by a temperature modulated technique T Hashimoto, W-D Jung and J Morikawa ............................................................

295

40. DSC studies on the structural change of water restrained by pectins M Iijima, K Nakamura, T Hatakeyama and H Hatakeyama .................................

303

39.

41. Thermal properties of wood ceramics by TG-MS and CRTG T Arii and M Momota ........................................................................................... 3 1 42. Application of environment controlled thermomechanical analysis system H Katoh, T Nakamura and N Okubo ....................................................................

3 7

43. Effect of water on molecular motion of alginic acid having various guluronic and mannuronic acid contents M Takahashi, Y Kawasaki, T Hatakeyama and H Hatakeyama ...........................

32 1

44. Effect of the initial state on the sorption isotherm and sorption kinetics of water by cellulose acetate H Gocho, A Tanioka and T Nakajima ..................................................................

327

45. Osmometric and viscometric studies on the coil-helix transition of gellan gum in aqueous solutions E Ogawa ................................................................................................................ 333

46. Weathering analysis of modified poly (2,6-Dimethyl-1,4-Phenylene ether) by thermal analysis Y Nishimoto, K Sato, Y Nagai and F Ohishi ........................................................

341

47. Non-desirable carbohydrate reactions in pulping and bleaching G Gellerstedt and J Li ...........................................................................................

347

Contents

ix

PART 6: BIOENGINEERING OF NEW MATERIALS 48.

49.

Precision analysis of biosynthetic pathways of bacterial cellulose by I3C N M R M Fujiwara, Y Osada, S Yunoki, H Kono, T Erata and M Takai .........................

359

Studies of transglycosylation of cellobiose by partially purified trichoderma viride R-Glucosidase H Kono, M R Waelchli, M Fujiwara, T Erata and M Takai .................................

365

50. Celsol - modification of pine sulphate paper grade pulp with

51.

52.

53.

54.

55.

56.

Trichoderma Reesei cellulases for fibre spinning P Nousiainen and M Vehvilainen .........................................................................

37 1

Formation and characterization of transformed woody plants inhibiting lignin biosynthesis N Morohoshi and Y Tsuji .....................................................................................

379

Characterization and utilization of ligninolytic enzymes produced by basidiomycetes M Kuwahara .........................................................................................................

387

Kinetics of biodegradation of n-alkanes by pseudomonas immobilised in reticulated polyurethane foam M G Roig, J F Kennedy, C J Knill, J M Sanchez, M A Pedraz, H Jerabkova and B Kralova ..................................................................................

397

Biocornpatible aspects of poly (2-methoxyethylacrylate) (PMEA) the relationship between amount of adsorbed protein, its conformational change, and platelet adhesion on PMEA surface M Tanaka, T Motomura, M Kawada, T Anzai, Y Kasori, T Shiroya, K Shimura, M Onishi, A Mochizuki and Y Okahata ............................................

405

Isolation of a lignin-degrading laccase and development of tranformation system in Coriolus Versicolor Y Nitta, Y limura, J Mikuni, A Fujimoto and N Morohoshi ................................

41 1

Effect of biodegradable plastics on the growth of Escherichk coli A Nakayama, N Yamano, S Fujishima, N Kawasaki, N Yamamoto, Y Maeda and S Aiba.. ..........................................................................................

.4 19

Index ....................................................................................................................

425

THE CELLUCON TRUST Incorporating

Cellucon Conferences International Educational Scientific Meetings on Cellulose. Cellulosics and Wood

Cellucon Conferences as an organisation was initiated in 1982, and Cellucon '84, which was the original conference, set out to establish the strength of British expertise in the international field of cellulose and its derivatives. This laid the foundation for subsequent conferences on carbohydrate etc. polymer topics in Wales (1986), Japan (1988), Wales (1989), Czechoslovakia (1990), USA (1991), Wales (1992), Sweden (1993), Wales (1994), Finland (1998), and Japan (1999). These conferences have had truly international audiences drawn from the major industries involved in the production and use of cellulose pulp and fibre derivatives of cellulose, plus representatives of academic institutions and government research centres. This diverse audience has allowed the cross-fertilisation of many ideas, which has done much to give the field of cellulose in its diverse forms the higher profile that it rightly deserves. Cellucon Conferences are organised by The Cellucon Trust, an official UK charitable Trust with world-wide objectives in education in wood and cellulosics. The Cellucon Trust is continuing to extend the knowledge of all aspects of cellulose, lignin, hyaluronan and other national polymers world-wide. At least one book has been published from each Cellucon Conference as the proceedings thereof This volume arises from the 1999 conference held in Tsukuba, Japan and the conferences planned to be held in the UK and in the USA etc, will generate further usehl books in this area

THE CELLUCON TRUST TRUSTEES AND DIRECTORS Prof G.O. Phillips (Chairman) Prof J.F. Kennedy (Deputy Chairman and Treasurer) Prof P.A. Williams (Secretary General)

Research Transfer Ltd, UK The North East Wales Institute, UK, and The University of Birmingham, UK The North East Wales Institute, UK

THE CELLUCON TRUST is a registered charity, UK Registration No: 328582 and a company limited by guarantee, UK Registration No: 2483804 with its registered offices at Chembiotech Laboratories, The University of Birmingham Research Park, Vincent Drive, Birmingham, B15 2SQ, UK.

The 1lthInternational Cellucon Conference

CELLUCON '99

Recent Advances in Environmentally Compatible Polymers

ACKNOWLEDGEMENTS This book arises fiom the International Conference - CELLUCON '99 - which was held at the Tsukuba Center for Institutes, Tsukuba. This Meeting owes its success to the invaluable work of its Organising Committees and its generous sponsors.

SPONSORS OF CELLUCON 99 Agency of Industrial Science and Technology (Japan) Ministry of International Trade and Industry (Japan) New Energy and Industrial Technology Development Organisation (Japan) The Cellucon Trust (UK)

MEMBERS OF THE ORGANISING COMMITTEES - CELLUCON '99

General Chairman Dr M Kubota National Institute of Materials and Chemical Research, Japan

Domestic Organising Committee Chairman Vice Chairman Members

Dr K Ueno National Institute of Materials and Chemical Research, Japan Dr Y Watanabe National Institute of Materials and Chemical Research, Japan Prof H Hatakeyama Fukui University of Technology, Japan Dr S Hirose National Institute of Materials and Chemical Research, Japan Dr M Funabashi National Institute of Materials and Chemical Research, Japan

International Organising Committee Chairman Vice Chairman Members

Prof H Hatakeyama Fukui University of Technology, Japan Prof J F Kennedy The University of Birmingham, UK Prof G 0 Phillips Research Transfer Ltd, UK Prof P A Williams The North East Wales Institute, UK Prof B Lonnberg Abo Akademi University, Finland Prof M Duran Universidad Nacional, Costa Rica Prof M A M Noor Universiti Sains Malaysia, Malaysia Prof E H M Melo Universidade Federal de Pernambuco, Brazil Prof T Hatakeyama Otsuma Women's University, Japan Dr S Hirose National Institute of Materials and Chemical Research, Japan Dr M Funabashi National Institute of Materials and Chemical Research, Japan

Local Committee

Chairman Vice Chairman Members

Dr Y Watanabe National Institute of Materials and Chemical Research, Japan Prof T Hatakeyama Otsuma Women’s University, Japan Prof K Nakamura Otsuma Women’s University, Japan Dr S Hirose National Institute of Materials and Chemical Research, Japan Dr M Funabashi National Institute of Materials and Chemical Research, Japan

Secretariat

Dr S Hirose National Institute of Materials and Chemical Research, Japan Dr M Funabashi National Institute of Materials and Chemical Research, Japan

THE CELLUCON CONFERENCES 1984 Cellucon '84 UK

CELLULOSE AND ITS DERIVATIVES Chemistry, Biochemistry and Applications

1986 Cellucon '86 UK

WOOD AND CELLULOSICS Industrial Technology, Biotechnology, Structure and Properties

1988 Cellucon '88 Japan

CELLULOSICS AND WOOD Fundamentals and Applications

1989 Cellucon '89 UK

CELLULOSE: SOURCES AND EXPLOITATION Industrial Utilisation, Biotechnology and Physico-Chemical Properties

1990 CeUucon '90 Czechoslovakia

CELLULOSE New Trends in the Complex Utilisation of Lignocellulosics (Phytomass)

1991 Cellucon '91 USA

CELLULOSE A Joint Meeting of: ACS Cellulose, Paper and Textile Division, The Cellucon Trust, and 1I* Syracuse Cellulose Conference

1992 Cellucon '93 UK

SELECTIVE PURIFICATION AND SEPARATION PROCESSES

1993 Cellucon '93 Sweden

CELLULOSE AND CELLULOSE DERIVATIVES Physico-Chemical Aspects and industrial Applications

1994 Cellucon '94 UK

CHEMISTRY AND PROCESSING OF WOOD AND PLANT FIBROUS MATERIALS The Chemistry and Processing of Wood and Plant Fibrous Materials

1998 Cellucon '98 F d a n d

PULP AND PAPER MAKING Fibre and Surface Properties and other Aspects of Cellulose Technology

1999 Cellucon '99 Japan

RECENT ADVANCES IN ENVIRONMENTALLY COMPATIBLE POLYMERS

2000 Hyaluronan 2000 UK

HYALURONAN 2000

The proceedings of each conference were formerly published by Ellis Horwood, Simon and Schuster International Group, Prentice Hall, Campus 400, Maylands Avenue, Hennel Hempstead, Herts, HP2 7EZ, UK and from 1993 are published by Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CB1 6AH, UK.

PREFACE Biopolymers such as polysaccharides, lignin, proteins and polyesters are a natural resource, being produced by living organisms. However, these compounds are not always useful for mankind. In order to compensate for the apparent unsuitability and inconvenience of natural polymers, various kinds of synthetic polymers have been developed by using petroleum and coal as raw materials. Recently, however, it has been found that most synthetic polymers are not compatible with the environment, since they cannot be included in the natural recycling system. They have therefore become less popular. Mankind is presented with serious contradictions between the convenience of human life and compatibility with natural circumstances. It is easy to say that we have to return to nature in order to solve the problems of man-made material. However, this means that we lose all the convenient features and materials which science has developed throughout human history. Accordingly, we have to accomplish a form of ‘sustainable development’, maintaining our present life, developed by science, along with compatibility.

In the polymer industry, utilization of plant and animal components is the key to sustainable development. Carbohydrates have already been used significantly in the food, medical and cosmetic industries. Plant materials such as cellulose, hemicellulose and lignin are the largest organic resources but with the exception of cellulose, they are not very well utilized. Hemicellulose is significantly under-utilized. Lignin, production of which is over twenty million tons per year worldwide, is mostly burnt as fuel and only increases the amount of carbon dioxide in the environment, although lignin is one of the most useful natural resources. We have to understand that nature constructs a variety of materials that can be used for human life. Physical properties of biomaterials cover the range from viscous liquids to solids. The complexity of biomaterials is based on the intricacies of their complex molecular architectures. However, scientific advances enable us to understand molecular features of biomaterials through modern analytical methods such as infrared spectroscopy, nuclear magnetic resonance spectroscopy, thermal and mechanical analysis and electron microscopy. Now is the time to consider that the compounds produced through biosynthesis can be used as “ready-made” raw materials for the synthesis of useful plastics and materials for human life. Is it possible for example, to convert plant components to high-performance and highly functional materials? Of course, the answer is ‘Yes’. Major plant components, such as carbohydrates and lignin, contain highly reactive hydroxyl groups that can be used as reactive chemical reaction sites. Using the reaction sites, it is possible to convert carbohydrates and lignin, for example to gels, membranes, functional polymers, engineering plastics and biodegradable polymers that are environmentally compatible. This book, which is the proceedings of the International Cellucon Conference 99 (Japan) is divided into several sections. It commences with the keynote lecture which offers an overview of basic reactions which occur in the degradation of important polymers. The section on Synthesis and derivatisation of biocompatible polymers

xvi

Preface

includes various reaction routes for the production of useful polymers and their derivatives from plant components. The section on production and use of biocompatible materials offers a material design lesson on the architectural methods to relate chemical structures of biocompatible polymers to their physical properties. The section on biodegradable polyurethane-based polymers reports the recent development in preparation and physical properties of polyurethanes from biomass. The section on analysis and characterisation of new polymers and materials covers the application of CPMAS NMR, X-ray analysis, differential scanning calorimetry (DSC), thermogravimetry (TG), TG-Fourier transform infrared spectrometry conversion, modification and characterisation of biopolymers. Collectively, the 56 papers cited in this book provide a perspective on the current state of knowledge of biomaterials science as it affects the structural, synthetic and biotechnological fields of environmentally compatible materials.

Hyoe Hatakeyama Chairman International Organising Committee for Cellucon '99

Part 1

An overview of the degradation of polymer materials

DEGRADATION OF IMPORTANT POLYMER MATERIALS AN OVERVIEW OF BASIC REACTIONS

-

Bengt RAnby Department of Polymer Technology, Royal Institute of Technology, SE-10044, Stockholm, Sweden

1. Introduction The main theme of this conference is related to environmentallycompatiblepolymers. Because most commercial polymer materials are of high molecular mass they have as such insignificantbiological effects. Their degradation products and the additives of low molecular mass may, however, affect the environment. Therefore, it is essential to know the basic reactions of degradation for the important polymer materials used in large amounts. Environmentaleffects of polymer materials are decreased when the materials are reused (recirculated) in some way. To maintain useful properties of the materials degradation should be under control and brought to a minimum, i.e. stability retained. Also during recirculation,it is important to know what basic degradationreactions may occur and affect the properties.

2. Degradation Reactions 2.1. Degradation reactions of polymer materials are initiated in various ways related to the conditions to which the materials are exposed A common first degradation step is radical formation by main valence bond scission which may be caused by high energy radiation, absorption of ultraviolet or even visible light, mechanical stress or a high velocity gradient, molecular motion at high temperature or electron injection at high voltage. The polymer radicals formed react easily with molecular oxygen in triplet (biradical) state which is the ground state for atmospheric oxygen.

2.2 Polymers containing or conjugated double bonds react easily by addition of molecular oxygen in excited singlet state and with ozone (0, which ) decomposes to singlet oxygen and atomic oxygen. This is jhe "ene" with singlet oxygen which causes oxidation and bond scission. Atomic oxygen may abstract hydrogen from the polymer which gives radical formation.

2.3.Polymers containing ester, amide and ether bonds in their main chain degrade by ' hydrolysis. This is which is catalyzed by acid and alkali in the presence of water and is faster at elevated temperature. 2.4 Many polymer materials are degraded in

andtheenzymes involved may have various initiation functions, e.g. catalyze hydrolysis, cause oxidation of C-Hgroups to C-OH,give proton transfer, etc.

The four types of basic degradation reactions will be further described and exemplified for the important commercial polymer makrials.

4

An overview of the degradation of polymer materials

3. Initiation bv Radical Formation 3.1. PhotodegI.adationof PoThe most frequent initiation of polymer degradation in v i m is radical formation by bond scission. Because most polymer materials are exposed to light, especially sunlight, when they are used, photodegradation is a very common and extensively studied reaction as reviewed (1,2,3). The spectrum of sunlight in clear weather extends from ultraviolet light (290-400 nm) to visible light (400-800 nm) with the relation of wavelengths and energy quanta shown in Fig. 1. The bond scission energies for single chemical bonds vary from about 110 kcal/mole for strong bonds to about 50 kcal/mole for weak bonds Fig. 1. This means that most common main valence bonds have bond scission energies correspondingto ultraviolet light quanta. The visible light quanta may only break weak chemical bonds (Fig. 1). Only light quanta which are absorbed may initiate a chemical reaction. The ultraviolet absorption spectra of thin polymer films and the spectral distribution of sunlight are given in Fig. 2. Polyolefms and poly(vinylch1oride)have low absorption of ultraviolet light but show high rate of photodegradation due to their chemical reactivity. Tertiary and allylic hydrogens are easily abstracted. The 0-0bonds in peroxides and hydroperoxides are very weak (Fig. 1). Photodegradation of polymers in air is largely a free radical process with a following oxidation (photo-oxidation)and leads to cleavage of the polymer backbone (chain scission), crosslinking,rearrangement, unsaturation and products of low molecular mass. All these processes may be responsible for the loss of mechanical or other physical properties of a polymer material such as color, gloss, impact strength, tensile strength, elongation at break and increase other properties, e.g., wettability, adhesion, etc. The polymer becomes brittle, cracks and holes are fonned on the polymer surface, oxygen and impurities penetrate into the bulk and the aging process is spreading through the sample.

150

i Fig. 1 Energy quanta ys wavelengths and dissociation energy of common chemical bonds.

I I

200

I

VISIBLE I

400

Wavelength (11111)

I

600

I

I

800

Degradation of important polymer materials

. ...

5

.

in photodegradation was given by Bolland and Gee in the 1940’s (5). It involves free radical formation followed by addition of molecular oxygen to peroxyl radicals which abstract hydrogen and form hydroperoxide groups (equ. 1). hv

+ Pa + Ha P. + o,+ P - 0 PH

P - 0- 0.+ RH-

0.

P - 0- OH + R* hv

PH + 02+

(1)

IPH - o,l-+

IPH

- o,l*+

P.+ H - o - 0-

The initiating free radicals are formed from absorption of light quanta by (i) impurities of low molecular mass, (ii) chromophoric groups in the polymer, or (iii) charge transfer complexes of polymer and oxygen, which upon irradiation or energy transfer break up into polymer and hydroperoxyl radicals (equ. 1).

200

2.0 Q)

3

z B

1.0

40 0

220

-

n

240 260 280 300 320 340 360 380 400 Wavelength (nm)

Fig. 2. Ultraviolet absorption spectra of polymer films (0.04 mm) and the spectral distribution of sunlight (at 41’ north in July at noon): aromatic polyester (AP),polyarylate (PAR), polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), polysulfone (PSF), poly(viny1 chloride) (PVC). Reproduced from ref. 4 by permission of Technomic. The impurities and the polymer chromophores may contain carbonyl (C=O) or aromatic groups or double bonds (C=C) which absorb light quanta. Continued irradiation leads to * . By energy transfer hydroperoxide groups decompose to alkoxyl and hydroxyl radicals which both are Eactive and abstract hydrogen and form new radicals (equ.2). The hydroxyl radicals are so reactive and shortlived that they an difficult to analyze by ESR spectroscopy.

P - 0- OH+

PO.+ HO.

PO*+PH+POH+P* HO.+ PH+

H,O + Pa

(2)

6

An overview of the degradation of polymer materials

In this way one initiated polymer radical (equ.1) may give an increasing number of polymer radicals in the propagation steps (equ.2).

Photooxidation of polyolefins leads to increased amounts of carbonyl groups dong the chains and also unsaturation at the chain ends. In spite of extensive research the mechanism of chain scission for polyethylene is not quite established. The formation of hydroperoxide groups is shown in equ.( 1). The photodecomposition of P-OOH in the polymer phase (PH) would give carbonyl groups and water (equ.3) or chain scission with one carbonyl chain end and one unsaturated chain end (equ.3). The presence of a carbonyl group on the polyolefin chain may also give chain scission (equ.3) by a nonradical rearrangement. Ketone groups have high absorption of UV light and transfer energy to hydroperoxide groups which have low UV absorption.

P - OOH

+P = 0 + H,O

(in cage reaction)

H /

+ v

P-OOH+P,-C

H,C=CH+H,O

(3)

\

P,

0

H /

P=O+P,-C \\

+

0

H,C=CH \

P,

During photodegradation of polyethylene there is an accumulation of carbonyl and vinyl groups, which are formed by "in cage" reactions and chain scissions. Polypropylene is rapidly photodegraded due to the tertiary hydrogens which are easily abstracted. The mechanism is well established by ESR studies of the intermediate radicals and analysis of carbon monoxide and methane during the degradation (6). The mechanism involves abstraction of tertiary hydrogen, formation of hydroperoxide groups as previously shown (equ.1). decomposition of the hydroperoxides and formation of carbon monoxide (CO) and methyl radicals in the cage (equ.4). The result is chain scission to two chain end radicals which together with the methyl radicals are analyzed by ESR spectra. The methyl radicals abstract hydrogen and form methane according to gas analysis. H

0

1

II

- CH, - C - CH, 1

CH,

0 H

- CH, - C - CH, -+- CH, + C + ~ H F CH,

(4)

CH,

The 1 * involves radical combination. Polyethylene radicals form stable C-C crosslinks (equ.5). With sufficient amounts of oxygen present peroxyl radicals form. One peroxyl and one polymer radical or two peroxyl radicals may combine (equ.5). The peroxide groups formed have low light absorbtion and are rather stable. When a polymer peroxyl radical is reacting with a hydroperoxyl radical, a polymer carbonyl group is formed which has high UV absorption and may initiate further photo-

Degradation of important polymer materials

7

degradation as shown in equ.(3). Tertiary peroxyl radicals on polypropylene chains may interact but do not terminate photodegradation by crosslinking. Instead new radicals are formed and the polypropylene chains degrade (equ. 4).

P.+ P-+

P-P

PO2.+Po,.+P-o0-P+O2

PO,.i. P.+P - 00 - P

PO; +Po,.+

P = O + POH + 0,

(5)

PO,.+ HO,---+P = O + H,O + 0,

. .

3.2 2 Most polymer materials degrade when irradiated with high energy radiation, e.g. an accelerated electron beam or gamma radiation from a T o cell. Some crosslinking may occur but chain scission is usually more rapid and prevails. In the case of polyethylene the crosslinking reaction is several times faster than the chain scission. Therefore, electron beam irradiation (EB) has been developed as an established commercial method for crosslinking of polyethylene in hot water pipes and electric wire insulation (7).

In mcdical and food technology high energy radiation is used increasingly for sterilization of disposable items in medical care and for packaging materials. For such applications the radiation dose is optimized to give desind sterilization effects at an acceptable degree of degradation. In these processes the energy of the radiation electrones and gamma particles is 10' to 10' times higher than the dissociation energy of the common chemical bonds. Therefon, a large number of different radicals are formed in the bulk of the polymer and the molecular mechanisms of degradation are difficult to establish as reported in the literature (8). A selected use of EB radiation to initiate polymer surface modification of medical devices has been developed and applied (9).

3.3. High mechanical stress of solid polymer samples and high velocity gradients are reported to cause chain scission. An example is ball milling of polyethylene (10) which gives degradation to a limiting chain length of about 100 carbon atoms. Shorter chains do not degrade by ball milling. It is also reported that polymers added to lubrication oil for viscosity control are degraded when used. Radical formation in stressed polyamide fibers has been observed by ESR measurements (11). Bond scission in a polymer exposed to air forms radicals which react by adding molecular oxygen observed by the emission of light. A simple experiment is rapid peeling a Scotch tape from a solid surface. In the dark a clear light emission is observed and interpreted as rJlemiluminescenceemitted from radical reactions occurring in the process.

.

.

Attempts to study expected chain scissions as of a solid polymer sample have been made (12). Rapid deformation giving a brittle crack of the sample causes emission of light which can be observed in the dark with the naked eye. Dogbone samples of 6.6-polyamide and polypropylene were mounted in an Instron tester and deformed at a rate giving necking of the sample. The stress-induced

8

An overview of the degradation of polymer materials

chemiluminescence at the necking position was measured with a very sensitive photometer constructed for the purpose (12). Simultaneous Insmn measurements of stress-induced chemiluminescence and temperature of a sample at the running neck position (Fig. 3) were interpreted as a reaction of thermally unstable hydroperoxide groups present in the samples (equ.3). Previous heating and treatment with sulfur dioxide which degrades hydroperoxide groups decrease the stress-inducedchemiluminescenceto low values. It is possible that the stress-inducedchemiluminescenceand the thermoluminescence of reacting hydroperoxide groups also initiate chain degradation of the polymer. The alkyl ketone groups (Pa)formed give chain scission by photo-oxidation (equ.3). 500

2ocW

400 -

0

Load

20

60

40 Exleiisioii

80

(IIIIII)

Fig. 3 Simultaneousstress chemiluminescence(SCL)and load-extension curve for an injection moulded polyamide (PA66) specimen. Load, photon counts and temperature sus extension. The temperature curve has been multiplied by a factor three for legibility (ref. 12).

4. Deeradation bv Sinelet Oxygen and Ozone While molecular oxygen in ground state is a triplet, i.e. a biradical state, which reacts with organic radicals according the quantum rules, excited singlet oxygen (lo2) and ozone (0,) react with double bonds in alkenes and dienes in organic molecules (14). Singlet oxygen has an excitation energy of 22,5 kcaVmole and a halflife of 45 min. in pure state. Singlet oxygen is produced, e.g. photochemically by energy transfer from excited dyes, by high frequency electric discharge in an atmosphere of molecular oxygen to2),and by decomposition of various peroxides. The "ene" reactions of singlet oxygen with akenes and dienes give endoperoxides and hydroperoxides (equ.6).

The "ene" reactioii

EticlopeIoxide

Degradation of important polymer materials

9

The hydroperoxides and endoperoxides react further and give chain scission as shown in equ. (3). Ozone (0,)reacts with alkenes by addition to double bonds (equ.7)and forms an intermediate endoperoxide which is unstable and causes chain scission to oxidized end groups (15).

This reaction is rapid and involves intermediateradical formation. Ozone is used as a reagent to test the stability of polydienes to oxidation.

5. Degradation bv Hvdrolvsis. Polyesters, polyamides, polyethers, polyanhydrides,etc. are formed by stepwise polymerization and contain hydrolyzable groups in the backbone chain. They are degraded by hydrolysis in common applications. Hydrolysis is an ionic reaction with rather high activation energy (20-30kcal/mole) which means that the naction rates increase rapidly at rising temper a t m (16).In aqueous solution the hydrolysis is catalyzed by protons (acid solution) and hydroxyl ions (alkaline solution). Also polysaccharidesare degraded by hydrolysis. Hydrolysis of polyesters, polyamides and polypeptides are extensively studied reactions. The reaction mechanisms are well presented in common textbooks. Briefly the bond scission occurs between the CO-group and the oxygen (0)in the polyester chains and between the CO and the amine (NH) group in the polyamide chains. The new endgroups are -COOH, -OH and

-NH,.

6. Biodegradation of Polymers Biodegradation is defined as a chemical decompositionwhich takes place through the action of enzymes associated with living organisms, e.g. bacteria, fungi, higher plants and animals, or their emitted products (17). All enzymes are proteins with active sites which are effective in close contact with the polymer substrate. Most enzymes are effective catalysts for reactions with certain chemical structures. Under favorable conditions, e.g. temperature, pH, added salts etc.. enzymes may increase the reaction rate by several powers of 10. For some common polymer structures like polyolefins, polystyrene and polyvinylchloride,then are no enzymes for direct reaction. In such cases the degradation may start with oxidation as a first critical step. One example is the extensively studied polyethylene which is biodegraded after chain or endgroup oxidation. Even some

10

An overview of the degradation of polymer materials

B

Table e1.v Enzyme class

Reaction catalysed

Oxidoreductase

Redox reactions

t (17). Reactive bonds

,c

1

=0

\

-C / - NH, Transfer of functional

Transferase

PUPS

Hydrolase

Hydrolysis

Lyase

Addition to double bonds

Isomerase

Isomerisation

Ligase

Formation of new bonds

One C-group Acetyl groups Esters Amides

-c=c-

>c=o

Racemaces (d, I-foms)

-c-0-c-s-

-C-N-

native polymers like lignin and polyisopren (cis and trans) are biodegraded only after initial oxidation. Some of the oxidation reactions are specific for the enzyme applied and may involve the insertion of one or two oxygen atoms.

6.1. Classification and Nomenclature for Enzvmes In a recent review of polymer biodegradation Albertsson and Karlsson (17) have classified the main groups of enzymes. The names give the nature of the chemical reaction catalysed and end with "ase". Shorter names are sometimes used for convenience. (Table 1). The mechanisms of enzyme mactions may involve free radical modifications of the substrate or alternative ionic reactions in other cases. Sometimes only the end products a known but not the reaction mechanisms and the intermediates.

6.2. Biological Oxidation enzymes (equ.8) Oxygen has an important role in many enzyme reactions. The incorporate one oxygen group (monooxygenases) while the ~ x v e e n a introduce ~e~ two oxygen atoms, i.e. molecular oxygen (equ.9).

PH, - 0,_$ PHOH + H,O BH,+

B

The oxidation (equ.8) requires a second substrate (BHJ which is oxidized simultaneously,e.g. nicotinamide adenine dinucleotide (NADH), a common hydrogen donor in the cells.

Degradation of important polymer materials

PH, + 0,+ P(OH),

+ PO + H,O or POOH

I1

(9)

The oxygenases are inserting whole oxygen molecules (03as dihydroxyl groups which split off water and form carbonyl groups CO or carboxyl groups (- CO - OH). With another type of oxidases molecular oxygen is not incorporated into the substrate but acts as a hydrogen acceptor (equ.10) and produces water (H,O) or hydrogen peroxide (H,OJ. PH, + M O,+P

PH, + 0, P‘

+ H,O

+ H202

(10)

Oxygenase enzymes may even catalyse the splitting of an aromatic structure like in lignin and produce two > C = 0 groups from each - HC = CH - group.

6.3. Biological Hydrolvsis Proteolytic enzymes catalyse various hydrolytic reactions like breaking of ester groups or amide groups. The mechanism may be analogous with the acid- and base- catalysed hydrolysis and could be written as equ. (1 1):

9

R, - c - o - R, + H,O+

0 R,- C - OH + HO - R,

0 0 (1 1) R, - 6- MI - R,+H,o+R, - OH+H,N- R, Amide groups in the polypeptide chains of proteins are hydrolysed like in synthetic polyamides.

e-

6.4. Oxidative Initiation of Biodegradation

The rate-determining initial step in biodegradation of many synthetic polymers with C - C chains was shown by Scott in 1975 (18) to be oxidation. Albertsson reported in her dissertation in 1977 that oxidized groups on the surface of polyethylene films were selectively removed by microorganisms (19). From the 20 years study of the biodegradation of polyethylene it is concluded that oxidation usually is the initial step (17). In laboratory experiments an atmosphere of molecular oxygen is used. Under anaerobic conditions certain microorganisms are able to utilize oxygen from nitrate, sulfate or carbonate groups for oxidation of a polymer substrate. A more complete mechanism for was presented in 1987 by Albertsson et al(20). The initial step is an * ,which may be and similar to the typical P-oxidation of fatty acids and paraffms (equ.12). Hydroperoxide groups are introduced. They degrade and form increasing amounts of keto groups which react further by adding water and give chain scission. The chain degradation is slow. When the chain length reaches the 40-carbon level, degradation of 3-oxo-carboxylic end groups occurs and gives progressive and complete mineralization (21). CH,

- +H,O+

- CH,

OH

(12)

12

An overview of the degradation of polymer materials

OH

- CH,- C = O + HO - CH, - CH, -

4

CH, = CH

- + H,O + CO,

(12)

The oxidised degradation intermediateproducts may be metabolized and enter the citric acid cycle of the microorganisms. Therefore, complete mineralization to carbon dioxide, water and other inorganic products e.g. of nitrogen, sulfur and phorphorons, does not always occur. Abiotic and enzymatic oxidations of a polymer may occur simultaneously and are not easy to distinguish. The hydrophobic surface of polyethylene is a major obstacle to microbial attack. Addition of surfactants in degradation experiments of polyethylene increased the rate (22). Certain microorganisms like with hydrophobic surface adhere to other hydrophobic surfaces, e.g. silicones, poly(tetrafluorethene),polydienes and LD polyethylene and may enhance biodegradation (23).

6.5. Hydrolysis of Synthetic Degradable Pofvmers Many scientist study hydrolysable polymers for replacement of the present commodity plastics. These are natural polymers like polysaccharides,proteins and polyuronides. There is a special interest in new polyesters like synthetic poly(1actic acid), poly(adipate) and poly(succinate) and their copolymers which are more or less easy to hydrolyse. Polyvinylalcohol (PVA) is degradable after oxidation according to an interesting mechanism reported by Huang et al(24). PVA samples in acid aqueous solution were oxidized with sodium hypochlorite. Ketone groups formed along the PVA chains to a PVA/PVK polymer. The original PVA degraded slowly and the PVAPVK much faster with inoculated microorganisms of which was the most active of four species tested. The mechanism is a P-cleavage between C = 0 and C - OH groups which is catalyzed by enzymes.

7. Discussion Oxygen is an important reagent in most degradation processes for polymers both in and in yirn. Initiation by radical formation of various means opens the polymers for oxidation by addition of molecular oxygen in ground state (triplet form, '0,) followed by series of degradation reactions. In radical formation by bond scission, the reactions depend on the dissociation energy of the various bonds. Oxidation is an important initiation reaction also for biodegradation of polymers. There are no enzymes active on long alifatic carbon chains. After initial oxidation which could be abiotic or enzymatic, enzymatic degradation is possible. Even native polymers like poly(is0prene) and lignin ate biodegraded after initial oxidation. The abiotic oxidation may be initiated by radical formation like in photooxidation of polymers. The enzymatic oxidation in yiyn involves specific reactions with insertion of either one oxygen atom (0)or one oxygen molecule to,)according to mechanisms which are not found in YitLQ.

Degradation of important polymer materials

13

Excited states of oxygen, singlet oxygen ('03and ozone (OJ. react with double bonds in akenes and certain aromatic compounds. The "ene" reaction and the formation of endoperoxides initiate chain scission and formation of new oxidized chain ends. Degradation of polymers by hydrolysis of ester, amide and ether bonds in the backbone chains are ionic reactions catalyzed both by acid and base catalysts.

In biological degradation the active enzymes involved catalyze oxidatiodreduction reactions, transfer of groups, hydrolysis, isomerization of d, 1-forms,addition to double bonds and formation of new bonds. The degraded polymer fragments may be incorporated in the metabolism of the living organism or mineralized to carbon dioxide, water and other products containing nitrogen, sulphur or phosphorous.

8. Conclusions The most important degradation reactions of polymers in yipn are inhiikd by radical formation and subsequent addition of molecular oxygen to the polymer radicals. The degradation of the oxidized polymer is p ' by radical combination.

from the modified groups and

Polymer degradation by excited oxygen in singlet form and by ozone are leading to chain scission and formation of oxidized endgroups. Degradation by hydrolysis of polyesters, polyamides, etc. are both by acid and alkali in aqueous medium

catalyzed

Biodegradation of C - C chain polymers hyirp is initiated by abiotic or enzymatic oxidation and by enzyme catalysis. Hydrolysis is catalyzed by enzymes. The enzymatic reactions may involve insertion of one or two oxygen atoms, transfer of groups, isomerization, addition to double bonds and fonnation of new bonds.

In biodegradation of polymers, fragments of the chains may be m&&gd ' inthe '' d to carbon dioxide, water and compounds of nitrogen, organismsor * sulphur and phosphorous.

Acknowledeements This paper is based on current literature and a review of research projects in the department supported by grants and fellowships from the State Board for Technical Development (STU and NUTEK), The Wenner-Gren Foundations, The Carl Trygger Foundation and several companies which all is gratefully acknowledged. My colleagues, Professors Ann-Christine Albertsson, Ulf Gedde and Sigbritt Karlsson and Drs.Bengt Stenberg and Anders WirsCn, have kindly supplied helpful information for the review.

14

An overview of the degradation of polymer materials

References 1.

2. 3. 4.

5.

6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

B.Rhby and J.F.Rabek, Photodegradation, Photo-Oxidation and Photostabilization of Polymers, J.Wiley, Chichester, 1975. B.Rhby and J.F.Rabek, Photodegradation of Polymer Materials in Comprehensive Polymer Science, First Supplement, (S.Aggarwal and S.Russo, eds), Pergamon Press, Oxford 1992, Chapter 12. J.F.Rabek, Photodegradation of Polymers, Spnnger-Verlag, Berlin 1996. N.D.Searle in Proceedings of the International Conference on Advances in the Stability and Controlled Degradation of Polymers, (A.V.Patsis, ed.), Technomic, Lancaster, PA, 1989, p.62. J.L.Bolland and G.Gee, Trans. Faraday Soc. 1946,42,236 and 244 and J.L.Bolland. Quart. Rev. Chem. Soc., 1949, 1,3. H.Yoshida and B.RAnby. J. Polym. Sci. B. 1964,2,1155 and Acta Chem. Scand. (1965). 19,72. Cf.A review by A.Chapiro, Radiation Chemistry of Polymer Systems, Wiley-Interscience, New York, 1962. See further K.Wunsch and H-J.Dalcolmo, Radiai. Phys. Chem., 1992,39,443. HKashiwabara, S.Shimada and Y.HorioJadiat. Phys. Chem., 1991, 37,43. A.WusCn, Heterogeneous Grajting of Acrylamide onto D P E : Kinetics, Morpology and Biomaterial Applications. Diss., KTH. Stockholm, 1995. J.Sohma, Dev.Polym. Deg. 1979,2,99. H.H.Kausch, Polymer Fracture, Springer Verlag, Heidelberg, 1978, Chapt. 7. K.Jacobson, B.Stenberg, B.Terselius and T.Reitberger, manuscript, 1999. K.Jacobson, G.F&nert, B.Stenberg, B.Terselius and T.Reitberger, Polymer Testing, 1999, in print. B.RAnby and J.F.Rabek (eds.), Singlet Oxygen Reactions with Organic Compounds and Pofymers, J.Wdey, Chichester, 1978. See further R.L.Clough, M.P.Dillon. K.K.Iu, P.R.Ogilby, Macromol. 1989,22,3620. J.F.Rabek. J.Lucki, B.Rhby, Europ. Polym. J. 1979,15,1089 and 1101. J.R.Danie1, Encycl. Polym. Sci. Technol. 1985.3, 105, A-C. Albertsson and S.Karlsson, in Chemistry and Biotechnology of Polymer Degradation, (G.J.L.Griffin, ed.),Blackie Acad. Prof., England, 1995, p.7. G.Scott, Polymer Age, 1975,6,54. A-C. Albertsson, Studies on the Mineralization of "C Labelled Polyethylenes, in Aerobic Biodegradation and Aqueous Aging, Diss. ,KTH, Stockholm, 1977. A-C.Albertsson, S.O.Andersson and S.Karlsson, Polym. Degrad. Stab., 1987,lS. 73. A-C-Albertsson and S.Karlsson, in Degradable Materials, (eds, S.A.Barenberg CRC Press, Boca Raton, 1990, p.263. S.Karlsson, 0.Ljungquist. and A-C.Albertsson, Polym. Degrad. Stab., 1988,21,237. U.Husmark, Packmarknaden, 1993,3,34. S.J.Huang, in Modification of Polymers, (eds, C.E.Carraher, Jr and J.A.Moore), Plenum Publ., 1983, p.75.

u,

Part 2

Synthesis and derivatisation of biocompatible polymers

CONJUGATED OLIGOMERS BEARING FURAN AND THIOPHENE HETEROCYCLES: SYNTHESIS, CHARACTERIZATION AND PROPERTIES RELATED TO ELECTRONIC CONDUCTION AND LUMINESCENCE Claire Coutterez & Alessandro Gandini' Matiriaux PolymPres, Ecole Francaise de Papeterie et des Industries Graphiques (INPG), LIP 65, 38402 Saint Martin d'Hdres, France

INTRODUCTION For a long time, the development of polymer science and technology based on petroleum-derived monomers has been the federative working hypothesis of most research on these topics. Today, the precarious state of these fossil materials whose future availability is inevitably limited has led to an alternative strategy based on the exploitation of renewable resources. Indeed, the biomass represents an important ecological and non-polluting raw material which can be submitted to specific transformations. Our laboratory took up this challenge several years ago with the general aim of exploiting these renewable resources as the starting point for the elaboration of original polymeric materials'. Three main areas were, and are being, explored, namely: (i) the chemical transformation of polysaccharides both as a surface or a bulk operation; (ii) the use of lignins as macromonomers for the preparation of polyesters and polyurethanes and (iii) the synthesis and polymerisation of furanic monomers derived from polysaccharides, hemicellulose and sugars. The interest of furanic monomers and the corresponding polymers and copolymers, as well as the chemical modification of macromolecules bearing furan heterocycles, was thoroughly described in a recent review2. This paper is devoted to our recent work on conjugated furan oligomers as novel materials for electronic conduction, luminescence and photoactivity. Conjugated polymers have attracted considerable interest in fundamental and applied research because of their potential use as electronic, optical, optoelectronic and, more recently, luminescence devices. On the one hand, the synthesis and processing methods of numerous conjugated polymers are well established and generally lead to controlled materials in terms of molecular weight and structures. On the other hand, however, the non-systematic reproducibility of some syntheses, the presence of intra- and inter-chain defects, the insolubility, infusibility and instability of many of these polymers, reduce considerably the possibilities of their detailed characterisation and their processability in view of their possible applications. As a consequence, the search for similar structures, albeit precisely defined, more soluble and more stable, such as oligomeric compounds based on the same features, has been the subject of many recent investigations. Indeed, soluble conjugated oligomers not only constitute useful models for the corresponding polymers, allowing the study of structure-properties relationships, but must also be considered a new source of processable materials because of their intrinsic electronic and optical properties.

I8

Synthesis and denvatisation of biocompatible polymers

EXPERIMENTAL Synthesis, purification and structural characterisation of the oligomers Series I oligomers

The experimental procedure was a modification of a method previously perfected in our laboratory for the preparation of poly(2,5-furylene vinylene)3 which was adapted specifically to the synthesis of oligomers. In a previous investigation in our laboratory', a new synthetic route to linear low molecular weight poly(2,5-furylene vinylene) was developed, which involved the basecatalysed polycondensation of 5-methylfuraldehyde (MF), using t-BuOK as a nucleophile. In this preliminary investigation, little attention was devoted to the possibility of preparing and studying isolated oligomeric structures (see Fig. I), except for the dirner 5-[2-(5methylfuryl vinylene)] furfural (MFVF). The main purpose of this study was the preparation and characterisation of the polymers. In the present article, the main emphasis is instead placed on the individual oligomers with regard to their electronic and photoluminescence properties. Moreover, a comparison with the corresponding behaviour of homologous unrnethylated oligomers bearing furan andor thiophene moieties (see Fig. 2)4 is briefly presented.

L

n

Ia: n = I , Ib: n = 2 , Ic n = 3, Id: n = 14-81

Figure 1. Methyl-terminated oligofurylenes vinylenes (series I).

IIe: X, Y, Z = 0 IIf x = s, Y,Z = 0 IIg: x, Y = 0 , z= s In: z = 0, Y = s

IIa: X,Y = 0 I n : x = s, Y = 0 IIc: x = 0, Y = s IId: X, Y = S

x.

IIi Figure 2. Heterocyclic unmethylated oligomers (series II)

Conjugated oligomers bearing furan and thiophene heterocycles

19

In a three neck flask provided with magnetic stirring and kept under a nitrogen atmosphere, 2 mol of monomer MF were mixed with 5 ml of dioxan and a small amount of calcium hydride, used as dehydrating agent. The mixture was then brought to 80°C. Since the potassium terbutoxide (t-BuOK) used as the basic catalyst was poorly soluble in dioxan, a homogeneous catalytic solution of t-BuOK (10 g I-') was prepared in that solvent, using a 18-crow-6 ether, viz. 1,4,7,10,13,16hexaoxacyclooctadecan, as solvating solubilising agent. 22.5 ml of this solution was then added dropwise to the monomer solution. The resulting reaction mixture was left for 1 h under stirring at 80°C. Then, successive quantities of 0.5 ml of monomer and 5 rnl of the catalyst solution were introduced dropwise at the same time every 30-min. and this until a total quantity of 10 ml of monomer had been added. At the end of these additions, the brown mixture was left under stirring once more for 4 h at 80°C. After cooling to room temperature, the reaction mixture was finally filtered, neutralised with concentrated acetic acid, shaken with 300 ml of water, and then extracted with methylene chloride (5 x 250 ml). The organic phase was dried over anhydrous sodium sulphate and concentrated at 1/10 of its initial volume by vacuum evaporation. The residue was then poured into a large excess of methanol and the ensuing precipitate redissolved and reprecipitated into a large excess of a 70/30 mixture of hexane/ethyl acetate. The resulting precipitate was composed of a mixture of higher oligomers (n = 4-8) exclusively (60 % yield) which mainly contained the hexamer and heptamer. The various filtrates, which contained the lower oligomers, were combined, concentrated and submitted to a separation procedure based on flash chromatography on silica gel (SiO2, 230-400 mesh, 60A), using (hexane/ethyl acetate) as eluent. The first fraction (90/ 10 hexane/ethyl acetate) contained exclusively the dimer (yellow, 10% yield), the second (85/15) exclusively the trimer (orange, -10% of yield), the third (80/20) exclusively the tetramer (red, 10% yield) and the last (75-25) consisted of an impure mixture of higher oligomers (brown, 10% yield). Each component was obtained as a crystalline powder. The characterisation of all these products called upon FTIR spectroscopy (KBr pellets, Perkin Elmer Paragon loo0 spectrometer), 'H-NMR spectroscopy (CDzC12, Bruker AC300 instrument at 300 MHz), GPC chromatography (THF, Styragel column for the molecular weight range 100-10,O00), detection by refractometry, elemental analysis (carried out at the Central Analysis Laboratory of the National Research Council of France), mass spectrometry (EI: 70 eV, Nermag R10-1OC spectrometer) and melting points (determined by DSC with a Setaram DSC-92 instrument). All structures were thoroughly confirmed and shown to bear exclusively trans conformations across the alkenyl moieties.

-

-

-

6

7

Trimer Ib

FTIR (vmdX) : Fu : 3121 (vCH), 1451 and 1392 (vFu), 1250 (6CH), 1017 (Fu breathing), 799 and 752 ( C H Fu); C=O : 1664 (vC=O); CH : 2820 (vCH); CH=CH : 1611 (vC=C), 957 (oCH trans); CH? : 2915 (vCH3)cm-'. Rh4N 'H (6) : 9.54 (lH, s, H12) ; 7,25 (lH, d, J = 3.7 Hz, H11) ; 7.09 (lH, d, J = 16.0 Hz, H9 trans) ; 6.92-6.86 (2H, rn, H8 and H5 trans) ; 6.71

20

Synthesis and derivatisation of biocompatible polymers

(IH, d, J = 16.0 Hz, H4 trans) ; 6.55-6.52 (2H, m, H7 and HIO) ; 6.38 (IH, d, J = 3.3 Hz, H6) ; 6.32 ( l H , d, J = 2.9 Hz, H3) ; 6.06 (IH, d, J = 2.1 Hz H2) and 2.34 (3H, s, CH3) ppm. UVvis,(kmJ : 325 (min) and 445 nm ( E : 3.39 ~ lo4 ~ I mol-' ~ cm-I). ~ M.p. : 1025°C. Mass ( d z ): 294 (M'), 165, 147, 115, 77, 43. Elem. An. calculed for CI8Hl4O4 : C : 73.49; H : 4.76; 0 : 21.75 96;found : C : 73.06; H : 4.89; 0 : 21.32 %.

Tetramer Ic

FTIR (vn,.J : Fu : 31 I 1 (vCH), 1442 and 1397 (vFu), 1252 (6CH), 1017 (Fu breathing), 776 and 752 ( K H Fu); C=O : 1669 (vC=O); -CH : 2858 (vCH); CH=CH : 1619 (vC=C), 940 and 960 (oCH trans); CH? : 2927 (vCH,) cm-'. RMN 'H (6) : 9.56 (lH, s, H16) ; 7.27 ( 1 H, d, J = 3.7 Hz, H15) ; 7.1 I (IH, d, J = 16.0Hz, H13 trans); 6.96-6.84 (4H, m, H12, H9, H8 and H5 trans) ; 6.71 (lH, d, J= 15.6 Hz, H4 trans) ; 6.57-6.56 (2H, m, HI1 and H14) ; 6.46 (2H, m, H7 and H10); 6.38 (IH, d, J = 3.2 Hz, H6); 6. 31 (IH, d, H3); 6.06 (lH, d, H2) and 2.35 (3H, s, CH?) ppm. W - v i s . ( L X ): 305(min), 475 and 495 (sh) nm (E475",,, : 4.08 lo4 I mol-' cmI). M.p. : 235°C. Mass ( d z ) : 386 (M"), 193, 165, 115, 77, 43. Elem. An. calculed for :C C24H1805 : 74.63:H : 4.66:O : 20.71 %; found :C : 73.03:H : 5.28:O : 19.50 %.

Oligomers Id FTIR (v,,,,,~): FU : 3113 (vCH), 1437 and 1395 (vFu), 1255 (6CH), 1017 (Fu breathing), 774 ( K H Fu); C=O : 1668 (vC=O); -CH : 2808 (vCH); CH=CH : 1614 (vC=C), 939 and 960 (wCH trans); CH3 : 2925 (vCH3) cm-'. RMN 'H (6):9.56 (lH, s, CHO) ; 7.26 ( I H, d, J - 3.5 Hz, He) ; 7.11 (IH, d, J = 16.0Hz, Hd trans) ; 6.96-6.84 (2pH. m, Htrans) ; 6.71 ( l H , d, J= 16.0 Hz, Hc trans) ; 6.57-6.36 ((2p+l)H, m, HFu) ; 6.29 (lH, d, J - 3.2 Hz, Hb) ; 6.06 (lH, d, Ha) and 2.34 (3H, s, CH3) ppm. UV-vis.(L,) : 295(min), 510 nm. Mass (dz): 846 (M+, p = 61,754 (M+, p = 5 ) , 662 (M", p = 4), 570 (M", p = 3), 478 (M", p = 2).

Series I1 oligomers These oligomers bearing furan and/or thiophene rings were devoid of the terminal methyl group and were prepared according to the synthetic technique described previously4. Their structural characterisation was conducted in the same way as described above for series I and again they all displayed trms conformations.

U.V.-visible absorption and emission spectroscopy Absorption spectra were taken from solutions of spectroscopic-grade methylene chloride with a Beckman DU-64 spectrometer. The photoluminescence spectra were obtained by exciting with either of two sources: (i) the 366 nm line of a high pressure 100 W Oriel mercury lamp or (ii) the 488 nm line of a 100 W Coherent argon-ion laser. The various oligomers were again dissolved in spectroscopic-grade methylene

Conjugated oligomers bearing furan and thiophene heterocycles

21

chloride and the solutions placed in a horizontal 2-mm thick glass cell. Samples were either in contact with air or maintained under an inert argon atmosphere. Their photoluminescence was measured at 90" using a Hamamatsu 10 dynodes AsGa photocathode inserted into a Head-on photomultiplier. The quantum yields were estimated from isoabsorbing solutions (A = 0.95) at the 366 nm excitation wavelength. Coumarin 152 was used as actinometer.

Electrical conductivity The higher oligomers were tested as possible electronic conductors. 200 mg of material were mixed with 570 mg of doubly-sublimed iodine and dissolved in a minimun amount of methylene chloride. The solvent was then vacuum-removed and the ensuing powder pressed into a 2 cm diameter, 0.5 mm thick cylindrical pellet. The conductivity was determined from the sheet resistance of these pellets measured using a four linear point Solem SQOHM-1 tester.

RESULTS AND DISCUSSION Electronic absorption spectra and conductivity The study of the electronic spectra of the series I oligomers confirmed the increasing conjugation as a function of the growing degree of polymerisation. Indeed, as shown in Table 1, a bathochromic shift was observed when going from the dimer (h,,, = 387 nm) to the trimer (?L",,~= 445 nm) and the tetramer (Lrn = 495 nm). This trend suggests that up to four units, the molecular structures remained essentially planar, but that this planarity was then progressively lost as the DP increased further, as indicated by a correspondingly lower bathochomic shift observed for the higher oligomers [n = 4-81 (h,,, = 5 10 nm). The polymeric species prepared previously had a broad visible spectrum centred around 520 nm, suggesting that this trend leads to an asymptotic situation, as in most conjugated polymers. The protonation of these oligomers by triflic acid induced a shift of each ,A to higher wavelengths (see Table 1) which reached the near-infrared with the higher members. This suggests that the charge distribution generated by the presence of the carbocations was more delocalised than that of the electrons in the neutral structures.

Compound

hmax (nm)

hmax (nm)

E (I mo1-l cm-')

after HSO$F:, addition

MFVF (n = I ) Ia

387

Trimer (n = 2) Ib

Tetramer (n = 3) Ic

I

Olieomers (n = 14-81) Id

1

3.y 104

527

445

3.39 lo4

615

415 and 495 (sh)

4.08 lo4

755

510

I

nd

I

> 900

Table 1. Maximum absorption wavelength and molar extinction coefficient in the UV-visible spectra(CH2Clz)of series I oligomers

22

Synthesis and derivatisation of hiocompatible polymers

Table 2. Maximum absorption wavelength of series II oligomers (CH2C12) The obvious implication of this behaviour is that it might be possible to obtain electronic conductivity with these materials. Indeed, a pellet of mixed oligomers [n = 4-81 exhibited, after doping with iodine, a conductivity of 0.4 S cm-' at room temperature, which indicated a typical semi-conducting behaviour. These good conducting properties, related to well-defined soluble low-DP structures, compare very favourably with those of many ill-defined insoluble conjugated polymers and open the way to their possible use as processable conducting materials. As for Series II oligomers, the same bathochromic shift was observed in their neutral electronic spectra, as a function of the DP, as shown in Table 2. The replacement of the furan heterocycle with a thiophene homologue produced a modest but systematic bathochromic shift.

Photoluminescence The photoluminescence of both series of oligomers was also studied. Given the range of absorption maxima reported above, two excitation sources were chosen, namely the 366 nm line of a mercury lamp and the 488 nrn emission of an argon-ion laser. When excited with the 366 nm line, all these compounds displayed some photoluminescence, even if the tetramer and the higher oligomers absorbed poorly at this wavelength. With the 488 nm excitation, the absence of luminescence from the dimer was simply due to the fact that this compound did not absorb above about 420 nm. The first relevant observation was that each compound which absorbed at both excitation wavelengths, gave two identical emission spectra (see Figs. 3 and 4 and Table 3). It is well known that fluorescence and phosphorescence occur essentially after the thermalisation of the excited species, i.e. when the excited molecules have lost their excess rotational and vibrational energy by collision and have thus returned to the 0-0 band level.

Conjugated oligomers bearing furan and thiophene heterocycles

3~

450

5~

6~

zo

7~

Ie (nm)

23

eo

Ie (nm)

Figure 3. Emission spectra of series I oligomers (CH2Ch. lcexc =366 nm)

Figure 4. Emission spectra of series I oligomers (CH2Ch. lcexc = 488 n

The second key observation is that. whatever the excitation wavelength. the emission Amax increased with the chain length of the excited oligomer. Indeed. the electronic emission spectra displayed a bathochromic shift of Amax as a function of the degree of conjugation, in the same fashion as already observed for the corresponding absorption spectra. The dimers emitted in the blue or blue-green region, whereas higher oligomers glowed in the red. The emission colours of the trimer and tetramer were intermediary (see Table 3). A decrease in the extent of this bathochromic shift was again observed as a function of a further DP increase, which was in tune with the similar trend discussed above for the absorption spectra. Compound

Amax of emission (nm)

lemax of emission (nm)

(for lcexc =366 nm)

(for lcexc =488 nm)

MFVF (n = I) la

493

-

Trimer (n = 2) Ib

590

590

Tetramer (n = 3) Ie

635

635

Oligomers (n =[4-8]) Id

650

650

Dimer lIa

470

-

Dimer lib

490

-

Dimer lie

485

Dimer lid

490

-

Trimer lie

565

565

Trimer Ilf

565

565

Trimer IIg

595

595

Trimer lib

560

560

Tetramer IIi

650

650

Table 3. Emission wavelength maxima of the oligomers (CH 2Cl2 )

24

Synthesis and derivatisation of biocompatible polymers

Compound MFVF (n = 1) Ia

Quantum yield 0.0 1s

Trimer (n = 2) Ib Tetramer (n = 3) Ic

0.003

Table 4. Emission quantum yields of series I oligomers Switching heterocycles produced the same trend as observed in the absorption spectra, with a small bathochromic effect induced by the thiophene ring. Thus, the use of two structural parameters, namely the DP of the oligomers and the sequence of heterocycles along their chain, constitutes an interesting source of a wide range of emission wavelengths (and therefore of colours) which could find promising applications. particularly if these trends were also displayed by electroluminescence. The emission quantum yields were estimated with a number of oligomers using Coumarine 152 as an actinometer. This reference was chosen for its high absorption at 366 nm, for its emission spectrum, which is intermediate among those of the various oligomers, and for its relatively high quantum yield of emission, viz. about 0.75. The results are reported in Table 4. This luminescence behaviour was unambiguosly assigned to fluorescence because of the good continuity, in all instances, between the highest absorption wavelength and the lowest emission wavelength. This indicated the existence of a 0-0 level frontier. Therefore, the results in Table 4 refer to fluorescence quantum yields. Moreover, the presence of atmospheric oxygen in the sample solutions (permanent contact with air during experimentation) inevitably quenched any possible phosphorescence. Indeed, oxygen is a well known powerful trap of triplet excited states. Experiments carried out in an argon atmosphere did not show any appreciable difference in the emission spectra, which suggested that the contribution of phosphorescence was negligible. However, the photomultiplier used for the detection of the emitted light was not sensitive beyond 900 nm and this leaves open the possibility of phosphorescence appearing in the infrared, if the triplet states of these molecules was associated with a particularly low energy level. Given the low values of the fluorescence quantum yields and the unlikelihood that any undetected phosphorescence would account for much higher quantum yields, the fate of most absorbed photons remains unclear. Two possible photochemical events come to mind: (i) whereas the dimerisation by cycloaddition of an excited species with a ground-state molecule through the external unsaturations was certainly negligible in the specific conditions of these emission experiments (high dilution'); (ii) the trans-cis isomerisation related to the alkenyl moieties seems a much more likely event in this context given the fact that this behaviour was previously observed in dilute solutions of the dimeric species5. No values are available as yet of the quantum yields of these isomerisations. The third type of pathway which could account for the missing contribution to primary quantum yields is that related to non-radiative photophysical events, viz. internal conversion from the first excited singled to the ground state and/or intersystem crossing from the first triplet state to the ground state. The study of the effect of the excitation wavelength on the emission quantum yield would give information about the role and relevance of possible photochemical processes. Except for coumarine, which was used for the 366 nm excitation

Conjugated oligomers bearing furan and thiophene heterocycles

25

wavelength, we did not find any other suitable actinometer common to all oligomers, which could respond adequately to the various excitation wavelengths corresponding to their different absorption spectra. Clearly, much remains to be done in order to gain a deeper insight into the photochemical and photophysical behaviour of these conjugated molecules. With both excitation wavelengths, when solutions of trirners were in contact with air, some degradation occurred within a few minutes. Thus, both the emission wavelength and the corresponding intensity declined during the exposure time (see Fig. 5). At the same time, the corresponding absorption spectra displayed a shift to lower wavelengths and a decline in the corresponding absorption intensities (Fig. 6). These changes did not occur when the samples were kept under an inert argon atmosphere, suggesting that when the excited molecules were in contact with air, photooxidation reactions took place leading to irreversible structural modifications. The decrease in the absorption and emission wavelengths, characteristic of a lower extent of conjugation, strongly suggests that the alkenyl unsaturations were the sites of these oxidation reactions. Curiously, no corresponding changes were detected when oligomers, other than the trirners, were studied in the context. The fact that the trimer also gave by far the highest fluorescence quantum yield indicates that this specific molecular structure was particularly apt to undergo radiative and photochemical pathways.

Figure 5. Evolution of the emission spectra of trimer under air (Lxc,tat,on = 366 nrn, CH2C12); (a): initial spectrum, (b) after 10 min, (c) after 3 h.

Figure 6. Absorption spectra of trimer (a) under argon, (b) under air after 3 h of exposure

26

Synthesis and derivatisation of biocompatible polymers

CONCLUSION The possibility of preparing well-defined oligomeric structures related to heteroarylene-vinylenes bearing furan and thiophene rings has provided a series of molecules which could be examined individually in terms of such properties as electronic spectroscopy, luminescence and electrical conductivity. A study of the possible application of these novel materials in advanced technologies is in progress.

Acknowledgements The authors wish to thank the Laboratoire de Spectromttrie Physique, UniversitC Joseph Fourier-Grenoble, and in particular J C Vial for his precious advice concerning the luminescence spectra.

REFERENCES 1. A. Gandini, Polymers from renewable resources, In: Comprehensive Polymer Science, 1" Suppl., G. Allen, S. L. Aggarwal & S. Russo (eds.), Pergamon Press, Oxford, 1992, pp.527-573. 2. A. Gandini & N. M. Belgacem, Furans in polymer chemistry, Progr Polyni Sci, 1997, 22, 1203-1379. 3. C. Mtalares, Z . Hui & A. Gandini, Conjugated polymers bearing furan rings: I . Synthesis ans characterization of oligo(2,5-furylene vinylene) and its thiophene homologue, Polymer, 1996, 37, 2273-2279. 4. C. Coutterez & A. Gandini, Synthesis and characterization of oligo(heteroary1ene viny1ene)s incorporating furan and thiophene moieties, Polymer, 1998, 39, 7009-70 14. 5 . V. Baret, A. Gandini & E. Rousset, Photodimerization of heteroarylene-vinilenes, J Photochem Photobiol, 1997, A103, 169-175.

POLYAMIDES INCORPORATING FURAN MOIETIES. 2. NOVEL STRUCTURES AND SYNTHETIC PROCEDURES Mejdi Abid', Souhir Gharbi', Rachid El Gharbi' and Alessandro Gandini** 'Laboratoire de SynthPse et Physicochimie Organique, Faculte' des Sciences, UniversitP de Sfar, 3038 Sfax, Tunisia 2'

Mate'riaux PolymPres, Ecole Francaise de Papeterie et des Industries Graphiques (INPG), BP65, 38402 Saint Martin d'H2res, France

INTRODUCTION Previous scientific work on furanic polyamides consisted mostly in the synthesis and characterization of structures bearing an alternation of aromatic and heterocyclic moieties, i.e. aramide-type polymers'. The sustained interest of one of us in macromolecular materials incorporating the furan ring2 prompted the present ongoing study devoted to a variety of polyamides in which the furan moiety appears in the polymer backbone either on its own (polymers 1,2, and 5 ) or alternating with aliphatic groups (polymers 3 and 4). The synthesis and characterization of some of the latter structures have already been reported3,but those described here are novel.

1

2

r

1

3

28

Synthesis and derivatisation of biocompatible polymers

5

EXPERIMENTAL Three different procedures were adopted to prepare the furanic polyamides, according to their structures, namely: (i) Conventional interfacial polycondensation for polymers 3-5, since it was found previously that this was the most appropriate way to optimize the molecular weight of furanic-aliphatic polyamides3. Thus, 2,2-bis(5-chloroformyl-furyl)propane(CFP) was dissolved in methylene chloride and the complementary diamine in a NaOH aqueous solution (the furfuryl diamine was synthesized by the condensation reaction of furfuryl amine with acetone in an acidic medium). The two solutions were vigorously stirred at room temperature in the presence of a phase-transfer agent (triethylbenzylammonium chloride) for two hours using a 5 % excess of diamine with respect to CFP. Polymers were isolated by filtration, then washed with acetone and ether before being dried . (ii) Polycondensation of N-hydroxymethyl-2-furamide(HF) for polymer 1. This synthesis was inspired by an old short communication4 and was significantly improved as described below. The major change consisted in carrying out the whole preparation in a single operation involving both the hydroxymethylation of 2-furamide and the ensuing polycondensation between the OH groups and the hydrogen atoms at the C5 position of the heterocycles. A thorough study of both the two-step synthesis (preparation of HF by basic catalysis involving paraformaldehyde followed by acidic promotion of its polycondensation) and the simpler one-step counterpart, revealed the advantages of the latter, particularly in terms of the much higher molecular weights of the resulting polyamide 1. Details of this investigation are given elsewhere', but the best conditions found up to now consist in working in a strong acidic medium at temperature of 60 to 80°C. These polyamides were isolated by precipitation in water. (iii) Polymer 2 was prepared by a totally different method which called upon the synthesis of N-furfuryl-2-fury1 amide (FFA) by the reaction of furfuryl amine with 2furoyl chloride and its subsequent polycondensation reaction with acetone carried out in sulphuric acid. Given the asymmetric character of the furanic comonomer, the reaction of acetone with two such molecules can give three different products and thus the ensuing polyamide is bound to possess a random assembly of these (c triads in its linear structure. However, it was thought that the basic features of these random copolymers could be assimilated, at least in the first approximation, to those of the simplified structure shown for polymer 2. These polyamides were also isolated by precipitation in an excess of water and washed to neutrality. All the polymers prepared in this study were characterized by FTIR and 'H-NMR spectroscopy and by inherent viscosity measurement of their solution in m-cresol (1.5 g/L at 25°C) in order to assess the validity of each structure and estimate the corresponding molecular weight. DSC and TGA analyses completed this preliminary ))

Polyamides incorporating furan moieties

29

evaluation of their properties in terms of crystallinity (if any), glass transition temperature and thermal stability.

RESULTS AND DISCUSSION The three procedures and their ensuing polymers will be treated separately before giving a more general appraisal of the present investigation.

Polyamide 1 The construction of this macromolecule follows the same basic idea as that exploited to polymerise furfuryl alcohol, viz. successive electrophilic condensations between OH and H5 moieties. However, whereas in the latter system the monomeric structure obtained, namely -CH2-2,5-Fu-, is deprived of polarity and is also extremely sensitive to side reactions involving the methylene bridge6, in the present context the polarity is greatly enhanced by the amide function and moreover its presence contributes to reduce very considerably the aptitude of the methylene moiety to promote unwanted structural modifications. Although formally polymer 1 was briefly described previously, its structure had not been proved and the DP obtained in that study was much lower than those reported here. In fact, by using the optimized conditions summarized above, we obtained a polymer with an inherent viscosity of 2.3 dl/g which suggests a reasonably high molecular weight. The FTIR spectrum of this sample was very similar to those of all other samples prepared under a variety of conditions, but which had all lower values of q, and displayed all the expected bands associated with structure 1'. The 'H-NMR spectra of some of these polymers, taken in DMSO-d6 at 300 MHz, were also entirely consistent with the expected structure both in the positions of each proton resonance and in their relative intensity. The strong intermolecular association arising from hydrogen bonding between C=O and N-H functions, typical of all primary polyamides, was verified here by the observation that only highly polar solvents like DMSO, or NMP with LiCl or strong acids like H2S04 dissolved these polymers. We noticed moreover that the higher the value of q, the lower the rate of dissolution. One drawback associated with the use of sulphuric acid as polymerization medium is the fact that the polymer has a dark-brown colour, whereas it has a creamy complexion when it is prepared in other acidic media. Obviously, some side reactions occur in the former conditions, although it is difficult to assess at present to what extent, since no spurious structure could be detected by spectroscopy and even the most deeply coloured samples remained entirely soluble. Work is in progress to determine the physical properties of this novel material which associates all the general features of a classical polyamide with the presence of furfuryl moieties as sole spacers.

Polyamide 2 In order to extend the realm of furanic polyamides incorporating a high content of heterocycles, we used an hitherto unexplored synthetic method, based on a standard reaction, albeit usually applied to simple furans. It is in fact well known that the particular reactivity of this heterocycle promotes a facile condensation of aldehydes and

30

Synthesis and derivatisation of biocompatible polymers

ketones with two rings through their H5 atoms because of the high nucleophilicity of that position. In the specific instance of furan itself, this leads to a series of oligomers arising from successive condensations and we thought therefore that this concept could be extended to the reaction of an amide bearing two furanic end-groups, viz. FFA. The polycondensation of FFA with acetone in sulphuric acid occurred as expected and the ensuing polyamide gave an inherent viscosity of 0.8, suggesting that, even within this preliminary context, the degree of polymerization was relatively high. Figure 1 shows the FTIR spectrum of this polyamide, which is entirely in tune with structure 2 with the typical free and hydrogen-bonded NH peaks at 3300-3400 cm-'; the amide carbonyl peaks at 1660, 1530 and 1320 cm-I; the in-plane and out-of-plane vibrations associated with the furan heterocycle at 3140, around 1600, 1020, around 900 and 780 cm-' and the peaks relative to the methylene and isopropyl groups just below 3000 cm-'. The work in progress on this system focuses on the optimization of the synthetic conditions, particularly with regard to the correct stoichiometry between the monomers, knowing that both monomers are likely to be also consumed in side reactions occurring in sulphuric acid. One possibility is to attempt the synthesis of an A-B type structure in order to dispose of an <( intrinsically stoichiometric >> monomer.

Polyamides 3-5 The experience acquired in our previous investigation on furanic-aliphatic and furanicaromatic polyamides suggested that the pursuit of this project should be conducted using the technique of interfacial polymerization'. The present addition of three novel polymeric structures was intended to extend our knowledge of structure-property relationships, already established in the homologous series of furanic polyesters', by including cycloaliphatic and furanic co-spacers in the polyamide chains.

-50

t

Y

Figure 1. FTIR spectrum of polyamide 2 (KBr pellet).

Polyamides incorporating furan moieties

31

Polymer 3 was prepared in good yields and, after careful purification of both monomers and optimization of the synthesis, an inherent viscosity of over 1.6 was achieved. However, despite this satisfactory molecular weight and spectroscopic evidence of a regular structure in tune with formula 3, the polymers did not exhibit any crystallinity, even after several heating-cooling cycles, as shown by the corresponding DSC tracings which only exhibited a glass transition occurring at 76°C. It is interesting to compare the morphology of this entirely amorphous polymer with that of its homologue with the same molecular weight, but bearing six methylene groups (instead of two), which readily crystallises, as indicated by the presence of clear melting peaks in the DSC thermograms) The difference in behaviour can hardly be attributed to any difference in macromolecular irregularities for 3, since the same monomer purification and polymer synthetic procedures were applied to both polyamides. It seems more likely that the bulky isopropyl group between the furan rings could play a detrimental role in being an obstacle to interchain organization, and this in a more dramatic way in the case of 3 because of the much shorter aliphatic spacer between the -Fu-C(CH3)2-Fumoiety. In other words, the establishment of regular sequences of N-H",C=O interchain hydrogen bonds, typical features promoting the crystallization of Nylons, would be attained much more easily with the polyamide bearing a sequence of six CH2 groups than with 3. The thermal stability of 3, shown in Figure 2, was entirely comparable to that of its homologues prepared previously3 which constitutes further evidence of its regular structure. The synthesis of polyamide 4 was less successful in terms of both yield and DP. This was attributed to the fact the the diamine used bore now secondary functions in a cyclic structure, i.e. less reactive entities. Despite these drawbacks, the FTIR spectrum of the polymer confirmed the postulated structure with the typical peaks of secondary amide (1630 cm" for the >N-C=O), of 2,5-disubstituted rings (3120, 1520, 1010, 960, 800 and 745 cm-') and of the aliphatic groups at 2860,2925 and 2975 cm-' and between 1300 and 1470 cm-'.

'.

I

t

'O

\

1

Figure 2. TGA thermogram of 3 ([q]=1.6 dug). Heating rate 2O0C/min,NZatmosphere.

32

Synthesis and derivatisation of biocompatible polymers

Finally, the novel polyamide 5 bearing furan rings in both spacers was prepared using a diamine whose synthesis and structure simulated that of the corresponding diacid chloride, except that, because furanic amines are thermodynamically unstable with respect to their tautomeric imines, a methylene group must be interposed between the heterocycle and the NH2 group. The resulting polymer 5 had an inherent viscosity of 1.3 dl/g, which is considerably higher than that obtained in the only previous study of the synthesis of entirely furanic polyamides’. This improvement stems from the detailed approach aimed at optimizing all the parameters related to the conditions of the interfacial polycondensation, as described elsewhere’. The ITIR spectrum of 5, showing again all the features related to the expected structure, is given in Figure 3. Among the five polyamides reported in this study, undoubtedly polymers 1 and 5 are the most interesting both because they possessed high molecular weights and because they were rich in furan moieties. Work is in progress to gain a deeper insight into these systems and to extend the characterization of the polymers to their physical polymers.

4000

3500

3000

2500

2000

1500

1000

500

Figure 3. FTIR spectrum of polyamide 5 (KBr pellet).

REFERENCES 1 A Mitiakoudis & A Gandini, ‘Synthesis and characterization of furanic polyamides’, Macromolecules 1991, 24, 830-841. 2 A Gandini & M N Belgacem, ‘Furans in polymer chemistry’, Progr Polym Sci, 1997, 22, 1203-1379. 3 S Gharbi & A Gandini, ‘Polyamides incorporating furan moieties 1. Interfacial polycondensation’, Acra Polym, 1999, 50, 293-298. 4 V M Mihajlov & N Peeva, ‘Polymerisation of furamide with formaldehyde’ Makromol Chem 1968, 116, 107-111. 5 M Abid, R El Gharbi & A Gandini, ‘Polyamides incorporating furan moieties. 2. Polycondensation of 2-furamide with paraformaldehyde’, Polymer, 2000,4 1, 355-362. 6 M Choura, M N Belgacem & A Gandini, ‘The acid-catalyzed polycondensation of furfuryl alcohol’, Macromolecules, 1996, 29, 3839-3854. 7 A Chaabouni, S Gharbi, M Abid, S Boufi, R El Gharbi & A Gandini, ‘Polyesters furaniques: transitions et stabilitt thermiques’, J Soc Chim Tunisie, 1999, 4, 547-558.

SACCHARIDE- AND LIGNIN-BASED POLYCAPROLACTONES AND POLYURETHANES Hyoe Hatakeyama*’, Yoshinobu Izutal, Takanori Yoshida’, Shigeo Hirose and Tatsuko Hatakeyama

’

‘Fukui University of Technology, 3-6-1 Gakuen, Fukui-city, Fukui 910-8505,Japan 2Nalional Institute of Materials and Chemical Research, 1-I Higashi, Tsukuba, Ibaraki 305-8565,Japan ’Otsuma Women’s University, I2 Sanbancho, Chiyoda-ku, Tokyo 102-8357,Japan

ABSTRACT Saccharide- and lignin-based polycaprolactones were synthesized from glucose-, fructose, sucrose, alcoholysis lignin (AL) and Kraft lignin (KL) by the polymerization of E-caprolactone (CL) which was initiated by the OH group of saccharides and lignins. The CWOH (moVmol) ratios of the saccharide-based PCL’s were changed from 1 to 5 and those for lignin-based PCL‘s were changed from 2 to 25. PU sheets were prepared from the above PCL derivatives by the reaction with diphenylmethane diisocyanate (MDI). Thermal properties of the prepared saccharide- and lignin-based PCL’s and PU sheets were studied by differential scanning calorimetry (DSC), thermogravimetry (TG) and TG-Fourier transform infrared spectroscopy (FTIR). Glass transition temperatures (T,’s), cold-crystallization temperatures (T,’s) and melting temperatures (T,,,’s) of saccharide- and lignin-based PCL’s and PU’s were determined by DSC, and phase diagrams were obtained. T,’s decreased with increasing CWOH ratio, suggesting that PCL chains act as a soft segment in the amorphous region of PU molecules. Two thermal degradation temperatures (T,,’s) were observed in TG curves of PU’s from saccharide- and lignin-based PCL’s with low CL/OH ratios. TG-FTIR analysis of PU’s from lignin-based PCL’s suggested that compounds having C-0-C, C=O and C-H groups are mainly produced by thermal degradation of PCL chains in lignin-based PCL’s and PU’s.

INTRODUCTION Since plant components such as cellulose, hemicellulose and lignin are fundamentally biodegradable, biodegradable polymers with plant components have been extensively studied by various research groups [1-15]. Biodegradable polyurethanes (PU’s), have been studied at our laboratory and at the Swedish Forest Products Research Laboratory since 1990 [3-10, 12-13, 151. Thermal and mechanical properties of PU’s derived from saccharide-based polycaprolactones (PCL’s) were also reported [14]. In the present study, PU’s from PCL derivatives which were synthesized from saccharides, such as glucose, fructose and sucrose, and also from alcoholysis lignin (AL) and &aft lignin (KL) were prepared by the reaction of the above PCL derivatives with diphenylmethane diisocyanate (MDI). Thermal properties of the obtained PU’s from saccharide- and lignin-based PCL’s were studied by differential scanning

34

Synthesis and denvatisation of biocornpatible polymers

calorimetry (DSC), thermogravimetry (TG) and TG-Fourier transform infrared spect ro metr y (FTI R) .

EXPERIMENTAL Sample preparation Saccharide- and lignin-based PCL's were synthesized by the polymerization of Ecaprolactone (CL) which was initiated by each OH group of glucose, fructose and sucrose, AL and KL. The amount of CL was varied from 1 to 5 moles per OH group of each saccharide, and was varied from 2 to 25 moles per OH group of each of the above lignins. The polymerizations were carried out for 12 hr at 150 "C with the presence of a small amount of dibutyltin dilaurate (DBTDL). PU's were obtained by the following procedure. Saccharide- and lignin-based PCL's (Sac- and Lig-PCL's) were dissolved in tetrahydrofuran (THF). MDI was reacted with each of the above solutions of Sac- and Lig-PCL's for 30 min at room temperature with stirring. Each of the obtained PU prepolymers was cast on a glass plate and the solvent was evacuated in a vacuum desiccator under dry conditions. The obtained PU's were cured at 120 "C for 2 hr.

Measurements Differential scanning calorimetry (DSC) was performed using a Seiko 220 at a heating rate of 10 "C/min under a nitrogen flow (flow rate = 30 ml/min). Sample mass was ca. 5mg. Aluminum open pans were used. The samples were heated to 120 "C and quenched to -150 "C. Melting temperature (TJ, melting enthalpy (AH,,,), cold crystallization temperature (T& glass transition temperature (TJ and heat capacity gap at Tg (AC,) were determined by the method reported previously [16]. Thermogravimetry (TG) was performed using a Seiko TG 220 at a heating rate of 10 "C/min in the temperature range from 20 to 800 "C under a nitrogen flow (flow rate = 200 ml/min). Sample mass was ca. 5mg. TG curves and derivatograms (DTG) were recorded. Mass residue (WR) was calculated according to the equation: WR = (mT/m,,) x 100 (%) where mT is mass at temperature T and mzois mass at 20 "C. In order to analyze gases evolved by thermal degradation, TG-Fourier transform infrared spectroscopy (FTIR) was performed using a Seiko TG 220 - JASCO ETIR-420 system at a heating rate of 20 "C in the temperature range from 20 to 800 "C under a nitrogen flow (flow rate = 200 ml/min).

RESULTS AND DISCUSSION Fig. 1 shows the schematic chemical reaction for the synthesis of saccharide-based PCL's. The results of the characterization of glucose-, fructose- and sucrose-based PCL's have been reported elsewhere [14]. The obtained saccharide-based PCL's were reacted with MDI according to the conditions mentioned in the Experimental Section. Fig. 2 shows the schematic chemical structure of the PU from sucrose-based PCL. The length of the PCL chains attached to the sucrose core structure is shown as "m" in the diagram. (The number "m" was controlled by the initial amounts of CL which are shown as "n" in Fig. 1.) TgyS were observed in all of the PU samples from saccharidebased PCL's. Tg decreases with increasing CL/OH ratio in PU's from ca. -15 to -60 "C in the case of PU's from glucose- and fructose-based PCL's and from ca. -40 to -60 "C

Saccharide- and lignin-based polycaprolactones and polyurethanes

3S

in the case of PU's from sucrose-based PCL's. The above facts suggest that PCL chains with saccharides act as soft segments in PU networks and that this softening effect of caprolactone chains was enhanced progressively with increasing chain length of CL chains.

Sucrose

E:

-Caprolactone

CH,OR

~~ ~~rn,o,

° b~L-ln( RO

Sucrose-based PCL CH20H O

~

H20 H O

~

OH

HO

HO

OH

HO

Glucose

Figure 1.

CH,OH -

OH

OH

Fructose

Schematic chemical reaction for the synthesis of saccharide-based PCL's (CO(CH2),O)m-CONHRNHCOO-

I

(CO(CH2)SO)m-CONHRNHCOO-

o

I

~H2

I

tR- ~ CH2

o

o

/

-oocm"'HNOC-JO(H,C),OC)

0

I

-OOCHNRHNOC-m(O(H2C),Oq

CHp

0

I

(CO(CH"O).-CONHRNHCOO-

I

(CO(CH2),O)m-CONHRNHCOO(CO(CH2),O)m-CONHRNHCOO(CO(CHi>sO)m-CONHRNHCOO-

Figure 2.

Schematic chemical structure of the PU from sucrose-based PCL

36

Synthesis and derivatisation of biocompatible polymers

The DSC curves representing each PU from saccharide-based PCL’s with CL/OH ratio 5 showed a prominent exothermic peak due to cold crystallization at around -20 “C. A peak of melting of crystals was also observed at around 40 “C. The DSC curve representing each PU from saccharide-based PCL’s with CL/OH ratio 4, which was annealed at room temperature, showed a melting peak around 40 “C. The above results suggest that the PU’s derived from the saccharide-based PCL’s with CL/OH ratios over 4 have a crystalline region in the molecular structure. Fig. 3 shows the schematic chemical reaction for the synthesis of lignin-based PCL’s. The obtained lignin-based PCL’s were reacted with MDI according to the conditions mentioned in the Experimental Section. Fig. 4 shows the schematic diagram for the preparation of lignin-based PCL’s and also for the preparation of PU’s from lignin-based PCL’s. The schematic chemical structure of the obtained PU from ligninbased PCL is shown in Fig. 5. The length of the PCL chains attached to the lignin core structure is shown as “m” in the figure. (The number “m” was controlled by the initial amounts of CL which are shown as “n” in Fig. 3.)

H

O

I-

-

/40CH3 C ~ ~ O

H

0

+ n

Figure 3.

0 I1

Schematic chemical reaction for the synthesis of lignin-based PCL’s

Saccharide- and lignin-based polycaprolactones and polyurethanes

&aft Lignin

*

Alcell Lignin Benzene

&aft Lignin

Alcell Lignin -caprolactone catalyst

E

Lignin-based Polycaprolactone

I

I-

Polyurethane Sheets

Dissolved in Dioxane

1

m’

NCO / OH ratio = 1,2 CL /OH ratio (mol/mol) = 2-5, 10,15,20,25 Figure 4. Schematic diagram for the preparation of lignin-based PCL’s and also for the preparation of PU’s from lignin-based PCL’s

O(CH2)~0)m-CONHRNHCOO-

I (CO(CH2)50)m-CONHRNHCOO-

(CO(CH2)50)m-CONHRNHCOO-

Figure 5. The schematic chemical structure of the obtained PU from lignin-based PCL

37

38

Synthesis and denvatisation of biocompatible polymers

Fig. 6 shows representative DSC curves of AL based PCL (ALPCL) with various CUOH ratios of 10, 15 and 20. A marked change in baseline due to glass transitjon was observed in each DSC curve. 7,’s were determined by the method reported previously [ 161. T, decreases with increasing CUOH ratio from 2 to 10 in PU’s from Lig-PCL’s, since caprolactone chains with lignin act as soft segments in PU networks. However, as shown in Fig. 6, when the CUOH ratio was 10 to 25, T, increased. In the case of the DSC curves representing AL-PCL’s with CUOH ratio 15, a prominent exothermic peak due to cold-crystallization of h P C L and also a prominent peak due to melting of crystals are observed at around 40 “C when CUOH ratio \was over 15. The above results suggest that the PU’s derived @omALPCL’s with CUOH ratios over 15 have a clear crystalline region in the molecular structure. A similar phenomenon was observed in PU from KL-PCL’s with CUOH ratio 10. Fig. 7 shows the changes of Tg’sofPU’s from A L and KLPCL’s. The TI markedly decreases with increasing CUOH ratio in the region where CUOH ratio below 15 and then the T, increases with increasing CUOH ratio in the region where CUOH ratio exceeds 15. The increase of T, over CUOH ratio = 15 suggests that by the introduction of long PCL chains the crystalline region increased and this restricted the motion of PCL chains. Fig. 8 shows the changes of T,’s, cold-crystallization temperatures (Tab) and melting temperatures (T,,,’s),against CUOH ratios of PU’s derived fiom A L and KLPCL‘s (KLPCL PU’s). The change of TI’Sis almost the same for the A L and KLPCL PU’s. Ta’s and T,’s slightly increase with increasing CUOH ratio in the region over CUOH ratios over 15, suggesting increasing crystallized area of PCL chains in the AL and KLPCL PU’s.

0 D

w“

I

I

-100

I

-50

T

I

I

0

50

/“C

Figure 6. DSC heating curves of PU’s from &based PCL Numerals in the figure show CUOH ratio and arrows indicate T“s. Tr glass transition temperature; T , cold-crystallization temperatures; Tm,melting temperature

Saccharide- and lignin-based polycaprolactones and polyurethanes

0 0

.

-20

,o M

h

-40 .

-60

-

-80 0

I

I

I

I

I

5

10

15

20

25

30

CL / OH ratio / (mol/mol) Figure 7. Relationship between Tg’sand CUOH ratios in AL and KL-PCL PU’S

0 AL-PCLPU

Figure 8.

0 KL-PCLPU

Phase diagram for AL-and KL-PCL PU’s showing Tp T, and T,,,

39

40

Synthesis and denvatisation of biocompatible polymers

--I0

-20 100

I

I

I

200

300

400

T I'C

Figure 9. TG and DTG curves of AL-PCL PU's with CUOH ratios of 10,15 and 20 rnolhnol 400

350 0

" R, 3m:%-8--

250

200

I

0

5

I

1

1

1

10

15

20

25

30

CL/ OH ratio / (rnol/rnol) Figure 10. Change of Tdland Td2with CUOH ratios of AL- and KL-PCL PU's.

0 AL-PCL PU T,,

w

0 AL-PCL PU Td2

0 KL-PCLPU T,j,

KL-PCL PU T,,

Saccharide- and lignin-based polycaprolactones and polyurethanes

41

Fig. 9 shows TG and DTG curves of AL-PCL PU’s with CUOH ratios of 10, 15 and 20. Two kinds of thermal degradation temperatures, Tdl and Tdz are observed. Similar TG and DTG curves of KL-PCL PU’s were also obtained. Fig. 10 shows the change of Tdl and Td2with CUOH ratios of AL- and KL-PCL PU’s. Matsuzak et al. reported that some urethane bonds in PU’s dissociate to form hydroxyl and isocyanate groups at about 200 “C [17]. Dornberg et al. proposed a mechanism where a dehydration reaction of hydroxyl groups in alkyl groups and heterolysis and homolysis dissociation of P-aryl ether bonds in lignin occur initially at about 200 “C [ 181. Accordingly, it is considered that Td, may reflect the degradation of lignin parts in AL- and KL-PCL PU’s. We reported that the Td of cellulose acetatebased polycaprolactones increased from 350 to 390 “C with increasing CUOH ratio from 2 to 20 [19]. The above change of Td accords well with the change of T d 2 of ALand KL-PCL PU’s, as shown in Fig. 10. Accordingly, it is considered that Tdz may reflect the degradation of PCL parts in AL- and KLPCL PU’s. Fig. 11 shows the change of WR at 420 “C with CUOH ratios of AL- and K L P C L PU’s. PU’s with various KL contents from 0 to 50 % in polyethylene glycol which was obtained by the reaction with MDI showed that the W R of the PU’s increased with increasing KL contents [18]. This suggests that the lignin part in PU’s derived fiom lignins constitutes a significant part of the residual products. It is also obvious that the WR’s of AL and KL-PCL PU’s decrease with decreasing lignin core structure in the lignin-based PCL PU’s. Accordingly, it is considered that a significant part of the residual products of AL- and KL-PCL PU’s may consist of core lignin structure.

50

40

t3 1

s

30

20 0

10

20

30

CL /OH ratio / (mol/mol)

Figure 11. Change of WR with CWOH ratios of AL- and KL-PCL PU’s WR at 420 “C A AL-PCLPU A KL-PCLPU

42

Synthesis and derivatisation of bioeompatible polymers

O.OS 0.04 III

-<

.0

0.02 800

00 \

Figure 12.

0.04

Stacked FfIR spectra of gases at various temperatures during thermal degradation of KL-PCL PU (CUOH ratio = 5 moVrnol)

r------------------------

0.03

.2 0.02

<

0,0)

o L4000

_ ___'_ _

-'-----=:=:::::::::~==::==___~

3000

2000

tOOO

~

600

Wavenumber / cm'

Figure 13.

TG-FTIR spectrum otthc KL-PCL PU (CLIOH ratio = C; rno l/rno l) corrcsrnnJing to 4~("C

Saccharide- and lignin-based polycaprolactones and polyurethanes

43

Fig. 12 shows the stacked FTI R spectra of gases at various temperatures during thermal degradation of KL-PCL PU (CWOH ratio = 5 rnol/moi). Fig. 13 shows the TGF H R spectrum of the above KL-PCL PU corresponding to 420 "C which was obtained from Fig. 12. The main peaks observed for the samples are as follows: 1128 cm" (V c0-C), 1260 cm-' [v C(=O)-C-1, 1517 and 1617 crn" (v C X ) , 1718 cm-' (v C=O), 2358 cm.' (v CO,, v NO,), 2892 cm-' (v CH) and 3700 cm" (v H,O) Figs. 14 and 15 show the changes of characteristic IR absorption peaks of evolved gases from AL- and KL-PCL PU's. The changes of IR absorption intensities calculated at 420 "C are almost similar. The IR absorption intensity of C 0 2gas from AL- and KLPCL PU's do not show the PCL chain length dependency, while the other IR absorption peaks show the PCL chain length dependency. This suggests that the evolution of CO, gas occurs randomly and is not specific to the chemical structure. The IR absorption intensities corresponding to C-0-C, C=O and CH peaks increase markedly with increasing CUOH ratios. This suggests that gases having C-0-C, C=O and CH groups are evolved from PCL chains. The above facts well accord with the decrease of the mass residue, WR,with increasing PCL chain length in lignin-based PCL PU's.

0.15

0.1

4 0.05

0

0

10 20 CL /OH ratio I (mollmol)

30

Figure 14. Changes of characteristic IR absorption peaks of evolved gases from AL-PCL PU's C-0-C

C=O

+ CO,

A CH

44

Synthesis and derivatisation of biocompatible polymers

0.12

10

0

20

30

CL /OH ratio / (mol/mol) Figure 15. The changes of characteristic IR absorption peaks of evolved gases from KL-PCL PU’s

O C-0-C

0 C=O

0 CO, A

CH

CONCLUSIONS (1) Saccharide- and lignin-based polycaprolactones were synthesized from glucose-, fructose, sucrose, alcoholysis lignin (AL) and &aft lignin (KL) by the polymerization of E-caprolactone (CL) which was initiated by the OH group of saccharides and lignins. The CUOH (mol/mol) ratios of the saccharide-based PCL’s were changed from 1 to 5 and those for lignin-based PCL’s were changed fiom 2 to 25. PU sheets were prepared from the above PCL derivatives by the reaction with diphenylmethane diisocyanate (MDI). (2) Glass transition temperatures (T,’s), cold-crystallization temperatures (T,’s) and melting temperatures (T,,,’s)of saccharide- and lignin-based PCL’s and PU’S were determined by DSC, and phase diagrams were obtained. Tg’s decreased with increasing CUOH ratio, suggesting that PCL chains act as a soft segment in the amorphous region of PU molecules. Two thermal degradation temperatures (T,,’s) were observed in TG curves of PU’s from saccharide- and lignin-based PCL’s with low CUOH ratios. (3) TG-FTIR analysis of PU’s from lignin-based PCL’s suggested that compounds having C-0-C, C=O and C-H groups are mainly produced by thermal degradation of PCL chains in lignin-based PCL’s and PU’s.

Saccharide- and lignin-based polyc:aprolactones and polyurethanes

45

REFERENCES 1

2

3

4

5 6

7

8

9

10

11

12

13

14

15

V. P. Saraf & W. G. Glasser, 'Engineering plastics from lignin. III. structure property relationship in solution cast polyurethane films', J. Appl. Polym. Sci., 1984,29, 1831-1841. V. P. Saraf & W. G. Glasser, 'Engineering plastics from lignin. VI. structure property relationship of PEG-containing polyurethane networks', J. Appl. Polym. Sci., 1985,30,2207-2224. H. Yoshida, R. Morek, K. P. Kringstad & H. Hatakeyama, 'Kraft lignin polyurethanes. II. effects of the molecular weight of Kraft lignin on the properties of polyurethanes from a Kraft lignin-polyether triol-polymeric MOl system', J. Appl. Polym. Sci., 1990,40,1819-1832. K. Nakamura, R. Morek, A Reimann, K. P. Kringstad & H. Hatakeyama, 'Mechanical properties of solvolysis lignin derived polyurethanes', Polym. Adv. Technol., 1991, 2,41-47. K. Nakamura, T. Hatakeyama & H. Hatakeyama, 'Thermal properties of solvolysis lignin-derived lignocellulose', Polym. Adv. Technol., 1992,3, 151-155. K. Nakamura, Y. Nishimura, T. Hatakeyama & H. Hatakeyama, 'Preparation of biodegradable polyurethanes derived from coffee grounds', In: Proceedings for International Workshop on Environmentally Compatible Materials and Recycling Technology, 1993,Tsukuba, Japan, pp. 239-244. H. Hatakeyama, S. Hirose, K. Nakamura & T. Hatakeyama, 'New types of polyurethanes derived from lignocellulose and saccharides', In: Cellulosics: Chemical, Biochemical and Material Aspects, J. F. Kennedy, G. O. Phillips and P. A Williams (eds.), 1993, Ellis Horwood, Chichester, pp. 524-536. H. Yoshida, K. Kobashigawa, S .Hirose & H. Hatakeyama, 'Molecular motion of biodegradable polyurethanes derived from molasses', In: Proceedings for International Workshop on Environmentally Compatible Materials and Recycling Technology, 1993, Tsukuba, Japan, pp.233-238. N. Morohoshi, S. Hirose, H. Hatakeyama, T. Tokashiki & K. Teruya, 'Biodegradability of polyurethane foams derived from molasses', Sen-i Gakkaishi, 1995,51,143-149. H. Hatakeyama, S. Hirose, T. Hatakeyama, K. Nakamura, K. Kobashigawa & N. Morohoshi, 'Biodegradable polyurethanes from plant components', J. Macromol. Sci., Pure Appl. Chem., 1995, A32, 743-750. M. J. Donnely, 'Polyurethanes from renewable resources. IV-properties of linear, crosslinked and segmented polymers from polytetrahydrofuran diols and their glucosides', Polymer International, 1995,37,297-314. K. Nakamura, Y. Nishimura, P. Zetterlund, T. Hatakeyama & H. Hatakeyama, 'TG-fTIR studies on biodegradable polyurethanes containing mono-and disaccharide components', Thermochmica Acta, 1996,282/283,433-441. P. Zetterlund, S. Hirose, T. Hatakeyama, H. Hatakeyama & A-C. Albertsson, 'Thermal and mechanical properties of polyurethanes derived from mono-and disaccharides', Polymer International, 1997,42,1-8. H. Hatakeyama, K. Kobahigawa, S. Hirose & T. Hatakeyama, 'Synthesis and physical properties of polyurethanes from saccharide-based polycaprolactones', Macromol. Symp., 1998, 130, 127-138. T. Hatakeyama, T. Tokashiki & H. Hatakeyarna, 'Thermal properties of polyurethanes derived from molasses before and after biodegradation', Macromol. Symp., 1998, 130, 139-150.

46

Synthesis and derivatisation of biocompatible polymers

16 S. Nakamura, M. Todoki, K. Nakamura & H. Kanetsuna, ‘Thermal analysis of polymer samples by a round robin method. I. reproductibity of melting, crystallization and glass transition t ernperatures’, Thermuchimica Acta, 1998, 136,

163-178. 17 M. L. Matsuzak & K. C. Frisch, ‘Termal degradation of linear polyurethanes and model ciscarbamates’, J . Polym. Sci.,Polym. Chem. Ed., 1973, 11, 637-648. 18 S. Hirose, K. Kobashigawa, Y. Izuta & H. Hatakeyama, ‘Thermal degradation of polyurethanes containing lignin studied by TG-FTIR’, Polymer International, 1998,47, 247-256. 19 H. Hatakeyama, T. Yoshida, S. Hirose & T. Hatakeyama, ‘Thermal and viscoelastic properties of cellulose- and lignin-based polycaprolactones’, In: Proceedings for Cellucon’ ’98, 1998, Turku, Finland, p.9.

CELLULOSE AS A RAW MATERIAL FOR LEVOGLUCOSENONE PRODUCTION BY CATALYTIC PYROLYSIS G . Dobele'(*), G. Rossinskaja', T. Dizhbite', G . Telysheva', S. Radtke*, D. Meier' & 0.Faix' I Latvian State Institute of Wood Chemishy, 27 Dzerbenes St.. LV-1006, Riga, Latvia 2 Institute for Wood Chemishy and Chemical Technologyof Wood, 91 Leuschnerstr. 0-21031 Hamburg, Germany

ABSTRACT This work concerns studies on the thermocatalytic dehydration of "Taircell" cellulose from the sulphate pulping process and microcrystalline "Munktel" cellulose in order to obtain 1,6-anhydro sugars (levoglucosane and levoglucosenone) in high yields by flash pyrolysis. It has been shown that the heterogeneous interaction of cellulose with phosphoric acid begins with the impregnating step already at room temperature and continues during thermal treatment of cellulose. The maximal yields of levoglucosenone in flash pyrolysis were 25% ("Taircell", 7% phosphoric acid, 100°C) and 30% ("Munktel", 3.5% phosphoric acid, 100°C).

INTRODUCTION Investigations in the field of thermocatalytic biomass conversions were carried out in various directions related to obtaining energy, valuable monomeric products for organic synthesis, carbon materials of a different type, etc. The present work is aimed at studying the pyrolysis process of cellulose catalyzed by phosphoric acid. The main volatile product of thermal cellulose degradation under acid catalytic conditions is levoglucosenone ( 1,6-anhydro-3,4-dideoxy-~-D-hexo-glycero-3enopyranose-2-ulose)' . Levoglucosenone (LGone) is an ideal monomer for the synthesis of optically active compounds for medicine, sulphur- and nitrogen-containing heterocycles, rare sugars being analogous to natural ones2. It is known that thermodegradation of cellulose impregnated with phosphoric acid proceeds in a lower temperature range and is accompanied by a decrease in the yield of organic volatile products and an increase in the yield of ate?'^. In this process the primary dehydration reactions control the c o m e of the subsequent thermal degradation. The mechanism of cellulose interaction with phosphoric acid was realized through multiple esterification steps, similarly to the case of other hydroxyl-containing polymers'. As a result of the esterification reaction and subsequent elimination of phosphoric acid, the amount of carbonyl groups and carbon double bonds in the degradation products tended to increase6. It has been demonstrated earlie?74that cellulose impregnated with phosphoric acid shows a pronounced tendency to form levoglucosenone already at 250°C under the conditions of slow pyrolysis (heating rate 510°C min-I). The maximum yield of LGone (22% based on cellulose) during slow pyrolysis (350°C)was obtained by adding 5% of phosphoric acid to cellulose. Under these conditions non-dehydrated 1,6-anhydro sugar, levoglucosan, being normally the main product of thermal cellulose depolymerization

48

Synthesis and denvatisation of biocompatible polymers

was not practically formed, although its formation in high yields is the usual case in

cellulose pyrolysis.

OH

Levoglucosan

Levoglucosenone

Under the conditions of catalytic flash pyrolysis (simulated by analytical pyrolysis gas chromatography, Py-GC) besides lev0 lucosenone a considerable amount of levoglucosan was found in volatile products . The latter may be explained by the short time of flash pyrolysis, which is insufficient for the dehydration action of phosphoric acid. It was suggested that an additional step of the low-temperature pretreatment of impregnated cellulose was necessary to increase the phosphoric acid dehydration effect, if the flash pyrolysis should be used for LGone production. This topic is in the focus of the present paper. The aim of the present work was to study the effect of pretreatment temperature and the amount of the phosphoric acid on 1,6-anhydro sugars formation in flash pyrolysis of sulphate and microcrystalline cellulose, characterized by different degrees of polymerization.

!

’

MATERIALS & METHODS “Taircell” cellulose from the sulphate pulping process (crystallinity index 78.5, polymerization degree 1040) and “Munktel” cellulose (crystallinity index 85.7, polymerization degree 200) were pyrolysed. Phosphoric acid (2, 3.5, 5 , 7 and 9% based on dry cellulose) was introduced into the material by impregnation: an aqueous solution of phosphoric acid was mixed with cellulose (cellulose/acid solution = 1/5), then the samples were dried at room temperature for 48 hrs. Impregnated samples were thermally treated for 1 hour at 100 or 16OoC in an inert atmosphere. A CDS Pyroprobe 100 combined with a gas chromatograph (CP 9000) was applied for Py-GC. Sample amount for pyrolysis: ca. 70 pg. Pyrolysis temperature: 350°C. Heating rate: 600°C s-’. Pyrolysis time: 10 s. GC column: DB 1701 (60 m x 0.25 mm, 0.25 pm film). Crystallinity index was calculated based on X-ray diffractometry data and the degree of polymerization was determined by intrinsic viscosity using codaxen as solvent.

RESULTS & DISCUSSION The interaction of cellulose with phosphoric acid was already detectable at room temperature and became very pronounced when the impregnated samples were thermally treated. The polymerization degree of “Taircell” cellulose decreased from 1040 to 445 and 380, respectively, just after its impregnation with 3.5 or 7% phosphoric acid (Table 1). The pretreatment of cellulose impregnated with 3.5% acid at 100°C resulted in the subsequent decrease of the degree of polymerization to 3 10. The increase in the amount of acid to 7% had no additional hydrolytic effect on cellulose (Table 1).

Cellulose as a raw material

49

Table 1. Variations in the degree of polymerization of Taircell cellulose depending on the amount of phosphoric acid and pretreatment temperature. Amount of H3P04, %

3.5 3.5 3.5 7 7 7

Temperature of thermal Degree of pretreatment, “C polymerization 20 1040 45.0 20 100,l h 310 160,l h 265 380 20 100,l h 380 160,l h 230

Solubility in codaxen, % 100 100 100 90.8 100 100 59.3

As a result of increasing the temperature to 160”C, a part of the cellulose became insoluble in cadoxen. In this case, increasing the acid concentration from 3.5 to 7% reduced the soluble moiety further from 90.8 to 59.3%. The samples impregnated with 3.5 or 7% phosphoric acid and treated at 160°C revealed a similar degree of polymerization in their soluble part (Table 1). The drop in solubility in cadoxen indicates that at elevated temperatures phosphoric acid, apart from the hydrolytic action, leads also to cross-linking. In general, the yields of the 1,6-anhydro sugars LGone and LG in flash pyrolysis of celluloses varied depending on the amount of phosphoric acid introduced and pretreatment temperature (Fig. 1, 2). However, in the case of the pyrolysis of impregnated “Taircell” cellulose (from 2 to 7% of acid) without thermal pretreatment, the LGone yield (16- 17% on cellulose) did not, in fact, depend on the amount of acid. Pretreatment of cellulose impregnated with 3.5% phosphoric acid also did not result in a change of the levoglucosenone yield (Fig. la). The increase in the acid addition to 5 and 7% under the pretreatment conditions at 100°C promoted the LGone yield in pyrolysis. In this case, a maximum yield of 25% was reached when adding 7% of acid. The elevation of the pretreatment temperature to 160°C had a negative effect on the LGone formation (Fig. 1a). The levoglucosan yield obtained by flash pyrolysis of “Taircell” cellulose impregnated with 3.5% of phosphoric acid, was the same (19%) as that obtained from the initial cellulose (Fig. 1b). At this level of acid impregnation the thermal pretreatment was not favourable for the LG formation. On the other hand, at higher levels of acid impregnation (5 and 7%), the thermal pretreatment resulted in some increase in the levoglucosan yield during the subsequent pyrolysis. In these cases there was an extreme relationship of LG yield vs the pretreatment temperature. However for different levels of acid impregnation the LG yield reached its maximum at different temperatures of pretreatment. At 5% of phosphoric acid the experiments rendered the highest LG yield (15%) for the samples pretreated at 16OoC, while in the presence of 7% acid the best LG yield (10%) was reached at 100°C pretreatment (Fig. 1b). Pyrolysis of the initial microcrystalline “Munktel” cellulose, in contrast to “Taircell” cellulose, proceeded with a higher levoglucosan yield, i.e. 79%, being close to the theoretically highest value at the formation of an 1,6-anhydro bridge in the glucopyranose unit. The dependencies of the LGone and LG yields in thermocatalytic flash pyrolysis, on the pretreatment parameters followed different functions for “Munktel”and “Taircell” cellulose (Figs. 1, 2).

50

Synthesis and derivatisation of biocompatible polymers

a b Figure 1. Yields of levoglucosenone (a) and levoglucosan (b) in flash-pyrolysis (350°C) of “Taircell” cellulose depending on the amount of phosphoric acid introduced and pretreatment temperature. The highest levoglucosenone yield (30%) was generated when cellulose was pyrolysed after impregnation with 3.5% of phosphoric acid and pretreatment at 100°C (Fig. 2a). Under the same thermocatalytic conditions also the highest amount of LG (1 1%) was formed (Fig. 2b). The catalytic pyrolysis of microcrystalline “Munktel” cellulose was most succesful by the addition of 3.5% of phosphoric acid and the subsequent pretreatment at 1OO”C, rendering 1,6-anhydro sugars in high yields. The highest total yields of LG and LGone in the catalytic flash pyrolysis of “Taircell” and “Munktel” celluloses were 37% (19 + 18) and 41% (1 1 + 30), respectively. This was the case when 3.5% of phosphoric acid were added both to “Taircell” and “Munktel” celluloses; no thermal pretreatment was needed for the former and heating at 100°C for the latter. ---__-

Amount of HIPOI, YO

a

r

1

Amount of HIPOI, %

b

Figure 2. Yields of levoglucosenone (a) and levoglucosan (b) in flash-pyrolysis (350°C) of “Munktel” cellulose depending on the amount of phosphoric acid introduced and pretreatment temperature.

