Anal. Chem. 1986, 58,3006-3010
3006
and in some instances one or two additional isocratic experiments, will be reported shortly.
APPENDIX Horvath et al. (10)have shown that the general plate height equation can be rearranged to yield an expression giving the dependence of reduced plate height, h, on solute k’. In its simplest form it can be written as
h = a(k’z/(l + kq2) + 2b(h’/(l + kq2) + c / ( l
+ kq2 (1)
where a, 6 , and c are determined by regression analysis of experimental data. From the definition of plate height we can write the following equalities
N = L/(hd,) = Tr2/02 =
+ k?2/a2
toZ(l
(2)
where L is column length, d, is the particle diameter of the solid support, and where Tr, o, and to represent the retention time, the standard deviation of the elution profile, and the column void time, respectively, In differential form we can define the peak capacity for resolution Rs separations as
dNc = dTr/4-Rs.o
(3)
Substituting eq 1 and 2 into eq 3 and taking the integral of both sides of eq 3 we obtain
[dNc =
Evaluated between the specific upper and lower limits of kl’ and k j this expression becomes (14)
+ c) + a1/2hl’+ aU1/’b + 2bhi + c) + al/Zkfr+ a-‘I2b -t 2bkl’
LITERATURE CITED (1) Deming, S.N.;Turoff, M. L. H. Anal. Chem. 1978, 5 0 , 546. (2) Otto,M.; Wegscheider, W. J . Chromafogr. 1983, 258, 11. (3) Berridge, J. C. J. Chromatogr. 1982, 244,1. (4) d’Agostino G.; Mitchell F.; Castagnetta L.; O’Hare M. J. J . Chromatogr. 1984, 305, 13.
(5) Spectra-Physics; Optim I1 Technical Bulletin GIs-01. (6) Glajch, J. L.; Kirkland, J. J.: Squire. K . M.; Minor, J. M. J . Chromatogr. 1980, 199, 57. (7) Drouen, A. C.J. H.;Biiliet, H. A. H.; Schoenmakers, P.J.; deGalan, L. Chromatographia 1982, 16, 48. (8) Schoenmakers, P. J.; Biliiet, H. A. H.; deGalan L. J . Chromatogr. 1981, 205, 13. (9) Schoenmakers. P. J.; Biiiiet H. A . H.; deGaian, L. J . Chromatogr. 1981, 218, 261. (10) Horvath, C.;Lin. H.J . Chromatogr. 1978, 149, 43. (11) Davis. J. M.; Giddings, J. C. Anal. Chem. 1983, 5 3 , 418. (12) Herman, D. P.;Gonnord, M. F.;Guiochon, G. Anal. Chem. 1984, 5 6 ,
995. (13) Martin, M.; Herman, D. P.; Guiochon, G. Anal. Chem. 1986, 5 8 , 2200. (14) Handbook of Chemistry & Physics, 42nd ed.: The Chemical Rubber Publishing Co.: Cleveland, OH, 1960.
RECEIVED for review January 14,1986. Accepted July 9,1986. The financial support of the Millipore-Waters Chromatography Division throughout the course of this work is gratefully acknowledged.
n -Alkyldimethylarnrnonium Propanesulfonates as Stationary Phase Modifiers in Reversed-Phase Liquid Chromatography Walter G. Tramposch’ and Stephen G. Weber* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
n-AkykWnethylammonbn propanedonates (C,DAPS) were studied as moblle phase addftlves in a reversed-phase liquid chromatographicsystem. When C,,DAPS is adsorbed on the column, the effective phase ratio of the native stationary phase is reduced while m e anlons and neutral specles exhlbft various degrees of enhanced retention. The retention of the anions in the presence of adsorbed C,,DAPS can be correlated wRh free energies of transfer from water to methanol, dhnethylformamkle, and acetonitrile. The fact that a correlatlon wHh all three solvents Is good lndlcates that the anion’s leaving water Is the major process governing the energetics of ion adsorption. I n independent experiments the association of anions with C,,DAPS micelles was determined by conductance measurements.
Mobile phase additives in reversed-phase liquid chromatography (RPLC) have become important tools in altering the retention selectivity of the separation system. Among the ’Present address: Calgon Corp., P.O.
