Para-hydroxybenzoic acid as an indirect detection buffer in capillary zone electrophoresis

Summary Para-hydroxybenzoic acid (p-HBA) has been used as indirect UV detection buffer in capillary zone electrophoresis (CZE). Being an UV-absorbing dibasic acid, p-HBA provides both the necessary buffering for pH control over a wide range and UV absorbance for indirect detection. With sodium dodecyl sulfate (SDS) as a probe, a CZE method using p-HBA solution as running buffer was developed to analyze anions, especially ones with low electrophoretic mobilities. The method was used to separate homologous series of sulfonates, SDS in a formulation sample, and SDS in a standard.     

Detection of carbohydrates in capillary electrophoresis

This review focuses on recent developments in sensitive detection modes for carbohydrates after separation by capillary electrophoretic methods. To bring detection sensitivity for carbohydrates analysis in line with current methods in protein sequencing, concentration detection limits of 10⁻⁶ molar or better are requires. A discussion of mass detection limits and concentration detection limits is followed by an overview of detection modes for natural and labeled carbohydrates. Amperometric detection and UV and laser-induced fluorescence detection after reductive amination, in particular with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), are discussed in more detail. Finally, the paper outlines developments to be expected in the near future, focusing on the needs in glycobiology such as improved sensitivity and selectivity.     

Paper electrophoresis of steroids

Summary A radiochemical method for the identification of some steroids on electropherograms is described. A comparison between this method and another modified spectrophotometric method is given. The radiochemical method can be used for quantitative work; the relationship between the concentration of the hydrazones and the radioactivity gained by the steroids is linear. This technique is superior to any colorimetric one, it needs less reagents and is not time consuming. Application of the method to other steroids is continued.

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Zusammenfassung Eine radiochemische Methode zur Identifizierung einiger Steroide auf Elektropherogrammen wird beschrieben. Ein Vergleich mit einer modifizierten spektralphotometrischen Methode wird gegeben. Die radiochemische Methode ist für quantitatives Arbeiten geeignet. Die Beziehung zwischen Aktivität und Konzentration der Hydrazone ist linear. Die Methode ist besser als die colorimetrischen; sie verbraucht weniger Reagentien und ist nicht so zeitraubend.

     

Screening of carbohydrates in urine by capillary electrophoresis

Carbohydrate analysis of urine is clinically important in assisting diagnosis of disorders of carbohydrate metabolism and understanding its pathologic significance. Paper chromatography and thin-layer chromatography are the techniques that are often employed for the determination of urinary carbohydrates. An aim of our experiments was to investigate the utility of capillary electrophoresis to develop a fast screening procedure of urinary carbohydrates. Simultaneous resolution of eight carbohydrates involving maltose, lactose, D-mannose, D-glucose, D-ribose, D-xylose, L-arabinose and D-galactose as markers was obtained with 130 mM borate (pH 10.2). Ethanol/water (80/20 v/v) and acetonitrile proved to be efficient reagents for urine sample clean-up which produced symmetrical peaks. The urine sample from a normal subject was determined to contain lactose, glucose, xylose and arabinose that fall within normal ranges of these carbohydrates in urine. The investigations made in this study may be potentially useful in carbohydrate screening, especially in neonatal urine screening.     

Separation of neutral carbohydrates by capillary electrophoresis

The basic strategies for analysis of neutral carbohydrates by capillary electrophoresis are summarized. Neutral carbohydrates are dissociated in strong alkali to give anions, hence they can be separated directly by zone electrophoresis based on the difference between their dissociation constants. However, neutral carbohydrates are not electrically charged under normal conditions. Therefore, they should be converted to ions prior to or during analysis. Precapillary introduction of a basic or an acidic group to a neutral carbohydrate gives the derivative positive (in acidic media) or negative (in alkaline media) charge, respectively. The derivatives thus obtained can be separated by zone electrophoresis. Analysis of carbohydrates in a carrier containing an oxyacid salt (such as sodium borate) or an alkaline metal salt (such as calcium acetate) causes in situ conversion to anionic or cationic complexes, respectively, which are separated by zone electrophoresis. The effective uses of electrokinetic chromatography in sodium dodecyl sulfate micelles for hydrophobic derivatives (such as 1-phenyl-3-methyl-5-pyrazolone derivatives) and size-exclusion electrophoresis in gel-packed capillaris for size different oligosaccharides are also discussed. Each separation mode has its inherent method(s) for detection, which are also described here.     

