Petersson Patrik, Frank Andre, Heaton James, Euerby Melvin R
AstraZeneca R&D Lund, Lund, Sweden.
J Sep Sci. 2008 Jul;31(13):2346-57. doi: 10.1002/jssc.200800064.
The practical effects of gradient time and flow rate on the peak capacities of a range of analytes of differing molecular weights (MWs) and physico-chemical properties have been evaluated using ultra high pressure LC instrumentation with sub-2 mum and superficially porous particle phases. Optimum peak capacity, in RP gradient LC, for small molecules, including typical pharmaceutical drugs and peptides with MWs up to 1300, was demonstrated at a maximum flow rate for a given gradient time (i.e. up to 40 min). Flow rates significantly higher than the optimum in the van Deemter plots and also higher than those typically employed by the majority of the chromatographers today are recommended for gradient LC (i.e. up to 1.0 mL/min on 50-150x2.1 mm 1.7 mum columns). This recommendation is applicable for temperatures above 40 degrees C, i.e. temperatures typically utilized for separations employing sub-2 mum particles to reduce column back pressure. Van Deemter and pseudo van Deemter plots were determined and combined with chromatographic gradient elution theory to explain our unexpected observations. The derived models exhibited good agreement between experimental and predicted peak capacities (absolute average error 4%, max. error 12%).
使用具有亚2微米和表面多孔颗粒相的超高压液相色谱仪器，评估了梯度时间和流速对一系列不同分子量（MW）和物理化学性质的分析物的峰容量的实际影响。在给定梯度时间（即长达40分钟）的最大流速下，展示了反相梯度液相色谱中对于小分子（包括典型药物和分子量高达1300的肽）的最佳峰容量。对于梯度液相色谱，建议使用显著高于范德姆特图中的最佳流速且高于当今大多数色谱工作者通常使用的流速（即在50 - 150×2.1 mm 1.7微米柱上高达1.0 mL/min）。该建议适用于40摄氏度以上的温度，即通常用于采用亚2微米颗粒以降低柱背压的分离的温度。测定了范德姆特图和伪范德姆特图，并结合色谱梯度洗脱理论来解释我们意外的观察结果。所推导的模型在实验和预测的峰容量之间表现出良好的一致性（绝对平均误差4%，最大误差12%）。
