Constant Potential Spectroelectrochemical Potentiometric Titration

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					     A Precipitate Redox Precursor of Analyte Resorufin and its

 Constant Potential Spectroelectrochemical Coulometric Titration

                            with a Novel Cell Design

                                      Chul Joo Kim*

      The Department of Chemistry, The University of Georgia, Athens GA 30602



Several experiments were conducted for the sake of optimizing the design of a

spectroelectrochemical cell towards an ability to generate reliable quantitative data for

the constant potential coulometric titration of analyte Resorufin employing Methyl

Viologen and Hydroxymethyl Ferrocene as reduction and oxidation catalysts.

Adjustments in the cell design consisted of improving upon the electrical contact between

the auxillary and reference chambers with that of the tin oxide working electrode

chamber as well as measures to shorten the height of the cell so as to enhance the

circulation of the reagents. The problems in sample preparation sourced from an

apparent allocation of the charge content to a compound other then Resorufin; this

compound may have either originated from an impure stock sample of Resorufin or was

formed through reactions of Resorufin with the solution constituents, facilitated by the

particular experimental conditions that were employed. It is proposed that this species

was a redox precursor of Resorufin which was initially present as an un-dissolved

precipitate, reacting predominantly with the reduced form of Methyl Viologen and


During a spectroelectrochemical coulometric titration, electrons are transferred between a

working electrode and the reductive or oxidative catalyst which in turn exchange

electrons with the analyte. Even when working with micromolar concentrations, the

potentiometric analysis can be performed in its entirety and reliable quantitative data can

be obtained. Also the titrant can be added at any rate, oxygen can be removed before and

excluded during the titration, and small volumes can be analyzed.

Determination of the formal potential through the electrochemical, constant potential,

titration is similar to methods employed in obtaining the pKa from an acid base titration

based on the Henderson Hasselbach equation pH=pKa+log[A-]/[HA] where A- and HA

are the conjugate base and acid components respectively. When the concentrations of the

two are equal on a graph where the acid is titrated with a strong base (with the pH on the

y axis and the volume of the titrant on the x axis), pH=pKa which is deemed as the half

equivalence point. With the Nernst equation E=E0'-(RT/nF)lnQ (where Q pertains to the

concentration of the products and reactants involved in the redox reaction); E is the cell

potential which is measured by the subtracting of the reference electrode potential from

the potentiometric electrode potential, E0' is the formal potential which may incorporate

pH dynamics or rather the pH dependence of the redox reaction. A plot of the cell

potential on the y axis versus the value of ln[(A max-A)/(A-A min)] has a linear

regression equation with the y intercept as the formal reduction potential for that

particular Nernst equation and a slope value that is equivalent to (RT/nF), from which the
value of n can be obtained assuming R=8.314 J/K and F=96,500 C/mole electron. "A" is

value of the absorbance unit for the analyte species for which the value decreases as it is

reduced and A max and A min are the highest and lowest values in the absorbance units

observed for that analyte species in the experiment.

Oxygen compromises the reliability in the methods of quantitative analysis which are

based upon experiments involving analyte species that are oxidized in its presence. In

such cases, an apparatus which suffices towards completely removing of the dissolved

oxygen as well as keeping it out is required. An open system situation is nearly

equivalent to an infinite reservoir of oxygen and procedures which are superficial with

respect to the degassing protocol are inadequate for some types of quantitative analysis

(e.g. such when determining the concentration of an analyte). When dealing with an

extremely small or large volume apparatus, adequate circulation of the reagents is a

common problem; careful measures must be taken to maintain the homogeneity of the

solution for the respective cell design. The best design of the cell should be spherical

with regard to circulation, yet this isn’t very feasible for spectrochemical experiments

since it is ideal that the main chamber of the apparatus resemble a cuvette with a path

length of 1 cm.

