Anal. Chem. 1987, 59,921-923
occurs for isomers with the double bond in the 3 and 4 position; these isomers give very similar spectra, showing differences only in the abundances of minor ions in the spectra. In addition, for isomers with the double bond in the 6 position or more remote, the ion signal that identifies the double bond position is relatively weak. Consequently, it is more likely that the present methods will find use in the identification of relatively pure samples rather than in the analyses of complex mixtures. In our experience, the negative ion CID mass spectra are considerably weaker than positive ion CID mass spectra, presumably because a significant fraction of the collisions result in electron detachment for negative ions. In our experiments we used a batch inlet system to admit the sample and, therefore, had the leisure to accumulate 20 to 40 2-s scans to improve the signal-to-noise ratio. Consequently, it is not clear whether the method could be applied to gas chromatography/mass spectrometry/mass spectrometry systems where the sample residence time in the source can be relatively short and the concentration of sample may be low, although, clearly, one does not require the signal-to-noise level exemplified by the figure for identification purposes. Since we do not have a gas chromatograph attached to our instrument, this aspect could not be explored.
LITERATURE CITED (1) Domrnes, V.; Wertz-Peiz, F.; Kunau, W. H. J. Chromafogr. Sci. 1978, 14, 360. (2) Suzuke, M.; Ariga, T.; Sekine, M.; Araki, E.; Miyatake, T. Anal. Chem. 1981, 53,995. (3) Kidwell, D. A.; Biemann, K. Anal. Chem. 1982,5 4 , 2462. (4) Buser, H.R.: Arn, H.: Guerin, P.: Rauscher, S. Anal. Chem. 1983,5 5 , 818. (5) Cervilla, M.; Puzo, G. Anal. Chem. 1983,55, 2100.
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(6) Budzikiewicz, H.; Busker, E. Tetrahedron 1980, 3 6 , 255. (7) Doolittle, R. E.; Tumlinson, J. H.; Proveaux, A. Anal. Chem. 1985,5 7 , 1625. (8) Hunt, D. F.; Harvey, T. M. Anal. Chem. 1975, 4 7 , 2136. (9) Busker, E.; Budzikiewicz, H. Org. Mass Specfrom. 1979, 14, 222. (10) Brauner. A.; Budzikiewicz, H.; Roland, W.Qrg. Mass Specfrom. 1982, 17, 161. (11) Ferrer-Correia, A. J. V.; Jennings, K. R.; Sen Sharma, D. K. Org. Mass Specfrom. 1978, 1 1 , 867. (12) Ferrer-Correia, A. J. V.; Jennings, K. R.; Sen Sharma, D. K. Adv. Mass Spectrom. 1978, 7A 267. (13) Greathead, R. J.; Jennings, K. R. Org. Mass Specfrom. 1980, 15, 431. (14) Chai, R.; Harrison, A. G. Anal. Chem. 1981, 5 3 , 34. (15) Peake, D. A.; Gross, M. L. Anal. Chem. 1985. 5 7 , 115. (16) Tomer, K. B.; Crow, F. W.; Gross, M. L. J. Am. Chem. SOC. 1983, 105, 5487. (17)Jensen, N. J.; Tomer, K. B.; Gross, M. L. Anal. Chem. 1985, 57, 2018. (18) Leonhardt, B. A. Anal. Chem. 1985, 5 7 , 1240A. (19) Lanne, B. S.;Appelgren, M.; Bergstrorn, G.; Lufstedt, C. Anal. Chem. 1985,57, 1621. (20) Harrison, A. G.; Mercer, R. S.; Reiner, E. J.; Young, A. 8.; Boyd, R. K.; I
March, R. E.; Porter, C. J. Int. J. Mass Specfrom. Ion Processes, in press.
(21) Cooks, R. G.;Beynon, J. H.; Capridi, R. M.; Lester, 0. R. Metesfable Ions; Elsevier: New York, 1973. (22)Jennings. K. R. Ionic Processes In the Gas Phase; Alrnoster Ferrelra, M. A., Ed.; Reidel: Dordrecht, 1984. (23) Jensen, N. J.; Tomer, K. B.; Gross, M. L. J. Am. Chem. SOC. 1985, 107, 1863. (24) Hunt, D. F.; Shabanowitz, J.; Giordani, A. B. Anal. Chem. 1980,5 2 , 386.
RECEIVED for review August 18, 1986. Accepted December 1,1986. This research was supported by the National Sciences and Engineering Research Council of Canada. A.G.H. gratefully acknowledges the award of a Killam Research Fellowship (1985-1987) by the Canada Council.
