Exploring the utility of complementary separations in liquid chromatography

An alternative strategy is explored for the separation of samples by liquid chromatography (LC). Unlike traditional approaches that aim to resolve all components in a given sample within a single LC separation, the proposed strategy uses two or more distinct separations carried out with a different gradient program and/or using different separation chemistries i.e., a different set of mobile and stationary phase. This set of complementary incomplete separations (CIS) is selected such that each component is at least fully resolved once, meaning the most critical pairs of each individual separation can be left unseparated. This allows for a significant time saving per separation. To investigate whether such an approach can lead to overall shorter analysis times than is possible with the fastest single-run gradient separation, a comprehensive in silico study covering a statistically significant number of samples is undertaken. The investigation shows that, for the presently considered sample sets and chemistries, CIS has a substantially higher probability, about two times greater for the simplest samples considered in this work and as much as 30 times greater for more complex samples, to fully resolve an unknown sample compared to a single gradient separation. Comparing separation speeds, the CIS approach can achieve complete sample resolution on average approximately four times faster than a single separation. Our findings thus demonstrate the potential of CIS in enhancing separation efficiency and offer insights regarding their use for solving analytical challenges.