On.column pre‐concentration of alcohol dehydrogenase in capillary electrophoresis

The analysis of alcohol dehydrogenase (ADH) at low concentration using capillary electrophoresis is described. Several simple and effective ways to improve detection limits and sensitivity are investigated. These include large volume sample stacking, head column field amplified sample stacking, and sweeping. Results indicate that by using a combination of head‐column field amplified sample stacking and sweeping, fluorescently labelled alcohol dehydrogenase can be pre‐concentrated online by dissolving samples in water or other low conductivity matrices, and injecting into a high conductivity micellar buffer. The abrupt changes in conductivity cause narrowing of the analyte length and thus enhance the detection sensitivity. Combination of this approach with laser induced fluorescence detection yields a limit of detection of 5×10^–13 M. Both qualitative and quantitative aspects of this method are investigated.     

Strategies for the on-line preconcentration and separation of hypolipidaemic drugs using micellar electrokinetic chromatography

Three strategies were investigated for the simultaneous separation and on-line preconcentration of charged and neutral hypolipidaemic drugs in micellar electrokinetic chromatography (MEKC). A background electrolyte (BGE) consisting of 20 mM ammonium bicarbonate buffer (pH 8.50) and 50 mM sodium dodecyl sulfate (SDS) was used for the separation and on-line preconcentration of the drugs. The efficiencies of sweeping, analyte focusing by micelle collapse (AFMC), and simultaneous field-amplified sample stacking (FASS) and sweeping, were compared for the preconcentration of eight hypolipidaemic drugs in different conductivity sample matrices. When compared with a hydrodynamic injection (5 s at 50 mbar, 0.51% of capillary volume to detection window) of drug mixture prepared in the separation BGE, improvements of detection sensitivity of 60-, 83-, and 80-fold were obtained with sweeping, AFMC and simultaneous FASS and sweeping, respectively, giving limits of detection (LODs) of 50, 36, and 38 μg/L, respectively. The studied techniques showed suitability for focusing different types of analytes having different values of retention factor (k). This is the first report for the separation of different types of hypolipidaemic drugs by capillary electrophoresis (CE). The three methods were validated then applied for the analysis of target analytes in wastewater samples from Hobart city.     

Two-step stacking in capillary zone electrophoresis featuring sweeping and micelle to solvent stacking: I. Organic cations

Two-step stacking of organic cations by sweeping and micelle to solvent stacking (MSS) in capillary zone electrophoresis (CZE) is presented. The simple procedure involves hydrodynamic injection of a micellar sodium dodecyl sulfate solution before the sample that is prepared without the micelles. The micelles sweep and transport the cations to the boundary zone between the sample and CZE buffer. The presence of organic solvent in the CZE buffer induces the second stacking step of MSS. The LODs obtained for the four beta blocker and two tricyclic antidepressant test drugs were 20-50 times better compared to typical injection.     

High-salt stacking principles and sweeping: comments and contrasts on mechanisms for high-sensitivity analysis in capillary electrophoresis

High-salt stacking in electrokinetic chromatography (EKC) is defined and contrasted to the sweeping method. A recent paper argued the two methods are identical, where high concentrations of micelle in the sample were intended to mimic the effect of high-salt stacking. However, high micelle concentration in the sample matrix in EKC is analogous to using a high-conductivity sample instead of a low-conductivity sample in field amplified stacking. High-salt stacking does not require a sample free of pseuostationary phase, only a sample with a high-mobility co-ion compared to the separation buffer electrokinetic vector. High-salt stacking uses a discontinuous buffer system and should not be confused with continuous buffer stacking systems such as sweeping.     

