THE CATALYTIC EFFECT OF FERRICYANIDE IN THE OXYGEN ABSORPTION OF OLEIC ACID

BY BACON F. CHOW AND S. E. KAMERLING

(From the Converse Memorial Laborator?/ of Harvard University, Cambridge)

(Received for publication, October 13, 1933)

Wright, Conant, and Kamerling (1) have shown that oleic acid in an alkaline potassium ferricyanide solution will absorb oxygen. They further suggested that the autoxidation of oleic acid in the presence of ferricyanide is a chain reaction.

In continuation of this problem we present in this paper studies with other catalysts with evidence for the chain reaction, and data concerning the relationship between the rate of oxygen absorption and the oxidation-reduction potentials of the catalysts.

Methods

All estimations of the oxygen absorption were made with the Warburg apparatus. The composition of the phosphate buffer was 2 parts of ~/3 KzHPOd and 1 part of M/S KOH. Such a buffer solution has a slight advantage over 1 per cent sodium carbonate not only for its buffering capacity but a.lso for producing a shorter induction period. Unless otherwise stated, the absorption vessel contained 2 cc. of 0.0575 M oleic acid (Kahlbaum, purified grade and linoleic acid-free) solution in the phosphate buffer, 0.20 cc. of 0.115 M catalyst, and 0.1 cc. of 0.023 M inhibitor or of the buffer, so that the resulting solution was 0.05 M in oleic acid and 0.01 M

in catalyst. All experiments were performed at 25’.

Results

The rate of oxygen absorption by oleic acid was catalyzed by the presence of ferricyanide, and the latter was reduced to ferrocyanide.

Oxygen was necessary for complete reduction of the ferricyanide. Thus when 100 cc. of 0.05 M oleic acid and 0.01 M potassium ferri-

69

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70 Oxygen Absorption of Oleic Acid

cyanide in the phosphate buffer were stored in nitrogen freed of oxygen by bubbling through a series of Fieser (2) solutions (so- dium hyposulfite plus /3-naphthoquinone sulfonate), about 28 per cent of ferricyanide was reduced in 70 hours according to the elec- trometric titration of an aliquot sample with potassium molybdi- cyanide. Further standing in nitrogen did not increase the reduc- tion. When oxygen was introduced over the solution, without shaking, the reduction took place with great rapidity. The results are summarized in Table I.

If more potassium ferricyanide, sufficient to restore its concen- tration to 0.01 M, was added to the completely reduced ferrocy-

TABLE I

Rate of Reduction of Ferricqanide by Oleic Acid be-fore and after Introduction of.Ozygen _

In nitrogen

After introduction of oxygen

Time

lkrs.

2Q 44 73 92

116 24 48 72 96

144

-

-

-

Reduction of ferricyanide

Redy;ip, in

per cent per cent 15.4 0.77 19.3 0.16 28.1 0.30 29.8 0.09 28.4 -0.06 39.0 0.44 61.0 0.92 85.0 1.00 94.0 0.38 95.0 0.02

-

-

anide solution, more oxygen was absorbed and the added ferri- cyanide was reduced. On the other hand, if the solution was kept in nitrogen after the addition of ferricyanide, only a slight reduc- tion of the latter to ferrocyanide was obtained; 9 per cent reduc- tion after 26 hours and also 9 per cent after 74 hours. This reduc- tion in nitrogen was probably due to some oxygen not removed. These results indicate that oxygen is necessary for the reduction of ferricyanide by oleic acid.

The ratio of oxygen utilization to ferricyanide reduction should give information on the stoichiometric relationship as well as on the length of the chain. To this end we first shook in nitrogen a 0.05 M oleic acid and 0.01 M ferricyanide solution in the phosphate

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B. F. Chow and S. E. Kamerling 71

buffer, until no further reduction of ferricyanide was obtained. In two experiments the concentration of ferricyanide was reduced to 0.0076 M (Experiment A) and 0.0066 M (Experiment B). Then air was admitted into the bottle, whiich was connected to a gas

TABLE II

Determination of Oz/KaFe(CN)6 Ratio

Ferrocyanide produced after the introduction of oxygen.