Cellulose as a raw material

51

The results obtained indicate that both 1,6-anhydro sugars, i.e. levoglucosan and levoglucosenone can be produced simultaneously with considerable yields when phosphoric acid impregnation followed by thermal pretreatment is applied before flash pyrolysis. However, if, in the case of sulphate “Taircell” cellulose, catalytic pyrolysis enhances the development of depolymerization reactions with the formation of an additional amount of 1,6 anhydro-sugars, in the case of microcrystalline “Muntkel” cellulose, the total amount of both 1,6-anhydro sugars is twice as low as the LG yield in the pyrolysis of the initial cellulose.

CONCLUSIONS The interaction of cellulose with phosphoric acid begins already with the impregnation step at room temperature and continues under the pretreatment and pyrolysis conditions. Under the conditions of flash pyrolysis, levoglucosenone yield could be increased with an additional low-temperature pretreatment (100°C) of the impregnated cellulose. The amount of phosphoric acid for impregnation has to be adapted to the initial degree of polymerization of cellulose in order to obtain optimal results. The maximum yields of levoglucosenone for “Taircell” cellulose (7% acid) and “Munktel” cellulose (3.5% acid) were 25% and 30%, respectively.

REFERENCES 1. F.Shafkadeh & P.P.S.Chin, ‘Preparation of 1,6-anhydro-3,4-dideoxy-~-D-glycero-

2.

3. 4.

5. 6.

hex-3-enopyranos-2-ulose (levoglucosenone) and some derivatives thereof, Carbohydr Res, 1977 56 (l), 79-87. Z.J.Witczak, Levoglucosenone and Levoglucosans, Chemistry and Application, ATL, 1994. G.Dobele, G.Rossinskaja, G.Telysheva, D.Meier & O.Faix, ‘Cellulose dehydration and depolymerisation reaction upon thermal treatment under the action of phosphoric acid’, JAnal Appl Pyrolysis, 1999 49, 307-17. G.Dobele, T.Dizhbite, G.Rossinskaja & G.Telysheva, ‘Thermocatalytic destruction of cellulose’, In: Cellulose and Cellulose Derivatives: Physico-chemical aspects and industrial application, J.F. Kennedy, G.O. Phillips, P.A. Williams (eds.), Woodhead, Cambridge, 1995, pp 125-130. KKatsuura , N.Inagaki, ‘Fire retardance in cellulose fabrics’, In: Developmenr of Polymer Degradation, vol. 4, London, 1982. G.Domburg, G.Dobele, G.Rossinskaja. ‘Cellulose dehydration under catalysis conditions’, Khim. Drev., 1988 (3), 97-102 (in Russian).

New ionic polymers by subsequent functionalization of cellulose derivatives M. Vieira, T. Liebert, and Th. Heinze* institute of Organic Chemisty and Macromolecular Chemistry, Friedrich Schiller University of jena, Humboldtstrasse 10, D-07743 Jena, Germany

ABSTRACT The selective oxidation of the primary OH functions of hydroxyethyl cellulose with sodium bromide/sodium hypochlorite in the presence of catalytic amounts of 2,2,6,6tetramethyl- 1-piperidinyloxy radical (TEMPO) is described. Depending on the reaction conditions, a oxidation of the OH groups of hydroxyethyl moieties or a complete oxidation of all OH functions is possible as revealed by I3C NMR spectroscopy and HPLC after complete chain degradation. A second approach for new ionic cellulosics is the subsequent esterification of the unmodified Off units of p-toluenesulfonyl cellulose. Very recent results show that besides the established sulfonation reactions tosylation is possible in a reactive microstructure resulting in an uneven distribution of substituents along the polymer. The esterification of the tosyl cellulose is carried out with di- and tricarboxylic acid anhydrides or with pyridine-SO3 complex.

INTRODUCTION Naturally occurring ionic polysaccharides are of enormous importance in biological systems mainly as structure forming units or bioactive transducers. They show a wide variety of sugar composition, types of functional groups and arrangements of these building segments [I]. Thus, polysaccharides like heparins, alginates, carrageenans or pectins can differ significantly in their amounts of anionic groups and the functionalizationpattern. These differences are caused by a whole variety of factors, e.g. the type of organisms, the stage of growth, the procedure of isolation or environmental conditions during the growth [ 2 ] . They have a strong impact on the polyelectrolyte characteristics, rheological behavior, interaction with ions, and the mentioned structure forming ability and biological activity. Consequently, the application of these naturally occurring materials with their valuable features is combined with a number of problems [3]. A very challenging alternative is the development of synthesis routes for the defined preparation of ionic polysaccharides starting from nonionic polymer backbones like cellulose. In the course of our own work in the field of semisynthetic ionic polysaccharides we were able to establish a new synthesis pathway for the specific introduction of ionic functions into polycarbohydrates like cellulose, starch, and dextran in a block-like manner [4}.These poIymers show a number of interesting new features including a different superstructurecompared to statistically functionalized polymers. This paper deals with the preparation of new ionic cellulose derivatives synthesized from hydroxyethyl cellulose by oxidation and from p-toluenesulfonyl (tosyl) cellulose by subsequent esterification reactions of the remaining OH groups (Fig. 1).

Synthesis and derivatisation of biocompatible polymers

54

*s=o

*s=o I

*o-

o *R

H

/

OH

R

R= S 0 3 b -CQ H + o

NaOOC OH

OR or

R

-Cg-cOONa NaOOC

1

*o

HO

~

OCH2CH2OH

*H

0 OCH2COONa

Figure 1. Synthesis pathways applied for the preparation of new ionic cellulosics

MATERIAL AND METHODS A commercial hydroxyethyl cellulose (HEC) were supplied, Tylose H4000P2 (1) from Clariant (Frankfurt a. M., Germany) with a molar degree of substitution (MS) of 2.5. 2,2,6,6-Tetramethyl-l -peperidinyloxy (TEMPO) and aqueous sodium hypochlorite (1 3%, wiv) was purchased from Fluka, Switzerland. Tosyl cellulose samples were obtained by homogeneous conversion of cellulose with a degree of polymerisation, DP=280 (2), 850 (3), 330 (4), and 1020 ( 5 ) in N,N-dimethylacetamideLiC1 using triethylamine and p-toluenesulfonyl chloride within 24 h at 8°C according to ref. [5]. Sulfation of tosyl cellulose (lla-12) was carried out according to ref. [6], esterification with phthalic anhydride (13a, 13b) and trimellitic anhydride (14a, 14b) according to ref. [71.

Oxidation, typical example (sample 7a) 2.00 g (7.3 mmol) HEC 1, 15 mg (0.096 mmol) TEMPO and 100 mg (0.97 mmol) NaBr were dissolved in 100 ml dist. water under stirring. A solution with a pH value of 5.8 results. The mixture was kept in an ice bath (temperature l i l ° C ) and the sodium hypochlorite solution was added until a pH value of 10.8 was obtained. When the pH dropped below 10.8, sodium hypochlorite was added. After complete consumption of 4.0 ml NaClO solution, the pH was adjusted to a value of 10.0 by successive addition of 1.5 ml of a 0.5 M NaOH solution. The clear solution obtained was precipitated in ethanol, filtered, washed and dried in vacuum. Yield: 1.53 g, degree of oxidation DO=0.38 (based on sodium analysis). FTIR (KBr): 3428 (v OH), 2912 (v CH), 1610 (v COONa), 1418, 1331 (6 COONa), 1109, 1060 (v COC) cm”. Alternative synthesis of p-toluenesulfonyl cellulose in reactive microstructure (6) A solution of 2 g cellulose in 70 ml DMA and 4 g LiCl was treated with 9.9 g solid NaOH (suspended in 20 ml DMSO). After 20 min 23.5 g tosyl chloride was added and the reaction mixture was kept for 5 h at 10°C. Isolation was carried out by pouring in 500 ml ethanol, washing with 200 ml ethanol four times, swelling of the product in

New ionic polymers

55

100 ml acetone for 24 h and washing with 150 ml ethanol. Yield 3.3 g, DS.~,,,,I=O.~ (calculated on the basis of the elemental analysis). FTIR (KBr): 3533 (v OH), 3070 (v C-Har,,,,,), 2888 (v CH), 1500 (v C-C,,,,), 1362 (v, SOZ), 1171 (v, S02) cm-'. The FTIR spectra were recorded on a Nicolet Impact 400 spectrometer using KBr pellets. I3C NMR spectra were acquired on a Bruker AMX 400 spectrometer, the accumulation number was between 1200 and 60000 scans. The HPLC measurements was undertaken according to ref. [8]. A JASCO GPC with refractive index, and two columns (type HEMA Bio 100,lOp; HEMA Bio linear lop) was used. Eluent was a 0.1% (w/v) aqueous NaN03 solution and the flow rate 1.0 ml/min. Calibration was carried out with dextran standards giving a optimal linear function between the lowest (1 80 Da) and highest standard (277 kDa). For sodium analysis a flame photometer of the type Flapho 41 was used. The amount of NaCl in the system was calculated on the basis of chlorine.

RESULTS AND DISCUSSION Oxidation of primary hydroxyl groups of polysaccharides Recently, de Nooy et al. succeeded in the selective oxidation of water-soluble polysaccharides such as starch, inulin and pullulan [9, 101. The oxidation is carried out in water homogeneously and is mediated by 2,2,6,6-tetramethyl- 1-peperidinyloxy radical (TEMPO) using hypobromite as the oxidizing agent. 6-Carboxyl polysaccharide derivatives with high degree of oxidation up to 0.87 were obtained. The selectivity was estimated to be complete. Selective oxidation of hydroxyethyl cellulose Compared to the most important ionic cellulose product, carboxymethyl cellulose (CMC), carboxyl celluloses (COC) show different properties caused by the fact that the ionic group is directly located at the polymer backbone. Due to this lower distance of the carboxyl groups, COC is more acidic [I 13 and the gelation with calcium ions is more effective [ 121. By introduction of differently located carboxyl groups, new cellulosic polyelectrolytes may be obtained. However, the carboxymethylation of COC seems to be not appropriate since a subsequent reaction of this polymer needs a time-consuming activation procedure in order to gain significant conversion [ 13, 141. In this context our interest was focused on hydroxyethyl cellulose, which is a commercially produced water-soluble cellulose ether and contains different types of primary hydroxyl groups (Fig. 1). The HEC sample used for our studies possesses a molar degree of substitution of 2.5. By means of I3C NMR spectroscopy it was revealed that not all of the hydroxyl groups at C-6 were included in the etherification reaction with ethylene oxide, since there are typical signals at 60 ppm for the C-6 position bearing an unmodified hydroxyl group. In a first series of oxidation experiments of HEC with TEMPO/NaBr/hypochlorite, the influence of the amount of NaOH in the reaction mixture was studied. A higher amount of NaOH results in a stronger depolymerization. Therefore, (Tab. 1) the pH was adjusted to 10.8 and the amount of NaOH applied was 0.1 to 0.5 mol NaOWmol

56

Synthesis and derivatisation of biocompatible polymers

modified anhydroglucose unit (AGU). To diminish side reactions and to obtain a good selectivity the hypohalite was added stepwise.

Table 1. Condition and results of the oxidation of HEC with TEMPO/NaBr/NaCIO Reaction conditions Oxidized Temperature Molar ratio Time No. Na NaCl NaClO NaOH ("C) (mol/mol) (mol/mol) (min) ("A) (Yo) 1 1.55 0.1 1 31 7a 3.07 5.35 3.08 0.23 35 7b 1 4.63 0.37 35 1 7c 8.21 0.34 7d 10.55 0.84 6.17 0.41 34 1 3.29 1.55 0.1 1 20 10 8a 5.58 3.08 0.23 28 8b 10 4.63 0.40 25 8.95 0.62 10 8c 6.17 0.44 29 10 8d 11.00 1.68 1.55 2.80 0.45 25 0.11 9 9a 25 3.08 0.23 9 9b 5.18 0.40 8.21 25 4.63 0.44 10 9c 25 6.17 0.48 10 9d 12.53 1.94 b) 2% in water a) calculated from the amount of Na c) insoluble

Polymer DOa) Viscosityb)

0.38 0.69 1.06 1.36 0.42 0.72 1.19 1.46 0.32 0.64 1.10 1.71

(Pas)x I o-* 2.68 1.13 0.74 0.43 4.55 0.60 0.32 0.75 C)

0.79 0.47 0.20

DP values for a number of oxidized samples were determined by means of GPC and are graphically displayed in Fig. 2. It can be seen that the DP is almost not influenced by the different amounts of NaOH used. The temperature seems to have a small effect on the DP at higher NaOH concentrations only (Fig. 2). An interesting finding was the drastic decrease in viscosity of aqueous solutions of the differently oxidized samples indicating a specific interaction of the polyelectrolyte chains. The DO can be adjusted directly by the amount of oxidizing agents in the range from 0.16 to 1.71 (1.7 1 corresponds to a complete conversion of all primary OH units for 1). This was confirmed by I3C NMR spectra acquired in D20 (Fig. 3).

7a-7d 8a-8d 9a-9d Figure 2. Graphic display of the DP values of differently oxidized HEC (7a-9d)

New ionic polymers

57

The spectrum of the starting HEC 1 shows in the range from 60 to 63 ppm the signals of C-atoms of CH2-groups adjacent to OH units. The C-1 signal for the anomeric carbon is not split which means that the adjacent C-2 position is completely functionalized. As the DO value of the samples is increased two groups of signals can be recognized in the region of the C=O functions. One at I78 ppm characteristic for carboxymethyl units and one at 175 ppm for carboxyl units, i.e. the oxidation product of the unmodified C-6

(COONa) c-9

-

.

.

'

I

.

,

,

I

.

.

,

~

.

'

/

.

p p m l 8 0 160 140 120 100

.

-80

60

6

3

OR

Figure 3. I3C NMR spectra of hydroxyethylcellulose(HEC 1) and oxidized HEC samples 8a,7b,8d,9d.

58

Synthesis and derivatisation of biocompatible polymers

position. With increasing DO the signal intensities for the C-atoms adjacent to the primary OH units are diminished. In the upper spectrum signals in the region 60 to 65 ppm disappear entirely indicating a complete oxidation of all primary OH units. At higher temperature and increasing amounts of oxidizing agents the occurrence of formic acid was observed (peak at 166 ppm) as a result of oxidative degradation. Further evidence for the discussed structural features were obtained by HPLC. A chromatogram of a degraded oxidized HEC (9d, DO=1.71) is shown in Fig. 4. In comparison with investigations on CMC [ 151 we can clearly assign the signals at 16.68, 17.95, and 19.65 min to the three basic functionalization patterns on the level of the repeating unit, i.e. with three-, two-, and one COO-groups (except glucuronic acid), obtained by oxidation of hydroxyethyl moieties. The signal at 21.83 min corresponds to non oxidized unit and the shoulder on that peak is the glucuronic acid formed by oxidation of the OH function at C-6. Preparation of new ionic cellulosics by esterification of free OH functions of tosyl cellulose p-Toluenesulfonyl (tosyl) esters of cellulose are of considerable interest in preparative cellulose chemistry because of their potential as starting material for a number of cellulosics with unconventional functional groups. Usually the tosyl group serves as a leaving group in nucleophilic displacement reactions [ 161. A new path for the application of tosyl cellulose as precursor for unconventional cellulosics is the functionalization of the remaining OH units. The introduction of ionic functions seems especially very challenging for the preparation of novel amphiphilic polysaccharides. The tosyl celluloses used in this study were on one hand synthesized under totally homogeneous conditions in DMA/LiCl yielded polymers preferably functionalized at C-6 [ 5 ] . On the other hand, for an alternative tosylation the solution of cellulose (DP 280) in DMA/LiCl was treated with solid NaOH particles suspended in DMA which leads to a reactive microstructure [4]. The conversion was carried out at 8°C for 5 h (sample 6, Tab. 2). In contrast to the totally homogeneous tosylation yielding products very soluble in dimethylsulfoxide (DMSO) the toslyl cellulose prepared in the reactive microstructure is insoluble in common organic solvents even at DS values as high as 0.9. Sulfation of the tosyl celluloses may be carried out with complexed SO3. For samples

16

20

24

Time [min]

Figure 4. Chromatogram of the depolymerized oxidized HEC (9d). The signals were assigned to three (A), two (B), one (C), and no COO group bearing glucoses (D) and glucuronic acid (E).

New ionic polymers

59

2a-3b the reactions were undertaken homogeneously in DMA. In the case of 6 the reaction started as a slurry in DMA but became homogeneous during the conversion. With 2 to 4 mol reagent per mol AGU and reaction times from 2 to 4 h at room temperature DSSulfatevalues up to 0.85 were accessible (Tab. 2). The products were water soluble starting from DSsulfatvalues of 0.57 except sample 12 which was prepared from the alternatively synthesized tosyl cellulose. Structure analysis was possible by means of FTIR and I3C NMR spectroscopy [6]. It can be concluded from these studies that the C-6 position is completely functionalized (no signal in the I3C NMR at about 60 ppm) and C-2 is partially tosylated and sulfated (splitting pattern in the 13CNMR at 98 to 101 ppm). Up to now, no detailed information about the functionalization pattern for sample 12 were accessible because of its insolubility. Phthalic and trimellitic anhydride were applied for the preparation of tosyl cellulose halfesters (Tab. 2 ) [7]. Determination of the DS values was possible via elemental analysis. In the FTIR spectra both the signals for the tosyl moiety (3048, 1500 cm-', aromatic region; 1362, and 1178 cm-', SO2 at the aromat) and the characteristic absorptions of the ester bonds (1 726 to 1756 cm-', depending on the DS value reached) were found. I3C NMR spectroscopy confirmed a complete functionalization of the C-6 position as well as tosylation and esterification of the OH group in C-2 [7]. It is worth mentioning that tosyl cellulose phthalates and trimellitates which are soluble in dilute aqueous sodium hydroxide solution form ionotropic gels by addition of a solution of multivalent metal cations like Ca2+ and A13+ ions as known for carboxyl groupcontaining polysaccharide polyelectrolytes. Table 2. Conditions and results of the subsequent esterification of tosyl cellulose Tosyl Derivatizing Agent Conditions Tosyl cellulose solubility cellulose derivative No. DS"' Type Molar t T No. 'Type DS') H2O DMSO ratiob) [h] ["C] 0.80 + 3a 0.46 SO3-pyridine 3.0 2.5 20 l l a sulfate 0.85 + 3b 0.89 S03-pyridine + 2.0 2.5 20 l l b sulfate 2a 1.43 SO3-pyridine + 0.57 + 2.0 2.0 20 l l c sulfate 2b 1.43 S03-pyridine 4.0 2.0 20 l l d sulfate 0.71 + + 2c 1.43 S03-pyridine 4.0 6.0 20 l l e sulfate 0.85 + + 6 O.9Od' SO3-DMF 2.0 5.0 10 12 sulfate 0.70 5a 0.46 phthalic anhydride 3.0 7.0 60 13a phthalate 0.90 +e) + 4a 1.39 phthalic anhydride 3.0 6.0 60 13b phthalate 0.63 +e) + 5a 0.46 TMA' 3.0 7.0 60 14a trimellitate 1.80 +') + 4a 1.39 TMA' 3.0 6.0 60 14b trimellitate 0.58 +e) + a) Degree of substitution of tosyl groups b) Mol reagent per mol free OH group c) Degree of substitution of additional esters groups d) Prepared via reactive microstructure e) Solubility in 1 N aqueous NaOH e) trimeliitic anhydride

-

REFERENCES [ 1J

R. L. Whistler & J. N. BeMiller, Industrial Gums:Polysaccharides and their

60

[2] [3] [4]

[5]

[6] [7]

[8]

[9]

[10]

[II]

[12]

[13]

[14]

[15]

[16]

Synthesis and derivatisation of biocompatible polymers

derivatives, Academic Press Inc., San Diego, 1993, pp. 1-19. G. Ebert, Biopolymere, Teubner, Stuttgart, 1993, pp. 354-427. E. Onsoyen, Commercial applications of alginates, Carbohydr. Eur., 1996, 14, 2631. T. Liebert & Th. Heinze, Induced Phase Separation: A New Synthesis Concept in Cellulose Chemistry, In: Cellulose Derivatives: Modification, Characterization, and Nanostructures, ACS Symposium Series No. 688, W.G. Glasser & Th. J. Heinze (eds.), American Chemical Society, Washington, DC, 1998, pp. 61-72. Th. Heinze, K. Rahn, M. Jaspers & H. Berghmans, Thermal Studies on Homogeneously Synthetized Cellulose p-Toluensulfonates, J Appl. Polym. Sci., 1996,60, 1891-1900. Th. Heinze & K. Rahn, The first report on a convenient synthesis of novel reactive amphiphilic polysaccharides, Macromol. Rapid Commun., 1996, 17,675-681. Th. Heinze, K. Rahn, M. Jaspers & H. Berghmans, p-Toluensulfonyl esters in cellulose modifications: acylation of remaining hydroxyl groups, Macromol. Chem. Phys., 1996,197,4207-4224. Th. Heinze, U. Erler, 1. Nehls & D. Klemm, Determination of the substituent pattern of heterogeneously and homogeneously synthesized carboxymethyl cellulose by using high-performance liquid chromatography, Angew. Makromol. Chem., 1994,215,93-106. A. E. 1. de Nooy, A. C. Besemer & H. van Bekkum, Highly selective TEMPO mediated oxidation of primary alcohol groups in polysaccharides, Reel. Trav. Chim. Pays-Bas, 1994, 113, 165-166. A. E. 1. de Nooy, A. C. Besemer & H. van Bekkum, Highly selective nitroxyl radical-mediated oxidation of primary alcohol groups in water-soluble glucans, Carbohydr. Res., 1995,269, 89-98. J. Katz, B. Philipp, I. Nehls, Th. Heinze & D. Klemm, Zum Polyelektrolytverhalten einer C-6-substituierten Carboxylcellulose im Vergleich zu Carboxymethylcellulose, Acta Polym., 1990,41,333-338. Th. Heinze, D. Klemm, F. Loth & B. Philipp, Herstellung, Struktur und Anwendung von ionotropen Gelen aus carboxygruppenhaltigen Polysacchariden, Acta Polym., 1990,41,259-269. K. Rahn, Th. Heinze & D. Klemm, Investigations of amidation of C-6 carboxy cellulose, In: Cellulose and Cellulose Derivatives: Physico-Chemical Aspects and Industrial Applications, 1. F. Kennedy, G. O. Philips, P. O. Williams, L. Piculell (eds.), Woodhead Pub!. Ltd., Cambridge, 1995, pp. 213-219. S. Vogt, D, Klemm, & Th. Heinze, Effective esterification of carboxymethyl cellulose in a new non-aqueous swelling system, Polymer Bull., 1996, 36, 549555. T. Liebert, Th. Heinze & D. Klemm, Synthesis and carboxymethylation of organosoluble formates and trifluoroacetates of cellulose, J Macromol. Sci-Pure Appl. Chem., 1996, A33, 613-626. W. G, Glasser & Th, Heinze, The Role of Novel Solvents and Solution Complexes for the Preparation of Highly Engineered Cellulose Derivatives, In: Cellulose Derivatives: Modification, Characterization, and Nanostructures, ACS Symposium Series No. 688, W.O. Glasser & Th. 1. Heinze (eds.), American Chemical Society, Washington, DC, 1998, pp. 2-18.

PREPARATION AND CHARACTERIZATION OF CARBAMOYLETHYLATED AND CARBOXYETHYLATED KONJAC MANNAN Shoji Takigami ', Yoshifumi Suzuki ', Akira Igarashi' and Kiyoshi Mlyashita'

'Techni('(/I Research Centerfor lnstrunrentul Analvsis. Gnnm« Universitv, Kirvu. Gunma 376-8518. Japan

2Gllnl11a Prefecture Industrial Technology Research Laboratory. Maebashi. Glllll11a 371-0845. Japan

INTRODUCTION

Konjac mannan (KM) is the main component of konjac flour obtained from the tuber of the konjac plant ( Amorphohal/us Konjac K. Kock).

It is a heteropolysaccharide

consisting of I3-D-glucose (G) and I3-D-mannose (M), with a G/M ratio of I to 1.61.2 or 2 to 3 3 _ KM forms irreversible gels by alkali treatment. It also interacts synergistically with xanthan gum

4.5

and kappa carrageenan" and makes thermoreversible gels.

Chemical modification of konjac mannan (i.e., acetylation 7,8 methylation':", and nitration 10) has been carried out in order to study the chemical structure and molecular weight. KM,

However, there are few investigations on the gelation of chemically modified In this study, KM was reacted with acry\amide in the presence of NaOH and the

substitution reaction was investigated. The gelation behavior of the carbamoylethylated and carboxyethylated KM with various degree of substitution (D.S.) was also examined.

EXPERIMENTAL

Materials

Commercial konjac flour (Seiko) supplied by Ogino Shoten Co. Ltd. (Gunma, Japan) was used as a starting material.

The flour was made from konjac tubers of the Akagi

Ohodama species. The flour was washed with 30% methanol aqueous solution twice

62

Synthesis and derivatisation of biocompatible polymers

and then with 70% iricthanol aqueous solution hefoi-e air-drying.

The konjac inannan

sample was thus obtained. Acrylamide was a rcagcnt grade and other chemicals used were special grade (Wako Pure Chemical Industries, Ltd.. Japan). They were used without further purification.

Carbamoylethylation and carboxyethylation of konjac mannan Konjac mannan ( 2 2 ) w a s dissolved i n dihtilltd water (198g) at 30°C and 20% sodium hydroxide aqueous solution (7OOg) was added.

A solution of acrylamide (6.2 g in 10 g

of water) was added and the mixture was allowed to stand for a programmed time at

30 "C with stirring. After the fixed time, the reaction mixture was neutralized with 6

M hydrochloric acid.

The solution was dialyzed against deionized water until the

dialysate was free of chloride ions and then freeze-dried. The nitrogen content of the reacted product was determined using a CORDER MT-5 elemental analyzer (YANACO, Japan).

The amount of carboxyl groups in the reacted

KM was determined by conductometric titration using a CM-60s conductivity meter (TOA, Japan) with 0.01M sodium hydroxide aqueous solution.

The amount of

carbamoylethyl and carboxyethyl groups in the product was determined as the degree of substitution (D.S.) per pyranose unit.

Fourier transform infrared (FT-IR) microscopy

FT-IR reflection spectra of carbamoylethylated and carboxyethylated KM were analyzed by the attenuated total reflection (ATR) method.

The FT-IR measurements

were carried out using a Magna750 FT-IR spectrophotometer equipped with a Nic-Plan infrared microscope (Nicolet).

A Ge polarizer was used as a high refraclive index

material.

Gel to sol transition temperature The gel to sol transition temperature of a mixture of reacted KM and xanthan gum aqueous solution was determined by the falling-ball method.

0.5% reacted KM and

0.5% xanthan aqueous solutions with equivalent weight were mixed at 75°C.

The

mixture was put into a glass tube (IOmm in diameter) with a stainless steel ball (0.1Ig

63

Carbamoylethylated and carboxyethylated konjac mannan

and 7 mm in diamcter) and then it was sealed. The position of the ball was mcasured with elevating temperature at the heating rate of 0. I " C h i n using a cathetornetcr.

The

transition temperature was defined as the initial temperalure that the ball began to fall and was estimated by the extrapolation of change of the height.

RESULTS AND DISCUSSION Addition reaction of acrylamide onto konjac mannan FT-IR spectra of KM and carbamoylethylated and carboxyethylated KM are shown i n Figure 1.

KM shows absorption due to the stretching vibration of the C=O bond the in

acetyl group at 1730 cm" and of OH groups of bound water and the pyranose ring at 1650 cm.' and near 1100 cm.' (spectrum a), respectively.

The acetyl group was

released under alkali conditions and a peak of the stretching vibration of the C=O amide bond appeared at 1670 cm-' (spectra b and c).

After 1 h, a new absorption due to

stretching vibration of the C=O bond in the carboxyl group appeared at 1720 cm" (spectrum d) and the intensity of the peak increased with increasing reaction time (spectra e to g).

The absorption due to the amide group was not observed after 6 h

(spectrum h) and the IR spectra of the KM reacted more than 12 h showed the same

2000

1500

1000

Wave number (cm-') Figure 1. FT-IR spectra of carbamoylethylated and carboxyethylated konjac mannan reacted for various time. a: original KM, reacted for: b: 15min. c: 30min, d: I h. e: 1.5 h, f: 2 h, g: 3 h, h: 6h, i: 12h, j: 24 h

64

Synthesis and derivatisation of biocompatible polymers

0

5

10

15

20

25

Reaction time (h) Relationships between degree of substitutions and reaction time for carbamoylethylated and carboxyethylated kon.jac mannan. 3:carbamoylethyl group, 0: carboxyethyl group. A:total

Figure 2 .

pattern (spectra i and j).

The amide group and carboxyl group belong to the

carbamoylethyl group and the carboxyethyl group, respectively. Figure 2 shows the relationships between degree of substitution (D.S.) and the reaction time

for carbamoylethylated and carboxyethylated KM.

The

D.S. of the

carbamoylethyl group increased with reaction time and showed a maximum at 2 h and then decreased rapidly. On the other hand, the carboxyethyl group was detected after 30 min.

The D.S. of carboxyethyl group increased remarkably and became almost

constant values after 6 h. This is due to hydrolysis of carbamoylethyl group to a carboxyethyl group. values after 3 h. complete in 3 h.

The total D.S. increased with reaction time and reached constant This means the addition reaction of acrylamide onto KM was

Carbamoylethylatlon and carboxyethylation of KM are carried out as

'

follows' .

KM-OH

+

CH?=CHCONH2

NaOH

______)

(konjac mannan) KM-O-CH,CH,CONH,

KM-O-CHlCH2CONH2 (carbamoylethylated KM)

NaOH

t-

KM-O-CHlCH2COOI-1 (carboxyethylated KM)

Carbamoylerhylatedand carboxyethylated konjac mannan

65

Gel formation of reacted konjac mannan with xanthan gum

The mixture of 0S% KM and 0.5% xanthan gum aqueous solutions formed a thei-inoreversible elastic gel and showed

; I

gel to sol Iransition tempei-ature (Tsol) a1

61 "C. The gel strength and T S Ofor ~ the mixture of reacted KM and xanthan gum with the same composition decreased with reaction time and the KM reacted for more than 3 h could not form a gel with xanthan gum.

Figure 3 shows the relationship between

TSOIand total D.S. of reacted KM. The Tsol decreased a little with increasing total D.S and then decreased rapidly when the total D.S. became greater than 0.1. presumed to be the influence of carboxyethyl group.

The relationship between Tsoi

and the D.S. of the carboxyethyl group is shown in Figure 4. remarkably

This can be

The TSOIdecreased

in the presence of a small amount of carboxyethyl

group and

carboxyethylated KM could not form a synergistic gel with xanthan gum. The gel formation is restricted by the electrostatic repulsion between the carboxyethyl groups on the reacted KM and the carboxyl groups on xanthan gum. However, since Tsol of the mixture of carbamoylethylated KM which is a nonelectrolyte and xanthan gum decreased a little, it was presumed that the gel formation is 70 60 0

v

f

'k a

50

U

2

40

30

C

.-0 *=

2

20

2

t-r . 10

0

I

I

I

I

0

0.05

0.10

0.15

0.20

Total D.S. per pyranose unit Figure 3. Relationship between gel to sol transition temperature and total degree of substitution for carbamoylethylated and carboxyethylated konjac mannan.

0

0.02 0.04 0.06 0.08 0.10 D.S. of carboxyethyl group per pyranose unit

Figure 4. Relationship between gel to sol transition temperature and degree of substitution of carboxyethyl group for carbamoylethylated and carboxyethylated konjac mannan.

66

Synthesis and denvatisation of biocornpalible polymers

a l w affected by the steric hindrance of the suhstituent groups introduced on KM by thc

addition reaction.

REFERENCES I. K. Kato and K. Matsuda. 'Chemical structure o f konjac mannan. I. Isolation and

characterization of oligosaccharides from the partial acid hydrolyzatc of manniln'. A y . . Biof. CImi.. 1969. 35, 1446-53.

2. H. Shiniahara, H. Suzuki. N. Sugiyama and K. Nishizawa. 'Mannan and related compounds. IV. Isolation and characterization of oligosaccharides from an enzymic hydrolysate of konjac glucomannan', Agc Biol. Cheru., 1975, 39. 293-9.

3. F. Smith and H. Srivastava. 'Constitutional studies on the glucomannan of konjac flour, J. AJIZ.Chern. Soc., 1959, 81. 1715-18.

4. G. Brownsey. P. Cairns, M.J. Miles, V.J. Morris, 'Evidence for intermolecular binding between xanthan and the glucomannan konjac mannan', Curhohvdrute Research, 1988, 176, 329-34. 5. P. A. Williams, D. H. Day, M. J. Langdon, G. 0. Phillips and K. Nishinari,

'Synergistic interaction of xanthan gum with glucomannans and galactomannans'. Food Hydrocolloids, 199I , 4,489-93. 6. K. Kohyama, H. Iida and K. Nishinari, 'A mixed system composed of different

molecular weights konjac glucomannan and kappa carrageenan: large deformation and dynamic viscoelastic study' Food Hydrocolloids. 1993, 7, 2 13-26.

7. N. Sugiyama, H. Shimahara, T. Andoh, M. Takemoto and T. Kamata, 'Molecular weights of konjac mannans of various sources', Agr. B i d . Chern., 1972, 36, 1381-87.

8. K. Kato and K. Matsuda, 'Isolation of oligosaccharides corresponding to the branching-point of konjac mannan' Agr. Biol. Chern., 1973,37, 2045-5 I .

9. N. Kishida, S. Okimasu and T. Kamata, 'Molecular weight and intrinsic viscosity of konjac gluco-mannan', Agr. Biol. Chem., 1978, 42, 1645-50. 10. H. Torigata, H. Inagaki and N. Kitano, 'Study of konjac mannan IV.

Molecular

weight and molecular form of nitrated konjac mannan', Nippotz Kngnku Znsslzi, 195 1,

73,30-32. I I . M. Shimada, H. Kuribara, S. Takigami and Y. Nakamura, 'Fine structure of

carbamoylethylated and carboxyethylated cotton cellulosic fibers', Sen-i Gnkkuishi,

1977.33, T- 109- 14.

PLASTIFICATION OF CELLULOSIC WASTES Marlen Durh', Manuel Moya', Eduardo Umala' & Guillermo Jimbez'

' Laboratorbde Polimeros (POIJWA), UniversidadNacional Ap. 863000 Herediia, Costa Ricu

ABSTRACT Cellulose cannot be thermally processed because degradation occurs before its melting temperature. Thermoplasticity can be obtained when chemical modifkation of hydroxyl groups decreases the polper interaction. Bemylation reaction was used to increase processability of several lignocellulose wastes such as saw dust, rice peel and pineapple peel. Benzylation increases the decomposition temperature (DT) of the materials. This indicates that this modification improves the thermal lignocellulose stability. In DSC a melting peak was observed for benzylated products, showing that the benzylation reaction increases lignocellulosethermoplasticity.

KEYWORDS Bemylation of ligocellulose,plastification, lignocellulose wastes

INTRODUCTION Costa Rica and other third world countries are increasing the industriahtion of their agricultural products such as oranges, coffee bean, ~~LWMS, pineapple and others. Usually, these products are exported and the wastes (Table 1) become a large environmentalproblem' . These wastes contain large quantities of lignocellulose that can be used to obtain several industrial products'. It is well known that cellulose cannot be thermally processed because severe thermal degradation occurs well before its melting point. The very high cohesive energy !?om inter- and intra-molecular hydrogen bonding places the melting temperature well above its degradation threshold. Chemical modification of lignocellulose is possible in order to obtain new materials with very interesting thennoplastic properties, that improve the processability and uses of these products3. Thermoplasticitycan be obtained only ifthe extent of hydrogen b o w is decreased by means of chemical modification of hydroxyl groups4. Introduction of non-polar substituentsresults in themplastic materials. Etherification and esterificationare very interesting reactions. Bemylation of lignocellulosecan be used in order to obtain thermoplastic materials. Pre-treatment conditions and reaction temperature were shown to have deep effects on the degree of benzylation 3*5.6. This paper describes the synthesis and some physical properties of benzylated materials fiom some wastes such as saw dust, rice peel and pineapple peel.

68

Synthesis and denvatisation of biocompatible polymers

Table 1. Agroindustd wastes in Costa Rica

Waste BaWltl Pineapple coffee bean parchment coffee bean pulp Rice peel Saw dust

Metric todyear lo3 3.900 100 25 228 18 300

MATERIALS & METHODS Materials

Lignocellulose raw materials were air dried and milled using a Wiley mill to reduce particle size to less than 0.25 mm. Cellulose was obtained by pulping with a 2% NaOH solution, 1O:l by vol., at 100 "C for 4 hours and bleached with a 2.5% Na C102solution (4 hours, room temperature). Lignin was precipitated by acidification of the pulping liquor with concentrated sulfuric acid. Benylation reaction

One gram of substrate was mixed with 3.5 g of a concentrated NaOH solution (5.3 g of NaOH and 8.4 mL of water) for 2 hours at room temperature. The temperature was raised to 100 "C, 7.5 mL of benzyl chloride were added and the reaction was allowed to proceed for &rent periods of time. The excess of benzyl chloride and salt were removed by extensive washing of benzylated pulp with ethanol and water. The pudied products were dried at room temperature under reduced pressure. Measurements

Infrared analysis was carried out using a Perkin Elmer 727 spectrometer. The degree of benzylation was evaluated by weight gain and the Relative Benzylation Degree (REID) was estimated by IR analysis considering the optical density (OD) at 2120 cm-' of an external standard of dicyclohexylcabodiimide(DCC) and the OD at 740 cm-' of the benzyl group. The thermal stability of samples was measured in a S h i m a b TGA, model 40M,from 25°C to 600 "C, at a heating rate of 20 "C/min in Nitrogen gas (30 mL/min). A Shimadzu DSC 40M was used to determine melting temperatures, working at a heating rate of 5 "C/min, under Nitrogen gas (30 mL/min). RESULTS & DISCUSSION

Two of the main components of hgnocellulose are cellulose and lignin. These hydroxy-rich macromolecules would compete during the benzylation reaction. To

Plastification of cellulosic wastes

69

evaluate the reactivity of eac4 benzylation was carried out on commercial Merck cellulose and ACC lignin. Figure1 shows the variation of RBD with the reaction t h e . Lignin is benzylated rapidly, and after 3 hours variation in RDB was not observed. In the case of cellulose, the maximum RBD was obtained after 4 hours. The data of the bemylation of celluloses obtained fiom agricultural waste are presented in Figure 2. The variation of RBD is diikent for each cellulose. Maximum RBD is obtained after 4 hours for rice peel cellulose, 6 hours for saw dust cellulose and 8 hours in the case of pineapple cellulose. The variation in RBD in each case is probably due to dil&rences in cellulose crystallinity'. Figure 3 shows that is possible to modify the whole lignocellulose material. The RBD was constant after 8 hours of reaction and the maximum RBD was observed in the case of pineapple peel. As shown in Figure 4 the extent of benzylation was evaluated through the determination of the recovered weight percentage. It can be seen that a loss of weight occurred during the first 3 hours. After this time a large increase of weight took place (recovered weight percentage higher than lOoO/o). The maximumyield was obtained after 8 hours of reaction and then a loss of weight was observed again. This behavior can be attributed to the occurrence of competitive processes: degradatiodsolubilisation and bemylation of ligwcellulose. In the first stages of the reaction, the first process seems to dominate. Alkaline degradation of polysaccMdes and solubilisation of liepin seems the most likely explanation for the loss of weight, and may involve the classical mechanisms of chain dissolution, loss of end-groups and alkaline hydrolysis together with the degradation of dissolved chains, hydrolyzed fiagments and released monosaccharides. The weight gained by the materials after 3 h could be reasonably attributed to the fact that lignin and the crystalline moieties of cellulose become progressively accessible to benzylation and become insoluble to the purification process. After 8 h the degradatiodsolubilisation process is important because of the long reaction time in strong alkaline conditions. Table 2 shows the thermal analysis results (TGA and DSC) of bemylated wastes. It can be observed that benzylation increases the decomposition temperature @T) of the materials. This indicates that benzylation improves the t h e d lignocellulose s t a b i i . In DSC a melting peak was observed for benzylated products, showing that the benzylation reaction increaseslignocellulose thermoplasticity.

Table 2. Thermal properties of benzylated agriculture wastes

Sample Pineapple peel Benzylated Pineapple peel Bananarachis BenzylatedBananarachis Benzylated rice peel

MP: Melting point

TGA AsW? DTPC 295 22 11 362 304 14 25 313

--

--

DSC M P , o n d ° C h4PpeaWOC

-_

--

143

154

141 14 1

153 150

--

--

70

Synthesis and derivatisation of biocompatible polymers

v __

1

0

2

24

8

6

4

Reaction Timehours ~-Cel.

ACC 1

Mack - L i d

Figure 1. Benzylation of commercial lignin and cellulose.

O '0l

I

-

0

1

2

4

6

24

8

Reaction Timehours 1 +Saw

dust +Rice

peel +Pineapple

peel 1

Figure 2. Benzylation of cellulose from agriculturalwastes

Plastification of cellulosic wastes

0.5

1

I

0,4

n

0,3

2 0,2 0,1

0 0

1

2

4

6

8 1 0 2 4

Reaction Timehours ~

-8-Pineapple peel

peel - 0 Saw - dust 1

-Rice

Figure 3. Benzylationof lignocellulosewaste without any separation

s.B

!.

140 120

loo

M

80 M

. I

P

O 0

1

2

4

6

8 1 0 2 4

Reaction Timehours

i +Pineapple

peel -h- Saw dust +Rice

peg

Figure 4. Weight recovered after benzylation of lignocellulosewastes

71

72

Synthesis and derivatisation of biocompatible polymers

CONCLUSIONS

-

-

Pineapple, banana rack, saw dust and rice peel residues are a good source of lignocellulosebut without treatment can produce ecologicaldamage. Plastilication of agroindustrial lignocellulose residues is possible using benzylation reactions to obtain new plastic materials. Benzylation reaction improves the thermal stability of materials

ACKNOWLEDGEMENTS The authors acknowledge financial support of the Universidad Nacional of Costa Rica, the project UNA-BID-CONICIT, the Iberoamerican Science and Technology Program (CYTED) and JICA of Japan.

REFERENCES 1. M. DutBn, M. Mop, M. Sibaja. Sintesis de productos quimicos especiales a partir de desechos agroindustriales,Zngenieria y Ciencia Quimica, 1993,3,53-62. 2. S. Nikolaev, M. Moya and M. Sibaja, Utilizacih de f i b de raquis de banano en materiales compuestos, III Congreso Interamericano sobre medio ambiente, Costa Rica, 1996. 3. R. Pereira, S. P. Campana, A. A. Curvelo, Benzylated Pulps fiom Sugar Cane Bagasse Cellulose, 1997,4,21-31 4. A. Gandini,Comprehensive Polymers Science, S.L. Aggarwal and Russo Eds, Oxford, Pergamon press, 1992. 5. D. Hon, Vicoelasticity Properties of Thennoplasticized Wood, Proceedings of the 5TbInternational Symposium on Wood and Pulping Chemistry, Atlanta, USA, 1989. 6. D. Hon, J. M. San Luis, Themplasticization of Wood. II, J Polym. Sc, 1989,27, 4143-41 60.

SYNTHESIS AND THERMAL PROPERTIES OF

EPOXY RESINS DERIVED FROM LIGNIN S. Hirose’, M. Kobayashi2, H. Kimura’ and H. Hatakeyama2 ‘National Institute of Materials and Chemical Research. 1-1 Higashi, Tsukuba. ibaraki 305-8565,Japan ‘Department of Applied Phvsics and Chernislry,Fukui IJniversity of Technology, 3-5-6 Gakuen. Fukui-City, Fukui YI0-8.50S,Japan

ABSTRACT Epoxy resin prepolymers were synthesized by the reaction of kraft lignin (KL) with polyethylene glycol diglycidyl ether (PEGDGE). The obtained prepolymer was cured with poly (azelaic anhydride) (PAA). The molar ratios of acid anhydride groups to epoxy groups ((AAI/(EPOXYI ratios) were varied at 6/10, 8/10, 10/10, 12/10 and 14/10. The thermal properties of the obtained epoxy resins were studied by differential scanning calorimetry (DSC) and thermogravimetry (TG). The glass transition temperatures (T,’s) of epoxy resins increased with increasing [AAI/[EPOXYI ratios. This suggests that main chain motion of epoxy resins is restricted when the cross-linking density of epoxy resin increases. Two thermal degradation temperatures (Tdl and Td2) were observed in TG curves of epoxy resins. They increased with increasing IAAI/[EPOXYJratios, suggesting that epoxy resins become thermally stable when epoxy and hydroxyl groups were changed to esters by curing reactions.

INTRODUCTION Lignin has highly branched chemical structure consisting of phenyl propane units as it can be easily chemically modified using reactive hydroxyl groups in the molecule. In these ten years, synthetic polymers, which were derived from lignin, were extensively studied in our laboratory. Recently, it was found that polyurethanes derived from lignin show excellent thermal and mechanical properties and also biodegradability . In the past, many researchers studied lignin-based epoxy resins which were prepared from lignin glycidyl ethers ’. In the present study, lignin-based epoxy resins with aliphatic polyester chains were directly synthesized from lignin by the reaction with polyethylene glycol diglycidyl ether (PEGDGE) and poly (azelaic anhydride) (PAA). The thermal properties of the obtained epoxy resins were studied by differential scanning calorimetry (DSC) and thermogravimetry (TG).

EXPERIMENTAL Materials Kraft lignin (KL) was kindly supplied by West Vaco Paper Company Ltd., USA. PEGDGE and PAA were also supplied by Toto Kasei Kogyo Ltd., Japan and ACI Japan Ltd., respectively. Dry N,N-dimethylformamide (DMF) was commercially obtained from Wako Chemical Industries Ltd.. Japan. All the materials were used without further purification.

Synthesis of epoxy resins Epoxy resin prepolymer was prepared as follows. K L was dissolved in DMF and PEGDGE was added. The reaction was carried out at 80 T for 24 hr. The obtained reaction mixture was poured into a large amount of methyl alcohol. The obtained epoxy

74

Synthesis and derivatisation of biocompatible polymers

prepolymer (Epoxy value was 0.60 eq/g) was cured with PAA at 150 “c3 for 24 hr in the presence of catalytic amount of dimethy laminophenol. The molar ratios of acid anhydride groups to epoxy groups ([AAI/[EPOXYl ratios) were varied at 6/10, 8/10. 10/10, 12/10 and 14/10. Epoxy resins were prepared also from AL in the same manner as mentioned above.

Measurements A Perkin-Elmer 2000 Fourier Transfer Infrared Spectrometer was used for infrared spectroscopy. The measurements were carried out using KBr pellets. A Seiko DSC 220 was used for differential scanning calorimetry (DSC). The measurements were carried out at a heating rate of 10 U m i n in nitrogen using ca 5 mg of samples. The glass transition temperatures (Tg’s) were determined according to a method reported by Nakamura et al. ’ . Thermogravirnetry (TG) was performed using a Seiko TG 220. The measurements were carried out at a heating rate of 20 U m i n in nitrogen using ca. 10 mg of samples.

RESULTS AND DISCUSSION Epoxy resins were successfully synthesized by the procedure described. The reactions for the synthesis of epoxy resins are as shown in Scheme I . Fig. 1 shows IR spectra of epoxy resins. The characteristic peaks at 2980 cm ’ (-CH2-), 1730 cm ’

/o\

0 Lignin-OH +

---t

LO-PEG-o

OH Lignin-O*O-PEG-0

/o\

Epoxy Prepolymer

I

c=o

6

I

o=c

1

c=o

Lignin-O--LO-PEG-O

Epoxy Resin

Scheme 1 . Reaction scheme for the synthesis of epoxy resins.

Synthesis and thermal properties of epoxy resins

75

(-COO-), 1610 cm ' and 1510 cm-' (phenyl) are observed. Fig. 2 shows the changes of relative optical densities of the peak at 1730 cm (-COO- groups) normalized by the intensities of the peak at 1600 cm (phenyl groups). As shown in Fig. 2, the relative optical density of the peak at 1730 cm increases with increasing IAAI/[EPOXYI ratios, indicating the formation of ester groups in epoxy resins after the curing reaction.

'

[AA]/[EPOXY] ratio

,

I

,

,

,

4600

I

I

,

,

3000'

,

,

I

I

I

--t

1000

2000

Wavenumber / cm-' Figure 1 . IR spectra of epoxy resins.

N

c UY

3 30a \

8 E 20 . v)

n

a

10 -

0.4 0.6 0.8 1

1.2 1.4 'I .6

[AA]/[EPOXY] ratio / mol/mo Figure 2

.

Relationship between Abs,,,,,/Abs,,,,, ratios and [AAI/IEPOXYI ratios for KL-based epoxy resins.

400

76

Synthesis and derivatisation of biocompatible polymers

The thermal properties of the obtained epoxy resins were studied by DSC and TG. The obtained results for KL-based epoxy resins are described below as the representative. The phase transition of epoxy resins was studied by DSC. Fig. 3 shows DSC curves of epoxy resins with various (AAI/(EPOXYI ratios. As shown in Fig. 3, a large gap in baseline due to the glass transition is observed in each DSC curve. The glass transition temperatures (T,’s) and also heat capacity difference due to the glass transition (AC,) were also determined using DSC Curves. Fig. 4 shows the relationship between Tg’s, heat capacity difference (AC,) and [AA]/[EPOXYI ratios of epoxy resins. As shown in Fig. 4, Ts’s slightly increase with increasing [AA[/IEPOXYI ratios, although the data are somewhat scattered. ACP values increase with increasing IAAI/IEPOXY I ratios. The above results suggest that main chain motion becomes restricted due to the increase in cross-linking density of epoxy resins.

ratio -60 -40

-80

I

-20

c)

T 1°C DSC curves of KL-based epoxy resins.

Figure 3 .

1

-30 1

1 .I

I l

h

U

U

a

‘

I I -90 0.4 0.6 0.8

I

1

I I I 0.5 1.2 1.4 1.6

[AA]/[EPOXyl ratio / mol/mol Figure 4.

Relationship between To’s, AC,, and IAAIIIEF’OXY I ratios for KL-based epdxy resins.

Synthesis and thermal properties of epoxy resins

77

The thermal degradation was studied by TG. Fig. 5 shows TG and TG derivative (DTG) curves of epoxy resins with various [AAI/[EPOXYI ratios. As shown in Figs. 5, thermal degradation proceeds in two steps. The thermal degradation temperatures (Td, and Td2)were determined using TG curves. Fig. 6 shows the relationship between Td’sand IAAJ/IEPOXYI ratios of epoxy resins. It is known that lignin is thermally unstable and it start to degrade at around 200 93 ’. It is also known that epoxy resins cured with acid anhydrides usually start to degrade at 300 “c: *. Therefore, it is reasonable to consider that T,, is mainly related to the degradation of lignin in epoxy resin molecules and Td2is related to the degradation of other components in epoxy resin molecules. As shown in Fig. 6, T,, and Td2increase with increasing (AA)/IEPOXYI ratios. This suggests that epoxy resins becomes thermally stable after curing with anhydride. Fig. 7 shows the relationship between weight residue at 500 SC (WR) and IAAJ/[EPOXYI ratios of epoxy resins. WR values decrease with increasing IAAI/IEPOXY] ratios. It is known that lignin molecules are readily condensed to form charcoal-like substances when it is heated in nitrogen. It is considered that the decrease in W R values is caused by the decrease in lignin contents in epoxy resins.

6/10

-.

[AA]/[EPOXY] ratio

k-

DTG,

A

100

200

300

400

!iOO

T 1°C

Figure 5 .

TG and mG curves of KL-based epoxy resins.

78

Synthesis and denvatisation of biocompatible polymers

500

450 400

~

y

350 300 -

?t

250

-

200

’

-

25 -

Td2

s

20

\

d‘ 1

100 I 0.4 0.6 0.8 I

1

1

I

1.2 1.4 1.6

[AA]/[EPOXY] ratio / mol/mol Figure 6. Relationship between Td’s and (AAIIIEPOXY I ratios for KL-based epoxy resins.

15 0

01 1 0.4 0.6 0.8

I

I

1.2 1.4 1.6 [AA]/[EPOXY] ratio / mol/mol

Figure 7

-

1

Relationship between weight residue at 500 “c (WR) and IAA I/IEPOXY I ratios for KL-based epoxy resins.

REFERENCES I . S. Hirose, S. Yano, T. Hatakeyama & H. Hatakeyama, ‘Heat-resistant polyurethanes derived from solvolysis lignin’, ACS Sym. Ser., No. 397,. , Washington D.C., Am. Chem. SOC., 1989, Chapter 29. 2. K. Nakamura, T. Hatakeyama & H. Hatakeyama, ‘Thermal properties of solvolysis lignin-derived polyurethanes ’, Polym. Adv. Technol., 1992, 3, 151- 155. 3. H. Hatakeyama, S . Hirose, T. Hatakeyama, K. Nakamura, K. Kobashigawa & N. Morohoshi, ‘Biodegradable polyurethanes from plant components’, J. Macromol. Sci., 1995, A32, 743-750. 4. S. Hirose, K. Kobashigawa, Y. Izuta & H. Hatakeyama, ‘ Thermal degradation of polyurethanes derived from lignin by TGFHR’. Polym. Intl., 1998, 47, 247- 256. 5. For example, H. It0 & N . Shiraishi, ‘Epoxy resin adhesives from thiolignin’, Mokuzai Gakkaishi, 1987, 33, 393-399, D Feldman and D Banu, ‘Kinetic data on the curing of an epoxy polymer in the presence of lignin’, J. Polym. Sci. Polym. Chem., 1989, 26, 973-983, K. Hofmann & W. G . Glasser, ‘Engineering plastics from lignin 23. Network formation of lignin-based epoxy resins’, Macromol. Chem. Phys., 1994, 195, 65-80. 6. S. Nakamura, M. Todoki, K. Nakamura & H. Kanetsuna, ‘Thermal analysis of polymer samples by a round robin method. 1. Reproducibility melting. crystallization and glass transition temperatures’, Thermochimica Acta, 1988, 163, 136. 7. S. Hirose & H. Hatakeyama, Mokuzai Gakkaishi, ‘Kinetic studies on thermal degradation of lignin by TG integral method’, 1986, 32, 62 1-625. 8. M Shinbo, in “Epoxy Resin Handbook”, Tokyo, Nikkan Kogyo Shinbun Ltd., 1987, p. 368.

EFFECT OF MODIFICATION ON THE FUNCTIONAL PROPERTIES OF RICE STARCH M A M Noor' and M N Islam2 1

School of Industrial Technologv. Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.

2

Department of Food Technologv. Bangladesh AgriculfurafUniversity. Mymensingh -2202, Bangladesh

INTRODUCTION Modified starches have long been developed by starch industries for food and nonfood application. Depending on type and extent of modification the functional properties of modified starches such as rheological and thermal behaviours as well as enzymic susceptibility are greatly affected. Changes occur both at granular and molecular levels as a consequence of relevant modification may affect some of the observed behaviours. Hydroxypropyl starch impart certain desirable functional properties notably the freezethaw cycles tolerence and resistance toward retrogradation. Rice starches in native form are widely consumed in food formulations but certain inherent physical properties such as the uniformly distributed granular size of relatively smaller than other cereal starches may enhance its usefullness upon chemical modification. In this study rice starches have been chemically modified through hydroxypropylation and certain functional properties upon complexation with calcium ion were monitored. Calcium complexation may influence the chemical and biochemical reactions catalysed by metal ions in starch containing foods. Rheological behaviour is an important characteristics determining the functionality and suitability of specific starch for specific application. Thermal behaviour of these starches required indepth studies for proper and effective process control and formulation of quality products of starch based processed foods. Thermal behaviour of starch granules is controlled by granular size, mylose content, molecular weight, crystallinity, internal granular organisation and type and extent of modification. MATERIALS AND METHODS Rice starch (Oryza sativa) of about 25% amylose content was obtained in an isolated form from rice flour of local variety (Mahsuri). Moisture, fat, protein, ash and calcium content were analysed according to the standard AACC methods (1984). Hydroxypropyl derivatives were prepared by varying mount of propylene oxide in the presence of NaOH and Na2S04 following the procedures described by Mohd. Azemi and Wootton (1984). The extent of hydroxypropylation was determined by the spectrophotometric method of Johnson (1969) and expressed in terms of molar substitution (MS). MS is defined as moles substituent per mole of anhydroglucose units (AGU). Calcium complex starches were prepared by reacting calcium chloride with native and hydroxypropyl starches as was described earlier by Nurul Islam and Mohd. Azemi (1992). A rotational viscometer (Contraves Rheomat 115) was used to determine the starch (native, derivative and complexes) viscosity at various temperatures and starch

80

Synthesis and derivatisation of biocompatible polymers

concentration. Plots of relationship between apparent viscosity and variable parameters (temperature, MS and starch concentration) were established. The flow behaviour of the starch slurry was evaluated by the power law equation : t=KYq where t=shear stress, K=consistency coefficient, Y=shear rate and q = behaviour flow index. The thermal properties of the starches were analysed by using a Du Pont 2910 DSC with a base of 2910 DSC cells coupled with Du Pont 2000 Thermal Analysis Program which measured glass transitions and other endothermic transition temperatures. A starch:water ratio of 1 : 1 by weight was used for the DSC studies. Two types of enzyme the Novo a-amyloglucosidase (1,4,oc-D-glucan-glucohydrolase E.C.3.2.1.3) with specific activity of 300 AGU/ml and porcine pancreatic a-amylase ( a 1,4-glucan-4-glycano-hydrolase,E.C.3.2.1.1) supplied by Sigma with specific activity of 1000 IU/mg protein were diluted with appropriate buffer for the digestibility study. Digestion procedures were adopted as described earlier (Nurul Islam and Mohd. Azemi (1998)). Scanning Electron Microscopy (SEM) was used to evaluate the morphological appearance of ungelatinised starch granules after enzymatic hydrolysis.

RESULTS AND DISCUSSION Upon modification the granular shape and sizes of rice starches did not show any significant changes as observed under SEM. The polygonal and angular shape of native rice starch granules remain fairly intact with no noticeable surface deformation. Chemical composition of hydroxypropyl rice starches (Table l(a)) did not vary significantly with the native counterpart. The low protein content in the hydroxypropyl derivatives is partly due to the modification procedures itself resulting in protein being washed out after alkaline treatment. MS increases with the amount of propylene oxide added (Table l(b)) with most of the substituent groups attached at C-2 and with possible substitution at C-3 and C-5 of the anhydroglucose unit (Wootton and Haryadi, 1992). The level of substitution is within the acceptable range for food application. The functional properties of the starch derivatives are greatly affected by MS whereby most of the undesirable inherent functional properties of native starches are somehow altered. The presence of OH groups on native starches and upon hydroxypropylation encourage formation of complexes with calcium ions. The presence of electron withdrawing substituent groups promotes acidity thus encourages cation (calcium) binding. The formation of the starch-calcium complex is time limiting reaching saturation point after about 20 minutes of reaction time. Alkaline condition and low temperature (Table 2(a) and (b)) favoured complexation. Similar trends have been reported and explained by other researchers working on starches of different origin (Hood and O’Shea, 1977 ). In native form gelatinisation encourages calcium binding and a similar trend was observed for the hydroxypropyl derivative. Such properties may facilitate the role of starch as a carrier for calcium for calcium-enriched preparations, releasing available calcium ion at ambient and acidic conditions. Viscosity increased exponentially with starch concentrations, MS and temperature and exibited pseudoplasticity under shear forces. Calcium suppresses the viscosity and MS enhances the viscosity indicating the tendency of hydroxypropyl groups to expand and extend the starch molecules thus providing higher hydration volume and consequently increased the viscosity. Calcium may squeeze the molecules by possible covalent bond formation with hydroxyl groups of the molecules providing lower hydration volume resulting in low viscosity. Both factors MS and calcium

Effect of modification on rice starch

81

complexation enhanced pseudoplasticity. As shown in Table 3(a) and (b) the value of n is always less than unity indicating the pseudoplastic nature of the starch slurry Mohd Nurul, Azemi and Manan (Nurul Islam and Mohd. Azemi (1997) and (1999». At a constant amount of calcium, MS enhanced the cosistency coefficient and the trend was somehow reversed in both native and hydroxypropyl starches as the amount of calcium bound increased. The shape of the viscosity-shear rate curves can be used to estimate the degree of sliminess, an important characteristic of gum solutions (Kramer & Szczesniak,1973). A slimy material ( as indicated by higher YJ values) is a thick solution which coats the mouth and is difficult to swallow. Calcium makes the starch less slimy and thus gives a clear mouthfeel and better consumer acceptance. In addition calcium complexed starch provides nutritional value along with other desirable textural properties of starches.

Table l(a). Chemical composition of native and modified starches Starch types

Moisture %

*

Crude Fat%

Ash %

Protein %

0.60±0.04

0.61±O.l6

6.61±0.21

30.62±0.67

Calcium ug/g

Native

5.26±0.30

0.02

5.03±0.18

0.68±0.05

1.93±0.26

0.68±0.11

29.85±0.56

0.05

5.01±0.11

0.62±0.06

2.53±0.46

0.72±0.09

30.25±0.43

0.07

5.05±O.l2

0.65±0.08

2.62±0.34

0.67±0.16

30.l2±0.37

0.09

5.00±0.11

0.60±0.10

2.54±0.55

0.89±0.27

19.88±0.23

0.12

4.96±0.16

0.61±0.09

2.77±0.65

0.64±0.13

29.71±0.56

*

Amylose content 24.45% Mean of the four measurements with ± standard deviation

Table l(b).

Effect of propylene oxide on molar substitution

% Propylene oxide added 0.00 2.00 4.00 6.00 8.00 10.00

% Propylene glycol equivalent 0.00 1.08 2.49 2.96 4.18 5.24

% Hydroxypropyl group 0.00 0.84 1.86 2.30 3.25 4.07

Molar substitution 0.00 0.02 0.05 0.07 0.09 0.12

82

Synthesis and derivatisation of biocompatible polymers

Table 2(a): Calcium bound (ug/g dry starch) by ungelatinized native rice starch under different treatments and conditions --------------- .. ------------------------ .. ---------------------------------------------------------------------------------------Calcium added ug/g starch

pH

Temperature 5°C 20m in. 60min. 120min.

Temperature 28°C 20min. 60min. 120min.

Temperature 40°C 20min. 6Omin. 120min.

--------------------------------------------------------------------------------------------------------------------------------50.00

3 7 9

31.61 35.23 37.06

31.83 35.91 37.54

32.05 36.36 37.88

26.11 28.31 32.63

26.55 29.33 32.88

27.18 30.21 33.57

23.99 25.69 28.91

24.23 26.07 29.32

25.03 26.57 29.85

tOO.OO

3 7 9

48.73 58.79 65.83

49.02 59.43 66.28

49.85 60.21 66.89

43.44 50.81 59.43

44.61 51.71 60.24

44.94 52.41 60.87

40.74 45.37 51.75

42.29 45.65 52.57

42.58 46.46 54.39

150.00

3 7 9

66.54 81.28 88.86

67.15 82.39 89.49

67.55 83.13 89.96

60.53 71.96 82.63

60.87 72.55 83.48

61.42 72.66 84.16

53.92 65.91 7784

54.65 66.61 78.37

56.39 67.44 78.52

200.00

3 7 9

79.41 80.0J 94.75 95.01 101.31 101.75

80.65 95.76 102.12

73.46 84.88 94.53

74.21 85.52 94.88

75.23 86.13 95.56

63.22 78.29 90.06

63.72 78.58 90.46

64.37 79.45 91.51

250.00

3 7 9

95.84 9654 106.64 107.23 117.13 117.63

97.17 107.92 118.19

88.86 99.11 110.27

89.41 99.84 111.36

89.93 101.56 112.14

74.89 89.63 101.93

75.81 76.45 90.15 91.43 102.41 10312

---------------------------------------------------------------------------------------------------------------------------------

Table 2(b). Calcium bound (u/g dry starch) by ungelatinized hydroxypropyl rice under different treatment and conditions

Hydroxypropyl starch (MS=O.02) ------------------------------------------------------------------------------------------------------------------------------Calcium added ug/g starch

pH

Temperature 5°C 20m in. 60min. 120min.

Temperature 28°C 20m in. 60min. 120min.

Temperature 40°C 20min. 60min. 120min.

------------------------------------------------------------------------------------------------------------------------------3 7 9

50.00

33.39 37.38 39.55

33.95 38.14 39.84

34.01 38.97 40.13

28.55 30.52 34.52

28.61 31.26 34.88

29.56 31.65 35.57

25.64 28.22 32.39

26.61 28.46 32.74

27.42 28.61 33.35

100.00

3 7 9

51.23 63.13 67.68

51.76 63.85 68.05

52.06 64.05 68.85

46.12 54.66 61.52

46.56 55.27 61.76

46.66 55.56 62.57

43.74 48.74 55.14

44.27 4936 55.45

45.11 49.56 56.29

150.00

3 7 9

71.54 86.78 94.54

71.94 87.01 94.84

72.66 87.64 95.04

63.53 75.26 85.76

64.06 75.61 86.33

64.57 76.51 86.67

58.12 70.89 82.17

58.55 71.44 82.71

59.36 71.59 83.37

200.00

3 7 9

84.38 98.91 106.98

84.94 99.84 107.76

85.55 100.03 107.99

77.46 88.67 96.78

77.71 89.39 97.55

78.41 89.88 97.77

66.92 82.03 93.92

67.65 82.67 94.61

68.31 83.45 9541

250.00

3 7 9

100.78 111.23 122.93

101.45 111.84 123.74

101.74 111.95 123.98

93.86 104.53 114.75

94.56 94.68 105.11 105.53 115.28 116.21

82.64 95.84 107.35

83.27 8359 96.42 96.62 107.89 108.46

---------------------------------------------------------------------------------------------------------------------------------

Effect of modification on rice starch

Table 3 (a):

Flow behaviour characteristics of starches influenced by different levels of MS Flow behaviour index

Without Ca2+

83

n K r

Ms 0.00

0.02

0.05

0.07

0.09

0.12

0.47 8.51 0.98

0.49 9.09 0.98

0.42 9.29 0.98

0.34 9.48 0.97

0.31 9.70 0.97

0.30 9.86 0.96

------------------------------------------------------------------------------------_ ... ---

--------- ..

With

ci+

0.41 7.20 0.99

n

K r

* r

0.38 7.48 0.99

0.35 8.19 0.99

0.33 8.79 0.99

0.31 9.11 0.98

0.30 9.27 0.98

Calcium added 250 ug/g dry starch Regression coefficient

=

Table 3 (b):

Starch types

Native Starch

Flow behaviour characteristics influenced by the amount of Calcium added

Flow index

n K r

Calcium added (ug/g dry starch) 0.00

50.00

100.00

150.00

200.00

250.00

0.47 9.14 0.99

0.39 9.08 0.99

0.36 8.83 0.98

0.36 8.50 0.98

0.35 8.07 0.98

0.33 7.20 0.99

*Hydroxy

n 0.46 0.43 0.34 0.41 0.35 0.31 propyl K 9.70 9.34 9.01 8.45 8.02 7.33 starch r 0.96 0.96 0.99 0.98 0.99 0.98 -----------------------------------------------------------------------------------------------------------* Level of Molar Substitution 0.09 r = Regression coefficient DSC analysis of native starch showed two endothermic melting temperature peaks but only a single peak appeared upon hydroxypropylation and calcium complexation. (Figures l(a),(b),(c) and (d) ) Both enthalpy and gelatinisation temperature decreased with MS. However it was observed that calcium increased the gelatinisation temperature of both native rice starch and its derivatives but the reverse for the enthalpy (Table 4). The treated starches rearranged themselves and stabilised to a new equilibrium with an

Synthesis and derivatisation of biocornpatihie polymers

84

2's

S a m o l c ' NS-1057 Size: ! 5 . 0 0 0 0 mg

necnaa:

F!!e: Run

A:

NS-1057.01

Date: 22-nor-92

09: 36

GELATINiiATION 0.006

i 0.004

'

,.

y

m

\

I

-0.004

1 -0.74

!

LO

KO

A -0.006 :20

:oo

80

140

3SC V S . 0 8 OuPont 2000

Temperature (0C)

Fig. I (a): DSC thermal curves of native starch with different transition temperature

CIS C

Samole: NS-1082 Size: 15.0000 mg n e r n o a : GELATINIZATION -0

File: A. : 0 6 2 C 7 . 0 2 Run D a t e : 25-nor-92

I:: 05

0.008

50.

0.006 -0.52

1st derivative

-

0.004

~

\

m

-

Heat flow

\

2

-Y 3

0.002

-0.54-

2

4

LL

U

m

LI

'0

I

I

0.000

.

.-I

-0

L

56-

c,

-0.002

-0.58

-r

50

60

80

Temperature

:oo

(oC)

-0.004

:20

2sE V 4 . 0 6 DuPonr 2 C O O

Fig. 1 (b): DSC thermal curve of liydroxypropyl starch (MS= 0.09)

Effect of modification on rice starch sample:

ii Fi'

15.0000 m g Metnod: G E L A T I N I Z A T I O N

F i i e : r.: !CECi:.02 iiun Date: 27-Aor-92

i:

NS-~O~S

Size:

v

:c: c;

C.^26

c . 008

0.00;

-

-C

\ 07

c.co2

v

3

---

0 4

LL

0 .o o c

U

a

-3

f

-c. 2:t

-C

-0.331 I

m

-c 7c

-C .3CE GO

80

50

12G

100

Temperature

2Ci ' 1 4 . 0 8 DuPan:

2'363

(OC)

Fig. 1 (c): DSC thermal curves of native starch treated with 250 pg calciurn/g dry starch

Ds

Caeple' N S - ? 3 E 6 S i z e : 15.COOO mS netnoa: G E L I K N I Z A T X N

File: A 1086CT.CZ Run Cat?: 27-AD,--92

1:

:2

O.OCf

---I

Tg

0 . OOE

A

-5.

-

0.304

m

-

-i

Heat f l o w

0.002

3 r(

L

cr a a

L

.

c . coo

-0.

I -0

?CZ

-c -0.co4

-c.

, -a.

79 LO

60

80

Temperature

100

(oC)

:2c

cs:

i c . 3 e aupon:

COB

290c

Fig. I (d): DSC thermal curves of hydroxypropyl starch (MS=0.09) treated with 250pg calciurn/g dry starch

86

Synthesis and derivatisation of biocompatible polymers

Table 4.

Gelatinization enthalpies of native. hydroxypropyl rice starches at different levels of calcium MS

00.00 150.00 200.00 250.00

•

0.00

0.02

9.68 8.36 7.98 6.55

8.58 7.46 6.59 5.53

0.05

7.52 6.34 5.94 4.32

0.07

6.37 5.23 4.89 3.30

0.09

0.12

5.16 4.06 3.97 2.85

4.13 3.63 3.12 2.17

Ilg/g dry starch

increased gelatinisation temperature and decreased enthalpy. The two melting profiles simply reflect melting and reorganisation processes occuring simultaneously during dynamic heating in the calorimeter (Biliaderis et al., 1992). Derivatisation and complexation may have disrupted the compactness and rigidity of the native starch granules thus disorganised the ordered conformation of the starch molecules. The hydroxypropyl groups also disrupted the intra and intermolecular hydrogen bonds, thereby weakening the granular structure of starch allowing the easy access of water to the starch granules. Lower enthalpy indicated looseness of bonding in the new equilibrium as a consequence of distabilised ordered molecular conformation. Calcium chloride used as complexing agent may act as a structure-stabilising agent bringing about melt-mediated interconversions between various polymorphic starch structures upon heating to yield states of lower free energy. The glass transition temperatue (Tg) is an important criteria for prolong storage with desired texture and quality of starchy foods. The glass transition of starch is strongly sensitive to the amount of diluent (solvent) present (Biliaderis, 1991); low molecular weight diluents cause a large depression of Tg of the amorphous matrix. Polymers (starch) in the glassy state are mechanically and chemically inert in realistic time-scales, quenching and storage of a product below its characteristic Tg would preserve most of the quality characteristics. It was observed that the glass transition temperature of native starch was recorded at 61.82°C and it increased gradually with increase of calcium: starch ratio (Table 5(a». However Tg decreased as a result of hydroxypropylation. For a particular level of MS, Tg increased as calcium:starch ratios increased. On the other hand Tg increases with an increase of MS for a fixed calcium:starch ratios. It was also observed that calcium complexed derivatives showed lower Tg compared to calcium complexed native starches. Calcium enhanced Tg of both native and hydroxypropyl derivatives which may subsequently suggest the possibility of maintaining the desirable properties of calcium treated starchy food upon storage at relatively higher temperature than the calcium free starchy food. Apparently binding of calcium to both types of starches reduced the tendency of cooked starches to retrograde upon storage at lower temperature as evidenced by the relatively lower enthalpy recorded for calcium complexed starches (Table 5(b».

Effect of modification on rice starch

87

Table 5 (a). Glass transition temperature of native and hydroxypropyl rice starches treated with different levels of calcium MS

00.00 150.00 200.00 250.00

•

0.00

0.02

0.05

0.07

0.09

0.12

61.62 62.46 65.45 69.27

61.03 62.34 63.28 64.63

57.06 58.40 60.50 61.93

56.34 57.36 58.92 61.77

54.92 56.06 57.04 58.89

53.42 55.06 56.11 57.96

ug/g dry starch.

Table 5 (b). Enthalpies (cal/g dry starch) of retrograded starches at different levels of MS with or without calcium treatment after 21 days storage at different temperatures

Temperature

MS

0.00 0.02 0.05 0.07 0.09 0.12

Without calcium

4.43 3.78 3.22 2.69 2.11 1.54

With calcium

Without calcium

With calcium

3.19 2.67 2.14 1.62 1.11 0.59

5.81 5.19 4.61 4.09 3.53 2.99

3.68 3.11 2.57 2.02 1.46 0.88

Without calcium

6.14 5.43 4.81 4.27 3.69 3.11

With calcium

4.35 3.71 3.05 2.36 1.77 1.15

Retrogradation itself is an undesirable inherent functional characteristic of starches and in most starch containing processed food this phenomena may affect the stability and textural properties of the products, thus rendering them unacceptable. It seems that at the molecular level, both substitution by hydroxypropyl groups and calcium complexation tend to stabilise the starch molecules and minimised retrogradation. In vitro digestibility of native cooked starch by PPA and AMG is relatively higher than the raw counterpart but the reverse for the hydroxypropyl derivatives (Table 6(a) and (b)). Similar trends have been reported earlier on other starches (Wootton and Chaudry, 1979; Mohd. Azemi and Wootton, 1984; and Yoshida and Kishikawa, 1984). This observation has been attributed to granular basis notably the disruption of starch granular compactness as a consequence of hydroxypropylation procedures. This may

88

Synthesis and derivatisation of biocompatible polymers

provide a larger surface area for enzymic attack. The higher amount of reducing sugar released by AMG may be due to its mode of action on starch whereby it hydrolysed all the glucosidic linkages including a 1,6 branch points in amylopectin. PPA attacked only the sterically available al,4 linkages endogenously. Cooking of starch increased the susceptibility to enzymatic hydrolysis. On cooking, the starch granules swelled, ruptured and lost their crystallinity and became amorphous. Under this condition the bonding system in the starch molecules weakens and consequently they are more accessible to enzymic attack. The reduction in in vitro digestibility of gelatinised hydroxypropyl rice starches as MS increases is explained on the basis of the greater restriction of enzyme attack by the increasing number of bulky substituent groups. It has been reported elsewhere that hydroxypropyl groups disrupted the natural compactness of starches. The presence of Table 6: Digestibility of Native and Hydroxypropyl Rice Starches (a) Porcine Pancreatic a-Amylase Raw starch

Starch Types

Native MS=0.02 MS=0.05 MS=0.07 MS=0.09 MS=0.12

Without calcium

Gelatinized starch

With calcium

Without calcium

With calcium

00.00

100.00

250.00

00.00

100.00

250.00

2.19* 2.54 2.67 2.82 3.92 4.09

2.21 2.85 3.40 3.70 4.80 5.09

2.45 2.93 3.58 4.86 5.06 6.50

7.40 6.07 5.93 4.57 3.35 2.29

8.55 7.31 6.15 5.93 4.74 3.65

9.81 8.65 7.49 6.25 5.16 4.00

* mg reducing sugar per gram substrate. (b) Amyloglucosidase Starch Types

Raw starch Without calcium 00.00

Native MS=0.02 MS=0.05 MS=0.07 MS=0.09 MS=0.12

3.55* 4.77 5.83 6.51 7.27 8.43

Gelatinized starch With calcium

Without calcium

With calcium

100.00

250.00

00.00

100.00

250.00

3.40 4.10 4.71 5.33 6.21 6.73

3.10 3.68 4.43 5.09 6.05 6.42

9.90 8.79 7.49 6.13 5.14 4.04

8.78 7.97 6.67 5.37 4.61 4.97

6.73 5.61 4.51 3.75 2.93 2.70

* mg reducing sugar per gram substrate.

Effect of modification on rice starch

89

hydroxypropyl groups keeps the starch molecules in an expanded form and subsequently reduces the degree of crytallinity (Takahashi et al. 1993). However the presence of bulky hydroxypropyl groups may act to sterically hinder enzymic hydrolysis of cooked hydroxypropyl starches. Calcium enhanced digestibility by PPA on both native and its derivatives raw and cooked, suggests the role of calcium iis a co-factor for PPA activity. AMG actions on both types of starches raw and cooked, was somehow restricted by calcium complexation. CONCLUSIONS The chemical composition of both native rice starch and hydroxypropy 1 derivatives varied little with modification. The rheological behaviour of both native and hydroxypropyl derivatives are affected by calcium ion concentration, MS, starch concentration and temperatures. MS enhanced psuedoplasticity but the effect was somehow reversed with calcium ions resulting in less slimy starch pastes thus imparting clear mouthfeel. The glass transition temperature (Tg) increased with the increase of calcium and decreased with MS and subsequently influenced the melting transition temperatures. Both calcium and hydroxypropyl groups tend to suppress the gelatinisation enthalpy of both the native rice starch and its derivatives. Calcium complexation and hydroxypropylation tends to restrict retrogradation as evidenced by the lowering of the enthalpy of retrograded starches. The in vitro digestibility by PPA of both gelatinised and raw starches was enhanced by the presence of calcium but was reduced for AMG. Attack by both enzymes on the gelatinised starch was somehow restricted as MS increased but the raw starch was more susceptible to enzymic attack as MS increased.

REFERENCES 1. A.A.C.C. Methods, ‘Approved Methods of Analysis’, St. Paul, Mannesota, American

Association of cereal Chemists Inc., 1984. 2. C.G. Biliaderis, “on-equilibrium phase transitions of aqueous starch systems’, In: Water Relationships in Foods (Levine, H. and Slade, L. eds.), Plenum Publ. Corp., New York. 1991, 251-273. 3. C. G. Biliaderis, ‘Structure and phase transitions of starch in food systems’, Food Technol. 1992, 46:98-109 & 145. 4. L. F. Hood & G. K. O’Shea, ‘Calcium binding by hydroxypropyl distarch phosphate and unmodified starches’, Cereal Chem. 1977, 54: 266-271. 5. D. P. Johnson, ‘Spectrophotometric determination of hydroxypropyl groups in starch ether’, Anal. Chem. 1969,41:859-860. 6. A. Kramer & A. A. Szczesniak, ‘Texture Measurement of Foods: Psychological, Fundamental Sensory, Mechanical and Chemical Properties and Their Interactions’, Dordrect, Holland, D. Reidel Publ. Co,. 1973. 7. M. Nurul Islam & B. M. N. Mohd Azemi, ‘Effect of molar substitution (MS) on Calcium Binding by Hydroxypropyl rice starches’, StarcWStarke, 1992, 44:332334. 8. M. Nurul Islam & B. M. N. Mohd Azemi, ‘Flow behaviour of calcium complexed hydroxypropyl rice starches’, Journal Texture Studies, 1997,28: 1-20.

90

Synthesis and derivatisation of’biocompatible polymers

9. M. Nurul Islam & B. M. N. Mohd Azemi, ‘Effect of calcium and hydroxypropylation on crystallinity and digestibility of rice starches’, Journal Sci. Food Agri., 1998,76:161-167. 10. M .Nurul Islam & B. M. N. Mohd Azemi, ‘Rheological Behaviour of Sago (Metroxylon sagu) Starch Paste’, Food Chemistry, 1999,64 501 -505. 1 1 . B. M. N. Mohd Azemi & M. Wootton, ‘Invitro digestibility of hydroxypropyl maize starches’, StarcWStarke, 1984,36(4):273-275. 12. S. Takahashi, C. C. Maningat & P. A. Seib, ‘Hydroxypropylated wheat starch in several foods in Japan’, Asean Food Journal, 1993,8(2):69-76. 13. M. Wootton & M. A. Chaudry, ‘Enzymic digestion of modified starch’, Starch/Starke, 1979,3 1(7):224-228. 14. M. Wootton & Haryadi, ‘Effects of starch types and preparation conditions on substituent distribution in hydroxypropyl starches’, J Cereal Sci. 1992, 15:18 1184. 15. M. Yoshida & T. Kishikawa, ‘A study of hydroxyethyl starch Part 11. Degradationsites of hydroxyethyl starch by pig pancreas a-amylase’, StarcWStarke, 1984, 6(5):167-169.

SUCCINYLATION OF CHEMICALLY MODIFIED WOOL KERATIN -THE EFFECT ON HYGROSCOPICITY AND WATER ABSORPTIONNatsuko Kohafal., Mayumi Kanei' and Toshinan Nakajima', I

Faculty of Practical Arts & Science, Show U'omen 's University, Taishido, Setagaya-ku, Tokyo 154-8533, Japan

Keywords: Succinylation, Wool, Hygroscopicity, Water absorbency, Sorption isotherm

INTRODUCTION Since wool is a natural protein having different reactive functional groups as well as a clothing material of excellent quality, it can be converted into a biodegradable and functional polymeric material by suitable chemical modification. Succinic anhydride generally reacts with a functional group such as an amino group', which can introduce a new carboxyl group into wool fibers as shown below. 0

Succinylation followed by additional modification with metal salts had been reported to retard felt shrinkage of wool fabric'. Toyoda et al. reported that succinylated collagen fibers showed remarkably higher water absorbancy than intact collagen3. Since wool contains more lysine (Lys) and arginine (Arg) residues than collagen, succinylation of wool is also expected to raise the water absorbability. In this work, intact wool and some kinds of wool keratin derivatives were succinylated and the effects on water absorption and hygroscopicity were studied for the purpose of re-utilization of waste or used wool fibers as a functional polymeric material. Chemical treatments such as reduction, oxidation and partial hydrolysis were made on wool fibers beforc succinylation to improve the reactivity with succinic anhydride. Cleavage of disulfide crosslinks by reduction or oxidation is expected to raise the accessibility of the reagent to the wool and hydrolysis increased the amount of amino groups as well as carboxyl groups. Powdery wool (Merry powder 30) and the partially hydrolyzed one were also used as other types of keratin derivatives, disulfide cross-linking of which had been ruptured.

EXPERIMENTAL Materials Wool und Merry powder 30 The wool fibers (New Zealand Corriedale) were degreased according to Nakamura et a14. Merry powder 30 which was obtained from wool by reduction and coagulation with alkali, was offered by Kyoeisha Chemical Co. Ltd.

Y2

Synthesis and derivatisation of biocompatible polymers

Chemical treatments Partial hydrolysis Wool libers (20 g) had been shaken in 1M or 3M HCl solution (liquor ratio, 1:50) at 50°C for 24 h. Merry powder 30 was similarly hydrolyzed with 1M or 2M HCl. Oxidution Wool fibers were shaken in a solution containing performic acid (99% formic acid : 30% hydrogen peroxide = 1:l) at 0°C for 24 h according to the procedure previously described5. Reduction Wool fibers (5 g) had been shaken in 1M 2-mercaptoethanol aqueous solution (liquor ratio, 1:100) at 40°C for 6 h. Succinylation Intact wool fibers, Merry powder and the chemically treated samples as described above were succinylated according to the procedure of Toyoda et a13. Succinic anhydride was added to the sample in 1/15M phosphate buffer solution at pH 8.0 (liquor ratio, 150) and the mixture was stirred at room temperature for 24 h. During the reaction, pH of the solution was kept at 8.0 by adding 10M NaOH. Succinylated sample was thoroughly washed with distilled water and the sample that set to gel by succinylation was dialyzed with water for over 72 h and lyophilized.

Measurements Add-on Add-on was evaluated as the amount of succinic acid in the hydrolyzed sample as follows: Succinylated sample (5-10 mg) was hydrolyzed in 6M HCl (5 mL) at 110°C for 48 h and evaporated to dryness. Amino acids in aqueous solution of the hydrolyzate were removed by passing the aqueous solution through a column of Amberlite IR120B (H' form). After the eluent had been evaporated to dryness, content of succinic acid in the sample was determined by colorimetry according to the method of Momose6. Water absorption Sample (0.2 g) dried at 110°C for 1 h was weighed and immersed in distilled water (5 mL) for 4 h at 30°C with shaking. After the sample in water had been centrihged at 3,000 r.p.m. for 5 min, the supernatant was removed. Water uptake (g/g * sample) was evaluated from the weight of the swelling sample. Hygroscopicity Moisture regain of samples was measured under 22.8, 43.3, 64.9 and 85.0% R.H. using the saturated solution of CHBCOOK, K2C03, NH4N03 and KCl. Sorption behavior of water vapor into the samples was measured at 20°C using the sorption apparatus described before7.

RESULTS AND DISCUSSION Succinylation Mainly amino groups of Lys residues and guanidino groups of Arg residues in the samples are thought to react with succinic anhydride under the conditions applied in this work'. Intact, partially hydrolyzed, oxidized and reduced wool fibers and Merry powder and partially hydrolyzed one were succinylated in aqueous solution at pH 8.0, where 23-92 mole of succinic anhydride was added for one cquivalcnt of A r g and Lys residues

6.8*' 6.8 2.0

Intact wool

Hydrolyzed wool*' Hydrolyzed wool*'

Hydrolyzed

Hydrolyzed wool*3

SC-Wc

SC-1 HW

3HW

SC-3HWa

SC-3HWb

SC-3HWc

Reduced wool

SC-RW

*'Sample was hydrolyzed in 2M HCI.

*3Samplewas hydrolyzed in 3M HCI. * h e product dissolved in water.

*'Sample was hydrolyzed in 1M HCI.

twice.

Reduced wool

RW

*' Succinylation was repeated

6.8*'

Hydrolyzed

SC-3HWe

6.8

8.0

Hydrolyzed wool*3

SC-3HWd

9.5

14.4

*5

SC-2HMPa Hydrolyzed Merry powder * 5 SC-2HMPb Hydrolyzed Merry powder * 5

11.4

6.8

- *4

Hydrolyzed Merry powder

2HMP

9.0

5.0

Hydrolyzed Hydrolyzed wool*3

*3

Hydrolyzed Merry powder **

1HMP

Hydrolyzed Merry powder

Merry powder Merry powder

Merry powder

Oxidized wool Oxidized wool

SC-MPb

SC-MPa

MP

SC-OWb

SC-OWa

Oxidized wool

Keratin derivative

SC-1HMP

9.3

11.8

6.7 8.2

ow

Abbr.

9.0

1HW

SC-Wb

5.0 6.8

Intact wool Intact wool

SC-Wa

k / P )

Intact wool

(%)

anhydride

W

Add-on

succinic

Keratin derivative

Abbr.

Table 1. Abbreviations and add-ons of the sample used in this work

6.E*

6.8

2.0

2.0 6.8

2.0 6.8

12.2 15.3

11.0

10.7 12.9

7.5

5.7

(%)

anhydride ( d g )

Add-on

succinic

c

w Q

8. z

8

??

2

B I: a

g:3

3

Y

i;'

5 CJ-

r

0

4

a

0

z.

Y,

a

0.

v)

94

Synthesis and derivatisation of biocornpatible polymers

in the samples. Abbreviations of the keratin derivatives used in this work and the add-ons were shown in Table 1. Add-ons of the samples were increased by increase of added amount of succinic anhydride. The wool fibers partially hydrolyzed in 3M HCI became soluble in water after succinylation with 8 g/g succinic anhydride (SC-3HWd). Add-ons of succinylated intact wool were in the rangc of 6.7-11.896. If amino groups of Lys and guanidino groups of Arg residues react with succinic anhydride except for the partially hydrolyzed samples, maximum add-on calculated from the contents of the amino acid residues in intact wool fibers' will be 8.75%. However, the add-ons of some succinylated wool samples were higher than this value. These facts suggest that succinic anhydride may have reacted with the other functional groups such as hydroxyl groups of the side chain or amino groups resulting from hydrolysis of main chain, which had occurred as a side reaction through succinylation.

Effect on water absorption Water absorption of intact wool was scarcely changed by the first succinylation, though succinic anhydride added to the samples to a certain extent, 5.0-6.7%. Both add-on and water absorption were increased by repeating the succinylation. The second succinylation of partially hydrolyzed wool (SC-3HWe) also raised the add-on but the water absorption did not exceed in the value of the corresponding sample succinylated for once (SC-3HWd). The add-ons of oxidized or reduced wool were scarcely different from that of intact-wool, while the water absorbabilities of the reduced o r oxidized and succinylated samples were much higher than that of succinylated wool (Fig. 1). In the case of Merry powder, water absorbability was not raised only by hydrolysis, but increased by a combination of hydrolysis and succinylation (Fig. 1). Though water

40.0

I I -

t

L

--0.0

5.0

I 10.0

15.0

20.0

Add-on (%,)

Figure 1. Rclationship between water absorption and add-on of succinylated wool keratin derivatives. O W O1HW A3HW A R W H O W + M P O l H M P X 2 H 4 ~-

__

Succinylation of chemically modified wool keratin

95

30 h

25

5 0

0

5

20

15

10

Relative humidity (%)

Figure 2. Sorption isotherms of wool and succinylated keratin derivative.

absorption of all samples increased with the add-on, the relationship between these values was not linear. This fact suggests that water absorbance is not influenced only by degree of succinylation but also by other changes in wool such as hydrolysis of the main chain, change in molecular geometry by the treatments and coupling of carboxylic groups in samples and Na’ from NaOH added during succinylation.

Effect on hygroscopicity and behavior of moisture sorption The chemical treatments made before succinylation had little effect on the hygroscopicities of the samples (Table.2). By succinylation the hygroscopicities were increased especially under the circumstance of high humidity. The behavior of moisture sorption of SC-3HWc was compared with that of intact wool (fig. 2). Moisture regain of intact wool fibers in circumstances of different humidities was the average of the values reported by Speakman and Cooperg, Bull” and Rowen and Blaine”. The B. E. T.’s plots were linear for both samples at relative humidities less than ca. 50%. From the slope and intercept of the plots the values of Vm (maximum volume of adsorbed water in a monolayer per one gram of dry material) and C (adsorptive energy factor) were Table 2. Moisture regain of succinylated keratin derivatives

Sample

Relative humidity (%) 22.8

43.3

64.9

85.0

SC-Wa

6.8 10.0 7.9 11.9

12.9 16.8

17.7 24.2

SC-WC

8.5

11.6

16.1

3HW

5.7

10.6

SC-3HWc

6.5

11.2

W

Sample

Relative humidity (%) 22.8

43.3

64.9

85.0

OW SC-OW

6.2 8.2

8.9 11.2

11.5 14.6

17.1 20.4

24.1

RW

5.2

10.1

10.4

15.3

11.2

16.6

SC-RW

4.8

10.5

11.5

20.1

12.2

21.2

MP

7.4

10.6

13.5

19.9

11.3 15.9

29.3

SC-MPb 7.4 -

Y6

Synthesis and derivatisation of biocompatible polymers

determined as 0.118 and 13.19, both o f which were higher than the corresponding values of intact wool (Vm: 0.064, C: 12.17). Isotherms calculated from the B.E.T.’s multilayer adsorption model by fixing the values of Vm and C determined above, and by varying the number of layers (i) from 2 to 5 were also compared with observed isotherms. The maximum number of layers can be found as 5 to 7 for intact wool fiber. On the other hand, the value of SC-3HWc seems to be over 6, though the observed isotherm was not completely in accord with the calculated isotherm.

CONCLUSION Water absorption of wool fibers was increased by succinylation and those of wool fibers and powdery wool were more remarkably increased by combinations of succinylation and the other chemical treatments such as partial hydrolysis, reduction or oxidation. Though the combination of these chemical treatments and succinylation had a lesser effect on the hygroscopicity than the water absorption, Vm and C values from the water sorption isotherms of partially hydrolyzed and succinylated wool were higher than the corresponding values of intact wool.

REFERENCES 1. I. M. Klotz, Succinylation, Methods in Enzymology, 1967, 11, 576-581. 2. N. H. Koenig & M. Friedman, Reaction of zinc acetate with wool carboxyl groups derived from cycljc acid anhydrides, Text. Res. .I., 1972, 42, 646-647. 3. H. Toyoda, Y. Chonan, T. Imai, A. Matsunaga & A. Kawamura, Chemical modification of collagen fibers (1) Preparation and some properties of succinylated collagen fibers, flikakukagaku, 1972, 18(3), 153-162. 4. Y. Nakamura, K. Kosaka, M. Tada, K. Hirota & S. Kunugi, Pecuriarity of enzyme inaccessible polypeptide present in the intercellular membrane cement, rhfnt. Wool Text. Res. Con$, Tokyo, The society of fiber science and technology, Japan,1985, Vol. 1, ~ ~ 1 7 1 - 1 8 0 . 5. I. J. O’Donnell & E. 0. P. Thompson, Studies on oxidized wool 11. Extraction of soluble protein from wool oxidized with performic acid, Aust. J. Biol. Sc.i, 1959, 12, 294-303. 6. T. Momose, Colorimetric determination of carboxylic acids with 2-nitrophenylhydrazine hydrochloride, Chem. Pharm. Bull., 1978,26(5), 1627-1628. 7. Y. Hirai & T. Nakajima, Moisture sorption of polyelectrolyte complex between poly(acry1ic acid) and poly(4-vinylpyridine), J. Appl. Polym. Sci., 1988, 35, 1325-1332. 8. H. Lindley, The chemical composition and structure of wool, In: Chemistry of natural protein fibers, R. S.Asquith (ed.), Prenum Prcss, New York, 1977, ~~147-187. 9. J. B. Speakman & C. A. Cooper, The adsorption of water by wool. Part 111 The influence of temperature on the affinity of wool for water, J . Text. Inst., 1936, 27, T191. 10 H. B. Bull, Adsorption of water vapor by proteins, J. Am. Chem. Soc., 1944, 66 1499. 11. J. W. Rowen & R. L. Blaine, Sorption of nitrogen and water vapor on textile fibers, Ind. Eng. Chem., 1949, 39, 1659.

NATURAL POLYMERS FOR HEALING WOUNDS John F. Kennedy, Charlea J. KniU & Michael Thorley Birmingham Carbohydrate & Protein Technolo.gyGroup, Chembiotech Laboratories, Universityof Birmingham Research Park, Vincent Drive, Birmingham. B15 2SQ, UK.

ABSTRACT

Some carbohydrate polymers have properties making them suitable for application as wound management aids. A variety of neutral (e.g. cellulose, dextran, & (1-'3)-P-D-glucans), basic (e.g. chitin & chitosan), acidic (e.g. alginic acid & hyaluronic acid), and sulphated polysaccharides (e.g. heparin, chondroiti, dermatan & keratan sulphates), have been the focus of interest with respect to biomedicdwound care applications. Recent investigations have also examined more unusual complex heteropolysaccharides, isolated fiom plant and microbial sources, which possess potentially usefid biological and/or physicochemical characteristics with respect to wound care applications. A review of the function and requirements of wound management aids, their physical forms, and the structural features of the polysaccharides that are commonly used for their preparation, is presented, along with a brief overview of selected commercially available products (specifically hydrogels). INTRODUCTION Function of a wound management aid

Wounds left untreated generally dry out and a hard protective coating, the scab, is formed. Early treatment methods therefore involved the application of a simple natural coating, such as cotton or lint gauze, which merely provided a protective absorbent layer under which the scab could develop. The treatment of wounds was revolutionised when it was found that Natural skin was wounds generally heal faster when a 'moist' dressing is applied recognised as the ideal wound dressing and therefore the development of 'moist' dressings was based upon the water content (- 85 % w/w) and inherent permeability of skin. The performance requirements for such dressings replicating skin properties and promoting wound healing are obviously higher than mere absorbent coverings. In order for the wound to remain moist during the contact period (which could be more than several days), and to aid the healing process, the wound dressing must fulfil several requirements 2,3, i.e. it should:

'.

0

0

0 0 0

0

maintain high humidity and water balance at the wound-dressing interface permit the exchange of gases and provide thermal insulation maintain a microorganism-impermeable layer to prevent secondary infection adhere satisfactorilyto maintain good wound-dressing contact not adhere too strongly to the wound and thus cause trauma upon removal remove via absorption excess wound exudate and associated toxic compounds maintain physical structure when wet (even after excessive fluid absorption) be biocompatible (e.g. not provoke adverse reactions through prolonged tissue contact) be producible in sterile form (sterilisation should not adversely effect strength)

98

Synthesis and derivatisation of biocompatible polymers

PHYSICAL FORMS OF WOUND MANAGEMENT AIDS Wound management aids are available in a wide range of physical forms including hydrateddehydrated gels, filmdsheets, wovednon-woven fibres, beaddmicrospheres, flakes, etc. Clearly, such formdproducts can be specifically targeted towards different classes of wounds, since they will require different types of products, for example a leg lesion may simply require a coating whilst a cavity wound requires an infill material. Hydrogels / xerogels

Hydrogels are one of the most important classes of commercially available wound management aids 4 . A hydrogel is a three-dimensional network of hydrophilic polymer chains in which at least 20% by weight is retained water (if the water content is greater than 95% by weight then the hydrogel is a superabsorbent). Hydrogels SwelYshrink in the presence/ absence of water, and collapse to form a xerogel if the water is completely removed '. The shrinkinghwelling process is reversible since the hydrogel structure can be reformed by absorption of water. In order for a hydrogel to maintain its three-dimensional structure in the presence of high retained water levels the hydrophilic polymer chains are cross-linked, either by covalent bonds, or non-covalently by electrostatic, hydrophobic or van der Waals interactions. Hydrogeldxerogels are generally used as wound dressing materials. They can be manufactured so that they are flexible, durable, non-antigenic and permeable to water vapour and metabolites whilst also securely covering the wound so preventing infection by bacteria. There are a number of commercially available hydrogel and xerogel wound dressing preparations 475, some of which are discussed throughout this paper. POLYSACCHARIDES Polysaccharides are high molecular weight condensation polymers composed of monosaccharide residues which can be neutral, basic, acidic, or combinations of the three, making the polysaccharide neutral, basic, acidic, or possessing the ability to have mixedvariable charges according to its environment. Homo-polysaccharides are composed of a single type of monosaccharide substituent, whilst hetero-polysaccharides contain two or more different monosaccharide substituents. Polysaccharide structures can also be linear or branched. The position and configuration of the linkages plays an important part in determining the three-dimensional structure. Polysaccharides are naturally occumng biomolecules that perform a number of different hnctions in living organisms, e.g. structural components, energy reserves, lubricating agents, etc. With so many potential variations in composition, structure and hnction, some polysaccharides may possess properties that would be beneficial as a wound management aid (i.e. physical properties), or even participate in the wound healing process (biochemical properties). A brief overview of the structural features of the polysaccharides utilised in wound management aids is presented '. Polysaccharides also contain a range of fbnctional groups (e.g. primary and secondary hydroxyl groups, free amino groups and carboxylic acid groups, etc.), which can be utilised as sites for chemical or enzymatic modificatiodderivatisation. Therefore, naturally occumng polysaccharides can be modified in order to alter their physicochemical characteristics, which may result in a material with enhanced applicability for a specific wound management application. The most common types of modification include esterification, etherification, oxidation, cross-linking (to increase molecular weight), and controlled hydrolysis (to reduce molecular weight).

Natural polymers for healing wounds

99

Neutral polysaccharides (D-glucans) The D-glucan homopolysaccharides are widely distributed amongst plants, animals and microorganisms, occumng mainly as structural components, and storage components. With four different glycosidic linkages ((1+Z), (1 +3), (I +4) and (1 +6)) and two configurations (a and p), a wide variety of polysaccharide forms are possible, with both linear and branched structures, the latter being sequential or random in nature. Such structural diversities, along with differences in molecular weight distribution and resultant molecular shapdsize give rise to a range of differing physicochemical characteristics. A number of D-glucans and their derivatives are utilised either directly, or are part of, wound management formulations.

Cellulose Cellulose is a linear structural polysaccharide composed of (1 +4)-a-D-Glcp residues (Figure 1) and its major sources are cotton and wood pulp. It is rigid, highly crystalline, and is therefore difficult to solubilise. It is therefore generally unsuitable for direct biomedical application in its native form since flexibility and/or solubility are often important characteristics. In order to overcome these problems methods of processing and/or chemical modification have been developed to produce flexible fibres 7w and water-absorbing swellable derivatives 'I@*'). Woven cellulose fibres (cotton and viscose) are used to prepare a wide range of basic wound dressings, such as retention bandages, support and compression bandages, absorbents, gauzes, tulle dressings, and wound dressing pads '. The primary derivatives produced for medical applications are biocompatible non-toxic cellulose esters and ethers (such as cellulose acetate and carboxymethyl cellulose) '. Commercial hydrogels containing cellulose derivatives include Intrasite@Gel (modified carboxymethyl cellulose, Smith & Nephew), Codeel@ parboxymethyl cellulose, Coloplast, available in sheet, powder and ad form), Granugel (pectin and sodium carboxymethyl cellulose, ConvaTec), and Aquacel ,a hydrocolloid dressing containing carboxymethyl cellulose, ConvaTec). '.

t

Dextran Dextrans are essentially linear (1+6)-a-D-glucans (Figure l), which differ only in chain length and degree of branching. Branching occurs via (13 3 ) - or (1+4)- a-D-Glcp linkages (and much less frequently by (1+3)-a-D-Glcp linkages). Many bacteria synthesise dextran from sucrose, Leuconostoc mesenteroides and Leuconostoc &xtranicurn are used commercially. Native dextrans have high molecular weights, whilst clinical dextrans are produced by synthesis or controlled degradation of native dextrans, thus having lower molecular weights lo. The characteristics of branching, length and frequency, are dependent upon synthesis temperature and molecular weight ll. Most of the physical and associated pharmacological properties of dextran fractions are dependent upon their molecular weight distribution. Evidence suggests that dextran accelerates polymerisation of fibrin in vivo, and influences the structure of the fibrin clot. Dextran appears to have beneficial activity in wound treatment but its high solubility is limiting. Reduction in solubility is achieved by making an emulsion polymerisation using epichlorohydrin as cross-linking agent to produce insoluble beads that swell in water. Such beads can be used in the treatment of skin lesions, to absorb wound exudate fiom secreting wounds with an associated reduction in wound healing time 12, and have been reported to assist the wound management process by stimulating macrophages 13. D e b r i d ' (Pharmacia & Upjohn) is the commercial product based on epichlorohydrin cross-lied dextran, and is available in bead and paste form '.

Synthesis and derivatisation of biocompatible polymers

100

O H [-

NH*c

I

i

n

n

hyaluronic acid ~)+DGIC@-(~-~)-P.DG~~NAC-(~-

Figure 1.

Neutral, basic and acidic polysaccharides used in wound management aids.

(1 +3)-/3-D-glucans (1 +3)-P-D-glucans are present in most plants and microorganisms and occur as major structural or storage components 14. Topical administration of yeast (1+3)-P-D-glucans has been shown to induce a more rapid rate of repair of experimental wounds compared with a range of other polysaccharides (carrageenan, levan, inulin, dextran and starch), which is attributed to induced RES (reticuloendothelial system) stimulation, i.e. stimulation of the lympho-reticular cells of the mammalian defence system including macrophages, endothelial and reticulum cells Is. By stimulating macrophages, several effects are observed, including an increased resistance to infection, an inhibition of tumour growth and an improvement in the wound repair process. A specific (1+3)-P-D-glucan that has been examined in some detail is lentinan, which also contains (1 +6)-p-D-Glcp branches, and has antitumour activity, suppressing chemical and viral oncogenesis and reportedly preventing cancer recurrence or metastasis after surgical intervention 14. There is also evidence that such (I+3)-P-D-glucans are also able to increase the host resistance to bacterial, viral and parasitic infections.

