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Effects of cell design on electrochemical measurements in submicroliter volumes
JOURNAL OF
ELSEVIER
Journal of Electroanalytical Chemistry 385 (1995) 157-162
Effects of cell design on electrochemical measurements in submicroliter volumes Mary Elizabeth Clark, Jennifer L. Ingram, Erin E. Blakely, Walter J. Bowyer Department of Chemistry, Hobart and William Smith Colleges, Geneva, IVY 14456, USA Received 14 April 1994; in revised form 6 September 1994
Abstract We describe cells of various geometries for electrochemical measurements in submicroliter volumes. Three microelectrodes are embedded in a fluoropolymer, and the drop of solution to be analyzed is placed on the three electrodes. In this paper we describe the effects of the geometry of the three electrodes. In particular, band and disk working electrodes are compared. Also, the effect of spacing of the electrodes is explored. In order to achieve analysis of very small volumes the electrodes must be only slightly separated. However, diffusion of products from one electrode to another may affect the voltammograms.
Keywords: Microelectrodes
1. Introduction
2. Experimental
There has been considerable interest in performing electrochemical analysis in very small volumes of solution, and the development of microelectrodes has greatly facilitated these m e a s u r e m e n t s [1-12]. Although spectroscopists have achieved analysis in much smaller domains, the minimum volume required for electrochemical analysis is limited by the need to place two or three electrodes in solution. The size of the diffusion layer may affect the volume requirements as well. Recently we described a method for performing electrochemical m e a s u r e m e n t s in drops as small as 0.02 ixl [12]. The solution was placed on a Tefzel cell containing three individually addressable band microelectrodes. Using this arrangement, differential pulse and cyclic voltammetry yielded voltammograms similar to those recorded in large volumes. However, under some conditions, the electrochemical behavior was more complicated than expected. In this p a p e r we describe experiments which elucidate the cause of the non-ideal behavior. Furthermore, we describe cells of various electrode geometries.
2.1. Construction of microcells
* Corresponding author. 0022-0728/95/$09.50 © 1995 Elsevier Science S.A. All rights reserved SSDI 0 0 2 2 - 0 7 2 8 ( 9 4 ) 0 3 7 8 6 - 8
Details of the construction of the microcells have already been described [12,13]. Briefly, three strips of foil (platinum, gold or silver) were cut to the appropriate dimensions and included in a multidecker sandwich of Tefzel film. After heat sealing (300°C for 9 min), the assembly was allowed to cool. It was then cut to expose the three foil strips as three band microelectrodes, and the face was polished with increasingly fine polish. Electrical contact to each electrode was made at the other end of the foil which had been left protruding from the sandwich by several millimeters. The face of the microcells was polished with 0.05 ~ m alumina (Buehler) immediately prior to each experiment. The electrode was then inverted, and the drops of solution were placed on the electrodes (Fig. 1). In this study, four microcells were studied in detail. In all cases, a silver band acted as a quasi-reference electrode and a platinum band acted as an auxiliary electrode. The working electrode was situated between these two bands. In two of the microcells the working electrode was a platinum band (4 I~m wide and either 1 or 1.3 m m long (see Table 1)). Alternatively, the working electrode in two of the microcells was a 25 l~m disk (obtained by using a 25 txm platinum wire in the
158
M.E. Clark et aL /Journal of Electroanalytical Chemistry 385 (1995) 157-162
Drop
Electrodes
- ~ -
-Tefzel
lll l
50 mV. High speed cyclic voltammetry (1-4000 V S -1) was performed with a P A R C 175 universal programmer and a home-built potentiostat (time constant, 2 txs) [131. Drops were placed on the microcell using Hamilton 1 ILl microsyringes. Typically, after a single voltammogram was recorded in a drop, the face of the microcell was wiped with a KimWipe and a new drop was introduced. Unless the volume is specified, voltammograms were recorded in 1 txl drops. Voltammetry in N,N-dimethylformamide (DMF) was usually performed with the electrode and the drop exposed to ambient atmosphere. When it was desirable to exclude oxygen (e.g. for reductions), a cell similar to that described by Baranski and Quon [4] was used. For voltammetry in aqueous solutions Baranski's cell was employed, and the nitrogen was passed through a fine frit and bubbled through 10 cm of aqueous electrolyte to "saturate" the atmosphere with water.
6 m m Glass Tubing 2.3. Reagents and materials
Leads Fig. 1. Diagram of microcell.
construction). Spacing between electrodes was controlled by the number of layers of Tefzel between the electrodes. The four microcells are summarized in Table 1.
Tefzel 500 LZ heat-sealing film was obtained from American Durafilm (Holliston, MA). All metal foils and wire were purchased from Aesar (Ward Hill, MA). Reagents were purchased from Aldrich and used as received. D M F (HPLC grade) was dried by passing down a column of activated alumina just before use. Tetrabutyl ammonium hexafluorophosphate (TBAHFP) was recrystallized three times from 95% ethanol and dried for 24 h at 100°C. The voltammetry of ((dimethylamino)methyl)ferrocene (DMAMF) was recorded in aqueous 0.8 M KNO 3 buffered to pH 7.4 with citrate.
