Large-volume Sample Stacking Research Articles

Simultaneous determination of effective components in Gualou Guizhi granules using microemulsion electrokinetic chromatography coupled with large volume sample stacking

A method using microemulsion electrokinetic chromatography (MEEKC) coupled with large volume sample stacking was developed for the quantitative analysis of five compounds in Gualou Guizhi granules.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2012-2014).

One of the most cited limitations of capillary (and microchip) electrophoresis is the poor sensitivity. This review continues to update this series of biannual reviews, first published in Electrophoresis in 2007, on developments in the field of on-line/in-line concentration methods, covering the period July 2012-July 2014. It includes developments in the field of stacking, covering all methods from field-amplified sample stacking and large-volume sample stacking, through to ITP, dynamic pH junction, and sweeping. Attention is also given to on-line or in-line extraction methods that have been used for electrophoresis.

Dendritic glycopolymers as dynamic and covalent coating in capillary electrophoresis: View on protein separation processes and detection of nanogram-scaled albumin in biological samples

Unique properties of dendritic polymers make them promising candidates for application as additives in various analytical methods. From this point, we investigated the potential use of maltose-modified hyperbranched poly(ethylene imine) (PEI-Mal) as a dynamically or covalently bound coating and a pseudostationary phase in capillary electrophoresis. The EOF mobilities were measured at different pH values (2.2, 8.5, and 10.2) using PEI-Mal in the background electrolyte (BGE) and desirable repeatability of the EOF with % RSD (n=50) ≤3.3 was obtained. The influence of pH, polymer concentration, and density of maltose shell on the separation properties of a model mixture of four proteins (albumin, lysozyme, myoglobin, insulin) were investigated. Applying PEI-Mal as a dynamic coating, improved separation of the protein mixture with a high repeatability was achieved. Applying PEI-Mal as a covalent coating for concentrating proteins in the large volume sample stacking (LVSS) combined with the field-enhanced sample injection (FESI), up to 1320-fold enhancement of sensitivity was achieved. The detection limit of 100–500ng/ml allowed successful analysis of albumin level both in blood and urine samples without additional preconcentration.

Progress in stacking techniques based on field amplification of capillary electrophoresis.

Numerous strategies have been developed to mitigate the intrinsic low detection sensitivity that is a limitation of capillary electrophoresis. Among them, in-line stacking is an effective strategy to address the sensitivity challenge, and among the different stacking techniques, stacking based on field amplification is the most effective and simplest method of achieving high sensitivity without special complicated mechanisms or operations. This review introduces several stacking techniques based on field amplification. Field-amplified sample stacking, large-volume sample stacking, matrix field-amplified stacking injection (FASI), head-column FASI, matrix FASI combined with head-column FASI, FASI coupled with extraction and clean-up methods, electrokinetic supercharging, cation-anion selective exhaustive injection-sweeping-micellar electrokinetic chromatography, and newly developed techniques based on field amplification combined with other methods are included, and examples of straightforward methods for solving the sensitivity problem are provided. We also present a brief overview of the advantages, limitations, and future developments of these techniques.

Large volume sample stacking for rapid and sensitive determination of antidiabetic drug metformin in human urine and serum by capillary electrophoresis with contactless conductivity detection

Two CE methods with contactless conductivity detection have been developed for determining the oral antidiabetic drug metformin in human urine and blood. The determination of metformin is performed on a separation capillary with an effective length of 14cm, using a maximum voltage of 30kV and with a small injection of 50-fold diluted urine into the capillary. Under these conditions, the migration time of metformin is 35s and the LOD is 0.3μM. Large-volume sample stacking was used to determine low metformin levels in serum. The injection of a sample of serum deproteinized with acetonitrile was 10 times greater compared to the injected amount of urine. This enabled reduction of the LOD to 0.03μM and the metformin migration time equalled 86s. The undesirable solvent from sample zone was forced out of the capillary to ensure rapidity and good repeatability of the determination. The RSD values for the migration time are 0.1% for urine and 0.7% for serum; RSD for the peak areas equalled 1.4% for urine and 2.6% for serum. The developed CE technique was tested on performance of routine analyses of metformin in the urine and serum of patients suffering from type II diabetes mellitus.

