Sample Stacking Technique Research Articles

A pH-mediated field amplification sample stacking technique based on portable microchip electrophoresis heavy metal ion detection system.

We built a portable microchip electrophoresis heavy metal ion detection system and proposed a pH-mediated field amplified sample stacking (pH-mediated FASS) online preconcentration method. The pH-mediated FASS focuses and stacks heavy metal cations by controlling electrophoretic mobilities with a pH change between the analyte and the background electrolyte (BGE) in solution to improve the detection sensitivity of the system. We optimized and adjusted sample matrix solution (SMS) ratios and pH to create concentration and pH gradients for SMS and BGE. Furthermore, we optimize the microchannel width to improve the preconcentration effect further. The system and method analyzed soil leachates polluted with heavy metals and separated Pb2+ and Cd2+ within 90s, obtaining their levels at 58.01mg/L and 4.91mg/L with sensitivity enhancement factors (SEF) of 26.40 and 43.73. Compared with inductively coupled plasma atomic emission spectrometry (ICP-AES), the detection error of the system was less than 8.80%.

Plasma levels of creatine, 2-aminobutyric acid, acetyl-carnitine and amino acids during fasting measured by counter-current electrophoresis in PAMAPTAC capillary

Determination of the minor nitrogen metabolites, creatine, 2-aminobutyric acid (2-AB), and acetyl-carnitine (Ac-carn), in human plasma is performed by capillary electrophoresis (CE) in a 6 % PAMAPTAC coated fused silica capillary. 6 % PAMAPTAC generates a stable electroosmotic flow (EOF) of 6.31 × 10−9 m2V−1s−1; and the nitrogen metabolites are separated in a counter-current mode, which increases electrophoretic resolution. Baseline separation of creatine, 2-AB, Ac-carn, and most proteinogenic amino acids is achieved in 8.5 M acetic acid (AcOH) at pH 1.37 as background electrolyte (BGE) at an effective capillary length of 14.4 cm with migration times ranging from 4.95 min for creatine to 5.43 min for Ac-carn. 20 μL of plasma is deproteinized with the addition of 60 μL of acetonitrile (ACN) and a 21.8 mm sample zone is injected into the capillary, which corresponds to 6.9 % of total and 15 % of effective capillary length. The zone is focused using a large volume sample stacking technique with LODs of 0.2–0.4 μM and LOQs of 0.8–1.3 μM relative to the untreated plasma sample. Inter-day reproducibility of migration time is 0.7–1.1 % and for peak area 2.5–3.9 %. The CE method is used to monitor plasma levels of nitrogen metabolites during 60 h of fasting in 10 healthy lean volunteers. T-test showed statistically significant differences in plasma levels of creatine, 2-AB, Ac-carn and most proteinogenic amino acids, which return to their original concentrations after subsequent 48 h recovery.

Ultrasensitive assay of O-GlcNAc transferase using capillary electrophoresis-laser induced fluorescence.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is directly associated with the level of O-GlcNAc glycosylation of biomolecules and various diseases, and it is expected to be a promising potential new therapeutic target. Here, we develop a robust and sensitive method for OGT assay based on capillary electrophoresis-laser induced fluorescence (CE-LIF) method. AF-488-modified peptide containing serine active group is designed as substrate for OGT-catalyzed reaction, and nonradioactive UDP-GlcNAc is employed as sugar donor to perform O-GlcNAc glycosylation modification. The enzyme activity of OGT is measured by quantitative determination of glycosylated peptide produced by the reaction. Large volume sample stacking technique for sample injection and a unique fluorescence collection system for LIF detection are adopted to greatly enhance the detection sensitivity, thus a low limit of detection down to 0.23 pM for OGT detection is achieved. The method is successfully applied to detect OGT activity in clinical blood samples with satisfactory accuracy. Our study provides a simple, accurate, and sensitive method with great potential application in clinical diagnosis of O-GlcNAc-related diseases.

