Characterization of HPLC columns for two-dimensional LC x LC separations of phenolic acids and flavonoids

The aim of the work is to contribute to rational selection of columns and mobile phases for 'orthogonal' two-dimensional LC x 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 x 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 x LC separations of natural antioxidants using a polyethylene glycol microcolumn in the first dimension and a short monolithic C18 column in the second dimension. Copyright (c) 2008 John Wiley & Sons, Ltd.