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At least 253 records · Page 14Linked to original sources
PubMed1994

Capillary zone electrophoresis determination of phenylalanine in serum: a rapid, inexpensive and simple method for the diagnosis of phenylketonuria.

A simple, rapid, and quantitative capillary zone electrophoresis method for phenylalanine analysis in serum has been developed, with the aim of providing an analytical tool, as an alternative to liquid and gas chromatography, for the routine laboratory diagnosis of phenylketonuria. Electrophoresis was carried out in a 65 cm long, 50 microns wide bare silica capillary, using 0.025 M borate (adjusted to pH 10 with 1 M NaOH) at a potential of 20 kV, with in-column UV detection at 214 nm. Under these conditions, the three aromatic amino acids (tryptophan, phenylalanine and tyrosine) migrated according to the pKs of the respective amine (and hydroxyl) groups. The efficiency of separation was about 150,000 plates/column for phenylalanine. Diprophylline was adopted as internal standard. The injection of ethanol-deproteinized normal control serum gave rise to only a few major peaks not interfering with phenylalanine; phenylalanine in serum at normal concentrations appeared in a clean region of the electropherogram as a symmetrical peak with a migration time of about 11 min. The sensitivity was > or = 3 micrograms/mL, with s/n ratio = 3. The linearity, in the range of 5-175 micrograms/mL, was described by the equation y = 1.407-0.583 x, r2 = 0.9998. Accuracy and precision were satisfactory, with intra-day and inter-day coefficients of variation lower than 4% and 7%, respectively. The injection of sera from five phenylketonuria patients gave electropherograms clearly showing huge peaks of phenylalanine, thus allowing an easy laboratory diagnosis of phenylketonuria.

Capillary Action↗
PubMed1994

Application of micellar electrokinetic capillary chromatography for monitoring of hippuric and methylhippuric acid in human urine.

The factors affecting micellar electrokinetic capillary chromatographic separation of hippuric and o-, m-, p-methylhippuric acid were investigated by changing the species of micelles, and adding urea to the micellar solution. The analysis of hippurates in human urine is demonstrated under optimum conditions using 20 mM phosphate buffer (pH 8.0) containing 100 mM dodecyltrimethylammonium bromide and 4 M urea at -22 kV applied voltage. This method proved suitable for the screening of hippurates in human urine following occupational exposure to toluene and xylene.

Buffers↗
PubMed1994

The determination of cocaine and related substances by micellar electrokinetic capillary chromatography.

A quantitative method for the determination of cocaine and related substances by micellar electrokinetic capillary chromatography (MECC) is described. Quantitative results obtained for both monitor materials and a number of actual drug seizures by this new capillary electrophoretic method are comparable to a gas chromatography (GC) run in parallel, both in the values and coefficient of variation achieved. The advantages of this new method include minimal use of solvents, the ability to readily automate the procedure and the ability to quantitate illicit heroin seizures under the same conditions with the exception of detector wavelength alteration. The method has proved rugged and reliable for both heroin and cocaine in a number of inter-laboratory proficiency studies.

Buffers↗
PubMedDate not supplied

The use of a hollow fiber membrane module in sample conditioning prior to electrophoresis.

A new method to continuously change the buffer conditions in samples prior to electrophoresis by counter-current dialysis has been developed using a hollow fiber membrane module (VariPerm M, bitop, Witten). It is demonstrated that the collected fractions of a column eluate after hydrophobic interaction chromatography cannot be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) due to the high content of ammonium sulfate. In contrast, when the hollow fiber membrane module is used for on-line desalting of the eluate during chromatography, it is possible to detect distinct bands on SDS-gels even after sample concentration by evaporation. Moreover, adding polyethyleneglycol 20,000 to the counter-current dialysis buffer the hollow fiber membrane module is suitable for combined on-line sample desalting and sample concentration of living organisms during free flow electrophoresis (FFE): a culture of Nitrosomonas eutropha which could not significantly be detected by FFE without conditioning was successfully analyzed when concentrated and desalted on-line during sample application.

Ammonium Sulfate↗
PubMedDate not supplied

Capillary zone electrophoresis of oligosaccharides derivatized with various aminonaphthalene sulfonic acids.

Malto-oligosaccharides were derivatized via their reducing ends with different aminoaphthalene mono-, di- and trisulfonic acids by reductive amination. The derivatives were then separated by capillary zone electrophoresis in uncoated fused silica capillaries, using 50 mM triethylammonium phosphate buffer, pH 2.5, as running electrolyte. The effect of degree of charge on speed of analysis and resolution was studied for different aminonaphthalene mono-, di- and trisulfonic acids. Under the conditions used, a higher degree of charge on the derivatives provided both faster analyses and higher resolution. Investigation of the electrophoretic behavior of derivatized oligosaccharides obtained from bovine pancreatic ribonuclease B gave insight into the possibility of applying such electrophoretic systems to the analysis of more complex carbohydrates. The resolution of positional isomers under the conditions described indicated that the high resolving power of this technique allows separations not strictly based on the effects of charge and mass of the analytes, but on structural characteristics as well. The relationship between electrophoretic mobility and molecular structure was investigated for the different derivatives.

