Capillary isoelectric focusing of native and inactivated microorganisms.

The research of microorganisms includes the development of methods for the inactivation of viruses and other microbes. It also means to efficiently eliminate the infectivity of microorganisms without damage of their integrity and structure. According to the results of the last 5 years the capillary electromigration techniques appear to be very perspective for the comparison of the methods applicable for inactivation in the diagnostics and study of the pathogens. In this paper we suggest the capillary isoelectric focusing of the model microorganisms, Escherichia coli, Staphylococcus epidermidis, Candida albicans and bacteriophage PhiX 174, native or inactivated by different procedures. UV detection and fluorometric detection for the dynamically modified microbes by pyrenebutanoate on the basis of the non-ionogenic tenside were used here. Isoelectric points of native and/or dynamically modified microorganisms and other properties were compared with those obtained after microorganisms inactivation. The segmental injection of the sample pulse enabled the reproducible and efficient capillary isoelectric focusing in different pH gradients. The low-molecular-weight pI markers were used for tracing of the pH gradient.

[Advances in enantioselective TLC of therapeutic agents]. / Pokroky v enantioselektivní TLC léciv.

Enantioselective TLC represents a still developing field of analysis of therapeutic agents. In recent years, similarly as in HPLC, mainly direct separations of enantiomers without their preceding derivatization have been published, preferentially using chiral stationary phases, separations with a chiral selector in the mobile phase being less frequent. In the future, a larger choice of commercially available chiral stationary phases could contribute to a more widespread use of enantioselective TLC.

Capillary isoelectric focusing with UV-induced fluorescence detection.

Low-molecular-mass fluorescent compounds excitable in the near UV region with suitable acidobasic and electrophoretic properties are suggested as isoelectric point (pI) markers for isoelectric focusing (IEF) with UV photometric and UV excited fluorometric detection. The experimental set-up of capillary IEF with UV excited fluorometric detection and properties of new UV-induced fluorescent pI markers are given. The pI values of 18 new pI markers determined independently of IEF methods range from 2.1 to 10.3. The examples of separation of new pI markers together with derivatized proteins by capillary IEF with photometric or fluorometric detection are presented.

Enrichment techniques and trace analysis with microbore columns in liquid chromatography.

The advantages of microbore columns for trace analysis by liquid chromatography are identified, with reference to on-column enrichment techniques performed on analytical micro-columns. The selectivity and high sensitivity of the amperometric detector are utilized in combination with a microbore column for a number of pharmaceutical and bioanalytical analyses, including phenothiazines, parabens, sulphonamides, catecholamines, tetracyclines, vitamins, amino acids and dipeptides.

Stability of acetylsalicylic acid in divided powders in the presence of aminophenazone.

The combination of acetylsalicylic acid with aminophenazone, often prescribed in divided powders, is considered to be incompatible. High performance liquid chromatography was applied to determine the content of salicylic acid which is the main known product from the decomposition of acetylsalicylic acid. The analysis of model samples containing a 1:1 (by weight) mixture of acetylsalicylic acid and aminophenazone, after having been stored under usual conditions in starch capsules, showed that the decomposition of acetylsalicylic acid into salicylic acid did not exceed 0.8% even after ten weeks.

Model of isotachophoresis (displacement electrophoresis) in tapered capillaries.

The principle and theoretical description of boundary focusing under electrophoretic migration in a tapered capillary are presented. The model describes the gradual volume decrease of a moving boundary between weak electrolytes or ampholytes. The relations between the departure from the local steady state, the ratio of inlet to outlet capillary cross section, the effective mobilities and the selectivity are formulated. They allow the selection of operating conditions such that the departure from the local steady state can be decreased to an acceptable level. The relations predict that isotachophoresis (displacement electrophoresis) in a capillary with a shallow taper can maintain a constant ratio of actual boundary width to local steady state width, provided that the local capillary cross section decreases linearly with length. The significance of some assumptions needed for the model formulation is analyzed. Numerical examples are given indicating the method feasibility.

Low-conductivity background electrolytes in capillary zone electrophoresis--myth or reality?

The asymmetric triangle (fronting or tailing) concentration profiles and their broadening are the typical results of the electromigrational zone dispersion characterizing a system of the analyte in the background electrolyte (BGE). The present contribution suggests the parameter named the relative velocity slope, SBGE,X, which was introduced here as a quantity characterizing the peak broadening and the asymmetry. SBGE,X VS. analyte ionic mobility diagrams are suitable for the comparison of BGEs of given pH and the conductivity composed of electrolytes of different pKaS and ionic mobilities. The concept of SBGE,X diagrams is verified by capillary zone electrophoresis of the model analytes, which involve (i) the series of sulfobenzoylated poly(ethylene glycols) as examples of the strong electrolytes with different ionic mobilities and (ii) the series of monobasic phenols as weak electrolytes with different pKaS and similar ionic mobilities. It follows from both theoretical predictions of peak symmetry and their experimental verification that the optimum composition of BGEs is determined mostly by the suitable ionic mobility of the coion in dependence on the ionic mobility of the analyte. The low-conductivity BGEs based on low-molecular carrier ampholytes are at best only comparable with the properly chosen monobasic electrolytes.

Determination of the isoelectric points of low and high molecular mass ampholytes by capillary electrophoresis.

A methodology for a reliable and absolute determination of isoelectric points of amphoteric species by capillary zone electrophoresis is proposed. The principle of this absolute method consists in the application of an additional electroosmotic and/or hydrodynamic flow of a background electrolyte. This allows the measurement of electrophoretic mobilities of substances close to their isoelectric points where the net electric charge is very low and electrophoretic mobility tends to zero. The mobilities are measured at various pH values of the electrolyte so as to find the pH at which the substance moves through the separation column at zero mobility under the action of the additional flow only. The number of required measurements is lowered to the minimum with the help of the iterative procedure based on the Regula Falsi algorithm. The precision of the proposed method is predicted theoretically. The feasibility is demonstrated on real samples. The isoelectric points of histidine, myoglobine and ribonuclease are found from their titration curves.

Screening of amphetamines by gradient microbore liquid chromatography and pre-column technology.

Amphetamine-type drugs with a wide polarity range have been screened in both human and horse urine using on-line pre-concentration on pre-columns packed with hydrophobic and cation-exchange sorbents in series and gradient microbore high-performance liquid chromatography. The underivatized amphetamines were identified by UV detection at 210 nm. The method has potential for the automated liquid chromatographic screening of amphetamines in urine, e.g., for doping control.