The issues related to the circulation and de-oxygenation of the experimental solution are

the most important when it is desired that the cell become optimized towards generating

reliable quantitative data when all other aspects of the setup have been appropriated to the

fullest extent e.g. issues related to the sampling methods). Anomalies in the
potentiometric data can arise from simple inadequacies in design with respect to these

two factors and such interesting observations may lead to superfluous inspirations and

endeavors. The issue of circulation and de-oxygenation are fundamental with respect to

the integrity of a novel spectroelectrochemical cell.


A stock solution consisting of 10 uM Resorufin, .1 mM Methyl Viologen, and .1 mM

Hydroxymethyl Ferrocene was prepared by dissolving the appropriate amount in grams

of each compound into a solvent consisting of 5% methanol and a pH 7 phosphate buffer

of .154 M ionic strength. The KCl was of 100 % purity and from J.T. Baker. The recipe

for preparing the buffer component was revealed through an online calculator invented by

the author of the site PFG [1] and J.T. Baker reagents were employed. The balance was

that of Mettller, model H51. The Methyl Viologen and ODA was obtained from Aldrich

and Acros Organics (97% purity) respectively. The Resorufin was also from Aldrich.

The method of de-oxygenating the solution in the smaller, primary, electrochemical cell

is described below. Purified Argon was passed through the main degassing setup

consisting of a Ridox catalyst (Fisher Scientific) at ambient temperature followed by a

water-filled bubbler to saturate the inert gas with water. The gas line and a vacuum line

were alternately connected to the electrochemical cell by a two-way glass valve. All

ground glass fittings at the junctions were greased with Apiezon N. The vacuum system

consisted of a liquid nitrogen trap connected by rubber tubing to a Drierite drying column

(to protect the vacuum pump from water evaporated during degassing), a vacuum flask
trap, and a mechanical vacuum rough pump. The mediator cell (a degassing bulb) was

connected to the spectroelectrochemical cell and the mediator end of the cell was

subsequently attached to the main degassing setup; both of these cells were initially

evacuated for 10 minutes with all valves open on the spectroelectrochemical cell. The

cells were then alternately purged with Argon and evacuated for a time of 1-2 minutes,

this procedure was repeated 5 times. The valves at the reference/auxillary arms were

closed off prematurely under Argon during the trials where they were a part of the design.

The system was then left under inert gas pressure for 2 minutes before closing the main

stopcock and subsequently the rest of the valves of the cell followed by those on the

degassing bulb. The cell was then removed from the degassing apparatus. The mediator

cell was filled with 5 mL of the solution and then reconnected back to the degasser. The

degassing protocol was applied as before to deoxygenate the solution. The experimental

cell was then connected, once again, to the mediator cell and the valve of the mediator

cell connecting to the experimental cell was opened to allow the solution to enter the

experimental cell. This cell was then assessed for the presence of air bubbles and refilled

accordingly if an air bubble was deemed to be an obstruction towards the electrical circuit

of the cell. Figure 1 diagrams the essential features of the initial version for the primary

electrochemical cell employed for the quantitation of the formal redox potential.
Figure 1 The initial design for the primary electrochemical cell employed for the

potentiometric titrations. Improvements were made so that the frits were replaced by

valves and the cell height was decreased to ~1 cm, while that for the original was ~3 cm.

The first of the trials was carried out in a larger cell, open system, apparatus with a

volume capacity of ~25 mL (Please refer to the Appendix). The electrode leads were

inserted into the sidearms along with the degassing glass tube which was connected to the

main degassing setup. The solution was purged with Argon for 5 minutes prior to adding

the charge and was kept above the solution during the actual experiment. With regard to

the experiments carried out in the smaller apparatus, all of the side chambers were closed

off with standard taper ground glass fittings. The initial configuration had a frit at the

base of the reference and auxillary compartments, separating them from the main

chamber; changes in design to this setup are described in the results section. Silver

chloride-coated silver wire electrodes were inserted through rubber septum caps into the