Large‐volume sample stacking with polarity switching for monitoring of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) reactions by capillary electrophoresis

Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is a membrane glycoprotein involved in the hydrolysis of extracellular nucleotides. Its main substrate is ATP yielding AMP and pyrophosphate. NPP1 has been proposed as a novel drug target, for diabetes type 2 and the treatment of calcium pyrophosphate dihydrate deposition disease leading to inflammatory arthritis. The monitoring of NPP1 reactions is difficult because its velocity is very slow requiring highly sensitive analytical procedures. In this study, a method of large-volume sample stacking with polarity switching was developed, and separations were optimized. Large sample volumes were loaded by hydrodynamic injection (5 psi, 13 s) followed by removal of a large plug of sample matrix from the capillary using polarity switching (-10 kV). The stacked analytes were subsequently separated in phosphate buffer (100mM, pH 9.2) at 20 kV. The validated method was found to be linear (R(2) = 0.9927) in the concentration range of 0.05-50μM of AMP, with high accuracy and precision. The determined LOD and LOQ of AMP were 18 nM and 60 nM, respectively. Compared to a previously reported CE procedure using sweeping technique, a fivefold improvement of sensitivity was achieved. Moreover, the new technique was faster, and reproducibility of migration times was improved (RSD value = 1.2%). Importantly, adenine nucleotide analogs and derivatives tested as NPP1 inhibitors could be completely separated from the substrate ATP and the enzymatic product AMP. The method was applied to NPP1 inhibition assays investigating nucleotide-derived inhibitors in the presence of ATP.

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.

On-line Sample Preconcentration by Large-volume Sample Stacking with an Electroosmotic Flow Pump (LVSEP) in Microscale Electrophoresis

This review highlights on-line sample preconcentration by large-volume sample stacking with an electroosmotic flow pump (LVSEP) in microscale electrophoresis. LVSEP requires only a hydrodynamic sample injection into the whole capillary/microchannel and a constant voltage application, i.e., without polarity switching, providing an efficient sensitivity enhancement with a relatively easy experimental procedure without loss of the separation efficiencies. Recent developments and applications of LVSEP are briefly reviewed from the first LVSEP study in 1999 with categorization on the basis of separation modes, including zone electrophoresis, cyclodextrin- and affinity-based separation modes and non-aqueous capillary electrophoresis.

Toward 10 000‐fold sensitivity improvement of oligosaccharides in capillary electrophoresis using large‐volume sample stacking with an electroosmotic flow pump combined with field‐amplified sample injection

A combination of two online sample concentration techniques, large-volume sample stacking with an electroosmotic flow pump (LVSEP) and field-amplified sample injection (FASI), was investigated in CE to achieve highly sensitive oligosaccharide analysis. In CE with LVSEP-FASI, analytes injected throughout the capillary were concentrated on the basis of LVSEP, followed by an electrokinetic introduction of concentrated analytes from the inlet vial by the FASI mechanism. After switching the inlet vial solution from the sample to running buffer, the concentrated analytes were then separated by CZE. In the present LVSEP-FASI-CZE, pressure was applied to the capillary inlet until the inlet vial solution was exchanged. The applied pressure generated a counterflow against the EOF. It kept the stacked sample zone within the capillary, minimizing loss of concentrated analytes. Fluorescein was first analyzed by LVSEP-FASI-CZE to optimize preconcentration condition. Up to 110 000-fold sensitivity increase was obtained with 200 μL of sample, compared to normal CZE with sample injection of 0.3psi for 3 s (ca. 1.7 nL). From the results, the pressure application improved the efficiency of the FASI-mode concentration significantly at total concentration time longer than 10 min. In the analysis of maltoheptaose, a 10 000-fold sensitivity increase was achieved, which is the highest concentration efficiency ever reported in CE of oligosaccharides. The relative standard deviations of the detection time and peak height were 2.4 and 11%, respectively. In the analysis of glucose oligomer, up to 8600-fold sensitivity increases were achieved without reducing the separation performance of conventional CZE.

