924
Anal. Chem. 1983, 55, 924-928
Efficiency Enhancement in Micellar Liquid Chromatography John G. Dorsey,” Marla 1.DeEchegaray,’ and John S. Landy Department of Chemistty, Universi@of Florida, Gainesville, Florida 326 1 I
The use of mlcellar mobile phases can provlde unlque selectivities In llquld chromatography. A major drawback In all published reports, however, Is a loss of efflclency when compared to tradltlonal hydroorganlc moblle phases. This inefflclency is shown to arise from slow mass transfer, which comes principally from poor wettlng of the stationary phase. Low concentrations of organic modlflers are useful for modlfylng the surface of the stationary phase and provlding the wetting needed for good mass transfer. Elevated temperatures are also shown to be useful In overcomlng the hlgher vlscoslty of mlcellar mobile phases and thereby serving to Improve peak shape. Mobile phases contalnlng about 3 % propanol and temperatures of about 40 OC provide efflclencies approachlng those of hydroorganic moblle phases.
The popularity of modern liquid chromatography continues to increase. One of the reasons for this is the unique selectivities that can be generated in the mobile phase by the addition of chemical modifiers. Retention in reversed-phase LC is dominated by solvent-solute interactions, with stationary phase-solute interactions making secondary contributions. The key to separation is then to be able to change the solvent-solute interactions in such a way as to shift the retention of any overlapping compounds. In some cases the selectivity of the chromatographic system can be modified by simply changing the eluent strength or ratio of the hydroorganic mobile phase. Most often, however, changes in selectivity reached in this fashion are accompanied by concomitant changes in the retention of all sample components, and an appreciable change in relative retention is achieved only with capacity factors that fall outside the practical range of 0.3 < k’ < 5 . It is desirable to find ways to change the selectivity of the chromatographic system for closely eluting sample components without drastic changes in the eluent strength as a whole. One way that this has been done, particularly for ionogenic solutes, is by the addition of low concentrations of ionic surface active agents having an opposite charge to the solute. The surfactant will then coulombically interact with the solute and drastically alter its retention characteristics. Because of the nature of the surfactant, this technique was dubbed “soap chromatography” ( I ) and is now more commonly known as ion-pair chromatography. Until recently, only low concentrations of surfactant were used, and investigators intentionally stayed below critical micelle concentrations. Armstrong and Henry first effectively demonstrated the usefulness of reversed-phase mobile phases containing surfactants above the critical micelle concentration (2). They showed that the micelle can provide a hydrophobic site for interaction with the solute in the mobile phase and can be used in place of an organic modifier. Since then other reports have appeared (3-8) and certain advantages have been shown, including the selectivity of the micellar interaction and economy of operating expense when compared to expensive Present address: Beckman Instruments, 5810 Hillcroft Av., Houston, TX 77036.
chromatographic grade organic solvents. Armstrong et al. and Cline Love et al. have also shown that micellar mobile phases can lead to increased selectivity of detection through the use of micelle-stabilized room-temperature phosphorescence (6,
