Anal. Chem. 1992, 64, 219-224
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High-Performance Capillary Isoelectric Focusing with a Concentration Gradient Detector Jiaqi Wu and Janusz Pawliszyn* Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1,Canada
An lnexpenshre and unhrersal concentration gradient detector based on Schlieren optlcs, conslstlng of a He-Ne laser and a light beam posltlon sensor, was combined wlth hlgh-performance capillary lsoelectrk focuslng (IEF) performed In a 12 cm long and 20 pm 1.d. coated caplllary. Because of the hlgh-concentration gradient generated by narrow proteln zones created Inside the capillary by concentratlon and focusing during the IEF separation, the concentratlon gradlent detection was a senSnlve detector for caplary IEF. A 50 pm wlde zone of separated ovalbumln, which corresponded to a 15-pL zone volume was monltored In the experlments. The sampk concenbatbn could be estbnated from the a n a lpeak helght wlth a dynamic range of 0.1-1.0 mg/mL. Femtomole-level protelns could be separated by caplllary IEF a d detected dlrectly by the concentration gradlent detector without derhratlzatlon. The senSnMty of the detector for high molecular welght proteins Is In the same order of magnltude as that of a UV absorbance detector, but It has a much smaller detectlon volume. ThIs capillary IEF/concentratlon gradient detector system Is the most lnexpenslve and convenient one among systems proposed to date.
First, it is an universal detector. In this detector, a laser probe beam is focused directly into the capillary, a beam deflection signal is generated when the probe beam encounters a refractive index gradient produced by migrating sample zones, and the deflection is detected by a light beam position sensor (3). This deflection signal can be generated by any substances that have a refractive index different from that of the buffer. Second, the concentrationgradient detector based on Schlieren optics is an inexpensive detector, which consists only of a low-power He-Ne laser or a laser diode and a photodiode position sensor (3). High-performance caplllary isoelectric focusing (IEF) is one of the HPCE techniques which possesses concentration and focusing properties (6). This technique is able to resolve proteins which differ in isoelectric point by less than 0.01 pH unit (6). Zone broadening in this technique is minimized; thus, a high-concentration gradient is created at each boundary of separated zones. In this report, we will demonstrate the unique compatibility of the concentration gradient detector based on Schlieren optics and the high-performance capillary IEF.
EXPERIMENTAL SECTION INTRODUCTION High-performance capillary electrophoresis (HPCE) offers fast and highly efficient separation of ionic species (1). The narrower capillary provides better separation efficiency and needs a smaller sample volume, which is desirable for analysis of biological materials; therefore, the latest HPCE studies have shown a tendency toward using small capillary diameters (2-30-pm i.d.1 (2). It is a challenging problem to develop on-column detectors with a picoliter detection volume for HPCE with narrow capillaries. Although W absorbance and fluorescence have been the most commonly used detection modes, and the fluorescence detector has high sensitivity, both modes require absorption or fluorescence activity for the analytes, which makes them Micult to use for direct detection of many biological samples. There exista a need for a universal and sensitive detector for HPCE. On the other hand, because of the high separation efficiency of HPCE with narrow capillaries, narrow sample zones are produced, which generate high-concentration gradients along the capillary. Therefore, concentration gradient detection was expected to be suitable for HPCE with narrow capillaries. The concentrationgradient detection method based on Schlieren optics (3)has already shown good sensitivity as the detector for some HPCE techniques, such as moving-boundarycapillary electrophoresis (4) and capillary isotachophoresis, which has self-concentration and focusing properties (5). In those techniques, high-concentration gradients created at the boundaries of separated zones inside the capillary made the concentration gradient technique a sensitive method of detection. Compared with other detectors, the concentration gradient detector based on Schlieren optics has two unique advantages.
