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  • 1
    ISSN: 0935-6304
    Schlagwort(e): Proteomics ; protein analysis ; multidimensional HPLC ; ion-exchange chromatography ; reversed phase chromatography ; comprehensive HPLC ; two-dimensional HPLC ; Chemistry ; Analytical Chemistry and Spectroscopy
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Chemie und Pharmazie
    Notizen: ---The interactive modes of High Performance Liquid Chromatography (HPLC) of proteins provide a platform for the construction of a multidimensional HPLC system coupled to mass spectrometry. We present a system composed of both anion and cation exchanger columns, in the first dimension, and n-octadecyl bonded 1.5 μm nonporous silica columns in the second dimension. Both columns are operated under gradient conditions. A system suitability test with standard proteins showed that the total analysis can be performed within about 20 minutes. The fractions taken from the ion exchanger column are directly analyzed within one minute on the reversed phase column at a high flow rate. Two reversed phase columns are applied and operated alternatively: while the first column performs the separation within one minute, the analytes leaving the first dimension are enriched in an on-column focusing mode on top of the second column. The sample clean-up and enrichment is performed on a novel type of restricted access cation exchanger column with internal sulfonic acid groups and external diol groups. The columns exhibit a molecular weight exclusion limit for globular proteins of about 15 kDa. Our next studies will be directed towards the analysis of proteins and peptides from extracts of fibroblasts.
    Zusätzliches Material: 4 Ill.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 2
    ISSN: 0006-3592
    Schlagwort(e): post-column switching ; microdialysis ; monitoring ; hydrolysates ; integrated pulsed electrochemical detection ; Chemistry ; Biochemistry and Biotechnology
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Biologie , Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
    Notizen: A quantitative evaluation of the hydrolysis of wheat starch using Termamyl, a thermostable α-amylase (endo-1,4-α-d-glucan, glucanohydrolase; EC 3.2.1.78), is reported. Data from the monitoring of the hydrolysis of wheat starch indicated that, after 1 h, glucose and maltooligosaccharides up to DP 7 were the main hydrolysis products and thus enabled optimization of a liquefication step during the production of L-lactic acid. The monitoring system used, both in the on- and off-line mode, was based on continuous flow microdialysis sampling (CFMS) coupled to anion exchange chromatography and integrated pulsed electrochemical detection (IPED). A microdialysis probe equipped with a 5-mm polysulfone (SPS 4005) membrane, with a molecular-weight cut-off of 5 kDa, was used to sample the hydrolysis products of native wheat starch at 90°C. Characteristic fingerprint separations were achieved by anion exchange chromatography after enzymatic hydrolysis. Post-column switching improved the detection and, consequently, also quantification of the hydrolysates as fouling of the electrode could be reduced. Maltooligosaccharide standards were used for quantification and to verify the elution of the hydrolysates by spiking the off-line samples. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 546-554, 1997.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 3
    ISSN: 1040-0397
    Schlagwort(e): Biosensor ; Amino acid ; Amino acid oxidase ; Carbon paste ; Chemistry ; Polymer and Materials Science
    Quelle: Wiley InterScience Backfile Collection 1832-2000
    Thema: Chemie und Pharmazie
    Notizen: An apparent direct electron transfer between various electrode materials and peroxidases immobilized on the surface of the electrode has been reported in the last few years. An electrocatalytic reduction of hydrogen peroxide promoted by the immobilized peroxidase starts at about +600 mV (vs. Ag/AgCl) at neutral pH. The efficiency of the electrocatalytic current increases as the applied potential is made more negative and starts to level off it about -100 mV (vs. Ag/AgCl). Amperometric biosensors for hydrogen peroxide can be constructed with these kinds of peroxidase modified electrodes. By coimmobilizing a hydrogen peroxide-producing oxidase with the peroxidase, amperometric biosensors can be made responding to the substrate of the oxidase within a potential range essentially free of interfering electrochemical reactions. Sensors for L- and D-amino acids are shown based on coimmobilizing HRP with L-amino acid oxidase (L-AAOD) or D-amino acid oxidase (D-AAOD). The effects of the immobilization procedure, buffer (flow carrier), pH, taken amounts of enzymes, flow rate, and injection volume on the response were investigated. The addition of polyethylenimine (PEI) into the paste was found to increase the response considerably. All 20 of the most common L-amino acids could be detected.
    Zusätzliches Material: 7 Ill.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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