Søren Michaelsen

Factors influencing the separation and quantitation of intact glucosinolates and desulphoglucosinolates by micellar electrokinetic capillary chromatography

Abstract Micellar electrokinetic capillary chromatography methods using cetyltrimethylammonium bromide as a surfactant have been developed for the qualitative and quantitative determination of intact glucosinolates and desulphoglucosinolates. The influence of changes in separation conditions has been investigated. A great number of different intact glucosinolates and desulphoglucosinolates have been used for the development of efficient separation conditions for the closely related, but structurally different, compounds. Repeatability and linearity of the quantitative analyses have been evaluated, and critical parameters have been determined. Rapid and efficient separations are possible for glucosinolates in crude extracts and for mixtures of glucosinolates isolated from seeds and the vegetative parts of plants.

Chromatography and capillary electrophoresis in food analysis

General Aspects of Experimental Biochemistry Buffers and Micelles Binding, Association, Dissociation and Kinetic Extraction of Native LMW and HMW Biomolecules Spectroscopy and Detection Methods Liquid Chromatography Ion-exchange Chromatography High Performance Liquid Chromatography and Fast Polymer Liquid Chromatography Electrophoresis High Performance Capillary Electrophoresis Analytical Determination of Low Molecular Weight Compounds Protein Purification and Analysis Immunochemical Techniques Analysis of Dietary Fibre Appendix: Supercritical Fluid Extraction (SFE) and Supercritical Fluid Chromatography (SFC) Subject Index.

Determination of phenolic carboxylic acids by micellar electrokinetic capillary chromatography and evaluation of factors affecting the method

Abstract Micellar electrokinetic capillary chromatography (MECC) based on cetyltrimethylammonium bromide (CTAB) was developed for separation of individual cinnamic and benzoic acid derivatives. This method was adapted to the separation of the phenolic carboxylic acids from other anions, phenolics and glucosinolates in samples prepared from plant materials. The influence of temperature, voltage, pH, electrolyte and detergent concentrations in the buffer on migration times for the compounds considered, peak areas, resolution and number of theoretical plates were investigated. It is shown that rapid and efficient separations (270 000 plates/m) are possible, even for structural closely related phenolics.

Determination of flavonoids by micellar electrokinetic capillary chromatography

Abstract HPCE based on cetyltrimethylammonium bromide (CTAB) or cholate micellar electrokinetic capillary chromatography (MECC) was found suitable for the separation of individual flavonoid glycosides following a rapid and simple technique of isolation, purification and group separation. Kaempferol and quercetin glycosides with varying degrees of glycosylation, and with or without additional esterification on the carbohydrate part, were included in the study. The influence of temperature and voltage as well as electrolyte, CTAB and organic modifier concentrations in the buffer on the migration order, migration times, and peak areas of the flavonoids was investigated. The method developed gives possibilities for the separation and specific determination of flavonoids isolated from vegetative parts of cruciferous plants.

Analysis of individual phospholipids by high-performance capillary electrophoresis

A method of analysis for determination of phospholipids by micellar electrokinetic capillary chromatography (MECC) has been developed. Sodium cholate (NaCh) was found suitable as the micellar phase, and 1-propanol was important in obtaining an efficient separation of individual phospholipids and as organic solvent required for acceptable solubility of the phospholipids. With equal volatility of water and organic modifier, only minor changes in effects from the modifier occur during analysis. The influence of changes in high-performance capillary electrophoresis-MECC separation conditions were evaluated in terms of migration time, relative migration time (RMT), relative normalized area (RNA), resolution and theoretical plates per meter. The method has several advantages compared to high-performance liquid chromatography: The total time of analysis is less than 25 min, and only small amounts of reagents and sample are required. Relative standard deviations were 0.26–0.48% for RMT, 1.7–2.9% for RNA, and in the linearity test correlation coefficients of 0.999 were obtained. Results from analyses of the phospholipid compositions of soybean and rapeseed lecithins are presented.

Determination of oligosaccharides by capillary zone electrophoresis

Abstract Oligosaccharides occur in various biological materials, and the α-galactosides raffinose, stachyose, verbascose and ajugose are important in relation to the quality or nutritive value of legume seeds. A simple technique for the isolation and group separation of oligosaccharides was developed as an appropriate purification step prior to determination of the individual oligosaccharides by high-performance capillary electrophoresis (HPCE). The HPCE technique adapted to the separation of α-galactosides was based on capillary zone electrophoresis (CZE) in borate buffers with UV detection at 195 nm. The influence of various separation conditions, including voltage, pH, temperature and buffer composition, on the resolution, migration times, number of theoretical plates and peak areas was studied by the use of galactinol and the mono-, di-, tri- and tetra-α-galactosides of sucrose. Up to about 375 000 plates/m were obtained with the described CZE method. Tests of repeatability and linearity and the use of internal standards and relative response factors were used for evaluations of the qualitative and quantitative aspects of the method. With the combined technique of group separation, purification and CZE, a rapid and efficient method for the determination of naturally occurring oligosaccharides is now available for even complex mixtures of these carbohydrates.

