Anal. Chem. 1992, 64, 1745-1747
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Whole Column Absorbance Detection in Capillary Isoelectric Focuslng Tiansong Wang and Richard A. Hartwick* Department of Chemistry, State University of New York at Binghamton, Binghamton, New York 13902
INTRODUCTION Isoelectric focusing (IEF) is a very important separation method, especially for protein mixtures. The recent advent of capillary electrophoretictechniques have spawned interest in capillary isoelectric Typical capillary IEF employs 25-200-pm4.d. and 12-20-cm-long capillaries with an on-column absorbance detector. Capillary IEF reflects the general advantages of high-performance capillary electrophoresis (HPCE), such as high efficiency,high speed, and small amount of sample. The capillaries are usually modified by the bonding of a polymer, such as polyacrylamide to the wall6 to eliminate electroosmotic flow during the focusing process. After focusing, mobilization can be achieved by replacement of a basic catholyte with acid or an acidic anolyte with base,l by the addition of salt to the catholyte or anolyte,2 or by hydrodynamic ~ u m p i n g .Alternatively, ~ Mazzeo and Krull' added methyl cellulose into the ampholyte to create a controllable electroosmotic flow to carry the bands to the detection region. Since a focused IEF separation represents an equilibrium situation, whole-columndetection without mobilization offers substantial potential advantages both in time and in the maintenance of separation integrity. Following their earlier work? Rowlen et al.9 utilized a series of diodes set about 1 cm apart to monitor the peak width of a zone on a chromatographic column during the separation process. Earlier, Evans and McGuffinlO used two-point detection to isolate extracolumn variance on an open capillary. Recently, Wu and Pawliszynll constructed a concentration gradient imaging detector in which a segment of capillary was illuminated by a defocused laser beam. Detection of the 2cm illuminated zone was accomplished either by a single photodiode which was mechanically moved across the detection region or by a photodiode array. We have investigated an alternative method of detection in which the entire column is transported past a single detection point, using an easily modified UV-vis detector, a technique we have called capillary scanning detection. A similar approach was taken by Brumbauch and Ackers12 in 1968to study protein separations during gel permeation chromatography. This application suffered from excessive noise levels, and no actual chromatograms were shown. According to the nomenclature of Rowlen et al.? capillary scanning falls under the rubric of whole-column detection (WCD), and we have maintained that terminology for this study to prevent a proliferation of similar terms. The first generation instrument fabricated in our laboratory produced noise levels (1)Hjerten, S.;Zhu, M. J. Chromatogr. 1985,346,265-270. (2)Hjerten, S.;Liao, J.; Yao, K.J . Chromatog. 1987,387, 127-138. (3)Hjerten, S.;Elenbring, K.; Kilar, F.; Liao, J.; Chen, A. J. C.; Siebert, C. J.; Zhu, M. J. Chromatogr. 1987,403,47-61. (4)Zhu, M.; Hansen, D. L.;Burd, S.; Gannon, F. J.Chromatogr. 1989, 480,311-319. (5)Zhu, M.; Rodriguez, R.; Wehr, T. J. Chromatogr. 1991,559,479488.
(6)Hjerten, S. J. Chromatogr. 1985,347,191-198. (7) Mazzeo, J. R.; Krull, I. S. Anal. Chem. 1991,63,2852-2857. (8)Rowlen, K. L.; Duell, K. A.; Avery, J. P.; Birks, J. W. Anal. Chem. 1989,6I,2624-2630. (9) Rowlen, K. L.; Duell, K. A.; Avery, J. P.; Birks, J. W. Anal. Chem. 1991,63,575-579. (10)Evans,C. E.;McGuffin, V. L. Anal. Chem. 1988,60,573. (11)Wu, J.; Pawliszyn, J. Anal. Chem. 1992,64,224-227. (12)Brumbaugh, E.E.;Ackers, G. K. 1968,24,6315-6324. 0003-2700/92/0364-1745$03.00/0
comparable to static detection. The use of whole-column detection permits the application of signal averaging and other signal to noise enhancement techniques. The preliminary results of the whole-column detection method applied to capillary isoelectric focusing are reported in this paper.
