Please cite this article in press as: F. Gritti, et al., Ideal versus real automated twin column recycling chromatography process, J. Chromatogr. A (2017), http://dx.doi.org/10.1016/j.chroma.2017.06.009 ARTICLE IN PRESS G Model CHROMA-358579; No. of Pages 14 Journal of Chromatography A, xxx (2017) xxx–xxx Contents lists available at ScienceDirect Journal of Chromatography A j o ur na l ho me page: www.elsevier.com/locate/chroma Ideal versus real automated twin column recycling chromatography process Fabrice Gritti ∗ , Mike Leal, Thomas McDonald, 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 20 April 2017 Received in revised form 1 June 2017 Accepted 2 June 2017 Available online xxx Keywords: Recycling chromatography Ideal versus real recycling process High-resolution liquid chromatography Reversed-phase liquid chromatography Planar polycyclic aromatic hydrocarbon isomers Pressure-dependent retention factor Partial molar volume a b s t r a c t The full baseline separation of two compounds (selectivity factors ˛ < 1.03) is either impractical (too long analysis times) or even impossible when using a single column of any length given the pressure limitations of current LC instruments. The maximum efficiency is that of an infinitely long column operated at infinitely small flow rates. It is determined by the maximum allowable system pressure, the column permeability (particle size), the viscosity of the eluent, and the intensity of the effective diffusivity of the analytes along the column. Alternatively, the twin-column recycling separation process (TCRSP) can overcome the efficiency limit of the single-column approach. In the TCRSP, the sample mixture may be transferred from one to a second (twin) column until its band has spread over one column length. Basic theory of chromatography is used to confirm that the speed-resolution performance of the TCRSP is intrinsically superior to that of the single- column process. This advantage is illustrated in this work by developing an automated TCRSP for the challenging separation of two polycyclic aromatic hydrocarbon (PAH) isomers (benzo[a]anthracene and chrysene) in the reversed-phase retention mode at pressure smaller than 5000 psi. The columns used are the 3.0 mm × 150 mm column packed with 3.5 m XBridge BEH-C 18 material (˛ = 1.010) and the 3.0 mm or 4.6 mm × 150 mm columns packed with the same 3.5 m XSelect HSST 3 material (˛ = 1.025). The iso- cratic mobile phase is an acetonitrile–water mixture (80/20, v/v). Remarkably, significant differences are observed between the predicted retention times and efficiencies of the ideal TCRSP (given by the number of cycles multiplied by the retention time and efficiency of one column) and those of the real TCRSP. The fundamental explanation lies in the pressure-dependent retention of these PAHs or in the change of their partial molar volume as they are transferred from the mobile to the stationary phase. A revisited retention and efficiency model is then built to predict the actual performance of real TCRSPs. The experimental and calculated resolution data are found in very good agreement for a change, v m = −10 cm 3 /mol, of the partial molar volume of the two PAH isomers upon transfer from the acetonitrile–water eluent mixture to the silica-C 18 stationary phase. © 2017 Published by Elsevier B.V. 1. Introduction For the lack of selectivity, numerous are the separations for which the use of a single column (operated at or below the pres- sure limits of either HPLC or UHPLC instruments) does not allow a complete baseline resolution within reasonable analysis times. They include the separation of planar polycyclic aromatic hydrocar- bon (PAH) isomers (ex: benzo[a]anthracene/chrysene), fullerenes (ex: C 84 /C 82 /C 80 ), isotopes (ex: deuterated benzenes), diastereoiso- meric steroids (ex: androstane-diol), and of derivatized amino acids ∗ Corresponding author. E-mail address: Fabrice Gritti@waters.com (F. Gritti). (ex: leucine/isoleucine) by RPLC on conventional silica-C 18 phases. Another example of tough separations is the isolation and char- acterization of low-concentrated impurities hidden below a main peak (ex: impurity mixed with active pharmaceutical ingredients, high-molecular-weight aggregates of degrading monoclonal anti- bodies). Also, regardless of the column chemistry and the nature of the mobile phase selected after screening and optimization pro- cedures, selectivity factors may still remain too small to achieve a complete baseline separation of some chiral compounds (ex: bro- macil and  -phenylbutyrolactone in RPLC). Another challenging separation problem is the fractionation of polymers and intact pro- teins by size-exclusion chromatography (SEC) because, in SEC, the resolution power is severely limited by the narrow separation space available. http://dx.doi.org/10.1016/j.chroma.2017.06.009 0021-9673/© 2017 Published by Elsevier B.V.