HEMICELLULOSES APPLICATION FOR SYNTHETIC POLYMER SURFACES FUNCTIONALISATION

Transcription

1 UNIVERSITY OF MARIBOR FACULTY OF MECHANICAL ENGINEERING HEMICELLULOSES APPLICATION FOR SYNTHETIC POLYMER SURFACES FUNCTIONALISATION Maribor, February 2016 Nena VELKOVA

2 HEMICELLULOSES APPLICATION FOR SYNTHETIC POLYMER SURFACES FUNCTIONALISATION Maribor, February 2016 Autor: Nena VELKOVA Mentor: red. prof. dr. Simona STRNAD Co-mentor: prof. dr. Bodo SAAKE Co-mentor: izr. prof. dr. Lidija FRAS ZEMLJIČ

3 Doktorska disertacija UPORABA HEMICELULOZ ZA FUNKCIONALIZACIJO POVRŠIN SINTETIČNIH POLIMEROV HEMICELLULOSES APPLICATION FOR SYNTHETIC POLYMER SURFACES FUNCTIONALISATION February 2016 Nena VELKOVA

4 ACKNOWLEDGEMENTS First of all I want to thank to my supervisor prof. dr. Simona Strnad for her great support, discussions and encouragements during my study. Thanks again for her help and guidance during the formation of this thesis. I want to acknowledge to my co-advisors prof. dr. Bodo Saake from the Centre of Wood Science, University of Hamburg, for his advices and allowing me to work in his research laboratory during my Ph.D studies and prof. dr. Lidija Fras Zemljič, University of Maribor, for her advises and great discussions. I m also grateful to prof. dr. Karin Stana-Kleinschek, University of Maribor, who gave me the opportunity to work in Laboratory for Characterization and Processing of Polymers and to all other co-workers from this laboratory, dr. Aleš Doliška and dr. Tijana Ristić (former co-workers), dr. Silvo Hribernik, dr. Tamilselvan Mohan, Matej Bračič, M. Cs., Petra Gašparič, M. Cs, Tanja Pivec, Jasna Tompa, M. Cs., for all discussions and help in the laboratory. Especially thanks goes to Tanja Kos for all the help regarding laboratory work during my pregnancy. Also thanks to Andreja Križanec for her help in the laboratory work. Thanks to all other professors from the group for their kind recommendations. I am grateful to prof. dr. Ivan Kosalec (Faculty of Pharmacy and Biochemistry, University of Zagreb) for providing MIC determination. Dr. Alenka Vesel (Josef Stefan Institute, Ljubljana) is acknowledged for performing XPS analysis. Finally I want to thank to my family, my parents, Elica and Blagoj, for their love and support and to my sister Netka and her family, for believeing in me all my life. At the end the most important, great thanks to my loves, my husband Alen and my lovely daughter Eva, who support me, love me and understand me all these years. I am grateful for receiving financial support for conducting this doctoral dissertation.»operation part financed by the European Union, European Social Fund. Operation implemented in the framework of the Operational Programme for Human Resources Development for the Period , Priority axis 1: Promoting entrepreneurship and adaptability, Main type of activity 1.1.: Experts and researchers for competitive enterprises.«

5 TABLE OF CONTENTS 1 INTRODUCTION THEORETICAL BACKGROUND Hemicelluloses Xylans Derivatisation and application of xylans Poly(ethylene terephthalate) Structure and properties of PET fibers Functionalisation of polymer surfaces for technical and medical applications Theoretical principal of analytical methods Attenuated total reflectance (ATR) and Raman infrared spectroscopy X-ray photoelectron spectroscopy Atomic force microscopy Surface wettability and surface free energy Quartz crystal microbalance with dissipation unit (QCM-D) Titration methods Methods for antimicrobial activity evaluation EXPERIMENTAL PART General experimental plan Materials Methods Chemical modifications of xylans Xylan film preparation Preparation and pretreatment of PET surfaces Preparation of model PET surfaces Pretreatment of real PET surfaces Adsorption of xylans Adsorption of xylans onto model PET surfaces Adsorption of xylans onto real PET surfaces Characterization of nonmodified and modified xylans Characterization of xylan films and functionalized PET fibers RESULTS AND DISCUSSION Characterization of nonmodified and modified xylans I-

6 4.1.1 Carbohydrate composition Elemental composition Total bound nitrogen Molecular weights and polydispersities Surface chemical composition Charging behaviour Antimicrobial properties Characterization of xylan films Surface chemical composition Surface morphology Hydrophilic/hydrophobic character and surface free energy Adsorption study of xylans onto PET model films using QCM-D Influence of ph Influence of ionic strength Influence of xylan concentration Adsorption of xylans onto PET model films using anchoring polymers X-ray photoelectron spectroscopy Hydrophilic/hydrophobic character and surface free energy Adsorption of xylans onto PET fabric Charging behaviour Surface Morphology Contact angles and wettability Antimicrobial activity of PET fabric samples SUMMARY AND OUTLOOK REFERENCES II-

7 HEMICELLULOSES APPLICATION FOR SYNTHETIC POLYMER SURFACES FUNCTIONALISATION Key words: hemicellulose, polyethylene terephthalate, glucuronoxylan, arabinoxylan, carboxymethylation, cationization, PET model films, quartz crystal microbalance, PET fabric, surface free energy, wettability, antimicrobial properties ABSTRACT The main aim of this thesis was development of thin functional layers from hemicelluloses xylans on the polyethylene terephthalate (PET) surfaces. Hemicelluloses, xylans, as renewable polymers, were chemically modified in order to introduce anionic and cationic functional groups. Two types of chemical modifications were performed: carboxymethylation in order to increase anionic nature of xylans and improve their hydrophilic character and cationization for introducing of amino groups and antimicrobial characteristics. Both types of modifications were successful, which was proved by ATR FTIR and raman techniques, elemental analysis, total bound nitrogen determination, size exclusion chromatography and polyelectrolyte titrations. Polyelectrolyte titration results showed increased amounts of deprotonated carboxyl groups in carboxymethylated xylans as well as increased amounts of protonated amino groups in cationized xylans. Antimicrobial activity of xylans was investigated by the determination of minimal inhibitory concentration (MIC) against S. aureus, E. coli, and C. albicans and it was found out that the samples with higher amounts of active amino groups showed lower MIC. Cationised glucuronoxylan showed significantly higher antimicrobial activities against S. aureus in comparison to cationised arabinoxylan and nonmodified xylan samples. However, none of xylan samples was active against fungi. In order to analyze surface properties of solid surfaces, films from xylan (nonmodified and modified) water solution was formed by casting method. The surface chemical composition of films were investigated by X-ray photoelectron spectroscopy (XPS), and the results showed that films made from carboxymethylated xylans had significantly higher amounts of carbon fraction involved in O=C-O bonds, compared to nonmodified xylans. Such surface chemical structure caused higher surface free energy with higher electron-donor contribution and thus high hydrophilicity of these films. Films made by cationized xylans had higher amount of carbon involved in C-C and C-H bonds compared to nonmodified and lower surface free -III-

8 energy with increase of dispersive Lifshitz Van der Waals contribution. In order to thoroughly investigate the adsorption of xylans onto synthetic surfaces Quartz crystal microbalance with dissipation unit (QCM-D) was used. For these measurements model films were prepared from PET by spin coating technique. Adsorption studies were performed at different conditions, such as ph, concentration and ionic strength of xylan solutions. For all the chemically modified xylans the adsorption was improved at ph 5 and with increased ionic strength with divalent ions. The adsorption increased as well with increasing of xylan solution concentration. In order to improve binding of adsorbed xylans so-called anchoring polymers were applied. When anchoring polymers were applied, better adsorption and fixation of adsorbed layer was confirmed, thus the adsorbed masses of xylans after rinsing with water were significantly higher in comparison to the adsorption without immediate anchoring layer. On the basis of these results, real PET fabric surfaces were treated using chemically modified xylans. The xylan solutions were applied onto PET fabric samples using spray coating technique, which is the best approximate to the large-scale procedures. In the first step, PET fabric was activated by alkaline hydrolysis and after that, anchoring agents and carboxymethylated and/or cationized xylans were adsorbed. The success of these treatments was evaluated by the determination of negative and positive charge of the treated PET fabric samples by titration techniques, methylene blue and acid orange 7 adsorption methods, water contact angles and wettability determination. From the potentiometric titrations results it was clearly seen that each new adsorbed layer onto PET fabric totally screened the charge of the former one. FESEM images showed rather thick layers covering the PET filaments in the fabric as well as partially filling the gaps between them. Water contact angles showed extremely high hydrophilicity of the PET fabric surfaces treated with chemically modified xylans with very fast spreading of water drops. Fabric samples were completely wetted in only half second. Results of antimicrobial activity tests showed that only PET fabric samples treated with cationised samples provided sufficient bacterial reduction (higher than 75 %). PET fabric samples, treated with cationised glucuronoxylan showed 94 % reduction of the gram negative E. Coli, while the reduction of gram positive S. aureus was only 42 % which is insufficient. However, the fabric sample treated with cationised arabinoxylan was sufficiently effective against both species, thus its reduction of S. aureus was 76 % and reduction of E. Coli was even 96 %. The presented work suggests that hemicelluloses xylans, derived from hard wood and/or oat spelt could be successfully applied as a coating material for synthetic surfaces as polyethylene terephthalate in order to significantly improve its hydrophilicity and antimicrobial properties. -IV-

9 UPORABA HEMICELULOZ ZA FUNKCIONALIZACIJO POVRŠIN SINTETIČNIH POLIMEROV Ključne besede: hemiceluloze, poletilentereftalat, glukuronoksilan, arabinoksilan, karboksimetilacija, kationizacija, PET modelni filmi, kremenova mikrotehnica, PET tkanina, površinska prosta energija, omakanje, protimikrobne lastnosti UDK klasifikacija: : (043.3) RAZŠIRJEN POVZETEK Glavni namen doktorske naloge je bil študij uporabe hemiceluloz ksilanov, kot obnovljivih biopolimerov (stranski produkti papirne in prehrambene industrije) za razvoj postopkov in tehnik za izdelavo tankih funkcionalnih slojev na površinah sintetičnih polimerov, kot je npr. polietilentereftalat, ki ga najdemo v najrazličnejših tehničnih in medicinskih aplikacijah. Pri teh aplikacijah so najpogosteje iskani lastnosti sintetičnih površin hidrofilnost in protimikrobnost. Uporaba sintetičnih polimerov se je v zadnjih desetletjih močno uveljavila predvsem zaradi nizkih cen in dobrih mehanskih lastnosti kot so elastičnost, trdnost, odpornost na drgnjenje [1, 2]. Vendar pa je v glavnem osnovna značilnost teh materialov, da imajo nizko površinsko energijo in visoko inertnost, kar pomeni hidrofobnost in zato slabe fiziološke lastnosti [2, 3]. Na področjih, kjer se materiali uporabljajo v stiku s človeškim telesom, je potrebno zadostiti številnim specifičnim zahtevam, od katerih je najpomembnejše fiziološko udobje. Glavni parametri, ki določajo fiziološko udobje so predvsem: prepustnost za vodno paro, hidrofilnost in termična prevodnost. Za medicinske namene je večkrat zelo zaželena odpornost na različne bakterije. V smislu preseganja teh težav se najpogosteje poslužujejo različnih površinskih obdelav, ki ob spreminjanju površinskih lastnosti ohranjajo mehanske lastnosti polimera. Za izboljšanje hidrofilnosti poliestrov se zelo pogosto uporablja alkalna hidroliza [1, 4, 5] ali obdelava s plinsko plazmo, ki ji je bilo v zadnjih desetletjih posvečeno veliko pozornosti [6, 7]. Slabost -V-

10 teh obdelav je predvsem nestabilnost funkcionalnosti (staranje). Poleg navedenih se za površinsko funkcionalizacijo uporabljajo encimatske obdelave, površinska kopolimerizacija ali adsorpcija polimerov in izdelava tankih slojev. Nadomeščanje izdelkov, ki temeljijo na petrokemičnih virih s tistimi, ki so izdelani iz obnovljivih materialov je eden od najpomembnejših inovacijskih in raziskovalnih ciljev zadnjih desetletij. Takšni obnovljivi materiali vključujejo filme, embalažo in celo vrsto drugih materialov, izdelanih iz biopolimerov [8, 9]. Ekonomski potenciali biopolimerov so ogromni, saj je to najpomembnejše področje za razvoj in doseganje vseh komponent obnovljive industrije. Realizacija je v tem trenutku še relativno slaba, saj trenutno le manj kot 1 % vse proizvodnje polimerov temelji na biopolimerih, vendar pa ti, okoljsko neškodljivi polimeri kažejo potencialno visoko rast. Zadnje študije kažejo, da bo globalno povpraševanje po obnovljivih polimerih naraščalo, in sicer od 180 Mio Ton v letu 2007 na 258 Mio Ton v letu 2010, do leta 2020 pa bi naj biopolimeri nadomestili 5 % celotnega povpraševanja po petrokemičnih polimerih. Pomembna pomanjkljivost naštetih obnovljivih polimerov je, da vsi temeljijo na surovinah, katerih pridobivanje zahteva uporabo kmetijskih zemljišč, ki bi lahko sicer bila uporabljena za pridelavo hrane. To dejstvo predstavlja enega glavnih motivov za poglobljene raziskave drugih potencialnih virov, predvsem stranskih produktov industrijskih procesov. Med takšnimi je vsekakor proizvodnja celuloze in papirja kot vir hemiceluloz in lignina. Hemiceluloze so polisaharidi, ki so v rastlinah tesno povezani s celulozo in predstavljajo tretjo največjo skupino biomaterialov. Hemiceluloze so izjemno heterogena skupina snovi, med katerimi so najpomembnejši ksilani, arabinoksilani, manani, galaktoglukomanani, glukomanani, arabinogalaktan II, ß-1,3-glukan in ß-1,3-ß-1,4-glukani [10, 11]. Hemiceluloze se nahajajo v glavnem v sekundarnih celičnih stenah in skupaj s celulozo in ligninom oblikujejo rastline na način, ki omogoča optimalno mehansko oporo in transport vode in hranilnih snovi. Ksilani predstavljajo 10 do 35 % vseh hemiceluloz, prisotnih v trdem lesu, medtem ko galaktoglukomanani predstavljajo okrog 15 do 20 % hemiceluloz mehkega lesa [12]. Ksilani so poleg celuloze najpomembnejša sestavina celične stene in so tako tudi največji vir obnovljive hemiceluloze na zemlji [11]. Ocenjuje se, da zagotavljajo tretjino vsega razpoložljivega obnovljivega organskega ogljika na zemlji. Njihova prisotnost je 10 do 35 % v mehkem in trden lesu in okoli 35 do 40 % celotne mase ličja kot je oves [13]. Velika -VI-

11 raznolikost in kompleksnost ksilanov daje možnost razvoja najrazličnejših novih stranskih produktov in jih torej lahko smatramo kot obetajoč vir biopolimerov. Ksilane so v zadnjih letih začeli pospešeno raziskovati kot obetajoč obnovljivi vir, saj se ne nahajajo samo v lesu, ampak tudi v vseh vrstah biomase. Sposobnost za ekstrakcijo je pri ksilanih omejena s fizikalnimi in/ali kovalentnimi interakcijami z drugimi sestavinami celične stene. Funkcionalne lastnosti hemiceluloz oz. ksilanov se lahko izboljšajo z različnimi kemičnimi in strukturnimi modifikacijami za dosego vodotopnosti, absorpcije/odbojnosti na vode, protimikrobne in protivirusne lastnosti itd. Izvedene so bile raziskave filmov in gelov iz različnih vrst ksilanov [8, 9]. Na drugi strani pa je zelo malo objav, ki govorijo o adsorpciji ksilanov na vlaknate površine, predvsem na celulozna vlakna in še nobene raziskave, ki bi govorila o adsorpciji ksilanov na sintetične površine, kot so PET vlakna. Natančna študija fizikalno-kemičnih in strukturno-morfoloških lastnosti ter njihovega vpliva na hidrofilno-hidrofobni karakter slojev ksilanov do danes še ni bila opravljena. Ksilani tudi še niso bili uporabljeni za funkcionalizacijo sintetičnih površin prav tako pa še ni bila opravljena natančna analiza adsorpcije ksilanov na sintetične površine pri različnih pogojih (ph, koncentracija, dodatek elektrolita). Doktorska naloga je razdeljena v štiri dele in sicer: (1) Kemična modifikacija ksilanov, in sicer glukuronoksilana iz lesa breze in arabinoksilana iz ovsa ter karakterizacija osnovnih in modificiranih ksilanov; (2) Priprava ksilanskih filmov s tehniko litja in karakterizacija površinskih lastnosti filmov s poudarkom na kemični sestavi, površinski morfologiji, površinski prosti energiji in stičnih kotih; (3) Študij adsorpcije osnovnih in kemično modificiranih ksilanov iz raztopin na modelne sintetične (PET) površine z uporabo kremenove mikrotehnice (QCM-D) in definicija optimalnih pogojev za adsorpcijo (koncentracija ksilanov, ph vrednost, uporaba elektrolitov) in (4) zadnji ter najbolj pomemben del naloge je prenos rezultatov optimizacije adsorpcije z uporabo QCM-D na realne sisteme - adsorpcija ksilanov na polietilen tereftalatno tkanino ter analiza njihove površine, hidrofilnosti in protimikrobnosti. V prvem delu sta bili obe vrsti ksilanov, glukuronoksilan (BX in BXL) in arabinoksilan (OX), sintetizirana. Karboksimetiliranje je bilo izbrano zaradi uvajanju karboksilnih skupin, saj višja vsebnost karboksilnih skupin, prisotna v materialu, prispeva k povečanju hidrofilnega -VII-

12 značaja. Kationizacija je bila uporabljena z namenom uvajanju kvarternih amino skupin, ki imajo protimikrobne lastnosti. Sledila je karakterizacija fizikalno-kemičnih in strukturnih lastnosti modificiranih ksilanov. Uspešnost karboksimetiliranja je bila za obe vrsti ksilana dokazana z ATR FTIR spektroskopijo, saj so v spektrih karboksimetiliranih ksilanov nastali novi absorpcijski maksimumi pri 1596 cm -1 in 1415 cm -1, ki so specifični za deformacijske vibracije karbonilnih C=O skupin v COO -. Rezultati polielektrolitskih titracij so potrdili količine na novo uvedenih deprotoniranih karboksilnih skupin pri ph 8, ki so bile za oba karboksimetilirana ksilana približno enake, in sicer 2 mmol/g. Višja povprečna molekulska masa modificiranih ksilanov v primerjavi z osnovnimi je dodatno potrdila uvedbo novih skupin. Kationizacija je bila dokazana z ramansko spektroskopijo, kjer je bil pri kationiziranih ksilanih zaznan zelo izrazit pik pri 763 cm -1, ki je tipičen za vibracijsko deformacijo kvarternih amonijevih skupin ((CH3)3N + ). Elementarna analiza (metoda CHN) je pokazala vsebnost dušika v obeh primerih in sicer 2.7 % pri kationiziranem glukuronoksilanu (CBX) in 1.5 % pri kationiziranem arabinoksilanu (COX). Enake vsebnosti dušika so bile ugotovljene tudi z metodo določanja celotnega vezanega dušika (TNb). V CBX je bila vsebnost protoniranih aminskih skupin pri ph 2 = 3.2 mmol/g in pri ph 8 = 1.5 mmol/g, medtem ko je v COX znašala 1.2 mmol/g pri ph 2 in 0.75 mmol/g pri ph 8. Pri ph = 2 smo določili vsebnost vseh aminskih skupin, medtem ko pri ph = 8 le kvatrerne aminske skupine. Protimikrobni učinki so bili spremljani z določanjem minimalne inhibitorne koncentracije (MIC). CBX je pokazal najvišji protimikrobni učinek proti S. aureus in sicer je bila že pri koncentraciji % zaznana 50 % manjša sposobnost preživetja bakterij. Enaka stopnja inhibicije E. Coli (50 %) je bila zaznana pri višji koncentraciji (0.125 %). COX je izkazoval nekoliko slabše protimikrobne učinke, saj se je inhibicija obeh vrst bakterij pričela pri koncentraciji 0,25 %. Nobena vrsta kationiziranih ksilanov pri preiskovani koncentraciji ni inhibirala glive C. albicans. V drugem delu so bili iz osnovnih in kemično modificiranih ksilanov izdelani filmi z metodo litja in raziskane njihove lastnosti, s poudarkom na kemični sestavi, površinski morfologiji, površinski prosti energiji in stičnih kotih. Površina filmov iz nemodificiranih in modificiranih ksilanov je bila preiskovana s pomočjo mikroskopa na atomsko silo (AFM). Slike površinske morfologije in profilne črte so pokazale vdolbine in izbokline na nano nivoju. Pri filmih iz karboksimetiliranega (CMOX) in -VIII-

13 kationiziranega (COX) arabinoksilana je bila izmerjena najvišja hrapavost (5.2 nm). Filmi iz kationiziranih ksilanov so imeli na površini večje aglomerate premera okoli 200 nm. Kemična sestava površine filmov iz karboksimetiliranega glukuronoksilana (CMBX) je pokazala za okrog 30 % večjo vsebnost ogljika C3, vezanega v C-O in C-OH skupinah v primerjavi z nemodificiranimi ksilani, v primeru arabinoksilanov pa je bila ta vsebnost večja celo za 35 %. Na površini filmov iz CMBX je bila ugotovljena za okrog 11 % večja vsebnost ogljika C4 (O=C-O) v primerjavi s filmov iz nemodificiranega glukuronoksilana, v filmih iz CMOX pa je bila ta vsebnost celo za okrog 225 % višja od filmov iz nemodificiranega arabinoksilana. Pri filmih iz kationiziranih ksilanov je bilo ugotovljeno znižanje vsebosti ogljika C4 (v O=C-O skupinah) v primerjavi z nemodificiranimi ksilanskimi filmi. V primeru CBX so bile te vsebnosti nižje za 54 % in v primeru COX za okrog 6 %. Na površini filmov iz kationiziranih ksilanov je bila dokazana prisotnost dušika, in sicer 2.6 at. % v primeru CBX in 1.5 at. % v primeru COX. Površinska prosta energija (SFE, γs TOT ) filmov iz karboksimetiliranih ksilanov se je značilno zvišala v primerjavi s SFE filmov iz osnovnih ksilanov. Ta povečanja so bila različna za posamezno vrsto ksilanov, in sicer 40 % v primeru CMBX in le 11 % v primeru CMOX. Prispevek nepolarne oz. Lifshitz-van der Waals komponente (γs LW ), je bil za te vzorce znižan, in sicer za 13 % pri CMBX in 30 % pri CMOX. Obenem pa se je polarna elektron-donorska komponenta (γs - ) zvišala za okrog 70 % v primeru CMBX in celo za okrog 270 % v primeru CMOX v primerjavi s filmi iz nemodificiranih ksilanov. Ti rezultati so pokazali pomembno povečanje površinske polarnosti ksilanskih filmov po karboksimetiliranju. Pri filmih iz kationiziranih ksilanov je bila dokazana za okrog 7 % nižja površinska energija v primerjavi s filmi iz nemodificiranih ksilanov, kar je bilo zaradi uvedbe kvarternih amino skupin pričakovano. Nepolarna komponenta SFE je bila pri teh vzorcih višja za okrog 4.5 % pri CBX in celo za 13 % pri COX v primerjavi z osnovnimi ksilani, medtem ko se je polarna elektronsko-donorska komponenta pomembno znižala, in sicer za 35 % pri filmu iz CBX in celo za 63 % pri filmu iz COX. O znižani hidrataciji priča tudi rezultat znižanja elektronakceptorske komponente (γs + ) pri CBX za 31 % in pri COX za 3.4 %. Ti rezultati so bili potrjeni z meritvami statičnega stičnega kota z vodo (WCA). Filmi iz karboksimetiliranih ksilanov so izkazovali pomembno zvišanje hidrofilnosti v primerjavi s filmi iz nemodificiranih ksilanov, saj so bili stični koti z vodo nižji za okrog 40 %. Stični koti z vodo, izmerjeni na filmih iz kationiziranih ksilanov pa so bili za okoli 10 % višji od tistih, izmerjenih na filmih iz nemodificiranih ksilanov. -IX-

