Pulsed Excitation Source Multiplexed Fluorometry for the

Jan 29, 2003 - The respective limits of detection are 25 pptv and 15 pptv. Design, performance details, and illustrative results from a field campaign...
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Anal. Chem. 2003, 75, 1203-1210

Pulsed Excitation Source Multiplexed Fluorometry for the Simultaneous Measurement of Multiple Analytes. Continuous Measurement of Atmospheric Hydrogen Peroxide and Methyl Hydroperoxide Jianzhong Li, Purnendu K. Dasgupta, and Gary A. Tarver†

Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061

Presently, solid-state sources such as light-emitting diodes (LEDs) provide for intense, nearly monochromatic light. They are available over a broad range of emission wavelengths. Unlike incandescent and discharge lamps, LEDs can be turned on and off at high speeds. The resulting light pulses are highly reproducible. This allows the use of a single photomultiplier tube (PMT), often the most expensive component in a high-sensitivity measurement system, as a multiplexed detector with multiple, fiber-optic-coupled, fluorescence-detection cells excited by solid-state sources. A time resolution of 1 min is adequate in many continuous detection schemes. This enables multiple-channel single-detector multiplexed measurement without any loss of S/N. On the basis of this principle, we describe a new automated continuous instrument for the simultaneous measurement of atmospheric hydrogen peroxide and methyl hydroperoxide (MHP). A Nafion membrane diffusion scrubber (DS) is used with hematin-catalyzed oxidation of thiamine to thiochrome for the measurement of H2O2, and an expanded poly(tetrafluoroethylene) (ePTFE) DS is used with a H2O2 destruction catalyst and horseradish peroxidasecatalyzed oxidation of thiamine to thiochrome for the measurement of MHP. The respective limits of detection are 25 pptv and 15 pptv. Design, performance details, and illustrative results from a field campaign (Philadelphia NEO3PS study, 2001) are presented. In recent years, light-emitting diodes (LEDs) have emerged as versatile inexpensive nearly monochromatic sources for use in analytical chemistry. New records of emitted light intensity in available commercial products are set almost every month. Currently, devices that can be operated with continuous forward currents of 700 mA with very high photon conversion efficiencies are available at wavelengths spanning the visible spectrum.1 In the past, we have described dedicated LED-excited fluorescence detection systems for H2S,2 HCHO,3 and H2O2.4 Many other papers † Current address: 410 11th St. NE, Washington, DC 20002. (1) www.lumileds.com (2) Toda, K.; Dasgupta, P. K.; Li, J.; Tarver, G. A.; Zarus, G. M. Anal. Chem. 2001, 73, 5716-5724.

10.1021/ac026234d CCC: $25.00 Published on Web 01/29/2003

© 2003 American Chemical Society

have appeared from other laboratories as well.5-7 The intensity ratios of emission bands of rare-earth-doped glasses change with temperature; this constitutes the basis for a point temperature sensor based on a LED-excited rare-earth glass-tipped fiber optic.8 LEDs can be the basis of high-frequency pulsed light sources; this has been exploited to improve detection sensitivity by using lock-in or time-discriminating detection.9 LEDs pulsed at high speeds have also been used for fluorescence lifetime measurement.10,11 We have extensively used LED-based instruments. In typical atmospheric measurement campaigns, multiple analytes are concurrently measured. In field campaigns, we routinely make simultaneous measurements of atmospheric H2O2, HCHO, and NH3,12 all by fiber optic (FO)-coupled, liquid core waveguide (LCW)-based fluorometry. Typically, the photomultiplier tube (PMT) detector is the most expensive component in each of these detection systems. The reproducible pulse intensities exhibited by LEDs and the ability to couple several individual FOs (if needed, with different terminal emission filters) from different fluorescence cells to a single PMT (even one with a small window) permit the time-multiplexing of different measurements by the same detector and requires a single data acquisition channel. In the present work, we apply this concept to the simultaneous measurement of H2O2 and MHP using novel dual-membrane diffusion scrubber (DS)-based collection systems and highly selective chemistry. Hydrogen peroxide is an important atmospheric oxidant that is photochemically produced. It plays a key role in the atmospheric oxidation of sulfur dioxide to sulfuric acid (3) Li, J.; Dasgupta, P. K.; Genfa, Z.; Hutterli, M. A. Field Anal. Chem. Technol. 2001, 5, 2-11 2001. (4) Li, J.; Dasgupta, P. K. Anal. Chem. 2000, 72, 5338-5347. (5) Mu ¨ ller, B.; Hauser, P. C. Analyst 1996, 121, 339-343. (6) Hauser, P. C.; Liang, C. L. C.; Mu ¨ ller, B.; Meas. Sci. Technol. 1995, 6, 10811085. (7) Hauser, P. C.; Tan, S. S. S. Analyst 1993, 118, 991-995. (8) Maurice, E.; Monnom, G.; Baxter, G. W.; Wade, S. A.; Petreski, B. P.; Collins, S. F. Opt. Rev. 1997, 4, 89-91. (9) Hillebrand, S.; Schoffen J. R.; Mandaji, M.; Termignoni, C.; Grieneisen, H. P. H.; Kist, T. B. L. Electrophoresis 2002, 23, 2445-2448. (10) Herman, P.; Maliwal, B. P.; Lin, H. J.; Lakowicz, J. R. J. Microsc. Oxford, 2001, 203, 176-181. (11) O’Hagan W. J.; McKenna, M.; Sherrington, D. C.; Rolinski, O. J.; Birch, D. J. S. Meas. Sci. Technol. 2002, 13, 84-91. (12) Li, J.; Dasgupta, P. K.; Genfa, Z. Talanta, 1999, 50, 617-623.

