The increased interest in the separation of small peptides, proteins, large immunoglobulins and whole DNA sequences, due to the development of a more biotechnological focused pharmaceutical industry, leads to a demand for appropriate analytical methodologies and instrumentation. As a result of its specific and unique separation mechanism, capillary electrophoresis (CE) is frequently used in the separation and analysis of those molecules. Additional assets for the use of CE are the small sample volumes, short separation times, method flexibility, online concentration possibilities, online detection and high separation efficiency. The known drawbacks are a lower precision and sensitivity compared to HPLC and a more complex analytical method transfer (AMT).
In a first step of this study, a CE method was developed (and compared to a HPLC method) for the separation of 6 angiotensins. In a second step, the method was transferred to another CE (Agilent) instrument applying earlier defined guidelines concerning analytical method transfer in CE [1, 2]. Although previously successful, the guidelines did not result in a complete baseline separation of this more complex and critical separation. Further research in the instrumental differences led to the discovery that the capillary resistance on both instrument differed. The resistance on the Agilent instrument was lower. This was also expressed in the lower observed applied voltage, while maintaining a constant current. A lower applied voltage could explain the lower efficiency and therefore non-baseline separation of two critical peaks on the Agilent instrument. Increase of the capillary resistance on the Agilent system was performed by lowering the applied capillary temperature with 5 °C leading to identical applied voltages on both instruments while maintaining a constant current.
The earlier developed guidelines were updated with an extra paragraph concerning the capillary resistance, leading to a successful separation and AMT for the 6 angiotensins.
Original languageEnglish
Title of host publicationIVth PhD day @ Jette, Vrije Universiteit Brussel, 31 March 2015, Brussels, Belgium
Publication statusPublished - 2015

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ID: 5193595