Corradini D, Kalghatgi K, Horváth C
Department of Chemical Engineering, Yale University, New Haven, CT 06520, USA.
J Chromatogr A. 1996 Mar 29;728(1-2):225-33. doi: 10.1016/0021-9673(95)01019-x.
The effect of two mobile phase additives, trifluoroacetic acid and phosphoric acid, on the energetics of peptide retention in reversed-phase chromatography was investigated using Hy-Tach C18 micropellicular and Vydac C4 and C18 totally porous stationary phases. The effect of the relatively low phase ratio of columns packed with micropellicular sorbents was also examined. The logarithmic retention factors, of two model peptides, Ac-RGGGGLGLGK-amide and Ac-RGAGGLGLGK-amide, were evaluated with different columns and additives in a practical range of eluent strength. The dependence of the logarithmic retention factor on the concentration of acetonitrile in the mobile phase was linear in all cases. The higher sensitivity of the retention to the organic modifier concentration in the case of the Hy-Tach C18 column is attributed to the relatively low phase ratio of this column. Pairwise plots of the logarithmic retention factors were linear. The plots of data obtained with the two additives has unit slopes and thus reveal homoenergetic retention behavior. On the other hand data obtained on two different columns manifest homeoenergetic retention, the slopes of plots are different from unity. The analysis has yielded consistent results and validated the assumption that the retention free energy can be divided into two components arising from mobile phase and stationary phase contributions. The approach also allowed an estimation of the relative phase ratios of the columns and the Vydac C18 column was found to have an 3 and 8 times higher phase ratio than the Vydac C4 and the Hy-Tech C18 column, respectively.
使用Hy-Tach C18微孔和Vydac C4及C18全多孔固定相，研究了两种流动相添加剂三氟乙酸和磷酸对反相色谱中肽保留能学的影响。还考察了填充微孔吸附剂的色谱柱相对较低相比的影响。在实际的洗脱强度范围内，用不同的色谱柱和添加剂对两种模型肽Ac-RGGGGLGLGK-酰胺和Ac-RGAGGLGLGK-酰胺的对数保留因子进行了评估。在所有情况下，对数保留因子对流动相中乙腈浓度的依赖性都是线性的。Hy-Tach C18色谱柱对有机改性剂浓度的保留具有较高的灵敏度，这归因于该色谱柱相对较低的相比。对数保留因子的成对图是线性的。用两种添加剂获得的数据图具有单位斜率，因此显示出同能保留行为。另一方面，在两种不同色谱柱上获得的数据表现出同能保留，图的斜率不同于1。该分析得出了一致的结果，并验证了保留自由能可分为由流动相和固定相贡献产生的两个组分的假设。该方法还允许估计色谱柱的相对相比，发现Vydac C18色谱柱的相比分别比Vydac C4和Hy-Tech C18色谱柱高3倍和8倍。
