Large volume sample stacking in capillary electrophoresis of weakly acidic compounds using coated capillaries at high pH

Large volume stacking using the electroosmotic flow (EOF) pump (LVSEP) in capillary electrophoresis under a reverse potential is a convenient and straightforward approach for on-line concentration of dilute anionic sample solutions. LVSEP achieves automatic sample matrix removal and subsequent separation without intermediate polarity switching nor complicated instrumental setup. Since anionic analytes should move against the EOF in LVSEP, EOF needs to be suppressed. We extended the range of LVSEP up to pH 11 using various EOF suppression methods, such as dynamic coating by polymer pretreatment and permanent coating. Weakly acidic organic compounds (pK_a<5.2), chlorinated phenols (pK_a=7-9), and aromatic amino acids (pK_a2∼9.3) were concentrated and separated. By hydrodynamically filling the whole capillary of 27 cm long with the sample solution, fast and reliable injection was achieved and sensitivity enhancement factors as large as 170 were readily obtained in less than 8 min.     

Improving sensitivity by large-volume sample stacking using the electroosmotic flow pump to analyze some nonsteroidal anti-inflammatory drugs by capillary electrophoresis in water samples

Large-volume sample stacking using the electroosmotic flow (EOF) pump (LVSEP) has been used to analyze some nonsteroidal anti-inflammatory drugs (NSAIDs) in water samples. With methanol as the run buffer solvent to suppress the EOF, sensitivity was enhanced by 80-100-fold. The sample for the analysis of real water sample was pretreated by solid-phase extraction (SPE). When the method was based on off-line SPE-LVSEP-CE, sensitivity improved by as much as 1000 times.     

肌肽类生物活性肽的毛细管电泳在线富集技术

建立了毛细管区带电泳(CZE)在线富集3种肌肽类活性肽(肌肽、鹅肌肽和高肌肽)的两种简便有效的方法。一种是大体积进样反向压力排除基体富集（LVSRP）技术,即通过流体动力学进样,在不改变电源极性的条件下,利用反向压力排除样品基体,电堆积富集后进行CZE分离;另一种是大体积进样电渗流排除基体富集（LVSEP）技术,即通过流体动力学进样,于运行缓冲液中加入溴化十六烷基三甲基铵(CTAB)动态修饰毛细管表面,通过电渗流排除样品基体,改变电源极性后进行CZE分离。与常规CZE相比,LVSRP技术和LVSEP技术使检测灵敏度提高了40~60倍。对影响两种富集过程的一些因素进行了研究,在最优富集条件下考察本方法的线性范围为0.080~5.0 μmol/L。对3种生物活性肽的检测限(S/N=3)分别为LVSRP 41~58 nmol/L,LVSEP 35~43 nmol/L。     

Combination of large‐volume sample stacking with an electroosmotic flow pump with field‐amplified sample injection on cross‐channel chips

A combination of two online sample concentration techniques, large‐volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field‐amplified sample injection (FASI), was investigated in microchip electrophoresis (MCE) to achieve highly sensitive analysis. By applying reversed‐polarity voltages on a cross‐channel microchip, anionic analytes injected throughout a microchannel were first concentrated on the basis of LVSEP, followed by the electrokinetic stacking injection of the analytes from a sample reservoir by the FASI mechanism. As well as the voltage application, a pressure was also applied to the sample reservoir in LVSEP‐FASI. The applied pressure generated a counter‐flow against the EOF to reduce the migration velocity of the stacked analytes, especially around the cross section of the microchannel, which facilitated the FASI concentration. At the hydrodynamic pressure of 15 Pa, 4520‐fold sensitivity increase was obtained in the LVSEP‐FASI analysis of a standard dye, which was 33‐times higher than that obtained with a normal LVSEP. Furthermore, the use of the sharper channel was effective for enhancing the sensitivity, e.g., 29 100‐fold sensitivity increase was achieved with the 75‐μm wide channel. The developed method was applied to the chiral analysis of amino acids in MCE, resulting in the sensitivity enhancement factor of 2920 for the separated d ‐leucine.     