Ag/AgCl/3 M KCl reference and auxillary electrode glass tubes fabricated from inner
ground-glass joints with porous Vycor frits epoxy sealed into the tips for electrolytic

contact and isolation from the main chamber. The working electrode was a 2.5 cm x 2.5

cm square piece of Sb-doped SnO2 glass epoxied to the bottom of the main chamber of

the cell with Loctite Marine Epoxy (which was supposed to settle after 50 minutes upon

application). The spectrophotometric optical pathlength through the main chamber of the

cell was 1.00 cm. A platinum wire was utilized for the potentiometric readings and it was

fused with the main chamber during the process of designing the apparatus. With

modifications in the design of the cell, a 5 mm glass protrusion at the side of the main

compartment had stabilized the platinum wire which was wrapped around it.

Arriving upon the final design of the cell consisted of replacing the frits at the side arms

with valves to improve the electrical contact and shortening the height of the main

compartment from 3 cm to 1 cm (For a detailed account, please refer to the Appendix).

As a result, the charge transfer to the analyte became more efficient; junction potentials,

represented by a layer of colorless solution at the working electrode and a pink layer at

the top, formed less readily during each charge increment.

All of the regression analysis involved the advanced regression tool on Quattro (Corel

Version 12). The potentiostat was homemade by professor James L. Anderson at the

University of Georgia.

The formal reduction potential of Resorufin was determined with the titration carried out

in an open system apparatus. With a simple qualitative inspection of Figure 3 this value

seems to be ~ -.2200 V versus the silver chloride reference electrode (3M KCl).

Figure 3 The potentiometric plot of cell potential versus the charge passed reading for the

experiment done with the open system apparatus (volume capacity of ~25 mL). The

relatively extended curve represents the oxidation trial that was subsequent to the


The charge that was required for titrating Resorufin was apparently in excess. This

aspect was understandable since the charge addition was carried out in an open system

and oxygen was essentially being titrated along with the analyte. Thus this trial had

confirmed the obvious; that the system had to be closed during the potentiometric

titration of Resorufin in an aerobic environment. The oxidation stage was carried out

soon after the end of the reduction stage and the additional charge required relative to the
reduction stage was somewhat inexplicable, since the presence of Oxygen would add to

the total charge content required for the completion of the reductive stage by also taking

up charge. The apparent dilemma was ultimately attributed to problems with the

potentiostat and its workings were refined accordingly.

A corresponding titration with the finalized version of the primary spectroelectrochemical

cell is shown in Figure 3 and the data is outlined in Table 1.

Figure 3 Potentiometric titration of Resorufin with the finalized cell; the height of the cell

at the main compartment was decreased to ~1 cm from ~ 3 cm. The second reduction

stage was subsequent upon the end of the first oxidation stage. The potentiometric plots

are at the top and the respective absorbance versus mC plots are below them.
Table 1 Potentiometric titration of the Resorufin stock solution with Hydroxymethyl

Ferrocene and Methyl Viologen with the finalized cell (its height at ~1 cm and valves at

the arms instead of frits). This table is in reference to the second reduction.

The results show a decrease in the amount of charge required for the reduction stage and

the shape of the plot is more refined. The most surprising feature from this experimental

data was that the absorbance at the 574 nm peak at the beginning of the second reductive

trial had increased by ~200 % relative to the initial absorbance value seen for the first

reduction. The concentration of had increased dramatically after the first reduction and it
seems that some additional content of Resorufin was liberated during the first reductive

stage. Also, the intensity at the 700 nm peak had decreased to a value of ~.30 from the

initial value of ~.46; this was an indication that a precipitate was present at the beginning

of the first reduction to cause scattering and had dissolved into the solution throughout

the titration. However, the value of the absorbance at 700 nm during the second trial was

much more constant, at ~.2750. These two trends suggest the existence of a redox

precursor of Resorufin, which dissolved as a precipitate throughout the first reductive

trial by taking up charge and undergoing a redox reaction to become Resorufin. The idea

of Resorufin being liberated during the first trial was evident in a more dynamic manner

when the absorbance at 574 nm was increasing during the initial period of the first

reduction (Figure 3, the plot at the bottom left representing the absorbance at 574 nm).

