Stacking in capillary zone electrophoresis

Due to the short light path of the capillaries, the CE detection limit based on concentration, is far less than that of HPLC and not sufficient for many practical applications. Several methods, based on different electrophoretic maneuvers, can concentrate the sample (stack) easily on the capillary before the separation step of capillary zone electrophoresis (CZE). These methods incorporate different types of discontinuous buffers as the means for invoking different velocities to the same analyte molecules to produce a sharpening of the band (stacking). In CZE, these buffers can be often very simple such as sample dilution or adding to the sample a high concentration of a fast mobility ion. However, in other applications these buffers can be as complicated as those required for isotachophoresis. Stacking can often yield a concentration factor of 5-30-fold, which can improve greatly in CZE the detection limits bringing them very close to those of HPLC. Different methods of stacking, the importance of discontinuous buffers and the different mechanism for concentration on the capillary are reviewed here. As there is a need for more practical applications, there will be more methods devised for stacking in CZE.     

Selected applications of cyclodextrin selectors in capillary electrophoresis.

Through the use of alpha-, beta-, gamma- and heptakis(2,6-di-O-methyl)-beta-cyclodextrin as stereospecific selectors or electrolyte modifiers, both in capillary zone electrophoresis and isotachophoresis, selected model isomeric compounds (including optical isomers) were resolved. Soluble alkylhydroxyalkylcellulose derivatives were further added to the cyclodextrin-modified background electrolytes under study. Their presence was found to be essential, as demonstrated by improvements in both enantioselectivity and separation efficiency. The results obtained in both electrophoretic modes, under optimized conditions, are compared and discussed.     

Computer simulation of transient states in capillary zone electrophoresis and isotachophoresis.

Transient states in the evolution of electrophoretic systems comprising aqueous solutions of weak monovalent acids and bases are simulated. The mathematical model is based on the system of nonstationary partial differential equations, expressing the mass and charge conservation laws while assuming local chemical equilibrium. It was implemented using a high resolution finite-difference algorithm, which correctly predicted the behavior of the concentration, pH and conductivity fields at low computational expense. Both the regular and the irregular modes of separation in capillary zone electrophoresis and isotachophoresis are considered. It is shown that the results of separation, particularly zone order, strongly depend on pH distribution. Simulation data as well as simple analytical assessments may help to predict and correctly interpret the experimental results.     

High speed electrophoresis simulation for optimization of continuous flow electrophoresis and high performance capillary techniques: Part I. Computer model.

A computer program for high-speed simulation and optimization of electrophoretic processes has been developed for carrier-free systems of all kinds. The calculations are based on the one-dimensional dynamic (transient-state) model. The three-dimensional geometry of the simulation space can be chosen deliberately. With a highly efficient transport algorithm instead of complicated integration schemes for the transport equations, the calculation time can be effectively spent on various important parameters such as ionic strength, temperature, Joule heat, activity coefficients and concentration changes due to membranes. The parameter set of any carrier free electrophoretic method (i.e., continuous-flow electrophoresis, capillary isotachophoresis and high performance capillary zone electrophoresis) can be imported directly into the computer program by means of a graphic user interface. The program performs overnight-simulation of any electrophoretic system containing up to 15 components.     

Evaluation of multidimensional capillary electrophoretic methodologies for determination of amino bisphosphonate pharmaceuticals

Alendronate and pamidronate are amino bisphosphonate analogues of pyrophosphate used for the treatment of a variety of bone diseases. Analysis of these compounds is problematic due to their polar ionic nature, lack of a suitable chromophore and chelation properties and current analytical approaches involve extensive sample preparation and derivatization procedures. The potential of multidimensional capillary electrophoretic methodological approaches, which eliminate sample preparation have been evaluated for the analysis of these compounds both in aqueous and urinary matrices. Capillary isotachophoresis (cITP) was employed as a pre-separation and on-line sample concentration step prior to analytical determination using either cITP or capillary zone electrophoresis (CZE) with conductivity detection. Single cITP, cITP-cITP and cITP-CZE approaches were partially validated with respect to repeatability, recovery and linearity of response for both compounds. The increases in sensitivity achievable through increasing injection volume from 30 to 300muL may render such strategies appropriate for determination of these agents at biologically relevant concentrations with minimal sample work-up.     

Application of capillary isotachophoresis and capillary zone electrophoresis to the determination of inorganic ions in food and feed samples

The purpose of this review is to summarise critically the possibilities of capillary isotachophoresis and capillary zone electrophoresis for the determination of inorganic ions in food and feed samples. This article covers papers published since 1977.     

