ARTICLE:

AN IMPROVED METHOD FOR THE

DETERMINATION OF ACETYL
VALUES OF LIPIDS APPLICABLE TO
HYDROXYLATED FATTY ACIDS

J. Biol. Chem. 1934, 104:627-634.

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Edward S. West, Charles L. Hoagland and

George H. Curtis

AN IMPROVED
METHOD
FOR THE DETERMINATION
OF ACETYL VALUES OF LIPIDS
APPLICABLE
TO HYDROXYLATED
FATTY ACIDS*
BY EDWARD
(From

S. WEST,

the Laboratory

(Received

CHARLES
L. HOAGLAND,
H. CURTIS

AND

of Biological
Chemistry,
Washington
of Medicine,
St. Louis)
for

publication,

January

GEORGE

University

School

10, 1934)

* Presented
in part before
logical
Chemists
at, Cincinnati,

the meeting
of the
April,
1933.
627

American

Society

of Bio-

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None of the methods that have been proposed for the determination of the acetyl values of lipids is generally satisfact.ory and none
is applicable to free hydroxylated
fatty acids.
Benedict and Ulzer
(1) first proposed a method in which the insoluble fatty acids of a
fat are acetylated and the acetyl value determined by estimating
the KOH bound before and after saponification
of the acetylated
acids. Lewkowitsch
(2), however, showed this method to be inaccurate and to give high results by demonstrating
that non-hydroxylated acids, as palmitic, stearic, and oleic, give considerable
acetyl values when treated according to Benedict and Ulzers
method.
This he found to be due to relatively stable mixed anhydrides formed by boiling the acids with acetic anhydride.
Lewkowitsch
(3) then proposed the method which in essentials is the
one most commonly used at the present time, and which consists
in acetylating the fat (or other lipid, not fatty acids) by boiling
with acetic anhydride, removing excess anhydride by boiling with
water, and determining the acetic acid liberated (as mg. of KOH
equivalent) per gm. of acetylated material.
A determination
of
volatile acids must be run on the original unacetylated
material
and subtracted from the above results to give the so called true
acetyl value.
The method is laborious and time-consuming
as
well as subject to a number of errors.
If the material acetylated
contains free fatty acids, the results may be too high, as in the

Acetyl Values of Lipids

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method of Benedict and Ulzer.

The prolonged boiling of the
acetylated product with water may lead to hydrolysis
and loss of
acetic acid with low results (4). The titrations of small amounts of
acid in the large volumes required are not accurate.
This is an
important
source of error when the acetyl value is low and the
volatile acid correction of rancid oils may be equal to or greater
than the true acetyl value.
Roberts and Schuette (4) have recently published an acetyl
method which consists in heating the sample with a weighed
amount of standardized
acetic anhydride in a sealed tube at 120
for an hour, followed by refluxing at reduced acidity, and final titration with alkali.
The writers have found the method tedious
and have been unable to obtain consistent results with it.
The evident need, stressed by Andrews
and Reed (5), for a
simple accurate method applicable not only to fats, waxes, and
sterols but also to free hydroxylated fatty acids has prompted the
writers to devise the procedure reported below.
It is being used
in this laboratory
in a study of the distribution
of hydroxylated
fatty acids in the animal body and their metabolism.
Verley and Biilsing (6) determined the hydroxyl groups of alcohols and phenols by acetylating with acetic anhydride and pyridine, decomposing excess anhydride with water, and titrating with
aqueous alkali.
Peterson and West (7) showed the method to be
applicable to hydroxyl groups of sugars and sugar derivatives.
A
thorough study of the method in attempts to apply it to accurate
determination
of the acetyl values of lipids in this laboratory has
failed, except with castor oil and some of the waxes.
The failure
was found to be due to the presence of free fatty acids in the acetylated mixtures which could not be efficiently removed by filtration
and which do not titrate accurately in aqueous solution, especially in the large volumes necessary.
These difficulties have now
been overcome in the procedure outlined below.
The method consists in acetylating the sample with a measured
quantity of a mixture of acetic anhydride and pyridine (either hot
or cold), decomposing excess anhydride with a little hot water,
and titrating with alcoholic alkali after the addition of sufficient
butyl alcohol to give a homogeneous solution.
The acidity of the
material is determined in the same way on a sample treated with
pyridine only and this value is subtracted from the above titration.

