Anal. Chem. 1994,66, 3060-3065
Auxiliary Electroosmotic Pumping in Capillary Electrophoresis Purnendu K. Dasgupta' and Shaorong Llu Department of Chemistry and Biochemistry, Texas Tech Universi& Lubbock, Texas 79409- 106 1
Followinga conventionalcapillary electrophoresis system with an optical detector, a conductive membrane can be used to connect a second capillary, the terminal end of which is connected to a second power supply, the membrane serving as the common ground for both high-voltage sources. The direction and magnitude of the field applied to the second capillary govern if the bulk flow in the first capillary is augmented, inhibited, or unaffected by the pumping action exerted by the second capillary. The majority of CE applications involve samples of ionic strength lower than that of the running electrolyte and optimum sample stacking is desirable. In such cases, auxiliary pumping of this type can be used to optimize the stacking profile and thus improve the separation efficiencies for charged solutes. This work also compares the characteristics of this type of auxiliary electroosmotic pumping with its hydrostatic counterpart. In capillary electrophoresis (CE), the electrophoretic movement of a charged analyte species is augmented or inhibited by the bulk electroosmotic flow (EOF) of the electrolyte medium. In conventional systems, both electrophoretic and electroosmotic movement results from the same applied voltage and cannot be independently controlled. It is relatively simple, however, to augment or inhibit the bulk flow by hydrostatic means, and models have been developed to describe flow profiles that include both these motive forces;' control of bulk flow by pressure had been experimentally implemented much earliere2 Although the mathematical developments are largely the same, the qualitative conclusions drawn by the different authors from the model differ in flavor. GrushkaIa predicts a rather dramatic increase in plate heights with the inclusion of even a small hydrostatic flow component. Datta and Kotamarthilb argue that under some circumstances actual efficiency may improve with some Poiseuille flow added. Keely et ale3carried out an exemplary study of dispersion in CE by various external flow control methods. It was found that with a pH 7 phosphate buffer carrier electrolyte, the plate heights for an uncharged solute follow theoretical expectations quite well and that pressureinduced bulk flow may be beneficial in reducing the time for a given separation with only a minor loss of efficiency. At pH 2.7, however, these authors found that plate heights actually decreased relative to theoretical expectations with pressuredriven flow augmenting the EOF. In systems where detection (1) (a) Grushka, E. J . Chromatogr. 1991,559,81-93. (b) Datta, R.; Kotamarthi, V. R. AIChE J . 1990. 36.. 916-926. (2) Everaerts,F. M.;Verheggen,T.P. E. M.;VandeVenne, J. L. M. J . Chromatogr. 1976, 123, 139-148. (3) Kcely, C. A.; Holloway, R.; Van de Goor, T. A. A. M.; Mcmanigill, D. J . Chromatogr. 1993, 652, 283-288. ~
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Analyilcal Chemistry, Vol. 66, No. 19, October 1, 1994
preferably takes place outside the high electric field (as in suppressed conductivity detection and some types of electrochemical detection4), flow in the post separation zone cannot be plug like. KokSahas reported that augmentation of EOF by a hydrostatic head minimizes the loss of separation efficiency in such systems; in studies with suppressed conductometric CE systems where flow must continue after the electric field ends, we have observed the same.5b An altogether different approach was developed independently by Ghowsi and Gale and Lee et a1.6 The magnitude of the EOF in a capillary is a function of the { potential of the capillary surface, and this can be manipulated by a second, radially applied, field. With a resistive coating or a liquid medium surrounding the outer side of the separation capillary and using this to uniformly dissipate the second field, the magnitude of the radial field can be maintained uniform across the length of the capillary. The radial field can also be applied more simply through a conductive sheath; in this case, the radial field is not uniform across the capillary. In either case, little or no plate losses occur;3a significant amount of additional work has been carried out to further develop this concept.' Hayes et al. have recently observed that it may not be necessary to apply the radial field across the entire external physical length of the capillary. Even if it is applied on a rather small external region (experimentally this makes it significantly more convenient), the radial field is distributed across the length of the capillary due to internal surface cond~ctance.~ It is not known at this time if plate losses are observed with such an approach. The major drawback of the radial field application approach is that the degree of control that can be exercised over the EOF is highly pH dependent and can be very limited at pH values far removed from the pKa of the surface ionizable groupse8 C. E.; Ewing, A. G. Elecrroannlysis 1991, 3, 587-596. Dasgupta, P. K.; Bao, L. Anal. Chem. 1993, 65, 10031011. Avdalovic, N.; Pohl, C. A.; Rocklin, R.; Stillian, J. R. Anal. Chem.