Green microemulsion electrokinetic chromatographic method for simultaneous determination of azelastine and budesonide

A facile, sensitive, and eco-friendly microemulsion electrokinetic chromatographic method coupled with field amplified sample stacking was developed for the simultaneous determination of azelastine (AZL) and budesonide (BUD) in their combined pharmaceutical preparations. The separation of both drugs was achieved using 13 mM borate buffer (pH 9.0) containing 1.2% sodium dodecyl sulphate (SDS), 2.4% n-butanol, and 1.0% ethyl acetate as the background electrolyte (BGE). A capillary tube (40 cm effective length; 75 μm internal diameter) of uncoated fused silica was used for separation of the studied analytes. A diode array detector was used to monitor the studied compounds at 195 nm. The applied voltage and temperature were adjusted at 28 kV and 25 °C, respectively. The method was linear over the concentration ranges of 0.5–50.0 μg/mL for azelastine and 1.0–20.0 μg/mL for budesonide. The sensitivity was improved by more than 10 folds by using field amplified sample stacking technique. Moreover, the method was applied for separation of budesonide epimers (22R-BUD and 22S-BUD) by increasing the concentration of SDS in the BGE to be 1.8%. Greenness evaluation was performed using analytical eco-scale, Green Analytical Procedure Index (GAPI) and Analytical GREEnness (AGREE) approaches. The excellent green profile of the developed method, confirmed by the three approaches, has supported its applicability in quality control laboratories.

Monitoring of amoxicilline and ceftazidime in the microdialysate of diabetic foot and serum by capillary electrophoresis with contactless conductivity detection.

Determination of the broad-spectrum antibiotics amoxicilline (AMX) and ceftazidime (CTZ) in blood serum and microdialysates of the subcutaneous tissue of the lower limbs is performed using CE withcontactless conductivity detection (C4 D). Baseline separation of AMX is achieved in 0.5 M acetic acid as the background electrolyte and separation of CTZ in3.2 M acetic acid with addition of 13% v/v methanol. The CE-C4 D determination is performed in a 25µm capillary with suppression of the EOF using INST-coating on an effective length of 18cm and the attained migration time is 4.2min for AMX and 4.4min for CTZ. The analysis was performed using 20µl of serum and 15µl of microdialysate, treated by the addition of acetonitrile in a ratio of 1/3 v/v and the sample is injected into the capillary using the large volume sample stacking technique. The LOQ attained in the microdialysate is 148ng/ml for AMX and 339ng/ml for CTZ, and in serum 143ng/ml for AMX and 318ng/ml for CTZ. The CE-C4 D method is employed for monitoring the passage of AMX and CTZ from the blood circulatory system into the subcutaneous tissue at the sites of diabetic ulceration in patients suffering from diabetic foot syndrome and also for measuring the pharmacokinetics following intravenous application of bolus antibiotic doses.

Separation of saikosaponins by on-line sample stacking CE method

Saikosaponins a, c, d, b1 and b2 which are the trace constituents in the crude extract of Bupleuri Radix can be separated successfully by the on-line sample stacking technique (Sweeping method) of capillary electrophoresis. The method uses aqueous phosphoric acid with a pH lower than 2 to prepare the extracting solvent and buffer solution of the same electric conductance. The former is prepared with methanol and the latter is prepared by adding SDS, methanol and acetonitrile. Bupleuri Radix extract analyzed by CE-sweeping method gives better result than by MEKC method with a higher separation efficiency, lower detection limit and ability to detect five saikosaponins. Applied to the analysis of two Chinese herbal formulas, CE-sweeping method is found to be able to determine the contents of saikosaponins a and b1 successfully in Minor Bupleurum Combination and Major Bupleurum Combination, both of which contain Bupleuri Radix as the imperial drug. However, the migration times and resolution of the two marker substances, saikosaponins a and b1, were changed obviously by the complicate constituents in Chinese herbal preparations.

