1746
Anal. Chem. 1985, 57, 1746-1751
Photoelectroanalytical Chemistry: Electrochemical Detection of a Photochemically Active Species, Tris(2,2’-bipyridine)ruthenium( I I) J. M. Elbicki, D. M. Morgan, a n d S. G. Weber* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
The sultablllty of the molecule trls( 2,2’-blpyrldlne)ruthenlum(11) (Ru(bpy):+) for use as the bask for a derlvatlzlng or labellng reagent Is under conslderatlon. Four Co( I I I ) complexes were tested for use as electron-transfer quenchers. The most analytlcally useful of the four Is Irls(glyc1nato)cobalt. Wlth thls quencher, the photooxldatlon/electrode reductlon cycle of Ru(bpy);+ was studied. Experimental dependences of photocurrent on llght Intensky, quencher concentratlon, and Ru(bpy):+ concentratlon are slmllar to, but not exactly, that predlcted for a slmple photosensltlve CE (regeneratlve) or “catalytic”mechanism. Studles of the flow rate dependence of the photocurrent demonstrated an apparent Increase In the effective rate constant for the photooxidation of Ru(bpy),*+ as the concentratlon of Ru( bpy):’ decreased. An electron transfer mediator, Ru(CN),~-, was used to mlnlmlze the Influence of slde reactlons of Ru(bpy)?+.
Photoelectroanalytical chemistry (PEAC) is the use of molecules that undergo excited-state electron transfer in analysis. This group (1)and that of Krull(2) are attempting to understand the usefulness and limitations of this technique. Electroanalytical investigations of photochemical reactions have been carried out for more than a decade notably by Johnson ( 3 , 4 ) ,Perone ( 5 , 6 ) ,and Albery (7-10). Since the now landmark report of Gafney and Adamson (11)on excited-state electron transfer reactions involving tris(2,2’-bipyridine)ruthenium(II) ( R ~ ( b p y ) ~ ~there + ) , has been a large body of work (recently reviewed (12)) on this and similar reactions. This paper addresses the feasibility of using these reactions in analytical systems. There are two groups of analytical procedures that may benefit from this novel approach. On one hand, one may wish to detect molecules that have intrinsic photoelectrochemical properties, after their separation by chromatography. Alternatively, one may choose to employ a particularly attractive photoelectrochemically active species as a specific label or derivatizing agent in chromatography or in binding assays, such as immunoassay. For either type of application, one must know how best to employ the chemistry involved. The work presented here is intended to explore the feasibility and limitations of the detection of a particular photoelectrochemically active molecule, Ru(bpy)32+. This molecule was chosen because it is well studied, stable, and available. The general conclusions that result from this work will be applicable to virtually any solute if the mechanism of photocurrent production is the same as that of the system studied. It is also true that a suitably derivatized R ~ ( b p y ) will ~ ~ +be useful as a label. Indeed it has recently been suggested that this molecule be employed as a chemiluminescent label (13). The reactions important to signal generation are given as eq 1-3c (D is the donor molecule; for this work D is Ru(b~~)3’+). 0003-2700/85/0357-1748$01 SO10
D
kph
Q + D* E !-,
D* A
D*
D+ + Q-
(24
D
(2b)
kl
+ D* .A, D + A*
-k + -+
D+
ke
+ D+ kr In + D+ Q-
D
D
D
(24 (3a)
Q
(3b)
In+
(3c)
D is excited with a first-order rate of k p h = (2.303 X 103)Ioe in the low absorbance limit (14). Since the concentrations of sensitizer D employed is usually micromolar or below, this is a valid approximation. Io is the photon flux (einstein cm-2 s-l) and z is the molar absorptivity (M-l cm-’) of D. There are three possible fates of D+ given by eq 2a-c. Equation 2b represents radiative and nonradiative decay with k1 = 1/7 + k,,, r being the excited state lifetime and k,, being the rate of decay due to nonradiative processes. Equation 2c represents energy transfer quenching and eq 2a represents electron transfer quenching. The latter reaction is of most interest in the present context. If the thermal back reaction of D+and Q- (eq 3b) is slow, or if Q-is unstable, then D+ may survive to reach an electrode surface where it may be reduced (eq 3a) yielding a photocurrent. Since the photocurrent is the analytical signal, it is worthwhile to consider its production. Using the steady state approximation for D*, one can combine reactions 1,2a-c, and 3b to yield kefi
