THE

MECHANISM

OF THE OXIDATION
BY BROMINE.
BY

(From

the Laboratory
(Received

H.

H.

BUNZEL.

of Biochemistry
for

of

publication,
I.

OF GLUCOSE

the

University

December

8,

of Chicago.)
rgq.)

INTRODUCTION.

In order to understand
metabolism better the behavior of the
different fuelstuffs under various conditions
outside the body
must first be investigated.
A series of studies in this direction
was accordingly
started,
particularly
on the physico-chemical
behavior of the sugars (1-7).
In a preceding investigation
by Mathews and the writer (7),
the following conclusions were drawn regarding the oxidation of
glucose by bromine:
(a) Glucose behaves both as a weak acid and as a weak base.
In an alkaline or neutral solution it dissociates into metal or
Hf ions and C,H,,O,- ions; in an acid solution it forms a salt of
the acid by addition, yielding C,H,,O,+ ions and the anion of the
corresponding
acid.
(b) Both of the glucose ions are oxidized by bromine side by
side, the oxidation of the first being depressed by the addition of
H+ ions, while the other remains unaffected.
Velocity equations were worked out on this basis, taking into
consideration
the two kinds of glucose ions, the concentration
of the H+ and OH- ions and of the bromine and the hypothesis
was tested under very diverse conditions by measuring the rate
of oxidation in the presence of various acids in different concentrations and varying amounts of bromine.
The hypothesis was
found to agree with all the facts observed.
In the experiments
just cited the concentration
of the sugar
was purposely taken SO large that its change of concentration
was negligible.
It seemed necessary, therefore, to vary also the
I.57

Oxidation

158

of Glucose

by Bromine

concentration
of the sugar, to observe its rate of disappearance
and
to ascertain
whether
the equations
alluded to in the above article
hold true under
these conditions.
It was desirable
also, to
ascertain
especially
whether
the oxidation
of the positive glucose
ions leads to the formation
of gluconic
acid exclusively.
The methods
were the same as those in the previous
investigation.
The acidity
developed
during
the reaction
was determined in some of the experiments
by titration
with 0.1 N sodium
hydroxide
using phenolphthalein
as the indicator,
and by subtracting
the amount
of hydrobromic
acid formed,
the amount
of gluconic
acid was determined.
II.

DEVELOPMENT

OF

EQUATIONS.

It was shown in the previous

paper on the subject that both
the positive
and negative
ion are oxidizable
and that in the
presence of sufficiently
high concentrations
of H ions the acid
ionization
of the glucose is so depressed
that only one of these
two reactions
needs to be considered,
i.e. the oxidation
of the
positive
glucose ion.
It was shown by experiment
that concentrations
of H ions above 0.1 N practically
completely
suppress
the oxidation
of the negative
glucose ion and in such conditions
we have to deal altogether
with the reaction:
C,HL0,

Cl-

Br,

HOH

C,H,,O,

In all of the following

experiments
studied,
an amount
of sulphuric
acid
solution
o. I s as regards
H,SO,.
The differential
equation
representing
ax

dt

=K.C&&

>

CoH-

HCl

+ 2 HBr

only this
being added
this

reaction

Cnrz(free)

reaction
to make

IVLLS

the

is
_.

_.

which means that the rate of the reaction

will depend
on three
factors:
(a) the concentration
of the positive glucose ion, C,H,,O,:
(b) the concentration
of the hydroxylions
in the solution;
(c)
the concentration
of the free bromine
in solution.
(a) The formation
and quantity
of the positive
glucose ion

H.

H.

is determined
by the amount
the case of hydrochloric
acid
C,H,,O,

I.59

of the acid present,

as shown
in the following
equation:

+ H++

CC6H1206+

Bunzel

Cl=

for

C,H,,+o, + Cl-

KCC~H~~O~

c+

Inasmuch
as the ionic dissociation
of the glucose is very small,
the amount
of C,H,,O,+ ions will be practically
directly
proportional
to the concentration
of the glucose
solution
and also
directly
proportional
to the concentration
of the H ions.
(11) The hydroxylions
are of course all derived from the water
present.
Their
quantity
is determined
by the equation
:
K

CHzO

H,

COH

from which it is evident

that COH- is inversely
proportional
to
the concentration
of the H ions in solution.
(c) The amount
of free bromine
may be computed
as follows:
CJ+~ X CB~- = o.oj CB~~CBr

(Total)

CBr
CBr-

CBrz

- (Total)

ZCBr

2 CBr

CBr,

.oj Br,
BrT - Br,

Br,

CBr,-

Consumed
Consumed

2 Br

05
%

2 Br

Total

Br

.=.5 +
.___~_

Consumed

Br

Total

Consumed

Br

Total

2 Br

+ 2 Br
_____.~
2

consumed
>

Consumed

By subtracting
able with sodium
the free Br,.
1 See literature

this value
thiosulphate

of Br, from the total bromine

titratsolution,
one obtains the value for

7, I 0, I 1, 12 at the end of the paper.