Large-volume sample stacking for in vivo monitoring of trace levels of γ-aminobutyric acid, glycine and glutamate in microdialysates of periaqueductal gray matter by capillary electrophoresis with contactless conductivity detection

A new variant of large-volume sample stacking injection (LVSS) was used in the capillary electrophoresis with capacitively coupled contactless conductivity detection (CE/C4D) determination of the neurotransmitters γ-aminobutyric acid (GABA), glycine (Gly) and glutamate (Glu) in microdialysates of periaqueductal gray matter (PAG). The separation capillary was filled to 98% from the injection side with a sample of microdialysate in acetonitrile. Simultaneously with turning on the separation voltage, the sample zone was forced out by the background electrolyte by increasing the pressure in the terminal capillary outlet vessel. As a consequence of the stacking effect, the analyte was concentrated from the large sample volume into a narrow zone at the sample/background electrolyte boundary close to the injection end of the capillary. Under these conditions, LOD values of 9, 10 and 15nM were determined in the model samples for GABA, Gly and Glu, respectively; RSD equalled 0.5% for the migration times and 1.0–1.9% for the peak areas, respectively. In analysis of microdialysates of PAG, LOD values of 29, 29 and 37nM were determined for GABA, Gly and Glu, respectively; RSD equalled 0.5–0.7% for the migration times and 2.6–8.2% for the peak areas, respectively. The determined basal levels of the neurotransmitters in PAG microdialysates are 0.08, 4.7 and 0.8μM for GABA, Gly and Glu, respectively. Carrageenan-induced hyperalgesia increases the Gly and Glu levels and reduces GABA in PAG microdialysate. Peroral administration of paracetamol in hyperalgesia effectively reduces the Gly value and has no effect on Glu and GABA.

Analysis of ten abused drugs in urine by large volume sample stacking–sweeping capillary electrophoresis with an experimental design strategy

A statistical tool equipped with Plackett–Burman design (PBD) and central composite design (CCD) was used for fast stacking analysis of ten frequently consumed drugs, namely codeine, morphine, methamphetamine, ketamine, alprazolam, clonazepam, diazepam, flunitrazepam, nitrazepam and oxazepam, by capillary electrophoresis (CE). This statistical design is expected to help quick analysis with few procedures, avoiding tedious work required because of the large number of variables or parameters. A large volume sample stacking (LVSS)–sweeping CE is developed for concentrating and analyzing the 10 abused drugs. First, phosphate buffer (50mM, pH 2.3) containing methanol was filled into a capillary and then the extracted urine sample was loaded (1psi, 200s) to enhance sensitivity. The sweeping and separating steps were completed simultaneously by phosphate buffer (50mM, pH 2.3) containing methanol and sodium dodecyl sulfate, within 15min. Better resolution was obtained by the experimental design than the “one factor at a time” (OFAT) approach. During method validation, calibration plots were linear (r>0.998), over a range of 25–1500ng/mL for the six benzodiazepines, methamphetamine and ketamine, and 50–3000ng/mL for codeine and morphine. The RSD of precision and absolute RE of accuracy in intra-day and inter-day assays were below 14.54% and 16.61%, respectively. The minimum limits for detection (S/N=3) of analytes were in the range of 7.5–30ng/mL. This stacking method increased sensitivity more than 200-fold and can be applied for detection of the presence of methamphetamine in an abuser's urine (3600ng/mL), which was confirmed by GC–MS. The method is considered feasible for fast screening of abused drugs in urine.

On‐line preconcentration and determination of six sulfonylurea herbicides in cereals by MEKC with large‐volume sample stacking and polarity switching

A new MEKC method with large-volume sample stacking and polarity switching was developed for on-line preconcentration and detection of sulfonylurea herbicide (SUH) residues in cereals, including nicosulfuron (NS), thifensulfuon (methyl) (TFM), tribenuron-methly (TBM), sulfometuron-methyl (SMM), pyrazosulfuron-ethyl (PSE), and chlorimuron-ethyl (CME). In order to achieve a high resolution and enrichment factor, several parameters were optimized, such as the pH of the running buffer, the concentration of the BGE and the SDS, the separate voltage, the sample size, the pH, and the electrolyte concentration of the sample. The optimal running buffer was composed of 30 mM borate and 80 mM SDS at pH 7.0. The borate concentration in the sample was 30 mM and the pH value of the sample was the same as that of the running buffer. The concentrating voltage and the separating voltage were -15 kV and 15 kV, respectively. The sample size was 1.455kPa × 780 s (33.11 cm). Under the optimum conditions, for NS, TFM, TBM, SMM, PSE, and CME, the enrichment factors were 613, 642, 835, 570, 709, and 599; the LODs were 0.29-0.50 ng/g, 0.22-0.36 ng/g, 0.60-0.89 ng/g, 0.39-0.72 ng/g, 0.28-0.56 ng/g, and 0.31-0.57ng/g; the LOQs of six SUHs were all 5 ng/g; the average recoveries of the spiked sample were 86.68-92.99%, 80.73-93.65%, 81.49-94.40%, 82.97-95.1%, 82.96-98.84%, and 80.41-92.94%, respectively.

