(15) Raikhbaum, Ya. D., Mal kh, V. D.,
opt. Spectry ( U S S R ) ( E n g i L transl.)
9, 223 (1960). (16) Raikhbaum, Ya. D., Malykh, V. D.,
Opt. Spectry. ( U S S R ) (English transl.)
10, 269 (1961). (17) Roes, R. L., Ph.D. thesis, University of Utrecht, 1962. (18) Snelleman, W., Ph.D. thesis, University of Utrecht, 1965. (19) Van Stekelenburg, L. H. M., Physicu 12, 289 (1946).
(20) Suits, G. C., J. Appl. Phys. 10, 730 (1939). (21 ) Vukanovie, V., “Emissionsspektroskopie,” p. 9, R. Ritschel, G. Holdt (edit.), . Akademie-Verlag, Berlin, 1964. (22) Walker, R. E., Monchick, L., West-
enberg, A. A., Favin, s., “Conference on Physical Chemistry in Aerodynamics and Space Flight,” p. 221, Pergamon Press, New York, 1961. (23) Walker, R. E., Westenberg, A. A.,
J . Chem. Phys. 29, 1147 (1958), 31, 519 (1959). (24) Walker, R. E., Westenberg, A. A., J. Chem. Phys. 32, 436 (1960). (25) Zaidel, A. N., Kaliteevskii, N. I.,
Lipis, L. V., Chaika, M. P., “Emission Spectrum Analysis of Atomic Materials” ( U S S R ) (English transl.), U. S. At. Energy Comm., AEC-tr-5745,1963.
RECEIVED for review December 10, 1965. Accepted February 7, 1966.
Unusual Adsorption Effects in the Electrochemical Reduction of Flavin Mononucleotide at Mercury Electrodes A. M. HARTLEY and G. S. WILSON Department o f Chemistry and Chemical Engineering, University of Illinois, Urbana, 111. 6 7 803 The adsorption of flavin mononucleotide (FMN) in acid solution has been studied by polarography, cyclic voltammetry, and chronopotentiometry. Conventional d.c. polarography shows a prewave which bears a superficial resemblance to the classical Brdicka prewave. Experiments which allow greater time for attainment of adsorption equilibrium result in the disappearance of the prewave. The observed behavior has been attributed to slow adsorption of the oxidized form resulting in the formation of a stable electroactive film, Estimation of equilibrium surface coverage of oxidized and reduced forms gave values for the latter which were almost twice that of the former. The mechanisms of the electrode processes are discussed in light of the evidence from cyclic voltammetry and chronopotentiometry.
T
CLASSICAL d.c. polarographic studies of Brdicka (2) provide a basis for the investigation of adsorption of electroactive species. The adsorption of products and/or reactants of a reversible polarographic reduction results in the appearance of an additional small anomalous adsorption controlled wave. If the product is adsorbed, this wave, referred to as a prewave, appears a t potentials more oxidizing than the main faradaic process; adsorption of the reactant results in the appearance of a postwave. The position of the adsorption wave with respect to the main wave will reflect the relative influence of both oxidized and reduced forms in the event that both are adsorbed. Interpretation of adsorption phenomena on the basis of the above HE
thermodynamic arguments is valid only if adsorption equilibrium is attained a t the electrode surface within the lifetime of a mercury drop. The presence of a polarographic prewave for FMN (5’-riboflavin phosphoric acid) in acid solution indicates the predominant influence of the reduced or leuco form. Breyer and Biegler (3) in an a x . polarographic study of riboflavin first showed that the oxidized form is also adsorbed. Senda, Senda, and Tachi (17’) have shown that FMN gives a x . and d.c. polarographic results similar to those obtained for riboflavin itself. Takemori (19) has investigated the adsorption of FMN by chronopotentiometry . The present investigation concerns the influence of adsorption of the oxidized form. Chronopotentiometry and cyclic voltammetry which can allow greater time for adsorption equilibrium are used to illustrate an effect which cannot be easily demonstrated by ordinary polarography. It will be shown that the prewave in this case resembles the Brdicka prewave only because during the lifetime of a mercury drop no appreciable adsorption of the oxidized form can take place. EXPERIMENTAL
Apparatus. A three-electrode polarograph of conventional design (5) constructed using Philbrick K B X A KSPA operational amplifiers (George A. Philbrick Researches, Inc., Dedham, Mass.) was employed for the linear sweep and polarographic studies. To perform cyclic linear sweep studies, a triangular wave generator, described previously by Weir and Enke ( M ) , was used. By using a square wave pulse of variable gate width, the sweep generator could be programmed to
