1140
Anal. Chem. 1992, 64, 1140-1144
E. A.; Kaplan M. L.; Cava, R. J. SolM State Ionics 1085, 77, 67. (e) Munshi. M. 2.; Owens, 8. B. Polym. J. 1088. 20, 577. (f) Ferloni, P.; Chlodelll, G.; Maglstrls, A.; Sanesl, M. SolM State Ionks 1988, 78 8 19, 265. (9) Sorensen, P. R.; Jacobsen, T. Electrochm. Acta 1082, 27, 1671. (h) Bhattacharja, S.; Smoot. S. W.; Whltmore, D. H. SolM State Ionlcs 1988, 78 & 79, 306. (I) Bruce, P.; Vincent, C. A. J . Elecfroenal. Chem. Interfeclel Elecb.ochem. 1087, 225, 1. Evans, J.; Vincent, C. A.; Bruce, P. 0. fo/ymer 1087, 28, 2324. (k) Watanabe, M.; Nagano, S.; Sanul, K.; Ogata, N. SolM State Ionics 1988, 28-30, 911. (I) Bowklah, A,; Dalard, F.; Deroo, D.; Armand, M. 8. SolM state ~on/cs1088, 78 & 79,287. (15) (a) Dahms, H. J. J . Phys. Chem. 1088. 72, 362. (b) Ruff, I.; Friedrich, V. J. J . Phys. Chem. 1071, 75. 3297. (c) Ruff, I.; Friedrich, V. J.; Demeter, K.; Cslllag, K. JPhp. Chem. 1071, 75. 3303. (d) Ruff, I.; Korosl-Odor, I. Inorg. Chem. 1070, 9 , 188. (e) Ruff, I . Ektrochlm. Acta 1070, 75, 1059. (f) Botar, L.; Ruff, I . Chem. Phys. Lett. 1988, 726, 348. (9) Ruff, I.; Botar, L. J . Chem. fhys.., 1985. 83, 1292. (h) References f-g correct the numerical prefactor in the quatlon from id4 to 116. (16) (a) Buttry, D. A.; Anson, F. C. J. Elecbpsnel. Chem. InterfacielElectrochem. 1001, 730, 333. (b) Buttry, D. A.; Anson, F. C. J . Am. them. soc. 1083, 705, 685. (c) White, H. S.; Leddy, J.; Bard, A. J. J. Am. Chem. Soc. 1082, 704, 4811. (d) Martin, C. R.;Rubinstein, I.; Bard, A. J. J . Am. Chem. SOC. 1982, 704, 4817. (e) Oyama, N.; Anson, F. C. J. Am. Chem. Soc. 1070, 707. 739, 3450. (f) Oyama, N.; Ohsaka, T.; Kaneko, M.; Sato, K.; Matsuda, H. J. Am. Chem. Soc. 1983, 705. 6003. (g) He, P.; Chen, X. J . Electroanal. Chem. Interfaclel Electrochem. 1088, 256, 353. (17) (a) Nlelson, R. M.; McManls, 0. E.; Golovln, M. N.; Weaver, M. J. J . Phys. Chem. 1988. 92. 3441. (b) McManis, G. E.; Weaver, M. J. them. Phys. Lett. 1088, 745, 55. (c) Zhang, X.; Leddy, J.; Bard, A. J. J. Am. Chem. Soc. 1085, 707, 3719. (d) Zhang, X.; Yang, H.; Bard, A. J. J . Am. Chem. Soc.1987, 709, 1916. (e) Weaver, M. J.;
u)
(18) (19) (20) (21) (22) (23)
Gennett, T. Chem. Phys. Lett. 1985. 773. 213. (f) Gennett. T.; Milner. D. F.; Weaver, M. J. J . Phys. Ct". 1985, 89, 2787. (9) McManls, G. E.;Golovin, M. N.; Weaver, M. J. J. Phys. Chem. 1086. 90, 6563. (h) Nielson, R. M.; Weaver, M. J. J . Elecbpsnal. Chem. Interfaciel Electrod". 1989, 260, 15. (i) Kapturklewlcz, A,; Behr, B. J. Electroanel. Chem. Interfaciel Electrod". 1084, 779, 187. (i) Kapturkiewicz, A.; Opallo, M. J . Electroanel. Chem. Intei-faciel Electrochem. 