The Electrochemistry of Micelle-Solubilized Ferrocene

1334 J. E~eetrochem. Soc.: E L E C T R O C H E M I C A L

b' - - h + a ( u -- #' + r [A-S]

b" = h -- (1 -- ~) (u -- ~b' + r [A-4]

The a s y m m e t r y p a r a m e t e r a m a y assume values be - tween 0 and 1. For ~ ---- 0 the energy ba r r i e r is located at the outer and for ~ =- 1 at the inner m e m b r a n e - s o l u - t ion interface. Fol lowing Eyr ing the s ta t ionary flux r of un iva len t cations f rom outside to inside is p ropor - t ional to

r ~_ c'm exp (--b') -- C"m exp (--b") [A-5]

The quant i t ies contained in the p ropor t iona l i ty factor ( jump length, f requency factor) are assumed to be the same on both m e m b r a n e sides and to be independen t of the ion concentrat ions and membrane surface po ten- tials. Inser t ing Eq. [ A - l ] , [A-S], and [A-4] into [A-5] y ie lds

* ~ c' exp [ - - (1 -- ~)~ ' - - ~ (u + ~" ) ]

-- c" exp [ ( 1 - - a) (u - - ~') - - a~"] [A-6]

This genera l express ion can be simplif ied for the fol - lowing special cases: (a) energy ba r r i e r located at the outer in terface (a = 0)

r __~ exp ( - -~ ' ) [c' - - c" exp (u) ] [A-7]

(b) energy ba r r i e r located a t the inner in ter face (~ = i )

V _____ exp ( - - # " ) [c' exp ( - - u ) -- c"] [A-8]

(c) symmet r i ca l ene rgy ba r r i e r (a = �89

/ r + ~" u ,. ( - y ) e x p

-- c " e x p ( 2 > ] [A-9]

If the energy ba r r i e r is located at one interface, the s ta t ionary ion flux is de t e rmined solely b y the surface potent ia l of this membrane surface. For a symmet r ica l energy ba r r i e r the flux r depends on the sum r + ~" of both surface potent ia ls (compare Eq. [A-9] which is ident ica l to [1])

SCIE N CE A N D T E C H N O L O G Y September 1976

REFERENCES 1. S. P. Verma, D. F. H. Wallach, and L C. P. Smith,

Biochim. Biophys. Acta, 345, 129 (1974). 2. H. Drouin and B. Neumcke, Pfl~gers Arch., $51, 207

(1974). 3. S. McLaughlin, J. Membrane Biol., 9, 361 (1972). 4. W. S. Chelack, A. Petkau, and T. P. Copps, Bio-

chim. Biophys. Acta, 274, 28 (1972). 5. B. Neumcke, Biophysik, 6, 231 (1970). 6. W. K. Chandler , A. L. Hodgkin, and H. Meves,

J. Physiol. (London), 18@, 821 (1965). 7. D. L. Gi lber t and G. Ehrenstein, Biophys. J., 9, 447

(1969). 8. G. Ehrens te in and D. L. Gilbert , ibid., 13, 495

(1973). 8a. T. Brismar, Acta Physiol. Scand., 87, 474 (1973). 8b. B. Hille, A. M. Woodhull , and B. I. Shapiro, Phil.

Trans. R. Soc. London, Set. Bo 270, 301 (1975). 9. J. M. Fox, Pfli~gers Arch., 355, R69 (1975).

10. S. McLaughl in and H. Hara ry , Biophys. J., 14, 200 (1974).

11. J. M. Fox, Biochim. Biophys. Acta, 426, 232 (1976). 12. G. Ehrens te in and D. L. Gi lber t , Biophys. J., 15,

847 (1975). 13. B. Neumcke, J. M. Fox, H. Drouin, and W. Schwarz,

Biochim. Biophys. Acta, 426, 245 (1976). 14. R. Fet t ip lace , D. M. Andrews, and D. A. Haydon,

J. Membrane Biol., 5, 277 (1971). 15. S. McLaughlin, G. Szabo, and G. Eisenman, J. Gen.

PhysioL, 58, 667 (1971). 16. M. Montal and C. Git ler , Bioenergetics, 4, 363

(1973). 17. P. F romherz and B. Masters, Biochim. Biophys.

Acta, 356, 270 (1974). 18. D. H. Haynes, J. Membrane Biol., 17, 341 (1974). 19. J. R. Macdonald and C. A. Barlow, Jr., This

Journal, 113, 978 (1966). 20. R. Fr iedenberg , A. Blatt , V. Gallucci, J. F. Daniell i ,

and I. Shames, J. Theoret. Biol., 11, 465 (1966). 21. K. S. Cole, Biophys. J., 9, 465 (1969). 22. J. R. Buysman and F. T. Koide, J. Theoret. Biol.,

32, 1 (1971). 23. O. H. Griffith, P . . J . Dehlinger, and S. P. Van,

J. Membrane Biol., 15, 159 (1974). 24. R. H. Brown, Prog. Biophys. Mol. Biol., 28, 343

(1974). 25. A. P. Nelson and D. A. McQuarrie , J. Theoret. Biol.,

55, 13 (1975). 26. B. Pa r l in and H. Eyring, in "Ion Transpor t Across

Membranes ," H. T. Clarke, Editor, Academic Press, New York (1954).