15230.
Box 1346, Pittsburgh, P A
many additives, organic solvents, such as methanol, acetonitrile, and tetrahydrofuran are the most widely used ( 1 ) . These organic modifiers not only alter the mobile phase but are imbibed by the stationary phase further influencing the separation ( 2 , 3 ) . Charged solutes are influenced by ion-interaction reagents (IIR’s), e.g., alkylsulfonates and alkyltrimethylammonium ions ( 4 , 5 ) . When IIR’s are used at concentrations above their critical micelle concentration (cmc), the solute may be partitioned between the mobile phase, micelles, and the stationary phase (6, 7). This type of system has been referred to as pseudo-phase chromatography. Other more solute specific additives have also been investigated. Crown ethers have been used for the chromatography of amines (8,9),zwitterion pair reagents have been used for the separation of zwitterionic solutes (10,l I ) , and cyclodextrins have been used in isomer separations (ref 12 and references therein). In this work, the use of zwitterionic surfactants as mobile phase additives is reported. n-Alkyldimethylammonium propanesulfonates (C,DAPS) comprise a class of compounds that are permanent zwitterions at the pHs commonly employed in RPLC (1 < pH < 8) (13). C,DAPS are similar to y-amino acids except that the sulfonate imparts a much wider
0003-2700/S6/0358-3006$01.50/0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58,NO. 14, DECEMBER 1986
neutral range due to the lower pK, of the sulfonate. Dielectric increment studies have determined that the dipole moment of these molecules is on the order of 25 D (14). However, unlike other polar molecules which contain ionic subunits, C,DAPS's do not contribute t o the conductance of solutions (13). C,DAPS also have the ability to solubilize hydrophobic membrane proteins without denaturation (15). Plots of log k'for the homologous series of C,DAPS ( n = 1, 6, 8, 10, 12) vs. percent methanol (16) are U-shaped. This is an indication of the interaction of the compounds with the silica and with the alkyl-bonded stationary phase (17-19). As the mobile phase becomes water-lean, the hydrophobic contribution to retention is reduced and a "silanophilic" retention dominates. This type of retention is most often observed with quaternary ammonium salts, protonated ammonium salts, amino acids, and peptides. However unlike the aforementioned species which are retained mainly by ion-exchange interactions (16, 20), C,DAPS have been shown to interact with silica probably due to dipole-dipole interactions. Note that C,DAPS are soluble in methanol. They do not alter the ion-exchange characteristics of the stationary phase when adsorbed (16). Although the polar head group associates with the polar silica, the polar head groups do not influence one another in micelles (21). When the mobile phase is largely aqueous, the sulfobetaines are retained hydrophobically. We were prompted to investigate these molecules as mobile phase additives since they contain the functionalities used as IIR's, Le., tetraalkylammonium and sulfonate. However, because they are electrically neutral, ionic strength effects and intermolecular coulombic interactions, which are unavoidable with IIR's, should be lower with C,DAPS. The aim was to determine the range of interactions that C,DAPS would have with solutes in an RPLC system.
