Anal. Chem. 1994,66, 3458-3465
Selectivity in Micellar Reversed-Phase Liquid Chromatography: C-18 and C-8 Alkyl Bonded Barry K. Lavine,' Sumar Hendayana, and Jonathan Tetreault
Department of Chemistryl Clarkson University, Potsdam, New York 13699-5810
Employing a set of six vanillin compounds as retention probes, the role of the surfactant-modified stationary phase in the micellar liquid chromatographic (MLC) separation process was investigated. Specifically, the effect of surfactant adsorption on retention and selectivity in MLC was evaluated by comparing the retention behavior of the vanillin compounds in different micellar media. Surfactant adsorption was found to produce distinct changes in the selectivity of the stationary phase. In fact, differences in selectivity between cetyltrimethylammoniumbromide (CTAB) and sodium dodecyl sulfate (SDS)micellar reversed-phase liquid chromatography were found to be due to the differing nature of the SDS and CTAB bonded phase association. For SDS, the hydrophobic tail is associated with the bonded alkyl phase, with the sulfate head group oriented away from the alkyl bonded phase surface. Adsorption of SDS in the manner described would lead to the formation of a hydrophilic layer on C-18 and C-8, which would explain the superior resolution achieved with SDSfor the vanillin compounds, which are also hydrophilic. For CTAB adsorbed on C-18, the nitrogen head group is probably incorporated partially or wholly into the bonded phase because of hydrophobic interactions between the methyl nitrogens and the bonded phase. Incorporation of CTAB in the manner described would ensure that much of the hydrophobic character of the original bulk phase is retained, which could also explain the longer retention time of the vanillin compounds with CTAB micellar mobile phases. The field of micellar liquid chromatography (MLC) has experienced unprecedented growth since the first report on the use of aqueous micellar solutions as mobile phase in reversed-phase liquid chromatography (RPLC) by Armstrong and Henry in 1980.' Interest in MLC can be attributed to the unique selectivities exhibited by micellar mobile phases.293 Micelles can organize and compartmentalize solutes at various sites within a surfactant a ~ s e m b l ythe ; ~ location of the solute in the assembly is determined by the nature of the solute and the surfactant system used. Each solubilization site in/on the micelle is unique, i.e., the properties of fluidity, acidity, and polarity vary from one site to the next and are distinctly different from those of the bulk solvent. The ability of micelles to selectively solubilize and interact with solute molecules is believed to be the basis of separation (1) Armstrong, D.; Henry,
S.J. J. Liq. Chromatogr. 1980, 3, 657-662.
(2) Armstrong, D. W.; Hinze, W. L.; Bui, K. H.; Singh, H. N. Anal. Lett. 1981, 14, 1659-1667. ( 3 ) Weinberger, R.;Yarmchuk, P.; Cline-Love, L. J. Anal. Chem. 1982,51,15521558. (4) Hinze, W. L.; Singh, H. N.; Baba, Y.;Harvey, N. G. Trends Anal. Chem. 1984, 3, 193.
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Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
CHO
Orthovanillin
Coumarin
CHO
CH 0
0 CH3
OH
I
Isovanillin
Ethylvanillin
CHO
COOH
I
I
p o c H 3
pKal = 4.5 pKa2 = 9.4
OH
OH
Vanillic acid
Vanillin
Flgure 1. Vanillin test mlxture. The pK. values are from ref 8.
in MLC.5 However, surfactant molecules readily adsorb on bonded stationary phases, e.g., C-18, (2-8, or cyanopropyl. Because many stationary phase properties are altered by the process of surfactant adsorption,6v7 the modification of the stationary phase by adsorbed surfactant can have profound implications with regard to retention and selectivity in MLC. Employing a set of six vanillin compounds as retention probes (see Figure I), the role of the surfactant-modified stationary phase in the MLC separation process was investigated. Specifically, the effect of surfactant adsorption on retention and selectivity in MLC was evaluated by comparing the retention behavior of the vanillin compounds in different micellar media. Surfactant adsorption was found to produce distinct changes in the selectivity of the stationary phase. Adsorption of sodium dodecyl sulfate (SDS) imparted a more hydrophilic character to the C- 18 and C-8 reversed-phase material, whereas cetyltrimethylammonium bromide (CTAB) ( 5 ) Hinze, W. L. In W. L. Hinze and D. W. Armstrong (Editors), OrderedMedia
in Chemical Separations; ACS Symposium Series 342; American Chemical Society: Washington, DC, 1987; pp 2-82. (6) Berthod, A.; Roussel, A. J . Chromatogr. 1989, 449, 349-360. (7) Borgerding, M. F.; Hinze, W. L.; Stafford, L. D.; Fulp, G. W.; Hamlin, W. C. Anal. Chem. 1989, 61. 1353-1358. (8) Kortum, G.; Vogel, W.; Andrussow, W. Dissociation Constants of Organic Acids in Aqueous Solutions; Butterworth: London, 1961.
