Anal. Chem. 1994,66,867-873
Dual Detection for Capillary Isoelectric Focusing with Refractive Index Gradient and Absorption Imaging Detectors Jlaqi Wu and Janusz Pawllszyn' Department of Chemistw, Univers@ of Waterloo, Waterloo, Ontario N2L 3G 1, Canada A unique imaging detector was constructed for capillary isoelectric focusing (CIEF)by combining a selective optical absorption imaging detector with a universal refractive index gradient imaging detector. AU sample zones focused inside a 100-pm-i.d., 4-cm-long capillary by isoelectric focusing were detected in a real-timefashion by the universalimaging detector, and their spectroscopic properties were measured by the absorption imaging detector almost simultaneously. The refractive index gradient imaging is based on a dark-field Toepler-Schlieren system. The optical alignment for both detection modes was optimized. The sensitivity of the detector was limited by noise due to opticaldefects in the capillary waU. Peptides without tyrosine and/or tryptophan can be detected by the universal mode of the detector. These peptides are difficult to detect by commercial CIEF instruments which use a UV-visible on-columnabsorption detector operated at a 280MI wavelength. The instrument can be used for fast analysis of protein samples, such as high-resolution separation and detection of human hemoglobin variants, and to investigate dynamic processes occurring inside capillary.
Capillary isoelectric focusing (CIEF) has become a powerful capillary electrophoretic technique for protein separations because of its high speed (2-20 min of separation time), resolution (0.01 pH unit), and small sample consumption (nL). Recent applications of CIEF have concentrated on the study of different isoforms of proteins2-" and on antibody separation^.^^ It has potential for in vitro observation of interactions between proteins7 and for peptide mappings8For these two applications, a universal detector using a longwavelength optical beam is needed to detect all analytes and avoid interference with the interaction between proteins, For peptide mapping performed in CIEF, a UV-visible absorption on-column detector must operate at 280-nm wavelength, instead of 180-240 nm, because of the UV absorption of the carrier ampholytes used in CIEF. At 280 nm, only those peptides which contain tyrosine and/or tryptophan can be (1) Yjerten, S.; Zhu, M. J . Chromatop. 1985, 346, 265-270. (2) Kilar, F.;Hjerten, S. Elecrrophonsls 1989, 10, 23-29. (3) Kilar, F.; Hjerten, S. J. Chromatogr. 1989, 480, 351-357. (4) Zhu, M.; Rodriguez,R.; Wehr, T.;Siebert, C. J. Chromarogr.1992,608,225237. (5) Silverman, C.; Komar, M.; Shields, K.; Diegnan, G.; Adamovics, J. J. Lfq. Chromarogr.1992,15, 207-219. ( 6 )costello, M. A.; Woititz. C.; De Feo, J.; Stremlo, D.; Wen, L.-F.;Palling, D. J.; Iqbal, K.; Guzman, N. A. J. ttq. Chromufogr. 1992, lS, 1081-1097. (7) Wu, J.; Pawliszyn, J. J. Chromarogr. 1993, 6S2, 295-299. (8) Mazzeo, J.; Martineau, J. A.; KNII, I. S. AMI. Blochem. 1993,208,323-329.
