Capillary isoelectric focusing with a universal concentration gradient

of a low-power He-Ne laser and a 1024 pixel charge-coupled device (CCD). The length of the capillary, the position of the. CCD sensor, and the separat...
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Anal. Chem. 1992, 64, 2934-2941

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Capillary Isoelectric Focusing with a Universal Concentration Gradient Imaging System Using a Charge-Coupled Photodiode Array Jiaqi Wu and Janusz Pawliszyn' Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada

The focused patternsof proteinsamplesby capillary lsoelectrlc focurlng (CIEF) were monitored without moblllzatlon by a concentratlon gradlent lmaglng detectlon system based on the Schlieren shadowgraph method. The system consisted of a low-power He-Ne laser and a 1024 plxel chargecoupled devlce (CCD). The length of the caplllary, the posttion of the CCD sensor, and the separatlon voltage were optlmized. Computer software for background correctlon and nolse reductlon was also employed. When a IOO-pm-Ld., 4cmlong square caplllary was Wed, the separation and detection of proteins havlng Isoelectric point (PI) ranglng from 4.7 to 8.8 could be completed In about 2 min. Thls Is the fastest reported speed for proteln separations by electrophoretlc technlques. The resolution of the system is 0.02 pH units. The PI values of sample components could be determined from thelr positions Inside the caplllary without internalPI markers. The reproduclbility of the focused pattern of proteln samples was better compared to conventional CIEF performed with a moblllzatlon process. The on-column mass detection limit for proteins reached the rub-plcomoie level for the lmaglng system constructed with Inexpensive, commonly available components. The use of the on-llne detectlon system allows for on-llneoptlmlzatlonof focudng voltage and ilme. Sekcttve detectlon of protelns based on molecular weight was also posslble using this system.

INTRODUCTION The newly developed capillary isoelectric focusing (CIEF) technique is a powerful tool for the separation of complex protein mixtures.' Because small-bore capillaries are used, CIEF is rapid (separations are typically completed within 15-20 min), and with high resolution, separates proteins with isoelectric point differences as small as 0.02 pH u n k 2 The narrow capillaries also require only small amounts of sample, which is desirable for the analysis of biochemical materials. Since 1985, there have been many reports on the optimization of CIEF parameters2r and applications of CIEF for the separation of protein sample~.~?5 However, CIEF has several drawbacks, mainly due to the mobilization process. In conventional CIEF, the focused pattern of protein samples is detected by an on-column detector during a subsequent mobilization process.1 Mobilization of focused protein zones is achieved by adding salt (1) Wehr, T.; Zhu, M.; Rodriguez, R.; Burke, D.; Duncan, K. Am. Biotechnol. Lab. 1990,8, 22-29. (2) Zhu, M.; Rodriguez, R.; Wehr, T. J. Chromatogr. 1991,559,479488. (3) Zhu, M.; Hansen, D. L.; Burd, S.; Gannon, F. J . Chromatogr. 1989, 480,311-319. ( 4 ) Kilar, F.; Hjerten, S. Electrophoresis 1989,IO, 23-29. (5) See, for example, separation of the isoforms of a monoclonal antibody by CIEF: Silverman, C.; Komar, M.; Shields, K.; Diegnan, G.; Adamovics, J. J . Liq. Chromatogr. 1992,15,207-219.

to one of the capillary ends. A pH shift occurs first at this end and then gradually progresses deeper into the capillary, causing sample zones to move toward the end. In the process, the distortion of the linear pH gradient is unavoidable, which results in deterioration of resolution. The distortion also makes it difficult to determine the PI values of the protein samples. Also, the mobilization process takes a long time (usually, 10-15 min) compared to the focusing process (4-6 min). Another problem with conventional CIEF is determining when the focusing process should be stopped to begin the mobilization process. The reproducibility for a focused pattern for a particular protein sample depends on properly selecting the length of the focusing process in CIEF. The optimum focusing length is different for different samples. Correctly judging the length requires a substantial amount of operator experience.' The performance of CIEF can be greatly improved in these aspects by eliminating the mobilization process. This can be done by using an on-line imaging detection system to replace on-column detectors. The use of an on-line imagingdetection system also permits on-line optimization of separation parameters and investigation of the focusing dynamics. Several on-line spectroscopic imaging systems have been developed for electrophoresis performed on slabs.697 However, they are difficult to apply to electrophoresis in narrow capillaries. For isoelectricfocusing in capillaries, the focusing process of blue dye stained proteins was monitored by photographic technique under the illumination of visible light. This optical absorption technique requires labeling of the proteins, and the use of photographic film makes it difficult to obtain good quantitative information. An electrode array detector could also be used as an on-line imaging system for CIEF;*however,the resolution obtained by such an imaging system is low due to the limited number of the electrodes. The shadowgraph based on Schlieren optics is a universal way for visualization of refractive index inhomogeneity in a medium. This method has already been used for observing the distribution of carrier ampholytes in gel slabs.9 An imaging instrument based on the method using a xenon lamp and TV camera has also been developed for electrophoresis on polyacrylamide gel slabs.10 Such an instrument cannot be applied to isoelectricfocusingin small-bore capillariesbecause it is difficult to focus a light beam from an incoherent light source into the capillaries. However,the use of modern optical components can greatly improve the performance of detection methods based on Schlieren optics. A single-point concentration gradient detection method based on Schlieren optics using a laser beam source has been proven to be a sensitive, (6) Jorgenson, J. W. Anal. Chem. 1986,58,743A-760A. (7) Chan, K. C.; Koutny, L. B.; Yeung, E. S. Anal. Chem. 1991,63,

