Effects of pH mismatch between the two dimensions of reversed-phase × reversed-phase two-dimensional separations on second dimension separation quality for ionogenic compounds—I. Carboxylic acids

Abstract

Two persistent impediments to wider adoption of two-dimensional liquid chromatography (2D-LC) are the perceptions that 2D methods are generally less sensitive than 1D ones, and that coupling of certain separation modes in a 2D system is difficult because of the negative impact of the effluent of the first separation on the second separation. In this work we address these problems in the specific case where reversed-phase separations are used in both dimensions of a 2D-LC system, but the pH is varied such that the ionization state of carboxylic acid analytes is different (i.e., neutral or negatively charged, in eluents buffered at pH 2 or 7) in the two columns. We first demonstrate that the effect of first dimension (1D) effluent on the performance of second dimension (2D) separation of ionogenic solutes is much more serious than it is for neutral compounds where the pH of the eluent does not play a role in retention. We have systematically varied the properties of the sample solution injected into the 2D column (i.e., the 1D effluent), as well as the 2D eluent, with the goal of establishing guidelines for conditions that yield acceptable 2D performance. We find that the organic solvent content of the 1D effluent and 2D eluent is not as important as the buffer concentrations in these two solutions, and that the greater the ratio of buffer concentration in the 1D effluent relative to the 2D eluent, the smaller the volume one can inject into the 2D column before dramatic peak splitting occurs. We have then used the information from these simple experiments to guide both 1D experiments that mimic the 2D separation, and actual 2D separations, to demonstrate that online adjustment of the properties of the 1D effluent by dilution with a buffered solvent prior to injection into the 2D column is a very effective solution to the pH mismatch problem. We find that when the buffer capacity of the diluent is high enough to effectively titrate the 1D effluent such that its pH approaches that of the 2D eluent, excellent 2D peak shape is obtained for the carboxylic acid analytes, even when the volume of injected sample solution exceeds the 2D column volume.