Kompletní sborník ke stažení (60 MB) - Fakulta chemická - Vysoké ...

Vysoké učení technické v Brně 

Fakulta chemická 

Sborník příspěvků 

soutěže Studentské tvůrčí činnosti „STUDENT 2006” 

a doktorské soutěže „O cenu děkana 2005 a 2006” 

Brno 2006

© Vysoké učení technické v Brně, Fakulta chemická, 2006 

ISBN: 80-214-3321-3 

Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006 

strana 2

OBSAH: 

Předmluva...........................................................................................................................................................7 

PRÁCE STUDENTŮ MAGISTERSKÝCH STUDIJNÍCH PROGRAMŮ 

SOUTĚŽE STUDENTSKÉ TVŮRČÍ ČINNOSTI 

SEKCE STČ 2006 

VLIV PROVOZNÍCH PARAMETRŮ FLOTACE NA SEPARAČNÍ ÚČINNOST ÚPRAVNY VODY MOSTIŠTĚ 

Jana Burianová..................................................................................................................................................9 

SYNTHESIS, CHARACTERIZATION AND ECOTOXICOLOGICAL ASSESSMENT OF NEW FLEXIBLE 

POLYURETHANE FOAMS WITH BIODEGRADABLE FILLERS 

Jan David..........................................................................................................................................................15 

NOVÝ SORPČNÍ GEL NA BÁZI SPHERON-OXINU PRO UŽITÍ V TECHNICE DGT 

Michaela Gregušová.........................................................................................................................................20 

MOLECULAR DYNAMICS SIMULATION OF POLYMER CHAIN IN THE VICINITY OF 

NANOPARTICLE 

Kateřina Hynštová............................................................................................................................................24 

APPLIKÁCIA NOVEJ EURÓPSKEJ NORNY NA STANOVENIE Cr(VI) V CEMENTE 

Tomáš Ifka.........................................................................................................................................................28 

DISSOLUTION OF HUMIC SUBSTANCES IN UREA IN LIGHT OF SUPRAMOLECULAR THEORY 

Jiří Kislinger.....................................................................................................................................................34 

WETTABILITY OF PLASMA POLYMERIZED VINYLTRIETHOXYSILANE FILM 

Soňa Lichovníková............................................................................................................................................39 

URČOVANIE DISTRIBÚCIE DOMÉN V SPIN CROSSOVER SYSTÉMOCH POMOCOU ANALYTIC- 

KEJ FUNKCIE 

Ján Pavlik.........................................................................................................................................................44 

A STUDY OF INTERFACIAL ASPECTS OF EPOXY-BASED HYBRID COMPOSITES REINFORCED 

WITH BASALT, CARBON AND CERAMIC FIBRES BY DMA ANALYZIS 

Lukáš Recman...................................................................................................................................................48 

ŠTÚDIUM ANTIOXIDANTOV A ICH ÚČINKOV PRI STABILIZÁCII POLYMÉROV 

Ján Rimarčík.....................................................................................................................................................54 

HOŘLAVOST A MECHANICKÉ VLASNOTSTI NANOKOMPOZITŮ EVA/Mg(OH) 2 

Jiří Sadílek........................................................................................................................................................59 

STUDIUM MECHANISMU KOORDINAČNÍ POLYMERACE HEXA-1,5-DIENU KATALYZOVANÉ 

FENOXYIMINOVÝM KOMPLEXEM TITANU A METHYLALUMINOXANEM 

Jan Ševčík.........................................................................................................................................................64 

MOLEKULOVÁ DYNAMIKA C-GLYKOZYLNITROMETÁNOV 

Stanislava Šoralová..........................................................................................................................................67 


strana 3

STUDIUM DEGRADACE TISKU NA TENKÝCH POLYMERNÍCH VRSTVÁCH 

Jiří Stančík.......................................................................................................................................................71 

ZMĚNY OBSAHU AMINOKYSELIN V BRAMBORÁCH V ZÁVISLOSTI NA HNOJENÍ DUSÍKEM 

Petra Valová.....................................................................................................................................................77 

PRECIPITATION OF DL – VALINE FROM AQUEOUS ISOPROPANOL SOLUTIONS 

Miroslav Variny................................................................................................................................................81 

VÝVOJ MIKROŠTRUKTÚRY SODNO-BORITO-KREMIČITÝCH SKIEL S PRÍDAVKOM TIO 2 

Oto Vojtechovský..............................................................................................................................................86 

VÝZNAM VOĽNÝCH RADIKÁLOV PRI POŠKODENÍ PAPIERA (EPR ŠTÚDIUM) 

Zuzana Vrecková..............................................................................................................................................91 

PRÁCE STUDENTŮ DOKTORSKÝCH STUDIJNÍCH PROGRAMŮ 

SOUTĚŽE O CENU DĚKANA 2005 

SEKCE DSP 2005 

CHITOSAN - A NEW TYPE OF POLYMER COAGULANT 

Marcela Borovičková.......................................................................................................................................97 

PREPARATION OF SELF-CLEANING PHOTOCATALYTICALLY ACTIVE SURFACES 

Jana Chomoucká............................................................................................................................................101 

RHEOLOGICAL, VISCOUS, AND SURFACE AACTIVE BEHAVIOR OF POLYSACCHARIDES IN 

AQUEOUS SOLUTIONS 

Martin Chytil..................................................................................................................................................106 

STUDY OF PHYSICOCHEMICAL AND ANTIOXIDATIVE PROPERTIES OF YEAST MEMBRANE 

COMPONENTS 

Michaela Drabkova........................................................................................................................................112 

DETERMINATION OF YEAST VIABILITY BY MEANS OF FLUORESCENCE MICROSCOPY AND 

IMAGE ANALYSIS 

Petra Jeřábková..............................................................................................................................................115 

FLOW BEHAVIOUR OF DILUTED SUSPENSIONS IN CARBOXYMETHYLCELLULOSE-WATER 

SOLUTION 

Michal Klimovič.............................................................................................................................................119 

USE OF VARIOUS POLYACRYLAMIDE DIFFUSIVE GELS FOR ASSESSMENT OF METAL AVAIL- 

ABILITY IN SOILS 

Vladěna Kovaříková.......................................................................................................................................123 

SPECTROPHOTOMETRIC STUDY OF SOME BIOLOGICALBUFFERS SOLUTIONS 

Miroslava Krčmová........................................................................................................................................128 

PHYSIOLOGICAL CHARACTERISATION OF XYLOSE-UTILISING SACCHAROMYCES CEREVI- 

SIAE STRAINS 

Jitka Kubešová................................................................................................................................................133 


strana 4

THE EFFECT OF STERILIZATION HEATING ON NUTRITION QUALITY OF PROCESSED CHEESE 

Blanka Loupancová........................................................................................................................................137 

INVESTIGATION OF ENVIRONMENTAL AND VARIETAL INFLUENCES ON DISTRIBUTION OF 

STARCH GRANULES IN BARLEY KERNELS BY GFFF AND LASER PARTICLE SIZE ANALYSIS 

Karel Mazanec................................................................................................................................................142 

FIBER ASPECT RATIO INFLUENCE ON FRACTURE IMPACT TOUGNESS OF POLYMETHYL 

METHACRYLATE/POLYVINYL ALCOHOL COMPOSITES 

Vladimír Pavelka............................................................................................................................................147 

DIAGNOSTIC OF THE DIAPHRAGM DISCHARGE IN WATER SOLUTIONS BY OPTICAL 

EMISSION SPECTROSCOPY 

Jana Procházková..........................................................................................................................................151 

SENSORIC PROPERTIES OF SOLUBLE PHTHALOCYANINES 

Kateřina Severová..........................................................................................................................................155 

INFLUENCE OF TRIETHYLALUMINIUM COCATALYST ON INITIAL KINETIC OF PROPENE 

POLYMERIZATION 

Miroslav Skoumal..........................................................................................................................................159 

PHYSICO-CHEMICAL PROPERTIES OF PLASMA-POLYMERIZED TETRAVINYLSILANE 

Jan Studýnka...................................................................................................................................................164 

PULSED 1 H-NMR OF IMPACT COPOLYMERS OF PROPYLENE (ICP) 

Ladislav Vilč...................................................................................................................................................169 

SPECIATION OF FIVE SELENIUM COMPOUNDS BY HPLC-HEATING-UV-HG-AFS 

Eva Vitoulová.................................................................................................................................................175 

EPITAXIAL GROWTH OF Ni AND Co ON (111) METALLIC SUBSTRATES 

Martin Zelený.................................................................................................................................................182 

ACETYLCHOLINESTERASE INHIBITOR FROM NOSTOC SLIZ. KOL. 

Petr Zelík........................................................................................................................................................187 

HUMIC ACIDS IDENTIFICATION BY PY-GCMS TECHNIQUE 

Pavla Žbánková..............................................................................................................................................191 

PRÁCE STUDENTŮ DOKTORSKÝCH STUDIJNÍCH PROGRAMŮ 

SOUTĚŽE O CENU DĚKANA 2006 

SEKCE DSP 2006 

PRODUCTION OF SELECTED SECONDARY METABOLITES IN TRANSFORMED BACTERIAL 

CELLS 

Jana Hrdličková.............................................................................................................................................197 

THE SIMPLE METHOD FOR THE RECOGNITION OF REDUCING AND NONREDUCING NEUTRAL 

CARBOHYDRATES BY MALDI-TOF MS 

Markéta Laštovičková....................................................................................................................................201 


strana 5

MINIATURE METALLIC DEVICE FOR COLLECTION OF HYDRIDE FORMING ELEMENTS 

Pavel Krejčí....................................................................................................................................................206 

INFLUENCE OF POLYUNSATURATED FATTY ACIDS INTAKE ON LIPID METABOLISM IN 

PATIENTS WITH HYPERLIPIDAEMIA 

Simona Macuchová........................................................................................................................................210 

HYDROPHOBIZED SODIUM HYALURONATE IN AQUEOUS SOLUTION - A FLUORESCENCE 

STUDY 

Filip Mravec...................................................................................................................................................213 

CHARACTERIZATION AND DEGRADATION BEHAVIOUR OF TRIBLOCK COPOLYMER 

Ludmila Nová.................................................................................................................................................218 

STUDY OF THE COPPER(II) IONS NON–STATIONARY DIFFUSION IN HUMIC GEL 

Petr Sedláček..................................................................................................................................................223 

LIVING RADICAL POLYMERIZATIONS INITIATED WITH SILSESQUIOXANES 

Ondřej Smrtka................................................................................................................................................228 

MEASUREMENT OF THERMOPHYSICAL PROPERTIES OF PMMA BY PULSE TRANSIENT METHOD 

Pavla Štefková................................................................................................................................................233 

THE ANALYSIS OF GAMMA LINOLENIC ACID IN EVENING PRIMROSE OIL 

Hana Štoudková.............................................................................................................................................238 

IMPROVED FLUORIMETRIC DETERMINATION OF Al, Ga AND In BY MICELLE ENHANCED 

8-HYDROXYQUINOLINE -5-SULPHONIC ACID COMPLEX 

Šimon Vojta.....................................................................................................................................................243 

PHYSICAL-CHEMICAL ASPECTS OF PAINT FILMS DEGRADATION 

Lucie Wolfová.................................................................................................................................................249 

USING LOW-MOLECULAR MASS PI MARKERS IN PROTEOMIC STAINING-FREE METHOD 

FOR STUDY OF POSTTRANSLATIONALLY MODIFIED PROTEINS 

Karel Mazanec, Karel Šlais and Josef Chmelík............................................................................................254 


strana 6

PŘEDMLUVA 

Tak jako v minulých letech, i letos se v měsíci říjnu na půdě Fakulty chemické VUT uskutečnila 

soutěž posluchačů bakalářských a magisterských studijních programů Student 2006, jakož 

i soutěž posluchačů doktorských studijních programů. V doktorských studijních programech 

proběhla tato soutěž již po deváté, soutěž Student 2006 se konala po šesté. Jako člen hodnotících 

komisí soutěže doktorandů v posledních letech mohu konstatovat významný kvalitativní růst 

její úrovně nejen po stránce odborné, ale také po stránce kvality prezentace příspěvků v anglickém 

jazyku. Rovněž hodnotící komise soutěže Student FCH 2006 konstatovala velmi dobrou 

úroveň prezentovaných prací. Předkládaný soubor příspěvků, zaslaných do obou soutěží, 

doplněný o některé příspěvky ze soutěže v minulém roce, dokumentuje tento trend. Stává se 

již tradicí, že se do studentské soutěže přihlašují také posluchači mimobrněnských chemických 

fakult, především Fakulty chemické a potravinářské technologie STU v Bratislavě. V tomto 

roce se však poprvé představil také zástupce UTB ve Zlíně. I v příštích ročnících bychom v této 

tradici chtěli pokračovat. Předpokládá se, že nejlepší účastníci soutěže z FCH VUT budou 

naši fakultu reprezentovat na podobných soutěžích v Bratislavě a Zlíně. Právě ve výsledcích 

vzájemné konfrontace s posluchači jiných fakult spatřuje vedení Fakulty chemické důležitý 

prvek, směřující ke zvýšení kvality pedagogického a vědecko-výzkumného procesu. Proto 

bude akce podobného typu i v budoucnosti všestranně podporovat. 

prof. Ing. Ladislav Omelka, DrSc. 

proděkan pro tvůrčí činnost 


strana 7

Příspěvky soutěže 

Studentské tvůrčí činnosti 

„STUDENT 2006” 

(Sekce STČ 2006)

VLIV PROVOZNÍCH PARAMETRŮ FLOTACE NA SEPARAČNÍ 

ÚČINNOST ÚPRAVNY VODY MOSTIŠTĚ 

Jana Burianová, 5.ročník 

vedoucí práce: doc. Ing. Petr Dolejš, CSc. 

konzultant práce: Ing. Pavel Dobiáš 

Vysoké učení technické v Brně, Fakulta chemická, 

Ústav chemie a technologie ochrany životního prostředí, Purkyňova 118, 612 00 Brno, 

e-mail: xcburianova@fch.vutbr.cz 

ÚVOD 

Práce se zabývá vlivem intenzity míchání v agregačním reaktoru flotace a délky a 

frekvence intervalu shrabování kalu na kvalitu upravené vody vycházející ze zařízení flotace 

rozpuštěným vzduchem (DAF - dissolved air flotation) na úpravně vody Mostiště. Tyto dvě 

studované proměnné mohou mít vliv jak na kvalitu upravené vody a také na ekonomiku 

provozu flotační jednotky a je proto nezbytné zjistit jejich optimální nastavení v provozu. 

TEORETICKÁ ČÁST 

Flotace rozpuštěným vzduchem je založena na snížení specifické hmotnosti vloček vzniklé 

suspenze vytvořením agregátů mezi vločkami a vzduchovými mikrobublinkami. Pak je 

hustota těchto agregátů nižší než vody. Agregáty tedy překonávají gravitaci a stoupají ve vodě 

směrem k hladině. 

Vlastní reaktor DAF se skládá ze dvou částí, ve kterých probíhají dva základní procesy 

(obr. 1). Do první části, zvané kontaktní zóna, vstupuje voda obsahující vyvločkované 

nečistoty. Do této vody je zaváděn proud kapaliny nasycené pod tlakem rozpuštěným 

vzduchem a z něj vzniká velké množství vzduchových mikrobublin. Mikrobublinky vznikají 

při vstupu nasycené recirkulační vody ve speciálně upravených tryskách. Poté dochází 

k agregaci vloček a těchto mikrobublin. Kontaktní zóna tedy zajišťuje kontakt a připoutání 

mikrobublin a vloček [1]. Náhlým snížením tlaku dochází k uvolnění vzduchových 

mikrobublin a jejich proud je míchán s hlavním proudem vody. Vyloučené mikrobublinky 

vzduchu způsobují „mléčný zákal“ vody, a proto se této vodě také říká „bílá voda“ („white 

water“). Vzduchem nasycená recirkulační voda je vytvářena v saturátoru, do kterého proudí 

část již upravené vody. Obvykle to bývá 6 – 12 % upravovaného proudu vody. Voda je 

sycena pod tlakem 0,5 – 0,6 MPa. 

Suspenze agregátů vločka-mikrobublina vytvořená v kontaktní zóně pak vstupuje do druhé 

části reaktoru, zvané separační zóna. Zde dochází k dalšímu růstu agregátů vločka-bublina a 

jejich pohybu k hladině. Tam tvoří souvislou vrstvu kalu, která je z hladiny shrabována 

mechanickým zařízením a odchází do kanálu pro odtah kalu. Upravená voda je odváděna ze 

dna separační zóny nádrže na další stupeň úpravny vody - filtraci. 

ÚPRAVNA VODY MOSTIŠTĚ 

Úpravna vody (ÚV) Mostiště má kapacitu 220 l/s a je z ní zásobován skupinový vodovod 

pro oblast Velké Meziříčí, Velká Bíteš, Olší nad Oslavou a Měřín, přivaděč do Třebíče a 

přivaděč do Žďáru nad Sázavou. Zásobováno je cca 75 000 obyvatel. 

Z vodní nádrže Mostiště je surová voda přiváděna na provzdušňovací kaskádu. Do odtoku 

z kaskády je dávkován koagulant. Na ÚV Mostiště se používá jako koagulant 41% roztok 


Sekce STČ 2006, strana 9

Fe2(SO4)3. Dále následuje hydraulický mísič. Odtud vzniklá suspenze pokračuje do DAF 

zařízení s celkovou kapacitou 110 l/s. DAF zařízení je tvořeno dvěma flotačními jednotkami. 

Souběžně s DAF zařízením mohou ještě fungovat dvě jednotky původních čiřičů v případě 

potřeby vyšší produkce upravené vody. Voda je dále vedena na pískové filtry. Desinfekce 

vody je prováděna pomocí Cl2 a ClO2 a pH je upraveno vápennou vodou. 

Flotace rozpuštěným vzduchem je na ÚV Mostiště novou technologií. Zařízení bylo 

uvedeno do provozu 28.11.2005. Důvodem zařazení nové technologie do procesu úpravy 

vody bylo zhoršení kvality surové vody vlivem havarijního stavu zdroje surové vody 

(přehrady nádrže Mostiště) a následných stavebních prací a s tím spojené nevyhovující jakosti 

surové vody. Stalo se prvním procesem flotace v zemích střední a východní Evropy [2,3]. 

Obr. 1. Schematické znázornění úpravy vody flotací rozpuštěným vzduchem 

EXPERIMENTÁLNÍ ČÁST 

Jako ukazatele kvality vody byla brána velikostní distribuce částic měřená pomocí 

analyzátoru velikostní distribuce částic (Water Particle Counter WPC-21), hmotnostní 

koncentrace Fe 3+ stanovovaná rhodanidem a absorbance při vlnové délce 387 nm měřené na 

spektrometru SPEKOL 11. Dále byly zaznamenávány hodnoty pH, absorbance při vlnové 

délce 254 nm a teplota vody měřené provozními měřícími přístroji ÚV Mostiště. 

Analyzátor částic: voda z výtoku flotace je přiváděna do analyzátoru z odtoku upravené 

vody na konci flotační jednotky. Analyzátor počítá částice o velikosti 2, 5, 7, 10, 15, 25, 50 a 

100 μm. Neustále je měřen počet částic o velikosti 2 μm, druhá hodnota udává postupně 

v určených intervalech počet částic ostatních velikostí. 

Použité metody stanovení a materiály 

Stanovení hmotnostní koncentrace Fe 3+ : připraví se zásobní roztok (NH4)2Fe(SO4)2.6H2O 

(0,7022 g do 1000 ml) okyselený 2 ml koncentrované H2SO4. Z něho se připraví pracovní 

roztok I (0,01 mg/ml) a roztok II (0,001 mg/ml). Z nich se připraví série roztoků pro sestrojení 

kalibrační křivky. Pracovní roztok se pipetuje do 25 ml odměrné baňky, přidá se 0,5 ml HCl 

(1:1) a 0,5 ml 3% H2O2. Po 5 min. se vzorek doplní asi na 15 – 20 ml, přidá se 2,5 ml KSCN a 

doplní se po rysku. Stejně se připraví roztoky neznámých vzorků. Absorbance se měří při 

vlnové délce 480 nm. 



Obr.2. Analyzátor částic 

VÝSLEDKY MĚŘENÍ A DISKUSE 

Změna intenzity míchání 

Výsledky jsou uvedeny pouze pro nádrž 2, ve které byla intenzita míchání měněna. Nádrž 

1 fungovala jako referenční, tj. se stálým provozním nastavením a výsledky naměřené pro 

nádrž 2 byly srovnány s hodnotami pro nádrž 1. Podmínky a výsledky měření při různé 

rychlosti míchání znázorňuje tabulka 1. Ve dvou případech byla použita stejná rychlost 

míchání, ale došlo ke změně průtoku. 

Tabulka 1. Podmínky a výsledky měření při různé rychlosti míchání 

č. měř. 

doba 

t (°C) 

intenzita 

míchání % 

rychlost míchání 

ot./min. 

D (mg/l) zbyková koncentrace Fe (mg/l) 

měření 

(2 

hod.) 

teplota 

vody 

M1 M2 M1 M2 

dávka 

síranu 

zákal 

(NTU) 

nádrž 

1 

nádrž 

2 

rozdíl 

koncentrací 

1 17,10 60 40 4,25 2,67 38,87 0,301 0,328 

2 17,10 60 0 4,25 0,00 38,87 0,317 0,401 0,321 0,080 

3 17,10 100 80 7,08 5,33 38,87 0,361 0,344 0,191 0,153 

4 17,00 100 60 7,08 4,00 38,81 0,335 0,486 0,234 0,252 

5 17,10 100 60 7,08 4,00 39,70 0,243 0,108 0,281 0,141 

6 17,20 100 40 7,08 2,67 38,64 0,247 0,449 0,362 0,087 



počet částic/ml 

120 

100 

80 

60 

40 

20 

0 

Porovnání počtu částic/ml při různé rychlosti míchání 

2 5 7 10 15 25 50 100 

rozmezí velikosti částic (μm) 

4,25; 2,67 

4,25; 0,00 

7,08; 5,33 

7,08; 4,00 

7,08; 4,00 

7,08; 2,67 

Obr. 3. výsledky měření analyzátorem částic při různé rychlosti míchání 

V legendě je vždy nejprve rychlost pro první míchadlo ve flotaci, kde je míchání rychlé a 

poté následuje rychlost pro druhé míchadlo, kde je míchání pomalé. Z porovnání počtu 

částic/ml je vidět, že nejvhodnější nastavení intenzity míchání, z těch, které jsem použila, je 

nastavení 7,08 ot/min (100 %) pro první míchadlo a 2,67 ot/min (40 %) pro druhé míchadlo. 

Změna intervalu shrabování kalu 

Podmínky a výsledky měření pro různé intervaly shrabování jsou uvedeny v tabulce 2. I 

v tomto případě je nádrž 1 referenční. 

Tabulka 2. Podmínky a výsledky měření při různých intervalech shrabování kalu 

č.měř. t ( °C) shrabování 

doba 

měření 

(2 

hod.) 

teplota 

vody 

prodleva 

(min.) 

hrabání 

(min.) 

rychlost 

shrabování 

% 

rychlost 

(cm/min.) 

D 

(mg/l) 

dávka 

síranu 

zákal 

(NTU) 

zbyková koncentrace Fe 

nádrž 

1 

(mg/l) 

nádrž 

2 

rozdíl 

koncentrací 

1 17,00 20 1 30 61,0 37,41 0,247 0,524 0,373 0,151 

2 17,00 15 1 30 61,0 37,41 0,250 0,424 0,363 0,061 

3 17,00 10 1 30 61,0 38,33 0,237 0,405 0,356 0,049 

4 17,00 5 1 30 61,0 37,65 0,249 0,539 0,490 0,049 

5 17,00 2 1 30 61,0 37,65 0,251 0,586 0,574 0,012 



počet částic/ml 


50 

40 

30 

20 

10 

0 

Porovnání počtu částic/ml pro různé intervaly shrabování 

2 5 7 10 15 25 50 100 

rozmezí velikosti částic (μm) 

20 + 1 min. 

15 + 1 min. 

10 + 1 min. 

5 + 1 min. 

2 + 1 min. 

Obr. 4. výsledky měření analyzátorem částic při různých intervalech shrabování kalu 

Při porovnání výsledků analyzátoru částic pro různé intervaly shrabování jsem zjistila, že 

nejvhodnější by byl interval 10 min. stání a 1 min. hrabání. Při nižších intervalech se počet 

částic zvýšil. To platí i pro zákal. Opět je tomu jinak pro koncentraci zbytkového železa, kde 

nejvhodnější interval je 20 min. + 1 min. Jako vhodný interval bych tedy zvolila 10 min. + 1 

min., protože stanovení ukázala dobrou kvalitu vody. Při nižších intervalech došlo pouze k 

nepatrnému zhoršení, ale zvýšila by se spotřeba energie. Při vyšších intervalech není také 

zhoršení příliš velké, ale později docházelo ke vzniku vrstvy kalu vysoké asi 20 cm, která byla 

velmi hustá a její váha způsobovala prohýbání lopatek hrabáku a nakonec jeho úplné 

zastavení. Kal musel být shrábnut ručně. Na intervalu 10 min. + 1 min. jsem se shodla i s 

obsluhou zařízení. 

ZÁVĚR 

Vlivy rychlosti míchání, délky intervalu a frekvence shrabování se projevily na kvalitě 

upravené vody. Větší vliv má změna intenzity míchání. Jako nejvhodnější se ukázalo 

nastavení, kdy první míchadlo má se otáčí rychlostí 7,08 ot./min. a druhé míchadlo 2,67 

ot./min. Při změně intervalu shrabování se ukázalo nejlepší nastavení 10 min. stání a 1 min. 

shrabování. Přihlédnutím k těmto výsledkům při provozu úpravny by tedy mohla být zvýšena 

kvalita upravené vod vycházející z flotace a tedy i vody dodávané ke spotřebiteli. 

Optimalizací studovaných provozních parametrů dochází především ke snížení počtu částic 

obsažených ve vodě z flotace, což má příznivý vliv na další stupně úpravy. Prodlužuje se doba 

provozu pískových filtrů, které nevyžadují tak časté praní a snižuje se i možnost průniku 

částic filtrem. 



CITOVANÁ LITERATURA 

[1] Haarhoff, J. and Edzwald, J.K.: Dissolved air flotation modelling: insights and 

shortcomings. Journal of Water Supply: Reseach and Technology-AQUA, 2004, vol. 53, pp. 

127-150. 

[2] Dolejš P., Dobiáš P., Mazel L.: Provozní výsledky první vodárenské flotace v ČR 

realizované na ÚV Mostiště. Sborník konference Vodárenská biologie 2006, s. 92-97. Praha 

31.1.-2.2.2006. VŠCHT Praha a Ekomonitor, s.r.o. Chrudim, Praha 2006. 

[3] Dolejš P.: Flotace rozpuštěným vzduchem (DAF) pro úpravu pitné vody a její první 

provozní realizace v ČR. Vodní hospodářství 56, č. 4, s. 99-101 (2006). 



SYNTHESIS, CHARACTERIZATION AND ECOTOXICOLOGICAL 

ASSESSMENT OF NEW FLEXIBLE POLYURETHANE FOAMS WITH 

BIODEGRADABLE FILLERS 

Jan DAVID 1 , 5. year 

Advisor: Prof. Milada VÁVROVÁ 1 ; Consultant: Dr. Lucy VOJTOVÁ 2 

1 Institute of Chemistry and Technology of Environmental Protection 

2 Institute of Materials Science 

Faculty of Chemistry, Brno University of Technology, Faculty of Chemistry, 

Purkynova 118, 612 00 Brno 

e-mail: xcdavid@fch.vutbr.cz 

ABSTRACT 

This work deals with the synthesis of new flexible biodegradable polyurethane foams 

(BIO-PU), in which non-degradable polyol polyether was partly substituted by bio-polyol, 

mainly the cellulose or starch derivatives. The bio-polyol replacements are expected to 

provide the PU foam cheaper, easily biodegradable and less toxic. 

Samples of the BIO-PU foams were characterized by means of Fourier Transform – Infra 

Red Spectroscopy (FTIR) and Thermogravimetric Analysis (TGA) methods and consequently 

hydrolyzed under reflux in freshwater. The ecotoxicological aspects of the foam leaches were 

determined by microbiotest screening toxkit „Thamnotoxkit F TM “. Values of toxicity were 

expressed as percentage mortality of the II-III larvae instars hatched from the cysts of 

freshwater fairy shrimps Thamnocephalus platyurus dependence on the effect criterion of the 

respective assay. 

INTRODUCTION 

Polyurethanes (PU) are polymers made by addition polymerizations of multi-functional 

isocyanates and multi-functional alcohols (polyols), e.g. polyether or polyester 1 , which 

functionality determine on linear or cross-linked structure. Various methods can be used to 

produce polyurethanes but for the PU foam production is often used the one-shot process, 

where direct mixing of polyols, catalysts, blowing agent and isocyanates are used. Foams 

(expanded plastics) are microcellular structures produced by gas (CO2) bubbles during the 

polyurethane preparation. PU foams properties are affected mainly by the raw materials and 

can be modified by a wide variety of additives, such as stabilizers, fillers, cross-linking agents 

and chain extenders 2,3 . PU foams are materials with a broad variety of applications like 

furniture, automotive and shoe industry, building construction and packaging, agriculture and 

medicine. Due to the recycling complications the PU foams are discarded after use, which 

represents contamination problems because of their difficult disintegration and incorporation 

to the environment. 2 Solution of this problem is including a natural material in the PU foam 

network in order to evoke easier biodegradation and lower ecotoxicity. Degradable natural 

additives containing –OH groups might be used as –OH providers to modify the PU 

properties and structure. 4 Various compounds of natural origin can be used instead of 

commercial polyol polyethers like starches, celluloses and their derivatives, soy flour, bark, 

castor oil and palm oil, wattle tannin or oil palm empty fruit bunch 2,3,4,5,6,7,8 . 

This work is focused on the use of carboxymethyl cellulose sodium salt (CMC-Na), 

acetylated potato starch (AS), cellulose acetate (CA), 2-hydroxyethyl cellulose (2-HEC) and 



wheat protein /gluten/ (WP-G) as bio polyols. Recently, the biodegradation of these flexible 

modified PU foams by thermophille bacteria Thermophillus sp. was published by our group of 

researchers from FCH BUT 9 . However, the levels of toxic substances must not exceed the 

permissible limit. 

The interest in biological testing is growing rapidly and toxicity testing is now gradually 

incorporated in environmental legislation in many countries. The most worldwide known and 

useful alternative toxicity biotests are Toxkits owing to their miniaturization, simplicity, quick 

availability, high sensitivity and reproducibility and low cost. The Toxkits tests are 

particularly suited for routine toxicity testing of chemicals, solid waste leaches or effluents 

released in aquatic environment 10 . The sensitivity of these microbiotests is equal to that of 

conventional bioassays, and the new alternative assays have already been validated in various 

studies 11,12,13,14 . 

In this proposed work, BIO-PU foams modified by AS, AC, CMC-Na,2-HEC and WP-G 

were characterized by FTIR, TGA, extracted in freshwater and used for ecotoxicological 

evaluation via Thamnotoxkit F. 

EXPERIMENTAL 

Raw materials 

Polyol polyether (hydroxyl number OH = 47) (PEP), tolylene diisocyanate 80/20 (TDI), 

tin-bis(2-ethylhexanoate) surfactant, and mixed catalyst were obtained from Gumotex, a.s., 

carboxymethyl cellulose sodium salt (CMC-Na), Mn = 250 000, cellulose acetate (CA), 

Mn = 30 000, and 2-hydroxyethyl cellulose (2-HEC), Mn = 90 000 were purchased from 

Aldrich, acetylated starch AMISOL HS (AS) was obtained from Starch and Potato Products 

Research Laboratory, Poland and wheat protein (gluten) (WP-G) was received from 

Amylon, a.s. 

Synthesis of BIO-PU foams and preparation of leaches 

The foams were synthesized according to a standard industrial recipe for furniture industry 

flexible foams, except of the partial PEP substitution (from 1 up to 30 w%) by starch or 

cellulose derivates or gluten, which were pre-mixed with the original PEP. Consequently the 

catalyst, surfactant and TDI were added, mixed up to cream time and quickly poured into a 

mould. BIO-PU foams were cured for 48 hours. 

0,5 g pieces of cured PU foams were cut to a smaller pieces and boiled under reflux with 

25 ml of standard freshwater (for Thamnocephalus platyurus) for 8 hours. 

Ecotoxicological assessment 

Alternative crustacean toxicity screening test Thamnotoxkit F based on the mortality of 

the instars II-III larvae of freshwater fairy shrimps Thamnocephalus platyurus hatched from 

the cysts after 24 hours exposure was applied to different concentrations of the PU foams 

freshwater leaches. Standard freshwater was prepared from solutions supplied with 

Thamnotoxkit F. The toxicity tests were performed according to the standard operational 

procedure manual of the Thamnotoxkit F producer 15 Shortly, cysts of Thamnocephalus 

platyurus were hatched for 23 hours before the use by pre-hydrating with diluted freshwater 

for 30 minutes and incubating at 25°C for 22 hours under continuous illumination. Then the 

hatched larvae were transferred to the test wells containing the toxicant dilution and incubated 

at 25°C in darkness for 24 hours. Then the immobilized/dead individuals of larvae were 



counted and toxicity data were expressed as percentage mortality of Thamnocephalus 

platyurus depending on the effect criterion of the respective assay. 

Characterization of foams 

Small pieces of cured PU foams were frozen by liquid N2 and pulverized. FTIR spectra 

were measured using Nicolet Impact 400D FTIR spectrometer with common KBr technique. 

The spectra analyses were performed on Omnic and MS Excel software. 

Thermogravimetric analyses were carried out via Perkin Elmer TGA 6, where 7 mg of 

samples were heated from 100°C to 500°C at a heating rate of 10°C.min −1 under N2 

atmosphere. The curve analyses were performed on PE Pyris 6 and MS Excel software. 

RESULTS AND DISCUSSION 

Synthesis and characterization of PU foams 

Five types of bio-polyols AS, AC, 2-HEC, CMC-Na and WP-G were successfully included 

in the 3D PU network in order to substitute as much of commercial polyether polyol as 

possible. It was found to substitute only 10 % of PEP by AC and 2-HEC, but 20 % by AS and 

even 30 % by CMC-Na. Resulting modified flexible foams were not distinguished at first 

sight from the respective CONTROL PU foam as for the size, shape, color, polymer network 

and flexibility, except the WP-G foam, where the wheat protein replaced only 5 % of 

commercial PEP. It was probably caused due to the side reactions of amino acids with the raw 

materials during the PU foam preparation resulting in collapsed hard foam. 

The chemical structure of the modified PU foams was characterized in terms of FTIR 

proving the natural polymers incorporation into the modified PU foam network (see Fig.1). 

The interesting point of view is shown at the FTIR spectra in the range of 2250 – 2400 cm −1 

where the peak at 2360 cm −1 was suppressed and on the other hand the peak at 2280 cm −1 was 

highlighted at modified PU foams evidential of forming new bonds between the filler and PU 

network. Other peaks corresponded to the urethane linkages. 

From the literature and previous experiments 2,3 it is known that modifying of PU foam 

with bio-polymer filler up to 10 % has no significant influence on thermal properties of the 

modified foam, which was confirmed. The TGA showed that the influence rises with the 

increasing bio-polymer filler amount in the foam when is higher than 10 %. 

Ecotoxicology 

Ecotoxicity of modified and control PU foams leaches was evaluated using alternative 

crustacean toxicity test Thamnotoxkit F. Values of toxicity were expressed as percentage 

mortality of Thamnocephalus platyurus dependence on the effect criterion of the respective 

assay. Dilution series of 100 %, 80 %, 60 %, 40 % and 20 % of the leaches were prepared 

according to the procedure of Thamnotoxkit F manual 15 . As for 100 % (non-diluted) 

leaches made of 10 % substituted foams, the lowest toxicity (80 %) performed the PU foam 

with 10 % CMC-Na. All 60 % diluted leaches showed lower toxicity than the CONTROL PU 

foam, except the leach made of 5 % WP-G PU foam. As for other dilution series, also the 

most toxic leaches were the CONTROL PU and 2-HEC leaches (see Fig.2). 



% of mortality 

4000,0 

120,00 

100,00 

80,00 

60,00 

40,00 

20,00 

0,00 

3500,0 

46,67 

3000,0 

SUBSTITUTED FOAMS - COMPARE ALL 

2500,0 

Control PUR 

10 % CMC-Na 

10 % ACS 

10 % CA 

10 % 2-HEC 

1 % WP(G) 

2000,0 

wavenumber [cm –1 ] 

1500,0 

Fig.1: FTIR spectra of modified PU foams. 

1000,0 

500,0 

Mortality graph - 60 % (diluted) leachates of various PU foams 

43,33 

50,00 

23,33 

56,67 

100,00 

10 % CMC-Na PU 10 % AS PU 10 % CA PU 10 % 2-HEC PU CONTROL PU 5 % WP-G PU 

foam sample 

Fig. 2: Percentage mortality graphs of diluted leaches of various modified PU foams. 

100,0 

95,0 

90,0 

85,0 

80,0 

75,0 

70,0 

65,0 

60,0 

0,0 

CONCLUSION 

Flexible modified PU foams with 10 % of PEP substituted by CMC-Na, AS, CA and 

2-HEC were successfully synthesized. The most suitable bio-polymer fillers for both physical 

and ecotoxicological properties were found to be CMC-Na and AS, which worked up to 30 % 

and 20 % of substitution, respectively. The worst properties presented the PU foam filled with 

WP-G. 

Moreover, based on our last research 9 the AS PU foam indicates also good 

biodegradability. 

Further ecotoxicological assessments and new TGA experiments of the modified PU foams 

are needed and planned as well as the analysis of the PU foams leaches on Gel Permeation 

Chromatography and High Pressure Liquid Chromatography in order to identify the products 

of hydrolysis of the foams for better understanding the ecotoxicological assessment. 


Sekce STČ 2006, strana 18 

transmittance [1]

ACKNOWLEDGEMENT 

I would like to thank to both of my tutors, Dr. Lucy Vojtová and Prof. Milada Vávrová. 

Special thanks are going to Dr. Karel Bednařík who gave me good advice and also to the 

manager of this research project Prof. Josef Jančář. 

This research was supported by the Ministry of Education, Youth and Physical Training of 

the Czech Republic under the research project MSM 0021630501. 

REFERENCES 

1 

Schyzer, M.: Schyzer's Handbook of Polyurethanes. CRC Press. 

2 

Vojtová, L., Zdilna, P., Jancar, J.: Degradace a recyklace polyuretanovych pen.[.doc 

document]. Brno: FCH Brno University of Technology, 2004 [cited 07.10.2006]. 

3 

Rivera-Armenta, J. L., Heinze, T., Mendoza-Martínez, A. M.: New polyurethane foams modified with 

cellulose derivatives. European Polymer Journal, 2004, vol. 40, pp. 2803-2812. ISSN 0014-3057/$ 

4 

Chang, L.-C., Xue, Y., Hsieh F.-H.: Comparative study of physical properties of water-blown rigid 

polyurethane foams with commercial soy flours. Journal of Apllied Polymer Science, 2001, vol. 80 (2001), pp. 

10-19. 

5 

Ge, J., Zhong, W., Guo, Z., Li, W., Sakai, K. : Biodegradable polyurethane materials from bark and starch I. 

Highly resilient foams. Journal of Applied Polymer Science, 2000, vol. 77 (2000), pp. 2575-2580. 

6 

Ge, J., Shi, X., Cai, M., Wu, R., Wang, M.: A novel biodegradable antimicrobial PU foam from wattle 

tannin. Journal of Applied Polymer Science, 2003, vol. 90, pp. 2756-2763. 

7 

Alfani, R., Iannace, S., Nicolais, L.: Synthesis and characterization of Starch-Based Polyurethane Foams. 

Journal of Applied Polymer Science, 1998, vol. 78, pp. 739-745. 

8 

Badri, K. H., Othman, Z. B., Razali I. M.: Mechanical properties of polyurethane composites from oil palm 

resources. Iranian Polymer Journal, 2005, vol. 14, no. 5, pp. 441-448. 

9 

Marova, I., Babak, L., Vavrova, M., Vojtova, L., Jancar, J.: Use of thermophillic bacteria to biodegradation 

of modified polyurethane foams, paper presented at The Sixth European Meeting on Environmental Chemistry, 

Belgrade, 6.-9. December (2005). 

10 th 

MicroBioTests Inc.: Background and general information [online]. Last update 30 of March 2006 [cited 

2006-10-07]. Available online at: . 

11 

Persoone, G., Van de Vel, A.: Report EUR 11342 EN, Commission of the European Communities (1987). 

12 

Latif, M., Licek, E.: Environmental toxicology, 19, 302 (2004). 

13 th 

MicroBioTests Inc.: Publications about microbiotests [online]. Last update 30 of March 2006 

[cited 2006-10-07]. Available online at: . 

14 

Torokne, A.: Sensitivity evaluation of the Daphtoxkit and Thamnotoxkit microbiotests on blind samples. 

Journal of Applied Toxicology, 2004, vol. 24, no. 5, pp. 323-326. 

15 TM 

MicroBioTests Inc., Belgium. (Ed.): Thamnotoxkit F . Standard Operation Procedure V310303. 



NOVÝ SORPČNÍ GEL NA BÁZI SPHERON-OXINU PRO UŽITÍ 

V TECHNICE DGT 

Michaela Gregušová, 5. ročník 

Vedoucí práce: prof. RNDr. Hana Dočekalová, CSc. 

Vysoké učení technické v Brně, Fakulta chemická, ústav chemie a technologie ochrany 

životního prostředí, Purkyňova 118, 612 00 Brno, e-mail: xcgregusova@fch.vutbr.cz 

ÚVOD 

Kvantitativní stanovení chemických forem kovů (specií) přítomných v přírodních vodních 

systémech je stále nedořešeným problémem environmentálních chemiků. V průběhu odběru 

vzorku a jeho zpracování dochází k fyzikálně-chemickým dějům, které mají za následek 

změny v rozdělení specií. Skutečnou situaci odráží některé in situ prekoncentrační postupy. 

Mezi tyto postupy patří technika difúzního gradientu v tenkém filmu (DGT), která byla 

poprvé popsána v roce 1994 [1]. Technika DGT umožňuje stanovení stopových koncentrací 

kovů, radionuklidů a rovněž některých anionů (fosfátů, sulfidů) [2,3] ve vodném prostředí, 

půdách a sedimentech. Technika DGT využívá vzorkovací jednotku tvaru pístu, ve které jsou 

uzavřeny dvě vrstvy polyakrylamidového gelu (APA) a membránový filtr (obr.1). Horní 

vrstvu tvoří polyakrylamidový hydrogel, přes který difundují ionty analytu. Ve spodní vrstvě 

PAM gelu se zakomponovaným iontoměničem se sorbuje analyt. 

Obr.1 Řez vzorkovací jednotkou DGT 

Technika DGT je založena na Fickových zákonech difúze. Po ponoření vzorkovací 

jednotky difundují mobilní ionty kovů přes difúzní gel známé tloušťky Δg k sorpčnímu gelu, 

kde se zachytávají na specifických funkčních skupinách sorbentu. V difúzním gelu se ve 

velmi krátké době ustaví lineární koncentrační gradient. Jestliže koncentrační gradient zůstává 

během měření konstantní (míchané vodné systémy), lze dle prvního Fickova zákona difúze 

vypočítat tok analytu F vrstvou Δg dle vztahu: 

M D ⋅ c 

F = = 

(1) 

A ⋅ t Δg 

kde M je množství kovu navázané na iontoměnič, A je plocha difúzního gelu, D je difúzní 

koeficient kovu a c je koncentrace kovu v měřeném roztoku. 



Kovy zachycené na sorbentu jsou eluovány kyselinou dusičnou a stanoveny metodami 

atomové spektrometrie. Při stanovení specií kovů technikou DGT je klíčové nalézt vhodný 

sorbent a difúzní gel. Technika DGT nejčastěji využívá iontoměnič Chelex 100 s vázanými 

funkčními skupinami kyseliny iminodioctové a polyakrylamidový difúzní gel. 

Tato práce se zabývá studiem vlastností nového sorpčního gelu s navázanými skupinami 

8-hydroxychinolinu (Spheron-Oxin) [4], které mají silné chelatační schopnosti. Sorpční gel 

se Spheron-Oxinem přináší nové možnosti pro speciaci labilních i méně labilních komplexů 

technikou DGT. 


Příprava gelů a jednotky DGT. Difúzní gel i sorpční gel se Spheron-Oxinem byly 

připraveny z gelového roztoku, který obsahoval 15%obj akrylamidu (Merck), 0,3%obj 

agarosového síťovadla (DGT Research Ltd., Lancaster) v ultračisté vodě. Směs 10 μl 

katalyzátoru polymerace TEMED (tetramethylendiamin) zředěného 1:1 ultračistou vodou, 

20 μl čerstvě připraveného 8%-ního roztoku peroxosíranu amonného, iniciátoru polymerace, a 

2 ml gelového roztoku byla promíchána a nadávkována mezi dvě skla oddělená distanční 

teflonovou fólií. Do směsi na přípravu sorpčního gelu s menším obsahem katalyzátoru a 

iniciátoru polymerace bylo přidáno 0,4 g Spheron-Oxinu 1000 (Lachema Brno, 40–63 μm). 

Směs byla ponechána mezi skly v sušárně po dobu 45 min, kde při teplotě 42 ± 2 °C 

zpolymerovala. Sesítěné gely byly hydratovány v ultračisté vodě 24 hodin, kde nabobtnaly 

do stabilní tloušťky 0,8 mm u difúzního gelu a 0,4 mm u sorpčního gelu. Několikanásobná 

výměna ultračisté vody během hydratace zajišťuje odstranění zbytků polymeračních činidel 

z gelů. Po nabobtnání byly z gelů vykrájeny disky o průměru 25 mm. Disky pak byly 

uchovávány v ledničce v roztoku 0,01M dusičnanu sodného v případě difúzního gelu 

a v ultračisté vodě v případě sorpčního gelu. 

Při sestavování jednotky DGT se na píst (obr.1) vložil sorpční gel, který byl pak překryt 

difúzním gelem, a nakonec membránovým filtrem (Supor®-450) k ochraně gelů. 

Příprava roztoků, testování DGT. Pro přípravu modelových roztoků byly použity 

standardní roztoky kovů Astasol®-Pb, Cd, Cu, Ni (Analytika, Praha), dusičnan sodný p.a. 

99,8% (Lachema Brno), hydroxid sodný čistý (Onex Rožnov p. R.) a ultračistá voda 

připravená přístrojem Milli-Q Academic firmy Millipore. Roztoky byly připraveny zředěním 

standardů na příslušné koncentrace. Do plastové nádoby s 2 L ultračisté vody bylo přidáno 

20 ml 1M dusičnanu sodného a příslušné množství standardů kovů. Roztok byl neutralizován 

0,1M hydroxidem sodným a následně 24 hodin míchán, aby se ustavila rovnováha v roztoku. 

Po vnoření DGT jednotek do modelového roztoku bylo odebráno malé množství roztoku 

k analýze. Po uplynutí doby expozice byl opět odebrán vzorek roztoku, vyjmuty jednotky 

DGT a rozebrány. Sorpční gel byl loužen v 1M kyselině dusičné (Suprapur® Merck) po dobu 

24 hodin. Metodou atomové absorpční spektrometrie s elektrotermickou atomizací 

(AAnalyst 600, Perkin Elmer Analytical Instruments, USA) byly v eluátu, vstupním a 

konečném roztoku stanoveny Cd, Cu, Ni, Pb při vlnových délkách 228,8; 324,8; 232,0 

a 283,3 nm za teplotního programu doporučeného výrobcem. 

Rychlost ustanovení rovnováhy eluce. DGT jednotky byly exponovány 6 hodin v roztoku 

o koncentraci 40 ppb každého z kovů Cd, Cu, Ni, Pb. Sorpční gely se Spheron-Oxinem byly 

eluovány 1ml 1M kyseliny dusičné v časových intervalech 0,5; 1; 2; 4; 8 a 16 hod. 



Eluční faktor. Ionty kovů byly opakovaně eluovány 1M kyselinou dusičnou ze sorpčních 

gelů z jednotek DGT exponovaných po dobu 24 h v roztoku kovů o koncentraci 10 ppb. Mezi 

jednotlivými po sobě následujícími elucemi byly gely pro odstranění vnitřního roztoku 

louženy ultračistou vodou. 

Sorpční kapacita. Z pentahydrátu síranu měďnatého p.a., Lachema Brno byly připraveny 

roztoky o koncentraci 25, 51, 146, 279 a 787 mg/l odpovídající 10, 20, 60, 100 a 300% 

teoretické kapacity. Do 2 ml roztoků mědi byly vloženy gely se Spheron-Oxinem po dobu 

24 hodin a občas promíchány. Sorbované množství mědi bylo určeno z rozdílů koncentrace 

připravených roztoků a roztoků s exponovanými sorpčními gely. Koncentrace mědi byla 

stanovena plamenovou atomovou absorpční spektrometrií na přístroji Perkin Elmer 3110 AA. 

Vliv pH. V plastové nádobě s 2 L ultračisté vody, dusičnanem sodným a 30 ppb kovů bylo 

upravováno pH pomocí kyseliny octové Suprapur® 96% (Merck) a hydroxidu sodného 

Suprapur® 30% (Merck). Nejprve bylo pH roztoku sníženo kyselinou, ustalováno po dobu 

24 h a nakonec měřeno. V následující periodě 22 h byly v roztoku exponovány jednotky 

DGT. Po jejich vyjmutí bylo pH roztoku změřeno a dále postupně zvyšováno přídavkem 

hydroxidu sodného a celý proces záchytu kovů v jednotkách DGT byl opakován. 

VÝSLEDKY 

Při přípravě sorpčního gelu se Spheron-Oxinem bylo nejprve optimalizováno množství 

sorbentu v gelovém roztoku. Ideální množství Spheron-Oxinu mělo být co největší z důvodu 

dostatečné kapacity disků, ale zároveň takové aby byla možná příprava homogenního gelu. 

Nalezené optimální množství činilo 0,4 g Spheron-Oxinu na 2 ml gelového roztoku. 

Při přípravě sorpčního gelu se částice Spheron-Oxinu usazovaly ke spodní straně odlévací 

skleněné formy, což vedlo k nehomogennímu rozdělení sorbentu v gelu. Bobtnání 

Spheron-Oxinu v gelovém roztoku po dobu minimálně tří dnů před přípravou gelu vedlo 

k potlačení tohoto negativního jevu. Pro přípravu homogenního gelu je rovněž důležitá 

důkladná homogenizace gelu po přidání iniciátoru polymerace a rychlé vlití polymerující 

směsi mezi skla formy. 

Pro vysoký obsah mědi a niklu v sorbentu bylo nutné Spheron-Oxin před použitím 

přečistit. Z důvodu možné kontaminace gelů během jejich přípravy byly proto vykrájené 

disky sorpčního gelu opakovaně několikrát eluovány v 1M kyselině dusičné (Suprapur® 

Merck) a následně několikrát hydratovány v ultračisté vodě, aby se zbytky kyseliny odstranily 

z vnitřního roztoku gelu. 

U připravených disků sorpčního gelu byl stanoven mrtvý objem a zároveň tloušťka gelu 

dvěma způsoby. Z úbytku hmotnosti gelů při sušení, který činil 0,19 g, a z průměru disků gelu 

25 mm byla odvozena tloušťka 0,4 mm. Přímo byla mikrometrem změřena tloušťka gelu, 

vloženého mezi dvě mikroskopická sklíčka. Přímé odečítání tloušťky bylo však 

komplikováno obtížností nastavení příslušného přítlaku na hlavičkách mikrometrického 

šroubu, při kterém již nedocházelo k nežádoucí deformaci (stlačování) gelu. Mikrometrem 

byla odečtena tloušťka 0,4 mm. 

Dalším zkoumaným parametrem byla rychlost ustanovení rovnováhy při eluci. Kinetika 

eluce kovů kyselinou dusičnou je procesem velmi rychlým. Během půl hodiny dochází 



k vyloužení cca 90% nasorbovaných kovů. Z toho vyplývá, že i velmi krátká doba eluce 

30 min postačuje k ustanovení rovnováhy. 

Eluční faktor vyjadřuje jaké množství (M1) z celkového sorbovaného množství (ΣM) se 

vyluhuje v jednom elučním kroku do roztoku 1M kyseliny dusičné: 

M1 

fe = (2) 

M + M + M + M 

1 

2 

3 

4 

Stanovení elučního faktoru bylo nezbytné pro následné ověření funkce techniky DGT 

s novým sorpčním gelem. Eluční faktor nabývá hodnot pro Ni 0,86; Cd 0,98; Pb 0,93 a Cu 

0,71. 

Sorpční kapacita má spíše informační charakter pro analytickou praxi. Slouží k odhadu 

přípustného zatížení a doby expozice. Sorpční kapacita disků Spheron-Oxinu pro měď činila 

3,2 μmol/disk. Tato hodnota je nižší oproti teoretické hodnotě 8 μmol/disk. Kapacita je však 

dostatečně vysoká pro několikatýdenní až několikaměsíční vystavení sorpčního gelu do 

přírodních vodních systémů. 

Vliv kyselosti vnějšího roztoku na záchyt Cd, Ni a Cu byl sledován v rozsahu hodnot 

pH 3-8. Z výsledků vyplývá, že sorpční gel se Spheron-Oxinem váže silně sledované kovy, 

což může být při srovnání s tradičním sorpčním gelem Chelex 100 s výhodou využito 

k charakterizaci labilních a méně labilních komplexů kovů v přírodních vodních systémech. 

Po nalezení všech pomocných parametrů jako jsou tloušťka sorpčního gelu, eluční faktor, 

sorpční kapacita a vliv pH mohla být ověřena funkce nového sorpčního gelu pro techniku 

DGT výpočtem koncentrace vnějšího roztoku z množství kovů získaných elucí sorpčního 

gelu. Nalezené hodnoty technikou DGT odpovídaly pro Ni 92 ± 4 % a pro Cd 98 ± 4 % 

očekávaných hodnot. Z toho lze usoudit, že technika DGT s využitím sorpčního gelu na bázi 

sorbentu Spheron-Oxin může poskytovat spolehlivé výsledky. 

LITERATURA 

Davison W., Zhang H., Nature 367, 546 (1994) 

Zhang H., Davison W., Gadi R., Kobayashi T., Anal. Chim. Acta, 370, 29-38 (1998) 

Teasdale, Hayward S., Davison W., Anal. Chem. 71, 2186-2191 (1999) 

Slovák Z., Bulletin n.p. Lachema Brno, 30-34 (1979) 



MOLECULAR DYNAMICS SIMULATION OF POLYMER CHAIN 

IN THE VICINITY OF NANOPARTICLE 

Kateřina Hynštová 1 

Advisor: prof. RNDr. Josef Jančář, CSc. 1 , Consultant: Mgr. Jan Žídek, Ph.D. 1 

1 Institute of Materials Science, School of Chemistry, Brno University of Technology, 

Purkynova 118, Brno, Czech Republic, e-mail: xchynstova@fch.vutbr.cz 

KEYWORDS Molecular dynamics, Rouse, Smoluchowski equation, viscoelasticity, model. 

ABSTRACT 

Molecular simulation of single chain in the vicinity of nanoparticle in comparison with pure 

system is presented. According to the Rouse theory, chains were considered as a sequence of 

beads connected together by harmonic springs. The motion of atoms was supported by thermal 

energy and retarded by the resistance of surrounding. New atom position, in given time, was 

determined by the Smoluchowski equation, that consists of two terms: first one includes the 

influence of the inter-atomic collisions, the sterical obstacles and the strong intermolecular 

interactions in friction coefficient, second one express the energy field aggregated from 

potentials of all atoms. Sinusoidal shear stress was applied to the chain. The output of the model 

was energy as a function of time. The energy course was also sinusoidal but shifted according 

to the deformation. The amplitudes and phase shifts were analyzed for the chains under 

different conditions .The chains were subjected to the model first as the standalone objects. 

Then, barrier was defined and chains placed in the vicinity of it. The barrier acted as a volume 

excluded hindrance. This type of chain molecular dynamics could be used as a stand-alone 

model or it could be suitable component for complex models, for example network model of 

polymer nanocomposite. 

INTRODUCTION 

The need for new materials has driven an increased interest in understanding the changes in 

structural, dynamic and melt properties caused by fillers and more recently, nanofillers. A 

considerable effort was devoted to understanding of viscoelastic properties of the materials. 

Phenomenological approach is not sufficient any longer; therefore the studying of the materials 

on multiple length scales is useful [1]. 

Many efforts have been made on description of polymer behavior at a molecular level. Even 

though instrumental methods have improved, there are still problems with exact measurement 

of the structural and mechanical properties at this level. Hence, computer simulations take 

place in this area being very powerful tool that enable direct insight into investigated system 

[2]. 

To define single chain motion in viscous surrounding the Rouse model is widely used [3,4]. 

Atoms or atom groups are considered as rigid beads connected together by harmonic springs. 

The diffusion coefficient of surroundings and the potential of the atom group determine its 

position. 

The simulation progressed from chains in polymer melt that are sufficiently far from filler 

across the glassy polymer to the chains in the interphase layer vicinity of particle. Two factors 

influenced the relation between the particle and chain. The first factor was excluded volume 



which caused immobilization of chain. This factor was observed rather in nanocomposites than 

in the microcomposites. For example, adding 1% by weight of ultra-fine, synthetic mica 

(30-nm diameter disks) to nylon gave super tough nylon, while adding the same amount of 

traditional mica (micron sized talc) gave only a slight improvement in toughness over unfilled 

polymer. Second factor was an acting of interactions, which were based on both physical and 

chemical mechanisms and their presence could cause diametrically different polymer response 

[5]. 

SIMULATION 

An application of ROUSE model was used to model viscoelastic response of the polymer 

chain. The chain was composed of single atoms which are distributed in space. Each atom 

position was limited by position of the neighbor atoms. System tried to achieve thermodynamic 

equilibrium, but its motion was retarded. To trace the position of atoms in time the Newtonian 

and Smoluchowski equations were solved. For non-interacting particle undergoing the thermal 

motion, the probability for finding the particle at x and t as ψ ( x, t) 

follows from 

∂ψ 

∂ 1 ⎛ ∂ψ 

∂V 

⎞ 

= ⎜kT 

+ ψ ⎟ 

(1) 

∂t 

∂x 

ς ⎝ ∂x 

∂x 

⎠ 

where resistance of surroundings is expressed in first member, energy influence including 

dihedral, bending and bond stretch potential are involved by latter one[8]. Potential functions 

and parameters adopted are proposed by Ryckaert-Bellemans for 4-body dihedral (torsion) 

potential V(φ) [6] and by by for 2-body bond length potential V(b) and 3-body bond angle 

potential V(θ), respectively [7], as shown in equations with parameters listed below in Table 1. 

