Gritti Fabrice, Guiochon Georges
Department of Chemistry, University of Tennessee, Knoxville, TN, USA.
J Sep Sci. 2008 Dec;31(21):3657-82. doi: 10.1002/jssc.200800472.
The parameters that affect the shape of the band profiles of acido-basic compounds under moderately overloaded conditions (sample size less than 500 nmol for a conventional column) in RPLC are discussed. Only analytes that have a single pK(a) are considered. In the buffer mobile phase used for their elution, their dissociation may, under certain conditions, cause a significant pH perturbation during the passage of the band. Two consecutive injections (3.3 and 10 microL) of each one of three sample solutions (0.5, 5, and 50 mM) of ten compounds were injected on five C(18)-bonded packing materials, including the 5 microm Xterra-C(18) (121 A), 5 microm Gemini-C(18 )(110 A), 5 microm Luna-C(18)(2) (93 A), 3.5 microm Extend-C(18 )(80 A), and 2.7 microm Halo-C(18) (90 A). The mobile phase was an aqueous solution of methanol buffered at a constant (W)(W)pH of 6, with a phosphate buffer. The total concentration of the phosphate groups was constant at 50 mM. The methanol concentration was adjusted to keep all the retention factors between 1 and 10. The compounds injected were phenol, caffeine, 3-phenyl 1-propanol, 2-phenyl butyric acid, amphetamine, aniline, benzylamine, p-toluidine, procainamidium chloride, and propranololium chloride. Depending on the relative values of the analyte pK(a) and the buffer solution pH, these analytes elute as the neutral, the cationic, or the anionic species. The influence of structural parameters such as the charge, the size, and the hydrophobicity of the analytes on the shape of its overloaded band profile is discussed. Simple but general rules predict these shapes. An original adsorption model is proposed that accounts for the unusual peak shapes observed when the analyte is partially dissociated in the buffer solution during its elution.
讨论了在反相液相色谱（RPLC）中，中等过载条件下（对于常规色谱柱，进样量小于500 nmol）影响酸碱化合物谱带轮廓形状的参数。仅考虑具有单一pK(a)的分析物。在用于洗脱它们的缓冲流动相中，在某些条件下，它们的解离可能会在谱带通过期间引起显著的pH扰动。将三种样品溶液（0.5、5和50 mM）中十种化合物的每一种进行两次连续进样（3.3和10 μL），进样到五种C(18)键合填充材料上，包括5 μm Xterra-C(18)（121 Å）、5 μm Gemini-C(18)（110 Å）、5 μm Luna-C(18)(2)（93 Å）、3.5 μm Extend-C(18)（80 Å）和2.7 μm Halo-C(18)（90 Å）。流动相是以磷酸盐缓冲液缓冲至恒定（W）（W）pH 6的甲醇水溶液。磷酸基团的总浓度恒定为50 mM。调整甲醇浓度以使所有保留因子保持在1至10之间。进样的化合物有苯酚、咖啡因、3-苯基-1-丙醇、2-苯基丁酸、苯丙胺、苯胺、苄胺、对甲苯胺、普鲁卡因氯化物和普萘洛尔氯化物。根据分析物pK(a)和缓冲溶液pH的相对值，这些分析物以中性、阳离子或阴离子形式洗脱。讨论了分析物的电荷、大小和疏水性等结构参数对其过载谱带轮廓形状的影响。简单但通用的规则可预测这些形状。提出了一种原始吸附模型，该模型解释了在分析物洗脱过程中在缓冲溶液中部分解离时观察到的异常峰形。
