Biomedical subjects
Publications and source records attributed to P Bocek.
The separation of 14 lanthanides by capillary zone electrophoresis was studied in the background electrolyte containing hydroxyisobutyric acid as complexing counter-ion and creatinine as a UV absorbing coion for indirect detection of lanthanide zones. A complete separation was achieved in less than 5 min and the applicability of the method for the analysis of real samples was demonstrated.
The influence of various parameters affecting separation of oligonucleotides by capillary zone electrophoresis has been examined. The effects of pH, ionic strength, and various additives, including highly charged cations such as spermine, were studied. Using polycytidines as model compounds, it was demonstrated that pH in the range of 5-8 and ionic strength in the range of 20-200 mmol/l do not influence the separation of oligonucleotides substantially. However, with the addition of spermine to the background electrolyte, migration order was inverted as the effective mobilities of the larger oligonucleotides were greatly decreased. With the addition of spermine and sodium dodecyl sulfate, the separation+ of these model oligonucleotides was also significantly affected. The best separation of a homologous series of polycytidines was obtained with a background electrolyte containing 60 mmol/l histidine, 30 mmol/l glutamic acid, 50 mmol/l sodium dodecyl sulfate and 3 mmol/l spermine.
A method is described for the determination of the triazine herbicides prometryne, desmetryne, terbutryne, OH-atrazine and OH-simazine in purified extracts of milk using analytical capillary isotachophoresis. The reproducibility of isotachophoretic analyses was 3.5% and the detection sensitivity reached 2 ng. Recovery of triazines from fortified samples of homogenized full milk (0.05 mg/L) was about 65%.
In contrast to common capillary zone electrophoresis, with a constant composition of the background electrolyte, a new method is described, based on a change of the ionic matrix composition during the migration of the sample components. The proposed method expands the scale of pK's of analyzed compounds in a single run and affords new potential for optimizing the separation. The step change of the ionic matrix composition was achieved by displacing the primary matrix by the modified ionic matrix (electrolyte) which migrated against the movement of the sample components. The separation thus proceeded in the primary ionic matrix (electrolyte) while the detection was performed in the modified matrix with resultant shortening of the analysis time and convenient quantitation. A model mixture of five compounds, with a difference of four units in pK could be well resolved.
The use of a bivalent counter anion for cationic isotachophoresis was studied both theoretically and experimentally. A mathematical model was proposed and solved to give the effective mobility of the hydrogen ion constituent, uH,H, in the respective electrolyte systems. The theoretical values agreed well with the experimental data in a series of electrolyte systems. The dependence of uH,H on such parameters as pH of the leading electrolyte, ionic mobilities of leading and counter ions, and dissociation constants was calculated and discussed. A bivalent counter anion will prove useful for the separation of low mobility cations and weak bases, as shown for the separation of substituted anilines in a succinate electrolyte system.
The practical usefulness of multicolumn isotachophoresis was demonstrated by the determination of oxalate levels in human serum. 10 mmol/L HCl and 100 mmol/L Na2HPO4 served as the leading and terminating electrolyte, respectively. For the stabilization of the isotachophoretic zones in large cross section channels a suspension of Bio-Gel P-300 in the leading electrolyte was used. For the analysis ca. 1 mL of fresh serum was required and 1.2 mL of 1:1 mixture of serum and the suspension of Bio-Gel P-300 in deionized water was applied as sample. The time of analysis ranged between 45-49 min. The detection limit of analysis was determined to be 50 nmol/L. Reproducibility of analysis of 1 mumol/L oxalate in water standard was found to be 2.6% (n = 6).
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This paper is summarizing the contributions to the analytical capillary isotachophoresis published during the period 1981-1984. It characterizes the present state of the method and covers theory, fundamental analytical aspects, instrumentation and applications. Special attention was payed to the fundamental analytical aspects, and a detailed discussion is given of the selection of electrolyte systems, stability of zones and separability of substances. The present commercial instrumentation is also briefly described.
An isotachophoretic device for the analysis of trace components in a sample with the efficient pre-separation and elimination of bulk components has been suggested and realized. A large volume of the sample in question is separated in a rectangular wide-bore channel packed with a suspension of a granulated polyacrylamide gel and analyzed in free electrolytes in a narrow-bore tube. Trace components, the concentrations of which are 10(-6) mol/l, can be analyzed in 60-70 min applying ca. 1 ml of the sample even in the presence of bulk excess of a major component the concentration of which is by 5 orders of magnitude higher.
The suitability of isotachophoresis for the analysis of metals in, e.g., environmental samples was studied. In a cationic operational system the heavy metals Fe, Cu, Ni, Cd, Co, Zn, Pb and Mn were simultaneously determined. The separation was achieved through complex formation with one of the counter ions, hydroxyisobutyric acid. The other counter ion was acetic acid, the leading ion was 0.02 M potassium or sodium (pH 4.1) and the terminator was H+. The analysis time was 15 min at 60 microA in a 0.2 mm I.D. capillary. Aqueous samples containing ppm and ppb amounts were enriched on a cation exchanger with an extremely low affinity for sodium (Chelex 100). Good recovery, linearity, precision and accuracy were obtained even down to the ppb range. Although the sensitivity of the method is not greater than that of some of the more established methods for the individual metals, a great advantage of isotachophoresis is the simultaneous determination of the metals, with equal response factors. An example is given of the determination of metals, including aluminium, in serum.
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In order to perform a required separation, an experimental procedure for the selection of suitable operating conditions is suggested and discussed. It is based on measurements of the dependences of the relative effective mobilities of the components under investigation on the pH of the leading electrolyte. The procedure was applied to a set of typical Krebs cycle acids and the values of the relative effective mobilities measured are given in tables and graphs. A pH of 3.8 was selected as the most suitable. At this value, the acids investigated were successfully separated in less than 4 min using 0.011 M hydrochloric acid + beta-alanine as the leading electrolyte.
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The upper limit of the size range of DNA amenable to separation in agarose solutions above their gelling temperature, using capillary zone electrophoresis apparatus, was increased to 12 kb. The plot of log(bp) vs. mobility derived from electrophoresis in 1.7% agarose solution is biphasic, exhibiting higher resolving power for DNA less than 1 kb in size than that of larger sizes. Resolving power for DNA larger than 1 kb increased when the agarose concentration was increased in the range of 1.0-2.6%. It was similar in solutions at 40 degrees C of SeaPrep and SeaPlaque agaroses as well as in Acrylaide (trade names are those of the manufacturer). However, the resolving power of SeaPrep agarose at 25 degrees C was inferior to that at 40 degrees C. Concave plots of log(mobility) vs. concentration of the agarose solutions are those predicted under the assumption that the effective "equivalent radius" of the DNA molecule diminishes with increasing agarose concentration in the investigated concentration range up to 2.6%.
A procedure is proposed for the calculation of the actual effective mobility of a zone from its migration time. It is based on the use of internal standards with known mobilities; the use of two internal standards provides reliable mobility data even if the magnitudes of the effects of sample composition, capillary temperature, capillary length, migration distance, used voltage, as well as the tube length occupied by the injected sample are unknown. Formulas have been derived for the calculation of the actual mobilities, and their experimental verification has been carried out by using a model set of anionic solutes with mobilities ranging from -56 to -20 x 10(-9) m2V-1s-1 and chloride as the ion modelling the effect of the sample matrix.
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.