D-D++Q-
(4)
where
-
as defined by Wight et al. (14). The efficiency of populating the luminescent state is 4’ (4’ 1 (15)). If more than species A and Q are present, or if A or Q quench by both processes, then suitable terms in the denominator must be present. Equation 5 is appropriate for optically dilute solutions. The absorbance due to Ru(bpy),2+does not exceed -0.02 in cases where eq 5 is employed. The absorbance due to quencher may be as high as 0.05 which yields an 11%decrease in kph from that calculated based on photon flux measurements made outside the cell. Since the slow step in the process is the diffusion step, the effect of the thermal back reaction will be to lower the efficiency of D+ formation. The fraction of D+ formed that survives by either escaping from Q- before reaction (“cage@ 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
1747
escape”) or due to Q-decaying to an unreactive species a t a rate k , is f
D -kD+ + Q-
(6)
Q(l-f)D+-D
(74
f D + Z D
(7b)
Iff = 1,then this mechanism is analogous to the EC catalytic mechanism, except that it begins with a “C” step and has a lighbdependent k. One goal of ow work, then, is to determine how closely the production of photochemical signal can be described by the catalytic mechanism. The theoretical basis for a hydrodynamic EC mechanism at a channel or thin-layer detector has been given by Aoki et al. (16). They give an expression for catalytic current, I , divided by normal Faradaic current for the reaction in the absence of a catalyst, Id, as a function of a dimensionless variable A
I/Id = f(A)
-
(8)
As the catalytic step becomes negligible, f ( A ) 1. In our system, eq 6 and 7, the first step in the reaction is the “catalytic” step. I t is easily shown that in this case
I/ld = f(A) - 1
(9)
We have used eq 9 in our theoretical analysis. When the catalytic reaction is very fast, then the current becomes independent of the hydrodynamics (reaction layer thickness < hydrodynamically assisted diffusion layer thickness) and one obtains
I = IZFCOA(~,@)’/~
(10)
the well-known expression for steady-state catalytic current (17). I in pA is related to n,the number of electrons involved, F is Faraday’s constant (9.65 X lo4 pC/pmol), C” is bulk concentration of sensitizer (pmol ~ m - ~A) ,is the electrode area (cm2),keffis the pseudo-first-order rate constant (s-l) described in eq 5, and D is the diffusion coefficient (cm2 s-l). In order t o increase the catalytic current generated based on this equation, one would predict that a large electrode area and large k,ffare necessary with a direct relation to area but square root dependence on k& Notice that the steady-state catalytic current is independent of the solution velocity. All of these factors must be considered in designing a detector that can be analytically useful. The chemistry of the system will determine quenching reaction rates, lifetime of excited state, and molar absorptivity, while the physical parameters of the detector will determine how much analytical signal is obtained from a certain light flux and electrode area. In addition to the chemical and physical aspects of the detection that determine sensitivity, selectivity may be gained over nonphotochemical reactions by using a modulated light source and a phase sensitive detector to monitor the modulated photocurrent.
EXPERIMENTAL SECTION Reagents. R ~ ( b p y ) ~ C l ~ ~was 6H~ purchased O from Strem Chemicals (Newburyport, MA) and was used without further purification. [(NH3)&oCI]C12,pentaamminechlorocobalt(II1) chloride was puchased from Alfa products (Danvers, MA) and was recrystallized (18)before use. K,[Co(C2O4),].3H20,potassium tris(oxa1ato)cobaltate was prepared by the method from Booth (19) and recrystallized twice from ethanol. The amino acid complexes of cobalt(III), tris(glycinato)cobalt(III), and tris(cysteato)cobalt(III) were prepared according to the literature (20) using amino acids from Research Plus Laboratories, Inc. (Denville, NJ), and Sigma Chemical Co. (St. Louis, MO), respectively. The tris(cysteato)cobalt is a complex of Co(II1) with cysteic acid. All
“t Figure 1. Cross section of PEAC cell: (a) inlet; (b) channel (defined by a spacer for width and height); (c) glassy carbon e l e c t r m (d) Teflon body; (e) polypropylene container; (f) stainless steel auxiliary electrodes; (9) glass windows.