27. B. Neumcke and P. L~uger, Biophys. J., 9, 1160 (1969).

The Electrochemistry of Micelle-Solubilized Ferrocene Peter Yeh* and Theodore Kuwana*

Department of Chemistry, The Ohio State University, Columbus, Ohio 43210

ABSTRACT

The electrochemistry of ferrocene solubilized by the use of nonionic detergent in aqueous phosphate solutions pH 7.0 was studied. The ferrocene molecules were incorporated as micelles which readily transferred electrons with a platinum electrode. Cyclic voltammetric and potentiometric data indicated that the electron transfer reaction was reversible. Micelle size calculated from electrochemical diffusion and light scattering data gave radii between 40-45A and molecular weights of ca. 130,000. The ferrocene-micelle served as an excellent mediator-titrant to couple electron transfer between an electrode and the heine proteins of cytochrome c, eytochrome e oxidase, and mixtures thereof.

Recently, ferricinium ion electrogenerated from ferrocene solubilized by the use of nonionic detergent has been employed Zor the redox titration of cytochrome c, cytochrome c oxidase, and mixtures thereof in aqueous pH 7.0 solutions (I). The detergent ap- parently formed micelles with the incorporation of ferrocene. The ferricinium/ferrocene couple is then

* E l e c t r o c h e m i c a l Soc ie ty Student Member. t E l e c t r o c h e m i c a l Soc i e ty Active Member. K e y w o r d s : electrochemistry, ferrocene , micel le , mediator-

titrants.

acting as a mediator to couple the electron transfer between the electrode and the heine proteins.

Ferrocene-micelles represent part of our effort to find water-soluble mediator-titrants (M-T's) with redox potentials suitable for use in the study of biological electron transport components. Requirements sought for these M-T's are: (a) both species of the redox couple of the M-T's need to be soluble in aqueous media buffered at or near pH 7; (b) the solubility of the M-T's should be i m_M or more; (c)

) unless CC License in place (see abstract). ecsdl.org/site/terms_use address. Redistribution subject to ECS terms of use (see 155.97.178.73Downloaded on 2014-11-27 to IP

http://ecsdl.org/site/terms_use

Vol. 123, No. 9 EC O F M I C E L L E - S O L U B I L I Z E D F E R R O C E N E 1335

the ra te of e lec t ron t ransfe r w i th both electrode and heme prote ins should be rapid ; (d) the chemical s ta - b i l i ty should be adequa te so tha t in te r fe rences wi th heme prote ins be avoided; (e) the redox reac t ion mechanism should be uncomplicated, p r e f e r ab ly in- volving one e lec t ron t rans fe r (n _-- 1); and (]) the opt ical absorbance of the M - T ' s should not in te r fe re wi th those of the heme proteins. Very few M-T 's fulfill al l of the above requirements .

Fo r work ing in the negat ive poten t ia l ranges, say --100 to --500 mV vs. NHE, the 4 ,4 ' -b ipyr idy l ium salts (commonly known as viologens or pa raqua t s ) have p roved to be e x t r e m e l y useful (2). A t po ten t ia l s posi t ive of ca. +300 mV vs. NHE, the n u m b e r of usable M-T's has been e x t r e m e l y l imited. F e r r i c y - an ide / f e r rocyan ide has been commonly used, but has been found to produce dele ter ious effects or to com- p lex wi th the hemes (2, 3). I t is des i rable to find M-T 's wi th s imi lar redox chemis t ry whose redox po - tent ia ls could be a l te red th rough s imple s t ruc tura l modifications.

F r o m previous exper ience (4) w i th the e lec t rochemis t ry of meta l locenes in nonaqueous solvents, it was known that ferrocene, for example , unde rwen t a fas t one e lec t ron redox reac t ion at a posi t ive po ten - tial. However , the low solubi l i ty of fer rocene (ca. 10-5 M) in aqueous solutions p reven ted its use. The re - fore, approaches to solubi l izat ion of these meta l locenes were explored. Tween 20 was chosen for the present w o r k because of our exper ience in the use of this sur fac tan t in solubi l iz ing heme prote ins (2).

The number of e lec t rochemical s tudies in which sur fac tan t concentrat ions were de l ibe ra t e ly increased above the cr i t ical micel le concentra t ion for the solu- b i l iza t ion of e lec t roact ive species has been l imited. Ha~ano and co-workers (5, 6) have repor ted the use of severa l k inds of sur fac tants for the solubi l izat ion of dye molecules which exh ib i ted reduct ion cur ren ts at d ropping mercu ry electrode. Thei r resul ts indica ted tha t d i f fus ion- l imi ted currents could be a t ta ined and tha t the ra te was l imi ted by the diffusing micel le par t ic les conta ining the dye. The effect of the sur- f a c t an t -dye concentra t ion ra t io to the s ize-volume of the micel le was ca lcula ted f rom the expe r imen ta l ly obta ined diffusion coefficient using the S tokes-Eins te in re la t ionship.

