Anal. Chem. 1995, 67,4376-4379
Zero-Dead-Volume Peroxyoxalate Chemiluminescence Detection in Liquid Chromatography Juana Cepas, Manuel Silva, and Dolores P6rez-Bendito* Department of Analytical Chemistiy, Faculty of Sciences, University of C6rdoba, E- 14004 C6rdoba, Spain
A simple, flexible, and inexpensive peroxyodate chemiluminescence detection reactor for liquid chromatography is reported. This postcolumn device permits the efficient mixing of the mobile phase and reactants, bis(2,4,6trichlorophenyl) oxalate and hydrogen peroxide, with a zero dead-volume and the following features: (a) highly flexible optimization of chemical variables affecting the peroxyoxalate chemiluminescence reaction, which can take place outside the chromatographicsystem; (b) use of high oxidantloxalate ester concentration ratios, which decrease background emission and increase the signal/ noise ratio; and (c) use of large reaction volumes in the detection cell without excessive additional band broadening (the chemiluminescencereaction is very rapid under the selected experimental conditions). The proposed detection system was tested in the high-performance liquid chromatographic determination of polycyclic aromatic hydrocarbons, where it exhibited higher sensitivity (limits of detection in the femtomole range) than existing alternatives. Peroxyoxalate chemiluminescence (PO-CL) is a very sensitive detection method for high-performance liquid chromatography (HPLC).' Its sensitivity is often 10-100 times higher than that of conventional fluorescence detection. To ensure a high sensitivity, the PO-CL reactants (oxalate and hydrogen peroxide) should be mixed with the column eluate at a point as close to the photomultiplier as possible. This seemingly simple condition poses several practical problems, however. Thus, the half-life of the PO-CL reaction is a very critical factor because, if the signal decays too rapidly, even a low dead-volume between the reactantsHPLC eluate mixture and the flow cell can result in considerable losses of emitted light. Reported devices for PO-CL detection in HPLC based on flow systems have non-zero dead-volumes, so some compromises must be made in their optimization. Such compromises include one or more of the following: (a) The relative concentrations of oxalate and hydrogen peroxide must be carefully selected owing to the low stability of the former in the presence of the latter,2-5 which precludes use of as high a hydrogen peroxide/oxalate ratio as desired. This, in turn, decreases the signal/noise ratio by a twofold effect, Le., a decrease (1) Kwakman, P. J. M.;Brinkman, C . A. Th. Anal. Chim.Acta 1992,266,175192.
( 2 ) Miyaguchi. IC: Honda, IC; Imai, K. J, Chromatogr. 1984,303, 173-176. (3) Honda, K.; Miyaguchi, K.; Imai, K. Anal. Chim.Acta 1985,177,103-110; 111-120. (4) Imai, IC;Nawa, H.; Tanaka, M.; Ogata, H. Analyst 1986, 111, 209-211. (5) Weber. A J.; Grayeski, M. L. Anal Chem. 1987,59, 1452-1457.
4376 Analytical Chemistry, Vol. 67,No. 23,December 7, 7995
in the PO-CL signal due to the instability of oxalate and an increase in background emission (according to Mann and Grayeski,6 minimizing background emission entails using excess hydrogen peroxide). (b)The optimum pH for PO-CL detection in HPLC is also critical on acccount of its influence on the reaction half-life.7 (c) The reactant flow rates relative to that of the mobile phase are also important, especially in packed capillary liquid chromatography. Other influential features of these chemiluminescence detection systems are the volume and configuration of the flow cell. However, owing to the special characteristics of the PO-CL reaction, increasing the flow cell volume does not signiiicantly increase band broadening; thus, flow cells with volumes between 5 and 125 pL have been useda-" in various configurations, such as spiral FTFE tubes,IOcoiled glass flow cells,l* etc., to collect the largest possible fraction of photons emitted during the reaction. These devices are usually custom-made, so any commercially available alternatives would be welcome in order to add greater flexibility to the detection system. Low-dispersion POCL detection has been reported only in relation to packed capillary columns with mixing of reactants and the eluate near a flow cell housed in an integrating sphere12 (the mobile phase flow and the configuration of the flow cell are critical). In this work, the above-mentioned shortcomings were circumvented by designing a very simple zero-dead-volume PO-CL detection system for liquid chromatography from commercially available parts. The device allows more flexible manipulation of the reactant concentrations and flow rates relative to that of the mobile phase. It uses a standard l.