Biomedical subjects

Petr Bocek

Publications and source records attributed to Petr Bocek.

At least 19 recordsLinked to original sources

PubMed2007

Recent progress in capillary ITP.

ITP has been attracting constant attention for many years due to its principal capability to concentrate trace analytes by several orders of magnitude. In the current capillary format, it is able to concentrate trace analytes diluted to several microliters of an original sample into concentrated zones having volumes in the range of picoliters. Due to this reason, ITP holds an important position in many current multistage and multidimensional separation schemes. This article links up previous reviews on the topic and summarizes the progress of analytical capillary ITP since 2002. Almost 100 papers are reviewed that include methodological novelties, instrumental aspects, and analytical applications. Papers using ITP and/or isotachophoretic principles as part of multistage and/or multidimensional separation schemes are also included.

Chemistry, Pharmaceutical↗

PubMed2007

Contemporary sample stacking in CE: a sophisticated tool based on simple principles.

Sample stacking is a general term for methods in CE which are used for on-line concentration of diluted analytes. During the stacking process, analytes present at low concentrations in a long injected sample zone are concentrated into a short zone (stack). The stacked analytes are then separated and individual zones are detected. Thus stacking provides better separation efficiency and detection sensitivity. Many papers have been published on stacking till now, various procedures have been described, and, many names have been proposed for stacking procedures utilizing the same principles. This contribution brings an easy and unified view on stacking, describes the basic principles utilized, makes a list of recognized operational principles and brings an overview of principal current procedures. Further, it surveys selected recent practical applications ordered according to their operational principles and includes the terms, nicknames, and acronyms used for these actual stacking procedures. This contribution may help both newcomers and experts in the field of CE to orient themselves in the already quite complex topic of sample stacking.

Electrophoresis, Capillary↗

PubMed2006

A new type of migrating zone boundary in electrophoresis: 2. Transient sample zone shapes.

A new type of migrating zone boundary in electrophoresis is presented theoretically and evidenced experimentally. This type of the boundary (called hybrid boundary) shows simultaneously a steady-state part with self-sharpening properties and an unsteady-state part with time-dependent electromigration dispersion. It is shown that a sample zone may possess a hybrid boundary both as its front and rear one simultaneously. In such a case, the evolution of a sample zone injected originally as a rectangular pulse exhibits very complex transient shapes before it reaches the well-known fronting or tailing triangular shape. Depending on the stage at which detection of such a sample zone occurs, variable and peculiar-shape peaks may appear in the electropherogram. Based on theoretical predictions, experimental examples of the above-mentioned phenomena are presented in this contribution for direct and indirect UV absorbance detection of sample zones. Excellent agreement of theoretical predictions with the experiments has been found. The knowledge of hybrid boundaries is of key significance for correct interpretation of records of CE analyses in practice.

Electrophoresis↗

PubMed2006

A new type of migrating zone boundary in electrophoresis: 3. The hybrid boundary and stacking criteria.

This article investigates the principles of stacking of analytes by a zone of a bulk sample component that possesses one hybrid boundary. The model system investigated comprises a transient stacking zone in a BGE where the rear boundary of this zone is a hybrid one. A theoretical description of such a system is given, and general rules and mathematical criteria for quantitative stacking are presented. These criteria are based on comparison of migration velocities of analytes to be stacked and velocities of the boundaries of the stacking zone. It is shown that the presence of a hybrid boundary brings about additional constraints for stacking of analytes due to the presence of the sharp part of the hybrid boundary that migrates faster than regular sharp boundary of this zone. An experimental example related to the described theory is also shown.

Computer Simulation↗

PubMed2005

A new type of migrating zone boundary in electrophoresis: 1. General description of boundary behavior based on electromigration dispersion velocity profiles.

Till now two principal types of electrophoretic boundaries have been recognized: a steady-state one showing self-sharpening properties and an unsteady-state one showing electromigration dispersion (EMD). In this contribution, the existence of a third (hybrid) type of electrophoretic boundary is revealed, that shows simultaneously: (i) steady-state character with self-sharpening properties in a certain part of the boundary, demarcated by a certain range of its composition; (ii) unsteady-state character with EMD in the resting part. A new theoretical approach to the study of the structure and properties of electrophoretic boundaries is presented, based on EMD velocity profiles representing the dependence of the EMD velocity on the composition in any point of the transition region across the boundary. According to this approach, the linearity or nonlinearity of the EMD velocity profile is recommended as the criterion for distinguishing the actual type of the boundary in question. It is shown that the new type of electrophoretic boundary is not an exotic case but may be observed even in simple systems as, e.g., formed by adjacent zones containing mixtures of current species such as picrate and acetate. Computer simulations are presented which confirm the theoretical conclusions and illustrate the three types of electrophoretic boundaries.

