Investigation into reversed phase chromatography peptide separation systems part II: An evaluation of the robustness of a protocol for column characterisation

Field, Jennifer K.; Euerby, Melvin R.; Petersson, Patrik

doi:10.1016/j.chroma.2019.05.037

Cited by 15 publications

(30 citation statements)

References 22 publications

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“…4 In order to investigate the hypothesis that TFA masks certain interactions between the column and the peptide, a study was conducted where 0.1% v/v formic acid was substituted with 0.1% v/v TFA in both the aqueous and organic phase on the reduced number of delta values. 13 columns were tested using a simplified and more robust version of the protocol described in reference [37] which used 8 of the 11 probes to increase the reliability of the methodology. Distinct groups can be observed in the formic acid biplot plot Fig.…”

Section: Principal Component Analysismentioning

confidence: 99%

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

Field

Euerby

Lau

et al. 2019

Journal of Chromatography A

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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.

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Section: Principal Component Analysismentioning

confidence: 99%

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

Field

Euerby

Lau

et al. 2019

Journal of Chromatography A

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“…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].…”

Section: Introductionmentioning

confidence: 99%

“…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]. The definitive protocol and the mitigation necessary to ensure robustness can be found elsewhere [10].…”

Section: Introductionmentioning

confidence: 99%

“…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]. The definitive protocol and the mitigation necessary to ensure robustness can be found elsewhere [10]. The mitigation included a further reduction of the number of peptides and delta values used in the definitive protocol applied in the current study [10].…”

Section: Introductionmentioning

confidence: 99%

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

Field

Euerby

Petersson

2020

Journal of Chromatography A

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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.

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“…In addition, pH 2.5 would minimise any contribution from free silanol groups of the base silica towards the phase's cation exchange capacity. An elevated temperature of 40 °C was selected to minimise differences in oven types [17]. A preliminary one factor at a time (OFAT) study over the temperature range of 36-44 °C highlighted that temperature had a minimal effect on the two hydrophilic bases (benzylamine and salbutamol, Δk (44-36 °C) = − 0.25 and − 0.19, respectively) whereas the two hydrophobic bases (nortriptyline and diphenhydramine, Δk (44-36 °C) = − 0.32 and − 0.41, respectively) demonstrated a greater reduction in retention as temperature was increased.…”

Section: Rationale For the Selection Of The Lc Conditions And Chromatmentioning

confidence: 99%

Column Classification/Characterisation of Strong Cation Exchange Phases for the Liquid Chromatographic Analysis of Small Molecular Weight Bases

et al. 2020

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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.

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Investigation into reversed phase chromatography peptide separation systems part II: An evaluation of the robustness of a protocol for column characterisation

Cited by 15 publications

References 22 publications

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

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

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

Column Classification/Characterisation of Strong Cation Exchange Phases for the Liquid Chromatographic Analysis of Small Molecular Weight Bases

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