Application of capillary isotachophoresis for fruit juice authentication

A method using capillary isotachophoresis (ITP) was developed and applied for the determination of the anionic profile of orange juices with the aim to obtain some useful information on the authenticity or adulteration of imported and native beverage products. An EA 100 electrophoretic analyser (Villa-LABECO, Slovak Republic) was used for capillary isotachophoretic determination of anions in tested samples. More systems of leading and terminating electrolytes were used. Detection conductivity and UV detection at 254 nm were used. Sample injection volume was 30 microl. These systems allow one to determinate inorganic anions, organic acids and some additives--adulterants in anionic forms in orange juices. By capillary isotachophoretic determination the lengths or areas of characteristic zones were established and compared to authentic orange juices of different species and origin and with RSK reference values (Code of Practice). Special emphasis was placed on D-isocitric acid ITP determination as a reliable fruit juice authentication marker. The presented multicomponent analysis of orange juice authenticity according to ITP anionic profiles obtained by capillary isotachophoresis presents an alternative information source necessary for deciding about authenticity of the products.     

Stacking and discontinuous buffers in capillary zone electrophoresis

Discontinuous buffers for capillary zone electrophoresis (CZE) can be used under less rigid conditions compared to those for isotachophoresis for stacking. They can be prepared simply by modifying the sample itself, either by addition of small inorganic ions, low conductivity diluents, or both, and also by adjusting its pH, meanwhile injecting a large volume on the capillary. Zwitterionic and organic-based buffers such as triethanolamine and tris(hydroxymethyl)aminomethane (Tris) are well suited for stacking due to their low conductivity, provided the buffer is discontinuous as demonstrated here. A simple mechanism based on discontinuous buffers is described to explain many of the observed stacking types in CZE, pointing out the many similarities to transient isotachophoresis.     

Analytical potential of enzyme-coated capillary reactors in capillary zone electrophoresis

Enzymes immobilized on the inner surface of an electrophoretic capillary were used to increase sensitivity and resolution in capillary zone electrophoresis (CZE). Sensitivity is enhanced by inserting a piece of capillary containing the immobilized enzyme into the main capillary, located before the detector, in order to transform the analyte into a product with a higher absorptivity. This approach was used to determine ethanol. In order to improve resolution, capillary pieces containing immobilized enzymes were inserted at various strategic positions along the electrophoretic capillary. On reaching the enzyme, the analyte was converted into a product with a high electrophoretic mobility, the migration time for which was a function of the position of the enzyme reactor. This approach was applied to the separation and determination of acetaldehyde and pyruvate. Finally, the proposed method was validated with the determination of ethanol, acetaldehyde, and pyruvate in beer and wine samples.     

Analytical capillary isotachophoresis: a routine technique for the analysis of lipoproteins and lipoprotein subfractions in whole serum

A capillary isotachophoretic separation technique was developed for lipoproteins in native serum which, compared with previous electrophoretic techniques, has negligible molecular sieve effects, does not need gel casting, is suitable for whole serum and has a high discriminative power for lipoprotein subfractions. The technique is based on pre-staining whole serum lipoproteins for 30 min at 4 degrees C before separation of 0.5 microliter of the sample in a free-flow capillary system (0.5 mm I.D.) with discontinuous buffer system. In normolipidaemic sera, high-density (HDL) and low-density lipoproteins (VLDL) are separated into two major subpopulations according to their net electric mobility. The identification of these fractions was confirmed by substitution with ultracentrifugally isolated lipoproteins and by their complete absence from Tangier and abetalipoproteinaemic serum. Triglyceride-rich very low-density lipoproteins (VLDL) revealed a defined zone between the HDL and LDL subpopulations. Our preliminary results indicate that the separation of human whole serum lipoproteins by capillary isotachophoresis is a promising method for the determination of lipoprotein subfractions.     

Recent progress in chiral separation principles in capillary electrophoresis

This review summarizes recent developments in the field of chiral separations by electromigration techniques including capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), isotachophoresis (ITP), electrokinetic chromatography (EKC), and capillary electrochromatography (CEC). This overview focuses on the development of new chiral selectors and the introduction of new techniques rather than applications of already established selectors and methods. The mechanisms of the different chiral separation principles are discussed.     

