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Use of liquid chromatography-diode-array-detection and mass spectrometry for rapid product identification in biotechnological synthesis of a chiral steroid
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Surface Biotechnology.
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2003 In: Journal of Chromatography A, ISSN 0021-9673, Vol. 992, 85-100 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2003. Vol. 992, 85-100 p.
URN: urn:nbn:se:uu:diva-91954OAI: oai:DiVA.org:uu-91954DiVA: diva2:164871
Available from: 2004-09-01 Created: 2004-09-01Bibliographically approved
In thesis
1. Development and Validation of HPLC Methods for Analytical and Preparative Purposes
Open this publication in new window or tab >>Development and Validation of HPLC Methods for Analytical and Preparative Purposes
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis concerns the development and validation of high performance liquid chromatography (HPLC) methods aimed for two industrially important areas: (i) analysis of biotechnological synthesis and (ii) determination of adsorption isotherm parameters. There is today a lack of detailed recommendations for analytical procedures in the field of biotechnological production of drugs. Therefore, guidelines were given for analytical development and validation in this field; the production of 9α-hydroxyprogesterone was used as model. In addition, a rapid method using HPLC coupled with diode-array-detection (DAD) and mass spectrometry (MS), was developed for the preliminary identification and quantification of the product. In addition, requirements and recommendations were developed for the selection of the internal standard and for its inclusion in the process liquid. By using this approach the precision and accuracy of the quantitative method were considerably improved.

Preparative chromatography is a powerful separation method for the purification of pure compounds from more or less complex sample mixtures. One such mixture can be the process liquid from a fermentation, another example can be a racemic mixture of compounds whose enantiomeric constituents must be isolated. Computer-assisted modeling can be used to optimize preparative chromatography. However, competitive adsorption isotherm parameters are required as input data for the computer simulations. In this thesis, a new injection technique, based on a firm theoretical basis, was developed for the peak perturbation (PP) method allowing the determination of binary competitive adsorption isotherm parameters from a broad concentration range. With the new method the determination of adsorption isotherm parameters from a quaternary mixture could be done for the first time. The profiles simulated with these parameters showed excellent agreement with the corresponding experimental profiles, validating the accuracy of the adsorption isotherm parameters derived by the new method.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 87 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 995
Chemistry, High performance liquid chromatography (HPLC), Analytical biotechnology, Sample preparation, Diode-array-detection (DAD), Mass spectrometry (MS), Validation, Preparative chromatography, Competitive isotherm parameters, Perturbation peak (PP) method, Kemi
National Category
Chemical Sciences
urn:nbn:se:uu:diva-4442 (URN)91-554-6005-4 (ISBN)
Public defence
2004-09-24, Room B42, BMC, Husargatan 3, Uppsala, 13:15
Available from: 2004-09-01 Created: 2004-09-01Bibliographically approved

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