K3C~(CZ04)3 solutions were kept in low actinic glassware or foil wrapped containers to prevent decomposition from room light. Potassium hexacyanoruthenate(I1) (Alfa Products, Danvers, MA) solutions were prepared daily in deoxygenated solutions. All other reagents were reagent grade chemicals from Fisher Scientific (Pittsburgh, PA). Water used for all solutions was deionized, passed through an organic removal cartridge, and then distilled. Apparatus. The pumping system consisted of a Constametric I11 reciprocating piston pump (Laboratory Data Control, Riviera Beach, FL) driving a Teflon piston in a glass fluid displacement chamber (built in-house to eliminate contaminants from organic or metal impurities in the fluid pumping system (21)). A syringe pump (Raze1 Scientific Instruments, Stamford, CT) could alternatively be used as the fluid pumping system but the flow noise is greater. After the pump, a Teflon six-port rotary valve (Rheodyne, Inc., Cotati, CA) with a 100-pLsample loop was used for injections. Immediately after the injector was an on-line vacuum degassing chamber (22),followed by the detector. The detector was constructed in-house to take advantage of the R~(bpy),~+-quencher chemistry. See Figylre 1. A rectangular glassy carbon electrode (15 mm X 2.5 mm) (Tokai Manufacturing, Tokyo, Japan) or a platinum disk electrode (r = 0.8 mm) (BAS, West Lafayette, IN) was glued into the face of a 1 in. diameter Kel-F disk mounted on a Teflon plug. The Kel-F disk has been omitted from Figure 1to avoid confusion. This cylindrical plug could then be used to hold the electrode while polishing or to anchor the electrode while in the detector. The plug containing the electrode is placed inside a hollow cylindricalpolypropylene detector body. A spacer is placed in the cell and a second cylindrical plug, this one containing the auxiliary electrodes, sandwiches the spacer, defining the channel width (2.5 mm) and height (50-500 pm). The detector body is equipped with entrance and exit windows for the light beam. Solution enters the detector and hits the electrode in the center and then flows along the channel and exits on either side of the electrode. The detector has a “wall-jet”configuration, but with the thin spacers employed, it behaves as a thin-layer detector (23). After leaving the detector, solution flows into the reference electrode chamber and on to waste. Signal monitoring was done amperometrically with a Bioanalytical Systems (West Lafayette, IN) LC-4 potentiostat or a PAR 174A Princeton Applied Research polarographic analyzer (Princeton, NJ). This signal was passed through an 852-01, eight-pole butterworth filter (Wavetek-Rockland,Inc., Rockleigh, NJ) before going to a stripchart recorder or a microcomputer (DEC LSI-11/03 Maynard, MA). Light was provided by a 40-mW, 441.6-nm HeCd CW laser (Liconix, Sunnyvale, CA). Dielectric or silvered front-surface reflectors were used to direct the beam into the detector. Modulation of the light beam was done with a shutter arm on a high torque motor, with the controller built in-house. The controller was driven by an external square wave pulse and could drive the shutter arm in the low frequency range (0-5 Hz) which is not easily accessible to normal choppers. When light modulation was used, a lock-in-amplifier (LIA) was placed after the potentiostat to detect the frequency dependent signal. The LIA’s used were either a Keithly Instruments 840 Autoloc (Cleveland, OH) or an Ithaco, Inc., 393 (Ithaca, NY).
1748
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
Focusing of the light was done with cylindrical plano-convex lenses of various focal lengths. These lenses were mounted in a holder (J. A. No11 Co., Pittsburgh, PA) that allowed rotation of the resulting wedge of light. The detector was fastened to a rotary table (Newport Research Corp., Fountain Valley, CA) that was mounted on an X-Y-Z positioner (J. A. No11 Co.) with the entire assemblage attached to an optical rail (Oriel Corp., Stamford, CT). This assemblage allows the electrode to be accurately positioned with respect to the light beam, as well as to make adjustments for different focal length lenses. Procedure. Before starting experimentation, a new electrode is sanded and polished with successively smaller grits until a final 0.5 wm diamond polish yields a flat, apparently scratch-free surface. The electrode is then washed with water and wiped with a piece of lens paper which was soaked in methylene chloride. An electrode that has already been used and has had materials adsorbed onto it can simple be wiped clean using the methylene chloride soaked paper. Once the electrode has been cleaned, the detector is assembled and placed on the rotary table. After the potentiostated electrode has relaxed, it is aligned with the laser beam until maximum analytical signal is acquired. The carrier solution is typically a 0.1 M acetate buffer at pH 5.0 and 1 mM in quencher. Injections of 100 rL of Ru(bpy)gP+ in variou matrices that have been prepared in the carrier solution can then be made. The analytical signal is recorded in either the dc or ac mode as a peak height and/or peak area. A syringe pump is used to control the flow rate in steady-state experiments in which one solution is continuously passed through the cell and the photocurrent is detected.