West, Hoagland,

and Curtis

629

TABLE

Acetyl

Acetyl
value by
,the LewIkowitsch
cletinition

Substance

Castor
oil..
Cottonseed
oil
Linseed
oil..
.
Croton
oil.
Neats-foot
oil.
Coconut
oil

146.1
5.5
6.3
8.3
20.0
4.4
-

Values

of Lipids
-

Acetyl
value by
xoposed I
*
efinition

d
-

Acetyl

Iralue

Substance

,d
--

(
-_

126
4.3
5.0
6.4
15.4
3.4

Olive oil..
Carnauba
wax.
Cholesterol.
Ricinoleic
acid.,
Lithium
lactate..
-

* According
to the proposed
definition,
acetyl
(CH&O)
taken
up per gm. of lipid
= (titration
alkali)
X 4.3)/(weight
of sample in sm.).

3.5
41.2
130.9
160.1
395.3

by

mpoacd
efinition.

2.7
32.6
111.5
134.7
431.7

value
= mg. of acetyl
difference
(cc. of 0.1 N

is not satisfactory
as a general definition.
It has specific reference to the Lewkowitsch
procedure of determining
the acetyl
value, and the necessity of calculating the weight of acetylated
material when other methods are used is an unnecessary
inconvenience.
Calculation
of the number of hydroxyl
groups in a
compound of known molecular weight from such acetyl values is
also somewhat complicated.
The writers propose as a more suitable definition of acetyl value, the mg. of acetyl taken up per gm. of
substance.
Such values have a definite chemical meaning which
should be applicable to all methods of determination
and easily
used in related calculations.
A comparison of values according
to the present and proposed definitions is given in Table I.

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This result subtracted from the titration of a blank on t.he acetic

anhydride-pyridine
mixture gives the necessary data for calculating the acetyl value.
The titration
of all of the acids, both
acetic and higher fatty acids, is very accurate when carried out in
the butyl alcohol solution with alcoholic alkali.
A large number
of determinations
may be run simultaneously
and the execution of
the method leaves little to be desired from the standpoint
of simplicity, accuracy, and time required.
Preliminary
experiments
indicate that the method may be applied satisfactorily
to semimicroquantities.
The definition of acetyl value as the milligrams of KOH required to neutralize the acetic acid from 1 gm. of acetylated fat

630

Acetyl Values of Lipids

Methods

Procedure
Two samples (0.5 t,o 1.0 gm. of materials with high acetyl values,
as castor oil, and 2 to 3 gm. of those having low acetyl values, see
Table 11) of material are weighed into each of two 250 cc. ground
glass-stoppered
Pyrex Erlenmeyer flasks.
5 cc. of acetic anhydride-pyridine
mixture (1 volume of anhydride and 7 volumes of pyridine) are added to one of the above
samples from a Folin-Ostwald
blood pipette, with careful measurement.
To the other, 5 cc. of pyridine only are added.
A blank
flask is set up with 5 cc. of the acetic anhydride-pyridine
mixture.
The stoppers are moistened with pyridine and placed loosely in
the flasks.
The flasks are then placed over well fitting holes (to
insure a minimum escape of steam around the flasks we have used
rubber rings over the holes) of a steam bath, and allowed to heat
about 5 minutes to permit expansion, the stoppers being slightly
unseated if necessary.
The stoppers are then turned in firmly
and the heating continued 40 to 45 minutes.
The flasks are removed from the bath and the stoppers quickly
loosened (to prevent sticking due to cooling) and placed at an
angle in the mouths of the flasks.
5 cc. of water are then added,
care being taken to rinse well the stoppers and necks of the flasks.
The stoppers are loosely replaced and the flasks heated 1.5 to

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Reagents
Pyridine.
Mallinckrodts
medicinal grade is dried by refluxing
for several hours with good barium oxide and then distilled.
The
fraction passing over above 114 is used.
Acetic anhydride, Mallinckrodts
reagent quality, redistilled.
Alcoholic NaOH.
This is prepared by dissolving sufficient 60
per cent aqueous NaOH (60 gm. per 100 cc.) in 95 per cent alcohol to make a 0.3 to 0.35 N solution.
The precipitated carbonate
is easily removed by adding a little norit, shaking, and filtering.
It should be standardized against standard acid (phenolphthalein)
daily.
The solution remains colorless for a long time if kept below 25.
Butyl alcohol, ordinary commerical butyl alcohol (Commerical
Solvents Corporation).
It generally contains a trace of acid but
this is taken care of in the blank titration.

