Indirect photometric detection in capillary zone electrophoresis

An indirect photometric detection method is described which is based on the use of an absorbing co-ion as the principal component of the background electrolyte. The zones of non-absorbing ionic species are revealed by changes in light absorption due to charge displacement of the absorbing co-ion. Theoretical considerations are given for selecting a suitable absorbing co-ion to achieve a high sensitivity of detection.The role of electromigration dispersion is illustrated by experiments and the effects of the differences in the effective mobilities of sample ions and that of the absorbing co-ion are discussed. The highest sensitivity can be achieved for sample ions having an effective mobility close to the mobility of the absorbing co-ion. In such a case, the concentration of the sample component in its migrating zone can be high while electromigration dispersion is still negligible. The useful dynamic range of the detection is then limited by the linearity and noise of the detector, the former parameter being given mostly by the shape of the on-column detection cell. The best sensitivities can be obtained in low-concentration background electrolytes containing a co-ion with high absorption at a given detection wavelength.It is shown that indirect photometric detection can be useful for detecting substances that have no optical absorption in the UV and/or visible region, provided that the composition of the background electrolyte is selected correctly.     

Indirect laser-induced fluorescence detection of diuretics separated by capillary electrophoresis

Indirect LIF detection was applied to the detection of four acidic diuretics separated by CZE. Semiconductor laser was employed to provide the stable excitation of 473 nm. With an optimized electrophoretic buffer system which contained 5 mM of triethylamine, 0.1 microM of fluorescein, and 5% of n-butanol, fast separation of four diuretics (ethacrynic acid, chlorthalidone, bendroflumethiazide, and bumetanide) can be performed within 3 min with the detection limits of 0.2-2 microg/mL. The impacts of buffer components including the concentrations of the electrolytes, fluorescence probe, and the organic additives were demonstrated. The method was applied for the detection of diuretics in urine. As an alternative way for the fast analysis of diuretics, this indirect detection method provided the technical support for future microchip performances, in which diuretics may be detected in the microchip by the common LIF detector without derivatization.     

Indirect fluorometric detection of tryptic digests separated by capillary zone electrophoresis

This work demonstrates the analytical utility of indirect fluorescence detection with capillary zone electrophoresis (CZE) for the analysis of trace quantities of macromolecular mixtures. Detection is based upon charge displacement and is not based upon any absorption or emission property of the analyte. No derivatization step is required. Indirect fluorescence is therefore a general detector for electrophoresis. Subfemtomolar quantities of tryptic digest mixtures are separated within three minutes, and reproducible peaks are obtained from the mixtures. Mass limits of detection are 3000 times lower than those of commercial high-performance liquid chromatography (HPLC) UV absorbance detectors and 180 times lower than those of UV absorbance detectors in CZE systems. This separation and detection system should be well suited to analysis of trace quantities of mixtures of peptides.     

Pulsed amperometric detection of calystegines separated by capillary electrophoresis

Calystegines are polyhydroxyalkaloids with a nortropane skeleton. They are oxidized by pulsed amperometry at a gold electrode due to their vicinal hydroxyl groups similar to monosaccharides, but at a slightly higher potential. Compared to carbohydrates, calystegines exhibit lower acidity, thus the effective electrophoretic mobility as anions in 0.1 M NaOH is lower, independent of their molecular mass. The acidity and mobility of calystegines increase with the number of hydroxyl groups. The influence of temperature and power dissipation in the capillary and changes of the inner surface on the migration times was eliminated by cooling and subtraction of the electroosmotic flow velocity. The high resolving power of capillary zone electrophoresis allows the separation of calystegines with the same number of hydroxyl groups. Detection is linear from 2 to 200 mg L(-1) with a 1 nL injection volume. Calystegines were determined in crude plant sap after filtration without further sample purification.     

