Buffer Modifier Research Articles

Analytical quality by design-based development of a capillary electrophoresis method for Omeprazole impurity profiling

Omeprazole (OME) is a proton pump inhibitor used to treat gastroesophageal reflux disease associated conditions. The current study presents an Analytical Quality by Design-based approach for the development of a CE method for OME impurity profiling. The scouting experiments suggested the selection of solvent modified Micellar ElectroKinetic Chromatography operative mode using a pseudostationary phase composed of sodium dodecyl sulfate (SDS) micelles and n-butanol as organic modifier in borate buffer. A symmetric three-level screening matrix 37//16 was used to evaluate the effect of Critical Method Parameters, including Background Electrolyte composition and instrumental settings, on Critical Method Attributes (critical resolution values, OME peak width and analysis time). The analytical procedure was optimized using Response Surface Methodology through a Central Composite Orthogonal Design. Risk of failure maps made it possible to define the Method Operable Design Region, within which the following optimized conditions were selected: 72 mM borate buffer pH 10.0, 96 mM SDS, 1.45 %v/v n-butanol, capillary temperature 21 °C, applied voltage 25 kV. The method was validated according to ICH guidelines and robustness was evaluated using a Plackett-Burman design. The developed procedure enables the simultaneous determination of OME and seven related impurities, and has been successfully applied to the analysis of pharmaceutical formulations.

Measurement of sweat lactate levels in exercise and non-exercise activities using capillary electrophoresis system with contactless conductivity detection and cyclodextrin-modified buffer

This study presents the analysis of lactate in sweat using capillary electrophoresis with contactless conductivity detection. Two cyclodextrin (CD) compounds, carboxymethyl-β-cyclodextrin sodium salt (CM-β-CD) and heptakis (2,3,6-tri-O-benzoyl)-β-cyclodextrin (TRIME-β-CD), were evaluated as buffer modifiers for the separation of chloride, nitrate, sulfate, oxalate, maleate, acetate, lactate, and phosphate anions. The concentrations of these modifiers were tested at 0.05, 0.1, and 0.5 mM for eight anions separation. The buffer was 40.0 mM MES/L-His (pH 6.0) with 0.05 mM CTAB, resulting in anodic separation. Due to separation efficiency in resolving lactate and phosphate peaks, TRIME-β-CD at a concentration of 0.1 mM was selected as the suitable buffer additive for sweat lactate analysis. The calibration of the developed method, using maleate as an internal standard, resulted in the linear ranges of 0.1–5.0 mM, r2 of 0.9999, and an instrumental LOD of 0.042 mM. The intra-day and inter-day precisions of the relative migration time (RMT) were 0.3–0.4% and 3–4% RSD, respectively. Lactate levels in sweat samples from the same group of volunteers were measured after two distinct activities: exercise (after running) and non-exercise (after sauna). For non-exercise activities, lactate concentrations ranged from 15 ± 1 mM to 36 ± 2 mM. While, after exercise, the concentrations were between 26 ± 1 mM and 136 ± 1 mM. Further evaluation of the stability of sweat storage for monitoring lactate contents, it was found that the changes in lactate levels were insignificant when stored for up to 24 weeks at 4 °C. This was confirmed by paired t-tests (t-stat ragnes from -0.59 to 2.49; t-critical = 2.57, P = 0.05). The measured lactate concentrations from volunteers were significantly higher, approximately 2–6 times, in exercise activities compared to non-exercise activities. This substantial difference in lactate secretion indicates a clear distinction in the factors that stimulate sweat lactate production. To evaluate the intensity of exercise based on lactate levels in sweat, a proposed cut-off level of 36.0 mM, with a decision limit of 40.0 mM, can be proposed.

