Multi-²D LC × LC as a Novel and Powerful Implement for the Maximum Separation of Complex Samples

Abstract

Achieving complete information about the chemical composition of complex samples requires the use of multianalytical platforms able to maximize the acquisition of high-quality data for unequivocal identification. However, this process requires long analysis times and several instruments. Food analysis is one of the analytical fields where the analysis of very complex samples has a huge impact. One of these complex samples is vermouth, a fortified wine based on the maceration of a large number of herbs, fruits, barks, seeds, and leaves. The application of conventional or even advanced analytical techniques like comprehensive two-dimensional (2D) liquid chromatography (LC × LC) does not provide enough separation power to resolve the complete profile of this sample. In this work, a novel 2DLC strategy called multi-2D LC × LC is developed. This new setup consists of the use of two different columns with different separation properties in the second dimension (2D) that can be selected during the LC × LC analysis accordingly to the chemical nature of the compounds eluted from the first dimension (1D). The vermouth sample was analyzed using a 1D-PFP and a combination of HILIC (from 0 to 30 min) and C18 (from 30 to the end) columns in the 2D. This setup increased both the peak capacity and the orthogonality of the analysis in comparison to the use of only one of the columns in the 2D. Multi-2D LC × LC is presented as an integrated 2DLC tool that maximizes the separation capacity for very complex samples.

Impact of conditioning runs on hydrophilic interaction chromatography repeatability and its application as a second dimension in online comprehensive two‐dimensional liquid chromatography

“…Previous studies recommending long equilibration times in HILIC implied that using this separation mode as a 2 D in LC×LC might be impractical. However, several promising studies using HILIC as a 2 D indicated that HILIC separations can be reproducible even with a very short equilibration time of less than one column volume [8,21,34]. Furthermore, studies on repeatability in HILIC suggested that it can be improved by increasing the number of conditioning runs [5,6].…”

“…For the analysis of phenolic compounds [21], samples from traditional Chinese medicine [32], and a chiral LC×HILIC method used for amino acid separation [33], equilibration volumes of one‐ to two‐column volumes, typical for second‐dimensional RPLC, were utilized. In a recent study, the 2 D HILIC column was equilibrated with less than one‐column volume (0.6 V c ) [34], without any distortion of the spots, which indicates repeatability between modulations. Nevertheless, evaluation of the repeatability of the 2 D, such as monitoring 2 tr between modulations or between repeated injections, was not conducted.…”