ISSN 1061-9348, Journal of Analytical Chemistry, 2016, Vol. 71, No. 8, pp. 755–758. © Pleiades Publishing, Ltd., 2016.

Original Russian Text © L.N. Borshchevskaya, T.L. Gordeeva, A.N. Kalinina, S.P. Sineokii, 2016, published in Zhurnal Analiticheskoi Khimii, 2016, Vol. 71, No. 8, pp. 787–790.

ARTICLES

Spectrophotometric Determination of Lactic Acid

L. N. Borshchevskaya*, T. L. Gordeeva, A. N. Kalinina, and S. P. Sineokii
State Research Institute of Genetics and Selection of Industrial Microorganisms,
Pervyi Dorozhnyi proezd 1, Moscow, 117545 Russia
*e-mail: larbor3@rambler.ru
Received November 5, 2015; in final form, February 11, 2016

Abstract—An efficient and inexpensive spectrophotometric method has been developed for the determina-
tion of lactic acid in the individual state and in food and biological and cultural liquids. The method is based
on the spectrophotometric determination of the colored product of the reaction of lactate ions with iron(III)
chloride at 390 nm. The optimum conditions of the reaction have been found, and a calibration curve in the
range from 0.3 to 10 g/L of lactic acid with the correlation coefficient 0.9999 has been constructed. The
method does not require complex sample preparation.

Keywords: spectrophotometry, lactic acid, iron(III) chloride

DOI: 10.1134/S1061934816080037

Lactic acid (CH3CHOHCOOH) is widely used in
food, cosmetic, pharmaceutical, leather, chemical,
and other industries; therefore, the development of
inexpensive methods for the determination of lactic
acid is of great importance. The world consumption of
lactic acid is several tens of thousands of tons and is
expected to increase significantly [1].
Efficient and inexpensive methods for the determi-
nation of lactic acid can be widely used, first of all, for
the control of the fermentation of bacterial strains of
lactic acid bacteria, which are traditionally used for the
production of lactic acid, as well as for the quality con-
trol of food, agricultural raw materials, etc. At present,
various versions of HPLC, including ion [2], ion-pair
[3], ion-exclusion [4], and reversed-phase [5] chro-
matography are the most accurate methods of the
determination of lactic acid. The use of HPLC in rou-
tine practice is limited by the necessity of complex
sample preparation and also by the need in expensive
equipment and skilled personnel.
A method based on the oxidation of lactic acid to
acetic aldehyde and the determination of the aldehyde
either by the bisulfite method [6] or by coloration with
p-hydroxydiphenyl in the presence of metal ions [7] is
also used for the determination of lactic acid. A disad-
vantage of this group of methods is the necessity of the
elimination of protein and carbohydrate impurities
using 12-tungsten phosphoric acid, CuSO4, and CaO
prior to the determination of lactic acid. KMnO4,
H2SO4, or Ce(IV), work with which requires special
precautions, are used as oxidants. The analysis takes
several hours, and the results of analysis can be insuf-
ficiently accurate because of the incomplete elimina-
tion of impurities from the test sample.
An enzymatic method based on the transformation
of lactic acid to pyruvate under the action of enzyme
lactate dehydrogenase in the presence of NAD+,
which in its turn is reduced to NADH, is also used for
the determination of lactic acid. The concentration of
lactic acid in the sample is proportional to the amount
of NADH formed in the enzymatic reaction; it is
determined by spectrophotometry at 340 nm [8]. The
use of expensive purified L- and D-lactate hydroge-
nases with the certain enzymatic activity is necessary
for the implementation of this method. Moreover, the
method is complex and requires high thoroughness in
the performance of measurements because of the
instability of NAD+ and NADH. There are commer-
cial reagent kits for the determination of lactic acid by
the enzymatic method; however, their cost is
extremely high and is approximately $400 for 100 tests
[9, 10].
Therefore, the problem of the development of effi-
cient and simple methods for the determination of lac-
tic acid is highly important. Spectrophotometric
methods of analysis are highly selective and sensitive.
They are frequently used in laboratories because of
their simplicity and availability.
The aim of the present work was the development
of a simple, inexpensive, and rapid spectrophotomet-
ric method for the determination of L- and D-isomers
of lactic acid suitable, in particular, for the microbio-
logical industry in the determination of lactic acid in
cultural liquids.

