Beyaza Ayse, Fana Wenzhe, Carr Peter W, Schellinger Adam P
Department of Chemistry, Smith and Kolthoff Halls, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN 55455, USA.
J Chromatogr A. 2014 Dec 5;1371:90-105. doi: 10.1016/j.chroma.2014.09.085.
The precision of retention time in RPLC is important for compound identification, for setting peak integration time windows and in fundamental studies of retention. In this work, we studied the effect of temperature (T), initial (ϕo) and final mobile phase (ϕf) composition, gradient time (tG), and flow rate (F) on the retention time precision under gradient elution conditions for various types of low MW solutes. We determined the retention factor in pure water ( [Formula: see text] ) and the solute-dependent solvent strength (S) parameters of Snyder's linear solvent strength theory (LSST) as a function of temperature for three different groups of solutes. The effect of small changes in the chromatographic variables (T, ϕo, ϕf, tG and F) by use of the LSST gradient retention equation were estimated. Peaks at different positions in the chromatogram have different sensitivities to changes in these instrument parameters. In general, absolute fluctuations in retention time are larger at longer gradient times. Drugs showed less sensitivity to changes in temperature compared to relatively less polar solutes, non-ionogenic solutes. Surprisingly we observed that fluctuations in temperature, mobile phase composition and flow rate had less effect on retention time under gradient conditions as compared to isocratic conditions. Overall temperature and the initial mobile phase composition are the most important variables affecting retention reproducibility in gradient elution chromatography.
在反相液相色谱（RPLC）中，保留时间的精密度对于化合物鉴定、设置峰积分时间窗口以及保留的基础研究都很重要。在本工作中，我们研究了温度（T）、初始（ϕo）和最终流动相（ϕf）组成、梯度时间（tG）以及流速（F）对各种低分子量溶质在梯度洗脱条件下保留时间精密度的影响。我们确定了三种不同溶质组在纯水中的保留因子（[公式：见原文]）以及斯奈德线性溶剂强度理论（LSST）中与溶质相关的溶剂强度（S）参数随温度的变化情况。利用LSST梯度保留方程估算了色谱变量（T、ϕo、ϕf、tG和F）的微小变化所产生的影响。色谱图中不同位置的峰对这些仪器参数变化的敏感度不同。一般来说，在较长梯度时间下，保留时间的绝对波动更大。与极性相对较小的溶质、非离子型溶质相比，药物对温度变化的敏感度较低。令人惊讶的是，我们观察到与等度洗脱条件相比，在梯度条件下温度、流动相组成和流速的波动对保留时间的影响较小。总体而言，温度和初始流动相组成是影响梯度洗脱色谱中保留重现性的最重要变量。
