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PubMed1993

Comparison of selectivities of reversed-phase high-performance liquid chromatography, capillary zone electrophoresis and micellar capillary electrophoresis in the separation of neurohypophyseal peptides and analogues.

A number of RP-HPLC systems, including those prescribed in several official monographs, have been evaluated for separating oxytocin, the vasopressins, some clinically important analogues and two additional neurohypophyseal nonapeptides. The separation has been compared with capillary zone electrophoresis and micellar electrophoresis in four micellar systems: cationic, anionic, zwitterionic and neutral. Complete separation was achieved by both RP-HPLC and micellar CE but the importance of charge as a major parameter of separation in CE confers a distinct and useful selectivity to micellar CE based separations.

Amino Acid Sequence↗

PubMed1992

Analysis of tubulin isoforms by two-dimensional gel electrophoresis using SDS-polyacrylamide gel electrophoresis in the first dimension.

A two-dimensional electrophoretic system using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the first dimension and isoelectric focusing (IEF) in the second dimension was devised. In spite of its simplicity, this method could show a markedly high resolution for tubulin isoforms and moreover could classify them into alpha- or beta-tubulin as a two-dimensional profile. With this method, seven alpha- and four beta-tubulin isoforms could be detected within axoneme from Tetrahymena cilia. Moreover this method could also resolve tubulin isoforms from the rabbit brain. These results indicate that the present two-dimensional gel electrophoresis is a useful tool for the electrophoretic analysis of tubulin isoforms from various sources.

Animals↗

PubMed2003

Two-dimensional capillary electrophoresis involving capillary isoelectric focusing and capillary zone electrophoresis.

Capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) was on-line hyphenated by a dialysis interface to achieve a 2D capillary electrophoresis (CE) system. The system was used with just one high-voltage power supply and three electrodes (one cathode shared by the two dimensions). The focused zone in the first dimension (i.e. the cIEF) was driven to the dialysis interface by electroosmotic flow (EOF), besides chemical mobilization from the first anode to the shared cathode. And then in the second dimension (i.e. the CZE), the separated zone was further separated and driven by an inverted EOF, which originated from the charged layer of a cationic surfactant adsorbed onto the inner wall of the capillary. Finally, a solution of ribonuclease was rapidly separated to assess the feasibility of the two-dimensional CE implement.

Electrolytes↗

PubMed2004

Characterization of recombinant human granulocyte colony stimulating factor (rHuG-CSF) by capillary zone electrophoresis, capillary isoelectric focusing electrophoresis and electrospray ionization mass spectrometry.

Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) is a hematopietic cytokine that stimulates and regulates the proliferation and differentiation of neutrophils. Glycosylated and non-glycosylated forms of rHuG-CSF cannot be distinguished by traditional biological assays. In addition, it is very difficult to characterize impurities of the same molecular weight in biologicals. In this study, non-glycosylated rHuG-CSF, two glycosylated rHuG-CSF isoforms and their commercial dosages were successfully separated by capillary zone electrophoresis (CZE) using 50mM Tricine containing 20mM NaCl and 2.5mM 1,4-diaminobutane (DAB) at pH 8.0, which could be employed for the qualitative discrimination assay of rHuG-CSF related products. CZE, capillary isoelectric focusing electrophoresis (CIEF), and mass spectrometry (MS) were used to effectively characterize non-glycosylated rHuG-CSF. It was found that proteins in the samples with different pIs in the CIEF profile could not be detected by CZE, while no difference was observed between these proteins and rHuG-CSF. Further analysis by electrospray ionization mass spectrometry with the resolution of 2000 showed that the components with different pIs in the non-glycosylated rHuG-CSF bulk sample are nearly equal in molecular weight. Therefore, it is necessary to combine several modern analytical techniques for quality control to get well-characterized biologicals.

Electrophoresis, Capillary↗

PubMed2001

Pre-concentration of non-uniform field electrophoresis for sample introduction of capillary electrophoresis.

A new sample introduction method of capillary electrophoresis, in which field-amplified sample injection was combined with a pre-concentration of non-uniform field electrophoresis, is presented in this paper. With an additional pre-concentration voltage applied to sample solution, a non-uniform electric field was generated, with which analytical cations or anions were pre-concentrated around an electrode adjacent to the injection end of capillary. After the pre-concentration, analytical ions were injected into the capillary and stacked at the boundary between sample and buffer solution inside capillary by field-amplified injection technique. In contrast to the conventional field-amplified injection, larger concentration factor and higher analytical sensitivity were obtained with the improved pre-concentration method. Its concentration factor was about 10 approximately 15 fold as that of field-amplified sample injection.