Application of capillary electrophoresis for assessing amino acids composition of Jujube from different geographical origins

A simple capillary electrophoresis (CE) method that using β-cyclodextrin (β-CD) as buffer modifier has been developed for the detection of ten amino acids of jujube from five different geographical origins. The CE methodology was optimized through the variation of type, pH and concentration of the buffer solution, concentration of the additive β-CD, applied voltage, injection time and wavelength of the UV detection. It was found that the best separation of ten kinds of amino acids was achieved within less than 6 min under the optimum separation conditions: 50 mmol L-1 pH 9.5 borate-phosphate buffer with 5.0 mmol L-1 β-CD, 15 kV applied voltage, 25 °C column temperature, 210 nm detection wavelength, and 5 s injection time. This method showed good repeatability with RSD values of 1.4-3.4% for peak area, and 1.4-4.7% for migration time, when β-CD was used as buffer modifier. Under the optimum conditions, the method has been successfully applied to the detection of actual jujube samples, which also verifies the effectiveness and practicability of the method. Recovery of real samples was in ranging of 90%-105%, which proved the feasibility of the method. It also proved that the method was successfully applied to the quantitative analysis of amino acids of interest in plant samples.

Simultaneous Determination of Phenolic Endocrine Disruptors in Water Samples by Poly(sodium 4-styrenesulfonate) Modified CE Coupled with Hollow-Fiber Liquid-Phase Microextraction

A CE combined with amperometric detection method has been developed for simultaneous determination of nine environmental endocrine disruptors, including bisphenol A, methylparaben, ethylparaben, propylparaben, 4-tert-butylphenol, diethylstilbestrol, 17α-ethinylestradiol, β-estradiol and estriol. In this work, various experimental parameters affecting enrichment factors, electrophoretic separation and detection were investigated in detail. And poly(sodium 4-styrenesulfonate) was selected as the buffer modifier, resulting in considerable improvement of the resolution of the target analytes. Under the optimum conditions, nine analytes could be well separated within 30 min in a 10 g/L poly(sodium 4-styrenesulfonate) modified 60 mmol/L Na2B4O7–H3BO3 buffer (pH 7.60). For each analyte good linear correlation between electrochemical response and its concentration at two or three grades was obtained, the method detection limits reached 0.047–13 ng/mL (S/N = 3) with the assistance of hollow-fiber liquid-phase microextraction, and the highest enrichment factor was up to 255-fold. This proposed method has been applied for the direct analyses of real environmental water and tap water samples without need of derivatization, the average recoveries were in the range of 81.4–117 %, and the corresponding relative standard deviations were lower than 6.2 %.

Optimization of Micellar Electrokinetic Chromatography Method for the Simultaneous Determination of Seven Hydrophilic and Four Lipophilic Bioactive Components in Three Salvia Species.

A micellar electrokinetic chromatography (MEKC) method was developed for the simultaneous determination of seven hydrophilic phenolic acids and four lipophilic tanshinones in three Salvia species. In normal MEKC mode using SDS as surfactant, the investigated 11 compounds could not be well separated. Therefore, several buffer modifiers including β-cyclodextrins (β-CD), ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF4) and organic solvents have been added to the buffer solution to improve the separation selectivity. Under the optimized conditions (BGE, 15 mM sodium tetraborate with 10 mM SDS, 5 mM β-CD, 10 mM [bmim]BF4 and 15% ACN (v/v) as additives; buffer pH, 9.8; voltage, 20 kV; temperature, 25 °C), the 11 investigated analytes could achieve baseline separation in 34 min. The proposed MEKC was additionally validated by evaluating the linearity (R2 ≥ 0.9965), LODs (0.27–1.39 μg·mL−1), and recovery (94.26%–105.17%), demonstrating this method was reproducible, accurate and reliable. Moreover, the contents of the 11 compounds in three Salvia species, including S. miltiorrhiza, S. przewalskii and S. castanea were analyzed. The result showed that the established MEKC method was simple and practical for the simultaneous determination of the hydrophilic and lipophilic bioactive components in Salvia species, which could be used to effectively evaluate the quality of these valued medicinal plants.

Simultaneous determination of oleanolic acid, ursolic acid, quercetin and apigenin in Swertia mussotii Franch by capillary zone electrophoresis with running buffer modifier.