0003-2700/94/036&0867~04.50/0 (Q 1994 Amerlcan Chemlcal Society
detectedq8As a result, for peptide mapping, fewer peaks were produced by CIEF as compared to capillary zone electrophoresis? Our group has developed two imaging detectors for CIEF performed in a short capillary (4 cm long): an optical absorption imaging detectorg and a universal concentration gradient imaging detector, which is based on the detection of the refractive index gradient created by the sample concentration gradient.lOJ1An imaging detection system has proved to be ideal for CIEF."' The use of it eliminates the mobilization procedure in CIEF and, thus, eliminates all problems associated with the mobilization procedure. The mobilization reduces the resolution of CIEF, prolongs the analysis time, and makes it difficult to determine the isoelectric points (p2) of protein samples due to the distortion of the linear pH gradient along the capillary during the process. The imaging detection system combines separation and detection into one step, which increases the analysis speed of CIEF (from 20 to 2 min).lO Our previous research on the concentration gradient imaging detector indicated some problems.IoJ Electropherograms were complicated because each zone produced one positive and two negative peaks, since the signal of the imaging detector was proportional to the second derivative of sample concentration along the capillary.ll The brightness of the probe beam was also limited due to total projection of the beam onto a charge-coupled device (CCD) sensor used in the detector, which becamesaturated at high light intensity. These two factors restricted the detection limit of the concentration gradient imaging detector to -50 pg/mL for protein samples.1° On the other hand, the selectiveabsorption imaging detector has good sensitivity for samples having strong absorption.9 It can also provide spectroscopicinformation of the sample zones. In this paper, we will report the construction of a unique imaging detection system for CIEF, which combines optical absorption with the universal concentrationgradient imaging detector. To improve the sensitivity of the concentration gradient detector, the dark-field Toepler-SchlierenmethodI2 was applied. Protein zones focused inside the capillary by CIEF can be detected by absorption and the concentration gradient imaging detectors almost simultaneously. (9) Wu, J.; Pawliszyn, J. J . Uq. Chromarogr. 1993, 16, 1891-1902. (IO) Wu, J.; Pawliazyn, J. AMI. Chem. 1992.61, 2934-2941. (11) Wu, J.; Pawliszyn. J. AMI. Chcm. 1992, 64, 224-227. (12) Stolzenburg, W. A. J. SMPTE 1965, 74,654-659.
A~,lytlcalChemistry, Vd. 66, No. 6, Mwch 15, IS94 067
CCD
Laserbeam (from expander) Flguro 1. Experimental setup of the CIEF lmaglng detection system: Ll, k m focal length cyllndrlcal kns; L2 and L3, &cm focal length cyllndrlcat lenses.
EXPERIMENTAL SECT1ON Instrumental Procedures. The capillary cartridge was similar to that of our previous experiment.1° A 100-pm-i.d., 4-cm-long square glass capillary (Vitro Dynamics Inc., Rockaway, NJ) was used for the separation. The capillary inner wall was coated with non-cross-linked acrylamide to eliminate electroosmotic flow.2 The light source was a 5 14.5-nm light beam from an argon ion laser (Model 265, Spectra-Physics, Cranbury, NJ). The power of the laser beam was 10 mW. The laser beam was first expanded to a 70-mm-diameter beam spot by a light beam expander which consisted of a 20-fold magnification microscopic objective lens, a 25-pm pinhole, and 25-cm focal length lens. In order to obtain a light beam with uniform intensity in its cross section, only the central part of the expanded beam was used by passing it through a 27 mm X 10 mm slit. As shown in Figure 1, the light beam was then focused into the capillary by a 5-cm focal length cylindrical lens (Ll) which was mounted on a three-axis stage. After the beam passed through the capillary, it was focused again into an optical stop (for concentration gradient detection mode) by an 8-cm focal length cylindricallens (L2) which was placed in a position vertical to the first cylindrical lens (Ll). The distance between the second cylindrical iens (L2) and the capillary was 10mm. Finally, as shown in Figure 1, the beam was projected by another 8-cm focal length cylindrical lens (L3) onto an 1024-pixel linear CCD sensor (S3903-1024Q, Hamamatsu, Hamamatsu City, Japan) which had a 25 mm X 0.5 mm sensing area and was mounted on a two-axis stage. This alignment monitors 25 mm of