14 V tretjem delu naloge, je bila opravljena obširna analiza adsorpcije nemodificiranih in modificiranih ksilanov na modelne PET površine. Uporabljena je bila kremenova mikrotehnica z disipacijsko enoto. Adsorpcija ksilanov je bila opravljena pri različnih pogojih. Preiskovani so bili vplivi ph, ionske jakosti in koncentracije raztopin ksilanov. Vpliv ph na adsorpcijo je bil preiskovan pri 4 različnih ph vrednostih, in sicer pri ph 4, ph 5, ph 7 in ph 9. Pri ph 5 je bila ugotovljena najvišja adsorpcija za večino vzorcev. Analiziran je bil vpliv prisotnosti dveh različnih elektrolitov (NaCl in CaCl2) in sicer pri različnih ionskih jakosti (0.05 M, 0.1 M, 0.3 M, 0.5 M in 0.7 M). S povečanjem ionske jakosti in ob prisotnosti dvovalentnih Ca 2+ ionov (CaCl2) se je adsorpcija povečala. Adsorpcija se je zviševala tudi z višanjem koncentracije ksilanov (od 50 mg/l do 500 mg/l). Pri teh analizah so bila potrjena dognanja nekaterih avtorjev v zvezi z adsorpcijo polimerov na hidrofobne površine [14], saj je bila najboljša adsorpcija dosežena v primeru nemodificiranega arabinoksilana (OX) zaradi najnižje vsebnosti karboksilnih skupin in posledično najnižjih odbojnih sil med makromolekulami in hidrofobno PET površino. V namenu izboljšanja adsorpcije in fiksacije ksilanov na PET površine je bila analizirana tudi uporaba t.i. sidrnih polimerov, kot sta polietilen imin (PEI) za izboljšanje adsorpcije karboksimetiliranih ksilanov in polivinilsulfonska kislina (PVSA) za izboljšanje adsorpcije kationiziranih ksilanov. Ob uporabi teh molekul je bila adsorpcija zvišana, najpomembnejše pa je dejstvo, da so bili adsorbirani sloji ksilanov tudi bolj odporni na spiranje z vodo, ki je sledilo adsorpciji. Ksilani adsorbirani na predhodno adsorbiran sloj sidrnega polimera, se težje ali sploh ne odstranijo po spiranju z vodo. V skladu s tem so bile adsorbirane mase pomembno zvišane, v primeru COX za okrog 50 %, v primeru CBX, za okrog 100 % in v primeru karboksimetiliranih ksilanov celo za okrog 200 %. Hidrofilnost PET modelnih filmov z adsorbiranimi ksilani se je zvišala v povprečju za 30 %. Zadnji (četrti) del naloge je predstavljal prenos rezultatov, dobljenih na modelnih PET površinah v realni sistem obdelavo PET tkanine s kemično modificiranimi ksilani. PET tkanina je bila najprej površinsko aktivirana s pomočjo alkalne hidrolize. Pred adsorpcijo karboksimetiliranih ksilanov je bila na tkanino adsorbirana sidrna molekula (PEI), medtem ko so bili kationizirani ksilani adsorbirani direktno na hidrolizirano PET površino tkanine. Negativni naboj vzorcev PET tkanine, obdelane s karboksimetiliranimi ksilani je bil spremljan s pomočjo potenciometričnih titracij in adsorpcijo barvila metilensko modro. -X-

15 Negativni naboj ugotovljen z obema metodama je znašal okrog 32 mmol/kg za obe vrsti ksilanov. Rezultati potenciometričnih titracij so pokazali, da vsaka faza adsorpcije polimerov popolnoma zakrije naboj prejšnje površine. Neobdelana PET tkanina je izkazovala slab negativni naboj v platoju, in sicer 21 mmol/kg, ki se je po hidrolizi dodatno povečal za 76 %. Po adsorpciji PEI na hidrolizirano PET površino le-ta izkazuje pozitivni naboj, in sicer 11 mmol/kg. Po adsorpciji karboksimetiliranih ksilanov se ta pozitivni naboj spremeni v negativnega v celotnem območju ph. Sprememba naboja vzorcev PET tkanin po obdelavi s kationiziranimi ksilani je bila spremljana s pomočjo potenciometričnih titracij in z metodo adsorpcije barvila Acid Orange 7. Rezultati potenciometričnih titracij so pokazali malo vsebnost pozitivnega naboja in sicer 9.96 mmol/kg v primeru obdelave s CBX in 2 mmol/kg v primeru obdelave s COX. V obeh primerih je bil še prisoten negativni naboj in sicer okoli 10 mmol/kg, kar nakazuje na nepopolno prekritje PET površine, kar je bilo ugotovljeno tudi z vrstično elektronsko mikroskopijo. V primeru metode Acid Orange 7 je bila ugotovljena zelo nizka vsebnost pozitivnih skupin, ki je znašala 3.34 mmol/kg v primeru obdelave s CBX in 2.05 mmol/kg v primeru obdelave s COX. Takšno nizko vsebnost pozitivnih skupin v obdelanih PET tkaninah je bilo pričakovati zaradi nizke stopnje substitucije, ki je bila dosežena pri kemični modifikaciji teh vzorcev. Vrstična elektronska mikroksopija je pokazala, da so adsorbirani sloji ksilanov na PET filamentih v tkanini prekriti z dokaj gladkimi in mestoma precej debelimi plastmi polimerov, ki na določenih mestih celo zapolnjujejo praznine med posameznimi filamenti. V primeru obdelave s CMOX, so te plasti bolj robate in vsebujejo inkorporirane večje skupke in delce. Takšna značilno večja hrapavost površin filmov iz CMOX je bila ugotovljena tudi z mikroskopom na atomsko silo (AFM). Protimikrobne lastnosti neobdelanih in obdelanih tkanin so bile spremljane v skladu s standardizirano metodo ASTM E-2149, in sicer na gram pozitivne (S. aureus) in gram negativne (E. Coli) bakterije. Najučinkovitejše inhibitorno delovanje je bilo ugotovljeno pri vzorcu tkanine, obdelane s kationiziranim arabinoksilanom COX. Pri tem vzorcu je bila ugotovljena učinkovita redukcija (> 75 %) obeh vrst bakterij, redukcija gram negativne (E. Coli) je bila celo 96 %. Tudi vzorec tkanine, obdelan s CBX je izkazoval zelo visoko stopnjo redukcije E. Coli, in sicer 94 %, medtem ko je bila redukcija S. aureus le 42 %, in torej nezadostna. Pri tkaninah, obdelanih s karboksimetiliranimi ksilani je bilo sicer ugotovljeno zvišanje redukcije S. aureus v primerjavi z neobdelano PET tkanino, za okrog 1.6-krat v primeru CMBX in za okrog 80 % v primeru CMOX, vendar pa je bila v obeh primerih -XI-

16 redukcija pod 75 %, in zato nezadostna. Redukcija E. Coli pa je bila v primeru tkanin obdelanih s karboksimetiliranimi ksilani pomebno nižja glede na neobdelano tkanino. PET tkanine, funkcionalizirane s kemično modificiranimi ksilani so izkazovale ekstremno visoko hidrofilnost površin. V primerjavi s hidrofobno površino neobdelane PET tkanine, kjer je izmerjen stični kot znašal 125, so se kapljice vode, položene na površine PET tkanin, obdelanih s ksilani, popolnoma razlile v izredno kratkem času (0.5 s). Doktorska disertacija podaja nekaj izvirnih znanstvenih prispevkov: 1. Poglobljena analiza fizikalno kemičnih površinskih lastnosti filmov iz kemično modificiranih ksilanov s poudarkom na površinskih prostih energijah. 2. Uporaba kremenove mikrotehtnice za poglobljeno analizo adsorpcije ter obnašanja in lastnosti adsorbiranih ksilanskih filmov (nemodificiranih in kemično modificiranih) na sintetičnih površinah (PET modelni filmi). 3. Najpomembnejši izvirni znanstveni prispevek pa je aplikacija ksilanov iz dveh različnih virov, iz lesa in lupin ovsa, za izdelavo tankih slojev na površini sintetičnih vlaknatih materialov, definicija pogojev izdelave slojev ter natančna analiza funkcionalnosti (hidrofilnosti in protimikrobnih učinkih) takšnih novih površin. -XII-

17 LIST OF ABBREVIATIONS ASTM ATR-FTIR BX BXL CBX CMBX CMOX COX DME DP DS EPTA MIC Mw OX PEI PET PVSA QCM-D RMS SEM SFE SMCA TB TNb XPS American Society for Testing and Materials Aattenuated total reflectance Fourier transform infrared spectroscopy Glucuronoxylan Glucuronoxylan Cationized glucuronoxylan Carboxymethylated glucuronoxylan Carboxymethylated arabinoxylan Cationized arabinoxylan 1,2-dimethoxyethane Degree of polymerization Degree of substitution (2,3-Epoxypropyl) trimethylammonium chloride Minimum inhibitory concentration Molecular weight Arabinoxylan Polyethylene imine Polyethylene terephtalate Poly(vinylsulfonic acid, sodium salt) Quartz crystal microbalance with dissipation Root mean squared roughness Scanning electron microscopy Surface free energy Sodium monochloroacetate Toluidine blue Total bound nitrogen X-ray photoelectron spectroscopy -XIII-

18 1 INTRODUCTION Fibrous forms and their composites, used in contact with human body, have to satisfy basic physiological demands, such as optimal hydrophilicity, water vapor permeability, thermal conductance, and in many cases antimicrobial properties. Technical textiles are fastest growing sector of the textile industry and account for over 40 % of the total textile production in many developed countries in year 2000 [1]. Fibrous materials for technical sector (medicine, civil engineering, car s interior ect.) are laminated composite materials, composed from two or more layers, and each of them play a specific role. At the moment laminates for car seats are composed mainly from synthetic materials, such as polyamides, polyesters, polyurethanes, and vinyls. Because of this composition of seats it often happens to accumulate an excessive amount of moisture on the surface of the seat [2]. To increase the hygroscopic function of laminates for seat s covers, in higher price range seats, as one component could be used natural fibers, like wool or hemp. For seats with lower price range, this is not an appropriate solution. Specific functions of these materials are dependent of its purpose of applying. All technical fibrous materials designed for human body contact applications encounter similar problems. On the one hand, they have to satisfy the demands for high mechanical stability and chemical inertness, and on the other hand the demands for physiological convenience such as absorption of moisture, hydrophilicity and resistance to microbial attack. In this group of textiles belong technical textiles and composites, like automotive sit covers, protection clothes, protection covers in medicine, masks, caps, surgical gowns ect., which are mainly made from synthetic fibers, like polyesters, polyamides, polyethylene, and polypropylene [2]. Synthetic polymers are relatively cheap and provide optimal mechanical properties (strength, elasticity, good stability, abrasion resistance) [3, 4], but they have also low surface tension, and high chemical inertness, so they have poor adhesion properties and high hydrophobic character [4, 5]. In order to preserve optimal mechanical properties necessary in highly demanding environments and to achieve functionalities, necessary for special applications, one of the solutions are functional layers/coatings on synthetic substrates. Coatings are used to make a proper finishing of the materials and produce multipurpose fabrics [6]. -1-

19 In these various applications, it is possible to improve the lack of functionalities of synthetic materials with their surface modifications. There are many procedures and techniques, which have been applied for synthetic surface physical and/or chemical modifications. Among them are: gas plasma treatments [7, 8], enzymatic treatments [9] surface grafting [10], alkaline hydrolysis, [3, 11, 12], polymer adsorption [13] etc. Renewable fibrous materials have a great potential [14]. They are investigated to produce biofuels, chemicals, textile materials and materials such as composites and pharmaceutical products [15]. One of the most innovative and researching aim nowadays are replacement of products, based on petrochemical resources with products made from renewable materials [15, 16]. Natural renewable polymers represented huge and still not very well investigated group of polymers, which have specific characteristics, suitable for variety of applications. This kind of renewable materials include fibers, films, foils, hydrogels, and a number of others materials, made from biopolymers [16, 17]. From the perspective of economical view there is a huge potential in biopolymers, because this is the most important field for development and growth of all components of renewable industry. At the moment realizations are relatively weak, because only less than 1 % of all production of polymers is based on the biopolymers, however, environmentally friendly polymers applications show continuous growth. From the last studies, it follows that demand for renewable polymers have grown from 180 Mil. Tons in 2007 to 258 Mil. Tons in The best success among renewable polymers, experienced polylactid (PLA) produced from corn. In the market, we can find many products made from PLA such as discs, cups, packaging foils, plastic bottles. Another successful biopolymer is starch from which are produced vessels, foils and bags. It was forecasted, that the most representative byoplastic in 2020 will be byoplastic from starch, PLA and polyethylene from bio-raw materials. The most important deficiency of all these renewable polymers is that they are made from sources, which demand additional fertile soil for their production, which could otherwise be used for food production. This factor is one of the main motives for detailed research of other potential resources, especially by-products in pulp and paper and cereal industry, such as hemicelluloses and lignin. Hemicelluloses are polysaccharides, which in plants are closely related with cellulose and represent third largest group of biomaterials. They are extremely heterogeneous group of substances, from which the most important are xylans, arabinoxylans, mannans, galactoglucomannans, glucomannans, arabinogalactan II, ß-1,3-glucan and ß-1,3-ß-1,4- glucans [18, 19]. Hemicelluloses are located in secondary cell walls and with celluloses and -2-

20 lignins form the plant in ways that provide optimal mechanical support and transport of water and nutritions. They constitute 20 to 30 % of the total bulk of annual and perennial plants [15, 20]. Xylans represent about 10 to 35 % of all hemicelluloses which are present in hardwood, while galactoglucomannans represent about 15 to 20 % of hemicelluloses in softwood [21]. Xylans are beside cellulose the most important component in cell walls and the largest source of renewable hemicelluloses on Earth [19]. They provide one third of all available renewable organic carbon on Earth. They are presented in about % in softwood and hardwood and in about % of the total mass in the residues of annual plants, such as oat spelts [22]. Big heterogeneity and complexity of xylans gives a chance to develop a different kind of new by-products and therefore can be considered like promising source of biopolymers. In recent years, xylans are increasingly investigated like promising source, because they are not presented only in wood, but also in all types of biomass. Ability of extraction is limited with physical and/or covalent interaction with other components in cell wall. In woody plants xylan is bounded to lignin by side chain of uronic acids with ester bonds. Recently, xylans had been investigated as renewable polymers for different applications, especially in nutrition [23, 24] medical [25, 26] and pharmaceutical [27] fields. Ebringerova and coworkers investigated mitogenic and comitogenic properties of xylans and compared it to the commercial immunomodulator zymosan [28]. Structurally and chemically modified xylans from birch wood were applied for nanoparticles preparation [29] and beech xylans were derivatised in order to prepare prodrugs for ibuprofen release [30]. Xylan sulfates are the only xylan derivatives, which have been continuously researched for their anticoagulant, antiviral and anticancer activities [31-33]. Some publications report about films and gels from different sources of xylans [15, 16]. However, until today, not one investigation has been done about the application of the xylans from hardwood and oat spelt for coating and functionalization of the synthetic polymers surfaces. There also still have not been performed detailed studies of physicalchemical and structural-morphological properties of different layers prepared from xylans and their influence on surface physical and chemical properties of synthetic surfaces. Until yet has also not been performed any analysis of adsorption behavior of xylans from solutions onto synthetic surfaces at different conditions (ph, c, ionic strength, etc.), as well as analysis of interdependence of the chemical composition and surface morphology of new xylan surfaces -3-

21 and their influence on hydrophilicity and antimicrobic characteristics of treated synthetic surfaces. The main aim of this dissertation is application of hemicelluloses xylans, as alternative renewable polymers (by-products of pulp and paper and/or cereal industry), for development of a procedure and technique for making a thin functional hydrophilic and/or antimicrobial layer on the synthetic polymer surfaces such as polyethylene terephtalate. The research work of this dissertation consisted of four phases. In the first phase two different types of xylans, glucuronoxylan derived from beech wood and arabinoxylan derived from oat spelt, were chemically modified, i.e.: carboxymethylated in order to introduce hydrophilic entities (carboxyl functional groups) and cationized in order to introduce amino groups, which are known to be a main carrier of antimicrobial properties in polymers. Chemical and structural analyses of nonmodified and chemically modified xylans were performed using carbohydrate analysis, elemental analysis, polyelectrolyte titrations, ATR-IR and raman spectroscopy as well as size exclusion chromatography. Total bound nitrogen determination was applied for determination of nitrogen bounded in cationic xylans. Antimicrobial properties of nonmodified and chemically modified xylans were analysed by minimal inhibitory concentration (MIC) determination of xylan solutions. In the second phase, films were prepared from nonmodified and chemically modified xylan samples by casting method and their surface properties were analysed. Surface morphologies of the films were characterized using atomic force microscopy, surface chemical compositions were analysed by X-ray photoelectron spectroscopy, and surface free energies (SFE), and their Lifshitz van der Waals and electron donor/acceptor contributions were determined using goniometry. In the third phase of the work, were defined optimal conditions of adsorption of xylans from water solutions onto polyethylene trephtalate surfaces on the basis of adsorption analyses using quartz crystal microbalance with dissipation unit (QCM-D). In this dissertation QCM-D -4-

22 was used for the first time for the analysis of adsorption behavior of nonmodified and chemically modified xylans from water solutions onto the synthetic surfaces (PET model films). Several parameters were analysed, i.e. ph, ionic strength, concentration of xylans and optimal adsorption conditions were defined. For the improvement of adsorption and fixation of xylans layers, the influence of the intermediate layers of anchoring polymers was investigated. Surface free energies and surface chemical composition of adsorbed layers were determined. In the last phase of this research, xylans were successfully applied in a real system, for functionalisation of PET fabric samples. Based on adsorption analyses using QCM-D, optimal conditions for xylan adsorption onto real PET fabrics/fibers surfaces were defined. Xylans were adsorbed onto PET fabric samples using spray coating technique, which was the best approximation to that one often applied on industrial level. In order to prove the success of surface functionalization using carboxymethylated and cationized xylans, charging behavior, amounts of negative and/or positive charge, surface morphology as well as wetting properties and antimicrobial effectiveness of PET fabric samples was analysed. The main hypothesis of this research was proved. Using chemical modifications of xylans and with the optimization of adsorption of xylans onto synthetic surfaces of polyethylene terephthalate, hydrophilic and antimicrobial layers were successfully developed on synthetic fabrics surfaces. -5-

23 2 THEORETICAL BACKGROUND 2.1 Hemicelluloses Renewable resources recently attract attention due to energy crisis and environmental concerns. Because of that, researchers striving for use renewable sources, instead of fossil resources. One of the most important renewable sources is biomass [34]. Hemicelluloses are the most complex components in the cell wall of woods. They form hydrogen bonds with cellulose, covalent bonds (mainly a-benzyl ether linkages) with lignins and ester linkages with acetyl units and hydroxycinnamic acids [21]. Hemicelluloses are white solid materials that are rarely crystalline or fibrous in nature. They have a linear polysaccharide backbone that is composed of β-1,4-linked glucose, mannose, xylose, or galactose units. They often have short side chains that may include xylose, glucose, arabinose, or glucuronic acid [18]. Monosaccharides that composed hemicelluloses have D configuration and occur in the six-member pyranoside forms, except arabinose, which has the L configuration and occurs as a five-member furanoside. They have an average DP about sugar units per hemicellulose molecule, which is much lower than cellulose ( 10,000) [18, 35]. Hemicelluloses are essentially linear polymers except for single-sugar side chains and acetyl substituent [35]. One of the most common hemicelluloses is xylan, constituting between % of the dry weight of hardwood and 5 10 % of softwood [18]. According to [19] four general classes of hemicelluloses with structurally different cellwall are observed: a) Xylans b) Mannans c) β-glucans with mixed linkages, and d) xyloglucans Wood fibers (wood cells) have a thick fiber wall. The fiber wall comprises several layers [36]. From the outside to the inside the layers are middle lamella (ML), primary wall (P), outer layer of the secondary wall (S1), middle layer of the secondary wall (S2), inner layer of the secondary wall (S3) and warty layer (W) (Figure 2.1) [37, 38]. -6-

24 ML middle lamela P Primary wall (lignin, pectin, hemicelluloses, small amount of cellulose) S1, S2, S3 Secondary wall (mostly cellulose, pectin, lignin, hemicelluloses) Figure 2.1: Structure of woody cell [37] The thin primary wall (P) and the middle lamella (ML) have high lignin contents and small amounts of cellulose, hemicellulose, pectin and extractives. The middle lamella is located outside the primary wall and links the wood cells together. The secondary wall consists of three sub layers (S1, S2 and S3), in each of which the fibrils run in a specific direction. The thicker layer is S2, account for the major part of the wood material and is the most important layer determining the physical properties of wood fibre [38]. Amounts of lignin, cellulose and hemicellulose are different in different types of wood. Both types of wood (hardwood and softwood) also have different morphology, cellular composition, and different fiber length. Softwood has relatively simple anatomy. Fibers are 2.5 to 7 mm length and thickness from 25 to 60 μm [35]. The major components in softwood are galaktoglucomannan, glucomannan and arabinoglucuronoxylan. Other hemicelluloses in softwood are arabinogalactan, xyloglucan and other glucans. Other polysacharides are pectins [37]. Hardwoods have complex structures including vessel elements, fiber tracheids, libriform fibers, rays cells, and parenchyma cells. Length of fiber in hardwood is 0.9 to 1.5 mm [35]. Main component in hardwood is xylan or O-acetyl-4-O-methylglucurono-β-D-xylan [35] representing about 15 to 30 % of the total dry-mass [39]. Glucomannan is also presented in hardwood [35, 37, 40]. Hardwood xylan does not contain arabinose side chains, such as -7-

25 softwood xylan [37]. Different composition of hardwood and softwood taken from Sjöström [36] are presented in the Table 2.1. Table 2.1: Main components, % of dry wood [35] Component Content (%) Hardwood Softwood Cellulose Hemicelluloses: (Galacto)glucomannans Xylans Lignin Other polysaccharides Isolated and purified hemicellulosic polysaccharides can be used for production of value added renewable polymers [34]. Current isolation and purification strategies including alkali peroxide extraction, organic solvent extraction, steam explosion, ultrasound-assisted extraction, microwave-assisted extraction, column chromatography, and membrane separation, which have been reviewed and summarized by Peng [41]. Hemicelluloses are available in huge amounts as by-products from wood, pulp, and paper industries. For using hemicelluloses, firstly they have to be separated from lignin. In case of pulp production, fibers are separated physically and/or chemically and are dispersed in water and reformed into a web [35]. Aim of pulp process is to get fibers separated from wood. There are many different types of pulp production: chemical, mechanical and their combinations: semi-chemical, semi-mechanical, thermo-mechanical pulping etc. Mechanical pulp constitutes % (1996) of world production and this is increasing because of high yield (90-98 %) [35]. Almost 40 % of total amount of pulp, manufactured in Finland in 2002 year, was acquired with mechanical pulping. Half of that was thermo-mechanical [42]. Thermo-mechanical pulping has two stages: refiners are at elevated temperature and pressure -8-

