Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1600, USA.
Anal Chem. 2009 Dec 15;81(24):9871-84. doi: 10.1021/ac902027t.
We recorded the overloaded elution band profiles of the dipeptide phenylalanine-alanine (Phe-Ala) on a column packed with C18-bonded organic (ethyl)/inorganic (silica) hybrid porous particles (BEH), eluted with a series of buffered methanol-water mobile phases (20/80, v/v). The (W)(S)pHs of the mobile phases were successively adjusted with addition of suitable buffers to values of 1.67, 2.44, 3.83, 4.94, 7.41, and 10.71 (where the notation (W)(S)pH means that the pH of the solution is directly measured in the solution (S) after the electrode was calibrated in pure water (W)). The ionic strength of the eluent was kept constant at 20 mM. The injected samples had different sizes and concentrations. The retention of the low-concentration samples was minimum at an intermediate (W)(S)pH (k' approximately 0.5) and maximum for the lowest (k' approximately 1.3) and highest (k' approximately 3.5) (W)(S)pHs showing that the zwitterion (+Phe-Ala-) is less strongly adsorbed than the positively (+Phe-Ala) and negatively (Phe-Ala-) charged species of the dipeptide onto BEH-C18. The elution profiles of the concentrated samples demonstrated that the adsorption isotherm of the zwitterionic species is an anti-Langmuirian Moreau isotherm due to significant adsorbate-adsorbate interactions, whereas the isotherms of the charged species are more conventional Langmuir isotherms. A simple ternary isotherm for the coadsorption of the three dipeptide species is proposed. It accounts well for the complex band profiles observed when large concentrated samples of the peptide are injected under controlled pH conditions. The slight departure between the calculated and the experimental band profiles obtained under uncontrolled pH conditions suggests the possible adsorption of the buffer components onto the packing material and the possible influence of microenvironment effects near the stationary phase surface, which would affect the local pH along the column. Preparative chromatography of peptides should preferentially be operated at intermediate (W)(S)pHs or with high ionic strength buffers under acidic or basic (W)(S)pHs.
我们记录了在填充有 C18 键合有机（乙基）/无机（硅）混合多孔颗粒（BEH）的柱上洗脱的二肽苯丙氨酸-丙氨酸（Phe-Ala）的过载洗脱带谱，用一系列缓冲甲醇-水流动相（20/80，v/v）洗脱。流动相的（W）（S）pH 值通过添加合适的缓冲液成功地调整至 1.67、2.44、3.83、4.94、7.41 和 10.71（其中（W）（S）pH 的表示方法是指在电极在纯水中校准后，直接在溶液（S）中测量溶液的 pH 值（W））。洗脱液的离子强度保持在 20 mM 不变。注入的样品大小和浓度不同。低浓度样品的保留率在中间（W）（S）pH 值（k'约为 0.5）时最小，在最低（k'约为 1.3）和最高（k'约为 3.5）（W）（S）pH 值时最大，表明二肽的两性离子（+Phe-Ala-）比带正电荷（+Phe-Ala-）和带负电荷（Phe-Ala-）的物种在 BEH-C18 上的吸附力较弱。浓缩样品的洗脱曲线表明，两性离子物种的吸附等温线是由于显著的吸附剂-吸附剂相互作用而形成的反朗缪尔莫尔等温线，而带电物种的等温线则更传统的朗缪尔等温线。提出了一种用于三种二肽物种共吸附的简单三元等温线。它很好地解释了在受控 pH 条件下注入大浓度肽样品时观察到的复杂带谱。在不受控制的 pH 条件下获得的计算和实验带谱之间的轻微偏差表明，缓冲成分可能吸附在填充材料上，以及固定相表面附近微环境效应的可能影响，这将影响沿柱的局部 pH 值。肽的制备色谱应优先在中间（W）（S）pH 值下或在酸性或碱性（W）（S）pH 值下用高离子强度缓冲液操作。
