On-line preconcentration methods for capillary electrophoresis

The limits of detection (LOD) for capillary electrophoresis (CE) are constrained by the dimensions of the capillary. For example, the small volume of the capillary limits the total volume of sample that can be injected into the capillary. In addition, the reduced pathlength hinders common optical detection methods such as UV detection. Many different techniques have been developed to improve the LOD for CE. In general these techniques are designed to compress analyte bands within the capillary, thereby increasing the volume of sample that can be injected without loss of CE efficiency. This on-line sample preconcentration, generally referred to as stacking, is based on either the manipulation of differences in the electrophoretic mobility of analytes at the boundary of two buffers with differing resistivities or the partitioning of analytes into a stationary or pseudostationary phase. This article will discuss a number of different techniques, including field-amplified sample stacking, large-volume sample stacking, pH-mediated sample stacking, on-column isotachophoresis, chromatographic preconcentration, sample stacking for micellar electrokinetic chromatography, and sweeping.     

Investigation of preconcentration strategies for the trace analysis of multi-residue pesticides in real samples by capillary electrophoresis

In this work, on-line preconcentration strategies were investigated for the multi-residue analysis of pesticides in drinking water and vegetables using micellar electrokinetic chromatography. Among the on-line strategies, sweeping and stacking with reverse migration of micelles (SRMM), with and without the insertion of a plug of water before sample injection, were contrasted. A new version of SRMM was also introduced. The modification consisted of momentarily applying a positive voltage at the inlet vial right after sample has been injected, increasing the efficiency by which the analytes are captured. Nine pesticides from different classes, carbendazim (benzimidazole), simazine, atrazine, propazine and ametryn (triazine), diuron and linuron (urea), carbaryl and propoxur (carbamate), were baseline separated in less than 6 min with a electrolyte composed of 20 mmol l(-1) phosphate buffer at pH 2.5, containing 25 mmol l(-1) sodium dodecyl sulfate and 10% methanol. Limits of detection (LODs) in the order of 2-46 microg l(-1) for the pesticides under investigation were obtained solely using the on-line strategies. Enrichment factors of 3-18-fold were obtained. These factors were computed as the improvement of the concentration LODs with respect to the reference condition (injection of 10 s at 2.5 kPa pressure). The proposed methodologies were applied to the analysis of pesticides in complex matrices such as carrot extracts where the detection of 2.5 microg l(-1) was illustrated. By combining off-line solid-phase extraction and the proposed on-line strategies, the detection of pesticides in drinking water at the 0.1 microg l(-1) level was conceived.     

Complementary on-line preconcentration strategies for steroids by capillary electrophoresis

Complementary on-line preconcentration strategies are needed when analyzing different classes of solutes in real samples by capillary electrophoresis (CE) with UV detection. The performance of three different on-line preconcentration (focusing) techniques under alkaline conditions was examined in terms of their selectivity and sensitivity enhancement for a group of steroids, including classes of androgens, corticosteroids and estrogens. Electrokinetic focusing of large sample injection plugs (up to 28% of effective capillary length or 22.1 cm) directly on-capillary can be tuned for specific classes of steroids based on changes in their mobility (velocity) using a multi-section electrolyte system in CE. A dynamic pH junction was applied for the selective resolution and focusing of weakly acidic estrogens using borate, pH 11.0 and pH 8.0 in the background electrolyte and the sample, respectively. Sweeping, using an anionic bile acid surfactant and neutral gamma-cyclodextrin (gamma-CD) under alkaline conditions (pH 8), resulted in focusing and separation of the moderately hydrophobic (non-ionic) classes of steroids, such as androgen and corticosteroids. Optimal focusing and resolution of all test steroids under a single buffer condition was realized by a dynamic pH junction-sweeping format using borate, pH 11.0 and bile acid surfactant with gamma-CD in the BGE, whereas the sample is devoid of surfactant at pH 8.0. The design of selective on-line focusing strategies in CE is highlighted by the analysis of microgram amounts of ethynyl estradiol derived from a female contraceptive pill extract using the dynamic pH junction method, which resulted in over a 100-fold enhancement in concentration sensitivity.     

