Simultaneous determination of 19 intracellular nucleotides and nucleotide sugars in Chinese Hamster ovary cells by capillary electrophoresis

Twelve nucleotides and seven nucleotide sugars in Chinese Hamster ovary (CHO) cells were determined by capillary electrophoresis (CE). The CE operating conditions of buffer pH value, ion strength, capillary temperature, polymer additive and cell extraction method were investigated. Optimum separation was achieved with 40 mM sodium tetraborate buffer (pH 9.5) containing 1% (w/v) polyethylene glycol (PEG) at a capillary temperature of 22 degrees C. Acetonitrile and chloroform were used for intracellular extraction. This method can be used to monitor intracellular carbohydrate metabolism.     

Large-volume sample stacking of selected drugs of forensic significance by capillary electrophoresis

Large-volume sample stacking capillary electrophoresis (LVSS-CE) and conventional capillary electrophoresis (CE) are compared for the separation of drugs of significance to forensic and clinical analyses. LVSS-CE for cations requires the use of an electroosmotic flow (EOF) modifier in conjunction with polarity switching to effect on-column concentration of an analyte and its subsequent migration in the capillary. The run buffer consists of 0.05 mol dm⁻³ disodium tetraborate adjusted to pH 2.2 with orthophosphoric acid, and the EOF modifier is 0.002 mol dm⁻³ cetyltrimethylammonium bromide. CE investigations used an identical run buffer minus the EOF modifier. LVSS-CE and CE investigations used injection times of 30 s and 3 s, respectively. Both modes of capillary electrophoresis are compared in terms of their limits of detection, efficiency, resolution and reproducibility. LVSS-CE is also applied to the analysis of a spiked urine sample.     

High-speed microchip electrophoresis method for the separation of (R,S)-naproxen

In this research, a capillary electrophoretic method for the fast enantiomeric resolution of (R,S)-naproxen was investigated. Method development involved variation of applied potential, buffer concentration, buffer pH, and cyclodextrin concentration. The optimum electrophoretic separation conditions were 110 mM sodium acetate run buffer (pH 6.0), 30 mM methyl-beta-cyclodextrin, 20% (v/v) acetonitrile, 25 degrees C. The total length of capillary was 48 cm, (50 microm I.D.) with ultra violet (UV) detection at 232 nm. Using these conditions, the number of theoretical plates was close to one million (896,000/m). The possibility of achieving a fast chiral separation of (R,S)-naproxen on a microchip of 2.5 cm in length was investigated. Complete enantiomeric resolution of naproxen was achieved in less than 1 min, on this microchip platform, with linear imaging UV detection. This system had the advantage of real-time separation monitoring, so that enantiomeric resolution could be visually observed, and high-speed chiral analysis was realized. The microchip electrophoresis (MCE) separation was compared with the capillary electrophoresis (CE) separation with regards to speed, efficiency, separation platform, and precision. This work highlights the potential of CE and MCE in future chiral separations.     

Determination of iothalamate in rat urine,plasma, and tubular fluid by capillary electrophoresis

A method for the quantitative determination of iothalamate (IOT) in rat urine, plasma and tubular fluid by capillary zone electrophoresis (CE) has been developed and validated. Samples of urine and tubular fluids were diluted with water and samples of plasma were deproteinized with two volumes of acetonitrile containing the internal standard, p-aminobenzoic acid (PABA). A BioFocus 2000 system (Bio-Rad, Hercules, CA, USA) was used. The UV detector was set at 254 nm. The samples were loaded into uncoated fused-silica capillary (40 cm×50 μm) by pressure injection. A borate buffer [20 mM, pH 12 (pH adjusted with 1.0 M NaOH)] was used as the electrophoretic buffer. The typical analytical conditions were: voltage, 22 kV; injection, 9 psi×s; capillary and carousel temperatures were 20°C and 18°C respectively. The linear relationship was observed between time-corrected peak area of IOT in water and urine or the corrected peak area ratio of IOT to PABA in plasma and the nominal concentration of IOT with correlation coefficient greater than 0.999. The intra- and inter-day coefficients of variation (CV) were less than 8%. The concentration of IOT in plasma, urine and tubular fluid determined by CE can be used for estimation of whole kidney and single nephron clearances.     

