Zdeněk Deyl

Microemulsion electrokinetic chromatography with different organic modifiers: separation of water- and lipid-soluble vitamins

Microemulsions prepared by using either sodium dodecyl sulfate (SDS) or trimethyltetradecylammonium bromide (TTAB) were tested with regard to the migration time, selectivity and number of theoretical plates using a set of both water-soluble and lipid-soluble vitamins. While in the separations where SDS was used as the surface-active agent for microemulsion preparation, better resolutions were obtained with both hydrophilic and lipophilic vitamins, with TTAB the best separations were obtained with the micelle-forming reagent only or with diethyl ether as the microemulsion-forming component. In the SDS-based separations nicotinic acid was moving with the same velocity (relative to the endoosmotic flow) regardless of the emulsion or micellar phase used. It is proposed that owing to its behaviour it can serve as an ideal internal standard in further studies on microemulsion separations.

Microemulsion and micellar electrokinetic chromatography of steroids

A mixture of ten steroids was separated by microemulsion and micellar (SDS and glycodeoxycholate) electrokinetic chromatography systems. Separations were done on a 50 cm (to the detector) x 50 microns I.D. fused-silica capillary. Complete separation of all the test compounds in the micellar mode was obtained with glycodeoxycholate (50 mM) in 25 mM borate buffer, pH 6.5, as the micelle-forming agent. The best results, however, were obtained using microemulsion electrokinetic chromatography in which higher aliphatic alcohols were used as the microemulsion-forming modifiers. The system consisted of n-hexanol (0.81%), SDS (3.31%) and n-butanol (6.61%) in 20 mM phosphate buffer, pH 10.0 (89.28%, w/w). In the microemulsion mode, linear calibration for steroid standards was obtained in the concentration range 3 x 10(-4)-3 x 10(-5) mol 1(-1) with a detection limit of 1 pmol. The method was validated and applied to an 11 beta-hydroxysteroid dehydrogenase assay in tissues.

Preparative procedures and purity assessment of collagen proteins

Collagens represent a large family (25 members identified so far) of closely related proteins. While the preparative procedures for the members that are ubiquitous and present in tissues in large quantities (typically fibre and network forming collagens types I, II, III, IV and V) are well established, the procedures for more recently discovered minor collagen types, namely those possessing large non-collagenous domain(s) in their molecule, are mostly micropreparative and for some collagenous proteins even do not exist. The reason is that the proof of their existence is based on immunochemical staining of tissue slices and nucleic database searching. Methods of preparation and identification of constituting alpha-polypeptide chains as well as collagenous and non-collagenous domains are also reviewed. Methods for revealing non-enzymatic posttranslational modifications (particularly of the fibre forming collagen types) are briefly described as well.

Microemulsion electrokinetic chromatography of diphenylhydrazones of dicarbonyl sugars

A mixture of diphenylhydrazones of dicarbonyl sugars was separated by microemulsion electrokinetic chromatography. Separations were carried out with a laboratory-made capillary electrophoresis apparatus using an untreated fused-silica capillary [70 cm (effective length 65 cm)×50 μm I.D.] at 20 kV; detection was at 220 nm. The microemulsion system was composed of 5 mM borate buffer, pH 8.0 (89.27%, w/w), sodium dodecyl sulphate (SDS; 3.31%, w/w), n-butanol (6.61%, w/w) and n-octanol (0.81%, w/w). Separation by microemulsion electrokinetic chromatography was significantly better then separation obtained by routine micellar electrokinetic chromatography with SDS and was characterized by a much higher efficiency (theoretical plates ranged from 100·103 to 254·103). SDS–micellar electrokinetic chromatography resolved only one compound from a joint peak of two sugars and from a cluster peak consisting of seven compounds. Applicability to the analysis of biological samples was also demonstrated.

Biomedical applications of capillary electrophoresis

After having grown through the stages of technique development and instrumentation availability, capillary electrophoresis has reached the stage of applications. This review attempts to show the potential of this technique for biomedical analysis. Rather than going into a detailed description of the technical details of the separation conditions suitable for the separation of a particular category of compounds, the focus is on the general principles and areas in which this technique can be applied and the prospects for the future. Particular emphasis is placed on the separation of complex matrices and their simplification, a daily task in biomedical laboratories. In addition, methods for the optimization of separation conditions are considered. Considerable prospects for capillary electrophoresis lie in profiling. The applicability of the technique in peptide and protein mapping is discussed in some detail. Finally, three other topics are dealt with, namely enzymic activity microassays, drug-protein binding assays and monitoring of drugs in body fluids.