Westmore land , Day, and Underwood (7) in 1972 r epor t ed the solubi l izat ion of azobenzene using sur - factants such as sodium lau ry l sulfate, ce ty lpyr id in ium chloride, and l a u r y l t r i m e t h y l a m m o n i u m bromide. Again, a d i f fus ion-control led wave a t t r ibu tab le to micel le was found, and the reduct ion of azobenzene appea red to approach revers ib le behavior as the sur - fac tant concentra t ion decreased. In the above e x a m - ples, r eve r s ib i l i t y and mechanism could be assessed only condi t ional ly because of the fa i r ly complex na - ture of the e l ec t ron- t r ans fe r reaction, i.e., n values g rea te r than un i ty and possible invo lvement of 'protons.

In add i t ion to our heme prote ins studies (1), f e r ro - cene (or d ibu ty i fe r rocene) has been used as a me- d ia to r to couple e lect ron t ransfe r th rough l ip id m e m - branes as a model sys tem for mi tochondr ia l ion t r anspor t and r e sp i r a to ry control (8, 9). Fe r rocene in these l ipids (b lack l ipid membranes and phosphol ip id vesicles) p rov ided the coupl ing of e lect ron t ransfe r th rough the m e m b r a n e wi th ex te rna l redox reactants . The env i ronment for fer rocene in the micel le is p rob - ab ly quite s imi lar to that in the hydrophobic l ip id membrane . Thus, a de ta i led s tudy of the e lec t rochemical and phys ica l p roper t ies of fe r rocene-mice l le was of cons iderable in te res t and has s t imula ted the p resen t work. The assessment of the formal r edox potent ials , E o', of cytochrome c and cy tochrome c oxidase using the fe r rocene-mice l le are discussed.

Experimental The e lec t rochemical cell and ins t rumen ta t ion were

s imi lar to those prev ious ly descr ibed (9). A p l a t i num optical t r anspa ren t e lect rode (OTE) wi th surface r e - sistance of ca. 10 a / s q was used for the e lec t rochem- istry. Fo r opt ical measurements , another P t OTE was placed in the reference beam of the spec t rophotometer (Cary Model 15). The cu r ren t -po ten t i a l (i-E) curves were recorded using a H e w l e t t - P a c k a r d (Moseley Div - ision) Model 7005B X - Y recorder . A si lVer-si lver chlor ide (1M KC1) reference e lect rode was used for measurements of cell emf. The e lec t rode poten t ia l of this reference ha l f -ce l l was eva lua ted to be 0.232V vs. NHE by measur ing its po ten t ia l vs. severa l r e fe r - ence sa tu ra ted calomel electrodes. Tempera tu r e was main ta ined ,at 20 ~ -+- I~ The s i lve r - s i lve r chlor ide reference e lect rode v~as used because of the desire to dupl ica te the e lec t rochemical cell used for heine pro te in s tudies (9).

Turb id i ty and re f rac t ive indices of the sur fac tan t solutions were measured wi th the Br i ce -Phoen ix l ight sca t ter ing pho tomete r (Universa l 1000 series) and a different ia l r e f r ac tomete r (Phoenix Precis ion In s t ru - ment ) . Viscosi ty measurements of solut ions were made using a Ubbelohde v iscometer (No. 1B-A356) which had been ca l ib ra ted as descr ibed b y Cannon (10). F i l t r a t ion exper iments were done wi th an u l t r a f i l t ra- t ion cell (Model 202, Amicon Corpora t ion) and Diaflo ul t raf i l ters XMS0 and XM100A under 15 psi of pressure .

An ul t rasonic v ib ra to r - c l eane r (Heat Systems U l t r a - sonics, Incorpora ted) was used to assist in the solubi l izat ion of fer rocene and to p re t r ea t g lasswares for cleaning purposes.

Fe r rocene (d icyc lopentad ienyl i ron) was obta ined f rom S t r e m Chemical Company and was t r ip ly re - crys ta l l ized f rom reagent grade ethanol. Tween 20 (Polysorba te 20, po lyoxye thy lene sorb i tan mono lau -

ra te) wi th average molecu la r weight of 1650 was obta ined from Sigma Chemical Company. I t was purif ied b y t r ea tmen t th rough an a lumina (a luminum oxide, basic, A l u p h a r m Chemicals) column (18 in. long, 0.5 in. d i amete r ) . A very l ight s t r aw-co lo red f ract ion was lef t on the column. Phosphate buffer, pH 7.00 • 0.02 (Buffer Titrosol, Merck and Company) was used for buffering all solutions. J. T. Baker and Com- pany reagent grade potass ium chloride and s i lver sulfate were used wi thout fu r the r purification. P r e p u r - ified grade ni t rogen gas (99.998%) was suppl ied b y Amer ican Oxygen Service Corporat ion. Doubly dis- t i l led wa te r was used to p repa re a l l solutions.

The so lubi l i ty of ferrocene is less than 10-SM in w a t e r at room tempera ture . I t was solubi l ized by adding purif ied Tween 20 and a few mi l l i l i te rs of phos- pha te buffer d i rec t ly to a weighed amount of f e r ro - cene in a 100 ml volumetr ic flask. The amount of Tween 20 added was de t e rmined by the percentage of Tween des i red in the final solution. The mix tu re was s t i r red and then agi ta ted in an ul t rasonic v ib ra to r for about 30 rain. The flask was filled to the m a r k and then s t i r red wi th a magnet ic s t i r re r for another 12 hr. A t t empt s to solubi]ize fer rocene in wa te r by dissolv- ing ferrocene in i t ia l ly in alcohol or benzene and then adding wa te r were unsuccessful since fer rocene p r e - c ip i ta ted as w a t e r was added.