1985, 785. 15. (k) Opallo, M. J. J . Chem. Soc.,Fafaday Trans. 7 1087, 8 3 , 161. Bard, A. J.; Fauikner, L. R. Electrochemhl Methods: Fundamntak and Applications; John Wlley 8 Sons: New York. 1980; Chapter 5. Shoup. D.; Szabo, A. J . Electroanel. Chem. Interfac&lElectrochem.ochem. 1982, 740, 237. Berthier. C.; Goreckl, W.; Mlnier, M.; Armand, M. B.; Chabagno, J. M.; Riguad, P. SolM State Ionlcs 1983, 7 7 , 91. (a) Williams, M. L.; Landel, R. F.; Ferry, J. D. J . Am. Chem. Soc. 1955. 77, 3071. (b) Cohen. M. H.: Turnbull. D. J . Chem. Phys. 1050, 37, 1164. Sears, J. K.; Darby, J. R. The Techncfogy of Plasrlclzers; John WHey 8 Sons: New York, 1982. Wooster, T. T.; Watanabe, M.; Murray, R. W. J . Phys. Chem., in Dress.
(24) kazeux, D.; Lupien, M. D.; Robitaille, C. D. J . Electrochem. Soc. 1987, 734. 2761. (25) Dexl, W.; Song, H.; Parcher, J. F.; Murray, R. W. Chem. Mater. 1980, 7 . 357. (26) Watanabe, M.; Ikeda,J.; Shinohara, I. Po/ym. J . 1983, 75, 65. (27) Wang, C. 8.; Cooper, S. L. Macromdecuks 1983, 76, 775. (28) Robitallle, C.; Prud'homme, J. Macrorolecules 1083, 76, 665.
RECEIVED for review December 2,1991. Accepted February 21, 1992.
Quantitation of Acridinium Esters Using Electrogenerated Chemiluminescence and Flow Injection Janet S. Littigt and Timothy A. Nieman* Department of Chemistry, University of Illinois, 1209 West California Street, Urbana, Illinois 61801
Acrklkrlun esters are used as chem#unlnescence (CL) labels
In hmunoasmy. Acrklkrkwn ester CL Is tradtthally triggered by addltlon of a solutlon of H202. Thls paper Is concerned wlth the genoratlon of the reaction-lnltlatlng specles In Sttu (electrochemically) to ellmlnate problems associated wlth clolutlon addttlon. Phenyl acrldlnlum9-carboxylateshows no electrochemldry over the range -1.0 to 4-1.0 V. I n the presence of dlswlved oxygen, as the applled potentlal Is stepped negatlvely, electrogenerated chemllumlnescence (ECL) emklon lntenrlty Increases to a plateau reglon correspodng to the peroxkk plateau of eM-1 oxygen reducth. ECL emlssh lntenrlty Increases as pH Increases from 9 to 12, but decreases at hlgher pH. The rate of form a t h of n o n c ~ l h l n e s c e npeeudobase i between sample h)ectbnand CL observation was 05Udled; at pH 12, that dday tlme should be llmlted to under 0.5 8. The worklng curve dynamlc range covers 4 decades In concentratlon. The detectlon llmlt for acrldlnlum ester-labeled lydne Is 10 fmol.
INTRODUCTION Recent interest in acridinium ester chemiluminescence (CL) has focused on ita application to imm~noassay.'-~Use of
* To whom correspondence should be addressed.
