Andrea Šlampová

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.

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).

Electric field-enhanced transport across phase boundaries and membranes and its potential use in sample pretreatment for bioanalysis.

Separation techniques, such as electrodialysis, electroextraction, electro‐membrane extraction and extraction across phase interfaces, are reviewed and discussed as methods for sample cleanup and preconcentration. This survey clearly shows that electromigration of ionic species across phase interfaces, especially across supported liquid membranes, may be very selective and is strongly dependent on the chemical composition of these interfaces. Thus, electric field‐enhanced transport across chemically tailored liquid membranes may open new perspectives in preparative analytical chemistry. This review offers comprehensive survey of related literature and discussion of the topic, which may stimulate interest of experts and practitioners in bioanalysis.

Electromembrane extraction using stabilized constant d.c. electric current--a simple tool for improvement of extraction performance.

This contribution presents an experimental approach for improvement of analytical performance of electromembrane extraction (EME), which is based on the use of stabilized constant d.c. electric current. Extractions were performed using a high voltage power supply, which provided stabilized constant d.c. current down to 1μA and facilitated current-controlled transfer of ions of interest from a donor solution through a supported liquid membrane (SLM) into an acceptor solution. Repeatability of the extraction process has significantly improved for EME at constant electric current compared to EME at constant voltage. The improved repeatability of the extraction process was demonstrated on EME-capillary electrophoresis (EME-CE) analyses of selected basic drugs and amino acids in standard solutions and in human urine and serum samples. RSD values of peak areas of the analytes for EME-CE analyses were about two-fold better for EME at constant electric current (2.8-8.9%) compared to EME at constant voltage (3.6-17.8%). Other analytical parameters of the EME-CE methods, such as limits of detection, linear ranges and correlation coefficients were not statistically different for the two EME modes. Moreover, EME at constant electric current did not suffer from SLM instabilities frequently observed for EME at constant voltage.

Recent progress of sample stacking in capillary electrophoresis (2014-2016).

The term “sample stacking” comprises a relatively broad spectrum of techniques that already form an almost inherent part of the methodology of CZE. Their principles are different but the effect is the same: concentration of a diluted analyte into a narrow zone and considerable increase of the method sensitivity. This review brings a survey of papers on electrophoretic sample stacking published approximately since the second quarter of 2014 till the first quarter of 2016. It is organized according to the principles of the stacking methods and includes chapters aimed at the concentration adjustment principle (Kohlrausch stacking), techniques based on pH changes, micellar methods, and other stacking techniques. Not reviewed are papers on transient ITP that are covered by another review in this issue.

Quantitative aspects of electrolysis in electromembrane extractions of acidic and basic analytes

Electrolysis is omnipresent in all electrochemical processes including electromembrane extraction (EME). The effects of electrolysis on quantitative aspects of EME were comprehensively evaluated for a set of acidic (substituted phenols) and basic (basic drugs) analytes. EMEs were carried out across supported liquid membranes formed by 1-ethyl-2-nitrobenzene at standard EME conditions, i.e., acidic analytes were extracted from alkaline into alkaline solutions and basic analytes were extracted from acidic into acidic solutions. Electric potential applied across the EME systems was 50 V and extraction recoveries of analytes as well as pH values of donor and acceptor solutions were determined after each EME. It has been proven that electrolysis plays a more significant role than has ever been thought before in EME. Electrolytically produced H(+) and OH(-) ions had a significant effect on pH values of acceptor solutions and variations of up to 8.5 pH units were obtained at standard EME conditions. pH values of donor solutions were affected only negligibly due to their significantly higher volumes. The observed variations in pH values of acceptor solutions had fatal consequences on quantitative EME results of weak and medium strong acidic/basic analytes. A direct relation was observed between the decrease in extraction recoveries of the analytes, their pKa values and the acceptor solution pH values. Acceptor solutions consisting of high concentrations of weak bases or acids were thus proposed as suitable EME operational solutions since they efficiently eliminated the electrolytically induced pH variations, offered stable EME performances and were easily compatible with subsequent analytical methods.