Basic polysaccharides Chitin and chitosan Chitin is a naturally occurring polysaccharide found in the outer shell of crustaceans. It is a (1+4)-P-D-glycan composed of 2-acetamido-2-deoxy-~-D-glucopyranoseresidues (N-acetyl-D-glucosamine residues, Figure 1). Chitosan is the name given to the partially deacetylated form of chitin and is therefore composed of 2-amino-2-deoxy-P-Dglucopyranose residues (D-glucosamine residues, Figure 1). Chitosan is biocompatible7I,'' (since its degradation products are natural metabolites) and can be produced in powder, film, bead, fibre and fabric forms 18*19(a). Chitosan has been evaluated in a number of medical applications including as a potential wound dressing were it has been shown to enhance wound healing and blood clot formation. Many of chitosans properties rely on its cationic nature, which allows it to interact with negatively charged biomolecules such as proteins, anionic polysaccharides and nucleic acids, many of which are located in skin.

Natural polymers for healing wounds

101

It has been shown that in the area of wound healing, chitosan and chitosan derivatives can reduce scar tissue (fibroplasia) by inhibiting the formation of fibrin in wounds, it is haemostatic and can form a protective fildcoating. One reason postulated for the ability of chitosan to enhance wound healing is its biodegradability. In addition, chitin, chitosan and chitosan derivatives affect macrophage activity, which will influence the wound healing process 17,20,21 The biomedical applications of chitosan require some physicochemical properties that chitosan itself does not possess, e.g. dissolution in water, gel-forming ability, etc. Modifcation is therefore required to make materials that in contact with body fluids locally form gels and then dissolve, such as N-carboxybutyl chitosan 22(ax23. Chemical modifications of the amino group and both primary and secondary hydroxyl grou s are possible 23. A commercial artificial skin material has been produced from chitin fibres 22% . ,

Acidic polysaccharides Alginic acid / alginate

The algal polysaccharide alginic acid is obtained from the cell walls of brown algae (Phaeophyta) such as the seaweeds Laminaria sp. and Ascophyllum sp. It is a linear block copolymer composed of two uronic acid residues, namely D-mannuronic and L-guluronic acid (Figure 1). The distribution of the uronic acids along the chain is non-random and involves relatively long sequences of each uronic acid. In the presence of divalent cations, such as calcium, alginate gels can be formed due to ionic cross-linking via calcium bridges between L-guluronic acid residues on adjacent chains. Alginates have historically been known to have a haemostatic hnction and to be capable of absorbing specific solutes. Calcium alginate gels have a large pore size and high water absorbency making them potentially usehl as hydrogel dressings. Hydrophilic sponges (xerogels) produced from calcium alginate are reported to have good absorptive properties for both blood and wound exudate Alginate gels are thermally stable and have been reported to deodorise wounds and absorb pain-stimulating compounds. However, if the calcium ions are exchanged for monovalent cations such as sodium (i.e. as a result of absorption) the cross-linking is lost and the gel loses its rigidity and stability after a period of time, however, this can be beneficial to wound healing since calcium exchange into the wound effects many cellular activities including adhesion, differentiation and proliferation. Alginate fibres can be prepared by injecting a sodium alginate solution into a calcium salt containing bath. Such fibres can be used to produce yarns and fabrics for medical applications 19(a)p2qawb), and as drug carriers for wound healing 19(b). There are a wide variety of commercial alginate products available. Dressings include Algisite@M (Ca al 'nate fibre non-woven, Smith & Nephew), Algosteril* (Ca alginate, Beiersdorf), Kaltogel (Ca Na alginate gelling fibre, ConvaTec), Kaltostat@(Ca alginate fibres in non-woven pads, ConvaTec), Melgisorb@(Ca Na alginate gelling fibre, Molnlycke), Seasorb@(Ca Na alginate gelling fibre, Coloplast), Sorbalgon@(Ca alginate, Hartman), and Sorbsan@(Ca alginate fibres in non-woven pads, Maersk). Hydrogels include Nu-Gel@(containing alginate, Johnson & Johnson), Fibracol@ (Ca alginate with collagen matrix, Johnson & Johnson), K.altocarb* (Ca alginate fibre, ConvaTec), and Purilon@Gel (Na alginate, Coloplast) *.

'.

Hyaluronic acid / hyaluronan / hyaluronafe/ hylans

Hyaluronic acid / hyaluronan is a naturally occurring polysaccharide, which is widely distributed in the connective tissue and vitreous and synovial fluid of mammals. It acts as a lubricant and shock-absorbing material in the fluid of joints. It is a linear polysaccharide

102

Synthesis and denvatisation of biocompatible polymers

consisting of a disaccharide repeating unit containing D-glucuronic acid and 2-acetamido-2deoxy-D-glucose (linked as shown in Figure 1). Hyaluronan has a very high molecular weight, affording very viscous aqueous solutions, even at low concentrations. Commercial sources include Cock's combs, human umbilical cords and fermentation (Streptococcus equi). Biologically, it is far more than just a high viscosity space filler, since it is capable of interacting with a wide range of biomolecules, including tissue components, proteins, proteoglycans, growth factors, etc. In a wound environment it acts as a free radical scavenger, so modulating inflammation 251a). It is recognised by receptors on a variety of cells that are associated with tissue repair and regeneration. It has also been reported that incorporation of hyaluronan into infected wounds (i.e. were the normal healing process is compromised) can accelerate the wound healing process, and that the hyaluronan can also act as a bacteriostat. Commercial preparations of sodium hyaluronate are available which are used in eye surgery (viscosurgery) and joint viscosupplementation 25(b). There are however, some l i t a t i o n s with respect to the direct use of hyaluronan in wound management due to its solubility, rapid resorption and short tissue residence time. Attempts have been made to overcome this by derivatisation, particularly esterification in the case of tissue engineering applications 26. Hyaluronan derivatives of varying solubility (from water soluble to insoluble biodegradable derivatives) have been produced by use of different ester types and controlling the degree of esterification. Such derivatives can be manipulated by extrusion, lyophilisation and spray d q h g to produce different physical forms including membranes, fibres, sponges and microspheres. The degree of esterification of benzyl and ethyl derivatives can be altered accordingly to produce materials that have a good absorbency capacity (> 100 % w/w) and that either dissolve rapidly or remain as a semi-solid hydrogel for long periods. Such materials have potential as tissue engineering supports where a biodegradable matrix is required to enable effective regeneration of skin in fullthickness bum injuries. Cross-linked hyaluronan derivatives are generally referred to as hylans. Water-insoluble soft hylan gels are suitable as viscosurgical implants to prevent postoperative adhesions and to control scar formation 25(b). Hyalofill@(ConvaTec) is a commercial non-woven fibrous material composed of Hyaff, a hyaluronan ester derivative '. OR

no. OH

NHk

n

NHh

n

chondroitin 4>B-rrGlcpn-(l-3>B-o-G.IIHAc-(~chondroitin: W=H. R'=H chondroitin+rulphate (A): R'-S03H. R'-H chondroitinhulphata (C): R'=H. R"= S q H

r

L

1

An

keratan sulphate 3 ~ p - D - G a l p ( 1 4 ~ p - D G l c p N A c ~ S 0 3-H ( l

4)-p-DGlcpA-(l-4~ u-DGk~SO~KBS03K(I-

heparin disaccharide repeating units

Figure 2.

Sulphated polysaccharides with wound management potential.

Natural polymers for healing wounds

103

Sulphated poiysaccharides 27

The group of naturally occumng sulphated polysaccharides including heparin , chondroitin (sulphate), dermatan sulphate and keratan sulphate (Figure 2) exhibit extensive biological activity. Some or all show anticoagulant activity, lipemia clearing activity, interaction with growth factors and fibronectin, and in some cases an anti-HIV effect. It is proposed that their biological activity is due to their anionic nature. Although there is little reported research with respect to their application in wound management, they will undoubtedly find application in the not too distant future since the healing of wounds is accompanied by an increased biosynthesis of the sulphate-containing glycosaminoglycans, within a zone adjacent to the edge of the wound '*. Complex heteropolysaccharides

Research is now focusing on the suitability of more complex polysaccharides for use in wound management aids. An example of this is branan ferulate, a substituted arabinoxylan isolated from high fibre corn bran by alkaline extraction (Figure 3) 3,29. Branan ferulate has also been incorporated into alginate fibres 24@). The ferulate ester groups in branan ferulate are enzymically cross-linked (using a peroxidase / hydrogen peroxide system) to form a commercial hydrogel product, Sterigel" (SSL International), which is used as a wound management aid ". U-L-AIaj 1

a-D-GlcpA 1

4

4

2 2 +4)-P-D-Xylp( 1+4>P-D-Xylp-( 1+$)-f%-D-Xylp-(l+4>P-D-Xylp-(1+ 3 3

t

t

1 a-D-Galp-( 1+2)a-L-Araf 5

1 U-L-kaf 5

Figure 3.

t

t

fedate

ferulate

The structural features / monosaccharide configuration in branan ferulate

REFERENCES 1.

2.

3. 4. 5. 6. 7.

G. D. Winter, Formation of the scab and the rate of epithelializationof superficial wounds in the skin of the young domestic pig, Nature, 1962,193, 293-294. S. Thomas, WoundManagement and Dressings, PharmaceuticalPress,London, 1990. L. L. Lloyd, J. F. Kennedy, P. Methacanon, M. Paterson & C. J. Knill, Carbohydratepolymers as wound management aids, Curbohydr. Polym., Special Issue Gluportwo, 199ft,z, 315-322. S. Dimitriu, P. F. Vidal & E. Chornet, Hydrogels based on polysaccharides, In: Polysucchurides in Medical Applications, S . Dimitriu (ed.),Marcel Dekker, New Yo&, 1996, pp. 125-241. R J. Schmidt, Xerogel dressings - an overview, In: Advances in WoundManugement,T. D. Turner, R J. Schmidi & K. G.Harding (eds.), Wiley, Chichester, 1986, pp. 65-71. P. M. Collins (ed.),Dictionary ofcurbohydrutes, Chapman & Hall,London, 1998. (a) E. E. Treiber, Formation of fibres from cellulose solutions, pp. 455479; (b) L. C. Wadsworth 8z D. Daponte, Cellulose esters, pp. 344-362; (c) M. D. Nicholson & F. M. Merritt, Cellulose ethers, pp. 363-383; In: Cellulose Chemistry and its Applications, T. P. Nevell & S. H. Zeronian (eds.), Ellis HorwOOQChichester, 1985.

-

104

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

20.

21. 22.

23. 24.

25.

26. 27. 28. 29.

30.

Synthesis and denvatisation of biocompatible polymers

British National Formulary, The Pharmaceutical Press. London, 41, March. 2001. D. N . 4 . Hon, Cellulose and its derivatives: structures: reactions, and medical uses, In:Polysacchandes i n Medical Applications, S . Dimitriu (ed.),Marcel Dekker, hc.. New York. 1996, pp. 87-105. A. N. de BeIder, Dextran, In: Industrial Gums:Polysaccharides and Their Derivatives, R. L. Whistler & J. N. BeMiller (eds.),Academic Press, New York, 1993, pp.399-425. T. Kuge. K. Kobayashi, S. Kitamura & H. Tanahashi, Degrees of longchain branching in dextran. Carbohydr. Rex, 1987,160,205-214. A. J. Howcroft, A controlled trial of Dextranomer (Debrisan) in burns of the hand, Bums. 1979,6, 12-14. C. A. Blanclaneister & D. H. Sussdorf, Macrophage activation cross-linked dextran, J. Leukoc. Biol., 1985. 37,209-219. B. A. Stone & A. E. Clarke, Chemistry and Biology of (1 -+3)-~Glucans,La Trobe University Press, Victoria, 1992. S. J. Leibovich & D. Danon, Promotion of wound repair in mice by application of glucm, J. Reticuloendothel. Soc., 1980,27, 1-11. P. C. Berscht, B. Nies, A. Liebendorfer & J. Kreuter, In vitro evaluation of biocompatibility of different wound dressing materials. J. Mat. Sci. Mater. Med.. 1995, fj,201-205. R A. A. Muzzarelli, M. Mattioli-Belmonte, A. Pugnaloni & G. Biagini, Biochemistry, histology and clinical uses of chitins and chitosans in wound healing, In: Chitin and Chitinases, P. JolKs & R. A. A. Muzzarelli (eds.),Birkhiiuser Verlag, Basel, 1999, pp. 25 1-264. Y. Q n & 0.C. Agboh, Chitin and chitosan fibres: unlocking their potential, Medical Device Technoloo, 1998, December,24-28. (a) Y. Qn. 0. C. Agboh, X. Wang & D. K. Gilding, Novel polysaccharide fibres for advanced wound dressings, pp. 15-20; (b) Y. Le, S. C. Anand & A. R Horrocks, Using algmate fibre as a drug carrier for wound heahg, pp. 21-26; In: Medical lextiles 96, S . C . Anand (ed.), Woodhead Publishing, Cambridge, 1997. R. A. A Muzzarelli, G. Biagini, A. Damadei, A Pugnaloni & J. Da Lio, Chitosans and other polysaccharidesas wound dressing materials, In: Biomedical and BiotechnologrcafAdvances in Industrial Polysaccharides, V. Crescenzi, I. C. M. Dea,S . Paoletti, S. S. Stivala & I. W. Sutherland (eds.), Gordon & Breach, Amsterdam, 1989,pp. 77-88. L. L. Balassa & J. F. Prudden, Applications of chitin and chitosan in wound-healing acceleration, In: Proceedings of The First International Conference on Chitin/Chitosan, R. A. A. Muzzarelli & E. R Pariser (eds.), Massachusetts Institute of Technology Sea Grant Report, MITSG 78-7,1978, pp. 296-305. (a) G.Biagini, R. A. A. Muzzarefi, R. Giardino & C.Castaldini, Biological materials for wound healing. pp. 16-24, (b) K. Kifune, Clinical application of chitin artificial skin (Beschitin W), pp. 9-15; In: Advances in Chitin and Chitosan, C. J. Brine, P. A. Sandford 62 J. P. Zikakis (eds.),Elsevier Applied Science, London, 1992. R. A A Muzzarelli, Chitin and chitosan: unique cationic polysaccharides,In: Towards a carbohydrutebased chemise, Report EUR 12757 EN, Commissionof the European Communities, Luxembourg, 1989, Pp. 199-231. (a) X. Chen. G. Wells & D. M. Woods. Production of yarns and fabrics fiom alginate fibres for medical applications; (b) M. Miraftab, Q. Qao, J. F. Kennedy, S. C. Anand & G. Collyer, Advanced materials for wound dressings: biofunctional mixed carbohydrate polymers, pp, 164-172; In: Medical Textiles, S . C. Anand (ed.),Woodhead Publishing Ltd, Cambridge, 2001. (a) P. H. Weigel, S. J. Frost, R. D. LeBoeuf & C. T. McGary, The specific interaction between fibrin(ogen) and hyaluronan: possible consequences in haemostasis, inflammation and wound healing, pp. 247-264; (b)E. A Balazs & I. L. Denlinger, Clinical uses of hyaluronan, pp. 265-280; In: The Biology of Hyaluronan (Ciba Foundation Symposium 143), John Wiley & Sons,Cluchester, 1989. D. Williams. The engineering of polysaccharides,Medical Device Technology, 1997, September, 8-11. B. Casu, Structure andbiological activity of heparin, Adv. Carbohydr. Chem. Biochem., 1985,43,51-134. R. A. Carlsen, P. Helin & G. Helin, Glycosaminoglycan formation around the linear wound, J. Invest. Dermatol., 1973,6l. 7-1 1. J. F. Kennedy, M. Paterson, C. J. Knill & L. L. Lloyd, The diversity of properties of polysaccharides as wound management aids, and characterization of their structures, In: Proceedings of the 5'h European Conference on Advances in WoundManagement, G. W. Cherry, F. Gottrup. J. C. Lawrence, C. J. Moffatt & T. D. Turner (eds.),Macmillan Magazines Ltd, London, 1996, pp. 122-126. J. F. Kennedy, P. Methacanon, L. L. Lloyd, M. Paterson & C. J. Knill, The chemical structure of a novel polysaccharide, Sterigel, suitable as a wound management aid, In: Proceedings ofthe 61h European Conference on Advances in WoundManagement, D. J. Leaper, G. W. Cherry, C. Dealey, J. C. Lawrence & T. D. Turner (eds.),M a d l l a n Magazines Ltd, London, 1997, pp. 141-147.

Part 3

Production and use of biocompatible materials

IMPROVEMENT OF ALGINATE FIBER MIXING WITH PHOSPHORYL POLYSACCHARIDES Seiichi Tokura'*, Hiroshi Tamura', Yukihiko Tsuruta', Chisato Nagaei2and Kouki Itoyama2 Faculo of Engineering and HKC. Kansai University, Suira, Osaka 564-8680, Japan Institute for Research and Development, Fuji Spinning Co. Ltd., Oyama, Shizuoka 410-1394, Japan

ABSTRACT Chitin and cellulose were converted to water soluble materials and to calcium specific adsorbents by introducing phosphoryl groups into sugar the residues. Phosphoryl chitin (P-chitin) or phosphoryl cellulose (P-cellulose) was mixed with alginate aqueous solution before spinning and then spun into calcium chloride aqueous solution under similar conditions as those for alginate filament spinning. The P-chitin mixed alginate filament was shown to have improved wet tensile properties in addition to the softness of filament, whereas P-cellulose mixed alginate filament showed less knot strength than that of Pchitin mixed filament.

,&!7+/&7&7j NHCOCH,

NHCOCH,

NHC0CH3

NHCOCH,

P-Chitin

'

,

COOH

Alginic acid

,g?&7&&-7 OH

OH

OH

P-Cellulose

Figure 1. Polysaccharides used in this study.

OH

108

Production and use of biocompatible materials

INTRODUCTION As alginate is known to be one of the biological polysaccharides biocompatible the fiber or membrane is used for biomedical purposes I . Chitin is also expected to be used as biomedical materials due to its biodegradability and low toxicity 2.3. However, the stiffness of alginate filaments and wet tensile properties of chitin filaments can be improved by substituting hydroxyl groups with functional groups. We have prepared chemically modified chitin derivatives, which possess good solubility in many kinds of solvents and characteristic functions, in order to promote the usefulness of this polysaccharide resource. In the course of this project, the acylation reactions in methanesulphonic acid were found to be efficient, and many kinds of acyl-chitins, such as sulfate, and carboxymethyl chitins, soluble in organic solvents were successfully prepared by this method 4.5. Recently, the reaction of chitin with phosphorus pentoxide by this method was found to give water-soluble phosphoryl-chitin (P-chitin) of sufficiently high degree of substitution (DS). In our preliminary experiments, it was found that P-chitin forms a gel in the presence of calcium ions as well as the alginate. We gave attention to the fact that both polysaccharides have the same coagulation condition and performed the mixed spinning of P-chitin with alginate using calcium chloride as coagulant. The properties of mixed spun filament were compared with those of the mixed spun filament of P-cellulose with alginate.

EXPERIMENTAL Materials P-Chitin fine powder originating from squid bone was obtained from Nippon Suisan Co. Ltd., and vacuum dried at 60 "C for 1 day. P-cellulose was purchased from Wako Pure Chemicals Co. Ltd. N,N-dimethylfomamide (DMF) was dried over potassium hydroxide and vacuum distilled before use. Orthophosphoric acid was prepared by adding diphosphorous pentoxide to 85% phosphoric acid followed by refluxing at 110 - 120 "C for 12 h. Other chemicals were purchased from Wako Pure Chemicals Co. Ltd. and used without further purification.

Synthesis of P-chitin and P-cellulose P-chitin was prepared by the orthophosphoric acid method using P-chitin from Squid bone. P-chitin of fine powder was stirred in urea-DMF solution, and reacted with orthophosphoric acid at 150°C for 3 h. P-cellulose was also prepared by the same method as the P-chitin applying P-cellulose of lower substitution.

Mixed spinning of filament Mixed solutions of P-chitin (DS=1.4) and alginate where P-chitin contents of 50,33,20 and 0 % were prepared. This solution was excluded out using air pressure through a nozzle (0.lmm diameter, 50 holes) into 3% calcium chloride solution to coagulate. The filament was wound up, using mini-spinning machine, with the first roller rate set at 4.9 d m i n and with a magnitude of elongation of 1.2 (Fig. 2). The obtained filament was extensively washed with water and methanol, and air dried at room temperature. Mixed spinning of P-cellulose with alginate was also performed in a similar manner as the Pchitin mixed alginate filament.

Improvement of alginate fiber mixing

109

PehStin(%) 0 20 33 50 Alglnate(%)lW 80 67 50 \

Wind-up rdler Stretching roller

1st roller rate :4.9 m/mln Stretching ratlo : 1.2

3% CaCh aq. soin.

Figure 2. Spinning machine.

Tensile strength The stress-strain diagram of the filament was measured by the JIS 1013-7.5 and 7.6 methods using Tendon RTA-250 apparatus. The initial sample length was 20.0 m m and the stretching rate was 20.0 d m i n . The force at the breaking point was taken as the tensile stress, which was transferred to tensile strength and Young's modulus.

RESULTS A N D DISCUSSION Synthesisof P-chitin and P-celldose

The introduction of phosphate groups was confirmed by FT-IR and I3C NMR spectra. The IR spectra of P-chitin with various DS show that as the DS increases there is a corresponding decrease in the absorption due to the hydroxyl group (1310cm-') and new absorption frequencies characteristic for stretching of the phosphate groups appear at 124Ocm-'and 920 cm". The proton decoupled 'CNMR spectra for two kinds of P-chitins (DS = 0.22 and 1.26) measured in D,O at pD 7.0 showed that the carbons which attach to the substituted hydroxyl groups are clearly distinguished from the non-substituted ones. It was also found that the introduction of phosphoryl groups into the 6 position took precedence over 3 the position. When the reaction was carried out using orthophosphoric acid, DS increased with increase of the urea-DMF. In contrast to the conventional methanesulfonic acid method, no decrease of MW of the P-chitin was found. There is a good correlation between MW and DS against Urea/DMF ratios suggesting the possibility of regulation of the DS by changing this ratio. This result suggests that destruction of the hydrogen bonds in p-chitin is performed by urea which is known as a general hydrogen bond breaking reagent. The commercially available water insoluble P-cellulose was further phosphorylated to become water soluble applying the similar procedure as the P-chitin.

I10

Production and use of biocompatible materials

Mixed spinning of filament Mixed spinning of P-chitin with alginate was successfully performed using calcium chloride as coagulant using the mini-spinning machine, because P-chitin forms gels in the presence of calcium ion as well as alginate. The obtained filaments were lustrous and smooth with a size of 3-6 denier/g. The P-chitin mixed alginate filament was more flexible than the alginate filament itself. Scanning electron microscope observations of the filaments with different contents of P-chitin indicated that the diameter was around 10 p m and the surface of the filament became rough as the content of the P-chitin increased. The same calcium absorption behavior of P-cellulose made it possible to perform mixed spinning of P-cellulose with alginate successfully to give the fine filament as well.

Tensile strength of filament Tensile strength of the P-chitin mixed alginate filament under dry conditions increased with the increase of the content of the P-chitin and that under wet conditions decreased. Thus, the dry-wet ratio in tensile strength was about 30-40% as shown in Fig. 3. Elongation of the filament in this condition is around 6 - 8 % both in dry and wet conditions with the same strength as rayon and wool. Knot strength was also measured applying the filament in the knot condition. Knot strength is an important characteristic factor for filaments because this character reflects characteristics such as stretching, compressing, bending and torsion. Although knot strength of the filaments decreased to 20-30 % compared to tensile strength, dry-wet ratios of the knot strength of them were 100 - 200 % (Fig. 3).

Figure 3. Dry-wet ratio of P-chitin mixed alginate filament measured in tensile strength and knot strength.

Improvement of alginate fiber mixing

I

0.50

P-chitin

0

11 1

I

P-cellulose

20 33 Content of P-saccharide (96)

50

Figure 4. Comparison of knot strength between P-chitin and P-cellulose mixed aiginate filaments.

These results suggest that the present mixed filament of alginate with P-chitin have excellent properties under wet conditions. The filament properties of alginate filament containing P-cellulose were also performed. The comparison of the knot strength between P-chitin and P-cellulose mixed alginate filament is shown in Fig. 4. It was found that the P-chitin mixed alginate filament is superior to the P-cellulose in knot strength. In addition, reduced antigenic, blood coagulation properties suggest that alginate filament containing P-chitin have the possibility of becoming a new biomedical material, such as wound dressing.

CONCLUSION Mixed spinning of P-chitin with alginate was successfully performed because both of the Pchitin and alginate coagulate under similar conditions. The obtained filament showed soft feeling and flexible, and has an advantage in knot strength especially in the wet condition. The comparison of the filament properties between P-chitin and P-cellulose mixed alginate filament indicates that the former is superior to the latter in the knot strength probably due to the strong interaction with alginate molecule.

ACKNOWLEDGEMENTS This research was partly supported by the Kansai University Special Research Fund, 1999 and also a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, and Sports, Japan (Grant 09240103).

112

Production and use of biocompatible materials

REFERENCES 1.

2. 3.

4.

5.

6.

S. Ohlson, P.-0. Larsson & K. Mosbach, Steroid Transformation by Living Cells Immobilized in Calcium Alginate; European J. Appl. Microhiol. Biotechnol., 1979,7, 103-107. F. G. Pearson, R. H. Marchessault & C. Y. Liang, Infrared Spectra of Crystalline Polysaccharides. V. Chitin, J. Polym. Sci. 1960, 38, 101-116. N. Nishimura, S . 4 . Nishimura, N. Nishi, F. Numata, Y. Tone, S . Tokura, & I. Azuma, Adjuvant Activity of Chitin Derivatives in Mice and Guineapigs, Vaccine, 1985, 3, 379-384. K. Watanabe, I. Saiki, Y. Uraki, S. Tokura & I. Azuma, 6-0-Carboxymethyl-chitin (CM-chitin) as a Drug Carrier, Chem. Pharrn. Bull., 1990, 38, 506-509. S. Tokura, Y. Miura, Y. Kaneda & Y. Uraki, Two-step Hydrolysis of a Polymeric Drug under a Model System, Carbohydr, Polym. 1992, 19, 185-190. Japanese Standards Association, Japanese Industrial Standard JIS L 1013, 1992, pp. 1-8.

PREPARATION OF CELLULOSE VISCOSE FOR VARIOUS MATRICES B Lonnberg’, S Ciovica’, T Strandberg’, T Hultholm’ and K Lonnqvist2

’ Abo Akademi University, Faculty of Chemical Engineering, Laboratoy of Pulping Technology, Porthansgatan 3, FI-20500 TurkdAbo, Finland Cellomeda @, Tykistokatu 6 A. FI-20520. Turku, Finland

ABSTRACT

New medical and clinical applications of regenerated cellulose matrices require also new and better properties. There are different ways to achieve them. Plant cellulose is a natural, pure and mostly crystalline material that can be dissolved and regenerated to form various products (threads, membranes and sponges). The properties of the cellulose viscose and hence the final regenerated cellulose will evidently be affected also by the cellulose source (wood, grass), the pulping method (sulphite, haft) including bleaching, One approach is to find acceptable products by just doing the processing in a better way than before, which implies process modification. In this case, the pulping was made by a new sulphur-free alkaline pulping method called the IDE- process ‘72. The first capital I stands for the ‘impregnation’of the cooking chemicals into the fibre material, D stands for ‘depolymerization’of the lignin and E for the ‘extraction’ of it. Thus IDE forms a special delignificationconcept providing controlled pulping. In this study, a commercial dissolving pulp and bleached IDE-pulps were processed into cellulose viscose and to membranes for comparison of certain chemical and physical properties. The work was considered an exploration of the most proper cellulose regeneration for certain matrix properties. INTRODUCTION

Currently, cellulose matrices are studied in the form of membranes or sponges for use in various clinical and medical products 3,4, since cellulose is a natural material and thus considered pure and biocompatible. Although it is very inert, due to its high degree of crystallinity, and subsequently resistant to acids and alkali, the cellulose may be modified after treatment with strong alkali with the aim of activating the cellulose. Thus the alcoholic hydroxyl groups become accessible and can be substituted with a number of functional groups to give the cellulose modified properties. The classical way of preparing a solution of cellulose is to treat the alkali cellulose with carbon disulphide to form a xanthate soluble in dilute alkali. The cellulose solution or viscose is finally coagulated and regenerated to provide a pure cellulose preferrably in a physical form easy to study. Therefore, the viscose was cast as a film or membrane for the regeneration. Since some medical products require biodegradability, new cellulose materials must be developed, and it is believed that IDE cellulose might fulfil such requirements.

114

Production and use of biocompatible materials

Table 1. Cooking conditions for the softwood IDE pulps. Cook

1 stage')

No.

Temp.

"C IDE-06 IDE-11 IDE-16

D stage Time min

Temp. "C

Time min

Temp.

"C

Time min

90

180 170 165

60 180 180

130 130 130

1x0 1xo 1x0

100 100 100

')

E stage

YO

90

Two-stage procedure under similar conditions

EXPERIMENTAL Pulping and bleaching Pulping conditions Softwood haft and IDE pulps were made in the laboratory under well controlled conditions. The haft pulping conditions were normal (kappa number 24), as the sulphurfree TDE pulping started with a two-stage alkali impregnation at 100" C and continued with a depolymerization stage containing alkali, anthraquinone as a catalyst, water and ethanol and carried out at different temperatures as shown in Table 1. The extraction stage finally carried out with water and ethanol for effective extraction of depolymerized residual lignin was constant in all experiments.

Bleaching conditions The pulps were bleached according to a common ECF bleaching sequence, which was DEDED (DI-I11 for chlorine dioxide, EI-I1 for alkaline extraction). The total chlorine dioxide charge was 13% as active chlorine and the alkali charge 4%, see Table 2.

Table 2. DEDED-bleaching conditions for the softwood haft and softwood IDE pulps.

Stage

act. C1 %

D1 EI DII El1 DIII

NaOH 5%

Pulp cons.

Temp.

%

"C

x 2

4 8 4 4

65 20 65 20

4 4

2 5

65

Time min 60

45 60 45 60

Preparations of cellulose viscose

115

Table 3. Unbleached and bleached softwood haft (K-2) and softwood TDE pulp properties. -

PULP SAMPLES K-2

IDE-06

IDE- 1 1

IDE- 16

-

UNBLEACHED PULPS: Total yield, % Screenings, % Kappa number Acetone extractives, % Viscosity’), mL/g

45.7 0.03 24.2 0. I 1007

49.1 0.08 25.6 0.2 746

47.5 0.02 23.5 0.2 798

47 .O 0.06 28.5 0.1 915

453 1078 8.3 6.6

325 773 9.4 7 .O

330 785 8.9 6.6

371 897 8.6 6.6

BLEACHED PULPS: Viscosity‘),mL/g DP” Slo Solubility3’,% S I S Solubility? %

SCAN-CM 15:88;

DP = (viscosity)/0.42; 3, SCAN-C 2:61

Pulp properties Table 3 presents the pulp yields and kappa numbers. All pulps, including the haft pulp (K-2) and the IDE pulps (IDE-06, IDE-11 and IDE-16), had an unbleached kappa number close to 25. It should be emphasized that the IDE pulp yields were significantly higher than those of the haft pulp, as again the viscosity was much lower, also after bleaching.

Viscose preparation The cellulose viscoses were produced in the laboratory starting with the bleached softwood haft and softwood IDE pulps described earlier, and with a bleached commercial softwood dissolving pulp as a reference pulp. The conditions are shown in Table 4. The alkalization was made with a 20% NaOH solution, which produced an alkali cellulose providing the degree of pressing Pd = 2.8 (weight of pressed alkali cellulose relative to the initial cellulose weight). The final viscose contained 9% cellulose and 6% NaOH. Some of the viscoses were coagulated and regenerated into membranes as to enable a study of the viscose process and the cellulose strength properties.

116

Production and use of biocompatible materials

Table 4. Conditions for preparation of the cellulose viscose.

Procedure

Conc./Charge %

Alkalization (NaOH-c.) - ageing Xanthogenation (CS2-ch.) - dissolution Viscose" ageing

')

To a final DP 400 (see Table 6 )

20

35

Temp. "C

Time

40 23

60

30

60

10 20

(24 h)

min

1)

90

Composition: 9% cellulose and 6% NaOH

Determinations

Fibre dimensions The original bleached pulps as well as the corresponding alkali celluloses were analyzed by the Kajaani FiberLabB Analyzer for fibre and particle dimensions as length, width, wall thickness and coarseness (weight/length). Before running the tests, the alkali celluloses were thoroughly washed, acidified as to exclude the bound sodium ions and washed again to complete neutrality. The fibre dimensions were determined following normal procedures implying that thousands of fibres were measured and reported as distribution curves and average values. The fibre length measurement interval covered the range born 0 to 7.6 mm and the fibre width accordingly from 0 to 200 pm, the resolutions being 50 and 1 pm respectively.

'

Infra-red spectra IR-spectra were taken on the bleached pulp samples (K-2, IDE-06, IDE-1 1 and IDE16, and the commercial dissolving pulp as a reference) as to determine the crystalline and amorphous domains of the cellulose, and to provide a measure of the crystallinity index. The absorption of the 1375 cm-' wavelength was considered reflecting the crystalline part and that of 2900 cm-' wavelength the amorphous part of the cellulose '; thus the ratio between these two absorptions would provide an approximation of the crystallinity index.

RESULTS AND DISCUSSION Cellulose crystallinity The IR spectra taken are given in Fig. 1, and the computed crystallinity indices are compiled in Table 5. It appears that the IDE pulps provided quite low crystallinity indices according to the method applied. The IDE-06 pulp which was cooked at a high temperature of 180' C appeared to be particularly low in crystallinity.

Prcparations of cellulose viscose

1 I7

I

I

Figure 1. IR spectra of the softwood DEDED-bleached haft (K-2) and IDE pulps as well as of the commercial reference pulp. Ageing of alkali cellulose Cellulose depolymerization took place during the ageing of the alkali celluloses obtained. The degree of depolymerization (DP) was computed from the viscosity divided by 0.42. Both DP and lnDP decreased linearly as a function of the ageing time with R2 clearly exceeding 0.95. Table 6 shows the ageing time required to obtain DP 400, and also the coefficients a and b of the equation lnDP = a - b t, as well as the R2 value. Fig. 2 provides an example with IDE-06, which had a low crystallinity index.

Pulp fibre dimensions The pulp fibre dimensions, i.e. fibre length, fibre width and fibre wall thickness and their respective distributions, were determined with the FiberLab Analyzer.

Table 5. Softwood haft (K-2) pulp, softwood IDE pulp and commercial dissolving pulp crystallinity indices evaluated from the IR spectra. Pulp

K-2 IDE-06 IDE- 1 1 IDE- 16 Reference

Al3WA29OO

0.8 1 0.35 0.67 0.57 0.7 1

118

Production and use of biocornpatible materials

Table 6. Ageing characteristics of the alkali celluloses including that made of the

dissolving pulp as a reference.

K-2

37.5 6.94 -0.025 0.98

Time to DP 400, h Coefficient a Coefficient b R2

IDE- I 1

IDE-06

27.5 6.66 -0.024 0.YY

26.3 6.66 -0.025 0.99

IDE- 16

Reference

27.7 6.76 -0.028 0.98

lY.O 6.29 0.016 0.96

Measurements were performed on the celluloses (initial pulps), on the alkali celluloses (celluloses after alkali treatment) and on the treated alkali celluloses (celluloses after alkali treatment, pressing and shredding). The ratio between fibre width and fibre wall thickness was computed and reported.

Fibre length The fibre length (weighted by length) is shown in Fig. 3 for the IDE-06 cellulose, the corresponding alkali cellulose, and the treated (pressed and shredded) alkali cellulose. It may be seen that the treatments decreased the average fibre length, which is further itlustrated in Fig. 4. If the cellulose was given the arbitrary average fibre length of 1, it was slightly exceeding 0.8 for the alkali cellulose and even lower after further treatment of the alkali cellulose (pressing and shredding). The trend was about the same for all other celluloses studied in this context, and thus the conclusion may be drawn that particularly the alkali treatment decreased the fibre length, as did also combined pressing and shredding, but to a lower extent, see Table 7.

IDE-06 Alkali cellulose

P

n

C

6.7 6.6 6.5 6.4 6.3 6.2 6.1 6

+ 6.6689 R 2 = 0.9951

y = -0.0246X

I

0

i

10

20

30

Ageing time, h

Figure 2. Ageing rate of the IDE-06 alkali cellulose.

Preparations of cellulose viscose

119

-

IDE-06

rn

1.o

0.0

I

2.0

3.0

4.0

5.0

6.1

Fibre length (length weighted), rnrn

Figure 3. Fibre length distributions for the IDE-06 cellulose, the corresponding

alkali cellulose and the further treated alkali cellulose.

IDE-06 ............

Cellulose

Alkali Treated cellulose alkali cellulose

Figure 4. Average fibre lengths for the IDE-06 cellulose, the corresponding alkali cellulose and the further treated akali cellulose. Table 7. Average fibre lengths (in arbitrary units: 1 for the initial cellulose) for the softwood haft (K-2) and IDE celluloses, and the dissolving cellulose, the corresponding alkali celluloses and the further treated alkali celluloses.

Pulp

Cellulose

K-2 IDE-06 IDE- 1 1 IDE- 16 Reference

1.oo 1.oo 1.o0 1 .00

1.oo

Alkali cell.

‘Treated alk. cell.

0.85

0.78 0.78

0.85 0.85 0.90 0.62

0.78 0.82 1x1

120

Production and use of biocompatible materials

0.0

Figure 5. Fibre width distributions for the IDE-06 cellulose, the corresponding alkali cellulose and the further treated alkali cellulose. Fibre width

A similar evaluation of the fibre widths was made on the celluloses, the corresponding alkali celluloses and the further treated alkali celluloses. Fig. 5 showing the fibre width distribution and Table 8 providing the average fibre widths - presented also in Fig. 6 indicate that the alkali treatment appeared to cause some s w e h g to the material, but not as much as to the reference dissolving cellulose.

Fibre wall thickness The fibre wall thickness index behaved in a similar way as the fibre width. The ratio between the fibre wall index and the fibre width may provide some further information

I

IDE-06

3 1.02 --

Cellulose Alkali Treated cellulose alkali cellulose

Figure 6. Average fibre widths for the IDE-06 cellulose, the corresponding alkali cellulose and the further treated alkali cellulose.

Prteparations of cellulose viscose

121

Table 8. Average fibre widths (in arbitrary units: 1 for the initial cellulose) for the softwood haft (K-2) and IDE celluloses, and the dissolving cellulose, the corresponding alkali celluloses and the further treated alkali celluloses. Pulp

K-2 IDE-06 IDE- 11 TDE-16 Reference

Cellulose

Alkali cellulose

Treated alkali cellulose

-

1.oo 1.00 1.oo 1 .oo 1.oo

1.02 I .0:3 0.99 1.04 1.13

0.98 0.96 0.94 0.97 nd

on the fibre swelling, see Table 9. It appears that the IDE celluloses would have a higher relative fibre wall swelling than the reference dissolving cellulose.

Strength properties Some strength properties were determined on membranes made of the cellulose viscoses produced of the softwood haft and IDE pulps. The filtration of the haft pulp viscose was difficult, and no membrane was thus prepared. The results compiled in Table 10 indicate that the membrane stretch, tensile work and stiffness were similar for the IDE celluloses and the reference, as the tensile strength showed some differences.

Table 9. Ratio between the fibre wall index and fibre width (in arbitrary units: 1 for the initial cellulose) for the softwood haft (K-2) and IDE celluloses, and the dissolving cellulose, the corresponding alkali celluloses and the further treated alkali celluloses. Pulp

Cellulose

Alkali Treated alkali cellulose cellulose

K-2

1.oo 1.oo 1.oo 1.oo 1.OO

1.06 1.02 1.oo 1.02 0.95

IDE-06 IDE- 11 IDE- 16 Reference

0.96 0.95 0.96 0.97 nd

Table 10. Strength properties of cellulose membranes made from softwood IDE pulp and dissolving pulp as a reference.

Property

IDE-06

IDE- 1 1

IDE- 16

Reference

Strength, kN/m Stretch, % Work, J/m' Stiffness, I r N h

3.4 1 4.8 162 286

3.21 4.4 144 292

4.57 3.6 156 312

4.26 4.8 158 304

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Production and use of biocompatible materials

CONCLUSIONS This study indicated that softwood pulps cooked to kappa number 25 by application of the sulphur-free IDE cooking concept, DEDED-bleached and finally converted into cellulose viscose (for medical and clinical membranes or sponges) might provide an interesting cellulose material. Dependent on the D stage temperature from 165" -180" C the crystallinity index and cellulose membrane strength may vary, but it appeared that a high temperature provided a low crystallinity index and a low temperature again a high membrane strength, In general, the pulp yield was very high for the IDE pulp, but the pulp viscosity (DP) was low compared with those of the haft pulp and the commercial dissolving pulp as il reference. The regenerated celluloses will in the future be developed towards good hydrophilicity and suitable biocompatibility.

REFERENCES 1. M. Backman, B. Lonnberg, K. Ebeling, K. Henricson & T. Laxen, Impregnation Depolymerization Extraction pulping, Paperi ja Puu, 1994,76 (lo), 644-64

2. T. E. M. Hultholm, K. B. Lonnberg, K. Nylund & M. Finell, The IDE process: a new pulping concept for nonwood annual plants, In: Proceedings of Pulping Conference, Chicago, Oct. 1-5, 1995, Book I , TAPPI Press, Atlanta, 1995, pp 85-89. 3. 0. Pajulo, B. Lonnberg, K. Lonnqvist & J. Viljanto, Development of a high grade viscose cellulose sponge, In: The M V I I Congress of the European Societyjor Surgical Research (ESSR),Turku-Finland, May 23-26, 1993. Abstract Book, P- 156. 4. S. Ciovica, B. Lonnberg & K. Lonnqvist, Dissolving pulp by the IDE concept, Cellulose Chem. Technol., 1998, 32 (3-4), 279-290.

5. Valrnet Automation Kajaani Ltd, FiberLab installation and operation manual W4230467 V1.3, June 1998, Kajaani, Finland. 6. O'Connor et al., Text. Res. J., 1958 28: 383, In: H. A. Krassig, Cellulose Structure, Accessibility and Reactivity, Gordon and Breach Sci. Publ., 1993, p. 125.

SYNTHESIS AND PROPERTIES OF NOVEL POLYELECTROLYTE ON THE BASIS OF WOOD POLYMER Galia Shulga'"', Girt Zakis', Brigita Neiberte', Janis Gravitis2 'Latvian State Institute of Wood Chemistry, 27 Dzerbenes St., Riga LV-1006, Latvia; 'United Nations Universi& Institute ofAdvanced Studies, 53-67Jingumae 5-chome. Shibuya-ku, Tokyo 150-8304, Japan;

ABSTRACT

In this work, diluted reaction aqueous mixtures, containing polymer cation (PC), a weak polymer base, and sodium salt of birch nitrolignin (Na-Mig) in a composition range of 0.1 5 Z I 5. where Z = [pC]/[Na-Nlig] have been studied. Nitrolignin is an environmentally compatible by-product of the nitrate pulping process and possesses pronounced biostimulating action. The presence of various ionogenic groups imparts polyelectrolyte properties to the lignin macromolecule. It has been shown that the interaction between the reaction mixture components proceeds according to an electrostatic mechanism and results in the formation of novel polyelectrolytes (NPE), differing from Na-Nlig and PC, in terms of their behavior in aqueous media. The water solubility of the W E formed is determined by the composition of the reaction mixture and depends on the extent of conversion in the interpolyelectrolyte reaction. An enhanced ability of adsorbing on the liquidgas and liquidhquid interfaces is conditioned by the presence of hydrophobic domains in NPE structure formed by the interacted regions of polycation and lignin-polyelectrolyte macromolecules. It has been shown that it is possible to regulate the hydrophilic-hydrophobic balance of the water soluble NPE structure by varying the extent of conversion in the interpolymer reaction. The last feature is of interest from the viewpoint of the use of NPE as a regulator of surface tension on various interfaces.

INTRODUCTION Reduction of available oil resources worldwide will gradually reveal lignin, a biomass constituent, as a very important starting material for production of polymers. Chemical modification will play a key role in the development of novel polymer products based on lignins - the by-products of various industrial processes of wood delignification. Therefore, environmental demands, economic realities and a high efficiency of the biomass conversion will be integrated in the process of creation of a new generation of high-performance, high quality and environmentally compatible polymers and plastics. The results reported may be regarded as one of the numerous steps made by the State Institute of Wood Chemistry in accordance with its program "New Materials of Wood and Plant Origin", and the United Nations University in UNU/ZERI (Zero Emissions Research Initiative) concept development based on the strategy of the high efficiency of the biomass conversion into value added chemical products [l]. Nitrolignin (Nlig) is a by-product of a pulping process with nitric acid. It has been shown that nitrolignin formed possesses pronounced biostimulating action. Owing to the

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Production and use of biocompatible materials

presence of a considerable amount of carboxyl and phenolic hydroxyl groups, Mig is one of the representatives of lignin-polyelectrolytes. One of the interesting reactions, which allow to modification of lignin-based polyelectrolytes, is an interaction of such polyelectrolytes with polymer or oligomer cations. These reactions proceed in aqueous, aqueous-salt, or aqueous-organic media at room temperature and normal pressure and lead to formation of polymer products [2,3). The goal of the present work was to study of the interaction between nitrolignin and polymeric cations in dilute aqueous mixtures, and to investigate the properties of novel polymer products formed.

MATERIALS & METHODS Mig was obtained as a result of a delignification process of birch wood 1431. The pulping process included the following stages: impregnation of birch chips with 12.5 g dl-' HNO3 at 323 K for 4 hours; cooking of chips in a sharp steam reactor at 323 K for 1 hour; leaching of chips with water and alkaline extraction with 2.5 g di-' NaOH at 323 K for I hour. Mig was isolated from the spent liquor by precipitating with the universal ion-exchange resin at 293K. Its purification was carried out by selective dissolution in an aqueousalcohol solution with further lyophilic drying. The elemental and functional analyses of Nlig have shown the following average formula of its phenyl-propane unit: C ~ H ~ . S ~ ( O C H ~ ) O . ~ ( O H ~ ~ . ~ ~ ( ~ ~ ~[S] ~. ~ The ~ content )~.~~(~O of ionogenic groups was estimated from potentiometric and conductometric titration curves of Nlig. Its average molecular mass value equal to 3500 was calculated from viscometry data [6]. As the polymer cation (PC), the weak polybase with a molecular mass 50,000 was chosen. It possessed a branched structure and contained up to 75% of primary and secondary amino groups. The novel polyelectrolyte products WE) were synthesized by mixing of diluted initial aqueous solutions of Na-salt of NLig and PC at 293K. The composition of the reaction mixtures was expressed by the Z=[PC]/wa-Nlig]] value, a ratio of the molar concentrations of oppositely charged fimctional groups. Surface tension equilibrium values IS of the aqueous polymer solutions were found according to the Wilhelmy method at 293K. A concentrated oiVwater type emulsion (75 mass p a d 25 mass part) was chosen as the liquid disperse system. The emulsion was obtained by mechanical dispersing of n-heptane in the stabiliser-containing water. The coalescence time (5, min) of equal-volume emulsion samples served as a criterion of aggregative stability. RESULTS & DISCUSSION Intermacromolecular interaction between wood and synthetic polyelectrolytes in aqueous solutions

It has been established that the interaction between the polymer components in the reaction mixtures has an electrostatic mechanism and results in the formation of the novel polyelectrolytes, differing from Nlig and PC, in terms of their behavior in aqueous solution.

Synthesis and properties of novel polyelectrolyte

125

An interpolyelectrolyte reaction of the NPE formation can be represented schematically in the following manner:

Na-Nlig

where A'

PC

NPE

- COO-, O'f, .

The NPE macromolecule formed can be regarded as a special macromoleculepolyampholyte, containing both hydrophilic chains with charged fiinctional groups of diverse nature and hydrophobic domains formed by macromolecule fragments of the interacted polymer cation and nitrolignin. The aggregative stability of the NPE is determined by the composition of the reaction mixtures Z and depends on the extent of conversion (0) in the interpolyelectrolyte reaction calculated fiom the potentiometric titration curves of the polyelectrolytes mixtures [2]. The profiles 0@H) of intermacromolecularreaction are characterized by the steep slope, which is generally typical for cooperative transitions. Initial values of 8 in the Na-NLig - PC interpolymer reaction for reaction mixtures with 0.5 5 Z_<2 vary in the range of 2.3-4.4%.With the decreasing of the pH value, 0 tends to increase and exceeds 30% already in alkaline media for the Na-Nlig - PC equimolar mixture. The solubility of the NPE formed in the diluted mixtures with compositions Z < 5 is retained both in alkaline and neutral media. In the acidic media (6+6,.x), dramatically increasing the content of hydrophobic domains in NPE macromolecule causes the loss of its aggregative stability, resulting in phase separation, i.e. the appearance of opalescence and the formation of colloidal suspensions, which, in time, tend to precipitate in the form of friable hydrated sediments. Therefore, it has been shown that by varying the extent of conversion in the intermacromolecular reaction, it is possible to perform the transition fiom water-soluble polymer products to insoluble ones. The turbidity of the reaction mixtures (see Fig. l), corresponding to the completion of the,interpolymer reaction (0+OmU),tends to decrease when moving away from the stoichiometric ratio of components. At the same time, the content of the residual lignin in the supernatants of the composition mixtures Z < 1 tends to decrease as PC content increases and is not actually changed in Supernatants of the reaction mixtures Z 2 1. The latter is an indicator of the full inclusion of Na-NLig molecules in NPE formed under conditions of their deficiency. Decrease in the turbidity of the supernatants of composition with 0.3 5 Z < 1 is, obviously, determined by the presence of a surplus content of Na-Mig, providing the pronounced lyophilization effect on the structure of NPE formed. At 2 < 0.3, the interaction between the polymer components leads to the formation of only water-soluble NPE characterized by an increased aggregative stability within a whole range of pH. It is remarkable to note that the reaction mixture with a considerable Na-NLig (Z=5) tends to opalesce immediately after initial polyelectrolytes are mixed, and a sufficient increase in turbidity with decreasing pH does not actually occur. Such behavior of the

126

Production and use of biocompatible materials

T, %

"

Figure 1.

-1

-0,3

0

The light transmittance coefficient (T) of the reaction mixtures at h = 590 nm (light bar) and mixture supernatants at h = 440 nm (dark bar) as a fbnction of the logarithm of the mixture composition (log Z); pH 4.5; 293 K; lpC] = 0.5 lo5 mole F'.

Ov5

Figure2.

C , g/dl

Dependence of the logarithm of the coalescence time (In T) of the nheptane/water emulsion stabilized by PC (I), Na-NLig (Z), Na-Mig - PC reaction mixture with the composition Z = 0.1 (3) on stabilizer content.

Synthesis and properties of novel polyelectrolyte

127

reaction mixture is possible only in the case of an uneven occupation of the long polycation chains by compact lignin molecules. The occupation according to the “everything or nothing” principle could be determined by specific conformation of the cation macromolecule stabilized by hydrogen bonds and hydrophobic interactions among amino groups in alkaline media. Properties of novel polymer products

The amphiphilic character as well as enhanced hnctionality of the NPE macromolecuIe predetermines its ability of adsorbing on various interfaces. According to surface tension isotherms measured on the water-air interface in terms of the maximal surface activity, Go, the polyelectrolytes and their reaction mixtures are presented in the following order: Na-NLig-PC (pH 9.6) > Na-NLig-PC (pH 10.7) > PC (pH 10.7) > Na-Nlig (pH 9.6) > PC (pH 5.0) > Na-Nlig (pH 10.7). As a decreases, the surface adsorption of nitrolignin increases, which is testified by the decrease of CT at the interface. At the same time, amino groups of PC in alkaline media have a negligible degree of ionization (a-+ 0), which tends to increase regularly upon passing to acidic media and to result in an increase in o. The pronounced ability of water-soluble NPE to dissipate fi-ee surface energy is, obviously, connected with the presence hydrophobic domains in NPE macromolecules, making their transfer fiom the solution volume to the water-air interface energetically profitable. As already noted, 8 of interpolyelectrolyte reaction tends to increase, as the pH values decrease, leading to an enhancement in the content of hydrophobic domains in the NPE macromolecule. Such hydrophobization is accompanied with a pronounced - ~ to 51.6 - 4 4 . 3 ~ 1 J/m2 0 ~ ~in 0.1-1.0 g dl-’ Nareduction ofo from 57.1 - 4 7 . 7 ~ 1 0down NLig-PC reaction mixtures at pH values of 10.7 and 9.6, respectively. Hence, varying the extent of conversion in the interpolyelectrolyte reaction, the regulation of the surface activity of the interpolymer products at the water-air interface can be achieved. According to the studies performed, water-soluble NPE is also capable of adsorbing at the interface of two non-mixing liquid phases in lyophobic disperse systems. The dependence of the coalescence time logarithm on the stabilizer content shows (see Fig.2) that the time of the complete phase separation of the direct n-heptane-water emulsion stabilized by the diluted Na-Nlig - PC reaction mixtures exceeds 2.5-14,5 times the value determined for the emulsion containing Na-Nlig or PC. It may be assumed that the formation of interfacial viscous-elastic layers occurs as a result of the hydrophobic interaction between the surface of emulsion drops and the hydrophobic domains of the water-soluble NPE macromolecule. In this case, the W E macromolecule hydrophilic segments in the form of loops and tails have to be transformed into the dispersive phase, lyophiliing the disperse phase surface and decreasing the interfacial tension. Similar to the case of the adsorption at the water-air interface, 8 growth facilitates and sufficiently promotes the NPE macromolecules transfer and fixation on the n-heptanewater interface. The revealed surface-active properties of NPE with a non-stoichiomehic composition, i.e. products of the essentially incomplete interpolyelectrolyte reaction, enable them to proceed for use as stabilizers of different lyophobic disperse systems. At the same time, the water insoluble NPE can be regarded as a soil conditioning agent for the improvement of the shvcture of sandy soils [7].

128

Production and use of biocompatible materials

CONCLUSION Thus, modification of nitrolignin as a result of the intermacromolecular reaction with polymer cation corresponds to an electrostatic mechanism and results in the formation of the novel polymer products, differing from initial polyelectrolytes, in terms of their behaviour in aqueous solutions and at interfaces. A pronounced ability of the polymer products to dissipate the free surface energy on various interfaces is conditioned by the peculiarities of their structure. By varying the extent of conversion in the interpolyelectrolyte reaction, it is possible to regulate surface activity of such polymeric products on various interfaces.

ACKNOWLEDGEMENTS The authors acknowledge financial support from the Latvian Council of Science (the grant for the research project No 96.0599).

REFERENCES: 1. J. Gravitis, T. Della Senta, E. D. Williams, ‘The conversion of biomass into fuels, fibers and value added chemical products from the perspective of the Zero Emissions Concept’, In: Biomass Conversion, Int. Symp., Sapporo, Japan, 1997, pp. 1-18. 2. G. Shulga, R. Kaljuzhnaja, L. Mozhedco, F. Rekner, A. Zezh, V. Kabanov, Cooperative reactions with lignosulfonates participation, Polymer Science, 1982, 24(7), 15 16-1 521. 3. G. Shulga, Modified lignosulfonates as component of interpolyelectrolyte complex, Latvian J. Chem., 1996,3-4, 118-123. 4. A. Mellcis, G. Zakis, Investigation of lignin obtained during nitric acid pulping of birch wood, WmdChemistry (in Russian), 1981, 1, 77-84. 5. A Melkis, G Zakis, M Meksha, On the structure of nitrolignin, Wood Chemistry (in Russian), 1983, 2, 72-79. 6. P R Gupta, D A I Goring, Physicochemical studies of alkali lignins, Can. J Chem., 1960,38(2), 270-79. 7. G. Shulga, G. Zakis, A. Melke, B. Neiberte, Method for Improvement of Soil Structure, LR Patent, No. 12 152, March 1997.

UTILISING THE POTENTIAL OF WOOD FIBRE Lennart Salmen* & Ulla-Britt Mohlin Swedish Pulp and Paper Research Institute (STFI) Box 5604, SE-114 86 Stockholm, Sweden

INTRODUCTION Wood fibres or cellulose fibres are a composite material with excellent strength properties which on a weight basis exceed those of steel. However, after they have been processed, it is often found that structural defects in the fibres render the material less effective in its strength bearing capacity. The reasons are found both in changes in the fibre structure as such and also in changes occumng at the molecular level within the fibre cell wall. Our understanding of the structural changes occurring when wood fibres are pulped is still limited, mainly due to the extremely complex nature of the fibre wall which, on the ultrastructural level, still poses a lot of questions. During recent years, a lot of research has been devoted to the ultrastructure of the cell wall, utilising new powerful techniques such as solid state NMR Atomic Force Microscopy 3, molecular modelling 4 and high resolution-Cry0 field emission SEM 5 . To some extent, these studies have only revealed that the structure of the cell wall is even more complex than was previously realised. In this paper, our present understanding of the ultrastructure and morphological aspects of wood fibres and of the potentials for improving the mechanical properties of the final paper product is examined. An understanding of'different structural features of the wood fibre wall and how they are changed during industrial processing may lead us to the development of less destructive pulping methods, and a better utilisation of the potential of the inherent mechanical properties of the wood fibre. MICROFIBRILLAR STRUCTURE Many years ago, Jayme 6 noticed that wood freshly cut and pulped produced a pulp with excellent ductile properties. Any drying of the wood or beating of the pulped wood reduced the ductility. Based on microscopic studies of the outer fibre walls, Jayme suggested that the native cellulose microfibrils have a very loosely arranged structure. When the wood is dried or the pulps are beaten, these loosely arranged microfibrils are brought closer to each other causing them to join and to form a more rigid less swelling structure. In the case of never-dried cotton fibres, they display an exceptional ability to stretch in the wet state which is totally lost in the first drying 7. The formation of strong water-resistant bonds between the cellulose microfibrils is said to explain this loss in extensibility. The cotton fibre never regains the high water sorption it has in its native biological growth state 7. With newer microscope techniques, high resolution-Cry0 field emission SEM, it is now possible to reveal the fibrillar structure of hydrated fibres without major artefacts 5 . The picture that emerges is one of a much more disordered structure than has traditionally been visualised; one in which fibrils are undulating along the main fibrillar axis of orientation as is schematically sketched in Fig. 1. This picture suggests a structure that could be extended substantially without loading the individual microfibrils

130

Production and use of biocompatible materials

Figure 1.

Cellulose fibrils of a wood fibre, schematically after a micrograph of Daniel and Duchesne 5, bar 100 nm.

in pure tension. The structure itself greatly resembles the one suggested by Boyd 8 in which the microfibrils have, as defined by Boyd, a lenticular trellis arrangement, Fig. 2. Between the microfibrils, the lignin-hemicellulose gel is distributed as lens-shaped platelets. The creation of pores in this structure by the removal of lignin, and the subsequent closure of these pores upon drying in the case of a chemical pulp or when the lignin is extensively removed as in a bleached pulp may thus easily be pictured as being due to the closure of the lens-shaped openings.

/

microfibrils in S,

in between the microfibrils, and lignin are located

Figure2.

Microfibrillar arrangement in the secondary wall based on the lenticular model of Boyd *.

Utilising the potential of wood fibre

131

isordered region

accessible surfaces

inaccessible surfaces

Figure 3.

Schematic representation of the microfibrillar structure after Rowland and Roberts 9.

More recent results based on NMR-studies suggest that the crystallinity of the cellulose in wood is extremely low (much of the structure being paracrystalline) 1. A large part of the cellulose signal was however assigned to inaccessible fibril surfaces and a minor part to accessible surfaces, confirming to a geat extent an earlier picture of the cellulose microfibrillar arrangement suggested by Rowland and Roberts 9, Fig. 3. When the material is processed the cellulose structure was found to rearrange into a more crystalline structure but also into a structure with more accessible surfaces l . This is in accordance with earlier X-ray diffraction data indicating changes of the ultrastructure of cellulose with increasing temperature lo. These X-ray results indicated that there was an increase in the degree of perfection of the cellulose crystals as well as a disordering consisting of an enlargement of the already defect areas of the crystallites. Thus, when wood is processed into pulp, a structural rearrangement of the cellulose apparently occurs, and this can be assumed to lead to a much more rigid structure. It may be this restructuring that gives the fibre material a more brittle appearance and thus results in a loss of those properties which would permit a versatile utilisation of the wood fibre material.

POLYMER ULTRASTRUCTURAL ARRANGEMENT The different wood polymers exhibit quite different mechanical properties and the chemical processing of fibres tries to use the composition to the advantage of the product performance. However, our knowledge of how the different polymers are arranged in the cell wall and of how they contribute to the overall mechanical behaviour of the wood fibre is still rather limited. Much of the strength properties of the fibre is related to that of the cellulose microfibrils. This is obvious not only in the dependence on the &-fibril angle of the longitudinal mechanical properties of the wood fibre 1 1 but also in the effect of temperature on the elastic properties of paper, the reduction with increasing temperature being proportional to the crystallinity of the cellulose microfibrils 12.

'

132

Production and use of biocompatible materials

2 , h

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0

hydrolysed

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Figure 4.

I

I

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70

Tg OC

Effects on the wet lignin glass transition temperature of spruce wood of different chemical treatments which alter the structure of the lignin 13.

Much of the moisture-related variations in fibre properties is related to the effects of the other components, the lignin and the hemicelluloses. In a lignin-rich pulp fibre, the lignin and the lignin softening play an important role for its properties. It is easily shown that a variation in the lignin structure, whether native l4 or induced 13, has a profound effect on the fibre properties. As seen in Fig. 4, various chemical treatments may substantially shift the wet lignin glass transition of spruce wood so that there is a variation in the onset of the loss of wood stiffness associated with the glass transition, between 70 and 130°C. Effects of variations in the hemicelluloses are however much more difficult to assess. It is suggested that, during the formation of the cell, the hemicelluloses act partly to regulate the pattern of cellulose aggregation and that later during lignification they control the assemblage and structure of the lignin being formed 15,16. Mechanical investigations 17 also suggest a fairly close association between the different hemicelluloses, xylan and glucomannan, and the other wood polymers, hindering the movement of these hemicelluloses. It has been suggested that the xylan occurs in a mixture with the lignin whereas the glucomannan has a close association to the cellulose. The proposed structure is schematically pictured in Fig. 5 17. This structure accounts for the swelling and softening effects of the hemicelluloses being spread over a large temperature and humidity interval, making them difficult to evaluate. The interlocking of the components will also make them contribute less to the overall fibre properties. Only when the lignin is removed and the xylan freed will its influence be readily apparent 17. 2D-FTIR may be useful to assess more fully this co-operation between the different polymeric components of the wood. Recent measurements 18 have shown that with 2D-FTIR it is possible to resolve the OH-band region of cellulose into different components, as shown in Fig. 6. Coupling between the cellulose molecular vibrations and those of hemicelluloses may then be possible to assess with this technique. A better knowledge of how the wood polymers contribute to fibre strength would provide a new way to optimise the pulping process.

Utilising the potential of wood fibre

133

-

c lignin 4-

-

4-

xylan

glucomannan

f -cellulose

Figure 5.

Schematic picture of the organisation of the different hemicelluloses in the secondary wall of wood fibres 17

in-phase ZD-spectra intermolecular 0(6)H------0(3) intramolecular 0(3)H-----0(5) intramolecular O(Z)H------0(6)

static absorbency 3500

3000

wave number Icrn-'

Figure6.

Separation of bands in the OH-region of cellulose by the 2DFTIR technique 18.

CELL WALL STRUCTURE At the cell wall level, a relatively clear picture is emerging of different cell wall layers with cellulose microfibrils at different angles. We have a fairly clear picture of the influence of the fibrils on the mechanical properties of the fibre in its length direction. Essentially, the microfibrillar angle of the Sz-layer determines the elasticity of the fibre wall l 1 9 l 9 . For the properties across the fibre, the picture is much less certain. Measurements of the local radial stiffness of fibre walls show that there are large variations, but there is no obvious relation to the fibrillar angle of the S2 layer, see Fig. 7 20. Many of the results suggest that the fibrillar angles of the S1 and S3 layers and the thickness of these layers should be the key variables controlling the transverse properties.

134

Production and use of biocompatible materials

m

:

&' 3000

-aa

0

U

0

0

E 2000

0

2

P c'

.-0

-ma

0

0 0

1000 0

Q)

2

a >

! ! L

0 0

E

C

Figure 7.

20

30 fibril angle S,, degrees

10

40

The transverse elastic modulus of the double wood fibre wall of spruce, measured in the radial direction, as a function of the microfibril angle of the Sz-layer 20.

Model calculations in the transverse direction of the fibre based on polymeric properties are still far from perfect. A key factor here is our lack of knowledge of the elasticity in the transverse direction of lignin and hemicelluloses, where only vague estimates are available. The present understanding is that both lignin and hemicelluloses have a structure oriented parallel to the microfibrils in the secondary wall 21-24. Thus, a knowledge of the orthotropic elasticity of both lignin and hemicelluloses is essential if it is to be possible to make any relevant model calculations With a better knowledge of the variables controlling the transverse properties of fibres, it is anticipated that more easily collapsible fibres may be produced.

FIBRE IRREGULARITIES In the pulp and paper mill, the treatment of the fibres is far from gentle and the fibres which are used in many paper grades are more or less distorted. In particular, microcompressions or kinked areas occur at regular intervals along the fibre. The result is that the properties of the fibres processed in the mill never reach the levels shown to be possible in a gentle laboratory preparation of the material. This applies to the cooking and bleaching procedures as well as to the beating of the pulp. How these irregularities come about is a question of great interest, i.e. whether they are induced by structural variations, such as pores or nodes, or whether they merely reflect stochastic effects of the mechanical actions on the fibre. When wood is treated mechanically, dislocations are often seen at regular intervals in the fibre 25, and these can well be due to a critical shearing stress of the fibre. The cellulose microfibrils are not perfect and more disordered structures exist along their lengths. It has been suggested that these occur at regular intervals and can be determined by chemical attack on the fibre, and this has led to the concept of the level-off DP 26. Such disordered areas could thus lead to damaged areas within the fibre wall. On the other hand, larger structural variations such as nodes of the fibre could be the areas where chemicals have easier access to the microfibril so that structural changes occur at regular intervals. It is

Utilising the potential of wood fibre

135

known that, when pulp fibres are totally dissolved by LiClDMAc, some small fragments of the fibre wall are still left, which suggests the possibility that there may be structural anomalies of a higher order along the length of the fibre 27. Fibre irregularities are the main reason why the effectiveness of the fibres in the sheet is impaired. Such inregularities have a two fold negative effect on the loadbearing ability of the fibres in the sheet. Firstly they are the centres for chemical attack during coolung and bleaching. Local degradation gives a local weakening of the fibre and this creates a centre for a fibre break when the fibre is loaded. Fibre strength measurement using the technique of the rewetted zero-span tensile index is a technique that is sensitive to local chemical degradation of the fibre zs. In the case of commercial pulp, there was a strong but not unique relationship between pulp degradation, measured as pulp viscosity, and rewetted zero-span tensile index measured on straight fibres (i.e. after PFI-mill refining), Fig. 8 28. The diagram shows results from six mills and includes samples of unbleached, oxygen-bleached and fully bleached pulps. Secondly, local irregularities are the origin of kinks and curls in the fibre which decrease the effective length of the load-bearing elements in the sheet. This is well illustrated in Fig. 9, where the tensile stiffness index is shown as a function of the number of defects (curl index) that change the direction of the fibre in the sheet i.e. kinks, twists 28. The diagram shows results for an unrefined pulp where the effect of fibre curl was very pronounced. Kink and curl can be introduced into the fibre both during pulping and during refining This is mainly an effect of the industrial process and is difficult to observe in Iaboratory-scale equipment. Refining can act both to increase and to decrease fibre curl. Laboratory refining and gentle industrial refining remove fibre curl, tougher industrial refining can introduce fibre curl.

f z

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N

d

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A

.

120 600

.

.

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.

800

.

.

I

. ..

900

I

.

. .

I

.

Mill 5 Mill

A Mill 6

I

1000 1100 1200 130

viscosity, cm 3 ~ g Figure&

The rewetted zero-span of handsheets as a function of pulp viscosity for pulps produced in different mills 28.

136

Production and use of biocompatible materials

1

Mill 1 0 Mill 2 . Mill 3 0 Mill 4 A Mill 5 A Mill 6 .

0

A 0

0

R

-

0 A

I

A 0

number of defectslfibre, kinks and twists

Figure9.

The tensile stiffness index for papers of fibres with different amounts of defects, kinks and twists, i.e. defects that change the direction of the fibre axis 28.

The fibre load-bearing ability is also affected by the drying conditions during papermaking. Drying under restraint stretches the fibre and activates the load-bearing ability of the fibres. As a consequence, the properties of a paper consisting of kinked and curled fibres will vary more if the drying restraint is varied at different parts of the paper machine than if the fibres were straight. With a better understanding of the origin of the fibre irregularities, the detrimental structural changes developed within the fibres during their processing may be avoided, giving the possibility of obtaining a stronger paper product.

CONCLUDING REMARKS It is evident that there are unresolved structural questions relating to many different structural levels of the fibre. A better knowledge of the ultrastructure of the fibre wall and the properties of its constituent wood polymers will lead to a better understanding of how the wood fibre is assembled. Such knowledge is essential if it is to be possible to assess the influence that structural anomalies have on the fibre properties, and to find ways of counteracting the detrimental development of these properties during the processing of wood fibres.

REFERENCES

K. Wickholm, P.T. Larsson, & T. Iversen, ‘Assignment of non-crystalline forms in cellulose I by CP/MAS 13C NMR spectroscopy’, Carbohydrate Research, 1998, 312, 123-129. 2. P.T. Larsson, K. Wickholm, 8~T. Iversen, ‘A CPMAS 1 3 NMR ~ investigation of molecular ordering in cellulose’, Carbohydrate Research, 1997,302, 19-25. 3. L. Kuutti, J. Peltonen, J. Pere, & 0. Teleman, ‘Identification and surface structure of crystalline cellulose studied by atomic force microscopy’, J. Microscopy, 1995, 178, 1-6. 1.

Utilising the potential of wood fibre

137

4. A.P. Heiner, J. Sugiyama, & 0. Teleman, ‘Crystalline cellulose la and ID studied by molecular dynamics simulation’, Carbohydrate Res., 1995,273,207-223. 5. G . Daniel & I. Duchesne. ‘Revealingthe surface ultrastructure of spruce pulp fibres using field emission-SEM’.In: 7th International Car$ on Biotechnology in the pulp andpaper industry. 1998. Vancouver, BC Canada: CPPA. pp. B81-B84. 6. G. Jayme, ‘Production and characteristics of spruce sulfate pulps with biological properties’, Tappi, 1963,46(7), 41 5-420. 7. P. Ingram, D.K. Woods, A. Peterlin, & J.L. Williams, ‘Never-dried cotton fibers partI: morphology and transport properties’, Text. Re.s.j . , 1974,44(2), 96- 106. 8. J.D. Boyd, ‘An anatomical explanation for visco-elastic and mechanosorptive creep in wood, and effects of loading rate on strength,’,In: Newperspective in wood anatomy, P. Baas, (ed.). 1982, Martinus Nijhoffmr W Junk Pub.: La Hague. pp. 171-222. 9. S.P. Rowland & E.J. Roberts, ‘The nature of accessible surfaces in the microstructure of cotton cellulose’, J. Polym. Sci., Part A-I, 1972, 10,2447-2461. 10. T. Hattula, Effects of heat and water on the ultrastructure of wood cellulose, Ph.D.thesis, Univ. Helsinki, Helsinki, 1985 1 1. D.H. Page & F. El-Hosseiny, ‘The mechanical properties of single wood pulp fibres. Part VI, Fibril angle and the shape of the stress-strain curve’, J. Pulp Pap. Sci., Trans T e c h Secr., 1983,9(4), TR 99-100. 12. N.L. Salmen, ‘Thermal softening of the components of paper: its effect on mechanical properties’, Trans. Tech. Sect. (Can. Pulp Pap. Assoc.), 1979,5(3), 4550. 13. A. Bjorkman & L. SalmCn, ‘Studies on solid wood. 11 The influence of chemical modifications on voscoelastic properties’, Cellul. Chem. Technol.,2000, In press, . 14. A,-M. Olsson & L. Salmen, ‘The effect of lignin structure on the viscoelastic properties of wood’, Nordic Pulp Pap. Res. J., 1997, 12(3), 140-144. 15. R.H. Atalla, J.M. Hackney, I. Uhlin, & N.S. Thompson, ‘Hemicelluloses as structure regulators in the aggregation of nativ cellulose’, Int. J. Biol. Macromol., 1993, 15, 109-1 12. 16. N. Terashima & H. Atalla. ‘Formation and structure of lignified plant cell wall factors controlling lignin structure during its formation’. In: The 8th International Symposium on Wood andpulping chemistry. 1995. Helsinki. pp. 69-76. 17. L. Salmen & A.-M. Olsson, ‘Interactionbetween hemicelluloses, lignin and cellulose: structure-property relationships’, J. Pulp Pap. Sci., 1998,24(3), 99-103. 18. B. Hinterstoisser & L. Salmen, ‘Two-dimensional step-scan FTIR: A tool to unravel the OH-valency-range of the spectrum of Cellulose 1’, Cellulose, 1999, 6(3), 251-263. 19. L. SalmCn & A. de Ruvo, ‘A model for the prediction of fiber elasticity’, Wood Fiber Sci., 1985, 17(3), 336-350. 20. A. Bergander & L. Salmen, ‘The transverse elastic modulus of the native wood fibre wall’, J. Pulp Pap. Sci., 2000,26(6), 234-238. 21. C.Y. Liang, K.H. Bassett, E.A. McGinnes, & R.H. Marchessault, ‘Infrared spectra of crystalline polysaccarides VII Thin wood sections’, Tuppi, 1960,43( 12), 10171024. 22. R.H. Atalla & U.P. Agarwal, ‘Raman microprobe evidence for lignin orientation in the cell walls of native woody tissue’, Sience, 1985,227,636-639. 23. N. Terashima, ‘A new mechanism for formation of a structurally ordered protolignin macromolecule in the cell wall of tree xylem’, J Pulp Pap. Sci., 1990, 16, J15O-Jl55.

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Production and use of biocompatible materials

24. N. Terashima, K. Fukushima, L.-F. He, & K. Takabe, ‘Comprehensive model of the lignified plant cell wall’, In: Forage cell wall structure and digestibility, H.G. Jung, D.H. Buxton, R.D. Hatfield, &J. Ralph, (eds.). 1993, ASA-CSSA-SSSA: Madison, WI. pp. 247-270. 25. P. Hoffmeyer, Failure of wood as influenced by moisture and duration of load, Ph.D.-thesis, State University of New York, Syracuse, 1990 26. D.H. Page, ‘The origin of the differences between sulphite and h a f t pulps’, J. P u b Pap. Sci., 1983,9( l), TRl5-TR20. 27. E. Sjoholm, K. Gustafsson, B. Pettersson, & A. Colmsjo, ‘Characterization of the cellulosic residues from lithium chloride/N,N-dimethylacetamidedissolution of softwood h a f t pulp’, Carbohydrate Polymers, 1997,32,57-63. 28. U.-B. Mohlin, J. Dahlbom, & J. Hornatowska, ‘Fiber deformation and sheet strength’, TAPPZJ., 1996,79(6), 105-11 1 .

COMPOSITES FROM BANANA TREE RACHIS FIBERS (jWUSA GIANT CAVENDISHII AAA)

',

Maria Sibaja Patricia Alvarado I

',Rocio Pereira ' t Manuel Moya

Luboratorio de Polimeros ( P O W A ) , UniversidadNational, Ap. 863000 HerediG Costa Rica

ABSTRACT Composite materials reinforced with synthetic fiber have been developed to produce high performance products. Utilization of natural fiber could be interesting due several characteristics as low density, wide availability and low cost. In Costa Rica, banana industrialization produces a great quantity of good quality fiber. In this study, this fiber was used to reinforce commercial polyesters. Results show no effect of fiber length in tensile strength or tensile modulus and fiber contents increases tensile modulus but decreases tensile strength. Besides, fiber addition improves flexion properties of polyester matrix.

KEYWORDS

Banana rachis, natural fibers, composites INTRODUCTION The development of composite materials reinforced with synthetic fibers has been successhl with the appearance of the glass fibers, carbon and aramide; due to the better mechanical, electrical and thermal properties that these fibers grant to the product. However certain disadvantages are presented in their use, which has promoted the search for other alternative materials. Among the disadvantages it is possible to mention: corrosion of the plastic processing equipment, increasing the density of the products, high cost of producing the fibers and specially the mayor problems caused to the environment. Natural fibers have a low density, a wide availability at low costs and they are renewable. For this reason, in the last 20 years, vegetable fibers have gained interest by researchers and designers of plastic products Costa Rica is one of the biggest banana producers in the world. The production has maintained a constant increase and is now the leading country in production per hectare and the second as banana tree exporter '. This agricultural activity generates a great quantity of organic waste with the The estimated amount of consequent problems of environmental contamination. banana wastes in Costa Rica, in metric tons by year is as follows: refbsed h i t , 260,000, leaves and stem, 2,874,000; and rachis 797,000 '. As shown in Table 1 the banana tree rachis has a high content of cellulose. This waste contains 5%-7'Yo of fiber that have been used in the production of handmade paper, cardboard boxes, biogas, organic compost and in textile fibers '. The purpose of the present work is to study the effect of the banana tree rachis fiber on the mechanical properties of composites using a polyester resin as matrix.

'.

140

Production and use of biocoinpatible materials

Table 1. Chemical composition of the banana's rachis Rachis Components

% (Drybase)

Hollocelulose a-cellulose Ash Lignin Extractives Humidity

85.5 66.7 2.3 15.3

5.4 6.2

MATERIALS & METHODS Materials. In both experiments a mechanically treated banana tree rachis fiber fiom the variety Muse Giant CavendishiiAAA was used. A polyester resin of general use was used as a polymeric matrix mixed with peroxymethylethyketoneas catalyst. Preparation of composites Method A.

Banana tree rachis fiber of different lengths (0.925 mm to 2.675 mm) was used up to 10% content. The fiber was added manually to the polyester resin at room temperature. The obtained mixture was dripped into a square laminated mould of 100 mm square and 3 mm thickness and it was dried at room temperature. Method B. The sheets of banana tree rachis fiber were obtained by sedimentation Erom a water fiber suspension with 3, 6, 8 and 10% fiber content. Then, it was pressed lightly and dried at room temperature. Finally, they were covered with the polyester resin using a brush. Measurements The instrument was a Tensilon Materials Tester (Model RTWlOO). The specimens were prepared using a cutter machine. Each group of samples contained 5 specimens. The tests were conducted at 20'C and 50 % relative humidity. The specimen dimensions had a thickness of 3.5 rnm and width of 11.0 mm. For tensile test, the cross-head distance was 50 mm and the speed was 5 d m i n . For the compression test, the two supports were open 100 mm with a cross-head speed of 2 mm/min.

Composites from banana tree fibers

141

7-

45

p o -

8%&a m f 25-

c 111

n

P

6

20-

21

15-

A900

10-

5-

!so0 2,675

Fiber length (mm)

Figure 3. Eff'ect of fiber length at 10% on flexural properties of polyester

'a' 50

4000 a

g

3000

E" ;; f

c

2

35 30 25

2000

20

1000

bil

15 10

a a

I

:

1

,,,

z8 I

1

I0 0

3

6

8

f

P El

10

Fiber content (wt%) Figure 4. Effect of fiber content on flexural properties of reinforced polyester using a fiber mat

142

Production and use of biocompatible materials

50

45 800

P a Q)

L

3 9)

g

25 20 15 10 5

600

m30

A450

400

200 0

0 0

0,925

1,44

1,85

2,675

fiber length (mm)

Figure 1. Effect of rachis fiber length at 1O?hon tensile properties of reinforced polyester

Figure 2. Effect of rachis fiber content on tensile properties of reinforced polyester using a fiber mat.

Composites from banana tree fibers

143

RESULTS & DISCUSSION Figure 1 shows the effect of banana rachis fiber addition on the tensile properties of polyester resin. It can be observed that there is apparent difference in tensile strength or modulus between the studied fiber lengths. However if it is compared with the resin without fiber addition a clear decrease in strength is observed. Figure 2 shows the influence of the rachis fiber content on tensile properties. A decrease on the tensile strength can be seen with the increase of fiber content, but the composite becomes more rigid with the addition of the rachis fiber (the tensile modulus

incms). Figures 3 and 4 show the efict of fiber length and fiber content on the flexural properties. As can be observed there is an increase in the flexural strength and modulus with the addition of fiber. In general, fiber addition improves the flexion properties of the matrix. However, these properties d i s h when the fiber contents increase in the sheet (Fig.4). The same behavior is presented for the flexural strength with the increase of fiber lengths (Fig. 3). In order to detect the fiber-matrix behavior, %ure zones on samples were studied by stereoscopic methods. The fiber suffered fiacture with the polymeric matrix. It suggests a good interfacial adhesion between the fiber and the resin.

CONCLUSIONS

- Banana tree rachis fibers increase the tensile and

-

flexural modulus of polyester resin. Fiber-matrix fkture showed good interfacial adhesion between the rachis fiber and the polymeric matrix. According to the studied fiber lengths and fiber content ranges, it is necessary to continue with the experiment using high fiber contents and different fiber lengths. The utilition of banana tree rack in the production of compound materials could be a good alternative in order to diminish the environmental impact.

ACKNOWLEDGMENTS

This work was supportedby the Universidad Nacional, the National Council for Science and Technology of Costa Rita (CONICIT), the Iberaamerican Science and Technology Program (CYTED) and (JICA). REFERENCES 1.

JL Gbmez, CJarones, P. Ganan. “Refuerzosnaturales para materiales plslsticos”, Pldsticos modernos, 1998,76,506-300.

2.

LM Alpfiar, “Evaluaci6n de la pulpa mecgDica obtenida a partir de raquis de banana", Thesis, Univ. de Costa Rica, 1997.

3.

M Moya, M Sibaja, R Pereua, S Nikolaeva, ‘‘Diagn6sticO y caracterizacibn de desechos s6lidos”. Informe ttScnico pmyecto Manejo sostenible de unafinca bananera, Heredia, 1997.

144

4.

Production and use of biocompatible materials

S Nikolaeva, M Moya, M Sibaja, “Utilizacionde raquis de banano como relleno en materides compuestos”. III Congreso Internacional de Medio Ambiente, San Jose, Costa Rica, Proceeding, 1997.

TEMPERATURE A N D CONCENTRATION DEPENDENCY O N EQUILIBRATION IN POLYSACCHARIDE ELECTROLYTE HYDROSOL ‘Masato Takahashi’, Masanobu Mishima’, ‘ratsno Yamanaka’, Tatsnko Hatakeyama’ and H y o e H ~ t a k e y a m a ~ ‘Department of Fme Mat&&

Engkeering, F a d t y of Textile Science aad Technology,

Shinshu Universq, 3-15-1 Tokik, Veda, N a p w 386-8567, Japan -‘I>epartmentof Textile Science, Faculty of Home Economics, Otswna Women’s University, 12 Sanbancho,Chqoda-h, Tokyo 102-8357, Japan

‘Department of Physics a.ud Chemistry, Faculty of Engineering, Fukui University of Technology, 3-6-1, Gakuen, Fukui910 -8505, Japan

ABSTRACT Polysacchde electrolyte aqueous solutions were annealed in the sol state before gelatian The gel-sol transition temperature (TJ was determined by the f a h g ball method (FBM).

It was found that

TB%shifted to the high temperature

side after

annedng, and the equilibrium value increased with the increase of the concentration of the solution and the annealing temperature.

% fact suggests that the structure whch

induces the gelation is formed when the system is armealed in the sol state. By differential scanning calorimetry (DSC), the melting enthalpy ( A H,) of the system was measured

A H, increased in the initial stage of anneahg, showing a varied osdation

pattern and then approached a constant value.

The rate of the change of A H, increased

with the increase of the annealing tempcratnre. However, an obvious dependency on the concentration was not observed in the concentration region where the measurements were camed out The anomalous behaviour of water was interpreted as the adsorption and desorption of water restrained by polysaccharide molecular chains. It is thought that the

molecular assemblies of polysaccharide chains dissociate in the above process, and following this the system was subsequently homogeneous. The phenomenological equation describing the anomalous behaviour of water was derived Temperature and concentration dependency of the structure formation in the
146

Production and use of biocompatible materials

INTRODUCTION

In our previous studies [I-51, it was found that xanthan and hyaluromc acid form hydrogels by annealing the aqueous solutions in the sol state and subsequent cooling (annealing and subsequent coohg induced gelation), although both saccharides are known as non-gelling polysaccharides [6-161.

F a h g ball method (FBM)

measurements were carried out to determine the gel-sol transition temperature (TJ

as a

function of anneahng time and it was found that Tgsincreases with the increase of anneahng time. From the viscoelastic measurements, it was revealed that the storage modulus (G’) of gels obtained from the solution annealed at 40 “c for various anneahng times increases with the increase of anneahng time [3, 171. Annealing the solution in the sol state was also effective for the gelation of gelling polysaccharides, such as gellan [ 18,

191. Further, in annealing and subsequent cooling induced gelation, the change of the enthalpy of mlting ( A H,) per 1 mg of water in systems was measured by differential scanning calorimetry (DSC).

Anomalous behaviour of water, i. e. oscillatory change of

A H, showing an initial increase in the early stages of the annealing and an approach to a constant value [2. 4, 51, was observed These findings suggested that the structure which enables the system to form gels is formed by anneahng. The anomalous behaviour of water is interpreted as the adsorption and desorption of water in polysaccharide chains. It is thought that polysaccharidemolecular assemblies dtssociate, and the system become homogeneous. Based on the experimental results, a phenomenological equation describing the anomalous behaviour of water was derived[3-

51. BnidtZ + (k, + &)dn/dt+ k, a nls= k, (11%,is

(1)

In this study, in order to investigate the effect of annealing conditions in detail on the structure formation of polysaccharide aqueous solutions in the annealing process, the falltng ball method (FBM) and differential scanning calorimetry (DSC) measurements were carried out

From the results of FBM and DSC, the temperature and concentration

dependency of the structureformation in the annealing process wiU be discussed.

EXPERIMENTAL S am pl e and sample preparation

Xanthan gum (XA) powder provided by Mitsubislu Chemical Co. and hyduronic

Temperature and concentration dependency

147

acid (HA) powder provided by Kibun Food Chemical Co. Ltd were used without further punfication The molecular weight of XA and HA were 1O5 and 2.2X 106, respectively. The limiting viscosity of HA and the content of protein contained in HA were 32 dl/g and 0.06%, respectively. Aqueous solutions of these polysaccharides were prepared using pure water provided by Wako Pure Chemical Industries Ltd and all glassware was sterilizedbefore use.

Falling ball method (FBM) A desired amount of XA or HA powder and 5 ml of pure water were sealed into a glass tube,which was ca. 35 cm in length and 10 mm in diameter, with a stirrer magnet After maintaining at room temperature for 3 days with occasional stirring, the sample tube was annealed at desired annealing temperatures for a predetermined anneahg time. Then, it was immersed in a thermostat at 5 ‘c for 1 day (2-3 days) for XA (HA) system to make a gel. After thus the glass tube was opened and a steel ball of ca. 0.5 mg weight with 0.8 mm darneter was inserted into the gel. The gel in the glass tube was heated from 5 “c to a temperature higher than the gel-sol transition temperature at a rate of ca. 0.5 “C imin in the thermostat. The height of the steel ball was measured by a cathetometer and

recorded The gel-sol transition temperature was determined as the temperature at whch the steel ball started to fall in the geL Details of the method to determine gel-sol transition temperature are described in our previous papers[21.

Differential scanning calorimetry (DSC) A S&o DSC-200 was used for all the thermal .analysis. Aqueous solutions weighing ca. 5 mg, which were annealed at a desired annealing temperature for a definite time, were cooled to -70 “C at 10 “Chin,and then the enthalpy of meltmg A H, per 1 mg of water in the system was measured in the heating pro~xssup to 55

“C at 10 “Chin

by DSC.

RESULTS A N D DISCUSSION Falling ball method (FBM) In XA system;, solutions were heterogeneous when prepared at room temperature. However, the solutions thus obtained were thermodynamically stable, and no precipitate was observed even though the solutions were kept in a refrigerator (5 “c) for more than one week.

When the solution was maintained at 40°C for a day, it was transformed into

148

Production and use of biocompatible materials

oa

06

Y

Y

Annealing Time (hr)

Annealing Time (hr) ~

~

--

_

_

Figure 2 Annealing time dependence of Figure 1 Annealing time dependence of Ts.s of 1 wt% X.4 gels obtained from Tg-sof XA gels obtained from solutions with concentration aqueous solutions annealed at temperature annealed at T = 40 c = 1 (a),2 (0) and 3 (A) wt%. T = 40 (a),50 (n)and 60 (A) “C.

“c

a homogeneous solution and by subsequent cooling at 5°C a firm gel was obtained.

Figure 1 shows the anneahng tune dependence of the gel-sol transition temperature (Tg.J measured by FBM of 1 wt% XA gels obtained from aqueous solutions annealed at temperature T = 40, 50 and 60°C. As shown in Figure 1, Tgs of the XA system gradually increased in the initial 10 hrs with increasing annealing time and then approached a constant value. At a constant annealing time, T,, increased with the increase of annealing temperature. Figure 2 shows the FBM raults for XA gels obtained from solutions annealed at T = 40 “c with concentration c = 1, 2 and 3 wt%. At a constant annealing time, Tgs increased with the increase of the concentration

On the other hand in HA system,

solutions prepared at room temperature were optically homogeneous and clear. FBM measurements of 1, 2, 3 and 4 wt% solutions were carried out Gelation was observed for solutions with concentrationlarger than3 wt% [5]. Figure 3 shows the anneahng tune dependence of Tgdmeasured by FBM of 4 wt% HA gels obtained from solutions annealed at 50 and 60 “C. Sirmtar to XA systems, Tgs gradually increased in the initial stage. However, Tg, decreased in the latter stage. It is apparent that the behaviour of T,* is Mferent from that of XA systems when Figure 3 is compared with Figures 1 and 2. Since HA is a thermolabile polysaccharide, the decrease of Tg, at long annealmg times seems to be due to t he scission of HA chains because of the annealing at relatively hgh temperature. As shown in Figure 1, such a decrease of Tgs is also seen in the XA system annealed at high temperahue T = 60 “c. Therefore, it can be said that the annealing time depmdence of Tg.sis essentially the same

Temperature and concentration dependency

Annealing Time

or)

Figure 3 Annealing time dependence of T, of 4 wt% HA gels obtained from solutions annealed at 50 ( 0 ) and 60 (0) "C.

149

Annealing Time @r) Figure 4 Annealing time dependenceof Tg.,of HA gels obtained from solutions annealed at 60 "c with c = 3 ( 0 )and 4

(0) wt%.

as that of HA systems although temperature and time do not overlap. At a constant anneakng time, TB, increased with the increase of annealing temperature. shows the FBM results for HA gels obtained from solutions annealed at 60

a=

Figure 4 with c =

3 and 4 wt%. At the initial stage of annealing time shorter than 10 hrs., T,, increased with the increase of the concentration FBM measurements revealed that T,, gradually increased with increasing anneahg time and approached a constant value. As shown in our previous papers, these experimentalfacts are interpreted as the results of a structlnal change, which enables the systems to form gels, during annealing of these polysaccllaride aqueous solutions [ 1-5, 171. Differential scanning calorimetry (DSC) Water molecules in polysaccharide electrolyte / water systems are categorized into three kinds of water, i.e. non-freezing water, freezing bound water and free water. Non-freezing water is tightly bound to polysaccharide electrolytemolecules, and therefore

it can not crystallize. electrolyte molecules.

Freezing bound water is weakly bound to pdysaccharide Consequently, it can crystahze, but the melting temperature is

Lower than that of bulk water. Free water is notlnfluenced by polysaccharide elecaolyte molecules, and the physical properties of free water are the same as those of bulk water. It is known that bound water plays an importantrole in forming the junction zones in gel

150

Production and use of biocompatible materials

forming polysaccharides[20-23].

Non-freezing water and freezing bound water are

included in bound water. In order to investigate the content of bound water in system, the enthalpy of melting A H, per 1 mg of water in systems was measured by DSC. In both exothermic and endothermic curves of water measured in cooling and heatmg processes, only a single peak was observed at around T = 0 "C, and therefore, the existence of freezing bound water was not confirmed. The observed value of A H, was smaller than A H, of bulk water and this fact shows that a part of the water in the systems behaves as non-freezing water. Annealing tune dependence of the amount of non-freezing water W,, (mg/mg) per 1 mg of water in the systems were calculated according to the equation

W,, (mg/mg) = (333 - A H, (mj/mg)) / 333

(6)

by assuming A Hm for bulk water is 333 mj/mg. Figure 5 shows the anneahg time

dependence of W,, of 1 wt% XA sols annealed at 40, 50 and 60°C.

W,, measured by DSC showed osdlatory change during anneahg passing through the minimum and maximum, and then approached a constant value. The final value of W,, seemed to be slightly larger than the initial value. From Figure 5, it is clearly shown that the rate of change of non-freezmg water increases with the increase of the annealing temperature. Figure 6 shows the annealing time dependence of W,, of XA sols annealed at T = 40 "c with concentration 1 and 2 wt%. Obvious dependency of the rate of the change of nonfreezing water on the concentration is not seen Figure 7 shows the annealmg rime dependence of W,, of 4 wt% HA sols annealed at T = 40, 50 and 60 "c, and Figure 8 0.1 2

0.08

<

% 0.08

9

0.06

2

0.04

-0.02

-5

0

5

10

15

20

25

30

Annealing time(hr)

0.02 -5

0

S

10

15

20

25

30

Annealing time(hr)

Figure 6 Annealing time dependence of Figure 5 Annealing time dependence of Wn,of 1 wt% XA sols annealed at 40 (0), Wn, of XA sols annealed at T = 40 with concentration 1 (0) and 2 ( 0 )wt%. 50 ( 0 )and 60 (A)"C. W, is the

"c

constant introduced in order to shift the data points along the ordinate. The 0.05 for values of W, are 0 for 40 50

"c and 0.10 for 60 "c.

"c,

Temperature and concentration dependency

15 I

shows the anneahg time dependence of Waf of 3 and 4 wt% HA sols annealed at T = 60 "c. A s d a r tendency to the results observed in XA system is also observed for both figures. Structure Formation i n Annealing Process In our previous studies, the structural change in the annealing process was discussed and interpreted as described below. XA and HA molecules in the pre-annealed samples

form molecular assemblies. In the initial stage of the anneahg, the assemblies of polysaccharide molecules hsociate by desorbing water molecules. The decrease of non-freezing water observed in the initial stage of the annealing can be explained as the results of such dissociation of the assemblies of polysacchande molecules. Subsequently, the solution becomes homogeneous by the Mfusion of polysaccharide molecules removed from the assemblies. After the homogenization or equilibraticm of the system is attained, XA and HA molecules form a junction structure which enables the system to form gels by adsorbing the non-freezing water. On the basis of the above physical process, eq. (1) was derived on the following three assumptions [4, 51. (i) Total number of repeating units of polysaccharide

molecules, N,can be divided into three numbers, i. e. the number of repeating units whch

0.25 n

M

-23

0.2

t t

E

0.1 5

0.1

.

0.05

t L

0

t TO

20

30 40

50

60

70

Annealing timefir)

Figure 7 Annealing time dependence of Wn, of 4 wt% HA sols annealed at T = 40 (V), 50 (@) and 60 (0)

"c.

i

Annealing time(hr)

Figure 8 rhealing time dependence of (0) and 4 (@) wt% HA sols annealed at T = 60

Wnf of 3

"c.

I52

Production and use of biocompatible materials

adsorbed water molecules No, those which do not adsorbed water molecules N,and those being incapable of adsorbing water molecules NS.

(ii) Time evolution of the number of water molecules n adsorbed in polysaccharide molecular assemblies is expected to obey

Here, it is assumed that s-repeating units are needed to adsorb one water molecule and the relations No

sn and N u = s q hold.

The constants k, (>O) and & (>O) represent the

(5) N - N, or No + Nu repeating units is expected to change with annealing &me t by the &sociation of polysaccharide molecular assemblies and subsequent homogemzation. We assume the simple relation to describe the time evolution of N - N,.

rate constants for adsorption and desorption of water molecules.

d(N - NJdt = - a! (n q,(T)) ~

Here,

a! (>O)

(4)

is the phenomenological constant, and n,(T) the equilibrium value of n at

the annealing temperature T.

The eq. (1) is derived from equations (Z), (3) and (4).

The solution of eq. (1 ) n=Cexp(-y t)cos(w t + 6 ) + n e ,

(5)

describes well the anomalousbehaviour of non-freezing water [4, 51. In h s study, Tg-s measured by FBM increased with the increase of the anneahg temperature and the concentration

On the other hand, the rate of change of non-freez&g

water increased with the increase of the annealing temperature, but the obvious dependency of the change of non-freezing water on the concentration was not observed in the measured concentration region. From the above physical pictures and the experimental results, the structure formed

in the annealing process is considered as follows. At low anneJng temperature, dissoaation of the assemblies of polysaccharide molecules is incomplete and the homogeneity partly remains in the system However, at h g h annealing temperatures, homogeneity m the systems almost disappears. The annealing temperature difference of the gel-sol transition temperature s e a m to be due to the ddference of the shucture in the systems or the Uference of the attainment of the equilibration of systems.

As for the

Temperature and concentration dependency

I53

concentration dependency, it is very difficult to discuss the concentration dependency because of the narrow region of measured concentrationsince it is generally impossible to prepare completely homogeneous aqueous solutions of plysaccharide electrolyte with concentration larger than 10 wt%. However, the dependency on concentration should be strong if the concentration fluctuation which causes the structural change in the

annealing process is based on the translational &fusion of plysaccharide electrolyte chains, i.e. the reptational motion of polysacchande electrolyte chams in concentrated aqueous solmons. Therefore, it may be concluded that the structural change in annealing process is caused by the molecular motion in the microscopic domain which is smaller than the dimension of plysaccharide electrolyte chains.

ACKNOWLEDGEMENTS This work was supported by Grant-in-Aid for COE Research (10CE2003) and that of (C) (No. 11650925)by the Ml~llstryof Education, Science and Culture of Japan.

REFERENCES 1) F. X. Qum,T. Hatakeyama, M. Takahashi and H. Hatakeyama, ‘The effect of annealing on the conformationalproperties of xanthan gum hydrogels’ , Polymer, 1994,

35, 1248-1252. 2) T Yoshida, M. Takahashi, T. Hatakeyama and H. Hatakeyama, ‘Annegelationof xanthadwater systems’ , Polymer, 1998, 39. 1119-1122.

induced

3) T. Yoshida, M. Takahash T Iwanami R. Tanaka, T Hatakeyama and H. Hatakeyama, In: StdsticrJ Physics. Experiments, Theotiesa d Computer Simulcrions, M Tokuyama and1 Oppenheim(eds.), 1998, World Scientific, Singapore, pp61. 4) M. Takahash, T. Hatakeyama and H. Hatakeyam, ‘phenomenological theory describing the behaviour of non-freezing water in structure formation process of plysaccharide aqueous solutions’ , Carbohydr. Polym., 2000, 41, 91-95. 5) J. Fujwara, M. Takahashi, T. Hatakeyama and H. Hatakeyama, ‘Gelation of HyaluronicAcid by Annealing’ , to be publishedin Polym. International. 6 ) S. B. Ross-Murphy, V. J. Morris and E. R Morns, ‘Molecular Viscoelasticity of XanthanPolysaccharide’ , Faraday Symposia of thechemical Society, 1983, 18, 115129. 7) K. Nishmari, ‘Gel Formation of Natural Polymers’ , Sen-i To Kogyo, 1993, 49(3), 84-93. 8) G. Cuveir and B. Launary, Carbohydr. Polym, 1986, 6,321. 9) R. K. Richardson and S . B. Ross-Murphy, Intenlahod Journal of Biological

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Macromolecules, 1987,9 , 257. 10) M M a s , M. Rmaudo, M Kmpper and J. L Shuppiser, ‘Flow and Viscoelastic Properties of Xanthan Gum Solutions’

,

Macromolecules, 1990,23, 2506-251 1.

11) P. A. Williams, S. M. Clegg, D. H. Day, K. Nishuran and G. 0. Phillips, In:

”

Food Polymers, Gels, and Colloids,” E. hckurson (eds.), RSC Publication. Cambridge,

1991,pp. 339-348. 12)P. A. Williams, D. H. Day, M J. Langdon, G. 0.Phillips and K. Nishinari, ‘Synergistic interaction of xanthan gum with glucornannans and galactomannans’ , Food Hydrocolloids, 199 1,4, 489-493. 13) P. A. William, P. Annable, G. 0. Phdlips and K. Nishinari, In: Food Hydrocolloids: Stmdure, Propetties cprd Functions, K. Nishinari and E. Doi (eds.), 1994,F’lenumF’resss, New York, pp.435-449.

14)S. C. De Srnedt, P. Bckeyser, V. Fhbitsch, A. Lauers, J. Demeester, Biorheology, 1993,30, 31. 15) T. Yanalu and T. Yamaguchi, ‘Temporary Network Formation of Hyaluronate Under a Physiological Condition 1. Molecular Weight Dependence’ , Biopolymers,

1990,30, 415-425. 16)J. E. Scott, C. CUmnnngs, A. Brass and Y . Chen, ‘Secondary and tertiary structures of hyaluronan in aqueous solution investigated by rotary shadowing-electron microscopy and computer simulation’ , Biochem J. , 1991,274,699-705.

17) J. Fujiwara, T. Iwanami, M. Takahash, R. Tanaka, T. Hatakeyama and H. Hatakeyama, ‘Structural Change of Xanthan Gum Association in Aqueous Solutions’ to be published in Thennochimica Acta.

18) F. X. Quinn, T. Hatakeyama, H. Yoshida, M. Takahashi andH. Hatakeyama, ‘The Conformational Properties of GeUan Gum Hydrogels’ , Polymer Gels and Networks,

1993,1 , 93-1 14. 19) H. Yoshda and M. Takahash, ‘Structural change of gellan hydrogel induced by annealing’ , Food Hydrocolloids, 1993,7,387-396. 20) T. Hatakeyama, K. Nakamura and H. Hatakeyama, ‘Determination of Bound Water Contents Adsorbed on Polymers by Dfferential Scanrung Calorimetry’ , Netsusokutei, 1979,6, 50-52. 21) K. Nakamura, T. Hatakeyama and H. Hatakeyama, ‘Studies on Bound Water of Cellulose by Differential Scanning Calorimetry’ , Text. Res. J _,1981,5 1, 607-613. 22) K. Nakarnura, T. Hatakeyama and H. Hatakeyama, ‘Relationship between Hydrogen Bonding and Sorbed Water in Styrene-Hydroxystyrene Copolymers’ , K o h b w h Ronbunshu, 1982,33,55-58, 23) K.Nakamura, T. Hatakeyama and H. Hatakeyama, ‘Effect of Water on Polymers’ Sen-i Gakkaishi, 1985,41, 369-378.

,

HYDROLYSED LIGNIN. STRUCTURE AND PERSPECTIVES OF TRANSFORMATION INTO LOW MOLECULAR PRODUCTS. M.Ja. Zarubinl, S.R. Alekseevl, S.M. Krutovl. 1

Department of Chemical Engineering, St. Petersburg Forestry Technical Academy 194021 St. Peterburg, Russia.

ABSTRACT Because of the large volume of wasty produced by the Russian wood hydrolysis industry the utilization of hydrolized lignin has emerged as a scientific problem. As of yet, this problem has not been solved due to the lack of detailed structural knowledge. Based on literature data and newly emerging experimental data, a hypothetical structure for hydrolysed lignin i s proposed which opens new opportunities for its practical use.

INTRODUCTION In Russia, wood hydrolysis is conducted by cooking wood in 0.5% HzS04 at I800 C. The hydrolysis process of hydrocarbons has been mastered sufficiently to consistently produce monomeric fragments which are quantitatively fermented to alcohol 111. During hydrolysis, the number of C-C bonds has been shown to increase. They are formed on account of the splitting of mainly a-n base groups, with carbocations forming benzyl type matemals and secondary condensation products [21. As a result the structure of hydrolysed lignin is more condensed in comparison with native lignin. The proposed reaction figure for the conversion of various lignin structure units, which contain a-n base linkages, is presented below (figure 1):

Figure 1. Proposed convertion figures for various lignin structural units containing a-n base groups.

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OC H3

r-

OH [OR1

Figure 1. (continued).