On-line preconcentration capillary electrophoresis for purity profiling of synthetic peptides

In this work, an on-line preconcentration capillary electrophoresis method was optimized and evaluated for the purity control of the biologically active synthetic peptide fas-F (a 28-residue long fragment of fasciculin-1) and applied for the purity profiling of angiotensin I, oxytocin, bradykinin and luteinizing hormone releasing hormone. The laboratory-made device of the analyte concentrator cartridge consisted of a fused-silica capillary piece (150 μm i.d.×8 mm in length) packed with silica-based C18 reversed-phase chromatographic material and coupled on-line near the inlet of the separation capillary (bare fused-silica capillary, 75 μm from the concentrator to the detector). Separation of impurities present in assayed samples was achieved by using 25 mM potassium dihydrogen phosphate, pH 3.5, as running buffer and a mixture of acetonitrile: running buffer, 75:25 (v/v), as elution buffer. The intra-day relative standard deviation (R.S.D.) values for migration times ranged from 3.4 to 4.2 and 2.2-2.6% for peak areas. The inter-day R.S.D. values were 5.6-7.1 and 4.6-5.1% for migration times and peak areas, respectively. The impurity profiles obtained for each peptide were compared by CZE and on-line preconcentration CE. The proposed method allowed the preconcentration and separation of impurities with greater selectivity and higher sensitivity (100-200-fold) with respect to capillary zone electrophoresis without on-line preconcentration.     

On-line cation-exchange preconcentration and capillary electrophoresis coupled by tee joint interface

An on-line preconcentration method based on ion exchange solid phase extraction was developed for the determination of cationic analytes in capillary electrophoresis (CE). The preconcentration-separation system consisted of a preconcentration capillary bonded with carboxyl cation-exchange stationary phase, a separation capillary for zone electrophoresis and a tee joint interface of the capillaries. Two capillaries were connected closely inside a 0.3 mm i.d. polytetrafluoroethylene tube with a side opening and fixed together by the interface. The preparations of the preconcentration capillaries and interface were described in detail in this paper. The on-line preconcentration and separation procedure of the analysis system included washing and conditioning the capillaries, loading analytes, filling with buffer solution, eluting analytes and separating by capillary zone electrophoresis (CZE). Several analysis parameters, including sample loading flow rate and time, eluting solution and volume, inner diameter and length of preconcentration capillary etc., were investigated. The proposed method enhanced the detection sensitivity of CE-UV about 5000 times for propranolol and metoprolol compared with normally electrokinetic injection. The detection limits of propranolol and metoprolol were 0.02 and 0.1 microg/L with the proposed method respectively, whereas those were 0.1 and 0.5 mg/L with conventional electrokinetic injection. The experiment results demonstrate that the proposed technique can increase the preconcentration factor evidently.     

Different strategies for the preconcentration and separation of parabens by capillary electrophoresis

Several strategies, namely, large volume sample stacking (LVSS), field‐amplified sample injection (FASI), sweeping, and in‐line SPE‐CE, were investigated for the simultaneous separation and preconcentration of a group of parabens. A BGE consisting of 20 mM sodium dihydrogenphosphate (pH 2.28) and 150 mM SDS with 15% ACN was used for the separation and preconcentration of the compounds by sweeping, and a BGE consisting of 30 mM sodium borate (pH 9.5) was used for the separation and preconcentration of the compounds by LVSS, FASI, and in‐line SPE‐CE. Several factors affecting the preconcentration process were investigated in order to obtain the maximum enhancement of sensitivity. The LODs obtained for parabens were in the range of 18–27, 3–4, 2, and 0.01–0.02 ng/mL, and the sensitivity evaluated in terms of LODs was improved up to 29‐, 77‐, 120‐, and 18 400‐fold for sweeping, LVSS, FASI, and in‐line SPE‐CE, respectively. These preconcentration techniques showed potential as good strategies for focusing parabens. The four methods were validated with standard samples to show the potential of these techniques for future applications in real samples, such as biological and environmental samples.     

