Infrared Spectrophotometric Determination of Small Amounts of Lactic and Pyruvic Acids Alfredo Riva and Mario Bisognani Isrituto di Tecnologie Chimiche Speciali, Uniuersiti di Bologna, 40136 Bologna, Italy
IN OUR EXPERIMENTAL WORK, we had the problem of the detection and quantitative analysis of lactic (2-hydroxypropanoic acid) and pyruvic (2-oxopropanoic acid) acids produced during our reactions. We used the method proposed by Dolinsky and Wilson ( I ) who analyze, by IR absorption, the selective binding of acids by long chain aliphatic amines (weak acids, as, for instance, atnino acids, are not bound). The main criticisms of this method are the following: solvent extraction of the compounds from the aqueous solution requires the use of carbon disulfide, which is inflammable and toxic; samples heating on a steam bath may cause the transformation of pyruvic acid into 4-hydroxy-5-oxo-2methyl-2,5-dihydro-furan-2-carboxylic acid ( 2 ) ; quick corrosion of the sodium chloride windows of the cells (low transparency makes the chemically inert Irtran windows unsuitable); IR absorption bands are too weak to give appreciable results due to the loss of acid during the solvent extraction and the heating steps. We set up a more suitable method based on the IRS (Internal Reflection Spectroscopy) technique (3) instead of IR absorption. EXPERIMENTAL Apparatus. Qualitative and quantitative spectra were recorded with a Perkin-Elmer IR Spectrophotometer Model 257, equipped with an FMIR unit (Frustrated Multiple Internal Reflection), bearing a KRS 5 crystal (thallium bromide and iodide); the angle of incidence was 45”. Reference beam attenuation was realized by an adjustable venetian blind type of aperture. Reagents. Lactic and pyruvic acids were analyzed to detect any impurities which would interfere with the successive experiments. All tests were negative. The required aliphatic amine was a liquid anionic resin Amberlite LA-2, supplied by Rohm and Haas Co. ( 4 ) , with acid binding capacity of 2.22.3 meq/ml. Procedure. A known amount of either acid was dissolved in distilled water and then a suitable volume of an ethanolic solution of Amberlite LA-2 (after a number of trials we were able to establish the most suitable quantity of the resin to be used in each test) was added. The solvents were then distilled under reduced pressure; the oily residue, spectrophotometrically tested on the IR region, was quite anhydrous. By this procedure, we were able to test the spectrum of Amberlite, as well as the spectra of combined Amberlite and either lactic or pyruvic acid. RESULTS AND DISCUSSION
IR-FMIR spectra of lactic and pyruvic acids are shown in Figure 1. Absorption bands at 2950-2850, 1460, 1380, and (1) M. Dolinsky and C. H. Wilson, ANAL.CHEM., 36, 1383 (1964). (2) M. Garino. G. Balletto, F. De Thierry, and C. Becchi, Gazz. Chint. Iiul., 60, 592 (1930). (3) N. J. Harrick, “Infrared Reflection Spectroscopy,” Interscience Publishers. New York, N. Y . . 1967. (4)Rohm & Haas Co., Philadelphia, Pa., Bull. I E 4 - 5 8 (1958).
Figure 1. IR-FMIR spectra of lactic and pyruvic acids bonded with Amberlite LA-2 - Lactic acid
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Pyruvic acid
720 cm-’ are due to Amberlite LA-2, whereas the remaining ones are to be assigned to the bonded lactic (1735, 1120, 1030, 845 cm-l) and pyruvic (1710, 1160, 965, 825 cm-l) acids. A number of tests allowed us to establish that the resin will not suffer any alteration or interference at any stage of the process. Reproducibility was checked many times and has been found fully satisfactory, the abscissa variations being below the limits of the spectrophotometer (k1-2 cm-I) employed. When either 19.3 mg of lactic acid or 20.5 mg of pyruvic acid were treated with 0.10; 0.15; 0.20; 0.25; 0.50; 0.75; 1.00; 1.25 ml of Amberlite LA-2, the reflectance of the acidresin compound decreased as the ratio between the two substances approached the theoretical one of 2.2-2.3 meq/ml. In these latter conditions the intensity of all the peaks was very strong, while any excess of the resin would result in dilution of the acid-resin compound and broader peaks. The sensitivity of the method was checked by making different quantities of either acid react with a fixed quantity of the resin; the reflectance of each reaction product was read at 1030 cm-I for lactic acid and at 1160 cm-l for pyruvic acid. By plotting log of the reflectance changes L‘S. mg of acid, we obtained two straight lines, with slopes of 0.26 for lactic acid and 0.22 for pyruvic acid, respectively. The smallest absolute amount detected was 2.5 mg of either acid. Sensitivity may be further improved by reducing the amount of resin for each initial concentration of acid and employing micro internal reflection accessories. On the other hand, we indicate an absolute value, which may be determined even in large solution volumes. RECEIVED for review February 22, 1972. Accepted June 12, 1972. This work has been carried out under a special grant of the Consiglio Nazionale delle Ricerche, Rome, Italy.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972
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