EXPERIMENTAL SECTION Apparatus. The chromatographic equipment used has been previously described (16). The column (5 X 30 mm) was packed with 5-wm RP-18 (Brownlee Labs) except where otherwise noted. A column water jacket was employed to maintain a constant temperature of 30.0 0.1 "C. Data were acquired and processed by a Digital Equipment Corp. LSI 11/03 microcomputer (22). All retention times are reported as the first statistical moment of the peak. Conductance determinations were performed with a Beckman Model RC18A conductivity bridge using platinum black electrodes. Reagents. C12DAPSwas prepared as previously described (16). The mobile phase consisted of 95/5 water/methanol (MCB Omnisolv) with the appropriate amount of H3P04added. The pH was adjusted with the addition of NaOH solution followed by dilution to a final volume of 1.0 L. To vary the ionic strength, differing amounts of H3P04were used and the ionic strength was estimated from the final pH and concentration. Reagent grade solutes were obtained from various sources and used without further purification. Procedures. Adsorption capacities of the column for C12DAPS were determined at a flow rate of 2.0 mL min-' and C,,DAPS breakthrough was determined by the change in column effluent refractive index. The void volume of the column was measured in the absence of C12DAPSby the injection of K2Cr0, and NaNO, in a buffered mobile phase. UV-visible spectra of solutes in the presence and absence of micelles were taken under the following conditions: Each of the solutes were 0.5 mM whereas the CIzDAPSconcentration was 200 mM. The solutions had the same pH and methanol concentration as that of the mobile phase used. Conductance experiments were carried out at 25.00 i= 0.05 "C. Ionic solutes (solutionsA) were 0.3 mM in water. Another solution (B) was 15.0 mM in C12DAPSand also 0.3 mM in ionic solute. A 15.0-mLaliquot of A was pipetted into the dried conductance cell and allowed to reach thermal equilibrium. A was then titrated with B, maintaining a constant ionic solute concentration. After each aliquot of B was added, the solution was stirred until thermal equilibrium was reached and then the stirrer was turned off and
*
0.05
3007
1f
U
a c
0.0
0 . 5 1.0 1.5 2.0 2.5
C12DAPS C o n c e n t r s t i o n ( m M )
Flgure 1. Adsorption isotherm for C,,DAPS: mobile phase, 95/5 water/methanol with 75 mM phosphate buffer, pH 6.1; flow rate, 2.0 mL/min; temperature, 30.0 f 0.1 O C ; 5 X 30 mm column packed with 5-pm
RP-18 (Brownlee Labs). 5.
,
' I
i
hdmber
of Carbon A'oms
Figure 2. In k'vs. carbon number for ethyl acetate, ethyl propionate, and ethyl butyrate: 0 , 0.0 mM C,,DAPS; +, 0.05 mM; U, 0.25 mM; A, 1.00 mM; 0, 2.5 mM. Conditions are given in Figure 1.
the conductance measurement was made. Identical results were obtained at 1 kHz and 3 kHz.
RESULTS AND DISCUSSION Stationary Phase Loading of C12DAPS. Figure 1 presents the loading of C12DAPS onto a RPLC stationary phase (Brownlee RP-18) as a function of C12DAPS concentration in the mobile phase. The compound shows an adsorptive capacity of 0.101 mmol on 0.2 g of stationary phase a t 2.5 mM ClzDAPS in the mobile phase. All mobile phase concentrations are below the cmc (3.2 mM in water (15)and as determined by laser light scattering in our laboratory). The column contains 0.11 mmol of C18 groups according to the manufacturer. On a second stationary phase (Spherisorb ODS 10 pm) under identical conditions, 1.75 mmol adsorbs onto 3.2 g of stationary phase. On comparison of the two phases in terms of coverage, 0.0027 mmol/m2 and 0.0025 mmol/m2 are obtained on the Brownlee and Spherisorb materials, respectively. Effect of Adsorbed C,,DAPS on the Hydrophobic Character of the Column. The homologous esters ethyl acetate, ethyl propionate, and ethyl butyrate were used to monitor the hydrophobic character of the stationary phase. Esters were used since the ester functional group contributes little to hydrophobic retention (23). In fact, the value of the ester functional group contribution to octanol-water partition coefficients indicates that it prefers water, but only slightly (log P = -0.77) (24). Figure 2 presents the results of these experiments as plots of In k' a t various ClzDAPS mobile phase concentrations vs. the number of carbon atoms contained in the esters. For a series of homologues, In h'is related to the free energy of adsorption of the functional group that is added
3008
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
: cc
25
0
EC
i
0 30
0 25
3 50
-*"
m"
-
*:
Ho: 0, ethyl acetate; 0 , ethyl propionate: e , Flgure 3. k' vs. 4 ethyl butyrate; A , bromoethyl acetate; 0,cyanoethyl acetate. Conditions are given in Figure 1 except flow rate was 1.0 mL/min.
