production of selected secondary metabolites in transformed ...

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, 

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

Sekce DSP 2006, strana 197

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. 

ACKNOWLEDGEMENTS 

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. 

ACKNOWLEDGEMENT 

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. 

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

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 


Sekce DSP 2006, strana 213 

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.) 

RESULTS AND DISCUSSION 

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). 



Temperature (°C) 

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 

6000 

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. 

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

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. 

EXPERIMENTAL PART 

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 

60 

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 

6000 

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. 

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

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. 

ACKNOWLEDGEMENTS 

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]. 

MATERIALS AND METHODS 

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 

60 

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 

60 

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 

60 

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 

60 

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 

60 

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 

60 

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 

60 

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. 

MATERIALS AND METHODS 

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.

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