Articles Anal. Chem. 1995, 67, 2315-2324
influence of Dodecyl Sulfate Counterion On Efficiency, Selectivity, Retention, Elution Range, and Resolution in Micellar Electrokinetic Chromatography Eric S. Ah*
and Joe P. Foley*
Department of Chemistiy, Villanova University, Villanova, Pennsylvania 19085-1699
In micellar electrokinetic chromatography (MEKC), the pseudostationary phase used most often consists of sodium dodecyl sulfate (SDS) micelles. We investigated the effects of using different counterions, E+and K+, with the dodecyl sulfate micelles in order to ascertain the influence of the counterion on aciency, selectivity, retention, elution range, and resolution in MEKC. A typically used concentration of 50 mM surfactant was employed for all of the surfactants studied. The effect of acetonitrilewas also investigated. Due to the high Kr& point of the potassium dodecyl sulfate (KDS), a minimum of 15% acetonitrile had to be used in order to solubilize the KDS surfactant monomers. Comparisons between the and K+ counterions are made with 15 and 20% Na+, E+, acetonitrile added, while comparisons between Na+ and E+ are also made with 0 and 10% acetonitrile added. Operating currents were lowest with the IiDS system. The IiDS system provided efficiencies almost 2.5 times that generated by SDS, while the KDS system generated efficiencies much lower than both the SDS and LDS micellar phases. The more hydrophobic test analytes exhibited the greatest enhancement in efficiency with the use of the IiDSmicelles. Retention factors for the various test analytes dropped appreciably with the use LiDS and acetonitrile in comparison to the acetonitrile-modified SDS system in which retention fadors appeared to stabilize with 15 and 20% acetonitrile. The KDS micelles provided the largest elution range followed by SDS and then EDS. (1)Terabe, S.;Otsuka, K.; Ichikawa, IC; Tsuchiya, A; Ando, T. Anal. Chem. 1984.56,111-113. (2) Terabe, S.; Otsuka, IC; Ando, T. Anal. Chem. 1985,57,834-841. 0003-2700/95/0367-2315$9.00/0 0 1995 American Chemical Society
Micellar electrokinetic chromatography (MEKC) was first introduced by Terabe in 19841*2for the high-resolution separation of neutral analytes. A subclass of capillary electrophoresis (CE), MEKC has also been extended to include the separation of charged analytes. Application of MEKC includes the separation of diverse compounds such as ,&blockers in urine,3 derivatized amino acids:+ gun shot residues?J0water- and fat-soluble ~itamins,~J~-13 herbicides,14 and Clearly MEKC shows considerable promise for the analysis of both charged and neutral analytes. The nature of the surfactant can have a significant effect on the separation process in MEKC. Of the four common classes of surfactants (anionic, cationic, nonionic, zwitterionic), anionic surfactants, especially sodium dodecyl sulfate (SDS),have seen the most use in MEKC. Aside from SDS, other anionic surfactants (3)Lukkari, P.;Siren, H.;Pantsar, M.; Riekkola, M. L. J. Chromatogr. 1993, 632,143-148. (4)Castagnola, M.; Rossetti, D. V.; Cassiano, L.; Rabino, R; Nocca, G.; Giardina, B.J Chromatogr. 1993,638,327-334. (5)Little, E. L.; Foley, J. P. J. Microcolumn Sep. 1992,4,145-154. (6)Miyashita, Y.;Terabe, S. Chromatogram 1990,11, 6-7. (7)Ong, C. P.;Ng. C. L.; Lee, H. IC; Li, S. F. Y. ]. Chromafogr. 1991, 559, 537-545. (8) Otsuka, IC;Terabe, S.; Ando, T.]. Chromatofl. 1985,332,219-226. (9)Northrop, D.M.; Martire, D. E.; MacCrehan, W. AAnal. Chem. 1991,63, 1038- 1042. (10)Northrop, D.M.; MacCrehan, W. A]. Liq. Chromatogr. 1992,15, 10411063. (11) Fujiwara, S.;Iwase, S.;Honda, S. J. Chromatogr. 1988,447,133-140. (12)Nishi, H.;Tsumagari, N.; Kakimoto, T.; Terabe, S. J. Chromatogr, 1989, 465,331-343. (13)Ong, C. P.;Ng, C. L.; Lee, H.IC; Li, S. F. Y. J. Chromatogr, 1991, 547, 419-428. (14)Wu,Q.;Claessens, H. A; Cramers, C. A. Chromafographia 1992,34,2530. (15)Kristensen, H.IC; Hansen, S. H. J. Chromatogr, 1993,628,309-315.