5 

1 

2 

1 

2 

i 

V ( b) 

= kb 

( b − b0 

) V ( θ ) = kθ 

( θ −θ 

0 ) V ( ϕ) 

= Ci 

cos ( ϕ) 

(2,3,4) 

2 

2 

Table 1: Parameters of Ryckaert-Bellemans multinominal [6] and Smit, Karaborni and 

Siepmann equations [7] for the polyethylene chain 

Parameter Value Units 

kb (bond) 3.3475 10 5 kJmol -1 

nm -2 

b0 0.153 Nm 

kθ(bending) 519.6 kJ mol -1 rad -2 

θ0 114 Deg 

C0(torsion) 9.2789 kJ mol -1 

C1 

C2 

C3 

C4 

C5 


12.156 

-13.120 

-3.0597 

26.24 

-31.495 

A set of 10 polyethylene chains with 50 segments was generated. A sinusoidal shear 

deformation was applied to each chain and its energy response was analyzed. The amplitude of 



∑ 

i= 

0

the deformation was 5% of the chain length and the frequency was 1,5,10 and 20 Hz 

respectively. Temperature was 298.15 K. The friction coefficient was 0.05. Parameters of the 

polymer chain were taken from Table 1. Deformation response is shown in the Figure 1. The 

amplitudes were taken as the height of energy peak and the phase shift was a shift between 

course of deformation and the energy. The results are shown in Table 2. In the second part, the 

half space barrier was defined. The chains were set in the vicinity of the barrier so its centers of 

gravity were in the distance 0.2, 0.3, and 0.4 nm from the surface (Figure 2). The chains were 

deformed with frequency 5 Hz. The calculated data were compared with measured data for the 

polyethylene and polyethylene/starch composites [8]. 

Deformation(nm) 

1,0640 

1,0635 

1,0630 

1,0625 

1,0620 

1,0615 

1,0610 

1,0605 

0,0 0,1 0,2 0,3 0,4 

Time(s) 

0,0045 

0,0040 

0,0035 

0,0030 

0,0025 

Figure 1: Deformation and energy course of sinusoidal deformation of set of 50 segments 

chains with frequency 5Hz, amplitude 0.05, T=298,15K, friction 0.05 

Amplitude of Energy (10 -15 J) 

1,4 

1,2 

1,0 

0,8 

0,6 

0,2 0,3 0,4 0,5 0,6 

Distance of chain from barrier (nm) 

Figure 2: Influence of barrier on the mechanical response of single chain. The 

mechanical response was represented by energy amplitude and phase shift. 



0,6 

0,5 

0,4 

0,3 

0,2 

0,1 

0,0 

tg(δ) 

Energy(pJ)

Table 2: Amplitudes and phase shifts of the energy course for different frequencies; 

M – results from our model; E – experimental values from the ref [9]. 

Frequency (Hz) Amplitude E (pJ) Phase shift: tg δ 

1 M– 50 segment PE chain 0.00150 ± 0.00002 0.063 ± 0.006 

5 M 0.00085 ± 0.00002 0.161 ± 0.01 

10 M 0.00139 ± 0.00004 0.203 ± 0.012 

20 M 0.00109 ± 0.00001 0.042 ± 0.002 

1 E- (LLDPE) - 0.126 

1 E- (composite LLDPE/Starch 60/40 wt) - 0.100 

CONCLUSIONS 

Sinusoidal deformation of single chain was modeled. Energy response of the model was 

analyzed. Amplitudes of the energy and phase shifts were taken from average energy course of 

ten chains ensemble. We found that the energy was shifted to the deformation of the model 

chain, as it was observed in real polymer. In comparison to real polyethylene, our calculated 

phase shift by the same frequency was still slightly small, but comparable. The phase shift 

increased by the frequencies 5-10 Hz. It was because not all of many parameters were in their 

confidence intervals. The correct viscoelastic response might be in a short interval of each 

parameter and its right combination should be determined. 

The impact of barrier in the vicinity of the chain was detectable. The energy amplitude in the 

vicinity of barrier increased by 50 percent from the amplitude of untreated chain, but in reality 

the modulus of the polymer increases orderly when it is adsorbed on the solid surface. The 

calculated phase shift of one sample increased and of another sample it decreased in 

comparison to the chain without the barrier. In reality, the phase shift of the composite is 

slightly lower than the phase shift of the pure polymer. The models reasonably described the 

behavior of real polymer chain. However, further improvement has been desirable. Moreover, 

the properties depend on the supermolecular structure, which would be introduced to the 

model. 


This research was supported by Ministry of Education of the Czech Republic under research 

project MSM 0021630501 

REFERENCES 

[1] F.W. Starr and S. C. Glotzer: Mat. Res. Soc. Symp. Proc. 661 (2001), p. KK4.1.1. 

[2] D. Frenkel, B. Smit: Understanding Molecular Simulation (Computational Science Series, 

Vol 1) Academic Press; 2 nd edition, USA (2001). 

[3] P.E. Rouse: J. Chem. Phys. 21 (1953) p.1273. 

[4] Yn-Hwang L.: Polymer viscoelasticity: Basics, molecular theories and experiments, 

World Scientific Publishing Co. Pte. Ltd., Singapore (2003). 

[5] F.W. Starr, T.B. Schrødr and S. C. Glotzer: Macromolecules 35 (2002), p. 4481. 

[6] J.P. Ryckaert and A. Bellemans: Mol. Phys. 44 (2001) p. 68. 

[7] B. Smit, S. Karaborni and J. I. Siepmann: J. Chem. Phys. 2126 (1995) p. 102. 

[8] A.G. Pedroso and D.S. Rosa: Carbohydrate Polymers 59 (2005) p. 1. 



APPLIKÁCIA NOVEJ EURÓPSKEJ NORNY NA STANOVENIE Cr(VI) 

V CEMENTE 

Tomáš Ifka, 4. ročník 

Vedúci práce: Doc. Dr. Ing. Martin Palou 

Oddelenie keramiky, skla a cementu, Ústav anorganickej chémie, technológie a materiálov, 

FCHPT STU Bratislava, Radlinského 9, 812 37 Bratislava, Slovenská Republika, 

e-mail: ifkatomas@centrum.sk 

ÚVOD 

Chróm je neodstrániteľný stopový prvok surovinového materiálu používaného vo výrobe 

cementárskeho slinku. Vyskytuje sa v prírodných surovinách (íloch, vápencoch a najmä 

železitých prísadách) vo forme Cr(III) a vo vymurovkách cementárskej rotačnej pece. V 

cementárskych peciach sa však pri vysokej teplote, pri oxidačnej atmosfére a alkalickej 

podmienke mení neškodný trojmocný chróm na šesťmocnú formu. 

Problematika obsahu chrómu v cemente je dnes vysoko aktuálna. Mnohé štáty, ako napríklad 

Nemecko či škandinávske krajiny, sprísňujú hygienické požiadavky na cementy najmä z 

hľadiska obsahu vodorozpustného chrómu. Škandinávska receptúra a Nemecké pravidlá pre 

nebezpečné materiály považujú 2 ppm Cr VI za maximálnu bezpečnú hodnotu na zabránenie 

výskytu ekzémov u murárov. 

Vedecké štúdie taktiež ukázali, že zlúčeniny Cr VI v cemente majú vysokú rozpustnosť vo 

vode a tak môžu ľahko prísť do kontaktu s pokožkou murárov. V samotnom Nemecku, 400 

murárov malo poškodenú pokožku zapríčinenú chrómanom (1997). Šesťmocný chróm, v 

podobe vodorozpustných chrómanov, môže spôsobiť precitlivené reakcie a alergické ekzémy 

pri priamom kontakte s pokožkou ľudí. Nebezpečenstvo hrozí aj z ich toxických a potenciálne 

karcinogénnych účinkov. Každé použitie cementu so sebou nesie riziko priameho dlhšieho 

styku s ľudskou pokožkou s výnimkou kontrolovaných uzatvorených a úplne 

automatizovaných procesov. Na predchádzanie kontaktu cementu s pokožkou sú potrebné, ale 

nie postačujúce, individuálne ochranné opatrenia. Z toho vyplýva, že na ochranu zdravia ľudí 

je potrebné obmedziť používanie cementu s vysokým obsahom vodorozpustného Cr(VI). 

Malo by sa obmedziť najmä uvádzanie na trh a používanie cementu alebo cementových 

stavebných materiáloch, ktoré obsahujú viac ako 2 ppm šesťmocného chrómu v prípade 

činností, kde existuje možnosť kontaktu s pokožkou. Preto podľa nariadenia Európskeho 

parlamentu platného od septembra 2001 je nevyhnutné označením na obale výrobku 

upozorniť zákazníkov kupujúcich cementy, resp. cement obsahujúce výrobky (suché 

omietkové zmesi, malty, betónové zmesi, cementové lepidlá, tmely a podobne) s obsahom 

vodorozpustného Cr(VI) nad 2 ppm, že ide o zdraviu škodlivý výrobok. Pri kontrolovaných 

uzavretých alebo úplne automatizovaných procesoch to nie je potrebné a preto im môže byť 

udelená výnimka. 

V rámci opatrení mnohé cementárske firmy vyvíjali a používajú činidlá na redukciu 

Cr(VI) v cemente. Medzi ne patria heptahydrát síranu železnatého, monosal, siderox alebo 

zlúčeniny na báze zeolitu. Cieľom použitia týchto činidiel je potlačiť obsah Cr(VI) na 

hodnotu pod 2 ppm. Intenzita redukcie môže závisieť od typu cementu, od činidla a jeho 

koncentrácií. Redukčné činidlá by sa mali použiť podľa možnosti čo najskôr, t.j. v mieste 

výroby cementu. 



Cieľom projektu je: 

• aplikovať nový harmonizovaný európsky postup na stanovenie Cr(VI) v cemente 

• skúmať účinok redukčných činidiel na cementoch pripravených v závode 

Rohožník (Holcim Slovensko a.s.) 

• dlhodobý monitoring obsahu Cr(VI) v cementoch (Rohožník Holcim Slovensko 

a.s.) pred a po použití redukčných činidiel 

• zistiť začiatok účinkovania redukčných činidiel 

• zistiť dobu starnutia redukčných činidiel. 

Na dosiahnutie vytýčených cieľov boli použité dve redukčné činidlá ( heptahydrát síranu 

železnatého a monohydrát síranu železnatého -monosal) a cementy pripravené v závode 

Rohožník Holcim Slovensko a. s. (CEM I 42, 5 R, CEM II 32,5/ B-S) 

Eliminácia toxických účinkov Cr (VI) obsiahnutých v cemente 

Problematikou imobilizácie toxických látok, zameranú na látky obsahujúce zlúčeniny 

Cr(VI) sa už dlhšiu dobu zaoberajú pracovníci OKSC FCHPT STU. Tieto látky sa považujú 

za príčinu kožných ekzémov a rozpustným zlúčeninám sa pripisujú aj karcinogénne účinky. 

Problematika zvýšeného obsahu Cr(VI) v cementoch sa rieši dvoma zásadnými 

postupmi 

1) je snaha znížiť celkový obsah chrómu už vo vstupnej surovinovej zmesi, čím sa 

dosiahne primárne zníženie obsahu chrómu vo výslednom produkte. 

2) sekundárne zníženie obsahu zlúčenín Cr(VI) v cemente možno dosiahnuť prídavkom 

aktivovaných železnatých solí, ktoré zabezpečujú redukciu vylúhovateľných škodlivých 

zlúčenín Cr(VI) na prakticky vo vode nerozpustné a zdraviu neškodné zlúčeniny Cr(III), 

pevne zabudované do matrice zhydratovaného cementu. 

Síran železnatý ako redukčné činidlo Cr VI 

Síran železnatý kryštalizuje z vodných roztokov zvyčajne so siedmimi molekulami vody 

ako ,,zelená skalica“ - FeSO4 . 7 H2O . Tvorí svetlozelené jednoklonné kryštály hustoty 1,88 

g.cm -3 , ktoré na vzduchu pomaly zvetrávajú a súčasne podliehajú oxidácii na žltohnedý 

zásaditý síran železitý. Pri zahrievaní ľahko stráca zelená skalica šesť molekúl vody; omnoho 

ťažšie sa oddeľuje posledná molekula. Pri ešte väčšom zahrievaní dochádza k rozkladu s 

odštepovaním oxidu siričitého: 

2Fe II SO4 = (Fe III O)2SO4 + SO2 

Technicky sa pripravuje zelená skalica buď rozpúšťaním železa v zriedenej kyseline 

sírovej alebo sa železný kyz nechá vetrať na vzduchu za častého vlhčenia. 

Zelená skalica sa získava ako vedľajší produkt pri výrobe kremenca chromitého. Používa 

sa pri výrobe atramentu, berlínskej modrej, vo farbiarstve, na konzervovanie dreva, na ničenie 

buriny, tiež príležitostne v lekárstve. Čistí sa zrážaním z vodných roztokov liehom. 

V prírode sa zelená skalica vyskytuje v malých množstvách v podobe výkvetu na železnej 

rude (pyrite). 



Princíp redukcie 

Cr 6+ + Fe 2+ → Cr 3+ + Fe 3+ . 

Cr 6+ + 3e - → Cr 3+ 

3Fe 2+ - 3e - → 3Fe 3+ 

Cr 6+ + 3Fe 2+ → Cr 3+ + 3Fe 3+ 

EXPERIMENTÁLNA ČASŤ 

Príprava vzoriek 

- Priemyselné vzorky. Vzorky boli pripravené v závode s prídavkom 0,3 % 

heptahydrátu síranu železnatého. 

- Laboratórne vzorky. Vzorky boli pripravené prídavkom redukčných činidiel – monosal 

a heptahydrát síranu železnatého v koncentráciách uvedených v tabuľke 1. 500g vzorky 

s príslušnou koncentráciou boli dokonale homogenizované. 

- Príprava vzoriek na stanovenie Cr(VI): 

Na stanovenie vodorozpustného Cr(VI) v cemente sme postupovali podľa Európskej normy 

prEN196-10:2004. Paralelne sme pripravovali vzorky bez štandardného piesku. 

Stanovenie bolo realizované pomocou UV – VIS Spektrofometer HEλIOS. 

VÝSLEDKY A DISKUSIA 

1. Účinok monosalu a heptahydrátu síranu železnatého pri rôznych koncentráciách na 

CEM I 42,5 R. 

Hlavným cieľom je, aby obsah vodorozpustného Cr(VI) v cemente bol pod 2ppm. Ak 

porovnáme účinok monosalu a heptahydrátu síranu železnatého pri rovnakej koncentrácii (tab. 

č.1), zistíme že s použitím heptahydrátu síranu železnatého je obsah Cr (VI) vo vzorke 

cementu nižší ako v prípade monosalu. Pri koncentrácii 0,1 hm. % monosalu bol obsah Cr 

(VI) v danej vzorke takmer ekvivalentný množstvu Cr(VI) stanovenému vo vzorke bez 

redukčného činidla. Účinok monosalu po týždni pri tejto koncentrácii je zanedbateľný. 

Z hľadiska dlhodobého účinku redukčného činidla ako aj z ekonomického hľadiska je podľa 

uvedených výsledkov (tab. č.1) vhodné používať na redukciu vodorozpustného Cr (VI) 

heptahydrát síranu železnatého pri koncentrácii 0,3 hm. %. 



Tab. č.1: Výsledky analýzy Cr(VI) v CEM 42,5 R pri aplikácii redukčných činidiel s rôznou 

koncentráciou 

CEM I 42,5R 

Chemické zloženie [ hm. %] 

CaO SiO2 Al2O3 Fe2O3 MgO SO3 Na2O K2O Troska 

62,52 22,01 4,95 2,96 2,48 2,74 0,4 0,75 6,6 

Stanovenie → 1 týždeň od prípravy vzoriek 

pH1 pH2 c [mg/l] A K [ppm] Kkor [ppm] 

Neredukovaný 12,92 2,39 0,4782 0,377 2,3910 2,5600 

Red. 0,1% FeSO4.7H2O 12,94 2,29 0,3292 0,259 1,6460 1,7623 

Red. 0,3% FeSO4.7H2O 12,63 2,29 0,0801 0,063 0,4005 0,4288 

Red. 0,5% FeSO4.7H2O 12,86 2,28 - - - - 

Red. 0,1% monosal 12,70 2,49 0,4187 0,330 2,0935 2,2414 

Red. 0,3% monosal 13,20 2,42 0,2139 0,169 1,0695 1,1451 

Red. 0,5% monosal 13,02 2,37 0,1413 0,111 0,7065 0,7564 

pH1 - pred prídavkom HCl, pH2 - po prídavku HCl, c - koncentrácia Cr(VI) z kalibračnej 

krivky v mg/l, A – absorbancia, K - obsah vodorozpustného Cr(VI) v cemente s obsahom 

trosky, Kkor - obsah vodorozpustného Cr(VI) v čistom cemente 

2. Začiatok pôsobenia redukčného činidla na Cr(VI) 

Na zistenie začiatku účinkovania redukčného činidla bol použitý heptahydrát síranu 

železnatého pri dvoch koncentráciách 0,3 a 0,5 hm. %. Stanovoval som obsah 

vodorozpustného Cr(VI) po dobu 1, 4 a 24 hodinách. Výsledky sú uvedené v grafe č.1, kde je 

evidentné, že po 1 jednej hodine sa obsah Cr(VI) redukoval na minimálnu hodnotu. To 

znamená, že heptahydrát síranu železnatého pri koncentrácii 0, 3 a 0,5 hm. % začína pôsobiť 

už po jednej hodine aplikácie. 

Tab. č.2: Chemické zloženie vzorky CEM II/ B-S 32,5R 

CEM II/ B-S 32,5R 

Chemické zloženie [ hm.%] 


56,20 27,44 6,02 2,57 3,64 2,77 0,45 0,77 28,1 



K [ppm] 

4 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

0 

CEM II / B-S 32,5R 

1 4 24 

t [hod] 

nered. 

red. 0,3% hepta 


Graf č.1: Začiatok pôsobenia redukčného činidla na Cr(VI) 

3 Doba starnutia redukčného činidla 

Na určenie doby pôsobenia redukčného činidla som použil cement CEM II/B-S 32,5R, do 

ktorého som pridal 0,5 hm. % heptahydrátu síranu železnatého. Po stanovení 

vodorozpustného Cr (VI) v tejto vzorke cementu som zistil, že pri danej koncentrácii 

redukčného činidla je obsah Cr (VI) vo vzorke veľmi nízky a to aj po 65. dňoch. Pretože 

obsah stanoveného Cr(VI) v cemente je veľmi nízky, kolísanie výsledkov je pravdepodobne 

spôsobené chybou pri meraní. 44 dní od prípravy danej vzorky sa začína Cr (III) spätne 

oxidovať na Cr(VI). 

Tab. č.3: Chemické zloženie vzorky cementu CEM I 42,5R 

CEM II/ B-S 32,5R 

Chemické zloženie [ hm. %] 


55,98 27,16 5,82 2,32 4,27 2,55 0,44 0,78 29,4 

K [ppm] 

3 

2.5 

2 

1.5 

1 

0.5 

0 

CEM II / B-S 32,5R 

9 16 44 

t[dni] 

51 65 

nered. 


Graf č.2: Monitorovanie doby starnutia redukčného činidla za obdobie 65 dní 



ZÁVER 

Podľa smernice 76/769/EHS Európskej Rady, každý balený cement musí obsahovať 

Cr(VI) pod 2 ppm. Z toho vyplýva, že cementársky priemysel musí vyvíjať metódy redukcie 

a kontroly obsahu Cr(VI) vo svojich výrobkoch. Aplikácia novej normy na stanovenie 

Cr(VI) v cemente bola na konktrétnych cementoch vyrábaných v závode Rohožník Holcim 

Slovensko, a.s. V práci som použil dve redukčné činidlá (heptahydrát a monohydrát síranu 

železnatého) a sledoval som ich účinok na Cr(VI) podľa koncentrácie (0,1; 0,3; 0,5 hm. %) 

a doby starnutia do troch mesiacov. 

Z laboratórnych výsledkov stanovenia Cr(VI) v cemente vyplýva: 

- Monosal začína účinkovať až pri (redukovať Cr(VI) pod 2 ppm) koncentrácii 0,3 hm. %. 

Pri koncentrácii 0,1 hm. % očakávaný efekt nebol dosiahnutý. 

- Heptahydrát síranu železnatého účinkuje už pri koncentrácii 0,1 hm. % 

- Výrazný účinok obidvoch redukčných činidiel je pri koncentrácii 0,5 hm. % 

- Redukčné činidlo začína pôsobiť už do jednej hodiny. 

- Po 45. dňoch začína spätná oxidácia redukovaného Cr(III) na Cr(VI). Obsah 

vodorozpustného Cr(VI) rastie s časom veľmi pomaly, takže sa nepredpokladá 

dosiahnutie kritickej hodnoty 2 ppm. 

- Monitorovanie obsahu Cr(VI) v cemente pred a po pridaní redukčného činidla prinieslo 

tieto výsledky: 

- Obsah Cr(VI) v cemente sa bez redukčného činidla menil podľa dátumu výroby. 

- Účinky redukčného činidla neboli v niektorých prípadoch uspokojivé, lebo obsah Cr(VI) 

nebol pod 2 ppm. 

- Aj v prípade vzoriek cementov zo závodu som zistil starnutie redukčného činidla 

(spätná oxidácia Cr(III) na Cr(VI)). 



DISSOLUTION OF HUMIC SUBSTANCES IN UREA IN LIGHT OF 

SUPRAMOLECULAR THEORY 

Jiří Kislinger, 5 th year 

Supervisor: Prof. Alessandro Piccolo* 

Brno University of Technology, Faculty of Chemistry, Institute of Physical and Applied 

Chemistry, Purkyňova 118, 612 00, Brno, e-mail: xckislinger@fch.vutbr.cz 

* Università degli Studi di Napoli Federico II, Facoltà di Agraria, Dipartimento di Scienze del 

Suolo, della Pianta e dell’Ambiente, Via Università 100, 800 55, Portici, Italy 

INTRODUCTION 

Humic substances (HS) are natural organic compounds present in all terrestrial and aquatic 

systems. Processes of their accumulation and decomposition in different environmental 

domains are still largely unclear [1]. Nevertheless, the characteristics and quantity of HS 

greatly affect the environmental fate of organic pollutants in soils and natural waters [2]. 

Molecular properties of dissolved HS have recently been recognized to influence removal of 

synthetic organic compounds from municipal and industrial wastewaters [3], and give rise to 

binding and environmental transport of both nonpolar and slightly polar organic contaminants 

[4]. 

Despite their prominent environmental role, the chemical structure of HS is still far from 

clear mainly because of their chemical heterogeneities and spatial variabilities. As a 

consequence the conformational structures of HS are also ill-defined. There is a general 

acceptance of the concept that HS are polydisperse, long-chain, randomly coiled molecules 

that may have a slight degree of crosslinking [5]. Negative charges arising from ionization of 

carboxyl groups are responsible for mutual repulsion and expansion of the coils [6]. The 

random coil model depicts HS as most densely coiled at high concentrations, low pH and high 

ionic strength, and as linear colloids at neutral pH, low ionic strength and low concentrations 

[7]. This model has helped several investigators to explain results obtained by gel permeation 

chromatography [8], or by diffusion through ultrafiltration membranes [9]. A common 

observation was that elution at larger permeation volumes was retarded considerably when the 

ionic strength of the mobile phase was increased. The changes were attributed to coiling of 

humic molecules with consequent decrease of hydrodynamic radius and enhanced diffusion 

through smaller gel pores. The same phenomenon was also observed by varying the ionic 

strength of the mobile phase in High Pressure Size Exclusion Chromatography (HPSEC) 

experiments with organic colloids from natural waters [10]. Another conformational model 

still represents humic materials as polymers but aggregated in micelle-like or membrane-like 

structures [11]. In this description, humic molecules would have inner hydrophobic domains 

with structural voids that could entrap hydrophobic organic compounds [12] and quench their 

fluorescence activity [13]. 

Recent results, mainly from low-pressure size-exclusion chromatography experiments, 

have indicated that humic aggregates are composed of relatively small subunits weakly held 

together by hydrophobic forces [4, 14]. The aggregation of humic material in high molecular 

dimensions is considered not to be permanent but found to be able to be reversibly disrupted 

by the presence of an organic acid in a rapid change from acidic to alkaline pH. These 

findings have suggested that HS in solution would appear to consist of randomly selfassembling 

supramolecular associations rather than polymeric coils, as in biological 



macromolecules [15]. This innovative explanation of the conformational behavior of HS had 

to be verified in other experimental conditions and HPSEC was utilized for further 

investigations. For a number of reasons, HPSEC is more valid chromatographic system than 

low-pressure gel permeation [16]. 

The evidence that humic molecules are suprastructural associations in aqueous solutions is 

given by the elution patterns of HS in HPSEC experiments in 8 M urea. Concentrated urea 

was successfully employed to disrupt protein-protein interactions and to solubilize 

aggregating hydrophobic proteins for their further separation [17]. Thus the aim of this work 

was to confirm and deepen the knowledge of behavior of HS in solutions in the term of 

supramolecular approach. 

EXPERIMENTAL PART 

Humic acids were obtained from 3 agricultural soils from: Denmark (Haplic Luvisol, 

Roskilde) – DK; Germany (Eutric Luvisol, Munich) – DE; and Italy (Eutric Regosol, Caserta) 

– IT. These humic acids were extracted by standard procedures [5] and were subsequently 

suspended in distilled water and titrated for 3 hours to pH 7 using CO2–free solution of 0.5 M 

NaOH in an atmosphere of N2. The resulting sodium-humates were than filtered through a 

Millipore 0.45 μm membrane, and freeze-dried. 

The HPSEC system used consists of a Shimadzu LC–10ADVP solvent pump and two 

detectors in series, a Perkin–Elmer LC–295 UV/VIS detector set at 280 nm and a refractive 

index (RI) detector (Refractomonitor IV, Fisons Instruments). A Rheodyne rotary injector 

equipped with a 100 μL sample loop was used to charge the humic solutions. A Biosep SEC– 

S–2000 column (300 by 7.8 mm i.d.) was adopted in these experiments. Humate solutions for 

HPSEC analysis were prepared by dissolving 15 mg of each sample in 25 mL of the eluent 

(8 M urea) to reach concentration 0.6 g×L –1 . 

In the UV/VIS spectroscopy experiments the Na-humates were redissolved in 8 M urea to 

reach concentration 0.2 g×L –1 . Such solutions were diluted 20 times in 8 M urea. These were 

scanned for the light absorbance from 200 to 800 nm in a Perkin–Elmer Lambda 25 UV/VIS 

spectrophotometer using quartz cuvettes of 1 cm pathlength against the solvent. Two 

replicates for each sample were run to obtain absorbance curves. 


The elution profiles of three different purified HS dissolved in 8 M urea and eluted in the 

same solution are shown in Figs. 1, 2, and 3. The experiment is illustrative of the capacity of 

urea to separate the hydrophobic from the hydrophilic components of HS. The 

chromatograms show a net separation of humic matter: a high-molecular-size fraction at the 

void volume visible only by the UV detector and a low-molecular-size fraction eluting at the 

total volume detected only by the RI detector. The humic hydrophobic molecules are strongly 

associated through a hydrophobic effect induced by the highly concentrated urea that interacts 

more favorably than hydrophobic molecules with the network of the water structure. 

Differences among HS can also be noted. The humic acid from Italian soil was the only 

one to show two well-separated peaks at the RI detector system, whereas only one very 

intense peak (>1000 mV) at the column void volume was shown by the UV detector (Fig. 1). 

This indicates that while light-absorbing chromophores (280 nm) with a high molar 

absorptivity were excluded rapidly from the column, their concentration in the humic sample 



was relevant and of similar order of magnitude to the nonabsorbing hydrophilic, probably 

ionized, compounds detected by the RI detector at the total exclusion volume. 

Fig. 1: UV- and RI-detected HPSEC chromatogram of humic acid from the Italian soil 

The HA from the Danish soil gave similar complete separation between the large-size 

hydrophobic chromophore fraction and the small-size ionized and hydrophilic components 

shown by the RI detector (Fig. 2). However, while the molar absorptivity of the excluding 

chromophores was still high (as suggested by an absorption intensity >500 mV), the lack of 

corresponding peaks at the RI detector indicated that their concentration in the sample was 

much less than that in the hydrophilic components excluded at large elution volumes. 

Fig. 2: UV- and RI-detected HPSEC chromatogram of humic acid from the Danish soil 



Identical behavior was shown by the humic acid extracted from the German soil (Fig. 3). 

Also for this sample, the chromophore association excluded at the column void volume was 

hardly representative of the mass of humic sample since the corresponding peak at the RI 

detector was almost irrelevant in comparison to the signal of the small-sized nonabsorbing 

hydrophilic constituents. 

Fig. 3: UV- and RI-detected HPSEC chromatogram of humic acid from the German soil 

All 3 HA samples diluted in 8 M urea were scanned for the light absorbance to gain some 

information about the optical properties. In all cases the absorption spectra (Fig. 4) decreased 

in an approximately exponential fashion with increasing wavelength and exhibited no distinct 

absorption bands. The differences in light absorption of HAs are related to their specific 

molecular composition. For the preceding HPSEC experiments the wavelength 280 nm has 

verified to be the optimal to monitor the separation of humic matter to excluding moieties. 

Fig. 4: Absorbance curves of soil humic acids dissolved in 8 M urea 



CONCLUSION 

These findings indicate that humic substances, rather than being polymeric macromolecules, 

may be more simply regarded as supramolecular associations of relatively small 

heterogeneous molecules stabilized mainly by dispersive forces. The model of dissolved 

humic substances based on the self-association of small components rather than on the 

polymeric random coil model should contribute to further understanding of the reactivity of 

humic substances in many ecological and environmental processes in which they are 

involved. 


Encouragement for work, help and useful comments of Prof. A. Piccolo and my Italian 

friends and colleagues Drs. P. Conte and R. Spaccini during my residence in Naples are 

gratefully acknowledged. Great thanks go also to Dr. Jiří Kučerík for his everlasting support. 

REFERENCES 

[1] Piccolo, A. 1996. Humus and soil conservation, p. 225-264 in Humic substances in 

terrestrial ecosystems. Elsevier, Amsterdam. 

[2] Carter, C.W., Suffet, I.H. 1982. Environ. Sci. Technol. 16: 735-740 

[3] Karanfil, T., Kilduff, J.E., Schlautman, J.A., Weber, W.J. Jr. 1996. Environ. Sci. 

Technol. 30: 2187-2201 

[4] Piccolo, A., Nardi, S., Concheri, G. 1996. Chemosphere 33: 595-602 

[5] Stevenson, F.J. 1994. Humus chemistry: Genesis, Composition, Reactions. 2 nd ed. 

Wiley-Intersci., New York. 

[6] Cameron, R.S., Swift, R.S., Thornton, B.K., Posner, A.M. 1972. J. Soil Sci. 23: 395-408 

[7] Ghosh, K., Schnitzer, M. 1980. Soil Sci. 129: 266-276 

[8] De Haan, H., Jones, R.I., Salonen, K. 1987. Freshwater Biol. 17: 453-459 

[9] Cornel, P.K., Summers, S.R., Roberts, P.V. 1986. J. Colloid Interface Sci. 110: 149-163 

[10] Chin, Yu-P., Gschwend, P.M. 1991. Geochim. Cosmochim. Acta 55: 1309-1317 

[11] Wershaw, R.L. 1993. Environ. Sci. Technol. 27: 814-816 

[12] Schlautman, M.A., Morgan, J.J. 1993. Environ. Sci. Technol. 27: 961-969 

[13] Engebretson, R., von Wandruszka, R. 1997. Org. Geochem. 26: 759-767 

[14] Piccolo, A., Conte, P. 2000. Adv. Environ. Res. 3: 508-521 

[15] Cantor, C.R., Schimmel, P.R. 1980. Biophysical chemistry, Part I: The conformation of 

biological macromolecules. Freeman, New York. 

[16] Conte, P., Piccolo, A. 1999. Chemosphere 38: 517-528 

[17] Hjelmeland, L.M. 1990. Solubilization of native membrane proteins. In Guide to 

Protein Purification. Methods of Enzymology, Vol. 182: 253-264. Academic Press, San 

Diego. 



WETTABILITY OF PLASMA POLYMERIZED 

VINYLTRIETHOXYSILANE FILM 

Soňa Lichovníková, 5 nd year 

Supervisor: Doc. RNDr. Vladimír Čech, PhD. 

Institute of Material Science, Faculty of Chemistry, Brno University of Technology, 

Purkyňova 118, 612 00 Brno, Czech Republic, e-mail: xclichovnikova@fch.vutbr.cz 

INTRODUCTION 

This paper deals with wettability of thin polymer films prepared by Plasma Enhanced 

Chemical Vapor Deposition (PE CVD) from vinyltriethoxysilane (VTES) monomer deposited 

on planar substrates. The sessile drop method was used to measure the contact angles and the 

surface free energy was evaluated using the approach of Owens-Wendt geometrical mean 

method, Wu harmonic mean method and the acid-base method. Changes in the surface 

structure were investigated by Fourier Transform Infrared Spectroscopy (FTIR) measurement, 

X-Ray Photoelectron Spectroscopy (XPS) measurement and Rutherford Backscattering 

Spectrometry (RBS). A relation between wettability and process parameters of the plasma 

deposition were discussed as well. [1] 

EXPERIMENTAL 

PLASMA POLYMERIZATION 

Plasma polymer films of VTES CH2-CH-Si(-O-CH2-CH3)3 (purity ≥ 98 %, Fluka) were 

deposited on glass substrates (special microscope slides without flaws, 1.0 × 26 × 76 mm 3 , 

Knittel Gläser, Germany, for surface free energy determination) and on infrared-transparent 

silicon wafers (0.8 × 10 × 10 mm 3 , Terosil Co., for film analyses by FTIR, XPS and RBS), 

using a helical coupling plasma system (13.56 MHz) in a vacuum tubular chamber (40 mm in 

diameter, made of Pyrex glass). Details of the apparatus are described in Ref. [2]. 

The vacuum system was evacuated to a pressure of 5 × 10 -3 Pa and then flushed with argon 

gas (10 sccm) for 10 min. A basic pressure of 2 × 10 -3 Pa was established in the deposition 

chamber after flushing. Monomer vapor at a mass flow rate of 0.45 sccm was introduced into 

the deposition chamber using a dose valve and the plasma then was ignited at a selected 

effective power of 0.1, 0.5, 2.5, 5.0, 10.0 and 25 W, using pulsed plasma with ton= 1 ms and a 

total power of 50 W. Deposition pressure was about 1.3 Pa and the film thickness (about 400 

nm) was controlled by deposition time ranging from 5-100 min. When the deposition process 

was finished, all the apparatus was flushed with argon gas and after 30 min the chamber was 

flooded by air to atmospheric pressure. Prepared samples were transported from the chamber 

into a desiccator to avoid contamination before measurements. [3] 

THIN FILM ANALYSIS 

The sessile drop method (tangent method) employing an OCA 10 goniometer 

(DataPhysics) with Software SCA 20 for Windows 9x/NT was used to measure the contact 

angles. The surface free energy was evaluated using the approach of Owens-Wendt 

geometrical mean method, Wu harmonic mean method and acid-base method [1]. The surface 

free energy, as well as the dispersive and polar components, the Lewis electron acceptor (γs (+) ) 

and Lewis electron donor (γs (-) ) components of VTES film were evaluated from contact angle 

measurements using distilled water, ethylene glycol, glycerol and methylene iodide. A small 

drop of liquid was carefully placed on the surface of substrate (glass slide). The contact angle 



is obtained by measuring the angle between the tangent to the profile at the point of contact 

with the solid surface. Software SCA 20 offers the calculation of the surface free energy of 

solids and their components. It also contains database of liquids and their necessary 

parameters. Owens-Wendt and Wu methods express the surface tension as a sum of 

components based on dispersion forces (γ d ) and polar forces (γ p ): γ=γ d +γ p . Van Oss, Good 

and Chaudhury´s acid-base theory is more detailed in the surface free energy specification and 

leads from Lewis theory, where Lewis acid is an electron-pair acceptor, Lewis base is an 

electron-pair donor. The surface tension is a sum of two components: the Lifshitz-van der 

Waals component (γs LW ) and acid-base component (γs AB ), 

γ + 

LW AB 

= γ γ . γs LW reflects the 

long-range interactions (to 100 Å), including dispersive interaction, the dipole-dipole and the 

dipole induced dipole interaction and it influences the total surface free energy at most. γs AB is 

associated with the transfer of electrons between an electron donor (γs (-) ) and an electron 

acceptor (γs (+) AB + − 

) in short range of < 3 Å and γ s = 2 γ s γ s . γs AB is relatively small and not so 

important when compared to γs LW . Values of the surface free energy parameters of probe 

liquids were taken according to different authors. Distilled water and diiodomethane were 

taken according to Erbil, formamide and glycerol according to Van Oss et al. Three different 

probe liquids have to be used for calculation by acid-base approach, averaged results of the 

surface tension calculation from the water-diiodomethane-formamide and waterdiiodomethane-glycerol 

triplets were used. The contact angle data of two different testing 

liquids are needed for the determination of γ d and γ p components according Owens-Wendt 

and Wu methods: water (author Strom et al.) and diiodomethane (author Janczuk et.al.). 

Several standard conditions have to be met to measure reproducible contact angles: drop 

volume (checked by micrometer screw), time (10 seconds) and laboratory temperature. More 

information about equations for surface tension calculation and contact angle measurements 

can be found in Ref. [1,6] 

The elemental composition (C, Si, O) in the surface region (top 6-8 nm) of the deposited 

layers was determined by XPS on an ADES 400 VG Scientific photoelectron spectrometer 

using MgKα (1253.6 eV) photon beams. XPS is sensitive surface selective method which 

provides information about chemical composition and electron structure of the surface of 

solids. The elemental composition has been also studied by conventional and resonant RBS 

and Elastic Recoil Detection Analysis (ERDA) methods using Van de Graaf generator with a 

linear electrostatic accelerator. The RBS spectrum contains information about elemental 

composition in depth levels of sample. [4,5] 

Infrared measurements were carried out using a Nicolet Impact 400 Fourier transform 

infrared (FTIR) spectrophotometer and the chemical bonds in thin films were determined by 

interpreting the infrared absorption spectrum. Samples were measured without the protective 

atmosphere. 


Plasma-polymerized VTES films were deposited on planar glass substrates at different 

effective powers (0.1-25 W) at conditions described above. Sessile drop measurements were 

employed to analyze the surface free energy of the film. Measured contact angles of the probe 

liquids (water, diiodomethane, formamide, glycerol) were changed according to various 

effective power (W). In Figure 1, trend of decreasing contact angles with the magnitude of the 

input RF power can be seen. Contact angles of all liquids are slightly decreasing before 

reaching 5 W effective power. Maximum decrement of contact angle can be seen between 2.5 



W and 5 W. The wettability of liquids generally increases with increasing effective power, 

except diiodomethane, where the maximum wettability is during 5 W effective power. In 

Figure 2, there can be seen the trend of increasing surface free energy (mJ.m -2 , average of 

both triplets) with the magnitude of the input RF power. The largest increase is during 5 W 

effective power. When the surface free energy increases, wettability is also increased. Total 

surface free energy has maximum during 25 W. As can be seen, the most important 

component which influences the total surface free energy is γs LW . The other part of the surface 

free energy is the γs AB divided in electron acceptor γs (+) (acid) and donor γs (-) (basic) 

components. The γs AB component is in comparison with the γs LW component small. γs (-) 

component is much higher than γs (+) with values near zero. 

Contact angle [deg.] 

90 

80 

70 


50 

40 

30 

20 

10 

0 

water 

diiodomethane 

formamide 

glycerol 

0,1 1 10 

W eff [Watt] 

Surface free energy [mJ/m 2 ] 

50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

0,1 1 10 

W eff [Watt] 

Figure 1, 2: Dependence of the contact angle of all testing liquids and dependence of the 

surface free energy components (acid-base approach) on the magnitude of the input RF 

power 

In Figure 3 can be seen calculated total surface free energy and its components (in mJ.m -2 ) 

for set of samples, by Owens-Wendt geometric mean (O-W) and Wu harmonic mean (Wu) 

methods, depending on the effective 

Surface free energy [mJ/m 2 ] 

50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

0,1 1 10 

W eff [Watt] 

γ TOT (O-W) 

γ TOT (Wu) 

(d) (O-W) 

γ 

s 

(d) 

γ (Wu) 

s 

(p) 

γ (O-W) 

s 

(p) 

γ (Wu) 

s 

Figure 3: Dependence of the surface free 

energy components (Owen-Wendt and Wu 

approach) on effective power 

γ TOT 

γ LW 

γ + 

γ − 

power. Total surface free energy is sum 

of γ d and γ p components. Values 

obtained by Wu method are not exactly 

equal to the values obtained by O-W 

method. It is caused by different γ d and 

γ p values of probe liquid diiodomethane. 

Total surface free energy (of both 

methods) and wettability increase with 

increasing effective power. The largest 

increase of surface free energy is during 

5 W. Surface free energy can be 

considered almost constant during higher 

effective powers. Dispersion component 

influences the total surface free energy at 

most, but influence of polar component 

can’t be neglected. Both components increase with increasing effective power. Total surface 

free energy of O-W, Wu and acid-base approaches is quite the same. The difference is caused 

by interpretation of the surface free energy components. Surface free energy values obtained 

by acid-base approach are closer to values at O-W method than Wu method. Wettability of 



pp-VTES thin films could be well controlled by process parameters, especially effective 

powers. 

To investigate relationship between wettability and chemical status of the thin VTES films, 

the data from FTIR, XPS and RBS measurements were used. In Figure 4 and 5 can be seen 

comparison of FTIR spectra of three samples with different effective powers. We can see 

some changes in chemical structure between the high-power and low-power deposited 

polymers. The absorption bands at 2973 cm -1 and 2924 cm -1 were assigned to CH3 and CH2 

stretching vibrations. With increasing of effective power, intensity of bands decreases as well 

as bands 1070 cm -1 assigned to Si-O-C stretching vibrations, 885 cm -1 assigned to Si-C, 790 

cm -1 assigned to Si-O, 1160 cm -1 and 965 cm -1 assigned to Si-O-C2H5. The dominant 

absorption bands in all spectra can be found at a range 1000-1200 cm -1 because the plasma 

polymer of VTES was formed by the Si-O-C network. The greatest decrease of peak area was 

measured in Si-O-C group. This group is only one from Si-groups which is apparent at 25 W 

effective power. The pp-VTES films seemed to be free of vinyl groups as there were no 

absorption bands at positions marked by dashed line (Fig. 4). With increasing power a new 

absorption band can be seen at 3480 cm -1 assigned to the OH stretching vibrations (strong 

polar group) and also at 1700 cm -1 corresponding to the C=O carbonyl stretching vibrations 

(medium polar group), so the presence of polar substances on the surface increases. It comes 

to that wettability also increases with increasing power which can be also connected with 

decreasing of stretch band intensity of apolar substances (especially CH3) and decreasing of 

inorganic Si-O-C substances. At lower effective powers (under 5 W), it seems that apolar 

groups (CH3, Si-Et, Si-C, Si-H) are linked to Si-O-C network and create more apolar surface 

in comparison with samples at higher effective powers where polar groups like OH and C=O 

are linked to the carbon network. 

Absorbance (a.u.) 

OH 

CH 3 

CH 2 

CH 2 

CH 3 

Si-H 

0.05 W 

5 W 

25 W 

Si-O-C 

Si-Et 

C=O CH2 Si-Et 

vinyl vinyl vinyl 

Si-O 

Si-C 

4000 3500 3000 2500 2000 1500 1000 500 

Wavenumber (cm -1 ) 

Figure 4, 5: Comparison of VTES FTIR spectra of three different effective powers (W) 

and dependence of the peak area (a.u.) from VTES FTIR spectra on the effective power (W) 

In Figure 6 and 7 can be seen the results from XPS and RBS measurements – element 

abundances of C, O, Si (at. %) and elements ratio which also depend on effective power. With 

increasing of effective power, C concentration increases, O and Si concentration decrease. 

Ratios C/Si, O/Si, C/O at a higher power of 25 W are very similar to those of the monomer 

molecule. As can be seen, dependences of atomic concentration and element ratio on film 

thickness could be neglected. C concentration increases with increasing power but it seems 

that in high powers C create cage and polar groups OH, C=O are linked to cage and stick out. 



Hence wettability is higher. O and Si concentrations decrease with increasing power, but in 

lower powers Si-O-C groups created polymer network, apolar groups are linked to network 

and stick out. Wettability is than smaller. This implies that wettability of VTES is influenced 

especially by groups which stick out the network and it depends if groups are polar or apolar. 

This conclusion corresponds to investigation in Ref. [7]. 

Atomic concentration (at%) 

80 

70 


50 

40 

30 

20 

10 

0 

Film thickness: ~400 nm 

XPS 

RBS 

Carbon 

Oxygen 

Silicon 

0,1 1 10 

Effective power (W) 

Effective power: 5 W 

Carbon 

Oxygen 

Silicon 

XPS 

RBS 

200 500 1000 

Film thickness (nm) 

80 

70 


50 

40 

30 

20 

10 

0 

Element ratio 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

Film thickness: ~400 nm 

C/Si (monomer) 

C/Si 

O/Si (monomer) 

O/Si C/O 

C/O (monomer) 

0 

0,1 1 10 

Effective power (W) 

Effective power: 5 W 

200 500 1000 

Film thickness (nm) 

Figure 6, 7: Comparison of RBS and XPS measurements: dependence of the atomic 

concentration (at %) on effective power (W) and dependence of the elements ratio on effective 

power (W) 

CONCLUSION 

Plasma-polymerized thin films of VTES were deposited on planar glass substrates and 

silicon wafers and investigated by contact angle measurements, FTIR, XPS and RBS 

methods. Process parameters, especially effective power can influence/control the chemical 

structure, elemental composition and wettability. Wettability increases with increasing 

effective power and it seems that wettability is influenced mainly by groups sticking out of 

the network and by polar groups. 

ACKNOWLEDGEMENTS 

This work was supported by the Czech Science Foundation, grant no.104/06/0437, and the 

Czech Ministry of Education, grant no. 1P05OC087 (COSTP12). The author would like to 

thank Dr. J. Zemek (XPS), Dr. V. Perina (RBS/ERDA) and J. Studynka (FTIR) for 

spectroscopic measurements and analyses. 

REFERENCES 

[1] F. Garbassi, M. Morra, E. Occhiello, Polymer Surfaces, John Wiley & Sons Ltd, New 

York, 1998. 

[2] R. Prikryl, O. Salyk, J. Vanek, V. Cech, Czech. J. Phys. 52 (2002), D816. 

[3] V. Cech, N. Inagaki, J. Vanek, R. Prikryl, A. Grycova, J. Zemek, Thin Solid Films 502 

(2006) 181-187. 

[4] Jiří Sova, Diploma Thesis, Brno University of Technology, Brno, 2005. 

[5] J.F. Watts, J. Wolstenholme, An Introduction to Surface Analysis by XPS and AES, John 

Wiley & Sons Ltd, London, 2003. 

[6] S. Wu, Polymer Interface and Adhesion, Marcel Dekker, Inc., New York, 1982. 

[7] V. Cech, J. Vanek, V. Perina, J. Zemek, Chemical properties of plasma-polymerized 

vinyltriethoxysilane, ISPC-17, Toronto, August 7-12, 2005, p.1-5. 



C/Si 

C/O 

O/Si 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0

URČOVANIE DISTRIBÚCIE DOMÉN V SPIN CROSSOVER 

SYSTÉMOCH POMOCOU ANALYTICKEJ FUNKCIE 

Ján Pavlik, 4. ročník 

vedúci práce: Prof. Ing. Roman Boča, DrSc. 

Slovenská technická univerzita v Bratislave, Fakulta chemickej a potravinárskej technológie, 

Oddelenie anorganickej chémie, Radlinského 9, 812 37 Bratislava, 

email: jan.pavlyk@gmail.com 

ÚVOD 

Niektoré komplexy prechodných kovov sa môžu predovšetkým v závislosti od teploty (ale 

napríklad aj tlaku, magnetického poľa a pôsobenia svetla) vyskytovať buď v nízkospinovom 

(LS) alebo vysokospinovom (HS) stave. Tieto dva stavy majú rozdielne optické, magnetické a 

objemové vlastnosti. Jav prechodu medzi týmito stavmi sa označuje ako spin crossover. Jeho 

možnosť je nutne podmienená tým, aby základným energetickým stavom molekuly v kryštáli 

bol nízkospinový. Ďalšou podmienkou je, aby entropia HS stavu bola vyššia ako LS. Pri 

splnení týchto podmienok bude existovať teplota (tzv. teplota prechodu), pri ktorej bude 

pozorovateľný prechod medzi nimi [6]. Spin crossover sa zaznamenáva najčastejšie formou 

závislosti zastúpenia HS molekúl od teploty. 

MODELY ISINGOVHO TYPU (MIKROSKOPICKÉ MODELY) 

Spoločnou črtou týchto modelov je predpoklad nerovnakého vzájomného pôsobenia 

susediacich molekúl, ktoré sú v rôznom spinovom stave a parametrizovanie tejto interakcie 

empirickou konštantou. Konkrétne sa pre spin crossover systém predpokladá hamiltonián 

nasledujúceho typu [9]: 

Hˆ= ( Δ ˆ ˆ 

0 /2) σ − J σ σ 

(1.1) 

kde Δ0 je energetický rozdiel molekuly v stave HS a LS, J je konštanta kooperativity, teda 

miera vplyvu stavov okolitých molekúl na stav sledovanej molekuly a ˆ σ je operátor tzv. 

fiktívneho spinu, ktorý rozlišuje medzi LS a HS stavom definičným vzťahom: 

ˆ σ LS = -1 LS (1.2) 

ˆ σ HS = +1 HS (1.3) 

Ďalej σ značí teplotný priemer fiktívneho spinu, teda 

∑ 

( E kT ) 

( −E 

kT ) 

σ = 

σ iexp − i / 

i 

∑ exp i / 

i 

(1.4) 

Tento model sa nazýva aj dvojhladinový model Isingovho typu, pretože hovorí o dvoch 

energetických stavoch molekuly, ktoré sú ďalej modulované priemerným stavom okolitých 

molekúl. Vlastné energie molekuly budú 

E ( σ =− 1) =− Δ 2+ 

J σ 

(1.5 a) 

1 0 

E2( σ =+ 1) = Δ0 2− 

J σ 

(1.5 b) 

Fyzikálne oprávnenejšie modely Isingovho typu sa získajú predstavou kryštálu zloženého z 

oblastí, v ktorých sa nachádzajú iba molekuly jedného stavu tzv. domény [2]. 

FORC A FORC DISTRIBÚCIA 

Jednou z novších perspektívnych metód umožňujúcich stanoviť niektoré parametre 

spomínaných modelov je meranie tzv. FORC (first-order reversal curve). Postup merania je 

nasledujúci: vzorka pri teplote kde je úplne v HS stave sa pomaly ochladzuje a stanovuje sa 



zastúpenie molekúl alebo domén v tomto stave. Pri určitej teplote (teplote obratu TA) sa 

ochladzovanie zastaví a začne sa znova s ohrevom až kým nie je vzorka opäť kompletne v 

pôvodnom stave. Zastúpenie HS stavu sa posudzuje ako funkcia TA a aktuálnej teploty TB. 

Vychádzať sa môže aj z LS stavu, kedy hovoríme o ohrevnom móde narozdiel od skôr 

uvedeného postupu, ktorý sa označuje ako chladiaci mód. Príklad typickej FORC v 

chladiacom móde uvádza obr. 1. 

Obr. 1. FORC v ohrevnom móde pre [Fe0.6Zn0.4(btr)2(NCS)2]H2O získaná 

pomocou optických metód. 

Dôležitý pojem spájajúci sa s FORC je tzv. FORC distribúcia [4] definovaná vzťahom 

2 

1 ∂ nHS( TA, TB) 

ρ( 

TA, TB) 

=− 

(2.1) 

2 ∂TA∂TB Existujú pádne dôvody pre stotožnenie takto definovanej veličiny s distribúciou domén v 

kryštáli. 

URČOVANIE FORC DISTRIBÚCIE 

V súčasnosti sa FORC distribúcia určuje numericky, čo znamená, že diferencie vo vzťahu 

(2.1) sa nahradia malými zmenami daných veličín. Ich hodnoty sú samozrejme determinované 

hustotou bodov v skúmanej FORC a presnosť takto určenej distribúcie je teda veľmi závislá 

na hustote merania. Ako alternatíva sa ponúka možnosť aproximovať namerané FORC 

vhodne zvolenou funkciou derivovateľnou do druhého stupňa a FORC distribúciu získať jej 

analytickým derivovaním, čím sa aj pri redších meraniach dosiahne interpolácia chýbajúcich 

bodov a hladšia FORC distribúcia. 

Obr. 2. FORC s rôznymi osami pre TA resp. TB 



Obr. 2. názorne uvádza FORC vo vyobrazení vhodnom pre predstavu o tvare takejto funkcie. 

Ako vidieť, závislosť je vzhľadom na TB v celom rozsahu TA približne sigmoidálna, čo sa dá 

vystihnúť všeobecným vzťahom 

1 

xHS = f ( TA, TB) + g( TA, T 

C 

B) 

(3.1) 

⎛ ⎛ TB−a ⎞⎞ 

1 

⎜1+ exp⎜− 

⎟⎟ 

⎝ ⎝ b1 

⎠⎠ 

kde a1, b1 a c sú parametre. Závislosť však bolo možné opísať tým lepšie, čím viac 

susediacich sigmoíd bolo použitých, v takom prípade vzťah (3.1) má tvar 

n 1 n 

xHS = f ( TA, TB) ∑ + g( TA, T 

C 

B) 

(3.2) 

i= 

1 ⎛ ⎛ TB − a ⎞⎞ 

1i 

⎜1+ exp⎜− 

⎟⎟ 

⎝ ⎝ b1 

⎠⎠ 

Priebeh funkcie pri konštantnom TB sa ukázalo výhodné opisovať závislosťou gaussovského 

typu, čo viedlo k nasledujúcim výrazom 

( ) 

( ) 

2. D 

⎛ TA−a ⎞ 

2 

f TA, TB 

= exp⎜− 

⎟ 

(3.3) 

⎜ b ⎟ 

⎝ 2 ⎠ 

( ) 

( ) 

2. D 

⎛ TA−a ⎞ 

2 

g TA, TB 

= 1−exp⎜− ⎟ 

(3.4) 

⎜ b ⎟ 

⎝ 2 ⎠ 

a výsledná závislosť nadobudla tvar 

( ) 2. 

⎧ ⎫ 

⎪ ⎪ 

D 

n ⎪ 1 n 

⎪ ⎛ TA−a ⎞ 

2 

xHS 

= 1+ ⎨∑ −1 exp 

C ⎬ ⎜− ⎟ (3.5) 

⎪ i= 

1 ⎛ ⎛ T b2 

B − a ⎞⎞ 

⎜ ⎟ 

1i 

⎪ 

1+ exp − 

⎝ ⎠ 

⎪ ⎜ ⎜ ⎟⎟ 

b 

⎪ 

⎩ ⎝ ⎝ 1 ⎠⎠ 

⎭ 

s voľnými parametrami a2, b1, b2, C, Da n parametrami a 1i , pričom D je celé číslo. Použitím 

vzťahu (3.2.6) dostávame pre FORC distribúciu analytický vzťah 

2D 

−C−1 ⎛ ( TA−a2) TB −a ⎞⎛ ⎛ 1i TB −a 

⎞⎞ 

1i 

2D−1 CDexp⎜− − ⎟⎜1+ exp 

( TA−a2) n ⎜ b2 b ⎟ ⎜ ⎟⎟ 

1 ⎝ ⎝ b1 

⎠⎠ 

ρ ( TA, TB) 

= 

⎝ ⎠ 

∑ (3.6) 

i= 

1 nb1b2 kde symboly majú už uvedený význam. 

ZÁVER 

Ako vidieť na obr. 3., existuje stále kvalitatívny rozdiel medzi navrhovanou analytickou 

závislosťou (3.6) a simulačne získanou FORC. V tomto ohľade tu zostáva priestor na vývoj 

uvedených vzťahov. FORC distribúcia získaná výpočtom zo vzťahu (3.6) však nadobúda tvar 

blízky očakávanému (obr. 4.). Na tomto mieste treba upozorniť na obmedzenú univerzálnosť 

navrhovaných vzťahov, nakoľko boli vyvíjané na základe dát získaných simuláciou správania 

sa Isingovho modelu pre jednojadrové komplexy, ktorý dobre opisuje síce podstatnú, nie však 

úplnú časť pozorovaných typov závislostí [7]. 



Obr. 3. Rozdiel hodnôt navrhovanej funkcie a FORC v percentách 

Obr. 4. Experimentálna FORC distribúcia k obr. 1 (vľavo) a vypočítaná podľa vzťahu (3.6) 

(vpravo) 

LITERATÚRA 

[1] Slichter CP, Drickamer HG (1972) J Chem Phys 56: 2142 

[2] Sorai M, Seki S. (1974) J Phys Chem Solids 35: 555 

[3] Boča R, Boča M, Dlháň Ľ, Fakl K, Fuess H, Haase H, Jaroščiak R, Papánková B, Renz F, 

Vrbová M, Werner R (2000) Inorg Chem 40: 3025 

[4] Enachescu C, Tanasa R, Stancu A, Codjovi E, Linares J, Varret F (2004) Physica B 343: 

15 

[5] Cambi L, Cagnaso A (1931) Atti Accad Naz Lintei 13: 809 

[6] Kahn O (1993) Molecular Magnetism. VCH, New York 

[7] Boča R, Linert W (2003) Monats Chem 134: 199 

[8] Zimmermann R, König E (1977) J Chem Phys Solids 38: 779 

[9] Boča R (1999) Theoretical Foundations of Molecular Magnetism. Elsevier, Amsterdam 

[10] Real J, Bolvin H, Bousseksou A, Dworkin A, Kahn O, Varret F, Zarembowitch J (1992) 

J Am Chem Soc 114: 4650 

[11] Nasser JA, Boukheddaden K, Linares J (2004) Eur Phys J B 39: 219 

[12] Herchel R, Boča R, Gembický M, Kožíšek J, Renz F (2004) Inorg Chem 43: 4103 



A STUDY OF INTERFACIAL ASPECTS OF EPOXY-BASED 

HYBRID COMPOSITES REINFORCED WITH BASALT, 

CARBON AND CERAMIC FIBRES BY DMA ANALYZIS. 

Lukáš Recman, 5. ročník 

Vedoucí práce prof. RNDr. Josef Jančář, CSc. 

Konzultant ing. Petr Poláček 

Vysoké učení technické v Brně, Fakulta chemická, ústav chemie materiálů, 

Purkyňova118, 612 00 Brno, e-mail: xcrecman@fch.vutbr.cz 

ABSTRACT 

This article introduces the recent knowledge on continious fibre reinforced epoxy 

based composites. The following topics are included: (i) theoretical and experimental 

studies of continious fibers – epoxy matrix interphase (ii) preparation of dual-fiber 

hybrid composites DFHC (iii) DMA measurement of DFHC. 

INTRODUCTION 

Interfacial properties and matrix-reinforcement (communication) adhesion plays a 

vital role in composite´s aplications, properties and his behaviour under stress 

conditions. According to this expression it is usefull to ensure carefull interphase 

connection hence the expression about the weakest link. The appropriate adhesion is 

defined by various physical and chemical parameters. [1-2] But it will be unreliable 

make ourselves satisfied that strong bonds between matrix and the reinforcement 

conquer all. So let discuss this phenomon detaily. 

Matrix and reinforcement can be connected by several types of bonding like 

mechanical bonding also known as keying, electrostatic bonding, interdiffusion 

bonding and finally chemical bonding. [1] When none of this types of connection can 

be established another parameter rises – a wettability. Wettability is the ability of a 

liquid to be spread over a solid surface. But for an epoxy based composites this is the 

key-word, the stumbling block of this phenomon. The epoxy resins are extremely 

viscous and hydrofobic liquids. It is not easy to ensure the appropriate connection 

between epoxy matrix and the reinforcement. 

Matrix transmits the applied load from its surface to its volume and then to the 

reinforcement. [2] Therefor a well-calculated and right connection has to be made. This 

matrix-fibre bond transmit the applied load to the stiffer and stronger reinforcement 

where is being gathered. After reaching a critical value of absorbed energy a several 

modes of its dissipation occures: Reinforcement breakage, pull out, interphacial 

debonding and reinforcement „elongation“. The better the connection is the advanced 

material properties are. 

