On-line Sample Preconcentration Techniques Research Articles

Recent Progress of On-line Sample Preconcentration Techniques in Microchip Electrophoresis

This review highlights recent developments and applications of on-line sample preconcentration techniques to enhance the detection sensitivity in microchip electrophoresis (MCE); references are mainly from 2008 and later. Among various developed techniques, we focus on the sample preconcentration based on the changes in the migration velocity of analytes in two or three discontinuous solutions system, since they can provide the sensitivity enhancement with relatively easy experimental procedures and short analysis times. The characteristic features of the on-line sample preconcentration applied to microchip electrophoresis (MCE) are presented, categorized on the basis of "field strength-" or "chemically" induced changes in the migration velocity. The preconcentration techniques utilizing field strength-induced changes in the velocity include field-amplified sample stacking, isotachophoresis and transient-isotachophoresis, whereas those based on chemically induced changes in the velocity are sweeping, transient-trapping and dynamic pH junction.

Recent approaches in sensitive enantioseparations by CE

The latest strategies and instrumental improvements for enhancing the detection sensitivity in chiral analysis by CE are reviewed in this work. Following the previous reviews by García-Ruiz et al. (Electrophoresis 2006, 27, 195-212) and Sánchez-Hernández et al. (Electrophoresis 2008, 29, 237-251; Electrophoresis 2010, 31, 28-43), this review includes those papers that were published during the period from June 2009 to May 2011. These works describe the use of offline and online sample treatment techniques, online sample preconcentration techniques based on electrophoretic principles, and alternative detection systems to UV-Vis to increase the detection sensitivity. The application of the above-mentioned strategies, either alone or combined, to improve the sensitivity in the enantiomeric analysis of a broad range of samples, such as pharmaceutical, biological, food and environmental samples, enables to decrease the limits of detection up to 10⁻¹² M. The use of microchips to achieve sensitive chiral separations is also discussed.

Hydrophobic labeling of amino acids: Transient trapping–capillary/microchip electrophoresis

Transient trapping (tr-trapping) was developed as one of the on-line sample preconcentration techniques to improve a low concentration-sensitivity in microchip electrophoresis (MCE), providing highly effective preconcentration and separation based on the trap-and-release mechanism. However, a poor performance to hydrophilic analytes limited the applicability of tr-trapping. To overcome this drawback, tr-trapping was combined with a sample labeling using a hydrophobic reagent in CE. Three commercially available fluorescent dyes, fluorescein isothiocyanate, succinimidyl esters of Alexa Fluor 488 and BODIPY FL-X, were tested as derivatization reagents to increase the hydrophobicity of amino acids (AAs) that were undetectable due to no fluorescence/UV-absorbance. As a result, it was confirmed that BODIPY labeling allowed various AAs to be analyzed in tr-trapping-micellar electrokinetic chromatography (tr-trapping-MEKC) by the increase in the hydrophobicity. In tr-trapping-MEKC, both the improvement of the resolution and 106-125-fold enhancements of the detectability of labeled AAs were achieved relative to the conventional capillary zone electrophoresis. The limit of detection of labeled phenylalanine was improved from 800 to 5 pM by applying tr-trapping-MEKC. In tr-trapping-microchip MEKC, furthermore, an 80-160-fold enhancement of the peak intensity and a baseline separation was also achieved within 30 s. These results clearly demonstrate that the tr-trapping technique with hydrophobic labeling will make CE/MCE more sensitive for various analytes.

Twenty-five years of micellar electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC), which can separate neutral analytes as well as charged analytes by the capillary electrophoretic technique, was developed in 1982 and the first paper was published in 1984. The authors’ group concentrated their effort into the characterization of MEKC as a separation technique until early 1990s. Most issues in MEKC separations were successfully solved and wide applicability of MEKC was verified in 1990s. In particular, sweeping, an on-line sample preconcentration technique, was very successful for the concentration of neutral analyte as well as ionic ones. In this paper, our studies on MEKC will be summarized from the personal viewpoint of the author.