Analytical Chemistry, Vol. 67, No. 14,July 15, 1995 2315
used include sodium decyl sulfate,lbl 7 which has been reported to generate high operating currents at surfactant concentrations greater than 50 mM resulting in Joule heating problems. Sodium tetradecyl sulfate has low solubility, and thus its use is limited to operation at high temperatures ('35 0C).21xThe surfactants sodium dodecyl sulfonate2 and sodium lauroylmethyl taurate12l9 have also been used. In all of these studies, the counterion was sodium while the surfactant moiety was varied. Although there have been many impressive separations utilizing SDS, little research has been conducted into the investigation of using different inorganic counterions with the dodecyl sulfate surfactant. Whereas counterion effects have been studied extensively in CE20-14 and are generally manifested in effects on electroosmotic flow, in MEKC, one might expect to see still larger effects due to changes in micellar structure. A recently published study2: investigated the effect of the divalent Mg2+ ion with dodecyl sulfate N g @ S ) d and showed that retention factors can be between 1.5 and 2.5 times greater with the Mg(DS)J micelles than with SDS. In addition, the elution range (tmJto)was independent of the percentage of acetonitrile in the buffer solution for the Mg(DS)2 system while the elution range for the SDS system increased with percentage of acetonitrile. This aided the retention-mediated optimization of resolution and resolution per unit time in the Mg(DS)L system. The results reported in the Mg(DS)j study, using a divalent inorganic counterion with the dodecyl sulfate surfactant, clearly indicate the need to further investigate the effect of other inorganic counterions on separations in MEKC. In this study, we chose to compare the separations obtained with SDS to that obtained using lithium dodecyl sulfate (LiDS) and potassium dodecyl sulfate (KDS). The focus was to explore the effect of the inorganic counterions Li-, Na+, and K+ on separation efficiency, selectivity, retention, elution range, and resolution These surfactants were commercially available and thus represent an advantage over the Mg(DS)l, which had to be synthesized in-house. The effect of the organic m o d ~ e acetor nitrile was also investigated with each of these micellar systems. EXPERIMENTAL SECTION Apparatus. A Quanta 4000 capillary electrophoresis system
(Waters Inc., Milford, MA) equipped with fixed-wavelength W detection at 254 nm was employed for all the separations performed in this study. All separations were performed in a 29.5 cm (injection to detection) x 50 ym i.d. (370 ym 0.d.) fused-silica capillary tube (Polymicro Technologies, Tucson, AZ). The total capillary length was 37.0 cm. Injections were made hydrostatically (16) Rasmussen, H. T.; Goebel, L. K.; McNair. H. M.J. Chromatogr. 1990.517, 549-555. (17) Burton, D. E.; Sepaniak. M. J.; Maskarinec. M. P. J. Chromatogr. Sci. 1987, 25.514-518. (18)Otsuka, K.; Terabe, S.; Ando, T. Chem. Abstr. 1986,105. 126484r. (19) %hi. H.: Tsumagari. N.;Kakimoto, T.; Terabe, S. J. Chromatogr. 1989, 477, 259-270. (20) Atamna. I. Z.: Metral, C. J.: Muschik, G. M.; Issaq. H. J. J. Liq. Chromutogr. 1990,13,2517. (21) Atamna. I. Z.; Metral, C. J.: Muschik. G. M.; Issaq. H. J. J. Liq. Chromatogr. 1990,13, 3201. (22) Issaq. H. J.; Atamna, I. Z.; Metral. C. J.; Muschik, G. M. J , Liq Chromatogr. 1990,13. 1247. (23) Issaq, H. J.; Atamna, I. Z.; Muschik. G. M.; Janini. G. M. Chromatogruphia 1991,32,155-161. (24) Issaq. H. J.: Janini, G. M.: Atamna, I. Z.; Muschik. G. M.; Lukszo. J. J. Liq. C h r o m ~ t o g 1992. ~. 15,1129-1142. (25) Xielsen. K. R.; Foley, J. P. J. Microcolumn Sep. 1993,5,347-360.
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Analytical Chemistry, Vol. 67, No. 74, July 75, 7995
for 1 s. The applied voltage was 15 kV, and operating currents were less than 35 yA unless otherwise noted in the text. The data were collected at a rate of 20 points/s and analyzed on a Macintosh IIci computer (Apple, Cupertino, CA) using a MacLab four-channel ADC with the appropriate vendor software (AD Instruments, Milford, MA). All experiments were done at ambient temperature (-25 "C). Materials. All the neutral test analytes were purchased from Aldrich (Milwaukee, WI) unless otherwise noted. The neutral test mixture consisted of benzyl alcohol (EM Science, Gibbstown, NJ), nitrobenzene, anisole, fi-nitrotoluene, m-nitrotoluene, benzophenone, biphenyl (MCB Reagents, Cincinnati, OH), and decanophenone. The homologous series of alkylphenones and nitrophenyl esters were purchased as a kits from Aldrich Milwaukee, WI). Sodium dodecyl sulfate was purchased from Sigma (St. Louis, MO), while lithium dodecyl sulfate and potassium dodecyl sulfate were purchased from Aldrich (Milwaukee, WI). All surfactants were used as received. The concentration of SDS, LiDS, and KDS was 50 mM for all the MEKC separations. The percentages of acetonitrile used were 0, 10, 15, and 20%. Stock buffer solutions were prepared with NaH2P04*H?0and sodium hydroxide to give a 100 mM phosphate buffer (PH 7.0). A phosphate buffer concentration of 5 mM was used in all the experiments. The micellar solutions were made by weighing appropriate amounts of SDS, LiDS, or KDS and diluting with the stock buffer solution, distilled water, and acetonitrile when applicable in a 100-mL volumetric flask to obtain the desired concentrations. All the micellar buffer solutions were filtered through 0.20-pm membrane filters obtained from Alltech Associates, Inc. (Deerfield, 11). HPLC grade distilled water used in the makeup of the micellar buffer solutions was obtained from J. T. Baker (Phillipsburg, NJ). Sample solutions were made up of 50% acetonitrile and 50%running buffer with solute concentrations at or below 0.75 mg/mL. Methods. The capillary was activated using a modification of a previously described procedure.26 The capillary was first rinsed with 1 M potassium hydroxide for 20 min followed by subsequent rinses of 0.1 M KOH and distilled water for 20 min each. A final 20-min rinse was performed with the operating buffer before the capillary was used. Purges with the operating buffer were done after each run for 5 min using a vacuum of -42 cm Hg at the detector reservoir. Acetonitrile was used as the electroosmotic flow marker. Electroosmotic velocities were determined by measuring the migration times at the start of the negative (acetonitrile) peak. The total length of the capillary was then divided by this value to yield the electroosmotic velocity. This method was discussed previously.2i The tnlCvalues, which represent the elution time of the pseudostationary phase for each separation, were measured using decanophenone and confirmed with the iterative computation method developed by Bushey and Jorgenson.28 Decanophenone was chosen as the t,, marker because it was soluble in the totally aqueous micellar systems studied. A higher homolog is only partially soluble and may have precipitated in the capillary with the totally aqueous micellar systems. Retention factors were calculated using the following equation? (26) Lauer, H. H., McManigill, D. Anal. Chem. 1986,58,166-170. (27) Ahuja, E. S.; Little, E. L.: Foley. J. P. J. Lzq Chromatogr. 1992,15. 10991113. (28) Bushey. M. M.: Jorgenson. J. W. J Microcolumn Sep. 1989,1. 125-130.
C+
x
where tr is the retention time of the analyte of interest, to is the retention time of an unretained analyte, and tmc is the retention time of the micelle. The selectivity of a separation is defined as a ratio of retention factors ( k 2 l k l where k2 > k l ) for two adjacent compounds. The selectivity in MEKC can be written as shown in eq 2, where , P
a = k,/k, = Pm,/Pmi
C+j
x C+
(2) x
is the water-micelle partition coefficient of the analyts. Selectivity can be altered by changing the micellar phase or using buffer additives like an organic solvent. The selectivities in this report are calculated using the ratio of retention factors. Efficiencies were calculated using the Foley-Dorsey equation?