7). A major drawback in all published reports, however, is a loss of efficiency when compared to traditional hydroorganic mobile phases. If micellar mobile phases are ever to be widely accepted as a viable chromatographic technique, the efficiency achieved must a t least approach that of conventional reversed-phase LC. This paper discusses the source of this inefficiency, as well as ways in which it can be improved. EXPERIMENTAL SECTION LC System. An Altex (Berkeley, CA) Model 322 gradient liquid chromatograph was used, incorporating two Model lOOA dual reciprocating pumps, a Model 210 sample injection valve with 20 and five microliter loops, and a Model 153 UV detector (254 nm) with an 8-pL flow cell and a time constant of 1.1s. The extra column volume of the system was calculated to be less than 60 pL. The column was either an Altex Ultrasphere ODS (250 X 4.6 mm) or an Alltech (Alltech Associates, Deerfield, IL) 5 fim CIS(250 X 4.6 mm) which was water jacketed and temperature controlled by a Techne (Princeton, NJ) TE-7 circulator. A thermostated silica precolumn was used before the injector to saturate the mobile phase with silicates and to thermally equilibrate the mobile phase before reaching the analytical column. Reagents. Reagents either were saturated solutions in water or were dissolved in methanol. Acetone, methyl ethyl ketone, toluene, and benzene (all Mallinckrodt) and anisole, acetophenone, phenol, and nitrobenzene (all Fisher Scientific Co.) were reagent grade and were used as received. Mobile phase components methanol and acetonitrile were HPLC grade (Fisher Scientific), propanol was reagent grade (Fisher), and ethanol was U.S.P. (US Industrial Chemicals Co.). Deionized water was purified with a Barnstead Nanopure System (Sybron Corp.). HPLC grade sodium dodecyl sulfate (SDS) (Fisher) was used as received. Procedure. The appropriate weight of surfactant was dissolved in water and the solution was then filtered through a 0.45-pm Nylon-66 membrane filter (Rainin Instruments, Woburn, MA). All capacity factor and plate count values are averages of at least duplicate determinations. RESULTS AND DISCUSSION Void Volume Determination. An accurate determination of column void volume is essential for meaningful capacity factor calculations. This problem has received some attention in the literature (9-12), but there is yet to be a consensus on a correct approach. The use of NaN03 gives a very reproducible value, is unretained by a hydrophobic stationary phase, and is small enough to be accessible to the pores of the silica (11). This method was used for nonsurfactant-containing mobile phases but, because of electrostatic effects, will not work for mobile phases containing ionic surfactants. For surfactant-containing mobile phases, the refractive index change from an injection of water gave comparable values to those determined with nitrate in nonsurfactant-containing mobile phases. This volume, approximately 2.4 mL, was used for all k’calculations. We are presently investigating the exact effect of surfactant concentration on void volumes. Effect of Surfactant Concentration. Figure 1is a plot of log k’ vs. log SDS concentration for nitrobenzene and
0003-2700/83/0355-0924$01.50/00 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 6, MAY 1983
925
-
Table I. aH"of Transfera AH",kcal/mol no SDS 2 X lo-' M SDS
\
\
a
\
acetone
MEK
-2.28 -2.99
-1.17
benzene anisole
-3.13
-1.59 -2.06
-4.68
Ultrasphere ODs, 10/90 methanol/water.
insight into the retention mechanism and helps in understanding the system as a whole. For a chromatographic process a van't Hoff plot is usually constructed. With a plot of log k' vs. 1/T, the standard enthalpies and entropies of transfer can be calculated. The capacity factor, k', is related to the thermodynamic distribution constant by
K ' = @K
(1)
where q5 is the volume phase ratio (stationary/mobile). Two expressions of the Gibbs free energy are
AGO = -RT In
\
K
and
AG' = AH" - TAS"
(3)
Combining these three equations, an expression for the capacity factor in terms of the thermodynamic properties AH" and AS" is obtained
-AHo RT
AS" R
I n k ' = -+ - + I n @
Figure 1. log k'vs;. log [SDS]:Uitrasphere ODS, moblle phase, 10/90 propanoVwater; flow rate, 1.5 mL/mln; (0)benzene, (A)nitrobenzene.