3. Results a n d d i s c u s s i o n 2.2. Voltammetry All voltammetry was performed in the three-electrode configuration. A P A R C 174A polarographic analyzer was used for differential pulse voltammetry (DPV), normal pulse voltammetry (NPV) and slow cyclic voltammetry (CV). Differential pulse voltammograms were recorded at a scan rate of 0.01 V s - l , a sampling time of 0.5 s and a modulation amplitude of
Table 1 Geometries of four microcells Microcell a Working (Pt)
Auxiliary (Pt)
Voltammetry recorded using the microcell with large spacing and a band working electrode (LSB) has been described [12], and so it is only briefly summarized here. Voltammetry was performed in volumes as small as 0.02 ~1. Capacitance values as well as faradaic currents were proportional to the length of the working electrode covered by the drop. In volumes greater than 0.5 t~1, the electrode was fully covered and voltammetry
Reference (Ag)
Spacing/l~m
SSB 1.3 mm × 4 ixm 1.3 mm x 100 ~m 1.3 mm x 100 ~m 90 SSD d = 25 ~m 1 mm × 25 wm 1 mm x 25 ~m 100 LSB 1 mm X 4 i~m 1 mm X 100 t~m 1 mm x 100 ixm 130 LSD d = 25 i~m 1 mm× 25 Izm 1 mm × 25 Ixm 250 a SSB, small space with band working electrode; SSD, small space with disk working electrode; LSB, large space with band working electrode; LSD, large space with disk working electrode.
M.E. Clark et al. /Journal of Electroanalytical Chemistry 385 (1995) 157-162 was not different from that observed in very large solution volumes. V o l t a m m o g r a m shape was independent of volume. By D P V a plot of current vs. concentration was linear between 5 and 200 IxM. In this p a p e r we discuss how the stability of the quasi-reference electrode depends upon electrode geometry. Also, we investigate the effect of volume on the electrochemical behavior using microcells of various geometries.
159
C
B
3.1. Quasi-reference electrode stability U n d e r normal conditions, quasi-reference silver electrodes are quite stable [7,12,14]. The quasi-reference electrodes in our microcells were typically stable to + 5 0 m V per day and varied by at most + 150 m V over several months. However, under certain conditions, the voltage of the reference electrode shifts dramatically on the time-scale of a single scan. For example, when performing slow scan cyclic voltammetry (CV) in ferrocene solutions greater than 1 m M using the LSB microcell, the El~ 2 of the reverse scan is as much as 200 m V negative of the E1/2 of the forward scan. Illustrated in Fig. 2(A) is a cyclic voltamm o g r a m recorded at 0.1 V s -1 in 20 m M ferrocene in DMF. At lower concentrations, the reverse scan follows the forward scan closely (see Fig. 2(B) for a v o l t a m m o g r a m of 1 m M ferrocene). We attribute this behavior to polarization of the quasi-reference electrode by diffusion (and in some cases convection) of the ferrocenium from the working electrode to the silver band. This results in a positive shift of the reference potential. Consistent with this interpretation, curve crossing is more dramatic at lower scan rates than at higher scan rates (because more time allows more ferrocenium to arrive at the quasi-reference electrode). If the microcell is inserted into 25 ml of solution and a saturated calomel electrode (SCE) is substituted as the reference electrode, no curve crossing is observed by CV even in concentrated solutions. The reference electrode instability is also apparent when performing D P V in solutions of high concentration using the LSB microcell. Fig. 3 shows a plot of D P V p e a k current vs. concentration between 0.005 and 66 m M ferrocene in DMF. Between 5 and 200 ixM, precision and linearity are excellent [12]. The sensitivity over this range is 0.63 I~A m M - 1 , and the points in this range are fitted by the Straight line in Fig. 3 (the correlation coefficient is 0.9998 with N = 17). However, above 1 m M the function is curved and the precision is much poorer. The attribution of this curvature to quasi-reference electrode instability is supported by the p e a k width of the differential pulse voltammograms. When the ferrocene concentration is less than 1 mM, the peak width
E~ Z D £)
y I
I
I
I
0.6
I
0.4
VOLTAGE/V
1
I
0.2
vs.
Ag
band
Fig. 2. Slow scan (0.1 V s-1) cyclic voltammograms of ferrocene in DMF using the LSB microcelh (A) 20. mM ferrocene, 0.1 M TBAHFP (y scale bar, 1 ~A); (B) 1.0 mM ferrocene, 0.1 M TBAHFP (bar, 0.1 p.A); (C) 1.0 mM ferrocene, no added electrolyte (bar, 0.1 ~A). at half-height is consistently 110 mV. At higher concentrations the peaks become much narrower (Table 2). Apparently, when the scan extends into the ferrocene oxidation region (but before the peak of the voltammogram is recorded), the quasi-reference electrode is shifted positively. This results in a shift in the
20. (
~ 10.0 Q. 5.0 / ~ I
h
I
L
I
I
10.0
20.0
30.0
40.0
50.0
60.0
Concentration
~ram
Fig. 3. Plot of peak current vs. ferrocene concentration up to 66 mM (open squares). The straight line is a linear regression for the 17 points at six concentrations between 5 and 209 IxM.
160
M.E. Clark et al. /Journal of Electroanalytical Chemistry 385 (1995) 157-162
Table 2 Dependence of peak width at half-height on ferrocene concentration for differential pulse voltammograms of ferrocene in DMF+0.1 M TBAHFP using the LSB microcell. [Cp2Fe]/mM Wi/2/mY < 1.0 110 1.0 90 1.8 85 5.1 75 11.0 60 31.0 62 66.0 45
effective voltage of the working electrode in the positive direction beyond the half-wave voltage of ferrocene. When the differential pulse voltammogram is recorded, this shift results in the return of the current to zero and the full peak is never observed. The shape of the D P V peaks is consistent with this interpretation (Fig. 4). To confirm that the narrow peaks are not due to migration (since under these conditions the ferrocene concentration is only slightly less than electrolyte concentration), we recorded differential pulse voltammograms at low electrolyte concentrations. With 0.5 m M ferrocene and T B A H F P concentrations of 0, 0.001 and 0.01 M, no peak narrowing was observed (W1/2 = 110 _+ 7 mV). Also, the absence of electrolyte does not induce curve crossing in cyclic voltammograms (see Fig. 2(C)). Migration effects are expected to be minimal when the reactant is uncharged as is the case for ferrocene [15]. Normal pulse voltammetry corroborates the interpretation of D P V peak widths. At 0.50 m M ferrocene, the slope of E vs. l o g ( I / I 1 - I ) is 56 mV, close to the expected value of 59 mV. At 5.0 mM this slope is only 34 mV and the plot is slightly curved downward. This is the expected result if the reference electrode is shifted positively during the rising part of the voltammogram.
