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Anal. Chem. 1991. 63,2508-2512
bution coefficients are related to the distribution of the ion pairs between the aqueous phase and the micellar pseudophase. However, for two catecholamines in the examined set of cationic compounds it was shown that some fraction in the aqueous phase was not directly associated with the free surfactant ions, resulting in a slight electrophoretic mobility of the solute in the aqueous phase. By means of the presented equations it is possible to obtain an estimate of the ion-pair formation constant of these solutes under the assumption that the solutes were fully dissociated at the examined pH. A full evaluation of eq 10 will require a set of experiments at various pH values and a comparison of the resulting correction factors. A study is presently underway in this laboratory to examine the effect of different important parameters on selectivity of acidic and basic compounds. In summary, it is possible to describe the observed migration and migration factors of solutes in MECC experiments with more or less linear relationships (eq 51, even in the case of charged (anionic or cationic) solutes, which reduces the number of experiments required in the method development stage.
ACKNOWLEDGMENT We thank C. Quang for his assistance in performing some of the experiments. LITERATURE CITED Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 5 3 , 1298. Tetabe, S.; Otsuka, K.; Ichikawa, K.; Ando, T. Anal. Chem. 1984, 5 6 , 111. Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, 834. Foley, J. P. Anal. Chem. 1990, 62, 1302. Ghowsl, K.; Foley, J. P.; Gale, R. J. Anal. Chem. 1990, 6 2 , 2714. Ewing, A. G.; Wallingford. R. A.; Olefirowicz, T. M. Anal. Chem. 1989, 6 1 , 292A. McCormick. R. M. Anal. Chem. 1988, 60, 2322. Snopek, J.; Jelinek, 1.; Smolkova-Keulemansova. E. J . Chromatwr. 1988, 452, 571. Otsuka, K.; Terabe, S.; Ando, T. J . Chromatogr. 1985, 348, 39-47. Cohen, A. S.; Terabe, S.; Smith, J. A,; Karger, B. L. Anal. Chem. 1987. 59. 1021. Walllngfcid, R. A.; Ewing, A. G. A M I . Chem. 1988, 60, 258. Khaledi, M. G.;Smith, S. C.; Strasters, J. K. Anal. Chem. 1991. 63, 1820.
RECEIVED for review April 24, 1991. Revised manuscript received August 5 , 1991. Accepted August 8, 1991. We gratefully acknowledge a research grant from the National Institutes of Health (FIRST Award, GM 38738).
High-speed Countercurrent Chromatography Used for Alkylbenzene Liquid-Liquid Partition Coefficient Determination Alain Berthod* and Madeleine Bully
Laboratoire des Sciences Analytiques, U A CNRS 435, Universite' de Lyon 1, 69622 Villeurbanne Cedex, France
Countercurrent chromatography (CCC) can be used to determine liquid-liquid partition coefficknts. A hydrodynamlc CCC apparatus was used to obtain methand-water-heptane partition coefficients, P, of the homologous serles of n-aikyibenzene from benzene to dodecyibenzene. the methanol-water compositions ranged from pure methanol to methanol 80%-water 20% v/v. They were used as ilquid statlonary phases. Heptane was the moblie phase. The parHtbn coefficients ranged from 8 X lo-' to 0.95, corresponding to heptane-methanol partition coefficients ranging from 1.05 to 1250. For every methanol-water stationary phase, the linear plots of log P versus the atom carbon number, n,, produced the free energy of transfer, A G O , for the methylene incremental group and the phenyl termlnai group. Both A G O energies increased linearly with the water molar fractlon whlch allowed development of an equatlon giving the methanolheptane P value of an alkyibenzene ( n c I 12) with water content 120% v/v.
Since the early Craig apparatus (I) for continuous extraction, countercurrent chromatography (CCC) was developed by Ito (2-4). Ito introduced high-speed CCC which allows separations within a few hours when classical CCC separations may need days (5). Today, high-speed CCC is moving from research laboratories to industrial laboratories (6). CCC uses two nonmiscible liquid phases: one is the mobile phase, the other one is the stationary phase (3-7). The CCC column is most often a continuous open tube coiled on a centrifuge rotor. The centrifugal field is used to hold tightly
the liquid stationary phase. CCC is mainly used in preparative separation and purification of samples. The basic retention equation is (5, 6 , 7)
VR =
vo + P v ,
(1)
Unlike in HPLC, in CCC the total internal volume, VT, can be used for partition process ( 5 )
v, = vo+ v,
(2)
Equation 1 can be rewritten as
VR =
v, + ( P - 1)v8
(3)
V,, VR, VT, and V, are respectively the mobile phase volume, the solute retention volume, the internal volume of the apparatus, and the liquid stationary-phase volume inside the apparatus. P is the solute partition coefficient, i.e. the ratio of the solute concentration in the stationary phase to the solute concentration in the mobile phase. The retention volume, VR, of a solute allows determination of its partition coefficient, P, in the biphasic liquid system used in the CCC apparatus. Partition coefficient determination is a critical measurement in quantitative structureactivity relationship (QSAR) studies. The classical shake-flask method for P measurements has a tedious procedure including accurate weighing, dissolution of the compound in one phase, -0.5-h shaking, centrifugation for several hours to separate the two liquid phases, and spectrophotometric analysis of both phases (8). It is critical to have a highly pure compound. CCC is able to produce reliable P values in a shorter time and with an easier operation that can separate impurities from the compound of interest during the run. Liquid chromatography on columns was used to obtain P values from capacity factors, k' (8). Linear regressions are used to correlate both log k'and log P param-