benzene in a mobile phase of 10/90 1-propanol/water. Two linear components of these plots are seen, one above and one below the critical micelle concentration (CMC). Curvature occurs in the region of the CMC, but extrapolation of the two linear portions should show an intersection at approximately the CMC of the particular system under: investigation. The CMC of SDS in pure water a t 25 "C is 8 X M and has been reported to first decrease and then increase with small additions of methanol (13). The value calculated from Figure 1is 0.013 M frorn both nitrobenzene and benzene data in the system of 10/90 1-propanol/water at 40 "C. The effect of the cosolvent on the micelle structure is then an important consideration. In solutions of 0.27 mole fraction methanol, micellar aggregation of SDS molecules is precluded (13). For this and other reasons it is important that the concentration of the cosolvent be kept as low as possible (vide infra). The slopes for both components are dightly negative for concentrations of SDS below the CMC. Other authors also have reported s m d decreases in k 'for nonionic solutes as SDS concentration is increased (3,14). Knox and Hartwick have shown that SDS adsorbs almost irreversibly onto a CI8phase (14), and it is liikely that adsorbed SDS remained on the column from previous experiments. The linear portion of the plot for concentrations above the CMC is evidence that the system is behaving in a well-defined reversed-phase manner and that the micelle concentration controls retention of these solutes in the same way as do organic modifiers (15). Thermodynamics. The effect of temperature on retention is related to the enthalpy of transfer of the solute from the mobile phase to the stationary phase (16). A study of the thermodynamics of transfer for a given solute gives some
(4)
If AHo and A S o are independent of temperature over the temperature interval studied, the van't Hoff plots will be linear. An unchanging slope will give a constant enthalpy of transfer, which can be interpreted as an unchanging retention mechanism over that temperature range. A van't Hoff plot was constructed for anisole over a temperature range of 30-50 "C in 5 "C intervals, with a mobile phase of 10/90 methanol/water. This plot shows excellent linearity (r = 0.998) and yields a AHo value of -4.7 kcal. Another van't Hoff plot was constructed for anisole over t,he same temperature range with a mobile phase of 10/90 M SDS, which is safely above the methanol/water + 2 X CMC of 7.9 X 10-3 M reported for SDS in a 12.6% methanol solution (13). This plot also shows excellent linearity (r = 0.997) and yields a AH" value of -2.1 kcal. The range of enthalpy values found for a variety of compounds is in agreement with the values expected for a reversed phase system, typically from -1 to -10 kcal/mol(17), and the values are shown in Table I. The large negative values of AHu for anisole describe its affiiity for the stationary phase, a favorable energetic process. A reduction in AHu of transfer is observed for the solutes from a micellar-free mobile phase to a micellar one, showing the ability of the micelle to compete with the stationary phase for the solute. The linearity of the van't Hoff plot for the micellar system is also evidence that the integrity of the micelle structure is maintained over the temperature range investigated. This is an important consideration as temperature changes can affect the aggregation number and shape of micellar structures (18). Efficiency Studies. The most serious problem in all published reports of micellar chromatography is a loss of efficiency when compared to traditional hydroorganic mobile phases. Plots of reduced plate height vs. reduced velocity can be very useful in comparing efficiencies of chromatographic systems. However, an accurate calculation of the number of
ANALYTICAL CHEMISTRY, VOL. 55, NO. 6, MAY 1983
926
1.4-
Table 11. Variation of Efficiency and Asymmetry with Methanol Concentrationa
A
% methanol 1.3-
0 2 5 10
a 5 1.2c 1.1-
1
5
10
6.67 4.83 4.00 3.50
15
J
Table 111. Variation of Efficiency and Asymmetry with Organic Modifiera organic modifier methanol acetonitrile ethanol propanol
N 1080 1990 2060 2230
a Alltech C,,,10/90 organiclwater t 5 X benzene solute.
n.oJ
BIA
a Ultrasphere ODs, mobile phase 2 X lo-’ M SDS, acetone solute.