Electrophoresis, Capillary↗

PubMed2005

System zones in capillary zone electrophoresis: moving boundaries caused by freely migrating hydrogen ions.

We demonstrate that system zones (SZs) can be expected in background electrolytes (BGEs) with a low buffer capacity. The mobilities of this type of SZ (m(SZ)) could be determined by calculations both based on a mathematical model and by a simulation program. The values of m(SZ) are increasing for decreasing buffer capacities and lower concentrations of the BGEs. For completely unbuffered BGEs with a pH below 7, the m(SZ) reached values up to 350x10(-9) m(2)V(-1)s(-1). This value indicates that the existence of this type of SZ originates from migrating hydrogen ions. Although both the mathematical model and simulation program do not consider the influence of the pH of the sample solution, experiments have shown that the pH of the sample solution is also very important. The lower the pH of the sample solution, the larger the m(SZ) in a specific BGE for cationic SZs. Using completely unbuffered BGEs the hydrogen ions present in the original sample start to migrate freely through the capillary tube with the mobility of a single hydrogen ion and cause stepwise disturbances in the base line of the detector trace. It is remarkable that this type of SZ appears not to affect the electromigration dispersion (EMD) in a strong way and so far only dips could be obtained experimentally.

Electrolytes↗

PubMed2005

System zones in capillary zone electrophoresis: moving boundaries caused by freely migrating hydroxide ions.

We present theoretical and experimental data indicating that anionic system zones (SZs), due to free migrating hydroxide anions, can be expected in background electrolytes (BGEs) with a low buffer capacity. In the system containing completely unbuffered BGEs the hydroxide ions derived from the sample start to migrate freely through the capillary tube with the mobility of single hydroxide ions and cause stepwise disturbances in the baseline of the detector trace. Remarkably, this type of SZs do not appear to contribute significantly to the electromigration dispersion (EMD) of the zones of the analytes.

Buffers↗

PubMed2004

Ionic boundaries in biological capillary electrophoresis.

Ion migration in electrophoresis always leads to the formation of ionic boundaries. While some types of the ionic boundaries can be formed intentionally, e.g., to improve separation efficiency or sensitivity of the analysis, other, naturally formed boundaries, may cause unexpected effects during the electrophoretic experiment. Some of the boundaries often go unnoticed in practice; however, many effects of the ionic boundaries formed by the sample matrix and background electrolyte components are frequently observed in capillary electrophoresis (CE). The interpretation of these effects may not be trivial and different explanations of the same phenomena may be found in the literature. This critical article attempts to review some of the important effects of the ionic boundaries observed or utilized during the CE of biologically important samples.

Electrophoresis, Capillary↗

PubMed2004

Interferon gamma enhances both in vitro and in vivo priming of CD4+ T cells for IL-4 production.

Classical studies have demonstrated that in vitro priming of naive CD4 T cells to become T helper (Th)2 cells is strikingly dependent on interleukin (IL)-4, whereas priming for interferon (IFN)gamma production is IL-12/IFNgamma-dependent. Therefore, it was quite surprising when we noted that priming of naive C57BL/6 CD4(+) cells to become IL-4 producers was substantially inhibited by the addition of anti-IFNgamma antibodies. This was true using immobilized anti-CD3 and anti-CD28 antibodies or soluble anti-CD3/anti-CD28 and antigen-presenting cells in the presence or absence of added IL-4. Priming of CD4 T cells from IFNgamma(-/-) C57BL/6 mice with immobilized anti-CD3 and anti-CD28 resulted in limited production of IL-4, even with the addition of 1,000 U/ml of IL-4. Titrating IFNgamma into such cultures showed a striking increase in the proportion of T cells that secreted IL-4 upon challenge; this effect was completely IL-4-dependent in that it was blocked with anti-IL-4 antibody. Thus, IFNgamma plays an unanticipated but substantial role in Th2 priming, although it is an important Th1 cytokine, and under certain circumstances a Th1 inducer.

Animals↗

PubMed2004

Experimental assessment of electromigration properties of background electrolytes in capillary zone electrophoresis.

Electromigration dispersion (EMD) properties of background electrolytes (BGEs) used in capillary zone electrophoresis (CZE) are of key importance for the success of an analysis. The knowledge of these properties may serve well for the prediction of the asymmetry of peaks of analytes, for the prediction of unsafe regions where a strong interference of system zones may be expected, and for the selection of optimum conditions where the analytes of interest may give sharp and practically symmetric peaks. Present theories enable one to calculate and predict EMD properties of many BGEs but there is also a lot of BGEs that are beyond the present theoretical models as far as their composition and equilibria involved are considered. This contribution brings a method for assessment of EMD properties of any BGE from easily accessible experimental data. The method proposed is illustrated by model examples both for cationic and anionic separations. Imidazole acetate, histamine acetate, and histidine acetate served as model BGEs for cationic separations; as the model BGE for anionic separations, Tris-borate and sodium-borate BGEs have been selected since these buffers are frequently used and borate is well-known for its complexing equilibria in aqueous solutions.

