2944
Anal. Chem. 1986, 58, 2944-2949
Asymmetric Double Potential Step Chronoabsorptometric Determination of Heterogeneous Electron Transfer Kinetic Parameters Eric E. Bancroft’ and Henry N. Blount*2
Brown Chemical Laboratory, T h e University of Delaware, Newark, Delaware 19716 Fred M. Hawkridge*
Department of Chemistry, Virginia Commonwealth University, Richmond, Virginia 23284
An asymmetrk double potential step chronoabsorptometric (ADPSICA) method for the determination of heterogeneous electron transfer khetlc parameters for redox systems exhibiting any degree of reversibility is described. This method complements the previously reported single potential step chronoabsorptometric technique by permitting the direct evaluation of the heterogeneous kinetics of the back electron transfer reaction from measurements made using the precursor for the forward reaction. The approach presented here utilizes dimensionless parameter working surfaces and has been verried by determination of the heterogeneous electron transfer kinetics for the reduction of ferricyanide in pH 7.00 solutions of ferrocyanide at fluorlde-doped tin oxide optically transparent electrodes. The resuHs obtalned by ADPS/CA ( l ~ , , ~= ” 5.45 (f0.05) X cm/s, a = 0.289 (f0.004)) are in excellent agreement with those independently determined by single potential step chronocoulometry for the dlrect reduction of ferricyanide (ks,ho’= 5.07 (f0.04) X lo4 cm/s, N
= 0.304 (f0.004)).
Single potential step chronoabsorptometry (SPS/CA) has been implemented for the determination of the heterogeneous electron transfer kinetic parameters of irreversible ( 1 ) and quasireversible (2) redox systems. This technique was developed to exploit the advantages of spectroelectrochemistry (3, 4 ) in such determinations. A limitation of any single potential step (SPS)method, either spectroelectrochemical or purely electrochemical, that arises in the study of a quasi-reversible redox reaction is the limited potential range over which heterogeneous electron transfer kinetic parameters can be reliably evaluated ( 2 ) . This is particularly problematic in the determination of the kinetic parameters of the “back” reaction. For the heterogeneous electron transfer reaction given by
SPS experiments carried out on solutions of 0 give rise t o observed responses that are dominated by the kinetic parameters of the “forward’‘ (0 to R) reaction. Although the analogous parameters of the “back” (R to 0) reaction do influence the observed response, their impact, like the flux of the back reaction, is small. When both members of a redox couple are readily available in chemically stable forms, separate reductive and oxidative single potential step experiments Present address: Dow Chemical Co., Midland, MI 48640. *Present address: National Science Foundation, Washington. DC 205.50.
can be carried out yielding responses that reflect principally the kinetics of the “forward” and “back” reactions, respectively. In this way, the dependencies of kfh and hb,hon potential, given by k,, = k,,ho’ exp[-unFa/RT]
can be precisely determined. In the Butler-Volmer formalism (5-7) shown in eq 2 and 3, the forward and back heterogeneous electron transfer rate constants are functions of the formal heterogeneous electron transfer rate constant, ks,ho’, the electrochemical transfer coefficient, a , and the overpotential 7 (7 = E - EOIRo’). The foregoing protocol for the “independent” evaluation of forward and back heterogeneous electron transfer kinetic parameters is inapplicable to many experimental systems, however. Both members of a redox couple are not always conveniently available and in situ electrochemical preparation of the complementary member by exhaustive electrolysis can be disadvantageous (8). Also, both members of a redox couple will not always exhibit experimentally accessible optical absorption bands such that their formation can be monitored. If a decrease in the absorbance of the precursor of the electrode reaction must be monitored, then the small diminution of the absorbance will give rise to measurements having poor signal to noise ratios (9). It is therefore desirable to have an experimental protocol that permits the independent evaluation of both “forward” and “back heterogeneous electron transfer kinetic parameters of redox systems (eq 1)but that requires the availability of only one form of that redox couple. The foregoing requirements can be met by the use of double potential step (DPS) methods. The DPS approach utilizes an initial potential step that drives the electrode reaction in one (e.g., forward or back) direction. A potential step back to a value that either completely or partially reverses the direction of the electrode reaction follows the initial