A highly sensitive method for enantioseparation of fenoprofen and amino acid derivatives by capillary electrophoresis with on-line sample preconcentration

A highly sensitive method for enantioseparation of trace fenoprofen and amino acid derivatives by capillary electrophoresis (CE) with vancomycin as the chiral selector was developed. Several CE techniques, such as the partial filling, large-volume sample stacking with EOF as pump plus anion-selective exhaustive injection (LVSEP-ASEI) were involved in the present method to improve the detection sensitivity. With on-column concentration, enantioseparation of racemic fenoprofen and six 9-fluorenylmethyl chloroformate (FMOC)-amino acid derivatives (at the concentration level of ng/mL) with the background electrolyte composed of 100 mmol/L Tris-H(3)PO(4) (pH 6.0) and 2 mmol/L vancomycin was detected readily with the UV detection at 214 nm. Successfully performing LVSEP-ASEI needs a very low EOF that could be depressed by coating the capillary with poly(dimethylacrylamide) solution. The coating also played a role to minimize the adsorption of vancomycin onto the capillary wall. Effect of the injected sample volume and the electrokinetic injection time on the peak area of the enantiomers and their resolution factor were investigated and optimized. Under the optimized conditions, more than 1000-fold enhancement in detection sensitivity compared with the normal injection was achieved.     

Analysis of tetrabromobisphenol A and other phenolic compounds in water samples by non-aqueous capillary electrophoresis coupled to photodiode array ultraviolet detection

Non-aqueous capillary electrophoresis (NACE) with large-volume sample stacking injection using the electroosmotic flow pump (LVSEP) has been developed for the determination of tetrabromobisphenol A (TBBPA) and other phenolic compounds in environmental matrices. Methanol has been used as run buffer solvent to reduce the electroosmotic flow (EOF). Identification and quantification of the analytes was performed by photodiode array ultraviolet detection. LVSEP-NACE improved sensitivity of the peak height by 90-300-fold. The method developed was applied to the analysis of TBBPA in river water and wastewater samples, using solid-phase extraction (SPE) as sample pretreatment process. The average recoveries of the analytes were in the range of 96-106% and 73-103% for 1 L of river water and 0.5 L of wastewater samples, respectively. When the method was based on off line SPE-LVSEP-NACE, sensitivity was improved by 3300-4500-fold and 1600-2200-fold for river water and wastewater samples, respectively.     

Large-volume sample stacking combined with separation by 2-hydroxypropyl-beta-cyclodextrin for analysis of isoxyzolylpenicillins by capillary electrophoresis

A simple, quick and sensitive capillary electrophoretic technique has been developed for the pharmaceutical analysis of isoxazolylpenicillins (oxacillin, cloxacillin and dicloxacillin) at trace levels for the first time. This method comprises large-volume sample stacking using the electroosmotic flow (EOF) pump (LVSEP), separation using 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as selective complex-forming background electrolyte additive, and direct UV detection. A complete resolution was achieved in the optimal background electrolyte containing 5.2 mM HP-beta-CD. LVSEP was successfully applied in their determinations to improve the sensitivity, where the EOF in the buffer zone was suppressed by using an acidic buffer with pH 3.6. The detection limits of the current technique were found to be 2.0 microg/L for each of the isoxazolylpenicillins based on the signal-to-noise ratio of 3. The curves of peak response versus concentration were linear from 5.0 to 400.0 microg/L with regression coefficients of 0.9982, 0.9986 and 0.9976, respectively. The interaction of isoxazolylpenicillins with HP-beta-CD was discussed. The association constants for complexes of HP-beta-CD with isoxazolylpenicillins were determined by electrophoretic method. The obtained association constants were 27.3, 34.9, and 48.5 M(-1), respectively, being proportional to their hydrophobic properties and steric hindrances. A simple and easy-manipulative sample preparation method was developed and validated by analyzing commercially available milk samples. It was found that with current sample preparation process and instrumentation system, 0.1 mL of milk sample is enough for the analysis of isoxazolylpenicillins to meet European Union (EU) guideline of 30 microg/kg.     

In-line coupling of two-phase single drop microextraction and large volume stacking using an electroosmotic flow pump in nonaqueous capillary electrophoresis

In order to improve the concentration sensitivity of capillary electrophoresis (CE), two sample preconcentration techniques, single drop microextraction (SDME) and large volume stacking using an electroosmotic flow pump (LVSEP), were coupled in-line in a commercial CE instrument. By simple programming of liquid handling sequences, a pentanol drop was prepared at the tip of a fused silica capillary over which a Teflon tube had been sleeved to serve as a hydrophobic support. After extraction of the analytes from an aqueous donor solution into the drop, the entire capillary column was filled with enriched pentanol extract. LVSEP, in which the sample matrix is automatically removed by the EOF, was then carried out using a methanolic run buffer. The overall enrichment factors for the analytes pentachlorophenol (PCP), 3-bromobenzoic acid (3-BBA), and 4-iodobenzoic acid (4-IBA), from a combination of 30 min SDME and LVSEP on a 27 cm capillary, were about 7000, even without agitation of the donor solution. The resulting limits of detection for PCP, 3-BBA, and 4-IBA were 0.7, 0.3 and 0.7 nM, respectively. Since no modification of the existing CE instrument is necessary and a bare capillary is used for LVSEP, this scheme can be adapted quite easily for many CE applications that require high concentration sensitivity.     