26 to promote fiber liberation and in the second refiners are at ambient temperature to treat the fibers for papermaking. In case of chemical pulping, fibers are product of chemically dissolving of wood component, mainly lignin [37]. Aqueous solutions of several alternative alkaline, neutral or acidic components at elevated temperature and pressure are used to dissolve lignin and some carbohydrates from wood chips [43]. The polysaccharides of wood react during alkaline cooking. The first industrial alkaline cooking process was soda cooking using sodium carbonate and sodium hydroxide as chemicals [43]. Nowadays, the Kraft process is the dominant chemical pulping method [35, 37, 44]. In Canada with this pulp process are produced 700 Tons of air dried pulp from hardwood in one day [44]. It is a strongly alkaline process, in which the active components are the hydroxide (OH - ) and the hydrosulfide (SH - ) ions [37] at ph above 12 and T = 160 to 180 C [35]. By Kraft process wood chips are transformed into paper pulp. A typical Kraft pulp mill uses biomass (wood chips), fuel (fossil and hog), chemicals, and water to produce market pulp, steam and power. The core of the Kraft process is the chemical delignification step in which the individual cellulosic fibers are separated from lignin to form pulp. Mixture of sodium hydroxyde (NaOH) and sodium sulfide (Na2S) are important delignification agents. After delignification, the fibers are washed and chemically bleached. Finally, they are drained, pressed, and thermally dried in a pulp machine [44] Xylans Xylans are hemicelluloses largely found in nature and are considered to be green polymers that may play an essential role in the renewability of waste products due to their biodegradable and biocompatible nature [45]. Xylans are heteropolymers possessing β-(1-4)-d-xylopyranose backbones, which are branched by short carbohydrate chains comprising: D-glucuronic acid or its 4-O-methyl ether, L-arabinose and/or various oligosaccharides [19]. Xylans are available in huge amounts as byproducts from the forestry, agriculture, wood, pulp, and paper industries [19] and thus also the largest source of renewable hemicellulose on Earth. Based on the hitherto-reported review papers on the primary structure of xylans from various plant tissues, xylan-type polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino) glucuronoxylans, (glucurono) arabinoxylans, arabinoxylans, and complex heteroxylans [19]. Most of the glucuronoxylans have single 4-O-methyl-α-d- -9-

27 glucopyranosyl uronic acid residues (MeGlcA) attached always at position 2 of the main chain Xylp units. This structural type is usually named as 4-O-methyl-D-glucurono-D-xylan (MGX). Both (arabino) glucuronoxylan (AGX) and (glucurono) arabinoxylan (GAX) have single MeGlcA and α-l-araf residues attached at position 2 and 3, respectively, to the β- (1 4)-D-xylopyranose backbone. These xylans are also the dominant hemicelluloses in the cell walls of lignified supporting tissues of grasses and cereals. They were isolated from sisal, corncobs and the straw from various wheat species [19, 46]. Arabinoxylans from oat spelt has a (1 4)-linked β-d-xylopyranosyl (Xylp) backbone to which a few α-l-araf grups are connected by (1 3)-glycosidic linkage at irregular intervals. This arabinoxylan also contain low amount of α-4-o-methylglucopyranosyluronic acid (MeGlcA) [47]. Structural formulas for both xylans were represented in Figure 2.2. HOOC H 3 CO 4 HO 3 5 O 2 OH 1 4 HO O OH 1 O HO O 2 O 1 O 4 HO 3 5 O 1 2 OH O HO OH 2 O 1 O a) HO O 4 OH O 5 O O O 4 OH 1 5 HOH 2 C 3 2 HO H 3 CO O 1 2 OH HOOC HO O 4 3 O 4 HO b) 5 5 O 2 O 1 O 4 HO 3 5 O 1 2 OH O Figure 2.2: Structural formula for 4-O-methyl-D-glucurono-D-xylan (a) and L-arabino- 4-O-methyl-D-glucurono-D-xylan (b) -10-

28 Potential resources of xylans are by-products produced in forestry and the pulp and paper industries (forest chips, wood meal and shavings), where Glucurono- and Arabino- Glucuronoxylans comprise 25 35% of the biomass as well as annual crops (straw, stalks, husk, hulls, bran, etc.), which consist of 25 50% arabino-, arabino-glucuronoxylans, (4-O- Methyl-D-glucurono)-L-arabino-D-xylan [46]. The molecular-weight values are different for different xylans. For cereal arabinoxylans, the Mw values were g/mol. The Mw values for arabino (glucurono) xylans and 4-O-Methyl-D-glucurono-D-xylan were g/mol and g/mol, respectively [19]. Cereals have low amount of lignin and % of hemicelluloses, mostly arabinoxylans. In Table 2.2 is represented arabinoxylan content in different cereals. Table 2.2: Amount of arabinoxylan in different cereals [48] Cereal material Arabinoxylan content (%) White flour 1-5 Whole grain 5-10 Bran Husks (spelts, hulls) Straw Oat spelt, mainly used as a low-value animal feed additive, contain up to 40 % of arabinoxylans, which is the major byproduct from oat milling [49]. Hemicelluloses from cereal straws have demonstrated to be a potential fermentation feedstock in production of ethanol, acetone, butanol, and xylitol [50]. Globally, however, the use of nonwood fiber is increasing faster than wood fiber [35]. -11-

29 2.1.2 Derivatisation and application of xylans Chemical modifications of xylans Due to low molecular weight and poor solubility of xylans, interest in their modification has been rather low in comparison to commercially available polysaccharides such as cellulose or starch [19]. Because of specific polysaccharide structure, and aim of improving the functional properties of xylans different functionalization and derivatisation have been investigated [51-56]. With different modifications, water solubility, antioxidant activities, antiviral properties have been achieved [53, 57-60]. Water absorbency could be achieved with introduction of carboxylic groups into xylans with different reagents namely citric acid, sodium monochloroacetate and succinic anhydride [53, 61]. Salam and co-workers [62] developed an absorbent foam material by the incorporation of carboxylic acid groups into hemicellulose based xylans with citric acid followed by cross linking with chitosan. Ebringerova and coworkers investigated mitogenic and comitogenic properties of xylans and compared it to the commercial immunomodulator zymosan [28]. They found out that disaccharide side chains could be important for expression of the immunostimulatory activity. Structurally and chemicaly modified xylans from birch wood were applied for nanoparticles preparation [29] and beech xylans were derivatised in order to prepare prodrugs for ibuprofen release [30]. Carboxymethylation of polysaccharides is one of the most versatile functionalization procedures [51]. The carboxymethylation of xylans has been studied in detail in order to obtain xylans with anionic properties [51, 63]. The structural characterization and carboxymethylation of arabinoxylan under different reactive conditions were described by Saghir et al. [52]. By Alekhina et al. [64] it was discovered that one of the advantages of using the carboxymethylation of xylans is the improvement of its film-forming properties. Films were flexible and highly transparent and have suitable oxygen barrier properties for packaging or coatings for fruits and vegetables. Cationisation is often used type of derivatisation. Cationic groups are usually introduced into xylan, similarly as to cellulose or starch, by the formation of ether bonds applying various reagents [19]. Cationisation was thoroughly investigated by Schwikal et al. [54, 65-68]. Etherification of hemicelluloses with cationic agents enhances their solubility and yields cationic or ampholytic polymers [66, 67]. Ebringerova end co-workers investigated -12-

30 antimicrobial activity against some Gram-negative and Gram-positive bacteria, depending on the DS and xylan type [68]. They found out that macromolecular backbone of the quaternized heteroxylans played an important role in the expression of their antimicrobial activity. Effectiveness in inhibiting the growth of bacteria was different at same degree of quaternization. They also found out that highly branched derivative showed a lower inhibitory property than the low-branched at about the same nitrogen content. Applications of xylans In last few decades, xylans had been investigated as renewable polymers for different applications, especially in nutrition [23, 24], food [69], medical [25, 26], pharmaceutical [70], [27], biomedical (especially as hydrogels) [71, 72] improvement of the pulps characteristics in the pulp and paper industries [38, 39]. Some researchers have discussed about films made from xylans, especially about their mechanical, oxygen, and moisture barrier properties. Escalante and coworkers [73] showed that addition of plasticizers resulted in less strong, but more flexible films. According to investigations of Goksu and coworkers [74] pure xylans were determined as non-film-forming polymers, with poor mechanical properties and hygroscopic nature, because of hydrophilicity of hemicelluloses [16]. For improving the film forming properties different additives and techniques were used. Goksu et al. [74] formed films from xylans using lignin as an additive to enhance film formation. Amount of lignin, necessary for forming of the film was 1 % (w/w lignin/xylan). With increasing of concentration of xylans the water vapor transfer rate decreased, but mechanical properties were improved. They also used plasticizer such as glycerol and these films were more stretchable with higher water vapor transfer rate and lower water solubility values. Films with glycerol were more suitable for coating purposes; films with lignin had similar characteristics as other biopolymer-based films. Other plasticizers used for improving the film-forming properties of xylans were: xylitol, sorbitol, propylene glycol [75-77]. Films from glucuronoxylan with xylitol and sorbitol were transparent, continuous, and flexible, with similar mechanical properties. Xylitol plasticized films were slightly less extensible at high plasticizer levels. Films with 35 wt. % of sorbitol had excellent oxygen barrier properties at 50 % relative humidity [75]. Mechanical properties of xylan films were different when -13-

31 different plasticizers were used [76, 77]. Using 10 % of glycerol, tensile strength was higher than films formed with same amount of sorbitol, but when 40 % of plasticizers were used results were inverse. When amount of sorbitol was 40 %, highest ability for transfer of water vapor and better oxygen barrier was achieved [77]. Films made with propylene glycol as plasticizer had poor mechanical properties [76]. Composite films from esters of arabinoxylan and cellulose [78] and biodegradable composite films from wheat gluten with xylans used as additives at different ph values were also investigated [79]. They showed that xylan could be used as an additive in wheat gluten film production, which is biodegradable. Due to their biocompatibility, biodegradability, and high stability, hemicelluloses were investigated for biomedical purposes [16]. Hemicelluloses/xylans have been intensively researched in last 10 years as material for making of hydrogels for medical uses. Gabrielii and Gatenholm [71, 80] investigated preparation of hydrogels from xylans with added chitosan at acidic condition. They analyzed swelling properties and influence of chitosan amounts in the gels. They found out that water uptake increased and crystallinity decreased with increasing of chitosan concentration. Beside hydrogels from xylan/chitosan mixtures, hydrogels from mixture of polyvinyl alchohol (PVA) and modified xylans were investigated by Tanodekaew and coworkers [81]. In this research it was discovered that gels from PVA and xylan modified with maleic anhydride (MA) have high ability to water uptake, higher strength, and are noncytotoxic and as such had a potential for biomedical application. Investigations of mixtures of xylans and kappa-carrageenan showed that with increase of concentration of xylans mechanical properties were improved. Gels from these mixtures were hard and elastic. Moisture content was increased when concentration of xylans was decreased [69]. Hettrich and coworkers found out that xylan gels and their hidrophobic/hydrophilic characteristic are dependent of substituent used during making of gels. Porosity of gels was different with differently used drying techniques [82]. Xylan from corn cobs was used for development of xylan-coated magnetic microparticles in order to protect magnetite from gastric dissolution [70]. Branan ferulate, arabinoxylan type, was blended and carried by an alginate-based fibre. In this research was shown that branan ferulate can improve the tensile and other mechanical properties of fibers at concentration 20 % and more [26]. -14-

32 Xylan-ibuprofen esters, dissolved in DMA (N,N-dimethyl acetamide), were used for formation of nanoparticles for controlled drug release [30]. Nanoparticles formation of the xylan mixed ester by nanoprecipitation from dilute solution was investigated. Esterification of xylans with ibuprofen was achieved via activation of the carboxylic acid with N,N - carbonyldiimidazole (CDI). Hydrophobicity of the xylan-ibuprofen esters could be influenced by introduction of sulfate groups. Sulfated xylans were studied for their anticoagulant, antiviral and anticancer activities. Pentosan polysulfate (PPS), derived from beechwood glucuronoxylan, had anticoagulant activity comparable to heparin as well as anti-tumor activity [56, 83]. There are very few investigations regarding adsorption of xylans onto different surfaces. Most of them are related to adsorption of xylans onto kraft pulp [39, 84, 85]. Adsorption of negatively charged xylans onto negatively charged mica and forces between xylan coated mica at ph 10 was studied by Österberg at al. [18]. Two different types of xylans were used, low charge density xylan with 0.5 % and high charge density xylan with 9 % of the segments carrying a carboxylic group. They found out that high charge xylan adsorbed in a more extended conformation than low charge, giving rise to stronger and more long-range repulsion. They also showed that, addition of electrolytes (CaCl2) reduced the range and magnitude of the repulsion [18]. Adsorption of xylans onto cellulose surfaces was investigated by different researchers [85-88]. Xylans interact with cellulose. They are irreversibly absorbed onto cellulosic surfaces and adsorption can occur in case of single molecules and in aggregates [88]. Adsorption of water soluble (glucurono) arabinoxylan onto cellulose fibers was studied by Köhnke et al. [87]. They demonstrated that adsorption of xylans depended of molecular structure. Xylans with lower degree of substitution more easily form aggregates in aqueous solutions and as such better adsorb onto cellulose surfaces. Köhnke and co-workers also found out that bleached softwood kraft pulp properties, such as tensile strength, beatability and resistance to hornification could be improved by the adsorption of xylans. They demonstrated as well that cationization process increased the adsorption, because of electrostatic interaction between anionic pulp fibers and cationic xylans [85]. Adsorption of xylans onto bleached pine kraft pulp was performed. Three types of derivatized xylans (xylan sulfate, carboxymethyl xylan and Xylan-4-[N,N,Ntrimethylammonium]butyrate chloride (XTMAB)) were used and their structure and solubility caracterized. Cationic xylans were adsorbed onto pulp fibers following Langmuir model, -15-

33 while in case of xylan sulfate and carboxymethyl xylan no sorption was observed, due to electrostatic repulsion of negatively charged surfaces [39]. Effect of the application of polyelectrolyte complexes (polyacrylic acid (PAA) and poly(allylamine hydrochloride)(pah), PAA/PAH, and Xyl/PAH for improving papermaking properties was characterized and feasibility of replacing PAA by xylan was investigated by Mocchiutti et al. [84]. It was found out that both complexes increased the papermaking properties and that natural polyelectrolyte such as xylan can be used instead of synthetic PAA. Until now there were no investigation reports on the adsorption of xylans onto synthetic surfaces. 2.2 Poly(ethylene terephthalate) Polyester was discovered by Carothers (Dupont), who found out that alcohols and carboxyl acids can be successfully mixed to create fibers. However, polyester made from mixing ethylene glycol and terephthalic acid was developed by British scientists Whinfield and Dickson. They patented polyethylene terephthalate or PET or PETE in Fibers had high melting points, chemical resistance and good hydrolytic stability and were commercialized through the 1950s as Dacron TM in the United States and as Terylene TM in the United Kingdom [3, 89]. Polyethylene terephthalate is by far the most widely used polymer in synthetic textile manufacture and serves as component for more than 95 % of all polyester textiles [3]. In 1990s polyester fiber amounted to 40 per cent of total man-made fibers [89]. In 2004 were produced 24 million Tons of polyester fibers and with this polyester approach to cotton [3]. PET is a linear macromolecular homopolymer (i.e., one repeating unit) formed from step reaction polymerization. It is nominally produced by the polymerization of terephthalic acid (C6H4(CO2H)2) and ethylene glycol (HO-CH2CH2-OH) monomers [3] in the presence of a catalyst and stabilizers. Polymerization of PET is a two-step process. The first step is a reaction between 2-to-1 ratio of ethylene glycol and terephtalic acid and formation of bis(hydroxyethyl)terephtalate (BHET). Second step is transesterification of BHET and formation of PET. PET is the first fibre-forming polyester of economic importance [90]. Schematic representation of reaction of polymerization is represented in the Figure

34 O O HO CH 2 CH 2 OH + HO C C OH Ethylene glycol Terephtalic acid O O * C C O CH 2 CH 2 O * n Poly(ethylene terephtalate) Figure 2.3: Schematic representation of reaction of formation of PET from ethylene glycol and terephtalic acid Structure and properties of PET fibers The conventional spinning and drawing process of commercial PET fibres involves the extrusion of the PET melt and winding-up at a different speed [90]. Depending of the spinning speed, can be obtained different kind of fibers: LOY (low oriented yarn); MOY (medium oriented yarn); POY (pre-oriented yarn); HOY (highly oriented) and FOY (fully oriented) [91]. Drawing is an essential fabrication process to achieve well-oriented structure with appropriate mechanical properties. Melt spun and drawn PET fibers have supermolecular structure, which is typical for majority of convetional synthetic fibers and consist of three different phases: amorphous and crystalline domains of the microfibrilar and the intermicrofibrillar regions. In model proposed by Prevoršek (1974) can be seen that crystallites extended amorphous molecules and disordered domains A (Figure 2.4) [90]. -17-

35 Figure 2.4: Fibrilar structure model of a PET fiber proposed by Prevoršek [90] Spun PET fibers, made by extrusion of the melt through spinnerets are characterized by little if any molecular orientation. Crystallization occurs only on drawing [90]. Commercial PET fibers are normally semicrystalline, highly oriented, but degree of orientation normally differ between more crystalline and less crystalline regions [90]. From the crystal structure of PET (Figure 2.5) can be seen that unit cell is triclinic and contain one repeating unit. The dimension of the unit cell varies with the processing history. Because of weaker attraction between chains and because of stiffening effect, crystallization of poly(ethylene terephtalate) is difficult [90]. -18-

36 Figure 2.5: Chrystal structure of polyester unit cell [90] PET fibres are poduced as monofilaments, multifilament yarns, staple fibre and tow, in a wide range of counts and staple lengths to suit virtually all textile requirements [92]. The standard PET fibres have mostly circular cross-section, some types have trilobal and smooth surface. The molecular mass of the fibreforming PET lies between and (DP ). The lowest molecular mass is (DP 65 70) [90]. The density of fibers is between 1.38 and 1.40 g/cm 3, the glass transition temperature (Tg) between 60 and 70 C and melting temperature is between C. They have excellent resistance to chemicals, better to acids and less resistance to alkali [90, 93]. PET is known as very hydrophobic and insoluble in water [3]. Because of its lacks of polar groups in molecular structure, such as -COOH and OH, PET has low surface-free energy and poor wettability [94, 95]. For the application of PET in sorption related industries surface activation is very important. Because of its high durability, strength, resilience, blend ability, dimensional stability, its great capacity for modification, good resistance to microorganisms and its low cost [3, 7, 96, 97] polyester is presented in many different areas from conventional clothing and garment applcations, medical applications, industrial applications, like automotive, home furnishing, geotextiles, etc. -19-

37 2.2.2 Functionalisation of polymer surfaces for technical and medical applications Functional polymers are macromolecules that have unique properties or uses. Most functional polymers are based on simple linear backbones. Properties of functional polymers can be determined by the presence of chemical functional groups [98]. Functional polymers are developed for a wide range of diverse applications, such as organic catalyst, medicine, optoelectronics, biomaterials, building materials, photographic materials etc. [98, 99]. Large group of functional polymer composite materials is applied in medicine, i.e. orthopaedics as spine rod, spine disk, nail, bone plate and screw, hip replacement, etc. [100]. Biomaterials in form of implants (sutures, bone plates, joint replacement, ligaments, vascular grafts, heart valves, intraocular lenses, dental implants etc.) and medical devices (pacemakers, biosensors, artificial hearts, blood tubes, etc.) typicaly are used to replace and/or restore the function of organs, to improve function, to assist in healing and to correct abnormalities [101]. Polyester is a widely used as packaging material such as bottles/containers, films, as a filament, fibers, fabrics and as a base material for photographic films and recording tapes application [102]. Polyesters used in architectural application such as architecture membranes or tensile membrane are normally woven, often coated or laminated on both sides [102]. Polyethylene terephthalate (PET) in the form of films is used in many technological fields for a wide variety of applications (packaging, decorative coatings, capacitors, ect.) since it has some excellent bulk properties, such as very good barrier properties, crease resistance, solvent resistance, high melting point, resistance to fatigue, and high tenacity as either a film or a fiber. However, PET is sometimes an unsuitable material to use due to its low surface free energy, leading to poor wettability and poor adhesion [95]. Fibers based on polyethylene terephtalate are used in textile industries for a variety of applications ranging from filtration, composites, tissue engineering and electronic textiles [7]. They are relatively cheap and in applications mostly provide optimal mechanical properties (strength, elasticity, abrasion resistance), but they are usually lacking in physiological properties and some specific functionalities. These problems could be solved by modifications of PET surfaces or bulk. There are many different strategies for modification of PET for a number of reasons. Chemical modification of PET can include co-monomers in the polymerization process, -20-

38 additives to the polymer melt before extrusion, which could in large extent change the supermolecular structure and as such, the physical properties of end-product. Therefore, much more preferable are surface treatments. Among the most conventional surface treatments are application of additives after fiber or foil is formed [3]. Among other surface specific tretaments of PET surfaces, most frequently reported are: surface grafting, gas plasma treatment, enzymatic treatments or coating/adsorption of polymers. Among the treatments for higher hydrophilicity alkaline hydrolysis was widely studied [11]. Hydrolysis leads to an increase of free hydroxyl- and carboxylate end groups changing the surface properties of the treated material, where smooth surface of PET become rough and with this adhesion of functional products for PET material is increased [3, 97]. With the progressive alkaline hydrolysis, the PET chains on the fiber surface are etched away and the fiber diameter is reduced. The mass loss from alkaline hydrolysis indicates the extent of hydrolysis and reduced fiber dimension [103]. Gas plasma treatment was widely studied in recent years for improving the wettability of PET surfaces [7]. Plasma is produced when a gas at low pressure and at room temperature is submitted to an electric field. The result is an atmosphere full of ions, atoms, molecules and free radicals [7, 104]. The mostly used gasses during plasma treatments are: O2, N2, NH3, H2O, CO2, air, noble gases or mixtures of gases [105, 106]. Plasma does not pollute the environment and with their uses water and energy can be saved [94]. During the gas plasma treatment surface is changed to a limited depth, while basic properties of materials stay unchanged [94, 104]. A major disadvantage of plasma treatment is the aging effect of the surface modification created. The hydrophilicity is often lost with time [107]. Using atmospheric air plasma treatment can be produced hydroxyl and carboxylic groups on the surface of PET materials, such as film, nonwoven and woven and oxidation can be generated. Polar groups at the polyester surface can enhance adhesion with silicon resin [108]. Because of increased polar component, achieved with plasma treatment onto PET surface, adhesion of zinc oxide coating can be improved [109]. Increasing of carboxyl groups was responsible for higher hydrophilicity of the nonwoven PET surface. With increase of the time of the plasma exposure were achieved higher hydrophilicity, but after a certain time saturation occured [106]. Pelagade et al. investigated improvement of hydrophilicity of PET surfaces, using Ar-plasma treatment [5]. Influence of different plasma onto wettability of PET -21-

39 fibers was investigated. Using different gasses different morphological changes were achieved as well. They also demonstrated that adsorption of water was increased with time of exposure [110]. Memhood et al. investigated adhesion of polypirrole onto PET surfaces in order to introduce surface conductivity. The adhesion onto PET thin films and fabrics was improved when low pressure plasma was used [111]. PET surfaces (untreated and pretreated with air atmospheric plasma) were functionalized with chitosan for improving the sorption of surfactin produced by Bacillus subtilis. High hydrophobicity of both surfaces was achieved. Immobilization of chitosan onto PET surfaces was achieved with ionic interactions [112]. Cazan et al. investigated preparation and adhesion of different components of rubber-pet- HDPE composite. The adhesion was controlled by functionalization of PET surfaces by PEG (polyethylene glycol) and SDS (sodium dodecyl sulphate). Mechanical properties such as tensile strength, impact strength and compression of composites were improved [113]. To achieve antibacterial effect and electrical conductivity of PET surfaces, wetchemical one-bath and two-bath metyllisation method was developed by Ongar et al. Silver layer with silver particles was attached onto PET surface by chosing suitable chemicals and by varying the internal and external process parameters for wet-chemical one-bath and twobath silvering [114]. Shin et al. functionalized PET films surfaces with poly-l-lysine solution or trichloro(1h,1h,2h,2h-perfluorooctyl) silane (FOTS) in order to change their triboelectric properties. Functionalization method changed the triboelectric sequence of the materials in a triboelectric series, which in turn greatly improved the output performance of the TEGs (triboelectric generators). PET surfaces indicated negatively charged state with opposite triboelectric polarity when they are in contact with each other [115]. Medical applications Using of Polyester in medical devices has started for more than 50 years ago, thus its excellent mechanical properties and processability provide wide variety of medical applications [3, 116, 117]. Among medical devices made from PET have to pointed out vascular grafts, surgical meshes, implantable sutures, sewing cuffs for heart valves to components for percutaneous access devices [118]. PET has been as well widely studied as polymeric matrices and supports for immobilization of cells and biomacromolecules [116, 119]. It is used in cardiovascular implants such as artificial heart valve sewing rings and -22-