(4) Curry, P. D., Jr.; Engstrom-Silverman,
1993, 65, 1470-1475. ( 5 ) (a) Kok, W. Th. Anal. Chem. 1993,65, 1853-1860. (b) Kar, S.; Dasgupta, P. K.; Liu, H.; Hwang, H. Anal. Chem. 1994,66, 1994. (6) Ghowski, K.; Gale, R. J. In Biosensor Technology; Buck, R. P., Hatfield, W. E., Umana, M., Bowden, E. F., Eds.; Proceedings of the International Symposium on Biosensors, University of North Carolina, 1989; MarcelDekker: New York, 1990; pp 55-62. Lee, C. s.;Blanchard, W. C.; Wu, C. T. Anal. Chem. 1990, 62, 1550-1552. (7)Ghowsi, K.; Gale, R. J. J . Chromarogr. 1991, 559, 95-101. Lee, C. S.; McManigill, D.; Wu,C. T.;Patel, B. A n d C h e m . 1991,63,1519-1523. Lee, C. S.;Wu, T.; Lops, T.; Patel, B. J . Chromarogr. 1991,159, 133-140. Wu, C. T.; Lopes, T.; Patel, B.; Lee, C. S . Anal. Chem. 1992,64,886-891. Hayes, M. A.; Ewing, A. G. Anal. Chem. 1992,64,512-516. Gajar, S. A.;Geis, M. J . Electrochem. Soc. 1992,139,2833-2840. Wu, C.-T.;Huang, T.-L.;Lee, C. S.; Miller, C. J. Anal. Chem. 1993,65,568-571. Hayes, M. A,; Kheterpal, I.; Ewing, A. G. Anal. Chem. 1993,65, 2010-2013. (8) (a) Hayes, M. A.; Kheterpal, I.; Ewing, A. G. Anal. Chem. 1993.65.27-31, (b) Culbertson, C. T.; Jorgenson, J. W. Anal. Chem. 1994, 66, 955-962.
0003-2700/94/0386-3060$04.50/0 0 1994 Amerlcan Chemlcal Society
Figure 1. Auxiliary electroosmotic pumping scheme. C1, Separation/ reaction capillary; D, detector; J1, grounding joint. C2, pump capillary; HV1 and HV2 are independent high voltage sources.
Controlling bulk flow in a conduit by electroosmotic means may be attractive in areas other than CE. The use of electroosmotic pumping is envisioned to be attractive, for example, in capillary flow injection a n a l y ~ i s .Because ~ two capillaries can be joined in some fashion (through porous glass or graphite, a porous frit, an ionically conductive membrane, or even a microcrack made in an otherwise integral capillary) such that an electrical connection can be established without interruption of f l o ~ , ~ JitOis possible to devise an independent bidirectional electroosmotic pump in a second capillary to affect bulk flow in a conjoined first capillary. Ready use for such auxiliary pumping can be envisioned where hydrostatic pressure is presently used.8b Consider the arrangement schematically shown in Figure 1. Capillary C1 is connected to a source vial housing the high-voltage electrode HV1 and to a grounding joint J via detector D. The joint J is connected tocapillary C2, hereinafter referred to as the pump capillary. C2 terminates in an electrolyte reservoir housing a second high-voltage source electrode HV2. J is the common ground. Other than the flow resistance posed by C2, when HV1 and HV2 are opposed in sign, the EOF generated in C2 augments the EOF in C1. When the signs are the same, one flow inhibits the other. In the present paper we wish to characterize such a pumping system. The effects on peak efficiency are reported and compared with the simpler expedient of hydrostatic pumping.