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in hydrometeors;13,14 it is typically present in the atmosphere at low- to sub-part per billion by volume (ppbv) levels; MHP is typically present at concentrations lower than that of H2O2. MHP and other organic peroxides are also of interest to atmospheric chemists because their concentrations reveal key mechanisms and pathways. Prevalent techniques for measurement of peroxides and field measurement results have been described in the literature.15-17 A variety of methods have been reported for the determination of H2O2. Chemiluminescence (CL) methods do have very high sensitivity,18-21 but other peroxides also react. Many other oxidants also generate CL when reacted with luminol, the most commonly used reagent in peroxide measurement. Most frequently, atmospheric peroxides are measured by liquid-phase fluorometry. Typically, a nonfluorescent substrate is oxidized by H2O2 to a fluorescent product in the presence of a peroxidase enzyme, usually horseradish peroxidase (HRP).22,23 Because HRP is expensive and unstable, attempts to use photocatalysis24 and nonenzymatic reactions25 have been made. Peroxidase mimics, especially synthetic metalloporphyrins, have been studied.26,27 Several Fe-porphine compounds, inexpensive commercial products from animal blood, display a greater peroxidatic activity per unit weight than the most purified HRP preparation available.28,29 Bovine hematin (Hmn), at a cost of ∼0.2% that of HRP in terms of comparable activity, is very effective. Unlike HRP, which catalyzes the reaction of both H2O2 and organic peroxides, Hmn is ∼25 times more selective for H2O2 over MHP.4,30 Although other methods have been advocated,31 presently, the best approach toward the speciation of different peroxides appears to be the determination of individual peroxides by postcolumn reaction, fluorometry after chromatographic separation.15,32-36 It (13) Gunz, W. G.; Hoffman, M. R.; Atmos. Environ. 1990, 24A, 1601-1633. (14) Sakugawa, H.; Kaplan, I. R.; Tsai, W.; Cohen, Y. Environ. Sci. Technol. 1990, 24, 1452-1462. (15) Saucer, F.; Limbach, S.; Moortgat, G. K. Atmos. Environ. 1997, 31, 11731184. (16) Deforest, C. L.; Kieber, R. J.; Willy, J. D. Environ. Sci. Technol. 1997, 31, 3068-3073. (17) Lee, M.; Heikes, B. G.; O’Sullivan, D. W. Atmos. Environ. 2000, 34, 34753494. (18) Price D.; Mantoura, R. F. C.; Worsforld, P. J. Anal. Chim. Acta 1998, 371, 205-215. (19) Yuan, J.; Shiller, A. M. Anal. Chem. 1999, 71, 1971-1980. (20) Stigbrand, M.; Karlsson, A.; Irgum, K. Anal. Chem. 1996, 68, 3945-3950. (21) Li, J.; Dasgupta, P. K. Anal. Chim. Acta 2001, 443, 63-70. (22) Lazrus, A. L.; Kok, G. L.; Giltin, S. N.; Lind, J. A. Anal. Chem. 1985, 57, 917-922. (23) Jacob, P.; Klockow, D. Fresenius’ J. Anal. Chem. 1993, 346, 429-434. (24) Genfa, Z.; Dasgupta, P. K.; Edgemond, W. S.; Marx, J. N. Anal. Chim. Acta 1991, 243, 207-216. (25) Lee, J. H.; Tang, I. N.; Weinstein-Lloyd, J. B. Anal. Chem. 1990, 62, 23812384. (26) Saito, Y.; Mifune, M.; Nakashima, S.; Odo, J.; Tanaka, Y.; Chikuma, M.; Tanaka, H. Talanta, 1987, 34, 667-669. (27) Saito, Y.; Nakashima, S.; Mifune, M.; Odo, J.; Tanaka, Y.; Chikuma, M.; Tanaka, H. Anal. Chim. Acta 1995, 172, 285-287. (28) Zhang, G.; Dasgupta, P. K. Anal. Chem. 1992, 64, 517-522. (29) Howell, R. R.; Wyngaarden, J. B.. J. Biol. Chem. 1960, 235, 3544-3550. (30) Zhang, G.; Dasgupta, P. K. Anal. Chim. Acta 1992, 260, 57-64. (31) Lee, J. H.; Tang, I. N.; Weistein-Lloyd, J. B.; Halper, E. B. Environ. Sci. Technol. 1994, 28, 1180-1185. (32) Hellpointner, E.; Ga¨b, S. Nature 1989, 337, 631-634. (33) Kurth, H. H.; Ga¨b, S.; Turner, W. V.; Kettrup, A. Anal. Chem. 1991, 63, 2586-2588. (34) Hewitt, C. N.; Kok, G. L. J. Atmos. Chem. 1991, 12, 181-194. (35) Kok, G. L.; McLaren, S. E.; Staffelbach, T. A.; J. Atmos. Ocean. Technol. 1995, 12, 282-289. (36) Wang, K.; Glaze, W. H. J. Chromatogr. 1998, 822, 207-214.

