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Issued September 13, 1911
United States Department of Agriculture,
BUREAU OF CHEMISTRY— Circular No. 76.
H. W. WILEY, Chief of Bureau.
DETERMINATION OF MALIC ACID.
By P. B. Dunbar and R. F. Bacon, Assistant Clicmist.s, Division of Foods.
The method described herein is based on the fact first observed by
Walden r that under certain conditions uranium salts produced a
very marked increase in the specific rotation of Z-malic acid.
Walden noticed that a similar but less marked effect is produced by
uranium salts on ^-tartaric acid, d-methyl tartrate, Z-quinic, and
Z-mandelic acids, the increased rotation being in the same direction
as the rotation of the aqueous solution of the substance. He con-
cluded that the effect is limited to the active oxy-acids. As stated
by Walden, the requisites of an agent which is to be used for the
detection of malic acid by optical means are: (1) That the direction
of rotation shall be constant, and (2) that the increase in rotation
shall be as large as possible. This, of course, applies also to the
quantitative estimation of malic acid. In addition, the agent in
question should have little or no influence on any other substances
which may occur in conjunction with malic acid. It has long been
known that certain substances such as the oxygen compounds of
boron 2 arsenic, antimony, molybdenum, and tungsten 3 have the
power of increasing the specific rotation of the oxy-acids. Uranium
salts, however, are the only ones which fulfill the requirements just
stated with any degree of satisfaction.
The authors have recently had the privilege of reading an article
by P. A. Yoder entitled "A Polariscopic Method for the Determina-
tion of Malic Acid and Its Application in Cane and Maple Prod-
ucts.'' 4 This paper was presented at the forty-second meeting of
J Ber. d. chem. Ges., 1897, 30 (3) : 2889.
2 Biot, Mem. de l'acad. roy. sci., 1838, 16: 229; Ann. chim. phys., 1884 (3), 11: 82;
1850, 29: 341, 430; 1860, 59: 229. Pasteur, Ann. chim. phys., 1860 (3), 59: 243.
8 Gernez, thr. Landolt's Optical Rotation of Organic Substances, tr. by Long, p. 248.
* J. Ind. Eng. Chem., 1911, 3: 563.
3123°— Cir. 76—11
2 DETERMINATION OF MALIC ACID.
the American Chemical Society in July, 1910, and contains the
results of a large amount of work. The method outlined depends
on the use of a uranium salt, but in other respects it differs mate-
rially from the one here proposed. With this exception, no one
appears to have attempted the quantitative determination of malic
acid by treatment with uranium.
THE METHOD IN BRIEF.
The method in brief consists in treating a portion of the neutral-
ized 1 solution containing malic acid with uranyl acetate and polar-
izing it. The algebraic difference between the reading so obtained
and that of the untreated solution is multiplied by the factor 0.036
to obtain the percentage of malic acid present. With the exception
of ^-tartaric acid, none of the common optically active substances
interfere with the determination, and consequently they need not be
removed. Uranyl acetate slightly decreases the rotation of sugars.
This may give rise to an error in the estimation of small amounts
of malic acid in the presence of large amounts of invert sugar.
Hence, when the rotation of the original solution is negative and the
approximate amount of sugar or malic acid is unknown, or when
more than 10 per cent of reducing sugars and less than 0.25 per cent
of malic acid are present, it is necessary to precipitate the malic acid
and treat the nitrate with uranyl acetate also. In this case the
polarization of this nitrate, instead of that of the untreated solu-
tion, is subtracted from the polarization of the solution containing
both malic acid and uranyl acetate, and the difference is multiplied
DETAILS OF THE METHOD.
Dilute a measured volume of the solution, usually 10 cc, with
quite a large volume of water, add phenolphthalein, and titrate with
standard alkali to a decided pink color. Transfer another measured
portion of the solution (75 cc is a convenient volume) to a 100 cc
graduated flask, and add enough standard alkali, calculated from the
above titration, to neutralize the acidity. A slight excess of alkali
is not objectionable. If the solution is dark colored, add 5 or 10 cc
of alumina cream. Dilute to the mark, mix thoroughly, and filter
if necessary through a folded filter.
