Development and Validation of HPLC Methods for Analytical and Preparative Purposes

• 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.

Ämnesord

NATURVETENSKAP  -- Kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

Nyckelord

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

Chemistry

Kemi

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