160

Oxidation

of Glucose

by Bromine

By examining
(a) and (b), it becomes evident
at once that,
provided
sufficient
acid is added
to suppress
completely
the
oxidation
of the negative
ion, the H ions hasten the reaction
according
to (a) exactly
to the same extent as they retard
it
according
to (b).
This allowed
us to neglect the concentration
of the H ions provided
it was over o. I N.
The velocity of the reaction
is accordingly
proportional,
under
these conditions,
to two variables
only; the concentration
of the
glucose and the concentration
of the free bromine,
and is represented by the equation
$
On integration

this
K=

= K (a - x) (b

X)

becomes
I
t (a -

b)

log

nat

b(a

x)

a (b -

x)

where
K is the velocity
constant,
t the time
elapsed
since the
beginning
of the exlzeriment,
a and b the concentration
respectively of the active bromine
and sugar at the start, and x the
amount
of bromine
and sugar consumed.
In a part of the experiments
a known amount of sodium bromide solution
was added, so that the concentration
of the Brions in the final mixture
was 0.3 N.
As the Br, in these experiments was only 0.01 N at the start, the slight increase on the concentration
of the Br- ions during the reaction
due to the formation of HBr, was negligible.
In the remainder
of the experiments, however,
where the concentration
of the free bromide
at
the start was larger or where the reactions
were so slow that it
seemed undesirable
to depress their rate still more by the addition of bromide
the value for Br, had to be calculated
separately
By taking
the constantly
changing
for each determination.
value for Br- in consideration
we obtained
a differential
eqation, which we were unable
to integrate.
For that reason the
values for K were calculated
for short time intervals,
always
using the time of the last preceding
determination
as a starting
point.
To approximate
the concentration
of the Br the writer
added to the concentration
of the HBr present at the preceding

H.

H. Bunzel

determination
(including
that
which
is present
as Br,) one
half of the HBr formed during the time interval
considered
and
subtracted
the concentration
of the bromine
present in the form
of Br,.
I

--

t _ .--------..
05 + Br-(,t

H Brt,
-~ ~-~--

&tart)+

The formula
used in the experiment
of the Br- ion was made .30 at the
1

K=
t %(a

III.

~~ --.-

- H Brt,
2

-~m~~~m-(a - b)
-

where the concentration

start, was
In b$G-Ei

.[III]

b)

EXPERIMENTAL

A. To detervnine the comtancy of K when the acidity, glucoseand

bromine are all varyitzg.
The figures given in Tables I and II indicate
by the constancy
of the values for K when the glucose and Br, are varied within
wide limits, that the course of the reaction
is actually
controlled
by those factors and in the manner
stated in our hypothesis
at
the beginning
of this paper.
B. Is gluconic acid the sole product of the oxidation of the posi-

tive glucoseion?
In all the experiments
just quoted the change in the concentraThe change in contion of the bromine
only was determined.
centration
of the sugar was assumed to be one molecule of sugar
oxidized
for each molecule
of Br, used up.
This assumption
gave a constant
value for K.
To test the hypothesis
further
that glucose went thus into gluconic
acid, it was necessary
to
measure at least in one experiment
the degree of acidity
of the
mixture
at varying
time intervals
and thus to determine
the
amount
of gluconic
acid formed.
It should of course be found
then that for each molecule
of bromine
which disappeared
from
the solution,
one molecule
of glucose has been used up and the

Oxidation

162

of Glucose
TABLE

Concentration

of H,SO,

0.37~;

by Bromine
I.

Concentration

of Na Br 0.30

I (MINUTES).

__

2.OOM* -t.-

1.00

0.50

0.25

0.10

0.05

0.00500

0.00520

0.00516

0.00496

0.00515

11.60
10.68
9.70
8.98
7.53
5.80
4.91
10.01
9.25
8.82
7.89
7.05
6.48
3.28
10.40
9.29
8.41
7.25
5.30
10.32
10.00
9.74
9.29
8.20
6.83
5.31
9.92
6.31
5.44
1.93
10.30
8.37
7.64
5.85

* nr refers

to a molecular

solution

K (log n&t).

