title: The importance of ion-pairing in peptide purification by reversed-phase liquid chromatography.
abstract: The adsorption mechanism for three peptides was studied under overloaded conditions through adsorption isotherm measurements in the presence of an ion-pairing reagent, trifluoroacetic acid (TFA), on an end-capped C18-bonded stationary phase. The overall aim of the study was to obtain a better understanding of how the acetonitrile and the TFA fractions in the eluent affected the overloaded elution profiles and the selectivity between peptides using mechanistic modelling and multivariate design of experiments. When studying the effect of TFA, direct evidence for ion pair formation between a peptide and TFA in acetonitrile-water solutions was provided by fluorine-proton nuclear Overhauser NMR enhancement experiments and the adsorption of TFA on the stationary phase was measured by frontal analysis. The adsorption isotherms for each peptide were then determined by the inverse method at eight TFA concentrations ranging from 2.6mM to 37.3mM (0.02-0.29vol-%) in isocratic elution. The equilibrium between the peptide ion and the peptide-TFA complex was modelled by coupling the mass-balance to reaction kinetics and determining separate adsorption isotherms for the two species. We found that a Langmuir isotherm described the elution profile of peptide-TFA complex well while the peptide ion was described by a bi-Langmuir adsorption isotherm since it exhibited strong secondary interactions. The elution profiles had an unfavorable shape at low TFA concentrations consisting of a spike in their front and a long tailing rear due to the secondary interactions for the peptide ion having very low saturation capacity. The acetonitrile dependence on the adsorption isotherms was studied by determination of adsorption isotherms directly from elution profiles obtained in gradient elution which enabled a broad acetonitrile interval to be studied. Here, it was found that the column saturation capacity was quickly reached at very low acetonitrile fractions and that there were significant variations in adsorption with the molecular weight. Finally, practical implications for method development are discussed based on an experimental design where gradient slope and TFA concentrations are used as factors.
authors.affiliation: Department of Engineering and Chemical Sciences, Karlstad University, SE-651 88 Karlstad, Sweden.
authors.forename: Dennis
authors.initials: D
authors.lastname: Åsberg
authors.affiliation: Respiratory, Inflammation and Autoimmunity, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, SE 431 83 Mölndal, Sweden.
authors.forename: Annika
authors.initials: A
authors.lastname: Langborg Weinmann
authors.affiliation: Respiratory, Inflammation and Autoimmunity, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, SE 431 83 Mölndal, Sweden.
authors.forename: Tomas
authors.initials: T
authors.lastname: Leek
authors.affiliation: Respiratory, Inflammation and Autoimmunity, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, SE 431 83 Mölndal, Sweden.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Lewis
authors.affiliation: Respiratory, Inflammation and Autoimmunity, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, SE 431 83 Mölndal, Sweden.
authors.forename: Magnus
authors.initials: M
authors.lastname: Klarqvist
authors.affiliation: Department of Chemical and Process Engineering, Rzeszów University of Technology, PL-359 59 Rzeszów, Poland.
authors.forename: Marek
authors.initials: M
authors.lastname: Leśko
authors.affiliation: Department of Chemical and Process Engineering, Rzeszów University of Technology, PL-359 59 Rzeszów, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Kaczmarski
authors.affiliation: Department of Engineering and Chemical Sciences, Karlstad University, SE-651 88 Karlstad, Sweden.
authors.forename: Jörgen
authors.initials: J
authors.lastname: Samuelsson
authors.affiliation: Department of Engineering and Chemical Sciences, Karlstad University, SE-651 88 Karlstad, Sweden. Electronic address: Torgny.Fornstedt@kau.se.
authors.forename: Torgny
authors.initials: T
authors.lastname: Fornstedt
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D000327:Adsorption; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification*; D014269:Trifluoroacetic Acid / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D007477:Ions; D010455:Peptides; D014269:Trifluoroacetic Acid; C032159:acetonitrile
keywords: Adsorption isotherm; Ion-pair formation; Nuclear Overhauser enhancement; Peptide separations; Preparative separation; TFA