Anal. Chem. 1991, 63, 1763-1766
semblies. It is worthwhile to note also, see Table IV, that provided that water is absent, the R T P triplet lifetime remains substantially constant whatever the solid support or the atmosphere may be. This could be attributed to shrinking of the matrix network during the drying step with the corresponding rigidity increase. It is well-known that the presence of a heavy atom is necessary to increase the triplet-state population (4). As an iodine substituent in the ferron molecule, acting as intramolecular heavy atom, is present in all the cases described here, intersystem crossing should not change substantially. The triplet decay profiles of this Al complex were measured in those order media mentioned above and in the FIA-SSRTP system (Table 11). We observed that the triplet lifetimes at room temperature were 0.186 ms on the resin, 0.156 ms for CTAB micelles, and 0.096 ms for DDAB vesicles. So,although RTP lifetimes in liquids could vary from system to system (24), these results support the idea that a higher rigidity of the complex into the resin along with higher protection of the phosphor by the resin was probably the main factor to prevent collisional deactivation of the excited triplet state and, hence, to secure analytically useful SSRTP signal in the aqueous flow.
ACKNOWLEDGMENT We are grateful to P. Mengndez Fraga for the ETAAAS analyses. Registry No. Al, 7429-90-5; Dowex 1x2, 9085-42-1.
LITERATURE CITED (1) Hurtublse, R. J. Anal. Chem. 1989, 61, 889A-894A. (2) Hutubke, R. J. h4dscuhr Llt&WSSpecboscopy: Melhods end Appllcetlons-Part I I ; Schuiman, S. J., Ed.; Wlley: New York, 1988; Chanter 1.. -..-I---. (3) CUM Love, L. J.; Habarta, J. G.; Dorsey, J. G. Anal. Chem. 1984, 56, 1133A-1148A.
1783
..
sis; Wiley: New York, .1984. (5) Sanz-Medel. A.; Martinez Garcia, P. L; Diaz Qarcia, M. E. Anal. Chem. 1987, 59, 774-778. (6) Ferndndez de ia Campa, M. R.; Diaz Gar&, M. E.; Sanz-Medei, A., Anal. Chim. Acta 1988. 212. 235-243. (7) Fernsndez de la Campa, M. R.; Llu, Y. M.; Dhz Garcia, M. E.; SanzMedei, A. Anal. Chlm. Acta 1990, 238, 297-305. (8) Liu, Y. M.; Fernindez de la Campa, M. R.; Dbz Gar& M. E.; SanzMedel, A. Anal. chkn.Acta 1990. 234, 233-238. (9) Nlshikawa, Y.; Hirakl, K.; Morlshige, K.; Murata, Y. Bunseki Kegeku 1989, 32, 729-735. (10) Endo, K.; Igarashi. S.; Yotsuyanagi, T. Chem. Lett. 1986, 1711-1714. (11) Ruzlcka, J.; Christian, G. D. Anal. Chlm. Act8 1990. 234, 31-40. (12) Valdrcel, M.; Luque de Castro, M. D. Anal. R o c . 1989, 26. 313-315. (13) Yoshimura, K. Anal. Chem. 1987, 59, 2922-2924. (14) Lizaro, F.; Luque de Castro, M. D.; Vaicarcel. M. Anal. Chim. ActB 1989, 219, 231-238. (15) Perelro, R.; Dlaz Garcia. M. E.; Sanz-Medei, A. Analyst 1990. 115. 575-579. (18) Woods, B. A.; Ruzlcka, J.; Christian, G. D. Anal. Chem. 1987, 59. 2767-2773. (17) Hool, K.; Nleman, T. A. Anal. Chem. 1988. 60, 834-837. (18) Camplgila, A. D.; Berthod, A.; Winefordner, J. D. Anal. Chim. Acta 1990, 231, 289-293. (19) Massey, R. C., Taylor, N. D., Eds. Aluminum in Foodand the Environment; Royal Society of Chemistry: Herts, U.K.. 1989. (20) Cannata, J. B.; Suarez. S. C.; Cuesta, V.; Rodriguez, R. M.; Allende, M. T.; Herrera, J.; Perez, J. Roc. Eur. Dial. Transpl. Assoc. Ew. Renal. Asspc. 1984, 21, 354. (21) Dlaz Garcia, M. E.; Sanz-Medel, A. A n d . Chem. 1988, 58, 1436- 1440. (22) Dlaz Gar& M. E.; Fedndez de la Campa, M. R.; Hinze, W. L.; Sanz-Medel. A. Mikmchim. Acfe 1988, 11 1 , 249-282. (23) Perelro Garcla, R.; Lopez Garcia, A.; Diaz Garcia, M. E.; Sanz-Medel, A. J . Anal. At. Spectrom. 1990, 5 , 15-19. (24) Kim, H.;Crouch, S. R.; Zabik, M. J.; Seiim, S. A. Anal. Chem. 1990, 62, 2365-2369.