The charge required for the titration of Resorufin in the second trial was determined to be

22 mC from a qualitative inspection of Figure 3. The value of “n” was determined from

the gran-plot for the second titration (Figure 4 and Table 2) to be 2.17±.02 (The

temperature was assumed to be 19 C. For sample calculations, please refer to the

supplementary Appendix for the detailed accounts of the experiments which were

conducted with the 3 cm height cell). The formal potential for Resorufin was -.2158 ±

(.0002) V.
Figure 4 and Table 2 Gran plot and statistical data for the experiment done with the

finalized spectroelectrochemical cell and associated with Figure 3. The analysis pertains

to the second reduction

Table 3 Raw data for the experiment performed with the finalized cell.
The value of “n” is in close agreement with the theoretical redox stoichiometry for

converting Resorufin to Dihydroresorufin, however there's still an indication that excess

charge is being allocated to another species. The presence of the precipitate was assumed

to be negligible for the second reduction (despite a change in absorbance of ~.005 at 700

nm throughout the second stage) and Resorufin was deemed as the sole analyte; 20 mC

was seen as the actual amount of charge allocated to reduce the Resorufin in the second

trial. To account for the 10% excess in the value of “n” (which was indicative of the

excess charge that was being passed off to a compound other then Resorufin) 2 mC was

subtracted from 22 mC of charge that was needed for the second reduction stage in its

entirety (since 10% of 22 mC is 2.2 mC~2 mC). The volume of the main compartment of

the cell was 1 mL and the concentration of Resorufin was calculated in the following


.020 C(1 mole e-/96,500 C)(1 mole Resorufin/2 mole e-)/(.001 L) =.1036 mM for the

concentration of Resorufin at the beginning of the second trial.

The oxidation stage after the first reduction had taken 22.95 mC of charge for a change in

the cell potential to take place from -.5191 V to .0475 V. The second trial had begun

subsequently, and it took 25.3 mC for the potential to change from .0460 V to -.5200 V.

The similar values in the charge content for the related oxidation and reduction stage

implicate the reversibility of the redox reaction of Resorufin to Dihydroresorufin and that

almost all of the precipitate content had been accounted for by the second reduction.
The amount of precipitate as compared to the Resorufin content that was dissolved in the

original solution in the cell before the titration was estimated in the following manner.

All of the Resorufin content was assumed to have been accounted for in the first

reduction (since the transitional equivalence point to the Nernst region of Methyl

Viologen is evident in Figure 2), thus the actual charge content, as it pertains to the

Resorufin to Dihydroresorufin conversion was 20 mC for the second as well as the first

reduction (since all of the Resorufin content was dissolved by the end of the first

reduction, it was assumed that the second reduction represents the full titration). For the

first titration, it was deemed that 35 mC was needed to reach the equivalence point from a

qualitative inspection of Figure 2. Since 20 mC was required for the conversion of

Resorufin to Dihydroresorufin for both of the trials, there was an excess charge allocation

of 15 mC during the first trial. If it is assumed that the cell did not contain any dissolved

oxygen during the titrations, this 15 mC of charge was used to redox and dissolve the

precipitate. Since all of the Resorufin was accounted for regarding the first stage, that is

all of the Resorufin content had been converted to Dihydroresorufin, the 15 mC of charge

was allocated to convert the all of the precipitate content to Dihydroresorufin. The 15

mC of the 20 mC Dihydroresorufin charge content for the second trial had sourced from

the supposed precipitate and thus it can be concluded that 5 mC was associated with the

actual quantity of Resorufin which was dissolved in the solution at the beginning of the

first trial. This amounts to a concentration of 26 uM for Resorufin that was dissolved

upon the start of the first reduction.
A trial, where the potentiometric titration of Resorufin was completed without the

catalysts, was conducted and the results suggests that neither of the catalysts (Methyl

Viologen and Hydroxymethyl Ferrocene) have roles in contributing to the excess charge

by taking up charge exclusively (by not fulfilling their essential catalytic functions for

with Resorufin) . The solution had the same solvent composition as the former stock

solution which contained Methyl Viologen and Hydroxymethyl Ferrocene. With a few

exceptions, the potentiometric plots with this sole Resorufin solution are identical to

those that have been generated for the main experiment.