ooo3-27oo~~4io3~~34sa~o4.so~o
@ 1994 American Chemical Society
adsorption on C-18 produced a more bulk hydrophobic stationary phase. Models depicting the structure of the SDS and CTAB surfactant-coated stationary phases are proposed from the retention data, and these models are in good agreement with solid state NMR data9 on SDS- and CTABmodified C- 18. EXPERIMENTAL SECTION The vanillin compounds were obtained from Aldrich and were used as received. Stock solution of the test solutes were prepared in methanol and then diluted to the appropriate working concentration (550 pg/mL) using 50% methanol in water. The surfactants, sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB), were purchased from BDH Chemicals, Ltd., and prior to chromatographic use were purified using Kratohvil's procedure,lO Le., the surfactant was dissolved in ethanol and then activated charcoal was added to the ethanolic solution. After the charcoal was separated from the mother liquor by filtration, the surfactant was recrystallized from the ethanol and dried in an oven at 65 OC. The micellar solutions were prepared in HPLC grade distilled water (J. T. Baker). The methanol-water mobile phases were also prepared with HPLC grade solvents. Both the micellar solutions and the methanol-water mobile phase solutions were filtered twice with a 0.45 pm Nylon membrane filter (Rainin Instruments, Woburn, MA) to remove particulate matter. Prior to use, the solutions were degassed and the pH was adjusted to 3 with hydrochloric acid to prevent ionization of the polar solutes in the mobile phase solutions. All high-performance liquid chromatographic (HPLC) measurements were made with a Rainin 81-20 M analytical HPLC system which incorporates two Rainin H P pumps (Rainin Instruments), an Apple Macintosh SE computer as the controller, a Model 7125 Rheodyne injection valve, and a Rainin Dynamax mixer. The detector was a Knauer variable-wavelength UV-visible spectrometer (Berlin, Germany). The analytical column was either an Apex I 5-pm (2-18 (10 cm X 4.6 mm i d . ) or an Apex 15-pm C-8 (10 cm X 4.6 mm i.d.). The C-18 and C-8 columns used in this study were purchased from Jones Chromatography (Golden, CO) and were made from the same 5 pm silica support. The analytical column and mobile phase reservoir were water jacketed and temperature controlled. A silica precolumn (Rainin Instruments) placed between the injector and the pump saturated the mobile phase with silicates to minimize dissolution of the analytical column packing. Separate columns were used for cationic and anionic surfactants (as well as the methanol-water mobile phases) because of the strong and irreversible adsorption of these surfactants on the stationary phase of the C-18 and C-8 columns. The dead volume of the system was determined by injecting different solutionssuch as methanol, methanol-water, or water onto the Jones columns. This volume, approximately 1.OmL, was used for all k'calculations. k'values determined in this study were averages of at least triplicate determinations. ~
(9) Lavine, B. K.; Cooper,W. T.; He, Y.; Hendayana, S.; Han, J. H.; Tetreault, J. J . Colloid Interfac. Sci. 1994, 165, 497-504. (10) Hsu, W. P. Micellar Growth in Solutions of Synthetic and Biological Surfactants. Ph.D. Thesis. Clarkson University, Potsdam, NY, 1985.
2
20%MeOH
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Figure 2. Chromatograms of the test mixture on Apex I C-18 with the following mobile phases: (a) 20% methanol In water, (b) 0.02 M SDS, and (c) 0.02 M CTAB. Flow rate was 1.O mL/min; pH of each mobile phase was 3; the temperature of the column was 25 O C for methanol-water and SDS, 30 OC for CTAB. The chromatograms were reproduced using Cord 3.0.