Stacking and separation of aspartic acid enantiomers under discontinuous system by capillary electrophoresis with light-emitting diode-induced fluorescence detection

We describe the stacking and separation of d- and l-aspartic acid (Asp) by capillary electrophoresis (CE) with light-emitting diode-induced fluorescence detection (LEDIF). In the presence of cyanide, d- and l-Asp were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) to form fluorescent derivatives prior to CE-LEDIF. The separation of NDA-derivatized d- and l-Asp was accomplished using a discontinuous system - buffer vials contained a solution of 0.6% poly(ethylene oxide) (PEO), 150 mM sodium dodecyl sulfate (SDS), and 60 mM hydroxypropyl-β-cyclodextrin (Hp-β-CD), while a capillary was filled with a solution of 150 mM SDS and 60 mM Hp-β-CD. The role of PEO, Hp-β-CD, and SDS is to act as a concentrating media, as a chiral selector, and as a pseudostationary phase, respectively. This discontinuous system could be employed for the stacking of 600 nL of NDA-derivatized d- and l-Asp without the loss of chiral resolution. The stacking mechanism is mainly based on the difference in viscosity between sample zone and PEO as well as SDS sweeping. The limits of detection at signal-to-noise of 3 for d- and l-Asp were down to 2.4 and 2.5 × 10⁻¹⁰ M, respectively. Compared to normal sample injection volume (25 nL), this stacking approach provided a 100- and 110-fold improvement in the sensitivity of d- and l-Asp, respectively. This method was further applied for determining d- and l-Asp in cerebrospinal fluid, soymilk, and beer.     

Sample stacking for the analysis of eight penicillin antibiotics by micellar electrokinetic capillary chromatography

We studied the use of micellar electrokinetic capillary chromatography for separating eight penicillins. The method consists of (i) an electrophoretic separation based on micellar electrokinetic capillary chromatography, which uses sodium dodecyl sulfate (SDS) as surfactant; (ii) a sample stacking technique called reverse electrode polarity stacking mode (REPSM); and (iii) direct UV detection. The background electrolyte that gave complete separation contained 20 mM sodium borate buffer and 60 mM SDS. The sensitivity of the method was improved by an enrichment step that used on-column stacking. The limits of detection were at the microg.L(-1) level for the penicillins and did not detract from the peak resolution.     

Comparison of two capillary electrophoresis online stacking modes by analysis of polycyclic aromatic hydrocarbons in airborne particulates

Naphthalene, fluorene, pyrene, anthracene, phenanthrene, and chrysene were successfully separated by CD-modified MEKC (CD-MEKC) using 20 mM borate (pH 9.0) containing 90 mM SDS and 75 mM beta-CD. Two online stacking methods, i.e., sweeping and field-enhanced sample injection (FESI), were explored to enhance the detection sensitivity. The influences of some crucial parameters in sweeping and FESI procedures were investigated. For FESI method, a plug of water and low-conductivity sample matrix was used to increase the stacking efficiency. Compared with the sweeping method, FESI can increase the sensitivity in the range of 10-20-fold. The proposed method was used for the analysis of polycyclic aromatic hydrocarbons in airborne particulates.     

准静态扫集胶束电动毛细管色谱法测定扇贝样品中的贝类毒素

采用准静态扫集胶束电动毛细管色谱(MEKC)法测定了扇贝样品中的2种贝类毒素。毛细管内首先充满含十二烷基硫酸钠(SDS)的缓冲溶液,调节缓冲溶液的pH值,使电渗流等于SDS胶束的电泳流速,电动进样时,带正电荷的贝类毒素离子被SDS扫集吸附,由于SDS在毛细管内处于准静止状态,可使进样时间延长至320 s。与常规电动进样MEKC相比,石房蛤毒素和软骨藻酸的检测灵敏度分别提高950和810倍。该方法对石房蛤毒素和软骨藻酸的检出限分别为0.05,0.12 ng/m L。方法可实现对扇贝样品中2种贝类毒素的快速、灵敏检测。     

Fluorescence imaging of sample zone narrowing and dispersion in a glass microchip: the effects of organic solvent (acetonitrile)-salt mixtures in the sample matrix and surfactant micelles in the running buffer