RESULTS AND DISCUSSION Many different quencher molecules have been used to oxidize or reduce excited-state Ru(bpy)gP+ (11,12,24-27). For use in producing an analytical signal, however, one must choose a quencher that will allow the largest amount of signal to be produced. A suitable quencher not only will have a large electron transfer rate with the excited state molecules (reaction 2a) but will not undergo thermal back reaction (reaction 3b) or produce any photobackground signal. The thermal back reaction is the reaction of Ru(bpy),3+ and the reduced quencher, Q-. This leads to the production of the original reactants and no production of signal. Initial studies on the analytical detection of Ru(bpy)gP+were done with the quencher tris(oxa1ato)cobaltate (27). This molecule has a high k,, for quenching and was thought to quench irreversibly. Performance of this quencher in high concentrations of Ru(bpy)32+seemed good, but two disadvantages became obvious. First, there was a large photoanodic current from the quencher itself. This large background made detection of small analytical signals difficult. Second, the reduction of tris(oxa1ato)cobaltate yields Co(I1) and free ligand. Since oxalate anions are very reactive toward Ru(bpy)gB+ (27, B), they markedly reduce the concentration of Ru(bpy)? and thus the signal. This led us to examine various quenchers for their effectiveness at producing analytical signal based upon the following qualifications: (1)reduced quencher (or ligands) must not be reactive toward R ~ ( b p y ) , ~ (2) + , background photocurrent must be minimal, (3) fast electron-transfer must occur with Ru(bpy)z+*, (4)quencher should be stable without special precautions (such as deoxygenation or keeping solutions in the dark). Since Co(II1) complexes undergo rapid aquation upon reduction, minimizing the effect of the process in eq 3b, we have examained them exclusively. Several different ligands were examined for the quencher complex in view of their anticipated behavior in regards to the four qualifications listed above. The results are listed in Table I. The Stern-Volmer constant, K,,, is the product of the bimolecular quenching rate constant and the lifetime for the excited state ruthenium complex. Since the latter is a constant, K,, is proportional to ket. Solely on the basis of the Stern-Volmer
Table I. Quencher Attributes
$“
K,:
El,,‘
photocurrent backgroundd S/Nd:
[CO(C,O~)~]~-3800 + [ C O ( N H ~ ) ~ C ~200 ]~+ + Co(gly), 600 -0.05 CO(CYSSO~)~~- 200 -0.05
90 22 32
5.4
SIN,:
+0.2 V. dThe shift in background current (nA) when the light goes from off to on in the analytical cell (Figure 1). “SignalM RuL?+ at conto-noise ratio for 100-pL injections of 5 X stant light intensity. While the physical conditions were consistent for the four experiments, they were not optimized for best detection limit. Noise is measured by integrating the noise power over the measurement bandwidth and taking the square root of that value. The noise data are acquired as base line fluctuations in time; then they are Fourier transformed. fSame as footnote e for a 1 Hz chopped light source with lock-in-amplifierdetection.
Table 11. Effect of Quencher Concentration on ken [ C ~ ( g l y ) ~ ]M, ”
peak area,bp C
keih( s-’
10-2
0.231 0.198 0.033
8.5
10-3
10-4
6.2 0.17
‘Carrier streams were 0.1 M acetate buffer pH 5 with indicated concentration of Co(gly)B. Peak areas determined by planimetry for 100-pL injections of 1 X M Ru(bpy),2’. ‘Calculated from eq 10 integrated over time.