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(8) Thormann, W.; Tsai, A.; Michaud, J.; Mosher, R. A.; Bier, M. J. Chromatogr. 1987,389,75-86. (9) Righetti, P. G.; Pagani, M. Gianazza, E. J.Chromatogr. 1975,109,

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(10) Takagi, T.; Kubota, H. Electrophoresis 1990,11,361-366.

0003-2700/92/0384-2934$03.00/0 0 1992 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992

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Probe beam Probe beam Flgure 1. Focal lengths of sample zones with dlfferent zone widths.

universal, and inexpensive detector for capillary electrophoretic techniques,” especially CIEF.12 We have shown the possibility of using a concentration gradient imaging system based on the Schlieren shadowgraph, consisting of a laser beam and a scanning photodiode or a small photodiode array as a CIEF detector.13 However,the use of the scanningmethod introduces additional noise to the detection, and one scan takes about 1 min. The small photodiode array can only monitor a 3-mm length of the 7h”mlong capillary.l3 This imaging system is not a real on-line detector. The performance of the CIEF/concentration gradient imaging system can be dramatically improved in sensitivity, speed, and reproducibility for PIdetermination by using a larger photodiode array with a short capillary. In this paper, we will discuss the construction and optimization of a CIEF/concentration gradient imaging system, which includes a low-powerHe-Ne laser, a low-cost 1024pixel charge-coupled device (CCD), and a short piece of capillary held by a specially designed cartridge.

EXPERIMENTAL SECTION Instrumental Procedures. A 100-pm-i.d., 3.8-4.2-cm-long square glass capillary (Dynamics Inc. Rockaway, NJ) was used for the separation. The capillary inner wall was coated with non-cross-linked acrylamide to eliminate electroosmosis by the reported way.’ The capillary cartridge was similar to that used in our previous e~periment,’~ which consisted of two pieces of glass and two polyethylene buffer reservoirs of 2-mm inner diameter and 1.5-cm length. The capillary was fixed between the two pieces of glass which were glued together to confine any movement caused by temperature inhomogeneity during the focusing process. Ita two ends were connected to the buffer reservoirs which were also glued to the two pieces of glass. The cartridge was mounted on a two-axis stage, the tilt angles of which were adjustable in the horizontal and vertical planes, so that the probe beam could be easily focused into the capillary. The high-voltagedc power supplywas the aame as that of previous experimenta.13 A light beam from a He-Ne laser (Uniphase, San Jose, CA) was used as the probe beam. The beam was first expanded to a 25-mm-diameter beam spot by a beam expander consisting of a 20-fold magnification microscopic objective lens, a pinhole, and a 25-cm focal length lens. It was then focused into the capillary by a 50-mm focal length cylindrical lens, which was mounted on a three-axis stage. The size of the beam spot at the focal point of the cylindrical lens was about 25 cm X 3 pm. After the probe beam passed through the capillary, it was directly (11)Pawliszyn, J. Spectrochim. Acta Rev. 1990, 13, 311-354. (12)Wu, J.; Pawliszyn, J. Anal. Chem. 1992,64, 219-224. (13)Wu, J.; Pawliszyn, J. Anal. Chem. 1992, 64, 224-227.