RECEIVED for review January 24,1991. Accepted May 17,1991. This research was supported by the ‘Fundacidn para el Foment0 en Asturias de la Investigacidn Cientifica Aplicada y la Tecnologia” (FICYT) and the “Comisidn Interministerial de Ciencia y Tecnologia” (CICYT) (PM88-0813-CO2-COl).
Oscillating-Plasma Glow Discharge as a Detector for Gas Chromatography Mikio Kuzuya’ and Edward H. Piepmeier* Department of Chemistry, Gilbert Hall 153, Oregon State University, Corvallis, Oregon 97331 -4003
The change In frequency of a 427-kHz osclllatlon In the current of a glow discharge cell at 1.8 Torr ls a linear and sendtlve measure of organk analyte concentration In the argon plasma support gas. When the plasma cell is used as a gas chromatography detector, an InJectlonof 3 X lo-‘’ mol of propand produces a peak dgnal maximum corresponding to twice the standard deviation In the background slgnal when 0.1-8 countlng tknes are used. Only 20 hnol Is present In the apparenUy acilve r q b n of the detector at the detecth Iknlt. The gas sample Is Introduced Into the plasma cell In the dlrectlon of the cathode vla a 0.34mm-dlameter hole In the center of the anode. The gas Jet that results Is surrounded near its natural outer boundary by a 0.34-cml.d. glass cylinder. Gentle sputterlng, alded by the jet strlklng the curved cathode, keeps the cathode clean.
* Author to whom correspondence should b e addressed.
Present address: De artment of Electronic Engineerin , College of Engineering, Chubu bniversity, 1200 Matsumoto-cho,kasugai-
shi, Aichi 487, Japan.
0003-2700/91/0363-1783$02.50/0
INTRODUCTION Self-sustaining oscillations in low-pressureelectrical plasmas have been known for a long time to be sensitive to plasma conditions ( I ) . Although these oscillations have been studied extensively for decades (e.g., see references in refs 2 and 3), a linear relationship between oscillation frequency and impurity concentration in the plasma support gas has not been demonstrated until now. The f i t use of a low-pressure glow discharge as a detector for gas chromatography was reported by Harley and Pretorius (4)in 1956. They observed the voltage change across an argon discharge and detected on the order of mol of hydrocarbons. Pitkethyl(5) observed the voltage change across a glow discharge in nitrogen and showed a nearly linear response over 3 decades of concentration for hydrocarbons. Although other workers usine different dasma cells had observed long-term drift due & the deposition of carbon on the cathode (6),he observed that no carbonaceous deposits had accumulated in several weeks Of use* He proposed that the discharge itself tends to clean the surface and assist in removal 0 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991 GAS F R O M GC
I
CATRIDGE THERMOCOUPLE HEATER \ 11 ,
LASS TUBE
T
C O O L I N G WATER
i
Figure 1. Schematic dlagram of the osclllatlngplasma glow discharge cell described in the text.
of adsorbed materials. Instead of observing the change in cell voltage, a change in average current or a change in oscillation frequency of an oscillating plasma can be used as a measure of impurity concentration in the plasma suppport gas. Commercial frequency meters can determine the frequency of a signal to within a few parts in 1Olo during a single 0.1-5gate time. Because of this outstanding precision and accuracy and the high sensitivity that the frequency of a plasma oscillation has to changes in plasma gas impurities, oscillations can be used to detect very small impurity concentrations. Therefore, an oscillating plasma can be a sensitive, general purpose detector for gas chromatography. The purpose of this study was to design a glow discharge cell with a stable baseline oscillating frequency and to show that under some conditions the oscillating frequency is linearly related to the concentration of an analyte impurity in the plasma support gas. This cell was interfaced to a gas chromatograph and is shown to be a useful detector for gas chromatography.
