Department of Chemistry, University of Tennessee, Knoxville, TN, USA.
Department of Chemistry, University of Tennessee, Knoxville, TN, USA.
J Chromatogr A. 2014 Jan 10;1324:155-63. doi: 10.1016/j.chroma.2013.11.036. Epub 2013 Nov 22.
Using a column packed with fully porous particles, four methods for controlling the flow rates at which gradient elution runs are conducted in very high pressure liquid chromatography (VHPLC) were tested to determine whether reproducible thermal conditions could be achieved, such that subsequent analyses would proceed at nearly the same initial temperature. In VHPLC high flow rates are achieved, producing fast analyses but requiring high inlet pressures. The combination of high flow rates and high inlet pressures generates local heat, leading to temperature changes in the column. Usually in this case a post-run time is input into the analytical method to allow the return of the column temperature to its initial state. An alternative strategy involves operating the column without a post-run equilibration period and maintaining constant temperature variations for subsequent analysis after conducting one or a few separations to bring the column to a reproducible starting temperature. A liquid chromatography instrument equipped with a pressure controller was used to perform constant pressure and constant flow rate VHPLC separations. Six replicate gradient separations of a nine component mixture consisting of acetophenone, propiophenone, butyrophenone, valerophenone, hexanophenone, heptanophenone, octanophenone, benzophenone, and acetanilide dissolved in water/acetonitrile (65:35, v/v) were performed under various experimental conditions: constant flow rate, two sets of constant pressure, and constant pressure operation with a programmed flow rate. The relative standard deviations of the response factors for all the analytes are lower than 5% across the methods. Programming the flow rate to maintain a fairly constant pressure instead of using instrument controlled constant pressure improves the reproducibility of the retention times by a factor of 5, when plotting the chromatograms in time.
使用填充有全多孔颗粒的柱，测试了在非常高的压力液相色谱（VHPLC）中控制梯度洗脱流速的四种方法，以确定是否可以实现可重复的热条件，使得随后的分析可以在几乎相同的初始温度下进行。在 VHPLC 中，流速很高，产生快速分析，但需要高入口压力。高流速和高入口压力的组合会产生局部热量，导致柱内温度发生变化。通常在这种情况下，会在分析方法中输入一个后运行时间，以使柱温度回到初始状态。另一种策略涉及在没有后运行平衡期的情况下操作柱，并在进行一次或几次分离以使柱达到可重复的起始温度后，保持柱温的恒定变化，以进行后续分析。配备压力控制器的液相色谱仪用于执行恒压和恒流速 VHPLC 分离。在各种实验条件下，对由溶于水/乙腈（65:35，v/v）的苯乙酮、丙苯酮、丁苯酮、戊苯酮、己苯酮、庚苯酮、辛苯酮、二苯甲酮和乙酰苯胺组成的九组分混合物进行了六次重复梯度分离：恒流速、两组恒压和恒压操作与程序流速。所有分析物的响应因子的相对标准偏差均低于 5%。与使用仪器控制的恒压相比，将流速编程以保持相当恒定的压力可将保留时间的重现性提高 5 倍，当以时间绘制色谱图时。