Large-volume sample stacking for analysis of ethylenediaminetetraacetic acid by capillary electrophoresis

A simple, quick, and sensitive capillary electrophoretic technique-large volume stacking using the electroosmotic flow (EOF) pump (LVSEP) - has been developed for determining ethylenediaminetetraacetic acid (EDTA) in drinking water for the first time. It is based on a precapillary complexation of EDTA with Fe(III) ions, followed by large-volume sample stacking and direct UV detection at 258 nm. The curve of peak response versus concentration was linear from 5.0 to 600.0 microg/L, and 0.7 to 30.0 mg/L. The regression coefficients were 0.9988 and 0.9990, respectively. The detection limit of the current technique for EDTA analysis was 0.2 microg/L with an additional 10-fold preconcentration procedure, based on the signal-to-noise ratio of 3. As opposed to the classical capillary zone electrophoresis (CE) method, the detection limit was improved about 1000-fold by using this LVSEP method. To the best of our knowledge, it represents the highest sensitivity for EDTA analysis via CE. Several drinking water samples were tested by this novel method with satisfactory results.     

Capillary electrophoresis of nonprotein and protein amino acids without derivatization

An efficient separation of eleven nonprotein amino acids (NPAAs) and three protein amino acids containing aromatic moieties was achieved by capillary electrophoresis without derivatization. The fourteen amino acids were well separated with a 100 mM sodium phosphate run buffer (pH 2.0) using a 57 cm fused-silica capillary (50 microm ID, 50 cm effective length) at 20 degrees C. With an electric field of 351 V/cm, the time needed for the separation was less than 20 min. Under optimum conditions, excellent linear responses were obtained in the concentration range of 5-100 microM, with the linear correlation coefficient ranging from 0.9785 or greater. The relative standard deviations of the migration times and the corrected peak areas were found to be 1.5-3.9% and 8.0-11.5%, respectively. In order to improve the limit of detection (LOD), simple stacking and large volume stacking using an EOF pump (LVSEP) methods were used. Improved LODs were about 300 nM in stacking and below 15 nM for five small NPAAs in LVSEP.     

Protein analysis with large volume sample stacking with an electrosmotic flow pump: a potential approach for proteomics

Large volume sample stacking using electrosmotic flow pump (LVSEP) in capillary electrophoresis is a useful tool for on-line concentration of low-abundant protein samples for proteomics research. While the stacking of only small anions with high pK_a values has been reported at low pH buffers (pH<4), protein samples (trypsin inhibitor and α-lactalbumin) with high pI values (pI>4) were stacked successfully in our system using LVSEP. Initially, the whole column of the SIL, or LPA-coated capillary was filled with protein samples (1 μg/ml), then a high voltage was applied without polarity switching with high pH buffer (pH 8.5). An enhancement factor of more than 100 times was achieved compared to the conventional pressure injection method.     

Large-volume stacking in capillary electrophoresis using a methanol run buffer

Highly sensitive nonaqueous capillary electrophoresis of weakly acidic organic compounds has been performed using methanol as the run buffer solvent. Methanol provided appropriate suppression of the electroosmotic flow and an increase in the electrophoretic mobilities of anionic solutes compared to water. These two effects allowed large-volume stacking using the electroosmotic flow pump (LVSEP) to be achieved for larger anions using a bare fused-silica capillary under an electric field of reverse polarity, whereas only fast-moving small anions were previously known to be suitable for LVSEP in aqueous media. A field-enhanced sample injection of an additional amount of analytes during the solvent plug removal further enhanced the limits of detection to below the nanomolar range with conventional UV absorption detection. Under optimum conditions, excellent linear responses and reproducibility in the migration times together with the corrected peak areas for ten analytes were obtained in the concentration range of 10-100 nM.     