Deviations in individual capacity factor values were never greater than 5%. All k'measurements were made at a flow rate of 1.O mL/min. The k'values were measured at 25 OC for SDS and 30 OC for CTAB. (Since the Kraft point of CTAB is 23 OC, it was necessary to carry out the CTAB studies at higher temperature.) RESULTS AND DISCUSSION A series of chromatograms were run to illustrate some of the advantages associated with micellar mobile phases. The test mixture consisted of six compounds: vanillin, the principal flavor component in vanilla extract, and isomers or analogues of vanillin. Food chemists have long been interested in isolating and quantifying these compounds in a variety of sample matrices which, in part, was our motivation in choosing these compounds to study via MLC. Figure 2 shows the separation of the mixture on Apex I C- 18with the followingmobile phases: methanol-water, 0.02 M SDS,and 0.02 M CTAB. The acidified 20% methanol/ 80% water mobile phase has been recommended for the separation of vanillin from the other compounds in extract of vanilla on C-18 by Supelco.ll For the anionic surfactant, the optimum separation is achieved with a 0.02 M SDS mobile phase. For the cationic surfactant, 0.02 M CTAB does not yield the optimum separation. However, it is representative of the type of separation which can be obtained for the test mixture using CTAB micellar mobile phases and is shown here for comparative purposes. Several things are apparent from an examination of the data. First, the test mixture is completely separated by the (1 1) Supelco. Technical Report, Vol. XI, No. 4, 1992; pp 15-16.
Analyfical Chemistry, Vol. 66,No. 20, October 15, 1994
3458
Table 1. Varlatlon of Efficiency. and Asymmetryb wHh Moblle Phase on C-18 Column 20180 CHpOH/H20 0.02 M SDS 0.02 M CTAB (PH = 3) (PH = 3) (PH = 3) compound N B/A N B/A N B/A
vanillic acid vanillin orthovanillin ethylvanillin isovanillin coumarin
684 1113 106 3897 834 2559
1.25 0.80 6.00 1.00 0.46 1.00
528 576 206 561 578 271
2.19 2.38 3.80 2.08 3.34 3.50
169 289 11 514 394 147
4.31 4.12 7.87 2.97 4.30 4.34
The Foley-Dorsey method12was used to compute N a n d determine the asymmetry of each peak. Plate count values reported in this study were the averages of at least triplicate determinations.
*
SDS micellar mobile phase but not by the hydroorganic or CTAB mobile phase. Second, the efficiency of the chromatographic process is poor for all three mobile phases (see Table l ) , and this implies that thevariability in the resolution of the test mixture among the three mobile phases is due to differences in selectivity. Third, the retention time of four of the six vanillin compounds is greater with 0.02 M CTAB (cmc of CTAB is 0.9 mM at 30 “ C in distilled water) than that with 0.02 M SDS (cmc of SDS is 8 mM at 25 “ C in distilled water), even though the CTAB mobile phase contains significantly more micelles, which suggests that the interaction between each of these four compounds and the stationary phase is greater with CTAB-modified (2-18 than that with SDS-modified (2-18. To better understand the reasons for the differences in selectivity among the three mobile phases, we first measured k’ on Apex I C-18 for the vanillin compounds using five different SDS mobile phases: (1) 0.01 M SDS, (2) 0.02 M SDS, (3) 0.03 M SDS, (4) 0.04 M SDS, and (5) 0.05 M SDS. Next, k’was correlated to surfactant concentration using the equation developed by Cline-Love and Arunyanart: l 3
where [MI is the concentration of surfactant, K2 is the solutemicelle binding constant per monomer of surfactant, 0 is the chromatographic phase ratio, [L,] is the concentration of ligate on the stationary phase, and Kl is the solute-stationary phase binding constant. A plot of llk’versus [MI should result in a straight line. In fact, excellent linearity was observed for all six compounds, i.e., r2> 0.98. A similar study with CTAB was undertaken, and again the agreement between 1/ k’ and [MI was very good, Le., r2 > 0.98. K2 and 8[L,]K1 (the reciprocal of the intercept) for the six vanillin compounds are listed for SDS in Table 2 and for CTAB in Table 3. (& is obtained by dividing the slope by the intercept.) For the hydroorganic mobile phase, the interaction of the vanillin compounds with the mobile and the stationary phase was evaluated using the following linear relationship: In k’= In k, -B@
(2)
Ink, is the natural logarithm ofthe capacity factor of a solute (12) Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983,55, 73C-737. (13) Arunyanart, M.; Cline-Love, L. J. Anal. Chem. 1984, 56, 1557-1561.