VOLTAGE/V vs.Ag 0.0
~=-
2.0
\/
0.5 i
0.3 I
0.5 I
0.3 I
0.00
0.04
i ~
0.08
4,0 11 m M
Fig. 4. Differential pulse voltammograms of ferrocene in DMF at two concentrations (LSB microcell).
Table 3 Curve crossing by cyclic voltammetry at 5.0 mM with a scan rate of 0.02 V s-i Micro E / m V cell CP2Fe+DM F DMAMF+ H20 a DMAMF+ H20 b LSB - 90 0 SSB - 140 - 30 SSD - 30 LSD - 70 a In water-saturated atmosphere. b In ambient atmosphere.
- 40 -
Finally, we note that this behavior is not peculiar to ferrocene. Narrow D P V peaks, curved plots of peak current vs. concentration and curve crossing were all observed for the reduction of p-nitrotoluene at high concentrations ( > 1 mM). 3.2. Quasi-reference polarization and cell geometry To test this interpretation of the data experimentally, we made two predictions: (1) curve crossing observed by CV will be less dramatic if the working electrode was a microdisk because the disk would generate less ferrocenium than a band, and (2) curve crossing will be more dramatic as the spacing between electrodes decreases. The curve-crossing data for the oxidation of 5 m M ferrocene at 0.02 V s-1 with the four microcell geometries are recorded in Table 3. (Curve crossing was quantified by subtracting the E l l 2 on the forward wave from that on the reverse scan.) The microcells with band working electrodes (LSB and SSB) exhibit significantly more curve crossing than those with disk electrodes (SSD and LSD). This is consistent with prediction (1). We then tested our prediction for electrode spacing. For the two microcells with band working electrodes, the one with larger spacing (LSB) has less curve crossing than the one with smaller spacing (SSB), as predicted. For the two microcells with disk working electrodes, the one with larger spacing has more curve crossing than the one with smaller spacing. Although this last observation is surprising, the difference is small (but reproducible). If the drop is maintained in an atmosphere saturated with solvent, curve crossing is not as severe as when the drop is exposed to ambient atmosphere. For example, when studying the oxidation of D M A M F in aqueous solutions with the LSB microcell, curve crossing was - 4 0 mV in ambient atmosphere (Table 3). However, if the drop is in an atmosphere saturated with water, very little or no curve crossing is observed. An explanation for the effect of the atmosphere was suggested when we observed a dust particle in a droplet exposed to ambient atmosphere. The particle was tumbling around inside the drop, indicating that convec-
M.E. Clark et al. /Journal of Electroanalytical Chemistry 385 (1995) 157-162
tion due to evaporative cooling of the surface can be a significant mode of mass transport from the working electrode to the reference electrode. However, consistent with what is expected for microelectrodes [16], the convection is not sufficient to affect flux at the microelectrode surface. We have never observed that currents depend upon exposure of the droplet to the ambient atmosphere. Diffusion, as well as convection, contributes to curve crossing since the SSB microcell exhibits - 3 0 mV of curve crossing even when in an atmosphere saturated with water (Table 3). Cyclic voltammetry experiments at single bands and band arrays indicate that diffusion over the distance separating the working and reference electrodes is significant on time-scales of about 1 s or more [13]. All these results indicate that convection and diffusion of electrogenerated products can cause quasi-reference instability when reference and working electrodes are only slightly separated.
3.3. Effect of volume and cell geometry The total solution volume may affect peak currents in three ways: (1) if the working electrode is not fully covered, (2) if evaporation is significant (since the surface-to-volume ratio is greater in smaller volumes) and (3) if the dimensions of the drop are too small to allow full development of the diffusion layer. To test the effect of solution volume on peak current, we recorded voltammograms of aqueous 1 mM D M A M F at volumes between 0.02 and 2 txl. The data for the four microcell geometries are recorded in Table 4. All these data were recorded in a nitrogen atmosphere which has been bubbled through an aqueous solution of the electrolyte. (The smallest volumes could not be tested using the microcells with disk electrodes because the droplets do not adhere to the hydrophobic Tefzel sufficiently to be removed from the syringe tip. Band working electrodes provide better adhesion to the drop, allowing voltammetry in smaller volumes.)
Table 4 Dependence of current on volume for four microcell geometries: DPV of 1 mM aqueous D M A M F in an atmosphere "saturated" with water
Volume/
I(LSB)/
I(SSB)/
/(SSD)/
I(LSD)/
~1
~A
ixA
nA
nA
2 1 0.5 0.2 0.1 0.05 0.02
0.94 0.98 0.93 0.93 0.63 0.48 0.52
1.25 1.26 1.25 0.91 0.83 0.73 0.34
55 57 57 65 78 91 -
54 55 49 56 60 -
(100) a (100) (100) (94) (74) (51) (37)
a Percentage of band covered.
