Behavior and Use of Nonaqueous Media without Supporting

Similar experiments were performed using hydroorganic mobile phases (ACN/water, MeOH/water) across the complete compositional range (100% ...
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Anal. Chem. 1997, 69, 3251-3259

Behavior and Use of Nonaqueous Media without Supporting Electrolyte in Capillary Electrophoresis and Capillary Electrochromatography Paul B. Wright,† Ashley S. Lister, and John G. Dorsey*,‡

Department of Chemistry, Florida State University, Tallahassee, Florida 32306-4390

Five nonaqueous solvents (acetonitrile, methanol, N,Ndimethylformamide, dimethyl sulfoxide, formamide) and deionized water were investigated for their ability to support electroosmotic flow (EOF) without electrolytic additives. In general, flow was found to be equal to or greater than flow with typical CE buffer systems. The magnitude of EOF was determined for each solvent by open tubular capillary electrophoresis (CE) and related to viscosity (η), dielectric constant (E), and the ratio of dielectric constant to viscosity (E/η). Zeta potentials (ζ) were derived indirectly from flow data and tabulated. Comparisons of flow behavior and ζ were made between pure solvents and conventional CE buffers, and questions of equilibration and reproducibility were addressed. Similar experiments were performed using hydroorganic mobile phases (ACN/water, MeOH/water) across the complete compositional range (100% water-100% organic), with flow characteristics and ζ reported for each mobile phase system. Packed capillary columns (5-µm ODS) were evaluated for flow and retention stability under capillary electrochromatographic (CEC) conditions. A separation of 11 polycyclic aromatic hydrocarbons was performed in under 13 min by CEC with an ACN/water mobile phase. Reduced plate heights (h) were calculated between 2.5 and 3.0 for solutes with capacity factors (k′) up to 4.5 for the most retained solute. Capillary electrophoresis (CE) has experienced rapid advancement since its introduction in 1981.1 The technique possesses both high resolving power and efficiencies with fast analysis times. These features have attracted researchers to the technique, resulting in many contributions in the field.2 Although CE is limited to only charged species, neutral compounds can be separated if chemical equilibria are invoked through interaction with some other charged species in the buffer. A notable contribution was made by Terabe, who introduced the use of micelles in CE.3 The technique was termed micellar electrokinetic chromatography (MEKC) and has enjoyed rapid growth and popularity. MEKC separates compounds on the basis of differential partitioning to the micelle. Most of the early † Current address: Rayonier Research Center, Analytical Development Department, 4474 Savannah Hwy., Jesup, GA 31545. ‡ E-mail: [email protected]. (1) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (2) St. Claire, R. L. Anal. Chem. 1996, 68, 569R-586R. (3) Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T. Anal. Chem. 1984, 56, 111-113.

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applications of MEKC focused on separation of polar compounds that are soluble in the surfactant-containing buffer. In order to solubilize and subsequently separate more hydrophobic compounds, several additives have been utilized in MEKC, including organic solvents, cyclodextrins, and bile salts.4-6 An inherent problem in MEKC is the limited elution range, or elution window. Extension of the elution range, defined here as tmic/t0, where t0 is the migration time for an uncharged and uncomplexed species and tmic is the migration time of the micelle, has been the focus of much research.4,7-20 Despite these efforts, basic limitations to MEKC still exist, specifically, coelution of hydrophobic compounds with the micelle resulting in a lack of separation and the insolubility of many classes of compounds in the surfactantcontaining MEKC buffer. These disadvantages preclude the utility of capillary electrophoresis as a routine analytical technique. Organic solvents as modifiers have been shown to dramatically affect both CE and MEKC separations.2,21-29 In our own studies, (4) Gorse, J.; Balchunas, A. T.; Swaile, D. F.; Sepaniak, M. J. J. High Resolut. Chromatogr. Chromatogr. Commun. 1988, 11, 554-559. (5) Copper, C. L.; Staller, T. D.; Sepaniak, M. J. Polycyclic Aromat. Compd. 1993, 3, 121-135. (6) Cole, R. O.; Sepaniak, M. J.; Hinze, W. L.; Gorse, J.; Oldiges, K. J. Chromatogr. 1991, 557, 113-124. (7) Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, 834-841. (8) Cohen, A. S.; Terabe, S.; Smith, J. A.; Karger, B. L. Anal. Chem. 1987, 59, 1021-1027. (9) Balchunas, A. T.; Sepaniak, M. J. Anal. Chem. 1987, 59, 1466-1470. (10) Balchunas, A. T.; Swaile, D. F.; Powell, A. C.; Sepaniak, M. J. Sep. Sci. Technol. 1988, 23, 1891-1904. (11) Balchunas, A. T.; Sepaniak, M. J. Anal. Chem. 1988, 60, 617-621. (12) Cai, J.; El Rassi, Z. J. Chromatogr. 1992, 608, 31-45. (13) Cole, R. O.; Sepaniak, M. J. LC-GC 1992, 10, 380-385. (14) Ahuja, E. S.; Little, E. L.; Nielson, K. R.; Foley, J. P. Anal. Chem. 1995, 67, 26-33. (15) Foley, J. P. Anal. Chem. 1990, 62, 1302-1308. (16) Kaneta, T.; Tanaka, S.; Taga, M.; Yoshida, H. J. Chromatogr. 1992, 609, 369-374. (17) Rasmussen, H. T.; Goebel, L. K.; McNair, H. M. J. Chromatogr. 1990, 517, 549-555. (18) Terabe, S.; Ishihama, Y.; Nishi, H.; Fukuyama, T.; Otsuka, K. J. Chromatogr. 1991, 545, 259-368. (19) Tsai, P.; Patel, B.; Lee, C. S. Anal. Chem. 1993, 65, 1439-1442. (20) Vindevogel, J.; Sandra, P. Anal. Chem. 1991, 63, 1530-1536. (21) Lee, Y. J.; Price, W. E.; Sheil, M. M. Analyst 1995, 120, 2689-2694. (22) Janini, G. M.; Chan, K. C.; Muschik, G. M.; Issaq, H. J. J. Liq. Chromatogr. 1993, 16, 3591-3607. (23) Schwer, C.; Kenndler, E. Anal. Chem. 1991, 63, 1801-1807. (24) Fujiwara, S.; Honda, S. Anal. Chem. 1987, 59, 487-90. (25) Shi, Y.; Fritz, J. S. J. High Resolut. Chromatogr. 1994, 17, 713-718. (26) Zemann, A. J. J. Capillary Electrophor. 1995, 2, 131-136. (27) Idei, M.; Mezo, I.; Vadasz, Z.; Horvath, A.; Teplan, I.; Keri, G. J. Liq. Chromatogr. 1992, 15, 3181-3192. (28) Potter, K. J.; Allington, R. J. B.; Algaier, J. J. Chromatogr., A 1993, 652, 427-429.