A mismatch in the EOF velocities between the sample zone and running buffer region is known to generate pressure-driven, parabolic flow profile of the sample plug in electrokinetic separation systems. In the present study, video fluorescence microscopy was employed to capture real-time dynamics of the sample plug (containing fluorescein as the probe molecule) in a discontinuous conductivity system within a glass microchip, in which the sample matrix consisted of a mixture of ACN and salt (NaCl), and the running buffer contained sodium cholate (SC) micelles as the pseudo-stationary phase (i.e., performing "ACN stacking" in the mode of MEKC). Upon application of the separation voltage, the video images revealed that zone narrowing and broadening of the probe molecules occurred as the sample plug headed toward the cathode during the initial time period, probably resulting in part from the stacking/sweeping, and destacking of the SC micelles at the boundaries between the sample zone and running buffer. Interestingly, a second sample zone narrowing event can be observed as the sample plug moved further toward the cathode, which could be attributed to the sweeping of the slower moving probe molecules by the faster moving SC micelles that originated from the anode. This phenomenon was studied as a function of pH, sample plug length, as well as the concentration of organic solvent and salt in the sample matrix. The data suggested that the presence of large amounts of an organic solvent (such as ACN or methanol) and salts in the sample matrix not only induces sample dispersion due to the formation of a pressure-driven (hydrodynamic) flow, but may also lead to the formation of a double sample zone narrowing phenomenon by altering the local EOF dynamics within the separation system.     

Analysis of ephedra-alkaloids using sweeping and cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography methods

Two stacking methods of capillary electrophoresis (CE) were developed for the separation of very dilute solutions of ephedra-alkaloids, namely ephedrine, pseudoephedrine, methylephedrine, methylpseudoephedrine, norephedrine, and norpseudoephedrine. A sweeping method which uses a carrier comprised of phosphoric acid, sodium dodecyl sulfate (SDS), diethylamine and acetonitrile permits the detection of the alkaloids down to the 10(-1) microg/mL level, and the cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-Sweep-MEKC) method using phosphoric acid, SDS, and acetronitrile as electrolytes can detect down to the 10(-3) microg/mL level. The former requires the conductance of the sample solution to be adjusted beforehand, and only five peaks were observed, two of which were overlapped. The latter is capable of separating the six alkaloids but has a somewhat poorer reproducibility. Using an optimized injection time, it was found that the more diluted a solution is, the greater the sweeping effect will be. The CSEI-Sweep-MEKC method with a 600 s injection time and a 10(-1) microg/mL solution concentration provides an amplification effect of approximately 10(4). The method is suitable for analyses of dilute herb drug extracts and mouse sera. The effect of buffers on the separation and validation of the methods in this study are also discussed.     

Two-step stacking in capillary zone electrophoresis featuring sweeping and micelle to solvent stacking: II. Organic anions

Two-step stacking of organic anions by sweeping and micelle to solvent stacking (MSS) using cationic cetyltrimethylammonium micelles in co-electroosmotic flow (co-EOF) capillary zone electrophoresis (CZE) is described. The co-EOF condition where the direction of the EOF is the same as the test anions was satisfied by positive dynamic coating of a fused silica capillary with hexadimethrine bromide. The strategy was as follows. After conditioning the capillary with the background solution (BGS), a micellar solution (MS) was injected before the sample solution (S). The BGS, MS and S have similar conductivities. Voltage was applied at negative polarity. The analytes in the micelle-free S zone were swept by micelles from the MS. The swept analytes were brought by the micelles to the MSS boundary where the second stacking step was induced by the presence of organic solvent in the BGS. Finally was the separation of concentrated analytes by CZE. The effect of electrolyte concentration in the S, injection time of the MS and the S and surfactant concentration in the MS were studied. A 20-29, 17-33 and 18-21 times increase in peak height sensitivity was obtained for the test hypolipidaemic drugs (gemfibrozil, fluvastatin and atorvastatin), non-steroidal anti-inflammatory drugs (diflunisal, naproxen, ketoprofen, indoprofen and indomethacin), and herbicides (mecoprop and fenoprop), respectively. The LODs (S/N=3) were from 0.05 to 0.55 μg/mL. The intraday and interday repeatabilities (%RSD, n=12) in terms of retention time, corrected peak area, and peak heights was less than 3.6, 8.9, and 10.8%, respectively. The application of sweeping and MSS in co-EOF CZE together with a simple extraction procedure to a waste water sample spiked with the test herbicides was also demonstrated.     