(1) Treat about 25 cc of the filtrate with powered uranyl acetate,
adding enough of the salt so that a small amount remains undissolved
after two hours. Two and one-half grams of uranyl acetate will
usually be sufficient, except in the presence of large amounts of
malic acid. In case all the uranium salt dissolves more should be
added. Allow the mixture to stand for two hours, shaking fre-
x Tf no mineral acids are present it is not necessary to neutralize the malic acid.
DETERMINATION OF MALIC ACID. 3
quently. Filter through a folded filter until clear and polarize if
possible in a 200 mm tube. If the solution is too dark to read in a
200 mm tube, a 100 or 50 mm tube may be used. It is desirable, how-
ever, to use the longest tube possible in order to obtain the maximum
rotation. This solution and reading will hereafter be designated
(2) Treat the remainder of the original filtrate with powdered
normal lead acetate until no further precipitation results. Cool in
an ice bath and filter through a folded filter until clear. Warm the
filtrate to room temperature and add a small crystal of lead acetate
to determine whether the precipitation is complete. If no further
precipitate results, remove the excess of lead completely with
anhydrous sodium sulphate, filter until clear, and polarize. Desig-
nate this filtrate as Solution (2) and its polarization as Reading
(2). Care should be taken to add no more lead acetate to the solu-
tion than is necessary for complete precipitation, as lead malate is
soluble in an excess of lead acetate. Solutions which are sufficiently
clear and contain less than 10 per cent of sugar may be polarized
directly without treatment with lead acetate.
(3) If Reading (2) is negative treat a portion of Solution (2)
with uranyl acetate in the manner described under (1) and polarize.
Designate this as (3). If Reading (2) is positive, Reading (3) need
not be made.
Polarize at room temperature with white light, taking care that
all solutions are polarized at the same temperature. Make at least
six readings in each case and take an average of these. In this work
a standard. Lippich type, triple field saccharimeter was used, the light
being furnished by an electric bulb placed behind a ground-glass
plate. Calculate all readings to the basis of a 200 mm tube. If
Reading (3) is numerically less than Reading (2), the latter should
be discarded; otherwise use Reading (2) in the subsequent calcula-
tion. Multiply the algebraic difference between this reading and
Reading (1) by 0.03G. The product will equal the percentage of
rCH 2 COOH l
malic acid \ | [in the solution as polarized.
EFFECT OF URANIUM SALTS.
The method just described was developed in the course of some
work on the composition of the apple. If uranium salts produce
an increase in the rotation of malic acid only, it would theoretically
be possible to estimate the amount of malic acid in a solution con-
taining sugars and other organic compounds by simply polarizing
4 DETERMINATION OF MALIC ACID.
the solution before and after the addition of the uranium salt and
multiplying the difference in the readings by a factor determined
experimentally. As was previously stated, the rotation of ^-tartaric
acid is also increased by uranium salts. Consequently, this method
can not, without modification, be applied to a solution containing
this acid. We have determined that, as would be expected, inactive
lactic, succinic, and citric acids are unaffected by salts of uranium.
There are, therefore, no serious difficulties in the way of applying
this method to the determination of malic acid in the absence of
^-tartaric acid. Work is now in progress on the estimation of tartaric
acid by a similar method.
In the earlier experiments of this investigation an attempt was
made to use uranyl nitrate and potassium hydroxid as reagents, as
recommended by Walden. This author states that a maximum rota-
tion is obtained by the use of 4 molecules of potassium hydroxid and
1 to 4 molecules of uranyl nitrate [U0 2 (N0 3 ) 2 -|-6H 2 0] for each
molecule of malic acid. He prefers the nitrate to the acetate because
of the greater solubility of the former salt.
