Anal. Chem. 1981, 53, 1118-1120
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CORRESPONDENCE Measurement of Surface Excess at a Dropping Mercury Electrode by Normal Pulse Polarography with Coulometry Sir: The usefulness of rapid normal and inverse normal pulse polarography for the study of an electroactive species which is adsorbed a t a dropping mercury electrode (DME)/solution interface has recently been reported (1). Since ita introduction (24,potential-step chronocoulometry has been used to determine the surface excess of an electroactive adsorbate at a stationary electrode/solutioninterface (5). The present communication describes the use of pulse polarographywith coulometry for measurement of the surface excess of an electroactive adsorbate at a DME/solution interface; the resulting method is essentially the marriage of potential-step chronocoulometry and rapid pulse polarography. The influence of adsorption of the electroactive species on normal pulse polarography (NPP), first reported by Barker and Bolzan (6),was studied in detail by Flanagan, Takahashi and Anson (7). When the reactant is adsorbed at the initial potential (Ei,potential at which the drop is held prior to pulse application), a “Barker-Bolzanpeak”, resembling a maximum of the first kind, is obtained on the normal pulse polarogram. For example, remarkably large “Barker-Bolzan peaks” are obtained in the initial one-electron (1 e) reduction of the coenzyme nicotinamide adenine dinucleotide (NAD+)when the delay time (time between pulse application and current measurement) is less than 1ms (Figure 1-11). Through the use of computer simulation, the surface excess of the electroactive species can be obtained from a normal pulse polarogram containing such a peak (7). In certain cases, normal pulse polarography with coulometry (NPP-C) allows estimation of the surface excess directly from the polarogram; a double potential-step or a separate polarogram run on a nonadsorbrng solution is not necessary for estimation of the charge consumed by the double layer (DL) capacitance. The present method of NPP-C differs from that of Temmerman, Abel, and Osteryoung (8) in which double potential-step chronocoulometrywas used with a DME to measure the surface excess of a reactant and measurement of the charge consumed by the DL capacitance was made with the second potential step. The double step method is difficult to apply, for example, to the investigation of NAD+ since the initial 1 e reduction product, a free radical, undergoes rapid and irreversible dimerization. On a second step, charge would pass from the oxidation of adsorbed and dissolved radical and/or dimer, obscuring the DL charge, The pulse train of NPP-C allows measurement of the charge consumed by the DL capacitance without the use of a double step or a separate nonadsorbing solution. A limitation of NPP-C is that, since only a single charge measurement is made, estimation of the surface excess of the electrode product is not possible. When the surface excess of the product is sought or when the variation of the DL capacitance across the reduction wave is not small or when adsorption equilibrium is not achieved, double potential-step chronocoulometry is the method of choice. INSTRUMENTATION NPP-Crequires integrating the current flow upon pulse application for each pulse, displaying the integrated current (charge) until the next pulse is applied, and resetting the integrator to zero 0003-2700/81/0353-1118$01.25/0
between pulses. To accomplish these tasks, an electronically resettable analog integrator was constructed by use of a suitable operational amplifier (Teledyne Philbrick 1026) and a solid-state switch (Fairchild F4016/34015 quad bilateral switch) (Figure 2). In the present study, the device was used with the current follower and sample and hold device (S/H) of a rapid response potentiostat (9) and a pulse polarographic function generator (I). The current flow through the DME is monitored, as usual, by the current follower in the potentiostat. For current display (polarography),the current follower output is fed to the S/Hand then to the recorder. For charge display (polarography with coulometry), the current follower output is fed serially to the integrator, the S/H, and the recorder (Figure 2). To reset the integrator between pulses, we used one transmission gate of the solid-stateswitch to short-circuitthe integrator feedback capacitor between pulses. The monostable MSI output in the potentiostat’s timing circuit (9)is used to control the reset switch. From drop birth until just prior to pulse application, the switch is closed and the integrator’s output is grounded. During pulse application, the switch is open and the output voltage of the integrator, Vout, is given by
where R2 is the feedback resistance of the current follower, R1 and C1are the input resistance and feedback capacitance of the analog integrator, respectively, and ihd is the current flow through the indicating electrode during time, t, which is the delay time (5).
