Radim Vespalec

Prospects of dissolved albumin as a chiral selector in capillary zone electrophoresis

Abstract It has been shown using as examples amino acids, mono- and dicarboxylic acids that the enantioselectivity of dissolved albumin may be employed with advantage in capillary zone electrophoresis (CZE) separations. Slow changes in the enantioselectivity of albumin dissolved in weakly alkaline aqueous solutions can be avoided by a short mild heating of the solution. The efficiency of a separation system for a given solute in CZE is at least one order of magnitude higher than that in LC based on bonded albumin. The good chiral separations obtained here and the high efficiency of CZE separation systems confirm that CZE separations based on dissolved albumin as a chiral selector can compete with analogous LC separations. Perfect qualitative agreement of both enantioselective properties of albumin and factors affecting its enantioselectivity in CZE and LC proves that the knowledge obtained in LC with bonded albumin may be transferred to CZE separation systems.

Performance of the capacitance detector for liquid chromatography

Abstract The performance and applicability of a detector based on the measurement of the dielectric constant of the eluate were investigated. Variations of the dielectric constants (e) are converted into frequency changes in the oscillator circuit and are further processed electrically. In the design described, the instrument is suitable for the detection of substances sufficiently soluble in the mobile phase, provided that the solution formed has no electrical conductivity and low dielectric losses. The basic frequency of the oscillators being 18 MHz and the stability 0.77·10 −6 , the linear range of the responses is 1.38·10 4 . When hexane was used as the mobile phase (e = 1.89), the minimum detectable concentrations varied from 2.6·10 −2 % for n -octane (e = 1.948) to 4.2·10 −5 % for acetone (e = 20.7).

Chromatographic properties of chemically bonded bovine serum albumin working as a chiral selector in alkaline mobile phases

Abstract By chemical bonding of bovine serum albumin to a hydrolytically stable Separon HEMA matrix, a sorbent with an excellent lifetime in mobile phases with pH ⩽ 11 is obtained. Maximum retentions of amino acids and separation selectivity of amino acids and monocarboxylic and dicarboxylic acids on chemically bonded albumin are attained around pH 9. By prolonged action of slightly alkaline mobile phases of pH

Interpretation of enantioselective activity of albumin used as the chiral selector in liquid chromatography and electrophoresis

Abstract The possibility of constructing a hypothesis on albumin enantioselectivity allowing consistent interpretation of retention and selectivity data for D.L-monocarboxylic acids, D.L-dicarboxylic acids and D.L-amino acids, measured by liquid Chromatography and capillary zone electrophoresis, is demonstrated. An explanation of effects caused by high temperature, mildly alkaline aqueous solutions and methanol, consistent with the explanation of the retention and selectivity data, is also possible. The hypothesis considers the conformational variability of albumin.

Measurement of exclusion volumes of packed columns by means of electrokinetic detection

Abstract A method is described for the measurement of the exclusion volumes of packed chromatographic columns by means of electrokinetic detection in liquid chromatography.

Improvement of the performance of the capacitance detector for liquid chromatography : Effects of the cell volume on band spreading and sensitivity of detection

Abstract Contributions to the zone width caused by band spreading in an injection block, connecting tubing and detection cell with volumes of 11.7–2.0 μl were measured at flow-rates of 0.03–0.85 ml/min. Cells that have the form of a flow capacitor do not contribute to the band spreading to a measurable extent if their volumes do not exceed 5.2 μl. Band spreading that is still acceptable can be obtained in an inlet tubing that has a total length of up to 62 cm if its internal diameter is 0.2 mm. The response of the capacitance detector was proved experimentally to be independent of the cell volume. If different mobile phases are used, the detector can be adapted by exchanging the detection cell or one of the plates. With noise corresponding to a change of 7.6 · 10 −7 unit of dielectric constant, the minimum detectable concentrations measured, expressed as volume per cent, varied from 3.2 · 10 −3 for n -nonane to 1.0 · 10 −5 for acetone in n -heptane as the mobile phase.