The method of capillary zone electrophoresis (CZE) with direct UV detection was developed for the determination of oleanolic acid, ursolic acid, quercetin and apigenin. and then for the first time successfully applied to the analysis of four analytes in Swertia mussotii Franch and its preparations. Various factors affecting the CZE procedure were investigated and optimized, and the optimal conditions were: 50 × 10(-3) mol/L borate-phosphate buffer (pH 9.5) with 5.0 × 10(-3) mol/L β-cyclodextrin, 15 kV separation voltage, 20 °C column temperature, 250 nm detection wavelength and 5 s electrokinetic injection time (voltage 20 psi). Under the conditions, oleanolic acid, ursolic acid, quercetin and apigenin could be determined within the test ranges with a good correlation coefficient (r(2) > 0.9991). The limits of detection for conditions, oleanolic acid, ursolic acid, quercetin and apigenin were 0.3415, 0.2003, 0.0062 and 0.2538 µg/mL, respectively, and the intra- and inter-day relative standard deviations were no more than 4.72%. This procedure provided a convenient, sensitive and accurate method for simultaneous determination of oleanolic acid, ursolic acid, quercetin and apigenin in S. mussotii Franch.

Evaluation of ionic liquids-coated carbon nanotubes modified chiral separation system with chondroitin sulfate E as chiral selector in capillary electrophoresis

Nanoparticles (NPs) and ionic liquids (ILs) have been extensively studied and have aroused considerable interest in separation science; however, the employment of ILs-dispersed NPs as buffer modifiers for CE chiral separation has not been previously studied. In this work, we describe a new CE method using ILs dispersed multi-walled carbon nanotubes (ILs-MWNTs) as a modifier for enantioseparation with a polysaccharide, chondroitin sulfate E (CSE), as the chiral selector. As observed, significantly improved separations, including better enantioselectivity and improved peak shapes, were obtained in the ILs-MWNTs modified separation system for all drug enantiomers compared to the single CSE system. Several parameters affecting the enantioseparation, such as the choice of ILs and carbon nanoparticles, ILs-MWNTs concentration, chiral selector concentration, buffer pH and applied voltage, were systematically investigated. Satisfactory separations were achieved when 2.4μg/mL ILs-MWNTs were introduced into the 20mM Tris/H3PO4 buffer solution containing 2.5% CSE at pH 2.8–3.4 with a 15kV applied voltage. A brief mechanism of the enhanced enantioseparation capability of the ILs-MWNTs modified chiral separation system was also discussed.

Simultaneous determination of phenylethanoid glycosides and aglycones by capillary zone electrophoresis with running buffer modifier

Although the separation efficiency of capillary electrophoresis (CE) is much higher than that of other chromatographic methods, it is sometimes difficult to adequately separate the complex ingredients in biological samples. This article describes how one effective and simple way to develop the separation efficiency in CE is to add some modifiers to the running buffer. The suitable running buffer modifier β-cyclodextrin (β-CD) was explored to fast and completely separate four phenylethanoid glycosides and aglycones (homovanillyl alcohol, hydroxytyrosol, 3,4-dimethoxycinnamic acid, and caffeic acid) in Lamiophlomis rotata (Lr) and Cistanche by capillary zone electrophoresis with ultraviolet (UV) detection. It was found that when β-CD was used as running buffer modifier, a baseline separation of the four analytes could be accomplished in less than 20min and the detection limits were as low as 10−3mgL−1. Other factors affecting the CE separation, such as working potential, pH value and ionic strength of running buffer, separation voltage, and sample injection time, were investigated extensively. Under the optimal conditions, a successful practical application on the determination of Lr and Cistanche samples confirmed the validity and practicability of this method.

Validation of Quetiapine Fumarate in Pharmaceutical Dosage by Reverse‐Phase HPLC with Internal Standard Method

A rapid, specific, and accurate isocratic HPLC method was developed and validated for the assay of quetiapine fumarate in pharmaceutical dosage forms. The assay involved an isocratic‐elution of quetiapine fumarate in Grace C18 column using mobile‐phase composition of 0.1% ortho phosphoric acid with triethyl amine as modifier buffer and acetonitrile in the ratio of 50 : 50 (v/v).The wavelength of detection is 294 nm. The method showed good linearity in the range of 2.0–50.2 × 10−3 g/Lt. The runtime of the method is 5 mins. The developed method was applied to directly and easily analyse of the pharmaceutical tablet preparations. The percentage recoveries were near 100% for given methods. The method was completely validated and proven to be rugged. The excipients did not interfere in the analysis. The results showed that this method can be used for rapid determination of quetiapine fumarate in pharmaceutical tablet with precision, accuracy, and specificity.