Highly Sensitive SDS Capillary Gel Electrophoresis with Sample Stacking Requiring Only Nanograms of Adeno-Associated Virus Capsid Proteins.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been the method of choice in the past decades for size-based protein analysis. However, in general it requires the protein concentration in mg/mL level and thus is not practical for trace level protein analysis, not to mention the lengthy labor-intensive procedures. The SDS capillary gel electrophoresis (SDS CGE) method reported herein requires only nanogram-sized proteins loaded onto the autosampler. A sample stacking technique (e.g., head-column field-amplified sample stacking (HC FASS)) was employed, providing three orders of magnitude sensitivity enhancement compared to conventional SDS CGE. This method has been used routinely in purity analysis and characterization of adeno-associated virus (AAV) intermediates and finished gene therapeutics of AAV vectors. The sensitivity achieved is comparable to the currently most sensitive size-based protein assay silver-stained SDS PAGE. The highly sensitive sample stacking SDS CGE can be used for other types of proteins as well.

Quality monitoring methods of initial and terminal manufacture of LiFePO4 based lithium ion batteries by capillary electrophoresis

Magnetic impurities of lithium ion battery degrade both the capacity and cycling rates, even jeopardize the safety of the battery. During the material manufacture of LiFePO4, two opposite and extreme cases (trace impurity Fe(II) with high content of Fe(III) background in FePO4 of initial end and trace Fe(III) with high content of Fe(II) background in LiFePO4 of terminal end) can result in the generation of magnetic impurities. Accurate determination of impurities and precise evaluation of raw material or product are necessary to ensure reliability, efficiency and economy in lithium ion battery manufacture. Herein, two kinds of rapid, simple, and sensitive capillary electrophoresis (CE) methods are proposed for quality monitoring of initial and terminal manufacture of LiFePO4 based lithium ion batteries. The key to success includes the smart use of three common agents 1,10-phenanthroline (phen), EDTA and cetyltrimethyl ammonium bromide (CTAB) in sample solution or background electrolyte (BGE), as well as sample stacking technique of CE feature. Owing to the combination of field-enhanced sample injection (FESI) technique with high stacking efficiency, detection limits of 2.5nM for Fe(II) and 0.1μM for Fe(III) were obtained corresponding to high content of Fe(III) and Fe(II), respectively. The good recoveries and reliability demonstrate that the developed methods are accurate approaches for quality monitoring of LiFePO4 manufacture.

Study on the potential application of salivary inorganic anions in clinical diagnosis by capillary electrophoresis coupled with contactless conductivity detection.

A capillary electrophoresis approach with capacitively coupled contactless conductivity detection method has been developed for the determination of inorganic metabolites (thiocyanate, nitrite and nitrate) in human saliva. Field amplified sample injection, as a simple sample stacking technique, was used in conjunction for online preconcentration of above inorganic anions. A selective separation for the target anions from other coexisting constituents present in saliva could be obtained within 14min in a 10mmol/L His-90mmol/L HAc buffer (pH 3.70) at the separation voltage of -18kV. The limits of detection and limits of quantification of the three analytes were within the range of 3.1-4.9ng/mL (S/N=3) and 10-16ng/mL (S/N=10), respectively. The average recovery data were in the range of 81-108% at three different concentrations. This method provides a simple, rapid and direct approach for metabolite analyses of nitric oxide and cyanide based on noninvasive saliva sample, which presents a potential fast screening tool for clinical test.

Large-Volume Sample Staking of Rice Polyphenols Prior to Their Determination by Non-aqueous Capillary Electrophoresis

A rapid non-aqueous capillary electrophoretic (NACE) method for the separation of (−)-epicatechin (EPI), (+)-catechin (−CAT), kaempferol (KAE), quercetin (QUR), naringanin (NAR), ferulic acid (FA), and p-coumaric acid (p-CA) has been developed and applied to the determination of these compounds in different rice varieties. All seven compounds were separated on capillary of 50 μm × 68 cm (60-cm effective length) using 20 mmol L−1 borate buffer of pH 9.0 and 5 % acetonitrile in methanol. Large-volume sample stacking (LVSS) technique was optimized and used to preconcentrate non-detectable polyphenols of white polished rice. Rice extracts were concentrated on-line by LVSS prior to separation by non-aqueous capillary electrophoresis. An improvement of 10–55 times in detectability was achieved with injection at 50 mbar for 30 s followed by voltage inversion (−20 kV) for 5 s. Linear calibration range of 1–300 μg L−1 and 0.01–60 μg L−1 was observed for NACE and NACE-LVSS method respectively, with the detection limit of 0.33–2.0 and 0.006–0.19 μg L−1. Good reproducibility with standard deviations of less than 5 % was achieved. Polyphenol contents of different rice varieties were determined using developed method.