Experiment Time 02 absorbed

KIF~(CN)G produced

hrs. nLM 7n.w

27 0.223 0.80

50 0.558 0.22 70 0.786 0.36

113 1.13 0.52 160 1.27 0.63 186 1.33 0.63 24 0.29 0.22 48 0.65 0.22 79 1.01 0.39 97 1.20 0.50

121 1.35 0.56 145 1.55 0.62 169 1.68 0.67 193 1.87 0.67

, --

-

Ratio Deviation h/KaFe(CN)e from mean

2.8 2.5 2.2 2.2 2.0 2.1 1.3 3.0 2.6 2.4 2.4 2.5 2.5 2.8

0.3 0.0

-0.3 -0.3 -0.5 -0.4 -1.2

0.45 0.1

-0.1 -0.1

0.0 0.0 0.3

FIG. 1. The rate of oxygen absorption of 0.05 M oleic acid in phosphate buffer with different concentrations of K,Fe(CN)G. Curve 1, 0.10 M; Curve 2,O.Ol M; Curve 3,O.OOl M. The dotted line represents the slope of the initial part of the curve, when only a small fraction of ferricyanide is reduced.

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72 Oxygen Absorption of Oleic Acid

burette and shaken. The volume of the absorbed oxygen was read, and the concentration of ferricyanide was determined by the electrometric titration. The results are given in Table II.

The average of the ratio is about 2.5. It indicates that the reduction of 2 moles of ferricyanide requires about 5 or 6 moles of oxygen .

To find how the rate of absorption is related to the concentra- tion of the catalyst, we carried out three experiments with varying concentrations of ferricyanide. The results are plotted in Fig. 1.

The rate of absorption increases with the concentration of the catalyst. However, although the concentration of ferricyanide in Curve 1 was 100 times that in Curve 3, yet the ratio of the rates of absorption, i.e. the slope of the curves, is only about 3. Thus Fig. 1 clearly indicates that the rate is not proportional to bhe first power of concentration of ferricyanide, and the following tabulation shows that the rate is approximately proportional to the t,hird root of the ferricyanide concentration.

Rate of 02 absorption, m>r 0s per hr. x 101

5.5 4.0 2.0

(K,Fe(CN)o) f (KsFe’e(CNM

M M

0.1 0.46 0.01 0.22 0.001 0.10

Effect of Oxidation-Reduction Potential of Catalyst on Rate of Oxygen Consumption-Experiments with other catalysts have shown that the oxidation-reduction potential of the catalyst is an important factor governing the rate of absorption. The potential is given by the electrochemical equation E = E. + (RT/nF) In ([Ol/[Rl), where E = the potential of the system, E. = the “nor- mal” oxidation-reduction potential characteristic of one substance, and [0] and [RI = the concentrations of the oxidant and reductant, respectively.

Variation in Potential by Varying the Ratio, K3Fe(CN)e:KcFe- (CN)e-It is obvious that the potential E is dependent on both Eo and the ratio of [Ol/[R]. Eo, of course, is a constant for one catalyst. Oxygen absorption velocities were measured in solu- tions containing ferricyanide to ferrocyanide in different ratios.

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B. F. Chow and S. E. Kamerling 73

The concentration of ferricyanide was constant at 0.01 M. Thus any variation in the rate must be due to the difference in potential, since potassium ferrocyanide alone does not catalyze the absorp- tion. The results in Table III show that O2 absorption decreases with decrease in the Fe+++:Fe++ ratio, and therefore wit.h the oxidation potential.

Variation in Potential by Use of a Diflerent Catalyst-A second method of varying the potential of the solution can be achieved by the use of a different catalyst. We have carried out experi- ments, using five catalysts of different normal potentials A’,, (see

TABLE III

Rate of Oxygen Absorption at Different Ratios of Potassium Ferricyanide to Ferrocyanide

The oxygen absorbed is expressed in mM X 103. The solutions were buffered with phosphate. The oleic acid concentration was 0.05 M.

