On-line Sample Concentration Research Articles

Determination of lysergic acid diethylamide (LSD) in mouse blood by capillary electrophoresis/ fluorescence spectroscopy with sweeping techniques in micellar electrokinetic chromatography.

The separation and on-line concentration of lysergic acid diethylamide (LSD) in mouse blood was achieved by means of capillary electrophoresis/fluorescence spectroscopy using sodium dodecyl sulfate (SDS) as the surfactant. Techniques involving on-line sample concentration, including sweeping micellar electrokinetic chromatography (sweeping-MEKC) and cation-selective exhaustive injection-sweep-micellar electrokinetic chromatography (CSEI-sweep-MEKC) were applied; the optimum on-line concentration and separation conditions were determined. In the analysis of an actual sample, LSD was found in a blood sample from a test mouse (0.1 mg LSD fed to a 20 g mouse; approximately 1/10 to the value of LD(50)). As a result, 120 and 30 ng/mL of LSD was detected at 20 and 60 min, respectively, after ingestion of the doses.

Determination of corticosterone in mouse plasma by a sweeping technique using micellar electrokinetic chromatography

The separation and on-line concentration of corticosterone in mouse blood was achieved by means of capillary electrophoresis/UV absorbance using sodium dodecyl sulfate (SDS) as a surfactant. The procedure involved the use of an on-line sample concentration method by sweeping-micellar electrokinetic chromatography (sweeping-MEKC). Optimal on-line concentration and separation conditions were determined. The detection limit for this method was 5 ng/ml ( S/ N=3) and photodiode array detection at 247 nm was used for identification. For the analysis of actual samples, corticosterones from blood samples of a non-stressed and stressed mouse were determined. The results show that only a minor amount of corticosterone was produced by a non-stressed mouse, whereas a significant amount was present in the blood sample from a stressed mouse. The method developed here can be used to examine corticosterone levels as a marker of stress in test animals and may also be used for estimating the effect of stress-release medications.

Recent progress in adsorbed stationary phases for capillary electrochromatography.

This review surveys the recent progress in the adsorbed stationary phases for capillary electrochromatography (CEC). Adsorption-based methods for preparation of stationary phase are novel approaches in CEC, which allow rapid and facile preparing stationary phases with desirable selectivity onto an open-tubular fused-silica capillary, a bare-silica or ion-exchange packed column or a monolithic silica or polymer column. A variety of adsorbing agents have been developed as adsorbed stationary phases, including ionic long-chain surfactant, protein, peptide, amino acid, charged cyclodextrin (CD), basic compound, aliphatic ionene, and ion-exchange latex particle. The adsorbed stationary phases have been applied to separation of neutral, basic and acidic organic compounds, inorganic anions and enantiomers. They have also been applied to on-line sample concentration, fast separation and study of the competitive binding of enantiomers with protein.

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.

Anion selective exhaustive injection-sweep–micellar electrokinetic chromatography

A selective on-line sample concentration technique of anion selective exhaustive injection (ASEI)-sweep–micellar electrokinetic chromatography (MEKC) was evaluated using a cationic surfactant. To suppress the electroosmotic flow, a polyacrylamide-coated capillary was introduced. Some aromatic carboxylic acids, dansyl amino acids, and naphthalenedisulfonic acids were used as test analytes. About 1000- to 6000-fold increases in detection sensitivity were obtained in terms of peak heights by ASEI-sweep–MEKC.

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.

Application of sweeping to micellar electrokinetic chromatography-atmospheric pressure chemical ionization-mass spectrometric analysis of environmental pollutants.