the 4-cm-long capillary. Because, in this experiment, a high-intensity argon ion laser was used as the light source, no collection lens was needed after the L3 lens to focus the beam again onto the CCD sensor. To perform the concentration gradient detection under dark-field conditions, an optical stop was used. The stop was the positive photographic film of a black line which was plotted on white paper by computer. The stop was placed just at the focal point of L2 lens, and the whole beam was blocked by the stop. The detector could be switched from the concentration gradient mode to the absorption mode by just removing the stop and moving the position of the CCD as described in 868
Analyticel Chemistry, Vd. 66, No. 6, March 15, 1994
the Discussion section, which only took -10 s. For the absorption mode, since it was performed under high background condition, the intensity of the 10-mW laser beam was too high for the CCD, a neutral attenuation filter (5% T) was employed to reduce the light intensity. 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 scanning speed of the 1024-pixel CCD was set at 10 Hz due to the 20-kHz maximum acquisition frequency of the DACA board. For each measurement, the CCD scanned 10 times in 1 s, and the 10 images were summed to reduce the random noise. The light intensity profile measured before the separation voltage was turned on was used as a background signal. Reagents. All chemicals were reagent grade, and solutions were prepared using deionized water. Solutions of 10 mM H3P04 and 20 mM NaOH were used as the anolyte and catholyte, respectively. Protein and peptide samples were purchased from Sigma Chemical Co. The samples included myoglobin (from horse skeletal muscle), carbonic anhydrase I1 (from bovine erythrocytes), apamin (from bee venom), and p l markers (IEF-100 marker kit). Human hemoglobin standard (hemo control AFSC) was purchased from Helena Laboratories, Beaumont, TX. Samples were mixed with the carrier ampholyte (Pharmalyte pH 3-10, Sigma) soIution to a final concentration of 2% ampholyte. All solutions were filtered using 0.2-pm pore size cellulose acetate filters (Sartorius, Gottingen, Germany) prior to use. In order to optimize the optical alignment, "fixed" zones were injected into the capillary by pressure when the alignment was being adjusted. These zones were made from polyacrylamide gel and 0.3 mg/mL myoglobin, which has absorption at a 514.5nm argon ion laser wavelength. During the alignment adjustment, no dc voltage was applied. IsoelectricFocusing Process. This procedure was the same as that used in our previous experiment.1° The separation voltage was 4 kV for the 4-cm-long capillary. All experiments were done in triplicate to ensure reproducibility.
RESULTS AND DISCUSSION In the optical alignment shown in Figure 1, if no sample zones are inside the capillary, the whole light beam will be focused at the focal point of the L2 lens (0point in Figure 2). However, if there is a sample zone inside the capillary, the beam rays passing through the refractive index gradient created by the zone will be deflected from their original directions. These rays cannot be focused at point 0 and will pass around the optical stop to reach the CCD sensor. Thus, the sample zones inside the capillary can be detected by the CCD. Sincethis detector is based on the detection of refractive index gradient created by concentrationgradient,it is universal. Compared to the concentration gradient imaging detector based on a Schlieren shadow graph,l0this alignment eliminates the negative peaks of signals, which simplifies the electropherogram. Each zone produces only a positive peak. For the absorptiondetector,thestopis removed from thealignment. The CCD detects the light beam intensity profile, and any sample zones inside the capillary having absorption at the beam wavelength can be detected by the system.
Flgm 2. Rlnclpb of the detection system. 0 , the focal point of the L2 kns.
a I b C
I
Y‘ e 12
18
111111 Flguro S, Images of flve zones lnsldethe capliiaryseen by the imaging daectkn s y m when the CCD b placed at dlfferent posltlons: (a) 7 mm from the L3 lens, without the UM) of the stop: (b) 7 mm from the L3 lens, with the stop; (c) 10 mm from the L3 lens, without the stop; (d) 10 mm from the L3 lens, with the stop; and (e) 10 mm from the L3 lens, the a m Image after background correction. Sample zones are pdyacrylamldo gel zones wlth 0.3 mg/mL myoglobln. All image8 except (e) are not corrected for background.
