Horváth Krisztián, Fairchild Jacob, Guiochon Georges
Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600, USA.
J Chromatogr A. 2009 Mar 20;1216(12):2511-8. doi: 10.1016/j.chroma.2009.01.064. Epub 2009 Jan 24.
A step by step strategy of optimization of comprehensive off-line two-dimensional liquid chromatography (2D-LC) separations is proposed. The goal of an optimization process in the separation sciences is either to achieve a given resolution (a target peak capacity in 2D-LC) within as short a time as possible or to reach the highest possible resolution in a given analysis time. The proposed method takes into account the characteristics of the columns used in the first and the second dimension and the number of fractions of the first dimension eluent that should be collected. The effect of the time spent during the analysis on the second dimension column to carry out necessary tasks that are not the separation itself (called the additional time) on the maximum peak capacity that is achievable was carefully investigated. It was shown that (1) an increase in the peak capacity of the first dimension column combined with the collection of larger volume fractions permits a significant reduction of the time needed to achieve the desired peak capacity; and (2) there is an optimum fraction collection ratio (or number of collected fractions per peak) which yields the target peak capacity in the minimum time. The proposed strategy was used for the optimization of the separation of samples of BSA tryptic digest by an off-line 2D-LC using an SCXmultiply sign in circleRP-HPLC method. As a result of this optimization, a peak capacity of 4000 could be achieved in about 5h with the two columns available. The time needed for the optimized analysis was less than two thirds of the analysis time that would have been needed had the conventional rule of thumb of sample collection in comprehensive on-line 2D-LC (4 samples/peak) been followed.
本文提出了一种优化综合离线二维液相色谱（2D-LC）分离的逐步策略。分离科学中优化过程的目标要么是在尽可能短的时间内实现给定的分离度（二维液相色谱中的目标峰容量），要么是在给定的分析时间内达到尽可能高的分离度。所提出的方法考虑了一维和二维中使用的色谱柱的特性以及应收集的一维洗脱液的馏分数量。仔细研究了在二维色谱柱上进行非分离本身的必要任务（称为额外时间）所花费的时间对可实现的最大峰容量的影响。结果表明：（1）一维色谱柱峰容量的增加与收集更大体积的馏分相结合，可以显著减少实现所需峰容量所需的时间；（2）存在一个最佳馏分收集率（或每个峰收集的馏分数），可在最短时间内产生目标峰容量。所提出的策略用于通过使用SCX×RP-HPLC方法的离线二维液相色谱优化牛血清白蛋白胰蛋白酶消化物样品的分离。通过这种优化，使用现有的两根色谱柱，在约5小时内可实现4000的峰容量。优化分析所需的时间不到遵循综合在线二维液相色谱中传统的样品收集经验法则（每个峰4个样品）所需分析时间的三分之二。
