Anal. Chem. 1992, 64, 1046-1050
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Sample Stacking of an Extremely Large Injection Volume in High-Performance Capillary Electrophoresis Ring-Ling Chien* and Dean S. Burgit
Ginzton Research Center, Varian Associates, Inc., 3075 Hansen Way, Pa10 Alto, California 94304
Sampk dacklng k a rlmple ontolumn concentration technlque In hlgh-perfonnancecapillary electrophorosk (HPCE). However, the amount d r(mpk whlch can be loaded Into the column In conventbnal sample dacklng k rather Ibnlted becaw0 of dktubrrncer cauwd by the lowconcentration sample buffer. This report describes a technique d dacklng an extremely large sample volume Into narrow bands. The technique k baeed on the prlnclpal that the local oiectrophoretlc vdodty of the Ions lndde the sample Mer k much raster than the bulk e k c t r m o t l c velocity d the rolutkn. By applyhg a voltage wlth rovemod pdarlty Inmodlately after kadlng the sample, one can remove the sample buffer prlor to separation of anaiytes. Slnce the peak-broadenlng mechanbm b removed from the system, better mo&Iion can be achbved for a large sampk vokme. S h e large amounts of ample can bo loaded onto the cokmn, a factor of several hundreds in rlgnal enhancement Is obtalnod.
INTRODUCTION High-performance capillary electrophoresis (HPCE) has been shown as an analytical instrument of high resolution with the number of theoretical plates reaching several hundred thousand.'V2 In order to achieve such a high resolution, the widths of the analyte zones have to be much smaller than the widths generated from either diffusion broadening or the detector window. These widths are on the order of 1 mm, which translates into an injection volume of 2 nL for a 50pm4.d. column. Such a small volume generates a host of difficulties for conventional chromatography technology. For example, the detection of a small volume using on-column W detaction is hindered by the short optical path. Although the mass limit in HPCE can be very low because of the small volume, the concentration limit is usually in the order of lo* M, which is much higher (worse)than the detection limit for high-performance liquid chromatography (HPLC). An obvious solution to achieve better concentration limits is to improve the detection system. Several different approaches have been studied, such as the use of laser-induced flu~rescence~~' or indirect fluorescences and the installation of a Z-shaped cells or a rectangular ~ o l u m n to , ~ list a few. However, those alternative techniques are either expensive or cumbersome and complicated to implement. It is possible to obtain enhanced detection limits in HPCE by using much simpler techniques like on-column concentration.+l0 Sample stacking is one of the on-column concentration techniques in HPCE."J2 In sample stacking, a long plug of low-concentration buffer (or water, which functions as an extremely diluted buffer) containing analytes for separation is introduced hydrodynamicallyinto the capillarycolumn filled with a support buffer of the same composition but of a higher
* To whom correspondence should be addressed. 'Current addreas: Spectra-Physics, 45757 North Port Loop West, Fremont, CA 94537. 0003-2700/92/0364-1046$03.00/0
concentration. A high voltage is then applied across the column to cause electrophoretic separation. Because the low-concentrationsample buffer has a higher resistivity, the electric field strength in the sample plug will be higher than the rest of the column. As a result, the ions migrate rapidly under this high fiild toward the boundary between the sample buffer and the support buffer. Once the ions pass the concentration boundary, they immediately experience the lower electric field and slow down, thus causing a narrow zone of analytes to be formed in the support buffer region. This stacking mechanism occurs for both positively and negatively charged species. In an untreated silica capillary, which usually has a large electroosmotic flow because of the negatively charged inside surface, the positive species stack up in front of the sample buffer plug and the negative species stack up in back of the plug. These thin zones of ions then move through the support buffer and separate into individud zones by conventional free zone electrophoresis. Theoretically, the degree of stacking and the narrowing of the sample zone is simply proportional to the ratio of resistivities between the sample buffer and the support buffer. Consequently, a sample prepared in water should achieve the maximum amount of stacking when injected into a column filled with a high-conductivitybuffer. However, since the local electroosmotic velocity in the sample plug is greater that the bulk electroosmoticvelocity