'Current address: T h e Procter & Gamble Co., Miami Valley Laboratories, P.O. Box 398707,Cincinnati, OH 45239-8707. 0003-2700/92/0364-1140$03.00/0
acridinium esters as CL labels for antibodies and other molecules seeks to exploit the characteristically low detection limits and wide dynamic ranges associated with the reaction. The CL reaction of acridinium esters is typically initiated by the addition of alkaline hydrogen peroxide. The hydrogen peroxide dissociates to form hydroperoxyl anion (H02-)which attacks the acridinium ring structure. The attack resulta in multiple bond cleavage to produce N-methylacridone in the excited state? Relaxation of the acridone to the ground state is via emission of light at 430 nm.2 Use of CL reactions in assay of clinical importance has been accomplished in a variety of confiiations. Initial work with CL immunoassay used static systems.' Recently, Wilson and co-workers have demonstrated that flow injection analysis (FIA) used in association with CL immunoassays offers advantages of greater speed and reproducibility. In our laboratory, we have been investigating analytical advantages which arise from electrogeneration of CL emission from several systems. This work has involved CL systems traditionally triggered electrochemically (Ru(bpy),2+)6as well as electrogeneration of CL emission from systems where CL emission has been traditionally triggered by chemical addition (lumin01).'8 CL immunoassay has a requirement that some reagent must be added to initiate the detection reaction (as opposed to radioactive or fluorescent tags where no such reaction is involved). This solution addition must be made in a reproducible way to minimize between-run variation, and the reagent volume added must be small to minimize the effects of dilution. Our research with acridinium esters is concerned 0 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
Inject,sample
1141
--
r Dark--Box --I
c=o
A A I
Reservoir
I c"a
Flgure 1. ECL flow system.
with the generation of the reaction initiating species in situ (electrochemically) to eliminatethese problems associated with solution addition. In this paper we illustrate the characteristica of acridinium ester electrogenerated chemiluminescence (ECL) with FIA. Introduction of an electrode into the flow cell eliminates the need to add hydrogen peroxide in solution and affords greater spatial and temporal control over reaction initiation. EXPERIMENTAL SECTION Materials. Phenyl acridinium-9-carboxylate(fluorosulfonate salt) and 4-(2-(succinimidyloxycarbonyl)ethyl)phenyl 10methylacridinium-9-carboxylatefluorosulfonate were purchased from London Diagnostics (Eden Prairie, MN). Cetyltrimethylammonium bromide (CTAB)was obtained from Aldrich Chemical Co. (Milwaukee, WI). m-Lysine monohydrochloride was from Nutritional Biochemicals Corp. (Cleveland, OH). All other chemicals were of reagent grade or better. All buffers and analyte solutions were prepared using water from a Milli-Q water system (Millipore, Bedford, MA). Instrumentation. Figure 1 is a schematic diagram of the apparatus used for the FIA-ECL measurements. The appropriate buffer was pumped using an Altex Model llOA HPLC pump (Berkeley, CA). Injections of analyte (acridinium ester or acridinium ester-labeledlysine) were made using a Valco injection valve (Houston, TX) equipped with a 10-pL injection loop. Because oxygen is required for the generation of HzOz,the SOlutions were not degassed; in order to eliminate problems with flow rate fluctuations from the pump, injections were synchronized to the pump cycle. The analyte was carried to the electrochemical flow cell through PEEK tubing of specified dimensions. The CL reaction was initiated in the electrochemical flow cell which consisted of a glassy carbon flow cell electrode (Bioanalytical Systems, West Lafayette, IN) separated from a Plexiglas window by a Teflon spacer to yield a cell volume of 10 pL. The electrochemical cell was completed with a screw-inAg/AgCl reference electrode (Bioanalytical Systems, West Lafayette, IN) and a stainless steel counter electrode. An AFRDE4 potentiostat (Pine Instrument Company, Grove City, PA) was used for potential control. The flow cell was enclosed in a light-tight box and placed directly in front of a PMT (1P28A, RCA) whose anode current was converted to voltage and amplified using a Pacific Precision Instruments Model 126 photometer (Concord, CA). The output signal was recorded using a strip chart recorder (Pederson, Lafayette, CA). Applied Potential Studies. Using phenyl acridinium-9carboxylate as a model system, the electroactivity of acridinium esters was examined via cyclic voltammetry. Experiments used a standard three-electrode cell with a glassy carbon working electrode. Potential scans were made over a range of +l.OOO to -1.OOO V (vs Ag/AgCl) using 2.5 pM acridinium ester in 5 mM ammonium acetate (pH 5). In a separate experiment, the effect of applied potential on CL emission intensity was investigated using the FIA set-up described above. Injections of 1p M phenyl acridinium-%carboxylatewere made into the carrier stream (0.15 M borate buffer at pHs 9,10,11, and 12) and transported at a rate of 2 mL/min to the flow cell through 35 cm of 0.25-mm-i.d. PEEK tubing. Instrument Optimization. The pH dependence of acridinium ester pseudobase formation is well-known.' Since the singlestream design of our FIA-ECL apparatus presents favorable conditions for this conversion, experiments were designed to minimize these effects. The duration of acridinium ester contact
0
R
(J
(bl
(cl
Flgure 2. (a) Lucigenin, (b) acridlnium ester, (c) N-methylacridone.