-
161
The data for the LSB microcell in Table 4 are similar to those that we have reported for ferrocene in D M F [12]. The peak current is approximately independent of volume down to 0.5 ~xl. However, smaller volumes do not cover the working electrode fully, and so only a fraction of the expected current is recorded. The peak current is approximately proportional to the fraction of the working electrode covered. The data for the SSB microcell are similar (absolute currents are larger because of the longer bands). With a disk working electrode, we expected the peak current to be independent of volume since the working electrode is fully covered by all volumes studied. However, with these microcells peak current increases slightly at the smallest volumes. This suggests that the surface-to-volume ratio is large enough for evaporation to be significant despite our attempts to saturate the atmosphere with solvent. To confirm the significance of evaporation, we recorded voltammograms either immediately after placing a drop on the microcell or after waiting for 120 s. Using a 1 ixl volume, peak currents were independent of time ( + 2 % ) . However, when using 0.1 ixl drops, peak current increased by more than 10% after 120 s (12 replicates for each experiment at both the SSD and LSD microcells). Presumably, evaporation is equally important when using the band microcells, but the effect is not so apparent because the fraction of electrode covered is the more important effect. Finally, the volume may affect faradaic currents if the drop is smaller than the diffusion layer which would normally form. For example, 0.2 ~1 of D M F solution covers the band of the LSB microcell fully 1, yet peak currents are about 90% of those recorded in larger volumes. The probability that a molecule with diffusion coefficient D arriving at the electrode at time t comes from beyond distance r assuming cylindrical diffusion can be described by [17]
W(r, t) = e x p ( - r Z / a D t )
(1)
We have measured the thickness of the 0.2 pA drop to be 0.023 cm and the D for ferrocene in D M F to be 9.8 x 10 -6 cm z s -1. Eq. (1) predicts that, after 5 s, 7% of the flux would be molecules arriving from beyond 0.023 cm (if the solution volume were larger). This is qualitatively consistent with our observation of a 10% decrease in peak currents as measured by DPV at a scan rate of 0.01 V s -1. Also consistent with this, if
i While 0.2 txl of aqueous solution forms a hemisphere of radius less than 1 ram, organic solvents spread out over the surface of the microcell and 0.2 ~1 of D M F fully covers the electrodes.
162
M.E. Clark et al. /Journal of Electroanalytical Chemistry 385 (1995) 157-162
peak currents are measured by high speed cyclic voltammetry, peak current is independent of volume down to 0.2 I~1 [12].
pensing the smallest volumes onto these electrodes is not possible. Acknowledgements
4. Conclusions (1) Tefzel microcells can be used to perform-electrochemical analysis in volumes as small as 0.02 ¢1. However, several experimental difficulties may be anticipated. (2) When the quasi-reference electrode is very close to the working electrode, products may arrive at the reference electrode by diffusion and convection. This induces reference electrode instability which may lead to curve crossing (cyclic voltammetry), narrow peaks (DPV) and curvature of calibration curves (DPV). This problem can be avoided by using a larger spacing between electrodes or by performing more rapid experiments. (3) Drop size may affect the development of the diffusion layer during long experiments and result in a decrease in faradaic currents. This can be avoided by performing more rapid experiments. Eq. (1) is useful for predicting when this effect is significant. (4) In very small volumes ( < 0.2 I~1), the surface-tovolume ratio is so large that evaporation of solvent may be a problem despite attempts to saturate the atmosphere with solvent. Faradaic currents are increased, and precision is degraded when evaporation is significant. This problem is, of course, less significant in less volatile solvents (e.g. DMF). (5) Under our conditions, little is to be gained by using disk working electrodes. Although the disk electrodes are fully covered by all volumes, evaporation is significant at the lowest volumes. Furthermore, dis-
We thank Brian Swift for bringing Eq. (1) to our attention. This research was supported by grants from Research Corporation and Hobart and William Smith Colleges. References [1] P.N. Bartlett and R.G. Whitaker, Anal. Chem., 61 (1989) 2803. [2] T. Miwa, Y. Nishimura and A. Mizuike, Anal. Chim. Acta, 140 (1982) 59. [3] J.O. Schenk, E. Miller and R.N. Adams, Anal. Chem., 54 (1982) 1452. [4] A.S. Baranski and H. Quon, Anal. Chem., 58 (1986) 407. [5] A.S. Baranski, Anal. Chem., 59 (1987) 662. [6] A.R. Harman and A.S. Baranski, Anal. Chim. Acta, 239 (1990) 35. [7] M. Morita, M.L. Longmire and R.W. Murray, Anal. Chem., 60 (1988) 2770. [8] T.T. Wooster, M.L. Longmire, H. Zhang, M. Watanabe and R.W. Murray, Anal. Chem., 64 (1992) 1132. [9] M. Wojciechowski and J. Balcerzak, Anal. Chim. Acta, 237 (1990) 127. [10] J. Kulys and E.J. D'Costa, Anal. Chim. Acta, 243 (1991) 173. [11] P.R. Unwin and A.J. Bard, Anal. Chem., 64 (1992) 113. [12] W.J. Bowyer, M.C. Clark and J.L. Ingram, Anal. Chem., 64 (1992) 459. [13] D.M. Odell and W.J. Bowyer, Anal. Chem., 62 (1990) 1619. [14] C.-L. Wang, K.E. Creasy and B.R. Shaw, J. Electroanal. Chem., 300 (1991) 365. [15] A.M. Bond, M. Fleischmann and J. Robinson, J. Electroanal. Chem., 168 (1984) 299. [16] P.M. Kovach, W.L. Caudill, D.G. Peters and R.M. Wightman, J. Electroanal. Chem., 185 (1985) 285. [17] W. Jost, Diffusion in Solids, Liquids, and Gases, Academic Press, New York, 1960, p. 17.