H+

Hydrolysed lignin

H

F

T

H3C

HOH2C-HC-HC'

-

C

0

Figure I . (continued). The last two steps are dominent in strongly acidic environments and, hence, are not produced during wood hydrolysis conducted at 0.50/0H S 0 4 . It is well-known from literature data [3,4], and as it is seen in figure 1, etheral bonds in lignin are well preserved during acid hydrolysis: mainly p-ether, 4-0-5 biphenylic, a-0-y in the pynoresinol structures and also a-0-4 bonds in phenylcumaran structures. Splitting of the final reaction product does not result in lignin like fragments. Rather the most likely fate for hydrolysed lignin is to be split into low molecular weight products at the p - 0 4 bonds. The splitting mechanism of these bonds is well-known for model compounds in lignin chemistry 151. We believe that hydrolysed lignin P-ether bonds are split by alkali via the proposed figure 2:

157

T H

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Figure 2. The P-ether bonds splitting in technical hydrolysed lignin. The P-ether bonds split in the presence of ZnClz are believed to proceed via the proposed mechanism shown in figure 3 161:

H [OR1

[OR1

[OR1

bH [OR1

Figure 3 The P-ether bonds splitting in the presence of ZnCh The proposed mechanism agrees with the results of experiments conducted on hydrolysed lignin fragments under basic conditions in alkaline solutions at high temperatures, and under acidic conditions in the presence of ZnCb in 00 C C H C O O H solutions.

EXPERIMENTAL Treatments of hydrolysed lignin samples (Klason lignin 72%) were conducted in 5% NaOH at 170 t o 180° C for 180 min. The yield of substances dissolved in alkali was more than 90%. Increases in the processing time did not result in increases in the yield of dissolved substances. Catalysts introduction in the form of soft-bases (HS- and anthraquinone) resulted in a reduced yield 161.

Hydrolysed lignin

159

The yield of dissolved substances for reactions conducted in the presence of ZnClz was found to be less than 10%. This can be explained by the steric hindrance experienced ZnClz in approaching the P-ether bond [6]. Further studies are planned to determine the influence of increasing concentrations of ZnClz and temperature treatments on the yield of soluble products (experiments will be conducted in autoclaves at increased boiling temperat ures of CH3COOH). The basis of calssification of alkali dissolved reaction products is shown in figure 4:

1 HYDROLYSED LIGNIN ethanol-benzene extract ion

(volatile fraction

-l UNDISSOLVED FRACTION

1

soluble fraction

II

soluble fraction

DISSOLVED FRACTION

I

HCl addition to p H 1

to p H 7 and evaporation separation to

C,H,OH (tainled with HCI) extraclion

r - i WATER-SOLUBLE FRACTION

Figure 4. The classification of alkali dissolved reaction products.

RESULTS AND DISCUSSION As shown in figure 4, alkali dissolved reaction products were separated into the following fractions: acetone soluble, acetone insoluble, and water soluble substances. The dominant fraction was found to be the acetone soluble fraction (36.4%). According to TLC data, this fraction consists of 6 major components. By comparison of the experimental Rf values with lignin model compound Rfvalues,

I

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a similarity was found between the most polar acetone soluble compound and diisoeugenol. Six preparative chromatographical separation zones were selected. IR-spectra of separated zones indicate that these compounds were of a phenolic nature, and possessed carbonyl and alcoholic groups. This interpretation agrees with those of other researchers 171. The TLC data for the water soluble fraction (18.1%) showed that this fraction is comprised of 5 major components. Hence, 5 preparative chromatographical separation zones were selected. The most polar compound in the water soluble fraction was found to be similar to guaiacylpropanol in Rfvalue. IR- and UVspectra indicated an aromatic nature of the analyzed compounds, and the presence of hydroxyl groups. The extractive substances (15.5%) were separated to a steam-volatile fraction (0.9%), an ether soluble fraction (27.30/9), and an acetone soluble fraction (58.8Y0). As preliminary reseach showed, the steam-volatile fraction consists of monoterpenes, ether-soluble substances (resin acids and polymerisation products of terpenes), acetone-soluble substances (likely gumification products of hydrocarbons). The extractives fraction, according to the TLC data, consists of 5 major comounds, or zones. Analysis by IR- and UV- spectra indicated the total absence of the aromatic compounds. The more polar compounds were shown to contain carbonyl groups.

CONCLUSION The results of this research showed the possibility of the fragmentation of hydrolysed lignin. The splitting of hydrolysed lignin under these conditions was found to proceed to a great extent. In the future, the products of the splitting reaction will be resolved and analyzed t o determine their molecular structures.

REFERENCES 1. U I Cholkin, m e hydrolysis manufactures technology, Forest industry, Moscow, 1989.

2. M Ja Zarubin, M F Kirushina, V V Troitskiy, K A Savov, V N Oparin, M I Ermakova, The lignin acid-base nature role in spite of the wood chemical process, Wood Chemistry, 1983, (9,3-24. 3. J Gierer, Svensk. Papperstidor. 1970, (73), 57 1. 4. J Gierer, I Noren, Acra Chem. Scand., 1976, (1962), ( I 6), 171 3. 5. K V Sarkanen, K H Ludvig, Lignins: structure, properties, reactions, (Translated into Russian by A.V. Obolenskaya et al.), Wood Industry, Moscow, 1975. 6. A Nikandrov, R M Sevillano, G Mortha, D Robert, M Ya Zaroubin, Lachenal

D, Characterisation of residual lignins from oak kraft pulps isolated by acetic acid and ZnClz.’ Conference Proceedings: Advances in Lignocellulosics Chemistry for Ecologically Friendly Pulping and Bleaching Technologies. August 30 - September 2, 1998, University of Aveiro, Portugal, 165-169. 7. S R Alekseev, Research into the structure of technical hydrolysed lignin, Masters Thesis, St. Petersburg Forestry Technical Academy, St. Petersburg, Russia. 1998. 8. Y L Stephen, Methods in Lignin Chemistry, C.W. Dence (eds), 1992, SpringerVerlag, Berlin.

PRODUCTS OF LIGNIN MODIFICATION: PROMISING ADSORBENTS OF TOXIC SUBSTANCES Tatiana Dizhbite(*), Anna Kizima, Galina Rossinskaya, Vilhelmina Jurkjane & Galina Telysheva Latvian State Institute of Wood Chemistry, 27 Dzerbenes St., Riga, LV-1006, Latvia

ABSTRACT In the present work, the interactions of water insoluble lignins (haft lignin and hydrolysis lignin) with oppositely charged water-soluble surfactants, quaternary ammonium salts (QAS), were investigated with the aim of producing new materials with enhanced adsorption and antiseptic properties. It has been shown, that lignin provides a matrix for holding alkylammonium cations owing to strong coluombic, complexing and hydrophobic interactions. Bonds formed are stable in aqueous and aqueous-basic media in the presence of low-molecular electrolytes. Using ESR (spin-probe technique), X-ray analysis and water vapour sorption it has been found that the main impact of modifications under investigation is an increase in hydrophobicity of lignin and its mesoporosity. Adsorbents obtained are characterized with a high adsorption capacity towards phenols, namely twice as large as that of a standard commercial active carbon and close to the well known polymer adsorbent Amberlite XAD-4.

INTRODUCTION The use of polymeric adsorbents is a promising option for the removal and recovery of organic contaminants from polluted water. It is known that the relatively low solubility and the presence of significant amounts of different oxygen-containing groups in lignins, the matrix of which is characterized with a cross-linked structure are prerequisites for sorption activity by mechanisms such as physical adsorption, hydrogen bonding, co-ordination and covalent linking, and acidicbasic interactions. Hydrolysis lignins, the major by-product of the conversion of lignocellulosic biomass to ethanol, were proposed as sorbents of various organic dyes, phenol from the wastewater of phenol-formaldehyde resins production, cleaning and discolouring of wastewater and enterosorbents for medicine and veterinary Regarding the porous structure, hydrolysis lignins can be characterized as sorbents whose porous structure is not enough developed. According to mercury porometry data the hydrolysis lignins contain pores with radii from 3.3 up to 3.6*104 nm and the maximal share belongs to pores with radii of 500-5000 nm. The total volume of hydrolysis lignin pores defined by the benzene vapours sorption, was found to be 1.7 cm3/g, and specific surface 3.7 m21g. The specific surface of hydrolysis lignins, found using nitrogen sorption, was 5-8m2/g '. It has been demonstrated that, among different methods increasing sorption activity of lignins towards organic compounds/contaminants,modification by organic cations is an easy and effective technique '. Use of quaternary ammonium salts (QAS) for

-

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modification enhances sorption activity of both insoluble lignins isolated from plant tissue and lignins in lignocellulose complexes. Besides, application of quaternary ammonium salts which exhibit the high bacteriostatichactericide effects there is also an opportunity to design sorbents with antimicrobial properties on the basis of biomass processing wastes. The main objective of the present work was an investigation of the regularities of heterophase interaction of lignins with quaternary ammonium salts differing by the structure of organic moieties, and assessment of the change in the lignin structure aimed at obtaining sorbents of high efficiency towards phenols which are wide spread contaminants. MATERIALS & METHODS Lignins Hydrolysis lignins: HL70 - commercially available lignin obtained by dilute sulhric acid hydrolysis, Klason lignin content 70%, carboxyl groups content 1.5%, phenolic hydroxyl groups content 5.0%; HL52 - lignin obtained in a pilot plant by the combination of diluted sulfbric acid hydrolysis and steam explosion procedure, Klason lignin content 52%, carboxyl groups content 0.4%, phenolic hydroxyl groups content 1.7%; HL98 - lignin obtained by concentrated hydrochloric acid hydrolysis in a pilot plant, Klason lignin content 98%, carboxyl groups content 4.3%, phenolic hydroxyl groups content 4.4%, Kraft lignin KL89 - commercially available lignin, Klason lignin content 78%, carboxyl groups content 3.2'0, phenolic hydroxyl groups content 4.6%. Quaternary ammonium salts (QAS) Hexadecyl trimethylammonium bromide (HDTMA-Br), trimethylbenzylammoniium bromide (TMBA-Br) and dimethylethylphenylarnmoniumbromide (DMEPhA-Br) were obtained from Sigma. Methods The modification procedure was carried out as described in 4, by stirring the lignin suspension in the QAS aqueous solution varying the duration, pH of the aqueous medium and temperature of interaction (6-120 h, pH 4-10 and 20-60°C). The degree of modification, a,expressed as the molar ratio of QAS cations introduced to the phenyl propane units of lignin was determined from the nitrogen content in the modified lignins. The nitrogen content in the samples was determined by the Kheldal method Klason lignin content was determined by the TAPPI method T 222 om-88. The sorption capacity of lignins and products obtained for phenol was examined under static conditions at 22-23OC from aqueous solutions of 10 g/Lconcentration. To characterize hydrophobic/hydrophilic properties of lignins and their derivatives, water vapour sorption isotherms were measured at 20-2 1"C. X-ray analysis and ESR spin probe measurements were used for monitoring changes in

Products of lignin modification

163

lignin microstructure as the result of modification. X-ray diagrams of lignin samples (pellets) were recorded within the range of 28 values fiom 6 to 50” using difiactometer DRON-2 (C& radiation, Ni-filter). An average distance, d, between planes of mesomorphous microregions was estimated as described in A spin probe, 2,2,6,6-tetramethylpiperidine-l-oxyl,was introduced into the lignin samples from gas phase ESR spectra were recorded at 20fl”C on an ES1006 X-band spectrometer using the following instrumental parameters: scan width, 125 G, time constant, 0.25 s, scan time, 4 min, modulation amplitude, 0.33 G, microwave power, 10 mW, modulation fiequence, 100 Hz. The rotational correlation time of the probe was estimated according the method

’.

‘,

’.

RESULTS & DISCUSSION Analytical data obtained have shown that electrostatic interaction and formation of heteropolar bonds involving carboxyl and phenolic (at pH>10) groups of lignin are the main type of interactions in the heterogeneous system lignin - aqueous QAS:

(Lignin)-COOH + R’R”.R”’3-. N H d 4 (Lignin)-COOR’R’’,,R”’3.. N’ + HHal (Lignin)- OH,h, + R’R”.R”’3.. NHal-+ (Lignin)-COR’R”.R’”3.. N’ + Hhal It has been established that at the reaction temperature 20”C, the amount of QAS coupled to lignin does not exceed 0.6 mole per 1 mole of phenyl propane units of lignin even if QAS is used in sufficient surplus. The pH change fiom 7.5 to 10, extension of the reaction duration to 120 h and temperature elevation within the interval 20-60°C does not influence the lignin modification degree (Fig. 1). The relationship between degree of modification and reaction time can be linearized in the coordinates of the Erofeev-Kolmogorov equation:

a=l-e

kt”

8

-__

0

I 0.1

0.3

0.5

0.7

0 .§

c , st.

Figure 1. Kinetic of interaction between hydrolysis lignin (HL98) and hexadecyltrimethylammonium bromide (HDTMA-Br). Reaction temperature: (1) 20°C, (2) 3OoC, (3) 40°C. pH=lO. EA=50.1 kJ*mole-’. Topolunetic parameters: (1) n=1.13, K=O. 12 h-’; (2) n=l. 10, K=O. 16 h’; (3) n=0.95, K=0.30 K’.

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Production and use of biocompatible materials

Topokinetic parameters of the process, n and k, were determined on the basis of the equation 111, while the rate constant K=nk”” and effective energy of activation were calculated using Sakovich and Arhenius equations, respectively In spite of their formal character, the results obtained (Fig. 1) revealed that the process is chemically controlledg. Based on the results obtained, the formation of a new insoluble lignin-quaternary ammonium salt can be described as a frontally extended ion exchange reaction. The organic cation transfer into the lignin matrix occurs via interchange between an already formed lignin-organic cation complex and free fragments of the lignin network, i.e. by “jumping” of the cation from one lignin segment to another localized further from the surface. The bonds between lignin and quaternary ammonium cations are rather stable to hydrolysis in water-alkaline medium containing 0.1 M NaCl (pH=lO): after a 12-h hydrolysis the content of quaternary ammonium cations in the products of reaction between TMBA-Br, DMEPhA-Br and HDTMA-Br was 93.5, 94 and 96% (in terms of the cation content in the products prior to the hydrolysis), respectively. The stability of lignin quaternary ammonium derivatives is determined not only by the electrostatic interaction but also by forming other types of bonds between the coreagents. The chemical structures of QAS under investigation are characterized by the presence of hydrophobic organic radicals, which enable QAS molecules to associate in aqueous solutions and immobilizing them in the Iignin matrix by hydrophobic interaction. Besides, formation of charge transfer complexes between lignin aromatic moieties and quaternary ammonium cations reveals itself in the 2 - 3 fold increase in the content of stable paramagnetic centres after lignin modification The stability to hydrolysis of lignin quaternary ammonium derivatives may result in a formation of a “barrier layer” (after establishing a certain polyaniodcation ratio in the reaction product), which hinders further penetration of new QAS molecules and formation of the product with an equimolar ratio of polyanion and cation units. Analysis of water vapour isotherms for initial and modified hydrolysis lignins (Fig. 2) gave evidence of increasing hydrophobicity of the lignin surface as a result of modification. Parameters of water adsorption by lignins decreased after modification: the specific water accessible surface by 8.5%, monolayer capacity by 19.7%, surface concentration of hydrophilic groups by 12.3% and BET equation energy constant by 37%, indicating that the energy of water interaction with sorption centres of the lignin surface diminished. The amount of inclusion water during desorption also decreased (the sharp narrowing of the hysteresis loop in the Fig. 2, b). X-ray analysis showed, that the lignin supramolecular structure changed significantly as a result of transport of voluminous QAS cations into the lignin matrix. The X-ray diagrams for both the initial lignins and their modified products showed one difisive reflex in the 28 20-21”. It has been shown ’,lo, that this reflex is attributed to the diffusion of the planes, formed by benzene rings. As a result of the modification, the average distance between the plains increased from 0.42 nm for lignin (HL98) up to 0.56 nm for HL98-HDTMA (a = 0.12) and sizes of the mesomorphous microregions decreased by 30%. Significant increase in the mesopore volume in the lignins after modification with QAS has been shown by the water sorption-desorption isotherms as well as ESR spin probe method. The average size of these mesopores has been estimated by the adsorption isotherms methods as - 100 - 150 A.

’.

Products of lignin modification

165

180

160 140

120 zll

100

E 6 80 60

40 20 0 0

02

OA

0.6

Od

1

0

0.2

0.6

0.8

1

PtPD

PfpD

Figure 2.

0.4

(a) (b) Adsorption-desorption isotherms of water on (a) lignin, HL98 and (b) modified lignin HL98 - HDTMA (a=O. 12).

The analysis of the ESR spectra of the modified lignln products containing the spin probe showed, that these spectra corresponded to the spin probe with relatively free movement within the microcavities. Taking into account the size of the spin probe, the average diameter of these microcavities could be estimated as 2 100 A. The sorption activity of lignins modified with QAS towards phenol, increases significantly (Fig. 3) as the result of lignin matrix hydrophobization and the changes in the lignin supramolecular structure. Efficiency of the long chain quaternary ammonium cation (HDTMA) is higher than shorter chain cations (TMBA and DMEPhA). Evidently, the more pronounced hydrophobic character of the surface of lignin-HDTMA favours adsorption of phenol from water.

KL89

HL52

HL70

HL98

Arnbdlte

BAC

m-4

Figure3. Evolution of phenol uptake onto lignins, as a result of modification - 0.20) with hexadecyltrimethylammoniumbromide (HDTMA-Br); comparison with commercial sorbents (Amberlite XAD-4 and BAC). (a=O. 18

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Production and use of biocompatible materials

In order to exceed the phenol sorption activity which has commercial birch active carbon (BAC) and obtain indices of Amberlite XAD-4, the well known high-efficiency towards phenol polymers, only a low degree of modification (a=O.14-0.17) of hydrolysis lignin with 78-98% content of Klason lignin is necessary.

CONCLUSIONS The proposed concept of modification of insoluble IigNns via heterophase interaction with cation surfactants, quaternary ammonium salts, gives the possibility of the design (synthesis) of high efficient sorbents for application in aqueous media. The modification, which is carried out under mild conditions and at low demand of a modifier, provides an increase of sorption efficiency owing to hydrophobization and development of mesoporosity as a result of quaternary ammonium cations diffusion into the lignin/lignocellulose matrix.

REFERENCES 1. M. Wayman & S. R. Parekh, Biotechnologv of Biomass Conversion, Open University Press, Milton Keyness, 1990. 2. N. A. Belyakov (ed.), Enterosorption, Center of the Sorption Technologies, Leningrad, 1991 (in Russian). 3 . V. P. Levanova, Medicine Lignin, Center of the Sorption Technologies, St.Petersburg, 1992 (in Russian). 4. T. Dizhbite, G. Zakis, A. Kizima, E. Lazareva, G. Rossinskaya, V. Jurkjane, G. Telysheva & U. Viesturs, ‘Lignin - a useful bioresource for the production of sorption-active materials’, Biores technol, 1999, 67, ( 3 ) , 22 1-28. 5. M. Ya. Ioelovich, G. M. Lebedeva, G. P. Veveris & G. M. Telysheva, ‘Study of the

supramolecular structure of organic-silicon lignin derivatives’, Khimiya drevesiny w o o d Chemistry), 1991, (I), 100-4. 6. T. Dizhbite-Scnpchenko, G. Domburg, J. Lebedev & V. Sergeeva, ‘A possibility of the spin probe method application for investigation of lignin microstructure’, Khimiya drevesiny (Wood Chemistry), 1975, (4), 75-9. 7. A. N. Kuznetsov, A. M Wasserman, A. U. Volkov, N. N. Korst, ‘Determination of rotational correlation time of nitric oyde radicals in a viscous medium’, Chem. Phys. Lett., ’, 1971, 12, (l), 103-6. 8. E N Eremin, Fundamentals of Chemical Kinetics, Vysshaya Shkola, Moscow, 1976 (in Russian). 9. G M Panchenkov and V P Lebedev, Chemical Kinetics and Catalysis, Khyrniya, Moscow, 1985 (in Russian). 10. J. Haggin, ‘Lignin in native wood tissue has ordered structure’, Chem. And Engng News, 1985, 63, (18), 33-4.

CHARACTERISATION AND ADSORPTION OF LIGNOSULPHONATES AND THEIR HYDROPHOBIZED DERIVATIVES ON CELLULOSE FIBRE AND INORGANIC FILLERS Galina Telysheva'(*), Tatiana Dizhbite', Anna Kizirna', Alexander Volpertsl & Elena Lazareva' 'Latvian State Institute of Wood Chemishy, 27 Drerbenes St., Riga, L V-1006, LATVIA 2Depar!menrof Chemism, Moscow State Universiv, Vorob 'evy goiy, Moscow, Russia

ABSTRACT The adsorption of lignosulphonates (LS) at the water-solid interface is characterized by a high value of the free energy of adsorption. The LS adsorption behaviour can be controlled by varying the LS macroion negative charge density, either changing the solution pH or increasing LS hydrophobicity, e.g. modifying with aluminum containing silicon-organic oligomers. At low concentrations,LS adsorb in a flat conformation owing to electrostatic (in the case of an oppositely charged surface) or chemisorption (in the case of identically charged surfaces) interactions. With an increase in the level of surface coverage LS macromolecules form a mobile pseudo-liquid microphase at the water-solid phase boundary. In the case of LS modified with silicon-organic oligomers, the microviscosity of the adsorption layer tends to increase. The adsorption behavior of LS and their hydrophobized derivatives at the water-solid interface corresponds to that predicted for polyelectrolyte adsorption by the self-consistent field model.

INTRODUCTION Lignosulphonates (LS), formed as a by-product of the process of cellulose manufacture, are widely used as technical surfactants with dispersing, stabilizing and adhesive abilities'32.In recent years the role of LS as auxiliary substances for papermaking and paper coating is increasing. A few types of LS based products, mainly LS purified and fractionated by ultrafiltration, now are proposed to improve coating properties and to retain fine cellulose material and fillers in the pape?&. In these cases LS adsorption on the solid components of paper composition is an important factor of their activity. Besides, LS can affect the adsorption of various auxiliary substances, e.g. rosins or starch, on fiber and filler particles during papermaking thus influencing paper strength. The majority of work concerning lignin ad~orption''~~ describe LS behavior as corresponding to the Langmuir model. Alongside that, the polylayer nature of LS adsorption on kaolin surfaces and the description of the process according to a network polylayer adsorption model have been proposed'. The present work develops our previous investigations aimed at generation of the background for purposeful alteration of the LS efficiency as auxiliary substances at different stages of papermaking'9'0. It is focused on the main features of LS adsorption onto solid components of paper composition and their relationship with characteristics of adsorption centers on solid surfaces under study.

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Production and use of biocompatible materials

MATERIALS & METHODS A commercial sodium lignosulphonate LS purified by ultrafiltration and containing less than 1% of carbohydrates as well as the products of their modification with sodium alumoethylsiloxanolate or sodium diethylalumoethylsiloxanolate(MLS) were used in the work as adsorbates. Bleached sulphate cellulose from coniferous wood (a-cellulose content 98%), kaolin, titanium dioxide (rutile) and calcium carbonate were applied as adsorbents. LS adsorption isotherms from aqueous solutions were obtained at 20fl°C and thermodynamic characteristics of adsorption as well as changes in thermodynamic functions along with increase in the level of sorbent surface coverage were calculated using the Aranovitch model of polylayer adsorption". The sorption of different organic compounds from aqueous solutions on solid surfaces under study was determined by measuring their equilibrium concentrations under conditions where the interaction between sorbate molecules could be disregarded (coverage 8<0.1). The experiments were performed in a 0.1N solution of the background electrolyte (NaC104)to overcome the forces of electrostatic origin. The concentration of the solution was 10" mol L-I, the volume was 10mL and the sorbent amount 0.1 g. After the addition of a sorbent to the solution, the system was kept for 24 h with periodic stirring. The phases were then separated by filtration and the equilibrium concentration of the organic compound was determined spectrophotometrically in the UV-region. Using the data obtained, the sorption (a) was plotted as a function of the ionization potentials ( I p ) of the sorbate. The data obtained were considered in terms of the method of resonance potentials'2. Microstructure of LS adsorption layer was characterized by a spin probe method13 using 4-benzoyloxi -2,2,6,6-tetramethylpiperidine-l-oxyl (further named "the spin probe"). When the spin probe was to be coadsorbed with LS, the probe, dissolved in methanol (a concentration of lo4 mol L-'), was added to a sorbent and methanol was removed by evaporation. LS aqueous solutions were added next, the mixtures were equilibrated for 24 h at 20fl°C. The sorbent particles were collected on a Bichner funnel, rrinsed with water, and then dried in air overnight. ESR spectra of sorbents with the adsorbed spin probe were recorded at 2O+l0C on an ES1006 X-band spectrometer using the following instrumental parameters: scan width, 125 G, time constant, 0.25 s, scan time, 4 min, modulation amplitude, 0.33 G, microwave power, 10 mW, modulation frequence, 100 Hz. The rotational correlation time (T~) of the probe was calculated from its ESR spectrum using equations derived from the theory of Ki~ e lso n ' ~ . The association of macromolecules in LS and MLS aqueous solutions was monitored by laser light scattering method using a standard system from EPIC equipped with an argon-ion laser (output power 135 mW at h=514.5 nm). Before light scattering experiments the solutions were purified with 0.5 pm pore size Millipore PTFE filter. All measurements were carried out at 20+0. 1"C. RESULTS & DISCUSSION The LS adsorption isotherm on cellulose indicates high affinity with complete adsorption of LS from dilute solutions. It also shows larger magnitudes of LS adsorption, in comparison with those obtained for inorganic solids, within the whole concentration region under investigation.

Characterisation and adsorption of lignosulphonates

169

In the case of LS adsorption on Ti02, the relationship of adsorption plateau vs. pH (ion strength is constant) has its maximum in the region of pH 4-5, where the effective pK for iignosulphonic acids is observed (Fig. 1). The shape of the experimental relationship corresponds to the theoretical one calculated according the self consistent field (SCF) modelI5 for polyelectrolyte adsorption on an oppositely charged surface. At the low values of pH the LS adsorbed amount is larger than at high pH due to a decrease of intersegmental repulsion. The presence of a maximum can be explained by the fact, that LS macroions with non-zero degree of dissociation, thus having some charge, more effectively compete with counterions to compensate the positive charge on the Ti02 surface. At these values of pH electrostatic forces between adsorbate and adsorbent are higher than intersegmental repulsion. The anionic form of LS adsorbs efficiently on the solid surfaces. At the pH 8 the kaolin surface charge density is about 6 pKl/cm2and specific adsorption of LS reaches up to 14 mg/g (about 1 mg/m2), which is a typical value for the adsorption of high molecular compounds on the oppositely charged surface. This proves that in the above-mentioned systems the LS adsorption is not governed by the electrostatic interactions only and nonelectrical contributions to the free energy of adsorption are significant. The structure and functional composition of LS provide the possibility for the sorption processes proceeding according to hydrogen bonding, coordination bond formation, ion exchange, and acid-base bonding mechanisms. Comparison of adsorption isotherms of LS samples with similar molecular masses but different polydispersity degrees has shown, that with an increase in polydispersity from 1.36 to 2.6 the isotherm becomes more rounded and adsorption monotonically increases without reaching a plateau under the conditions investigated. In the terms of SCF theory, this can be explained by the phenomenon of preferential adsorption of macromolecules with high molecular weight, i.e. adsorption fra~tionation'~. The experimental evidence of the involvement of the macromolecular associates of LS and MLS in the formation of an adsorption layer on the solid surface has been obtained. The concentration of the associates in the bulk solution decreases four times after the adsorption. Alongside with that, comparison of associates size distribution histograms shows that associates remaining in bulk solution have significantly lower hydrodynamic radii compared to those in the initial solution. ___ 3 13

wllT
~

210-

E

i 9---8---. 7~ 0

- -

-1

- ~ _ _ - _ _

2

~-

-

____

0,5 0

-

6

4

a

10

FH Figure 1.

Comparison of the LS experimental adsorbed amount r onto Ti02 (1 D ) with the theoretical Oa(2 -), as a function of solution pH.

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Production and use of biocompatible materials

Comparison of experimental values of areas occupied per adsorbed monomer (the phenyl propane unit) at the adsorption plateau level for LS with different molecular masses and MLS (Table 1) with values predicted16for horizontal and vertical projections of the phenyl propane unit (57A2and 24 A’ correspondingly) has shown, that with an increase in LS molecular mass and hydrophobicity the conformation of adsorbed polymer macromolecules becomes less flat. Table 1. Characteristics of LS and MLS adsorption on kaolin. Adsorbate LS, Mw=23000 LS, Mw=46000 MLS. M,=25000

Adsorption plateau, mg/g 11 14 18

Adsorption area, A2/monomer 55 36 30

Typical ESR spectra of the spin probe coadsorbed with LS are shown in the Figure 2. At low levels of the sorbent surfaces coverage two-component spectra were observed (Fig. 2 a). The outer peaks in these spectra, representing the spin probe with correlation time of rotational movement, T~ = 10-6-10-8s,could be attributed to flat immobilized position of the probe surrounded by the polymer molecules. The intensity of inner peaks characteristic for the spin probe having rotational movements with small correlation time, ‘tC< s, increased with growth of adsorbed LS amount. One-component spectra with rC=2*10‘”s, were observed for sorbents prepared from LS solutions above a concentration value, corresponding to the adsorption plateau (Fig. 2b). These ESR spectra represented the spin probe with relatively free movement among tails and loops of adsorbed LS molecules. This indicates a gradual decrease of the fraction of adsorbed polymer segments laying flat on the surface of adsorbent and an increase in the amount oriented into the solution loops and tails forming pseudo-liquid microphase at the adsorbent surface. Modification of LS with the silicon-organic oligomers increases the structural density of macromolecular associates (light scattering data) and, consequently, microviscosity of the adsorption layer. Thermodynamic characteristics of the adsorption process (Table 2 ) and changes in thermodynamic functions along with an increase in the level of sorbent surface coverage indicate, that solid surfaces under investigation can be estimated in the following order based on their affinity to LS: cellulose>>titaniumdioxide2kaolin>>calcium carbonate.

TOTAL S C A N WIDTH.

125 G

\t

Figure 2. ESR spectra of 4-benzoyloxi -2,2,6,6-tetramethylpiperidine-1-oxyl coadsorbed with LS on cellulose surfaces at coverage 0 = 0.2 (a) and 0 = 0.9 (b).

Characterisationand adsorption of lignosulphonates

171

Higher enthalpy values for LSMLS adsorption on the kaolin and cellulose compared to CaC03 (weaker surface negative charge) indicates specific interaction of LS with adsorptive centers on the formers. High values of the nonionic energy of the LS segment adsorption interaction with kaolin and cellulose (4.8 kT and 7.4 kT, respectively) calculated on the basis of the experimental data give evidence of possible Coulomb repulsion forces compensation. Thermodynamic characteristicsof LS and R4LS adsorption on splids. Table 2. Adsorbate

LS

MLS

Adsorbent Cellulose Kaolin Ti02 CaCO3 Cellulose Kaolin Ti02 CaCOj

-AG~~, kJ/moll 9.5 5,6 6-8 296 10,7 798 470 3,7

-m0I,

kJ/moll 21,9 14,3 16,3 10,5

28,2 19,6 123 14,4

42, kJ/moll 48,l 32,9 33,2 n.d. 51,2 34,3 25,4 n.d.

' -AGO,-AHo ((pure))values of the free Gibbs energy and adsorption enthalpy, correspondingly

'Aq,-

energy of bonding between adsorbate and adsorbent surface

The adsorption centres of the surfaces under investigation were characterized via the relationship of maximum adsorption of organic compounds vs. their ionization potentials, a=f (I,,). The results obtained suggest a specific: interaction of sulphonate and carboxyl LS groups (ionization potential 9.5 eV) with adsorption centers of kaolin and cellulose, which have a resonance potential of 9.5 eV. High content of aluminum in kaolin permits formation of surface metal-LS sr-complexes. The possibility of such an interaction is due to the resonance potential at 10.1 eV for kaolin and values close to it of the first and the second ionization potentials of the aromatic compounds relative to lignin. In the case of cellulose, the maxima on the curve of the relationship a=f (I,,) at 7.8 and 8.1 eV, which were not observed on the correlation dependencies for the nonorganic surfaces, correspond to potentials of x-ionization of some lignin model compounds, namely aromatic acids, alcohols and ketones. A significant increase in the adsorption plateau and heat and free energy of adsorption interaction as the result of LS modification by the silicon-organic oligomers indicates the possibility of regulation the adsorption behavior and structure of the adsorption layer of LS. The presence of the polyvalent metal in the silicon-organic block permits the adsorbate molecules approaching closer to the negatively charged adsorbent surface than in the case of LS. CONCLUSIONS The adsorption of the LS at the water-solid interface is characterized by high values of the non-electrical portion of the free energy of adsorption, which provide effective adsorption on the surfaces charged similarly to LS. Balance of the LS adsorption forces can be regulated by the change of the density of the negative macroion charge, variation of solution pH, or by modification, for example, by silicon containing organic oligomers. At the low concentrations in the solution, LS adsorbs in a flat conformation due to electrostatic (oppositely charged surfaces) or chemisorption (similarly charged surfaces) interactions. With an increase in the degree of coverage LS macromolecules adsorb

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Production and use of biocompatible materials

forming a mobile pseudo-liquid micro-phase. Modification of the LS by the siliconorganic oligomers increases microviscosity of this layer. Depending on the specific characteristics of the adsorbent surface, lignin adsorption occurs via ion exchange or chemisorption due to hydrogen bonding and formation of donor-acceptor complexes with participation of Ic-orbitals of the aromatic lignin structures.

REFERENCES I . G. M. Telysheva & N. I. Afanas’ev, ‘Surface-active properties of lignosulphonates aqueous solutions’, Khim. Drev., 1990 (l), 3-1 1 . 2. V. Hornof, ‘Lignosulphonates application for enhanced oil recovery”, Cell Chem Technol, 1990.24 (3), 407-15. 3. P. Sennert, Use of high molecular weight sulfonate as auxiliary dispersants for structured kaolins, US Patent, No. 4 859 246, August 1989. 4. G. Telysheva, T. Dizhbite, J. Hrol, M. Akim & E. Kurkova, ‘Application of modified lignosulphonates in paper production’, In: Int. con$ PaprFor ’93, VNIIB, St.Petersburg, 1992, 103-4. 5. F. A. Adamsky & B. J. Williams, ’Effect of new drainage, retention, and formation technology for improving production rates and runnabihty of recycled fiber cylinder machines’, Tappi J , 1996, 79 (8), 175-82. 6. G. Telysheva. T. Dizhbite & M. Akim, ‘Lignin based auxiliary substances for paper and board production’, In: Proc. 4Ih Europ. Workshop Advances in characterization andprocessing of wood, non-woody and secondaryfibers, Streza, 1996,5 18-23. 7. J. C. Le Bell, B. Bergroth, P. Stenius & B. Stenlund, ‘The adsorption of sodium lignosulphonates on kaolin’, Paperi j a Puu, 1974,56 ( 5 ) , 463-71. 8. P. Dilling & H. Eicke, “Adsorption of lignosulphonates to disperse dye substrates’, Colourage, 1990, (3), 37-47. 9. N. I. Afanas’ev, G. M. Telysheva. N. A. Makarevich & Yu. S. Hrol, ‘Adsorption of the fractionated lignosulphonates on kaolin’, Khim Drev, 1990, (2), 85-92. 10. G. Telysheva. T. Dizhbite, E. Paegle & A. Kizima, ‘The regularities of lignosulphonates behaviour on different interfaces and its alteration by purposehl modification’, In: The Chemistry and Processing of Wood and Plant Fibrous Materials, J. Kennedy, G. 0. Phillips & P. A. Williams (eds.), Woodhead, 1996, 3394. 1 1 . G. L. Aranovich, ‘The determination of adsorption heat from adsorption isotherm at infinitively little coverage’, Zhurn Fiz Khim, 1990,64 (I), 161-65. 12. Yu. Tarasevich, E. Nechaev, V. Rudenko, Z. Ivanova & B. Kats, ‘Preparation and properties of carbon-mineral sorbents’, Kolloidn Zhurn, 1995,57 (2), 240-46. 13. J. Yao & G. Strauss, ‘Adsorption of quaternary ammonium surfactants on poly(tetdluoroethy1ene) surfaces‘, Langmuir, 1991,7 (lo), 2353-57. 14. C. G. Pin, J. W a g , S. S. Shab, R. Sik & C. F. Chignell, ‘ESR spectroscopy as a probe of the morphology of hydrogels and polymer-polymer blends’, Macromolecules, 1993,26 (9), 2 159-64. 15. G. J. Fleer & J. M. H. M. Scheutjens, ‘Modeling polymer adsorption, steric stabilization and flocculation’, In: Coagulation and Flocculation, B. Bogus (ed.), Schpringer, Basel, 1994, 105-83. 16. P. Luner & U. Kempf, ‘Properties of lignin monolayers at the air-water interface’, Tappi J, 1970, 53 (1 l), 2069-76.

Part 4

Biodegradable polyu rethane-based polymers

BIODEGRADABLE AND HIGHLY RESILIENT POLYURETHANE FOAMS FROM BARK AND STARCH J-J Gel, W Zhong’, Z-R Guol, W-J I,il and K Sakai2* Laboratory of Molecular Engineering of Polymers, Department of Macrmlecuiar Science, Fudan UniversiQ Shanghai 200433. P. R . chi^ Department of Forest Products, Faculty of Agriculture. Kyushu University, Fukwka 812-8581,Jqxm

ABSTRACT Liquefaction of the Acacia meamsii bark (BK) and cornstarch (CS) has been conducted by using a solvent mixture consisting of polypropylene glycol (PPG), glycerol and sulfuric acid with a weight fmction of (9445/1) at 150 C. Solubilities of BK and CS were about 80% for 60 min and 1 W o for 20 min in the same solvent, respectively. Highly elastic or highly resilient polyurethane foams (PLIFs) for car-seat cushions have been prepared from the liquefied BK and CS without removing insoluble residue from the liquefaction mixture. About 20% insoluble residue from BK contributed remarkably to the improvement of flame resistance of the resulting PUFs. PUFs having better resilience properties were prepared using PPG of molecular weight around 4OO0, as compand with polyethylene glycol having the same hydroxyl value. PUFs were synthesized from three BKs with different tannin contents to evaluate the effect of tannin content on their performances of resilience. Both the resilience value and density of the PUFs increased with increasing BK content for all BK systems. A BK with the largest tannin content, 48.5%, provided PUFs possessing tbe best resilient property. Density and flame resistance are the important properties for commercialization, f m cost and burning safety points of view, respectively. When CS replaced partly BK, the density and compressive strength of the PUFs decreased with increasing CS proportion whereas resilience value had its maximum value when the weight ratio of CS:BK was 1:l. The PUFs were to some extent biodegradable, the average weight loss of samples buried in soil for 6 months was 15.6 wt %. Keyword: bark, starch, polyurethanefoams, biodegradability.

INTRODUCTION Polyurethane foams (PUFs) are used widely in many fields as structural, cushion, insulation, electrical, flotation and packaging materials. Much attention is paid on introducing plant sources into PUFs production. The materials prepared from biomass not only open a new and efficient way to use renewable natural sources, but also possess a great potential for bio- and photodegradability. The latter advantage is more striking in the urgent need of environmental protection.” Natural polymers containing more than one hydroxyl group in the main chain are expected to be utilird as polyols for polyurethane preparation*;(’. We have prepared PUFs with moderate strengths and biodegradab~ties from wattle tannin (WT) or the Acacia meanrrii bark (BK) and diisocyanate in the presence of synthesized polyester.CXI

176

Biodegradable polyurethane-based polymers

It was proved that WT which is the main component of BK m t e d with diisocyanate as crosslinking agent in polyurethane molecules through careful studies on model

reactionM'and that the W component improves the compressive properties of the PUFs."" However, the density of tannin PUF was higher than those of the commercially produced PUFs; this may lead to higher production cost. Recently, Nakashima et aL9) prepared low-density rigid PUFs from polyol mixtures of barks of Acacia mearmii or Crypfomeriujaponicu with polyethylene glycol (PEG) 400. Yao et al.'" repoited the method to prepare PUFs from combined liquefaction of wood and starch in a PEG 400/glycerol blended solvent using sulfuric acid as catalyst. But those methods are not suitable to prepare flexible PUFs because polyol with larger molecular weight is indispensable in preparation of the flexible PUFs. Flexible PUFs for car cushion use has attracted more and more attention recently. For this purpose PUFs need a high crosslinking density to afford a high resilient property. Component with benzene rings and flexible configurations such as diethanolamine are usually introduced into PUFs formulations as crosslinking agents to improve the compressive strength and resilience."' In this study, a new kind of effective, environmentally safe and low-cost crosslinking agent BK was introduced in the preparation of high resilient flexible PUFs. The liquefaction of BK in a solvent mixture consisting of larger molecular weight polyether polyols and glycerol with sulfuric acid as catalyst has been investigated. We also report the preparation of high resilient flexible PUFs from the obtained liquefaction mixture. The properties needed for cushion material are discussed.

EXPERIMENT Materials and chemicals BK (Acacia meamsii De Wild 80 mesh pass) and cornstarch (CS) were dried in an oven at 105@ for 24 h before use. Polypropylene glycol (PPG) (GEP-330N, Hydroxyl group value 33.5-36.5 mgKOWg, Viscosity: ~ 1 o o cP/25@) O was kindly supplied by No.3 Chemical Plant of Shanghai Gao Qiao petrochemid Co. Other chemicals used were reagent grade and obtained from commercial sources.

Liquefaction solvent and procedure Liquefaction was conducted by the acid catalyst method given by Yao et al.,"' using a liquefaction mixture consisting of PPG, glycerd and sulfuric acid with a weight fraction of (94/S/1). The solvent and the catalyst were premixed thoroughly in a three-necked flask equipped with a mechanical stirrer and a nitrogen inlet. Then the BK powder in the flask was heated to 150T within 30 minutes. The liquefaction mixture was maintained at this temperature with stimng and refluxing under a nitrogen atmosphere for 30 min and followed by adding a predetermined amount of the CS as the second biomass component at this temperature for another 20 minutes if needed. After that, the flask was cooled down to mom temperature, the excess sulfuric acid was neutdized with an equivalent amount of sodium hydroxide aqueous solution (48 wt 96). The hydroxyl value of a liquefaction mixture was determined by the method described by Yao et al.'')

Determination of the liquefaction extent of the biomass The liquefaction extent of the biomass was determined after a prescribed liquefaction

Biodegradable polyurethane foams

177

time by the diome/water binary diluent method described by Yao et d.') That is, the liquefied mixture was diluted by an adequate amount of dioxandwater (8/2), stirred with a magnetic stirrer for more than 4 h, and then vacuum filtrated through a Q/XHJ3017 filter paper. The residue was rinsed by the diluent repeatedly until a colorless filtrate was obtained, and then the residue was dried to a constant weight. The residue content of biomass was calculated by the following equation. Residue content = (weight of residudweight of total biomass) x 1 W o Preparation and characterization of PUFs

A standard formulation for PUF syntheses was as follows, unless otherwise noted Isocyanate index 1IWO;diethanolamine 0.5%; dibutyltin dilaurate 0.32%; water 0.4%; triethylenediamine 0.03%; silicone oil (Y10366) 2.0%. The definte amounts of biomass polyol, catalyst, surfactant, water, and other additives, if any, were premixed thoroughly in a paper cup with a mechanical stirrer. To this mixture, a calculated amount of tolylene diisocyanate (TDI) was added and stirred at 2400 rpm for 10-15 seconds. The mixture was then poured immediately into a 12~12x10 cm mould and was allowed to rise at room conditions. The resulting foam was removed from the mould after one hour and was allowed to cure at room temperature for one week before cutting into test specimens. The ball rebound resilience value, flammability and compressive strength of the PUFs were measured according to ASTM D 357481,GB 8410-94 and the method described in a previous report," respectively. The isocyanate index are defined as follows: Isocyanate index = [MTDI x Wmd (Mmx WID+ 2118 x WW)]x 100 Where MTDIis the isocyanate group contents in TDI (moUg), Wm is the weight of TDI (g), Mu, is the hydroxyl group contents in liquefied biomass (moVg), WIDis the weight of liquefied biomass (g), and W w is the weight of water in the foam formulation (8).

RESULT AND DISCUSSION Effect of liquefaction solvent composition

PUF prepared from polyether such as PPG or polyethylene glycol has comparatively low density and high resilience because of the low viscosity of polyether prepolymer and the low rotation hindrance of ether bonds in the main chain. However, the high chain flexibility also leads to low compressive strength. Usually there ~IEtwo ways to improve the compressive strength; first, introduction of aromatic groups to the main chain to improve the chain rigidity, and second, increasing the amount of crosslinking agent such as diethanolamine and trimethylol propane to increase the crosslinking density of the polymer chain."' In previous work, we reported that tannin and tannin-containing BK became effective crosslinking agents in preparing PUFs6". In this paper, it is expected that BK can impmve the strength and resilience of PUFs because tannin has not only phenyl groups for the improvement of chain rigidity and ether bonds for the change in the chain configuration but also enough active hydroxyl groups to form crosslinks as shown in Fig.1. BK should be liquefied in larger-molecular-weight poXyols so as to produce flexible PUFs. Two kinds of polyether polyol, PEG and PPG having the same hydroxyl value, 35 mgKOWg, were used for preparation of PUFs from BK. The PUFs from PPG gave higher resilience values than those from PEG at given BK contents (data not

I78

Biodegradable polyurethane-based polymers

Figure 1 Structure of condensed tannins.

Figure 2 Effect of molecular weight of PPG on the solubility of BK and on the resilience values of PLJFs.

shown). On the other hand, solubilities of BK were about 85%, 80% and W o in PPG with molecular weight 3000,4000 and 5oO0,respectively. However, the resilience values of PUF showed a maximum value at molecular weight of 4OOO as shown in Fig. 2. Thus, poly-ether PPG GEP-330N with Mn 4OOO and viscosity 800 CPwas selected to prepare highly resilient PUFs in the further work.

Effect of tannin content in bark on resilience value and density of PUFs in order to evaluate the effect of tannin content on the resilience of PUFs, we synthesized PUFs from three BKs with different tannin contents, that is BKO (tannin: 0%, residue after tannin extraction), BK1 (tannin: 43.0%),and BWtannin: 48.5%);the results are shown in Fig. 3. Both the resilience values and densities of the PUFs increased with increasing BK contents for all BK used. The densities of PUFs from BK2 were slightly larger than those of BK1 and BKO at given BK contents. However, the former had larger resilience values than those from BK2 and BKO. Since both resilience value and density are important properties of PUFs, the resilience valuddensity (RID) ratio was calculated. The larger the RID ratio, the better the PUF properties for car-cushion use. RID of PUFs from BK2 possessed the best values at given BK content (data not shown). This can be ascribed to the presence of the aromatic structure in tannin. BK2 was chosen for all the synthesis described hereinafter.

Effect of cornstarch content in biomass on resilience of PUFs. Cornstarch can easy be dissolved in a PEG 4Wglycerol mixture’), and there are a lot of glycoside bonds in its main chain that may contribute to the resilience of PUF, so we tried to use CS as the second biomass component. It can be totally dissolved in the liquefaction mixture within 20 rnin. at 1 5 0 C From Figs. 4 and 5, it can be seen that when CS replaced a part of BK, densities and compressive strengths of the PUFs decreased with increasing CS proportion whereas resilience value showed a maximum at CS/(CS+BK) 50%, that is 1: 1 of CS:BK .

Effect of insoluble bark residue on flammability of PUFs Flame resistance is an important property from safety points of view. We find that

Biodegradable polyurethane foams

179

62 60-

L

2 c O

P

A . 56' 54-

C

52-

O"

50-

.

20 io

30 i

i40o ia do 50 00 70 CS/(CS+BK) (WT%)

80 do

Figure 4 Effect of CS contents in total biomass on resilience values and densities of PUFs. J 80-

6040 -

Figure 3 Effect of BK contents on resiiience values and densities of PUFs.

20 -

0CS/(CS+BK) (wt96)

Figure 5 Effect of CS contents in total biomass on compressive strengths of PUFs.

8070 -

*\

-+PUFS with BK

8050-

40-

30-

0 4 8 12 16 20 Insoluble Reaidw in Lquefklion M i u m (M%)

Figure 6 Effect of the amount of insoluble bark residue in liquefaction mixture on the flammability of PUFs. flammability of the PUFs derived from BK is remarkably better than PUF without BK. More interestingIy, the insoluble BK residue plays an important role on flame resistance properties of BK-derived PUFs. The insoluble residue removed from a liquefaction mixture of BK was partly added back to the liquefaction mixture containing no insoluble residue, then PUFs were prepared from the mixture. The flammability was plotted against insoluble residue contents in the liquefaction mixture as shown in Fig.6. It can be seen that the flammability of PUFs decreased with increasing the insoluble residue in PUF. This improvement of flame resistance may be caused by the inorganic components in BK, though more detailed study is necessary for clarifying its mechanism.

I80

Biodegradable polyurethane-based polymers

Biodegradability Three categories of PUFs, the first one derived from 30% BK, the second one from 15%BK and 15%CS, and the third one commercially purchased, were buried in soil. Their weight loss was observed every month. Weight losses of two PUF samples derived from biomass increased with increasing periods of soil-microbial treatments (data not shown). The PUFs containing BK and CS showed a slightly faster weight loss behavior than those only containing BK. These results indicate that the CS component in PUFs is easier to be decomposed by soil microorganisms than the BK component. On the other hand, almost no change was observed in the weight of commercially obtained PUF. The above-mentioned results indicate that the biomass components contribute mainly to the biodegradability of the PUFs. 4. CONCLUSION

Highly elastic or highly resilient polyurethane foams (PUFs) suitable for car-seat cushions can be prepared from the liquefied BK and CS. Insoluble residue from BK liquefaction contributed remarkably to the improvement of flame resistance of the resulting PUFs. When CS replaced part of BK, the density and compressive strength of the PUF decreased with increasing CS proportion whereas resilient ratio had its maximum value when the weight ratio of CS:BK was 1:1. The PUFs were to some extent biodegradable, the average weight loss of samples buried in soil for 6 months was 15.6 wt %. Acknowiedgment

This work was aided by N o 3 Chemical Plant of Shanghai Gao Q a o petrochemical Co and Shanghai Yan Feng automotive trim Co., Ltd. in material supplying and testing of mechanical properties of PUFs, respectively. We are indebted to the financial aid from the Scientifc Research Fund of the Shanghai Educational CommiW and Shanghai Environment Protecting Ministry. The authors thank Mr. R. M. Gu for the synthesis of polyether polyol. REFERENCES 1 Y Yao, M Yoshioka and N Shiraishi, Mokuzai Gakkaishi, 19!B 39, 930-938. 2 S Hirose, T Tokashiki, H Hatakeyama, 39&Lignin Symposium, Fukuoka, 1994 p.59-62. 3 K Nakamura, R Morch, A Reinmann, K Kringstad, H Hatakeyama, Wood Processing and Utilization, J F Kennedy, Ed, Ellis Horwood, 1989 p. 175-180. 4 R L Cunningham, M E Cam, E B Bagley, JAppl Polym Sci, 1992 44, 1447- 1483. 5 N Shiraishi, Cellulose Utilization;Research and Rewards in Cellulosics, H Inagaki, and G 0 Philips,.Ed, Elsevier Appl Sci, 1989 p.97-109. 6 J-J Ge and K Sakai, Mokuzui Gukkuishi, 1993 39,801-806. 7 J-J Ge and K Sakai, Mokuzai Gakkaishi, 1996 4 2 , 8 7 9 4 . 8 J-J Ge and K Sakai, Mokuzai Gukkuishi, 19% 42,417-426. 9 Y Nakashima, J-J Ge and K Sakai, Mokuzui Gdkuishi, 1996 42, 1105-1112. 10 Y Yao, M Yoshioka, N Shiraishi, Mokuzui Gukkuishi, 1995 41, 659-668. 11 Y S Fang, R M Zu: "Polyurethanefoam (in Chinese)",J Q Cai Ed. Beijing,1996 p .74-77.

BIODEGRADABLE POLYURETHANES DERIVED FROM WASTE IN THE PRODUCTION OF BEAN CURD AND BEER Kunio Nakamural, Mika Iijimal, Emiko Kinoshital arid Hyoe Hatakeyamaz

’ Otsuma Women‘s Universi@, 12, Sanban-cho, Chiyoah-ku, Tokyo 102-8357, Japan Fukui University of Techrwlogy, 3-6-1, Gakuen, I W u i 910-0028, Japan

SYNOPSIS From the viewpoint of the recycling of bio-based resources and development of environmentally friendly polymers, polyurethanes (PU’s) were prepared from polyethylene glycol (PEG) containing fine powder of bean curd refuse (BCR) or beer grains (BG) (waste derived from beer production) , and diphenylmethane diisocyanate (MDI). Mechanical and thermal properties of the PU’s were measured and biodegradability of the PU’s was also examined. Stress at break and Young’s modulus of the PU’s increased with increasing waste contents of BCR and BG in PU’s. The maximum stress at break was observed at about 0.5 (gig) of BCRPolyol and BGPolyol ratios. Glass transition temperature (TJ increased with increasing BCRPolyol and BGPolyol ratios. It is found from these results that the fine powder of BCR and BG acts as a hard segment in PU’s and that BCR and BG fine powder contributed effectively to an improvement in the mechanical and thermal properties of PU’s. The weight loss percentage of PU samples after biodegradation tests in soil increased with testing time and depended on the waste materials. SEM observation suggested that PU’s were degraded by microorganisms in soil.

1. INTRODUCTION World production of plastics is about 140 million tons a year and about 65% will be waste [l].These waste plastics are ordinarily burned or buried. It is considered that waste plastics cause global environmental problems, such as global warming, and acid rain caused by sulfur dioxide (SO,) and nitrogen oxides (NO,). Recently, there have been many reports concerning endocrine-disrupting chemicals [21 such as dioxin, nonylphenol and bisphenol A which affect human beings and the ecological system. Polyurethanes (PU’s) are one of the most useful multi-purpose polymers, since they can be used in various forms such as sheets, foams, paints etc. From the ecological viewpoint, PU’s containing natural polymers are beneficial, since natural polymers are generally recognized as biodegradable polymers. We have developed biodegradable polyurethanes (PU’s) derived from various natural polymer wastes, such as lignins [3-61, brewer’s grains [7], coffee grounds [8,9], edible fat and oil [lo] and other waste materials Ill-141. It has been reported that the mechanical and thermal properties of PU’s derived from waste materials increased with the introduction of plant components, which have pyranose rings and phenyl groups. From the viewpoint of the recycling of bio-based resources and development of environmentally friendly polymers, polyurethanes (PU’s) were prepared from polyethylene glycol (PEG) containing fine powder derived from bean curd refuse (BCR) or beer grains (BG), and diphenylmethane diisocyanate (MDI). Mechanical and thermal properties of the PU’s were measured and biodegradability of the PU’s was also examined.

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2. EXPERIMENTAL

2.1 Chemical reaction of PU’s Table 1 shows the production and components of bean curd refuse and beer grains. The production of bean curd (TOFU) and beer is 70 thousand tons / year and 7 million kl / year in Japan and the amounts of bean curd refuse and beer grains produced by the industry are 80 thousand and 20 thousand tons/year, respectively. Bean curd refuse and

Table 1. Production and components of bean curd refuse and beer grains.

Resources

Production Waste (looot) (1Ooot)

Protein Lipid Saccharide Ash (%)

(a)

(%I

(%)

Bean-curd refuse

700

800

25.4

19

51.4

4.2

Beer grains

7000

200

27.1

9.8

58.7

4.4

beer grains have not been used since no methods were found for effective utilization. The main chemical components of dry bean curd refuse and beer grains are polysaccharide 50-60%, protein 26%, lipid 10-20%, and ash 4 % as shown in Table 1. Figure 1 shows the reaction scheme of PU consisting of PEG-MDI system. PU’s have been synthesized using various types of polyols and isocyanates. In the reaction to form the urethane linkage between isocyanate and waste materials, the hydroxyl groups in wastes act as reaction sites. The MDVpolyol ratio and BCR and BG contents (BCR/polyol and BG/polyol) were defined as the following equations. MDVpolyol = mass of MDI / mass of polyol BCWpolyol = mass of BCR / mass of polyol BG/polyol = mass of BG / mass of polyol

Figure 1.

(1) (2) (3)

Reaction scheme of PU derived from PEG and MDI.

Biodegradable polyurethanes from waste

183

2.2 Samples Bean curd refuse and beer grains were obtained from Sugimoto Tofuten, Tokyo and Asahi Beer Co. Ltd., respectively. Polyethylene glycol (PEG) with molecular weight of 400 and crude diphenylmethane diisocyanate (MDI) were obtained from Mitsui Toatsu Chemical Co. Ltd. Figure 2 shows the preparation scheme of PUS containing waste materials. In order to prepare PU films containing waste materials, a certain amount of waste materials which passed through 200 mesh filter of diameter = 75 pm was mixed well with PEG (Mw = 400). The polyol mixture was mixed with a trace amount of catalyst (di-nbutyltin dilaurate) and MDI in tetrahydrofuran (THF).MDI/polyol ratio was 1.0. The mixture was reacted at room temperature for about 1 2 hours under stirring. Then the mixture was cast on a glass plate and THF was slowly removed in a desiccator. PU films were dried in air, cut for testing and treated at 120 "C for 5 hours in an electric oven. The thickness of PU films derived from various wastes was about 0.1 0.3 mm. The colors of PU films were derived from the original colors of the wastes.

-

-

I

Waste materials

Drying at 1Zo'C

I

-

Crushed by mixer Filtering

Original polyol PEG 400

Polyol Sn catalyst

-

L-THF

StirringStirring cast

MDI

-

Homogenized

-

Homogenized

-

Surfactant + H@

MDI

Heat treatment Drying Humidity control

P Measurement

Figure 2. Preparation scheme of PU's derived from various wastes.

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Biodegradable polyurethane-based polymers

2.3 Measurement

Tensile properties of PU films were determined using a tensile test machine (TENSILON RTA-500, Onentec Co. Ltd.) at 25°C. The strain rate was 5 mm/min. The gauge length was 30mm. Tensile stress at break and Young's modulus were calculated from stress-strain curves. The width and thickness of samples were 5 mm and 0.1 to 0.3 mm, respectively. Thermal properties of PU's were measured by a differential scanning calorimeter (DSC 220C, Seiko Instruments Inc.). Scanning rate was 10"C/min. 2.4 Biodegradation tests

Biodegradation tests were canied out in a field at Otsuma Women's University, Sayamadai campus where there is good sunshine and good drainage. PU films were kept between stainless steel nets and were buried under the ground.

3. RESULTS AND DISCUSSION 3.1 Mechanical properties Figure 3 shows the stress-strain curves (S-S curve) of PU films derived from BCR (BCR-PU) obtained from tensile tests. The similar S-S curves were obtained in the case of BG-PU systems.

50

I

I

BCR/Polyol=0.6

40

d 30 I

. m

0.1 (22.3MPa,6E.l0h)

m

g 20

v)

0.05(22.7MPa,104.3%)

10

PEG-MDI (28.2MPa,l W h )

0

0

2

Figure 3.

4

6 Strain / Yo

8

10

Stress-strain curves of BCR-PU systems.

Biodegradable polyurethanes from waste

50

5000

185

50

5000

(b) 1B P U G --

p”;

0

0

0.6 0.8 (BCWPolyol) I 8.9-’

0.2

Figure 4.

0.4

1.0

0

0.2

0.4

0.6

0.8

1.0

(BGIPolyol) I pQ1

The relationship between stress at break (a,)and Young’s modulus (E) of (a) BCR-PU and (b) BG-PU films, and BCWpolyol and BGfpolyol ratios.

and Young‘s modulus Figure 4 shows the relationship between stress at break (q) (E) of PU films and BCWpolyol and BG/polyol ratios. In both cases of BCR-PU and BG-PU systems, both 0, and E show similar relations. q, and E increased until about 0.5 g/g and then decreased with increasing BCWpolyol and BG/polyol ratios. However, oband E of BCR-PU were higher than those of BG-PU. The results indicate that the BCR and BG components act as a hard segment and that PU’s in the glassy state are hardened with increasing waste content. Moreover, BCWpolyol of 0.5 g/g represents the borderline between ductile fracture and brittle fracture of PU films. Polyols of BCR-PU and BG-PU consisted of PEG for soft segments and BCK and BG fine powder for hard segments. An appropriate balance between molecular flexibility from soft segments and rigidity from hard segments in PU’s was obtained at 0.5 g/g of BCWpolyol and BG/polyol ratios.

3.2 Thermal properties Figure 5 shows the DSC curves near the glass transition (T,) of BCR-PU systems. T,’s of BCR-PU systems increased with increasing waste content of BCR. In the case of BG-PU systems, similar DSC curves were obtained. Figure 6 shows the glass transition temperature (T,) estimated from DSC heating curves of BCR-PU and BG-PU systems as a function of BCWpolyol and BG/polyol ratios. The values of Tg gradually increased until about 0.5 g/g and then rapidly increased with increasing BCWpolyoI and BG/polyol ratios. The inflection point of 0.5 g/g agreed well with the borderline between ductile and brittle fractures which is obtained from tensile test of PU films.

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Biodegradable polyurethane-based polymers

5 BCR/P0lyol=0.05 0.2

0 Y

7 BCR-PU 0

-50

Figure 5.

50 100 150 Temperature / "C

200

250

DSC curves near T, of BCR-PU.

(b) BG-PU 200

200

0 - 0

0

0.2

0.4

0.6

0.0

(BCWPolyol) / g.g-7

Figure 6.

1.0

(BG/Polyol) I g.g-1

T,'s and ACp's of (a) BCR-PU and (b) BG-PU plotted against waste content.

Biodegradalble polyurethanes from waste

187

3.3 Biodegradability of PU's Figure 7 shows the relationship between weight loss by biodegradation test in soil and testing time. The weight loss rapidly increased until about 50 days and then gradually increased with increasing testing time. Weight loss also increased with increasing waste contents of BCR and BG. However, comparing the weight loss at the same waste contents of BCR and BG, weight loss of BCIR-PU was higher than that of BG-PU. 30

30

I (a) BCR-PU I

/

(b) BG-PU

I

=E0

BGlpolyol=O.?

01-

O

15

gm10 5

5

I

0

0

I

I

5 0 1 0 0 1 5 0 2 0 0 2 5 0 Time / day

Figure 7. The relationship between weight loss by biodegradation test in soil and testing time. Figure 8 shows the relationship between weight loss and particle size of BCR fine powder and depth under the ground of BG-PU test films. Weight loss decreased with increasing particle size of BCR fine powder because the. surface area of wastes in PU films increased with decreasing particle size. From Figure 8(b), the weight loss changed in the order of 15cm > 5cm > 30cm depth in soil. The maximum weight loss was obtained at the depth of 15 cm under the ground. It is considered that good conditions for microorganismsare formed at this depth.

(b) BG/POIYOI~O.~

deDth=l5cm

J 'Ah

1

5cm

PEG-MDI PU

50

100 Time I day

150

Figure 8. The relationship between weight loss and (a) particle size of BCR fine powders and (b) depth under the ground of BG-PU films.

200

1 88

Biodegradable polyurethane-based polymers

Before biodegradation tests

After 90 days

(a) PEG-MDI PU

(b) BCR-PU

I

(c) BG-PU

Figure 9.

The SEM photographs of (a) PEG-MDI PU, (b) BCR-PU and (c) BG-PU films before and after biodegradation test in soil.

Figure 9 shows the photographs of PEG-MDI, BCR-PU and BG-PU films before and after biodegradation tests in soil. The surface of the films gradually changed with increasing testing time. In the first stage, microorganisms come into contact with the wastes in PU films. Finally, many small holes in the films were observed. A clear

Biodegradable polyurethanes from waste

189

difference was not observed in PEG-MDI PU without wastes, but a clear difference was observed in BCR and BG PU’s before and after biodegradation tests.

3.4 Physical properties of the PU films after biodegradation Figure 10 shows the relationship between stress at break and Young’s modulus of BG-PU films after biodegradation tests and the testing time. Both stress at break and Young’s modulus obtained from the stress-suain curves of PU’s after biodegradation tests increased with increasing testing time until about 30 days and then decreased. Figure 11 shows the relationship between T of BCR-PU films after biodegradation tests and testing time. T slightly decreased with increasing testing time in the case of BCR-PU. While Tg of PhG-MDI PU increased with increasing testing time. Tg of BGPU also increased until about 60 days and then decreased although the figure is not shown here. It is considered from these results that PU films became stiff during biodegradation tests. This means that PU molecules were affected not only by microorganisms but also by water in soil. Concerning these results, further investigation is necessary. 5

600

BGPU

70

BG/PolyoI=O.5

60

r t

i

50

9 40 \

p30 20

10 0- 0

0

I BCR-PU 1st run

0

30

60 90 Time I day

120

0

50

100 150 Time I day

I 200

~

~~

Figure 10. The relationships between Figure 11. The relationship between Tg stress at break and testing of BCR-PU films and testing time of biodegradation. time of biodegradation. 4. CONCLUSIONS

From the above results, the following conclusions are obtained. (1) Bean curd refuse and beer grains can be used as a part of polyols in polyurethanes. (2) Bean curd refuse and beer grains act as hard segments in polyurethanes. (3) Stress, Young’s modulus and glass transition temperature of polyurethanes derived from bean curd refuse and beer grains have almost the same values as those of ordinary polymers. (4) It is considered that environmentally friendly polyurethanes can be prepared from bean curd refuse and beer grains.

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Biodegradable polyurethane-based polymers

5. REFERENCES

1. S. Yamauchi, Statistics of Plastics and Related Materials, Tokyo, NIKKAN PLASTICS, 1996. 2. T. Colbom, D. Dumanoski, and J. P. Myers, Our Stolen Future, New York, PLUM PENGUIN, 1996.

Compression 3. K. Nakamura, R. Morck, A. Reimann and H. Hatakeyama, Properties ofpolyurethane foam derived from kraft lignin. in "Wood Processing and Utilization", J. F. Kennedy, G. 0. Phillips and P. A. Williams Eds., P.175, Ellis Horwood, Chichester , 1989. 4. K. Nakamura, R. Morck, A. Reimann, K. P. Kringstad and H. Hatakeyama, Mechanical Properties of Solvolysis Lignin-derived Polyurethanes , Pol ym. Adv. Technol., 1991,2,41. 5. S. Hirose, K. Nakamura H. Hakakeyama, J. Meadouws, P. A. Williams and G. 0. Phillips, Preparation and Mechanical Properties o f Polyurethane Foams From Lignocellulose Dissolved in Polyethylene Glycol, CELLULOSICS : Materials for Selective Separations and Other Technologies, Ellis Horwood, London, 1993. 6. K.Nakamura, H.Hatakeyama, J. Meadows, P. A. Williams and G. 0. Phillips, Mechanical Properties of Polyurethane Foams Derived From Eucalyputus Kraft Lignin, CELLULOSICS : Materials for Selective Separations and Other Technologies, Ellis Horwood, London, 1993. 7. K. Nakamura, E. Kinoshita and H. Hatakeyama, Physical properties and Biodegradability of Polyurethanes Derived from Brewers grains, Sen-i Gakkai Preprints, 1998, G-254. 8. K. Nakamura, Y. Nishimura, T. Hatakeyama and H. Hatakeyama, Viscoelastic properties of biodegradable polyurethanes derived from coffee grounds, The chemistry and processing wood and plant fibrous materials (J. F. Kennedy, G. 0. Phillips, P. A. Williams), 1996. 9. K. Nakarnura, Y.Nishimura, T. Hatakevama and H. Hatakevama. Preparation of biodegradable polyurethanes derived from coffeegrounds, International 'Workshop, Tsukuba. 1993.239. 10. K. Nakakura &d Y. Nishimura, Polyurethane Foam Derived from Waste Vegetable Oil, Kobunshi Ronbunshu, 1993,50,881. 11. Y. Tamai, Y. Sasaki and K. Nakamura, Utilization of By-product(Shir0-Kasu)from Wheat Starch lndusfryfor Polyurethane, SEN-I GAKKAISHI, 1997,53, 381. 12. H. Hatakeyama, S. Hirose, K. Nakamura and T.Hatakeyarna, New types o f polyurethanes derived from lignocellulose and saccharides, CELLULOSICS, 1993. 13. K. Nakamura, Y. Nishimura, T. Hatakeyama and H. Hatakeyama, Mechanical and Thermal Properties o f Biodegradable Polyurethanes Derived f m m Sericin, S EN-I GAKKAISHI, 1995,5 1 , 11 1 . 14. K. Nakamura and Y. Nishimura, Thermal Properties o f Polyurethanes Derived from Tea Grounds,Netsu Sokutei, 1995,22, 114 15. M. Iijima and K. Nakamura, Mechanical Properties o f Polyurethanes Derived from Bean-Curd Refuse, Nihon Kasei Gakkaishi (J. Home Econ. Jpn.), 1999,50, 6

BIODEGRADABLE POLYURETHANE COMPOSITES CONTAINING COFFEE BEAN PARCHMENTS Hyoe Hatakeyama”, Daisuke Kamakura’, Hideyuki Kasahara’, Shigeo Hirose’ and Tatsuko Hatakeyama’ I

Fukui Universiry of Technology, 3-6-1 Gakuen, Fukui-city, Fukui 910-8505, Japan

’National Institute of Materials and Chemical Research, 1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan

’Otsuma Women’s University, 12 Sanbancho, Chiyoda-hi, Tokyo 102-8357, Japan ABSTRACT Polyurethane composites were prepared using coffee bean parchments (CBP) mixed with a molasses-polyol (MP) consisting of molasses and polyethylene glycol (PEG-200). The content of CBP in the polyol was varied from 0 to 90 wt %. The above mixture was reacted with diphenylmethane diisocyanate (MDI) with the presence of a catalytic amount of dibutyltin dilaurate (DBTDL) to form polyurethane composites. The compression strength ( u ) and the compression modulus (E) was almost constant in the region of CBP content lower than 50 9%. When the CBP content exceeds 60 %, u and E increase prominently with increasing CBP content, reaching a maxima at CBP content = ca. 70 %., and then decreasing with increasing CBP content. The derivative thermogravimetry (DTG) curve of the obtained CBP composites showed two kinds of the thermal degradation temperatures: DTd, and DTd2.DTdldecreased with increasing CBP content. DTdz increased slightly with increasing CBP content, reaching the degradation temperature of coffee bean parchments.

INTRODUCTION In the past 15 years, various synthetic polymers, whic:h can be derived from plant components such as saccharides and lignin, have extensively been studied by various research groups [l-81. We have paid attention to composites which were obtained from fine parchments the diameter of which was less than 2 ~ l land l thickness was 0.09 m. In the present study, PU composites were prepared from ground plant particles, such as coffee bean parchments, mixed with a molasses-polyol (MP) solution consisting of molasses and polyethylene glycol (PEG-200). Mechanical and thermal properties of the above composites were studied. EXPERIMENTAL Sample preparation Coffee bean parchments were kindly provided by the National Federation of Coffee Growers of Colombia. The particles were 2 111111 in diameter and 0.09 mm in thickness for coffee bean parchments. A molasses polyol (MP, molasses mixed with polyethylene glycol 200, Tropical Technology Center Co.) was used as a polyol and diphenylmethane

192

Biodegradable polyurethane-based polymers

diisocyante (MDI, Mitsui Chemical Co.) was used an isocyanate.

Measurements Apparent density ( ,n ) was measured using a Mitsumoto A B S digital solar caliper and an electronic balance. The size of the sample was 40-60 rnm (length), 20-30 (width) and 20-30 nun (thickness). The unit of apparent density was g / cm3. Compression measurements were carried out using a Shimadzu Autograph AG 2000D at room temperature. Test specimens were a rectangular solid, and the added stress was less than 10 MPa / min. Compression strength ( 0 )was defined as the value of highest point of the linear part in the stress-strain curve. Static Young's modulus ( E ) was calculated using the initial stage of compression curves. Conditions in detail accorded with Japanese Industrial Standards (JIS 2-2101). Thermogravimetry (TG) was carried out in nitrogen flow using a Seiko TG 220 at a heating rate of 20 "C / min in the temperature range from 20 to 500 "C. Sample mass was ca. 5 mg. TG curves and DTG curves were recorded. Mass residue was indicated as ( mT / m m ) x 100 (%), where mT is mass at temperature T and m, is mass at 20 "C. Mass residue was evaluated at 450 "C.

RESULTS AND DISCUSSION

As shown in Fig. 1, CBP was mixed with polyol and suspensions were obtained with various mixing ratios from 10, 20, 30, 40, SO, 60, 70, 80 and 90 wt 96. Acetone was added to each mixture in order to control the viscosity of the suspension. MDI was added to the suspension under stirring and coffee bean parchment-PU composites were obtained. After drying for 3 days at room temperature, the sample was cured at 120 "C for 2 hrs. Fig. 2 shows the relationship between density ( p ) of PU composites and coffee bean parchment contents. The density reaches a maximum when the content of parchments in the composites is ca. 70 76. Coffee bean parchments

I added Molasses polyol mixed Suspension

I reacted with MDI

rn PU composites

NCO/OH =1.2 Material content = 10-90 wt%

Figure 1.

Preparation scheme of polyurethane composites (PU composites)

Biodegradable polyurethane composites

0.80

0.00

,

193

-

' 0

20

40

60

80

100

coffee bean parchments content I %

Figure 2.

Relationship between density ( p ) of PU composites and coffee bean parchment contents

Fig. 3 shows the relationship between compression strength ( u ), modulus of elasticity ( E ) and coffee bean parchment content in PU composites. As seen from the figure, both compression strength ( CJ ), and modulus of elasticity ( E ) increase with increasing coffee bean parchment content in PU composites and reach a maximum when powder content is ca. 70 $5. 20.0

800.0

16.0 600.0 rJ

12.0

2

2.

400.0

2 --. rrl

8.0 200.0 4.0

0.0

0

20

40

60

80

1Oil

coffee bean parchments content 1 %

Figure 3.

The relationship among compression strength ( u ) modulus of elasticity (E) of PU composites and coffee bean parchment contents 0 compression strength ( u ), o modulus of elasticity (E)

194

Biodegradable polyurethane-based polymers

20.0

800.0

15.0

600.0

cd

rj

a

a

5 10.0

400.0

5 4

b

5 .O

200.0

0.0

0.2

0

0.4

0.6

0.8

P ~g-cm-~

Figure 4.

The change of compression strength ( u ) and modulus of elasticity ( E ) with density ( p ) of PU composites from CBP 0 compression strength ( u ), o modulus of elasticity ( E )

content I %

3

200

300

400

5

T i "C TG heating curves and derivative curves of CBP-MP type PU composites containing various amounts of coffee bean parchment

Biodegradable polyurethane composites

1-

370.0

195

370.0

330.0

9 \

cn 290.0

'------I

250.0

0

20

40

60

80

250.0

100

coffee bean parchments content / 9%

Figure 6.

Change of Td and DT, with coffee bean parchment content in PU composites a DT,,, CBP 100%DT,,, A Tdl, + CBP 100% T,, 0 DT&, 0 CBP 100% DTa, A Ta, C) CBP 100% Tdz

Fig. 4 shows the change of compression strength ( u ) and modulus of elasticity (E) with density ( p ) of PU composites obtained from coffee bean parchments. As seen from the figure, both compression strength ( u ), and modulus of elasticity (E) increase with increasing density ( p ) of PU composites. The above results suggest that the highest mechanical properties of PU composites from CBP properties are observed when the density of PU composites reaches the highest value. Fig. 5 shows TG curves and DTG curves of PU composites from CBP. As seen from Fig. 5, DTG curves show the presence of two kinds of thermal degradation corresponding to DTdland DTc DTdz seem to be specific to the degradation of CBP, since the DT,,peak becomes prominent when CBP content in PU composite are over 30 5% and this is very clear when coffee bean parchment content is 100 %. Fig. 6 shows the change of Td,,Ta, DT,, and DTdZwith the coffee bean parchment contents. Both degradations may be mainly caused by the degradation of CBP, since both are observed when the coffee bean parchment content is 100%.

CONCLUSIONS (1) Polyurethane composites were prepared using coffee bean parchments (CBP) mixed with a molasses-polyol (MP) consisting of molasses and polyethylene glycol (PEG200). The content of CBP in the polyol was varied from 0 to 90 wt%. (2) The compression strength ( (T ) and the compression modulus (E) was almost constant in the region of CBP content lower than SO 5%. When the CBP content exceeds 60 96,CJ and E increase prominently with increasing CBP content, reaching maxima at CBP content = ca. 70 %, and then decreasing with increasing CBP

196

Biodegradable polyurethane-based polymers

content. (3) The DTG curves of the obtained CBP composites showed two kinds of thermal degradation temperatures: DTdl and DTa. DTdl decreased with increasing CBP content. DTdz increased slightly with increasing CBP content, reaching the degradation temperature of coffee bean parchments.

ACKOWLEDGEMENT The authors would like to thank the National Federation of Coffee Growers of Colombia, for providing coffee bean parchments.

REFERENCES 1. V. P. Saraf and W. G. Glasser, ‘Engineering plastics from lignin. 111. Structure Property Relationship in solution cast polyurethane films’, .I. Appl. Polym. Sci., 1984, 29, 1831-1841. 2. V. P. Saraf, W. G. Glasser, G. L. Wilkes and J. E. McGrath, ‘Engineering plastics from lignin. a.Structure Property Relationship of PEG-containing polyurethane networks’, J . Appl. Polym. Sci., 1985, 30, 2207-2224. 3. W. H. Newman and W. G. Glasser, ‘Engineering plastics from lignin. XI. Synthesis and performance of lignin adhesives with isocyanate and melamine’, Holzforschung, 1985,39,345-353. 4. A. Reimann, R. Morck, H. Yosida, H. Hatakeyama and K. P. Kringstad, ‘Kraft lignin in polyurethane. 111. Effects of the molecular weight of PEG on the properties from a kraft lignin-PEG-MDI system’, J . Appl. Polym. Sci., 1990, 41, 39. 5. K. Nakamura, R. Morck, K. P. Kringstad, H. Hatakeyama, ‘Compression properties of polyurethane foam derived tiom kraft lignin’, Wood Processing and Utilization (J. F. Kennedy, G. 0. Phillips and P. A. Williams, Eds.), Ellis-Honvood, Chichester, 1989, 175-180. 6. H. Hatakeyama, S. Hirose, K. Nakamura and T. Hatakeyama, ‘New type of polyurethanes derived from lignocellulose and saccharides’, in Cellulosics: Chemical, Biochemical and Material Aspects ( J . F. Kennedy, G. 0. Phillips and P. A. Williams, Eds.), Ellis-Honvood, Chichester, 1993, 525-536. 7. N. Morohoshi, S. Hirose, H. Hatakeyama, T. Tokashiki and K. Teruya, ‘Biodegradabability of polyurethane foams derived from molasses’, Sen-i Gakkuishi, 1995, 51(3), 143-149. 8. K. Nakamura, Y. Nishimura, T. Hatakeyama and H. Hatakeyama, ‘Viscoelastic properties of biodegradable polyurethanes derived from coffee grounds’, the Chemishy and processing of wood and plunt firous materials (J. F. Kennedy, G. 0. Phillips and P. A. Williams, Eds.), Woodhead Publishing Ltd., Cambridge, England, 1996. 283-290.

BIODEGRADABLE POLYURETHANE SHEET DERIVED FROM WASTE COOKING OIL Sumalai Srikumlaithong*, Chulaporn Kuwarananchrroen* and Narongdej Asa* Thailand Instilute of Scientif?cand Technological Research, 196 Phahonyothin Rd, Chatuchak, Bangkok 10900, Thailand

ABSTRACT Experiments on the preparation of biodegradable polyurethane sheet derived from waste cooking oil were carried out. The major factors affecting its properties were casting conditions, the amount of waste cooking oil, additives used and NCO/OH ratio. The products prepared by casting into a mould at ambient temperature gave more even properties and higher hardness than that using hot mould. When silica and fibre glass were applied as reinforcement, the result of polyurethane sheet containing fibre glass possessed higher hardness and lower elongation at break than those with silica at amounts of 2, 4, 6 and 10 parts by weight (pbw). The additives used improved the quality of products prepared at NCO/OH ratio 1 to a certain extent but they were not good enough for commercial applications. The effect of NCO/OH ratio on mechanical properties was also studied. The product obtained at NCO/OH ratio of 1.6 containing 30 pbw waste cooking oil gave properties that complied with the requirement. Preparation of polyurethane sheet with properties complying to standard was accomplished by blending 30 pbw oil, 70 pbw PEG and 79.7 pbw MDI (NCO/OH ratio of 1.6), casting into a mould at ambient temperature and hot air post-curing at 120'C for 4 h.

INTRODUCTION Waste cooking oil from the food industry and restaurants is increasing substantially every year. The waste which could be collected was 42,000 metric tons in 1995, thus resulted in environmental pollution. Fats and oils are the ester of glycerol consisting of hydroxyl groups (Schauerte. 1985). Hatakeyama (1991) and Nakamura (1993) preliminary investigated its utilization for production of biodegradable polyurethane. Polyurethanes composing of carbonate groups in their backbone structure (Wood 1987) were produced by the reaction of isocyanate with more than one reactive isocyanate group per molecule (a diisocyanate or polyisocyanate) and alcohols having two or more reactive hydroxyl groups per molecule (diols or polyols). The molecular weight of the effective polyols should be 200 -10,000, depending on their applications (Schauerte 1985). All types of polyurethanes are based on the exothermic reaction of diisocyanate or polyisocyanate with polyols. Mechanical properties of urethane are influenced directly to NCO/OH ratio and the excess of isocyanate groups is able to take part in the crosslinking reaction through the formation of allophanate or biuret linkages (Nierzwichi et al. 1980). It was found that the increase in NCO/OH ratio results in the increase of hardness, tensile modulus, tensile strength and elongation properties. To enhance a wide range of applications, additiies - catalyst, chain extenders, crosslinking agents and fillers - may be used to control and modify both the reaction and properties of the final polymer. In this study, waste cooking oil from the food industry

198

Biodegradable polyurethane-based polymers

utilized as polyols for production of biodegradable polyurethane. The effect of NCO/OH ratio, and amount of oil and additives on its properties were assessed.

EXPERIMENTAL

Materials 1. Polyols 1.1 Waste cooking oil fiom a restaurant was filtered and its properties are presented in Table 1.

Table 1. Properties of waste cooking oil Characteristics Moisture, % w/w Acid value, mg KOWl g oil Saponificationvalue, mg KOWl g oil Hydroxyl number Molecular weight (av.)

Waste cooking oil

0.1 3.39 207.57 61.80

2,723

1.2 Polyethylene glycol (PEG) with properties are shown in Table 2.

Table 2. Properties of polyethylene glycol (PEG) Characteristics Molecular weight (av.) Hydroxyl number Acidity pH (5% aq. soln.) Degree of polymerization (n)

PEG

400 28 1 0.1 5.5 9

2. Isocyanate Diphenylmethanediisocyanate (MDI) with average molecular weight of 250 and NCO content of 3 1% 3. Additives 3.1 Silica with particle size less than 45 micron as reinforcement 3.2 Fibre glass as reinforcement 3.3 Silane A 1100 as coupling agent 3.4 Vulkanox BMT as antioxidant 3.5 DioctylphthalateP O P ) as plasticizer 3.6 Releasing agent A-F- 1

Methods 1. Material preparation Waste cooking oil, PEG and MDI were dried and deaerated in an vacuum oven with a reduced pressure of 5- 10 inch Hg at 105-110°C for 15 min, 1 h and 2 h respectively. MDI was cooled in a vacuum chamber at a reduced pressure of 1-2 inch Hg for 2 h.

199

Biodegradable polyurethane sheet

2. PUsheetpreparation

PEG solution was prepared by mixing dried oil and hot PEG at 90°Cfor 5-10min and cooled in a vacuum chamber with a reduced pressure of 1-2inch for 2 h. PU sheet was achieved by prepolymerization of PEG solution and MDI at ambient temperature. The speed and time consumed are indicated in Table 3, 4,and 5. The solution was casted on glass plates coated with releasing agent at the size of 10~15~0.20 cm3, followed by curing at 120°Cfor 1.5-4h. The product was released out of the mould and left for 2 weeks to complete the crosslinking reaction. Table 3. Weight of oil and casting conditions at NCO/OH ratio 1 Ingredients Experiment No. 1 2 3 4 30 20 30 @bw) 20 Oil

5 40

6 50

PEG400

(pbw)

80

80

70

70

60

50

MDI

@bw)

55.10

55.10

49.80

49.80

44.70

40.70

Speed of mixing (rpm) Mixing time

Curetime

30

70

55-80

80

hot

cold

hot

cold

.

Cure temperature (“C)

115-120 115-120 hot

hot

120 1.50

(h)

200-225 200-230 200-230

200

(sec.)

Mould

200

200

b

1 .so

4

1.50

1.50

4

Table 4. Additives for property improvement of PU sheet at NCO/OH ratio 1 Ingredients Experiment No. 1 2 3 4 5 6 7 8 9 10

NCO/OH

1

1

1

1

1

1

1

1

1

1

(pbw) 20

20

20

20

20

20

20

20

20

20

30

PEG400

(pbw) 80

80

80

80

80

80

80

80

80

80

70

MDI

(pbw) 55.1 55.1 55.1 55.1 55.1 55.1 55.1 55.1 55.1 55.1 49.80

Silica

(pbw)

-

Fibreglass

(g)

-

SilanellOO

(pbw)

-

ratio Oil

1

11

V U ~ ~ ~ O (pbw) X BHT DOP (Pbw) Speed of mixing Mixingtime

2

2

4

6

10

20

-

2.75 1.67 2.88 2.50

0.12 0.12 0.24 0.36 0.36 0.12 0.24 0.24 0.24 0.24

-

0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25

(rpm.)200 200 200 200 200 210 450 (sec.)

60

60

70

95

100 70

70

-

1

-

200-250 70

60

60

b

75

200

Biodegradable polyurethane-based polymers

Mould Cure temperature Cure time

("C)

.

hot mould

120 b

1.50

(h)

3.50 1.00 t

4 4

t

Table 5. Variation of NCO/OH ratio and oil Ingredients Experiment No. 1

2

3

4

5

6

7

8

9

10

1

1

1.2

1.2

1.4

1.5

1.6

1.6

1.6

1.6

NCO/OH ratio Oil

(pbw)

20

30

20

30

30

30

20

30

40

50

PEG 400

(pbw)

80

70

80

70

70

70

80

70

60

50

MDI

(pbw) 55.10 49.80 66.20 59.80 69.80 74.80 88.20 79.70 71.10 65.12

Speed of mixing Mixingtime

(rpm.) 200

200

200

200

200

200

200

200

200

200

70

80

72

76

70

78

66

78

84

90

(sec.)

Mould Cure temperature Cure time

4

Cold mould

t

("C)

4

120

+

(h)

4

4

b

3. Study on the effect of oil, additives and NCO/OH ratio on properties of PUsheet 3.1 Oil

PU sheets were prepared by using an isocyanate to hydroxyl group (NCO/OH) ratio of 1 and 1.6 while the weight of oil was varied from 20% to 50% of PEG solution. The experimental conditions are presented in Table 3 and 5 . 3.2 Additives

Silica and fibre glass were used as reinforcement in PU sheets. Prior to mixing with PEG solution, silica was pretreated with water (6%) and dried at 110°C for 24 h. Fibre glass was coated with silane containing a small amount of water (8% of fibre glass) before application. 3.3 NCO/OH ratio

PU sheets were prepared at a NCO/OH ratio of 1, 1.2, 1.4, 1.5 and 1.6. The weight of oil, PEG and MDI used are shown in Table 5. 4. Testing

Tensile strength and elongation at break were measured according to ASTM D 412-80. Hardness and Izod impact strength were determined by the method of ASTM D 2240-81 and ASTM D 256-92 respectively.

Biodegradable polyurethane sheet

201

RESULTS AND DISCUSSION

Utilization of cooking oil waste for the preparation of PU sheet has been accomplished, resulting in both environmental protection and value added of the waste. The effects of various parameters consisting of casting conditions, the amount of oil, additives and NCO/OH ratio used were studied.

Casting conditions Polymerizationis an exothermic reaction which leads to heat evolution and results in a high rate of reaction. Hence, the setting took place rapidly before homogeneous mixing was ready while casting with hot mould. Whereas PU sheet casted with cold mould could yield homogeneousproperties (Table 6).

Table 6. Effect of oil and casting condition on PU properties at NCO/OH ratio 1 Experiment Mould Oil Tensile strength Elongation at Hardness (Pbw

No. 1

2 3 4 5 6

hot cold hot

20

Wa) 0.84-1.44 1.08-1.44 1.08-2.54

cold

30

1.82-1.89

hot hot

40

0.54- 1.22 0.44-1.35

50

(”/)

57-90 49-68 33-1 15 43-46 47-1 13 63-99

(shore A) 33-42 51-52 33-63 62-63 10-40

7-20

Effect of oil on PU properties at a NCO/OH ratio of 1 Table 3 shows that the higher the amount of oil used, the longer the time of mixing. The oil increased with the decrease of PEG and MDI at NCO/OH ratio of 1, thus resulting in a slower rate of reaction. Hardness of PU sheet substantiallydecreased when the weight of oil used increased as presented in Table 6.

Additives for property improvement Silica and fibre glass were added as reinforcements to increase the tensile strength and hardness, an antioxidant to prevent product degradation caused by reaction with atmospheric oxidation, and a coupling agent to increase level of bondmg in polymer. (Lutz 1989). PU sheets prepared 6pm NCO/OH ratio of 1,20 pbw oil and silica at the weight of 2, 4,6, 10 pbw possessed no significant difference in tensile strength and elongation at break but hardness decreased as shown in Table 7. At 20 pbw of silica, tensile strength of the sheet was relatively high. Fibre glass ranging fiom 1.67 to 2.88 pbw strengthened the hardness of PU sheets but decreased in elongation at break. However, the property of PU sheet embedded with either silica or fibre glass was improved to some extent but not sufficient for commercial applications.

Table 7. Effect of silica and fibre glass on PU properties at NCO/OH ratio 1 Experiment Additives Tensile strength Elongation at Hardness break No. (pbw) @Pa) (%) (shore A) 1 0.841.44 57-90 33-42

202

Biodegradable pol yurethane-based polymers

2

Silica 2

0.33-0.68

52-66

15-19

3

2

0.40-0.72

82-107

15-22

4

4

0.67-1.12

65-84

14-20

5

6

0.65-1.40

65-87

14-25

6

10

1.07-1.17

34-37

19-50

7

20

1.94-2.16

38-45

40-45

8

FG 2.75

1.92-5.22

4-6

37-62

9

1.67

1.25-4.16

5-6

63-68

10

2.88

0.91-2.13

6-15

46-58

11

2.50

1.70-4.13

5-10

55-59

Effect of NCO/OH ratio and oil on PU properties The ratio of NCO/OH influences the ultimate properties of polymer (Nakamura et al. 1992). The PU sheets were prepared at a NCO/OH ratio of 1, 1.2, 1.4, 1.5 and 1.6 with 30 pbw of oil. Tensile strength and hardness increased with the increase of NCO/OH ratio (Table 8 and Fig. 1). The product obtained at NCO/OH ratio of 1.6 containing 30 pbw waste cooking oil gave properties that complied with the requirement. To make use of more waste cooking oil, PU sheets at NCO/OH ratio of 1.6 were produced from the oil at a weight of 20,30,40 and 50 pbw. Their mechanical properties decreased with the increase of oil used as shown in Fig. 2. Oil at 30 pbw possessed the properties of PU sheets that complied with the requirement.

Table 8. Effect of NCO/OH ratio and oil on PU properties Experiment NCO/OH Tensile Elongation Hardness No. ratio strength at break (MPa) (YO) 49-68 51-52 0 1 1 1.08-1.44 2

1

1.82-1.89

43-46

62-63 0

3

1.2

5.32-6.57

44-49

81-84

4

1.2

6.60-8.23

29-40

83-87

5

1.4

11.94-14.22

18-33

44

6

1.5

18.50-19.88

7-9

52-53 A

7

1.6

23.8 1-31.08

5-12

58-63 A

8

1.6

22.81-33.82

6-9

62-64 A

9

1.6

21.66-25.15

3-6

52-59 A

10

1.6

16.20-19.62

3-4

51-52 A

~

0 shore A A

shore D

Impact strength (J)

A

0.1

Biodegradable polyurethane sheet

NCO/OH ratio Figore 1.

Effect of NCO/OH on PU properties at 30 pbw oil.

Figure 2.

Effect of oil on PU properties at NCO/OH ratio of 1.6

203

204

Biodegradable polyurethane-based polymers

CONCLUSION The effect of casting conditions, the amount of waste cooking oil, additives and NCO/OH ratio on mechanical properties of PU sheets was studied. PU sheets with properties complying to the standard was accomplished by blending 30 pbw oil, 70 pbw PEG and 79.7 pbw MDI (NCO/OH ratio of 1.6), casting into a mould at ambient temperature and hot air post-curing at 120°C for 4 h.

REFERENCES 1 . K. Schauerte, M. Dahm, W. Diller, and K.Uhlig, Raw Materials in Polyurethane Handbook By Gunter Oretel, Munich, Copyright Carl Hauser, 1985. 2. H. Hatakeyama, S . Hirose and K. Nakamura, Biodegradable Polyurethanes and Manufacture, Jpn., Kokai Tokyo Koho, P 05, 186, 556,1991. 3. K. Nakamura and Y. Nishimura, Polyurethane foam derived fiom waste vegetable oil, Kobunshi Ronbunsh, 1993,50(1 l), 881-886. 4. G. Wood, ICI Polyurethane Book, New York, John Wiley & Sons Inc., 1987. 5. W. Nierzwicki and E. Wysoaka, Microphase separation and properties of urethane elastomer, J Appl Polym Sci,1980,25,739-746. 6. J . T. Lutz, Jr., lllennoplastic Polymer Additives, New York, Marcel Dekker, IncJ989. 7. K. Nakamura, T. Hatakeyama, and H. Hatakeyama, Thermal properties of solvolysis lignin-derived polyurethanes, Polymersfor Advanced Technologies,1992,3, 15 1- 155.

BIODEGRADABLE POLYESTERS PKEPARED WITH DIMETHYL SUCCINATE, BUTANEDTOL, AND MONOGLYCERIDE Yoichi Taguchi', Akihiro Oisht, Ken-ichi Fujita', YosMazu Ikeda' and Takashi Masuda' I National Institute of A4aterials and Chemical Research. 1-1, Higashi. Tswkuha-shr. Iharah-ken. 305-8565, Japan

INTRODUCTION

Aliphatic polyesters are well known to be biodegradable and environmentally compatible polymers. Above all, poly(buty1ene succinate) which is produced by the polymerization between succinic acid derivative and 1,4-butanediol is known to be an excellent biodegradable polyester with a high melting point and good mechanical strength, while the properties are not always optimal properties for use. Therefore, many kinds of improved copolymers based on poly(buty1ene succinate) have been reported [I]. On the other hand, monoglycerides such as monolaurin, monostearin, and monoolein can be derived from natural oils and fats whch are renewable resources, and have two reactive alcohols per molecule. It was reported that a high molecular weight poly(buty1ene succinate) could be obtained from transesterification between dimethyl succinate and 1 ,Cbutanediol [2]. Therefore, the copolymers prepared with dimethyl succinate, butane diol, and monoglyceride was expected to be biodegradable polymers with good properties for use (Scheme I). In this paper, copolymers were produced from dimt9hyl succinate, 1,4-butanediol, and monoglyceride, and their properties were compared with the properties of poly(buty1ene succinate) homopolymer.

EXPERIMENTAL Materials

Dimethyl succinate, 1 ,Cbutanediol, and titanium tetraisopropoxide were reagent grade chemicals from Wako Pure Chemical Industries Ltd., and monolaurin, monoolein, and monostearin were reagent grade chemicals from Tokyo Kasei Kogyo Ltd.. Measurement

GLC was carried out using a Shmazu GC-14 chromatograph (FID) with a capillary column (J&W Scientific DB-I, 0.541 mm x 30 m). Distillates of the

206

Biodegradable polyurethane-based polymers

H2c-CooMe + HOfCH&OH H2C-COOMe

+

HO-CHp-CH-OH 942

li(O-i-Pr)4 c

- MeOH

0I C=O c1lH23

0 C=O CllH23 Scheme 1

polymerization were analyzed at a temperature increasing from 100°C to 300°C at a rate of S"C/min (injection and detector temperature: 320°C). Number (Mn) and weight (Mw) average molecular weights of the polymers were measured by Toso GPC-8010 system using TSK gel column (G2000HR + G3000HR + G4000HR + G5000HR) and monodisperse polystyrenes as standards at 40°C. Chloroform was used as an eluant at 1 ml/min. The differential scanning calorimetric and thermogravimetric studies were carried out using Seiko SSC-5200. Tensile tests were carried out according to SS-207-EP on Toyo Baldwin tensile testing machine. 'H NMR spectra were measured in CDCL using a Bruker AC200 (200 MHz) spectrometer. Preparation of copolymer including monoglyceride The typical procedure was as follows. The solution of dimethyl succinate (180 mmol), butane diol (187 mmol), monolaurin (0.9 mmol), and titanium tetraisopropoxide (0.1 mmol) as a catalyst was heated at 160 "C for 1 h, and then the temperature increased gradually to 200 "C and was maintained for 30 min. The

methanol generated by transesterification was removed through a glass condenser. In the next stage, the prepolymer was further polymerized at 215 "C under 0.1 mmHg for 5 h A part of the obtained copolymer was dissolved in chloroform, and the solution was poured into MeOH to induce the precipitation of polymer. The precipitant was h e d under vacuum at 60°C for one day, and it was used for 'H Nh4R and GPC measurements.

RESULTS Table 1 shows the effect of the amount of monolaunn on the molecular weight and thermal properties of the copolymer. Monolaurin in the range of 0.1 % to 10 % molar ratio against methyl succinate was used for copolymerization. 'H N M R spectra of copolymers showed peaks for methylene and methyl protons of laurate respectively at about 1.2 ppm and 0.9 ppm. This fact shows that monolaurin component is

Biodegradable polyesters

Transesterification

~yH-O~

207

H3C-O-~-C11H23

o

CH2

6

c=o

C"H23

Scheme 2

included in the copolymer. A little amount of methyl laurate was detected by GLC analysis of distillates during the polymerization reaction. When more than 2 % of monolaurin to methyl succinate was used for copolymerization, gelation took place (Run 6-8). These results show that dodecanoate in copolymer is replaced with a corboxylate of the other polymer by transesterification to give a three dimensional polymer network and a methyl laurate (Scheme 2). The molecular weight distribution increased with increasing amount of monolaurin in the copolymer. These copolymers had similar thermal properties to homopolymer (Run I). Table 1. Properties of copolymers including monolaurin *: Dimethyl succinate: butanediol : monolaurin

Run I

2 3 4 5 6 7 8

Molar Ratio* 180 : 188 : 0 180: 187: 0.21 180 : 187 : 0.53 180 : 187 : 0.91 180 : 185 : 1.81 180: 184: 3.61 180: 178: 9.00 200 : 188 : 20.0

Mn 32,400 35,000 46,200 52,200 22,200 gel gel gel

Mw Mn 1.63 1.80 2.10 2.91 3.93 x x x

Tg

Tm

·C

·C

- 39.9 - 37.6 - 35.9 - 38.8 - 40.0 - 35.4 - 39.8 - 42.0

114.1 114.1 113.7 114.1 114.9 112.8 105.4 96.7

aHm Td mJ/mg (2%) 110.1 322.2 92.4 314.3 72.0 311.8 106.4 320.0 107.7 309.0 100.5 311.0 106.2 306.5 65.5 297.6

Table 2. Tensile test of copolymers including monolaurin *: Dimethyl succinate: butanediol : monolaurin

Run Molar Ratio* 180: 188 : 0 I 180: 187: 0.21 2 180: 187: 0.53 3 180: 187: 0.91 4 180 : 185 : 1.81 5 180: 184: 3.61 6 180: 178: 9.00 7 200 : 188: 20.0 8

Elastic MPa 333 375 292 344 330 brittle brittle brittle

Yield Stress Break Stress MPa MPa 29.9 32.4 29.2 29.8 28.5 31.4 32.9 30.0 25.8 28.0 x x x x x x

Break Strain % 119.3 14.3 250.0 190.4 42.6

x x x

208

Biodegradable pol yurethane-based polymers

Table 2 shows the results of the tensile tests of copolymers including monolaurin, elastic strain, yield stress, and break stress of copolymers were not so different to thehomopolymer. However, the break strain of copolymers was greatly dependent on the amount of monolaurin. Copolymers including 0.3 % and 0.5 % of monolaurin had larger break strains than the homopolymer (Run 3 and 4). Table 3 shows the number average molecular weight and molecular weight distribution of copolymers including 0.3 % and 1.0 % monostearin (Run 3 and 6) and monoolein (Run 4 and 7). 'H N M R spectra of copolymers including monostearin and monoolein also showed that stearate and oleate were included in the obtained copolymers. These copolymers had similar molecular weight to copolymers including monolaurin (Run 2 and 5). Table 4 shows thermal properties of copolymer including monostearin (Run 3 and 6) and monoolein (Run 4 and 7 ) . These values were similar to the values of FHp-OH CH-OH CHZ-O-C-C~ iH23

0 Monolaurin

I

Run 1 I 2 3 4 5 6 7

CHp-OH CH-OH

CHp-OH

CH-OH CHp-O-C-Cq7H35

H H

-CH*

CH2 - 0 - C

,c=c,

CHz-CH3

0

0 Monostearin

Monoolein

Molar Ratio* 180 : 188 : 0 (Homowlvmer) 180 : 187 : 0.53 (Monolaurin) 180 : I86 : 0.55 (Monostearin) 180 : 186 : 0.55 (Monoolein) 180 : 185 : 1.81 (Monolaurin) 180 : 186 : 1.81 (Monostearin) 180 : 186 : 1.83 (Monoolein)

f

I

MwfMn 1.63 2.10 1.80 1.77 3.93 2.19 1.48

Mn 32.400 46,200 37,200 34,400 22,200 33,900 25,000

Table 4. Thermal properties of copolymers including monoglyceride *: Dimethyl succinate : butanediol : monoglyceride

I

I

Run Molar Ratio* 180 : 188 : 0 (Homopolymer) 1 I 2 1 180 : 187 : 0.53 (Monolaurin) 1 180 : 186 : 0.55 (Monostearin) 3 4 180 : 186 : 0.55 (Monoolein) 180 : 185 : 1.81 (Monolaurin) 5 6 180 : 186 : 1.81 (Monostearin) 7 180 : 186 : 1.83 (Monoolein)

Tg "C - 39.9 - 35.9 - 38.9 - 38.5 - 40.0 - 35.3 - 37.0

I 1

Tm "C 114.1 113.7 115.0 115.0 114.9 113.7 116.1

1 I

AHm mJ/mg 110.1 72.0 76.7 74.2 107.7 71.3 80.6

I I

Td(2%) 322.2 314.3 301.9 304.4 309.0 298.3 304.4

1

Biodegradablepolyesters

209

Table 5. Tensile test of copolymer including monoglyceride *: Dimethyl succinate: butanediol : monoglyceride

Run Molar Ratio· 180: 188 : 0 (Homopolymer) I 2 180 : 187 : 0.53 JMonolaurin) 3 180 : 186 : 0.55 (Monostearin) 4 180: 186: 0.55 (Monoolein) 5 180: 185 : 1.81 (Monolaurin) 6 180 : 186 : 1.81 (Monostearin) 7 180 : 186 : 1.83 (Monoolein)

Elastic MPa 333 292 316 309 330 330 350

Yield Stress Break Stress

MPa 324 28.5 26.3 26.. 0 28.. 0 26.. 9 29.6

MPa 29.9 31.4 32.2 37.7 25.8 26.4 23.1

Break Strain

% 119.3 250.0 386.8 384.3 42.6 201.1 33.6

homopolymer (Run I) and copolymers including monolaurin (Run 2 and 5). Table 5 shows the results of tensile test of copolymers including monolaurin, monostearin, and monoolein. Although elastic strain, yield stress, and break stress ofcopolymers including 0.3 % monoglyceride were not so different with homopolymer, the break strain of copolymers was larger than the homopolymer (Run 2, 3, and 4). In particular, copolymers including monostearin and monoolein had superior values of break strain. This means that break strain of copolymer is dependant not only on the amount of monglyceride but also on the chain length of the fatty acid of the monoglyceride. CONCLUSION Aliphatic polyesters were produced from dimethyl succinate, butanediol, and monoglyceride with a long-chain fatty acid. IH NMR spectra of copolymers showed that the monoglyceride component was included in the copolymers. Three dimensional networks were built up by transesterification of a part of the monoglyceride component, and gelation occurred if more than 2 % of monolaurin was used for copolymerization. The number average molecular weight and thermal properties of copolymers were unchanged by the amount and type of monoglyceride if less than 2 % of monoglyceride was used. Molecular weight distribution increased with increasing amount of monoglyceride in the copolymer. The elastic strain, yield stress, and break stress of copolymers were similar to the homopolymer. The tensile tests showed that the break strain of the copolymer was very dependent on the amount of monoglyceride. Copolymers including 0.3 % of monoglyceride had larger break stresses than the homopolymer. In particular, break stress of copolymers with 0.3 % monostearin and monoolein were superior. REFERENCES [1] For example, M. Kadobayashi, I. Takahara, Biodegradable polyesters with high molecular weight and their manufacture, Jpn. Kokai Tkkyo Koho JP 09 40,762,

2 10

Biodegradable polyurethane-based polymers

February 1997; E. Takiyama, T. fujimaki, Y. Hatano, and R Ishioka, Manufacture of biodegradable aliphatic polyesters with hlgh molecular weight and good transparency, Jpn. Kokai Tokkyo Koho JP 09 31,176, February 1997; I. Takahashi, K. Kawamoto, A. Matsuda, and T. Masuda, Biodegradable high molecular weight aliphatic copolyesters with good modability and their preparation, Jpn. Kokai Tokkyo Koho JP 08 31 1,181, May 1995; T. Ooyama, H. Isozaki, S. Morita, and K. Sueoka, Thermal contractive aliphatic polyester films and their manufacture, Jpn. Kokai Tokkyo Koho JP 09 57,849, March 1997. [2] Y. Kawaguchi, N. Migita, H. Shiraharna, and H Yasuda, Synthesis and biodegradability of aliphatic polyesters prepared by polycondensation, Polymer I'reprmfs, .Jupun, 1994, 43 (1 I), 4048-49; Y. Imada, Y. kajikawa, M. Taniguchi, K. Koumoto, I. Takahashi, and T. Masuda, Synthesis of aliphatic polyester and Enzymatic Hydrolysis, Kohunshr Ronhunshu, 1998, 55 (8), 497-99.