Results and Discussion Cyclic vo l tammet ry of ferrocene-mice~le . - -Since fe r -

rocene was being inves t iga ted as a possible med ia to r - t i t rant , the work ing range of concentra t ion selected for s tudy was be tween 0.1 and 0.5 raM. At Tween 20 concentrat ions be low about 0.5% by volume of final solution, ferrocene was not to ta l ly solubil ized as was evidenced by the nonreproduc ib le e lec t rochemical r e - sults. In the concentra t ion range of 1.0-5.0% of Tween 20, ferrocene was comple te ly solubi]ized and the cyclic vo l tammetr ic i -E curves were reproducible . A t y p -



1336 J. Electrochem. Soc.: ELECTROCHEMICAL SCIENCE AND TECHNOLOGY Sep tember 1976

~ [0 A

2

J I t I I i I 0.6 015 0 4 0.3 0 2 O. I 0 -0.1 - 0 . 2

Petentio~ , E ( Volts v e r s ~ s Ag/AgCI Reference Electrode)

Fig. 1. Cyclic voltammetric i-E curve of ferrocene (0.5 mM) and Tween 20 (3% v/v) in phosphate buffer (pH 7.00 + 0.02). Dotted curve is computer simulated according to Nicholson and Shain (11).

Table II. D and ip values and dependence on Tween 20 concentration and scan rate

P e a k c u r r e n t s * (/LA) at var ious Tween 20 concentrations (% vol)

Scan rate (V/sec) 1% 2% 2.5% 3.0% 3 . -~

0.023 8.75 12.0 12.1 14.3 12.$ 0.047 13.5 16.5 17.1 19.0 16.0 0,071 15,8 21.2 20.7 22.5 20.7 0.096 20.8 23.5 24.3 24.8 22.8 0.120 24.5 26.9 26.3 29.0 26.0

Calculated s lopes • 10~ 79.8* * 76.7 74.7 73.2 71.2

Intercepts - 4 . 0 3 0.11 0.70 2.93 1,07 Calculated D • 10~

(cm2/sec) 4.87 4.50 4.27 4.10 2.88

* Each value is average of 3 or 4 separate runs. ** Slopes and intercepts are least square values .

Table III. Viscosities of Tween 20 solutions

Concentration Tween 20 (% v/v) Viscosity (cs)*

ical {-E curve for micelle-solubilized ferrocene in phosphate buffer at pH 7.00 is shown in Fig. I.

The exper imenta l curve is well defined and is in close agreement with one which is computer-s imulated for a reversible, one-electron transfer, electrode reac- t ion using the relationship of Nicholson and Shain ( l l ) . The background i-E which was added to the theoretical curve, was obtained for a Tween 20 solu- t ion in the absence of ferrocene. The value of the diffusion coefficient, D, chosen for the calculation was one which gave the closest match of the simulated i-E curve to the exper imental one. More will be said about the exper imenta l D values shortly.

The dependence of Tween 20 concentrat ion on the reversible electrode potential (E0.s5 for cyclic vol tam- merry) and the separation of anodic and cathodic peak potentials (AEp) is summarized in Table I. The ferrocene concentrat ion was constant at 0.5 raM, the scan rate, ~, was 96 mV/see, and the Tween 20 concentration was varied between 1 and 3.5% by volume. The E0.85 and AEp values appear quite independent of Tween 20 concentrat ion at this part icular scan rate. The ~Ep of 60 mV indicates that the electrode reac- t ion is essentially reversible under these par t icular exper imental conditions.

The plots of the anodic peak current, ip, vs. ferrocene concentrat ion were l inear for any given Tween 20 concentrat ion at a constant scan rate. Also, ip in - creased l inear ly proport ional to the square root of scan rate as expected for a diffusion-limited electrode react ion for a constant Tween 20 and ferrocene concentration. However, the ip was dependent on the Tween 20 concentration. In Table II, these ip values (corrected for background current) are tabulated for Tween 20 concentrat ion varying between 1 and 3.5% by volume. The ferrocene concentrat ion was kept constant at 0.5 raM. The D values given in Table II were calculated from the slopes of the plots of ip vs. v I/2 for each Tween 20 concentration. The calculation assumed the validity of the we l l -known Randle- Sevcik relationship

Table I. Dependence of E0.s5 and ~Ep on Tween 20 concentration

Tween 20 concert- Eo.B~ (mV v s . tration (% vol) Ag/AgC1)* AEp (mV)

1.0 + 180 60 1.5 190 63 2.0 195 65 2.5 190 63 3.0 190 60 3.5 208 60

A v g 1 9 2 ~ 0 6 2 •

* E a c h v a l u e i s t h e a v e r a g e os 3 separate runs.

