Anal. Chern. 1990, 62, 1550-1552
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Direct Control of the Electroosmosis in Capillary Zone Electrophoresis by Using an External Electric Field Cheng S. Lee,**lWilliam C. Blanchard,* a n d Chin-Tiao Wu' Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County Campus, Baltimore, Maryland 21228, and Blanchard & Co., Inc., 27 Glen Alpine Road, Phoenix, Maryland 21131-2407 INTRODUCTION After years of development since the first invention of capillary electrophoresis in 1974 ( I ) , capillary electrophoresis performs such functions as quality control of recombinant proteins, evaluation of the purity of synthetic peptides, studying serum proteins, checking biological degradation, analyzing drugs, monitoring antibodies, and studying bioactive peptides with the resolving power of electrophoresis and the ease and speed of HPLC (2). The low-volume capability, high separation efficiency, and sensitive detection schemes make capillary electrophoresis a powerful method for analytical biotechnology, a critical need for today's bioindustry. A fundamental problem in capillary zone electrophoresis (CZE), one of the most frequently used separation modes in capillary electrophoresis, is controlling electroosmosis, the flow of solvent in an applied potential field. Under normal aqueous conditions with small binary electrolytes, the silica surface has an excess of anionic charge resulting from ionization of surface functional groups. The cationic counterions to these anions are in the diffuse layer adjacent to the capillary walls. The potential across the diffuse layer is termed the {potential. These hydrated cations migrate toward the cathode and drag solvent with them. Thus, the direction and velocity of electroosmotic flow are dependent on the polarity and magnitude of the { potential at the capillary walls ( 3 ) . Electroosmotic flow affects the amount of time a solute resides in the capillary, and in this sense both the separation efficiency and resolution are related to the direction and flow rate of electroosmosis ( 4 ) . If the rate of electroosmotic flow is greater in magnitude and opposite in direction to the electrophoretic mobilities of all anions in the buffer, then all ions will migrate in the same direction. Thus, electroosmosis results in better resolution of anions such as DNA fragments which migrate against the electroosmotic flow. Conversely, cations will be more poorly resolved under these conditions. In fact, improved resolution of substances having very similar mobilities can be achieved by balancing electroosmotic flow against electrophoretic migration. In addition to controlling electroosmosis, application of CZE to the separation of proteins is complicated by adsorption of the minute quantities of the protein sample onto the walls of the capillary. Such interactions result in band broadening and tailing, with greatly reduced separation efficiency. Reported attempts (4-7) to eliminate this adsorption involve deactivation of the silica capillaries by physically coating the capillary wall with methylcellulose, as well as via silane derivatization. Because of the inherent difficulty of reproducibly deactivating the capillary surface (4-7), alternative methods employing dynamic reduction of protein/capillary interactions have been developed. These include the addition of chemical reagents to the separation buffer (4), as well as manipulation of the charges on the proteins and the silica capillary wall to prevent adsorption by Coulombic repulsion (8, 9).
* To whom all correspondence should be addressed University of Maryland. 2Blanchard& Co.
To enhance separation resolution and to prevent protein adsorption, it is clearly of interest to have the ability to directly and dynamically control the polarity and magnitude of the boundary condition at the aqueous/capillary wall, the { potential. A novel concept involving the use of an additional electric field from outside the capillary is proposed in this study. This technique vectorially couples the externally applied potential with the potential inside the capillary. Thus, the { potential can be controlled with a definite value and made to be positive, zero, or negative. Further, the {potential can be changed a t any time during the analysis to achieve innovative separation results. EXPERIMENTAL SECTION The experimental setup is shown in Figure 1. A 20-cm-long capillary (Polymicro Technologies, Inc., Phoenix, AZ) with 75-pm i.d. (375-pm 0.d.) was placed inside a larger capillary (530-pm id., 630-pm 0.d.) that was 17 cm long. The smaller (inner) capillary was attached between reservoir 1 and reservoir 4 while the larger (outer) capillary was attached between reservoir 2 and reservoir 3. The polyimide coating on the exterior surfaces of both inner and outer capillaries was removed by using concentrated sulfuric acid solution. A syringe was used as reservoir 1and as a pumping device for flushing out air bubbles in the inner capillary. Platinum wire electrodes were affixed to all four reservoirs. Reservoir 2, reservoir 3, and the annulus between the inner and outer capillaries were filled with 0.002 M potassium phosphate buffer at a pH of about 6. One high-voltage power supply connected to reservoir 2 or reservoir 3 so that an electric field (outer) was applied to the annular space between the two capillaries. A pipet vacuum pump was used to accelerate a fluid flow in the annulus between the inner and outer capillaries. This fluid flow is to enhance the heat transfer in the annulus for removing the additional heat generated by the application of external electric field. Another high-voltage power supply connecting reservoir 1 with reservoir 4 applied an electric field (inner) inside the inner capillary. With adjustable resistor R3, we were able to establish different electric field gradients between the inner and outer fields along the 17-cm-longannulus between reservoir 2 and reservoir 3. The resulting changes in the direction and speed of electroosmotic flow in the inner capillary due to these electric field gradients was monitored by using the current-monitoringmethod (10).