Instrumental Procedures. A 20 pm i.d. and 350 pm 0.d. fused-silica capillary (Polymicro Technologies, Tucson, AZ) was used for separation. The capillary inner wall was coated with non-cross-linked acrylamide to eliminate electroosmosis by the reported method (7). The total length of capillary for the separation was 12 cm. The cartridge holding the capillary and the detection system are shown in Figure 1. The capillary was fixed on the Plexiglass plate cartridge using epoxy glue, and its two ends were connected to small buffer reservoirs made of polyethylene. The focusing and mobilization of samples were driven by a high-voltage dc power supply (Spellman, Plainview, NY). The current passing through the capillary was monitored at the cathodic end of the capillary. As shown in Figure 1, a laser beam from a He-Ne laser (Uniphase,San Jose, CA) was used as the probe beam and was focused directly into the capillary by a 30 mm focal length lens which was mounted on a three-axis stage. The beam deflection was monitored by a position sensor consisting of a dual silicon photodiode with a differentialamplifier (3). The probe beam was arranged so that the far-field intensity profile points to the center between the two photodiodes placed close together in the position sensor. When a concentration gradient is encountered inside the capillary, the probe laser beam is deflected and the amount of light reaching the photodiode is not equal. The difference in photocurrent associated with the two photodiodes corresponds to the magnitude of deflection of the probe laser beam. The whole system was mounted on a vibration isolation table. The data were collected by an IBM DACA board, in a PC-AT personal computer, using the software ASYST (Asyst Software Technology Inc., Rochester, NY). Reagents. All chemicals were reagent grade, and solutions were prepared using deionized water. Solutions of 10 mM H3P04and 20 mM NaOH were used as anolyte and catholyte, respectively (8). NaOH solution was degassed before use, by sparging with helium. Samples used include a-chymotrypsin (type 11,Sigma), phosphorylase b (Sigma),insulin (Sigma), and ovalbumin (grade
0003-2700/92/0364-0219$03.00/00 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992 e
~
a2 K
*-
~
Direction i l ; of migration ~ ~ ~
I
a
n nin
Irni
a
Photodiode
~
1.
+ Plexiglass plate
X
A
C 1
CaPflW
\
\
1
Probe beam
I
Time
Figure 1. Diagram demonstrating the cartridge holding the capillary, and the probe beam and the position sensor.
V, Sigma). Samples were mixed with carrier ampholyte (Pharmalyte pH 3-10, Sigma) solution for a final concentrationof 2% ampholyte (8). Solutions were filtered using 0.2-pm pore size cellulose acetate filters (Sartorius, Gottingen, Germany). The sample concentrations introduced into the capillary ranged from 0.1 to 1 mg/mL. IEF Process. As shown in Figure 1, the samples were introduced into the capillary by pressure generated by a syringe. Then 8-kV dc voltage was applied, and current passing through the capillary was monitored to follow the focusing process. Typically, the current dropped from 1p A to about 0.1-0.2 p A in 4-7 min. The final step was mobilization. In the present experiment, cathodic mobilization was employed, which required exchanging catholyte with a solution containing 20 mM NaOH and 80 mM NaCl (8). The voltage for mobilization was 10 kV. During the process of mobilization, the protein samples moved through the detector in order of decreasing PI.The zone width of a protein during the focusing process could be estimated from the capillary length, the pH range of the ampholytes used, the p1 value of the protein, and the retention time of the zone during the mobilization process. 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. Caution should be taken to prevent the laser probe beam from reflecting or scattering to the operator's eyes while the optical alignment is adjusted.
RESULTS AND DISCUSSION When a voltage is applied to two ends of a capillary filled with carrier ampholytes and protein samples, a pH gradient is established by isoelectric stacking of the ampholytes, arranged under the electric current in order of increasing p1 from anode to cathode (9). At the same time, proteins will also migrate to the point in the capillary where their PIare equivalent to the pH and migration ceases, creating discrete narrow zones. Figure 2a illustrates the separation in the capillary of a protein mixture consisting of phosphorylase b and ovalbumin. These two proteins are expected to focus a t the respective isoelectric points inside the Capillary. The concentration distribution C(x) of the protein in a zone can be written as (9)
C = C,
exp(-x2/2aX2)
(1)
500
600
Time (s) Figure 2. Capillary isoelectric focusing with concentration gradient detection: (a) focused zones of phosphorylase 6 (peak 1) and ovalbumin (peak 2) in a capillary; (b) hypothetical refractive index trace along the capillary associated with the focused protein zones; (c) signals detected by the concentration gradient detector during mobilization; (d) integral of the signals from (c).