RESULTS AND DISCUSSION The “Barker-Bolzan peak” at -1.0 V in the rapid normal pulse polarogram of NAD’ (Figure 1) indicates that NAD+ is adsorbed at the Ei,-0.10 V. The corresponding normal pulse polarogram with coulometry using the same delay time of 0.57 ms is also given. The total charge passed, Qba, in a potential-step experiment at a stationary electrode is given by &total
=
Qdl
+ Qa1+
Qs
(2)
where Qal is the charge consumed by the electrode DL capacitance, QJ is the charge consumed by reduction of the adsorbed layer, and Q, is the charge consumed by reduction of dissolved reactant which diffuses to the electrode (4). The same general expression applies to a DME (8). The integrated Cottrell equation
Q, = 2 d 7 A C o ( D $ / ~ ) l ’ ~
(3)
can be used (8)to estimate Q,,which, for the conditions used in Figures 1 and 3, is less than 5% of Qd + Q,. Thus, by assuming that Q, is negligible with respect to the measured Qd + Q,,a maximum 5% overestimationof the surface excess is introduced. The use of eq 3 is not strictly correct because, whenever a “Barker-Bolzan p e a k appears, the subsequent limiting current is depressed (7)so that the integrated Cottrell equation will overestimate the true value of Q,. Qd flows immediately after the pulse is applied to the DME and decays exponentially. It is dependent on both the magnitude and the initial potential of the pulse and differs in the 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 7, JUNE 1981
k
POTENTIAL,Vvs. SCE
Flgure 1. Normal pulse polarography with coulometry (NPP-C) (I) and normal pulse polarography (NPP) (11) of 0.20 mM NAD' (A) In 0.4 M KCI and 63 mM carbonate (pH 9.1) and of the background alone (B) at 25 O C : drop time, 2.00 s; m , 0.988 mg s-'; delay time, 0.57 ms; lnitlal potential, -0.10 V. Positive feedback used for iR compensatlon. US1
LNTROL
SO-IOOpF
IOK
0.01uF dme
IN S/H
Lind
OUT Y AXIS
Flgure 2. Circuit for normal pulse polarography with coulometry: F, current follower; I, analog integrator; SW, quad bilateral switch; S/H, sample and hold device (c:omponents are described In the text). MS I and MS I1 are monostable outputs and are described in ref 9.
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wave, Qd is equal to the height of the wave and can be reliably used to estimate the surface excess. For the conditions used in Figure 1, NAD+ is adsorbed at potentials positive of ita 1 e reduction at -1.0 V (10, 11). The dimeric product is adsorbed at potentials positive of -1.4 V, where a tensammetric peak for its desorption occurs in the ac polarogram. Beyond -1.4 V, the background electrolyte cation is adsorbed (10). The slope of curve A (Figure 1-11is slightly different before and after the wave at -1.0 V because the DL capacitance is depressed differently by adsorption of NAD+ before the wave and by adsorption of dimer after the wave. Thus, the double-headed arrow in Figure 1 representa Qd and charge due to a change in the DL capacitance, A&,, caused by the different adsorbates. Another change in the curve A slope a t -1.4 V can be attributed to adsorption of dimer before -1.4 V and of background electrolyte beyond -1.4 V. The parallel nature of curves A and B beyond -1.4 V confirms that the background electrolyte is adsorbed in the latter potential region, even when NAD+ and dimer are present. In order to estimate the surface excess from curve A, we must know the magnitude of AQd. If the segment of curve A from the wave to -1.4 V is extrapolated to -1.6 V, AQa for desorption of the dimer can be found to be less than 0.01 pC over the 0.2 V extrapolation. The wave at -1.0 V spans about 0.2 V. AQ, across the wave is probably less than that due to dimer desorption, since NAD+ and dimer depress the DL capacitanceto nearly the same extent, which is well below #at measured for the background alone (10). It is likely that AQd across the wave at -1.0 V is less than 0.01 pC, which is about 9% of Qd + AQ, = 0.113 pC. Assuming that AQ, and Q, are negligible with respect to Qd, a surface excess, I' = 8.7 X 10-l' mol cm-2; is estimated from the double-headed arrow in Figure 1, using
A = 0.00853m2/3t2/3
(4)
I' = Qd/nFA
(5)
and
0
-0.4 -08 -1.2 POTENTIAL, V VI SCE
-16
Figure 3. Normal pulse polarography with coulometry (NPP-C) of 0.20 mM NAD+ (A) and 0.20 mM 1,CNADH (C) in 0.4 M tetraethylammonium chloride and 63 mM carbonate (pH 9.1) and of the background alone (B) at 25 O C : drop time, 2.00 s; m , 0.988 mg s-'; delay time, 0.57 ms; initial potenitlal, -0.10 V. Positive feedback was used
for
iR
compensatlon.