b
1.0
N 300 350 7 50 1120
b
IO
5
15
3
Flgure 2. Reduced parameter van Deemter plot’for acetone: Ultra-
sphere ODS, mobile phase, 5/95 methanoVwater. (B) Same as A but moblle phase also 2 X M SDS. theoretical plates is necessary for this to be accomplished. The commonly used equation
2) 2
N = &54(
(5)
has been shown to be highly inaccurate for skewed peaks (19). Foley and Dorsey have recently described a simple yet accurate equation for plate count which corrects for the asymmetry of skewed peaks (20). This equation
was used in this work where B / A is the asymmetry ratio measured at 10% peak height (19). The reduced plate height was calculated by
h = -H dP
(7)
where H is the plate height and d p is the particle diameter. The reduced velocity was calculated as
where u is the linear velocity and D, is the diffusion coefficient of the solute in the mobile phase. For the nonsurfactantcontaining mobile phase, the diffusion coefficient of acetone was calculated to be approximately 1.3 X lo* cm2/s, estimated by the Wilke-Chang equation (21). For solutes in micellar mobile phases the diffusion coefficient is harder to estimate. The diffusion coefficient of SDS in 0.2 M aqueous solution has been reported as about 2 X lo4 cm/s (22). These values
BIA 3.51 1.98 2.08 1.61
lo-’
M SDS,
then represent upper and lower limits, as the solute spends some fraction of time in the bulk mobile phase and some fraction of time in the micelle itself. An average value of 7 X lo4 cm/s was then used for these calculations. Figure 2A is a reduced parameters plot for acetone in a mobile phase of 5/95 methanol/water. Figure 2B is for acetone in the same mobile phase with 2 X M SDS. The steep slope of the plot at high reduced velocities for the micellar system is indicative of high resistance to mass transfer (23). It is likely that this poor mass transfer is a slow transfer step from the micelle to the stationary phase, as it is wellknown that the interior of micelles exhibit somewhat higher viscosity than bulk solution (24). Two approaches have been taken to improve these mass transfer characteristics. Most reports of micellar LC have used totally aqueous mobile phases. It is known that hydrophobic stationary phases exhibit very poor mass transfer characteristics under these conditions (25). This is presumably because of the poor wetting of the stationary phase, resulting in slow equilibrium across the interface of the two highly dissimilar phases. Indeed, when a gradient program with a CIScolumn is run, it has been suggested that the initial aqueous component of the mobile phase contains some minimum concentration of organic solvent to improve wetting of the stationary phase (25). For this reason, it was felt that the addition of small amounts of an organic solvent to the aqueous micellar mobile phase would greatly improve the efficiency of the system. Table I1 shows plate counts for a solute of acetone in a mobile phase of 2 X M SDS as methanol is added to the mobile phase. While both the symmetry of the peak and the plate count increased dramatically over the range of methanol studied, this certainly does not represent a usable analytical system. It should be noted, however, that these represent true plate counts and would have appeared much higher using the common half-height equation. Obviously even better wetting of the stationary phase is necessary. Studies have shown that as much as 40-50’70of organic solvent may be required to allow some reversed-phase packings to be operated at highest efficiency (26). This represents an unrealistic situation for micellar chromatography, so an attempt was made to find an organic solvent which would give good wetting at low concentrations. Table I11 shows plate counts and asymmetries for benzene in a mobile phase of 5 X M SDS in which the 10% organic component was varied. Again an improvement is seen in both peak
ANALYTICAL CHEMISTRY, VOL. 55, NO. 6, MAY 1983
927
Table IV. Variation of Efficiency and Asymmetry with Temperaturea temp, "C 30 35 40 45 50 a
N 15 000 15 500 14 800 15 000 13 700
BIA 1.16 1.09 1.06 1.00 1.07
Ultrasphere ODs, 10/90 propanol/water t 5 X
I
M
SDS,benzene solute.