40 artificial blood vessels [120]. Currently, PET (Dacron) grafts are used as a replacement for all major large vessels (the ascending, thoracic and abdominal aorta), and many extra-anatomic bypass grafts [121]. The main problem of materials suitable for implantation is biocompatibility, which is defined as acceptance/rejection of an artificial material by the surrounding tissues and by the body [92]. In the field of PET devices used in blood contact thrombosis is still a huge problem, even after 60 years of extensive study of blood compatibility [122], therefore the surfaces of all the blood contacting surfaces of devices made from PET had to be functionalized with antithrombotic agents. Surface functionalization of PET materials was applied in many cases in order to tune its reactions when in contact with tissues, blood, and/or biological liquids. Alkaline hydrolysis was used for fabricationg nonwoven fibrous matrices from polyethylene terephtalate (PET) with predictable porosity, pore size, and fiber diameter. It was demonstrated that with increase of surface roughness of PET fibers, cell adhesion enhanced, but decrease in fiber diameter caused an increase in the surface curvature of the fibers and a decrease in available surface area for cell attachment. PET matrices are biocompatible and can used as scaffolds for cell culture and tissue engineering [11]. Pre-treated PET films using alkaline hydrolysis, was used to modify its surface with layer-by-layer assembly of chitosan and chondroitin sulfate. This functionalized PET fabric was used for stronger ability to adhere endothelial cells and to maintain the endothelial function [12]. Technical applications Technical applications of fibrous materials in a contact with human body have similar problems. They have to satisfy in one side high mechanical stability and chemical inertness, and on the other side physiological convenience such as absorption of moisture and hydrophilicity. This group comprises for example protective textiles in medicine, where are the most important request beside physiological convenience, resistance and impermeability for microorganisms. About 25 % of the total worldwide use of fibre in textiles is used for technical textiles. In advanced countries this amount is about 40 %. Automotive sector is the largest user of technical textiles. The amount of fibre used for a standard passenger car is about 25 kg [102]. Polyester is one of the most used fibers in automotive industry with about 42 % of total fiber amount [102]. Automotive elements, where polyester is used are: seat-covers, liners, -23-

41 side-panels, seat-backs, door and door-panels covering, roof-liners, door pillars etc. PET material mostly in fibrous woven forms is used also in civil engineering in cases when concrete external reinforcement is required. Woven, knitted and needle punched nonwoven polyester materials are also available in geotextiles and geogrids. Monofilaments, roving (tows) and fibrillated fibres are specially used to reinforce cementitious or polymeric matrices [102]. 2.3 Theoretical principal of analytical methods Attenuated total reflectance (ATR) and Raman infrared spectroscopy Selection rule for infrared spectroscopy is changing of an electric dipole moment of the molecule during the vibration [123]. Infrared spectroscopy is a versatile experimental technique and it is relatively easy to obtain spectra from samples in solution or in the liquid, solid or gaseous states. Attenuated total reflectance spectroscopy ATR (attenuated total reflectance) spectroscopy utilizes the phenomenon of total internal reflection [123]. The absorption band maxima are similar to infrared spectroscopy in transmission mode but usually different absolute intensities and peak broadening are observed. For the measurement setup, an ATR-IR crystal is used which is made out of high refractive index materials such as germanium and zinc selenide. The beam of infrared light is passed through the ATR crystal (Figure 2.6) in such a way that it reflect at least once off the internal surface in contact with sample. During reflection, an evanescent wave is formed and extends into the sample. The penetration depth of infrared light depends on the material and on the wavelength and is typically in the range of μm. The beam is then collected by a detector as it exits the crystal [122]. The resultant attenuated radiation is measured and plotted as a function of wavelength by the spectrometer and gives rise to the absorption spectral characteristics of the sample [123]. -24-

42 Figure 2.6: Internal reflection ATR plate showing path of light [124] Raman spectroscopy Raman spectrometry is an alternative, and often complementary, way to measure vibrational spectra to infrared spectrometry [125, 126]. The Raman effect occurs when a sample is irradiated with intense monochromatic light, usually from a laser [127]. Raman scattering occurs when a molecular vibration in a certain mode alters the electronic distribution of the molecule and consequently the optical polarizability for the incident light [127]. In Raman scattering, the light interacts with the molecule and distorts (polarizes) the cloud of electrons round the nuclei to form a short-lived state called a virtual state. This state is not stable and the photon is quickly re-radiated (Figure 2.7) [128]. Figure 2.7: Diagram of the Rayleigh and Raman scattering processes [128] -25-

43 The elastic or Rayleigh scattering arises from a transition that starts and finishes at the same vibrational energy level. The shifts to lower and higher frequencies are known as Stokes and anti-stokes Raman scattering, respectively. Stokes Raman scattering arises from a transition that starts at the ground state vibrational energy level and finishes at a higher vibrational energy level, whereas anti-stokes Raman scattering involves a transition from a higher to a lower vibrational energy level [129]. The intensity of bands in the Raman spectrum of a compound are governed by the change in polarizability, that occurs during the vibration [125] X-ray photoelectron spectroscopy X-ray photoelectron spectroscopy (XPS or ESCA-electron spectroscopy for chemical analysis) is surface sensitive technique and is used for measuring the elemental composition of the surface (top of the surface with depth 2-10 nm). Principle of this technique is based on the photoelectric effect, where surface of material is irradiated with X-rays (monochromatic or unfiltered Mg Kα or Al Kα) and photoelectrons are injected. Energy of emitted electrons is kinetic energy and is measured in an electric spectrometer. The binding energy of the electron Eb can be obtained using the Einstein relation, where emitted photoelectrons are collected by an electrostatic energy analyzer as a function of their kinetic energy (Ek) [130]. E h E (2.1) b k where h is the Planck s constant, ν is the frequency of the exciting radiation, and is the work function of the spectrometer. The binding energy is characteristic of the atom and orbital from which the electron was emitted. In the Figure 2.8 are presented photoelectron emitting during the injection. -26-

44 Figure 2.8: Schematic presentation of X-ray photoelectron emitting [131] Atomic force microscopy Atomic force microscopy is mostly used technique for investigation of morphology of surfaces. The basic principle is presented in Figure 2.9. Sharp tip (r~50 nm), which scans in the near-field of the surface (usually < 10 nm), is attached to the loose end of an oscillating cantilever (force-measuring spring < N/m). Normal (cantilever deflection) and lateral (cantilever torsion) forces can be detected as movements using a four-segment photodiode [130]. These movements can be used to obtain the morphology of the surface as three dimensional pictures. The lateral (x-y direction) and horizontal (z-direction) resolution of AFM instrument are usually better than 10 nm and 1 nm, respectively [132]. AFM can be used in tapping and contact mode. In tapping mode, cantilever is externally driven to oscillate around its resonance frequency with constant amplitude. The interaction between the tip and the sample determines the change of oscillation amplitude. In contact mode, the tip is in constant contact with the surface of the sample during scanning and can cause strong lateral forces between the tip and the sample, which can destroy the surface and the image. Atomic force microscopy can be performed in many environments, including ultrahigh vacuum, ambient conditions and liquids [127]. -27-

45 Figure 2.9: Schematic representation of AFM basic principle [133] Surface wettability and surface free energy Wetting of solid by a liquid could be determined quantitatively with contact angle measurements [134]. When a drop of liquid is placed on solid surface, three phase are create: solid-liquid, solid-gas and liquid-gas (Figure 2.10) [134]. Geometrically, contact angle is defined as an angle, formed by the intersection of the two planes tangent to the liquid and solid surfaces at the perimeter of contact between the two phases and the third surrounding phase. Typically, the third phase will be air or vapor [135]. Perimeter of contact between three phases is called three-phase contact line or the wetting line [135]. Contact angle could be determined from Young's equation (2.2) [135, 137]: SV SL cos (2.2) LV where γsv is interfacial tension between solid and vapor, γsl is interfacial tension between solid and liquid and γlv between liquid and vapor. -28-

46 Figure 2.10: Vectorial equilibrium for a drop of a liquid resting on a solid surface to balance three forces [136] Surface free energy of solids can be determined from contact angle values through different approaches or methods. Most commonly used approach is Van Oss and Good, where total surface free energy (SFE - γs TOT ) is calculated using acid-base approach [138]. With this approach the total surface free energy can be divided into Lifshitz-van der Waals interactions (γs LW ), comprising dispersion, dipolar, and induction interactions, and the acid-based interactions (γs AB ), comprising all the electron donor-acceptor interactions, like hydrogen bonding [138]. TOT S (2.3) LW S AB S The acid-base interactions were subdivided into electron donor γs - (Lewis base) and electron acceptor γs + (Lewis acid) parts. AB S 2 (2.4) When determining the components of surface free energy (SFE) and parameters (γs LW, γs + and γs - ) of the solid surfaces, contact-angle measurements of at least three different liquids -29-

47 with known surface tension components (γl LW, γl +, γl - ) had to be performed [138]. The SFE components of the solid surface were then calculated using Young s equation [139]: LW LW (1 cos ) 2( ) (2.5) L S L S L S L Quartz crystal microbalance with dissipation unit (QCM-D) Quartz crystal microbalance with dissipation unit (QCM-D) is a sensitive and practical technique for measurements of macromolecule adsorption in several solid-liquid interface studies at real-time [140]. It is a simple and high-resolution mass sensing technique, based on the piezoelectric effect of a quartz sensor. With this method the mass adsorption could be observed on solid surfaces, i.e. thin films, while simultaneously providing the information on the viscoelastic properties of the adsorbed layer [141]. The QCM-D device enables measuring the mass of thin film deposition onto quartz crystal sandwiched between two electrodes [122]. By applying an AC voltage across the electrodes, because of piezoelectric properties of quartz, could excite the crystal oscillation. Usually electrodes are made from gold, but can be thin coated with other materials. Using QCM-D technique, changes in frequency and energy dissipation caused by an oscillation of piezoelectric crystal could be measured, during the adsorption (mass changing) on the crystal surface. Frequency of the crystal is decreased when mass, viscosity and roughness of the electrode is increased [122]. In the Figure 2.11 is presented a typical QCM-D frequency and dissipation response during adsorption of molecules. -30-

48 Figure 2.11: Frequency ( f) and dissipation ( D) response during the QCM-D measurement [122] In the Figure 2.11 three phases of adsorption/desorption are represented: 1. When small amount and small molecules are adsorbed on the surface, the change in frequency ( f) and dissipation ( D) occurs. The change in D is small owing to the rigidity of the adsorbed layer. 2. More complex (elongated) molecules, binded on the surface of the first layer, caused higher frequency decrease and much higher dissipation increase. This indicates that much softer and thicker layer is adsorbed. The reason for such behavior could be in the incorporation of the water molecules into the adsorbed layer. 3. When adsorbed amount is rinsed and when elonged molecules are removed, frequency and dissipation reduce to the first level. The frequency change of the crystal is in correlation to the adsorbed mass according to the Sauerbrey relationship [142]: C f m (2.6) n -31-

49 where m is change in mass of the crystal, C is the mass sensitivity constant (17.7 ng Hz -1 cm -2 for a 5 MHz quartz crystal), n is the overtone number (1, 3, 5, etc.) and f is the frequency change Titration methods Titration is an analytical technique which allows the quantitative determination of a specific substance (analyte) dissolved in a sample. It is based on a complete chemical reaction between the analyte and a reagent (titrant) of known concentration which is added to the sample. The titrant is added until the reaction is complete [143]. The analyte reacts with the titrant according to the stoichiometric ratio defined by the corresponding chemical equation. The equivalence point corresponds to the point at which the ratio of titrant, added to the analyte, originally present equals the stoichiometric ratio of the titrant to the analyte, defined by the chemical equation [127]. End of the titration reaction has to be easily observable. This means that the reaction has to be monitored (indicated) by appropriate techniques, e.g. potentiometry (potential measurement with a sensor) or with colour indicators [143]. Polyelectrolyte titration Polyelectrolyte titrations are based on stoichiometric reaction between oppositely charged particles. The equivalence point can be identified by measuring fluorescence, absorbance, conductivity, potential, current, etc. Usually the titration s end point is determined spectrophotometrically with the change in color of an indicator [144, 145]. Cationic polyelectrolytes are titrated with an anionic polyelectrolyte in the presence of a cationic indicator, typically toluidine blue (TB). The color during titration change from blue in the beginning, to pink, when it complexes with excess anionic polyelectrolyte [146]. Anionic polyelectrolytes are titrated with a cationic polyelectrolyte in the presence of a toluidine blue (TB). The color during titration change from pink in the beginning, to blue, when it complexes with excess cationic polyelectrolyte. The complex formation can be written as: A + C AC (K1) (2.7) -32-

50 A + I AI (K2) (2.8) where A is an anionic polyelectrolyte, C is a cationic polyelectrolyte and I the indicator. In order for this method to work, the complex formation of AC must be preferred over the AI complexation, i.e. K1» K2 [146]. ph potentiometric titration Potentiometric titration is a method which is used for determination of the surface charge of polymers. Polymer sample is dissolved or dispersed in a known excess of acid (e.g. HCl) and then titrated with base (e.g. NaOH, KOH), while the ph value of the solution is monitored with a calibrated ph-sensitive glass electrode. The titrating system contains ionic species from the acidic and alkaline titrant such as K +, Cl -, H +, OH - and some conjugated species of the analyte or contaminants, Ak n, where n is the charge number and k is an enumerator. Total ionic charge, Q, due to presence of Ak n can be calculated using the eq. 2.9 [147]. n ph FV n A FV Cl K OH H Q (2.9) t k k t where square brackets denote the concentration of ionic species in mol 1-1, F is the Faraday constant and Vt is the total volume. The concentration of K + and Cl - are known from the added volume and the concentration of the burette solutions. The concentration difference of H + and OH - is determined by the ph. For the blank titration and in the absence of contamination, only K +, Cl -, H + and OH - are present, thus Q becomes zero for any measured ph value. Therefore, the excess charge present on the titrated sample can be calculated (eq. 2.10). ph FV Cl K K Cl Q (2.10) t blank blank where [K + ]blank and [Cl - ]blank imply the blank concentration of K + and Cl -, respectively. The values of ([K + ]blank - [Cl - ]blank) at the ph values measured for the analyte system can be -33-

51 obtained by interpolating the blank titration curves. The Q(pH) curves are referred to as the charging isotherms [147] Methods for antimicrobial activity evaluation Antimicrobial activity of functionalized textiles can be tested qualitatively and quantitatively with many standardized test methods. Qualitative tests are: AATCC TM147, AATCC TM30 (American Association of Textile Chemists and Colorists Test Method), ISO 20645, ISO (International Organization for Standardization), and SN , SN (Swiss Norm). Basic of these methods is agar diffusion test. Agar plates, with the test bacteria and with tested textile on its surface are incubated for 24 to 48 h, depending on microorganisms used. After incubation, the plates are examined for bacterial growth directly underneath the fabric and around its edges. Antimicrobial agent was acting inhibitory on the surface with no bacterial growth [148]. These methods are relatively quick, simple, cheap and well defined. Weakness of these methods is its unappropriation for all kind of textiles and for analyses of efficacy of different antimicrobial agents [148, 149]. Quantitative tests are: AATCC TM100, ISO 20743, SN , JIS L 1902 (Japanese Industrial Standards), and ASTM E These methods are more broadly applicable because they can be used for all types of textiles and antimicrobials, but are expensive [148, 149]. One of the most used quantitative method is ASTM E 2149 (Shake flask method). This is a standard test method for determining the antimicrobial activity of immobilized antimicrobial agents under dynamic contact conditions [150]. It was developed for routine quality control and screening tests, in order to overcome the difficulties of using classical static antimicrobial test methods during the evaluation of substrate-bound antimicrobials. These difficulties are about ensuring contact of inoculum with treated surface, use of inappropriately applied static conditions, sensitivity, and reproducibility. The dynamic shake flask method is particularly appropriate for non-leaching antimicrobials whilst the dynamic contact conditions are applied to the samples [151]. Quantitative testing can be used for all antimicrobials. Methods are long and expensive, because they require a number of manipulations to the sample and organisms [149]. -34-

52 3 EXPERIMENTAL PART 3.1 General experimental plan Experimental work of this dissertation consisted of four phases. In the first phase xylans were chemically modified: carboxymethylated and cationized. In second phase films were prepared from nonmodified and chemically modified xylans and characterized regarding chemical and physical surface properties. In the third phase interactions between xylans and PET model surfaces were studied using QCM-D technique and in the fourth phase adsorption of xylans onto real surfaces - PET fabrics was performed and chemical and physical properties of treated samples analysed. Xylans (Glucuronoxylan, Arabinoxylan) Chemical modification - Carboxymethylation - Cationization Substrate (PET) Thin model film preparation Adsorption/desorption studies (QCM-D) Film preparation Solutions XPS, goniometry Chemical and physical analysis: Elemental analysis, SEC, ATR- FTIR, Raman, Titration methods, goniometry, AFM, XPS Definition of adsorption/desorption conditions (ph, electrolytes, concentration, anchoring polymers) Real surface treatment (PET fabric, hydrolysis, anchoring polymers) Titration techniques, Acid Orange, Methylene blue, SEM, goniometry, ASTM E 2149 Definition of optimal system Figure 3.1: Shematic illustration of the experimental work performed in the thesis -35-

53 (PET - polyethylene terephthalate, SEC - Size exclusion chromatography, ATR-FTIR - attenuated total reflectance Fourier transform infrared spectroscopy, AFM - atomic force microscopy, XPS - X-ray photoelectron spectroscopy, QCM-D Quartz crystal microbalance with dissipation unit, SEM Scanning electron spectroscopy, ASTM American society for testing and materials) 3.2 Materials Xylans Three types of xylans were applied in this research. Two types of glucuronoxylans, marked as BX and BXL and one type of arabinoxylan, marked as OX. 4-O-methyl glucuronoxylans from beech wood (BX with Mw = g/mol, Mw/Mn = 1.9 and BXL with Mw = g/mol, Mw/Mn = 1.4) were obtained by the extraction of beech wood holocellulose. The holocellulose was prepared by treatment of the starting material with 4.5 % sodium chlorite for 120 h at room temperature [152]. BX type of xylan was applied as a source sample for carboxymethylation, BXL type of xylan was applied as a source sample for cationisation. Arabinoxylan from oat-spelts (OX) (Mw = g/mol; Mw/Mn = 2.3) was obtained by sodium hydroxide extraction and precipitation as described in [153]. This type of xylan was used for both types of modification. PET fabric As a real surface and substrate for functionalization, woven PET fabric was used. The fabric s weave was canvas with a weave density of 21 threads/cm. Fabric thickness was 0.51 ± 0.0 mm and weight per unit area of ± 1.4 g/m 2. Chemicals and reagents Carboxymethylation: Sodium monochloroacetate SMCA (Mw = g/mol; HCLO4 98 %), Ethanol (absolute, 99.8 %; Mw = g/mol), Methanol (Mw = g/mol; 80 wt. % in H2O), sodium hydroxide - NaOH (Mw = 40 g/mol, 25 wt. %), Acetic acid CH3CO2H (Mw = 60.05; 40 wt. % in H2O). -36-

54 Cationization: 1,2-dimethoxyethane DME (Mw = g/mol, anhydrous, 99.5 %), (2,3-Epoxypropyl) trimethylammonium chloride EPTA (Mw = g/mol; 70 %), Ethanol (absolute, 99.8 %; Mw = g/mol), sodium hydroxide NaOH (Mw = 40 g/mol, 4.5 wt. %), hydrochloric acid HCl (Mw = g/mol, 1 M in H2O). Preparation and pretreatment of PET surfaces: 1,1,2,2-tetrachloroethane (Mw = g/mol; 98 %), sodium hydroxide NaOH (Mw = g/mol, 4 M in H2O), hydrochloric acid HCl (Mw = g/mol, 1 M in H2O). Adsorption of xylans onto PET surfaces: Sodium chloride NaCl (different molar concentrations), calcium chloride CaCl2 (different molar concentrations), Polyethylene imine PEI (Mw = g/mol; branched), Poly(vinylsulfonic acid, sodium salt) PVSA (25 wt. % in H2O). Determination of surface free energies (SFE): Diiodomethane DM ( g/mol; 99 %), Formamide (Mw = g/mol; puriss, p.a., ACS reagent; 98 %). Determination of Acid Orange 7 dye adsorption: C.I. Acid orange 7 dye, Mw = g/mol. Determination of Methylene blue dye adsorption: AlliedSignal Riedel-deMaen dye, Mw = 319,86 g/mol Milli-Q (MQ) water from a Millipore water purification system (Ma, USE, resistivity 18.2 MΩ cm, ph 6.8) was used for the preparation of all aqueous solutions. 3.3 Methods Chemical modifications of xylans Two types of chemical modifications were used in order to introduce specific functional groups into basic xylan chemical structure. Carboxymethylation was applied for introduction -37-

55 of carboxyl groups, thus it is known that higher amounts of the carboxyl groups present in the materials structure contribute to improvement of its hydrophilicity. Cationization was applied for introduction of quaternary amino groups, which are known as a carrier of materials antimicrobial properties. Carboxymethylation Xylans were carboxymethylated according to [52]. About 2.5 g of purified xylan sample was suspended in 180 ml of 99.8 % ethanol, and 9 ml of 25 % aqueous NaOH solution was added. The mixture was vigorously stirred for 1 h at room temperature. Sodium monochloroacetate (6.6 g) was added, and the temperature was raised to 55 C. After 5 h of etherification, the product was filtered off, suspended in 80 % aqueous methanol, neutralized with 40 % acetic acid, and washed five times with 50 ml of ethanol. Finally, the product was dried at 40 C in vacuum overnight. The samples were labeled as follows: CMBX for carboxymethylated glucuronoxylan and CMOX for carboxymethylated arabinoxylan. Possible structural formulas of carboxymethylated xylans are presented in Figure HO O O - O O Na + H 3 CO 4 HO 1 HOOC O 3 HO 4 5 O 2 1 OH O O O O 6 7 O - 4 O Na O OH 1 O HO 4 3 O - Na + O 7 6 O 2 5 O 1 O a) -38-

56 HO O 4 3 O 5 O 2 O O 4 OH 5 HOH 2 C 3 O 7 O O - Na + O 2 Na + O O O 1 2 OH HOOC H 3 CO 4 5 O HO O HO O O 1 O 4 HO 3 O O - O O Na + 1 O b) Figure 3.2: Structural formulas of carboxymethylated 4-O-methyl-D-glucurono-D-xylan (CMBX) (a) and carboxymethylated L-arabino-4-O-methyl-D-glucurono-D-xylan (CMOX) (b) Cationization Xylans were cationized according to modified procedure of Schwikal et al. [65]. Procedure was modified in order to achieve higher degree of substitution (DS). 2 g of xylan was suspended in 15 ml of water and heated under reflux for 15 min. The mixture was cooled to a room temperature and 2 ml of NaOH (4.5 %) was added. After 45 min 1,2-dimethoxyethane (DME) (1.5 ml : 1 ml in relation DME : water) were added to the dissolved xylan. After that, (2,3-Epoxypropyl) trimethylammonium chloride (EPTA) (70 %) was added drop wise (EPTA was for OX ml; for BXL was ml). This mixture was stirred for 24 h at room temperature. After this time mixtures were neutralized with 1 M HCl and the product was precipitated with ethanol (absolute) and washed three times with ethanol. Product was dissolved again in water and reprecipitated with ethanol and washed again three times with ethanol. Product was dried under vacuum (at 40 C for 24 h). The samples were labeled as follows: CBX for cationized glucuronoxylan and COX for cationized arabinoxylan. Possible structural formulas for cationized xylans are presented in Figure

