Novel Comprehensive Two-dimensional LC and Related Application for Complex Samples

A novel two-dimensional LC (2D LC) has been developed and successfully applied to phospholipids, polyphenols and medicinal compounds in biological, food and crude drug samples, respectively. A comprehensive 2D LC is recognized as very effective strategy for both quantitative and qualitative analyses, especially for natural and biological samples in complex matrices. However, the combined effect of the necessity to minimize band broadening in the 1st dimension and the very short analysis time in the 2nd dimension has been a difficulty for spreading this technology. Recent progress in the technology of solvent delivery at very low flow rate and ultra-high speed HPLC (UHPLC) analysis realizes a practical and comprehensive 2D LC. Our new, truly comprehensive 2D LC system, "Nexera-e", combines our latest UHPLC Nexera X2 product line with photo diode array and/or mass spectrometric detectors. Comprehensive 2D LC allows for highly efficient separation of a variety of samples, even in complex matrices, as well as ordinary quantitative analysis. The orthogonal separation selectivity between 1st and 2nd dimensions is a key issue for this technique but difficult to accomplish due to solvent compatibility and column selection in the 1st and 2nd dimensions. In our study for improving the limited orthogonality, it showed dramatic improvement when employing a shifted gradient profile in the 2nd dimension, where initial and final mobile phase composition increase stepwisely, to match the 1st dimension gradient profile. Raw data is mathematically manipulated by dedicated software, resulting in enhanced peak capacity and identification ability compared to those of independent 1D and 2D separations.