Matrix – reinforcement adhesion is a complicated manner, which can not be 

designed and described easily. It depends on wide physical and chemical parameters. 

But also another parameters play vital role: like homogenity, uniderectional 

orientation, avoiding fibre – fibre contact. All these parameters mentioned above 

influence composite´s parameters. 



Let answer the question from the beginning of the chapter. Is strong bond always a 

desired manner? Sometimes yes and sometimes no. First it is necessary to know that 

the bond breakage serves as an energy dissipating process. Second it all depends on the 

applied stress. When the material is being stressed with increasing force during a long 

time period it retains a greather value of the force, then stress during a short time 

interval. A strong bonding for this case is a mortal habit. So the conclusion is materials 

are designed for one purpose only. When designing material for concrete purpose then 

confining his field of application! It is called tailor-made or tailoring. 

EXPERIMENTAL 

Materials 

Basalt, Carbon and Ceramic fibres. 

The basalt fibre was made from naturally occuring basalt rock from Charberec 

location. The chemical composition of the fibre is determinated by the native basalt 

rock, which was used as a raw material. The main composition is SiO2 43%, Al2O3 

14%, CaO 10%, MgO 9%, Na2O 4%, TiO2 2%, Fe2O3/FeO 12%, K2O 1%. Material 

was melted in alumina-mullite crucialbe at 1400 °C. 

The HS Carbon Celion fibres were obtrained form Union Carbide Company. 

The Nextel 440 and Nextel 550 fibres were obtained form 3MNextelIndustrial 

fibers & 3MNextelComposite fibres. The composition and mechanical data are 

included in table 1. 

Table 1. Composition and selected properties of used fibres and resin 

Property Units Nextel Nextel HS- Basalt Atlac 430 

440 550 Carbon 

composition % Al2O3 Al2O3 - - 

SiO2 (28) 

B2O3 (2) 

SiO2 (27) 

diameter μm 10-12 10-12 - - - 

Young´s 

modulus (E) 

GPa 190 193 230 89 2 

Density (ρ) g.cm -3 3.05 3,03 1,75 2,7 1060 

Tensile 

strenght (σ) 

GPa 2 2 3,4 4,8 2 

Polymer matrix 

An epoxy resin ATLAC 430 was obtained from DSM corporate. It is based on 

unsaturated polyesters. It is a solution of linear unsaturated polyesters, containing a 

reactive doble-bond in polymerizable solution – styrene. Some useful data are 

mentioned in table 1. 



METHODS 

DMA measurement 

To gain detailed informations about DFHC properties a complex DMA 

measurement on Dynamic Mechanical Analyser 2980 (TA Instruments) was designed. 

From a wide range of DMA modes a DMA single-cantilever multifrequency mode was 

selected (DMA-SC-MF). DMA-SC-MF is an application of a constant amplitude as a 

function of time, temperature and frequency of oscillation. The set parameters: 

Frequency 10Hz, equilibrate at 40 °C, ramp 3 °C/min to 180 °C and amplitude 2μm. 

Loss modulus storage modulus and their mutual ration tanδ were obtained. 

Preparation of dual-fibre hybrid composites (DFHC) specimens. 

The dimensions of the specimen was 2mm thick, 40mm long and 10mm wide. The 

fibres were embedded in flexibilized epoxy resin in a rectangular-shaped (Lukopren) 

silicon-rubber mold. They were than heated at 65 °C for 2h, because of gasses and as a 

bubbles prevention. The specimens were than post-cured at 90 °C for 6h in a kiln. 

To obtain rigid epoxy specimens with DBP, the resin and curing agent 1wt% DBP 

was thoroughly mixed and degassed in a vacuum oven to remove air. The mixture was 

poured slowly in the mold and first continious fibres were put in, then another layer of 

resin was appplied and so the fibres until complete amount of fibres has been used. 

After taking the specimens out of the mold (when finished curing process) a standard 

metallographic techniques were used to obtain a smooth surface, when necessary. The 

two best specimens of each species and ratios were chosen and performed a 

measurement. Finaly pure Nextel 440, Nextel 440 – Basalt in three different ratios and 

pure basalt has been measured and so Nextel 550 – Carbon. See table 2. 

Table 2. Prepared composites Nextel 440 – Basalt, Nextel 550 – Carbon 

Nextel 440 Basalt Nextel 550 Carbon 

% Strand % Strand % Strand % Strand 

10 66 0 0 10 54 0 0 

8 53 2 11 8 43 2 2 

5 33 5 28 5 27 5 5 

2 13 8 44 2 11 8 8 

0 0 10 55 0 0 10 10 


DMA-SC-MF analyzis of DFHC 

To obtain the data sheet of mechanical properties a three values were gathered. The 

storage modulus, loss modulus and tanδ was gathered. For each type of manufactured 

DFHC and resin a two figures were constructed. 



Storage modulus 

(MPa) 

5000 

4500 

4000 

3500 

3000 

2500 

2000 

1500 

1000 

500 

0 

Storage modulus comparison 

40 65 90 115 140 165 

Temperature (°C) 

N440 N4B1 N1B1 N1B4 Basalt Resin 

Figure 1. DMA-SC-MF analyzis of composites Nextel 440 – Basalt 

Storage modulus (MPa) 

6000 

5000 

4000 

3000 

2000 

1000 

0 

Storage modulus comparison 

40 60 80 100 120 140 160 180 200 


C C4N1 C1N1 C1N4 N550 Resin 

Figure 2. DMA-SC-MF analyzis of composites Nextel 550 – Carbon 

Law of mixtures assessement 

Composite´s features must obey a law of mixtures (see equation 1), where E 

represents an appropriate property of composit, ν volume fraction and subscripts 1, f1, 

2 refers to each type of reinforcement (1 for Nextel 440, 550 and 2 for basalt and 

carbon respectively) and m to the matrix respectively. η represents a parametr of 

ideality of adhesion between matrix and reinforcement. Varying this parameters allowe 

us tailoring the material properties. 

Equation 1. 

E = η1νf1E1 + η2(1-νf1)E2 + νmEm 

η1 = η2 = 0,8 



The following figures represents the corelation between theoretical prediction 

represented by equation 1 and DMA-SC-MF measurement of each composite. (see 

figure 3 and 4). By varying the η1, η2 parameter the equation 1 can be fit to the DMA- 

SC-MF (45 °C) dependence. Practicaly it means changing the matrix-reinforcement 

interphase features! (see introduction for further informations and conclusion). 

E (GPa) 

20,00 

15,00 

10,00 

5,00 

0,00 

Composite Nextel 440/Basalt 

0 0,25 0,5 0,75 1 

v f1 0,75 0,5 0,25 v f2 

Equation 1 DMA-SC-MF (45°C) 

Figure 3. Equation 1 vs. DMA-SC-MF (45 °C) Nextel 440 – Basalt composite 

E (GPa) 

20,00 

15,00 

10,00 

5,00 

0,00 

Composite Nextel 550/Carbon 

0 0,25 0,5 0,75 1 

vf2 0,75 0,5 0,25 vf1 

Equation 1 DMA-SC-MF (45°C) 

Figure 4. Equation 1 vs. DMA-SC-MF (45 °C) Nextel 550 – Carbon composite 

Epoxy matrix properties. 

The DMA-SC-MF measurement was performed for pure ATLAC 430 resin to 

receive the same data sheet as for the DFHC. But another measurement was performed 

to gain resin´s curing point – DSC. This was done to get know the safe temperature 

interval for heating the resin as a bubbles prevention (see also chapter Preparation of 

dual-fibre hybrid composites DFHC specimens). 

CONCLUSIONS 

DMA-SC-MF analyzis of Nextel 440 – Basalt composites indicates a 

correlation between increasing basalt content and decreasing Nextel 440 fibres content. 

The prediction of storage modulus was not fulfilled. It should be truly opposite. 



DMA-SC-MF of Nextel 550 – Carbon displays a correlation between increase 

in Nextel 550 content and dicrease of carbon fibres. The theoretical prediction prevails 

the best mechanical data for carbon reinforced composites. 

The explanation of non-corresponding data can be found in the following 

sentences. First inhomogenity. It is no easy to ensure an appropriate playcement of 

fibres hence they are well sized and keep their manufactured strand well. Also the 

mutual distribution between ceramic basalt or ceramic-carbon fibres wasn´t ideal and it 

is one of important assignment that has to be solved in industry too. And third reason 

lies with the wettability basalt fibres interact with resin better than the other ones so the 

connection between matrix and reinforcement occurs frequently than for carbon or 

ceramic fibres where occurs rarely. The non-connected fibres does not sustain any 

stress or energy. 

The corresponding data between theoretical prediction and practical realization 

(figure 3,4) lies with more careful preparation of composites and as well as with 

increasing the wettability by varying η. This could be done by a sizing agent, which 

permanently binds matrix and the reinforcement, or increasing the time period of 

contact between reinforcement and resin befor curing it. 

REFERENCES 

[1] Composite materials: Engineering and science /F. L. Matthews and R. D. 

Rawlings, 1995. p. 1-72, 168-219,251-282. 

[2] Introduciton to physical polymer science L. H. Sperling, 2001. 604-612. 



ŠTÚDIUM ANTIOXIDANTOV A ICH ÚČINKOV PRI STABILIZÁCII 

POLYMÉROV 

Bc. Ján Rimarčík, 5. ročník 

Vedúci práce: Ing. Erik Klein, PhD 

Ústav fyzikálnej chémie a chemickej fyziky, Fakulta chemickej a potravinárskej technológie, 

Slovenská technická univezita v Bratislave, Radlinského 9, SK-812 37 Bratislava, 

Slovenská republika, e-mail: hadica@gmail.com 

ÚVOD 

Problematika spojená s negatívnym vplyvom prostredia na životnosť polymérov sa 

študuje od začiatku ich komerčného využívania. Životnosť polymérov determinuje oxidácia 

a mechanická degradácia. Zlúčeniny redukujúce alebo inhibujúce termickú oxidáciu sú 

všeobecne nazývané antioxidantmi. Ako špeciálny typ aditív sa pridávajú do polymérov 

individuálne alebo ako súčasť kompozitného stabilizačného systému. Cieľom tejto práce bolo 

overiť vhodnosť semiempirických kvantovochemických metód AM1 a PM3 na výpočty 

disociačných entalpií väzieb N–H, O–H, S–H v molekulách substituovaných anilínov, fenolov 

a tiofenolov. Tie slúžia ako modelové štruktúry antioxidantov. Predpoveď vlastností 

potenciálnych antioxidantov, ktoré ešte neboli syntetizované, na základe kvantovochemických 

výpočtov dokáže ušetriť ekonomické i ľudské zdroje nevyhnutné na syntézu a testovanie 

týchto látok. Preto je dôležité zistiť, ako spoľahlivo jednotlivé výpočtové metódy reprodukujú 

dostupné experimentálne výsledky. 

STABILIZÁCIA – ANTIOXIDANTY 

Radikálový mechanizmus navrhnutý Bollandom a Geeom vysvetlil termickú oxidáciu 

mnohých polymérov [1]. Rýchlosť oxidácie závisí od najpomalšieho kroku – tvorby voľných 

radikálov. Oxidačné reakcie sa vo všeobecnosti uskutočňujú rôznymi spôsobmi. Sú zrýchlené 

tepelnou energiou a mechanickým namáhaním pri spracovaní. Často sú katalyzované 

prítomnými zvyškami katalyzátora polymerizácie a nečistotami. Využívajú sa 3 hlavné typy 

stabilizácie [1, 2]: predstabilizácia, stabilizácia počas procesu spracovania polyméru, 

dlhotrvajúca stabilizácia. Antioxidanty môžu ovplyvniť každý krok termickej oxidácie, čo 

záleží od ich zloženia a štruktúry. Pôsobenie stabilizátorov – antioxidantov (InH) počas 

radikálovej oxidácie možno opísať rovnicami (P – polymér) [1, 2]: 

• 

PO 2 + InH ⎯→ ⎯ POOH + In • (1) 

PO • + InH ⎯→ ⎯ POH + In • (2) 

Radikál na molekule antioxidantu je rezonančne stabilizovaný, čo v konečnom dôsledku 

znamená, že tieto radikály prakticky nie sú schopné uskutočniť prenosovú reakciu 

na polymér. Preto radikál obyčajne zaniká v terminačnej reakcii s ďalším oxy- alebo 

peroxylovým radikálom. Účinnosť antioxidantov sa zvyšuje so znižovaním disociačnej 

entalpie (BDE) väzby O–H vo fenolových, resp. N–H amínových antioxidantoch, keďže prvý 

krok terminácie reaktívnych radikálov vznikajúcich počas oxidácie polymérov predstavuje 

transfer vodíkového atómu z molekuly antioxidantu na reaktívny radikálový intermediát 

[1, 2]. Ďalšou dôležitou charakteristikou antioxidantov je ionizačný potenciál (IP). Nízke 

hodnoty IP prispievajú k zvýšeniu pravdepodobnosti prenosu elektrónu, čím sa zvyšuje riziko 

vzniku superoxidového anión-radikálu prenosom elektrónu priamo na molekulu O2 [1]. 



ŠTUDOVANÉ MOLEKULY 

Vhodnosť semiempirických kvantovochemických metód AM1 a PM3 na výpočty 

disociačných entalpií väzieb (BDE) N–H, O–H, S–H budeme testovať na molekulách 

substituovaných anilínov, fenolov a tiofenolov (obr. 1) slúžiacich ako modelové štruktúry 

v praxi používaných antioxidantov. Tieto zlúčeniny boli vybrané nielen preto, že existujú 

pre ne experimentálne dáta, ale umožňujú nám aj preštudovať efekt substituentov v meta 

a para polohách na veľkosť BDE. 

X 

X 

NH 2 

NH 2 

X 

X 

SH 

SH 

X N H COMe X N H NH 2 

Obr. 1: Študované molekuly, X = NO2, CF3, CN, Cl, Br, MeCO, MeSO2, PhCO, Me, MeO, 

OH, MeO2C, CHO, NMe2, NH2. 


Tabuľka 1 uvádza spracované výsledky pre tiofenoly. Hodnota ΔexpBDE je roziel medzi 

experimentom a vypočítanou hodnotou pre BDE. Ostatné série zlúčenín boli spracované 

analogicky. 

Tabuľka 1. Tiofenoly 

BDE [kJ/mol] ΔexpBDE [kJ/mol] IP [eV] 

názov exp PM3 AM1 PM3 AM1 PM3 AM1 σm, p 

p–NO2PhSH 341 394 381 –53 –40 9,53 9,20 0,78 

m–CF3PhSH 338 381 367 –43 –29 9,19 8,85 0,43 

p–BrPhSH 332 381 369 –49 –37 8,94 8,61 0,23 

p–ClPhSH 331 381 366 –50 –35 8,78 8,54 0,23 

m–MePhSH 330 378 363 –48 –33 8,75 8,40 –0,07 

p–MeO2CPhSH 329 385 375 –56 –46 9,06 8,79 0,45 

p–MePhSH 328 377 362 –49 –34 8,66 8,32 –0,17 

p–MeOPhSH 322 372 355 –50 –33 8,52 8,18 –0,27 

p–NH2PhSH 292 372 348 –80 –56 8,29 7,93 –0,66 

m–ClPhSH 335 380 366 –45 –31 8,92 8,63 0,37 

PhSH 331 377 363 –46 –32 8,78 8,43 

Hodnoty disociačných entalpií väzieb boli vypočítané pomocou programu HYPERCHEM [3]. 

Experimentálne hodnoty týchto entalpií boli získané metódou termodynamického cyklu. 

Autori uvádzajú smerodajnú odchýlku určených hodnôt ±8,5 kJ/mol [4]. 



X 

X 

OH 

OH

Na základe experimentálnych údajov pre anilíny sme zistili, že PM3 metóda podhodnocuje 

BDE v priemere o 10 kJ/mol. Naopak metóda AM1 entalpie nadhodnocuje v priemere 

o 11 kJ/mol. V prípade fenolov obe metódy poskytujú nižšie hodnoty BDE než experiment 

pre väčšinu študovaných substituentov. Výnimkou sú meta –NH2, para –OH, para –NH2 

pre PM3 a v prípade AM1 aj para –MeO substituent. Priemerná odchýlka vypočítaných BDE 

od experimentu je 16 kJ/mol pre PM3 a 9 kJ/mol pre AM1 metódu. Pri substituovaných 

tiofenoloch obe metódy významne nadhodnocujú experimentálne entalpie – metóda PM3 

v rozmedzí 43–80 kJ/mol, AM1 29–56 kJ/mol. Priemerné odchýlky od experimentálne 

určených hodnôt BDE sú 52 a 37 kJ/mol pre PM3 a AM1. Príčinou nepresných výsledkov je 

zrejme atóm síry s vyšším počtom elektrónov oproti atómom H, N, alebo O a problémy 

súvisiace s výpočtom radikálu, keďže vypočítaná tvorná entalpia nesubstituovaného tiofenolu 

(115 kJ/mol – PM3, resp. 107 kJ/mol – AM1) dobre súhlasí s experimentálnou hodnotou 

112 kJ/mol [5]. 

ZÁVISLOSŤ ΔBDE A ΔIP OD HAMMETTOVÝCH KONŠTÁNT 

L. P. Hammett zistil, že pre meta a para substituované deriváty existuje lineárna závislosť 

medzi disociačnou konštantou a rýchlosťou hydrolýzy esterov substituovaných benzoových 

kyselín [6]. Podobný vzťah platí aj pre rovnovážne konštanty a rýchlostné konštanty. Pratt, 

DiLabio a kol. zistili, že ΔBDE (kde ΔBDE = BDEsubstituovaná – BDEzákladná) hodnoty para 

substituovaných anilínov a fenolov [7] lineárne závisia od Hammettových konštánt. Všetky 

závislosti ΔBDE od σm, σp sú približne lineárne (obr. 2). Najlepšia lineárna korelácia sa 

získala pre fenoly a tiofenoly, kde korelačné koeficienty dosiahli hodnoty v rozmedzí 0,916 až 

0,954. V prípade substituovaných anilínov sú jednotlivé body rozptýlené okolo regresnej 

priamky viac (R=0,813 pre PM3, R=0,873 pre AM1). V prípade substituovaných anilínov 

a tiofenolov lepšie korelujú s Hammettovými konštantami výsledky AM1 metódy. Pre fenoly 

sú obe metódy rovnocenné – korelačné koeficienty sú zhodné. Podobne sme študovali aj série 

para substituovaných acetanilínov a fenylhydrazínov. Aj tu sa potvrdila lineárna závislosť 

ΔBDE od Hammettových konštánt. Parasubstituované acetanilíny korelujú lepšie ako 

fenylhydrazíny s korelačnými koeficientami podobnými ako pri substituovaných fenoloch 

a tiofenoloch. Okrem hodnôt ΔBDE sme korelovali s Hammettovými konštantami aj 

vypočítané hodnoty ΔIP. Ionizačné potenciály IP sa rovnajú zápornej hodnote energie 

najvyššieho obsadeného orbitálu (HOMO). Hodnoty ΔIP boli určené ako rozdiel IP 

substituovanej a nesubstituovanej zlúčeniny. Zistili sme, že tiež lineárne závisia od 

Hammettových konštánt. Hodnoty ΔIP korelujú s Hamettovými konštantami lepšie ako 

hodnoty ΔBDE (obr. 3). Potenciálnu nepresnosť oboch semiempirických metód možno 

pripísať problémom s výpočtom radikálov. Optimálna geometria nemusí zodpovedať 

geometrii s minimálnou energiou kvôli metóde polovičného elektrónu, ktorú používajú mnohé 

programy [8]. 



ΔBDE/kJ mol -1 

20 

15 

10 

5 

0 

-5 

-10 

-15 

-20 

-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 

σp, σm 

Obr. 2: Závislosť ΔBDE substituovaných 

tiofenolov od Hammettových konštánt σm, 

σp pre AM1 metódu. 

-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 

ZÁVER 

Táto práca bola zameraná na posúdenie vhodnosti semiempirických kvantovochemických 

metód na predikciu vlastností hlavných skupín antioxidantov. Vypočítané hodnoty 

disociačných entalpií väzieb N–H, O–H a S–H sa porovnali s publikovanými 

experimentálnymi hodnotami. Odchýlky vypočítaných hodnôt BDE N–H (okrem p –CF3 

v prípade PM3) a O–H väzieb od experimentálnych sú blízke smerodajnej odchýlke BDE 

z experimentov. Výpočty BDE S–H väzby v tiofenoloch ukázali, že obe metódy významne 

nadhodnocujú experimentálne hodnoty, hoci trendy vyplývajúce z efektu substituentov 

na BDE vystihujú uspokojivo. Hodnoty ΔBDE pre všetky série zlúčenín lineárne korelujú 

s Hammettovými konštantami σp, σm. Rozdiel medzi IP substituovaných zlúčenín a základnej 

tiež lineárne závisí od Hammettových konštánt. 

Možno teda konštatovať, že PM3 a AM1 semiempirické kvantovochemické metódy 

predstavujú vhodnú alternatívu k DFT (Density Functional Theorem) a ab-initio metódam, 

vhodnú predovšetkým pre výpočty molekúl amínových a fenolových antioxidantov s vyšším 

počtom atómov. Obe metódy poskytujú výsledky v signifikantne kratšom čase (trvanie 

výpočtu niekoľko minút na bežnom PC) ako DFT alebo ab-initio metódy. 

LITERATÚRA 

[1] Gugumus F.: Oxidation Inhibition in Organic Materials, Vol I, CRC Press, Boca Raton 

1990. 

[2] Wolf R., Kaul B. L., Plastics Additives, Ullman’s Encyclopedia of Industrial 

Chemistry, Vol. A20, VCH, Weinheim 1992. 

[3] HYPERCHEM, rel 7.5 for Windows, Hypercube, Inc., 2003. 

[4] Zhu Q., Zhang X. M., Fry A. J.: Polym. Degrad. Stab. 57, 43 (1997). 

[5] Scott D. W., McCullough J. P., Hubbard W. N., Messerly J. F., Hossenlopp I. A., 

Frow F. R., Waddington G.: J. Am. Chem Soc. 78, 5463 (1956). 

[6] Hrnčiar P.: Organická chémia, SPN, Bratislava 1990, str. 110. 

[7] Pratt D. A., DiLabio G. A., Mulder P., Ingold K. U.: Acc. Chem. Res. 37, 334 (2004). 

ΔIP/eV 

0.8 

0.6 

0.4 

0.2 

0.0 

-0.2 

-0.4 

-0.6 

σp , σm 

Obr. 3: Závislosť ΔIP substituovaných 

tiofenolov od Hammettových konštánt σm, σp 

pre AM1 metódu. 



[8] Stewart J. J. P.: J. Comp-Aided Mol. Design 4, 1 (1990). 



HOŘLAVOST A MECHANICKÉ VLASNOTSTI NANOKOMPOZITŮ 

EVA/Mg(OH)2 

Sadílek Jiří, 5. ročník 

Vedoucí práce: prof. RNDr. Josef Jančář, CSc. 

Vysoké učení technické v Brně, Fakulta chemická, Chemie, technologie a vlastnosti 

materiálů, Purkyňova 118, 612 00 Brno, e-mail: xcsadilek@fch.vutbr.cz 

ÚVOD 

Práce se zabývá studiem vlivu velikosti částic hydroxidu hořečnatého (MH) na tepelnou 

stabilitu a mechanické vlastnosti poly(ethylen-co-vinyl acetátu), EVAc. Hlavní použití EVAc 

je v oblasti kabelové a elektroizolační techniky. Plniva na bázi hydroxidu hořečnatého 

představují způsob, jak snížit hořlavost a zároveň modifikovat mechanické vlastnosti EVAc 

kopolymeru. 

CÍL PRÁCE 

• příprava nanočástic Mg(OH)2 

• příprava kompozitů 

• stanovení tepelné stability, hořlavosti a mechanických vlastností 

• interpretace dat 


Proces hoření je souborem fyzikálních a chemických procesů, které působí ve třech 

rozdílných fázích: plynná (plamen), mezifáze a kondenzovaná (pevná) fáze. Mezifáze je 

fázovým rozhraním mezi plynnou a kondenzovanou fází. Plynnou fázi tvoří plynné produkty 

degradace polymeru spolu se vzduchem [2]. Kondenzovanou fází je degradující polymer, 

případně polymerní kompozit. Vedením tepla z plamene dochází k roztavení pevné fáze, 

degradaci kondenzované fáze a k vytvoření hořlavých plynných produktů. 

Tepelná degradace EVAc probíhá ve dvou krocích. První krok (300 – 400°C) zahrnuje 

odštěpení kyseliny octové a vznik ethylenové struktury na původním řetězci kopolymeru. V 

rozmezí teplot 425 – 470 °C nastává rozklad polyethylenového zbytku. 

Retardéry hoření lze rozdělit podle mechanismu, kterým působí, na fyzikální inhibitory a 

chemické inhibitory. Mg(OH)2 patří mezi fyzikální inhibitory hoření. Fyzikální inhibice 

v plynné fázi je založena na působení inertních plynů (H2O), které zřeďují hořlavé plyny a 

snižují jejich koncentraci v zóně hoření. Uvolnění H2O je spojeno s výrazným endotermickým 

účinkem, který je schopen pohlcovat teplo vznikající v procesu hoření. 

Hydroxid hořečnatý se rozkládá na oxid hořečnatý a vodu. Endotermický rozklad 

(1450 J/g) začíná při teplotě 300°C a probíhá podle uvedené rovnice: 

Mg(OH) 2 

2 

→ MgO + H O 

(1) 

Na základě publikovaných výsledků [4] lze usuzovat, že v kompozitech s nanoplnivy 

dochází k výrazné imobilizaci polymerních řetězců v důsledku velkého specifického povrchu 

plniva. Tato imobilizace má přímý dopad na mechanické vlastnosti nanokompozitů. 

V případě tepelné stability a hořlavosti se ovšem mohou imobilizačním efektům konkurovat 

katalytické účinky velkého povrchu nanoplniva. 



PRAKTICKÁ ČÁST 

Hydroxid hořečnatý byl připraven precipitací síranu hořečnatého a vodného roztoku 

amoniaku: 

MgSO 4 + 2NH 

4OH 

→ Mg(OH) 2 + ( NH 4 ) 2SO 

4 

Kompozitní vzorky byly připraveny míšením kopolymeru EVAc a plniva v roztoku 

cykolhexanu. Byly připraveny vzorky s obsahem nanometrického MH připraveného 

laboratorně a s obsahem komerčního mikrometrického hydroxidu hořečnatého - FR–20 

(DEAD SEA PERICLASE Ltd., Israel). Obsah plniva v kompozitu byl 13obj.% (26hmot.%). 

Snímky plniv z transmisního elektronového mikroskopu jsou uvedeny v obr. 1. Snímky 

lomových ploch kompozitů připravených v tekutém dusíku jsou na obr. 2. 

Obr. 1: TEM použitých MH plniv, nano-MH (vlevo) a mikro-MH FR-20 (vpravo). 

Použitá plniva byla charakterizována pomocí následujících metod: (1) BET, (2) rozptyl 

světla, (3) transmisní elektronová mikroskopie (TEM), (4) pyknometrické stanovení hustoty, 

(5) rentgenová difrakce, (5) termogravimetrická analýza (TGA). Základní charakteristiky 

použitých plniv jsou v tab. 1. 

Tabulka 1: Charakterizace všech použitých plniv. 

Metody stanovení Nanoplnivo FR – 20 

TEM (nm) 32 – 34 800 – 900 

Spec. povrch (m 2 /g) změřený 43 – 48 5 – 6 

Spec. povrch (m 2 /g) počítaný 44 – 47 1 – 2 

Teplota rozkladu (°C) 336 – 433 310 - 430 


Sekce STČ 2006, strana 60 

(2)

Pro charakterizaci vlastností kopozitních vzorků byly použity následující metody: (1) 

termogravimetrická analýza, (2) stanovení kyslíkového čísla, (3) dynamicko-mechanická 

analýza, (4) tahové testy. 

Obr. 2: SEM snímky lomových ploch kompozitů s nano-MH (vpravo) a s mikro-MH FR-20 

(vlevo) . 

VÝSLEDKY A DISKUSE 

Z průběhu křivek izotermální degradace při 430°C, obr. 3 vlevo, je zřejmé, že vzorky 

EVAc plněné nanometrickým hydroxidem hořečnatým vykazovaly rychlejší pokles hmotnosti 

v porovnání se vzorky EVAc plněné mikrometrickým MH. 

Limitní kyslíkové číslo, kyslíkový index je stanovení relativní hořlavosti plastů podle ČSN 

EN ISO 4589-3. LOI je test zpětného hoření tenkých vzorků polymerních proužků v proudu 

plynu a vyjadřuje nejnižší koncentraci kyslíku ve směsi s dusíkem (v %), která ještě stačí na 

to, aby materiál při podmínkách zkoušky hořel. Nízká hodnota LOI znamená, že materiál hoří 

i při malém podílu kyslíku ve směsi. 

Obr. 3: TGA skeny čisté matrice a obou kompozitů při konstantní teplotě 430°C a LOI test. 

Uvedené výsledky naznačují, že velký specifický povrch nanometrického MH vede k 

výraznému katalytickému působení a snížení retardační schopnosti MH. 



Výsledky tahových zkoušek jsou uvedeny na obr. 4. Z výsledků je patrné, že došlo 

k výraznému zvýšení modulu pružnosti (směrnice tahové křivky při hodnotě deformace 0,15 

%) u nanokompozitního vzorku ve srovnání s čistou matricí a EVAc plněného 

mikrometrickým MH. Na druhé straně, nedošlo k výrazné změně houževnatosti EVAc 

kopolymeru s přídavkem MH plniv. 

Obr. 4: Mechanická tahová zkouška. 

ZÁVĚR 

Na základě termogravimetrické analýzy a stanovení limitního kyslíkového čísla bylo 

zjištěno, že v případě velký specifický povrch nanometrického hydroxidu hořečnatého může 

působit katalyticky na procesy termooxidační degradace. Tento jev je schopen efektivně 

snižovat endotermický efekt hydroxidu hořečnatého daný rozkladem na vodu a MgO. 

Z mechanických zkoušek vyplynulo, že použití nanometrického MH jako plniva vede 

k výraznému zvýšení modulu pružnosti ve srovnání s mikrometrickým hydroxidem 

hořečnatým. Tento jev je pravděpodobně dán výraznou imobilizací polymerních řetězců 

v přítomnosti velkého specifického povrchu plniva. 

LITERATURA 

[1] Rychlý, J., Veselý, K., Gál, E., Kummer, M., Jančář, J., Rychlá, L.: Use of Thermal 

Methods in the Chracterization of the High-Temperature decomposition and Ignition of 

Polyolefins and EVA Copolymers Filled with Mg(OH)2, Al(OH)3 and CaCO3. Polymer 

Degradation and Stability, 1990, 30, 57 – 72 

[2] Troitzsch, J.: Plastics Flammability Handbook: Principles, Regulations, Testing, and 

Approval. 3. vydání. Mnichov: Hanser, 2004. 748 s. ISBN: 1-56990-356-5 

[3] Halikia, I., Neou-Syngouna, P., Kolitsa, D.: Isothermal kinetic analysis of the thermal 

decomposition of magnesium hydroxide using thermogravimetric data determination of 

polymerization shrinkage kinetice in visible-light-cured materials: methods developement. 

Thermochimica Acta, 1998, 320, 75 – 88. 



[4] Kalfus, J.: Viscoelastic properties of polyvinylacetate-hydroxyapatite nanocomposites. 

Brno : Vysoké učení technické, 2005. 106 s. 



STUDIUM MECHANISMU KOORDINAČNÍ POLYMERACE HEXA- 

1,5-DIENU KATALYZOVANÉ FENOXYIMINOVÝM KOMPLEXEM 

TITANU A METHYLALUMINOXANEM 

Jan Ševčík, 4. ročník 

Vedoucí práce: Mgr. Soňa Hermanová, Ph.D. 

Vysoké učení technické v Brně, Fakulta chemická, ústav chemie a technologie materiálů, 

Purkyňova 118, 612 00 Brno, email: xcsevcikj@fch.vutbr.cz 

ÚVOD 

Koordinační polymerace dienů přitahují pozornost především z důvodu vzniku strukturně 

rozmanitých typů monomerních jednotek v polymerních řetězcích. Izolované dieny mohou 

podstoupit cyklopolymeraci, ve které je inzerce dvojné vazby monomeru do vazby Mt–C 

následována intramolekulární inzercí zbývající dvojné vazby za vzniku methylen-1,3cyklopentanové 

jednotky (MCP). 1 Doi a kol. polymerovali hexa-1,5-dien v toluenu pomocí 

V(acac)3 a Al(C2H5)2Cl při -78 °C za vzniku homopolymeru obsahujícího 46 mol % 1vinyltetramethylenových 

(VTM) jednotek a 54 mol % 1,3-cyklopentylenmethylenových 

(MCP) jednotek. 2 

Cílem této práce je studium mechanismu polymerace nekonjugovaného dienu 

katalyzované fenoxyiminovým komplexem titanu (FITi) a methylaluminoxanem (MAO) jako 

kokatalyzátorem. Uvažované způsoby zabudování molekul hexa-1,5-dienu do rostoucího 

řetězce jsou znázorněny na obr. 1. Adice dienu může probíhat buď 1,2-(primární) nebo 2,1- 

(sekundární) inzercí. Po 1,2-inzerci může následovat inzerce zbývající dvojné vazby v poloze 

1,2, čímž dojde k vytvoření cyklopentanového kruhu. Po inzerci dvojné vazby dienu v poloze 

2,1 může také následovat 1,2 inzerce zbývající dvojné vazby pří níž vzniká 

cyklobutylmethylový meziprodukt, který se β-alkyleliminací přemění na jednotku s boční 

vinylovou skupinou. 

monomer 

FITi 

Polymeračně aktivní centrum 

FITi 

P 

(1,2) 

MCP 

P 

P 

FITi 

+ 

(2,1) 

hexa-1,5-dien 

FITi 

FITi 

cyklobutan 

VTM 

P 

β-alkyl elim. 



P

Obr. 1 Navržené mechanismy inzerce hexa-1,5-dienu 


Všechny práce s látkami citlivými na vzduch a vlhkost byly prováděny pod inertní 

atmosférou, za použití standardních Schlenkových technik a vakuové linky pracující v režimu 

vakuum/inert. Dusík (99,999%, Siad) byl dočištěn pomocí sušících věží naplněných 

molekulovými síty (4 Å) a měďným katalyzátorem. Toluen (p.a., Lachema) byl před použitím 

sušen pomocí směsi sodík/benzofenon. Hexa-1,5-dien (99 %, Fluka) byl sušen na CaH2 

a čerstvě destilován. Kokatalyzátor methylaluminoxan (10 % wt. v toluenu, Crompton 

GmbH) byl před použitím zbaven volného trimethylaluminia (TMA) a znova rozpuštěn 

v toluenu. Bis[N-(3-terc-butylsalicylidene)-2,3,4,5,6-pentafluoroanilinato]titanium (IV) 

dichlorid byl syntetizován dle publikovaného postupu. 3 

Polymerace 

Polymerace byly prováděny v opláštěném reaktoru (250 ml) s magnetickým míchadlem. 

Jednotlivé komponenty byly přidávány v následujícím pořadí: toluen (50 ml), kokatalyzátor 

(6 mmol) a monomer (2 mmol). Reaktor byl temperován na příslušnou teplotu po dobu 20 

minut a poté byl dávkován roztok katalyzátoru (10 μmol) pro iniciaci polymerace. 

Polymerace byla ukončena za 60 min terminací ethanolem (10 ml), polymer byl vysrážen 

v roztoku methanolu okyseleném HCl, izolován filtrací a sušen za vakua do konstantní 

hmotnosti. 

Analýza polymeru 

GPC analýza byla provedena na SEC zařízení použitím Polymer Laboratories PL gel 

10 μm MIXED-8, 300×7,5 mm koloně, RI detektor RUBY 05. Analýza byla provedena při 

25 °C za použití THF jako rozpouštědla. Pro měření 1 H NMR spektra poly(hexa-1,5-dienu) 

byl připraven roztok rozpuštěním 20 mg polymeru v 0,5 cm 3 1,1,2,2,-tetrachlorethanu-d2 při 

120 °C, který byl následně při této teplotě homogenizován po dobu 8 hodin. Měření NMR 

spekter katalyzátorů bylo provedeno na přístroji Bruker Avance o pracovní frekvenci 

300 MHz. 

VÝSLEDKY A DISKUZE 

Polymerace hexa-1,5-dienu byla provedena v toluenu (50 ml) při teplotě 0 °C za katalýzy 

bis[N-(3-terc-butylsalicyliden)-2,3,4,5,6- pentafluoranilinato]titan (IV) dichloridem a MAO 

jako kokatalyzátorem. Vzniklý polymer (Mn = 41 kg·mol -1 , Mw/Mn = 1.5) byl po celou dobu 

polymerace rozpustný v polymeračním médiu, což indikuje absenci zesítěných struktur. 

K zesítění makromolekul by přispívaly nezreagované dvojné vazby monomerních jednotek ve 

formě butenylových postranních větví. Během polymerace bylo dosaženo vysokého stupně 

konverze (96 %). Počet vzniklých polymerních řetězců však neodpovídal počtu potenciálních 

aktivních center. Pouze 39 % aktivních center přispělo k propagaci řetězce, možným 

vysvětlením je nedostatečný přebytek kokatalyzátoru, jehož esenciální funkcí je aktivace 

katalytického prekurzoru a ochrana aktivního centra. Šířka distribuce molárních hmotností 

(Mw/Mn = 1.5) naznačuje, že v systému se uplatňují nežádoucí boční reakce (přenos). 

Mikrostruktura řetězce byla analyzována pomocí 1 H NMR spektroskopie. Procentuální 

zastoupení VTM jednotek ve vzorku polymeru bylo kvantifikováno na 36,5 % (Obr.2). 

Teplota skelného přechodu polymeru (Tg = -15,3 °C) byla stanovena pomocí DSC. 



Obr. 2 1 H NMR poly(hexa-1,5-dienu), zastoupení jednotek VTM/MCP = 36,5 % / 63,5 % 

ZÁVĚR 

Prvotní studie prokázala, že systém FITi/MAO je katalyticky aktivní pro polymeraci hexa- 

1,5-dienu. Větší podíl MCP jednotek v připraveném poly(hexa-1,5-dienu) při 0 °C naznačuje 

upřednostnění adice molekul monomeru na aktivní centrum mechanismem primární 1,2inzerce. 

Další výzkum bude zaměřen na studium mechanismu a kinetiky propagace hexa-1,5dienu 

za různých polymeračních podmínek (T, vliv koncentrace monomeru, vliv poměru 

katalyzátor/kokatalyzátor). 

LITERATURA 

1. Marvel C. S., Stille J. K.: J Am Chem Soc 80, 1740 (1957). 

2. Doi Y., Tokuhiro N., Soga K.: Makromol. Chem. 190, 643 (1989) 

3. Fujita T., Mitani M.: J. Am. Chem. Soc. 124, 3327 (2002) 



MOLEKULOVÁ DYNAMIKA C-GLYKOZYLNITROMETÁNOV 

Stanislava Šoralová, 5. ročník 

Vedúci práce: Mgr. Juraj Kóňa, PhD.* 

Ústav fyzikálnej chémie a chemickej fyziky, FCHPT STU, Radlinského 9, SK-812 37 Bratislava, 

Slovenská republika, e-mail: stanislava.soralova@gmail.com 

*Chemický ústav SAV, Dúbravská cesta 9, 845 38 Bratislava, SR 

ÚVOD 

Rezistencia patogénov na existujúce lieky sa stáva čoraz väčším problémom nie len pre 

medicínu. To zapríčinilo rozsiahly výskum nových inhibítorov. Pretože výskyt D-rabinofuranózy 

a D-galaktofuranózy je v prírode špecifický pre mykobaktérie, protozoa a huby, môžu zlúčeniny 

im podobné prekážať enzymaticky katalyzovaným cestám ich spracovania a vytvárať tak 

kompetetívnu inhibíciu. Preto sa stále viac stáva žiadanou syntéza C-glykofuranózových 

štruktúr. 

Monosacharidy v roztoku podliehajú vnútromolekulovej reakcii medzi karbonylovou 

skupinou a jednou z hydroxylových skupín. To dáva rôzne možnosti cyklických štruktúr. Ak 

daný monosacharid môže poskytovať päť i šesťčlánkové kruhy, sú v rovnovážnom roztoku 

zastúpené predovšetkým šesťčlánkové kruhy, relatívne zastúpenie päťčlánkových kruhov je 

veľmi nízke, pretože šesťčlánkové kruhy sú oproti päťčlánkovým termodynamicky výhodnejšie 

[2]. 

Napriek tomu bola ako pracovná hypotéza prijatá predstava, podľa ktorej ak má molekula 

schopnosť tvoriť oba kruhy, tvorba päťčlánkového cyklu prebieha s väčšou rýchlosťou ako 

šesťčlánkového, je kineticky preferovaná. Zrejmým dôvodom je vzdialenosť reakčných 

partnerov cyklizačnej reakcie, ktorá je pri tvorbe päťčlánkového kruhu asi o 20% kratšia a z toho 

vyplývajúca vyššia vnútromolekulová koncentrácia ako pri šesťčlánkovom cykle. 

Pravdepodobnosť cyklizácie klesá s rastúcou dĺžkou reťazca medzi reakčnými partnermi. Táto 

hypotéza sa samozrejme nedá aplikovať na menšie kruhy, pretože nárastajúce napätie väzbových 

uhlov vo vznikajúcom kruhu by výrazne zvýšilo aktivačnú energiu. [3] 

Pri cyklizácii 1,2-dideoxy-1-nitroalk-1-enitolu nie je možné zachytiť primárne vznikajúci 

kineticky preferovaný produkt päťčlánkový kruh C-glykozylnitrometánov, pretože sa v dôsledku 

rýchlej vratnej konverzie mení na termodynamicky výhodnejší šesťčlánkový kruh. Z tohoto 

dôvodu boli rozpracované metódy nerovnovážnej prípravy C-glykozylnitrometánov, ktoré môžu 

poskytovať netermodynamické C-glykofuranozyl-nitrometány. [3] 

Táto práca naväzuje na výskum prípravy anomérov C-L-arabinofuranozylnitro-metánov 

z dostupného 3,4,5,6-tetra-O-acetyl-1,2-dideoxy-1-nitro-L-arabino-1-enitolu 1. V prvom kroku 

O-deacetylácia látky 1 v kyslom metanole poskytuje nitroalkén 2, jeho cyklizácia prebieha ako 

1,4-adícia poskytujúca C-(L-arabinofuranozyl)-metylnitrónové kyseliny 5 a 6, ktoré po následnej 

protonizácii podliehajú novej modifikácii Nefovej reakcie. Táto reakcia zastaví ďalšie zmeny 

v cykle, čo umožňuje izolovať kineticky preferované päťčlánkové cykly. Výsledkom sú 

dimetylacetálovo chránené C-(L-arabinofuranozyl)metanaly 7, 8 a zmiešaný vnútro-metylový 

acetál 9. [3, 4] 



AcO 

AcO 

Obr. 1 

1 

NO 2 

OAc 

OAc 

MeOH 

+ 

H 

MeOH 

H+ 

HO 

O 

H 

2b 

N 

OH 

OH 

HO 

O 

H 

HO OMe 

O 

OMe 

HO OH 

7 

O 

O 

2a 

NO 2 

OH 

OH 

+ 

HO NO H 2 

O 

HO OH 

HO OMe 

O 

OMe 

HO OH 

HO NO2 O 

+ 

+ 

HO OH 

3 

HO NO2H O 

HO OH 

5 6 

H + 

MeOH 

O OMe 

O 

HO OH 

8 9 

+ 

HO NO2 O 

HO OH 

METÓDA 

Pri voľbe teoretického prístupu bol použitý predpoklad, že aktivačné energie vzniku 

anomérov päťčlánkových a šesťčlánkových kruhov sú približne rovnaké a tak nemajú vplyv 

na experimentálne získané množstvá produktov. Takisto sa predpokladá, že kineticky 

preferované produkty budú mať za rovnaký časový interval menšiu priemernú vzdialenosť 

reakčných partnerov, a zároveň bude preferovaná hydroxylová skupina častejšie pod správnym 

uhlom k väzbe C=C. U preferovaného anoméru sa bude hydroxylová skupina častejšie 

približovať k elektrofilu zo strany typickej pre tento anomér, prípadne bude mať kratšiu 

priemernú vzdialenosť reakčných partnerov ako pri približobaní z opačnej strany. 

Vďaka týmto predpokladom bolo možné pre teoretickú analýzu použiť relatívne jednoduché 

a rýchle výpočty molekulovej dynamiky pre rôzne C-glykozylnitrometány v rôznych formách 

(1,2-dideoxy-1-nitroalk-1-enitolu s protonizovanou nitro skupinou, per-acetylovaný a pod.). 

Vzhľadom na nepoužiteľnosť parametrov programu Amber 8 SANDER v tomto prípade, 

bolo potrebné ich vyrobiť [1]. Geometrie molekúl boli zoptimalizované v programe 

HYPERCHEM, získané geometrie slúžili ako vstup do programu GAUSSIAN G03 verzia D1 

Windows. Výsledné geometrie a rozloženia nábojov boli po solvatácii metanolom podrobené 

minimalizácií energie počas 300 krokov. Výpočty molekulovej dynamiky boli robené pre čas 



4

10 ns, geometria systému sa zapisovala každé 2 fs. Použitá teplota pri výpočtoch bola 273 K a 

243 K. 


Tab. 1 

Názov a forma 

Protonizovaný 1,2-dideoxy- 

1-nitro-L-arabino-1-enitol 

Protonizovaný 3,4,5,6-tetra- 

O-acetyl-1,2-dideoxy-1-nitro- 

L-arabino-1-enitol 

1,2-dideoxy-1-nitro- 

D-galakto-1-enitolu 

Protonizovaný 1,2-dideoxy- 

1-nitro-D-galakto-1-enitolu 

1,2-dideoxy-1-nitro- 

D-gluko-1-enitolu 

Päťčlánkový 

Teplota 

273 K 243 K 

Priemerná vzdialenosť reakčných 

partnerov pre vznik kruhu [Å] 

Šesťčlánkový 

Päťčlánkový 

Šesťčlánkový 

4.41 4.99 4.41 4.62 

3.76 5.09 4.26 6.34 

4.36 4.76 4.17 4.80 

4.95 5.05 4.95 5.04 

3.95 5.39 4.38 4.62 

Ako vidieť z Tab. 1 priemerné vzdialenosti reakčných partnerov sú za rovnakých podmienok 

menšie pri vzniku päťčlánkových cyklov. Nejedná sa len o priemerné vzdialenosti ale 

i o okamžité vzdialenosti, ktoré sú u päťčlánkových kruhov kratšie (Graf 1). 



vzdialenosť reakčných 

partnerov ( ) 

6.4 

5.9 

5.4 

4.9 

4.4 

3.9 

3.4 

2.9 

2.4 

Protonizovaný 3,4,5,6-tetra-O-acetyl-1,2-dideoxy-1nitro-L-arabino-1-enitol 

päťčlánkový 

šesťčlánkový 

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 

čas (ns) 

Graf 1 

Tento výsledok podporuje pôvodnú predstavu kineticky preferovaných päťčlánkových kruhov, 

zároveň i vyhovuje experimentálnym poznatkom získaných z analýzy reakčných produktov 

metanolýzy 3,4,5,6-tetra-O-acetyl-1,2-dideoxy-1-nitro-L-arabino-1-enitolu, kde výťažky 

päťčlánkových kruhov 7-9 tvoril až 65-78%. Ďalší súhlas výpočtov s experimentom bol 

dosiahnutý v určení preferencie jedného anoméru za rôznych teplôt. Z experimentu i výpočtov 

vyplýva, že pri nízkej teplote je u rôzne veľkých cyklov tendencia vytvárať len jeden anomér. 

Záverom môžeme tvrdiť, že použitá metóda je vhodná na opis kinetického správania 

C-glukozylnitrometánov. 

LITERATÚRA 

[1] T. M. Glennon, K. M. Merz Jr.: J. Mol. Struc. (Theochem), 395-396 (1997) 157-171. 

[2] G. Illuminati, L. Mandolini: Acc. Chem. Res. 14 (1981) 95-102. 

[3] M. Petrušová, M. Vojtech, Pribulová B., Lattová, E., Matulová M., Poláková M., BeMiller 

J. N., Kren V. Petruš L. Carbohydr Res. 341 (2006) 2019-2025. 

[4] Ballini R., Petrini M.: Tetraedron 60 (2004) 1017-1047. 



STUDIUM DEGRADACE TISKU NA TENKÝCH POLYMERNÍCH 

VRSTVÁCH 

Jiří Stančík, 5. ročník 

Vedoucí práce: doc. Ing. Michal Veselý, CSc. 

Vysoké učení technické v Brně, Fakulta chemická, ústav fyzikální a spotřební chemie, 

Purkyňova 118, 612 00 Brno, e-mail: xcstancik@fch.vutbr.cz 

ÚVOD 

Cílem práce bylo zkoumání vlastností potisků prováděných na polymerních vrstvách 

s anorganickými plnivy. Jako médium byl použit filtrační papír, polymerním nosičem byl 

modifikovaný polyvinylalkohol (dále jen PVAL). Do roztoku PVAL byl dispergován 

modifikovaný oxid titaničitý (TiO2). TiO2 je často používaným polymerním plnivem pro svou 

snadnou dostupnost a nízkou cenu. Zlepšuje vlastnosti polymerních vrstev při tisku, ale může 

urychlovat negativní vlivy, které způsobují degradaci použitých inkoustů vlivem slunečního 

záření. 


Na kvalitu výtisku mají vliv tři stěžejní faktory: tisk, skladování a potiskované médium 

(papír). Kvalita papírů používaných pro tisk je hodnocena hlavně z hlediska jejich textury, 

světlosti a opacity. 

• Textura: může být hrubá nebo hladká. 

• Světlost: vyjadřuje se v jednotkách od 1 do 100, přičemž hodnota 100 odpovídá 

nejsvětlejšímu papíru. 

• Opacita: je mírou neprůhlednosti či odrazivosti média. Vypočítá se jako poměr 

světelného toku dopadajícího na papír ku světelnému toku, který papírem projde 

(odrazí se). Čím je opacita vyšší, tím je papír neprůsvitnější (matnější), má lepší 

kvalitu. 

Opacitu lze zjistit pomocí veličiny průsvitnost (ν) podle vztahu 100 − ν. Průsvitnost lze 

vypočítat ze vztahu 

ν = 100 

Rb 

− Rč 

, 

R ´ −R 

´ 

(1) 

b 

kde Rb´ a Rč´ jsou hodnoty reflektance pro referenční bílou a černou podložku a hodnoty Rb a 

Rč jsou hodnoty reflektancí vzorků měřených na těchto podložkách [1] . Použitý filtrační papír 

nelze nazvat kvalitním médiem pro inkoustový tisk, avšak pro studium procesu je výhodné 

použití nosiče složeného z čistých celulózových vláken bez jakéhokoli přídavku plniv a 

aditiv. 

Kvalitu výtisku lze mimo jiné hodnotit podle optické hustoty a ostrosti vytištěných prvků 

(sledování nárůstu rastrového bodu). Vysoká optická hustota a ostrost jsou známkami 

kvalitního výtisku. 

Skladování je také velmi důležité. Blednutí výtisku může být způsobeno účinkem 

chemických látek ze vzduchu (ozón), vlhkostí, ale převážně světlem. Největší podíl na 

degradaci barviv má přímé sluneční světlo, hlavně jeho UV složka. Podobný účinek, i když 

mnohem menších dopadů, má také umělé osvětlení (žárovka). Další negativní faktory 

představují teplo a vlhko. Žádné médium by se tedy nemělo vystavovat příliš vysokým 



č

teplotám a nebo nechávat na vlhkých místech Chyba! Nenalezen zdroj odkazů. . Všechny uvedené 

faktory způsobují blednutí, či nežádoucí změny odstínů vytištěných barev. 

Světlo odrážející se od pozorovaného objektu je modulované vlastnostmi tohoto objektu 

(schopnost povrchu pohlcovat či odrážet světlo určitých vlnových délek). Takto modulované 

světlo dopadá do oka a ve zrakovém systému vyvolává určitý barevný vjem. Mezinárodní 

komise pro osvětlení (Commision Internationale de l’Eclarage – CIE) v roce 1931 

standardizovala systém měření barev a definovala standardní zdroje osvětlení (definice 

spektrálních charakteristik sady světelných zdrojů, se kterými se nejčastěji pracuje, světelný 

zdroj A až F, kdy nejpoužívanější pro grafiku jsou D50 a D65), podmínky osvětlování vzorku 

a detekce odraženého světla. Definovala také spektrální citlivost detektorů zavedením funkcí 

standardního pozorovatele a doporučila způsob vyhodnocování získaných údajů. 

Standardní pozorovatel představuje spektrální citlivost průměrného zdravého lidského oka 

na tři základní barvy – červenou, zelenou a modrou. Funkce standardního pozorovatele se 

také označují jako CIE trichromatické členitelé x(λ), y(λ), z(λ) nebo 2° standardní 

pozorovatel CIE 1931, protože odpovídají pozorování barevného pole v úhlu 2°. V roce 1964 

CIE definovala a doporučila tzv. CIE 1964 doplňkové trichromatické členitele pro 10° 

standardního pozorovatele. 

Ve smyslu barevného vjemu je možné jednoznačně definovat barvu pomocí tří čísel – 

trichromatických složek X, Y a Z, vypočítaných z remisních křivek barevného vzorku R(λ), 

spektrální distribuce osvětlení Φ 0 (λ) a funkcí trichromatických členitelů x(λ), y(λ) a z(λ). Pro 

příklad bude uveden výpočet trichromatické složky X, přičemž dvě ostatní trichromatické 

složky se vypočtou podle stejného vzorce, pouze se zaměněním patřičného trichromatického 

členitele 

730nm 

380nm 

0 

X = K ∫ Φ ( λ) ⋅R( λ) ⋅x( 

λ)dλ, (2) 

kde konstanta K je dána vztahem 

100 

K = 

. (3) 

0 

∫Φ 

( λ) ⋅ y( 

λ)dλ Normováním těchto trichromatických složek lze získat trichromatické souřadnice x, y, z, ze 

kterých byl vytvořen barevný model CIE xyY. Mnohem důležitější pro tuto práci je barevný 

model, který CIE navrhla v roce 1976 a je označován jako barevný prostor CIE 1976 L*a*b*. 

Hodnoty souřadnic udávají polohu barvy v třírozměrném barevném prostoru a získají se 

přepočtem z trichromatických složek dle vzorců 

* 

1 3 

L = 116 ⋅ Y Y − , (4) 

( n ) 16 

( X 

1 3 

X n ) ( Y 

1 

n ) 

1 3 ( Y Y ) ( Z 

1 ) 

3 

[ ] 

3 

[ ] 

∗ 

a = 500 ⋅ − Y 

* 

b ⋅ n − 

n 

, (5) 

= 200 Z , (6) 

kde Xn, Yn, Zn jsou trichromatické složky pro referenční bílou při zvoleném osvětlení a 

pozorovateli. 

Měrná světlost L* reprezentuje vertikální osu a osy a*, b* tvoří chromatickou rovinu. 

Prostor L*a*b* je téměř uniformní, a proto se používá hlavně k vyhodnocování barevných 

diferencí a tolerancí (Obr. 1). 

Barevnou odchylku ΔE*ab lze vypočítat ze souřadnic L*, a* a b* podle vzorce 

* 

Eab ( ) ( ) ( ) 2 

* 2 * 2 * 

ΔL 

+ Δa 

+ Δb 

Δ = 

. (7) 



Při měření barev prakticky všechny barevné prostory vycházejí při definici svých 

souřadnic z trichromatických složek X, Y a Z. Každý přístroj používaný pro měření barvy by 

měl zabezpečit: osvětlení měřeného povrchu zvoleným standardním osvětlením, detekci a 

měření změny vlastností odraženého světla od měřeného povrchu a samozřejmě vyhodnocení 

a vyčíslení trichromatických složek ve smyslu jejich definice [2] . 

−a 

zelená 

L = 100 

bílá 

−b 

modrá 

+b 

žlutá 

L = 0 

černá 

Obr. 1 Chromatická rovina barevného prostoru CIE 1976 L*a*b* a osa světlosti L. 

POUŽITÉ PŘÍSTROJE 

• Barvivová tiskárna Epson R220 obsahující cartridge: C, M, Y, K, LM, LC, 

• sonda Spectrolino využívající software Gretag Macbeth Measure Tool 4.1, 

• mikroskop Intrecomicro, 

• fotoaparát Nikon D70, 

• UV metr Lutron 340, 

• spektrofotometr Datacolor 3890. 


Připravený 5% roztok modifikovaného PVAL byl rozdělen na 5 dílů, do těchto 5 dílů byla 

dispergována různá množství modifikovaného TiO2 se sníženou fotokatalytickou účinností. 

K nanášení disperzí na filtrační papír byla použita metoda sítotisku. První vzorek byl ovrstven 

roztokem PVAL bez přídavku TiO2, u dalších vzorků byl poměr sušiny PVAL k TiO2 zvolen 

následovně: 1:1, 1:2, 1:4, 1:8 a 1:16. Připravené vzorky byly ponechány k volnému vysušení. 

U nepotištěných vzorků byla hodnocena světlost a opacita. Poté byla na vzorky vytištěna 

sada barevných škál obsahujících azurovou (C), purpurovou (M), žlutou (Y) a kompozitní 

černou barvu (K). Škály mají 11 políček na kterých klesá pokrytí inkoustem po 10 % od 

100 % po 0 % (Obr. 2). Neexponované vzorky byly vyfotografovány přes mikroskop, na 

výsledných snímcích byla hodnocena textura média a ostrost tisku. 

Obr. 2 Škála odstínů šedé, tištěná také v barvách C, M, a Y. 

+a 

červená 

Po proměření L*, a*, b* souřadnic a optických hustot (D65, 2° pozorovatel) pro jednotlivé 

barvy byly vzorky připevněny na podložku a umístěny na okno nacházející se na slunné 

straně budovy. Byly tedy vystaveny extrémním podmínkám pro degradaci potisku. Během 13 

dnů po které trvala expozice byla v okně se vzorky prováděna měření intenzity ozáření. Poté 



yly u vzorků opět proměřeny L*, a*, b* souřadnice a optické hustoty. Z výsledků byla 

vypočítána barevná odchylka ΔE*ab po první expozici (7). Vzorky byly poté vystaveny druhé 

expozici a opět proměřeny. 

VÝSLEDKY A DISKUSE 

Pro fotografování přes mikroskop byla vybrána políčka s 20% krytím inkoustu, protože 

jednotlivé rastrové body jsou na nich snadno rozlišitelné (neslívají se). S rostoucí koncentrací 

TiO2 se zlepšuje ostrost tisku. Podrobnějším zkoumáním těchto fotografií bylo zjištěno, že ve 

stejném směru se zlepšuje také textura a světlost. 

Měření opacity bylo provedeno na přístroji Datacolor 3890 tak, že byla nejprve proměřena 

reflektance referenční bílé a černé podložky a poté byly proměřeny jednotlivé vzorky na 

těchto podložkách. Ze změřených reflektancí byla vypočítána průsvitnost dle vztahu (1), 

z této veličiny pak byla spočtena opacita. Vypočtené hodnoty opacity shrnuje Tabulka 1. 

