Comparison of the practical resolving power of one- and two-dimensional high-performance liquid chromatography analysis of metabolomic samples
Comparison of the practical resolving power of one- and two-dimensional high-performance liquid chromatography analysis of metabolomic samples
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DOI：
10.1021/ac701676b
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发表时间：
2008-01-01
影响因子：
7.4
通讯作者：
Carr, Peter W.
中科院分区：
文献类型：
--
作者：
Stoll, Dwight R.;Wang, Xiaoli;Carr, Peter W.
二维液相色谱 (2DLC) 因其比一维液相色谱 (1DLC) 更高的峰容量而成为蛋白质组学研究的支柱。由于 2DLC 的分析时间通常较长（数十小时）且主要用于蛋白质组学应用，因此与更常见的等度和梯度洗脱 1DLC 技术相比，一般 HPLC 中的 2DLC 被视为一种利基技术，可用于分析含有数百至数千种成分的混合物。 2DLC 分析开发的下一步重要步骤是考虑使用其更高的分辨率来减少相当“简单”的混合物（仅包含数十到数百种化学成分）的分析时间。本文的主要目的是为需要决定 1DLC 还是 2DLC 在给定分析时间内提供卓越分离效果的从业人员提供指导。使用基于代表低分子量物质实际混合物的化合物的色谱特性的模型，预测玉米种子提取物的低分子量成分在多个分析时间的完全优化的 1DLC 和实际 2DLC 分离的条件峰容量。考虑到分离空间的不完全使用和一维欠采样的严重影响，对理想 2DLC 峰值容量进行了两项重要修正；我们相信，这第一次可以在现实条件下对 1D 和 2DLC 的分辨率进行公平比较。然后使用预测的最佳条件对低分子量玉米种子提取物进行实验分离，并对每个 1D 和 2DLC 色谱图中的峰进行计数。根据预测峰容量和实验色谱图中观察到的峰数量的比较，我们相信，对于持续时间超过约 10 分钟的分离，实用的 2DLC 将优于完全优化的梯度 1DLC。这个交叉时间比直观预期的要短得多，我们相信这一发现将不可避免地对 2DLC 在液相分离中的实践产生重大影响。
Two-dimensional liquid chromatography (2DLC) has become a mainstay of proteomics research due to its higher peak capacity compared to one-dimensional LC (1DLC). Because of the long analysis times typically associated with 2DLC (tens of hours) and its use primarily in proteomics applications, 2DLC in the context of general HPLC has been regarded as a niche technique for use in analysis of mixtures containing hundreds to thousands of components compared to the far more common techniques of isocratic and gradient elution 1DLC. A significant next step in the analytical development of 2DLC is to consider using its higher resolving power to reduce the analysis time of rather "simple" mixtures, in the range of only tens to hundreds of chemical constituents. The chief objective of this paper is to provide guidance to practitioners who need to decide whether 1DLC or 2DLC gives the superior separation in a given analysis time. Conditional peak capacities are predicted for fully optimized 1DLC and practical 2DLC separations of the low molecular weight constituents of an extract of corn seed at several analysis times using a model based on the chromatographic properties of compounds that are representative of real mixtures of lower molecular weight species. Two important corrections to the ideal 2DLC peak capacity are made to account for both incomplete usage of the separation space and the serious effect of first-dimension undersampling; this allows, we believe for the first time, a fair comparison of the resolving power of 1D- and 2DLC under realistic conditions. The predicted optimum conditions are then used to carry out experimental separations of low molecular weight corn seed extract, and peaks are counted in each 1D- and 2DLC chromatogram. Based on comparisons of both the predicted peak capacities and number of peaks observed in experimental chromatograms, we believe that practical 2DLC will be superior to fully optimized gradient 1DLC for separations lasting more than only about 10 min. This crossover time is much shorter than intuitively expected, and we believe this finding will inevitably have a major impact on the practice of 2DLC in liquid-phase separations in general.