Tomáš Křížek

Monolithic columns based on a poly(styrene-divinylbenzene-methacrylic acid) copolymer for capillary liquid chromatography of small organic molecules.

A very simple and readily performed method is described for the preparation of poly(styrene-divinylbenzene-methacrylic acid) monolithic columns for capillary liquid chromatography. The effect of the methacrylic acid content on the morphological and chromatographic properties has been investigated. Methacrylic acid is shown to be essential for isocratic separations of small organic analytes by capillary liquid chromatography. Column efficiencies of about 28,000 theoretical plates/m have been obtained for all the test compounds. The batch-to-batch and run-to-run repeatability of the retention times is better than 1.5%.

Separation of inorganic and small organic anions by CE using phosphonium‐based mono‐ and dicationic reagents

The separation of relatively small, fast‐migrating anions by CE is not possible under standard conditions with cathodic EOF of relatively high velocity. Therefore, separation approaches for these analytes usually include suppression or reversal of the EOF using various cationic additives to the BGE or covalent coating of the capillary walls. In this study, novel phosphonium‐based mono‐ and dicationic reagents (originally synthesized as ionic liquids) were evaluated as additives to the BGE for the separation of inorganic and small organic anions. The effects of these reagents on the EOF and their interactions with anionic analytes were compared. The effects of the additives structure and concentration as well as the separation buffer pH have been studied. Propane‐1,3‐bis(tripropylphosphonium) fluoride proved to be the most effective additive, as it suppressed the EOF in the system and enabled remarkable manipulation of separation selectivity. A method for fast, reliable, and efficient separation of six inorganic and seven organic anions within 7 min time was developed using propane‐1,3‐bis(tripropylphosphonium) fluoride as an additive to the separation buffer.

Microscale separation methods for enzyme kinetics assays

Miniaturization continues to be one of the leading trends in analytical chemistry and one that brings advantages that can be particularly beneficial in biochemical research. Use of a miniaturized scale enables efficient analysis in a short time and requires very small amounts of samples, solvents, and reagents. This can result in a remarkable decrease in costs of enzyme kinetics studies, especially when expensive or rare enzymes and/or substrates are involved. Free zone electrophoresis is without a doubt the most common microscale separation technique for capillary and on-chip enzyme assays. Progress and applications in this field are reviewed frequently whereas other modes of separation, although successfully applied, receive only marginal interest in such publications. This review summarizes applications of less common modes of separation in capillary or chip formats, namely micellar electrokinetic chromatography, liquid chromatography, gel electrophoresis, isoelectric focusing, and isotachophoresis. Because these techniques are based on separation mechanisms different from those of free zone electrophoresis, they can be, and have been, successfully used in cases where zone electrophoresis fails. Advantages and drawbacks of these alternative separation techniques are discussed, as also are the difficulties encountered most often and solutions proposed by different research groups.

Differentiation among various kinds of cheese by identification of casein using HPLC-chip/MS/MS†

In proteomics, proteins can be identified by enzymatic cleavage of the target protein using an enzyme of the known specificity (primarily trypsin), sequencing the obtained specific peptides by MS and comparing the amino acid sequence of the peptides with a protein database. The sophisticated approach described above was used in this study to determine and verify the original species of cheeses. Proteins were extracted from three different cheese samples which were produced from cow, sheep and goat milks. The isolated proteins were cleaved with trypsin and the peptides obtained were sequenced and identified by a HPLC-chip/MS/MS microfluidic system. Two different extraction methods and two various chromatographic sorbents packed in plastic chips were studied. Beta-lactoglobulin and four kinds of casein were found in the cheese samples. The species of kappa-casein were identified unambiguously in all the three cheese samples and, thus, kappa-casein can be used to determine the origin of milk of the cheese. The other proteins found in the samples show very similar primary structures and cannot be recommended for identification of the cheese milk origin.

Counterion condensation in short cationic peptides: limiting mobilities beyond the Onsager-Fuoss theory.

We investigated the effect of the background electrolyte (BGE) anions on the electrophoretic mobilities of the cationic amino acids arginine and lysine and the polycationic peptides tetraarginine, tetralysine, nonaarginine, and nonalysine. BGEs composed of sodium chloride, sodium propane‐1,3‐disulfonate, and sodium sulfate were used. For the amino acids, determination of the limiting mobility by extrapolation, using the Onsager–Fuoss (OF) theory expression, yielded consistent estimates. For the peptides, however, the estimates of the limiting mobilities were found to spuriously depend on the BGE salt. This paradox was resolved using molecular modeling. Simulations, on all‐atom as well as coarse‐grained levels, show that significant counterion condensation, an effect not accounted for in OF theory, occurs for the tetra‐ and nonapeptides, even for low BGE concentrations. Including this effect in the quantitative estimation of the BGE effect on mobility removed the discrepancy between the estimated limiting mobilities in different salts. The counterion condensation was found to be mainly due to electrostatic interactions, with specific ion effects playing a secondary role. Therefore, the conclusions are likely to be generalizable to other analytes with a similar density of charged groups and OF theory is expected to fail in a predictable way for such analytes.