potential step. This problem has been addressed by several workers. The solution of Smit and Wijnen (10) to this boundary value problem is restricted in that the kinetic parameter Q , where (4) is constrained to be the same for both initial and reverse potential steps (i.e., the use of potential steps that are symmetric about EOIRof for systems where the transfer coefficient, CY,is equal to 0.5, is dictated). Kimmerle and Chevalet (11) have presented a solution for the asymmetric double potential step (ADPS) case, wherein the initial step drives the forward reaction at the mass transfer limited rate and the reverse step is to any potential at which the rate of the electrode reaction is limited by kinetics of the back electron transfer. This
0003-2700/86/0358-2944$01.50/0 C 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
treatment, however, is restricted to systems that are electrochemically irreversible during the reverse step. A more recent report by Evans and Kelly (12) details the first tractable solution for the asymmetric double potential step case wherein quasi-reversible behavior holds during the reverse step. However, the reported function, derived from a series solution of the boundary value problem, can diverge for various practical combinations of time and heterogeneous kinetic parameters. In those instances where divergence is imminent, the suggested series truncation (12) will give rise to residual errors in the calculated responses. Although limiting forms of these solutions are attractive, they are applicable for a restricted range of combined time and kinetic parameters. At present an exact and general analytical solution describing responses observed during asymmetric double potential step perturbations remains unavailable. The use of asymmetric double potential step chronoabsorptometry (ADPs/ CA) in the investigation of heterogeneous electron transfer kinetics of quasi-reversible processes a t optically transparent electrodes (OTEs) has been previously described (13). In that work, kinetic parameters were determined by digital simulation of each absorbance-time transient with adjustment of k f , h and k h , h to obtain optimal agreement between simulation and experiment. This approach requires extensive recursive simulation and is subject to the individual bias inherent in operator judgement of the "best-fit" agreement between simulated responses and experimental transients. Clearly a method that eliminates this bias is desirable. A general ADPS/CA method using digitally simulated dimensionless parameter working surfaces has been developed in this present work for the evaluation of heterogeneous electron transfer kinetics of quasi-reversible systems. This method is not subject to errors arising from operator bias as described above and is applicable to electrode reactions exhibiting any degree of reversibility.
EXPERIMENTAL SECTION Materials. Potassium ferrocyanide (Mallinckrodt,Analytical Reagent) and potassium ferricyanide (Baker) were used as received. All solutions were prepared in pH 7.00 phosphate buffer (Buffer-Titrisol, EM Reagents) containing 0.100 M sodium chloride (Fisher) using deionized (Barnstead D-0803),glass distilled water. Immediately prior to use, solutions were purged of oxygen by a stream of prepurified nitrogen that had been passed over hot copper turnings and then saturated with water. Apparatus. The electrochemical, optical, and computer data acquisition systems employed in this work have been previously described ( I , 14,15). The amplitude of the reverse potential step for ADPS/CA experiments was set using a separate bias potential with a solid-state relay (PRME 1A05) that was switched at the end of the forward potential step. Fluoride-doped tin oxide OTEs (PPG Industries) were cleaned by an established procedure (16, 17) and then stored in buffer solution for 14 h prior to use. OTEs were fitted to a Pyrex transmission cell of conventional design (3, 4). A saturated calomel electrode was employed in all measurements and was routinely calibrated with a platinum wire immersed in saturated solutions of quinhydrone (18). All kinetic measurements were made at room temperature, 22 (k2) "C. Digital simulations were performed after the manner of Feldberg (19, 20). RESULTS AND DISCUSSION Method. For the heterogeneous electron transfer process defined by eq 1, application of a potential step of sufficient amplitude to cause the 0 to R conversion to proceed a t the maximum diffusion limited rate results in the time-dependent optical absorbance of the product, A R ( X , t ) , that is given by
I
2945
i
t lI
-E
EA
l
I
I
I
0
r
27
TIME
Figure 1. Potential step profiles as a function of time for asymmetric double potential step chronoabsorptometry.