@cm spectrofluorometric quartz cell in which the reactant solutions delivered by a peristaltic pump and the eluate from the column are mixed, the resulting chemiluminescence signal being simultaneously monitored by the photomutiplier. One additional channel is used to keep the volume of the reaction mixture constant in the cell. This chemiluminescence detection system, which can be optimized separately from the chromatographic system, was evaluated in terms of sensitivity and band broadening in the HPLC determination of polycyclic aromatic hydrocarbons (PAHs) using bis(2,4,& trichlorophenyl) oxalate (TCPO) . Mann. B.; Grayeski, M. L. Anal. Chem. 1990, 62, 1532-1536. Weinberger. R. J. Chromatogr. 1984, 314, 155-165. Grayeski, M. L.; DeVasto, J. K. Anal. Chem. 1987, 59, 1203-1206. Kwakman. P. J. M.; van Schaik, H.-P.: Brinkman, U. A. Th.; de Jong G. J. Analyst 1 9 9 1 , 116, 1385-1391. (10) Imai, IL: Higashidate, S.; Nishitani, A; Tsukamoto, Y.; Ishibashi, M.; Shoda, J.; Osuga, T. Anal. Chim Acta 1989, 227, 21-27. (11) Yan, B.; Lewis, S. W.; Worsfold, P. J.; Lancaster, J. S.; Gachanja, A. Anal. Chim.Acta 1991,250, 145-155. (12) De Jong, G. J.; Lammers, N.; Spruit. F. J.; Dewaele, C.; Verzele, M. Anal. Chem. 1987, 59, 1458-1461. (6) (7) (8) (9)
0003-2700/95/0367-4376$9.00/0 0 1995 American Chemical Society
Column TCPO H 2 0 2 / Trls Waste
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Peristaltic Pump Flgure 1. Schematic depiction of the proposed zero-dead-volume chemiluminescence detection system for liquid chromatography.
EXPERIMENTAL SECTION
Chemicals. All chemicals used were of analytical-reagent grade. A 3.5 x M TCPO solution was made by dissolving 157 mg of the chemical (Aldrich) in 100 mL of ethyl acetate. A 0.15 M tris (hydroxymethy1)methylamine (Tris, Merck) buffer solution was prepared by dissolving 1.8 g of reagent in water and adding enough hydrochloric acid to adjust the pH to 9.5 in a final volume of 100 mL. The oxidant/pH solution was prepared by mixing 40 mL of concentrated hydrogen peroxide and 1.0 mL of Tris buffer solution and diluting the mixture to 100 mL with 2-propanol. All PAHs were obtained from Aldrich and used without further purification. Standard solutions containing 50pg/ mL of each, except for pyrene (500 pg/mL), were prepared by dissolving the required amount in acetonitrile (chromatographic grade, Romil Chemicals). All dilute solutions were prepared in 75% (v/v) acetonitrile/water. It should be noted that some of the chemicals used are highly irritant and toxic, so they must be handled carefully. HPLC and Detection System. The instrumental setup used is depicted in Figure 1. The HPLC system consisted of a Waters W-600E pump, a Rheodyne 7125 injector (20-pL loop), a 4pm C18 Nova-pack (15 cm x 3.9 mm) cartridge column, and a PerkinElmer 650-10s spectrofluorometer with its light source switched off, the bandpass of emission monochromator set at 20 nm and the monochromator wavelength at 438 nm for chemiluminescence detection. Data were acquired and processed by a NEC PC-AT 33-MHz compatible computer equipped with a Maxima 820 chromatographic workstation from Waters. The chemiluminescence detection system consisted of a Gilson Minipuls-3peristaltic pump and two displacement bottles, one for pumping the TCPO solution and the other leading to waste, which ensured constancy in the volume of the reaction mixture in the cell. Poly(viny1 chloride) pumping tubes were used for all reactants except hydrogen peroxide, which contained some 2-propanol and must therefore be pumped through Solvaflex tubing. All other connections were established through stainless steel tubes. The sample compartment of the spectrofluorometer accommodated
(a) the l . k m spectrofluorometer quartz cell between an Oriel 441321 1-in.-diameter mirror and the photomutiplier in order to acquire as much emitted light as possible and (b) a small magnetic stirrer for efficient, reproducible mixing of streams in the cell. RESULTS AND DISCUSSION