(3) where tr is the migration time of the analyte, W0.lis the width of the peak at 10%of the peak height, and B/A is the asymmetry factor. THEORY Micelle shapes can vary from relatively small spherical structures to elongated cylindrical, flat lamellar, inverted, or even disk-shaped structures. Micelles are made up of individual surfactant monomers that, at a specilic concentration, termed the critical micelle concentration (cmc), will aggregate to form micelles. The number of surfactant monomers required to form a micelle is known as the aggregation number. Ionic surfactants like LiDS, SDS, and KDS with aggregation numbers less than 100 will form small spherical micelles.30 In general, the surfactant monomer consists of a hydrophobic tail and a hydrophilic head group. Upon aggregation, the monomers will orient themselves such that the hydrophobic tails are in the interior or core of the micelle and the hydrophilic head groups are on the exterior or surface of the micelle. Counterions like Li+ or Na+ bind to the micelle in different degrees (see Results and Discussion) and strongly influence the surface chemistry of the micelle in terms of water structure and charge density. The association of counterions to the micelle can be represented by the following model:
M + n(C+) * M(C+). where M is the micelle, n is the number of bound counterions, and (C') is the counterion. Figure 1 illustrates a section of the interfacial region of a dodecyl sulfate micelle. The location of the head groups and counterions is arbitrary. The hydrophobic tail and hydrophilic head group are the same in the case of LiDS, SDS, and KDS; the only difference is the identity of the counterion and the degree to which the counterion binds to the micelle. (29) Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983, 55, 730-737. (30) Rosen, M. J. Sulfactanfsand Intetfacial Phenomena, 2nd ed.; John Wiley & Sons, Inc.: New York, 1989 pp 431.
C+
C+ Core of Micelle
Interfadsl Rdon
Aqueous
Phase
Figure I. A section of the interfacial region of a dodecyl sulfate and head groups are micelle. The locations of the counterions (C+) arbitrary. The various regions and positions within that region where counterions may be present are shown.
The inherent chemical properties of the counterion will influence the overall chemistry on surface of the micelle. Even though the general structure of these micelles is small and spherical in nature, the chemical properties of these micelles will change. In terms of MEKC, the effect of monovalent inorganic counterions Lit, Na+, and K+ on separation parameters like efficiency and resolution has never been examined. The separation mechanism in MEKC is based on the differential partitioning of analytes between the micellar and aqueous mobile phases. It seems reasonable that the choice of surfactant counterion could influence the partitioning process of analytes, since they have a great effect on chemical properties of the micelles themselves. One way to ascertain the influence of the surfactant counterion in MEKC and directly relate it to the partitioning mechanism would be to examine the retention factor of the analytes in terms of eq 4,2where the water-micelle partition coefficient is given as
k=Pd
(4)
P, and the chromatographic phase ratio as ,9. If separations were done at or near a constant phase ratio, the changes in retention would be solely due the water-micelle partition coefficient. The phase ratio in MEKC is governed by three parameters as shown in eq 5,2
V([surfI - cmc) (5)
where P is the partial molar volume of the micelle and [surf] is the surfactant concentration. A review of the literature indicates that the partial molar volumes for LiDS, SDS, and KDS are approximately the same.3l Aggregation numbers and cmc values for the surfactants are shown in Table 1. Since there is very little variation in both the partial molar volume and the cmc of these surfactants, if the concentration of surfactant used is same, then (31) Mukejee, P.; Mysels, K. J.; Kapauan, P. J. Phys. Chem. 1967, 71, 41664175.
Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
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Table 1. Critical Micelle Concentrations,. Aggregatlon Numbers,band the Hydrated Radius of the Counterlone for the Dodecyl Sulfate Surfactantsd
cmc, mM aggregation no. temp, "C hydrated radius, A LDS SDS KDS
8.9 8.2 7.8
63 62 60
25 25 40
3.40 2.76 2.32
Reference 30. Reference 31. Reference 35. The reported aggregation numbers are subject to great variation. For example, reported aggregation numbers for SDS range from 40 to 100. For more information, see ref 43.
the phase ratio should not be significantly altered. This would mean that changes in retention factor would primarily result from changes in the water-micelle partition coefficient. This was the approach taken in this investigation. All surfactant concentrations were 0.050 M with a sodium phosphate buffer concentration of 0.005 M. The ratio of counterions from the surfactant, [C+Isud, to the Na+ ion concentration from the running buffer, [NatIbuffer was 7.1. This value includes the concentration of sodium countenons from the addition of sodium hydroxide necessary to adjust the buffer pH. Retention optimization can be done through the optimization of the water-micelle partition coefficient. The addition of an organic modifier such as acetonitrile to the running buffer will lower high water-micelle partition coefficients in MEKC. Although this has been the most common way of changing the water-micelle partition coefficient (PAin MEKC, other methods have included changing surfactant counterion or operating at a different t e m ~ e r a t u r e .In~ ~this study, we investigated the change in the surfactant counterion. The resolution equation for neutral compounds in MEKC is2
Equation 6 can further be approximated, if the two peaks are such that kl w k2 = k,, = k, to yield2
It is readily seen that any enhancement in efficiency, N , the number of theoretical plates generated by a column of a given length, or selectivity, a, given by kzlkl, can improve resolution. It has also been demonstrated that it is important to couple improvements in selectivity with retention optimization as this will provide the greatest increase in r e s o l u t i ~ n .A~ separation ~~~~ is ultimately judged by the resolution that can be obtained. In order to understand the role of the surfactant counterion on resolution in MEKC, we specifically examined the effect on efficiency, retention, selectivity, and the elution range. RESULTS AND DISCUSSION
Influence of Counterion. The structure of a micelle is dependent on the micellar counterion e n v i r ~ n m e n t . ~The ~.~~ (32) Foley, J. P.Anal. Chem. 1990,62, 1302-1308. (33) Almgren, M.: Swamp. S. J, Phys. Chem. 1983,87, 876.
