Selectivity in CE.
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Chromatography, High Pressure Liquid↗
Biomedical subjects
R Vespalec
Publications and source records attributed to R Vespalec.
Determination of dissociation constants of cytokinins by capillary zone electrophoresis.
A method for the pKa determination, based on mobility data measured by capillary zone electrophoresis, was applied to cytokinins and their analogs. The combination of charged mobility standards with an uncharged electroosmosis marker, injected in the uncoated capillary simultaneously with the measured substances, allows one to minimize the number of runs, reduce their duration and, in addition, to inform on the run-to-run stability of electroosmosis and on contingent side-effects. pKa values of investigated cytokinins and their analogs ranged from 2.8 to 4.0 at 25 degrees C in the phosphate and acetate buffers of ionic strength 0.015 M. Standard deviations of the constants, obtained by the non-linear fitting of equations for the pKa calculation, were 3-5-times lower than standard deviations from the linear fitting or from the point-to-point calculation utilizing the Hendersson-Haselbalch equation. The equation of Boltzman sigmoid offers two checks on reliability of effective mobilities that serve as the raw data in the pKa calculation.
Cytokinins↗
Calculation of stability constants for the chiral selector-enantiomer interactions from electrophoretic mobilities.
Interactions of chiral selectors with enantiomers or with other chiral analytes, underlying their electrophoretic separations, are particular cases of interactions of dissolved species. The interaction model that describes these reversible, fast interactions is simple. Equations describing the model are also simple and applying equations for the calculation of respective equilibrium constants to experimental data is easy using computers. Obtaining the experimental mobility data, necessary for the calculation, is the critical step on the way to stability constants quantitating the strengths of interactions of chiral selectors with enantiomers and other analytes. These data are decisive for both the accuracy and precision of the calculated constants. The meaning and applicability of the particular constant depend on the type of the constant. The common method for the determination of stability constants from electrophoretic migration data is reasonable for low and medium stability constants. For stronger complexing, characterised by stability constants of the order of 10(4) l/mol, typical of affinity chiral selectors, the method becomes unreliable. For strong complexing giving constants of the order of 10(5) or higher of or higher the method is not applicable in its commonly used form.
Electrophoresis↗
Chiral separations in capillary electrophoresis.
The marked increase in the number of communications on the utilization of electrophoresis for practical chiral separations within the last three years is the most evident, and the most important fact. It reveals that the basic period of intensive research in the field is finished. The search for chiral selectors discriminating racemates in a reasonably analytical manner and the study of both the mechanism and physicochemical aspects of the chiral discrimination process were the main features of that period. Here, we review the state of the art in the field and state the references of the related literature up to the end of 1998.
Electrophoresis, Capillary↗
Methods for determination of electrophoretic mobility and stability of complexes originating in solutions during the chiral discrimination process.
An equation for the calculation of electrophoretic mobility of kinetically labile complexes originating in solutions during the chiral discrimination process is derived. The mobility of the complex is calculated from that of a fully ionized racemic compound, measured in absence of the chiral selector, and from the effective mobilities of its enantiomers, corresponding to the concentration of the chiral selector causing their maximum difference. Correct values of stoichiometric stability constants of both enantiomers may be calculated from the mobility of the complex obtained in this way. Both the mobility and the stability values hold only for the experimental conditions used and the selected background electrolyte. The proposed method is demonstrated for the separation of the fully ionized N-t-BOC-DL-tryptophan with beta-cyclodextrin in 20 mM aqueous solution of alpha-hydroxyisobutyric acid, adjusted with NaOH to pH 4.5. Mobility of the complex is 8.4 X 10(-9) m2V[-1]s(-1) at 25 degrees C. The stability constants of D- and L-enantiomers of N-t-BOC-DL-tryptophan with beta-cyclodextrin, KD and KL obtained from migration data using this mobility of the complex, are 374+/-37 M(-1) and 336+/-31 M(-1), respectively. The geometrical mean value of calculated stability constants, 355 M(-1), agrees perfectly with the value of 350 M(-1), calculated from the same experimental data by another procedure recently.
Cyclodextrins↗
Chiral separations by capillary zone electrophoresis: present state of the art.
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Electrophoresis, Capillary↗
Vancomycin as a chiral selector in capillary electrophoresis: an appraisal of advantages and limitations.