Do and Co are the diffusion coefficient and bulk molar concentration of the precursor, 0. If after some time T the potential is stepped back to a less extreme value, then a portion of the product formed during the initial step ( t C T ) will be converted back to precursor. When the back reaction proceeds at the maximum diffusion limited rate during the reverse step, then a continual decrease in the absorbance of R a t times greater that T , A R ( X , ~ > T ) , that is linear in [ t 1 I 2- ( t - T ) ' / ' ] observed (21)
The temporal and potential dependencies of the change in absorbance of R during the reverse step enables the direct determination of the heterogeneous electron transfer kinetics of the back (R to 0) reaction. For ADPS/CA the potential step profile as a function of time is illustrated in Figure 1. Initially the working electrode is poised a t a potential EA such that the 0 to R conversion does not proceed. The experiment is initiated by a potential step to E* which causes R to be produced a t the maximum diffusion limited rate. At time T the potential is stepped back to a value (e.g., Ec) a t which the rate of electron transfer is limited by the kinetics of electrode reaction. The temporal dependence of A R ( X , ~ > T )under conditions of kinetically limited rates of heterogeneous electron transfer are then used to evaluate kh,h. A series of such asymmetric step experiments in which the reverse step potential is varied (Ec through E I , Figure 1) permits determination of kb,b as a function of overpotential. The absorbance observed during the reverse step ( t > T ) in an ADPS/CA experiment is dependent upon the overpotential applied a t t > T ( q h ) , the kinetics of the electrode reaction, the total time of the experiment ( t ) ,and the duration of the initial step ( 7 ) . The overpotential fixes the ratio k f , h / k h h as can be seen by combining eq 2 and 3
To facilitate kinetic determinations, it is desirable to express the experimental measurement in the form of a dimensionless absorbance parameter that is a function of both a dimensionless kinetic parameter and a dimensionless time parameter. A convenient form for this experimentally derived absorbance parameter is
(211 AR(X,t)
= (2/a1/2)€R(X)Do'izCot'iz
(5)
where C R ( X ) is the molar absorptivity of the product, R, and
The dimensionless kinetic parameter chosen is (hb,hs'/2/DR1/2)
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
Table I. A b / A f at an Overpotential of 100 mV as a Function of (kb,hT1/2/DR1/2) and ( t / r ) for Varied Combinations of and DRa
kb,h, 7,
AhlA,' 10.0 1.o
0.10
10-3 10-2 10-1
10-4 10-3 10-2 10-5 10-4 10-3
10.0 10.0 0.10 10.0 10.0 0.10 10.0 10.0 0.10
10-7 10-5 10-5
10-7 10-5
10-6
10-7 10-5 10-5
0.317 0.317 0.317 0.124 0.124 0.124 0.017 0.017 0.017
0.420 0.420 0.420 0.199 0.199 0.199 0.030 0.030 0.030
0.482 0.482 0.482 0.254 0.254 0.254 0.042 0.042 0.042
0.526 0.526 0.526 0.298 0.298 0.298 0.051 0.051 0.051
"Equivalence of diffusion coefficients used in these simulations. bValues input for simulation, kf,h determined by eq 7. 'According to eq 8.
a.
0
b.
4 -03 AI
2' 0
-0 6
1
C. -
0 -03
k
-06
1
c
e.
f.
-0
- -03 Ah 2' 0
-
AI
-0G 10
Flgure 2. Working surfaces for determination of heterogeneous electron transfer rate constants by asymmetric double potential step chronoabsorptometry: (a) P,, = 120 mV, viewing angle = 45'; (b) same as (a), viewing angle = 135'; (c) vb = 0 mV, viewing angle = 45'; (d) same as (c), viewing angle = 135'; (e) qb = -120 mV, viewing angle = 45'; (f) same as (e), viewing angle = 135'. All surfaces plotted for a viewing elevation of 15'.