The aim of this work was to develop a simple, flexible, and inexpensive chemiluminescence detection system for liquid chromatography in order to circumvent the problems posed by reported chemiluminescence postcolumn detection flow systems. The device was designed with a twofold aim in mind, namely (a) to ensure a zero dead-volume in the mixing of the individual reactant streams and the eluate from the column, which was carried out in a commercially available l . k m spectrofluorometer quartz cell, and (b) to use an inexpensive delivery system such as a peristaltic pump (potential bubbling and pulsation in the flow did not affect the performance of the detection system, so no dumper was required), with no detriment to the sensitivity or resolution of the chromatographic process. To test the performance of the proposed device, the TCPOI hydrogen peroxide reaction was used for the HPLC detection of several PAHs, including perylene, 9,1@diphenylanthracene, anthracene, benzo [ a ]pyrene, and pyrene, by reversed-phase separation with an acetonitrile/water mobile phase prior to CL detection. Optimization of the PO-CL Detection System. Based on the differential sensitivity exhibited by PAHs in this postcolumn reaction, we chose perylene to study the effect of experimental variables on the peroxyoxalate CL detection. Both chemical and flow variables potentially influencing the system performance were investigated by using the univariate method. Experiments were carried out using the instrumental setup depicted in Figure 1,less the column (see dashed line in the figure). Solvent Efects. One important consideration in adapting POCL detection to HPLC is the solvent composition of the mobile phase relative to postcolumn CL reagents: the solvent used for separation should be compatible with those required for efficient CL production. In this work, a 75% acetonitrile/water (v/v) Analytical Chemisity, Vol. 67, No. 23, December 1, 1995
4377
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Figure 2. Effect of (A) the concentration(0)and pH (0)of the Tris buffer solution and (B) the hydrogenperoxide (0)and TCPO (0)concentrations on peak height. The injected amount of perylene was 1 ng in all cases, except when the effect of pH was studied (4 ng).
mixture was used as the mobile phase to separate PAHs. Because the CL detection system was evaluated using the TCPO peroxyoxalate reaction and taking into account that TCPO is most stable and efficient in organic solvents such as ethyl acetate, a cosolvent was required to improve its water miscibility. Acetone and acetonitrile are the usual choices for this purpose; however, both damaged the peristaltic pump tubes (even Solvaflex tubes). Based on the results of preliminary experiments, these solvents were replaced with 2-propanol, which can be pumped through Solvaflex tubing with no appreciable detriment to the sensitivity (the results obtained in batch experiments revealed the PO-CL signal to decrease by only -15% relative to the use of acetone in the same proportion). This is the first reported use of 2-propanol as cosolvent in PO-CL determinations. The resulting decrease in the PO-CL signal was offset by the high hydrogen peroxide/TCPO concentration ratio used, as shown below. Concentration and pH of the Buffer Solution. The pH s i g n 5 cantly influenced PO-CL detection in HPLC; on the other hand, the concentration of the Tris buffer solution used to adjust it was not so marked (Figure 2A). Although increasing the concentration and pH of the buffer solution decreased the half-life of the PO-CL reaction7 and hence the peak width of the CL signal, at the Tris concentrations and pH values tested, the latter scarcely changed (-2 min in all cases). Based on results obtained, a 0.15 M Tris buffer of pH 9.5 was chosen for subsequent experiments (1.0 mL of this solution was used to prepare 100 mL of the oxidant/pH solution). Reuctunt Concentrations. The CL intensity increased almost linearly with increasing the concentration of hydrogen peroxide from 0.4 to 5.2 M (referred to the final concentration in the oxidant/pH solution), whereas the response to the TCPO concentration peaked at -5.5 x M (Figure 2B). Taking into account that the maximum TCPO concentration resulting in no solubility problems in the cell (irreproducible results after mixing with other reaction ingredients) was 3.5 x M, this concentration was adopted for further work. With regard to hydrogen peroxide, a 4.3 M concentration, obtained by using 40 mL of concentrated hydrogen peroxide to prepare 100 mL of the oxidant/pH solution, was selected for further experiments. These 4378 Analytical Chemistry, Vol. 67, No. 23, December 7, 7995
