© Copyright 1996 by the American Chemical Society
VOLUME 100, NUMBER 39, SEPTEMBER 26, 1996
LETTERS Measurement of Partition Coefficients of Substituted Benzoic Acids between Two Immiscible Solvents by Hyper-Rayleigh Scattering Paresh Chandra Ray and Puspendu Kumar Das* Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012, India ReceiVed: April 30, 1996; In Final Form: July 10, 1996X
We demonstrate that the hyper-Rayleigh scattering technique can be employed to measure the partition coefficient (kp) of a solute in a mixture of two immiscible solvents. Specifically, partition coefficients of six substituted benzoic acids in water/toluene (1:1 v/v) and water/chloroform (1:1 v/v) systems have been measured. Our values compare well with the kp values measured earlier by other techniques. The advantages offered by this technique are also discussed.
The measurement of the distribution of various solutes between two immiscible liquids has a long history in physical and biological chemistry beginning with Nernst.1 Nernst defined the partition coefficient as the constant kp ) Corg/Cwater, where Corg and Cwater are the concentrations of a solute in the organic layer and in water, respectively. In many instances solute molecules can exist in different forms in two phases and the partition coefficient has been utilized in the study of intermolecular forces in them.2 Interests are now growing for studying partitioning of biomaterials in aqueous two-phase systems for purification of various cell constituents including proteins, nucleic acids, membranes, and cell organelles. The two-phase system often provides an environment where activities of such materials are retained and preserved.3 Weak organic acids such as substituted benzoic acids, when partitioned between the aqueous and an organic media, ionize in the aqueous phase and dimerize in the organic layer. The dimerization constant (Kdim) of these acids is e10-3 M-1 in common organic solvents, and therefore dimerization is not significant at low concentrations of the acid. In water, these acids ionize and the ionization constants (Ka) are ≈10-4 M. We have shown recently4,5 that the ionization constant of these acids can be obtained from the first hyperpolarizabilities (β) of X
Abstract published in AdVance ACS Abstracts, August 15, 1996.
S0022-3654(96)01213-0 CCC: $12.00
their acidic and basic forms in water. The extent of ionization depends on the initial concentration of the acid, and a mean hyperpolarizability is measured in solution when both the forms are present. This ionization and dimerization of solutes in different phases remains the most vexing problem in understanding partition coefficients. In this letter, we report hyperRayleigh measurements on six different para-substituted benzoic acids between two sets of immiscible (water and chloroform or toluene) solvents. Partition coefficients of these acids in these solvents have been obtained and compared with data available by other methods. Compounds 1-6 (Figure 1) were purchased from Aldrich, and all the solvents were obtained locally. Prior to use all solvents were purified by known methods. Two sets of solvents (1:1 (v/v) water/chloroform, and 1:1 (v/v) water/toluene) were chosen for the partitioning experiments. Glass-stoppered bottles containing mixture of water, the organic solvent (chloroform or toluene) and the solute (10-5-10-7 M) were shaken vigorously for 6 h and then allowed to equilibrate for 16 h. After equilibration the organic layer was separated from the aqueous layer using a separating funnel. Two-photon Rayleigh intensities were then measured in separated solutions using 1064 nm light from a Q-switched Nd:YAG laser (Spectra Physics, e 8 mJ/pulse). The experimental apparatus has been described © 1996 American Chemical Society
15632 J. Phys. Chem., Vol. 100, No. 39, 1996
Letters TABLE 1: Partition Coefficients (kp) of Compounds 1-6 in Water/Chloroform and Water/Toluene Mixtures Measured by the HRS Techniquea compound
kp(water/chloroform)
kp(water/toluene)
1 2 3 4 5 6
8.04 (7.94) 3.28 (3.31) 6.61 (6.53) 51.88 (52.40) 30.60 (31.00) 7.54 (7.50)
3.42 (3.46) 2.20 (2.21) 4.51 (4.56) 18.24 (18.10) 12.45 (12.60) 3.20 (3.20)
a
Numbers in parentheses are partition coefficients taken trom the literature and are shown for comparison.
experimentally measured HRS intensity (I2ω). However, this procedure for data analysis is not valid for aqueous medium since the substituted benzoic acids ionize in water and the concentration of the specie(s) exhibiting NLO activity in solution changes. In water, these acids exist in two different forms (acid and basic) and the double-quantum Rayleigh scattering intensity, I2ω is related to the incident intensity Iω by7,8
Figure 1. Structures of compounds 1-6.
