Department of Chemistry, University of Tennessee, Knoxville, TN, USA.
Department of Chemistry, University of Tennessee, Knoxville, TN, USA.
J Chromatogr A. 2014 May 23;1343:79-90. doi: 10.1016/j.chroma.2014.03.054. Epub 2014 Mar 28.
A critical aspect in fast gradient separations carried out under constant pressure, in the very high pressure liquid chromatography (VHPLC) mode is that time-based chromatograms may not yield highly reproducible separations. A proposed solution to improve the reproducibility of these separations involves plotting the chromatograms as functions of the volume eluted vs. UV absorbance instead of time vs. UV. To study the consequences of using the volume-based rather than the time-based chromatograms, separations were first performed under low pressures that do not generate significant amounts of heat and for which the variations of the eluent density along the columns are negligible. Secondly, they were performed under very high pressures that do generate heat and measurable variations of the local retention factor and eluent density along the column. Comparison of the results provides estimates of the improvements obtained when volume based chromatograms are used in gradient analyses. Using a column packed with fully porous particles, four different types of methods and several sets for each method were used to perform the gradient elution runs: two sets of constant flow rate operations, four sets of constant pressure operations, two sets of constant pressure operations with programmed flow rate, and one set using the constant heat loss approach. The differences between time-based and volume-based chromatograms are demonstrated by using eight replicates of early, middle, and last eluting peaks. The results show that volume-based chromatograms improve the retention time reproducibility of the four constant pressure methods by a factor of 3.7 on average. If the column is not thermally conditioned prior to performing a long series of separations, flow controlled methods (constant flow rate, programmed constant pressure, and constant wall heat approaches) are more precise. If one gradient run is used to bring the column to a relatively stable temperature, constant pressure separations have a factor of 3 times better reproducibility of retention times with respect to constant flow rate gradient separations.
在恒压下进行快速梯度分离时，一个关键的方面是基于时间的色谱图可能无法产生高度重现的分离。为了提高这些分离的重现性，提出了一种解决方案，即将色谱图绘制为洗脱体积与紫外吸光度的函数，而不是时间与紫外吸光度的函数。为了研究使用基于体积而不是基于时间的色谱图的后果，首先在不会产生大量热量的低压下进行分离，并且对于这些分离，洗脱液密度沿柱子的变化可以忽略不计。其次，在会产生热量并且可以测量柱上局部保留因子和洗脱液密度沿柱子变化的非常高压下进行分离。比较结果提供了当在梯度分析中使用基于体积的色谱图时获得的改进的估计值。使用填充有全多孔颗粒的柱子，使用四种不同类型的方法和每种方法的多组来进行梯度洗脱运行：两组恒流速操作，四组恒压操作，两组带程序流速的恒压操作，以及一组使用恒热损失方法。通过使用八个早期、中期和最后洗脱峰的重复来证明基于时间和基于体积的色谱图之间的差异。结果表明，基于体积的色谱图将四种恒压方法的保留时间重现性提高了平均 3.7 倍。如果在进行一系列长的分离之前不对柱子进行热调节，则流量控制方法（恒流速、程序恒压和恒壁热方法）更精确。如果使用一个梯度运行将柱子带到相对稳定的温度，则相对于恒流速梯度分离，恒压分离的保留时间重现性提高了 3 倍。