hold a specific potential followed by a desired number of cyclic scans. An operational amplifier current source (Philbrick USA-3) in the operational feedback current control configuration (10) was used for chronopotentiometric measurements. A 1.35volt mercury battery in series with a variable resistor served as the current reference source. For current reversal applications, the mercury battery was replaced with a controlled amplitude square wave obtained from the triangular wave generator. The controlled current was determined by measuring the iR drop across a precision resistor in series with the electrolysis cell. To eliminate large anodic potential excursions prior t o the application of cathodic current, a double pole relay system similar to that employed by Osteryoung (15) was used for the current switching operation. This arrangement allowed control of the indicating electrode potential during equilibration prior t o the electrolysis. Such control is especially important when the adsorption being measured is potential dependent. Unless otherwise indicated, a potential of f 0 . 1 volt us. the reference electrode was maintained during equilibration. Transition times for an electrode of approximately 4 x 10-2 cm.2 ranged from 15-150 msec. so that the spherical diffusion contribution was neglected. Current-voltage or voltage-time curves were observed with a Houston Instruments Model HR-97 x-y recorder or a Tektronix 561A oscilloscope. The latter, which was equipped with two identical Model 3A72 differential preamplifiers for the vertical and horizontal axes, was used for the linear sweep experiments above 0.3 volt/ second. For the chronopotentiometric investigations, a Model 3B3 time base was substituted for the horizontal amplifier. The oscilloscope traces produced were photographed using a VOL. 38, NO. 6, M A Y 1966
681
water prepared by filtration through activated charcoal and distillation from alkaline permanganate in an all-glass still. Mercury was purified by vacuum distillation.
A
RESULTS
E VS SSCE
Figure 1. Polaroarams of a 2.38 X el;ctrochemical red;ction in 0.1 M HCIOd.
Tektronix C-12 camera with a Polaroid back and Type 47 Polaroid film. Where it was desired to enlarge the trace, positive transparency Type 46L Polaroid film was used. The resulting slide was projected on graph paper on which a tracing could be made. This technique was found to be particularly useful for the graphical integration of areas under current peaks using a planimeter. The cell used for a variety of electrochemical experiments was constructed of a 75/50 ball and socket joint, the former part being sealed off to provide a flat-bottomed container of 70 ml. volume; the latter was fitted with a Teflon cover into which six outer standard taper joints were inserted and sealed with epoxy cement. The cell was immersed in a separate water jacket connected to a constant temperature bath which was maintained a t 25 f 0.5' C. A dropping mercury electrode (DME) of conventional design with an open circuit drop time of about 4.6 seconds at 50 cm. mercury column height served as the indicating electrode or as a source of mercury drops for a hanging mercury drop electrode (HMDE). The latter was of conventional design (4) except for a 30 mm. horizontal offset in the glass tubing above the platinum wire seal. This modification allowed the placement of the tip of the electrode in various parts of the cell by rotation of the electrode about a fixed vertical axis provided by a 10/30 standard taper joint and a Teflon thermometer adapter (Kontes Glass Co., Vineland, N. J.). To perform time dependent adsorption studies while applying a potential, a microburet hanging drop electrode (MHDE) was 682
0
ANALYTICAL CHEMISTRY
10-4M solution of FMN produced b y Increasing reduction from' A-D
employed. This was constructed by modification of a 0.25-ml. microsyringe buret (California Laboratory Equipment Co.); details are given elsewhere (25'). The counter electrode was a large (3-4 cm.2) or small (1 cm.2) platinum flag. The latter was isolated in a separate compartment constructed by cutting a fritted glass (10 mm.) sealing tube flush with one side of the frit. The reference electrode was a saturated sodium chloride calomel electrode (SSCE). This was connected to the cell with a U-shaped salt bridge of 10 mm. diameter which terminated a t each end with sintered glass frits and was filled with supporting electrolyte. Materials. All chemicals were of reagent grade unless otherwise specified. Stock solutions were prepared by determinate weighing. The FMN (sodium salt), obtained through the courtesy of the Sigma Chemical Co., was used without further purification. To avoid decomposition, a dilute aqueous stock solution of FMN was prepared so that aliquots of less than 10 ml. could be frozen and stored in 50-ml. volumetric flasks. These were then diluted to the appropriate volume with supporting electrolyte as needed. Experiments were carried out in a darkened room to avoid photolytic decomposition. Solutions were deaerated with nitrogen purified by passage through two wash towers containing acidic vanadous sulfate over zinc amalgam followed by a final wash through distilled water. Perchloric acid solutions were obtained by dilution of double vacuum distilled 70% perchloric acid (G. F. Smith Chemical Co.) with distilled
