[II]
THE ANALYSIS OF MIXTURES OF VOLATILE
FATTY ACIDS
BY F. V. GRAY
From the Division of Biochemistry and General Nutrition of the Council for
Scientific and Industrial Research, University of Adelaide, South Australia
{Received 4 October 1946)
In the course of investigating certain aspects of carbohydrate fermentation in the
rumen of the sheep it became necessary to estimate the individual concentrations of
formic, acetic, propionic and butyric acids in aqueous solutions containing all four
components.
Two main procedures are available for such analyses: the distillation method of
Hillig & Knudsen (1942), which is based essentially on the classical Ducleaux
(1874, 1900 a, b, 1908) procedure, and the method of Osburn, Wood & Werkman
(1936) which involves partition of the acids between water and ether phases. In
each of these methods formic acid is estimated by a separate procedure.
Preliminary examination of the partition method proved its effectiveness for the
assay of mixtures containing acetic, propionic and butyric acids except when one of
the components constituted either a very large or a very small proportion of the
mixture. As it was expected that such mixtures would be encountered, an improvement in the method was sought.
No difficulties were experienced in the analysis of solutions prepared from
mixtures of two acids, when one of them was present in much larger proportion than
the other; but when analyses were made of mixtures containing known concentrations
of three acids, the amounts of each, determined by the partition procedure, indicated that serious sources of error are inherent in the method. This is to be expected;
the estimation involves essentially a calculation by difference and so in certain
circumstances the implied error is large. Assuming, for example, an accuracy of
+ 0-5% in the titration of the acid content of the aqueous phase of each partition,
and of ±o-i % in the titration of the original acid mixture, the theoretical maximum
error involved in the estimation of propionic acid in a mixture containing acetic acid
1-5 g./l., propionic acid 0-25 g./l. and butyric acid 0-25 g./l. would be 67%;
whereas, with the same manipulative accuracy, the maximum error involved in the
estimation of this component of a binary mixture containing only acetic and
propionic acids in the above concentrations would be reduced to 10%.
If the concentration of butyric acid could be determined by a separate estimation
with an accuracy of even 5 %, which may readily be accomplished, then, by applying
the partition procedure and substituting the figure for butyric acid in solving the
equations, the maximum error involved in the estimation of the propionic acid in the
former mixture would be reduced to 19%.
12
F . V.
GRAY
It is clear, therefore, that the accuracy of the determination of the concentrations
of each of three acids in a mixture should be very considerably increased if the
concentration of one of them is determined by an independent method and the
resulting data introduced prior to solving the partition equations. It is also clear
that if such a combined procedure is to improve the precision of the method, the
accuracy of the independent determination of the third acid must increase with the
accuracy of the measurement of the partitions.
The case for improved accuracy arising from the use of combined methods of this
sort would apply equally well to both partition and distillation procedures, and so
the method of distillation at constant volume (Hillig & Knudsen, 1942), or, since the
collection of only one fraction of distillate would be necessary, the simpler semidistillation procedure (Virtanen & Pulkki, 1928) could be adopted. In spite of the
fact that the distillation constants of the acids are less widely different than are their
partition constants, such a combination of a distillation method for acetic and
propionic acids with separate independent estimations of formic acid and of
butyric acid, may be preferred: partition calls for considerable quantities of very
pure ether and a critical control of the temperature at which equilibrium is established, whereas the distillation procedure involves nothing other than simple,
careful manipulation.
. EXPERIMENTAL
A number of 3-acid mixtures containing acetic, propionic and butyric acids, and
some 4-acid mixtures containing formic acid in addition, were made up from carefully purified components. Partition constants of the pure acids between water and
pure ether at 200 C. were determined; the findings were in accord with the constants
reported by Osburn et al. (1933).
In the first series of analyses the results from the partition method when applied
to 3-acid mixtures containing acetic, propionic and butyric acids, were compared
with those derived from solving partition equations after introducing the data
obtained from a separate estimation of the concentration of butyric acid by the
method of Kline (1934). The results of analyses of the 4-acid mixtures which
contained formic acid are included in this series, the concentration of formic acid in
the mixtures being determined by oxidation with mercuric oxide by a procedure
slightly modified from that of Osburn et al. (1933), and the data so obtained substituted in the partition equations.