High-performance capillary electrophoresis for the determination of trypsin and chymotrypsin inhibitors and their association with trypsin, chymotrypsin and monoclonal antibodies.

High-performance capillary electrophoresis (HPCE) was adapted for the determination of Kunitz soybean trypsin inhibitor, Bowman Birk inhibitor from soybean and protein-type proteinase inhibitors from pea (Pisum sativum L.). The method was developed for the determination and characterization of the inhibitors, the enzymes trypsin and chymotrypsin and the monoclonal antibodies (mAbs) raised against the inhibitors, and also the inhibitor-enzyme and inhibitor-mAb association complexes. The results from studies involving the use of various types of buffers revealed the advantages of having zwitterions such as trimethylammoniumpropyl sulphonate (AccuPure) or taurine included in the buffer. The use of capillaries dynamically coated with zwitterions efficiently reduced the interactions of the proteins with the silica capillary surface, which was important for the analyses for trypsin, chymotrypsin and mAbs and their association complexes with the inhibitors. The influence of temperature, voltage, pH and buffer type on migration times, resolution, peak areas and number of theoretical plates was investigated for the proteins studied. The proposed HPCE method is very suitable for studies of proteinase inhibitors compared with traditional inhibitor studies, and it gives efficient protein separations with the possibility of 245,000 plates/m.

Separation and determination of glycosaminoglycan disaccharides by micellar electrokinetic capillary chromatography for studies of pelt glycosaminoglycans.

Capillary electrophoresis based on cetyltrimethylammonium bromide micellar electrokinetic capillary chromatography (MECC) was developed for the separation and determination of glycosaminoglycan (GAG) disaccharide units without derivatization. The influence of changes in several separation conditions was studied, and the separation mechanisms are discussed. Tests of repeatability and linearity were performed for qualitative and quantitative evaluation of the method. The described procedure gives a rapid and efficient determination of GAG disaccharides. Samples of chondroitin sulphates and mink skin were treated with proteases, and the extent of protein cleavage was followed by free zone capillary electrophoresis. The result of the chondroitinase ABC treatment following the protease treatment was evaluated by the MECC method.

Analysis of dansyl amino acids in feedstuffs and skin by micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MECC) using sodium dodecyl sulphate (SDS) and sodium cholate have been used for analyses of 30 dansylated (Dns) amino acids. The influences of sample preparation, Dns/amino acid ratio, sample solvent composition, and separation conditions including voltage, temperature, pH and buffer composition were investigated. Complete separations of acidic and neutral amino acids were obtained within 45 min in the SDS system. The efficiency expressed as number of theoretical plates for the applied capillary 0.52 m long were between 210,000 and 343,000, and the repeatability was very good with relative standard deviations on relative migration times between 0.09 and 0.70% and on relative normalised peak areas (RNPAs) between 0.85 and 3.41%. The linearity studies gave correlation coefficients between 0.9957 and 0.9993 for RNPAs against concentration. Detection limits were between 3 and 6 fmol or approximately 2 pg of each amino acid. Basic amino acids were separated in a MECC system using sodium cholate. Procedures and problems using Dns derivatisation for amino acids analysed by the MECC methods are described. Finally, examples of analyses of hydrolysates of real complex samples show, that this method can be applied to determine the amino acid composition of proteins in feedstuffs and skin.

Separation of desulphoglucosinolates by micellar electrokinetic capillary chromatography based on a bile salt

Abstract Micellar electrokinetic capillary chromatography (MECC) has been developed as a promising method for the determination of 40 desulphoglucosinolates. A sodium cholate based MECC method was found to be efficient for the qualitative and quantitative analysis of desulphoglucosinolates produced in an on-colum, enzymatic step form the corresponding intact glucosinolates. Separation conditions and sensitivity of the method have been optimised with respect to different parameters, including capillary types, where the 75-μm I.D. capillary increased the sensitivity 2.5 times over that of a 50-μ capillary. With use of a high-sensitivity optical cell assembly (Z-cell), the sensitivity was further increased ten times, resulting in detection of picogram amounts, or concentration levels corresponding to 10−6 M. Repeatability with a 75-μm capillary was good, with the relative standard deviation varying between 0.2% and 0.9% for relative migration times and for relative normalised areas between 1.0% and 3.0%. Linearity of the optimised method gave correlation coefficients between 0.99 and 0.9999 for the 50-μm capillary and 0.99 and 0.9997 for the 75-μm capillary. Separation efficiency expressed as number of theoretical plates (N/m) was in the range 250 000–300 000 for the 50-μm capillary and 210 000–250 000 for the Z-cell. Limitations and possibilities of the MECC method here presented are discussed with respect to analyses of glucosinolates occuring in a wide range of cruciferous seed, vegetative plant parts including cabbage varieties, feed and food.