Subzero Temperature Chromatography for Reduced Back-Exchange and Improved Dynamic Range in Amide Hydrogen/Deuterium Exchange Mass Spectrometry

Amide hydrogen/deuterium exchange is a commonly used technique for studying the dynamics of proteins and their interactions with other proteins or ligands. When coupled with liquid chromatography and mass spectrometry, hydrogen/deuterium exchange provides several unique advantages over other structural characterization techniques including very high sensitivity, the ability to analyze proteins in complex environments, and a large mass range. A fundamental limitation of the technique arises from the loss of the deuterium label (back-exchange) during the course of the analysis. A method to limit loss of the label during the separation stage of the analysis using subzero temperature reversed-phase chromatography is presented. The approach is facilitated by the use of buffer modifiers that prevent freezing. We evaluated ethylene glycol, dimethyl formamide, formamide, and methanol for their freezing point suppression capabilities, effects on peptide retention, and their compatibilities with electrospray ionization. Ethylene glycol was used extensively because of its good electrospray ionization compatibility; however, formamide has potential to be a superior modifier if detrimental effects on ionization can be overcome. It is demonstrated using suitable buffer modifiers that separations can be performed at temperatures as low as -30 °C with negligible loss of the deuterium label, even during long chromatographic separations. The reduction in back-exchange is shown to increase the dynamic range of hydrogen/deuterium exchange mass spectrometry in terms of mixture complexity and the magnitude with which changes in deuteration level can be quantified.

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.

In‐channel amperometric detection for microchip electrophoresis using a wireless isolated potentiostat

The combination of microchip electrophoresis with amperometric detection leads to a number of analytical challenges that are associated with isolating the detector from the high voltages used for the separation. While methods such as end-channel alignment and the use of decouplers have been employed, they have limitations. A less common method has been to utilize an electrically isolated potentiostat. This approach allows placement of the working electrode directly in the separation channel without using a decoupler. This paper explores the use of microchip electrophoresis and electrochemical detection with an electrically isolated potentiostat for the separation and in-channel detection of several biologically important anions. The separation employed negative polarity voltages and tetradecyltrimethylammonium bromide (as a buffer modifier) for the separation of nitrite (NO₂⁻), glutathione, ascorbic acid, and tyrosine. A half-wave potential shift of approximately negative 500 mV was observed for NO₂⁻ and H₂O₂ standards in the in-channel configuration compared to end-channel. Higher separation efficiencies were observed for both NO₂⁻ and H₂O₂ with the in-channel detection configuration. The limits of detection were approximately two-fold lower and the sensitivity was approximately two-fold higher for in-channel detection of nitrite when compared to end-channel. The application of this microfluidic device for the separation and detection of biomarkers related to oxidative stress is described.

Preparation and Characterization of Gold Nanoparticle-Modified Silica Monolith for Capillary Electrochromatography

Abstract A novel silica monolithic stationary phase functionalized with octadecanethiol gold nanoparticles (GNPs) was prepared for capillary electrochromatography (CEC). The bare silica monolith was prepared by a sol-gel process and then modified chemically with 3-mercaptopropyltrimethoxysilane, followed by immobilization of GNPs and modification by octadecanethiol. The relationship between plot of plate height and the linear velocity on octadecanethiol GNPs-modified silica monolith (Silica-GNPs-C18) was investigated, and a minimum plate height of about 7.8 μm for toluene was obtained. The electroosmotic flow characteristics of the Silica-GNPs-C18 and bare silica monolith (Silica) were studied by varying the percentage of organic modifier in buffer and buffer pH. To evaluate the column performance, hydrophobic alkylbenzenes as model compounds were tested on the prepared Silica, thiol-modified silica monolith (Silica-SH), and Silica-GNPs-C18. The electrochromatographic retention behavior of alkylbenzenes on Silica-GNPs-C18 column was also investigated. The experiment results showed that Silica-GNPs-C18 column exhibits typical reversed-phase electrochromatographic behavior toward neutral solutes. Reversed-phase CEC separations of four selected polar phenols were also demonstrated on Silica-GNPs-C18 column using mobile phase containing 15% methanol.

