Anal. Chem. 1986, 58,479-481
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CORRESPONDENCE Capillary Zone Electrophoresis of Neutral Organic Molecules by Solvophobic Association with Tetraalkylammonium Ion Sir: High-voltage zone electrophoresis performed in open tubular capillaries offers several advantages over more conventional approaches to electrophoresis (I,2). Efficient cooling of the separation chamber allows the use of high voltages, resulting in high-resolution separations in a short period of time. Since no stabilizers are used, zone broadening effects such as "eddy migration" and adsorptive interactions between solutes and stabilizers are eliminated. On-column detection devices permit accurate measurement of migration times and zone concentration profiles in a format similar to a chromatogram. It is desirable to extend capillary zone electrophoresis to separations in nonaqueous and mixed waterlorganic media, in order to broaden the range of compounds to which this technique can be applied. Electrophoretic separations in nonaqueous media have received little attention in the literature (3-7), perhaps because electrophoresis has been traditionally employed in separations of water-soluble compounds of biological origin. Nonaqueous and mixed media will be particularly useful in electrophoretic separations of compounds that are insoluble in water. Nonaqueous solvents permit a wider range of acidlbase strength than is available in water, and thus a wider range of compounds may be ionized through the use of acid/base chemistry. Nonaqueous and mixed media also allow the use of other means of placing charge on solutes. In the case of this work, a mixed water/acetonitrile medium is used to promote a solvophobic interaction between the solute and a tetraalkylammonium ion, to place a charge on an otherwise neutral molecule. The use of nonaqueous and mixed media should allow electrophoretic separations of many more classes of compounds, if the solutes can be rendered ionic. Electrokinetic separations of uncharged organic molecules in aqueous micellar solutions in open tubular capillaries have been reported recently (8,9). While the micelles migrate by electrophoresis, the separation mechanism involved is a form of partition chromatography. This technique makes use of some of the advantages of capillary electrophoresis in separations of molecules not traditionally analyzed by electrophoresis. It would seem that electrokinetic micellar chromatography is limited to solutes that have some degree of solbuility in water, since the addition of significant amounts of organic modifiers will likely cause the micelles to disintegrate. An electrophoretic method has been devised to effect separations of less hydrophilic compounds. The electrophoretic medium used in these experiments consists of tetrahexylammonium perchlorate (THAP) dissolved in acetonitrile modified with varying amounts of water. When analyte molecules are dissolved in this medium, they undergo a solvophobic interaction with the tetrahexylammonium (THA+) ion, forming a positively charged species that can migrate in an electric field. Thus electrophoretic separations of "neutral" organic molecules can be accomplished. EXPERIMENTAL SECTION Apparatus. Electrophoresis was performed in a fused silica capillary that was 100 cm long and had a 75 fim i.d. (Polymicro 0003-2700/86/0358-0479$01.50/0
Technologies, Inc., Phoenix, AZ). A regulated high-voltage dc power supply (Spellman High Voltage Ekctronics Corp., Plainview, NY) delivering from 0 to *30 kV was used to drive the electrophoresis. Detection was carried out by use of a fixedwavelength on-column UV absorption detector (IO). The detedion wavelength was 229 nm. The electrophoresis setup has been described in detail elsewhere ( I , 2). Reagents. Tetrahexylammonium perchlorate was obtained from Alfa Producta (Danvers, MA) and was used without further purification. Reagent grade acetonitrile was obtained from J. T. Baker Chemical Co. (Phillipsburg, NJ). Benzo[ghi]perylene, perylene, 9-methylanthracene, and mesityl oxide were obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI). Pyrene was obtained from Eastman K d a k Co. (Rochester,NY). Naphthalene and formamide were obtained from Fisher Scientific Co. (Fair Lawn, NJ). Procedure. The procedure has been described in greater detail elsewhere ( I , 2). The capillary was filled with the electrophoretic medium via suction. Once the capillary was filled, both ends of the capillary were dipped into reservoirs containing the electrophoretic medium. The reservoirs were specially designed with covers to minimize evaporation of the nonaqueous solvent. The reservoir at the positive end of the capillary, where samples were introduced, was connected to the high-voltage power supply with a platinum wire electrode. The other reservoir at the opposite end of the capillary was grounded in a similar fashion. The detector was located, on-column, 15 cm from the grounded end of the capillary. Samples were injected by electromigration at 2.5-5 kV for 7-20 s. Electrophoresis was carried out with an applied voltage of 20 kV. In Figures 2 and 3, each data point was the average result of at least three measurements. The error bars represent 1 standard deviation. RESULTS AND DISCUSSION The electropherogram in Figure 1 shows a separation of five aromatic hydrocarbons, mesityl