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Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
degree of counterion binding to the micelle is reflected in the critical micelle concentration in aqueous solution. Increased binding of the counterion, in aqueous systems, results in a decrease in the cmc of the surfactant. The degree of counterion binding increases with increasing polarizability and valence and decreases with increasing hydrated radius.30 Therefore, in an aqueous medium, for the anionic dodecyl sulfates, the cmc decreases in the order Li+ > Na+ > Kt > Cst > Ca2+ > Mg2+. Table 1 lists the cmc's of the surfactants and hydrated radii of counterions studied here. It is important to note that the cmc of KDS was determined at 40 "C, which is the Krafft point of this surfactant. The Krafft point is defined as the temperature at which the cmc is equal to the solubility of the surfactant in aqueous solution. Below this temperature, the surfactant will precipitate from solution. Consequently, in order to evaluate this surfactant with our CE instrument, a minimum of 15% acetonitrile was required to solubilize KDS. Smaller percentages of acetonitrile were tried but found to be inadequate as KDS precipitated from solution. Comparisons of KDS versus LiDS and SDS are thus made with 15 and 20% acetonitrile added to the running buffer, but comparisons between LiDS and SDS were possible without any acetonitrile added to the running buffer since the Kraftl point of both of these surfactants is below room temperature (25 "C). The hydrated radii of the counterions, shown in Table 1, indicate that Lit with the largest hydrated radius should bind to a lesser degree than Nat or Kt. In terms of the separation mechanism in MEKC, this lower degree of counterion binding on the surface of the micelle should manifest itself in an increased kinetic transfer of the analyte between the aqueous mobile phase and the pseudostationary micellar phase. With a smaller degree of counterion binding on the surface of the micelle there is more electrostatic repulsion of the adjacent hydrophilic head groups, and therefore, access to the surface or interior of the micelle by an analyte should be less hindered. In other words, the rate of mass transfer for the analyte between the aqueous mobile phase and the micelles should be larger. If this is indeed the case, band broadening of the analytes should be less, and consequently, separation efficiencies of the analytes with LiDS should be greater than those obtainable with SDS or KDS. This was our hypothesis at the onset of this investigation. Joule Heating Considerations. Whenever attempting a separation with MEKC, one must consider the proper operating voltages at which to perform the analysis. Electrokinetic energy is released as heat during the electrophoretic process, and this has been termed the Joule effect.35 If operating currents are too high, proper heat dissipation will not occur and the temperature inside the capillary will rise to a point that the solution in the capillary may start to boil. The subsequent formation of bubbles will inhibit further passage of ~urrent.3~ Initially we began this study using an applied voltage of 25 kV. Figure 2 shows the dependence of current density on field strength for the micellar buffer solution consisting of 50 mM KDS with 20% acetonitrile. Deviation from linearity occurs at a field strength of -450 V/cm. This corresponds to an applied voltage of -17 kV for a capillary of 37 cm total length. In order to eliminate the possibility of any Joule heating effects, all further analyses were done with an applied voltage of 15 kV, which falls (34) Huang, J. F.; Bright, F. V. Appl. Spectrosc. 1992,46, 329. (35) Sandra, P.; Vindevogel, J. Introduction to Micellar Electrokinetic Chromatography; Hiithig: Oxford, CT. 1992.
Table 3. Retention Factors of Various Anaiytes with LIDS,SDS, and KDS
. O.Ooe+O
1 0
retention factors benzobibenzyl nitrosurfactant % acetonitrile alcohol benzene phenone phenyl
..
.
LiDS
0 10 15 20
SDS
0 10 15 20 15 20
KDS 200
400
600
0.63 0.42 0.35 0.28 0.59 0.53 0.45 0.47 0.29 0.28
1.43 1.09 0.95 0.78 1.40 1.32 1.17 1.19 0.78 0.77
27.40 9.88 6.23 1.59 25.31 16.87 11.11 10.79 4.48 3.80
43.23 23.23 15.29 8.90 43.80 35.77 26.40 25.92 11.36 9.43
800
Field Strength (V/cm)
Figure 2. Current density (uNcm2)versus field strength (V/cm) for 50 mM KDS with 20% acetonitrile. This running buffer solution
on the linear portion of the plot in Figure 2. Table 2 lists the operating currents for all the micellar running buffer solutions at an applied voltage of 15 kV. The currents generated with the LDS micellar system were lower than those obtained with the SDS and KDS systems. The amount of current generated is dependent upon the amount of available charge carriers present in the buffer. In all of the micellar systems, a 5 mM sodium phosphate buffer was present. The currents reported in Table 2 will reflect the contribution of the 5 mM sodium phosphate buffer, but since the concentration of the surfactant counterions is much greater, the current generated will primarily be due to these counterions. It is well-known that the greater the mobility of an ion in solution, the greater its contribution to c~nductivity.~~ The mobilities for Lit, Na+, and K+ are 38.7,50.5, and 73.5 (x lo5 cm2/V*s),re~pectively.3~ It easy to see then from these data why the trend in operating currents is LiDS < SDS < KDS. Operation with the LiDS system represents a significant advantage over both SDS and KDS in terms of operating currents generated. An--Micelle Interactions. The interactions between the test analytes and LiDS and KDS were quite different from that of SDS. Table 3 contains the retention factors for some of the neutral analytes studied. The retention factors for the LiDS and SDS systems without the addition of organic modifer were a p proximately the same. However, as the percentage of acetonitrile increased from 0 to 20% in the running buffer, the retention factors for the LiDS system were signifkantly less than those obtained with SDS. In fact, the retention factors calculated with the SDS system appear to level off with the addition of 15 and 20%
acetonitrile. The reductions in retention factor with the addition of acetonitrile are a result of the decreasing water-micelle partition coefficient for the analytes. The only other possibility to alter the retention factors would have been a change in the phase ratio. For all practical purposes, the phase ratios in this study were constant due to similarities in the cmc values of the three surfactants and the expected similar aggregation numbers. In addition, if the phase ratio significantly influenced retention, then any changes in retention factor should have been proportional for all the surfactant systems, but as shown in Table 3, the addition of only 10%acetonitrile results in a much larger reduction in retention factor for LiDS than SDS. The LiDS micellar system offers an advantage with the sharp reduction in retention factor with the addition of acetonitrile. This should facilitate resolution optimization in comparison with SDS, especially for more hydrophobic analytes that tend to stay in the core of the micelle and elute at or near the tmcmarker. The KDSbased separations resulted in retention factors of a lower magnitude than SDS, but a similar leveling off of the retention factors with 15 and 20% acetonitrile was observed for benzyl alcohol and nitrobenzene. However, it appears it may be solute dependent for the more hydrophobic analytes as evidenced by the retention factors of benzophenone and biphenyl. It is important to remember that the KDS analyses were only possible with the addition of 15 and 20% acetonitrile. Organic Modifier Effects on Retention. The use of organic modifers in MEKC can have a significanteffect on the separation process. In terms of counterion binding to the micelle, the addition of organic modifier may increase the degree of counterion binding due to a decrease in the dielectric constant of the micellar solutions. This would destabilize the micelle and result in a larger cmc for the surfactant.37The increase in counterion binding could vary with the identity of the counterion. The use of organic modifiers can also decrease the cmc if the organic m o d ~ e r reduces the electrostatic repulsion between the ionic head groups in the micelle. In addition to these effects, the use of organic modifier in MEKC will increase the operating currents and change the magnitude of the electroosmotic flow and electrophoretic mobility of the micelle. As mentioned previously, the watermicelle partition coefficient of the analytes will also change. In order to get a better understanding of the effect of acetonitrile with the LiDS and SDS micellar systems, we have
(36) Atkins, P. W. Physical Chemisty, 3rd ed.; W. H. Freeman and Co.: New York. 1986.