Capillary Separation: Micellar Electrokinetic Chromatography

Micellar electrokinetic chromatography (MEKC), a separation mode of capillary electrophoresis (CE), has enabled the separation of electrically neutral analytes. MEKC can be performed by adding an ionic micelle to the running solution of CE without modifying the instrument. Its separation principle is based on the differential migration of the ionic micelles and the bulk running buffer under electrophoresis conditions and on the interaction between the analyte and the micelle. Hence, MEKC's separation principle is similar to that of chromatography. MEKC is a useful technique particularly for the separation of small molecules, both neutral and charged, and yields high-efficiency separation in a short time with minimum amounts of sample and reagents. To improve the concentration sensitivity of detection, several on-line sample preconcentration techniques such as sweeping have been developed.

Neutral analyte focusing by micelle collapse in partial‐filling MEKC with UV and ESI‐MS detection

Analyte focusing by micelle collapse is investigated as an on-line sample preconcentration technique in partial-filling MEKC with detection using UV and ESI-MS. Enhancement in peak height sensitivity of more than a 100 was obtained for model alkylphenylketones and dialkylphthalates using analyte focusing by micelle collapse-partial-filling MEKC with UV or ESI-MS detection.

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.

Micelle to solvent stacking of organic cations in capillary zone electrophoresis with electrospray ionization mass spectrometry

The direction of the effective electrophoretic mobility of small organic cations in micellar electrokinetic chromatography using sodium dodecyl sulphate in a low-pH electrolyte can be reversed in the presence of organic solvent. This effective electrophoretic mobility change is presented here as a new dimension for on-line sample preconcentration of cations in capillary zone electrophoresis (CZE) using a background solution (BGS) modified by an organic solvent. The sample is prepared in a micellar solution without organic solvent. The focusing effect relies on the reversal in the effective electrophoretic mobility at the boundary zone between the micellar matrix and the BGS modified with organic solvent. This on-line sample preconcentration technique, called micelle to solvent stacking (MSS) afforded more than an order of magnitude improvement in concentration sensitivity compared to typical CZE-UV or CZE-electrospray ionization (ESI) MS analysis. The calculated limit of detection (S/N = 3) for pindolol and metoprolol analysed by MSS-CZE-ESI-MS was found to be 0.03 and 0.01 μg/mL, respectively.

Neutral analyte focusing by micelle collapse in micellar electrokinetic chromatography

The operating parameters that affect the performance of analyte focusing by micelle collapse (AFMC) to neutral analytes (i.e., dialkyl phthalates) in normal migration micellar electrokinetic chromatography (NM-MEKC) are examined. NM-MEKC is characterized by an electroosmotic flow greater than the electrophoretic velocity of the micellar pseudostationary phase, and was performed using sodium dodecyl sulfate (SDS) with neutral to high pH electrolytes in fused silica capillaries. AFMC is a recently introduced on-line sample preconcentration technique in capillary electrophoresis that can provide hundreds fold improvement in detection sensitivity. The mechanism of AFMC is based on the analyte transport, release, and accumulation by the moving surfactant micelles [e.g., SDS at concentrations closer to the critical micelle concentration (CMC)] in the sample that dilutes below its CMC into a liquid phase zone. The sample is prepared in a matrix that contains SDS micelles and high mobility anions, where the conductivity of the sample matrix is higher compared to that of the separation solution. The sample injection length, sample and separation solution conductivity ratio, and surfactant micelle concentration in the sample were found to affect the AFMC performance, as well as the effective separation length in NM-MEKC. The use of a different electrolyte salt in the sample and separation solution also affected AFMC NM-MEKC results. In particular, sodium chloride in the sample matrix can induce a micelle-mediated neutral analyte isotachophoretic concentration that is detrimental to the technique.

Recent progress of online sample preconcentration techniques in microchip electrophoresis

Microchip electrophoresis (MCE) has been advanced remarkably by the applications of several separation modes and the integration with several chemical operations on a single planer substrate. MCE shows superior analytical performance, e.g., high-speed analysis, high resolution, low consumption of reagents, and so on, whereas low-concentration sensitivity is still one of the major problems. To overcome this drawback, various online sample preconcentration techniques have been developed in MCE over the past 15 years, which have successfully enhanced the detection sensitivity in MCE. This review highlights recent developments in online sample preconcentration in MCE categorized on the basis of "dynamic" and "static" methods. The dynamic techniques including field amplified stacking, ITP, sweeping, and focusing have been easily applied to MCE, which provide effective enrichments of various analytes. The static techniques such as SPE and filtration have also been combined with MCE. In the static techniques, extremely high preconcentration efficiency can be obtained, compared to the dynamic methods. This review provides comprehensive tables listing the applications and sensitivity enhancement factors of these preconcentration techniques employed in MCE.