N.

YE*?4
(log nat)

.--

0
10
20
30
50
82
100
0
15
25
50
75
100
260
0
50
100
165
300
0
30
50
100
200
350
570
0
1060
13%
3825
0
970
1185
2350

0.0290

0.0312
0.0298
0.0301
0.0295
0.0301

/
I

0 0300

0.0342
0.0338
0.0330
0.0310
0.0304
0.0300

0.0276
0.0298
0.0306
0.0314

I
1
i

0.0299

0.0293
0.0312
0.0291
0.0322
0.0330
0.0328

)
:
I
/
I
,

0 0314

I
I

~
0.03'21

0.0301
0.0298
0. 0334

0.0310

0.0303
0.0357
0.0343

0.0x35

TABLE

No NaBr

added.

II.

C,

= 0.

N H,

SO,

I
T.

Tot.

Br.

CB*-

I-

0.77

.l.Ol
8.88

0.55

0.275

0.110

0.55
Calculated
ineach
determination from
thebeginning of
the experiment.

0.100

5.78
4.20
3.19
2.67
11.02
8.82
6.50
4.04
1.94
.Sl
11.71
10.40
9.04
7.14
4.85
3.05
10.95
9.38
8.05
6.72
11.41

9.59
8.59
x.19

45.40
36.45
31.50
20.30
17.00
14.98

K (log nat)

IEAN K.

-_

0
10
30
45
60
70
00
15
35
65
120
185
0
15
35
65
120
185
0
50
100
160
0

3.00550

3.00444
3.00289
3.00210
0.00160
0.00134
0.00551
0.00441
0.00325
0.00202
0.00097
0.00041
0.00586
0.00520
0.00452
0.00357
0.00243
0.00153
0.0054s
0.00469
0.00403
0.00336
0.00571

( ).00017
( j.00026

C). 00089
c j.00342

( I.0283

(I.00024
().00021
().00020

CI.00578
Cj.00710
Cj.00788

( I.0308

().00017
(1.00024
(3.00024
I3.00015
I3.00006

( ). 00093
( j.00312
( I.00575
( ).00788

(I.0274

( I.00959

I I.

0297

I3.00010
I0.00022
I0.00029
I0.00028
I0.00022

( I.00055
( I.00177

(I.

0290

( I.00333
( I.00544
(I.00776

I 3.0306

,0.00010
/0.00021
/0.00025

(I.00069
(1.00203
(1.00332

I0.0288
10.0299

0.0292

100
170

0.00480
0.00430
0.00410

0.00015
0.00020
0.00023

(3.00076
I3.00121

I3.00161

0.0315
0.0310
0.0313

0.0313

0.090s
0.0729
0.0630
0.0406
0.0340
0.0300

0.0066
0.02185
0.0263
0.0233
0.0240

0.0113
0.0238
0.0517
0.0837
0.0936

0.0300
0.0282
0.027C
0.027E
0.026t

200

0
100

200
775
150
480

--

( I.0299
( I.0272
( I.0269

3.0286

(I.0290
(J.0322
(1.0282

D .02X3

I 3.0265

0.0285

I I
I 3.0304

0.0290

ICl.0290

Oxidation

164

of Glucose
TABLE

by Bromine

II-Continued

lK(log

nst)

MEAN

K.

I0.05

Meanvalue

7.49
4.30
31.12
28.50
24.62

100
320
0
100
320

of K (log nat).

0.03745
0.0215
0.1556
0.1425
0.1231

/
0.0110

0.01255
0.0300

0.0253
0.0313

0.0131

0.0258

0.0283
0.0296

).0302*

*The
cause of the divergence
of this constant
from the one found in the previous
paper by
Mathews
and Bunael,
i. e., 0.0169 is twofold.
There the sugar wau always present
in the same
concentration,
0.5 M, which wits regarded
iw unity;
moreover
it was thought
then that the glucose
used conkdined
one molecule
of water of crystallization,
which was proved
to be incorrect
by
polarimetric
measurements
carried
on later.
If the previous
value
of the constant
is transformed
on this basis, we obtain
as mean value for K (log nat) from our former
paper 0.0308,
which
agrees with the one found
above.