Intenzita ozáření vzorků byla proměřována pravidelně ve stejnou dobu (13:00 SLČ) 

v období od 29. 5. do 10. 7. 06. V rámci jednoho dne v daném období byla navíc provedena 

měření intenzit ozáření každou hodinu, aby bylo možno udělat si představu o rozložení 

intenzit ozáření v průběhu celého dne. Výsledky těchto měření jsou zaznamenány na Obr. 3. 

Intenzita ozáření ( μW cm -2 ) 

300 

250 

200 

150 

100 

50 

0 

29.5 12.6 26.6 10.7 

Datum 

Obr. 3 Výsledky měření intenzit ozáření 

Intenzita ozáření ( μW cm -2 ) 

300 

250 

200 

150 

100 

a) intenzita ozáření vzorků ve dnech experimentu (13:00 SLČ) 

b) průměrná intenzita ozáření vzorku během jednoho dne 

50 

0 

7 9 11 13 15 17 19 

Čas (h) 



ΔE * ab 

50 

40 

30 

20 

10 

0 

1:0 

1. expozice 2. expozice 

Azurová 

1:1 

1:2 

1:4 

1:8 

PVAL:TiO2 

1:16 

ΔE * ab 

50 

40 

30 

20 

10 

0 

1:0 


Purpurová 

1:1 

1:2 

1:4 

1:8 

PVAL:TiO2 

Obr. 4 Grafická znázornění barevných odchylek pro azurovou a purpurovou barvu. 

Z proměřených barevných škál byla vyhodnocována pouze políčka se 100% krytím 

inkoustem. Jejich barevné odchylky ΔE*ab po první i po druhé expozici byly graficky 

zaznamenány pro všechny koncentrace TiO2 (Obr. 4, Obr. 5). 

Tabulka 1 Nárůst hodnot opacity s koncentrací TiO2. 

Poměr PVAL:TiO2 1:0 1:1 1:2 1:4 1:8 1:16 

Opacita 57,6 69,4 72,6 74,3 81,8 85,9 

ΔE * ab 

50 

40 

30 

20 

10 

0 

1:0 


Žlutá 

1:1 

1:2 

1:4 

1:8 

PVAL:TiO2 

1:16 

ΔE * ab 

50 

40 

30 

20 

10 

0 

1:0 


Černá 

1:1 

1:2 

1:4 

1:8 

PVAL:TiO2 

Obr. 5 Grafická znázornění barevných odchylek pro žlutou a černou barvu. 

Bylo zjištěno, že TiO2 má jako plnivo do polymerních vrstev pozitivní vliv na kvalitu 

tisku. Nanášením disperzí na filtrační papíry byla zlepšována jeho textura a světlost, zároveň 

docházelo k nárůstu opacity. Všechny tyto faktory se podepsaly na zlepšení kvality výtisku 



1:16 

1:16

což bylo prokázáno pozorováním rastrových bodů a textury papíru pod mikroskopem a 

následným měřením opacity. 

ZÁVĚR 

Použitý TiO2 měl mít vlastnosti potlačující jeho fotokatalytické účinky, i přesto však 

výrazně přispíval k degradaci potisků což potvrdily vypočtené barevné odchylky. Barevné 

odchylky narůstaly s rostoucí koncentrací TiO2. Odchylky také narůstají s počtem expozic, 

proto byly po 2. expozici větší než po 1. expozici. 

Intenzita ozáření v měřeném období vzrůstala, ke konci období její nárůst poklesl. 

Průměrná hodnota intenzity ozáření byla 133 μW cm −2 . V průběhu jednoho dne intenzita 

ozáření vzrůstala až do 16 hodin, kdy dosáhla svého maxima. 

Polymerní vrstvy obsahující TiO2 vykazují zlepšené tiskové vlastnosti, zároveň však 

dochází k výrazné degradaci potisků. V další práci tedy bude věnována pozornost použití UV 

stabilizátorů za účelem zvýšení archivní stálosti výtisků. 

LITERATURA 

[1] Nároky na papír při tisku. Univerzita Pardubice [online]. [cit. 6. října 2006]. Dostupné 

na www: http://www.upce.cz/priloha/kpf-tiskovepapiry3 

[2] Hájek, M., Fade Resistance aneb blednout či neblednout? Fotografování [online]. 2005, 

[cit. 6. října 2006]. Dostupné na www: 

http://www.fotografovani.cz/art/fotech_fototisk/Fade-resistance-p.html 

[3] Veselý, M., Králová, I., Dzik, P., Zita, J., Vnímání barev a jejich měření, Vysoké učení 

technické v Brně, Fakulta chemická, Purkyňova 118, 612 00, Brno 2004 



ZMĚNY OBSAHU AMINOKYSELIN V BRAMBORÁCH 

V ZÁVISLOSTI NA HNOJENÍ DUSÍKEM 

Bc. Petra Valová, 5. ročník 

Vedoucí práce: Ing. Otakar Rop, Ph.D. 

Univerzita Tomáše Bati ve Zlíně, Fakulta technologická, Ústav potravinářského inženýrství 

a chemie, Náměstí T.G. Masaryka 275, 762 72 Zlín, e-mail: petra.flajsmanova@email.cz 

ÚVOD 

Dusík (N) je pro rostliny nejvýznamnější živinou (1). Po přihnojení dusíkatými hnojivy 

se zvyšuje intenzita tvorby organických sloučenin v rostlinném organismu. Příliš vysoké 

dávky dusíku však mohou inhibovat syntézu zásobních polysacharidů na úkor zvýšené tvorby 

některých jiných chemických sloučenin (2). Při hnojení dusíkem se každopádně mění 

chemické složení rostlin v důsledku zvýšené schopnosti rostlin přijímat anorganické 

i organické složky půdního roztoku (3). 

Správně volené hnojení dusíkem má kladný vliv na výnosy a kvalitu brambor. Se zvyšující 

se dávkou dusíku však jeho účinnost klesá. To znamená, že v rámci nízkých dávek N na 1 

hektar (50 kg) na 1 kg dusíku připadá přírůstek výnosu kolem 100 - 120 kg hlíz, ale u dávek 

nad 120 kg N.ha -1 již jenom 20 - 30 kg hlíz (4). Velmi vysoké dávky dusíku snižují obsah 

sušiny, škrobu a zhoršují chuť hlíz po uvaření. Existuje i nebezpečí zvýšeného obsahu 

dusičnanů v hlízách i když tento jev je více záležitostí průběhu počasí v ročníku a délky 

vegetační doby jednotlivých odrůd brambor (5). 

Jedním z nejzajímavějších důsledků aplikace dusíku k bramborám může být změna 

v obsahu aminokyselin v bramborové hlíze (6). Hnojení dusíkem zvyšuje v bramborových 

hlízách obsah celkového dusíku a obsah bílkovinného dusíku i když při velmi vysokých 

dávkách N může dojít ke stresu rostliny, který má naopak za následek pokles množství 

bramborové bílkoviny (7). Přestože se u brambor rostoucích na pozemcích, kde byla 

aplikována dusíkatá výživa, většinou setkáváme s vyšším množstvím bílkovin, jejich 

výživová hodnota jednoznačně klesá. Tento fakt je zapříčiněn přednostním zabudováváním 

dusíku v neesenciálních aminokyselinách (8). 

METODIKA 

Cílem práce bylo sledovat vliv aplikace dusíku na obsah aminokyselin v dužnině 

bramborových hlíz. Pokus byl prováděn v plastových nádobách, do kterých bylo navažováno 

po 10 kg stejné zeminy. Nádoby byly umístěny na pokusném pracovišti Ústavu 

potravinářského inženýrství a chemie FT UTB ve Zlíně. 

Do pokusu byly zařazeny varianty v 8 opakováních, a to bez přídavku dusíku do půdy 

a s přihnojením dusíkem na úrovni 20 mg N.kg -1 (odpovídá 60 kg N.ha -1 ) a 40 mg N.kg -1 

zeminy (odpovídá 120 kg N.ha -1 ). Dusík byl aplikován ve formě dusičnanu amonného. 

K výsadbě byly použity velmi rané brambory odrůdy KORUNA, jejichž hlízy byly pro 

analýzu sklízeny v 90 dnech vegetace, kdy jsou v konzumní zralosti. 

Pro stanovení celkového dusíku bylo využito metody podle Kjeldahla. Množství dusíku 

v hlízách bylo přepočteno na obsah hrubé bílkoviny. Hydrolýza vzorků pro stanovení 

aminokyselin byla provedena c (HCl) = 6 mol⋅dm -3 . Aminokyseliny methionin a cystein byly 

stanoveny pomocí oxidativně kyselé hydrolýzy ve směsi 85 % kyseliny mravenčí a 30 % 

peroxidu vodíku (9). Chromatografická analýza hydrolyzátu byla provedena na přístroji AAA 



400 (INGOS Praha) pomocí sodnocitrátových pufrů a ninhydrinovou detekcí. Obsah 

aminokyselin byl vyjádřen a mezi variantami porovnán standardní hodnotou - v g 

aminokyseliny na 16g N (10). 

Výsledky chemických analýz byly zpracovány metodou analýzy variance. 

Pro vyhodnocení průkaznosti rozdílů byl použit Scheffého test při 95 % hladině významnosti 

(11). 

VÝSLEDKY 

Výnosové parametry a výsledky chemických analýz jsou uvedeny v tabulkách I – III. 

Se zvyšujícím se přídavkem dusíku do půdy vrůstal výnos bramborových hlíz. U varianty 

s 40 mg N.kg -1 zeminy byl ve srovnání s kontrolní variantou výnos větší o 83 %. U této 

varianty bylo také množství hlíz získaných z jedné rostliny statisticky průkazně vyšší než 

u varianty kontrolní a varianty s 20 mg N.kg -1 zeminy. Naopak průměrná hmotnost 1 hlízy 

se snižovala (tab. I). 

Stupňované dávky dusíku snižovaly množství sušiny v bramborových hlízách (tab. I). 

Statisticky významně se snižovalo také celkové množství aminokyselin a hrubé bílkoviny 

(tab. II). Přihnojení dusíkem na úrovni 20 mg N.kg -1 zeminy mělo za následek snížení obsahu 

všech aminokyselin v čerstvé hmotě bramborových hlíz ve srovnání s kontrolou (pokles 

o 27 %). Další přídavek dusíku (tj. 40 mg N.kg -1 zeminy) opět způsobil nižší obsah většiny 

aminokyselin s výjimkou histidinu a tyrosinu jejichž množství se mírně zvýšilo. Ve srovnání 

s variantou 20 mg N.kg -1 zeminy byl celkový obsah aminokyselin u varianty s vyšší dávkou 

dusíku snížen o 17 % (tab. II). 

Při vyjádření obsahu aminokyselin v bramborové hlíze standardní hodnotou v gramech 

aminokyseliny na 16 g hlízového dusíku je také patrný trend snižování obsahu všech 

aminokyselin v závislosti na stupňovaných dávkách dusíku v půdě (tab. III). Výjimkou je opět 

vyšší obsah histidinu a tyrosinu u varianty s vyšší dávkou dusíku v půdě ve srovnání 

s variantou s 20 mg N.kg -1 půdy. Zatímco u kontrolní varianty bylo zaznamenáno přibližně 

90 % aminokyselin vázaných v bílkovině, u brambor hnojených dusíkem na úrovni 

20 mg N.kg -1 zeminy byl jejich obsah jen 67 % a v bramborových hlízách získaných z půdy 

s 40 mg N.kg -1 byl obsah aminokyselin v bílkovině pouze 65 % (tab. III). 

Tabulka I. Průměrná hmotnost bramborových hlíz v gramech, průměrný počet hlíz v kusech 

a průměrná hmotnost 1 hlízy v gramech připadající na 1 nádobu a průměrný obsah sušiny 

v bramborových hlízách (v hmotnostních %) 

Parametr Kontrolní varianta Varianta s 20 mg 

N.kg -1 zeminy 

Varianta s 40 mg 

N.kg -1 zeminy 

Výnos 118,33 g 168,33 g 217,50 g 

Počet hlíz 5,5 ks 6,33 ks 10,75 ks 

Hmotnost 1 hlízy 21,51 g 26,59 g 20,23 g 

Sušina 23,5 % 22,18 % 21,14 % 



Tabulka II. Obsah aminokyselin a hrubé bílkoviny v bramborových hlízách (g.kg -1 čerstvé 

hmoty) 

Aminokyselina Obsah v kontrolní 

variantě 

Obsah u varianty s 20 

mg N.kg -1 zeminy 

Obsah u varianty 

s 40 mg N.kg -1 

zeminy 

Valin 0,84 0,68 0,55 

Leucin 1,06 0,88 0,72 

Isoleucin 0,63 0,53 0,45 

Threonin 0,70 0,57 0,45 

Methionin 0,33 0,31 0,26 

Lysin 1,01 0,81 0,66 

Fenylalanin 0,73 0,61 0,51 

Histidin 0,33 0,20 0,25 

Arginin 1,06 0,73 0,57 

Cystein 0,37 0,33 0,25 

Kyselina asparagová 2,23 1,84 1,32 

Kyselina glutamová 2,19 1,67 1,39 

Serin 0,69 0,60 0,48 

Prolin 0,96 0,75 0,59 

Glycin 0,65 0,52 0,43 

Alanin 0,62 0,47 0,43 

Tyrosin 2,02 1,41 1,72 

Celkové množství 

aminokyselin 

16,42 12,91 11,03 

Hrubá bílkovina 18,20 17,59 16,86 

Tabulka III. Obsah aminokyselin v bramborových hlízách (g.16 g -1 dusíku - vyjadřuje 

přibližně procentické zastoupení příslušné aminokyseliny v bílkovině) 

Aminokyselina Obsah v kontrolní Obsah u varianty s 20 

variantě mg N.kg -1 Obsah u varianty s 40 

zeminy mg N.kg -1 zeminy 

Valin 4,63 3,54 3,28 

Leucin 5,80 4,56 4,23 

Isoleucin 3,43 2,74 2,64 

Threonin 3,81 2,97 2,65 

Methionin 1,82 1,61 1,55 

Lysin 5,52 4,21 3,90 

Fenylalanin 3,99 3,19 3,00 

Histidin 1,82 1,06 1,47 

Arginin 5,84 3,81 3,37 

Cystein 2,06 1,69 1,48 

Kyselina asparagová 12,25 9,59 7,83 

Kyselina glutamová 12,04 8,69 8,27 

Serin 3,75 3,12 2,87 

Prolin 5,31 3,9 3,49 

Glycin 3,54 2,72 2,56 



Alanin 3,47 2,45 2,54 

Tyrosin 11,07 7,36 10,20 

Celkové množství 

aminokyselin 

90,15 67,21 65,33 

REFERENCES 

1) PURVES W. et al., 2004. Life: The Science of Biology. Sunderland: Sinauer 

Associates:1121 p. 

2) WESTERMANN D. T. et al., 1994. Nitrogen and potassium fertilization on potatoes – 

sugars and starch. American Potato Journal, 71 (7): 433 - 453. 

3) LIN S. et al., 2004. Influence of nitrogen nutrition on tuber quality of potato with special 

reference to the pathway of nitrate transport into tubers. Journal of Plant Nutrition, 27 (2): 

341-350. 

4) ROP O., 1999: The toxic elements content in early potato varieties. Thesis. Brno, MZLU: 

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5) VOKÁL B., RADIL B., 1996. Effects of row spacing on tuber yield, dry matter content 

and starch in potatoes. Rostlinna vyroba, 42 (1): 5 – 9. 

6) FRIEDMAN M., 2000. Nutritional value of proteins from different food sources. 

A Review. Journal of Agricultural and Food Chemistry, 70 (1): 6 – 29. 

7) TALAAT M., 2003. The effect of mineral and compost fertilization on the nitrogen content 

and amino acid composition of potato and oat grains. Thesis. Wien, Universität Wien: 154 p. 

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and amino acids in the potato tubers. Plant Soil and Environment, 49 (3): 131 – 134. 

9) JANDASEK J., KRÁČMAR S., MILERSKI M et al., 2003. Comparison of the contents 

of intramuscular amino acids in different lamb hybrids. Czech Journal of Animal Science, 48 

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10) Official Journal L 206. Eighth Commission Directive 78/633/EEC of June 15, 1978. 

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29, 1978, 0043 – 0055. 

11) UNISTAT : Statistical Package for Windows. London : Unistat House, 2002, s. 406 – 

419. 



PRECIPITATION OF DL – VALINE FROM AQUEOUS ISOPROPANOL 

SOLUTIONS 

Bc. Miroslav Variny, 5. ročník 

Vedúci práce: Ing. Ján Šefčík, PhD. 

Slovenská Technická Univerzita, Fakulta Chemickej a Potravinárskej Technológie, oddelenie 

chemického a biochemického inžinierstva, Radlinského 9, 812 37 Bratislava, Slovenská 

Republika, e-mail: laurelindorenan@zoznam.sk 

INTRODUCTION 

Protein–coated microcrystals (PCMCs) 1,2 show potential in providing a novel, inexpensive 

method of administration of modern drugs. Their formation involves mixing of aqueous 

excipient and protein solution with excipient saturated solvent, miscible with water, in which 

both protein and excipient are less soluble. However, mechanism and kinetics of PCMC 

nucleation and growth are poorly understood. There are some preliminary indications from 

previous experimental work that the precipitation process does not proceed through classical 

nucleation/growth mechanism, but rather an intermediate liquid-liquid phase separation step 

was postulated 3,4 . In order to better design, scale-up and control PCMC formation processes, 

better understanding of underlying physico-chemical mechanisms is needed. Therefore, in this 

project I focussed on experimental studies of precipitation of a suitable excipient 3,4 , the amino 

acid DL–valine, from aqueous isopropanol solutions. 


Materials used in experiments were of laboratory reagent grade: 2-propanol (isopropanol), 

DL-valine, deionised water from in–house Millipore Water System and were used without 

further purification. Laboratory instruments used for measurements were DU 800 

Spectrophotometer (Beckmann Coulter) for turbidimetry and Malvern Mastersizer MS 2000 

(Malvern Instruments) for small angle static light scattering measurements. Procedure of 

solution preparation can be found elsewhere 3 . 

Standard operation procedure of a spectrophotometric measurement included using light 

of the wavelength 600 nm and manual control of cuvette placement. The experiment itself 

consisted of taking blank measurement, sample preparation, injecting a small volume of 

prepared sample in cuvette and measuring the sample sufficiently long to achieve a plateau in 

absorbance, or for at least 600 – 800s. Typical sample preparation included injecting small 

volume of valine solution in valine saturated isopropanol and mixing for 20 s on magnetic 

stirrer. Isopropanol solution volume ranged from 10 to 20 ml, volume of valine aqueous 

solution was between 30 and 100 μl. For one set of experimental conditions, at least five 

measurements were performed. In one set of experiments I deliberately changed the initial 

mixing time (leaving the mixing intensity unchanged). In dilution experiments, a 1–2 ml 

sample was diluted by dilution ratio from 1:1 to 1:4 after 20 s of mixing. 

Raw data from Mastersizer measurements were analyzed with standalone to obtain the 

mean radius of gyration. Radius of gyration is equal to the geometrical radius divided by 1,41 

(=2 1/2 ) for thin disks, which correspond to crystal shape of DL-valine observed in these 

experiments. 

In batch experiments, samples were prepared identically to those measured by the 

spectrophotometer. From samples mixed either for 20 s or all time, at certain times a small 



sample was taken. It was immediately injected in a glass beaker filled with bigger volume of 

quenching solution and well stirred for a couple of seconds. Small Volume Dispersion Unit, 

attached to measurement cell, had to be wetted with isopropanol, pumped through it for a 

couple of minutes, to ensure bubble removal from the cell window. Only after this procedure, 

the measurement itself could be started. Each measurement included also background scan. 

For the purpose of stopped flow experiments 5 , a complete apparatus has been kindly 

provided by XstalBio Inc. This apparatus is typically used to produce PCMCs in amounts of 

several hundreds of grams per hour. I managed to obtain a complete data set, having used 50 

mg/ml and 70 mg/ml valine aqueous solution, initial isopropanol flow rates 100, 200 and 400 

ml/min and resulting water content of 1 or 1,5%. In order to assess the effect of added protein 

on kinetics of the process and particle size, I repeated these measurements with addition of 5 

% w/w albumine in aqueous valine solution. 


Average absorbance 

1 

0,8 

0,6 

0,4 

0,2 

0 

0 200 400 600 800 1000 

Time (s) 

Fig. 1 Spectrometric data for samples with 100 μl of 

aqueous valine solution with valine concentration 50 

mg/ml added to a certain volume of saturated 

isopropanol solution (ml): solid – 10, dash – double 

dotted – 12, dotted – 15, dash - dotted – 17, dashed - 

20. 

Fig. 1 shows sensitivity of 

precipitation kinetics to the 

volume ratio of aqueous and 

isopropanol solutions. In terms 

of both initial absorbance 

increase as well as the final 

absorbance reached, it can be 

seen that even a small difference 

in the volume ratio has extensive 

effects. Absorbances for two of 

volume ratios of aqueous and 

isopropanol solutions are 

depicted separately in Fig. 2, 

together with the measured radii 

of gyration. In the case of 

classical nucleation and growth 

mechanism, many small nuclei 

would form in first moments of 

precipitation and in later stages 

only their size increase would 

contribute to absorbance increase. 

But while absorbance doubles or even triples, particle size grows only by 15 % (diamonds) or 

by 60 % (squares). Furthermore, absorbance dependence on particle size is of order 1/3 - 2/3 

(A ~ dp 0,33 - 0,67 ) so that it is not particle size growth, but particle number increase that is 

responsible for the observed absorbance increase. This confirms that non-trivial precipitation 

mechanism is present in this system. 

Spectrophotometric data were subjected to supersaturation analysis based on DL – valine 

solubility 4 data. In Fig. 3 data from several sets of experiments are shown. (S-1) equals to 

supersaturation driving force for crystal growth, where S=C/Cs, C is concentration of valine in 

supersaturated solution and Cs is its solubility in corresponding solution. “Seeded” data relate 

to solutions with addition of small amount of valine crystals, which resulted in higher 



absorbance growth rate and its lesser sensitivity to amount of valine available in the 

supersaturated solution. 

Data from dilution experiments are included as well. Generally, tremendous change in 

Ln (Absorbance growth rate) 

-3 

0 

-4 

0,5 1 1,5 2 2,5 

-5 

-6 

-7 

-8 

-9 

-10 

-11 

-12 

Ln (S - 1) 

Fig. 2 Average absorbance and radii of gyration vs. 

time for samples prepared batch wise on magnetic 

stirrer by injecting 100 μl of 50 mg/ml aqueous valine 

solution in: solid line, diamonds - 15 ml; dotted line, 

squares – 10 ml of valine saturated isopropanol. 

Average absorbance 

1 

0,8 

0,6 

0,4 

0,2 

0 

0 100 200 300 400 500 600 

Time (s) 

Fig. 3 Ln (Absorbance growth rate) vs. Ln (S – 1) 

analysis with linear regression of batch wise prepared 

samples by using aqueous solution with 

concentration: full diamonds – 50 mg/ml, empty 

circles – 50 mg/ml, seeded, empty diamonds – 70 

mg/ml, triangles – dilution of more supersaturated 

solution, squares – dilution of less supersaturated 

solution. 

5 

4 

3 

2 

1 

0 

Rg (um) 

absorbance growth rate 

dependence on (S-1) value can be 

seen. Slopes of linear regression 

fits in this chart give exponents x 

in the dependence of the 

absorbance growth rate on (S-1). 

The values of exponent x range 

from 6-7 (full and empty 

diamonds) through 4,5 (circles) to 

2-3 (triangles and squares). This 

observation is again inconsistent 

with simple nucleation and growth 

mechanism, where nucleation 

should cease or at least slow down 

after dilution, thus leaving the 

already formed nuclei to grow. In 

order to obey absorbance 

dependence on particle size 

(absorbance grew several times), 

nuclei would have to grow to tens 

or even to hundreds of μm size, 

which immediately would cause 

sedimentation. However, this was 

not observed. One can therefore 

conclude that nuclei formed even 

after dilution. Moreover, the 

absorbance growth rate after 

dilution was higher than the one 

for indiluted samples at the same 

supersaturation. Mechanism 

involving liquid-liquid phase 

separation of solution prior to 

nucleation and growth of solid 

particles would explain observed 

phenomena. 



Ln (time of clouding) 

9 

8 

7 

6 

5 

4 

3 

1 2 3 4 5 

(Ln(S)) 2 

Fig. 4 Ln(Time of clouding) vs. (Ln(S)) 2 

analysis without and with linearization. 

Samples prepared by using aqueous 

solution with concentration (mg/ml): 

diamonds – 70, squares – 60, triangles – 

50, full circles – 30, empty circles – 20. 

Concentration of valine in 

aqueous solution (mg/ml) 

A B 

70 -1,273 9,896 

60 -1,230 9,630 

50 -1,650 10,345 

30 -1,957 10,624 

20 -2,973 11,643 

Tab. 1 Regression coefficients of 

linearization 

2 

Ln ( time of clouding) 

= A( 

Ln( 

S ) ) 

ding to Fig. 4. 

+ B accor 

Fig. 4 provides further evidence for the 

role of liquid-liquid phase separation and 

related mixing effects in the precipitation 

mechanism. It shows the induction time 

(estimated as time of clouding of 

supersaturated solutions) dependence on 

squared logarithm of supersaturation. Data 

for valine concentration in aqueous solution 

60, 30 and 20 mg/ml can be found 

elsewhere 3 . In classical nucleation and growth theory, such chart should show a single line 

regardless what was valine concentration in the original aqueous solution before mixing with 

the isopropanol solution. Clearly, it is not the case here. Linearization 8,9 regression 

Rg (um) 

20 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

0 100 200 300 

Time (s) 

Fig. 5 Radii of gyration and laser obscuration as a function of elapsed time for samples 

prepared by stopped flow experiments, initial isopropanol solution flow rate 400 ml/min, 

aqueous solution with 5% w/w albumine First number – volume flow rate ratio (aqueous 

solution/isopropanol solution), second number – concentration of valine in aqueous 

solution: diamonds – 1 %, 50 mg/ml, big squares – 1,5 %, 50 mg/ml 1. Run, small squares 

– 1,5 %, 50 mg/ml 2. Run, triangles – 1 %, 70 mg/ml, circles – 1,5 %, 70 mg/ml. 

Laser obscuration (%) 

100 

80 


40 

20 

0 

0 100 200 300 

Time (s) 



coefficients, summed up in Tab. 1, show a clear trend of slope and intercept as valine 

concentration changes. 

In Fig. 5 clear evidence of precipitation rate dependence on solutions flow rate can be 

seen, which is caused by imperfect mixing in static mixer. No significant particle size changes 

can be observed with changing solutions flow rate or solutions flow rate ratio in this chart, but 

generally particles grow quicker as supersaturation increases. Albumine presence has only 

little influence on both particle size and precipitation rate, except an interesting effect that in 

some measurement particle size at first decreased. This happened entirely when using aqueous 

valine solution with concentration 70 mg/ml and, except one case, always in the presence of 

albumine. It remains an open question how did albumine presence modify the precipitation 

process. But regardless of albumine addition, valine particle sizes are much bigger than those 

seen in batch experiments (Fig. 2), their sizes being similar to those valine particles formed by 

mixing in the vortexer unit (without stirring bar present). This indicates existence of 

additional shear stress effects 10 on precipitation kinetics. 

CONCLUSIONS 

During this research project, I obtained a wide range of data characterising precipitation of 

DL – valine from aqueous isopropanol solutions under various conditions. 

The spectrophotometric data show that the precipitation process is very sensitive both in 

respect to supersaturation and to water content in final solution. Seeding the solution with 

valine crystals can further speed up the precipitation kinetics. Mixing and dilution 

experiments show different absorbance vs. time dependence, compared to standard samples 

with the same supersaturation. The conclusion from the spectrophotometric measurements 

and supersaturation analysis is that DL-valine precipitation is strongly mixing-dependent 6-8 

with indications pointing to intermediate liquid-liquid phase separation, confirming earlier 

preliminary experiments. 

Small angle static light scattering measurements and the following data analysis show 

particle size dependence on the type and length of mixing. Particle sizes from magnetic stirrer 

agitated batch experiments are smaller than those from either vortexer mixed batch 

experiments or stopped flow experiments. In some cases the visible particles (domains) at first 

decrease in size and after passing through a minimum, they start to grow, which is consistent 

with existence of liquid particles in precipitation process formed by liquid-liquid phase 

separation. 

REFERENCES 

(1) Kreiner, M.; Moore, B. D.; Parker, M. C. Chem. Commun. 2001, 1096 – 1097 

(2) Kreiner, M.; Moore, B. D.; Parker, M. C. J. Mol. Cat.B 2005, 65 – 72 

(3) De Miguel, S. A. Effects of composition and mixing on formation of valine microcrystals 

2006 

(4) Vos, J. Understanding the formation mechanism of protein coated microcrystals 2006 

(5) Roelands, C. P. M.; Roestenberg, R. R. W. Cryst. Growth Des. 2004, 4, 921 – 928 

(6) Vekilov, P. G. Cryst. Growth Des. 2004, 671 – 685 

(7) Laferrère, L.; Hoff, C.; Veesler, S. J. Cryst. Growth 2004, 550 – 557 

(8) Brick, M. C.; Plamer, H. J; Whitesides, T. H. Langmuir 2003, 19, 6367 – 6380 

(9) Mahajan, A. J.; Kirwan, D. J. J. Phys. D.: Appl. Phys. 1993, 26, B176 – B180 

(10) Kaneko, S.; Yamagami, Y.; Tochihara, H. J. Chem. Eng. Jpn. 2002, 35, 1219 – 1223 



ÚVOD 

VÝVOJ MIKROŠTRUKTÚRY SODNO-BORITO-KREMIČITÝCH 

SKIEL S PRÍDAVKOM TIO 2 

Oto Vojtechovský, 5. ročník 

Vedúci práce: RNDr. Jana Kozánková 

FCHPT, Ústav anorganickej chémie, technológie a materiálov, STU Bratislava, 

Radlinského 9, 812 37 Bratislava, Slovenská Republika, 

e-mail: OtoVojtechovsky@zoznam.sk 

Sklo tvorí celý rad anorganických a organických látok, ktoré ochladzovaním z kvapalného 

stavu určitou rýchlosťou nestačia vytvoriť pravidelné štruktúry. Z anorganických látok 

môžeme uviesť: 

• prvky: S, Se, Te, P 

• oxidy: B2O3, SiO2, GeO2, P2O5, As2O3 

• boritany a kremičitany: Na2B4O7, Na2Si2O5 

• iné zlúčeniny: sulfidy, selenidy, teluridy [1] 

Makrolikvácia v magme 

Sklá boli považované za homogénne izotropné hmoty. Avšak s objavom sklokryštalických 

hmôt sa v súvislosti s cieľavedomým štúdiom nukleácie ukázalo, že mnoho skiel nemožno 

zďaleka považovať za skutočne homogénne, ale že sa u nich prejavujú mikrohomogenity, 

ktoré sa podarilo dokázať modernými výskumnými metódami. Zistené nehomogenity majú 

obdobné charakteristiky, aké sa prejavujú u nemiešateľných kvapalín. Na odmiešanie (tj. na 

likváciu) roztavenej lávy upozornili petrológovia Vogt a Levinson Lessing. Odmiešaním sa 

snažili vysvetliť mechanizmus magmatickej diferenciácie a petrogenézy. Okrem 

makrolikvácie poznáme aj mikrolikváciu. Obidva tieto pojmy úzko súvisia s teplotou liquidus. 

K makrolikvácii dochádza nad teplotou liquidus a k mikrolikvácii pod touto teplotou. 

Odmiešanie sa prejavuje aj u skiel typu Vycor. 

Teória micelárnej štruktúry skla 

Podľa Moriyovej teórie, teórie micelárnej štruktúry skla,sa sklo skladá z dvoch fáz: z 

vysoko molekulárnych buniek (mikromiciel) a početných iónov či molekúl, ktoré ich 

obklopujú. Táto kombinácia vysokomolekulárnych buniek a ďalších iónov či molekúl 

nevykazuje pravidelnosti kryštálov, pretože bunky sú amorfné alebo kvázikryštalické. Medzi 

týmito bunkami a obklopujúcimi iónmi existuje rovnováha za každej teploty. Za vysokých 

teplôt tavenia sa sklovina skladá z disociovaných iónov a molekúl, ktoré sa vyznačujú veľkou 

pohyblivosťou [2]. Pri znížení teploty takéto častice začínajú asociovať a tvoria sa zárodky 

mikrofázy. Množstvo a rozmery mikrofázy závisia na tepelnej histórii skla.Pri veľmi rýchlom 

ochladzovaní vznikajú pomerne malé zárodky mikrofázy, okolo nej zostáva veľa 

disociovaných iónov. Naproti tomu, u pomaly ochladzovaného skla sú rozmery mikrofázy 

značne väčšie, pretože je dostatok času k asociácii iónov do väčších útvarov. Za nižších teplôt 

sa kvapky navzájom zrážajú (kondenzujú) vo väčšie útvary a sklo tuhne. 



Spinodálny rozklad 

• metastabilná oblasť - a 

• metastabilná oblasť - b 

• nestabilná oblasť – c 

Základné charakteristiky Vycor skla 

Vycor sklo je zaradené medzi alkalickoboritokremičité sklá. V našom experimente sme 

sledovali sklo s prídavkom a bez prídavku TiO2 Toto sklo je zložené z B2O3, SiO2, Na2O. 

B2O3 

Oxid boritý je bezfarebná sklovitá látka, ktorá len veľmi obtiažne kryštalizuje. Topí sa pri 

teplote 450°C. Svoju vysokú sklovitosť a fluiditu si zachováva aj ako zložka viaczložkových 

skiel a zlepšuje ich taviteľnosť. Teplota liquidus vstupom oxidu boritého klesá. Skladá 

saz nepravidelne usporiadaných skupín BO3 trojuholníkového tvaru, spojených tak, že každý 

atóm kyslíka je viazaný s dvoma atómami bóru. V kryštalickom stave sa skladá z dvoch typov 

navzájom spojených reťazcov, tvorených tetraedrickými skupinami BO4[4]. 

SiO2 

Oxid kremičitý je veľmi ťažko taviteľná látka. Je to podmienené tým ,že atómy kremíka 

a kyslíka tvoria v oxide kremičitom polymérne priestorové útvary. V oxide kremičitom je 

každý atóm kyslíka viazaný na dva atómy kremíka. V bežných podmienkach je každý atóm 

kremíka viazaný so štyrmi atómami kyslíka, ktoré sú okolo neho usporiadané tetraedricky. 

Vzájomné naviazanie tetraédrov umožňuje vznik rôznych modifikácií tuhého oxidu 

kremičitého. 

Obr. 1. Fázový diagram sústavy Na2O- 

B2O3-SiO2 [2]. 

Obr. 2. Schematické znázornenie 

spinodálneho rozkladu pod teplotou 

liquidus u modelových sústav SiO2-R2O 

(R= Li, Na)[2] 



Pri tepelnom spracovaní odmiešaných skiel dochádza ku zväčšovaniu odmiešaných častíc. 

Po počiatočnom stave odmiešania, ktorý môže byť buď nukleácia kvapiek a ich ďalší rast., 

alebo spinodálny rozklad, sa sústava snaží zmenšiť svoje medzipovrchy zväčšovaním častíc. 

K tomu môže dôjsť zrážaním (koalescenciou) kvapôčok v dôsledku Brownovho pohybu, 

viskóznym tokom a pod. ďlší možný proces vedúci k rastu odmiešaných častíc, ktorý sa 

uplatňuje predovšetkým u tuhých skiel je Ostwaldov proces zrenia. Tento proces je založený 

na rozdielnej rozpustnosti malých a väčších častíc. Najmenšie častice majú snahu sa 

rozpúšťať a rozpustené ióny potom difundujú k väčším stabilnejším časticiam, ktoré rastú. 

K zhrubnutiu mechanizmom Ostwaldovho zrenia dochádza u sústavy SiO2 - B2O3 

EXPERIMENTÁLNA ČASŤ 

V náväznosti na výskum sodno-borito-kremičitého skla (NBS) a sodno-boritokremičitéhoskla 

s prídavkom TiO2 (NBST) sme sa podrobnejšie zamerali na sledovanie 

vývoja mikroštruktúry NBS a NBST skla v počiatočnom úseku oblasti fázovej separácie pri 

tepolte 700°C a teplotnej výdrži v intervale od 0 do 120 minút. Cieľom práce bolo štúdium 

zmien v mikroštruktúre NBS a NBST skla temperovaných na teplotu 700°C (pri ktorej 

dochádza k fázovej separácii) metódou rastrovacej elektrónovej mikroskopie (REM) .Cieľ 

práce bol zvolený na základe pozorovaných zmien optickej priepustnosti NBS skla v prvých 

hodinách výdrže pri 700°C. 

Zloženie sledovaných skiel v hmotnostných %: 

NBS: 9,09% Na 2O, 26,18% B 2O 3, 64,43 SiO 2 NBST: 9,09% Na 2O, 26,18% B 2O 3, 60,43 SiO 2, 

4,00 TiO 2 

Vychladené sklo sme narezali na vzorky veľkosti 5x10x10mm na diamantovej píle. Vzorky 

utaveného skla sa fázovo separovali pri teplote 700°C po dobu: 0 min., 30 min., 60 min., 90 

min, 120 min. Zmeny v mikroštruktúre vzoriek sme sledovali v rastrovacom elektrónovom 

mikroskope REM - TESLA BS – 300. Vzorky skiel boli vodivo upravené naprášením vrstvy 

Au v naprašovacej aparatúre BALZERS SCD 050. Sledované boli neleptané a leptané (2,5% 

HF) lomové plochy skiel. 

Optická priepustnosť (a.u.) 

2000 

1600 

1200 

800 

400 

0 

3 

2 

1 

0 5 10 15 20 25 30 

Čas (h) 

Obr. 3. Zmena optickej priepustnosti vzoriek NBS skla počas ich temperácie pri 

špecifikovaných teplotách: 1 – 700 °C, 2 – 720 °C, 3 – 740 °C [3]. 



Obr. 3 poukazuje na pokles priepustnosti vzorky skla v intervale 0-120 min. Tento jav 

môžeme vysvetliť tým, že v priebehu spinodálneho odmiešania sa v pôvodnom skle 

vytvárajú dve vzájomne penetrujúce fázy s charakteristickou vermikulárnou mikroštruktúrou. 

Fázy sa líšia chemickým zložením, a teda aj veľkosťou indexu lomu. Svetelný lúč na každom 

fázovom rozhraní mení smer podľa veľkosti indexu lomu prostredia, do ktorého vstupuje. 

Opakovanými zmenami smeru lúčov sa časť z nich presmeruje takým spôsobom, že buď 

nedochádza k ich vyústeniu na spodnej ploche vzorky, alebo dochádza k ich vyústeniu pod 

uhlami divergentnými voči pôvodnému smeru prechádzajúceho žiarenia. Dôsledkom je 

zodpovedajúci pokles intenzity registrovaného žiarenia. Veľkosť poklesu žiarenia je úmerná 

počtu a veľkosti fázových domén, cez ktoré svetlo prechádza. To súhlasí s 

mikroštruktúrou vzoriek (obr. 4, 5) a počtom vytvorených domén (tab.1, 2). 

Tabuľka 1 

Leptaná vzorka NBS skla 

zahrievaná po dobu ( v min.) 

Celkový počet domén na ploche 

5 μm * 5 μm 

Tabuľka 2 

Leptaná vzorka NBS skla 

zahrievaná po dobu ( v min. ) 

Celkový počet domén väčších 

jako 1 μm *1 μm na ploche 

5 μm * 5 μm 

Výsledky sledovania mikroštruktúry 

0 30 60 90 120 

140 47 3 ojedinelé Vermikulárna 

štruktúra 

0 30 60 90 120 

0 5 3 ojedinelé Vermikulárna 

štruktúra 

Vybrané obrázky z riadkovacej elektrónovej mikroskopie REM pri zväčšeniach 10000x a 

20000x dokumentujú mikroštruktúru skiel tak, ako bola pozorovaná na väčšine pripravených 

lomových plôch skiel. 

Obr.4. Mikroštruktúra NBS skla po fázovej separácii pri 700°C a) 0 min, b) 30 min, c) 60 

min, d) 120 min 



Obr.5. Mikroštruktúra NBST skla po fázovej separácii pri 700°C a) 0 min, b) 30 min, c) 60 

min, d) 120 min 

ZÁVER 

Obrázky 4, 5 vývoja mikroštruktúry v sklách NBS a NBST poukazujú na výrazný vplyv 

prídavku TiO2 na rýchlosť fázového odmiešania a tvorby vermikulárnej mikroštruktúry. 

Vývoj mikroštruktúry NBS skla (obr. 4) je v súlade s Moriyovou teóriou micelárnej štruktúry 

skla. Fázové odmiešanie sledované v sodno-borito-kremičitých sklách umožňuje vylúhovaním 

boritanovej fázy pripraviť pórovitý materiál s riadenou veľkosťou pórov a značnou 

chemickou a tepelnou odolnosťou kremičitého skeletu. Tento typ pórovitého skla má 

významné uplatnenie v oblasti membránových filmov aj pre biologické aplikácie a vzhľadom 

na sorpčné vlastnosti aj ako materiál vhodný pre oblasť mikroelektroniky. 

LITERATÚRA 

1. Hlaváč J., Základy technologie silikátů, str. 135-2712. Voldán J., Odmísení skel, str. 4-13, 

20-253. Mojumdar S. C., Kozankova J., Chocholusek J., Majling J. and Nemecek V., Jour. of 

Ther. 

Analysis and Calorimetry (2004) 145–152. 

4. Volf M. B., Technická skla a jejich vlastnosti, SNTL, Praha 1987, p. 214 



VÝZNAM VOĽNÝCH RADIKÁLOV PRI POŠKODENÍ PAPIERA 

(EPR ŠTÚDIUM) 

Bc. Zuzana Vrecková, 5. ročník 

Vedúca práce: prof. Ing. Vlasta Brezová, DrSc. 

Ústav fyzikálnej chémie a chemickej fyziky, Fakulta chemickej a potravinárskej 

technológie, Slovenská technická univerzita v Bratislave, Radlinského 9, SK-812 37 

Bratislava, Slovenská republika, e-mail: zuvrro@gmail.com 

ÚVOD 

Historické dokumenty na papierových podložkách zahrňujú okrem archívnych a 

knižničných materiálov aj ostatné práce, ako sú obrazy, grafiky, kresby, plagáty, plány, mapy 

a pod., ktoré predstavujú súčasť kultúrneho dedičstva každého štátu. Veľká časť materiálov, 

ktoré boli napísané v posledných 150 rokoch trpí neustálym procesom poškodzovania, ktoré 

pri dlhodobom pôsobení môže viesť k úplnej strate tlačených a písaných textov v knižniciach 

a archívoch [1, 2]. 

Pre hodnotenie stability vlastností papierových materiálov je definovaná stálosť 

(permanence), ktorá označuje zachovanie pôvodných vlastností papiera s dôrazom na 

chemickú stabilitu a jej zmeny sa opisujú chemickými parametrami. Z chemických 

parametrov sa sleduje povrchové pH, obsah α-, β-, γ-celulózy, rozpustnosť v zriedených 

alkáliách, stupeň polymerizácie, obsah redukujúcich látok (meďné číslo), číslo kappa, obsah 

karboxylových skupín, obsah síry resp. rozpustných síranov, obsah dusíkatých látok. 

Trvanlivosť (durability) označuje zachovanie mechanických a optických vlastností papiera, 

ktoré si uchováva napriek používaniu a pôsobeniu vonkajšieho prostredia. Monitorujú sa 

mechanické parametre ako odolnosť v prehýbaní, pevnosť v dotrhnutí, pevnosť v ťahu 

a v prietlaku, nulové tržné dĺžky. Z optických parametrov sa hodnotí belosť, špecifický 

koeficient svetelnej absorpcie, relatívne dekoloračné číslo, opacita alebo špecifický koeficient 

rozptylu svetla [1, 2]. 

Keďže základný polymérny materiál tvoriaci papier predstavuje celulóza, je proces 

degradácie papiera determinovaný poškodením molekulovej a nadmolekulovej štruktúry 

celulózy (obr. 1). Poškodenie papiera pri starnutí je 

proces, kedy dochádza k značnej zmene 

mechanických, optických, chemických a fyzikálnych 

vlastností. Proces starnutia papiera pri dlhodobom 

skladovaní je determinovaný rôznymi faktormi, ktoré 

môžeme rozdeliť na vnútorné (druh, kvalita, chemické 

zloženie vlákna, lepidlá, glejidlá, plnidlá, prítomnosť 

kyslých skupín a iónov kovov) a vonkajšie 

(podmienky uskladnenia, teplota, relatívna vlhkosť 

okolitého vzduchu, svetlo, atmosférické znečistenie 

Obr. 1. 

Štruktúra celulózy a vodíkových väzieb 

medzi polymérnymi vláknami [3] 

a biologická kontaminácia papiera), pričom počas 

dlhodobého skladovania môže dochádzať k vzájomnej 

interakcii ako je schematicky znázornené na obr. 2 [4]. 

Pri laboratórnom hodnotení degradácie papiera je 

nevyhnutná simulácia podmienok prirodzeného starnutia metódami urýchleného starnutia, pri 

ktorých sa proces degradácie urýchľuje aplikáciou vyššej teploty a rôznej relatívnej vlhkosti 



v klimatizačných komorách [5, 6]. Metódami urýchleného starnutia môžeme zmeny vlastností 

papiera zodpovedajúce niekoľkoročnému prirodzenému starnutiu pozorovať už po niekoľkých 

dňoch (napr. 3 dni umelého starnutia papiera pri 105 °C zodpovedajú 25 rokom prirodzeného 

starnutia [6]). 

ENDOGÉNNE FAKTORY 

pH 

Kovové ióny 

Lignín 

Produkty degradácie 

EMISIA 

Prchavé produkty degradácie 

ABSORPCIA 

Kyslé plyny (SO2, NOx) 

EXOGÉNNE FAKTORY 

Teplota 

Vlhkosť 

Kyslík 

Svetlo 

Znečistenie (prach) 

Obr. 2. 

Faktory a procesy ovlyvňujúce 

degradáciu papiera [4]. 

Poškodenie polymérneho reťazca celulózy môže prebiehať rôznymi mechanizmami (kyslá 

hydrolýza, alkalická degradácia, oxidácia, autooxidácia iniciovaná iónmi kovov, 

fotodegradácia, mikrobiologická depolymerizácia), ktoré sa môžu vzájomne ovplyvňovať [4]. 

Významnú úlohu pri procesoch oxidačného poškodenia papiera zohrávajú kyslíkomcentrované 

voľné radikály, ktorých tvorba je iniciovaná prítomnosťou iónov prechodných 

kovov a kyslíka (1–4) [7, 8]. Generované reaktívne hydroxylové radikály ( • OH) sú schopné 

iniciovať depolymerizáciu celulózy, ako aj neselektívne reakcie s ďalšími zložkami papiera, 

napr. s lignínom (5). 

Fe(II) + O2 ↔ Fe(III) + O2 •– (1) 

Fe(III) + O2 + RH → • R + HOO • + Fe(III) (2) 

Fe(II) + HOO • + H + → Fe(III) + H2O2 (3) 

Fe(II) + H2O2 → Fe(III) + HO • + OH – (4) 

ROH + • OH → RO • + H2O (5) 

Výsledkom interakcie reaktívnych radikálov s lignínovými zložkami papiera je vznik 

paramagnetických semichinoidných štruktúr, ktoré sa vyznačujú dostatočne dlhým časom 

života aj pri laboratórnej teplote a ich prítomnosť v papierovom materiáli je možné priamo 

monitorovať pomocou EPR spektroskopie. Predpokladá sa, že koncentrácia semichinoidných 

radikálových štruktúr môže reflektovať oxidačné poškodenie papiera [9]. 

Naša práca je orientovaná na kvantitatívne EPR štúdium semichinoidných radikálových 

štruktúr v kyslom papieri, ktorý bol vystavený pôsobeniu urýchleného starnutia v rôznych 

experimentálnych podmienkach. 

EXPERIMENT 

Meranie EPR spektier sme realizovali pri teplote 22 °C na EPR spektrometri EMX 

(Bruker, SRN) s príslušným programovým vybavením. Do okrúhlej kremennej kyvety 

(vnútorný priemer 3 mm) na meranie v tuhej fáze sme umiestnili vzorku papiera so známou 

hmotnosťou nastrihanú na tenké pásiky (28 mm×1 mm). Kyvetu sme umiestnili do 

štandardnej dutiny TE102 (ER 4102 ST) a zmerali sme EPR spektrum. Pri meraniach všetkých 

vzoriek sme používali tú istú kyvetu, pričom sme ju vkladali do dutiny spektrometra vždy 

rovnakým spôsobom, aby sme zabezpečili reprodukovateľnosť meraní. Pri tejto manipulácii 



s kyvetami je chyba pri určení relatívnej integrálnej intenzity EPR signálu približne 5 %. 

Hodnotu g-faktorov radikálov sme určili s presnosťou ±0,0001 simultánnym meraním EPR 

spektra semi-stabilného radikálu 1,1-difenyl-2-pikrylhydrazyl (DPPH, Sigma). Na určenie 

koncentrácie spinov v jednom grame vzorky sme použili pripravený štandard so známou 

koncentráciou DPPH v tuhom NaCl (0,0002 mg DPPH g –1 ). 

Na meranie sme použili čiastočne drevitý, kyslý písací papier (80 g m –2 , pH = 4,4); 

Slavošovské papierne, Slavošovce, Slovenská 

republika, ktorý sme získali z Oddelenia polygrafie 

a aplikovanej fotochémie ÚPM FCHPT STU. Papier 

bol vystavený pôsobeniu umelého starnutia v rôznych 

podmienkach (suché teplo pri teplotách 105 °C 

a 120 °C s; vlhké teplo pri teplote 80°C a relatívnej 

vlhkosti 25%, 45% a 65%) počas rôznych časových 

intervalov (0, 8, 24, 72, 168, 336 a 672 hodín). Reálny 

objekt poškodeného papierového materiálu 

predstavovala kniha (rok vydania 1977), ktorá bola 

v roku 1980 poškodená kyselinou sírovou a 26 rokov 

bola uložená v knižnici pri laboratórnej teplote 

Obr. 3. 

Kniha poškodená v roku 1980 kyselinou 

sírovou. 

(obr. 3). Z knihy sme odobrali vzorky s rôznym 

stupňom poškodenia a hore uvedeným spôsobom sme 

v nich určili koncentráciu spinov g –1 . 


Obrázok 4 zobrazuje EPR spektrum namerané vo vzorkách papiera pri laboratórnej teplote, 

ktoré predstavuje singlet charakterizovaný parametrami gef = 2,0054; polšírkou ΔHpp = 0,85 

mT, ktorý je typický pre semichinoidné štruktúry 

nachádzajúce sa často v materiáloch rastlinného 

pôvodu [10]. Počas starnutia papiera sme 

Relatívna intenzita EPR signálu 

g ef =2,0054 

326 330 334 338 342 

Magnetické pole, mT 

Obr. 4. 

EPR spektrum vzorky papiera merané 

pri teplote 22 C. 

v podmienkach EPR experimentov nepozorovali vznik 

iných paramagnetických signálov. Problémom 

stanovenia koncentrácie semichinoidných 

paramagnetických štruktúr je stabilita EPR 

spektrometra a subjektívne chyby a nepresnosti pri 

nastavení EPR spektrometra, ktoré sme minimalizovali 

použitím identickej kyvety a jej rovnakého 

umiestnenia v dutine a EPR merania sme realizovali 

v čo najkratšom časovom úseku. 

Obrázok 5 vyjadruje závislosť koncentrácie spinov 

v jednom grame vzorky papiera od času urýchleného 

suchého starnutia pri teplote 120 °C () a pri teplote 

80°C s relatívnou vlhkosťou (RH) 45% (tzv. mokré 

starnutie) (). Už po 8 hodinách pozorujeme nárast 

koncentrácie spinov u oboch metód starnutia, ktorá 

dosahuje maximum po 336 hodinách (14 dní). Rozdiel v absolútnej koncentrácii spinov pri 

suchom (120°C) a mokrom (80°C/45% RH) starnutí dobre korešponduje s degradačným 



efektom metódy starnutia. Pokles koncentrácie spinov pozorovaný po 672 hodinách (28 dní) 

starnutia je pravdepodobne spôsobený premenami paramagnetických semichinoidných 

štruktúr na diamagnetické produkty v súlade s predpokladaným 2-elektrónovým 

mechanizmom oxidácie fenolických zlúčenín [9]. 

Spiny, g -1 

1.6x10 16 

1.4x10 16 

1.2x10 16 

1.0x10 16 

8.0x10 15 

6.0x10 15 

0 48 96 144 192 240 288 336 384 432 480 528 576 624 672 

Urýchlené starnutie, hodiny 

Obr. 5. 

Závislosť koncentrácie spinov v jednom 

grame vzorky papiera od času urýchleného 

suchého starnutia pri teplote 120 °C () 

a pri teplote 80 °C s relatívnou vlhkosťou 

45% (). 

Spiny, g –1 

1 2 2 6 7 8 10 

Stupeň poškodenia papiera 

EPR merania realizované na papieri získanom z knihy poškodenej kyselinou sírovou ukázali, 

že koncentrácia paramagnetických semichinoidných zlúčenín je približne 10-násobne vyššia 

(obr. 6), pričom najvyššiu koncentráciu vykazovali silne poškodené časti papiera, ktoré sa pri 

manipulácii rozpadali na prach. Stupeň poškodenia chápeme ako rozsah poliatia jednotlivej 

vzorky kyselinou. Je pravdepodobné, že koncentrácia semichinoidných štruktúr demonštruje 

komplexný mechanizmus poškodenia papiera kyslou hydrolýzou a radikálovými oxidačnými 

procesmi. 

ZÁVER 

EPR spektroskopiou sme monitorovali zmeny koncentrácie paramagnetických 

semichinoidných zlúčenín v drevitom papieri obsahujúcom lignín, ktorý bol vystavený 

pôsobeniu suchého a vlhkého tepla (120 °C a 80 °C/45% RH) alebo poškodený kyselinou 

sírovou. Predpokladáme, že plánovaná kombinácia EPR spektroskopie s inými technikami 

sledovania poškodenia papiera (meranie mechanických, optických a chemických vlastností) 

môže priniesť zaujímavé informácie o degradačných mechanizmoch a tým aj ich 

predchádzaniu. 

POĎAKOVANIE 

Predovšetkým chcem poďakovať prof. Ing. Vlaste Brezovej, DrSc. za odbornú 

a pedagogickú pomoc. Zároveň ďakujeme Slovenskej grantovej agentúre za finančnú podporu 

EPR výskumu (projekt VEGA/1/3579/06) a RNDr. Bohuslave Havlínovej, CSc. za 

poskytnutie vzoriek papiera. 

1.2x10 17 

1.0x10 17 

8.0x10 16 

6.0x10 16 

4.0x10 16 

2.0x10 16 

Obr. 6. 

Závislosť koncentrácie spinov v jednom 

grame vzorky papiera od stupňa poškodenia 

papiera H2SO4. 



LITERATÚRA 

[1] B. Havlínová, V. Brezová, F. Belányi, G. Szeiffová, Papír a Celulóza 57 , 338-342 

(2002). 

[2] B. Havlínová, V. Brezová, Ľ. Horňáková, J. Mináriková, M. Čeppan, Journal of 

Materials Science 37, 303-308 (2002). 

[3] http://www.steve.gb.com/science/molecules.html 

[4] http://www.heritage.xtd.pl/pdf/full_strlic.pdf 

[5] ISO 5630. Paper and board – accelerated ageing. – Part 1: Dry heat treatment at 105° C. 

Last revision 1991 – Part 2: Moist heat treatment at 90° C and 25% RH. Last revision 

1985 – Part 3: Moist heat treatment at 80° C and 65% RH. Last revision 1986 – Part 4: 

Dry heat treatment at 120° or 150° C. Last revision 1986. Part 1 is equivalent to the US 

American Standard ASTM (1987). Standard Test Method for Determination of Effect of 

Dry Heat on Properties of Paper and Board. American Society for Testing and Materials 

(ASTM-D776-87; 72 hours at 105±2 °C). 

[6] http://www.uni-muenster.de/Forum-Bestandserhaltung/kons-restaurierung/agebansa.html 

[7] L. Botti, O. Mantovani, D. Ruggiero, Restaurator 26, 44-62 (2005). 

[8] D. F. Guay, B. J. W. Cole, R. C. Fort Jr., M. C. Hausman, J. M. Genco, T. J. Elder, 

Tappi Fall Conference & Trade Fair, 2002), http://www.umche.maine.edu/pilot/docs/ 

2002%20TPC%20O2%20Delig%20Reactions.pdf. 

[9] C. Felby, B. R. Nielsen. P. O. Olesen, L. H. Skibsted, Appl. Microbiol. Biotechnol. 48, 

459-464 (1997). 

[10] M. Polovka, V. Brezová, A. Staško, Biophys. Chem. 106, 39-56 (2003). 




doktorských studijních programů 

„O cenu děkana 2005” 

(Sekce DSP 2005)

CHITOSAN - A NEW TYPE OF POLYMER COAGULANT 

Ing. Marcela Borovičková 1 , 3. DSP 

Supervisor: doc. Ing. Petr Dolejš, CSc. 1,2 

1 Institute of Chemistry and Technology of Environmental Protection, Faculty of Chemistry, 

Brno University of Technology, Purkyňova 118, 612 00 Brno, Czech Republic, 

e-mail: borovickova@fch.vutbr.cz 

2 W&ET Team, box 27, Písecká 2, 370 11 České Budějovice, Czech Republic 

ABSTRACT 

Results of laboratory experiments into the removal of humic substances by cationic 

biopolymer chitosan are presented. Chitosan is a natural product derived from chitin, a 

polysaccharide found in the exoskeleton of shellfish like shrimp or crabs. The high content of 

amino groups provide to chitosan very interesting heavy metals chelating properties. Chitosan 

is partially soluble in dilute mineral acids such as HNO3, HCl, H3PO4. We have used 1% 

solutions of chitosan diluted in 0,05M; 0,1M and 0,15M HCl. Aggregates of humic 

substances after inorganic coagulant or chitosan addition were separated by centrifugation. 

Tests were made with two model humic waters having different concentration of humic 

substances. Residual concentration of coagulant (Fe and Al) and absorbance at 387 nm and 

254 nm were evaluated. 

INTRODUCTION 

Impurities present in raw water are in suspended, colloidal, and dissolved forms. These 

impurities are dissolved organic and inorganic substances, microscopic organisms, and 

various suspended inorganic materials. It is necessary to destabilize and bring together 

(coagulate) the suspended and colloidal material to form particles. Afterwards these particles 

are removed by filtration. 

Coagulation is accomplished by the addition of ions having the opposite charge to that of 

the colloidal particles. Since the colloidal particles are almost always negatively charged, the 

ions which are added should be cations or positively charged. Typically, two major types of 

coagulants are added to water. These are aluminium salts and iron salts. The most common 

aluminium salt is aluminium sulphate, the most comon iron salt is ferric sulphate. 

Iron and aluminium salts are used as primary coagulants and the reactions that occur after 

addition of these coagulants are fairly well elucidated. More recently, organic polyelectrolyte 

coagulants have become also used. Organic coagulants are sometimes used in combination 

with inorganic coagulants. Depending on the specific chemistry of the target water, polymer 

use can vary from as little as 5 % of the total coagulant dosage to as much as 100 % [1]. 