PREPARATION AND THERMAL PROPERTIES OF POLYURETHANE COMPOSITES CONTAINING FERTILIZER Nobuyuki Yamruchi', Shigeo Hirose', Hyoe Hatrkeyama3 'Taki Chemical Co.,Ltd,2-1-6Sengeih Tsukuba, Ibaraki 305-0047,Japati ZNatio,ia[ ItistitUte of Materials atrd Chemical Research,l-1 Higashs Tsukuba,

[baraki 305-8565, Japan 'Fukui UiiiVersilyof Technology, 3-6-1 Gnkuen, Fukui-ciry, F i h i 910-8505,

Japan

ABSTRACT Polyurethane foam composites containing fertilizer were prepared as follows. Fertilizer particles (urea, diameter:0.2-2.0mm) were mixed with a polyol consisting of molasses polyol, polyethylene glycol, with a molecular weight of 200 (PEG200) and polypropylene glycol with a molecular weight of 3000 (PPG3000 triol). The above mixture was reacted with diphenylmethane diisocyanate (MDI) in the presence of a catalyst to foam polyurethane form composites. The content of fertilizer in the composites was varied from 0 to 15wtY0. The thermal properties of composites were studied by differential scanning calorimetry (DSC) and thermogravimetry (TG). Glass transition temperatures (7'' 's) were determined by DSC. T, 's increased with increasing fertilizer content and with decreasing particle size. Thermal decomposition temperatures (Td's) were determined by TG. Three Td's were observed in TG curves. Td)swere almost constant regardless of fertilizer content. KEYWORDS Polyurethane, composites, degradation temperature

fertilizer,

glass transition

temperature, thermal

INTRODUCTION Polyurethanes are recognized as one of the most important polymeric materials since they can be produced in various forms such as fibers, films and sheets. We have extensively studied the biodegradable polyurethanes which can be derived from plant components such as saccharides and lignin[ 1-51, In the above studies, polyurethanes from molasses showed excellent thermal and mechanical properties and also showed biodegradability with relatively high degradation rates. In the present study, molasses

2 12

Biodegradable polyurethane-based polymers

based PU foams containing solid fertilizer of urea were prepared. The thermal properties of the obtained PUF composites, were studied by DSC and TG. The relationship between thermal properties of PUF and content and particle size of fertilizer is discussed in this study

EXPERIMENTAL Materials Molasses pol yo1 (MOL) consisting of sucrose, glucose and other saccharides was obtained from Tropical Technology Center Ltd. Water in obtained MOL was removed by evaporation and, PPG and PEG were used as received. Commercial grade polymeric MDI was obtained from Mitsui Chemical Industries Co.

Preparation of Polyurethane Composites Polyurethane composites were prepared by the following procedure. MOL (lowto/) was mixed with PPG (8Owt%), PEG (lOwt%)(MOL solution). Fertilizer (urea) or Barium Sulfite (BaSO, as a standard), small amounts of silicone oil (surfactant), 1,8Diazabicyclo [5,4,0] -7-undecene (catalyst : 0.2wtY0against total polyol weight) were mixed with MOL solution using a mechanical stirrer. The obtained solution was reacted with MDI (NCO/OH=I . l ) at room temperature. After the foam sample was obtained in a vessel, the sample was allowed to stand overnight at room temperature. In the above process, the NCO/OH ratio was calculated as follows: where NCOiOH is the molar ratio of isocyanate NCO/OH=(M,,,xWm,) / (MhZOLxWivlOL) and hydroxyl groups per gram of MDI (7.4mmol/g), W,, the weight of MDI, M,, the number of moles of hydroxyl groups per gram of MOL solution.

Measurements DSC measurements were carried out using a Seiko DSC 220. Samples of ca.5mg were heated at a heating rate of 10"C/min in nitrogcn. A Seiko TG 220 was used for TG measurements. Samples of ca. 5mg were heated in nitrogen at a heating rate ol' 10°C /min.

RESULTS & DISCUSSION Phase transition of polyurethane composites was studied by DSC Fig.1 shows DSC curves of PU composites having various fertilizer contents. In each DSC curve, the gap in the baseline due to glass transition is observed. The glass transition temperatures

Polyurethane composites containing fertilizer

2 I3

(T,’s) were determined by a method reported by Nakamura et a1 [ 6 ] . Fig.2 shows the change of Tg’s plotted against filler contents. As shown in Fig.2, TR’s increase more markedly with increasing fertilizer contents when the particle size of fertilizer is decreased i.e. surface area of urea fertilizer is increased. The increase in 7‘’’s of PU composites with BaSO, is smaller than those with fertilizer. The above results suggest that the polyurethane chains interact with urea molecules on the surface of the fertilizer and the main chain motion. The relationship between A : f s of polyurethane composites and filler content is shown in Fig.3 T was calculated as follows[7] : A T = T, - Zg I“, g: Extrapolated End Temperature Tg:Extrapolated Initial Temperature AT increases with increasing filler contents and the walues of PU composites with fertilizer are larger than those with BaSO,. A T values increase with the decrease in particle size of fertilizer i.e. with the increase in surface area of fertilizer. This result suggests that main chain motion of PU’s is a effected by the interaction with urea molecules and the distribution of the units for main chain motion of PU’s becomes broader.

I

I

-70

I

-50

-30

Temperature / “C

Figure 1.

-52

DSC curves ofPU composites containing fertilizer

J

L 0

..

5

10

15

Filler Contents (wt%)

Figure 2. Change of T,’s plotted against filler contents

0

5

10

1s

Filler Contents(wt%)

Figure 3. Change of A T s plotted again st fi 11er contents

214

Biodegradable polyurethane-based polymers

The thermal decomposition behavior of polyurethane composites was studied by TG. Fig.4 shows TG and differential TG (DTG) curves for fertilizer. TG curves show a twostep decrease in the temperature range below 500 . Fertilizer decomposed completely at 500°C. Fig.5 shows TG and DTG curves for polyurethanes containing fertilizer. TG curves show a three-step decrease in the temperature range below 500°C. Thermal decomposition temperature ( Td) was determined as the temperature of the crosspoint of extrapolated baseline and tangent line at the peak temperature of DTG curve as shown in Fig.4 The first decomposition temperature (Td,)at around 160-170°C is the decomposition temperature of fertilizer as shown Fig.4. It is known that saccharides such as sucrose and glucose start to decompose at around 200°C and to form caramel[8]. It is also known that urethane bonds dissociate to form hydroxyl and isocyanate groups at around 250°C [9]. Therefore, it considered that the second decomposition temperature (T,) is mainly related to the decomposition of urethane bonds and molasses, while the third decomposition temperature (Ta) is related to the decomposition of the remaining components. Fig.6 shows TG and DTG curves for polyurethane containing BaSO,. TG curves show a two-step decrease in the temperature range below 500°C. It is considered that Td is mainly related to decomposition of urethane bonds and molasses, T, is related to decomposition of remaining components, since BaSO, is stable up to 500°C.

"c

I 20

I 1W

13 75

p

so

50

175

n

0

I 0

I

I

I

I

IW

200

ml

rm

l 5 yn

Telllprralure I 'C

Figure 4. TG and DTG curves of fertilizer

Figure 5. TG and DTG curves of Polyurethane containing fertilizer (diarneter:O.O2mm)

Polyurethane composites containing fertilizer

400 10

im

-__I-

-

-

-

350

\

2 15

Td3

13 75

z .

E 75

YE4

I25

o

0

<

I

i

f

im

100

m

rm

Temperalure I

0

5

t

0

sm

'C

5 '

Figure 6. TG and DTG curves of polyurethane containing BaSO, (diameter0.02mm)

15

10

Filler Contents(wt%)

Figure7. Change of Td's plotted against fertilizer contents

26

\-":1

p

320 300

1 F(2 Omm)

220

12

200 0

5

10

15

10 0

Bas04 Contents(wt%)

Figure 8. Change of Td'5 plotted against BaSO, contents

5

10

15

Filler Contents(wt%)

Figure 9. Change of WR at 500°C plotted against filler contents

2 16

Biodegradable polyurethane-based polymers

Fig.7 shows the relationship between Z i s and fertilizer content of polyurethane composites. Fig.8 shows the relationship between T i s and BaSO, content. T,‘s of polyurethane composites slightly increase with increasing fertilizer contents. This suggest that some reaction occurs between degradation products of urea and polyurethane during heating. Fig.9 shows the weight residue (WR) at 500°C for polyurethane composites having a different mixture content and particle size. WR (fertilizer) decreases with increasing fertilizer content, while WR (BaSO,) increases with increasing BaSO, content. It is known that BaSO, is stable at 500°C. Therefore, it is considered that the increase in WR is caused by the increase in BaSO, content in polyurethane composites.

CONCLUSION

A l‘s increased with increasing mixture content of polyurethane composites. T,’s and A T s also increased Polyurethane foams were reinforced with urea powder. Ta’s and

with decreasing particle size. The reinforcement effect of urea powder was larger than that of BaSO, powder. WR decreased gradually with fertilizer content, since urea is completely decomposed on heating at 500°C. REFERENCES

1 . S Hirose, K Kobashigawa, H Hatakeyama, ‘Preparation and Physical Properties of Polyurethanes Derived from Molasses’, SEN-I GAKKAISHI, 1994 SO 11 538-542 2. H Hatakeyama, S Hirose, T Hatakeyama, ‘Biodegradable Polyurethanes from Plant Components’, JMacrornol Sci, 1995 A32 (4) 743-750 3. S Hirose, K Kobashigawa, H Hatakeyama, ‘Preparation and Physical Properties of

Biodegradable Polyurethanes Derived from the Lignin-Polyester-pol yo System’, The chemistry tmnd processing qf wood irnd p i m f jibrorrs mnferials, England, Woodhead publishing Ltd. 1996 4. P Zetterlund, S Hirose, T Hatakeyama, ‘Thermal and Mechanical Properties of Polyurethanes Derived from Mono-and Disaccharides’, Polrn Itifl, I997 42 1-8 5. S Hirose, K Kobashigawa, Y Izuta, H Hatakeyama, ‘Thermal Degradation of Polyurethanes Containing Lignin Studied by TG-FTIR’, Polm In?/, 1998 47 247-256 6. S Nakamura, M Todoki, K Nakamura, H Kanetsuka, Therrnochimica Actir, 1988 163 136 7. JIS K 7121 1987 8. H Sugisawa, H Edo, J. FoodSci., 1966 31 561 9. J H Saunders, K C Frish, Poij,wrethaties : Chemistry arid Technologp rn High Polymers, New York, Interscience, 1962 ,

BIODEGRADABLE POLYMERS DERIVED FROM LACTIDE AND LACTIC ACID So0 Hyun Kim'" and Young Ha Kim'

' Division of Polymers, Korea Institute of Science and ;Tecltnology, RO.Box 131, Clteongtyang, Seoul, Korea.

INTRODUCTION Lactic acid that can be made by chemical synthesis oc by fermentation technique is nontoxic and harmless to animals, plants and human beings. Poly(1actic acid)s, which are polymers of lactic acid, can be easily hydrolyzed in the presence of moisture. Due to these properties, poly(1actic acid)s have been used in biodegradable medical applications'.'' such as suture or staples for surgery, sustained release polymers for drug delivery, etc., as well as in agricultural applications for soil treatment including herbicides, soil disinfectants. and the like. In recent years, poly(1actic acid)s have attracted commercial interest for solving environmental pollution problems caused by plastic wastes. The application of poly(1actic acid)s have been extended for use as general purpose biodegradable polymeric materials for packaging materials, food containers, coating materials, and so forth. The polymeric materials for these uses must have a high molecular weight in order to provide the desired strength to the resulting products. Two processes for preparing a poly(1actic acid)s have been known in the arts. One is the ring-opening polymerization of lactide". This process involves two steps: the conversion of lactic acid feed stock to lactide and the polymerization of lactide to give poly(1actic acid)s product. This process is complicated, and time and labor consumptive. But relatively high molecular weight poly(1actic acid)s could be obtained. The another process is the direct condensation polymerization of lactic acid8'. This process is relatively simpler than that mentioned above, but it has a defect in that the resulting polymers has a low molecular weight and thus, shows poor physical properties which are not useful as a general purpose biodegradable material. For the modifications of physical properties of poIy( lactic acid)s, we synthesized star-shaped poly(1actic acid)s by ring-opening polymerization of lactide. Multifunctional hydroxyl compounds were used as initiator while stannous octoate used as catalyst. Multifunctional initiation of lactide by hydroxyl compound was studied and the star-shaped architecture was confirmed. The star-shaped poly(1actic acid)s could be synthesized to have a higher molecular weight than linear poly(1actic acid)s did under certain conditions. The in-vitro degradation of poly(1actic acid)s was performed in a phosphate buffer at 37 OC. This concept that the higher molecular weight poly(1actic acid)s could be synthesized by star-shaped architecture was introduced to the direct condensation polymerization of' lactic acid. Multihnctional hydroxyl compounds were used as star-shaping agent also. . The star-shaped polymer in this article means the polymer in which many polymeric chains are attached to a polyfiinctional substance in a radial arrangement.

21 8

Biodegradable polyurethane-based polymers

EXPERIMENTAL PART Material L-lactide(L) from Boehringer Ingelheim was purified by recrystallization from thoroughly dried toluene under dry nitrogen atmosphere. Pharmaceutical grade L-lactide acid (LA) from Purac was a 90% aqueous solution of the monomer that was minimum 95% optically pure. Tin-2-ethylhexanoate(stannous octoate. (Sn-oct)) was distilled under reduced pressure at 175 O C before use. Pentaerythritol(PEr) was purified by sublimation at 200 O C under reduced pressure. Antimony trioxide (Sb,O,), tin oxide (SnO), dipentaerythritol with 99+% purity, respectively. were purchased from Aldrich Chemical co. and used without purification. All other chemicals and solvents were of analytical grades and used without further purification. Measurements The structure of polymers was analyzed by means of a Varian 300 MHz 'HNMR(Gemini-300) apparatus in deuterated chloroform solution and by an Alpha Centauri FT-IR (Mattson Instruments) apparatus. The intrinsic viscosity of the polymers was measured in chloroform at 25 "C using an Ubbelohde viscometer. Gel permeation chromatography (GPC) measurements were carried out at 35 "C using a Waters ALCiGPC 150C equipped with micro-styragel columns and calibrated with polystyrene standards, which covered a MW range of 1,000- 1,000.000. Chloroform was used as eluent at a flow rate of 1.0 ml/min. Differential scanning calorimeter (DSC) was performed on a Du Pont Thermal Analyzer 2000 using a heating rate of 20 "Cimin in a nitrogen atmosphere. Second heating runs were carried out after sample had been quenched from the melt. Polymerization of L-lactide Polymerization of L-lactide was carried out in a glass ampoule containing a tefloncoated magnetic stirring bar. Freshly recrystallized L-lactide(7.207 g, 0.05 mol) and various amounts of PEr were transferred into the ampoule. Sn-oct in toluene was added to the ampoule using a syringe through a rubber septum under nitrogen atmosphere. The ampoule was sealed in vacuum after 3 times nitrogen purging at 90 OC. The ampoule was heated up to 130 O C in an oil bath for 24 h. The reaction medium was stirred by a magnetic bar as long as stirring was possible. After the reaction was completed. the ampoule was broken and the products were dissolved in chloroform. The polymers were precipitated dropwise into methanof and then dried in a vacuum oven at ambient temperature. For comparison. the polymerization was also carried out with Sn-oct in the absence of PEr.

In-vitro degradation PLLA films were cast on glass surfaces from 2.5%(wiv) chloroform solution. The solvent was slowly evaporated for at least 2 days. and subsequently dried at 40OC for 24 hrs under reduced pressure. Thickness of the films were approximately 150 pm. PLLA films of 1.5cm x 2cm were totally immersed in vials containing 20 ml of phosphate buffer at pH 7 . The vials were placed in a shaker bath at 37 h 2 OC. At varying times,

Biodegradable polymers from lactide

2 19

the vials were taken from the shaker bath. On removal, the films were rinsed thoroughly in distilled water to remove any buffer solution remaining on the film surface, and then dried in a vacuum oven for 24 hrs. Loss of molecular weight was determined by viscometry in chloroform at 25 OC. Polymerization of L-lactic acid

150 g of a 90 % aqueous L-lactic acid solution and various amount of dipentaerythritol (diPEr) were added to a 4-neck flask equipped with a thermometer, a condenser, and a N, inlet tube. The mixture was dehydrated by heating it in a nitrogen stream, while stepwise varying the temperature and the pressure from 105 "C and 350 mmHg to 150 "C and 30 mmHg. After removing about 38 g of water, 0.1 g of Sb,O, was added. The resulting mixture was then polycondensated at an elevated temperature of 200 "C under a reduced pressure of 3 to 5 mmHg. The viscosity of the reaction system increased as the polycondensation proceeded. After heating for 27 hr under reduced pressure. 106 g of a colorless polymer was obtained. RESULTS AND DISCUSSION Model reaction L-lactide was polymerized with Sn-oct in the presence of pentaerythritol(PEr). The 4 primary hydroxyl groups are expected to initiate the polymerization of lactide. The resulting polymer is expected to have 4 arms ended with secondary hydroxyl groups. Whether all 4 hydroxyl groups of PEr are reacted to fixm 4 armed polymer can be investigated by analyzing the unreacted primary hydroxyl groups or the secondary hydroxyl groups at the polymer chain ends. If the molecular weight of the resulting PLLA is not too large, such an analysis can be performed by means of NMR technique. Table 1 shows the results of the polymerizations of L-lactide. The added amounts of PEr and Sn-oct were relatively high in order to obtain low molecular weight polymers. The polymerization was carried out with various amounts of PEr. We assigned the methyl, methine, hydroxyl and methylene groups by the NMR spectrum and determined Table 1. Results of L-lactide oligomerization (at 13O0CC, 5 hrs, 8.648 g (0.06 mole) lactide(L)) with PEr No Mole ratio Mole ratio T,, Mua) DP"' Teoretical [L]/[PEr] [L]/[Sn-oct) "c DPb' 1 2 200 -6 460 2.3 2 1 2 4 200 960 5.7 4

3

8

4

16

5 6 7

11

1360

8.5

8

38

2340

15.3

16

32

2 00 200 200

117

4600

30.9

32

No PEr

4

163

8600

59.7

4

No PEr

200

178

96800

672.0

200

Mw and DP (degree of polymerization) were evaluated from Mark-Houwink equation of branched and linear PLLA based on light scattering data. b ' Theoretical DPs were calculated under the assumption of complete conversion of pentaerythritol (PEr) and lactide.

220

Biodegradable polyurethane-based polymers

the contents of each groups. Also, 1-dodecanol. 1,6-hexanediol. glycerol, pentaerythritol dipentaerythritol (diPEr). tripentaerythritol (triPEr) was used to yield linear and several kind of star-shaped PLLA. From the conversion of alcohols reacted with lactide the number of chain arms could be evaluated, as shown in Fig. 1. Under the assumptions that each alcohol molecules takes part in the lactide initiation and all lactide monomers reacted , when the ratio of lactide to alcohol is 4-8 all alcohol hydroxyl groups should react to form each armed polymers theoretically. However, it is interesting to find out that the polymer contains about 15 - 20% of unreacted hydroxyl groups when lactide to alcohol is 4-8. As the ratio of lactide to alcohol is increased up to 8 or 16, the unreacted fractions of alcohol is rapidly decreased down to a few %. When the ratio is above 32, the alcoholic groups are completely reacted to form each armed star-shaped molecules. When the ratio of lactide to alcohol is above 32 the intensity of CH, or OH end group peaks is too small to be observed so that the NMR technique can not be used any more. As a conclusion, we could confirm the formation of star-shaped polymers when enough lactide was applied for polymerization. The incomplete participation of alcoholic groups in initiation at low concentration of lactide to alcohol may result from steric hindrance.

Polymerization of L-lactide High molecular weight star PLLAs were synthesized and compared with linear PLLAs made in the absence of alcohols. All the polymerizations were carried out by ring-opening polymerization in bulk. Table 2 shows the results of melt polymerization of L-lactide by Sn-oct with or without PEr. For the preparation of linear PLLAs various amounts of Sn-oct were applied. The mole ratios [L]/[Sn-oct] were varied from 1,000 to 50,000 to yield high MW polymers. All the linear PLLAs showed melting points(Tm) of 180 "C. Their conversions were high when the amount of Sn-oct was not extremely small. In the polymerization of lactide, glycolide, or caprolactone initiated by Sn-oct. there is a still controversy on the initiator species whether it is Sn-oct truely or impurities

9 triPEr

0

c

0

10

20

30

Mole ratios Fig. 1. Plot of no. of arms of PLLA evaluated from conversion of polyol series

Biodegradable polymers from lactide

22 1

Table 2. Bulk polymerization of L-lactide at 13O'C for 30 hrs (amount of lactide: 7.207 g (0.05 mole))") Mole ratio Mole ratio Yield Trn 1O-.'.M,,,a) in % OC dL/g [L]/[PEr] [L]/[Sn-oct] 135 0.28 (1 1) 100 5000 78.5 172 0.66 38 5000 1000 177 1.77 146 5000 95.6 2000 180 1.57 95 5000 94.6 5000 180 3.06 267 5000 98.4 10000 98.5 20000 5000 180 5.76 (620) 179 4.55 460 95.5 40000 5000 97.2 179 50000 5000 4.58 (460) 125 95.8 179 1.99 -b) 1000 289 97.8 180 3.75 5000 87.3 178 10000 4.00 (315) 84.5 179 4.02 320 20000 - 3.55 235 50000 35.6 180 T,: melting temperature; [R 1: intrinsic viscosity in CHCl, at 25 'C; the weight-average mol. w t (Mbu)

u

I'

was measured by light scattering in hexatluoro-2-propanol (HFIP), numbers in parentheses were calculated by the obtained [R 1-M,, relationships. b , In absence of pentaerythritol (PEr).

containing hydroxyl groups such as water or certain hydrolyzed products of the monomers. In this study the Mw were analyzed based. on the Sn-oct concentration because tiny water or impurity contents could not be measured (all the polymerization were actually carried out in anhydrous conditions). The Mw of linear PLLA was increased with increasing [L]/[ Sn-oct] at low Sn-oct concentrations, but leveled up and rather decreased when the ratio was above 20,000. The maximal Mw was about 320,000 at 10,000 - 20.000 [L]/[Sn-oct]. The star PLLAs polymerized with Sn-oct/PEr were compared in Table 2. In this case the amount of Sn-oct was kept constantly at 5,000 [L]/[Sn-oct], while the ratio of [L]/[PEr] were varied from 100 to 50,000. Their melting points were not so much different from linear PILAs except ones prepared with large PEr concentrations. In the polymerization of lactide or glycolide initiated by Sn-oct/alcohol, it is generally accepted that alcohol may be a initiator for a cationic polymerization. The relationship between M,, and [L]/[PEr] was shown also in Table 2. That the M,+ of star PLLAs were varied upon PEr concentrations at constant Sn-oct amounts indicates also the role of PEr as an initiator. The M,, of star PLLAs were also increased with increasing [L]/[PEr] at low PEr concentrations, but decreased to exhibit a maximal M, of 620,000 at about 20.000 [L]/[PEr]. The both relationships of linear and star PLLAs were very similar. Such bell-shaped patterns, indicating a maximal M, along the change of initiator concentrations, have been explained by the effect of transesterification in the later stage of polymerization. In Table 1. M,, of oligomeric star PLLAs coincided with the [L]/[PEr] ratio indicating a living character. Jerome et a]. reported in the case of D,Llactide polymerization that the polymerization is no :longer quantitative when the theoretical molecular weight exceeds certain limits. These authors suggested that the transesterification proceed via intramolecular and intermolecular reaction. It is highly desirable to be able to prepare higher Mw PLLAs by designing a star architecture in this study. Such architecture may have four polymerizing centers in one molecule to yield a higher MW molecule. Star shaped molecules are compact. thus to have a smaller coil dimension than linear

222

Biodegradable polyurethane-based polymers

ones of equivalent molecular weight. The coil dimension of polymers can be estimated by either light scattering or viscometry. All the linear and star PLLAs were characterized by light scattering method. The weight average molecular weight(Mw), the root mean square radius of gyration(Rg), and the second virial coefficientt Aj) were evaluated by the Zimm plot methods. Fig. 2 shows the intrinsic viscosity and molecular weight relationship for linear and star PLLAs in chloroform solution(4 arms). It is evident that the branched PLLAs show a smaller intrinsic viscosity of than that of the linear ones at the equivalent molecular weight. This is a solid demonstration of the branched architecture of the PLLA polymerized with pentaerythritol since the branched polymers are known to exhibit a smaller hydrodynamic volume than the linear counterpart. The second vi rial coefficient, A" can also be used as a measure for the branching architecture. In the case of anionically polymerized star shaped multiarms polystyrene(PS), it is well established experimentally and theoretically that A? decreases with the increase of the number of arms. As displayed in Fig. 3, the A, values of star (J)

8

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-' -0

<,

-;::.

6 5 ~

3

2

0.8 0.6 3

10-5 ,

5

«

Fig.2. Plot of intrinsic viscosity as a function of molecular weight for linear ( 0 ) and star PLLA (*) '" 5 r - - - - - , - - - - - - - - - - , ~

~

"

6

10

Nw

FigJ. A2 for !inear( 0 ) and star PLLA (*) in hexaf1uoro isopropanol

Biodegradable polymers from lactide

223

PLLAs were smaller than those of linear ones, which is again consistent with the theoretical prediction to confirm a branched architecture of the PLLAs.

In-vitro degradation In-vitro degradation of PLLAs was investigated in terms of the relationship between degradation behavior and PLLA structure. Fig. 4 summarize the percentages of the molecular weight remained of PLLAs as a function of the immersion time at 37 "C, pH 7. The samples of linear(open ones) and 4-arms star PLLAs(fi1led ones) were selected to have similar molecular weights. As can be seen in Fig. 4, there are distinct differences between linear and star PLLAs in their degradation behavior. which was observed also at other various pH levels. Apparently the star PLLAs seems to be more resistant against hydrolysis than linear PLLAs at the initial stage of degradation. But this trend was reversed as the degradation times elapsed. I t is thought that there are two reasons for the slow decrease of molecular weight for star PLLAs at the initial stage of degradation. One may be due to the specific structure of star PLLAs. Namely, if a bond cleavage is occurred at the center of the molecules statistically, the molecular weight of star PL,LAs would decrease to about three-fourth of it's original molecular weight whereas linear PLLAs decrease to about half of it's original value. Second. we found that the crystallinity of the star PLLAs were higher than linear PLLAs. It is reported by Leenslag et.al that the degradation of PLLAs takes place predominantly in the amorphous region at the initial stage. We observed that the heat of fusion by DSC of star PLLAs(3.4 J/g) were higher than that of linear PLLAs(3.0 J i g ) . Crystallinity differences are confirmed by density measurement using density gradient column. where star PLLA films showed a density of 1.274 but linear PLLA films of 1.267. That star PLLA showed a faster degradation at the later stage of degradation would be explained by the influence of chain end concentration. The degradation of PLLAs would be accelerated by the hydroxyl end groups. The chain end concentration of star PLLAs should be higher than linear PLLAs of equivalent molecular weight. Therefore, the slope of degradation rate of star PLLAs became stiffer than linear PLLAs. In this study, the star architecture of the polymers prepared by PEr/Sn-oct was confirmed to show that the physical properties and the degradation rate of the star PLLA are different from those of linear PLLA. M 100

."C ."C a

-

8

I

95

90

v

0

10

20

30

40

50

60

70

80

90

Time (days)

Fig. 4. Molecular weight loss data for PLLA at pH 7, 37 "C; ( 0 ) M,,=2.8 x lo', ( V ) M , , = 3 . 9 ~ 1 0 ' . (0 )M,,=2.4x105,( )M,,=4.1x105

224

Biodegradable polyurethane-based polymers

Polymerization of L-lactic acid In general, the polycondensation of lactic acid proceeds stepwise in a similar manner to the esterification of a diacid with a diol. The molecular weight of the resulting polymer increases in proportion to the reaction time. During the polycondensation. an amount of water is produced as a by-product, in which hydrolyzes the resulting polymer, and thus, decreases the molecular weight of the polymer. Therefore, it is very important to choose a method to remove the water formed effectively from the reaction system. A variety of dehydration techniques have been known which include. for example. increasing the stirring rate during the reaction. using much reduced pressure. and introducing a nitrogen gas flow. However, these techniques have fundamental limitations as a method to remove water. because the viscosity of the reaction system increases with the increase in the molecular weight of the resulting polymer. Therefore, it is a key point to reduce the viscosity of the reaction system as much as possible in order to obtain a high molecular weight of polymers. The star-shaped polymer has been known in the art and is reported to have a lower melting viscosity than that of linear chain polymers of the same molecular weight”. The poly(L-lactic acid)s. PLLA. in a star-shape. which show a lower melting viscosity, enable efficient removing the water formed during the reaction. In principle. two processes are available for preparing a star form of PLLA, namely, a direct polymerization of lactic acid using a multi-functional reagent. and a two-step process comprising preparing a straight chain PLLA followed by coupling. Among these. the former process is preferred to produce the star-shaped PLLA. In this study, we used the former process to obtain higher molecular weight PLLA by direct condensation pol) merization, and dipentaerythritol (diPEr) having six primary hydroxyl group was used as the multifunctional branching monomer. The reaction mechanism for forming the star-like molecular structure depends on the difference in the reactivities of polyhydroxyl compounds and lactic acid used in the reaction. In the initial stage of the reaction. all primary hydroxyl groups of polyhydroxyl compounds are first reacted with lactic acid to form a small star-shaped structure. The molecular chains grow while maintaining their star-shaped structure by the subsequent reaction of lactic acid, resulting in the desired polymer in a star form having a high molecular weight. This type of reaction is expected to have a higher reaction rate in the growth of molecular weight when it is compared with that of linear polymer at the same conversion of reaction. The results of polymerization are summarized in Table 3. The polymerization was conducted in bulk with continuous stirring. The molecular weight of a polymer slowly increases upon initiation of the polymerization. But after 5 hour, the increase of the molecular weight of the star-shaped PLLA is much faster than that of the corresponding linear PLLA. Such a high molecular weight of the star-shaped PLLA is a distinctive feature in view that the PLLA obtained by the conventional polycondensation technique shows a lower molecular weight. These results reveal that the process of the present work can produce a PLLA having a higher molecular weight in a facile and economically efficient manner as compared with the conventional techniques. From GPC data in table 3 , linear PLLA showed a molecular weight distribution ranging form 1.5 to 2 with unimodal distribution, while star-shaped PLLA showed a molecular weight distribution ranging form 3 to 5 with bimodal distribution. This is a typical demonstration of the branched structure of the PLLA. Various kind of catalyst can be used as the polycondensation catalyst for lactic acid. Table 4 shows the molecular weight of PLLA polymerized with various catalysts. When titanate catalysts were used, the higher molecular weight PLLA was obtained whereas

Biodegradable polymers from lactide

225

Table 3. Condensation polymerization of L-lactic acid at 200 "C (amount of lactic acid: 135g, Sb,O,: 0.2g). Mole ratio [LA]/[diPEr]

Reaction Time (hour) 0.5 1 2

458 764 1600

-

3

3500

-

4 7 10 24 2 5 7 9 23 28 32

5900 13800 16000 17100 773 1900 3000 4900 42200 45 100 56600

-C ) -

-

1800 1800 1800 1800 1800 1800 1800

M 2 114 345 775 2700 8100 251700 25900 31100 196 1100 3300 6600 39400 45700 67600

136 503 2900 6800 16200 42500 50200 58400 626 4000 9200 16400 134600 224000 388000

1.2 I .46 3.73 2.52 1.99 1.65 1.94 1.88 3.19 3.54 2.79 2.49 3.41 4.9 5.74

a) M v was calculated from [q] = 3.48xIO-'Mvo7' b) GPC data (polystyrene calibrated) c ) In absence of diPEr.

obtained polymers showed dark color. SnO showed a similar result. Sb?O;, which is well known as an active catalyst in the esterification reaction. showed the higher molecular weight PLLA. Although catalytic activity was less than titanate catalysts, the obtained PLLA had a good color. The PLLA samples were analyzed using 'H NMR and FT-IR spectroscopies. The NMR and FT-IR spectra of the linear and star polymers were not so much different to each other, as expected. For the characterization of structural differences between linear and star PLLAs, their thermal behavior as well as their mechanical properties were investigated. Tg of linear and star-shaped PLLA was observed at 52.9 "C and 54.8 "C respectively. Star-shaped PLLA has a lower melting point (147 "C) compare to ring opening Table 4. The molecular weight of star-shaped PLLA's polymerized at 200 "C with various kind of catalyst. ~

Catalyst

Reaction Time (hour)

Yield

Mv

Color

p-Toluene sulfonic acid Sb,O, SnO Sn Stannous octoate Dibutyl tin oxide Tetraphenyl tin Titanium-n-butoxide Titanium isopropoxide Zr(OBu),

20 27 12 16 20 20 71 47 55 20

80 93 80 85 71 60 80 60 85 70

14400 48000 48000 35000 16700 12000 17000 67000 47000 14200

medium good poor poor medium medium medium poor poor Medium

226

Biodegradable polyurethane-based polymers

Table 5. Mode Uniaxial Uniaxial Uniaxial Uniaxial Uniaxial Biaxial Biaxial

PLLA film drawing at 80°C Draw Strain Draw at break rate ratio (%) (%) (%/min)

400 400 200 300 500 3OOX300 400x400

325 400 475 475 475 3300 3300

22.820 21.920 18.900 13.400 15.620 4.175 13.850

(Yo)

Stress At peak (kg/mm')

Young's Modulus (kgimm')

14.150 19.450 4.808 4.850 9.850 3.638 3.550

11.020 12.410 5.649 6.551 9.832 4.699 5.467

271.1 311.1

Stress at break (kg/mm')

Strain at peak

8.773 8.655 1.770 2.263 7.192 4.488 3.702

187.2 244.1 286.6 196.6 217.9

polymerized linear PLLA. while condensation polymerized linear PLLA has no melting point. Also the cold crystallization temperature w-as shown at 115 "C. which was compared with 102 "C of ring opening polymerized linear PLLA. The lower melting point and higher cold crystallization of star-shaped PLLA is another typical demonstration of the branched structure of the PLLA. From TGA thermograms of PLLA in nitrogen, linear PLLA started to decompose at about 219 "C. indicating relatively poor thermal stability than star-shaped PLLA. Tensile strength and ultimate elongation of PLLAs were evaluated from films, which was made by hot melt pressing followed by drawing of melt pressed sheets. Star-shaped PLLAs were successfully processed into strong film, whereas linear PLLAs were not processed due to the lower molecular weight of polymer. Drawing of films at different speeds and ratios were performed at 80 "C. The mechanical data for uniaxial and biaxial drawn star-shaped PLLA films are summarized in Table 5. As expected. the higher the drawing speed of film. the higher strain and strength was obtained. Film collected at 400%/min and drawn 4 times reached a tensile strength of 12.41 Kg/ mm', a tensile strain of 21% and tensile modulus higher than 300 Kg/ mm'. Considering that the molecular weight of polymers prepared in this study were not high enough compared to the commercial polylactide (-Mn 130,000), the prepared PLLAs exhibited good tensile strength and modulus values.

REFERENCES 1. J. W. Leenslag and A. J. Pennings, 'Synthesis of high molecular weight poly(Llactide) initiated with tin 2-ethylhexanoate'. Mukromol. Chem., 1987, 188, 1809-

1814. 2. R. K. Kulkarni. E. G. Moore, A F Hegyeli and F Leonard. 'Biodegradable poly(1actic acid) polymers', J. Biomed. Mafeu. Res.. 1971, 5 , 169-181. 3. R. L. Kronenthal. 'Polymer Science and Technology'. New York. Plenum Press. 1975. 4. C. Schugens. R. Gerome and Ph. Teyssie. 'Polylactide macroporous biodegradable implants for cell transplantation'. J. Biomedical Material Res., 1996,30. 449-46 1 5. D. L. Wise, 'Biopolymeric Controlled Release System', Florida, CRC Press. 1985. 6. D. C. Tunc. 'Absorbable bone fixation devices'. US Patent, No. 4 539 981, Sep., 1985. 7. H. R. Kricheldorf, 'Poly(1actones)'. Macromolecules, 1988, 21. 286-293 8. T. Sakai and N. Hashimoto. 'Direct melt polymerization process from lactic acid to biodegeadable polyflactic acid)'. 13'" Polymer Processing Proems, NJ. USA: 1997. 9. J. E. L. Roovers and S. Bywater. 'Preparation and characterization of four-branched star polystyrene'. :24acromolecules, 1972, 5. 385-388.

BIODEGRADABLE POLYURETHANE FOAMS FROM MOLASSES Yuzo Hazulani' I

Aisen Industries Co. Lld., 258 Otioda, Natrkai, Ywakayama 642-0014, Japan

We have developed mainly household articles for use in the kitchen, bath and toilet, from synthetic and also natural polymers. Recently, polymer waste has caused environmental problems. In order to addrcss this problem, our attention h a y been focuses in biodegradable polymers having excellent physical properties. We have successfully developed soft kitchen sponges,

using biodegradable

polyurethane foams from molasses, which can be used in kitchen cleaners and for other purposes. The above biodegradable polyurethanes, which were originally developed by Professor H yoe Hatakeyama, Fukui University of Technology, have saccharide residues in polyurethane chain network. The basic physical properties and also biodegradability of the polyurethanes derived from molasses can be easily controlled by changing the molasses content in polyurethanes. The developed soft sponge products show excellent physical properties and biodegraded when placed in soil. A photograph of a sponge sample is shown in Photograph 1. It was found that the foams show sufficient durability such as resistance to wear and figure in the presence of water and surfactants, after repeated use. It was also found that our products are safe and hygienic. As shown in Fig. 1, they can be biodegraded by microorganisms, when placed in soil.

Photograph 1 .

Photograph of a sample of polyurethane sponge.

Conversion of Environmentally Compatible

and Recyclable Materials into Soil by Microorganisms

w r e 1. m N

h,

BIODEGRADABLE POLYURETHANE FOAMS DERIVED FROM MOLASSES K. Kobashigawa’, T. Tokashiki’, H. Naka’, S. Hirose’ and H.Hatakeyama4 I Tropical Technology Center Ltd.. 5-1 Suzaki, Gushikawa, Okinawa ‘904-2234,Japan 2 Yamato Concrete Industry Ltd.. 1839-1 Konbu, Gushikawa, Okinawa 904-2201, Japan 3 National Institute of Materials and Chemical Research,I - I Higashi, Tsukuba. Ibarnki 305-8565, Japan 4 Fukui University of Technology. 3-6-1 Gduen. Fukui-city, Fukui 91 0-0028, Japan

ABSTRACT Rigid polyurethane(PU) foams containing molasses were prepared according to the following procedure. The molasses-polyol(MP), which is a solution of molasses in polyethylene glycol(PEG) having a molecular weight of 200, was mixed with commercial polyols such as polypropylene glycol(PPG) and sucrose based polyol. The obtained mixture was reacted with diphenylmethane diisocyanate(MD1) after the addition of silicone surfactant, catalyst and blowing agent under vigorous stirring at room temperature. During the reaction, the cream time and the rise time of PU foams were measured. The cream time and the rise time of PU foams decreased with increasing MP contents due to the influence of primary alcohol in PEG. Thermal and mechanical properties of PU foams were studied by thermogravimetry, differential scanning calorimetry and compression tests. The thermal decomposition temperatures of PU’s containing sucrose based polyols decreased slightly with increasing MP content in the polyols. These results suggest that relatively thermally unstable saccharide components in PU molecular chains reduce the thermal stability of PU’s. The glass transition temperatures, the compressive strength and elasticity of PU’s containing sucrose based polyols decreased with increasing MP contents, and those of PU’s containing PPG increased with increasing MP contents in PU’s. This suggests that sucrose in molasses and sucrose based polyols act as hard segments in PU molecules. Biodegradability of PU foams was studied by a soil I>urial method. The PU foams derived from molasses had hgher degradability than that of PU foams without molasses. INTRODUCTION Recently, synthetic polymer wastes are causing serious environmental ploblems since they do not match the ecological system. The needs for biodegradable polymers, for example, aliphatic polyesters and synthetic polymers containing natural polymers have been increasing. Therefore, biodegradable polymers have been studied by many researchers[ 1-61, We have studied preparation, physical properties and biodegradation of polyurethanes(PU’ s) derived from molasses, which were by-products of the cane sugar industry. It was found that saccharides such as sucrose, glucose and fructose in molasses act as hard segments, and they increase glass transition temperature, mechanical strength and elasticity of PU’s[7-111. Concerning the biodegradation, it was found that the rate of biodegradation of PU foams was between that of cryptomeria and beech [7,11,12].

230

Biodegradable polyurethane-based polymers

In the present study, biodegradable rigid PU foams containing molasses were studied by the combination of commercial polyols such as polypropylene glycol (PPG: trio1 type) and sucrose-based PPG(SU4SO: brand name). Thermal and mechanical properties, and biodegradation in the soil of the rigid PU foams were investigated. EXPERIMENTAL Materials Molasses was obtained from Shounan Seito Co. Ltd. Commercial grade polyethylene glycol (PEG) having molecular weights of 200, PPG having molecular weights of 3,000 and SU450 were obtained from Dai-ichi Kogyo Seiyaku Co. Ltd., commercial grade crude-MDI from Mitsui Chemical Industries Co., silicone surfactant from Nihon Unicar Co. Ltd., and catalyst from Kao Co. Water was used for the blowing agent. Preparation of PU foams Molasses-polyol (MP) was prepared according to the following procedure. Molasses was mixed with PEG, and then a small amount of insoluble material was separated by centrifigation. Excess water was removed by evaporation. MP was mixed with commercial polyols such as PPG and SU450. The obtained mixture was reacted with MDI after the addition of silicone surfactant, amine catalyst and water under vigorous stirring at room temperature. The obtained rigid PU foams were allowed to stand overnight at room temperature. Measurements The hydroxy value, the acid value and water content in MP were determind by the method of Japanese Industrial Standard, JIS K 1557, which was the testing method of polyether for PU. In the foaming reaction, the cream time and the rise time of PU foams were measured : the cream time is the time-lapse from the beginning of the mixing process until a visual discernment of change, and the rise time is the time-span from the beginning of mixing to the end of the rise of the foam. Differential scanning calorimetry (DSC) measurements were performed using a Seiko DSC 220C. Samples of ca. 5mg were heated at a heating rate of 10"C/min in nitrogen. A Seiko TG/DTA220 was used for thermogravimetry (TG) measurement. Samples of ca. 5mg were measured in nitrogen at a heating rate of lO"C/min. Compression tests were performed using a Shimadzu Autograph AGS S00D according to JIS K 7220, which is the testing method for compressive properties of rigid cellular plastics. Dimensions of foam samples were as follows : length ca. SOmm, width ca. 50mm and thickness ca. 30mm. Biodegradation of PU foams

PU foams derived from molasses were cut into specimens of 50mm x 25mm x 10mm. The specimens were buried in the cultured soil and they were kept in this state for predetermined periods. Then a certain number of the specimens were taken from the soil, washed, and dried. The average weight loss of ten specimens of each kind of sample was calculated according to the following equation :

Biodegradable polyurethane foams from molasses

23 1

Weight Loss (%) = (Ws - Wd) / Ws x 100 where Ws is the sample weight and Wd is the sample weight after the sample was kept in the soil for a certain time.

RESULTS AND DISCUSSION Preparation of PU foams

The reactivities of PU foams containing molasses were studied by the cream time and the rise time. Fig. 1 shows the change of the cream time and the rise time plotted against MP content in MP-PPG system. The cream time and the rise time of PU foams decreased with increasing MP contents due to the influence of primary alcohol in PEG. As shown in Fig. 2, the cream time and the rise time: of PU in MP-SU450 system increased with increasing MP contents. SU450 contains sucrose-based PPG and aminebased PPG. Therefore, the reaction rates of SU450 with MDI were considered to be fast due to the catalysis of tertiary amine.

Thermal Properties of PU foams Phase transition of PU foams was studied by DSC.Fig. 3 shows the change of glass transition temperatures (Tg’s) plotted against MP content in MP-SU450 system for PU foams. Tg’s of PU foams decrease with increasing MP content in polyols. Molasses is a mixture of sucrose, glucose and fructose. These saccharides have eight, five and five hydroxyl groups per molecule, respectively. However, MP is mainly composed of PEG having two hydroxyl groups per molecule. SU450 contains sucrose-based PPG having eight hydroxyl groups per molecule. These molecules of saccharide and sucrose-based PPG exist as cross-linking points in the polyurethane network. Therefore, the chain length of PU’s between cross-linking points decreases with increasing SU450 content in polyols.

200 CI

3

1 a,

E

i= 100 C 0

.-*

3

i= 100

b

0

C

.-0

1

4-

Cream Time

50-

350

U

w

0 50

I

60

I

70 80

I

90 100

MP Content I O% Fig. 1. Change of the cream time and the rise time plotted against MP content in MP-PPG system for PU foams.

* -

m -

4)

Cream Time

11 I

W

0 50

I

I

I

I

60 70 80 90 100 MP Content I %

Fig. 2. Change of the cream time and the rise time plotted against MP content in MP-SU450 system for PU foams.

232

Biodegradable polyurethane-based polymers

110 -

50 60 70 80 90 100

60 70

80 90 MP Content / %

50

MP Content / Yo Fig. 3. Change of glass transition temperature (Tg) plotted against MP content in MP-SU450 system for PU foams.

100

Fig. 4. Change of starting temperature of thermal decomposition (Tdi) and thermal degradation temperature (Td) plotted against MP content in MP-SU450 system for PU foams.

The thermal decomposition behavior of PU foams was studied by TG. Fig. 4 shows the change of starting temperatures of thermal decomposition (Tdi’s) and thermal decomposition temperatures (Td’s) plotted against MP content in MP-SU450 system for PU foams. Tdi’s and Td’s of PU foams decreased slightly with increasing MP content in polyols. In previous studies[9,10], it was found that molasses decompose at around 150200°C. It is known that the dissociation of the urethane linkage formed between the hydroxyl groups and the isocyanate groups occurs at around 20OoC[13]. As shown in Fig. 4, Td’s of PU’s are at around 280°C and decrease with increasing MP contents. It is considered that relatively thermally unstable saccharide components in PU molecular chains reduce the thermal stability of PU’s.

500

20

I

50

MP-SU450 I

60 70 80 90 100 MP Content / o/o

Fig. 5. Change of density plotted against MP content in MP-PPG and MP-SU450

system for PU foams.

0‘

50

10

I

I

I

I

70 80 90 MP Content I YO

60

JO 100

Fig. 6. Change of compressive strength and elasticity plotted against MP content in MP-PPG system for PU foams.

Biodegradable polyurethane foams from molasses

40

1- rn

233

MP+SU450

0 PEG

0

100 50

I

I

60 70

1

80

I 0 90 100

MP Content I O/O Fig. 7. Change of compressive strength and elasticity plotted against MP content in MP-SU450 system for PU foams.

0

3

6

9

1

2

Time / month Fig. 8. Relationship between weight loss of PU foams and degradation time in cultured soil

Mechanical Properties of PU foams

Fig. 5 shows the change of density plotted against MP content in MP-PPG and MPSU450 systems for PU foams. The density of PU foams in MP-PPG system increases with increasing MP content in polyols. The reactivities of hydroxyl groups with isocyanate groups are as follws[ 14,151: R-CHZ-OH > H,O > RZ-CH-OH > R,-C-OH The reactivity of secondary hydroxyl is lower than that of water. Water acts as the blowing agent. Accordingly, the density of PU foams decreases with increasing PPG content due to the influence of reactivity of water. As shown in Fig. 5, the density of PU foams in MP-SU450 system was at around 40kg/m3. Mechanical properties of PU foams were studied by compression test. Fig. 6 shows the change of compressive strength and elasticity plotted against MP content in MPPPG system for PU foams. The compressive strength and elasticity of PU foams increased with increasing MP content in polyols. Chain lengths of PPG having a molecular weight of 3,000 are longer than those of MP. As a result, it is considered that PPG acts as a soft segment, and MP acts as a hard segment in PU molecules. Fig. 7 shows the change of compressive strength and elasticity plotted against MP content in MP-SU450 system for PU foams. The compressive elasticity of PU foams decreases with increasing MP content in polyols. These results suggest that SU450 composed of sucrose-based PPG acts as cross-linking points and hard segments in PU molecules. Biodegradation of PU foams

Biodegradation of PU foams containing molasses was studied by a soil burial method. Fig. 8 shows the weight loss of PU foam which were kept in the cultured soil for certain periods up to 12 months. In this figure, MP shows the weight loss of PU foams prepared from polyol containing 100% of MP, MP+SU450 that of PU foams prepared from combination of MP and SU450, PEG200 and SU450 those of PU foams prepared without MP, and Bioporl that of the commercial biodegradable polymer samples. As

234

Biodegradable polyurethane-based polymers

seen from Fig. 8, PU foams derived from molasses had higher degradability than that of PU foams without molasses. This suggests that the PU foam prepared from molasses has good biodegradability.

REFERENCES 1 M. Mocluzuki, S. Murase, M. Inagaki, Y. Kanmuri and K. Kudo, ‘Structure and biodegradation of fibers made from poly (butylene succinate-co-ethylene succinate)s’, Sen’i Cakkaishi, 1997,53(9), 348-355. 2 A. Nakamura, N. Kawasaki, S. Aiba, N. Yamamoto, H. Sakai, K. Yamasaki, Y. Maeda, T. Takeuchi and T. Higashi, ‘Evaluation of biodegradability of poly (E-

caprolactone-co-&-caprolactam)and poly (&-caprolactone-co-w-laurolactam)’,Sen’i Gakkaishi, 1997, 53(9), 373-380. 3 J. Kylmae and J. V. Seppaelae, ’Synthesis and characterization of a biodegradable thermoplastic poly (ester-urethane) elastomer’, Macromolecules, 1997, 30( lo), 2876-2882. 4 R. Miyoshi, N. Hashimoto, K. Koyanagi, Y. Sumihiro and T. Sakai, ‘Biodegradable poly (lactic acid) with high molecular weight’, fnt. Polym. Process, 1996, 11(4), 320-328. 5 J. Yu, J. Gao and T. Lin, ‘Biodegradablethermoplastic starch’, J. Appl. Polym. Sci., 1996,62(9), 1491-1494. 6 H. Pranamuda, H. Tanaka and Y. Tokiwa, ‘Physical properties and biodegradability of blends containing poly (E-caprolactone) and tropical starches’, J. Environ. Polym. Degrad., 1996,4( l), 1-7. 7 H. Hatakeyama, S. Hirose, T. Hatakeyama, K. Nakamura, K. Kobashigawa and N. Morohoshi, ‘Biodegradable polyurethanes from plant components’, J. Macromol. Sci.,1995, A32(4), 743-750. 8 T. Tokashiki, S. Hirose and H. Hatakeyama, ‘Preparation and physical properties of polyurethanes from oligosaccharides and lignocellulose system’, Sen’i Cakkaishi, 1995,51(3),1 18- 122. 9 S. Hirose, K. Kobashigawa and H. Hatakeyama, ‘Preparation and physical properties of polyurethanes derived from molasses’ Sen’i Gakkaishi, 1994, SO( 1 l), 538-542. 10 S. Hirose, K. Kobashigawa and H. Hatakeyama, ‘Thermal properties of biodegradable polyurethanes’, Netsu Sokutei, 1994,21(3), 144-146. 11 H. Hatakeyama, S. Hirose, K. Nakamura, K. Kobashigawa, T. Tokashiki and N. Morohoshi, ‘Biodegradable polyurethanes derived from plant materials’, in Proceedings for Intemationul Workshop on Environmentally Compatible Materials and Recycling Technology, Tsukuba, Japan, 1993. 12 N. Morohoshi, S. Hirose, H. Hatakeyama, T. Tokashiki and K. Teruya, ‘Biodegradability of polyurethane foams derived from molasses’, Sen’i Gakkaishi, 1995, 51(3), 143-149. 13 J. H. Saunders and K. C . Frisch, Polyurethane Chemistry and Technology in High Polymers Vol.XVI Part I, New York, Interscience, 1962. 14 K. Iwata, Polyurethane Resin Handbook, Tokyo, Nikkan Kogyo Shinbun, 1987. 15 Y. Imai, Polyurethane Foams, Kyoto, Koubunshi Kankoukai, 1987.

POLYURETHANES FROM PINEAPPLE WASTES Manuel Moya', JoSe Vega', Maria Sibaja' k Marlen D u d s '

'Loboratorio & Polimeros (powCnvA)),UniversihdNacional, Ap. 86-3000 Heredie Costa Ricu

ABSTRACT Agriculture is an important activity in many countries. Besides the final product, many sub-products are generated. In Costa Rica the total production of pineapple was higher than 200,000tons in 1997. Industrialization of this crop produces large amounts of lignocellulose waste like pineapple peel (PAP). 1% natural materia1 contains hydroxyl groups and it was used as polyol for polyurethane synthesis. A solution of PAP in PEG with a hydroxyl content of' 3.6 mmoVg was prepared and polyurethane foams were obtained by reaction with MDI. The compressive and thermal properties of these foams were measured. It was found that strength and strength modulus increase with increasing PAP contents. A decrease in thermal stability of lignocellulosepolyurethane was observed.

KEYWORDS Polyurethanes,pineapple wastes, lignocellulose

INTRODUCTION Agriculture k a very important economic activity in Costa Ria. In 1997 the total amount of pineapple (PA) production was higher than 200 000 tons produced in almost 8000 ha. The industrialization of PA produces large amounts of wastes, in the order of lo5 tons per year (60% of the whole crop), containing mainly pineapple peel (PAP,33%), foliage (21%) and others (46%). The PAP mah components are biopolymers like cellulose (70%) and lignin (16%) which contain hydroxyl groups. These natural polymers are hdamentally biodegradable. Accordingly, synthetic polymers containing plant components, such carbohydrates and lignins, are essentially biodegradable. PU is one of the most usefbl three-dimensional polymers, commonly prepared by reacting diisocyanates, as diphenyhethane diisocyanate (MDI), and polyols as polyethylene glycol (PEG). Several researchers have described the use of renewable materials as polyol~fir polyurethane (PV)preparation'". From ecologicai considerations PU's containing natural polymers such PAP are beneficial, since it is possible to convert an agricultural waste into useful biodegradable materials. In the present study the use of PAP in PU foam preparation and its mechanical and thermal properties was investigated.

236

Biodegradable polyurethane-based polymers

MATERIALS t METHODS Preparation of PAP dissolution in PEG Using a mechanical sieve shaker 4 fractions of PAP were obtained. Their particle size range was between 425-250 microns, 250-180 microns, 180-150 microns and 150125 microns. After oven drying at 110 "C for 4 hours, one part of PAP was heated for 4 hours at 225 OC with one part of PEG having a molecular weight of 400. The obtained black pasty solution was vacuum dried for 24 hours at 80 "C. In order to measure the PAP solubility in PEG, 0.5 g of this solution were mixed with 10 ml of dimethylformamide and filtered through a Whatman PTFE membrane having a pore size of 1 micron. The effect of several conditions (the particle size, reaction temperature, reaction time, molecular weight of PEG and PAPPEG ratio) on dissolution of PAP was investigated. Determination of hydroxyl content The OH content of PAP solution was determined according to the Japan Industrial Standard (JIS) K 1557. The acid group content was determined by potentiometric titration using 0.1 N NaOH aqueous solution Preparation of PU foams The obtained PAP solution was mixed with 3-5 drops of catalyst (di-n-butyltin dilaurate), 0-0.3 g of water and MDI using a mechanical stirrer. The stirrer was removed itom the mixture after foams were observed, and then the mixture was allowed to stand at room temperature. Measurements Compressive properties of the PU foams were measured according to JIS K 7220 using a Tendon model RTM-100 h m Orientec Corporation. The cross-head speed was 2 mm/min. The sample dimensions and its apparent density was calculated before mechanical measurement. The decomposition temperatures and the weight residue (WR) at 585 "C of PU foams were measured with a Shimadzu TGA, model 40M.The thermogravhnetric (TG) curves were obtained at a heating rate of 20 OC/min in Nitrogen gas (30 mL./min atmosphere). RESULTS & DISCUSSION The chemical composition of PAP is similar to wood that is thermally degraded at 260 "C *. It is also known that hemicellulose and lignin are degraded by hydrolysis catalyzed by the acetic acid formed fiom the acetyl groups of hemicellulose 9 . Therefore, these two degradation reactions are considered to contribute to the degradation of lignocellulosewhen PAP is heat-treated in PEG. Solubility of PAP in PEG was dependent on several conditions such as particle size of PAP powder, heat-treatment temperature and time, PAPPEG ratio and molecular

Polyurethane from pineapple wastes

237

Table 1. Hydroxyl contents of PAP solution in PEG400

Substrate

OH groups

Pineapple peel Coffee bean Parch

(mm0Vg) 3.6 f 0.2 3.4 f 0.1

COOH groups (mmoVg) 1.1 f 0.1

TOTAL OH (mmoVg) 4.7 f 0.2

1.2 f 0.1

4.6 f 0.1

weight of PEG. The results show that the insoluble materials decreased with the decrease in particle size. It seems that 150-125 microns are small enough to produce good solubility. This is a smaller particle size than in the case of wood lo and coffee bean parchment (CBP) 'I. Maximum solubility is reached at 225 "C, less drastic conditions than in the case of CBP (250 "C)'I. At higher temperatures the insoluble materials increased due probably to the carbonization of lignocellulose. The reaction time was 4 hours at a PEGPAP Solubility of PAP increased with the decrease in M.W. of PEG, probably ratio of 1. due to the greater diffUsion of low M.W. PEG molecules into the PAP. According to these results, the optimum conditions of dissolution were considered as follows: particle size of PAP powder, 125-150 microns; heat treatment temperatute, 225 OC; heat treatment time, 4 hours; PEGPAP ratio, 1 and PEG Molecular weight, 400.

It was shown that the molecular ratio of total amount of isocyanate groups to the total amount of hydroxyl groups (NCO/OH) is a very important parameter for PU synthesis and its properties '. So it is necessary to know the hydroxyl content of the dissolution of PAP in PEG. This information is shown in Table 1. In this study the NCO/OH ratio was 1.2. PAP is a mixture of polysaccharides and lignin containing primary, secondary and phenolic hydroxyl groups. Therefore, the reaction rates in lignocellulosemolecules with isocyanate groups were considered to be slow, so di-n-butyltin dilaurate was used as catalyst. Homogeneous semi-rigid foams having densities between 84 kg/m3 and 115 kg/m3 can be obtained (Figure 1). Foam density was similar to that reported for PU fiom coffee b a n lignocellulose ". Figure 2 shows the change of Q and E with PAP contents of PU foams. It can be observed that Q and E increase with increasing PAP contents. This suggests that PAP components act as a hard segment in PU molecules. It was found that it is possible to control PU compression properties by varying PAP content of foams. Hatakeyama et al." found the importance of considering density (p) in estimating mechanical properties of PU foam,since cell wall thickness of PU foam is dependent on density when the pore size of each cell is assumed to be almost the same. In order to remove the effect of foam density in compression properties, Figure 3 shows the relationship between PAP content and o/p and E/p of PU foams. Again, it is clear that strengthand modulus of PU foams increase with increasing of PAP contents. The thermal degradation of PU foams was studied using Thermogravimetry (TG). The decompositiontemperature (Td) is defined as the temperature corresponding to the tangent drawn at the inflexion point in the curve. The Table 2 shows the decrease of Td and an increase in the residual components at 585 "C in the PU foams containing PAP lignocellulose. A similar behavior was observed with PU obtained fiom coffee bean lignocellulose ' I .

238

Biodegradable polyurethane-based polymers

120 110 100 90 80

70

~

0

5

8 11 PAP content/%

14

17

Figure 1. Densities of PU foams with different PAP content.

100

0

r

0

5

I

8

I1

T-4 0 14

17

PAPcontent/% Figure 2. Change of compression strength and compression modulus of PAP PU foams

Polyurethane from pineapple wastes

0

5

8

11

239

17

14

PAP confenfjo/o Figure 3. Change of compression strength@ and compression moduludp of PAP PU foams

Table 2. Decomposition temperature and ash contents of PAP polyurethanes

PAP content/% 0 5 8 11 14 17

T d "C 400 370 370 362 363 360

WR at 585 "CPh 15 23 22 24 22 23

WR: Weight Residue CONCLUSIONS

-

The solubility of PAP in PEG was dependent on conditions such as particle size of PAP powder, heat-treatment temperature and time, molecular weight of PEG, PEGPAP ratio. The optimum conditions for the highest solubility were as follows: particle size 125150 microns, temperature 225 "C, time 4 hours, PEG-400 and PEGPAP ratio 1/1. The OH content of the solution of PAP in PEG is 4.7 InmoVg. PU foams were successllly prepared fiom PAP solutions. Lignocelluloseacts as a hard segment in PU molecules. The PAP decreases the thermal stability of polyurethanes.

240

Biodegradable polyurethane-based polymers

ACKNOWLEDGEMENTS

The authors acknowledge financial support from the Universidad Nacional of Costa Rica, the project UNA-BID-COMCIT, the Iberoamerican Science and Technology Program (CYTED) and JICA of Japan.. REFERENCES 1. H. Yoshida, R. Morck, K. P. Kringstad and H. Hatakeyama, J. Appl. Polym. Sci., 1990,40, 1819. 2. A. Reimann, R. Morck, H. Yoshida, H. Hatakeyama and K. P. Kringstad, J Appl. Polym. Sci., 1990,41,39.

3. H. Yoshida, R. Morck, K. P. Kringstad and H. Hatakeyama, J Appl. Polym. Sci., 1997,34, 1187. 4. S. Hirose, S. Yano, T. Hatakeyama and H. Hatakeyama, In: Lignin, Properties and Materials. Eds W. Glaser and S. Sarkanen. ACS Symposium series, 397. Ellis Horwood Ltd., UK, 1989.

5. S. Yano, S. Hirose and H. Hatakeyama, In: Wood Processing and Utilization,Eds J. F. Kennedy, G. 0. Phillips and P. A. Williams, Ellis Horwood Ltd, UK, 1989. 6. K. Nakmura, T. Hatakeyama and H. Hatakeyama, Pot'ym. A&. Technol., 1991, 2, 41. 7. K. Nakamura, T. Hatakeyama and H. Hatakeyama, Polym. A h . Technol., 1992, 3, 151. 8. M.V. Ramiat,J. Appl. Polym. Sci., 1970, 14, 1323. 9. M. Waymanand M.G.S. Chua, Can. J. Chem., 1979,57,2612. 10. S . Hirose, K. Nakamura, H. Hatakeyama, J. Meadows, P. A. Williams and G. 0. Phillips, In: Cellulosics; Materials for Selective Separations and Other Technologies,Eds J. F. Kennedy, G. 0: Phillips and P. A. Williams. Ellis Horwood Ltd, UK, 1993. 11. H. Hatakeyama, K. Kasuga, M. Aikawa, S. Hirose, M. Duran, M. Moya, R. Pereka and M. Sibaja, Research on Polyurethanes fiom Lignocellulose, Report of ITIT Project, Japan, 1995. 12. H. Hatakeyama, S. Hirose, K. Nakamura and T. Hatakeyama, In: Cellulosics: Chemical, Biological and Material Aspects, Eds. J. F. Kennedy, G. 0. Phillips and P. A. Williams, Ellis Horwood Ltd, UK, 1993.

PREPARATION AND PHYSICAL PROPERTIES OF SACCHARIDE-BASED POLYURETHANE FOAMS Yasu him Asano’, Hyoe Hatakeyama*’, Shigeo Hirose’ and Tatsuko Hatakeyama’ Fukui University of Technology, 3-6-1 Gakuen, Fukui-city, Fukui 910-8505, Japan National ItistitUte of Materials and Chemical Research, I -I Higashi, Tsukuha, Ibaraki, 305-8565, Japan

’Otsuma Women’s Universi& I2 Sanbancho, Chiyoda-ky Tokyo 102-8357, Japan ABSTRACT Biodegradable polyurethane (PU) foams were prepared from a polyol mixture containing a molasses polyol (mixture of molasses and polyethylene glycol, MP), polypropylene glycol (PPG, diol type, molecular weight :3000),a graft polyol (GP) and a polyester polyol (PE, diol type, molecular weight 2200). The above mixtures were reacted with polyphenyl polymethylene polyisocyanate (MDI), toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI), lysine diisocyanate (LDI) and/or lysine triisocyanate (LTI) in the presence of small amounts of silicone surfactant, catalysts, blowing agent and water. The mechanical properties of the obtained PU foams were measured by compression tests. The values of compression strength ( 0 ) and compression elasticity (E) increased with increasing amount of isocyanates. Some PU’s containing inorganic fillers such as barium sulfate (BaSO,) were prepared in order to control the ridity of the samples.

INTRODUCTION Since natural polymers are basically biodegradable, they are part of the ecological system. Alcoholic OH groups in mono-, poly- saccharides and phenolic OH groups in lignin are able to react with isocyanate. Polyurethanes (Pll’s), which have the molecular structure show in Fig 1,can be prepared[ 1-63. In this study, polyurethane (PU) foams were prepared using polyols such as PEG, PPG, GP and PE. Molasses contains sucrose, glucose and fructose. In our research, several isocyanates such as MDI, TDI, HDI, LDI and LTI were used. Thermal properties of the obtained PU’s were studied by differential scanning calorimetry (DSC) and thermogravimetry (TG). Mechanical properties of PU’s were also studied by compression tests.

EXPERIMENTAL Materials Molasses polyol (molasses + polyethylene glycol, MP) was obtained from Tropical Technology Center, Ltd. The above polyol was mixed with PPG, GP (styrene- and acrylonitrile-grafted polyether, Asahi Glass Ltd., EX941), PE (molecular weight 2200 diol-type, Nippon Polyurethane Ltd., N-2200), small amounts of water, silicone surfactant, catalyst (dibutyltin dilaurate and amines) and blowing agents. The mixture was reacted with aromatic isocyanates (polymeric-MDI and toluene

242

Biodegradable pol yurethane-based polymers

diisocyanate, TDI) and aliphatic isocyanates (hexamethylene diisocyanate, HDI, lysine diisocyanate, LDI, and lysine triisocyanate, LTI) under vigorous stirring at room temperature. After foams were obtained in cups, the samples were allowed to stand overnight at room temperature.

Measurements DSC measurements were carried out using a Seiko DSC 220C. Samples of ca. 5 mg were heated at a rate of 10 "C/min in nitrogen. A Seiko TG/DTA 220 was used for TG measurements. The measurements were carried out in nitrogen at a rate of 20 " C h i n . Compression tests were performed using a Shimadzu AG-2000D according to JIS K6401 and K7220 methods at room temperature. Number of test pieces was 3 / each test. Sample size was 40mm X 40mm X 30mm. Compression speed was 3.0mm/min.

RESULTS AND DISCUSSION Preparation of PU foams

In this investigation, we used molasses as saccharides. At first in order to prepare PU foams, it was necessary to dissolve molasses into PEG. The preparation scheme is shown in Fig. 2. Silicone surfactant, catalyst and water were added to the solvent before mixing. This premixture was reacted with several isocyanates and blowing agents were added. In some cases inorganic material such as barium sulfate (BaSO,) was also added in order to control density and mechanical properties. Density Fig. 3 shows the relationship between density ( 0 ) and BaSO, content in PU's foams from the PE-GP-MP-LDI+BaSO, system. The density of PU foams increased with increasing BaSO, content.

v

0-CONH--R,-HNOC-O-(l2

(H~C)*-O+

i

p3 -HNOC

Figure 1.

'CONH-

Schematic molecular structure of saccharide- based PU (For example, glucose and PEG, PPG, PE with 2,4-TDI)

Saccharide-basedpolyurethane foams

243

I Polvurethane foams I Figure 2.

Preparation of polyurethane foams

DSC Fig. 4 shows DSC curves of PU’s with various MDIADI ratios in the PE-PPG-MP(MDI/TDI) system. MDIRDI ratio is indicated in each DSC curve. Glass transition temperature (Tg)increases with increasing MDI content, since MDI has rigid phenyl methane units. This suggests that polymeric-MDI reduces the mobility of the main chain of PW molecules. At the same time, the increase of MDI contents increases the cross-linking density of obtained PU’s.

TG Fig. 5 shows TG curves of PU’s with various BaSO, contents in the PE-GP-MPLDIt BaSO, system. Two degradation temperatures (Tdla.nd Td2)are observed. Fig. 6 shows the relationships between mass residue at 450°C (WR),Td’sand BaSO, content in PU’s. WR increases with BaSO, content. TG measurements were carried out from room temperature to 600°C.In this heating range:, BaSO, can not be thermally decomposed.

s

0.2

--. Q

0.1 0

Figure 3.

20

40

60

80

BaSO, content 1 wt % Relationship between density and BaSO, content of PU’sfrom PE - GP - MP - LDItBaSO, system

244

Biodegradable polyurethane-based polymers

M D I / T D I ratio

1010 812 6 I4

416

-

218

0110 -100

-5 0

0

50

100

T /’ “C Figure 4.

DSC curves and MDIEDI ratio of PU’s from PE-PPG-MP-(MDImDI) system

BaSO, content (%)

100

200

300

400

500

T/‘C Figure 5.

TG curves with various BaSO, contents of PU’s from PE-GP-MP-LDI+BaSO, system BaSO, content (95)= [BaSO, (g) I polyol (g)] X 100

Saccharide-based polyurethane foams

245

450

50

40

400

g

20

350

10 0 I 0

Figure 6.

I

I

300

20 40 60 80 BaSO, content / wt 96 Relationship among WR (450°C), Td'sand BaSO, content of PU's from PE-GP-MP-LDI+BaSO, system 0 :WR, M: Tdl, 0:T d 2

Results of mechanical analysis Mechanical properties of these PU foams were also studied by compression tests. Fig. 7 shows the changes of compressive strength (a)/apparent density ( Q) and compressive elasticity (E)/Q as a function of MDI additive ratio. These results agree well with DSC results and suggest that polymeric-MDI acts as a hard segment in PU molecules. Fig. 8 shows the results of compressive tests for PE-GP-MP-LDI+BaSO, system. 0 / 0 and E/ p increase with increasing BaSO, content, since BaSO, is a rigid material. PU foams are very flexible. Accordingly, the addition of BaSO, is effective in controlling the mechanical properties of PU foams. 1500

15

1

h

3

E

Y

1

lo00

10

h

3

E I.'

\" 2

tL,

z

\

\

a

500

5

0

0 100

b

$

40 60 80 MDI ratio / 5% Relationships among (T / p , E / 6) and MDI ratio / 96 of Figure 7. PU's i?om PE-PPG-MP-(MDI/TDI systems a:a / p O:E /) P MDI ratio / % was calculated according to the following equation MDI ratio / % = [MDI/(MDI+TDI)] X 100

0

20

246

Biodegradable pol yurethane-based polymers

1.5

200

n

h

-7

3

E i-'

\" cd

a

3

\

100

\

\"b

0.5

\" lu

0 0 Figure 8.

40 60 80 BaSO, content / wt %I Relationships between u / p , E / p and BaSO, content of PU's from PE-GP-MP-LDI+BaSO, systems 20

0:a

/ p

O:E/p

CONCLUSIONS (1) Flexible PU foams were successfully prepared. (2) Saccharide-based polyurethane foams can be prepared from several isocyanates (3) Rigidity of PU foams can be controlled by addition of inorganic material such as BaSO,.

REFERENCES 1 T Tokashiki, S Hirose and H Hatakeyama, 'Preparation and physical properties of polyurethanes from oligosaccharides and lignocellulose system', Sen-i Gakkaishi, 1995, 51(3) 66-70. 2 S Hirose, K Kabashigawa and H Hatakeyama, 'Preparation and physical properties of polyurethanes derived from molasses', Sen-i Gakkaishi, 1994,50111) 78-82. 3 H Hatakeyama, S Hirose and T Hatakeyama, 'Biodegradable polyurethanes horn plant components', J Mol Sci Pure & Appl Chem, 1995, A32(4) 743-750. 4 H Hatakeyama, 'Biodegradable plastic derived from plant resources', Mokuzai KOUQOU,1993,48(4) 161-165. 5 S Hirose, K Kobashigawa and H Hatakeyama, 'Preparation and physical properties of biodegradable polyurethanes derived from the lignin-polyester-polyol system', The Chemistry and Processing Wood Plant Fibrous Materials (J. K. Kennedy et al. Eds), Woodhead, Great Yarmouth, UK, 1996. 6 P Zetterlund, S Hirose, T Hatakeyama, H Hatakeyama and C AAlbertsson, 'Thermal and mechanical properties of polyurethanes derived from mono- and disaccharides', Polym Znt, 1997, 42(1) 1-8.

BIODEGRADABLE POLYMER IN SEED PROTEIN FROM CORN Jun Magoshi' & Shigeo Nakaniura' 'National Institute of Agrobiological Resources, Science and Technology Corporation Tsukuba, Ibaraki 305-8602, Japan, Japan 'Department of Applied Chemistry, Faculty of Engineering, Kanagawa University, kanagawa-ku, Yokohama 221-0802, Japan

ABSTRACT Zein film cast from aqueous ethanol and methanol is amorphous in the random coil conformation. In the DSC measurement water in the specimen is lost by evaporation at about 100°C. The glass transition is observed at 165°C. The amorphous zein crystallize to B -crystals at about 210°C accompanied by the random-coil

3

f3 -

form conformational transition. The thermal degradation of zein occurs at about 320°C. Steam treatment of zein film results in the conformational change to the a - and f3 forms, simultaneously, irrespective of treating temperature.

KEYWORDS Corn, protein, biodegradable polymer, thermal properties, glass transition

INTRODUCTION The seed of corn contains several kinds of proteins. These proteins are classified into four components according to their solubility: prolamine (soluble in alcohol), albumin (soluble in water), globulin (soluble in saline), arid glutelin (soluble in aqueous acid and alkali solutions). Prolamine, the alcohol-soluble fraction, is designated as zein. However, very few scientific studies have been reported on seed proteins, especially ones directly concerned with the structure and physical properties of these proteins. In the present study, the thermal properties such as glass transition and crystallization of zein films was examined in detail.

248

Biodegradable polyurethane-based polymers

MATERIALS & METHODS

Substrates Zein protein was isolated from maize seed. Corn meal was ground in a mill and then by pestle in a mortal. The corn meal was stored without prior defatting with petroleum ether. The corn meal was extracted with either 60 or 70% (by weight) aqueous ethanol at room temperature for 12 h. The extract was dialyzed against distilled water. The precipitated protein was centrifuged and lyophilized. The protein prepared was a white yellow granular solid. The protein was again dissolved in 70% ethanol and washed repeatedly with dichloroethane or petroleum ether until all color was removed. After concentrating by removal of a part of ethanol by vacuum distillation, the solution was poured into a large volume of 1% sodium chloride solution. The gummy substance obtained was washed with water to remove sodium chloride and then freeze-dried after the remaining ethanol was allowed to evaporate. The film specimen was obtained by casting the protein solution in aqueous ethanol on a glass plate at 20°C.

Measurement Differential scanning calorimetry (DSC) scans were recorded on a Seiko DSC-100 at a heating rate of 10 Wmin. Thermogravimetry was done with a Rigaku TG-DSC 8085E 1. Linear thermal expansion was measured using a Rigaku TMA type CN 8095 by recording the change in length of the film specimen under constant tension at a heating rate of 10 Wmin. X-ray diffraction patterns were obtained with a Rigaku D3F X-ray diffraction apparatus. Ni filtered CuK (Y radiation was used at 35 kV and 20 mA. Infrared spectra were recorded on a Nicolet Model 60SCR infrared spectrometer. The specimen placed between two NaCl plates was heated at 5 Wmin with a Hitachi HPC-

300 temperature-programming controller. The temperature was measured by a thermocouple attached to the NaCl window. Band intensity was determined by the baseline method.

RESULTS & DISCUSSION The amino acid composition of zein was determined by an amino acid analyzer to

Biodegradable polymer in seed protein

249

consist of 28% glutamic acid, 23% valine, 14% lysine, 11% proline, and 9% alanine. Zein was separated into four fractions by SDS-PAGE according to molecular weight. The molecular weights were 24 kD (strong), 27 kD (strong), 38 kD (weak), and 42 kD (weak), respectively. Figure 1 shows X-ray diffraction patterns of zein film cast from aqueous ethanol solution that annealed at 190°C for 10 min and at 210'C for 30 min. An amorphous halo is observed for the as-cast film, whereas diffraction rings appear by annealing films and these diffraction patterns are identified to be caused by B -form crystals. In Figure 2 are shown the infrared spectra of zein film before and after heat treatment at 210°C for 30 min. The as-cast film shows absorption bands at 1660, 1540, 1240, and 650 cm-1, which are assigned to amide 1 , ]I.a, and V bands for the random-coil conformation, respectively. By annealing the film at 210°C for 30 min, new absorption bands appear at 1630, 1535, 1265, and 700 cm-1. They are assigned to amide [ ,

,a, and v

bands for the

B -form conformation, respectively. From the infrared spectra and X-ray diffraction patterns, zein film cast from

I

a

b

C

Figure 1 X-ray diffraction patterns of zein films from corn (a) untreated and (b) treated at 190°C for 10 min and (c) treated at 210°C for 30 min.

250

Biodegradable polyurethane-based polymers

:

b

v,

2 E

a

srn L

4 4 SJ

c

Q c, L Q,

4 I

I

2000

1600

1200

I

800

400

wavenumber (crn- 7) Figure 2

Infrared spectra of zein films from corn (a) untreated and (b) treated at 210°C

for 30 min. aqueous ethanol is amorphous and has a random-coil conformation and crystallizes to the /3 -crystal form by heat treatment accompanied by the random coil

+

P -form

conformational transition. Curve a of Figure 3 shows DSC curves of amorphous zein film in the random coil conformation under nitrogen. Two endothermic peaks appear at 100 and 320°C. An exothermic peak at 210°C and an endothermic shift at 165°C are also observed. The endothermic peak at 320°C is prominent, suggesting the degradation of zein, since an abrupt weight loss is observed in the thermogravimetry (TG) curves of zein film at about 300°C under nitrogen (Fig.4). The TG curve shifts to higher temperature by heat treatment, indicating the increase in thermal stability. The broad endothermic peak observed at 100°C is attributed to the evaporation of water in the specimen, since the peak became smaller when the specimen was annealed at 210°C for 30 min (Fig.3, curve b).

Biodegradable polymer in seed protein

165 I

25 1

210

100

b

320 I

I

I

100

200

300

400

t/c Figure 3 30 min.

DSC curves of zein films from corn (a) untreated and (b) treated at 210"Cfor

0 20

-

0 0

60

80

a

I00 0

I00

I

1

200

300

I

400

t/"C Figure 4 min

TG curves of zein films from corn (a) untreated (b) treated at 210°C for 30

252

Biodegradable polyurethane-based polymers

0

Figure 5

100

200

Thermal expansion curves of zein films from (a) untreated and (b) treated at

210°C for 30 min. Figure 5 shows the linear thermal expansion curves of zein film. The as cast specimen contracts slowly up to 130°C with increasing temperature, and at 165"c, the length of the specimen begins to increase abruptly (curve a). The initial contraction is attributed to evaporation of water, since prior drying at 100°C makes the contraction smaller. The length of zein film heat-treated at 210°C starts to increase abruptly at 167°C and then the specimen started to contract at 186°C (curve b). Therefore, the endothermic shift of the DSC curves at 165°C is due to the glass transition of zein. The exothermic peak at 210°C almost disappears by heat treating the specimen at

210°C for 30 min, as shown in curve b of Figure 3. Considering the results of X-ray diffraction and infrared spectroscopy mentioned above, this exothermic peak is attributed to the crystallization of amorphous zein to random-coil

-, B -form conformational transition.

0-crystals accompanied by

the

Biodegradable polymer in seed protein

253

0.4 Q,

u

C

rp

20

2 0.2 -?

Figure 6

Temperature dependence of infrared absorption bands of amorphous zein

film. Random-coil bands at

(0) 1660 and (A) 1540cm" and

B -conformation band at

(0)1535cm-'.

To further examine the random-coil

+

B-form transition, three infrared bands of

amorphous zein film were measured with stepwise increasing of temperature, as shown in Figure 6. The absorbance of the bands at 1660 and 1.540 cm-1, the amide I and

fl

bands of the random-coil conformation, decreased linearly with increasing temperature, until an abrupt change in slope occurs at about 189°C. The absorbance of the amide

II

band of the B -form at 1535 cm-1 also

decreases slowly with increasing temperature and begins to increase abruptly at 188°C. Therefore, the conformational change of random-coil to the 188°C.

13 -form takes place above

Biodegradable polyurethane-based polymers

'254

125°C

120°C

100°C

untreated

1800

1600

1400

800

600

400

Wavenumber (cm-1) Figure 7

Infared spectra of zein films steam-treated at various temperature for 15 min.

Figure 7 shows the change of infrared spectra in the range of 400-2000 cm 1 of zein films steam-treated for 15 min at various temperatures. In addition to the amide 1 ,

fl , and amide

v

bands at 1660,1540, and 650 cm-1 due to random-coil conformation, the

I ,II , and V

bands at 1630, 1530, and 700 cm-1 due to the

amide V band at 610 cm-1 due to the

Q

0 -form and the

form appear by steam treatment irrespective

of steam-treating temperature. The same results were obtained by extending the duration of steam treatment to 2 h.

By steam treatment, the random-coil conformation was converted to the a - and

P -forms simultaneously. Water molecules play an important role in the transformation. Presumably, water molecules cleave intra- and / or intermolecular hydrogen bonds of zein molecules, resulting in the transformation to the treating temperature.

Q-

and /3 -forms irrespective of

Biodegra,dablepolymer in seed protein

b

a Figure 8

255

X-ray diffraction patterns of zein film (a) untreated and (b) steam-treated at

120°C for 15 min.

From the X-ray diffraction pattern (Fig&, the amorphous zein film was converted to

13 -form crystals by steam treating at 120°C

for 15 min..

Figure 9 shows DSC curves of the amorphous zein films before and after being steam-treated for 1 h at various temperatures. The endothermic shift at 165°C due to the glass transition slightly moved to higher temperatures by steam treatment and the exothermic peak due to thermal degradation also shifted to higher temperature. These results are induced by the partial crystallization of amorphous zein.

CONCLUSIONS Zein film cast from aqueous ethanol is amorphous in the random-coil conformation.

256

Biodegradable polyurethane-based polymers

I

trc

I

Figure 9 DSC curves of zein films steam-treated at various temperature for 1 hr.

Water in the specimen is lost by evaporation at about 100°C. The glass transition is observed at 165%. The amorphous zein crystallizes to B -crystals at about 210°C accompanied by the random-coil

-+

B -form conformational transition. The thermal

degradation of zein occurs at about 320°C. Steam treatment of zein film results in the conformational change to the

(kl

- and B -forms, simultaneously, irrespective of

treating temperature.

ACKNOWLEDGEMENTS This research is supported by the Ministry of Agriculture, Forestry and Fisheries.

(BRP-2000- a -B-2). The authors wish to acknowledge Mr. Yoshinari Yamamoto (Kanagawa University) in part of this work.

REFERENCES 1.

C . B. Kretschmer, Infrared Spectroscopy and Optical Rotatory Dispersion of Zein,

Wheat Gluten and Gliadin, J. Phys. Chem., 1957,61, 1627-1631 .

Biodegradablepolymer in seed protein

2.

257

S. Akabori, Tanpakushitu, Zein, Kagaku, Kyoritsu Shuppan, Tokyo, 1951, Vol. 3, p.

46-48. 3.

A. Esen, Separation of Alchol-Soluble Proteins (zein) from Maize into Three

Fraction by Differential Solubility, Plant Physiol., 1986, 80,623-627. 4. I.D. Mason, J. A. Boundy, and R. J. Dimler, Prepararion of Whire Zein from Yellow

Corn, J.Biol.Chem. 1934, 131, 107-108. 5.

C. C.Watson, S. Arrhenius, and J. W. Williams, Physical Chemistry of Zein, Nature,

1936, 137,322-323 .

6.

T. Miyazawa, T. Shimanouchi, and S. Mizushima, Perturbation of the

Characteristic Vibrations of Polypeptide Chains in Various Configurations, J. Chem. Phys., 1958,32, 1647-1652.

Part 5

Analysis and characterisation of new polymers and materials

THE COMPLETE ASSIGNMENT OF THE 'k CPMAS NMR SPECTRA OF NATIVE CELLULOSE BY USING I3C LABELED GLUCOSE Tomoki Erata', Tamio Shikano' ,Masashi Fujiwara', Shunji Yunoki' & Mitsuo Taka? 'Division of Biochemistry, Graduate School of Engineering, Hokkaido University, Sapporo, 060-8628, Japan 2R and D Institute, Ohji Yuka Gouseishi Co. ltd Kashima, Ibaraki, 3 14-0102, Japan

ABSTRACT The complete assignment of the I3C CP/MAS spectrum of the native cellulose were performed by using the synthesized cellulose by Acelobacter xylinum from the culture which contains D( l-13C)glucose or D(2-I3C)glucose. On the cellulose from D(2-13C)glucose,the observed I3C CP/h4AS NMR spectrum shows that the transition of the labeling to C1, C3 and C5 as reported by A. Kai et.al. The quantitative analysis of the I3C normal high resolution NMR spectrum of the glucose hydrolyzed from the labeled cellulose indicates that the transition ratio during the biosynthesis of the cellulose. With this obtained ratio and the comparing the CPMAS spectra of the normal cellulose and the labeled cellulose, it is possible to assign the complex part of the cellulose spectrum (C2,C3,Cs part) which was not completely assigned before. The doublet of lower field was assigned as C3, the higher field main line as C2 singlet and the one of C5 doublet, and middle line as the other line of Cs doublet. From those results, the careful quantitative analysis of I3C CPMAS spectra of cellulose I (Tch-Tlp relaxation analysis of the CPMAS spectrum) showed that the spectrum is composed by two equal-intensity subspectra and can be explained as both cellulose Ia and Ip crystals contain two inequivalent glucose chains. Although the clear transition of the labeling from C, carbon was not observed, the intensity of the CI line of the cellulose from D( 1-13C)glucose were much reduced from initial labeling rate of glucose, which suggests that the rate of the I3C-C1 of the cellulose approximately indicate the direct polymerization rate from the fed glucose, and the left were from metabolized and resynthesized glucose.

262

New polymers and materials

INTRODUCTION It has continuously been investigated on the crystal structure of the native cellulose experimentally and theoretically. The successful elucidation was obtained by the electron diffraction method by Sugiyama et al.') The cellulose, however, does not look to have the regular structure as the other polysaccharides, so that it is not easy to elucidate on the exact structure or chain conformations. In the similar polysaccharides such as chitin, the I3C CP/MAS NMR spectra look much simpler than that of cellulose.

In the case of chitin, the each spectral line assigned to each carbon which does not show the multiplet structure like in the case of cellulose. In addition to this fact, it was revealed that the native cellulose (called as cellulose I) has two crystalline forms (called as cellulose la and ID ) with different proportions by the precise investigations of its I3C CPIMAS NMR spectra223). With those situations, it makes more confusion on the interpretation of the NMR spectra of the cellulose, although the NMR spectroscopy is generally one of the powerful tool for studying the conformation and dynamics of molecules. First of all, the complete assignment of the spectrum of cellulose is still not had tried to assign this clear, especially C2, C3 and C5 carbons. Lippmaa et complex part of spectrum indirectly with the careful consideration on the I3C spin-lattice relaxation time of each resonance line. And Bardet et al." made 2-dimensional spin diffusion measurements on the wood which contains I3C labeled cellulose. Both group interpreted that the 74 ppm doublet of the complex part might be C2 carbon. This is, however, not direct evidence of the complete assignment of the spectrum. The other carbons, C1,C4 and C6 were well assigned, however the interpretation of the multiplet of each spectral lines of those carbons is not successful. For instance, la has singlet in C1 and C6 carbons and doublet in C4 carbon, while Ip has all doublet. Many models on this problem has been discussed and the most plausible explanation might be two chain per unit cell model. On the other hand, the diffraction studies, X-ray or electron, say that Ia has 1 chain per unit cell and ID two chains per unit cell. Thus, for elucidating the structure of the cellulose, it is absolutely necessary to assign the CPIMAS spectrum completely. One of the way to analyze the biopolymer such as polypeptide, protein or nucleic acid through one- or multi-dimensional NMR methods is the labeling technique with I3C and 15 N, which is widely applied in the field of protein structure research recently. In the polysaccharide, Kai et aL6' applied this method to cellulose and curdlan for analysis of the biosynthetic processes of those polysaccharides by microorganism such as Acetobacter or Agrohacterium. During the synthesis of the cellulose from glucose which is specifically labeled by ''C, the transition of labeling were observed, and it is

“C CPlhIIAS MMR spectra of cellulose

263

clarified that the cellulose is synthesized not only directly but also from resynthesized glucose which comes from the some specific metabolic path like the pentose phosphate cycle. In this study, we have also synthesized I3C labeled cellulose by using Acetohacter xylinum with culture containing 99 at % site specifically labeled glucose and characterized the specific transition rate of labeling from which carbon to which carbon during the metabolic pathway with I3C NMR quantitative measurements on the glucose which is obtained by completely hydrolysis from labeled synthesized cellulose. With the obtained transition rate of labeling and comparing the 13C CP/MAS spectra of non-labeled and labeled cellulose, we had tried to assign the complex part, namely C2, C3 and C5 carbons, and examine the spectral structure of those resonance lines for further discussion of the structure of cellulose.

EXPERIMENTAL DETAILS By Acetobacter xylinum with the culture media according to Hestrin and Schramm” containing 99 at % of D-(l -13C), D-(2-’3C)glucose, or ( 1,3-’3C)glycerol, ‘k labeled cellulose was biosynthesized. The used strain for biosynthesis of cellulose was ATCC10245. After 7 days of cultivation, the biosynthesized cellulose was washed and dried in normal way and cut into small pieces for solid state high resolution NMR measurements. The NMR measurements were performed with Bruker MSL 400 spectrometer equipped with solid state high resolution apparatus. The operating frequencies of proton and carbon are 400.13MHz and 100.63MHz, respectively. The conventional CP/MAS method was used for high resolution solid state I3C measurements. The rotors which contain the cellulose were spun at ca. 4 k k , and the 90 degree pulse, contact time and repetition times were 4ps, 1S m s and 4s, respectively. 13Cchemical shifts were calibrated indirectly from the carbonyl carbon signal of glycine (1 76.03ppm). For quantitative discussions, the Tch-Tl,,(H) measurements were also performed. The contact time in the CPMAS measurements were vaned from loops to 100ms. The obtained ratio of TchjTlpwere from 0.01 to 0.1, which means the rather quantitative discussion can be possible on the cellulose when the contact time is properly chosen. As references, the I3C CPMAS NMR spectra on the as prepared and annealed(260degree) celluloses from Cladophora sp., Ia- and 10-rich celluloses, respectively, were recorded. Also, same measurements on the labelled cellulose (laand 10-rich) from A. xylinum were carried out, and those cellulose were completely hydrolyzed to glucose with cellulase (ONOZUKA R-lLO, by Yakult co. ltd.), and dissolved in D20 for the normal high resolution NMR measurements. The gated decoupling mode was engaged at high resolution NMR measurements for determining

264

New polymers and materials

the I3Ccontents for each carbon of the glucose from labeled cellulose. The obtained data were transferred to the PC for the line fitting. Non-linear least-square methods were performed for line-fitting with Lorentzian function.

RESULTS AND DISCUSSIONS A. Derivation of subspectra oj'cellulose la and lp and line:fitrings One of the reason which makes the analysis of the solid state NMR spectra of cellulose complex is that the native cellulose always appears as the composite of two crystalline forms, namely Ia and Ip. 'The pure lp cellulose can be obtained from tunicate, while the pure Ia cannot obtained from any species of plants. Hence it is necessary to obtain the NMR spectrum of at least pure la cellulose. We have derived the la and ID subspectra from original spectrum with simple mathematical treatment such as linear combination of spectra o f Ia-rich and ID-rich celluloses, as following; Subspectrum(1a or Ip) = a x spectrum(1u-rich)+ h x spectrum(lp-rich), where constants a and b were chosen properly with trial and error. Fig. 1 shows the original spectra of the (a)Ia- (as prepared) and (b)IP-rich (annealed) celluloses from Cladophora, and the mathematically derived subspectra of (c)la and (d)IP cellulose.

'I ,,., .. . . - .,I.

I,%

. I

,.-

Figure 1. Solid State 13C C P M S NMR spectra of (a)Ia-rich(as prepared) and (b) ID-rich(annea1ed) celluloses from Cladophora, and subspectra of la(c) and Ip(d) components. Solid lines in (c) and (d) represents the results of line-fittings. Numerical results is shown in Table 1.

CPlhthS N M R spectra of cellulose

265

Ip seems to be very similar to that of pure Ip cellulose from tunicate, it can be said that this derivation is adequate. The dotted line in Fig. l(c) and (d) represents the results of the line fitting by least square method. The overall fitting results were summarized in table 1. In the part of C2, C3 and C5, I a spectrum has 5 resonance lines with intensity ratio as 1 :1:1:1:2(from low field), and Ip spectrum has 4 lines with I:l:l:3(same way). Since three carbons should be contained in this region, this line fitting results indicates:that some carbon lines should be also split as other carbon like C4 or C1 in IP-rich cellulose. Since the derived subspectrum of

Line fitting results of I a subspectrum 3

4

5

6

7

8

9

105.1 89.9

89.0

74.7

74.1

72.6

71.7

70.8

65.3

2.05

0.84

0.96

1.23

1.00

1.10

1.08

2.05

1.69

C1

C4

C4

C3

C3

C5

C2

C2/C5

C6

Line No.

1

Position(ppm)

Integral Assignment

2

Line fitting results of ID subspectrum Line No.

1

2

3

4

5

6

7

8

9

10

~

~~

Position(ppm)

105.7

104.0 88.9 88.1

74.9 '74.1

72.5

Integral

1.07

1.16

0.99 0.88

1.57 0.85

1.14 2.73

0.60

1.00

Assignment

CI

C1

C4

C3

C5

C6

C6

C4

C3

71.2

C2K5

65.6 64.9

Table 1.

The summary of the line-fittin,g (deconvolution) results. The integral value was estimated as total integration to be 12 for convenience. The discussion on the ;mignment will be shown following text.

B. Cellulosefiom '3CI,3-Glycerol Fig.2 shows the I3C CPMAS NMR spectra of the cellu1o:seIa-rich(a) and Ip-rich(b) biosynthesized from ('3C-1,3)glycerol. The resolution of the resonance line is not so good, however it is obvious that four main resonance lines were observed. In 1957, Greathouse et a1.*) had already made same experiments with I4C labeled glycerol to synthesize the cellulose by A. xylinum and they reported that the labelings transfer to C1,C3, C4, and C6 of synthesized cellulose through biosynthetic pathway. The resonance lines of C I , C4 and C6 are already known and the left line which has 74 ppm chemical shift should be C3 carbon in both Ia and Ip.

266

New polymers and materials

Figure 2. Observed NMR Spectra of the synthesized cellulose la(a) and I(b) from 1,3-I3C labeled Glycerol.

Fig. 3 shows the NMR spectra of C2-cellulose. The specific transition of labeling from C2 to C1, C3 and C5 was observed obviously as reported by Kai et al.

...

... ... .. .. .. ..

.,

1.-

. ... ...

..

,,

.

. .,..~~, .

,

.. .. .. .... ..

.

. .. .. .,

Figure 3. Observed I3C CP/MAS NMR spectra of C2-cellulose. (a) I a -rich (as prepared) and (b) Ip -rich (annealed). At not only C2 carbon, but also other C1, C3 and C5 part, the spectral intensity can be observed. C1 ~ 1 u c o s e - 2 - ' ~o~

Cellulose

1s

C2 99 9.2

C3 0 12

C4 0 0

CS 0 3

C6 0 0

Table 2. The obtained transition rate of labeling determined by 13C high resolution quantitative measurements on the hydrolyzed glucose from synthesized cellulose. Transition rate was estimated with the method by Kai et aL6'

Total 99 36.2

"C CPMAS NMR spectra of cellulose

267

The labeling transition from C2 to the other site caused by gluconeogenesys from pentose-phosphate cycle. It is important to determine the transition rate precisely. In order to determine this transition rate, we try to regenerate the glucose from the C2-cellulose by hydrolysis and made quantitative NMR measurements on the regenerated glucose. The obtained transition rates were summarized in table 2. According to this table, C1 and C2 have nearly equal and most population, C3 has less population and C5 has slight population. The intensity of each carbon resonance line of solid state NMR spectrum of C2-cellulose should reflect this population ratio. Thus we can expect that intensity order in C2,3,5 complex part should be C2>C3>>C5 in C2-cellulose. We could assign already C3 as 74 ppm lines, so only C2 and C5 which should exist in from 70 to 73 ppm should be considered. The enlarged spectra of this part are shown in Fig.4. In this figure, (a) and (b) are subspectra of la and ID from Cludophoru sp., respectively, and (c) and (d) subspectm of C2-cellulose(derived with same manner as mentioned above). In the case of la, comparing (a) and (c), 72.5ppm line are missing and 7lppm line (most high field peak) is losing in intensity nearly to half in C2-cellulose, which indicates that C5 carbon is splitting to 71 and 72.5 ppm and the remaining lines, half of 72.5ppm and 72ppm, are to be C2 carbon. {I,

..

... ...

~

..l

~

... ... .....- ...

, . . 1

I.-

. .,. ._I"..I"... ... ....

... .,. ...

.

... ... .,, ..-

/.

... ... ... *.* ... ...

.,. .... ... ...-_

-_ ..-.._.r._.._ ,..

Figure 4. Expanded subspectra of C2,3 and 5 part of the native cellulose Ia(a) and Ip(c), and of the c2-labeled cellulose Ia(b) and IP(d), respectively. It is clear that the intensity change with labeling, which corresponds to the transition rates. On the other hand, in the case of lp, 72.5ppm line is missing in C2-cellulose and this should be C5 carbon. However, the intensity of this line is originally only 1, while the most high field line has intensity as 3. It is, therefore, that C5 carbon line also split into doublet and the other line should lie upon the high field line which is remaining C2 carbon singlet.

268

New polymers and materials

CONCLUSION

Through these analysis, the complete assignment of the I3C CP/MAS NMR spectra of native cellulose was performed. Two remarkable points can be noticed as following; Both NMR spectra of Ia and ID are composed by equal-intensity doublet of each carbon except for C1,C6 in la and C2 in Ip. And C5 carbon splitting in Ia is relatively larger than that of I@. The former fact suggests that the native glucose is constructed by two kind of glucose residues. The latter fact suggests that the difference of conformation of C5 or C6 can be the key point of the difference of la and I@.

ACKNOWLEDGEMENTS Authors indebted to Mr. Eiji Yamada (NMR instruments lab., Graduate school of Engineering, Hokkaido University) for his technical support of NMR measurements. This research was supported in part by a grant from the ministry of Education.

REFERENCES

4 5 6 7 8

J. Sugiyama, R. Vuong and H. Chanzy, Macromolecules, 1991,20,4168. D. L. VanderHart and R. H. Atalla, Macromolecules, 1984, 17, 1465. F. Horii, H. Yamamoto, A. Hirai and R. Kitamaru, Macromolecules, 1987, 20, 2946. R. Teeaar and E. Lippmaa, Polymer Bulltine, 1984, 12, 3 15. M. Bardet, L. Emsey and M. Vincendon, Solid State Nuclear Magnetic Reson., 1997, 8, 25. A. Kai, T. Arashida, K. Hatanaka, T. Akaike, K. Matsuzaki, T. Mimura and Y. Kaneko, Carbohydrate Polymers, 1994,23, 235. S. Hestrin and M. Schramm, Biochem. J., 1954,58,345 G. A. Greathouse, J. Am. Chem. SOC., 1957,79,4505

13CCPMAS N M R AND X-RAY STUDIES OF CELLOOLIGOSACCHARIDEACETATES AS A MODEL FOR CELLULOSE TRIACETATE Hiroyuki Kono*, Yukari Numata, Nobuhiro Nagai, Masashi Fujiwara, Tomoki Erata and Mitsuo Takai

' Division of Molecular Chemictrv. Graduate School of Engineering, Hokkaido Universiq, Kiru I S . Nishi 8. Kita-ku. Sapporo, Hokkaido &lo-8628.Japan.

ABSTRACT The series of crystalline CTA oligomers (DS = 2-6) were prepared and characterized by I3C CP/MAS NMR spectroscopy and X-ray analyses in order to obtain structural models of cellulose triacetate (CTA) in the solid state. Progressing toward hexamer, the NMR spectra of the oligomers, in comparison with CTA I and CTA 11, gradually approached that of CTA I. In addition, X-ray diffractograms of the oligomers determined that the crystalline pentamer and hexamer have the CTA I lattice in spite of recrystallization from ethylacetate-n-hexane. We therefore concluded that the higher oligomers (DS > 4) of f f A would be useful models for CTA I structure.

INTRODUCTION When compared to the other synthetic polymers, the fundamental properties I of cellulose triacetate are poorly understood because there are two allomorphs of CTA, denoted CTA I and CTA 11. In X-ray crystallographic studies of CTAs, diffraction patterns of C I A I and CTA 11 can be distinguishable from each ~ t h e r ~and . ~ it, has been explained that the structural model of CTA I has parallel-chain packing4 and that CTA I1 has an anti-parallel structure '. CTA I is only produced by heterogeneous acetylation from cellulose I ', while the crystalline state of CTA produced by homogeneous acetylation or by heterogeneous acetylation from cellulose I1 is C I A I1 *, which are widely accepted. However, there are recent opinioiis 3 . 6 8 conflicting with the polymorphism relationships between CTA and cellulose in the solid state. Further, the most important question whether reversals of chain direction on neighboring area are required in the transformation from CTA I to CTA I1 has not been explained. In order to recognize correct crystal structures of CTAs, it is important to determine structures of crystalline oligomers of ( T A because some polymer properties has been obtained by examining the homologous series of their oligomeric compounds which asymptotically approach the polymer structure '. Regarding CTA, Buchanan et al. l o compared physical properties of a-D-cellooligosaccharide per-acetates (DP = 2-9) with those of CTA by DSC analysis. However, surprisingly few studies have so far been made regarding the relationship between the crystal structures of CTAs and those of CTA oligomers. In this study. we report on preparation of the CTA oligomers (DS = 2-6) by homogeneous acetylation of the corresponding cellooligosaccharide and on characterization of their crystal structures by solid-state N M R and X-ray diffraction. The comparisons of the CfA oligomers with the polymorphs of (3-A in solid-state are also described herein.

270

New polymers and materials

EXPERIMENTAL Samples CTA I (DS=2.95) and CTA I I (DS=2.93) were prepared from Whatman CFI 1 cellulose powder and mercerized cellulose powder, respectively, by heterogeneous acetylation using the method of Tanghe I ] and then recrystallized by heat-treatment' under nitrogen at 210 "C for 15 min. Mercerization of cellulose were previously reported '. CTA oligomers were prepared from the corresponding D-cellooligosaccharides (Seikagaku Co., Japan) by homogeneous acetylation as follows: A reaction mixture containing 200 mg of the cellooligosaccharide in 20 mL pyridine-acetic anhydride (3:2 %(v/v)) was stirred at 100 "C. After 3 h, the mixture was poured into ice water, filtered, and evaporated. The oligomer acetate was obtained by recrystallization twice from ethylacetate-n-hexane ( I : 1). All CTA oligomers were assayed for homogeneity by TLC, ',C NMR, and elemental analyses.

