Analytical method transfer of capillary electrophoretic methods

: development of guidelines to overcome interinstrumental differences.

Student thesis: Doctoral Thesis

Abstract

The increased interest in the separation of peptides, proteins, large
immunoglobulins and DNA sequences leads to an increased demand
for appropriate analytical methodologies. Because of its specific and
unique separation mechanism, capillary electrophoresis (CE) is
frequently used in the analysis of these molecules. The more
frequent application of capillary electrophoretic methods requires
that method transfer (AMT) between laboratories/instruments
should be simple and straightforward. Due to the lower precision
and robustness of CE methods, the instrumental differences and the
greater impact of multiple parameters on the separation, the AMT of
CE methods is more complicated and prone to failure than HPLC
methods. In order to facilitate and increase the use of CE as
separation technique, AMT guidelines, were defined to increase the
AMT rate. In a first case study a simple chiral separation of betablockers
was studied to improve the repeatability of a CE method by
a constant current application. This furthermore generated more
analogous results during interinstrumental AMT. In a second study
instrument-different settings were selected as robustness-test
parameters. Critical parameters, prone to variation were identified.
The developed guidelines were evaluated for a more complex
separation of angiotensin II and five derivatives. However, loss of
baseline separation occurred during this AMT and an update of the
developed guidelines was needed by equalizing the electrical
resistances on both instruments to overcome separation efficiency
differences. In a next study, the differences of the detector and
data-handling specifications were analysed by means of a second
robustness test. Finally, the occasional interinstrumental differences
in effective length that occur with fast short-end injection methods
were studied and added to the guidelines.
Date of Award18 Apr 2018
Original languageEnglish
Awarding Institution
  • Vrije Universiteit Brussel
SupervisorYvan Vander Heyden (Promotor), Debby Mangelings (Promotor), Ann Massie (Jury), Ann Van Eeckhaut (Jury), Francis Puttemans (Jury), Ann Van Schepdael (Jury) & Rawi Ramautar (Jury)

Keywords

  • separation of peptides
  • proteins
  • immunoglobulins
  • DNA
  • capillary electrophoretic methods
  • guidelines

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