Polarographic Data. I n 0.1M perchloric acid (pH 0.85), F M N exhibits two polarographic waves: a prewave followed a t more negative potentials by the main reduction wave. Adsorption control of the prewave and diffusion control of the main wave were verified by mercury column height dependence studies and agreed with those reported previously (7'). A typical polarogram obtained with a solution containing only the oxidized form of FMN is shown in Figure 1.4. The current-time behavior on the prewave plateau is indicative of adsorption (8). At concentrations less than 6 X 10-6M the height of the prewave is linearly dependent on conthe centration; above 2 X lO-'M limiting current remains essentially constant. In the concentration range of 3-8 X 10-'M the prewave is very close to the main wave which in turn is quite measurably distorted. With increasing concentration, however, the prewave shifts to more positive potentials, and the upper portion of the main wave returns to a more ideal appearance. The products of the reversible electrochemical reduction are a semiquinone resulting from the addition of one electron and a completely reduced two electron product (FMNH2) ( 7 ) . The concentrations of the oxidation states of FMN are defined by a chemical equilibrium involving all three species. As Michaelis (11) and Muller ( l a ) have shown, the intermediate or semiquinone state is not sufficiently stable in this case to result in the observation of two one-electron steps. On the rising portion of the polarographic wave, however, a significant concentration of the semiquinone is present a t the electrode surface thus yielding a slope which reflects more closely a one rather than a two electron process. The Michaelis index potential (11) which is a measure of this distortion has been used to evaluate the extent of semiquinone formation. The total cathodic current (i*) including that of the prewave was found to be proportional to the bulk concentration of the oxidized form from 3.81 X 10-3 to 4.00 x lO-SM FMN. The diffusion current constant ( I = i/Cm21a W) was found to be invariant within experimental error over this range. Polarograms of mixed FMN-FMNH2 solutions produced by controlled potential electrolysis are shown in Figure 1B-D. With increasing reduction the height of the anodic wave increases
while that of the cathodic wave decreases. The sum of the cathodic (&) and anodic currents ( i A ) after the usual correction for residual current, is a constant within experimental error a t all points during the reduction. Exhaustive controiled potential electrolysis gives a value of n = 2 (1.98) for the overall process in agreement with Ke (7). Since it was not possible to evaluate the contribution of the direct oxidation or reduction of the semiquinone to ic or i d by electrochemical techniques, an independent nonelectrochemical method was used to determine the concentration of the semiquinone. Simultaneous measurements of solution absorbance a t a wavelength characteristic of the semiquinone and polarographic determination of the extent of solution reduction were used to evaluate the semiquinone concentration a t any point during electrolysis (6). Spectral measurements in the ultraviolet and visible region verified that the products of the electrochemical reaction were the same as those produced chemically. This method gives results for semiquinone concentration in good agreement with the alternative procedure of evaluating the s2miquinone formation constant through the index potential (11). For example, the spectral method indicates that 35y0 of the total flavin in a haIfreduced solution exists as the semiquinone while the index potential measurements yield a calculated value of 41% (Table I). In Figure 2 the electrocapillary curves for FMN in 0.1M perchloric acid are given. Lowering of mercury drop surface tension in both faradaic and nonfaradaic regions indicates significant adsorption of both reduced and oxidized forms, respectively, in unstirred solution during the lifetime of the drop. The abrupt change in the surface tension a t about -0.050 volt is coincident with the prewave process. The polarographic data are summarized in Table I. Cyclic Voltammetry. Linear sweep chronoamperometry or cyclic voltammetry was employed t o allow greater variation in the time scale of the electrochemical experiment. The discussion will be restricted to the reversible case first derived by Randles (16) and Sevcik (18) and more recently by Nicholson and Shain (14). The system is expected to obey the Nernst Equation and hence the concentration ratios a t the electrode surface will be determined by the potential. Since rapid equilibrium is achieved, the shape of the current-voltage curve should be independent of scan rate. Therefore two diagnostic criteria for the simple reversible case are the invariance of the peak potential with scan rate and a theoretical separation of anodic and cathodic peak potentials of 57.00jn mv. where n is the number of electrons
46
t
39'
Figure 2.
I
I
0
-0.2
I
I
-0.4 -0.6 E vs. SSCE
I
-0.8
-
1
1.0
Electrocapillary curves for FMN in 0.1M HCIO4
(0)0.1M HClOa, (e) 1.33 X 10-4M FMN, IB)3.81 X lO-'M FMN
transferred in the reversible process. Cyclic voltammetric studies of FMN in 0.1M perchloric acid were carried out using the MHDE previously described. Two sets of peaks were observed; one corresponding to the prewave in polarography, the other to the main faradaic process. The shape and position of the prepeaks, however, were dependent on scan rate and on the elapsed time between the formation of the mercury drop and the electrolysis experiment. Figure 3 shows the effect of equilibrium time on the prepeak a t constant scan rate. As mentioned earlier, the potential of the electrode was maintained at +0.1 volt during equilibration. When a scan is begun immediately after forming a drop, a sharp cathodic prepeak is observed which is reasonably well separated from the main process.