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a CE separation of nortriptyline and protriptyline, which are positional isomers, is shown to improve dramatically by the addition of 20% acetonitrile to the buffer.30 At a buffer pH of 9, these two compounds are both positively charged but are not resolved by CE alone. Schwer and Kenndler studied the general effect of a series of organic solvents on the electrokinetic properties of fused silica, i.e., the electroosmotic velocity (νeo) and zeta potential (ζ).23 They observed that the electroosmotic velocity in fused silica capillaries was always reduced by an increasing percentage of organic modifier in the buffer. This effect was not attributed solely to changes in viscosity of the organic modifier-buffer mixed solvent because the well-known viscosity vs percent organic solvent curves possess a maximum.23,31-33 If viscosity was the principle cause of the decrease in electroosmotic velocity, a minimum should occur in the plots of electroosmotic velocity vs percent organic modifier in the CE buffer; however, a steady decrease was seen. Changes in the electroosmotic velocity, therefore, were attributed to decreases in the ζ with an increase in percentage organic modifier.23 The use of nonaqueous media is prominent in classical electrophoresis techniques, such as gel electrophoresis and paper electrophoresis. Korchemnaya provided an excellent review of the use of nonaqueous and mixed solvent media in classical electrophoresis techniques.34 A report of CE separations utilizing 100% nonaqueous media first appeared in 1984.35 The subject has been the focus of more in depth research more recently, however, by several groups.36-48 Nonaqueous mobile phases have also been utilized in capillary electrochromatography (CEC).49-51 With a few notable exceptions,39,49-51 most of the nonaqueous studies referenced above utilize a supporting electrolyte in the nonaqueous media in order to provide a conductive medium that can produce currents in the microampere range, as are typical in capillary zone electrophoresis. Three studies involved nonaqueous mobile phases in CEC,50 while another utilized 100% Nmethylformamide (NMF) as a separation medium in CE to achieve separation of charged carboxylic acids.39 In the latter study,39

however, there were no descriptions of acid/base properties for the carboxylic acids in NMF. Organic solvents are of interest in capillary electrophoretic separations for several reasons. Most obviously, their use extends the range of applications for the technique by enhanced solubility of analytes, addressing one of the main limitations of CE techniques. In addition, organic solvents offer greater flexibility in selectivity adjustment and permit a wider range of acid/base strength than in water, extending the range of compounds that can be ionized. Chemical and physical properties of nonaqueous solvents are much different from those of water and can be exploited in the optimization of CE separations. Other advantages include reduced interaction of hydrophobic compounds with the negatively charged capillary wall, ion-pairing capabilities, and the ability to invoke various forms of chemical equilibria in order to place a charge on compounds and thereby allow for a separation.37 The use of typical organic solvents, such as acetonitrile (ACN), results in very low currents compared to typical CE buffers. As a result, the amount of Joule heat produced under nonaqueous CE conditions vs buffered aqueous systems is greatly reduced, allowing much higher electric field strengths than are currently used in CE. This should result in faster, more efficient separations, considering the simple relationship between electric field strength and efficiency given in eqs 1-3.1 The time, t, for a solute to migrate the length of a capillary is given by

