744
Anal. Chem. 1986, 58, 744-747
Reversed Micellar Mobile Phases for Normal-Phase Chromatography Maria A. Hernandez-Torres, John S. Landy,' and John G. Dorsey* Department of Chemistry, University of Florida, Gainesville, Florida 32611
Reversed micelles of aerosol OT, formed in hexane, can be used in place of polar organic solvents to control the strength of normal-phase mobile phases. The effect of surfactant concentration on retention, both below and above the critical micelle concentratlon, is Investigated and found to be similar to the behavior of normal micelles in reversed-phase chromatography. A unique characterlstlcof reversed micelles is their ability to tightly bind water in the lnterlor of the micelle. This then serves to greatly reduce solute retention dependence on water content of. the moblie phase. Retention of polar test solutes is shown to be approximately constant above a water concentration of ca. 0.1 % (vlv). Chromatographic efficiency of reversed micellar mobile phases is also discussed.
Micelle structures have been heavily studied by physical chemists and biochemists for many years ( I ) . It is only recently, however, that many analytical chemists have realized that micellar systems can often be advantageously applied to chemical analysis. These systems offer the unique capability to solubilize hydrophobic compounds in aqueous solution and also to organize reactants on a molecular level so as to increase the proximity of reagents and analytes. These properties offer advantages in virtually every field of chemical analysis. A recent tutorial on the application of micellar systems to chemical analysis has appeared (2) as well as a comprehensive review of analytical applications of organized molecular assemblies (3). One of the areas of greatest interest has been in the use of micellar solutions as mobile phases in reversed-phase liquid chromatography. Since the first report by Armstrong and Henry (4),several groups have been actively investigating the use of micellar mobile phases to overcome inherent limitations of hydroorganic mobile phases. One of the most dramatic examples is the ability to perform a gradient elution separation with no column reequilibration necessary at the end of the gradient (5,6).Increasing the concentration of micelles does not change the structure or composition of a reversed-phase stationary phase as does a hydroorganic gradient. Micellar gradient elution has also recently been shown to be highly compatible with electrochemical detection, in marked contrast with the very limited compatibility with hydroorganic mobile phases (7). Cline Love and co-workers have shown a highly useful property of micellar mobile phases allows the direct injection of serum onto a reversed-phase column for the monitoring of drugs and metabolites, with no protein precipitation or subsequent pressure buildup (8,9). Chromatography with micellar mobile phases is also extremely information rich, providing data for the determination of micelle/solute partition coefficients as well as information about the structure of the micellar assembly itself (IO,11). To date, virtually all of the analytical applications of micellar solutions have utilized normal micelles, Le., micelles formed in polar solvents. When surfactants are dissolved in Present address: Revlon Health Care Group, 1 Scarsdale Rd., Tuckahoe, NY 10707. 0003-2700/86/0358-0744$01.50/0
nonpolar solvents the micellization process will often still occur, depending on the choice of surfactant, but now the polar head groups are oriented toward the interior of the aggregate, and the hydrophobic chains are in contact with the solvent. These reversed micelles are more complex, and less studied and understood than normal micelles (12,13), but offer the same potential advantages to analysis as do normal micelles: that is the ability to solubilize polar species which would be excluded from normal micelles, and thus to alter their analytical properties. The only chromatographic application of reversed micelles has been as a mobile phase in TLC, but this was on reversed-phase plates (14). All of the column chromatographic uses of micellar mobile phases have been in a reversedphase mode. This is not in variation with usage utilizing traditional mobile phases, however. It has been recently estimated that approximately 72% of modern LC is performed in a reversed-phase mode (15). That this figure is so high is both a reflection of the wide applicability of the technique and also is indicative of a fundamental problem of normal-phase separations. Normal-phase chromatography would often be the separational mode of choice for the analysis of polar compounds, but there is a serious problem with day-to-day retention reproducibility. Since the stationary phase is polar, the strongest mobile phase is a polar solvent. This means that any variation in water content of the mobile phase can cause dramatic differences in retention, and as the water content of typical organic solvents can vary even with room humidity, it is extremely difficult to control. This change in solvent strength alone will cause retention variations with bonded-phase columns, but in adsorption chromatography with silica or alumina stationary phases, the problem is actually twofold. Here, along with the change in the strength of the mobile phase there is also a change in the activity of the silica or alumina stationary phase itself. Caude et al. have extensively studied the influence of water on retention data in liquid-solid chromatographic systems (16-18) and found in one instance that capacity factors changed up to 1 order of magnitude as the water content of the mobile phase changed from 100 to 600 ppm (16). Somewhat surprisingly the use of adsorption (mainly silica gel) columns still dominates bonded polar phases by approximately 2:l (15). The use of reversed micelle mobile phases for normal-phase chromatography may offer a unique solution to this problem. One of the more interesting aspects of reversed micelles is their ability to solubilize water in the interior of the micelle structure. In many instances water actually promotes the formation of larger and more stable reversed micelles. Indeed, in the absence of traces of water, aggregation is sometimes precluded (19). Here we report the application of reversed micellar mobile phases for normal-phase chromatography, using both silica and bonded-phase columns, and discuss retention changes as a function of water content of the mobile phases.
