Abstract
A simple, rapid and robust protocol for the characterisation of strong cation exchange columns for the analysis of small molecular weight bases is described. A range of ten different phases were characterised, and the resultant selectivity and retention factors analysed using Principal Component Analysis. The score plots for the first and second principal components described 83% of the variability within the dataset. Score plots highlighted the large chromatographic differences observed between the phases, the validity of which was established using a larger range of bases. All the strong cation exchange materials demonstrated a synergistic mixed mode (i.e. ion exchange and hydrophobic) retention mechanism. Principal Component Analysis also highlighted the potential difficulty in locating suitable strong cation exchange “back-up” columns for the analysis of small molecular weight bases in that the characterised columns all displayed very different selectivities. The robustness of the protocol was confirmed by a factorial design experiment.
Highlights
Over the last 20 years, considerable efforts have been made to assist the chromatographer in selecting reversed-phase LC (RP-LC) columns for various purposes such as identifying ones with differing chromatographic selectivity for method development proposes and those with very similarElectronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, UKNovo Nordisk A/S, Novo Nordisk Park, 2760 Måløv, DenmarkAstraZeneca, Silk Road Business Park, Macclesfield SK10 2NA, UKShimadzu UK, Milton Keynes, Buckinghamshire MK12 5RD, UK selectivity and retention as equivalent columns for “back up” purposes
A small range of basic probes was desirable for the column characterisation protocol to rapidly assess the cation exchange capacity, hydrophobicity and potential aromatic character of the strong cation exchange (SCX) phases
Based on initial one factor at a time (OFAT) studies and the likely errors associated with the procedure, the following design of experiment (DoE, see Table 2 and “Factorial design” section) was performed on a Luna SCX column which was chosen as it was located fairly close to the origin in the Principal Component Analysis (PCA) score plot (Fig. 2a) and, can be considered to have average properties
Summary
Over the last 20 years, considerable efforts have been made to assist the chromatographer in selecting reversed-phase LC (RP-LC) columns for various purposes such as identifying ones with differing chromatographic selectivity for method development proposes and those with very similar. Modde Pro software (see “Software and calculations” section) was employed to create and evaluate the factorial designs, which utilised eight experiments with various + 1 or − 1 levels for the different variables, and three repeat experiments for the central nominal conditions to ascertain the reproducibility of the procedure (see Online resource 2). The DoE study aims to prove the robustness of the method when slight differences in operating parameters are assessed that could be imparted by different instruments, laboratories and operators when performing the experiment These experiments were performed using pre-mixed mobile phases made up by weight to minimise errors associated with dispensing large quantities of solutions volumetrically
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