Improving sensitivity by large-volume sample stacking combined with sweeping without polarity switching by capillary electrophoresis coupled to photodiode array ultraviolet detection

An easy, simple, and highly efficient on-line preconcentration method for polyphenolic compounds in CE was developed. It combined two on-line concentration techniques, large-volume sample stacking (LVSS) and sweeping. The analytes preconcentration technique was carried out by pressure injection of large-volume sample followed by the EOF as a pump pushing the bulk of low-conductivity sample matrix out of the outlet of the capillary without the electrode polarity switching technique using five polyphenols as the model analytes. Identification and quantification of the analytes were performed by photodiode array UV (PDA) detection. The optimal BGE used for separation and preconcentration was a solution composed of 10 mM borate-90 mM sodium cholate (SC)-40% v/v ethylene glycol, without pH adjustment, the applied voltage was 27.5 kV. Under optimal preconcentration conditions (sample injection 99 s at 0.5 psi), the enhancement in the detection sensitivities of the peak height and peak area of the analytes using the on-line concentration technique was in the range of 18-26- and 23-44-fold comparing with the conventional injection mode (3 s). The detection limits for (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (+)-catechin (C), (-)-epigallocatechin gallate (EGCG), and (-)-epicatechin gallate (ECG) were 4.3, 2.4, 2.2, 2.0, and 1.6 ng/mL, respectively. The five analytes were baseline-separated under the optimum conditions and the experimental results showed that preconcentration was well achieved.     

Determination of nerve agent degradation products by capillary electrophoresis using field-amplified sample stacking injection with the electroosmotic flow pump and contactless conductivity detection

In the present study, field-amplified sample stacking injection using the electroosmotic flow pump (FAEP) was developed for the capillary electrophoretic separation of the four nerve agent degradation products methylphosphonic acid (MPA), ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA) and cyclohexyl methylphosphonic acid (CMPA). Coupled to contactless conductivity detection, direct quantification of these non-UV active compounds could be achieved. Sensitivity enhancement of up to 500 to 750-fold could be obtained. The newly established approach was applied to the determination of the analytes in river water and aqueous extracts of soil. Detection limits of 0.5, 0.7, 1.4 and 2.7 ng/mL were obtained for MPA, EMPA, IMPA and CMPA, respectively, in river water and 0.09, 0.14, 0.44 and 0.22 μg/g, respectively, in soil.     

Comparative study of aqueous and non-aqueous capillary electrophoresis in the separation of halogenated phenolic and bisphenolic compounds in water samples

Capillary zone electrophoresis methods, based on either aqueous and non-aqueous solutions as running buffers and UV spectrophotometric detection, have been developed and optimized for the separation of several halogenated phenolic and bisphenolic compounds, suspected or proved to exhibit hormonal disrupting effects. Both aqueous capillary electrophoresis (CE) and non-aqueous capillary electrophoresis (NACE) methods were suitable for the analysis of compounds under study. The separation of the analytes from other 25 potentially interfering phenolic derivatives was achieved with NACE method. Large-volume sample stacking using the electroosmotic flow pump (LVSEP) was assayed as on-column preconcentration technique for sensitivity enhancement. LVSEP-CE and LVSEP-NACE improved peak heights by 5-26 and 16-330 folds, respectively. To evaluate their applicability, the capillary electrophoresis methods developed were applied to the analysis of water samples, using solid-phase extraction as sample pre-treatment process.     

Parallel separation of multiple samples with negative pressure sample injection on a 3-D microfluidic array chip

A simple and powerful microfluidic array chip-based electrophoresis system, which is composed of a 3-D microfluidic array chip, a microvacuum pump-based negative pressure sampling device, a high-voltage supply and an LIF detector, was developed. The 3-D microfluidic array chip was fabricated with three glass plates, in which a common sample waste bus (SW(bus)) was etched in the bottom layer plate to avoid intersecting with the separation channel array. The negative pressure sampling device consists of a microvacuum air pump, a buffer vessel, a 3-way electromagnet valve, and a vacuum gauge. In the sample loading step, all the six samples and buffer solutions were drawn from their reservoirs across the injection intersections through the SW(bus) toward the common sample waste reservoir (SW(T)) by negative pressure. Only 0.5 s was required to obtain six pinched sample plugs at the channel crossings. By switching the three-way electromagnetic valve to release the vacuum in the reservoir SW(T), six sample plugs were simultaneously injected into the separation channels by EOF and electrophoretic separation was activated. Parallel separations of different analytes are presented on the 3-D array chip by using the newly developed sampling device.     