data, tris(oxalato)cobaltate would be the quencher of choice due to its large K,,. The large background photosignal produced by tris(oxa1ato)cobaltate with the resulting high detection limits shows that while a large K,,. is desirable, it is not the only criterion. With its relatively small K,,, tris(g1ycinato)cobalt yields a small photobackground and a large SNR. Thus, this quencher has been used in the majority of experiments. Table I1 shows how kerf changes as the concentration of tris(g1ycinato)cobalt changes. For this table, keBwas calculated from experimentally measured peak areas (coulombs) using eq 10 integrated over time (23),i.e., coulombs as a function of mass injected. These results show that kerf (signal) drops rapidly when quencher concentrations below the millimole per liter level are used. Solutions prepared with the quencher concentration greater than millimole per liter cause kerf to increase but the enhancement is not large enough to warrant the use of higher concentrations of quencher. Such a dependence is predicted by eq 5. All other experiments were done in millimole per liter quencher. Equation 10 predicts a square root dependence of photocurrent on photon flux. For these experiments, it was assumed that the focused laser beam would have the same area regardless of intensity, allowing intensity (einstein s-’ or power) measurements to suffice in place of flux (einstein cm-* s-l) measurements. The log of the signal (nA) obtained for an injection of Ru(bpy),*’ is plotted against the log of light intensity (mW) between 45 mW and 1.6 mW in Figure 2. The plot is linearly increasing at low light intensities, while it flattens above 20-25 mW. Thermal lensing could be causing a defocusing effect which changes the light flux in the reaction layer and thus leads to the decrease in slope. The linear portion of the plot does have a slope of 0.509 0.002 in
*
1749
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8,JULY 1985
Table 111. Concentration Dependence of Rate Constant [Ru(bpy)32+1, M
8-
expt no.
electrode material GC"
10"
.cS
1 2 3
GC GC GC
10"
8
0
4
0
10-8 10-8
Ptb Pt
5 6
0
2.35 f 0.17 3.66 f 0.74 5.25 f 0.90 9.84 f 2.78 5.18 f 0.64 44.2 f 9.0
10" 10-8
Glassy carbon, rectangular electrode, 37.5 mm2. bPlatinum, disk electrode, 2.0 mm2. CErrorsare 95% confidence intervals.
0 I
,
.
.
.
I
.
.
.
,
I
.
.
.
/
. 4~
0.5
0.0
1.0
2.0
1.5
log(Power,mW) Figure 2. log (peak current) (nA) vs. log (light intensity) (mW) for the system shown In Figure 1. CondRions were as follows: HeCd laser light source at 441.6 nm; BAS LC-4 potentlostat at +0.25 V vs. Ag/AgCI (3 M NaCI); glassy carbon electrode, 15 mm X 2 mm; flow, 0.3 mL/min; pumping solution, 1.0 X lo-, M Co(gly), in 0.1 M pH 5 acetate buffer. Samples were 100 pL of 5 X lo-' M R~(bpy),~+ In the same solution as Is pumped. Two experiments were done on different days under similar conditions (0 and 8).
2
1
i
..
c1 0
b
I
0
0
4
,
E
$
2
l
B
2
Q
2
A
2
0
3
2
3
6
4
0
Lambda 0 3 0
-u
-
Flgure 4. Plot of A vs. III, for theoretically predicted data (-) and Pt electrodes experimental data with (+ and X) glassy carbon and (0) for a lo-' M Ru(bpy),'+ solution.
102 10'
3
100
0 L W
4
10-1
Y W O
a.
10-2 10-8
10-7
10-6
10-5
10-4
10-3
C o n c e n t r a t i o n R u ( b p y ) 3 2+ ( M )
Flgure 3. Calibration curves for varying spacer thicknesses: (0) 51 pm; (0) 125 pm; (0) 250 pm; (A)510 pm. Llne drawn has a slope of 1. Injections were 100 pL of R~(bpy),~+(in carrier solution) into a carrier solution of 1 X IO-, M Co(gly), in 0.1 M pH 5 acetate buffer at 0.5 mL/min. Electrode was aligned after each spacer change. The electrode was not polished except before the first run. Electrode potential was 0.25V vs. Ag/AgCI (3 M NaCI); laser power was 16 mW continuous.