Microemulsion electrokinetic chromatography of fatty acids as phenacyl esters

Abstract A mixture of saturated fatty acids containing even number of carbon atoms was successfully separated as phenacyl esters by microemulsion electrokinetic chromatography. Separations were done by using an untreated fused-silica capillary [57 cm (effective length 50 cm)×75 μm] at 15 kV; detection was done by UV at 243 nm. The microemulsion system was composed of 10 m M borate buffer (87.93%, w/w), cholate (4.87%, w/w), heptane (0.66%, w/w) and n -butanol (6.55%, w/w), pH 10.2. Separation by microemulsion electrokinetic chromatography was characterized by high efficiency (theoretical plates ranged from 142·10 3 to 428·10 3 ) with peak resolution 1.25 and better. Micellar electrokinetic chromatography under the standard conditions with sodium dodecyl sulfate as micellar phase was unsuccessful in separating all long-chain fatty acids. The described method can be used for monitoring of saturated fatty acids containing even number of carbon atoms in the molecule in a broad spectrum of chain length (C 2 –C 20 ).

Determination of the thermodynamic equilibrium constants of water-insoluble (sparingly soluble) compounds by capillary electrophoresis

Abstract A method based on migration time estimation in capillary electrophoresis for the determination of aqueous p K a th values of water-insoluble compounds of pharmacological (or environmental) interest is presented. The method requires apparent p K a estimation at least at two different pH values and at different methanol concentrations in the background electrolyte (usually more points of the methanol concentration vs. p K a th dependence are determined). Two automated operational modes were used; in the first, a stepped pH change was applied in the ascending mode (starting with low pH — usually 3.5) reaching, in 0.5 pH units steps, the final value of 5.5, followed by a similar descending pH sequence. This procedure was repeated for different methanol concentrations (w/w). The other operational mode was shortened in the sense that only two buffers were used, typically pH 5.5 and 3.0 (the former being assumed to yield the pH at which the solutes investigated are fully ionized). In all cases, the apparent pH of the methanolic solutions was estimated and the p K a value was determined by extrapolating the set of data obtained to zero methanol concentration.

Advances in capillary electrophoresis

This review summarizes the advancement in operational modes and selected applications of the title technique over the past five years. Regarding operational modes particular emphasis is put upon increasing selectivity and resolution, hyphenation of capillary electrophoresis with techniques based on other than electromigration principles, the so-called chip technology and new ways of detection. In applications selected examples of chiral separation and separation of biopolymers (proteins, nucleic acids) are emphasized. It is demonstrated that capillary electrophoresis represents a complementary technique to high-performance column chromatography and in a number of cases it offers better separations than standard chromatographic procedures.

Separation of proteins and peptides by capillary electrophoresis in acid buffers containing high concentrations of surfactants

Separations of proteins at acid pH in the presence of a high concentration of surfactant [sodium laurylsulfate (SDS), 50 mmol/l] was investigated. The purpose of using high concentrations of SDS as background electrolyte modifier was threefold: First, the surfactant exerts a washing effect upon the capillary wall thus preventing binding of analytes and possible clogging of the capillary. Second, it was revealed that even under very acid conditions (below pH 3) the surfactant is capable of forming associates with protein analytes which still bear considerable negative charge and can be separated on this basis. Third, the system can be applied not only for protein mixtures sufficiently soluble in neutral to alkaline media (leukocyte lysates, standard proteins), but it can be used also with proteins, that are under such conditions virtually insoluble and their solubilization is possible in acid buffers only (eggshell proteins or collagen CNBr fragments). The result was that adsorption to the capillary wall was minimized and the analytes were separated as negatively charged associates with high efficiency. With collagen fragments partition was possible on the affinity differences of the peptides to the surfactant micelles and inner wall of the capillary. Theoretical plate counts approaching 100,000 were easily achieved even with proteins which under the more conventional operation conditions exhibit considerable sticking to the capillary wall. The other feature of this system is that the associates move very rapidly to the anode. Owing to the low pH, endoosmotic flow is negligible, and therefore the system has to be operated at reversed polarity.

Post-translational non-enzymatic modification of proteins. II. Separation of selected protein species after glycation and other carbonyl-mediated modifications.

There are two strategies applicable to revealing non-enzymatic post-translational modifications of proteins; while assaying of the hydrolytically stable adducts was the subject of our previous communication [1], here we attempted to review separation technologies for the unfragmented modified proteins. There are a few standard procedures used for this purpose, namely Laemmli gel electrophoresis, different modes of gel permeation chromatography and boronate affinity chromatography. The latter approach makes use of the vicinal hydroxy groups present in glycated proteins. Some (but not all) arising adducts exhibit typical fluorescence which can be exploited for detection. In most cases fluorescence is measured at 370/440 nm for the so-called advanced glycation products or at 335/385 nm for the only so far well characterized glycation marker (pentosidine). Some indication exists that, e.g., synchronous fluorescence detection will probably in the future add to the selectivity and allow the distinction of the different adducts arising during non-enzymatic post-translational modifications (glycation). The proteins reviewed are serum albumin, collagen and lens proteins while glycation of hemoglobin is the subject of another review within the present volume.