An application study of sweeping, an on-line sample concentration technique, to micellar electrokinetic chromatography (MEKC) directly combined with mass spectrometry (MS) using an atmospheric pressure chemical ionization (APCI) interface, namely MEKC-APCI-MS, was investigated to enhance the concentration sensitivity for the analysis of environmental pollutants. Under a neutral condition, around 100-fold increase in the concentration sensitivity was achieved for several aromatic amines and alkyl phthalates as test samples by sweeping-MEKC-APCI-MS compared to conventional MEKC-APCI-MS, whereas under an acidic condition, 100 to 600-fold sensitivity enhancement was gained for similar solutes. Linearity of the corrected peak area obtained in the mass chromatogram against the sample concentration was examined for 3,4-dichloroaniline and diisopropyl phthalate (DIPP). The estimated limits of detection for 3,4-dichloroaniline and DIPP were 0.6 and 0.4 ppm, respectively, in terms of the injected sample concentration.

On-line sample concentration in micellar electrokinetic chromatography using cationic surfactants

Two on-line sample concentration techniques, sample stacking and sweeping, were evaluated using cationic surfactants as pseudostationary phases in micellar electrokinetic chromatography. As cationic surfactant micelles, tetradecyltrimethylammonium bromide and cetyltrimethylammonium chloride were employed. About 10-fold and 1000-fold increases in detection sensitivity in terms of peak heights were observed by sample stacking and sweeping, respectively, without suppression of the electroosmotic flow. In particular, the concentration limits of detection ( S/ N=3) for test naphthalenesulfonic acids obtained with sweeping were from 0.96 to 0.47 ppb with UV detection without any preconcentration procedure.

On-line sample concentration in micellar electrokinetic chromatography with cationic micelles in a coated capillary

The electroosmotic flow was successfully suppressed even in the presence of cationic surfactants, when a polyacrylamide-coated capillary was employed. Two on-line sample concentration techniques of sample stacking and sweeping were evaluated in micellar electrokinetic chromatography (MEKC) using the polyacrylamide-coated capillary. Cationic surfactants were used as pseudostationary phases in MEKC. At least 60-fold and about 600-fold increases in detection sensitivity were obtained in terms of peak heights by sample stacking and sweeping, respectively.

Direct chiral resolution of lactic acid in food products by capillary electrophoresis

Chiral resolution of native dl-lactic acid was performed by capillary electrophoresis using 2-hydroxypropyl-β-cyclodextrin as a chiral selector. Various factors affecting chiral resolution, migration time, and peak area of lactic acid were studied. The running conditions for optimum separation of lactic acid were found to be 90 m M phosphate buffer (pH 6.0) containing 240 m M 2-hydroxypropyl-β-cyclodextrin with an effective voltage of −30 kV at 16°C, using direct detection at 200 nm. In order to enhance the sensitivity, sample injection was done under a pressure of 50 mbar for 200 s. On-line sample concentration was accomplished by sample stacking. With this system, d- and l-lactic acids in food products were analyzed successfully.

Head-column field-amplified sample stacking in binary system capillary electrophoresis: Preparation of extracts for determination of opioids in microliter amounts of body fluids

Head-column field-amplified sample stacking (head-column FASS) is an efficient, on-line sample concentration technique that can easily provide a sensitivity enhancement of three orders of magnitude. Application of head-column FASS to the capillary electrophoretic analysis of opioid extracts prepared from 20 to 100 μl of human plasma, serum or urine is reported. In the described approach, efficient concentration of cationic opiates from low conductivity extracts of body fluids is effected across a water plug, with separation taking place in a binary buffer comprising 60% (v/v) ethylene glycol, 75 mM Na2HPO4 and 25 mM NaH2PO4 (pH 7.9), and detection is effected at 210 nm. Sample extracts are prepared in 55% (v/v) ethylene glycol containing 100 μM H3PO4. Application of mixed-mode polymer solid-phase resins is shown to provide extracts that are either too salty or contain quite a large number of endogenous substances that could interfere with certain opioids. Liquid–liquid extraction with hexane, dichloromethane, ethyl acetate and dichloromethane–isopropanol is shown to provide extracts that are sufficiently clean. At a given pH, however, only closely related opioids can be extracted. Using ethyl acetate at alkaline pH, dihydrocodeine and nordihydrocodeine can reproducibly be recovered from 20–100 μl of plasma, serum and urine. Application of head-column FASS and UV absorption detection thereby leads to the determination of ppb concentrations (≥1 ng/ml) of these compounds, an approach that only requires microliter amounts of sample and organic solvents.