a032 AU
Table V. Variation of Efficiency and Asymmetry with Propanol Concentrationa % propanol
0 1 2 3 4
6 8 10
N
B/A
9 070
1.62 1.15 1.03 1.02 1.00 1.01 1.01 1.01
14 100 14 280 14 540 15 310 14 930 14 710 15 340
M SDS, 40 '(2,
Ultrasphere ODs, mobile phase 5 X :LO'' benzene solute. a
symmetry and efficiency as the polarity of the organic modifier is decreased and wetting of the stationary phase is enhanced. Because of the great improvement shown in going from methanol to propanol, a new Altex Ultrasphere ODS column was installed and the lo/% propanol/water 5 X M SDS experiment was repeated. A plate count of 15000 with an asymmetry factor of 1.16 was found for benzene at a flow rate of 1.5 mL/min, showing that the data had been limited by the efficiency of the Alltech column. This represents a reduced plate height of 3.3, which is well within the range of efficiencies shown by traditional hydroorganic mobile phases. While the actual values in 'Table I11 may therefore be low, the trend is still obvious. A second parameter which can be adjusted to improve mass transfer characteristics is temperature. As the viscosity of a micellar solution will be somewhat greater than that of an identical solution without surfactant, diffusion coefficients will be smaller, thLereby slowing mass transfer. Table IV shows asymmetries and plate counts for benzene in 10/90 propanol/water + 5 X 110" M SDS as the temperature is varied from 30 to 50 O C . While the efficiency remains virtually the same, an improvement in peak shape is seen a t the elevated temperatures. Above a temperature of 40 "C it is necessary to preheat the mobile phase to prevent peak distortion from thermal gradients (27). It is desirable to keep the concentration of organic modifier as low as possible both to maintain the integrity of the micelle and to minimize expense. Furthermore, micellar chromatography may represent m almost ideal way in which to study certain physical parameters of micelles. As the structure and behavior of micelles are much better understood in purely aqueous systems, added organic modifier can greatly complicate interpretation of data. Table V shows asymmetries and plate counts for benzene in 5 X M SDS a t 40 "C as the concentration of propanol is varied. Little improvement is seen above 3% propanol. Scott and Simpson have studied modification of CIS phases by organic modifiers and have shown that over 90% of the surface is covered with the alcohol at a concentration of 3% (w/v) propanol, but there is only about 50% coverage with the same concentration of methanol (28). This modification of the surface allows fast mass transfer, resulting in improved efficiencies. In addition to better
I
-I
+
V
4
6
t (min)
Figure 3. Chromatogram of five-component test mixture: Ultrasphere ODS; mobile phase, 70/30 methanoVwater; flow rate, 1.5 mL/min; (A) phenol, (B) acetophenone, (C) nitrobenzene, (D) benzene, (E)toluene.
+
I
0.008 AU
Figure 4. Same as Figure 3 but mobile phase 10/90 propanoVwater.
wetting of the stationary phase, the added alcohol may be "loosening" the micellar structure so that the local microviscosity experienced by a solubilized solute is diminished. This effect however is probably small in comparison to the improved wetting. Addition of low concentrations of organic modifiers also lowers the k' of the test solutes, but this has a negligible effect on the efficiency calculations. Table V shows essentially a constant plate count from 3 to 10% addeld
028
ANALYTICAL CHEMISTRY, VOL. 55, NO. 6, MAY 1983 B
I
On16 AU
n
i
!-----c(min)
Figure 5. Same as Figure 3 but mobile phase 10/90 propanoVwater
+ 5 X lo-* M SDS.
ACKNOWLEDGMENT The authors are grateful to Ken A. Dill for many helpful discussions and to Nelson H. C. Cooke, Altex Scientific, for a gift of the column. Registry No. 1-Propanol,71-23-8;methanol, 67-56-1;ethanol, 64-17-5; acetonitrile, 75-05-8. LITERATURE CITED
T
Figure 6. Same as Figure 3 but mobile phase 10/90 propanoVwater
+ 1 X lo-'
and efficiency. Figure 6 is the same mixture with a mobile phase of 10/90 propanol/water + 1 X 10-1 M SDS. The retention of toluene has been decreased to 24 min. Increasing the concentration of micelles then serves to adjust retention in the same manner as increasing the amount of organic modifier. With careful attention paid to optimizing mass transfer characteristics, micellar chromatography can give equivalent efficiencies to hydroorganic mobile phases. This optimization study has been performed with a "worst case" situation, a C18 phase. The use of shorter alkyl bonded phases or polar bonded phases should inherently yield better mass transfer, as the wetting problem will be less severe. Cline Love et al., using a C1 column, have shown that the selectivity of micellar mobile phases can be quite different than that of the commonly used organic modifiers (8). This technique then represents a unique and useful approach to separation problems.
