Gustavus Adolphus College, Department of Chemistry, St. Peter, MN, USA; Universidade de São Paulo, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Department of Chemistry, Ribeirão Preto, SP, Brazil.
Restek Corporation, 110 Benner Circle, Bellefonte, PA, USA.
J Chromatogr A. 2019 Oct 25;1604:460484. doi: 10.1016/j.chroma.2019.460484. Epub 2019 Aug 27.
In recent years there has been increasing interest in the use of HILIC separations in two-dimensional liquid chromatography (2D-LC), mainly because the selectivity of HILIC separations complement that of reversed-phase separations for a variety of molecules. Historically, the re-equilibration of HILIC phases following gradient elution has been perceived as too slow to be useful in the second dimension of 2D-LC separations in particular. Recent studies of re-equilibration of HILIC phases by McCalley and coworkers using a limited set of conditions showed that highly repeatable gradient separations could be obtained with re-equilibration times as short as 4.3 min [1,2]. In this study we aimed to study re-equilibration of HILIC phases under a broader set of conditions, and at much shorter re-equilibration times, in the interest of determining whether or not HILIC separations can be generally considered as a viable option for use in the second dimension of 2D-LC separations. To this end we studied the effects of mobile phase pH, buffer concentration, and preparation method, flow rate, analyte and stationary phase chemistry, column length, and re-equilibration time on retention of a variety of small molecule probe solutes following gradient elution. In general, we have found that excellent separation repeatability can be obtained with quite short (≪10 min) re-equilibration times, even when progress toward full equilibration of the column is quite slow (≫10 min). In other words, even if the stationary phase is not fully equilibrated, as long as it is partially equilibrated in a highly precise manner, highly repeatable retention times can be obtained. Higher flow rate has a positive effect on both the rate of progress toward full equilibration and the repeatability of separation. No significant, consistent effects of eluent pH or buffer concentration on repeatability of gradient separation were observed for the stationary phases studied. Excellent gradient separation repeatability was obtained with shorter columns (30 mm length) with re-equilibration times as short as 3 s. A proof-of-concept 2D-LC separation of several small molecule probes using HILIC separations in both dimensions was performed to illustrate that re-equilibration of these columns can be fast enough for HILIC columns to be considered as a viable option for the second dimension of comprehensive 2D-LC separations.
近年来，人们对在二维液相色谱（2D-LC）中使用亲水相互作用色谱（HILIC）分离越来越感兴趣，主要是因为 HILIC 分离的选择性可以补充反相分离对各种分子的选择性。从历史上看，HILIC 相在梯度洗脱后的再平衡被认为太慢，在二维液相色谱分离的第二维中不太有用。最近，McCalley 及其同事使用有限的条件研究了 HILIC 相的再平衡，结果表明，使用再平衡时间短至 4.3 分钟即可获得高度可重复的梯度分离[1,2]。在这项研究中，我们旨在在更广泛的条件下，在更短的再平衡时间下研究 HILIC 相的再平衡，以确定 HILIC 分离是否可以普遍认为是二维液相色谱分离第二维的可行选择。为此，我们研究了流动相 pH 值、缓冲液浓度和制备方法、流速、分析物和固定相化学、柱长和再平衡时间对各种小分子探针溶质在梯度洗脱后的保留的影响。一般来说，我们发现即使柱的平衡过程非常缓慢（>10 分钟），通过非常短（<10 分钟）的再平衡时间也可以获得出色的分离重现性。换句话说，即使固定相没有完全平衡，只要它以高度精确的方式部分平衡，就可以获得高度可重复的保留时间。更高的流速对完全平衡的速度和分离的重复性都有积极的影响。在所研究的固定相中，没有观察到洗脱液 pH 值或缓冲液浓度对梯度分离重复性有显著一致的影响。使用较短的色谱柱（30 毫米长度）和 3 秒的再平衡时间即可获得出色的梯度分离重现性。通过在两个维度上使用 HILIC 分离进行了几种小分子探针的二维液相色谱分离的概念验证，证明这些色谱柱的再平衡速度足够快，可以将 HILIC 柱视为全面二维液相色谱分离的第二维的可行选择。