57 HOOC H 3 CO 4 HO O 1 4 HO 3 5 O 1 2 O 6 7 OH N + Cl - 9 O HO 4 3 OH O 2 5 O 1 O HO O 1 2 O 6 7 OH 8 N Cl - O HO OH 2 O 1 O a) HO O O N OH 9 Cl - OH 2 1 O O O O 4 OH HO O 2 O N + OH 9 9 HOOC H 3 CO 4 5 O HO O 3 2 HO 1 1 O O Cl - O 4 HO 3 5 O 1 2 O 6 7 OH 8 N Cl b) Figure 3.3: Structural formulas of cationized 4-O-methyl-D-glucurono-D-xylan (CBX) (a) and cationized L-arabino-4-O-methyl-D-glucurono-D-xylan (COX) (b) Xylan film preparation In order to analyze surface properties of solid non-modified and chemically modified xylans formed from water solutions, films were prepared by casting method. Nonmodified and -40-

58 modified xylans were dissolved in milli Q water (0.5 % (w/v)), heated to boiling point, and filtrated through polytetrafluoroethylene filters with pore size 1.0 μm. Samples were filtrated in order to remove rests of impurities from modification process, such as fibers from filter paper during filtration of products. Xylan solution (0.5 %) was casted into glass Petri dishes (3.5 cm in diameter). Solution was dried under vacuum over night at 40 C Preparation and pretreatment of PET surfaces Preparation of model PET surfaces Spin coated PET films were prepared by dissolving 1 wt % of PET foil (Mylar foil, with a thickness of 175 μm) in 1,1,2,2-tetrachloroethane and heating (T 150 C) until the foil was dissolved. When the solution was cold, it was filtered through a 0.2 μm Acrodisc GHP filter. 30 μl of solution was spread on a 14 mm gold quartz crystal and spin coated using spin coater Polos (Figure 3.4) at a maximum of 2000 rpm for 60 s. Figure 3.4: Spin coater Polos (Germany) Pretreatment of real PET surfaces Prior to the adsorption of xylans, PET fabric was washed for 30 min at 60 C with non-ionic detergent (1 g/l) and Na2CO3 (1 g/l). After that fabric was washed with distilled water until conductivity of pure water was reached and air-dried over night. -41-

59 In order to activate PET fabric surface, washed PET fabric was hydrolysed in 4 M NaOH solution for 35 min at 70 C. After that, the fabric was immersed in 1 M HCl for 10 min to neutralize the NaOH and to stop the hydrolysis. After neutralization process PET fabric was demineralised with 0.1 M HCl. Fabric was air-dried after rinsing with distilled water until constant conductivity of rinsing water. O O * C C O CH 2 CH 2 O * n Na + HO - O * C COO - Na + + OH CH 2 CH 2 O * Figure 3.5: Shematic representation of alkaline hydrolysis of PET [3] Adsorption of xylans Adsorption of xylans onto model PET surfaces In order to define an optimal adsorption process for the real PET surfaces, adsorption of differently chemically modified xylans as well as anchor polymers were studied using quartz crystal microbalance with dissipation unit (QCM-D). The apparatus E4 instrument (Q-Sense, Gothenburg, Sweden), coupled with peristaltic pump (Ismatec) was applied (Figure 3.6). The -42-

60 analysis is based on the measurement of changes in resonance frequency of a thin AT-cut piezoelectric quartz crystal disc [ ]. It allows simultaneous measurement of change in resonance frequency and energy losses (dissipation) when the mass adsorbed on an oscillated piezoelectric crystal changes. The resonant frequency (f0 5 MHz) of the crystal decreases when additional mass is adsorbed on its surface. All adsorption analyses were performed at flow rate of 0.1 ml/min. In the pre rinsing step flow rate were higher, because of cleaning the surface of coated crystals. The influence of ionic strength, ph and concentration of xylans' solutions as well as the influence of the precense of anchoring polymers on the adsorption of nonmodified and chemically modified xylans onto PET model films was investigated. For the determination of the influence of ionic strength two types of electrolytes were applied: NaCl and CaCl2 with different ionic strengths (0.05 M, 0.1 M, 0.3 M, 0.5 M and 0.7 M). Influence of ph was investigated from acidic to alkaline region at ph 4, ph 5, ph 7 and ph 9. Concentration of xylans was changed from 50 mg/l, 100 mg/l, 200 mg/l to 500 mg/l. In order to improve adsorption and resistivity of adsorbed carboxymethylated xylans on PET surfaces, positively charged polymer polyethyleneimine (PEI, Figure 3.7), was used as an anchoring polymer layer. For improving the adsorption of cationic xylans two types of anchoring polymers, positively charged (PEI) and negatively charged (PVSA-Poly(vinylsulfonic acid, sodium salt, Figure 3.8) were used. Figure 3.6: E-4 QCM-D apparatus from QSense (left) and flow module QFM 401 (right) -43-

61 H 2 N N NH 2 N H N N H N NH 2 H N NH 2 H 2 N N NH 2 * n Figure 3.7: Structural formula of Polyethyleneimine (PEI) * * n O S O ONa 3.8: Structural formula of Poly(vinylsulfonic acid, sodium salt), PVSA Adsorption of xylans onto real PET surfaces In order to optimize consumption of xylans and to imitate large scale processes, spray coating technique was applied for functionalization of real PET surfaces. Prior to adsorption of carboxymethylated xylans, anchoring agent PEI was adsorbed using spraying technique. Two layers of PEI solution in concentration of 0.05 % were sprayed with intermediate drying in vacuum dryer at 40 C. Afterwards, xylan solutions were sprayed onto PET fibers. Cationic xylans were sprayed directly onto hydrolyzed PET surface. According to the optimal process conditions defined by QCM-D analyses, solution concentration of xylans was 1 % with addition of 0.1 M CaCl2 at ph 5. Each sprayed xylan layer was dried in vacuum drier at 70 C for 1 hour. At the end PET fabrics with xylans were rinsed with distilled water until conductivity of rinsing water was reached and dried in vacuum oven at 40 C overnight. Dual -44-

62 Action airbrush SP-575 (Sparmax) was used for spray coating (Figure 3.9). The pistol is equipped with a 0.5 mm nozzle, making it ideal for fine work. Figure 3.9: Airbrush SP-575 (Sparmax) Characterization of nonmodified and modified xylans Carbohydrate composition determination At the begining was determined the moisture content. 50 mg of each sample was weighed and dried during the night. Next day sample was weighed again and moisture content was calculated. For determination of the carbohydrate composition was used mild hydrolysis. About 100 mg ± 1 mg of xylan (oven dry basis) were weighed into an erlenmeyer flask (conical flask) and suspended with 8 ml destilated water and for few second immersed in the ultrasonic bath. After that 2.04 ml of 0.5 M H2SO4 were added and the flask was closed with a little condenser. Samples were hydrolysed under pressure (0.12 mpa/1.2 bar) in an autoclave for 40 min. After cooling down to room temperature the flask was filled up to exactly 100 ml and flask was shaked. The condensed lignin residue was removed by filtration over a G4 sinter glass crucible. 1 ml of the liquid was transferred into a sample vial for the analysis in the Borat system and 1.5 ml was frozen (for HPLC analysis). -45-

63 Elemental analysis Elemental analysis was performed using Vario EL Cube (Elementar, Hanau, Germany) analyzer. The experiment was performed at a temperature of 1150 C in the combustion tube and 850 C in the reduction tube, using a helium flow of 230 ml min -1 and an oxygen flow of 35 ml min -1. The detection limit for the analysis of the 5 mg sample was around 0.1 wt. % for C, 0.3 wt. % for H, 0.02 wt. % for N and 0.4 wt. % for S. All the samples were dried for 1 week over P2O5 in a desiccator prior to analysis. Attenuated total reflectance-fourier transform infrared spectroscopy ATR-FTIR was performed on Perkin Elmer Spectrum GX apparatus with Golden Gate ATR attachment and diamond crystal. The samples (non-modified and modified xylans) were placed on the ATR crystal and spectra were collected without any additional sample preparation. FTIR spectra of 32 scan at 4 cm -1 resolution were performed to collect the transmission spectra of the samples. Spectra were recorded between 4000 cm -1 and 600 cm -1 and studied using Spectrum software. The experiments were performed at room temperature. Raman spectroscopy Raman spectra were recorded on a Perkin Elmer Spectrum GX apparatus. The absorbance measurements were carried out within the range of 3500 cm -1 and 300 cm -1, with 16 scans and a resolution of 4 cm -1. The samples (non-modified and modified xylans) were collected without any additional sample preparation. The experiments were performed at room temperature. Size exclusion chromatography Size-exclusion chromatography of non-modified and modified samples performed on GRAM Columns (30, 100, 300 A ; each 8x300 mm; guard column 8*50 mm GRAM; Polymer Standard Service) using the eluent of dimethyl sulfoxide (DMSO):H2O (9:1) with an addition of 0.05 M LiBr at 60 C and a flow rate of 0.4 ml/min was used for determining the molecular weight distribution [157]. The detection was performed with the combination of a viscosimetric detector (PSS Eta 2010) and a refractive index detector (RI-71, Shodex o) using universal calibration with pullulan standards (Polymer Standards Service) [158]. -46-

64 Total bound nitrogen determination Total bound Nitrogen (TNb) determination was performed using multi N/C 2100 analyzer. The method covers the measurement range 0.5 mg/l 200 mg/l. In the TNb determination, the nitrogen-containing constituents in water are thermocatalytically converted to nitrogen monoxide at a temperature > 700 C, which is quantitatively detected with a suitable method; the TNb in the sample is then calculated in mg/l. Polyelectrolyte titration Polyelectrolyte titrations were carried out within an aqueous media (xylan solution) at different ph values (ph 2, 4, 8), adjusted by the addition of NaOH (0.1 M) and HCl (0.1 M). Two milliliters of the dissolved xylan (for carboxymethylated) and 0.5 milliliters (for cationized) in water (0.1 % (w/v)) was pipetted into a titration vessel, and 0.5 ml (for carboxymethylated) and 2 ml (for cationized) of 0.1 mm solution of o-toluidine blue indicator was added. The cationic indicator toluidine blue (TB) was used to photometrically determine the equivalence point. The vessel was then filled up with distilled water to a volume of 40 ml. ph of the solution was adjusted. A Mettler-Toledo DL 53 Titrator with a 10-mL burette was used for the incremental addition of the cationic polyelectrolyte as titrant (pdadmac; c ~ 1 mm) for negatively charged xylans and anionic ethylenesulfonate of PES- Na for positively charged xylans. Incremental additions of 0.1 ml were added every 3 10 s. The absorbance was measured as a potential change in mv, using a Mettler Toledo Phototrode DP660 (Figure 3.10) at a wave length of 660 nm. The amount of deprotonated anionic groups (carboxyl groups) was determined from the equivalent volume of added pdadmac solution, detected as the steepest rise in the curve of absorbance vs. volume of pdadmac, and by assuming a 1 : 1 binding stoichiometry between the ammonium and negatively charged groups. The amount of positively charged amino groups in cationic xylans can be determined from the volume of the titrant in the solution at the point of equivalence by estimating a 1 : 1 binding stoichiometry between the amino groups and ethylenesulfonate of PES-Na [143]. The experiments were performed at room temperature, and the titration results were expressed as an average value of three measurements. -47-

65 Figure 3.10: Mettler Toledo DL 53 titrator with a Mettler Toledo Phototrode DP660 Antimicrobial properties determination Determination of minimal inhibitory concentration (MIC) Inoculum suspensions were prepared by dispersing a fresh culture of microbial strains (Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 10536, and Candida albicans ATCC 10231) with physiological saline adjusted to 0.5 McFrland units (Densimat, Biomerieux, France). Suspensions for the tests were prepared as 1 to 10 dilution in physiological saline containing CFU/mL. Minimal inhibitory concentration (MIC) was determined according to the Clinical and Laboratory Standards Institute (CLSI) M-27A recommendations [159]. Serial twofold microdilution method in Müller-Hinton broth (Merck, Germany) for bacteria (Staphylococcus aureus and Escherichia coli) and RPMI 1640 broth containing 2 % (w/v) glucose for yeast (Candida albicans), in microtiter 96-well plates was used. MIC was defined as the lowest concentration of the tested product (CS/CSNP/ TMC/TMCNP) that allowed no more than 20 % growth of microbes after re-incubation of a 10 µl sample from each dilution on the tryptic-soy agar plate (for S. aureus and E. coli), and Sabourad 2 % glucose agar plate (for C. albicans) at 37 C for 48 h. -48-

66 3.3.6 Characterization of xylan films and functionalized PET fibers Goniometry The OCA35 contact-angle measurement system from Dataphysics (Germany) (Figure 3.11) was applied for contact-angle measurements and surface-free energy calculations. A drop of liquid was released onto xylan film and photographed. The tangent of the sessile drop profile at the three-phase contact point drawn onto the photo-print and the value of the contact-angle was determined. Three different liquids were used for measuring the static contact-angle: Milli Q water (with a higher polar part), diiodomethane (with a higher dispersive part), and formamide (with similar amounts of polar and dispersive parts). The basic data of the test liquids are represented in the Table 3.1. The total SFE (γs TOT ) was calculated using the acid-based approach of van Oss and Good [138]. All the measurements were conducted at room temperature with a drop volume of 3 μl. Each SCA value was the average of at least three drops of liquid per surface. Figure 3.11: Goniometer OCA35 (Dataphysics, Germany) -49-

67 Table 3.1: Total surface free enrgies γ TOT and their components (γ LW, γs +, γs - ) for the test liquids used in this thesis [160] γ TOT γ LW γs + γs - Water Formamide Diiodomethane Atomic force microscopy (AFM) Topographical features of the xylans films surfaces were characterized by atomic force microscopy (AFM) in tapping mode with an Agilent 5500 AFM multimode scanning probe microscope (Digital Instruments, Santa Barbara, CA). The images were scanned using silicon cantilevers (ATEC-NC, Nanosensors, Germany) with a resonance frequency of khz and a force constant of N/m. The scanned image size was 1 1 μm. All measurements were performed at ambient temperature in air. Software SPIP (Image Metrology A/S) was used for processing of images. Scanning electron microscopy (SEM) Surface morphology of untreated and with xylans treated PET fabric was observed with the scanning electron microscope (FESEM SUPRA 35 VP, Carl Zeiss). Sample holder with an adhesive carbon tapewere was used for fixing of the fabrics on the sample holders. Different magnifications were used. X-ray photoelectron spectroscopy (XPS) Spectra were recorded using a PHI model TFA XPS spectrometer [161]. The base pressure in the XPS analysis chamber was about mbar and the samples were excited with X-rays over a specific 400 µm area using monochromatic Al Kα1,2 radiations at ev. The photoelectrons were detected by a hemispherical analyzer, positioned at an angle of 45 with respect to the sample s surface. Energy resolution was about 0.6 ev. An electron gun was -50-

68 used for the surface-charge neutralizations of the samples. Spectra were recorded from three locations on each sample using an analysis area of 400 µm. XPS survey spectra were measured at a pass-energy of 187 ev using an energy step of 0.4 ev, while high-resolution C1s spectra were measured at a pass-energy of 29 ev using an energy step of ev. Surface elemental concentrations were calculated from the survey -scan spectra using the Multipak program. The same program was also used for determining the concentrations of different bonds at high-resolution carbon spectra. Potentiometric titration The ph-potentiometric titrations were performed with a two-burette instrument (Mettler Toledo T70) with a combined glass electrode (Mettler T DG 117), Figure 3.12, in an inert atmosphere (N2 bubbling). The burettes were filled with 0.1 M HCl and 0.1 M KOH. All the solutions were prepared with Mili-Q water with low carbonate content (< 10-5 M). Low carbonate content was achieved by boiling and cooling under constant nitrogen atmosphere. The xylan solutions were titrated in a forth and back manner between the ph = 2.5 and ph = The titration experiments were carried out at 0.1 M ionic strength, set to its appropriate value with KCl. The titrant was added dynamically within a preset interval of [0,001 0,25] ml. The equilibrium criteria for the timed addition was set to de/dt = 0.1/10 s. Where 10 s was the minimum time to reach equilibrium conditions between two additions of the titrant, and the maximum time was set to 180 s. A blank HCl-KOH titration was carried out under same conditions as above. The fibrous samples were suspended in 0.1 M KCl solution and titrated under the same conditions as the solutions only with a prolonged timed interval between two additions of the titrant. The equilibrium criteria for the timed addition was set to de/dt = 0.1/150 s. Where 150 s was the minimum time to reach equilibrium conditions between two additions of the titrant, and the maximum time was set to 7200 s. More detailed description of the charge calculations can be found elsewhere [162]. -51-

69 Figure 3.12: Mettler Toledo T70 with a combined glass electrode Determination of C. I. Acid orange 7 dye adsorption For determination of the amounts of cationic groups present in PET fibers, treated with cationic xylans the C.I. Acid Orange 7 dye adsorption method was applied. The adsorption of C.I. Acid Orange 7 dye (purified by recrystallization) by PET fabric samples treated with cationised xylans was evaluated by determining the dye concentration in solution in the presence of PET fabric samples. PET fabric samples were immersed in an acidic dye solution (dye concentration 4x10-4 M and ph = 3.6). The solution was thermostated at 25 ± 0.5 C and kept under constant magnetic stirring. The adsorption kinetics was monitored by on-line measurements of absorbance at the wavelength of maximum absorbance (484 nm). For these measurements was used Cary 50 computer-controlled UV/VIS spectrometer (Varian). As a result, the amounts of amino groups per kg of fibers were calculated. Methylene blue sorption method For determination of charging behavior of fibers, treated with carboxymethylated xylans the methylene blue dye absorption method was used. 0.5 g of PET fabric samples treated with carboxymethylated xylans with known moisture content were suspended in 25 ml of aqueous solution (300 mg/l) of C.I. Basic Blue 9 dye (methylene blue bis(dimethylamino) phenazatonium chloride) and 25 ml of borate buffer at ph = 8.5 in Erlenmeyer flask and was -52-

70 stirred for 1 hour at 20 C. After that fibers were filtered through a sintered-glass disk. 5 to 10 ml of the filtrate was transferred to a 100 ml calibrated flask and 10 ml of 0.1 N HCl was added. After that, water up to 100 ml was added. Methylene blue content of the liquid was determinated spectrophotometrically, employing a calibration plot (at nm) and from the result the total amount of free, i.e. nonsorbed, methylene blue was calculated. Carboxyl groups content of the sample was calculated according to: mmol COOH/g absolute dry sample = 7.5 A * E (3.1) where: A - total amount of free methylene blue (mg) E - weight of absolute dry sample (g) Antimicrobial properties determination Determination of antimicrobial activity of PET fibers according to ASTM E 2149 ASTM E 2149 method, a quantitative antimicrobial test method, performed under dynamiccontact conditions was applied to evaluate antimicrobial activity of functionalized PET fabric samples. Gram-positive and gram-negative bacteria were used as test organisms. As a working bacterial dilution was used an incubated test culture within a nutrient broth, diluted using a sterilised 0.3 mm phosphate buffer (KH2PO4; ph = 6.8) in order to give a final concentration of x 105 CFU/mL. Small pieces (1 x 1 cm) from 0.5 to 2 g of each sample were transferred to a 250 ml Erlenmeyer flask containing 50 ml of the working bacterial dilution. All flasks were loosely capped, placed in the incubator and shaked for 1 h at 37 C and 120 rpm using a Wrist- Action incubator shaker. After a series of dilutions using the buffer solutions, 1 ml of the diluted solution was plated in nutrient agar. The inoculated plates were incubated at 37 C for 24 h and the surviving cells were counted. The average values of the duplicates were converted to CFU/mL in the flasks, by multiplying with the dilution factor. The antimicrobial activity was expressed as R = the % reduction of the organism after contact with the test specimen, and compared to the number of bacterial cells surviving after contact with the control [147]. -53-

71 4 RESULTS AND DISCUSSION 4.1 Characterization of nonmodified and modified xylans In order to characterise nonmodified source xylan samples and to investigate the success of chemical modification processes, following analyses were applied: carbohydrate composition, elemental composition (CHN), polyelectrolyte titration, ATR-FTIR and raman spectroscopy, size exclusion chromatography, and total bound nitrogen determination. Minimal inhibitory concentration (MIC) determination was applied for antimicrobial properties investigation of nonmodified and cationized xylans Carbohydrate composition The carbohydrate content in the xylan hydrolyzates was analyzed by borate-anionexchange-chromatography with post-column derivatization and detection at 560 nm, as described previously [163]. The compositions regarding neutral carbohydrates of samples are represented in Table 4.1. The hydrolysis residue represents the residual lignin and cellulose of the sample. For BX and BXL was lower than OX and was 1.9 % for BX and 1.8 % for BXL. OX contained larger amounts of lignin impurities, which was 6.4 %. The mass balance of the analysis amounts only to about 66 % for BX, 69 % for BXL and 88 % for OX. This is due to the fact that some components are not considered in the analysis e.g. ashes, extractives and acid soluble lignin. From the Table 4.1 it follows that after both chemical derivatisations, carboxymethylation and cationisation, the total carbohydrate content decreased. The main reason for that is substitution. In glucuronoxylan the majority of degradation occurred on the side chains. The ratios of xylose with other carbohydrates (Ara, Gal, Glc), which were rather low already in reference sample (app ), dropped after carboxymethylation and cationisation by about 50 %. In nonmodified arabinoxylan the xylose/carbohydrate ratios were higher (Xyl/Ara = 0.14; Xyl/Gal = 0.03 and Xyl/Glc = 0.04) and the drop after carboxymethylation was only 20 %. Amounts of impurities were also reduced by about 50 %. -54-

72 Table 4.1: Carbohydrate composition of nonmodified beech wood (BX and BXL) and oat spelt (OX) and chemically modified (CMBX, CMOX, CBX, COX) xylan samples Xylan sample Rham. Mann. Arabin. Galac. Xylose Gluc. Sum Res. %abs %abs %abs %abs %abs %abs %abs %abs BX BXL OX CMBX CMOX CBX COX Elemental composition The elemental composition of nonmodified and chemically derivatised xylans are represented in the Table 4.2. In the reference samples the content of carbon was around 42 % (41.11 % for BX, % for BXL and % for OX). Hydrogen was also same for all three cases and was around 5.7 % (5.67 % for BX, 5.75 % for BXL and 5.89 % for OX). In all three cases could be observe some nitrogen, as result of impurities presented into samples. After carboxymethylation amounts of carbon were only slightly reduced, in the case of CMBX by 0.3 % and in the case of CMOX by 6.6 % in comparison to reference samples. At the same time hydrogen content was reduced, thus the H/C ratio after carboxymethylation remained unchainged. -55-

73 Table 4.2: Elemental composition of nonmodified (BX, BXL, and OX) and chemically modified xylans (CMBX, CMOX, CBX, COX) Xylan sample Nitrogen [%] Carbon [%] Hydrogen [%] BX 0.08 ± ± ± 0.03 BXL 0.17 ± ± ± 0.03 OX 0.14 ± ± ± 0.03 CMBX 0.16 ± ± ± 0.03 CMOX 0.10 ± ± ± 0.03 CBX 2.68 ± ± ± 0.03 COX 1.46 ± ± ± 0.03 After cationisation, derivatised xylan samples contained nitrogen. The content of nitrogen was 2.7 % for CBX and 1.5 % for COX sample. As expected, due to reaction with 2,3-epoxypropyltrimethylammonium chloride and introduction of methylene groups, carbon content increased after cationisation by about 6.8 % in the case of glucurono- and by about 5 % in the case of arabinoxylan, as well as hydrogen content was increased (by about 21 % for CBX and by about 12 % for COX in comparison to reference samples) Total bound nitrogen Nitrogen content in cationised samples was determined by total bound nitrogen determination. Results are represented in the Table 4.3. The results of total bound nitrogen determination confirmed the results of elemental analysis. Amounts of nitrogen were almost same like nitrogen content determined with elemental analysis. CBX contained nearly 100 % more nitrogen in comparison to COX. -56-