EXPERIMENTAL SECTION Except as otherwise stated, capillary C 1was 40 cm in length and capillary C2 was 20 cm in length. Both were fused silica (75 pm i.d., 375 pm o.d., Polymicro Technologies, Phoenix, AZ). A butt-joint was made using a stretched piece of a Nafion 014 tubing (Perma-Pure Products, Toms River, NJ). The latter was swelled in methanol, slipped over the capillary tubing, and tied in place firmly with nylon or Kevlar thread. For all of the present experiments, the same electrolyte was used in both C1 and C2 and was contained in each terminal vial. The joint was also immersed in the same electrolyte contained in another vial and served as the common ground for two identical power supplies (CZE 1000R, Spellman Inc., Plainview, NY). The test analytes consisted of (a) negatively charged solutes, 40 pM concentration of sulfonephthalein dyes in 100pM Na2B407, pH -9; (b) neutral solute, benzyl alcohol (100 pM in water); and (c) positively charged solute, 500 pM (9) (a) Liu, S.;Dasgupta, P . K. Anal. Chim. Acta 1992, 268, 1-6. (b) Liu, S.; Dasgupta, P. K. Anal. Chim. Acta 1993,283,739-745. (c) Dasgupta, P.K.; Liu, S.Anal. Chem. 1994, 66, 1792-1798. (LO) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1988.60, 258-263. Yik, Y. F.; Lee, H. K.; Li, S.F. Y.; Khoo, S . B. J. Chromatogr. 1991,585, 139-144. Huang, X.; Zare, R.N. Anal. Chem. 1990,62,443446. Linhares, M. C.; Kissinger, P.T. Anal. Chem. 1991,63,2076-2078. O'Shea, T. J.; Greenhagen, R. D.; Lunte, S. M.; Lunte, C. E.; Smyth, M.R.; Radzik, D. M.; Watanbe, N. J. Chromatogr. 1992, 593, 305-312.
FeS04 and' 200 pM hydrazine sulfate (as an antioxidant) in 20 mM 1,lO-phenanthroline (pH 6.1). For the anionic and neutral solute experiments, a 2 mM Na2B407, pH 9.2 electrolyte was used with +9 kV being applied on C1. For the cationic solute experiment, the carrier electrolyte contained 20 mM, 1,lo-phenanthroline, 20 mM NBudC104,2 mM Na2HP04, and had a pH of 7.9. The EOF with this electrolyte is much less than with the borate; up to + I 8 kV was applied on C1. In both cases, the voltage applied on C2 was varied both in sign and magnitude. Detector D was a LINEAR PHD (Therm0 Separation Systems) equipped with a ball lens for capillary detection. Samples were introduced hydrostatically, typically for 5 s, with a height difference of 10.5 cm. Plate counts were computed from N = 5.54(tm/ W I ~ ) ~ where t, is the migration time and W1/2 is the half-width of the peak. In assessment of the results presented, it should be noted that each experiment was repeated three to five times with a plate count reproducibility of 5 4 % in relative standard deviation, unless otherwise stated.
