Applications of high-resolution recycling liquid chromatography: From small to large molecules

A twin-column recycling separation process (TCRSP) is assembled and used to generate higher speed and/or higher resolution levels than those of the usual non-recycling process at the same back pressure. It enables the users to solve very challenging separation problems caused by too small selectivity factors and/or too low column efficiencies. The relative gain in speed-resolution performance increases with increasing the number of cycles in the TCRSP, decreasing the maximum allowable pressure imposed by the LC system, decreasing the column permeability, and with reducing the separation speed. TCRSP is then particularly attractive for conventional LC systems (5000 psi maximum) and columns packed with sub-2 μm to 3.5 μm particles. The performance of the real TCRSP was compared to that of the ideal TCRSP for which the retention factor is strictly pressure-independent. A broad range of separation problems encountered in conventional non-recycling chromatography can be easily solved by using a TCRSP assembly based on two 15 cm long columns. Under adsorption conditions, the TCRSP enables the full baseline separation of polycyclic aromatic hydrocarbon (PAH) isomers (benzo[a]anthracene and chrysene) on a 3.5 μm XSelect-HSS T3 phase, the complete or improved resolution of racemic mixtures (4-phenylbutanol and bromacil) using the same 2.5 μm cellulose-1 chiral stationary phase, and the full resolution of isotopic compounds (benzene/1,3,5-benzene-d3/benzene-d6) on a 2.7 μm Cortecs-C18 phase. Under non-adsorption conditions or in size-exclusion chromatography (SEC), the fractionation of a polystyrene standard mixture (molecular weights of 35, 66, 130, 277, 552, 1210, and 2500 kDa) was completed after only 8 cycles on a 1.7 μm BEH 200 Åphase. Similarly, a mixture of intact proteins with molecular weights of 16.7, 66.4, 150, 660, and 1320 kDa was fully resolved on a 2.5 μm BEH 450 Åphase after only 6 cycles. Finally, TCRSP enables the complete separation of a few high-molecular-weight species (monoclonal antibody aggregates, small relative abundance of 1 for 250) from the intact monomeric monoclonal antibody (Vectibix).