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Analytical Chemistry. Vol. 66,No. 20, October 15, 1994
Table 2. K2 and O[L& for the Vanlllln Compounds In Sodlum Dodecyl Sulfate on C-18. compound ~[L~IKI K2
vanillic acid vanillin isovanillin ethylvanillin orthovanillin coumarin
4.2 f 0.18 9.6 f 0.92 10.5 f 0.99 17.5 f 1.23 31.2 f 1.95 41.7 f 3.13
28.9 f 2.07 39.5 f 6.12 37.6 f 4.75 45.7 f 3.65 41.3 f 3.0 59.0 f 4.60
Determined on an Apex IC-18 column (10 cm X 4.6 mm i-d.); flow rate of 1.0 mL/min; pH = 3. Uncertainties in 8[L,]K1 and K2 were determined from the statistical parameters of the least-squares fitting and from propagation of error. Table 3. K2 and e[L& for the Vanlllln Compounds In Cetyltrlmethylammonlum Bromlde on C-18. compound eLs1K1 K2
vanillic acid vanillin isovanillin orthovanillin ethylvanillin coumarin
16.9 f 1.43 17.2 f 1.18 17.4 f 1.21 26.5 f 1.40 36.0 f 2.59 29.5 f 3.48
58.6 f 5.23 32.7 f 2.73 29.7 f 2.70 31.7 f 2.31 58.8 f 4.47 37.3 f 5.30
Determined on an Apex I C-18 column (10 cm X 4.6 mm i.d.); flow rate of 1.0 mL/min; pH = 3. Uncertainties in 8[L,]K, and K2 were determined from the statistical parameters of the least-squares fitting and from propagation of error. Table 4. Kw and Slope for the Vanlllln Compounds In Methanol-Water Moblle Phase on C-18. compound KW slope
vanillic acid isovanillin vanillin orthovanillin ethylvanillin coumarin
31.8 f 2.5 36.8 f 1.5 35.4 f 1.4 70.9 f 2.1 101 f 4.0 103 f 2.1
-0.035 -0.035 -0.033 -0.034 -0,037 -0.035
f 0.002 f 0.001 f 0.001 f 0.001 f 0.001 f 0.001
Determined on an Apex I C-18 column (10 cm X 4.6 mm id.); flow rate of 1.O mL/min; pH = 3. Uncertainties in Kw and the slope were determined from the statistical parameters of the least-squares fitting and from propagation of error.
in a purely aqueous medium and is a measure of the interaction of the solute with the stationary phase. @ is the volume percentage of organic modifier (i.e., methanol) in the mobile phase. B is directly related to the solvent strength of the organic modifier but is used here as a measure of the strength of interaction between the solute and the mobile phase. For C-18 alkyl bonded phases, this equation14 is valid when the modifier content is in the range of 10-5076. Retention data for the vanillin compounds were generated using methanol-water mobile phases containing 10-50% methanol. The data were fitted to eq 2. Over this range of modifier content, reasonably good linear retention behavior was observed (r2 > 0.98) for the vanillin compounds. Table 4 lists the values of K, and B for each compound. In Tables 2-4, the vanillin compounds are listed in the order that they elute from the C-18 column. For SDS (see Table 2), elution order clocks 0[LS]K1,implying that solutestationary interactions define the selectivity of the separation. (If the micelle-solute binding constant defined the selectivity of the separation, K2 would be expected to decrease as the retention time of the compounds increased, which is not the (14) Kaliszan, R.:Osmialowski, K. J . Chromatogr. 1986, 352, 141-150.
Before SDS adsorption
0.03M
After SDS adsorption
20%MeOH
0.04M
SDS
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Flgure 3. Chromatograms of the test mixture on Apex I C-18 with the following micellar mobile phases: 0.02 M SDS, 0.03 M SDS, 0.04 M SDS, and 0.05 M SDS. Flow rate was 1.0 mL/min; pH of each mobile phase was 3; column temperature was 25 OC. The chromatograms were reproduced using Cord 3.0.
case.) Solute-stationary phase interactions also appear to play an important role in the separation process with the methanol-water mobile phase (see Table 4). (If solute-mobile phase interactions defined the selectivity of the separation, the slope would be expected to decrease as the retention time of the compound increased, which is not the case.) For CTAB, the elution order can be correlated to changes in both e[L,]K1 and K2 (see Table 3), which suggests that it is a combination of the interaction of the micelles and the surfactant-modified stationary phase with the solute that defines the selectivity of the separation of the test mixture on C-18 when a CTAB mobile phase is used. Although SDS adsorption enhances the selectivity of the stationary phase toward thevanillin compounds, SDS micellesolute interactions also contribute to the selectivity of this separation. For example, SDS micelles interact morestrongly with vanillin than with isovanillin, as evidenced by the greater K2 value for vanillin, and this interaction is responsible, at least in part, for the 6cr or base line resolution of these two compounds. Nevertheless, the successful separation of the vanillin compounds with the 0.02 M SDS mobile phase is primarily due to solute-stationary phase interactions, which is also the reason why the separation of thevanillin test mixture is more favorable at lower SDS concentrations (see Figure 3). At higher SDS concentrations, there are more micelles in the mobile phase, and the net result is that micelle-solute interactions play a greater role (which is not altogether beneficial) in the separation of the vanillin compounds. [Note that surfactant stationary phase interactions are independent of micelle concentration because the amount of surfactant
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Figure 4. Chromatograms of the test mixture on an Apex I C-18 column using a 20% methanol in water mobile phase (pH = 3,25 'C, flow rate of 1.OmL/min) prior to and after separationswith SDS micellar mobile phases. Since it is generally accepted that retention of ail solutes in RPLC decreases as the polarity of the stationary phase increases, the decrease in retentiontime is probably due to an increase in the polarity of the C-18 phase as a resuit of SDS adsorption. The chromatograms were reproduced using Cord 3.0.