Figure 5 The plot at the left is with respect to the cell potential versus the charged passed

reading. The linear portion for the plot at the right is a seconds versus mC function (to

assess the current of the potentiostat) and the other is an absorbance (at 574 nm) versus

mC function. The experiment was conducted with a sole Resorufin solution. . It should

be noted that this experiment was performed with the cell when its height at the main

compartment was still 3 cm.

The transfer of charge seemed less effective in general without Methyl Viologen as a

reduction catalyst for this trial as compared to the main experiment. This claim is

particularly evident during the Nernst region of Resorufin in that the change in the cell
potential was drawn out with respect to the amount of charge that was expected to

completely reduce Resorufin. Also the absorbance at 574 nm had stabilized

unexpectedly after 15 mC of charge was incorporated (Figure 5 at 17 mC on the plot of

absorbance versus mC), whereas it was expected to decrease in a linear fashion. The

absorbance at 574 nm had become more responsive with the initial charge addition, that

is it had decreased more readily, which may be because Methyl Viologen wasn't present

to cause a “homogenizing” effect for the precipitate; the absorbance did not tend to

increase as in Figure 2 which means that the precipitate was less involved in taking up the

charge at the start of the reduction process. Also, the reduction kinetics of the aggregate

doesn't seem to have influenced the equivalence transition (between the ~.1000 V and ~-

.2200 V) towards the Nernst region of Resorufin as substantially as when Methyl

Viologen was present; it is abrupt as compared to the gradual transition that is observed

in Figure 2. An explanation for these two observations is that the precipitate had taken

up relatively less charge during the former portions of the entire reduction stage and that

perhaps its redox kinetics was more favorable with higher concentrations of

Dihydroresorufin. The Nernst region for Resorufin was prolonged in Figure 6 because

the rate at which the precipitate was consuming charge had become substantial with the

increased concentration of Dihydroresorufin. If the reduction of the precipitate take place

through a surface mechanism, Dihydroresorufin would be the main agent to redox and

dissolve it. The kinetic interplay between these three species is quite interesting in

hypothesis and the matter should be investigated with further, well designed,

The cell was assessed for leakage by observing the rate at which the Methyl Viologen

peaks decreased versus the time when the cell was left to itself shortly after the end of the

reduction stage of Resorufin. Figure 7 displays the results for the application of this

method to the cell when its cell height was still at ~3 cm.

Figure 6 and Table(s) 4 The absorbance peaks of the Methyl Viologen and the 700 nm

baseline absorbance versus the time. The time measurement was started when the

reduction of Resorufin was complete and when the absorbance of the Methyl Viologen

peak at 396 nm had a significant presence.
The 396 nm peak absorbance data reveals a rate of decline in the concentration of the

reduced Methyl Viologen at a rate of .00031 ± (.00005) absorbance units/second. The

analysis indicated that the cell had a leakage that was allowing oxygen to enter and

oxidize the Methyl Viologen. At this rate, it would have taken a little over 5 minutes for

the absorbance to decrease by .1 units. An additional suggestion pertains to the

precipitate which may have oxidized both Dihydroresorufin as well as Methyl Viologen

at the end of the reduction stage if charge was added relatively continuously (without

stabilizing at the system’s equilibrium state) and large increments to expedite the titration

process; if the redox kinetics of the precipitate was slower as compared to that of the

analyte Resorufin. Because the same experiment performed with the drastically revised

version of the cell produced a similar result in the value of the rate, Figure 7 and Table 5

displays the result of the method when it was ~1 cm in height at the main compartment.