West, Hoagland,
TABLE

Acetyl Values of Lipids,

and Curtis

631

II

Etc., by Pyridine-Acetic
Method

Anhydride

Titration

Tem-

SUbSt3IlCQ

W~i~htOf

Kit-

A&y1
values
(in order of
sample)

Given in
literature

AWIage

_ -

am.
Castor

oil..
I

Hot
Cold

0.596,
0.630,

Cottonseed
fresh.

oil,
. .. .

Hot

2.069, 1.542,
2.940
1.383, 1.835

5.3, 5.5,
5.2

5.5

3.670, 2.083,
1.465
3.424, 2.515,
1.694
2.692, 1.770,
3.288
3.156, 2.264,
1.563
2.350, 3.044,
1.609
3.055, 2.303,
1.545

31.4, 30.6,
29.8
5.8, 6.2, 6.1

30.6

Cottonseed
old............

oil,

Linseed

fresh

oil,

Croton

Cold

Hot
Cold

. .

Hot

Nests-foot

oil.

Cocoanut
fresh.

oil,
.. .. ..

Cold

Cocoanut

oil, old

Olive

oil..

.. ...

Carnauba
Cholesterol.
Ricinoleic

Lithium
Palmitic

wax.
..
acid.

lactate
acid.

.
..
.
..
..

Hot

Cold
Hot*
I

1.155
1.226

2.052, 1.974,
1.932
3.612, 2.482,
1.150
3.498, 2.556,
1.774
2.822, 1.228
0.3352, 0.3826
0.3401, 0.3339
0.3463, 0.3463
0.3734, 0.3000
0.4755,0.3135,
0.3547

146.1,
147.1,

5.9,

144.6
147.3

45.3
47.2

5.4

5.4

146

5.5-

6.0

6.3, 6.5,

6.3

6.31 6

8.2, 8.8,

8.7

8.51 6

8.2,

8.3

8.21 6

8.3,

20.4,
19.8
4.4,

4.1,

20.0,

20.1

4.8

4.4

19.4,
20.4,
20.1
3.5, 3.9, 3.3

20.0

41.2,
40.9
130.9, 133.5
158.5,158.8
159.6, 160.1
394.4, 395.3
No acetyl
value

41
32.2
58.6
59.8
94.8

65 minutes

on the steam

bath.

25

19.8-

8.5

32

22

3.6

o.!+-

12

4.9-

10.6

44.755.2
131.1 (theory)
164.6

406.1

None

* Heated

-150.5

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. . . . .

. . . . .

632

Acetyl Values of Lipids

Calculations
According to Lewkowitschs
definition, the acetyl value equals
the mg. of KOH required to neutralize the acetic acid from 1 gm.
of acetylated substance.
If we let A represent the weight of the
sample acetylated, B the acidity (cc. of 0.1 N) of the blank, C
the acidity (calculated from titration of the sample treated with
pyridine alone) of the sample used, and D the acidity of the acetylated sample, the calculations are as follows: acidity equivalent
of anhydride bound, E = B - (D - C) ; weight of acetylated
sample, F = A + (E X 0.0042) ; mg. of KOH equivalent to acetyl
bound, G = 5.61 X E; acetyl value = G/F.
According to the proposed definition, the acetyl value equals
the mg. of acetyl taken up per gm. of substance.
With the necessary symbols above the calculation is acetyl value = (E X 4.3)/A.
DISCUSSION

Our statement that the method is applicable to free hydroxylated fatty acids and not subject to the error of the BenedictUlzer procedure is based upon several points of evidence.
We
prepared ricinoleic acid of neutral equivalent 298.5 (theory 298.36)
according to the directions of Rider (8) and determined its acetyl
value by our method both with heating and at room temperature
with good agreement between the two determinations
as shown in
Table II.
Our values, however, are a little lower than the theoretical, amounting to about 97 per cent of the theory.
This is