Recent progress of sample stacking in capillary electrophoresis (2016–2018)

Electrophoretic sample stacking comprises a group of capillary electrophoretic techniques where trace analytes from the sample are concentrated into a short zone (stack). This paper is a continuation of our previous reviews on the topic and brings a survey of more than 120 papers published approximately since the second quarter of 2016 till the first quarter of 2018. It is organized according to the particular stacking principles and includes chapters on concentration adjustment (Kohlrausch) stacking, on stacking techniques based on pH changes, on stacking in electrokinetic chromatography and on other stacking techniques. Where available, explicit information is given about the procedure, electrolyte(s) used, detector employed and sensitivity reached. Not reviewed are papers on transient isotachophoresis which are covered by another review in this issue.     

Capillary zone electrophoresis separations of enantiomers present in complex ionic matrices with on-line isotachophoretic sample pretreatment.

Analytical capabilities of capillary zone electrophoresis (CZE) with on-line coupled capillary isotachophoresis (ITP) sample pretreatment in the column-coupling capillary electrophoresis equipment to separate and determine enantiomers present in multicomponent ionic matrices were studied. Tryptophan was used as a model analyte in the ITP-capillary zone electrophoresis experiments performed in this context while a 90-component model mixture of UV-light absorbing organic anions and urine served as multicomponent sample matrices. Various working modes in which the on-line coupled capillary isotachophoresis-capillary zone electrophoresis combination in the column-coupling separation system can operate were employed in the anionic regime of the separation with direct injections of the samples. Advantages and limitations of these working modes in the separations of enantiomers present in model and urine matrices were assessed. Experiments with model mixtures of tryptophan enantiomers revealed that the two were resolved in the capillary zone electrophoresis stage with the aid of alpha-cyclodextrin also when their concentration ratio in the sample was 1:200 while the concentration of L(-)-tryptophan was 25 nmol/l. The limits of detection for the enantiomers were at approximately 10 nmol/l (approximately 1.5 ng/ml) concentrations for a 220 nm detection wavelength of the UV detector employed in the capillary zone electrophoresis stage and for a 30 microliters sample load. A high sample load capacity of the on-line coupled capillary isotachophoresis stage was effective in separating the samples corresponding to 3-6 microliters volumes of undiluted urine. The results from the runs with urine samples showed that only the capillary isotachophoresis-capillary zone electrophoresis combination with a post-column on-line coupled capillary isotachophoresis sample clean-up (responsible for a removal of more than 99% of the sample anionic constituents migrating in the on-line coupled capillary isotachophoresis stack and detectable in the capillary zone electrophoresis stage) provided a universal alternative for the detection and quantitation of the model analyte (L(-)-tryptophan).     

Comparison of hydrodynamically closed two‐dimensional capillary electrophoresis coupled with ultraviolet detection and hydrodynamically open capillary electrophoresis hyphenated with mass spectrometry in the bioanalysis of varenicline

Two capillary electrophoresis methods for monitoring renally excreted varenicline, a highly effective drug prescribed for smoking cessation, in human urine were developed and compared. A method combining capillary electrophoresis with mass spectrometry was proposed for the fast analysis of varenicline (analysis time up to 7 min). Here, mass spectrometry was a prerequisite for achieving high sensitivity and selectivity of the analysis suitable for the quantification of a 15 ng/mL level of varenicline in un‐pretreated urine matrices. An alternative approach, two‐dimensional (column‐coupled) capillary electrophoresis with enhanced sample load capacity and ultraviolet detection, was proposed as a low‐cost alternative to capillary electrophoresis with mass spectrometry. The isotachophoresis on‐line sample treatment included simple elimination of the major matrix constituents and stacking of the sample in a large volume so that threefold lower quantitation limits could be easily achieved in comparison to the capillary electrophoresis with mass spectrometry. On the other hand, longer analysis time (ca. 4.5‐fold) and more complex electrolyte system in the coupled zone electrophoresis step (including two additives enhancing separation selectivity, i.e. isopropanol and cyclodextrin) were prerequisites for the complete separation of varenicline from the sample matrix. Anyway, both the developed methods were validated according to the Food and Drug Administration guidelines showing favorable performance parameters, suitable for their routine biomedical use.     

