Pavla Pantůčková

Isotachophoresis in zone electrophoresis

Abstract Conditions for existence of transient isotachophoresis (ITP) in zone electrophoresis are quite common. Transient ITP can either be induced by the composition of the sample or by the composition of the electrolyte system or result from the first step during capillary ITP–capillary zone electrophoresis (CZE) combination. This paper brings a comprehensive analysis of the problem and description of the effects of transient ITP on the migration time, separation efficiency and the detection sensitivity of the CZE analysis. Theoretical considerations are accompanied by model experimental examples. It is shown that in cases where transient ITP can be controlled, the effects of transient ITP can be employed for improvement of the performance of the analysis. Further, it is shown that the combination of capillary ITP–CZE is by far superior. It enables one to inject large sample volumes, to reach efficient sample clean-up, and to separate and to detect trace analytes under optimum conditions.

Sample self‐stacking in capillary zone electrophoresis: Behavior of samples containing multiple major coionic components*

It is a frequent phenomenon in practice that a sample contains bulk levels of more than one coionic component that affect the stacking behavior of minor analytes and in this way also the sensitivity of the method. Here, attention is paid to stacking resulting from the presence of a macrocomponent of leading type that is deteriorated by the presence of another macrocomponent of like charge in the sample. Based on the isotachophoretic model of migration in the initial period of separation, a theoretical approach was elaborated both for strong and weak electrolytes which describes the separation process and finds the conditions that define whether transient isotachophoretic stacking of the analyte takes place or not. It is shown that the crucial parameter is the ratio of the concentrations of macrocomponents migrating in front and behind the analyte of interest. The destacking effect can also be expected when the coion of the background electrolyte is present in the sample. Rules how to cope with effects of destackers present in the sample are given. Theoretical considerations are illustrated by computer simulations and verified experimentally. Examples of antagonistic effects of macrocomponents are demonstrated for model serum samples.

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).

Recent advances in CE-MS: Synergy of wet chemistry and instrumentation innovations.

CE with MS detection is a hyphenated technique which greatly improves the ability of CE to deal with real samples, especially with those coming from biology and medicine, where the target analytes are present as trace amounts in very complex matrices. CE‐MS is now almost a routine technique performed on commercially available instruments. It faces currently a tremendous development of the technique itself as well as of its wide application area. Great interest in CE‐MS is reflected in the scientific literature by many original research articles and also by numerous reviews. The review presented here has a general scope and belongs to a series of regularly published reviews on the topic. It covers the literature from the last 2 years, since January 2008 till June 2010. It brings a critical selection of related literature sorted into groups reflecting the main topics of actual scientific interest: (i) innovations in CE‐ESI‐MS, (ii) use of alternative interfaces, and (iii) ways to enhance sensitivity. Special attention is paid to novel electrolyte systems amenable to CE‐MS including nonvolatile BGEs, to advanced CE separation principles such as MEKC, MEEKC, chiral CE, and to the use of preconcentration techniques.

Electrolyte systems for on-line CE-MS: detection requirements and separation possibilities.

In contemporary analytical practice, the on‐line CE–MS technique has established as a powerful separation and identification analytical tool. Its major instrumental aspects can be considered as solved and its routine use with specific applications to be implemented is the typical task at present. In that context, the chemical (wet, electrophoretic) part of the technique becomes very important as here the separation battle of the overall success of the developed method is fought. The review brings an overview of the electrolyte systems used in the CE part of the CE–MS technique together with a detailed insight into their pros and cons from the electrophoretists viewpoint. Advanced electrophoretic approaches including sample stacking, ITP and use of non‐aqueous solvents are also discussed. An overview of the electrolyte systems used in over 200 selected applications is given, sorted according to the analytes of interest.

Capillary zone electrophoresis in phosphate buffer - known or unknown?

It has been shown recently that the analysis records in capillary electrophoresis may involve regions with extremely strong electromigration dispersion of peaks. Such a fundamental effect is due to the existence of more centers of symmetry in a given electrolyte system. This paper shows that even such a simple and frequently used electrolyte system as phosphate buffer may exhibit more than one center of symmetry. By using the peak shape diagram approach we have revealed that neutral and alkaline phosphate buffers have two centers of symmetry and one center of extreme dispersion. Model experiments confirmed this new important discovery.

Advanced electrolyte tuning and selectivity enhancement for highly sensitive analysis of cations by capillary ITP-ESI MS.

In this contribution we present an innovative way to easy, fast, and highly sensitive analyses by CE with ESI‐MS detection. The new method is designed to be applied to ESI‐compatible electrolytes (e.g. ammonium acetate) and offers advanced tuning of selectivity conditions within a wide range of analyte mobilities. We use a full capillary ITP format to provide powerful on‐line analyte stacking at the ITP boundary all the way to detection and introduce the model of extended ITP where a controlled concentration of the leading ion is added to the terminating zone. Such systems preserve all properties of an ITP system and the velocity of the stacking ITP boundary can be tuned by the composition of both the leading and terminating zone. In this way, the system properties can be controlled flexibly and the mobility window of stacked analytes can be tailored to actual needs. The presented theory and the newly defined concept of zone‐related boundary mobility allow easy assessment of system selectivity using simple diagrams. We demonstrate the model and its potential on the example of simple acidic cationic systems composed of only two substances (ammonium and acetate) including the example of thiabendazole analysis with a detection limit of 10−10 M (20 ng/L) and its determination in orange juice by direct sampling after filtration, selective stacking by a tuned extended ITP system, and ESI‐MS detection.

