Separation and determination of some stereoisomers by capillary gel electrophoresis with cyclodextrin incorporated in polyacrylamide gel

Capillary gel electrophoresis (CGE) was successfully applied to the separation of optically active isomers and position isomers by incorporating a suitable cyclodextrin chiral selector in polyacrylamide gel. A commercially available ß-cyclodextrin (ß-CD) was used for enantioselectivity towards o-, m- and p-nitrobenzoic acid, o-, m- and p-hydroxybenzoic acid, o-, m- and p-toluic acid and the optical isomers of dansyl-D,L-leucine and R,S-1,1-binaphthyl-2,2-dihydrogenphosphate. Especially the effect of organic solvents, such as acetonitrile, methanol, dimethylsulphoxide and others were examined in detail. The resolution varied to some extent with the addition of the organic solvent to the polyacrylamide gel and the running buffer solution. The possible mechanism has also been discussed. In addition, quantitative aspects of the separation of stereoisomers using CGE have been studied, showing that both the resolution and accuracy of the determinations were affected by the ratio of the enantiomers.     

Separation of drug stereoisomers by capillary electrophoresis with cyclodextrins

Using capillary electrophoresis, the enantiomers and isomers of several chiral drug molecules were resolved with cyclodextrins. Parameters affecting the resolution between (+)- and (−)-epinephrine, such as pH, cyclodextrin concentration, buffer concentration, and capillary dimensions were investigated. In addition to this, the effect of cyclodextrin type (β and several derivatized β-cyclodextrins) on resolution between stereoisomers of several chiral drug was also investigated. This study showed that the structural features of the molecule, the derivative groups on the cyclodextrin, the buffer composition and the capillary dimensions influence resolution. The chiral drugs used in this study were propranolol, atenolol, betaxolol, dipivefrin, AL03152 (an aldose reductase inhibitor), AL03363 (an oxidation product of AL03152) and the cis/trans isomers of pilocarpine.     

Separation of cocaine stereoisomers by capillary electrophoresis using sulfated cyclodextrins

A capillary electrophoretic method for the separation of cocaine and its stereoisomers was developed. In this study, the effect of organic modifier was also investigated. The separation was achieved using 1% sulfated cyclodextrin, 10 mmol L(-1) phosphate buffer, 10% methanol at pH 3. The method provides good reproducibility and easy application.     

Band broadening of DNA fragments isolated by polyacrylamide gel electrophoresis in capillary electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is used frequently for isolation and purification of DNA fragments. In the present study, DNA fragments extracted from polyacrylamide gels showed significant band broadening in capillary electrophoresis (CE). A pHY300PLK (a shuttle vector functioning in Escherichia coli and Bacillus subtilis) marker, which contained nine fragments ranging from 80 to 4870 bp, was separated by PAGE, and each fragment was isolated by phenol/chloroform extraction and ethanol precipitation. After extraction from the polyacrylamide gel, the peaks of the isolated DNA fragments exhibited band broadening in CE, where a linear poly(ethylene oxide) was used as a sieving matrix. The theoretical plate numbers of the DNA fragments contained in the pHY300PLK marker were >10⁶ for all the fragments before extraction. However, the DNA fragments extracted from the polyacrylamide gel showed decreased theoretical plate numbers (5–20 times smaller). The degradation of the theoretical plate number was significant for middle sizes of the DNA fragments ranging from 489 to 1360 bp, whereas the largest and smallest fragments (80 and 4870 bp) had no obvious influence. The band broadening was attributed to contamination of the DNA fragments by polyacrylamide fibers during the separation and extraction process.     

Separation of hyaluronic acid by ultrahigh-voltage capillary gel electrophoresis

Hyaluronic acid was separated using 95 kV applied potential in a polyacrylamide gel-filled capillary. The results of this separation were compared to those obtained using a capillary electrophoresis instrument operated at a more conventional potential of 15 kV. For lower-molecular-weight oligomers, the separation efficiency was found to improve by about tenfold, and the resolution by about threefold. However, the improvement in resolution declined as the polymer molecular weight increased.     

Formamide modified polyacrylamide gels for DNA sequencing by capillary gel electrophoresis.

