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P Bocek

Publications and source records attributed to P Bocek.

At least 19 recordsLinked to original sources
PubMed2001

Why robust background electrolytes containing multivalent ionic species can fail in capillary zone electrophoresis.

In this paper it is demonstrated that system peaks are induced by multivalent weak ionic species in background electrolytes. Their existence is derived from SystCharts and from Peak Shape Diagrams and the theory is confirmed experimentally. If analytes are present in a sample, with mobilities approximately equal to the mobility of a system peak, they interact, resulting in a strong increase of electromigration dispersion. This leads to strong peak broadening, peak deformation and a loss of resolution. Typical background electrolytes containing multivalent ionic species, e.g. phosphate and phthalate buffers, often reported to be robust electrolytes, are therefore not always universally applicable and can fail for the application of specific analytes. This paper reports a systematic study of the above phenomena and shows both theoretical and experimental results for background electrolytes containing phosphoric acid and phthalic acid.

Electrolytes↗
PubMed2001

Success and failure with phthalate buffers in capillary zone electrophoresis.

Phthalate buffers are currently used in capillary electrophoresis as robust electrolyte systems for indirect detection. This contribution demonstrates that these buffers show regularly not only successful regions of mobilities of analytes (sample window) but also regions of failure where the migration of analytes is strongly deteriorated due to the presence of a system zone. System zones in phthalate buffers may be easily detected by UV detection and manifest themselves as peaks or dips. Peak shape diagrams are advantageously used for the prediction of the migration behavior of system zones in phthalate background electrolyte (BGE) systems at various pH. It is shown that the mobility of the system zone varies strongly with pH, is practically zero at pH values below 4 and above 7, and shows a maximum at pH 5. Thus, the system peak may coincide either with the peaks of various analytes or with the electroosmotic flow (EOF) peak. Experiments are given showing the effects of such coincidences as, e.g., zigzag detection patterns, double EOF peaks, and/or unusually broad peaks/dips. The message of this contribution is to show how to understand the electrophoretic properties of phthalate BGEs that, regardless of possible failure regions, may be successfully used in the analytical practice of capillary zone electrophoresis (CZE).

Buffers↗
PubMed2001

Fast separation of DNA sequencing fragments in highly alkaline solutions of linear polyacrylamide using electrophoresis in bare silica capillaries.

An optimized procedure for the fast separation of DNA sequencing fragments in short bare fused-silica capillaries filled with highly alkaline solutions of replaceable linear polyacrylamide is presented. High denaturing abilities of the separation media at pH values over 12 are the main reason for their applications in analyses of ssDNA fragments. Moreover, the alkaline solutions of polyacrylamide provide other advantageous properties: three times higher electrophoretic mobility of ssDNA fragments in comparison to those in urea, negligibly low electroosmotic flow in uncoated capillaries, and an adequate stability to a fast alkaline hydrolysis. The separation power of this procedure is enhanced strongly by using monocarboxy poly(ethylene glycol), a terminator for transient isotachophoresis, which eliminates the electromigration dispersion. A high separation efficiency of our system enables to reduce analysis time to several minutes by decreasing the effective lengths of capillaries to 7 cm. A special sample introduction by diffusion is successfully applied. The experimental results demonstrate a potential of the alkaline electrolytes for an implementation in diagnostic sequencing practice.

Acrylic Resins↗
PubMed2000

Capillary zone electrophoresis in phosphate buffer--known or unknown?

It has been shown recently that the analysis records in capillary electrophoresis may involve regions with extremely strong electromigration dispersion of peaks. Such a fundamental effect is due to the existence of more centers of symmetry in a given electrolyte system. This paper shows that even such a simple and frequently used electrolyte system as phosphate buffer may exhibit more than one center of symmetry. By using the peak shape diagram approach we have revealed that neutral and alkaline phosphate buffers have two centers of symmetry and one center of extreme dispersion. Model experiments confirmed this new important discovery.

Buffers↗
PubMed2000

Selectivity in CE.

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Chromatography, High Pressure Liquid↗
PubMed2000

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↗
PubMed2000

Sweeping of neutral analytes in electrokinetic chromatography with high-salt-containing matrixes

The concept of sweeping neutral analytes using a high-conductivity matrix or under a reduced electric field in micellar electrokinetic chromatography (MEKC) using anionic micelles and in the presence of electroosmotic flow is presented. Three important processes are identified. First, stacking of the micelles at the cathodic interface between the sample solution (S) and background solution (BGS) zones is identified. This is then followed by the sweeping of analyte molecules by the stacked micelles that enter the S zone. Finally, the destacking of the stacked micelles at the anodic interface between the S and BGS zones occurs. The stacking of the micelles improves the focusing effect of sweeping by a factor approximately equal to the ratio of conductivities between the S and BGS zones (ratio = enhancement factor = gamma'). However, the destacking of the stacked micelles broadens the swept zones by a factor approximately equal to 1/gamma'. In effect, the focusing effect of sweeping using a matrix with equal or higher conductivity compared to the BGS will be roughly the same. The micelle stacking and destacking mechanisms are verified experimentally. This paper also provides comments on the mechanism of neutral analyte focusing under similar conditions proposed by another group.