The procedure is to fill the inner capillary tube and reservoir
4 with 0.001 M potassium phosphate buffer and to fill reservoir 1with the same electrolyte but at 0.002 M. A positive potential is then applied to reservoir 1with respect to reservoir 4. Thus, the phosphate buffer in reservoir 1with the higher concentration
migrates into the inner capillary tube by electroosmosis and displaces the lower concentration phosphate buffer in the tubing. As a result, the total resistance of the fluid in the inner capillary tube decreases. This change can be followed by recording the increase in current during the experiment. A 10-kQ resistor is inserted between the reservoir 4 electrode and ground. This means that a 1-pA current increase would increase the potential drop across the resistor by 10 mV. A chart recorder is connected directly across the 10-kQresistor for monitoring such changes. This increase in current continues until the entire inner capillary becomes filled with 0.002 M phosphate buffer. The inner capillary length, 20 cm, divided by the time required to complete the filling of capillary tube with 0.002 M phosphate buffer gives the electroosmotic flow rate. Because the electroosmotic flow rate is a function of the electrolyte concentration, our measured values are the average electroosmotic flow rates.
0 1990 American Chemical Society 0003-2700/90/0362-1550$02.50/0
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
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Table I. Effect of External Electric Field on the Electroosmosis outer potentials,b kV
inner potentials," kV Vi1
Vi,
Vi3
VO,
5.5 5.5 5.5 5.5 5.5 5.5
5 5 5 5 5 5
0.4 0.4 0.4 0.4 0.4 0.4
0
0 5 8 10 11
V, -4.6 0 0.4 3.4 5.4 6.4
electroosmotic flow rate: cm/min
potential gradientcAB, kV -5
r potential, mV
+4.48 f 0.22 +3.73 f 0.19 +2.29 f 0.16 +1.68 f 0.08 -0 -0.87 k 0.02
no external field 0 +3 +5 +6
-35 -29
-18 -13
-0 +7
Vi,, the inner potential at reservoir 1; Vi,, the inner potential at the beginning of the annulus (in reservoir 2) between the inner and outer capillaries; Vi3, the inner potential at the end of the annulus (in reservoir 3). Vi, and Vi, are estimated by assuming a linear potential gradient inside the inner capillary. Voz,the outer potential at the beginning of the annulus; V,, the outer potential at the end of the annulus. V,, - Vi, or V , - Vi,. The gradient is uniform through the annulus. Electroosmosis in the inner capillary with the cathode end in reservoir 4. +, from reservoir 1 to reservoir 4; -, from reservoir 4 to 1.
I
RESERVOIR NO. I
RESERVOIR NO. 2
RESERVOIR NO. 3
RESERVOIR
NO. 4
I
I
I
I
I
I
30
25
zo
Urm(mJ""Ua) 15
10
5
0 0
Figure 2. Electropherogram showing the change of the direction and flow rate of electroosmosis in the inner capillary with the application of external electric field.
RESERVOIR NO I
RESERVOIR NO 2
RESERVOIR NO 3
RESERVOIR
NO
4
Figure 1. Test setup for examining the effect of (a) positive, (b) negative external field on the electroosmosis.
