Anal. Chem. 2007, 79, 1262-1265
Adaptation of a Commercial Capillary Electrophoresis Instrument for Chemiluminescence Detection Jonathan C. Dumke and Mark A. Nussbaum*
Chemistry Department, Hillsdale College, 33 East College Street, Hillsdale, Michigan 49242
Modification of a commercial CE instrument (HP 3D-CE, or Agilent G1602A) to adapt it for chemiluminescence (CL) detection is described. The reversible modification involves interchanging the deuterium lamp (used for standard absorbance detection) with a sidearm photomultiplier tube in the lamp housing. Power supply and amplification circuitry is added via an external breadboard, and the amplified CL signal is recorded via a recorder/integrator. The viability of the CL detector was demonstrated with both pressure rinses and electrophoresis of chemiluminescent samples. Peak area precision was 1.5% RSD for pressure rinses of a peroxyoxalate (“lightstick”) sample and 4.0% RSD for electrophoresis of 100 µM luminol (catalyzed by horseradish peroxidase). The CL peak area was linear with concentration for electrophoresis of luminol (10-s injections) from at least 1.0-50 µM. Due to simplicity, high efficiencies, mass sensitivity, and low operating cost, capillary electrophoresis (CE) has become an important analytical separation tool. Separation of analytes by CE is based on size, charge, and retarding forces (e.g., adsorption and viscosity). The analytes pass a capillary window where they are typically detected by ultraviolet/visible (UV-vis) absorbance. Although UV-vis absorbance detection is rugged and reliable, some analytes have no chromophore and are thus undetected. Moreover, for many analytes, the absorbance limit of detection is poor because of the small path length across the capillary window. Since most capillary internal diameters are only 20-100 µm, whereas, for example, most liquid chromatography flow cells have path lengths of 1.0 cm, absorbance sensitivity can be a significant limitation in CE. Therefore, enhanced sensitivity of detection is an area of active interest in CE. Various alternatives for CE detection have been investigated to increase the sensitivity, including fluorescence, electrochemical detection, mass spectrometry, and chemiluminescence.1-5 * Corresponding author. E-mail:
[email protected]. Fax: (517)-6072252. (1) Landers, J. P., Ed. Handbook of Capillary Electrophoresis, 2nd ed.; CRC Press: Boca Raton, FL, 1997. (2) Szulc, M. E.; Krull, I. S. J. Chromatogr., A 1994, 659, 231-245. (3) Bardelmeijer, H. A.; Lingeman, H.; de Ruiter, C.; Underberg, W. J. M. J. Chromatogr., A 1998, 807, 3-26. (4) Staller, T. D.; Sepaniak, M. J. Electrophoresis 1997, 18, 2291-2296.
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Chemiluminescence (CL) detection is useful when the analytes cannot be detected with adequate sensitivity by other means.4,5 Typically, a CL reagent reacts with the analyte (or derivative) after it migrates through the separation capillary, producing emission of light. Although gas-phase CL has been interfaced with CE,6 most CL detection methods use solution-phase reagents such as peroxyoxalate esters, acridinium esters, firefly luciferase, tris(2,2′bipyridine)ruthenium, and luminol (5-amino-2,3-dihydro-1,4phthalazinedione).4,5,7-10 Coupling CL detection with CE can significantly increase sensitivity over absorbance measurements, since CL emission is measured against a dark background. Furthermore, even broadly applicable indirect CL detection, in which the analytes are detected by a decrease in background CL intensity, has been found to be very sensitive.11,12 CL detection in CE can be done via on-line detection or postseparation addition of reagent solutions. Postseparation addition of reagents is complicated owing to the electrophoresis voltage, small volumes, and mixing involved. In on-line detection, the CL reaction occurs within the separation capillary, often by using an immobilized reagent. On-line detection eliminates capillary-to-capillary junctions and associated complications in mixing, dilution, and voltage control. On-line detection is also amenable to positioning the CL reaction directly in front of the photodetector for maximum intensity. However, immobilization of CL reagents can add to the complexity of capillary preparation. In some cases, a microelectrode held at the appropriate potential can either serve as or generate the localized CL reagent.5,7,13 CL detection has also been obtained by migrating the analytes from the CE capillary into an outlet reservoir containing CL reagents where the emission is observed. Such an arrangement circumvents the need for capillary-to-capillary mixing or immobilization but requires the construction of the outlet vessel and its interface.14,15 (5) Campana, A. M. G.; Baeyens, W. R. G.; Zhao, Y. Anal. Chem. 1997, 69, 83A-88A. (6) Sokolowski, A. D.; Vigh, G. Anal. Chem. 1999, 71, 5253-5257. (7) Wang, X.; Bobbitt, D. R. Anal. Chim. Acta 1999, 383, 213-220. (8) Barnett, N. W.; Hindson, B. J.; Lewis, S. W.; Purcell, S. D. Anal. Commun. 