With increasing equilibration time, the prepeak is observed to merge with the main peak and ultimately disappear entirely. No shift is observed in the main cathodic or anodic peak potentials; however, a slight increase in the peak currents is observed. The process is slow, requiring a t least 15 minutes for the disappearance of the prepeak. If the mercury drop is allowed to equilibrate until no prepeak is observed and the drop size is increased by forcing additional mercury out of the capillary, the prepeak is again observed. This supports the notion that the equilibration results in the formation of a stable film not reducible in the prewave region. Exposing a fresh mercury surface on the same drop permits the prepeak electrolysis on the uncovered surface. Figures 3A and 4 show the effect of
Table I. Polarographic Parameters of FMN in 0.1 M Perchloric Acid Half-wave potential (us. SSCE)s -0.025 f 0.005 v. (Prewave) Half-wave potential (us. SSCE) -0.112 f 0.002 v. (Main Wave) Diffusion current constant 3.03 f 0.02 Diffusion coefficient* 6.25 X 10-6 cm.2 sec.-1 Saturation surface coveragec 1.25 f 0.08 x 10-10 mole cm.-2 Index potentiald 24.0 f 1 . 0 mv. 1.23 f 0.01
ne
Measured for 2.38 X 1 O - W FMN. Calculated from Ilkovic Equation. See Reference (2). Calculated for 2.66 X lO-4M FMN. See Reference (11). * Determined from log [(id i)/i]plot.
-
VOL 38, NO. 6, MAY 1966
683
P I
o
0.1
0.2
-0.1
-0.1
L
Figure 4. 01
0
-0.1
I -02 '03 E V I SSCE
Figure 3.
1.90 X
Effect of equilibration time 1 O-4M FMN in stirred solution
-05
Table II.
Scan rate
-0s
on cyclic scan for
A, immediately; B, after 5 minutes; C, after 15 minutes. voIt/sec.
scan rate on current-voltage curves taken immediately after formation of the drop. With increasing scan rate, the cathodic prepeak shifts to more negative potentials while all other peaks remain essentially invariant. More important than the prepeak potential is the broadening of the prepeak itself. At 25 volts/sec., for example, the half peak potential on the cathodic side of the prepeak corresponds to a point which is a t least 25y0 up the main peak. Representative data are summarized In Table TI. (The peaks are numbered as indicated in Figure 4.) At low scan rates (less than 0.3 volt/ sec.) the anodic prepeak (IV) splits to give another peak (V) a t slightly more anodic potentials. The presence of this peak appears t o be scan rate dependent but is actually related to the time during which the scan potential is in a region more cathodic than the main process. If the potential is held a t such negative values for about 30 seconds prior to the anodic scan, the shape of peaks (IV) and (V) is practically independent of the anodic scan rate over a fivefold range. The area under the prepeaks
1.90
I
I
-0.
x
ANALYTICAL CHEMISTRY
' 0 . '
Scan rate 0.250 volt/rec. (Peaks numbered to correspond with Table II Dotted lines define integrated peak area)
.
Scan rate 0.050
Peak area (pcoulomb)
I1 I11 IV I IV V 0.160 0.108 0.45 0,162 0.112 0.038 0.43 0.158 0.110 0.038 0.44c 0.72d 0.158 0.109 0.038 0.44 0.7Zd 0,160 0.111 0.037 0.45 0.72d 0.158 0.108 0.037 0.56 0.40 0.29 0.158 0,108 0,037 0.56 0.38 0.45 0.159 0.110 0.037 0.63 0.29 0.42 10-4M in 0.1M HClOa. Electrode area 0.038 cm.* From peak 1.14. b Roman numerals refer to numbered peaks (see Figure 4). c Corresponds to surface coverage of r = 0.6 X 10-lo mole cm.-* d Indicates sum of areas of peaks IV and V.
