MOBILE PHASE EFFECTS IN REVERSED-PHASE CHROMATOGRAPHY .2. ACIDIC AMINE PHOSPHATE BUFFERS AS ELUENTS

In order to meet the particular requirements with regard to the eluent in liquid chromatography of ionogenic substances on non-polar stationary phases, various buffers composed of phosphate and a vicinal diamine having a pKa value in acidic pH domain were examined and characterized by their physico-chemical properties. The heat of ionization for such amines is generally much greater than that of ac idic compounds having commensurable pKa values. Consequently changing temperaturre may strongly affect the degree of ionization of the buffer and as a result the temperature dependence of retention can be drastically altered for certain sample components. This caloric effect of the buffer on retention in reversed-phase chromatography was theoretically treated by taking into account the enthalpies for all processes involved, the constants of the protonic equilibria in solution and the limiting retentiion factors of both the fully ionized and the neutral of the eluite. Thus thermal analysis of a chromatographic system containing a buffer in the eluent and ionogenic solutes by using Van't Hoff plots of chromatographic data may not yield the enthalpy for solute transfer between mobile and stationary phases due to secondary equilibria. Experimental results did not always agree the theoretical predictions. The aberration was attributed to complex formation between sample molecules and buffer species, that is to a hetaeric effect of the buffer. Some dramatic changes were observed in retention behavior with concomitant improvement in peak shape and chromatographhic efficiency upon replacing sodium phosphate by piperazine or tetramethylethylene- diamine phosphate in the eluent at pH 6.0. The propitious buffer effect, however, was attributed, at least in part, to masking the silanol groups at the stationary phase surface by the weak amine component of the buffer. The results demonstrate that besides their classical static role to maintain the pH of a solution constant, buffers may play a variety of other roles and affect significantly the properties and efficiency of a chromatography system. © 1979.