Measurement of hold-up volumes in reverse-phase liquid chromatography: Definition and comparison between static and dynamic methods

The hold-up volumes, VMVM of two series of RPLC adsorbents were measured using three different approaches. The first method is based on the difference between the volumes of the empty column tube (150×4.6150×4.6 mm) and of the material packed inside the column. It is considered as giving the correct value of VMVM. This method combines the results of the BET characterization of the adsorbent before packing (giving the specific pore volume), of carbon element analysis (giving the mass fraction of silica and alkyl bonded chains), of Helium pycnometry (providing silica density), and of inverse size exclusion chromatography (ISEC) performed on the packed column (yielding the interparticle volume). The second method is static pycnometry, which consists in weighing the masses of the chromatographic column filled with two distinct solvents of different densities. The last method is based on the thermodynamic definition of the hold-up volume and uses the dynamic minor disturbance method (MDM) with binary eluents. The experimental results of these three non-destructive methods are compared. They exhibit significant, systematic differences. Pycnometry underestimates VMVM by a few percent for adsorbents having a high carbon content. The results of the MDM method depend strongly on the choice of the binary solution used and may underestimate or overestimate VMVM. The hold-up volume VMVM of the RPLC adsorbents tested is best measured by the MDM method using a mixture of ethanol and water.