On-line preconcentration of perfluorooctanoic acid and perfluorooctanesulfonic acid by nonaqueous capillary electrophoresis

Separation of major environmental pollutants as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) by capillary electrophoresis is reported for the first time. It is not possible to resolve the solutes in an aqueous media. However, the use of methanol and acetonitrile as the background electrolyte (BGE) solvents allowed their rapid separation in an uncoated capillary. A major effort was put into BGE optimization in respect to both separation efficiency and detection for further on‐line preconcentration. 5 mmol.L^?1 naphthalene‐1‐sulfonic acid and 10 mmol.L^?1 triethylamine dissolved in ACN/MeOH (50:50 v/v) provided best separation and detection conditions. Next, the large‐volume sample stacking and the field‐amplified sample injection were applied and compared. Large‐volume sample stacking improved limits of detection (LODs) with regard to the standard injection by 69 times for PFOA and 143 times for PFOS with LODs of 280 and 230 nmol.L^?1, respectively. Field‐amplified sample injection improved LODs 624 times for PFOAand 806 times for PFOS with LODs 31 and 40 nmol.L^?1, respectively. Both preconcentration methods showed repeatabilities of migration times less than 1.2% RSD intraday and 6.6% RSD interday. The method was applied on PFOA and PFOS analysis in a sample of river water treated with solid‐phase extraction, which further improved LOD toward 5.6 × 10^?10 mol.L^?1 for PFOS and 6.4 × 10^?10 mol.L^?1 for PFOA and allows the method to be used for river water contamination screening or decomposition studies.     

On-line sample preconcentration by sweeping with dodecyltrimethylammonium bromide in capillary zone electrophoresis

On-line sample preconcentration of oligonucleotides with a new sweeping carrier was developed by using dodecyltrimethylammonium bromide (DTAB) below the critical micelle concentration (CMC). The sweeping results with DTAB below and above the CMC were compared. The use of DTAB below the CMC benefits the preconcentration of the oligonucleotides, while the use of DTAB above the CMC is good for hydrophobic small molecules. The factors affecting the sweeping results were optimized and this method was evaluated by constructing calibration curves for thrombin aptamers. The sweeping scheme produced a 112-fold sensitivity enhancement for the oligonucleotides relative to that run in a running buffer without DTAB. The sweeping method developed here can be a good reinforcement of the preconcentration scheme by sweeping when less-hydrophobic analytes or large negatively-charged molecules need to be preconcentrated.     

On-line sample preconcentration in capillary electrophoresis. Fundamentals and applications

On-line preconcentration is one of the aspects of analytical method development using capillary electrophoretic techniques. The choice of the sample matrix alone can significantly alter both method sensitivity and separation efficiency. The recent trend to detect samples in narrower separation vessels also necessitates the need to improve detection sensitivity. The desire to detect very low levels of analytes using limited amounts of sample from biological specimens and the high separation efficiency obtainable using very large injections compared to classical small size injections also adds to this list. Indeed, one of the rich areas of research in the capillary electrophoresis field is on on-line sample preconcentration. More than 400 published research articles gathered from the http://www.webofscience.com from the year 2000 described a form of on-line preconcentration in capillary electrophoresis. This review provides a comprehensive table listing the applications of on-line preconcentration in capillary electrophoresis.     