Thus the rate of decline in the concentration of the reduced form of Methyl Viologen may

have been more intrinsic to the reaction dynamics that were going on in the sample

solution and not due to the degree of leakage that could be attributed to the inadequacy in

the design of the cell itself.
Figure 7 and Table(s) 5 Regression data for the Methyl Viologen peaks versus the time

after the second reduction to assess for leakage when the cell height at the main

compartment was reduced to ~ 1 cm. The statistical analysis were based on the

396 nm peak of Methyl Viologen. The rate of the decline in the 396 nm peak was .00033

± (.00002) abs/mC . The slope value, .000310 ± (.000005) abs/mC, was almost identical

to that obtained with the more “primitive” version of the cell, thus the rate may be based

on particular reaction kinetics of Methyl Viologen rather then due to leakage.

A couple of points should be stressed in regards to the proper employment of this cell.

The most obvious factor is that the concentration of the reagents should be relatively

lower then what was implemented in the trials; 25 uM seemed to be a suitable value with

respect to the shape and volume of the cell. Errors associated with the experiment

sourced primarily from sample preparation and also from the potentiostat itself; since the

errors that were inherent to its functioning (or some unknown malfunctioning perhaps)

were not taken into account in the calculations. The redox precursor of Resorufin may

have originated from the impurity of the Resorufin stock sample itself or it could be that
the methanol component of the solvent facilitated an ether reaction of a hydroxyl group

of Resorufin with any constituent in the solution that could feasibly interact with it. The

solution would cool considerably during the vacuum applications in the degassing

protocol, this may have further promoted side reactions of Resorufin and may have

stabilized adducts. A constant temperature setting should be implemented during the

degassing protocol with the bulb. The actual intent of the experiment was to titrate

Resorufin without any interference from other compounds towards the quantitative

analysis of the analyte. However, if the presence of the precipitate is inevitable, to

whatever degree it is to be present in a solution with Resorufin, a complete reduction and

oxidation cycle may be required to dissolve of the precipitate. With the precipitate

dissolved, the cell potential was much more stable between the charge increments and the

amount of charge required for a specific change in the voltage value for the reduction

stage should match that for the subsequent oxidation stage. Further improvements to the

design of the cell should apply to the elimination of any isolated corners, which were near

the valves with this cell, so that small junction potentials would not develop in these

spaces and the modifications which would lower the resistance between the arms of the

cell and the main compartment. In addition, further measures should be taken to ensure

that there is no mass transport between them.

The formal potential for Resorufin was determined to be -.2158 ± (.0002) V versus a 3M

KCl Silver Chloride reference electrode with the finalized design of the cell. This

particular value of the formal potential may be indirectly related to the solubility of a

precipitate redox precursor of Resorufin. The value of “n” was determined to be 2.17±

(.02) when the temperature was assumed to be 19 C. The precipitate had taken up charge

throughout the initial reduction stage conducted with the finalized cell design and much

of it had dissolved into the solution upon the end of the subsequent oxidation stage of the

first redox cycle. Its redox kinetics seemed to have slowed considerably when Methyl

Viologen was excluded towards a sole Resorufin solution and it may have depended on

the concentration of Dihydroresorufin. The charge content for the reductive titration of

Resorufin was always greater for the first when two trials were conducted and the current

was noted to have been considerably greater when the precipitate was present. This

condition should be investigated further to elucidate the nature of the fundamental rate

dynamics behind it. Hydroxymethyl Ferrocene and Methyl Viologen didn't seem to

cause any direct problems with the usage of this cell, although Methyl Viologen and

Resorufin did adsorb to the glass surface of the cell as well as the degassing bulb. The

finalized cell was ~ 1 cm in height and had valves separating the arms from the main tin

oxide chamber; the result was significant improvements in the circulation and the passing

of charge efficiency.