Capillary Action↗
PubMedDate not supplied

Electrophoretic mobilities of the complexes between sodium dodecyl sulfate and various peptides or proteins determined by free solution electrophoresis using coated capillaries.

Electrophoretic mobilities of various proteins or peptides complexed with sodium dodecyl sulfate (SDS) were determined by free solution capillary electrophoresis. The complexes formed between SDS and protein polypeptide showed electrophoretic mobilities virtually insensitive to the protein molecular weight. On the other hand, scattered and larger negative electrophoretic mobilities were observed for peptides of molecular weights less than 10000. Mobility differences as small as 0.3-3% among the three differently modified derivatives of bovine serum albumin could also be successfully determined due to the high resolving power of capillary electrophoresis.

Amino Acid Sequence↗
PubMedDate not supplied

Micellar electrokinetic capillary chromatography analysis of water-soluble vitamins and multi-vitamin integrators.

Micellar electrokinetic capillary chromatography (MECC) was applied to the determination of water-soluble vitamins (WSV) in pharmaceutical preparations. The analytical procedure employed in the WSV separation by MECC showed a satisfactory average column efficiency (195,000 N) and resolution (more than 100 theoretical peaks separable). The mean reproducibility in the retention time of consecutive and day-to-day runs was 1.1% and 5.1%, respectively. Analyses were performed after extraction of active ingredients by solid phase extraction. Recoveries (92-103%) and relative variation coefficients (less than 5%) confirmed the potential of MECC in the field of pharmaceutical analysis.

Ascorbic Acid↗
PubMedDate not supplied

Separation of cis/trans isomers of a prolyl peptide bond by capillary zone electrophoresis.

On capillary electrophoresis of the chemically pure thioxo peptide Ala-Phe-psi[CS-N]-Pro-Phe-4-nitroanilide a peak splitting was observed at a capillary temperature of 25 degrees C. By contrast, the oxo peptide analogue exhibits a single, sharp peak under these conditions. Both peaks of the thioxo compound coincided gradually when the temperature was increased to 60 degrees C. Peak fusion was reverted by cooling down the heated sample. This behavior could be attributed to the electrophoresis-mediated separation of the cis/trans prolyl bond isomers of the thioxo peptide, allowing data of this conformational equilibrium to be determined. Derived from computational data about molecular volume and the hydration energy of low-energy cis and trans isomeric structures, the more rapid migration of the cis form in comparison to trans may be explained by structural parameters.

Algorithms↗
PubMedDate not supplied

Quantitative studies of different injection systems in capillary electrophoresis.

For a rigorous assessment of the precise amount of sample loaded, for quantitation purposes, different sample injection systems were evaluated with two commercially available units, Waters Quanta 4000 and Beckman P/ACE 2100. In the first system, sample introduction by hydrostatic means (i.e., placing the sample vial at some height, usually 10.1 cm, above the other capillary end) was evaluated. It was found that in this system there is a constant positive bias, i.e. the amount of sample loaded lies on a curve parallel and above the theoretically predicted loading curve. However, the excess of mass loaded was constant along the injection times explored (covering from 5 to 35 s) and, for a 75 microns capillary, was found to be of the order of +6 nL (above the expected injected value). Thus it is easy to correct for this sample bias. In the electrokinetic mode, a very good correlation between expected and predicted sample loads was obtained for both units. In the pressure system (by positive pressure from a nitrogen tank, Beckman unit) a substantial discrepancy was found between experimental and predicted values (13.5% overload). Since the manufacturer claims a constant pressure of 0.5 psi, i.e. 3447.5 Pa, it would appear that the injection pressure is higher than the given value.(ABSTRACT TRUNCATED AT 250 WORDS)

Capillary Action↗
PubMedDate not supplied

Fractionation of human serum proteins by capillary and recycling isotachophoresis.