of the support buffer, the pressure difference caused by the mismatch in eledroosmotic velocities will generate a laminar flow inside the column which will broaden the sharp zone generated by the stacking process and sharply reduce the res01ution.l~ This laminar flow is proportional to both the resistivity and the volume of injected low-concentration buffer. To increase the amount of material loaded onto the column, a balance must be made between the length of the sample plug injected into the column, the concentration of the sample buffer, and the resolution of the separation. Another problem with introducing a long plug of low-conductivity sample buffer into the column is the redistribution of the electric field strength. Since almost all the field strength is dropped a c r w the sample buffer due to its high resistivity, the field strength in the support buffer region where the separation occm approacheszero. Therefore,once the analyte ions migrate out of the sample plug, the electrophoretic velocity of the ions will approach zero. Consequently, all ions will stack up at the concentration boundary and will only move through the column with the electroosmotic flow; no further separation can occur. To stack an extremely long sample plug while retaining high resolution, one needs to remove the sample buffer after the stacking process is completed to eliminate the nonuniform distributions of both the field strength and the electroosmotic velocity. One method is to use a switching valve to move the stacked sample zone physically away from the sample buffer into the support buffer. This report presents a different method which removes the sample buffer by pumping it out of the column using the electroosmotic flow while the sample stacking process is in progress. 0 1992 Amerlcan Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 64, NO. 9, MAY 1, 1992
The on-column concentration technique of pumping out the sample buffer only applies to ions that have a negative electrophoretic mobility with respect to the electroosmotic flow. While the electrophoretic velocity of a charged species is simply the product of its electrophoretic mobility and the local electric field strength, the electroosmotic velocity of the bulk solution has to be averaged over the whole c01umn.’~ Thus, some of the negative ions which usually migrate in the direction of the bulk solution’s electroosmotic flow will obtain a much higher electrophoretic velocity inside the low-concentration buffer and migrate against the bulk flow. To apply the method to a positive analyte, which has a positive electrophoretic mobility with respect to the electroosmotic flow, one has to reverse the direction of the electroosmotic flow, either by coating the inside of the column or by adding an organic modifier to the buffer reservoir. In any event, the use of the sample buffer backout method can yield a more than several hundredfold increase in the amount of sample injected into the column without loss of resolution. In addition, we introduce the idea of whole column sample stacking, which can be very useful in real life applications.
EXPERIMENTAL SECTION The experiments were performed on a research version CE instrument similar to the one reported by Jorgenson and Lukacs? The high-voltage power supply (Glassman High Voltage, Whitehouse Station, NJ) delivered -30 kV in 5-kV incrementa. The column was a 100-cm-long, 50-pm-i.d., 365-pm-0.d. fused-silica capillary (PolyMicro Technologies, Phoenix, AZ)with a detector window at 35 cm from one end of the column. Either end of the column was used as the injection side, giving a detector window at 35 or 65 cm. The detector window was formed by burning off a 1-mm section of the outer polyimide coating. The UV absorbance detector was a Varian 2550 (Walnut Creek, CA) with a 100-pm slit in a modified microcell holder. The wavelength of analysis was 265 nm. The data were collected by an A/D board run by the Varian STAR Integrator and presented in the figures after minimum smoothing by a subroutine in the graphics program Igor (WaveMetrics, Eugene, OR). Stock solutions of two negative ions: 2.3 X 10“ M phenylthiohydantoin (PTH)-asparticacid (PTH-Asp) and 1.7 X 10” M PTH-glutamic acid (PTH-Glu) were made up in HPLC-grade distilled water (Aldrich,WI) and then diluted to 4.6 X and 3.4 X M, respectively. The positive ions were PTH-arginine (PTH-Arg) and PTH-histidine (PTH-His) at concentrations of 1.7 X lo-‘ and 5.0 X 10” M, respectively, prepared in distilled water. The support buffer for separation was 100 mM 24Nmorpho1ino)ethanesulfonic acid (MES) brought to a pH of 6.1 by adding 100 mM histidine (His) to the solution. All chemicals were purchased from Sigma (St. Louis, MO). Sample injection