74 Table 4.3: Total bound nitrogen for the nonmodified (BXL and OX) and cationized (CBX and COX) xylan samples Xylan sample TNb [%] BXL 0.15 OX 0.24 CBX 2.81 COX Molecular weights and polydispersities Molecular weight distributions were determined for carboxymethylated samples using size exclusion chromatography. The insolubility of cationised samples in the eluent dimethyl sulphoxide prevented performance of this method for cationized samples. The average molecular weights (Mw) and polydispersities (Mw/Mn) of nonmodified and carboxymethylated samples are presented in Table 4.4. Table 4.4: Average molecular weights (Mw) and polydispersity indices (Mw/Mn) of the nonmodified (BX, BXL and OX), and carboxymethylated xylans (CMBX, CMOX) Xylan sample Mw [g/mol] Mw/Mn BX BXL OX CMBX CMOX

75 The molecular weights of the nonmodified samples determined by size-exclusion chromatography were 19,400 g/mol for BX, g/mol for BXL and 23,500 g/mol for the OX sample. After carboxymethylation, the molecular weights for both samples increased in comparison with the nonmodified, by 2600 g/mol for glucuronoxylan and by 4100 g/mol for the arabinoxylan sample. Based on the assumption that no chain degradation occurred during derivatization, the introduction of new (-CH2COO - Na + ) groups can be estimated from the increase in the molecular weight (Mw). The calculation results into 1.45 or 1.81 mmol/g of carboxymethyl groups for glucuronoxylan or arabinoxylan, respectively. These increases in Mw after carboxymethylation were a consequence of the introduction of the CH2COONa groups, thus the differences in molecular weights correspond to about 32 or 50 introduced groups respectively, which is in accordance to the deprotonated groups amounts in carboxymethylated samples determined by polyelectrolyte titrations (Table 4.5). Therefore, it can be concluded that the increase in molecular weights correspond well with the introduction of the new carboxymethyl groups, which proves that no significant degradation of polymer chains has occurred during the derivatization. The polydispersities did not change after carboxymethylation process, which could be seen as well from the Table Surface chemical composition ATR-FTIR spectroscopy The efficiency of different modifications was estimated as well by ATR-FTIR spectroscopy. Chemical composition and typical functional groups of nonmodified and carboxymethylated xylans were evaluated. This method was not successful for cationic samples, because IR spectroscopy is more successful for chemical groups which contain highly polar bonds, or bonds whose dipole moment changes during vibration, e.g. the C=O and OH groups [164]. Therefore, chemical composition and typical functional groups of nonmodified and cationized xylans were determined using Raman spectroscopy, because Raman is more sensitive for the fundamental vibrations of less polar molecular groups and bonds like C-C and for symmetric vibrations [164]. -58-

76 Figures 4.1 and 4.2 represent the spectra of the nonmodified and carboxymethylated glucuronoxylan and arabinoxylan samples respectively. BX % T CMBX /cm Figure 4.1: FTIR-ATR spectra of nonmodified (BX) and carboxymethalated (CMBX) glucuronoxylan sample OX %T CMOX /cm Figure 4.2: FTIR-ATR spectra of nonmodified (OX) and carboxymethalated (CMOX) arabinoxylan sample -59-

77 Typical peaks for xylan samples can be seen from both samples: spectrum peak at 1160 cm -1 indicated glycosidic linkage, the one at 1039 cm -1 is assigned to C-OH bending and at 897 cm -1 a band typical for the β-configuration of C1 in xylans (β-(1 4)) was detected [ ]. At 720 cm -1 an intensive absorption band could be observed for OX sample, which is typical for a methylene CH2 rocking band [123] that originated from CH2 group present in the α-l-arabinofuranose residues. After carboxymethylation of xylans, new functional groups appeared, indicating successful derivatization of xylans. Three new typical bands for carboxymethylated xylans had appeared at 1596 cm -1, 1415 cm -1, and 1326 cm -1. The absorption bands at 1596 cm -1 and 1415 cm -1 arose due to C=O stretching of the COO - ion [168, 169], proving the presence of new carbonyl groups within the carboxymethylate (-COO - Na + ) species [169]. In addition, the proof for xylan carboxymethylated groups is the absorption band at 1325 cm -1 either caused by C=O stretching of the COO - ion [168, 169] or was related to the symmetric angular deformation of the C-H bond [123, 170]. Raman spectroscopy Raman spectroscopy was applied for determining the efficiency of cationization. Raman spectra of nonmodified and cationized xylan samples are represented in Figures 4.3 and 4.4. Typical peaks for nonmodified xylans can be seen at: 2926 cm -1 assigned to C-H stretching vibration [164, 166], spectrum peak at 1125 cm -1 indicated glycosidic linkage [166] and at 899 cm -1 a band typical for β-configuration of C1 in xylans (β-(1 4)) can be observed [166, 169, 171]. -60-

78 Intensity CBX BXL /cm Figure 4.3: Raman spectra of nonmodified (BXL) and cationized (CBX) glucuronoxylan samples Intensity COX OX /cm Figure 4.4: Raman spectra of nonmodified (OX) and cationized (COX) arabinoxylan samples -61-

79 After cationization of xylans three new typical bands appeared in both cases. The first one at 3025 cm -1 characteristic for CH3 antisymmetrical stretching vibration, and the second one, absorption band at 2973 cm -1 which is assigned to CH3 and CH2 stretching vibration. The third one was high intensity peak which appeared at 763 cm -1. This absorption band is according to [164] assigned to quaternary ammonium group ((CH3)3N + ) symmetric stretching vibration. All the new absorption bands in the raman spectra of cationised glucurono- and arabinoxylan samples were strong evidence for successful introduction of quaternary ammonium groups Charging behaviour Polyelectrolyte titration Polyelectrolyte titration technique was applied for deprotonated/protonated groups determination of nonmodified, carboxymethylated, and cationised xylans. The results are represented in the Table 4.5 where the amounts represent deprotonated carboxyl groups for nonmodified and carboxymethylated xylans and protonated amino groups for cationised xylans. Nonmodified samples (BX, BXL and OX) contained small amounts of weak acidic groups in the form of 4-O-methylglucuronic acid units. The titration results at ph = 8 (Table 4.5) showed that samples BX, BXL and OX contained 0.68, 0.80 and 0.25 mmol/g of deprotonated carboxylic groups, respectively. At ph = 8 all of the carboxylic groups were fully deprotonated. Thus, at this conditions, the amount of deprotonated groups represent total amount of anionic charge. Due to the protonation of carboxylic groups at lower ph, the amount became lower. It has to be pointed out that with lowering of the ph, also conformation changes occurred (more coiled structure) and thus accessibility of anionic charge is limited. After carboxymethylation, the amounts of deprotonated carboxyl groups had increased (Table 4.5). At ph = 8 glucuronoxylan (CMBX) contained 2.72 mmol/g of deprotonated carboxyl groups, which was four times more than reference sample BX, whereas the amount of deprotonated carboxyl groups in arabinoxylan (CMOX) was 2.19 mmol/g, which was

80 times higher than in the reference sample OX. It can be seen from the results that, by the carboxymethylation procedure, almost the same amounts of carboxylic groups were introduced for both xylan samples (2.04 mmol/g for glucuronoxylan and 1.94 mmol/g for arabinoxylan). Table 4.5: Amounts of deprotonated/protonated (carboxyl/amino) groups in nonmodified (BX, BXL and OX) and chemically modified (CMBX, CMOX, CBX and COX) xylan samples at different phs Xylan sample Amounts of deprotonated/protonated functional groups [mmol/g] ph 2 ph 4 ph 8 BX ± ±0.02 BXL ± ±0.00 OX ± ±0.00 CMBX ± ±0.03 CMOX 0 2± ±0.03 CBX 3.2±0.2 2± ±0.12 COX 1.2±0.2 1± ±0.1 From the Table 4.5 it follows that new positively charged groups were introduced by cationization process. At lower ph values all primary ammonium groups are fully protonated and deprotonation at higher ph values occurs. Quaternary ammonium groups are protonated in whole ph range and can be individually detected at ph 8, where others (primary, secondary, tertiary) ammonium groups are deprotonated. From the table could be seen that CBX have bigger amount of positively charged groups (3.2 mmol/g all and 1.53 mmol/g quaternary) compared to COX, where amount of positively charged groups were 1.2 mmol/g and amount of quaternary groups was 0.75 mmol/g. -63-

81 4.1.7 Antimicrobial properties In order to analyse antimicrobial properties of cationised xylans minimal inhibitory concentration (MIC) determination of xylan solutions by using microdilution assay was performed. Antimicrobial activities were tested against S. aureus, E. coli, and C. albicans and results are represented in Table 4.6. Lower MIC values signify higher antimicrobial activity. Cationised glucuronoxylan (CBX) showed the highest antimicrobial activity against S. aureus, thus 50 % or less viability of species was detected already at the solution concentration of %. Against E. Coli the same degree of inhibition (50 %) was achieved at the concentration of %. In the case of cationised arabinoxylan COX for both bacteria S. aureus and E. Coli the concentration at which 50 % or less viability was detected was 0.25 %. As it was expected, the samples with higher amounts of active amino groups showed higher effectiveness against selected bacteria. Table 4.6: Minimal inhibitory concentration for nonmodified (BXL and OX) and cationized (CBX and COX) xylan solutions MIC (% m/v), N=3 Xylan sample S. aureus E. coli C. albicans ATCC ATCC ATCC BXL > 0.25 OX > 0.25 CBX (DS=0,36) > 0.25 COX (DS=0,17) > 0.25 Higher antimicrobial activity of the sample CBX is most probably caused by the highest amount of ammonium groups. As it was confirmed by polyelectrolyte titrations at ph 8 this sample contained 1.54 mmol/g protonated groups in comparison to COX, which contained only half of that amount. The MIC results also showed that neither of cationised samples are active against fungi, thus there was no inhibition of C. albicans detected at the concentration 0.25 %. -64-

82 4.2 Characterization of xylan films In order to investigate surface properties of solid structures, which are formed from xylans water solutions, films were prepared from nonmodified and chemically modified xylan samples by a casting method. X-ray photoelectron spectroscopy was applied for the analyses of surface chemical compositions. Surface morphologies of the films were characterized using atomic force microscopy, and surface free energies (SFE), and their Lifshitz van der Waals and electron donor/acceptor contributions of the films surfaces were determined using goniometry Surface chemical composition Surface elemental composition and the relative surface concentrations of different functional groups within the carbon spectra of the nonmodified and differently derivatized xylan films were obtained by XPS spectra measured at two different spots on the surfaces of each sample (Tables 4.7 and 4.8). Table 4.7: Surface elemental composition of films made from nonmodified (BX, BXL and OX) and chemically modified (CMBX, CMOX, CBX, COX) xylans Xylan sample Surface elemental composition (at. %) C O N Na O/C BX BXL OX CMBX CMOX CBX COX

83 Surface elemental compositions (at. %) of nonmodified xylans showed similar amounts of carbon, oxygen and nitrogen. In case of glucuronoxylan (BXL) higher amount of nitrogen could be observed. Amount of nitrogen in this samples is most probably due to impurities presented into samples. After carboxymethylation of glucurono xylan (CMBX) amount of carbon decreased by about 3.7 % and oxygen by about 2 % therefore, O/C ratio increased by about 15 % compared to nonmodified sample BX, as well as, sodium appeared on the surface with a surface share about 2.6 %. In case of carboxymethylated arabinoxylan (CMOX) however, surface amount of carbon increased by about 6.4 %, and amount of oxygen decreased even by about 23 % in comparison to nomodified OX sample. O/C ratio decreased by about 25 % in comparison to the OX. Surface proportion of sodium remained unchainged in comparison to nonmodified arabinoxylan. In case of cationized xylans amount of carbon increased by about 3 % in case of CBX and decreased by about 1.4 % in case of COX when compared to nonmodified samples. Surface portions of oxygen decreased significantly after cationisation, by about 23 % and 4 % in case of CBX and COX respectively. As expected, O/C ratio decreased in both cases, by 25 % and 2 % in case of CBX and COX respectively. On the surfaces cationized xylans films increased portions of nitrogen could be observed. Amounts of differently bound carbons for xylan samples are represented in the Table 4.8. There are practically no differences in surface amounts of C1, C2 and C3 between both nonmodified glucuronoxylan samples (BX and BXL). BXL showed only slightly higher amount of C4, which indicated slightly higher surface portion of carboxyle groups for this film sample. Arabinoxylan showed lower surface portions of carbon involved in C-C/C-H and O-C-O/C=O bonds, but higher portion of carbon in C-O/C-OH compared to nonmodified glucuronoxylans. The relative surface carbon amounts involved in the C-C bonds decreased after carboxymethylation for both xylan samples (by about 18 % in case of CMBX and 52 % in case of CMOX), and at the same time, the C-O and C-OH carbon fractions increased for glucuronoxylan by about 30 % and for arabinoxylan by about 35 %. At the same time, relative amounts of carbon belonging to the carboxyl O=C-O groups increased in the case of glucuronoxylan by about 11 %, and in the case of arabinoxylan by about 225 %. This increase in the presence of carboxylic groups on the surfaces of carboxymethylated arabinoxylan in comparison with the nonmodified sample as well as in comparison with the -66-

84 carboxymethylated glucuronoxylan film was quite remarkable. It can be assumed that the main reason for such a large amount of carbon belonging to O=C-O bonds in comparison with other film samples was the much higher CMOX films surfaces roughness, which rendered higher amounts of different elements and/or groups on the surface area, as examined by XPS. In addition, it has to be taken into account that this technique provides limited information regarding the carboxylic groups amount. The results of C4 have to be linked only to the surface carboxyl groups positioned within a thin ~10 nm layer. The total charge of the films was, regarding their preparation (no crosslinking or chemical bonding), equal to the amount of those groups determined by polyelectrolyte titration (Table 4.5). Obviously, in the case of CMBX, the majority of charge was distributed within the inner part of the film, whereas in the case of CMOX much more in the (10 nm) surface layer. Table 4.8: Relative amounts of differently bound carbons obtained from the XPS survey spectra for the films made from nonmodified (BX, BXL, OX) and chemically modified (CMBX, CMOX, CBX, COX) xylans Xylan sample C1 (%) C-C; C-H C2 (%) C-O; C-OH C3 (%) O-C-O; C=O C4 (%) O=C-O BX BXL OX CMBX CMOX CBX COX After cationization the relative surface carbon amounts involved in the C-C and C-H bonds increased for both xylan samples, for glucuronoxylan by about 6.5 % and for arabinoxylan by about 8.2 %. The C-O and C-OH carbon fraction also increased for -67-

85 glucuronoxylan by about 11 % but decreased slightly (1 %) in case of arabinoxylan. Relative amounts of carbon belonging to the carboxyl O=C-O groups decreased in the case of glucuronoxylan by about 54 %, and in the case of arabinoxylan by about 6 %. These results are the consequence of the presence of higher amounts of methyl groups, which were introduced by quaternary ammonium groups into cationised xylans Surface morphology The results of atomic force microscopy are represented in the Figures 4.5 to 4.11 in the form of AFM height images and AFM profile lines, and in the Table 4.9 where root mean square roughness of the nonmodified and chemically modified xylan samples are represented. From Figures 4.5 to 4.7 could be seen that surface of BX film had highest roughness between nonmodified xylans, with surface roughness of 3.3 nm (Table 4.9). BXL film was smoother compared to BX, with smaller hollows or embossments and with surface roughness of 1.4 nm. Nonmodified (OX) showed uniformly grained surface morphology without larger hollows or embossments and with root mean-squared roughness of 1.2 nm, which was the lowest surface roughness among all samples (Table 4.9). Such a uniformly grained structure was most probably a consequence of a higher presence of lignin (Table 4.1) within this sample, as already shown by Notley and Norgren [172]. Figure 4.5: AFM height image (left) and AFM profile line (right) of 1x1 μm 2 surface area of nonmodified glucuronoxylan (BX) -68-

86 Figure 4.6: AFM height image (left) and AFM profile line (right) of 1x1 μm 2 surface area of nonmodified glucuronoxylan (BXL) Figure 4.7: AFM height image (left) and AFM profile line (right) of 1x1 μm 2 surface area of nonmodified arabinoxylan (OX) Surface of carboxymethylated glucuronoxylan (CMBX) had smaller embossment and hollows, with same size, and with average roughness of 3.3 nm (Table 4.9). Surface of carboxymethylated arabinoxylan (CMOX) had much more irregular surface structure with larger hollows and embossments, which were confirmed by a root mean-squared roughness value of 5.2 nm (Table 4.9). From the Figures 4.5 and 4.8 could be seen that there were no remarkable difference between the surface morphology of films made from nonmodified and carboxymethylated glucuronoxylans (BX and CMBX), which was shown by their root meansquared roughness (Table 4.9). In case of arabinoxylan films (Figures 4.7 and 4.9), the -69-

87 differences were significant with 4 times higher roughness in case of CMOX compared to OX. Figure 4.8: AFM height image (left) and AFM profile line (right) of 1x1 μm 2 surface area of carboxymethylated glucuronoxylan (CMBX) Figure 4.9: AFM height image (left) and AFM profile line (right) of 1x1 μm 2 surface area of carboxymethylated arabinoxylan (CMOX) It can be seen from the Figures 4.10 and 4.11 there are significant differences in the surface morphology of films from cationized xylans compared to the nonmodified ones (Figures 4.6 and 4.7). Cationised xylans formed films with larger agglomerates with a diameters up to app. 200 nm. These films have more irregular surface structure with large hollows and embossments, which were confirmed by a root mean-squared roughness value of -70-

88 5.2 nm in case of CBX and 3.8 nm in case of COX (Table 4.9). Surface roughness of CBX film was 3.7 times higher and that one of COX film was 3 times higher, compared to nonmodified BXL and OX respectively. Figure 4.10: AFM height image (left) and AFM profile line (right) of 1x1 μm 2 surface area of cationized glucuronoxylan (CBX) Figure 4.11: AFM height image (left) and AFM profile line (right) of 1x1 μm 2 surface area of cationized arabinoxylan (COX) -71-

89 Table 4.9: Root mean square roughness of nonmodified and differently modified xylan films Xylan sample Root mean square roughness Sq [nm] BX 3.3 BXL 1.4 OX 1.2 CMBX 3.3 CMOX 5.2 CBX 5.2 COX Hydrophilic/hydrophobic character and surface free energy Surface free energies and their Lifschitz Van der Waals and electron-donor/-acceptor contributions of nonmodified and chemically modified xylan films were calculated from contact-angle measurements with three different test liquids (water, formamid and diiodomethane), which were performed using goniometry. In the Figure 4.12 are represented surface free energies (γs TOT ), Lifshitz van der Waals contributions (γs LW ), electron-donor contribution (γs - ), and electron-acceptor contribution (γs + ) of nonmodified and carboxymethylated xylans. -72-

90 Surface free energy [mj/m 2 ] γslw: γs+: γs-: γstot 0 BX CMBX OX CMOX Xylans Figure 4.12: Surface free energy values (γs TOT ) and their components (γs LW, γs - and γs + ) for films made from nonmodified (BX, OX) and carboxymethylated (CMBX, CMOX) xylans BX film with surface free energy γs TOT of ± 4.36 mj/m 2 could be considered as a low energy surface with high content of dispersive Lifshitz van der Waals contribution (γs LW = ± 1.5). The electron-donor contribution (γs - = ± 4.12) was higher than the electron acceptance contribution (γs + = 0.59 ± 0.38) because of the presence of certain amounts of carboxyl groups (0.68 mmol/g) and other nonsubstituted hydroxyl groups. Film made from OX with total SFE of γs TOT = ± 4.67 mj/m 2 expressed by about 11 % higher content of Lifshitz van der Waals contribution (γs LW = ± 1.08) in comparison to the BX film. For this sample, as well, the electron-donor contribution (γs - = ± 3.86) was much higher than the electron-acceptor part (γs + = 4.15 ± 0.75), and according to the lower content of carboxylic groups in the source sample (0.25 mmol/g), much smaller in comparison with the BX film. The film formed by carboxymethylated glucuronoxylan (CMBX) showed higher total surface energy by about 40 % (γs TOT = ± 4.77 mj/m 2 ) in comparison with the film formed from nonmodified glucuronoxylan. The same trend was observed in the case of -73-

91 carboxymethylated arabinoxylan with 11 % higher total surface energy (γs TOT = ± 2.72 mj/m 2 ). In both cases (CMBX and CMOX), the dispersive Lifshitz van der Waals contributions (γs LW ) were lower (24.58 ± 1.53 for CMBX and ± 0.7 for CMOX) and the electron-donor contribution higher by about 69 % for CMBX (γs - = ± 2.82) and even by about 2.7 times for CMOX (γs - = ± 1.62), in comparison with the nonmodified xylan films. This was a consequence of the increased amounts of carboxyl groups present on the surfaces of the carboxymethylated samples, which was also proved by XPS (Table 4.8). Additionally, much larger differences could be observed in the electron-donor contribution of SFE for the CMOX film sample. According to the XPS results, the surface amounts of the carbon fraction within the O=C-O groups for CMOX increased in comparison with the nonmodified OX film sample by about 225 % and within the C-OH groups by about 35 %, which had an influence on the increase in the electron-donor contribution of its SFE by about 270 %. In the case of the CMBX film, the surface carbon fraction belonging to the O=C-O groups was higher by about 11 %, and the carbon fraction belonging to the C-OH bonds was higher by about 30 %. This was rather low in comparison with its increase in electron-donor contribution of SFE, which was higher by about 70 % in comparison with the nonmodified BX film sample. Similar relations could be discovered by comparing the amounts of the surface carbon fractions belonging to the C-C bonds with dispersive Lifshitz van der Waals contributions of SFEs (γs LW ). In the case of CMBX, the amount of carbon fraction within the C-C bonds decreased by about 18 % (Table 4.8), and the dispersive part of its SFE decreased by about 13 %. The carbon fraction of CMOX within C-C bonds decreased by about 50 % (Table 4.8) and the dispersive part (γs LW ) decreased by about 31 %. The much larger differences in the case of carboxymethylated arabinoxylan in comparison with glucuronoxylan film were, to a greater extent, supported by higher surface roughness (Table 4.9), which was threefold higher in comparison with the nonmodified arabinoxylan film and about 50 % higher in comparison with both glucuronoxylan films. Carboxymethylated xylans formed films surfaces with significantly higher polarity and total surface free energies. In the Figure 4.13 are represented Lifshitz van der Waals contributions (γs LW ), electron-donor contribution (γs - ), electron-acceptor contribution (γs + ) and total surface free energies (γs TOT ) of nonmodified and cationized xylans. -74-

92 60 Surface free energy [mj/m 2 ] γslw: γs+: γs-: γstot 10 0 BXL CBX OX COX Xylans Figure 4.13: Surface free energy values (γs TOT ) and their components (γs LW, γs - and γs + ) for films made from nonmodified (BX, OX) and cationized (CBX, COX) xylans Similar to other reference samples, nonmodified glucuronoxylan (BXL), formed low energy film surface with surface free energy γs TOT of ± 3.02 mj/m 2 and the highest content of dispersive Lifshitz van der Waals contribution (γs LW = 32.1 ± 1.08) among all nonmodified xylan film samples. The electron-donor contribution (γs - = 8.49 ± 1.62) was by about 46 % higher than the electron-acceptance contribution (γs + = 5.8 ± 0.49) because of the presence of certain amounts of carboxyl groups (0.80 mmol/g) and other nonsubstituted hydroxyl groups. Film made from nonmodified arabinoxylan (OX) showed surface properties with total surface free energy of ± 4.67 mj/m 2 and Lifshitz van der Waals contribution γs LW = ± 1.08 similar to the sample BXL. As described earlier, for this sample the electron-donor contribution (γs - = ± 3.86) was by about 170 % higher than the electronacceptor component (γs + = 4.15 ± 0.75). The film formed by cationized glucuronoxylan (CBX) showed by about 7 % lower total surface energy (γs TOT = ± 2.07 mj/m 2 ) in comparison with the film formed from nonmodified one (BXL). The same trend could be observed in the case of cationized arabinoxylan (γs TOT = ± 4.29 mj/m 2 ). In both cases (CBX and COX), the dispersive Lifshitz van der Waals contributions (γs LW ) were increased in comparison with the -75-

