Journal of Chromatography A, 1524 (2017) 108–120 Contents lists available at ScienceDirect Journal of Chromatography A jo ur nal ho me pag e: www.elsevier.com/locate/chroma Applications of high-resolution recycling liquid chromatography: From small to large molecules Fabrice Gritti ∗ , Sebastien Besner, Sylvain Cormier, Martin Gilar Waters Corporation, 34 Mapple Street, Milford, MA 01757, USA a r t i c l e i n f o Article history: Received 3 August 2017 Received in revised form 11 September 2017 Accepted 23 September 2017 Available online 25 September 2017 Keywords: Twin-column recycling chromatography Isomers Enantiomers Isotopes Polymers Biomolecules a b s t r a c t 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 m to 3.5 m 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 separa- tion 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 m XSelect-HSS T 3 phase, the complete or improved resolution of racemic mix- tures (4-phenylbutanol and bromacil) using the same 2.5 m cellulose-1 chiral stationary phase, and the full resolution of isotopic compounds (benzene/1,3,5-benzene-d 3 /benzene-d 6 ) on a 2.7 m Cortecs- C 18 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 m BEH 200 ˚ Aphase. 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 m BEH 450 ˚ Aphase 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). © 2017 Elsevier B.V. All rights reserved. 1. Introduction There are many practical cases for which a single chromato- graphic run is insufficient to achieve full baseline separation. They include the separation of isomers, isotopes, or enantiomers by adsorption chromatography because the compounds present in these mixtures have very similar physico-chemical properties. They also concern the fractionation of larger molecules by SEC such as polymers, bio-polymers (proteins), and aggregated/fragmented species of monoclonal antibodies (IgG) because both the sep- aration space and the column efficiency are extremely limited in exclusion chromatography. In all cases, even after intensive ∗ Corresponding author. E-mail address: Fabrice Gritti@waters.com (F. Gritti). optimization of column and mobile phase chemistry using any design-of-experiment or quality-by-design methods, selectivity factors and/or column efficiencies are not large enough to enable a complete separation with a single column. The first and obvious solution would consist in increasing the column length by coupling a large number of commercial columns. However, speed inevitably decreases due the limitation in the operating pressure of standard LC instruments. The plate height eventually increases and the col- umn efficiency cannot exceed a maximum value, which is imposed by the allowable system pressure, the particle size (or the specific column permeability), the eluent viscosity, and by the effective diffusivity of the analyte along the column. The full separation is then either physically impossible or unacceptably long and poorly sensitive. Alternatively, a second and more elaborated solution based on the known principle of recycling chromatography can alleviate this https://doi.org/10.1016/j.chroma.2017.09.054 0021-9673/© 2017 Elsevier B.V. All rights reserved.