RESULTS AND DISCUSSION Effect on Migration Time. If the applied voltage on the L1 cm long separation capillary C1 is VI volts and that on the L2 cm long pump capillary C2 is V2 volts, the corresponding electric field strengths E1 and E2 are given by El = VlILl
(1)
E2 = VdL2
(2)
If the intrinsic electroosmotic mobility for the combination of the capillary and the specific carrier electrolyte used is pLco cm2 s-l V-I, according to Chien et al.," the over all electroosmotic velocity, uW, is the weighted average of the electroosmotic velocities in the two components of the capillary ensemble:
or
In our experiments, the electric field E l on C I is invariant, and thus the electrophoretic velocity, uep,is given by the product of El and the electroosmotic mobility, pep:
Where pepcan be either positive or negative, depending on the charge of the ion. The overall migration velocity u, is thence given by the vector sum of ueoand uep,as given respectively by eqs 4 and 5 : u, = P,(Vl
+ V,)/(L, + 4)+ EIPLcp
(6)
In the absence of a second capillary and a second electric field (11) Chien,R.-L.;Helmer, J. C. Anal. Chem. 1991,63.1354-1361. Burgi,D.S.;
Chien, R.-L. Anal. Chem. 1991, 63, 2042-2047.
Analytical Chemistry, Vol. 66, No. 19, October 1, 1994
3081
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EXXD Bromthymol blue, pH 9 borate QMDBenzyl alcohol,2pH 9 borate W Fe(o-Phen)3 , pH 7.9, o-Phen,
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Flgure 2. Control of flow in a conjoined Separation capillary (40 cm, 9 kV) by an external electroosmotic pump (20 cm) for anlonlc, neutral, and cationic solutes. See text for experimental conditions.
Flgure 3. Migration of three anlonlc dyes (C1 = 40 cm, 9 kV) belng controlled by an external electroosmotic pump. 2 0.04
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a 0.01 Voltage applied on the pump capillary can dramatically affect the migration rate in the separation capillary. Experimental data are shown in Figure 2 for (a) an anionicsolute bromthymol blue (BTB), detected at 610 nm, (b) an uncharged solute, benzyl alcohol (detected at 208 nm), and (c) a cationic solute, [Fe(o-phen)312+(detected at 510 nm), all experiments being conducted with HV1 = 9 kV. In each case, in accordance with eq 6, the overall migration velocity changes linearly with the voltage applied on the pump capillary as evidenced from the linear dependence of the reciprocal of the migration time upon the pump capillary voltage. The slope of an individual line is equal to p e o / ( L 1 L2); it is a measure of the intrinsic electroosmotic mobility for the particular combination of the capillary and the carrier electrolytesolution. This is essentially the same for cases (a) and (b); the slight difference can be ascribed to the individual experiments being conducted at different times with different capillaries. The slope for (c) is much lower; the carrier electrolyte has a very different composition and pH. In favorable cases, the use of auxiliary pumping can bring about large changes in the migration time. In case (a), the migration time changes from 1.7 min at HV2 = -23 kV to >20 min at HV2 = +1.7 kV. With HV2 approaching +5 kV, the analyte never elutes. This degree of bulk flow control is substantially larger than that attainable with radial field effects, especially at this operating pH. As eq 6 also indicates, the ordinate intercepts for the individual lines in Figure 2 are dependent both on pm and pep,the latter being different for each individual analyte. The reproducibility of auxiliary pumping attained in this fashion is as good as the reproducibility of EOF in a single capillary. Figure 3 shows the combined results of an
+
3062 AnaiyticalChemisrry, Vol. 66, No. 19, October 1, 1994
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Flgure 4. Peak positions and shapes obtained wlth thymol blue as the solute (C1 = 40 cm, HV1 = +9 kV) as a function of auxillary pumplng (C2 = 20 cm). Electrolyte: 5 mM NanB407, 10 mM sodium anthraquinone sulfonate.