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intercepted by a 1024 pixel CCD sensor (Type S3903-1024Q, Hamamatau, Hamamatau City, Japan), which had a 26-mm X 0.5” sensing area. The distance between the capillary and the CCD was about 5 mm. In this way, a 26-mm length of the capillary could be monitored by the detection system. The position of the cartridge w i adjustable so that different parts of the capillary could be monitored. The whole system was mounted on a vibration isolation table. The data was collected by an IBM DACA board, in a PC-AT personal computer, using ASYST software (Asyst Software Technology Inc., Rochester, NY).The CCDs trigger signal was employed as the external clock pulse for the board, and the start pulse from the CCDs clock was used as the trigger for the computer. The frequency of the CCD (trigger pulse) was set at 15.6 lrHz due to the 20-kHz maximum acquisition frequency of the A/D board, and the scanning frequency (start pulse) of the CCD sensor was 10Hz. For each measurement,the CCD scanned for three times in 0.3 s, and the light profies of these three measurements were summed to reduce the random noise. First, the background signals, which were recordedbeforethe separation voltage was turned on, were subtracted from the measured data. The measured data was then normalized by the background signals. As a result of those procedures, the measured data corresponded to the relative light intensity changes created by the refractive index changes inside the capillary during the focusing process. Reagents. All chemicals were reagent grade, and solutione were prepared using deionized water. Solutions were fitered using 0.2-pm pore size cellulose acetate fiters (Sartorius,Gob tingen, Germany). Solutionsof 10mM H a 0 4 and 20 mM NaOH were used as anolyte and catholyte, respectively? All proteins were purchased from Sigma Chemical Co. Proteins used include a-chymotrypsin (type 11, from bovine pancreas), human hemoglobin (75% methemoglobin,balance primarily oxyhemoglobin), human carbonicanhydraseI (from erythrocytes),@lactoglobulin B (frombovine milk),ovalbumin (gradeV),and human transferrin (iron-poor). Samples were mixed with the carrier ampholytee (Pharmalyte pH 3-10, Sigma) solution to a f i concentration of 2% ampholytes.’ The protein concentrationsintroduced into the capillary ranged from 0.2 to 2 mg/mL. The iron-free hemoglobin was reacted with ferric ions using the conditions described in ref 4. Isoelectric Focusing Process. The sampleswere introduced into the capillary by pressure. A plug of an 1%agarose gel (prepared in the anolyte, 10 mM H ~ O I was ) placed in the reservoir of the anodic end of the capillaryto avoid hydrodynamic flow inside the capillary. A 3-3.5-kV dc voltage was applied to the two ends of the capillary. The current which paseed through the capillary dropped from 30 to about 3 f i in 2 min before stabilizing. All experimentswere done in duplicate or triplicate to ensure reproducibility. Safety Considerations. A Plexiglass box should be used to isolate the anodic end of the capillary because of the high dc voltage applied to this end.

RESULTS AND DISCUSSION The relative changes of the probe beam intensity after the beam passes through the capillary are proportional to the second derivative of the refractive index distribution inside the capillary.’lJ3J4 Since the relationship between the magnitude of refractive index change and the protein sample’s concentration is approximately linear, the relative changes of the probe beam intensity are also proportional to the second derivative of the protein sample’s concentration inside the capillary.ll Thus, the image recorded by the CCD sensor is expected to correspond to the second derivative of the concentration distribution of the samples inside the capiliq.13

First,the capillary length must be optimized for our CIEF/ concentration gradient imaging system. The length of the (14)Menkirch, W.Flow Viswlization;Academic Preee: New York, 1987.

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capillary usually affects the separation efficiency of the capillary electrophoresis. For most of the reported applications of CIEF performed on commercial instruments, 1460-cm-long capillaries were used. Most commercial instruments use a relatively long capillary because of their design of capillary cartridge and the size of the UV absorption detector. The detection point of the capillary has to be positioned at some distance from capillary end connected to the buffer reservoir, which restricts the use of short capillaries. In zone electrophoresis, a longer capillary may have a better separation efficiency due to the larger plate number. In CIEF, however, the capillary length should have no direct effect on the separation efficiency. The efficiency can be calculated from the standard deviation of the concentrationdistribution of a sample zone, u:15

a

I

b

c = G E (1) where D is the diffusion coefficient of the samde. E = Vll. where V is the separation voltage and 1 is oveh'length of the capillary, and p is given as

where x is direction along the capillary, and du/d(pH) is the change in the mobility of the sample with regard to change in pH. For a linear pH gradient along the capillary, the term d(pH)/dz can be written as A(pH)/Z, where A(pH) is the pH range of the carrier ampholytes. From eqs 1and 2, u can be written as (3)

As described by eq 3, the separation efficiency will not change when a short capillary is used. For example, if 1 decreases to half of ita original length, E will be doubled, and the zone width will also decrease to half of its original length. Therefore, theoretically, it is possible to use shorter capillaries in CIEF to achieve the same resolution asthose of conventional CIEF instruments. A shorter capillary also decreases focusing time and enhances the sensitivity of the concentration gradient detector due to increased pH gradient.12 On the other hand, a long capillary prolongs the focusing and mobilization times and causes distortion in the pH gradient during the mobilization process.2 The best results to date have been reported for CIEF performed on commercial instruments with a 12-14-cm capillary.'S2 However, there has been no report of using even shorter capillaries in CIEF, although they might give better result. For our design of cartridge and detection system discussed p r e v i ~ u s l y the ,~~ limiting factor for using a short capillary is the resolution of the detector, the CCD sensor, since narrower sample zones are created in shorter capillaries as described by eq 3. The ClEF method itself should have a resolution of le2 pH units.'S2 For the CCD sensor with a 25+m pixel width used in the experiments, its spacial resolution ie expected to be 50 gm. If carrier ampholytes of pH 3-10 are employed, it can be calculated that the capillary should be longer than 3.5 cm to obtain a resolution of pH unita. Therefore, in the experiment, a 4-cm-long capillary was used. The use of this short capillary is also expected to increase the focusing speed. A 4-7-min-long focusing time was needed for a 14-cm-long capillary under a separation voltage of 600-800 V/cm.l The time decreased to 3-4 min for a 6-cm-long capillary in our previous experiments. The focusing time for the 4-cm-long capillary was expected to be 1-2 min. (15) Righetti, P. G. Isoelectric Focusing: Theory, Methodology and Applications; Elserier Press: Amsterdam, 1983.