684
-0.1
Effect of scan rate on prepeak process for
was estimated by graphical integration using a planimeter. It was assumed that the prepeak and the main process could be separated by integrating the peak areas enclosed by the dotted lines (Figure 4). As indicated in Table 11, the area under peak (I) is independent of scan rate a t the higher scan rates but increases in the lower range studied. Characteristics of F M N Adsorbed Films. The adsorbed oxidized film was examined directly by equilibrating a H M D E in a solution of F M N . After about 10 minutes the electrode was removed, washed with 0.1M perchloric acid, and transferred to a solution containing only perchloric acid. The transfer operation was performed under a nitrogen atmosphere in the cell previously described. Several electrodes were checked to ensure that the results obtained were not artifacts. Only one set of peaks was observed which corresponded very closely (-0.050 volt us. SSCE) to the prepeak mentioned above. The separation of the cathodic and anodic peaks was less than 10 mv. as was the variation of peak
( -Peak potential us. SSCE)b
I 0.077 0.057 0.051 0.048 0.048 0,045 0.042 0,039 0 FMN concn. 4.65 x (11-111) separation n =
-0.4
SCCC
1 0 - 4 ~FMN
Linear Sweep Studies of FMN Prewaveo
(v. set-1) 25.0 5.0 2.5 1.0 0.50 0.25 0.10 0.05
va.
-0.1
potential with scan rate from 0.050 to 25.0 volts/sec. No anodic peak splitting was observed, contrary to the previous experiments. Integration of peak areas showed no detectable desorption as a result of the electrolysis process. Desorption was slow a t an applied potential of 0.0 volt us. SSCE, requiring more than 10 minutes. The film transfer method was used to measure the rate of attainment of adsorption equilibrium of an FMN solution. A HMDE was immersed for a specified time in a rapidly stirred solution of FMN, transferred as previously described, and the surface coverage measured by chronopotentiometry using the relationship r = &/2F Figure 5 shows the time dependence of surface coverage and indicates that less than half the equilibrium coverage would be obtained within the lifetime (about 5 sec.) of a mercury drop from a DME. This value probably represents a maximum limiting value since equilibration was accelerated in this case by stirring. A final equilibrium surface coverage of 0.7 X 10-'0 mole cm.-2 was obtained in this manner. Chronopotentiometry. A series of chronopotentiometric experiments analogous to the linear sweep studies was performed using the M H D E . I n Figure 6 the effect of varying the current density on solutions run immediately is indicated. As expected, the adsorption prewave dominates the overall process a t high current densities although the prewave transition is ill-defined. An examination was made of the transition times, 71, for the prewave and r2 for the main wave. The chronopotentiometric model for reactant adsorption reduction followed by diffusion controlled depletion of the solution reactant predicts a constancy of iorl and &r2lI2with varying current density (io). The system did not conform to this model. At low current densities there is a diffusion contribution to the prewave indicated by a slight
8.0
7.0
80 N I 0
2
LO
-
L
- 40 0
X
L
30
Figure 6. Influence of current density on chronopotentiometric prewave of
&
1-90 x i o - 4 ~FMN
2c
IC'
1
I
I
I
I
I
1
I
0
2
4
6
8
10
12
14
No equilibration. electrolysis +O.l
Applied potential prior to v. VI. SSCE. Curve 113.63 X amp. cm-2, 5 msec./crn.; Curve 2-4.55 X 1 0-4 amp. cm.-*, 10 msec./ cm.; Curve 3-0.91 X amp. ern.-*, 0.2 sec. j c m .
t , (MINUTES)
Figure 5. Effect of equilibration time on transferred film coverage for 3.81 X 1 O-4M FMN in 0.1M HCIOI
increase in r1 with solution stirring. With increasing current density, &rl increases as does ~ o T ~ resulting ~ ' ~ , very likely from a mixed process involving the simultaneous reduction of adsorbed film and diffusing species. At low curamp. cm.+) rent densities (2.27 X measurement of iorlgives a surface coverage of 0.7 x 10-lOmole cm.-2which is in good agreement with the values obtained from the linear sweep experiments a t low scan rates (see Table 11). A series of experiments using current reversal mere employed in the study of the nonequilibrium situation. Reversal of the current a t any time in the prewave region results in a reverse (anodic) transition (0.17 see.) approximately equal to that for the forward (cathodic) prewave electrolysis for equal current densities in both directions. This indicates that all of the material reduced must remain on the electrode. If the current is reversed in the region of the main process a distortion of the anodic portion of the chronopotentiogram just prior to the prewave transition results. This phenomenon appears to be associated with the process which produced peak (V) in the linear sweep studies. The effect of equilibrating the mercury drop with the FMN solution is shown in Figure 7. With increasing equilibration time the prewave becomes less distinct and the overall wave steeper. The disappearance of the prewave through equilibration probably esplains the ab5ence of a prewave in the work of Tatwawadi and Bard (20). As the prewave disappears, the total measured transition time becomes significantly shorter. Incomplete attainment of equilibrium with respect t o adsorption of the oxidized form will lead to high values of the measured transition time. For this reason the HMDE used for equilibrium