Analysis of enantiomers in biological matrices by charged cyclodextrin-mediated capillary zone electrophoresis in column-coupling arrangement with capillary isotachophoresis

The possibility to apply charged chiral selector as buffer additive in capillary zone electrophoresis (CZE) on-line coupled with capillary isotachophoresis (CITP) was studied. Enantioseparations and determinations of trace (ng/ml) antihistaminic drugs [pheniramine (PHM), dimethindene (DIM), dioxopromethazine (DIO)] present in samples of complex ionic matrices (urine) served as model examples. A negatively charged carboxyethyl-β-cyclodextrin (CE-β-CD) was used as a chiral selector in analytical CZE stage following upon a sample pretreatment by CITP (preconcentration of the analytes from 5 to 20-times diluted urine samples, partial sample clean up removing macroconstituents from the sample matrices). A high recognition capability of the oppositely charged CE-β-CD was demonstrated by enantioselective retardation of the drugs in presence of micro-and semi-macroconstituents migrating in CZE stage and detectable by UV detector. In this way, enantiomers of the drugs could be easily separated and determined. Due to lack of interferences between the drugs and sample-matrix constituents in presence of charged CE-β-CD, demands on both spacers in CITP step and multiple column-switching were minimized. CITP-CZE method with charged selector appeared to be a useful analytical approach for the trace enantiomers in complex ionic matrices as it combined enhanced separation selectivity and sample loadabitlity with high separation efficiency and provided favorable performance parameters including sensitivity, linearity, precision, accuracy/recovery and robustness with minimal demands on sample preparation. Analysis of urine sample taken from a patient treated by PHM, showing concentration profile of PHM enantiomers and their metabolites, illustrated potentialities of the method in clinical research.     

Determination of impurities and counterions of pharmaceuticals by capillary electromigration methods

The review presents a survey of recent applications of high‐performance capillary electromigration methods—capillary zone electrophoresis, nonaqueous capillary electrophoresis, capillary isotachophoresis, micellar electrokinetic chromatography, microemulsion electrokinetic chromatography and capillary electrochromatography—for the determination of impurities of pharmaceuticals, including chiral impurities, for the period 2007–2013. In addition, due to the missing evaluation of the determination of counterions of pharmaceuticals by capillary electromigration methods in the last 20 years, the publications dealing with this topic since 1995 are included in this review. General aspects of both these types of applications of capillary electromigration methods in pharmaceutical analysis are discussed, and detailed experimental conditions used for determination of various chemical impurities and counterions of many particular drugs are described.     

Capillary electrophoretic determination of S-carboxymethyl-L-cysteine and its major metabolites in human urine: feasibility investigation using on-column detection of non-derivatized solutes in capillaries with minimal electroosmosis

The capillary electrophoretic analysis of S-carboxymethyl-L-cysteine and some of its metabolites in human urine is reported using (i) on-column detection of underivatized solutes, (ii) minimal sample pretreatment and (iii) capillary columns with minimized electroosmosis. Experimental results obtained with two apparatus, the HPE-100 (Bio-Rad), featuring coated fused silica capillaries of 25 microns i.d., and with the Tachophor 2127 (LKB), having Teflon capillaries of 500 microns i.d. are discussed. Drug concentrations down to 0.2 mg/mL on capillary isotachophoresis and 0.03 mg/mL on capillary zone electrophoresis can be monitored with these instruments.     

Determination of adenosine deaminase activity in human erythrocytes by on-column capillary isotachophoresis-capillary zone electrophoresis in the presence of electroosmotic flow

Transient capillary isotachophoresis (CITP)-capillary zone electrophoresis (CZE) in presence of electroosmotic flow (EOF) was utilized for the measurement of adenosine deaminase activity in human erythrocytes. Phosphates, dominant anions of the sample matrix, were used as leading ions for transient isotachophoresis, and borates (0.3 M, pH 10) were used as terminating ions and background electrolyte for CZE. Final experimental conditions made it possible to inject 70% of the total capillary volume (1.45 microL) with the sample. Enzymatic conversion products (inosine and hypoxanthine), present in the sample in the low-micromolar range, were determined using optimized conditions. The limit of detection was 28 nM using UV detection at 202 nm. The presented data shows that CITP-CZE can be performed in uncoated capillaries in the presence of strong EOF.     

Applications of capillary electrophoresis for industrial analysis

Capillary electrophoresis techniques, particularly isotachophoresis and zone electrophoresis, are useful for the determination of various analytes in a wide range of sample matrices. This paper reports applications of capillary electrophoresis developed for detergent, food additive, herbicide, animal nutrition and biotechnology samples. Major advantages of capillary electrophoresis include versatility and speed of method development. Options for small ion analysis using modern, fused silica capillary instruments are discussed.