with the alkaline buffer was varied in three ways: (i) variation of tubing length between the injector and flow cell (22-145 cm), (ii) variation of tubing diameter between the injector and flow cell (0.25-0.75-mm i.dJ, and (iii) variation of the flow rate (0.5-5 mL/min). Analyte Preparation. Phenyl acridinium-9-carboxylatesolutions were prepared in 5 mM ammonium acetate (pH 5). This pH was used because it had been successfully used in previous studies.' Over the time scale of the experiments described here, that is sufficiently acidic to prevent conversion of the ester to the pseudobase. Acridinium ester-lysine complexes were prepared using the N-hydroxysuccinimide ester as described in the literature.' A 6OOO-foldexceas of lysine over the N-hydroxysuccinimide ester was used to guarantee complete binding. Studies using CTAB were conducted with 2 mM surfactant added to the analyte solution. RESULTS AND DISCUSSION Electrogenerated Chemiluminescence (ECL). The phenomenon of ECL has been used both for mechanistic studies of solution-phase reactions and for determination of analytes. The ECL reaction of lucigenin is of particular interest here due to structural similarities between lucigenin and acridinium esters (Figure 2, parta a and b) and the identical nature of the emitter in both cases (Figure 24. The electrochemistry of lucigenin has been studied in aqueous solution using both platinumg and mercury'O electrodes. There is agreement that the reaction involves initial 1-electron reduction of lucigenin to the monocation radical at -300 mV vs Ag/AgCl. The radical is then subject to further reaction with a second electrochemical product (possibly peroxideg) to yield CL. Our initial ECL studies of acridinium esters examined the possibility of similar direct electrochemical conversion of the acridinium ester to the emitter, N-methylacridone. Cyclic voltammetry experiments showed no electroactivity for the compound over a range of +l.OOO to -1.OOO V (vs Ag/AgCl). Both in the presence of dissolved oxygen and in nitrogenpurged solutions, current/potential plots for the acridinium ester solution and the supporting electrolyte blank were identical in appearance. The only differencenoted in the four scans wae the disappearance of the oxygen reduction wave in the two purged solution voltammograms. In the absence of direct acridinium ester ECL, we proceeded to show that the chemiluminescent reaction may be initiated via electrochemical generation of hydrogen peroxide. Reduction of oxygen to hydrogen peroxide proceeds readily in basic solution at a negatively biased glassy carbon electrode.ll Visual observation of ECL in the flow cell showed that the light-producing reaction is effectivelyswitched on and off by successive application and removal of peroxide-producing potentials a t the electrode. This fact suggests that the ECL reaction can be initiated at precisely controlled times and in precisely known locations. It should be noted with regard to spatial control, however, that although light emission begins a t the electrode, the lifetime of the reaction is long enough
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ANALYTICAL CHEMISTRY, VOL. 84, NO. 10, MAY 15, 1992
Table I. Relative ECL Intensities for Various Flow Rates, Tubing Lengths, and Tubing Diameters flow rate (mL/min)
tube id. (mm)
tube length (cm)
0.5
1.0
2.0
3.0
4.0
5.0
0.25
22 40 75 145 40 75 145 40 75 145
887 430 45.2 1.52 7.3 1.08 0.56 0.88
3510 2530 646 24.3 138 4.03 0.82 2.52 0.8
7120 4040 1880 239 957 125 13.2 127 9.53
13700 7980 3680 957 2790 583 110 685 54.1 1.19
13500 7090 4730 1270 3260 1130 364 1080 142 3.89
11300 6100 4680 1570 4170 1290 543 1460 242 11.2
0.50
0.75
-
15000
P
2F
0 0.25 A 0.50 0.78
1
0
-1 -1.2
"
'
I
'
-0.8
"
1
"
'
I
-0.4
~
0.0
AppUed Potential, V
Flgure 3. Effect of potentlei and pH on ECL intensities. Solution composition and measurement conditions are described in the Experknentai Section. (0)pH 9.0; (0)pH 10.0; pH 11.0 (0) pH 12.0; (A)pH 13.0.