Equation 1 expresses a Gaussian concentration distribution with a standard deviation a,, which is given as (9)
Here, E = V/1,where V is the voltage applied to the two ends of the capillary, D is the diffusion coefficient of the protein, and p is given by
where du/d(pH) is the change in the mobility of the protein with regard to change in pH. These separated zones have associated characteristic properties, such as refractive index, temperature, conductivity, and electric field strength, which can be detected by corresponding sensors. A refractive index profile, corresponding to the hypothetical situation in Figure 2a, is generated by the concentration distribution of the separated phosphorylase b and ovalbumin along the capillary, as illustrated in Figure 2b. This profile can be detected by a concentration gradient detector based on Schlieren optics (3). When the probe beam of the detector, which is focused into the capillary, encounters a refractive index profile inside the capillary, the direction of the probe beam is deflected by the gradient. The deflection signal 6 of the probe beam can be given as (3)
e = - L- =d-n- - L dn dC n dx
n dC dx
(5)
here, n is the refractive index of the solution in the capillary,
L is the inner diameter of the capillary, and dC/dx can be
where x is the direction along the capillary and ,C is the maximum concentration in the zone, which is determined by the peak width, the concentration of introduced sample which fills the capillary, Co, and the overall length of the capillary,
calculated from eqs 1and 2:
I:
Equation 5 shows a linear relationship between the amplitude of the deflection signal and the first derivative of the refractive index profile in the capillary. As expected, two high deriva-
c,,,
= C01/~2Ta,
ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992
a 'I
b
4
280
140
420
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Time (s) Figure 3. (a) Mobilization electropherograms of femtomole-level phosphorylase b (peak 1) and ovalbumin (peak 2); (b) integral of signals from (a). Sample concentrations are 0.25 mg/mL, which correspond to 75 fmol of phosphorylase b and 1.6 X lo2 fmol of ovalbumin in the 12 cm long, 20 pm i.d. capillary. Focusing time, 4 min.
tives are observed in the trace detected by the concentration gradient sensor (Figure Zc), which correspond to the focused phosphorylase b and ovalbumin zones shown in Figure 2a and b. Besides two high signals, several small peaks are also visible in the detected signals shown in Figure 2c, which are considered to be the minor components of the samples. Figure 2d shows the integral of the signal from Figure 2c, which is the refractive index trace along the capillary. Two high peaks are observed in Figure 2d, which correspond to phosphorylase b and ovalbumin focused in the capillary. Contrary to the hypothetical trace shown in Figure 2b, in which only the refractive index changes due to the focused proteins are considered, refractive index fluctuations due to the carrier ampholytes and/or heat gradienta are also observed in Figure 2d. From eqs 1-6, the peak height of the signals detected by the concentration gradient detector, corresponding to the focused proteins, can be written as (3)
emax= f0.24-Ln
d n CoZ dc x,-2
--= 1 dn d u d(PH) LCOV f0.24- - --- (7) n dC d(pH) dx D
In this equation, dn/dC is approximately constant for a given solute (3);therefore, eq 7 shows that the peak height of the signal detected by the concentration gradient detector based on Schlieren optics is approximately proportional to the concentration of the introduced sample. It can be seen from eq 7 that the zone width is the major factor affecting the sensitivity of the concentration gradient detector. Decreasing zone width results in increasing concentration gradients at the boundaries of zones, and the sensitivity of the concentration gradient detector as indicated by eq 7. The upper limit for the voltage used depends mainly on the capillary inner diameter (1). The enhanced heat dissipation by using a smaller inner diameter capillary, which has a higher surface-to-sample volume ratio, permits application of a high focusing voltage in the capillary IEF. From eqs 1 and 3, a narrower zone is expected when a higher voltage is used for the focusing. In our experiments, the focusing voltages of 5,