dicate that the photocurrent is velocity independent. It is hard to imagine a chemical effect caused by changing b, thus we can only guess that altering b may alter the light distribution within the cell. The results presented so far are in general agreement with anticipated results, but there are discrepancies. In order to understand the system more completely a test of the mechanism was carried out. One of the most important issues is the nonlinearity of the calibration curve since eventual analytical applications are anticipated. Measurements of photocurrent at two different concentrations of Ru(bpy):+, 1.0 X lo4 M and 1.0 X M, were made. Two different electrodes were used, chiefly because they differed in size: glassy carbon (0.375 cm2) and platinum (0.020 cm2). Steady-state photocurrents, I , were obtained as a function of solution flow rate. Steady-state dark currents, Id, for K,Fe(CN), were also obtained as a function of flow rate in order to quantitatively determine the diffusion limiting current. These latter data were within 5% of that predicted by Weber and Purdy (29) for a channel-type thin-layer cell. The dimensionless parameter A in eq 9 is given by
agreement with the predicted square root dependence from eq 10. We had previously found a reproducible, though nonlinear, calibration curve from M to lo4 M injected R ~ ( b p y ) ~ ~ + (I). The relationship between signal and spacer thickness, b, and higher concentrations of Ru(byp)g2+ was tested. where A is the electrode area, 0 is the average flow rate, and Equation 10 predicts a photocurrent independent of spacer D is the diffusion coefficient of R ~ ( b p y ) ~ The ~ + . data (I/Id) thickness and linearly dependent on concentration of Ruwere used to find k,ff knowing 6, A , 0(from physical mea(bpy),2+. The data are shown in Figure 3. Also shown is a surement), D (estimated as 4 X lo4 cm2&), and eq 9. Values line of slope 1for reference. Two things are worthy of note. from this single-parameter nonlinear least-squares fit are given The calibration curves are linear for lo4 M < [ R ~ ( b p y ) ~ ~ + ] in Table 111, and plots of the data are shown in Figures 4 < M, and the photocurrent depends on b. Except for the ( [ R ~ ( b p y ) ~=~ +1.]0 x lo4 M) and 5 ( [ R ~ ( b p y ) ~=~ +1.0 ] x smallest (50 pm) spacer the signal increases (by a factor of M). The values of keff are smaller and more precise for about 1.4)as b decreases (by a factor of 4). A large quantity the 1.0 X lo4 M data than those for the 1.0 X lo-* M data. of scattered light is observable when the small spacer is used The magnitude of the difference in rate constants is statisindicating that in this case, the decrease in the signal is due tically significant. One can estimate about a factor of 2 into a decrease in light intensity. For the other spacers, the crease in k,ff in going from the higher to the lower conceneffect is not hydrodynamic since other data (see below) intration. This is entirely consistent with the experimentally
1750
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
observed relationship (1,Figure 3)
i C"; a = 0.8 (12) The difference in precision can be viewed in the figures. In Figure 4, two of the three sets of data conform to the curve with randomly scattered residuals, whereas for all of the data in Figure 5 the data are simply not well represented by the line of best fit; the residuals when plotted show a distinct pattern. The implication is that the mechanism (equation) is not what we thought it was at low [R~(bpy),~+]. In the absence of further experimentation, the difference between the Pt and C electrodes cannot be understood in detail. These experiments were performed at various potentials, all of which were very reducing compared to Eo(Ru( b p ~ ) , ~ + / ~so+it) ,is fair to view the electrode as an "electron source" and ignore ita chemistry. One can then speculate that the difference is merely due to the fact that the large carbon electrode is not uniformly illuminated, thus the entire electrode is not active in producing catalytic current. This, of course, lowers the observed kefP TOdetermine what kind of advantage has been gained from the Ru(bpy),2+catalytic cycle, the experimental efficiency was compared to the theoretical efficiency calculated from geoM metric considerations (29). A 100-pL sample of 5 x Ru(bpy)?