2 75
::
1
Figure 4. k' vs. 4 H/4Ho for aromatics: 0,benzonitrile; 0,methyl benzoate: 0, chlorobenzene; A, bromobenzene. Conditions as in Figure 3.
to each homologue (CH,- or -CH2-) AGc, the free energy of transfer of the similar functional group AGF and the phase ratio 4 RT In k' = -nAGc - AGF RT In
+
+
Plotting In k'vs. the number of methyl and methylene groups in the molecule yields a slope of -AGc and an intercept of -AGF +- RT In 4. As the ClzDAPS concentration increases, the function remains linear with carbon number and the slopes remain constant. The intercept, however is reduced. In Figure 2, the slopes for the esters could only remain constant if the free energy of adsorption for the methylene group is unaltered. The decrease in the intercept points to a reduction in the effective hydrophobic phase ratio (4) or to a shift to a less favorable adsorption energy for the ester functional group. The latter is unlikely since the stationary phase becomes more waterlike as surfactant, with its hydrated (14 H20 per head-group (21))head-group is adsorbed. Thus a phase ratio reduction is the most likely cause for the observed decrease in retention. Solute Retention w i t h C,,DAPS i n the Mobile Phase. It is not unreasonable to expect a simple relationship to exist between surface coverage and solution retention. The otherwise attractive model of Deming (25,26)must be considered untenable in this circumstance. This is because if C1,DAPS had altered the retention by changing the interfacial surface tension, then this would have been revealed by a change in the energy of hydrophobic retention. Such a change was not observed. Initially the model of Wahlund and Beijeresten (27) was employed in which the retention of solutes in the presence of 1-pentanol could be understood based upon the surface coverage of 1-pentanol and two partition coefficients, one to the native stationary phase and one to the pentanol. Plotting k'for the solutes vs. 0 (determined by breakthrough curves) of C,,DAPS yields nonlinear plots in most instances indicating that the system being studied is more complex than a simple two-site stationary phase system in which one phase replaces the other as the mobile phase additive adsorbs. The attempt has been made to rationalize the data based on the phase ratios calculated from the data in Figure 1. The fractional hydrophobic phase ratio was used as an independent variable in examining the effects of C,,DAPS on the retention of solutes in the system. It was calculated as exp[int, - into] where int, is the intercept (Figure 2) for the ith concentration of C,,DAPS and into is the intercept for [C,,DAPS] = 0. On this abscissa &H/q5Ho = 1 corresponds to native stationary phase, while @ H / 4 H 0 = 0 corresponds to a fictitious surface dominated by the sulfobetaine functional group. Retention data (k') for a number of esters, anions, and substituted benzenes are plotted vs. the fractional hydrophobic phase ratio & H / & H O at various C,,DAPS concentrations
Figure 5. k'vs. 4H/$Hofor aromatics: 0, phenol: 0, benzene; 0, nitrobenzene: A , anisole. Conditions are given in Figure 3. P
i t
L . 3 Y
I100
I / ,
I
I
i
'50
I
2 3
0
3 c
G cr,
c 25 c 50
0 71
1
cc
@"
Figure 6. k'vs. 0, NaNO,: A,NaOAc; 0,NaI; 0, NaSCN: 0 , picric acid; e , benzoic acid. Picric acid and benzoic acid use the right side axis label. Conditions are given in Figure 3.
(Figures 3-6). For many of the substituted benzenes and all of the esters (Figure 3 and 4) a linear increase of k' with
hydrophobic phase ratio is observed and an extrapolation to 4H/4Ho= 0 yields an intercept near zero. This indicates that for these solutes there is very little interaction between them and adsorbed C,,DAPS; the retention seems to be governed by hydrophobic forces. With different substitution on the benzene, varying effects on k'are seen. For example (Figure 5) benzene retention is virtually unaffected by the presence of ClzDAPS on the stationary phase while phenol shows almost a 4-fold increase in retention a t the highest ClzDAPS concentration compared to the retention in the absence of C,,DAPS. An extrapolation to 4H/4Ho= 0 for the solutes represented in Figure 5 yields an intercept significantly greater than zero indicating that there is an association between the
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
I /
2i
3009
i
0.00
0.10
0.05
Cl2DAPS C o n c e n t r a t i o n ( m t 4 )
Ionic Strength(M)
Flgure 7. k’vs. ionic strength: 0 , NaNO,; m, NaX; A,NaSCN: 1.O mM C,pAPS; phosphate buffer concentration adjusted to vary the ionic strength; pH 6.1; other conditions as in Figure 1 except the column packed with Spherisorb ODS 10 pm (5 X 250 mm).