surface coverage studies was freshly prepared and allowed to equilibrate with solution stirring a t least 15 minutes between determinations. The surface coverage for the oxidized form was estimated assuming the applicability of several adsorption mechanisms. These have been classified by Murray (IS) according to whether the adsorbed layer is reduced prior to (AR, SR), following (SR, AR) or simultaneously (SAR) with the diffusing species. Graphical evaluation of surface coverage was accomplished by the methods given by Laitinen and Chambers (9). The data are summarized in Table 111. In addition a value for the surface coverage by the reduced form was obtained in 1.33 X lO-4M FMN. This was accomplished by holding the potential of the indicating electrode a t - 0.4 volt followed by the application of an anodic current of high current density in a stirred solution. A surface coverage of 1.22 X 10-lo mole em.-) was calculated from the relationship r = i0rl/2F which is in good agreement with the value obtained from the Brdicka prewave theory (Table I). Anomalous Effects in Polarography. Although somewhat less obvious, the influence of adsorption also leads to anomalous results in polarography, If a polarogram of F M N is obtained a t a scan rate of 0.1 volt/min. in the
cathodic direction, half-wave potentials for the prewave and main wave are both 30 mv. more positive than those found by starting on the diffuqion plateau and scanning in the anodic direction. This hysteresis effect is shown in Figure 8. During the course of bulk solution reduction by controlled potential electrolysis, the polarographic half-wave potentialq obtained by scanning in the cathodic direction remain essentially invariant. Thoqe measured by anodic scanning shift in the anodic direction with increahg re-
>-
I 0
0-
t
W 1
3 2
I
Figure 7. Effect of equilibration time on chronopotentiometric prewave of 1.90 X 1OP4MFMN in stirred solution Current density-4.55 X amp. cm.?; Time base-1 0 msec./cm. Curve 1 -1mmedi1 minute, Curve 3-after ately, Curve 2-after 10 minutes
Table 111. Chronopotentiornetric Adsorption Data AR, SR SR, AR
Concn. (mmol.) 0.381 0.190 0.133 0,095 0.048
r"
Db
1.53 32.9 1.34 3.8 1.29 5.8 1.24 0.60 a Surface coverage X 1010 mole cm.-2 b Diffusion coefficient X 108 cm.2 sec.-1
ra
Db
ra
1.00 1.04 1.10 1.02
1.2 0.8 0.9
1.16 1.09 1.09 0.93
SAR
VOL. 38, NO. 6, MAY 1966
0 6
2.5 2.4 4.1
e
685
duction of bulk solution finally becoming identical with the values obtained from the cathodic scan. The zero current potentials also show a similar displacement depending on scan direction, but this difference is less dependent on the extent of solution reduction. A comparison was made of zero current potentials measured a t various stages of reduction with several methods for determining the solution potential. The equilibrium potential of the mercury el ctrolykis pool was measured with a potcntiometer isolated from the electrode u. nd a voltage follower to prevent p( larization during measurement. The equilibrium potrntial of the mercury pool and the zero current potential taken from the polarogram (cathodic scan) always agreed quite closely. These potentials, especially a t early stages of reduction, were 20-30 mv. more negative than the potential expected on the basis of the ratio of cathodic to anodic limiting currents or the amount of material reduced as determined coulometrically. The expected potentials were calculated using the Michaelis Equation (11) for the potentiometric titration curve assuming a semiquinone formation constant of K = 2. The latter two methods gave potentials which agreed with each other within 5 mv. The discrepancy between the zero current and the ratio measurements decreased with increasing extent of solution reduction. A comparison was also made between polarographic zero current potentials and those measured with a platinum electrode. Again the potentials on mercuiy were consistently more negative. DISCUSSION
It is not possible to fit the adsorption behavior of FMN to one mechanism for the different conditions under which the experiments were run. Both the chronopot( ntiometric and linear sweep studies indicate that the characteristics of the reduction of adsorbed FMN are deper dent upon the attainment of adsol ption equilibrium and the relative influence of film electrolysis with respect to the diffusion contribution. Under conditions where a prewave or prepeak is observed, the following reactions can take place in the prewave region :
+ Oxoda.+ 2e-
O X ~ ~ I2e~ . + Redad,. +
Red,&.
(1)
(2)
Reaction 1 is the classical Brdicka reaction in which diffusion controlled adsorption is assumed. The increase of prewave transition times with stirring and the increase in the coulombs associated with the prewave process by lowering current densities and scan rates 686
ANALYTICAL CHEMISTRY
!-
6 K K
= 0 I
0
t0,l
0
-0.2
-0.1
E
VS.
