Joselito P. Quirino

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2010-2012)

CE has been alive for over two decades now, yet its sensitivity is still regarded as being inferior to that of more traditional methods of separation such as HPLC. As such, it is unsurprising that overcoming this issue still generates much scientific interest. This review continues to update this series of reviews, first published in Electrophoresis in 2007, with updates published in 2009 and 2011 and covers material published through to June 2012. It includes developments in the field of stacking, covering all methods from field amplified sample stacking and large volume sample stacking, through to isotachophoresis, dynamic pH junction and sweeping. Attention is also given to online or inline extraction methods that have been used for electrophoresis.

Sample stacking of cationic and anionic analytes in capillary electrophoresis.

The behavior of charged species along concentration boundaries in capillary zone electrophoresis (CZE) that was first described in detail by Everaerts et al. in 1979 assured the possibility of concentrating charged solutes inside the capillary. The concentration effect is based on the sudden change in analyte electrophoretic velocity brought about by the difference in the magnitude of the electric field. Furthermore, this on-line method could be the needed solution to the problem of low concentration sensitivity in CZE. Sample stacking, which is now its well known name, has then found valuable use in applying CZE in many fields, especially after the in-depth studies performed in the early 90s by Chien and Burgi. This article reviews the theory and methodological developments of sample stacking developed for charged analytes in CZE and also in electrokinetic chromatography. A table conveying the reported applications especially in the biomedical and environmental fields is given. On top of this, other on-line concentration methods for charged species, namely, sample self-stacking, acetonitrile stacking, sweeping, cation selective exhaustive injection-sweeping, and use of a pH junction, are briefly discussed.

Sweeping: concentration mechanism and applications to high-sensitivity analysis in capillary electrophoresis

Sweeping in capillary electrophoresis (CE) involves the interaction of a pseudostationary phase (PS) in the separation solution and a sample in the matrix that is free of the PS used. The PS includes not only the PSs employed in electrokinetic chromatography, but also complexation reagents such as borate. The sample matrix could have a lower, similar, or higher conductance than the separation solution. Thus, the basic condition for sweeping is a sample matrix free of the additive. The accumulation of analyte molecules during the interaction makes this interesting phenomenon very useful as an on-line preconcentration method for CE. Preconcentration occurs due to chromatographic partitioning, complexation, or any interaction between analytes and PS. Contact between analyte and PS is facilitated by the action of electrophoresis and is independent of electroosmosis. The analyte, PS, or both should have electrophoretic velocities when an electric field is applied. The extent of preconcentration is dictated by the strength of the interaction involved. From tens to several thousand-fold improvements in detector response for many neutral and charged analytes have been achieved with this technique, suggesting sweeping as a general approach to on-line preconcentration in CE. The mechanism and applications of the sweeping phenomenon under different experimental conditions are discussed in this review, with particular emphasis on a better understanding of the sweeping mechanism under reduced electric field (high conductivity) in the sample zone.

Sweeping and new on-line sample preconcentration techniques in capillary electrophoresis

Sweeping is a powerful on-line sample preconcentration technique that improves the concentration sensitivity of capillary electrophoresis (CE). This approach is designed to focus the analyte into narrow bands within the capillary, thereby increasing the sample volume that can be injected, without any loss of CE efficiency. It utilizes the interactions between an additive [i.e., a pseudostationary phase (PS) or complexing agent] in the separation buffer and the sample in a matrix that is devoid of the additive used. The accumulation occurs due to chromatographic partitioning, complexation or any interaction between analytes and the additive through electrophoresis. The extent of the preconcentration is dependent on the strength of interaction involved. Both charged and neutral analytes can be preconcentrated. Remarkable improvements—up to several thousandfold—in detection sensitivity have been achieved. This suggests that sweeping is a superior and general approach to on-line sample preconcentration in CE. The focusing mechanism of sweeping under different experimental conditions and its combination with other on-line preconcentration techniques are discussed in this review. The recently introduced techniques of transient trapping (tr-trapping) and analyte focusing by micelle collapse (AFMC) as well as other novel approaches to on-line sample preconcentration are also described.

