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HALF-\‘AVE. PCTE‘NTiALS OF
V‘ITAMWS K. AND SOME VITAMIN B
COMPLEXES

Thesis for the Degree 0{ M. S
MICHEGAN STATE COLLEGE

Winn-um John Abraham
3943

 

 

 

 

 

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HALF-WAVE POTENTIALS OF VITAMINS K,
AND SOME VITAMIN B COMPLEXES

BY

William John Abraham

A THESIS

Submitted to the Graduate School of Michigan
State College of Agriculture and Applied
Science in partial fulfilment of the
requirements for the degree of

MASTER OF SCIENCE
Department of Chemistry
1945

Tffia‘ll
arse

 

ACKNOWLEDGMENT

The writer wishes to express his appreciation to
Dr. D. T. Ewing, Professor of Physical Chemistry, for
his guidance and the materials furnished.

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INTRODUCTION

Polarographic methodssllhave been successfully used ‘
in the study of the kinetics of reactions; and the evalu-
ation of the stability, and polymerization of organic sub-
stances. In numerous instances it has given definite
assistance in establishing the organic structure of some
complex compounds. It has been fairly well determined
that there is a close relationship between the polaro-
graphic reduction potential of a compound and its chemi-
cal constitution. The polarOgraph is well suited for
such measurements.

Much less is known of the interpretation of current-
voltage curves of organic compounds than of most inorganic
compounds, because, generally, reduction of organic com-_
pounds are irreversible reactions. In irreversible re-
ductions the nature of the reduction product is often
unknown, due to the exceedingly small amount formed during
the electrolysis. However, much information regarding
the structure of the compound can be gathered by deter-
mining the number of electrons involved in the reduction
at the drapping mercury electrode.

The main difference between the oxidation or reduc-
tion of inorganic substances and organic substances is
that in the reduction or oxidation of the organic sub-

stance hydrogen ions are usually involved in the electrode

reaction. The equation

R + nH+ + ne :1: iRHh
represents a general case in which the oxidant and reduc-
tant are both uncharged molecules. Ordinarily n is equal
to 2.

It is desirable to work with buffered solution in
the studies of the reductions or oxidations of organic
compounds, because electrons are involved in the elec-
trode reactions. If buffered solutions weren't used the
pH of the solution would be changing continually. Never-
theless, some compounds form better waves in unbuffered
solutions and in these cases unbuffered solutions are
used.

In this investigation experiments were carried out
in an endeavour to determine the stability and rate of
decomposition, on eXposure to air, of various vitamin K

solutions.
{ATERIALS

The mercury which is used must be exceedingly pure,
and may be used only once before being repurified. A
high grade of mercury, which had been previously used in
polarographic work, was used for first experiments. After
being used once it was purified by first shaking vigor-
ously with distilled water in order to remove all water

soluble material. This Operation is repeated four times

and then the mercury is shaken four times with 8% nitric
acid. Next it is passed through a cone of filter paper
with a hole in the apex and through a four foot length of
glass capillary. This Operation removes all solid par-
ticles not soluble in water or 5% nitric acid. The mercury
is now allowed to dr0p in a stream of fine drOplets through
a five foot column of 3% nitric acid. After this the mer-
cury is distilled in vaccuum and is then ready for use.

The nitrogen used is obtained from a commercial tank
of nitrogen. Oxygen and other impurities which may be pre-
sent are removed by first passing the nitrogen through a
solution of pyrogallic acid, next through a solution of
alkaline potassium permanganate, and finally washed by

passing through water.