m
that the acridinium ester reagent flows out of the cell before light emission is complete. Figure 3 illustrates the results of FIA-ECL experiments in which ECL emission was monitored as a function of both applied potential and reaction pH. As the applied potential is stepped in the negative direction, ECL emission increases to a plateau region which corresponds to the peroxide plateau of electrochemical oxygen reduction. When ECL is measured over this potential range using deaerated samples and mobile phase, the ECL "wave" is absent. This result, coupled with the absence of acridinium ester electroactivity at these potentials, indicates that the ECL reaction is in fact initiated by the presence of electrochemically generated peroxide. The shift in the half-wave potential to more negative values at lower pH is a function of the buffer system employed (0.15 M borate) and is in agreement with the electrochemistry of Oz/Hz02as has been explained in the literature.12 Subsequent experiments were conducted with the glassy carbon working electrode at -1.OOO V. From Figure 3 it is seen that the ECL intensity increases going from pH 9 to pH 11or 12 and then decreases to pH 13. The reason for this behavior is that pH affects several processes: loss of acridinium ester to form the pseudobase, electrochemical generation of H202,and the CL reaction itself. Over the pH range studied, the acridinium ester concentration (at any time) decreases with pH and the CL intensity (for constant acridinium ester concentration) increases with pH. The result is the intensity vs pH behavior seen in Figure 3. Solution pH affects electrochemical production of Hz02,11J2 so the profiles of emission intensity vs pH would not be expeded to be identical for use of solution addition of H202and electrochemical generation of H202over a broad pH range. However, over the pH 11-13 range, which is the analytically
2
4
6
Reaction Tfme k c ) Flgure 4. Effect of changing analyte/buffer reaction time.
useful region, those two emission intensity vs pH profiles are essentially the same. For the flow system employed here, we find pH 12 to be optimum for acridinium ester ECL. In our ECL experiments, electrogenerated H202concentrations are less than 0.5 mM, but conventional triggering of acridinium ester CL uses H202concentrations over 10 times greater. Recent work has shown that the optimum pH and Hz02 concentrations for acridinium ester CL are not independent, but are inte~elated,'~ and conventional use of acridinium ester CL is typically reported at around pH 13. FIA-ECL. The use of flow injection analysis (FIA) in association with the ECL measurements facilitates greater sample throughput and generates very reproducible observation times. It is essential, however, that the appropriate observation time window be established experimentally. Two times of importance are the delay time between sample injection and entrance of the sample into the observation cell and the residence time of the sample bolus within the observation cell. Because the volume of the observation cell was fixed, the flow cell residence time is inversely proportional to the flow rate. The delay time after mixing is inversely proportional to flow rate and directly proportional to the tubing volume between injection and observation. Hence, optimization of the FIA system involved variation of flow rate and the volume between the injector and the flow cell. The singlestream design of the FIA apparatus requirea that the analyte be injected into a buffer stream that defines the reaction pH. Diffusional mixing occurs after injection and prior to the observation in the electrochemical flow cell. Since acridinium esters form their pseudobase (nonchemiluminescent) form upon contact with alkaline solutions, the amount of time between injection and observation becomes a vital parameter. Table I is a summary of results from experiments in which this delay time was varied by changing flow rate, tubing length, or tubing diameter. All values represent the ECL intensities observed for injection of 500 nM phenyl acridinium-9carboxylate into a pH 12 (0.15 M phosphate) carrier stream.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 10, MAY 15, 1992
0
1149
l o B ~ h W s d 1 Flgwe 6. Working curve for lysine labeled with 4-(2-(succlnimldyC oxycarbonyl)ethyl)phnyl l O - m e t h y l a c r M i n i u m - g ~ ~ ~ t e .