The properties of the macrocyclic antibiotic vancomycin, used as a chiral selector, were studied with aminoquinolycarbamate derivatives of amino acids, containing sulfur and selenium, as well as with other organic ions. Vancomycin combines the ability to resolve fully ionized anionic enantiomers, typical of proteins, with excellent separation efficiency, exceeding that of cyclodextrins. It allows better than baseline chiral separations of several anionic analytes within 3-5 min. The resolving power of vancomycin results from its great skill in discriminating enantiomers rather than from high affinities to the separated enantiomers. The association constants of vancomycin are of the same order of magnitude, 10(2) L/mol, as that found for beta-cyclodextrin (beta-CD). The difference in association constants of separated cystine enantiomers with vancomycin, 2 x 10(2) L/mol, is one order of magnitude higher than that of enantiomers separated with beta-CD. Analytically convenient mobility differences up to 1-2 x 10(9) m2V-1s-1, with only one of the enantiomers appreciably decelerated, are obtained at submillimolar vancomycin concentrations. Typical separation efficiencies are close to 250,000 theoretical plates per meter of capillary. Deceleration of various organic ions by millimolar vancomycin implies that chiral separations with vancomycin need not be restricted to carboxylic acids. The vancomycin-analyte interactions are strongly affected by the chemical composition and concentration of the buffer. An additional experimental variable, highly effective in manipulating the separation selectivity of analytes, is the buffer pH.
Amino Acids↗
Chiral separations by capillary electrophoresis: present state of the art.
Capillary electrophoresis is a powerful tool for chiral separation of ionogenic enantiomers in solutions. This article brings an overview of the theory of electrophoretic separations with special emphasis on enantiomer-chiral selector equilibria, followed by a survey of indirect separations, based on formation of diastereomers with different electrophoretic mobilities, and a comprehensive appraisal of direct separations when the chiral recognition stems from (i) host-guest interactions using cyclodextrins and crown ethers as hosting agents, (ii) ligand exchange mechanism, (iii) affinity interactions or (iv) a combination of solute-chiral selector interactions with micellar electrophoretic transport. Finally, some trends in chiral separations by capillary electrophoresis are discussed.
Cyclodextrins↗
Consequences of a maximum existing in the dependence of separation selectivity on concentration of cyclodextrin added as chiral selector in capillary zone electrophoresis.
The maximum in the dependence of the separation selectivity on the concentration of cyclodextrin may be utilized for the determination of the mean value of host-guest interaction constants of the separated enantiomers. For the hosting of N-t-BOC-DL-tryptophan by beta-cyclodextrin, the mean value of these constants, found by drop line estimated from the cyclodextrin concentration corresponding at the maximum, is 350 L.mol-1. The separation at this concentration of cyclodextrin offers the highest resolution in the shortest separation time. It is shown that commonly used simple preliminary experiments, testing the capability of a cyclodextrin to resolve chiral compounds, and based on relatively high cyclodextrin concentrations, may easily lead to incorrect conclusions in cases of enantiomers strongly interacting with the cyclodextrin used.
Cyclodextrins↗
Capillary zone electrophoresis with indirect photometric detection in the visible range.
Sensitivity and applicability of commonly used indirect photometric detection in the UV region can be adversely affected by the absorption of the UV light by detected solutes and/or by matrix constituents migrating with them. If the visible light is exploited instead of the UV light, both these disadvantages may be for the most part eliminated, and, moreover, the indirect photometric detection will extend its versatility and sensitivity. Prospects as well as main problems of the approach were tested using inorganic ions as analytes. If organic dyes with a molar light absorption coefficient of the order of 10(6) mol-1 L m-1 was selected as the light-absorbing constituents of the background electrolyte, detection sensitivity comparable with the best reported results was reached for anions; for cations, the detection limit was even lower by two orders of magnitude.
Anions↗
Problems connected with HPLC separation of pilocarpine from its degradation products by means of silica gel and water-based mobile phase.
HPLC separation of pilocarpine from its degradation products (isopilocarpine, pilocarpic acid and isopilocarpic acid) was studied on unmodified silica gel stationary phase. An acidified aqueous solution of inorganic salts with addition of methanol served as the mobile phase. The influence of silica gel used, methanol content, mobile phase pH, anion of acids and/or inorganic salts and column temperature on solute retention, separation selectivity as well as peak shape was studied and partly explained. A significant dependence of the properties of the resulting separation system on the silica gel used was found. A test was proposed for evaluation of silica gel suitability.
Chromatography, High Pressure Liquid↗
Identification of peaks in capillary zone electrophoresis based on actual mobilities.
A procedure is proposed for the calculation of the actual effective mobility of a zone from its migration time. It is based on the use of internal standards with known mobilities; the use of two internal standards provides reliable mobility data even if the magnitudes of the effects of sample composition, capillary temperature, capillary length, migration distance, used voltage, as well as the tube length occupied by the injected sample are unknown. Formulas have been derived for the calculation of the actual mobilities, and their experimental verification has been carried out by using a model set of anionic solutes with mobilities ranging from -56 to -20 x 10(-9) m2V-1s-1 and chloride as the ion modelling the effect of the sample matrix.
Electrolytes↗