On-line sample preconcentration of cationic analytes by dynamic pH junction in capillary electrophoresis

To improve detection sensitivity of cationic analytes, a dynamic pH junction technique was examined. Dynamic pH junction is an on-line focusing method in capillary electrophoresis (CE) based on the difference in the analyte's mobility between the background electrolyte (BGE) and sample matrix. The effects of pH values and concentrations of the BGE and the sample matrix on dynamic pH junction were examined. Optimization of analyte focusing resulted in enhanced detection responses of about 100-160-fold in terms of peak heights for some anilines in comparison to conventional injections. In particular, the concentration limits of detection (LOD) (S/N = 3) for the test anilines obtained with dynamic pH junction were from 1.9 to 3.7 ppb with UV detection without any pretreatment procedure.     

On-line preconcentration and ICP determination for trace metal analysis

The use of a closed-loop on-line enrichment procedure in combination with an ICP plasma emission spectrometer has been developed for the analysis of trace metal ions, such as Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn. The procedure utilizes a preconcentration column filled with an anion exchange resin and 8-hydroxy-7-iodoquinoline-5-sulphonic acid is added to the sample prior to preconcentration. Details on the optimization of pretreatment and instrumental conditions are described. Results obtained for the analysis of river water and antarctic seawater are reported.Presented in part at the 1989 European Winter Conference on Plasma Spectrochemistry, Reutte, Austria     

On-line stacking capillary electrophoresis for analysis of methotrexate and its eight metabolites in whole blood

Therapeutic drug monitoring of methotrexate (MTX) and its metabolites is significant for the evaluation of treatment response of acute lymphoblastic leukemia and the prevention of adverse effects. Those eight metabolites including 7-hydroxymethotrexate, MTX-polyglutamate derivatives (MTX-(Glu)(n), n=2-7), and 2,4-diamino-N(10)-methylpteroic acid, especially the MTX-(Glu)(n), only exist in blood cells and also present antifolate effects. Therefore, whole blood analysis has importance in clinical therapy. This study combined solid-phase extraction and on-line stacking capillary electrophoresis to examine the levels of MTX and its eight metabolites in whole blood. A higher conductivity buffer (HCB) was used to accumulate large amount of samples into a narrow zone, which resulted in effective stacking and sharp peaks. A fused-silica capillary was filled with phosphate buffer (80 mM, pH 2.5) containing 15% v/v tetrahydrofuran and 100 mM sodium dodecyl sulfate. Then HCB (100 mM phosphate, pH 2.5; 1 psi, 60 s) was injected for accumulating large volume of analytes (1 psi, 200 s). Owing to the pH difference between sample zone and HCB, dynamic pH junction was generated for focusing. Meanwhile, sweeping made further stacking by using sodium dodecyl sulfate in phosphate buffer. The limits of detection (S/N=3) were 0.1 microM for MTX, 0.2 microM for 7-hydroxymethotrexate and 2,4-diamino-N(10)-methylpteroic acid, 0.3 microM for MTX-(Glu)(n=2--5), and 0.5 microM for MTX-(Glu)(n=6--7). During validation, this method showed good linearity (r> / =0.9914) and reproducibility (relative standard deviation and relative error, both less than 13%). The applications were performed to monitor blood MTX and its metabolites in acute lymphoblastic leukemia patients. The blood concentrations could provide some information related to therapeutic response and adverse effects.     

On-line two-step stacking in capillary zone electrophoresis for the preconcentration of strychnine and brucine

An on-line sample preconcentration method by two-step stacking i.e., sweeping and micelle to solvent stacking, in capillary zone electrophoresis (CZE) has been developed for the determination of strychnine and brucine in traditional Chinese herbal medicines. After experimental optimizations, the best separation was achieved by using 75 mM phosphate buffer (pH 2.5) with 30% methanol (v/v). Compared with normal CZE injection, 51- and 38-fold improvement in concentration sensitivity was achieved for strychnine and brucine, respectively. The calibration curve was linear in the range of 0.1–5.0 μg mL⁻¹ for both strychnine and brucine, with the correlation coefficients of 0.9998 and 0.9997, respectively. The limits of detection (S/N = 3) for both alkaloids were 0.01 μg mL⁻¹. The inter-day (n = 8) and intra-day (n = 5) reproducibilities expressed as the relative standard deviations for corrected peak area were less than 9.5%. The method was applied to determine strychnine and brucine in two Chinese herbal medicines, with recoveries ranging from 94.2% to 105.4%. The results indicated that the method is simple, rapid, reliable, and can be applied to determine strychnos alkaloids in traditional Chinese herbal medicines.     

