Department of Chemistry, University of Tennessee, Knoxville, TN, USA.
Department of Chemistry, University of Tennessee, Knoxville, TN, USA.
J Chromatogr A. 2014 Jan 17;1325:99-108. doi: 10.1016/j.chroma.2013.11.058. Epub 2013 Dec 11.
Five methods for controlling the mobile phase flow rate for gradient elution analyses using very high pressure liquid chromatography (VHPLC) were tested to determine thermal stability of the column during rapid gradient separations. To obtain rapid separations, instruments are operated at high flow rates and high inlet pressure leading to uneven thermal effects across columns and additional time needed to restore thermal equilibrium between successive analyses. The purpose of this study is to investigate means to minimize thermal instability and obtain reliable results by measuring the reproducibility of the results of six replicate gradient separations of a nine component RPLC standard mixture under various experimental conditions with no post-run times. Gradient separations under different conditions were performed: constant flow rates, two sets of constant pressure operation, programmed flow constant pressure operation, and conditions which theoretically should yield a constant net heat loss at the column's wall. The results show that using constant flow rates, programmed flow constant pressures, and constant heat loss at the column's wall all provide reproducible separations. However, performing separations using a high constant pressure with programmed flow reduces the analysis time by 16% compared to constant flow rate methods. For the constant flow rate, programmed flow constant pressure, and constant wall heat experiments no equilibration time (post-run time) was required to obtain highly reproducible data.
为了确定快速梯度分离过程中柱子的热稳定性，测试了五种用于梯度洗脱分析的流动相流速控制方法，这些方法均使用非常高压液相色谱（VHPLC）。为了实现快速分离，仪器在高流速和高入口压力下运行，导致柱子之间的热效应不均匀，并且需要额外的时间来恢复连续分析之间的热平衡。本研究的目的是通过测量在没有后运行时间的情况下，在各种实验条件下对九组分反相高效液相色谱（RPLC）标准混合物的六个重复梯度分离结果的重现性，研究最小化热不稳定性和获得可靠结果的方法。在不同条件下进行梯度分离：恒定流速、两组定压操作、程序流量定压操作和理论上应在柱子壁上产生恒定净热损失的条件。结果表明，使用恒定流速、程序流量定压和柱子壁恒定热损失都可以提供重现性分离。然而，与恒定流速方法相比，使用高恒定压力和程序流量进行分离可将分析时间缩短 16%。对于恒定流速、程序流量定压和恒定壁热实验，无需平衡时间（后运行时间）即可获得高度重现的数据。
