Enhancing the Possibilities of Comprehensive Two-Dimensional Liquid Chromatography through Hyphenation of Purely Aqueous Temperature-Responsive and Reversed-Phase Liquid Chromatography.

Abstract

Comprehensive two-dimensional liquid chromatography (LC × LC) allows for substantial gains in theoretical peak capacity in the field of liquid chromatography. However, in practice, theoretical performance is rarely achieved due to a combination of undersampling, orthogonality, and refocusing issues prevalent in many LC × LC applications. This is intricately linked to the column dimensions, flow rates, and mobile-phase compositions used, where, in many cases, incompatible or strong solvents are introduced in the second-dimension (2D) column, leading to peak broadening and the need for more complex interfacing approaches. In this contribution, the combination of temperature-responsive (TR) and reversed-phase (RP) LC is demonstrated, which, due to the purely aqueous mobile phase used in TRLC, allows for complete and more generic refocusing of organic solutes prior to the second-dimension RP separation using a conventional 10-port valve interface. Thus far, this was only possible when combining other purely aqueous modes such as ion exchange or gel filtration chromatography with RPLC, techniques which are limited to the analysis of charged or high MW solutes, respectively. This novel TRLC × RPLC combination relaxes undersampling constraints and complete refocusing and therefore offers novel possibilities in the field of LC × LC including temperature modulation. The concept is illustrated through the TRLC × RPLC analysis of mixtures of neutral organic solutes.