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A methodology for the estimation of the different phase volumes in HILIC is presented. For a ZIC-HILIC column the mobile phase volume (hold-up volume) is determined in several acetonitrile-and methanol-water compositions by a Linear Free Energy Relationships (LFER) homologous series approach involving n-alkyl-benzenes, -phenones, and -ketones. We demonstrate that the column works as a HILIC column when the mobile phase contains high and medium proportions of methanol or acetonitrile. However, for acetonitrile contents below 20%, or 40% for methanol, same column works in RPLC. In between, a mixed HILIC-RPLC behavior is observed, and solutes of low molecular volume are retained as in HILIC mode, but the largest ones show RPLC retention. From the homologous series retention data and pycnometric measurements involving the pure organic solvents and their mixtures with water, the mean solvent composition of the water-rich transition layers between column functionalization and the bulk mobile phase, which act as stationary phase, is estimated. Finally, the phase ratio between stationary and mobile phases is also estimated for each eluent composition, allowing the calculation of the corresponding stationary phase volumes. All volumes are strongly dependent on the water content in the eluent, especially when acetonitrile is selected as mobile phase constituent. In HILIC mode, when the water content in the hydroorganic mobile phase increases, the volumes of mobile phase decrease, but the volumes of stationary phase (mainly the water layer adsorbed onto the bonded-phase and the water-enriched interface) increase. However, at high water concentrations, where the column works in RPLC mode, the mobile phase volume increases and the stationary phase (which is now the bonded zwitterion) decrease when increasing the water percentage in the mobile phase.

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Common methods for hold-up time and volume determination in Reversed-Phase Liquid Chromatography (RPLC) have been tested for Hydrophilic Interaction Liquid Chromatography (HILIC). A zwitterionic ZIC-HILIC column has been used for the testing. The pycnometric determination method, based on differences in column weight when filled with water or organic solvent, provides the overall volume of solvent inside the column. This includes the volume of eluent semi-sorbed on the packing of the column, which acts as the main stationary phase. The homologous series approach, based on the retention behavior of homologues in relation to their molecular volume, allows the determination of accurate hold-up volumes. However, the application of this method is time-consuming. In some cases, large neutral markers with poor dipolarity/polarizability and hydrogen bonding interactions can be used as hold-up volume markers. This is the case of dodecylbenzene and nonadecane-2-one in clearly HILIC behaving chromatographic systems, the use of decanophenone as a marker can be even extended to the boundary between HILIC and RPLC. The elution volume of the marker remains nearly unaffected by the concentration of ammonium acetate in the mobile phase up to 20 mM. The injection of pure solvents to produce minor base-line disturbance as hold-up markers is strongly discouraged, since solvent peaks are complex to interpret and depend on the ionic strength of the eluent.

In the present study a pressure-assisted MEEKC method with reversed-polarity using a conventional CE instrument with UV detection and uncoated fused silica capillaries is validated as a high-throughput methodology for the lipophilicity determination of the neutral species of acidic compounds (pK a > 3.5). After the calibration of the system with four standard compounds of known log P o/w , mass distribution ratios (log k MEEKC ) of new molecules can be directly converted into log P o/w values by means of a simple linear equation (log P o/w =a·log k MEEKC +b). The method was internally and externally validated for a log