ELSEVIER
Journal of Electroanalytical Chemistry 385 (1995) 157-162
Effects of cell design on electrochemical measurements in submicroliter volumes Mary Elizabeth Clark, Jennifer L. Ingram, Erin E. Blakely, Walter J. Bowyer Department of Chemistry, Hobart and William Smith Colleges, Geneva, IVY 14456, USA Received 14 April 1994; in revised form 6 September 1994
Abstract We describe cells of various geometries for electrochemical measurements in submicroliter volumes. Three microelectrodes are embedded in a fluoropolymer, and the drop of solution to be analyzed is placed on the three electrodes. In this paper we describe the effects of the geometry of the three electrodes. In particular, band and disk working electrodes are compared. Also, the effect of spacing of the electrodes is explored. In order to achieve analysis of very small volumes the electrodes must be only slightly separated. However, diffusion of products from one electrode to another may affect the voltammograms.
Keywords: Microelectrodes
1. Introduction
2. Experimental
There has been considerable interest in performing electrochemical analysis in very small volumes of solution, and the development of microelectrodes has greatly facilitated these m e a s u r e m e n t s [1-12]. Although spectroscopists have achieved analysis in much smaller domains, the minimum volume required for electrochemical analysis is limited by the need to place two or three electrodes in solution. The size of the diffusion layer may affect the volume requirements as well. Recently we described a method for performing electrochemical m e a s u r e m e n t s in drops as small as 0.02 ixl [12]. The solution was placed on a Tefzel cell containing three individually addressable band microelectrodes. Using this arrangement, differential pulse and cyclic voltammetry yielded voltammograms similar to those recorded in large volumes. However, under some conditions, the electrochemical behavior was more complicated than expected. In this p a p e r we describe experiments which elucidate the cause of the non-ideal behavior. Furthermore, we describe cells of various electrode geometries.
2.1. Construction of microcells
* Corresponding author. 0022-0728/95/$09.50 © 1995 Elsevier Science S.A. All rights reserved SSDI 0 0 2 2 - 0 7 2 8 ( 9 4 ) 0 3 7 8 6 - 8
Details of the construction of the microcells have already been described [12,13]. Briefly, three strips of foil (platinum, gold or silver) were cut to the appropriate dimensions and included in a multidecker sandwich of Tefzel film. After heat sealing (300°C for 9 min), the assembly was allowed to cool. It was then cut to expose the three foil strips as three band microelectrodes, and the face was polished with increasingly fine polish. Electrical contact to each electrode was made at the other end of the foil which had been left protruding from the sandwich by several millimeters. The face of the microcells was polished with 0.05 ~ m alumina (Buehler) immediately prior to each experiment. The electrode was then inverted, and the drops of solution were placed on the electrodes (Fig. 1). In this study, four microcells were studied in detail. In all cases, a silver band acted as a quasi-reference electrode and a platinum band acted as an auxiliary electrode. The working electrode was situated between these two bands. In two of the microcells the working electrode was a platinum band (4 I~m wide and either 1 or 1.3 m m long (see Table 1)). Alternatively, the working electrode in two of the microcells was a 25 l~m disk (obtained by using a 25 txm platinum wire in the
158
M.E. Clark et aL /Journal of Electroanalytical Chemistry 385 (1995) 157-162
Drop
Electrodes
- ~ -
-Tefzel
lll l
50 mV. High speed cyclic voltammetry (1-4000 V S -1) was performed with a P A R C 175 universal programmer and a home-built potentiostat (time constant, 2 txs) [131. Drops were placed on the microcell using Hamilton 1 ILl microsyringes. Typically, after a single voltammogram was recorded in a drop, the face of the microcell was wiped with a KimWipe and a new drop was introduced. Unless the volume is specified, voltammograms were recorded in 1 txl drops. Voltammetry in N,N-dimethylformamide (DMF) was usually performed with the electrode and the drop exposed to ambient atmosphere. When it was desirable to exclude oxygen (e.g. for reductions), a cell similar to that described by Baranski and Quon [4] was used. For voltammetry in aqueous solutions Baranski's cell was employed, and the nitrogen was passed through a fine frit and bubbled through 10 cm of aqueous electrolyte to "saturate" the atmosphere with water.
6 m m Glass Tubing 2.3. Reagents and materials
Leads Fig. 1. Diagram of microcell.
construction). Spacing between electrodes was controlled by the number of layers of Tefzel between the electrodes. The four microcells are summarized in Table 1.
Tefzel 500 LZ heat-sealing film was obtained from American Durafilm (Holliston, MA). All metal foils and wire were purchased from Aesar (Ward Hill, MA). Reagents were purchased from Aldrich and used as received. D M F (HPLC grade) was dried by passing down a column of activated alumina just before use. Tetrabutyl ammonium hexafluorophosphate (TBAHFP) was recrystallized three times from 95% ethanol and dried for 24 h at 100°C. The voltammetry of ((dimethylamino)methyl)ferrocene (DMAMF) was recorded in aqueous 0.8 M KNO 3 buffered to pH 7.4 with citrate.