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6, and 8 kV were used for 100,50, and 20 pm i.d., 12 cm long capillaries, respectively, without an overheating problem. As expected, the zone widths for focused ovalbumin are 300,150, and 70 pm, respectively. However, the experimental zone widths for 100 and 50 pm i.d. capillaries are larger than those predicted theoretically by eq 3. This is because sample convection in the capillary due to temperature differences is not considered in eq 3. The convection may be a major zone broadening factor when high voltage is applied for large inner diameter capillaries, while it can be well confined in narrow capillaries. Although the capillary inner diameter, which is also the light path for the probe beam, decreases from 100 to 20 pm, the experimental sensitivity shows a 4-fold increase as predicted by eq 7 because of the decreasing zone width for smaller inner diameter capillary. This clearly demonstrates the advantage of using a concentration gradient detector for detection in IEF with narrow capillaries. As predicted by eqs 3 and 4, the peak width can also be reduced by employing a shorter capillary, and the sensitivity of the detector will increase linearly with the decreasing length of the capillary, which increases the d(pH)/& term in eq 7. To verify this, a 6 cm long capillary was employed for separation of the same proteins, and the applied voltage was still 8 kV. As expected, the signal magnitude for the 6 cm long capillary is almost doubled compared with that for the 12 cm long capillary, as predicted by eqs 3 and 7. However, the zone width is -50 wm, which does not decrease to as predicted by eq 3. One of the major reasons for this is that the diameter of focused probe beam spot is in the same range as the zone width. For the laser probe beam, the beam spot diameter A can be calculated from the following equation:
A = 4AF/aB
(8)
where F is the focal length of lens used for focusing the probe beam into the capillary, X is the wavelength of the probe beam, and B is the diameter of the probe beam before focusing. For the 30 mm focal length focusing lens used in the experiments, the beam spot is calculated to be -30 pm in diameter. This means that the probe beam spot size is similar to the zone width, and further decrease of the zone width w i l l not improve overall spatial resolution. Although use of short focal length lens will decrease the beam spot and therefore improve the resolution, sensitivity will decrease with the decreasing focal length of the probe beam focusing lens since the sensitivity of the detector is proportional to the focal length of the probe beam focusing lens (3). From the experimental results, a 30-mm focal length has proved to be the most suitable for capillaries with 20-100 pm i.d. (4, 5). From the above discussion, it is evident that the zone width of ovalbumin focused by the 6 cm long, 20 pm i.d. capillary and 8-kV voltage is near the resolution limit of the detector under the present experimental conditions. The detection volume of the detector can be calculated from the 30-pm probe beam spot, and it is 9 pL. Because a laser beam is used as the probe beam in the detector, which is easily focused into a small beam spot, its detection volume is of the same order of magnitude as that of a laser-induced fluorescence detector, and much less than that of a UV absorbance detector with an incoherent light beam. It should be noted that the moving carrier ampholytes in the capillary during the mobilization process can also be detected by the concentration gradient detector based on Schlieren optics, because of the universal nature of the detector (9). They are visible especially in the mobilization electropherogram of proteins at a low-concentration level. The integral of the mobilization electropherogram of phosphorylase b and ovalbumin at a low-concentration level is shown in Figure 3b, which can be considered to be the refractive index
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992
a
+
450
510
I2
500
b
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Figure 4. Mobilization electropherograms of focused phosphorylase b (peak 1) and ovalbumin (peak 2) for different focusing times: (a) 1 min; (b) 15 min. Sample concentrations, 1.0 mg/mL.
profile in the capillary. Many high and wide peaks are observed in Figure 3b which correspond to the mixture of carrier ampholytes. Phosphorylase b and ovalbumin are focused between these ampholytes and form narrow and low peaks. The concentrations of carrier ampholytes are -100 times higher than those of the proteins. Therefore it is difficult to detect those protein zones by the refractive index detectors or other universal sensors based on concentration detection. However, the derivative nature of the concentration gradient detector distinguishes those narrow, small zones from the high and broad background produced by carrier ampholytes (3). From eqs 2 and 6, the ratio of the sensitivities of the detector based on concentration gradient detection and the detector based on concentration detection may be expressed as a function of the zone width ux (IO):
Cmax
=-.-&F 1
b
C
I
d
Figure 5. Mobilization electropherograms of focused phosphorylase b of different concentrations: (a) 0.10 mg/mL; (b) 0.30 mg/mL; (c) 0.50 mg/mL; (d) 1.0 mg/mL. Focusing time, 4 min.