presence and absence of an electroactive adsorbate. The use of curve B as a measure of Qd in the presence of the adsorbed layer would introduce considerable error. With NPP-C, Qal is fiound directly from the polarogram by extrapolation from the potential region where no faradaic activity occurs to the region of faradaic activity. The double-headed vertical amow in Figure 1 shows Qd obtained by such extrapolation. For the extrapolation to be valid, it is necessary that any variation in charge due to a change in DL capacitance across the reduction wave is small with respect to Qd. For example, the change in Qdl brought about by desorption of the reduction product must be small with respect to Qd. In the special case where the product is adsorbed and depresses the DL capacitance at potentials beyond the wave to the same extent as reactant adsorption does before the
Given the described assumptions, I' could be high by no more than 14%. When 0.4 M tetraethylammonium ion (Tea+, a weak surfactant) is presented in solution, NAD+ is only adsorbed in the region positive of -0.6 V (10, 11); the enzymatically 2 e reduced form of NAD+, l,GNADH, is also adsorbed in this region (12). On NPP-C, the curves for NAD+ and NADH (curves A and C, respectively, in Figure 3) are less than the background (curve B) before the reduction wave. In the region from -0.6 V to the wave, curves A and C are equal since Tea+ is adsorbed in this region. Except for the wave, curves A, B, and C are parallel beyond -0.8 V, confirming that Tea+ is adsorbed in all three solutions beyond -0.8 V. Curve C contains no faradaic component since NADH is polarographically inactive in the potential region involved. Since the slope of curve A does not change immediately before and after the faradaic wave, the double-headedarrow in Figure 3 represents only Qd and is a reliable measure of the surface excess, I' = 6.7 X mol cm-2, for the conditions given. The present results for 0.2 mM NAD+ in KCl and Et4NC1 solutions at pH 9.1 are in good agreement with those reported by Wilson and Epple (13)and based on chronopotentiometry with different background electrolytes and higher NAD+ concentrations. In a Tris-HC1 buffer (ionic strength, p = 0.5; pH 8.0), I? varied from 4.9 to 8.8 X mol cm-2 for 0.51 to 1.0 mM NAD'. Slightly higher surface excesses, 7.1 to 19 X 10-l' mol cm-2 for 0.51 to 1.5 mM NAD+, were measured in a phosphate buffer ( p = 0.27; pH 8.0) (13). Flanagan, Takahashi, and Anson (7)have emphasized that adsorption equilibrium is never truly achieved under condi-
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Anal. Chem. 1981, 53, 1120-1 122
tions where a “Barker-Bolzan peak” and depressed limiting current in the plateau region of the wave occur. However, under the conditions used in Figures 1 and 3, the surface excess was measured at drop times of 2, 3, and 5 s and was found to decrease by less than 3% with increasing drop time. The present results have convinced us that the adsorption equilibrium of NAD+ is nearly truly achieved. It should be noted that in many cases Qd values large enough to measure accurately are obtained only when adsorption equilibrium is not reached; in such cases, NPP-C with a DME is not reliable. Obviously,NPP-C is valid when results are in agreement with those based on conventional techniques, Le., chronocoulometry or chronopotentiometry. When adsorption equilibrium is not achieved a t a DME, the NPP-C pulse train can be applied to a stationary electrode or a static mercury drop electrode (14);with the latter, the advantage of a renewable electrode surface is not lost. LITERATURE CITED (1) Cummlngs, T. E.; Bresnahan, W. T.; Suh, S. Y.; Elving, P. J. J . Electroanal. Chem. 1080, 106, 71-83. (2) Chrlstle, J. H.; L a w , G.; Ostwoung, . - R. A,; Anson, F. C. Anal. Chem. 1983, 35, 1979. (3) Christie, J. H.; Lauer, G.; Osteryoung, R. A. J. Nectroanal. Chem. 1.-M-A.., 7. . , 60-72. - - . -. (4) Anson, F. C. Anal. Chem. 1084, 36, 932-935.
(5) Murray, R. W. in “Techniques of Chemlstry”; Weissberger, A., Rosslter. E., Eds.; Wllev-Intersclence: New York, 1971; Vol. I. Part IIA, Chapter 8. (6) Barker, 0. C.; Bolzan, J. A. Z . Anal. Chem. 1988, 216, 215-238. (7) Flanagan, J. E.; Takahashi, K.; Anson, F. C. J . €/ectroanal. Chem. 1977, 65, 257-286. (8) Temmerman, E.; Abel, R.; Osteryoung, R. A. J. Electroanal. Chem. 1974, 55, 173-186. (9) Cummlngs, T. E.; Jensen, M. A.; Elvlng, P. J. €lectroch/m. Acta 1978, 23, 1173-1184. (10) Schmakel, C. 0.; Santhanam, K. S. V.; Elvlng, J. P. J . Am. Chem. SOC. 1975, 97, 5083-5092. (11) Bresnahan, W. T.: Elvlng, P. J. J . Am. Chem. Soc.,In press. (12) Bresnahan, W. T.; Elvlng, P. J., unpubllshed results. (13) Wilson, A. M.; Epple, D. 0. Siochemistty 1968, 5 , 3170-3175. (14) Peterson, W. M. Am. Lab. (Fairflew, Conn.) 1070, 11 (Dec), 69-78.
