Application of supplementary flow in comprehensive 2D liquid chromatography combining SEC and RPC

Abstract

A comprehensive two-dimensional HPLC system (SEC x RPC) was evaluated. Various model compounds with differing hydrophobicity (log D: -0.08 to 2.22) and size (MW: 194 to 66.0 x 10(3)) were used. In order to reduce the run time of the second dimension, and thereby optimize the number of runs per unit volume from the first dimension, short RPC columns were used (7.5 mm). This column size demanded a low concentration of methanol in the mobile phase from the first dimension, in order to avoid severe band broadening and solute loss. Secondary interactions on SEC make a high methanol concentration in the mobile phase a necessity. Up to 40% methanol was required to diminish non-ideal SEC behavior. These conditions were non-compatible with trapping of hydrophilic compounds on RPC. The use of a supplementary flow (0.1% TFA) mixed after the first dimension led to better peak shape and trapping of hydrophilic compounds in the second dimension. This demanded flow adjustment in SEC, which in turn caused performance improvement and an increase in analysis time, making more RPC separations possible. These factors contributed to a larger peak capacity.