was performed in one of two ways. The fist method was to position the sample vial 15 cm above the floor of the buffer reservoir. The injection end of the capillary was inserted into the vial and held there from 1 to 10 min. A 1-min injection was 0.7 cm in length; a 5-min injection was 3.5 cm in length; and a 10-min injection was 7 cm in length. After sample injection, the end of the capillary was returned to the buffer reservoir containing the support buffer and the electrodes were set up so the sample buffer was pushed out into the buffer reservoir. No significant dilution of the support buffer in the buffer reservoir from the sample plug was seen. The second method of sample injection was to use a syringe and fill the column until the detector responded to the sample passing by the window. This method allowed 35 cm, 65 cm, and the whole column to be filled rapidly. The sample buffer was then pushed out as described below. The electric current was monitored to indicate when the sample buffer was almost removed from the column. Before injection, the current of a column completely fiied with support buffer was measured. After injection, the current level decreased due to the increase in resistivity caused by the presence of the sample buffer. As the sample buffer was pushed out of the column, the current would increase; the current was monitored until it was within 1% of the original support buffer value. The voltage was then turned
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Migration time (min) Figure 1. Electropherogramsof dmerent injection lengths without the sample buffer removed from the W m n . Run conditions: 50 pm, 100 cm long untreated fused-sllica capillary column, 100 mM MES/Hls buffer a! pH of 6.1, detector window located at 35 cm, analysis wavelength 265 nm, -30 kV, 8 pA. Peak A Is 4.8 X lo4 M PWAsp; peak B is 3.4 X M PTH-OIu. Sample injection lengths: (a) 35. (b) 7, (c) 3.5, and (d) 0.7 cm. Panel d is expanded vertically 3 tlmes for comparison purposes.
off, and the electrodes were switched to the separation configuration. To reverse the electroosmotic flow, which allows the concentrating method to be applied to positive ions, an organic modifier, tetradecyltrimethylammonium bromide (TTAB),was placed into the support buffer. There was some diffusion broadening due to the amount of time it took for the sample to translate the column but a hundredfold improvement in sample injection was accomplished. A 1mM solution of TTAB was added to the 100 mM MES/His buffer system. When injections greater than 10 min were performed, a 1mM solution of TTAB was added to the sample buffer.
RESULTS AND DISCUSSION Sample Stacking without Sample Buffer Backout. For hydrodynamic injections there exists an optimal point as to the amount of material loaded onto the column without loss of the high resolution of HPCE. The optimal point is dependent on the length of the sample plug and the concentration ratio between the sample buffer and the support buffer.” Figure 1 is a comparison between four different injection lengths without the sample buffer removed from the column. The sample is prepared in water for all injections. Figure l a is a 35-cm injection; Figure 1b is a 7-cm injection; Figure ICis a 3.5-cm injection; Figure Id is a 0.7-cm injection. Figure Id is within the optimal conditions for sample stacking, thus showing the resolution obtainable under these separation conditions. The negative species PTH-Asp (peak A) and PTH-Glu (peak B)are baseline resolved, peak widths are 4.5 and 5.2 s, respectively, and they migrate pass the detector window at the correct time for these separation conditions. The electropherogram is enhanced 3X to allow comparison with the other runs. The four electropherograms in Figure 1clearly indicate how the redistribution of the electroosmotic flow and the depletion of the support buffer electric field influence the migration time of the analytes. As indicated by the migration time of the water plug, the average bulk electroosmotic velocity increases as the volume of the sample buffer increases. At the same time, the electrophoretic velocity of the ions decreases in the support buffer region resulting in the two negative species peaks getting closer to the water plug and to each other. In addition, the initial sample volume will also change the migration length to the detector window. The combination of
ANALYTICAL CHEMISTRY, VOL. 64, NO. 9, MAY 1, 1992
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Comparison of peak shape continuously at three different injection lengths: (a) 05, (b) 7, and (c) 0.7 cm. Panels b and c are vertically enlarged 10 and 30 times, respectively. The sample buffer is removed after the stacking is completed. Figure 3.