and the dimensionless time parameter is t / T . Results of digital simulations of ADPS/CA responses, summarized in Table I, show that the experimentally derived Ab/Af ratio is uniquely dependent upon kb,hT1/2/DR'/2and t / T and is independent of the numerical values of (kb,J,, DR1j2,t ) and T from which these dimensionless parameters are formed. The tabulated values of Ab/Af were derived from simulations using the associated combinations of kb,, 7, and DR1l2for q, equal to 100 mV. From such simulated responses, working curves of Ab/Af as a function of (kb,hT'I2/DR1/') were obtained, each for a specific
ratio of ( t / T ) and value of vb. With a collection of such curves corresponding to different values of ( t / T ) at the same q,, a working surface was constructed that relates Ab/Af to both (kbhT1/'/DR1/2) and ( t / ~ )Examples . of such surfaces are shown in Figure 2 for reverse step overpotentials of 120,0, and -120 mV. These surfaces are presented for ( t / T ) ranging from 1.00 ( t = T ) to 2.10. The upper limit is purely a matter of choice, however, and is unrestricted. Operationally, Ab/Af ratios and corresponding values of t / T are determined from an ADPS/CA experimental transient.
ANALYTICAL CHEMISTRY, VOL. 58,NO. 14,DECEMBER 1986
2947
Table 11. Relative Error in k b , h as a Function of Overpotential and log ( k b , h T 1 ’ z / D R L i z )Resulting from an Uncertainty of H k O O 1 in A b/A Values Reported for ( t / r ) = 1.5” log
relative error at the following vb (mV) values
(kb.hr‘/2/
DR1lz)
180
120
80
60
40
20
10
0
-10
-2.00 -1.75 -1.50 -1.25 -1.00 -0.75 -0.50 -0.25 0 0.25 0.50 0.75 1.00 1.25 1.50
60.5 32.3 20.4 11.9 7.08 4.34 2.82 2.01 1.65 1.63 1.96 2.77 4.38 7.36 12.8
60.7 35.4 20.5 12.0 7.12 4.36 2.84 2.03 1.67 1.65 1.99 2.83 4.48 7.53 13.1
61.6 35.9 20.8 12.2 7.25 4.46 2.92 2.10 1.75 1.76 2.15 3.08 4.91 8.29 14.4
62.9 36.7 21.3 12.5 7.46 4.61 3.04 2.22 1.88 1.93 2.40 3.49 5.61 9.53 16.7
66.1 38.6 22.5 13.2 7.95 4.97 3.33 2.49 2.19 2.34 3.04 4.55 7.45 12.8 22.6
74.2 43.6 25.5 15.2 9.23 5.91 4.11 3.26 3.09 3.60 5.03 7.95 13.5 23.7 42.9
83.2 49.0 28.9 17.3 10.7 6.99 5.04 4.21 4.26 5.32 7.90 13.1 22.8 41.4 77.4
106 60.0 35.7 21.7 13.7 9.30 7.09 6.43 7.23 10.1 16.6 29.9 56.4 116
164 91.7 54.3 34.0 22.5 16.4 14.0 15.2 22.6 47.5
-20
-b -b -b -b -b
-b
-40
-60
-80
-120
21.7 13.2 8.58 6.11 4.96 4.79 5.54 7.52 11.4 18.8 32.4 58.1 113
9.62 6.41 4.74 4.07 4.17 5.12 7.25 11.4 18.9 32.9 59.6 119 500
3.77 3.65 4.24 5.77 8.80 14.5 24.7 44.0 81.7 209
-b
65.3 39.1 24.0 15.3 10.5 8.02 7.22 7.79 10.0 14.7 23.6 40.3 72.6 165
-b
-b
-b
-b
-b
-b
-b
-b
-b
-0 -b
-b -b -b -b
57.9 35.7 34.2 43.2 65.0 114
-b -b -b
Values reported correspond to d(kb,h)/kb,hin percent. bRelativeerror greater than 1000%. -160 -140
I
I
I
10
08
06
I
I
I
I
I
E r
-120 -100
- 80 -60
$
m n
m
8 m f X
- 20 15’
11-
O
04 02 E [Volts vs SCEI
0
-0.2
-04
Flgure 3. Dependence of reiative error in kb,h (d[k,,,]/k h)arising from an uncertainty of f0.001 in Ab/A, on log (kb,hT”’/~~”’) and ( f / T ) for vb = 120 mV.