EXPERIMENTAL SECTION Figure 1 shows a schematic diagram of the glow discharge cell whose body was machined from a 5- X 5- X 9.75-cm block of Delrin. The eluate from the gas chromatograph enters the cell through a brass nozzle that has a 1.5-mm-long,0.34-mm-diameter orifice. The nozzle is soldered onto the end of a 1/8-in.-o.d.stainless steel tube. The stainless steel tube is 10 cm long and is connected with an Ultra-Torr union (Cajon Co.) to a 2.5-cm length of a l.&in.-0.d. Teflon tubing. The Teflon tube is connected with an an Ultra-Torr union to the '/*-in.-o.d. outlet of a gas chromatograph (Model 5710A, Hewlett-Packard) fitted with a SP2250 column. The Teflon tube electrically insulated the stainless steel anode tube from the gas chromatograph. The tubes between the chromatograph and the plasma cell are wrapped with heating tape to help maintain a temperature of 100 "C. Inside the plasma cell, a 1/8-in.-i.d.glass tube 18 mm long is slipped onto the end of the nozzle. Two turns of Teflon tape are wrapped around the outside of the nozzle to provide a press fit to help aecure the glass tube. The highly visible part of the positive column of the glow discharge appears in the glass tube. Since oscillations are known to occur in the positive column, the purpose of the glass tube is to restrict the eluate to this region to improve the sensitivity of the detector. The distance from the tip of the nozzle to the open end of the glass tube is 5 mm.
A small notch is ground into the other end of the glass tube to expose a l-mm2 area of the anode. This area provides an alternativeelectrical anode surface, where an a n d e glow is clearly visible when high currents are used. This modification provides a more stable oscillation frequency. The end of the '/&.-diameter stainless steel cathode rod is a hemispherical dome. The cathode rod can be advanced along the axis of the cell by turning a micrometer adjustment. The cathode rod can be extended into the glass tube around the anode nozzle to aid in starting the discharge at lower pressures. The cathode is then backed off to the desired distance from the anode, 17 mm for these experiments, and the distance is finely adjusted to obtain a stable frequency as observed on an oscilloscope. Two meters of 1/2-in.-o.d. Teflon tubing connect the cell to a vacuum pump. A valve in this tubing is used to help adjust the cell pressure to 1.8Torr. The 1/2-in.-o.d.Teflon tube is connected to the cell with an Ultra-Torr male connector sealed to the cell body with an O-ring. Opposite this connector is an Ultra-Torr male connector for a 3/4-in.-diameter tubing, in which a glass window is fitted instead of a tube. An Ultra-Torr male connector (not shown on the back side of Figure 1)for l/,-in.-O.D. tubing provides a port for a capacitance manometer (Validyne Engineering Corp.) that monitors the cell pressure. Cooling water at room temperature is circulated in the brass block around the cathode. A 50-W cartridge heater and thermocouple in the brass anode block maintain the block at 90 O C with the help of an electronic temperature control unit (Model 9111, Omega Engineering Inc.). However, because minor baseline fluctuations occur when the heater switches on and off, the controller is usually turned off temporarily just before each run. Argon gas is supplied from a tank that has a two-stage regulator, with the second stage pressure set at 40 psig. A 3-m length of l / & ~tubing leads to a 30-pig full scale pressure regulator, which is normally set at 10 psig to obtain a flow rate of 6 mL m i d STP through the cell. A 5-m length of 1/4-in.tubing leads to a mass flowmeter (Tylan Corp.), which is connected with a short length of 1/4-in.tubing to the input of the gas chromatograph. Later a "T" was placed in this short length of tubing and a valve connected between the T and the atmosphere to speed up purging the air from the argon supply system. The plasma is powered by two regulated, adjustable 400-V dc power supplies (Model EUW-15, Heath) connected in series with each other. A 68-kR resistor is connected between the power supply and the anode to help limit the current. A 10-kR resistor is connected between the cathode and power supply ground to sample the cell current. The cell current is now monitored by connecting the cathode to a follower-with-gain operational amplifier circuit with a gain of 9.6 connected to a strip chart recorder. To monitor the oscillation frequency of the cell, the cathode is connected via a 0.01-pF, 5.6-kR high-pass fiiter to a gain-of-two follower operational amplifier circuit. This is followed by a second-order low pass cutoff filter of the Sallen-Key type (7) with a damping factor of unity and a cutoff frequency of 3.7 MHz to reduce the counting jitter caused by very high frequency signals superimposed on the approximately sinusoidal main oscillation wave form. The output of the filter amplifier is connected via a 820-kR resistor, in parallel with a 330-pF capacitor, to the base of a high-gain NPN transistor (2N3391) in a simple inverter circuit (grounded emitter). The 820-kQresistor was selected to give a square-wave output at the collector of the transistor. A 1-kQload resistor is connected between the collector of the transistor and a 5-V power supply. The rounded square-wave output at the collector of the transistor is connected to the counting input of counter 0 of a DASH-16 (MetraByte Corp.) computer interface board capable of counting up to 10-MHz signals. An IBM-PC computer controls the board and stores and manipulates the data. Counter 1of the DASH-16 is configured to have a square-wave output, and counter 2 is configured to have what the manual identifies as a "rate counter" configuration so that its output produces 90-ms positive gate signals alternating with 10-ms off-times. The output of counter 2 is connected to the gating input of counter 0 in order to produce counting times of 90 1119 at a rate of 10 Hz. The computer program is synchronized to the gating sequence by monitoring the gating input of counter 0 (also identified as digital input IP2). The near-megahertz frequencies cause the 16 bits of counter