adsorbed on the stationary phase remains constant once the concentration of surfactant in the mobile phase is above the cmc.15] The observed differences in selectivity between CTAB and SDS micellar RPLC can be explained by the differing nature of the SDS and CTAB bonded phase association. In the case of SDS, it appears that the hydrophobic alkyl tail of the surfactant is associated with the (2-18 bonded phase, with the sulfate group oriented away from it.9 SDS incorporated into the bonded phase in the manner described, Le., with its polar head group projecting away from the C-18 bonded phase toward the mobile phase, would greatly affect the polarity of the bonded phase (see Figure 4 and Table 5 ) and would also lead to the formation of a hydrophilic layer, which would explain the superior resolution achieved by SDS for the vanillin compounds, which probably undergo some type of selective hydrogen-bonding interaction with the layer. The increase in polarity of the C- 18 bonded phase as a result of SDS adsorption is evident from an examination of Figure 4, which shows chromatograms of the test mixture on C-18 using a 20% methanol in water mobile phase prior to and after separations involving SDS micellar mobile phases. The shorter retention times of the vanillin compounds on the SDS-modified C-18 are due to an increase in the polarity of the stationary phase that has occurred as a result of surfactant adsorption. In addition, the correlation coefficient, r, for log KI(SDS) and log of the octanol-water partition coefficient or log P (which is a well-known index of hydrophobicity) is 0.166 (see Table 5 ) , which is substantively smaller than the correlation coefficient for log Kw vs log P. Clearly, the smaller log P correlation coefficient suggests that a decrease in the hydrophobicity of the bonded phase has occurred as a result of surfactant adsorption. (15) Hung,
C.T.;Taylor, R. B.J . Chromofogr. 1981, 209, 175-190.
Analytical Chemistfy, Vol. 66,No. 20, October 15, 1994
3481
Before CTAB adsorption
Table 5. Solute Hydrophoblcky as Represented by the of the OctanoCWater Partillon Coefficient (log P) versus log K, or log K, tor the Vanlllln Compounds on C-18 compound log P log Kwc log Kl(sDsf log K I ( C T A B ~
isovanillin vanillin orthovanillin coumarin vanillic acid ethylvanillin
0.97 1.21 1.37 1.39 1.43 1.88
1.57 1.55 1.85 2.01 1.50 2.00
1.02 0.98 1.49 1.62 0.62 1.24
1.24 1.24 1.42 1.47 1.23 1.56
a log P is a well-known index of hydrophobicity. log P values were obtained from the CLOGP Program, Medicinal Chemistry Project, Pomona College, Claremont, CA. c The correlation coefficient for log P and log Kw is 0.638. The correlation coefficient for log P and log K l ( s ~ s ) is 0.188. The decrease in r is not surprising since SDS adsorption imparts significant hydrophilic character to the C- 18 bonded phase. e The correlation coefficient for log P and log KI(CTAB) is 0.758. The high correlation between log P and log K ICTAB) is not altogether surprising since incorporation of CTAB into &e bonded phase in the manner described would ensure that much of the hydrophobic character of the original bulk C-18 phase is retained.