Time (s)
(dc/dx)max
a
(9)
For the carrier ampholytes having mobility u and first and second acid dissociation constants K1 and K2, from eq 4, p can be written as 4 . 6 ~ d(PH)
PP1O(PK2-PK')dX The ratio of sensitivies becomes
(dC/ dx 1max Cmax
Equation 9 predicts a linear relationship between the ratio and the zone width. Since the pK2- pK1 values of the carrier ampholytes usually differ by several pH units (9), they form much wider bands than those of proteins. Equation 9 shows that the concentration gradient detector is relatively insensitive to these wide bands, compared with the detector based on concentration detection. In contrast, high signal peaks of the concentration gradient detector can be generated only by a high-concentration gradient at the narrow protein zones inside the capillary. Two sharp high peaks corresponding to the narrow zones of phosphorylase b and ovalbumin, which are almost invisible in Figure 3b, are observed in the signals detected by the concentration gradient detector, shown in Figure 3a, with good signal-to-noiseratios. Because the focused protein zones are narrower than the carrier ampholyte bands, the refractive index gradients produced by the carrier ampholytes are small compared to the protein zones. The
derivative nature of the detector makes it the best technique among the universal methods for the detection of protein samples separated by the capillary IEF. As expected, the concentration gradient detector is unable to detect the focwed wide zones of amino acids in the capillary IEF, since most amino acids have large pK2- pK, values which are in the same range as those of carrier ampholytes. As indicated by eq 7, the peak height emaxof the signal detected by the concentration gradient is proportional to the concentration of the introduced sample; the electropherogram detected by the Concentration gradient detector can be used for quantitative determination. However, to obtain quantitative information of the focused proteins from the signal peak height, the focusing time should be carefully controlled since the concentration distribution of the focused zone, i.e., peak shape, can be affected by the focusing time (8). Figure 4 shows the mobilization electropherogram of phosphorylase b and ovalbumin under different focusing times. As shown in Figure 4a, when the focusing time is 1 min, each zone is not well focused, and only a broad band is produced. Two separated high peaks are observed for each protein, which correspond to two boundaries of the band. When the focusing time is 15 min, as shown in Figure 4b, a strong interference signal is observed near the peak, which is considered to correspond to the scattering light of the probe beam generated by precipitation or denaturization of the protein because of the long focusing time (6). If a suitable focusing time is chosen, a reproducible peak height can be obtained. From the experimental results under the conditions, the best focusing time was found to be in 4-7 min. Signal peaks of focused phosphorylase b for different sample concentrations, after 4-min focusing, show reproducible peak shape (Figure 5 ) . As predicted by eq 7 , a approximate linearity between the peak height and the introduced sample concentration is shown in the concentration range of 0.1-1.0 mg/mL. The detection limit of the method corresponding to the concentration of introduced sample can be estimated theoretically. The concentration detection limit, Comln,can be calculated from eq 7: COmin= 4. 10minnax'/L1(dn/dC) where Bmi, is the smallest angle change that can be detected by the detection system, which theoretically depends on the pointing noise of the laser probe beam (11). Here Omin is -10-5 rad for the He-Ne laser beam (II), dn/dC is estimated to be about 10-2-10-1 M-' for the proteins (3),n equals -1, and ux is 15 km for the ovalbumin zone and 60 km for phosphorylase b zone. From eq 12, the concentration detection limits of introduced samples are calculated to be in 10-s-10-7 M range. The experimental data of detection limits for phosphorylase b and ovalbumin can be estimated from 3 times the signalto-noise ratios of Figures 3a and 5a, which correspond to 2 X M (8 fmol absolute amount for the 12 cm long, 20 pm
ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992
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a00
400
4
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Time (s) Figure 6. Mobilization electropherogram of focused a-chymotrypsin (peak l), phosphorylase b (peak 2), insulin (peak 3), and ovalbumin (peak 4). Sample concentrations,0.75 mg/mL; focusing time, 4 min.