-0.3
-0.4
SSCE
Figure 8. Effect of scan direction on polarographic wave for 2 Scan direction indicated by arrows
are used to verify the diffusion contribution (Reaction 1) to the prewave process. It is necessary to postulate the alternative path of Reaction 2 because the oxidized form is not in rapid adsorption equilibrium with the electrode surface. The equilibrium thermodynamic arguments of Brdicka cannot be strictly applied in this case. The possibility of the occurrence of Reaction 2 in the prewave region has been shown by the film transfer studies. No adsorption-desorption equilibrium is established after the equilibrated film is transferred to the supporting electrolyte containing no depolarizer. I n this case the prior equilibration process merely serves as a means for forming the adsorbed film. The characteristics of the transferred film-e.g., peak potentials and peak shapes-are essentially independent of the fraction of surface coverage. It seems reasonable, therefore, that the film transfer is analogous to the nonequilibrium case and establishes the possibility of Reaction 2. Because of the apparent irreversibility of the cathodic prepeak process, it is not possible to increase the scan rate or current density to separate the components due to Reactions 1 and 2. The estimate of sur ace coverage given in Table I1 must be regarded as a maximum value for ro, under the conditions of these experiments since it may include a contribution from Reaction 1. With increasing scan rate the cathodic prepeak shifts to more negative potentials so that the main faradaic process given by Reaction 3 below can occur simultaneously.
+
O X ~ ~ I2e~.
* Red.,l,.
(3)
It should be noted that the anodic pre-
X 10-4M FMN.
peak process (IV) does not show the irreversibility of the corresponding cathodic process (Table 11). The extent of prepeak electrolysis is not sufficient for reduced form monolayer saturation when compared with the calculated value from polarography. There is good evidence (1) that electrolysis will proceed under conditions where monolayer saturation is expected, to complete coverage of the electrode with the reduced form without the occurrence of desorption and that the reduced form adsorption equilibrium is very rapid. In order to justify further electrolysis in the presence of a completely covered surface, either rapid desorption of leuco FMN to accommodate the product of the continuing reduction or reduction through an adsorbed layer must be postulated. Equilibrium surface coverage measurements lead to higher values for the reduced form than for the oxidized form (1.22 X 10-l0 and 0.7 X 10-10 mole cm.2). If desorption equilibrium were attained in the electrolysis of a saturated monolayer of leuco FMN only 57y0 of the product (FMN) would remain adsorbed on the electrode surface. Conversely a saturated coverage of FMN could be completely reduced to leuco FMN a t less than full coverage for the latter. The appearance of peak (V), Figure 3, and the anodic distortion of the current reversal chronopotentiogram may reflect the film reorientation process associated with the oxidation of leuco FMN. The shift in the prepeak process resultingfrom equilibration of the electrode with the solution causes the electrolysis of the oxidized film in the potential region of Reaction 3. Therefore, it is concluded that the equilibrated film is reduced either a t the same time (SAR)
or following (SR, AR) the diffusion process. By allowing sufficient time for adsorption equilibrium of the oxidized form to be achieved, the potential for its reduction is as least as cathodic as the main process. At very low concentrations, complete constancy of io7 with varying current density is observed and the system approaches the behavior of transferred film reduction in which there is no diffusion contribution. The above conclusions are in good agreement Kith the results reported by Takemori (19) who obtained a value of 0.84 X 10-lo mole cm.? for the oxidized form in 1.0 x 10-4M FMK with 0.1M citric acid as the supporting electrolyte. His calculations were made on the basis of the SR, AR mechanism. In the polarographic case, the variation of half-wave potential with scan direction and the extent of solution reduction is a clear indication of a mixed potential process. The electrode potential is affected not only by the main faradaic process but also by the adsorption phenomena associated with it. Lacking detailed information regarding the adsorption isotherms, molar free energies of adsorption, adsorptiondesorption rates of the individual species and their influence on each other, it is not possible a t present to quantitatively justify the polarographic potential shifts observed. This representation of the adsorption process as one involving only the oxidized and reduced forms has neglected the possible influence of the semiquinone. At present little is known about the adsorption behavior of this intermediate form which could prove important in view of its significant concentration in solution during
electrolysis. Calculations from molecular models indicate a surface coverage for the oxidized form of 1.5 X mole cm.+ assuming a planar orientation of the electrode and the isoalloxazine ring system. In the leuco form, however, the entire ring system cannot be coplanar owing to the quinoid structure of the middle ring. Under such conditions the reduction of an adsorbed layer would also imply reorientation. The observed slow adsorption of the oxidized form suggests the possibility of a preferred electroactive orientation resulting from the reorientation of initially adsorbed material. Such a process would account for the shift of the prepeak to more negative potentials and the creation of an electroactive film sufficiently stable to survive film transfer in the latter studies. It is possible that the initial approach of FMN occurs “edge on” with the plane of the rings normal to the electrode surface to produce a weakly bound unstable state which slowly reorients t o a stable film in which the ring system is coplanar with the electrode. The existence of a polarographic prewave even for a reversible electron transfer does not prove the predominant influence of the reduced form on an equilibrium basis. To correctly evaluate the overall adsorption process, isotherms for the individual forms must be known. The prewave alone cannot be used as a basis for deciding on the mechanism of film electrolysis since the mechanism can change as the experimental parameters are varied. The combination of several electrochemical methods has facilitated the formulation of a consistent picture of FMN adsorption.