Determination of melatonin in biological samples by capillary electrophoresis

The determination of melatonin (MLT) in physiological samples was investigated using capillary electrophoresis (CE). Mouse blood was collected in tubes containing EDTA, centrifuged at 1500 g for 20 min at 4°C, and stored at −20°C. Plasma samples were extracted with dichloroethane, centrifuged and the aqueous phase was discarded. Then the organic phase was evaporated to dryness. The residue was dissolved in deionized water and filtered with a microfilter (0.22 μm). Separations were carried out using a CE system equipped with a fused silica capillary [80 cm (effective length 52 cm)×75 μm I.D.] and an ultraviolet–visible detector (200 nm), and programmed to provide 25 mM 2-(N-morpholino)ethanesulfonic acid (pH 5.7). Injection was performed hydrostatically by elevating the sample by 10 cm at the cathodic side of the capillary. The calibration curve, reproducibility, recovery and limit of detection were examined, and validation of the method was performed. The result showed that MLT in blood could be easily determined with the new method.     

Indirect thermo-optical detection for capillary electrophoresis

Indirect thermo-optical detection for capillary electrophoresis is described first. A 20 mW helium-neon laser (632.8 nm) was used to provide the pumping beam and a 2 mW helium-neon laser (632.8 nm) supplied as the probe beam; Methylene Blue dye was used as a background absorber. The addition of ethanol to the background electrolyte solution can be performed to reduce adsorption of Methylene Blue onto the capillary wall. The detection method was applied to the detection of amino acids separated by capillary electrophoresis. The detection limit for lysine was 5 × 10⁻⁶ mol l⁻¹ (signal-to-noise ratio, 2).     

Direct tris(2,2'-bipyridyl)ruthenium (II) electrochemiluminescence detection of polyamines separated by capillary electrophoresis

Capillary electrophoresis (CE) with tris(2,2'-bipyridyl) ruthenium (II) (Ru(bpy)3(2+)) electrochemiluminescence (ECL) detection technique was developed for the analysis of four polyamines (putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm)) analysis. The four polyamines contain different amine groups, which have different ECL activity. There are several parameters which influence the resolution and ECL peak intensities, including the buffer pH and concentrations, separation voltage, sample injection, electrode materials, and Ru(bpy)3(2+) concentrations. Polyamines are separated by capillary zone electrophoresis in an uncoated fused-silica capillary (50 cmx25 micro m (ID) filled with acidic phosphate buffer (200 mmol/L phosphate, pH 2.0) - 1mol/L phosphoric acid (9:1 v/v) and a separation voltage of 5 kV (25 micro A), with end-column Ru(bpy)3(2+) ECL detection. A 5 mmol/L Ru(bpy)3(2+) solution plus 200 mmol/L phosphate buffer (pH 11.0) is added into the reagent reservoir. The calibration curve is linear over a concentration range of two or three orders of magnitude for the polyamines. The analysis time is less than 25 min. Detection limits for Put and Cad are 1.9x10(-7) mol/L and 7.6x10(-9) mol/L for Spd and Spm, respectively. Intraday and interday relative standard deviations of ECL peak intensities are less than 8%. The main advantages of this CE-ECL detection technique for polyamines analysis presented herein are the omission of chemical derivatization of the analytes and the high selectivity.     

Indirect detection of organic acids in non-aqueous capillary electrophoresis.

Non-aqueous capillary electrophoresis (NACE) with indirect detection has been applied to the determination of fatty acids (FAs) and ascorbic acid (AA), respectively. C2-C18 FAs have been separated in less than 12 min using 8-hydroxy-7-iodoquinoline sulfonic acid as chromophores in NACE with indirect absorbance. The dissociation constant (pKa) values of C8-C18 FAs obtained from the slope of the linear plot -log [(mu 0/mu)-1] vs. pH, using 20% isopropanol and 40% acetonitrile as the organic modifier in NACE, are all above about two units than those obtained in aqueous solution. NACE with indirect laser-induced fluorescence, using merocyanine 540 (MC540) as fluorophores, has been performed to the analysis of AA and its stereoisomer, isoascorbic acid (IAA), and the limits of detection of AA and IAA are 0.30 microM and 0.17 microM, respectively. This method has been applied to the determination of AA in a lemon juice spiked with IAA as the internal standard in less than 3 min and its concentration is 76.7 +/- 0.4 mM.     