Simultaneous Determination of Nitrate and Nitrite in Fish Products with Improved Sensitivity by Sample Stacking-Capillary Electrophoresis

A fast and reproducible method for the simultaneous determination of nitrate and nitrite ions in canned fish samples by capillary zone electrophoresis has been developed. The sensitivity of the method was increased by applying a sample stacking technique. Optimal separation conditions were selected as 30 mmol L−1 formic acid and 30 mmol L−1 sodium sulfate at a pH of 4.0. The separation of nitrate and nitrite ions was achieved within 2.5 min. The limits of detection obtained at a signal-to-noise ratio of 3 for nitrate and nitrite were 0.55 and 0.82 μmol L−1, while the relative standard deviations of intra-day corrected peak areas were 0.99 and 2.74 %, respectively. Recovery values ranged between 88.7 and 104 % for both ions. The method was successfully applied to canned fish samples, namely tuna, mackerel and sardine.

Repetitive injection field-amplified sample stacking for cationic compounds determination

The development of a field-amplified sample stacking technique is presented. Sensitivity enhancement in this technique was obtained by repetitive injections of a sample followed by steps of sample matrix removal through the application of counter-pressure. Under optimized conditions the background electrolyte (BGE) was composed of 80mM H3PO4 while the sample matrix contained 0.5mM H3PO4 and 30% (v/v) methanol. The elaborated method enabled a 4-fold effective injection of the sample (53s, 0.5psi). Each injection was followed by a focusing step during which the application of a voltage (2kV) and counter-pressure (−1psi) was performed for 0.65min. The method was developed for the determination of six psychiatric drugs (opipramol, hydroxyzine, promazine, amitriptyline, fluoxetine, and thioridazine). The elaborated method was applied for analysis of human urine samples after a simple liquid–liquid extraction procedure. The detection limits obtained were in the range of 2.23–6.21ng/mL.

Off-line hyphenation of molecularly imprinted magnetic nanoparticle-based extraction with large volume sample stacking capillary electrophoresis for high-sensitivity detection of trace chloro-phenols

The present work developed a sensitive and selective method for the determination of 4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4-DCP) and 2,4,6-trichlorophenol (2,4,6-TCP) in an environmental water sample by molecularly imprinted magnetic nanoparticle (Fe3O4@MIP) based extraction coupled with capillary electrophoresis (CE). The highly selective, fast and effective sample pretreatment method with Fe3O4@MIPs can overcome the low sensitivity of the highly efficient capillary electrophoresis-ultraviolet detection method (CE-UV). In order to further improve the detection sensitivity, the analytes eluted from the Fe3O4@MIPs were analyzed by the on-line sample preconcentration large volume sample stacking (LVSS) technique with MEKC. Under optimal concentration conditions, signal enhancements of 781, 3276 and 8916-fold were achieved for 4-CP, 2,4-DCP and 2,4,6-TCP, respectively. The average recoveries of the three analytes in spiked water samples were 99.33–104.5% with a relative standard deviation (RSD) below 6.8%. The detection limits for 4-chlorophenol, 2,4-dichlorophenol and 2,4,6-trichlorophenol were 1.1 × 10−3, 2.13 × 10−4 and 6.68 × 10−5 μg mL−1, respectively.