First, the best position of the CCD in the detector was determined. As shown in Figure 2, the focused protein zone inside the capillary acts as a lens,l0and the beam rays passing through the zone are focused at its “focal point”. The focal length of the protein zone under these conditions is 3-5 mm, depending on the zone width and its concentration.’O For the absorption detector, the changes in the beam intensity profile caused by the refractive index gradient created by the sample zones interferes with the detection. They can be eliminated by placing the CCD at the imaging point of the capillary (P2 in Figure 2). At this position, all the deflected beam rays caused by the sample zones return to their original positions as shown in Figure 2. Figure 3 shows the images of five “fixed” zones (Figure 3e) inside the capillary measured by the CCD at different positions. The sample zones are polyacrylamide gel with myoglobin, prepared using the method described in
Experimental Section. These zones absorb light at a 514.5nm wavelength. Many positive and negative peaks can be observed in Figure 3a, which is the image of the sample zones inside the capillary without the use of the stop. These peaks are created by the absorption of the sample zones and refractions produced by the sample zones and optical defects in the capillary wall. The peaks generated by the refractive index gradients caused by sample zones cannot be eliminated by simply subtracting background image from the signal images because there is no sample zone in the background image. When the CCD is placed at a position 10 mm from the L3 lens, which corresponds to the imaging point of the capillary, as shown in Figure 3c, compared to Figure 3a, the beam profile is simplified around the absorption peaks. At this position, the peaks generated by the zone refractive index gradients are almost eliminated; five negative absorption peaks can be observed for the five sample zones as shown in Figure 3c (peaks 1-5). Besides the peaks created by the sample absorption, other peaks can still be observed in Figure 3c. They are created by the refractiveindex defects in the capillary wall, which cannot be totally eliminated at this position because it is not the imaging position for these defects. However, those peaks can be eliminated simply by background correction. Figure 3e is the absorption image of the five zones after background correction. The CCD position corresponding to Figure 3c-e (P2 in Figure 2) is the best for the absorption detection mode. However, the best position for the absorption mode is not the best position for the concentration gradient detection mode. As shown in Figure 3d, in the concentration gradient image, the peaks are split at the center. This is because the light intensity profile at the P2 plane, as shown in Figure 2, is proportional to the deflection angle of the beam rays passing through the zone, as will be discussed later. The deflection angle at the center of the zone is 0 so that split peaks are observed. As mentioned above, to obtain the best peak shape, the CCD should be at the focal point of the sample zone (P1 position in Figure 2). The focal point depends on the concentration and the width of the zone.1° All the focused zones have different focal lengths because of the different zone widths and concentrations. For detection of zones having focal lengths longer than that of the zone shown in Figure 2, the best position should be between the P1 and L3 lens. As discussed in our previous paper,1° to obtained a nondistorted image of the all sample zones inside the capillary, the best position for the CCD is at the focal point of the narrowest zone with the highest concentration in the experiment. Too far or too near will give a distorted image of the zones inside the capillary.’O The focal length of the maximal zone was estimated in our previous experiment to be 3 which corresponds to the position 7 mm from L3 lens. This will be the best CCD position for the concentration gradient imaging detector, as shown in Figure 3a,b. In the experiment, the CCD position is set at 10 mm from the L3 lens for the absorption detection mode and 7 mm for the concentration gradient detection mode. At these two positions, the concentration gradient image and the absorption image agree well with each other, as shown in Figure 3b,e. The application of the dark-fieldToeplerSchlieren method in the concentration gradient imaging detector results in AnaWcaI Chemistry, Vol. 66, No. 6, Mrch 15, I994
009
blocked undeflected light rays and also the rays with small deflection angles caused by defects in the capillary wall. It reduces the background and facilitates the use of high-intensity light beam to increase the signal-to-noiseratio of the detector. The signal-to-noise ratio depends on the width of the optical stop, a, in the alignment shown in Figure 1. The relationship between signal amplitude and the stop width can be calculated theoretically:
4 x 1 = lefl
(1)
where x is the direction along the capillary, d is the distance between the deflected light ray and the point 0 in Figure 2, 8 is the deflection angle of the ray passing through the sample zone as shown in Figure 2, andfis the focal length of the L2 lens. For the stop width a, when the CCD is at the position P2 in Figure 2, the light intensity reaching CCD caused by the deflection angle 8 can be written a d 3 I(x) = 0