0 1.04 0.5 1.07 1.0 1.08 1.5 1.11 2.0 1.14 2.5 1.17 3.0 1.21 3.5 1.24 4.0 1.28

* V i s c o s i t y , ~, in cent is tokes was calculated us ing ~ = k t - - b/t, w h e r e k is viscometer constant, b is cal ibration constant , and t i s the t i m e r e q u i r e d for the test solut ion meniscus to pass b e t w e e n the t w o f low m a r k s on the v i scometer s tem ( k = 0.05193 and b -- 0.60). A l l v a l u e s are the average os 10 results .

{p = knS/2AD1/2C%I/2 [i]

A value of 2.64 X I05 was used for k in the computa- tion of D (12). The D values decreased as the Tween 20 concentrat ion increased.

The {p and D value dependence on Tween 20 concentrat ion was assumed to be due to the changing viscosity of the solutions. Thus, viscosities of Tween 20 solutions at concentrat ions between 0.5 and 4.0 volume percent (v/o) were determined using an Ub- belohde viscometer. The viscosity was found to vary l inear ly with concentrat ion between 1.0 and 4.0 v/o Tween 20. The data are summarized in Table III.

In a simple diffusing system, the D value should be inversely proport ional to the viscosity, ~I, assuming val idi ty of the Stokes-Einstein relationship (13)

D = IcT/Ta~Ir [2]

In Fig. 2, D value is plotted vs. 1/~r. The l inear i ty of the plot is excellent.

Calculation of micelle size.--The radius and molecular weight of the micelle can be computed from know-

5.0

c~ E o

~ ) 4 . 5

x

,5

~ 4.0

m

i5

5.5 0.017

I I I I I

I I [ [ [ 0.018 0.019 0 0 2 0 0.021 0 .022

(~r ) - I x I0 - [ ~ ( seo /g )

Fig. 2. Diffusion coefficient vs. reciprocal of ~lr

0.023



Vol . I23 , No. 9 EC OF M I C E L L E - S O L U B I L I Z E D FERROCENE 1 3 3 7

ing the values of diffusion coefficients and viscosities. The shape of the micelle is assumed as spherical. The volume fraction, ~b, of the dispersed spherical micelles is de termined by using the Guth and S imha equa- t ion (14, 15)

~1/~o "- 14.1~ 2 + 2.5~b -~ 1 [3]

where ~l/~lo is the re la t ive viscosity of the solution. The value of viscosity of the phosphate buffer solu- t ion in the absence of Tween 20 and ferrocene was used for 00. The effective specific volume, V, of one gram of surfactant, including any hydrated water, can be expressed as

V = r [4]

where C' is the concentrat ion of surfactant in the solution (grams/cubic cent imeter) .

The micelle weight, M, can be given in terms of the calculated 9"

(kt):sN M = [5 ]

162~sDsV

where N is Avogadro's number . The radius can be calculated from the value of V, again assuming a spherical particle. Values of D, V, r, and M as a func- t ion of Tween concentrat ion are tabula ted in Table IV. The average radius and molecular weight are 43A and 127,000 for the data listed in Table IV. From the data in this table, a slight increase in the radius and molecular weight of the micelles as the concentrat ion of Tween increases can be seen. Whether or not this increase actual ly reflects size change due to increased amount of Tween 20 per micelle cannot be accurately ascertained. Similar increases of measured radius or molecular weight wi th increase of nonionic surfactant concentrat ion have been previously reported (16). However, these increases may very well reflect changes in the micelle shape from spherical to ellipsoidal or in the number of hydrated water molecules associated with the micelle as a funct ion of Tween 20 concentration.

The molecular weight of the micelle was also determined independent ly using t.he Debye method of light scattering (17). Measurements of turbidi ty of Tween 20 between 0 and 5% concentrat ion in phosphate buffer were made with a Brice-Phoenix light scattering photometer at 0 ~ and 90 ~ This photometer was cali- brated with a dilute solution of Ludox (SM, du Pont) in water. The solute turbidity, or excess turbidity, upon which the molecular weight depends, is the apparent turbidity, z, of the solution with micelle minus the apparent turbidi ty, To, of the solvent, i.e., phosphate buffer without Tween 20 or ferrocene. The apparent difference in turb id i ty caused by the micelles and the refractive indices of the surfactant solutions can be related to molecular weight, M, through Eq. [6] and [7] as follows (17)

HC'/T = I / M q- 2BC' [6] a n d

32~no 2 (n -- no) 2 H = [7]

3~4N (C') 2

B is a constant depending on the solvent. All measurements were made with a blue filter (~ = 463 rim).

Table IV. Micelle parameters as a function of Tween 20 concentration

Tween 20 C' "V ~" x l0 s (% v/v) (g/cm a) (emS/g) (cm) M

1 0.010 1.48 41.2 119,000 2 0.020 1.68 42.2 113,009 2.5 0.025 1.57 43.4 132,000 3 0.030 1.67 43.7 127,000 3 . 5 0 . 0 3 6 1.60 45.1 144,000

Table V. Apparent turbidity and refractive index of Tween 20 solutions

T w e e n 20 ~- x 108 ( % v / v ) ( c m -1) ~ m

0 0.09* 1.34127" * 1 1.59 1.34221 2 2.35 1.34314 3 3.33 1.34421 4 2.49 1.34500 5 3.00 1.34600

* Ligh t s ca t t e r ing p h o t o m e t e r c a l i b r a t i o n us ing di lute s o l u t i o n of Ludox (SM, du Pon t ) .

