Ludmila Křivánková

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.

On-line isotachophoresis–capillary zone electrophoresis versus sample self stacking capillary zone electrophoresis: Analysis of hippurate in serum

Abstract This paper is aimed at the problem of sensitive analysis of a microcomponent in a sample with a bulk macrocomponent and complex matrix by capillary electrophoresis. In such samples, the simple way to increase the sensitivity by increasing the injected sample volume is counterworked by the stacking–destacking processes due to the presence of transient isotachophoresis (ITP) and a certain maximum sample load exists which gives useful results. The important analytical task of the analysis of hippuric acid (benzoyl glycine) was selected as the model problem. The aim was to reach the highest possible sensitivity in the analysis of hippuric acid in serum of healthy persons where hippuric acid represented a microcomponent and serum was a representative of a sample matrix containing varying amounts of macrocomponents. In order to find the maximum allowable sample load (volume) that enabled maximum sensitivity, various stacking techniques in two types of instrumentation for capillary zone electrophoresis (CZE) and a commercially available instrumentation for the on-line ITP–CZE combination were compared. The systems for CZE differed in detectors and in using an open or closed capillary of different diameter and material, which resulted in different sensitivity of the analyses of model samples. In analyses of serum samples, however, comparable limits of detection were obtained using both a single ITP and CZE with the open or closed capillary (6·10−5 M hippurate). A limit of detection two-orders of magnitude lower (7·10−7 M hippurate) was reached in the instrumentation for the ITP–CZE combination even though the parameters of the capillary and the detector that were available for the CZE step were substantially worse than those of the open CZE system that was used.

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.

Some practical aspects of utilizing the on-line combination of isotachophoresis and capillary zone electrophoresis

The on-line combination of isotachophoresis (ITP) and zone electrophoresis is a very effective tool for increasing the separation capability and sensitivity of capillary zone electrophoresis (CZE). Its most effective version is performed in column coupling instrumentation and is characterized by isotachophoresis in the first capillary serving as the efficient preseparation and concentration stage followed by on-line transfer of the sample cut into the second capillary where analytical zone electrophoresis proceeds as the second stage. The on-line transfer of the sample from the first capillary into the second is always accompanied by the segments of some additional amounts of the leader and/or terminator from the ITP step. This results in transient survival of isotachophoretic migration during the second stage. Hence the separation process during the second stage can be characterized as the destacking of analytes followed by zone electrophoretic separation. In this paper, both a theoretical and an experimental study is presented showing that destacking of analytes is a selective process, which affects strongly the most important final analytical parameters of the detected zones, namely the detection time and zone variance, and thus it makes the simple use of detection data for qualitative analysis misleading. It was shown that both the detection time and variance of a zone of an analyte depend strongly on the amount of the accompanying segments of the leader/terminator transferred and on the actual type of electrolyte system selected from the generally possible types. These types are (i) L-S-L, where the leading electrolyte (L) from ITP stage serves as the background electrolyte (BGE) during CZE (S = sample), (ii) T-S-T, where the terminating electrolyte (T) from the ITP stage serves as the BGE during CZE, and (iii) BGE-S-BGE, where L, T and BGE are mutually different. Explicit equations were derived enabling one to predict migration time and zone variance for actual working conditions, and, for model systems, the theoretical data were calculated and verified experimentally. Further, it is shown that the systems T-S-T and L-S-L are user friendly and a simple standardization procedure was proposed, allowing correct qualitative evaluation of the analytical data in these systems. Finally, a theory is presented predicting the existence of anomalous variances of zones in the record of analysis in BGE-S-BGE systems and its experimental verification is given.

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.

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.

Analysis of orotic acid in human urine by on-line combination of capillary isotachophoresis and zone electrophoresis.

The techniques of the on-line combination of capillary isotachophoresis with zone electrophoresis in two coupled capillaries (ITP-CZE) and a single capillary zone electrophoresis (CZE) were used for the sensitive determination of orotic acid (OA) in human urine. The simple CZE system was successfully applied for fast and reliable analyses of urine of healthy adult volunteers (the detection limit 1.7.10(-6) M OA, the total time of analysis 6 min). However, this method failed in analyses of OA in urine of ill children due to more complex matrix of the samples. Here, the ITP preconcentration and preseparation step coupled on-line with CZE proved to serve well with an electrolyte system developed and optimized for this purpose. The maximum selectivity and resolution of OA from other sample constituents in ITP-CZE was achieved by use of an electrolyte system of very low pH 2.15 both for ITP and CZE stage. The sensitivity of detection and simplicity of OA identification were enhanced by use of an external UV scanning detector. High sensitivity of ITP-CZE combination (limit of detection 3.10(-7) M OA), low sample consumption (1 microliter), good reproducibility of migration times (inter-day RSD < 1.86%) and acceptable reproducibility of the determination of OA in urine samples (average RSD = 7.27%) make this technique suitable for routine determination of trace concentration of OA especially in urine of ill children under various pathological conditions and medication.

Determination of ethyl glucuronide in human serum by hyphenation of capillary isotachophoresis and zone electrophoresis

The determination of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in human serum by hyphenation of capillary ITP (CITP) and CZE is reported. For CITP step, 1×10−2 M hydrochloric acid adjusted with ϵ‐aminocaproic acid (EACA) to pH 4.4 was used as the leading electrolyte, and 1×10−2 M nicotinic acid with EACA, pH 4.4, was used as the terminating electrolyte (TE). All electrolytes contained 0.2% hydroxypropylcellulose to suppress electroosmosis. In CITP, EtG was separated from fast serum macrocomponents chloride, phosphate, lactate, and acetate. Zones of microcomponents including EtG that migrated between acetate and nicotinate were forwarded to the second capillary filled with a BGE identical with the TE. Conductivity detection was used in the CITP step. Sensitive detection in the CZE step was performed using indirect spectrophotometric detection at 254 nm. The assay is based on a 1:5 dilution of serum with deionized water and has a concentration LOD for EtG in diluted sample of 9.8×10−9 M. The method was used for the determination of EtG in sera of volunteers consuming alcohol.

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.