(37) Meguro, IC; Esumi, K Surfactunts in Solution; Plenum Press: New York, 1991; Vol. 11.
generated the highest operating currents. See Experimental Section for operating conditions. Table 2. Operating Currents for LiDS, SDS, and KDS. % acetonitrile
LiDS
SDS
KDS
0 10 15 20
17.8 19.9 21.4 23.8
22.0 24.1 26.2 28.7
28.0 30.0
Applied voltage, 15 kV.
Analytical Chemistty, Vol. 67, No. 14, July 15, 1995
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Table 4. Solvent Strength Values for Various Analytes in SDS and LiDS
SDS
analyte
Sabs‘
log kw
benzyl alcohol
nitrobenzene benzophenone biphenyl nitrophenyl acetate nitrophenyl propionate nitrophenyl butyrate nitrophenyl valerate nitrophenyl caproate acetophenone propiophenone butyrophenone valerophenone hexanophenone
0.57 (10.14) 0.36 (10.11) 1.96 (10.26) 1.13 (10.24) 0.59 (10.15) 0.73 (10.17) 0.87 (10.20) 1.01 (10.24) 1.16 (10.28) 0.88 (10.17) 0.97 (k0.17) 1.07 (10.19) 1.17 (10.18) 1.23 (10.11)
-0.23 0.15 1.40 1.64 0.32 0.66 1.05 1.44 1.83 0.26 0.58 0.92 1.31 1.72
av
0.98 & 0.38
LiDS SElb
Sabs
log k w
0.57 0.36 2.02 1.16 0.60 0.74 0.89 1.03 1.18 0.90 0.99 1.09 1.20 1.27
1.64 (10.06) 1.22 (50.05) 4.18 (f0.13) 3.23 (10.30) 0.98 (h0.05) 1.21 (f0.07) 1.48 (f0.09) 1.69 (10.14) 1.92 (10.21) 1.65 (10.07) 1.82 (10.08) 2.05 (10.11) 2.28 (10.15) 2.46 (k0.22)
-0.20 0.16 1.44 1.64 0.39 0.73 1.12 1.51 1.90 0.30 0.62 0.96 1.34 1.75
1.00 & 0.40
1.99 h 0.85
&el
0.82 0.61 2.10 1.62 0.49 0.61 0.74 0.85 0.96 0.83 0.91 1.03 1.15 1.24 1.00 f 0.44
Absolute S value. * Relative S value.
calculated the Snyder “S” values developed originally for liquid chromatography (LC).38,39The magnitude of the Svalue indicates the strength of the solvent in the separation medium of interest. In reversed phase liquid chromatography (RPLC), the S values can be calculated using
that they are significantly different. This is similar to what is seen in RPLC with different types of columns. In order to compare each analyte with both micellar systems, a relative S value can be calculated using eq 9. Here, Sanalfle is the absolute S value
Sr = SanalytJSav log k = log k, - S@
where Q, is the volume fraction of organic modifier, k is the retention factor in the mobile phase containing Q, organic modifier, and k, is the retention factor in a totally aqueous system. The KDS system was not included in this section because kw values could not be obtained due to the minimum amount of acetonitrile required just to solubilize the surfactant. Acetonitrile was chosen as the organic modifier for use in this study for three reasons. First, we wanted to compare our results on counterions with those obtained in a previous study using the divalent Mg2+counterion in which the organic modifier employed had been acetonitrile. Second, acetonitrile interacts less with the capillary wall than methanol to affect the electroosmotic flow. Thiid, it does not change the properties of the micelle significantly because the interactions with the micelle are not as great.37 Table 4 lists the S values for the SDS and LiDS micellar systems. For the homologous series of alkylphenones and nitrophenyl esters, the S value increases with the carbon number, nc. This follows the same trend as that seen in RPLC.39,40In RPLC, there is a linear correlation between S and In k, or log k’ and Q, in a certain concentration range of organic modifier. This linear relationship is also observed in MEKC as the correlation coefficients (12) for the plots done were all greater than 0.985 (n L 5) for the concentration range of acetonitrile used in this study. The higher average absolute S value (1.99 f 0.85) obtained for the LiDS system indicates that acetonitrile is a stronger solvent in this micellar system than in the SDS system, where the average absolute S value was 0.98 f 0.38. If the S value for each analyte is compared statistically in both micellar systems, it can be seen (38) Reversed Phase Chromatography; Horvath, C., Ed.; Academic Press: New York, 1980; Vol. 2. (39) Snyder. L. R.; Dolan, J. W.; Gant, J. R. J. Chromatogr. 1979, 165, 3. (40) Tanaka. N.; Thornton, E. R. /. Am. Chem. Sot. 1977, 99,7300.