** Cal ibra ted wi th KCI solut ion (0.09635 g / m l ) .

The apparent turbidi t ies and the dependence of the refractive indices on Tween 20 concentrat ion are tabulated in Table V. The refractive indices of the surfactant solution at tempera ture of 25~ was found empirically to follow the relat ionship

= no + 0.000944C' [8 ]

where the value of no was determined to be 1.34127. The molecular weight of micelle was determined by

calculating HC'/~ for the surfactant solutions and by plott ing HC'/~ vs. C' (Fig. 3). The extrapolated curve to zero concentrat ion gave an intercept whose reciprocal value was equal to the molecular weight, which was 143,000. The over-al l error was estimated to be • The light scattering method assumes that the micelle size is independent of surfactant concentration and that the number of micelles is increasing with increased C'. There appears to be reasonable agreement between the molecular weight of the micelle as evaluated from both the electrochemical and the l ight scattering data.

Assuming the average molecular weight of Tween 20 to be 1650, the average aggregation number of micelle (data of Table IV) is in the order of 77 • 5 (molecules/micelle) . An order of magni tude estimate of the ferrocene to micelle ratio is about 3:1 for a 2% by volume Tween 20 and 0.5 mM ferrocene concentration. For all of the experiments reported herein, the number of moles of ferrocene was greater than the number of moles of micelle present in the solution.

E o' v a l u e s . - - A n accurate assessment of the formal redox potential, E o', is impor tant for the use of the ferrocene-micelle as a M-T (2). The E ~ value (assuming E o' = E0.ss) evaluated from cyclic vo l tammetry gave an average of 424 • 6 mV vs. NHE. As a fur ther confirmation, E o' values were determined from potentiometric data for t i t rat ion using Ag2SO4 as oxidant and from A-E data obtained dur ing the exhaustive coulometr ic oxidations. The concentrat ion of ferr i - c inium ion was monitored at wavelength of 615 nm

5O

4O

HC' 6 -T- x 10

3O

2O

IO

I I I I

I I I I 0 I 2 3 4

Concentrotion of Tween 20 (g/ lOOm1)

Fig. 3. Reciprocal specific turbidity of Tween 20 solutions



1338 J . E l e c t r o c h e m . S a c . : E L E C T R O C H E M I C A L SCIENCE AND TECHNOLOGY S e p t e m b e r 1976

for the la t ter experiments. Nernst ian slopes of 58 _ 2 mV were obtained and the average value of E ~ was 425 ___ 10 mV vs. NHE. The E o' values de termined by these methods are in excellent agreement with that evaluated by cyclic vol tammetry. Ferrocene had been proposed (18) as a redox standard from solvent to solvent because both ferrocene and ferr ic in ium ion supposedly were min imal ly solvated. The formal potent ial for ferrocene has been reported to be 400 mV vs. NHE (18).

Hinkle (8) in his l ipid work found the addit ion of small amounts of the anion te t raphenylboron to greatly enhance the effectiveness of ferrocene as a mediator. He suggested that the te t raphenylboron increased the solubil i ty of ferr ic inium cation in the lipid phase of the membrane. Thus, the effect of te t raphenylboron to the cyclic vol tammetry of ferrocene- micelle was examined. In a solution containing 2% Tween, 0.74 mM ferrocene, and 0.40 mM sodium te t raphenylboron, the cyclic i - E curves were completely reversible with a zlEp of 60 mV • 2 inV. The E0.s5 was 390 mV vs. NHE. The shift of potent ial to less positive value is consistent with stabil ization of fer- r ic in ium ion by the anion, te t raphenylboron. Whether fer r ic in ium ion interacted with te t raphenylboron inside of the micelle and thus shifted the equi l ibr ium of react ion [11] could not be ascertained from the present data.

F e r r i c i n i u m ion . - -Fer r i c in ium ion is known to be quite water soluble. Its par t i t ioning be tween the micelles and solvent was qual i ta t ively examined by filtering a fer r ic in ium solution through a Diaflo filter (Amicon, XMb0; M.W. separat ion 50,000) at 15 psi pressure in a st irred cell. The blue ferr ic in ium ion was electrogenerated from ferrocene by controlled potential electrolysis in the Tween 20/phosphate buffer solution. The blue-colored filtrate indicated that fer- r ic in ium ion went through the filter and was not total ly micelle bound. A similar exper iment was performed with the fer rocene-Tween 20 solution. The filtrate in this case was clear and did not contain ferrocene, suggesting that ferrocene is indeed micelle bound. The reactions for the fe r rocene/ fer r ic in ium system in the presence of micelle are suggested as

Fe(CbH~)2 ~- micelle : Fe(CbHb)~. . . micelle [9]

Fe (C~HD2. . . micelle -- Fe (C5H5)2 + . . . micelle ~ e - [10]

Fe (CbHb)2 + . . . micelle = Fe (C~Hb)2 + ~- micelle [11]

The Keq'S are assumed to be greater than un i ty for both reactions [9] and [11].

The optical absorbance band at 615 nm was monitored dur ing controlled potential coulometric oxida- t ion of ferrocene to fer r ic in ium ion in a st irred solution. The optical absorbance, A, at 615 nm changed l inear ly with the number of coulombs of charge.

