Jaroslav Churáček

Comparison of retention behaviour of aromatic sulphonic acids in reversed-phase systems with mobile phases containing ion-pairing ions and in systems with solutions of inorganic salts as the mobile phases

The retention of 34 naphthalene and anthraquinone sulphonic acids, which are important as dye intermediates, was measured on C18 and C8 columns in mobile phases containing either an ion-pairing reagent or an inorganic salt in aqueous methanolic solvents. The influence on retention of the length of the bonded alkyl chains, of the nature and concentration of the ion-pairing reagent (an alkylammonium ion), of the inorganic salt (sodium sulphate) and of methanol in the mobile phase was investigated. Systems containing inorganic salts offer distinct advantages for the separation of sulphonic acids with different numbers of sulphonic groups and allow some efficient separations that are not feasible in the mobile phases with ion-pairing reagents. An explanation of the retention mechanism is attempted.

Oriented immobilization of galactose oxidase to bead and magnetic bead cellulose and poly(HEMA-co-EDMA) and magnetic poly(HEMA-co-EDMA) microspheres

In order to obtain an active and stable oxidation reactor for daily use in biochemical laboratory we decided to immobilize galactose oxidase orientedly through a carbohydrate chain to the magnetic carriers. We used hydrazide derivatives of non-magnetic and magnetic bead cellulose and of magnetic and non-magnetic poly(HEMA-co-EDMA) microspheres. Activation of the enzyme molecules was done by sodium periodate in the presence of supplements (fucose, CuSO4, catalase). Orientedly immobilized galactose oxidase presents high storage stability and lower susceptibility to inappropriate microenvironmental conditions. Reactor reactivated by three pulses of D-galactose retained practically 100% of its native activity after 6 months. The positive properties of both magnetic carriers were entirely confirmed.

Enzymes immobilized on magnetic carriers: efficient and selective system for protein modification

In order to obtain an economical, efficient and selective system for glycoprotein modification we prepared reactors with immobilized neuraminidase or (and) galactose oxidase. High storage and operational stability of the enzyme reactors was obtained by their immobilization through the carbohydrate parts of the enzyme molecules to hydrazide-modified supports. Magnetic and non-magnetic forms of bead cellulose and poly(HEMA-co-EDMA) microspheres were used for immobilization. These reactors can be used almost universally for the activation of ligands and for labelling of substances having a carbohydrate moiety.

A simple, optimized method for the determination of sulphide in whole blood by GC–MS as a marker of bowel fermentation processes

Hydrogen sulphide is produced in human large intestine by anaerobic fermentation and may play a pathogenic role. An analytical method for determination of sulphide in whole blood using an extractive alkylation technique was optimised and validated for this purpose. The sample was mixed with organic phase containing pentafluorobenzyl bromide as an alkylating agent. The benzalkonium chloride was used as a phase-transfer catalyst. The quantitative determination was performed using GC-MS technique in selected ion monitoring mode. The blood levels of sulphide of healthy controls were measured (35-80 microM/l). The method is versatile, reproducible (RSD=2.7%) and suitable for research of anaerobic fermentation in vivo.

Comparison of various stationary phases for normal-phase high-performance liquid chromatography of ethoxylated alkylphenols

Abstract Unmodified silica and chemically bonded diol, nitrile and amino phases were tested as column packings for normal-phase liquid chromatographic separations of ethoxylated alkylphenols in mobile phases consisting of aliphatic alcohol(s) and a non-polar hydrocarbon. The performance of unmodified silica is improved when ethanol is used instead of propanol in binary mobile phases or when ethanol—propanol—aliphatic hydrocarbon ternary mobile phases are applied. Diol and nitrile bonded phases show regular retention behaviour only in mobile phases with a low content of propanol, where 6–8 ethoxymers can be resolved in a reasonable time. In propanol-rich mobile phases, a mixed retention mechanism causes a non-linear increase in log k′ with increasing number of oxyethylene units, which hinders the prediction of retention by calculation and deteriorates the separation of the individual oligomers in propanol—n-alkane mobile phases. The amino-bonded stationary phase shows regular retention behaviour even for higher ethoxymers in mobile phases containing high proportions of propanol in n-alkane, so that the capacity factors can be predicted by calculation. The amino-bonded phase offers better separation possibilities for the individual ethoxymers than the other stationary phases tested, in both the isocratic and gradient modes of elution.

Retention characteristics of some volatile compounds on Tenax GR

Abstract Breakthrough volumes (maximum sample volumes, Vmax.) of low-molecular-mass compounds on graphite-filled Tenax have been measured. Using Tenax GR the sampling volumes are greater than with Tenax GC or Tenax TA. Tenax GR can be used in environmental analysis for trapping and enriching traces of low-molecular-mass organic compounds.

Oriented immobilization of chymotrypsin by use of suitable antibodies coupled to a nonporous solid support.

In order to eliminate the kinetic limitation of chymotryptic hydrolysis of proteins due to diffusion, nonporous hydroxyalkyl methacrylate solid support was developed and used for oriented immobilization of chymotrypsin by means of suitable polyclonal antibodies. Nonporous microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in an alcohol-toluene mixture stabilized with cellulose acetate butyrate. The resulting particles were 1.2 microm in diameter and possessed narrow size distribution. After modification with adipic acid dihydrazide they contained 2 micromol of reactive groups available for coupling of anti-chymotrypsin antibodies. Prepared immunosorbent adsorbed 166.7 microg of chymotrypsin per 1 g of dry carrier. Immobilized chymotrypsin retained practically 100% of its native proteolytic activity. Kinetic parameters of catalysis by chymotrypsin immobilized via this way were improved due to the good steric accessibility of the enzyme active site for high-molecular-mass substrates, when digestion of proteins in batch experiments was used.

Separation of phenoxyacid herbicides using liquid column chromatography on chemically bonded phases

Abstract Possibilities for the liquid chromatographic (LC) separation of phenoxyacid herbicides were tested. Two complementary LC systems are suggested for the separation and identification of these compounds. In the first system, an octadecylsilica column is used together with aqueous-methanolic mobile phases containing an inorganic salt or a buffer, such as sodium sulphate or potassium dihydrogen phosphate. The second system makes use of a chemically bonded amino phase and of mobile phases containing acetic acid in water, methanol or other organic solvents and their mixtures. The influence of the mobile phase composition and of the structure of phenoxyacid herbicides on retention and selectivity of separation was investigated. The separation methods suggested can be used in the analysis of commercial herbicide formulations and in environmental analyses.

Glass capillary gas chromatography of homologous series of esters : V. Separation of halogenoacetates of C1C16 aliphatic alcohols and 2-halogenoethyl esters of C2C16 aliphatic acids with the same molecular weight on an SP-400 glass capillary column

Abstract The gas chromatographic separation of halogenoacetates of C 1 C 16 aliphatic alcohols and 2-halogenoethyl esters of C 2 C 16 aliphatic acids and comparisons of retentions of pairs of these esters having the same molecular weights has been studied on SP-400 glass capillary columns. The influence of the type of halogen atoms, their number and their positions in esters on their retention was examined. Kovats retention indices were used to interpret the results.

Chromatography of monomers : II. Glass capillary gas chromatography of c1-c18 alkyl esters of acrylic and methacrylic acids

Abstract Retention indices of 38 acrylate and methacrylate monomers at different column temperatures are reported. The temperature dependences of the retention indices and the incremental effects of the methylene group were determined on non-polar (OV-101, SE-54) and a polar (SP-1000) capillary column, operated isothermally be tween 80 and 200°C.