On-line sample enrichment for the determination of proteins by capillary zone electrophoresis with poly(vinyl alcohol)-coated bubble cell capillaries

Field-amplified sample stacking (FASS) is used to separate basic proteins in a poly-(vinyl alcohol)-coated bubble cell capillary. To our knowledge, this is the first paper describing the on-column stacking of proteins (as cations) using FASS in bubble cell capillary. The bubble cell capillary is fabricated using a one-step method. Cetyltrimethylammonium chloride is added into the running buffer to reverse the EOF and, thus, to pump the water plug out during the sample stacking step. The effect of the water plug lengths and sample injection durations were investigated and optimized. The results obtained were compared with those for the normal capillary without bubble cell in terms of resolution and sensitivity enhancement. Under the optimal condition, this method can improve the sensitivity of the peak areas ranging from 5000- to 26 000-fold. The RSDs (n = 5) of the migration time and peak area are satisfactory (less than 0.6 and 12%, respectively). Application of the capillary electrophoresis method with bubble cell, FASS, and UV detection thereby leads to the determination of these proteins at concentrations ranging from 3 to 10 ng/mL, based on a signal-to-noise ratio of 3:1.     

Solid-phase extraction and large-volume sample stacking with an electroosmotic flow pump in capillary electrophoresis for determination of methotrexate and its metabolites in human plasma

This paper describes approaches for large-volume sample stacking (LVSS) with an EOF pumpin CE for the determination of methotrexate (MTX) and its metabolites in human plasma. After pretreatment of plasma through a SPE cartridge, a large sample volume was loaded by hydrodynamic injection (3 psi, 70 s) into the capillary filled with phosphate buffer (70 mM, pH 6.0) containing 0.01% polyethylene oxide. Following removal of a large plug of sample matrix from the capillary using polarity switching (-25 kV), the separation of anionic analytes was subsequently performed without changing polarity again, achieving an improvement of sensitivity of around a 100-fold. The method was applied to therapeutic drug monitoring of MTX in one acute lymphoblastic leukemia patient. This study is one of very few applications showing the feasibility of LVSS in analysis of biological samples by CE.     

An electrical pumping approach to eliminate sample bias in capillary electrokinetic injection

A general pumping injection (PI), which involves the use of two capillaries with different diameters, was taken to evaluate systematically the effects on eliminating sample bias associated with the electrokinetic injection process in CE. One end of the separation capillary of the smaller diameter was inserted into another pumping capillary of larger diameter. When a high voltage was applied to the pumping capillary, the EOF generated inside will act as a pump to drive the solution stream in the separation capillary. The results have demonstrated that PI is suitable for both normal and reverse EOF situations. Second, the bias degree (BD) and SD of bias we presented were used to evaluate the degree of the bias under different conditions, and the factors of bias elimination have been investigated. Under optimal conditions, the bias was satisfactorily eliminated by PI. This EOF pumping system was successfully applied to the analysis of samples in CEC for a bias-free injection. Moreover, this two-capillary pumping system did not significantly affect the EOF, current, and the column efficiency of the separation process. Finally, a PI with grounded electrode was proposed and shown to be suitable for samples with low conductivity and ions with different mobility.     

Quality evaluation of six bioactive constituents in goji berry based on capillary electrophoresis field amplified sample stacking

Goji berry, fruits of the plant Lycium barbarum L., has long been used as traditional medicine and functional food in China. In this work, a simple and easy‐operation on‐line concentration capillary electrophoresis (CE) for detection flavonoids in goji berry was developed by coupling of field amplified sample stacking (FASS) with an electroosmotic (EOF) pump driving water removal process. Due to the EOF pump and electrokinetic injection showing different influence on the concentration, the analytes injection condition should be systemically studied. Thereafter, the verification of the analytes injection conditions was achieved using response surface experimental design. Under the optimum conditions, 86–271 folds sensitivity enhancement upon normal capillary zone electrophoresis (CZE, 50 mbar × 5 s) were achieved for six flavonoids, and the detection limits ranged from 0.35 to 1.82 ng/mL; the LOQ ranged from 1.20 to 6.01 ng/mL. Eventually, the proposed method was applied to detect flavonoids in 30 goji berry samples from different habitats of China; and the results indicated that the flavonoids were rich in the eluent of 30–60% methanol, which provided a reference for extraction of goji berry flavonoids.