The separation selectivity of octadecyl-silica (C18) and of bonded pentafluorophenylpropyl-silica (F5) and PEG-silica columns was compared for natural phenolic antioxidants. The separation selectivities for phenolic antioxidants on C18 and F5 columns are strongly correlated, but low selectivity correlation indicating strong differences in the retention mechanism was observed between the C18 and PEG columns. Hence, the combination of a C18 and a PEG column is useful for separation of phenolic antioxidants that are not fully separated on single columns. Two-dimensional comprehensive liquid chromatography using a short PEG-silica column in the first dimension and a conventional C18-silica in the second dimension has the advantage of on-column focusing of the fractions transferred onto the C18 column in the second dimension, as a weaker mobile phase is used in the first dimension than in the second dimension. However, a stop-flow set-up in the first dimension system is necessary after the transfer of each fraction to the second dimension. Peak capacity is considerably larger but the separation time is much longer than with serially coupled PEG and C18 columns, which were employed for separation of beer and hop extract samples in connection with coulometric detection.

Three different comprehensive 2-D HPLC systems for the separation of phenolic antioxidants have been developed on the basis of different selectivities of a PEG-silica column in the first dimension and a packed or monolithic C18 or a ZR-CARBON column, respectively, in the second dimension. Two-dimensional comprehensive liquid chromatography using a serially connected short PEG-silica column and a conventional C18-silica or a ZR-CARBON column in the second dimension was tested to improve the resolution of the earlier eluting compounds in the first dimension. Various types of interface were used to connect the columns in the first and in the second dimension: i) two injection sampling loops of 100 microL in conventional arrangement; ii) a 10-port 2-position valve equipped with two trapping X-Terra columns instead of loops; and iii) two analytical D2 columns in parallel. The mobile phase in the first dimension has a lower elution strength than in the second dimension, allowing band compression of the solutes transferred from the first to the second dimension. This effect was enhanced using trapping columns instead of sampling loops as the interface between the two dimensions, thus allowing a decrease in the time of analysis. These systems were used for the analysis of beer samples. The relative location of the components in the 2-D retention plane varied in relation to their chemical structure in each instrumental set-up and allowed positive peak identification.

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