PROCEDURE

A Leeds and Northrup Electro-ChemOgraph was used in
this investigation and in the following paragraph the
procedure used is outlined. mercury is poured into the
dry electrolysis cell until the bottom is covered. A
measured amount, usually four mililiters, of the solution
to be analyzed is added. Five drOps of 0.05% gelatin is
added to the solution in the cell. The gelatin is added
to prevent the formation of current maximas. The cell is
placed in position and the electrodes inserted. Nitrogen

is bubbled through the solution for three minutes, in order

to remove any dissolved oxygen. Oxygen is easily reduced
at the drOpping mercury electrode and if not removed it
will give a reduction wave which.will interfere with the
wave of the substance to be analyzed. The solution is

kept under an atmOSphere of nitrogen throughout the elec-
trolysis. During the time the nitrogen is bubbling through
the solution the three electrical balances, described in
the directions for operation of the instrument, are made.
The preper shunt, depending on the concentration of the
solution, is inserted. The height of the mercury column

is adjusted so that the drOp time is from 2 to 4 seconds.
In most cases it was adjusted so that one drOp of mercury
fell every three seconds. The galvanometer is now released
and allowed to come to rest. The polarizing unit is now
started, this steadily increases the potential between the
electrodes, and at the same time the chart is set in motion.
The current-voltage curve is now automatically traced on
the moving chart and nothing remains to be done except

turn the instrument off at the conclusion of the run.

INVESTIGATION OF VITaMIN K COMELEXES

The substances used in this investigation were:
2-methyl-l,4-naphthoquinone, 2,3-dimethyl-l,4-naphtho-
quinone, l-hydroxy-Z-mtthyl-d-naphthol amine hydrochlo-
ride, and the dimer of 2-methyl-l,4-naphthoquinone.

Stock solutions of each compound were prepared by dis-

solving in either oxygen free water or oxygen free alcohol,
depending on the solubility of the compound. The oxygen
was removed from the water and alcohol by suction. The
stock solutions were kept tightly stoppered and in the

dark except when a sample was being withdrawn. Then a
determination was to be made a sample was withdrawn and
diluted 1 to l with 0.1 N KCl or buffer, gelatin was added
and it was then electrolyzed.

Z-Methyl-l,4-naphthoquinone is practically insoluble
in water, so ethyl alcohol was used as a solvent. The
alcoholic solution was found to be perfectly stable. The
2-methyl-l,4-naphthoquinone gave an excellent cathodic
wave in both an unbuffered KCl solution and a buffered
solution of a pH of 5.0. The buffer used was composed

of equal parts of l M NaCZHEO2 and l M HCZH O The

3 2’
buffer alone had a pH of 4.6. When it was diluted with
an equal amount of water it had a pH of 4.6. When it was
diluted with an equal amount of ethyl alcohol it had a pH
of 5.0. Fig. 1 shows a cathodic wave of 2-methyl~l,4-
naphthoquinone, and Table 1 summarizes the results. The
unbuffered solution exhibited a current maxima which
could not be suppressed.

2,5-Dimethyl-l,4-naphthoquinone is quite similar to
2-methyl-l,4-naphthoquinone. it is insoluble in water

but soluble in ethyl alcohol. The cathodic wave of 2,5-

dimethyl-l,4-naphthoquinone is exactly like the one ob-
tained with 2-methyl-l,4-naphthoquinone except that it
has a higher reduction potential and a higher half-wave
potential as can readily be seen from Table 7. Fig. 5
shows a typical wave. Cathodic waves were formed from
both buffered and unbuffered solutions. The wave from
the unbuffered KCl solution was the better of the two.
The wave formed from the buffered solution exhibited a
current maxima which could not be suppressed by either
gelatin or glue solution.

1-Hydroxy-2-methylo4-naphthol amine hydrochloride
is soluble in water thus permitting use of an aqueous
solution. l—Hydroxy-Z-methyl-d-naphthol amine hydrochlo-
ride was found to be very easily oxidized upon being
exposed to the air for only a very few moments. The
cathodic wave formed from a buffered solution of l-hydr-
oxy-Z-methyl-4-naphthol amine hydrochloride, pH of 5.0,
changed considerably on aging. At first the reduction
potential remains constant and the diffusion Current
increases steadily. When the solution was one hour old
the diffusion current had reached a limiting value and
now the reduction potential started to increase with the
diffusion current remaining constant. When the solution
was three hours old the reduction potential and half-wave
potential had reached limiting values. After this the

cathodic wave did not change as the solution continued

to age. This effect is shown in Table 4 and Fig. 5.