Glucose-β-CD interaction assisted ACN field-amplified sample stacking in CZE for determination of trace amlodipine in beagle dog plasma

A simple, sensitive and low-cost method using CE coupled with glucose-β-CD interaction assisted ACN stacking technique has been developed for quantification of trace amlodipine in dog plasma. The plasma samples were extracted with methyl tert-butyl ether. The separation was performed at 25°C in a 31.2 cm × 75 μm fused-silica capillary with an applied voltage of 15 kV. The BGE was composed of 6.25 mM borate/25 mM phosphate (pH 2.5) and 5 mg/mL glucose-β-CD. The detection wavelength was 200 nm. Because CD could diminish the interaction between drugs and matrix, and derivation groups of CD play an important role in separation performance, the effects of β-CD, and its derivatives on the separation were studied at several concentrations (0, 2.5, 5.0, 10.0 mg/mL). In this study, organic solvent field-amplified sample stacking technique in combination with glucose-β-CD enhanced the sensitivity about 60-70 folds and glucose-β-CD could effectively improve the peak shape. All the validation data, such as accuracy, precision extraction recovery, and stability, were within the required limits. The calibration curve was linear for amlodipine from 1 to 200 ng/mL. The method developed was successfully applied to the pharmacokinetic studies of amlodipine besylate in beagle dogs.

Determination of alkaloids inSinomenium acutumby field-amplified sample stacking in capillary electrophoresis with chemiluminescene detection

A simple and rapid capillary electrophoresis (CE) with an acidic potassium permanganate chemiluminescence (CL) detection method was developed to determine three alkaloids (curine, sinomenine and magnoflorine) simultaneously. A laboratory-built CE-CL detection interface was used. The field-amplified sample stacking technique was applied to the online concentration of alkaloids. Experimental conditions for CE separation and CL detection were investigated in detail to acquire optimum conditions. Under optimal conditions, the three alkaloids were baseline separated within 6 min, and the detection limits (S/N = 3) ranged from 0.03 µg/mL to 0.49 µg/mL. This method was successfully applied to determine the above three alkaloids in Sinomenium acutum, and the result of the determination of sinomenine was in good agreement with those given by high-performance liquid chromatography and CE methods. In addition, a possible CL reaction mechanism of sinomenine-KMnO4-H2SO4 was proposed.

Improved nonaqueous capillary electrophoresis for tetracyclines at subparts per billion level

A NACE method with laser-induced fluorescence detection was modified for sensitive detection of 4 tetracyclines (TCs) in biological samples and feeds. The changes in injection mode, injection times, id of capillary, excitation wavelength, and the use of surfactant and sample stacking technique all contributed to improved LODs of TCs to sub-ng/mL level. With the optimized conditions, the instrumental LODs could reach 1.33 ng/mL for chlorotetracycline (CTC) and 13.3 ng/mL for TC, oxytetracycline (OTC), and doxycycline (DC), an improvement of 10-100-fold over past studies. A simple SPE procedure was further developed for the extraction and concentration of TCs in plasma, urine, feed, and milk. Taken together, the instrumental LOD and feasible SPE concentration factors the overall LODs for CTC could reach 65 pg/mL in feed and milk and 260 pg/mL in plasma and urine. Detection limits for TC, OTC, and DC at sub-ng/mL level were also achieved. The modified CE-LIF method was found to be less complicated and more sensitive than the best current methods using UV or LIF detection, and has been applied successfully to assess oral absorption of DC in swine and chickens and to confirm suspected TC-positive bovine serum samples.

Review of recent developments of on-line sample stacking techniques and their application in capillary electrophoresis

Abstract Capillary electrophoresis (CE) has become one of the most useful tools in separation science because of its high separation efficiency, low cost, versatility, ease of sample preparation and automation. However, some limitations of CE, such as poor concentration sensitivity due to its lower sample loading and shorter optical path length, limits its further applications in separation science. In order to solve this problem, various on-line sample preconcentration techniques such as transient isotachophoresis preconcentration, field-enhanced sample stacking, micelle to solvent stacking, micelle collapse, dynamic pH junction, sweeping, solid phase extraction, single drop microextraction and liquid phase microextraction have been combined with CE. Recent developments, applications and some variants together with different combinations of these techniques integrating in CE are reviewed here and our discussions will be confined to the past three years (2008–2011).