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would be very helpful if H2O2 and MHP, the two dominant atmospheric hydroperoxides (based on data thus far available) could be measured directly without the need for chromatographic separation. Preferably, such a method should involve direct signals for each analyte rather than a subtraction approach. To measure gaseous peroxides by liquid phase reactions, the analytes must first be transferred to the aqueous phase. The DS and a wetted glass coil are the most commonly used approaches. In one comparison study, the authors concluded that the DS is superior.37 Straight-inlet DS devices have the advantage of low particle deposition and high collection efficiency. They are suitable for near-real-time peroxide measurement while combined with flow-analysis systems. In our last paper4 on this topic, we described a fully automated instrument for the determination of H2O2 and hydroxymethylhydroperoxide (HMHP). A straight inlet Nafion membrane based DS was used to collect H2O2. A liquid core waveguide flow-through cell, transversely illuminated by an InGaNbased LED (370 nm), was used with Hmn-catalyzed oxidation of thiamine hydrochloride (TH) by H2O2 (and also any HMHP present; HMHP rapidly dissociates into H2O2 and HCHO under the alkaline conditions of the fluorogenic reaction). The method very selectively determines H2O2 over MHP because (a) MHP does not permeate through Nafion, a highly polar membrane and (b) Hmn does not significantly catalyze the oxidation of TH by MHP. It was also shown that at realistic concentrations, SO2 does not interfere. The interference from O3 is small and can be readily corrected for when O3 is simultaneously measured. In this paper, we extend the general approach to concurrently, selectively and separately measuring MHP by using a two-channel DS, one channel identical to that previously reported and the other channel based on an ePTFE DS that collects both H2O2 and MHP. The effluent from the second channel passes through a miniature MnO2 column that selectively destroys H2O2 without appreciable destruction of MHP. The HRP-catalyzed TH oxidation by MHP is then used as the fluorogenic reaction. The two fluorescence detection cells are identical LED-illuminated FO-coupled LCW devices in which the LEDs are turned on/off under personal computer (PC) control. The two FOs carrying the individual fluorescence signals are multiplexed to the same PMT. The same channel of the PC acquires the signals. Since the PC itself turns the LEDs on, it obviously “knows” which LED is on. It is, thus, simple for appropriate software to assign the collected data to individual analytes. EXPERIMENTAL SECTION Reagents. Hematin (from bovine blood, Aldrich) stock solution was prepared by dissolving 60 mg of Hmn in 200 mL of 0.1 M NaOH. Refrigerated at 4 °C, this solution is stable for at least 2 months. Working Hmn solution (10 µM) was prepared by dilution of 10.5 mL of stock to 500 mL with phosphate buffer (50 mM K2HPO4 adjusted to pH 12 with 2 M NaOH). The optimum TH concentration in the two determination systems was different. For the H2O2 channel, the TH stock solution (10 mM) was prepared by dissolving 337 mg of TH (Sigma) in 100 mL of water. Refrigerated, this solution is stable at least 1 month. The TH working solution (100 µM) was prepared by dilution with water and kept protected from light. Both Hmn and TH working (37) De Serves, C.; Ross, H. B. Environ Sci. Technol. 1993, 27, 2712-2718.

Figure 1. Analytical system schematic representation: N, Nafion tube; E, ePTFE Teflon membrane tube; V1,V2, six way injection valve; P, peristaltic pump; AP, air pump; AC, activated carbon column; SV, three way solenoid valve; FM, flow meter; NL, 23-gauge hypodermic needle (supplementary flow); FC, flow controller; F1, F2, flush/debubble ports (normally closed); TP, trap column packed with active carbon to protect reagents and water from contamination; T1-T4, mixing tees; M, MnO2-packed column; R1, R2, thermostated reactors; D1, D2, LCW fluorescence detectors.

solutions were stable for at least 14 days at room temperature; there was no detectable sensitivity loss. The fluorescence background did increase marginally over this period but it did not affect the sensitivity for H2O2 measurement. For the HRP-MHP system, the optimum TH concentration (3 mg/mL) was nearly 2 orders of magnitude higher than that used in the H2O2 system. The working solution was directly prepared by dissolving 1.5 g of TH in 500 mL of water. HRP (Sigma, P-8125, 148 purpurogallin units/ mg) working solution (20 U/mL) was prepared by dissolving 67 mg of HRP in 500 mL of 50 mM phosphate buffer (adjusting pH of 50 mM KH2PO4 with 2 M NaOH to 8.0). This reagent is not stable at room temperature. During field use, the reagent was maintained in a miniature Peltier cooler-based refrigerator (5-7 °C). A 5-µm sterile Acrodisc syringe filter was connected in the line to remove any particles in HRP solution. In the absence of the filter, erratic spikes were sometimes observed in the signal, probably as a result of the presence of particles in the solution. Five hundred milliliters of the HRP reagent can be used for ∼3.5 days without appreciable changes in sensitivity.