Influence of high‐conductivity buffer composition on field‐enhanced sample injection coupled to sweeping in CE

The aim of this work was to clarify the mechanism taking place in field‐enhanced sample injection coupled to sweeping and micellar EKC (FESI‐Sweep‐MEKC), with the utilization of two acidic high‐conductivity buffers (HCBs), phosphoric acid or sodium phosphate buffer, in view of maximizing sensitivity enhancements. Using cationic model compounds in acidic media, a chemometric approach and simulations with SIMUL5 were implemented. Experimental design first enabled to identify the significant factors and their potential interactions. Simulation demonstrates the formation of moving boundaries during sample injection, which originate at the initial sample/HCB and HCB/buffer discontinuities and gradually change the compositions of HCB and BGE. With sodium phosphate buffer, the HCB conductivity increased during the injection, leading to a more efficient preconcentration by staking (about 1.6 times) than with phosphoric acid alone, for which conductivity decreased during injection. For the same injection time at constant voltage, however, a lower amount of analytes was injected with sodium phosphate buffer than with phosphoric acid. Consequently sensitivity enhancements were lower for the whole FESI‐Sweep‐MEKC process. This is why, in order to maximize sensitivity enhancements, it is proposed to work with sodium phosphate buffer as HCB and to use constant current during sample injection.     

Use of short chain alkyl imidazolium ionic liquids for on‐line stacking and sweeping of methotrexate,flinic acid and folic acid: Their application to biological fluids

Methotrexate (MTX) is widely used for the treatment of many types of cancer. Folinic acid (FNA) and folic acid (FA) were usually simultaneously supplemented with MTX to reduce the side effects of a folate deficiency. This study, for the first time, included on‐line sample preconcentration by stacking and sweeping techniques under reduced or enhanced electric conductivity in the sample region using short chain alkyl imidazolium ionic liquids (ILs) as micelle forming agents for analyte focusing. Both analyte focusing by micelle collapse (AFMC) and sweeping‐MEKC had been investigated for the comparison of their effectiveness to examine simultaneously MTX, FNA and FA in plasma and urine under physiological conditions. In sweeping‐MEKC, the sample solution without micelles was hydrodynamically injected as a long plug into a fused‐silica capillary pre‐filled with phosphate buffer containing 3.0 mol/L of 1‐butyl‐3‐methylimidazolium bromide (BMIMBr). Using AFMC, the analytes were prepared in BMIMBr micellar matrix and hydrodynamically injected into the phosphate buffer without IL micelles. The conductivity ratio between BGE and sample (γ, BGE/sample) was optimized to be 3.0 in sweeping‐MEKC and 0.33 in AFMC resulting the adequate separation of analytes within 4.0 min. To reduce the possibility of BMIMBr adsorption, an appropriate rinsing protocol was used. The limits of detection were calculated as 0.1 ng/mL MTX, 0.05 ng/mL FNA and 0.05 ng/mL FA by sweeping‐MEKC and 0.5 ng/mL MTX, 0.3 ng/mL FNA and 0.3 ng/mL FA by AFMC. The accuracy was tested by recovery in plasma and urine matrices giving values ranging between 90 and 110%. Both stacking and sweeping by BMIMBr could be successfully used for the rapid, selective and sensitive determination of pharmaceuticals in complex matrices due to its fascinating properties, including high conductivity, good thermal stability and ability to form different types of interactions by electrostatic, hydrophobic, hydrogen bonding and π–π interactions. In sweeping‐MEKC, the using of BMIMBr enhanced the γ factor, k retention factor and the injected amount of sample. Consequently, this technique offers particular potential for higher sensitivity by giving 22‐ and 5‐fold sensitivity enhancement factors (SEFs) of MTX compared to CZE and AFMC, respectively.     