(29) Monnig, C. A.; Kennedy, R. T. Anal. Chem. 1994, 66, 280R-314R. (30) Wright, P. B. Ph.D. University of Cincinnati, Cincinnati, OH, 1996. (31) D'Aprano, A.; Fouss, R. M. J. Phys. Chem. 1969, 73, 400-406. (32) Melander, W. R.; Horvath, C. High Performance Liquid Chromatography; Academic Press: New York, 1980. (33) Tourky, A. R.; Abdel-Hamid, A. A. Z. Phys. Chem. 1971, 274, 289-301. (34) Korchemnaya, E. K.; Ermakov, A. N. J. Anal. Chem. USSR (Engl. Transl.) 1978, 33, 635-639. (35) Walbroehl, Y.; Jorgenson, J. W. J. Chromatogr. 1984, 315, 135-143. (36) Sahota, R. S.; Khaledi, M. G. Anal. Chem. 1994, 66, 1141-1146. (37) Salimi-Moosavi, H.; Cassidy, R. M. Anal. Chem. 1995, 67, 1067-1073. (38) Ng, C. L.; Lee, H. K.; Li, S. F. Y. J. Liq. Chromatogr. 1994, 17, 3847-3857. (39) Jansson, M.; Roeraade, J. Chromatographia 1995, 40, 163-169. (40) Okada, T. J. Chromatogr., A 1995, 695, 309-317. (41) Bjornsdottir, I.; Hansen, S. H. J. Chromatogr., A 1995, 711, 313-322. (42) Bjornsdottir, I.; Hansen, S. H. J. Pharm. Biomed. Anal. 1995, 13, 14731481. (43) Benson, L. M.; Tomlinson, A. J.; Reid, J. M.; Walker, D. L.; Ames, M. M.; Naylor, S. J. High Resolut. Chromatogr. 1993, 16, 324-326. (44) Tomlinson, A. J.; Benson, L. M.; Naylor, S. LC-GC 1994, 12, 122-130. (45) Tomlinson, A. J.; Benson, L. M.; Naylor, S. Am. Lab. 1994, 26, 29-36. (46) Tomlinson, A. J.; Benson, L. M.; Naylor, S. J. High Resolut. Chromatogr. 1994, 17, 175-177. (47) Tomlinson, A. J.; Benson, L. M.; Gorrod, J. W.; Naylor, S. J. Chromatogr., B 1994, 657, 373-381. (48) Hansen, S. H.; Tjornelund, J.; Bjornsdottr, I. TrAC, Trends Anal. Chem. 1996, 15, 175-180. (49) Jorgensen, J. W.; Lukacs, K. D. J. Chromatogr. 1981, 218, 209-216. (50) Whitaker, K. W.; Sepaniak, M. J. Electrophoresis 1994, 15, 1341-1345. (51) Tsuda, T.; Nomura, K.; Nakagawa, G. J. Chromatogr. 1982, 248, 241-247.

where D is the diffusion coefficient of the solute. Using the statistical equivalence of variance and the number of theoretical plates, the maximum efficiency, N, for a CE separation is given by

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t ) L2/µeV

(1)

where L is the capillary length, µe is electrophoretic mobility of the solute, and V is the applied voltage across the capillary. Therefore, higher voltages and shorter capillaries result in faster analysis times. Assuming the only contribution to band broadening is longitudinal diffusion, variance of the width of the migrating solute zone, σ2, is described by Einstein’s diffusion law:

σ2 ) (2DL2)/(µeV)

N ) L2/σ2 ) (µeV)/2D

(2)

(3)

Note that, for a capillary of given length, theoretical plates increase with applied voltage.52 The high-voltage power supply available in most commercial instruments delivers a maximum of 30 000 V. Electric field strength can be varied by the use of different capillary lengths, to an upper limit of ∼1500 V/cm, corresponding to 30 000 V applied across a 20-cm capillary. However, a field strength of this magnitude is rarely, if ever, achieved in CE using buffered aqueous systems because the amount of Joule heat produced causes temperature gradients within the capillary, resulting in significant band broadening and efficiency degradation.53-57 Nonaqueous (52) Weinberger, R. Practical Capillary Electrophoresis; Academic Press Inc.: San Diego, 1993. (53) Grushka, E.; McCormick, R. M.; Kirkland, J. J. Anal. Chem. 1989, 61, 241246.

EXPERIMENTAL SECTION Apparatus. Capillary electrophoresis was performed using a modular Spectra Phoresis 100 from Thermo Separations Products (Fremont, CA). Detection was carried out using a Spectra FOCUS scanning detector set at 254 nm. Both CE and CEC were performed on a home-built system made of a Plexiglas box outfitted with an interlock system to prevent electrical shock. All experiments were performed at ambient temperatures, where the temperature varied by no more than 3 °C during the course of this study. Both CE systems were equipped with a fused-silica capillary purchased from Polymicro Technologies, Inc. (Phoenix, AZ), with an inner diameter of 50 µm and outer diameter of ∼365 µm. Capillary length was 75 cm in total length, with an effective length of 45 cm. UV detection was at 254 nm using an Isco CV4 capillary electrophoresis absorbance detector (Lincoln, NE) in the home-built system, while a Glassman High Voltage Inc. power supply, series EH (Whitehouse Station, NJ), with 0-30 kV range, was used to apply potential across the capillary columns. Electropherograms and electrochromatograms were collected through PE Nelson Turbochrom 4.0 (Cupertino, CA) and/or PC1000 (ThermoSeparations Products) software. Reagents. All samples, solutions, and buffers were prepared using analytical reagent grade chemicals and ∼18 MΩ deionized

