Liquid–liquid extraction procedures for sample enrichment in capillary zone electrophoresis

Abstract

This review article presents an overview of applications of liquid–liquid extraction (LLE) for analyte enrichment and clean-up of samples prior to capillary zone electrophoresis (CZE). The basic principles of LLE are discussed with special emphasis on analyte enrichment. In addition, attention is focused on the requirements for the final extract to be compatible with CZE. The paper discusses selected examples from the literature with special emphasis on detection limits in drug analysis and in environmental chemistry. Finally, the paper focus on alternative liquid-phase extraction concepts based on electroextraction, supported liquid membranes, and liquid-phase microextraction.

Introduction

In capillary zone electrophoresis (CZE), cationic and anionic analytes may be separated based on differences in their charge-to-size ratio, and subsequently measured on-column by UV detection. Compared with the traditional chromatographic techniques, CZE provides an alternative separation principle characterized by high separational efficiency, rapid separations, and by a low consumption of reagents as well as solvents. Based on these advantages, CZE has been implemented in a broad range of application areas including pharmaceuticals, proteins, peptides, agrochemicals, raw materials, water, DNA, surfactants and fine chemicals [1].

Unfortunately, CZE suffers from relatively high concentration detection limits because the sample volumes injectable under standard conditions are limited to the low-nl level, and because UV detection is accomplished directly on the capillary with a short optical path length. Thus, for trace analysis applications, the amount of analyte injected into the capillary or the detector sensitivity has to be increased. The latter aspect may be accomplished by utilizing extended light paths in connection with UV detection, or alternatively by utilizing laser-induced fluorescence (LIF) detection [2]. Both bubble cells and z-shaped cells have been utilized as extended light paths for UV detection, which typically provides an enhancement of the signal-to-noise by a factor of 3–6 [2]. Although the detector sensitivity theoretically may be further improved with the z-configuration, loss of electrophoretic resolution limits the light path in practical work. Thus, UV detection in capillary electrophoresis (CE) with extended light paths provides no substantial improvements in sensitivity. With LIF detection, extremely high mass sensitivity has been reported [2], but currently direct LIF detection is only applicable for some analytes as the number of wavelengths available with the commercial LIF detectors is limited.

The second approach, to increase the amount of analyte injected into the capillary, may be accomplished either by analyte enrichment during a sample preparation step or by extended volume injections followed by analyte focusing during the CZE analysis. The latter concept involves sample stacking, and sample stacking may take place when the sample plug is sandwiched between leading and terminating electrolytes (isotachophoretic sample stacking) [3] or when the sample is of lower conductivity than the running buffer (field-amplified sample stacking) [4], [5]. Although detection limits may be improved dramatically, extensive sample stacking may be difficult in routine analysis due to several practical limitations. Isotachophoretic sample stacking suffers from difficulties in selecting proper electrolytes meeting the requirements for stacking and separation, while field-amplified sample stacking suffers from a mass loading dependency on the conductivity of the sample [5].

Although improvements of detection limits may be accomplished both by detector optimization and by sample stacking as discussed above, analyte enrichment during a sample preparation step normally is the most practical concept to overcome the sensitivity problems of CZE. Several sample preparation concepts have been utilized in combination with CZE, including solid-phase extraction (SPE), solid-phase microextraction (SPME), microdialysis, on-line preconcentration with small beds of packing material inside the separation capillary, and liquid–liquid extraction (LLE) [6]. In the present review, attention will be focused on the latter type of sample preparation for CZE. In addition to general theory and some practical points of high importance, a literature review of LLE combined with CZE has been included in the paper. Focus has been limited to analyte enrichment from aqueous samples; thus, liquid extraction of solid samples has not been included in the present work.