Compressions are occasionally found during the separation of DNA sequencing fragments, particularly in G/C-rich regions and in gels operated at room temperature. Addition of at least 10% formamide to urea/polyacrylamide sequencing gels improves the denaturing capacity of the gel, minimizing compressions. Addition of 20% or more formamide decreases the separation rate, theoretical plate count, and resolution for normally migrating fragments. An optimum concentration of 10% formamide improves resolution of compressed regions without degrading the other characteristics of the gel. Operation of gels at room temperature simplifies the engineering associated with automated sequencers based on capillary gel electrophoresis.     

Separation and determination of enkephalin-related peptides by capillary electrophoresis with amperometric detection

A simple, reliable, and reproducible method for separation and determination of five enkephalin-related peptides based on CE with amperometric detection (AD) is described in this paper. A potential of 1.0 V was applied to the carbon disk electrode, which was used as a working electrode in this system. At 15 kV of applied voltage, the five compounds were separated within 18 min in a 20 mmol/L phosphate buffer solution (PBS, pH 7.6) including 2.5% methanol v/v. LOD for five enkephalins were ranged from 6.31 to 54.3 nmol/L. The method was applied successfully to determine the five compounds added in the human cerebrospinal fluid (CSF) and the recoveries were in the range of 95.8-98.2%.     

Separation and determination of phenolic compounds by capillary electrophoresis with chemiluminescence detection

A methodology for multi-class pesticide determination at trace level in lanolin is presented. Gel permeation chromatography on a Bio-Beads SX-3 column followed by a dual GC chromatographic determination has been developed. The effluent of the analytical column (50% diphenyl–methyl- or 14% cyanopropyl–phenylpolysiloxane) was split into an electron-capture and a nitrogen–phosphorus detection system. The chromatographic system was optimised for 28 pesticides commonly used to control sheep pests and corresponding to organochlorine, organophosphorus and pyretroid classes. Identification has been carried out by gas chromatography coupled to negative chemical ionization mass spectrometry. Recoveries ranged from 72 to 94% and the detection limits from 20 to 97 ng/g depending on the pesticide class, the RSDs were below 10%. Finally, the developed analytical methodology has been successfully applied to the determination of pesticides in several lanolin samples.     

高效毛细管电泳-电导检测对四环素衍生物分离测定的研究

运用毛细管电泳-电导检测方法对4种四环素衍生物——土霉素(OTC)、金霉素(CTC)、强力霉素(DOC)和四环素(TC)的分离进行了研究。在3．5mmol/L三羟基氨基甲烷(Tris)-7．5mmol/L柠檬酸(Cit)pH4．0的运行缓冲液中，4种四环素衍生物在15min内获得完全分离。四环素衍生物的线性范围分别为5．0-500μg/mL OTC，3．6-420μg/mL CTC，4.5-470μg/mL DOC和2.5-400μg/mL TC。检测限(S／N＝3)分别为OTC2．0μg/mL，CTC 1．8μg／mL，DOC2．5μg/mL和TC1．0μg/mL。采用本法对实际样品强力霉素片中强力霉素和土霉素片中土霉素进行测定，回收率分别为97．2％和96.4％。     

毛细管电泳-电化学发光法分离检测饮料中亮氨酸

基于亮氨酸对化学发光试剂三联吡啶钌(Ru(bpy)32+)在铂电极上电致发光信号的增敏作用,建立了一种快速测定亮氨酸的毛细管电泳-电化学发光分析方法.对实验条件进行优化,最佳实验条件如下:检测电位为1.15 V;Ru(bpy)32+浓度为7 mmol·L-1(pH=8.5);进样时间为10 s;进样高压为10 kv;分...     

Separation and determination of flavonoids in Ixeridium gracile by capillary electrophoresis

A simple and rapid capillary electrophoresis method has been developed for the quantitative analysis of three active compounds: (3R)-7,2'-dihydroxy-3',4'-dimethoxy-isoflavan, kaempferol, and quercetin in Ixeridium gracile. The buffer solution used in this method is 20 mmol/L borate at pH 9.5. The effects of pH value, borate concentration, and applied voltage on migration behavior are systematically investigated. Regression equations reveal good linear relationships (correlation coefficients: 0.9986, 0.9997, and 0.9999) between the peak area of each compound and its concentration. The relative standard deviations of the migration time and peak area are less 1.12% and 3.11% (intraday), and 1.43% and 3.52% (interday), respectively, under the optimized separation conditions. The contents of the three flavonoids in I. gracile are successfully determined within 7.8 min, with satisfactory repeatability and recovery.     