3. Results a n d d i s c u s s i o n 2.2. Voltammetry All voltammetry was performed in the three-electrode configuration. A P A R C 174A polarographic analyzer was used for differential pulse voltammetry (DPV), normal pulse voltammetry (NPV) and slow cyclic voltammetry (CV). Differential pulse voltammograms were recorded at a scan rate of 0.01 V s - l , a sampling time of 0.5 s and a modulation amplitude of
Table 1 Geometries of four microcells Microcell a Working (Pt)
Auxiliary (Pt)
Voltammetry recorded using the microcell with large spacing and a band working electrode (LSB) has been described [12], and so it is only briefly summarized here. Voltammetry was performed in volumes as small as 0.02 ~1. Capacitance values as well as faradaic currents were proportional to the length of the working electrode covered by the drop. In volumes greater than 0.5 t~1, the electrode was fully covered and voltammetry
Reference (Ag)
Spacing/l~m
SSB 1.3 mm × 4 ixm 1.3 mm x 100 ~m 1.3 mm x 100 ~m 90 SSD d = 25 ~m 1 mm × 25 wm 1 mm x 25 ~m 100 LSB 1 mm X 4 i~m 1 mm X 100 t~m 1 mm x 100 ixm 130 LSD d = 25 i~m 1 mm× 25 Izm 1 mm × 25 Ixm 250 a SSB, small space with band working electrode; SSD, small space with disk working electrode; LSB, large space with band working electrode; LSD, large space with disk working electrode.
M.E. Clark et al. /Journal of Electroanalytical Chemistry 385 (1995) 157-162 was not different from that observed in very large solution volumes. V o l t a m m o g r a m shape was independent of volume. By D P V a plot of current vs. concentration was linear between 5 and 200 IxM. In this p a p e r we discuss how the stability of the quasi-reference electrode depends upon electrode geometry. Also, we investigate the effect of volume on the electrochemical behavior using microcells of various geometries.
159
C
B
3.1. Quasi-reference electrode stability U n d e r normal conditions, quasi-reference silver electrodes are quite stable [7,12,14]. The quasi-reference electrodes in our microcells were typically stable to + 5 0 m V per day and varied by at most + 150 m V over several months. However, under certain conditions, the voltage of the reference electrode shifts dramatically on the time-scale of a single scan. For example, when performing slow scan cyclic voltammetry (CV) in ferrocene solutions greater than 1 m M using the LSB microcell, the El~ 2 of the reverse scan is as much as 200 m V negative of the E1/2 of the forward scan. Illustrated in Fig. 2(A) is a cyclic voltamm o g r a m recorded at 0.1 V s -1 in 20 m M ferrocene in DMF. At lower concentrations, the reverse scan follows the forward scan closely (see Fig. 2(B) for a v o l t a m m o g r a m of 1 m M ferrocene). We attribute this behavior to polarization of the quasi-reference electrode by diffusion (and in some cases convection) of the ferrocenium from the working electrode to the silver band. This results in a positive shift of the reference potential. Consistent with this interpretation, curve crossing is more dramatic at lower scan rates than at higher scan rates (because more time allows more ferrocenium to arrive at the quasi-reference electrode). If the microcell is inserted into 25 ml of solution and a saturated calomel electrode (SCE) is substituted as the reference electrode, no curve crossing is observed by CV even in concentrated solutions. The reference electrode instability is also apparent when performing D P V in solutions of high concentration using the LSB microcell. Fig. 3 shows a plot of D P V p e a k current vs. concentration between 0.005 and 66 m M ferrocene in DMF. Between 5 and 200 ixM, precision and linearity are excellent [12]. The sensitivity over this range is 0.63 I~A m M - 1 , and the points in this range are fitted by the Straight line in Fig. 3 (the correlation coefficient is 0.9998 with N = 17). However, above 1 m M the function is curved and the precision is much poorer. The attribution of this curvature to quasi-reference electrode instability is supported by the p e a k width of the differential pulse voltammograms. When the ferrocene concentration is less than 1 mM, the peak width
E~ Z D £)
y I
I
I
I
0.6
I
0.4
VOLTAGE/V
1
I
0.2
vs.
Ag
band
Fig. 2. Slow scan (0.1 V s-1) cyclic voltammograms of ferrocene in DMF using the LSB microcelh (A) 20. mM ferrocene, 0.1 M TBAHFP (y scale bar, 1 ~A); (B) 1.0 mM ferrocene, 0.1 M TBAHFP (bar, 0.1 p.A); (C) 1.0 mM ferrocene, no added electrolyte (bar, 0.1 ~A). at half-height is consistently 110 mV. At higher concentrations the peaks become much narrower (Table 2). Apparently, when the scan extends into the ferrocene oxidation region (but before the peak of the voltammogram is recorded), the quasi-reference electrode is shifted positively. This results in a shift in the
20. (
~ 10.0 Q. 5.0 / ~ I
h
I
L
I
I
10.0
20.0
30.0
40.0
50.0
60.0
Concentration
~ram
Fig. 3. Plot of peak current vs. ferrocene concentration up to 66 mM (open squares). The straight line is a linear regression for the 17 points at six concentrations between 5 and 209 IxM.
160
M.E. Clark et al. /Journal of Electroanalytical Chemistry 385 (1995) 157-162
Table 2 Dependence of peak width at half-height on ferrocene concentration for differential pulse voltammograms of ferrocene in DMF+0.1 M TBAHFP using the LSB microcell. [Cp2Fe]/mM Wi/2/mY < 1.0 110 1.0 90 1.8 85 5.1 75 11.0 60 31.0 62 66.0 45
effective voltage of the working electrode in the positive direction beyond the half-wave voltage of ferrocene. When the differential pulse voltammogram is recorded, this shift results in the return of the current to zero and the full peak is never observed. The shape of the D P V peaks is consistent with this interpretation (Fig. 4). To confirm that the narrow peaks are not due to migration (since under these conditions the ferrocene concentration is only slightly less than electrolyte concentration), we recorded differential pulse voltammograms at low electrolyte concentrations. With 0.5 m M ferrocene and T B A H F P concentrations of 0, 0.001 and 0.01 M, no peak narrowing was observed (W1/2 = 110 _+ 7 mV). Also, the absence of electrolyte does not induce curve crossing in cyclic voltammograms (see Fig. 2(C)). Migration effects are expected to be minimal when the reactant is uncharged as is the case for ferrocene [15]. Normal pulse voltammetry corroborates the interpretation of D P V peak widths. At 0.50 m M ferrocene, the slope of E vs. l o g ( I / I 1 - I ) is 56 mV, close to the expected value of 59 mV. At 5.0 mM this slope is only 34 mV and the plot is slightly curved downward. This is the expected result if the reference electrode is shifted positively during the rising part of the voltammogram.