Figure 8. Relative changes in resistance of various salt solutions as a function of C,,DAPS concentration: 0 , NaI: A,NaSCN; m, NaNO,. Conditions were as follows: aqueous solutions, temperature 25.00 f 0.05 ‘C.
Table I. Functional Group Selectivities for Substituted Benzenes
ClzDAPS with solute molecules. The interaction of organic molecules with micelles may be investigated with UV spectrophotometry. None of the solute spectra showed any increase in fine structure, which is associated with the inclusion of molecules into the hydrophobic interior of micelles (29,30). However all solutes except benzoate do exhibit spectral shifts with ClzDAPSto varying degrees and in the correct direction for interaction with micelles (29-31). The only other study of solute partitioning into CIzDAPS is that by Fendler and Fendler (32). In this study proton NMR was used to determine the location of solubilized benzene and nitrobenzene. Both molecules interact with the charged headgroups but the dynamic solubilization site for nitrobenzene is closer to the micelle interior. Both of these species are solubilized in the water-rich region of the micelle. Our chromatographic data indicate that both of these species interact with the C12DAPS-modified surface. The lack of vibronic structure in the UV absorbance spectra for these species is consistent with the location of the solutes as determined by Fendler and Fendler. Solution conductance experiments were performed using NaI, NaSCN, and NaN03 in water at constant concentration while increasing the concentration of ClzDAPS (Figure 8). All conductance data are normalized by dividing by the conductance of the salt in the surfactant-free water system and are plotted as a relative resistance, ‘%/A. For all the salts there is a decrease in resistance as the concentration of C,,CAPS increases below the cmc. Although ClzDAPSdid not produce any solution conductance, this decrease in resistance may be due to some change in the bulk properties of water (14) although changes in solution viscosity of surfactants below the cmc is minimal (33). After the cmc however, NaI and NaSCN showed a dramatic increase in resistance whereas NaN03 shows weak or no response. For any ionic species in solution, an increase in the ion’s hydrodynamic radius, due to association with a neutral micelle, would cause a increase in the measured resistance due to solvent drag on that species (34). The resistance data of solutions of these ions with the micelles reflect their chromatographic response. NaI and NaSCN show a dramatic response not only to C,,DAPS in the RPLC system but also to C12DAPSmicelles in solution. NaN03, which has some limited retention in the chromatographic system, seems to associate weakly with the micelles. Note that all the ions appear to behave similarly a t [ClZDAPS]< cmc. It can be inferred from the similarity, and from the direction of the shift in resistance, that ion-monomeric ClzDAPSassociations are negligible. Further attempts to rationalize retention data were made. Plots of single ion free energies of transfer from water to various solvents (35)vs. In k i, where k is the solute retention
substituent -H
-c1
-Br -NO2 -0Me -CO&HB -COOH -OH -CN picric acid
alia
1.0 6.14 11.6 1.32 2.01
5.00 1.37 0.27 1.41 0.50
aAb
1.0
0.73 3.85 1.02 1.47 1.98
0.57 1.00
0.55 4.96c
Hydrophobic selectivities calculated from 12’ data using a mobile phase of 9515 water/methanol with 75 mM phosphate buffer, pH 6.1. bClzDAPSselectivities calculated at 2.5 mM ClzDAPS in the moble phase. Selectivity calculated at 1.0 mM CI2DAPS. a
surface-bound ClzDAPS and these solutes. An increase in retention is observed for all of the anions (Figure 6) except for benzoic acid. Picric acid shows a large response, demonstrating a k’ value greater than 100 with ClzDAPS in the mobile phase at the 1.0 mM level. Remarkably, the k’values of SCN- and I- increase from approximately 0 to 6.80 and 1.95, respectively. All of the anions however yield nonlinear k’ vs. 4H/4H0 plots. The clear indication is that the anion’s K’values are not linearly related to the surface coverage of ClzDAPS. This is consistent with surfactant aggregation on the stationary phase and with the anion’s having a particular sensitivity to the presence of aggregates. Anions are retained when ClzDAPS is adsorbed, therefore the ionic strength (I) of the mobile phase was varied (Figure 7 ) to determine the extent of the ion-exchange effects. In each case, with an increase in ionic strength up to 1.1M an increase in retention is observed. The quantity of adsorbed Cl,DAPS remains constant throughout the range of ionic strengths tested. For an ion-exchange process to be present, it is expected that as the ionic strength increases, k’should decrease; thus ClzDAPS is not acting as an ion-exchanger (28). This is consistent with the previous findings from ion-exchange studies with C,DAPS in the mobile phase (16). Functional group selectivities (a)for the substituted benzenes are shown in Table I. Picric acid is also included in this table. Many of the functional group selectivities (relative to -H) are reduced with ClzDAPS present, the exceptions being the phenols. Thus, the modified stationary phase shows a selectivity for anions and phenols. Nonchromatographic Investigations. Alternate methods were sought to determine the nature of the interaction of
3010
--
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986 18.