Acetates↗

PubMed2004

Calibrationless quantitative analysis by indirect UV absorbance detection in capillary zone electrophoresis: the concept of the conversion factor.

A new, fast and efficient procedure is described for the simultaneous quantitative analysis of various non-UV absorbing species in a sample, by capillary zone electrophoresis with indirect UV absorbance detection. The procedure is based on the concept of the conversion factor (CF). The CF of an analyte is defined as the ratio of the measured temporal peak area and the product of its migration time and transfer ratio (TR). Thus defined, the CF is of general validity for all analytes separated and detected in a given background electrolyte (BGE), since it has the same value for the same amounts of various analytes. If a sample is enriched with a known concentration of a standard component and analyzed by CZE, the CF of this standard component can be calculated and then the concentrations of all other analytes can be determined, without the use of any calibration graph. The individual TRs can be determined a priori from tabulated ionic mobilities and pK values of the analytes and of the constituents of the BGE or, for strong analytes, by using experimental data from the electropherogram of the analysis itself. The practical procedure of the analysis includes enrichment of the sample with a known quantity of a suitable standard and a single CZE run of the resulting mixture. The injected volume does not need to be known and thus the procedure also eliminates the injection error. The proposed procedure has been verified experimentally and reproducible and accurate values were obtained by using four different CZE apparatus for the analyses of standard mixtures of cations in three different BGEs.

Calibration↗

PubMed2004

Importance of the counterion in optimization of a borate electrolyte system for analyses of anions in samples with complex matrices performed by capillary zone electrophoresis.

Borate buffers are common background electrolytes for analyses of anions in capillary zone electrophoresis. Usually, sodium borate at a given pH is used and this specification seems to be sufficient for a successful analysis. In this paper, we show that free migration of OH(-) may deteriorate the analysis of a typical anionic analysis of clinical samples due to uncontrolled migration of OH(-) throughout the systems of analyzed zones and may damage the stacking of anionic analytes of interest. We have proven that the use of ammonium borate may remedy the situation where the presence of ammonium may selectively stop the free migration of OH(-) ions, slow down their effective mobility and bring their safe behavior resulting in reproducible stacking of clinically important anions. Results of real analyses of human serum samples confirmed the proposed method and proved that substitution of sodium for ammonium in borate buffers offers reliable analyses of clinical samples having chloride as the bulk component. The experimental results given in this paper are supported also by computer simulation, which can not only support the positive results but also show the dynamics of the separation that is otherwise hidden to any detection possibilities.

Borates↗

PubMed2003

Capillary zone electrophoresis in methanol: migration behavior and background electrolytes.

Nonaqueous (NA) solutions are often used as background electrolytes (BGEs) and NA solvents are added to aqueous BGEs as organic modifiers in capillary zone electrophoresis (CZE), in order to optimize the separations. This can be tricky, however, because the pH* and pK* concepts may be totally different in NA solvents, whereas often less knowledge is available concerning phenomena, such as system zones, applying NA solvents. In this paper, the concepts of pH* and pK* are considered for methanol as a solvent and pK* values are determined for several components in mixtures of water and methanol. With a mathematical model, adapted for calculations in methanol, parameters are calculated describing the fronting or tailing character of peaks and the question of peaks or dips, and the existence of system zones is discussed for pure methanol as a solvent. These aspects are experimentally verified, applying BGEs useful for the separation of cationic species in the indirect UV mode. It can be concluded that the mathematical model developed for aqueous BGEs is applicable to BGEs in methanol, too, and that the behavior of BGEs in methanol is comparable with that in water concerning the fronting or tailing character of peaks and the question of peaks and dips, although the mobilities and pK values can change significantly.

Electrolytes↗

PubMed2003

Stacking phenomena in electromigration: From basic principles to practical procedures.

The review describes principles of procedures and techniques used in capillary zone electrophoresis for enhancement of sensitivity that are based on increasing analyte mass in its zone during the electromigration process, for which the term stacking is generally used. Attention is paid to intrinsic stacking in samples with low conductivity, transient isotachophoretic stacking applied in samples with high conductivity, and sweeping in micellar electrokinetic chromatography. Principles of these stacking schemes are explained, new procedures and instrumental arrangements are discussed, and all contributions involving stacking principles that have been published since the year 2000 are surveyed.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2003

Chloride present in biological samples as a tool for enhancement of sensitivity in capillary zone electrophoretic analysis of anionic trace analytes.