2320 Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
(9)
(8)
for the individual analytes and Sa, is simply the average S value for all the analytes in a particular micellar system. These data are also presented in Table 4. Inspection of the data in Table 4 reveals that the Sr values for the hydrophilic analytes are greater in the LiDS micellar system than in the SDS system. As the analytes become more hydrophobic in nature, the Sr values get closer in magnitude and in some cases for the more hydrophobic analytes the Sr value is greater in SDS than in LiDS. The sensitivity of each analyte to acetonitrile in each micellar system, in general, is dependent on the structure of the analytes. Comparison of average Sabsolute values for LiDS, SDS, and Mg@S)2, the latter obtained from a recently published provides further insight into the effect of counterions in MEKC in the presence of organic modifier. As reported in Table 4, the average Sabsolute value for LiDS is 1.99 f 0.85 and for SDS it is 0.98 f 0.38. The conclusion reached here is that acetonitrile is a stronger solvent in LiDS than in SDS. The previous report comparing SDS with Mg@S)2 revealed that acetonitrile is a stronger solvent in SDS than in Mg@S)2. In terms of counterion binding to the micelle, the Mg2+ ions experience the tightest binding to the micelle, followed by the Na+ ions, and finally the Li+ ions will be the least tightly bound to the micelle. The solvent strength of acetonitrile in MEKC is influenced by the counterion binding to the surface of the micelle. In terms of the dodecyl sulfate counterion, the solvent strength of acetonitrile is greatest in the order of Li+ > Na+ > Mg2’. This is significant because through judious control of the ratio of counterions in the separation medium it should be possible to control the strength or influence of an organic modifer, acetonitrile in this case, on the separation process. In terms of resolution enhancement, it should be possible to achieve baseline resolution of pairs of analytes simply by controlling the ratio of counterions in the running buffer. Previous ways of improving resolution have often
Table 5. Methylene Selectlvlty for LiDS, SDS, and KDS Using Alkylphenones and Nitrophenyl Esters
surfactant
% acetonitrile
log(methy1ene SelectiviQ)
0 10 15 20 0 10 15 20 15 20
0.370 (fO.001) 0.365 (fO.OO1) 0.356 (f0.003) 0.332 (f0.003) 0.372 (fO.OO1) 0.366 (2zO.001) 0.364 (f0.002) 0.363 (f0.002) 0.382 (10.004) 0.313 (f0.007)
0 10 15 20 0 10 15 20 15 20
0.377 (fO.OO1) 0.362 (f0.002) 0.342 (f0.003) 0.331 (f0.003) 0.381 (fO.OO1) 0.374 (10.001) 0.348 (f0.003) 0.350 (10.004) 0.486 (h0.005) 0.394 (fO.009)
a1kdDhenones hDS LiDS LiDS LiDS SDS SDS SDS SDS KDS KDS
nitrophenyl esters LiDS LiDS LiDS LiDS SDS SDS SDS SDS KDS KDS
included increasing surfactant concentrations or increasing the organic modifier content. Although these methods can provide the desired resolution improvements, they often come at the cost of increased analysis time and a much higher operating current. By controlling the ratio of counterions in solution, these types of disadvantages may be minimized. Selectivity. Methylene or hydrophobic selectivity (uaJ can be obtained from the ratio of retention factors for members of a homologous series that differ by only one CH2 group or from the slope of a plot of log retention factor versus carbon number (nc). Examination of the methylene selectivities can provide information on the polarity of the micelles. If the methylene selectivities are higher in one micellar medium, then the polarity of that medium will be less. Table 5 lists the log of the methylene selectivity for two homologous series, alkylphenones and nitrophenyl esters, with each micellar system and percentage of acetonitrile. As the difference in the polarity of the mobile phase and micellar phases is reduced through the addition of acetonitrile, the methylene selectivity is reduced. In general, the methylene selectivities were lower for both homologous series with the addition of acetonitrile. Methylene selectivities generated with the LiDS system were lower than with the SDS system, which indicates that LiDS has a greater polarity than SDS. Since the Lit counterions are not as tightly bound to the dodecyl sulfate micelle, the number of water molecules on the micelle surface (known as waters of hydration) will be greater.41The increased electrostatic repulsion of the ionic head groups due to the decreased counterion binding for LiDS allows for the inclusion of more water molecules on the surface and interfacial region (see Figure 1) of the micelle. As a result, LiDS is more polar than SDS or KDS. It is interesting to note that the methylene selectivities for both homologous series continued to decrease in LiDS with the addition of acetonitrile while with SDS they appear to level off with the addition of 1520% acetonitrile. This follows the same trend seen with retention factors in both systems. (41)Myers, D.Surfactant Science and Technology,2nd ed.; VCH Publishers: New York. 1992.