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.     

Paper electrophoresis of growth hormone

Summary A simple method is described for the identification of bovine growth hormone crude fractions using paper electrophoresis. The mobilities of the fractions are tabulated and shown on the extinction curves. The separation of the bands was carried out at room temperature using glycine buffer at pH 10 and Elphor Horizontal apparatus.

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Zusammenfassung Die Rohfraktionen von Rinder-Wachstumshormon wurden papierelektrophoretisch untersucht (Elphor Horizontalgerät; Glycinpuffer pH 10; Zimmertemperatur). Die Versuchsergebnisse für die einzelnen Fraktionen werden tabellarisch und durch eine Abbildung dargestellt.

     

Separation of derivatized carbohydrates by co-electroosmotic capillary electrophoresis

Summary The separation of derivatized carbohydrates has been performed by co-electroosmotic capillary electrophoresis. Derivatization was performed by reductive amination of the carbohydrates with ethylp-aminobenzoate or withp-aminobenzonitrile. Separation selectivity is optimized using buffer electrolytes containing high concentrations of borate, organic solvents, and mixtures thereof; this enabled separation of the carbohydrate derivatives then direct UV detection. Co-directional migration of the anionic analytes with the electroosmotic flow was achieved by adding a cationic polyer (hexadimethrine bromide, HDB) to the electrolyte. With this method it is possible to determine specific carbohydrates, such as arabinose, mannose, and glucose, which are difficult to separate by other CE methods. The applicability of the method is demonstrated for the analysis of plant hydrolyzates     

Separation of negatively charged carbohydrates by capillary electrophoresis

Capillary electrophoresis (CE) has recently emerged as a highly promising technique consuming an extremely small amount of sample and capable of the rapid, high-resolution separation, characterization, and quantitation of analytes. CE has been used for the separation of biopolymers, including acidic carbohydrates. Since CE is basically an analytical method for ions, acidic carbohydrates that give anions in weakly acid, neutral, or alkaline media are often the direct objects of this method. The scope of this review is limited to the use of CE for the analysis of carbohydrates containing carboxylate, sulfate, and phosphate groups as well as neutral carbohydrates that have been derivatized to incorporate strongly acidic functionality, such as sulfonate groups.     

Separation of carbohydrates on electrophoresis microchips with controlled electrolysis

This study reports the separation of fructose, galactose, glucose, lactose and sucrose on glass microchip electrophoresis (ME) devices using a microfluidic platform adapted with external reservoirs for controlling the electrolysis phenomenon. The connections between external reservoirs and microfluidic platform were performed by saline bridges created using silicone tubing filled with BGE. The separation conditions were optimized and the best results were achieved using a BGE containing 75 mmol/L NaOH and 15 mmol/L trisodium phosphate. Electrophoretic separations were monitored using a capacitively coupled contactless conductivity detection system. The controlled electrolysis has successfully allowed the application of a higher voltage on the separation channel promoting the baseline separation of five carbohydrates within 180 s with great run‐to‐run repeatability (RSD < 1%). The achieved efficiencies ranged from 45 000 ± 6000 to 70 000 ± 3000 plates/m demonstrating a performance better than ME devices without controlled electrolysis. The proposed system offered good linearity from 1 to 10 mmol/L and LODs between 150 and 740 μmol/L. The use of external tubes for controlling the electrolysis phenomenon on ME devices has solved common problems associated to run‐to‐run repeatability and analytical reliability required for routine and quantitative analysis.     

Paper electrophoresis with surface-enhanced fluorimetric detection

Amorphous fumed silica was used as a fluorescence sensitizer coated on a filter-paper substrate for use in paper electrophoresis. The usefulness of the separation method was illustrated for a mixture of two polynuclear aromatic compounds of biological interest: benzo[a]pyrene (BaP) and benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol, a hydrolysis product of BaP—DNA adduct. These two compounds were well separated and an enhancement of the fluorescence intensity was also observed when the sensitizer was applied to filter-paper prior to electrophoretic separation.     