Fine-tuning of electromembrane extraction selectivity using 18-crown-6 ethers as supported liquid membrane modifiers

Selectivity of electromembrane extractions (EMEs) was fine‐tuned by modifications of supported liquid membrane (SLM) composition using additions of various 18‐crown‐6 ethers into 1‐ethyl‐2‐nitrobenzene. Gradually increased transfer of K+, the cation that perfectly fits the cavity of 18‐crown‐6 ethers, was observed for EMEs across SLMs modified with increasing concentrations of 18‐crown‐6 ethers. A SLM containing 1% w/v of dibenzo‐18‐crown‐6 in 1‐ethyl‐2‐nitrobenzene exhibited excellent selectivity for EMEs of K+. The established host–guest interactions between crown ether cavities in the SLM and potassium ions in donor solution ensured their almost exhaustive transfer into acceptor solution (extraction recovery ∼92%) within 30 min of EME at 50 V. Other inorganic cations were not transferred across the SLM (Ca2+ and Mg2+) or were transferred negligibly (NH4+, Na+; extraction recovery < 2%) and had only subtle effect on EMEs of K+. The high selectivity of the tailor‐made SLM holds a great promise for future applications in EMEs since the range of similar selective modifiers is very broad and may be applied in various fields of analytical chemistry.

Standard systems for measurement of pKs and ionic mobilities: 1. Univalent weak acids

Determination of pK values of weak bases and acids by CZE has already attracted big attention in current practice and proved to offer the advantage of being applicable for mixtures of analytes. The method is based on the measurement of mobility curves plotting the effective mobility vs. the pH of the background electrolyte, and following computer-assisted regression involving corrections for ionic strength and temperature. To cover the necessary range of pH for a given case, both buffering weak acids and bases are used in one set of measurements, which requires implementing computations of individual ionic strength corrections for each pH value. It is also well known that some components of frequently used background electrolytes may interact with the analytes measured, on forming associates or complexes. This obviously deteriorates the reliability of the resulting data. This contribution brings a rational approach to this problem and establishes a standard system of anionic buffers for measurements of pKs and mobilities of weak acids, where the only counter cation present (besides H(+)) is Na(+). In this way, the risk of formation of complexes or associates of analytes with counter ions is strongly reduced. Moreover, the standard system of anionic buffers is selected in such a way that it provides, for an entire set of measurements, constant and accurately known ionic strength and the operational conditions are selected so that they provide constant Joule heating. Due to these precautions only one correction for ionic strength and temperature is needed for the obtained set of experimental data. This considerably facilitates their evaluation and regression analysis as the corrections need not be implemented in the computation software. The reliability and the advantages of the proposed system are well documented by experiments, where the known problematic group of phenol derivatives was measured with high accuracy and without any notice of anomalous behaviour.

Additional considerations on electrolysis in electromembrane extraction.

Optimized acceptor solutions, which eliminate electrolytically induced variations in their pH values, have been shown to improve electromembrane extraction (EME) performance. Acceptor solutions containing 500 mM formic acid (pH 1.97) ensured stable EME process for three basic drugs extracted at 50 V across 1-ethyl-2-nitrobenzene and constant extraction recoveries (66-89%) were achieved for 40-80 min EMEs. Back-extraction of analytes into donor solutions has been eliminated by application of optimized acceptor solutions, moreover, saturation of acceptor solutions with analytes had no additional effect on their back-extraction; the presence of up to 300-fold excess of analytes in optimized acceptor solutions led to slightly reduced but stable enrichment of analytes over the entire extraction time. Stable EME performance has been also achieved for extractions into 100mM HCl, note however, that seriously compromised performance of subsequent capillary electrophoretic analyses has been observed due to high conductivities of resulting acceptor solutions. Electrolytically produced H(+) and OH(-) ions have mostly remained in corresponding operating solutions, have determined their final pH values and have not been subjects of EME transfers across selective phase interfaces as was experimentally verified by pH measurements of anolytes and catholytes at various EME times.

Regular properties of simple electrophoretic BGEs with multiprotic weak acids: Discovery of complex hybrid system boundaries

In electrophoresis, the introduction of a sample into the BGE creates two new zone boundaries, the front and rear boundary of the sample zone which is sandwiched between the BGE. During an electrophoresis run, these boundaries move and split into other new boundaries demarcating the zones of analytes. Besides the analyte zones migrating out of the original sample location, system zones may be also formed. Such zones are formed by the BGE components and differ from the BGE only in their concentrations. The front and rear boundaries of such system zones evolve during electromigration and may show sharp (S), dispersed (D), and/or hybrid character. This contribution brings the results of theoretical and experimental investigation of system properties of very simple BGEs formed by one weak acid and one weak or strong base provided that the acid is polyprotic. It is shown that system boundaries of the hybrid type occur even in these simple systems represented, e.g., by the very common phosphate buffer. Theory reveals that a phosphate buffer system (formed by phosphoric acid and a strong cation) may exhibit a very unusual complex shape of the electromigration dispersion velocity curve showing three turns. This leads to the formation of complex hybrid boundaries having D–S–D, S–D–S, or even S–D–S–D or D–S–D–S concentration profile types that have not been reported so far. The velocity diagram method allows theoretical analysis of such complicated profiles and the results are in good accordance with both computer simulation and experiments.