MHP was synthesized from H2O2 and (CH3)2SO4, as described by Davies and Deary,38 with final purification by distillation under reduced pressure. The MHP thus synthesized was stored frozen. MHP thus prepared is not completely free from H2O2. H2O2 present in the MHP working solution was removed by passing the solution through an MnO2 column immediately before use. The concentration of MHP in the effluent was determined by measuring the absorbance of I3- at 352 nm (Agilent model 8453A spectrophotometer) produced by the oxidation of KI in the presence of acetic acid under a CO2 blanket.39 H2O2 stock solution (∼1 M) was prepared by dilution of the 30% reagent (Fisher) with water and standardized by titration with secondary standard KMnO4. Analytical System. The liquid and gas flows in the system are shown schematically in Figure 1. In the liquid flow system, water was aspirated by a peristaltic pump (P, Dynamax, Rainin) through the individual Nafion DS (N) and ePTFE DS (E) units. (38) Davies, D. M.; Deary, M. E. J. Chem. Soc., Perkin Trans. 1992, 559-561. (39) Wibaut, J. P.; van Leewuen, H. B.; van der Waal, B. Recl. Trav. Chim. PaysBas, 1954, 73, 1033-1036.

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The liquid effluent from N, bearing H2O2 (and any HMHP), was mixed with the TH-Hmn reagent mixture in tee T2 and allowed to react in a serpentine-II knit PTFE reactor, (R1, 0.65-mm i.d. × 1700 mm, residence time ∼ 180 s). The liquid effluent from E, bearing both H2O2 and MHP, was pumped through a miniature MnO2 column M (granular MnO2, Mallinckrodt, packed in 0.87mm i.d. × 10-mm 20 ga. PTFE tube) to destroy H2O2 and allow MHP to pass through. The effluent was then mixed with the THHRP mixture in tee T4 and allowed to react in reactor R2 identical to R1. Two 6-port loop injectors (V1 and V2, respectively) were connected in the DS effluent lines for introduction of solutionbased standards or samples. Both reactors were potted in a lowmelting bismuth-tin alloy (Cerrobase, 5550-1, Canfield Technologies, Sayreville, NJ) in a poly(vinyl chloride) container containing two flexible silliconized heaters (Watlow, St. Louis, MO) in series (22.5 W total). A 100-Ω platinum RTD monitored the temperature, and a miniature temperature controller (type CN 1632 GNR, Omega Engg, Stamford, CT) provided temperature control. The temperature of the reactor was maintained at 30 °C. The liquid effluent from the respective reactors then flowed individually through two identical LCW-based LED-driven fluorescence detectors, and the fluorescence signals from both detectors were coupled to the same PMT (vide infra). Flush ports F1 and F2 are provided via tee arms on detectors D1 and D2, respectively, to remove recalcitrant bubbles and to wash and clean the cells; these ports are plugged in normal use. A miniature air pump (AP, T2-03 HP, Tee-Squared Mfg, Fairfield, NJ) was used to aspirate the sample and zero gas through the DS. The pump outlet was connected to a 3-way valve SV (MBD002, Honeywell) via flow meter FM. The valve was controlled by digital output from a PC via a logic level N-channel MOSFET switch (RFM8N18L, Harris Semiconductor). Ability to control this manually or by an independent programmable timer was also provided. When the valve was energized, the pump exhaust was vented, and the DS sampled ambient air. When SV was turned off, the pump exhaust was directed to pass through an activated carbon column that removed all H2O2 and MHP. This was fed as the sample to the DS. Effectively, this was the zero period. To ensure that the zero gas flow to the DS was at least as much as the air pump aspirated through it, a 23 gauge needle NL was incorporated in a tee between the DS and the AP inlet. The needle allowed ∼2% of the total flow (∼1.6 L/min) as ballast to be drawn by AP. As a result, during the zero procedure, a small amount of zero air actually flowed out of the sample inlet. The sample and zero periods were set at 3 and 7 min, respectively, the instrument thus working in a 10-min measurement cycle. Effectively, the gaseous sample was thus “injected” into a zero gas stream acting as the carrier for 3 of every 10 min, and flowinjection (FI)-type signals were obtained. The zeroing function is important and very useful in the field where it was inconvenient or impossible to carry a large zero-air cylinder. However, the instrument baseline was very stable. For all but very low concentration measurement applications, the instrument could be operated continuously without zeroing. Diffusion Scrubber. The design and principle of the diffusion scrubber used was basically the same as that reported previously,4 except that two separate diffusion scrubber membranes were incorporated within the same external jacket. The two-channel 1206