Analytical method Elemental analyses were carried out with a Hewlett-Packard model 185 analyzer. I3C NMR spectra of the samples were recorded on a Bruker MSL-400 NMR spectrometer at 23 "C in CDCI,. ',C chemical shifts were referenced to the center peak of the triplet resonance of CDCI,, 77.0 ppm. I3C CP/MAS NMR spectra were obtained at "C frequency of 400 MHz. Rotating frequency of 4000 cps, contact time of 2 ms, and recycle delay of 5 s were employed 1 2 . Chemical shifts were calibrated through the carbonyl carbon resonance of glycine as an external reference at 176.03 ppm. X-ray diffractograms were measured on a Rigaku Rint2000 diffractometer by the refraction method using nickel-filtered CuKa radiation operated in o-20scanning mode between 5 and 30 (20). Slit system were I for divergence, 0.15 mm for receiving, and 1 for scatter. O

O

O

O

RESULTS AND DISCUSSION Solid state NMR spectra The "C CP/MAS NMR spectra of CTAs and CTA oligomers are shown in Figure 1 with indications of the assignments of resonances to various carbons. The assignments were based on I3C chemical shift data for the oligomers in CDCI,. Progressing toward the hexamer, the spectra of the oligomers become to be simple because the signal intensities of the internal residues of the oligomers were gradually increased relative to those of reducing end and non-reducing end units with the increase of their DP. The numbers of the "C signals in the ring carbon region (60-106 ppm) were converged to five when the DP of the oligomers reached the pentamer. Strong similarities were observed between the pentamer and hexamer in the chemical shifts of the predominant five resonances. The CI region (88-106 ppm) for the oligomers shows a remarkable tendency in that CI resonance(s) of internal residue at about 102 ppm becomes dominant while two resonances at 89 ppm and 99 ppm, which were derived from nonreducing and reducing end, respectively, become to be negligible. On the other hand, because carbonyl carbon and methyl regions of the oligomers show a more complex of the variation, the trends in the spectral feature of the methyl regions were not obtained.

"C CPMAS NMR and X-ray studies

271

CTAl

CTAII

Hexamw

Pentarner

4..

+.J

Tetrarner

A

Trlmer I

Figure 1. I3CCP/MAS Spectra of CTAs and the oligomeric CTA (DP=2-6) Chemical shifts of the CTAs and the hexamer are summarized in Table 1. The two allomorphs of CTA could be distinguishable from each other by observing the chemical shifts and crystallographic splittings, as previously reported 6. 12. In comparing the CPMAS spectrum of hexamer with those of =As, there are conspicuous similarities between the orientation of acetyl groups in the hexamer and those in CTA I. The sharper doublet for methyl carbon (19-24 ppm) of the hexamer and CTA I with splitting of 0.91 and 1.01 ppm, respectively, were observed, while for that CTA I I spectrum has a broad single peak. At the carbonyl carbon resonance regions (169-172 ppm), there is a doublet

Table 1. Solid-state I3CNMR chemical shifts (ppm) for CTAs and hexamer Compound

CIA I

C I A 11

Carbonyl carbon 170.9 172.2

I69.8

C1

103.2

170.9 173.0

C6 62.7

Methyl carbon 23.2 22.3

101.8

80.8 78.2 76.0 75.0 73.2

65.8 61.9

21.8

103.2

80.6

62.6

23.2 22.3

170.6 172.6 172.9

hexamer

Ring carbon c2-cs 80.6 76.3 72.9

76.3 73.0

-

272

New polymers and materials

r n . 5 10

I

,

I

15

,

,

,

, 20

,

, ,

I

I

25

,

,

.

30

20 I degree

Figure 2. X-ray diffractograms of CTAs and the oligomeric CTA (DP=2-6) splitting for both CTA I and the hexamer. Carbonyl carbons of CTA 11, on the other hand, gave shoulder peaks characteristic of the partially resolved quartet. Significant multiplicity of CTA I and the hexamer was also confirmed in the spectral feature of C2C5 (70-83 ppm) and C6 (66-62 ppm) region. In C2-C5 regions of these spectra, numbers of the predominant signals were three for both CTA I and the hexamer, in contrast to five for CTA If. At the C6 regions of the spectra, both CTA I and the hexamer show a single peak. On the other hand, triplet splitting for C 6 carbon of CTA I were detected. In addition, Table I also showed the close correspondence of chemical shifts of ring carbon for the hexamer and CTA 1 at crystalline phase. From these results, it was suggested that the both crystalline pentamer and hexamer have the crystal CTA I lattice, which was confirmed by X-ray analysis.

X-ray diffraction In order to firmly establish the crystal structures of the oligomers suggested by the solid-state NMR analyses, we measured X-ray powder diffractograrns for the two allomorphs of CTA and the CTA oligomers by refraction method as shown in Figure 2. Heat treatment' of the CTAs results in sharp and typical X-ray patterns' for CTA I and CTA 11, respectively. CTA I gave four sharp equatorial diffractions at 11.6 (2e = 7.6 "), 5.6 (15.9 "), and 4.37 (20.3 "). while CTA 11 gave strong diffractions at 10.48 (20 = 8.4"),8.5(10.4"),6.7(13.4"),5.4(16.3o),5.3(16.7"),4.8(18.6"),and3.8(23.4"). These diffraction spots of the both crystalline CTAs were in complete agreement with those previously reported '. On the other hand, although the diffractograms of both the CTA dirner and trirner show a number of sharp peaks, the number of diffractions of CTA

"C CPiMAS NMR and X-ray studies

273

Table 2. Interplaner spacing values (d) and relative intensity of equatorial diffractions for CI'As and cellooligosaccharide per-acetates (DP = 4 - 6) Compound ____

2@(")

d (A)

Intensity"

7.64 14.58 15.90 17.80 20.30 22.37 26.50

11.56 6.07 5.57 4.95 4.37 3.97 3.36

V.S.

CIA 11

8.43 10.42 13.14 16.28 16.74 18.59 21.39 23.39 26.50

10.48 8.48 6.73

V.S.

5.44

S.

5.29 4.77 4.15 3.80 3.36

S.

Hexamer

-

Compound

__

-~

CTA 1

Miller

V.S.

.

S.

w.

w.

V.S. V.S.

S.

w. S.

\v ,

V.S. 7.64 11.56 \\' . 8.75 10.09 14.63 6.03 IV . 15.91 5.55 V .s. \V . 17.76 4 97 S. 20.35 4.34 22.48 3.92 \v , . 26.42 _ _3.34 _ ~ \V_ _

d (A)

___ 7.64 1 I .56 8.75 10.09 14.60 6.05 5.55 15.92 4.97 17.76 4.33 20.40 22.65 3.90 3.5 1 25. I7

Intensity"

-.

Pentamcr

w. \V

28(")

Tetramcr

7.49 9.56 14.3I 15.91 17.61 18.19 20.40 22.60 23.61 24. I 2 25.3 1 28.70

I I .78 9.23 6.17 5.5.5 5.01 4.85 4.33 3.91 3.74 3.66 3.49 3.08

V.S.

w. W.

V.S

w. S.

w. \v. V.S.

w,

w. V.S.

w. \v , S. \V

,

w. S. S.

w

.

.~

"The abbreviations used are as follows: v.s., very strong; s., strong; w . , weak.

oligomers were decreased with increase of their DP. The :<potsand relative intensities of diffractions detected on the crystalline CTA and the oligomers (DP = 4-6) are summarized in Table 2 with their Miller indexes. In comparing the X-ray diffractograms of these samples, many similarities were observed between the oligomer and CTA I. There are three remarkably strong diffractions at approximately 1 I .6 (20= 7.6 "), 5.5 (16.1 and 4.3 (20.3") in the CTA oligomers, which correspond to the (OOI), (202), and (500)plane for the crystal CTA I. Although very weak diffractions are observed at some spots in the diffractograms of the tetramer, there are no diffractions at the spots in the both CTA I and CTA 11. The difference of the weak diffractons between the tetramer and CTAs may be arisen from the effect of the end units form of these samples. CTA are composed of a number of 2,3,6-triacetyl-P-D-glucosylunits, while the reducing and non-reducing end of the oligomers are I ,2,3,6-tetraacetyl-a-D-glucosylresidue and 2,3,4,6-tetraacetyl-p-D-glucosylresidue, respectively. However, the pentamer and hexamer gave the approximately similar diffractograms for CTA I, and, especially, the hexarner were in complete agreement with CTA 1 in tht: sense of interplaner spacing O),

274

New polymers and materials

values and diffraction intensities except for the very weak diffraction at 10.1 (8.8 "). On the basis of the X-ray analysis data in conjunction with the results of the I3C CP/MAS spectra, it is clear that the crystal pentamer and hexamer has the crystalline CTA I lattice. The assumption that CTA I is not at all obtained from the homogeneous acetylation of cellulose I as well as cellulose I1 is now widely accepted whereas our studies here prove clearly that crystal CTA oligomers (DS = 5, 6) take the crystalline CTA I structure despite the homogeneous acetylation of the corresponding cellooligosaccharides. Regarding the contradiction, in our previous X-ray study ', a mixed pattern of CTA I and CTA 11 was obtained from CTA which were prepared by heterogeneous acetylation of a previously acid-hydrolyzed cellulose 11. Although the distinct relationship between the crystal structure of CTA and its DS has not yet been proved, our studies herein also suggest that the crystal structure of oligomeric CTA may be the same as that of CTA I. We therefore conclude that the crystalline states of CTA hexamer and pentamer have been identified with the crystalline CTA I lattice. Especially, the crystal hexamer would be useful model for CTA 1. If the oligomers could be grown to appropriate crystal size, this single crystal would help in the elucidation of the molecular order of CTA I.

'.

ACKNOWLEDGEMENTS This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. The authors are grateful to Mr. Eiji Y amada for the help of NMR measurements.

REFERENCES I.

Ceilutose Derivatives: Mod$cation, Characterization, and Nunustructures, T.J. Heinze and W.G. Glasser, Ed., American Chemical Society, Washington, DC, 1998. 2. B.S. Sprague, J.L. Reley, and H.D. Noether, Text. Res. J., 28 (1958) 257. 3. S. Watanabe, M. Takai, and J. Hayashi, J . Polyrn. Sci.,Part C. 23 (1968) 825. 4. A.J. Stipanovic and A. Sarko, Polymer, 19 (1978) 3. 5. W.J. Dulmage, J. Paly. Sci.,26 (1957) 277. 6. M. Takai, K. Fukuda, and J. Hayashi, J . Polyrn. Sci., Part C: Polyrn. Lett., 25 (1987) 121. 7. J.J. Creely and C.M. Conrad, Text. Res. J., 35 (1965) 184. 8. E. Roche, J.P. O'Brien, and S.R. Allen, Polyrn. Cornmun., 27 (1986) 138. 9. J. Schaefer. E.O. Stejskal, and R. Buchdahl, Macromolecules, 8 (1975) 291. 10. C.M. Buchanan, J.A. Hyatt, S.S. Kelly, and J.L. Little, Macromolecules, 23 (1990) 3747. 1 I. L.J. Tanghe, in Methods in Carbohydrate Chemistry und Biochemistry, Vol. 111, R.L. Whistler, Ed., Academic Press, New York, 1963, pp. 193. 12. M. Hoshino, M. Takai, K. Fukuda, K. Imura. and J. Hayashi, J . Polyrn. Sci., Part A : Polyrn. Chern., 27 (1989) 2083.

THERMAL AND MECHANICAL PROPERTIES OF CELLULOSE ACETATES WITH VARIOUS DEGREES OF ACETYLATION IN DRY AND WET STATES Tanemi Asail', Hiroki Taniguchi' ,Emiko Kinoshita' and Kunio Nakamura2 Daicel Chemical Industries, LTD., I239 Shinzaike, Aboshi - lac Himeji, Hyogo, 671-1283, JAPAN Otsuma Women's Universiw, 12 Sanban-cho, Chiyoda-hi, Tokyo 102-8357, JAPAN

INTRODUCTION Cellulose diacetate (CDA, Degree of acetylation = 2.4) and cellulose triacetate (CTA, Degree of acetylation = 2.9) are widely used as the raw material of acetate fibers, photographic films and cigarette filters. However the physical properties of a cellulose acetate (CA) with degree of substitution (DS) between CDA and CTA have not been studied. In this study, CA with various degrees of acetylation (DS = 2.42 2.92) were prepared. The glass transition temperature (Tg) of the films at dry and wet states was measured by differential scanning calorimetry (DSC) in order to study the effect of water on the physical properties of CA films containing various DS. The stress at break of CA films was also measured by tensile test at dry and wet states. It is clarified from the results that the intermolecular hydrogen bond influences on physical properties of CA films.

-

EXPERIMENTAL, Sample preparation Cellulose acetate (CA) was prepared from wood pulps by two steps. First, CTA was prepared from cellulose by acetylation with acetic-anhydride / acetic acid in the presence of sulphuric acid, and second, CA with required DS was obtained by hydrolysis of the CTA. The samples used in this study are listed in Table 1.

Preparation of CA film CA films were prepared by casting on a glass plate from 12 wt % CA solution in dichloromethane / methanol (91 / 9, wt / wt %), followed by drying in an oven at the range of 45°C to 100°C. The thickness of the CA films were between 45 and 49 m.

Table 1. DS and DP of CA samples

2.42

2.75

2.81

2.92

Degree of polymerization (DP) 183

303

323

274

Degree of substitution (DS)

276

New polymers and materials

Measurement Differential Scanning Calorimetry (DSC)

DSC measurements of CA films was carried out by Seiko Instruments Inc. DSC220C. Sample weight was about 3 mg and scanning rate was 10°C / min. A wet sample was prepared by adding an excess amount of water with a micro syringe in an aluminum pan. The water content (Wc) of the sample was calculated from the equation: Wc, (g / g) = [weight of added water, (g)] / [weight of dry sample, (g)]. Phase transition temperature and enthalpy of sorbed water were calibrated using pure water as a standard. After a DSC measurement, no weight change during the measurement was checked. The bound water content was calculated by the method reported previously [l]. Tensile Test (Stress at break)

Stress at break (a) of CA films with various relative humidity (RH) was measured by tensile test using Orientec co. Ltd TENSIRON RTAdOO equipped with load cell of 5 Kg. Gauge length was 20 mm and head speed was 5 mm / min. The samples were cut for length of about 50 mm and width of 3mm. The sample was kept in a desiccator controlled at a selective relative humidity for a week before testing. True Density

True density was measured using a gas pycnometer, Shimadzu Corp. Accupyc 1330. The 0.5 g of dried CA films chopped into small pieces was put into a cell, and measured in the following conditions. The temperature was 25°C and fill pressure was 19.5 psig and equilibration rate is less than 0.005 psig / min using He gas. Sorption Isotherms

The sorption isotherms of CA films were measured according to the following procedure. A dry sample was kept in a desiccator controlled with a selective relative humidity for a week. After that, it was weighed again and water regain was calculated from the increase of sample weight.

RESULTS AND DISCUSSION Sorption Isotherms Figure 1 shows the sorption isotherms curves of CA films. It was found that the amount of retained water in CA films is more than that expected based on one mole water per every one mole unsubstituted hydroxyl groups of CA, e. g. 1.2 % for CA containing DS = 2.81. Water regain depends on DS of CA, that is, water regain of CA decreased with increasing DS.

F'roperties of cellulose acetates

*O

-.-

7

OS5 p-s;P.*l

*

15

8 C

wl wl

K :lo

0.4 .

0.3

\

E

3

z

277

c

5

0

0.2

0.1

0

20

40

60

80

100

RHI% Figure 1. The sorption isotherms of CA films.

0

0.5

I

1

I

1.0

1.5

2.0

.5

m I g g-1 Figure 2. Wnf calculated from enthalpy of crystallization or melting of sorbed water on CA with various Wc.

Non-freezing Water The amounts of free water calculated from the enthalpies of phase transition peaks obtained by DSC measurement are less than those of added water. It is considered that the difference between the amounts of added water and calculated water is caused by the presence of non-freezing water (Wnf) in CA. Wnf is categorized into two different kinds, that is, one is calculated from enthalpy of crystallization (WnfJ and the other is calculated from that of melting (Wnf,). Wnf, and Wnf, are calculated from the following equations, where wf, is freezing free water and Wf, is melting free water: Wnf,= Wc-wf, Wnf,= Wc-wf, Figure 2 shows the Wnf, and Wnf, of CA with DS = 2.81 plotted against water content (Wc). Wnf increased with increasing Wc at low Wc and then plateaued at Wc values more than 1.0 g / g. Figure 3 shows the relationship between Wnf at Wc = 2.0 and DS of CA. Wnf, and Wnf, decreased with increased DS of CA. Wnf, is higher than Wnf,. This means that super-cooled water exists in higher order structures of CA.These results suggest a difference in the binding mechanism between non-freezing water and CA molecules. Figure 4 shows the relationship between Wnf, (mol / mol and mol / OH group) and DS of CA. Wnf, (mol / mol) slightly decreased from 3 to 2 because of the increase of hydrophobicity. However, Wnf (mol / OH. group) rapidly increased from 5 at DS = 2.42 to 25 at DS = 2.92. This value is considerably larger than those of other cellulose derivatives, e. g. in the case of viscose rayon, Wnf is 1.8 (mol / OH group).

278

New polymers and materials

0.5 r - - - - - - - - - - - - - - ,

25 r - - - - - - - - - - - - , 3.0

0.4

20

2.5

2.0 ...!-

~

"';' 0.3 Cl Cl

iO.2

'0 1.5 E

-

1.0 0.1

5

0'-------'-------1.-----' 2.8 2.4 2.6 3.0

os

Figure 3. The relationship between Wnf and OS ofCA.

i

0.5

0'------'------'--------'0 2.4 3.0 2.6 2.8

os

Figure 4. The relationship between Wnfm (mol/mol and mol / OH group) and OS of CA.

1.335,-------------, True density Figure 5 shows the relationship 1.330 between true density of CA and DS. The true density linearly decreased with '? 51.325 increasing DS. This suggested that the Cl free volume in CA increased with f1.320 increasing DS of CA. It is considered ii 't:I that the amount of Wnf (mol/mol) ~ 1.315 ... restricted by a monomer unit decreased I1.310 with increasing DS of CA. However, the amount of Wnf (mol / OH) restricted by --'-' 1.305 '--_ _----1. a hydroxyl group was extremely large as 3.0 2.6 2.8 2.4 os shown in Figure 4. From these results, it Figure S. Relationship between OS and true was ascertained that there are two types density of CA. of adsorption mechanisms of water. One is restricted by hydroxyl groups and the other is restricted by the free volume of CA. Figure 6 shows the molecular models of interaction between CA and water. The number of water molecules restricted by hydroxyl groups decreased with increasing DS of CA (Figure 6a). However the number of water molecules restricted by the free volume of CA increased (Figure 6b). This means that the higher order structure of CA containing high DS forms three dimensional networks, which are advantageously bound to water molecules.

•

•

Properties of cellulose acetates

279

> : restricted water by hydrophilic

>freerestricted water in the volume of CA :

Figure 6a. The low DS CA

Figure 6.

Figure 6b. The high DS CA

The molecular models of interaction between CA and Water.

Glass Transition Figure 7 shows the DSC curves of the dry CA films and Table 2 shows the Tg values. The Tg's decreased with increasing DS of CA. This means that the increase of Tg is caused by the increase of hydrogen bonds between hydroxyl groups of CA and that molecular motion was easily occurred by introduction of large acetyl groups. Figure 8 shows the relationship between Tg of CA and Wc. Tg rapidly decreased at low amounts of sorbed water and then gradually decreased in the region of Wc more than 2.0 g / g. The effect of water on the Tg's of CA decreased with increasing DS because of the decrease of hydrogen bonds.

Stress at break Figure 9 shows the relationship between stress at break (a,)of CA films at various RH and DS. Figure 10 shows the relationship between the a b ratio of wet and dry states of CA films and RH. As shown in Fig. 9 and Fig. 10, u b decreases in the presence of water, and the decrease of ab is much more significant in the case of CA containing low DS. It can be ascertained that intermolecular hydrogen bonds are broken by water molecules and hence ub decreases. Table 2.

Tg of dry CA films.

DS

2.42

2.75

2.81

2.92

Tg("C)

185.0

181.2

177.3

173.7

New polymers arid materials

280

200

1

1 fllm (dry)

160

I

170

180

200

190

0'

0

Temperature I 'C

I

0.2

0.6

0.3 0.4 0.5

wc I g l'g Figure 8. The relationshipbetween Tg of CA films and Wc.

Figure 7. The DSC curves of dry CA films. 120

0.1

110,

m 100

n

. g, z Y

c 0 u) u)

2

60

/OO%

40 2.4

2.6

2.8

! :t

3.0

DS Figure 9. The relationshipbetween q,of CA and DS.

4u ' 0

,

20

film

. 40 60 RH 1'3'0

80

100

Figure 10. The relationshipbetween awe, /udvratios of CA and RH.

CONCLUSION It is concluded that there are two types of sorbed water in CA molecules. One is restricted by the hydrogen bonds between hydroxyl groups and water molecules, and the other is restricted by the free volume of CA which is formed by acetyl and hydroxyl groups. The results of the increase of u b and Tg of CA at dry state suggest that the intermolecular hydrogen bonds between free hydroxyl groups of CA decrease with increasing DS. However, intermolecular hydrogen bonding is broken by sorbed water.

REF'ERRENCE 1. T. Hatakeyama, K. Nakamura, H. Yoshida and H. Hatakeyama, ThermochimicaAcfa, 'Phase transition on the water-sodium poly (styrenesulfonate)', 1988, 88,223 - 228.

DSC AND TG STUDIES ON CELLULOSE-BASED POLYCAPROLACTONES Hyoe Hatakeyarna', Hitoshi Katsurada', Nobuhide Takahashi',Shigeo Hirose', and Tatsuko Hatakeyama' 'Fukui University of Technology, 3-6-1 Cakuen,Fukui-cify,Fukui 910-8505, Japan 'National Institute of Materials and Chemical Research,1-1 Higashi,Tsuhba, Ibaraki 305-8565, Japan 'Otsuma Women's Wniversify,12 Sanbancho,Chi+-ku,

Tokyo 102-8357, Japan

ABSTRACI' Cellulose-based polycaprolactones(CellPCL's) were synthesized from alkali cellulose by the polymerization of E-caprolactone (CL) which was initiated by the OH group of cellulose in the presence of crown ether (18-crown-6) in dimethylsulfoxide at 80 "C. The ratios of CL to OH groups in cellulose (moVmol) were varied fiom 1 to 20 mol/moI. Differential scanning calorimetry (DSC) and thennogravimetry (TG) were performed using the CellPCL samples. The relationship between the chemical structures and thermal properties is discussed in this study.

INTRODUCTION In the previous studies [l-51,we have prepared saccharide-, lignin and cellulose acetatebased polycaprolactones and their molecular properties have been studied. In the present study, we have tried to prepare cellulose-based polycaprolactones (CellPCL's) directly from cellulose. They are expected to have molecular properties such as thermoplasticity and biodegradability. Thermal properties of the obtained CellPCL's were analyzed by differential scanning calorimetry (DSC) and thennogravimetry (TG).

EXPERIMENTAL Sample preparation Fig. 1 shows the preparation scheme in order to obtain CellPCL's. Cellulose powder (Arbwell) was kindly provided by Miki Sangyo Industries. The cellulose powder was immersed in 30 76 potassium hydroxide (KOH)aqueous solution and kept for 5 hr. at room temperature. The obtained alkali cellulose was washed with ethanol several times and then it was dried in vacuum. The dried alkali cellulose powder was dispersed in dimethylsulfoxide (DMSO) and was reacted with distilled & -caprolactone (CL) using crown ether (18-crown-6) (Merck Co. Ltd.) as a catalyst at ca 80 "C for 24 hrs. The CJJhydroxyl group (OH) ratio was changed from 1 t o 20 mol/mol. The obtained CellPCL's were precipitated by adding water and then were dried in vacuum for 48 hrs at room temperature. The obtained crude CellPCL's were pursed by the precipitation method which was carried out as follows: CellPCL's were dissolved into DMSO and then

282

New polymers and materials

[

Cellulose (Arbocel)

Alkali Cellulose DMSO E

-caprolactone

crown ether

Cellulose-based Polycaprolactone CL / OH Ratio (moVmol) = 1, 2, 3, 5, 10, 15, 20 Fig. 1 Preparation of cellulose PCL derivatives

3-f' 1 \ X

=

8

-fC(CH&d)Ti

caprolactone chain

n = 1,2,3,5,10,15,20 Fig. 2 Synthetic scheme of cellulose PCL derivatives were precipitated by putting their solution in water dropwise. The obtained Cell-PCL was dried in vacuum for 48 hrs at 60 "C. Fig. 2 shows the schematic chemical structure of the obtained cellulose PCL derivatives.

Measurements Differentialscanning calorimetry (DSC) measurements were carried out in nitrogen flow using a Seiko DSC 220C at a heating rate of 10 "C/min under a nitrogen flow. Sample mass was ca. 5 mg. Aluminum open pans were used. At first the CellPCL samples were heated to 120 "C and then quenched to -150 "C. DSC heating curves of the quenched

Cellulose-based polycaprolactones

283

samples were used for analysis. The melting temperature (T,), melting enthalpy (AH,), cold crystabtion temperature (TJ,glass transition temperature (TJ and heat capacity gap (AC,)were determined by the methods reported prewiously [ 6 ] . Thermogravimetry (TG) was carried out in nitrogen flow using a Seiko TG 220 at a heating rate of 20 "C/min in the temperature range 6om 20 to 500 "C. Sample mass was ca. 5 mg. TG curves and derivatograms were recorded. Mass residue (WR) was indicated as (mT/ mm)x 100 (%), where mT is mass at temperature T and mm is mass at 20 "C.

RESULTS AND DISCUSSION fig. 3 shows DSC heating curves of CellPCL derivatives with CUOH ratios of 1, 10 and 20 mol/mol. Numerals in the figure show CUOH ratio. The samples were heated to 120 "C and quenched at the cooling rate of 40 "C/min to -120 "C. Glass transition is recognized as the endothermic deviation of each DSC curve. Glass transition temperatures (T,'s) of CellPCL derivatives were observed in the temperature range from ca. -70 to -60"C, depending on CUOH ratios. A broad exothermic peak observed in the temperature range 6om -50 to -10 "C in each CellPCL derivative shows cold c r y s t a t i o n . By introduction of PCL chains, it is clear that a part of the amorphous chains rearranges and crystallizes. Peak temperature of cold crystallization (Ta)was observed from -38 to -25 "C depending on CUOH ratio. Melting peak (Tm)was observed as the endothermic peak of each DSC curve in a temperature range 6om ca. 30 to 40 "C. It is known that cellulose shows neither glass transition nor melting in the dry State 171 . The fact that T, is observed by the introduction of CL chains suggests that the main chain becomes less restricted and that some parts of the main chain can rotate 6eely. At the m e time, the introduced PCL chains associate and form the crystalline region. When the sample is quenched in the conditions as outlined above, a part of the amorphous chains rearranges during heating. It is also observed that cold crystallization occurs in a broad temperature. This indicated that the higher order structure of amorphous chains of CellPCL's is distniuted in a wide range when the sample is quenched from the molten state. As shown in our previous study [3] , it was found that cellulose acetate-based PCL showed T,at around -70 to do "C. In cellulose based polycaprolactones (CAPCL's) 3 hydroxyl groups per each glucose unit of cellulose are substituted by C L Accordingly, it is reasonable to consider that the molecular chains of CellPCL are bulky enough to show low T,value, even if CUOH ratios are low. Fig. 4 shows the relationship between T, , d C pand ClJOH ratio of CellPCL samples which were cooled at 40 "C 6om 120 "C to -120 "C. This figure shows T i s of CellPCL's increased and reached a maximum at 5 moVmol and then decreased with increasing CUOH ratios over 5. However, dC, of CellPCL's decreased in the CUOH ratios below 5 and then increased in the CUOH ratios over 5. This suggests that the matrix of CellPCL's becomes slightly flexible due to side chain association when CUOH ratios are over 5. When CL /OH ratios are below 5 the crystalline region increases by the increase of side chain, molecular packing. A slight decrease of T, at a high CUOH ratio can be explained by the molecular distortion of molecular chains in the amorphous region that exists between the crystallites. Fig.5 shows the relationship between T,, dH,and CUOH ratio of CellPCL samples which were cooled at 40 "C /min from 120 "C to -220 "C. This figure shows T , ' s with CellPCL's slightly increase with increasing CUOH ratio. The increase of both T,,, and AH, indicates that the crystalline regionincreases with increasing CWOH ratio.

284

New polymers and materials

1 -so

-100

o T /·C

so

100

Fig. 3 DSC heating curves of cellulose PCL derivatives (Cooled at 40°C /min to -120°C)

-55

0.5 0.4

-60

blI

E

p <,

......"

0.3 ~

-65

Q.

o

0.2~

-70

0.1

o

-75

o

5

10 15 20 CL/OH Ratio (mol/mol)

25

Fig. 4 Relationship between Tg , L1 Cp and CUOH ratio in

PCL derivatives of cellulose (Cooled at 40 DC Imin to 120 DC)

Cellulose-based polycaprolactones

285

30 50

.d' 0'

20

40

,o

i?

\

kE

2

1

E

30 10

&.d' 20

0

10

0

5

10

.I 5

20

25

CUOH Ratio (mol/mol) Fig. 5 Relationship between T, , AH, and CUOH ratio in

PCL derivativesof cellulose (Cooled at 40 " C h i n to 120 "C)

0.5

-55

0.4

-60

,o \

0.3 -65

0.2 0"

7

-70

0.1

-75 0

I

I

5

10

0 1!j

20

25

CL/OH Ratio (mol/mol)

fig. 6 Relationship between T p AC, and CUOH ratio in PCL derivativesof cellulose (Cooled at 10 "Chin to 120 "C)

286

New polymers and materials

Fig. 6 shows the relationship between Tg, d Cpand CWOH ratio for samples cooled at 10 "C/min to -120 "C. This figure shows Tg's and d C p of CellPCL samples cooled slowly are observed in a smaller temperature range compared with CellPCL samples cooled at 40 "C /min. This suggests that during the cooling process of CellPCL derivatives at 10 "C /min, crystallization of CellPCL's occurred more effectively compared with cooling at 40 "C /min. Fig. 7 shows the relationship between T,, AH,,,and CUOH ratio for samples cooled at 10 "C /min to /120 "C. This figure also supports the development of the crystalline region of CellPCL's during slow cooling at 10 "C /min. From the DSC results shown above, it is clearly seen that cellulose chains show thermoplasticity by the introduction of PCL chains. This suggests that ordinary plastic processing can be applied to CellPCL's. In order to examine the thermal stability of the samples, TG was carried out in a temperature range from 20 to 500 "C. Fig. 8 shows TG and DTG curves of CellPCL derivatives with CUOH ratios of 1 , l O and 20. The sample decomposes in 2 stages when CUOH ratio is lower than 3, and in 3 stages when CUOH ratio is higher than 5 . Peak temperatures of DTG curves were defined as DTdl,DTa and DT,. As shown in Fig. 9, DTdl decreases from ca. 350 "C to 320 "C with increasing CUOH ratio. Since the degradation of cellulose was observed at ca. 330 "C, DT,, is considered to reflect the degradation cellulose part in the CellPCL molecules. DT, is observed at ca. 410 "C and slightly increases with increasing CUOH ratio. DT, may reflect the thermal degradation of the PCL chain part in CellPCL's. DTa appears when CUOH exceeds 10 rnol/mol. The reason for the appearance of DTa is now under survey.

I 50

1 30

t

-

40

20

,o

P

s

\

kE

2

\

30

-

10

'

I

I

1

I

0

5

10

15

20

10

'0 25

CL/OH Ratio (mol/rnol)

Fig. 7 Relationshipbetween T, , A& and CUOH ratio in PCL derivatives of cellulose (Cooled at 10"C/min to 120°C) T,

0 AH,

7

Cellulose-based polycaprolactones

287

Fig. 8 TG and DTG curves of cellulose PCL derivatives

450

G

---4---*- +

400

9.

&...

b

c-: n

00..0

350

.-*................. ....... 0 ................ 0 0 ---*-*.m

300

0

5

10 15 20 CL/OH ratio (mol/rnol)

25

Fig. 9 Relationship between DT,,, DTa, DT, and CWOH ratio in PCL

derivatives of cellulose

0 DTdl

DTa

DT,

288

New polymers and materials

CONCLUSIONS The above results lead to the following conclusions (1) CellPCL‘s were successfully obtained from natural cellulose powder. (2) The substitution of PCL chains to the OH group of cellulose resulted in the appearance of Tg’s in the temperature range from -70 to -60 “C and also the appearance of Tm’s in the temperature range from ca 30 to 40 “C. (3) The higher Td of Cell-PCL than cellulose suggests that natural cellulose was successfully converted to thermally more stable materials by the substitution of PCL chains.

REFERENCES 1. H. Hatakeyama, Y. Izuta, K Kobashigawa, S. Hirose, and T. Hatakeyama, ‘Synthesis and physical properties of polyurethanes 6om saccharide-based polycaprolactones.’, Manomol Symp., 1998 130 127-138. 2. Y. Izuta, H. Hatakeyama, S. Hirose, and T. Hatakeyama, ‘Preparation and Thermal Properties of Polyurethanes Derived 6om Llignin-Based PCL.’, in 42nd Lingin Symposium, Sapporo, October, 1997. 3. H. Hatakeyama, S. Hirose, and T. Hatakeyama, ‘Synthesis and Thermal Properties of Cellulose-based Polycaprolactones.’, Stockholm, June, 1998. 4. H. Hatakeyama, T. Yoshida, S. Hirose, and T. Hatkaeyama, ‘Molecular Properties of Saccharide-,Cellulose-andLignin-based Polycaprolactones.’, Cellucon ’98, Truk/Abo, Fiiand, December, 1998. 5. T. Hatakeyama, T. Tokashiki, and H. Hatakeyama, ‘Thermal properties of polyurethanes derived from molasses before and after biodegradation.’, Macromol. Symp., 1998 130 139-150. 6. T. Hatakeyama, and F. X. Quinn, Thermal Analysis, Chicherster, UK, John Wiley and Sons, 1994. 7. T. Hatakeyama, K Nakamura, and H. Hatakeyama, ‘Studies on Heart Capacity of Cellulose and Lignin by Differential Scanning Calorimetry.’, Polymer, 1982 23 1802.

TG-FTIR STUDIES ON CELLULOSE .ACETATE-BASED POLY CAPROLACTONES Takanori Yoshida', Hyoe Hatakeyama*', Shigea Hirose*and Tatsuko Hatakeyama' Fukui University of Technology, 3-6-1 Gakuen, Fukui-city, Fukui 910-8505, Japan 'National Institute of Materials and Chemical Research, 1-1 Higashi TsukubaJbaraki 305-8565, Japan

'Olsuma Womenf Universit)i, 12 Sanbancho, Chiyoda-ky Tokyo 102-8357 Japan ABSTRACT Cellulose acetate-based polycaprolactones (CA-PCL's) were synthesized from cellulose acetate (CA, acetyl content, 39.87 9%) by the polymerization of E-caprolactone (CL). The molar ratios of CL to OH groups (CUOH ratios) were varied from 2 to 20. The average molecular weights were measured by gel permeation chromatography. Thermogravimetry (TG) and TG-Fourier transform infrared spectrometry (FTIR) were performed using the CA-PCL samples. The samples with a low CUOH ratio &om 2 to 5 moVmol decomposed in one stage and the peak temperature of derivative TG (DTG) curves were observed at ca. 375 "C. When the CUOH ratio exceeded 8 rnol/mol, two DTG peaks were observed at 375 - 380 and 420 425 "C. Mass residue (mT/mm) x 100 at 450 "C decreased from 14 to 3 % with increasing CUOH ratio. The results obtained by TG-lTIR analysis of CA-PCL showed that gases with OH, CH, C=O, C-0-C groups were evolved by thermal degradation.

-

INTRODUCTION Cellulose molecules lack thermal plasticity, since dry cellulose shows neither glass transition nor melting before thermal decomposition. At the same time, it is also known that cellulose is stable in a large variety of organic solvents. On this account, various types of cellulose derivatives have been synthesized in order to add solubility to cellulose molecules. Among them, cellulose acetates (CA) have been produced in order not only to give solvent accessibility but also to control hydrophobicity of cellulose. Partially acetylated cellulose is soluble in organic solvent, at the same time, the remaining hydroxyl groups attached to glucopyranose rings can be used as active reaction sites. In our previous studies, we developed cellulose acetate based polycaprolactones (CA-PCL) [l-31 in order to obtain processability and biodegradability. It was established that transition temperatures of CA-PCL's could be controlled when molecular mass of PCL chains is varied. When biocompatible polymers are used it is necessarily taken into consideration, that the polymers were degraded in soil or decomposed thermally. In this study, the thermal decomposition process of CA-PCL was characterized by simultaneous thermogravimetry (TG)- Fourier Transform Infrared Spectroscopy (mIR).

290

New polymers and materials

EXPERIMENTAL

Sample preparation As shows in Fig. 1, CA-PCL's were prepared from cellulose acetate (Kodak GI. Ltd., CA; acetyl content, 39.87 76;M, = 6.32 x lo4;MJMn=2.27) which was dehydrated in benzene by refluxing. Distilled E-caprolactone (CL) was added to dried CA and the polymerization was carried out for 22 hrs at 150 "C with the presence of a small amount of dibutyltin dilaurate (DBTDL) as a catalyst. CWOH (mol/mol) ratios were varied from 2 to 20 (moVmo1) (CWOH ratio = 2, 5, 8, 10, 15, 20). When the polymerization of the samples with CLiOH ratio was 2 and 5, N-methyl-2-pyrrolidinone was used as a solvent in order to provide a viscous media. The above CA-PCL's were dissolved in hot acetone and then added to methanol dropwise. Precipitates in flake shape were obtained. Samples were dried in an oven in vacuum at 55 "C for 12 hrs. Flake shaped samples were pressed at 100 kg/cm* at 180 "C for 10 min. Fig. 2 shows the schematic chemical structure of prepared CA-PCL.

Cellulose Acetate ( N-methyl-2-pyrollidone)

Benzene

E -caprolactone

+ catalyst

Cellulose-based Polycaprolactones heat press

CA-PCL Sheets CL / OH Ratio (mol/mol) = 2.5, 8. 10, 15, 20

Figure 1.

Preparation of CA-PCL Sheets

R=

fCOCH2CH2CH2CH2CH2-ObH n

n=2, 5 , 8 , 1 0 , 1 5 , 2 0 Figure 2.

Schematic chemical structure of CA-PCL

Cellulose acetate-based polycaprolactones

29 1

Measurements

Thermogravhetry was carried out in nitrogen using a Seiko TG 220 at a heating rate of 20 "C/min in the temperature range from 40 to 800 "C. Sample mass was ca. 10 mg. Mass residue was indicated as (m, /mm)x 100 (%). where mT is mass at temperature T and m m is mass at 20 "C [4,5]. Mass residue was evaluated at 450 "C. TG curves and derivative TG (DTG) curves were accumulated. Decomposition temperature (T.,), which was d e h e d as an intercept of two extrapolated lines; where one line is the mass where no degradation occurred and the other is where mass decreased abruptly. Peak temperatures of DTG and temperatures where the mass attains m,/2 were also evaluated. Gases evolved by thermal degradation were transferred to an infrared spectrometer, JASCO FT/IR-420. Heating rate of TG was 20 "C/min in the temperature range from 40 to 800 "C. The temperature of the gas transfer system, directly connected to an IR sample cell, was maintained at 270 "C. The flow rate of carrier nitrogen gas was 200 dmin and the sample weight was ca 10 mg. The resolution of FTIR was 4 cm-'. The number of data acquistion was ten and the data incorporation time was 30 sec.

RESULTS AND DISCUSSION

Fig. 3 shows TG and DTG curves of CA-PCL's with various CUOH ratios. The samples with a low CUOH ratio from 0 to 5 moVmol decompose in one stage and the peak temperature of DTG is observed at ca. 375 "C.When the CUOH ratio exceeds 8 moVmol, two peaks can be observed at 375 380 and 420 425 "C. Mass residue [ ( q / m m ) x 1001 at 450 "C decreased from 14 to 3 9% with increasing CUOH ratio, although it is not shown in the figure.

-

-

100

200

300

400

500

T 1°C Figure 3.

TG heating curves and derivative curves of CA-PCL

292

New polymers and materials

3 .

440 430 -

440

430

420

420 -

-

410 -

- 410 ; 400

- 390

,o \

:

LE

- 380 -

360

0

5

10

15

20

'

370 360

25

C Y O H Ratio/(mol/mol)

Figure 4.

Relationship among DT,, T,,and

CUOH ratio in CA-PCL

Fig. 4 shows the relationships between peak temperature of DTG curves and the CUOH ratio. The low temperature peak (DTd,)maintains an almost constant value with increasing CUOH ratio. The high temperature peak (DTd2)is almost constant, as shown in the figure. At the same time, the height of DTG of the low temperature side peak markedly decreased at CUOH ratio = 8. The temperature where the mass residue reaches 50 % shows similar behavior to that of DTU

Figure 5.

TG-FTIR of CA-PCL (CWOH Ratio: IOmol/mol)

Cellulose acetate-based polycaprolactones

t

293

G O

n

c-0-c

il

CH

A

0.05

3000

2000

Wavenumber / ~ m - '

Figure 6.

TG-FTIR of CA-PCL (CUOH Ratio: lOmol/mol)

Fig. 5 shows a representative three-dimensional diagram of CA-PCL with CUOH ratio 10 mol/mol. Three-dimensional diagrams of the samples were also obtained. Wave number ranges born 600 to 4000 cm-' and temperature ranges ffom 40 to 800 "C. As shown in the diagram, IR-absorption bands can mainly be observed from 250 to 450 "C. FTIR spectra obtained at 380 "C, corresponding to DT,, of DTG, and those at 430 "C, corresponding to DTU are shown in Fig. 6.

0

5

10

15

20

25

C V O H Ratio/(mol/mol)

Figure 7.

Relationship between C-0-C, C-0, CO,, CH, OH and CUOH ratio in CA-PCL at 380 "C

+ c-0-c,

c-0,

A

co,,

CH,

X

OH

294

New polymers and materials

0

5

10

15

20

25

C V O H Ratio/(rnol/rnol)

Figure 8.

+ c-0-c,

Relationship among C-0-C, C-0, CO, CH, OH and CUOH ratio in CA-PCL at 430 "C

c-0,

A

co,,

0 CH,

X

OH

Figs. 7 and 8 show the change of IR peak intensities corresponding to C-0-C, C-0, CO, CH and OH bands with CUOH ratios in CA-PCL at 380 and 430 "C respectively. The above results suggest that only the cellulose structure in CA-PCL degrades at 380 "C and that PCL chains degrade at 430 "C.

REFERENCES H Hatakeyama, Y Izuta, K Kobashigawa, S Hirose and T Hatakeyama, 'Synthesis and Physical Properties of Polyurethanes from Saccharide-based Polycaprolactones' Macromol. Symp, 1998 130 127-138. Y Izuta, H Hatakeyarna, S Hirose and T Hatakeyama, 'Preparation and Thermal Properties of Polyurethanes Derived from Lignin-based PCL' in 42" Lingin Symposium, Sapporo, 1997, 77-80. H Hatakeyama, S Hirose and T Halakeyama, 'Synthesis and Thermal Properties of Cellulose-based Polycaprolactones' in Sh International Scientific Workshop on Biodegradable Plastics and Polymers, Stockholm, 1998 T Hatakeyama and F X Quinn, Thermal Analysis, Chichester UK, John Wiley and Sons, 1994. T Hatakeyarna and Z Liu, Handbook of Thermal analysis, Chichester UK, John Wiley and Sons, 1998 Chap.7 Appendix.

THERMAL ANALYSIS OF FUNCTIONAL PAPER BY A TEMPERATURE MODULATED TECHNIQUE Toshimasa Hashimoto*, Woo-Duck Jung, and Junko Morikawa Department of Organic and Polymeric Materials. Tokyo institute of Technology, 2-12-1, 0-okayama. Meguro-ku, Tokyo 152-8552. Japan Tel&Fax + 8/ -.?-.5734-2435.email tushimas~l7.cc.titech.ac.jp

ABSTRACT Thermal diffusivity of paper, which is characterized as porous and thin material, is determined directly with a temperature modulation techn.ique developed by the authors. With this technique the measurement can be done without any special pre-treatments. The theoretical background and the principle of this method are presented with a data table of thermal diffusivity of various materials. Furthermore this method is applied to the measurement of functional paper and synthetic paper.

INTRODUCTION Thermal conductivity, thermal diffusivity, and heat capacity are the basic thermal properties of materials. Many studies have been accomplished'-' for the measurement technique of thermal properties, but for a thin porous film such as cellulose paper or functional paper it is still necessary to establish a new technique for the measurement of thermal conductivity or thermal diffusivity. A method for thermal diffusivity measurement based on the precise analysis of a sinusoidal temperature wave'-'', which propagates in the thickness direction of thin film is shown in this manuscript. Thermal diffusivity is directly calculated from the phase delay of temperature wave detected on the rear surface of the film. The features of this method are summarized as follows:( 1 ) a high-sensitivity sensor with thin metal resistor with negligible heat capacity; (2) the temperature wave that is actually propagating in the specimen can be observed using two thin gold resistors located at a distance d (dthickness) from each other, one acting as a heater, and the other as a sensor; and (3) the thermal property of thin film (order of vm) can be determined without any destruction or pretreatment. Following the description on the theoretical backgtound and the principle of this method, the thermal diffusivity of thin porous film such a:$cellulose paper and functional paper are shown as a function of temperature.

THEORY The technique we have developed exploits the fact that the solution to the heat diffusion equation is simple near a heater with a very simple geometry, such as a thin, flat plane. The specimen with thickness d is sandwiched between the substrates with semi-infinite thickness and known thermal properties. Assuming the one-dimensional heat flux j,],the temperature wave generated on the front surface (.x=O> by a.c. Joule heating propagates in the direction of thickness and is detected by the sensor attached on the rear surface (x=d), on which the amplitude decay and the phase delay can be observed. The one-dimensional diffusion equation leads to the solution of temperature oscillation at x=d as follows;

296

New polymers and materials

4-

A : thermal conductivity, a : thermal diffusi! ity, k = 0 , (0 = 2nf 2a Here, p=Ilk is a thermal diffusion length, determined with measurement frequency and thermal diffusivity . Subscript 7 means the property of substrates and the other for the specimen. If the conditions:(i) kd>>/, or(ii) Ak-iL,k,, are satisfied, eq.(i) becomes to a simple form of

and we can get the result as :

Then thermal diffusivity a can be obtained from the slope when A 0 is plotted against o’” in a step change of temperature. The frequency of temperature wave is selected considering the value of kd.

EXPERIMENTAL Equipment Schematic diagrams of the measurement system is shown in Fig.1. A thin gold layer is used as a heater for generating temperature wave and as a sensor for thermometer. The temperature wave is detected as a voltage oscillation on the sensor. T o improve the signal to noise ratio a lock-in amplifier (EG&G5210) is used to detect a temperature oscillation. The electrical resistance of the gold layer was controlled at approximately 50-100~2. A temperature wave is generated by the passage of sine wave current supplied from a function generator (NF1940). The specimen with IOX IOmm’ in size and 10-IOOpm in thickness was inserted between the substrate plates on which the thin gold layers were sputtered across an area 1 X 5mm’ as a sensor and a heater respectively (Fig.2). The thickness of the specimen is maintained by the insertion of the spacers to avoid deformation during measurement. The temperature on the hot stage was scanned by a step. The function synthesizer, lock-in amplifier, and temperature controller are all automatically controlled by personal computer.

Thermal analysis of functional paper

297

Temperature controller

............................... . __.

1

i

t i

Function synthesizer Ref. Input i ..............................................................

L!

....................................... Rs.232. .........................

....................

2

GP-I9 ...........

Fig.1 Schematic diagram of measurement system.

Heater and sensor The heater and the sensor are thin gold layers sputtered on the substrates directly. Gold was chosen because of its large temperature coefficient of resistance and the stability in the process of repeated heating and cooling (Fig.3). The thickness of the sputtered gold layer is estimated less than 500 A . The heat flux generated on the heater is controlled at 50mW, or at least less than IOOmW. The temperature variation on the heater is estimated less than 0.1K. The influence of thermal flow vertical to the thickness direction of the specimen is neglected because the area of the gold sputtered sensor (lmm x Smm) is much larger than the thickness of the specimen (50pm). The ratio of length and width of the rectangle of the resistance has no influence on the measurement when the generated heat flow is kept constant. This method is based on the measurement of a x . temperature, and the amount of heat flux of lOOmW does not influence on the phase shift measurement. Heater

F-T/

................................

................................ , .. ...............................

.i

.............................. \

,Spacer

Gold layer leads

Fig3

Schematic diagram of measurement cell

298

New polymers and materials

Io5

100

90

5

I 0

100

50

150

200

T/'C

Fig. 3 The temperature dependence of resistor of the gold-sputtered layer. Frequency range It is examined that the time constant of gold sensor is sensitive enough up to 14 KHz. The frequency range to which this method is applicable is only limited by the condition kd >> 1. In order to extend the frequency below I Hz, digital lock-in amplifier is used for a thick specimen. Substrate The influence of the thermal property of substrates on the propagation of temperature wave in the specimen is taken into consideration in 5. Fig. 4 is a simulation result of a relationship between dB and kd. The linearity in the lower frequency (or smaller kd) depends on f . Pyrex 7740 is chosen as a substrate in this study. Precision The standard deviation to the average value of thermal diffusivity is less than 2% including the error of the thickness measurement. Comparison with the conventional method Table 1 shows the thermal conductivity of thermoplastic in the molten state by the conventional method (hot wire method) and by this method. The identical value was obtained for the thermal conductivity in the both methods.

Table I Material

Comparison with the conventional methods. TPC

h JWm 'K ' this method

PP ABS PBT PA66

220 260 220 250

0.1 1 0.12 0.23 0.20

h IWm 'K ' conventional method

0.10 0.12 0.15 0.20

Therm.nl analysis of functional paper

299

-0.5 -1

-1.5

-2.5

-3 -3.5 -4

0

0.5

1.5

1

2

2.5

3

kd Fig. 4 The effect of Eon the relationship between A0 and kd.

RESULTS & DISCUSSION Fig. 5 shows a relationship between A 0 and UPof various materials. As predicted by eq.(4) AO and u”*show a linear relationship in the condition kd>>l, with the intercept - d 4 . The thermal diffusivities obtained by the slopes are shown in Table 11. 0

1

- x/2

I

I

I

I

1

40

60

80

1

sapphire (126 pm) polyimide (26prn) polystyrene (28 pm) borosilicate glass (78 urn) E. polyethylene (92 pm) F. borosilicate glass (I51 pm)

A. B. C. D.

-n

-3d2

I

0

I

20

1/2

I 100

0

Fig. 5 A 0 vs. c”of various materials.

300

New polymers and materials

Table I1

Thermal diffusivity o f various materials.

a / 107ms'

sample sapphi re borosilicate glass

grade

polyimide polyethylene polypropylene

Kapton Sholex Noblen Y up0

130 (30°C)

4.8 (25°C)

'I'R-K

poly(ethy1ene tercphthalate)

oriented foaming Toporcx 1,itac

polystyrene polq(st4.r"nc-co-acrilonitrile) poly(ary1ether aryl)sull'one poly(ether imidc)

lldel Ultem

n-Tetracosane n-Henacosanc

/ IO'm's' 63 ( I 50°C) 4.6 (140°C) CL

I .7 (25°C) 4.3 (30°C) I .5(30°C) 0.57 (30°C) 1.7 (30°C) l.0(3O0C) 0.52 (20°C)

0.90 (310°C) 0.92 (180°C) 0.49 ( 180°C) 0.50(130"C) 0.61 (260°C) 0.63 (210°C)

I .2 (30°C) 1.2 (30°C) 1.3 (30°C) I .J (30°C)

0.68 (140°C) 0.61 (185°C) 0.53 (305°C) 0.61 (250°C)

2.1 (30°C) 2.2 (30°C)

0.63 (70°C) 0.66 (70°C)

Fig.6 shows the linear relationship between A# and w''' detected in the cellulose paper. It means that even for the complex material such as cellulose fiber and air, a good linearity i s observed and the slope leads to an apparent thermal diffusivity of paper. The density is controlled by the roll pressure on the same base paper on which a small amount of kaolinite is coated. In Table 111 the apparent thermal diffusivity of the paper with the various density are listed. Furthermore the thermal conductivity was calculated with the relationship A=nc,p, where C, is the heat capacity determined by DSC and pis the density determined by another method. The thermal diffusivity decreases with increasing the density, on the other hand the thermal conductivity shows an almost constant value.

-2

-

2 -2.5

-

-3

t 2

4

6 w

1/ z

8

10

Fig. 6 The phase delay A 0 of paper. The density is controlled by the roll pressure.

Thermal analysis of functional paper

Table 111 a and h of cellulose paper. p / g ~ r n - ~ d //.L m a/107m's I h/Wm-'K-I 0.77 91 0.81 0.1 1 0.79 0.1 1 0.78 90 0.1 1 0.9 I 78 0.69 0.1 I 0.94 74 0.66 0.1 1 0.96 73 0.65 0.1 1 0.99 71 0.63 1.01

-

0.7

?

0.1

0.6

70

\

no coating

2 \

CI 0.65 -

I

I

I

Cb 0

2 -

0

-

0

<-

cooling 0

0

?

21 \

o o m

U

~

o

o

o

o

o

0 o

0

heating 0 -

Fig. 8 Thermal diffusivity of synthetic paper.

301

302

New polymers and materials

As a second step this method is applied to the measurement of functional paper and synthetic paper. The heat-sensitive recording paper (or thermal paper) is one of the functional papers which consists of three layers, the base layer, the adiabatic layer and the color-developing layer. In general the inorganic filler (or the pigments) mixed with the binder polymer is coated on the base paper as an adiabatic layer. Fig.7 shows the thermal diffusivity of heat-sensitive recording paper with the different coated amount of the adiabatic layer as a function of temperature. A study on the relationship between the thermal property and the color-developing sensitivity is also examined”. The thermal diffusivity of synthetic paper of polypropylene is examined on heating and cooling as shown i n Fig.8. The thermal diffusivity decreases with increasing temperature in the first heating and above 140°C it starts to increase up to the value of the molten state of polypropylene. On the other hand in the cooling run from the molten stare a increases slightly with decreasing temperature and below 140°C it goes up because of a crystallization. After the slow crystallization a goes up to four times larger value than that in the oriented state at room temperature. This measurement technique is sensitive enough to detect the difference of the physical state of functional papers and synthetic papers.

CONCLUSION A temperature modulated technique was applied for the measurement of apparent thermal diffusivity of thin and porous film such as cellulose paper and heat-sensitive recording paper in addition to thin films of thermoplastics and synthetic paper. The density, the coated amount, and the oriented state are sensitively detected by the thermal diffusivity, which is also detected as a function of temperature. This method is one of the new techniques for the study of paper and functional papers.

REFERENCES I . W. J. Parker, R. S. Jenkins, C. P. Butler, G. L. Abbott, J. Appl. Phys., 1961, 32 1679. 2. P. F. Sullivan, G. Seidel, Phys. Rev., 1968, 173, 679. 3. A. Rosencwaig, A. Gersho, J. Appl. Phys., 1976,47, 64. 4. M. J . Adams, G. F. Kirkbright, Analyst, 1997, 102, 678. 5. N. 0. Birge, S. R. Nagei, Phys. Rev. Lett., 1985, 54. 2674. 6. M. Reading, D. Elliott, V. L. Hill, 10th ICTAC August 24-28 1992, ibid, J. Therm. Anal., 1993, 40, 949. 7. T. Hashimoto, A. Hagiwara, A. Miyamoto. Thermochim. Acta, 1990, 163, 317. 8. T. Tsuji, T. Hashimoto, 10th ICTAC August 24-28 1992, ibid, J. Therm. Anal., 1993 40,721. 9. T. Hashimoto, The Data Book for Thermal Diffusivity, Specific Heat, and Thermal Conductivity of Polymers, Youtes, Tokyo, 1994. 10. J. Morikawa. J. Tan, andT. Hashimoto, Polymer, 1995, 36,4439. 11. J. Morikawa. A. Kobayashi, and T. Hashimoto, Thermochim. Acta, 1995, 267 289 12. T. Kurihara, J. Morikawa, andT. Hashimoto, Int. J. Thermophys., 1997, 8, 505. 13. T. Hashimoto. J. Morikawa, T. Kurihara, and T. Tsuji, Thermochim. Acta, 1997 3041305, 15 I . 14. J. Morikawa, and T. Hashimoto, Polymer, 1997,38, 5397. 15. J. Morikawa, T. Hashimoto, and G . Sherbelis, Thermochim. Acta. 1997, 299, 95100. 16. J. Morikawa, and T. Hashimoto, Polymer Int., 1997, 45, 207. 17. T. Hashimoto, J. Morikawa, M. Kamei, and T. Watanabe, Polymer lnt., in press.

DSC STUDIES ON THE STRUCTURAL CHANGE OF WATER RESTRAINED BY PECTINS M Iuima", K Nakamural, T Hatakeyama' and H Hatakeyama'

' Otsuma Women's University, Sanban-cho, Chiymiu-ku, Tokyo 102-8357 ' Fukui University of Technology, Gakuen, Fukui 910-8505 SYNOPSIS The phase transition behaviour of pectins having various degrees of methyl esterification (DE)in the presence of sorbed water WiXj investigated by differential scanning calorimetry (DSC). In the DSC cooling curves in a temperature from 40 to -150 "C of pectin-water systems, a crystallization exothermic peak (T,) of water was observed. In the DSC heating curves of pectin-water systems, glass transition temperature (T,) of pectin-water systems and a melting endothermic peak ( T J of water were observed. T, and T , of sorbed water were not observed when water content (W, = mass of sorbed water / mass of dry pectin (g-g-I)) was less than ca. 0.5 g-g-'. This amount of water corresponds to the non-freezing water of the system. In a W, ranging from ca. 0.4 to ca. 0.7 g-g-', cold crystallization was observed. Melting peak range decreases with increasing W,. T , of the pectin-water systems decreased remarkably with increasing W, when W , was lower than 0.5 g-g-'. After reaching a minimum value, T slightly increased and approached a constant value. The heat capacity difference (ACpj between the glassy and the rubbery states at T, increased with increasing W , ranging from 0 to ca. 1.0 g-g-', and ACp decreased when W, increased more than 1.0 g-g-I. Non-freezing water (WnJ increased with increasing DE. The above results suggest that non-freezing water molecules break the hydrogen bonds and markedly affect the molecular motion of the pectin-water system. 1 . INTRODUCTION Pectin is a block copolymer consisting of galacturonic acid and rhamno galacturonan, i.e. unbranched blocks of galactouronan where rhamnose units are rare and branched blocks containing a main galactouronan chain are interrupted and bent by frequent rhamnose units [l-91. Pectins are 1,4 linked polysaccharides, as the hydroxyl groups at carbon atom 1 and 4 are on the axial position. It is known that there two types of pectins; one is water soluble and the other is water-insoluble [4]. Water solubility is markedly affected by the amount of methyl esterified carboxyl groups (degree of methyl esterification = DE), although the solubility depends on molecular weight, molecular weight distribution, pH, temperature, concentration and nature of the original material. It is reported that pectin molecules in aqueous solution are not homogeneous and that a small amount of aggregates is observed [6]. It is thought that pectin contains a certain number of junction zones. even if divalent cations are not present r71. These facts suggest that not only the higher-order structure of pectins but also the role of water in the p,ectin molecules is impprtant in order to investigate the nature of pectin hydrogels, since the role of water in the junction zone is closely related with structure formation. In this study, water-pectin interaction is investigated in a narrower water content range, since the structural change of both pectin and water molecules is considered to be affected by the monolayer order of water. In our previous studies, we reported that a trace amount of water markedly affects the phase transition behaviour of polysaccharides in both neutral and electrolyte polysaccharides [10-121. In this study, the phase transition behaviour of pectins having various degrees of methyl esterification (DE)in the presence of bound water was investigated by differential scanning calorimetry

(DSC).

304

New polymers and materials

2 . EXPERIMENTAL

2.1 Sample Preparation Pectins in powder form were obtained from Taiyo Kagaku Co. Pectin samples with various degrees of methyl esterification (DE), 24% (Pl), 31% (P2), 66% (P3) and 72% (P4) were used. The above samples were in crystalline form [ 131. Sample weight was 3 - 5 mg and an aluminum sealed type sample pan was used. Amorphous pectin samples were prepared by melting at 150 "C for 10 minutes in a DSC sample holder. A determined amount of water was added to each amorphous sample by a micro-syringe and then the pan was sealed. The sample pan was hermetically sealed and the total weight of the pectin and water recorded.

2.2 Measurements A Seiko Instruments lnc. differential scanning calorimeter (DSC) EXSTAR 6000 equipped with a cooling apparatus was used for all the thermal analysis experiments. Temperature and enthalpy calibrations were carried out using indium and water. Dry nitrogen was used as a purge gas and the flow rate was 30 mllmin. The scanning rate was 10 "Clmin. The sample was cooled from 40 "C to -150 "C and heated from -150 "C to 80 'T (fst-run measurement). Then the 2nd-run was cooled from 80 "C to -150 "Cand heated from -150 "C to 80 "C. Liquid nitrogen was used a coolant. A Sartorius ultramicro-balance (t 0.1 x g) was used for sample weight measurements. The accurate water contents (W,) of the samples were calculated according to the following equation. Wc = (mass of sorbed water) I (mass of dry pectin)

(g.g-' )

(1)

The sample pans were pierced with a pin after DSC measurements in order to remove water from the measured sample. The pan was then dried at 120 "C for 90 minutes. The dried sample was then quickly reweighed and the intrinsic water content determined. The crystallization temperature (TJ of water was defined as DSC starting temperature of crystallization exothermic peak. Starting temperature of melting (T,,J, peak (Tp,) and end temperature of melting (Tedwere defined. The crystallization (AH,) and the melting enthalpy (AH3were calculated area of crystallization exothermic peak and that of melting endothermic peak, respectively. The glass transition temperature (T,) was defined as the intersection of extrapolations of the baseline and slope, and the heat capacity difference (ACp)was defined between the glassy and the rubbery state at T . Non-freezing water (W,,) was calculated follows; from the Ah, and AHmof water, the total amount of freezing water (WJ in the sample was calculated using equation (2). W , and Wn, were calculated using equation (3) and (4). In this equation, W , is mass of dry pectin and 334 is enthalpy of water.

w, = AH f 334 1 w,

w,=w,tW", w,, = w,- w,

Observed AH, values were smaller than 334 J g ' due to super-cooling. On this account, AH, values were obtained from ACp between water and ice at Tc's. W,, ,'s were calculated using the above calibrated AH, values. 3 . RESULTS AND DISCUSSION

Figure 1 shows schematic DSC curves of pectin (P4)-water systems. The DSC curves show a representative sample with Wc=l.l1 g g " . When the sample is cooled from 40 to - 150 "C, a crystallization exothermic peak (TJ of water was observed at - 27.6 "C. In the DSC heating curve, a melting endothermic peak (Tp,,,)of water was observed at - 4.0

Water restrained by pectins

305

"C. As clearly seen in Figure 1, a melting endothermic peak starts at around - 40 OC, suggesting that the structure of ice formed in the system. In the 2nd-run DSC curves of pectin-water systems, T, and Tpmwere observed at -27.3 'C and -4.2 T, respectively. T, and Tpmvalues and the patterns of DSC curves were reproducible by repeated runs. AH, obtained by the 2nd-run was slightly larger than that of the 1st-run. In contrast, AH,,, obtained the 1st-run and the 2nd-run was maintained at a tmstant value. Figure 2 shows representative DSC curves of pectin (P4)-water systems containing various water contents. DSC cooling and heating curve:j of pure water measured at the same conditions are also shown as a broken line in Figure 2 (a). The first order transition (Tc and Tm) of pure water was observed at about -25 "C and 0 C, respectively. It is clear that a large supercooling is observed for crystallization of water. When W , of pectinwater system is less than ca. 0.5 gg-', the first order transition (T, and T,,,) of sorbed water was not observed. AH of sorbed water is categorized as non-freezing water. When W , was higher than 0.5 gg-', T,,, of water in the system was observed. DSC heating curves near glass transition are magnified, as shown in Figure 2 (b). Tg of pectin-water systems was observed at a temperature lower than melting of water. Tg was clearly observed when W , of the system was smaller than 3.0 gg-I. In a W , range from ca. 0.4 to ca. 0.7 g-g-', a shallow exothermic peak due to cold crystallization is observed. The cold crystallization peak temperature was - 50 - 25 "C. As shown in the DSC curve of the sample with W , = 0.60 gg-', small and broad melting peaks were observed, though no crystallization peak was observed. The lower temperature peak was merged into the high temperature peak with increasing W , and disappeared when W , reached 1.50 gg". It is found that both exothermic and endothermic peaks of pectin-water systems are broader than pure water until W,reaches 3.0 gg-I. Ts and TPmshifted to the high temperature: side with increasing W , and approached 0 C.

-

fi

W

1 0 '0 E

W

-150

Fig. 1

-50 0 50 Temperature / 'C

-100

100

Schematic DSC curves of pectin-water systems. W,=l.ll gg-', I; 1st-run cooling, 11; 1st-run heating, 111; 2nd-run cooling, IV; 2nd-run heating.

New polymers and materials

306

s w

0 X

Wc=0.39g/g

w

w

1

1,=0.39gIg 0 0.60 U E 1.11 w 1.48 1.99

-150

1

0.60 0.39

-

-100 -50 0 50 Temperature / 'C

d mE

w

100

-150

-100 -50 0 Temperature / "C

50

Fig. 2 DSC curves of pectin (P4)-water systems containing various water contents. From the above facts, it is thought that water in the systems differs from pure water. When the number of water molecules are ca. 5 / repeating unit of pectin, all of water molecules are frozen as an amorphous state. Amorphous ice frozen in the systems crystallizes during heating. The lower temperature peak or shoulder observed for the samples with Wc7slower than 1.5 g g - ' is attributed to irregular ice whose normal structure formation is disturbed by the presence of dense pectin molecules. The amount of amorphous ice becomes negligible when the amount of free water in the system increases. Figure 3 shows relationships between the melting temperatures of pectin (P4)-water systems and W,. Starting temperature of melting ( T J , melting peak temperature (Tp,,,) and end temperature of melting (Tern)were obtained from the DSC heating curves shown in Figure 2 . Tpmwas observed at -15 "C at W c= 0.4 gg-', shifted to 0 "C with increasing W, until W c= ca. 1.0 gg-' and maintained at a constant value. T,, was observed at - 70 "Cat W , = 0.4 g-g" and shifted remarkably to the high temperature side with increasing W , below 1.0 g-g-'. When W , exceeds 1.0 g g - ' , T,, was a constant value at - 35 "C. When cold crystallization was observed, Ttmwas difficult to estimate, since exothermic and endothermic transitions occur simultaneously. On this account, T,,,,of the samples with W,'s ranging from 0.4 to 0.7 pg-', was conventionally defined as the temperature starting of cold crystallization, hence T,, of this system is observed at a low temperature. T,, was observed at a constant value at 12 20 "C. This suggests that melting peak range decreases with increasing Wcand approaches the melting peak of pure water. As clearly seen in Figures 2 and 3, the high temperature peak increases with increasing W c and accords with the melting peak of pure water. On this account, the high temperature peak is attributable to free water in the system. Figure 4 shows relationships between melting (AHJ and crystallization (AHc) enthalpies of water in the pectin (P4) and W c . Both Gl, and AH,,, increased with increasing W , linearly. AHmis larger than AH,in the W , range examines in this study. In particular, AH,,, values in a W , range from ca. 0.4 to ca. 0.7 gg-' is not evaluated accurately since the endotherm of melting overlaps with the exotherm due to cold crystallization. It is thought that real AH,,, value is larger than the observed value. From the extrapolation of linear lines to x axis, the maximum amounts of Wn, where no melting or crystallization take place can be calculated, i.e. 0.53 gg-' from the AH,,, and 0.50 from

-

Affc-

Water restrained by pectins

307

0

0 l-

E

-20 40

""I

;,

-80

0

I

0.5

1.0 wc19 g"

1.5

2.0

Fig. 3 Relationships between melting temperature (T,,,) of pectin (P4)-water systems and water content (WJ. 0; melting peak temperature, 0; start temperature of melting, 0; end temperature of melting. Figure 5 shows relationships between amount of non-freezing water of pectin (P4) (Wnf, gg-') and W , (g-g-'). Non-freezing water calculated from crystallization enthalpy of pectin (Wnf J and that from melting enthalpy (Wnf,,) increase with increasing W , in the region below 0.7 g-g.' and maintains almost constant value of W , = ca. 0.6 g-g-I. Figure 6 shows relationships between non-freezing water (Wnf, g*g") and degree of methyl esterification (DE) of pectins. W,, values calculated at W E= 3 are shown in Figure 6. Wnfincreases with increasing DE. This suggests that the large side-chain -COCH, expands inter-molecular distance. The number of non-freezing water molecules bound by one repeating unit of pectin is 4.6 6.4 mol. The number of hydrophilic groups present in one repeating unit of pectin is 2.28 0.84 according to DE. On this account, it is found that the number of water molecules bound by one hydrophilic group is larger than 1 . It is clear that several water molecules are restrained by the hydrophilic group. At the same time, it is seen that W,, values calculated by crystallization enthalpy are larger than those from melting enthalpy. As already mentioned in our previous paper [ 131, a part of bound water freezes as amorphous state. Amorphous ice markedly affects the molecular motion of polysaccharide molecules. Figure 7 shows relationships between glass transition temperature (T,) of pectin (P4)water systems, heat capacity difference (ACp) at T, and W,. T of the pectin-water systems decreased remarkably with increasing W , ranging from d to 0.4 pg". After reaching a minimum value of T ( for example - 92 "C for P4), T, slightly increased and approached a constant value. h e minimum value of T , corresponded to W , where a melting peak of free water appeared. T of pectins shows it minimum value (T, m,n) at -91 "C to -97 "C in the W , ranging from d.6 to 0.8 g-g-I. T, m,n shifted to the low W , side with increasing DE, though the data of other pectins are not shown here 13 . ACp remarkably increased with increasing W , ranging from 0 to ca. 1.0 g g -I, and 1 ACp

- -

decreased when W , increased more than 1.O gg-'.

1.o

lo00

I I

I

800

-

600

-

0.8

I

Tm0.6

cn

0, 3

\ v-

\

-

/,-

2oo

I /$' 0

t

sC0.4

-

$400

1

i

0.2

wc I 9 9-'

2

.

Fig 4 Relationships between enthalpy of sorbed water with pectin (P4) (AH) and water content (WJ. 0; crystallization enthalpy (AH,), @; melting enthalpy (AH,,,).

0.8

0

3

1

0

wcI 9 g-'

2

Fig. 5 Relationships between non-freezing water of pectin (P4) (Wnf) and water content (W,). 0; non-freezing water at crystallization enthalpy of pectin (Wnf J, @; non-freezing water at melting enthalpy (Wnf3.

1

0

wnfc

0.4 0

20

3

40

60

80

100

DE/%

Fig. 6 Relationships between non-freezing water (Wnf)and degree of methyl esterification (DE) of pectins. 0; non-freezing water at crystallization enthalpy of pectin ( W , ,), @; nonfreezing water at melting enthalpy (Wnf,,,).

Water restrained by pectins

- 0.8

0

0.5

1.0

1.5

wc19 g”

2.0

Fig. 7 Relationships between glass transition temperature (T ) of pectin (P4)-water systems, heat capacity difference (A&) at T , and water content (WJ.

0.8 ”

r

-100

.

-

-50

TgI ‘C

0

50

Fig 8 Relationship between A Q and T,.

309

310

New polymers and materials

Figure 8 shows the relationship between ACp and T, in a W range from 0 to 0.5 gg-'. The reported ACp value of pure amorphous ice is 1.94 J-g-'*Kd at 134 K [14]. ACp value of pectins in the dry state are 0.072 J-g-'-K-' (Pl), 0.150 J-g-'K' (P2), 0.379 J.g-'eK-' (P3) and 0.249 Jog- *K ' (P4). On this account, the results shown in Figure 8 indicate that the amount of amorphous ice is a major factor which contributes to the molecular motion.

CONCLUSION The phase transition behaviour of pectins having various degrees of methyl esterification in the presence of sorbed water was investigated by differential scanning calorimetry (DSC). It was found that non-freezing water markedly affects the glass transition, cold crystallization temperatures. Phase diagram of pectin-water systems with water content from 0 to 3.0 g.g-' was established in a temperature from -150 to 70 "C.

REFERENCES 1 Beli R. Thakur, Rakesh K . Singh, Avtar K . Handa, 'Chemistry and Uses of Pectin - A Review', Crit. Revi. Food Sci. Nufr., 19973 7 47-73 2 Reginald H. Walter, The Chemistry and Technology of Pectin, New York, Academic Press, 1991 3 Robert L. Davidson, Handbook of Wafer-SolubleGums and Resins, USA, McgrawHill Book Company 1980 4 Robert C. Jordan, David A. Brant, 'An Investigation of Pectin and Pectic Acid in Dilute Aqueous Solution', Biopolymers, 1978 17 2885-2895 5 E. R. Morris, M. J. Gidley, E. J. Murray, D. A. Powell, D. A. Rees, 'Characterization of pectin gelation under conditions of low water activity, by circular dichroism, competitive inhibition and mechanical properties', Int. J . Biol. Macromol, 1980 2(10) 327-330 6 M. A. F. Davis, M. J. Gidley, E. R. Morris, D. A. Powell, D. A. Rees, 'Intermolecular association in pectin solutions', Inf.J . Biol. Macromol, 1980 2(10) 330332 7 M. .J. Gidley, E. R. Morris, E. J. Murray, D. A. Powell, D. A. Rees, 'Evidence for two mechanisms of interchain association in calcium pectiate gels', Int. J . Biol. Macromol, 1980 2( 10) 332-334 8 M. A. V. Axelos, M. Branger, 'The effect of the degree of esterification on the thermal stability and chain conformation of pectins', Food Hydrocolloids, 1993 7(2) 91-102 9 D. Oakenfull, 'A method for measurements of shear modulus to estimate the size and thermodynamic stability of junction zones in noncovalently cross-linked gels', J . Food Sci., 1984 49 1103-1110 10 H. Hatakeyama, T. Hatakeyama, 'Interaction between water and hadrophilic polymers', Thermochima. Acta, 1998 308 3-22 11 T. Hatakeyama, F. X. Quinn, H. Hatakeyama, 'Change in freezing bound water in water-gellan systems with structure formation', Carbohydrate Polymers, 1996 30 155160 12 T. Hatakeyama, H. Yoshida, H. Hatakeyama, 'The liquid crystalline state of watersodium cellulose sulphate systems studied by DSC and WAXS', Thermochima. Acta, 1995 2 6 6 343-354 13 M. Iijima, K . Nakamura, T. Hatakeyama, H. Hatakeyama, 'Phase transition of pectin with sorbed water', Carbohydrate Polymer, 2000 41 101-106 14 M. Sugisaki, H. Suga, S. Seki:, 'Calorimetric study of the glassy state. IV. Heat capacities of glassy water and cubic ice.', B u f f .Chem. Soci. Jpn., 1968 41 2591-2599

THERMAL PROPERTIES OF WOOD CERAMICS BY TG-MS AND CRTG Tadashi Ariil*and Michihiko Momota'

'

Thermal Analysis Division, Rigaku Corporation, 3-9-12 Matsubara, Aki.shima, Tokyo 196-8666, Japan *corresponding author e-mail: [email protected] Fax.: 81 -42-544-9630

ABSTRACT Thermal property of wood ceramics developed as eco-materials has been carried out successively by means of TG-MS and CRTG. The decomposition process strongly depended upon the curing temperature of the specimen. TG-MS was available for characterization and identification of evolution gases, and CRTG provided more reliable information for themostability of the material.

INTRODUCTION Recently, eco-materials harmonizing with an ecological system have been the focus of much attention from the viewpoint of global environmental problems. "Wood ceramics" has been developed as one such new eco-material, and is made from a c:omposite precursor of natural lignin and phenol resin carbonized at high temperature in vacuum '. Now, the material has been examined as use for various purposes, e.g., spaceship material, electromagnetic shield, neutron moderation material, etc. In this study, to confirm the thermal properties in more detail, the thermal decomposition in both inert and oxidative atmospheres has been carried out by means of controlled-rate thermogravimetry (CRTG) and simultaneous method of coupling TG with evolved gas analysis using mass spectrometry (TG-MS) 5-6.

EXPERIMENTAL

Specimen The following three types of specimens were used; 1) wood ceramics from medium density fiber board (MDF) infiltrated with phenol resin at mass ratio 1:l using ultrasonic vibration, 2) wood ceramics cured at 400°C, and 3) wood ceramics cured at 80O0C, respectively. Before curing it was dried and hardened in a oven at 135°C for 10 hours.

Apparatus A Rigaku Thermo Plus Dynamic TG-DTA 8120 apparatus is connected to a Rigaku Thermo Mass system, using a capillary interface to create an integrated simultaneous TG-MS. The ion source of the quadrupole mass spectrometer (Q-MS) consists of a thoria-coated iridium filament to prevent oxidative-deterioration. This Q-MS was operated in the electron-bombardment

3 12

New polymers and materials

ionization (70eV) mode and the m/z ranging from 10 to 100. In the CRTG experiments, a specimen is heated at first at a constant heating such as S'C-min.', then at a proper switchover temperature the automatic control of the heating rate starts so that the rate of the mass loss is kept at a desired constant value. The powder specimens were heated in a platinum pan in inert (pure He and N2) and oxidative (20%0,-He mixture) atmospheres, with a purge flow rate of 200ml.min-'.

RESULTS AND DISCUSSION Inert (He) atmosphere Fig.1 shows the three-dimensional representation of TG-MS data in a He atmosphere on the non-cured wood ceramics. The change of fragment ions in the mass spectra for the evolved gasses is clearly observed during the decomposition of the specimen. Fig.2 shows TG, total ion current (TIC) and main mass chromatogram (MC) curves extracted from Fig.1. The total mass loss at 1000°C was ca.70.0%. The thermal process proceeds successively and seems to be complicated. It was easily recognized that the mass spectrum obtained during a first mass loss (peak-top temperature 86°C) in the TIC corresponded to only dehydration which was assigned by the simultaneous detection of m/z 18 (H,O) and 17 (OH'). After dehydration the specimen decomposed continuously with a maximum decomposition rate at 380°C. The mass spectrum obtained at 380°C included so many fragment ions (see Fig.l), and implied that several gaseous products may be evolved simultaneously. Production of water vapor (miz 18), carbon monoxide (m/z 28) and carbon dioxide (miz 44) were observed almost the whole range of the reaction, while benzene (miz 78) and phenol (m/z 92) were observed throughout later, as clearly shown in Fig.2. The results suggested that at first the MDF gives rise to evaporation of lignin oil due to dehydration and the cutting reaction of cellulose carbon fiber, and subsequently phenol resin gives rise to dehydration and bridge-building reactions within and between molecules. Figs.3 and 4 shown the results of TG-MS for wood ceramics cured at 400°C and 800°C. The mass loss until 150°C was approximately identical with those of the non-cured wood ceramics. With increasing the curing temperature for the specimen, the decomposition shifts toward the high temperature region, and the amount of mass loss is decreased. In the case of the wood ceramics cured at 400"C, after dehydration the gradual mass loss around 350°C was attributed to evolution of almost entirely carbon dioxide. The mass loss rate accelerates from around 500"C, and rises to the peak at 588°C. Detected gaseous products were approximately similar to those obtained from the non-cured specimen. Evolution of benzene and phenol was still detected, and was observed at higher and narrower temperature regions. In the case of the specimen cured at 800°C on the contrary, after dehydration it was followed by two reaction stages, and the rates of production of the detected gases were different to each other; carbon dioxide was mainly observed at the initial stage and was gradually evolved, while carbon monoxide was observed in the later stages. Benzene and phenol were not observed anymore. This implied that slow separation reaction of elements by cabonization processes proceeded gradually.

Oxidative (20%0,-He) atmosphere Practically, it is essential to know the influence of the thermal process for the atmospheric

Thermal properties of wood ceramics

3 13

Figure 1. Three-dimensional representation of TG-MS in He on non-cured wood ceramics.

I

200

400

600

BOO

Temperature / C

Figure 2. Results of TG-MS in He for thermal decomposition of non-cured wood ceramics.

J 1000

25 & o-

Temperature I

C

Figure 3. Results of TG-MS in He for thermal decomposition of wood ceramics cured at 400°C.

difference. Fig.5 shows the comparison of TG-DTA curves in the oxidative atmosphere for the three types of specimens. All specimens decomposed completely without residues, and the mass losses accompanying the rapid exothermic DTA peaks were observed. The decompositions occurred rapidly at higher temperature with increasing curing temperature of the specimen. Fig. 6 shows the results of TG-MS for the specimen cured at 400°C. The decomposition was initiated and completed at lower temperature than that in the inert atmosphere. Detected gaseous products were only water vapor (m/z 18) and carbon dioxide (m/z 28 and 44) without poisonous gases, regardless of the curing temperatures. Therefore, it was explicable that all volatilized products were combusted due to the high temperature and oxidative atmosphere. These facts mean that the thermo-stabilityof the specimen is increased with increasing curing temperature.

3 14

New polymers and materials

0

50

? 3 100 (II

-7

3 150

200

400

600 Temperature i"

c

800

1000

200

Temperature i" C

Figure 4. Results of TG-MS in He for thermal decomposition of wood ceramics at 800°C.

Figure 5. TG-DTA curves in 20%02-Hefor three types of wood ceramics. non-cured wood ceramics (solid line), wood ceramics cured at 400°C (dotted line),wood ceramics cured at 800°C (broken line).

i 0 10

. 2

$ 20

30

c

2

40

0

50

60

wood ceramics

70

80

160

240

320

400

480

560

640

Temperature i 0 C

Figure 6. Results of TG-MS in 20%0,-He For thermal decomposition of wood ceramics at 400°C.

200

400

600

800

Temperature 1 a C

Figure 7. Comparison of mass loss curves for thermal decomposition of wood ceramics in He using TG (solid line) with CRTG (broken line).

Thermal properties of wood ceramics

3 I5

CRTG analysis Fig.7 shows the comparison of mass loss curves for decomposition using conventional TG of 2OoC.min-’ with those using CRTG of 0.12%.min-’, and indicates the difference of thermal stability between three types of specimens. In the conventional TG, the specimen was forced to heat up at a constant rate, ignoring whether a certain decomposition occurs or not, so that the thermal process was observed in a relatively wide temperature range. On the contrary, the CRTG caused significant decomposition steps at lower and narrower range temperatures. In addition, the results implied that features of the thermal process observed by the conventional TG were different from those by CRTG, so that the reaction seems so as to proceed through a different mechanism in TG and CRTG.

CONCLUSIONS Thermal property of wood ceramics consisting of three differing curing temperatures was clarified in both inert and oxidative atmospheres by means of TG-MS and CRTG. Both TG-MS and CRTG permitted a detailed insight into the course of decomposition reactions and yielded significant information about the physical-chemical nature of the thermal process. The decomposition process strongly depended upon the curing temperature of the specimen. With increasing curing temperature of the specimen, 1) therrno-stability improved and the specimen was stable. 2) volatile products were simplified such as water vapor, carbon monoxide and carbon dioxide, and evolution of benzene and phenol was negligibly decreased. 3) combustion caused by the oxidative atmosphere occurred steeply at higher and over a narrower range of temperature. Because CRTG allows the decomposition steps at lower and narrower over a temperature window, 4) it provided more reliable data for the themo-stability of the specimen than the conventional TG.

ACKNOWLEDGEMENS The authors would like to acknowledge Professor Makoto Kano of Science University of Tokyo, in providing guidelines for the scope and supplying the materials.

REFERENCES 1. M. Kano, M.Momota, T. Okabe, K. Saito and R. Yamarrtoto, ‘Thermogravimetric and differential analysis of wood ceramics’, Trans. Muter: Res. SOC.Jpn, 1996, 20,40-43. 2. F. Paulik and J. Paulik, ‘Kinetic studies of thermal decomposition reactions under quasiisothermal and quasi-isobaric conditions by means of the derivatograph’, Thermochim. Acta, 1972,4,189-198. 3. J. Rouquerol, ‘Ccontrooled transformation rate thermal analysis : The hidden face of thermal analysis’, Thermochim. Acta, 1989, 144,209-224. 4. T. Arii and N. Fujii, ‘Controlled-ratethermal analysis : Kinetic Study in Thermal dehydration of Calcium Sulfate Dihydrate’,J. Anal. Appl. Pyrolysis, 1997,39, 129-143. 5 . T. Arii and Y. Masuda, ‘Thermal decomposition of calcium copper acetate hexahydrate by simultaneous measurement of controlled-rate thermogravintetry and mass spectrometry

3 16

New polymers and materials

(CRTG-MS)’, Thermochim. Acra, 1999, 342, 139-146. 6. T. Arii, Y. Sawada, N. Kieda and S. Seki, ‘TG-DTA-MS and Controlled-Rate TG of Ammonium Oxalate Monohydrate (NH4),C20; H,O’, J. Muss Spectrom. SUC.Jpn., 1999, 47, 354-359.

APPLICATION OF ENVIRONMENT CONTROLLED THERMOMECHANICAL,ANALYSIS SYSTEM Hidetaka KATOH', Toshihiko,NAKAMURA' & Nobuaki OKUBO'

' Scientific Instruments Division, Seiko Instruments Inc. 1-8 Nakase, Mihama-ku, Chiba-shi, Chiba 261-8507, Japan

INTRODUCTION Dynamic mechanical analysis (DMA) and thermomechanical analysis (TMA) are widely used to characterize the mechanical properties of various materials. The evaluation of moisture absorption properties by changing relative humidity on a constant temperature has been reported by Yano, Kodornari and Okubo '. Viscoelastic properties of rubber under different organic fluids have also been reported previously *. Research interest and demand for these types of measurements have increased over the years. Dh4A or TMA systems which can be used under a variety of environments (for example ; in controlled humidity environments, water or organic solvent environments, etc.) were newly developed'. In this report, we introduce the Environment Controlled Thennomechanical Analysis System. This system can be applied not only I:O isothermal measurements at humid conditions but also to temperature scanning under a constant humidity. Furthermore, this system is available for measurements under liquid environments, such as water, organic solvents and oil. Viscoelastic properties of nylon 6 studied under humidity controlled environment as a function of tempera.ture is discussed.

Measurement Module

Sample

Ou.terChamber

/-Transfer tube

I1

U Monitor Vapor Generator

,

t l

U I -

Bath Circulator Bath Circulator for Vapor control for Sample temp. control

Figure 1.

Schematic diagram of the Environment Controlled Thermomechanical Analysis System

3 18

New polymers and materials

SYSTEM CONFIGURATION Fig. 1 shows a schematic diagram of the Environment Controlled Thermomechanical Analysis System, This system can be adopted to work with both, the Seiko DMS6100 Dynamic Mechanical Analyzer and the TMNSS6100 Thermomechanical Analyzer. A special furnace is connected with two water bath circulators capable of temperature control. One bath circulates temperature-adjusted water into the outer chamber and the other bath is used to introduce humidity-controlled air to the inner chamber. Temperature of the transfer tube connecting the inner chamber with the vapor generator is controlled in order to prevent vapor condensation. Both temperature and humidity close to the sample are monitored using sensors installed in the inner chamber. When measurements are carried out in fluids, such as water, organic solvents and oil, the fluid is directly introduced into the inner chamber. In this case, the water vapor generating unit is closed.

STABILITY OF RELATIVE HUMIDITY Both isothermal measurements at humid conditions and temperature scanning under constant humidity can be carried out using this apparatus. Fig. 2 shows variation of relative humidity (RH) values of 20, 40, 60 and 80% on heating at 0.5"C/min. Area enclosed by the dotted line in figure 2 showed the range where reliable results were obtained using this system. For 20, 40 and 60%RH, variability was within 21.5%. For 80%RH, it was within 23%.

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Stability of Relative Humidity as a function of temperature

Thennomechanical analysis system

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0.02 10

20

30

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Figure 3.

Dynamic viscoelastic properties of nylon 6 conditioned at various humidity

MEASUREMENT EXAMPLE Fig. 3 shows the DMA curves of nylon 6 measured with the DMS6100 under humidity controlled environments. Storage modulus E' and tan 6 measured at 1 Hz are plotted as a function of temperature at 0, 20, 40, 60, and 80%RH, respectively. It is evident from the data that glass transition temperature decreases with increasing relative humidity. The moisture absorbing properties of nylon have been widely studied and are well known. The amide group (-NH-CO-) acts as a hydrophilic: functional group. A humidity controlled system to study such properties not only provides a critical means for quantitative measurement of this shift, but also gives an insight to plasticization effects on such polymers.

REFERENCES 1. S . Yano, M. Kodomari, N. Okubo, The dynamic viscoelastic properties of hydroxypropyl cellulose/silica-gel hybrid, In: The Pacific Conference on Rheology and Polymer Processing '94, 1994, pp. 309-310 2. Y. Ichimura, In: Handbook of Calorimetry Measurement and Thermal Analysis, The Japan Sociaty of Calorimetry and Thermal Analysis, Maruzen, Tokyo, 1998, p. 240 3. H. Kato, T. Nakamura, N. Okubo, Application of Thermomechanical analysis with controlled atmosphere system, In: The 341hJapanese Conference on Calorimetry and Thermal Analysis, 1998, pp. 236-237

EFFECT OF WATER ON MOLECULAR MOTION OF ALGINIC ACID HAVING VARIOUS GULURONIC AND MANNURONIC ACID CONTENTS *Masato Takahashi'), Yuka Kawasaki", Tatsuko Hatakeyama') and Hyoe Hatakeyama" ')DepartmentofFine Materials Engineering, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ue& Nagano 386-8567, J q o n

''Department of Textile Science, Faculty of Home Economics, Otsuma Women's University, 12 Sanhancho, Chiyoda-ky Tokyo 102-8357, Japan

"Depmfment of Physics and Chemistry, Farulty of Engineoing, F u h i University of Technology, 3-6-1, Gakuen, Fukui 910 -8505, Japan

ABSTRACT The effect of water on the molecular motion of alginic acid (Alg) was investigated by differential scanning calorimetry. Alg is a copolysaccharide consisting of guluronic (G) and mannuronic acid (M). A phase diagram of water-Alg with various M/G ratios, such

as 1.20, 0.88 and 0.18 , was established over a water content (= W, = gram of water/gram of dry Alg) ranging from 0 to 3.0 g/g. When the system was quenched from 50 to -1OO"C, glass transition, cold crystallization, pre-melt crystallization, melting and liquid crystal transition were observed from the low to high temperature side. The Wc range where the glassy state and liquid crystalline state are formed increased with increasing M/G ratio. This suggests the molecular flexibility of M constituent is higher than that of the G constituent, since the hydroxyl groups of the M acid are located to facilitate easy contact with water molecules. This is also supported by the fact that the amount of bound water in the systems increases with increasing M/G ratio.

INTRODUCTION Alginic acid (Alg) is a polysaccharide extracted from seaweed. Morns and coworkers studied the conformational structure of Alg and suggested that Alg is the copolysaccharideconsisting of guluronic acid (G) and mannuronic acid (M) [ 1-31. The

322

New polymers and matenals

physical properties of Alg/water systems depends on the M/G ratio.

In our previous

studies, phase diagrams of various polysaccharide-water systems have been investigated

as a function of water content W, (= weight of water in gram / weight of dry plysaccharide in gram) by differential scanning calorimetry (DSC) [4-201. In general, glass transition of water-plysaccharide complex domain, cold crystallization, melting of water and liquid crystal transition were observed from low to high temperature side in the phase diagrams.

Therefore, it is very important to investigate the effect of M/G ratio on

the transition temperatures described above. In this study, phase diagrams of water-Alg with various M/G ratios were determined by differential scanning calorimetry (DSC), and the effect of water on the molecular motion of Alg with various M/G ratio is discussed.

EXPERIMENTAL Alg was supplied by &bun Food Chemical Co. The values of G/M-ratio and limiting viscosity Q are listed in Table 1. The sample was prepared in an aluminum

(Al)sealed pan. The solvent used was pure water, provided by Wako Pure Chemical Industries L.t.d. The desir'ed amount of dry Alg and excess amount of water were put into an Al sealed pan, and then sealed when the water content Wc reached the desired amount by the evaporation of water.

The experimental procedure is reported elsewhere

in detail[6, 8, 9, 111. The water content is defined by

Wc = weight of water in grams 1weight of dry Alg in gram (1) The sample was annealed at 40 "C for 1 hr in order to ensure homogeneity before DSC measurements.

Table 1. Values of M/G ratio and limiting viscosity Code Name

M/G

350M 500M

0.88

350G

0.18

1.28

Q

of Alg samples used. 7) (CP)

380 550 355

In DSC measurements, the measurement was carried out in a N2 atmosphere. The sample was cooled to -150 at 10 "Clmin and then heated to 85 at 10 "C/min after

"c

holding at -150 "C for 10 min.

"c

After the measurement was completed, a small pinhole

was made and the sample dried in an oven.

The final value of Wc was determined by

Effect of water on alginic acid

4

323

.1 Y

w

w

120

175

230

285

- 175

I

I

120

T(K)

230 T(I0

285

340

Figure 1. DSC curves of SOOM/water systems with W, = 0.56: (a) and 1.83: @) measured in heating process at 10 "C/min.

sz

250

200 150 100

i

0 0.5

A

1

1.5

2

2.5

Tpc Tcc

3 3.5 4

wc (dg)

300 jC"250

F 200 150 100

Figure 2. Phase diagrams of Aldwater systems composed of 350G: (a), S00M (b) and 3SOM: (c) determined by DSC measurements.

324

New polymers and materials

weighing the dried sample.

RESULTS AND DISCUSSION Figure 1 shows DSC heating curves of SOOM/water systems with Wc = 0.56 g/g.

As shown in Figure

l(a),

glass transition

(T,),

cold

crystallization

(To),

premeltcrystallization (TJ, melting of water (TJ and liquid ~ ~ y s t to a l isotropic liquid transition (T*) temperatures were observed from low to high temperature side. T,,,,, are hardly observed for the sample with Wc as shown in Figure. l(b).

T,, and

Figure 2 shows phase diagrams of water/Alg systems with three different M/G ratios. From the phase diagrams thus obtained, the following results were obtained. transition temperature T, increased with the increase of M/G ratio.

Glass

Wc range, where the

glassy state and liquid crystalline state are formed, increased with increasing M/G ratio. On the other hand, the Wc range where the premelt crystallization temperature Tpcwas

observed decreased with the increase of MIG ratio. 350

300 250

-.

200

ol

1

150

I

a

100

so 0 0

0.5

1

1.5

2

2.5

3

3.5

4

wc (919)

3 50

300 250

-.

Figure 3 . Relationship between the enthalpy of melting, A H,, of Aldwater

200

0

LI

2

systems composed of 3SOG: (a), 500M: @) and 3SOM: (c) and Wc.

150

r

a

100

50 0

0

0.5

1

1.5

2

2.5

wc (919)

3

3.5

4

Effect of water on alginic acid

325

Figure 3 shows Wc dependence of the enthalpy of melting of 1 mg of water in systems with various M/G ratios. From Figure 3, it is concluded that the W, range where all

-

-

water molecules behave as non-freezing water increased with the increase of M/G ratio, i.e. 0 0.3 for 350G, 0 0.4 for 500M, 0 -0.5 for 350M. The experimental results obtained here seem to show that the molecular flexibility of the M constituent is higher than that of the G constituent, since the hydroxyl ;groupsof M acid are located to facilitate easy contact with water molecules.

ACKNOWLEDGEMENT This work was supported by Grant-in-Aid for COE3 Research (10CE2003) by the Ministry of Education, Science and Culture of Japan.

REFERENCES 1). E. D. T. Atkins, I. A. Nieduszynkins, W. Mackie, K.. D. Parker and E. E. Smolko, ‘Structural Components of Alginic Acid. , The Cryslalline Structure of Poly-P-D Mannuronic Acid. Results of X-ray Diffraction and Polarized Infrared Studies’,

Biopolymers, 1973, 12, 1865-1878. 2). E. D. T. Atkins, I. A. Nieduszynkins, W. Mackie, K. D. Parker andE. E. Smolko, ‘Structural Components of Alginic Acid. . The Crystalline Structure of Poly-a-LGuluronic Acid. Results of X-ray Diffraction and Polarized Infrared Studies’, Biopolymers, 1973, 12, 1879-1887. 3). A. Haug, B. Larsen and 0. Smidsrod, Acta Chem. Scand., 21,691 (1967). 4). K. Nakamura, T. Hatakeyama, and H. Hatakeyama, ‘Studies on Bound Water of Cellulose by Differential Scanning Calorimetry’, Text. Res. J., 1981, 51, 607-613. 5). K. Nakamura. T. Hatakeyama, and H. Hatakeyama ‘Effect of Bound Water on Tensile Properties of Native Cellulose’, Text. Res. J., 1983, 53, 683-688. 6): T. Hatakeyama, K. Nakamura, H. Yoshida and H. Hatakeyama, ‘PHASE TRANSITION ON THE WATER-S ODIUM P IDLY(STYRENESU LFONATE) SYSTEM’, ThermochimicaActa, 1985, 88,223-228. 7). T. Hatakeyama, H. Yoshida, and H. Hatakeyiuna, ‘A differential scanning Calorimetry study of the phase transition of the water-sodium cellulose sulphate system, Polymer, 1987, 28, 1282-1287. 8). T. Hatakeyama, K. Nakamura and H. hatakeyama, ‘DETERMINATION OF BOUND WATER CONTENT IN POLYMERS BY DTA, DSC AND TG’, Thermochimica Acta, 1988, 123,153-161. 9). T. Hatakeyama, K. Nakamura, H. Yoshida, and H. Hatakeyama, ‘Mesomorphic

326

New polymers and materials

properties of highly concentrated aqueous solutions of polyelectrolytes from saccharides ’, Food Hydrocolloids, 1989, 3, 301-31 1. 10). H. Yoshida, T. Hatakeyama and H. Hatakeyama, ‘Glass Transition of Hyaluronic Acid Hydrogel’, Kobunshi Ronbunshu, 1989, 46, 597-602. 11). H. Yoshida, T. Hatakeyama, and H. Hatakeyama, ‘Phase transition of the waterxanthan system’, Polymer, 1990, 31, 693. 12). K. Nakamura, T. Hatakeyama, and H. Hatakeyama, ‘Formation of the Glassy State and Mesophase in the Water-Sodium Alginate System’, Polym. J . , 1991, 23, 253. 13). T. Hatakeyama and H. Hatakeyama, Polym. Adv. Technol., 1,305 (1991). 14). T, Hatakeyama, N. Bahar, and H. Hatakeyama, ‘LIQUID CRYSTALLINE STATE OF WATER-CARBOXYMETHYLCELLULQSE SYSTEMS SUBSTITUTED WITH MONO- AND DIVARENT CATIONS’, Seni-Gakkaishi, 1991, 47,417-418. 15). K.Nakamura, T. Hatakeyama, and H. Hatakeyama, ‘FORMATION OF THE LIQUID CRYSTALLINE STATE IN THE WATER-SODIUM ALGINATE SYSTEM’, Sen4 Gakkaishi, 1991, 47,421-422. 16). T. Hatakeyama, H. Hatakeyama, and K. Nakamura, ‘Non-freezing water content of mono- and divarent cation salts of polyelectrolyte-water systems studied by DSC’, Thermochim. Acta, 1995, 253, 137-148. 17). T. Hatakeyama, H. Yoshida, and H. Hatakeyama, ‘The liquid crystalline state of water-sodium cellulose sulphate systems studied by DSC and WAXS’, Thermochim. Acta, 266,343 (1995). 18). K. Nakamura, Y. Nishimura, T. Hatakeyama, and H. Hatakeyama, ‘Thermal properties of water insoluble alginate films containing di- and trivalent cations’, Thermochim. Acta, 1995, 267,343-353. 19). J . Ratto, T. Hatakeyama, and R. B. Blumestein, ‘Differential scanning calorimetry investigation of phase transitions in waterkhitosan systems Polymer, 36, 2915 (1995). 20). T. Hatakeyama, K. Nakamura and H. Hatakeyama, ‘Glass Transition of Polysaccharide Electrolyte-Water Systems’, Kobunshi Ronbunshu, 1996, 53, 795-802.

EFFECT OF THE INITIAL STATE ON THE SORPTION ISOTHERM AND SORPTION KINETICS OF WATER BY CELLULOSE ACETATE Hiromi Gocho', Akihiko Taniokaz*, and Toshinari Nakajima3 'Department of Food and Nutrition, Seitoku Jr. College of Nutrition 1-4-6 Nishishinkoiwa, Khtsushika-ku, Tokyo 124-8530, Japan 'Department of Organic and Polymeric Materials, TokyoInstitute of Technology 2-12-1 Ookayama, Meguro-ku Tokyo 152-8S52, Japan 3Doctoral Course of Science for Living System, Showas Women's University 1-7-57 Taishido,, Seiagaya-ku, Tokyo 154-8S33, Japan

INTRODUCTION Cellulose acetate is in a glassy state near room temperature, since its glass transition temperature is about 150°C. Sorption experiments of water by cellulose acetate are performed at 20°C to 30°C because cellulosic materials are usually used around these temperatures. If sorption kinetic measurements of water using this polymer are done in this state, the shape of the sorption kinetic curve strongly depends on the initial state of the sample and the sorption history of the sample. After repeating the same sorption experiments several times, we can obtain data which can be used for the analysis. The sorption isotherm of water using a glassy polymer shows a sigmoid shape' and strongly depends on the state at 0% relative humidity, which is determined by the drying procedure. These problems are due to the fact that the state of the glassy polymer is determined not only by the state variables, i.e. pressure (p) and temperature (T), but also requires a third state variable, the so-called ordering parameter (5)for full characterization of its statezv3. In this study, the sorption kinetics and sorption isotherm phenomena of water using cellulose acetate are examined based on non-equilibrium thermodynamics' though there are no optimum methods to exactly determine the 5 parameter at this time.

EXPERIMENTAL Samples A commercial cellulose triacetate film, whose acetyl group degree of substitution was 2.71, was partially hydrolyzed in a 1N NaOH aqueous solution at 25°C. The reaction times were 0, 1, 3, 5 and 7 days in order to obtain samples which had various degree of acetylation, that is 42.2%, 38.9%, 32.2%, 27.6% and 11.8%, respectively. After the hydrolysis, the cellulose acetate film was immersed in benzene for 1 hr and ethanol for 1 hr, and subsequently washed in distilled water. The prepared samples were then kept in water at 80°C for 3 hr before air drying. The degree of acetylation was determined using the acetyl group microanalysis method based on saponification and successive titration by NaOH.

328

New polymers and materials

Measurements of sorption kinetics The measurement of the sorption kinetics was made using the spring balance method. Seventy mg rectangular-shaped samples were hung at the bottom of the quartz spring balance in the vacuum chamber made of Pyrex glass connected to a vacuum pump and humidity controller via glass cocks. The temperature in the vacuum chamber and the humidity controller was kept at 20°C by circulating water around them. First the vacuum chamber was evacuated by the vacuum pump for several hours and successively the water vapor at 44% R.H. was introduced into it. The relativc humidity (R.H.) was controlled by a saturated K2C0, aqueous solution. The change in the length of the quartz spring balance according to the water vapor sorption by cellulose acetate was measured with a cathetometer as a function of time and converted to the weight change from the calibration curve between length and weight. After a sorption kinetics measurement, the sample was dried in order to make the next measurement of sorption kinetics. The measurements were repeated several times before obtaining a reproducible curve.

Measurements of sorption isotherm Samples dried by three different procedures (A, B and C) in weighing bottles were placed in desiccators for 10 days where the humidities were controlled by various saturated aqueous salt solutions. The weighing bottles with the samples were removed from the desiccator for quick weighing. After weighing, the bottles were This placed back in the desiccator. After 10 days, they were again weighed. procedure was repeated every 2 or 3 days until the weight change was within t0.05%. The measurement temperature was 2O0CkO.2"C. The water content in the polymer (water regain), V, was represented by the weight of sorbed water (8) per weight of dried polymer (g) as a function of Telative vapor pressure, x. Sample A is the sample that was dried by P,O, at 20°C. Sample B is the sample that was evacuated for 2hr at 105°C and Sample C is the sample that was first dried by P,O, at 20°C and successively cvacuated for 2hr at 105°C after once sorbing water. The degree of acetylation for the sample, which was used in this experiment, was 27.6%.

RESULTS AND DISCUSSION In Fig. 1, the changes in the adsorbed water (Q: g/g-dry polymer) by the cellulose acetate film with a 42.2% degree of acetylation are shown as a function of time (t: min). The experiments were repeated 7 times before obtaining a reproducible curve. Sorption kinetic curves for the samples with 38.9%, 32.2% and 27.6% degrees of acetylation are very similar to those in Fig.1. In Fig. 2, Q is shown for the film with an 11.8% degree of acetylation as a function of t. The repeated experiments were done only 4 times. Since the glass transition temperature of cellulose acetate is about 150"C, these samples are in glassy state before water sorption and still with a low water content. The shape of the sorption kinetic curve strongly dcpends on the initial state of the sample and the sorption history of the sample. Aftcr repeating the experiments several times, the curve is summarized in its final form. This situation is affected by the acetyl content. The state of the glassy polymer is determined not only by the state variables, is., pressure (p) and temperature (T), but also requires third state variables, the so-called ordering parameters (t,,E2 5,) for full characterization of its state.