VOLTAGE/V vs.Ag 0.0
~=-
2.0
\/
0.5 i
0.3 I
0.5 I
0.3 I
0.00
0.04
i ~
0.08
4,0 11 m M
Fig. 4. Differential pulse voltammograms of ferrocene in DMF at two concentrations (LSB microcell).
Table 3 Curve crossing by cyclic voltammetry at 5.0 mM with a scan rate of 0.02 V s-i Micro E / m V cell CP2Fe+DM F DMAMF+ H20 a DMAMF+ H20 b LSB - 90 0 SSB - 140 - 30 SSD - 30 LSD - 70 a In water-saturated atmosphere. b In ambient atmosphere.
- 40 -
Finally, we note that this behavior is not peculiar to ferrocene. Narrow D P V peaks, curved plots of peak current vs. concentration and curve crossing were all observed for the reduction of p-nitrotoluene at high concentrations ( > 1 mM). 3.2. Quasi-reference polarization and cell geometry To test this interpretation of the data experimentally, we made two predictions: (1) curve crossing observed by CV will be less dramatic if the working electrode was a microdisk because the disk would generate less ferrocenium than a band, and (2) curve crossing will be more dramatic as the spacing between electrodes decreases. The curve-crossing data for the oxidation of 5 m M ferrocene at 0.02 V s-1 with the four microcell geometries are recorded in Table 3. (Curve crossing was quantified by subtracting the E l l 2 on the forward wave from that on the reverse scan.) The microcells with band working electrodes (LSB and SSB) exhibit significantly more curve crossing than those with disk electrodes (SSD and LSD). This is consistent with prediction (1). We then tested our prediction for electrode spacing. For the two microcells with band working electrodes, the one with larger spacing (LSB) has less curve crossing than the one with smaller spacing (SSB), as predicted. For the two microcells with disk working electrodes, the one with larger spacing has more curve crossing than the one with smaller spacing. Although this last observation is surprising, the difference is small (but reproducible). If the drop is maintained in an atmosphere saturated with solvent, curve crossing is not as severe as when the drop is exposed to ambient atmosphere. For example, when studying the oxidation of D M A M F in aqueous solutions with the LSB microcell, curve crossing was - 4 0 mV in ambient atmosphere (Table 3). However, if the drop is in an atmosphere saturated with water, very little or no curve crossing is observed. An explanation for the effect of the atmosphere was suggested when we observed a dust particle in a droplet exposed to ambient atmosphere. The particle was tumbling around inside the drop, indicating that convec-
M.E. Clark et al. /Journal of Electroanalytical Chemistry 385 (1995) 157-162
tion due to evaporative cooling of the surface can be a significant mode of mass transport from the working electrode to the reference electrode. However, consistent with what is expected for microelectrodes [16], the convection is not sufficient to affect flux at the microelectrode surface. We have never observed that currents depend upon exposure of the droplet to the ambient atmosphere. Diffusion, as well as convection, contributes to curve crossing since the SSB microcell exhibits - 3 0 mV of curve crossing even when in an atmosphere saturated with water (Table 3). Cyclic voltammetry experiments at single bands and band arrays indicate that diffusion over the distance separating the working and reference electrodes is significant on time-scales of about 1 s or more [13]. All these results indicate that convection and diffusion of electrogenerated products can cause quasi-reference instability when reference and working electrodes are only slightly separated.
3.3. Effect of volume and cell geometry The total solution volume may affect peak currents in three ways: (1) if the working electrode is not fully covered, (2) if evaporation is significant (since the surface-to-volume ratio is greater in smaller volumes) and (3) if the dimensions of the drop are too small to allow full development of the diffusion layer. To test the effect of solution volume on peak current, we recorded voltammograms of aqueous 1 mM D M A M F at volumes between 0.02 and 2 txl. The data for the four microcell geometries are recorded in Table 4. All these data were recorded in a nitrogen atmosphere which has been bubbled through an aqueous solution of the electrolyte. (The smallest volumes could not be tested using the microcells with disk electrodes because the droplets do not adhere to the hydrophobic Tefzel sufficiently to be removed from the syringe tip. Band working electrodes provide better adhesion to the drop, allowing voltammetry in smaller volumes.)
Table 4 Dependence of current on volume for four microcell geometries: DPV of 1 mM aqueous D M A M F in an atmosphere "saturated" with water
Volume/
I(LSB)/
I(SSB)/
/(SSD)/
I(LSD)/
~1
~A
ixA
nA
nA
2 1 0.5 0.2 0.1 0.05 0.02
0.94 0.98 0.93 0.93 0.63 0.48 0.52
1.25 1.26 1.25 0.91 0.83 0.73 0.34
55 57 57 65 78 91 -
54 55 49 56 60 -
(100) a (100) (100) (94) (74) (51) (37)
a Percentage of band covered.