Assay of melamine in milk products with a pH‐mediated stacking technique in capillary electrophoresis

A pH‐mediated stacking method in capillary electrophoresis as an assay for low concentrations of melamine in milk products was established. Real samples were treated with acetone and sodium acetate and injected directly after centrifugation and filtration. Several experimental factors, such as buffer pH, buffer concentration, sample matrix, injection/sweeping ratio, sweeping time/voltages, separation voltages, as well as sample pretreatment, which affected stacking and separation, were investigated and optimized. Under the selected condition, a low LOD of 0.01 μmol/L (S/N = 5) and a wide range of linearity of 0.01～1.0 μmol/L could be easily achieved with a good reproducibility (RSDs < 5.8% for both migration time and peak area) and an acceptable recovery of 94.0～103.2% (for milk, infant formula, yogurt, and milk products). The proposed method was suitable for routine assay of melamine in real milk samples.     

Sample stacking by field-amplified sample injection and sweeping for simultaneous analysis of acidic and basic components in clinic application

In this study, online sample concentration method, which coupled field-amplified sample injection (FASI) and sweeping technology with micellar electrokinetic chromatography (MEKC), was used to detect and analyze acidic and basic components in a single run. In order to concentrate the acidic and basic components simultaneously in a single run sweeping step, a combination of successive anion- and cation-selective injections were used. Before sample loading, a rinse buffer containing 50 mM Tris buffer (pH 3) with 41% MeOH and 0.1% polyethylene oxide (PEO) was injected in order to suppress the electroosmotic flow (EOF). Sample loading of anionic components was achieved by electrokinetic injection at a negative voltage of -2.5 kV for 80 s, and then the cationic components were injected at a positive voltage of +5 kV for 120 s. Finally, sweeping with SDS micelles from the separation buffer (25 mM Tris buffer with 60 mM SDS, pH 3) was performed at a negative voltage of -20 kV. This capillary electrophoretic methodology was applied to the quantification of acidic and basic drugs in commercial tablets and in plasma samples. The precision and accuracy of the proposed method at different concentrations ranging from high, medium, to low were evaluated on spiked plasma samples. The intra and interday precision and accuracy values at three concentrations were all below 6.1%. The method was also successfully applied to monitor the tested drugs in the plasma of nine elderly cardiovascular and/or Alzheimer's disease patients after oral administration of the commercial products.     

胶束在线扫集毛细管电泳法测定三聚氰胺

研究胶束在线扫集毛细管电泳法测定三聚氰胺的可行性,结果表明,与区带毛细管电泳相比,胶束在线扫集毛细管电泳法富集倍数提高约60倍。缓冲体系为140 mmol/L SDS+20 mmol/L NaH2PO4(pH 2.20)+10%(体积分数)甲醇,分离电压-18 kV,进样时间30 s,测量波长214 nm。考察了SDS浓度、pH、进样时间、运行电压等因素对分离测定的影响情况。在优化条件下,三聚氰胺在9 min时出峰,峰面积RSD≤3.7%。方法检出限、线性范围、相关系数分别为:0.13μg/mL、0.50~32.0μg/mL、0.9997。方法可用于奶粉中三聚氰胺的分离测定。     

On-line microheterogeneity analysis and rapid phenotyping of haptoglobin by capillary electrophoresis using sodium dodecyl sulfate as additive