Uranyl nitrate, however, has one serious disadvantage. Its solu-
tion in water always contains nitric acid due to hydrolysis. It is
impossible to obtain the maximum polarization in the presence of
free mineral acid and it is therefore necessary, as Walden pointed out,
to neutralize the solution when uranyl nitrate is used. The color of
the solution always makes this operation very difficult. Many at-
tempts were made to use uranyl nitrate, but with indifferent results.
After addition of the uranyl nitrate solution to that containing
malic acid, a solution of potassium hydroxid was added drop by
drop until a precipitate just began to form. This was dissolved
either by addition of a drop of nitric acid or of uranyl nitrate solu-
tion. The results, however, were very irregular. In some cases the
theoretical recovery of malic acid was obtained, while in others the
results were very much too low. Attempts to neutralize the solution,
using various indicators, were made with similar results. No better
success was attained when the amount of alkali required to neutralize
both the uranyl nitrate and the malic acid solution was determined
in advance and the calculated amount added to the mixture.
Uranyl acetate was then substituted for the nitrate in the hope
that the presence of a weak organic acid like acetic acid would not
influence the rotation of the uranium-malic complex. As a further
precaution the solution to be examined was carefully neutralized
before the addition of the uranium salt. The concentration of IT
ions in a solution containing acetic acid and sodium acetate is so
slight that it was believed its influence on the rotation would be
DETERMINATION OF MALIC ACID. 5
negligible. This procedure proved >atisfactory and no further diffi-
culty was experienced in this direct ion.
A number of experiments were made to determine what effect, if
any, is produced by an excessive amount of uranyl acetate. The re-
sults all showed that such an excess does not influence the rotation of
the solution. On the other hand, it is quite possible to add too small
an amount of the salt to the solution under examination. In the case
of solutions containing a comparatively large amount of malic acid,
several hours may elapse before a maximum change in rotation is ob-
tained. To test this point a solution containing 2.94 per cent of malic
acid as sodium malate was treated with an excess of uranyl acetate,
shaken frequently and polarized at intervals. The polarizations ob-
tained were as follows:
. After 10 minutes —60.4
85 minutes —68.7
1 hour^! —74.1
2 hours, 10 minutes —82.0
2 hours, 45 minutes — 82.8
4 hours, 10 minutes — 83.0
This indicates that after two hours the reading is practically con-
stant, since a difference of 1° corresponds to only 0.036 per cent of
malic acid. It must be remembered, however, that frequent agitation
is also needed if a maximum is to be attained in this time.
An approximate determination of the solubility of uranyl acetate
at ordinary temperatures gave the following results:
1. In water 8.5
2. In a solution containing 1.25 per cent of malic acid as sodium malate 11. 96
3. In a solution containing 2.5 per cent of malic acid as sodium malate— 13. 32
REMOVAL OF THE MALIC ACID.
"Walden reports [a] D for a 0.65 per cent solution of Z-malic acid
as approximately —0.77 circular degrees, as compared with —475°
(circular) for the uranium complex. It is evident, therefore, that in
ordinary concentrations the rotation of free malic acid is negligible.
However, as has been said, it is sometimes necessary to remove the
malic acid from the solution before making reading (2) for the reason
that the addition of uranyl acetate causes a decrease in the rotation of
strong sugar solutions. 1 To determine the extent of this effect, 13
grains of uranyl acetate were added to 100 cc of various sugar solu-
tions and allowed to stand one hour with frequent shaking. The so-
1 Rembach and Weber (Zts. physikal. Chem., 1905. 51:491) have noticed that uranyl
nitrate produces a slight increase in the specific rotation of solutions of dextrose and
levulose containing 7.2 grams of the sugar in 100 cc of solution.
DETERMINATION OF MALIC ACID.
lutions were then polarized in 200 mm tubes with white light and the
readings compared with those of the untreated solution. After stand-
ing overnight the readings were repeated.