M SDS.
propanol while the k'of benzene was reduced from 18.2 to 11.7 over this range. Separations. To fully illustrate the efficiencies attainable with micellar mobile phases, a series of chromatograms were run. Figure 3 is a separation of a five-component mixture using a mobile phase of 70/30 methanol/water. The small amount of tailing on the peaks is from the somewhat slow time constant of the detector used and is only apparent for fast eluting compounds. Figure 4 is the same mixture with a mobile phase of 10/90 propanol/water. The fifth component (toluene) had still not eluted after 2 h. Figure 5 is the same mobile phase but with 5 X lom2 M SDS added. Toluene now elutes a t about 39 min, and all peaks show good symmetry
(1) Knox, J. H.; Laird, G. R. J . Chromatogr. 1978, 122, 17-34. (2) Armstrong, D. W.; Henry, S. J. J . Ll9. Chromatogr. 1980, 3 , 657-672. (3) Graham, J. A.; Rogers, L. B. J . Chromatogr. Scl. 1980, 18, 614-621. (4) Sorel, R. H. A.; Hulshoff, A.; Wiersema, S . J . Ll9. Chromatogr. 1981, 4 , 1961-1985. (5) Armstrong, D. W.; Nome, F. Anal. Chem. 1981, 53, 1662-1666. (6) Armstrong, D. W.; Hinze, W. L.; Bui, K. H.; Singh, H. N. Anal. Left. 1981, 14, 1659-1667. (7) Weinberger, R.; Yarmchuk, P.; Cline Love, L. J. Anal. Chem. 1982, 54, 1552-1558. (8) Yarmchuk, P.; Welnberger, R.; Hirsch, R. F.; Cline Love, L. J. Anal. Chem. 1982, 54,2233-2236. (9) McCormlck, R. M.; Karger, B. L. Anal. Chem. 1980, 52,2249-2257. (10) Berendsen, G. E.; Schoenmakers, P. J.; de Galan, L.; Vlgh, G.; VargaPuchony, 2.; Inczedy, J. J . Llq. Chromatogr. 1980, 3 , 1669-1686. (11) Wells, M. J. M.; Clark, C. R . Anal. Chem. 1981, 5 3 , 1341-1345. (12) Neidhart, 8.; Krlnge, K. P.; Brockman, W. J . Llq. Chromatogr. 1981, 4 , 1875-1886. (13) Magld, L. I n "Solution Chemistry of Surfactants"; Mlttal, K. L., Ed.; Plenum Press: New York, 1979; Vol. 1, pp 427-453. (14) Knox, J. H.; Hartwlck, R. A. J . Chromatogr. 1981, 204,3-21. (15) Melander, W. R.; Horvath, C. I n "High Performance Liquid Chromatography: Advances and Perspectives"; Horvath, C, Ed.; Academic Press: New York, 1980; Vol. 2, p 176. (16) Sander, L. C.; Field, L. R. Anal. Chem. 1980, 52, 2009-2013. (17) Reference 15, p 198. (18) Fisher, L. R.; Oakenfull, D. G. Chem. SOC. Rev. 1977, 6 , 25-42. (19) Kirkland, J. J.; Yau, W. W.; Stoklosa, H. J.; Dilks, C. H., Jr. J . Chromatogr. Scl. 1977, 15,303-316. (20) Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983, 55,730-737. (21) Karger, B. L.; Snyder, L. R.; Horvath, C. "An Introduction to Separation Science"; Wiley: New York, 1973; p 78. (22) Weinhelmer, R. M.; Evans, D. F.; Cussler, E. L. J . Colloid Interface Scl. 1981, 80, 357-368. (23) Brlstow, P. A,; Knox, J. H. Chromatographla 1977, 10, 279-289. (24) Menger, F. M. Acc. Chem. Res. 1979, 12, 111-117. (25) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography", 2nd ed.; Wiley-Interscience: New York, 1979; p 298. (26) Scott, R. P. W.; Kucera, P. J . Chromatogr. 1977, 142, 213-232. (27) Perchalskl, R. J.; Wilder, B. J. Anal. Chem. 1979, 51, 774-776. (28) Scott, R. P. W.; Slmpson, C. F. Faraday Symp. Chem. SOC. 1980, 15, 69-82.
RECEIVED for review November 30,1982. Accepted February 1,1983. J.G.D. acknowledges partial support of this work by an Unsolicited Young Faculty Development Grant by Eli Lilly and Co. This work was presented in part at the 184th National Meeting of the American Chemical Society, Kansas City, MO, Sept 4,1982.