CORRESPONDENCE Correlation of OctanoVWater Partition Coefficients with Hydrophobicity Measurements 0btained by Micellar Chrornatography Sir: The hydrophobic character of a chemical is usually expressed in terms of its partition coefficient between l-octanol and water (PoIw). This parameter, for which large compilations already exist ( I ) , is probably the one most frequently used in the analysis of quantitative structure-activity relationships (QSAR) for the prediction of pharmacological activity in a series of test compounds. Attempts to determine log Poi,., by other means different to the classical shake-flask method (2) have included reversed-phase high-performance liquid chromatography (RP-HPLC) with C-8 ( 3 ) ,C-18 ( 4 ) , 1-octanol coated (2-18 (5),and phenyl (6) stationary phases and very recently droplet countercurrent chromatography (DCCC) (7). In both techniques, however, organic solvents have to be used. In the former case, the organic modifier is added in different proportions to water or aqueous buffers in order to increase the eluent strength, thus shortening the retention times. In the latter, mixtures of methanol, chloroform, and water or buffer were used to achieve a continuous flow of droplets. Retention in RP-HPLC is mainly due to hydrophobic interactions originating from a net repulsion between water and the stationary phase as well as the unpolar moiety of the solute molecule (8). The magnitude of this nonpolar interaction depends, among other factors, on the surface tension of the
eluent. Neat aqueous eluents are the weakest in RP-HPLC due to the very high cohesive density of water (9) and would make the chromatographic system impractical in many cases. Increasing concentrations of an organic modifier such as methanol or acetonitrile result in a systemic decrease of the surface tension of water which is accompanied by lower capacity factors, although dependence is strongly nonlinear (8). In ion-pair chromatography low concentrations of ionic surfactants (always below the critical micellar concentration, cmc) having an opposite charge to that of the solute of interest are added to the eluent, thus altering its retention in the chromatographic system. Sodium dodecyl sulfate (SDS) has probably been one of the most frequently employed, its main function being to mask the function of the eluite that interacts with the residual free silanol groups present at the stationary phase surface. Adsorption of some of the surfactant to the stationary phase also plays a significant role in altering the elution behavior mainly of charged solutes. Hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide, CTAB), on the other hand, has been found to be one of the most potent silanol scavengers (IO)and is currently used at low concentrations (1-5 mM) to reduce the silanophilic interactions that cause excess band spreading, especially in the case of basic compounds.
0003-2700/87/0359-0921$0 1.50/0 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987
Table I. Solutes Used in the Correlation Studies, Literature log Polw Values, and log k' Values in the Different Micellar Mobile Phases
log k' no. 1 9
3 4 5 6 I
I
8 9 10 11
0.1 M
0.016 M
X
SDS"
CTABb
0.05 M CTAB*
CTAB"
0.016 M Brij 35*
0.05 M Brij 35b
MeOH/ bufferC
log P d
H Br OMe CHO COMe C02Me CN
1.173 1.420 1.122 0.930 0.966 1.149 0.876 1.008 1.358 0.656 0.499
1.526 1.912 1.559 1.221 1.245 1.568 1.235 1.434 1.813 1.029 0.803
1.232 1.430 1.220 1.039 1.008 1.220 1.009 1.137 1.393 0.850 0.658
1.023 1.158 0.994 0.829 0.824 0.983 0.830 0.926 1.141 0.701 0.527
1.458 1.826 1.483 1.062 1.152 1.481 1.128 1.366 1.762 0.711 0.530
1.192 1.377 1.179 0.893 0.940 1.165 0.930 1.079 1.384 0.578 0.400
-0.021 0.195 0.027 -0.312 -0.251 -0.062 -0.311 -0.147 0.171 -0.434 -0.673
2.13 2.99 2.11 1.48 1.58 2.12 1.56 1.85 2.69 1.10 0.64
NO2
Me CH20H CONH,
0.1 M
"Flow rate = 1.0 mL/min. bFlow rate = 1.5 mL/min. "Comparison with log k' values reported for a hydroorganic mobile phase (MeOH/pH 7 0.01 M sodium phosphate buffer (75/25); 10-pm C-18 Perkin-Elmer column 25 cm X 4.6 mm i.d.; flow rate = 1 mL/min; ref 18). Reference I . Table TI. Linear Regression Data of the Correlation between log P o , , and log k'"
a h r2 \
F "log k ' = a
0.1 M SDS
0.016 M CTAB
0.05 I d CTAB
0.1 M CTAB
0.270 0.404 0.977 0.045 375.1
0.500 0.486 0.994 0.027 1435.9
0.493 0.335 0.978 0.036 400.2
0.399 0.274 0.975 0.031 345.4
0.016 M Brij 35 0.05 M Brij 35 0.185 0.589 0.972 0.072 306.7
0.194 0.444 0.944 0.078 150.6
MeOH/buffer -0.872 0.381 0.981 0.038 455.5
+ h log. P: r2 = squared correlation coefficient: s = standard error: F = variance.