Application of 2,3-naphthalenediamine in labeling natural carbohydrates for capillary electrophoresis

Neutral and acidic monosaccharide components in Ganoderma lucidum polysaccharide are readily labeled with 2,3-naphthalenediamine, and the resulting saccharide-naphthimidazole (NAIM) derivatives are quantified by capillary electrophoresis (CE) in borate buffer. Using sulfated-α-cyclodextrin as the chiral selector, enantiomers of monosaccharide-NAIMs are resolved on CE in phosphate buffer, allowing a simultaneous determination of the absolute configuration and sugar composition in the mucilage polysaccharide of a medicinal herb Dendrobium huoshanense. Together with the specific enzymatic reactions of various glycoside hydrolases on the NAIM derivatives of glycans, the structures of natural glycans can be deduced from the digestion products identified by CE analysis. Though heparin dissachrides could be successfully derived with the NAIM-labeling method, the heparin derivatives with the same degree of sulfation could not be separated by CE.     

Large-volume stacking in capillary electrophoresis using a methanol run buffer

Highly sensitive nonaqueous capillary electrophoresis of weakly acidic organic compounds has been performed using methanol as the run buffer solvent. Methanol provided appropriate suppression of the electroosmotic flow and an increase in the electrophoretic mobilities of anionic solutes compared to water. These two effects allowed large-volume stacking using the electroosmotic flow pump (LVSEP) to be achieved for larger anions using a bare fused-silica capillary under an electric field of reverse polarity, whereas only fast-moving small anions were previously known to be suitable for LVSEP in aqueous media. A field-enhanced sample injection of an additional amount of analytes during the solvent plug removal further enhanced the limits of detection to below the nanomolar range with conventional UV absorption detection. Under optimum conditions, excellent linear responses and reproducibility in the migration times together with the corrected peak areas for ten analytes were obtained in the concentration range of 10-100 nM.     

Microchip capillary electrophoresis with amperometric detection for several carbohydrates

Microchip capillary electrophoresis (μCE) with amperometric detection at Cu electrode benefited fast separation and direct detection of carbohydrates. The working electrode of 50-μm Cu wire attached nearly against the channel outlet—4 μm, where it benefited collecting detection current and suppressing overwhelming noise. The use of alkaline medium was essential to separating and detecting carbohydrates, which dissociated into the sensitive alcolate anions. The 10-cm serpentine chip, though lengthening the migration time, it provided better efficiency. Sucrose, cellobiose, glucose, and fructose migrated from the outlet in 400 s +2000 V. The linear calibration plots ranging from 10 to 1000 μM with regression coefficients better than 0.996 were obtained. The injection-to-injection reproducibility of 1.24% (n=7) for glucose in peak current and 0.6% for migration times were excellent. The detection limit was low, down to 2.3 μM for glucose (S/N=3) or 27.6 attomole in mass detection.     

Capillary electrophoresis of ANTS labelled oligosaccharide ladders and complex carbohydrates

The detection limits of the ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid) label and ANTS maltose as a model carbohydrate conjugate were investigated with on-column UV and laser induced fluorescence detection. Under capillary electrophoresis conditions, the concentration and mass detection limits were found to be 5×10^–7 mol/l or 8 femtomole with UV and 5×10^–8 mol/l or 400 attomole with laser induced fluorescence detection, respectively. Including the derivatization reaction, the best concentration detection limit increases to 1×10^–6 mol/l carbohydrate. A model calculation shows that these detection levels are still insufficient to match those of current protein sequencing protocols.Derivatization conditions for dextran and polygalacturonic acid ladders are described with subsequent fast separation in a capillary electrophoresis system under acidic pH buffer conditions. Up to 30 oligomers could be separated in less than 10 min. The application of ANTS labelled carbohydrate analysis in the food industry is demonstrated with the carbohydrate fraction of sweets and the kinetic monitoring of the hydrolysis of polygalacturonic acid.The described ANTS derivitization protocol works with as little as 5 g carbohydrate as demonstrated with a complex oligosaccharide labelled in a reaction volume as little as 2 l. To demonstrate the applicability of this approach to complex carbohydrate analysis, an oligosaccharide mixture derived from human Immunoglobuline G was labelled and separated within 5 min. Separation efficiency and speed are superior to state-of-the-art chromatographic methods. Both electrophoretic and chromatographic methods are complementary because of their different separation mechanism. The implications of using capillary electrophoresis with laser induced fluorescence and appropriate labelling strategies for structural and compositional analysis of complex carbohydrates are discussed.Dedicated to Professor Dr. Dieter Klockow on the occasion of his 60th birthday     