1998, 35, 321-324. (9) Tsukagoshi, K.; Nakahama, K.; Nakajima, R. Anal. Chem. 2004, 76, 44104415. (10) Zhao, J. Y.; Labbe, J.; Dovichi, N. J. J. Microcolumn Sep. 1993, 5, 331339. (11) Ren, J.; Huang, X. Anal. Chem. 2001, 73, 2663-2668. (12) Liao, S.; Whang, C. J. Chromatogr., A 1996, 736, 247-254. (13) Gilman, S. D.; Silverman, C. E.; Ewing, A. D. J. Microcolumn Sep. 1994, 6, 97-106. (14) Dadoo, R.; Seto, A.; Luis, C.; Zare, R. Anal. Chem. 1994, 66, 303-306. 10.1021/ac061885l CCC: $37.00
© 2007 American Chemical Society Published on Web 01/03/2007
Commercial CE instruments do not typically offer CL detection. The most widely used and simplest CE instruments are designed specifically for UV-vis absorbance detection, and many of these, because of monochromator optics or type of detector, are not directly amenable to CL detection (e.g., by simply turning off the lamp). Spectral dispersion and wavelength selection are often superfluous for CL detection and decrease light throughput; also, typical photodiode array detectors are not sensitive enough for CL detection. Therefore, most work using CL detection in CE has used in-house-built detection equipment, often involving external reagent pumps, mixing regions, or CL emission collection devices that must be constructed. In addition, such CL detectors generally require either an in-house-built electrophoresis instrument or substantial, irreversible modifications to a commercial CE instrument. In this work, a widely used commercial CE instrument was adapted for on-line CL detection. The modification involved replacement of the deuterium lamp with a photomultiplier tube and associated electronics, as well as minor software and hardware considerations. The adaptation is relatively simple and easily reversible, while eliminating the optical path throughput and photodiode array issues noted above. The feasibility of the CL detector was demonstrated using both pressure and electrophoretically driven solutions. EXPERIMENTAL PROCEDURES Apparatus. The CE instrument used was a Hewlett-Packard 3D-CE G1600AX (currently marketed as Agilent G1602A). The software used for capillary electrophoresis was 3D-CE Chemstation (Rev.B.02.01 [244]) from Agilent Technologies. Measurements of pH were performed using an Accument Portable AP61 pH meter (Fisher Scientific). The bare silica capillary (75 µm i.d. × 65 cm (57 cm to window)) was from Polymicro Technologies. The capillary was rinsed sequentially with 0.10 M NaOH, water, and run buffer (2-5 min each) prior to electrophoresis and stored in water when not in use. The sidearm photomultiplier tube (PMT type R1527 SN: MK5226), and the PMT socket and power supply (type C6270 No. 504360) used with the CE instrument were purchased from Hamamatsu. The power supply ((5, (15 V) that powered the breadboard was from JameCo Electronics. The signal from the PMT was amplified by using an LT1001 operational amplifier (Linear Technology). The two trim potentiometers were 5 kΩ from JameCo, and 47 kΩ (horizontal PCT-mount Catalog No. 271-283) from Radio Shack. Resistors and capacitors were also obtained from JameCo and Radio Shack. The recorder/integrator was a Hewlett-Packard (Agilent) model 3396A. Modification of the Instrument. The detector cover on the CE is easily removed and set aside. Appropriate caution should be exercised in manipulating internal components to avoid contact with optics or live voltages, especially during the initialization stage, described below. The HP (Agilent) instrument has its lamp housing positioned directly in front of the capillary window, so this housing is ideal for installation of a sidearm PMT. Note that no modifications to the original diode array detector or optics are (15) Tsukagoshi, K.; Nakamura, T.; Nakajima, R. Anal. Chem. 2002, 74, 41094116. (16) Hamamatsu Corp. C6270: High Voltage Power Supply Socket Assembly; http://usa.hamamatsu.com/assets/pdf/parts_C/C6270.pdf, accessed Aug 2006.