On-Line Sample Preconcentration and Separation Technique Based on Transient Trapping in Microchip Micellar Electrokinetic Chromatography

This paper describes a novel on-line sample preconcentration and separation technique named transient trapping (tr-trapping), which improves the efficiencies of separation and concentration by using a partially injected short micellar plug in microchip electrophoresis. Although a longer separation length often provides a better resolution of complexed or closely migrating analytes, our proposed theoretical model indicated that a trap-and-release mechanism enables a short micellar zone, which was partially injected into the separation channel, to work as an effective concentration and separation field. Application of the tr-trapping technique to microchip micellar electrokinetic chromatography (MCMEKC) was performed on a newly fabricated 5-way-cross microchip by using sodium dodecyl sulfate and rhodamine dyes as test micelle and analytes, respectively. When the injection times of micelle (t(inj),M) and sample solution (t(inj),S) were 1.0 and 2.0 s, respectively, both the preconcentration and separation of the dyes were completely finished within only 3.0 s. At t(inj),S of 8.0 s, a 393-fold improvement of the detectability was achieved in comparison with conventional MCMEKC. The resolution obtained with tr-trapping-MCMEKC was also better than that with conventional MCMEKC in spite of the 160-fold shorter length of the injected micellar zone at t(inj),M of 1.0 s. These results clearly demonstrated that the tr-trapping technique in MCMEKC provides a rapid, high-resolution and detectability analysis even in the short separation channel on the microchips.

New advances in on-line sample preconcentration by capillary electrophoresis using dynamic pH junction

The small injection volumes and narrow dimensions characteristic of microseparation techniques place constraints on concentration sensitivity that is required for trace chemical analyses. On-line sample preconcentration techniques using dynamic pH junction and its variants have emerged as simple yet effective strategies for enhancing concentration sensitivity of weakly ionic species by capillary electrophoresis (CE). Dynamic pH junction offers a convenient format for electrokinetic focusing of dilute sample plugs directly in-capillary for improved detection without off-line sample pretreatment. In this report, we highlight new advances in dynamic pH junction which have been reported to enhance method performance while discussing challenges for future research.

最新のキャピラリー電気泳動とチップ電気泳動 マイクロチップミセル動電クロマトグラフィーにおける新規オンライン試料濃縮法：トランジェント‐トラッピング法の開発

最新のキャピラリー電気泳動とチップ電気泳動 マイクロチップミセル動電クロマトグラフィーにおける新規オンライン試料濃縮法：トランジェント‐トラッピング法の開発

Simple on-line sample preconcentration technique for peptides based on dynamic pH junction in capillary electrophoresis–mass spectrometry

We report an on-line sample preconcentration technique based on dynamic pH junction in capillary electrophoresis–mass spectrometry (CE–MS). For peptide analysis, the samples were dissolved in a solution with higher pH than the background solution (BGS), and were injected into the capillary as a long plug. The pH difference between the sample matrix and BGS caused changes in analytes’ mobilities during electrophoresis, resulting in narrowing of their bands at the boundary. Around 550–1000-fold sensitivity enhancement could be achieved in terms of peak intensity without degrading peak shape and resolution. This technique is easy to perform and will be useful for peptide mass fingerprinting in protein analysis.

Capillary electrophoretic techniques toward the metabolome analysis

Abstract Metabolome analysis is a systematic chemical analysis of metabolites, which may be used to investigate the metabolic activity in the cell. Capillary electrophoresis (CE) is one of the most promising techniques for the metabolome analysis, because it gives high-resolution separations in a reasonable time and requires a minimum amount of samples. General characteristics of CE are discussed from the viewpoint of metabolome analysis. Micellar electrokinetic chromatography (MEKC), a separation mode of CE, enables the separation of neutral analytes by using micelles as pseudostationary phases. MEKC is also powerful for the separation of ionic analytes to improve selectivity. To solve relatively poor concentration sensitivity with UV absorbance detection, on-line sample preconcentration techniques were developed resulting in up to few thousand-fold increases in sensitivity. Laser-induced fluorescence detection is another solution to increase concentration sensitivity, but most analytes are not natively fluorescent. Therefore, several derivatization reactions were performed to selectively detect a class of analytes with high sensitivity. Some preliminary results are shown with formic acid extracts of Bacillus subtilis.