i.d. capillary) for phosphorylase b and 4 X lo-' M (20 fmol absolute amount) for ovalbumin. These results are near the theoretical value even though there exists a background signal, as shown in Figure 3, generated by band boundaries of the carrier ampholytes, which have 100 times higher concentrations than those of proteins. Equation 12 also shows that the concentration gradient detector based on Schlieren optics has a higher sensitivity for larger molecular weight samples. The molecular weight of sample, M, can affect the values of dn/dC, which is proportional to the molecular weight (3),and the sample's diffusion coefficientD, which is proportional to l/Mo.5 (12). An increase of molecular weight will decrease D, and as shown in eq 3, a, will decrease, resulting in narrower sample zones. Because protein samples usually have high molecular weight, and separation of proteins is the main purpose of the capillary IEF, from this point of view, the concentration gradient detection is also one of the most suitable detectors for protein samples separated by the capillary IEF. Here, the sensitivity of the concentration gradient detector as the detector for the capillary IEF is compared with UV absorbance and laser-induced fluorescence detectors. From the peak width of the focused phosphorylase b in Figure 5, and the concentration detection limit of the detector, the maximum concentration of the protein, C,,,, in the focused phosphorylase b zone at the detection limit can be estimated to be 8 X M by eq 2. This means that the detection limit of the concentration gradient detector for C, in the phosphorylase b zone is 8 X M under the conditions reported. The absorbance of the phosphorylase b at concentration C,, 8X M, for the 20 pm i.d. capillary is estimated to be from the absorption coefficient of proteins, lo4 cm-l M-'. absorbance is usually considered to be the detection limit of the optical absorption methods. This calculation indicates that 8 X M is also a t the concentration detection limit of the conventional UV absorbance detector. This sensitivity of the W absorbancedetedor will decrease when the detector is used with narrower capillaries. Until now there have been no reports of applying a UV absorbance detector for HPCE with a capillary narrower than 25 pm i.d., which indicates the difficulties in focusing an incoherent light beam into small inner diameter capillaries. In the capillary IEF with a UV absorbance detector, the introduced sample concentration usually ranges from 0.2 to 2 mg/mL for 25-100 pm i.d. capillaries (7,8).Therefore, the sensitivity of the detector proves to be of the same order of magnitude as that of a UV absorbance detector. In addition, the sensitivity of the concentration gradient detector can be improved by using a dual-beam configuration (3). For detection of proteins by the UV absorbance detector, the best wavelength range is 180-240 nm (13), since most proteins have the strongest optical absorption in this range.
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However, for capillary IEF detection, a wavelength of 280 nm has to be used due to the high background absorption of carrier ampholytes in the short-W wavelength range (6). The UV detector suffers sensitivity loss at 280 nm, and even so, the concentration of ampholytes still cannot exceed 2%. Although the distribution of carrier ampholytes creates refractive index fluctuations inside the capillary, as discussed above, the derivative nature of the concentration gradient detector effectively reduces those fluctuations; the limit for the carrier ampholyte concentration can be much higher when the concentration gradient detector is used. Although laser-inducedfluorescence detection is among the most sensitive detectors for HPCE and shows a detection limit of lo-" M (14),it requires derivatization for most analytes, which makes the direct