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  • 1.
    Åsberg, Dennis
    et al.
    Karlstads universitet, Fakulteten för hälsa, natur- och teknikvetenskap (from 2013), Institutionen för ingenjörs- och kemivetenskaper (from 2013).
    Weinmann, Annika Langborg
    AstraZeneca, Resp Inflammat & Autoimmun, Innovat Med & Early Dev Biotech Unit, SE-43183 Molndal, Sweden..
    Leek, Tomas
    AstraZeneca, Resp Inflammat & Autoimmun, Innovat Med & Early Dev Biotech Unit, SE-43183 Molndal, Sweden..
    Lewis, Richard J.
    AstraZeneca, Resp Inflammat & Autoimmun, Innovat Med & Early Dev Biotech Unit, SE-43183 Molndal, Sweden..
    Klarqvist, Magnus
    AstraZeneca, Resp Inflammat & Autoimmun, Innovat Med & Early Dev Biotech Unit, SE-43183 Molndal, Sweden..
    Lesko, Marek
    Rzeszow Univ Technol, Dept Chem & Proc Engn, PL-35959 Rzeszow, Poland..
    Kaczmarski, Krzysztof
    Rzeszow Univ Technol, Dept Chem & Proc Engn, PL-35959 Rzeszow, Poland..
    Samuelsson, Jörgen
    Karlstads universitet, Fakulteten för hälsa, natur- och teknikvetenskap (from 2013), Institutionen för ingenjörs- och kemivetenskaper (from 2013).
    Fornstedt, Torgny
    Karlstads universitet, Fakulteten för hälsa, natur- och teknikvetenskap (from 2013), Institutionen för ingenjörs- och kemivetenskaper (from 2013).
    The importance of ion-pairing in peptide purification by reversed-phase liquid chromatography2017Inngår i: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1496, s. 80-91Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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 2.6 mM to 37.3 mM (0.02–0.29 vol-%) 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. (c) 2017 Published by Elsevier B.V.

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