for
20f < a
I ( x= ) (2ef - a)Iod3/SAf
for
(2a) 2ef > a
(2b)
where X is the wavelength of the light beam, B is the beam spot diameter before it is focused by L1 lens, and IO is the intensity of the light beam. Equation 2 is only valid when 28f is near the stop width a. For larger angles, the signal intensity will not increase with increasing deflection angle; the signal is saturated. For a sample zone inside the capillary, the light intensity at the maximum deflection angle reaching the CCD can be calculated from the sample concentration, CO,the capillary length, I, and the zone width, 4 d 4
I=
'"8Af"(
0.4%
--
dn coif 2 a)
(3)
where n is the refractive index of the solution inside the capillary, L is the inner diameter of the capillary, and dn/dC is the change in the refractive index of the sample with regard to change in its concentration, which is approximately constant for a protein ~amp1e.l~ For the CCD positions other than P2, the deflected light beam rays are focused or defocused, but the maximum signal intensity generated by the sample zone is still proportional to the I value in eq 3. The best stop width is theone which lets signal pass through and cuts off the low-amplitude noise. It was difficult to determine the optimum width theoretically. In our experiments, we used the following method. From our previous research for the single point concentration gradient detector, the concentration detection limit, under the best conditions, was theoretically estimated to be 10-8 M for a very narrow The stop chosen should cut off 60-pm-wide protein all beam rays deflected by a sample zone having a concentration lower than 10-8 M and a width larger than 60 pm since all these deflections are considered to be noise in the experiment. By applying eq 3, the stop width can be estimated to be in the
-
(13) Merzkirch, W.Flow Visuallzarlon;Academic Press: New York, 1987. (14) Wu, J.; Pawliszyn, J. Anal. Chrm. 1992, 64, 219-224. (15) Sober,H. A., Ed. Handbook ofBiochemlsrry,2nd cd.;The Chemical Rubber Co.: Cleveland OH, 1970.
870 AmWcaI Chembtry, Vol. 66, No. 6, Mrch 15, 1994
Table 1. Ndro Level ol tho COnoMtratlon Badknt ImDetector glass l$w quartz noise level capillary 13 der tubing stop (mV)
b
43
X
C
d
X
e f g h i
X
X X X
X X X
X
0.5
173 1.4 83 1.0 78 0.9
rangeof 102pm. In the experiment, 400-pm stop widths were employed for samples having concentrations in the range of l e 7M. Stops narrower than 50 pm cannot totally cut offthe whole beam spot in the optical alignment. When a larger stop is used, signals will also be blocked. Larger stops may be employed when higher concentration samples are analyzed. The noise sources of the concentration gradient imaging detector and absorption imaging detector were investigated under the conditions set above. Signals, without and with capillary, and without and with stop, were measured. The noise levels produced under different conditions were measured by the followingmethod. Two images are obtained at 1-minute intervals under the same conditions. Then one image is subtracted from another, and the resulting image represents the noise under the conditions used. Table 1 shows noise levels under various conditions. For the experimental setup, when the laser is off (condition a), the dark noise signal of the CCD cannot be detected without further amplification. Only quantization noise is recorded. The noise level in the laser beam intensity profile is measured to be 43 mV, as shown in Table 1 (condition b). When the stop is used, the noise level becomes 100 times lower (Table 1 (c)). When the capillary is inserted into the alignment, the noise level increases -4 times (173 mV in Table 1 (d)). This is because the fluctuations in beam profile after it passes through the capillary are not reproducible in the two consecutive measurements due to the reasons discussed in the following paragraph. For a glass slide (f) and a larger i.d. (1 mm) fused quartz capillary (h) which have better optical quality in their walls than a glass capillary, the situations are almost the same, except that their noise levels (83 and 78 mV, respectively) are about half of that of the capillary. The reasons for the high noise level caused by the capillary, glass slide, and quartz capillary are optical defects in their walls. Because there is pointing noise for each ray in the probe beam (43 mV in Table 1 (b)), when the whole beam illuminates the capillary wall, beam rays are deflected by the defects. The resulting beam profile in far field is the mixture of deflection by the defects and interference of the rays. The deflection and interference amplify the pointing noise of each ray in the probe beam. This noise due to deflection and interference cannot be completely eliminated by simply subtracting one image from another measured under the same conditions. As shown in Table 1 (f, h), decreasing the noise level requires a capillary with goad optical quality in its wall. The noise levels (83 and 78 mV) were 2 times lower when the glass slide and fused quartz capillary were used. But they are still higher than the light beam noise (43 mV).