755
756 BORSHCHEVSKAYA et al.
A curve. The processing of the results and the construc-
1.4
1.2
3 lactic acid. A test solution (50 μL) containing lactic
1.0
0.8
0.6
2
0.4
0.2
1 ing. The supernatant was diluted 20-fold with deion-
0 ized water. Lactic acid in the sample was determined
350 360 370 380 390 400 410 420 430 440 450
λ, nm
Absorption spectra of (1) lactic acid (10 g/L) distilled
water; (2) iron(III) chloride (2 g/L) against distilled water;
and (3) iron(III) lactate against iron(III) chloride.
EXPERIMENTAL
Equipment. A 10S UV-Vis spectrophotometer
(Genesys, United States) (l = 1 cm) was used.
Materials, reagents, and solutions. FeCl3 ⋅ 6H2O,
reagent grade (Khimmed, Russia), DL-lactic acid, cp
grade (89%, VAG Chemie, Germany), glacial
CH3COOH, cp grade (99.8%, Khimmed, Russia),
ethanol, cp grade (Reakhim, Russia), glycerin,
reagent grade (Khimmed, Russia), and bovine serum
albumin (98–99% Sigma, United States) were used.
Solutions were prepared by dissolving of correspond-
ing weighed portions of substances and aliquot por-
tions in deionized water.
Construction of calibration curve. Lactic acid
(1.2 g) with the know concentration (89%, ρ =
1.2 g/mL) was placed in a 10-mL volumetric flask and
diluted with water. A stock solution with the x concen-
tration of lactic acid 89 g/L was obtained. A series of
lactic acid solutions was prepared from the stock solu-
tion using two-fold dilutions.
A solution of iron(III) chloride (0.2%) was
prepared. Iron(III) chloride (0.3 g) was placed in a
100-mL volumetric flask, diluted to the mark with
water and stirred to the complete dissolution of the
salt. The solution must be of room temperature 25 ±
5°C. A solution of lactic acid (50 μL) of a correspond-
ing concentration was added to 2 mL of a 0.2% solu-
tion of iron(III) chloride and stirred. The absorbance
of the obtained colored solutions was measured at
390 nm. The reference solution contained 2 mL of a
0.2% solution of iron(III) chloride. The dependence
of the absorbance of colored solutions on the concen-
tration of lactic acid taken for the reaction was used for
the calculation of the parameters of linear equation
corresponding to the linearity range of the calibration
JOURNAL OF ANALYTICAL CHEMISTRY
tion of the calibration curve were done using the Sta-
tistica 6.0 software.
Method of the spectrophotometric determination of
acid was added to 2 mL of a 0.2% solution of iron(III)
chloride and stirred and absorbance was measured at
390 nm against the reference solution (2 mL of a 0.2%
FeCl3 solution). The reaction and measurements were
performed at 25 ± 5°C. The color of the solution was
stable for 15 min.
Determination of lactic acid in cultural liquid. Cul-
tural liquid was separated from the cells by centrifug-
by the spectrophotometric method for the determina-
tion of lactic acid. The concentration of lactic acid was
calculated using a calibration curve taking into
account the 20-fold dilution of the test sample.
Determination of lactic acid in the composition of
fermented dairy product. Whey was separated from the
precipitate by centrifuging and tenfold diluted with
deionized water. Lactic acid in the sample was deter-
mined by the spectrophotometric method for the
determination of lactic acid. The concentration of lac-
tic acid was calculated using a calibration curve taking
into account the 10-fold dilution of the test sample.
RESULTS AND DISCUSSION
Absorption spectrum. The reaction of iron(III)
chloride with lactic acid in an aqueous solution results
in the formation of yellowish-green iron(III) lactate in
the solution. Absorption spectra of the following com-
ponents of the reaction mixture were recorded:
iron(III) chloride solution and a solution of lactic
acid, as well as the product of their interaction
iron(III) lactate (figure). It can be seen that the
absorption maximum of iron(III) lactate was observed
at 390 nm. The absorbance of lactic acid in this region
is close to zero and is substantially lower for the solu-
tion of iron(III) chloride than for the product of reac-
tion iron(III) lactate. As a result of the study, we
selected the analytical wavelength at 390 nm.
Effect of iron(III) chloride concentration. The
effect of the concentration of iron(III) chloride on the
absorbance of the reaction product was studied at
390 nm. Iron(III) chloride solution with the concen-
tration from 0.05 to 0.3% was added to a lactic acid
solution with a constant concentration of 10 g/L.
Measurements were performed against iron(III) chlo-
ride solutions of corresponding concentrations. The
absorbance of the solution increased with an increase
in the concentration of iron(III) chloride, reaching
the optimum value at the concentration 0.2%. The
further increase in the concentration of the reagent did
not increase the color intensity of the formed iron(III)
lactate solution.
Vol. 71 No. 8 2016
 SPECTROPHOTOMETRIC DETERMINATION OF LACTIC ACID