93 nonmodified xylan films by about 4.5 % for CBX and even by about 13 % for COX (33.57 ± 0.72 for CBX and 35.6 ± 1.51 for COX), which was most probably owing to the presence of higher amounts of C-C/C-H bonds on the surface (Table 4.8). Additional proof of lower surface polarity was a decrease of electron-acceptor component after cationization by about 31 % in case of CBX and by about 3.4 % in case of COX. Electron-acceptor component is in the first place a consequence of hydration [173]. At the same time the electron-donor contributions decreased by about 35 % for CBX and even by 63 % for COX (5.55 ± 0.4 for CBX and 4.11 ± 2.27 for COX) in comparison with the nonmodified films samples. This was a consequence of the decreased amounts of carboxyl groups present on the surfaces of the cationized samples, which was also proved by XPS (Table 4.8). In the case of CBX film, the surface carbon fraction belonging to the O=C-O groups was decreased by about 54 %, which resulted in a decrease of electron-donor contribution by about 35 %. According to XPS results (Table 4.8) the surface amounts of the carbon fraction within the O=C-O groups for COX decreased in comparison with the nonmodified OX film by about 6 %, while electron-donor contribution of this film was decreased by about 63 % compared to nonmodified OX sample. Good correlation between surface carbon fractions belonging to the C-C/C-H bonds and dispersive Lifshitz van der Waals contributions of SFEs (γs LW ) can be observed in both cases (CBX and COX). In the case of CBX, the amount of carbon fraction within the C-C/C-H bonds increased by about 6.5 %, and the dispersive part of its SFE increased by about 4.6 %, while carbon fraction within the C-C/C-H bonds of COX film increased by about 8.2 % and its γs LW increased by about 13 %. In general cationised xylans formed films with lower polarity and total SFE in comparison to nonmodified xylan films. Hydrophilic/hidrophobic character of films from nonmodified and differently derivatized xylans were additionally studied by the determination of water contact angles (WCA). Results are represented in Figure

94 WCA / BX BXL OX CMBX CMOX CBX COX xylans Figure 4.14: Water contact angles (WCA) of films made from nonmodified (BX, BXL and OX) and chemically modified (CMBX, CMOX, CBX, COX) xylans From the diagram in the Figure 4.14 it could be clearly seen that all the samples are from the point of view of water contact angles, hydrophilic, thus all the film samples WCAs are lower than 90. However, nonmodified and cationised xylan films showed less hydrophilic character with higher water contact angles in comparison to carboxymethylated xylan film samples. Water contact angles of carboxymethylated xylan films were by about 35 % lower when compared to nonmodified xylan films. Such results were expected and were the consequence of higher amounts of carboxyl groups in these samples, which was proved by XPS analysis (Table 4.8). Films from cationized xylans showed by about 10 % higher WCAs in comparison to nomodified samples and as such less hydrophilic nature. 4.3 Adsorption study of xylans onto PET model films using QCM-D Quarz crystal microbalance with dissipation unit was applied in order to study adsorption of nonmodified and chemically modified xylans onto model PET surfaces under different -77-

95 conditions. In the first phase it was investigated the influence of ph, ionic strength, and concentration of xylans. The adsorption studies were performed at ph 4, ph 5, ph 7 and ph 9, with added electrolyte. For the determination of the influence of ionic strength on the amounts of adsorbed xylans, two types of electrolytes were applied: monovalent NaCl and divalent CaCl2 in different ionic strengths (0.05 M, 0.1 M, 0.3 M, 0.5 M and 0.7 M). Concentration of xylans solutions was varied from 50 mg/l to 500 mg/l. In the second phase intermediate layers of polymers like polyethylene imine (PEI) and Poly(vinylsulfonic) acid, sodium salt (PVSA) were applied as so-called anchoring polymers to improve adsorption and resistivity of adsorbed xylan layers. Adsorbed masses (xylan, electrolyte ions and entrapped water molecules) were calculated according to the Sauerbrey equation Influence of ph Changes in frequencies and dissipations of nonmodified glucuronoxylan (BX) with different ph values during adsorption onto PET model films are represented in the Figure Figure 4.15: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of nonmodified glucuronoxylan (BX) adsorbed onto PET model films at different ph values (4, 5, 7 and 9) -78-

96 In case of non-modified glucuronoxylan (BX) (Figure 4.15) highest decrease in frequency to -97 Hz could be observed at ph 5. Decreases in frequencies were at other ph values lower and amounted to -90 Hz at ph 7, -88 Hz in case of ph 9 and -77 Hz in case of ph 4. After rinsing with MQ water frequencies increased in all cases, so desorption occur. Highest frequency increase after rinsing step could be observed at ph 7 (frequency increased to -47 Hz) and the smallest change in frequency caused rinsing step at ph 4 (-50 Hz). Rinsing steps at ph 5 and 9 caused similar changes in frequencies. Higher adsorbed mass (880 ng/cm 2 ) could be observed at ph 5 (Figure 4.20). At other ph values adsorbed masses were lower and were 845 ng/cm 2, 744 ng/cm 2 and 655 ng/cm 2 at ph 4, 7 and 9 respectively. Changes in dissipation were small and changed in accordance to changes in frequency. In the Figure 4.16 changes in frequencies and dissipations of nonmodified glucuronoxylan (BXL) during adsorption onto PET model films at different ph values are represented. Figure 4.16: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of nonmodified glucuronoxylan (BXL) adsorbed onto PET model films at different ph values (4, 5, 7 and 9) -79-

97 Frequency changes during the adsorption of BXL at different ph values of solutions showed negligible differences and similar results as for BX. The highest decrease in frequency could be observed at ph 5 (decrease to -80 Hz) and the changes at other ph values were in the same range (at ph 7-79 Hz, at ph 9-74 Hz and at ph 4-70 Hz). Rinsing step caused an increase in frequencies in all the cases. The highest change in frequency after a rinse step could be observed at ph 9 with frequency increase to -40 Hz. The changes in dissipation were in accordance to changes in frequencies and similar to dissipation changes during the adsorption of BX. Higher adsorbed mass was again determined at ph 5 (841 ng/cm 2 ) (see Figure 4.20). In the Figure 4.17 are represented changes in frequencies and dissipations during the adsorption of nonmodified arabinoxylan (OX) at different ph values. Figure 4.17: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of nonmodified arabinoxylan (OX) adsorbed onto PET model films at different ph values (4, 5, 7 and 9) -80-

98 In case of arabinoxylan (OX) (Figure 4.17) the highest change in frequency occurred during the adsorption at ph 7 when frequency decreased to -141 Hz. At other ph values changes in frequencies were similar and were around -124 Hz. After rinsing with MQ water frequency increased (desorption occur) for the same degree in all cases (by about 11 %). At ph 7 after rinsing frequency increased to -127 Hz and at other ph values to -110 Hz. Changes in dissipation during the adsorption were in accordance to the changes in frequencies at all ph values. During the rinsing step however, the changes in dissipation were significantly lower than in the case of glucurono xylan samples (BX, BXL), which indicated better persistence of adsorbed arabinoxylan layer. Arabinoxylan had the lowest amount of carboxyle groups and therefore less hydrophilic nature among the reference samples. Higher adsorption of this sample in comparison to other two was most probably driven by its lower solubility in water and lower repulsion between arabinoxylan macromolecules and PET surface. The adsorbed mass in the case of this sample was the highest at ph 7 and amounted to 2219 ng/cm 2 (Figure 4.20). a) b) Figure 4.18: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of carboxymethylated glucuronoxylan (CMBX) a) and arabinoxylan (CMOX) b) onto PET model films at different ph values (4, 5, 7 and 9) -81-

99 There were only negligible differences in frequencies changes during the adsorption of carboxymethylated glucuronoxylan sample (CMBX) at different ph values (Figure 4.18 (a)). In all cases, frequencies decreased to around -50 Hz. After rinsing with MQ water frequency increased for around 15 Hz (to -35 Hz). Adsorbed mass at ph 5 was 610 ng/cm 2 and lowest (520 ng/cm 2 ) at ph 4 (Figure 4.20). When compared to the adsorption of nonmodified glucuronoxylan (BX) (Figure 4.15) the frequencies changes during the adsorption and adsorbed masses of nonmodified xylan were higher. The main reason for that could be lower solubility in water owing to lower content of carboxyl groups and therefore lower repulsion between PET surface and BX macromolecules. Carboxymethylated arabinoxylan (CMOX) as well showed small differences in frequencies changes during the adsorption at different phs. Highest decrease in frequency (to -60 Hz) could be observed at ph 5. At other ph values frequencies decreases were up to -55 Hz. After rinsing with MQ water increase in frequency (desorption of xylans) was the lowest at ph 5 (for 13 Hz). Adsorbed mass of CMOX (Figure 4.20) was the highest at ph 5 (820 ng/cm 2 ). However, the adsorbed mass of CMOX was by about 60 % lower than the adsorbed mass of nomodified arabinoxylan (OX). Higher content of carboxyl groups and therefore higher water solubility of carboxymethylated samples was the main reason for poorer adsorption of these samples onto hydrophobic PET surface. For the same reason the adsorption of CMOX was better in comparison to CMBX. Nonmodified xylans comprise small amounts of carboxylic groups, which was demonstrated with polyelectrolyte titration (part 4.1.6). Deprotonation of carboxylic groups starts at around ph 4. At lower ph molecules have more coiled structure and better adsorption was expected. To prove this the same experiment was performed at ph 2 and the results proved the hypothesis, thus in all the cases where carboxylic groups were present (nonmodified and carboxymethylated xylans) adsorption at ph 2 was the highest. The same effects were not observed in the case of cationized xylans. -82-

100 a) b) Figure 4.19: Frequency (third overtone) (above) and dissipation (below) change as a function of time during the dsorption of cationized glucuronoxylan (CBX) a) and arabinoxylan (COX) b) onto PET model films at different ph values (4, 5, 7 and 9) There were only negligible differences in frequencies changes when cationised xylans were adsorbed to PET surfaces at different phs (Figure 4.19). During the adsorption of cationised glucuronoxylan (CBX) the decreases in frequencies were around -50 Hz and after rinsing frequencies increased to around -30 Hz. At ph 5 the highest adsorbed mass of CBX was noted (584 ng/cm 2 ) and there were only negligible differences in adsorbed masses at other ph values. In case of cationised arabinoxylan (COX) frequencies decreased to -65 Hz during the adsorption at all ph values and after rinsing with MQ water the frequencies increased to -32 Hz. The differences in adsorbed mass at different phs were negligible and amounted around 600 ng/cm 2 (Figure 4.20). The adsorption of COX was higher in comparison to CBX (Figures 4.19 and 4.20). From Figure 4.19 could be seen that adsorption of CBX was faster and equilibrium was -83-

101 reached already after app. 15 min, while in the case of COX the adsorption equllibrium was reached in app. 1 hour mass [ng/cm2] ph4 ph5 ph7 ph BX BXL OX CMBX CMOX CBX COX Figure 4.20: Adsorbed masses after rinsing with MQ water of nonmodified (BX, BXL and OX) and chemically modified (CMBX, CMOX, CBX and COX) xylans onto PET model films at different ph values (4, 5, 7 and 9) Summary Highest adsorption and amount of adsorbed mass among all xylan samples could be observed in case of arabinoxylan (OX), most probably due to lowest amount of carboxylic groups (Table 4.5) present in the sample and therefore lowest solubility in water. Adsorption of majority of xylan samples was the highest at ph 5, with highest frequencies decrease and highest amounts of adsorbed mass. Although xylans were negatively charged at ph 5, the presence of electrolyte caused coiled conformation of molecules and in this way, the adsorption onto PET surfaces was easier. Based on these results ph 5 was chosen as the most appropriate. -84-

102 4.3.2 Influence of ionic strength The results of frequencies and dissipation changes during the adsorption of xylans at various ionic strengths using NaCl or CaCl2 are represented in the Figures 4.21 to For the investigation of the influence of ionic strength on the adsorption kinetics different molarities (0.05, 0.1, 0.3, 0.5 and 0.7 M) of both electrolytes were applied. The concentration of xylans solutions was 100 mg/l and ph was adjusted to 5 on the basis of previous findings (part 4.3.1). As it was expected in all the cases, in the presence of divalent electrolyte (CaCl2) the changes in frequencies and dissipations were higher as in the case of monovalent one. The main reason for such behaviour was the success of shielding of the charge of polymer molecules, especially when it was negative, thus the hydrophobic PET surface, which is at ph higher than 3.1 negatively charged due to preferential adsorption of anions from solutions (OH -, Cl - ) [174]. Figure 4.21: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of nonmodified glucuronoxylan (BX) onto PET model films in the presence of electrolytes (NaCl and CaCl2) -85-

103 Figure 4.22: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of nonmodified glucuronoxylan (BXL) onto PET model films in the presence of electrolytes (NaCl and CaCl2) Figure 4.23: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of nonmodified arabinoxylan (OX) onto PET model films in the presence of electrolytes (NaCl and CaCl2) -86-

104 When low concentration (0.05 M) of monovalent (NaCl) electrolyte was used, the frequency change was only -15 Hz for BX and BXL and -35 Hz for OX. At higher NaCl concentration (0.7 M), frequency change was around -60 Hz for BX and BXL and -86 Hz for OX. When divalent electrolyte (CaCl2) was applied the frequencies changes were higher at all concentrations in comparison to presence of NaCl. At low concentration (0.05 M) frequencies changes amounted to -50 Hz for glucuronoxylans (BX and BXL) and to -80 Hz for OX. At the highest CaCl2 concentration (0.7 M) frequency decreased to around -100 Hz for BX and BXL and even to -140 Hz in the case of OX adsorption. The main reason for such differences in glucurono- or arabinoxylans adsorption was most probably in different contents of carboxylic groups in reference samples and therefore in different water solubility as well as conformations of macromolecules in water solutions. In the cases when xylans contained higher amounts of carboxylic groups (samples BX and BXL) the polymer is more hydrophilic (see chapter 4.2.3) and molecules occupy larger hydrodynamic volume than in the cases, when there are less carboxylic groups (OX). The sample OX showed the highest Lifshitz Van der Waals and the lowest electron-donor component of SFE (Figure 4.12). Low water solubility of polymer is the main driving force of adsorption onto hydrophobic surfaces [175]. As it was expected, after rinsing with MQ water, frequency change dropped to lower values, which indicated, that significant amount of adsorbed layer was removed from the surface and that was confirmed as well by the significant decrease of dissipation after rinsing step (Figures 4.21 to 4.23 (below)). Beside polymer molecules and electrolyte ions the adsorbed layer contained also some amounts of entrapped water molecules. Furthermore, there were no adequate binding sites on the PET surface, on which polymer molecules could successfully attach. For successful binding of negatively charged polymer onto hydrophobic PET surface a drying step would be necessary, which would enable polymer molecules to approach enough to the surface and to attach via Van der Waals forces. Adsorbed masses of nonmodified xylans were calculated using Sauerbrey equation from the frequencies changes in the equllibrium after rinsing with MQ water. Results showed higher adsorbed masses when divalent electrolyte (CaCl2) was used. In case of BX there were only negligible differences in adsorbed masses in presence of different electrolytes. Adsorbed mass when both electrolytes were used was almost same (around 600 ng/cm 2 ). In case of BXL and OX adsorbed mass was higher (for around 70 % and 40 % in case of BXL and OX respectively) when CaCl2 was used compared to adsorbed masses when NaCl was used. -87-

105 Adsorbed masses when CaCl2 was used were 408 ng/cm 2 in case of BXL and 1382 ng/cm 2 in case of OX. OX showed highest adsorbed mass in both cases (NaCl and CaCl2) compared with all other (nonmodified and modified) xylans. Carboxymethylated xylans showed similar behavior during the adsorption onto PET model films as nonmodified ones when application of two electrolytes was compared (Figure 4.24). a) b) Figure 4.24: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of carboxymethylated glucuronoxylan (CMBX) a) and arabinoxylan (CMOX) b) onto PET model films in the presence of electrolytes (NaCl and CaCl2) When NaCl was applied at lower concentration (0.05 M) frequency change amounted only to -14 Hz for both glucurono- (CMBX) and arabinoxylan (CMOX). At the higher concentration of NaCl (0.7 M), frequency changes were around -50 Hz. Rinsing steps caused the same drop of frequency change for both xylans to about -15 Hz in case of NaCl and about -88-

106 -30 Hz in case of CaCl2. In comparison to reference samples frequencies changes for carboxymethylated xylans were significantly lower, by about 10 % when NaCl was applied and even by about 36 % for the sample CMOX in comparison to the processes when CaCl2 was applied. The same trend could be observed after rinsing. This indicated lower adsorption of carboxymethylated xylans, which was due to higher hydrophilicity and better solubility in water. Lower adsorption of these samples in comparison to reference samples was confirmed also by smaller dissipation changes during the adsorption of carboxymethylated xylans. In the case of adsorption of nonmodified and carboxymethylated xylans (all the samples comprised some amounts of carboxyl groups) onto PET surface, the polymer and the PET surface had the same charge, and the driving force of adsorption originated from attractive van der Waals forces between the polymer and the surface [175]. Addition of salt increased the adsorption due to shielding of the charge, however the profound influence on the adsorption has the solvent and solubility of the polymer. This effect had also the crucial role when carboxymethylated xylans were adsorbed. Sample CMOX with the lowest Lifshitz Van-der Waals component and the highest electron donor component of surface free energy showed the largest differences in adsorption behavior when compared to the reference sample (OX), which showed the highest Lifshitz Van der Waals and the lowest electron donor component of SFE (Figure 4.12). Adsorbed masses of carboxymethylated xylans (Figure 4.26) in the presence of CaCl2 were higher when compared to the adsorption in the presence of NaCl and amounted to around 570 ng/cm 2. According to the results of polyelectrolyte titrations the amounts of carboxylic groups in both carboxymethylated samples was nearly the same (chapter 4.1.6), therefore similar behavior during the adsorption in the presence of electrolytes was expected. When cationized xylans were adsorbed no differences in frequencies and dissipation changes could be observed when lower concentrations of both electrolytes were applied (Figure 4.25). -89-

107 a) b) Figure 4.25: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of cationized glucuronoxylan (CBX) a) and arabinoxylan (COX) b) onto PET model films in the presence of electrolytes (NaCl and CaCl2) At higher concentrations of both electrolytes the differences in frequencies and dissipation changes increased slightly and at the maximum concentration (0.7 M) reached the highest values (-64 Hz for CBX and -73 Hz for COX in the presence of NaCl and -90 Hz for CBX and -108 Hz for COX in the presence of CaCl2). At the adsorption of cationised xylans surface had opposite charges and the addition of the salts caused the driving force of the adsorption at the presence of counterions [175]. The entropy caused by the release of the counterions from both the surface and the polymer, brings the system into the lower free energy state and so it drives adsorption in this case. Surface free energies of the cationised samples were lower and Lifshitz Van-der Waals components higher in comparison to the reference samples, which indicated lower hydrophilicity and water solubility. This was most probably the main cause of higher adsorption of these samples in comparison to references as well as to carboxymethylated samples. After rinsing step frequency change decreased by -90-

108 about 60 % for both samples and both electrolytes, which was nearly the same as in all other cases. There was no significant influence of different electrolytes on the adsorbed masses of CBX and COX after rinsing (Figure 4.26) NaCl CaCl2 mass [ng/cm2] BX BXL OX CMBX CMOX CBX COX Figure 4.26: Adsorbed masses after rinsing with MQ water of nonmodified (BX, BXL and OX) and chemically modified (CMBX, CMOX, CBX and COX) xylans onto PET model films in the presence of electrolytes (NaCl and CaCl2) Summary With higher ionic strengths, adsorptions of all xylans onto PET model films were higher. The differences were higher in the case of negatively charged polymers in comparison to cationic ones, which were caused by the mechanism of adsorption of negatively charged polymers onto negatively charged PET surface. In general the adsorption of chemically modified xylans was lower in comparison to the nonmodified samples. The main reason for that were the hydrophilicity and/or solubility of polymers in water. Less soluble polymers adsorb better onto hydrophobic surfaces. The other reason was changed conformation of polymers from flat -91-

109 to coil-like structure, which was more effective, when molecule comprised less hydrophilic carboxyl groups. Repulsive forces between negatively charged PET surface and negatively charged polymers could be shielded with added salt and adsorption could be increased. This shielding was stronger when divalent calcium cations were added [176] Influence of xylan concentration For the investigation of the influence of concentration of xylans solutions on adsorption onto PET films, xylans were adsorbed from solutions with concentrations 50 mg/l, 100 mg/l, 200 mg/l and 500 mg/l. 0.1 M CaCl2 was added and ph was adjusted to 5. Figure 4.27: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of nonmodified glucuronoxylan (BX) at different solution concentrations onto PET model films -92-

110 Figure 4.28: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of nonmodified glucuronoxylan (BXL) at different solution concentrations onto PET model films Figure 4.29: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of nonmodified arabinoxylan (OX) at different solution concentrations onto PET model films -93-

111 From the Figures 4.27 to 4.29 could be seen that for the samples BX and OX at the highest concentration (500 mg/l) adsorption was higher, thus the frequencies and dissipation changes at equilibrium were significantly higher in comparison to the adsorption from lower solutions concentrations. When xylan concentration was 50 mg/l frequency decreased to -60 Hz and -72 Hz for BX and OX respectively. After rinsing with MQ water frequency increased to -40 for BX, but there was no change in frequency in case of OX. At highest concentration frequency decreased to -72 Hz in case of BX and to -87 Hz in case of OX and after rinsing increased to -42 for BX and -70 for OX. Adsorbed mass (Figure 4.32) of BX after rinsing with MQ water was highest at highest xylan concentration and it amounted to 654 ng/cm 2. The mass of adsorbed OX was again the highest compared to all other samples, furthermore the highest mass of this sample (1323 ng/cm 2 ) was adsorbed from the lowest solution concentration 50 mg/l. For the glucuronoxylan (BXL) sample when compared the adsorptions at the lower concentrations (form 50 to 200 mg/l) there were no significant differences in frequencies (around -60 Hz) and dissipation changes at the equilibrium. Adsorbed mass (Figure 4.32) in this case was highest when solution concentration 50 mg/l was applied. However, with the higher polymer concentration the time when equilibrium was reached was shorter, thus at the highest concentration (500 mg/l) the equilibrium was reached already after 5 min, compared to lower concentrations, where the equilibria were reached after 30 min or more. These differences were more obvious for the arabinoxylan sample as for glucuronoxylans. Dissipation changes support the results of frequency changes for all nonmodified samples. With the highest frequency change, dissipation change was the highest, which confirmed growth in adsorbed layer thickness during the adsorption. At the ph 5 nonmodified xylans were negatively charged but the presence of electrolyte shielded the charge and caused more or less coiled conformation of molecules, which were deposited onto hydrophobic PET surface. At the lower concentrations the adsorption was lower than in the case of higher concentrations. Rinsing step removed app. 30 % of adsorbed layer in the case of glucuronoxylans and app. 20 % of adsorbed layer in the case of arabinoxylan. Better resistance of the adsorbed layer of arabynoxylan is most probably connected to the lower hydrophilicity of this polymer and higher Lifshitz Van der Waals component of surface free energy (see the Figures 4.12 and 4.13). -94-

112 a) b) Figure 4.30: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of carboxymethylated glucuronoxylan (CMBX) a) and arabinoxylan (CMOX) b) at different solution concentrations onto PET model films In case of carboxymethylated xylans (Figure 4.30) could be clearly seen that with increase of concentration, frequency changes decreased. Larger differences in frequencies changes at equillibria as well as in velocities of these changes during the adsorption could be observed when different concentrations of CMBX and CMOX samples were applied in comparison to nonmodified ones. In general the frequencies changes at equilibria were by about 30 % lower in comparison to nonmodified xylans. The main reason for that could be the higher amounts of carboxyl groups present in these samples and therefore higher water solubility. Larger differences could be observed for the sample CMBX. At lowest concentration frequency decreased to -20 Hz and -30 Hz for CMBX and CMOX respectively. The highest adsorption occurred at the concentration of 500 mg/l. Frequency decreased to - 52 Hz for CMBX and to -46 Hz for CMOX. After rinsing with MQ water frequency increased to -25 for CMBX and to -36 Hz for CMOX. Adsorbed mass, as could be seen from Figure 4.32 was also lowest and with increase of xylan concentration, adsorbed mass increased. At -95-

