Petr Boček

Indirect photometric detection in capillary zone electrophoresis

Abstract An indirect photometric detection method is described which is based on the use of an absorbing co-ion as the principal component of the background electrolyte. The zones of non-absorbing ionic species are revealed by changes in light absorption due to charge displacement of the absorbing co-ion. Theoretical considerations are given for selecting a suitable absorbing co-ion to achieve a high sensitivity of detection. The role of electromigration dispersion is illustrated by experiments and the effects of the differences in the effective mobilities of sample ions and that of the absorbing co-ion are discussed. The highest sensitivity can be achieved for sample ions having an effective mobility close to the mobility of the absorbing co-ion. In such a case, the concentration of the sample component in its migrating zone can be high while electromigration dispersion is still negligible. The useful dynamic range of the detection is then limited by the linearity and noise of the detector, the former parameter being given mostly by the shape of the on-column detection cell. The best sensitivities can be obtained in low-concentration background electrolytes containing a co-ion with high absorption at a given detection wavelength. It is shown that indirect photometric detection can be useful for detecting substances that have no optical absorption in the UV and/or visible region, provided that the composition of the background electrolyte is selected correctly.

Use of charged and neutral cyclodextrins in capillary zone electrophoresis: enantiomeric resolution of some 2-hydroxy acids

Abstract Enantiomers of racemic 2-hydroxy acids, namely 2-phenyllactic, 3-phenyllactic, mandelic, m-hydroxymandefic, p-hydroxymandelic and 3,4-hydroxymandelic acid, were resolved by capillary zone electrophoresis. The separation was achieved by using the background electrolyte with addition of cyclodextrins. The effects of the type of cyclodextrin, the pH of the electrolyte and the shape of analyte compounds on the migration time and resolution were studied. Good resolution was obtained with background electrolytes in the pH range 5–7, supplemented with 2-hydroxypropyl, 6A-methylamino- and 6A 6D-dimethylamino- β-cyclodextrin.

Contemporary sample stacking in CE

Sample stacking techniques remain an important tool for enhancement of the selectivity and sensitivity of analyses in contemporary CZE. This contribution reviews new knowledge on this topic published since 2006. It is organized according to the operational principles used, which include concentration adjustment, application of a pH step, MEKC and sweeping, and transient ITP. Techniques combining several of these principles and comparative studies are also included.

Prospects of dissolved albumin as a chiral selector in capillary zone electrophoresis

Abstract It has been shown using as examples amino acids, mono- and dicarboxylic acids that the enantioselectivity of dissolved albumin may be employed with advantage in capillary zone electrophoresis (CZE) separations. Slow changes in the enantioselectivity of albumin dissolved in weakly alkaline aqueous solutions can be avoided by a short mild heating of the solution. The efficiency of a separation system for a given solute in CZE is at least one order of magnitude higher than that in LC based on bonded albumin. The good chiral separations obtained here and the high efficiency of CZE separation systems confirm that CZE separations based on dissolved albumin as a chiral selector can compete with analogous LC separations. Perfect qualitative agreement of both enantioselective properties of albumin and factors affecting its enantioselectivity in CZE and LC proves that the knowledge obtained in LC with bonded albumin may be transferred to CZE separation systems.

Options in electrolyte systems for on-line combined capillary isotachophoresis and capillary zone electrophoresis

Abstract A theoretical description of the electrolyte systems that can be used in the on-line combination of isotachophoresis and zone electrophoresis is given. A classification of these systems is presented, based on the type of electrolyte used for the zone electrophoretic separation step. It is shown that transient sample stacking effects always persist from the isotachophoretic step to the beginning of the zone electrophoretic step and that they may negatively influence the zone electrophoretic separation and detection of the sample components. A mathematical description of these effects is given that allows the calculation of their magnitude and consequently the selection of operating conditions such that the stacking is decreased to an acceptable extent. In order to verify the reliability of the theoretical model, a modified PC simulation pack was prepared and used for investigating the behaviour of some model systems.