Table 1. Effect of impurities on the determination of lactic acid (5 g/L)
Concentration
Impurity
of impurity, g/L
Acetic acid 100
Ethanol 96
Glycerol 100
Bovine serum albumin 100
Effect of time. The color intensity of the reaction
product, iron(III) lactate, in solution was studied at
390 nm in the range from 1 to 30 min. To perform the
reaction, a 0.2% solution of iron(III) chloride and a
lactic acid solution of the concentration 10 g/L were
used. The color of iron(III) lactate was stable in the
range from 1 to 15 min, then a slight decrease in absor-
bance occurred, which can be due to the partial
decomposition of the reaction product.
Effect of impurities on determination of lactic acid.
The effect of ethanol, proteins, glycerin, and acetic
acid, side products of the enzymatic process present in
cultural liquids in the microbiological production of
lactic acid and also in food, was studied. The studied
impurity substances (100 µL) were added into the
reaction mixture containing 2 mL of a 0.2% solution of
iron(III) chloride and 50 µL of a 0.5% solution of lac-
tic acid, and the absorbance of the obtained solutions
was measured at 390 nm. The results of analysis of the
obtained solutions and a solution containing 2 mL of a
0.2% solution of iron(III) chloride, 50 µL of a 0.5%
solution of lactic acid, and 100 µL of water were com-
pared. The results are presented in Table 1. It can be
seen that the effect of the studied compounds is insig-
nificant and does not exceed 2%. Thus, the developed
method ensures the determination of lactic acid in
complex mixtures and does not require the purifica-
tion of the target product at the sample preparation
stage.
Calibration curve. The absorbance of iron(III) lac-
tate solution (A) is proportional to the concentration
of lactic acid (c) in the range from 0.5 to 11 g/L.
The equation of the calibration curve has form A =
0.1414c – 0.0222, correlation coefficient is 0.9999,
confidence level 0.000001, and statistical assurance of
approximation 0.9998.
Analytical application. The developed method was
applied to the determination of lactic acid in an aque-
Table 2. Comparison of the results of determination of lactic acid (g/L) by the spectrophotometric method and HPLC
Spectrophotometric
Sample
method
Aqueous solution of lactic acid 89.8 ± 1.6
Cultural liquid 91.8 ± 1.9
Fermented dairy product 10.8 ± 0.2
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 71
757
Measured concentration
Error, % (n = 3)
of DL-lactic acid, g/L
4.94 1.3
4.90 2.0
4.99 0.2
5.07 1.4
ous solution, in the composition of a cultural liquid in
microbiological production, as well as in the composi-
tion of a fermented dairy product obtained by the
inoculation of milk using lactic acid bacteria.
To evaluate the accuracy of the developed method,
we compared the results of studies obtained using the
proposed spectrophotometric method and (Table 2).
The statistical processing of the data (n = 3) showed
that the results of the spectrophotometric method fell
into the confidence interval of the results of measure-
ments by HPLC. The difference of the results did not
exceed 3%. The proposed method gives sufficiently
accurate results of the determination of the amount of
lactic acid both in the individual state and in the com-
position of complex mixtures, such as cultural liquids
or food.
Thus, the presented spectrophotometric method is
simple, sensitive, inexpensive, and not time consum-
ing, does not require complex sample preparation, and
gives sufficiently accurate results. Moreover, the pro-
posed method ensures the determination of both L-
and D-isomers of lactic acid.
The method can be used in routine practice of
genetic and microbiological laboratories in the cre-
ation and testing of strain-producers of lactic acid,
elaboration of cultivation methods, etc. The proposed
method is also necessary for the control of the produc-
tion of lactic acid at the plants of microbiological pro-
duction of lactic acid.
It should be noticed that, though the wavelength
390 nm is optimum for the measurement of the absor-
bance of iron(III) lactate, a possibility of using this
method at 400–405 nm (figure) also exists, which
slightly reduces its sensitivity; however, allows the ana-
lyst to avoid the use of expensive spectrophotometric
equipment.
HPLC Error, % (n = 3)
88.9 ± 1.6 1.0
93.6 ± 1.7 1.9
11.1 ± 0.2 2.7
No. 8 2016
758 BORSHCHEVSKAYA et al.

ACKNOWLEDGMENTS 4. Lesh, M.J., Wilkinson, E.L., Zolfaghari, M.R., and

This work was supported by the Ministry of Educa-
tion and Science of the Russian Federation (Unique
identifier of the project RFMEFI57914X0013) using
the National Bioresource Center “Russian National
Collection of Industrial Microorganisms” (Unique
identifier of the project RFMEFI59214X0002).
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Translated by I. Duchovni

JOURNAL OF ANALYTICAL CHEMISTRY Vol. 71 No. 8 2016