Analysis of plant hormones by microemulsion electrokinetic capillary chromatography coupled with on-line large volume sample stacking

A novel method of microemulsion electrokinetic capillary chromatography (MEEKC) coupled with on-line large volume sample stacking was developed for the analysis of six plant hormones including indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, 1-naphthaleneacetic acid, abscisic acid and salicylic acid. Baseline separation of six plant hormones was achieved within 10 min by using the microemulsion background electrolyte containing a 97.2% (w/w) 10 mM borate buffer at pH 9.2, 1.0% (w/w) ethyl acetate as oil droplets, 0.6% (w/w) sodium dodecyl sulphate as surfactant and 1.2% (w/w) 1-butanol as cosurfactant. In addition, an on-line concentration method based on a large volume sample stacking technique and multiple wavelength detection was adopted for improving the detection sensitivity in order to determine trace level hormones in a real sample. The optimal method provided about 50-100 fold increase in detection sensitivity compared with a single MEEKC method, and the detection limits (S/N = 3) were between 0.005 and 0.02 μg mL(-1). The proposed method was simple, rapid and sensitive and could be applied to the determination of six plant hormones in spiked water samples, tobacco leaves and 1-naphthylacetic acid in leaf fertilizer. The recoveries ranged from 76.0% to 119.1%, and good reproducibilities were obtained with relative standard deviations (RSDs) less than 6.6%.

Gas dispersion concentration of trace inorganic contaminants from fuel gas and analysis using head-column field-amplified sample stacking capillary electrophoresis

The presence of inorganic elements in fuel gas generally accelerates the corrosion and depletion of materials used in the fuel gas industry, and even leads to serious accidents. For identification of existing trace inorganic contaminants in fuel gas in a portable way, a highly efficient gas-liquid sampling collection system based on gas dispersion concentration is introduced in this work. Using the constructed dual path gas-liquid collection setup, inorganic cations and anions were simultaneously collected from real liquefied petroleum gas (LPG) and analyzed by capillary electrophoresis (CE) with indirect UV absorbance detection. The head-column field-amplified sample stacking technique was applied to improve the detection limits to 2-25 ng mL(-1). The developed collection and analytical methods have successfully determined existing inorganic contaminants in a real LPG sample in the range of 4.59-138.69 μg m(-3). The recoveries of cations and anions with spiked LPG samples were between 83.98 and 105.63%, and the relative standard deviations (RSDs) were less than 7.19%.

Determination of benzimidazole residues in animal tissue samples by combination of magnetic solid-phase extraction with capillary zone electrophoresis

Benzimidazole drugs (BZDs) comprise a large number of synthetic anthelmintics, which are widely used in food-producing animals for prophylactic and therapeutic purposes. To protect consumers from the risks related to BZDs residues, a simple, rapid, and efficient method for simultaneous determination of ten BZDs in animal tissues samples was developed. This analytical procedure involved extracting samples with magnetic solid-phase extraction (MSPE) using magnetite/silica/poly (methacrylic acid-co-ethylene glycol dimethacrylate) (Fe3O4/SiO2/poly (MAA-co-EGDMA)) magnetic microspheres, and determination by capillary zone electrophoresis (CZE). To improve the sensitivity of the method, we employed the electrokinetic injection with field-amplified sample stacking technique (FASS). Berbine solution was used as internal standard to minimize the fluctuation of analytical results. Under the optimized extraction conditions, good linearities were obtained for the ten BZDs with the correlation coefficients (R2) above 0.9920. The limits of detections (LODs) for ten BZDs were 1.05–10.42ng/g in swine muscle and 1.06–12.61ng/g in swine liver, respectively. The intra- and inter-day relative standard deviations (RSDs) of the developed method were less than 13.6%. The recoveries of the ten BZDs for the spiked samples ranged from 81.1% to 105.4% with RSDs less than 9.3%.