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mm Flguro 2. Images of focused hemoglobin for different CCD positlone: (a) 5 mm from the capillary; (b) 10 mm; and (c) 15 mm.

In the imaging system, as shown in Figure 1,a sample zone inside the capillary (its concentration distribution along the capillary can be assumed to be Gaussian12J6)can be approximately considered as a "lens". The focal length of the "lens" depends on the width and concentration of the zone. A wider zone or lower concentration creates a longer focal length and a narrower one or higher concentration gives a shorter focal length. From Figure 1,it can be seen that the position of the CCD sensor is important for obtaining good sensitivity and a correct image of focused samplezones. Ideally, for obtaining a correct image of all focused zones, the distance between the CCD and the capillaryshould be 0. However, at this position, the sensitivity of the detector is also 0. For our system, the CCD sensor should be placed at the position where the distance between the capillary and the CCD is about the same as the focal lengths of focused sample zones. If the zone widths or concentrations vary in a wide range, it should be placed at the point where its distance from the capillary equale the focal length of the narrowest zone or the highest concentration expected under the experimental conditions. Otherwise, the sensitivity of the imaging system will be low, and the light beam intensity profile recorded by the CCD sensor will give a distorted image of the sample zones inside the capillary because of light beam interference. This can be verified by Figure 2 which shows the electropherogramsof hemoglobin for different detector positions. Peaks 1and 2, which correspondto methemoglobin and oxyhemoglobin,are the narrowest zones in the capillary. When the detector position is 5 mm, two high, narrow peaks are generated by those two zones. When the detector is moved to the position of 10 mm, peak 2 becomes lower and wider more quickly than peak 1does since zone 2 is narrower than zone 1. At the same time, many other wider peaks appear, which correspond to the wider zones of carrier ampholytes. When the detector is moved to the position of 15 mm, peaks 1 and 2 almost disappear, and only the wider peaks remain. The results suggest that the position of the CCD sensor should be determined by the zone width of the sample that determines "focal length" of the zone.

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The focal length of a zone can be estimated theoretically. The relative light intensity for a given zone, with a 4a width, along the F axis as shown in Figure 1can be approximately given by

i(0)=

/-

La

-1 (4) d2(h0 - In (n.4)) where, n is the refractive index of the solution inside the capillary, L is the i.d. of the capillary, and A is given as

b

(5)

where C is the concentration of sample along the x direction and COis the sample's concentration introduced into the capillary. The point of strongest light intensity can be considered as the focal point of the zone as shown in Figure 1. As described by eq 4, the focal point of the 'lens" is at the point where F equals nA. From the previous experimental results, the lengths of protein zones were 200-300 pm for a 7-cm-long capillary.13 For the 4-cm-long capillary used in the present experiment, the zone width is expected to be about 100 pm. For such a zone, the focal length is estimated to be 5 mm, from the highest sample concentration used in the experiments,2 X 10-6 M, dnldCvalue of 1W/M for protein samples.12 In our system, the distance between the CCD sensor and the capillary was usually set at 5 mm. At this position, the imaging system is expected to be sensitive only for protein zones which are much narrower than zones of carrier amph01ytes.l~A correct image of all protein zones is expected to be obtained because of the short distance between the capillary and the CCD and the small deflection angle of the probe beam. As shown in Figure 1and eq 4, sample zones with different widths can be emphasized by moving the detector away or toward the capillary. In the experiments, some protein zones are found to be narrower than 100 pm, such as some bands of ovalbumin. In those cases, the distance between the capillary and CCD is set at 3-4 mm. However, further decrease in the distance is impossible for the present instrument design because of the thicknesses of the CCD window and the glass piece fixing the capillary. Background correction and noise reduction methods are used to obtain enhanced image data in the experiments. Without these methods, the images recorded by the CCD sensor are difficult to use for the analysis of complicated protein samples. Figure 3a,b shows the images of capillary filled with a hemoglobin sample mixed with the carrier ampholytes before and after the separation voltage is turned on, respectively. A lot of peaks and baseline fluctuation can be observed, which make the identification of sample peaks difficult. The baseline fluctuation and peaks are caused by the refractive index defects inside the capillary wall and the intensity profiie of the probe beam itself. They can be corrected by f i s t subtracting the background image (Figure 3a), recorded before the voltage is on, from the measured data (Figure 3b). Then, the data is normalized by the background image (Figure 3a) to correct the baseline fluctuation caused by the intensity profile of the beam itself. After this procedure, as shown in Figure 3c, the image is proportional to the refractive index changes inside the capillary caused by the sample.13 The noise level in Figure 3c can be reduced by averaging data recorded by the fast scanning CCD sensor. Figure 3d is the image obtained by averaging the data from three scans of the same image as those of parts b and c of Figure 3. Figure 3d shows better signal-to-noise ratio compared to that of Figure 3c. It should be noted that the more correct background image is that of pure carrier ampholytes after focusing. However, the ex-

