Investigation into reversed phase chromatography peptide separation systems part III: Establishing a column characterisation database

Abstract

The Peptide RPC Column Characterisation Protocol was applied to 38 stationary phases, varying in ligand chemistry, base silica, end capping and pore size, which are suitable for the analysis of peptides. The protocol at low and intermediate pH is based on measuring retention time differences between peptides of different functionality to calculate selectivity delta values. The characterisation was designed to explore increases / decreases in positive or negative charge (deamidation), steric effect (i.e. racemisation / switch in amino acid order), oxidation and addition / removal of aromatic moieties. The necessity of developing a characterisation protocol specifically for peptide analysis was highlighted by the fact that the small molecule databases (Snyder's Hydrophobic Subtraction Model and the extended Tanaka protocol) failed to correlate with the Peptide RPC Column Characterisation Protocol. Principal Component Analysis was used to demonstrate that the protocol could be used to identify columns with similar or dissimilar chromatographic selectivity for the purpose of selectivity back-up or method development columns respectively. This was validated using peptide fragments derived from the tryptic digest of bovine insulin and carbonic anhydrase. It was also demonstrated that the presence of positively charged functional groups on the stationary phase was advantageous as it yielded very different chromatographic selectivity and improved peak shape.

Investigation into reversed-phase chromatography peptide separation systems Part IV: Characterisation of mobile phase selectivity differences

“…The first principal component described approximately 62% of the variability of the data and, as expected, contained the delta values Δ(9,1) and Δ (26,13) (i.e. variables) associated with electrostatic interactions [29], which were diametrically opposite to one another in Fig. 1.…”

“…Each ion-pair utilised a dedicated column to avoid memory effects between mobile phases. The Polaris Amide C18 (150 x 2.0 mm, 3 µm, Agilent Technologies, Santa Clara, CA, USA), Acquity CSH Fluoro Phenyl (150 x 2.1 mm, 1.7 µm, Waters, Milford, MA, USA) and Ascentis Express Biphenyl (150 x 2.1 mm, 2.7 µm) were selected as chromatographically diverse stationary phases to ascertain the applicability of the Ascentis Express C18 results on other types of stationary phases [29]. The MS response comparison was performed on an Acquity CSH C18 (150 x 1.0 mm, 1.7 µm) which utilised a translated gradient from the 150 x 2.1 mm column format.…”

“…The MS response comparison was performed on an Acquity CSH C18 (150 x 1.0 mm, 1.7 µm) which utilised a translated gradient from the 150 x 2.1 mm column format. A brief description of each of the stationary phases can be located in Table 2 in [29]. The peak apex of a water injection was used as the dead time marker [32].…”

“…This paper is the fourth in a series which deals with maximising the chromatographic selectivity of peptide separations using reversed-phase chromatography (RPC). Papers one to three of the series focussed on the development of a column characterisation protocol using 26 specifically designed peptide probes [27], the robustness of the optimised protocol [28] and the characterisation of 38 disparate stationary phases [29], respectively. The characterisation protocol, which determines seven retention time differences between nine selected probes based on the 33 amino acid peptide, [Ile27]-Bovine GLP-2 (see Table 1 and references [27][28][29][30][31]), has been demonstrated to successfully discriminate between differing types of RP stationary phases through the peptide probe's hydrophobic, electrostatic, hydrogen bonding and aromatic interactions with the stationary phases, in addition, to the stationary phase's ability to separate diastereoisomeric or isomeric probes [27].…”

“…Initially, a large range of mobile phases on a single representative new generation C18 column were investigated. It was then established if these findings could be extrapolated to a range of other disparate RP columns (shown to provide maximal selectivity with two mobile phases [29]) using a limited number of mobile phases (shown to possess large selectivity differences on the C18 phase). It is hoped that the findings can be utilised to identify suitable initial mobile and stationary phase combinations in a RP-LC method development strategy that will provide optimal separation of peptides.…”