Overloaded elution band profiles of ionizable compounds in reversed-phase liquid chromatography: influence of the competition between the neutral and the ionic species.
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.
Gritti
Fabrice
F
Department of Chemistry, University of Tennessee, Knoxville, TN, USA.
Guiochon
Georges
G
D000327:Adsorption; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry*; D008956:Models, Chemical; D015394:Molecular Structure; D004364:Pharmaceutical Preparations / Q000737:chemistry; D012996:Solutions / Q000737:chemistry*; D013816:Thermodynamics
D007477:Ions; D004364:Pharmaceutical Preparations; D012996:Solutions