Amount w e d (femtomolea) Flgura 5. Working curve for phenyl acrMlnium9-carboxylate with CL Initiated electrochemically and by conventional solution additlon of
hydrogen peroxide. Figure 4 shows the intensity va time response associatad with these data. It can be noted that regardless of the mechanism for changing the delay time, emission intensity incream with a decrease in delay (or pseudobase formation) time except at the higheat flow rates. As the flow rate becomes very fast (for any set of tube parameters), the residence time in the flow cell becomes shorter. Because the CL reaction is somewhat slow (recall the visual observations), a decreased flow cell reaidence time results in measurement of lower emission intenaities. All of these observations are consistent with the necessity to minimize the delay time without unduly shortening the flow cell reaidence time. A plot of peak area (instead of peak height) va reaction time yields the identical shape as that shown in Figure 4 and therefore supporh the same conclusion. Because this study was done at constant pH (and thus there could be no pH-dependent variation in the electrochemical generation of HzOz), these observations and conclusions are the same as would be reached with conventional solution addition of HzOz,so have more general applicability than simply to the electrochemical generation of acridinium ester CL as described here. Figure 5 (top) shows the log-log working curve obtained for phenyl acridinium-9-carboxylateusing 22 cm of 0.25mm4.d. PEEK tubing and a flow rate of 3 mL/min (corresponding to a delay time of 0.22 s and a flow cell residence time of 0.2 8). Also shown in this figure is a working curve generated in the same apparatus using 10 mM HzOzin the same pH 12 phosphate buffer carrier stream solution to initiate the CL reaction. Visual inspection shows the two working curves to be essentially identical in shape, slope, dynamic range, and detection limit. Over the 10-lLIO-ll-molrange the
least-squares values for the slopes (with standard error of the estimate values) are 0,924 (0.026) for solution HzOzand 0.967 (0.065) for ECL; within the precision of the measurements, the two slopes are equal. Also shown at the bottom of Figure 5 is the 0-100-fmol section of that working curve presented on linear axes; averaged over the entire 1-fmol to 10-pmol linear range, the ECL intensities are 65% of the CL intensitiea for solution HzOP Added Surfactant. It has been shown that surfactants greatly enhance the emission intensity of the acridinium eater CL reaction in aqueous sol~tion.'~In our ECL experiments, addition of 2 mM cetylammonium bromide to phenyl acridinium-9-carboxylatesolutions exhibited a 2-fold increase in CL intensity which is in agreement with previous work using conventional addition of H2O2.l4 The critical micelle concentration for CTAB is 0.94 mM.16 Labeled Analytes. Application of acridinium ester ECL to immunoassay requires that this detection method be compatible with detection of acridinium ester-labeled analytes. Toward this end, the ECL response of 4-(2-(succinimidyloxycarbony1)ethyl)phenyllO-methylacridinium-Q-carboxylate fluorosulfonate and lysine labeled with this ester were examined. In order to achieve the lowest possible detection limits and to minimize variation in sensitivity between various labeled analytes, it is preferable to strip the label from the analyte prior to CL detection. This w a accomplished by acidifying the sample with 0.1 M HN03prior to measurement in order to achieve hydrolysis of the coupling bond. Figure 6 shows the working curve