Electrophoresis, Capillary↗

PubMed2002

Analysis of catecholamines by capillary electrophoresis and capillary electrophoresis-nanospray mass spectrometry. Use of aqueous and non-aqueous solutions compared with physical parameters.

Catecholamines were analysed in aqueous and alcoholic non-aqueous solutions by capillary electrophoresis and capillary electrophoresis-mass spectrometry using sheathless nanospray coupling. Decreases in the electrophoretic mobilities of the catecholamines and in the electroosmotic mobilities were observed from water to 1-propanol. Separations were more efficient in all non-aqueous media than in water. The diffusion coefficients of the catecholamines in the different media were determined. The solvent had little effect on the sensitivity of the UV or MS detection. Both methods were successfully applied to the analysis of urine samples.

Catecholamines↗

PubMed2002

Interaction between netropsin and double-stranded DNA in capillary zone electrophoresis and affinity capillary electrophoresis.

Capillary zone electrophoresis (CZE) and affinity capillary electrophoresis (ACE) were applied to study the interaction between netropsin and a 14mer double-stranded DNA (dsDNA). The use of a polyacrylamide coated capillary can suppress the electroosmotic flow (EOF) and the adsorption of DNA onto the wall. Better analysis of the DNA was achieved in a coated capillary upon Tris-acetate. In CZE, the peak width broadened due to the affinity interaction between dsDNA and netropsin. In ACE, o-toluic acid, a negatively charged molecule was used as the indicator to monitor the changes of EOF when netropsin was added to the running buffer. The 14mer dsDNA showed different mobilities upon various concentrations of netropsin due to the affinity interaction between the dsDNA and netropsin. The binding constants of this interaction were (1.07 +/- 0.10) x 10(5) M(-1) calculated from CZE and (4.75 +/- 0.30) x 10(4) M(-1) from ACE using a Scatchard plot. The binding stoichiometry was 1:1 calculated from CZE which was superior to ACE in this study.

Base Sequence↗

PubMed2001

Electrophoresis separation in open microchannels. A method for coupling electrophoresis with MALDI-MS.

The separation of biological mixtures in open micro-channels using electrophoresis with rapid and simple coupling to mass spectrometry is introduced. Rapid open-access channel electrophoresis employs microchannels that are manufactured on microchips. Separation is performed in the open channels, and the chips are transferred to a matrix-assisted laser desorption/ionization (MALDI) source after the solvent is evaporated. The matrix (2,5-dihydroxybenzoic acid) is placed in the solution with the run buffer before the separation of the analyte components. After separation, the solvent is evaporated and the microchip is ready for MALDI-MS analysis. The microchip is placed directly into a specially designed ion source of an external source Fourier transform mass spectrometry instrument. Separation of simple mixtures containing oligosaccharides and peptides is shown.

Animals↗

PubMed2004

Using nonequilibrium capillary electrophoresis of equilibrium mixtures for the determination of temperature in capillary electrophoresis.

Until now, all methods for temperature sensing in capillary electrophoresis (CE) relied on molecular probes with temperature-dependent spectral/optical properties. Here we introduce a nonspectroscopic approach to determining temperature in CE. It is based on measuring a temperature-dependent rate constant of complex dissociation by means of a kinetic CE method known as nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). Conceptually, a calibration curve of "the rate constant versus temperature" is built using NECEEM and a CE instrument with a reliable temperature control or, alternatively, a nonelectrophoretic method, such as surface plasmon resonance. The calibration curve is then used to find the temperature during CE in the same buffer but with another CE apparatus or under otherwise different conditions (cooling efficiency, length and diameter of the capillary, electrical field, etc.). In this proof-of-principle work, we used the dissociation of a protein-DNA complex to demonstrate that the NECEEM-based temperature determination method allows for temperature determination in CE with a precision of 2 degrees C. Then, we applied the NECEEM-based temperature determination method to study heat dissipation efficiency in CE instruments with active and passive cooling of the capillary. The nonspectroscopic nature of the method makes it potentially applicable to nonspectroscopic detection schemes, e.g. electrochemical detection. A "kinetic probe" can be coloaded into the capillary along with a sample for in situ temperature measurements. Higher order chemical reactions can also be used for temperature sensing, provided a kinetic CE method for measuring a corresponding rate constant is available.

DNA↗

PubMed1996

Silver(I)-mediated separations by capillary zone electrophoresis and micellar electrokinetic chromatography: argentation electrophoresis.