Exploration of the electrophoretic behaviour of borane cluster anions and of the capability of capillary electrophoresis to separate them chirally

Mobilities of investigated boron cluster compounds in 3-(N-morpholino)propanesulfonic and phosphate buffers adjusted to pH 7 either with sodium hydroxide or with tris(hydroxymethyl)aminomethane depend on both buffer ions. The zone width and zone asymmetry, which are usually markedly higher than those of organic or common inorganic ions of comparable size, depend on the type of the borane cluster anion. Unusual shapes of zones of two investigated compounds have been found in tris phosphate buffer. Acetonitrile was superior to methanol as an organic additive to separation systems from the viewpoint of the zone symmetry and separation speed. Narrow trigonal zones, typical of organic ions non-interacting with the capillary wall, have been observed for some bridged sandwich cobalt complexes in run buffers with the addition of acetonitrile. The interaction of borane cluster anions with beta-cyclodextrin cavity is excessively strong in purely aqueous solutions. Methanol and acetonitrile, which generally weaken the interaction, sometimes affect the separation enantioselectivity of various compounds in different ways in addition to the weakening effect. Chiral discrimination was reached for all ten investigated anions, which belong to four different structural types of cluster boranes. Stability constants estimated for some analyte-beta-cyclodextrin complexes range between 100 and 1800 l/mol in acceptable separations. The relative difference of the constants was from 3 to 20%.

Emerging subject for chiral separation science: cluster boron compounds.

The structural chirality is an inherent feature of fully synthetic boron cluster compounds that sometimes exhibit unique biochemical effects. HPLC studies with zwitter-ionic cluster boron compounds and electrophoretic studies with boron cluster anions reveal that the chiral separability of these species is remarkably dissimilar to that of organic species, if uncharged cyclodextrins are used as chiral selectors. Furthermore, marked differences were found between the analytical characteristics of the chiral separations of the boron cluster species and those of the organic species with uncharged cyclodextrins. The present-day experimental database indicates that the rules valid for the chiral separations of the organic species cannot be applied to the chiral separations of the boron cluster species without experimental verification. The current extent of research work devoted to the investigation of chirality and chiral separations of boron cluster species is negligibly small in comparison with that devoted to the investigation of chirality and chiral separations of organic species. This makes difficult a reliable explanation of both the particularities observed in chiral separations of boron cluster species with cyclodextrins as chiral selectors and the strange effects related to these separations at the moment.

Separation of inorganic anions by liquid chromatography

Abstract The influence of the presence of an organic counter ion, mobile phase pH and ionic strength on the retention of inorganic ions was studied in a reversed-phase chromatographic system with ODS-silica gel as the stationary phase. Retentions of all tested anions could be reached by acidifying the aqueous solutions of inorganic salts used as the mobile phase. The addition of tetrabutylammonium cations to the system led to the increase in retentions of all the studied anions within the whole investigated range of pH 7-3. However, it did not affect the elution order both inside the group of metal oxo anions and non-metallic anions (Cl-, Br-, I-, NO2- NO3- and ). The ionic strength of the mobile phase influenced retention, selectivity and elution order. Retentions of anions in systems without organic counter ion were explained by Coulombian interaction of anions with protonized residual silanols. Differences in the behaviour of oxo anion of Cr, Mo, W and V from the behaviour of non-metallic anions were explained by the formation of complexes between hydrated metal oxo anions and residual silanols.

Electrokinetic detection at different points in a narrow-bore glass column in liquid chromatography

Abstract The measurement of the electrokinetic current at different points in a narrow-bore glass column (0.4–0.5 mm I.D.) packed with sorbent is described. The origin of a “derivative” signal and the dependence of its shape on the capacity ratio of the solute and the flow-rate of the mobile phase are explained and verified experimentally. The values of the height equivalent to a theoretical plate, were determined at two places in the column, and a value of Hmin = 0.11 mm (i.e., 3.1 dp) was calculated.