The molar absorptivities, ~, calculated for fer r ic in ium ion and also ferrocene-micel le are tabulated in Table VI and compared to l i terature values (19). The large discrepancy of the molar absorptivi ty between our value and the l i te ra ture value at wavelength of 250- 235 nm is present ly unexplored.

Fer r ic in ium ion in aqueous media has been reported (19) to undergo decomposition. Thus, the stabil i ty of ferr ic inium ion was determined by following the de- crease of the absorbance of the 615 nm band. A half- life of 14 hr was obtained for the ion in the pH 7.0 phosphate-buffered solution at tempera ture of 25~ At pH 2.0, the half- l i fe increased to 26 hr. This loss of ferr ic inium ion can affect results of long- te rm experiments or those where the ion concentrat ion is high.

Table VI. Spectral properties of ferrocene-micelle and ferricinium ion

Molar Wavelength absorptivity

maxima (rim) (M-l-era -I)

Ferrocene-micelle 325 (325)* 96 (52)* 440 (440) 120 (91)

Ferriciniumion** 250 (235) 3,860 (12,000) 615 (617) 335 (340)

* Literature values (19) in brackets; ferricinium ion data re- portedly taken in solution alcohol-water containing tetraphenyl- borate anion.

** Ferricinium ion generated by controlled potential coulom- etry.

F e r r o c e n e - m i c e l l e as m e d i a t o r . - t i t r a n t f o r herne pro- t e i n s . - - A s previously mentioned, methyl viologen was used as the M-T for the reduct ion of cytochrome c, cytochrome c oxidase, and mixtures thereof (2). In the respiratory chain, c (n = 1) is the component which transfers electrons To the enzyme bound cytochrome c oxidase (n -- 4). Oxidase is the enzyme which is responsible-~or the fast tu rnover of molecular oxygen to water and for coupling to oxidative phosphorylation.

The charge dis tr ibut ion between these heme c o m - p o n e n t s has been evaluated from the change in the optical absorbance, hA, at 550 n m (cytochrome c) and 605 nm (cytochrome c oxidase) dur ing indirect coulometric t i t ra t ion experiments (2). The plot of the oxidative ~A-q (q : electrochemical charge) was not a m i r r o r image of the reduct ive one (20). The oxidant was O2(n -- 4) and the reductant was the viologen radical cation (n = 1; E ~ - - --446V vs . NHE) (9). Besides the obvious difference in the n values, a difference in the react ivi ty toward cytochrome c exists between the reductant and oxidant. The viologen radical reduces both the cytochrome c and cytochrome c oxidase rapidly (21). On the other hand, O3 oxidizes na tured cytochrome c slowly. Thus, in a mix ture of cytochrome c and cytochrome c oxidase, cytochrome c must be oxidized pr imar i ly by cytochrome c oxidase when O2 is used as the oxidant.

With use of electrogenerated ferr ic in ium ion from the ferrocene-micelle, the AA-q curves were mir ror images of the reductive ones ( reductant :v io logen radical) (1). These results suggest that the charge dis t r ibut ion between cytochrome c and the redox centers of cytochrome c oxidase are at equi l ibr ium when the redox properties of the reductant and oxidant are similar.

Results to date indicate that the Eo' values for cytochrome c oxidase are 215 __. 15 and 345 • 15 mV vs . NHE. Each value of Eo' involves two electrons and the metal centers of one i ron and one copper. Since these E o' values are for cytochrome c oxidase isolated from the membrane (low lipid concentrat ion) there is a question whether these values accurately r e f l e c t the cytochrome c oxidase potent ial in the mitochon- drial system. It is also a question of why na ture would have the E o' of cytochrome c (E o' = 257 • 17 mV vs. NHE) (9) midway between the two potentials of cytochrome c oxidase.

Solubilization of M-T's by micelle formation pro- vides (a) access to a wider var ie ty of M-T's with oppor tuni ty for graded E o' values; (b) means of de- signing experiments to model biological electron t ransfer mechanisms a la Hinkle (8); and (c) an approach to kinetic studies to test whether e lect ron- t ransfer rates to components in membranes can be acceler- ated by interactions with micelle bound M-T's.

There has been suggestion (22) that " . . . the detergent solution, which consists of l ipoidal micel lu lar regions dispersed throughout an essentially aqueous



Vol. 123, No. 9 EC OF M I C E L L E - S O L U B I L I Z E D F E R R O C E N E 1339

phase, resembles the col loidal na tu re of biological envi ronment . "

Acknowledgment This inves t iga t ion was suppor ted by P H S - N I H Re-

search Gran t GM 19181 and NSF Gran t No. MPS73- 04882.

Manuscr ip t received June 30, 1975. This was Pape r 366 presen ted at the Toronto, Canada, Meet ing of the Society, May 11-16, 1975.

A n y discussion of this p a p e r wil l appear in a Discus- sion Sect ion to be publ i shed in the June 1977 JOURNAL. Al l discussions for the June 1977 Discussion Section should be submi t ted by Feb. 1, 1977.

Publication costs of this article were assisted by The Ohio State University.