water filtered through a Barnstead Nanopure II system (Dubuque, IA). Organic solvents such as acetonitrile, methanol, formamide, DMF, dimethyl sulfoxide (DMSO), benzene, and acetone were purchased from Fisher Scientific (Fair Lawn, NJ) and used as received. No further purification steps or drying steps were performed. Buffer salts, such as sodium acetate, ammonium acetate, sodium phosphate, sodium tetraborate, and CAPS, and the solutes acridine and phenazine were obtained from Sigma Chemicals (St. Louis, MO). Benzo[h]quinoline and phenanthridine were purchased from Fluka (Ronkonkoma, NY). Test solutes such as naphthalene, acenaphthene, anthracene, pyrene, fluoranthene, 1,2-benzanthracene, benzo[k]fluoranthene, benzo[a]pyrene, 1,2,5,6-dibenzanthracene, and indeno[1,2,3-cd]pyrene were purchased from Chem Service (West Chester, PA). Sample solutions of the various PAHs for analysis by CEC were prepared by mixing the solutes in 65:35 acetonitrile/water mobile phase which was placed in an ultrasonic bath for ∼5 min. Acetone samples were prepared by adding several drops to a vial containing the mobile phase to be examined. For buffer solutions, the pH was adjusted with an appropriate acid or base to the desired pH. The solution pH was measured by a pH meter for buffered solutions and estimated using pH paper for the acetonitrile-containing buffer. To ensure adequate mixing, all prepared solutions were treated using an ultrasonic bath for 30 s. All solutions, both samples and solvents (or buffers), were filtered through a 0.45-µm nylon Acrodisk filter (Gelman Sciences, Ann Arbor, MI) prior to use. Procedure. All detection windows were formed by burning off a small section of the polyimide coating using a heated loop of nichrome wire. For open tubular experiments, a new capillary was cut for each solvent system examined and pretreated by flushing with solvent for several column volumes through connection to a glass syringe. The use of a fresh capillary eliminates the possibility of capillary “memory effects”, as the solvents (or buffers) are changed. All connections employed PEEK tubing sleeves with stainless steel fittings and a zero dead-volume junction. For equilibration studies, capillaries were placed into the instrument and run at 20 kV for ∼5 min. For other studies, capillaries either were flushed with solvent by pressure for 10 min or a potential was applied to allow EOF to proceed for 30 min or until the capillary was determined to be equilibrated. Samples were introduced by electrokinetic injection in the home-built system (5 s) and hydrodynamic injection in the TSP instrument (0.5-2.0 s). Electroosmotic mobilities were calculated from the observed migration time of an uncharged, uncomplexed solute, such as benzene or acetone. A 50-µm-i.d.packed-column capillary was prepared for use in CEC with the detection window at 25 cm and total length of 50 cm. The capillary was slurry packed using Shandon 5-µm ODSHypersil (Cheshire, UK) for a length of 10 cm by an ABI Analytical Spectroflow 400 solvent delivery system (Ramsey, NJ) at a pressure of ∼250 bar. The capillary was flushed with water at high pressure, and a retaining frit was formed by heating the end of the stationary phase packing with a glowing wire. The column was then filled with mobile phase and placed into the homemade instrument for equilibration at 20 kV.

(54) Jones, A. E.; Grushka, E. J. Chromatogr. 1989, 466, 219-225. (55) Knox, J. H. Chromatographia 1988, 26, 329-337. (56) Liu, K. K.; Davis, K. L.; Morris, M. D. Anal. Chem. 1994, 66, 3744-3750. (57) Davis, K. L.; Liu, K. K.; Lanan, M.; Morris, M. D. Anal. Chem. 1993, 65, 293-298. (58) Tjornelund, J.; Hansen, S. H. Chromatographia 1997, 44, 5-9.

RESULTS AND DISCUSSION Nonaqueous Solvent Properties Governing Electroosmotic Flow. The governing equation for electroosmotic flow in CE is given by

media for CE separations would allow higher applied voltages, without contributions to band broadening caused by Joule heating, yielding higher efficiencies. Some uncertainties exist in the use of nonaqueous solvents in CE, most notably, solvent purity.37 Solvent purification steps are often complicated, time-consuming, and incomplete, especially with hygroscopic solvents, and therefore undesirable for use in CE. If electroosmotic flow and/or migration time reproducibility is a problem with nonaqueous solvents as separation media in CE, the use of mixed organic/aqueous media containing a small, well-defined, and reproducible water content (0.1-0.5%) might be a good compromise.58 Destacking effects when no supporting electrolyte is used can lead to band broadening. Other drawbacks of organic solvents include volatility, viscosity, and toxicity factors. In addition, appreciable ion pairing in organic solvents can affect peak shape and detection sensitivity.37 This research is the first to critically examine the use of nonaqueous media without the use of supporting electrolyte. The goals of this research are to perform fundamental CE studies in order to examine the electroosmotic flow characteristics under nonaqueous conditions, investigate the reproducibility of electroosmotic flow using nonaqueous solvents, and compare these results to typical CE buffers. We have found that flow velocities in 100% organic media [i.e., ACN, MeOH, N,N-dimethylformamide (DMF)] are similar to those of typical buffers used in CE. This paper also reports the ζ when an electric field is applied under 100% nonaqueous conditions. We also attempt to explain the source of the high electroosmotic velocities obtained with organic solvents, in particular, acetonitrile. Finally, we have used nonaqueous media, without supporting electrolyte, for separations by capillary electrochromatography.

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νeo ) 0rζE/η

(4)