KzFe CN) t

6,rnY 10 10 K,Fe CN)a, “ 0.1 1

I iz ;i

0.00 10

Time Oxygen absorbed - - hrs.

2

4 6 8

10 15 20 30 50

3.6 2.2 0.7 5.6 4.0 1.1 7.2 5.4 1.8 8.9 6.7 2.7

10.1 8.0 3.4 12.0 10.7 5.3 16.1 13.4 7.2 21.0 17.3 10.7 25.6 22.8 17.9

0.4 0.8 1.3 1.7 2.1 3.1 4.5 6.2 8.9

0.4

0.5 0.6 0.8

Table IV). The concentration of the catalysts, as before, is 0.01

M. Indophenol [O==<z==N(IT)--0-Na] having the same

molarity is twice as strong as ferricyanide in the oxidizing equiv- alent.

The normal potentials (E,J are all referred to the “normal” hy- drogen electrode. The EO of the indophenol at pH 11 of the phos- phat,e buffer is -0.08 volt. The normal potentials of the copper- glycine complex and of the copper-pyridine complex at pH 11 were measured electrometrically by the method of titration with sodium hydrosulfite in the phosphate buffer. The copper complexes were

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74 Oxygen Absorption of Oleic Acid

prepa.red by adding 0.01 mru of CuS04*5Hz0 to the phosphate buffer, 0.05 M in oleic acid and 0.05 M in pyridine or glycine respec- tively. In the determination of &, oleic acid was omitted. The average value of the normal potentials of the two complexes is 0.01 volt,.

Table IV is arranged in such a way that the normal potential decreases from left to right. The rate also decreases in the same direction.

Since the copper complexes and the indophenol have very low potent.ials, the atmospheric oxygen will oxidize their reduced forms

TABLE IV

Relationship between Rate of Absorption and EO of Diflerent Catalysts

The oxygen absorbed is expressed in mM X 103.

Time

--__ hrs.

t i:

1 2 4 6 8

10 15 20 30 50

Control. Il”

catalyst

0.1

0.2

0.2

0.4

TsMo(CN)s Fo =yo~too.72

1.9 3.3 6.8

12.2 13.4

-

;

-

KzFe(CNh so ;,f,".""

1.8 0.4 0.4 0.2 3.6 0.9 0.8 0.4 5.7 1.8 1.4 1.1 7.6 2.7 2.4 1.8 9.3 3.6 3.0 2.6

10.7 4.4 3.6 3.1 13.7 6.4 5.3 4.8 16.0 8.9 7.2 6.6 21.0 13.0 11.0 10.2 25.0 22.3 17.9 16.1

(

-

Zu-pyridine

Eo =vot,““’

-

v

Indophenol Eo = -0.08 olt at pH 11

as soon as they are formed. For that reason, the concentration of catalysts in the oxidized form is practically constant during the oxygen absorption by oleic acid. If this is true, the rate of ab- sorption will be constant, as long as oxygen and oleic acid are in excess. Thus we would expect to get a straight line, if we plot the oxygen absorbed against time. This is found essentially to be the case in Fig. 2. For comparison, the results with ferricyanide as catalyst are also included. The ferricyanide curve shows definite decrease in rate as the concentration of ferricyanide diminishes. The copper complexes and the indophenol curves give essentially

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B. F. Chow and S. E. Kamerling 75

constant slopes. Control experiments on pyridine and glycine (Le. without CuS04) are included in Fig. 2 to show the increase of the catalytic effect by the presence of copper sulfate. The formation of the copper complexes is indicated by the character- istic deep blue color.