I2ω ) G(Nsolventβsolvent2 + N0βm2)Iω2
(2)
where N0 is the total acid concentration and βm is the measured average first hyperpolarizability in solution. βm is related to the first hyperpolarizability of the acid form, βa and that of the basic form, βb and R by4
βm2 ) (1 - R)βa2 + Rβb2
(3)
Combining eqs 2 and 3, we can write
I2ω ) G(Nwaterβwater2 + Naβa2 + Nbβb2)Iω2
Figure 2. I2ω/Iω2 vs the number density of compound 1 in pure toluene.
elsewhere.6-9 Since we have used very low concentrations of acids (10-5-10-7), we can ignore the possibility of their dimerization in the organic solvents. Usually the dimerization constant10 of the benzoic acids is ≈10-2-10-3 M-1. At acid concentrations of ca. 10-7 M (used in our experiments), the concentration of the dimer will be ≈10-17 M, which can be neglected without introducing much error. Ionization constants (Ka) of these acids in chloroform and other organic solvents11 are of the order of ≈10-12 M, which implies a degree of ionization R ≈ 10-3 at an acid concentration of 10-7 M. Therefore, the substituted benzoic acid molecules exist mainly in the acid form in chloroform and toluene. The intensity of the second harmonic scattered light in solution, I2ω, and the incident light Iω are related by7,8
I2ω ) G(Nsolventβsolvent2 + Nsoluteβsolute2)Iω2
(1)
where G is an instrument factor, β is the average first hyperpolarizability, and N is the number density. First we measure the second harmonic scattered light intensity as a function of solute concentration in a solvent. Since Nsolvent, Nsolute, and βsolvent are known, we obtain βsolute from the slope of I2ω/Iω2 vs Nsolute plot (see Figure 2). In a partitioning experiment, we measure I2ω/Iω2 and find the number density of the acid, Nsolute from eq 1, since all other quantities in eq 1, that is, Nsolvent, βsolvent, and βsolute are known. We have thus obtained Nsolute in chloroform and toluene phases from the
(4)
where N0 (1 - R) ) Na, number density of acidic form and N0R ) Nb, number density of the basic form in water. The equilibrium ionization constant, Ka can be expressed in terms of the number densities as
ka ) Nb2/Na
(5)
By substituting the value of Na in terms of Ka which is constant at a fixed temperature, eq 4 can be rewritten as
I2ω ) G[Nwaterβwater2 + (Nb2/Ka)βa2 + Nbβb2]Iω2
(6)
In a partitioning experiment, if βwater, βa, βb, and Ka are known, it is straightforward to obtain the number density of the basic form in the aqueous layer from eq 6. In fact, for the substituted benzoic acids investigated in this letter, we know the above quantities from a previous study.4 Thus, after obtaining Nb, calculating N0 is trivial. It turns out that for the substituted benzoic acids studied here, Ka ≈ 10-4 M and the degree of ionization, R ≈ 0.99 at an acid concentration of ≈10-7 M. In other words, the benzoic acids exist entirely in their basic form in the aqueous layer, that is, N0 ) Nb. From all these concentration measurements via the second harmonic generation in solution, we thus calculate the partition coefficient, kp, using the following relationship:
kp ) Norganic/Naqueous
(7)
kp values obtained from these measurements are listed in Table 1. A comparison between the partition coefficients obtained from the HRS method and the same2,10,12 reported by other methods is also made. The agreement between our values and the reported values is excellent and within (2%. Therefore,