Several variables are encountered, among which the actual error of the titrations
themselves, the influence on the partition exerted by the concentrations of the
individual acids, and changes in the physical conditions under which the partitions
are estimated, are probably the main contributors. The observed partitions differed
from the theoretical by amounts varying between o and 0-5 %.
The results from this first series, summarized in Table 1, showed quite definitely
that under the conditions of this test, the combined procedure afforded a more
accurate analysis when one of the acids was present in relatively high proportion.
In the second series analysed, the results of a distillation procedure were compared
with those from the partition method, each being used in conjunction with the
*
Propionic
Butyric
Formic
Acetic
Propionic
I
Acids found in mixtures
Butyric
Acetic
Propionic
Butyric
% error in each determination
Formic
Analysis of mixed fatty acids by two procedures
Method
used*
I.
( a ) Partition method for acetic, propionic and butyric acids. Separate method for formic acid. (6) Partition method for acetic and
propionic acids. Separate methods for formic and butyric acids.
0.480
0.240
0'240
1'44
0'525
= -47
0.147
0'147
Acetic
present in mixtures
(€511.)
Table
14
F. V. GRAY
separate determination of butyric acid. The semi-distillation method was adopted,
but only 50 ml. of the mixtures were distilled instead of the 200 ml. recommended by
Virtanen & Pulkki (1928). The distillation constants were determined at known
concentrations of approximately 0-2 g./ioo ml. for each acid. The results which are
set out in Table 2 render it clear that the simpler manipulations involved in this
distillation may, without loss of accuracy, be employed in place of the more exacting
partition procedure.
Table 2. Comparison between partition and semi-distillation methods
Acids present in mixtures
Acetic
Propionic
OO
O
0-128
0N
0-261
GO vj
0128
Butyric
0-261
i'54
3-13
% error in each
determination
Acids found in mixtures
(g./D
Method
used*
Acetic
a
b
a
b
a
b
i'5S
O-I2O
0-134
0-131
0-127
Propionic
Butyric
07
07
62
47
7-2
0-8
o-8
o-s
0-4
23
SO
2-8
Propionic
Butyric
Acetic
0194
0-170
0259
0259
218
2-21
0-262
0-262
OI72
0-176
313
313
o-8
o-4
0
0
* (a) Partition method for acetic and propionic acids. Separate method for butyric acid. (6) Semidistillation method for acetic and propionic acids. Separate method for butyric acid.
SUMMARY
1. The analysis of mixtures of acetic, propionic and butyric acids, in which one
of the acids is present in either very large or very small proportions has been
examined.
2. A combination of a separate method for butyric acid, with the partition method
for acetic and propionic acids, provided more accurate estimations than those
obtained directly from the application of the partition method to all three acids.
3. Semi-distillation proved a suitable alternative to partition in the combined
procedure.
The author is indebted to Mr H. R. Marston, Chief of the Division, for
originally suggesting the problem and for his help and criticism.
REFERENCES
DUCLEAUX, E. (1874). Ann. chim. phys. [5], 2, 289.
DUCLEAUX, E. (1900a). Traitf de Microbiologie, 3, 384. Paris: Masson et Cie.
DUCLEAUX, E. (19006). Z. anal. Chem. 39, 376.
DUCLEAUX, E. (1908). Z. anal. Chem. 47, 615.
HILLIG, F. & KNUDSEN, L. F. (1942). J. Ass. Off. Agric. Chem. 25, 176.
KLINC, L. (1934). Biochem. Z. 273, 1.
OSBURN, O. L., WOOD, H. G. & WERKMAN, C. H. (1933). Industr. Engng Chem. (Anal, ed.), 5, 247.
OSBURN, O. L., WOOD, H. G. & WERKMAN, C. H. (1936). Industr. Engng Chem. (Anal, ed.), 8, 270.
VIRTANEN, A. I. & PULKKI, L. (1928). J. Amer. Chem. Soc. 50, 3138.