Advantages of the use of monolithic stationary phases for modelling the retention in sub/supercritical chromatography. Application to cis/trans-beta-carotene separation.

The low viscosity of supercritical fluids enables the coupling of columns, which favours both the high efficiency of separation and the ability of tuning the selectivity. However, it increases the inlet pressure then modifies the fluid density, i.e. the eluotropic strength of the mobile phase. In this case, the latter is rather different depending on the number of coupled columns. This fact prevents the calculation of the chromatographic parameters for coupled columns from the results obtained from one. In subcritical conditions, by using silica rod columns, which have bimodal porous structure, the flow resistance parameter is dramatically reduced. Consequently, the addition of monolithic columns induces only slight internal pressure changes and the fluid density does not vary with the column length. In this case, the calculation of retention factor and selectivity based on retention values obtained on each separate column provides accurate results allowing to determine the optimum column length in regard to the studied separation. After a better characterisation of the stationary phase included in the Chromolith column, this paper describes the beta-carotene isomers separation obtained by coupling up to six Chromolith columns to an octadecyl bonded particulate one. These compounds were studied because of the difficulty to separate these cis/trans isomers. No abnormal apparent dead volume change due to fluid density variation was reported, and good correlations between experimental and calculated retention factors and selectivities were observed. The optimum separation requires five highly porous columns coupled to a YMC Pack Pro. Moreover, the use of monolith packing allows to decrease both the retention factor and the analytical time by comparison to previous studies.