Monitoring of transferrin isoforms in biological samples by capillary electrophoresis

Work dealing with the monitoring of transferrin isoforms in human serum and other body fluids by capillary electrophoresis is reviewed. It comprises capillary zone electrophoresis and capillary isoelectric focusing efforts that led to the exploration and use of assays for the determination of carbohydrate‐deficient transferrin as a marker for excessive alcohol intake, genetic variants of transferrin, congenital disorders of glycosylation and β‐2‐transferrin, which is a marker for cerebrospinal fluid leakage. This paper provides insight into the development, specifications, strengths, weaknesses, and routine use of the currently known capillary electrophoresis based assays suitable to detect transferrin isoforms in body fluids. The achievements reached so far indicate that capillary zone electrophoresis is an attractive technology to monitor the molecular forms of transferrin in biological specimens as the assays do not require an elaborate sample pretreatment and thus can be fully automated for high‐throughput analyses on multicapillary instruments. Assays based on capillary isoelectric focusing are less attractive. They require immunoextraction of transferrin from the biological matrix and mobilization after focusing if instrumentation with a whole‐column imaging detector is not available. Interactions of the carrier ampholytes with the iron of transferrin may prevent iron saturation and thus provide more complicated isoform patterns.     

Indirect thermal lens detection for capillary electrophoresis

Thermal lens detection with a 325.0 nm He-Cd excitation laser is used for thermooptical indirect detection in combination with the capillary electrophoretic separation of organic anions. The optimization of indirect thermooptical detection is discussed. With Mordant Yellow 7 (an azo dye) chosen as a probe ion limits of detection for 1-heptane-, 1-pentane-, 1-butane-, 1-propanesulfonic, and acetic acid at a level of n × 10⁻⁷ M were achieved with a separation electrolyte containing 50 μM of the probe ion and 5 mM Tris pH 9.90. A further increase in the detection sensitivity (twofold decrease in the limit of detection ) was obtained with a separation electrolyte containing a volume fraction of 20% acetonitrile.     

Determination of agmatine in biological samples by capillary electrophoresis with optical fiber light-emitting-diode-induced fluorescence detection

A capillary electrophoresis (CE)/optical fiber light-emitting diode (LED)-induced fluorescence detection method is developed for the determination of agmatine in biological samples. The agmatine was precolumn-derivatized with fluorescence tagging reagent, fluorescein isothiocyanate (FITC). Optimal separation and determination for agmatine were obtained with an electrophoretic buffer of 20 mM sodium borate (pH 9.2). Under the optimal conditions, the determination of agmatine was achieved in less than 4 min, and the detection limit was 4.1x10(-9) M (S/N = 3). The relative standard deviation (RSD) for 11 parallel determination of agmatine was less than 3.0%. The present CE-LED induced fluorescence detection method has been applied to detect agmatine in rat brain tissue, rat stomach tissue, human serum, and human urine. The level of agmatine in human urine was quantified by CE for the first time and found to be in the range 2.5-4.1x10(-7) M.     

Determination of monoamine transmitters and tyrosine in biological samples by capillary electrophoresis with electrochemical detection

Summary Capillary electrophoresis (CE) has been employed for the separation of monoamine transmitters (MAs) and tyrosine (Tyr), combined with electrochemical detection (ED) at a carbon disc electrode. The effects of several factors such as the acidity and concentration of running buffer, the separation voltage, the potential applied to the working electrode and the injection time were investigated to find the optimum conditions. Detection limits (S/N=3) ranged from 48.8 to 315.4 nmol·L⁻¹, and the response was linear over 3 order of magnitude for MAs and Tyr. The proposed method was successfully applied to determine MAs and Tyr in the cerebral cortex, thalamus and spinal cord of rats with satisfactory assay results.     

Electrofilament deposition and off-column detection of analytes separated by capillary electrophoresis

Capillary electrophoresis interfaced with electrospray is a convenient technique for continuously transferring column effluent from capillary-to-planar format. Conditions are optimized to produce a narrow (approximately 20 microm) liquid filament (electrofilament), which is capable of depositing spatially focused bands with track widths that are routinely 100 microm. A fiber optic-based, laser-induced fluorescence cell is employed to monitor the separation on-column while the separated bands are deposited onto a moving substrate. The photodetection of deposited bands is accomplished by using either a charge-coupled device camera or a photomultiplier tube. Deterioration of on-column separation performance is observed when the electrofilament voltage is applied. Elevating the inlet of the capillary column, to provide hydrodynamic flow, restores separation performance. Substrate temperature and translational rates are optimized with respect to both off-column separation efficiency and signal intensity. Off-column separation efficiencies of 65 000 plates per meter were achieved. A linear dynamic range of 10(3) and a limit of detection of 10(-8) M were obtained for kiton red deposited onto a reversed phase thin-layer chromatography plate. To demonstrate the applicability of this technique to more complex separation solutions, a dye mixture was successfully separated and deposited with sodium dodecyl sulfate in the running buffer.     