On-Line Concentration of Neutral Analytes for Micellar Electrokinetic Chromatography. 3. Stacking with Reverse Migrating Micelles

On-line concentration of neutral analytes by sample stacking in reversed migration micellar electrokinetic chromatography is presented. Micellar separation solutions of sodium dodecyl sulfate are prepared with acidic buffers to reverse the direction of the migration velocity of neutral analytes owing to a reduced electroosmotic flow. Samples are prepared in nonmicellar matrixes of low conductivity (i.e., water, diluted buffer, or dilute organic/aqueous solvent) to achieve field enhancement in the sample zone. Without polarity switching inherent in large-volume sample stacking, narrowing of analyte bands, removal of sample matrix, and separation of focused analyte bands are achieved. A model is proposed to describe the stacking technique and is supported by experimental results. In addition, equations are derived to describe band broadening associated with the technique. Detector response improvements reaching a 100-fold are confirmed experimentally. Concentration detection limits on the order of low-ppb levels (S/N = 3) are realized with model steroidal compounds.

On-line concentration of neutral analytes for micellar electrokinetic chromatography. I. Normal stacking mode

Using two anionic high-molecular-mass surfactants, butyl acrylate-butyl methacrylate-methacrylic acid copolymers, sodium salt (BBMA), and sodium 10-undecylenate (SUA) oligomer, and an anionic low-molecular-mass surfactant, sodium dodecyl sulfate (SDS), almost an order of magnitude improvement in concentration detection limit for a neutral analyte was made feasible by normal stacking mode (NSM). Stacking efficiency and recovery were independent of analyte retention factors and were slightly dependent on the nature of the pseudostationary phases. Furthermore, fundamental conditions for on-line sample concentration by NSM were theoretically and experimentally examined.

On-Line Concentration of Neutral Analytes for Micellar Electrokinetic Chromatography. 5. Field-Enhanced Sample Injection with Reverse Migrating Micelles

Elementary conditions for the on-line concentration of neutral analytes by field-enhanced sample injection with reverse migrating micelles for micellar electrokinetic chromatography is presented. Acidic phosphate buffers containing micelles of sodium dodecyl sulfate are utilized as both sample solvent and separation solution. After the capillary is conditioned with a separation solution, a water plug is hydrodynamically injected to achieve field enhancement at the injection end of the capillary during injection by application of voltage. A model is provided to give insight into the stacking scheme. Significant detector response improvements are confirmed experimentally. Moreover, utility of the technique for the analysis of a real sample is tested using urine spiked with testosterone and progesterone.

Online Sample Preconcentration in Capillary Electrophoresis using Analyte Focusing by Micelle Collapse

A dimension for online sample preconcentration in capillary electrophoresis (CE) without modification of current CE commercial instrumentation is introduced. The focusing mechanism is based on the transport, release, and accumulation of molecules bound to micelle carriers that are made to collapse into a liquid phase zone. More than 2 orders of magnitude improvement in detection sensitivity for model steroidal compounds using sodium dodecyl sulfate micelles as carrier is demonstrated.

Sample concentration by sample stacking and sweeping using a microemulsion and a single-isomer sulfated β-cyclodextrin as pseudostationary phases in electrokinetic chromatography

Two of the powerful on-line sample concentration techniques, sample stacking and sweeping under pH-suppressed electroosmotic flow, have been evaluated using a microemulsion and a single-isomer sulfated β-cyclodextrin derivative in electrokinetic chromatography. Several clinically relevant steroids have been separated and concentrated using a microemulsion consisting of 100 mM sodium dodecyl sulfate, 41 mM n-heptane and 700 mM 1-butanol in 50 mM phosphoric acid (pH 1.9). Three environmentally relevant phenoxy acid herbicides and their enantiomers have been separated and concentrated using a background electrolyte consisting of 20 mM hepta-6-sulfato-β-cyclodextrin in 15 mM phosphoric acid (pH 1.9). Significant detector response improvements have been achieved and utilized for analysis of a relatively clean matrix, lake water.

On-line concentration of neutral analytes for micellar electrokinetic chromatography II. Reversed electrode polarity stacking mode

Basic conditions for the on-line neutral sample concentration by reversed electrode polarity stacking mode for micellar electrokinetic chromatography are developed theoretically and experimentally verified. Stacking is mainly dependent on analyte retention factors and nature of the pseudostationary phases. More than an order of magnitude improvement in concentration detection limit is achieved for resorcinol using high molecular mass surfactants.