The noise caused by defects in the capillary wall can be greatly reduced by using the stop as shown in Table 1 (e, g, i) because the stop cuts off low-amplitude deflection noise signal and improves the signal-to-noise ratio. With the use of the stop, the noise levels under all conditions are -100 times lower than those without use of the stop. However, although the noise levels of the glass slideand large i.d. capillary (1 .O and 0.9 mV) are lower than that of the glass capillary, they are still 2 times higher than the noise in the light source when the stop is used (0.5 mV in Table 1 (c)). The conclusion is that the main noise source in the detector is the defects in the capillary wall, under the present conditions. If a capillary with a perfect wall is used, the noise level should be the same as those from the light source and detector. Under this condition, the noise from the light source would be the main factor affecting the sensitivity of the detector. The situation is almost the same for the absorption imaging detector. Because the i.d. of the capillary is 100 pm, the noise generated by the defects in the capillary wall is difficult to eliminate totally by placing the CCD at the imaging points of the capillary wall. This noise and the noise from the light beam profileare again the main factors affecting thesensitivity of the detector. This is confirmed by experimental results. When the CCD is placed at the best position for absorption mode, the noise level, when the capillary is inserted into the optical alignment, is 2 times higher than that without the capillary. Also, when a halogen lamp, which has a much lower noise level in intensity profile than a laser, is used as the light source, the noise level is only about one-third of that when the laser is used. However, the lamp is difficult to apply for the imaging detector when narrow capillaries (C200pm i.d.) are used because of the difficultyof focusing the incoherent light beam into the ~ a p i l l a r y .The ~ defects in the capillary wall and noise in the light source are the main sources of noise in the absorption imaging detection mode. Thus, the sensitivitiesof the two detectors can be enhanced by using a good optical quality capillary and a light source which is stable in its intensity profile. Thecapillaries employed in the application can be replaced by narrow channels in quartz plates, which are created by an etching method.16 The optical quality of the narrow channels is expected to be better than that of a glass capillary, so use of the channels would increase the signal-to-noise ratio of the detector. Equation 3 shows that the signal amplitude of the concentration gradient detector is proportional to the concentration of sample, CO,in the low-concentration range which should be near the "cutoff concentration", 10-8 M. As shown in Figure 4, the linearity was confirmed by the experimental results in the concentration range of 10 (3 X lo-' M) to 100 pg/mL (3 X 1O-a M). The detection limit of the detector under the present conditions can be estimated from 3 times the noise level in Figure 4c. It is 5 pg/mL for the carbonic anhydrase I1 sample. This detection limit is about one-seventh of our previous results obtained by theSchlieren shadow graph method even though the argon ion laser used in this experiments has a higher noise level than the He-Ne laser used previously.10 As discussed above, the enhancement of the sensitivity is attributed to thedark-field method and the suitable stop width. (16) Harrison, D. J.; Manz, A.; Fan, 2.;Ludi, H.; Widmer, H. M.Anal. Chcm. 1992,64, 1926-1932.
la
Figure 4. Concentratbn gradient Images of carbonic anhydrase 11: (a) 100, (b) 25, and (c) 10 )cg/mL.
4 m
I
17
26
35
mm Figure 5. Concentration gradient Image of plmarkers. plvalues: (1)
5.9; (2) 6 . 6 (3) 6.8; and (4) contalns m e than three peaks, 8.3,8.4, and 8.6. Concentration Is 40 pg/mL for each proteln.