C

c

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mm Flguro 9. (a)Probe beam intensity profile recorded by the CCD before the voltage Ison; (b) probe beam intensity profilefor hemogkMn sample after -in focusing; (c) the profile of b after subtracting a; (d) the same intensity profile as c by averaging three data scanned by the CCD in 0.3 8.

perimental data did not give a higher signal-to-noise ratio or better baseline by subtracting thie background compared to the above procedure. This is because the signal fluctuation caused by the carrier ampholytes is low due to the short distance between the CCD and the capillary. The procedure involvingsubtracting focused ampholytebackground requires two runs per sample, and the results can be affected by the position error between the two runs. Therefore, in our experiments,only the image measured before the voltage was applied is used as the background. The factors affecting the signal-to-noise ratio of the detection were investigated. The noise levels of the detector were measured when the laser beam was on and off. The noise level, when the laser was on and the capillary was not mounted, was found to be 5 times higher than that when the laser was off and about 3 times higher than that when a flashlight was used as the light source. Also, the noise level is proportional to the light beam intensity. The results indicate that the major factor affecting the signal-to-noise ratio of the detection system is the stability of the laser beam intensity profile. Under the present conditions, a signal-tonoise ratio of 80 can be achieved by the detection system for the peaks of hemoglobin protein in Figure 3d which has total concentration of 1mg/mL. This results in detection limit of about 50 pg/mL for the protein, which corresponds to 8.0 X M. The theoretical detection limit of the concentration gradient detector performed in the single-point mode was estimated to be about 1Cr8M for CIJ3F.12 The present experimentaldetection limit for the imaging system is at least 1 order of magnitude above the theoretical limit. Improvement of the sensitivity for the detection system can be achieved by using a beam-stabilized He-Ne laser and a good optical quality capillary, which should results in a detection limit close to the theoretical value. In addition, the averaging approach with multiple scans can further improve signal-to-noise ratio. As shown in Figure 3, the averaging method proved to be very effective. The CCD can scan 10oO times in 300 ms. Therefore, if a fast A/Dboard is used, the averaging method would result in an additional

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signal-to-noise ratio increase of a factor of 30. After these improvements, the concentration detection limit of the imaging system is expected to be below 1 pg/mL. It should be noted that because of the good resolution of the instrument, usually several peaks appear for a protein, which correspond to isoforms. The real concentration of one peak is lower than the total concentration of the sample. For example, the concentration of hemoglobin in Figure 3d is 1 mg/mL. However, as discussed in following paragraph, there are two isoforms in the sample. The concentration of the isoform correspondingto the lower peak in Figure 3d is only about 0.25 mg/mL. The separation voltage was also optimized. Using a higher voltage increases the focusing speed. For example, when a 1.5-kV voltage is applied to the 4-cm-long capillary, the focusing process was completed in 3 min for the hemoglobin sample. The process lasts only 1.5 min when a 3.5-kV voltage is applied. However,the voltage level is limited by the current passing through the capillary at the start of the focusing process. The current is more than 0.1 mA if a 5-6-kV voltage is used and can create enough heat to boil the sample inside the capillary. On the other hand, high voltages provide better separation efficiency as describedby eq 1and, therefore, good sensitivity for the concentration gradient detector. This problem can be solved by a voltage gradient because the current decreases with time in the focusing process in CIEF separation.16 In this gradient, the voltage is first set at a low level to prevent overheating. Then, when the current becomes lower, the voltage is gradually adjusted to a higher level. The focused zones can be observed by the on-line imaging system, and the focusing process is stopped when satisfactory resolution is obtained. This procedure eliminates the need for running repetitive optimization experimentsto determine the best focusing conditions for a particular sample. Here, focusing of ovalbumin sample is taken as a example. The voltage is initially set at 1.5 kV for 2 min. When the current decreasesto 3 pA, the voltage is then increased in stages from 1.5 to 6 kV and is held for 1min at each stage (2.0,3.0,4.0, and 6.0 kV). The on-line detector records all images of the focused patterns. From all images, the result obtained at 4 kV shows the best resolution. The electropherogramfocused under 4 kV can be used as the focused pattern for the samples although the focusing voltage finally increases to 6 kV in the experiment. This unique on-line optimizationis only possible when an on-line imaging detector is used. The changes in the images can also be observed simply with an oscilloscope. The ability of on-line monitoring can also be extended to applications for the selective detection of proteins. Some proteins may focus well at a low voltage or in short time, whereas others may need a high voltage or long time. When a voltage gradient is used, each protein may be selectively determined under ita own optimum conditions. This is impossible in conventional CIEF instruments. This is illustrated in Figure 4. &Lactoglobulin B can be focused much faster than ovalbumin under low voltage probably due to its lower molecular weight and larger diffusion coefficient. Ovalbumin can only be focused under a high voltage. In Figure 4, @-lactoglobulinB is selectively determined under a 1.5-kV voltage after 1min focusing since ovalbumin cannot be focused under these conditions. Then, the voltage is increased to 4 kV, where both proteins can be determined. The on-line detection system may also be combined with pulsed electrophoresis16to obtain better protein separations based on molecular weight. Although, theoretically, higher separation voltage creates better resolution, in practical separations, overheating still (16)Jorgenson,J. W.; Phillips,M. New Directions in Electrophoretic Methods; American Chemical Society; Washington, DC, 1987.