Table I. — Effect of uranyl acetate on the rotation of sugars.
Sugar solution, after 1
Sugar solution, after
standing over night.
tion of ura-
tion of ura-
The results show a decided reduction in rotation which would have
a serious effect on the determination of small amounts of malic acid
in the presence of large amounts of sugar. To avoid inaccuracy from
this cause it is necessary in some cases to remove the malic acid as
completely as possible from solution (2) and then to add uranyl
acetate to the filtrate. For reasons which will be given later this
procedure is never followed when the rotation of solution (2) is
positive. Normal lead acetate is used to precipitate malic acid.
Even when the removal of the malic acid is not necessary it is ad-
visable to clarify the solution with lead acetate and then remove the
excess of lead in order to obtain an easily readable solution. In the
earlier work a solution of lead acetate was added to the malic-acid
solution before diluting to the mark. It was found, however, that
the volume of the precipitate in some cases seriously changed the
concentration of the solution, especially where sugar solutions in-
verted with hydrochloric acid were used, and the solid normal lead
acetate, Pb(C 2 H 3 2 ) 2 +3H 2 0, was therefore substituted. This salt
contains 4.7 per cent of water of crystallization and introduces a
negligible dilution error in the amounts used. An attempt was made
to use Home's dry lead subacetate, but the results were not satis-
Dry neutral potassium oxalate was at first used to remove the ex-
cess of lead from the solution. It was later found that the rotation
of sugar solutions is affected to some extent when this salt is added
to an invert sugar solution containing lead acetate. An approxi-
mately 25 per cent solution of invert sugar polarized —29° before
and after addition of lead acetate. After precipitating the lead with
potassium oxalate, however, the filterate polarized —29.6°. Conse-
DETERMINATION OF MALIC ACID. 7
quently anhydrous sodium sulphate was substituted for potassium
oxalate with satisfactory results.
Lead malate is somewhat soluble in water, lead acetate, and sugar
solutions. This fact has caused difficulty at two points during the
development of the method. It was early discovered that when a
solution of sodium malate is treated with lead acetate the filtrate has
a positive polarization amounting to as much as one degree. Previous
to this discovery it was not thought necessary to remove the lead from
solution (2) before polarization. A more serious difficulty was en-
countered when it was found necessary to treat solution (2) with
uranyl acetate. The presence of even a slight amount of malic acid
in this solution after removal of the lead will of course cause a change
in rotation when the uranium salt is added. To obtain the best pos-
sible separation of lead malate, therefore, the solution is cooled in an
ice bath before filtering. This procedure is sufficient to remove most
of the malic acid when quantities less than 0.2 per cent are present,
but if large amounts of the acid are present a very considerable error
may be introduced by the change in rotation caused by the formation
of the uranium-malic complex. This is illustrated by the following
Four solutions containing known amounts of malic acid, as sodium
malate, were treated with lead acetate at room temperature, the lead
removed with potassium oxalate, and the filtrate treated with uranyl
acetate for one-half hour, with frequent shaking. The filtrates were
polarized with the following results:
Per cent of malic acid: °v.
0.099 — 0.1
.199 — 1.1
1. 99 —15. 5
When the solution, which has been treated with lead acetate, is
cooled in ice water before filtering, the rotation with uranyl acetate
is much decreased, so that if small amounts of malic acid occur in
the presence of large amounts of invert sugar, Reading (3) is less than
Reading (2). When comparatively large amounts of malic acid are
present. however, Reading (3) is likely to be greater than (2), and for
this reason both readings are made when the approximate amount of
invert sugar or malic acid are unknown, and the smaller is used in
calculating the percentage of malic acid present.
In the presence of large amounts of sucrose the error due to the
effect of the uranium salt on sugar is in the same direction as that
due to the presence of traces of malic acid. This will be clearly seen
from the following determinations on an approximately normal solu-
tion of sucrose containing 0. 24 per cent of malic acid .