corresponding
amount
of gluconic
acid formed.
Furthermore
there ought to be for each atom of bromine
transformed
into
hydrobromic
acid, an acidity
produced
which is one and a half
that of the HBr calculated,
one-third
of this acid being due to
the gluconic
acid formed and two-thirds
of it to the HBr.
The
experiment
was performed
under the usual conditions.
As the
temperature
was not absolutely
uniform
in this experiment
but
oscillated
between
25
and 25.5O, no attempt
was made to calculate the velocity constants
in this experiment
and only the parallelism between
the changes of concentration
of the two compoFor this purpose
at various
time
nents named
were studied.
intervals
two portions
of the reaction
mixture
were removed
from the reaction
flask.
One of these portions
of IO cc. volume
was run into KI solution
and
titrated
with 0.1 N sodium
thiosulphate
solution
to determine
the Br, used up.
The other
portion
of 50 cc. had air blown through
it for 15 minutes
at room
temperature
to remove the Br,.
Then IO cc. of this portion
was
titrated
with 0.10
sodium
hydrate
and then with 0.1 N silver
nitrate
using a few drops of a saturated
potassium
chromate

H.
solution
as indicator
to
and the bromine
in the
To make certain that
ing the passage of air, the
solution
of hydrobromic
Time.
3,3j.............................
3.55
4.10 . . . . . . . . .

H. Bunzel

1%

determine
respectively
the total acidity
form of bromide.
none of the hydrobromic
acid is lost durfollowing
experiment
was carried out: .4
acid was made and air drawn through
it.

.._.

._.

_..__

Titer of 10 cc.
of HBr solution
j.ZSCC.
0.1 NNaoHsol
5.30
(

j.Zj

From this experiment

it was clear that there was no appreciable
loss of hydrobromic
acid.
A vigorous current
of air was blown through
the bromine-containing
solutions
in tall cylinders
and by separate experiments
it
was determined
that a complete
removal
of the bromine
was
affected.
Table III gives the results of the experiment.
The strength
of the sodium hydrate solution used was o. I o I 2 N, the silver nitrate
solution
was 0.09703
N, the sodium
thiosulphate
solution
was
0.1000

N.

From the figures in Table III the following

relationships
become
evident :
(I)
The total acidity at any time in the course of the reaction
determined
by titration
with 0.1 N sodium hydrate solution
after
removal of the free bromine,
is, \yithin experimental
errors, equal
to the acidity calculated
from the amount
of bromine
which has
disappeared
from the solution
assuming
that each molecule
of
Br, thus used up has oxidized
one nolecule of glucose to form one
molecule of gluconic acid and two molecules
of hydrobromic
acid.
(2)
Two-thirds
of the acidity at any particular
time is due to
the hydrobromic
acid formed and one-third
to the gluconic
acid
produced.
(3)
The amount
of bromide
found in the solution
at varying
times agrees closely with the amount calculated
from the acidity.
(4)
The amount
of glucose oxidized
calculated
from the gluconic acid produced
agrees closely with the amount
calculated
from the assumption
that one molecule
of Br, oxidizes
one molecule of glucose to gluconic
acid.

Oxidation

of Glucose

by Bromine

666666
+

0
0

H. H.
IV.

DETERMINATION

OF

THE

Bunzel

TEMPERATURE

COEFFICIENT

OF

THE

REACTION.

Cklucose 0.5 M; CH, so1 0.3 N; CN~B~ 0.30 N. Temperature 0.

12.66
11.50
10.01
7.40
5.45

j
I
I

0'
1000
2430
5410
8200

0.00633
0.00575
0.00500
0.00369
0.00273

&lean K oog nat).

K, clog natI. ..................................
K, clognatj ..................................
Temp. Coeff ..................................
v.

0.00134
0.00136
0.00140
0.00144
0.00139
I
o. OVJOZ
.o.oo~gg
.j.sz for Id

CONCLUSIONS.

The results of the present investigation

have shown:
(I)
that
the amount
of acid and of bromide
produced
and of glucose and
bromine
disappearing
in the oxidation
of glucose by bromine
in
acid solution
is equal to that calculated,
on the hypothesis
that
one molecule
of bromine
oxidizes
one positive
glucose ion to
gluconic acid ; and (2) that the velocity of the reaction is correctly
represented
by equation
I, (p. 154) as shown
by the constancy
of
the velocity
constant,
K, under widely
varying
conditions
of
acidity
and of different
concentrations
of bromine
and glucose.
These results
strongly
support
the conclusions
of our former
paper, that glucose, like other alcohols and aldehydes,
forms two
series of salts; the first in which glucose behaves as an acid, the
salt dissociating
into metal and negative glucose ions; the second,
in which glucose behaves
as a base, forming
an oxonium
salt
dissociating
into C,H,,O,+
ions and the negative
acid radicle.
Both of these ions are more easily oxidized
than the neutral
glucose molecule and they give rise on oxidation
to different
acids.
The I)ositive ion, as this research sho*,vs, goes quantitatively
into