Indirect UV detection of carbohydrates in capillary zone electrophoresis

Summary A new system for the rapid and sensitive analysis of underivatized carbohydrates has been established using capillary zone electrophoresis with indirect UV detection. At an applied potential of 28 kV, sugars and sugar acids could be separated by the combined effects of electroendosmosis and electrophoresis within 20 minutes in a fused silica capillary of 50 m internal diameter and an effective length of 100 cm using 6mM sorbic acid, pH 12.1, as both carrier electrolytie and chromophore. The alkaline pH ensured ionization of the sugars and, hence, their detection by means of charge displacement. Furthermore, the chosen concentration of sorbic acid allowed the smallest fractional change in the background signal to be measured. While the electrophoretic mobilities of the sugars were found to increase within a pH range of 11.9 to 12.3, those of the sugar acids were not affected. Due to the increasing competition of hydroxide ions in the displacement of the chromophore with rising pH, a significant loss of sensitivity is observed at pH values higher than 12.1 and this pH was found to provide sufficient resolution, optimum sensitivity, and a acceptably short analysis time. Under these conditions, a lower detection limit of 2 pmol was obtained for glucose.     

Indirect photometric detection of anions in nonaqueous capillary electrophoresis employing Orange G as probe and a light-emitting diode-based detector

A method based on indirect photometric detection (IPD) in CE employing a blue LED (473 nm) as a light source and the highly absorbing (478 nm) anionic dye, Orange G, as the probe ion was developed for the sensitive analysis of inorganic and organic anions. The use of nonaqueous solvents was examined as a simple way to reduce the adsorption of the dye onto the capillary wall and to thereby improve the baseline stability. The benefits of this approach were confirmed by experiments using BGEs in methanol (MeOH) and DMSO in which superior baselines were obtained relative to those achieved using aqueous electrolyte systems. A range of commercial LEDs was tested to improve the detection performance, with a difference of 25% in sensitivity being observed between the best and worst performing LED. The final system (4 mM Orange G, 0.05% w/v hydroxypropylcellulose (HPC), 20 mM triethanolamine (TEA) in pure MeOH) exhibited stable baselines and very low LODs (0.10-0.18 microM) for a test mixture comprising nine inorganic and organic anions. These values represent a two- to six-fold improvement over previous studies and the proposed method provides the most sensitive IPD method for the determination of anions using CE published to date. RSDs for ten replicates were in the ranges of 0.42-0.62% for migration time, 1.41-3.46% for peak area and 3.20-5.78% for peak height.     

Analysis of proteins in biological samples by capillary sieving electrophoresis with postcolumn derivatization/laser-induced fluorescence detection

Previously, we have demonstrated postcolumn derivatization of proteins separated by capillary sieving electrophoresis (CSE), in which naphthalene-2,3-dicarbaldehyde was employed as a fluorogenic labeling reagent. Standard proteins separated by CSE were reacted with naphthalene-2,3-dicarbaldehyde in the presence of 2-mercaptoethanol (2-ME) which plays a role of a reducing agent in the derivatization reaction. To improve the sensitivity, we attempted the use of ethanethiol instead of 2-ME. Ethanethiol showed 1.4- to 4.5-fold lower limits of detection for proteins than 2-ME. Furthermore, we found that 8-aminopyrene-1,3,6-trisulfonate (APTS) is a good marker for relative electrophoretic mobilities of proteins in CSE. Since APTS is a fluorescent and trivalent anion, it generates strong fluorescence and migrates faster than any of the proteins. Therefore, we employed APTS as a marker to obtain the relative electrophoretic mobilities of proteins. The present method was applied to the analyses of proteins in biological samples. Human Ewing's family tumor cell line 'RDES' was used as a sample. The cultured cells were lysed with a buffer containing Tris-HCl, NaCl, sodium dodecyl sulfate, and 2-ME. After denaturation, the lysate was directly introduced into the capillary. Several peaks, which would correspond to proteins with molecular mass ranging from 10 to 93?kDa, were found in the cell lysate. In addition, we measured a milk sample by the CSE with postcolumn derivatization. The electropherogram showed five major peaks which corresponded to α-lactalbumin, β-lactoglobulin, κ-casein, bovine serum albumin, and mixture of α- and β-casein.     

