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Validation of the Tracer-Pulse Method for Multi-Component Liquid Chromatography. A Classical Paradox Revisited
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Surface Biotechnology.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry.
2006 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 78, no 13, 4615-4623 p.Article in journal (Refereed) Published
Abstract [en]

The tracer-pulse method was extended and validated for the determination of multicomponent adsorption isotherms in liquid chromatography. Competitive adsorption isotherms can be determined for any number of solutes, up to the column resolution limit. The basic principle is to equilibrate the column with an eluent containing a mixture of the solutes and then measure the migration velocity of each of them through the column. It is easy to calculate the stationary phase concentrations from these velocities, given the eluent composition. As in frontal analysis, real competitive isotherm data are measured using this method, unlike other methods, which only produce parametric estimates. The method was used to measure the binary isotherms of beta-blockers on a Kromasil C8 column. The data were fitted to competitive bi-Langmuir adsorption isotherm functions and was found to agree well with the results of frontal analysis and the perturbation method. Computer simulations based on the isotherm parameters were performed and displayed very good agreement with the experimental chromatograms. An intriguing and seemingly paradoxical property is visualized and discussed: the fact that the injected molecules are not found in the detected peaks.

Place, publisher, year, edition, pages
2006. Vol. 78, no 13, 4615-4623 p.
National Category
Analytical Chemistry
URN: urn:nbn:se:uu:diva-95289DOI: 10.1021/ac0601260ISI: 000238665200051PubMedID: 16808473OAI: oai:DiVA.org:uu-95289DiVA: diva2:169450
Available from: 2006-12-22 Created: 2006-12-22 Last updated: 2016-10-27Bibliographically approved
In thesis
1. Development and Validation of Methods for Characterization of Multi-Component Systems in Preparative LC
Open this publication in new window or tab >>Development and Validation of Methods for Characterization of Multi-Component Systems in Preparative LC
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Utveckling och Validering av Metoder för Karaktärisering av Flerkomponentsystem vid Preparativ Vätskekromatografi
Abstract [en]

This thesis concerns the development and validation of methods for characterization of multi-component preparative LC systems. Measurements of competitive adsorption isotherms are performed to gain detailed information about the interactions inside the chromatography column. This information increases our understanding of the separation process and makes it possible to perform computer simulations and numerical optimizations to find optimal operating conditions.

The methods under focus are called “the tracer-pulse method”, “the inverse method”, and “the inverse method on plateaus”. They are extensions of existing methods, with new experimental and numerical procedures to enable rapid and accurate multi-component adsorption isotherm determination. In the validation it was shown that they can produce results agreeing with traditional methods and that the acquired adsorption isotherm parameters can be used in simulations to accurately predict the outcome of preparative LC separations.

The methods were used to characterize several complex LC systems and two phenomena were discovered and theoretically treated: 1) The presence of invisible deformed peaks in single-component systems. 2) Peak deformations encountered with modern chiral stationary phases, caused by strongly adsorbed eluent additives. The latter type of deformation was highly tuneable and it was possible to adjust the enantiomer peak shapes so that the peaks tailed in opposite directions with the sharp sides in between, yielding baseline resolution at remarkably high sample loads.

In a final applied study both the LC-based perturbation peak method and a biosensor method based on surface plasmon resonance (SPR) were used for the first time for detailed characterization of chiral drug-protein interactions. The fundamental properties of the two very different methods were compared and it was found that the LC method is more suitable for multi-component analysis and that the SPR method is more suitable for stronger interactions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 48 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 256
Analytical chemistry, Liquid chromatography, Preparative LC, Chiral LC, Multi-component LC, Adsorption isotherm, Tracer-pulse method, Inverse method, Inverse method on plateaus, Peak deformations, Chiral drug-protein interactions, Analytisk kemi
urn:nbn:se:uu:diva-7422 (URN)91-554-6762-8 (ISBN)
Public defence
2007-01-19, B22, Biomedicinskt centrum, Husargatan 3, Uppsala, 13:15
Available from: 2006-12-22 Created: 2006-12-22Bibliographically approved

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