McCalley David V
Centre for Research in Biomedicine, University of the West of England, Frenchay, Bristol BS 1QY, UK.
J Chromatogr A. 2005 May 6;1073(1-2):137-45. doi: 10.1016/j.chroma.2004.10.023.
The performance of a purely polymeric and a Type B silica-based C18 reversed-phase column was compared for the analysis of the basic peptide bradykinin and some analogues in order to assess the contribution of silanol interactions to peak shape. Good peak shapes were obtained for small masses of these peptides (0.1 microg or less) using acidic mobile phases on both columns; however, both showed a similar and serious deterioration in peak shape with increasing sample mass. Loss of efficiency on both columns as sample mass increased was considerably more serious when using formic acid rather than trifluoroacetic acid (TFA) as a mobile phase additive. For example, the peak capacity for a 2.5 microg load of one bradykinin on the polymeric column was reduced to only 0.38 times its value for 0.1 microg when using 0.02 M formic acid, compared with 0.77 times its value when using the same concentration of TFA. This result can be attributed to the ion pair effect of TFA and its higher ionic strength, which reduce mutual repulsion of charged peptides when held on the hydrophobic surface of the phase. Addition of salt (KCl) to the formic acid mobile phase caused dramatic increases in retention on the polymeric column, which can also be attributed to ion-pairing effects between halide ions and peptides. The increase in retention with salt addition also confirms that there are no ionic retention sites on the polymeric phase at low pH. The general similarity in behaviour between the polymeric and silica column suggests that silanol groups have little involvement in the retention and overload behaviour of these peptides when using highly inert Type B silica phases.
为了评估硅醇相互作用对峰形的影响，比较了纯聚合物型和B型硅胶基C18反相柱在分析碱性肽缓激肽及其一些类似物时的性能。使用酸性流动相时，在两根柱子上对这些小质量肽（0.1微克或更少）都获得了良好的峰形；然而，随着样品质量增加，两根柱子的峰形都出现了类似且严重的恶化。当使用甲酸而非三氟乙酸（TFA）作为流动相添加剂时，随着样品质量增加，两根柱子的效率损失要严重得多。例如，在聚合物柱上，当使用0.02 M甲酸时，2.5微克缓激肽负载的峰容量降至0.1微克时的0.38倍，而使用相同浓度TFA时为0.77倍。这一结果可归因于TFA的离子对效应及其较高的离子强度，这减少了带电肽在固定相疏水表面时的相互排斥。向甲酸流动相中添加盐（KCl）导致聚合物柱上的保留显著增加，这也可归因于卤离子与肽之间的离子对效应。加盐后保留增加也证实了在低pH值下聚合物固定相上不存在离子保留位点。聚合物柱和硅胶柱行为的总体相似性表明，当使用高惰性的B型硅胶固定相时，硅醇基团对这些肽的保留和过载行为影响很小。