The results reported for the KDS micellar system indicate that this was the least polar of the micellar systems studied. The methylene selectivities were higher in KDS with 15%acetonitrile than either the SDS or LiDS system. Although we are not completely sure why the methylene selectivities are so much higher in KDS, especially for the nitrophenyl esters, this increase was also observed for Mg(Ds)~.2~ Overall, the methylene selectivity for the homologous series increased with the degree of counterion binding to the micelle in the order Li+ < Na+ < K+. The hydrophobicity of the dodecyl sulfate micellar systems increases as the counterion is changed from Lit to Na+ to K+. The effect of the various counterions on adjacent analyte selectivity is shown for the neutral analytes in Table 6. The effect of the addition of acetonitrile and the type of counterion can be better viewed in F i r e 3 (see Table 6 for analyte pair identi6cation). The changes in adjacent analyte selectivityare very similar for LiDS and SDS, but there is almost no change in selectivityfor the KDS system with the addition of 15-20% acetonitrile. It is worth noting that even though the adjacent analyte selectivities are almost the same in LiDS and SDS without any acetonitrile, the addition of acetonitrileinfluences the selectivities of the analyte pairs for the LiDS system much more than the SDS system. This should facilitate resolution optimization of analytes in LiDS since the selectivity of the analytes is more affected in LiDS by the addition of acetonitrile than in SDS. Counterion Effects on Efficiency and Resolution. The efficienciesof the neutral analytes were calculated by use of eq 3 for all three micellar systems and with the additions of acetonitrile. From the results shown in Figure 4, it is easy to see the signiftcantly higher efficiencies obtained with the LiDS system as opposed to the SDS and KDS systems. Even with the addition of 10%acetonitrile, the efficiencies generated with LiDS are still significantly higher than that in SDS with 10%acetonitrile. With the addition of acetonitrile the efficiencies of the more hydrophw bic analytes in the SDS system are not significantly altered, but in the LiDS system the efficiencies continue to increase as the hydrophobicity of the analyte increases. This represents a significant advantage in MEKC where hydrophobic analytes are often dif6cult to resolve. Hydrophilic analytes such as benzyl alcohol will primarily interact with the surface or interfacial region (see Figure 1)of the LiDS micelle. Since these types of analytes do not partition deep into the core of the micelle, they will not benefit as much from the improved mass transfer kinetics of the LDS system as would a more hydrophobic analyte. Consequently, enhancements in efficiency will be greatest for the more hydrw phobic analytes. As mentioned in the Theory section, any improvement in efficiency will enhance resolution (see eq 7). Figure 5 shows the separation of a homologous series of nitrophenyl esters. The LiDS system allows for partial resolution of homologs 6 and 7 while with the SDS system only one peak is visible, indicating complete peak overlap. Even though the elution range is much smaller for the LiDS system, the much higher separation efficiencies allow for an almost 10%improvement in resolution for the most hydrophobic solutes. Clearly the enhancement in resolution can be attributed to the higher efficiencies generated with LiDS. Under totally aqueous conditions, the efficiencies that can be obtained with LDS and SDS are very different. Figure 6 illustrates these differences quite clearly. The number of theoretical plates Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
2321
Table 6. Adjacent Analyte Selectivity in the Various Micellar Systems. % acetonitrile
NB-BA
0 10 15 20
2.29 2.59 2.71 2.74
1.31 1.22 1.17 1.12
0 10 15 20
2.37 2.50 2.60 2.58
1.28 1.26 1.22 1.21
15 20
2.68 2.70
1.13 1.13
M-BA
pNT-AN
m NT-p NT
BZ-mNt
BP-BZ
1.04 1.06 1.07 1.06
6.82 3.72 2.91 2.36
1.58 2.35 2.44 2.37
1.04 1.06 1.07 1.06
6.50 4.87 3.86 3.71
1.73 2.11 2.38 2.40
1.07 1.07
2.64 2.36
2.54 2.43
Surfactant, 50 mM LiDS 2.06 1.87 1.79 1.71 Surfactant, 50 mM SDS 2.08 1.98 1.89 1.87 Surfactant, 50 mM KDS 1.79 1.72
a Analyte pair identification: NB-BA, nitrobenzene-benzyl alcohol; AN-NB, anisole-nitrobenzene; pNT-AN, p-nitrotoluene-anisole; mNTpNT, m-nitrotoluene-p-nitrotoluene; BZ-mNT, benzophenone-m-nitrotoluene; BP-BZ, biphenyl-benzophenone.
I
1
0
5
10
15
Benzyl alcohol Nltrobenzene Benzophenone
Biphenyl
Decanophenone
Benzyl alcohol Nltrobcnzene Benzophenone
Biphenyl
Drcnnophenonc
20
% Acetonitrile 1AWNB
1
+ pNT.AN
6-
-0- “pNT -ABZ:mNT
5-
40000
4-
30000
h . c1I
>
. I
c1 u
= I x $ 3
1
0
5
10
15
20
-
N ~0000 10000
-
nBenzyl alcohol Nitrobenzene Benzophenone
Biphenyl
Decanophenone
Analyte
% Acetonitrile Figure 3. Adjacent analyte selectivity in LiDS (top) and SDS (bottom). Identification of analyte pairs is given in Table 6.
Figure 4. Separation efficiencies for selected neutral test analytes in LiDS (top),SDS (middle), and KDS (bottom).
approaches 300 000 for some of the neutral analytes with LiDS. Efficiencies rose sharply in LiDS for the analytes as the hydrophobicity of the analyte increased, whereas in the SDS system, the efficiencies remained approximately the same as the hydrophobicity of the analyte increased. For the analytes benzophenone, biphenyl, and decanophenone, the efficiencies obtained in LiDS were 2-3 times that obtained in SDS. The KDS micellar system provided the lowest separation efficiencies (see Figure 3). The magnitude of the efficiencies generated with each of these micellar systems can be explained in terms of the counterion
binding to the micelle. Since the Lit counterions are not as tightly bound as the Naf and K‘ ions to the micelle, the polar head groups of the micelle will not be as rigid. This allows for an increase in the water penetration of the micelle surface and an increase in the polarity of the outer core region of the micelle. Consequently, moderately hydrophobic to very hydrophobic analytes will not interact as strongly with the micelle. The result is an increase in the rate of transfer in and out of the micelle. In terms of the MEKC separation process, the net result will be higher separation efficiencies due to a decrease in the amount of band broadening possible for the analytes.
2322 Analytical Chemistry, Vol. 67,No. 74, July 75, 7995
I -Benzyl alcohol 2-Nilrobenzene 3-Anisole
-
4-p-N~tmroiucnr 5 m-Nitratolucnc 6 -Benzophenone 7 -Biphenyl
2
2
IO.
'
SDS l
8-Decanophenone 8
3
L
0 .b I
O
3
2
4
5
6
6
7
Time (minutes)
I
o
2
4
6
6
10
12
10
12
I4
Time (minutes)
I .I
1
4
I
6
14
Time (minutes)
Figure 5. Separation of the nitrophenyl ester homologous series
in 50 mM LiDS-20% acetonitrile (top) and 50 mM SDS-20% acetonitrile (bottom). There is a slight improvement in resolution for peaks 6 and 7 in the LiDS system. Peak identification of nitrophenyl esters: (1) acetate, (2) propionate, (3) butyrate, (4) valerate, (5) caproate, (6) caprylate, and (7)caprate.
z
2 - Nitrobenzene 3 -Anisole
6
3' 0I
2
4 - p-Niuotoluene
7
5 - m-Nitrotoluene 6 . Benzophenone 7 - Biphenyl 8 - Decanophenone
a 8
10
1 2
I
300000
MI
6 7
4 5
I
3
I
SDS I
8
A
200000
Time (minutes)
N
I":.