A simple sample pretreatment device with supported liquid membrane for direct injection of untreated body fluids and in‐line coupling to a commercial CE instrument

A simple sample pretreatment device was developed employing extractions across supported liquid membranes (SLMs) and in‐line coupling to a commercial CE instrument. The device consisted of two polypropylene conical units interspaced with a polypropylene planar SLM, which were impregnated with 1‐ethyl‐2‐nitrobenzene. The two units and the SLM were pressed against each other, donor unit was filled with 40 μL of an untreated body fluid and acceptor unit with 40 μL of DI water. The device was then placed into conventional CE vial fitted with a soft spring, which was depressed during injection into CE capillary and ensured that the SLM was not ruptured. Position of separation capillary injection end and high‐voltage electrode in the CE instrument was optimized in order to ensure efficient injection of pretreated body fluids. The device can be easily assembled/disassembled and SLMs can be replaced after each extraction thus minimizing sample carry‐over, avoiding tedious SLM regeneration, and reducing total pretreatment time and costs. The pretreatment device was examined by direct injection of human urine and serum spiked with nortriptyline, haloperidol, and loperamide. The basic drugs were diffusionaly transported across the SLM within 10 min and were injected into the separation capillary directly from the SLM surface in the acceptor unit, whereas matrix components were retained by the SLM. The in‐line SLM‐CE method showed good repeatability of peak areas (3.8–11.0%) and migration times (below 1.4%), linear relationship (r2 = 0.990–0.999), and low LODs (12–100 μg/L).

Supported liquid membrane extraction coupled in-line to commercial capillary electrophoresis for rapid determination of formate in undiluted blood samples.

A cheap, disposable sample pretreatment device with planar supported liquid membrane (SLM) was proposed, assembled and placed into an autosampler carousel of a commercial capillary electrophoresis (CE) instrument for automated pretreatment and analysis of formate in undiluted whole blood and serum samples. All analytical procedures except for filling the pretreatment device with donor and acceptor solutions, i.e., extraction across SLM, injection of the extracted sample and CE-UV determination of formate, were performed fully automatically. The pretreatment device required only μL volumes of blood sample and organic solvent per extraction and was disposed off after each extraction. Good repeatability of peak areas (≤7.7%) and migration times (≤1.5%), linear relationship (r(2)=0.998-0.999) and limits of detection (≤35μM) were achieved. The overall analytical process including blood withdrawal, filling the SLM device with respective solutions, extraction of blood sample, injection into separation capillary and CE separation of formate from other anions took less than 4min. The method was proved useful by direct determination of elevated formate concentrations in undiluted serum samples of a methanol intoxicated patient. Due to its compatibility with currently commercially available CE instrumentation, disposability of extraction devices, minimum sample handling/consumption, and short extraction/analysis times, the developed method might be attractive for rapid diagnosis of methanol poisoning in clinical and toxicological laboratories.

Sensitivity enhancement in direct coupling of supported liquid membrane extractions to capillary electrophoresis by means of transient isotachophoresis and large electrokinetic injections

Enhanced sensitivity for determination of basic drugs in body fluids was achieved by in-line coupling of extraction across supported liquid membrane (SLM) to large electrokinetic injection and transient isotachophoresis-capillary zone electrophoresis (tITP-CZE) in commercial CZE instrument. Twelve cm long tITP plug of 300mM ammonium acetate was formed in the separation capillary just before the electrokinetic injection of acceptor solution containing nortriptyline, haloperidol and loperamide extracted across the SLM. The tITP plug ensured efficient stacking and preconcentration of the injected basic drugs due to the tITP action of ammonium and the drugs were then separated by CZE using 5.2M acetic acid as background electrolyte. No interferences were observed from highly-abundant body fluid species (NaCl and human serum albumin) due to the excellent clean-up properties of SLMs and analytical sensitivity increased up to 340 times compared to SLM extractions coupled in-line to CZE with standard hydrodynamic injections. The SLM-tITP-CZE method was characterized by good repeatability (RSDs of peak areas below 7.8%), linearity over two orders of magnitude (r(2) better than 0.994) and limits of detection (defined as 3×S/N) between 3 and 45μg/L. Interfacing of SLM extractions to CZE instrumentation was achieved by low-cost, disposable micro-extraction devices, which can be routinely prepared in every analytical laboratory. These devices eliminated sample carry-over, minimized the need for manual sample handling and ensured fully automated determination (including extraction, injection, preconcentration and separation) of the three basic drugs in 20μL of untreated body fluids.