detection of small amounts of biological samples difficult. The derivatization is usually made under high-concentrationlevels for the samples, ranging from M (14) and then the derivatized samples are to diluted to low concentrationfor determination. By the existing techniques, it is impossible to derivatize small-amount, lowconcentration biological samples, such as cytoplasm in a single cell. In the present experiments, the sample concentrations introduced into capillary IEF range from to lo+ M, which can be detected directly without derivatization. It is possible that in the future a sample injection method for the capillary IEF system with a universal concentration gradient detector will be developed for direct analysis of cytoplasm in a single cell. As mentioned above, the unique advantage of concentration gradient detection based on Schlieren optics over other present detectors for HPCE is its universal nature, which makes it possible to detect all eluted analytes without derivatization. Figure 6 shows a mobilization electropherogramof femtomole levels of four proteins separated by the capillary IEF system and directly detected by the concentration gradient detector without derivatization. Because of the small detection volume of the detector, the narrow zones of the proteins are detected without resolution losses. The experimental results demonstrate that the concentration gradient detector not only has good sensitivity, but also maintains the high resolution obtained by the capillary IEF. The combination of capillary IEF with the concentration gradient detector based on Schlieren optics appears to be a perfect match. In addition to all these advantages, the concentration gradient detection method is also an inexpensive detector, especially when a diode laser is used as the probe beam source (11). Although it is difficult to focus a light beam from a contemporary diode laser into a 20 pm i.d. capillary because of its non-Gaussian beam profile, the diode laser is still expected to be a major probe beam source for the concentration gradient detector in the future, because of its small pointing noise and the rapid development of laser devices. The price for the whole capillary IEF system is expected to be less than $2000, which is much lower than those of UV absorbance and fluorescence detectors, in which an expensive monochromator or UV laser and photomultiplier must be used. The use of a diode laser will also further decrease the size of the whole capillary IEF system. It may be possible in the future to construct a whole capillary IEF system on a single wafer which could include a narrow capillary, the universal concentration gradient detector consisting of a diode laser and a silicon position sensor.
REFERENCES (1) Jorgenson, J. W. Anal. Chem. 1986, 58, 743A-760A. (2) Olefirowicz, T. M.; Ewing, A. G. Anal. Cbem. 1990, 62, 1872-1876. (3) Pawliszyn, J. Spectrochim. Acta Rev. 1990, 73,31 1-353. (4) Pawliszyn, J.; Wu, J. J . Cbromatcgr. 1991, 559, 111-118. (5) McDonnell, T.; Pawliszyn, J. A d . Chem. 1991, 63, 1884-1889.
Anal. Chem. 1992, 6 4 , 224-227 Wehr, T.; Zhu, M.; Rodriguez, R.; Burke, D.; Duncan, K. Am. Biotechno/. Lab. 1990, 8,22-29. Kilar, F.; Hjerten, S. Nectrophoresis 1989, 70, 23-29. Zhu, M.; Hansen, D. L.; Burd, S.; Gannon, F. J. Chromatogr. 1989, 480, 311-319. Righetti, P. G. Isoelectric Focusing: Theory, Methodology and Applications; Elsevier Press: Amsterdam, 1983. Pawliszyn, J. Anal. Chem. 1988, 5 8 , 243-246. Pawliszyn, J. Rev. Sci. Instrum. 1987, 5 8 , 245-248. Fuller, E.; Schettler, P.; Giddings, J. Ind. f n g . Chem. 1988, 5 8 , 19-23.
(13) Cobb, K. A.; Novotny. M. Anal. Chem. 1989, 67, 2226-2231. (14) Cheng, Y. F.; Dovichi. N. J. Science 1988, 2 4 2 , 562-564.
RECEIVED for review July 25,1991. Accepted October 18,1991. This work was supported by the Natural Sciences and Engineering Research Council of Canada.
