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:
[email protected] Received November 5, 2015; in final form, February 11, 2016
Abstract—An efficient and inexpensive spectrophotometric method has been developed for the determination 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 built. 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 determination 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 control 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] chromatography are the most accurate methods of the determination of lactic acid. The use of HPLC in routine 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 disadvantage 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 insufficiently accurate because of the incomplete elimination 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 hydrogenases 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 commercial 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 efficient and simple methods for the determination of lactic 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 spectrophotometric method for the determination of L- and D-isomers of lactic acid suitable, in particular, for the microbiological industry in the determination of lactic acid in cultural liquids.
755
756
BORSHCHEVSKAYA et al.
A 1.4 1.2 3 1.0 0.8 0.6
2
0.4 0.2 1 0 350 360 370 380 390 400 410 420 430 440 450 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), lactic acid, cp grade (89% DL, 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 corresponding weighed portions of substances and aliquot portions 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 concentration of lactic acid 89 g/L was obtained. A series of lactic acid solutions was prepared from the stock solution using two-fold dilutions. A solution of iron(III) chloride (0.2%) was prepared. Iron(III) chloride (0.3 g) was placed in a 100mL 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 corresponding concentration was added to 2 mL of a 0.2% solution 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 concentration 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
curve. The processing of the results and the construction of the calibration curve were done using the Statistica 6.0 software. Method of the spectrophotometric determination of lactic acid. A test solution (50 μL) containing lactic 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. Cultural liquid was separated from the cells by centrifuging. The supernatant was diluted 20-fold with deionized water. Lactic acid in the sample was determined by the spectrophotometric method for the determination 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 determined by the spectrophotometric method for the determination of lactic acid. The concentration of lactic 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 components 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 solution of iron(III) chloride than for the product of reaction 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 concentration 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) chloride 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.
JOURNAL OF ANALYTICAL CHEMISTRY
Vol. 71
No. 8
2016
SPECTROPHOTOMETRIC DETERMINATION OF LACTIC ACID
757
Table 1. Effect of impurities on the determination of lactic acid (5 g/L) Impurity Acetic acid Ethanol Glycerin Bovine serum albumin
Concentration of impurity, g/L
Measured concentration of DL lactic acid, g/L
Error, % (n = 3)
100 96 100 100
4.94 4.90 4.99 5.07
1.3 2.0 0.2 1.4
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 absorbance 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 lactic 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 compared. The results are presented in Table 1. It can be seen that the effect of the studied compounds is insignificant and does not exceed 2%. Thus, the developed method ensures the determination of lactic acid in complex mixtures and does not require the purification of the target product at the sample preparation stage. Calibration curve. The absorbance of iron(III) lactate 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-
ous solution, in the composition of a cultural liquid in microbiological production, as well as in the composition 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 measurements 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 composition of complex mixtures, such as cultural liquids or food. Thus, the presented spectrophotometric method is simple, sensitive, inexpensive, and not time consuming, does not require complex sample preparation, and gives sufficiently accurate results. Moreover, the proposed method ensures the determination of both Land D-isomers of lactic acid. The method can be used in routine practice of genetic and microbiological laboratories in the creation and testing of strain-producers of lactic acid, elaboration of cultivation methods, etc. The proposed method is also necessary for the control of the production of lactic acid at the plants of microbiological production of lactic acid. It should be noticed that, though the wavelength 390 nm is optimum for the measurement of the absorbance of iron(II) lactate, a possibility of using this method at 400–405 nm (Figure) also exists, which slightly reduces its sensitivity; however, allows the analyst to avoid the use of expensive spectrophotometric equipment.
Table 2. Comparison of the results of determination of lactic acid (g/L) by the spectrophotometric method and HPLC Sample Aqueous solution of lactic acid Cultural liquid Fermented dairy product
Spectrophotometric method
HPLC
Error, % (n = 3)
89.8 ± 1.6 91.8 ± 1.9 10.8 ± 0.2
88.9 ± 1.6 93.6 ± 1.7 11.1 ± 0.2
1.0 1.9 2.7
JOURNAL OF ANALYTICAL CHEMISTRY
Vol. 71
No. 8
2016
758
BORSHCHEVSKAYA et al.
ACKNOWLEDGEMENTS
2. Miura, Y., Ohno, H., and Koh, T., Bunseki Kagaku, 1994, vol. 43, no. 10, p. 745.
4. Lesh, M.J., Wilkinson, E.L., Zolfaghari, M.R., and Schreiber, M.A., J. Liq. Chromatogr., 1993, vol. 16, no. 11, p. 2415. 5. Ohara, H., Hiraga, T., Katasho, I., Inuta, T., and Yoshida, T., J. Ferment. Bioeng., 1993, vol. 75, p. 470. 6. Frledemann, T.E., Catonlo, M., and Shatter, P.A., J. Biol. Chem., 1927, vol. 73, p. 335. 7. Barker, S.B. and William, H., J. Biol. Chem., 1941, vol. 138, p. 535. 8. Paradis, D. and Nadeau, D., J. Tissue Cult. Methods, vol. 7, no. 4, p. 175. 9. Lactate colorimetric/fluorometric assay kit, BioVision. www.biovision.com/lactate-colorimetric-fluorometric-assay-kit-2903.html. Cited November 5, 2015. 10. Amplite colorimetric L-lactate assay kit, AAT Bioquest. http://aatbio.com/gen4prst.pl?Cid=13815. Cited November 5, 2015.
3. Lebuhn, M. and Hartmann, A., J. Chromatogr. A, 1993, vol. 629, p. 255.
Translated by I. Duchovni
This work was supported by the Ministry of Education 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). REFERENCES 1. Abercade Consulting. cade.ru/research/analysis/7626.html. November 5, 2015.
http://aberCited
SPELL: 1. OK
JOURNAL OF ANALYTICAL CHEMISTRY
Vol. 71
No. 8
2016