+ (30 mW laser power, 0.5 mL/min flow rate, 0.5 V Eappl,1.4 cm X 0.25 cm electrode, and 510 pm channel height) was injected. The area under the resulting peak was then used to find the number of moles of species reduced at the electrode surface (Q= nF mol). The area (0.43 pC) gives 4.5 X mol seen at the electrode surface. Comparing this value to the actual number of moles injected (5 x mol) gives a coulometric efficiency of 0.9. A normal Faradaic response for this electrode (no catalytic behavior) under the same conditions would give a conversion efficiency of 0.025 (calculated from ref 29 for the particular physical parameters used). Thus, an increase in the signal by a factor of 36 has been achieved by the cyclical nature of the scheme. This can be compared to what is expected from our steady-state measurementa (Figure 5). Using a k,E of 9 (Table 111), one estimates a A of 10 and I/& of 11. The measurement using the integration of the peak yields a value higher than the steady-state measurement. The difference is reconciled if it is realized that the sample is diluted upon injection and that the actual concentration measured during the evolution of the peak is of the order of 10-l' M to 5 x M for the . we obtain more injection of 5 X lop8M R ~ ( b p y ) , ~ +Hence, charge than expected due to the relationship in eq 12; low concentrations yield a higher equivalent signal than high concentrations. Our investigations have not been detailed enough to ascribe a mechanism to explain all of our data. Recent work (30) from the Brookhaven group has shown that Ru(bpy),3+ undergoes several side reactions. In one reaction Ru(bpy)S3+oxidizes a minor form of Co(II), CO(OH)~, resulting in the formation of peroxide and oxygen. R ~ ( b p y ) is ~ ~also + photosensitive (30-32). It may react, from the excited state, with oxygen to yield the superoxide ion (3I),or it may react from the excited state with water (32). These processes may be responsible for the flatness of the photocurrent vs. light intensity curve (Figure 2) at high light intensities. We have previously shown that serum contains species that R ~ ( b p y ) ~can , + oxidize ( I ) . Therefore, it is appropriate to consider means by which the lifetime of the reactive Ru(II1) can be minimized to minimize ita participation in side reactions. The use of a mediator can accomplish this. Scheme I illustrates only three of the many (30)possible reactions of R ~ ( b p y ) , ~ +reduction : at the electrode, reaction with an interferent, In, and reaction with a mediator, M. The mediator reaction can be made to predominate over the others by having the highest rate in com-
Scheme I
p!
Ru(bpy)32t
Ru(bpy)s3+
kICInl
Ru(bpy)32+
+
other products
k,[MI
Ru(bpy)32+
+
Mt
Table IV. Effects of Mediator and Interferent
mediator
+
-d
oxalateb Ru2+ nett
+
-
oxalateb
-
+
-
+
6.P -1.2 7.7
-45
29 9 20
21 -4
-10 -35
25
Mediator concentration 1 X lo-' M Ru(CN)~~-.Oxalate conM H2C204. 'RuZt concentration 1 X 10" M centration 5 X is not in sample. Ru(bpy)Szt. d " + n is present in sample. e Signals are from steady-state concentrations of those species present flowing at 0.3 mL/min. The light on to light off value is shown in nA. Positive signals are cathodic. E,,, = 0.250 V vs. Ag/AgCl (3 M NaC1). Laser power 40 mW. 'Signal attributable to the presence of Rdt. U-n
parison to the other parallel paths. Characteristics required of a mediator are given by Fultz and Durst (33) in their compilation of mediator compounds. In addition to those given by them, three further constraints must be adhered to for PEAC detection. There must be no quenching of excited state Ru(bpy):+, the reduction potential of the mediator must be below that of Ru(bpy),,+, and there should be no mediator photosignal. Advantages gained by use of a mediator are that (1) a less powerful oxidant is formed (EO(M+/O)< E O ( R U ( ~ ~ ~ ) , ~ + / ~ + ) decreasing the driving force for an unwanted reaction with an interferent so that it becomes more likely that a signal producing electron transfer will occur, (2) more cycling of Ru(bpy)?+ can take place since the mediator is in such excess that it will react with Ru(bpy),,+ before it diffuses to the electrode surface. The mediator that has been under investigation is the hexacyanoruthenate(I1) complex, Ru(CN)*-. The specific advantage of this mediator is that the oxidizing power of Ru(CN)l- (Eo = 0.97 V vs. NHE) is 0.3 V lower than that of Ru(bpy):+ (Eo = 1.26 V vs. NHE). This should help to lower the amount of Ru(CN)& reduction by interferents. RU(CN)~"is capable of reductively quenching R~(bpy),~+*, but the rate is -10' M-l s-l (I2),1 to 2 orders of magnitude lower than that for the Co(II1) quenchers used; therefore it is only of minor importance. Steady-state signals from a solution containing a known interferent, (oxalate), mediator, (Ru(CN)*-), and Ru(bpy),'+ were compared in a three-factor, two-level experiment. These resulta are given in Table IV. Without mediator, blank signals with and without oxalate are anodic, with a significant increase when oxalate is added. When R u ( b p ~ ) , ~was + added to the solutions, cathodic (reductive) current was obtained with no oxalate, but large anodic current was obtained with oxalate. The evidence suggests that oxalate is reacting with Ru(bpy):+ and producing another species that is oxidizable at the electrode (COz.?) (13,27,28). The same four experiments were then done with mediator included. Solutions without oxalate show an increase in signal for the blank (cathodic due to photosignal from the mediator), as well as an increase in cathodic signal due to Ru(bpy),2+. When oxalate is added the blank is anodic (as it was in the experiment without mediator), but of smaller magnitude. The analytical signal, however, is still cathodic, and about the same as that without the oxalate.
