The importance of ion-pairing in peptide purification by reversed-phase liquid chromatography
The importance of ion-pairing in peptide purification by reversed-phase liquid chromatography
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DOI：
10.1016/j.chroma.2017.03.041
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发表时间：
2017-05-05
影响因子：
4.1
通讯作者：
Fornstedt, Torgny
中科院分区：
文献类型：
--
作者：
Asberg, Dennis;Weinmann, Annika Langborg;Fornstedt, Torgny
在离子对试剂三氟乙酸(TFA)存在的情况下，在端盖的C-18键合固定相上进行等温测量。这项研究的总体目的是通过机械建模和多变量实验设计，更好地了解洗脱液中的乙腈和TFA组分如何影响超载洗脱曲线和多肽之间的选择性。在研究TFA的作用时，通过氟-质子核磁共振增强实验提供了多肽与TFA在乙腈-水溶液中形成离子对的直接证据，并通过前沿分析测量了TFA在固定相上的吸附。然后用反相法测定了8种TFA浓度范围为2.6 mM~37.3 mM(0.02~0.29体积%)的等度洗脱条件下的吸附等温线。通过将质量平衡与反应动力学相耦合，并测定了两种物种的分离吸附等温线，模拟了多肽离子与多肽-TFA络合物之间的平衡。我们发现，多肽-TFA络合物的洗脱曲线符合Langmuir等温式，而多肽离子具有较强的二次相互作用，其吸附等温式符合双向朗缪尔吸附等温式。在低TFA浓度下，由于与饱和容量很低的多肽离子的二次相互作用，洗脱图谱呈现出由前端的尖峰和较长的尾部组成的不利形状。通过直接从梯度洗脱获得的洗脱曲线测定吸附等温线，研究了乙腈对吸附等温线的依赖关系，从而使研究乙腈的较宽区间成为可能。在这里，发现在很低的乙腈馏分时，柱很快就达到饱和容量，并且吸附随着分子量的变化有很大的变化。最后，基于一个以坡度、坡度和TFA浓度为因子的实验设计，讨论了方法开发的实际意义。(C)2017年，由爱思唯尔出版。
isotherm measurements in the presence of an ion-pairing reagent, trifluoroacetic acid (TFA), on an end-capped C-18-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.