Schellinger Adam P, Stoll Dwight R, Carr Peter W
Department of Chemistry, Smith and Kolthoff Halls, University of Minnesota, 207 Pleasant Street S.E., Minneapolis, MN 55455, USA.
J Chromatogr A. 2008 May 23;1192(1):41-53. doi: 10.1016/j.chroma.2008.01.062. Epub 2008 Jan 31.
We studied the run-to-run repeatability of the retention times of both non-ionizable and basic compounds chromatographed using buffered eluents. The effect of flow rate, organic modifier and other additives, buffer type/concentration, stationary phase type, batch-to-batch preparation of the initial eluent, gradient time, sample type and intra-day changes on retention repeatability were examined. We also assessed the effect of column storage solvent conditions on the inter-day repeatability. Although retention repeatability is strongly influenced by many parameters (flow rate, solvent compressibility compensation, precision of temperature control, and buffer/stationary phase type), our primary finding is that with a reasonable size column (15cmx4.6mm (i.d.)) two column volumes of re-equilibration with initial eluent suffices to provide acceptable repeatability (no worse than 0.004min) for both non-ionizable and basic analytes under a wide variety of conditions. Under ideal conditions (e.g. the right buffer, flow rate, etc.) it is possible to obtain truly extraordinary repeatability often as good as 0.0004min. These absolute fluctuations in retention translate to worst case changes in resolution of 0.2 units and average changes of only 0.02 units.
我们研究了使用缓冲洗脱液进行色谱分析时，非离子化化合物和碱性化合物保留时间的批间重复性。考察了流速、有机改性剂及其他添加剂、缓冲液类型/浓度、固定相类型、初始洗脱液的批次间配制、梯度时间、样品类型和日内变化对保留重复性的影响。我们还评估了柱储存溶剂条件对日间重复性的影响。尽管保留重复性受到许多参数（流速、溶剂压缩性补偿、温度控制精度以及缓冲液/固定相类型）的强烈影响，但我们的主要发现是，对于尺寸合理的色谱柱（15cm×4.6mm（内径）），用初始洗脱液进行两个柱体积的再平衡，足以在各种条件下为非离子化和碱性分析物提供可接受的重复性（不超过0.004分钟）。在理想条件下（如合适的缓冲液、流速等），有可能获得通常高达0.0004分钟的极佳重复性。这些保留时间的绝对波动在最坏情况下导致分离度变化0.2个单位，平均变化仅为0.02个单位。
