Measurement of Partition Coefficients in Waterless Biphasic Liquid Systems by Countercurrent Chromatography Alain Berthod,* Anne Isabelle Mallet, and Madeleine Bully Laboratoire des Sciences Analytiques, Universite ´ Claude Bernard, Lyon 1, UA CNRS 435 (J. M. Mermet), 69622 Villeurbanne cedex, France Countercurrent chromatography (CCC) is a chromato- graphic separation technique that uses a liquid as a stationary phase. Centrifugal forces are used to im- mobilize the liquid stationary phase when the liquid mobile phase is pushed through it. In CCC, the solutes are separated according to their liquid-liquid partition coefficients. The solutes studied were the alkylbenzene homologues from benzene to hexylbenzene and some polyaromatic hydrocarbons (PAHs) from naphthalene to coronene. Their liquid-liquid partition coefficients were measured in the five waterless biphasic systems formed by heptane, as the apolar liquid phase of the five biphasic systems, and four dipolar aprotic solvents, dimethyl sulfoxide, dimethylformamide, furfural, and N-methylpyr- rolidone, and the polar proton-donor solvent methanol. The coefficients were compared to the corresponding capacity factors obtained by classical liquid chromatog- raphy on octadecyl-bonded silica. For the five biphasic solvent systems studied, linear relationships were found between the partition coefficients and the sp 3 and sp 2 hybridized carbon atom number for the alkylbenzene and PAH series, respectively. The sp 2 and sp 3 transfer ener- gies were estimated, and their ratio was used to quantify the solvent selectivity toward aromatic extraction. Countercurrent chromatography (CCC) is a chromatographic separation technique that uses a liquid stationary phase. 1-3 The mobile phase and the stationary phase form a biphasic liquid system. The very name of the technique was chosen to mean that the technique was a hybrid between the countercurrent distribution method (Craig machines) and liquid chromatography. 1 It may be misleading: there is no countercurrent liquid circulation in CCC. The technique name was coined by Yoishiro Ito, who invented the technique and developed it. 1,5,6 He published more than 160 papers using the CCC acronym, well establishing it. The advantages of having a liquid stationary phase in chro- matography are (i) a high loading capability, (ii) a very simple solute retention mechanism, (iii) that either phase of the biphasic system can be used as a mobile phase, (iv) no irreversible solute adsorption, (v) no pH problem, and (vi) less biological solute denaturation. The high loadability is possible because the solutes reach the volume of the liquid stationary phase and not just the surface of the solid phase as in classical liquid chromatography (LC). In CCC, the solutes are retained according to their liquid-liquid partition coefficient: V R ) V M + PV S (1) where P is the solute partition coefficient expressed as the ratio of the solute concentration in the stationary phase to the solute concentration in the mobile phase. The subscripts R, M, and S refer to the retention, mobile phase, and stationary phase volumes, respectively. The dual-mode use of CCC, i.e., the stationary phase becomes the mobile phase and vice versa, precludes any irrevers- ible adsorption inside the CCC column. It is well described in the literature. 3,4,7,8 The CCC columns are made of perfluorinated polymers that can withstand extreme pHs, unlike the classical silica skeleton of LC packings. 9 Aqueous two-phase systems were used for protein or cell separations without denaturation. 10 The direct and accurate measurement of liquid-liquid partition coefficients is another important capability of CCC. 11 Octanol- water partition coefficients as high as 20 000 (log P ) 4.3) were determined by CCC. 12,13 The dual-mode reversal of the phase roles of octanol and water was proven useful in such determina- tions. 14 The retention volume of the solute is directly related to its liquid-liquid partition coefficient (eq 1). The vast majority of the biphasic liquid systems used in CCC contain water as one solvent. In this work, the capability of CCC to work with waterless biphasic liquid systems, which we introduced for fullerene (1) Ito, Y. Adv. Chromatogr. 1984 , 60,181-226. (2) Mandava, N. B., Ito, Y., Eds. Countercurrent Chromatography, Theory and Practise; Chromatographic Science Series 44; M. Dekker: New York, 1988. (3) Conway, W. D. Countercurrent Chromatography; VCH Publishers Inc.: Weinheim, 1990. (4) Foucault, A., Ed. Centrifugal Partition Chromatography; Chromatographic Science Series 68; M. Dekker: New York, 1995. (5) Ito, Y.; Weinstein, M. A.; Aoki, I.; Harada, R.; Kimura, E.; Nunogaki, K. Nature 1966 , 212, 985-987. (6) Ito, Y.; Conway, W. D. High Speed Countercurrent Chromatography; J. Wiley & Sons Inc.: New York, 1995. (7) Gluck, S. J.; Martin, E. J. J. Liq. Chromatogr. 1990 , 13, 3559-3570. (8) Berthod, A.; Chang, C. D.; Armstrong, D. W. In Centrifugal Partition Chromatography; Foucault, A., Ed.; Chromatographic Science Series 68; M. Dekker: New York, 1995; Chapter 1, pp 1-24. (9) Berthod, A. J. Chromatogr. 1991 , 549,1-28. (10) Van Alstine, J. M.; Synder, R. S.; Karr, L. J.; Harris, J. M. J. Liq. Chromatogr. 1985 , 8, 2293-2313. (11) Berthod, A. In Centrifugal Partition Chromatography; Foucault, A., Ed.; Chromatographic Science Series 68; M. Dekker: New York, 1995; Chapter 7, pp 167-198. (12) Berthod, A.; Dalaine, V. Analusis 1992 , 20, 325-331. (13) Berthod, A.; Menges, R. A.; Armstrong, D. W. J. Liq. Chromatogr. 1992 , 15, 2769-2779. (14) Gluck, S. J.; Martin, E.; Benko, M. H. in Centrifugal Partition Chromatog- raphy; Foucault, A., Ed.; Chromatographic Science Series 68; 1995; M. Dekker: New York, Chapter 8, pp 199-218. Anal. Chem. 1996, 68, 431-436 0003-2700/96/0368-0431$12.00/0 © 1996 American Chemical Society Analytical Chemistry, Vol. 68, No. 3, February 1, 1996 431