Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography
Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography
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DOI：
10.1016/j.chroma.2004.10.092
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发表时间：
2004-12-24
影响因子：
4.1
通讯作者：
Gebler, JC
中科院分区：
文献类型：
--
作者：
Gilar, M;Daly, AE;Gebler, JC
柱峰容量被用来衡量梯度洗脱条件下的柱效率。通过实验评估反相 (R-P) 和阳离子交换高效液相色谱 (HPLC) 柱的峰容量，并与 RP-HPLC 梯度理论预测的值进行比较。人们发现该模型对于预测单维和二维 (2D) 色谱中的峰容量和生产率非常有用。理论预测和实验数据都表明，尽管使用高效色谱柱或非常浅的梯度，HPLC 中分离的峰数量仍达到上限。最先进的 RP-HPLC 色谱柱的实际峰容量值约为数百。将色谱柱长度加倍（效率）可提高。峰容量仅增加 40%，并按比例增加分离时间和背压。同样，极浅的梯度对峰容量有积极影响，但分析时间会变得令人无法接受。该模型预测，使用第一维中的多个馏分收集，然后在第二维中使用短而高效的色谱柱进行快速 RP-HPLC 梯度，可在 8 小时内实现 15,000 的 2D-HPLC 峰容量。 (C) 2004 Elsevier B.V. 保留所有权利。
Column peak capacity was utilized as a measure of column efficiency for gradient elution conditions. Peak capacity was evaluated experimentally for reversed-phase (R-P) and cation-exchange high-performance liquid chromatography (HPLC) columns, and compared to the values predicted from RP-HPLC gradient theory. The model was found to be useful for the prediction of peak capacity and productivity in single- and two-dimensional (2D) chromatography. Both theoretical prediction and experimental data suggest that the number of peaks separated in HPLC reaches an upper limit, despite using highly efficient columns or very shallow gradients. The practical peak capacity value is about several hundred for state-of-the-art RP-HPLC columns. Doubling the column length (efficiency) improves the. peak capacity by only 40%, and proportionally increases both the separation time and the backpressure. Similarly, extremely shallow, gradients have a positive effect on the peak capacity, but analysis becomes unacceptably long. The model predicts that a 2D-HPLC peak capacity of 15,000 can be achieved in 8 h using multiple fraction collection in the first dimension followed by fast RP-HPLC gradients employing short, but efficient columns in the second dimension. (C) 2004 Elsevier B.V. All rights reserved.