ANALYTICAL CHEMISTRY, VOL. 03, NO. 17, SEPTEMBER 1, 1991
1785
0
.rl
4J
0.4 .c
or
.I-
F %
81 0.2
0 Concentration o f propanol (%) Flgure 3. Oscillation-frequencyworking curve for propanol In butanol. The concentration axis indicates the % vlv of propanol in the liquid. Liquid inj&kms of 0.5 p l were used. The chromatogam peak W t s
were measured In frequency units, and the propanollbutanol peak height ratio is plotted on the vertical axis.
Flgure 2. Chromatograms showing (a)cvrent signal and (b) frequency signal recorded simultaneously. Peak 1 is air, peak 2 Is propanol, and peak 3 is pentanol. The 1-V vertical bar In A corresponds to 0.01 1
mA. The time bases are dlsplayed on different scales.
0 to overflow during the 90-ms counting time. Therefore, before a chromatogram is run, the actual frequency is observed on an oscilloscope or determined with a shorter counting time, and then the recorded counts during a chromatogram are adjusted by adding the appropriate number of increments of 216 = 65536.
RESULTS AND DISCUSSION Figure 2 shows simultaneous chromatograms using average cell current (top) and frequency response (bottom) for a mixture of 3 x mol of 2-propanol and 3 x IO* mol of 2-pentanol. The baseline frequency is 427 kHz, and the baseline average current is 0.2 mA. The 1-V indicator bar in Figure 2A corresponds to 0.011 mA. The cell voltage is 535 V. The frequency-to-current peak-height ratio differs from one compound to the next because the mechanisms that determine frequency are at least partially different from those that determine current. The standard deviation in the frequency signal for 100 points of the baseline is 0.8 count, close to the standard deviation of 0.5 count expected from frequency counting error alone. An injection of 3 X lo-" mol of propanol produces a peak whose height is twice the standard deviation of the baseline. With a peak width at half-height of 3 s, this corresponds to a detection limit of 1 X lo-" mol s-l for the flow and pressure conditions used in this particular experiment. The detection limit corresponds to a propanol/argon mole ratio of 1part in 4 X 106,or 6 X lo4 g of propanol/ (STP)mL of argon entering the detector. This is better than the best thermal conductivity detectors and is comparable to the detection limits of a flame ionization detector (8, 9). If it is assumed that the detector responds mainly to the impurity present in the 0.07-cm3glowing volume of the plasma between the anode and cathode, then there is 20 X mol of propanol in the detector a t the detection limit. Since an oscillating
plasma does not require a flowing gas, this represents the smallest amount of propanol that could be detected if much smaller flow rates were used (e.g., with a capillary column) or if the cell were temporarily sealed off during the measurement time. Figure 3 shows the linearity of the frequency response for propanol in butanol. The concentration axis indicates the % v/v of a mixture of propanol and butanol in 1 mL of liquid added to a 5-mL glass vial sealed with a septum cap and kept at 25 "C. Samples of 0.5 p L were removed from the vial with the injection syringe, and then 10 pL of air was pulled into the syringe before injection into the chromatograph. Because it is difficult to reproducibly inject such small samples, the butanol frequency peak is used as an internal reference. Since the highest point in Figure 3 corresponds to 28 pg of propanol, the linear dynamic range is 4 decades above the detection limit of 2 ng. Over several months of operation, a discoloration was noticed on the anode and inside the cathode end of the glass tube around the anode. The cathode was kept clean, apparently by very mild sputtering. Argon was used as the plasma gas because of our extensive experience with glow discharges in argon. Helium is more useful for gas chromatography (II), and studies using He are planned, as are ways to improve the detection limits by optimizing the shape and operating conditions of the glow discharge cell. The advantages of low pressure at the outlet of a gas chromatograph have been studied experimentally (IO)and in theory ( I 1) . The main improvement is in reduced analysis time for easy separations requiring relatively few plates, say less than lo3 plates for a packed column and IO6plates for a capillary column (11). Even then an improvement in analysis time by as much as a factor of 2 would be unusual. The main advantage of this detector is its ability to be used with capillary columns or in other situations where fmol detection limits are required.
