A RAPID COLORIMETRIC METHOD FOR THE DETERMINATION

OF L A C T I C A C I D I N M I L K
BURDET IIEINEMANN
Chief Chemist, Producers Creamery Company, Springfield, Mo.

~[any methods for determining lactic acid in biological fluids have been
reported. Several have been applied successfully to d a i r y products. Most
of these methods, however, are too time consuming to be used r e g u l a r l y in
the control laboratory. A search was made, therefore, for a method which
was both r a p i d and reliable: A method using a simple procedure for the
removal of interfering substances; a simple oxidation p r o c e d u r e ; and a
simple means of estimating the quantity of lactic acid present.
The method Mendel and Goldscbeider (5) used for determining the lactic
acid in blood seemed to be the most promising, b u t when applied to milk
gave unreliable results. M a n y variations were tried, one of which is re-
ported below. The method for removing interfering substances is almost
identical with that described b y T r o y and S h a r p (7). Their work on pre-
cipitation procedures was repeated to determine the most satisfactory tech-
nique to be used in conjunction with the eolorimetric method, but no change
was f o u n d necesssary.
PROCEDURE
Weigh 5 grams of milk in a 50 ml. volumetric flask. A d d 30 ml. of
cold water and mix. Dilute a portion of a 25 per cent solution of copper
sulphate 1 : 3 a n d add dropwise with agitation 2-8 drops of the dilute solu-
tion to the water and milk m i x t u r e until precipitation occurs. W a r m the
flask in hot water until the contents reach 45 C. A d d 6 ml. of 25 per cent
copper sulphate solution and mix thoroughly b y rotation. Hold the con-
tents of the flask at 45 to 47 C. for 8 to 10 minutes. Next loosen a n y c u r d
particles which m a y adhere to the flask with a bent wire. A d d 6 ml. of cal-
cium hydroxide suspension. This suspension is p r e p a r e d b y slaking 300
g r a m s of the best quality calcium oxide using 1400 ml. water and working
through a 24 mesh screen a f t e r 30 minutes standing. Before mixing the
lime with the other constituents in the flask, w a t e r should be added to bring
the contents to exactly 50 ml. Then mix until the contents are homogeneous
and allow to stand at 45 to 47 c. for 10 minutes. Cool to 25 C. and filter,
discarding the first few ml. of filtrate.
Place 1.0 ml. of the filtrate in a thoroughly clean and d r y test tube.
Hold the test tube in ice w a t e r and allow 5.0 mI. of cold concentrated sul-
phuric acid (arsenic free) to r u n down the side wall of the test tube. Mix
gently while cooling. Place mixture in boiling water for exactly 5 minutes~
Received for publication March 28, I940.
969
970 BURDET HEINEMANN

Cool in ice water for three. A d d 4 drops of a 0.1 per cent solution of v e r a t r o l e
in water and mix. H o l d in ice w a t e r for 60 minutes and compare colors with
artificial or n a t u r a l standards.
N a t u r a l standards m a y be p r e p a r e d according to Hil!ig (3). Dissolve
106.6 rag. of purified lithium lactate in 100 ml. of distilled water. 1.0 ml.
of this solution contains the equivalent of 0.1 p e r cent lactic acid. Conse-
quently, to p r e p a r e a 0.01 lactic acid standard, 5.00 ml. of this solution is
diluted to exactly 50.0 ml. and mixed. One ml. of this dilution is then
placed in a test tube and 5 ml. of concentrated sulphuri acid added. The
m i x t u r e is held in boiling water 5 m i n u t e s and cooled in ice water 3 minutes.
F o u r drops of a 0.1 per cent solution of veratrole is then added and the color
observed a f t e r 60 minutes. Additional standards m a y be made b y diluting
the original lithium lactate solution as required.
Artificial standards which are fairly satisfactory m a y be p r e p a r e d ac-
cording to the method of Nordb5 (6). Mix 25.0 ml. of a 0.01 per cent solu-
tion of fuchsin in w a t e r a n d 4.75 ml. of a 0.01 per cent solution of Tropaolin
000. (The m i x t u r e m u s t be diluted immediately for a precipitate f o r m s on
standing.) One tenth ml. of this m i x t u r e diluted to 50.0 ml. of solution is
equivalent to a p p r o x i m a t e l y 0.005 p e r cent lactic acid; 0.25 ml. diluted to 50
ml. is equivalent to a p p r o x i m a t e l y 0.01 per cent and ~0.9 ml. diluted to 50
ml. is equivalent to 0.02 per cent. NordbS's standards are only suitable f o r
concentrations below 0.03 per cent. Above this percentage, the yellow color
increases in intensity. Consequently, f o r a 0.05 per cent lactic acid stan-
dard, it is necessary to add 0.4 ml. of .01 p e r cent Tropaolin and 1.8 ml. of
.01 p e r cent fuchsin to 47.8 ml. of water; for a 0.07 per cent standard, 1.0
ml. of Tropaolin and 2.0 ml. of fuchsin to 47.0 ml. of w a t e r ; and f o r a 0.10
per cent standard, 2.0 ml. of Tropaolin and 2.0 ml. of fuchsin to 46.0 ml. of
water. These artificial standards should be checked against n a t u r a l stan-
dards before using.
DISCUSSION