Potassium molybdicyanide is metastable to the oxygen elec- trode at pH 11. The decomposition is catalyzed by light; there- fore the molybdicyanide experiment (Table IV) was performed in

6 he 8 I I I I I

3urs 10 al 30 40 50 60

FIG. 2. The rate of oxygen absorption of 0.05 M oleic acid in phosphate buffer under the influence of 0.01 M catalysts. Curve 1, ferricyanide; Curve 2, copper-glycine complex; Curve 3, copper-pyridine; Curve 4, indophenol; Curve 5,0.05 M pyridine; Curve 6,0.05 M glycine. The dotted line represents the slope of the initial part of the curve, when only a small fraction of ferricyanide is reduced.

the dark. Even then decomposition took place. For that reason no measurements after 4 hours were recorded.

Reproducibility of Results-During the whole course of the work one sample of pure oleic acid was used. Other reagents were pre- pared from time to time. In some thirty experiments with po- tassium ferricyanide the results checked with each other within 10 per cent or less. Data in Tables III and IV are the typical results. In three experiments with indophenol the results also checked with one another within less than 10 per cent.

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76 Oxygen Absorption of Oleic Acid

The results of copper complexes are more variable. Their catalytic activities are much more affected by impurities. Im- pure reagents gave higher rates. To avoid as much as possible the presence of impurities, the phosphate buffer was prepared from Kahlbaum’s highest grade potassium hydroxide and phos- phoric acid. Copper sulfate was repeatedly crystallized. The ordinary distilled water was fractionated several times. Pyri- dine was distilled over pure potassium hydroxide and refraction- ated. The results of the oxygen absorption obtained by using ordinary and purified reagents are presented in Table V.

TABLE V

Difference between Rates of Oxygen Absorption Catalyzed by Ordinary and by Carefully PuriJied Copper-Pyridine Complexes

The figures represent oxygen absorbed in rnM X 103.

Time, hrs.. 5 10 20 40

Ordinary reagents 3.1 6.2 12.5 25.0 3.1 6.2 10.7 22.8 3.0 5.6 11.6 17.9

Purified reagents 2.2 3.6 7.2 14.3 2.1 3.5 6.7 12.5 1.8 4.0 6.9 12.5 1.8 3.4 6.3 12.5 1.9 4.0 6.9 12.5 1.8 3.8 6.7 13.0 1.9 3.8 6.9 13.2

The experiments with the purified reagents were performed at seven different, times. The results, as given in Table V, show a reasonable accuracy and reproducibility. Whether the higher rate with the ordinary reagent is due to impurities or not is an open ques- tion.’ Nevertheless, the experiments with the purified reagents apparently show the catalytic effect of the copper-pyridine com- plex.

1 The fact that purified pyridine is also a catalyst may also be due to im- purities. The presence of 0.000002 M KCN completely inhibits the oxygen absorption.

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B. F. Chow and S. E. Kamerling 77

Inhibitors

Preliminary work (1) indicated that the reaction rate was de- creased by the addition of compounds known to function as in- hibitors of chain reactions. Further experiments of this kind are now reported. Obviously the inhibitors for the oxygen absorp- tion by oleic acid in the presence of potassium ferricyanide must not reduce the catalyst. For that reason, the inhibitors such as hydroquinone, pyrogallol, etc., which are extremely effective in other chain reactions cannot be used. We have tried several in- hibitors on the absorption catalyzed by ferricyanide and copper- pyridine complex. Because of the limitation stated above, the

TABLE VI

Results of Inhibition on Ferricyanide Catalyst

The figures represent oxygen absorbed in mM X 103.

Time

(1)

hrs.

5 10 15 20 30 50 70

Phenol 0.001 M

(2)

2.7 4.9 7.1 8.9

11.7 14.3 17.0

-

I

0

_ _

-

Saturated solution of dimethyi-

aniline

.@%I (ca.

2.4 4.5 5.8 7.3 9.4

11.2 15.3

Ethanol amine 0.001 M

F&&n;1

(4) (5) ~___

2.7 3.1 4.5 4.5 6.2 6.9 7.4 7.5 9.0 10.7

12.5 14.8

0.005 M KdFe(CN)s 0.005 M KzFe(CN)a 0.050 M oleic acid

(6)

2.7 4.5 5.8 6.9 7.8

11.6 12.0

N0ne

(7)

6.7 10.7 13.7 16.0 21.0 25.0

inhibition effects obtained are not as pronounced as in reactions definitely known to have a chain mechanism.