Chitosan is a derivative of chitin, a polysaccharide that is the major component of the 

shells of crustaceans and insects. Chitin consists of long chains of acetylated D-glucosamine, 

that is, glucosamine with acetyl groups on the amino groups (N-acetylglucosamine). Chitosan 

is N-deacetylated chitin, although the deacetylation in most chitosan preparations is not 

complete (see Fig. 1). Chitin itself is usually prepared from crab or shrimp shells or fungal 

mycelia. Treatment with an alkali then produces chitosan with about 70% deacetylation. 


Sekce DSP 2005, strana 97

Fig. 1 Sructures of chitin and chitosan 

Chitosan has been widely used in vastly diverse fields, ranging from waste management to 

food processing, medicine and biotechnology. It becomes an interesting material in 

pharmaceutical applications due to its biodegradability and biocompatibility, and low toxicity. 

The protonization of amino groups in solution makes chitosan positively charged, and 

thereby very attractive for flocculation and different kinds of binding applications. Since most 

natural colloidal particles, including bacteria and macromolecules, are negatively charged, 

attractive electrostatic interactions may lead to flocculation [2-5]. 

EXPERIMENTAL 

Chitosan solutions 

Chitosan is partially soluble in dilute mineral acids such as HNO3, HCl, H3PO4. We have 

used 0,05M; 0,1M and 0,15M HCl solutions. Five hundred milligrams of chitosan powder 

was dissolved in a glass beaker and mixed with 30 mL of HCl solution. The dissolution of 

chitosan was slow. Afterwards, it was further diluted to 50 mL with HCl solution to obtain a 

solution containing 10,0 mg chitosan per mL of solution. The solutions were prepared fresh 

before each set of experiments for consistency. 

Metal-based coagulants 

Ferric sulphate [Fe2(SO4)3] and aluminium sulphate [Al2(SO4)3.18 H2O], supplied by 

Kemifloc a.s. (Přerov, Czech Republic) or Kemwater ProChemie s.r.o., (Bakov nad Jizerou, 

Czech Republic) respectively were used for the experiments. 

Model humic waters 

Tests were made with two model humic waters (A, B) having different concentration of 

humic substances. Model waters were mixtures of distilled water, tap water and natural 

concentrate of humic substances sampled from a peatbog near Radostín. Selected model 

humic water parameters are given in Table 1. Absorbance at 254 nm was measured in 1 cm 

cell and absorbance at 387 nm was measured in 5 cm cell. Absorbance at 387 nm is 

proportional to colour by multiplying by factor 255 [6]. 



Table 1 Parameters of model humic waters 

Model 

water 

pH χ [mS/m] ANC4,5 A254 A387 

A 6,5 18,2 0,4 0,163 0,148 

B 6,5 13,8 0,4 0,307 0,295 

χ - conductivity; ANC4,5 – acidic neutralising capacity to pH 4,5; A254 – absorbance at 254 

nm; A387 - absorbance at 387 nm 

Coagulation tests 

Coagulation test using centrifugation as the separation method was employed. This test did 

allow us to study formation of NOM particles by Brownian motion (perikinetic coagulation) 

and has the highest possible degree of reproducibility (in any place in the world) as 

temperature is the only parameter influencing the kinetics of particles formation. Aggregation 

time was 10 and 40 minutes. Residual concentration of coagulant (Fe and Al) and absorbance 

at 387 nm and 254 nm were evaluated. 

RESULTS 

Fig. 2 shows comparison of removal of UV absorbance between ferric sulphate; aluminium 

sulphate and chitosan, which was dissolved in different concetrations of HCl. Optimum 

coagulant dose ranged in the order of several units of mg/l for chitosan, and tens of mg/l 

respectively for aluminium and ferric sulphate. 

Model water A: optimum dose was between 21 and 28 mg/l of Fe2(SO4)2, 25 - 35 mg/l 

of Al2(SO4)3 and about 5 mg/l of chitosan. 

Model water B: optimum dose ranged in interval 30 - 39 mg/l of Fe2(SO4)2, 

41 - 48 mg/l of Al2(SO4)3 and about 10 mg/l of chitosan. 

Generally, the results show relatively good removal of humic substances by chitosan, 

which is comparable with Al and Fe coagulants. Main disadvantage of chitosan is its price, 

which should be compensated by low dosage. The optimum pH value for chitosan coagulation 

was between 5 – 7. 

The results show that chitosan is a promising substitute of metal-based coagulants, which 

are traditionally applied in treatment of humic waters.. 



A254 

A254 

A254 

0,25 

0,20 

0,15 

0,10 

0,05 

Ferric sulphate versus Chitosan in 0,05 M HCl 

Fe(A)-10´ Fe(A)-40´ Fe(B)-10´ Fe(B)-40´ 

Chit(A)-10´ Chit(A)-40´ Chit(B)-10´ Chit(B)-40´ 

0,00 

0 10 20 30 40 50 60 

0,25 

0,20 

0,15 

0,10 

0,05 

Dose [mg/l] 


0,00 

0 10 20 30 40 50 60 

0,25 

0,20 

0,15 

0,10 

0,05 

Fe(A)-10´ Fe(A)-40´ Fe(B)-10´ Fe(B)-40´ 


Dose [mg/l] 


Fe(A)-10´ Fe(A)-40´ Fe(B)-10´ Fe(B)-40´ 


0,00 

0 10 20 30 40 50 60 

Dose [mg/l] 

A254 

A254 

A254 

0,25 

0,20 

0,15 

0,10 

0,05 

Aluminium sulphate versus Chitosan in 0,05 M HCl 

Al(A)-10´ Al(A)-40´ Al(B)-10´ Al(B)-40´ 


0,00 

0 10 20 30 40 50 60 

0,25 

0,20 

0,15 

0,10 

0,05 

Dose [mg/l] 


Al(A)-10´ Al(A)-40´ Al(B)-10´ Al(B)-40´ 


0,00 

0 10 20 30 40 50 60 

0,25 

0,20 

0,15 

0,10 

0,05 

Dose [mg/l] 


Al(A)-10´ Al(A)-40´ Al(B)-10´ Al(B)-40´ 


0,00 

0 10 20 30 40 50 60 

Dose [mg/l] 

Fig. 2 UV254 removal comparison between metal-based coagulants and chitosan (black 

curves for model water A, grey curves for model water B) 

REFERENCES 

1. Gulbrandsen: Organic polymers [online]. 2002 [cit. 2005-08-23]. 

. 

2. Dalwoo Corporation: Chitin, chitosan and chitosan oligomer from Crab Shells. [online]. 

Last updated April 12, 1999 [cit. 2005-07-31]. http://members.tripod.com/~dalwoo/. 

3. Beaulieu E.B.: Marinard Biotech [online]. [cit. 2005-07-31]. http://www.marinard.com/. 

4. Li J. et al.: Polymer Degradation and Stability, 87, 441, 2005. 

5. Vander P. et al: Plant Physiol., 118, 1353, 1998. 

6. Dolejš P.: Spektrofotometrické stanovení barvy huminových vod. In Sborník konference 

"Hydrochémia ‘83". Bratislava: ČSVTS VÚVH, 1983, s. 361-370. 

























STUDY OF PHYSICOCHEMICAL AND ANTIOXIDATIVE 

PROPERTIES OF YEAST MEMBRANE COMPONENTS 

Michaela Drabkova, 3 rd year of PGS 

Supervisor: Prof. Ing. L. Omelka, DrSc. 

Doc. RNDr. I. Márová, Ph.D. 

Department of Food Chemistry and Biotechnology, Faculty of Chemistry, Brno University of 

Technology, Purkynova 118, 612 00, Czech Republic, email: drabkova@fch.vutbr.cz 

INTRODUCTION 

Carotenoids are isoprenoid membrane-protective antioxidant pigments produced by plants, algae, 

bacteria and fungi. They belong to the most widespread natural pigments with many important 

biological activities. Production of carotenes, the great variety of natural carotenoids, is more than 

100 million tons per year. They are produced by specific branch of common isoprenoid biosynthetic 

pathway occuring in all types of organisms. With regard to different applications in food and feed 

industry most attention is now being focused on the natural production of carotenoids by microbial 

technology using yeast and/or bacteria. 

There are two major ways to influence the microbial production of carotenoids: i) by 

modification of cultivation conditions and ii) by construction of genetically modified 

overproducers. Qualitative and quantitative changes in a cell metabolite complement can be induced 

by environment, stress and other factors. Thus, identification of metabolic marker characteristics for 

certain events provides important insight into the mechanisms of pathways occurring in the organism 

and can also lead to the regulation of production of industrially significant metabolites. Especially in 

microorganisms, production of metabolites is strongly influenced by various external factors. 

Environmental stress surrounding of yeast cells evokes various changes in their behaviour in order to 

survive under unfavourable conditions. Under stress, various specific compounds including lipidic 

substances are overproduced (e. g. glycerol, phospholipids, carotenoids, ergosterol etc.). However, 

more information is needed about regulation of production of these substances. Factors that influence 

efficiency of natural carotenoid biosynthesis are important for commercial applications. 

Carotenoids are membrane-bound lipid-soluble pigments, which can act as effective 

antioxidants and scavenge singlet oxygen. In red yeast they probably act as adaptive and/or 

protective mechanism against exogenous oxidative stress and UV-irradiation. Those compounds 

are accumulated in particular cell organelles. It is not clear so far, whether carotenoids are present 

in plasma membrane only or in other inner membrane systems as well as in cell wall. Also distribution 

of individual carotenoid derivatives in individual sub-cellular fractions has not been studied 

yet. Moreover, significant changes of these parameters under exogenous stress could occur, which 

could influence potential biotechnological use of red yeasts to industrial production of carotenoids. 

In this work some techniques for isolation and separation of sub-cellular fractions (cell wall, 

membrane fraction, cytosol) of red yeast cells grown in optimal conditions and under osmotic and 

oxidative stress were tested. Further, analysis of carotenoids in these fractions as well as in 

whole cells was done. Results of antioxidant properties of sub-cellular fractions were compared 

with carotenoid composition and antioxidant activity of some standard carotenoids. The aim of this 

work is to study, what is the distribution and trafficking of carotenoids in the cell and which 

carotenoids are the main contributors of antioxidant activity in individual cell compartments. 



MICROORGANISMS 

Yeast strains Rhodotorula glutinis CCY 20-2-26, Sporidiobolus salmonicolor CCY 19-4-8, 

Phaffia rhodozyma CCY 77-1-1 and Saccharomyces cerevisiae CCY 21-4-88 were used. 

Saccharomyces cerevisiae was used as comparative strain because of very high importance in 

genetic engineering. 

CULTIVATION CONDITIONS 

Yeasts were cultivated on glucose medium aerobically with light at 28 °C to the exponential phase 

of growth. Exogenous stress was induced by 2-5 % NaCl added into inoculation media and 2-5 mM 

H2O2 added into production media. 

Saccharomyces cerevisiae was cultivated on YPD medium anaerobicaly at 28 °C for 24 hours . 

PREPARATION OF SPHEROPLASTS 

Spheroplasts were prepared by incubation of yeast cells with some enzymes to softly disrupt 

plasmatic membrane. Different concentrations of lyticase and glucuronidase, other detergents (e.g. 

SDS, beta-merkaptoethanol, etc.) and osmotic lysis were used. Membrane fraction and cytosol were 

separated by ultracentrifugation. Spheroplasts were detected by light-microscopy and with addition of 

fluorescein dye by UV-microscopy. Further, spheroplasts were used for isolation of chromosomal 

DNA from red yeasts. Isolation was performed in 1% low melting agarose plugs and analyzed then 

by PFGE. 

a) Spheroplasts of R. glutinis – Light microscopy b) Bursting cells in isotonic environment 

ISOLATION OF SUB-CELLULAR FRACTION 

Sub-cellular fractions of cells were obtained by gradually separation using combination of 

enzymes and detergents. Cell wall fraction (surface layer) was obtained using sonification followed 

by ethanol precipitation. In selected fraction lipid profiles were analyzed using TLC. Levels of 

carotenoids - lycopene, alpha-carotene, beta-carotene, torulen and phytoene were obtained from yeast 

cells using acetone extraction and saponification by ethanolic KOH solution. The sample was 

repeatedly extracted by diethylether, evaporated and dissolved into ethanol for HPLC. We analyzed 

those samples using HPLC/MS. Antioxidant activity of individual sub-cellular fractions was tested 

using ABTS Metod (Randox kit). Protein profiles under stress conditions were compared too. Those 

were analyzed by PAGE-SDS. 




The most important industrial red yeast Rhodotorula glutinis was used as tested strain for 

identification of carotenoids and other membrane compounds. Dry biomass of these yeasts is used as 

feed complement for improvement of appearance and nutritional properties of animal products (e. g. 

the color of eggs and milk, the color of salmon meat). 

This microorganism was chosen based on results of previous screening study on selected yeast 

strains which produce carotenoids as natural pigments. Carotenogenic cultures were influenced by 

several exogenic chemical and physical stress factors added into cultivation media to clear the role of 

carotenoids in general stress response. We found that all types of stress conditions led to increased 

production of beta-carotene according to phase of application or concentration of stress factor. Crossprotection 

and production of similar metabolites in various types of stress conditions suggests the 

existence of an integrating mechanism that senses and responds to different forms of stress. 

Preparation of individual sub-cellular fractions from red yeast cells was strongly complicated by 

lipotrophic character of this strain. Especially preparation of spheroplasts was very difficult because of 

rigid character of cell wall membrane and yields of membrane fractions were therefore very low. 

Under stress conditions, about hundred proteins were overproduced by this strain. Expecting shock 

proteins, it could be some enzymes that catalyze overproduction of stress metabolites including 

carotenoids. Under both oxidative and osmotic stress pigments were overproduced, higher amount 

was detected in surface cell structures (cell wall and plasma membrane). Ergosterol was found both 

in cytosol and membrane lotions and its production under stress changed simultaneously with 

carotenoid formation. Glycerol was detected above all in cytosol fraction and its production under 

stress was inversely to carotenoid and ergosterol production. 

As surprising finding can be noted high content of carotenoids found in upper cell wall fraction. 

Presence of carotenoids, mainly beta-carotene, was detected in plasma membrane as well as in inner 

membrane fraction. The highest antioxidant activity was found in surface structures. 

This work was supported by project MSM 0021630501 H/'Czech Ministry of Education and by 

project IAA400310506 f Grant Agency of the Academy of Sciences of the Czech Republic. 

REFERENCES 

.l. Marova I., Breierova E., Koci R., Friedl Z., Slovak B., Pokorna J.: Annals Microbiol. 54, 73 

(2004). 

2. Farina J. I., Molina O. E., Figueroa L. I.. C.: Journal of Applied Microbiology 96, 254-262 (2004) 

3.Pardo M. Monteoliva L., Pla J. Sanchez M., Gil C. Nombela C.: Yeast 15, 459-472 (1999) 

4. Misawa N. Shimada H.: J. Biotechnol. 59, 169 (1997). 

5. Armstrong G. A., Hearst J. E.: FASEB J. 10, 228 (1996). 



DETERMINATION OF YEAST VIABILITY BY MEANS OF 

FLUORESCENCE MICROSCOPY AND IMAGE ANALYSIS 

Ing. Petra Jeřábková 

Supervisor: doc. Ing. Oldřich Zmeškal, CSc. 

Institute of Physical and Applied Chemistry, Faculty of Chemistry, Brno University of 

Technology, Purkyňova 118, 612 00 Brno, e-mail: jerabkova@fch.vutbr.cz 

1. INTRODUCTION 

In a condition assessment of microbiological populations, the important factor is a 

representation of live and dead individuals that it is possible to differentiate by means of 

fluorescent labelling. A direct microscopic count is usually used for direct determination of 

the cell number in counting chambers (by Thom, Bürker). The number of cells of microorganisms 

can frequently be determined indirectly with the assistance of cultivation when it is 

assumed that one colony will grow up from a viable cell and these colonies will be counted 

after that. It is possible to use an image analysis for the detection of defined objects 

(e.g. yeasts) without the necessity to count them. 

In this work, the wavelet transformation at a fractal analysis of an image, the so-called box 

counting method, was utilised. The wavelet transformation (or the Haar one) makes the 

calculation of squares of different sizes of a laid mesh more effective with the box counting 

method provided that a square area is being analysed. An analysed image with a size that is 

given by the power of a definite number (2 i ) is transformed into the space of spectra. On this 

spectra, the filter of a low pass type with the size of square 2 i , where i = 1, 2, 4… n – 1 is 

gradually applied and the number of black, white and black and white squares is determined 

in images that arise from the inverse transformation in the same way as with a box counting 

method. It is possible to substitute the process of transformation and inverse transformations 

of an image by the addition of neighbouring foursome pixels and to analyse a relevant 

sequence of these pictures. Three fractal dimensions of the black and white area and their 

interface (DBBW, DBWB, DBW) and fractal measures (KBBW, KBWB, KBW) will again be the result. 

When analysing a black and white picture that may be obtained from a coloured picture by the 

process called thresholding, a method identical with a box counting method is described [1]. 

With the assistance of the fractal analysis, characteristic data about an analysed structure, 

namely the fractal dimension D and the fractal measure K are determined. These parameters 

can be used for picture ordering evaluation as well as e.g. for specifying the number of 

defined objects or for specifying their radius [2]. 

Pictures for the analysis were obtained by means of fluorescence microscopy because this 

method is quick and simple. Results are obtained within minutes, whereas classic cultivation 

methods require the minimum of 24 hours. 

2. EXPERIMENTAL PART 

The fractal analysis was used for specifying the number and dimension of images of live 

and heat-killed yeast cells. The fluorescent dye of acridine orange (AO) was used for the 

simultaneous distinction of live and dead cells (dead cells give out red or orange fluorescence 

and live cells yellow-green; 180 µM AO, pH 6). Fluorescein diacetate (FDA) was used for the 

distinction of live cells (greenish fluorescence; 6 mM FDA, pH 7.2). With the assistance of 



fluorescent labelling it is possible to threshold either dead or live cells on a black colour in 

one picture. Images of cells were thresholded by the intensity or coloured components of the 

RGB space. To detect the convenient thresholding, it is possible to use the fractal spectrum, 

i.e. fractal dimension dependence on the intensity or selected RGB component, which is 

accessible in the HarFA software as a tool referred to as Fractal Analysis – Range [3]. The 

number of cells x and their radius r were determined provided that images of cells were of a 

circular shape, similar in size and distinguishable on the background according to relations 

x = 

4π 

N 

2 

( ) , 

BW 

N + N 

B 

BW 

r = 

( N + N ) 

B 

where NB is the number of black boxes, NBW is the number of black and white boxes with the 

size of ε × ε pixels. The resulting cell number x is derived from the value x, which is the 

maximum from the calculated values for different sizes of the mesh. The maximum value is 

selected because the fractal structure is bordered most conveniently for the given mesh size. 

Yeasts from Culture Collection of Yeasts – CCY, the Institute of Chemistry, Slovak 

Academy of Sciences in Bratislava and from the Faculty of Chemical and Food Technology 

of the Slovak University of Technology in Bratislava were used for the image analysis. Yeast 

Saccharomyces cerevisiae FV1 and yeast Saccharomyces cerevisiae subsp. cerevisiae CCY 

21-4-102 have ellipsoidal and spherical cells with width about 2.6–6.4 µm and with length of 

3.7–9.7 µm. Yeast Kloeckera apiculata CCY 25-6-22 has the shape of a lemon and size of 

(1.5–5) µm × (2.5–11) µm. Vegetative cells of yeast Hansenula anomala CCY 38-1-30 are 

spherical or ellipsoidal with size of 2–4 and 2–6 µm [4]. 

Yeasts that were cultivated on an inclined agar by the room temperature were, then, 

inoculated into a liquid culture medium (glucose peptone yeast extract medium) where they 

were cultivated for a minimum of 24 hours again by the room temperature under aerobic 

conditions. After that, a half of the volume of the culture medium with cells was exposed to 

95°C for 3–6 hours (according to the yeast species). A drop of live and dead cells mixture was 

put on a slide and a drop of fluorescent dye was added. Microscopical preparation was 

observed immediately at staining by fluorescein diacetate and after 30 minutes at staining by 

acridine orange with 40× objective. 

The number of cells was evaluated in 35 visual fields. A suitable neutral density filter 

(ND4, ND8, ND16), which decreases the intensity of the excitation light, was inserted 

between the excitation light (mercury lamp) and excitation filter, when required. For the 

recording of pictures, the combination of the epifluorescence microscope Nikon Eclipse E200 

with the filter cube and a digital camera Nikon Coolpix5400 with the resolution of 2592 × 

1944 was used. For the storage of pictures, the software Lucia Net 1.16.6. was used. The 

obtained pictures with the size of 1024 × 1024 were analysed by means of the software 

HarFA [3]. 


Sekce DSP 2005, strana 116 

πx 

BW 

ε 2

Table 1 

Average numbers of cells from 35 pictures of sample of different yeast species that were 

obtained by fractal analysis 

AO FDA 

Yeast 

species 

Saccharomyces 

cerevisiae 

Saccharomyces 

cerevisiae FV1 

Kloeckera 

apiculata 

Hansenula 

anomala 

Dead 

cells 

number 

Average 

error 

(%) 

Live 

cells 

number 

Average 

error 

(%) 

Total 

number 

of cells 

Average 

error 

(%) 

Live cells 

number 

Average 

error 

5.42 8.05 6.60 8.66 11.97 6.10 11.40 4.02 

4.19 7.23 7.00 4.85 11.19 4.61 8.96 3.74 

4.94 6.61 10.34 6.94 14.90 5.66 10.53 5.93 

10.17 8.44 10.38 6.92 20.57 5.22 8.05 8.11 

Table 2 

Average radii of cells from 35 pictures of sample of different yeast species that were obtained 

by fractal analysis 

AO FDA 

Yeast 

Dead cells 

Live cells Live and dead cells Live cells 

species 

radius 

radius 

radius 

radius 

Saccharomyces 

cerevisiae 

Saccharomyces 

cerevisiae FV1 

Kloeckera 

apiculata 

Hansenula 

anomala 

3.25 4.12 3.74 3.78 

3.47 3.70 3.55 3.75 

2.88 2.72 2.96 2.62 

2.61 2.65 2.72 3.17 

3. CONCLUSION 

The fluorescence microscopy in the connection with the image analysis seems to be a 

suitable method for the counting of yeast cells. For the staining of dead and live cells 

simultaneously, the acridine orange dye is suitable, for the staining of live cells it is the 

fluorescein diacetate. When counting live and dead cells of different yeast species by means 

of the fractal analysis it was determined that the average error of determination of the yeast 

number from 35 pictures is less than 10 % with the number of cells up to 20 in a picture 



(%)

(Table 1). This error can also be caused by an unequal size of cells and shapes, differences in 

colouring of the individual cells and quality of the recorded picture. If cells are killed by heat, 

the cytoplasm is sometimes stained greenly as well, which complicates the thresholding 

process with cells that were stained by the acridine orange. Budding cells were counted as one 

cell identically as in the direct determination of the cell number whereas if a daughter cell 

carried another bud still not separated from a mother cell, it was counted as two. 

Obtained radiuses of cells image provided that they have a circular shape are listed in 

Table 2 and are within the range of 2.61–4.12 µm. The sizes of the radii fall within values that 

are listed in literature or, in some case, are slightly greater [4]. 

4. REFERENCES 

[1] Tománková, K. Studium vlastností hrubě disperzních soustav pomocí metod obrazové 

analýzy. Brno, 2004, 42 s. Diploma thesis, Faculty of Chemistry, Brno University of 

Technology. Supervisor: Oldřich Zmeškal. 

[2] Zmeškal O., Sedlák O., Nežádal M.: Metody obrazové analýzy dat, Digital Imaging in 

Biology and Medicine, Czech Academy of Science České Budějovice: May 13., 2002, pp. 

34 - 43, ISBN 80-901250-8-5. 

[3] Zmeškal O., Nežádal M., Buchníček M., Bžatek T.: HarFA 5.0, Harmonic and Fractal 

Image Analyzer, http://www.fch.vutbr.cz/lectures/imagesci, Brno, 2003. 

[4] Kocková-Kratochvílová, A.: Taxonómia kvasinek a kvasinkovitých mikroorganizmov. 

1. vyd. Bratislava: Alfa, 1990. ISBN 80-05-00644-6. 



FLOW BEHAVIOUR OF DILUTED SUSPENSIONS IN 

CARBOXYMETHYLCELLULOSE-WATER SOLUTION 

MICHAL KLIMOVI 


Chemistry, Purkyova 118, 612 00 Brno, Czech Republic, e-mail: 

klimovic@fch.vutbr.cz 

Introduction 

Recently we have reported on rheological problems in processing lignite pastes to form 

elements for lignite alternative applications outside the power generation industry [1, 2]. 

We have also shown how these problems can be overcome using cellulosic derivative as 

a rheological aid [3]. Further our work was concerned with study of much more diluted 

suspensions of liquid-like appearance. We have found that small amount of lignite 

surprisingly lowered apparent viscosity of the solution comparing with the pure solvent. 

This work reports on flow behaviour of inorganic particle dispersions in the same 

medium. The aim was to find whether this thinning effect is specific to lignite or not. 

Experimental 

Carboxymethylcellulose (in the form of sodium salt; CMC) was supplied by Aldrich. 

Two preparations were used with following producer’s specifications: high molecular 

weight, Mw = 700 000 g mol -3 , degree of substitution 0.9; low molecular weight, Mw = 

90 000 g mol -3 , degree of substitution 0.7. Moisture content was determined as 8.2 and 

9.4 %. 

Two types of filler were used for preparation of dispersions, the foundry sand from 

laboratory of FSI VUT Brno and microsil from Silchem. The fraction of foundry sand 

captured between 0.15 and 0.30 mm sieves was used for subsequent experiments. 

Microsil is Silchem trademark of glass microspheres; type G080 was used. 

The peparation of suspensions consisted in suspending weighted amounts of filler and 

CMC in corresponding amount of warm (70 °C) deionized water. Mixture was stirred 

for one hour, temperature was maintained for the first 30 min only. 



Measurements of flow properties were carried out at 25 °C on Haake RS100 rheometer 

equipped with the double cylinder sensor Z20 DIN both in constant rate and in constant 

stress mode. All reported data points are averages from three replicates. 

Results and discussion 

Figure 1 shows flow curves for increasing sand contents measured immediately after 

preparation with suspensions based on the high molecular weight cellulose derivative. 

Higher sand loadings (7 % and more) shift flow curves up, to higher shear stress values, 

on increasing sand contents and also apparent viscosity rises. On contrary, flow curves 

for low sand loadings (5 %) lie bellow the curve of blank CMC solution giving also 

lower apparent viscosities. Thus, adding sand to the carboxymethylcellulose solution 

does not increase its (apparent) viscosity as expected, unless the sand concentration is 

sufficiently high. The same effect was observed for lignite as a filler. 

Suspensions prepared from the low molecular weight cellulose derivative did not show 

this untypical behaviour. 

The same behaviour of suspensions was observed at carboxymethylcellulose-microsilwater 

dispersions (Fig 2). 

Fig 1 The dependance of shear stress on shear rate for dispersions of pure CMC 

solution and different concentrations of sand in CMC solution (2 %, 5 %, 7 %, 10 %). 



Fig 2 The dependance of shear stress on shear rate for dispersions of pure CMC 

solution and different concentrations of microsil in CMC solution (0.5 %, 1 %, 3 %, 

5 %). 

Measured flow curves can be adequately fitted by the Ostwald-de Waele model 

n 

= K 

Model parameters reflected the influence of lignite on suspension flow behaviour. 

Results of this study indicate that lowering apparent viscosity of suspensions prepared 

from water solution of high molecular weight carboxymethylcellulose may be quite 

common phenomenon when concentration of dispersed particles is low (approximately 

in orders of tenths up to ten weight percent). The cause is probably in easier movement 

of particles entrapped within long biopolymer chains like balls in ball-bearing. 

Conclusion 

Flow curves determined in this study showed that lowering suspension apparent 

viscosity at moderate amount of particles suspended in high molecular weight 

carboxymethylcellulose solution is not specific effect of lignite particles. The same 

effect was observed also for sand and even for glass microspheres, which are usually 

used as thickener. Results thus confirm our hypothesis on some “ball-bearing” effect, 

which enables easier flow of long macromolecular chains then when they are in closer 

contact in pure solution. 



References 

1. Lapík ., Lapíková B., Filgasová G.: Colloid Polym. Sci. 278, 65 (2000) 

2. Peka M.: Flow behaviour of concentrated lignite dispersions. In Proceedings of the 

XIIIth International Congress on Rheology, Cambridge, vol.4. British Society of 

Rheology, Glasgow, p.4, 105, 2000. 

3. Peka M., Diviová P., Klimovi M.: submitted to Colloid. Polym. Sci. (2005) 













SPECTROPHOTOMETRIC STUDY OF SOME BIOLOGICAL 

BUFFERS SOLUTIONS 

Ing. Miroslava Krčmová a , 3 rd year of DSP 

Supervisor: doc. Ing. Radim Vespalec, DrSc. b 

a Brno University of Technology, Faculty of Chemistry, Institute of Physical and Applied 

Chemistry, Purkyňova 118, CZ 612 00 Brno, Czech Republic, 

e-mail: xckrcmova@fch.vutbr.cz 

b Institute of Analytical Chemistry, Czech Academy of Science, Veveří 97, CZ 611 42 Brno, 

Czech Republic, e-mail: vespalec@iach.cz 

INTRODUCTION 

Biological buffers are predominantly synthetic compounds serving widely to the pH 

stabilization and for the pH control in the pH range of 5.5–11.4 1 . They are predominantly 

white crystallized powders that are good soluble in water and they have a high chemical 

stability and compatibility in various biological systems. Today, the biological buffers are 

predominantly utilized as chemical agents for analytical chemistry. They are used in buffer 

exchange processes and as mobile phases during some chromatography steps, too 2 . These 

buffer compounds provide alternate buffer compounds with pKa values near physiological 

conditions for biological applications 2, 3 . The biological buffers are free of chromophores 

absorbing light above 200 nm. In spite of this, it was reported recently that some of the 

buffers weaken markedly the passing light at some experimental condition 4 . 

GOOD BUFFERS 

In the year 1966 discovered, developed and introduced Goods research team the special 

group of zwitterionic biological buffers, called Good buffers 2 . Later, in 1980, was patented 

and introduced a new generation of biological buffer compounds based on the zwitterionic 

character of Good buffers 5 . Good buffers stabilise pH of solution and they are used as protein 

solubilization agents in aqueous solutions 6 , too. The aforesaid buffers are non-toxic to cells, 

they are not interferenced through biological membranes (for example: penetration, 

adsorption surface, solubilization, etc.). The pKa values of these biological buffers lie at or 

near physiological pH area 2, 5 . 

Popularity of using of biological buffers in analytical chemistry follows from two 

phenomenons. At first it is a photometric detection that is accounted as a very frequently and 

popular in contemporary modern instrumental analytical methods. The second phenomenon is 

the fact that biological buffers are free of chromophores absorbing ultraviolet light above 

200 nm. Regardless it was detected modify of intensity of the passing UV-light beam that 

passed through aqueous solutions of same biological buffers in the short wavelength area 4, 7 . 

The strain of our study is the verification of this finding for three most frequently and popular 

used zwitterionic Good buffers, 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(Nmorpholino)ethanesulfonic 

acid (MES), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic 

acid (CAPSO), see Fig. 1 1, 7 and Table 1 1 . 



A B C 

Fig. 1: The structure of research biological buffers: A) MOPS, B) MES and C) CAPSO 1 . 

Table 1: Inquired zwitterionic GOOD buffers 1 . 

Buffer Formula M W 

(g.mol -1 ) 

pKa 

(25 °C) 

pH range 

MOPS C7H15NO4S 209.26 7.2 6.5 – 7.9 

MES C6H13NO4S 195.24 6.1 5.5 – 6.7 

CAPSO C9H19NO4S 237.32 9.6 8.9 – 10.3 


The UV-Vis spectrophotometer VARIAN – CARY 50 Probe (Varian BV, The 

Netherlands) equipped with the quartz cell of 1 cm optical path length served for photometric 

measurements in the range of 190-300 nm. The acquired experimental data were processed by 

the program Cary WinUV, the program equipment to the spectrometer Varian (Varian BV, 

The Netherlands). The examined buffers (MES, MOPS and CAPSO) are from Sigma-Aldrich 

(Prague, Czech Republic). The buffer solutions were prepared from redistilled water and 

theirs concentration was between the value 4 and 200 mmol.l -1 . The pH of researched 

solutions was adjusted with sodium hydroxide (Lachema, Brno, Czech Republic). The 

experimental temperature has the value 25 °C (common laboratory temperature). 

RESULTS 

Investigated zwitterionic biological Good buffers, MES, MOPS and CAPSO cover almost 

completely pH range of the Good buffers applicability. The investigated buffers are free of 

chromophores absorbing light above 190 nm disregarding their chemical identity and pH. 

Weakening of a light beam passing through their aqueous solutions cannot be therefore 

classified the light absorption even if it is displayed in absorbance units by the used 

instrument. Three pH units below their pKa values, the buffers are only in the zwitterionic 

form considering the measurement precision. At pH = pKa, the concentration of the 

zwitterionic form and of the anionic form is identical. At pH = pKa + 3, practically only 

anions exist in solution. 

Experiments with purely zwitterionic solutions of MES, MOPS (Fig. 2) and CAPSO 

revealed that their formal absorbance increases with both increasing concentration and with 

the decreasing wavelength of the passing light. The maximum measured absorbance depended 

on the dissolved compound. In the range of usually utilized concentrations of Good buffers, 

ranging from 20 to 60 mmol.l -1 , the light absorption was acceptably low above 200 nm. Zero 

absorbance was obtained at the wavelength of 250 nm and higher disregarding the compound 

and its concentration. 



Fig. 2: Typical course of the dependence of the formal absorbance of dissolved 

zwitterionic buffers on the passing light wavelength documented on the example of MOPS. 

The MOPS concentration is given as a parameter: 1) 4.1 mmol.l -1 , 2) 50 mmol.l -1 , 3) 

100 mmol.l - 1 and 4) 200 mmol.l -1 . 

The (pH-pKa) difference affects the light beam weakening dramaticly at concentrations up 

to pH = pKa (Fig. 3). At pH > pKa, the absorbance rises up much less. The dependences of 

formal absorbance at pH = pKa and pH = pKa + 3 on the light beam wavelength differ 

therefore only slightly (Fig. 4). The steepness of the concentration dependence of the apparent 

absorbance rises up with the decreasing wavelength (Fig. 5) as may be expected from 

previous experiments. 


zwitterionic buffers on the buffer pH documented on the example of MOPS. The wavelength of 

the passing light is given as a parameter: (A) 1) 200 nm, 2) 220 nm and 3) 250 nm, 

(B) 1) 210 nm and 2) 230 nm. 




zwitterionic buffers on the buffer pH at 1) pH = pKa and 2) pH = pKa + 3 documented on the 

example of MOPS. 


zwitterionic buffers on their concentration documented on the example of MOPS. The 

wavelength of the passing light is given as a parameter: (A) 1) 200 nm, 2) 220 nm and 3) 

250 nm, (B) 1) 210 nm and 2) 230 nm. 

CONCLUSIONS 

Photometric experiments evidenced general aggregation capability of investigated 

zwitterionic Good buffers MES, MOPS and CAPSO and, in agreement with the previous 

communication 4 , dramatic increase of this capability in the pH range in which the 



concentration of the anionic form of the buffer approaches 50 % of its total concentration. 

Formation of aggregates from dissolved MES, MOPS and CAPSO, which disperse the 

passing UV-light beam, is a possible interpretation of presented experiments. Conductivity 

measurements not presented here evidence that the aggregates are not micelles. This finding 

controverts the explanation proposed in the article 4 . Presented results of photometric 

experiments evidence correctness of experiments given in Ref. 4. The results also substantiate 

continuation of the presented research. 

REFERENCES 

1. Sigma-Aldrich catalogue 2000-2001. 

2. Accessible in the Internet: http://www.jtbaker.com/biopharm/biopharm_buffer_bio.html 

[citation 22 February 2005]. 

3. Ferguson, W.J., Braunschweiger, K.I., Braunschweiger, W.R., Smith, J.R., McCormick, 

J.J., Wasman, C.C., Jarvis, N.P., Bell, D.H. and Good, N.E.: Anal. Biochem. 104 (2), 300 

(1980). 

4. Vespalec, R., Vlčková, M., Horáková, H.: Aggregation and intermolecular interactions of 

biological buffers observed by capillary electrophoresis and UV photometry. Journal of 

Chromatography A, 2004, vol. 1051, p. 75 - 84. 

5. Good, N.E., Winget, G.D., Winter, W., Connolly, T.N., Izana, K., Singh, R.M.M.: 

Biochemistry 5, 467 (1966). 

6. Hames, B.D., Rickwood, D.: Gel Electrophoresis of Proteins. 2-nd edition, p. 153, OIRL 

Press, Oxford, 1990. 

7. Beynon, R.J., Easterby, J.S.: Buffer Solutions: The basics. p. 48, 69 – 70, 74, 78, OIRL 

Press, Oxford, 1996. 





















Investigation of environmental and varietal influences on distribution of 

starch granules in barley kernels by GFFF and laser particle size analysis 

Ing. Karel Mazanec 

Supervisor: RNDr. Josef Chmelík, CSc. 

Institute of Material Chemistry, Faculty of Chemistry, Brno University of Technology, 

Purkyňova 118, 612 00 Brno, Czech Republic, e-mail: mazanec@iach.cz 

INTRODUCTION 

The beer production has great tradition and is much extended in this country. Because the 

production of beer is very energy-consuming, any decrease of input and process loads keeping 

taste and qualities of resulting product may play a significant role at volumes of production. 

Therefore understanding of processes joined with brewing and malting may give promising 

results. 

Starch is formed in green plants as the final product of photosynthesis. Starch is one of the 

barley components important for its malting quality. The starch present in barley kernels is in 

form of large (type A) and small (type B) granules [1]. Distribution of starch granules in 

barley kernels plays important role in technological processes of beer production. Because the 

small granules are less susceptible to enzymatic digestion and causing technological problems 

during brewing (they can form starch haze and can block filtration beds in lauter tuns) for a 

long time it was presumed that the malting varieties experimentally selected as 

technologically suitable for brewing should have only minimal content of small starch 

granules. The so-called malting varieties of spring barley have different distribution of size of 

starch granules in comparison to other varieties. Measurement of starch particles size 

distribution gives good idea of technological quality of individual malting varieties. Several 

techniques have been generally employed for this purpose. Nevertheless implementation of 

field-flow fractionation (FFF) fitted the needs of low cost and short separation time. 

MATERIALS AND METHODS 

Plant material and sample preparation 

Twelve spring and winter barley varieties (obtained from the testing stations of the Central 

Institute for Supervising and Testing in Agriculture (CISTA) of the Czech Republic) were 

used for isolation and size measurements. Each year the above given variety assortment was 

grown in three testing stations. External conditions influenced all varieties equally. 

Monitoring was conducted for three years (2001-2003) and thus 108 samples were processed 

during whole project. This work shows data from the last harvest of this three years project. 

Starch granules were isolated by way described in [2]. Barley grains were ground on the 

adjusted milling equipment, stirred with 0.02M HCl and refrigerated for 15 hours (4 °C). 

When taken out pH was adjusted with 0.2M NaOH to 5.0 ± 0.2 and cellulase and β-glucanase 

were added, content was shaken in bath (30 °C for 3 hours), then pH was adjusted to 

11.0 ± 0.2 (50 °C for 90 minutes). After this pH was adjusted to 7.0 ± 0.2 and the content was 

filtered. Filtrate containing starch was poured into a storage pot. Obtained starch was 

repurified with an aqueous solution of CsCl (80%). Isolated starch was purged with acetone 

and dried out. Suspension of starch granules in solvent (10 -3 % SDS – sodium dodecyl sulfate) 

were measured on GFFF and with commercial particle size and shape analyzer. 



Gravitational Field-Flow Fractionation 

Gravitational FFF is the experimentally simplest and cheapest among the family of FFF 

techniques. Separation in GFFF is based on combination of gravitational field and a nonuniform 

flow velocity profile of carrier liquid in channel (fig 1). Gentle experimental 

conditions (low pressure and weak force field) and the possibility to use isotonic buffer 

solutions as carrier liquids make GFFF unique possible also for separations and purifications 

of biological samples [3]. 

Plexiglass 

Glass 

Detector 

Spectra 100 

(λ = 470 nm) 

Sample injection 

High-pressure 

pump 

HPP 4001 

Fig.1: Principe of Gravitational FFF Fig.2: Arrangement of GFFF channel 

The arrangement of GFFF channel is shown in the fig. 2. The separation channel was cut in 

0.150 mm thick foil and inserted between two glass plates. The channel dimensions were 360 

mm x 20 mm x 0.150 mm. The carrier liquid and the sample were introduced into the channel 

via an inlet capillary situated at the channel head, and taken out via an outlet capillary located 

at the end of the channel. The high-pressure pump HPP 4001 (Laboratory Instruments, 

Prague, Czech Republic) was used to introduce the carrier liquid into the channel. As the 

detector the UV/VIS Spectra 100 (Development Workshop AS CR, Prague, Czech Republic) 

operating at 470 nm was used. Data were collected on 386’s (Intel, Santa Clara, CA, USA) 

PC equipped with digitalization card. 

Starch samples were suspended (the concentration of 10 mg/ml) in 10 -3 % SDS and soaked 

for at least 24 h. Then the sample was sonicated for 1h prior to the FFF experiment. Samples 

were injected during the stopped flow. Just after injection, a loading flow at rate 0.2 ml/min 

was applied for 10 s. Relaxation time period (stopped-flow period) was for 1.5 min. After this 

a linear flow rate of 0.8 ml/min was applied. 

CIS-100 Particle Size and Shape Analyzer 

The CIS-100 (Ankersmid Ltd., Yokneam, Israel) is a Computerized Inspection System for 

Particle Size Analysis (PSA) in the range of 0.5-3600 micron employing laser and video 

measurement channels. Together, both channels provide all necessary information to analyze 

and characterize spherical, non spherical and elongated particles of large variety of materials. 

A He-Ne laser beam in the first channel is scanned circularly by a rotating wedge prism and 

focused down to a 1.2 μm spot, which scans the sample measurement volume. As the particles 

(moving or stationery) within the sample volume are individually bisected by the laser spot, 



interaction signals are generated. These signals are then detected by a PIN photodiode. Since 

the beam rotates at a constant speed, the duration of interaction provide a direct measurement 

of each particle's size. The interaction signals are collected by a data acquisition card and 

analyzed in 600 discrete size intervals. Pulse analysis algorithms are employed to reject outof-focus 

and off-center interactions. 

The shape analysis channel uses a CCD video camera microscope to provide an optimal 

image for processing. Illumination is provided by a synchronized strobe light. Acquired 

images are passed to a frame grabber card for analysis, and then displayed on a monitor for 

viewing. 

Samples were prepared at the concentration of 50 mg/ml in 10 -3 % SDS. In storing reservoir 

of instrument they were diluted to 0.5 l and were sonicated with nominal frequency 65 KHz 

during whole analysis. The stirring was set up to 226 rpm. The pump drove the suspension to 

the channel at flow rate 190 ml/min. 

RESULTS 

The results of both methods presented in this work give principally the same interval 

ranges of starch granule size in barley kernels as in the preceding works [4]. The main size 

fractions of starch granules were detected in the areas of 0-8 μm with peak around 3 μm (9 

min in case of GFFF) and 8-30 μm with maximum around 20 μm (4 min). This classification 

resulted from the natural bimodal distribution of starch granule size. The smallest amount of 

starch particles is among 7.5 and 8 μm, so 8 μm was taken as a boundary between small (A) 

and large (B) particles. Due to the different principle of measurement of both methods, the 

results are not comparable in absolute values. Nevertheless the starch distributions of 

individual barley varieties obtained by both methods correspond well. 

Cultivar Winter/Spring Row Quality GFFF PSA 

CHT LIB STA CHT LIB STA 

Tolar Spring 2 VG 0,612 0,584 0,753 3,921 3,228 4,453 

Orthega Spring 2 Feed 0,503 0,560 0,970 6,358 6,123 5,988 

Heris Spring 2 Feed 1,009 0,709 0,992 7,496 4,015 9,799 

Jersey Spring 2 VG 1,388 0,913 1,252 7,091 5,317 5,083 

Prestige Spring 2 VG 0,748 0,739 1,205 4,534 4,086 5,631 

Scarlett Spring 2 G 0,891 1,042 0,720 6,199 7,834 7,711 

Luran Winter 6 Feed 0,961 0,684 1,215 5,460 5,277 5,911 

Luxor Winter 6 Feed 1,021 0,903 1,543 7,842 7,123 9,020 

Nelly Winter 6 Feed 1,089 1,098 1,328 8,328 7,354 7,150 

Vilna Winter 2 Feed 1,589 1,536 1,467 6,402 6,117 7,039 

Camera Winter 2 Feed 2,052 2,844 1,250 8,921 8,662 6,764 

Tiffany Winter 2 G 2,974 2,541 2,745 9,163 10,521 6,918 

Tab.: shows results of individual barley varieties. Quality means: Feed, good malting quality 

(G) and very good malting quality (VG). Values are A/B peaks areas from individual test 

stations. 



The set of the studied varieties was split into three groups according to the results obtained 

by the GFFF and PSA methods. Winter, two-row varieties Tiffany, Camera and Vilna 

belonged to a group of varieties with the smallest ratio of small starch granules. Conversely, 

the two-row varieties of spring barley Heris, Jersey, Tolar and Orthega formed a group with 

the largest ratio of small starch granules. This study did not confirm the assumption that the 

malting barley varieties in the studied set compared to the non-malting varieties had a lower 

number of small starch granules. 

The variety of barley affects the starch granule size distribution most significantly. Effect 

of locality and local environmental conditions in the testing stations during growing of the 

seeds and year is significantly lower. 

CONCLUSIONS 

The isolated starch granules from kernels of twelve barley varieties cultivated in three 

different regions were analyzed using gravitational FFF instrument and particle size and shape 

analyzer. Significant intervarietal and also some interregional and year differences were 

detected in starch granule size distribution. Because of different principle of both methods the 

results are not the same in absolute values, but show the same tendencies. This study did not 

confirm the assumption that the malting barley varieties in the set observed compared to the 

non-malting varieties had a smaller number of small starch granules. 

FUTURE WORK 

Because malting varieties of barley have other enzymatic apparatus quality, future work will 

be focused on characterization of enzymatic activity and starch degradation by defined 

enzymes by combination of separation techniques (IEF, GPC) with MALDI-TOF/TOF MS. 

REFERENCES 

[1] May L.H., Buttrose M.S.: Physiology of cereal grain. II. Starch formation in the 

developing barley kernel. Aust. J. Biol. Sci. 12, 146 - 159, 1959. 

[2] Psota V., Bohačenko I., Pytela J., Vydrová H., Chmelík J.: Determination of size 

distribution of size distribution of barley starch granules using low angle laser light scattering. 

Plant production 46 (10), 433-436, 2000. 

[3] Giddings J.C.:Field-Flow Fractionation – Analysis of macromolecular colloidal, and 

particulate materials. Science 260 (5113), 1456 – 1465, 1993. 

[4] MacGregor A.W., Fincher G.B.: Carbohydrates of the barley grain. MacGregor, A.W., 

Bhatty, S.R. (eds.) Barley: Chemistry and Technology. AACC St.Paul, MN, USA. 1993. 



A granules 

(8 – 30 μm) 

0 5 10 

Fig.3: GFFF curves of a) Tiffany, b) Luxor and c) Tolar varieties 

a) 

b) 

0 10 20 30 

Size [μm] 

B granules 

(0 – 8 μm) 

Time [min] 

0 10 20 30 

Size [μm] 

Fig.4: Differential and integral PSA volume distribution curves of a) Tiffany, and b) Tolar 

varieties 

a) 

b) 

c) 

100% 



























INFLUENCE OF TRIETHYLALUMINIUM COCATALYST ON 

INITIAL KINETIC OF PROPENE POLYMERIZATION 

Ing. Miroslav Skoumal 1 , 3 th year Ph.D. student 

Supervisor: Ing. Jan Kratochvíla, CSc. 2 

1 Institute of Materials Chemistry, Faculty of Chemistry, Brno University of Technology, 

Purkyňova 118, 612 00 Brno, Czech Republic, e-mail: skoumal@fch.vutbr.cz 

2 Polymer Institute Brno, Tkalcovská 2, 656 49 Brno, Czech Republic 

INTRODUCTION 

The triethylaluminium (TEA) influence on the initial stage of propene polymerization with 

heterogeneous TiCl4/MgCl2-supported Ziegler-Natta catalysts is presented in this study. It is 

generally known that alkylaluminium compound (TEA is the most common) plays the 

essential role in active center formation which is able to produce poly-α-olefins under the low 

temperature and pressure. A wide range of experimental studies, summarized in several 

reviews [1,2], was performed for the evaluation of the alkylaluminium role in Ziegler-Natta 

catalysts. It was defined that the activation of the catalyst proceeds in two steps. First, 

alkylaluminium reduces Ti (IV) to Ti (III). Then it alkylates the Ti (III) forming the first 

metal-polymer bond accessible for monomer insertion. Due to high reactivity of TEA, it was 

also found that a large number of side reactions with TEA proceeds during the polymerization 

such as transfer (broadening of MWD) or termination (further reduction to non-propagative 

Ti (II) species). 

The heterogeneous nature of MgCl2-supported catalyst, the presence of different types of 

active centers on its surface, the activity decay during the polymerization, influence of 

catalyst compounds and experimental conditions on the rate of polymerization make difficult 

to study the polymerization kinetics with this catalysts. These problems are significant 

especially during the first seconds of polymerization, where the MgCl2-supported catalyst 

exhibits high activity. The investigations of the initial kinetics recently became a subject of 

intensive research, assuming that the catalyst behavior during the first seconds of 

polymerization determines its efficiency and consequent polymer particle morphology. 

For the better understanding of the reactions involved in the initial period, the novel 

method for the assessment of initial polymerization kinetic and determination of the number 

of active sites was developed [3]. This method allows us to evaluate the kinetic profiles from 

1 to 300 seconds with high accuracy and reproducibility. 


The presented study was performed with commercial high-activity TiCl4/MgCl2-supported 

Ziegler-Natta catalysts commonly used for polypropene production in industry. Catalyst was 

slurried in mineral oil and stored in glass vessels covered by a nitrogen flow. Polymerization 

grade propene originated from Chemopetrol Litvínov. Contents of critical impurities CO and 

COS were less than 10 ppb, water and oxygen levels were under 0.1 ppm. Triethylaluminum 

(TEA) originating from Witco, GmbH (Germany) was dissolved in heptane and kept in glass 

vessels similarly as the catalyst. n-Heptane used as the polymerization medium was purchased 

from Aldrich (99 % spectrophotometric grade). Prior to polymerization, removal of water and 

oxygen was conducted inside the reactor by stripping. About 10 % of heptane was stripped- 



off by high-purity nitrogen (70°C, 40 min) to remove water and oxygen. The contents of O2 

and H2O in the nitrogen were under 0.5 ppm. 

The slurry polymerizations were carried out in a water-jacked glass reactor (240 ml). The 

reactor was equipped with magnetic stirrer and connected to a low-pressure polymerization 

apparatus. The apparatus was under the PC control, operating the thermostatic circuit, 

switching-on the magnetic stirrer, injection of a quenching agent and measuring the 

polymerization time. 

The PC control operates the automatic injection of a quenching agent into the reactor at a 

pre-set time. The main part of the injector was a syringe connected by tube to a solenoidoperated 

valve. Before the polymerization, the syringe was loaded with ca. 0.5 ml of a 

quenching agent (methanol, 2-propanol, and concentrated hydrochloric acid, volume ratio 

4:4:1) and connected to the neck of the glass reactor. After the preset polymerization time had 

elapsed, the solenoid valve was automatically opened. Water pressure pushed down the 

syringe piston and injected the quenching agent into the reactor. This ensured a fast and 

complete termination of the polymerization reaction. 

Polymerizations were carried out in ca. 180 ml of previously stripped and cooled heptane 

saturated with propene at 30°C (0.61 mol C3H6/l) under a propene flow blanket at 

atmospheric pressure. The defined amount of TEA was introduced via a syringe and the 

content of the reactor was homogenized by short stirring. Next, the stirrer was turned off and 

the appropriate amount of catalyst oil slurry was slowly dosed into the reactor by means of a 

syringe with a long metal injector leading close to the bottom of the reactor. The application 

of viscous mineral oil prevents sedimentation of homogenized catalyst slurry during the 

charging procedure. Thanks to a nearly zero mass transfer between the oil and the heptane 

phases, the catalyst remained separated from cocatalyst (TEA) dissolved in the heptane phase 

until the stirrer was turned on (Figure 1). 

Figure 1: Schematic drawing of the oil/heptane diffusion interface before the start of stirring. 

Typically, 10-15 s after the catalyst was dosed the magnetic stirrer was turned on. The 

stirrer speed was high enough to homogenize the oil/heptane mixture within ca. 0.2 s. The 

stirrer was operated at 600 rpm during polymerization and polymerization conversion 

corresponded to a max. 5 % of the monomer dissolved in the polymerization slurry; therefore 

the effect of mass transfer through the gas-liquid interface was negligible. It was proven that 

the reproducibility, in the range of polymer yields 10 – 300 mg and polymerization times 

1 − 300 s, was always within about 7 %. For more information see [3]. 


Sekce DSP 2005, strana 160


The presented study was focused on influence of starting TEA concentration on the 

catalyst behavior during first seconds of propene polymerization. Using the recently 

developed method for the initial kinetic determination, it was observed that the applied 

MgCl2-supported catalyst exhibits the decay kinetic profile with high initial activity followed 

by rapid deceleration to the level of 15% of starting value during 10 seconds 

([TEA] = 0.0092 mmol/ml). The high level of initial activity is a negative phenomenon, 

accompanied with intensive releasing of heat, which could cause an overheating of polymer 

particles and consequent adverse changes in particle morphology. Typical example is 

formation of solid polymer particle agglomerates dangerous mainly in industrial reactors. 

Yield [g-PP/mmol-Ti] 

Activity [g-PP/(mmol-Ti*h)] 

30 

25 

20 

15 

10 

5 

0 

[TEA] = 0.0092 mmol/ml 

[TEA] = 0.0044 mmol/ml 

[TEA] = 0.0007 mmol/ml 

0 20 40 60 80 100 120 

Time [s] 

7000 


5000 

4000 

3000 

2000 

1000 

0 

[TEA] = 0.0092 mmol/ml 

[TEA] = 0.0044 mmol/ml 

[TEA] = 0.0007 mmol/ml 

0 20 40 60 80 100 120 

Time [s] 


Activity [g-PP/(mmol-Ti*h)] 

10 

8 

6 

4 

2 

0 

7000 


5000 

4000 

3000 

2000 

1000 

[TEA] = 0.0092 mmol/ml 

[TEA] = 0.0044 mmol/ml 

[TEA] = 0.0007 mmol/ml 

0 2 4 6 8 10 12 14 16 18 20 

Time [s] 

0 

[TEA] = 0.0092 mmol/ml 

[TEA] = 0.0044 mmol/ml 

[TEA] = 0.0007 mmol/ml 

0 2 4 6 8 10 12 14 16 18 20 

Time [s] 

Figure 2: Kinetic profiles for different starting TEA concentrations presented as dependence 

of the polymer yield and its derivative (activity) on polymerization time. Polymerization 

conditions: Atmospheric pressure, temperature 30°C, heptane 180 ml, propene: 0.61 mol/l. 

The kinetic profiles from the first 120 s of polymerization indicate that the initial activity 

directly depends on the starting TEA concentration in the solution. Changes in TEA 

concentration cause significant effect on the initial activity and the following deceleration 

period. The Figure 2 shows that the 50% decrease in TEA concentration reduces the initial 

activity to one-half while the activity after 120 s is comparable. It suggests that the 

unfavorable high initial activities of MgCl2-supported catalyst can be controlled by 

application of a suitable starting TEA concentration or by catalyst prepolymerization under 

the mild conditions. Further lowering of the cocatalyst concentration reduces the initial 

activity to 15%. However, such TEA lowering also causes more significant decrease in the 

catalyst productivity. 



Obtained polymer samples were utilized for the determination of molecular weight 

distribution by GPC (gel permeation chromatography) analyses. Then the number of active 

sites was evaluated from the number-average molecular mass values by a method based on 

the number of macromolecules [3-5]. The active sites are determined from the dependence of 

the number of macromolecules versus polymer yield (Figure 3), where the intercept of the 

linear extrapolation to zero corresponds to the number of propagative centers formed at the 

beginning of polymerization. 

N [mol/mol-Ti] 

0.25 

0.20 

0.15 

0.10 

0.05 

0.00 

y = 0.0105x + 0.0259 

R 2 = 0.9977 

y = 0.0107x + 0.012 

R 2 = 0.9955 

y = 0.0102x + 0.0045 

R 2 = 0.9965 

[TEA] = 0.0092 mmol/ml 

[TEA] = 0.0044 mmol/ml 

[TEA] = 0.0007 mmol/ml 

0 5 10 15 20 


Figure 3: Dependence of the number of macromolecules N versus polymer yield extrapolated 

to zero. 

It was found that the number of active sites decreases linearly in dependence on the 

starting TEA concentration. From the determined values of initial catalyst activity A(0) and 

number of active sites [C*] the propagation rate constant kp can be calculated for each TEA 

concentration. The equation for the kp evaluation follows from the expression [4]: 

( 0) 

= k ⋅[ 

M] 

[ C* 

] 

A p ⋅ 

where [M] is the monomer concentration. 

Table 1: Calculated values of propagation rate constant kp for each TEA concentration. 

[TEA] 

A(0) 

[C*] 

kp 

[mmol/ml] [mol-C3/(mol-Ti*s)] [mol-%] 

[l/(mol*s)] 

0.0092 47.8 2.6 3000 

0.0044 22.7 1.2 3100 

0.0007 7.2 0.5 2700 

The Table 1 shows that contrary to the number of active sites the kp value remains almost 

unchanged under the different cocatalyst concentrations. From the above-presented results are 

obvious that the TEA effect on the catalyst activity is more affected by the change of the 

number of active sites than by propagation rate constant. This finding is in a good agreement 

with results obtained by other researchers using different procedures [6]. 



The slope of linear equation in Figure 3 depends on the intensity of transfer reactions 

during polymerization. The higher extent of the transfer reactions causes the increase of the 

slope value, at negligible level of the transfer reactions the linear dependency (N vs. yield) 

would be parallel with x-axis. The results in Figure 3 show that the TEA concentration did not 

affect the slope of the fitted lines. This observation suggests that TEA does not act as an 

active transfer agent during the polymerization under the mild conditions (atm. pressure, 

30°C). So, it could be assumed that the polymer chain transfer reaction proceeds mainly with 

monomer or solvent. 

LITERATURE 

[1] Dusseault J. J. A., Hsu Ch. C., J. Macromol. Sci., Rev. Macromol. Chem. Phys. C33, 103 

(1993) 

[2] Albizzati E., Giannini U., Collina G., Noristi L., Resconi L., Polypropylene Handbook 

Part I., ed. Moore E. P., p. 11, Hanser Publishers, Munich Vienna New York 1996 

[3] Skoumal M., Cejpek I., Cheng C. P., Macromol. Rapid Commun. 26, 357 (2005) 

[4] Kashiwa N., Yoshitake J., Makromol. Chem., Rapid Commun. 3, 211 (1982) 

[5] Chu K.-J., Eur. Polym. J. 34, 577 (1998) 

[6] Liu B., Matsuoka H., Terano M., Macromol. Rapid Commun. 22, 1 (2001) 



PHYSICO-CHEMICAL PROPERTIES OF PLASMA-POLYMERIZED 

TETRAVINYLSILANE 

Mgr. Jan Studýnka 

Supervisor: doc.RNDr. Vladimír Čech, Ph.D. 