EXPERIMENTAL SECTION A Spectra-Physics (Santa Clara, CA) 8700 ternary liquid chromatograph was used and was equipped with a Model 7125 Rheodyne (Cotati, CA) sample injection valve with a 10-pL loop, 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
745
- 180
- 150
- I 70 - I60
--\
- 150 -
I40
130
- 120 - I10 -
100
-090
-
-
080 070
L9
s
- 060 - 050 - 040 -
030
- 020 - 010 -50
-40
-30
-20
-10
LOG [AOT]
Flgure 1. log k'vs. log [AOT]: column, Ultrasphere-Si; mobile phase, AOT; (0)naphthol, (0)phenol. hexane
+
a Model 153 Beckman (Berkeley, CA) UV detector operating at 254 nm and a Model 1210 Linear (Irvine, CA) strip chart recorder. The columns were an Altex Ultrasphere-Si (150 X 4.6 mm, 5-pm particle diameter) and an Altex Ultrasil-NH, (250 X 4.6 mm, 10-pm particle diameter). The columns were thermostated at 30 i 0.2 "C using a water jacket and a Haake (Saddle Brook, NJ) D1 circulator. Operating pressures never exceeded 500 psi at a flow rate of 1.0 mL/min. HPLC grade n-hexane (Fisher Scientific) was used as the primary mobile-phase component and was dried by using type 3A Linde Molecular Sieves (Union Carbide). The surfactant was reagent grade Aerosol OT (AOT, sodium bis(2-ethylhexy1)sulfosuccinate) (Fisher Scientific) and was used as received. The micellar mobile phases were prepared by dissolving the appropriate weight of Aerosol OT in hexane and filtering through a 0.45-pm Nylon-66 membrane filter (Rainin Instruments, Woburn, MA). Water used in the mobile phase was purified with a Barnsted Nanopure system (Sybron Corp.). The solutes 2,4dinitrotoluene, naphthol (both Matheson, Coleman & Bell), and phenol (Mallinckrodt) were dissolved in hexane and were reagent grade and used as received. Reagent grade n-pentene (J.T. Baker) was assumed to be unretained and was used for the determination of the void volume. Plate counts were accurately determined by using an equation that corrects for tlk asymmetry of skewed peaks (20): N = 41.7(t~/Wo.J~/(B/A+ 1.25)
(1)
where tRis the retention time, W,,, is the peak width at 10% peak height, and B / A is the asymmetry ratio measured at 10% peak height. This equation was recently found to be the most accurate manual method for calculation of column efficiency (21). The reduced plate height was calculated by h = H/d, (2) where H i s the plate height, H =L/N
(3)
Flgure 2. log k'vs. log [AOT]: column, Ultrasil-NH,; mobile phase, hexane 4- AOT; (0)phenol, (0)naphthol.
where L is the column length and d, is the stationary-phase particle diameter.