Pluronic F‐127 as the buffer additive in capillary entangled polymer electrophoresis: Some fundamental aspects

Polymeric macromolecules of well-designed structures and specific properties open promising directions in the capillary entangled polymer electrophoresis. Pluronic F-127, as a thermoassociating polymer, possesses some unique properties that can be utilized in capillary entangled polymer electrophoresis of amino acids, peptides and proteins. In this study, we studied properties of Pluronic F-127 polymer as an additive to BGE for the separation of peptides and proteins. The influence of the thermoassociation on separation selectivity was studied. The addition of Pluronic caused severe instabilities of the electrical current and the signal of the UV detector. This study reveals remarkable positive effect of a low pressure applied to the inlet buffer vial during the analysis, which apparently stabilizes the electrical current and the detector signal. The effect of hydrodynamic flow induced by the pressure applied on the separation efficiency was studied and the significance of this effect was discussed. Pluronic F-127, as a representative of synthetic macromolecules, was compared with dextran, as a representative of natural polymers, in terms of separation power, selectivity and repeatability of migration times.

Characterization of polyaniline-coated stationary phases by using the linear solvation energy relationship in the hydrophilic interaction liquid chromatography mode using capillary liquid chromatography: Taraba et al.

A polyaniline coating was used to modify the surface of bare silica gel and octadecyl silica stationary phases to characterize the properties of altered materials. It was assumed that the mixed-mode retention was established on the basis of the polyaniline chemical structure and its combination with the original sorbents. Polyaniline was deposited onto the original surfaces during the chemical polymerization of aniline hydrochloride. The prepared materials were slurry packed into capillary columns and systematic chromatographic characterization was performed using the linear solvation energy relationship, also employing descriptors that allow inclusion of ionic interactions in the proposed retention mechanism. The retention times of 80 solutes with various chemical structures were measured in the hydrophilic interaction liquid chromatography mode. The obtained results demonstrated the significant contribution of the polyaniline coating to the retention mechanism under the given conditions; the assumed mixed-mode retention was confirmed. The dominant retention interaction for both modified stationary phases was based on the protonation of nitrogen atoms in the polyaniline structure, leading to suitable retention and selectivity for the hydrophilic analytes, especially anionic and zwitterionic species. Thus, especially, the polyaniline-coated bare silica gel sorbent seems to be promising for potential applications related to the separation of polar compounds.

Electrophoretic mobilities of neutral analytes and electroosmotic flow markers in aqueous solutions of Hofmeister salts

Small neutral organic compounds have traditionally the role of EOF markers in electrophoresis, as they are expected to have zero electrophoretic mobility in external electric fields. The BGE contains, however, ions that have unequal affinities to the neutral molecules, which in turn results in their mobilization. In this study we focused on two EOF markers—thiourea and DMSO, as well as on N‐methyl acetamide (NMA) as a model of the peptide bond. By means of CE and all atom molecular dynamics simulations we explored mobilization of these neutral compounds in large set of Hofmeister salts. Employing a statistical mechanics approach, we were able to reproduce by simulations the experimental electrophoretic mobility coefficients. We also established the role of the chemical composition of marker and the BGE on the measured electrophoretic mobility coefficient. For NMA, we interpreted the results in terms of the relative affinities of cations versus anions to the peptide bond.

Binding of Divalent Cations to Insulin: Capillary Electrophoresis and Molecular Simulations

In the present study, we characterize the binding of divalent cations to insulin in aqueous salt solutions by means of capillary electrophoresis and molecular dynamics simulations. The results show a strong pH dependence. At low pH, at which all the carboxylate groups are protonated and the protein has an overall positive charge, all the cations exhibit only weak and rather unspecific interactions with insulin. In contrast, at close to neutral pH, when all the carboxylate groups are deprotonated and negatively charged, the charge-neutralizing effect of magnesium, calcium, and zinc, in particular, on the electrophoretic mobility of insulin is significant. This is also reflected in the results of molecular dynamics simulations showing accumulation of cations at the protein surface, which becomes smaller in magnitude upon effective inclusion of electronic polarization via charge rescaling.

Study of polyaniline-coated silica gel as a stationary phase in different modes of capillary liquid chromatography

Separation potential of a stationary phase based on polyaniline-coated silica gel porous spherical particles was investigated in different chromatographic modes of capillary liquid chromatography. l-Tryptophan and three of its structural derivatives with modified amino or carboxylic functionality were used to investigate the retention factor curves under different mobile phase composition including pH variation. Some retention curves pass minimum which indicates that more than one chromatographic mode can be employed to retain the relevant solute. The positional isomers of aminoacetophenone and caffeine and its demethylated analogs—theobromine and theophylline—were used as separation probes in normal phase, hydrophilic interaction liquid chromatography, and reversed phase modes. The stationary phase exhibits the mixed-mode retention mechanism; therefore, it is not easy to predict the elution order of the solutes only according to their polarity. Even slightly hydrophilic compounds can be sufficiently retained in reversed phase mode using polyaniline-coated silica gel stationary phase. All positional isomers of aminoacetophenone were separated for the first time in normal phase and reversed phase modes.Graphical abstract