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mm Flgwo 4. Electropherograms of &lactoglobulinB (1) and ovaibumln (2) when a voltage gradient is used. Concentratkins of protelns are 1 mg/mL: (a) 1.5 kV for 1 min; (b) 1.5 kV for 1 min and 4 kV for 1 mln; and (c) 1.5 kV for 1 min; 4 kV for 2 min.

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Voltage (kV) Fbwr 1. Relationship between zone width and theseparationvoltage. Sample is 1 mg/mL ovalbumin. Solid line is the theoretical results according to eq 1. occurs even voltage gradient is used. This causes deterioration in the resolution and increases noise for the universal concentration gradient detector due to temperature inhomogeneity inside the capillary under high current. Figure 5 shows that the best resolution is obtained under a 3-4-kV separation voltage which correspondsto 0.8-1 kV/cm for the 4-cm-long capillary. The resolution starta to deteriorate when the voltage increases to 5-6 kV due to increasing temperature imide the capillary since the current reaches 10-12 PA at the end of focusing process under this voltage. The signal-tonoise ratio also stops increasing when a 5-6 kV voitage is applied, as illustrated by Figure 6, because of the thermal gradient. The optimum separation voltage is about 3.5 kV. Because of application of the short capillary, this applied voltage is much lower and safer than that for capillary zone electrophoresis (CZE) in which a 10-30-kV voltage is commonly used. Under all set conditions, the focusing process can usually be completed in 1-3 min by the 4-cm-long capillary for most protein samples, which is faster than that of conventional

ANALYTICAL CHEMISTRY, VOL. 04, NO. 23, DECEMBER 1, 1982

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Voltage (kV) Flgwe 6. Relationship between signal-tonoise ratlo of the imaging detectionsystem and the separationvoltage. Conditlonsare the same as Figure 5.

CIEF. The length of the focusing process for different samples can also be determined in on-line fashion by the imaging system. Figure 7 shows electropherograms of hemoglobin obtained under a constant separation voltage in a 4-min focusing process. From their positions in the capillary, the two peaks observed in Figure 7 are assigned to methemoglobin (peak 1,PI 7.201, which comprises about 75% of the sample, and oxyhemoglobin (peak 2, PI6.95). These two peaks become higher during the focusing process and stabilize after 2 min. The separation is completed after 2 min for this sample. These results are also confirmed by the changes in current passing through the capiUary duringthe focusing process. The current dropped from 25 to 3 p A in 2-3 min and then stabilized. As expected, a short capillary facilitates fast isoelectric focusing separation. The heights of the signal peaks recorded by the imaging system are approximately proportional to the second derivatives of the concentrations of the protein samples focused inside the capillary."J3J4 The maximum of the second derivatives can be written to be

