Dellinger M, Brault D
Laboratoire de Biophysique, INSERM U 201, CNRS UA 481, Paris, France.
J Chromatogr. 1987 Nov 27;422:73-84. doi: 10.1016/0378-4347(87)80441-3.
The isocratic separation of dicarboxylic porphyrins (hematoporphyrin, hydroxyethylvinyl-deuteroporphyrin and protoporphyrin) and their isomers by normal-phase high-performance liquid chromatography is described. The stationary phase is unmodified silica and the mobile phase consists of acetone-ethyl acetate (1:1, v/v) mixtures containing water and inorganic acids. Retention (capacity factor, k') was found to decrease exponentially with the mole fraction of water (NH2O) and to increase linearly with the concentration of hydrochloric acid, following the relation k' = A [HCl] NH2O-4.85, where A is a constant characteristic of the porphyrin. The effects of the concentration and the nature of the acid used strongly suggest that retention involves a form of the porphyrin in which the inner nitrogens are protonated. The retention is thus partly determined by the basicity of the inner nitrogens, which depends on the electron-donating power of the porphyrin side-chains. Good resolution of the various components of hematoporphyrin derivative was obtained. In comparison with reversed-phase chromatography this method shows a different retention mechanism, appears to yield results of comparable reproducibility and provides complementary information. Possible retention mechanisms based on partition or adsorption equilibria are discussed.
本文描述了通过正相高效液相色谱法对等二羧酸卟啉（血卟啉、羟乙基乙烯基 - 次卟啉和原卟啉）及其异构体进行等度分离。固定相为未改性硅胶，流动相由含有水和无机酸的丙酮 - 乙酸乙酯（1:1，v/v）混合物组成。发现保留值（容量因子，k'）随水的摩尔分数（NH₂O）呈指数下降，随盐酸浓度呈线性增加，遵循关系k' = A [HCl] NH₂O⁻⁴.⁸⁵，其中A是卟啉的一个特征常数。所用酸的浓度和性质的影响强烈表明，保留涉及卟啉的一种形式，其中内部氮原子被质子化。因此，保留部分由内部氮原子的碱性决定，而碱性取决于卟啉侧链的给电子能力。获得了血卟啉衍生物各组分的良好分离度。与反相色谱法相比，该方法显示出不同的保留机制，似乎能产生具有可比重现性的结果，并提供补充信息。讨论了基于分配或吸附平衡的可能保留机制。