those three effects causes a dramatic change in migration times of the analytes, which is very undesirable in HPCE. Figure 1also shows how the laminar-broadeningmechanism generated from the mismatch in the electroosmotic flows destroys the resolution of the separation. The peak widths in Figure ICincrease to 6.5 and 7.5 s for PTH-Asp and PTH-Glu, respectively. The resolution of the separation continues to degrade as the volume of sample buffer increases as shown in Figure 1. Finally, the negative ions can no longer resolve themselves from the sample plug as seen the Figure la. In this electropherogram, the sample plug is so long, the average bulk electroosmotic velocity is dominated by the sample plug and the electric field in the support buffer is near zero; thus the ions cannot be separated from the sample plug and resolve themselves. Sample Stacking with Sample Buffer Backout. To increase the amount of analytes injected into the column while retaining high resolution, one has to be able to remove the sample buffer after the stacking process is completed. In a normal separation configuration with a negatively charged column wall, the positive voltage is applied at the injection end of the capillary column to cause the support buffer to migrate electroosmotically toward the detector end of the column. The positive analyte ions will then stack in front of the sample plug, and the negative analyte ions will stack at the back of the plug after the high voltage is applied. In our experiment,we applied a negative high voltage on the injection end immediately after the sample is loaded into the column. The support buffer can now migrate out of the column from the injection end instead. Under this reversed polarity, the positive analyte ions are the first to be pushed out of the column by the electroosmotic flow followed by the neutral sample buffer. When the sample buffer is almost completely out of the column, the polarity of the electrodes is reversed again to the normal separation configuration with the positive voltage on the injection end. The negative analyte ions will remain in the column and turn around toward the detection end, and separation of the negative species can then occur. Figure 2 is a comparison of four different initial injection lengths with the sample buffer removed as described above. Figure 2a is 65 cm; Figure 2b is 35 cm; Figure 2c is 7 cm; and Figure 2d is 3.5 cm. All the electropherograms are plotted on the same scale to show the increase in amount of material
loaded onto the column. All electropherograms are baseline resolved. Since the sample buffer has been pushed out of the column, the electric field strength and the electrmmotic flow are constant during sample separation regardem of the initial sample volume. Consequently, the migration times are reasonably reproducible, the slight difference is probably due the different amounts of water which remain in the column for each run. Figure 2c shows how the presence of a little bit more water will change the bulk electroosmotic velocity and the migration time of the separation. Nevertheless, both Figure 2a and 2b indicate that large amounts of analyte can be loaded onto the column without a great loss in resolution. Figure 3 is a comparison of the PTH-Asp and PTH-Glu peaks in the 65- (a), 3.5- (b), and 0.7-cm (c) injections with the sample buffer removed after the stacking process is completed. The electropherogram of the 0.7-cm injection is enhanced 30X, and the 3.5cm injection is enhanced 1OX to allow comparison between peak shape and migration time. As seen, the difference in migration time between PTH-Asp and PTH-Glu in all three runs is the same. The peak shapes are similar in all three runs except the 65-cm injection shows the characteristic triangular peak shapes of overloading. In a conventional injection in HPCE, the sample length is limited to less than 0.1 cm. Consequently, B 65-cm injection means a 650-fold increase in the amount of sample injected into the column. The sample zones are so concentrated that isotachophoresis is occurring in the sample zone, which leads to broadening of the peaks.' Whole Column Sample Stacking. Since two-thirds of the column can be fiied with negative analytes and separated into individual bands without a great loss of resolution, the next step is to fill the whole column with the sample in water and concentrate the entire column length of sample into a narrow zone. Figure 4 is an electropherogram of the entire separation process from sample stacking and removal of the sample buffer to the separation of the negative ions. The detector is located at 35 cm from one end of the column where the sample buffer was pushed out by the supporting highconcentration buffer. At the beginning, the whole column, including the detector position, is fiied with sample buffer. The negative ions start to stack at the end of the sample plug after the high voltage is applied while the sample buffer is pushed out from the column. As seen in the figure, the negative species are following the sample plug and passing by the detector window after about 4.5 min. However, the negative species are not separating into individual zones because the electric field in
ANALYTICAL CHEMISTRY, VOL. 64, NO. 9, MAY 1, 1992
species i and u b is the bulk electroosmotic velocity of the solution. The negative sign indicates the electrophoretic velocity has to be in a direction opposite to the electroosmotic flow. The electrophoretic velocity of the analytes inside the sample buffer region is simply proportional to the local field strength and could be expressed ad4
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Flgure 4. Electropherogram of a whole column lnjectlon. The whole column was fllled wtth the sample solutlon at tlme zero and Inserted Into vials contalnlng the support buffer. A voltage with the reversed polarity was then applied and the sample buffer mlgrated out of the column from the hjectlon end. At about the 4.5-mln mark, all negative analytes that stacked Into a single zone passed the detector for the first tlme and contlnuously mlgrated toward the lnjectlon end. The polarity of the electrodes was swttched to normal conflguratlon at the 7-mln mark and caused the support buffer to flow in the oppostte direction. The negathre analytes were then separated and passed the detector again between the 15- and 20-min mark.