According to the authors, the original test which was applied to blood is
accurate to -+- 0.004 per cent between 0.001 and 0.05 per cent (5) a n d 1 p a r t
in 400,000 lactic acid could be detected. Mendel (4) later made two im-
provements which he stated made possible the detection of one p a r t in
1,000,000. I-Iowever, when applied to milk, the original test was sensitive
to 0.01 per cent and had a limited degree of accuracy ( 0.04 p e r cent).
W i t h the modification outlined above, the test is sensitive to 0.005 p e r cent
and accurate to 0.004 per cent between the ranges Of 0.005 and 0.12 per
cent lactic acid providing the procedure is closely followed.
Mendel (4) has pointed out t h a t small changes in the concentration of
the sulphuric acid m a y lead to inaccurate results. A difference of 2 to 3
per cent in the concentration m a y result in as much as a 15 to 20 p e r cent
 DETERMINING L A C T I C ACID I N MILK 971

error. It is wise, therefore, to check frequently the color of the artificial

standards. In this respect, it should be noted that the individual analyst
should t r y several different quantities of sulphuric acid (i.e., 4.5, 4.75, 5.0
ml. etc.,) in the preparation of natural standards in order to ascertain the
exact amount to add to the 1.0 ml. filtrate to obtain the greatest degree of
color development for each batch of sulphuric acid. Derviz (1) describes a
method for purifying and storing sulphuric acid so as to maintain a con-
stant concentration.
1Vfendel (4) makes the recommendation that after the mixture of acid and
filtrate has been held in boiling water five minutes, it be cooled 2 minutes in
ice water, veratrole added, and set at 25 C. After 20 minutes, the color
may be compared with standards similarly treated. This method may be
employed whenever results of great accuracy are not desired. ~
Mendel and Goldscheider (5) used 0.1 ml. of a 0.125 per cent veratrolc
in 99.8 per cent alcohol. Other investigators suggested 0.05 ml. of a 1 : 800
solution in 96 per cent alcohol (6), 0.1 ml. of 20 per cent veratrole solution
in glacial acetic acid (1) and 0.1 ml. of 20 per cent in absolute alcohol (2).
These were all tried, but none gave as satisfactory results as 4 drops of a
0.1 per cent solution in water. Various solutions of guaicol, hydroquinone,
and Schiffs reagent were also tried and considered unsatisfactory.
Although the original method called for a 4 minute heating period, 0.5
ml. filtrate and 3.0 ml. of conc. tt2SO, it was found that 5 minute heating
period, 1.0 ml. filtrate and 5.0 ml. of concentrated sulphuric acid gave more
uniform results.
The same substances interfere with this method as those Troy and Sharp
( 7 ) found to interfere with theirs. Formaldehyde, overneutralization,
rancidity, sucrose, and products resulting from the heating of milk at or
near the boiling point for one half hour or longer resulted in high values.
Mendel and Goldscheider state, however, that acetone, ~-oxybutyric acid,
acetic acid, urea, uric acid, creatin, creatinin, glycocol, alanine, and pro-
pionic acid, do not interfere with the color development.
Traces of organic matter interfere with proper color development and
consequently the test tubes must be thoroughly clean and kept stoppered
as much as possible during the determination.
When greater rapidity of testing is required, the precipitate of proteins,
lactose and fat, may be centrifuged. In this case, cream test bottles (18
gram body, 9 gram neck, graduated in 1 per cent between 0 and 55 per cent)
were selected from stock and graduated to contain 50.0 ml. at 20 C. The
milk was weighed in these bottles which, after precipitation and cooling,
were placed in a Babcock tester and centrifuged 4 minutes. The centrifu-
gate was then decanted through a fluted filter, the first few ml. of filtrate
being discarded.
972 BURDET HEINEMANN

SUMMARY

A rapid colorimetric method for the quantitative estimation of lactic

acid in milk is described. The steps involved are precipitation of fat, lac-
tose and protein with copper sulphate and calcium hydroxide, filtering,
adding concentrated sulphuric acid (arsenic free) to the filtrate, heating,
cooling, and adding veratrole. A red color develops on standing which is in
proportion to the amount of lactic acid present.
The test is sensitive to about 0.005 per cent and is accurate to ___ 0.004
per cent between the ranges of 0.005 and 0.12 per cent lactic acid.
:REFERENCES
(1) DERWZ, G.V. The colorimetric lactic acid determination according to Mendel-Gold-
scheider. Zhur. Exptl. Biol. Med. 12: 147-50. 1929. (Original not seen.
See Chem. Abstr. 24- 1880. 1930.)
(2) FUCHS, HANS J. Einige Verbesserungen der kolorimetrischen Milchsfiurcbestim-
mung nach Mendel und Goldscheider. Biochem. Ztschr. 217: 405-8. 1930.
(3) HILLIG, FRED. The colorimctric determination of lactic acid in milk and milk prod-
ucts. J . A . O . A . C . 20: 130-40. 1937.
(4) MENDEL, BRUNO. Zur Methode der k01orimetrischen Milchs~urebestimmnng. Bio-
chem. Ztschr. 202: 390. 1928.
(5) MENDEL, BRUNO AND GOLDSCYIEIDER, INGEBORG. Eine kolorimetrische Mikromethode
zur quantitativen Bestimmung der Milchsiiure im Blut. Biochem. Ztschr. 164:
163-74. 1925.
(6) NORDBh,I:~AGNVALD. Zur Methode der Milchs~urebestimmung nach Mendel und Gold-
scheider. Biochem. Ztschr. 271: 213-215. ]934.
(7) TROY, It. C. AND SHARP, P. F. Quantitative determination of lactic acid in dairy
products. Corner Univ. Ag. Exp. Sta. Memoir 179. 1935.