where νeo is the electroosmotic velocity, 0 is permittivity of a vacuum, r is the dielectric constant of the medium, ζ is the zeta potential, E is the applied field, and η is viscosity of the medium. From eq 4, one notes that flow velocity depends upon properties such as viscosity and dielectric constant. It becomes clear that the ratio of dielectric constant to viscosity (r/η) becomes important, which has been suggested previously.50 Therefore, solvents with r/η similar to that of water should provide flow in capillary electrophoresis, provided that a significant ζ also exists, such that the velocity does not become prohibitively slow. Several solvents possess these desirable properties and were chosen as candidates for use in nonaqueous CE. Table 1 shows these solvents and respective properties at 25 °C.36,59,60 It should be noted, however, that since viscosity is very temperature-dependent, this parameter should be monitored when performing capillary electrophoresis is performed in either aqueous or nonaqueous media. Reproducibility of Electroosmotic Flow (EOF). Initially, one area of concern was that impurities, such as water, a common contaminant in organic solvents, may affect electroosmotic flow reproducibility and, hence, separation time and selectivity. A general examination of electroosmotic mobility reproducibility was performed in order to provide a qualitative assessment of the effect of water impurities on EOF reproducibility. The initial set of experiments utilized the same capillary for the series of organic solvents. Day-to-day reproducibility using the same capillary for different solvents was generally found to be good. For ACN, less than 2% RSD (N ) 25 runs over three nonconsecutive days) in electroosmotic mobility, µeo, was observed. Data for ACN were collected after several other solvents had been run through the same capillary, indicating that previous rinsing steps performed, i.e., 20-30-min rinsing with a new solvent, were sufficient for equilibration and regeneration of the capillary surface. We found that reproducible migration times (RSD less than 3%, N ) 15) could be achieved for ACN even after the use of several other organic solvents in the same capillary. It should be noted from this qualitative study that reproducibility was much worse after trying to return to 100% ACN (or other solvents) following CE experiments with 100% water or buffered aqueous media. In addition, longer equilibration times (longer rinses) are required when switching from water or buffer to organic solvents, such as ACN. Because of the possibility of memory effects, especially in the case just described, a second set of experiments was conducted where a new capillary was used for each new solvent. In order to obtain a fair qualitative estimate of the equilibration time of the capillary with varying solvents, defined here as the time it takes to achieve reproducible run-to-run migration times, the fresh open capillary was filled either by syringe or hydrodynamically. Some solvents equilibrated slowly, and pressure rinsing between runs with the solvent became necessary in order to observe reproducible electroosmotic velocities. Capillaries were not flushed with sodium hydroxide, as is typical in aqueous CE. (59) Weast, R. C., Ed. CRC Handbook of Chemistry and Physics, 70th ed.; CRC Press, Inc.: Boca Raton, FL, 1989. (60) Covington, A. K.; Dickenson, T. In Physical Chemistry of Organic Solvent Systems; Covington, A. K., Dickenson, T., Eds.; Plenum Press: London, 1973; pp 1-23.

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Table 1. Dielectric Constants and Viscosities for CE Solvent Candidates solvent ACNa watera MeOHa DMFa formamidea DMSOb a



η (cP)

/η

37.5 80 32.7 36.7 111 46.7

0.34 0.89 0.54 0.80 3.3 1.96

110 90 60 46 34 23.8

Reference 59. b Reference 36.

Figure 1. Effect of pure solvent carriers on equilibration of electroosmotic flow in open capillaries (solute acetone): (A) pure ACN; (B) pure MeOH.

Figure 1 provides an example of the equilibration times for ACN and MeOH, respectively. Both studies were performed without rinsing with solvent between injections under identical experimental conditions. Acetonitrile allows for fast equilibration, while methanol shows greater variability in migration times after 100 min. Rinsing with solvent between runs produced better EOF reproducibility for MeOH. Behavior similar to that for MeOH was seen for formamide and DMSO, while water and DMF behaved more like ACN in terms of equilibration times. Table 2 provides electroosmotic mobilities calculated according to

Table 2. Comparison of Electroosmotic Velocity and Mobility between Solvents and Buffers solvent

νeo (cm/s)

µeo × 10-4 (cm2/V‚s)a

ζ (mV)

ACN water MeOH DMF formamide DMSO pH 10.9 CAPS buffer (30 mM) pH 9.0 borate buffer (40 mM) pH 7.5 phosphate buffer (30 mM) pH 4 acetate buffer(50 mM)

0.533 0.210 0.153 0.093 0.078 0.048 0.181 0.171 0.156 0.072

19.9 ( 0.2 7.80 ( 0.05 5.82 ( 0.01 3.48 ( 0.02 2.86 ( 0.01 1.83 ( 0.01 6.72 ( 0.03 6.36 ( 0.01 5.80 ( 0.03 2.68 ( 0.01

207 99 108 86 96 85 85 80 73 34

a Errors represent the standard deviations of the calculated electroosmotic mobilities, where n g 5.

µeo ) (Ll)/(t0V)

(5)

where µeo is the electroosmotic mobility (cm2/V‚s), L is the total capillary length (cm), l is the effective length of the capillary, or length to the detector window (cm), t0 is the migration time of a neutral marker (s), and V is the applied voltage (V). The parameters on the right-hand side of eq 5 are determined experimentally. Although the equilibration times for some of the solvents are long, once equilibration is reached, the run-to-run variation is small, supporting the notion that solvent impurities, such as trace water, do not significantly affect the reproducibility of electroosmotic flow in nonaqueous CE.58 Comparison of Electroosmotic Flow Characteristics of Nonaqueous vs Typical Buffered CE Systems. Using the data in both Tables 1 and 2, the parameters dielectric constant, viscosity, and ratio of dielectric constant to viscosity were plotted against electrophoretic mobility (µeo) for each solvent, as shown in Figure 2. Although not entirely predictive, as shown by Figure 2C, this provides a clear illustration of the importance of this ratio in estimating electroosmotic flow characteristics of the solvent. Panels A and B of Figure 2 show poor correlation between the observed electroosmotic mobilities and either dielectric constant or viscosity. However, upon plotting electroosmotic mobilities vs the ratio of dielectric constant to viscosity (Figure 2C), there is much better correlation (r2 ) 0.813). Nonlinearity is most likely due to the absence of the ζ contribution to electroosmotic mobility. To put these results in perspective, it is useful to compare the flow characteristics obtained in nonaqueous media with those obtained when typical CE buffers were used. Studies similar to those using 100% organic solvents were performed with typical CE buffers for comparison. It should be noted that the various aqueous buffers achieved equilibrium very quickly (within a couple of runs), compared to most of the organic solvents. Several buffers were prepared at varying pH, including acetate at pH 4.0, phosphate at pH 7.5, borate at pH 9.0, and CAPS at pH 10.9. The comparison of flow velocities and electroosmotic mobilities is provided in Table 2. Flow velocities and electroosmotic mobilities, as determined experimentally, are similar between aqueous and nonaqueous systems. Therefore, the use of nonaqueous solvents does not have detrimental effects on the potential speed of analysis. In fact, 100% acetonitrile provides the fastest electroosmotic flow velocities of the systems studied. The flow velocity obtained with a high-pH