variables had virtually no effect on the peak width of the CL signal, which was -2 min. The selected hydrogen peroxide and TCPO concentrations resulted in increased sensitivity in the PO-CL detection in HPLC, owing to the high oxidant/oxalate ester concentration ratio used, -500, and hence a decreased background emission6 and increased signalhoise ratio. These working conditions can readily be achieved thanks to the special features (a zero dead-volume) of the proposed detection device. In classical postcolumn PO-CL detection in HPLC (with a non-zero dead-volume), the hydrogen peroxide/TCPO concentration ratio is typically 150, so background emission detracts from sensitivity.I3 Postcolumn Flow Rates. The effect of postcolumn flow rates on the signal intensity was examined using a reaction volume of 450 pL, as in the abovedescribed study of chemical variables. The flow rate of the oxidant/pH solution was set at 0.2 mL/min, because higher flow rates increased the water content in the reaction medium (to the detriment of sensitivity) and smaller flow rates decreased the signal intensity through a decreased hydrogen peroxide concentration and posed solubility problems arising from diminished supply of 2-propanol (cosolvent) to the reaction medium. Changes in the flow rate of the TCPO solution between 0.5 and 0.8 mL/min increased the PO-CL signal only slightly, so for economy, a flow rate of 0.5 mL/min was chosen. Effect of the Cell Volume. The cell volume or, specikally, the volume of reaction mixture in the cell, was the most influential variable on the performance of the proposed device for PO-CL detection in HPLC. The peak height and width profiles shown in Figure 3 are consistent with predicted results: the peak width increased and the signal intensity decreased with an increase in the reaction volume. The minimum volume used to ensure reproducible results was 100 pL. A reaction volume of 350 pL was chosen for further experiments as a compromise between high sensitivity and minimal band broadening (peak width only -1.5 min) for the determination. Determination of PAHs by HF’LC. The optimized zero-deadvolume CL detector was evaluated in the determination of several (13) Sigvardson, IC W.; Birks, J. W.Anal. Chem. 1983,55,432-435. (14) Christensen, R. G.; May, W. E. J. Liq. Chromatogr. 1978,1, 385-399.
Table 1. Characteristic Parameters of the Calibration Graphs and Analytlcal Figures of Merlt of the -termination of PAHs
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linear range (ng)
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PAHs, viz., perylene, 9,1@diphenylanthracene,anthracene, benzo[alpyrene, and pyrene. Figure 4 shows a typical chromatogram for a mixture of the analytes. Table 1 gives the least-squares parameters for the working curves of the PAHs, their detection limits (calculated as the mass of analyte providing a signal equal to two times the peak-to-peak noise)14and the precision, expressed as the relative standard deviation (RSD) and obtained by analyzing 11 samples containing a concentration within the range of the working curve for each PAH. The dynamic ranges shown in Table 1 can be expanded to higher PAH concentrations by changing the instrumental response of the spectrofluorometer used for POCL detection. The proposed CL detector is a very simple and useful alternative for the postcolumn determination of PAHs by HPLC, (15) Xi, X.; Yeung, E. S. Anal. Chem. 1991,63, 490-496. (16) Li, 8.;Fu, C. Fenri Ceshi Tongbao 1990,9,33-37. (17) Mazzeo, J. R;Krull, I. S.; Kissinger, P. T.]. Chromatogr. 1991,550, 585594. (18) Robbat, A, Jr.; Liu, T. Y.; Abraham, B. M. Anal. Chem. 1992,64, 358-
364. (19) Goates, S. R; Sin, C. H.; Simons, J. K; Markides, K E.: Lee, M. L. /. Microcolumn Sep. 1989,1, 207-211.
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Time, mln Figure 4. Chromatogram for PAHs: 1, anthracene (40 ng); 2, pyrene (1000 ng); 3, perylene (1 ng); 4, benzo[a]pyrene (15 ng); and 5, 9,lO-diphenylanthracene(12 ng).
as it provides similar or even higher sensitivitythan other recently reported alternatives involving magnetooptical rotation,15 chemiluminescence,16and electrochemical dete~ti0n.l~ It also surpasses the gas chromatography/mass spectrometry tandem18 and supercritical fluid chromatography with high-resolution laser-induced fluorescence detectionlg in PAH determinations in terms of sensitivity. ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the Spanish Direccibn General Interministerial de Ciencia y Tecnologia (DIGICy”) for the realization of this work as part of Project PB91-0840. Received for review June 14, 1995. Accepted August 25, 1995.a AC950586L @Abstractpublished in Advance ACS Absfracts, October 1. 1995.
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