The pioneering work of Armstrong and Henry (11) first showed the utility in RP-HPLC of mobile phases containing surfactants above the cmc. Resolution of the peaks and efficiency were low when compared to hydroorganic mobile phases, but these authors set the trend that other investigators have later followed and from which several successful applications have derived (12, 13). These systems provide the unique ability to solubilize hydrophobic compounds in aqueous solution. The solutes can then partition to the micelles and a decreasing retention time is generally observed with increasing surfactant concentration in the mobile phase (once above the cmc), although inversions in retention orders have been reported for a small number of substituted benzenes (14). Molecules of surfactant can bind to the stationary phase and cause profound changes in the capacity factor of the solute. In order to explain the elution behavior of a solute in micellar chromatography, apart from certain column parameters and the characteristics of the micelles, three different partition coefficients must be considered for the solute (15),namely, those between the micelle and water (K,,), between the micelle and the stationary phase (Ksm),and between the stationary phase and water (Ksw).Bulk water in these systems acts as a barrier through which the solute must pass, and it slows down mass transfer between the micelles and the stationary phase (13). The combined effects of these primary equilibria would lead to the different elution patterns of different solutes. By use of different micellar mobile phases, measuring capacity factors of a compound on a single stationary phase would result in different values of K,, and K,, but identical values of K8,. On the contrary, were they measured in the same micellar mobile phase, K,, values should be the same for two different stationary phases but K,, and K,, would differ. Janini and Attari (16) studied the partition of the three dihydroxybenzene isomers between octanol and aqueous micellar solutions of SDS. determining solute concentration
in both phases by RP-HPLC. The apparent octanol/water partition coefficients were found to decrease linearly with increasing concentrations of surfactant in the micelles. The resulting plot yielded a correlation coefficient near unity. The POiwvalues were obtained from the intercept and were significantly lower than the corresponding literature "shake-flask values (1). Our objective was to study the influence of hydrophobicity on the chromatographic retention data of a set of 11 wellcharacterized monosubstituted benzenes (1) under different experimental conditions, evaluating their possible correlation with the l-octanol/water partition coefficient. Critical micellar concentrations for the three surfactants used in this work have been reported (14, 17).
EXPERIMENTAL SECTION Apparatus. A Perkin-Elmer 10 liquid chromatograph was used equipped with a Rheodyne sample injection valve with a 20-pL loop, a Perkin-Elmer fixed-wavelength detector operating at 254 nm, and a Hewlett-Packard 3390A integrator. The column used was 10 cm long and 4.6 mm i.d., and contained Hypersil, 5-km particle diameter octadecylsilane packing. Operating pressures never exceeded 1200 psi at a maximum flow rate of 1.5 mL/min (see Table I). Room temperature was 25 f 3 "C. Materials. Electrophoresis-grade sodium dodecyl sulfate (SDS),obtained from Merck, and hexadecyltrimethylammonium bromide (CTAB)and polyoxyethylene(23)dodecanol (Brij 35) from Fluka were used as received. Monosubstituted benzenes of analytical grade were collected from laboratory stock and were the following: (1) benzene, (2) bromobenzene, (3) anisole, (4) benzaldehyde, (5) acetophenone, (6) methyl benzoate, ( 7 ) benzonitrile, (8) nitrobenzene, (9) toluene, (10) benzyl alcohol, and (11) benzamide. Procedures. The micellar mobile phases for RP-HPLC were prepared by dissolving the surfactant in distilled water in an ultrasound bath, followed by filtration under normal pressure through a 0.65-pm Millipore cellulose ester filter. Vacuum is not adequate to speed up filtration as it favors foam production.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 6, MARCH 15, 1987
Solutions for each test compound were prepared by taking a few micrograms of the product into the same micellar mobile phase that was to be used for the separation. Dissolution was accomplished by vortex mixing for a few seconds and concentrations were adjusted to permit their UV detection with injections of up to 20 r L yielding similar areas under the peaks. The column dead time, to,was determined by injecting 10 pL of pure water and was used for the k’ calculations in the usual way by using the ratio k’ = ( t , - t o ) / t owhere , t, is the retention time of the test solute.