oxide, and formamide. The solvent composition of the electrophoretic medium was 50% acetonitrile and 50% water (v/v). The concentration of THAP was 0.025 M. The applied voltage was 20 kV, and the current was 12 PA. The larger, more nonpolar solutes migrate fastest, indicating that they have the strongest interactions with the THA+ ion. Mesityl oxide, a small, polar molecule has a low mobility, indicating that it interacts more weakly with the THA+ ion. Formamide is used as a neutral marker. Ideally, the neutral marker is a species that is completely uncharged and migrates by electroosmotic flow alone. The migration time of the neutral marker, therefore, can be used to determine the rate of electroosmotic flow. Formamide is a small, polar molecule, which is similar in size to acetonitrile. It is unlikely that it will interact more strongly with the THA+ ion than the solvent does, and thus it is a reasonable choice for the neutral marker. One can imagine several operating parameters that can affect the migration behavior of solute molecules in this system. One of these is the solvent composition. Electrophoretic mobilities were measured for all six test solutes in mixed acetonitrile/water media, which varied in water content from 0 to 50% (v/v) water. The concentration of THAP was 0.025 M in each case. Solute concentrations were adjusted to give reasonable peak heights and were at least 50 times less 0 1986 American Chemical Soclety
480
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986 F
101
1
'~
T 0?5 AU
0
5
I 0
20
15
I
3t
25
Time (min) Figure 1. Zone electrophoretlc separation of organic compounds: (A)
benzo[ghi]perylene (1 X lo4 M),(B) perylene (1 X lo4 M), (C) pyrene (1 X IO-' M), (D) 9-methylanthracene (1 X lo-' M), (E) naphthalene (5 X IOm4 M), (F) mesityl oxide (1 X M), (G) formamlde.
Figure 2. Electrophoretic mobility as a function of percent water in electrophoretic medium for a series of test solutes: (1) benzo[ghi]perylene, (2) perylene, (3) pyrene, (4) 9-methylanthracene, (5) naphthalene, (6) mesityl oxide.
concentrated than THAP, except for the neutral marker formamide, which was 0.5% (v/v) in concentration. In capillary zone electrophoresis, mobilities can be measured directly from an electropherogram. The observed migration velocity of a solute ion is the s u m of ita migration velocity and its velocity due to electroosmosis. Both electrophoretic migration and electroosmosis are related to electric field strength in the following way: Vobsd
= pelE + p
e s
=
bel
+ peo)E
(1)
where E is the electric field strength, pel is the electrophoretic mobility of the solute, and peo is the electroosmotic flow coefficient. It is convenient to refer to the sum (pel + peo)as the observed mobility. Observed mobility can be calculated from the migration time of a solute using the following equation:
where L is the total length of the capillary, 1 is the length from the end of the capillary where sample is introduced to the detector, t is the migration time, and Vis the applied voltage. The electroosmotic flow coefficient can be calculated from the migration time of a neutral marker using the following equation: Peo
=
L1
t,v
(3)
where t,, is the migration time of the neutral marker. Then peocan be subtracted from the observed mobility of the solute to obtain electrophoretic mobility pel
= pobsd - peo
Flgure 3. Electrophoretic mobility as a function of concentration of THAP in the electrophoretic medium. Solutes are Indicated as in Figure 2.
of THA+ may be decreasing. This is most likely because, at high water concentrations, the hydrophobic solutes are forced to interact more strongly with the THA+ ion. Further supporting this hypothesis is the fact that the mobilities of the most hydrophobic solutes increase most rapidly, while the mobilities of the more hydrophilic species increase less quickly. It is important to note that in no case does mobility appear to level off as water content increases. This suggests that the mechanism for migration involves a dynamic equilibrium between an associated species with a positive charge and dissociated species with no charge, rather than a saturable binding between the solute and THA+ ion. This mechanism can be represented by the following model:
s + L+ + SL+ SL+ + L+ + SL22+
(4)
Figure 2 shows that as the water content of the electrophoretic medium increases, the electrophoretic mobilities of the solutes first decrease and then increase. There are several factors that could account for this behavior. As the water content of the electrophoretic medium increases, ita viscosity increases. This could account for the initial decrease in mobility for all test solutes. However, as the water content of the medium increases, so does the dielectric constant. Theoretically, this should increase the effective charge on the solute ions, thus increasing their mobility. Experimentally, this does not seem to be the case. As the water content of the medium increases, the current decreases, indicating that the mobility of the THA+ decreases. Since the THA+ ion and the perchlorate ion are not well-solvated in water, they probably tend to form ion pairs, causing a decrease in the mobility of THA+. However, when the water content of the medium reaches 30%, the mobilities of the test solutes start increasing steadily, despite the likelihood that the mobility
SL:+
+ L+ +=SL33+ ..., etc.