Electrically controlled electrofocusing of ampholytes between two zones of modified electrolyte with two different values of pH

Abstract A method is described for the separation and concentration of ampholytes from their mixtures with other ionic species. In a quadrupole electromigration column, two zones of different pHs are created by using controlled flows of the solvolytic ions (H + and OH − ) from appropriate electrode chambers at opposite sides of the column. Thus, a time-variable interface is created between two zones with a sharp change of pH. The position of the interface in the column, the direction and velocity of its movement and the difference in pH across the interface-pH gap can be adjusted by electric currents. This arrested interface is reasonably stable with time and has the following separation properties: ampholytes with p I values between the pHs of both zones are focused into a zone at interface; and other types of ampholytes and other weak or strong ions are not trapped at the interface. The basic properties of the above system are described and experiments showing the effects of the type of sample (ampholyte, weak ion), time, the concentration of primary electrolyte and the additives changing the viscosity, solubility or p I of ampholyte are given. The method proposed offers the following advantages: the ampholytes in the sample may be concentrated several hundred-fold; the focused zones have sharp boundaries (zones 0.1 mm in length were prepared) and high concentrations of the trapped species; the zone of a trapped ampholyte contains the ampholyte proper and simple ions of primary electrolyte (KCI) only; and the zones can be shifted to any selected position in the column (potentially to the location of a detector cell or a collection device).

Use of complex formation equilibria in the analytical isotachophoresis of strong electrolyte ions : Separation of halides and sulphates

Abstract The possibility of effecting isotachophoretic separations by means of complex formation equilibria between a counter ion and the components being separated was investigated. This procedure was shown to be of potential significance mainly for the separation of ionic species of strong electrolytes, and the separation of sulphates and halides was examined. The use of Cd(II) as a counter ion and NO3− as a leading ion is suggested. The migration of sulphate and nitrate zones is considered theoretically, and data calculated for actual operating conditions are verified experimentally.

Contemporary sample stacking in analytical electrophoresis: CE and CEC

Sample stacking is a term denoting a multifarious class of methods and their names that are used daily in CE for online concentration of diluted samples to enhance separation efficiency and sensitivity of analyses. The essence of these methods is that analytes present at low concentrations in a large injected sample zone are concentrated into a short and sharp zone (stack) in the separation capillary. Then the stacked analytes are separated and detected. Regardless of the diversity of the stacking electromigration methods, one can distinguish four main principles that form the bases of nearly all of them: (i) Kohlrausch adjustment of concentrations, (ii) pH step, (iii) micellar methods, and (iv) transient ITP. This contribution is a continuation of our previous reviews on the topic and brings an overview of papers published during 2010–2012 and relevant to the mentioned principles (except the last one which is covered by another review in this issue).

Selection of the background electrolyte composition with respect to electromigration dispersion and detection of weakly absorbing substances in capillary zone electrophoresis

Abstract A characteristic feature of the migration of sample ions in zone electrophoresis is the formation of either fronting or tailing zones. This so-called electromigration dispersion can generally be suppressed by keeping the sample concentration more than two orders of magnitude below the concentration of the background electrolyte (BGE). In capillary zone electrophoresis the low sample concentration decreases the reliability of on-column UV absorbance detection, especially when substances having low molar absorption coefficients are to be detected. By the proper selection of the electrophoretic mobility of the background electrolyte co-ion, both electromigration dispersion and detection can be optimized. The migration behaviour of phenyllactic acid and four phenyl derivatives of acetic acid was studied in buffered electrolytes with different mobilities and concentrations of the BGE co-ions. For a better understanding of zone broadening, a simplified model of the electromigration together with some model calculations is presented.

Instrumentation for high-speed isotachophoresis

Abstract The design and operating parameters of the individual elements of an isotachophoregraph were examined from the viewpoint of rapid quantitative analysis. An arrangement comprising an inlet port, separation column and detection cell was constructed, in which dead spaces were eliminated and the capillary and detection cell were efficiently cooled. This arrangement permits the time of isotachophoretic analysis from the instant of sample introduction to be shortened to 3-5 min, the development of the isotachophoregram proper taking 20-40 sec. The basis of the arrangement is a monolithic block of Perspex that comprises both electrode compartments, control stopcocks of the sampling device and the capillary. The latter is formed by a flat groove in the block, closed with a PTFE foil overlapping the groove and compressed on to the block with a thermostatted metallic plate. The detection cell is formed by part of the capillary and consists of two platinum contacts that extend into the groove in the monolithic block. The detector functions by sensing the electric gradient of the migrating zone. Recording was effected by means of a home-made voltmeter of high input resistance, with the detector input insulated electrically from the output of the recorder. The high-voltage source employed was also home made and yielded a stabilized current of up to 400 μA at 16 kV.