113 highest xylan concentration adsorbed mass was 459 ng/cm 2 for CMBX and 569 ng/cm 2 for CMOX. Dissipations changes were in accordance to the changes in frequencies. At the highest concentration dissipation changes were the highest, which confirmed building of thicker layer owing to higher amounts of polymer electrolyte ions and water molecules deposited. The results of frequency and dissipation changes during the adsorbtion of cationized xylans (CBX and COX) onto PET model films at different concentrations are represented in the Figure a) b) Figure 4.31: Frequency (third overtone) (above) and dissipation (below) changes as a function of time during the adsorption of cationized glucuronoxylan (CBX) a) and arabinoxylan (COX) b) at different solution concentrations onto PET model films Cationized glucuronoxylan (CBX) did not show any differences in frequency or dissipation changes at equilibrium with changing the concentration. In all cases, frequency decreased to around -40 Hz and after rinsing with MQ water increased to around -25 Hz. The -96-

114 solution concentration had strong influence on the time when equilibrium of adsorption was reached. At the highest concentration the equilibrium of adsorption was reached very fast, (in about 5 to 7 min), in the case of the lowest concentration that time was significantly longer, at the lowest concentrations longer than 30 min. Adsorbed mass (Figure 4.32) was similar at all xylan concentrations with differences between 20 and 30 ng/cm 2. Highest adsorbed mass (422 ng/cm 2 ) was achieved when 100 mg/l of xylan was used. Dissipations changes were in accordance to the changes in frequencies. However, after rinsing dissipations results showed opposite effect than frequencies. At lower concentrations dissipations after rinsing for CBX were higher than at higher concentrations. This means that at low concentrations the adsorbed layer after rinsing was thicker and more viscous. In the case of cationized arabinoxylan (COX) the differences in adsorption at different concentrations were more significant and changes in frequencies and dissipations during the adsorption more pronounced when compared to cationised glucuronoxylan. At lowest concentration frequency decreased to -44 Hz and after rinsing increased to -39 Hz. When the concentration of 500 mg/l was applied frequency decreased to -60 Hz and after rinsing, frequency increased to -40 Hz. At concentration 100 mg/l adsorbed mass of COX was the highest and amounted to 701 ng/cm 2. Dissipations changes were in accordance to the changes in frequencies. At the highest concentration dissipation changes were the highest, which confirmed building of thicker layer owing to higher amounts of polymer molecules, electrolyte ions and water molecules deposited. -97-

115 mg/l 100 mg/l 200 mg/l 500 mg/l mass [ng/cm2] BX BXL OX CMBX CMOX CBX COX Figure 4.32: Adsorbed masses after rinsing with MQ water of nonmodified (BX, BXL and OX) and chemically modified (CMBX, CMOX, CBX and COX) xylans onto PET model films at different solution concentration Summary From the results of adsorption of nonmodified and modified xylans it follows that frequencies and dissipation changes at equilibrium were significantly higher when higher concentrations were applied in comparison to the adsorption from lower concentrations. With increase of concentration, adsorption increased in almost all cases, except in case of BXL and cationized (CBX) where no differences in frequency between different polymer concentrations were observed. Adsorbed masses (mass of xylan, electrolyte ions and entrapped water molecules) calculated after rinsing with MQ water, showed that in case of BXL and OX, highest adsorbed mass was achieved when 50 mg/l of xylan concentration was used. In case of arabinoxylan this amount was highest compared to all other samples. This is most probably due to more branched structure of this xylan which could cause larger sterical hindrances at higher solution concentrations and therefore lower adsorption. -98-

116 4.3.4 Adsorption of xylans onto PET model films using anchoring polymers In order to improve the adsorption and persistence of adsorbed xylan layers after rinsing, two polymers with specific functional groups were applied as anchoring polymers. In the case of carboxymethylated xylans positively charged polymer Polyethyleneimine (PEI) was applied in concentration of 0.05%. For improvement of the adsorption of cationic xylans two types of anchoring polymers were applied, in the first phase positively charged (PEI) and then negatively charged (PVSA-Poly(vinylsulfonic acid, sodium salt). After each adsorption stage the samples were dried in vacuum oven. PVSA was applied in concentration of 0.25%. Xylan solutions were prepared in concentrations of 500 mg/l with 0.1M CaCl2 at ph5. Schematic representation of adsorption of xylans onto PET model surface using anchoring agents is represented in the Figure PET PEI PET CMBX/CMOX PET Ca 2+ Ca Ca 2+ Ca - 2+ PEI PET PET PET PVSA - CBX/COX Ca Ca 2+ Ca2+ Ca 2+ Figure 4.33: Schematic representation of adsorption of xylans onto PET surfaces using PEI and PVSA as anchoring agents. In the Table 4.10 are represented frequencies values at equillibrium of adsorption and after rinsing of carboxymethylated glucurono- and arabinoxylan (CMBX and CMOX) and -99-

117 cationized glucurono- and arabinoxylan (CBX and COX) without and with anchoring agents applied. Presented are only frequency values when xylans are adsorbed, values of anchoring agents are subtracted. Without PEI, frequency decreases were -25 Hz for CMBX and -38 Hz for CMOX. When PEI was used frequencies at equllibrium after rinsing showed higher changes: -62 Hz for CMBX and -109 Hz for CMOX. These values showed that anchoring agents significantly improve the adsorption of xylans onto PET model surface and especially the persistence after rinsing with water. Adsorption of xylan layers was higher for 2.5 times in case of CMBX and 2.9 times in case of CMOX when PEI was used. Dissipation changes for CMBX were in accordance with frequency changes and were smaller compared to CMOX. Most probably the reason for this could be more branched molecular structure [176] and higher molecular weight of arabinoxylans (Table 4.4). For cationic xylans frequencies changes at equilibrium after rinsing without anchoring polymers were similar to that ones of carboxymethylated xylans. However, when PEI and PVSA layers were applied frequency changes at equillibrium were 2.5 times higher for CBX and 1.4 times higher for COX (Table 4.10). Table 4.10: Frequency (df3 - third overtone) values at adsorption equillibrium and after rinsing for carboxymethylated and cationized xylans with and without application of anchoring polymers (PEI, PVSA) Xylan sample df3 (Hz) without anchoring polymers adsorption equillibrium after rinsing df3 (Hz) with anchoring polymers (PEI, PVSA) adsorption equillibrium after rinsing CMBX CMOX CBX COX

118 The application of intermediate anchoring polymer layers was proved to have large influence in the first place on the persistance of adsorbed xylans. In the Figure 4.34 are represented adsorbed masses of xylans at equillibrum after rinsing with and without using anchoring polymers. In all the cases, the adsorbed masses of anchoring polymers were subtracted. Only masses of adsorbed xylans, water and electrolyte molecules were taken into account mass [ng/cm 2 ] CMBX CMOX CBX COX w ith w ithout Figure 4.34: Adsorbed masses after rinsing with MQ water of carboxymethylated (CMBX, CMOX) and cationized (CBX and COX) xylans with and without anchoring polymers (PEI, PVSA) From this results could be clearly seen that anchoring polymers significantly contribute to better adsorption and fixation of xylans onto PET surfaces. The masses of adsorbed xylans when anchoring polymers were not applied were significantly lower in comparison to the masses of adsorbed layers when anchoring polymers were used. Adsorbed masses after rinsing of carboxymethylated xylans when PEI was not used were similar. Masses were 458 ng/cm 2 for CMBX and 569 ng/cm 2 for CMOX. When anchoring polymers was used adsorbed masses after rinsing were around three times higher and amouted to 1490 ng/cm 2 for CMBX and 1751 ng/cm 2 for CMOX. For cationised xylans adsorbed masses after rinsing were increased when intermediate layers of anchoring polymers were applied for about two times in case of CBX (to 822 ng/cm 2 ) and 1.5 times in case of COX (to 1006 ng/cm 2 ) in comparison to the procedures whitout anchoring layers

119 Electrostatic interaction are driving force for this adsorption, which also provide stability of adsorbed layers X-ray photoelectron spectroscopy Surface elemental composition of the PET model films, before and after the adsorption of anchoring polymers and modified xylans, are represented in the Table Table 4.11: Surface elemental composition of PET model films before and after the adsorption of anchoring polymers (PEI, PVSA) and chemically modified xylans (CMBX, CMOX, CBX, COX) Xylan sample Surface elemental composition (at. %) C O N S O/C PET PET+PEI PET+PEI+PVSA PET+PEI+CMBX PET+PEI+CMOX PET+PEI+PVSA+CBX PET+PEI+PVSA+COX Pure PET model film surface contained only carbon and oxygen in O/C ratio of 0.35, which was in accordance to PET chemical structure (Figure 2.3). After PEI adsorption 6.6 at. % of nitrogen was presented on the surface and at the same time the surface O/C ratio droped to This was an evidence that PEI was succesfully adsorbed onto PET surface. After next step, adsorption of PVSA onto PEI layer, 1 at. % of sulphur is detected on the surface and -102-

120 accordingly the surface portion of nitrogen decreased by about about 73 % due to covering of PEI layer. After the adsorption of carboxymethylated xylans onto PET+PEI surface, surface share of nitrogen was decreased by about 20 %, which means that a covering of PEI layer was not complete. However, the additional evidence of the presence of carboxymethylated xylans' layers on the PET+PEI surface was the significant increase (by about 70 %) of the O/C surface ratio in comparison to PET-PEI surface. Adsorption of cationized xylans onto PET+PEI+PVSA layer, caused significan increase in surface share of nitrogen (by about 194 % for CBX and 177 % for COX). These results proved a successful adsorption and fixation of cationised xylans onto previously adsorbed PVSA layer Hydrophilic/hydrophobic character and surface free energy In order to follow the changes in surface properties caused by the adsorption of different polymers, QCM crystals were dried in vacuum after each adsorption process and contact angles with three chosen liquids were determined by goniometry. Surface free energies and their components of carboxymethylated xylans calculated on the basis of these measurements are represented in the Figures 4.35 and PET model film showed low total surface energy γs TOT of 36.6 ± 1.6 mj/m 2 with high content of Lifshitz van der Waals contribution (γs LW = 35.2 ± 0.8). Electron acceptance contribution is not presented onto surface and electron donating contribution (γs - ) was 12.8 ± 1.5 because of carboxyl groups present in PET. After PEI adsorption total surface energy (γs TOT = 41.5 ± 3.8 mj/m 2 ) and its Lifshitz van der Waals component slightly increased in comparison to pure PET surface (γs LW = 38.0 ± 1.24). Electron acceptance contribution (γs + ) in this case appeared (γs + = 5.9 ±0.5) and electron donor (γs - ) contribution decreased to

121 70 Surface free energy [mj/m 2 ] γslw: γs+: γs-: γstot 0 PET PET+PEI PET+PEI+CMBX PET+PEI+CMOX Figure 4.35: Surface free energies and their components of PET model films before (PET) and after adsorption of anchoring polymer (PEI) and carboxymethylated xylans (CMBX, CMOX) After the adsorption of carboxymethylated xylans onto PET+PEI surface total surface energies as well as its electron-donor components significantly increased, while the Lifshitz van der Waals contribution decreased. Total surface free energies increased after CMBX and CMOX adsorption for around 35 % compared to PET-PEI surfaces. Electron donating components of SFE increased by about 65 and 80 times in case of CMBX and CMOX respectively. Lifshitz van der Waals contributions dropped by about 20 %. This results clearly confirmed successful adsorption and fixation of carboxymethylated xylans on the PET+PEI surface

122 70 Surface free energy [mj/m 2 ] γslw: γs+: γs-: γstot 0 PET PET+PEI PET+PEI+PVSA PET+PEI+ PVSA+CBX PET+PEI+ PVSA+COX Figure 4.36: Surface free energies and their components of PET model film before (PET) and after the adsorption of anchoring polymers (PEI+PVSA) and cationized xylans (CBX, COX) After the adsorption of PVSA on the PET+PEI surface total surface energy (γs TOT = 46.6 ± 3.37 mj/m 2 ) increased by about 12 % and Lifshitz van der Waals component increased by about 10 % (γs LW = 42 ± 1.2). Owing to introduction of sulphonic groups (from the PVSA) to the PET+PEI surface electron-donor contribution (γs - ) increased significantly from 0.5 to 5.35 ± 2.42 mj/m 2 and electron acceptance contribution (γs + ) dropped from 5.9 ±0.5 to 0.97 ± 0.49 mj/m 2 compared to PET +PEI layer. An increase of total SFE could be observed after the adsorption of cationized xylans onto PET+PEI+PVSA surface, by about 6 % for CBX and by about 14 % for COX. Lifshitz van der Waals components decresed by about 25 %, while electron donor and electron acceptor contributions increased in both cases, which indicated more polar surface character [173]. Hydrophilic/hidrophobic characters of differently treated PET surfaces were analysed using water contact angle WCA determination. Results are represented in the Figure

123 80 60 WCA / PET PET+PEI PET+PEI+ CMBX PET+PEI+ CMOX PET+PEI+ PVSA PET+PEI+ PVSA+CBX PET+PEI+ PVSA+COX Figure 4.37: Water contact angles (WCA) of pure PET model film (PET) and PET model films after adsorption of anchoring polymers (PEI, PVSA) and chemically modified xylans (CMBX, CMOX, CBX and COX) It could be clearly seen that surfaces of pure PET model film (WCA = 73.4 ± 1.3) and the film after the adsorption of PEI (WCA = 76.9 ± 3.1) and PVSA (WCA = 73.2 ± 2.9) showed less hydrophilic character in comparison to all other surfaces with adsorbed xylans' layers. After xylan adsorption, contact angles decreased in general for more than 30 %. After the adsorption of carboxymethylated xylans onto PET+PEI surfaces contact angles decreased for more than 50 % in comparison to the WCA of PET+PEI surface. Adsorption of cationised glucuronoxylan (CBX) caused smaller decrease of WCA (by about 28 %) in comparison to COX (by about 60 %). 4.4 Adsorption of xylans onto PET fabric In the last phase of this research, chemically modified xylans were applied for real PET fabrics surface functionalisation. As a most conventional form of PET fibrous material used in technical sector, woven PET material was chosen as a real system

124 For the activation of PET fabrics surfaces hydrolisis was performed prior to the treatments with chemically modified xylans. Spraying technique was applied for the surface treatments with xylans solutions. Surface properties of PET fabric treated with xylans were analysed using Scanning Electron Microscopy (for characterizing of surface morphology) and goniometry (for determination of wettability). The amounts of charged groups in the fabric samples were determined using potentiometric titration and spectrophotometric (Methylene blue and Acid Orange 7 adsorption) methods. Antimicrobial properties were determined by the method according to ASTM E PET PET PET NaOH PET PET + + CMBX/CMOX PEI COO- OH COO- OH COO- OH COO- + OH COO- + OH COO- + OH COO- + OH COO- + OH COO Ca 2+ Ca 2+ - Ca 2+ - Ca 2+ - CBX/COX OH COO- + OH Ca 2+ COO- + OH COO Ca 2+ Ca 2+ Figure 4.38: Schematic representation of adsorption of xylans onto PET fabric samples -107-

125 4.4.1 Charging behaviour Determination of negative charge Potentiometric titration and Methylene blue adsorption method were used for the determination of negative charge of untreated PET fabric sample (PET), hydrolysed PET fabric sample (PET-H) PET fabric samples treated with PEI (PET+PEI) and further adsorbed by carboxymethylated xylans (PET+PEI+CMBX and PET+PEI+CMOX). In the Table 4.12 the amounts of charged groups of untreated, hydrolysed and differently treated PET fabric samples are represented. The amounts of charge was calculated according to the Eq 2.10 from charging isotherms obtained by potentiometric titrations (see Figure 4.39). Charge per mass [mmol/g] PET PET-H PET+PEI PET+PEI+CMBX PET+PEI+CMOX ph Figure 4.39: Potentiometric charge titration values as a function of ph for nontretaed PET fabric (PET), hydrolysed PET fabric (PET-H) and PET fabric samples with adsorbed PEI and carboxymethylated xylans (CMBX, CMOX) -108-

126 Table 4.12: Amount of charged groups in untreated, hydrolysed and differently treated PET fabric Xylan sample Amount of charge [mmol/kg] positive negative PET / PET-H / PET+PEI 11.0 / PET+PEI+CMBX / PET+PEI+CMOX / Untreated PET fabric showed small amount of negative charge (21.06 mmol/kg) owing to the carboxyl end-groups present in PET. Negative charge of hydrolysed PET fabric was mmol/kg and this amount was more than 75 % higher compared to untreated fabric sample. This was expectable because new carboxyl end groups should be introduced after hydrolysis procedure. After a treatment with PEI onto hydrolysed PET fabric surface, amino groups were introduced. The results indicate almost total covering of hydrolised PET surface with PEI. Further treatment of PET+PEI fabrics with carboxymethylated xylans converted fabrics into anionic character. From charging isotherms which show the deprotonation/protonation behaviour typical for presence of carboxyl groups one can see that attachment of CMBX onto PET-PEI resulted into mmol/kg ionic charge, whilst the attachment of CMOX resulted in mmol COO - groups/kg of fabric. Even more, carboxymethylated xylans totally cover PET-PEI surface; i.e. positive charge is not anymore available, most probable due to electrostatic interactions between PEI and CMOX or CMBX, respectively [177]. For determination of the presence of negative charge caused by COO - groups methylene blue adsortion method was performed in order to support potentiometric titration results (Figure 4.40)

127 40 negative charge [mmolcooh/kg] PET PET-H PET+PEI+CMBX PET+PEI+CMOX Figure 4.40: Amounts of negatively charged groups in untreated PET fabric and PET fabrics treated with carboxymethylated xylans determined by methylene blue adsorption method From the Figure 4.40 it could be again clearly seen that hydrolization of PET fabric introduced carboxyl end-groups (27.62 mmol/kg). Amount of -COOH groups in untreated PET was mmol/kg. When carboxymethylated xylans were sprayed onto PET+PEI fabric samples it could be seen that amount of -COOH groups was increased. In the case of CMBX application, the amount of -COOH groups accounted 31.4 mmol/kg and in the case of CMOX amount of - COOH groups was 32.7 mmol/kg. When results of both techniques are compared it can be concluded that results followed the same trend as well as they are good correlating. Same results for both techniques were already demonstrated by [178]. Determination of positive charge Potentiometric titration and spectrophotometric C.I. Acid Orange 7 method were used to determine the amount of accessible amino groups in the PET fabric samples treated with cationised xylans

128 In the Table 4.13 the amounts of charged groups of untreated, hydrolysed and differently treated PET fabric samples are represented. The amounts of charge were calculated from charging isotherms (Figure 4.41) obtained by potentiometric titration using Eq Charge per mass [mmol/g] PET PET-H PET+CBX PET+COX ph Figure 4.41: Potentiometric charge titration values as a function of ph for untretaed PET fabric (PET), hydrolysed PET fabric (PET-H) and PET fabric samples with adsorbed cationized xylans (CBX, COX) Table 4.13: Amount of charged groups in hydrolysed and differently treated PET fabric Xylan sample Amount of charge [mmol/kg] positive negative PET / PET-H / PET+ CBX PET+ COX

129 It has to be pointed out that quaternary ammonium groups which were introduced with the cationisation procedures into xylan structures could not be detected by potentiometric titrations. Some positive charge that appeared under ph 5 at the samples treated with cationised xylans are most probably due to the presence of small amounts of primary ammonium groups. Positive charge was 9.96 mmol/kg in case of PET fabric treated with CBX and 2 mmol/kg for fabric treated with COX. This small amount of primary ammonium groups was also detected in cationic samples by the polyelectrolyte titrations (Table 4.5). In both cases some amounts of negative charge were still present (9.40 mmol/kg for CBX and mmol/kg for COX), most probably due to nonequal covering of hydrolysed PET fabric surface, which was also observed by SEM microscopy (chapter 4.4.2). However, the amounts of negative charge was significantly decreased compared to charge of hydrolysed PET fabric. For the sample treated with CBX the negative charge decreased by about 75 % and for COX by about 60 % when compared to PET-H. In order to detect quaternary amino groups in PET fabric samples treated with cationised xylan samples, anionic C.I. Acid Orange 7 dye adsorption method was performed. Amino groups were detected by monitoring of the concentration of anionic C.I. Acid Orange 7 dye, when material containing amino groups was immersed in the dye bath at ph 3.6, where full protonation of amino groups was expected. The results are represented in the Figure Positively charged groups [mmol/kg] PET PET+CBX PET+COX Figure 4.42: Amounts of positively charged groups in untreated PET fabric and PET fabrics treated with cationized xylans determined by Acid Orange adsorption method -112-

130 The amount of amino groups presented on PET+CBX sample was 3.34 mmol/kg and PET+COX was 2.05 mmol/kg. The sample CBX contained slightly higher amounts of positively charged groups compared to the sample COX, which is in good accordance to the higher degree of substitution in the sample CBX (part 4.1.2). These amounts are too small and are not in accordance with results of potentiomentric titration. With potentiometric titration, as discussed above, can be detected only primary ammonium groups, but with this method can be detected total amount of charged amino groups (including quaternary ammonium gorups), so with this method, higher amount of charged groups was expected Surface Morphology Surface morphologies of PET fabric samples before and after the treatment with chemically modified xylans were studied by scanning electron microscopy (SEM). SEM images are represented in the Figures 4.43 to Figure 4.43: Surface morphology of untreated PET fabric at two different magnifications (5000 X-left and 1000 X-right) The surface morphology of untreated PET fabric showed smooth filament surfaces with some separate small particles on it, which most probably arise from impurities and/or dust. After the treatment of PET fabric with carboxymethylated glucuronoxylan (CMBX) rather thick layer covering filaments as well as filling the gaps in between can be observed -113-

131 (Figure 4.44). The coating is relatively smooth without larger particles or embossments. Some cracks could be seen in the adsorbed layer, which fills up the gap between filaments. This cracks are most probably the result of lower flexibility of the coating in comparison to the substrate - PET fabric. Figure 4.44: Surface morphology of PET fabric treated with carboxymethylated glucuronoxylan (CMBX) at two different magnifications (5000 X-left and 1000 X-right) Figure 4.45: Surface morphology of PET fabric treated with carboxymethylated arabinoxylan (CMOX) at two different magnifications (5000 X-left and 1000 X-right) The surface of PET filaments treated with carboxymethylated arabinoxylan (CMOX) was much more irregular and rough with some amount of larger clods. In comparison to CMBX layer, the surface morphology of CMOX layer is more corrugated and thicker, which -114-

132 is in accordance with the morphologies of the xylan films analysed by AFM, where the CMOX film surface morphology had much more irregular surface structure with larger hollows and embossments, and the highest root mean-squared roughness. The main reason for these differences could lay in different molecular structures (higher amount of side chains in arabinoxylan) and therefore different supermolecular arrangements during the formation of a film. Figure 4.46: Surface morphology of PET fabric treated with cationized glucuronoxylan (CBX) at two different magnifications (5000 X-left and 1000 X-right) Figure 4.47: Surface morphology of PET fabric treated with cationized arabinoxylan (COX) at two different magnifications (5000 X-left and 1000 X-right) The surfaces of the PET filaments after the adsorption of cationised xylans are pretty much the same. The adsorbed layers morphologies were rather smooth with some separate clods -115-

133 and particles incorporated. Individual cracks of the adsorbed layers, are most probably the result of different mechanical properties of the layers and substrates Contact angles and wettability In order to found out whether the hydrophilic properties of xylans were successfully introduced to PET fabric after the treatments with xylans, static contact angles with water or water contact angles (WCA) were measured. The measurement technique using goniometry is represented in the Figure The image proves the hydrophobic nature of nontreated PET fabric surface, thus the WCA between water drop and the fabric surface is larger than 90. Figure 4.48: Photograph of a drop of water on the surface of nonmodified PET fabric Water contact angles values for untreated PET fabric sample and PET fabric treated with carboxymethylated and cationized xylans at different contact times (from 0 to 0.5 second) are represented in the Figure WCA measurements were performed using Milli- Q water. High speed camera allowed accurate determination of contact angles for different liquid drops as a function of time