0003-2700/9110383-2508$02.50/0 0 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 21, NOVEMBER 1, 1991
eters. The regression coefficients are significant. However, for a given compound, the log P value obtained from the log k’value of the compound can be off by 0.3 unit or more. The estimated P value can be 2 times higher or lower than the actual P value. CCC is better than HPLC since the partition coefficients are obtained with no approximation. The same two liquids used in the shake-flask method are also used in the CCC apparatus. For this reason, CCC is a promising technique for liquid-liquid partition coefficient determination. Octanol-water partition coefficients, P& were measured directly by CCC over the range -2 < log P& < 2.5 (9). Hexane was added to octanol to decrease the viscosity. The partition coefficients, obtained by CCC with octanol-hexane-water biphasic mixtures, correlated well with the Pmtliterature values (9,lO). Several technical improvements were used to extent the measurable Pact range: use of a octanol mobile phase instead of a water mobile phase (11), dual-mode or back-flushing (12, 13), cocurrent mode in which both the mobile and the stationary phase move at different speeds (14), and use of modified apparatus (15). CCC can measure liquid-liquid partition coefficients in any biphasic liquid system (4,5,6,16). The goal of this work is to show that CCC can be used to measure accurately the liquid-liquid partition coefficients of the homologous series of n-alkylbenzenes, from benzene to dodecylbenzene, in the ternary biphasic liquid system heptane-methanol-water. The n-alkylbenzene homologous series is widely used in liquid chromatography to obtain information on solute-stationaryphase interaction and the structure-retention relationship (I7-20). The heptane-methanol-water partition coefficients of alkylbenzenes, which are not found in the literature, could be useful in structure-retention studies with identical methanol-water mobile phases.
EXPERIMENTAL SECTION Chemicals. Benzene, toluene, ethylbenzene, butylbenzene, hexylbenzene, and dodecylbenzene were obtained from Merck, Shuchardt, Germany, and Aldrich, Steinheim, Germany. Methanol and heptane were obtained from Prolabo, RhbnePoulenc, France. Heptane was used instead of hexane because its toxicity is lower. Water was deionized and distilled before use. Countercurrent Chromatograph. The CCC apparatus was the Model CPHV 2000 from Societ6 FranCaise de Chromato Colonne (SFCC), 93360 Neuilly-Plaisance, France. It is a coil planet centrifuge apparatus first designed by It0 (4). It contains three multilayer coils connected in series and spinning with a planetary motion around a central axis. The original design described by Ito contained 10 gears (21). It was simplified to obtain a similar motion with only 7 gears. Figure 1shows the gear arrangement used to produce the planetary motion of the three spools without rotating seals. Each spool bears a gear (dotted) that meshes into the central stationary gear (hatched). The connecting tubing is twisted by the spool rotation. It is unwinded passing through the antitwisting connector that rotates in the opposite way (open gear). With such a gear arrangement, if the rotor makes one full rotation, each coiled spool makes two rotations. All rotation speeds given in the text correspond to rotor rotation. Each spool was filled with 133 turns of PTFE tubing, i.d. 5/s in. (1.6 mm), length 29 m, coiled in 7 layers of 19 turns. The Ito fl value is the ratio of the coil radius, r, to the spool revolution radius, R. The /3 ratio was 0.37 for the inner first layer with r = 2.2 cm and R = 6 cm. It was 0.75 for the most outer visible layer with r = 4.5 cm and R = 6 cm. The average /3 value for this CCC apparatus was 0.56. The internal volume of one coiled spool was 58 mL. The three-coil apparatus had a total internal volume, VT, of 175 mL. The total PTFE tube length was 87 m, with a total of 400 turns. The whole system was housed in a air-thermostated box. The temperature was regulated at 22 f 0.5 “C. Chromatograph Preparation. The heptane-methanol-water system is a normal biphasic system. It means that the best stationary-phase retention in a CCC apparatus is obtained when the lighter mobile phase (heptane) is pushed through the denser
antitwisting connector-
2509
rotor
gear
be
-,L
connecting tubing
coiled tube spool
Figure 1. CCC threemil gear arrangement. The seven gears are one central stationary gear (hatched), three gears coupled to the spool (dotted), and three gears used to unwind the connecting tube (open). The arrows indicates the mobile-phase motion when entering through the head of the apparatus.
stationary phase (methanol phase) entering through the tail of the apparatus (3-6). The apparatus is first filled with the heptane-saturated methanol phase using a Model LC 6A Shimadzu pump. This takes about 25 min at a 8 mL/min flow rate. Then, the rotor is started and the rotation allowed to stabilize at the desired speed. The heptane phase is pushed through the methanol phase at the working flow rate of 2 mL/min, unless otherwise indicated, entering through the tail of the apparatus. As long as the apparatus is not equilibrated,the methanol phase is pushed off, exiting by the head of the apparatus. The methanol phase displaced by the heptane phase is collected in a graduated cylinder. Once the heptane phase appears at the apparatus head, two liquid layers are seen in the graduated cylinder. The mobile-phase-stationary-phaseequilibrium is reached. The CCC “column”is ready. The displaced methanol phase is measured. The volume is V, (eq 1). The mixture of alkylbenzenes solubilized in heptane can be injected using an in-line Rheodyne 7125 valve with a 300-pL sample loop. At a flow rate of 2 mL/min, the working pressure was always lower than 1 kg/cm2 (