After CTAB adsorption
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The hydrophilic layer that is formed on the stationary phase would also affect the penetration depth of the vanillin compounds into the bonded phase because of strong hydrogenbonding interactions between these compounds and the layer. The expected result would be a decrease in hydrophobic interactions between the vanillin compounds and the C- 18 stationary phase. Clearly, this is part of the answer as to why the retention time of the vanillin compounds is greater with 0.02 M CTAB than with 0.02 M SDS or 0.03 M SDS. (The concentration of micelles in the 0.02 M CTAB solution is approximately the same as that in the 0.03 M SDS solution using the cmc and aggregation number data on page 224 of ref 5.) This simplified model of the SDS-modified C-18 bonded phase has also been used by Armstrong and BerthcdI6 to explain poor stationary phase mass transfer in MLC. According to Armstrong, a hydrophobic solute has to traverse the hydrophilic boundary layer formed by the sulfate head groups, ions, and associated waters in order to gain access to or to leave the stationary phase. This boundary layer can, therefore, obstruct mass transfer between the bulk solvent and the bonded phase. Furthermore, both the thickness and the viscosity of the polar layer can be increased by adsorption of more surfactant onto the bonded phase. Any or all of these factors can contribute to poor stationary phase mass transfer in SDS micellar RPLC. For CTAB, the nitrogen head group is probably oriented closer to the silica surface due to hydrophobic interactions between the N-methyl groups and the bonded phase.9 In fact, the nitrogen head group of CTAB is probably incorporated partially or wholly into the bonded phase, giving rise to a modified bulk phase that is significantly denser. Incorporation of CTAB in the manner described would ensure that much of the hydrophobic character of the original bulk stationary phase is retained. This conclusion is reinforced by Figure 5, which shows chromatograms of the test mixture on C- 18 using a 20% methanol in water mobile phase prior to and after separations involving CTAB micellar mobile phases. The longer retention times of thevanillin compoundson the CTABmodified C- 18 suggests that an increase in the hydrophobicity (16) Armstrong, D. W.; Ward, T. J.; Berthod, A. Anal. Chem. 1986,58,579-582.
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Figure 5. Chromatograms of the test mlxture on an Apex I C-18 column using a 20% methanol In water mobile phase (pH = 3, 25 OC, flow rate of 1.0 mL/mln) prior to and after Separations with CTAB micellar mobile phases. The increase in retention time is probably due to an increase in the hydrophobicity of the (3-18 phase as a result of CTAB adsorption. The chromatograms were reproduced using Core1 3.0.
of the bonded phase has occurred as a result of surfactant adsorption. We believe that a small amount of CTAB adsorbs “head-first” on the bonded phase. (The technique of solid state NMR which was used in a previous studyg to examine the nature of surfactant bonded phase associations in MLC is probably not sufficiently sensitive to pick up the signal generated by the small amount of CTAB monomer which is adsorbed head-first on (2-18.) The CTAB that is adsorbed head-first is probably irreversibly adsorbed on (2-18. Hence, it is conceivable that a sizable fraction of CTAB irreversibly sorbed on the C- 18 exists in the so-calledhead-first orientation, which would explain the greater retention time of the vanillin compounds with a 20% methanol in water mobile phase after, rather than prior to, separations involving CTAB micellar mobile phases because of the decrease in the silanol activity of the bonded phase. In other words, the decrease in silanol activity due to head-first adsorption by some CTAB monomers might compensate for the increase in the polarity of the C-18 phase due to the presence of N-alkyl head groups at the surface of the stationary phase, which would explain the observed differences in retention times for the chromatograms shown in Figure 5 . The correlation coefficient for log P and log K ~ ( C T A B )is 0.758, whereas r is 0.638 for log Kw and log P (see Table 5). However, the correlation coefficients are small enough that we judge them to be approximately equal, which reinforces our conclusion that much of the hydrophobic character of the original bulk phase is retained because of the manner in which CTAB is adsorbed onto the bonded phase. Models that depict the structure of CTAB- and SDS-modified (2-18 are shown in Figure 6. Our studies of the modification of bonded alkyl phase by the adsorption of SDS or CTAB monomers were not limited to C-18 alkyl bonded phases. In Figure 7, the separation of the vanillin test mixture on Apex I C-8 is shown with the same
2-
2-
SDS adsorbed
CTAB adsorbed
Figure 8. Models deplctlng the structures of SDS- and CTAB-modified C-18. ~
Table 7. K, and Slope for the Vanlllln Compounds In 20% Methanol In Water on C-8.
compound
K W
slope
vanillic acid isovanillin vanillin orthovanillin ethylvanillin coumarin
15.7 f 1.6 21.1 f 0.42 20.7 f 0.41 41.2 f 1.2 54.7 f 1.1 59.7 f 1.8
-0.030 f 0.004 -0.030 f 0.0005 -0.029 f 0.0007 -0.030 0.0008 -0.033 f 0.0007 -0.032 f 0.0008
*
a Determined on an Apex I C-8 column (10 cm X 4.6 mm id.); flow rate of 1.0 mL/min; pH = 3. Uncertainties in Kw and slope were determined from the statistical parameters of the least-squares fitting and from propagation of error.