The results shown in Table 2 were obtained using an
old solution of l-hydroxy-z-methyl-4-naphthol amine hydro-
chloride in which the reduction potential and diffusion
current had become constant. Good curves were formed
from the buffered solution of a pH of 5.0. A slight cur-
rent maxima, which could not be suppressed by either gel-
atin or glue, was exhibited both in the buffered and the

unbuffered solution. Fig. 2 illustrates a wave from an

old solution. If a sample of a fresh solution of l-hydr-
oxy-Z-methyl-é-naphthol amine hydrochloride is taken and
electrolized, then left exposed to air and electrolyzed
again, etc., the same change in reduction potential and
diffusion current, as described above, is noted; only this
time it occurs much faster. The change depends on the
time and intimacy of the contact of the solution with the
air. It is probably due to oxidation of the l-hydroxy-Z-
methyl-4-naphthol amine hydrochloride by the oxygen in the
air. I

The effect of irradiation, by ultra-violet light, on
a solution of 2-methyl-l,4-naphthoquinone was studied.
The solvent used was absolute ethyl alcohol. The solution
was irradiated for a definite interval of time and a
sample was withdrawn. The sample was diluted 1 to l with
0.1 N K01 and electrolyzed.

The results of this irradiation are shown in Table 8.

At first the curve broke at -0.44 v. After being irrad-

iated for ten minutes there were two curves; one broke at
-0.44 v. and the other at -0.74 v. The first curve dis-
appeared gradually and the second increased as the irrad-
iation continued. After the solution had been irradiated
for a period of twenty minutes the first curve had com-
pletely disappeared and a third wave had appeared. The
second wave broke at -O.70 v. and the third at -l.18 v.
As irradiation continued the second curve began to lose
its shape and after the solution had been irradiated for
forty minutes only the third wave remained. As irradia-
tion continued the half-wave potential of the third wave
remained constant but the diffusion current increased
slowly. It is readily apparent that the irradiation with
the ultra-violet light changed the structure of the
2-methyl-l,4-naphthoquinone.

A solution of the dimer of 2-methyl-l,4-naphthoqui-
none was prepared by dissolving the dimer in absolute
alcohol. Upon being reduced at the drOpping mercury
electrode the dimer gave a double cathodic wave. Results
are given in Table 6. The dimer was diluted 1 to l with
0.1 N KCl before being electrolyzed.

Commercial liquid samples of l-hydroxy-Z-methyl-4-
naphthol amine hydrochloride were measured. The samples
were obtained in sealed ampoules; each contained 1 mg. of
the compound. The ampoule was Opened immediatly before
it was to be electrolyzed. The contents of the ampoule

was diluted 1 to l with acetate buffer and measured, then

left eXposed to the air for a short time and remeasured.

When ampoule No. 199 (50271) was used, two cathodic
waves were formed. The first increased in sized steadily
until reaching a maximum value when the solution was
about three hours old. The first wave had a reduction
potential of -0.02 v. at first. When the solution was
about two hours old the reduction potential started to
increase. It reached a maximum value of -0.20 v. when
the solution was six hours old. The reduction potential
of the second wave was ~0.70 v. at first. It increased
steadily to a value of -l.0 v. by the time the solution
was three hours old. The size of the second wave varied
hardly at all. Table 20 summarizes the results.

When ampoule No. 199 (49771) was used, two waves
were again formed, which were of about equal size at first.
One broke at -0.39 v. and the other at -0.65 v. During the
first one and one-half hours the first wave decreased in
size while the second increased in size and the final
limiting current remained approximately constant. By
stating that the uwave increases in size" is meant that
the diffusion current of that particular wave is increased.
The reduction potential of the first wave increased to
-0.45 v. whereas the reduction potential of the second
did not change. At the end of four and one-half hours
both full waves had increased in size. The reduction

potential of the first wave fell to -0.02 v. and the sec-

10

0nd had risen to -0.75 v. When the solution was five and
one-half hours old the diffusion current of the first wave
had increased slightly and the diffusion current of the
second wave had remained constant. The reduction poten-
tial of the first wave had risen to -0.08 v. and the reduc-
tion potential of the second wave had risen to —0.95 v.
When the solution was forty-eight hours old the values

for the half-wave potential and the diffusion current of
both waves were approximately the same as when the solu-
tion was only five and one-half hours old. This showed
that limiting values had probably been reached. The re-
sults are tabulated in Table 21.

