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 (D) 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.

Recent Developments in Two-Dimensional Liquid Chromatography: Fundamental Improvements for Practical Applications

“…Baert et al recently advocated the use of temperature-responsive LC (TRLC) in purely aqueous environments as 1 D separation, in combination with RPLC in the second dimension. 77 Their concept relied on the use of temperature-responsive stationary phases, which exhibit strong changes in retention behavior upon small changes in temperature. The purely aqueous 1 D mobile phases were ideally compatible with a 2 D RPLC separation.…”

Towards spatial comprehensive three-dimensional liquid chromatography: A tutorial review

“…An alternative approach to locally generate different selectivities in a spatial separation device is by integrating responsive materials that can undergo reversible conformational changes after applying specific stimuli [63][64][65]. A well-known example of a thermo-responsive material frequently applied in separation science is poly(Nisopropylacrylamide) [66][67][68].…”

Maximizing two-dimensional liquid chromatography peak capacity for the separation of complex industrial samples

“…An early demonstration of comprehensive 2D-LC was conducted for the separation of a 14-component mixture of proteins using a combination of size exclusion chromatography and ion exchange chromatography [14]. Recently, 2-D LC has become more and more attractive due to its ability to separate complex samples, such as proteomic [15][16][17][18][19][20][21][22][23][24], metabolomics [25,26], surfactant/ polymer/nanoparticles [27][28][29][30], and pharmaceutical molecules [31][32][33]. The wide use of 2-D LC is facilitated by the commercialization of 2-D LC instrumentation which makes data acquisition and analysis user-friendly.…”

“…The wide use of 2-D LC is facilitated by the commercialization of 2-D LC instrumentation which makes data acquisition and analysis user-friendly. Although one main area focuses on developing 2-D LC method with short total run time [19,21,32,[34][35][36][37], there is also a strong need to develop more generic 2D-LC methods with a large peak capacity, especially for the industrial samples that might only be analyzed once for process troubleshooting or process improvement. Hence, a maximum peak capacity is desirable while the total time can be slightly sacrificed as long as it is not excessively long.…”