-
161
The data for the LSB microcell in Table 4 are similar to those that we have reported for ferrocene in D M F [12]. The peak current is approximately independent of volume down to 0.5 ~xl. However, smaller volumes do not cover the working electrode fully, and so only a fraction of the expected current is recorded. The peak current is approximately proportional to the fraction of the working electrode covered. The data for the SSB microcell are similar (absolute currents are larger because of the longer bands). With a disk working electrode, we expected the peak current to be independent of volume since the working electrode is fully covered by all volumes studied. However, with these microcells peak current increases slightly at the smallest volumes. This suggests that the surface-to-volume ratio is large enough for evaporation to be significant despite our attempts to saturate the atmosphere with solvent. To confirm the significance of evaporation, we recorded voltammograms either immediately after placing a drop on the microcell or after waiting for 120 s. Using a 1 ixl volume, peak currents were independent of time ( + 2 % ) . However, when using 0.1 ixl drops, peak current increased by more than 10% after 120 s (12 replicates for each experiment at both the SSD and LSD microcells). Presumably, evaporation is equally important when using the band microcells, but the effect is not so apparent because the fraction of electrode covered is the more important effect. Finally, the volume may affect faradaic currents if the drop is smaller than the diffusion layer which would normally form. For example, 0.2 ~1 of D M F solution covers the band of the LSB microcell fully 1, yet peak currents are about 90% of those recorded in larger volumes. The probability that a molecule with diffusion coefficient D arriving at the electrode at time t comes from beyond distance r assuming cylindrical diffusion can be described by [17]
W(r, t) = e x p ( - r Z / a D t )
(1)
We have measured the thickness of the 0.2 pA drop to be 0.023 cm and the D for ferrocene in D M F to be 9.8 x 10 -6 cm z s -1. Eq. (1) predicts that, after 5 s, 7% of the flux would be molecules arriving from beyond 0.023 cm (if the solution volume were larger). This is qualitatively consistent with our observation of a 10% decrease in peak currents as measured by DPV at a scan rate of 0.01 V s -1. Also consistent with this, if
i While 0.2 txl of aqueous solution forms a hemisphere of radius less than 1 ram, organic solvents spread out over the surface of the microcell and 0.2 ~1 of D M F fully covers the electrodes.
162
M.E. Clark et al. /Journal of Electroanalytical Chemistry 385 (1995) 157-162
peak currents are measured by high speed cyclic voltammetry, peak current is independent of volume down to 0.2 I~1 [12].
pensing the smallest volumes onto these electrodes is not possible. Acknowledgements
4. Conclusions (1) Tefzel microcells can be used to perform-electrochemical analysis in volumes as small as 0.02 ¢1. However, several experimental difficulties may be anticipated. (2) When the quasi-reference electrode is very close to the working electrode, products may arrive at the reference electrode by diffusion and convection. This induces reference electrode instability which may lead to curve crossing (cyclic voltammetry), narrow peaks (DPV) and curvature of calibration curves (DPV). This problem can be avoided by using a larger spacing between electrodes or by performing more rapid experiments. (3) Drop size may affect the development of the diffusion layer during long experiments and result in a decrease in faradaic currents. This can be avoided by performing more rapid experiments. Eq. (1) is useful for predicting when this effect is significant. (4) In very small volumes ( < 0.2 I~1), the surface-tovolume ratio is so large that evaporation of solvent may be a problem despite attempts to saturate the atmosphere with solvent. Faradaic currents are increased, and precision is degraded when evaporation is significant. This problem is, of course, less significant in less volatile solvents (e.g. DMF). (5) Under our conditions, little is to be gained by using disk working electrodes. Although the disk electrodes are fully covered by all volumes, evaporation is significant at the lowest volumes. Furthermore, dis-
We thank Brian Swift for bringing Eq. (1) to our attention. This research was supported by grants from Research Corporation and Hobart and William Smith Colleges. References [1] P.N. Bartlett and R.G. Whitaker, Anal. Chem., 61 (1989) 2803. [2] T. Miwa, Y. Nishimura and A. Mizuike, Anal. Chim. Acta, 140 (1982) 59. [3] J.O. Schenk, E. Miller and R.N. Adams, Anal. Chem., 54 (1982) 1452. [4] A.S. Baranski and H. Quon, Anal. Chem., 58 (1986) 407. [5] A.S. Baranski, Anal. Chem., 59 (1987) 662. [6] A.R. Harman and A.S. Baranski, Anal. Chim. Acta, 239 (1990) 35. [7] M. Morita, M.L. Longmire and R.W. Murray, Anal. Chem., 60 (1988) 2770. [8] T.T. Wooster, M.L. Longmire, H. Zhang, M. Watanabe and R.W. Murray, Anal. Chem., 64 (1992) 1132. [9] M. Wojciechowski and J. Balcerzak, Anal. Chim. Acta, 237 (1990) 127. [10] J. Kulys and E.J. D'Costa, Anal. Chim. Acta, 243 (1991) 173. [11] P.R. Unwin and A.J. Bard, Anal. Chem., 64 (1992) 113. [12] W.J. Bowyer, M.C. Clark and J.L. Ingram, Anal. Chem., 64 (1992) 459. [13] D.M. Odell and W.J. Bowyer, Anal. Chem., 62 (1990) 1619. [14] C.-L. Wang, K.E. Creasy and B.R. Shaw, J. Electroanal. Chem., 300 (1991) 365. [15] A.M. Bond, M. Fleischmann and J. Robinson, J. Electroanal. Chem., 168 (1984) 299. [16] P.M. Kovach, W.L. Caudill, D.G. Peters and R.M. Wightman, J. Electroanal. Chem., 185 (1985) 285. [17] W. Jost, Diffusion in Solids, Liquids, and Gases, Academic Press, New York, 1960, p. 17.