Vysoké učení technické v Brně, Fakulta chemická, Ústav chemie materiálů, Purkyňova 

118, 612 00 Brno, email: xcstudynka@fch.vutbr.cz 

INTRODUCTION 

Plasma-Enhanced Chemical Vapor Deposition (PE CVD) is a suitable technique for 

preparation of thin films on a basis of organosilicones [1,2]. The technique enables 

reproducible deposition of plasma polymer films with desired physico-chemical properties [3] 

by changing the deposition conditions (power, monomer flow rate, pressure). 

Tetravinylsilane (TVS) was used as a monomer for deposition of pp-TVS films for the 

first time.The helical coupling system [4] was applied to deposit single films using an RF 

glow discharge operated in pulsed mode. Various spectroscopic methods were used to 

evaluate atomic composition and content of the chemical bonds. Ellipometric measurements 

were employed to determine thicknesses of the films and optical constants of the layers. 

Selected mechanical properties of single films were determined from nanoindentation 

measurements. 

EXPERIMENTAL DETAILS 

Tetravinylsilane, Si–(CH=CH2)4 

(TVS), was used as the monomer. The 

silicon wafer was pretreated by O2 

plasma (5 sccm, 4 Pa, 25 W) for 10 min 

in order to improve the film adhesion. 

Pulsed plasma was operated at 

conditions given in Table 1. 

The elemental composition of thin 

films was studied by conventional and 

Table 1. Deposition conditions tested for preparation of 

pp-TVS films using a stable plasma. 

Frequency 13.56 MHz 

ton, toff 

1 ms, 1 – 999 ms 

Effective power 0.05 – 10 W 

Effective power density 8 ×10 -4 – 1 ×10 -1 W cm -3 

Basic pressure 2 × 10 -3 Pa 

Process gas pressure 0.1 – 4.4 Pa 

Monomer vapor flow rate 0.45 sccm 

resonant Rutherford Backscattering Spectrometry (RBS) and Elastic Recoil Detection 

Analysis (ERDA) methods. Chemical structure of plasma polymer was investigated using a 

NICOLET IMPACT 400 Fourier transform infrared (FTIR) spectrophotometer. Measuring 

range was from 400 to 4000 cm -1 . We employed a Nano Hardness Tester (NHT) by CMS 

Instruments in order to carry out nanoindentation tests. The Young’s modulus and hardness of 

films were determined from unload-displacement curves measured at 12% of the film 

thickness. 




A) Ellipsometry 

Ellipsometric measurements were performed in order to determine the film thickness and 

optical constants, i.e. refractive index and extinction coefficient, for pp-TVS films. 

Dispersions of refractive index and extinction coefficient for pp-TVS films deposited at 

different effective power are given in Fig. 1. The results correspond to pp-TVS films with a 

thickness about 400 nm deposited at the same deposition conditions but different power. The 

value of refractive index at the wavelength of 633 nm increased with power from 1.63 

(0.05 W) to 1.75 (10 W). There is a growth of the refractive index and extinction coefficient 

in ultraviolet region. A 

different shape of 

dispersion in ultraviolet 

region for the refractive 

index corresponding to a 

film deposited at higher 

power (10 W) may be 

caused by higher 

absorption in UV region. 

The extinction coefficient 

rose sharply with the 

power from 0.09 

(0.05 W) to 0.37 (10 W) 

at the wavelength of 240 

nm. 

300 400 500 600 700 800 

B) Elemental composition and chemical structure of films 

Atomic concentration [at%] 

Refractive index 

1.95 

1.90 

1.85 

1.80 

1.75 

1.70 

1.65 

1.60 

0.40 Film thickness: 0.4 μm 

0.35 

0.05 W 

0.5W 

0.30 

5 W 

0.25 

10 W 

300 400 500 600 700 800 

Wavelenght 

Wavelenght 

Figure 1. Dispersions of refractive index and extinction coefficient 

for pp-TVS films deposited at different power. 

The pp-TVS films of a film thickness about 1 µm were used to characterize chemical 

70 


50 

40 

30 

20 

10 

0 

C 

O 

Si 

H 

C/Si 

O/Si 

0.1 1 10 

Effective power [W] 

Figure 2. Elemental composition of pp-TVS films deposited 

at different power. 

14 

12 

10 

8 

6 

4 

2 

0 

Extinction coefficient 

0.20 

0.15 

0.10 

0.05 

0.00 

-0.05 

properties of the plasma polymer. 

Elemental composition of films was 

determined from RBS and ERDA 

spectra. The RBS spectra were 

evaluated by computer code GISA 3 

[5] and the ERDA ones by 

SIMNRA code [6] both using crosssection 

values from SigmaBase. An 

estimated accuracy of evaluated 

concentration was up to 5 at%. A 

dependence of atomic 

concentrations on the effective 

power is given in Fig. 2. The 

concentration of hydrogen atoms 



was about 56 at% and did not vary with the effective power. The same trend or a slight 

decrease was observed for the concentration of silicon atoms in plasma polymer deposited at 

different power. A mean concentration of silicon atoms was about 5 at%. However, the 

oxygen concentration decreased rapidly at the expense of carbon atoms with increasing 

power. The concentration of oxygen atoms descended from 13 at% (0.05 W) to 1 at% (10 W), 

while carbon concentration rose from 27 at% (0.05 W) to 42 at% (10 W). The C/Si ratio 

indicated great changes in organic/inorganic character of plasma polymer with enhanced 

power. The film deposited at a power of 10 W was hydrogenated amorphous carbon 

containing a small amount of silicon-carbide phase. 

Table 2. Assignment of IR absorption bands. 

Absorbance [a.u.] 

Adsorption 

band 

Wavenumber 

[cm -1 ] 

Assignment 

A 3650 – 3200 O–H stretching 

B 3000 – 2800 CH2, CH3 stretching 

C 2122 Si–H stretching 

D 1714 C=O stretching 

E 1591 C=C stretching in vinyl 

F 1461 CH2 scissoring 

G 1412 CH2 deformation in vinyl 

H 1255 CH2 waging in Si–CH2–R 

I 1100 – 1000 Si–O–C stretching 

J 1015 =CH wagging in vinyl 

K 959 =CH2 wagging in vinyl 

L 845 Si–H bending 

M 732 Si–C stretching 

A 

B 

0.1 W 

0.5 W 

2.5 W 

10 W 

C 

D 

G 

E 

F 

J 

L 

M 

I K 

3500 3000 2500 2000 1500 1000 500 

Wavenumber [cm -1 ] 

H 

TVS molecule does not contain 

any oxygen and thus, the oxygen 

atoms incorporated in plasma 

polymer could have their origin in 

pretreatment procedure using O2 

plasma. Most likely, the residual 

oxygen was desorbed from the 

wall of plasma chamber during 

the deposition process and was 

incorporated in plasma polymer. 

We estimated the flow rate of 

desorbed oxygen at < 0.05 sccm 

from the pressure increase in 

separate deposition chamber. The 

results on elemental composition 

revealed that the residual oxygen 

was effectively built in plasma 

polymer only if a lower power (< 

5 W) was used. The lower power 

the higher oxygen concentration 

in pp-TVS film. 

Typical infrared spectra of pp- 

TVS films deposited at different 

power are given in Fig. 3. 

The assignment of IR 

absorption bands using Ref. 7 and 

8 was summarized in Table 2. One 

of the dominated peaks, the 

absorption band B, was assigned 

to CH2, CH3 stretching vibrations. 

However, both the species cannot 

be distinguished with respect to 

symmetric character of the band 

Figure 3. Infrared spectra of pp-TVS films prepared at 

Sborník different soutěže effective Studentské power. tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006 


Young's modulus [GPa] 

B. The intensity and area of absorption bands A, D, and I, corresponding to species such as 

OH, C=O, and Si–O–C, respectively, descended with increased power and the trend was in 

good agreement with a descent of oxygen concentration in plasma polymer revealed by RBS 

measurements. A decrease of bands C, H, L, and M assigned to species containing silicon 

atoms was observed as well and the trend corresponded to an increase of C/Si ratio with 

enhanced power. An occurrence of vinyl groups in plasma polymer was evident from 

IR spectra corresponding to pp-TVS films deposited at lower power (≤ 2.5 W). Quantity of 

vinyl groups decreased with enhanced power as a descent of bands E, J, and especially G, K 

indicated. 

C) Nanoindentation 

Load-displacement curves were measured at a depth of 50 nm for all the films with the 

film thickness about 400 nm. Five indents were produced on each sample with spacing 

between the indentations of 5 µm. Nanoindentation measurements were evaluated by Oliver 

& Pharr method [9] in order to determine the Young’s modulus and hardness of pp-TVS 

40 

30 

20 

10 

0 

Film thickness: 0.4 μm 

0.1 1 10 

Effective power [W] 

films. The Poisson’s ratio used for 

plasma polymer was 0.3. The mean 

modulus and hardness was 

calculated from five values for each 

film. The results on mechanical 

properties of pp-TVS films are 

summarized as power dependence in 

Fig. 4. The modulus increased 

almost 3 times and the hardness 

increased almost 4 times with 

enhanced power. The increase of 

modulus could be related to a higher 

crosslinking and/or an alteration of 

microstructure with increasing 

organic character of plasma polymer. 

CONCLUSIONS 

Plasma polymer films of tetravinylsilane were deposited on silicon wafers by plasmaenhanced 

chemical vapor deposition. Pp-TVS films deposited at a flow rate of 0.45 sccm 

(process pressure 1.5 Pa), and an effective power of 0.05 – 10 W were analyzed by 

spectroscopic techniques extensively to elemental composition and chemical structure (RBS, 

ERDA, FTIR). A phase-modulated spectroscopic ellipsometer was employed to determine the 

film thickness and optical constants (refractive index, extinction coefficient) of deposited 

films. The refractive index increased by 7.4% at the wavelength of 633 nm when the power 

was enhanced from 0.05 to 10 W. The extinction coefficient in ultraviolet region (240 nm) 

rose sharply by 4 times with enhanced power (0.05 – 10 W). The results revealed that an 

14 

12 

10 

8 

6 

4 

2 

0 

Hardness [GPa] 

Figure 4. Power dependence of mechanical properties for pp- 

TVS films. 



organic/inorganic character (C/Si ratio) of films and a content of vinyl groups could be 

controlled by deposition conditions precisely. Nanoindentation measurements enabled us to 

evaluate the elastic modulus and hardness of films prepared at different power. The elastic 

modulus and hardness could be varied from 11 GPa (0.05 W) to 30 GPa (10 W) and from 1.4 

to 5.9 GPa for an increased power, respectively. 

Acknowledgements: This work was supported in part by the Czech Ministry of Education 

(contracts COST 527.110 and P12.001) and the Czech Science Foundation (contract 

104/03/0236). 

References 

1. A.M. Wrobel and M.R. Wertheimer, in Plasma Deposition, Treatment, and Etching of 

Polymers, (R. d’Agostino, Editor), Academic Press, New York (1990) p.163. 

2. Y. Segui, in Proceedings of NATO ASI Plasma Processing of Polymers (R. d’Agostino, P. 

Favia, F. Fracassi, Eds.). Acquafredda di Maratea, Kluwer Academic Publ. (1997) p.305. 

3. V. Cech, J. Vanek, J. Zemek, L. Zajickova, and R. Prikryl, Variety of functional 

interlayers utilizable for polymer composites, Proc. 11 th Int. Conf. on 

Composites/NanoEngineering, August 8-14, 2004, Marriott Hilton-Head Beach & Golf 

Resort, South Carolina, USA, pp. 2. 

4. V. Cech, R. Prikryl, R. Balkova, J. Vanek, and A.Grycova, J. Adhesion Sci. Technol. 17 

(2003) 1299. 

5. J. Saarilahti and E. Rauhala: Interactive personal-computer data analysis of ion 

backscattering spectra, Nucl. Instrum. and Methods in Physics Research B64, 734 (1992). 

6. M. Mayer, SIMNRA User`s Guide, Forschungscentrum Julich, Inst. fur Plasmaphysik, 

1998. 

7. D. Lin-Vein, N.B. Colthup, W.G. Fateley, J.G. Grasselli, The Handbook of Infrared and 

Raman Characteristic Frequencies of Organic Molecules, Academic Press, San Diego, 1991. 

8. V.P. Tolstoy, I.V. Chernyshova, V.A. Skryshevsky, Handbook of Infrared Spectroscopy 

of Ultrathin Films, John Wiley & Sons, New Jersey, 2003. 

9. W. C. Oliver, G.M. Pharr, J. Mater. Res. 7 (1992) 1564. 















SPECIATION OF FIVE SELENIUM COMPOUNDS BY 

HPLC-HEATING-UV-HG-AFS 

Ing. Eva Vitoulová 

Supervisor: doc. Ing. Miroslav Fišera, CSc. 

Brno University of Technology, Fakulty of Chemistry, Department of Food Chemistry and 

Biotechnology, Purkyňova 118, 612 00 Brno, e-mail: xcvitoulova@fch.vutbr.cz 

INTRODUCTION 

Selenium is an essential nutrient at low concentrations, but is toxic for humans and animals at 

high doses. It is a component of the enzyme glutathione peroxidase, which is one of the antioxidant 

defence systems of the body, catalyses intermediate metabolic reactions, and inhibits the toxicity of 

some heavy metals. 1 

The effect of the element on human health is highly dependent on the chemical species under 

which it is consumed. Selenium exists in different chemical forms, as inorganic (selenite, selenate) 

and as organic species (selenoaminoacids, selenoproteins), in environmental and biological 

matrices. The nutritional bioavailability and cancer chemoprotective activity of selenium depend on 

the concentration and the chemical form in which it is present. 2, 3 

The absorption of selenium is greater in the case of organic species and the possibility that the 

selenium supplementation of food might protect against the development of cancer in humans has 

generated great interest in the speciation of the various chemical forms in foodstuffs and in dietary 

supplements. 1 Each species adsorbed differently by the human body and has a different tendency to 

bioaccumulate in organisms. 4 

The availability of analytical techniques for the separation and determination of the compounds 

of an element at trace level has gained considerable importance. In this context, hyphenated 

techniques are those most frequently used. For selenium, speciation is necessary because of the 

differing mobilities, toxicities and bioavailabilities of its compounds. 5 

Analytical systems developed for the speciation of selenium species employ a powerful highperformance 

liquid chromatography (HPLC) coupled to a specific atomic detector with a high 

efficiency sample introduction system. 6 Spectrometry methods are those most widely used as a 

detection system. Atomic fluorescence spectrometry (AFS) and inductively coupled plasma - mass 

spectrometry (ICP-MS) have been incorporated with very good results. Atomic spectrometry 

methods are the most widely used because of their high selectivity and sensitivity. 5 

EXPERIMENTAL 

Instrumentation 

The HPLC system consisted of an HPLC pump Gynkotech P580 (Gynkotech, Softron, Germany) 

equipped with a six-port sample injection valve (C&D, Ecom, Prague, Czech Republic) and a 20 μl 

loop for sample introduction. The separation of the selenium species occurred in column Hamilton 

PRP X-100 (250 x 4.1 mm, 10 μm) strong anion exchange column. 

Hydride generation atomic fluorescence spectrometry (AFS) was performed using PSA 



Millenium Excalibur continuous system (PS Analytical, Orpington, Kent, UK) with a PSA 10.570 

UV cracker and heating system (Chromspec, Czech Republic) (Fig. 1.). Measurements were carried 

out using a boosted discharge hollow cathode lamp for selenium (Photron, Pty. Ltd., Australia) at 

196,0 nm line, with a 20,0 mA primary current and 25,0 mA boost current. 

A Heidolph Instruments – Promax 1020 and a Heidolph Instruments – Incubator 1000 

(Germany) were used for extracting the Se-compounds from the samples. The samples were 

centrifuged in Boeco U 32 – R (Hettich zentrifugen, Germany). 

The supernatants were filtred through a 0,45 μm PTFE membrane Minisart SRP 15 (Sartorius, 

UK). 

Fig. 1 The scheme of interconection between HPLC-Heating-UV-HG-AFS 

Reagents and materials 

All the solutions were prepared with deionised water (18,2 MΩ.cm) purified through a Purelab 

Classic purification system (Elga LabWater, UK). 

Stock solutions of 100 μg.ml -1 were prepared by dissolving sodium selenite (Sigma-Aldrich, 

Germany), sodium selenate (Sigma-Aldrich, Germany), Seleno-DL-ethionine (Sigma-Aldrich, 

Germany), L-selenomethionine (Merck, KGaA, Germany) and Se-(methyl)-selenocystein (Sigma- 

Aldrich, Germany). Diluted solutions of concentration 500 ng.ml -1 for analysis of standard mixtures 

were prepared immediately before using. 

Protease from Bacillus licheniformis (13,1 units/mg) (Fluka, Denmark), Protease XIV from 

Streptomyces griseus (4,4 units/mg) (Sigma-Aldrich, Germany) and Subtilisin A, type VIII, from 

Bacillus licheniformis (8 units/mg) (Sigma-Aldrich, Germany) were used for enzymatic hydrolysis 

of the samples. 

Sodium tetrahydroborate solution (1%) was prepared by dissolving the solid product (Sigma- 

Aldrich, Germany) in 0,1M NaOH (Riedel de Haën, Germany). This reductive solution was 

prepared daily. 

The mobile phase was a 40 mmol.l -1 ammonium dihydrogenphosphate solution of pH 6.2 

prepared daily from the commercial product (Sigma-Aldrich, Germany) and pH was adjusted by 

adding dropwise 25 % ammonium hydroxide solution (Sigma-Aldrich, Germany). 

Hydrochloric acid 32% solution was prepared from 37% hydrochloric acid p.a. (Analytika, 

Prague, Czech Republic). 



Samples 

The samples were different nutritional selenium supplements, two of them were based on 

selenized yeast and one on the sodium selenite, all of which were commercially obtained. All the 

supplements were in tablets and labeled with the quantity of selenium in the each tablet. 

1. Sample was a nutritional suplement based on selenized yeast 

2. Sample was a nutritional suplement based on selenized yeast 

3. Sample was a nutritional suplement based on sodium selenite 

PROCEDURES 

Enzymatic hydrolysis 

Tablets of nutritional supplements were properly ground in a agate mortar. Approximately 0,5 g of 

the powdered sample and 20 mg of enzyme: 

1. Protease XIV from Streptomyces griseus (4,4 units/mg) 

2. Subtilisin A, type VIII, from Bacillus licheniformis (8 units/mg) 

3. Protease from Bacillus licheniformis (13,1 units/mg) 

4. Without enzyme 

were placed in a 25 ml polypropylene bottle. After addition of 5 ml of deionised water, the samples 

were shaken at 170 rpm for 24 hours using mechanical shaker at 37 °C. 

After extraction, the extracts were separated from the samples by centrifugation for 15 min at 

3000 rpm. The supernatants were filtered through 0,45 μm membrane to eliminate suspended 

solids. The clear filtrate was injected into the chromatograph. 


Optimisation of chromatographic separation of Se species 

For the separation of the five selenium species we used a polystyrene-divinylbenzene-based 

anion exchange column Hamilton PRP X-100. 

Firstly we studied the dependence of the retention time of the five species on the pH of the 

phosphate solution used as mobile phase. The flow of the mobile phase was 1 ml.min -1 . Inorganic 

species selenite and selenate required the use of an alkaline solution as mobile phase to obtain low 

retention times. Selenoaminoacids showed high retention times at alkaline pH, whereas at lower pH 

values retention times were shortened. We tried to change pH values between 5.5 to 7.5 and the best 

results were obtain by working at pH 6,2. The species were eluted in the following order: SeEt, 

SeCys, selenite, SeMet and selenate (Fig. 2.). 

Secondly we varied the concentration of the mobile phase. Several concentrations of the mobile 

phase were tested, ranging from the 20 mmol.l -1 to the 80 mmol.l -1 . The concentration 40 mmol.l -1 

was chosen as the best, because this concentration gave sufficient resolution. 



Thirdly we tried to change the composition of the mobile phase, by adding of the methanol to the 

mixture. This addition of methanol had no significant influence on the separation. 

Extraction of selenium species from nutritional supplements 

The common procedures used for the speciation of selenium in yeast, plants and vegetables have 

been hot water extraction, enzymatic hydrolysis, buffers, water-methanol and HCl extraction. 7 In 

this study, protease XIV, Subtilisin A and Protease from Bacillus licheniformis (13,1 units/mg) were 

used for enzymatic extraction to cleave peptide bonds in proteins. The examples of chromatograms 

obtained for the samples are given in Fig. 3 -5 

Fig. 2 The HPLC-Heating-UV-HG-AFS chromatogram of mixed selenium standard solution: (A) 

SelenoEthionine, (B) selenocysteine, (C) selenite, (D) selenomethionine, (E) selenate 



Fig. 3 The HPLC-Heating-UV-HG-AFS chromatogram of selenium species after enzymatic 

extraction (Protease XIV) - Sample 1 : (A) selenocysteine, (B) selenite, (C) selenomethionine, (D) 

selenate 

Fig. 4 The HPLC-Heating-UV-HG-AFS chromatogram of selenium species after enzymatic 

extraction (Protease XIV) - Sample 2 : selenate 



Fig. 5 The HPLC-Heating-UV-HG-AFS chromatogram of selenium species after non enzymatic 

extraction - Sample 3 : selenite. 

CONCLUSION 

The speciation of selenite, selenate, selenoethionine, selenocysteine and selenomethionine was 

satisfactory when using the combination HPLC-Heating-UV-HG-AFS. This method is relatively 

free of interferences, involving low cost and maintenance. The procedure was applied to the 

speciation of Se-species in nutritional supplements. In the sample number 1 (suplement contained 

selenised yeast) selenoethionine, selenocysteine, selenite, selenomethionine and selenate were 

identified. In the sample number 2 (suplement contained selenised yeast) only selenate was 

identified. In the sample number 3 (suplement contained sodium selenite) selenite was identified. 

Three different enzymes for enzymatic extraction were tested. The best results were obtained with 

Protease XIV from Streptomyces griseus (4,4 units/mg). Forthcoming work will be directed to the 

evaluation of selenium recoveries. 


This work was supported by Ministry of Education (MŠMT) of the Czech Republic (Project 

G4/1259/2005 FRVŠ). 

REFERENCES 

[1] Vińas P., López-García I., Merino-Merońo B., Campillo N., Hernández-Córdoba M.: Anal. 

Chim. Acta 535, 49 (2005). 

[2] Dumont E., De Cremer K., Van Hulle M., Chéry C., Vanhaecke F., Cornelis R.: Journal of 

Chrom. A 1071, 191, (2005). 

[3] Gómez-Ariza J.L., Caro de la Torre M., Giraldez I., Sánchez-Rodaz D., Velasco A., 

Morales E.: App. Organometallic Chem. 16, 265, (2002). 



[4] Tsopelas F. N., Ochsenkühn-Petropoulou M. T., Mergias I. G., Tsakanika L. V.: Analytica 

Chimica Acta 539, 327 (2005) 

[5] Vilanó M., Padró A., Rubio R., Rauret G.: Journal of Chrom. A 819, 211, (1998). 

[6] Ipolyi I., Stefánka Z., Forot P.: Anal. Chim. Acta 435, 367, (2001). 

[7] Kahakachchi Ch., Boakye H. T., Uden P.C., Tyson J. F.: Journal of Chrom. A 1054, 303, 

(2004) 



EPITAXIAL GROWTH OF Ni AND Co ON (111) METALLIC 

SUBSTRATES 

Ing. Martin Zelený, 2 nd year 

Supervisor: Prof. RNDr. Mojmír Šob, DrSc. 

Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00 Brno, 

Czech Republic, zeleny@fch.vutbr.cz 

INTRODUCTION 

Transition-metal thin films and multilayers have recently attracted a lot of interest because of 

their technological importance in data storage and sensor applications as well as in fundamental 

research of magnetism. 

Bulk Co exists in two ferromagnetic thermodynamically stable phases at ambient pressure: 

hcp α-Co below 388 °C and fcc β-Co above 450 °C. In case of Ni, only the fcc modification is 

known. It turns out that Ni and Co thin films exhibit similar structures as in the bulk. For 

example, only distorted fcc films of Ni have been observed [1, 2]. On the other hand, cobalt thin 

films with the hcp structure [3] as well as with a trigonally distorted fcc structure [4] were found 

on fcc (111) substrates. In some cases, both structures were reported [5]. 

The goal of this contribution is to advance our fundamental understanding of epitaxial growth 

of Ni and Co thin films on various fcc(111) substrates. For this purpose, we employ ab initio 

electronic structure calculations based on fundamental quantum mechanics. Pseudomorphic 

overlayers adopt the lattice dimensions of the fcc substrate in the (111) plane and relax the 

interlayer distance. There is a stress in the (111) plane keeping the structure of the film and of the 

substrate coherent, and the stress perpendicular to this plane vanishes due to relaxation (Fig. 1). 

On the (111) surface, there are two possible stackings of the layers in the film: ABABAB..., 

giving the hcp structure, and ABCABC..., providing the trigonally distorted fcc structure. The 

nearest-neighbor distance DNN in the plane of the substrate is equal to asub 2 /2, where asub is the 

lattice constant of the substrate. If there is a large difference between the lattice dimensions of the 

substrate and material of the film (lattice mismatch), the stress in the (111) plane is too large. As 

a consequence, the film becomes incoherent and has a structure similar to the ground state of the 

bulk of the film material. 

Our investigations allow us to understand, from the first principles, the structure of Ni and Co 

thin films on various fcc(111) substrates. In particular, we explain why some of those films 

exhibit the hcp structure and some of them the trigonally distorted fcc structure. 

METHOD 

To simulate the epitaxial growth, we first calculate the total energy of the film material (i. e. 

Ni or Co) in equilibrium hcp and fcc structures. Here a transformation of fcc cell to hexagonal 

coordinates is useful (Fig. 1). Then, in the second step, we apply some elongation of the bulk 

crystal along both crystallographic axes by a fixed amount ε that provides the same atomic 

spacing as in the (111) plane of substrate, DNN. For each ε, we minimize the total energy, relaxing 

the stress in the direction perpendicular to the loading axes. This is a very simple model, but it 

turns out that it is quite realistic. 

For the total-energy calculations, we employ the full-potential linearized augmented plane 

waves method incorporated in the WIEN2k code [6]. The calculations are performed using the 

generalized gradient approximation (GGA). The muffin-tin radius of atoms of 2.0 a.u. is kept 



Figure 1. Epitaxial growth of thin films on the fcc(111) substrate. The fcc cell is characterized 

also by hexagonal coordinates (ahex, chex). 

constant for all calculations, the number of k-points in the whole Brillouin zone is equal to 8000, 

and the product of the muffin-tin radius and the maximum reciprocal space vector, RMTkmax, is 

equal to 9. For Ni, the maximum value of l for the waves inside the atomic spheres, lmax, and the 

size of the largest reciprocal vector G in the charge Fourier expansion, Gmax, is set to 9 and 16, 

respectively. For cobalt, lmax is set to 11 and Gmax is set to 16. 


The total energy of bulk Ni as a function of DNN is shown in Fig. 2(a). Triangles correspond 

to trigonally distorted fcc structures and circles to hcp structures. There are several interesting 

points on these dependences. Each curve has the global minimum, which corresponds to the 

equilibrium state. The equilibrium state with the lowest energy is the fcc ground state of Ni. The 

structural energy difference between the fcc and hcp structures at this point is 2.0 mRy/atom. 

Second interesting point on each curve is the inflexion point. The inflexion point with the lowest 

energy corresponds to the maximum strain in the (111) plane and to maximum DNN (5.31 a.u.), 

where the Ni film may be expected to be coherent. Next interesting point is a crossing point 

between the curves at 5.25 a.u. It represents the position where a phase transition between 

trigonally distorted fcc structures and hcp structures may take place. 

It follows from the analysis of Fig. 2(a) that the fcc layer stacking (ABCABC...) is stable in 

the neighborhood of the ground state up to DNN equal to 5.25 a.u. This is in a good agreement 

with the experimental data for Ni film on Cu(111) [5] and on Pt(111) [7] substrates. If the 

substrates have the DNN larger than 5.25 a.u., the film should exhibit the hcp layer stacking 

(ABAB…). However, a maximum value of DNN, where the film is coherent, is equal to 5.31 a.u. 

(the inflexion point). The value of DNN for Pt of 5.24 a.u. is nearly equal to 5.25 a.u., so we can 

expect hcp islands in Ni films on Pt(111). Because Au and Ag substrates have larger DNN than the 

value corresponding to the inflexion point, the Ni thin films on these substrates have not the hcp 

structure, but are incoherent and have a structure similar to fcc Ni in the ground state [8]. 



Figure 2. Total energy of Ni (a) and Co (b) as a function of nearest-neighbor distance DNN in the 

fcc(111) substrate. Full symbols denote our predictions of structures and coherence of Ni and Co 

thin films on different fcc(111) substrates. The inflexion point is shown only for the stable 

structures. 

Cobalt films behave differently (Fig. 2(b)). Total energies of hcp structures are lower than 

energies of trigonally distorted fcc structures, which means that hcp phases are energetically 

more favorable along the whole deformation curve. The structural energy difference between the 

fcc and hcp Co at the energy minimum is only 1.1 mRy/atom, which is half the value for Ni. Hcp 

structure is experimentally found in Co films on Cu(111) substrate [9], but islands with trigonally 

distorted fcc structures have also been reported [5]. This is due to a small structural energy 

difference between the hcp and fcc phases of Co, so that a transition between these phases is very 

easy. To the best of our knowledge, the only coherent films of cobalt were found on Cu(111) 

substrate. Our calculations predict also coherent Co layers on Pt(111) and Pd(111) substrates, 

because the DNN for these substrates is smaller than the value corresponding to the inflexion point 

(5.30 a.u.) – see Fig. 2(b). However, experimentally prepared films on these substrates are 

incoherent. Co thin films on Pd(111) substrate have the hcp structure [10], and the structure of Co 

thin films on Pt(111) strongly depends on the way of preparation [4, 11]. The difference between 

our prediction and experiment is probably due to the diffusion of Pt into the Co film and 

formation of alloys based on CoPt [12]. For Co on Au(111) substrate we predict incoherent film 

with the hcp structure, which is in agreement with available experimental data [3]. 

The interlayer distance d in the film as a function of the nearest-neighbor distance DNN in the 

substrate is shown in Fig. 3 for Ni (a) and Co (b). Comparison of numerical values is given in 

Table 1. Our predictions are in a good agreement with the experimental data. 

CONCLUSIONS 

We have calculated the total energies of Ni and Co as a function of nearest-neighbor distance 

DNN in the fcc(111) substrate for two different structures – trigonally distorted fcc structure and 

hcp structure, and applied these results to understand the atomic configuration of Ni and Co thin 

films. We have found an inflexion point on the dependence of total energy on DNN. It corresponds 

to the maximum value of DNN, where the film keeps to be coherent with the substrates. 



Figure 3. Interlayer distance d of Ni (a) and Co (b) as a function of nearest-neighbor distance 

DNN in the fcc(111) substrate. Full symbols represent the values from experimentally prepared 

thin films. The inflexion point is shown only for the stable structures. 

Our predictions for Ni films are in a good agreement with available experimental data. For 

Co films the agreement is not so good, because the structural energy difference between the hcp 

and fcc structures is too small and the structure of films strongly depends on the way of 

preparation. Some deviations may be also due to the simplicity of our model of epitaxial growth. 

Our investigations show that the results of ab initio calculations may be used to understand 

and predict the structure of nickel and cobalt overlayers on various fcc(111) substrates. 


This research was supported by the Grant Agency of the Czech Republic (Projects No. 

202/03/1351 and 106/05/H008), by the Grant Agency of the Academy of Sciences of the Czech 

Republic (Projects No. IAA1041302 and S2041105), and by the Research Projects 

AV0Z20410507 and MSM0021622410. The use of the computer facilities at the MetaCenter of 

the Masaryk University, Brno, is acknowledged. 

Table 1. Comparison between theoretical results and experimental data. Predictions of interlayer 

distance d are shown only for coherent films. 

film/sub. 

Ni/Cu(111) 

asub 

[a.u.] 

DNN 

[a.u.] d [a.u.] 

theory 

struc. film 

experiment 

d [a.u.] struct. film 

a 

6.82 4.82 3.78 fcc coh. 3.82 fcc coh. 

Ni/Pt(111) b 

7.40 5.24 3.57 fcc/hcp coh. 3.61 fcc coh. 

Ni/Au(111) c 

7.69 5.44 - fcc incoh. 3.78 fcc incoh. 

Ni/Ag(111) c 

7.71 5.45 - fcc incoh. 3.78 fcc incoh. 

Co/Cu(111) d 

6.82 4.82 3.76 hcp coh. 3.82 hcp coh. 

Co/Pd(111) e 

7.34 5.19 3.57 hcp coh. 3.72 hcp incoh. 

Co/Pt(111) f 

7.40 5.24 3.52 hcp coh. 3.68 hcp/fcc incoh. 

Co/Au(111) g 

7.69 5.44 - hcp incoh. 3.84 hcp incoh. 

a b c d e f g 

Ref. [5], Ref. [7], Ref. [8], Ref. [9], Ref. [10], Ref. [4, 11], Ref. [3]. 



REFERENCES 

1. D. W. Gidley, Phys. Rev. Lett. 62, 811, (1989). 

2. M. Sambi, E. Pin, G. Granozii, Surf. Sci. 340, 215 (1995). 

3. N. Marsot, R. Belkhou, H. Magnan, P. Le Fevre, C. Guillot, and D. Chandesris, Phys. Rev. B 

59, 3135 (1999). 

4. M. Galeotti, A. Atrei, U. Bardi, B. Cortigian, G. Rovida, and M. Torrini, Surf. Sci. 297, 202 

(1993). 

5. F. Huang, M. T. Kief, G. J. Mankey, R. F. Willis, Phys. Rev. B 49, 3962 (1994). 

6. P. Blaha, K. Schwarz, G. K. H. Madsen, D. Kvasnicka, and J. Luitz: WIEN2k, An 

Augmented Plane Wave + Local Orbitals Program for Calculating Crystal Properties 

(Karlheinz Schwarz, Techn. Universität Wien, Austria), 2001. 

7. M. Sambi, G. Granozii, Surf. Sci. 400, 239 (1998). 

8. S. Morin, A. Lachenwitzer, F. A. Möller, O. M. Magnussen, and R. J. Behm, J. Electrochem. 

Soc. 146, 1013 (1999). 

9. P. Le Fevre, H. Magnam, O. Heckmann, V. Briois, and D. Chandesris, Phys. Rev. B 52, 

11 462 (1995). 

10. S. K. Kim, Y. M. Koo, V. A. Chernov, and H. Padmore, Phys. Rev. B. 55, 114 (1996). 

11. J. Thiele, R. Belkhou, H. Bulou, O. Heckmann, H. Magnam, P. Le Fevre, D. Chandesris, 

C. Guillot, Surf. Sci. 384, 120 (1997). 

12. S. Ferrer, J. Alvarez, E. Lundgren, X. Torrelles, P. Fajardo, and F. Boscherini, Phys. Rev. B 

56, 9848 (1997). 



ACETYLCHOLINESTERASE INHIBITOR FROM NOSTOC SLIZ. KOL. 

Ing. Petr Zelík 

Supervisor: Ing. Jiří Kopecký, CSc. 


Technology, Purkyňova 118, 612 00 Brno, e-mail: zelik@fch.vutbr.cz 

INTRODUCTION 

Alzheimer´s disease is the most frequent dementia with progressive degeneration of central 

nervous system 1 . Selective decrease of nerve cells in the brain is associated with cognitive 

disorders and memory lost. There have been proposed several ways how to improve cognitive 

functions. Current symptomatic treatment use especially acetylcholinesterase (AChE) 

inhibitors, eg. donepezil, galanthamine and rivastigmine. Requirement for a new AChE 

inhibitors leads either to screening of natural sources, mainly higher plants, or modification of 

chemical structures of known AChE inhibitors via organic synthesis. Only a few works have 

been focused on screening of microorganisms (actinomycetes and fungi). Surprisingly, there 

isn´t any publication dealing with screening of autotrophic microorganisms – algae and 

cyanobacteria. 

About 250 species of algae and cyanobacteria have been tested for AChE inhibitory 

activity in attempt to find a new AChE inhibitors of nature origin. The most active strain, 

Nostoc sliz. kol., has been used for the next study. Fraction with AChE inhibitory activity was 

determined in the crude biomass extract of Nostoc sliz. kol. and subsequently isolated. 

Purified AChE inhibitor has been used in preliminary determination of the inhibition nature of 

AChE and for structure elucidation by IR, MS and NMR techniques. 

METHODS 

Assay of AChE activity 

Measuring of AChE activity was modified from the assay described by Ellman et. al. 2 

Method was optimized for microplate assay. Reaction mixutres containing 25 μl of 0,76 mM 

DNTB, 50 μl of 0,04 U/ml AChE, 20 μl of sample, and 125 μl of buffer were incubated for 

30 min at 30°C. After incubation, 30 μl of 6,22 mM ATCI (substrate) was added and the 

increase of absorbance was read at 412 nm every 18 s for twenty times. The results were 

corrected for spontaneous hydrolysis of the substrate. Enzyme activity were calculated as a 

percentege compared to an assay without any sample. Every experiment was done in 

triplicate. 

HPLC- MS analysis 

The active extracts were analysed by HPLC/ESI-MS/MS on Agilent 1100 MSD SL-Ion 

Trap mass spectrometer. The ZORBAX Eclipse XDB-C8 column (150x4,6 mm, 5 μm) was 

used for HPLC analyses. Elution was realized in gradient mode of methanol – water with 

addition of formic acid and UV detection was performed at 280 nm. 

Nostoc sliz. kol. – determination of active fraction (IN6) 

The crude methanolic extract was fractionated using HPLC and every fraction was tested 

for AChE inhibitory activity. 



Nostoc sliz. kol. – isolation of active fraction (IN6) 

The scheme of the isolation procedure is shown in Fig. 2. Freeze-dried biomass was 

desintegrated with sea sand and extracted with methanol. The crude extract was evaporated 

under reduced pressure to the dryness and dissolved in the mixture of acetone-hexane (3:7, 

V/V). This solution was chromatographed in the system of acetone-hexane (gradient mode; 

3:7→7:3, V/V) on silicagel. All fractions were analyzed by HPLC/MS. Middle fraction 

containing mainly AChE inhibitor was evaporated to the dryness at low pressure. Residue of 

evaporation was dissolved in the minimum volume of methanol. Final separation of the active 

compound was realised by preparative HPLC. Watrex Reprosil C8 column (250x10 mm, 

5 μm) and mobile phase of methanol-water in gradient mode were used. UV detection was 

performed at 280 nm. Peak belongs to active compound was collected manually. 

Determination of AChE inhibition nature by IN6 

Initial reaction velocities were measured at two fixed concentrations of ATCI (0,175 mM 

and 0,75 mM) and different IN6 concentrations (final range in reaction mixure was 0-30 μM). 

Data were graphically processed according to Dixon. 


Approximately 250 species of algae and cyanobacteria were screened in quest to find 

potential producers of new AChE inhibitors among autotrophic microorganisms. The 

dichloromethane extracts of cultural mediums, methanolic and methanolic-tetrahydrofuran 

biomass extracts were tested for AChE inhibitory activity. There was found out AChE 

inhibitory activity higher than 90 % in biomass extracts of Monodus subteratus, Nostoc sliz. 

kol. (SV-mol, ISB 93, tok Soj, 5/97, DE) and Nostoc ellipsosporum (2, GM, Štěbal). Extract 

from the biomass of Nostoc clipsespor proved 85 % AChE inhibitory activity. Geminella 

terricola and Monodopsis subterranea biomass extracts proved inhibitory activity of 50 – 

60 %. The rest of biomass extracts and dichloromethane medium extracts showed AChE 

inhibitory activity either no or lower than 50 %. 

The crude extract of the most active strain Nostoc sliz. kol. (SV-mol, ISB 93, tok Soj, 5/97, 

DE) was analyzed by HPLC-MS and fraction proved anti-AChE activity was determined. The 

only fraction responsible for AChE inhibitory activity of the extract was the peak with 

retention time of 23,6 min (Fig. 1). 

Isolation of IN6 was developed (Fig. 2). At first, methanol extract of the freeze-dried 

biomass is prepared. Concentrate of IN6 is obtained in second isolation step, through the 

column chromatography of the crude extract in the system of acetone-hexane on silicagel. 

Thereafter is IN6 purified using preparative HPLC. Sufficient amount of IN6 (in 96% and 

higher purity) was isolated for structural analysis and determination of the inhibition nature in 

this way. 



mAU 


300 

0 

0 20 23,6 

min 

40 

Fig. 1: HPLC chromatogram of the crude methanolic extract from biomas of Nostoc sliz. kol.; 

I – fraction with AChE inhibitory activity 

Freeze-dried biomass of Nostoc sliz. kol. 

Crude methanolic extract 

Concentrate of IN6 

IN6 of > 96 purity 

Fig. 2: Scheme of IN6 isolation procedure. 

Isolated AChE inhibitor was analyzed by MS, IR and NMR but structure is still unknown. 

Nature of AChE inhibition by IN6 was also studied. Initial reaction velocities were 

measured at two fixed concentrations of substrate and different concentrations of IN6 (0- 

30μM). Data were graphically processed according to Dixon (Fig. 3). Two possible types of 

inhibition resulting from shapes of curves in Fig. 3 - either reaction of inhibitor with substrate 

or non-competitive inhibition with strongly bounded inhibitor to enzyme. 

Current interest is focused on more detailed kinetic study of IN6 and structure elucidation 

of IN6. 

I 

extraction with methanol 

column chromatography 

preparative HPLC 



[v (μmol.l -1 .s -1 )] -1 

120 

100 

80 


40 

20 

0 

ATCI - 0,75 mM 

ATCI - 0,175 mM 

0,0E+00 5,0E-06 1,0E-05 1,5E-05 2,0E-05 2,5E-05 3,0E-05 3,5E-05 

IN6 [mol.l -1 ] 

Fig. 3: Dixon plot for AChE inhibition by IN6 at two substrate concentrations. 

REFERENCES 

1. Jirák, R., Obenberger, J., Preiss, M.: Alzheimerova choroba. 1998. 15. ISBN 80-85800- 

88-8. 

2. Ellman, G.L., Courtney, K.D., Andres, V., Featherstone: A new and rapid colorimetric 

determination of acetylcholinesterase activity. Biochem. Pharm., Vol. 7, 1961, 88-95. 














doktorských studijních programů 

„O cenu děkana 2006” 

(Sekce DSP 2006)

PRODUCTION OF SELECTED SECONDARY METABOLITES IN 

TRANSFORMED BACTERIAL CELLS 

Ing. Jana Hrdličková, 3. ročník DSP 

Vedoucí práce: Doc. RNDr. Ivana Márová, Csc. 

Vysoké učení technické v Brně, fakulta chemická, ústav chemie potravin a biotechnologií, 

Purkyňova 118, 612 00 Brno, e-mail: hrdlickova@fch.vutbr.cz 

INTRODUCTION 

Polyketides are a large group of secondary metabolites of varied structure. They exhibit 

many physiological actions on organisms. Some OF them are useful drugs for humans, 

including antibacterials (e.g. erythromycin, tetracycline), anticancer agents (e.g. daunomycin), 

antifungal agents (e.g. amphotericin), cholesterol-lowering agents (e.g. lovastatin), 

immunosuppressants (e.g. rapamycin) and veterinary products (e.g. the antiparasitic, 

avermectin; the feed additive, monensin), others can be allergenic, toxic, and in some cases 

carcinogenic. Genetic engineering may lead to new polyketide drugs. 

The tetracyclines were the first major group of antimicrobial agents for which the term 

broad-spectrum was used, they exhibit activity against both gram-positive and gram-negative 

bacteria. Most natural tetracyclines have a common structure with the β-diketone system in 

rings B and C. Streptomyces rimosus is used for the production of natural tetracyclines by 

commercial fermentation. In S. rimosus a mixture of tetracycline and oxytetracycline is 

produced, but the 5-hydroxylase enzyme is extremely active, thus, the equilibrum is far in 

favor (>95%) of oxytetracycline production. Oxytetracycline (OTC) is a broad-spectrum 

antibiotic produced by S. rimosus. OTC is a member of the "polyketide" class of secondary 

metabolites biosynthesized by condensation of coenzyme A derivatives of metabolic 

precursors. The backbone of the antibiotic, consisting of 19 carbon atoms, is thought to be 

derived from an aminated starter unit (most likely malonamyl-CoA), to which eight acetyl 

(malonyl-CoA) extender units are added sequentially. 

Streptomyces rimosus has a linear chromosome of about 8 Mb. The chromosome has 

inverted repeats of 550 kb, which are the longest yet reported for a Streptomyces species.The 

otc biosynthetic gene cluster is located about 600 kb from one of the chromosome ends, just 

outside the inverted repeat structure. 

Carotenoids are naturally occuring membrane-protective antioxidant pigments, that 

efficiently scavenge singlet oxygen and peroxyl radicals. Carotenoids are produced in higher 

plants, algae and phototrophic bacteria as well as in non-phototrophic bacteria, yeasts and 

fungi. In recent years, there is evidence that accumulating of carotenoids plays an important 

role in human health by preventing degenerative diseases. From a commercial point of view, 

there is an increasing demand of special carotenoids in nutrient supplementation, for 

pharmaceutical purposes, as food colorants and in animal feeds. Biotechnological production 

of carotenoids by exploiting the carotenoid genes cloned from different species is of 

increasing interest. 

Lutein, lycopene and beta-carotene belong to industrially important carotenoids, widely 

used in food and feed industry as natural pigments, provitamins and food/feed supplements. 

Carotenoids act as antioxidants and protect organism from photooxidative damage. 

Availability of carotenoids for industrial usage is limited by partial problems associated with 

their chemical synthesis as well as with isolation from natural sources. According to this fact, 



in last years some ways for overproduction of carotenoids including modern methods of 

molecular cloning and genetic engineering are studied. 

Erwinia carotovora is nonphotosynthetic bacterium pathogenic for higher plants. Beside 

its agricultural significance, Erwinia strains are of increasing interest as industrial microbes 

producing pectinolytic, cellulolytic and proteolytic enzymes as well as antileukaemic 

asparaginase. Erwinia strains form carotenoids which cause yellow-orange coloured 

phenotype. 

METHODS 

Bacterial strains. For production of oxytetracyclines bacterium Streptomyces rimosus 4018 

was used. For production of carotenoids bacterium Erwinia carotovora CCM 1008 was used. 

For transformation DH5α, DH10β and ET 12567 Escherichia coli competent cells were 

prepared. 

Cultivation. Escherichia coli was cultivated in 2TY medium at 37ºC. 2TY medium 

contained, per litre: tryptone, 16 g; yeast extract, 10 g; NaCl, 5 g . 

Plasmids. pHSG298, pGEM-T (for PCR product), pSGset2 (containing Erm, OriC, Apr, 

OriT, attP, phi-C31 int), pTS55 (containing Amp, tsr, att, int, repSA) and pIJ 4026 

(containing Erm, bla) were used as transformation vectors. 

Isolation. Plasmid DNA isolation was performed using commercial kits (Gen Elute 

Plasmid Miniprep Kit). 

Transformation. Transformation of E. coli cells was done using electroporation by BioRad 

GENEPULSER apparatus at following conditions: voltage 2500 V, resistance 200 Ω, 

capacitance 25 μF. 

Electrophoresis. DNA was analysed using agarose electrophoresis and pulsed field gel 

electropohoresis (PFGE). 

Analysis of carotenoids. Production of carotenoids by transformants was analysed 

chromatographically. Carotenoids were extracted from E. coli transormant cells by ethanol. 

Individua pigments were separated and quantified by RP-HPLC using a Nucleosil 100 C18 

column and methanol (analysis of lycopene, lutein and carotenes) or mixture 

acetonitril:methanol 95:5 (phytoene analysis) as eluent. 

RESULTS 

Presented work was focused on production of selected secondary metabolites in 

transformed bacterial cells. First, regulation of polyketide antibiotik production in Escherichia 

coli cells transformed by otc genes from Streptomyces rimosus was studied. Further, isolation 

and cloning of crt gene cluster from bacteria Erwinia carotovora in E.coli DH5α cells was 

tested. Most OF experiments were performed in co-operation with Biotechnical Faculty, 

University of Ljubljana (Socrates/Erasmus exchange). 

The DNA sequence of Streptomyces rimosus was analysed by FramePlot (FramePlot is a 

web-based tool for predicting protein-coding regions in bacterial DNA with a high G+C 

content, such as Streptomyces). Primer structure was derived from sequence analysis results. 

The genes were amplified by PCR. Recommended sizes of PCR products were 714 bp and 

470 bp. The sequence of 714 bp was named CEL and the sequence of 470 bp was named 

MUT. After purification by Gen Elute PCR Clean-Up Kit the PCR products were ready to be 

cloned. CEL and MUT PCR products were ligated into the pGEM-T and introduced into 

E. coli competent cells. The cells with CEL or MUT were selected on LB agar plates 



containing ampicillin, IPTG and x-gal (blue-white test). Plasmids incorporated into 

transformants were isolated by Gen Elute Plazmid Miniprep Kit. To confirm that genes CEL 

and MUT were successfully cloned into pGEM, CEL+pGEM and MUT+pGEM were 

digested with EcoRI. Electrophoretic separation showed that inserts CEL and MUT were 

present in transformed cells. Verification of CEL and MUT sequences was done in cooperation 

with Macrogen Inc. Company (Soul, Korea). 

The CEL gene was extracted from pGEM-T using Nde I. After electrophoretic separation 

and extraction from the gel genes were ligated into the pSGset2/Nde I-deP and introduced 

into Escherichia coli competent cells. The cells containing CEL+pSGset2/NdeI were selected 

on LB agar plates with apramycin. 

The MUT gene was extracted from pGEM-T using Eco RI and after electrophoretic 

separation and extraction from the gel. After ligation into the pIJ 4026/Eco RI-deP 

transformation vestors were introduced into ET 12567 Escherichia coli competent cells. The 

cells containing MUT+pIJ 4026/Eco RI were selected on LB agar plates with 

ampicillin+chloramphenicol. 

We can summarize, that in this part of work PCR amplification of obtainable sequence and 

cloning and incorporation to expression vector was performed. Further, target sequences were 

incorporated into transformation vectors and recombinant plasmids were then incorporated 

into Escherichia coli recipient cells. The presence of recombinant plasmids was verified at the 

level of genotype and phenotype. Transformation of Streptomyces rimosus recipient cells by 

recombinant plasmids will be conduct in the future. 

In second part of this work, regulation of carotenoid production using genetic 

engineering was tested. Several methods of isolation and transfer of crt genes from bacteria 

Erwinia carotovora to recipient strain Escherichia coli DH5α cells were tested and 

optimized. Identification of carotenoids produced by recombinant cells was verified by HPLC 

analysis. 

In individual Escherichia coli transformants production of lutein, lycopene and βcarotene 

was demonstrated. Production of carotenoids in Escherichia coli cells transformed 

by several recombinant vectors pHSG298/crt was substantially higher then those found in 

Erwinia carotovora cells. Further, the posibility of regulated high-yield carotenoid production 

in laboratory fermentor was tested. Production of lutein in Escherichia coli transformants was 

about 8x higher then amount of lutein found in Erwinia carotovora cells, which were 

cultivated in the same conditions. 


I would like to thank Dr. Hrvoje Petkovič and Ms. Urška Lešnik for help and technical 

assistance. Both from Biotechnical Faculty, University of Ljubljana, Slovenia. 

REFERENCES 

1. Petkovič H., Thamchaipenet A., Zhou L-H., Hranueli D., Raspor P., Waterman P.G., 

and Hunter I. S.: Disruption of an Aromatase/Cyclase from the Oxytetracycline 

Gene Cluster of Streptomyces rimosus Results in Production of Novel Polyketides 

with Shorter Chain Lengths. The Journal of Biological Chemistry 46, p.32829 

-32834, 1999. 



2. Pandza K., Pfalzer G., Cullum J. and Hranueli D.: Physical mapping shows that the 

unstable oxytetracycline gene cluster of Streptomyces rimosus lies close to one end 

of the linear chromosome. Microbiology, p.1493-1501, 1997. 

3. Bentley R.: Polyketides. Encyclopedia of life sciences, 2001. 



THE SIMPLE METHOD FOR THE RECOGNITION OF REDUCING AND 

NONREDUCING NEUTRAL CARBOHYDRATES BY MALDI-TOF MS 

Ing. Markéta Laštovičková a,b , 5. DSP 

Supervisor: RNDr. Josef Chmelík b 

a Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00 Brno, Czech 

Republic; e-mail: lastovickova@iach.cz 

b Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, 611 42 Brno, 

Czech Republic 

1. INTRODUCTION 

The Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry 

(MALDI-TOF MS) can provide valuable information on several aspects of carbohydrate 

structural analysis, such as the determination of sequence, branching, and linkage. For the 

analyses of neutral oligosaccharides, more frequently the positive-ion MALDI-TOF mode has 

been performed. The negative-ion mode has been applied mainly in the analysis of charged 

carbohydrates that more simply form the deprotonated molecules ([M–H] – ) [1–4]. 

Inulin and maltooligosaccharides (MOSs) are the representatives of neutral carbohydrates. 

Inulin belongs to the fructan group. It is a nonreducing polysaccharide containing Dfructofuranosyl 

units linked by α–1–2 glycosidic bonds and ended with one glucose unit. 

MOSs are the linear glucose oligomers containing only 1–4 linkages. Glucose syrups are 

concentrated, aqueous solutions of reducing, low molecular mass oligosaccharides (containing 

1–4 and 1–6 glycosidic bonds) obtained by hydrolysis of starch. These starch hydrolysates 

can be trespassed as the cheap sweeteners at the adulteration of food, e.g., fruit juices, 

therefore they are important for food industry [5]. 

This study demonstrates a great potential of the negative-ion mode MALDI-TOF MS for 

the characterization of underivatized neutral oligosaccharides. 

2. EXPERIMENTAL 

PREPARATION OF NEUTRAL CARBOHYDRATES STANDARD SAMPLES: Inulin from 

Dahlia tubers Mw 5000 (Fluka, Buchs, Switzerland) and MOSs G4-G10 (Sigma, St. Louis, 

MO) were prepared at concentrations of 1 mg/mL in deionized water. 

EXTRACTION OF NEUTRAL CARBOHYDRATES FROM REAL SAMPLES: Red onion and 

Jerusalem artichokes were acquired from a private producer from the Czech Republic. A 

procedure for the carbohydrate extraction from fresh samples was described previously [4, 6] 

Low glucose syrup (LGS) from 80% wheat starch (w/v), obtained from Amylon Co. 

(Havlickuv Brod, Czech Republic), was used at a concentration of 4 mg/mL in deionized 

water. 

MALDI-TOF MS: The reflectron negative-ion mode experiments were performed with an 

Applied Biosystems 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA) 

utilizing a Nd:YAG laser (355 nm). The optimal laser power was selected from the relative 

scale 0-8800. 2,4,6-trihydroxyacetophenone (THAP; 100 mg/ml acetone) was used as a 

matrix. 



3. RESULTS AND DISCUSSION 

The main task of this study was the application of the negative-ion mode MALDI-TOF MS 

to the determination of a structure of storage oligosaccharides isolated from plants. 

The proper experimental conditions (the most convenient matrix, optimal concentrations of 

samples and matrix, optimal laser power etc.) were determined for inulin and MOSs standard 

samples (the details are not shown). Moreover, these initial experiments showed a potential of 

negative-ion mode MALDI-TOF MS for the differentiation of reducing (MOSs) and 

nonreducing (inulin) oligosaccharides, because of easy fragmentation of reducing end ring 

(the production of in-source fragment ions [M – H – 120] – ; see Figure 1). This in-source 

fragmentation has already been described for negative-ion mode MALDI-TOF mass spectra 

of dextrans (the polymer containing the main chain with 1–6 glycosidic bonds and different 

degree of branching) [7]. This information is very useful for the identification of storage 

carbohydrates isolated from plants as is shown below. 

All real samples (LGS and oligosaccharides isolated from Jerusalem artichoke and red 

onion) were analyzed with THAP that was selected as the most convenient matrix. While 

[M – H] – ions formed the dominant distribution for oligosaccharides from both vegetables, the 

main distribution of LGS was formed by the in-source fragment ions [M – H – 120] – (see 

Figure 2). LGS showed this fragmentation because of its structure that contains the main 

chain formed by 1–4 glycosidic bonds and branches with 1–6 glycosidic bonds and a reducing 

end group. The mass spectra differed in the quantity of the adducts which was dependent on 

the amount of ions in the samples. 

Thus there are some important conclusions on the mass spectrometric behavior of the 

neutral oligosaccharides. Although the negative-ion mode MALDI-TOF MS is ignored in 

connection with neutral carbohydrates it is possible to determine the main characteristics of 

their distribution without any carbohydrate derivatization (see Table 1). In addition, the 

negative-ion mode MALDI-TOF mass spectra is able to differentiate reducing 

maltooligosaccharides and nonreducing fructooligosaccharides extracted from real samples, 

because of easy fragmentation of reducing end ring, which is not evident in the positive-ion 

mode MALDI-TOF MS where both types of oligosaccharides form the alkali-ion adducts. 

REFERENCES 

[1] Harvey, D. J. Matrix-assisted laser desorption/ionization mass spectrometry of 

carbohydrates and glycoconjugates. Int. J. Mass Specrom. 2003, 226, 1-35. 

[2] Zaia, J. Mass spectrometry of oligosaccharides. Mass Spectrom. ReV. 2004, 23, 161-227. 

[3] Harvey, D. J. Mass Specrom. Reviews. 2006, 25, 595-662 

[4] Štikarovská, M.; Chmelík, J. Analytica Chimica Acta 2004, 520, 47–55 

[5] Robyt, J.D. Essentials of carbohydrate chemistry, Springer, New York, 1998 

[6] Laštovičkova,M; Chmelik, J. J. Agric. Food Chem. 2006, 54,5092-5097 

[7] Čmelík, R.; Štikarovská, M.; Chmelík, J. J. Mass Spectrom. 2004, 39, 1467-1473. 



Figure 1 Negative-ion mode MALDI-TOF mass spectra of standard oligosaccharides: inulin 

(A) and MOSs (B) where 



Figure 2 Negative-ion mode MALDI-TOF mass spectra of oligosaccharides from the real 

samples: Jerusalem artichoke (A); red onion (B); and LGS (C). 



Table 1 Evaluation of negative-ion MALDI-TOF mass spectra of LGS and oligosaccharides 

isolated from red onion and Jerusalem artichoke; where the range between the shortest and 

highest detected oligomers = the range of the degree of polymerization (DP), the total 

number of detected oligomers = np, number-average molecular mass = Mn, weight-average 

molecular mass = Mw and polydispersity = δ. 