Determination of trace levels of copper(II), aluminium(III) and iron(III) by reversed-phase high-performance liquid chromatography using a novel on-line sample preconcentration technique

A column switching high-performance liquid chromatographic technique for the determination of copper(II), aluminium(III) and iron(III) as their 8-hydroxyquinolinate complexes using spectrophotometric detection at 400 nm is described. On-line preconcentration of the metal chelates was effected using a 10 × 1.5 mm i.d. pre-column packed with C18 reversed-phase sorbent. The pre-column was mounted before the analytical column; by manual actuation of a six-port switching valve it could be switched on-line. The metal chelates were resolved by reversed-phase high-performance liquid chromatography on the analytical column following elution from the pre-column with the appropriate mobile phase. A systematic approach to this type of trace enrichment is outlined, and the results show an increase in both sensitivity and selectivity over previous methods. The linear dynamic range is from 5 ppb to 10 ppm for AlIII and from 40 ppb to 5 ppm for both CuII and FeIII. Application of the technique to waste water from a mine and to beverage samples is reported.

Advances in ion chromatography: speciation of μ 1 −1 levels of metallo-cyanides using ion-interaction chromatography

Fe(CN) 4− 6, Cu(CN) 3− 4, Co(CN) 3− 6, Fe(CN) 3− 6, Ni(CN) 2− 4 and Cr(CN) 3− 6 are determined by ion-interaction chromatography using a C 18 column and methanol-tetrahydrofuran-10 mM phosphate buffer (pH 7.9) (25 + 1 + 74, v/v/v) containing 5 mM tetrabutylammonium hydroxide as mobile phase, with spectrophotometric detection at 214 nm. Detection limits are in the range 0.01–0.5 mg 1 −1. In an alternative approach, an automated on-line sample preconcentration technique is used wherein a 2-ml volume of sample containing metallo-cyanides is loaded onto a C 18 precolumn which has been equilibrated with the above mobile phase. The bound solutes are then eluted from the precolumn to a C 18 analytical column where they are separated using the same mobile phase as employed to equilibrate the precolumn. Detection limits are in the rate 0.08–1.58 μg 1 −1 and calibration graphs are linear up to 200 μg 1 −1. The preconcentration step is shown to give quantitative recoveries for all species except Fe(CN) 4− 6 and (CN) 3− 4. The iron(II) complex does not bind quantitatively to the precolumn, and extensive studies with the copper complex suggested that low recoveries were due to dissociation and ligand-exchange reactions occurring during the chromatographic separation process. Negative interference effects were observed for Cl − and SO 2− 4 when present at a level of 250 mg 1 1−, and UV-absorbing anions such as Br −, SCN −, NO − 2 and NO − 3 caused positive interference when present at concentrations as low as 1 mg 1 −1. The negative interferences could be reduced by diluting the sample and the positive interferences could be eliminated by incorporating an additional step in the preconcentration process, in which UV-absorbing anions bound to the precolumn after sample loading were eluted selectively using an eluent consisting of 10 mM NaCl in phosphate buffer (pH 6.7).

Determination of polychlorinated dibenzofurans in surface water by means of liquid chromatography using on-line sample preconcentration and different spectroscopic detection techniques

Three different spectroscopic techniques, UV absorbance, fluorescence and sensitized room temperature phosphorescence are examined for the detection of polychlorinated dibenzofurans. A pre-column enrichment step is used prior to reversed-phase liquid chromatographic separation. The fluorescence detection mode offers the best sensitivity and selectivity but UV detection in combination with pre-column technology provides also a powerful means for trace analysis of the dibenzofurans. The method presented allows the determination of ppt-ppb concentration levels in polluted surface waters with good recoveries and reproducibility.