obtained for the ester-labeled lysine species. Due to the acid hydrolysis step the working curve for the ester-labeled lysine (Figure 6) is essentially identical to that for the acridinium ester itself (Figure 5). Without the acidification step, ECL signals were over 1000-times lower; the fact that this prehydrolysis signal is so low is good indication that the coupling reaction between lysine and the N-hydroxysuccinimide acridinium ester proceed quantitatively. The detection limit for ECL detection of acridinium ester-labeled lysine is 10 fmol injected. Over the 0.03-Wpmol range a pooled estimate (based on seven data points each replicated 6 1 3 times) of the relative standard deviation for replicate injections is 4.2% .16J7 Conclusions. We have demonstrated that electrogenerated chemiluminescence of acridinium esters is a viable approach for quantitation of those esters whether free or as a label. Electrochemial generation of the hydrogen peroxide neceaeary for CL reaction within the observation cell eliminates the need for addition of unstable peroxide solutions and additional solution streams. The electrode further yields a greater degree of spatial and temporal control over reaction initiation. Use of acridinium ester ECL in conjunction with FIA allows for handling of small samples and should facilitate easier automation of complicated assays. With respect to added sur-
Anal. Chem. lQQ2,6 4 , 1144-1 153
1144
factant and labeled analytes, the behavior of the system appears to parallel that of the solution-initiated reaction.
ACKNOWLEDGMENT This research was supported by grants from Merck Sharp & Dohme Research Laboratories and from the Biotechnology Research and Development Corporation. We also thank Bioanalytical Systems for the loan of a potentiostat used in the early phase of this work. Registry No. Cetylammonium bromide, 68810-16-2; phenyl lO-methylacridinium-9-carboxYlate, 123632-55-3; 4-(2-(succinimidyloxycarbony1)ethyl)phenyl 10-methylacridinium-9carboxylate, 87198-88-7. R li!li'RR li'.NP.li!!S L l Y I YIlYI. V Y U
Weeks. I.; Beheshtl, I.; McCapra. F.; Campbell, A. K.; Woodhead, J. S . Clh. Chem. 1983. 29. 1474-1479. Weeks, 1.; Sturgess, M.;Brown. R. C.; Woodhead. J. S. Methods EnZymOl. 1988, 133, 366-387. McCapra, F.; Beheshtl, I . Bldomnescence end Chemllumlnescence: Instnrments and Appllcetions;CRC Press: Boca Raton, FL, 1985, pp 1, 9-42.
(4) McCapra, F. Acc. Chem. Res. 1878, 9 , 201-208. (5) Llu, H.; Yu. J. C.; Blndra, D. S.; Wens. R. S.; Wilson. G. S. Anal. Chem. 1981, 63, 666-669. (6) Downey. T. M.; Nleman, T. A. Anal. Chem. 1802, 64. 261-268. (7) VanDyke, D. A., Ph.D. Thesls, University of Illlnois at Urbana-Champalgn. 1986. (8) Nieman, T. A. Mkfochlm Act8 W88, 111, 239-247. (9) Haapakka, K. E.; Kankare, J. J. Anal. Chlm. Acta 1981. 130. 415-4 18. (10) Murphy, R. J.; Svehla, G. Anal. Chlm. Acta 1081, 125, 73-83. (11) Taylor, R. J.; Humffray, A. A. J . Electraenal. (2".Interfaclel Elechochem. 1075, 64, 63-84. (12) Taylor, R. J.; Humffray, A. A. J . Elechoanal. Chem. InterfeclelElech o d " . 1875, 64, 95-105. 63, 586-595. (13) Hage, D. s.; Kao, p. C. Anal. Chem,, (14) Bagazgohia, F. J.; Garcia, J. L.; Diequez. C.; Weeks, I.; Woodhead, J. S . J . Biolumln. Chemllumln. 1988, 2 , 121-128. (15) Taylor, D. W.; Nleman, T. A. Anal. Chem. 1084. 56, 593-595. (16) Skooa. D. A.: West. D. M.: Holler. F. J. Fundementals of AnaMicel CheGshy, 5th ed.; Saunders: New Yolk, 1988 pp 25-26. (17) Box, G. E. P.; Hunter, W. 0.;Hunter, J. S. Statistics for Ekperhentters, Wilav. ....-, . Naw ..-.. Vnrk . _..., 197R. .- . -, nn rr 76 . and -. .- 919 .-.