-

Sorption isotherm and sorption hnetics of water

329

These ordering parameters are defined by the affinity (A,) of the actual state of the polymer membrane where A, is equal to the chemical potential difference of the polymer in its actual state, ~.lp(T,p,E,),minus the chemic:al potential of an equilibrium state, pp(T,p,C=O), of the polymer. The sorption isotherms and the sorption kinetics of water by cellulose acetate were analyzed by the model based on nonequilibrium thermodynamics. If the degree of acetylation decreases, we can reduce the number of

0.05 C

a

E n

0.04

0.03

2

.

P 0.02 CI)

z

CI)

0.01

0 0

50

100

150

200

t (min)

Fig. 1 Sorption kinetic curves of water by cellulose acetate film with 42.2% degree of acetyla-tion as a function of time (min) at 20 "C. Q is the amount of sorbed water ( g ) per dry polymer (g). The sorption experiments are repeated 7 times with the same sample and under the same conditions. 0 indicates the 1st sorption experiment, the 2nd, X the 3rd, 0 the 4th, A the 5th, A the 6th and 0 the 7th. 0.05 L h L

'

0.04

21 -

2

P

0.03 0.02

0)

v

0

0.01 0 0

50

100

150

200

t (min)

Fig. 2 Sorption kinetic curves of water by cellulose acetate film with 11.8% degree of acetyla-tion as a function of time (min) at 20 "C. Q is the amount of sorbed water (8) per dry polymer (g). The sorption experiments are repeated 4 times with the same sample and under the same conditions. 0 indicates the 1st sorption experiment, the2nd, X the3rdand 0 the4th.

330

New polymers and materials

!i

-

0.08 -

) .

g

0.06

-

2

u 0.04 m m

. * ;

0.02

1

0 0

10

20

30

40

50

Degree of Acetylation (%)

Fig. 3 Amount of saturated sorbed water per dry polymer (Q,) during the final stage of the repeated sorption experiment as a function of the degree of acetylation. repeated experiments before obtaining the final curve. Fig.2 shows that the curves for four trials had the same value, which is similar to the case for a rubbery polymersolvent system. In Fig. 3, the amount of saturated sorbed water Q, (gig-dry polymer) during the final stage of the repeated experiments are shown as a function of the degree of acetylation. R decreases with increasing degree of acetylation because the sample, which has the high acetyl content, is hydrophobic. Sorbing the water by cellulose acetate decreases the glass transition temperature. The sample with degree of acetylation 11.8% seemed to have become rubbery with the high water content because we cannot observe a large difference between each trial. In Fig. 4, the ratio of the sorbed water at time t to the saturated sorbed water for various samples, QJQ,, with different degrees of acetylation are plotted as a function of tin. These curves are not linear at the early sorption times, which indicates that they are not Fi~kian'.~. However, we can estimate the apparent diffusion coefficient of water through the cellulose acetate, Dapp,from the curve whose degree of acetylation is 38.9% because the linearity is higher than that of the other curves. Therefore, Dapp was determined to be about 3 ~ 1 O ' ~ c m ~ / s . Taking into account the QJQ, -tlR curve for the 11.8% degree of acetylation, it shows a typical non-Fickian figure though we cannot find the deviation among the curves as shown in Fig. 2. In this case, it is suggested that the distribution of the ordering parameter is very low. Fig. 5 shows the sorption isotherms for the cellulose acetate films for various drying procedures. The degree of acetylation for this sample is 27.6%. A shows the sample which was dried using P,O, at 20"C, B shows the sample which was evacuated for 2hr at 105"C, and C shows the sample which was first dried using P,O, at 20°C and successively evacuated for 2hr at 105°C after sorbing water one time. The amount of water sorbed by each sample is in the order C>B>A. The shape of the sorption isotherm strongly depended on the initial state of the sample, which was determined by the drying procedure. Applying the modified n-th layer BET equation as shown in

Sorption isotherm and sorption kinetics of water

331

equation (1) below to the sorption isotherm, the total number of adsorption sites in the

0.8 0.6

~

9

U-

0.4

0.2 0

5

0

t 112

10

15

Fig. 4 Sorption kinetic curves as a function of tln at 20°C for various samples with different degree of acetylation. QJQ, indicates the amount of sorbed water at time, t, shows the sample whose degree of per amount of saturated sorbed water. accetylation is 42.2%, 38.9%, X 32.2%, 0 27.6% and A 11.7%.

0.1

L

0.08

a,

0.06 Q

z

6 0.04

. 0)

0)

iF 0.02 0

0

0.2

0.6

0.4

0.8

X

Fig. 5 Sorption isotherms for the cellulose acetate films using various drying procedures. R is the amount of sorbed water per dry polymer at the sorption equilibrium. The degree of acetylation for this sample is 27.6%. .(A) shows the sample which was dried for 10 days using P,O, at 20°C, H(B) evacuated for 2hr at 105"C, and X(C) first dried for 10 days by P,O, at 20°C and successively evacuated for 2hr at 105°C after sorbing water one time.

332

New polymers and materials

polymer, V,, the interaction energy between water and adsorption sites, K, and the number of adsorbed layers, n are calculated4,’and listed in Table 1.

(1) where x corresponds to the relative vapor pressure (= p/p,), and (2) K = fmoHC,oH + fmAcCiac fmOHand fmAcare the fractions of unadsorbed sites of the hydroxyl groups and acetyl groups relative to the total number of unadsorbed sites, respectively, and C,, and CIA, are the C parameter in the original BET equation. The following relationship is found between fmOH and fmAc, (31 fmOH + fmAC= 1 According to Table 1 , K and n are constants for drying procedures, A, B and C. On the other hand, V, increased in the order of A, B and C. It suggests that the adsorption site increases if the sample in a glassy state is completely dried as much as possible. In eq.(2) C,, and CIA, should be constant even if the drying procedure is altered. Therefore, fmoHand fmAcare also constant though the number of adsorption sites is changed according to the drying procedure. This result indicates that the existing ratio of the hydroxyl groups and the acetyl groups in the film is not affected by the drying procedure.

Table 1 Parameters of modified BET equation (V,,,, K and n) for the cellulose acetate films’)for three drying procedures*)at 20°C

B 0.034 3.1 3

A v m

K n

0.028 3.2 3

C 0.037 3.2 3

1) The degree of acetvlation this sarnole is-27.6%. . .for .~ ~ ~. ~ . ~ . . .~ 2) A m e s s thesample which was dried for 10 days usin P 0 at 20°C B evacuated for 2hr at 105°C. and C first1 dried for 10 days using P,& at 2 k and kcessively evacuated for 2hr at 105.6 after one water sorptxon. ~~~~~

~

~

REFERENCES 1. T Nakajima and H Gocho, ‘Sorption of the water vapor by vinyl acetate-vinyl alcohol copolymers’, Nihon Kagakukaishi, 1978, 10, 143 1- 1436 2. S Motamedian, W Pusch, A Tanioka and F Becker, ‘Sorption isotherms of gases by polymer membranes in the glassy state; An explanation based on the nonequilibrium thermodynamics’, J. Colloid and Interface Sci., 1998,204, 135-142 3. R N Haward, Ed., The Physics of Glassy Polymers, Applied Science, London, 1973 4. H Gocho, A Tanioka and T Nakajima, ‘Sorption isotherm analysis of water by hydrophilic polymer composed of different adsorption sites using modified BET equation’,J. Colloid and Interface Sci., 1998,200, 155-160. 5. J Crank and G S Park, Ed., Diffusion in Polymers, Academic, London, 1968 6. J Crank, The Mathematics of Diffusion, Clarendon, Oxford, 1975 7. H Gocho, H Shimizu, A Tanioka, T -J Chou and T Nakajima, ‘Effect of acetyl content on sorption isotherm of water by cellulose acetate: Comparison with the thermal analysis results’, Carbohydrute Po(ymers, 1999,41,83-86

OSMOMETRIC AND VISCOMETRIC STUDIES ON THE COIL-HELIX TRANSITION OF GELLAN GUM IN AQUEOUS SOLUTIONS Etsuyo Ogawa' I Showagakuita Jr. College, Higashisugano, Ichikawa, Clriba 272-0823, fopan

ABSTRACT Conformational behavior of sodium type gellan gum in aqueous solutions was studied by osmometry and viscometry. Osmotic pressure and intrinsic viscosity mcasuremcnts were carried out in the range from 45 to 15"Cfor aqueous solutions with NaCl (concentration Cs=25, 50, and 75 mmoVdm3). It was found that the Mn values obtaincd at 45, 40, 36, 28, and 25°C with different Cs agreed with cach othcr and the averagc Mn values above 36°C wcrc almost half the valucs obtained at 28 and 25"C, suggcsting association of two molecules. At 32°C unassociated molecules seems to be in simultaneous equilibrium with associated molecules. By lowering tempcrature, the viscosity numbers, vsp/c, of three solutions with different Cs remained almost constant over higher temperature regions but increased rapidly between the regions of 40-35, 3832, and 34-28 "c for the NaCl solutions of Cs= 75,50, and 25 mmoVdm 3 , respectively, and below these temperature regions the values remained again almost constant (NaCI solutions of Cs=75 and 50 mmoVdm3) or increascd gradually (NaCI solutions of Cs=25 mmoVdm3). These variations of vsp/c could be interpreted as a reflection of the conformational transition and association of helices observed from osmometry .

INTRODUCTION Gellan gum is an extracellular microbial polysaccharide produced by fermentation of the organism Pseudomonas Elodea. It has potential applications in the food and biotech-

nological industry because it forms transparent and heat- and acid resistant gels.1 Jansson ct al. established that the chemical structure has a tctrasaccharide rcpcating unit,-.3)- B -D-Glcp-(l-4)- P -D-GlcpA-(14)- B -D-Glcp-(l-+4)- a -L-Rhap-(1-, as shown in Fig. 1. The gelation mechanism of gellan gum solutions has been the subject of controversy, but now it is accepted that gellan gum shows a thermorevwsible conformational transition from a disordered state (single coil) at high tempcrature to an ordercd state (doublc hclix) at low tcrnperature, and junction zones of gellan gcls arc formcd by aggregation of doublc helical gellan molec~les.~-11 Thus, helix formation is a pre-requisite for gel formation.4 The conformational transition temperature of gellan gum has been reported to be around 30"C.s The detailed mechanism, however, has not bccn clarified sufficiently. *13

COI M

CHPH

~

o

&

o

&

o

CHPH

~

o

-

-

HO OH

OH

OH

HO

OH

Figure 1. Rcpcating units of a gcllan gum M =Na molecule.

334

New polymers and materials

Previously we studied the coil-helix transition of tetramethylammonium gellan gum (TMA-gellan) and sodium gellan gum (Na-gellan) in aqueous solutions by osmometry.12-16 In the present study, the temperature dependence of the conformational properties of Na-gellan in aqueous NaCl solutions by osmometry and viscometry has been investigated.

MATERIALS & METHODS Substrates The sample of Na-gellan were prepared from deacetylatcd gcllan gum, kindly supplied by San-Ei Gen F.F.I., Inc. Osaka, Japan (Lot 62058A), by passing through a column of cation exchangc rcsin (Ambcrlitc IR120B) at 6Ooc.17-J6 Thc conversion to Na salts was checked by measuring the ionic contents of the Na-gelIan samples (Table 1). The Na-gellan was dissolved in aqueous NaCl and stirrcd 2 hours at 60°C. In the case of the osmotic pressure measurements, the Na-gellan solutions were dialyzed for 3-5 days at 45°C against aqueous NaCl and diluted with this dialyzing solvent. Measurements Osmometry was carried out using a Hewlett-Packard High-speed Membrane Osmometer, Model 503, having a special type of glass tube. 12-16 Viscometry was made using an Ubbelohde-type viscometer. The flow time for water in the viscometer utilized was about 250 sec at 20°C.

RESULTS & DISCUSSION Osmotic pressure measurements were carried out at 45 to 25°C for the three solutions (NaCI concentration Cs= 25, 50, and 75 mmovdm3). The n / C plots are shown in Figures 2a-c. x is the osmotic pressure and C the polymer concentration. The 7r /C values increased almost linearly at 45, 40, and 36°C (except for the solution of Cs=75 mmoVdm3 at 36°C) in the observed concentration region. While at 28 and 25°C (and 36°C at CS=75mmoVdm3), the n /C values deviated downward above around C=O.20.4 (lOKg/m3) and the lowest polymer concentrations at which these deviations were observed decreased with increasing NaCl concentrations. In these solutions, we noticed a small increase in the solution viscosities at the same concentration regions. It is supposed that interchain aggregation, which should be responsible for gel formation, may occur at least partly. During the measurements, howevcr, thc solution was stablc as a whole without gelation and good reproducibility of the data was obtaincd. Thcrcfore,

Table 1 . Metal Contents in the Gellan Gum and Na-Gellan Samples.

Sample

Na

K

ca

Mg

Gellan g u m ( h t 62058A)

4300

5YY00

8000

( fl g/g) 1600

Na-gellan gum

300 300 30 36800 Metal contents were measured by flame spectrophotometry (Na,K) and flame atomic absorption spectrometry (Ca,Mg).(Perkin Elmer Model 3100)

Coil-helix transition of gellan gum

a, 25

20

CJrnniol dm-3

335

I

i-.

50

3

15 75

d 10

0

7s

k

45°C

O

- 5

m

0 0 0

28°C

L . . . . . . & A

0 0.0

b,

c/lOKg m-3

CJmmol dm-3

0.2 0.4 c/lOKg m-3

25°C

0.6

Figure 2. (a) Plots of ~r/c VS. C for NaCl solutions of Na-gellan at 45, 40, and 36°C. The solid lines denote the values calculated from eq.2 using the values of M, and A2 at 40°C shown in Table 2. (b)Plots of n / C vs. C for NaCl solutions of Na-gellan at 32°C. The solid line is the values calculated from eq.2 using the values of M, and A2 at 32 "C shown in Table 2 (25mmoVdm3) and an empirical fit to the data (50 and 75 mmol/dm:i). (c) Plots of 7r /C vs. C for NaCl solutions of Na-gellan at 28 and 25°C. The solid lines denote the values calculated from eq.2 using the values of M, and A.2 at 28°C shown in Table 2.

336

New polymers and materials

below 28°C (and the solution of Cs=75mmoVdm3 at 36°C ) the data in the low concentration region below C = 0.25-0.4 (10Kdm3) were used for the following calculations. It is known that osmotic pressure for the polymer solutions is expressed by the following equation with appropriate value of g.17 E /C = (RT/Mn)[l+A2MnCtg(A2Mn)'C'] t11 Here R is the gas constant, T the absolute temperature, Mn the number average molecular weight, and A2 the second virial coefficient. The parameter g in equation [I] is related to the third virial coefficient, & by: g=A3/A2M. To diminish the third virial contribution, the empirical value g=1/4 is often used. For the Na-gellan aqueous NaCl solutions of Cs= 25, 50, and 75 rnmoUdm3, we showed previously that g values obtained by osmometry were close to 1/4.13-15 By assuming g=1/4, equation 1 can be rewritten in the following form. ( r/C)"2 = (RT/Mn)l/' (1+A2MnC/2) PI Plots of ( i-r /C)1/2 VS. C for the Na-gellan solutions are shown in Figs. 3a and b. The Mn and A2 obtained respectively from the intercepts and slopes of the straight lines are shown in Table 2. The M n values obtaincd above 36°C in three different NaCl solutions were almost coincident and the average values obtained above 36°C were almost half the

b, 5

CJ mmol dm

3 2

3

c/lOKg

m-3

Figure 3 . (a)Plots of ( ~ / C ) 1 / 2vs. C for NaC1 solutions of Na-gellan at 45, 40, and 36°C. @)plots of ( i-r /C)l/2 vs. C for NaCl solutions of Na-gellan at 28 and 25°C.

Coil-helix transition of gellan gum

337

Table 2. Number-Average Molecular Weights and Second Virial Coefficients for the Na-Gellan in NaCl Solutions. CS (mmoUdm3) 25 50 75 CS (mmoUdm3)

25 50 75

M,,

~10-4

-

25

28

32

36

40

9.6 9.4

9.3 9.2 9.6 <9.4>

5.6

4.3 4.4 4.7

4.5 4.5 4.9 <4.6>

<9.5>

<4.5:*

-

A2 xlO3

45 4.1 4.4 5.0 <4.5>

(mrrioV Kg-2 m3)

25

28

32

36

40

45

8.0 5.6

7.8 5.5 4.1

7.8

8.0 5.6 4.1

8.0 5.7 4.1

8.2 5.7 4.0

_-

values obtained below 28°C suggesting, that two molecules associated below 28°C. In the previous studies of the osmometry of TMA-gellan in W C l solutions, we also observed that the Mnvalue (9.8~104)obtained at 28°C was almost twice the value (5.0 xlO4) at 40°C, suggesting an association of two molecules.13 At 32"C, the M,, value (5.6~104)obtained for the solutions at Cs = 25mmoVdm3 is slightly higher than those obtained above 36°C but lower than those below 28°C. In addition, as shown in Fig. 2, the plots of ?r/C vs. C for the solutions of G= 50 and 75 mmoVdm3 showed upward curvatures below C=-0.2 (10 Kg /m3), and M n and A2 value could not be determined due to non linearities of the ( x / C ) ~ /vs. ~ C plots (Fig. 3). These results suggest that unassociated molecules (unimer) may be in simultaneous equilibrium with associated molecules (dimer) in these solution at 32"C.I4 Therefore, a conformational change of Na-gellan molecules may occur at the temperature region between 28 and 36"C.14,15 Several authors reported that the molecular conformation of gellan gum changed from double helix (rodlike form) to flexible mil with increasing temperature and the transition temperature was -3O"C.4-11 It is considered that the unimer-dimer transition observed from osmometry corresponds to the oil-double helix transition. The values of A2 obtained between 45 and 25 "C (except for the value obtained at 32°C) were almost the same at each Cs,while the AZ values decreased with increasing Cs. This could be interpreted as showing that even if the coil to double helix conformational transition of Na-gellan occurred with decreasing temperature, the flexible coil chain is highly extended and takes more or less a rod like form due to electrostatic repulsions. Therefore, the observed A2 values remain almost unchanged over the temperature range 4525°C and are described qualitatively by the theory of Donnan and Guggenheim.18.19 In order to obtain further information for the temperature dependence of the conformational behavior of Na-gellan in aqueous solutions, viscosity measurements were carried out over the temperature range from 50 to 5 "C for the Na-gellan at four polymer concentrations (C=O.l, 0.15, 0.25, 0.35, and 0.5 wt%) in aqueous solutions of three NaCl concentrations (C,= 25, 50, and 75 mmoUdm3). (Fig. 4).

338

New polymers and materials

1

CiWlk

F

0.35

0

10

15

20

25

30

35

40

45

50

10

15

20

25

30

35

40

45

50

I

I

I

I

I

I

I

'

I

I

- 0.35 0.25

u,a

10

F

-

-0.16

5

-

0

L

C/WlPc I

I

I

. I . . l . . . l

I

Figure 4. Plots of vsp/C vs. Temprature for NaCl solutions of Na-gellan. (a) C,=75 mmoVdm3, (d) Cs= SOmmoVdrn3, (c) CS=25mmoVdm3.

The viscosity number, flSp/Cwas determined from measurement of the flow -times of solutions, ts and of solvents, to in the capillary viscometer. Dsp/C = ( 77s- a a ) / 77oc [3 1 = (ts- to) / t a c Here, 7 7 ~ ,and V 0 are the viscosities of solution and solvent, respectively.

Coil-helix transition of gellan gum

339

As shown in the figures, on lowering the temperature, the vsp/C values increased rapidly between the temperature regions of 34-28, 37-32, and 3935°C for the NaCl solutions of Cs= 25, 50, and 75 mmoVdm3, respectively, and below these temperature regions the vsp/C values increased gradually (NaCI solutions of Cs=25mmoVdm3)or remained again almost constant (NaCI solutions of Cs= 75 and 50 mmoUdm3). It is noted that these temperature regions showed rapid increases of the flsP/C values corresponding to the temperature (32%) at which, in osmometry, the abnormal Mn (=5.6x104) value was obtained or ( 15 /C)1/2 vs. C plots showed nonlinearities. The variation of osp/C is a reflection of the conformational change of Na-gellan molecules and can be interpreted as follows. In NaCl solutions, on lowering the temperature, the coil to double helix transition took place at the critical temperatures and was promoted with decreasing temperature. Below these: temperature regions of 34-28, 37-32, and 39 -35°C for the NaCl solutions of Cs = 25, 50, and 75 mol/dm3, respectively, helices aggregated gradually. Helix formation is a pre-requisite for gel f~rmation.~However, dilute solutions do not form a gel. even if the aggregation occurs due to an insufficient number of helices to percolate a three dimensional nctwork. As shown in Fig. 4, the critical temperatures leading to rapid increase of the vsp/C shift to higher temperatures with increasing NaCi concentration, and in Fig. 2 the critical concentrations at which the K /C values deviated downward decrease with increasing NaCl concentrations. These results suggest that thi: solutions of higher NaCl concentrations have a greater tendency to form a double helix and moreover promote aggregation of helices than those of lower Cs.

CONCLUSION Conformational behavior of the Na- gellan in aqueous solutions was studied by osmometry and viscometry. The M,, values obtained at 45,40,36,28, and 25°C with C,= 25, 50, and 75mmoVdm3 agreed with each other and the average M, value (=4.6x104) obtained above 36% were just half the values obtained below 28% (=9.5~104), suggesting association of two molecules. At 32%, unassociated molecules may be in simultaneous equilibrium with associated molecules. The viscosity numbers, vsP/c, of the three solutions with different C, remained almost constant over higher temperature regions by lowering temperature but increased rapidly between the temperature regions of 40-35, 38-32, and 34-28°C for the NaCl solutions of C,=25, 50, and75 mmoVdm3, respectively, and below these temperature regions the vsp/c values remained again almost constant. The variation of vsp/C can be interpreted as the reflection of the conformational transition and the aggregation of helices observed in osmometry. The solutions of higher N aC1 concentrations have a greater tendency to promote double helix formation and moreover aggregation of helices.

ACKNOWLEDGMENT This work was partly supported by a Grant-in-Aid from the Ministry of Education, Science, Culture and Sports.

340

New polymers and materials

REFERENCES 1) Sanderson,G.R. Food Gels, Harris,P.Ed. Elsevier Applied Science, London &

New York, 1990, 201. 2) Jansson,P.E; Lindberg,B.;Sandford,P.A.Carbohydr.Res., 1983, 124, 135. 3) O'NeilI, M.; Selvendran,R.R.;Morris,V.J.Curbohydr. Res., 1983, 124, 123. 4) Nishinari,K. Collid Polym.Sci., 1997, 275, 1093. 5) Milas,M.; Shi,X.; Rinaudo,M. Biopolymers, 1990, 30, 451. 6) Dentini,M.; Coviello,T.; Burchard,W.; Crescenzi,V. Macromolecules, 198 8 , 21, 3312. 7) Chapman,H.D.; Chilvers,G.R.; Milas,M.; Morris,V.J. In Gums arid Stabilizers for the Food Industry, Phillips,G.O.; Wedlock,D.J.; Williams,P.A.Eds. Pergamon Press, Oxford &New York, 1990, vo1.5, 147. 8) Robinson,G.; Manning,C.E.; Morris,E.R. In Food Polymers, Gels arid Colloids. Dickinson,E. Ed., Royal Society of Chemistry, UK, 1991, 22. 9) Yuguchi,Y.; Mimura,M.; Urakawa,H.; Kitamura,S.; Ohno,S.; Kajiwara,K. Carbohydr.Polym. 1996, 30, 83. 10) Miyoshi,E.; Takaya,T.; Nishinari,K. Carbohydr. Polym. 1996, 30, 101). 11)Morris,E.R.; Gothard,M.G.E.; Hember,M.W.N.; Manning,C.E.; Robinson,G. Curbohydr. Polym. 1996, 30, 165. 12) Ogawa, E. Food Hydrocolloidr, 1993, 7 , 397. 13) Ogawa, E. Polym. J., 1995, 27,567. 14) Ogawa, E. Macromolecules, 1996, 29, 5178. 13) Ogawa, E. Reserrt Res. Devel. in MacromoLRes., 1997, 2, 81. 15) Ogawa, E. Bull. Showagukuiri Jr. College, 1994, 31, 78. 16) Yamakawa,H. Modern Theory of Polymer Solutions, Harper & Row, New York, 1971, Chap. 7. 17) Donnan, F.G.; Guggenheim,E.A. 2. Physik. Chem., 1932, 162, 346. 18) Donnan, F.G. 2. Physik. Chem., 1934, 168,369.

WEATHERING ANALYSIS OF ETHER) MODIFIED POLY (2,6-DIMETHYL-1,4-PHENYLENE BY THERMAL ANALYSIS Y. NISHIMOTO*', K. SATO', Y. NAGAI', F. OHISHI' 'Faculty of Science, Kanagawa University.Hiratsuka. Kcmagma 259-1293, Japan

ABSTRACT

Weathering analysis of a modified poly (2,6-dimethyl-1,Cphenylene ether) using thermal analytical methods was studied. It was found that the evolution of C02 and HzO was detected at the initial decomposition of degraded modified PPE. We report the application of dynamic load Th4A (DL-TMA) wing compressive oscillating stress to the analysis of surface degradation of plastics. 'The results from the DL-TMA method have close relation with the molecular weight of' the region of the surface. It was proved that the surface degradation is detectable with h g h sensitivity without any pretreatment by applying DL-TMA. Keywords: Degradation, Thermal analysis, Dynamic load TMA, modified poly (2,6dimethyl-1,Cphenylene ether) , Weathering analysis INTRODUCTION

Thermal analysis methods were used for analysis of polymers degraded thermally, and some investigations of the thermal degradation of various polymers have shown the value of the combined technique of thermogravimetric analysis coupled with infrared spectroscopy TG-FT IR [1,2]. In the previous paper [3], we have reported the new analytical methods of weathering of elastomers and application to outdoor -exposed SBR.It has been found that thermal analysis methods such as TMA using R were useful for weathering analysis of compressive oscillating load and TG-FT l degraded elastomers. In this paper, we report the application of TG-FT-IR and dynamic load TMA using compressive oscillating stress to the analysis of surface degradation of plastics. Because of its good mechanical and thermal properties, poly(2,6-Qmethyl-1,Cphenylene ether), usually abbreviated as PPE, is widely used in commercial fields, especially as a blend with high impact polystyrene. The blended polymer of PPE and high impact polystyrene was called modified PPE (m-PPE). Weathering analysis of m-PPE has been of our interest because of its practical use. So we took up the m-PPE as the sample in this study.

-

EXPERIMENTAL

Materials

Modified polyphenylene ether (m-PPE) whch is a blend polymer of

342

New polymers and material5

50% PPE and 50% high impact polystyrene (HIPS) was injection molded. All the samples were provided by GE Plastics Japan Ltd. Exposure test Outdoor exposure test was carried out at Choshi exposure laboratory by Japan Weathering Test Center. The exposure conditions are shown in Table 1. Instruments The static or dynamic load is automatically applied by the TMA probe, Dynamic load TMA ( DL-TMA ) is herein defined as the technique which measures the deformation of a sample under periodxally changing load as a function of the temperature. Exposed and unexposed samples were measured in a compression mode using DL-TMA and in a bending mode using dynamic mechanical analysis @MA). The samples were heated at a rate of 20Wmin and an Ar gas purge using TG/DTA. Evolved gases were transferred to a heated gas IR cell by a heated quartz transfer line. Molecular weight measurement was done in a GPC system. A fraction of the exposed and unexposed samples soluble in chloroform were measured. The FT IR measurements were carried out by means of the diffuse reflectance method. The exposed surface of the sample were scraped from surface.

Table 1 Exposure Conditions of the outdoor exposure test at Choshi Latitude N. Longitude E. Above sea level Direction Angle of elevation Exposure period (year) Start date

35 43' 37" 140 45' 02" 53.6m toward the south 30 ' 0.5,1.0, 1.5, 2.0, 3.0, 5.0 April 1. 1988

RESULTS AND DISCUSSIONS 1. Determination of measuring conditions of DL-TMA The elastic behavior is numerically expressed in terms of the complex modulus E*, which is given by the following equation: E * = (o/&)(cos6+isin6)(Fo / A) / (DL / Lo ) (cos6+isin6) where; G = stress ; E = strain ; 6 = the phase difference between stress and strain ; i = a imaginary number ; Fo = the load applied to a specimen ; A = the area of the specimen which is applied the load ; DL= the amplitude of a deformation ; Lo = the thickness of the original specimen. Fig.1 shows the DL-TMA curves with oscillation load for elastic behavior measurement of m-PPE. The glass transition is the temperature range, where E* decreases and strain increases rapidly. The Lissajous figure is prepared from 1 cycle

Poly (2.6-llimethyl-1.4-Phenylene ether)

343

of DL-TMA curves fiom glass transition temperature 1~ softening temperature. Loss energy can be calculated from the area of the Lissajous figure. From the results of investigations, optimum measuring condition was determined as oscillation loads of changing every 50 sec between 0.32 and 2.86 gYmm’. And optimum specimen size for this measurements was determined as 5 6mm cube.

-

Figure 1. Typical DL-TMAcurve and typical Lissajous figure prepared from DL-Th4A curve. 2. Weathering analysis of modified PPE using DL-TMA, DMA and TG-FT IR. Fig.2 illustrates the changes in apparent complex modulus and apparent loss energy of the m- PPE with outdoor exposure. The apparent complex modulus of the sample shows a maximum between 0.5 year and 1 year exposure. It was proved that both of the apparent complex modulus and loss energy changed with outdoor exposure. Fig.3 indicates the apparent complex modulus and loss energy of the unexposed and the 3 years exposed samples of which the surfbe layer was scraped from the exposed surface. The 3 years exposed sample shows remarkable variations in both of apparent complex modulus and loss energy within 1OOpn of scraping thickness from the exposed surfice, whereas in the case of the unexposed specimen, these values were maintaiued almost constant. From these results, it was proved that surface degradation of the m-PPE is detectable without pretreatment with high sensitivity using this DL-TMA method. In addition, as the values of complex modulus at 150 “c of the unexposed and the 5 years exposed specimens were 9x10’ Pa and 8xlV Pa, respectively, in a bending mode using DMA, the complex modulus of the 5 years exposed specimen with surface agreed closely with the sample after scraping the surface. It was found that the value obtained in this DL-TMAmethod did not indicate the complex modulus of whole sample, but reflects the surface degradation of sample. Many investigations of the thermal degradation of various polymers and rubbers have shown the value of combined technique of thermogravimetric analysis coupled with h d S ~ ~ ~ ~ ~ O S -C TG-FT O P Y IR [4,5I.By eXarmnaa . ’on of the IR absorption spectra of the gases evolved fiom the unexposed and the 5 years exposed samples from exposed surface to l o o m depth at 300 “c and 400 “c. The evolution of PPE

344

New polymers and materials

and HIPS was not observed at 300 'Co From the IR spectra at 300 'C, it was shown that the evolution of H20 and C02 from the exposed sample was found to be more than that from the unexposed sample in the initial thermal decomposition state. The aromatic C-H(1630 em", 1495em·') of PPE and mono substituted benzene (700 em", 770em- ') of PS were found at 400 'Co

2 '2 o, ~ II>

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Figure 20 Changes in apparent complex modullus and loss energy with outdoorexposure period.

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Figure 3. Apparent complex modulus and loss energy of samples without surface.

Figure 40 Infrared spectra of the gases evolved from the unexposed and 5 years exposed samples from exposed surface to 100JUIl depth at 300 'C and 400 'Co 3. Weathering analysis of m-PPE using FT IR and SEC SEC is one of the most useful methods for determination of average molecular weights and molecular weight distributions of polymers. Fig.5 shows the changes in number average molecular weight of the scraped powder sample from the exposed

Poly (2,h-Dimethyl- 1,4-Phenylene ether)

345

surface and apparent complex modulus of specimen whose surface layer scraped from the exposed surface. The 3 years exposed sample shows remarkable changes in number average molecular weight within l o o p from the exposed surface, whereas in the case of the unexposed sample, these values were almost constant. These results show good agreement with the result from the DL-TMA method. It was demonstrated that the apparent complex modulus and loss energy from the DL-TMA method correlated fairly we11 with the number average molecular weight or weight average molecular weight (Fig.6).

-! r 1.2

0

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5

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Figure 8. Relationship between IR absorbance ratio (173Ocm"/ 7OOcm-') and outdoor exposure period.

It was observed that the gelation ratio of the whole Sample increased remarkably at the initial degradation period (Fig.7). In the case of m-PPE, it was reported that the

346

New polymers and materials

increase in the absorption at 3400cm.' (OH) or 1730cm" ( G O ) owing to thermal degradation or photo oxidation [6,7]. In this study, the increasing in absorbance at 173Ocm.' with outdoor exposure period was found. It was observed that the IR absorbance ratio of the sample from the surface to 2Op.m thickness increased remarkably at the initial degradation period, while the absorbance ratio increase was found only after 3 years exposure in the case of the thickness of 20pn to 80pm (Fig.8).

CONCLUSION We measured the degraded surface of the m-PPE by number average or weight average molecular weight, IR spectrum and gelation ratio. It was shown from these experiments that the degradation of the m-PPE used in this study occurred in the range of about l00p.m depth from the exposed surface. It was found that the results from this DL-TMA method have close relation with the molecular weight. The molecular weight of exposed sample changes are associated with the cross linking reaction or main chain scission by degradation in the region of the surface. The gelation ratio of whole exposed samples increased according to the outdoor exposure period. The evolution of H20 and C02 from the exposed sample was found to be more than that from the unexposed sample in the initial thermal decomposition state. From these results, it was considered that both the cross linking and main chain scission occurred in the region of the surface of exposed m-PPE. It was proved that surface degradation is detectable with high sensitivity by this DL-TMA method.

ACKNOWLEGEMENTS The authors wish to thank GE Plastics Japan Ltd. for gifts of samples. The authors would like to thank Japan Weathering Test Center for outdoor exposure test. The authors are indebted to Mr. Y. Ichirnura of SEIKO Instruments Inc. for obtaining the DMA and TG-FT IR data.

REFERENCES l)I.C.McNeill, M.H. Mohammed, Polym. Degrad. Stab., 1997,56,191 2)S.Zulfiqar, M.Rizvi, A.Ghaffar, I.C.McNeill,I. Polym. Degrad. Stab.. 1996,52,341 3)Y.Nishimoto, H.Nagata, Y.Nagai, F.Ohshi, Kobunshi Ronbunshu, 1997,54,119 4)S.H.Hamid, and W.H.Prichrd, Polym. Plast. Technol. Engng, 1988,27,303 5)M.Suzuki, and C.A.Wilhe, Polym. Degrad. Srab., 1995,47,217 6)T.Tanakq T.Fujimoto, K .Shibayama, Kohunshi Ronhunshu, 1977,34,377 7)J.D.Cooney, Polym. Eng. Sci., 1982,22,492

NON-DESIRABLE CARBOHYDRATE REACTIONS IN PULPING AND BLEACHING Goran Gellerstedt'. and Jiebiiig Li' 'Department of P u b and Paper Chernishy and Technologv Royai Instiiute of Technology,SE-100 44 Stockholm. Sweden

ABSTRACT

The remaining lignin content in chemical pulps is usually measured as the "Kappa Number" and, fiequently, this number has been transformed into a corresponding amount of residual lignin. In this paper it is demonstrated that the kappa number does not correlate exactly with the amount of remaining lignin Other oxidizable groups present in the pulp carbohydrates can also contribute and the extent of this contribution can vary largely depending on wood species and pulping procedure. In a subsequent bleaching operation these carbohydrate derived structures may or may not react depending on the bleaching agent(s) employed. This, in tim can result in bleached pulps still having considerable amounts of reactive but colourless structures being chemically attached to the fibre polysaccharides. The major contribution fiom the carbohydrates to the kappa number is hexenuronic acid which is formed under alkaline pulping conditions. In addition, other, still unknown, carbohydrate structures formed in the pulping process can contribute to various extents depending on the process and wood species. In the paper, a summary of our present knowledge concerning the structure of the carbohydrate derived reactive structures in haft pulp fibres, unbleached and bleached, is presented.

INTRODUCTION In haft pulping, wood is delignified at around 160-170 "(3 by the action of strong alkali which, together with hydrogen sulfide ions, promote cleavage of the lignin macromolecule into smaller alkali soluble hgments. The resulting unbleached kraft pulp is brownish in colour and still contains around 2-5% lignin attached to the fibres. In a subsequent bleaching operation, this lignin is removed leaving a colourless cellulosic fibre. The colour of haft pulps has usually been attributed to the presence of lignin which in the pulping process is modified such that chromophoric groups are introduced (1). In a model experiment with cellulose this view has, however, been modified (2). Thus, haft pulping of pure cellulose results in a certain formation of chromophores in the resulting "pulp" although the same experiment carried out in the presence of lignin gives an even more discoloured pulp. In the same investigation it was also shown that krafi pulping of cellulose in the presence of either xylan or glucomannan likewise gave rise to a discoloured cellulose. The results are summarized in Fig. 1 which also shows the UV-absorption curves of the pulping liquors corresponding to the various experiments described above. From the UV-spectra it can be seen that experiments carried out with only polysaccharides present result in an absorption maximum around 300 nm whereas the experiment with lignin gives a maximum around 280 nm. The latter

348

New polymers and materials

is well-known and attributed to the lignin aromatic ring while the identity of the former

Polymer

Brightness of "pulp"

UV-spectra of liouors Absorbance

A. Cellulose

80.1

B. Cellulose t Lignin (25%)

65.6

C. Cellulose + Xylan (25%)

69.5

r

\

D. Cellulose + Glucomnnnan (25%) 72.2

230

250

270

290

310

1.nm

Figure 1. Brightness of the resulting "pulp" as well as UV-spectra of the resulting pulping liquors after kraft pulping of pure cellulose (cotton linters; original brightness 90.5%) in the absence/presence of either lignin, xylan or glucomannan (2). is unknown. The figure also shows one further absorption maximum, viz. at around 260 nm,in the experiment with xylan (curve C). In fiuther work, Theander et a1 were able to demonstrate that simple sugar molecules like glucose or xylose to a small extent can be converted to a variety of aromatic and olefinc structures by treatment with alkali at an elevated temperature (3). These structures are shown in Fig. 2. Such and similar structures have also been found in the black liquor from kraft pulping of pine (4) and it was recently shown that at least the cyclopentenone structures are formed early in the h a f t cook (5), i.e. in that part of the cook when the majority of the hemicelluloses are degraded (Fig. 3). Treatment of birch cnm

won

on

Figure 2. Conversion of either D-glucose or D-xylose to aromatic and olefinic structures by the action of alkali at an elevated temperature (3).

Non-desirable carbohydrate reactions

Krnft rook

+ Black liquor

Pine wood +White liquor --->Pulp R!

349

,OH

Figure 3. Cyclopentenones found in the black liquor after kr& pulping of pine wood (4, 5).

kraft pulp with strong alkali at an elevated temperature has also been shown to result in the formation of cyclopentenone structures. In this case it is not known, however, if these compounds were present in the pulp or formed during the treatment (6). The ability of 4-0-methyl-uronic acids to yield the corresponding unsaturated hexenuronic acid by loss of methanol has been known b r a long time. In pulping, this reaction was fmt described in a model experiment by the use of 2-0-(4-O-methyLP-Dglucopyranosyluronic acid)-D-xylitol(7). When treating this compound with alkali at 150 OC, the corresponding unsaturated compound was found and identified as hexenuronic acid-D-xylitol. In its protonated form, this acid exhibits a UV-absorption maximum at around 230 nm whereas in alkali the maxinium is shifted to 260 nrn (8), i.e. the value found by Theander (see Fig. 1). More recently, the presence of hexenuronic acid as part of the xylan in krafi pulps has been thoroughly investigated by Buchert et al(9). By employing enzymatic techniques coupled with N M R the hexenuronic acid moiety could be identified and quantified directly on pulp samples. Contrary to the traditional sugar analysis based on acid hydrolysis, this type of analysis does not result in any degradation of the acid sensitive hexenuronic acid. Alternative analytical methods for the quantificationof hexenuronic acid in pulp samples have been developed and give results in good agreement with each other (10). In chemical pulping, the kappa number is fiequently used as a tool for process control. The method is based on the oxidation of a pulp .sample with an excess of acidic potassium permanganate, which, under specified conditiims, is allowed to react with the pulp. After 10 min, the unreacted permanganate is determined and the permanganate consumption is calculated. Although the method states that there is no direct relationship between kappa number and lignin content, a conversion factor is often used to calculate the lignin content of the pulp. Kappa number measurements are also used fiequently in technical investigations on pulping and bleaching processes in order to evaluate the degree of delignification. The results are used in e.g. comparisons of the influence of different pulping and bleaching parameters imd of different bleaching agents. As discussed above, there are, however, reasons to believe that not only lignin but also a variety of carbohydrate derived structures may contribute to the kappa number

350

New polymers and materials

measurement since permanganate is a powerful oxidant. Furthermore, the carbohydrate derived structures may have a different reactivity as compared to lignin thus giving rise to suboptimization of e.g. a bleaching stage if only the kappa number is used for process evaluation. Therefore, an attempt has been made to identify and quantifL different types of structures that can contribute to the kappa number in unbleached and bleached chemical pulps thereby facilitating a more thorough understanding of the chemical structures and changes that take place in the fibres when going from wood to bleached chemical pulp.

RESULTS AND DISCUSSION In order to determine the contribution to the kappa number h-om different types of structural units in chemical pulps, a series of oxidation experiments were carried out using lignin model compounds as well as isolated lignins and a variety of other compounds containing oxidizable functional groups. Based on these experiments, it was found that lignin, irrespective of type and origin, gave a consumption of permanganate in the kappa number method of approximately 11.6 equivalents of permanganate per mole of phenylpropane units. For hexenuronic acid, the corresponding value was found to be around 8.5 equivalents; a value based on both model experiments and on experiments with pulp samples (1 1). Other types of structuresKunctiona1groups can, however, also contribute as shown in Fig. 4. Although the presence of some of these in unbleached pulp fibres have not been unequivocally identified, they all constitute possible structures based on the discussion above. In a series of unbleached chemical pulps, the contribution to the kappa number was determined using the values for lignin and hexenuronic acid shown in Fig. 4.The amount of lignin in the pulps was determined as Klason lignin and recalculated as kappa number. For hexenuronic acid, the recalculation was based on the amount of hexenuronic acid, analysed as described in Ref 12. On all pulps, the kappa number was

1-

1 1 ~ . polymeric lignin

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Figure 4. Different types of structuredfunctional groups that consume permanganate

when subjected to a kappa number determination.

Non-desirable carbohydrate reactions

35 1

0 other non-lignin mHaxaneuronic acid

Figure 5. The contribution to kappa number fiom Klason lignin and hexenuronic acid with all values re-calculated in kappa number units. The values for "other non-lignin" structures are taken by difference to the analysed kappa numbers in the pulps. also measured directly. As shown in Fig. 5, all pulps gave calculated values that were lower than the actual measured ones with a discrepancy in the order of 2-4 kappa number units, referred to in the figure as "other non-lignin" structures. It can also be seen that the contribution fiom hexenuronic acid varies widely depending on both pulping process and wood species. Thus, soda based processes seem to promote a dissolution or degradation of the hexenuronic acid which, consequently, is present in a very small amount. In birch kraft pulps, on the other hand, the contribution fiom hexenuronic acid is substantial and may, in fact, exceed that fiom lignin. In birch haft pulps, the total contribution to the kappa number fiom lignin is rather small and, consequently, the kappa number does not at all reflect the degree of delignification. Based on the results presented above, a m h e r series of pulp samples was analysed with respect to the contribution to kappa number fiom hexenuronic acid (Table 1). In agreement with the well-known fact that birch kr& pulps usually contain much more xylan and, consequently, more uronic acid groups than pine and spruce pulps it was found that the kappa number contribution was higher for the former. When these pulps

Table 1.

The contribution to kappa number fiom hexenuronic acid (HexA) groups present in some unbleached and bleached hiiftpulps. Bleaching with oxygen (0),ozone (Z) and hydrogen peroxide (P). Q denotes a treatment with chelating agent; n.d. = not determined.

Pulp Unbleached, pine 0-bleached, pine OZQP-bleached, pine OQPQ(PO)-bleached, pine Unbleached, birch OQP-bleached, birch

Kappa No 18.4 10.4 n.d. n.d.

14.5 4.5

HexA contribution 2.3 2.3 0.3 1.9 4.9 3.4

352

New polymers and materials

-+

Unbleached pulp bleaching

h,,H COOH

Equivalentslrnole

10.8

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4.2

2.0

Figure 6. Consumption of permanganate in the oxidation of various structures assumed to be present in bleached chemical pulps. were subjected to bleaching, none of the investigated sequences was able to completely eliminate the hexenuronic acid, however. Thus, despite being a powerhl oxidant for hexenuronic acid groups in addition to lignin (13), ozone bleaching (2)of a pine kraft pulp sample did not result in a complete elimination of these groups. For bleaching agents like oxygen (0)and hydrogen peroxide (P) which are used in alkaline medium, the kappa number reduction proceeds smoothly but the amount of hexenuronic acid in the pulp is only affected to a small extent (12). The fact that bleaching of krafi pulps does not result in a complete removal of hexenuronic acid (and possibly of other non-lignin structures) is supported indirectly by studies on the heat-induced yellowing tendency of such pulps. Thus, it has been demonstrated that the yellowing tendency of a variety of bleached pine and birch kraft pulps can be reduced after subjecting the pulps to treatment with xylanase (14). In agreement with the discussion above, the birch pulps showed the greatest reduction of yellowing after this treatment. The chemical reactions of lignin in bleaching have been elucidated in a large number of studies both with lignin model compounds and with isolated lignin samples (1 5 ) . In summary, it can be concluded that the aromatic rings generally are the most reactive structures present in the lignin. Chlorine and ozone as well as chlorine dioxide and oxygen are all oxidants which are able to degrade the aromatic ring albeit with very large differencies in reactivity. Hydrogen peroxide, on the other hand, does not react with aromatic rings, whether phenolic or not, unless the peroxide is allowed to decompose into radical species. This bleaching agent is, however, superior in eliminating chromophoric groups and it may also degrade lignin through oxidation reactions in the side chains. In the bleaching of chemical pulps, a successive oxidation of the remaining lignin will thus take place with formation of degraded and partly degraded aromatic rings and/or side chains. At the same time, it can be anticipated that oxidizable non-lignin structures and hexenuronic acid will or will not react depending on the chosen bleaching agent. In the kappa number determination, intact aromatic rings consume 1 1.6 equivalents of permanganate per mole of phenylpropane units as shown in Fig. 4. The contribution &om some other possible structures in unbleached pulps are also shown in that figure. Some other types of structures assumed to be present in bleached or partly bleached pulps give, when subjected to permanganate oxidation, the results shown in Fig. 6. The frst two of these structures are known to be formed when lignin is oxidatively degraded whereas the presence of the furan derivative is more uncertain. The a-ketoacid structure may be present in oxidized carbohydrates. All of these structures contribute to a

Non-de:sirable carbohydrate reactions

353

consumption of permanganate although the number of equivalents is different from that of aromatic rings. The fact that even fully bleached pulps, irrespective of the bleaching sequence, usually have a measurable kappa number can, however, be expIained. In order to further detail the contribution fiom various,structures to the kappa number in chemical pulps, a modified kappa number determination procedure has been developed (16). This is based on the fact that mercury (11) can be used to eliminate double bonds in the so-called oxymercuration reaction. Ifthis is followed by a demercuration step, i.e. a treatment with sodium borohydride, almost all interfering structures can be eliminated leaving the aromatic rings as the sole source of permanganate consumption in the kappa number determination. The reaction sequence, denoted Ox-Dem kappa number, is outlined in Fig. 7. In the first reaction step, hexenuronic acid is eliminated fiom the pulp and dissolved. Other types of double bonds also add mercury(T1)-ions and, after hydroxylation, the mercury(I1)-ions are reductively eliminated by the action of borohydride. At the same time, keto groups present in aldehydes and ketones are reduced to the corresponding alcohol groups. The effects of applying the Ox-Dem kappa number to some unbleached birch krafl pulps are shown in Fig. 8. In these two series of pulping experiments, birch wood was used and the krafi cooks performed under identical conditions with exception of the concentration of alkali. The reaction time at the maximum temperature was varied and is expressed as the H-factor. From both series of cooks, the normal as well as the OxDem kappa number was measured on the resulting pulps. It can clearly be seen that a

Llgnin containing pulp + exceaa of KMnO, Determination of unreacted KMnO,

Figure 7. The reaction sequence employed to selectively analyse the content of lignin in chemical pulps by kappa number determination;the "Ox-Dem" kappa number.

354

New polymers and materials

160 "C,(HO-]= 0.6 M

160 "C, [HO-J= 1.0 M

Figure 8. Normal and Ox-Dem kappa numbers of two series of birch kraft pulps prepared with variation of cooking time at two different alkalinity levels. large discrepancy exists between the two measured values in any given pulp. An increased alkalinity during the cook results, as expected, in an enhanced delignification rate but it is also obvious that, at a prolonged cooking time, there is a tendency for an increase of the apparent lignin content in the pulp. The reason for this is not known but reactions of the type discussed above (see Fig. 1 and 2) may well be responsible. Two of the birch pulps, both having a normal kappa number around 16, were chosen and subjected to bleaching in an OQP sequence. As before, both the normal and the OxDem kappa number was measured after each bleaching stage. The results are shown in Fig. 9 and demonstrate that the large difference between the two types of kappa number, 18

2 '

I

Unbleached

* 140 C,

(OH-)= 0 . a cook Kormal Kappa No.

* 160 C,

(OH-)= I.0M cook K o m l Kappa No.

1

Aftcr 0 2

I

After OQP 140 C, (OH-I=0.6M COOL

Ox-Dcm-Kappa No.

* Ox-Dcm-Kappa 160 C, (OH-]=1.OM COO^ No.

Figure 9. Normal and Ox-Dem kappa number of birch krafi pulps after bleaching in an OQP sequence.

Non-desirable carbohydrate reactions

355

observed after the cook, still remain after the subsequent bleaching operation. Thus, the oxygen (0)bleaching stage is an efficient delignification stage, particularly if the preceding cook is carried out at the lower alkalinity 1evt:l. The hydrogen peroxide (P) stage, on the other hand, does not give much lignin dissolution but it can be seen that the normal kappa number is lowered indicating the presence of structures in the pulp being non-aromatic but reactive towards hydrogen peroxide. These structures are still unknown since it has been shown in other work that hexenuronic acid does not react with hydrogen peroxide under bleaching conditions (1 1, 12).

CONCLUSIONS Based on the results presented in this work as well as the earlier work on conversion reactions of carbohydrates in alkaline pulping, it can be concluded that carbohydrate structures contribute to the kappa number and possibly also to the colour of unbleached chemical (haft) pulps. A major portion of the non-lignin related kappa number originates from hexenuronic acid, formed through elimination of methanol from the uronic acid moieties in xylan. Other non-lignin structures are, however, also present in the pulp in amounts that vary with pulp type and wood species. Indications have been obtained that pulping under non-optimal delignification conditions may result in a formation of new structures in the pulp which behave like lignin in the kappa number measurement. Oxygen is an efficient delignification agent but neither oxygen nor hydrogen peroxide is able to degrade hexenuronic acid thus giving bleached chemical pulps still containing a considerable amount of this structure.

ACKNOWLEDGEMENTS Financial support to one of us (JL) f?om The Swedish Pulp and Paper Research Foundation, Grants No 87 and 21 1, is gratefully acknowledged. The authors are also much indebted to professor Olof Theander for generously sharing his knowledge about carbohydrate reactions in pulping with us.

REFERENCES 1.

J Gierer, The reactions of lignin during pulping, Svensk Papperstidn, 1970 73 571-596.

2.

0 Theander, in S.S. Stivala, V. Crescenzi and I.C.M. Dea (Eds.), Industrial Polysaccharides. The Impact of Biotechnology and Advanced Methodologies, New York, Gordon and Breach Science Publishers, 1987, pp 481-492.

3.

I Forsskal, T Popoff and 0 Theander, Reactions of D-xylose and D-glucose in alkaline aqueous solutions, Carbohydr Res, 1976 48 13-2 1.

4.

K Niemelii,The formation of 2-hydroxy-2-cyclopenten-1-ones from - polysaccharides during haft pulping of pine wood, Carbohydr Res, 1988 184 13 1-137.

5.

F Berthold and G Gellerstedt, Reactive structures formed during the initial phase of a haft cook, 7" int conf Wood and Pulping Chemistry,Beijing 1993. Proceedings 3 160- 163.

356

New polymers and materials

6.

G Gellerstedt and J Li, Extraction and fractionation of residual lignin from birch kraft pulp, 3'* European Workshop on Lignocellulosics and Pulp, Stockholm 1994. Proceedings 2 15-218.

7.

M H Johansson and 0 Samuelson, Epimerization and degradation of 2-0-(4-Omethyl-a-D-glucopyranosyluronicacid)-D-xylitol in alkaline medium, Carbohydr Res. 1971 54 295-299.

8.

A Torngren and G Gellerstedt, The nature of organic bound chlorine fiom ECFbleaching found in kraft pulp, 9" int symp Wood and Pulping Chemistry, Montreal 1997. Proceedings 1M2- 1--4.

9.

J Buchert, A Teleman, V Harjunpw M Tenkanen, L Viikari and T Vuorinen, Effect of cooking and bleaching on the structure of xylan in conventional pine krafi pulp, Tappi J, 1995 78:ll 125-130.

10.

M Tenkanen, G Gellerstedt, T Vuorinen, A Teleman, M Perttula, J Li and J Buchert, Determination of hexenuronic acid in softwood krafi pulps by three different methods, J. Pulp Paper Sci. 1999 25 306-3 11.

11.

J Li and G Gellerstedt, The contribution to kappa number from hexenuronic acid groups in pulp xylan, Carbohydr Res, 1997 302 213-2 18.

12.

G Gellerstedt and J Li, An HPLC method for the quantitative determination of hexenuronic acid groups in chemical pulps, Carbohydr Res, 1996 294 41-51.

13.

N - 0 Nilvebrant and A Reimann, Xylan as a source for oxalic acid during ozone bleaching, 4h European Workshop on Lignocellulosics and Pulp, Stresa 1996. Proceedings 485-491.

14.

J Buchert, E Bergnor, G Lindblad, L Viikari and M Ek, The role of xylan and glucomannan in yellowing of krafi pulps, 8' int symp Wood and Pulping Chemisw, Helsinki 1995. Proceedings 3 43-48.

15.

C W Dence and D W Reeve, Pulp Bleaching. Principles and Practice, Atlanta, TAPPI PRESS, 1996.

16.

J Li and G Gellerstedt, Oxymercuration-demercuration-kappanumber; A more accurate estimation of lignin content in pulps, 5h European Workshop on Lignocellulosics and Pulp, Aveiro 1998. Proceedings 28 1-284.

Part 6

Bioengineering of new materials

PRECISIOh ANALYSIS OF BIOSYNTHETIC PATHWAYS OF BACTERIAL CELLULOSE BY l 3 C NMR Masashi Fujiwara, Yoshiko Osada, Shunji Yunoki, Hiroyuki Kono, Tomoki Erata and Mitsuo Takai Division of Molecuhr Chemistry, Graduate School of Engineering, Hokkaido University, Sapporo 060-8628, Japan

ABSTRACT The biosynthetic pathways of bacterial cellulose (BC) in Acetobacter xyfinum were precisely examined by using culture media containing D-( 1- ” C)glucose, (2- l 3 C)glucose or (6-’’C)glucose as the carbon source. Quantitative analysis of the NMR spectra of the glucose hydrolyzed from synthesized BC allows us to estimate the percentage of which metabolic pathway the fed glucose pass through, such as the pentose phosphate cycle (PC) and Entner-Doudoroff (ED) pathway. The results indicated that the rate of direct polymerization (DP) of glucose intensely increased to 47% by ethanol addition compared to the standard (16%). The other pathways (PC and ED) decreased to 30% from 35%, to 8% from 41%, respectively. From these results, it is considered that the role of ethanol is to act as the energy source for proliferation of cells instead of PC or ED pathways and a large part of the glucose which does not pass through PC or ED is used for BC production through DP. Keywords; Bacterial cellulose, Acetohucter xylinum, Biosynthetic pathways, Labeled ethanol, Labeled glucose, I’ C NMR spectroscopy

INTRODUCTION Bacterial cellulose is an extracellular polysaccharide produced by some species of Acetobacter xylinum. Recently, bacterial cellulose is expected to be one of the novel industrial materials due to its excellent properties, such as high mechanical strength, high biodegradability and so on. However, the production cost for bacterial cellulose is considerably high in the present state to realize mass production. For its efficient production, it would be n,ecessary to elucidate the mechanism of cellulose biosynthesis in microorganisms. The biosynthetic process of cellulose in Acetobacter xylinum has been investigated using 14C-specificallylabeled carbohydrates by Minor and Greathouse et al. in the 195Os[1-5]. Minor et a l . [ l ] found that the presence of ethanol in the culture medium increased the yield of cellulose and the quantity of l 4C labeling in cellulose. Recently investigations using I’ C-labeled glucose as the carbon source with or without addition of ethanol have been reported by Arashida and Kai et af.[6-71.They succeeded in the quantitative I’ C labeling analysis of cellulose dissolved in N-methyl morpholine oxide / dimethy sulfoxide-d6 by I’ C NMR spectroscopy. They showed that the direct polymerization l(DP) of introduced glucose was mainly found, especially with the addition of ethanol. Their technique is useful for estimating the ” C labeling on cellulose, however. there has been a problem that the resonance of the C-6 carbon of anhydroglucose units of cellulose which completely overlapped with that ofN-morpholine

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Bioengineering of new materials

(a) Glucose from standard BC

I

I

concluded that the carbon of the added-ethanol did not directly incorporated into cellulose molecule.

I

....................

Cellulose from the medium containing l 3 C-glucose

L

(b)Glucolu from labeled BC aynthuled from (1-1JC)dhaMl.

Fig. 2 shows the ''C NMR spectra of the labeled glucoses prepared by cellulase-hydrolysis of the I I . i . ir. i. ." . ". i. . . c . P. . . .n . b. . .A i b h L. labeled cellulose produced w from the culture medium Fig. 1 1 X NMR spectra of glucose obtaincd from hydrolysis of bacterial cellulose. Bacterial cellulose is designated as BC. containing D-(1- I' C) glucose, D-(2- C)glucose or D-(6- I ' C)glucose. All the l 3 C signals for each carbon of glucose were previously assigned[9], and their assignments are indicated in Fig. 2. For calculating the percentage of the biosynthetic pathways of cellulose, I' C-labeling ratio (LR, the ratio of introduced C intensity, i.e. observed intensity minus the natural abundance of carbon to that of I

Y

1.

I I

.

.

a

.

!

n

i

n

s

Chemical shift (ppm)

Fig. 2 1 X NMR spectra of glucose obtained from hJdrolysis of bacterial cellulose. The culture medium contained (a) unlabeled glucosc, (h) D-(l-lJC)glucose.(c) I)-(2-l3C)glucose. (d) D-(6-13C)glucose.

Precision analysis of biosynthetic pathways

361

oxide, suggesting that no information was obtained from the C-6 carbon resonance of cellulose. In order to avoid the overlaps between the signals for cellulose and those for the solvents and to estinisite the correct labeling of C-6, we adopted ” C labeled glucoses hydrolyzed from I’ C labeled cellulose by cellulase enzyme for ” C NMR spectroscopic analysis in deuterium oxide. In this paper, the possibility of direct incorporation of added ethanol into cellulose was examined using (1 -I’ C)ethanol. The percentage of the respective pathways for cellulose biosynthesis in .4cetobucter xylinum were precisely determined by our method using D-(l-” C)glucose, Cb(2-I’ C)glucose or D-(6-” C)glucose as the carbon source with or without ethanol. Frorn these results, the mechanism of the increase of cellulose production by ethanol is discussed. EXPERIMENTAL For biosynthesis of C-labeled cellulose, Acetohucter xylinum ATCC 10245 was grown statically in 15ml of Hesrrin-Schramm medium[8] containing D-( I-‘‘ C)glucose, D-(2”C)glucose or 0-(6-”C)glucose (Isotec Inc., Ohio, USA) as 10% of the carbon source. The isotopic purity of the labeled glucoses was 99.0, 98.7, and 99.8%, respectively. In the case of “C-labeled dhanol, 1 (v/v) % of ethanol containing 10 (w/w)% of ( I I’ C)ethanol (Isotec Inc., Ohio, USA) was added in the Hestrin-Schramm medium. After 7days of cultivation at 28 “C, the biosynthesized cellulose was purified by boiling in 1 ( w h ) YOaqueous NaOH solution, thoroughly washed with distilled water, and freeze-dried. The purified cellulose were completely hydrolyzed to glucose units with cellulase (Cellulase ONOZUKA R- 10, manufactured by Yakult Co. Ltd.). The solution of labeled glucose was concentrated using a rotary evaporator, filtered through a <5000MW molecular weight separation membrane, purified twice with HPLC using TSK GEL Amido-80 culumn (24 x 300mm, Tosoh Co., Japan), and dried under vacuum. The labeled glucose from cellulose was dissolved in 600ml of DzO (99.996 atom % D, Isotec Inc., Ohio, USA.) containing one drop of dimethylsulfoxide-d,, and subjected to the I’C NMR measurement with a Bruker MSL 400 spectrometer at 23 “C operating at 100.63MHz for carbon with a 5mm wide C-H dual probe. Non-NOE (nuclear overhauser effect) gated-decoupling technique with a pulse-repetition time of 10.0 sec and with up to 5000 transients was engaged for quantitative-mode ’’C NMR measurement. The chemical shifts were calibrated from the signal of dimethylsulfoxide-d, (39.50 ppm). RESULTS AND DISCUSSIONS

Cellulose from the medium containing (1-I’ C)ethanol By the addition ofethanol i n the medium. the yield ofbacterial cellulose at 7days-incubation increased about 1.5 fold compared to the standard condition (data not shown). Glucose as a carbon source had remained even after 6 days under the ethanol-added conditions whereas it was completely consumed within three days under the standard conditions (data not shown). This observation indicates that the added ethanol might substitute glucose. In order to determine whether the added ethanol leads to cellulose directly through metabolic pathways, bacterial cellulose was produced in Hestrin-Schrmm medium containing ( I - ” C ) labeled ethanol. Fig. 1 shows the NMR spectra of the glucose from the cellulose produced in the presence of ( 1 -I’ C)ethanol. No l 3 C-specific labeling was observed in the glucose molecule (Fig. 1). From this result, it can be

362

Bioengineering of new materials

the labeled carbon in the original labeled glucose) of each carbon in labeled glucose was determined by integrating the peak intensity on the NMR spectra. the results being shown in Table 1 (the standard condition; without ethanol) and Table 2 (with ethanol).

Transfer and loss of

l3

C labeling of ~ - ( l -C)glucose '~

As can be seen from Table 1 and 2, in the cellulose from D-( I-" C)glucose. I ' C labeling into cellulose was solely found in the original C-l position. In several papers on the biosynthesis of cellulose from D-( 1-14 C)glucose by Acetohacter xylinum, transfer of labeling from C-I to C-3 as well as to C-4 was reported[l], [ 5 ] . The transfer has been explained by rearrangement of (I-" C)dihydroxyacetone 1-phosphate to (3l 4 C)dihydroxyacetone 1-phosphate in the Embden-Meyerhof (EM) pathway. On the other hand, in the C NMR experiment of cellulose from D-( 1- ' I C)glucose by Acerobucfer xylznum IF013693, the small amount of transfer of labeling from C-1 to C-6 was observed by Kai et a1 [7]. In our experiment, however, no transfer of ' I C labeling from C-I was observed. This observation indicates that the EM pathway does not exist in the metabolic pathways of Acefohacferxylinum ATCC 10245. The result that transfer of C-1 to C-5 was not observed also supports the lack of the EM pathway. This strain takes the Entner-Doudoroff (ED) pathway in place of the ED pathway for glycolysis. Therefore, the dilution of the LR of C-l carbon in glucose from D-(I-"C) can be explained by glycolysis via the ED pathway and the pentose phosphate cycle (PC), and the LR of the C- 1 carbon indicates the percentage of cellulose synthesized via direct polymerization (DP) from introduced cellulose. Table 1 1 X labeling ratio on each carbon in cellulose synthesized from 13C laheled glucose without the addition of ethanol. (Standard) Position D-~~UCOSX CI

c2

c3

GI

c5

c6

0 0 0

0

Labeling ratio (%) (1-130

16

0

0

(2- 13 C ) (6.13 C)

24

22

30

0

11 0

0 0 0

0

92

Table 2 1 X labeling ratio on each carbon in cellulose synthesized from 1 X labeled glucose with the addition of ethanol. (Ethanol) Position D-glucose

CI

c2

c3

c4

c5

c6

0

Labeling ratio ( % ) (1-13C)

47

0

0

0

0

c)

24

53

14

0

0

24

0

0

0 0

0

85

(2.13

(613C)

Precision analysis of hiosynthetic pathways

363

Transfer and loss of l3 C labeling of D-(2-” C)glucose

In the case of cellulose produced from D-(2-” C)glucose, a large amount of transfer of “ C labeling from C-2 to C-1 and C-3 were observed in both conditions (with and without ethanol). This type of transfer can be explained by the PC, as described by Arashida et ul. [6]. The LR (22%, 53%(ethanol)) of C-2 from D-(2- C)glucose remained higher than that (16%, 47%(ethanol)) of C- 1 from D-( 1- I’ C)glucose. This result indicates the existence of a route via which the C-1 labeling disappears but the C1-2 labeling is preserved. That route may be due to the second turnover of the PC. As mentioned above, the transfer from C-2 to C-5 was not observed, which indicates the lack of the EM pathway in our microorganism. Transfer and loss of

C labeling of D46- l3 C)glucose

In the experiment using D(6-I’ C)glucose, considerable amount of transfer is found at the C-1 carbon. This phenomenon is already reported in cellulose produced by Acetobacfer xylinum[5], [lo], [ l l ] and cotton cellulose[l2]. It has been explained by reversible isomerization of D-(3- I’ C)glyceraldehyde 3-phosphate formed via the EM pathway, the ED pathway ,or the PC to (1-”C)dihydroxyacetone 1-phosphate. However, because the EM pathway was not found in Acetobacter xylinum ATC‘C10245, the LR of C-1 carbon from C-6 is determined as the transfer through the EM pathway and the PC. If cellulose is synthesized only via DP. the PC and the ED pathway, the LR of the C-6 position in cellulose from D-(6- I’ C)glucose would be expected to be 100%. However, the small dilution of C-6 labeling (92%. 85%(ethanol)) was observed in our experiment. This result might be explained only by contamination of unlabeled substrates into biosynthetic pathways of cellulose. Kai et ul. [7] reported that the substrate is derived from protein or lipids in yeast extract and pepton of the medium. Therefore. the dilution of LR at C-6 carbon from 100% indicates the percentage of cellulose synthesized via neogenesis of glucose from medium components (designated as GN). Biosynthetic pathways for bacterial cellulose From the results obtained above, it was determined that the biosynthetic pathways of Acetohacter xylinum ATCC 10245 consisted of the following four routes; (1) direct polymerization (DP), (2) the pentose phosphate cycle (PC), (3) the Entner-Doudoroff pathway (ED), (4) glucose neogenesis from medium conponents (GN). Based on discussions of loss and transition of ” C labeling via various pathways, the percentage of the above four routes (DP. PC, ED, and GN) was estimated from the observed LR of each carbon, as shown in Table 3 (without ethanol) and Table 4 (with ethanol). As shown in Table 3 and 4, the rate of DP of introduced glucose intensely increased to 47% with the addition of ethanol compared to the standard (16%). And both the percentage of the PC and that of the ED pathways decreased to 30% from 35%, and to 8% from 41%, respectively, indicating that glycolysis of the fed glucose decreased by the addition of ethanol. As the carbon of ethanol did not incorporate into produced cellulose, it can be concluded that the added ethanol is consumed as an energy source for proliferation of cells instead of glucose, and a large part of glucose which does not pass through the PC and the ED pathway is utilized for biosynthesis of cellulose through

364

Bioengineering of new materials

Table 3 The percentage of the biosynlhetic pathways of bacterial cellulose without the addition of ethanol. (Standard)

Pathway

DP

PC

ED

GN

Percentage of pathway (%)

16

35

41

8

Table 4 The percentage of the biosynthetic pathways of bacterial cellulose with the addition of ethanol. (Ethanol) ~

Pathway

DP

PC

ED

GN

Percentage of pathway ( % )

47

30

8

15

DP. For this reason. it can be explained that Acetobacter xylinum ATCC 10245 produces cellulose in higher yield in the presence of ethanol.

ACKNOWLEDGEMENTS This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science. Sports. and Culture of Japan. The authors are grateful to Mr. E. Yamada (NMR lnstruments Lab., Hokkaido University) for his technical support of NMR measurements. REFERENCES 1 F W Minor. G A Greathouse, H G Shirk, A M Schwartz and M Harris, .J. Am. Chem. Soc., 76 (1 954) 1658. 2 F W Minor, G A Greathouse, H G Shirk, A M Schwartz and M Harris, J. Am. Chem. Soc., 76 ( 1 954) 5052. 3 G A Greathouse, H G Shirk and F W Minor, J. Am. C'hem. Soc., 76 (1954) 5157. 4 F W Minor. G A Greathouse and H G Shirk, J. Am. Clhem. Soc.. 77 (1955) 1244. 5 G A Greathouse. .I Am. C'hem. Soc., 79 (1957) 4505. 6 T Arashida. T Ishino. A Kai, K Hatanaka, T Akaike, K Matsuzaki, Y Keneko and T Mimura. J. C'arbohydr. C'hem., 12 (1993) 641. 7 A Kai. T Arashida. K Hatanaka. T Akaike, K Matsuzaki. T Mimura and Y Keneko.

Curhohydr. Polym., 23 ( 1994) 235. 8 S Hestrin and M Schramm. Biochem. J., 58 (1954) 345. 9 T Usui. N Yamaoka, K. Matsuda and K Tuzimura, J. C'hem. Soc. Perkin Truns. I , (1 973) 2425. 10 R G Everson and J R Colvin. C'un. J. Biochem.. 44 ( 1 966) 1567. 11 D Y Gagnaire and F K Taravel, Eur. ./. Riochem.. 103 (1980) 133. 12 F Shafizadeh and M L Wolfrom, 1.Am. C'hem. Sor.. 77 ( I 955) 5 I 82.

STUDIES ON TRANSGLYCOSYLATION OF CELLOBIOSE BY PARTIALLY PURIFIED TRICHODERMA VIRIDE fbGLUCOSIDilSE Hiroyuki Keno*', Markus R. Waelehl?, Masashi Fujiwara', Tomoki Erata' and Mitsuo Takai'

' Division of Molecular Chemistry. Gruduuie School of Engi,atwing. Hokkaido University, Kim 13, Niihi 8. Kitu-ku. Sapporo. Hokkaido 660-8628, Japan. Bruker Japan Co. Ltd.. Tsukubu. Ibaraki. Jupun.

ABSTRACT In order to obtain enzyme catalyzing regioselective p-glucosyl transfer to hydroxyl group on sugar acceptor, p-glucosidase, which was active in producing trisaccharide from cellobiose, was fractionated from a 7richoderma viride ( T . viride) cellulase complex by chromatographic methods. In the transglycosylation with excess of cellobiose as an initial substrate, the p-glucosidase exhibited not only cellobiose hydrolysis but also conversion of the cellobiose into GI@( 1-16)Glcp( 144)Glc and GI@( 1+6)Glc p( 1+6)Glcp( 1 4 ) G l c . The P-glucosidase catalyzed the high regio- and stereoselective glucosyl transfer to C-6 position of non-reducing end of the acceptor, and thus could be expected to apply to syntheses of functional carbohydrates.

INTRODUCTION With the discoveries of biologically active carbohydrates, there is high current interest in application of cellulase to the method of oligosaccharide ' and polysaccharide syntheses, because the cellulase exhibits not only cellulolytic activities but also catalysis activities of transglycosylation of cello-oligosaccharides. The enzymatic approach to obtain oligosaccharide does not require various elaborate procedures for protection. glycosylation, and deprotection as in the chemical approach. Furthermore, because the cellulase is more available in sufficient quantities and less expensive, it is attractive for oligosaccharide syntheses. Cellulase is generally characterized by three main components ':endoglucanase, exoglucanase, and p-glucosidase. Regarding the transglycosylation, it is recognized that p-glucosidase plays an important role in the reaction, because various p-glucosidase 4.6 have been reported to catalyze transglycosylation while there are few reports on the reaction by endo- or exoglucanase. Most p-glucosidases with catalytic activity of transglycosylation do exhibit high stereoselectivity toward the acceptor. However, wider use of the p-glucosidase for oligosaccharide syntheses has been limited because the transglycosylation products are composed of a number of isomers in many cases 46. From a practical viewpoint, it is required for carbohydrate synthesis that p-glucosidase could catalyze regioselective glucosyl transfer toward sugar acceptors. In this study, we report on the transglycosylation reaction of cellobiose by pglucosidase prepared from a commercial T. viride cellulase We also carried out a

366

Bioengineering of new materials

structural analysis of the products to elucidate the regioselectivity of the p-glucosidase, which is also described herein.

EXPERIMENTAL Enzyme purification and assays T. viride cellulase ONOZUKA R-10 (Yakult Pharmaceutical Ind. Co.) was concentrated by (NH&SO, at 80 % saturation. After the centrifugation, the precipitate dissolved in a small amount of water was dialyzed against a large amount of water and then concentrated by ultra-filtration. The resultant enzyme solution was directly applied to a column of Hi-Load Q HP (Pharmacia) equilibrated with 50 mM acetate buffer, pH 5.0 (buffer A ) and eluted with linear gradient (0-0.55 M) NaCl in buffer A at 3.0 mllmin. Transglycosylating active fractions were collected and concentrated to 2-3 mL by ultrafiltration. The concentrate was loaded onto a Superdex 75 HR10/30 column (Pharmacia) equilibrated with buffer A , and eluted with buffer A. Fractions containing the transglycosylating active fraction were collected, concentrated, lyophilized and then stored at 4 "C. 1U (unit) of endo- and exoglucanase activities were defined as the amounts of enzyme liberating 1 pmol of reducing sugar per min from carboxymethylcellulose and Avicel, respectively. The amount of reducing sugar was estimated by the Nelson '-Somogyi method using glucose as a standard. I U of p-glucosidase was defined as the amount of enzyme liberating I pmol of p-nitrophenol from p-nitrophenylp-11-glucopyranoside per min. The amount of the released p-nitrophenol was determined by absorbance at 405 nm.

Transglycosylation of cellobiose A reaction mixture i n buffer A containing 500 mM cellobiose and 0.141 U of pglucosidase was incubated at 40 "C. After 24 h, the mixture was heated in a boilingwater bath for 5 min, to inactivate the enzyme, and was analyzed by HPLC. For fractionation. Bio-Gel P-2 (2.5 x 120 cm) was used with water as eluent set at a flow

-

-

30

-1 0

E

l

c

:

I -

j

0 -

0

10

20

30

40

Fraction number

Figure 1. Elution profile of Trichoderma viride cellulase by anion-exchange chromatography on Hi Load Q HP. The sample was obtained by ammonium sulfate precipitation. p-glucosidase ( 0 )and transglycosylating activities (0)were assayed. 25-mL fractions were collected.

Studies of trransglycosylation of cellobiose

367

rate of 60 mL/h.

Analytical methods Protein concentration was estimated spectrophotometrically at 280 nm or by the method of Bradford lo, using BSA as a standard. SDS-PAGE was carried out according to Laemmli ' I , using 12.5 % gel. Methylation analysis was carried out by the Hakomori method 13. The resultant methylated alditol acetates were analyzed by GUMS. NMR spectra were recorded on Bruker avance 600 spectrometer at 23 "C. The 'H and I3C chemical shifts were referenced to internal 4,4-dimethyl 4-silapentane sodium carboxylate (DSS), 0.015 ppm, and acetone, 31.55 ppm, respectively. Assignments of all the protons and carbons was determined by DQF-COSY, TOCSY, HSQC, and HMBC spectra. For inter-residue correlation, ROESY with a spin-lock time of 300 ms was used. All the NMR experiments were performed according to standard pulse sequence.

RESULTS AND DISCUSSION Purification of pglucosidase In order to fractionate p-glucosidase that could catalyze transglycosylation, a cellulase complex was applied to successive chromal:ographies. Transglycosylating activity was estimated by the amount of trisaccharide synthesized from cellobiose by the action of cellulose. As shown in Figure I , one symmetrical protein peak associated with the transglycosylating activity was observed in chromatographic elution pattern of Hi Load Q column. The active fractions were combined and then loaded onto Superdex 75 HR10/30 column. Finally, P-glucosidase with high rransglycosylating activity and molecular weight of 68,000 was obtained. A summary of the purification procedures of the P-glucosidase are presented in Table 1. The optimum pH for cellobiose hydrolysis was found to be 5.0 in sodium acetate buffer, and the maximum activity of cellobiose hydrolysis was observed at 45 "C.

Synthesis of oligosaccharides A high concentration of substrate favors the transglycosylation because this reaction is competitive with hydrolysis. In this study, aqueous 500 mM cellobiose was used for the transglycosylation. As shown in Figure 2. the chromatogram gave two remarkable large peaks associated with trisaccharide and tetrasaccharide deriving from

Table 1. Purification of Trichoderma viride P-glucosidase Steps (NH,),SO, precipitation

Hi Load Q Superdex 75

Total Protein

p-glucodidase

'mi?

lunitimg-protein

-

266 45.6 16.6

-

0.051 0.057 0. I41

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Bioengineering of new materials

G5 G4 G3

I

l

l

G2

GI

I

I

-I

t 120

60

80

100

Fraction No (4-mL each)

Figure 2. Gel-chromatographic separation of transglycosylation products of cellobiose. The chromatography of carbohydrates was carried out on a column of Bio-Gel P-2. The elution positions of G,-G,, glucose (G,) and a series of cellooligosaccharides (G,-G,) are shown. the action of p-glucosidase on cellobiose. Fraction G1-G5 appeared at the same filtration volume compared with standard glucose and cello-oligosaccharides. Fractions G3 and G 4 were collected and lyophilized to give a 32 mg and 6.4 mg, respectively, from 171 mg of initial cellobiose, which were used for the structural analyses, described later.

Structural analyses of the transglycosylation products In methylation analysis of the products as shown i n Figure 3, GLC pattern of the partially methylated alditol acetates derived from trisaccharide and tetrasaccharide gave 2,3,4,6-tetra-U-methyI glucitol, 2,3,6-tri-U-methyl glucitol, and 2,3,4-tri-O-methyl glucitol, i n the relative proportions 1 : 0.96 : 1.02, and 1 : 1.05 : 1.94, respectively. 1 2

0

5

10

3

15

20

25

30

35

Retention time / min

Figure 3. Methylation analysis of trisaccharide ( A ) and tetrasaccharide (B). ( 1 ) 2,3.4,6-methyl glucitol; (2) 2,3,6-methyl glucitol; (3) 2,3,4-6-methyl glucitol

Studies of traiisglycosylationof cellobiose

369

3z 0 .Y

1

I

1

I

T"I

'

100

" I '

" I ' " ' I " " l ' ' ' ' I

90

80

/z

e B

N

I

-

-

70

60

PPm

Figure 4. I3C NMR spectra of trisaccharide (A) and tetrasaccharide (B) Because no di-0-methyl glucitols were observed, the possibilities of these results indicated that the trisaccharide was composed of one 6-substituted-D-Glcp and one 4substituted-D-Glcp while the tetra-saccharide contained two 6-substituted-D-Glcp residues and one 4-substituted-D-Glcp residue. Figure 4 shows I3C NMR spectra of the products. In I3C NMR spectra, chemical shifts of the glycosidic carbon with an alinkage are in the region 97.5-101 ppm 13, while those with a p-linkage are downfield in the region 104-106 ppm 1 3 . The spectra of the both products gave no resonances at 97.5101 ppm with the exception of CI anomeric carbons of reducing-end units. When a glycosyl residue is bound to a certain carbon 14, it causes a significant downfield shift (5-10 ppm) to the I3C resonance of the carbon. A comparison with C6 chemical shift of Glc 1 (see Figure 4 for numbering of the mono-saccharide residues) and Glc 3 reveals that the C6 signals of Glc 2 is observed in downfield region. I n the spectrum of tetrasaccharide, C6 resonances of both Glc 2 and Glc 3 easily recognized in major downfield region compared with those of Glc 1 and Glc 4. These structural analyses identified the tri- and tetra-saccharide structures as P-D-Glcp-( I +6)-P-D-Glcp-( 1 4 ) D-Glcp and P-D-Glcp-( 1 +6)-P-D-Glcp-( I +6)-P-D-Glcp-( 1.-+4)-D-Glcp,respectively.

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Bioengineering of new materials

Disaccharides such as sophorose Is, cellobiose 16, gentiobiose ”, as well as the other oligosaccharides composed of p-glucosyl residues, are believed to regulate the cellulase synthesis in the metabolism of cellulase-producing microorganisms. Regarding the fungus T. viride, sophorose I s was found to be the inducer of its cellulase synthesis, whereas glucose caused repression of the enzyme synthesis. The oligosaccharides obtained in the experiments described herein, though their biological function are not clear, may have effects on the metabolism of the cellulase-producing microorganisms. Many researchers have reported that cellulases or p-glucosidases originating from a wide variety of microorganisms and higher plants catalyze transglycosylation. Although the principal linkage synthesized by the action of these enzymes were p( 1-6) 5 , p-glycosidic linkages with ( 1 +2), ( I +3), and (1-4) were also formed to a lesser extent ’,6. In our experiments, p-( 1+2), p-( 1+3), and p-( 1-4) linkages were not formed in the reaction products. This indicates that purified p-glucosidase obtained from T. viride cellulase in this experiment has high regioselectivity as well as stereoselectivity toward the hydroxyl group of the sugar acceptor in the transglycosylation. It is concluded that T. viride cellulase contains /3-glucosidase which could catalyze not only cellobiose hydrolysis but also synthesis of oligosaccharides up to tetrasaccharides from cellobiose in aqueous buffer solution. Furthermore, strict regioselectivity was observed in the transglycosylation of cellobiose, which may prove highly significant in the regio- and stereoselective synthesis of oligosaccharides.

REFERENCES 1

2 3 4 5 6 7 8 9 10

11 12 13

14 15

16 17

SP. Shoemaker and R.D. Brown Jr., Biochim. Biophis. A m . , 523 (1978) 133-46. S. Kobayashi. K. Kashiwa, T. Kawasaki, and S. Shoda, J . Am. Chem. Soc., 113 (1991)3079-84. M.C. Flickinger, Biotechttol. Bioeng., 22 (Suppl.1) ( 1980) 27-48. T. Watanabe, T. Sato, S. Yoshioka, T. Koshijima, and M. Kuwahara, Eur. J . Biothetn., 209 ( 1992)65 1-59, K. Ohmiya and S . Shimizu, Methods Enzvmol., 160 (1986) 408-13. H. Fujimoto. H. Nishida, and K. Ajisaka, Agric. B i d . Chern., 52 (1988) 1345-5 I . SP. Shoemaker and R.D. Brown Jr., Biochim. Biophis. Acta., 523 (1978) 147-61. N . Nelson, J . Biol. Chem.. 153 (1944) 375-80. M. Somogyi, J . B id. Chem., 195 (1952) 19-23. M.M. Bradford, Anal. Biochem., 72 (1976) 248-54. U.K. Laemmli, Nature, 227 (1976) 680-85. S. Hakomori, J . Biochem., 55 (1964) 205-8. T. Usui, N. Yamaoka, K. Matsuda, and K. Tuzumura. J . Chem. SOC.Perkin I , ( 1973) 2425-2432. K. Bock and C. Pedersen, Adv. Curhohvdr. Chem. Biochem. 41 (1983) 27-66. J.R. Loewenberg and C.M. Chapman, Arch. Microbiol., 113 (1977) 61-64. M. Rao. S. Gaikwad, C. Mishra, and V. Deshpande., Appl. Biochetn. Biotechnol. 19 (1988) 129-37. T. Kurasawa, M. Yachi, M. Sato, Y. Kamagata, S. Takao, and F. Tomita, Appl. Environ. Microbiol., 58 (1992) 106-10.

CELSOL - MODIFICATION OF PINE SULPHATE PAPER GRADE PULP WITH TRICHODERMA RLCESEI CELLULASES FOR FIBRE SPINNING Pertti Nousiainen and Marianna Vehvilainen* Fiber, Textile and Clothing Science, Tampere University of Technology, P.0.Box 589, FI-33101 Tampere, Finland http://www.tut.j3units/ms/teva

ABSTRACT Never-dried pine sulphate paper grade pulp was treate'd with specific enzyme mixture from Trichoderma reesei, which increased the alkaline ,c,olubilityof pulp from 20 % to 69 % and decreased the DP, from 1124 to 622. The effect of different viscose process additives and zinc oxide on solubility was studied. The best combination for producing a good spinning dope was chosen. The fibres were spun using a wet spinning machine and titre, tenacity and elongation of the fibres were determined. The strongest fibres obtained have tenacity of 1.2 cN/dtex and elongation of 12-13 %, which are comparable to the properties of cotton.

KEYWORDS Cellulases, Trichoderma reesei, paper grade pulp, directly alkaline soluble cellulose, enzymatic treatment

INTRODUCTION The aim of the present CELSOL-research is to devehp an environmentally friendly method for producing cellulosic fibres compared to the viscose process. In this method the polluted chemical treatments, which are needed in the viscose process, are replaced with environmentally harmless enzymatic treatment. We have studied the effect of enzymes on dissolving pulps and paper grade pulp and noticed, that the paper grade pulp suits for enzymatic processing, also. It is common knowledge that cellulose having a higher degree of polymerisation is extremely difficult to dissolve into a simple and cheap aqueous alkali solution. For this reason the viscose method is widely used for producing cellulosic fibres in spite of the environmental problems associated with it. Difficulties in dissolving cellulose have also caused intensive studies in this field over the decades. Hergert el al.' have classified the cellulose solvent systems in the four main categories that are 1) cellulose acting as a base, 2) cellulose acting as an acid, 3) cellulose complexes and 4) cellulose derivatives. There are only two other industrial scaled method,$ besides the viscose process, which are used for producing cellulosic fibres, those are the cuprammonium and NMMO methods. In the viscose method cellulose has to be converted to a certain cellulose derivative prior to dissolution, but in the NMMO and cuprammonium methods cellulose is dissolved without derivatisation.

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Bioengineering of new materials

Most of the other cellulose dissolving methods are still at the laboratory or small

pilot scale level and many of them are based on a metal complex as cadmium, zinc or nickel with ethylene diamine and alkali or iron with tartaric acid and alkali. However, these methods are neither economical nor more environmentally friendly than the viscose process. In some other methods the cellulose is converted to a derivative prior to dissolution. For example in the cellulose carbamate method cellulose is first treated with urea in liquid ammonia and thereafter dissolved in the form of carbamate into alkali ', and in the DMSO / (CH20), system cellulose is dissolved as methylol cellulose. It is also possible to prepare nitrogen-based unstable derivatives by use of N 2 0 4 with either DMF or DMSO '. Schleicher and Lang have studied the parameters influencing the solubility of cellulose in aqueous sodium hydroxide and came to the conclusion that the solubility is strongly influenced by the supermolecular structure of cellulose. In that the solubility increases with decreasing degree of order, decreasing DP, and increasing accessibility of cellulose. The dissolving conditions, such as temperature, concentration of sodium hydroxide and possible additives affect the solubility, also. Kamide and Okajima ' have done profound studies concerning the correlation between structure parameters of cellulose and its solubility in sodium hydroxide. According to them it is possible to dissolve cellulose completely in aqueous alkali solution after preliminary steam explosion treatment. They claim the effect is based on the weakening of C3-0,' and C2C,' intramolecular hydrogen bonds. However, Schleicher and Lang state that it is difficult to separate the influence of intermolecular and intramolecular hydrogen bonds from each other. We have studied the alkali solubility of cellulose, also, and proved that enhanced solubility is obtained after suitable enzymatic treatment. The ability of cellulases to degrade cellulose has been common knowledge for decades '-I3. In nature the action is based on synergism of endoglucanase, cellobiohydrolase and P-glucosidase, which are the most typical enzymes produced by cellulolytic fungi. However, in the reaction cellulose Is hydrolysed into short, easily dissolved glucose chains and monomers, being thus unusable for fibre production. The cellulases used in our method are specially formulated mixtures, which result in improved alkaline solubility with controllable degradation of cellulose. In this work pine sulphate paper grade pulp was treated with Trichoderma reesei cellulase mixture, the effect of different kinds of additives on alkaline solubility of enzyme treated cellulose was also investigated and the best parameters for producing a spinnable dope was chosen. Fibres were spun using a laboratory wet spinning machine, and the effect of spinneret type, coagulation bath temperature and coagulation time on fibre properties were studied.

MATERIALS AND METHODS The cellulose used was never-dried paper grade sulphate pulp from Finnish pine and was kindly supplied by Metsa-Sellu Oy, Aanekoski, Finland. The cellulase mixture used was a culture filtrate from genetically modified Trichoderma reesei strain and was obtained from Primalco Ltd, Biotec, Finland. The genes producing cellobiohydrolase (CBH) proteins were removed from the culture and the production of endoglucanase II (EG II)protein was enriched. The enzyme activities were measured as previously I 4 , I 5 . The additives used in the dissolving stage were process additives developed for the

Celsol - modification of pine sulphate pulp

373

viscose process and were kindly supplied by Akzo Nobel. Additionally the zinc oxide was used for better solubility and stability. The never-dried form pulp was mechanically shredded prior to any other processing. The enzyme treatment was carried out in distilled water, pH 4.8-5.0, at 50 "C for 5 hours at cellulose consistency of 2 %. The enzyme dosage was 500 ECU / g of cellulose. Treated and washed cellulose was dried at room temperature for storage and wet with distilled water prior to dissolution. The average molecular weights (DP,) and the molecular weight distributions of the untreated and enzyme-treated pulps were measured in the Institute of Chemical Fibres, Lodz, Poland by the Hewlett-Packard HP1050 apparatus and GPC method based on LiCIDMAc dissolving system. The alkali solubility of cellulose samples was measured by adding 0.70 g (based on dry weight) of wet cellulose into 20 ml of 9 wt % aqueous sodium hydroxide. The mixture was stirred at -5 O C for 10 minutes with the speed of 750 -1000 rpm. The solution obtained was poured into a centrifuge tube and diluted with 20 ml of 9 wt % NaOH used for rinsing the vessel and stirrer head. The mixture was centrifuged at 3000 g for 10 minutes, the clear portion was poured off andl the insoluble part washed by adding 20 ml of 9 wt % NaOH and centrifuging for a further 10 minutes at 3000 g. Thereafter the insoluble part was treated with 10 % sulphuric acid, washed with distilled water until neutral and dried at 105 O C over night. The solubility of cellulose was calculated as a percentage using the following equation: S = 100 x [(0.70 - Wi)/0.70]

r11

where Wi= weight of insoluble cellulose. The spinning dope was prepared by adding ca. 5 wt 4) of enzyme-treated pulp into 9 wt % NaOH containing the additives required and stirred at -5 "C for 10 hours. The solution was filtered through AMN 546K filter felt, dega.sed and stored in a refrigerator over night prior to spinning. The cellulose content of filtered solution was measured by coagulating films from the weighed amount of solution. The films were washed until neutral and dried at 105 "C for at least 4 hours. The cellulose content (a)was calculated as a percentage using the following equation:

a = 100 x (Wf/ W,)

[21

where Wf = weight of dried films and W,= weight of cellulose solution. The spin bath contained 10 % of sulphuric acid and 201% of sodium salt, temperature being 20 "C or 50 "C. The stretching bath contained hot (90 "C) water and the washing baths cold water. The spinnerets used were either 36 x 100 pm or 40 x 80 pm having a length to diameter of 4 and 5, respectively. A reservoir of 1000 ml and a gear metering pump of 0.3 mllr were used. The fibres were collected on a fibre collector after three godets, one stretching and two washing baths. The fibres were additionally washed with distilled water until neutral and dried from water/acetone mixture. Titre, tenacity and elongation were determined using the Lenzing Vibroscop and Vibrodyn testing machines.

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Bioengineering of new materials

RESULTS AND DISCUSSION

Preparation of the spinning dope The mechanically shredded, never-dried, pine sulphate paper grade pulp was treated with 500 ECUIg of EG II rich cellulase mixture. The alkali solubility, DP, and molecular mass distribution of untreated and enzyme-treated samples were analysed, Table 1 and Figure 1. The enzyme treatment used is based on our preliminary work, when we studied the effect of enzyme type, dosage and treatment time on the solubility of never-dried pine sulphate paper grade pulp. The significant drop in DP, was unexpected based on the results we have obtained with dissolving type pulps. However, the original DP, of pine sulphate pulp was too high for obtaining a spinnable viscosity for the dope, wherefore the reduction of DP, was essential, in any case. The solubility of 69 % obtained for pine sulphate pulp was not high enough for the spinning dope, which made it necessary to study other ways to increase it. The effect of selected viscose process additives on the alkali solubility was studied first. The dosage used was 1 g I kg of cellulose, the additive was added to sodium hydroxide and the mixture was cooled to -5 "C prior to addition of pulp. The characterisation of additives and the alkaline solubility obtained are presented in Table 2.

Table 1. The DP, and alkali solubility in 9 wt % NaOH at -5 "C, [S(-5)], of untreated and enzyme-treated pine sulphate pulps. Sample Untreated pine sulphate Enzyme-treated pine sulphate

DP, 1124 622

S(-5), % 20 69

dw1dlogM

4.0

5.0 6.0 logM Figure 1. The molecular weight distributions of untreated and enzyme-treated pine sulphate paper grade pulps.

Celsol - modification of pine sulphate pulp

375

Table 2. The effect of additives on the alkali solubility, [S(-5)], of enzyme-treated pine sulphate pulp. Additive

Characterisation

V 4045 V 4047 V 4044 BV 444 BV 44

non-ionic surfactant, polyethylene oxide derivative non-ionic surfactant, dialkyldimethyl ammonium chloride surfactant, alkylpolyethyleneglycol alkyl polyglycol ether, improve reactivity of pulp aliphatic polyoxy ethylene glycol derivative, improve filterability polyethylene derivative of an aromatic compound. improve processability of pulp and filterability of viscose alkylamine polyoxy ethylene glycol, improve proccssability of pulp and filterability of viscose

BV 388

BV 32

66 66 67 65 65

66 68

None of the additives used improved the alkali solubility. On the other hand none of them decreased the solubility significantly either, which proved, that the process additives can be used in the enzyme-aided system far their own purpose, without loosing the effect of enzymes. The increased swelling action of sodium hydroxide solution containing zinc oxide (sodium zincate) on cotton was discovered already over a century ago by John Mercer 16. A few decades later Davidson " made profound studies on the dissolution of chemically modified cotton cellulose in alkaline solutions. He studie:d the solvent action of sodium zincate solution and found that the molar ratio of zinc oxide to sodium hydroxide in the solution is important regarding the properties of the solution obtained. The maximum solubility of a given material proved to be the greater the higher the molar ratio of zinc oxide to sodium hydroxide was in the solution, except at high sodium hydroxide concentrations the solutions tended to form gels on stantling and with high molar ratio ZnO/NaOH precipitation of zinc oxide took place. Davidson also found that the solvent action was greatly increased by lowering the temperature from 15 "C to -5 "C. He concluded the optimum solvent for modified cotton was a 2.5 N or 2.75 N sodium hydroxide having a molar ratio ZnO/NaOH of 0.1. This kind of solution was stable both with respect to gel formation and precipitation of zinc oxide. We studied the action of sodium zincate solutions on the solubility of untreated and enzyme-treated pulps. A solution having a molar ratio 0.1 in 2.5 N sodium hydroxide equals to 1.8 wt % of zinc oxide in 9 wt % NaOH. We dissolved 0.9 wt % and 1.8 wt % of zinc oxide into 9 wt % NaOH and studied the alkali solubilities of the pulps. Results are presented in the Fig. 2. The solubility increased remarkably with the aid of zinc oxide, as was expected. However, the action of enzymes is required also, since the highest solubility of untreated pulp was only 38 % compared to 98 % of enzyme-treated pulp with 1.8 wt % ZnO. The solubility obtained is high enough for appropriate spinning trials. Two spinning dopes were prepared by dissolving ca. 5 wt % of enzyme-treated pulp into 9 wt % aqueous sodium hydroxide containing 1.8 wt '% of ZnO. The first dope did not contain any other additive, but 1 g I kg cellulose of Berol Visco 44 was added in the second dope.

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Bioengineering of new materials

100

80

60

40 20

I

of

I

1,s

03 Amount of zinc oxide, %

0

Figure 2. The solubility of untreated (-) grade pulps with the aid of zinc oxide.

and enzyme-treated (----) pine sulphate paper

The spinning trials The dopes were stored in a refrigerator over night prior to spinning. The cellulose content of the filtered dopes was 4.8 %. The parameters varying during the trials were the use of additive, spinneret type, temperature of the spin bath, coagulation time and draw of the fibres. Titre, tenacity and elongation of the fibres obtained were measured. The results are given in the Table 3. The function of Berol Visco 44 additive is to increase: a) filterability, b) dispersion of gel and resins, and c) spinning stability '*. Slightly stronger fibres were obtained from trial 6 than trial 2, in which the only difference is the presence of Berol Visco 44 additive in the trial 6. Otherwise the effect of additive is not very clear, which is probably due to the fairly short spinning time with one type of parameter. Temperature of the spin bath was increased only for one trial, because the solution turned to a gel with increasing temperature. However, a small amount of fibres with low tenacity but higher elongation was obtained. The difference in l/d ratio of the spinnerets was not significant and did not affect the fibre properties. The effect of coagulation time was not studied systematically, but it was altered for practical reasons, nevertheless, it seems that the longest time, 1.1 seconds, results in strongest fibres, 1.2 cN/dtex.

Table 3. The parameters in the spinning trials and the properties of the fibres obtained. Trial

1

2

3

4

5

6

I

Berol Visco 44 Spinneret Vd Spin bath temp. ["C] Coag. time [s] Draw [ %] Titre [dtex] Tenacity [cN/dtex] Elongation [%I

No 4 20 1.1 29 4.0 1.21 12.5

No 5

No 4 50 0.8 25 9.1 0.71 17.9

Yes 4 20 0.4 20 5.4 0.93 12.6

Yes 4 20 0.7 II 3.9 1.10 13.7

Yes 5 20 0.R 15 4.2 1.13 16.8

Yes 5 20 0.3

20 0.8 29 3.8 1.02 13.4

1s 5.1 1.06 15.8

Celsol - modification of pine sulphate pulp

377

CONCLUSIONS The pine sulphate paper grade pulp was treated with specific enzyme mixture, which increased the alkali solubility from 20 % to 69 % in 9 wt % aqueous sodium hydroxide and from 38 % to 98 % in 9 wt % NaOH containing 1.8 wt % of zinc oxide. Two spinning dopes with and without Berol Visco 44 additive were prepared dissolving the enzyme-treated pulp into 1.8 wt % ZnO / 9 wt % NaOH. The dopes were filtered and degased prior to spinning and the fibres were spun using a laboratory wet spinning machine. The strongest fibres obtained have tenacity of 1.2 cN/dtex. The first requirement for fibre spinning is to obtain a spinnable dope, which means good viscosity, filterability and stability of the spinning solution. The most important factor affecting on spinnability in this case is the solubility of cellulose. As Schleicher and Lang stated the solubility increases with decreasing DP,, at the same time the viscosity decreases and the cellulose concentration increases. The balance between DP,, viscosity and cellulose concentration is important. Yamashiki and co-workers l9 obtained an alkaline solubility of 100 % with 5 wt % of steam exploded soft wood cellulose, however the DP, of cellulose was only 33 1, comparing to the DP, of 622 in our trials with almost the same cellulose concentration and solubility. Nevertheless, Yamashiki et ul. have successfully spun fibres from the dope prepared from steam exploded cellulose. They used a wet spinning method and an acidic bath containing 20 wt % of sulphuric acid. Titre of the fibres ranged from 53 - 84 denier ( = 5.9 - 9.3 tex) and tensile strength from 1.53 - 1.82 g/d (=1.38 - 1.63 cN/dtex). In spite of much lower DP, of cellulose they obtained stronger fibres than we did. Yamane and co-workers ”-*’ have continued the work with steam exploded cellulose studying closely the dissolving and spinning conditions. They found that especially the revolution number of agitator affects greatly on the solubility of steam exploded cellulose. When the speed increased from 1000 rpm to 12000 rpm the solubility increased from 60 % to 96 %. The result is important for us, since the speed used in the solubility measurements of the enzyme-treated cellulose was only around 1000 rpm. It means there might be a possibility to increase the solubility even without the addition of zinc oxide. Considering the spinning parameters, Yamane et al. found that the concentration of sulphuric acid affects strongly on the structure and tensile properties of the spun fibres. They obtained cellulose filaments with tensile strength in the dry state higher than that of regular viscose, using sulphuric acid with concentration of 50-65 %. The sulphuric acid concentration used in our trials was only 10 %, which might explain the weaker tensile properties of our fibres compared to the fibres Yamashiki et al. and Yarnane et al. have produced. Taking into consideration the origin of the pulp used in our trials and the possible improvements on solubility and spinning conditions based on the work of the Japanese scientists, we conclude the enzyme-aided production of cellulosic fibres will be a noteworthy method in the future.

REFERENCES 1. Hergert, H.L., Hammer, R.B. and Turbak, A.F., New methods for preparing rayon, Tuppi,1978,6l(2) 2. Ekman, K., Eklund, V., Fors, J., Huttunen, J.I., Selin J-F., and Turunen O.T., Fibres by wet-spinning of cellulose carbamate solutions, In: International Dissolving and

Speciality Pulps Conference, 5-8, April, Boston, MA 1983, pp. 99-104

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3. Nicholson, M., and Johnson, D.C., Tappi 8th Dissolving Pulp Conference, Syracuse, N.Y., 1975 4. Clermont, L.P., Can. Pat. 899,559, May 1972 5. Williams, H.D., U.S. pat 3,236,669, Feb. 1966; Hergert. H.L., and Zopolis, P.N., French pat. 1,469,890, Feb. 1967 6. Schleicher, H. and Lang, H., Solutions of cellulose in sodium hydroxide lye, International Conference on Advanced Polymer Materials, Dresden, Sept. 6-9, 1993 7. K.Kamide and K.Okajima Cellulose dope, process for preparation and method for application thereof, US Patent, No. 4634470, January 1987 8. Li, L.H., Flora, R.M. and King, K.W., Arch.Biochem., 11 1,1965, p.439 9. Selby, K. and Maitland, C.C., Bi0chem.J. 104,1967, p. 716 10. Wood, T.M., Biochem.J., 109,1968, p.217 11. Erikkson, K.E. and Rzaedowski, W., Arch.Biochem.Biophys. 129,1969, p. 683 12. Wood, T.M. andMcCrae, S.I., Bi0chem.J. 128,1972, p. 1183 13. Reese, E.T., in "Biological Transformation of Wood", ed. Liese, W., Springer Verlag, Berlin, 1975, pp. 165-181 14. Bailey, M.J. and Nevalainen, K.M.H. Induction, isolation and testing of stable Trichodenna reesei mutants with improved production of solubilizing cellulase. Enzyme Microb. Technol., 3,1981, pp. 153 - 157 15. Chose, T.K, Measurement of Cellulase Activities, IUPAC Comission on Biotechnology, 1984 16. Parnell, "Life and Labours of John Mercer", London 1886, p. 201 17. Davidson, G.F., The dissolution of chemically modified cotton cellulose in alkaline solutions, Part 3 - In solutions of sodium and potassium hydroxide containing dissolved zinc, beryllium and aluminium oxides, 1937, J.Text.lnst., 28, pp. 27-44 18. Use of Process Additives in the Viscose Manufacturing Chain, Akzo Nobel Surface Chemistry AB, Viscose Chemistry, Sweden, May 1993 19. Yamashiki, T., Matsui, T., Kowsaka, K., Saitoh, M., Okajima, K. and Kamide K., New class of cellulose fiber spun from the novel solution of cellulose by wet spinning method, J.Appl.PoL.Sci., 1992,44, pp. 691-698 20. Yamane, C., Saito, M. and Okajima, K., Production of New Cellulosic Filament Spun from Cellulose / aqueous NaOH Solution (part l), Sen'i Gakkaishi, 1996, 52(6) pp. 310-317 21. Yamane, C., Saito, M. and Okajima, K., Production of New Cellulosic Filament Spun from Cellulose / aqueous NaOH Solution (part 2), Sen'i Gakkaishi, 1996, 52(6), pp. 3 I8 - 324 22. Yamane, C., Saito, M. and Okajima, K., Production of New Cellulosic Filament spun from Cellulose / aqueous NaOH Solution (part 3)", Sen'i Gakkaishi, 1996, 52(7), pp. 369 - 377 23. Yamane, C., Saito, M. and Okajima, K., Production of New Cellulosic Filament spun from Cellulose / aqueous NaOH Solution (part 4)", Sen'i Gakkuishi, 1996, 52(7), pp. 378 - 384

FORMATION AND CHARACTERIZATION OF TRANSFORMED WOODY PLANTS INHIBITING LIGNIN BIOSYNTHESIS Noriyuki Morohoshi and Yukiko Tsuji Graduate School of Bio-Application and Systems Engineering Tokyo Vniversiv of Agriculture & Technology, 2-24-16 Nakamachi, KoganeLhi, Tokvo 184-8588,Japan

ABSTRACT We have tried to form a super tree with new biotechnological and genetic engineering techniques. First subject of our research is to form a lower lignin content tree by controlling the lignin biosynthesis genes using the antisense KNA method. We have succeeded in isolating and sequencing the phenylalanine ammonia-lyase (PAL), 0-methyltransferase (OMT) and peroxidase (PO) genes from hybrid aspen (Populus kitukumiensis), and also isolating the promoter region of these genes. These results show that the genes of PAL, OMT and PO involved in lignification are palg2a, homtl and prxA3a, respectively. We have been able to construct the system that includes transducing a foreign gene to the hybrid aspen by use of Ti-plasmid and infecting with Agrobacterium tumefaciens by the leaf desk method. In t h i s paper, we focus on the peroxidase gene. First, transgenic poplars were made with the prxAl of a peroxidase gene and CaMV35S promoter by the antisense RNA method. They could not grow to young plants, because the promoter can not control its expression in situ. Therefore, a new vector having the original peroxidase @rxA3a) promoter and the antisense prxk3a gene involved in ligrufication was constructed. The transformants with this vector can grow as well as non-trntnsformants. The transgenic poplars have lower total peroxidase activity (10-25%) than that of the control. From the result of peroxidase isozyme analysis by isoelectric focusing a peroxidase band (PI 3.8) involved in lignification disappeared in the transgenic plants. Lignin content in transgenic plant decreases 40-80% compared with control, on the basis of the results by the potassium permanganate oxidation method. The amount of glucose determined by the alditol acetate method in transformants increases 5- 10% compared with non-transformants (control). These results show it is possible to form transgenic; poplars having lower lignin content and higher glucose content which indicates the cellulose content.

