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Development and Validation of HPLC Methods for Analytical and Preparative Purposes
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Surface Biotechnology.
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. , p. 87
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 995
Keywords [en]
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
Keywords [sv]
Kemi
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-4442ISBN: 91-554-6005-4 (print)OAI: oai:DiVA.org:uu-4442DiVA, id: diva2:164877
Public defence
2004-09-24, Room B42, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2004-09-01 Created: 2004-09-01Bibliographically approved
List of papers
1. Use of liquid chromatography-diode-array detection and mass spectrometry for rapid product identification in biotechnological synthesis of a hydroxyprogesterone
Open this publication in new window or tab >>Use of liquid chromatography-diode-array detection and mass spectrometry for rapid product identification in biotechnological synthesis of a hydroxyprogesterone
Show others...
2003 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 992, no 1-2, p. 85-100Article in journal (Refereed) Published
Abstract [en]

In exploratory scale biotechnological process development, the product must be rapidly identified although a reference compound may not always be available. LC-diode-array detection and MS were used for this purpose in a process producing 9alpha-hydroxyprogesterone from progesterone as substrate. The electrospray ionization mass spectrometer was combined with an ion trap mass spectrometer for the second generation MS. The preliminary identification, which could be carried out within the course of a day, confirmed that the product was a hydroxyprogesterone. The final identification step, which was much more material intensive and hence time consuming, involved a two-step preparative separation to yield quantities necessary for definitive product identification based on 1H- and 13C NMR.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-91954 (URN)10.1016/S0021-9673(03)00314-5 (DOI)12735465 (PubMedID)
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2018-01-23Bibliographically approved
2. Guidelines for analytical method development and validation of biotechnological synthesis of drugs. Production of a chiral steroid as model
Open this publication in new window or tab >>Guidelines for analytical method development and validation of biotechnological synthesis of drugs. Production of a chiral steroid as model
2003 In: Journal of Chromatography B, ISSN 1570-0232, Vol. 791, p. 323-336Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91955 (URN)
Available from: 2004-09-01 Created: 2004-09-01Bibliographically approved
3. Investigation of the adsorption behaviour of a chiral model compound on Kromasil CHI-TBB
Open this publication in new window or tab >>Investigation of the adsorption behaviour of a chiral model compound on Kromasil CHI-TBB
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-91956 (URN)
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2010-01-13Bibliographically approved
4. Theoretical and experimental study of binary perturbation peaks with focus on peculiar retention behaviour and vanishing peaks in chiral liquid chromatography
Open this publication in new window or tab >>Theoretical and experimental study of binary perturbation peaks with focus on peculiar retention behaviour and vanishing peaks in chiral liquid chromatography
2003 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 991, p. 31-45Article in journal (Refereed) Published
Keywords
perturbation method, adsorption isotherms, enantiomer separation, retention behaviour
National Category
Analytical Chemistry Computational Mathematics
Identifiers
urn:nbn:se:uu:diva-91957 (URN)10.1016/S0021-9673(03)00213-9 (DOI)
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2018-01-23Bibliographically approved
5. Validation of the Accuracy of the Perturbation Peak Method for Determination of Single and Binary Adsorption Isotherm Parameters in LC
Open this publication in new window or tab >>Validation of the Accuracy of the Perturbation Peak Method for Determination of Single and Binary Adsorption Isotherm Parameters in LC
2004 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 76, p. 4856-4865Article in journal (Refereed) Published
National Category
Analytical Chemistry Computational Mathematics
Identifiers
urn:nbn:se:uu:diva-91958 (URN)10.1021/ac0497407 (DOI)
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2018-01-23Bibliographically approved
6. Validation of the Accuracy of the Perturbation Peak Method for Determination of Multicomponent Adsorption Isotherm Parameters in LC
Open this publication in new window or tab >>Validation of the Accuracy of the Perturbation Peak Method for Determination of Multicomponent Adsorption Isotherm Parameters in LC
2004 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 76, p. 5472-5478Article in journal (Refereed) Published
National Category
Analytical Chemistry Computational Mathematics
Identifiers
urn:nbn:se:uu:diva-91959 (URN)10.1021/ac049632k (DOI)
Available from: 2004-09-01 Created: 2004-09-01 Last updated: 2018-01-23Bibliographically approved

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