Journal Article↗
PubMed2000

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↗
PubMed2000

Ultrafast detection of microsatellite repeat polymorphism in endothelin 1 gene by electrophoresis in short capillaries.

The methodology and instrumentation for fast denaturing electrophoresis in short capillaries was developed and exemplified by detection of short tandem repeat polymorphism in the endothelin 1 gene. The resolution of two nucleotides, which is required for the detection of a dinucleotide repeat polymorphism, was achieved in a capillary of an effective length of 2.5 cm at a temperature of 600C and an electric field strength of 600 V/cm in 42 s. Thus, the use of denaturing electrophoresis in short capillaries with laser-induced fluorescence detection resulted in a reduction of analysis time by a factor of 200 when compared to the conventional slab gel electrophoresis. The developed methodology and instrumentation is advantageous for an implementation in clinical diagnostics and genetic population screening where fast analytical instrumentation amenable to automation is of paramount importance.

DNA↗
PubMed2000

Capillary zone electrophoresis and micellar electrokinetic chromatography of solution of polyaniline particles.

The behavior of two different fractions of the electrically conducting polymer polyaniline (PANI) was studied in order to characterize them using fast, simple, and cheap methods of capillary zone electrophoresis. In contradistinction to the expectations that the particles will bear a positive charge on their surface, in the milieu of electrolytes used for electrophoresis (0.01-0.05 M buffers of pH 4-8.5), the surface was demonstrated to be slightly negatively charged, which led to a slow migration to the anode. An evident charge was given to the particles after their interaction with micelles formed from surface active components, cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS). An active electrophoretic migration and full separation of the PANI fractions was achieved by micellar electrokinetic chromatography (MEKC) with negatively charged SDS micelles. With this method, a reliable qualitative as well as quantitative analysis was performed in the concentration range of 0.1-1.6 mg/mL with a reproducibility of migration times better than 1.5%.

Aniline Compounds↗
PubMed2000

Sample stacking in capillary zone electrophoresis: principles, advantages and limitations.

The principles of stacking procedures are described and their properties are discussed, including the fundamentals of the behavior of zone boundaries and the consequences of the self-correcting properties of boundaries in moving boundary electrophoresis, isotachophoresis, and zone electrophoresis. Further, the diverse possibilities of stacking procedures and the unavoidable destacking are described, and several examples of practically applied stacking procedures are given, besides many references to applications. Some limitations in the use of stacking procedures are discussed. The paper is arranged in such a way that it can serve both as an introduction into the field and as a reference overview.

Animals↗
PubMed2000

Sample self-stacking in capillary zone electrophoresis: behavior of samples containing multiple major coionic components.

It is a frequent phenomenon in practice that a sample contains bulk levels of more than one coionic component that affect the stacking behavior of minor analytes and in this way also the sensitivity of the method. Here, attention is paid to stacking resulting from the presence of a macrocomponent of leading type that is deteriorated by the presence of another macrocomponent of like charge in the sample. Based on the isotachophoretic model of migration in the initial period of separation, a theoretical approach was elaborated both for strong and weak electrolytes which describes the separation process and finds the conditions that define whether transient isotachophoretic stacking of the analyte takes place or not. It is shown that the crucial parameter is the ratio of the concentrations of macrocomponents migrating in front and behind the analyte of interest. The destacking effect can also be expected when the coion of the background electrolyte is present in the sample. Rules how to cope with effects of destackers present in the sample are given. Theoretical considerations are illustrated by computer simulations and verified experimentally. Examples of antagonistic effects of macrocomponents are demonstrated for model serum samples.

Electrophoresis, Capillary↗
PubMed2000

New aspects of buffering with multivalent weak acids in capillary zone electrophoresis: pros and cons of the phosphate buffer.

Phosphate buffer is frequently used as background electrolyte in capillary electrophoresis. It can cover a broad range of pH due to the three dissociation constants (pK1 = 2.0, pK2 = 7.2, and pK3 = 11.0) of phosphoric acid and because it is UV-light transparent. This contribution brings a theoretical study of the analytical separation performance (sample window, regions of peak symmetry, regions of fronting and tailing peaks) of phosphate buffer, serving as a model of buffering with multivalent weak acids. The study is based on the use of peak shape diagrams and covers the pH range 2.55-11.43. New important general knowledge has been revealed that single multivalent weak acids mimic the performance of background electrolytes with multiple coanionic species for anionic analyses. It is shown that simple phosphate buffers prepared by mixing phosphoric acid and potassium hydroxide may exhibit up to two regions of symmetry, of fronting as well as of tailing zones, on the mobility scale inside the sample window. Moreover they may exhibit a "schizophrenic" region of enormous electromigration dispersion.