Recent advances in on-line concentration and separation of amino acids using capillary electrophoresis

This article highlights recent methodological developments in the on-line concentration and separation of amino acids and their enantiomers using capillary electrophoresis. Sections are dedicated to recent contributions to on-line concentration strategies such as field-amplified sample stacking, large-volume sample stacking, dynamic pH junction, transient isotachophoresis, sweeping, and the combination of two methods. The main applications, advantages, and limitations of these procedures in the biological, food, and pharmaceutical fields are addressed. Comprehensive tables listing on-line techniques for the concentration and separation of amino acids and their enantiomers, categorized by the stacking strategies used, background electrolytes, sample matrix, limit of detection, and enhancement factor, are provided.

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.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2010–2012)

CE has been alive for over two decades now, yet its sensitivity is still regarded as being inferior to that of more traditional methods of separation such as HPLC. As such, it is unsurprising that overcoming this issue still generates much scientific interest. This review continues to update this series of reviews, first published in Electrophoresis in 2007, with updates published in 2009 and 2011 and covers material published through to June 2012. It includes developments in the field of stacking, covering all methods from field amplified sample stacking and large volume sample stacking, through to isotachophoresis, dynamic pH junction and sweeping. Attention is also given to online or inline extraction methods that have been used for electrophoresis.

Chiral speciation and determination of selenomethionine enantiomers in selenized yeast by ligand-exchange micellar electrokinetic capillary chromatography after solid phase extraction

A new phenylalanine derivative (l-N-(2-hydroxy-propyl)-phenylalanine, l-HP-Phe) was synthesized and its chelate with Cu(II) (Cu(II)–(l-HP-Phe)2) was used as the chiral selector for the ligand-exchange (LE) chiral separation of d,l-selenomethionine (SeMet) in selenized yeast samples by micelle electrokinetic capillary chromatography (MEKC). In order to improve the sensitivity of MEKC–UV, two-step preconcentration strategy was employed, off-line solid phase extraction (SPE) and on-line large volume sample stacking (LVSS). d,l-SeMet was first retained on the Cu(II) loaded mesoporous TiO2, then eluted by 0.1mL of 5molL−1 ammonia, and finally introduced for MEKC–UV analysis by LVSS injection after evaporation of NH3. With the enrichment factors of 1400 and 1378, the LODs of 0.44 and 0.60ngmL−1 for l-SeMet and d-SeMet was obtained, respectively. The developed method was applied to the analysis of d,l-SeMet in a certified reference material of SELM-1 and a commercial nutrition yeast, and the results showed that most of SeMet in the SELM-1 selenized yeast was l isomer and the recovery for l and d isomers in the spiked commercial nutrition yeast was 96.3% and 103%, respectively. This method is featured with low running cost, high sensitivity and selectivity, and exhibits application potential in chiral analysis of seleno amino acids in real world samples.

Analysis of carbamazepine and its five metabolites in serum by large-volume sample stacking–sweeping capillary electrophoresis

This study establishes a method, using different buffer conductivities and large-volume sample stacking (LVSS)-sweeping capillary electrophoresis, for analysis of carbamazepine (CBZ) and its five metabolites in serum. The capillary (50/60 cm) was filled with a high concentration of background electrolyte (150 mM phosphate, pH 3.5, containing 15 % methanol), followed by a large volume of samples (10 psi, 20 s) with low-concentration buffers (5 mM phosphate, pH 3.5, with 5 % methanol). When high voltage was applied (-20 kV), the sodium dodecyl sulfate (SDS) started to sweep the analytes to an outlet. Meanwhile, the analytes decelerated at the boundary between low- and high-conductivity buffers. Finally, a narrow sample zone was formed. The procedure of sweeping and separation was simultaneously carried out by a sweeping buffer (150 mM phosphate, pH 3.5) with 15% methanol and 50 mM SDS added, and the detection was performed by UV at 214 nm. The method was validated for linearity (r >/= 0.997), precision, and accuracy. The calibration curves were established for CBZ and its five metabolites between 0.03-25 and 0.03-3 μg/mL. The limits of detection (S/N = 3) were 0.01 μg/mL for each analyte. Compared with simple MEKC (0.5 psi, 5 s), this system can improve the sensitivity about 300-fold. Finally, this method was successfully applied to five patients, who had taken 200 mg CBZ daily, and CBZ levels were found to be from 3.72 to 5.82 μg/mL.