Flgure 4. Cyclic voltammetry of 5.20 mM K,Fe(CN), in pH 7.00 phosphate buffer at fluoride-doped tin oxide OTE. Area = 0.30 cm2; sweep rate = 50 mVls. For asymmetric double potential step experiments, initial potential (A) = -400 mV, diffusion step limit (B) = 900 mV, kinetic step limits: C = 134 mV, D = 114 mV, E = 84 mV, F = 54 mV, G = 14 mV, H = -27 mV, I = -66 mV.
Together with the values of T and D R 1 I 2 , this array of experimental pairs is then input to a two-dimensional lookup program that searches the working surface corresponding to the overpotential employed in the experiment. Linear interpolation is utilized where necessary in the working surface search algorithm. Values of k b b are returned without operator intervention. This procedure is then repeated for ADPS/CA experiments conducted with various other reverse-step overpotentids to determine the dependence of kb,h on 7 (eq 3). The nonlinear dependence of the dimensionless absorbance parameter &/Af on k b ~ h T 1 ~ 2 / D R 1 / 2dictates that attention be given to the uncertainty in the derived absorbance parameter. By analogy to SPS/CA ( Z ) , the sigmoidal shape of Ab/Af cuwes as functions of log (kb,hT1/2/DR1/2) for any value of t / T indicates that the initial step duration, T , should be chosen such that data to be used for kinetic analysis fall on the working surface in a region where d(Ab/Af)/d(log (kb,hT’/’/ &‘I2)) is greatest. Figure 3 shows the relative error in k b , h ( d [ k b b ] / k b , h ) resulting from an uncertainty of f0.001 in Ab/Af for v b = 120 mV. This representation clearly shows the importance of selecting values of T that force log (kb,hT’/2/DR’iz) to a region where the relative error in k b , h is minimal. Comparison of the working surfaces in Figure 2 shows that less variation in Ab/Af over the range of log (kbbT1/’DR1/’) occurs a t reverse step overpotentials near zero. Hence the proper
selection of T is most critical for experiments conducted under these conditions where the working surfaces are flattest. An example of proper T selection is provided by Table 11, which summarizes values of the relative error in k b , h as a function of log (kb,hT112/DR1iz) for various values of v b a t t / T = 1.50. With these data and an estimate of k b , h at a given overpotential, preferred T values can be defined for the ADPS/CA characterization of a given redox system. For positive overpotentials, the data shown in Table I1 indicate that the relative error in k b , h will be minimal for values of log (kb,hT112/DR”2) ranging from ca. -0.25 to +0.25. For a redox system having kb,h = 1.0 X cm/s and DR = 5 x IO4 cm2/s, T values ranging from ca. 1.6 to 16 s would be optimal for characterization of k b , h using data obtained at t / T = 1.5. Ferri-/Ferrocyanide System. The ADPS/CA method described here was experimentally verified by using the ferri-/ferrocyanide redox couple at fluoride-doped tin oxide OTEs in pH 7.00 phosphate solution ( 2 ) . The cyclic voltammetry of this system and the ADPS/CA potential step sequence employed are shown in Figure 4. The potential is initially stepped from A to B, which results in the oxidation of ferrocyanide at the maximum diffusion limited rate. At 4.50 s the potential is stepped back to each of the values indicated by C through I. The resulting time-dependent absorbance of ferricyanide observed for this series of potential steps (C
2948
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
35t 30
3
1
-
25
-
23
-
15
-
Q
E
I
W 0
z
4
m
X 0 Lo
I
m Q
7
I
5 lo
0
i
- 34
0
-10
-80
7b 2
&
G
8
TIME ( s e d
Figure 5. Absorbance-time behavior of ferricyanide observed at X