The fractionation of human serum proteins using capillary isotachophoresis (CITP) and recycling isotachophoresis (RITP) in presence of low molecular mass spacer compounds is reported. Anionic CITP was performed in an instrument equipped with a Teflon capillary of 0.5 mm ID as well as in an apparatus which features an open-tubular fused-silica capillary of 75 microns ID. RITP was performed in a recycling fast flow focusing apparatus in which fluid flows rapidly through a narrow, rectangular cell and the effluent from each outlet port is reinjected into the electrophoresis chamber through the corresponding input port. Typically, 1 mL serum was processed batchwise within about 2.5 h prior to collection of 30 fractions of about 4 mL each. The fractions were analyzed separately for conductivity, pH and UV absorbance and selected fractions were characterized by an immunoassay for transferrin, as well as by gel isoelectric focusing, two-dimensional gel electrophoresis, CITP and capillary zone electrophoresis. The search for suitable electrolyte systems and spacers was executed by CITP and by computer simulation. For simple configurations, separations predicted by simulation are shown to qualitatively agree with fractionation performed by CITP and RITP. Configurations producing three protein subgroups, the first containing mainly albumin, the second transferrin and the third the globulins, are discussed.

Blood Proteins↗
PubMed1995

Feasibility study of a drug immunoassay based on micellar electrokinetic capillary chromatography with laser induced fluorescence detection: determination of theophylline in serum.

This paper presents principle and first results of a novel competitive binding immunoassay for monitoring of theophylline in human serum. The assay is based upon short time incubation of a mixture of antiserum, containing the antibody raised against theophylline, fluorescein labelled theophylline (tracer) and serum prior to injection of a few nanoliter of this mixture onto a fused-silica capillary for subsequent separation and analysis of free tracer and the antibody-tracer-complex by micellar electrokinetic capillary chromatography with laser induced fluorescence detection. Quantitation based upon multi-level calibration using the height of the peak produced by the free tracer is shown to provide theophylline serum levels which are in agreement with those obtained by a commercial fluorescence polarization immunoassay and with those determined by micellar elektrokinetic capillary chromatography with direct serum injection and on-column UV absorption detection.

Binding, Competitive↗
PubMed1995

The use of solid phase concentrators for on-line preconcentration of metallothionein prior to isoform separation by capillary zone electrophoresis.

UV absorbance is a convenient detection method for monitoring a wide variety of different components separated by capillary electrophoresis (CE). However, a significant disadvantage of UV detection is its low sensitivity and this is a major factor limiting the application of CE as an analytical technique. In order to improve sensitivity, several methods of on-line sample preconcentration by electrofocusing, including "stacking" and isotachophoresis, have been investigated and the potential of on-line solid phase preconcentrators for this purpose has also been demonstrated. We have developed methods for the separation of isoforms of metallothionein (MT), a low M(r) metal-binding protein which functions as a detoxification system and cellular buffer for heavy metals, primarily in liver and kidney. While a variety of modifications to the capillary surface and electrolyte chemistry have been used to resolve many MT isoforms in liver samples from a variety of species, there are few available alternatives to UV detection and thus sensitivity limits the application of these methods to samples with low MT levels, such as urine or plasma. In this study we have investigated the use of an inexpensive and easy to assemble C18 concentrator which was fitted to the capillary inlet to extend the detection limits for MT isoform analysis. Samples were pressure loaded onto the concentrator for up to 5 min, eluted using 33% acetonitrile and then subjected to electrophoresis in borate or phosphate buffer. The purified isoforms MT-1 and MT-2 from rabbit liver were best resolved using an acidic acetonitrile eluent and a phosphate buffer electrolyte at pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Nonlinear "Ferguson curves" by two runs of the commercial automated HPGE-1000 gel electrophoresis apparatus with intermittent scanning of fluorescence.

The exploitation of gel electrophoretic migration distance to gain information of molecular and gel fiber properties depends on the functions relating mobility with gel concentration. To the degree that these are nonlinear, the definition of those functions by past methods has been excessively laborious or, in application of gel concentration gradients, based on a number of assumptions. The recent commercial introduction of gel electrophoresis apparatus capable of intermittent scanning of the pattern promised to solve these problems. The present study shows that such apparatus allows for a precise definition of a nonlinear "Ferguson curve" (mobility vs. gel concentration) in two experiments, using different gel concentrations in the eight channels of the HPGE-1000 apparatus and 5-29 scans in each during the course of an electrophoretic run. Simultaneously, these Ferguson curves are obtained for five components of a DNA ladder ranging in DNA length from 121 to 1857 bp.

Autoanalysis↗
PubMed1995

An improvement of restriction analysis of bacteriophage DNA using capillary electrophoresis in agarose solution.

