Theoretical and practical guidelines for solvent dilution between the two dimensions in online comprehensive two-dimensional liquid chromatography

Online comprehensive two-dimensional liquid chromatography (online LC x LC) has become increasingly popular. Among the different chromatographic modes that can be combined, hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) are particularly interesting because they offer a high degree of orthogonality. However, this combination remains complex due to the incompatibility of the solvents in the two dimensions. To avoid this problem, it is possible to dilute the first dimension (¹D) effluent with (z_dilution -1) volumes of a weaker solvent added to one volume of ¹D-effluent, where z_dilution represents the extent to which the fraction volume has been multiplied. This can be done using either active solvent modulation technology or an additional pump, prior to the second dimension analysis. The objective of this study was to develop theoretical models to predict whether or not dilution can be effective, and, if so, what is the minimum z_dilution value required. This approach is based on the calculation of the ratio (called x_dilution) between the peak standard deviation due to the injection process and the peak standard deviation in the absence of extra-column dispersion. x_dilution was calculated from theoretical relationships and plotted as a function of z_dilution, to predict the value required to obtain good peak shapes for the compound of interest. The maximum x_dilution value was found to be of the order of 1 for chromatographically acceptable peak shapes. The proposed theoretical approach was experimentally validated on a number of representative small molecules and peptides. Agreement between experimental results and theoretical models was very high, especially for small molecules. Finally, it is shown that this approach helps to predict the most appropriate set of conditions in HILIC x RPLC, depending on the compounds to be separated.