This detection limit will be sufficient for analysis of minor protein componentsin biological cells without preconcentration steps. Proteins having isoelectric point values in a wide range can be detected by the CIEF instrument in 2 min. Figure 5 shows the electropherogram of p l markers in the pH range of 4.99.3. The p l values of all focused zones can be determined directly from their positions inside the capillary,1° as shown in Figure 5 . The resolution of the instrument reached 0.02 pH unit in our previous experiment.1° The resolution of the detector when the dark-field method is used still reaches 0.02 pH unit, which is estimated from the width of peak 3 and the distances between peaks 2 and 3 in Figure 5. Figure 6 is the electropherogram of a peptide, apamin. Since it contains no tyrosine and tryptophan,Is it is impossible to detect by the conventional absorption detectors for CIEF. However, it can be easily detected by the universal concentration gradient imaging detector. Its plvalue was determined to be 5.2. Another way to detect the peptides without tyrosine and tryptophan is to perform CIEF under carrier ampholyte free conditions. The pH gradient could be formed by a temperature gradient created in a cone-shaped capillary.17 Besides the universal nature of the detector, absorption spectroscopic information can also be obtained by the detector. Figure 7 shows the electropherograms of carbonic anhydrase I1 and myoglobin as detected in the universal and selective (17) Pawliszyn, J.; Wu,J. J. Microcolumn Sep. 1993, 5, 397-401.
Ana!~tIcalChemkiW, Vol. 66,No. 8, March 15, 1994
071
I
C +
12
21
30
I
mm Figure 8. Electropherogramof apamin obtained by the concentration gradient Imaging detectlon mode. Concentration is 1 mg/mL.
b 4
20
29
3%
I
mm Flgure 8. Absorptlon (a) and concentration gradient (b) images of human hemoglobin standard (AFSC) made in human blood plasma matrix. The sample is diluted by 100 times. The images are obtained 2 mln after the dc separation voltage is turned on.
4
17
26
35
t
mm Flgure 7. Absorptlon (a) and concentration gradient (b) images of carbonic anhydrase I1 (1) and myoglobin (2). Sample concentrations are 50 pg/mL for carbonic anhydrase I1 and 15 pg/mL for myoglobin.
modes. Because myoglobin absorbs at the 514.5-nm argon laser wavelength, it can be detected in the absorption mode. However, the carbonic anhydrase I1 zone has no absorption at this wavelength, so it cannot be sensed by the absorption detector. Because the CCD position is optimized, the performance of the absorption imaging detection mode is also improved compared to our previous r e ~ u l t s . ~Its detection limit is calculated to be 1 pg/mL, from 3 times the noise level in Figure 9a. The detection limit of absorbance can be estimated from the zone concentration and absorption coefficient of the myoglobin. The maximum concentration in a focused zone will bel4 (4)
where CO is the concentration of the injected sample. 872
Analytcal Chemistry, Vol. 66,No. 6,March 15, 1994
According to eq 4, the myoglobin sample of 1 pg/mL will form a zone (peak 2 in Figure 7a; zone width, 300 pm; CT = 70 pm) with a maximum concentration of 230 pg/mL. The absorbance of 230 pg/mL myoglobin was measured to be 0.06 AU/cm-' at a 514.5-nm wavelength, which corresponds to 6 X lo4 AU for the 100-pm capillary i.d. This is the detection limit of the absorption imaging detection mode under the present conditions. As mentioned above,it can be improved by using a good optical quality capillary and an intensitystabilized laser as its light source. Because of its high resolution and fast analysis speed, this instrument is expected to have many applications in clinical analysis, such as fast screening of human hemoglobin variants. Several techniques are currently available for this application, including gel electrophoresis, immunoassay, and high-performance liquid chromatography (HPLC) based on cation exchange.I8 In all of these methods, electrophoresis has the highest resolution, but its speed is much slower than that of the HPLC method (3 min/sample).I8 Electrophoresis performed in capillary which has high resolution and fast speed provides a promising method for clinical application. But the speed of commercial CIEF instruments (20 min/sample) is still slower than that of the HPLC methode4 Our CIEF imaging detection system would be the best instrument for the analysis of hemoglobin. It has the same resolution as that of gel electrophoresis and faster speed than that of the HPLC method. Figure 8 shows two electropherograms of hemoglobin standard separated by CIEF and detected by the imaging detection system. The electropherograms were obtained in 2 min after the start of separation. All variants (A, F, S , C) in the sample were well separated, and the resolution is higher (18) Loomis, S. J.; Go, M.; Kupeli, L.; Bartling, D. J.; Binder, S. R. Am.Clin.Lab. 1990, 10, 33-40.