105-54-4; bromoethyl acetate, 927-68-4; cyanoethyl acetate, 5325-93-9; benzonitrile, 100-47-0; methyl benzoate, 93-58-3; chlorobenzene, 108-90-7;bromobenzene, 108-86-1;phenol, 10895-2; benzene, 71-43-2; nitrobenzene, 98-95-3;anisole, 100-66-3; picric acid, 88-89-1; benzoic acid, 65-85-0.
LITERATURE CITED
-
-2.
Figure 9.
AG,,,,, H,O
-
0.
2.
4.
E;
l p I * ’ e j MeOH VS.
In k’a.
at 2.5 mM C,,DAPS in the mobile phase, were made. The best correlation exists with methanol (Figure 9) where R2 = 0.981. Slightly poorer correlations result with dimethylformamide and acetonitrile (R2= 0.890 and 0.755, respectively). The increase in an anion’s k ’ that results from CIzDAPS presence on the stationary phase is of course due to the polar portion of the ClZDAPS. The significant correlation with the single-ion free energies of transfer is evidence that the energetic process responsible for retention is related to an intersolvent transfer. The general correlation with three different solvents indicates that the ion’s leaving water is of major importance in the overall energetics. This affinity of C12DAPS for ions is not without precedent. Others have obtained evidence for ions “salting in” ClzDAPS (36). Other regressions of retention on molecular properties including ionic and molecular polarizabilities, acid/ base strength, nucleophilicity, and electrophilicity were done. In all cases correlations were poor. The similarity between the behavior of solutes toward CI2DAPSmicelles and the C12DAPS-modifiedstationary phase is suggestive of a micellelike environment on the surface. The highly nonlinear dependence of k’on phase ratio shown by the most strongly influenced solutes (Figures 5 and 6), and the fact that the adsorption of the C12DAPSseems to leave a portion of the stationary phase in its native state (Figure 2) are suggestive of C,,DAPS aggregation. This is not unprecedented. In a recent study a nonionic surfactant adsorbed on silica was found to form discrete aggregates or islands for surface coverages between 0.17 and 0.80 (37). The C12DAPS-modifiedsurface shows interesting selectivity. Further studies are under way in micellar systems to attempt to better understand the reasons for this selectivity. Registry No. CI2DAPS,39536-51-1;PIC-, 14789-26-6;OAc-, 71-50-1;NOS-, 14797-55-8;I-, 20461-54-5;SCN-, 302-04-5;NaN03, 7631-99-4; NaOAc, 127-09-3;NaI, 7681-82-5; NaSCN, 540-72-7; ethyl acetate, 141-78-6;ethyl propionate, 105-37-3;ethyl butyrate,