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DS is schematically shown in Figure 1. The H2O2-channel DS is based on a Nafion tube.4 The MHP-channel DS was based on a thin-wall (0.005 in.), large internodal distance (30-50-µm) expanded poly(tetrafluoroethylene) (ePTFE) tube (Zeus, P/N 2E040 005E*FJ).2 To reduce the internal volume, the lumen of the tube was filled with a nylon monofilament (0.021-in. diam fishing line, 25 lb strength). The general connection strategy is to insert the solution inlet and outlet tube within the membrane tube, put a sleeve tubing (∼1/16-in. o.d.) atop the membrane tube, and make a compression seal with a flangeless ferrule and nut (Upchurch, Oak Harbor, WA). The active length of each membrane tube was 46 cm. LCW Fluorescence Detection Cells and Signal Acquisition. Identical LCW fluorescence detectors3,4,40 were used in the two channels, and both of the fluorescence collection fibers were connected to the same PMT. The excitation LEDs (NSHU 590E, center wavelength 370-375 nm, fwhm 12 nm, Nichia America Corp.; Pittsburgh, PA) in the two detectors were switched on and off by two individually programmed digital outputs from a Pentium III class laptop PC via two MOSFET switches (RFM8N18L). Individual LED currents are ∼13 mA when on. When LED1 was on, LED2 was off, and vice-versa. The fluorescence signals from the two detector cells were coupled with two 1.0-mm-core plasticcoated silica FOs to the window of the same PMT (H5784, Hamamatsu Phtonics K. K., Edgewater, NJ) Because the fluorescence excitation and emission characteristics are the same in both detectors, a small circle of blue plastic filter (no. 861, Edmund Scientific, Gloucester, NJ) was put in front of the PMT window to filter out stray excitation radiation. The duration of the LED on and off periods can be easily changed in the PC-based software. For the present experiments, each LED was turned on for 5 s. The data collection frequency was set at 1 Hz. The PMT output was the sequential signal from the two LCW detectors. Instrument Calibration and Standard Gas Generation. The instrument can be calibrated by injection of liquid H2O2 or MHP standards through respective loop injectors (200-µL volume, V1, V2, Figure 1). Gaseous H2O2, MHP, and SO2 sources were set up and calibrated as previously reported.4,41 Ozone was generated with a high-pressure mercury lamp, and its concentration was calibrated and monitored as reported previously.4 During the 5-week-long field study in Philadelphia, PA, the instrument was calibrated daily with solution-phase H2O2 and weekly or more often, with gaseous H2O2. In solution, MHP slowly decomposes to H2O2; the solution-phase MHP concentration had to be iodometrically measured (after catalytic destruction of H2O2) immediately before the Henry’s law-based MHP gas source was set up. In the field, it is too difficult to calibrate with MHP. The stability of the MHP measurement channel was checked with H2O2 after temporarily removing the MnO2 reactor. The fact that the MnO2 reactor removes H2O2 completely was checked by reinstalling the reactor. Field Studies. The instrument was used continuously in the Philadelphia NEO3PS experiment from June 27 to August 30, 2001. The site is adjacent to a major urban area. The instrument was set up inside an air-conditioned trailer. The sample inlet, an U-extension to a 4-in.-i.d. poly(vinyl chloride) (PVC) pipe, was (40) Dasgupta, P. K.; Zhang, G.; Li, J.; Borring, C. B.; Jambunathan, S.; Al-Horr, R. Anal. Chem. 1999, 71, 1400-1407. (41) Hwang, H., Dasgupta, P. K. Environ. Sci. Technol. 1985, 19, 255-258.