On-line drug metabolites generation and their subsequent target analysis by capillary zone electrophoresis with UV-absorption detection

This study presents the in-capillary enzymatic biotransformation of dextromethorphan, an antitusive drug and opioid receptor antagonist, and subsequent electrophoretic separation of its products. The study includes the optimization of separation parameters to fulfill the requirements of an online microreaction. The analyses were performed in a bare fused-silica capillary using 100 mM sodium tetraborate (pH 10.0) mixed with linear polyacrylamide (20%, v/v) and 2-propanol (10%, v/v). This BGE was suitable for monitoring both off-line and in-capillary incubations. The partial filling technique enabled the enzymatic reaction to be carried out in its optimal environment (20 mM sodium phosphate, pH 7.4). Finally, in-capillary microreaction in the presence of cytochrome P450 3A4 gave satisfactory outcomes.     

On-line sequential preconcentration of inorganic anions by anion-selective exhaustive injection and base-stacking in capillary zone electrophoresis

A sequential electrostacking method based on anion-selective exhaustive injection (ASEI) and base-stacking (BS) is presented for the preconcentration and determination of inorganic anions by capillary zone electrophoresis (CZE) in this paper. Tetradecyltrimethylammonium bromide as an electroosmotic flow (EOF) modifier was added into the buffer to suppress EOF of the capillary. Firstly, a water plug was hydrodynamically injected into the capillary. During ASEI under negative high voltage, the sample anions migrated quickly towards the boundary between the water plug and buffer in the capillary. Then an alkaline zone was injected electrokinetically to concentrate the anions further. With the sequential electrostacking method, the preconcentration factor of (0.8-1.3) x 10(5) was obtained compared with the conventionally electrokinetic injection and the relative standard deviation of peak area was 3.3-5.3% (n = 5). The detection limits of ASEI-BS-CZE for six inorganic anions were 6-14 ng/L. The proposed method has been adopted to analyze six anions in cigarette samples successfully.     

On-line preconcentration of organic anions in capillary electrophoresis by solid-phase extraction using latex-coated monolithic stationary phases

Quaternary ammonium functionalised polymeric latex particles were coated onto the wall of a fused-silica capillary or onto a methacrylate monolithic bed synthesised inside the capillary in order to create ion-exchange stationary phases of varying ion-exchange capacity. These capillaries were coupled in-line to a separation capillary and used for the solid-phase extraction (SPE), preconcentration and subsequent separation of organic anions by capillary electrophoresis. A transient isotachophoretic gradient was used for the elution of bound analytes from the SPE phase using two modes of separation. The first comprised a low capacity SPE column combined with a fluoride/octanesulfonate discontinuous electrolyte system in which peak compression occurred at the isotachophoretic gradient front. The compressed anions were separated electrophoretically after elution from the SPE preconcentration phase and resolution was achieved by altering the pH of the electrolyte in which the separation was performed. In the second approach, a latex-coated monolithic SPE preconcentration stationary phase was used in combination with a fluoride/perchlorate electrolyte system, which allowed capillary electrochromatographic separation to occur behind the isotachophoretic gradient front. This method permitted the removal of weakly bound anions from the SPE phase, thereby establishing the possibility of sample clean-up. The effect of the nature of the strong electrolyte forming the isotachophoretic gradient on the separation and also on the preconcentration step was investigated. Capillary electrochromatography of inorganic and organic species performed on the latex-coated monolithic methacrylate column highlighted the presence of mixed-mode interactions resulting from the incomplete coverage of latex particles onto the monolithic surface. Analyte preconcentration prior to separation resulted in compression of the analyte zone by a factor of 300. Improvement in the limit of detection of up to 10400 times could be achieved when performing the preconcentration step and the presented methods had limits of detection (S/N=3) ranging between 1.5 and 12 nM for the organic anions studied.     