Effects originating from the variability of the sample matrix can be efficiently eliminated when the separation conditions are selected so that compounds of like charge with high concentration referred to as macrocomponents are embodied into the system of transient isotachophoresis. For stacking and separation of anionic trace analytes in biological samples, the presence of chloride is shown to be important to balance out effects of other macrocomponents that act against isotachophoretic stacking. Having acetoacetate, malate, citrate, and some drug metabolites in untreated human serum samples, the stacking mechanism of these compounds in an electrolyte system comprising 5 mM mandelic acid and epsilon -aminocaproic acid, pH 3.8, is explained. Analytes are monitored by indirect UV-absorption detection. Attention is paid to the minimum chloride concentration required with respect to the concentration ratio of phosphate (stacker) and lactate (destacker) present in the sample so as to ensure both stacking and separation of trace analytes. Insight into the separation process is given both with computer simulations and experiments. For selected analytes, the effect of chloride concentration on quantitative evaluation, sensitivity and limit of detection is demonstrated as well. Moreover, the applicability of the mobility window between phosphate and lactate for an additional group of metabolites is sketched.

Anions↗

PubMed2003

The preparation of background electrolytes in capillary zone electrophoresis: golden rules and pitfalls.

In this article the methodology of the design of suitable background electrolytes (BGEs) in capillary zone electrophoresis (CZE) is described. The principal aspects of the role of a BGE in CZE are discussed with respect to an appropiate migration behavior of analytes, including the transport of the electric current, the buffering of pH, the Joule heat, the electro-endosmotic flow (EOF) and the principal migration and detection modes. The impact of the composition of the BGE upon migration and detection is discussed. It is shown that the total concentration of the BGE is a principal factor and the adjustment of migrating analyte zones according to the Kohlrausch regulating function (KRF) is the principal effect in most of the sample stacking techniques. The number of co-ions and their properties are of key importance for peak shapes of the analyte peaks and for the existence of system zones. The detection of UV-transparent analytes may advanteously be done in the indirect UV mode, by using UV-absorbing co-ions, however, both peaks and dips may be expected in the UV trace in case of multiple co-ionic BGEs. Properties of BGEs can be predicted applying mathematical models and it is shown that with SystCharts, predictions can be given concerning the existence of system zones, detection modes and the peak shapes of analytes for a given BGE. Practical examples of methodological considerations are given in the design of suitable BGEs for four principal combinations of migration and detection modes. The properties of the BGEs selected are exemplified with experimental results. Golden rules are summarized for the preparation of suitable BGEs in CZE.

Electrolytes↗

PubMed2002

Theory of system zones in capillary zone electrophoresis.

In the last years, it has been shown that the formation and migration of system zones is an inherent feature of capillary zone electrophoresis (CZE) and that it depends predominantly on the composition of an actual background electrolyte (BGE). In most of the currently used BGEs, the SZs are invisible by the UV absorbance detection system, however, the comigration of SZs with the zones of analytes deteriorates the analytical performance of CZE and may be fatal for its utilization. Therefore, the theoretical predictions of the existence and migration of SZs is of key importance for the expediency of CZE. This is a review of the theoretical treatments of SZs which reveals the origin and the properties of SZs and shows how to cope with them. Also, a table of some typical BGEs is presented where the existence and mobilities of SZs are given.

Electrophoresis, Capillary↗

PubMed2002

Quantitation of trace analytes in capillary zone electrophoresis with UV-absorbance detection: a critical study of approaches based on peak height.

This contribution is aimed at providing a survey of the limitations of the peak-height technique. It is shown that the shape and the slope of calibration curves give a precise insight into the separation mechanism and can warn the analyst against improper selection of background electrolyte or sample dilution. This work investigates three typical situations where the sample contains (i) the minor analyte only, (ii) the minor analyte and a nonstacking bulk component, (iii) the minor analyte and a stacking bulk component, and various modes of measurement of the calibration curves differing in the medium used for the dilution of the original sample (water, background electrolyte, solution of the bulk component). Based on simple theoretical models, the shapes of the calibration curves are derived and elucidated for all modes, clearly demonstrating that only a few modes can provide useful results while most of them seem useless for quantitation. The knowledge of regularities affecting the character of calibration curves can be used for optimization of the analysis so that reliable results and highest attainable sensitivity can be reached. The outcome of this study brings a clear instruction how to successfully quantitate trace analytes even in samples with a complex and variable matrix*.

Electrolytes↗