experiment in which HV2 was scanned from 0 to -7.5 kV both going up and going down in four separate runs on different days. Also shown on these plots are the observed migration velocities (um0)when there is no second capillary connected. These points are shown plotted at HV2 = -4.5 kV (V2 = OSVI) and are seen to fall on the corresponding line, in accordance with eq 7. The three analytes have different pep, but the pm value is the same in all three cases. In accordance with eq 6 , a set of three parallel straight lines with three different ordinate intercept values are observed. Peak Area and Asymmetry. The change in migration behavior brought about by auxiliary pumping affects both peak efficiency and asymmetry. A portion of the data representing experiment (a) in Figure 2 is used to generate Figures 4 and 5 . Increasing migration times correspond to increasingly positive voltages applied as HV; it is easily observed from Figure 4 that efficiency is not a monotonic function of the migration time. This is examined in greater detail in the next section. Meanwhile it should be noted that the apparent peak areas are not constant as a function of the applied pump capillary voltage. The apparent peak areas (the absorbancedime product, i.e., the physical peak areas in
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P e a k width, a r b i t r a r y u n i t s Figure 5. Peak asymmetry as a function of auxiliary pumping for a subset of the peaks shown In Figure 4. Height and wldth have been normalized(seetextfordetails)suchthat peak positiondirectly Indicates asymmetry.
Figure 4) are related directly to the migration time. Because the migration velocity varies, a constant value is obtained only when the apparent area is multiplied by the migration rate. A second aspect is illustrated in Figure 5 in which the different responses have been normalized to the same height and the same width at the 10% height point. In this format the change in asymmetry of the eluite band at the 10% point ( A s , ~ . lcan ) be readily ascertained. The vertical line corresponds to the peak maximum of a perfectly symmetrical peak (As,0.l = 1.0). As the peak maximum is located more and more toward the right of this line, the As,0.lvalue decreases. What is really striking here is that the asymmetry does not change monotonically with the applied voltage. The peak symmetry is nearly unity at HV2 = 0 (not shown) and this continues at least to HV = -1.5 kV. As,0.l decreases down to HV2 = -5.1 kV. At even more negative HV2 values, the asymmetry begins to increase, until at HV2 = -7.5 kV the peakshapeisalmost thesameas thatat-1.5 kV (itisimportant to realize that the data in Figure 5 are width and height normalized: the peaks obtained with HV2 = -1.5 and -7.5 kV are considerably different in efficiency). Nature of the Charged Solute and Peak Efficiency. The observed peak efficiency varies as the extent of auxiliary pumping is varied, whether induced hydrostatically or electroosmotically. In both cases, the peak efficiencies typically display a maximum. As mentioned in the Introduction, the case for hydrostatic pumping superimposed on electroosmotic flow in a single capillary has been well studied both experimentally and theoretically; maximum plate counts are obtained when the hydrostatic potential is zero. At least for the experimental systems studied in this work (neutral, anionic, and cationic solutes with corresponding electrolyte systems), the maximum plate counts were always found within f5 mm of zero hydrostatic head. The degree of reproducibility in plate count measurements precludes further narrowing of this limit. For the experimental results shown in Figure 6, the results with zero hydrostatic head and no hydrostatic resistance correspond to about 65 000 plates (single asterisk, note logarithmic ordinate). If a second capillary is connected to the separation capillary, the latter presents some resistance
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,,,,
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Migration time, min Figure 6. Change in peak efficiency as a function of auxiliary electroosmotic or hydrostatic pumping. Conditions as in Figure 4 HV2 and the heightdifferenceof the source and destlnatlonvials are indicated on the right and the left of the respective plotted points. Note the iogartthmic ordinate. The asterisk representsthe conventional singlecapillary results (no C2, no HV2).
to the flow, and the maximum number of plates is obtained with added hydrostatic potential, similar to the results reported by K o ~ .Although ~ there is a configurational difference between the experiments of Kok and those herein in that the hydrostatic resistance element is downstream from the detector, a hydrostatic backflow is nevertheless generated and the same considerations as those outlined by Kok hold. The maximum plate count obtained with an optimum amount of hydrostatic head is less than that when no flow resistance is present. For the experiment shown in Figure 6 , the maximum plate count obtained with auxiliary hydrostatic pumping is obtained with a hydrostatic head of ca. 20 cm (not shown) and is