Separation of 1,4-benzodiazepine Derivates by Micellar Electrokinetic Capillary Chromatography Using Cyclodextrines as Buffer Modifiers

The applicability of micellar electrokinetic capillary chromatography (MEKC) for the separation of benzodiazepine (BZD) derivates with different structural characteristics has been studied. Efforts were first focused on the optimization of the analytical conditions, on the effects of buffer concentration, surfactant concentration and buffer pH on the separation. To manipulate selectivity of the separation βcyclodextrin (CD) was added to the buffer solution, and urea was used to improve the solubility of the CD in water. The CD-modified MEKC separation of the eight BZDs was achieved within 15 minutes using 25 mmol dm natrium tetraborate-50 mmol dm sodium dodecyl sulphate-15 mmol dm β-CD-2 mol dm urea (pH 9.3) as the running buffer. The proposed separation method was evaluated on the basis of precision, linearity and limit of detection. (doi: 10.5562/cca1763)

Separation of Dihydroergotoxine on Polar-Embedded Reversed-Phase Stationary Phase

Abstract The four components of dihydroergotoxine show different pharmacodynamic properties. An analytical method is required to check the compliance of ingredient and dosage forms with their specifications. Traditionally, the baseline separation of dihydroergotoxine can only be achieved with alkaline mobile phases, which damage the silica matrix badly. In this study, a polar-embedded reversed-phase stationary phase-octadecylamide (C 18 amide) bonded silica has been prepared and used to separate the four components of dihydroergotoxine with neutral mobile phases. The effects of the mobile phase composition including pH (3.0–8.0), buffer, and organic modifier on the retention behavior of the four components are investigated. A baseline separation of four components has been realized for the first time with a mobile phase, composed of acetonitril-20 mM Na 2 HPO 4 (30:70, v/v, pH 7.0), on a C 18 amide bonded silica column. Compared with the traditional methods, the method described here offers several advantages such as improving the separation performance of basic compounds and prolonging the service-time of the HPLC column.

Enhanced analysis of triterpenes, flavonoids and phenolic compounds in Prunella vulgaris L. by capillary zone electrophoresis with the addition of running buffer modifiers

A cyclodextrin-modified capillary zone electrophoresis method was developed for the separation and determination of three isomeric compounds (ursolic acid, oleanolic acid and betulinic acid), caffeic acid, p-coumaric acid, rosmarinic acid, rutin and quercetin. Without the addition of β-cyclodextrin (β-CD) and methanol, the separation of these analytes was poorly resolved. These eight compounds, however, were well separated from each other within 20 min with a borax running buffer (40 mM of borax, pH 9.4) containing 2 mM β-CD and 4% (v/v) methanol at the voltage of 25 kV, temperature of 25 °C and detection wavelength of 210 nm. The relative standard deviations (RSDs) of migration time ranged from 0.16 to 0.74% while those of the peak area ratios ranged from 2.17 to 4.61% for six determinations of the analytes at concentration of 10 and 25 μg mL −1. The correlation coefficients of the calibration curves of the analytes were all >0.998, and the recoveries were from 96.8 to 103.6%. The method was successfully applied to determine these bioactive components in the samples of Prunella vulgaris L. and its beverage drink products. Our results reveal that only the isomeric compounds and rosmarinic acid could be detected in the spikes of P. vulgaris L.; other components were either too low to be detected or not present while only rosmarinic acid was detected in the beverage products.