located 1 m from the trailer roof line and ∼5 m above ground level. From the bottom of the PVC pipe, a blower fan aspirated the sample through the main tube at ∼1400 L/min. The center of the flowing stream under these conditions does not see the walls. The sample was drawn by a 0.125-in.-i.d. PFA Teflon tube angled downward, from the exact center of the PVC tube. This sampling inlet served the present combined peroxides monitor, two formaldehyde monitors, and an ammonia monitor. The total sampling flow was ∼5.5 L/min and would be isokinetic if the inlet was angled in the direction of the airflow. Angling the inlet downward showed no loss of analyte gases and was preferred because there was much less particle deposition in the lines. Although the particle deposition was low, the Teflon sampling line and the DS jacket tube were washed every week with water (and dried with cylinder nitrogen before recommencing sampling). The detector cell D1 for H2O2 was washed with 2 M HCl every day by injecting it through debubble port F1 to remove any Hmn that tends to slowly deposit on surfaces. RESULTS AND DISCUSSION DS Characteristics. The Nafion DS has a very high collection efficiency for H2O2, but not for MHP. Because the solubility of MHP in this highly polar membrane is poor, it does not collect detectable amounts of MHP.4 To measure MHP, we utilized the porous ePTFE tube. This membrane leaks easily under significant positive pressure. It also collapses under significant negative pressure because the walls are very thin. The DS liquid is water. The water reservoir bottle is put at an optimum height of ∼60 cm above the DS to provide a positive pressure. Without some positive pressure, air bubbles are aspirated into the system. Despite these apparent disadvantages, the ePTFE tube has the outstanding advantage over other porous hydrophobic membranes in that solutions with a high dissolved solids content can be easily used over prolonged periods without blocking the pores of the membrane. No other porous hydrophobic membranes that we have previously used (Celgard, Accurel, Gore-Tex) can tolerate significant salt concentrations without showing a loss of collection efficiency with time, presumably due to evaporation and deposition of solid material in the pores. We have pumped 0.2 M NaCl through the ePTFE membrane tube continuously for 7 days while sampling 2 ppbv H2O2 and observed no significant change in collection efficiency during the experiment. Removal of H2O2 from the ePTFE DS Liquid Effluent. The ePTFE DS collects both H2O2 (and any HMHP) and MHP. Since H2O2 reacts in the HRP-TH system, it will be preferable to destroy the H2O2 selectively over MHP before the measurement reaction, rather than measuring the total and then subtracting the H2O2 measured in the Nafion DS-Hmn-TH reaction system. Of granular iron, stainless steel, and MnO2, MnO2 was most effective in destroying H2O2. However, although it can completely destroy H2O2, it also destroys some of the MHP. Overdesigning the conditions, that is, allowing too long a residence time in contact with the MnO2 catalyst at the operating flow rate (86-87 µL/ min), thus leads to greater loss of MHP than necessary and should be avoided. We tested MnO2 columns in different sizes. On the basis of such experiments, we made the choice of a relatively small column (0.87-mm i.d. ×10-mm long) as the optimum. Over 3 days of use, the column destroyed 100% of the H2O2, leaving no detectable residue while allowing 86 ( 1% (n ) 3) of the MHP to

Figure 2. Parametric dependence in the MHP determination system (HRP-Thiamine-MHP). (a) Effect of 50 mM sodium phosphate buffer pH used to constitute the HRP solution (20 U/mL) on the signal. 1 µM MHP injected, 3 mg/mL thiamine. (b) Effect of thiamine concentration. HRP, 20 U/mL in 50 mM pH 8.0 Na2HPO4 buffer; 1 µM MHP injected. (c) Effect of HRP concentration on the fluorescence intensity. Thiamine, 3 mg/mL; MHP, 1 µM.

pass through. The reproducibility of making the MnO2 column (from aqueous slurry, gravity packing, glass wool retainers in plastic barbed Luer connectors (Ark-Plas, Flippin, AR)) was examined. Three identically made columns allowed 86.4 ( 0.8% of 1 µM MHP to pass through. In the field, the MnO2 column was replaced each time the reagents were replaced (every 3 days). Reaction Condition Optimization. The reaction conditions of the Hmn-TH-H2O2 system was previously optimized.4 The HRP-TH-MHP system was optimized as follows. Effect of pH and Ionic Strength. A portion of 20 U /mL HRP was prepared in 50 mM Na2HPO4 buffer; the pH of the buffer was adjusted with 2 M NaOH and 2 M HCl as needed. The dependence of the fluorescence signal upon injecting 2 µM MHP and the pH of the phosphate buffer is shown in Figure 2a. It can be seen that the fluorescence intensity is maximum at pH 9.0; however, the change between pH 8 and 10 is not large. Considering the much greater buffer capacity of the system at a pH of 8, a 50 mM Na2HPO4 buffer of pH 8.0 was used throughout. The concentration of phosphate buffer in the range of 50-300 mM had no significant effects on the fluorescence intensity. Effect of TH Concentration. In the Hmn-TH-H2O2 system, the optimized TH concentration was 100 µM.4 In the present system, the optimum concentration of TH was much higher, as shown in Figure 2b. The fluorescence intensity increased with the TH concentration from 0.1 to 2 mg/mL, reaching a virtual plateau at 2 mg/mL and increasing little thereafter. TH is relatively inexpensive; we opted to use a TH concentration of 3 mg/mL (8.9 mM). Effect of HRP Concentration. The effect of HRP concentration on the signal elicited by 1 µM MHP is shown in Figure 2c. On Analytical Chemistry, Vol. 75, No. 5, March 1, 2003

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Figure 3. Instrument response. (a) PMT response as seen by the PC, consisting of the composite signal from two channels. The sample contained 2.0 ppbv H2O2 and 3.4 ppbv MHP. The lower and upper envelopes of the composite trace constitute the individual signals for H2O2 and MHP, respectively. (b) Software-isolated signal for the two channels.