O.02M SDS on C-8
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Figure 7. Chromatograms of the test mixture on Apex I C-8 with the following three moblle phases: 20% methanol in water, 0.02 M SDS, and 0.02 M CTAB. Flow rate was 1.0 mL/mln; pH of each mobile phase was 3; column temperature was 25 OC for methanol-water and SDS, 30 OC for CTAB. The chromatograms were reproduced using Corel 3.0. Table 8. Ka and e[L,]K, for the Vanlllln Compounds In Sodium Dodecyl Sulfate on C-8.
compound vanillic acid vanillin isovanillin ethylvanillin orthovanillin coumarin
~[LIKI
* *
4.4 0.4 12.5 1.6 14.2 f 2.0 24.1 f 0.6 40.5 f 1.6 49.8 f 1.2
K2 21.2 f 2.9 41.1 f 8.1 37.9 f 7.8 58.9 f 1.6 66.6 f 2.9 86.0 f 2.4
a Determined on an Apex I C-8 column (10 cm X 4.6 mm id,); flow rate of 1.0 mL/min; pH = 3. Uncertainties in 8[L,]K1 and K2 were determined from the statistical parameters of the least-squares fitting and from propagation of error.
three mobile phases used in the C-18 study: 0.02 M SDS, 0.02 M CTAB, and 20% methanol in water. Several things are apparent from an examination of the data. First, the test mixture is completely separated by the SDS micellar mobile phase but not by the hydroorganic mobile phase. As with C-18, solute-stationary phase interactions again appear to play an important role in the separation of the test mixture with these two mobile phases (see Tables 6 and 7). Second, the retention time of the vanillin compounds with the 0.02 M SDS mobile phase is longer on C-8 than on
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30
Flgure 8. Chromatograms of the test mlxture on a E 1 8 and C-8 Apex I column with a 0.02 M SDS mobile phase. Flow rate was 1.O mL/mln; pH of each mobile phase was 3; column temperature was 25 OC. The chromatograms were reproduced uslng Corel 3.0.
(2-18 (see Figure 8), even though the C-18 phase has a higher carbon loading. (According to the manufacturer, the carbon loading of Apex I C-18 is 13.396, whereas it is only 9.0% for Apex I (2-8.) Since the (2-18 and C-8 columns used in this study were the same except for the bonded phase, the longer retention time of the vanillin compounds on C-8 can probably be attributed to lower SDS sorption on octylsilane because of the higher bonding chain density of the C-8 phase used in this study. (Berthod, Ginnard, and Gonnet have also reported that significantly more SDS adsorbs on C-18 than on C-8 alkyl bonded phases.17) Lower SDS sorption would result in a more hydrophobic phase. Since the hydrophilic layer formed by sorbed SDS is responsible for the superior resolution (17) Berthod, A.; Girard, I.; Gonnet, C. Anal. Chem. 1986, 58, 1356-1361
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I
Table 8. K1 and e[L& for the Vanlllln Compounds In Cetyltrhnethyiammoniwn Bromide on C-8*b compound e[LslK~ K2
O.OZM CTAB on C-8
vanillin isovanillin vanillic acid coumarin orthovanillin ethylvanillin
1
46.4 f 2.3 41.3 f 1.5 126.2 f 24.2 43.9 & 1.3 36.5 i 3.6 69.0 i 2.9
20.4 f 0.8 21.2 f 3.2 31.9 i 1.2 25.1 i 0.6 21.3 f 2.2 38.6 f 1.5
Determined on an Apex I C-8 column (10 cm X 4.6 mm i.d.); flow rate of 1.0 mL/min; pH = 3. Uncertainties in e[L,]Kl and K2 were determined from the parameters of the least-squares fitting and from ropagationof error. Thecorrelation between e[L& and elutionorder !or the vanillin compounds is stronger on C-8 than it is on C-18. (If vanillic acid is removed from the table, the correlation between 8[L,]Kl and elution order is very strong.)
0.02MCI'ABon C-18
20% MeOH on C-8
0
2
4
6
8
10
12
14
16
18
20
22
24
26
minutes
L
Y
Figure 9, Chromatograms of the test mixture on a C-18 and C-8 Apex I column with a 0.02 M CTAB mobile phase. Flow rate was 1.0 mL/ min; pH of each mobile phase was 3; column temperature was 30 OC. The chromatograms were reproduced using Corel 3.0.