= 420 nm for asymmetric double potential step chronoabsorptometry of 5.20 mM K,Fe(CN), in pH 7.00 phosphate buffer at a fluoridedoped tin oxide OTE. Solid lines are experimental transients, solid circles are from digital simulations using q,, and k,,hvalues from Table I11 and kb,h values determined from eq 7. Letters adjacent to transients correspond t o kinetic steps noted in Figure 4. Table 111. Heterogeneous Electron Transfer Rate Constants for the Reduction of Ferricyanide Evaluated by Asymmetric Double Potential Step Chronoabsorptometry at Fluoride-Doped Tin Oxide" qb,b mV
1 0 % , , cm/s ~,~
qb,b mV
103kf,,,' cm/s
-20.0 -40.0 -70.0 -100.0
0.70 (k0.03)d 0.84 (10.03) 1.17 (Et0.03) 1.64 (k0.04)
-140.0
2.76 (k0.06) 4.09 (10.07) 6.48 (Et0.10)
-181.0 -220.0
Initial step to 900 mV vs. SCE, T = 4.50 s, [K,Fe(CN),J = 5.20 mM. Q, = E!,, - E", both E,,, and Eo' measured relative to the same reference electrode potential. Values reported are the mean of two absorbance-time transients independently acquired and analyzed, 45 points between 4.50 s and 9.00 s treated from each transient. Parentheses contain one standard deviation. through I) is shown in Figure 5. For each of these ADPS/CA transients, Ab/Af ratios were determined at various values of ( t / T ) and analyzed by the method outlined above using working surfaces constructed for the experimental overpotentials. In this experimental system the precursor is the reduced form of the redox couple and the reverse step corresponds to kf,h in eq 1. The results of these analyses are summarized in Table 111. These rate constants were then used to simulate ADPS/CA transients for comparison with the experimentally determined ones. The solid circles shown in Figure 5 are absorbance values that were digitally simulated and 7 values reported in Table 111. using Independent verification of these ADPS/CA results was provided by the single potential step chronocoulometric (SPS/CQ) characterization of the kinetics of the reduction of ferricyanide under the same solution conditions. The chronocoulometric data were treated according to the previously reported model for quasi-reversible electrode reactions (2, 22). The dependencies of k f , h on 7 determined by both ADPS/CA experiments using ferricyanide and SPS/CQ experiments using ferrocyanide are shown in Figure 6. The formal heterogeneous electron transfer rate constant and electrochemical transfer coefficient derived from SPS/CQ (ks,ho' = 5.07 (f0.04) x cm/s; a = 0.304 (f0.004)) are in
I
-120
I
,
I
-1GO
I
I
,
-200
(mV1
Figure 6. Dependence of log kl,hon overpotential, from asymmetric double potential step chronoabsorptometry (open circles) and from single potential step chronocoulometry (open squares). Values from chronocoulometricdata are as follows: 7 = -1 mV, k,,h = 5.42 X cm/s; 7 = -19 mV, k , , = 6.07 X cm/s; 7 = -39 mV, k,,, = 8.02 X lo-, cm/s; 7 = -71 mV, k , , = 1.14 X cm/s; 7 -101 mV, kI,, = 1.72 X cm/s; 7 = -141 mV, kf,, = 2.71 X cm/s; 7 = -180 mV, k , , , = 4.21 X cm/s; 7 = -220 mV, kf,h= 6.97 X cm/s. Linear regression analysis of chronocoulometricdata is as follows: ks,hor = 5.07 (f0.04) X lo4 cm/s, a = 0.304 (10.004), R = 0.9994. Values from chronoabsorptometric data in Table 111. Linear regression analysis of chronoabsorptometricdata is as follows: k s h o '= 5.45 (10.05)X lo-,, (Y = 0.289 (f0.004), R = 0.9994.
excellent agreement with those determined from ADPS/CA (ks,ho' = 5.45 (zk0.05) x w4; a = 0.289 (f0.004)). The utility of spectroelectrochemical methods in studies of heterogeneous electron transfer kinetics has been previously established ( I , 2 , 4 , 8 , 9 , 1 3 ,16,23-25). The development of ADPS/CA described here complements the SPS/CA technique reported earlier ( I , 2 ) and enables comprehensive characterization of heterogeneous electron transfer system exhibiting any degree of reversibility.