To improve the detection sensitivity and determine phenotypes of haptoglobin (Hp), a prefilling technique was developed and tested in capillary electrophoresis (CE) with UV–vis absorbance detection. Adding 0.01% sodium dodecyl sulfate (SDS) to the protein sample and 0.1% SDS to the prefilling buffer solution, on-line stacking and microheterogeneity separation of Hp were achieved. In addition, the influences of pH, buffer concentration, sample and prefilling buffer SDS concentration upon resolution were examined. Under optimized conditions, Hp-microheterogeneity was well resolved and two phenotypes of Hp (Hp 1-1 and Hp 2-2) were differentiated. This method was applied to the analysis of sera from normal individuals and β-Thalassemia patients. After the depletion of albumin (HSA) and immunoglobulin G (IgG), this method allowed to determine two phenotypes in different individuals and to detect the decrease of Hp in β-Thalassemia patients. Featuring high efficiency, speed and simplicity, the proposed method shows great potential for use in clinical diagnosis and proteome research.     

Sweeping on a microchip: concentration profiles of the focused zone in micellar electrokinetic chromatography.

On-line sample concentration by sweeping was investigated in microchip micellar electrokinetic chromatography (MEKC), By changing the distance between the injection cross and the detection points, the profile of the concentration process and the diffusion process in sweeping was elucidated. Rhodamine B injected for 4 s was best concentrated by sweeping at 9.4 mm from the injection cross and the enhancement factor was 450. At the longer distance from this point the peak of Rhodamine B was broadened and diluted by diffusion. The diffusion constant of Rhodamine B calculated from the experiment was 5.7 x 10(-6) cm2s(-1). The mixture of rhodamine B, sulforhodamine B, and cresyl fast violet was concentrated by sweeping and separated by MEKC at the same time.     

Electrophoretic analysis of amines using reversed-phase,reversed-polarity,head-column field-amplified sample stacking and laser-induced fluorescence detection

This paper describes the use of reversed-phase, reversed-polarity head-column field-amplified sample stacking (HCFASS) for on-line sample concentration in conventional capillary electrophoresis. The effective stacking efficiency was determined as a function of sodium hydroxide concentration in the sample matrix. Results concur with theoretical predictions where stacking efficiency depends on the conductivity (electric field strength) and electrophoretic mobility in the sample matrix solution. Fluorescein isothiocyanate-derivatized aniline and 2,4-dimethylaniline were dissolved in sodium hydroxide (800 microM), separated in a phosphate running buffer (0.05 M, pH 9.0) and detected utilising laser-induced fluorescence. The use of reversed-phase, reversed-polarity HCFASS with laser-induced fluorescence detection yielded sensitivity improvements with respect to normal injection schemes in excess of three orders of magnitude, and a limit of detection as low as 10(-13) M.     

Direct and sensitive analysis of methamphetamine, ketamine, morphine and codeine in human urine by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-Sweep-MEKC) was directly used to test some abuse drugs in human urine, including morphine (M), codeine (C), ketamine (K) and methamphetamine (MA). First, phosphate buffer (50 mM, pH 2.5) containing 30% methanol was filled into uncoated fused silica capillary (40 cm, 50 microm I.D.), then high conductivity buffer (100 mM phosphate, 6.9 kPa for 99.9 s) was followed. Electrokinetic injection (10 kV, 500 s) was used to load samples and to enhance sensitivity. The stacking step and separation were performed at -20 kV and 200 nm using phosphate buffer (25 mM, pH 2.5) containing 20% methanol and 100 mM sodium dodecyl sulfate. Using CSEI-Sweep-MEKC, the analytes could be simultaneously analyzed and have a detection limit down to ppb level. It was unnecessary to have sample pretreatments. During method validation, calibration plots were linear (r>or=0.9982) over a range of 150-3,000 ng/mL for M and C, 250-5,000 n g/mL for MA, and 50-1,000 ng/mL for K. The limits of detection were 15 ng/mL for M and C, and 5 ng/mL for MA and K (S/N=3, sampling 500 s at 10 kV). Comparing with capillary zone electrophoresis, the results indicated that this stacking method could increase 6,000-fold sensitivity for analysis of MA. Our method was applied for analysis of 28 real urine samples. The results showed good coincidence with immunoassay and GC-MS. This method was feasible for application to detect trace levels of abused drugs in forensic analysis.