Investigation into reversed phase chromatography peptide separation systems part I: Development of a protocol for column characterisation

Abstract

A protocol was defined which utilised peptides as probes for the characterisation of reversed phase chromatography peptide separation systems. These peptide probes successfully distinguished between differing stationary phases through the probe's hydrophobic, electrostatic, hydrogen bonding and aromatic interactions with the stationary phase, in addition, to more subtle interactions such as the phase's ability to separate racemic or isomeric probes. The dominating forces responsible for the chromatographic selectivity of peptides appear to be hydrophobic as well as electrostatic and polar in nature. This highlights the need for other types of stationary phase ligands with possibly mixed mode functionalities / electrostatic / polar interactions for peptide separations rather than the hydrophobic ligands which dominate small molecule separations. Selectivity differences are observed between phases, but it appears that it is the accessibility differences between these phases which play a crucial role in peptide separations i.e. accessibility to silanols, the hydrophobic acetonitrile / ligand layer or a thin adsorbed water layer on the silica surface.

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

“…The sequences for the peptide probes followed either the base sequence for Bovine GLP-2 (1-15) "HADGSFSDEMNTVLD" or Bovine GLP-2 (16-33) "SLATRDFINWLIQTKITD". A further description of the peptide probes can be found in references [9,10].…”

“…Each stationary phase was assessed using nine peptide probes; three from the 1-15 portion of Bovine GLP-2, and six from the 16-33 portion of Bovine GLP-2, all nine probes differed by just one amino acid residue in order to evaluate subtle selectivity differences of the stationary phases towards these probes. The full 33 amino acid sequence had the propensity to form higher order structures, thus the sequence was divided into two, where the first base sequence contained amino acids 1-15 and the second contained amino acids 16-33 (Bovine-GLP-2 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) and Bovine-GLP-2 (16-33)). Selectivity (referred to as delta, Δtg * ) was determined by the normalised retention difference ( [9,10]) between certain modified peptides and its corresponding original base sequence (either Peptide Numbers 1 or 13, Table 2).…”

“…peptide probes to assess columns used for peptide separations [8]. With the recent development of the Peptide RPC Column Characterisation Protocol, it is now feasible to characterise stationary phases which highlight interactions, relevant for peptide separation methods, which was not feasible before [9,10]. This protocol characterises the stationary phase at both low and intermediate pH using gradient chromatography, where the solvents used were rationalised in reference [9].…”

“…With the recent development of the Peptide RPC Column Characterisation Protocol, it is now feasible to characterise stationary phases which highlight interactions, relevant for peptide separation methods, which was not feasible before [9,10]. This protocol characterises the stationary phase at both low and intermediate pH using gradient chromatography, where the solvents used were rationalised in reference [9]. Twenty-six probes were designed to interrogate the phases, before being iteratively assessed to reduce the number of peptides required but still adequately describe the column with minimal loss of information [9].…”

“…This protocol characterises the stationary phase at both low and intermediate pH using gradient chromatography, where the solvents used were rationalised in reference [9]. Twenty-six probes were designed to interrogate the phases, before being iteratively assessed to reduce the number of peptides required but still adequately describe the column with minimal loss of information [9]. The robustness of the protocol was critically assessed using a reduced factorial design to establish which variables must be carefully controlled to ensure the integrity of the protocol [10].…”