Capillary electrophoretic behavior of milk proteins in the presence of non-ionic surfactants

The electrophoretic behavior of α-lactalbumin and β-lactoglobulins (A and B) in the presence of non-ionic surfactants was studied by capillary electrophoresis (CE), using a poly(ethylene glycol) coated capillary column. The surfactants (Tween 20, Brij 35 and 78) were used as buffer additives. The separation is based on the difference in the strength of protein–surfactant association complexes, which results in a change of the effective electrophoretic mobility. The modification of the electrophoretic mobilities of proteins was observed and this variation permitted the estimation of the interaction between protein and surfactant. The effect of surfactant type and concentration on the migration behavior of protein in CE is discussed. It is found that the retention behavior of the milk proteins (the α-lactalbumin and the β-lactoglobulins) in CE is very different. The pH of the buffer and the surfactant type influence significantly the protein–surfactant interactions.     

Determination of creatinine and other uremic toxins in human blood sera with micellar electrokinetic capillary electrophoresis

We have been interested in the clinical use of capillary electrophoresis (CE) to monitor low-molecular-mass uremic toxins in body fluids. Creatinine, an important clinical marker for renal failure, is zwitterionic over a fairly wide pH range (pH 5–9) and can not be resolved from neutral components using free solution CE under these conditions. We report here a micellar electrokinetic capillary chromatography method using an sodium dodecyl sulfate-borate buffer system at pH 9.0 to determine creatinine levels in human serum. This method, performed on deproteinized sera, is also suitable for determining multiple ionic components. Moreover, this method compares favorably with an enzymatic method for creatinine performed in a clinical laboratory and thus appears to be a promising method in terms of potential clinical use.     

Enantioselective separation of phenylglycidates by capillary electrophoresis employing sulfated beta-cyclodextrin as chiral selector

A capillary electrophoresis (CE) method for the enantioseparation of phenylglycidates has been developed. Successful enantioseparation was achieved using sulfated beta-cyclodextrin as chiral selector in a phosphate buffer. The effects of varying pH, sulfated beta-cyclodextrin concentration and electrophoresis voltage were systematically investigated and the optimized separation conditions were thus obtained. When the migration time was set at the threshold value, it was found that the best enantioseparation was obtained at 10 kV with 3% (w/v) sulfated beta-cyclodextrin at pH 6.5. A range of substituted phenylglycidates were successfully separated using the method and the results shown to be superior to those obtained using gas chromatography (GC).     

Investigation of maltodextrin‐based synergistic system with amino acid chiral ionic liquid as additive for enantioseparation in capillary electrophoresis

The combined use of chiral ionic liquids (ILs) and chiral selectors in capillary electrophoresis (CE) to establish a synergistic system has proven to be an effective approach for enantioseparation. In this article, tetramethylammonium‐L‐arginine, a kind of amino acid chiral IL, was applied to investigate its potential synergistic effect with maltodextrin in CE enantioseparation. The established maltodextrin‐based synergistic system showed markedly improved enantioseparations compared with the single maltodextrin system. Parameters such as the chiral IL concentration, maltodextrin concentration, buffer pH, applied voltage, and capillary temperature were optimized. Satisfactory enantioseparation of the five studied drugs, including nefopam, duloxetine, ketoconazole, cetirizine, and citalopram was achieved in 50 mM Tris‐H₃PO₄ buffer solution (pH 3.0) containing 7.0% (m/v) maltodextrin and 60 mM tetramethylammonium‐L‐arginine. In addition, the chiral configuration of tetramethylammonium‐L‐arginine was also investigated to demonstrate the existence of a synergistic effect between chiral ILs and maltodextrin.     

Comparison of different techniques for the analysis of metallothionein isoforms by capillary electrophoresis

We have investigated free-solution capillary electrophoresis (FSCE) and micellar electrokinetic capillary chromatography (MECC) separations of metallothionein (MT) isoforms conducted in uncoated and surface-modified fused-silica capillaries. At alkaline pH, FSCE rapidly resolves isoforms belonging to the MT-1 and MT-2 charge classes. At acidic pH, additional resolution of MT isoforms is achieved. The use of high-ionic-strength (0.5 M) phosphate buffers can result in high peak efficiencies and increased resolution for some MT isoforms. Interior capillary surface coatings such as polyamine and linear polyacrylamide polymers permit separation of MT isoforms with enhanced resolution through their effects on electroosmotic flow (EOF) and protein-wall interactions. Improvements in MT isoform resolution can also be achieved by MECC using 100 mM borate buffer pH 8.4 containing 75 mM SDS. Deproteinization of tissue cytosol samples with acetonitrile (60–80%) or perchloric acid (7%) produces extracts that can be subjected to direct analysis of MT by FSCE or MECC. We conclude that optimal separation of MT isoforms by capillary electrophoresis (CE) can be achieved with the appropriate combination of different capillaries, buffers and sample preparation techniques.     