A control experiment was performed to follow the reduction of ferricyanide by the inhibitors (0.001 M) alone without the presence of oleic acid. In 20 hours not more than 25 per cent of ferricyanide was reduced, as determined by electrometric titration with molyb- dicyanide. Another experiment was carried out with 0.05 M oleic acid in the buffer solution which contained both K4Fe(CN)B and K,Fe(CN)B in 0.005 M concentration. These would be the con- centrations if an inhibitor had reduced 0.01 M ferricyanide 50 per cent at the very beginning of the experiment. Column 6 of Table

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78 Oxygen Absorption of Oleic Acid

VI gives the result of absorption of such a solution. The com- parison between this column and the inhibitor columns (Nos. 2 to 5) gives evidences that inhibition is real and not due to the decrease of ferricyanide concentration. Two points are worth mentioning in this connection: (1) If there were no real inhibition, the rates shown by the inhibitors during the first 10 hours could be accounted for by the reduction of ferricyanide only of 50 per cent or more. Such reduction did not occur, since the electrometric titration of the control sample with potassium molybdicyanide showed that only about 10 per cent ferrocyanide was produced in 10 hours. (2) In Column 6 after 50 hours the absorption was very slight

TABLE VII

Inhibitors of Copper-Pyridine Catalyst

The oxygen absorbed is expressed in mM X 103.

Time NOW2

hrs.

5

10 20 40 70

100

2.2 3.6 7.2

14.3 24.8

T -

$ L

-

Dimethylaniline p-Bromo- phenol

Solution Bt 0.001 M blution A*

0.4 1.3 1.7 2.0 2.9 3.6

0.9 0.8 1.1 1.3 2.2 2.7 4.2 4.5 5.8 7.2

-

-

Phenol

0.8 1.7 1.8 3.3 3.8 5.8 8.3 11.6

14.3 20.6 17.8

* Solution A = saturated solution of dimethylaniline in the phosphate buffer.

t Solution B = Solution A diluted lo-fold with buffer.

and an end-point of 12.0 X 10m3 mM of oxygen was soon reached. The electrometric titration of the solution after 70 hours gave the presence of ferricyanide in less than 5 per cent. On the other hand, the rate of absorption of inhibitor solutions was not changed considerably. The yellow color of ferricyanide in the solutions still existed.

Since the copper-pyridine complex has a very low oxidation- reduction potential, there is no possibility of complication from reduction of Cu++ by the inhibitors. Hence the evidence of in- hibition is especially definite in the case of this catalyst. The results are given in Table VII.

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B. F. Chow and S. E. Kamerling 79

Dimethylaniline apparently has a greater inhibiting power than either one of the phenols.

Besides oleic acid other unsaturated compounds have been tried. Preliminary experiments have shown that linoleic acid and linseed oil suspended in the phosphate buffer absorbed oxygen much more rapidly than oleic acid when equal concentration of ferricyanide was present. However, undecylenic acid under the same conditions absorbed oxygen more slowly than oleic acid.

The authors express their appreciation to Professor James B. Conant for the suggestion of this problem and for his guidance, without which this work would not have been possible; and to Dr. D. D. Van Slyke for criticizing the manuscript.

BIBLIOGRAPHY

1. Wright, G. P., Conant, J. B., and Kamerling, S. E., J. Biol. Chem., 94, 411 (1931-32).

2. Fieser, L. F., J. Am. Chem. Sot., 46, 2639 (1924).

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Bacon F. Chow and S. E. KamerlingABSORPTION OF OLEIC ACID

FERRICYANIDE IN THE OXYGEN THE CATALYTIC EFFECT OF

1934, 104:69-79.J. Biol. Chem. 

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