CORRESPONDENCE Universal Detection for Capillary Isoelectric Focusing without Mobilization Using a Concentration Gradient Imaging System Sir: Isoelectric focusing (IEF) has been widely employed for separation of proteins based on differences in their isoelectric points (PI)(1).Recently, the development of capillary electrophoresis (CE) techniques has generated interest in performing this separation method in capillaries, since efficient dissipation of Joule heat from the 10-100 ym diameter capillary eliminates convection effects and enables highly efficient separations (2). Since 1985, there have been many reports on the developments of isoelectric focusing procedures performed inside capillaries (3-5). In a conventional capillary IEF system, focused zones must be moved through the flow cell, which is usually located at one end of the capillary, by the mobilization process which follows the focusing step ( 5 ) . During the mobilization process, distortion of zones and the loss in resolution are unavoidable. The mobilization process also takes -20 min compared to a few minutes required for focusing (5), which makes the capillary IEF a relatively slow separation method compared to other capillary electrophoretic techniques. Therefore an imaging on-line detection method is critical to improve the speed and performance of this separation technique. Several on-line scanning spectroscopic and radiometric detection methods have been developed for electrophoresis performed on slabs (2,6). However, they cannot be directly used with the electrophoresis carried out in microbore capillaries because of their small size. Recently there were attempts made to continuously monitor capillary IEF separation. Photographs of the focusing process of blue dye stained proteins inside 0.4-0.6-mm-i.d. tubes were taken, and the focused zones of proteins could be investigated (7).However, this technique required labeling of the proteins and could not give good quantitative information, because of the use of photographic film. Further development of this optical absorbance technique for proteins without derivatization in narrow capillaries (10-100-ym i.d.) requires a photodiode array and a coherent light beam from a UV laser, which is very expensive and operates at limited wavelengths. The focusing process was also monitored by an electrode array detector (8). Although a complicated 100-electrode array was used, the resolution obtained in these experiments was very poor. Sharply focused analyte zones are formed in the capillary IEF, which create high-concentration gradienh in the system. It is logical to consider application of the concentration gradient type detector. A simple Schlieren shadowgraph system has already been used for observing the distribution of carrier ampholytes focused in the gel slabs (9). However, it is difficult to obtain quantitative information about analytes inside a
narrow capillary by the conventional method based on incoherent light source and a screen. This situation can be dramatically improved by using modern optical instruments, such as a laser and photodiodes or a photodiode array. Such a single-point concentration gradient detection system (10) has proven to be a sensitive, universal, and inexpensive detector for the CE techniques (11-13) and has also been demonstrated to be a suitable detector for the high-performance capillary IEF (14). Therefore, a concentration gradient imaging system along the capillary is also expected to be a powerful tool for on-line monitoring and detection. In this report, the feasibility of capillary IEF with such an imaging system was demonstrated by performing separations of proteins inside a 100 pm i.d. square capillary.
EXPERIMENTAL SECTION Instrumental Procedures. A 100 pm i.d., 6.5 cm long square glass capillary (Dynamics Inc. Rockaway, NJ) was used for spearation. The capillary inner wall was coated with noncross-linked acrylamide to eliminate electroosmosisby the reported method ( 4 ) . The cartridge holding the capillary and the highvoltage dc power supply were the same as those of previous experiments (14). The cartridgewas mounted on a two-axis stage, the tilt angles of which were adjustable in the horizontal plane and in the vertical plane, so that the probe beam could be easily focused into the capillary. As shown in Figure 1, a laser beam from a He-Ne laser (Uniphase,San Jose, CA) was used as the probe beam. The probe beam was expanded to a 2 cm diameter beam spot, and then it was focused into the capillary by a 6 mm focal length cylindrical lens, which was mounted on a three-axis stage. A 20 cm diameter probe beam spot in the detector plane was formed by a 25 mm focal length lens mounted just behind the capillary. In this way, 1-cmlength in the detector plane corresponded to a 1-mm length of the capillary, and the probe beam intensity profile could be measured in the detector plane. A photodiode was used to measure the light intensity profile in the detector plane, and a 0.1-mm slit was placed before it. The photodiode was mounted on a one-axis stage driven by the moving part of a syringe pump (Model 341B, Orion Research Inc. Cambridge, MA), so that it could scan in the detector plane. The scanning distance of this system was -150 mm, which corresponded to a 15-mm length on the capillary. The probe beam intensity profile was also measured by a photodiode array of 128 pixel (Type S2301-128Q, Hamamatsu, Hamamatsu City, Japan), by which fast electrophoretic processes in the capillary could be recorded. The whole detection system was mounted on a vibration isolation table. The data were collected by an IJ3M DACA board, in a PC-AT personal computer, using the software ASYST (Asyst Software Technology Inc., Rochester, NY).
0003-2700/92/0364-0224$03.00/00 1992 American Chemical Society
