OPTIMIZATION OF A REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY SEPARATION USING AN ION-PAIR REAGENT FOR THE DETERMINATION OF CARBOXYLIC-ACIDS IN PLANT MATERIALS

A method was developed to separate a number of mono-, di- and tri- car boxylic acids present in plant materials, using a C-18 column and a si mple eluent containing a phosphate buffer and tetra-n-butylammonium ph osphate, an ion-pairing agent. The effects of the ion-pairing agent, p H, presence or absence of organic modifier and the concentration of th e buffer, on the chromatographic response function (CRF), were investi gated using a two level factorial experimental design. The results ind icated that a number of main, two and three factor effects were import ant. The largest effect was due to the interaction of pH with ion-pair ing concentration and was further investigated using univariate experi ments. The mobile phase developed consisted of 10 mM tetra-n-butylammo nium phosphate and 20 mM potassium dihydrogen orthophosphate at pH 3.3 . Formic, glutamic, quinic, succinic, malic and tartaric acids were ex tracted from 3 different grass ecotypes, the extracts were cleaned-up using a C-18 Sep-Pak(R) cartridge and then identified using the develo ped procedure.