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Why ultra high performance liquid chromatography produces more tailing peaks than high performance liquid chromatography, why it does not matter and how it can be addressed.
AstraZeneca R&D Lund, Sweden.
Department of Chemistry and Biomedical Science, Karlstad University, Sweden.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
AstraZeneca Nordic Headquarters, Södertälje, Sweden.
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2011 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1218, no 39, 6914-21 p.Article in journal (Refereed) Published
Abstract [en]

The purpose of this study is to demonstrate, with experiments and with computer simulations based on a firm chromatographic theory, that the wide spread perception of that the United States Pharmacopeia tailing factor must be lower than 2 (T(f)<2) is questionable when using the latest generation of LC equipment. It is shown that highly efficient LC separations like those obtained with sub-2μm porous and 2.7μm superficially porous particles (UHPLC) produce significantly higher T(f)-values than the corresponding separation based on 3μm porous particles (HPLC) when the same amount of sample is injected. Still UHPLC separations provide a better resolution to adjacent peaks. Expressions have been derived that describe how the T(f)-value changes with particle size or number of theoretical plates. Expressions have also been derived that can be used to scale the injection volume based on particle size or number of theoretical plates to maintain the T(f)-value when translating a HPLC separation to the corresponding UHPLC separation. An aspect that has been ignored in previous publications. Finally, data obtained from columns with different age/condition indicate that T(f)-values should be complemented by a peak width measure to provide a more objective quality measure.

Place, publisher, year, edition, pages
2011. Vol. 1218, no 39, 6914-21 p.
National Category
Chemical Sciences
URN: urn:nbn:se:uu:diva-159085DOI: 10.1016/j.chroma.2011.08.018PubMedID: 21872875OAI: oai:DiVA.org:uu-159085DiVA: diva2:443637
Available from: 2011-09-26 Created: 2011-09-21 Last updated: 2014-04-29Bibliographically approved
In thesis
1. Adsorption Studies with Liquid Chromatography: Experimental Preparations for Thorough Determination of Adsorption Data
Open this publication in new window or tab >>Adsorption Studies with Liquid Chromatography: Experimental Preparations for Thorough Determination of Adsorption Data
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Analytical chemistry is a field with a vast variety of applications. A robust companion in the field is liquid chromatography, the method used in this thesis, which is an established workhorse and a versatile tool in many different disciplines. It can be used for identification and quantification of interesting compounds generally present in low concentrations, called analytical scale chromatography. It can also be used for isolation and purification of high value compounds, called preparative chromatography. The latter is usually conducted in large scale with high concentrations. With high concentrations it is also possible to determine something called adsorption isotherms.

Determination of adsorption isotherms is a useful tool for quite a wide variety of reasons. It can be used for characterisation of chromatographic separation systems, and then gives information on the retention mechanism as well as provides the possibility to study column-column and batch-batch reproducibility. If a protein is immobilised on a solid support, adsorption isotherms can be used for pharmacological characterisation of drug-protein interactions. Moreover, they can be used for the study of unexpected chromatographic phenomena.

If the adsorption isotherm is known it is also possible to simulate chromatograms, and subsequently optimise the separation process numerically. The gain of a numerically optimised separation process is higher purity or yield of valuable compounds such as pharmaceuticals or antioxidants, as well as reducing the solvent usage. Taken all together, it saves time, money and the environment.

However, the process of the adsorption isotherm determination requires a number of careful experimental considerations and preparations, and these are the main focus of the thesis. Important steps along the way include the choice of separation system and of suitable analytes, preparation of mobile phases and sample solutions, calibration, determination of injection profiles and column void, and of course the adsorption isotherm determination method itself. It is also important to keep track of parameters such as temperature and pH. These issues are discussed in this thesis.

At the end, a description of useful methods for processing of the raw adsorption isotherm data is presented, as well as a brief passage on methods for numerical optimisation.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 56 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1118
Liquid chromatography, HPLC, UHPLC, Reversed phase, Preparative chromatography, Adsorption isotherm, Injection profile, Sample pH, pH stable conditions, Peak deformation, Band distortion, Overloaded band, Chiral preparative chromatography
National Category
Chemical Sciences Analytical Chemistry
Research subject
Analytical Chemistry
urn:nbn:se:uu:diva-216235 (URN)978-91-554-8858-1 (ISBN)
Public defence
2014-03-14, B22, BMC, Husargatan 3, Uppsala, 10:15 (English)
Available from: 2014-02-14 Created: 2014-01-20 Last updated: 2014-04-29

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