Buffers↗
PubMed2000

Recent progress in capillary isotachophoresis.

This article is a continuation of previous reviews and summarizes the progress of analytical capillary isotachophoresis in the years 1997-1999. Papers reviewed include theoretical and methodological aspects as well as analytical applications. Included are also papers using isotachophoresis and/or isotachophoretic principles as part of multidimensional separation schemes.

Animals↗
PubMed2000

Ranitidine or dobutamine alone or combined has no effect on gastric intramucosal-arterial PCO(2) difference after cardiac surgery.

OBJECTIVE: To test the hypothesis that ranitidine, either alone or in combination with dobutamine, modifies the gastric intramucosal-arterial PCO(2) difference. DESIGN: Full factorial design (double-blinded for ranitidine). SETTING: Intensive Care Unit of a university hospital. PATIENTS: Sixty-four haemodynamically stable coronary artery bypass surgery patients. INTERVENTIONS: Ranitidine (150 mg preoperatively per os and 50 mg intravenously postoperatively) and dobutamine (4 micro g. kg(-1). min(-1) for 3 h postoperatively) were administered in four randomised groups of patients: preoperative and postoperative ranitidine, either alone (n = 15) or in combination with dobutamine (n = 17), dobutamine alone (n = 15) or neither ranitidine nor dobutamine (n = 17). MEASUREMENTS AND RESULTS: Gastric intramucosal-arterial PCO(2) difference was measured during the first 5 postoperative hours. No differences in the postoperative pattern of gastric intramucosal-arterial PCO(2) difference were found among the groups. CONCLUSIONS: Ranitidine and dobutamine have no effect on the gastric tonometry results on intramucosal-arterial PCO(2) difference after uncomplicated cardiac surgery. Hence, the routine use of H(2)-antagonists for gastrointestinal tonometry is not warranted. Our results must be limited to results obtained by tonometry; they do not allow any conclusions on the effects of these drugs on splanchnic blood flow or its distribution.

Adrenergic beta-Agonists↗
PubMed2000

Genomic relatedness of Staphylococcus aureus phages of the International Typing Set and detection of serogroup A, B, and F prophages in lysogenic strains.

On the basis of HindIII-restriction digest analysis of genomic DNAs, the S. aureus bacteriophages of the International Typing Set were divided into five clusters designated as A, F, Ba, Bb, and Bc. The clusters A and F include all the phages of serogroups A and F and correspond to species 3A and 77 proposed by Ackermann and DuBow (1987). On the other hand, the phages of serogroup B were divided into three clusters designated as Ba, Bb, and Bc that differ significantly each from the other in their restriction patterns. The clusters Ba and Bb may represent two separate species, while the cluster Bc may include more than one phage species. For each of the phage serogroups A, B, and F, common HindIII-restriction fragments of phage 3A (1700 bp), of 53 (4060 bp), and of 77 (8300 bp) were used for the preparation of probes specific to the phages of serogroups A, B, and F. These probes were very effective, making it possible to detect up to three different prophages in a given lysogenic strain at the same time. Restriction enzyme maps of phages 3A, 53, and 77, each representing a different serogroup, were constructed. The restriction maps of phage 3A and that of phage 77 are linear, whereas that of phage 53 is circular and exhibits a circular permutation. DNAs of the phages of serogroups A and F have cohesive ends. On each restriction map, the sites corresponding to specific probes are indicated. The size of intact genomic DNA of all phages estimated by PFGE varies within the range of 41.5-46.2 kb.

Bacteriophage Typing↗
PubMed1999

Highly alkaline electrolyte for single-stranded DNA separations by electrophoresis in bare silica capillaries.

A new, highly denaturing electrolyte system based on a solution containing 0.01 M NaOH, 0.0015 M Na2B4O5(OH)4 and a replaceable polymer sieving medium was designed for the separation of single-stranded DNA fragments in bare fused-silica capillaries. Extreme denaturing power, together with the optimized composition of the electrolyte, allows for a separation efficiency as high as 2,300,000 height equivalents to a theoretical plate per meter. Sample denaturation in alkaline solutions provides single-stranded DNA fragments without any intra- or intermolecular interactions at room temperature. Their electrophoretic mobilities were found to be twice those of fragments denatured by dimethylformamide or HCl. This can be interpreted in terms of an increased effective charge on the DNA molecules. The surprisingly weak electroosmosis (6 x 10(-10) m2 V-1 s-1) of polymer solutions at pH 12 or higher is considered to be the result of the dissolution of the silica capillary wall. A highly viscous thin layer of dissolved silica probably causes a shift of the slipping plane further away from the wall to the lower value of the zeta potential. Applications of the electrolyte in clinical diagnostics demonstrate its remarkable properties.

DNA, Single-Stranded↗