Calibration of polyacrylamide gel columns for the separation of oligonucleotides by capillary electrophoresis

Polyacrylamide-filled gel columns are used to separate oligonucleotide samples. For homopolymeric standard samples, plots of migration time versus molecular size are presented over a range of 30-160 bases. With 2.5-4% T and 3.3% C gels, good resolution over the examined mass range, with peak width at half height of 3 to 6 s, is obtained by applying electrical fields of 200-400 V/cm. The separation of heteropolymeric nucleotides by slab gel electrophoresis under routine conditions was compared with capillary gel electrophoresis. Using the same column and the same separation conditions, the plot of migration time versus base number is linear with an identical slope for three oligonucleotide samples which were examined, allowing a calibration of a gel-filled capillary for molecular mass determination.     

Separation and analysis of DNA sequence reaction products by capillary gel electrophoresis

This paper demonstrates the potential of capillary gel electrophoresis with laser induced fluorescence detection as a tool for DNA sequence determination. Both synthetic oligonucleotides and single-stranded phage DNA were utilized as templates in the standard chain termination procedure. Primer molecules were tagged at the 5' end with the fluorescent dye, JOE. First, baseline resolution of a dA extended primer from 18 to 81 bases long, a total of 64 fragments, was observed. A second synthetic template was designed to yield alternating stretches of dA and dT extensions of the primer. Thirdly, the sequence reaction products from a synthetic oligonucleotide template containing all four bases was analyzed in four independent runs, one for each of the four base-specific reactions. In all cases, the expected number and patterns of peaks were observed by capillary gel electrophoretic analysis. Finally, separation of sequence reaction products generated with single-strand M13mp18 phage DNA as template exhibited baseline resolution of fragments differing in length by a single nucleotide and from 18 to greater than 330 bases total length.     

Separation of amino acid and peptide stereoisomers by nonionic micelle-mediated capillary electrophoresis after chiral derivatization

Enantiomers of amino acids and peptides were derivatized with a fluorescent chiral reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole [R-(−)- or S-(+)-NBD-PyNCS] and the resulting diastereomeric derivatives separated by capillary electrophoresis (CE). The CE running buffer consisted of 25 mM acetate buffer (pH 4) and 10 mM of the nonionic surfactant Triton X-100. The excitation maximum of NBD-PyNCS at 480 nm matches the major Ar-ion emission line at 488 nm allowing sensitive laser-induced fluorescence detection with limits of detection around 50 nM.

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-Proline and

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-aspartate spiked (at 10⁻⁴ M and 10⁻⁵ M concentrations, respectively) into complex biological matrices (rabbit serum and homogenate of Aplysia californica buccal ganglion) are detected without matrix interferences. This method has also been applied to the determination of

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- and

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-amino acid residues in peptides after acid hydrolysis. Results from the chiral analysis of the naturally-occurring peptide, gramicidin D, are shown.     

Separation of double-stranded DNA fragments by capillary electrophoresis in interpenetrating networks of polyacrylamide and polyvinylpyrrolidone

Mixtures of two polymers with totally different chemical structures, polyacrylamide and polyvinylpyrrolidone (PVP) have been successfully used for double-stranded DNA separation. By polymerization of acrylamide in a matrix of PVP solution, the incompatibility of these two polymers was suppressed. Laser light scattering (LLS) studies showed that highly entangled interpenetrating networks were formed in the solution. Further systematic investigation showed that double-stranded DNA separation was very good in these interpenetrating networks. With a concentration combination of as low as 2% w/v PVP (weight-average molecular mass Mr = 1 x 10(6) g/mol) + 1% w/v polyacrylamide (Mr = 4 x 10(5) g/mol), the 22 fragments in pBR322/HaeIII DNA, including the doublet of 123/124 bp, have been successfully separated within 6.5 min. Under the same separation conditions, similar resolution could only be achieved by using polyacrylamide (Mr = 4 x 10(5) g/mol) with concentrations higher than 6% w/v and could not be achieved by using only PVP (Mr = 1 x 10(6) g/mol) with a concentration as high as 15% w/v. It is noted that the interpenetrating network formed by 2% PVP and 1% polyacrylamide has a very low viscosity and can dynamically coat the inner wall of a fused-silica capillary. The separation reached an efficiency of more than 10(7) theoretical plate numbers/m and a reproducibility of less than 1% relative standard deviation of migration time in a total of seven runs. The interpenetrating network could stabilize polymer chain entanglements. Consequently, the separation speed was increased while retaining resolution.     