LIST OF SYMBOLS p scan ra te Ip peak cu r ren t hEp separa t ion be tween anodic and cathodic peak

poten t ia l s n n u m b e r of electrons t r ans fe r red pe r ferrocene

molecule A a rea of electrode, cm 2 D diffusion coefficient, cm2/sec k Bol tzmann constant T t empera tu re , ~ r radius, A ~] viscosi ty ~]o re ference viscosi ty

vo lume f rac t ion C' concentra t ion (g /ml ) M micel le molecu la r weight

V effective specific volume N Avogadro ' s number

appa ren t t u rb id i t y To appa ren t t u rb id i ty of solvent

wave leng th of l ight, nm n re f rac t ive index no re f rac t ive index of solvent

s molar absorp t iv i ty A optical absorbance

REFERENCES 1. Y. Fuj ih i ra , T. Kuwana , and C. R. Hartzel l , Bio-

chem. Biophys. Res. Commun., 61, 488 (1974). 2. T. Kuwana and W. R. Heineman, Bioelectrochem.

Bioenergetics, 1, 389 (1974). 3. L. N. Mackey, T. Kuwana , and C. R. Hartzel l , Un-

publ i shed results. 4. T. Kuwana, D. E. Bublitz, and G. Hoh, J. Am.

Chem. Soc., 82, 5811 (1960). 5. S. Hayano and N. Shinozuka, Bull. Chem. Soc.

Japan, 42, 1469 (1969); 43, 2083 (1970); 44, 1503 (1971).

6. H. Suzuki, N. Shinozuka, and S. Hayano, ibid., 47, 1093 (1974).

7. P. G. Westmoreland, R. A. Day, and A. L. Under - wood, Anal. Chem., 44, 737 (1972).

8. P. Hinkle, Biochem. Biophys. Res. Commun., 41, 1375 (1970); Federation Proc., 32, 1988 (1973).

9. F. M. Hawkr idge and T. Kuwana , Anal. Chem., 45, 1021 (1973).

10. M. R. Cannon, Ind. Eng. Chem. Anal. Ed., 16, 708 (1944).

11. R. S. Nicholson and I. Shain, Anal. Chem., 36, 706 (1964).

12. R. N. Adams, "Elec t rochemis t ry at Sol id Elec- trodes," Marcel Dekker , New York (1969).

13. Robinson and Stokes, "Elect ro ly te Solutions," Academic Press, New York (1959).

t4. T. Nakagawa and K. Shinoda, "Phys iochemical Studies in Aqueous Solut ions of Nonionic Surface Act ive Agents, in "Colloidal Surfactants ," Shin- oda, Nakagawa, Tamamushi , and Isemma, Edi - tors, chap. 2, Academic Press, New York (1963).

15. E. Guth and R. Simha, Kolloid Z., 74, 266 (1936). 16. C. Tanford, J. Phys. Chem., 78, 2469 (1974). 17. P. Debye, J. Phys. Colloid Chem., 51, 18 (1947). 18. H. M. Koepp, H. Wendt, and H. Strehlow, Z. Elec-

trochim., 64, 483 (1960). 19. M. Rosenblum, "Chemis t ry of the I ron Group Met-

allocenes, P a r t 1," chap. 2, John Wi ley & Sons, Inc., New York (1965).

20. Wm. R. Heineman and T. Kuwana , Biochem. Bio- phys. Res. Commun., 50, 892 (1973).

21. L. Mackey, Ph.D. Thesis, Ohio Sta te Univers i ty (1975).

22. M. B. Lowe and J. N. Phil l ips, Nature, 190, 262 (1961).

Investigation of Adsorbed Hydrogen on Platinum Electrode by Means of Dynamic Impedance Measurement

Tetsuya Ohsaka, Yoshimitsu Sawada, and Tadashi Yoshida* Department of Applied Chemistry, Waseda University, Tokyo 160, Japan

and Kohji Nihei OKI Electric Industry Company, Limited, Fundamental Working Technology Laboratory, Tokyo 108, Japan

ABSTRACT

Adsorbed hydrogen phenomena on a p l a t inum electrode were inves t iga ted by an impedance method which draws po ten t iodynamica l ly the in -phase and quadra tu re components of e lec t rode admit tance. The kinetic behavior of h y - drogen adsorbed on p l a t inum was examined in ful l wi th the complex ca- paci tance representa t ion. The kinetic expe r imen ta l resul ts were wel l expla ined on the basis of the proposed equivalent circuit which was composed of the pseudocapaci tance, Warbu rg impedance, and ohmic component associated wi th the adsorp t ion-desorp t ion process of adsorbed hydrogen, together wi th the reac t ion resis tance due to the Volmer reaction.

Impedance measurements at the e lec t rode /so lu t ion in ter face have been deve loped recent ly (1) and used

�9 Electrochemieal Society Active Member. Key words: dynamic u'npedance measurement, hydrogen adsorp-

tion phenomena.

extens ive ly in solid e lec t rode /so lu t ion systems (2-4). The impedance measurements can be classified into two groups: that using a-c br idges and tha t d i rec t ly m e a - sur ing the a l t e rna t ing cur ren t th rough a c e i l The former enables us to de te rmine the in -phase and quad-



Documents

The Electrochemistry of Micelle-Solubilized Ferrocene