Departament de Química Analítica I Institut de Biomedicina, Universitat de Barcelona, Martí I Franquès 1-11, 08028, Barcelona, Spain.
Departament de Química Analítica I Institut de Biomedicina, Universitat de Barcelona, Martí I Franquès 1-11, 08028, Barcelona, Spain; Serra Húnter Programme, Generalitat de Catalunya, 08002, Barcelona, Spain.
Anal Chim Acta. 2019 Oct 31;1078:200-211. doi: 10.1016/j.aca.2019.05.063. Epub 2019 May 29.
The effect of the ionization in the RP-HPLC retention of 66 acid-base compounds, most of them drugs of pharmaceutical interest, is studied. The retention time of the compounds can be related to the pH measured in the mobile phase (pwsH) through the sigmoidal equations derived from distribution of the neutral and ionic forms of the drug into the stationary and mobile phases. Fitting of the obtained retention vs. pH profiles provides the retention times of the ionic and neutral forms and the pK values of the drugs in the mobile phase (pwsK). The obtained pwsK values are linearly correlated to the pK values in water (pwwK) with two different correlations, one for neutral acids and another for neutral bases that reflect the different influence of the dielectric constant of the medium in ionization of acids and bases. The retention of the neutral species is well correlated to the octanol-water partition coefficient of the drugs as measure of the lipophilicity of the drug, which affects chromatographic retention. Also, the retention time of the ionized forms is related to the retention time of the neutral forms by two different linear correlations, one for anions and the other for cations. These last correlations point out the different retention behaviour of anions and cations: anions are less retained than cations of the same lipophilicity, as measured by the octanol-water partition coefficient of the neutral form. The different retention behaviour of anionic, cationic and neutral forms is confirmed by the hold-up times obtained from different approaches: pycnometry and retention times of anionic (KBr and KI) and neutral (DMSO) markers. Hold-up times obtained by pycnometric measurements agree with those obtained by retention of neutral markers (0.83-0.85 min), whereas hold-up time for anions is mobile phase pH dependent. At acidic pH it is similar to the hold-up time for neutral markers (0.83 min), but then it decreases with the increase of mobile phase pH to 0.65 min at pH 11. The decrease can be explained by the ionization of the silanols of the column and exclusion of anions by charge repulsion. Although not directly measured, the obtained retention data and correlations indicate hold-up time for cations are similar or slightly lower than hold-up time for neutral compounds (0.77-0.83 min). The model proposed and the correlations obtained can be very useful for its implementation in retention prediction algorithms for optimization of separation purposes.
研究了在反相高效液相色谱中 66 种酸碱化合物（大多数为药物）保留的电离效应。通过药物中性和离子形式在固定相和流动相之间分布的拟合曲线，可将化合物的保留时间与流动相中的 pH 值（pwsH）相关联。从获得的保留时间与 pH 值关系图中可以得到离子和中性形式的保留时间以及药物在流动相中的 pK 值（pwsK）。获得的 pwsK 值与水中的 pK 值（pwwK）呈线性相关，有两种不同的相关性，一种适用于中性酸，另一种适用于中性碱，这反映了介质介电常数对酸和碱电离的不同影响。中性物质的保留与药物的辛醇-水分配系数密切相关，作为药物亲脂性的衡量标准，它影响色谱保留。此外，离子化形式的保留时间与中性形式的保留时间通过两种不同的线性相关性相关，一种适用于阴离子，另一种适用于阳离子。这些最后的相关性指出了阴离子和阳离子的不同保留行为：阴离子的保留比具有相同亲脂性的阳离子小，这可以通过中性形式的辛醇-水分配系数来衡量。通过不同方法（比重瓶法和阴离子（KBr 和 KI）和中性（DMSO）标记物的保留时间）获得的保留时间证实了阴离子、阳离子和中性形式的不同保留行为。比重瓶法测量得到的保留时间与中性标记物的保留时间（0.83-0.85 min）相符，而阴离子的保留时间则与流动相 pH 值有关。在酸性 pH 值下，它与中性标记物的保留时间（0.83 min）相似，但随后随着流动相 pH 值的增加而降低，在 pH 11 时降至 0.65 min。这种下降可以通过柱子上硅醇的电离和电荷排斥来排除阴离子来解释。虽然没有直接测量，但获得的保留数据和相关性表明，阳离子的保留时间与中性化合物的保留时间相似或略低（0.77-0.83 min）。所提出的模型和获得的相关性对于在保留预测算法中的实现非常有用，以优化分离目的。