The solutions from the two different ampoules behaved
differently on being exposed to the air but it is interest-
ing to note that the final curves obtained from both
solutions are approximately the same, as can readily be
seen from a comparsion of Table 20 and Table 21. The
reason for the two samples passing through a different
series of steps is probably due to a difference in sol-
vent, which would lead to a difference of pH between the
two solutions. It is also to be noted that the half-wave
potential of the first curve in its final form is the same
as the half-wave potential of and old solution of l-hydr-
oxy-Z-methyl-4-naphthol amine hydrochloride which was

made up from the solid.

Table l.

of ZéMethyl-l,4-Naphthoquinone

Effect of pH on the Half-Wave Potential

 

 

 

 

 

 

 

 

 

 

 

 

Cone. Shunt Reduction alf-Weve
mm was a “masters
100 22222 5.0 -0.54 -O.42
200 22222 5.0 -0.54 -0.45
100 50000 5.0 -O.20 -0.42
200 50000 5.0 -0.53 -0.44
100 22222 7.0 -0.44 —0.57
200 22222 7.0 -0.44 -0.58
100 50000 7.0 -0.41 -0.57
200 50000 7.0 -0.40 -O.59
Table 2. Effect of pH on the Half-Wave Potential
l-Hydroxy-24Methyl-4-Naphthol Amine HCl
Conc . Shunt Feduction Ealf-Wave
W was) PH was was
100 22222 4.6 -0.24 ~0.54
200 22222 4.6 -0.22 -0.55
100 50000 4.6 -0.22 -0.56
200 50000 4.6 -O.23 -0.56
100 22222 7.0 '-o.o7 -o.5o
200 22222 7.0 -0.55 ~0.52
100 50000 7.0 -O.54 -0.50
200 50000 7.0 -0.54 -0.52

 

 

 

 

 

 

ll

12

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 3. Effect of Aging on a Solution of
2-Methyl-l,4-Naphthoquinone
Cong. Shunt Age heduction Half-Wave Diffusion
(mg/l) (ohms) (hrs) TotentialIPotential Current
(volts) tvolts) (microamp)
200 22222 0 —0.40 -0.57 5.9
200 22222 2 -0.45 -0.57 4.0
200 22222 8 -0040 -0057 0.9
200 22222 24 -0.40 -0.58 5.7
Table 4. Effect of Aging on a Solution of
l-Hydroxyo24Methyl-4-Eaphthol Amine
Hydrochloride
gone. Shunt 383 Eeduction.balf-Wave Diffusion
(mg/l) (ohms) (hrs) otential otential Current
A (volts) (volts) tmicroamp)
200 50000 0 -0.02 ~0.09 1.65
200 50000 1 -0.05 -0.ll 2.7
200 50000 2 ~0.09 -0.11 2.6
200 50000 5 -0.25 -0.55 2.6
200 50000 6 -0.24 -0.55 2.7
‘200 50000 7 .0.24 -0.35 2.6

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5. Effect of Aging on a Solution of
2,5-Dimethyl-l.4-Kaphthoquinone
Conc. Shunt Age Eeduction Half-Wave Diffusion
(mg/l) (ohms) (hrs) otential Potential Current
(volts) (volts) (microamp)
200 22222 0 ~0.48 -0.65 5.5
200 22222 2 -0.48 -0.65 5.5
200 22222 6.5 -0.48 ~0.65 5.5
200 22222 51.5 -0.49 -0.65 5.5
Table 6. Measurement of the Half-Wave Potential of the
Dimer of 24Methyl-l,4-Naphthoquinone
Cone. Shunt I First Wave I Second Wave
(me/ll looms} . ,
heduction alf-Wave eduction.Ealf-Wave
JEotential otential otential otential
(volts) (volts) (volts (volts)
100 22222 ~0.99 -1.06 ol.55 -l.66
100 50000 -0.98 -l.06 -l.54 -l.68
200 22222 -0.98 -l.07 -l.57 -l.69
200 50000 -0.96 -l.07 -l.57 -l.7l