‘
Present address: 19899.
Research Center, Hercules, Inc., Wllmlngton, DE
William T. Bresnahan’ Philip J. Elving*
Department of Chemistry University of Michigan Ann Arbor, Michigan 48109
RECEIVED for review May 8, 1980. Resubmitted December 15, 1980. Accepted February 23, 1981. The support of the National Science Foundation is gratefully acknowledged.
Ribbon Storage Techniques for Liquid Chromatography-Mass Spectrometry Sir: Liquid chromatography-mass spectrometry (LC-MS) is a rapidly developing technique for the analysis of complex mixtures not amenable to gas chromatography-mass spectrometry (GC-MS) techniques ( I ) . Existing commercial LCMS interfaces use either (a) a direct introduction of a small flow of the LC effluent directly into a heated chemical ionization (CI) source ( 2 , 3 )or (b) deposition of the effluent on a moving ribbon with evaporation of the volatile mobile phase on progression through a series of vacuum locks and volatilization (or pyrolysis) of the material in (or adjacent to) a conventional CI (4) or electron impact (EI) (5) ion source. A number of other LC-MS interfaces using a variety of approaches have also been reported (6-10). The moving ribbon technique has the advantage that the LC flow rate is, in principle, not restricted and relatively large samples can be transported to the ion source due to nearly complete removal of the mobile phase. One difficulty with this approach, however, is that the source (or “flash heater”) temperature must be optimized for each compound of interest. Too high a temperature may cause the sample to pyrolyze or be volatilized outside the source while too low a temperature will result in only partial volatilization. We have developed a new LC-MS interface which allows semipermanent storage of the chromatographically separated material on a moving ribbon permitting multiple temperature analyses of a single LC separation. The new interface removes the major disadvantage of conventional moving ribbon devices by allowing analysis of a single LC separation at several different temperatures. EXPERIMENTAL SECTION Figure 1is a schematicillustration of the LC-MS interface. The system incorporates a number of departures from conventional practice including a new aerosol liquid deposition device (11), SIMS analysis as an alternate ionization mode (12),and a triple quadrupole mass spectrometer for improved selectivity (12). The 0003-2700/81/0353-1120$01.25/0
effluent from a Spectra Physics Model 8700 HPLC is sprayed on a slowly moving (5-60 cm/min) continuous ribbon (0.63 cm wide, 0.008 cm thick, 320 cm long). One unique feature of the interface is the inclusion of a 120 cm long region before the first vacuum lock. This increased ribbon length allows the semipermanent The aerosol deposition method requires only gas to effect evaporationof the liquid effluent in nearly all cases. Tests show that one can readily evaporate a variety of liquids (hexane, methylene chloride, 2-propanol,etc.) deposited at more than 2 cma/min and at a ribbon speed of 5 cm/min prior to the f i t vacuum chamber (11). The evaporation of the mobile phase is essentially instantaneous under most flow conditions (11)allowing the use of very slow ribbon speeds (i.e., >10 cm/min). At the slowest speeds, some loss of chromatographic resolution appears unavoidable due to the size of the spray deposition area (-0.3 cm2)and the length of ribbon in the volatilization region (1cm). Thus, for a ribbon speed of 10 cm/min one would expect a maximum peak broadening of approximately 8 s, Most “sharp” LC peaks in our work are 10-30 s wide at half-height, and comparison of UV and reconstructed ion chromatograms shows that at ribbon speeds >20 cm/min such effects are usually negligible. Three regions of differential pumping are employed prior to the high-vacuumregion. The first two regions are pumped at 10 L/s by “hot pumps”, maintained at >lo0 O C to limit the effects of condensablevapors during long-term pump operation, and the third by a 1500 L/s turbomolecularpump. Vacuum slits (-0.03 cm X 0.68cm) are machined from Teflon. The main drive wheel is also used to adjust ribbon tension and is motor driven through three universal joints; at no point does the sample surface of the ribbon contact another surface. Typical working pressures are torr in the three differentially approximately 40, 1, and pumped regions. The pressure in the high vacuum chamber is approximately lo-’ torr. The interface also incorporatesa rhenium “cleanup” heater for removal of material after completion of the analysis. The flash heater is designed to rapidly heat a 1cm length of ribbon; heat shields and two copper wheels serve as thermal “drains” and prevent heating of the ribbon outside of the flash 0 I981 American Chemlcal Society