Oxidation

of Glucose

by Bromine

gluconic acid. The gluconic acid thus formed is presumably

converted into lactone and in the course of time would be still
farther oxidized by the bromine.
This reaction is, however, a
slow one compared to that which leads to the formation
of the
gluconic acid. It probably accounts for the slight tendency of
the constant K to rise toward the end of the experiment, in long
experiments,
and in those cases where Br, is present in large
amounts and the glucose in small amounts.
Whether gluconic acid is produced in small quantities by the
oxidation of the negative glucose ion, or whether it is produced
by slow oxidation also of the unionized molecule, if there is such
an oxidation, cannot be said without further investigation.
The negative glucose ion when oxidized by bromine probably
gives rise to the large variety of acids, described by many observers and studied particularly
by Nef (13) in his splendid investigation of the transformation of the sugar molecules.
The fact that glucose oxidizes both as an oxonium salt and the
salt of a metal explains the dependence of the rate and character
of the oxidation on the reaction acid or alkaline of the medium.
Glucose being a weak acid (14-15)
dissociates into H+ ions and
negatively charged C,H,,O,- ions and in the presenceof acidsinto
positive C,H,,O,+ ions and the negative acid radical. The first
kind of dissociation is the principal one in the presenceof alkalies.
The effect of the alkali, therefore, is to increase the number of
the active negative sugar particles in solution. Acids, on the
other hand, by means of the large number of H ions they furnish,
greatly depress the dissociation of so weak an acid as glucose,
and at the same time they form salts with the positive ion. In
a neutral solution both of the oxidations of both positive and
negative glucose ions go on side by side at their respective velocities, which have been determined in a former paper (7). The
addition of small amounts of acid will decrease the rate of oxidation of the glucose, because, for the reason just given, acids
diminish the extent of the negative ion oxidation which is
rapid and leave unaltered the extent of the slower positive ion
oxidation,
Increasing quantities of acid will slow the rate
of oxidation farther and cause more and more a predominance
of the latter reaction; but this decrease in rate due to increasing
acidity holds true only up to a certain point i.e., to the point

H. H. Bunzel
where

the negative ion oxidation

is entirely
suppressed
(appr. .02
ions); beyond
that additional
amounts
of all acids tried,
except HCl and HBr have no noticeable
depressing
effect.
The
particular
action of these acids was discussed in the first communication.

NH+

The writer
wishes to express his gratitude
to Prof. A. P.
Mathews,
for the theoretical
formulation
of the reaction
and for
his uninterrupted
interest
in the execution
of this work.
LITERATURE.

I.

Mathews

P. 1997 1907.
2. McGuigan,
3. Mathews
909.
4. Mathews
909.
5.
6.
7.
xxxi,
8.
909.
9.
1909.
IO.
544,
I I.
12.
905.
13.
14.
15.

and

McGuigan:

American

Journal

of

Physiology,

xix,

H.: American
and Walker:

Journal
of Physiology,
xix, p.
Journal
of Biological
Chemistry,

175, 1907.
vi, p. 2 I.

and

Journal

vi,

Walker:

of Biological

Chemistry,

p.

29,

Mathews:
Journal
of Biological
Chemistry,
vi, p. 3, 1909.
Bunzel,
H. H.: American
Journal
of Physiology,
xxi, p. 23, 1908.
Bunzel
and Mathews:
Journal
of the American
Chemical
Society,
p. 464, 1909.
Mathews
and Walker:
Journal
of Biological
Chemistry,
vi, p. 289,
Mathews

and

Walker:

Journal

Richards
and Stull : Zeitschrift
1902.
Roloff : Zeitsckrijt
fiir physikalische
Worley
: Journal
of the American
Nef: Annalen
der Ckemie,
cc&ii,
Cohen:
Zeitsckrift
fzir pkysikaliscke
Osaka:
Zeitsckrijt
ftir pkysikaliscke

of Biological
ftir

Chemistry,

physikaliscke

vi,
Chemie,

Chemie,
xiii, p. 353,
Chemical
Society,
lxxxvii,
p. 214, 1907.
Ckemie,
xxxvii,
Ckemnir,
xxxv,

P. 299,
xli,

p.

1894.
p. I 107,

p. 69, 1909.
p. 661, 1900.