The addition of Ag(I) to the run buffer in capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), containing sodium dodecyl sulfate, applies the principles of argentation chromatography to electrophoretic separations and is termed "argentation electrophoresis". This technique is shown to provide a complementary method to CZE and MEKC for the separation of specific types of solutes that selectively complex with Ag(I). Baseline resolution in the CZE separation of nine sulfonamides is achieved by the addition of 50 mM silver nitrate to the run buffer. Retention mechanisms in MEKC separations can also be manipulated by the addition of Ag(I) to the micellar solution. Only slight resolution of a pair of sulfonamides was achieved under normal MEKC conditions. Upon the addition of Ag(I) to the mobile phase containing SDS micelles, baseline resolution of the compounds is shown. The retention order and resolution of five sulfonamides changed significantly when 25 mM Ag(I) was added to the SDS-containing buffer. The use of Ag(I) addition in MEKC is also applied to the separation of various other compounds that show selectivity toward Ag(I) complexation, including S-containing heterocycles and vitamin D compounds. The effect of the addition of Ag(I) on the elution range in MEKC is also investigated. A steady increase in the elution range is seen upon increasing the concentration of Ag(I). With a constant percentage of organic modifier (15%), the addition of higher concentrations of silver nitrate (25-50 mM) results in elution ranges greater than 12. The results using Ag(I) as buffer additives in MEKC are also compared to studies utilizing a mixed counterion surfactant of sodium/silver dodecyl sulfate.

Electrochemistry↗

PubMed1998

Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology.

Here, the state of the art of the application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology will be presented. Furthermore, the potentials and limitations of these techniques will be discussed, and it will be indicated why their use in ecological studies has become so important.

Bacteria↗

PubMed1987

Characterization of serum alkaline phosphatase isoenzymes by affinity electrophoresis in agarose gel containing lectin combined with agar gel electrophoresis.

The combined use of affinity electrophoresis in agarose gel containing lectin and of agar gel electrophoresis for the quantitation of liver, bone, biliary and intestinal alkaline phosphatase isoenzymes is described. Sera from patients with various diseases and from normal subjects (blood donors) have been analyzed. Data from normal subjects show that the bone isoenzyme is the predominant fraction (about 62%) in adults. The relative proportions of the alkaline phosphatase isoenzymes are similar in both sexes in adulthood (21-50 years). The higher alkaline phosphatase activity found in men than in women (ages 21-50 years) is due to higher values for both liver and bone isoenzymes. The difference between men and women tends to decrease after the age of 50 mainly due to an increase of the bone isoenzyme in women.

ABO Blood-Group System↗

PubMed1996

Analysis of antisense oligonucleotides by capillary electrophoresis, gel-slab electrophoresis, and HPLC: a comparison.

Three techniques are evaluated for assessing the purity of synthetic oligodeoxyribonucleotides: reversed phase high-performance liquid chromatography (RP-HPLC), polyacrylamide gel-slab electrophoresis (PAGE), and capillary zone electrophoresis highly concentrated (18% T) entangled polymer networks (CZE). RP-HPLC does not seem to be able to discriminate and resolve the spectrum of failed sequences expected to accompany an oligonucleotide of a given length. The purity data, as given by the manufacturer, are most often close to 100%. PAGE in 20% T matrices, followed by ethidium bromide staining, gives a good resolution of failure sequences and purity assessments decidedly more realistic. CZE in 18% liquid polyacrylamide is able to resolve to baseline all shorter fragments and to give a precise evaluation of the amount of impurities, based on the intrinsic DNA absorbance at 254 nm. Most of the 18 mer oligonucleotides (and of their phosphorothioate derivatives) analyzed by us, as supplied by three different manufacturers, were found to be contaminated by a spectrum of failed and truncated sequences, ranging in size from 7 mer to 17 mer. The purity data rarely exceeded 80% and most often were of the order of 60%-70%. Conditions for a good routine performance of the CZE technique are described.

Base Sequence↗

PubMed1995

Electrophoresis for genotyping: temporal thermal gradient gel electrophoresis for profiling of oligonucleotide dissociation.