 

 

 

 

Table 7. Effect of pH on the Half-Wave Potential
of 2,5-Dimethyl-l,4-Naphthoquinone

Conc. Shunt Reduction Half-Wave

(mg/l) (ohms) pH Potential Potential

(VOltS) (volts)
100 22222 5.0 -0.41 -0.48
200 22222 5.0 ~0.40 -O.50
100 50000 5.0 ~C.40 ~0.49
200 50000 5.0 -0.40 -0.52
100 22222 7.0 -0.5 -0.64
200 22222 7.0 -0.50 -O.66
100 50000 7.0 -0.51 -0.66
200 50000 7.0 -0.44 -0.67

 

 

 

 

 

 

 

l4

Table 20. Effect of Aging on a Solution of l-Hydroxy-
24Methyl-4~Naphthol Amine Hydrochloride*

Ampoule No. 50271

 

 

 

 

Shunt Age First Wave Second Wave
(Ohms) (hrs) Half-Wave Diffusioanalf-Wave iffusion
Potential Current Potential Current
(volts) Imicroamp)(volts) (microamp)
22222 .5 ~0.06 1.9 -0.85 10.8
22222 .75 -0.07 2.6 -0.84 10.8
22222 1.5 -0.08 5.2 -0.87 10.8
22222 3.0 ’0026 {Job “1011 1008
22222 6.0 -O.29 5.5 -l.12 11.0
22222 9.0 -0051 508 -1012 1100

 

 

 

 

 

 

 

*Conc. is 500 mg/l.

Table 21. Effect of Aging on a Solution of l-Hydroxy-
2-Nethyl-4-Naphthol Amine Hydrochloride*

Ampoule No. 49771

 

 

 

 

Shunt Age | First Wave l Second Wave
(ohms) (hrs) Ealf-Wave Diffusio1galf-Wave Diffusion
otential Current otential current
(volts) (microamp) (volts)(microamph
22222 0 -0.48 4.2 -0.75 5.0
22222 .25 -0048 5.2 -0076 604
22222 105 -'0051 102 -0077 8.4
22222 5.5 -0.17 5.4 -l.08 9.2
22222 48.0 -0.o2 5.7 -l.ll- 10.4

 

 

 

 

 

 

 

*Conc. is 500 mg/l.

/6

 

 

 

 

 

 

 

 

 

 

 

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Fig. 2
l-HydroxJ-z-xethyl-4-Rbphthol Amine Hydrochloride
200 mg. per liter
Shunt - 22.222 ohms

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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56

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Fig. 3

2.3-Dimgthyi-L4-NGphthoquinone

200 mg. per liter
Shunt - 22.222 ohms

 

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Microamperes

Fig. 4

Dimer of 2-Methyl-l.4-Naphthoquinone
200 mg. per liter
Shunt - 50,000 ohms

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Fig. 5

l-Hydroxy-z-Methyl-4-N0phthol Amine Hydrochloride
200 mg. per liter
Shunt - 50,000 ohms

l7

INVESTIGATION OF VITAMIN B CLNELEXES

Riboflavin, thiamin hydrochloride, nicotinic acid,
and nicotinic amide were the complexes used. JgJ. Lin-
gane‘2)did most of the early work in the application of
the polarOgraph to the Vitamin B complexes.