LITERATURE CITED
(1) Biegler, T., Laitinen, H. A., J . Phys. Chem. 68, 2374 (1964). (2) Brdicka, R., Collection Czech. Chem. Commun. 12, 522 (1947). (3) Breyer, B., Biegler, T., Zbid., 25, 3348 (1960). (4) DeMars, R. D Shain, I., ANAL. CHEM.29,1825 (19k7). (5) Enke, C. G., Bsxter, R. A., J. Chem. Educ. 41,202 (1964). (6) Hartley, A. M., Wilson, G. S., unpublished results, 1966. (7) Ke, B., Arch. Biochem. Biophys. 68, 332 (1957). (8) Koryta, J., Collection Czech. Chem. Commun. 18,206 (1953). (9) Laitinen, H. A., Chambers, L. M., ANAL.CHEM. 36, 5 (1964). (10) hfalmstadt, H. V., Enke, C. G., Toren, E. C., “Electronics for Scientists,” p. 370, W. A. Benjamin, New York, 1962. (11) Michaelis, L., Schubert, &I P., . Smythe, C. V., J. Biol. Chem. 116, 587 (1936). (12) Muller, 0. H., Ann. N . Y . Acad. Sci. 40,91(1940). (13) Murray, R. W., J . Electroanal. Chem. 7. 242 (1964). (14) Nicholson, R. S., Shain, I., ANAL. CHEM. 36,706 (1964). (15) Osteryoung, R . A,, Ibid., 37, 429 (1965). (16) Randles, J. E. B., Trans. Faraday SOC.44.327 11948). (17) Senda, hi., Senda, M., Tachi, I., Rev. Polarog. (Kyoto) 10, 142 (1962). (18) Sevcik, A., Collection Czech. Chem. Commun. 13, 349 (1948). (19) Takemori, Y., Rev. Polarog. (Kyoto) 12,63 (1964). (20) Tatwawadi, S. V., Bard, A. J. ANAL.CHEM.36, 2 (1964). (21) Weir, W. D., Enke, C. G., Rev. Sci. Instr. 35, 833 (1964). (22) Wilson, G. S., Ph.D. Thesis, University of Illinois, 1965. ~
RECEIVED for review Jan. 7, 1966. Accepted February 25,1966. This work was supported by the National Institutes of Health Grant USPH GM 12009.
Cryoscopic Titrations Principles of a N e w Method of End-Point Detection STANLEY BRUCKENSTEIN and NICHOLAS E. VAN DERBORGH School of Chemistry, University of Minnesota, Minneapolis, Minn.
b An experimental apparatus for continuously recording the variation in the freezing point depression during the course of a titration is described and applied to several model aqueous systems. Close approach to equilibrium conditions is attained in the titration of a strong and a weak acid and their mixture with strong base, and in the titration of chloride ion with silver ion. A number of bases were also titrated in benzene as a solvent using trifluoroacetic, trichloroacetic, and acetic acids as titrant. The analytical accuracy was approximately 1 yoin all titrations, and the experimental titration curves agreed with the theoretical predictions.
A
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solution parameters have been successfully investigated using a variety of colligative properties. For example, the association of carboxylic acids, nitrogen bases, and their adducts has been studied by cryoscopic (2,S, 7‘) and ebuillioscopic (1, 8) methods. More recently, studies have demonstrated the utility of the differential vapor pressure method for the investigating acid-base reactions in the solvent benzene (4) as well as solute association in several other solvents (6). In nonaqueous acidbase studies using cryoscopy, it w&s desirable to continuously monitor the equilibrium freezing temperature as the QUEOUS AND NONAQUEOUS
mole ratio of acid to base was varied. Experimentally, this was accomplished by adding acid from a motorized buret to a rapidly stirred mixture of the solvent plus base in the presence of finely divided frozen solvent, while the temperature was measured continuously using a thermistor bridge, This paper reports the resulte of the initial investigations of this technique with both water and benzene as solvents. The measurement of equilibrium freezing temperature of a two-phase (solid-liquid) mixture permits determination of the total number of solute particles in the liquid phase. This measurement offers a universal end-