-
RECEIVED
-
for review September 307 lggl*
6, 1992.
Vaporization and Atomization of Lead and Tin from a Pyrolytic Graphite Probe in Graphite Furnace Atomic Absorption Spectrometry Glen F. R. Gilchrist, Chuni L. Chakrabarti,*Jeff T. F. Ashley, and Dianne M. Hughes Centre for Analvtical and Environmental Chemistrv. DeDartment of Chemistry, Carleton University, Ottaua; Canadd KlS 5B6
A novel technique has been used to lnvestlgate the proceeses of vaporlzatlon and atomlzatlon of lead and tln In graphtte probe furnace atomic absorption rpectrometry. Udng this technique lt ha8 been found that, for lead and tln, vaporlzatbn and atomlzatlon are separate processes and lead and tin are vaporized a8 molecular specks. Furthermore, rate constants and acthratlon energies for the vaporization of the molecular specks of lead and tln have been detennlned. The acthratlon energies for vaporization have been found to be 86 f 4 kJ mol-' for the lead molecular specles and 120 f 6 kJ mol-' for the tin molecular specles. It Is wggested that these acthratlon energks represent the energy for desorption of PbO and SnO from the graphlte surface. Experhrental resulte have been compared wlth those predicted by an earlier model based on homogeneow gas-phase thermodynamic equlllbrlum. The etfects d the gas-phase chemlcal modifiers, H, and 0,, on the atomlc absorption a n a l proflk d lead and th are presented and dlscu88ed. Atomlzatlon meChanl8m8 have been proposed for lead and tin and compared wlth tho8e proposed by other Workers. PbO and SnO have been proposed as the mort Ilkely gaseous molecules of lead and tin, respectively, formed by vaporlzatlon.
INTRODUCTION Several have studied mechanisms of formation and loss of analyte atoms in graphite furnace AAS. Fuller2% *To whom all correspondence should be addressed.
has introduced the kinetic approach to predict the shape of copper atomic absorption signal profiles. The kinetic approach2s3is based on the supply and removal of atoms by consecutive first-order reactions. Thus, the rate of change in the number of gas-phase atoms is equal to the difference between the supply function and the removal function. Sturgeon et aL4have proposed a combined thermodynamic and kinetic model and have used the leading edge of the atomic absorbance signal profie to determine the activation energy of the rate-determining step for atom formation. To elucidate reaction mechanisms, Sturgeon et al.' have compared experimental activation energies with the standard enthalpy of some appropriate reactions. On the basis of the standard enthalpy of reaction, they4 have concluded that lead and tin are atomized direct from the surface of the graphite atomizer after PbO(1) and SnO(s) have been reduced by carbon at high temperatures to Pb(1) and Sn(s). L'vov et aL5 have used a kinetic approach and have obtained results similar to those of Sturgeon et al.4 for atomization of lead and tin. Smeta6has applied a kinetic approach which has allowed use of the whole atomic absorbance signal profie to determine the activation energy of atom formation process. Sturgeon and Arlow' have used a kinetic approach, similar to that of Smetd to investigate vacuum atomization and have concluded that PbO(g) is reduced on the wall of the graphite atomizer to yield Pb(g) and CO(g). Using the kinetic approach, Akman and co-workers8 have proposed that the condensed-phase lead monoxide is vaporized directly from the graphite surface and is then dissociated in the gas phase. The kinetic approach has proved useful in predicting the behavior of the atomic absorption signal profdes of many elements under a variety of conditions.
0003-2700/92/0364-1144$03.00/00 1992 American Chemical Society