RESULTS AND DISCUSSION Figure 2 is the electropherogram showing the change of the direction and flow rate of electroosmosis in the inner capillary with the application of external electric field. The changes in the potential drop across the 10-kQresistor inserted between the reservoir 4 electrode and ground were recorded. In regions a and b on Figure 2, the anode end for the inner electric field is in reservoir 1 and reservoir 4, respectively. Regions a and b represent the typical electroosmotic flow without the influence of external electric field. From region c to region h, the anode end for the inner electric field is always in reservoir 1. The positive potential gradients from 0 to 5 kV between the inner and outer fields were applied between regions c and e. The decrease in the flow rate of electroosmosis from reservoir 1 to reservoir 4 was observed as the slope of electropherogram decreased with the application of an external electric field. In region f, the direction of electroosmosis was reversed with a 6-kV positive potential gradient between the inner and outer fields. The flow rate of electroosmosis was enhanced in region g with the application of a 5-kV negatiue potential gradient. This enhancement can be easily seen by comparing the slopes of the electropherogram in regions g and
h. There was no external electric field in regions h and i. To evaluate the effect of external electric field on the direction and magnitude of electroosmotic flow more accurately, the electroosmosis was monitored as the time required to completely change the buffer concentration in the inner capillary (see Experimental Section). The quantitative results from six runs are summarized in Table I. The flow rate of the electroosmosis from reservoir 1 to reservoir 4 increases from 3.73 f 0.19 cm/min without an external electric field to 4.48 f 0.22 cm/min with the application of a -5 kV potential gradient between the inner and outer fields along the 17-cm-long annulus. Applying positive potential gradients from 0 to 5 kV between the inner and outer fields reduces the rate of the electroosmotic flow. The direction of electroosmotic flow can be reversed (from reservoir 4 to reservoir 1) at an even higher positive potential gradient, 6 kV. The measured electroosmotic flow rates are lower than the values reported in the literature (10). This is because our solution pH is lower than the p H used in the reported experiments (10). The absolute value of { potential a t the aqueous/inner capillary interface is calculated on the basis of LJ
= (C/dE{
(1)
where u is the linear velocity of the electroosmotic flow, t is the permittivity of the solution, 7 is the viscosity, E is the electric field strength, and {is the {potential ( I I , 1 2 ) . The cathode end of the inner electric field is set in reservoir 4. Thus, the {potential would be negative if the direction of the electroosmosis is from reservoir 1 to reservoir 4. The { po-
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
tential changes from -29 mV without an external field to -35 mV with a -5 kV potential gradient. The absolute value of the { potential decreases from -29 mV without an external field to about 0 mV with a +5 kV potential gradient. The polarity of the {potential can be reversed at +6 kV potential gradient. These preliminary experimental results indicate that the electroosmosis can be enhanced, decreased, eliminated, and even reversed by simply using an external electric field to control the f potential at the aqueous/inner capillary interface. The proposed concept involving the use of an additional electric potential from outside the capillary for controlling the electroosmosis will be verified again by using the neutral marker method (13,14). The studies for enhancing separation resolution of biomolecules in CZE and in micellar electrokinetic capillary chromatography with the control of electroosmosis will be followed.
RECEIVED for review February 9, 1990. Accepted April 18,
ACKNOWLEDGMENT We thank Andrew G. Ewing for his helpful discussions.
1990. Support for this work by Minta Martin Foundation and Engineering Research Center of the University of Maryland is gratefully acknowledged.
LITERATURE CITED (1) (2) (3) (4)
Virtanen, R. Acta Polytech. Scand. 1974, 723, 1. Wailingford, R. A.; Ewing, A. G. A&. Chromtogr. 1989, 2 9 , 1. Rice. C. L; Whitehead R. J. phvs. Chem. 1985, 7 1 , 4017. Jorgenson, J. W.: Lukacs, K. D. Science 1983, 222, 266. (5) Hjerten, S. Chromatogr. Rev. 1987, 9 , 122. (6) Herren, B. J.; Shafer, S. G.; Aistlne, J. V.; Harris, J. M.; Snyder, R. S. J . Colloid Interface Sci. 1987, 175, 46. (7) Hjerten, S. J . Chromatog. 1985, 347, 191. (8) Lauer, H. H.; McManigiii, D. Anal. Chem. 1988, 5 8 , 166. (9) McCormick, R. M. Anal. Chem. 1988, 6 0 , 2322. (10) Huang, X.: Gordon, M. J.; Zare, R. N. Anal. Chem. 1988, 60, 1837. (11) Pretorius, V.; Hopkins, 8.J.; Schieke, J. D. J. Chromatogr. 1974, 264, 385. (12) Tsuda, A.; Nomura, K.; Nakagawa, G. J. Chromatogr. 1983, 2 6 4 , 385. (13) Tsuda, A.; Nakagawa, G.; Sato, M.; Yagi, K, J. Appl. Biochem. 1983. 5 , 330. (14) Waibroehl, Y.; Jorgenson, J. W. Anal. Chem. 1988, 58, 479.
CORRECTION Nile Red as a Solvatochromic Dye for Measuring Solvent Strength in Normal Liquids and Mixtures of Normal Liquids with Supercritical and Near Critical Fluids Jerry F. Deye, T. A. Berger, and Albert G. Anderson (Anal. Chern. 1990, 62, 615-622). Table I contains two errors. Solvent 53 should be N,Ndimethylaniline and solvent 74 should be C02/methanol (9O:lO) (v/v). On p 618, the next to the last sentence in the first column should read: It also produces a bathochromic wavelength shift consistent with stabilization of the excited state in n-a* or T-H* electronic transitions and comparable with the a* scale of Kamlet and Taft (16).