Table 9 shows some of the results obtained with
riboflavin. Riboflavin was the easiest of the complexes
to determine. It gave excellent cathodic waves in an
unbuffered 0.1 N KCl solution. It also gave good waves
in buffered solutions. The riboflavin was dissolved
directly in the 0.1 N KCl. Riboflavin did not exhibit

a current maxima and it was not necessary to add gelatin.
The reduction potential of riboflavin was found to vary
with the pH of the solution. Fig. 6 shows a good cath-
odic wave of riboflavin.

It was necessary to neutralize the nicotinic acid
with sodium hydtoxide before it would give a good cath-
odic wave in 0.1 N KCl. A good cathodic wave was obtained
if the nicotinic acid was dissolved directly in 0.1 R
NaHCOE. The sodium bicarbonate neutralized the nicotinic
acid and also acted asva buffer. ‘Results are shown in
Sable lO. Nicotinic acid did exhibit a current maxima
and so it was necessary to add gelatin as a suppressor.

The curve obtained is actually the cathodic wave of sodium

18

nicotinate. The pH of the solution greatly affects the
reduction potential of the nicotinic acid. A cathodic
wave of nicotinic acid is shown in Fig. 8.

It was difficult to get a good cathodic wave of thi-
amin hydrochloride. Best results were obtained when the
thiamin hydrochloride was dissolved directly in 0.1 N KCl.
These results are given in Table 11. Fig. 7 shows a typ-
ical curve.

Nicotinic amide was easier to measure than nicotinic
acid. The cathodic wave of nicotinic amide had a slightly
higher half-wave potential than the wave of nicotinic acid.
Fig. 9 shows a cathodic wave of nicotinic amide. This
wave was obtained by dissolving the nicotinic amide direct-
ly in 0.1 R KCl. Results are given in Table 12.

Riboflavin and neutralized nicotinic acid may be si-
multaneously determined from the same solution. The re-
duction potentials of the two substances are far enough
apart so that the individual waves do not interfere with
each other. Only fair results, as shown by Fig. 10, Iere
obtained. Table 13 gives the results of the determination.
Table 14 summarizes results from a simultaneous determin-
ation of riboflavin and thiamin hydrochloride. Riboflavin
and nicotinic amide can also be determined together. Re-
sults are given in Table 17.

A simultaneous determination of riboflavin, thiamin

19

hydrochloride, and nicotinic acid was made from the same
solution. Fair results were obtained, as can be seen from
Fig. 12 and Table 15. The results from a simultaneous
determination of riboflavin, thiamin hydrochloride, and
nicotinic amide were not very satisfactory; these results
are given in Table 16. Great care had to be taken in the
neutralization of the nicotinic acid. Either an insuffic-
ient amount or an excess of sodium hydroxide would ruin
the curve. The whole determination was very sensitive to

the pH of the solution. The best results were obtained

at a pH of 6.8.

20

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 9. Measurement of the Half-Wave Potential
of Riboflavin
Conc. Shunt fieduction Half-Wave
'm 1) (ohms) H’ otential Retential
( g/ P (volts) (volts)
200 50000 7.0 -0.54 -0.64
100 400000 7.0 -0.48 -0.6b
87.5 200000 7.0 -0.51 -0.62
50 400000 7.0 -0.08 -0.62
Table 10. Measurement of the Half-Wave Potential
of Nicotinic Acid
Reduction elf-Wave
gnyi) $3332) pH’ I?otentiali¥otential
g (volts) (volts)
200 200000 8.9 -l.68 -l.77
200 50000 8.9 -l.70 -l.77
100 400000 8.9 -l.06 -l.77
Table 11. Measurement of the Half-Wave Potential
of Thiamin Hydrochloride
eduction elf-Wave
(£32711) $333; pH botential Potential
A (volts) (volts)
200 50000 7.0 ~l.20 -l.54
100 50000 7.0 -l.25 -l.55

 

 

 

 

 

 

21

 

 

 

 

 

 

 

 

 

Table 12. Keasurement of the Half-Wave Potential
of Nicotinic Amide
. S t Reduction Half-Wave
(3271) (2233) PH: PotentialIPotential
(volts) (Volts)
50 200000 7.0 -1.67 -1.81
Table 15. Eeasurement of the Half-Wave Potential of a