where S is the solute and L+ is the THA+ ion, loosely referred to as a ligand. However, the likelihood of a solute combining with more than one THA+ is probably small, due to charge repulsion. Figure 3 illustrates the relationship between electrophoretic mobility and concentration of THAP. Electrophoretic mobilities were measured for all six test solutes in a medium consisting of 70% acetonitrile and 30% water (v/v) and varying in THAP concentration from 0.005 to 0.05 M. The graph shows that as the concentration of THAP in the medium increases, so do the mobilities of the solutes. Initially, the increase of mobility with concentration of THAP is rapid, but as the concentration of THAP increases, the mobilities begin to level off. The slopes of the curves are larger for the large, solvophobic solutes and small for the smaller, more polar solutes. This again indicates stronger interactions between
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
the larger solutes and the THA+ ion, as would be expected. It is also noteworthy that at the lowest THAP concentration tested, the concentration of THAP ie still 10 times greater than that of naphthalene and 50 times greater than that of the other solutes, and yet the mobilities of the solutes are Iow. As more THAP is added to the medium, the mobilities increase steadily. This is additional evidence supporting the dynamic equilibrium mechanism, since if an irreversibly bound complex were being formed, one would expect that a 10-fold excess of THAP would be enough to saturate the solute with THA+ ions and that further increases in THAP concentration would not result in a large increase in electrophoretic mobility. It is important to discuss the role that electroosmosis plays in the separations. It has been shown previously that as electroosmotic flow becomes faster, resolution in capillary zone electrophoresis is diminished (I). Optimum resolution is achieved if the electroosmotic flow coefficient and electrophoretic mobility are equal in magnitude, but opposite in sign. The obvious cost of this approach to high resolution is long migration time. Electroosmosis becomes faster as the viscosity of the electrophoretic medium decreases. For a medium containing pure acetonitrile, the viscosity is low, resulting in a fast electroosmotic flow rate. This combined with the fact that the mobilities of the solutes are very low in this medium results in a very poorly resolved electropherogram. As the water content of the medium increases, viscosity also increases (over the range tested). Therefore, the increased resolution observed because the electrophoretic mobilities are increasing at different rates is enhanced by reduced electroosmotic flow. It is possible to exercise some control over electroosmotic flow through surface treatment of the fused silica capillary. Perhaps better control of electroosmosis will lead to higher resolution separations, albeit at the expense of analysis time. Some parallels can be drawn between this technique and reverse-phase liquid chromatography (RPLC). The solvophobic interaction between solute and THA+ ion is similar to that between a solute and an RPLC stationary phase. The larger nonpolar solutes are more tightly bound to the THA+ ion than the smaller or more polar solutes. Similarly, the larger solutes would spend more time in a reverse-phase LC
481
stationary phase than the smaller soIutes. The effect of solvent composition on retention in RPLC and migration in this electrophoretic technique is also analogous. As the water content of the solvent mixture increases, solutes interact more strongly with the THA' ion, just as they would spend more time in a RPLC stationary phase. However, this technique is significantly different from liquid chromatography in that it involves a one-phase system. It is an electrophoretic technique in which a charge is placed on the solutes by association with a tetraalkylammonium ion. Further experiments will be carried out to formulate a more precise model for the mechanism responsible for migration and to investigate fully the capabilities of this form of electrophoresis. Registry No. T W , 4656-81-9;benzo[ghi]perylene, 191-24-2; perylene, 198-55-0;pyrene, 129-00-0;9-methylanthracene,779-02-2; naphthalene,91-20-3;mesityl oxide, 141-79-7;formamide, 75-12-7.
LITERATURE CITED Jorgenson, J. WL;Lukacs, K. D. Anal. Chem. 1981, 5 3 , 1298-1302. Jorgenson. J. W.; Lukacs, K. D. Science 1983, 222, 266-272. Paul, M. H.; Durrum, E. L. J . Am. Chem. SOC.1952, 7 4 , 4771-4773. Leighton, D.; Moody, G. J.; Thomas, J. D. R. Analyst (London) 1974, 99, 442-452. (5) Korchemnaya, E. K.; Ermakov, A. N.; Bochkova, L. P. Zh. Anal. Khim. 1978, 3 3 , 816-821. (6)Tshabalala, M. A.; Schram, S. 6.; Gerberlch, F. G.; Lowman, D. W.; Rogers, L. 6. J . Chromatogr. 1981, 207, 353-363. (7) Parekh, N. J.; Fatmi, A. A.; Tshabalala, M. A.; Rogers, L. B. J . Chromatogr. 1984, 314, 65-82. (8) Terabe, S.;Otsuka, K.; Ichikawa, K.; Tsuchiya, A,; Ando, T. Anal. Chem. 1984, 56, 113-116. (9) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 5 7 , 834-841. (10) Walbroehl, Y.; Jorgenson, J. W. J . Chromatogr. 1984, 315, 135-143. (1) (2) (3) (4)
Yvonne Walbroehl James W. Jorgenson" Department of Chemistry University of North Carolina at Chapel Hill Chapel Hill, North Carolina 27514 RECEIVED for review June 6,1985. Accepted September 23, 1985. Support for this work was provided by a grant from the Alfred P. Sloan Foundation and by the National Science Foundation under Grant CHE-8213771.