ACKNOWLEDGMENT We thank Hyo J. Kim for his kind assistance and Robert Boyer for his help in the design and skillful machining of the cell.
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Anal. Chem. 1991, 63, 1766-1771
LITERATURE CITED (1) Pekarek, L., Ion Waves and Ionization Waves.
10th I n t m t k n e l 1971;Donald Parsons 8
Conterenoe on pheromene In Ionized Gases Co. Ltd.: Oxford, England, 1971;pp 385-403. (2) Franklin, R. N. plesma phenomene In Gas Dlschargas; Claredon Press: Oxford, England, 1976. (3) Oleson, N. L.; Cooper. A. W. Movlng Strlatlons. In Advences k, ElechonlCS end €&iron physlcs; Marton, L., Ed.; Academic Press: New York, 1988;Vol. 24. (4) Harley, J.; Pretorlus, V. Nature 1956, 178, 1244. ( 5 ) Pltkethly, R. C. Anal. Chem. 1958, 8 , 1309-1314. (6)Lovelock, L. E. Anal. Chem. 1961, 33, 182-178. (7) Maimstadt, H. V.; Enke, C. G.; Crouch, S. R. Ekpctronics end Instru-
mentetbn for SdenHSts;The Benjamin/Cummlngs Co., Inc.: Reading, MA. 1981;p 210. (8) Davld, D. J. Gas chrometograph/c Detectors; John Wiiey 8 Sons: New York, 1974; p 4. (9) Christlan, G. D.; ORellly, J. E. Insfrumentill Ana!mis. 2nd ed.; Ailyn and Bacon, Inc.: Newton, MA. 1986;p 749. (10) Locke, D. C.; Brardt, W. W. Reduced Pressure Gas Chromatography. In Gas chromatography; Fowler, L.. Ed.; Academic Press: New York,
1963. (11) Giddings, J. C. Anal. Chem. 1962, 51, 314-319.
”R
for review March 11,1991. Accepted June 10,1991.
Micellar-Enhanced Aqueous Peroxyoxalate Chemiluminescence Niya Dan, Miu Ling Lau, and Mary Lynn Grayeski* Department of Chemistry, Seton Hall University, South Orange, New Jersey 07079
The aqueous peroxyoxalate (CL) reactbn of blr[N-[2-(N’-methyl-2‘-pyrldlnlumyl)ethyl~N-[(trHluoromethyl)sulfonyl]~xamlde(METQ) wlth hydrogen peroxtde In the presence of a fluorophor was characterlzed In dlfferent mlcellar and homogeneous media, and the effects of the various types of surfactants on chemiluminescencelntenelty and quantum efflclencles were assessed. The surfactants examlned Include catlonlc, anlonlc, neutral, and zwfflerionlc types. I n the presence of hexadecyltrlmethylammonlum chlorlde (CTAC), polyoxyethylene(23) dodecanol (Brl) 35), and 3-( N~decyCN,Ndlmethylammonlo)-l-propanecwHonk acid (SB-12) micelles, enhancement of the CL lntenslty by factors of 1.6-130 was observed relatlve to that In buffer. I t was found that In mlcellar medkm not onty were Huoresccmce efflclencles Improved but also the reaction excltatlon efflckncy. The v a r h experhmtal varlabks that Influence the magnltude of the CL enhancements were studied. Lastly, the mkellar CL reactlon systems were evaluated for the analysis of fluorophors and hydrogen peroxide, and analytkal flgures of merlt were generally Improved In micellar medlum.
