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A comprehensive 2-D LC x LC system was developed for the separation of phenolic and flavone antioxidants, using a PEG-silica column in the first dimension and a C(18) column with porous-shell particles or a monolithic column in the second dimension. Combination of PEG and C18 or C8 stationary phase chemistries provide low selectivity correlations between the first dimension and the second dimension separation systems. This was evidenced by large differences in structural contributions to the retention by -COOH, -OH and other substituents on the basic phenol or flavone structure. Superficially porous columns with fused core particles or monolithic columns improve the resolution and speed of second dimension separation in comparison to a fully porous particle C(18) column. Increased peak capacity and high orthogonality in different 2-D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2-D separation time range. Multi-dimensional set-up combining the LC x LC separation on-line with UV and multi-channel coulometric detection and off-line with MS/MS technique allowed positive peak identification. The Coularray software compensates for the effects of the baseline drift during the gradient elution and is compatible with parallel gradient comprehensive LC x LC technique. Furthermore, it provides significant improvement in the sensitivity and selectivity of detection in comparison to both UV and MS detection. The utility of these systems has been demonstrated in the analysis of beer samples.

The aim of the work is to contribute to rational selection of columns and mobile phases for 'orthogonal' twodimensional LC T LC operation of specific sample types, providing maximum increase in the peak capacity with respect to single-dimension HPLC. As a criterion of suitability, the dissimilarity of the separation selectivity of the phase separation systems was selected. For this purpose, correlations between the retention and molecular structure descriptors were used to characterize and compare the columns with respect to various types of selective interactions using the interaction indices as the general polarity scale characteristics and the linear free energy relationships (LFER) model employing selective molecular structural descriptors. Chemometric methods-cluster analysis using similarity dendrograms and multiple regression analysis-were used to classify several types of bonded C18, amide and polyethyleneglycol columns on the basis of differences in separation selectivities for selected phenolic and flavone standards. Systematic development of a comprehensive LC T LC method for two-dimensional separation of phenolic acids and flavone natural antioxidants was based on the selection of the combination of first-and second-dimension columns with low correlation of retention of representative standards providing a highly orthogonal two-dimensional separation system. The mobile phases providing best separation selectivity for phenolic compounds on each column tested were optimized using window-diagram optimization strategy. The final optimized conditions were employed for comprehensive LC T LC separations of natural antioxidants using a polyethylene glycol microcolumn in the first dimension and a short monolithic C18 column in the second dimension.

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