Simultaneous determination of flavonoids in chrysanthemum by capillary zone electrophoresis with running buffer modifiers

Despite the separation efficiency of capillary electrophoresis (CE) is much higher than other chromatographic methods, it is sometimes difficult to perfectly separate the complex ingredients in biological samples. One possible and simple way to develop the separation effect in CE is to add some modifiers in the running buffer. In this paper, the suitable running buffer modifiers were explored to simultaneously separate and detect six typical flavonoids (apigenin, luteolin, kaempferol, quercetin, (+)-catechin and (−)-epicatechin) which are the main active ingredients in chrysanthemum by capillary zone electrophoresis with amperometric detection (CZE-AD). It was found that when β-cyclodextrin (β-CD) and the mixture of methanol and ethanol were used as running buffer modifiers, a baseline separation of the six analytes could be accomplished in less than 20 min and the detection limits were as low as 10 −7 or 10 −8 g ml −1. Other factors affecting the CZE separation, such as working potential, pH value and ionic strength of running buffer, separation voltage and sample injection time were extensively investigated. Under the optimum conditions, a successful practical application on the determination of chrysanthemum samples confirmed the validity and practicability of this method.

Use of cyclodextrins as modifiers of fluorescence in the detection of mycotoxins

Cyclodextrins, cyclic oligosaccharides composed of amylose subunits, are known to interact with mycotoxins. The interactions may be useful to analytical chemists by altering the properties of the mycotoxin of interest, namely the chromatographic properties, electrophoretic properties, fluorescence, or absorption of these fungal metabolites. Practical applications of these effects have been the incorporation of cyclodextrins into high-performance liquid chromatography and capillary electrophoresis methods for mycotoxin detection. Specific mycotoxins include those with a native fluorescence such as the aflatoxins, ochratoxin A (OTA) and zearalenone (ZEN) as well as those that can be rendered fluorescent through derivatization, such as T-2 toxin. The literature describing the applications of cyclodextrins in mycotoxin analysis is reviewed and an attempt to extend the use of cyclodextrins to the detection of labelled T-2 toxin is presented. Twenty cyclodextrins were evaluated for their ability to enhance the fluorescence emission of T-2 toxin derivatized with pyrene-1-carbonyl cyanide (T2-Pyr). This evaluation revealed that heptakis (2,6-di-O-methyl)-β-cyclodextrin (DIMEB), in particular, enhanced T2-Pyr fluorescence. DIMEB was used as a buffer modifier in a capillary electrophoresis-laser-induced fluorescence (CE-LIF) method for detecting T-2 in maize. Because of the effects that certain cyclodextrins have, especially under aqueous conditions, they may make useful additives for a variety of mycotoxin analytical methods.

A Stability-Indicating LC Assay Method for Pemetrexed Disodium

This present work describes the development of a stability-indicating high performance liquid chromatographic method for the quantitative determination of pemetrexed disodium. Pemetrexed disodium is an antifolate antineoplastic agent that exerts its action by disrupting folate-dependent metabolic processes essential for cell replication. The chromatographic separation was achieved on an ACE 3 C18 HPLC column using a mobile phase consisting of a mixture of buffer (solvent A) and organic modifier acetonitrile (solvent B). Forced degradation studies were performed on bulk sample of pemetrexed disodium using acid (0.5 N hydrochloric acid), base (0.5 N sodium hydroxide), oxidation (10% v/v hydrogen peroxide), heat (60 °C) and UV light (254 nm). Degradation of the drug substance was observed in base hydrolysis. Degradation product formed under acid and base hydrolysis was found to be starting material. The stressed samples were assayed using the developed LC method and the mass balance found was close to 99.5%, thus proving its stability-indicating power. The developed method was validated with respect to linearity, accuracy, precision and robustness.

Rapid identification of purified enteropathogenic Escherichia coli by microchip electrophoresis

In this work, the potential of PDMS-based microchip electrophoresis in the identifications and characterizations of microorganism was evaluated. Enteropathogenic E. coli (EPEC) was selected as the model microorganism. In this study, separation parameters such as applied voltage, concentrations of buffer and buffer modifier, injection voltage, and duration of injection had been investigated and optimized. Determination of EPEC bacteria could be completed within 2 min with good reproducibility. RSDs were less than 0.5 and 5% in migration time and peak area, respectively. Separation efficiency corresponding to plate number of more than 100,000 was achieved. In order to obtain reproducible separations, sample pretreatment was found to be essential. Microchip electrophoresis with LIF detection could potentially revolutionize certain aspects of microbiology involving diagnosis, profiling of pathogens, environmental analysis, and many other areas of study.