Schlabach T D, Wilson K J
J Chromatogr. 1987 Jan 9;385:65-74. doi: 10.1016/s0021-9673(01)94622-9.
Reversed-phase chromatography of proteins on microbore columns can achieve sensitivities that exceed those for standard-bore columns by a factor of 10-20, when operated at the same linear velocities. These gains in sensitivity are accompanied by proportional reductions in peak volume. Sensitivities on standard- (4.6 mm I.D.) and narrow-bore (2.1 mm I.D.) columns have been further improved by reducing the flow-rates to those typical for microbore (1 mm I.D.) columns. We have investigated the role of flow-rate in determining peak volumes for a constant time gradient and found that flow-rate affects peak volume to a much greater extent than column diameter. Column length was not found to have a significant effect on either peak volume or sensitivity. We have found that a four-fold reduction in flow-rate results in at least a two-fold reduction in peak volume over the flow-rate range from 25 to 400 microliters/min. Recovery of proteins in smaller volumes should prove beneficial to subsequent protein characterization methodologies.
当以相同的线性流速操作时，在微径柱上进行蛋白质的反相色谱分析可实现比标准内径柱高10至20倍的灵敏度。灵敏度的提高伴随着峰体积成比例的减小。通过将流速降低到微径柱（内径1mm）的典型流速，标准内径（4.6mm）柱和窄内径（2.1mm）柱的灵敏度得到了进一步提高。我们研究了流速在恒定时间梯度下对峰体积的影响，发现流速对峰体积的影响远大于柱直径。未发现柱长对峰体积或灵敏度有显著影响。我们发现，在25至400微升/分钟的流速范围内，流速降低四倍会导致峰体积至少降低两倍。在较小体积中回收蛋白质对后续的蛋白质表征方法应该是有益的。