    Beynon, Rob. PFG. The University of Liverpool

    Tratnyek, Paul G. “Visualizing Redox Chemistry: Probing

    Environmental Oxidation–Reduction Reactions with Indicator Dyes” The

    Chemical Educator. V 6. No 3

    Anderson, James L. “Spectroelectrochemical Investigations of

    Stoichiometry and Oxidation-Reduction Potentials of Cytochrome c

    Oxidase Components in the Presence of Carbon Monoxide: The

    “Invisible” Copper”’. Biochemistry. 1976. V. 15. 3847-3855

    Encinas, M. V. “The Excited-State Interaction of Resazurin and Resorufin

    with Aminesin Aqueous Solutions. Photophysics and Photochemical

    Reaction”. Photochemistry and Photobiology. V. 76. No 4. 385-390

Figure A1 The apparatus was open to the atmosphere and the volume capacity was

~25 mL. The tin oxide bar was ~1 cm by ~.5 cm by ~7 cm. The de-gasser glass tube was

inserted with the platinum wire partially wrapped around it. The constant temperature

feature of this apparatus was not utilized.
Experiments applied with the cell when the height of its main compartment was ~ 3 cm

The initial cell height was 3 cm - 4 cm with a 1 cm square base and the cell had two frits

separating the auxillary and reference electrodes from the main compartment. The

formal reduction potential for this setup turned out to be -.215 ± (.003) V from a Gran

plot analysis of the potentiometric data (Figure A2).

Figure A2 The corresponding potentiometric and Gran plot prepared with data obtained

with the initial cell where the height of its main compartment was at ~3 cm and both side

arms were separated from the main compartment by a simple frit.

The charge that had to be passed for the entire titration was in excess; however, there was

significant improvement on this matter as compared to the experiment that was conducted

with the open system apparatus.

The next revision in the cell design consisted of replacing the frit at the side arm of the

reference electrode compartment with a valve in its place to place it in better electrical
contact with the main compartment. In addition, the platinum wire was reattached to the

main chamber in a more secure fashion to prevent any more leakage of oxygen through

the area of its junction with the main compartment (in a manner described in the

Experimental). Two stages of reduction was applied to the spectroelectrochemical cell.

As with the main experiment, the initial amount of charge to completely titrate Resorufin

was substantially greater for the first as compared to the second reduction (Figure A3).

Figure A3 The potentiometric data for the two reduction experiments done with a

modified version of the cell; the frit at the reference electrode compartment was removed

and replaced with a valve and the platinum wire was fused in a more secure fashion so as

to further seal off the system from outside air.
The 14 mC of charge that was required to titrate the Resorufin in the second trial was

more along the lines of what was expected with regard to the concentration of the stock

solution. Since there are 96,500 C per a mole of electron and with the consideration that

2 moles of electrons are required to completely titrate one mole of Resorufin to

Dihydroresorufin, the following calculation reveals a concentration of 24.18 uM for the

Resorufin in the stock solution with the data for the second trial (The cell apparatus was

still 3 cm in height at its main compartment and the volume was 3 mL. It should also be

noted that the circulation was still inadequate at this stage).

14 mC(1 C/1000 mC)(mole of electron/96,500 C)(mole of Resorufin/2 moles of

electron)(1/.0030 L)=24.18 uM

Analyzing the data by preparing a Gran plot revealed a value of -.2322 ± (.0009) V. The

slope of the regression equation was .0100 ± (.0005). The results show that the new

design improved the reliability of the experiments since the placement of the digit in the

error moved to 10th of a mV. Since this value is equivalent to the product value of nF

divided by that of RT (where the temperature was assumed to be 19 C), the value of “n”

can be determined to understand the electron equivalence for the redox reaction when

Resorufin was primarily being reduced. Equating .01000 to RT/nF resulted in an “n”

value of 2.5 ± .1. The formal potential of Resorufin for the second trial was -.2328 ±

(.0003) V and the value of “n” was 2.8 ± (.1). The value of “n” for both of these trials

was excessive since the circulation was worse off when the height of the main

compartment of the cell was relatively greater at 3 cm.

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