Letters we propose that the HRS technique is an alternative method for measuring the partition coefficient of a solute between two immiscible solvents. We have varied the concentrations of the solute from 10-5 to 10-7 M, and we have noted that the error is (6%. Determination of partition coefficient by the HRS technique has several advantages over other methods such as the acidbase titration10 and linear optical spectroscopic methods.13-14 In the titration method only acids and bases can be used, and higher concentrations (ca. 10-3 M) of solute are necessary. The latter causes dimerization in organic solvents (specifically for benzoic acids in nonpolar solvents such as benzene, toluene, chloroform, etc.). Therefore, without the knowledge of the dimerization constant in a nonpolar solvent, partition coefficient of a weak organic acid cannot be determined by the titration method.2,10 Linear absorption spectroscopic techniques such as absorption or fluorescence can be used at low concentrations. However, complications arise when the solute ionize in solution. The benzoic acids employed here ionize in water and the absorption spectra of their acidic and basic forms are not well separated in wavelength (the absorption spectrum of the basic form is red-shifted relative to that of the acid form).4 Consequently, it is difficult to measure the total acid concentration (acid + basic forms) accurately in aqueous solution by absorption.5 To utilize this method for determination of concentrations of molecules in solution, the molecules must absorb in the region of the light source. Therefore, the choice of the source is important. The sensitivity of the absorption technique depends on the molar extinction coefficient, , of the solute. Typically (other than dyes) concentrations of ≈10-5 M are used. The HRS technique employed here is highly sensitive. Although we have used concentrations g10-7 M, this limit can be lowered further without much difficulty. This technique is applicable to all noncentrosymmetric molecules including proteins and
J. Phys. Chem., Vol. 100, No. 39, 1996 15633 other biologically important systems.15,16 Any powerful light source can, in principle, be used. In other words, the HRS technique seems more promising for partition coefficient measurements both in chemistry and biology, than other conventional techniques used in the literature. Acknowledgment. The laser used in these experiments was purchased with a grant from the Department of Science and Technology (DST), Government of India. P.C.R. thanks the Council for Scientific and Industrial Research, Government of India, for a senior research fellowship. References and Notes (1) Nernst, W. Z. Phys. Chem. 1891, 8, 110. (2) Leo, A.; Hansch, C.; Elkins, D. Chem. ReV. 1971, 71, 515. (3) Methods in Enzymology: Aqueous two-phase systems; Walter, H., Johansson, G., Eds.; Academic Press: New York, 1994; Vol. 228. (4) Ray, P. C.; Das, P. K. J. Phys. Chem. 1995, 99, 17891. (5) Ray, P. C.; Munichandraiah, N.; Das, P. K. Chem. Phys., in press. (6) Terhune, R. W.; Maker, P. D.; Savage, C. M. Phys. ReV. Lett. 1965, 14, 681. (7) Clays, K.; Persoons, A. Phys. ReV. Lett. 1991, 66, 2981. (8) Clays, K.; Persoons, A.; De Maeyer, L. AdV. Chem. Phys. 1994, 85, 455. (9) Ray, P. C.; Das, P. K. J. Phys. Chem. 1995, 99, 14414. (10) Smith, H. W.; White, T. A. J. Chem. Soc. 1955, 1953. (11) Ritchie, C. D.; Uschold, R. E. J. Am. Chem. Soc. 1968, 90, 2821. (12) Hassan, A. O.; Isaacs, N. S. J. Chem. Soc., Parkin Trans. 2 1983, 834. (13) Ruth, W.; Mullikin, L. J.; Yoshimura, T.; Helmkamp, M. G. Biochemistry 1984, 23, 6086. (14) Anderson, H. C. Annu. ReV. Biochem. 1978, 47, 359. (15) Clays, K.; Hendrickx, E.; Triest, M.; Verbiest, T.; Persoons, A.; Dehu, C.; Bre’das, J.-L. Science 1993, 262, 1419. (16) Hendrickx, E.; Clays, K.; Persoons, A.; Dehu, C.; Bre’das, J.-L. J. Am. Chem. Soc. 1995, 117, 3547.
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