Mixture of Riboflavin and Nicotinic Acid

 

 

Cone. Shunt Reduction elf-Wave

Substance (mg/1) (ohms) pH Potential 'otential

(volts) (volts)
Riboflavinl 100 400000 8.9 -0.54 -0.62
Riboflavinz 200 400000 8.9 -0.47 -0.59
Nicotinic 100 400000 8.9 -l.66 -l.77

Acidg

Nicotinicl 200 400000 8.9 -l.64 —l.77

 

 

 

 

 

 

 

 

1 3 Same Solution 2: Same Solution

 

 

 

 

 

 

 

 

Table 14. Measurement of the Half-Wave Potential of a
Kixture 0f Riboflavin &.Thiamin Hydrochloride
Cone. Shunt Reduction Half-Wave
(me/n «ohms: p. meme meme
Riboflavin 100 50000 7.0 -0.35 -0.47
Riboflavin 50 200000 7.0 -0.54 -0.45
Thiamin H01 100 50000 7.0 -1.26 -l.04
Thiamin H01 50 200000 7.0 -1.22 -1.06

 

 

22

Table 15. Measurement of the Half-Wave Potentials of a
Mixture of Riboflavin and Nicotinic Acid.&Thiamin

 

 

Reduction Half-Wave
Substance €2n7iI igfiggl pH JBotential Potential
5' (volts) (Volts)
Riboflavin 50 200000 6.8 -0.36 -0.45
Thiamin H01 100 200000 6.8 -1.18 -l.3
Nicotinic 100 200000 6.8 -l.60 -l.74
Acid

 

 

 

 

 

 

 

 

Table 16. Measurement of the Half-Wave Potentials of a
Mixture of Riboflavin, Thiamin HCl & Nicotinic

 

 

Amide
eduction alf-Wave
Substance (gn7l) §22§§I pH Eotential.Eotential
, g (volts) (volts)
Riboflavin 100 50000 7.0 -0.56 -O.46
Thiamin H01 100 50000 7.0 -l.24 -l.38
Nicotinic 100 50000 7.0 ~1.6l -l.81
Amide

 

 

 

 

 

 

 

 

Table 17. Measurement of the Half-Rave Potentials of a
Mixture of Riboflavin and Nicotinic Amide

. Reduction lf-Wave
0 . S un
Substance %m271I (ghmEI pH lPotentiallEotential

(volts) (volts)

 

 

Riboflavin 87.5 200000 7.0 -O.5l o0.62

Amide

 

 

 

 

 

 

 

 

 

 

 

 

 

03

 

 

 

 

 

 

 

 

 

02

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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r15. 6
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000 ohm.

per liter

200 I8-
ahunt - 50

 

 

 

 

 

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200 mg. per liter
Shunt - 50.000 ohms

 

Thiamin Hydrochloride

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

-108

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400 mg. per liter

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98

06

94
Hicroenmeree
Fig. 9
licotinic Amide
200 mg. per liter
Shunt - 22.222 ohms

 

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10

Fig.

Riboflavin 100 mg. per liter
Nicotinic Acid 200 mg. per liter

Shunt - 200,000 ohms

 

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l--- I"“"“
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Thiamin Hydrochloride 100 mg. per liter

 

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-100

 

Fig. 12

Riboflavin 50 mg. per liter
Nicotinic Acid 100 mg. per liter

Thiamin Hydrochloride 100 mg. per liter

Shunt - 200,000 ohms

INVESTIGATICK OF ASCORBIC ACID

Ascorbic acid did not give a cathodic wave. It is
.a strong reducing agent and, as a result, gives a good
anodic wave. in the determination of ascorbic acid the
drOpping mercury electrode is the anode and the pool of
mercury in the bottom of the cell is the cathode.

Ascorbic acid is soluble in water and an aqueous
solution of ascorbic acid is stable. However, ascorbic
acid is readily oxidized by oxygen and so oxygen must be
removed from the water before the ascorbic acid is dis-
solved in it, and the solution must be kept in an air
tight container. The oxygen was removed from the water
by suction.