INTRODUCTION The peroxyoxalate chemiluminescence (CL) reaction, involving the oxidation of an oxalic acid derivative by hydrogen peroxide in the presence of a suitable fluorophor (I+), has been used analytically for the measurement of dansylated amino acids (3,hydrogen peroxide (8), biological reducing agents such as glucose and uric acid (9),fluorophors or f l u e rophor-labeled compounds (10, 1I ) , and polynuclear aromatic hydrocarbons (12-14). However, in many of these applications, some major problems are encountered. Most oxalic acid derivatives are soluble and efficient only in organic solvents, causing irreproducible mixing when measuring aqueous samples. Although water-soluble derivatives have been reported (15),efficiencies are very low. In many cases, the rates of the chemiluminescence reaction in an aqueous system are very fast, resulting in imprecise measurements. Some approaches have been reported to overcome these difficulties. Microemulsions have been used with nonaqueous peroxyoxalate CL (16). In our laboratory, cyclodextrins were used to enhance the aqueous peroxyoxalate CL quantum yield (17). Here we report the study of micelle effects on the aqueous peroxyoxalate CL. Micelles have been demonstrated to influence the chemistry and photophysics of molecules by altering the microenviron0003-2700/91/0383-1768$02.50/0
ment in which the molecules reside. Micelles can change microviscosity, local pH, polarity, reaction pathway or rate, etc. Different aqueous micelles have been used to improve lucigenin (18,191 and luminol CL reactions (20). Surfactants at concentrations below their critical micelle concentrations (cmcs) have been used to increase the efficiency of aqueous peroxyoxalate CL by a factor of 5 (21). The effects of four different types of micelles (anionic, cationic, zwitterionic, and nonionic) on the aqueous peroxyoxalate CL reaction of bis[ N -[ 2-(N’-methyl-2’-pyridiniumyl)ethyl]-N[ (trifluoromethyl)sulfonyl]]oxamide (METQ) with hydrogen peroxide are reported here. The effects of micelles on the excitation efficiency and fluorescence efficiency are studied. The potential analytical implications of these effects will be dimmed.
EXPERIMENTAL SECTION Apparatus. All CL measurements were obtained by using a Turner Designs (Mountain View, CA) Model TD20-e photometer equipped with a manual injector; a Fisher strip-chart recorder Model 2000 printer was used to record the CL intensity-time profiles. Some data were collected and stored on disks by an Apple IIe and graphed on a Gemini printer. The absorption spectra for fluorescence quantum yield determination were carried out with a Varian 2200 spectrophotometer. The fluorescence spectra were recorded on a Spex Fluorolog 2+2 spectrofluorometer. Reagents. The following reagents were used: hexadecyltrimethylammonium chloride (CTAC; Eastman Kodak Company), polyoxyethylene(23) dodecanol (Brij 35; IC1 Americas Inc.), 3(N-dodecyl-N,N-dimethylammonio)-l-propanesulfonic acid (SB12; Hoechst Calbiochem Behring Diagnostics), sodium dodecyl sulfate (SDS; BDH Chemical Ltd.), polyoxyethylene(20) cetyl ether (Brij 58; Aldrich Chemical Company), polyoxyethylene(20) stearyl ether (Brij 78; Aldrich), polyoxyethylene(20) oleyl ether (Brij 99; Aldrich), 8-anilino-1-naphthalenesulfonicacid (ANS; Sigma Chemica Company), rhodamine B (RH B; Aldrich), 1(dimethylamino)naphthalene-5-sulfonyl chloride (dansyl chloride; Aldrich), avidin-rhodamine isothiocyanate (avidin-RH B Sigma), human albumin (Sigma), and 30 w t % hydrogen peroxide (Ald(trifluororich). Bis [N-[2-(N’-methyl-2’-pyridiiumyl)ethyl]-N-[ methyl)sulfonyl]]oxamide (METQ) was prepared by the method of Tseng and Rauhut (22). Albumindawyl chloride was prepared by Jacobsen’s method (23).HPLC grade acetonitrile(Fisher) was used as obtained. All fluorophors and surfactants were wed as received. Buffers were prepared from ACS reagent grade sodium phosphate. The surfactants were dissolved in 0.1 M phosphate buffer at pH 7.0. hEXQ solution was prepared by dissolving 75 mg of dried METQ in 5 mL of acetonitrile (0.017 M). Hydrogen peroxide was diluted to 0.1 M in distilled water. ANS, RH B, avidin-RH B, and albumin-dansyl chloride were dissolved in distilled water. 0 I991 American Chemical Society