Seven representatives of the serogroup B Staphylococcus aureus bacteriophages, 29, 53, 55, 83A, 85, phi 11 and 80 alpha, were examined by capillary electrophoresis (CE) for genomic homology using DNA restriction analysis. Genomic DNA of individual bacteriophages was cleaved by HindIII restriction endonuclease, and the resulting restriction fragments were separated by standard horizontal agarose slab gel electrophoresis (SGE) as well as by CE in low-melting-point agarose solutions. The number and size of restriction fragments identified by both methods were compared. The high separation power of CE makes it possible to extend the restriction fragment patterns. In most of the restriction patterns, some additional restriction fragments as small as 150 bp, not identified by SGE, were detected. With respect to speed, high separation efficiency, low sample consumption and automation, CE offers a simple procedure for processing of multiple samples cost-effectively in a reasonable time. The comparison of the complemented restriction patterns of the different phage strains and the subsequent identification of their common fragments leads to a deeper understanding of their phylogenetic relationships. The genome homologies expressed for individual phage pairs in terms of coefficient F values ranged from 15 to 69%. These values are in good accordance with the degree of DNA homology of these phages as determined by DNA hybridization studies and thermal denaturation analysis of DNA by other authors. The total size of each phage genome was estimated by adding the sizes of individual restriction fragments.

Bacteriophages↗
PubMed1995

Sequence dependent migration behavior of double-stranded DNA in capillary electrophoresis.

Capillary electrophoresis (CE) with a replaceable linear polyacrylamide (LPA) sieving matrix was used to examine sequence-dependent migration of double-stranded DNA fragments. It has been found that DNA conformational effects were significant under high electric field separations, especially using high resolution matrices. Compared to linear DNA-ladder standards, both anomalously slow and rapid DNA fragments were observed, with the degree of anomalous migration depending on the electric field strength, polymer concentration, column temperature, and background electrolyte (denaturants, sodium and magnesium ions, DNA-intercalating dyes). By selecting a combination of electrophoretic conditions (e.g. 3% T LPA, elevated capillary temperature, lower electric field strength and addition of DNA intercalating dyes), molecular weight dependent separations were closely restored.

Bacteriophage phi X 174↗
PubMed1995

On-line capillary electrophoresis-electrospray ionization mass spectrometry of nucleotides.

Capillary zone electrophoresis (CZE) and reversed anionic capillary isotachophoresis (CITP) conditions have been developed for the separation of mixtures comprised of monophosphate nucleosides, pyridine and flavin dinucleotides, and monophosphate dinucleosides. Results for the on-line coupling of CZE and CITP with electrospray ionization mass spectrometry (MS) are presented. CITP-tandem MS is utilized to provide both molecular weight and structural information of monophosphate dinucleotides. The fragmentation pattern of dinucleotides in the low collision energy range is described. The resulting mass spectra are readily interpreted in terms of dinucleotide structures. These results demonstrate the new capability for applications for the study of DNA and RNA.

Capillary Action↗
PubMed1995

Hydrolytically stable cellulose-derivative coatings for capillary electrophoresis of peptides, proteins and glycoconjugates.

A method for the preparation of versatile, hydrolytically stable coatings of fused silica capillaries is described. First, the HNO3-pretreated silica surface is reacted with 7-oct-1-enyltrimethoxysilane to form a stable self-assembled alkylsilane monolayer. Secondly, the copolymer of hydroxypropylcellulose and 2-hydroxyethyl methacrylate is statically coated onto a hydrophobized wall. This allows the formation of a highly crosslinked copolymer layer which is tightly bound to the wall surface. Hydrolytical stability in the pH range of 2-10 for several weeks has been demonstrated. The coating generates a low electroosmotic flow at alkaline pH, which can be easily masked by using special buffers. The versatility of this column technology is documented by separations of peptides, glycoproteins and derivatized oligosaccharides.

Capillary Action↗
PubMed1998

Molecular imprint polymers as highly selective stationary phases for open tubular liquid chromatography and capillary electrochromatography.

Chiral separations employing molecular imprint polymer (MIP) stationary phases in both open tubular liquid chromatography (OT-LC) and capillary electrochromatography (OT-CEC) are demonstrated. MIPs are highly crosslinked polymers containing spatial and functionality memory of template molecules which provide a higher degree of selectivity when used as stationary phases for chromatographic separations. Thin films of molecular imprinted polymers bonded to the inner walls of 25 microm ID fused-silica capillaries were prepared using an in situ polymerization technique developed in our laboratory that allows the use of conventional fused-silica capillaries with polyimide outer coatings. The success rate in preparing such open tubular columns was about 70%. Methacrylic acid and 2-vinyl pyridine were chosen as functional monomers, and either ethylene dimethacrylate or trimethylol propane trimethacrylate was used as the crosslinker. Toluene was employed as the porogen. Effects of polymerization conditions on column preparation and chromatographic performance were studied. Enantiomeric separations of D- and L-dansyl phenylalanines were achieved in both OT-LC and OT-CEC modes with good selectivity and efficiencies. Both types of separations may be performed on the same column using a single commercial instrument.

Capillary Action↗