ACKNOWLEDGMENT We acknowledge numerous enlightening discussions with Dennis H. Evans. Registry No. K,Fe(CN),, 13943-58-3; ferricyanide, 13408-62-3; tin oxide, 1332-29-2;fluoride, 16984-48-8. LITERATURE CITED Albertson, D. E.; Blount. H. N.; Hawkridge, F. M. Anal. Chem. 1979, 5 1 , 556. Bancroft, E. E.; Blount, H. N.; Hawkridge. F. M. Anal. Chem. 1981, 5 3 , 1862. Kuwana, T.; Winograd. N. I n Nectroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1974; Vol. 7, pp 1-78. Heineman, W. R.; Hawkridge, F. M.; Blount, H. N. I n Electroanalytical Chemistry; Bard, A. J.. Ed.; Marcel Dekker: New York, 1984; Vol. 13, pp 1-113. Butler, J. A. V. Trans. Faraday SOC. 1924, 19, 729. Butler, J . A. V. Trans. Faraday SOC. 1924, 19, 734. Erdey-Gruz, T . ; Volmer, M. Z . Phys. Chem., Abt. A 1930, 750, 203. Bowden, E. F.: Hawkridae. F. M.: Biount. H. N. Bioelectrochem. Bioenerg. 1980, 7 , 447. Crawley. C. D.; Hawkridge. F. M. Biochem. Biophys. Res. Commun. 1981, 9 9 , 516. Smit, W. M.; Wijnen, M. D. Recl. Trav. Chim. Pays-Bas 1980, 7 9 , 5 . Kimmerle, F. M.; Chevalet. J. J. Electroanal. Chem. 1969, 2 1 , 237. Evans, D. H.; Kelly, M. J. Anal. Chem. 1982, 5 4 , 1727. Bancroft, E. E.; Blount, H. N.; Hawkridge, F. M. Adv. Chem. Ser 1982, No. 20 1 23. Evans, J. F.; Blount, H. N. J. A m . Chem. SOC. 1978, 100, 4191. Seelig, P . F.; Blount. H. N. Anal. Chem. 1978, 48 252. Bowden. E. F.; Hawkridge, F. M.; Blount, H. N. Adv. Chem. Ser. 1982, No. 2 0 1 , 159. Armstrong. N. R.; Lin, A. W. C.; Fujihira, M.; Kuwana, T. Anal. Chem. 1978. 48. 741. Hili, H. R . 'In Modern Methods of Plant Analysis; Peach, K., Tracey, M. V., Eds.; Springer-Verlag: New York. 1956; Vol. 1, p 393.
Anal. Chem. 1988, 58, 2949-2954 (19) Feldberg, S.W. I n Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1969; Vol. 3, pp 199-296. (20) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1980; p 675. (21) Winograd, N.; Blount, H. N.; Kuwana, T. J. Phys. Chem. 1969, 7 3 , 3456. (22) Christie, J. H.; Lauer, G.; Osteryoung, R. A. J. Nectroanal. Chem. 1984, 7 , 60. (23) Bowden. E. F.; Hawkridge. F. M.; Chlebowski, J. F.; Bancroft. E. E.; Thorpe. C.; Blount, H. N. J. Am. Chem. SOC. 1982, 104, 7641. (24) Cohen. D. J.; Hawkridge, F. M.; Blount, H. N.; Hartzell, C. R. I n Charge and Field Effects in Biosystems; Allen, M. J., Usherwood, P. N. R.,
2949
Eds.; Abacus Press: Tonebridge (U.K.), 1984; pp 19-31. (25) Bowden. E. F.; Hawkridge, F. M.;Blount, H. N. I n Comprehensive Treatise of Electrochemistry; Srinivasan, S . , Chizmadzhev, Yu. A,, Bockris, J. O’M., Conway, B. E., Yeager, E. B., Eds.; Plenum: New York, 1985; Vol. 10, pp 297-345.
RECEIVED for review March 13,1986. Accepted July 24,1986. This work was supported by the National Science Foundation (CHE-8208291).