Figure 2. Relationship of electroosmotic mobility (µeo) to (A) dielectric constant (), (B) viscosity (η), and (C) dielectric/viscosity ratio (/η).

buffer (pH 10.9), where the surface silanols are essentially completely dissociated and a large ζ exists, is one-third lower than that obtained with 100% ACN. Analytical Chemistry, Vol. 69, No. 16, August 15, 1997

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Another point worth noting is that deionized water provides a faster flow rate than any of the buffered aqueous systems. Unfortunately, because the water is unbuffered, it is very susceptible to pH changes from temperature effects or absorption of carbon dioxide from the environment. Slight changes in the pH of the unbuffered water result in the increased variance in the observed electroosmotic mobilities. It is not surprising, however, that the use of pure water, without buffer salts present, results in an increase in the electroosmotic flow velocity, considering the well-established effect of increased ionic strength on the flow velocity, due to changes in the nature of the double layer and resulting ζ.61 Zeta Potentials. ζ were calculated indirectly using eq 4 with literature values for dielectric constants and viscosities of the medium. For the purposes of this discussion, dielectric constants and viscosities of aqueous buffers are assumed to be equal to the those of water.62 Rearrangement of eq 4 allows calculation of the ζ in volts. Table 2 also compares the calculated ζ for various solvents and CE buffers. The comparison of MeOH with the pH 7.5 phosphate buffer shows that although the flow velocities are nearly identical, the ζ is much higher in the MeOH system. DMF, despite carrying a lower ζ than formamide, possesses a faster flow velocity. DMF, DMSO, and the CAPS buffer have nearly identical ζ, but the flow velocity with the CAPS buffer is more than twice that of the DMF system, and almost 4 times larger than that of DMSO. As with aqueous buffered systems,4 no single parameter can predict the electroosmotic flow characteristics of a given solvent. What remains elusive, however, is the nature of the capillary wall chemistry and double-layer formation in solvents such as ACN or MeOH, where calculated ζ are very high. Studies of small-particle dispersions in nonaqueous media may help shed some light on the subject of double-layer generation and ζ in these solvents. It has been determined that ionic species are present in nonaqueous solvents to a greater extent than is commonly thought, especially at a high surface area-to-volume ratio. The process of ion formation differs from that found in aqueous systems in that dissociation is inhibited by solvents of low dielectric constant. Actual charge generation is an interfacial process that takes place at the solid surface.63 Acid/base reactions between the solvent and solid determine the charge that is imposed on the particle. For a solid with acidic surface groups (such as SiOH), a solvent with basic properties will impose a negative charge by a three-step process. The basic group first adsorbs onto the solid surface at an acidic functional group. Proton transfer takes place from the acid to the base, after which, the base partially desorbs into the bulk solution leaving a negative potential at the solid surface. The opposite is true for solids with basic groups dispersed in solvents with acidic groups, generating positive surface potential.63 This behavior has been well characterized and is used extensively for creating electrostatically stabilized dispersions of solid particles in nonaqueous solvents.64 Although aprotic, acetonitrile has both acidic and basic functionalities, generating negative charges on particles with acidic (61) Salomon, K.; Burgi, D. S.; Helmer, J. C. J. Chromatogr. 1991, 559, 69-80. (62) Debye, P.; Pauling, L. J. Am. Chem. Soc. 1925, 47, 2129-2134. (63) Fowkes, F. M. In Advances in Ceramics; Messing, G. L., et al., Eds.; The American Ceramic Society, Inc.: Boston, MA, 1987; Vol. 21, pp 411-421. (64) Fowkes, F. M.; Jinnai, H.; Mostafa, M. A.; Anderson, F. W.; Moore, R. J. In Colloids and Surfaces in Reprographic Technology; Hair, M. L., Croucher, M. C., Eds.; ACS Symposium Series 200; American Chemical Society: New York, 1982; pp 307-324.