3-Aminobenzamide and 3-aminobenzoic acid, tags for capillary electrophoresis of complex carbohydrates with laser-induced fluorescent detection

The efficiencies in derivatization of reducing carbohydrates were compared by capillary electrophoresis using maltose as a model with nine monoaminobenzene derivatives by reductive amination in the presence of sodium cyanoborohydride. We found that aminobenzene derivatives substituted at the 3-position showed good reactivity with reducing carbohydrates as expected from the reaction mechanism, although the fluorescence intensities and molar absorptivities of these derivatives were not as high as those of 2- and 4-aminobenzene derivatives. The reagents, 3-aminobenzamide and 3-aminobenzoic acid, which showed the highest reactivity, were applied to the labeling of carbohydrate chains obtained from some sialic acid-containing glycoprotein samples, and also high-mannose and hybrid-type oligosaccharides. Capillary electrophoresis of these labeled carbohydrate chains in an inner surface-modified capillary with (50% phenyl)methylpolysiloxane allowed excellent separation of sialic acid-containing carbohydrate chains derived from fetuin and thyroglobulin as well as high mannose-type and hybrid-type carbohydrates derived from bovine pancreas ribonuclease B, soybean agglutinin and hen ovalbumin. The lower limit of calibration was as low as the 10(-16) mol (injected amount) with helium-cadmium laser induced detection.     

Application of binary buffer systems to free flow cell electrophoresis

The applicability of free flow electrophoresis (FFE) was expanded towards processing of sensitive cells. The chamber medium was adjusted to a physiologic pH of 7.35 by a mixture of N-(2-hydroxyethyl)piperazine-N'-(3-propanesulfonic acid) (EPPS) and 2,2-bis(hydroxymethyl)-2,2'2"-nitrilotriethanol (BISTRIS). These substances proved to be nontoxic to sensitive cells such as human smooth muscle or thyroid cells. They enhanced the electrical conductivity of the medium only slightly so that a new cell electrophoresis separation medium could be prepared, which contained 30 mM NaCl together with or without 1 mM CaCl2 but did not generate problems of overheating the fluid. Suspended in this medium, human smooth muscle cells as well as human thyroid carcinoma cells remained viable single cells for at least 120 min. After this period they could be recultured to form monolayers. If electrophoresed in the Octopus preparative FFE device, they migrated as single cells and did not clot; therefore, their electrophoretic behavior could be determined exactly.     

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 (r² > 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. Copyright © 2014 John Wiley & Sons, Ltd.     

Effect of buffer flow on DNA separation in a microfabricated electrophoresis system

An adequate buffer reservoir is one essential component of an electrophoresis system, providing current carrying ions and maintaining constant pH. In a microfabricated DNA separation system with on-chip electrodes, the amount of buffer used is limited by the design of the device; the buffer continuity can be easily disturbed by the production of bubbles. Continuously flowing 1 x Tris-borate-EDTA (TBE) buffer over the electrodes at the cathodic end solves both problems. This flow increases the resolution for ssDNA primer separations (21 and 25 bases) to a maximum value of 1.4 within a distance of 1.2 cm, about four times higher than that without flow. Similar improvement has been achieved for dsDNA separation (20 bp ladder; BioRad) at a distance of only 0.4 cm, giving baseline resolution for bands from 20 to 240 bp. We have also investigated the effect of buffer concentration on resolution, and no similar improvement can be obtained by merely increasing the buffer concentration without flow.