Continuous-Flow Sample Probe for Fast Atom Bombardment Mass Spectrometry Richard M. Caprioli* and Terry Fan
T h e Analytical Chemistry Center and Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030
John S . Cottrell Kratos Analytical Instruments, Urmston, Manchester, United Kingdom
The deslgn and performance of a sample probe that allows a contlnuowr fbw of sdutlon to be Introduced Into a fast atom bombardment (FAB) Ion source are descrlbed. Samples can be Injected Into a solvent flow that contains water/glycerol (8:2) and dllute buffers. Samples contalnlng 13.5 ng of peptides Injected In 0.5-pL portlons show peaks In the total Ion chromatogram emerging over 30 8, correspondlng to a volume of 2.5 pL. Ion lntensltles recorded with varylng sample amounts show a llnear relatlonshlp from 0.7 to 200 ng. QuantHatlvely, calculatlons of peak areas from repllcate lnJectlonsshow standard deviations of approximately * I O % of the mean. Wlth regard to sensltlvlty, the peptlde substance P (mol wt 1347) at 0.3 ng gave a slgnal-to-nolse ratio of 51. Comparlson of background chemlcal noise between the continuous-flow probe and the standard FAB probe (uslng an 80 % glycerol matrix) showed a slgniflcant Improvement In signal-to-chemlcal nolse using the flow probe. High mass performance Is demonstrated by showlng the resolved molecular Ion regions of Injected samples of oxldlred bovine Insulin B chain (mol wt 3493) and Intact bovlne lnsulln (mol wt 5730). CondHions requlred for the stable operatlon of the probe are discussed.
Samples are usually introduced into the ionization chamber of a fast atom bombardment (FAB) mass spectrometer through the use of a direct insertion probe. The sample is first dissolved in glycerol or some other suitable viscous matrix, and then several microliters of the solution are placed on the probe tip. Exposure of this sample to a beam of energetic xenon atoms inside the mass spectrometer source causes surface layers of molecules to be sputtered and the resulting ions are subsequently analyzed (1). Although this type of sample introduction is simple and easy to use, it has several shortcomings. First, it does not easily lend itself to following dynamic processes especially where rapid changes in reactants or products are expected. Since each sample becomes an isolated analysis, the number of samples taken is a matter of speed and/or endurance both in the sampling process and also
in the subsequent analyses. Second, comparisons of ion intensities from sample to sample are difficult and the results uncertain without the use of internal standards. Third, substantial amounts of glycerol (or other matrix material) are required, usually 80-9570, so that the liquid droplet can survive introduction into the vacuum system. This precludes direct sampling of reactions that must proceed in a substantially aqueous environment such as, for example, enzyme reactions. Several investigators have reported work involving the continuous introduction of liquid samples into a FAB source of a mass spectrometer, although these were mostly aimed at on-line HPLC applications. One approach involves use of a moving belt onto which is deposited fractions of the HPLC eluant ( 2 , 3 ) . The belt is then continuously cycled into the source of the mass spectrometer where the sample spots are bombarded. More recently, Ito et al. ( 4 ) reported on the use of a capillary inlet device for the direct connection of a microbore HPLC column to a FAB ionization source. These workers demonstrated the separation and analysis of bile acids using a mobile phase of glycerol/acetonitrile/water (1027:63) at a flow rate of 0.5 wL/min. A stainless-steel mesh frit was used a t the terminus of the capillary in the ion source to disperse the mobile phase and concentrate the solute and glycerol. Other approaches to continuously flowing aqueous solutions into mass spectrometers, although not involving FAl3 ionization sources, include thermospray (5,6)and direct liquid injection (DLI) methods (7, 8). We have constructed and tested a sample introduction probe for use with mass spectrometers equipped with fast atom bombardment sources that permits a continuous flow of solution to be brought directly into the source. The solution can be essentially aqueous, containing as little as 10% glycerol and is brought into the source at a flow rate of about 5 pL/min. Samples may be injected into this flow of solvent or included in the solvent, depending on the application. Dilute buffers, acids, and salts can be used in the aqueous solvent. Total ion current chromatograms obtained with injected samples in the nanogram range produce sharp peaks with little tailing and no significant memory effects. Further, since the spectrometer can operate a t full accelerating po-
0003-2700/86/0358-2949$01.50/00 1986 American Chemical Society