Electrophoretic analysis of cations using large-volume sample stacking with an electroosmotic flow pump using capillaries coated with neutral and cationic polymers

To realize the high-performance and simple-operation analysis of cationic compounds in capillary electrophoresis, we investigated large-volume sample stacking with an electroosmotic flow pump (LVSEP) using capillaries with hydrophilic and weakly cationic inner surface. Three capillary modification methods were employed: thermally passivated physical coating with polymer mixture of poly(vinyl alcohol) and poly(allylamine); covalent modification with random copolymer of acryl amide and 3-(methacryloylamino)propyltrimethylammonium chloride; easily preparable physical coating with dimethyldioctadecylammonium bromide and polyoxyethylene stearate. In these capillaries, the electroosmotic flow (EOF) was well suppressed in the high ionic strength (I) electrolyte under the acidic and basic pH, whereas the EOF was enhanced in the low I electrolyte, indicating a suitable EOF property for the rapid LVSEP and following separation. In the LVSEP-capillary zone electrophoresis (CZE) analyses of benzylamine and 1-naphthylethylamine, up to 550-fold sensitivity increases were successfully obtained in the three capillaries without significantly reducing the repeatability and resolution. LVSEP-cyclodextrin-modified CZE of chlorpheniramine and brompheniramine was also carried out, resulting in up to 380-fold sensitivity enhancement with keeping the baseline separation for the enantiomers. Finally, we performed the LVSEP-CZE analysis of basic proteins, where up to 100-fold sensitivity increases were achieved, but a peak broadening was observed due to the sample adsorption in the low I sample matrix.

Headspace‐single drop microextraction with a commercial capillary electrophoresis instrument

Automated coupling of headspace-single drop microextraction (HS-SDME) and CE has been demonstrated using a commercial CE instrument. When a drop hanging at the inlet tip of a capillary for CE is used as the acceptor phase, HS-SDME becomes a simple but powerful sample pretreatment technique for CE before injection to facilitate sample cleanup and enrichment. By combining HS-SDME with an on-line sample preconcentration technique, large volume sample stacking using an electroosmotic flow pump, the sensitivity can be improved further. The overall enrichment factors for phenolic compounds were from 1900 to 3400. HS-SDME large volume sample stacking using an electroosmotic flow pump was successfully applied to a red wine sample to obtain an LOD of 4 nM (0.8 ppb) for 2,4,6-trichlorophenol which is a precursor for 2,4,6-trichloroanisole causing the foul odor in wine called cork taint.

Nonaqueous capillary electrophoresis of imatinib mesylate and related substances

In the present study, nonaqueous capillary electrophoretic separation of imatinib mesylate (IM) and related substances, N-(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-pyrimidinamine (PYA), N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-((piperazin-1-yl)methyl) benzamide (NDI) and 4-chloromethyl-N-(4-methyl-3-((4-(pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) benzamide (CPB) was developed. The influential factors affecting separation, including type and concentration of the electrolyte, applied voltage, and buffer modifier were investigated. Baseline separation of the studied analytes was obtained using a buffer of 50 mM Tris and 50 mM methanesulfonic acid in methanol at a apparent pH (pH*) of 1.65. To enhance the sensitivity, large-volume sample stacking was employed for online concentration. The strongest analytical signal with a suitable separation was achieved when the injection time was 100 s. The linearity ranges of PYA and NDI were 0.100-2.50 μg mL(-1), and that of CPB was 0.125-2.50 μg mL(-1), with good coefficients (r(2) > 0.9948). The relative standard deviations of intra- and interday were satisfactory. Under the optimized conditions, seven batches of the synthesized samples were analyzed and CPB was detected in two batches. Owing to its simplicity, effectiveness, and low price, the developed method is promising for quality control of IM.