Thus, the peak height in the image can be used for quantitative determination of protein samples. Figure 8 shows that concentration of protein samples can be estimated from their signal peak height in the concentration range of 0.2-1.0 mg/ mL. As expected, the detection limit of the system reaches 50 pg/mL, which can be estimated from the signal-to-noise ratio in Figure 8d. This correspondsto a low or sub-picomole on-column mass detection limit for the capillary volume, 400 nL, and molecular weight of proteins, 10L109 The detection limit is in the same order of magnitude as that of a UV-vis absorption d e t e ~ t o r .Although ~ laser-induced fluorescence detectors have the highest sensitivity among spectroscopic detectors, they are difficult to use for CIEF because they require the derivatization of proteins which is not suitable for the direct analysis and may change PI values of the proteins. The resolution of the system was evaluated for the 4-cmlong capillary. The PI difference between peaks 1and 2 in Figure 7e is 0.25 pH units, and the distance between the two peaks is about 1 mm. The resolution of the system can be estimated to be about 0.02 pH units from the peak width of peak 2, which is about 75 pm. This resolution is in the same order of magnitudeas those of commercial CIEF instruments? The use of the short capillary does not decrease the resolution in the system because of the high resolution of the CCD sensor and the derivative nature of the signal.12 All above results suggest that protein samples can be separated and detected rapidly by our CIEF/concentration

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mm Flguo7. Images of focused hemogkbin. Concentratbnof the sample Is 1 mg/ml: (a) 3 8 after focusing; (b) 0.5 mln; (c) 1 mln; (d) 2 mln; and (e) 4 mln.

a

b

d

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mm F@MO8. Images of focused hemoglobin for different concentrations: (a) 2 mg/ml; (b) 1 mg/mL; (c) 0.5 mg/mL; and (d) 0.2 mg/mL.

gradient imaging system with good resolution and signalto-noise ratio. Because of the use of a large CCD sensor, a 2.5-cm length of the 4-cm-long capillary can be monitored at a time, which correspondsto 4.4 pH units for PIvalues when carrier ampholytss of pH 3-10 are used. Proteins having PI values in this range are expected to be separated and detected by the system in about 2 min. Figure 9 shows the images of five focused proteins having PI values from 4.70 to 8.76. Only 1.5 min are needed for separation and detection of those proteins. This is the fastest speed reported to date for the separation of proteins by any electrophoretic technique. In conventionalCIEF, mobilization of the focused proteins is achieved by applying an anion or a cation to one end of the capillary.' The mobilization efficiency at the opposite end

ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992

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c

a

a b

I

mm Images of focused transferrlng after 2-min focusing. Concentration of protein Is 1 mg/ml: (a)iron-poor form and (b) after reacted wlth lod M iron ion. Flguro 10.

4

3

+

9

5

27

18

mm Flgure 9. Images of five focused proteins: (1) &chymotrypsin; (2) hemoglobin; (3) carbonic anhydrase I; (4) &lactoglobulin B; and (5) ovalbumin. Concentratlon of sample Is 0.5 mg/mL: (a) 3 s after focusing; (b) 0.5 min; (c) 1 min; (d) 1.5 min; and (e) 2 min.

Table I. Reproducibility of Positions of Focused Protein Zones Inside the Capillary positions of PI values the zones (from cathodic standard of the deviation zones end of proteins (PHunit) capillary) (mm) (mm) a-chymotrypsin 8.76 7.71,7.78,7.81 0.05 hemoglobin

hemoglobin carbonic anhydrase I @-lactoglobulin ovalbumin

7.20 6.95 6.60 5.31 4.70

14.78,14.85,15.06 15.62,15.55,15.76 21.99,22.05, 22.10 27.59,27.65, 27.70 32.28,32.35,32.50

0.21 0.15 0.06 0.06 0.11

of the capillary is low, inducing distortion in the pH gradient. The protein zones focused at this end may become wider, decreasing the resolution, even making those zones undetectable.2 The resolutions for proteins focused at different positions inside the capillary are different in conventional CIEF. Those problems prevent the technique from analyzing protein mixtures with a wide PI value range.2 However, the on-line imaging system can detect all proteins with the same resolution regardless of their positions inside the capillary. Conventional CIEF is also difficult to use to determine protein's PI value from its retention time without internal standards since the mobilization speed is not a constant during the whole process. Also, the protein's retention time in the mobilization process is dependent on the duration of the focusing pr0cess17which can be different for different samples. However, for the CIEF/concentration gradient imaging system, the PI values of proteins may be determined from the position of their zones inside the capillary since a steady pH gradient is established along the capillary during the focusing process. Table I lists the reproducibility of the positions of six focused protein zones inside the capillary after 2 min of (17) Kilar, F. J . Chromatogr. 1991,545, 403-406.