On-line stacking and sweeping capillary electrophoresis for detecting heroin metabolites in human urine

Heroin metabolites including morphine, codeine, and 6-acetylmorphine were determined by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI–sweep-MEKC). Liquid–liquid extraction was used for urine pretreatment. An uncoated fused silica capillary (Ld = 30 cm, 50 μm ID) was filled with phosphate buffer (50 mM, pH 2.5) containing 30% methanol, then high conductivity buffer (100 mM phosphate, 41.3 kPa for 18 s) was followed. Samples were injected electrokinetically (20 kV, 300 s). The sweeping and separation were performed at −25 kV using phosphate buffer (20 mM, pH 2.5) and 80 mM sodium dodecyl sulfate. The baseline separation was done within 10 min. During method validation, the calibration curves were linear over a range of 50–500 ng/mL (r ≧ 0.994). The RSD and RE values in intra-day and inter-day assays were all below 20%, which showed good precision and accuracy. Their detection limits were 10 ng/mL (S/N = 3). The optimized method was applied to determine real urine samples from addicts. These samples were confirmed by liquid chromatography/mass spectrometry.     

Sample preconcentration by field amplification stacking for microchip-based capillary electrophoresis

A microchip structure for field amplification stacking (FAS) was developed, which allowed the formation of comparatively long, volumetrically defined sample plugs with a minimal electrophoretic bias. Up to 20-fold signal gains were achieved by injection and separation of 400 microm long plugs in a 7.5 cm long channel. We studied fluidic effects arising when solutions with mismatched ionic strengths are electrokinetically handled on microchips. In particular, the generation of pressure-driven Poiseuille flow effects in the capillary system due to different electroosmotic flow velocities in adjacent solution zones could clearly be observed by video imaging. The formation of a sample plug, stacking of the analyte and subsequent release into the separation column showed that careful control of electric fields in the side channels of the injection element is essential. To further improve the signal gain, a new chip layout was developed for full-column stacking with subsequent sample matrix removal by polarity switching. The design features a coupled-column structure with separate stacking and capillary electrophoresis (CE) channels, showing signal enhancements of up to 65-fold for a 69 mm long stacking channel.     

On-line preconcentration of protein in capillary electrophoresis with an end-column cellulose acetate-based porous membrane

A simple on-line preconcentration method of protein for capillary electrophoresis (CE) using a cellulose acetate (CA)-coated porous membrane was proposed. CA membrane is fabricated at one of the ends of the column that allows the passage of buffer ions but excludes larger protein molecules. Protein sample is continuously electrokinetically loaded and trapped by the membrane. When injection is completed, the direction of the electric field is switched and the trapped proteins are then separated by conventional CE procedure. The results achieved showed that the preconcentration mechanism of this method was based on size-exclusion effect. Bovine serum albumin (BSA) was used for model protein sample, and signal enhancement of 550-fold with 15 min injection time was achieved.     

A continuous preconcentration/extraction method for organic trace analysis by capillary gas chromatography

Summary A slightly modified steam distillation-extraction device is described for the continuous extraction and preconcentration of organic traces in aqueous samples, prior to capillary G.C.-analysis. The quantitative performance, both theoretically and practically, is studied using phenols as the test substances. The final recovery is determined by the flow-ratio of the water and the extracting solvent and by the extraction coefficient. The process is found to be highly reproducible even at low concentration levels (ppb’s). Using 30 ml. samples with a concentration of 30 ppb (1:10^9), 100 % recoveries are obtained for the phenolic substances studied, with a relative standard deviation of about 3 %, both for methylene chloride and ethylacetate as the extracting solvents. Using methylene chloride as the extracting solvent, for phenol a maximum recovery of 80 % was obtained. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984