EXPERIMENTAL SECTION Apparatus. Figure 1is a schematic diagram of the capillary scanning apparatus. The detector was a Spectra Physics Model 100, using a Spectra Physics 4400 integrator (Spectra Physics, Freemont, CA). The original cell was substituted with an adjustable aperture cell constructed in our laboratory, and ita design has been described e1~ewhere.l~ The aperture was 37 pm X 0.5 mm. In order to reduce friction,the groove on the detector cell body was covered by a Teflon film, and the rigid capillary retainer was substituted by a foam ring. A DA-1 synchronous motor (1turn/min) was obtained from Hurst MFG (Princetone, IN) and was used to pull the capillary. The pulling speed could be changed by selectingthe diameter of the drivingwheel. Fusedsilica capillaries of 75-wm i.d. X 360-pm 0.d. with either a UVtransparent coating or a polyimide coating (Polymicro Technologies, Phoenix, AZ) were used, and the typical length was 20 cm. A Model PS/MK30P02.5 30-kV power supply (Glassman High Voltage, Whitehouse Station, NJ) was used for isoelectric focusing. Signal averaging experiments were conducted using a Maxima data acquisition system (Waters Associates, Milford, MA) with a 16-bit AD converter, with transferral of the ASCII data to Quattro software, rev. 3.0 (Borland International, Inc., Scotts Valley, CA) for final reduction and plotting. Procedures. For dynamic noise measurements,the capillaries were filled with water, and an 8-cm segment was pulled through the cell. The capillaries with UV-transparent coating were used without any treatment. The polyimide coating of regular capillaries was stripped using hot sulfuric acid. In the IEF experiment, the regular capillary was first modified by PEG 8M10 according to the temperature protocols recommended by the chemical manufacturer, and then the polyimide coating was stripped by hot sulfuric acid. For IEF separations, proteins were dissolved in 2 % FisherBiotech ampholytes with an individualconcentration of 0.05 mg/ mL, and the capillary was filled with this protein solution. The anolyte was 20 mM H3P04;the catholytewas 20 mM NaOH. The focusing voltage was 400 V cm-l. After focusing, the capillary was transferred to the detector for moving column detection.All experiments were conducted at 280-nm wavelength with a 1.0-s rise time on the detector. Chemicals. Bovine albumin (A-21531, ovalbumin (A-5503), @-lactoglobulin B (L-8005),and carbonic anhydrase ((2-7500)were obtained from Sigma Chemical Co. (St. Louis, MO), PEG 8M-10 from Innophase Corp. (Portland, CT), sulfuric acid and phosphoric acid from J. T. Baker Chemical Co. (Phillipsburg,NJ), and pH 3-10 ampholytes and sodium hydroxide from Fischer Scientific (Fair Lawn, NJ). Safety Considerations. Care must be taken when the hot sulfuric acid is used to strip the capillary. Operation must be conducted in a fume hood, while the operator's hands are protected by long rubber gloves. Suitable eye and clothing protection is also necessary.
RESULTS AND DISCUSSION Dynamic Noise. For whole-column absorbance detection, since the capillary functions as a lens with a very short focal length, small changes in its position will produce large shifts in the light path through the cylindrical capillary, usually (13)Wang, T.; Hartwick, R. A,; Champlin, P. B. J.Chromatogr. 1989, 462,147-154. 0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 84, NO. 15, AUGUST 1, 1992 1
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5 Figurr 1. Apparatus for whole column scanning detectlon: (1) Integrator;(2)detecm;(3)caplilary,(4)wlper;(5)foamrhg;(8)adjuetable aperture cell body; (7)spring wire; (8) connector: (9) thread; (10) guMe wheel; (11) drhrlng wheel; (12) motor.