Separation and determination of nitroaniline isomers by capillary zone electrophoresis with amperometric detection

In this paper, capillary zone electrophoresis with amperometric detection (CZE-AD) was firstly applied to the simultaneous separation and determination of nitroaniline positional isomers. The three analytes could be perfectly analyzed by using the buffer of extreme pH. The effects of several important factors were investigated to find optimum conditions. A carbon-disk electrode was used as working electrode. The optimal conditions were 40 mmol/L tartaric acid-sodium tartrate (pH 1.2) as running buffer, 17 kV as separation voltage and 1.10 V (versus saturated calomel reference electrode, SCE) as detection potential. Under the optimum conditions, o-, m- and p-nitroaniline were separated successfully and good linearity, reproducibility and recovery results were obtained. The detection limit for m-nitroaniline was as low as at 9.06 × 10⁻⁹ mol/L. This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 1.8% for migration time and 1.1% for peak areas. The utility of this method was demonstrated by monitoring dyestuff wastewater and the assay results were satisfactory.     

Separation and simultaneous determination of quinolone antibiotics by capillary zone electrophoresis

Summary The potential of capillary zone electrophoresis has been investigated for the separation and quantitative determination of some quinolone antibiotics. The influence of different conditions, such as the nature and concentration of the electrophoretic electrolyte, on migration time, peak symmetry, efficiency and resolution was studied. A buffer consisting of 100mm HEPES adjusted to pH 8.5 containing 10% (v/v) acetonitrile was found to furnish a very efficient and stable electrophoretic system for the separation of exoxacin, ciprofloxacin, ofloxacin, oxolinic acid, nalidixic acid and pipemedic acid. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.25–40 μg mL⁻¹; detection limits were approximately 0.25 ng mL⁻¹. It was demonstrated that the method can be used for the simultaneous determination of these six antibiotics in serum and urine samples.     

Separation and determination of phospholipids in plant seeds by nonaqueous capillary electrophoresis

A method has been developed for the separation and determination of phospholipids by nonaqueous capillary electrophoresis in a separation medium of acetonitrile-2-proponol (3:2, v/v), 0.3% acetic acid and 60 mM ammonium acetate. To optimize the separation conditions, the composition of separation medium including alcohols, acetic acid, n-hexane and ammonium acetate was studied. The solvation interaction and ion-dipole interaction were also investigated. The contents of phospholipids in soybean, sunflower, peanut, apricot kernel, filbert and walnut were determined by the recommended method. The results obtained by the nonaqueous capillary electrophoreses were in good agreement with those determined by micellar electrokinetic chromatography.     

Separation and determination of cations in beverage products by capillary zone electrophoresis

Cation determination is important for quality control of beverage products. To determine a large group simultaneously, a capillary electrophoresis procedure is developed with indirect UV at 214 nm in a three-complex buffer system (10 mM N,N-dimethylbenzylamine (DBA), 8 mM lactic acid and 2 mM 18-crown-6) with good mobility matching with desired cations. Under optimized conditions with pH adjusted to 4.65, a baseline separation is achieved for 14 cations (Rb(+), NH(4)(+), K(+), Ca(2+), Na(+), Mg(2+), Mn(2+), Co(2+), Fe(2+), Cd(2+), Cr(3+), Ni(2+), Zn(2+) and Cu(2+)) within 7 min using an uncoated silica column. To cover ng/l to mug/l range, both hydrostatic and electrokinetic sampling are studied, showing working ranges within (0.05-50)/(0.005-2) microg/l and detection limits (13-78)/(1.4-10) ng/l, respectively with satisfactory repeatability (RSD 0.31-0.47% for migration time, and 3.0-4.0% for peak height measurement). Agreeable results with established inductively coupled plasma-atomic emission spectrometry method have been obtained for orange juice and tea samples.