the basis of these results, 20 U/mL HRP was chosen for further experiments. Data Acquisition and Processing. The composite signal output of the PMT while sampling a mixture of 2 ppbv H2O2(g) with 3.4 ppbv MHP is shown in Figure 3a. In this example, the baselines for the H2O2 and MHP channels were deliberately offset to different values, ∼0.2 and ∼0.8 V, respectively. Because of the baseline offset, the H2O2 and MHP signals constitute the lower and upper envelopes of the composite trace. Such an offset is not necessary for the software to accurately process the data. The software itself turns the LEDs on and off and thus “knows” which LED is on and accordingly can “tell” which cell the signal is coming from. The algorithm takes the average of 5 data points acquired during the period each LED is on. It then separates the sequentially mixed signal into individual channels. The separated signals are shown in Figure 3b. The system operates on a 3/7 min sample/zero cycle. The temporal resolution of the signal in each channel is 10 s, but the ultimate determining factor is the sampling cycle, 10 min in duration in the present experiments. However, the time constant in the signal processing/amplification circuitry must be significantly less than 1 s. Otherwise, the signals from the two channels will affect each other, especially when the signal from one channel is much larger than that from the other. The software can also be instructed to ignore the first datum when each LED is turned on to avoid this signal “carryover”. Instrument Performance. Liquid Phase. When injected in to the liquid phase, the HRP system has no response to H2O2 because the MnO2 column completely destroys the H2O2. In the hematin system, the fluorescence intensity was linear with H2O2 concentration.

signal, mV ) 653.4 [H2O2]

µM + 25.4, r2 ) 0.9995 (1)

On the basis of a S/N of 3, the limit of detection (LOD) was 8 1208

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nM. The response of the hematin reaction system to MHP was 25-fold lower, as compared to equimolar concentrations of H2O2.4 The response of the HRP system to MHP solutions could be expressed with the equation

signal, mV ) 412.3 [MHP]

µM - 32.2, r2 ) 0.9987 (2)

On the basis of a S/N of 3, the LOD was 16 nM. Gas Phase. The response of the hematin system to gaseous H2O2 could be expressed as

signal, mV ) 261.0 [H2O2]

ppbv -156.0, r2 ) 0.9934 (3)

On the basis of a S/N of 3, the LOD was 25 pptv. The response of MHP(g) in the Nafion DS-hematin system is practically insignificant, ∼1.5% that of H2O2(g). The relevant response equation is

artifact H2O2 signal, mV ) 3.8 [MHP]

ppbv + 9.8, r2 ) 0.9869 (4)

The H2O2 concentrations obtained from eq 3 can be corrected using the measured MHP concentrations via eq 4; however, ambient MHP concentration are typically only one-tenth or less that of the H2O2 levels, so the interference posed by MHP in the determination of H2O2 for practical purposes is insignificant and is often within experimental error. Because of the effectiveness of the MnO2 catalyst, the ePTFEMnO2-HRP system has no measurable response to H2O2(g). The

Table 2. Interference of SO2 in the Determination of 1 ppbv H2O2 with the EPTFE DS SO2 (ppbv)

signal, mV (( SD)

error %

0 5 10 20 40 80

237.2 ( 2.3 226.9 ( 3.0 201.8 ( 5.8 142.6 ( 3.2 75.9 ( 6.1 37.7 ( 8.3

-4.3 -14.9 -39.9 -68.1 -84.1

Table 3. Interference of O3 in the Determination of 2 ppbv MHP, EPTFE DS

Figure 4. System output for 2 ppbv H2O2 and 0.23-3.44 ppbv MHP. 3-min sample, 7-min zero. Table 1. Interference of Ozone in the Determination of 1 ppbv H2O2 with the EPTFE DS O3 (ppbv)

signal (mV ( SD)

error %

0 40 80 120 160

268.5 ( 3.6 787.5 ( 26.1 619.1 ( 53 552.0 ( 31.2 573.1 ( 36.1

+193 +130 +106 +113

response to MHP(g) is given as

signal, mV ) 443.4 [MHP]

ppbv + 20.2, r2 ) 0.9966 (5)

On the basis of a S/N of 3, the LOD was 15 pptv. Typical system output for the two software-processed channels to different concentrations of MHP(g) mixed with 2 ppbv H2O2(g) is shown in Figure 4. Interference Studies. At the low sampling rate used in this study, the ePTFE DS exhibits a quantitative collection efficiency for H2O2 and 89.5 ( 1% collection efficiency for MHP. Thus initially, we wanted to use a single ePTFE DS and split the liquid effluent into two streams and then carry out differential chemistry. However, studies on interferences of O3 and SO2 showed that both gases very seriously interfere in the determination of H2O2 when collected by an ePTFE DS. The data are presented in Tables 1 and 2. The ozone interference is highly variable. As a first approximation, past some minimum concentration, the ozone interference is zero order in ozone. Minor hydrocarbon contamination of the membrane surface and its variability with time may be responsible for the erratic nature of the interference. In a real sampling situation, it is impossible to predict what adventitious impurities will adsorb onto the ePTFE surface and thereby lead to artifact H2O2 production. Although sulfur dioxide does not interfere on an equimolar basis (presumably because of its lower