Sample stacking of fast-moving anions in capillary zone electrophoresis with pH-suppressed electroosmotic flow

On-line sample concentration of fast moving inorganic anions by large volume sample stacking (LVSS) and field enhanced sample injection (FESI) with a water plug under acidic conditions is presented. Detection sensitivity enhancements were around 100 and 1000-fold for LVSS and FESI, respectively. However, reproducibility and linearity of response in the LVSS approach is superior compared to the FESI approach.

Quantitative analysis of 4-aminobiphenyl-C8-deoxyguanosyl DNA adducts produced in vitro and in vivo using HPLC-ES-MS.

Electrospray mass spectrometry (ES-MS) is a powerful tool for analysis of carcinogen-adducted DNA. In this study, we developed a quantitative isotope dilution method for analysis of N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-4-ABP), the principal nucleoside adduct derived from enzymatic hydrolysis of 4-aminobiphenyl (4-ABP)-modified DNA. The method used column switching valves to perform on-line sample concentration and cleanup, which permitted direct analysis of enzymatic DNA hydrolysates using narrow-bore liquid chromatography (LC). ES-MS detection was performed using a single quadrupole instrument by monitoring M+H+ and two fragment ions characteristic for dG-C8-4-ABP, along with M+H+ and a fragment ion for the deuterated internal standard. The detection limit for dG-C8-4-ABP in DNA hydrolysates was approximately 10 pg on-column, equivalent to 0.7 dG-C8-4-ABP adducts in 10(7) normal nucleotides for a sample containing 100 microg DNA. The method was applied to the analysis of calf thymus DNA modified in vitro through reaction with N-hydroxy-4-ABP and of hepatic DNA isolated from mice treated in vivo with two dose levels of 4-ABP.

Sample concentration by sample stacking and sweeping using a microemulsion and a single-isomer sulfated β-cyclodextrin as pseudostationary phases in electrokinetic chromatography

Two of the powerful on-line sample concentration techniques, sample stacking and sweeping under pH-suppressed electroosmotic flow, have been evaluated using a microemulsion and a single-isomer sulfated β-cyclodextrin derivative in electrokinetic chromatography. Several clinically relevant steroids have been separated and concentrated using a microemulsion consisting of 100 m M sodium dodecyl sulfate, 41 m M n-heptane and 700 m M 1-butanol in 50 m M phosphoric acid (pH 1.9). Three environmentally relevant phenoxy acid herbicides and their enantiomers have been separated and concentrated using a background electrolyte consisting of 20 m M hepta-6-sulfato-β-cyclodextrin in 15 m M phosphoric acid (pH 1.9). Significant detector response improvements have been achieved and utilized for analysis of a relatively clean matrix, lake water.

On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins.

We have used on-line sample clean-up, concentration, and chromatography with electrospray ionization mass spectrometry (ESI-MS), to characterize and determine the presence of disulfide bonds in recombinant full-length rat brain calbindin D28K and two deletion mutants of the protein, one lacking EF-hand 2 (calbindin delta 2) and the other lacking EF-hands 2 and 6 (calbindin delta 2,6). The molecular weights of the expressed proteins dissolved in biological buffers were determined with high accuracy using a low-flow, pressurized chamber infusion system, that allows on-line protein clean-up by removing buffers/salts incompatible with ESI-MS. The molecular weight determinations showed that the amino-terminal methionine residues had been cleaved during the expression and isolation of the recombinant proteins. Approximately 85-90% of the protein sequences were confirmed by on-line HPLC-ESI-MS analysis of peptides generated by a lysyl endoproteinase C digestion. Comparisons of ESI-MS spectra of native and reduced calbindin D28K and delta 2 show that the full length- and delta 2 mutant-protein contain one disulfide bond. Molecular mass determinations of calbindin delta 2,6 showed that this protein contains a highly active cysteine residue that covalently binds a mercaptoethanol group, or forms a homodimer via a disulfide bond. The results show surprising differences amongst the deletion mutants of calbindin D28K with respect to the formation of disulfide bonds. These differences are not readily detected by other techniques and show that ESI-MS is a powerful, rapid method by which to detect disulfide linkages for intact proteins.