necessary since the PMT is placed in the lamp housing, on the side of the capillary window opposite the original detector. It is therefore straightforward to switch between CL detection and diode array absorbance detection simply by interchanging the PMT and the deuterium lamp. PMT. To protect the PMT from stray light, a thin (1.6-mm) piece of black rubber, (available from a local hardware store) was used to line the inside of the lamp housing unit with only a small hole cut out at the capillary window location (Supporting Information, Figure S-1). The PMT and associated socket were inserted into the lined housing and held in place by a rubber band. A breadboard is required in order to interface the PMT output to the recorder and to control the power supply for the PMT. The PMT power supply is integrated into the socket and controlled by a 0-5 V input.16 Circuitry. The LT1001 operational amplifier was configured as a current-to-voltage converter, and the subsequent 10 kΩ, 100 µF RC circuit served to filter out high-frequency noise (circuit diagram shown in Supporting Information, Figure S-2). This RC time constant of 1.0 s reduced noise while not noticeably affecting CE peak shape, as verified by comparison with resistors of 1 and 5 kΩ (time constants of 0.1 and 0.5 s, respectively). A control voltage of 3.5 V was applied to the PMT socket, corresponding to a PMT power supply voltage of -875 V. Initialization. The HP (Agilent) instrument must be initialized (using the System INIT command) at power-up, before any rinses or electrophoretic separations are performed. Initializing this particular CE instrument requires the CE top access door to be closed and the deuterium lamp to be present. That is, although the lamp is replaced with the PMT for CL detection, the lamp needs to be in place during initialization of the instrument. The instrument has a clear viewing window on the top access door, which is easily removed to allow for access inside the instrument without having to open the door. The lamp may be inserted or removed from the housing without removing the black rubber lining used with the PMT. The lamp is turned off after initialization, removed, and replaced with the PMT in the lamp housing unit with the detection side facing the capillary window. The lamp may simply be set aside within the instrument and does not need to be disconnected. The housing and PMT are covered with black cloth and the PMT turned on to stabilize for ∼30 min before a run. Photographs of the modified instrument are available in Supporting Information, Figures S-3 and S-4. Reagents. All reagents were used as received without further purification. Hydrogen peroxide (3% w/w) and sodium hydroxide (50% w/w) solutions were from Fisher Scientific; methanol, 3-aminophthalhydrazide (luminol), and horseradish peroxidase type I (HRP) were from Sigma-Aldrich. Boric acid was obtained from Fluka, and the blue lightsticks were purchased from Flinn Scientific. Purified water from a Milli-Q system (Millipore) was used for all solution preparation and capillary rinses. Solution Preparation. A stock solution of 0.10 M boric acid, adjusted to pH 10.0 by addition of sodium hydroxide, was used in the preparation of luminol and HRP solutions. A stock 1.00 mM luminol solution in 25 mM borate was prepared by dissolving 18.0 mg in 25 mL of the 0.10 M borate with ∼2 mL of methanol and diluting to 100 mL with water. The resulting pH was measured to be 9.7. Further dilutions of this stock solution were prepared Analytical Chemistry, Vol. 79, No. 3, February 1, 2007
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Figure 1. Triplicate pressure rinses of a peroxyoxalate (lightstick) sample past the CL detector (offset for clarity). Numbers on the peaks are elution times in minutes after injection.
in water to obtain 1.0-100 µM luminol solutions. The HRP catalyst solution was prepared by dissolving 0.8 mg in 1.0 mL of the 0.10 M borate and 3.0 mL of water (i.e., final concentration 25 mM borate, pH ∼9.7). The electrophoresis run buffer was 0.050 M boric acid, adjusted to pH 9.1 with sodium hydroxide, containing 2 mM H2O2. The two lightstick reagent solutions were removed from the lightstick and stored in separate glass vials until use, at which time 1.00 mL of the outer (clear) solution was mixed with 0.50 mL of the inner (colored) solution in a CE vial. All solutions other than the lightstick reagents were stored at ∼5 °C when not in use. Procedure. The CL detector was tested by two different methods of transporting the chemiluminescent sample through the capillary window: pressure or electrophoresis. Pressure. The lightstick reagent aliquots were combined in a CE vial and mixed. The capillary was flushed with water, and then the lightstick mixture was injected for 10 s at 50 mbar. This chemiluminescent mixture was then rinsed through the capillary with water at 50 mbar for several minutes until the peak in light intensity was observed and baseline reestablished. The same lightstick mixture was run in triplicate and peak areas measured. Electrophoresis. Under the conditions used, the luminol has a more negative electrophoretic mobility (i.e., opposing the electroosmotic flow) than does HRP. Therefore, the luminol was injected and driven ahead of the HRP so that the two reagents would combine near the capillary window as they moved at their differing electrophoretic velocities. Following a run buffer flush, the luminol solution was injected (10 s at 50 mbar), run buffer vials were placed at both the inlet and outlet, and 30 kV was applied for 75 s. At that point, the HRP solution was injected (50 s at 50 mbar), and then electrophoresis at 30 kV with run 1264 Analytical Chemistry, Vol. 79, No. 3, February 1, 2007
Figure 2. Electrophoresis with CL detection of (a) 5.0 and (b) 1.0 µM luminol samples (10-s injection). Numbers on the peaks are migration times in minutes following injection of HRP catalyst (see Experimental Section for detailed conditions).