ANALYTICAL CHEMISTRY, VOL. 57,NO. 8,JULY 1985 14
P
Lamb08
Figure 5. Same as Figure 4 except a lo-*M Ru(bpy);+
solution was
1751
in deaerated solutions (N, sparging) and kept blanketed with N2 between use. This mediator is adequate for proof of principle for the advantage gained by use of a chemical method for enhancing selectivity in photoelectrochemical detection. A better mediator is required, however, so that some of the major disadvantages of Ru(CN),~-can be eliminated. This technique can be useful for doing precolumn derivatization in applications involving liquid chromatographic separations where small quantities of analyte need to be determined or for use in an immunoassay where R ~ ( b p y ) , ~is+ used as a photochemical label. Work on the synthesis of a complex R~(bpy),(bpy')~+ is currently under way in our laboratory where (bpy') is a bipyridine ring that has been substituted to allow attachment to molecules of interest. Extension to molecules other than the ruthenium complexes is possible.
used.
ACKNOWLEDGMENT
Table V
[Med]" 1x 1x 4x 4x 1x 1x
10" 10-6 10-6 10" 10-6 10-6 3 x 10" 3 x 10-6
[ R ~ ( b p y ) ~ ~ + ] "photocurrentb 0 4 x 10-8 0 4 x 10-8 0 4 x 10-8 0 1 x 10-8
1 39 I 40
We wish to thank BAS, Inc., for the donation of one of the potentiostats used in this work. Registry No. Ru(bpy),2+,15158-62-0; [CO(C~O~)~]*, 15053-34-6; [C0("3)&1]~+, 14970-14-0;Co(gly)3,14221-43-3;CO(CYSSO~):-, 96445-27-1; RU(CN)B~-, 21029-33-4.
LITERATURE CITED
6.5 58 30 48
Concentrations (M) indicated were flowing through detector at 0.3 mL/min in the steady state. bSignalis steady-state photocurrent minus steady-state dark current from light going on to off, in nA.
In the presence of mediator, the net analytical signal not only is preserved from the interferent, but is actually enhanced. Therefore, a greater selectivity is obtained with the mediator as well as a gain in signal. There are several disadvantages of this mediator. One problem that limits its usefulness is that there is a significant photosignal from the mediator and quencher alone. Thus, the concentration of R u ( C N ) ~ must ~ - be optimized in order to obtain effective mediation with an acceptable background signal. The photosignals for R u ( C N ) ~ and ~ - quencher are shown in Table V. On the basis of these results, the concentration of mediator can be no larger than 1X lo* M, where there is a tolerable photobackground with good signal enhancement. This concentration was used in most mediator experiments (including the oxalate experiment, above). Another problem with this mediator is the instability of R u ( C N ) ~ (34). ~ - At high concentrations, M or M, electrode response to solution changes was irreproducible and sluggish. Visual inspection of the electrode after this had occurred revealed that there was a thick layer on the surface, possibly a ruthenium analogue of Prussian blue (35). This layer could not be rinsed off with water but was readily removed with methylene chloride. When the electrode was wiped clean with a Kimwipe soaked in methylene chloride and the electrode assembly was reinserted into the detector, the response was returned to its normal character. This response showed that a reasonable concentration of mediator could not be allowed to flow through the system continuously since the prolonged contact at the electrode would result in the same sluggish response. If mediator was injected with each sample instead of being continuously pumped through the cell, good electrode response was maintained for a week. One other minor problem with RU(CN),~is that it is slowly oxidized by air. All solutions containing Ru(CN)~" were prepared fresh and used immediately, or they were prepared
(32) (33) (34) (35)
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RECEIVED for review June 18,1984. Resubmitted April 8,1985. Accepted April 8,1985. It gives us pleasure to acknowledge the support of the National Institutes of Health through Grant GM 28112.