100000
f
10
6
I
0
I
,I
2
,
4
6
4
7
A 10
12
4 8 12
16
111
20
22
Time (minutes)
Figure 8. Comparison of separations obtained with 50 mM surfacBenzyl alcohol
Nitrobenzene
Bcnmphenone
Biphenyl
Deeanophenone
tant and 15% acetonitrile. LiDS (top), SDS (middle), and KDS (bottom).
m y t e
Figure 6. Comparison of separation efficiencies obtained under totally aqueous conditions with 50 mM LiDS and 50 mM SDS for selected neutral test analytes. Efficiencies were -2.5 times greater
in LiDS for the hydrophobic analytes than in SDS.
Figure 7 is a comparison of the separations obtained with LiDS and SDS under totally aqueous conditions. In both micellar systems all of the analytes are resolved except for p- and m-nitrotoluene. The resolution of these peaks is similar but slightly better with SDS. The most glaring difference is the sharpness of the analyte peaks with LiDS (bottom), especially for the more hydrophobic analytes. In terms of analysis time, the separation performed in SDS was almost twice as long as the LiDS analysis. This is particularly advantageous for both method development and routine analysis. Figure 8 compares the separation obtained for all three micellar systems with the addition of
15% acetonitrile. The improvement in signal-to-noise ratio for peaks 6-8 when LiDS is used is a result of the much sharper peaks (higher efficiencies) generated. The analytes are fully resolved in each system but at a cost of longer analysis times in both the SDS and especially KDS systems. Elution Range. It is important to keep in mind the magnitude of the elution range &/to) in MEKC. From eq 7, we see that a decrease in the elution range will result in a decrease in resolution. Table 7 lists the t,,/t,, values for each surfactant system with the various percentages of acetonitrile added. Comparing the results with the addition of 15%acetonitrile to each system shows that KDS provides the largest elution range followed by SDS and finally LiDS. The smaller elution range associated with LiDS may offset the advantage of higher efficiencies in terms of resolution when a large number of analytes are present in a sample. However, Analytical Chemistty, Vol. 67, No. 14, July 15, 1995
2323
"..- I
I
0.20
0
5
10
1.5
20
% Acetonitrile
Figure 9. Electroosmotic velocities as a function of the percentage
of acetonitrile added in LiDS and SDS. Table 7. Elution Ranges for LiDS, SDS, and KDS
surfactant
% acetonitrile
tmclto
LiDS
0 10 15 20 0 10 15 20 15 20
2.72 3.35 3.88 4.38 3.91 4.91 5.85 6.81 9.75 10.52
SDS
KDS
the improved resolution for the most hydrophobic analytes shown in Figure 5 with LiDS as opposed to SDS indicates that the higher efficiencies generated with LiDS override the loss in resolution due to the smaller elution range. The addition of acetonitrile from 0 to 20%increases the elution range by almost 40%with LiDS and almost 60%with SDS. In order to understand why the elution range is significantly altered by simply using a different counterion, we have to consider counterion effects on electroosmotic flow and, more importantly, on the electrophoretic mobility of the micelle due to differences in the degree of counterion binding. Ignoring the relatively small effects on electroosmotic flow (see Figures 5,7, and S), one might expect, since the Li+ ions are not as tightly bound, a more negatively charged micelle and hence a greater absolute micelle electrophoretic mobility. Since the electroosmotic flow differs by less than 20%in pure/aqueous buffer and is almost identical with 15-20% acetonitrile (Figure 9), a greater absolute micelle electrophoretic mobility would mean a larger elution range (flow is from anode to cathode). This is not the case, however. Three possible explanations can be offered. First, the solution may lie in the number of surfactant monomers, aggregation number, necessary for the formation of the LiDS, SDS, and KDS micelles. (42) Treiner, C.; Makayssi, A /. Colloid Interface Sci. 1992,150, 314. (43) Shanks. P. C.; Franses, E. I. J. Phys. Chem. 1992,96, 1794-1805.
2324 Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
The aggregation number generally increases with a higher degree of counterion binding.41 From electrostatic models relating counterion binding to micelle structure, it is known that as the micelle aggregation number increases, the electric field created by the head groups increases and the fraction of ions condensed at the micelle surface also increases.42This would be one explanation why the SDS micelle had a larger absolute electrophoretic mobility than the LiDS micelle. Due to the higher aggregation number of SDS, there were more negatively charged polar head groups on the surface of the micelle and hence a greater absolute micelle electrophoretic mobility. The same rationale can be applied to the KDS system, which exhibited the largest elution range. A second explanation is that the LiDS micelle is more solvated as a result of the decreased counterion binding. Greater solvation with the LiDS micelle would help to explain why the absolute electrophoretic mobility of a LiDS micelle was less than an SDS micelle. The frictional drag would be larger for LiDS, thus reducing its electrophoretic mobility. A third possibility is that it is a combination of a lower aggregation number and higher degree of solvation for the LiDS micelle that reduces its absolute electrophoretic mobility. CONCLUSIONS
The results of this study provide further insight into the role of inorganic dodecyl sulfate micelle counterions in MEKC. Clearly, the nature of the counterion can significantly influence separations in MEKC. It should be possible to vary the ratio of counterions in the separation medium to attain the desired selectivity and resolution for hydrophilic to moderately hydrophobic analytes. The analysis of very hydrophobic analytes may be best in a running buffer consisting primarily of Li+ ions. The KDS analyses performed in this investigation were only possible with the addition of at least 15% acetonitrile. Future studies will include analysis of this micellar system under totally aqueous conditions by operating at temperatures above its Krafft point. The advantages of using LiDS as opposed to the often used SDS include (i) higher separation efficiencies, (ii) shorter analysis times, (iii) lower operating currents, (iv) better signal-to-noise ratios, and (v) easier resolution optimization. In situations where a large number of analytes are present in a sample, the use of SDS may be desirable since the peak capacity is slightly larger in comparison to LiDS. The compatibility of LiDS with the sample of interest should also be monitored since LiDS is not as frequently used as SDS. ACKNOWLEDGMENT
The authors thank Waters Inc. for their support of this investigation. We are also grateful to the Department of Chemistry and the Graduate School at Villanova University for providing financial support necessary to present portions of this research at HPCE '94 in San Diego, CA Received for review April 20, 1995. Accepted April 21, 1995.@ AC950388U
* Abstract published in Advance ACS Abstracts, June
1, 1995.
