Evaluation of a linear free energy relationship for the determination of the column void volume in hydrophilic interaction chromatography

Abstract

The application of a linear free energy relationship (LFER) to a variety of hydrophilic interaction chromatography columns with different bonded ligands and pore sizes was studied in order to determine their void volume Vm. The method was based on the determination of the elution volume of a series of alkylbenzene standards from C1 (toluene) to C17 (heptadecylbenzene). Results were compared with those obtained by injection of toluene alone, which has traditionally been used as a simple Vm marker. Vm was smaller when derived from the LFER plot than when measured with toluene with differences between the two methods ranging from 2.7 to 12.7 % for the columns studied. This result could be due to the small but appreciable retention of toluene due to its solubility in the water rich layer, which partially constitutes the stationary phase in HILIC. Larger pore size columns showed less difference in Vm between LFER and toluene procedures. This result may be due to size sieving effects of non-excluded solutes in the pores of the stationary phase, or to differences in phase ratio between columns of different pore size.