Using multiple heart-cutting valves and stop-flow operation to enable variable second dimension gradient times in online comprehensive two-dimensional liquid chromatography

Online comprehensive two-dimensional liquid chromatography (LC xLC) is the preferred method currently for the separation of highly complex mixtures of non-volatile compounds. With fully automated com-mercial instrumentation and software making the method appealing to both researchers and industry, the demand for systems with improved separation capabilities for highly complex mixtures such as those found in natural products or proteomics has increased. In this study we report an approach that en-ables variable second dimension analysis times based on the use of multiple heart-cutting valves and stop-flow operation to circumvent the requirement for very fast second dimension ( 2 D) analyses in on-line LC xLC. As application, the HILIC xRP-LC analysis of condensed tannins (proanthocyanidins) in cocoa, grape seed and quebracho extracts is used to demonstrate the performance on the proposed methodol-ogy. The method offers increased flexibility compared to conventional online LC xLC separations in that longer 2 D gradients can be used to accommodate more complex portions of the chromatogram, while shorter 2 D gradients can be used in sections containing fewer peaks, while largely maintaining the ben-efits of comprehensive separation. We present an evaluation of the performance of the variable gradient time stop-flow HILIC xRP-LC method compared to a comparable, conventional online HILIC xRP-LC sys-tem in terms of practical peak capacities using established 2D-LC theory. The improved separation of especially low-level intermediate molecular weight proanthocyanidin oligomers by the former method demonstrates the benefits of the developed approach.(c) 2022 Elsevier B.V. All rights reserved.