Wang Nu, Boswell Paul G
Department of Plant and Microbial Biology, University of Minnesota, 1479 Gortner Ave., St. Paul, MN 55108, USA.
Department of Plant and Microbial Biology, University of Minnesota, 1479 Gortner Ave., St. Paul, MN 55108, USA.
J Chromatogr A. 2017 Oct 20;1520:75-82. doi: 10.1016/j.chroma.2017.08.050. Epub 2017 Aug 26.
Gradient retention times are difficult to project from the underlying retention factor (k) vs. solvent composition (φ) relationships. A major reason for this difficulty is that gradients produced by HPLC pumps are imperfect - gradient delay, gradient dispersion, and solvent mis-proportioning are all difficult to account for in calculations. However, we recently showed that a gradient "back-calculation" methodology can measure these imperfections and take them into account. In RPLC, when the back-calculation methodology was used, error in projected gradient retention times is as low as could be expected based on repeatability in the k vs. φ relationships. HILIC, however, presents a new challenge: the selectivity of HILIC columns drift strongly over time. Retention is repeatable in short time, but selectivity frequently drifts over the course of weeks. In this study, we set out to understand if the issue of selectivity drift can be avoid by doing our experiments quickly, and if there any other factors that make it difficult to predict gradient retention times from isocratic k vs. φ relationships when gradient imperfections are taken into account with the back-calculation methodology. While in past reports, the accuracy of retention projections was >5%, the back-calculation methodology brought our error down to ∼1%. This result was 6-43 times more accurate than projections made using ideal gradients and 3-5 times more accurate than the same retention projections made using offset gradients (i.e., gradients that only took gradient delay into account). Still, the error remained higher in our HILIC projections than in RPLC. Based on the shape of the back-calculated gradients, we suspect the higher error is a result of prominent gradient distortion caused by strong, preferential water uptake from the mobile phase into the stationary phase during the gradient - a factor our model did not properly take into account. It appears that, at least with the stationary phase we used, column distortion is an important factor to take into account in retention projection in HILIC that is not usually important in RPLC.
梯度保留时间很难从潜在的保留因子（k）与溶剂组成（φ）的关系中推算出来。造成这种困难的一个主要原因是，高效液相色谱泵产生的梯度并不完美——梯度延迟、梯度扩散和溶剂配比不当在计算中都很难考虑进去。然而，我们最近表明，一种梯度“反算”方法可以测量这些不完美之处并将其考虑在内。在反相液相色谱（RPLC）中，当使用反算方法时，基于k与φ关系的重复性，预测的梯度保留时间误差低至预期水平。然而，亲水作用色谱（HILIC）带来了新的挑战：HILIC柱的选择性会随时间强烈漂移。保留情况在短时间内是可重复的，但选择性在数周内经常会发生漂移。在本研究中，我们着手探究快速进行实验是否能避免选择性漂移问题，以及在使用反算方法考虑梯度不完美因素的情况下，是否还有其他因素使得从等度k与φ关系预测梯度保留时间变得困难。在过去的报告中，保留预测的准确率>5%，而反算方法将我们的误差降低到了约1%。这个结果比使用理想梯度进行的预测精确6 - 43倍，比使用偏移梯度（即仅考虑梯度延迟的梯度）进行的相同保留预测精确3 - 5倍。不过，我们在HILIC预测中的误差仍然高于RPLC中的误差。基于反算梯度的形状，我们怀疑较高的误差是由于在梯度过程中流动相中的水大量优先被固定相吸收导致显著的梯度畸变造成的——这是我们的模型没有适当考虑的一个因素。看来，至少对于我们使用的固定相而言，柱畸变是HILIC保留预测中一个需要考虑的重要因素，而在RPLC中通常并不重要。