RESULTS AND DISCUSSION The retention times of the test compounds selected for this study were measured at different concentrations of each of the aqueous surfactant mobile phases. Their experimental capacity factors (expressed in logarithmic f o p ) , together with their log Poi,, are tabulated in Table I and compared to those obtained with a hydroorganic mobile phase on a similar stationary phase (18). From the statistical treatment of the data it is apparent that the relationship between log k’ and log Poiw is linear and yields very good regression coefficients. The results of linear regression analysis of these data are given in Table 11. Compounds like pyridine, which are very sensitive to residual free silanol sites in the stationary phase, were anomalously retarded and gave rise to asymmetrical peaks in systems other than CTAl3, as could be expected. On the contrary, phenol was accurately placed in the 0.016 and W5 M Brij 35 systems, fairly well placed in 0.016 M CTAB, and definitely out of place in 0.05 M CTAB (results not shown). Use of lower concentrations meant longer capacity factors for a given flow rate and slightly better regression coefficients. It is intriguing the parallelism observed between these findings and the fact that an increase in organic component of the hydroorganic mobile phase in RP-HPLC also results in worse correlations of capacity factors with octanol/water partition coefficients. In this case better correlates are usually obtained when the capacity factors extrapolated to 100% water composition are used instead (3, 19). Retention times were remarkably reproducible in all of the micellar mobile phases used and resolution of the peaks was comparable to that achieved with hydroorganic mixtures. Elution order reversals took place for the same surfactant a t different concentrations. This can be clearly seen in the trios acetophenone-benzonitrile-benzaldehyde and benzene-methyl benzoate-anisole, both of very similar log Po,, values (1.58-1.56-1.48 and 2.13-2.11-2.12, respectively).
CONCLUSIONS The chromatographic behavior of this series of monosubstituted benzenes eluted with different aqueous micellar mobile phases appears to be well correlated with the hydrophobic character of these chemicals as determined in the standard system 1-octanol/water.
923
Better knowledge of the nature of the partition processes involved may make it possible to design systems in which the chromatographic retention of a certain solute depends solely on its relative hydrophobicity, being electrostatic interactions with the solvent and stationary phase neglected. The interest of organized surfactant systems as models for biological membranes supports our belief that micellar chromatography is a useful technique to collect hydrophobicity data which could be later used in QSAR analysis instead of the popular log Poi,. Work along this line of research is already in progress in our laboratory. Registry No. 1-Octanol, 111-87-5.
LITERATURE CITED Hansch, C.; Leo, A. Substituent Constants for Correlation Analysis in Chemistry and Biology; Whey: New York, 1979. Fujita, T.; Iwasa, J.; Hansch, C. J. A m . Chem. SOC. 1964, 86, 5175-5 180. Garst, J. E.; Wilson, W. C. J. Pharm. Sci. 1984, 73, 1616-1623. Carlson, R. M.; Carlson, R. E.; Kopperman, H. L. J. Chromatogr. 1975, 107, 219-223. Unger, S.H.; Cook, J. R.; Hollenberg, J. S. J . Pharm. Sci. 1978, 67, 1364- 1367. Thus, J. L. G.; Kraak, J. C. J. Chromatogr. 1985, 320,271-279. Gago, F.; Alvarez-Builla, J.; Elguero, J. J. Chromatogr. 1986, 360, 247-25 1. Horvath, C.; Melander, W. J. Chromatogr. Sci. 1977, 15, 393-404. Horvath, C.; Melander, W.; Molnar, I. J . Chromatogr. 1978, 125, 129-1 56. Bij, K. E.; Horvdth, C.; Melander, W. R.: Nahum, A. J. Chromatogr. 1961, 203, 65-84. Armstrong, D. W.; Henry, S. J. J. Liq. Chromatogr. 1980, 3 , 657-662. ... .._ DeLuccia, F. J.; Arunyanart, M.; Cline Love, L. J. Anal. Chem. 1985, 57, 1564-1568. Cline Love, L. J.; Zibas, S.;Noroski, J.; Arunyanart, M. J. Pharm. Biomed. Anal. 1985, 3 , 511-521. Cline-Love, L. J.; Habarta, J. G.; Dorsey, J. G. Anal. Chem. 1984, 56, 1132A-1148A. Armstrong, D. W.; Nome, F. Anal. Chem. 1981, 53, 1662-1666. Janini, G. M.; Attari, S.A. Anal. Chem. 1963, 55,659-661. Armstrong, D. W. Sep. Purif. Methods 1985, 14, 213-304. Gago, F.; Alvarez-Builla, J.; Elguero, J. J. Liq. Chromatogr., in press. Hammers, W. E.; Meurs, G. J.; De Ligny, C. L. J. Chromatogr. 1982, 247, 1-13.
’ Departamento de Farmacolo&a, Universidad de Alcald de Henares.
* Departamento de Qdmica Orgdnica, Universidad de Alcald de Henares. Instltuto de Quimica MiKlca, C.S.I.C.
‘Instituto de Qdmica Orgdnica, C.S.I.C. Federico Gaga*' Julio Alvarez-Builla2 Departamentos de Farmacologia y Quimica Orginica Universidad de Alcali de Henares Madrid, Spain
Jose- Elguero3 Jose C. Diez-Masa4 Institutos de Qujmica MBdica y Quimica Orginica C.S.I.C., Juan de la Cierva, 3 28006 Madrid, Spain
RECEIVED for review August 11, 1986. Accepted November 4, 1986.