focusing in three runs. In each run,5 proteins are introduced into the capillary. The relationship between zone position and PI value is approximately linear. The relative standard deviation for the position of each zone is less than 1% from the standard deviation in Table I for the 4O-mm-longcapillary, which corresponds to 0.06 pH units. This reproducibility is much better than that of conventional CIEF, in which the relative deviation of the retention times of sample zones is usually 10-15 % in a 12-minmobilization process when carrier ampholytes of pH 3-10 are used.6 In the central portion of the capillary, the data in Table I shows deviations from a straight line. This is due to the nonlinearity of the pH gradient.16 In Table I, a drift toward high value can also be seen in positions of individual zones for the three comcutive runs. This is most likely produced by the deterioration of coating of the capillary, which results in increase in electroosmotic flow.' The coating prepared by the present technique can last about 20-30 runs. The reproducibility of zone position can be improved by using more stable coatings. Because of the good reproducibility of the peak position, the PI values of protein samplescan be determined directly from their zone positions in the capillary without intemal PI markers once the relationship between the position and PI is calibrated by PI markers. The CIEF/concentration gradient imaging system is expected not only to be applicable to all sampleswhich now can be analyzed by conventional CIEF instruments, but also to have its own unique applications. Next, some examples of unique applications of the system to protein analysis will be discussed. Serum transferrin is an iron-transport protein with different isoforms. These isoforms may exist in iron-free and ironcomplexed molecular forms, each of which have different PI values.' The pattem of the transferrin by isoelectric focusing has been an important tool in the study of genetic variations in different population^.^ However, we may use the iron-free transferrin as a probe for the detection of ferric ions. Figure 10showsthe focused pattems of iron-freeand iron-complexed transferrin after 2-min focusing. Figure 10b shows totally different pattern for the protein in the presence of 60 ppb Fe3+from that of Figure loa. Although this protein ferric ion probe is not very practical compared with other existing methods, it shows a new idea for sensitive and fast screening and detection of existence of a target substance which does not need to be an ampholyte. The selectivity and sensitivity of the detection method are expected to be high with the development of new antibodies and new reagents.18 The method may be developed into new assays for fast and sensitive clinic and environmental analysis. (18)Van Emon, J. M.; Viorica, L.Anal. Chem. 1992, 64,79A-MA.

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,Ovalbumin

c

,P-lactoglobulin

B

Y

Time (sec) Figure 11. Relationships between P and t for ovalbumln and @-lactoglobulinB. Data are results of three measurements.

Another possible application of the system is selective determination of proteins based on their molecular weights. When the separation voltage is off, a focused protein zone will broaden. The peak height and width can be used to determine the diffusion coefficient of the proteinlg which is related to the molecular weight of the protein. Thus, the CIEF/concentrationgradient imaging system may distinguish proteins with different molecular weights. For a focused protein zone in free solution inside the capillary after the separation voltage is turned off, the concentrationdistribution can be written a s I 2 (7) where Q is the quantity of the protein in the zone and t is time. The peak height of the signal detected by the concentration gradient imaging system, P, can be given aa

(8) where R is a constant for agiven solution. R can be calculated from the slope of the P - t-3/2 line. Figure 11 shows the relationship between P and t for focused zones of ovalbumin (molecular weight 45 OOO) and @-lactoglobulinB (molecular weight/35 OOO) after the separation voltage is turned off. The measured R values are 1.5 X 104 for ovalbumin and 7.0 X 103 for @-lactoglobulinB, respectively. The difference in diffusion coefficients can be distinguished by this method. Accurate determination of the absolute values of the molecular weights for an unknown protein by this method is still difficult. However, this method may be used for selective measurement of proteins with larger molecular weights in the presence of proteins with smaller molecular weights. Figure 12 shows electropherograms of @-ladoglobulin B and ovalbumin. When t = 0, both ovalbumin and @-lactoglobulinB peaks can be observed. A t 15 s, the peaks of @-lactoglobulinB (peak 1) almost disappear while the peaks of ovalbumin (peak 2) are still visible. One advantage of using the concentration gradient detection for this purpose, compared to detectors (19)Murugaiah, V.;Synovec,R.E.Anal. Chim. Acta 1991,246,241249.

d

I

43

23

mm Figun 12. Images of focused zones of ovalbumin (peak 2) and @4actogbbullnB (peak 1) for different times after the separatkn voltage is tumed off (a) 0 8; (b) 15 s; (c) 30 8 ; and (d) 60 8.

based on concentration detection, is that the peak height is more sensitive to the diffusion coefficient. The signal peak height is proportional to D3I2in the detection system as described by eq 8,whereas, in detectors baaed on concentration detection, the signal peak height is proportional to D1I2 aa shown in eq 7. The above applications illustrate the potential of the system. In addition to all these advantages, the CIEF/ concentration gradient imaging system is also an inexpensive instrument, compared with instruments with laser-induced fluorescence or UV absorption detectors, since its detector mainly consists of a low-power He-Ne laser and low-coet CCD. Further enhancementof the imaging system is still posaible. For example, a narrower capillary can be used in the system if a recently developed diode laser array is applied. T h e imaging system can also be utilized to monitor more than one capillary at a time if a two-dimensional CCD is used, which will increase the throughput of CIEF.

ACKNOWEEDGMENT This work was supported by the National Sciences and Engineering Research Council of Canada.

RECEIVED for review May 29, 1992. Accepted September 10, 1992. Ragistry No. a-Chymotrypsin,9004-07-3;carbon anhydrase, 9001-03-0.