O3 (ppbv)

signal (mV ( SD)

error %

0 40 80 120 160 200

875.2 ( 14.4 798.4 ( 3.2 825.6 ( 12.8 876.4 ( 14.0 908.4 ( 3.2 911.6 ( 3.2

-8.8 -5.7 +0.14 +3.4 +4.2

Table 4. Interference of SO2 in the Determination of 2 ppbv MHP, EPTFE DS SO2 (ppbv)

signal (mV ( SD)

error %

0 5 10 20 40 80

878.2 ( 7.9 878.6 ( 15.0 860.3 ( 7.5 851.5 ( 3.3 715.2 ( 12.1 499.1 ( 11.7

0.05 -2.04 -3.04 -18.6 -43.2

Henry’s law solubility and lower diffusion coefficient), it still interferes very seriously in a predictably negative fashion. In contrast, previous experiments established that the Nafion DS exhibits a negative, approximately linear, interference from SO2 amounting to 1.4 ( 0.3 pptv H2O2 per ppbv SO2 and a similar positive interference from O3 amounting to 2.7 ( 0.5 pptv H2O2 per ppbv O3. These interferences are far smaller and, if desired, can be corrected for if SO2 and O3 data are concurrently available (as is commonly the case). For the determination of MHP, the data in Table 3 show that O3 has no interference within experimental error because the artifact it produces is H2O2 and not MHP; the H2O2 is effectively removed by the MnO2. SO2 interferes in a significant fashion only at concentrations higher than 20 ppbv. At SO2 concentrations higher than this, the interference increases linearly (r2 ) 0.9963) and can be accordingly compensated for. The relevant data are listed in Table 4. Field Measurement. A portion of the data from the Philadelphia field campaign in the summer of 2001 is shown in Figure 5. To our knowledge, these represent the first sustained measurements of both H2O2 and MHP. On all days, the diurnal cycle in both peroxides is evident. In general, MHP concentration was much lower than that of H2O2, the one sustained exception being the week beginning July 21. It will be noted that during this period, the peak MHP concentrations did not occur precisely at the same times when H2O2 peak concentrations were observed. Thus, the MHP peak could not have been an artifact, due to, for example, the failure of the MnO2 catalyst to destroy H2O2. Both MHP and Analytical Chemistry, Vol. 75, No. 5, March 1, 2003

1209

Figure 5. H2O2 and MHP data from Philadelphia, PA, June 27, 4:00 p.m., to July 31, 7:40 a.m.

H2O2 concentrations were high during this period. There was no rain during this week. It was not merely that the [MHP]/[H2O2] ratio was high because of rainout or washout, but the absolute concentrations of both species were high. At the present time, the auxiliary chemistry data that we have available from measurements made by us and others at the site do not provide a clue as to why the MHP concentration was so much higher during this period. Clearly, future measurements that will include the determination of reactive hydrocarbons are necessary. A complete listing of the Philadelphia data will appear in the NARSTO database.42 During the field measurement, of a total deployment period of 62 860 min, the instrument collected data 96.68% of the total time; calibration required 2.21%, and reagent replacement/cleaning required 1.11% of the total time. The instrument was inoperative at no time. In summary, we have described a facile, compact, simple, source-multiplexed fluorometric instrument for the measurement (42) http://www.cgenv.com/Narsto/. (43) Chen, Q.-Y.; Li, D.-H.; Zhu, Q.-Z.; Zheng, H.; Xu, J.-G. Anal. Lett. 1999, 32, 457-469. (44) Matsubara, C.; Kawamoto, N.; Takamura,K. Analyst 1992, 117, 1781-1784. (45) Li, J.; Dasgupta, P. K. Anal. Sci., submitted.

1210 Analytical Chemistry, Vol. 75, No. 5, March 1, 2003

of H2O2 and MHP. The present arrangement cannot differentiate between H2O2 and HMHP. There is scant data in the literature as to whether HMHP is a significant constituent of atmospheric peroxides. The present Nafion DS collects both H2O2 and HMHP; however, HMHP decomposes under the alkaline conditions to H2O2. There are at least two reactions that permit measurement of H2O2 under acid conditions. One uses fluorometric measurement with an iron phthalocyanine catalyst;43 the other uses the ligation of H2O2 to Oxo[5,10,15,20-tetra(4-pyridyl)porphyrinato]titanium(IV), followed by absorptiometric measurement.44,45 Consideration of the chemistries indicates that it is highly likely that both reactions will be insensitive to HMHP. In the future, we therefore hope to report on a three-channel instrument. ACKNOWLEDGMENT This research was made possible in part by a contract from Man-Tech Environmental. We gratefully acknowledge the help and interest of William A. McClenny, U.S. Environmental Protection Agency. Received for review December 22, 2002. AC026234D

October

14,

2002.

Accepted