RESULTS AND DISCUSSION Surfactant Choice and Concentration. Muller has distinguished between two types of surfactant associations in organic solvents (22). Type I association (e.g., dodecylammonium propionate in benzene) is characterized by sequential, indefinite self-association. Here aggregation numbers are small (e.g., 3-7) and increase progressively with increasing concentration of surfactant without reaching a limiting value. There are then no pronounced critical micelle concentration (cmc) vales for these systems, and these surfactants would appear to be of little analytical utility, as the variability in aggregation number would likely cause variability in partition coefficientsas well. Type I1 surfactants, however, behave quite differently. They have reasonably well-defined cmc values and larger aggregation numbers that reach constant limiting values. The aggregation numbers are, however, dependent on water content (vide infra). They are then much more similar to normal micelles and would appear to be the surfactants of choice for chemical analysis. Aerosol OT is a type I1 surfactant (22). Figures 1and 2 are plots of log k'vs. log AOT concentration for the solutes phenol and naphthol on silica and an amino bonded phase, respectively. Two linear components are seen for both solutes on both columns, one above and one below the cmc. Curvature occurs in the region of the cmc, but extrapolation of the two linear portions should show an intersection at approximatey the cmc of the particular system under investigation. The average value determined is 3.5 X M in dry hexane at 30 "C. We have previously used this method to determine cmc values for normal micelles used in reversed-phase chromatography (23,24) and have obtained excellent comparisons with literature values. The value obtained here, however, is more difficult to compare,'both be-
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
Table I. Critical Micelle Concentration (cmc) Values of Aerosol OT in Different Hydrocarbon Solvents (from ref
'Or
12)
solvent cyclohexane
temp, "C 37
28 f 4 benzene
CC14
37
RT 28 20 25 37
*4
20
technique'
cmc, M
VPO
3.9 x 10-4 1.3 x 10-3
LS VPO PA LS
3.5 x 2.2 x 2.7 x 2.0 x
TCNQ
10-4 10-3
10-3
10-3 1.6 X 10"' 4.0 x 10-4 6.0 x 10-4
VPO VPO TCNQ
LS 4.9 x 10-4 VPO, vapor pressure osmometry; LS, light scattering;PA, positron annihilation; and TCNQ, solubilization of 7,7,8&tetracyanoquinodimethane. pentane
25
Ye H,O
~n 5 X lo'' MA,, in hexone
-
Figure 3. Effect of water content (v/v) on retention: column, Ultrasphere-Si; mobile phase, hexane 5 X lo-' M AOT; (0)2,4-dinitrotoluene, (A) phenol, (X) naphthol.
+
,-
/
Table 11. k'Value for Phenol and Naphthol in Dry Hexane and 5 X LO-' M AOT in Hexane column
mobile phase
Ultrasphere- dry hexane Si 5X M AOT in hexane Ultrasil-NH, dry hexane 5X M AOT in hexane
k' k' (phenol) (naphthol) 72.5
47.5
27.9
20.3
47.5
53.7 40.9
29.4 ~
~~
cause of the paucity of data concerning reversed micelles and because the cmc of these systems is much more highly dependent on solvent, water content, and temperature than normal micelle systems (12, 13). Table I shows cmc values for AOT in various hydrocarbon solvents, taken from ref 12, and careful inspection shows that the values seem to be more dependent on measurement technique than either temperature or solvent. Caution is then advised in close comparison of cmc values obtained by different techniques. At concentrations above the cmc, the retention of the test solutes is clearly affected by the surfactant concentration, and the behavior is similar to solutes eluted with normal micelles in reversed-phase chromatography. There is also a significant change in retention of the test solutes as surfactant below the cmc is initially added to the hexane mobile phases. Table I1 shows the change in k'as the mobile phase is changed from dry hexane to hexane + 5 X lo4 M AOT. As shown in Table I1 the change is much greater for the silica column than for the polar bonded phase. This is likely caused by the same mechanism as the large change seen as trace water is first added to a dry mobile phase (16). That is, the surface adsorption sites on silica are not energetically homogeneous. At concentrations below the cmc, the polar head groups of the surfactant are likely tightly bound to the active sites on the silica surface, thereby reducing retention of solutes that would otherwise be attracted to these sites. The much smaller change with the amino bonded phase is likely due to a combination of two effects. Again, surfactant may be tightly bound to any residual silanol groups on the stationary phase, thereby masking these adsorption sites. Also, the reduction in surface tension (and interfacial tension) caused by the low concentration of surfactant is likely contributing as well (25). Water Content. One of the more interesting aspects of reversed micelles is their ability to solubilize water in the interior of the micelle structure. In many instances water actually promotes the formation of larger and more stable reversed micelles. Indeed, in the absence of traces of water, aggregation is sometimes precluded (19). For the surfactant AOT, the first water molecules in the micelle are bound tightly
.L X-x-x
0 0 OM01
02
% H,O
03
05
04
06
07
08
09
100
in 5 XIO-' Mnor in hexane
Flgure 4. Effect of water content (v/v) on retention: column, Ultra5 X lo-' M AOT; (0)naphthol, (A) sil-NH,; mobile phase, hexane phenol, (X) 2,4dinitrotoluene.