INTRODUCTION Today we have serious problems concerning the exhaustion of energy and resources, and the aggravation of environment. One of the means to solve these problems is to achieve an increased yield of biomass and to develop useful techniques of biomass utilization, being able to take the place of ancient energy sources. As trees are a major biomass, it is very important to aim to improve the trees by use of genetic engineering techniques and to establish some useful conversion systems of biomass in the near future. First of all, we focused on the lignin biosynthetic pathway including main secondary

380

Bioengineering of new materials

metabolites in trees, and tried to inhibit the lignin biosynthesis and to form a useful tree consisting of lower lignin content and being able to use polysaccharide materials effectively. Popultts kitakamiensis is used as a plant material and a target gene in lignin biosynthesis pathway is a peroxidase gene involved in lignification. It was inhibited by the antisense RNA method.

DEVELOPEMT OF THE TECHNICAL REQUISITES TO INHIBIT LIGNIN BIOSYNTHESIS We need three technical requisites to form an improved plant by genetic engineering. These are as follows: 1) To establish the developmental and regenerative techniques from the callus to a mature plant.( 1) 2) To establish a stable transformation system to integrate into plants with a foreign gene.(2) 3) To isolate and analyze some target genes and their promoters.

Development and regeneration system of hybrid aspen The callus of hybrid aspen grows in Murashige-Skoog (MS) medium containing 2 , 4 dichlorophenoxyacetic acid (2,4-D: 0.5 mgll). Formation of adventitious buds from the callus is carried out in MS medium containing Benzyladenine (BA: O.lmg/l) and Zeatin (1.0mdl). The root is developed from the shoot in MS medium containing 1Naphthaleneacetic acid (NAA: 1.Smg/l).

PP sc

HPALl3 (PCR clone)

SI

E

E - 1

-2

- 6

-3

I

P

X - 4

X - 5

Figure 1. Restriction enzyme maps of PAL genes from P . kitahmiensis. Bars indicate the probes used for southern and northern blot analyses. E:EcoRI, B:BumHI, PPst I, RV:Eco RV, SI:Snl I, Sc:Suc I, X:Xho I.

Formaticin of transformed woody plants

381

Integration with foreign genes

A vector used for the transduction is pBI121 which has some target genes. The transformation system is performed by the Ti-plasmid method. A transgenic A. tumefuciens integrated with the vector were formed by the triparental conjugation system among the E. coli HBlOls containing the vector and a helper plasmid (pRK2013), respectively, and A. tumefuciens L B A W (not virulent strain). The infection of wood samples with transformed A. tumefaciens was camed out by the leaf disk method. Isolation and analysis of target genes Many enzymes and genes are involved in the pathway of lignin biosynthesis in plants. We focusedon the genes of phenyialanine ammonia-Iyase (PAL), a first step enzyme in the general phenylpropanoid synthesis in plants, 0-methyltransferase ( O W ) , an enzyme deciding the characteristics of the lignin structure between soft and hard woods, and peroxidase (PO) an enzyme for the polymerization monomer units of lignin. In PAL genes, palgl, palgZa, palg2b and palg4 were isolated from P. kitakamiensis (Fig. 1)(3) andpulg2a was found to be a PAL gene involved in lignin biosynthesis on primers1

+

4.

primcrA2

primer A 1

Fignre 2.

Restriction enzyme maps of OMT genes from P. kitakamiensis. Black and white boxes indicate exon and introns, respectively. Arrows indicate the positions of primers for PCR. EEco RI,B:BmHI, H:HindIII, Bg:Bgl 11, P P s t I

Sa P

I AT0

?!TI3

pnrA3a

Figure 2.

pnrA4a

Restriction enzyme maps of PO genes from P. kitukamiensis. EEco Rl,H:HindIII, PPst I, RV:Eco RV, Sa:Sul I, Sc:Sac I, Sh:Sph I, Xh:Xho 1.

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Bioengineering of new materials

the basis of the results of northern hybridization analysis and the expression analysis of the promoter. Homtl from homtl and homt2, (Fig.2) (4) and prxA3a (Fig.3) (5,6) from prxAl, prxA2a, prxA2b, prxMa, prxA4a and HPOX14 were also determined as each candidate of OMT and PO genes involved in lignin biosynthesis by use of the same analytical methods.

IDENTIFICATION LIGNIFICATION

OF

A

PEROXIDASE

ENZYME

INVOLVED

IN

There are many isozymes of peroxidase in plants, therefore it is very important to identify the enzyme involved in lignification in order to inhibit the lignin biosynthesis only. Crude enzyme fractions were extracted from one-year old hybrid aspen samples which are prepared on every month and analyzed by isoelectric focusing. It was found that the acidic peroxidase isozyme obviously expresses when the synthesis of lignin vigorously occurs in July, August and September. It occurs in regenerated shoots and one-year old trunk samples in which the lignification occurs, but not in the callus, green callus and leaf. This isozyme band has a PI value, 3.8-4.2. Therefore, the crude enzymes from leaf and stem in July were analyzed by anion-exchange column chromatography. The results are shown in Fig.4. The difference in enzyme patterns between leaf and stem is in the S-4 fraction, which appears in the stem, but not in the leaf. These fractions were subjected to isoelectric focusing analysis. The S-4 fraction was isolated and had a PI value, 3.9-4.0 (Fig. 5). Finally, we assumed that the acidic peroxidase enzyme is involved in lignification. Furthermore, in order to ascertain whether the peroxidase involved in or not, its monoclonal antibody was made and the histochemical localization of this enzyme was analyzed. We can find that immunogold-silver staining is detected in the xylem near the cambial zone where the lignin biosynthesis occurs in July. The lignification in July was also ascertained by ultraviolet microscopic observation. Conclusively, this result indicates that this acidic peroxidase isozyme is involved in the lignification of the hybrid poplar.

El

B

l'p

-5

, I

d 4J

m

vl

Tube number (16 g/tube)

Figure 4.

Anion-exchange chromatograms of the crude enzymes from leaf and stem. A : Shoot, B: Leaf

Formation of transformed woody plants

383

Isolated fractions

t

5; 2

4

5;-5

U

8-4

s-3

I

9

L-5

I

F!-1 Cmde enzyme from leaf Crude enzyme from stem

sw * Figure 5.

a

$

b

Isoelectricfocusing patterns of purified and crude enzymes of peroxidases.

PROPERTIES OF THE POPLAR CONTROLLING THE PEROXIDASE GENE Peroxidase is a final step enzyme in the synthetic pathway of lignin biosynthesis. There is no other bypass in this enzymatic step and it will be a rate-limiting step for the dehydropolymerizationof lignin. Therefore, we thought that it is possible to inhibit the lignin biosynthesis effectively when the peroxidase gene is used for the control of lignin content. A vector having the CaMV35S promoter which is followed by a reversed prxA1 sequence, was constructed and introduced into the hybrid poplar. Transformants obtained with POXl have died because the peroxidase enzyme activity of the transformants with the POXl was strongly inhibited in all part of transformants. This fact shows that CaMV35S promoter that can express everywhere in plants can not be used for the inhibition experiment by the antisense RNA method in the case of the peroxidase gene. Therefore, we constructed an antisense vector having an original promoter of prxA3a (Fig.6) and transformants were formed. These transformants have grown similar to the control without death. Some transformants have characteristics, for example, the delay of root growth and having more branches. Fig. 7 shows the peroxidase activity of the transformants. Obtained results indicate that peroxitlase activity in the stem of transformants decreases 7590% compared with control a.nd in the leaf decreases 20-

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Bioengineering of new materials

LB

RB

ATG

Figure 6.

prxA3a

Structure of a plasnlid coutaining anti-prxA3a.

100

50

0

Figure 7.

Peroxidase activity of transgenic aspen in stem and leaf, Control : Wild Transformants: POX43, POX44, POX49, POX53.

Formation of transformed woody plants

Figure 8.

385

Lignin content of transformed hybrid aspen estimated by permanganate oxidation method. Control :Wild Transfomants: POX43, FQX44, POX49, POX53.

80%. This result suggests that the prxA3a gene specifically expresses in the stem. From the analysis of isoelectric focusing we found that the transformants lack an acidic peroxidase band, PI 3.8, which mainly expresses in the wild stem. In order to analyze the lignin chemical structure of transformants in detail, the samples were subjected to potassium permanganate oxidation (7) and thioacidolysis analyses(8). These results show that the lignin content of transformants decreases 2060% in comparison with the control (Fig.8) and the characteristic of the chemical structure of transformants is the decrease of p-hydroxyl, guaiacyl and syringyl units. From this result, it seems that there is no effect for the process of polymerization among aromatic units when the peroxidase inhibits by antisense RNA method. As the amount of uncondensed units is almost similar between transformants and control, it is concluded that the inhibition of peroxidase in transformants has the main effect on the decrease of the lignin content in plants. To clarify the amount and quality of polysaccharides in transformed aspens, the composition of monosaccharide degraded by alditol acetate method (9) was analyzed. This result indicates an increasing tendency of glucose, which forms the cellulose and decreasing tendency of xylose, which forms the hemicellulose. These results might mean that transformed aspens increase cellulose content and decrease hemicellulose fractions in comparison with the wild.

CONCLUSION In order to reduce lignin content in aspen trees, we tried to isorate the phenylalanine ammonia-lyase, 0-methyltransferase and peroxidase genes involved in lignin biosynthesis from the hybrid aspen. palg2a, HomtlandPrxA3a were isolated and the

386

Bioengineering of‘ new materials

DNA sequence and promoter were analyzed. As the promoter of prxASa expressed in the stem specifically, it was assumed that prxA3a has involved in lignin biosynthesis. By using the antisense RNA method, some transformed aspens inhibiting in peroxidase activity and lignification were formed. These transformants have lower peroxidase activity (70-90% in stem) and lower lignin content (15-60%), and more increasing tendency of the glucose than the wild type has. Especially, the characteristics of the lignin chemical structure in the transformants is lower content of p-hydroxyl, guaiacyl and syringyl units, increased tendency of the degree for condensed type and decreased degree of uncondensed type in comparison with the wild. These results will give us hope that we may succeed in forming useful trees by means of genetic engineering techniques.

REFERENCES 1. H. Ebinuma, K. Sugita, E. Matunaga et al., ‘Selection of marker-free trnsgenic plant using the isopentenyl transferase gene’, Proc Natl Acad Sci USA , 1997,94,21172121. 2. S. Kajita, K. Osakabe, Y. Katayama et al., ‘Agrobacterium-mediated transformation of poplar using a disarmed binary vector and the overexpression of a specific member of a family of poplar peroxidase gene in transgenic poplar cell ’, Plant Science, 1994, 103,231-239. 3. Y. Osakabe, Y. Ohtsubo, S . Kawai et al., ‘Structure and tissue-specific expression of genes for phenylalanine ammonia-lyase from a hybrid aspen, Populus kitahmiensis.’, Plant Science, 1995, 105,217-226. 4. T. Hayakawa, K. Nanto, S, Kawai et al., ‘Molecular cloning and tissue-specific expression of two genes that encode caffeic acid and 0-methyltransferases from Populus kitakamiensis. ’, Plant Science, 1996, 113, 157-165. 5. K. Osakabe, H. Koyama, S . Kawai et al., ‘Molecular cloning and the nucleotide sequences of two novel cDNAs that encode anionic peroxidases of Populus kitakamiensis. ’, Plant Science , 1994, 103, 167-175. 6. K. Osakabe, €4. Koyama, S. Kawai et al., ‘Moiecular cIoning of two tandemfy arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem ’, Plant Molecular Biology , 1995,28,677-689. 7. M. Erickson, S. Larsson and G. E. Miksche. ‘Gaschromatographische Analyse von Ligninoxydationsprodukten. VII. Ein verbessertes Verfahren zur Charakterisierung von Ligninen durch Methylierung und oxydativen Abbau ’, Acta Chemica Scandinavica , 1973,27,127-140. 8. C. Lapierre, B. Monties, and C. Rolando. ‘Thioacidolysis of poplar ligninIdentification of monomeric syringyl products and characterization of guaiacylsyringyl lignin fractions ’, Hokforschung , 1986,40, 113-118. 9. A. B. Blakeney, P. J. Hams and R. J. Henry et al., ‘A simple and rapid preparation of alditol acetates for monosaccharide analysis ’, Carboh.y& Res , 1983, 113,291299.

CHARACTERIZATION AND UTILIZATION OF LIGNINOLYTIC ENZYMES PRODUCED BY BASIDIOMYCETES Masaaki Kuwahara

Wood Research Institute, Kyoto University, VJi, .Kyoto 611-0011, Japan ABSTRACT Lignin peroxidase (Lip) and manganese peroxidase (MnP) purified from the culture filtrate of the basidiomycetes Bjerkandera adusta catalyzed the oxidation of various phenolics and aromatic amines in the reaction mixture containing water-miscible organic solvents such as ethylene glycol, methylcellosolve and acetone. Aromatic compounds that have low ionization potential and high hydrophobicity were found to be preferable substrates of the enzymes in these solvents. Parameters that characterize the activity of Lip and MnP in organic solvents were analyzed. By using MnF’, coniferyl and sinapyl alcohols were polymerized to c-DHP and s-DHP, respectively, with a high yield in the mixed solvent of acetone and methylcellosolve. NMR analysis showed s-DHP synthesized by MnP contained higher content of p-0-4 substructure than the DHP synthesized by horseradish peroxidase. Using this reaction, polymerization products were obtained from various phenols, aromatic amines and other compounds in the media containing acetone or dioxan.

INTRODUCTION Lignin is an amorphous and polyphenolic material and account for more than 20% of weight of angiosperms (hardwoods) and more than 25?6 of that of the gymnosperms (softwoods). Lignin is synthesized by an enzyme-mediated dehydrogenative polymerization of three different phenyl propanoids, coniferyl, sinapyl and p-coumaryl alcohols. These structural units are linked by a variety of chemical linkages including carbon-carbon and ether bonds which are found in the substructures arylglycerol p-arylether, biphenyl, phenylcoumaran, diarylpropane and others. In nature, plant and wood biomass are mineralized by tnicroflora in soil. Wood-rot fungi are the main degraders of wood and its components. White-rot fungi, mostly basidiomycetes, degrade lignin selectively or non-selectively. More than 100 phenolic compounds were isolated and characterized as the metabolites of lignin degradation by a fungus, Phanerochaete chyosporium . Main degradation monomeric products soluble in methanol were recovered in acidic fractions, which contained vanillic and veratric acids. Thus far, three different phenol oxidizing enzymes, lignin peroxidase (Lip), manganese (11) peroxidase (MnP) and laccase (Lac) have been isolated from the culture of white-rot fungi and characterized (Table 1) The genes encoding the production of reaction of these enzymes is basically one electron oxidation of the substrates. Lip oxidizes non-phenolics using hydrogen peroxide as the electron acceptor forming cation radical. M n p oxidizes Mn(I1) to Mn(II1) which oxidizes phenolic compounds to form

‘

’.

388

Bioengineering of new materials

Table 1. Basidiomycetes producing Lip and MnP *.a, Enzyme activity; b, Hybridization; c, Cloning; d, Antibody Lip or MnP Bjerkandra adusta (Polyporous adustus) Ceriporiopsis subvennispora Chrysonilia sitophila Chrysosporium pruinosum Coriolopsis occidentalis Coriolopsis polyzona Coriolus consors Coriolus hirsutus Daedaleopsis confragosa Dichomitus squalens Fomes lignosus (Rigidoporous microsporous) Ganoderma valesiacum Lentinula edodes Panus tigrinus Phanerochaete chrysosporium Phellinus pini PNebia brevispora PNebia radiata Pleurotus ostreatus Polyporus ostreifonnis Pycnoporus cinnabarinus Rigidoporus lignosus Steieum lursutum Streptomyces viridosporus Trametes gibbosa Trametes versicolor (Coriolus versicolor) Trametes villosa

Lip

a, b, c

MnP, Lip Lip Lip Lip MnP Lip Lip MnP MnP Lip

a, b a a a a b a a a a, b

MnP MnP MnP Lip, MnP Lip

MnP, Lip Lip, MnP Lip Lip MnP MnP MnP Lip Lip, Mnp LB, M n p a

Native Fe 111

Native Fe 111

n \ /

Compound I FeIv=o[P]; Mn AH: Substrate

Lignin peroxidase (Lip)

Compound II FeIv=o

Mn 111

x

(Phenoxy radical) A'

Figure 1.

Detection ___

AH

Mn(ll) peroxidase (MnP)

Catalytic cycle and reaction mechanism of LiP and MnP

*

Characterization of ligninolytic enzymes

389

phnoxy radical. Laccase oxidizes phenolic substrates using molecular oxygen as the electron acceptor and the first reaction product is also the phenoxy radical. Catalytic cycles of Lip and MnP are shown in Fig. 1 '. UTILIZATION OF LIGNIN-DEGRADING ENZYMES

Lignin-degrading hngi and enzymes can be potentially used in various fields of industries as summarized bellow. Chemical indust?: a. Biological pulping and bleaching of pulp b. Delignification of lignocellulosicmaterials for the production of chemical feedstock and &el alcohol c. Involvement in chemical processes Agriculture: a Enhancement of digestability of ruminant feed b. Composting of agricultural wastes c. Production of edible mushrooms Environment technolow: Bioremediation of soils polluted by toxic chemicals Others: Synthesis of polymers using enzymes

Various polymers have been synthesized in vitro using, mainly hydrolytic enzymes, cellulose by cellulase, poly(g1ucose-xylose) by xylanase, polyesters and polyamides by lipase and protease. These reactions are basically caused by the reverse reaction of hydrolysis. The first attempt to synthesize polymers by using oxidases was the synthesis of lignin model polymer using laccase or peroxidase. In the utilization of enzymes for the chemical processes, reaction atmospheres are thought to influence the yield of the polymers. Although enzymes are active in aqueous systems, following advantages are expected in the usage of organic solvents for enzymatic reactions: 1. Increased solubility of non-polar substrates 2. Shifting thermodynamic equilibria to favor synthesis over hydrolysis 3. Suppression of water-dependent side reactions 4. Alteration of optimal pH and temperature and other catalytic parameters 5. Enhanced thermostability and pH stability 6. Alteration in substrate and enantioselectivity 7. Ease of enzyme recovery by simple filtration 8. Ease of product recovery tiom low boiling high vapor pressure solvents 9. Elimination of microbial contamination By employing these advantages, enzymes can be potentially used directly within new or existing chemical processes. A. Reaction of Lip andMnP in organic solvents Since most of the compounds related to lignin are insoluble in water, development of reaction systems in organic media is necessary for extending the catalytic action of

390

Bioengineering of new materials

Table 2. Activity of LIP in organic solvents One unit of enzyme produces an absorbance of 1.O/min. Substrate: 3,3'-dimethoxybenzidine.Specific activity of Lip: 125U/mg (in queous buffer). Abbreviations: DMF, N,N-dimethylformamide; DEGME, diethylene glycol monomethyl ether; DEGDE, diethylene glycol diethyl ether. Solvents (YO) Water 70% Acetone 70% Methanol 70% 2-PropanoI 70% Dioxane 70% Dh4F 70% -dine 70% Ethylene glycol 70% Methylcellosolve 70% 1,2-Dimethoxyethane 70% Diethylene glycol 70% DEGME 70% DEGDE Chloroform Ethyl acetate Benzene Toluene

Relative activity 100 90 2 10 13 0 3 76 1 165 35 93 68 38

0 0 0 0

lignin degrading enzymes. We are trying to activate these enzymes to degrade and oxidize lignin and related aromatic compounds in organic solvents. Lip purified from the culture of€? chrysosporium or B. adusta was found to oxidize 3,3'-dimethoxybenzidine (3,3'-DMB) in the reaction mixture containing 70% water-miscible organic solvents including ethylene glycol, diethylene glycol, methylcellosolve and acetone. Various compounds tested were oxidized by LiP in these solvents (Table 2) '. Higher ET(30) , a parameter concerning conformational changes of enzymes in organic solvents, influence the activity of the enzyme. Lower ET (30) values were reported to indicate higher denaturation capacity of the solvents. Low ionization potential and high hydrophobicity of the substrates resulted in higher activity in organic solvents '. Activities of MnP were also measured using various aromatic compounds as substrates in organic solvents. MnP retained its activities in 70% aqueous solutions of several water-miscible organic solvents, ethylene glycol, diethylene glycol, acetone and acetonitrile (Table 3). Except for acetonitrile, absorption spectra of MnP in these solvents were similar to those observed in water, indicating that the heme of MnP was little affected by addition of these water-miscible organic solvents. MnF' also exhibited oxidation activities of Mn(1I) to Mn(II1) in several 70% aqueous solutions of the water-miscible organic solvents. Measurement of ET(30) value for the oxidation of h4n(II) suggested that conformational change of MnP by the solvents enable Mn(I1) to approach heme easily '.

Characterization of ligninolytic enzymes

39 1

Table 3. Oxidation of aromatic compounds by MnP in 70% aqueous organic solvents

Abbreviations: HQME, hydroquinone monomethyl ether; ABTS, 2,2'-azino-bis (3-ethylbenzthiazoline)-6-sulfonicacid -

Substrates 3,3-Dimethoxybenzidine o-Phenylenediamine p-Phenylenediamine m-Phenylenediamine Aniline o-Anisidine p-Anisidiie

m-Anisidine 0-Aminophenol p-Aminophenol m-Aminophenol Phenol Catechol Hydroquinone Resorcinol Pyrogallol 1,2,4-Benzenetriol Guaiacol

HQME 3-Methoxyphenol 2,6-Dirnethoxyphenol Vanillyl alcohol Veratryl alcohol ABTS

MnP activity in Water (U/mg) 518 614 842 98

Relative activitv (%) Acetone 168 0 42 33

0

0

152 696 18 434 0 86 132 106 930 88 504 0 404 0 0 1987 0

20 3 111 106 0 42 9 45 n.d. n.d. n.d.

0

1468

0

44 0 0 82 n.d. n.d. 5

Acetonitril

118 102 46 20 0 8 12 89 84 0 28

I5 57 108 125

13 0 60 0

Diethylene d y w l 219 134 61 84 0 16 8 67 58 0 23 3 42 30 45 7 0

18 0

0

0

50

18

0 0

0 0 22

5

Different types of aromatic compounds were oxidized by MnP in 70% aqueous acetone, acetonitrile and diethylene glycol medium. Hydrophobic aromatic compounds were more reactive than hydrophilic compounds in these solvents. Especially, in the oxidation of 3,3'-dimethoxybenzidine,the activities in several 70% organic solvents were found to be higher than that in water. MnP activities in water-miscible solvents were also higher than those of Lip from I! chrysosporium. Therefore, it is concluded that MnP is more tolerant than Lip in organic solvents '.

B. Production of dehydrogenationpolymers (DHP) using MnP Dehydrogenation polymers (DHP)have been synthesized from monolignols by using laccase or peroxidase of plant origin. This polynler has been used as a model compound of lignin. Monomeric sugars added into the reaction mixture were found to bind to the polymer, which is the model compound of lignin-carbohydrate-complex (LCC) found in native wood and plants components. MnF' was found to degrade DHP synthesized from coniferyl and sinapyl alcohols synthesized by peroxidase with conventional synthetic process. In this reaction, repolymerization of the substrate and the product occurred besides depolymerization'.

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Bioengineering uf new materials

A:

C-DHP

6: S - D H P

70% Acetone

70% Acetone

Vo 90,000 9,000 2,500 731

I/

(//

VO 90,000 9,000 2,500

I(

731

///

Water

Retentiontime (min)

Figure 2.

Retention time (min)

GPC of c-DHP (A) and s-DHP (B) synthesized by MnP and HRF’ in acetone and water (Column: Shodex GPC KD-803; Eluent: DMF, 0.05M LiCl)

This suggested the possibility of using MnP to the production of polymers from phenolic compounds. As expected, coniferyl and sinapyl alcohols were polymerized to give c-DHP (coniferyl-DHP or softwood type DHP) and s-DHP (sinapyl-DHP or hardwood type DHP), respectively, with a high yield in the mixed solvent of acetone and methylcellosolve by using MnP purified from the culture of B. udusta. In the acetone media, although s-DHP was not synthesized by HRP, this DHP was synthesized by MnP. Molecular weights of c- and s-DHPs formed by MnP were higher than those synthesized conventionally by horseradish peroxidase (HRP)(Fig. 2). The chemical structure of MnP-DHP was found to differ from HRP-DHP in that MnP-DHP was rich in p-0-4 linkage and less in p-p linkage than HRP-DHP ’.

C. Production of phenolic polymers by using MnP Using the similar reaction system to that of DHP synthesis, polymerization of various phenolic compounds including guaiacol and catechol and aromatic amines was examined in aqueous acetone and other solvents by using MnP of B. adusta. The reaction was carried out in the reaction mixture containing I50 mM substrate, 20 mM of hydrogen peroxide and 5 m M MnS04. The reaction was carried out for 24 hr. Polymerization products were recovered in water insoluble fraction. This fraction was dissolved in methanol and the insoluble fraction was solubilized again in demethyl formamide (DMF). Polymers with higher molecular weight were obtained as DMF insoluble precipitate.

Characte:rization of ligninolytic enzymes

393

Table 4. Yield and molecular weight of poly-guaiacol synthesized by MnP in organicsolvents

M m e i g h t average molecular weight, Mn=number average molecular weight d=Mw/Mn=degree of dispersion Solvents

Polymer yield (%)

Water Acetone DMF Ethanol Dioxane

33 22

Acetonitrile Methanol

5 4 2 (DMF-insoluble 0.5 2 (DMF-insoluble 0.5 2 (DMF-insoluble 0.5

Ethylene glycol Metbvlcellosolve

2 2

h4w

,

11,400 30,300 . 19,900 15,600 38,000 >> 100,000) 26,800 >> 100,otmo) 14,000 >> l00,oC~) 13,800 15,500

Mn

d

5,400 12,200 10,800 8,600 13,600

2.1 2.5 1.8 1.8 2.4

16,500

1.6

8,500

1.6

7,800 9,100

1.8 1.7

The highest yield of polymer from guaiacol, 22 % of the substrate, was obtained in 50% acetone (Table 4). The weight average and the number average molecular weights of the product were about 30,000 and 12,000, respectively, as determined by gel filtration chromatography using polystyrene as a molecular weight standard. In the reactions in dioxane, acetonitrile and methanol, DMF insoluble polymer, with higher molecular weight of over 100,000, was obtained. However, the yields were much lower than that obtained in acetone. MULDI-TOF-MS analysis gave lower molecular weight than GFC analysis. This information showed that the polystyrene standard is possibly not applicable to the determination of molecular weight of phenolic polymers. Thermo-degradation GC-MS, "C-NMR and IR analyses of the polymer of guaiacol indicated the presence of C-C and C-0 linkages and benzoquinone structure in the polymer. As shown in Table 5, o-cresol and 2,3-dimethoxyphenol gave polymers insoluble in DMF, whose molecular weights were over 100,000 as measured by GFC. The yields of these polymers were over 20% against the substrate. Other utilization of lignin degrading fungi and enzymes A. Production offie1 alcoholfi.om lignocellulosic biomass

Cellulose and hemicellulose can be fermented to produce alcohol after hydrolysis by cellulases or acids. However, lignin is the physical barrier against the enzymatic attack. In the saccharification and fermentation of cellulosic materials, the efficiency of the overall process is strictly dependent on the efficiency of removing lignin from biomass and disrupting their hard structure. It was shown that pretreatment of wood biomass by steam explosion, which is effective both to remove Iignin and destruct the structure of wood, enhance the enzymatic saccharification of the biomass. Treatment of biomass

394

Bioengineenng of new materials

Table 5.

Polymerization of aromatic compounds by M n p in aqueous 50 % acetone

Abbreviation: 2,6-DMP, 2,6-dimethoxyphenol Substrates

o-Cresol 2.6-DMP Guaiacol Varullin o-Ethoxyphenol

Acetovanillon Catechol o-hsidine Synngic acid o-Phenylene&amine

Polymer yield ("10) 25 (DMF-insoluble 22 24 (DMF-insoluble 23 22 8 7 (DMF-insoluble 2 6 4 2 1 (DMF-insoluble 0.2 1

Mn

d

7,300

17

4,400

1.2

12,200 4,600 1 1,800

2.5 I .5 3.0

3,700 7,900 7,800 16,300 >> 100,000)

2,200 3,900 5,600 5.600

1.7 2.0 1.4 3.0

24,100

11,400

2.2

MW

12,500

>> 100,000) 5,200

>> 100,000) 30,300 6,400 34,500

>> 100,000)

by the ligninolytic hngi prior to explosion caused additional enhancement for the enzymatic production of fermentative sugars lo. To promote the degradation of lignin by fungi, genetic engineering of the genes encoding lignin degrading enzymes is promising. As shown in Table 1, Lip and MnP genes have been cloned from c-DNA and chromosomal DNA of various white rot fungi. Heterologous and homologous transformation systems have been developed in I? chrysosporium ' I . As to other white-rot fungi, construction of expression vectors are being carried out in Coriolus hirsutus, Pleurotus ostreatus, Lentinula edodes and other fungi

B. Degrachtion of chemical pollutants by lignin-degradingfingz Various aromatic compounds and their halides have been used as pesticides, herbicides, fungicides, explosives, dyes and other purposes. Most of these chemicals are carcinogenic and suspected to be endocrine disturbing materials and cause serious chemical pollution of environment. Lignin degrading activity of fungi can be applied to remediate the soils polluted by these toxic chemicals. The mechanism of the degradation of these chemicals is thought to be one electron oxidation as in the case of lignin degradation. Degradation of these chemicals and bioremediation of polluted soil has been carried out by using i? chyosporium and the results have been summarized in many article^'^''^. We are trying to use other basidiomycetes including I? ostreatus and Bjerkundera adusta. These hngi decolorized various aromatic dyes and dechlorinated mono- and dichlorophenols within two weeks of the culture. Lip and MnP purified from the culture of i? chrysosporium and B. adusta oxidized dimethoxybenzidine [4], indicating these fungi can be used for the degradation of biphenyl compounds such as polychlorinated biphenyls (PCB). MnP of P ostreatus was also found to degrade bisphenol A and nonylphenol both are leaked from plastics.

Characterization of ligninolytic enzymes

395

References 1 . K. -E. L. Eriksson, R. A. Blanchett & P. Ander, Microbial and Enzymatic Degradation of Wood and Wood Components, Springer-Verlag, Berlin, 1990, pp. 225-333, 2. A. B. Orth & M. Tien, Biotechnology of lignin degradation, In: The Mycota, II, K. Esser & P. A. Lemke (eds), Springer-Verlag, Berlin, 1995, pp287-302. 3. M. H. Gold & M. A h , ‘Molecular biology of the lignin-degrading basidiqmycete Phanerochaete chrysosporium,Microbiol. Rev., 1993, 57, 605-622. 4. M. H. Gold, H. Wariishi & K. Valli, Extracellular peroxidases involved in lignindegradation by the white rot basidiomycete Phanerochaete chrysosporium, In: ACSSymp. Ser. 1989, 389, 127-140. 5. S. Yoshida, T. Watanabe, Y. Honda & M Kuwahara, Effects of water-miscible organic solvents on the reaction of lignin peroxidase of Phanerochaete chrysosporium, J. Mol. Catalysis B; Enzymatic, 1997,2,243-251 6. S Yoshida, Degradation and synthesis of lignin imd its related compounds by fungal ligninolytic enzymes, WoodRes., 1997, 84, 76-129 7. S. Yoshida, A. Chatani, Y. Honda, T. Watanabe & M. Kuwahara, Reaction of manganese peroxidase &om Bjerkandera adusta in aqueous organic solvents, J. Mol. Catalysis B: Enzymatic, in press 8. S. Yoshida, A. Chatani, Y. Honda, T. Watanabe & M. Kuwahara, Reaction of manganese peroxidase of Bjerkandera adusta with synthetic lignin in acetone solution, J. Wood Sci., 1998,44, 486-490 9. S. Yoshida, A. Chatani, M. Tanahashi, Y. Honda, T. Watanabe & M. Kuwahara, Preparation of synthetic lignin by manganese peroxidase of Bjerkandera adusta in organic solvents, Holzforschung, 1998, 52, 282-286 10. T. Sawada, Y Nakamura, F. Kobayashi, M. Kuwahara & T. Watanabe, Effects of fungal pretreatment and steam explosion pretreatment on enzymatic sacchanfication of plant biomass, Biotechnol. Bioeng., 1995, 48, 7 19-724 1 1 . M. B. Mayfield, K. Kishi, M. Alic & M. H. Gold , Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium, Appl. Environ. Microbiol., 1994, 60, 4303-4309 12. P Barr and S D Aust, Mechanisms white rot fungi use to degrade pollutants, Environ. Sci. Bchnol., 1994,28, 78A-87A 13. A. B. Orth, E. A. Pease & M. Tien, Properties of lignin-degrading peroxidases and their use in bioremediation, In: Biological Degraahtion and Bioremedation of Toxic Chemicals, G. R. Chaudhry (ed.), Chapman and Hall, London, 1994, pp 345-402

KINETICS OF BIODEGRADATION OF n-ALKANES BY PSEUDOMONAS IMMOBILISED IN RETICULATED POLYURETHANE FOAM Manuel G. Roig', John F. Kennedy2,Charles J. Knill', Jose M. Sanchez', Miguel A. Pedraz', H a m Jerabkova3& Blanka Kralova3

I

Departamento de Quimica Fisica, Facuhd de Quimiccz, Uniwrsidad de Salamanca, P h u de la Merced dn, 37008 Salammca, S p i n . 'Birmingham Carhahydrate & Protein Techno& Group. Chemhiotech L.aboratories, 1Jniversiy of Birmingham Research Park, Vincent Drive, Birmingham, B I5 2SQ, UK.

' Ileparhnent of Bimhemisny andMicrohiolo~,Inshtule of Chemical Zechnoloa, Prague, Technic& 5, 166 28 Prague 6, Czech Republic.

ABSTRACT The n-alkane-degrading bacteria Pseudomonas C 12B and Pseudomonas A3 were immobilised by physical entrapment of cells within reticulated polyurethane foam. Such immobilised biocatalysts have been shown to be appropriate for the effective removal (sorption on the support plus biodegradation) of n-decane and n-hexadecane. Support particles exhibit a positive fractionating effect of the substrate on the polymer matrix. This effect involves sorption of the n-alkane onto the polyurethane foam, saturating it and synergistically enhancing the biodegradative actwity of the immobilised cells. Comparison of sets of kinetics for the overall n-alkane (n-decane and n-hexadecane) removal from water for polyurethane foam with and without (controls) entrapped cells showed that the overall kinetics for the immobilised biocatalyst were well fitted to a biphasic process. A rapid sorption step of the substrates onto the cell-loaded support 111, and the intrinsic primary biodegradation slower step [2], both a m g synergistically:

([n-alkane]

=

Ae-ki'

+

(kl

=

0.092 min",

(t%)l =

(k,

=

4.9 x 104 min-', (t%k =

Ae-k:

,+

C)

7.5 minutes) ... ... ... ... ... ... ... 111 23.6 hours)

_ _ _ ... . . __ _. _. . _ _ . _121 .

INTRODUCTION Extensive soil and groundwater contammation with oils, surfactants and related hydrocarbons consbtute a significant environmental problem throughout Europe. Any in srtu bioremediation approach should exploit biodegradative orgmsms in high One population densities, preferably with enhanced activities, at the polluted sites of the ways to concentrate biodegradation reagents for water decontamination is by their immobilisation onto appropriate support particles.

398

Bioengineering of new materials

Most micro-organisms grown with an n-alkane as (part of) the carbon source convert the alkane into the corresponding fatty acid. The fatty acid may be incorporated directly into cellular lipids, Thus an organism grown on an alkane with an odd number of carbons might have an unusual amount of odd-carbon fatty acids in its lipids '. The ability to grow on hexadecane is prominent among arenicolous hngi, is less prevalent among lignicolous fungi, and is rate limiting among caulicolous organisms '. Marine hngi have been exposed to hydrocarbons in sea foam and in surface slicks for thousands of years. Consequently, marine fungi could be considered as natural agents for bioremediation of oil spills, due their ability to grow on single hydrocarbons as their sole source of energy and of organic carbon. Methanogenic and sulphate-reducing bacteria living in oil deposits are able to modify the original hydrocarbons producing methane and hydrogen sulphide. Other microorganisms, especially the anaerobic fermentative bacteria, could be responsible for other transformations, such as the degradation of n-alkanes from 12 to 35 carbons. However, the biodegradation reactions of hydrocarbons in anaerobic conditions exhibit slow rates of loss of specific activity (half lifetimes of several months) *, The ability to utilise hydrocarbons by underground bacteria could contribute to solving the pollution of underground waters by hydrocarbons. Study of the metabolism of such bacteria will permit a better understanding of the reasons why the hydrocarbons are modified and an improvement of methods for their biodegradation. Various methods and supports have been described for the immobilisation of bacteria and entrapment within polymeric matrices has often been successful 539. A wide range of supports (organic and inorganic) has been screened for the immobilisation of Pseudomonas cells. Specifically, biofilm growth, cell entrapment and chemical binding (silanisatiodglutaraldehyde) have been the methods of immobilisation evaluated by Roig el a f .(1999) I". They found that reticulated polyurethane foam provides high cell retention capacity, small or no decrease in enzymatic activity, and satisfactory biodegradation activity and operational stability during bioreactor performance. In addition, the cost of this support is low and its synthesis straightforward ". The mechanisms involved in microbial attachment to solid surfaces have not been completely elucidated. However, it is apparent that some types of organisms secrete macromolecules that initiate microbial-surface interactions and this could be the case of Pseudomonas, which grows readily in the biofilm-attached state. Physical entrapment of organisms inside a polymer matrix is one of the most widely used techniques for cellular immobilisation since it does not depend significanily on cellular properties. Examples of matrices commonly used are K-carrageenan, alginate, and polyacrylamide. For the present purposes, carrageenan is of no use because it is degraded by Pseudomonas C12B 12. Alginate is likewise unsuitable because the presence of phosphates in the intended substrate medium would precipitate the Ca2' ions essential for maintaining the gel structure. Polyacrylamide provides a potential for significant losses in cell viability and enzyme activity during free radical polymerisation of the gel, low mechanical strength and resistance to chemical and microbial attack. In this case, the disadvantages appear to be greater than the advantages. Polyurethanes, on the other hand, constitute a group of polymers with highly versatile properties for the immobilisation of cells and have therefore been chosen for testing as supports for the entrapment of P.wudomonas sp. cells 13. The urethane moiety is created by reacting an isocyanate with an alcohol. In the case of polyurethane foams, a diisocyanate solution is added to a 1-2 kDa molecular weight prepolymer with terminal hydroxyl groups to establish the polyurethane linkages.

Kinetics 0 1 biodegradation of n-alkanes

399

The foaming agent is carbon dioxide generated in situ by controlled addition of water to the reaction. The water transforms isocyanates into amines, releasing carbon dioxide. The amine then reacts with other isocyanates to afford. urea linkages. The polyol to diisocyanate ratio is essential for obtaining an appropriate reticulated foam with good consistency, porosity and mechanical properties. Plug-flow bioreactors packed with immobilised cells of Pseudomonus C12B and A3 were therefore constructed to follow the kinetics of the removal of n-alkanes from aqueous solutions. The physicochemical and biochemical processes involved were assessed for rr-decane and n-hexadecane and Pseudomom C12B and A3 physically entrapped within xeticulated polyurethane foam.

MATERIALS & METHODS Substrates

Substrates utilised €or this work were n-decane (Mw 142.28, density 0.7300 g/mL) and n-hexadecane (Mw 226.44, density 0.7730 g/mL), and were all of analytical grade.

Basal salt medium (BSM) BSM contained dibasic potassium phosphate (dipotassium hydrogen phosphate, monobasic potassium phosphate (potassium dihydrogen Mw 174.18, KzHPO4, 3.5 phosphate, Mw 136.09, K H 8 0 4 , 1.5 g/L); ammonium chloride (NH4C1, MW 53.49, 0.15 sodium chloride (NaCI, MW 58.44, 0.5 @,); sodium sulphate (NazSO4, MW142.04, 0.15 magnesium chloride hexahydrate (MgCl2.6Hz0, MW 203.30, 0.15 a l l reagent grade, giving an overall pH of 7.1.

a);

a); a); a),

Micro-organisms Pseudomonm C12B (NCIMB 11753) bacteria were kindly supplied by Prof. N. J. Russell, Wye College, University of London, London, UK and Dr. G. F. White, School of Molecular & Medical Biosciences, University of Wales College of Medicine, Cardiff, Wales, UK. Pseudomonas A3 bacteria were kindly supplied by Prof K. Demmerova and Dr. J. Pdarov6, Department of Biochemistry & Microbiology, Institute of Chemical Technology, Prague, Czech Repubilic. Inoculum was batchgrown for 24 hours at 28°C with orbital shaking (2.5 &) in nutrient broth. BSM (200mL) with 1 % v/v of n-alkane (CIO for Pseudomonus C12B, Cll-Cl6 for Pseudomom A3) and inoculum (2 mL) were cultivated at 28°C with orbital shaking. The growth of the microbial culture was monitored by measuring optical density at 650 nm. Bacteria were harvested routinely from cultures in early stationary phase (48 hours for Pseudomonas C12B, 13 hours for Pseudomom A3) by centrifkgation (8520 G, 10 minutes, 4°C) and resuspended in BSM lacking magnesium chloride (BSM-Mg) to a concentration of 0.1 g wet celldmL. Immobiliied cell supports

Commercial polyurethane reticulated foams were a gift from Calther (Salamanca, Spain). Prepolymer Hypo1 FHE' 2002 (hydrophilic polyisocyanate containing < 10 % v/v fiee toluene diisocyanate, 2.35 meq/g NCO, Grace Service Chemicals GmbH, Heidelberg, Germany) was used for reticulated polyurethane foam synthesis.

400

Bioengineering of new materials

Immobilisation of Pseudomonus Cl2B cells

A. Passive immobilisation on reticulatedpolyurethanefoam Biofilm formation on polyurethane reticulated foam was achieved by growing Pseudomonas sp. cells in a batch culture medium in the presence of 100 cubes (0.125 cm3 each) of commercial polyurethane foam. AAer 48 hours of growth (&5,,,,, = 0.8), the biofilm coated supports were removed. The coated supports were washed in BSM and stored at 4°C for subsequent use in biodegradative experiments.

R. Cell entrapment in reticulated polyurethane foam Polyurethane was synthesised by quickly mixing prepolymer Hypo1 FHP 2002 (5 g) with the aqueous cell solution (5 mL, 0.1 g wet weight/mL) and allowing the reticulated foam to cure for 30 minutes 13. The resultant block of polyurethane reticulated foam entrapped cells was consecutively washed with BSM-Mg (200 mL), a mixture of deionised water and BSM-Mg (1: 1 v/v, 200 mL) and deionised water (200 mL). The washings were collected and the protein content in the total volume was determined using a modified Lowry assay 14, the loss of bacterial protein after immobilisation and washings being 15- 18 YOof the total protein offered to the foam. In order to increase the n-alkanes biodegradative specific activity of the cell-loaded supports, additional cell growth proliferations were carried out for a hrther 20-40 hours, a slight increase in activity being detected. The blocks were cut into cubes (0.125-1.0 cm3) and the resulting particles of reticulated foam were stored in BSM-Mg solution at 4°C. Plug-flow bioreactors Several plug-flow bioreactors were made as water jacketed methacrylate columns (2.5 cm inner diameter (i.d.), 8 cm height (h), design ratio (i.d./h) 0.31). Each reactor was packed with support particles (7 g) loaded with immobilised cells and BSM-Mg and decane or hexadecane (1 % v/v) was continuously pumped (1 mL/minute) in ascending mode with total recirculation. Samples (2 mL) were removed from the top of the bioreactor and supplemented with internal standard (n-undecane or n-pentadecane, 5 pL), and extracted by shaking with diethylether (2 mL) for 1 hour. Diethylether layers were analysed using gas chromatographic (GC) techniques to determine residual n-alkanes. Controls were performed using support particles without immobilised cells. Biodegradation assays in batch mode

a)

BSM-Mg (10 mL) and n-alkane (100 were placed together with polyurethane immobilised cells (1.5 g, 5 x 5 x 5 mm particles) in flasks (50 mL) and covered with cotton stoppers. Considering that 1 YOv/v n-alkane is a higher alkane concentration than their solubility in water, uniform concentrations were ensured by constant shaking at laboratory temperature. At intervals, the entire contents of the flasks (liquid with polyurethane foam and cells) were extracted with diethylether (10 mL) after addition of internal standard (20 G). By this procedure, all portions of alkanes, even those adsorbed onto polyurethane foam and/or flask walls, were extracted into ether and their concentration measured by GC, thus allowing estimation of the real concentration of n-alkane in the system. This was confirmed by extraction of known amounts of n-decane added to polyurethane foam. Repeatability of the assays was within f 5 YO.

Kinetics of biodegradation of n-alkanes

401

n-Alkane analysis Quantitative analysis was pedormed using a Labio GC 92 (Labio, Czech Republic)

GC equipped with a flame ionisation detector (FID). Separation was achieved using a fused silica capillary column NB-20M (0.32 mm id. x 50 m, HNU-Nordion, Finland) with a temperature gradient program of 70°C (for 120 seconds) increasing at lO"C/minute up to 240°C. Nitrogen was used as the carrier gas at 25 mWminute flow rate, and the injection port and detector tempetatures were 240°C and 25OoC, respectively. Quantification of individual hydrocarbons was made by peak area measurement using a CSW integrator, and by comparison with standards.

Curve fitting Non-linear regression of data on remaining substrate (n-alkane) concentration versus time was performed using the EXFIT program in the SIMFIT software package.

RESULTS & DISCUSSION Pseudomonas C 12B bacteria were immobilised by physical entrapment in reticulated polyurethane foam and evaluated for stability and suitability for large scale use, with a view to obtaining an appropriate operational biodegradative activity towards n-alkanes.

Characteristics of growth of biodegraders Pseudomonas C12B displays optimum growth on the mineral medium with n-decane (1 % v/v) as a sole source of carbon 6 ; strain A3 on mineral medium with n-hexadecane (1 % v/v) 15. Under optimum growth conditions strain A3 grew considerably quicker than strain C12B, due to the long lag period of the latter Is. However, the actual growth rates during the exponential period were similar (0.25 hour-'). Pseuahmonas cells entrapped in reticulated polyurethane foam

Preliminary assays with Pseudomonas bound to commercial polyurethane foam by passive immobilisation showed that cell loading was not very high and some washout of cells occurred. This was shown by scanning electron micrographs of exposed surfaces15. However, a sigdicant degree of n-alkane removal was observed. This simple method of immobilisation was discarded in favour of cell entrapment due to the higher degree of cell loading and repeatability of immobilisation of the latter. Cell entrapment within polyurethane foam has other advantages including the facility to choose the size, chemical and physical resistance, stability ;and firmness of particles.

Sorption of n-alkanes & biodegradative activity of entrapped cells Long kinetic runs (up to 6 days) were camed out, testing the remaining n-alkane in water versus time of operation of the recirculation bioreactor packed with polyurethane reticulated foam particles with and without (controls) Pseudomonas entrapped cells (flow rate 1 mL/minute, pH 7.1, known weight of supporl, initial n-alkane concentration 50 mM). Figs. 1 and 2 show the n-hexadecane and n-decane removal kinetics for the cell loaded reticulated polyurethane foam, respectively. The decrease of substrates in experiments where no biodegraders are present is due to the volatility of the n-alkanes.

Bioengineering of new materials

402

I

o cell-free foam

I I

A cell suspension

1

0

3

2

4

5

I

6

time (days) Figure 1.

Remaining n-hexadecane in the presence of immobilised Pseudomonas A3 cells and of foam without cells. [Protein conc., 3.9 mg of protein per g of foam; weight of foam, 1.5 g per flask; original concentration of n-alkane, 10 pUmL.1

10 9

8

T

0 cell-free foam

E 7

l

3

6

a , 5 C

Q

0

4

$ 3 C

2 1

0 0

2

1 -

Figure 2.

~

3

4

5

time (days)

~

_

~

_

~

7

6 _

~~~

~._

Remaining n-decane in the presence of immobilised Pseudomonus C 12B cells and of foam without cells [Protein conc , 3 9 mg of protein per g of foam, weight of foam, 1 5 g per flask, original concentration of n-alkane, 10 &/mL ]

lnetics of biodegradation of n-alkanes

403

n-Decane and n-hexadecane are both relatively volatile, thus part of their decrease during the experiment is due to evaporation (5 mg n-decane was evaporated within 10 minutes from an interfacial area of 10 cmz under similar experimental conditions as described for biodegradation assays). This can be clearly seen fiom Figs. 1 & 2 where next to experiments with immobilised cells also experiments with polyurethane foam without cells are shown. Adsorption is excluded (see 'Bidepation assays in batch mode ' section in 'Materials and Methods 7. Fig. 3 shows the decrease of n-decane and n-hexadecane as a function of time in the presence of immobilised Pseudomonas sp. cells. It is evident that in 4 days 99% of originally present alkane has been removed via a combination of evaporation and biodegradation. The level of biodegradation participation can be estimated for a particular length of time from the data for the control foams (without immbolised cells) displayed in Figs. 1 and 2. Therefore, after 4 days 20 YOof n-decane, and 3 5 'YOof n-hexadecane, are removed by biodegradation. More detailed investigations and discussion of results relating to this topic are presented elsewhere 15. From the data collected during the experiments detailed in this research it may be concluded that the n-alkane-removing activity of these immobilised biocatalysts is rather effective. Mathematical analysis (curve fitting) of the data revealed that the overall kinetics are well fitted to a biphasic process. Firstly a rapid sorption step of the substrates onto the cell-loaded suppcrt [I], followed by the intrinsic primary biodegradation slower step [Z],both acting synergistically:

-

-

(In-alkane]

=

+

Adkl'

(kl

=

0.092 min-',

(kz

=

4.9 x lo4 min-', (t%)Z

(t%), = =

Ae'kzf

+

C)

7.5minutes) .....................

[l]

23.6 hours) .....................

[2]

I -100%

80% E

0 ._ U

x

60%

W 0

40%

0

L

5 20%

0% 1

2

3

4

5

time (days) -__

Figure 3.

Biodegradation of n-hexadecane and n-decane by immobilised cells Pseudomonas A3 and C 12B, respectively. [Data calculated fiom the results presented in Figs. 1 & 2.1

6

404

Bioengineering of new materials

CONCLUSIONS

As a consequence of the simultaneous and synergistic action of both adsorption and biodegradation of n-alkanes by Pseudomonas cells immobilised in polyurethane reticulated foam, an action which provides enhanced kinetics of removal of the substrate, and in view of the low cost and ease of synthesising the support, the system studied demonstrates potential for efficiently removing these characteristic pollutants from the environment. ACKNOWLEDGEMENTS

The authors acknowledge financial support from the European Union (Third Framework Program 1990-1994 R&D, PECO program for Cooperation in Science and Technology with Central and Eastern European countries; contract CIPA CT-3020). REFERENCES 1. 2. 3.

4.

5.

6. 7.

8. 9. 10.

11. 12.

13.

14. 15.

R. M. Atlas, ‘Microbial degradation of petroleum hydrocarbons: an environmental perspective’, Microbiol. Rev.,1981, 45, 180-209. P. W. Kirk, ‘Direct enumeration of marine arenicolous fungi’, Mycologia, 1983; 75, 670-682. J. G. Leahy & R. R. Colwell, ‘Microbial degradation of hydrocarbons in the environment’, Microbiol. Rev., 1990, 54, 305-3 15. P. W. Kirk, B. J. Dyer & J. Noe, ‘Hydrocarbon dlisation by higher marine fungi from diverse habitats and localities’, Mycologia, 1991, 83, 227-230. J. F. Kennedy 62 M.G. Roig, In: Handbook of Enzyme Biotechnology, A. Wiseman (ed.), 1995, Ellis Horwood, Chichester, pp. 235-310. J. Kostil, M. Mackovi, J. Pazlarola & K. Demnerova, ‘Alkane assimilation ability of Pseudomonas C 12B originally isolated for degradation of alkylsulphate surfactants’, Biotechnol. Letts., 1995, 17, 765-770. J. J. Cooney, S . Wuertz, M.M.Dooiittle, M. E. Miller, K. Henry & D. hcca, In: Trends in Microbial Ecology, R. Guerrero, R. and C. Pedros-Alio (eds.),1993, Spanish Society for Microbiology, Barcelona, p. 640. F. Aeckersberg, F. Back, & F.Widdel, ‘Anaerobic oxidation of saturated hydrocarbons to C02by a new type of sulphate reducing bacterium’, Arch. Microbiol., 1991, 156, 5-14. A. Rosevear, In: Molecular Biology and Biotechnology, J. M. Walker and E. B. Gingold, (eds.),1988, Royal Society of Chemistry, London, pp. 235-258. M. G. Roig, M. A. Pedraz & J. M. Sanchez, ‘Sorption isotherms and kinetics of the primary biodegradation of alkyl sulphate and linear alkyl benzene sulphonate surfactants by immobilised Pseudomonas C12B’, Appl. Catal. B: Environ., 1999, in press. Z. Wirpsza, In: Polyurethanes - Chemistry, Technology and Applications, Ellis Horwood, Chichester, 1993, pp. 60-68. G. F. White & 0. R. T. Thomas, ‘Immobilisation of the surfhctantdegrading bacterium P. C12B in polyacrylamidegel beads’, Enzyme Microb. Technol., 1990, 12, 697-705. H. M. Pinheiro & J. M. S . Cabral, ‘Activity and stability of an entrappedcell system for the A’dehydrogenation of steroids in organic media’, Enzyme Microb. Technol., 1992, 14, 619-624. C. M. Stoscheck, In: Guide to Protein PurrJication, M.P. Deutshcer, (ed.), Academic Press, London, 1990, pp. 50-68. H. Jerabkova, M. G. Roig, B. Kralova, J. M. Sanchez, M. A. Pedraz, J. F. Kennedy & C. J. Knill, Biodegradation of n-alkanes by immobilised Pseudomonas sp. cells: a kinetic investigation,J. Chem. Technol., 2001 (in preparation).

-BIOCOMPATIBLE ASPECTS OF POLY(2-METHOXYETHYLACRYLATE) (PMEA)THE RELATIONSHIP BETWEEN AMOUNT OF ADSORBED PROTEIN, ITS CONFORMATIONAL CHANGE, AND PLATELET ADHESION ON PMEA SURFACE Masaru Tanaka', Tadahiro Motomura', Miho Kawada', Takao Anzai', Yuu Kasori', Toshifumi Sbiroya', Kenichi Shimura',Makoto Onishi', Akira Mochizuki' & Yoshio Okahata2 'Research and Development Center, Terumo Corporation, 1500 Inokuchi, Nakai-machi, Ashigarakomigun, Kanagawa 259-0151, Japan 'Department of Biomolecuiar Engineering, Tokyo h i i t u t e of Technology, Nagatsuda, Midori-ku, Yokohama226-8507, Japan

ABSTRACT Poly(2-methoxyethylacrylate) (PMEA) surface suppresses platelet adhesion and spreading when compared with other polymer surfaces. To clarify the reason, the relationship between the amount of the plasma protein adsorbed on PMEA, its secondary structure and platelet adhesion were investigated. Poly(2hydroxyethylmethacrylate) (PHEMA) and polyacrylate analogous were used as references. The amount of the protein adsorbed on PMEA was very low and almost equal to that of PHEMA. Circular dichroism (CD) spectroscopy was applied to examine the changes in the secondary structure of the proteins, resulting fiom adsorption on the polymer surface. The conformation of the proteins adsorbed on PHEMA changed considerably, but that adsorbed on PMEA differed a little fkom the native one. These results suggest that low platelet adhesion and spreading have a close relation to the low degree of denaturation of the protein adsorbed on PMEA. PMEA is a promising material to produce for blood contacting surfaces for medical devices. Keywords: Poly(2-methoxyethylacrylate) ;protein adsoiption; conformational change; platelet adhesion; blood compatibility

INTRODUCTION Foreign material which comes into contact with blood will rapidly adsorb proteins onto its surface, and the adsorbed protein layer determines all further events in platelet adhesion, aggregation and coagulation (1-3). Therefore, to understand the mechanism of protein adsorption is very important for surface design for excellent biomedical materials. Protein adsorption on various kinds of polymer surfaces has been investigated extensively, and a lot of work have focused on the development of synthetic materials for blood compatible devices (4). Several studies suggest that both the amount of

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adsorbed protein and the conformational change play an important role in platelet adhesion. In particular, the conformational change influences the functions of proteins itself. Thus, determining the degree of conformational change in adsorbed protein is considered to be one of the important aspects affecting blood compatibility (5-7). Fibrinogen (FNG) is one of the adhesive proteins as well as fibronectin, von Willebrand factor, and thrombospondin, to regulate or modulate the adhesive response of platelets. FNG has been well investigated as the mediater of platelet adhesion via direct interactions with platelet receptors such as glycoprotein GPIIahIIb. Several groups reported a monoclonal antibody that binds to the hnctional region against the platelet receptor (e.g. RGDS or C-terminal dodecapeptide of the chain) ,reacting with

FNG adsorbed on a surface, but not reacting with FNG in solution (8,9). They concluded that the conformation of FNG adsorbed on the surface is changed. Moreover, other studies suggest that not only the total amount of adsorbed FNG but also the conformation or orientation of adsorbed FNG play an important role in determining the platelet adhesion on biomaterials (10,11). In order to produce a novel surface for artificial organs such as artificial lung, we newly prepared a poly(2-methoxyethylacrylate) (PMEA) surface. The advantageous properties of PMEA for biomedical applications are mentioned as follows; blood compatibility, low toxicity, adhesive property, economical production by radical polymerization on large scale, easiness to copolymerize, and easiness to control quality. However, we have not proven the reason for blood compatibility of PMEA yet. In this study, to understand the reason for blood compatibility of PMEA, we investigated the amount of plasma protein adsorbed on PMEA. We focused primarily upon the relationship between the protein adsorption and the platelet adhesion behavior on PMEA surface. The degree of conformational change of the adsorbed protein on the surface was estimated by determining the a -helix content from the CD measurements. We report here the conformational change of serum albumin and FNG adsorbed on PMEA surfaces and on the various analogous poly(meth)acrylates polymer surfaces. EXPERIMENTAL METHODS Materials Various poly(meth)acrylates were prepared by radical polymerization using azobisisobutyronitrile (AIBN) as an initiator. The monomers used were 2methoxyethylacrylate (MEA), ethylacrylate (EA), 2-phenoxyethylacrylate (PEA), 2hydroxyethylmethacrylate (HEMA), 2-hydroxyethylacrylate (HEA), and 2ethylhexylacrylate (EHA). Methanol (MeOH) and tetrahydrokan (THF) used as solvents were analytical grade. Human plasma fibrinogen (FNG), was purchased from

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30 I

PPEA

PEHA

PEA

PMEA PHEMA PHEA

Figure 1. Number of platelets adhered to poly(meth)acrylates

Sigma Co. Ltd., (USA). Protein solutions were made up in phosphate buffer (PBS, pH7.4). The test polymers (PMEA, PHEMA, PHEA, PEA) were cast fiom O.lwt% MeOH solution, and PPEA and PEHA were cast from O.lwt?? 'THF solution onto PP or PET plates, dried under air at room temperature, and then dried in vacuo for 24h. The wettability of polymer surfaces was characterized by contact angle measurement. For CD spectroscopy measurement, a quartz plate (40mm X 9.5mm X lmm) was used instead of polymeric substrate. PMEA and PHEMA were cast from O.lwf?? MeOH solution, and PEHA was cast from O.lwt?! THF solution, followed by drying in vacuo for 24h. Prior to experiment, the plate surfaces were washed three times with PBS .

RESULTS AND DISCUSSION Protein adsorption and platelet adhesion

The number of platelets adhered to poly(meth)acrylate surfaces was shown in Fig. 1. The platelet number on the PMEA surface was least when compared with other polymer surfaces. Fig. 2 shows the comparison of the adsorption of human plasma proteins onto six meth(acry1ate)polymet-s. This figure indicates that the adsorption profiles are different to one another, being influenced by the different nature of the polymer surfaces. The total amount of plasma proteins adsorbed on PMEA was 0.26 fl g/cm2, and was lower than those on PEA, PEHA, PPEA and PHEA. The amount of that adsorbed on

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PHEMA was almost the same as that of PMEA. These results indicate that PMEA and PHEMA surfaces are effective in preventing plasma protein adsorption, compared with other polymer surfaces. The platelet number on PMEA is considerably smaller than that on PHEMA. The platelets adhered on PMEA maintain their original round shape compared with those on PHEMA. The adhered platelets on PHEMA were spreading. These findings indicate that the activation of platelets significantly was inhibited on PMEA but accelerated on P H E W and also indicate that the conformation of adsorbed FNG have a close relation to platelet adhesion behavior. Conformational change of adsorbed FNG FNG is known as a blood-clotting protein and serves as a universal cofactor for platelet aggregation and adhesion. The dissolved, native FNG does not bind to the adhesion receptors of platelets unless the platelet is appropriately stimulated. Whereas unstimulated platelets can adhere to the FNG adsorbed on polymer surfaces, it appears that adsorption of FNG to the surface accentuates and modulates the adhesion receptor and FNG interaction (2). Moreover, platelet adhesion and activation will strongly depend not only on the preliminary adsorption of FNG, but also on the conformational change of the adsorbed FNG (12). In order to provide the support for the hypothesis that the FNG adsorbed on PMEA is a key player in determining platelet adhesion, the state of FNG adsorbed on PMEA was examined by measuring the conformation of adsorbed fibrinogen. The Q! -helical

6.P

1.2

% 0.6

f

s

2

0.4

k

0

PPEA

Figure 2.

PEHA

PEA

PMEA

PHEMA PHEA

Total amount of adsorbed proteins from human plasma onto polymer surfaces.

Poly (2-methoxyethylacrylate) 409

PMEA

PHEMA

I

I

PEHA Native FNG

0

5

10 15 20 25 Q! -Helix content(%)

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Figure 3. Percentage of a -helix in FNG adsorbed on polymer surface and in PBS

content of native FNG in PBS was calculated at 27%. The degree of conformational change of the adsorbed protein was dependent on the property of the polymer surfaces. The a-helix contents decrease from 27 to 22, 8 and 6% for PMEA, PHEMA and PEHA, respectively (Fig. 3). FNG adsorbed on PHEMA and PEHA reduced a-helix content strongly. On the other hand, the adsorbed FNG on PMEA is similar to the native state which will be not recognized by platelets. Results from these observations support the view that the conformation of the adsorbed FNG is an important factor for the adhesion of platelets onto polymer surfaces. These results also support the view that platelet adhesion is dominated by both the conformational change and the amount of the adsorbed FNG. In summary, PMEA surface suppresses platelet adhesion and spreading as compared to other poly(meth)acrylates. These results indicate that PMEA surface performs the minimization of the interactions between the surface and blood. The reason why PMEA shows excellent compatibility for platelet is considered to be due to the fact that it maintains the higher-order structure of the adsorbed plasma proteins. Therefore, PMEA is a useful polymer for surface modification of artificial materials for improvement of blood compatibility.

ACKNOWLEDGMENTS We deeply thank Prof. Dr. Haruma Kawaguchi and Dr. Keiji Fujimoto (Keio university.) for their helphl discussion and support in the use of the CD system. We also thank Dr. Kazuhisa Senshuu, Dr. Noboru Saito, Dr. Naoki Ishii and Dr. Takayuki Kido (Terumo Co.) for their helpful comments.

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REFERENCES 1. J. L. Brash & T. A. Horbett, In: Proteins at interfaces : Physicochemical and Biochemical Studies, vol. 343, J. L. Brash, T.A. Horbett (eds.), 1987, Washington, DC, ACS Symposium Series, pp. 1-33. 2. J. D. Andrade, In: Principles of protein adsorption : surface and interfacial aspects of biomedicalpolymers, J . D. Andrade (ed.), 1985, New York, Plenum Publ, pp. 180. L. Vroman & A. L. Adams, ‘Identification of rapid changes at plasma-solid 3. Interfaces’, J. Biorned. Mater. Res., 1969,3,43-67. 4. Tsuruta T, ‘Contemporary topics in polymeric materials for biomedical applications’,Adv. Polyrn. Sci., 1996, 126, 1-51. 5. T. A. Horbett, In: Biomaterials Science, ‘An Introduction to Materials in Medicine’, B. D. Ratner, F. J. Schoen & J. E. Lemons (eds.), 1996, London, Academic Press, pp. 133- 14 1. 6. T. A. Horbett & J. L. Brash (eds.), 1995, Proteins at interfaces 11. Fundamental and applications. vol. 602, ACS Symposium Series, Washington, DC. 7. H. Nagai, M. Handa, Y. Kawai, K. Watanabe &Y. Ikeda, ‘Evidence that plasma fibrinogen and platelet membrane GPIIb-IIIa are involved in the adhesion of platelets to an artificial surface exposed to plasma’, Thromb. Res., 1993, 71, 467477. 8. E. Shiba, J. N. Lindon, L.Kushner, M. Kloczewiak, J. Hawiger, G. Matsueda, B. Kudryk & E.W. Salzman, ‘Conformational changes in fibrinogen adsorbed on polymer surfaces detected by polyclonal and monoclonal antibodies’, In: Fibrinogen, vo1.3, M. W. Moseson, D.L. Ambani, DiOrio JR. Siebenlist (eds.), 1988, New York, Elsevier Science, pp.239-244. 9. C Zamarron, M. H. Ginsberg, E. F. Plow, ‘Monocronal antibodies specific for a conformationaly altered state of fibrinogen’, Thromb.Haemostas, 1990,64,41-46 10.J. N. Lindon, G. McManama, L. Kushner, E. W. Merrill & E. W. Salzman, ‘Does the conformation of adsorbed fibrinogen dictate platelet interactions with artificial surfaces?’, Blood, 1986,68,355-62. 11. D. Kiaei, A. S. Hoffman, T. A. Horbett & K. R. Lew, ‘Platelet and monoclonal antibody binding to fibrinogen adsorbed on glow-discharge-deposited polymers’, J. Biorned. Mater. Res., 1995,29, 729-39. 12. T. A. Horbett, ‘Principles underlying the role of adsorbed plasma protens in blood interactions with foreign materials’, Cardiovascular Pathology, 1993,2, 137s-148s.

ISOLATION OF A LIGNIN-DEGRADING LACCASE AND DEVELOPMENT OF TRANSF'ORMATION SYSTEM IN CORIOLUS VERSZCOLOR Y.Nittal, Y.Iimura2

,J. Mikuni2, A. Fojimotoz and N. Morohoshi2

Koshida Corporation, 2-2-9Higashi-shinbashi, Minato-ku, Tokyo 105-8642.Japan.

2Graduate School of Bw-Application and Systems Engineering Tokyo University of Agriculture & Technology. 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan.

ABSTRACT A White-rot fungus Coriolus versicolor is a typical lignin-biodegrading fungus and secretes laccases. We could isolate three laccase fractions, Laccase I, I1 and 111 by gelfiltration, ion-exchange, carboxymethyl cellulose and hydroxyapatite chromatographies. These three laccases were characterized by substrate specificity for several simple phenols, electrophoresis patterns, ultraviolet spectra, electron spin resonance spectra, copper content, immunological similarity, and the biodegradability of several lignin model compounds, wood meals and thin cross-sections of woods. It was clear that laccase 111 is the most abundant secreted isozyme among them and is involved in the depolymerizationof lignin. We obtained a candidate gene of laccase 111 from the cDNA library. The genomic library of C. versicolor was screened with the cDNA probes of laccase 111 and two positive clones were selected. One was a laccase gem: encoding laccase 111, another clone was an unknown laccase gene. Finally, we succeeded in the isolation of the Laccase I11 gene. We tried to establish the transformation system by the electroporation method to make a new C.versicolor secreting more laccase 111. Protoplasts were prepared from the mycelia harvested from C. versicolor. Green Fluorescent Protein Vector (GFP)was used as a marker gene to determine the efficiency of transformation. Finally, the introduction of foreign genes by electroporation was confirmed by fluorescent microscope and these transformants could be screened on media containing G418.

INTRODUCTION A lignin-biodegradation system of the fungi, Basidiomycetes contains non-specific oxidative reactions catalyzed by phenoloxidase. Since white-rot fungi have a system for the degradation of lignin, it has been proposed that these fungi have to be useful for bioremediation and bioconversion of raw materials. A white-rot fungus, C. versicolor can typically degrade lignins. Its lignindegradation system consists of non-specific oxidative reactions catalyzed by multiple isozymes of phenoloxidases. We assumed that the ligninolytic systems of C.versicolor can also degrade a variety of aromatic pollutants. Laccase, a kind of phenoloxidases, was purified from C. versicolor and finally laccases I, I1 and I11 enzyme fractions were isolated. There laccases were characterized by substrate specificity for several simple phenols, electrophoresis patterns, ultraviolet spectra and copper content [l], and it was shown that laccase I11 is the most abundant secreted isozyme among them and plays an important role in the degradation of lignin. We have cloned and sequenced a gene coding for the ligninolytic laccase I11 and the enzyme activity of laccase 111 for lignin degradation was investigated by the method of ultraviolet microscope. In this study, we also tried to establish a transformation system by the electroporation method to make a new C. versicolor secreting more laccase 111. Protoplasts were prepared from the mycelia harvested from C. versicolor. Green Fluorescent Protein Vector (GFP)constructed by use of a fluorescent protein derived from Aequorea

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,

Pvu I I /

AlwNl

,

. Bsr GI

\

Fsp I Aat II Figure 1. pEGFP (Green Fluorescent Protein (GFP) Vector, blue-SGFP-TYG-nos SK) victoria was used as a marker gene to test the efficiency of transformation. The introduction of the foreign gene by the electroporation was confirmed by fluorescence microscopy, and the transformants were screened on media containing G418 (Geneticin) resistance. Finally using this approach, we will be able to understand the lignin degradation mechanism in this fungus precisely.

MATERIAL AND METHOD Isolation of laccase We fractionated crude laccase fractions and isolated three pure enzyme fractions. C. versicolor was incubated in the glucose-peptone medium including copper. An extracellular crude enzyme was isolated from the culture medium by precipitation with ammonium sulfate (90% saturation) and subsequently fractionated by gel-filtration using Sephadex G-50. A high molecular weight fraction having phenoloxidase activity was isolated by gel filtration and subjected to anion-exchange chromatography using DEAE TOYOPEAL 650M [I].

Properties of lignolytic laccase 111 Preparation of cross-sections of wood samples Cross-sections (OSmicrometer thick of beech) were dehydrated with ether and acetone. Samples were soaked in acetone-epoxy resin over night and were treated for 28 hrs at 40,50 and 60 C. Samples were washed with 28% sodium methylate: benzene: methanol ( 2 5 3 ) solution for 40 min and benzene: methanol (1: 1) solution for 10 min, and finally with water. Treatmentfor wood samples with laccase III

Lignin-degrading laccase

413

Tube number (8ghbe) Figure 2.

Fractionation with DEAE TOYOPEAL 650.

Samples were treated with laccase 111 in sodium acelate buffer (pH 4.0) for 48 hrs at 30 C. After that, they were washed with sodium acetate buffer (pH 4.0), dried and observed by ultraviolet microscopy (280nm).

Isolation of laccase I11 gene Analysis of laccase genes in C. versicolor Genomic DNA was isolated according to the procedure of Yelton et al. [2], digested with various restriction enzymes and analyzed by Southern blot hybridization with a 32P-labeled Laccase I11 cDNA fragment as a probe.

Construction of a genomic DNA library from C. versicolor, isolation of laccase gents and determination of nucleotide sequence High molecular weight genomic DNA was isolated from frozen mycelia. The genomic DNA was partially digested with Sau 3A1, and then ligated with Bam HI site into AEMBL-3. The genomic library was constructed by transfection of E.coli €2392 with the packaged mixture. The genomic hEMBL-3 library was screened by plaque hybridization. DNA fragments were isolated corresponding to the genomic DNA inserts of phage DNAs from positive plaques and subcloned into pUC19. DNA sequencing was canied out by the dideoxy-chain termination method.

Transformation Preparation ofprotoplasts The fungal strain used in this study was C. versicolor (FES1030 (IF 08753)). This fungus was grown at 28C in potato dextrose agar (PDA) plate. After plate culture for one week, the mycelia were moved into a glucose-peptone medium [glucose 30g, peptone log, KH2PO4 1.5g, MgSO4-7H20 5OOmg, thiamine hydrochloride 2mg,

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A

B

C

Figure 3.

Cross-section photograph by ultraviolet microscope. A:Before enzymatic treatment B:Laccase I treatment C:Laccase 111 treatment

CuS04-5HzO 16mg /1] and incubated at 28C for 3 days. C. versicolor protoplasts were prepared by using a modification of the procedure of Binninger el aZ[3].The cultured C. versicolor mycelia were harvested and washed with 0.05M maleic anhydride - NaOH buffer solution (pH 5.5) containing 0.5M mannitol (MMbuffer) by centrifugation (1,500g for l h i n at room temperature). The harvested mycelia were treated with an enzymic solution containing 2% Yatalase, 0.5% Uskizyme, 0.2% Chitinase and 0.1% Cellulase Onozuka R-10 in MMbuffer at 30C for 2h with filtered shaking (60rpm). One hundred mg mycelia were treated with lml. After the treatment, the suspension was filtrated through Mira-Cloth and washed with MMbuffer by centrifugation (1,500g for lOmin at room temperature). The precipitate was added to about 3ml of 0.5M sucrose and the solution centrifuged (1,500g for lOmin at room temperature). A white layer under the interface appeared in the centrifugation-tube. It was isolated and called protoplasts. Electroporation Electroporation was used to introduce foreign genes. Protoplasts were washed in electroporation buffer [5mM MgC12,70mM KCl, 0.1% MES, 0.5M mannitol] twice

Lignin-degrading laccase

Figure 5.

415

Construction of plasmid for transformation.

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and resuspended in the same buffer. The suspension (approximately 6 8 X 106/ml) was mixed with 30 microgradmi of blue-SGFP-TYG-nos SK (pEGFP, Fig.1) and kept on ice for l0min. The suspension was transferred to electroporationcuvettes having 0.2cm interelectrode distance and 120 microliter sample capacity, and electroporated (6kV/cm). The treated mixture was chilled on ice for l0min. The apparatus used for electroporation was Gene h l s e r I1 (BIO RAD). The protoplast-electroporation procedure was carried out according to the method of Ward et al. [4]. The introduction of marker genes by electroporation was confirmed by fluorescent microscopy and screened on medium containing G418.

Improvement of the transformation plasmid In order to integrate the promoter, terminator regions and restriction sites of pEGFP, this EGFF site was inserted into pLFT as an expression vector.

Regeneration The suspension of protoplasts was plated on OSSMY medium [OSM sucrose, 1% malt extract, 0.4% yeast extract, 0.7%low gelling agar] containing G418 (25mg/l) for 3 to 4 days at 28C.Wild protoplasts were 50,OOO per plate.

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n

b

n

Figure 6.

Protoplast after electroporation.

RESULTS A ND DISCUSSION We isolated three laccase fractions, laccase I, I1 and 111 (Fig.2). These three laccases were characterized, and their influence on the biodegradation of thin wood crosssections was invertigatede. In the cross-section treated with laccase 111 (Fig.3-B), there was slightly higher absorbance compared with control. In other hand, the cross-section with laccase 111 was broken into a woody tissue structure and the absorption at 280nm was remarkably decreased. These results show that the function on the lignin

Figure 7.

After electroporation, protoplast by fluorescence microscopy photograph.

Lignin-degrading laccase

417

biodegradation between laccases 1 and 111 is completely different. Laccase I doesn’t degrade the protolignin, but laccase I11 can do. Laccase 111 might have some characteristic enzyme functions, for example the depolymerization of lignin high polymer and the solubilization of degraded lignin fragments having more hydrophilic functional groups caused by chemical oxidation by laccase 111. It is concluded that laccase 111 is involved in the lignin biodegradation and laccase I is not. This fact supports our result obtained by the chemical analysis on the distribution of molecular weight previously. We have cloned laccase I11 gene. The nucleotide sequence of the gene and the deduced amino acid sequence are shown in Fig.4. The deduced amino acid sequence is completely consistent with the partial amino acid sequence of the NH2 terminus of the purified laccase 111, determined by the Edman degradation method [5].The nucleotide sequence of the laccase 111 gene is also different to that of another laccase gene isolated from C. versicolor. Concerning the regeneration of protoplast, the rate of regeneration of nontransformed protoplast was 0.004 1.7%, which is similar to the results obtained from other fungi. The rate of the transformed one is 0.0003 --O.o006%. The introduction of the marker gene could be confirmed by Fluorescence microscopy (Fig.7,8). Therefore, we could construct a system to integrate the foreign gene into C.vmsicolor, though the regeneration rate of transformants is low. We could construct a new plasmid integrated GFP into pLpT having promoter and terminator regions. C. versicolor was transformed with the constructed plasmid and could be regenerated on the culture medium containing G418. These results show it is possible to make useful transformant having higher lignin degrading ability.

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REFERENCE 1. N.Morohoshi, ‘Laccases of ligninolytic fungus Coridus versicolor’,American Chemical Society, 1991,207-224. 2. M.Yelton, J.E.M.Hamer and WETimberlake, ‘Transformation of Aspergillus nidulam by using a trpC plasmid’, Proceedings of the National Academy of Science, 1984,81,1470-1474. 3. D.M.Binninger, C.Skrzynia, P.JPuMla and L.A.Casselton, ‘DNA-mediated transformation of the basidiomycete Coprinus cinereus’, The EMBO Journal, 1!387,6, 835-840. 4. M.Ward. L.J.Wilson, C.L.Carmona and C.Turner, ‘The oliC3 gene of Aspernillus niger :isolation, sequence and use as a selectable marker for t&nsforma&on’,Curr. Genet. , 1988, 14,3742. 5. Y .Iimura, K.Takenouchi, M.Nakamura, S.Kawai, Y .Katayama and N.Morohoshi, ‘Cloning and sequence analysis of laccase genes and its use for a expression vector in Coriolus versicolor’,International conference on biotechnology in the pulp and paper industry, 1992.

EFFECT OF BIODEGRADABLE PLASTICS ON THE GROWTH OF ESCHERICHU COLI A Nakayama', N Yamano', S Fujishima', N Kawasaki', N Yamamoto', Y Maeda' and S Aiba' 1 Osaka National Research Institute, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan 2 Industial Technology Center of Wakayama Prefecture. 60 Ogura, Wakayama 6496261, Japan

ABSTRACTS In order to make sure of biological safety of biodegradable polymers before and after use, the growth of Escherichia coli K-12 in the existence of biodegradable polymers or their degraded products was investigated. E. coli has a worldwide distribution, therefore it is an appropriatemicrobe as an environmental indicator. Among the series, E.coli K12 was used because its genetic and biochemical characterizationswere wellknown. The tested samples were copolyesters, copolyesteramides, copolyesterethers, and their oligomers and monomers. In the case of a paper-disc method as a screening test, the samples were solved in DMSO (0.01 %(w/v) to l%(w/v)), and the DMSO solution was added on a paper disc on the surface of agar on which E.coli was inoculated in advance. After over-night incubation, most of monomers showed inhibitory zones in the case of 1% solution, however no monomers showed the zone at 0.01%. Furthermore, most of copolymers did not show the zone even at 1%. Relationships between the results obtained in DMSO and those in fresh water has been also studied from the viewpoint of polymers or degraded products concentration to evaluate the safety. When using Bacillus subtilis (gram-positive) as an indicator, a similar tendency was observed.

INTRODUCTION Most synthetic plastics are inert toward micro-organisms in the form in which they are initially produced. The long-term properties of synthetic and natural polymers have attractedmore interest during recent years as environmental concern has increased1S2. Biodegradable polymers have been studied in an attempt to solve the problems of waste management for conventional plastics3-'. Most of researchers in this field have been studying new synthetic methods, syntheses of novel polymers, physical properties, morphology, biodegradability, biodegradationfactors and biodegradation mechanism. As a result, biodegradable plastics are expected to be used in a variety of fields. However, there is a restricted number of reports on safety assessment of biodegradable plastics. For this purpose, degradation mechanism or identification of degraded products should be studied8-' l . Before mass production / consumption of biodegradable plastics, it is necessary to estimate the acceptable accumulation mass level of plastics and their degraded products, since a certain amount level of polymers and degraded products might be stay during a limited period in the environment. If they are beyond a threshold of the accumulation level, they might be show harmful effect on

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Bioengineenng of new materials

environmental livings including microorganisms. In this paper, we investigate the safety of biodegradable polymers and degraded products toward environmental microorganisms using Escherichia coli.. The knowledge as for a concentration factor could be referred to one of a standard concerned about the acceptable accumulation level of polymers or degraded products in environment and it would be useful to establish a method of safety assessment as for biodegradable polymers.

EXPERIMENTAL The samples tested were polyesters (ex. poly(L-lactide), poly(ecaprolactone), copolyesters containing Llactic acid unit, copolyesters having long methylene chain, copolyesters having side chain), copolyesterethers ( poly(succinic anhydride / ethylene oxide), poly(succinic anhydride / ethylene oxide / propylene oxide)), copolyesteramides ( copoly(l1actic acid / E-caprolactam)), polyethers ( polyethylene glycol, poly(ethy1ene oxide-block-propylene oxide)), and their oligomers and monomers ( Llactic acid, sodium L-lactate, L-lactide, dicarboxylic acids, w -hydroxyl carboxylic acids, succinic anhydride, succinic acid, sodium succinate, w-amino carboxylic acid, ethylene glycol, ethylene glycol oligomers). These samples and medium were sterilized by autoclaving (121°C, 20 min) or ethylene oxide gas. As a screening test, a paper-disc method (PD method) was used. On a surface of LB agar medium, pre-culture Escherichia coli K 12 or BacilZus subtilis IF0 13719 was inoculated. Paper discs (5mm0) were lined up on the surface of the agar in a petri dish. The samples were solved in DMSO (0.01 to 1% (w/v)), and 10111of the DMSO solution was added on the paper discs. After over-night incubation, the agar became opaque with the growth of E.coli, however, when a tested sample was shown harmful effect on E.coli growth, an inhibitory mne around the sample was observed. The samples which recognized toxicity were studied the E.coli growth in a LB solution in the presence of the sample (LB method). Samples were suspended in LB medium (peptone 1%, yeast extract 0.5%, NaCl 1%) with a certain concentration. After inoculation of E.coli, incubation was carried out for a certain time at 37 "C with shake of 120 rpm, then the growth was evaluated by absorbance at 600 nm of the LB culture. In the case of polymer sample, the growth was evaluated by count colonies which were incubated on agar plate after dilution (los - lo6).

RESULTS AND DISCUSSION

Screening results by PD method Most of polymers were soluble in DMSO around lwt%, and they were insoluble in water. Therefore, it seems to be difficult that polymer samples diffuse in a agar well after immersion of DMSO solution to a paper disc. However, it is not a serious matter because these phenomena are not so different from a natural environment. The results of screening test of the polymers were shown in Table 1. Harmful effect was not observed for most of polymers with any concentration besides a few exception. Copolymers of Nos.3 and 7 in Table 1 showed positive (+) with l%(w/v). These results seems to be dependent upon a small amount of propiolactone or polypropyleneglycol as a

421

Effect of biodegradable plastics on escherichia coIi

Table 1 Screening results of polymers by paper disc method Conc. of DMSO soln. (%,w/v) Run Samples no. 0.01 0.1 1 I

Copolyesters

1 2 3 4

pol y (e-caprolactone) poly(l1actide) copoly(p-propiolactone/llactide)

-

copoly(glycorideb1actide)

-

-

-

Cop1y esterethers copoly(SNethy1eneoxide)

2 -

-

+ k

6

copoly(SNethy1eneoxide)-block-PPG

-

k

7

copoly(SNethy1eneoxide)-block-PLN

-

+

-

-

-

-

k -

5

8

Copolyesteramides copoly(l1actic acidb-caprolactam)

-

Polyethers 9

polyethylene glycol

10 11

PLN copoly(L1actic acid/€-caprolactam)

-

+ -

SA :succinic anhidride PPG :polypropylene glycol PLN : polyethylene glycol-block-polypropyleneglycol (-A) contamination. It is difficult to evaluate the effect with higher concentration because of their limited solubilities to DMSO. In the cases of monomers and oligomers, not a few samples showed inhibitory effect. LLactic acid, e-hydroxy caproic acid dimer, eicosa dicarboxylic acid, ethylene glycol (EG), propylene glycol, EG trimer, EG tetramer showed considerable inhibitory zone with l%(w/v), and some of them showed the zone even with O.l%(w/v). The reason is not clear that EG dimer did not show the effect. 2,4-Dichlorophenoxyacetic acid (2,4-D) is well-known as a herbicide, and it is noteworthy that even O.Ol%(w/v) of 2,4-D inhibited the E.coli growth. From these results, a definite tendency could not find from the view point of chemical structure, for example hydroxy carboxylic acid, amino carboxylic acid, or diol etc. Therefore, these results seem to become one of guideline for an evaluation of safety assessment for biodegradable plastics. Nevertheless 8. subtilis is gram positive, the screening results were a similar tendency to those of E. coli which is gram negative. ‘The differences are 1) polymers which effect the E.coli growth in some cases (Nos. 28 imd 29 in Table 3) did not show any effect up to lwt%, 2) most of monomers (Llactic acid, sodium succinate, e-hydroxy caproic acid dimer, ethylene glycol trimer, and tetramer) showed a similar tendency to those of E-coli, however €-aminocaproic acid and ethylene glycol were opposite results. We thought that this method is effective to examine the safety assessment of biodegradable polymers for environmental microorganisms.

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Bioengineering of new materials

Table 2 Screening results of monomers and oligomers by paper disc method Conc. of DMSO soln. (%,w/v) Run Samples 0.01 0.1 1 no. Hydroxy carboxylic acids 12 Llactic acid +_ 13 s-hydroxy caproic acid dimer k o-hydroxy hexadecanoic acid 14 Dicaroxylic acids 15 succinic acid sodium succinate 16 succinic anhydride 17

++

++

18 19 20 21 22

eicosa dicarboxylic acid Amino carboxylic acids y-aminovaleric acid E-amino caproic acid Diols ethylene glycol (EG) propyrene glycol

23 24 25

+

++

-

-

-

k

k k

-

EG dimer EG trimer EG tetramer

+_

2,4-dichrolophenoxy acetic acid

+

Others

26

-

+_

-

+ + +

+_

++ ++ ++ ++ +

Table 3 Screening results by paper disc method with Bacillus Subtilis Conc. of DMSO soln. (%,w/v) Run Samples no. 0.01 0.1 1 Polymer 27 poly (a-caprolactone) 28 copoly (p-propiolactoneLlactide) 29 copoly(SNethy1ene oxide)-block-PLN 30 poly(ethy1ene glycol)

31 32

Monomers and oligomers Llactic acid sodium succinate

-

-

-

-

-

f

k

++ ++ ++ ++

33 34 35

E-hydroxy caproic acid dimer €-aminocaproic acid ethylene glycol (EG)

Ik

36 37

EG trimer EG tetramer

k

SA : succinic anhidride

+_

k -

+ +

-

Effect of biodegradable plastics on escherichia coli

423

Growth test results by LB method with E.coli PD method found to be convenient for screening, however it has a defect. In PD method, agar medium can regard as a soil in environment. However, DMSO solution is far from real environment, therefore, it is difficult to imagine the concentration of tested samples in environment in PD method. To compare with natural environment, investigation in LB liquid medium was carried out. The suspension or solution of tested samples corresponds to soil water or fresh water of pond or river containing sample, therefore it seems to be of great value for quantitative safety assessment of biodegradable polymers. With E. coli growth, the incubated solution becomes opaque, so the evaluation of the growth was carried out with measurement of absorbance at 600 nm. Table 4 shows the results which normalized by control values. LLactic acid and succinic acid showed obviously the inhibition beyond 0.1%(w/v) of concentration However, sodium Llactate did not show definite effect. LB solution contains 1% of sodium chloride. Therefore, sodium ion does not contribute tht: results. From this reason, the difference of the results between Llactic acid and Llactate seems to be dependent upon pH value. The value is as follows; succinic acid: 10%2.55, 1%3.45, 0.1% 4.43, LB solution 6.51 Llactic acid: 10%2.07, 1%3.07, 0.1% 4.46, 0.01% 5.97, LB solution 6.51. Llactate : 10% 6.97, 1%6.65, 0.1% 6.58, 0.01% 6.57, LB solution 6.51. In PD method, 1%solution of some monomers in DMSO showed the effect more or less (Nos.12-25 in Table 2). On the contrary, in LB method, the effect is clear with 10% of them (Nos. 40-43) except for Nos. 38 and 39 in which pH seems to be effective. The obvious difference of the border concentration of harmful effect between PD and LEi methods may explain that monomers can not diffuse in a large area in an agar medium because of their poor migration rates in the agar compared with LB solution; monomers exist concentrically in short distance from a paper disc. From the tendency, the circumstance as a 1%of DMSO solution in an agar ( PD method) corresponds to that of a 10%concentration in natural environment model ( LEi method ). Therefore, PD method with up to 1%DMSO solution is meaningful method for safety assessment. To verify the Table 4 Results of E.coZi growth test by LB method for 4 hours by absorbance measurement of incubated medium and by colonycount Conc. of samples in LB so h . (%,w/v) Run Samples no. control 0.01 0.1 1 10 Absorbance 38 Llactic acid 1 1.o 0.35 0.23 0.18 39 succinic acid 1 0.67 0.21 0.15 40 sodium Llactate 1 1.1 0.84 0.04 41 E-aminocaproic acid 1 1.1 1.0 0.48 42 ethylene glycol 1.1 1 0.96 0.99 0.33 43 diethylene glycol 1 0.97 0.97 0.91 0.28 Colony count 44 sodium Llactate 1 0.74 0.32 0.18 ethylene glycol 1 0.80 1.0 0.90 0.31 45 diethylene glycol 1 0.76 0.47 0.61 46

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Bioengineering of new materials

results obtained by absorbance of the incubated medium, a part of the incubated medium was cultivated on agar plate after dilution (lo5 - lo6) and the grown colonies were counted (Nos. 44-46). The obtained results showed a similar tendency to those of the former. However, the values were smaller than the corresponded values by absorbance measurement. The reason is explained as follows; 1) the results obtained from colonycount are based on viable cell count, whereas those obtained by absorbance measurement are based on total cell count; 2) the results are ratheramplified in the case of measurement of viable cell count; 3) therefore, the results derived from colony-count were sensitive to concentration of chemicals in medium. To summarize, PD method is worthwhile to screening to examine harmful suspect, however, LB method i s more authentic to consider of sample concentaration in environment. Especially, the evaluation based on colony-count shows viable cell count which seems to reflect on harmful effect of chemicals. However, evaluation by absorbance is convenient and the data are similar tendency to those by colony-count.

CONCLUSIONS From the results of PD method, most of monomers showed inhibitory zone with 1% solution, however no monomers showed the zone at 0.01%. Few polymers showed the mne even at 1%. With Bacillus subtilis (gram-positive), a similar tendency was observed. LB method gave similar results to those of PD method, however, LB method was affected by pH. Therefore, some results were inconsistent with those of PD method.

REFERENCES 1) R. V Wilder, Mod. Plasf. Znt., 19, 74 (1989). 2) G. Scott and D. Gilead, DegradablePolymers, pp.1-87 (1995), Chapman & Hall London (U.K.). 3) R. W. Lenz, Adv. Polym. Sci,. 10, 73 (1993). 4) S . J. Huang, Spec. Pub1.R.Soc. Chem., 109, 149 (1992). 5) 2.Gan, D. Yu, 2. Zhong, Q. LiangandX. Jing, Polymer, 40, 2859(1999). 6) Y Maeda, A. Nakayama, I. Arvanitoyannis, N. Kawasaki, K.Hayashi, S. Aiba, N. Yamamoto, J. Appl. Polym. Sci. , 69 ,303 (1998). 7) G. Yu, F. G. Morin, G. A. R. Nobes and R. H. Marchessault, Macromolecules, 32, 518 (1999). 8) G. W. Fuhs,Arch. Mikrobiol., 39, 374(1961). 9) I.Arvanitoyannis, E. Nikolaou and N. Yamamoto, Polymer, 35, 4678 (1994). 10) F. Kawai, CFC Crit. Rev. Biotech., 6, 273 (1987). 11) K. Sakai, N. Hamada and Y. Watanabe,Agric. Biol. Chem., 49, 1901 (1985).

INDEX absorbants, of toxins 161-166 acetate ester Acetobacterxylinum 261-268, 359364 adsorption, of lignosulphonates 167172 albumin 247 alcohol, fuel 393 alginate 107-112 alginic acid, molecular motion of 321-325 aliphatic polyesters 205-210 alkali cellulosie 118 anion-exchange 382 anion exchange chromatography 366 annealing 149 antisense RNA method 379-386 antibody 388 aspen 379-386 back-based foams 175-180 bacterial cellulose 359-364 banana tree fibres 139-144 Basidiomycetes 387-395, 41 1 bean curd waste 181-190 beer waste 181-190 benzylation 68, 70 -71 BET equation 330,332 biodegradability 21 1 biodegradable, foams 175-180, 227228,229-234,241-246 biodegradable composites 191 -196 biodegradableplastics 419-424 biodegradablepolymers 217-226, 247-256 biodegradable polyurethanes 181190, 197-204 biodegradation9 biodegradation, of n-alkanes 397403 biosynthesis, lignin 379-386 biosynthetic pathways, of bacterial cellulose 359-364 biotechnologicaltechniques 379 bjer kandera adusta 387-395 black liquor 349

bleaching 347-355 blood dotting protein 408 brightness, of pulp 348 calcium binding 82 carbamoylethylation62 carboxyethylation62 carbohydrate reactions 347-355 13Ccarbohydrate polymerslMAS NMR spectrum, of cellulose 261268 catalytic pyrolysis 47-51 cellobiose 365 - 370 cello-oligosaccharides269-274 cellulose 47-51, 113 - 122, 393 cellulose I a 264 cellulose I 264 cellulose, I4Clabelled 359-364 cellulose, 13CN M R 359-364 cellulose, NMR spectra of 261-268 cellulose acetates 269-274,275280, 327-332 cellulose derivatives 53-60 cellulose fibre 167-172, 371 -378 cellulose viscose 113 cellulose-based, poly caprolactones 281-288,269294 cellulosic wastes 67-72 CELSOL 371-378 ceramics, wood 311-315 characterisation, polyamide 27-21 chitin 107 cloning 388 coffeebean237 coffee bean parchments 191-196 coil-helix transition 333-339 composites 139-144,191 -196 composites, polyurethane foam 21 1216 compression modules 238, 239 compression strength 238, 239 compression tests 242- 246 conduction, electronic 17-26 conformational transition 247 -257 copolymerisation 205-210 Coriolus venicolor 41 1-417 corn, seed protein from 247-256

426

Index

CPlMAS NMR 269-274 cuprammonium 371 cyclopentenones 349

decomposition temperature 237 dehydrogenation polymers 391 degradation 3-14 degradation, of polylactic acid 223 degraded surface 346 demecuration stage 352 density, true 276,278 derivatisation of starch 79-90 differential scanning calorimetry 211216, 242-246, 248-257, 276-280, 281-288, 303-310,321-325 diphenylmethane diisocyanate 211, 235-241 dissolving pulp 113-122 dynamic load TMA 342-346 dynamic mechanical analysis 317320 eco-materials 311-315 elastomers 341 electrolyte hydrosol 145-154 electronic conduction 17-26 electroporation 414 endoglucanase 365-370 enzymes 10 enzyme activity 388 enzyme, cellulase 365-370, 371-378 enzyme treatment 371-378 enzymes, ligninolytic 387-395 enzymes, mapping of 379 -386 epoxy resins 73 -78 Escherichia coli 419-424 ESR, on lignin 161-166 exo-glucanase 365-370 exposure test 344 fertiliser 2 11-216 fibre spinning 371-378 fibres 116, 129-138, 139-144, 167172 fibrils 130 fillers 167-172 films, cellulose acetate 275-280, 327-332 films, polylactic acid 218-226 film, zein 247

fluorescent microscopy 416 fluorescent protein 412 foam composites 211-216 foams 175-180 foams, polyurethane 227-228. 229234, 241-246, 397-403 FT-IR spectroscopy 42,63,75 fuel alcohol 393 functional paper 295-302 fungi, lignin degrading 394 fungus, lignin biodegrading 41 1-417 furan 17-26, 27-31 gelatinisation of starch 86 gelation 65 gelation mechanism 333 gelation ratio 345 gellan gum 333-339 gels, polysaccharide 97-104 genes 379-386,414 genetic engineering 379-386 glass transition 212-216 glass transition, of cellulose acetate 279, 327-332 globulin 247 glucose, labelled 261-268 p-D-glucosidase 365-370 glutelin 247 guluronic acid 321-325 heat diffusion 295 hemicellulose 393 heterocycles 17-26 HPLC 366 humidity, relative 318 hyaluronic acid 146 hybridisation 388 hydrogels 97-104 hydrolysis, of lignin 155-160 hydrophilic-hydrophobicbalance 123-128 hydrophobicity, of lignin, 161-166 hydrosol 145-154 hydroxyethyl cellulose 53-60 hydroxyl content 236 hygroscopicity 92-95 immobilisedcell supports 399-403 infrared spectra, of Zein films 250257

Index

ionic polymers 53-60 IR spectra, of pulp 116 isothermal measurements 318 isotherms, sorption 276-280 kappa number 347-355 keratin, wool 91-96 kinetics of biodegradation 397-403 konjac mannan 61-66 kraft pulping 347-355

14Clabelled cellulose 359-364 laccase 387,411-417 lactic acid 217-226 lactide 217-226 levoglucosenone47-51 lignosulphate 167-172 lignin 37 lignin, epoxy resins from 73-78 lignin, structure 132, 155 -160 lignin biosynthesis 379-386 lignin cellulosic biomass 393 lignin degrading enzyme 389 lignin degrading laccase 411-417 lignin hydrolysis 155-160 lignin modification 161-166 lignin peroxidase 387-395 ligninolytic enzymes 387-395 lignocellulose239 luminescence 17-26 manganese peroxidase 387-395 maize seed 248 mannan 61-66 mannuronic acid 321-325 mechanical analysis 245, 246 mechanical properties, of cellulose acetates 275 280 mesoporosity, of lignin 161-166 O-methyl transferase 379-386 methylation analysis 368 molasses, polyurethane foams from 21 1-216, 227-228,229-234, 241246 molecular weight, of gellan 337 nitrolignin 123-120 NMMO method 371 N M R spectra, of cellulose 261-268 N M R spectroscopy 57,269-274

427

13CNMR,analysis of cellulose by 359-364 ‘3C NMR,analysis of saccharides 369 non freezing water 277-280 nylon 6 319 oligomers 17-26 oligosaccharides, synthesis of 367 osmometric studies 333-339 osmotic pressure 334 oxidation of hydroxyethyl cellulose 53-60 oxymercuration stage 352 paper, functional 295-302 paper disc method 420-424 paper grade pulp 371-378 pectins, water restrained by 303-310 pectins, DSC studies on 303-310 peroxidase 379-386 phase diagram, waterlalginic acid 321-325 phase transition behaviour, of pectins 303-310 phase transition 212-216 phenolic polymers 392 phenylalamine ammonia-lyase 379386 phosphoryl polysaccharides 107-112 pineapple waste, polyurethanes from 235-240 plasma protein 405-416 plasmid 415 platelet adhesion 405-410 plastics, biodegradable 419-424 plastiiication of cellulosics 67-72 polyamides 27-31 poly(buty1ene succinate) 205 pofy(capro1actones) 33-46, 281-288, 289-294,420-424 poly(2, 6-dimethyl-l,4-phenylene ether) 341-346 poly(L,-lactide)420-424 poly(2-methoxyethyl (acrylate) 405410 polyelectrolyte 123-128 polyester resin 140 polyesters420-424

428

Index

polyesters, biodegradable205-210 polymerisation, of lactic acid 224226 polymers, biodegradable217-226, 247-256 polymers, degradation of 3-14 polymers, natural 97-104

spinning, fibre 371-378 spinning dope 374 sponge, polyurethane 227-228 spruce wood 132 star polylactic acid 217-226 star-shaped 2 17 starch 79-90, 175-180

polymers, ultra structure 131

starch-based foams 175-180

polymethacrylates406 polysaccharide electrolyte 145-154 polysaccharides 97-104 polysaccharides, phosphoryl 107112 polyurethane composites 181-190, 191-196, 211-216 polyurethanefoams 175-180, 227228, 229-234, 241-246, 397-403 polyurethane sheet 197-204 polyurethanes 33-46, 181-190 polyurethanes,from pineapple waste 235-240 prolamine 247 protein, adsorbed 405-410 protein, fluorescent 412 proteins, corn 247-257 protoplasts 413 Pseudomonas 397-403 Pseudomonas elodea 333 pulp, paper grade 371-378 pulping 347-355 pulps 115 pyrolysis 47-51

succinylation 92 sulphate 371-378 sulphate pulp 371-378

radical initiation 4 radical polymerisation406 random coil conformation 247-257 reactions, degradation 3-14 relative humidity 318 resins, epoxy 73-78 reticulated polyurethane foam 397403 rice starch 79-50

TEMPO 54 tensile test 276, 279 TG-FTIR studies 289-294, 341-346 TG-MS, on wood ceramics 312-315 fibrinogen, human plasma 406 thermal analysis 341-346 thermal analysis, of paper 295-302 thermal conductivity 295-302 thermal degradation 191-196 thermal diffusivity 295-302 thermal properties 33-46, 73-78, 7990, 206-207 thermal properties, of cellulose acetates 275-280 thermal properties, of composites 21 1-216 thermal properties, of wood ceramics 311-315 thermocatalytic dehydration 47-51 thermogravimetry 30, 40, 77, 191196, 21 1-216, 237, 242-246, 248257, 281-288 thermogravimetry, controlled rate 31 1-315 thermomechanical analysis 317-320 transformation, of lignin 155-160 transglyosylation 365370 Trichoderma reesei, 37 1-378 Trichoderma vjride 365-370 ultra structure 131

saccharide-based, polyurethane foams 241-246 seed protein 247-256 sheet, polyurethane 197-204 solutions, polyelectrolyte 145-154 sorption kinetics 327-332 sorption isotherms 276-280,327-332

vanillic acid 387 veratric acid 387 viscometnc studies 333-339 viscose process 371 waste, cellulosic 67-72

Index

waste cooking oil 197-204 waste materials 181-190 water, effect of 321-325 water, non freezing 277-280 water, restrained by pectin 303-310 water absorption 92-95 weathering analysis 34 1-346 Wood 129-138 wood, delignified 347 wood, treatment with laccase 412417 wood ceramics 31 1-315 wood fibre 129-138 woody plants 379-386 wound healing 97-104 x-ray diffraction patterns, of zein films 248-257 x-ray analysis, of lignin, 161-166 x-ray studies 269-274 xanthan gum 146 tein films 247-257

429