2 min H Figure 9. Isoelectropherogram obtalned uslng movlng column detection. Condltlons: capillary dimensions, 75-pm 1.d. X 380-pm 0.d. and 20 cm long, modlfled by cross-llnked PEG 8M-10; 2% ampholyte (pH 3-10); development voltage 8 kV; 6mln focusing tlme; detectlon wavelength 280 nm; movlng speed 0.3 mm/s. Key: (1) ovalbur&; (2) bovinealbumin; (3)~4actogbbullnB (4a and 4b) carbonlc anhydrate (precipitation). 0.05 mg/mL each.
1 min Figuro 2. Noise levels from dlfferent capillaries and conditions: (a) UV-transparentcoated capillary, movlng speed 0.30 "1s; (b)strlpped silica capillary, moving speed 0.30 "1s; (c)strlpped silica caplllary, static. All capillaries were 75-pm-1.d. X 380-pm-0.d. caplllarles fllled
with water, wavelength 280 nm.
resulting in output signal changes (dynamicnoise). Capillary positioning accuracyis not a problem in fixed-pointdetection, as long as the capillary position is constant. Success of the capillary scanning technique depends upon reducing this mechanically-induced dynamic noise when the capillary is pulled through the detector. Initial experiments with the capillary scanning method using an unpolished metal groove produced unacceptable noise levels. Not surprisingly, it was found that the reduction of friction between the capillary and the groove played a critical role in reducing dynamic noise. Reduction in friction was accomplished by polishing the groove and, ultimately, by masking the groove with a thin layer of Teflon. In addition, the use of a wiper assembly was necessary to prevent dust particles from being registered as false peaks. Figure 2 presents three baselines obtained from two kinds of capillaries, the UV-transparent coated and the stripped types, using the Teflon-masked cell. The mechanical noise from the transparent coated capillary was found to be 3 times higher than that observed under static conditions. Occasional peaklike signals were observed, which may be caused by scratches on the coating. In addition, dynamic noise from the transparent coated capillary was also sensitive to the pressure applied to the capillary through the foam, with too high and too low a pressure resulting in increased noise.
Dynamic noise from the stripped capillary was found to be significantly lower than the coated capillary (6.4X 10-4vs 16 X au). The dynamic noise from the stripped capillary was only ca. 20% higher than the static noise level of 5.4 X au. In addition, the stripped capillary was found to be insensitive to the foam pressure, and no peaklike signals were observed. It is not known whether these observed differences are a result of the optical properties of the UV-transparent coating andlor nonuniformity of the coating, of the friction between the coated capillary and the cell setting up vibrations, or both. Further attempts to optimize the groove material for the coated capillaries were not conducted. All subsequent studies were conducted on stripped silica capillaries. Another important factor that contributed to the dynamic noise was the pulling operation. The mechanical vibration generated in pulling will be conducted through the capillary, with a resulting increase in dynamic noise. It was found that this noise source could be reduced by pulling the capillaries with a thread, and by using a relatively heavy (5-g)connector for noise damping. The effect of pulling speed was examined, and no obvious differences in dynamic noise levelswere found between 0.30 and 0.55 mm/s. The choice of pulling speed will be determined by the detector time constant, and by the data system acquisition rate. IsoelectricFocusing. One of the advantages of capillary IEF over conventionalIEF is high speed, since field strengths of several hundred volts per centimeter can be used. Figure 3 shows an IEF electropherogram obtained by capillary scanning detection. The focusing took 6 min, and the detection took another 6 min. The sharp split peaks of carbonic anhydrase were caused by precipitation, which could be directly observed as cloudy material under a microscope. An advantage of whole-column detection is that the solute zones are readily recoverable at will by sectioningthe capillary. Whole column detection also avoids the problems associated with zone mobilization, such as nonuniform mobilization efficiency and peak 10sses.~
ANALYTICAL CHEMISTRY, VOL. 64, NO. 15, AUGUST 1, 1902 MOVING COLUMN DETECTION - IEF SIGNAL AVERAGING VS SINGLE SCAN 2a
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z k " ' 1st Scan (X 6)
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Flgurr 4. Signalaveraglngof an isoelectropherogram. The conditions are the same as those of Figure 3 except a moving speed of 0.55 "1s. The lower trace Is the first scan (muitlplied by 6 to normalize the signal levels); the upper trace is the sum of seven scans. Peak klentificatlon: (1) bovlne albumin; (2a and 2b) 84actogiobuiin B.