Jerusalem 

artichoke 

Red onion LGS 

np 17 6 19 

DP 6–22 6–11 7–25 

Mn 1805 1392 1763 

Mw 1962 1426 1966 

δ 1.09 1.02 1.11 

Peak types 

[M–H] – 

[M+K–2H] – 

[M–H] – 

[M+K-2H] – 

[M–H–120] – 

[M+Na–2H–120] – 

[M+K–2H–120] – 

[M+HSO4] – 

The most abundant peak 

Mr 1313.06 1313.06 1193.31 

Intensity 471.2 mV 371.8 mV 534.5 mV 

DP 8 8 8 



MINIATURE METALLIC DEVICE FOR COLLECTION OF HYDRIDE 

FORMING ELEMENTS 

Pavel Krejčí 1,2 

Bohumil Dočekal 2 

1 Department of Environmental Chemistry and Technology, Faculty of Chemistry, 

Brno University of Technology, Purkyňova 118, CZ-61200 Brno, Czech Republic, 

e-mail:krejci-p@fch.vutbr.cz 

2 Institute of Analytical Chemistry, Czech Academy of Sciences, Veveří 97, CZ-60200 Brno, 

Czech Republic, e-mail: docekal@iach.cz 

INTRODUCTION 

Collection of hydride forming elements (As, Bi, Ge, In, Pb, Sb, Se, Sn and Te) has become 

a simple and useful tool for pre-concentration and separation purposes in the trace and 

ultratrace analysis by atomic spectrometric methods [1,2]. It is typically performed "in situ" 

using conventional graphite (GF) or tungsten (WETA) heated atomizers with subsequent 

analyte detection by electrothermal atomic absorption spectrometry (ETAAS) method [1]. 

Nevertheless, the trapping technique can also be applied in other spectrometry methods 

(atomic emission spectrometry, atomic fluorescence spectrometry and mass spectrometry). 

For this purpose, conventional electrothermal atomizers are modified and/or special, mostly 

laboratory made devices are designed [2-5]. 

Capability of trapping of hydrides on a prototype of a miniature electrothermal 

vaporization (ETV) device was studied employing antimony, arsenic, bismuth and selenium 

hydrides as volatile species of analytes. This device is based on a strip of the molybdenum 

foil (which is typically used in production of "halogen" bulbs) and combined with miniature 

hydrogen diffusion flame for specific analyte detection in atomic absorption spectrometry. 

Influence of trapping temperature, modification of the molybdenum surface with noble metals 

– Ir, Pt and Rh, distance between the orifice of the injection capillary and the strip and 

composition of the gaseous phase (argon-hydrogen-oxygen) was studied in order to clarify the 

general hydride trapping mechanism. 

EXPERIMENTAL 

A Perkin–Elmer (Norwalk, USA) model 3110 atomic absorption spectrometer equipped 

with a deuterium background correction system was employed in this study. The Photron 

Super Lamps® (Photron, Victoria, Australia) of antimony, bismuth, arsenic and selenium 

were used as a specific radiation sources. 

The device for electrothermal induced collection of hydrides and their subsequent 

electrothermal release was based on a piece of the molybdenum foil (Metallwerk Plansee, 

Reutte, Austria), 85 µm thick, 2.15 mm wide and 56 mm long. It was bent to form a U– 

profile. Both ends of the strip were pressed between boron nitride cylindrical body (6 mm in 

diameter) and two brass contacts. A part of the bent strip (9 mm) remained free. Boron nitride 

electric insulation and the brass contacts maintained also an efficient cooling of the strip 

providing reproducible temperature setting in series of experiments. In this arrangement, 

maximum power of about 130 W was supplied at the highest temperature applicable 

(2600°C). 



Fig. 1: Scheme of the hydride generation aparature coupled with the heated molybdenum-foil 

trap system and simple hydrogen diffusion flame atomic absorption system. 

The implementation of the electrothermal trapping/vaporisation (ETV) device in the 

instrumental arrangement is shown in Fig. 1. Only a very simple atomiser based on 

a miniature hydrogen diffusion flame was used for atomic absorption spectrometry detection 

in trapping experiments. The flame was supported by the gas mixture of hydrogen and argon 

at a flow rate of 215 ml min -1 and 800 ml min -1 , respectively. The injection capillary, made of 

a wide bore quartz GC–capillary (8 cm × 0.53 mm id), was inserted through the narrow hole, 

drilled in the axis of the boron nitride body. The tip of the capillary was precisely positioned 

by means of an adjusting screw made of PEEK. A laboratory made pulse-width modulation 

power supply was used for heating the molybdenum strip. It was based on a high power 

MOS-FET transistor and a car battery (12 V, 60 Ah). The power supply was controlled by 

a PC by using software created in Visual Basic ver.3. In trapping experiments, the actual 

temperature of the heated central part of the molybdenum strip was simultaneously measured 

by pyrometers. 

A laboratory made flow injection hydride generation system is depicted also in Fig. 1. The 

peristaltic pump (model 72624–71, Ismatec, Switzerland) was fitted with Tygon tubes. The 

generation system was based on 3-channel peristaltic pump, PTFE-reaction loop and gasliquid 

separator with a forced outlet for liquid phase and with a PTFE-filter in an outlet for 

gaseous phase. The flow rates were 1.1, 3.6 and 5.0 ml min -1 for 0.5% m/v NaBH4 solution, 

sample solution in 1 mol l -1 HCl and waste solution, respectively. The sample channel was 

equipped with a Knauer (Berlin, Germany) 6–port injection valve made of PEEK with 

a 100 μl sampling loop for performing flow injection of the sample solution. Argon was 

introduced in two channels, upstream of the reaction loop as reaction gas and into the gas– 

liquid separator as stripping gas at a flow rate of 55 ml min -1 and 10 ml min -1 , respectively. 



The gaseous hydrides were directed towards the center of the bent part of the molybdenum 

foil via a wide bore quartz GC–capillary. See Ref. [5] for more details. 

RESULTS 

The influence of the molybdenum foil temperature and concentration of hydrogen in the 

gaseous phase on trapping behavior of bismuth and antimony were investigated for a bare 

molybdenum surface and argon-hydrogen atmosphere of the flame supporting gas mixture. 

Maximum trapping efficiencies were found for antimony and bismuth in the temperature 

ranges of 650-750 °C and 500-600 °C, respectively. These optimum temperatures are 

approximately 300-500 °C lower than those found for arsenic and selenium (1100-1200°C). 

Capability of trapping antimony and bismuth hydrides on a modified surface of the 

molybdenum trap was also investigated. Rhodium, platinum and iridium were chosen as 

permanent modifiers and were introduced stepwise on the surface in amounts of 10, 30, 100 

and 200 μg. Significant depletion of signals of collected antimony and bismuth was observed 

when the amount of any modifier used exceeded 30 μg. Evidently, all modifiers inhibit the 

interaction of both analytes with active sites on the molybdenum surface. In contrary, these 

modifiers do not significantly affect trapping of arsenic and selenium on the molybdenum 

surface. The maximum trapping efficiency of arsenic and selenium was independent of the 

modifier amount applied in the range from 0 to 200 μg. Their signal profiles were higher, 

more reproducible and symmetrical when increasing modifier amount. 

The overall efficiency of generation of hydrides and their transport into the trapping 

chamber is independent on the injection gas flow rate between the minimum and the 

maximum achievable rates of 40 ml min -1 and 260 ml min -1 , respectively. Maximum trapping 

efficiency was reached at a flow rate close to 70 ml min -1 , and at a distance of 2 mm between 

the tip of the introduction capillary and the foil surface. Obviously, aerodynamic conditions 

prevailing near the capillary orifice and the molybdenum foil during the trapping step play the 

same role in trapping of all analytes studied. 

Vaporization experiments showed that antimony, arsenic and selenium are strongly bonded 

to the molybdenum surface. Collected antimony is completely released at temperatures above 

2200 °C and arsenic and selenium at temperatures above 2400 °C. To the contrary, bismuth 

exhibits a different behavior. A relative low temperature of 1200 °C is sufficient for complete 

vaporization of trapped Bi. The heating vaporization pulse should be very short to prevent 

losses of analyte on the inner quartz wall of the trap chamber and to perform an efficient 

transport of the analyte into the diffusion flame. In the present experimental arrangement, the 

optimum heating pulse in duration of 0.4 s was found. 


This work was supported by The Grant Agency of the Czech Republic (Project 

No. 203/06/1441) and by Ministry of Education, Youth and Sports of the CZ 

(FRVS 1054/2006). 

REFERENCES 

[1] J. Dedina, D. L. Tsalev: Hydride Generation Atomic Absorption Spectrometry, 

Wiley & Sons, Inc., Chichester (1995). 

[2] H. Matusiewicz and R. E. Sturgeon, Spectrochim. Acta, Part B, 51 (1996) 377-397. 

[3] F. Barbosa Jr., S. Simiao de Souza, F.J. Krug, J. Anal. At. Spectrom., 17 (2002) 382-388. 



[4] H. Matusiewicz, M. Kopras, J. Anal. At. Spectrom., 18 (2003) 1415-1425. 

[5] P. Krejci, B. Docekal, Z. Hrusovska, Spectrochim. Acta, Part B, 61 (2006) 444-449. 



INFLUENCE OF POLYUNSATURATED FATTY ACIDS INTAKE 

ON LIPID METABOLISM IN PATIENTS WITH HYPERLIPIDAEMIA 

Simona Macuchová 

Mentor: Doc. RNDr. Ivana Márová, CSc. 

Brno University of Technology, Faculty of Chemistry, Department of Food Technology 

and Biotechnology, Purkyňova 118, 612 00, Brno, email: macuchova@fch.vutbr.cz 

INTRODUCION 

Cardiovascular diseases are the main cause of mortality in most of industrialized countries. 

Risk factors include smoking, diabetes, obesity, high level of blood cholesterol, a diet high in 

fats, and having a personal or family history of heart disease. Cerebrovascular disease, 

peripheral vascular disease, high blood pressure, and kidney disease involving dialysis are 

disorders that may also be associated with atherosclerosis (1). 

Atherosclerosis is characterized by deposition of cholesterol rich plaques in the 

endothelium. This observation stimulated research on the metabolism of cholesterol and 

revealed that cholesterol is transported in esterified form to cells by the low density 

lipoprotein (LDL). LDL is recognized by an endothelial cell receptor and introduced into the 

cell by endocytosis. There the esters are cleaved. The resulting free cholesterol is transferred 

to the cell walls. The process is strictly regulated. In atherosclerotic patients LDL is altered by 

oxidation. This altered LDL is taken up in unlimited amounts by macrophages. Dead 

macrophages filled with cholesterol esters are finally deposited in arteries (1). 

The fact that LDL is rendered toxic by oxidation raises the question which constituents of 

LDL are prone to undergo oxidation. LDL consists of a core of cholesterol esters which is 

surrounded by a phospholipid membrane in which the protein is inbedded. The latter is 

required to recognize the LDL cell receptor. 

Polyunsaturated fatty acids (PUFAs) esterified to cholesterol or present as phospholipids 

represent the most oxygen sensitive compounds of all these LDL constituents. 

Dietary polyunsaturated fatty acids (PUFA) have effects on diverse physiological 

processes impacting normal health and chronic diseases, such as the regulation of plasma lipid 

levels, cardiovascular and immune function, insulin action, and neural development and 

visual function (1). 

Ingestion of PUFA would lead to their distribution to virtually every cell in the body with 

effects on membrane composition and function, eicosanoid synthesis, and signaling as well as 

the regulation of gene expression. 

Cell specific lipid metabolism, as well as the expression of fatty acid-regulated 

transcription factors likely play an important role in determining how cells respond to changes 

in PUFA composition. 

Chemically, PUFA belong to the class of simple lipids, as are fatty acids with two or more 

double bonds in cis position. There are two main families of PUFA: n-3 and n-6. These fatty 

acids family are not convertible and have very different biochemical roles. 

Dietary n-3 PUFA have several beneficial properties: 

- act favorably on blood characteristics by reducing platelet aggregation and blood 

viscosity; 



- are hypotriglyceridemic; 

- exhibit antithrombotic and fibrinolytic activities; 

- exhibit antiinflammatory action; 

- reduce ischemia/reperfusion-induced cellular damage. This effect is apparently due to the 

incorporation of eicosapentaenoic acid in membrane phospholipids. 

Linoleic acid (n-6) (LA) and alfa-linolenic acid (n-3) (LNA) are two of the main 

representative compounds, known as dietary essential fatty acids (EFA) because they prevent 

deficiency symptoms and cannot be synthesized by humans (1). 

Reduction of blood lipids and inhibition of LDL oxidation are the main therapeutic 

approaches to the treatment of atherosclerosis. Antioxidant agents, alone or in combination 

with hypolipidaemic drugs are considered useful for this treatment. Food supplements 

containing such substances can serve as additional therapeutical agents (1). 

CLINICAL EXPERIMENT 

The aim of this work was to contribute to current knowledge of the influence of an 

antioxidant supplement type on metabolic and antioxidant status in a group of hyperlipidemic 

patients. Influence of complex food supplement containing tocopherol as antioxidant 

component and polyunsaturatd fatty acids as hypolipidaemic component on antioxidant status 

and parameters of lipid metabolism in 30 patients with hyperlipidaemia was studied. Food 

supplement (180 mg of eicosapentaneic acid EPA, 120 mg of docosahexaneic acid DHA, 1.12 

mg of vitamin E in 1 tbl.) was taken for 3 months, two tbl. daily; blood samples of each 

subject were taken in regular intervals. 

METHODS 

Determination of antioxidant activity 

Total antioxidant status was determined using ABTS method (Randox Laboratories, USA). 

Serum AGE (Advanced Glycation End Products) were analysed fluorimetrically at 

350 nm/440 nm. Total amount of serum oxidation products „AOPP“ (Advanced Oxidation 

Protein Products) was analysed spectrophotometrically according to Witko-Sarsat et al. 1998 

(2), in Kalousová et al. 2001 modification (3). 

Biochemical parameters 

A set of biochemical parameters characterizing lipid metabolism was measured 

at Department of Clinical Biochemistry in the Kyjov Regional Hospital. Levels of total 

cholesterol, triacylglycerols, HDL and LDL – cholesterol, apolipoproteine A and B, urea, 

creatinine, uric acid, alaninaminotransferase, aspartataminotransferase, albumin and glycated 

haemoglobin were determined using automatically system HITACHI 717. 

HPLC analysis 

As parameters of antioxidant status levels of serum carotenoids, α-tocopherol and retinol 

were measured using HPLC method. Separation of carotenoids, retinol and α-tocopherol was 

carried out using the Biospher column C18 (4,6 mm × 150 mm, particulation size 7 μm), 

methanol as the mobile phase and flow rate 1.1 ml.min -1 . Content of trans-all-retinol was 

detected at 325 nm, α-tocopherol at 289 nm and carotenoids at 450 nm. 



Determination of fatty acids 

A simplified method for analysis of fatty acids in human serum was used according to 

Kang at al. 2005 (4). Fatty acids were methylated using BF3/methanol reagent and extracted 

to hexane phase. Fatty acids methyl esters were measured using GC-FID method. In each 

sample 30 different fatty acids were detected using external standards. 

RESULTS 

After 3-month intake OF PUFA/tocopherol supplemet a significant decrease of serum 

cholesterol, LDL-cholesterol (10-15%) and mainly triglycerides (about 35%) in 

hyperlipidaemic patients was observed. No similar changes in controls was shown. While the 

decrease of cholesterol as well as LDL-cholesterol levels was caused predominantly by 

tocopherol effect, TAG levels could be influenced by combined effect of PUFA and 

tocopherol. Further, PUFA/tocopherol intake led to a significant increase of tocopherol levels 

and TAS and, thus, to corresponding decrease of serum AGEs and AOPPs. 

The profiles of serum fatty acids (FA) shown also some changes after supplementation. A 

significant decrease of saturated FA (myristic, palmitic, stearic acid) was observed in all 

groups. Different changes of unsaturated FA were observed in hyperlipidaemics when 

compared with controls. A significant increase of PUFA mixture, EPA and DHA was found 

in controls, while no changes of PUFA, EPA and moderate changes of DHA were observed 

in hyperlipidaemics. 

The main problem with any epidemiological study is that correlation does not imply 

causation. There are many other factors, that could be responsible for biological effect. 

Additional problems are connected with group composition as well as with biomarker 

selection (1). Moreover, in Czech population basal levels of antioxidants were changing in 

the course of time. 

Despite these problems, our results indicated, that intake of food supplement containing 

PUFA and tocopherol can positively influence lipid metabolism and antioxidant status in 

patients with hyperlipidaemia. Very important is composition of vitamin preparative; many 

commercial PUFA are extensively oxidized and, thus, isoprostan formation can occur. 

Acknowledgements: This work was supported by project FRVS 3150/G1/2006 the Czech 

Ministry of Education, Youth and Sport. 

References: 

1. Halliwell B., Gutterdige J.M.C.: Free Radicals in Biology and Medicine. 3rd Edition, 

Oxford University Press, 1999 

2. Witko-Sarsat et al.: Advanced Oxidation Protein Products as Novel Mediators of 

Inflammation and Monocyte Activation in Chronic Renal Failure. The Journal of 

Immunology 2524-2532, 1998 

3. Kalousová M. et al.: Advanced Glycation End-Products and Advanced Oxidation 

Protein Products in Patients with Diabetes Mellitus. Physiol. Res. 51: 597-604, 2002 

4. Kang J.X., Wang J.: A simplified method for analysis of polyunsaturated fatty acids. 

BMC Biochemistry 6:5, 2005 



HYDROPHOBIZED SODIUM HYALURONATE IN AQUEOUS 

SOLUTION - A FLUORESCENCE STUDY 

Ing. Filip Mravec, 3 rd year PGS 

Supervisor: doc. Ing. Miloslav Pekař, CSc. 


Chemistry, Purkyňova 118, 612 00 Brno, e-mail: mravec@fch.vutbr.cz 

INTRODUCTION 

Polysaccharides and their derivatives have become as major components for the 

development of biocompatible and biodegradable materials with many areas of interests (e.g. 

tissue engineering, drug delivery). Chemical modification, which no affected 

biodegradability, can lead to the expansions of medicine and engineering applications. 

Hyaluronan is major component of pericellular and extracellurar matrices. It is a linear 

polymer of the disaccharide D-glucuronic acid-1-β-3-N-acetylglukosamine (Figure 1a). It 

plays important role in stabilizing the extracellular matrix in many tissues by binding to 

specific proteins called hyaladherines. The main hyaluronan fraction is localized in skin 

tissue. 

The preparing of the hyaluronan derivatives are generally based on the esterifcation on the 

D-glucuronic subunit. Our derivatives were modified on the second carbon on the glucuronic 

subunit (Figure 1b). Because carboxylic groups are still free, we obtained the amphiphilic 

polyelectrolyte - hydrophobized hyaluronan (hHA). From its structure we predict in aqueous 

solution modified hyaluronan will aggregate to form micelle-like structures with non-polar 

core. This aggregation behavior can be study by non-polar fluorescence probes solubilized to 

H 

H 

O 

O 

HO 

a 

O 

O 

HO 

b 

O 

O - 

O - 

O 

OH 

O 

Na + 

Na + 

O 

NH 

R 

HO 

O 

HO 

O 

C 

H 3 

C 

H 3 

Figure 1 Structure of the native hyaluronan (a) and its hydrophobized derivative (b), 

R = C10. 

this core. 

Pyrene, benzo[d,e,f]fenanthrene is the even and alternating hydrocarbon (Figure 2a). The 

“Pyrene I1:I3 ratio method” is widely used method to determine the critical aggregation 

concentration (cac) for a lot of surfactant-based systems. Its unique response to the 



OH 

OH 

O 

NH 

O 

O 

NH 

O 

n 

n 

OH 

OH

microenvironment polarity is well known and described (Aguiar 2003). We evaluated 

experimental data using non-linear fitting with Boltzman’s curve with four parameters – 

maximum (a), minimum (b), inflex point (x0), and width of the gradient (Δx) (Equation 1). 

We voted and subsequently confirmed the “x-coordinate” of the inflex point as the cac value. 

For the confirmation we used the perylene’s fluorescence measurements. 

a − b 

y = + b 

1) 

( 0 

x − x ) 

Δx 

1+ 

e 

Perylene, dibenz[de,kl]anthracene, is also the even and alternating hydrocarbon (Figure 

2b). The perylene’s measurements are quite simple for evaluation. The fluorescence intensity 

of the perylene rise with the number of non-polar domains in the hHA’s solution. Earlier than 

domains are presented in solution no fluorescence is observed. When domains are formed we 

observe sharp increasing of the fluorescence. These two trends can be fitted by the linear 

curves and the x-coordinate from their point of intersection defines the cac value directly. 

a) b) 

Figure 2 Fluorescence probes - a) pyrene b) perylene 

MATERIALS AND METHOD 

The hyaluronan and its derivatives were obtained from CPN Ltd. (Dolní Dobrouč, Czech 

Republic). Hyaluronans were in these molecular weights: 97, 560, and 1630 kg·mol -1 . 

Derivatives were in molecular weights 134, 183, 360, and 1470 kg·mol -1 , respectively, and 

theirs substitution degrees were in range from 10 to 70 %. The substitution degree is defined 

as the ratio of the number of the monomer with and without the alkyl chain per polymer 

chain, and it was determined from 1 H NMR spectra. All molecular weights were determined 

by SEC-MALLS (Mlčochová, 2006). Pyrene and perylene were obtained both from Fluka 

GmbH. Acetone p.a. was obtained from Lachema Ltd. 

The samples were dissolved in doubly distilled water to the concentration 2 g l -1 . This 

stock solution was stabilized by addition sodium azide (NaN3) in final concentration 10 -3 M. 

Sample nomenclature. The samples are named in correspondence to their characteristics. The 

first come alkyl-type abbreviation, next are basic molecular weight (before derivatization), 

and after the solidus the substitution degree. For example D 134/10 means C10-derivate with 

the molecular weight 134 kg·mol -1 and the substitution degree 10 %. 

Fluorescence Method. The acetone stock solutions of the pyrene and perylene were prepared. 

Probes stock solution was introduced into a flask and acetone was evaporated. The stock 

solution of hHA was introduced into a flask with evaporating probe, it was diluted to the 

desirable concentration, and the resulting solution was sonificated during 4 hours and stored 

during next 20 hours. The fluorescence emission spectra were monitored with a luminiscence 



spectrophotometer (AMINCO-Bowman, Series 2) at 293.15 ± 0.1 K. The excitation and 

emission slit widths were set to 4 nm, where for pyrene and perylene the excitation 

wavelength was 335 nm and 408 nm, respectively. 

By the pyrene way, the ratio of the fluorescence intensity at 373 nm (I1) and at 383 nm (I3) 

was plotted against the logarithm of the concentration. These data was fitted by sigmoid curve 

with the nonlinear curve fitting with Origin 75. From non-linear fitting we obtain two possible 

cac points - directly cac1-point as the inflex point. Second one, cac2-point, is defined as 

cac2 = x0 + 2Δx 

. 2) 

Perylene’s data evaluation was based on fit of two linear trends. From equations related to 

these linear curves was evaluated “x-coordinate”, cacPe, of the point of intersection. 

Fluorescence (a.u.) 

1 

0.8 

0.6 

0.4 

0.2 

0 

Fluorescence (a.u.) 


It is useful to use only one probe for the CAC determination. Pyrene’s data contain not 

only information about aggregation but even polarity information. Because pyrene is partially 

water soluble, it is necessary to know exactly which cac-point is the right. 

F int. norm. 

250 300 350 400 450 500 

1.0 

0.8 

0.6 

0.4 

0.2 

I3 

a) Emission 0.8 b) 

Excitation 

0.6 

I1 

wavelength (nm) 

1 

0.4 

0.2 

0 

Excitation 

Emission 

320 370 420 470 520 570 

wavelength (nm) 

Figure 3 Spectral properties of the pyrene (a) and the perylene (b) 

Perylene 

Pyrene 

R 2 = 0.9702 

R 2 = 0.9998 

0.0 

0.8 

-3 -2 -1 0 1 

Log C 

1.6 

1.4 

1.2 

1.0 

I 1 /I 3 

Figure 4 The plot of the normalized integral fluorescence and the I1/I3 vs. the Log C. The 

perylenes data are separate and fit with the linear curves. The pyrenes data are fit by sigmoid 

curve with marked cac1 (×). The point of intersection (↑) from perylenes dependence(x-coordinate 

- 0.747) is identical with the pyrenes cac1 point (x-coordinate - 0.750). 



Table 1 Sum of the cac values for all samples 

sample SD cac 

- % x10 6 mol·l -1 

D 44 

D 134 

D 183 

D 360 

D 1470 

In Figure 4 there are typical 

dependencies of the pyrene and perylene 

in hydrophobized hyaluronan solution. 

Pyrene ratio shows typical decreasing Stype 

curve. Perylene, on the other hand, 

shows two line system. For pyrene it was 

used equations 1) and 2) to solve 

parameter cac1 and cac2, respectively. 

Perylene’s data, cacPe was solved by 

combination of two linear equations as 

point of intersection. Final values (in 

g·l -1 ) are: cac1 = 0.179, cacPe = 0.178, 

cac2 = 0.758. So it was established the 

best value for the cac determination is 

cac1 point, realized as the inflex point x0. 

The cac values are showing two 

trends. First, the cac values are 

decreasing with increasing SD (except for 

samples D 1470). Second, the cac values 

are decreasing with increasing M. These trends are obvious in Figure 5 and summarized in 

Table 1. The greatest decreasing of the cac values show D 44 samples. It is possible to relate 

this behavior to the fact that D 44 samples have the shortest chains and new types of 

interactions can be founding. On the other hand, heavy weighted chains show stable behavior 

against the SD changes. These results can be explained if we accept that next addition of the 

alkyls to the chain only fortify chain-chain interaction. And in fact, it does not lead to the 

formation of new hydrophobic cores. 

cac (10 -6 mol l -1 ) 

100.00 

10.00 

1.00 

0.10 

0.01 

10 

30 

50 

10 

30 

50 

70 

10 

30 

50 

10 

30 

50 

30 

50 

70 

12.27 

1.02 

0.06 

4.35 

1.21 

0.76 

0.20 

0.99 

0.73 

0.47 

0.51 

0.19 

0.18 

0.15 

0.06 

0.10 

D 44 D 134 

D 183 D 360 

D 1,470 

0 20 40 60 80 

SD (%) 

Figure 5 The dependences of the cac values on the SD. Except for D 1470 all samples 

show the decreasing tendencies with increasing M and SD. 

CONCLUSION 

Fluorescence determination of cac for the novel hyaluronate derivatives was presented. It 

was showed cac1 as the best point for determination of the critical aggregation concentration. 



Hyaluronate derivatives showed with increasing SD decreasing tendency of the cac through 

the light weighted chains, and stable value for heavy weighted ones. 

REFERENCES 

Aguiar J. et al.: J. Colloid Interface Sci 2003, 258, 116-122 

Angelescu D., Vasilescu M.: J. Colloid Interface Sci 2001, 244, 139-144 

Dong, D. C.; Winnik, F.: Can. J. Chem. 1984, 62, 2560-2564 

Mlčochová P. et al.: Biopolymers 2006, 82, 74-79 

Molina-Bolívar J.A. et al.: J. Phys. Chem. B 2004, 108, 12813-12820 



CHARACTERIZATION AND DEGRADATION BEHAVIOUR OF 

TRIBLOCK COPOLYMER 

Ing. Ludmila Nová 

Supervisor: Prof. RNDr. Milada Vávrová, CSc. 

Consultant: Ing. Lucy Vojtová, PhD. 

Institute of Chemistry and Technology of Environmental Protection, Faculty of Chemistry, 

Brno University of Technology, Purkyňova 118, 612 00 Brno 

E-mail:nova@fch.vutbr.cz 

ABSTRACT 

This work is focused on the studying of the thermoreversible behaviors of two copolymers, 

PLGA-PEG-PLGA and the same but modified with itaconic acid (ITA-PLGA-PEG- 

PLGA-ITA). The critical gel concentrations (CGC) and the critical gel temperatures (CGT) 

were determined. As for PLGA-PEG-PLGA the CGC and CGT equal to 19,2 w% and 34,5 

°C, respectively, was observed. Second polymer of ITA-PLGA-PEG-PLGA indicated the 

shift of the sol-gel transition curve down to the lower values of both CGC (15,3 w%) and 

CGT (25 °C). The degradation behaviors of PLGA-PEG-PLGA in a phosphate buffer (pH 

7.4) at 37 °C were investigated. A significant decrease in the molecular weight and increase in 

the polydispersity within 10 days (until the samples have dissolved) was observed. 

INTRODUCTION 

Poly(lactic acid) and poly(glycolic acid) have been under extensive study since they were 

introduced as biodegradable polymers having hydrolytically unstable backbones. Therefore, 

these biopolymers can be used for a certain type of biomedical application such as injectable 

polymer drug delivery systems, tissue implants or resorbable bone adhesives. The prevailing 

mechanism for the biopolymer degradation is a simple random chemical hydrolysis [1]. The 

most common explanation for this heterogeneous degradation process comes from the 

absorption of water, followed by a hydrolytic cleavage of ester bonds, which generates chain 

fragments with the acidic groups (Fig. 1). This process is characterized by a decrease in 

molecular weight, an increase in polydispersity (PD = Mw/Mn) and polymer mass loss 

accompanied by an increase in low molecular chain compound concentration in the 

surrounding medium [2 – 7]. 

H 

CH 3 

O 

O 

CH 3 

O 

O 

x 

O 

poly(lactic-co-glycolic acid) 

O 

O 

y 

OH 

CH 

H 3 

OH 

HO 

O 

lactic acid 

Fig. 1: Scheme of the PLGA degradation. 

+ 

2x 2y 

H 

HO 

H 

O 

OH 

glycolic acid 

The injectable biodegradable thermosensitive ABA triblock copolymers consisting of 

hydrophobic biodegradable copolymer of poly(lactic acid-co-glycolic acid) (PLGA) and 

hydrophilic poly(ethylene glycol) (PEG) acting as A and B block, respectively, were 

synthesized via ring-opening polymerization method in a bulk at 155 °C. The ABA 



copolymers were additionally functionalized by itaconic acid (ITA), which can be gained 

from renewable resources by pyrolysis of citric acid or by fermentation of polysaccharides. 

ITA brings reactive double bonds and functional carboxylic acid groups to the end of 

copolymer resulting in preparation of biodegradable ITA/PLGA-PEG-PLGA/ITA 

macromonomer. Successful end-capping of ITA to PLGA-PEG-PLGA copolymer was proved 

by 1H NMR and FT-IR analysis followed by characterization with GPC method. The above 

mentioned thermosensitive polymers are soluble in water forming free-flowing solution that 

spontaneously gels as the temperature increases generating a water-insoluble physical 

hydroge. The sol-gel transition behaviors were studied by the test tube inverting method. The 

tests of degradation behaviors of PLGA-PEG-PLGA copolymer were carried out in vitro in 

the phosphate buffer medium at 37 °C. 

EXPERIMENTAL WORK 

Materials 

The triblock copolymers of PLGA-PEG-PLGA and ITA- PLGA-PEG-PLGA-ITA were 

synthesized at our faculty by Dr. Lucy Vojtová. 

Tetrahydrofurane for GPC analyses (THF for HPLC, gradient grade, Merck, Germany), 

was used as received. Polystyrene standards (Mp= 316 500 – 162) were purchased from 

Polymer Laboratories, Germany. Milli-Q water, phosphoric acid (p.a., 85 %, Czech Republic 

and potassium phosphate dibasic (p.a., for HPLC, Fluka, USA) were used for the polymer 

degradation studies. 

Method 

The molecular weight and the molecular weight distribution of the copolymers were 

determined by GPC method using Agilent Technologies 1100 Series instrument equipped 

with a refractive index detector, PLgel Mixed C column of 300 x 7.5 mm with particle size 5 

μm, degasser, pump, auto sampler and fraction collector. Tetrahydrofurane was used as the 

mobile phase at a flow rate equal to 1 ml.min -1 . The average molecular weight was calculated 

using a series of polystyrene standards (Mp = 316 500 – 162). Samples of triblock copolymers 

were prepared in the concentration of 0.5 mg/ml in tetrahydrofurane for HPLC. 

The sol-gel transition was determined by the test tube inverting method. 4 ml vials 

containing 1 ml of the triblock copolymer were heated from 10 to 60 °C in a water bath. The 

transition temperatures were determined by a flow (sol) – no flow (gel) criterion when the vial 

was inverted with a temperature increment of 1 °C per step. 

The degradation behavior study was performed using 0.3 ml of 23 w% polymer aqueous 

solutions. The 1.8 ml vials with polymer solutions were put into an incubator at 37 °C (the 

temperature of a human body) to formed the hydrogels. Consequently, 0,5 ml of the 

phosphate buffer (pH 7.4, 37 °C) was added to the vials and the samples were placed into the 

incubator for 10 days with a view to degrade during this time. At regular intervals, the 

samples were withdrawn from of the incubator, lyophilized and analyzed. 


The sol-gel transition diagrams of the triblock copolymers (PLGA-PEG-PLGA, ITA- 

PLGA-PEG-PLGA-ITA) were created on the base of the test tube inverting method (Fig. 2). 




45 

40 

35 

30 

25 

20 

PLGA/PEG/PLGA 

ITA-PLGA/PEG/PLGA-ITA 

white viscous 

suspension 

suspension 

white gel 

white viscous cloudy gel 

cloudy viscous 

cloudy viscous 

amber sol 

amber sol 

15 

0 5 10 15 20 25 

Concentration (w% ) 

white gel 

amber viscous 

cloudy gel 

amber viscous 

amber gel 

amber gel 

Fig. 2: The sol-gel transition phase diagram of the triblock copolymers PLGA-PEG-PLGA 

and ITA-PLGA-PEG-PLGA-ITA 

The diagram demonstrates three basic areas - sol, gel and suspension (precipitate). The 

phase diagram of PLGA-PEG-PLGA shows the critical gel concentration (CGC) of 19.2 w% 

and the critical gelation temperature (CGT) of 34.5 °C. For the next study, the solution of 23 

%wt was used because of forming the amber hydrogel at around 37°C. As for the copolymer 

modified by ITA, CGC and CGT were determined to be 15.3 w% and 25.0 °C, respectively. 

The values were shifted below the curves of the polymer without the itaconic acid which 

pointed that the presence of hydrophilic –COOH groups causes better interaction between the 

polymer and water molecules. Therefore, CGC and CGT were observed to be lower than 

those of the copolymer without ITA. 

The change from sol to gel, which occurred by increasing the temperature, was not sharp. 

At the temperature much lower than the critical gel temperature; unimers, individual micelles, 

and grouped micelles coexisted in the sol state (Fig. 2 amber sol). The unimer fraction 

decreased with the temperature increasing (Fig. 2 amber viscous). At the same time, the 

grouped micelle size grew rapidly resulting in sol-gel transition (Fig. 2 amber gel). The 

aggregation and packing interactions between micelles increased to form denser gel with the 

raising temperature (Fig. 2 cloudy viscous state, cloudy gel). When the temperature was 

further raised, the hydrophobic chains in the micelle core shrank tightly. Also, the hydrophilic 

PEG block underwent dehydration and the second gel-sol transition arisen (Fig. 2 white 

viscous state, white gel, suspension). The over shrunk micelle groups precipitated in water 

and the solution separated into two phases of water and precipitated polymer (Fig. 2 

precipitation) [8]. 



Each micelle has a hydrophobic core and a hydrophilic shell, and can move relatively 

freely without any bridging connection between the micelles. Sol-gel transition occurs when 

the total volume of micelles fraction is larger than the maximum packing fraction volume [8]. 

The degradation behavior of PLGA-PEG-PLGA was investigated. Four samples of the 

copolymer in 0.7 ml of the phosphate buffer (pH 7.4) were placed into the incubator at 37 °C 

(the normal temperature of a human body). The samples were taken out and lyophilized after 

3, 5, 7 and 10 days. The rest of the copolymer was analyzed by GPC. The decrease in the 

molecular weight during these periods can be seen in Fig. 2. After 3, 5 and 7 days two phases 

in the vials can be observed, the copolymer gel and the buffer. The total dissolving of the 

copolymer occurred after 10 days when the measured molecular weight (Mn) was found to be 

4170 (Tab. 1). 

Tab. 1: The change of the molecular weight and the polydispersity during the degradation 

in the phosphatic buffer (pH 7.4, 37 °C) 

Time (day) Molecular weight Polydispersity 

0 6010 1.21 

3 5840 1.23 

5 5220 1.31 

7 4770 1.33 

10 4170 1.35 

The change of polydispersity (D) was related to the change of Mn. Mn decreased while D 

increased with the growing number of the shorter copolymer chains. 

Molecular weight 

6500 


5500 

5000 

4500 

4000 

3500 

0 2 4 6 8 10 12 

Time (day) 

Fig. 3: The change of the molecular weight (Mn)● and the polydispersity (D)∆ after 0, 3, 

5, 7, and 10 days. 



1,36 

1,34 

1,32 

1,30 

1,28 

1,26 

1,24 

1,22 

1,20 

Polydispersity

CONCLUSION 

The copolymers of PLGA-PEG-PLGA and ITA-PLGA-PEG-PLGA-ITA were synthesized 

and characterized by GPC and the test tube inverting method. The presence of the itaconic 

acid in the polymer chain caused the decrease in both CGC and CGT. 

The degradation of PLGA-PEG-PLGA was attended by the decreasing of molecular 

weight during ten days until the gel has dissolved. The number of the low molecular weight 

chains increased with time causing the increasing in polydispersity of the polymer. 

Acknowledgement 

I would like to thank Dr. Lucy Vojtová for the copolymers preparation and expert advice. 

This work was supported by the Ministry of Education of the Czech Republic under the 

research project MSM 0021630501. 

REFERENCES 

[1] A. Göpferich, Biomaterials 1996, 17, 103 – 114. 

[2] P. Giunchedi, B. Conti, S. Scalia, U. Conte: J. Contr. Rel. 1998, 56, 53 – 62. 

[3] S. Li,: Biomed. Mater. Res. 1999, 48, 342 – 353. 

[4] I. Grizzi, H. Garreau, S. Li, M. Vert: Biomaterials 1995, 16, 305 – 311. 

[5] B. Marcato, G. Paganetto, G. Ferrara, G. Cecchin: J. Chromatogr. B. 1996, 682, 147 – 

156. 

[6] T. G. Park: J. Contr. Rel. 1994, 30, 161 – 173. 

[7] G. Schliecker, C. Schmidth, S. Fuchs, T. Kissel: Biomaterials 2003, 24, 3835 – 3844. 

[8] M. S. Shim, H. T. Lee, W. S. Shim, I. Park, H. Lee, T. Chang, S. W. Kim, D. S. Lee: 

J. Biom .Mat. Res. 2002, 61, 188 - 196 



STUDY OF THE COPPER(II) IONS NON–STATIONARY DIFFUSION 

IN HUMIC GEL 

Ing. Petr Sedláček, 1 st year of study 

Supervisor: doc. Ing. Martina Klučáková, PhD. 


Chemistry, Purkyňova 118, 612 00 Brno, e–mail: sedlacek@fch.vutbr.cz 

INTRODUCTION 

Severe reduction of use of solid fossil fuels in the power–producing industry has markedly 

encouraged the investment to alternative applications of these materials. Humic acids (HA), 

which are one of their key components, are because of their rich natural sources, simple 

isolation methods and profitable chemical ad physical behavior predetermined for the wide– 

spread use in different industrial and agricultural branches. Although the intensive study of 

this material lasts for several decades the knowledge level (mainly concerning the structure of 

HA) is still quite low (it is often compared with the knowledge of proteins fifty years ago). 

Application of HA in a form of humic gel (see [1]) is quite new and unexplored field of 

their study. The gel form of HA is easy to prepare, suitable for the exploration of transport 

phenomena and mainly simulates the natural conditions; HA are usually found in the highly 

humid environment (water sediments, peat etc.) and thus in the swollen form. 

One of most famous and promising HA properties is the ability to bind metal ions. They 

can form stable complexes among others with heavy metals, which influences their toxicity in 

environment and this fact encourages potential applications of humic substances mainly in 

environmental industry, in the production of fertilizers and in pharmacy. Most important 

binding sites in HA molecule are carboxylic and phenolic groups and aromatic cycles. 

Besides the lower mobility of metal ions in humic gels comparing water solution, the 

diffusion in gels are influenced also by the retention or immobilization of the ions, both 

caused by chemical reaction between metals and HA. The result is that mathematical 

apparatus used in the description of diffusion phenomena is very complicated. 

Copper(II) ion is well–known for its high affinity to humic substances [2]. Besides this the 

HA–Cu(II) binding is among the highest strengths. Therefore and also because of easy 

quantification of copper content by means of 

spectroscopy, the copper(II) ions has been chosen 

as model metal ions for this work. 

The main aim of this research was the study of 

copper(II) ions diffusion from solutions with 

different Cu 2+ concentration into humic gel across 

the phase interface and the diffusion in the gel 

itself. Other experiment interested in the influence 

of other properties of the copper(II) source 

solution, namely the type of anion of copper salt, 

solution pH and ionic strength. 


HA were obtained from South-Moravia lignite by means of the alkaline extraction (see 

[2]). Humic gel was prepared by the technique optimized in [3]: HA were diluted in 0.5 M 

NaOH in the 8 g HA in 1 dm 3 Fig. 1 Humic gel sample 

NaOH ratio. This sodium humate solution was acidified by 



concentrated HCl to pH ~ 1 and leaved in 

refrigerator overnight. After the centrifugation (10 

min., 4000 rpm, cooling for 15 °C) the 

supernatant was poured out and the gel was 

washed three times by deionized water. Each 

washing was followed by centrifugation, the last 

one took 30 minutes. Finally the gel was 

deposited in dessicator with water to stabilize its 

humidity. The picture of prepared gel sample is 

shown on the Fig. 1. 

The HA sample was characterized by means of 

table 1 Elementary analysis results 

content in dried 

element ash-free HA 

[atomic %] 

H 42,12 

C 41,16 

O 15,64 

N 0,91 

S 0,17 

the elementary analysis (Microanalyser Flash 1112, Carlo Erba), ash content analysis (sample 

contained 6.8 weight % of water and 28.5 weight % of ash), and UV–VIS and FT–IR spectral 

analysis (Hitachi U 3300 and Nicolet impact 400 respectively). From the exact results of these 

analyses, which are listed elsewhere ([4]), it is clear, that used HA were of high degree of 

humification. The humic gel sample was analyzed for its solid matter content 

(14.5 weight %), total gel acidity was determined by potentiometric titration (gel contained 

8.12 mmol acid equivalents per 1 g of HA). The FT–IR spectroscopy analysis of dried gel 

sample results in fact that no chemical structure changes occur while gelation of HA. 

Diffusion experiment was divided into several parts; in the first one the pre-determined 

(see [3]) value of the diffusion coefficient of copper(II) ions in humic gel was verified by the 

stationary diffusion from the saturated copper(II) chloride solution. In detail, experimental 

procedure is listed in [4]. 

In all remaining parts the non-stationary diffusion was studied. All experiments took place 

in apparatus presented on Fig. 2. Humic gel was packed into plastic tube as 5 cm long 

cylinder and 2 ml of both copper ions solution and deionized water were filled into side 

containers. First of all diffusion dependency on Cu(II) initial concentration in solution was 

monitored by changing the initial concentration of CuCl2 solution placed in the apparatus 

container, while the duration of experiment was maintained at 24 hours. After the end of the 

experiment, each gel sample was sliced and each slice was separately extracted by 1 mol.dm –3 

HCl. By the UV–VIS quantification of Cu(II) content in each extract, the concentration 

profile of the gel cylinder and the total diffusion flux across the solution–gel interface were 

determined. 

Next part deals with the influence of time duration of diffusion experiment. The 1 day, 

3 days and 5 days periods have been chosen. The experiment was repeated for three Cu(II) 

initial concentration values: 0.1 mol.dm –3 , 0.3 mol.dm –3 and 0.6 mol.dm –3 . Following 

experimental technique was adopted from previous experiment without changes. 

In the last part, the influence of other copper(II) solution properties has been investigated. 

The influence of the type of copper(II) salt anion was studied by using CuCl2, Cu(NO3)2, 

Cu(II) 

plugged solution 

containers 

HUMIC GEL 

plastic tube 

H2O 

Fig. 2 Scheme of the aparatus used in all 

diffusion eperiments 



CuSO4, Cu(ClO4)2 and Cu2P2O7 of the same Cu 2+ concentration (0.1 mol.dm –3 ) as a stock 

solution for diffusion experiments of the same time duration (24 hours). Finally, the solution 

pH – dependency and solution ionic strength dependency of the diffusion process was 

checked by diluting CuCl2 salt using the differently concentrated hydrochloric acid and 

sodium chloride, respectively. For all of these experiments the same Cu 2+ concentration 

(0.1 mol.dm –3 ) as well as the time duration (24 hours) was used and the same experimental 

technique (see above) was adopted. 


The value of calculated effective diffusion coefficient of copper(II) ions in humic gel was 

7.96×10 -10 m 2 .s –1 . This value is in good agreement with the one pre–determined by another 

experimental method (see [3]) and was used for all following calculations. As can be seen on 

Fig. 3 and Fig. 4, total diffusion flux of Cu 2+ through solution–gel phase interface linearly 

increases with the initial concentration of copper(II) in solution and with the square root of 

time duration of diffusion experiment. The later dependency is however much more 

complicated; the nonzero intercept of the regression line indicates that the linearity is just 

illusory and in fact the dependency is curved. Klučáková et al. in [5] stated the equation for 

the total diffusion flux across the solution–gel interface: 

ε c D τ 

0 

g 

m = (1) 

1+ 

ε D / π 

g D 

in which m stands for the total diffusion flux, c0 is initial concentration of the ion in solution, 

τ is the time duration of the diffusion, ε is ratio of ion concentration in the gel and in the 

solution in final equilibrium (at the “end of diffusion”), Dg and D is diffusion coefficient of 

cupric ions in humic gel and in solution, respectively. Following this equation the value of ε 

was calculated from total diffusion flux corresponding to c0 and τ values. It was found that for 

the same duration of diffusion experiment this value is constant for different initial 

concentrations of the solution but it varies for different duration of experiment (Fig. 5). This 

fact could be explained by the apparatus construction (small solution volumes – gel weight 

ratio) or by the time–consuming formation of some stable structural or chemical complexes 

between gel and ions. This affects the mobility of ions (retention of ions can lead up to their 

immobilization in gel) and their equilibrium in gel and solution. This fact could explain 

mentioned deformation of total flux time dependency as well. 

The knowledge of the ε value allows 

the calculation of theoretical 

y = 6.454E-03x 

concentration profiles of the copper (II) 

R 

ions in the humic gel (used 

mathematical apparatus is presented in 

detail in reference [5]) for individual 

experiment conditions (initial 

concentration, time duration). The 

example on Fig. 6 shows very good 

agreement between calculated and 

measured concentration profiles. 

2 50 

40 

= 1.000E+00 

30 

20 

10 

0 

0 2000 4000 6000 8000 

total flux [mol.m -2 ] 

c0 [mol.m -3 ] 

Fig. 3 The total diffusion flux dependency on 

the initial concentration of Cu 2+ in the 

solution (diffusion duration 24 hours) 



ε 


7 

6 

5 

4 

3 

2 

1 

y = 0.0099x 

R 2 = 0.9997 

y = 0.0066x 

R 2 = 0.9983 

y = 0.0084x 

R 2 = 0.9985 

0 

0 200 400 600 800 

c0 [mol.m -3 ] 

1 day 

3 days 

5 days 

The results of the part which deals with the influence of another copper(II) solution 

properties shows dramatically smaller effect of the type of anion in the copper(II) salt and 

solution ionic strength and pH compared with initial copper(II) concentration and time 

duration of diffusion. On Fig. 7, it can be seen that for all anions with unit valence (CuCl2, 

Cu(NO3)2 a Cu(ClO4)2) the total diffusion flux is similar even if for example anion size differs 

markedly. On the other hand, anions with higher valence (CuSO4 and Cu2P2O7) show 

inconsiderable decrease of total diffusion flux. The pH and ionic strength measurement 

excluded these properties as causer of this 

decrease, so it can be supposed that this shift 

can be explained as a consequence of higher 

charge of multivalent anions. 

Total diffusion flux is also affected by the 

copper solution pH. Fig. 8 shows that with 

higher acidity of the solution total diffusion 

flux decreases. This could be explained for 

example by well–known decrease of HA 

sorption ability in acid media (see [2]) or by 

the change of solution–gel equilibrium. 

Higher acidity affects salt hydrolysis and 

thus actual ion concentration in solution. 


7 

6 

5 

4 

3 

2 

1 

y = 5.517E-03x + 2.250E+00 

R 2 = 9.950E-01 

y = 2.449E-03x + 1.343E+00 

R 2 = 9.994E-01 

y = 1.095E-03x + 3.265E-01 

R 2 = 9.999E-01 

0 

200 300 400 500 600 700 

t 1/2 [s 1/2 ] 

Fig. 4 The total diffusion flux dependencies on the initial concentration of Cu 2+ and on the 

duration of the experiment 

2 

1.5 

1 

0.5 

0 

0 1 2 3 4 5 6 

τ [days] 

Fig. 5 The time shift of ε 

copper(II) ions concentration 

[mol/dm 3 gel] 

0.15 

0.1 

0.05 

0 

experimental results 

(0.3 M; 3 days) 

calculated 

concentration profile 

(0.3 M; 3 days) 

0.1M 

0.3M 

0.6M 

0 10 20 30 40 50 

distance from the interface [mm] 

Fig. 6 Concentration profiles of Cu 2+ 

in humic gel 

total flux [mol.m –2 ] 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0 

CuCl2 

0.614 0.620 0.608 

Cu(NO3)2 

Cu(ClO4)2 

CuSO4 

0.476 

Cu2P2O7 

Fig. 7 Total diffusion flux for different 

copper(II) salts 



0.355


0.7 

0.65 

0.6 

0.55 

0.5 

0.45 

0.4 

0 1 2 3 4 5 

pH 


0.4 

0 1 2 3 4 5 6 

Finally the total diffusion flux showed complex dependency on the ionic strength of 

copper(II) solution. For small NaCl additions, the decrease of total flux comes with the 

increase of ionic strength, on the contrary substantial increase appeares in higher NaCl 

concentration range. Former decrease can be caused by decrease of HA sorption ability, 

competitive diffusion of Na + ions or by affecting Cu 2+ solvation. The later increase is not very 

clear. It can be supposed that in the solution with high ionic strength bigger Cu 2+ clusters are 

solvated and easily took out from the solution. 

CONCLUSIONS 

This paper deals with the study of copper(II) ions non–stationary diffusion in humic gel. 

Main aim was to determinate the influence of individual copper(II) solution properties on the 

total diffusion flux across the solution–gel interface. 

It was proved that copper(II) ions diffusion across the phase interface is affected mainly by 

the initial concentration of copper in the solution and by the time duration of the diffusion. 

The influence of other solution properties, such as its pH or ionic strength as well as the type 

of copper(II) salt anion is far less important however show interesting dependencies. 

Although the applied apparatus was very simple and could be improved for following 

experiments (the higher solution volumes should be used) experimental data are fitted well by 

the theoretical calculations, so it can be said that this method presented itself suitable for the 

wide spectrum of diffusion experiments using humic gel as a very usefull natural–like model. 

REFERENCES 

[1] Klučáková, M.: Huminový gel jako model pro studium transportu těžkých kovů 

v přírodních systémech. CHEMagazín 2004, Vol. 14, No. 3, p. 8–9. ISSN 1210–7409 

[2] Klučáková, M., Kaláb, M., Pekař, M., Lapčík, L.: Study of Structure and properties of 

Humic and Fulvic Acids. II. Study of Adsorption of Cu + ions to Humic Acids Extracted 

from Lignite, J.Polym.Mater 19 (3) 2002 

[3] Malenovská, M.: Studium difúzních procesů v huminových gelech. 55 stran. Diplomová 

práce na VUT, FCH Brno 2005. Vedoucí diplomové práce Ing. Martina Klučáková 

PhD. 

[4] Sedláček, P..: Difúze kovových iontů v huminových gelech. 70 stran. Diplomová práce 

na VUT, FCH Brno 2006. Vedoucí diplomové práce Doc. Ing. Martina Klučáková PhD. 

[5] Klučáková, M., Pekař, M.: Diffusion of Metal Cations in Humic Gels. In Humic 

Substances: Nature´s most Versatile Materials (E. Ghabbour, G. Davies Eds.), Francis 

& Taylor, New York, 2004, p. 263–74 

0.7 

0.65 

0.6 

0.55 

0.5 

0.45 

ionic strength [mol.dm –3 ] 

Fig. 8 Total diffusion flux dependency on solution pH (left) and ionic strength (right) 



LIVING RADICAL POLYMERIZATIONS INITIATED WITH 

SILSESQUIOXANES 

Author: Ing. Ondřej Smrtka – 3 rd year of DSP 

Supervisor: Prof. RNDr. Josef Jančář, CSc. 

Brno University of Technology, Faculty of Chemistry, Institute of Material Chemistry 

Purkyňova 118, 612 00 Brno, Czech Republic; email: smrtka@fch.vutbr.cz 

INTRODUCTION 

It has been discovered that small variations in molecular structure at the nano-meter scale 

level allow substantial changes in macroscopic behavior of nanostructured systems. 

Nanocomposites with polymeric matrix belong among the most intensively investigated 

materials since they promise extensive industrial and biomedical applications. 

The polyhedral oligomeric silsesquixanes (POSS) can be included into the large group of 

nano-particles used for nanocomposite preparation. POSS, due to their well-defined structure, 

are considered to simulate the surface of silica nano-particles as the smallest possible silica 

particle. Nanocomposites containing POSS nanoparticles are considered extremely interesting 

class of nanostructure organic-inorganic materials. 

Polyhedral oligomeric silsesquioxans are compounds of general formula (RSiO1.5)x, where 

x is an even number higher than 4, mostly being 8. R is any of large number of organic 

groups, or e.g. hydrogen or halogen atom. 

R 

R 

O 

Si 

O 

R 

Si 

O 

O 

Si 

O 

R 

Si 

O 

Fig. 1: Polyhedral oligomeric silsesquioxan (POSS) 

POSS are prepared preferentially by hydrolytic condensation of trifunctional silanes [1]. 

Organic groups are carried into the molecule either directly during the synthesis (as the fourth 

non-reactive group of the silane precursor), or by a transformation of existing groups [2]. 

Monofunctional POSS are synthesized from incompletely condensed molecules of 

silsesquioxane by condensation of another silane with appropriate functional group [3]. 

POSS particles can be incorporated to the polymeric materials directly by blending with 

polymer as the additive, or as the component of polymeric chain. The POSS containing 

macromolecules can be synthesized using monomers with POSS pendant groups, or 

afterwards, by grafting to the polymer using various condensation or addition reactions (e.g. 

hydrosilylation). Another way how to connect POSS with the polymer chain is to use it as the 

initiator for ATRP. 

ATRP (Atom Transfer Radical Polymerization) has been developed in 1995 by 

Matyjaszewski [4]. It is based on rapid attainment of dynamic equilibrium between low 

amount of growing free radicals and much higher amount of dormant species. Because the 



O 

Si 

O 

O 

Si 

R 

R 

O 

O 

Si 

Si 

O 

R 

R

number of free radicals is very low, the termination is reduced and the polymerization proves 

living character. 

P –X + Cu –Y/2L ⎯⎯→ P + M + X–Cu –Y/2L 

I kakt 

• 

II 

n ←⎯ ⎯ k 

n 

deakt 

Pn + Pm 

Fig. 2: Scheme of ATRP 

The dormant species are mostly alkylhalides, from which the halogen atom is transferred to 

catalyst (copper halide + organic ligand); the specie then becomes a radical, efficient to 

propagate radical polymerization. The oxidation state of copper increases by one with 

admitting the halogen; with the transfer back to the polymer the original oxidation state is 

retrieved. Other transition-metal salts can be used as the ATRP catalyst, with the requirement 

to the metal center to have at least two readily accessible oxidation states separated by one 

electron. 

A variety of monomers have been successfully polymerized using ATRP, typical 

monomers include styrenes, methacrylates, acrylamides and acrylonitrile. 

In ATRP, alkylhalides are typically used as the initiator. A variety of transition-metal 

complexes have been studied as ATRP catalyst, but copper catalysts are superior in ATRP in 

terms of versatility and cost. Presence of organic ligand is necessary for dissolution of copper 

salt in reaction mixture. Above all, the nitrogen-based ligands are used (e.g. bipyridine, 

PMDETA, HMTETA). 

Initiation of ATRP with the POSS-based initiator has not been intimately described so far, 

only some notes were published [5, 6]; however initiation of ATRP with polysiloxane-based 

initiators has been described [7, 8] much more and due to the silimilar structures the depicted 

techniques might be used also for POSS-based initiators. 

EXPERIMENTAL 

Materials used 

Styrene was distilled from calcium hydride prior to use. Tetrahydrofurane was distilled 

from purple sodium/benzophenon solution. Copper (I) bromide was stirred in glacial acetic 

acid overnight, decanted, then washed with absolute ethanol and diethyl ether and vacuum 

dried at 60°C. Ethyl-α-bromo-isobutyrate (EBIB), p-dimethoxybenzene (DMB), POSShydride, 

allyl-bromoisobutyrate, pentamethyldiethylene triamine (PMDETA), and Karstedt 

catalyst were used as received. 

Synthesis of POSS-Br 

POSS-Br was synthesized using hydrosilylation reaction. 1.37 g (1.44 mmol) of POSS-H, 

16 ml dried THF, 0,24 ml (1.44 mmol) allyl-α-bromoisobutyrate and 55μL of Karstedt 

catalyst solution in xylene (3 wt.%, 3,64 μmol) were placed into a 50 mL two-neck roundbottom 

flask under nitrogen blanket and stirred. The flask was fit with rubber septum and a 



kp 

k´t 

P • m 

kt 

Pn+m

eflux condenser equipped with bubble flask filled with silicon oil. Then 55μL of Karstedt 

catalyst solution in xylene (3 wt.%, 3,64 μmol) was injected to the reaction flask and the 

mixture was heated in 80 °C oil bath under reflux for 24 hours. THF was evaporated and the 

product POSS-Br was vacuum dried. 

R 

R 

R 

Si 

O 

O 

Si O 

O Si 

O 

R 

Si 

O 

O 

R 

O 

Si 

Si O 

O Si 

O 

O 

Si 

R 

H 

C 

H 3 

R 

+ 

C 

H 2 

RH3 

= C 

O 

O 

CH 3 

Br 

CH 3 

Karstedtův Karstedt's katalyzátor catalysator catalyst 

THF, reflux 

24 hours 

Fig. 3: Synthesis of POSS-Br initiator 

Polymerizations 

Polymerization 1: 

A typical polymerization is as follows: 31 mg CuBr was placed into a 50 mL double-neck 

flask; the flask was evacuated and filled with nitrogen. Under a nitrogen blanket 5 mL of 

styrene, 40 μL of PMDETA and 4.6 g of p-dimethoxybenzene was added. The mixture was 

stirred until a homogenous solution formed and was placed into a 120°C oil bath under 

nitrogen. After heating to the reaction temperature 0,48 g of POSS-Br (initiator) was added. 

The solution turned dark green as the reaction began. Periodically, small amounts of reaction 

mixture were removed for kinetic and molecular weight analysis (conversion was 

determinated by weight analysis, molecular weight of polymers was determined using gel 

permeation chromatography). Molar ratios: Styrene:POSS-Br:CuBr:PMDETA - 

100:1:0.5:0.5. 

Polymerization 2: 

This polymerization is similar to the polymerization 1, except the POSS-Br initiator is 

replaced with 65 μL of ethyl-α-bromo-isobutyrate, which was injected to the hot mixture via 

rubber septum. Molar ratios: Styrene:EBIB:CuBr:PMDETA - 100:1:0.5:0.5. 

RESULTS 

Reason for using POSS as an initiator of ATRP is to get a polystyrene macromolecule with 

one bulky POSS group at one of the chain ends; the initiator of ATRP remains the inseparable 

part of the macromolecule. 

Molecular weight plots prove living character of polymerizations; molecular weight grows 

linearly with conversion, with conversion growing to almost 100%. Linear dependence of 

conversion on time in semi logarithmic coordinates (Fig. 5) shows the first-order kinetic with 

respect to monomer. 