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2 minutes on the steam bath, after which they are allowed to cool
10 minutes with the stoppers at an angle. The stoppers and necks
of the flasks are rinsed down with 25 cc. of butyl alcohol (more if
necessary to give a homogeneous solution) and the solutions titrated with 0.3 to 0.35 N alcoholic NaOH from a good 50 cc. burette,
with phenolphthalein
as indicator (3 to 4 drops of 0.1 per cent in
alcohol).
The titrations are all reduced to cc. of 0.1 N alkali.
Instead of heating on the steam bath, the stoppers of the flasks
may be sealed in with pyridine and the flasks permitted to stand
24 hours or longer at room temperature
and the procedure continued as outlined above.
Table II shows that results obtained
with heating and in the cold agree well.
The values here are expressed according to the Lewkowitsch
definition.

West, Hoagland,

and Curtis

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probably due to the presence of a small amount of impurities and

possibly to a slight polymerization
of the ricinoleic acid similar to
that pointed out by Meyer (9). The fact that the results obtained
with heating and in the cold checked within the limits of error of
the method indicates that no undecomposable mixed anhydrides
were formed, because if this were true the heated samples would
have undoubtedly
been most affected and given higher acetyl
values. This source of error also would have caused the acetyl
values to be higher than the theory instead of lower, as found. In
order to check this point several samples of palmitic acid were run
with heating and in every case no acetyl value was found, the
titration values checking closely with the acidity of the anhydride
plus that of the acid. We have found very low acetyl values on a
sample of ricinoleic acid kept under conditions favoring polymerization.
Carnauba wax and lithium lactate did not dissolve well in the
acetylating mixture, the latter rather gelatinizing.
Despite this
fact both were completely acetylated as shown by the concordant
results on different sized samples. The value found for lithium
lactate is 97.33 per cent of the theory.
Lactic acid determination
on the salt by oxidation to nldehyde (10) showed it to be 96.9 per
cent pure. This result also shows the method to be applicable to
hydroxylated acids.
In most of our determinations
we have purposely used samples
of widely varying weights. This was done in order that we might
be sure that acetylation proceeded to completion.
We consider
this, along with the general agreement with published values recorded in the literature as sufficient check on the method.
The
acetyl value for castor oil is essentially a constant owing to the
presence of a rather definite large proportion of hydroxylated
acids. Lewkowitsch (11) has pointed out that this is not true for
most fats or oils since they may contain sterols and mono- and
diglycerides owing to rancidity in varying proportions in addition
to hydroxylated fatty acids. Table II shows the large effect of
rancidity upon the acetyl values of cottonseed and coconut oils.
We believe that the above method of analysis now makes it
possible, by determination
of the acetyl values of the fatty acids
in fats, to arrive at values which may be considered as true characteristics.
This is being investigated.

Acetyl Values of Lipids

SUMMARY

A new method for the determination

of the acetyl values of
lipids has been developed.
It is simple, accurate, and rapid.
The method differs from those previously
proposed in that it is
applicable to free hydroxylated
fatty acids.
A new definition of acetyl value more suitable for calculation
than the present one is proposed.
BIBLIOGRAPHY

5.
6.
7.
8.
9.

10.
11.

Benedict, R., and Ulser, F., Monatsh. Chem., 8, 41 (1887).

Lewkowitsch,
J., Proc. Chem. Sot., 8, 72, 91 (1890).
Lewkowitsch,
J., .I. Sot. Chem. Ind., 18, 503 (1897).
Roberts, W. L., and Schuette, H. A,, Ind. and Eng. Chem., Anal. Ed.,
4, 257 (1932).
Andrews, J. T. R., and Reed, R. M., Oil and Soap, 9, 215 (1932).
Verley, A., and Bijlsing, F., Ber. them. Ges., 34, 3354 (1901).
Peterson, V. L., and West, E. S., J. Biol. Chem., 74,379 (1927).
Rider, T. H., .I. Am. Chem. Sot., 63,413O (1931).
Meyer, H., Arch. pharm.,
236, 184 (1897), quoted in Lewkowitsch
((11)
p. 164).
Wendel, W. B., .I. Biol. Chem., 192, 47 (1933).
Lewkowitsch,
J., Chemical technology and analysis of oils, fats and
waxes, London, 4th edition, 1, 345 (1909).

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1.
2.
3.
4.