DETERMINATION OF MALIC ACID.
Table II. — Determinations on a normal solution of sucrose containing 0.24 per
cent of malic acid.
1 Difference between (1) and (2). 2 Difference between (1) and (3).
It will be seen that when the difference between readings (1) and
(2) is used in the calculations a somewhat high recovery is obtained.
This is to be expected since the decrease in rotation due to the effect
of the uranium salt on sugar is added to that due to the rotation of
the uranium malic complex. Consequently, Reading (1) is lower,
and the difference between (1) and (2) is higher, than it should be.
Reading (3) is decreased by the combined effect of the action of the
uranium salt on sugar and also on the traces of malic acid present.
Hence, the difference between (1) and (3) is too small. The per-
centage recovery obtained by using the difference between readings
(1) and (2) is not excessively high and this is the largest error that
would be obtained due to the action of uranium salts on sucrose,
assuming that no more concentrated solution than one of normal
sugar is examined. Therefore, while not theoretically correct, it is
practically permissible to dispense with Reading (3) entirely when
working with solutions in which Reading (2) is positive. It would,
of course, be possible to avoid error from this source by inverting
the solution before beginning the determination, then neutralizing,
and diluting to a definite volume. This has not been found to be
necessary in practice, however. When it is known that the solution
under examination contains either less than 10 per cent of invert
sugar or more than 0.25 per cent of malic acid it is unnecessary to
make Reading (3).
Solutions are frequently encountered which are too dark to polarize
without clarification. It was found that alumina cream can be used
for this purpose without affecting the results. 1
The question arises as to whether uranium salts produce the same
effect on levo, dextro, and inactive malic acid. For ordinary work on
natural products this question is of little importance, since, as far as
the literature goes, malic acid appears to occur in nature only in
the levo form. W. Gintl 2 reports the presence of the inactive form
in the leaves of Fraxinus excelsior L., but this discovery does not
appear to have been confirmed since that time.
1 With very dark solutions such as fruit juices, C. W. Clark, of this laboratory, has
found that a few drops of bromin may often be used to decolorize without affecting the
2 Jahresber. Chem., 1868, p. 800.
DETERMINATION OF MALIC ACID.
It is well known that the specific rotation of aqueous solutions of
?-malic acid varies greatly with concentration. 1 changing from minus
to plus in the higher concentrations. The addition of concentrated sul-
phuric acid 2 will also cause a change in rotation. Some experiments
were made to throw light on the question as to whether such dif-
ferences in the specific rotation of £-malic acid affect the rotation of
the uranium malic complex. A solution containing 13.95 grams of
Z-malic acid in 28 cc of solution polarized +1.1°. This solution was
diluted and the malic acid determined by the uranium method, the
theoretical recovery being obtained. This same solution was treated
with concentrated sulphuric acid and after standing over night
polarized +2.7. After standing four days the rotation was found
to be the same. On dilution the theoretical recovery was again
obtained by the uranium method.
DETERMINATION OF THE FACTOR.
A large number of experiments were made to determine the factor
for calculating the percentage of malic acid from the polarization.
Neutral solutions containing known amounts of malic acid were
treated with uranyl acetate, polarizing in a 200 mm tube with white
light and dividing the percentage of malic acid present by the read-
ing in degrees Ventzke. In some cases this determination was made
in the presence of invert sugar. The details of the experiment are
given in Table IV, page 10. It will be observed that there is a
gradual decrease in the factor with increase in concentration of malic
acid. The increase is not sufficient, however, to seriously affect re-
sults in the ordinary concentrations found in natural products. The
average of 27 determinations gave a factor 0.036, and this was accord-
ingly adopted for use in all calculations. A number of determinations
in which sodium light was used instead of white light showed a
slightly smaller specific rotation with the former.
