Relationship Study Between Reversed Phase HPLC Retention and Octanol/Water Partition Among Amphoteric Compoundsw

The retention of eight non-congeneric amphoteric compounds was followed in a reversed phase (RP HPLC) system (C18/methanol-water vol. 50:50) in the pH range 4–9. The chromatographic behavior of the amphoterics is explained by means of their species distribution diagram (pH profile of the protonation macro- and microspecies) in the same pH interval. Maximum retention was observed at the isoelectric point of the compounds even in cases when the zwitterionic species was in a great excess over the neutral (uncharged) one. This finding reveals, that the retention at the isoelectric point (ie.p.) must be generated by the retention of the less polar, neutral form. The great similarity of pH dependent partition (octanol-water) and retention (C18/methanol-water) pattern provides further proof to the concept, that in case of amphoteric compounds the neutral species is transferred into the octanol phase during octanol/water partition. Linear correlation analysis showed that logPoct/w and Iogk'c18 are analogue lipophilicity parameters i.e.: the physico-chemical content of logk'c18 corresponds to that of the octanol/water true partition coefficient.