From the molecular weight plot of polymerization 1 (Fig. 6) it is obvious, that initiation 

efficiency is low and only about one half of the POSS initiator molecules is used for 

initiation; this causes the molecular weight to be twice as high as is should be according to the 

theoretical value determined from molar ratio monomer:initiator. In contrast, polymerization 

2 initiated with low molecular weight initiator EBIB behaves in accordance with theory 

(Fig. 7). 

R 

R 

O 

Si 

O 

R 

Si 

O 

O 

Si 

O 

R 

Si 

O 



O 

Si 

O 

O 

Si 

R 

R 

O 

O 

Si 

Si 

O 

R 

O 

O 

CH 3 

Br 

CH 3

Differences can be caused by several reasons; no one of these reasons was studied in this 

work in detail, so the following ideas must be taken as a speculations. 

One factor could be different solubility of catalyst system due to the presence of different 

initiator in reaction mixture; it is also important to rule out the possibility of conventional free 

radical polymerization taking place to form polystyrene homopolymer, although the catalyst 

system should inhibit this reaction; part of POSS based initiator molecules could be 

deactivated with some side reaction, or there is just a steric block for approach of molecules 

of catalyst or monomer to the bulky POSS molecule; these ideas must be taken into 

consideration in subsequent work on this project. 

Convers ion [%] 

ln([M]0/[M]t) 

M 

100 

2 

1 

80 


40 

20 

0 

0 200 400 600 800 1000 

t [min] 

Fig. 4: Kinetic plots 

0 

0 100 200 300 400 500 

25000 

20000 

15000 

10000 

5000 

t [min] 

Fig. 5: Semilogarithmic kinetic plots 

0 

1.00 

0 20 40 60 80 100 

Co nve rs io n [%] 

1.70 

1.60 

1.50 

1.40 

1.30 

1.20 

1.10 

D 

Polymerization 1 




M(th e o r.) 

Fig. 6: Molecular weight plot for polymerization 1 



M 

D

CONCLUSION 

M 

12000 

10000 

8000 


4000 

2000 

0 

0 20 40 60 80 100 

Conve rs ion [%] 

1.3 

1.25 

1.2 

1.15 

1.1 

1.05 

1 

D 

M(theor.) 

Fig. 7: Molecular weight plot for polymerization 2 

ATRP has been employed to produce polystyrene macromolecules containing polyhedral 

oligomeric silsesquioxanes (POSS). Polymerizations initiated with POSS prove slower 

growth of polymers than polymerizations initiated with low-molecular initiators, and also 

lower initiation efficiency, which causes the molecular weight to be much higher than 

expected. Further work is underway to optimize the conditions of the polymerizations with 

respect to the efficiency of initiation, polydispersity of polymer and polymerization rate. 

REFERENCE 

[1] Agaskar P.A., Inorg. Chem., 30 (1991), p. 2707-2708 

[2] Zhang C., Laine R.M., J. Organomet. Chem., 521 (1996), p. 199-201 

[3] Lichtenhan J.D. et al., Mat. Res. Soc.Symp. Proc., 435 (1996), p. 3-11 

[4] Wang J.S., Matyjaszewski K., J. Am. Chem. Soc., 117 (1995), p. 5614-5615 

[5] Pyun J. et al., J. Am. Chem. Soc. 123 (2001), p. 9445-9446 

[6] Matyjaszewski K. et al., ACS Symp. Ser. 729 (2000), p. 270-283 

[7] Brown D.A., Price G.J., Polymer 42 (2001), p. 4767-4771 

[8] Miller P.J., Matyjaszewski K., Macromolecules 32 (1999), p. 8760-8767 



M 

D

MEASUREMENT OF THERMOPHYSICAL PROPERTIES OF PMMA 

BY PULSE TRANSIENT METHOD 

Ing. Pavla Štefková 

Supervisor: Prof. Ing. Oldřich Zmeškal, CSc. 


Technology, Purkynova 118, 612 00 Brno, Czech Republic, email: stefkova@fch.vutbr.cz 

INTRODUCTION 

Polymethyl methacrylate (PMMA) is the synthetic polymer of methyl methacrylate. This 

thermoplastic and transparent plastic was developed in 1928 in various laboratories and was 

brought to market in 1933 by the German Company Rohm and Haas. This material is used as 

the standard reference material for thermal conductivity measurements in metrology. 

Chemical analysis and materials testing are becoming ever more important as science, 

trade and society are getting more complex worldwide. The number and significance of 

decisions based on the results of chemical analysis and materials’ testing is ever increasing in 

all spheres of life. For this purpose results of analysis and testing have to be reliable and 

comparable as well as acceptable worldwide. The use of certified reference materials is an 

efficient and proper tool to achieve these goals [1]. 

Measurement of the thermophysical properties of PMMA shows that some effects 

influencing the measurement process have to be known when one want to use it as laboratory 

reference or standard reference material (SRM). This material should be used for validation of 

apparatuses upon well-known experimental conditions, to obtain reliable data [2]. 

The pulse transient method allows investigating the thermal diffusivity, specific heat and 

thermal conductivity within single measurement. The principle of this method and the 

arrangement of the measured sample are shown in Figure 1. The heat pulse is generated by 

the passing of the electrical current through the plane electrical resistor made of metallic foil. 

A sensor measures the time development of the temperature field (temperature response) in a 

point of the tested body. Then the temperature is characterized by a function [3] 

⎟ 2 

Q S ⎛ h ⎞ 

ΔT = 

⋅ exp ⎜ 

⎜− 

. (1) 

( E− 

D) 

/ 2 

cp 

ρ ( 4π 

at 

) ⎝ 4a 

t ⎠ 

The thermophysical parameters are calculated from the characteristic parameters of the 

temperature response (time and the maximum of temperature response to the heat pulse). 

The thermal diffusivity is given by 

2 

h 

a = 

2tmax f a 

the specific heat by 

2 

h 

= 

2( 

E − D) 

tmax 

, (2) 

Q 

cp = ⋅ 

ρ ΔTmaxh 

and thermal conductivity by 

f c 

= 

2π exp( 1) 

Q 

E− 

D 

ρ ΔTmaxh 

( E −D 

) / 2 

⎛ E − D ⎞ 

⋅ ⎜ 

2 exp( 1) 

⎟ 

⎝ π ⎠ 

(3) 

Q 

λ = cp ρ a = 

E− 

D−2 

2( 

E − D) 

ΔTmaxt 

maxh 

( E −D 

) / 2 

⎛ E − D ⎞ 

⎜ 

2 exp( 1) 

⎟ . 

⎝ π ⎠ 

(4) 



It is possible to definite the coefficient fa (fractal dimension D respectively) for every point of 

the experimental dependence 

2ln( 

ΔTmax 

ΔT 

) 

f a = E − D = 

. (5) 

ln( t t ) + ( t t −1) 

t t 

specimen 

tm t m 

Figure 1 The principle of measurement of thermophysical parameters by the pulse transient 

method. 

max 

planar source thermocouple 

current current pulse pulseplanar 

source 

h 

thermocouple 

I I 

t t0 o 

I II III 

max 

T 

temperature response response 

EXPERIMENT AND RESULTS 

For measuring of the responses to the pulse heat the Thermophysical Transient Tester 1.02 

was used. It was developed at the Institute of Physics, Slovak Academy of Science. The 

specimen of 30 mm in diameter and 6 mm thick was used for the pulse transient method. Its 

density is ρ = 1184 kg m –3 . Thermophysical properties of material were measured in air. 

1. Comparison between experimental and recommended data of the thermophysical 

parameters of PMMA measured at 25 °C 

The pulse width of 4 – 40 s, the heat power of 0.18 up to 3.03 W was used and adequate 

the pulse heat energy of 3000 – 42000 J m –2 was obtained. The typical heat energy of pulse 

was about 13000 J m –2 that is low enough to avoid temperature damage of this material. The 

temperature response ΔTmax in the range of 0.1 up to 1.4 °C was obtained. Analysis of these 

sets of data was carried out to find optimal experimental conditions. 

ΔT (°C) 

1.0 

0.8 

0.6 

0.4 

0.2 

0.0 

T m 

0 150 300 450 600 750 

t (s) 

15065 J m 

20197 

23753 

28706 

40081 

-2 

J m -2 

J m -2 

J m -2 

J m -2 

Figure 2 Temperature responses of PMMA measured by the PTM for different heat powers 

and various pulse widths; see Table 1. 



ΔTm

The typical time responses of temperature for the rectangle (Dirac) pulse of different input 

power of heat with various pulse widths are presented in Figure 2. The heat power and width 

of pulse were changed for find the optimal measurement conditions and subsequently 

determine the reliable data of thermal diffusivity, specific heat and thermal conductivity of 

studied material. These results are summarized in Table 1. 

Table 1 Thermophysical parameters of PMMA measured in optimum exp. conditions. 

Q/S (J m –2 ) P (W) ΔTmax (°C) tmax (s) a (m 2 s –1 ) cp (J kg –1 K –1 ) λ (W m –1 K –1 ) 

13 069 0.58 0.307 144 0.113 1425.0 0.190 

17 325 0.67 0.398 146 0.110 1457.1 0.190 

17 267 0.67 0.397 148 0.109 1456.9 0.188 

19 789 0.76 0.460 144 0.112 1440.5 0.190 

17 990 0.92 0.424 148 0.113 1420.0 0.190 

20 197 1.21 0.477 141 0.120 1419.1 0.202 

23 753 1.75 0.563 139 0.124 1414.1 0.207 

28 706 2.88 0.680 141 0.123 1415.2 0.207 

40 081 2.88 0.955 148 0.115 1405.6 0.192 

(0.115 ± 0.005) (1428.2 ± 17.8) (0.195 ± 0.007) 

The thermophysical parameters were calculated from the parameters of the temperature 

response to the heat pulse. Typical temperature increases for reliable data were between 

0.3 °C and 1.0 °C that were equivalent to the heat powers of 0.58 W up to 2.88 W and to the 

heat energies of 13000 J m –2 up to 40000 J m –2 . 

The pulse transient method gives data within the experimental error less than 4.35 % for 

thermal diffusivity, less than 1.25 % for specific heat and 3.59 % for thermal conductivity. 

Average values a = 0.115 m 2 s –1 , cp = 1428.2 J kg –1 K –1 and λ = 0.195 W m –1 K –1 are in 

reasonable coincidence with the recommended values, see Table 2. 

Table 2 Recommended values of thermophysical parameters of PMMA at 25 °C [4]. 

D (–) 

ρ (kg m –3 ) a (m 2 s –1 ) cp (J kg –1 K –1 ) λ (W m –1 K –1 ) 

3.0 

2.5 

2.0 

1.5 

1.0 

0.5 

0.0 

1188 0.112 1450.0 0.193 

0 50 100 150 200 250 

t (s) 

15065 J m 

20197 

23753 

28706 

40081 

-2 

J m -2 

J m -2 

J m -2 

J m -2 

Figure 3 Fractal dimension of the heat distribution in the specimen determined from 

increased part of characteristics. 



The coefficient fa (fractal dimension D respectively) of the fractal heat source for every 

point of the experimental dependence was calculated using the Eq. (5). 

The fractal heat source characterizes the distribution of the temperature in the specimen in 

specific time. The character of these dependences differs for different thicknesses of measured 

sample. Dependencies of the fractal dimension on the time are plotted in Figure 3. 

Generally we can say that all measured results were started from the value of the fractal 

dimension D ≈ 3 and then they were saturated at the some constant value of the fractal 

dimension. This value of the fractal dimension depends on the losses during the transport of 

heat throw the sample. 

2. Study of thermophysical parameters of PMMA at 25 °C and 30 °C 

The measurements were carried out with the sample temperature stabilized at 25 °C and 

30 °C in atmosphere of air and in the same experimental conditions. The temperature 

response occurred in the range of 0.1 up to 1.4 °C. Experimental data was obtained for the 

pulse width of 4 – 40 s and the pulse heat energy of 6000 up to 36000 J m –2 . 

a (mm 2 s -1 ) 

c p (J kg -1 K -1 ) 

λ (W m -1 K -1 ) 

0.17 

0.16 

0.14 

0.13 

0.11 

15705000 

11000 17000 23000 29000 

1510 

1450 

1390 

1330 

0.28 

0.26 

0.24 

0.22 

0.20 

5000 11000 17000 23000 29000 

5000 11000 17000 23000 29000 

Q /S (J m -2 ) 

25 °C 

30 °C 

Figure 4 Thermophysical parameters of PMMA measured at 25 °C and 30 °C. 



Figure 4 illustrates the typical dependencies of the thermophysical parameters of PMMA 

on the heat energy for the heat power 1.75 W. This figure shows that thermal diffusivity of 

PMMA slightly decreases with the increasing temperature as well as thermal conductivity. On 

the contrary, the specific heat increases with the increasing temperature of measured sample. 

CONCLUSIONS 

This paper presents the results of the measurements in optimal experimental conditions and 

of the study of the dependency of the thermophysical parameters on the temperature of 

studied specimen that were obtained on PMMA. The measurements were made in air. To 

interpret the outcomes, the simplified heat conductivity model is used [3]. Results show the 

image of heat distribution in the specimen, in various time intervals after the heat supply from 

the source. 

The analysis of experimental data measured by the pulse transient method for various heat 

power and pulse width of measurement was performed on polymethyl methacrylate (perspex). 

The optimization of the procedure of conditions metering was used to find the optimal range 

of measuring process at 25 °C where data stability interval exists, e.g. the values of 

thermophysical parameters are reliable. The value of thermal diffusivity calculated from the 

data stability interval was determined as 0.115 m 2 s –1 , 1428.2 J kg –1 K –1 for the specific heat 

and the value of thermal conductivity was calculated as 0.195 W m –1 K –1 and they are close to 

the recommended values; see Table 2. The pulse transient method gives data within the 

experimental error less than 4.35 % for thermal diffusivity, less than 1.20 % for specific heat 

and 3.59 % for thermal conductivity. These evaluations could be used for more accurate 

determination of the thermal parameters of studied (homogeneous and heterogeneous) 

matters. 

Data measured on PMMA clearly show difference of thermophysical parameters measured 

in different temperatures for the same specimen and experimental conditions. Thermal 

diffusivity of PMMA slightly decreases with the increasing temperature as well as thermal 

conductivity. On the contrary, the specific heat increases with the increasing temperature. 


This work was supported by the Grant Agency of the Czech Republic, contract No. 

2239/2006/G1. 

REFERENCES 

[1] Steiger T., Pradel R.: Update on COMAR – the Internet Database for Certified 

Reference Materials. Journal of Metrology Society of India. Vol. 19. No. 4. 2004. 

p. 203-207. 

[2] Boháč V., Kubičár L.: Investigation of Surface Effects on PMMA by Pulse 

Transient Method. In Thermophysics 2001, Meeting of the Thermophysical 

Society, Working Group of the Slovak Physical Society. Račkova dolina. October 

23, 2001. p. 21-27. ISBN 80-8050-491-1. 

[3] Stefkova, P., Zmeskal, O., Capousek, R. Study of Thermal Field in Composite 

Materials. In Complexus Mundi. WS. London, World Scientific. 2006. p. 217 – 

224. ISBN 981-256-666-X. 

[4] Vohlídal J., Julák A., Štulík K.: Chemické a analytické tabulky. Grada Publishing 

1999; 1. vydání; Praha 1999; 652 s. ISBN 80-7169-855-5. 



THE ANALYSIS OF GAMMA LINOLENIC ACID IN EVENING 

PRIMROSE OIL 

Hana Štoudková, 2.ročník DSP 

Školitel: doc. Ing. Miroslav Fišera, CSc. 

Vysoké učení technické v Brně, Fakulta chemická, Ústav potravinářské chemie 

a biotechnologie, Purkyňova 118, 61200 Brno, email: stoudkova@fch.vutbr.cz 

INTRODUCTION 

Evening primrose oil (Oenothera biennis L.) is rich source of ω-6 series of polyunsaturated 

fatty acids. One of these fatty acids is gamma linolenic acid – GLA (cis-6,cis-9,cis-12octadecatrienoic 

acid). The content of GLA is the single most important parameter to be 

determined [1,2]. 

The oil content of evening primrose seeds varies with such factors as the age of the seed, 

cultivar and growth conditions, and typically varies between 18 and 25 %. The oil consists of 

about 98 % triacylglycerols, with small amounts of other lipids (free acids, diacylglycerols, 

phospholipides) and about 1–2% unsaponifiable matter, of which sterols and tocopherols are 

of some importace [1]. 

Evening primrose oil is used in increasing amount in nutritional and pharmaceutical 

preparations. Essential fatty acids are vital components of all membrane structures in the body 

and there are also involved in the production of prostaglandins, which regulate the immune 

system. A prostaglandin deficiency can cause skin rashes, hair loss, lowered immunity [3,4]. 

One of the most important unsaturated fatty acids involved in beneficial prostaglandin 

production is gamma linolenic acid. 

Gamma linolenic acid and evening primrose oil have been the focus of many medical 

studies. Some of these studies claim that evening primrose oil supplementation boost natural 

immunity, help lower cholesterol levels, reduce blood pressure, can help ease premenstrual 

syndrome, eczema, diabetes, osteoporosis [3,4]. 


Samples 

A total of eight evening primrose oils were tested. Samples were obtained from Aromatica, 

v.o.s. and the other producers. Common lipophilic compounds with stabilized properties were 

chosen as antioxidants: coenzyme Q10, β-carotene, vitamin E and Origanox - extract of 

Origanum Vulgare containing flavonoids. Various ways of storage were chosen for 

assessment of stability of oils. Maximum time of storage was 183 days from opening the vial 

with sample. 

Methanol esterification method 

The fat sample (1,0 g) was saponified with 15 ml methanolic solution of potassium 

hydroxide (c = 0,5 mol⋅dm -3 ) for 30 minutes in distilling flask with condenser and was 

esterified after neutralization by sulphuric acid on methyl orange for 30 minutes again. 

After cooling methyl esters were shaken with 10 ml of heptane three times. The extract 

was dried by anhydrous sodium sulphate and filtered to a 50 ml volumetric flask again. Both 

heptane portions were rinsed with 20 ml of water twice. The extract was dried by anhydrous 



sodium sulphate and filtered to a 50 ml volumetric flask and filled up to the mark with 

heptane [5]. 

GC analysis 

The GC method – gas chromatography was used for identification of the fatty acids. So 

prepared heptane methyl esters solutions were injected to gas chromatograph using 

autosampler. The compounds were identified according to available standards. 

GC conditions: gas chromatograph TRACE GC (ThermoQuest Italia S. p. A., I) equipped 

with flame ionization detector, split/splitless injector and capillary column SPTM 2560 

(100 m × 0,25 mm × 0,2 μm) with the temperature programme 60 °C held for 2 min, ramp 

10 °C⋅min -1 up to 220 °C, held for 20 min. The injector temperature was 250 °C and the 

detector temperature was 220 °C. The flow rate of the carrier gas N2 was 1,2 ml⋅min -1 . 


Gamma linolenic acid (GLA) is found naturally to varying extents in the fatty acid fraction 

of some plant seed oils. The content of GLA is the one of the most important parameter for 

tested evening primrose oils. It is comprised of 18 carbon atoms and three double bonds. 

Double bonds can be cause of the reduce GLA and it leads to changes GLA during time of 

storage and used antioxidants. 

Virgin evening primrose oils 

Virgin evening primrose oils samples (Arnaud, Gustav Heess) were stored at 4 °C during 

time of storage. Evening primrose oil Arnaud was also stored under laboratory conditions. 

The percentage numbers GLA are shown in producer´s certificates (Arnaud – minimal 9 %, 

Gustav Heess - in range from 8 to 12 % GLA). The GLA amount changed significantly 

during storage. In the case of virgin evening primrose oil Arnaud the measure values GLA 

were lower for up to 1 %. In the case evening primrose oil sample Gustav Heess the content 

of GLA was about 8 % and it´s in the certificate range. 

No-stabilized sample of evening primrose oil which was held on laboratory conditions 

recorded a considerable loss GLA (decrease of the GLA content – 2,9 %). 

Commercially produced oil samples 

Samples of evening primrose oils with antioxidants (vitamin E, vitamin E and coenzyme 

Q10, vitamin E and β-carotene) obtained from Aromatica, v.o.s. The GLA content decreased 

during time of storage slightly. The loss GLA was observed independently of added kind, 

combination or amount antioxidants. 

Dependent the content of GLA on time of storage three samples commercially produced 

evening primrose oils with the addition of antioxidants is shown in Fig. 1. In the case evening 

primrose oil with coenzyme Q10 a vitamin E a decrease was 0,6 % GLA, the sample with 

β-carotene and vitamin E 1,3 % GLA and in the case evening primrose oil with vitamin E a 

loss was 1,0 % GLA. The decrease GLA related to the total content of fatty acids. 



GLA (%) 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

coenzyme Q10 + vitamin E β-carotene + vitamin E vitamin E 

1 23 52 91 106 147 183 

time (days) 

Fig. 1: Variation over time of storage in gamma linolenic acid content of commercially 

produced evening primrose oils with antioxidants 

Laboratory prepared samples 

Two samples (evening primrose oil with Origanox TM and evening primrose with mixture 

antioxidants coenzyme Q10, β-carotene and vitamin E) were laboratory prepared. In the case 

both of samples the GLA content changed slightly over time of storage (Fig. 2). 

GLA (%) 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

coenzyme Q10 + β-carotene + vitamin E Origanox 

1 30 51 84 125 161 183 

time (days) 

Fig. 2: Variation over time of storage in gamma linolenic acid content of laboratory 

prepared evening primrose oils with antioxidants 



Compare all tested evening primrose oils 

The GLA content was similar for the all tested evening primrose oils and range of GLA 

was from 5 to 11 % of total fatty acids. The content of GLA decreased gradually during time 

of storage. Compare of the GLA content in various tested evening primrose oils at first and 

last (183) day of storage is shown in Table 1. 

Maximum level GLA recorded virgin evening primrose oil Arnaud in 147 day of storage 

(10,40 ± 0,17 %). Minimal amount GLA was found in the sample evening primrose oil at 183 

day of storage, which was stored under laboratory conditions (5,65 ± 0,06 %). Evening 

primrose oil with antioxidant Origanox changed minimally. Whereas, most losses recorded 

no-stabilized evening primrose oil which was hold on laboratory conditions. 

Table 1: The content of GLA in evening primrose oils (first and last day of storage) 

Time of storage [days] 

Evening primrose oils 

1 183 

GLA [%] sr [%] GLA [%] sr [%] 

+ Q10 + vitamin E 8,00 ± 0,10 1,27 7,39 ± 0,03 0,38 

+ β-carotene + vitamin E 8,47 ± 0,09 1,07 7,24 ± 0,10 1,39 

+ vitamin E 8,77 ± 0,00 0,01 7,75 ± 0,04 0,53 

+ Q10 + β-carotene + vitamin E 7,26 ± 0,18 2,44 7,00 ± 0,06 0,86 

+ Origanox 8,84 ± 0,04 0,43 8,72 ± 0,18 2,07 

virgin, Arnaud 8,50 ± 0,14 1,63 8,01 ± 0,13 1,66 

virgin, Gustav Heess 8,06 ± 0,05 0,68 7,63 ± 0,02 0,26 

virgin, laboratory conditions 8,55 ± 0,00 0,04 5,65 ± 0,06 1,01 

CONCLUSION 

Primrose is a pure natural vegetable oil processed from the seeds of the evening primrose 

plant. Evening primrose oil is higher in total essential fatty acids than any other vegetable oil. 

The oil contains 74 % linolenic acid (LA) and 8-10 % gamma linolenic acid (GLA). 

The purpose of this work was to find out and compare the influences of used antioxidants 

and time of storage in respect of the content of fatty acids in evening primrose oil. Results of 

gas chromatography were statistically compared to determine the relationship between the 

stability of oils and the type of the antioxidant or the way of storage. 

A total of eight evening primrose oils (virgin or with addition of antioxidants - coenzyme 

Q10, β-carotene, vitamin E and Origanox) were considered. 

Various ways of storage were chosen for assessment of stability of oils. Methanol 

esterification method using potassium hydroxide catalysis was applied to oil for preparing 

fatty acids methyl esters. Gas chromatography (GC) was applied for the determination of fatty 

acids content (especially gamma linolenic acid - GLA). The compounds were identified by 

their retention times relative to authentic standards 

Gamma linolenic acid was present most often in concentration 7 – 9 % of total fatty acids 

in the samples of evening primrose oils. The content of GLA decreased gradually depending 

on increasing time of storage. Evening primrose oil, which was stored under laboratory 

conditions, showed the greatest losses GLA. Whereas, sample evening primrose oil 

containing Origanox had minimal changes. It was observed great dissimilarity GLA during 

another way of storage of oils with addition antioxidants. Virgin evening primrose oils have 

comparable levels of GLA. 



Addition of antioxidants, their amounts or combination have no influence on changes in 

oils during the storage. However, the way of storage can influence on the content of GLA. 

REFERENCES 

[1] Christie, W.W.: The analysis of evening primrose oil. Industrial Crops and Products, 

1999, vol. 10, pp. 73-83. ISSN 0926-6690. 

[2] Court, W. A., Hendel, J.G., Pocs, R.: Determination of fatty acids and oil content of 

evening primrose (Oenothera biennis L.). Food Reearch International, 1993, vol. 26, 

pp. 181-186. ISSN 0963-9969. 

[3] Fan,Y.-Y., Chapkin, R.S.: Importance of dietary γ-linolenic acid in human health and 

nutrition. Recent Advantages in Nutritional Science, 1998, vol. 128, pp.1411-1414. 

ISSN 0022-3166. 

[4] Horrobin, D. F.: Nutritional and medical importance of gamma-linolenic acid. Progress In 

Lipid Research, 1992, vol. 31, pp. 163-194. ISSN 0163-7827. 

[5] ČSN EN ISO 5509: Animal and vegetable fats and oils – preparing of methyl esters of 

fatty acids, 2000. 



IMPROVED FLUORIMETRIC DETERMINATION OF Al, Ga AND In 

BY MICELLE ENHANCED 8-HYDROXYQUINOLINE -5-SULPHONIC 

ACID COMPLEX 

Šimon Vojta, 3. ročník DSP 

Prof.RNDr.Lumír Sommer, DrSc. 

Faculty of Chemistry, Brno University of Technology, Institute of Chemistry and Technology 

of Environmental Protection, Purkyňova 118, 61200 Brno, e-mail: vojta@fch.vutbr.cz 

INTRODUCTION 

The enhancement (sensitization) of fluorescent reactions of metal ions with chelating dyes 

by the presence of surfactants provides an inexpensive alternative to fluorimetric methods, 

when determination of lower concentrations of elements is required. 

The complexes formed in micellar media are often characterized by high molar 

absorptivities, high stability over a wide pH range, and usually by large bathochromic shift 

caused by addition of surfactants to the binary complex formed in water. Moreover, some 

fluorescent binary chelates may dramatically increase their quantum yield in micellar media, 

providing exceptionally sensitive fluorimetric methods. 

The 8-hydroxyquinoline-5-sulphonic acid (QSA) should provide surfactant-sensitized 

reactions with Al(III), Ga(III) and In(III), because it is strong acid readily yielding 

a negatively charged group (sulphonate) to interact with the positive head of cationic 

surfactants. 

The importance of monitoring aluminium concentrations in the blood of patients 

undergoing haemodialysis has been realised because of the suspicion that aluminium 

intoxication is responsible for encephalopathy, oesteopathy and Alzheimer's disease. 

In this work, the influence of various surfactants on the reaction of QSA with Al, Ga and 

In is studied in details. Improved method of determination of Al, Ga, In by QSA in presence 

of cationic surfactant Zephyramine is brought and optimized. 

EXPERIMENTAL 

REAGENTS 

Aluminium standard, containing 1,000±0,002 g l -1 of Aluminium in 5% HCl, purchased 

from Analytica, LTD. Prague 

Gallium standard, containing 1,000±0,002 g l -1 of Gallium in 10% HCl, purchased from 

Analytica, LTD. Prague 

Indium standard, containing 1,000±0,002 g l -1 of Indium in 10% HCl, purchased from 

Analytica, LTD. Prague 

8-hydroxyquinoline-5-sulphonic acid – Sigma-Aldrich Co. 

Benzyldimethyltetradecylammonium chloride (Zephyramine ® ) – Sigma-Aldrich Co. 

1-ethoxykarbonylpentadecyltrimethylammonium bromide (Septonex ® )–Sigma-Aldrich Co. 

Dodecylbenzyldimethylammonium bromide (Ajatin ® ) – Sigma-Aldrich Co. 



Didodecyldimethylammonium bromide – Sigma-Aldrich Co. 

Polyoxyethylene(23) lauryl ether (Brij 35) – Sigma-Aldrich Co. 

Hexadecyltrimethylammonium chloride – Sigma-Aldrich Co. 

4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100) – Calbiochem Co. 

APPARATUS 

A single beam spectrofluorimeter Aminco Bowman ® , series 2 with a xenon lamp working 

in a continuous regime, 4 nm slits and constant photomultiplier voltage was used for the 

measurements. 

RESULTS 

The 8-hydroxyquinoline-5-sulphonic acid forms with Al, Ga and In fluorescent complex with 

emission maxima at 495 nm (Al), 503 nm (Ga) and 505 nm (In). Excitation maxima for Al is 

360 nm, 365 nm for Ga and 367 nm for In. There is no fluorescence observed for isolated 

QSA. Excitation and emission spectra of Al-QSA in dependence on concentration of Al and 

reaction blank without Al are shown in Fig.1. 

The formation of fluorescent complex is instantaneous and fluorescence is stable at least for 

twelve hours (Fig.3). Fluorescence intensity grows from In to Al and strongly depends on pH 

(Fig.2). 

F i 

80 

70 


50 

40 

30 

20 

10 

1a 

2a 

3a 

4a 

5a 

6a 

6b 

0 

270 320 370 420 470 520 570 620 

Fig.1.: Excitation(a) and Emission(b) spectra of Aluminium(III) in the presence of 7,4.10 -5 

mol.dm -3 QSA in dependence on concentration of Al(III). 

1-1,6μg.cm -3 ,2-0,8μg.cm -3 , 3-0,4μg.cm -3 ,4- 0,2 μg.cm -3 ,5-0,05 μg.cm -3 ,6–0μg.cm -3 



λ (nm) 

1b 

2b 

3b 

4b 

5b

Al 

F i 

80 

70 


50 

40 

30 

20 

10 

0 

0 

1 2 3 4 5 6 7 8 9 10 

Fig.2.: Fluorescence intensity dependence on pH for each complex 

pH 

The fluorescence intensity can be significantly increased in the presence of cationic 

surfactant and small bathochromic shift in excitation spectra is also observed. The 

fluorescence is increased in one hour (Fig.3) and after it reaches maximum, it’s stable at least 

10 hours. The best positive effect was found for Zephyramine (Fig.4.). No positive effect was 

observed for anionic and nonionic surfactants. 

20 

Ga 

In 

18 

a b 

Fig.3.: Comparison of emission spectra time trace for Al-QSA complex without surfactant (a) 

and in presence of cationic surfactant Zephyramine (b) 



Al 

In 

Ga 

16 

14 

12 

10 

8 

6 

4 

2

F i 

90 

70 

50 

30 

10 

-10 

-30 

Zephyramin 

Septonex Ajatin 

DDAB 

Triton 

SDS 

-50 

0 0,002 0,004 0,006 0,008 0,01 0,012 0,014 0,016 

Fig.4.: Surfactants influence on Ga-HQS complex at pH 3. Septonex, Zephyramine, Ajatin, 

SDS – mol.dm -3 , CTAC (Hexadecyl trimethyl ammonium chloride)– c/4 (mol.dm -3 ), 

DDAB (Didodecyldimethyl ammonium bromide) – c/5 (mol.dm -3 ), Triton X-100 – 

c/4.10 -5 (ppm), Brij – c/15 (mol.dm -3 ). 

Critical micellar concentrations are highlighted by the fulfilled marks. 

The nature of the complex strongly depends on the pH. The molar ratio of Me to QSA in 

the binary complex determined by the continuum variations method is 1:1 in acidic range and 

1:3 in alkalic range (Fig.5). In the presence of cationic surfactant Zephyramine, the molar 

ratio of ternary complex was found to be always 1:3 (Fig.5). 

F i 

100 

80 


40 

20 

0 

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 

x L 

1 

2 

3 

c 

80 


40 

20 

CTAC 

Brij 

a 100 

b 

F i 

0 

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 

Fig.5.: (a) Continuum variations of Al-HQS complex at pH 4. 1-c0= 1,1.10 -4 M, 2- c0= 5,6.10 -5 M, 

3-5,6.10 -5 M, 0,0012M Zephyramine 

(b) Continuum variations of In-HQS complex. 1-c0= 3,5.10 -5 M, pH8, 2- c0= 1,8.10 -5 M, 

pH8, 3-1,8.10 -5 M, 0,0012M Zephyramine, pH8, 4- c0= 1,8.10 -5 M, pH4 

Finally, the improved method with enhanced sensitivity by the presence of cationic 

surfactant Zephyramine was optimized with concentration of Zephyramine 0,0012M and five 

times excess of QSA to highest molar concentration of Me. Optimal pH is 4 for Al, 3 for Ga 

and 8 for In. 



x L 

1 

4 

2 

3

F i 


50 

40 

30 

20 

10 

Data 

UCL 

LCL 

A 

B 

Calibration Plot 

y = 51,982x + 0,9186 

R 2 = 0,9991 

0 

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 

c Al (μg.cm -3 ) 

Fig.6.: Calibration plot for Al in the presence of 1,4.10 -4 M QSA and 0,012M Zephyramine, 

pH4 

F i 

80 

70 


50 

40 

30 

20 

10 

Data 

UCL 

LCL 

A 

B 


y = 78,46x + 1,1235 

R 2 = 0,9992 

0 

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 

c Ga (μg.cm -3 ) 

F i 

200 

180 

160 

140 

120 

100 

80 


40 

20 

Data 

UCL 

LCL 

A 

B 


y = 185,06x + 10,75 

R 2 = 0,9995 

0 

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 

Fig.7.: Calibration plots for Ga (In) in the presence of 5,75.10 -4 M (resp.3,5.10 -4 ) QSA and 

0,012M Zephyramine, pH3 (resp. 8) 

Tab.1.: Comparison of calibration parameters for each complex. U is photomultiplyer voltage 

and detection limits were calculated according to Graham 4 

a 

c In (μg.cm -3 ) 

Al-HQS Ga-HQS In-HQS 

Concentration range (μg.cm -3 ) 0,004-1 

U(V) 610 700 680 

α 

X D (μg.cm -3 ) 7,476E-05 0,0149 6,720E-05 

blank 

X (μg.cm -3 ) 0,0304 0,0349 0,0333 

β 

D 

−3 

( g ⋅ cm ) 

X b μ 

x + 3s 

blank 

6, a 0,00015 0,00022 0,0042 



CONCLUSIONS 

Cationic surfactants sensitize the reaction of Al, Ga and In with 8-hydroxyquinoline-5sulphonic 

acid. The effect of various surfactants was studied in details. The best results were 

obtained for Zephyramine and Septonex. The nature of the complex was investigated by the 

continuum variations method. Improved method for determination of Al, Ga and In in 

aqueous solutions was optimized and calibration plots were constructed. 

REFERENCES 

1. Vercruysse A.: Hazardous Metals in Human Toxicology, Part B, Elsevier Amsterdam 

1984 

2. Garcia Alonzo J. I., Diaz Garcia M. E., Sanz Medel A.: Talanta. 31, 361 (1984) 

3. Hinze W. L., Singh H. N.: Trends in Analytical Chemistry. 3, 193 (1984) 

4. Graham R. C.: Data Analysis for the Chemical Sciences. A Guide to Statistical 

Techniques. VCH Publishers, New York 1993 

5. ČSN ISO 8466-1: 1993, 15. Praha, 1993. 

6. Anonyme: Anal.Chem. 52, 2245 (1980) 



PHYSICAL-CHEMICAL ASPECTS OF PAINT FILMS DEGRADATION 

Ing. Lucie Wolfová 

Supervisor: prof. RNDr. Zdeněk Friedl, CSc. 

Brno University of Technology, Purkyňova 118, Brno 612 00, e-mail:wolfova@fch.vutbr.cz 

INTRODUCTION: 

My dissertation thesis deals with the physical-chemical aspects of paint film degradation. 

This work is focused on the study and examination of physical-chemical aspects of paint film 

degradation by selected solvents. Mainly it is considered to the determination of solubility 

parameters of used polymers δ2 and the examination of the influence of especially polymer 

structure to the value of this δ2. The main goal of this study is to verify the coating solubility 

theory based on the knowledge of their solubility parameters in practise and its application for 

coatings. The other goals include the projection and formulation some new solvent’s mixture 

and additives intended for removing paint films and graffiti. Such formulation need to be well 

performing and ecological acceptable at the same time, which is the precondition for their 

wide application in practise. 

THEORETICAL BACKGROUND: 

Chemical degradation of paint films could be defined as the physical and chemical 

interactions between film-forming components of the paint and chemical surroundings. 

During the chemical degradation of polymer a disruption of chains and reticulation, a change 

of chemical structure of chains, a change of side groups or combination of all these 

phenomena take place. This leads to changes in paint qualities and coats lose their visual 

appearance and original qualities upon the exposure to organic solvents. 

From the thermodynamic point of view, a solubility of polymer and a degree of solvent 

affinity to polymer is given by the Gibb´s energy change, which is determined by the 

interaction between solvent molecules and macromolecular chain elements. ΔGmix = ΔHmix - 

TΔSmix < 0. 

The more negative is value of ΔGmix the better is solubility of polymer in solvent. Then the 

dissolving of polymers is mainly a question of value ΔHmix because during swelling or 

dissolving always increase system’s disorderliness. According to thermodynamic conditions 

than is necessary that ΔHmix

Then, the solubility parameter can be estimated from general equations: 

ΔHmix = Vs(δ1 – δ2) * φ1 φ2 . 

- This is one of the simple expressions of solubility parameter deduced for nonpolar 

systems. In practise, we have solvents and solutions with different polar forces such as 

dispersion, polar and hydrogen ones. Therefore the cohesive energy Ecoh is divided into three 

parts, corresponding to three types of interaction forces Ecoh = Ed + Ep + Eh (contribution of 

dispersion forces Ed, polar forces Ep and hydrogen-bonding Eh). Then every substance could 

be characterized according to free contributions of total solubility parameter 

2 2 2 

δtotal.= σ + σ + σ . 

disp. 

polar. 

hydrogen. 

By this is predicted that if δ1 = δ2 the ethalpy of mixing is ΔHmix = 0 and ΔGmix = ΔHmix - 

TΔSmix < 0 and this is in accorrdance with the general rule for thermodynamic view of 

solubility and mixing. Then, the smaller is the difference of value δ of solvent and polymer, 

the better is possibility of its dissolving. The polymers should be mostly soluble in that 

solvents, which their < δ > correspond to the middle of the interval of polymer solubility or 

approximately ± 2 units. 

AIM OF STUDY: 

The aim of conducted research is the evaluation of influence of several aspects – especially 

structure of samples – on the chemical resistance of PUR coatings and the value of their 

solubility parametr δ2. As far as these aspects are concerned, these include especially the type 

of used monomers (in case of PUR polymers for example like type and funcionality of used 

alcohols, acids and isocyanates, type of bonds, etc.), the weight, size and type of film forming 

polymer and the type of interactions between individual chain parts. 

METHOLOGY: 

The solubility parameter δ2 of polymer could be determined according to its interaction 

with solvents of known solubility parameter δ1. For example, the solubility parameter of a 

linear polymer can be determined from its limiting viscosity number and of a cross-linked 

one according to the degree of swelling, both of these values being the function of solubility 

parameter of used solvent δ1. In case of best solvent, is the value of [η] or Q of polymer the 

heights and solubility parameter of this solvent δ1 correspondents with solubility parameter of 

polymer δ2 

As far as measuring of viscosity is concenred, the samples were dissolved in selected 

group of solvents whose solubility parameters δ1 varied from 18 to 24 (30). Then their 

dynamic viscosity η were masured. It was done using capillar or Ubelohde viscosimeter. 

η sp 

Subsequently, specific viscosity ηspec and the limiting viscosity number [η] = lim were 

c→0 

c 

calculated. 

The degree of sample’s swelling Q = (m-m0/m0)*1/ςsolv was calculated by weighting 

samples after soaking in chosen solvents for 5 days. 



Finally, the solubility parameter of sample δ2 was determined as a function of [η] or Q and 

compared with values of these δ2 obtained by calculation from sample’s structures. The 

calculation of δ2 from sample’s structures is possible from the contibutions of dispersion Ed, 

polar Ep and hydrogen-bonding Eh forces of the polymer. 

RESULTS AND DISCUSSION: 

The exact sample composition can not be published at the moment because of their 

intended patent protection. Therefore, the samples are named by the manufacturer code. 

The result of influence of different structure of PUR samples to the value of their 

solubility parameter δ2 (10 -3 J 1/2 m -3/2 ): 

- Differences in sample structures were especially polymer’s molecular weight and size of 

chain, type and functionality of used alcohols, acids and isocyanates. 

Limiting viscosity number of 

samples [η] (m 3 /kg) 

Solubility parametrs of PUR samples δ2 determined according to 

0,09 

limiting viscosity number [η] 

U14EG2000 

0,08 

Diexter G214 

C36EG34 

0,07 

Priplast 3192 

0,06 

0,05 

0,04 

0,03 

0,02 

0,01 

0,00 

19,0 19,5 20,0 20,5 21,0 21,5 22,0 22,5 23,0 23,5 24,0 24,5 25,0 25,5 26,0 

Solubility parametr of solvent δ 1(10 -3 J 1/2 m -3/2 ) 

U14EG56 

U14BD2000 

According to the theory, the solubility parameter δ2 value corresponds to the point where 

the plot of [η] as a function of δ1 has its maximum. 

Determination of solubility parameter δ2 as the function of limiting viscosity number 

and by calculation from structure: 

Solvents: 

Solubility 

parameter 

δ1 

Limiting viscosity number of tested samples [η] (m 3 /kg) 

U14EG2000 Diexter C36EG34 Priplast U14EG56 U14BD2000 

Dichloromethane 19,8 0,0076 0,0173 0,0094 0,0071 - 0,0266 

1,4-dioxane 20,5 0,0135 0,0117 0,0093 0,0048 0,0515 0,0540 

Acetyl acetone 22,1 0,0180 0,0087 0,0199 0,0068 0,0856 0,0570 

N,Ndimethylacetamide 22,7 0,0618 0,0337 0,0677 0,0243 0,0638 0,0580 

N methylpyrrolidone 22,9 0,0150 0,0425 0,0193 0,0163 0,0686 0,0514 

N,Ndimethylformamide 24,7 0,0400 0,0144 0,0000 0,0185 - 0,0413 

Solubility parameter δ2(10 -3 J 1/2 m -3/2 ) 

Determined from measuring 22,7 22,9 22,7 22,7 22,1 22,7 

Determined from structure 21,37 23,87 21,31 21,78 20,92 21,25 

According to the obtained results and the theory, we can predict that the total solubility 

parameters δ2 of our tested PUR samples are about 22,7×10 -3 J 1/2 m -3/2 and in case of U14EG56 



22,1×10 -3 J 1/2 m -3/2 . However, if we calculate the value δ2 from the structure, we obtain 

approximately similar values of δ2 in a range of 20,9 to 23,9×10 -3 J 1/2 m -3/2 . The biggest 

differences are in case of Diexter G214 and U14EG56 where the biggest differences between 

their structures are also. 

According to these results, it should be possible to dissolve these samples in solvents with 

total solubility parameter value of δ1 about 20 to 24 units.- And in most cases, experimental 

results comply with theory and samples could be dissolved in solvents with value of δ1≈δ2 ± 2. 

Solubility of PUR sample U14EG56 in various type of solvent: 

solubility U14EG56 

solubility U14EG56 

Solvent: p. δ1 δ2 ≈ 22,1 Solvent: 

p. δ1 δ2 ≈ 22,1 

Toluene 18,2 * N,N dimethylacetamide 22,7 + 

Ethylmethylketone 19 + N methylpyrrolidone 22,9 + 

Dichloromethane 19,8 + Isopropanol 23,6 - 

Acetone 20 * N,N dimethylformamide 24,7 + 

1,4-Dioxane 20,5 + Ethanol 26 - 

Acetyl acetone 22,1 + Butyrolactone 27 - 

Ethylene glycol 30 - 

In the case, when sample is not soluble in right one solvent according to the theory, it can 

be interpreted by different chemical nature of used solvents and it relates to different value of 

individual components of their total solubility parameter. 

The result of study of influence of different values of rigid segment fraction and crosslinked 

fraction content of sample U14BD2000 to its solubility parameter, according its 

parameter of swelling: 

During the study of the influence of rigid segment content and cross-linked fraction 

content to the solubility parameter, it was observed that the more rigit segment fraction and/or 

cross-linked fraction is present, the more resistent the sample was. All these changes should 

be connected with a changes of sample solubility parametr and should be observable 

experimentally. 

Study of the influence of different percentage distribution of rigid segment fraction in 

case of sample U14BD2000: 

Parameter of swelling Q 

(Type A with the less quantity, type D with the most quantity of rigid segment content) 

16 

14 

12 

10 

8 

6 

4 

2 

Influence of different quantity of rigid segment to the 

solubility parametr of U14BD2000 (according its 

parameter of swelling) 

type A 

type B 

type C 

type D 

0 

19,0 20,0 21,0 22,0 23,0 24,0 25,0 

Solubility parameter of solvent δ1 (10-3J1/2m -3/2 ) 

Type of 

sample: 

δ2 - from 

measuring 

δ2 - from the 

structure 

A 19,8 20,11 

B 20,5 20,72 

C 22,9 21,34 

D 22,9 21,96 



Study of the influence of different percentage distribution of cross-linked fraction in 

sample U14BD2000: 

(Type AA with the less quantity, type DD with the most quantity of cross-linked fraction 

content) 

Parameter of swelling Q 

14,00 

12,00 

10,00 

8,00 

6,00 

4,00 

2,00 

Influence of different quantity of cross-linked to the 

solubility parametr of U14BD2000 (according its 

parameter of swelling) 

type AA 

type BB 

type CC 

type DD 

0,00 

18,0 19,0 20,0 21,0 22,0 23,0 24,0 25,0 

Solubility parameter of sample δ1 (10-3J1/2m -3/2 ) 

Obtained results show that in the case of the increasing quantity of rigid segment the 

solubility parameter δ2 is changing and moving to higher values. In the case of increasing of 

cross-linked fraction, the value of δ2 is more or less stable and similar and changes only 

parameter of swelling. – Both of these agree with the theory. 

CONCLUSION: 

Type of 

sample: 

δ2 - from measuring 

AA 19,8 

BB 20,5 

CC 20,5 

DD 20,5 

According to the obtained data, experimental results comply with the theory about possible 

evaluation of coating’s solubility on the base of knowledge their solubility parameters and 

also structures in most cases. 

In next experimental part of my dissertation work I would like to improved, extend and 

apply this theory and information to the area of dissolving and removing especially water 

based coats and modify all of this for use in practice. 

Acknoledgments: 

I would like to thank to C. B. Coreia and Prof. J. C. Bordado, Instituto Superior Technico, 

Lisboa for their help and support during this research. 

LINKS: 

1. Barton A. F. M., Handbook of solubility parameters and other cohesion parameters, 

London 1991 

2. Van Krevelen D. W., Hoftyzer P. J., Properties of polymers – their estimation and 

correlation with chemical structure, Amsterdam 1976 

3. Bicerano J., Prediction of polymer properties (second edition), New York, 1996 

4. Munk P., Aminabhavi T.M., Macromolecular science (second edition), New York, 2002 

5. Mleziva J., Šňupárek J., Polymery-výroba, struktura, vlastnosti a použití, Praha, 2002 



Using low-molecular mass pI markers in proteomic staining-free method 

for study of posttranslationally modified proteins 

Karel Mazanec 1,2 , Karel Šlais 2 and Josef Chmelík 2 

1 Institute of Material Chemistry, Faculty of Chemistry, Brno University of Technology, 

Purkyňova 118, 612 00 Brno, Czech Republic, e-mail: mazanec@iach.cz 

2 Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veveří 97, 

602 00 Brno, Czech Republic 

INTRODUCTION 

At present the knowledge of proteome plays very important role in the study of living 

processes. Special attention in our lab is paid to the investigation of the proteome of barely 

grains. The aim is to contribute to understanding of malting processes and to selection of 

malting barley cultivars. Proteins are the major functional molecules of life made of amino 

acids arranged in a linear chain linked together by peptide bonds. The residues in a protein are 

often chemically altered in a process known as post-transcriptional modification (PTM): 

either before the protein can function in the cell, or as part of control mechanisms. The 

identification of the PTM is experimentally difficult. 

Due to the fact that many samples, mainly of biological origin, are complex mixtures of 

proteins with a wide range of molecular masses, salts and other compounds, proteins should 

be separated and purified prior to their identification by mass spectrometry. Traditionally, 

most proteomics researches are based upon two-dimensional gel electrophoresis involving 

isoelectric focusing (IEF) as one dimension. Separated compounds are focused into very 

sharp bends according to their pI values during IEF. They are twice or more concentrated in 

this zones. However, ampholytes present in gel and creating pH gradient during IEF 

complicate the visualizing the separated proteins by staining and make this method 

impracticable for common proteomic utilization. 

The aim of this work was to develop and optimize the novel staining-free proteomic 

procedure for separation of intact proteins by gel IEF in presence of low-molecular mass pI 

markers and subsequent determination of molecular masses of separated compounds in the 

gels by MALDI-TOF/TOF MS. The selected set of pI markers includes both colored and 

colorless compounds with known low-molecular mass. Thus, the identification of both pI 

marker and proteins in the same excised piece of gel by MS technique is expected to give 

reliable information about the correct pI values of analyzed proteins even in complex samples. 


pI markers – The substituted phenols (I - Mw 314.08, pI 3.9, yellow color; II - Mw 359.10, 

pI 4.3, orange; III - Mw 272.06, pI 5.3, yellow; IV - Mw 252.12, pI 5.7, colorless; V - Mw 

285.09, pI 6.4, yellow; VI - Mw 315.10, pI 7.0, yellow; VII - Mw 337.17, pI 7.5, yellow; VIII 

– Mw 265.14, pI 7.9, yellow; IX - Mw 363.23, pI 8.4, yellow; X - Mw 267.16, pI 8.9, yellow; 

XI - Mw 352.20, pI 9.0, colorless; XII - Mw 333.21, pI 10.1, yellow) used as pI markers were 

prepared by the procedure described by Šlais and Friedl 1 at the Institute of Analytical 

Chemistry (Brno, Czech Republic). The pI values were determined by potentiometric 

titration. The chemical structures of pI markers are shown in Fig. 1. 



Fig.1: Chemical structures of pI markers used in this work. 

Polyacrylamide gels and IEF - The protein samples (2 mg/ml) were mixed with pI markers 

(10 mg/ml each) and the 4 μl of the mixtures were loaded onto the polyacrylamide gel (16%). 

The carrier BioLyte 3/10 ampholyte (final concentration 2%) (Bio-Rad, CA, USA) were 

added prior to gel polymerization. Separation was performed with constant electric power of 

0.6 W for 2 h supplied by VNZ 22 power supply (CSAV Development Workshop, Czech 

Republic) using a model 111 Mini IEF Cell (Bio-Rad, CA, USA). After focusing completion 

the gels were gently removed from electrodes and scanned. 

Ladders of color pI markers simplify the orientation in the gel even when the separated 

proteins cannot be seen. Thus, the bands with proteins were excised according to positions of 

pI markers. The excised gel pieces were then transferred to 0.5 ml tubes. The pI markers were 

then eluted from gel by 30 μl water/ethanol 1:1 (v/v) for 15 min. 

Protein digestion and identification - Excised gel pieces after the extraction of the pI markers 

were washed twice with water/acetonitrile 1:1 (v/v) for 15 min. Then proteins were identified 

according to standard proteomic protocol utilizing in-gel digestion of reduced and alkylated 

proteins with trypsin. 2 

For comparison, the other IEF focused gels were stained with Coomassie Brilliant Blue R 

250. Due to the carrier 3/10 ampholytes used for creating of pH gradient in gels precipitated 

the Coomassie dye during staining causing strong blue haze on the background, the washing 

step (10% TCA in water/methanol (10/3 v/v); 12-14 h) had to be inserted prior to own 

staining procedure. 

Mass spectrometry - MS measurements were carried with 4700 Proteomics Analyzer 

(Applied Biosystems, USA) MALDI TOF/TOF mass spectrometer (equipped with Nd/YAG 

laser; 355 nm). Argon was used as a collision gas. A solution of sinapinic acid (SA) (20 

mg/ml in acetonitrile/water (3/2 v/v)) was used as matrix for markers. Alpha-cyano-4hydroxycinnamic 

acid (CHCA) (both Sigma-Aldrich, Germany) was used as matrix for 

peptides in concentration of 10 mg/ml acetonitrile/water (3/2 v/v). 



RESULTS 

More than forty pI markers of different types of structures were studied by MALDI(LDI)- 

TOF MS. Twelve selected suitable pI markers are used and more closely characterized in this 

work. 

At analysis of low-molecular mass pI markers, several features were observed that help to 

their identification. In the positive ion mode mass spectra of nitro-substituted pI markers the 

characteristic peaks at -16 and -32 Da caused by loss of oxygen atoms during the process of 

ionization (also reminded by Strohalm et al. 3 ) can be seen (Fig. 2, spectrum a, c). A 

characteristic double-peak pattern is obtained for markers containing a chlorine atom (Fig. 2, 

spectrum b, c). These groups of peaks are very helpful for identification of pI markers 

especially in complex mixtures where one peak may be hidden with a cluster or by another 

well-ionizable substance. 

Fig.2: The positive ion mass spectra of the pI markers XII (a); II (b) and I (c) obtained after 

extraction from the IEF gel. A characteristic peak patterns are clearly seen. 

Isoelectric focusing and gel treatment - The 3/10 carrier ampholytes were used to create pH 

gradient in this work. At 50-mm distance of electrodes it means that the slope of the gradient 

is 0.14 pH/mm. The color pI markers enable the orientation in gels and supervision of the IEF 

process. Each abnormality in pH gradient can be immediately and very easily recognized. 

Moreover, the gels can be cut up according to the position of the visible markers. The IEF 

gels were immediately after focusing scanned (Fig. 3A) and the bands with focused pI 

markers were excised. For comparison, another IEF gel was stained with Coomassie Brilliant 

Blue R 250. The focused proteins visualized by Coomassie staining are shown in Fig. 3B,C. 



Identification of pI markers - First, the pI markers were extracted from each excised gel 

piece and identified from extracts by MALDI-TOF/TOF MS. The experiments confirmed that 

pI markers are easily and unambiguously identifiable in extracts from excised gel pieces after 

IEF. 

Fig.3: IEF gel of pI markers and proteins scanned immediately after focusing (A); standards 

(B) and beta-amylase extract (C) after Coomassie staining. The positions of electrodes are 

marked with E. Letters at right show positions where the proteins were identified (see Tab. 1). 

Identification of proteins - First experiment was provided with standard proteins (cytochrome 

c, myoglobin and albumin). After extraction of pI markers from particular gel pieces, the 

proteins in the same gel pieces were treated by enzyme digestion and identified by MS. These 

proteins were identified in positions according to their real pI values and validated our 

approach. 

Then, the extract from barley malt was used as a model mixture of glycated proteins. The 

separated protein mixture after the referential Coomassie staining is shown in Fig. 3C. Table 1 

shows the proteins identified in the gel pieces from which the pI markers were extracted and 

identified. Nine proteins were identified in eight excised gel pieces of twelve ones excised 

according to the locations of the pI markers used. Some proteins were found in two different 

gel pieces. During malting process the barley proteins are gradually cleaved and modified. 

This is the reason why some proteins may be found in several gel pieces with different pI 

values. The differences observed for some other proteins might be caused either by 

posttranslational modifications or by discrepancies between experimental and theoretically 

calculated pI values. For more detailed studies, the gels can be excised into a higher number 

of narrower pieces and the pI values of proteins identified between the locations of pI markers 

can be interpolated or a higher number of pI markers can be used. The identifications of the 

proteins were confirmed from the stained gel by the same proteomic protocol. 



Gel 

pos. Name 

Protein 

Number 

pI marker 

Theor. 

pI 

No. pI 

a Protein Z (Z4) (Major endosperm albumin) P06293 5.70 II 4.3 

b Beta-amylase P16098 5.58 III 5.3 

Glucan endo-1,3-beta-glucosidase GV Q02438 6.92 

c Protein Z (Z4) (Major endosperm albumin) P06293 5.70 IV 5.7 

d Granule-bound starch synthase 1 P09842 7.06 VI 7.0 

Alpha-amylase/subtilisin inhibitor precursor P07596 7.78 

e Glucan endo-1,3-beta-glucosidase GV Q02438 6.92 VII 7.5 

f Lichenase II precursor P12257 9.0 IX 8.4 

Elongation factor 1-alpha Q40034 9.15 

g 26 kDa endochitinase 2 precursor P23951 8.83 XI 9.0 

Histone H3 (Fragment) P06353 9.73 

h 26 kDa endochitinase 2 precursor P11955 8.54 XII 10.1 

Elongation factor 1-alpha Q40034 9.15 

Tab.1: The list of proteins identified directly in the same gel piece as the pI marker. Standard 

proteins are bolded. 

CONCLUSIONS 

The method is based on separation of intact proteins by gel IEF along with low-molecular 

mass pI markers and subsequent sequential determination of molecular masses of separated 

compounds by MALDI-TOF/TOF MS. The identification of both pI markers and proteins in 

the same gel piece give reliable information about the correct pI values of particular identified 

proteins in complex samples. Moreover, it allows omitting protein staining and destaining 

procedure, because the Coomassie procedure is in the case of IEF gels complicated by 

presence of ampholytes causing dark background. The suggested procedure shortens roughly 

by half the time of analysis. The choice of appropriate ampholytes allows to modify the pH 

gradient and to study only the certain area of the pH scale. 

There are only twelve mainly colored pI markers shown in this work. Nevertheless, the 

number of pI markers used may be much higher. The color of pI markers is not relevant for 

MS determination. Focused pI markers may cover whole pH range and from gels narrow 

pieces can be cut out, which will allow more precise determination of identified proteins. 

Utilization of low-molecular mass pI markers with IEF gels was chosen as a first example of 

their applicability because the gel process can be easily observed visually. There is a good 

chance to use this approach also in other IEF techniques (e.g. in capillaries or on chips). 

REFERENCES 

[1] Šlais K, Friedl Z. Low-molecular-mass pI markers for isoelectric focusing. Journal of 

Chromatography A 1994; 661: 249. 

[2] Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins 

from silver stained polyacrylamide gels. Analytical Chemistry 1996; 68: 850. 

[3] Strohalm M, Santrucek J, Hynek R, Kodicek M. Analysis of tryptophan surface 

accessibility in proteins by MALDI-TOF mass spectrometry; Biochemical and 

Biophysical Research Communications 2004; 323: 1134. 



Název: Sborník příspěvků soutěže Studentské tvůrčí činnosti „STUDENT 2006” a doktorské 

soutěže „O cenu děkana 2005 a 2006” 

Editor: Mgr. Radek Přikryl, Ph.D., doc. Ing. Martin Weiter, Ph.D., prof. Ing. Ladislav Omelka, DrSc. 

Vydání: První 

Počet stran: 259 

Vydavatel: Vysoké učení technické v Brně, Fakulta chemická 

Výroba: Vysoké učení technické v Brně, Fakulta chemická 

ISBN: 80-214-3321-3 

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