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character.63 The fused-silica capillary surface may act in a manner similar to a high concentration of dispersed particles, which would generate a substantial ζ, and at the same time introduce counterions from the desorbed basic components in solution, creating charge carriers that constitute the double layer. Because all solvents were of reagent grade, small amounts of impurities (up to 0.1%) are present in each, consisting mainly of water and titratable acid and/or base. This impurity level may increase slightly as water is absorbed from the atmosphere. Other sources of dilute ionic contaminants have been reported elsewhere.36,65 If we consider all “pure” solvents to have a very low ionic strength, it is interesting to compare ζ with more concentrated buffer solutions. Acetonitrile and methanol are observed to have the largest ζ (207 and 108 mV, respectively), and each should have a very large double layer thickness (δ) according to theory.66 The lowest ζ is found for acetate buffer, which is at the highest electrolyte concentration (50 mM) and should have the smallest δ. Assuming double-layer theory holds for all solvents, this would indicate that, as double-layer thickness increases, the resulting ζ also increases. Solvation of ionic species in nonaqueous solvents is complicated and difficult to determine, especially when one is attempting to estimate the effect on equilibration of electroosmotic flow. Solvent properties such as aprotic/amphiprotic character and protophobicity/protophylicity influence solvation, hydrogen bonding, dissociation, and formation of ion pairs, ion triplets, and higher complexes.67 The effects of these properties on the double layer and resulting EOF remain unclear and require better understanding through further experimental observation in nonaqueous CE. Studies on Binary Mixtures of ACN/Water and MeOH/ Water. The previous results led to an extension of this work involving mixed hydroorganic solutions. Our efforts were limited to acetonitrile and methanol as organic modifiers since these solvents are the most popular choice for MEKC and CEC, as well as HPLC. Experimental conditions were identical to those used for pure solvents. Schwer and Kenndler attributed changes in electroosmotic velocity with the addition of organic modifier to decreases in the ζ. Their studies examined the effect of organic solvents only as buffer additives, up to 80% organic modifier on the electroosmotic velocity and ζ.23 We performed a similar study using a pH 6.9 phosphate buffer (30 mM) (Figure 3), showing a steady decrease in electroosmotic mobility with increasing organic modifier in the buffer, which agrees well with the work of Schwer and Kenndler.23 Figure 4 shows the relationship of EOF to percent organic modifier for ACN and MeOH. Acetonitrile shows flow behavior varying with organic composition up to 80% ACN, beyond which a large increase in flow is observed up to 100% ACN. Again, reproducibility studies show RSD is e1% at these compositions (N ) 5). This is in contrast to earlier reports that show an almost linear decrease in EOF when ACN is added to an aqueous buffer solution up to 80% (v/v).23 Suppression of EOF due to the presence of electrolyte is the most likely explanation between the two studies but is difficult to determine given the multiple factors that can influence EOF. Methanol shows a parabolic relationship between µeo and percent organic with a flow minimum occurring (65) Blades, A. T.; Ikonomou, M. G.; Kebarle, P. Anal. Chem. 1991, 63, 21092114. (66) Knox, J. H.; Grant, I. H. Chromatographia 1987, 24, 135-143. (67) Kholtoff, I. M. In Pure and Applied Chemistry; Butterworths: London, 1971; Vol. 25, pp 305-325.

Table 3. Parameters for Mixed Mobile Phase Compositions % ACN

a

ηb

0 20 40 60 80 100

78.5 67.5 56.0 46.5 41.0 36.1

0.89 0.91 0.98 0.76 0.58 0.43

/η ζ (mV) %MeOH 88 74 57 61 71 84

97 101 134 142 115 226

0 20 40 60 80 100

c

ηb

78.5 70.0 61.1 51.8 42.8 32.6

0.89 1.26 1.42 1.40 1.01 0.57

/η ζ (mV) 88 56 43 37 42 57

108 122 117 103 92 127

a 25 °C; see ref 69. b 25 °C; values in centipoise; see ref 71. c 25 °C; see ref 70.

Figure 3. Effect of increasing acetonitrile concentration in aqueous buffer on electroosmotic mobility (µeo). Buffer is 30 mM phosphate, pH 6.9.

Figure 4. Relationship of electroosmotic mobility (µeo) to volume percent organic in water: (A) ACN; (B) MeOH.

near 70% organic modifier. Comparing the two solvent systems, there is a greater decrease in flow for MeOH than ACN at low organic compositions (80%), this is strikingly similar to flow behavior for acetonitrile reported here (Figure 4), where µeo greatly increases from 80 to 100% acetonitrile. Although purely speculative, there seems to be an inverse relationship between solvent polarity and electrophoretic mobility at high acetonitrile concentrations. If this relationship holds true, it suggests for two solvents of sufficiently different solvent polarity, as the mixture composition becomes predominantly nonpolar in nature, solvent polarity becomes a determining factor in electroosmotic flow. ET(30) solvent polarity values for methanol show a gradual, almost linear decrease from 0 to 100% methanol. The dynamic range of methanol/water ET(30) values (∼7 kcal/mol solvatochromic shift) is more than 2 times smaller than that for acetonitrile (∼17 kcal/mol).68 Because the difference in solvent polarity is not as pronounced between methanol/water as in acetonitrile/water, the effect on EOF will not be as severe, explaining why methanol/water flow values more closely track their respective /η values than do those for acetonitrile/water. ζ were calculated by substituting literature values for 69,70 and 71 η into eq 4 with experimentally determined νeo. Table 3 shows the respective literature values of , η, and /η with our calculated ζ values for the different compositions of each mobile phase system. The ratio of /η for both solvent systems is plotted against organic percentage in Figure 5. It should be noted that EOF determinations were performed at ambient temperature for both solvent systems, which may explain differences in ζ for pure water. (68) Johnson, B. P.; Khaledi, M. G.; Dorsey, J. G. Anal. Chem. 1986, 58, 23542365. (69) Horvath, C.; Melander, W. J. Chromatogr. Sci. 1977, 15, 393-404. (70) Timmermans, J. The Physico-Chemical Constants of Binary Systems in Concentrated Solutions; Interscience: New York, 1960. (71) Snyder, L. R. J. Chromatogr. Sci. 1977, 15, 441-449.

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Figure 6. Effect of pure ACN as mobile phase on equilibration of EOF in CEC separation of test PAH mixture: (b) acetone; (+) 1,2benzanthracene; (9) indeno[1,2,3-cd]pyrene.

Figure 5. Relationship of dielectric/viscosity ratio to volume percent organic in water.

Although measured at the same temperature by separate sources, solvent properties listed in Table 3 differ slightly from those in Tables 1 and 2, most notably in the viscosity for acetonitrile (0.34 vs 0.43). This will affect the exact calculation of ζ, but should allow us to make qualitative estimates of trends for ζ with percent organic composition. Calculated ζ as a function of percent organic modifier for ACN and MeOH show a nonlinear relationship, indicating that ζ cannot be predicted simply from binary mixture solvent properties. Solvent structure in binary mixtures may explain this behavior. It is well-known that mixtures of ACN/water and MeOH/water exist in several compositions on the microscopic scale. Methanol/ water mixtures have been found to consist of a ternary mixture with free methanol, methanol/water-associated molecules, and free water molecules.72 Acetonitrile/water mixtures are also capable of forming up to six different compositions on the microscopic scale.73 It is reasonable to assume that as the microscopic structure of these solvent mixtures changes with composition, the double-layer structure will be affected in some manner, causing fluctuations in ζ. (72) Katz, E. D.; Ogan, K.; Scott, R. P. W. J. Chromatogr. 1986, 352, 67-90. (73) Rowlen, K. L.; Harris, J. M. Anal. Chem. 1991, 63, 964-969.