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where ~ . l is~ the ~ i electrophoretic mobility of ion species i, y is the ratio of the resistivities between sample buffer and the support buffer, Eo = V / L is the field strength of a uniform system with voltage V across the column length L, and x is the length of the sample buffer region normalized with respect to L. For yx >> 1, eq 2 can be simplified as
(3) The electroosmotic velocity of the solution on the other hand is a bulk property and has to be averaged over the whole column. It generally changes rather insignificantly as the sample buffer is replaced by the support buffer.13 It can be approximated by (4)
where bBois the electroosmotic mobility of the sample buffer. Substituting eqs 3 and 4 into eq 1 yields -Pepi/X
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the support buffer is near zero. The pushing out of the sample buffer continues until the current reading reaches 99% of the support buffer current level, which occurs at about 7 min. The electrodes are then switched, the electroosmotic flow is reversed, and separation of the negative species with baseline resolution occurs. Although the whole column was loaded with the sample solution, some of the analytes would not be able to be loaded onto the column for separation. Figure 5 is a plot of the peak areas of PTH-Asp versus injection length. The amount of analyte left inside the column begins to roll over at about 65-cm injection length. A simple model is presented here to explain this phenomenon. In the previous section, as the sample buffer is pushed out of the column by the electroosmotic flow, the negative ions overcome it and stack inside the column. To achieve that goal, one relies on the principle of field enhancement inside the sample buffer region such that -’epi ub (1)
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where -uepi is the local electrophoretic velocity of the analyte
For example, the electrophoretic mobility of PTH-Asp calculated from the electropherogram in Figure 2 is about 55% of the electroosmotic mobility of the sample buffer. We can then f i i up to more than half of the column with the sample solution and stack them into a sharp zone as shown in Figure 3. In reality, it is easier to fill the whole column with sample/water and then insert the column into the high-concentration buffer reservoir to perform sample stacking. At the beginning, there is no field enhancement because the whole column is filled with water. Consequently, some of the analytes will be carried out of the column by the electroosmotic flow. However, as the high-concentration buffer slowly replaces the water inside the column, the electric field in the region with water will begin to increase rapidly.’* As the ratio of the length of the water drops to the maximum fill length x,,, the analytes will then migrate opposite to the electroosmotic flow and stack at the rear end of the sample buffer. Positive Ion Separation. The separation of positive species can be done by placing an organic modifier, which changes the direction of the electroosmoticflow, into the buffer reservoir. Figure 6 is a series of electropherograms which shows the separation of positive ions; peak C is PTH-His and peak D is PTH-Arg. The elution order is reversed because the electric field is in normal configuration but the electroosmotic flow is reversed; thus, the more positive species elute out last. Figure 6a is a 0.7-cm injection of the positive species prepared in water. The support buffer is 100 mM MES/His with 1 mM of TTAB to reverse the electroosmotic flow. The electropherogram is shown to indicate that separation of positive species is possible using TTAB. Figure 6b is a 7-cm injection with the water plug backed out of the column. The
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ANALYTICAL CHEMISTRY, VOL. 64, NO. water,
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Migration time (min) Flgure 6. Electropherograms of positive ion separation. Panel a is a 0.7cm InjecUon without the sample buffer removed. Run condklon: 1 mM n A B in support buffer, water in sample buffer, -30 kV, 25 PA. PeakC ispTKHs; peek D is PW-Arg. The Rgve Isverticellyenlarged 2 times for comparison purposes. Panel b is a 7.0-cm injection with the sample buffer removed. Run conditions are the same as in panel a. Panel c Is a 35cm InjecUon wlth the sample buffer removed. There is 1 mM TTAB included in the sample buffer.