+
to the sodium counterion (6 molecules of water/Na+), and subsequent water goes to make up the water "pool". Addition of water results in a rapid increase in the aggregation number and the size of the surfactant-entrapped water pool (26). However, the average size of aggregates at a given water-tosurfactant ratio is essentially independent of the concentration of AOT and of the solvent used. This is again beneficial for chromatographic application, as surfactant concentration gradients used to speed elution of highly retained solutes will not appreciably change the micelle structure. This unique water entrapment property of reversed micelles provides a possible solution to the worst problem of normal-phase chromatography; that being the large retention dependence on water content of the mobile phase. Since water is solvated in the interior of reversed micelles, it is possible that the use of reversed micellar mobile phases will greatly reduce this problem. Figures 3 and 4 show k' changes on a silica and amino bonded phase column, respectively, for the solutes naphthol, phenol, and 2,4-dinitrotoluene7 as water is added to a mobile phase of 5 X M AOT in hexane. The solubility of water in dry hexane at 20 "C has been reported as 0.0111 g of water/100 g of hexane, or 0.0073% v/v (27). Here we have added as much as 1% water, which is evidence of the great solvating power of the reversed micelles. The small changes seen at very low water content are likely from a combination of changes in both the mobile and stationary phases. Water is very tightly bound to the active sites of silica (16-18), and small amounts of water will greatly affect k' values. With amino bonded phases it is also likely that trace amounts of water will solvate the stationary-phase structure and thereby change its polarity. With reversed micelles, the aggregation number and structure are dependent on the surfactant-to-water ratio (vide supra), which will likely change partition coefficients of solutes to the micelle. Both of these effects would be most important at very low water concentrations, and that is in agreement with Figures 3 and 4. Above a water content of ca. 0.1% (v/v) there is virtually no change in the retention of the test solutes, leading us to believe that reversed micellar mobile phases may provide a much more
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986 T a b l e 111. P l a t e C o u n t s (N), P l a t e H e i g h t s (H, mm), R e d u c e d P l a t e H e i g h t s ( h ) ,and A s y m m e t r y R a t i o s ( B / A ) for P h e n o l in 5 X M AOT in H e x a n e and 5/95 2-Propanol/Hexane
5/95
M AOT
5 X
parameter
in hexane
2-propanol/ hexane
Ultrasphere-Si N H h BIA
3300
0.045 9.1 1.50
Ultrasil-NH2 N H h BIA
900
0.28 28 2.18
problem. One of the major differences between normal and reversed micelles is that solutes do not penetrate appreciably into normal micelles, but in reversed micelles polar solutes are localized in the hydrophilic core (19). This leads us to believe that the source of inefficiency is a slow transfer step out of the micelle. We are presently investigating the possibility of using elevated temperatures to increase the exit rate of solutes from the micelle core, as well as other possible solutions to the efficiency problem.