/a
-b
7
After 1 min, several absorption peaks corresponding to the iron-complexed isoforms appear and become higher with time.
\
C
13
23
mm Figure 9, Interactions of Fe-free bovine transferrin and iron InsMe a ceplllary. The InteractionIsmonitoredby the absorptlonimagingdetector operated at 496 nm. (a) Four minutes after the iron-free transferrin Is focused, ferric Ions are Introduced into the capillary; (b) 10s,(c) 1 mln, (d) 2 mln, (e) 3 mln, and (0 4 mln after fenic Ions are introduced.
than that of the HPLC method.ls Also, because of the good sensitivity of the detection, a diluted sample (diluted by 100 times from standard) is used in the experiment, which makes it practical for direct analysis of blood samples without a desalting process. The real-time imaging dual detection can also be applied to investigationof dynamic processes insidea capillary column. One example is the in vitro study of interactions of iron and transferrin by the refractive index gradient imaging detector, which was described in our previous paper.7 The formation of iron-complexed isoforms of transferrin during the interaction can be observed since the iron-complexed and iron-free isoforms are focused at different positions inside the capillary due to their different plvalues. However, the iron-complexed isoforms formed during interaction can also be distinguished by the absorption imaging detector because only ironcomplexed isoforms of transferrin have absorption around 500 nm. Figure 9 shows the interactions between transferrin and ferric ion observed by the absorption imaging detector operated at 496 nm. The experimental conditions and procedure are the same as those used in our previous r e s e a r ~ h . ~ The iron-free transferrin is first focused inside the capillary by isoelectric focusing. Since the iron-free isoforms have no absorption at 496 nm, no peaks are observed in Figure 9a. Then a plug of ferric ion is introduced into the capillary.' (19) Wu, J.; Pawliszyn, J. Electrophoresis 1993, 14, 469474.
CONCLUSIONS Simultaneous refractive index gradient and absorption detection using the dual detection scheme is possible by simply modifying the alignment. The light beam can be split into two beams after it passes the L2 lens. Only one beam is blocked by the stop. Then the two beams are projected onto a two-dimensional CCD sensor. To study the spectroscopic properties of all sample zones by the absorption mode, a light source with adjustable wavelength will be used in the detection system. It can be a dye laser or a halogen lamp.9 Light sources with any wavelength are applicable to the concentration gradient imaging detection mode. In some applications, as shown in Figure 9, the wavelength may be fixed only to monitor interested species inside the capillary column. The present CIEF is a single-channel separation method. In a run, only one sample can be analyzed. Its sample throughput is much smaller than that of gel electrophoresis. However, our CIEF imaging detection system can bedeveloped into a multichannel method,Ig and the imaging detection system has proved to be applicable to the CIEF performed in multi~hannels.~~ The multichannels can be made on quartz plate with an etching method.16 This instrument will greatly increase the sample throughput of the CIEF technique, making it comparable to gel electrophoresis in sample throughput, but it will have a much faster analysis speed. Also, the separation channels made by etching have better optical quality than those of capillaries, which will enhance the signal-tonoise ratio of the imaging detection system. Isoelectric focusing performed in multichannels, detected by an imaging detector, is expected to be a powerful tool for routine analysis in biological research or clinical laboratories. ACKNOWLEDGMENT This work was supported by the Natural Sciences and Engineering Research Council of Canada. Professor D. Irish loaned us the argon ion laser. We thank M. Adams for his assistance in preparing the manuscript. Received for review August 16, 1993. Accepted December 15, 1993.' Abstract published in Advance ACS Absrracts, February 1 , 1994.
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