Rebirth of recycling liquid chromatography with modern chromatographic columns : Extension to gradient elution

Twin column recycling semi-preparative liquid chromatography (TCRLC) is revived to prepare small amount (similar to 1 mg) of a pure targeted compound, which cannot be isolated by conventional preparative liquid chromatography. In this work, TCRLC is extended to gradient elution. The first step of this modified process consists of a gradient step, which eliminates both early and late impurities. If not discarded, some late impurities could echo during the second isocratic recycling step of the process and compromise the purity level required for the targeted compound. Additionally, the entire gradient TCRLC (GTCRLC) process is automated regarding the eluent composition programmed and the actuation times of two valves: one two-position four-port divert valve enables to shave the targeted compound from early and late impurities during the initial gradient step. The second two-position six-port recycling valve ensures the complete baseline resolution between the band of the targeted compound and those of the closest impurities, which are not fully eliminated after the initial gradient step. The automation of the whole GTCRLC process is achieved by running four preliminary scouting gradient runs (at four different relative gradient times, t(g/)t(0) = 2, 6, 18, and 54, where t(0) is the hold-up column time) for the accurate determination of the thermodynamics (ln k versus.plots of the retention factor as a function of the mobile phase composition) of the first impurity, the targeted compound(s), and of the last impurity. The automated GTCRLC process was successfully applied for the isolation of a polycyclic aromatic hydrocarbon (PAH), chrysene, from a complex mixture of PAHs containing two nearly co-eluting impurities (benzo[a]anthracene and triphenylene) and nine other early/late impurities (sample volume injected: 1 mL, 7.8 mm x 150 mm Sunfire-C-18 column, acetonitrile/water eluent mixtures, T = 55 degrees C, 20 cycles, baseline separation in less than two hours). Additionally, the GTCRLC process is advantageously used to isolate and baseline separate the vitamins D-2 and D-3 initially present in a milk extract mixture (0.3 mL sample injection volume, 7.8 mm x 150 mm Sunfire-C-18 column, methanol/water eluent mixtures, T = 65 degrees C, 14 cycles needed in 1.5 hours). These results open promising avenues toward an effective preparation of unknown targeted compounds before further physico-chemical characterization and unambiguous identification. (C) 2021 Elsevier B.V. All rights reserved.