Sample conditions to avoid pH distortion in RP-LC

Band deformations might take place for acids and bases in preparative applications and adsorption studies where it is necessary to use overloaded injections. In this study, we focus on how deformations can be prevented without using highly concentrated buffers that may precipitate in the eluent. We have systematically investigated how the elution zones depend on which protolytic form the analyte has when it is dissolved. Basic and acidic model compounds are investigated using eluents with different pH values and the resulting elution profiles are compared when the analytes are dissolved in their protonated and deprotonated form, i.e., in uncharged form or as different kinds of salts. Depending on the analyte's protolytic form, a sample zone is created at the column inlet whose pH deviates, more or less, from the bulk eluent's. If the local adsorption strength in this sample zone is greater than the bulk eluent's, the elution profiles are compressed. Under opposite conditions, the eluted bands are more or less deformed and may even be split; completely different deformations can even take place for different kinds of salt combinations. Explanations of these, and other, effects, together with detailed guidelines for proper sample preparation to avoid peak deformations, are given.