Traditional use of an oligonucleotide probe to determine genotype depends on perfect base pairing to a single-stranded target which is stable to a higher temperature than when imperfect binding occurs due to a mismatch in the target sequence. Bound oligonucleotide is detected at a predetermined single temperature 'snapshot' of the melting profile, allowing the distinction of perfect from imperfect base pairing. In heterozygotes, the presence of the alternative sequence must be verified with a second oligonucleotide complementary to the variant. Here we describe a system of real-time variable temperature electrophoresis during which the oligonucleotide dissociates from its target. In 20% polyacrylamide the target strand has minimal mobility and released oligonucleotide migrates extremely quickly so that the 'freed' rather than the 'bound' is displayed. The full profile of oligonucleotide dissociation during gel electrophoresis is represented along the gel track, and a single oligonucleotide is sufficient to confirm heterozygosity, since the profile displays two separate peaks. Resolution is great, with use of short track lengths enabling analysis of dense arrays of samples. Each gel track can contain a different target or oligonucleotide and the temperature gradient can accommodate oligonucleotides of different melting temperatures. This provides a convenient system to examine the interaction of many different oligonucleotides and target sequences simultaneously and requires no prior knowledge of the mutant sequence(s) nor of oligonucleotide melting temperatures. The application of the technique is described for screening of a hotspot for mutations in the LDL receptor gene in patients with familial hypercholesterolaemia.

Autoradiography↗

PubMed2004

Simultaneous determination of the main metabolites in rice leaves using capillary electrophoresis mass spectrometry and capillary electrophoresis diode array detection.

The study of the metabolomics of primary metabolites using conventional chemical analyses requires a high-throughput method. Chemical derivatizations are a prerequisite for gas-chromatographic separation, and a large sample quantity is needed for liquid-chromatographic separation and nuclear magnetic resonance detection systems. Recently, we have developed a capillary electrophoresis-mass spectrometry (CE-MS) technology that can simultaneously quantify a large number of primary metabolites, using only a small quantity of samples, and without any chemical derivatizations. Parallel use of a capillary electrophoresis-diode array detector (CE-DAD) system further enables almost all water-soluble intracellular metabolites to be analyzed. We demonstrate, with rice leaves, a simple and rapid method of sample preparation for CE analysis; using this method, we have successfully measured the levels of 88 main metabolites involved in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, photorespiration, and amino acid biosynthesis.

Electrophoresis, Capillary↗

PubMed1995

Analysis of Enterococcus faecalis isolates from intercontinental sources by multilocus enzyme electrophoresis and pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) and multilocus enzyme electrophoresis (MLEE) were compared in this study of 65 Enterococcus faecalis isolates recovered over a 20-year period from diverse geographic sources. Clonal relationships recognized by PFGE were also recognized by MLEE; however, MLEE recognized as greater number of isolates as belonging to clonal groups than did PFGE. Both techniques were reproducible and discriminatory, but MLEE more readily recognized relationships among large numbers of isolates. MLEE confirmed the previously reported clonal spread of beta-lactamase-producing E. faecalis to six hospitals in five states. MLEE provided a useful population framework of the E. faecalis isolates in this sample, while PFGE was able to differentiate among isolates within some MLEE clonal groups.

Alleles↗

PubMed2002

Method development of enantiomer separations by affinity capillary electrophoresis, cyclodextrin electrokinetic chromatography and capillary electrophoresis-mass spectrometry.

Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection sensitivity was observed under high concentration of the protein in running solutions. The partial filling technique was practically useful to solve the serious problem. It allowed operation at high protein concentrations, such as 500 mumol/L, without the detection problem. Charged CDs had several advantages for the enantiomer separations over neutral ones. Strong electrostatic interactions between a charged CD and oppositely charged analytes should be effective for the formation of the complex. A large difference in electrophoretic mobility between the free analyte and the inclusion complex should also enhance the enantiomeric resolution. In CE-mass spectrometry (CE-MS), the partial filling technique was applied to avoid the introduction of nonvolatile chiral selectors into the CE-MS interface. By replacing the nonvolatile electrolytes in the running buffer by volatile ones, the separation conditions employed in CE with the UV detection method could be transferred to CE-MS.

Chromatography, Micellar Electrokinetic Capillary↗

PubMedDate not supplied

Oral Candida albicans and Candida dubliniensis differentiation by multilocus enzyme electrophoresis and sodium dodecylsulphate-polyacrylamide gel electrophoresis.

Five oral strains of Candida albicans and five C. dubliniensis, as well as their respective type-strains, were analyzed by multilocus enzyme electrophoresis (MLEE) and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoreses and numerical analyses, we obtained two distinct species-specific taxa, which may justify the use of MLEE and SDS-PAGE as reliable methods for differentiation and complementary identification of C. dubliniensis.

Alcohol Dehydrogenase↗