Snyder, L. R.; Kirkland, J. J. I n Introduction to Modern Liqukl Chroma tography, 2nd ed.; Wiley-Interscience: New York, 1979. Yonker. C. R.; Zwier, T.A.; Burke, M. F. J. Chromatogr. 1982, 241, 257. Yonker, C. R.; Zwier, T. A,; Burke, M. F. J. Chromafogr. 1982, 247, 269. Tomlinson, E.; Jefferies, T. M.; Reiley, C. M. J. Chromatogr. 1978, 159,315. Stranahan, J. J.; Deming, S. N. Anal. Chem. 1982, 5 4 , 2251. Cline Love, L. J.; Habarta, J. G.; Dorsey, J. G. Anal. Chem. 1984, 56. 1132A. Armstrong, D. W.; Stine, G. Y. Anal. Chem. 1983, 55, 2317. Nakagawa, T.; Mizunuma, H.; Shibukawa, A,; Uno, T. J. Chromatogr. 1981,211, 1. Nakajima, M.; Kimura, K.; Shono, T. Anal. Chem. 1983, 5 5 , 463. Knox, J. H.; Jurand, J. J. Chromatogr. 1981, 203, 85. Knox, J. H.; Jurand, J. J. Chromatogr. 1981, 218, 341. Alak, A.; Armstrong, D. W. Anal. Chem. 1988, 5 8 , 582. Konig, H. Z . Anal. ChBrn. 1972, 259, 191. Kirchenorova, J.; Farrell, P. G.;Edwards, J. T. J. Phys. Chem. 1976, 8 0 , 1974. Gonenno, A.; Ernst, R. Anal. Biochem. 1978, 8 7 , 28. Weber, S. G.; Tramposch, W. G. Anal. Chem. 1983, 55, 1771. Melander, W. R.; Stoveken, J.; Horvdth, C. J. Chromatogr. 1980, 799, 35. Nahum, A.; Horvith, C. J. Chromatogr. 1981, 203, 53. Bij, K. E.; Horvdth, C.;Melander, W. R.; Nahum, A. J. Chromatogr. 1981, 203, 65. Van Der Houwen, 0. A. G. J.; Sorel, R. H. A,; Hulshoff, A.; Teeuswsen, J.; Indemans, A. W. M. J. Chromatogr. 1981, 209, 393, Muller, S.C.;Pottel, R. I n Solution Behavior of Surfactants; Mittal, K. L., Fendler, E. J.. Eds.; Plenum Press: New York, 1982; Vol. 1. Tramposch, W. G.; Chen, J. C. Paper presented at the Pittsburgh Conference, Atlantic City, NJ, 1981; paper 792. Tanaka, N.; Goodeli, H.; Karger, B. L. J. Chromatogr. 1978. 158, 233. Hansch, C . ; Quinlan, J. E.; Lawrence, G. L. J. Org. Chem. 1988, 33, 347. Tang, M.; Deming, S. N. Anal. Chem. 1983. 55, 425. Wu, G.;Deming, S . N. J. Chromatogr. 1984, 302, 79. Wahland, K.-G.; Beijersten, I.Anal. Chem. 1982, 5 4 , 128. Helfferich, F. ion Exchange; McGraw-Hill: New York, 1962; p 424. Riegelman, S.;Allawala, N. A.; Hrenoff, M. K.; Strait, L. A. J. Colloid Sci. 1958, 73, 208. Rehfeld, S.J. J. Phys. Chem. 1970, 74, 117. Organic Nectroflic Spectral Data : Kamlet, H . J., Ed.; Interscience: New York, 1960; Vol. 1. Fendler, E. J.; Fendler, J. H. J. Phys. Chem. 1972, 76, 1460. Ekwall, P. I n Chemistry. Physics and Applications of Surface Active Substances; Overbeek, J. T. G. Ed.; Gordan and Breach Science: New York, 1967; Vol 11. Bockris, J. 0.; Reddy, A. K. N. I n Modern Nectrochernistry; Plenum Press, New York, 1970; Vol. 1, Chapter 4. Marcus, Y. Pure Appl. Chem. 1983, 5 5 , 977. Tsujll, K.; Mino, J. J. Phys. Chem. 1978. 8 2 , 1610. Levitz, P.; Van Damme, H.; Keravis, D. J. Phys. Chem. 1984, 8 8 , 2228.
RECEIVED for review August 22, 1985. Resubmitted July 9, 1986. Accepted July 15,1986. We wish to thank the Research Corporation and the National Institutes of Health (Grant GM28112) for sunnort of this work.