Analysis of oxycodol and noroxycodol stereoisomers in biological samples by capillary electrophoresis

A capillary electrophoresis (CE) method for the separation of the diastereoisomers of 6-oxycodol (6OCOL) and nor-6-oxycodol (N6OCOL), the 6-keto-reduced metabolites of oxycodone (OCOD) and noroxycodone (NOCOD), respectively, is reported and employed to assess the stereoselectivity of these metabolic steps in vivo, in vitro, and in chemical synthesis. CE in an untreated fused-silica capillary with acidic buffers containing 2-hydroxypropyl-beta-cyclodextrin, randomly sulfated beta-cyclodextrin, or single isomer heptakis(2,3-diacetyl-6-sulfato)-beta-cyclodextrin (HDAS-beta-CD) is shown to permit the simultaneous separation of the stereoisomers of 6OCOL and N6OCOL. A 100 mM phosphate buffer of pH 2.0 containing 2.05% w/v HDAS-beta-CD provides a medium for rapid analysis and unambiguous identification of these stereoisomers in solid-phase extracts of (i) urines stemming from patients under pharmacotherapy with OCOD, (ii) incubations of OCOD and NOCOD with human liver cytosol and the human liver S9 fraction, and (iii) after chemical synthesis from OCOD and NOCOD using NaBH(4). In all cases, alpha-N6OCOL is shown to be the predominant stereoisomer of N6OCOL. For 6OCOL, the same is true for in vitro formation and for chemical synthesis. In urine, however, beta-6OCOL is observed to be excreted in a higher amount than alpha-6OCOL. For the urinary alpha-/beta-isomer ratio of 6OCOL and N6OCOL, there are no differences between the data obtained for nonhydrolyzed and enzymatically hydrolyzed urines. The data document the stereoselectivity of the 6-keto-reduction of OCOD and NOCOD in man.     

Indirect determination of Fe(III) in synthetic samples and iron dextran tablets by capillary electrophoresis with ultraviolet detection

A capillary electrophoresis method based on the oxidation of ascorbic acid is proposed for the indirect determination of Fe(III). Fe(III) concentration corresponds to the decrease in ascorbic acid peak area. The calibration graph was linear in the range of 1.68-112 mg/L for Fe(III), which was easily detected at a concentration of 1.12 mg/L at 3 times the standard deviation of the blank divided by the slope of the calibration graph. The lack of interferences from Fe(II) in synthetic samples and 3 excipients (starch, magnesium strearate, and microcrystalline cellulose) in dextran tablets in the determination of Fe(III) confirmed the high selectivity of the proposed method. Its application to the determination of Fe(III) in several synthetic samples and iron dextran tablets produced excellent results.     

Determination of bromate ion in drinking water by capillary zone electrophoresis with direct photometric detection

Bromate ion in drinking water was determined by capillary zone electrophoresis (CZE) with direct photometric detection. Bromate ion in the sample solution was introduced and concentrated into the capillary by electrokinetic injection for 50s at -10 kV. Electrophoretic separation was made at an applied voltage of -25 kV and bromate ion was detected at wavelength 193 nm, at which the baseline was stabilized with less UV-absorbing acidic phosphate buffer. Bromate ion was detected within 5 min in the electropherogram. By increasing the electric conductivity in the migrating solution with 10 mM Na2SO4, a limit of detection (LOD) of 9 x 10(-10)M (0.1 microg/L BrO3-) was achieved. The proposed method was applied to the analysis of tap water and river water samples, but bromate ion was not detected. Because the practical samples contain relatively large amount of foreign ionic substances, the tap water sample was diluted to avoid the matrix ions. Bromate ion added in a tap water at the concentration of 8 x 10(-8)M was quantitatively recovered by diluting it 1/10.