heighta of the peaks are 10 times larger than those seen in Figure 6a; the resolution is the same in both electropherograms, in fact, the peaks are slightly narrower than those in Figure 6a. Figure 6c is a 35cm injection with the sample buffer backed out of the column. To perform this separation, 1mM "AB had to be added to the sample buffer. If the sample is prepared in water, the zeta potential in contact with the sample plug region is negative and the sample plug moves counter to the rest of the column. A standoff occurs in the column and the sample plug is not pushed out of the column; no separation occurs. By adding the "TAB to the sample buffer, the (potential in the sample plug is similar to the rest of the column, and the sample plug can be pushed out of the column. However, the mismatch in the {potential due to a difference in ?TAB concentration caum band broadening and resolution of the separation decreases. Also, the enhancement factor, Le., the ratio of concentration between the sample plug and the support buffer, has decreased; thus,band broadening from
CONCLUSION In this report, we have demonstrated an on-column concentration technique which stacks an extremely large sample volume into narrow bands. By removing the sample buffer out of the column prior to separation, the high-resolution separation capabilities in HPCE are preaerved. This technique applies to a fused-silica, uncoated column which has a large electroosmotic flow. In an extreme case, the whole column can be filled with sample solution and then stacked into narrow bands. This whole column sample stacking is attractive and simple and requires no additional equipment over a conventional CE instrument. When the amount of analyte injected is compared to a conventional injection with 0.1-cm sample length, a several hundredfold improvement is seen. This technique is excellent in separating and deteding minute amount of analytes and impurities in the sample solution. However, it could be problematic in some real solutions where matrix components may also be concentrated. Large amounta of background matrix might degrade separation and mask the analytes of interest. Nevertheless, it is our opinion that our concentration technique can be used as a stand-alone microsample preparation device and can be coupled to other analytical instruments. REFERENCES (1) Mlkkers, F. E. P.; Everaerts. F. M.; Verheggen, Th. P. E. M. J . Chromtogr. 1979, 769, 11-20. (2) Jwgenson, J.; Lukacs, K. D. Anal. Chem. W81, 53, 1298-1302. (3) Cheng, Y. F.; Dovichi, N. J. Scknce 1988, 242, 562-564. (4) Pentoney, S. L., Jr.; Hung, X. H.; Burgl, D. S.; Zare, R. N. Anal. Chem. 1988, 60, 2625-2629. (5) Kuhr, W.; Yeung, E. S. Anal. Chem. 1988, 60, 2642-2646. (6) Chervet, J. P.; Van Soest, R. E. J.; Ursem, M.; Salzmann, J. P. Presented a t the Thhd International Symposlum on High Performance Caplliary Electrophoresis. San Diego, CA, Feb 3-6. 1991. (7) Tsuda, T.; Sweedler, J. V.; Zare, R. N. Anal. Chem. 1900, 62, 2149-2152. (8) Everaerts, F. M.; Verheggen. Th. P. E. M.; Mikkers, F. E. P. J . ChromtOgr. 1979, 760, 21-38. (9) Hjerten, S.; Jerstedt, S.; Tiselius, A. Anal. B/ochem. 1965, 7 7 , 219-223. ( I O ) Foret, F.; Sustacek, V.; Bocek, P. J . Mlcrocdumn Sep. 1900, 2 , 229-233. (11) Burgl. D.S.; Chien, 13.-L. Anal. Chem. 1991. 63,2042-2047. (12) Burgl, D. S.; Chlen, R . I . J . Mlcrocoumn Sep. 1991, 3, 199-202. (13) Chlen, R.-L.; Helmer, J. C. Anal. Chem. 1991, 63, 1354-1361. (14) Chlen, I?.-L. Anal. Chem. 1991, 63,2868-2689.
RECEIVED for review November 11,1991. Accepted February 10, 1992.