Table III. — Specific rotation in circular degrees (10 cm tube).
Per cent malic
r«i D .
With very clear solutions containing large amounts of uranium-
malic complex difficulty may be experienced in obtaining the neutral
point on account of the decided blue color developed in one segment
of the field. This can be eliminated to a great extent by the use of
a 3 cm cell containing a 3 per cent solution of potassium bichromate.
i Schneider, Ann. Chem. (Liebig.) 1881, 207: 262.
2 Ibid., p. 279.
DETERMINATION OF MALIC ACID.
The use of such a screen has the additional advantage of lowering
the specific rotation of the uranium-malic complex somewhat and con-
sequently partially correcting for the normal rise in specific rotation
due to concentration.
-Determination of factor.
Number of deter-
DISCUSSION OF RESULTS.
The results which have been obtained in the application of the
method are tabulated in Tables V and VI, pages 11 and 12, which are
in the main self-explanatory. Table V contains the results of de-
terminations in which known amounts of malic acid were added by
the analyst, while the determinations recorded in Table VI were
made on solutions in which the amount of malic acid was unknown.
The percentages of invert sugar given in the second column of
Table V are only approximate. This accounts for the fact that in
some cases solutions containing the same amount of sugar and malic
acid show a larger difference in polarization than can be accounted
for by errors in reading. Stock solutions containing 5 or 10 per cent of
carefully weighed C. P. malic acid were made up and the percentages
of acid present were determined by titration against standard
alkali. A measured portion of the stock solution was then added to
a definite volume of invert-sugar solution and the mixture diluted
DETERMINATION OF MALIC ACID.
to the desired concentration. The sugar solutions always contained
more or less sodium chlorid resulting from the neutralization of the
acid used for inversion. New malic acid stock solutions were made
up every two or three days, as a growth of mold usually appears after
a few days' standing. In many cases Reading (3) was not made.
Wherever possible it is given, however. It will be noted that in
many cases Reading (3) is higher than (2) for the reason previously
explained. When the former is lower, the percentage of malic acid
is calculated from it. The most favorable concentrations of malic
acid appear to be between 0.2 and 2.5 per cent. Below 0.2 per cent
the increase in polarization due to the uranium-malic complex is so
small that a small error in reading may make a relatively large error
in the final result, while above 2.5 per cent the increase in specific ro-
tation tends to give high recoveries.
The results shown in Table VI illustrate the applicability of the
method to natural products. The strawberry juice used as a solvent
was highly colored and most of the readings had to be made in 50
or 100 mm polariscope tubes.
Table V. — Determinations on solutions containing known amounts of malic acid.
Number of deter-
1 Determinations by C. W. Clark.
12 DETERMINATION OF MALIC ACID.
Table VI. — Malic acid determinations on unknown solutions.
1 Determinations by C. W. Clark.
(1) When a neutralized solution of malic acid is treated with
uranyl acetate, its rotation is increased approximately 28° V. for each
per cent of malic acid in the solution; 6?-tartaric acid is the only
other common acid which is affected in this way by uranyl acetate,
Hence, in the absence of ^-tartaric acid, malic acid may be deter-
mined quantitatively by treating its solution with uranyl acetate,
polarizing, and multiplying the difference between this reading and
that of the untreated solution by 0.036. The product equals the per-
centage of malic acid present.
(2) In the presence of more than 10 per cent of reducing sugars
and less than 0.25 per cent of malic acid, the results may be affected
by the action of uranyl acetate on the rotation of the sugar. Hence,
in this case or when the amounts of sugar or malic acid are unknown,
certain simple modifications are necessary.
(3) For this determination, the most favorable limits of concen-
tration of malic acid are between 0.2 to 2.5 per cent. The percentage
error seldom amounts to more than 5 per cent of the malic acid
present. Twelve complete determinations may easily be made in
four hours time, including two hours during which the solutions
require no treatment other than frequent shaking.
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