Development of a capillary electrophoretic method for the separation of the macrolide antibiotics, erythromycin, josamycin and oleandomycin

Capillary electrophoresis (CE) provides high separation efficiency and thus is suitable for the analysis of complex mixtures of structurally similar compounds. The versatile nature of CE can be realised by controlling the chemistry of the inner capillary wall, by modifying the electrolyte composition and by altering the physicochemical properties of the analyte. A CE method has been developed for the separation of three macrolide antibiotics, erythromycin, oleandomycin and josamycin. A systematic approach was used to maximise analyte differential electrophoretic mobility by manipulating electrolyte pH, molarity and composition. In addition, some instrumental parameters such as capillary length and diameter and applied voltage were varied. The effect of the sample solvent and on-capillary concentrating techniques such as field amplified sample injection were investigated. Also, the influence of the injection of a water plug on the quantity of sample injected was demonstrated. The macrolides were completely resolved in less than 30 min in a 100 cm×75 μm I.D. fused-silica uncoated capillary with a Z-shaped flow cell of path-length 3 mm. The analysis was performed in a 75 mM phosphate buffer (pH 7.5) with 50% (v/v) methanol and an applied voltage of 25 kV was selected to effect the separation.     

Optimized sample preparation for tandem capillary electrophoresis single-stranded conformational polymorphism/ heteroduplex analysis

Here we describe DNA sample preparation methods that allow the rapid, simultaneous generation of both single-stranded conformational polymorphism (SSCP) and heteroduplex DNA elements from a single sample in a single tube, which are suitable for direct injection into a capillary electrophoresis (CE) instrument with excellent sensitivity of genetic mutation detection. The p53 gene was used as a model DNA region for this study, which was performed on a high-throughput MegaBACE 96-capillary array electrophoresis instrument. We found that, contrary to the practice common in slab-gel SSCP analysis, denaturants such as formamide are incompatible with this novel technique because they result in homo- and heteroduplex peak broadening in CE (possibly as a result of incomplete dsDNA re-hybridization) that reduces the peak resolution and hence the sensitivity of mutation detection. We also have found that PCR buffers, which are typically used to suspend samples for slab-gel heteroduplex analysis (HA), but which are less suitable for CE because of the presence of extra salt that reduces the efficiency of electrokinetic injection, may be substituted with a 10 mM Tris-HCI buffer (pH 8.5). The use of this Tris-HCl buffer for sample preparation provides both a high sensitivity of mutation detection by tandem SSCP/HA and high efficiency ofelectrokinetic injection by CE. In a related study (published elsewhere), we have applied this optimized protocol to the screening of a set of 32 mutant DNA samples from p53 exons 7 and 8 and recorded 100% sensitivity of mutation detection for tandem CE-SSCP/HA, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA).     

Determination of metformin hydrochloride using precolumn derivatization with acetaldehyde and capillary electrophoresis coupled with electrochemiluminescence

A novel method was developed using capillary electrophoresis (CE) coupled with tris(2,2′‐bipyridyl)ruthenium(II) electrogenerated chemiluminescence (ECL) for highly sensitive detection of metformin hydrochloride (MH) derivatizatized with acetaldehyde. The precolumn derivatization of MH with acetaldehyde was performed in phosphate buffer solution (0.3 mol/L, pH 7.5) at room temperature for 120 min. The effects of acetaldehyde concentration, buffer pH, electrokinetic voltage and injection time were investigated. Under optimized detection conditions, the MH ECL detection sensitivity was more than 120 times that without derivatization. The linear concentration range for MH was 0.001–15.00 μg/mL (with a correlation coefficient of 0.9992). The detection limit was 0.31 ng/mL with a signal:noise ratio of 3. The recoveries of MH in human urine were in the range 98.50–99.72%. Copyright © 2011 John Wiley & Sons, Ltd.     

Hyphenated thermal field flow fractionation--capillary electrophoresis

The possibility was considered to use the transverse thermophoresis of analytes in the capillary for capillary electrophoresis (CE) to control the separation process, decrease the peak width due to thermal effects and provide new separation parameters in CE. As the examination has shown, in non-aqueous buffers the Joule heating in the capillary for CE can provide transverse temperature gradients comparable with the temperature gradients in conventional devices for thermal field flow Fractionation (ThFFF). It was proposed to use the non-uniform velocity profile of analytes caused by the transverse temperature gradient and the temperature dependence of the buffer viscosity for the FFF-like separation of analytes besides CE separation. The expressions for the peak parameters have been derived, where the non-uniform transverse analyte concentration distribution due to the thermophoresis is taken into account, and the possibilities based on FFF-CE principles are discussed. As possible objects of this hyphenated technique, macromolecules and particles are considered.     