achieved by the 0.02 M SDS micellar mobile phase for the vanillin test mixture, the decrease in the resolution of the polar test mixture which occurs when a C-8 column is used (see Figure 8) can probably be attributed to lower SDS sorption on octylsilane as well. Third, there is a change in elution order when an Apex I C-8 column is used, in lieu of an Apex I C-18 column, with the 0.02 M CTAB mobile phase (see Figure 9). The retention time of the vanillin compounds with the 0.02 M CTAB mobile phase is also longer on C-8 than on C- 18. On the basis of these two effects, reversals in elution order and longer retention times, we can conclude that differences in the selectivity of CTAB-modified C- 18 and C-8 alkyl bonded phases toward the vanillin compounds are due to the differing nature of the CTAB monomer C-18 and C-8 bonded phase association. In all likelihood, small surfactant aggregates form within the C-8 stationary phase, and these aggregates are responsible for the longer retention times and reversals in elution order of the polar test mixture. (We define the stationary phase in MLC as a ternary combination of silica substrate, bonded organic moiety, and adsorbed surfactant.) These aggregates are probably similar in nature to the surfactant clusters that form in the presence of watersoluble polymers, e.g., poly(ethy1ene oxide) or poly(viny1 pyrrolidone) in aqueous media.I8 Our previous studies19 in MLC have shown that CTAB aggregates exhibit strong selectivity toward phenols and other compounds containing acidic functional groups. This selectivity can be attributed to a secondary chemical equilibrium process involving the transfer of a proton from the ionogenic solute to water molecules in the Stern region of the surfactant aggregate. In fact, a decrease of 0.5-3.0 in the pKa value of a dissociable amphiphile can occur upon incorporation of the (18) Goddard, E. D. Colloids Surf. 1986, 19, 255-300; 301-329. (19) Lavine, B. K.; White, A. J.; Han, J. H . J . Chromatogr. 1991, 542, 29-40.
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20% MeOH on C-18
1
8
l
0
2
4
~
6
8
t
10 12 minuus
~
14
16
l
18
20
22
~
24
26
Flgure 10. Chromatograms of the test mixture on a C-18 and C-8 Apex I column with a 20% methanol in water mobile phase. Flow rate was 1.0 mL/min; pH of each mobile phase was 3; column temperature was 25 OC. The chromatograms were reproduced using Corei 3.0.
guest molecule into a cationic micelle.20 The aforementioned acid-base effect would explain the strong interaction of vanillic acid with the CTAB-modified C-8 phase (see Table 8). In other words, a decrease in the pK, of the vanillic acid probably occurs as a result of its incorporation into the CTAB aggregates that we postulate to exist within the C-8 bonded phase. Theexistenceof surfactant aggregates within the C-8 stationary phase would also explain the longer retention times and the differences in elution order between C- 18 and C-8 bonded phases which occur when CTAB micellar mobile phases are used. The importance of solutestationary phase interactions in the separation of the polar test mixture on C-8 with CTAB micellar mobile phases (see Table 8) further reinforces this conclusion. Although lower CTAB sorption on the C-8 bonded phase could also be responsible for the longer retention time of the vanillin compounds, it cannot be thecauseof the reversals in the elution order of the test mixture. Because of the greater silanol activity of the C-8 phase, the possibility that silanol groups are responsible for the reversals through their interactions with the vanillin compounds was also considered. If the silanol groups were responsible for the (20) Underwood, A. L. Anal. Chim. Acta 1982, 140, 89-97.
~
~
reversals, then there would be a marked difference in the retention behavior of the vanillin compounds on C- 18 and C-8 with a methanol in water mobile phase. However, the vanillin compounds on C-18 and C-8 exhibit similar retention behavior with a 20% methanol in water mobile phase (see Figure lo), as well as with other methanol-water mobile phases, e.g., 30%, 40%, or 50% methanol in water solvent mixture. Hence, the reversals in the elution order of the test mixture must be due to the differing nature of the CTAB monomer (2-18 and C-8 bonded phase association. CONCLUSION Solute-stationary phase interactions in MLC are very important. The differing nature of the SDS and CTAB bonded phase association is largely responsible for the observed differences in selectivity between CTAB and SDS micellar reversed-phase liquid chromatography. Perhaps some of the reported differencesZ1J2in selectivity between MLC and (21) Khaledi, M. G. Anal. Chem. 1988, 60, 876-887. (22) Khaledi, M. G.; Peuler, E.; Ngwainbi, J. N. Anal. Chem. 1987, 59, 27382747.
reversed-phase HPLC with hydroorganic mobile phases are also due in some measure to the modification of the stationary phase by adsorbed surfactant. ACKNOWLEDGMENT
This work was supported by a grant from the Reynolds Tobacco Company. Jonathan Tetreault acknowledges the generous support of the National Science Foundation (NSFREU awardee), and Sumar Hendayana acknowledges the financial support of the Ministry of Education of Indonesia. Critical discussions on amphiphilic association structures and surfactant adsorption with Stig Friberg, Petr Zuman, and Josip Kratohvil of the Chemistry Department of Clarkson University are also acknowledged. Received for review February 10, 1994. Accepted June 30, 1994." *Abstract published in Advance ACS Absrracrs, August IS, 1994.
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