8200 0.018 3.7 1.14
2400
0.10 10 1.22
robust methodology for normal-phase chromatography than traditional nonpolar hydrocarbon mobile phases. Efficiency. As with any mobile-phase additive, it is important to assess the effect of the additive on column efficiency. This problem was recognized early in the use of normal micelles for reversed-phase chromatography, and the decrease in efficiency was found to arise from slow mass transfer of the solute between an aqueous mobile phase and a hydrophobic stationary phase (23). This problem was solved for sodium dodecyl sulfate surfactant by the addition of 3% 1-propanol to the micellar mobile phase, which serves to wet the hydrophobic surface and speed mass transfer across the phase boundary. Subsequently this solution was found to work for cationic and nonionic surfactants as well and appears to be a universal solution for hydrophobic stationary phases (24). As was found initially with normal micelles, there appears to be a loss in efficiency when using reversed micellar mobile phases in normal-phase chromatography. Table I11 shows plate counts, reduced plate heights, and asymmetry values for both columns for a micellar mobile phase of 5 X 10" M AOT in hexane and for a mobile phase of 5:95 2-propanol/ hexane. For both columns there is a severe loss of efficiency associated with the use of micellar mobile phases. Here the poor efficiency is likely caused by a different fundamental
747
LITERATURE CITED Tanford, C. "The Hydrophobic Effect: Formation of Micelles and Biological Membranes", 2nd ed.; Wiley: New York, 1980. Cline Love, L. J.; Harbarta, J. G.; Dorsey, J. G. Anal. Chem. 1084, 56, 1132A-1148A. Peiizzetti, E.; Pramauro, E. Anal. Chlm. Acta 1085, 169, 1-29. Armstrong, D. W.; Henry, S. J. J. Llq. Chromatogr. 1080, 3 , 657-672. Landy, J. S.; Dorsey, J. G. J. Chromatogr. Scl. 1984, 2 2 , 68-70. Dorsey, J. G.; Khaledi, M. G.; Landy, J. S.; Lin, J-L. J. Chromatogr. 1984, 316, 163-191. Khaledi, M. G.; Dorsey, J. G. Anal. Chem. 1985, 57, 2190-2196. DeLuccla, F. J.; Arunyanart, M.; Cline Love, L. J. Anal. Chem. 1085, 57, 1564-1568. Arunyanart, M.; Cline Love, L. J. J. Chromatogr. Homed. Appl. 1085, 342, 293-301. Arunyanart, M.; Cline Love, L. J. Anal. Chem. 1984, 56, 1557-1561. Pellzzetti, E.; Pramauro, E. J. fhys. Chem. 1984, 88, 990-996. Eicke, H.-F. Top. Curr. Chem. 1980, 87, 85-145. Fendler, J. H. "Membrane Mimetic Chemistry"; Wiley-Interscience: New York, 1982; Chapter 3. Armstrong, D. W.; Terrill, R. Q.Anal. Chem. 1079, 5 1 , 2160-2163. Majors, R. E.; Earth, H. G.; Lochmuller, C. H. Anal. Chem. 1984, 56, 300R-349R. Szepesy, L.; Combelias, C.; Caude, M.; Rosset, R. J. Chromatogr. 1982. 237. 65-78. Souteyrand, C.; Thibert, M.; Caude, M.; Rosset, R. J. Chromatogr. 1983, 262, 1-18. Souteyrand, C.; Thibert, M.; Caude, M.; Rosset, R. J. Chromatogr. 1084, 316, 373-388. Fendler, J. H. Acc. Chem. Res. 1978, 9 , 153-161. Foley, J. P.; Dorsey. J. G. Anal. Chem. 1983, 55, 730-737. Bidlingmeyer, B. A.; Warren, F. V., Jr. Anal. Chem. 1984, 56, 1583A- 1596A. Muller, N. J. Colloid Interface Sci. 1078, 63, 383-393. Dorsey, J. G.; DeEchegaray, M. T.; Landy, J. S. Anal. Chem. 1083, 55, 924-928. Landy, J. S.; Dorsey, J. 0.Anal. Chim. Acta, in press. Tang, M.; Demlng, S. N. Anal. Chem. 1983, 55, 425-428. Day, R. A.; Robinson, B. H.; Clark, J. H. R.; Doherty, J. V. J. Chem. Soc., Feraday Trans. 1 1979, 75, 132-139. Black, C.; Joris, G. G.; Taylor, H. S. J. Chem. Phys. 1948, 16. 537-543.
RECEIVED for review October 3, 1985. Accepted November 11, 1985.