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Figure 7. Effect of pure ACN as mobile phase on k′ for retained solutes. ([) 1,2-benzanthracene; (b) indeno[1,2,3-cd]pyrene.

Application of Nonaqueous Media in Separations by CEC. Although uncertainty exists about the nature of the double layer in a nonaqueous environment, Whittaker and Sepaniak have demonstrated that separations using CEC are possible with 3-µm ODS stationary phase particles using pure ACN mobile phases.50 Their work investigated separations of C60-C70 fullerenes. Because these solutes were heavily retained even in pure acetonitrile, tetrahydrofuran (THF) and methylene chloride (MeCl2) were used as mobile phase additives to decrease capacity factors (k′), which was accompanied by a reduction in EOF velocity. Our studies have focused on using pure acetonitrile to achieve fast separations of the larger hydrophobic PAH solutes, while adding water to the mobile phase in order to increase capacity factors and enhance separation for solutes that are weakly retained in pure acetonitrile. A 50-cm column was packed with 5-µm ODS Hypersil for a length of 10 cm for experiments investigating separations of PAHs. A test solution was prepared with acetone, 1,2-benzanthracene, and indeno[1,2,3-cd]pyrene in pure ACN. The

Figure 8. Separation of PAH mixture in 65:35 ACN/water. Peaks: (1) acetone, EOF marker; (2) benzene, k′ ) 0.23; (3) naphthalene, k′ ) 0.47, h ) 2.8; (4) acenaphthene, k′ ) 0.83, h ) 2.5; (5) anthracene, k′ ) 1.0, h ) 2.6; (6) pyrene, k′ ) 1.4, h ) 2.7; (7) fluoranthene, k′ ) 1.4, h ) 2.5; (8) impurity; (9) 1,2-benzanthracene, k′ ) 2.0, h ) 2.8; (10) benzo[k]fluoranthene, k′ ) 2.9, h ) 3.0; (11) benzo[a]pyrene, k′ ) 3.1, h ) 2.8; (12) 1,2,5,6-dibenzanthracene, k′ ) 4.0, h ) 2.8; (13) indeno[1,2,3-cd]pyrene, k′ ) 4.5, h ) 3.0.

column was equilibrated electrokinetically for 1 h with ACN. Repeated injections of the test solute were made, and retention time was monitored for equilibration of flow and reproducibility. Acetone was used as the EOF marker. Figure 6 shows the equilibration of flow under these conditions. There is a marked leveling off of retention times at the seventh injection, corresponding to a total equilibration time of ∼50 min. When combined with the electrokinetic flushing done previous to separations, the total equilibration time for this column was estimated to be ∼2 h. Figure 7 shows a plot of capacity factor (k′) vs injection number for the two retained solutes. RSD was calculated to be 3.5 and 2.3% for 1,2-benzanthracene and indeno[1,2,3-cd]pyrene, respectively (N ) 15), indicating retention is relatively stable even during equilibration of electroosmotic flow. Compared to open tubular experiments, we observe EOF to equilibrate much more slowly due to the presence of the stationary phase. Several possible explanations exist for this behavior. The nonpolar alkyl chains may require a longer period of time to become solvated by the acetonitrile and allow the solvent access to the silica surface of the stationary phase. Because water is flushed through the column under pressure during the frit-making process, it is also possible that equilibration is slowed due to the expulsion of this water from the pores of stationary phase particles. Figure 8 shows a capillary electrochromatographic separation utilizing an unbuffered mixed organic/aqueous mobile phase (typically used in reversed phase liquid chromatography). The isocratic separation of 11 PAHs was achieved using a mobile phase of 65:35 ACN/H2O. Near-baseline resolution is observed for all (74) Foley, J. P.; Dorsey, J. G. Anal. Chem. 1983, 55, 730-737.

solutes in under 13 min, with an EOF velocity of 2.1 mm/s at 20 kV using acetone as the unretained marker. Efficiencies were determined using an exponentially modified Gaussian estimation of peak shape.74 Reduced plate heights (h) were found to be between h ) 2.5 and h ) 3.0 for peaks 3 -12. Peaks 1 and 2 (acetone, benzene) were inadequately sampled during data collection, causing efficiency calculations to be in error. The small peak eluting after peak 7 was reproducible and thought to be an impurity from the fluoranthene standard (∼98% purity). Capacity factors were calculated, with k′ ) 4.5 for the latest eluting peak. Peak symmetry is excellent with asymmetry ratios (B/A) between 1.01 and 1.06 for all solutes. Although not optimized, these separations show the utility of unbuffered mobile phases for achieving fast analysis of complex mixtures. It is worth noting that all separations were performed under ambient conditions without pressurization of the mobile phase and bubble formation was not encountered. ACKNOWLEDGMENT This work was financially supported by the National Institutes of Health, Grant GM-48561, and the Air Force Office of Scientific Research (AFOSR). J.G.D. is grateful to Merck Research Laboratories for continued support of our research.

Received for review December 31, 1996. Accepted June 12, 1997.X AC9613186 X

Abstract published in Advance ACS Abstracts, August 15, 1997.

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