On-line comprehensive two-dimensional separations of charged compounds using reversed-phase high performance liquid...

Comprehensive on-line two-dimensional liquid chromatography is expected to generate impressive peak capacities, which makes it a method of choice for the analysis of complex samples such as pharmaceutical or biological ones. A comparative study of different sets of chromatographic conditions including stationary phase, mobile phase and column temperature was carried out with mixtures of representative solutes in order to find out the best two-dimensional analytical conditions for charged compounds. Our approach focused on ultra-fast gradient runs in second dimension using HT-UHPLC conditions. The choice of volatile buffers was intended for future coupling with mass spectrometry in order to get another relevant dimension. The potential of various pairs of chromatographic systems was examined by means of two-dimensional gradient data. An attempt is made to rationalize the concept of orthogonality and a method is proposed to assess, for a given pair of chromatographic systems, both the degree of orthogonality and the practical peak capacity. It is shown that the degree of orthogonality between both dimensions is a critical factor but it is not sufficient to definitely appreciate the potential of a given pair of systems. The combination of HILIC and RPLC (HILIC×RPLC or RPLC×HILIC), although providing a very high degree of orthogonality, is disappointing due to the poor peak capacities obtained in HILIC especially with peptide samples. RPLC systems offer a large variety of analytical conditions, some of them leading to appropriate degrees of orthogonality when they are combined. More importantly, due to high column efficiencies along with large separation power, some combinations of RPLC systems leads to very high practical peak capacities.