Sodium ascorbate is also water soluble and it gave
an anodic wave almost identical with that obtained from
ascorbic acid. Sodium ascorbate is, also, easily oxidized
by oxygen, so the same precautions were used in the case
of sodium ascorbate as were used in the preparation of a
solution of ascorbic acid. i

Both ascorbic acid and sodium ascorbate gave best
results in a buffered solution. A bufier of pH of 7.0
and a buffer of pH 4.6 were used. The bufier of a pH of
7.0 was composed of 0.1 M NaOH and 0.1 M KH2P04. The

buffer of a pH of 4.6 was made up of equal parts of l M

24

2
ascorbic acid and sodium ascorbate were diluted 1 to l

NaC HgO9 and l M HCDHHOZ. The aqueous solutions of the

with the buffer, gelatin was added, and the solution was
then electrolyzed. Excellent curves were obtained of
both compounds as can be seen from Fig. lb and Fig. 14.
The buffered solution of a pH of 4.b gave the more satis-
factory results. Sodium ascorbate gave good anodic waves
in both buffers. The waves of ascorbic acid in a buffer
of pH of 7.0 had a poorly defined limiting current. The
results obtained with these two compounds are tabulated

in Table 18 and Table 19.

 

 

 

 

 

 

 

 

 

 

 

Table 18. Effect of pH on the Half-Wave Potential
of Ascorbic Acid
Conc. Shunt . indation Half-Wave
«mg/n (Ohms: DH “23:32:? ”was:
.100 22222 4.6 0.02 0.09
100 22222 7.0 0.02 0.08
100 50000 4.6 0.02 0.09
100 50000 7.0 0.02 0.08
200 22222 4.6 0.02 0.09
200 22222 7.0 0.02 0.09
Table 19. Effect of pH on the Half-Wave Potential
of Sodium Ascorbate
Cone. Shunt Exidation.Half-Wave
W we PH was was
100 22222 4.6 0.02 0.08
100 22222 7.0 0.02 0.07
100 50000 4.6 0.02 0.09
100 50000 7.0 0.02 0.08
200 22222 4.6 0.02 0.08
200 22222 7.0 0.02 0.08

 

 

 

 

 

 

 

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0 ‘2 O4
n1croonpcrco

Fig. 13
Ascorbic Acid

200 I8- per liter
shunt - 22,222 ohm-

-2 0 92 64 96
licroa-pcrea

Fig. 14

Sodium Ascorbote
200 mg. per liter
Shunt - 22.222 ohms

 

08

26.

SULKfiRY

Z-Methyl-l,4-naphthoquinone forms excellent cathodic
waves in an unbuffered 0.1 N K01 solution. 2,5-Dimethy1-
1,4-naphthoquinone, also forms excellent cathodic waves
in an unbuffered 0.1 N K01 solution. l-Hydroxy-Z-methyl-
4-naphthol amine hydrochloride forms good cathodic waves
in a buffered solution. Solutions of 2-methyl-l,4-naph-
thoquinone and 2,3-dimethyl-1,4-naphthoquinone are found
to be stable upon prolonged eXposure to the air. A solu.
tion of l-hydroxy-Z-methyl-4-naphthol amine hydrochloride
was found to be rapidly decomposed upon being exposed to
the air.

The best cathodic waves of riboflavin, thiamin hydro-
chloride, nicotinic acid, and nicotinic amide were obtain-
ed in an unbuffered 0.1 N KCl solution. Riboflavin, thia-
min hydrochloride, nicotinic acid were determined simultan-
eously from the Same solution. Riboflavin was the easiest
to determine.

Excellent anodic waves of ascorbic acid, and sodium
ascorbate were obtained from buffered solutions of these

compounds.

BIBLIOGRAPHY

(ll Kolthoff and Lingane; Chemical Reviews, 24, l (1929)

(2) Lingane and Davis; J. Biol. Chem., 137, 567 (1941)

 

-6

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