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 20 min and the detection limits were as low as 10⁻³ mg L⁻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.     

Capillary electrophoretic enantiomeric separations using the glycopeptide antibiotic,teicoplanin

Teicoplanin is the third in a series of macrocyclic glycopeptide antibiotics that has been evaluated as a chiral selector in capillary electrophoresis (CE). It was used to resolve over 100 anionic racemates at low selector concentrations. Like the other related glycopeptide antibiotics, its enantioselectivity tends to be opposite to that of the ansa-type antibiotics which prefers cationic compounds, particularly amines. Factors that affect teicoplanin-based enantioseparations include the selector concentration, pH, and the concentration of the organic modifier. The temperature and the nature and strength of the buffer are also known-to affect the stability of the chiral selector as well as the enantioseparation. Teicoplanin exhibited some features that were not noted with the other glycopeptide antibiotics. For example, it aggregates (forms micelles) in aqueous solutions and this influences its enantioselectivity. Unlike the other studied glycopeptides, teicoplanin precipitates in alcohol-water mixtures. It also binds less to the capillary wall than vancomycin as evidenced by the faster electroosmotic flow velocity. The micellization of teicoplanin is pH dependent so that the effect of pH on enantiorecognition is more complex for teicoplanin than for other chiral selectors. Also it is shown that the simple model proposed to explain the role of organic modifiers in cyclodextrin-based CE enantioseparations may not apply to these and other systems. © 1996 Wiley-Liss, Inc.     

Estimation of pK(a) values using microchip capillary electrophoresis and indirect fluorescence detection

Microchip capillary electrophoresis (CE), coupled with indirect fluorescence detection was investigated for estimating the pK(a) values of non-fluorescent compounds. The CE method is based on the differences in electrophoretic mobility of the analyte as a function of the pH of the running buffer. Nine compounds were tested, including several of pharmaceutical importance, with pK(a) values from 10.3 to 4.6. All buffers contained 5-TAMRA as the fluorescent probe for indirect detection. Calculated pK(a) values agreed well with literature values obtained by traditional methods, differing not more than 0.2 from the literature value. The current work on single lane chips demonstrates the principle of microchip CE with indirect detection as a viable method for estimating pK(a) values. However, increased throughput will be required using a multilane chip to enable the approach to be used practically.     

Analytical and micropreparative capillary electrophoresis of the peptides from calcitonin.

The capillary electrophoresis (CE) of peptide fragments from the tryptic digest of salmon calcitonin and elcatonin has been carried out in H2O- and D2O-based buffer solutions. Analysis in heavy water was found to be superior to that in H2O especially at a pH or pD of 7.93. From a single CE run on elcatonin digest we were also able to harvest three pure cleavage peptides in sufficient quantity to determine each amino acid residue by protein sequencing. The order of elution from CE agreed with that predicted on the basis of net charge calculated for each peptide.     

Separation by capillary electrophoresis followed by dynamic elution

In this study we have explored the behaviour of peptides after capillary electrophoresis (CE) followed by elution under pressure. The use of D2O- rather than H2O-based buffer solutions appears to restrict the diffusion of peptides after CE, resulting in little loss of resolution when peptides are eluted by dynamic flow. In this paper we present results showing that a simple two-step process, involving CE at a low voltage, switching off the power supply, and connecting the fused capillary at the anode end to a syringe pump for dynamic flow, can retain separation characteristics and can be used for the isolation of picomole quantities of peptides for sequence determination.     

The stretched DNA geometry of recombination and repair nucleoprotein filaments

The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA x tsDNA complex), has been elucidated by classical methods. Here we review the systematic characterization of the helical geometries of the three DNA strands of the RecA x tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. Measurements of the helical parameters for the RecA x tsDNA complex are consistent with the hypothesis that this complex is a late, poststrand-exchange intermediate with the outgoing strand shifted by about three base pairs with respect to its registry with the incoming and complementary strands. All three strands in the RecA x tsDNA complex adopt extended and unwound conformations similar to those of RecA-bound ssDNA and dsDNA.