buffer was continued until the CL peak was detected. This procedure simulated the use of an immobilized CL catalyst. RESULTS AND DISCUSSION Pressure. The viability of the CL detector for simple pressure rinses is shown by the reproducible peaks observed for triplicate injections of the lightstick mixture (Figure 1). Given that the peroxyoxalate CL generated by the lightstick reaction is readily visible to the unaided eye, sensitivity would not be expected to be an issue, and the strong signal-to-noise ratio confirms that. Peak-area precision was excellent with a relative standard deviation (RSD) of 1.5%, approximately the same as for strong UV absorbance peaks and likely limited by the reproducibility of injection, not by the detector. Electrophoresis. Application of the 30-kV electrophoresis voltage had no noticeable affect on the PMT baseline noise under the conditions used. Samples of luminol from 1.0 to 100 µM were injected and detected following electrophoresis as described in the Experimental Section. The signal-to-noise ratio for the 1.0 µM luminol was ∼10 and would presumably be higher with optimization of the location of mixing with the CL catalyst (e.g., with use of an immobilized reagent in the detection window) and with electrical shielding. Of course, the signal can also be increased with larger injection volumes. These conditions suffice, however, for the purposes of demonstrating feasibility of the CL detector. Figure 2 shows the electropherograms resulting from injections of 1.0 and 5.0 µM luminol. The peak area was linear with luminol
concentration from 1.0 to 50 µM (R2 ) 0.9996), with a slight decrease in linearity if the 100 µM point was included (R2 ) 0.9948); see Supporting Information Figure S-5. Triplicate injections of 100 µM luminol gave a peak area RSD of 4.0%. CONCLUSION This work has demonstrated the feasibility of adapting a widely used commercial CE instrument for CL detection. Interchange of the deuterium lamp and a sidearm photomultiplier tube allows one to easily switch between CL and the standard UV-vis diode array absorbance detection. Although luminol and peroxyoxalate (“lightstick”) solutions were used here for demonstration of viability, the detection method obviously applies to other luminescent analytes as well. Furthermore, use of an immobilized reagent or enzyme can simplify the mixing and localization of the CL reaction and broaden the application of the methodology. By immobilizing a CL enzyme in the capillary window, analytes labeled with a CL reagent (e.g., luminol derivatives) can then be separated by CE and sensitively detected by CL. In preliminary (17) Dumke, J. Development of a Chemiluminescence Detector for Enhanced Sensitivity in Capillary Electrophoresis. B.S. Thesis, Hillsdale College, Hillsdale, MI, May 2005.
work, we have successfully immobilized HRP in a capillary window using a polyacrylamide gel and have obtained CL signal from luminol migrated by electrophoresis (using a borate/H2O2 run buffer) using the modified CE instrument described here. Additional work is necessary regarding the reproducibility and longterm stability of the immobilized enzyme for routine application to CL-labeled analytes.17 However, the instrument adaptation described here has been shown to be a straightforward and practicable extension to allow for CL detection in addition to UVvis diode array absorbance. SUPPORTING INFORMATION AVAILABLE Figures S-1, diagram of PMT housing liner; S-2, circuit diagram; S-3 and S-4, photographs of the modified instrument; and S-5, plot of CL peak area as a function of luminol concentration. This material is available free of charge via the Internet at http://pubs.acs.org.
Received for review October 6, 2006. Accepted November 17, 2006. AC061885L
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