Improvement of detection sensitivity in micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) as well as other capillary electrophoretic modes suffers from low concentration sensitivity due to low sample volume and limited pathlength for on-capillary photometric detection. On-line sample concentration techniques have been developed by simple manipulation of sample and background solution constituents without alteration of MEKC instrumentation, which are sample stacking and sweeping. These focusing techniques provide from 10 to more than 5000-fold improvement of the detector response. The enhancements are equivalent to 1-3 orders of magnitude improvement in the limit of detection. They are simple and are easily transferable technologies.

Micellar Electrokinetic Chromatography

Abstract A review of the research on micellar electrokinetic chromatography (MEKC), carried out mainly in our laboratory, is described from the viewpoints of (1) fundamental characteristics of MEKC, such as the separation principle, chromatographic parameters, selectivity and resolution, thermodynamic parameters, retention index, and band broadening; (2) selectivity manipulation, including effects of surfactants, temperature, pH, and various additives; (3) on-line coupling of MEKC with mass spectrometry; (4) applications, such as the separation of closely related compounds and enantiomer separations; and (5) on-line neutral sample concentration techniques.

On-column sample preconcentration using sample matrix switching and field amplification for increased sensitivity of capillary electrophoretic analysis of physiological samples.

An on-line sample concentration method using sample matrix switching and field amplification peak stacking has been developed. A microbore LC guard column is used to slightly retain the analytes in order to switch from a high ionic strength sample matrix (the physiological fluid) to a low ionic strength matrix (the LC mobile phase). The eluted LC peak is then trapped in a CE system and preconcentrated by field amplification peak stacking. The concentrated sample peak is then analyzed by CE. Compared to normal hydrodynamic injection, the sensitivity was increased by more than 500-fold without loss in resolution. A limit of detection of less than 10 nM for a physiological sample was achieved using UV adsorption detection. This method can be used for negatively or positively charged analytes.

Improved photometric detection of metal ions by capillary zone electrophoresis after precapillary complexation

Precapillary reactions using complexing reagents are increasingly being used to carry out separations of metal ions by capillary zone electrophoresis. To compensate for the sensitivity limitations of on-column photometric detection, several approaches were evaluated. Parameters affecting the sensitivity response of a direct detection system, such as the detection wavelength, sample size, applied voltage, and electrolyte concentration, were examined to produce the best possible sensitivity for metal ions complexed with cyclohexane-1,2-diaminetetraacetic acid. Using injection in the electrokinetic mode, a 2-fold increase in detectability over hydrodynamic (gravity) injection was obtained for certain metal analytes that corresponds to the detection limits in the sub-μg/l range. A characterization and optimization of photometric detection in an indirect format have been conducted. An organic dye, combined with the carrier electrolyte to impart an absorbance background in the visible range and selected with due account for its spectral and electrophoretic characteristics, was found to provide micromolar-level sensitivities. Feasibility of on-line (stacking) sample concentration for metal complexes has also been demonstrated.

On-line concentration of neutral analytes for micellar electrokinetic chromatography: II. Reversed electrode polarity stacking mode

Basic conditions for the on-line neutral sample concentration by reversed electrode polarity stacking mode for micellar electrokinetic chromatography are developed theoretically and experimentally verified. Stacking is mainly dependent on analyte retention factors and nature of the pseudostationary phases. More than an order of magnitude improvement in concentration detection limit is achieved for resorcinol using high molecular mass surfactants.