Signal Averaging. Another advantage of whole-column detection is the possibility of signal to noise ratio enhancement by signal averaging. Figure 4 shows the increase in the signal to noise ratio obtained by averaging seven scans of a single capillary. An increase in the signal to noise ratio of 2.3 was observed, which is close to the theoretically expected value of 2.6. In order to average the data, it was necessary to normalize the times of each data set by referencing the time of a single sharp peak. Ultimately, this can be more easily accomplished by having two sharp reference lines scribed on the capillary at each end. This would also help to locate zones for recovery, since accurate measurements from each reference line could be correlated to the isoelectropherogram. A limitation of signal averaging is also obvious in Figure 4,in that the peaks have broadened considerably over the course of the seven scans. Some of this is due to diffusion, because there is no voltage applied during detection, while some is undoubtedly caused by mechanical movement of the fluid within the capillary during the scanning process.
CONCLUSIONS The results have shown that whole-columndetection using the capillary scanning mode with UV detection is feasible. The f i t generation instrument produced dynamic noise levels only 20% higher than the noise observed for the same system under static conditions. The moving column detection method offers many advantages over elution separations using single-point detection, and these advantages may bring benefits to other capillary electrophoresis separation modes,
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not just IEF. The application of signal averaging can help to offset any noise increases in detection. It should be possible to employ novel detection methods, such as radioactivity, by being able to monitor the column over time at any position. Detector and data system time constants become less of a constraint even for high efficiency peaks, since the scanning speed can be adjusted as required. Also, columns can be rescanned using several wavelengths, or even using fluorescence, without the need for diode arrays or multiple detectors on a single column. This could offer advantages when applied to DNA sequencing and other biological separations. Another major set of advantages lies in the ability to perform the separation without compromise. In IEF, since the mobilization step is removed, focusing can be performed without compromise and be compared to conventional focusing systems. When PAGE gel separations are used, the use of a development, rather than an elution, mode likewise permits direct comparison of data between conventional slab gel methods and HPCE. The use of gel gradients becomes possible, since it is not necessary for the zone to migrate past any particular point on the column to be detected. This should also permit the use of immune response based separations and detection modes to be applied to the capillary format. Finally, since development of the separation takes place outside the detector, multiple capillaries can be prepared simultaneously,and separations can be easily conducted under refrigerated or other conditions. * Limitations of moving column detection methods include the need to handle the capillary and molecular diffusion. Any movement of fluid in the capillary during handling will result in parabolic flow, with resulting peak distortions. In the data presented here, even when one end of the capillary was plugged, some distortion of the zones was observed. Molecular diffusion will also limit the time over which a capillary can be scanned and stored. One possible solution to these problems, which is currently under investigation in our laboratory, is the introduction of an innocuous monomer into the electrolyte, which can be cross-linked after separation development, thus effectively "freezing" the separation for subsequent handling and long-term storage.
ACKNOWLEDGMENT This research was supported by Spectra Physics Analytical Instruments and by the New York Science and Technology Foundation through the Center for Biotechnology of SUNY at Stony Brook. We wish to thank Scott Weinberger (formerly of Spectra-Physics) for his stimulating discussions on the instrumental design on this project.
RECEIVED for review February 13, 1992. Accepted May 4, 1992. Registry No. Carbonic anhydrase, 9001-03-0.
