A Colorimetric Method for Quantitative Determination ...

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East Rutherford, New Jersey and 2,7-dihydroxy- naphthalene from Aldrich Chemical Compa- ny, Milwaukee,, Wisconsin. r-Carboxymethyl ester of L-glutamic ...
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/. Biochem., 71, 563-565 (1972)

A Colorimetric Method for Quantitative Determination of Glycolic Acid with 2, 7-Dihydroxynaphthalene Kenji TAKAHASHI The Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, Hongo, Tokyo Received for publication, August 11, 1971

VoL 71, No. 3, 1972

inside wall of the vessel was collected by centrifugation. The color produced, which has a maximum absorption at 540 mp (Fig. 1), is stable for several hours. The absorbance at 540 m/i was read against a reagent blank in 1-cm cells in a Zeiss PMQII spectrophotometer. The absorbance was found to be linear up to at least 50 nmoles of glycolic acid as can be seen from Fig. 2, and 10 nmoles of the acid gave an absorbance of 0.22±0.01. The blank 0.600

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Fig. 1. Absorption spectrum of the color produced by reaction of glycolic acid with the 2,7-dihydroxynaphthalene reagent. —•—, sodium glycolate (2.1 MS); - - • - - , reagent blank.

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Glycolic acid is known to give a reddish color when heated with 2,7-dihydroxynaphthalene in concentrated sulfuric acid and this reaction has been utilized for qualitative detection of glycolic acid {1,2). In the experiments described in this paper, conditions have been investigated to make use of this color reaction for quantitative determination of glycolic acid. No colorimetric method for such purpose has ever, to the author's knowledge, been reported. Sodium glycolate (98%) was purchased from Matheson Coleman and Bell Company, East Rutherford, New Jersey and 2,7-dihydroxynaphthalene from Aldrich Chemical Company, Milwaukee,, Wisconsin. r-Carboxymethyl ester of L-glutamic acid was prepared as previously described (3). DL-Mandelic acid was obtained from Fisher Scientific Company, New York, New York. In the standard conditions ultimately employed, a solution containing 1 to 100 nmoles of glycolic acid (about 0.1 to 10 ng as sodium glycolate) was taken into a test tube (1.3x12 cm). Samples larger than 50 ^*1 were dried. To the test tube was added 1.0 ml of 0.01% 2,7-dihydroxynaphthalene.in concentrated sulfuric acid. The tube was covered with a polyethylene or glass cap and heated in a 100°C water bath for 20 min. After the color development, any water that had condensed on the

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value was usually about 0.1 or less. The 2,7dihydroxynaphthalene reagent should be prepared every 2 weeks since the reagent tends to give a higher blank value when stored for more than 2 weeks. The time course of the color development is shown in Fig. 3. For the maximum color development, the reaction mixture should be heated for over 15min and therefore 20-min heating is satisfactory.

mination of glycolic acid with the 2,7-dihydroxynaphthalene reagent. Samples containing various amounts of sodium glycolate in 50 /J! each of water were heated with the reagent at 100°C for 20 min.

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Fig. 3. Time course of color reaction of glycolic acid with the 2,7-dihydroxynaphthalene reagent. Samples containing 10 nmoles each of sodium glycolate in 50 pi of water were heated with 1.0 ml of the reagent at 100°C for different time lengths.

Fig. 4. Effect of water in samples on the color yield in the determination of glycolic' acid with the 2, 7dihydroxynaphthalene reagent. Samples containing 10 nmoles each of sodium glycolate in different volumes of water were heated with 1.0 ml of the reagent at 100°C for 20 min. Correction was made for the difference in volume of the reaction mixtures. The color yield obtained with a dried sample was taken as 100%. / . Biochem.

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Figure 4 shows the effect of water on the color development. The presence of water more than 5% by volume in the reaction mixture significantly interferes with the color development. The volume of water in a sample should not exceed at most 10% of that of the reagent solution. Therefore when the volume of a sample solution is larger than 50 (A, it should be dried before addition of the reagent solution, or otherwise the volume of the reagent solution should be increased 2.000 accordingly. This color reaction is known to depend on condensation of formaldehyde split off from glycolic acid with 2, 7-dihydroxynaphthalene in cone. H2SO4 (2). Therefore formaldehyde and the compounds that liberate glycolic acid LJ 1,000 or formaldehyde under the assay condition may also be determined directly by this method. Indeed, this method was utilized for determination of glycolic acid liberated from r-carboxymethyl ester of L-glutamic acid (5) and a-N-glycolyl-D-arginine (4). DL-Mandelic acid (phenylglycolic acid) was found to be negative 0 50 100 to .this color reaction (3). AMOUNT OF GLYCOLIC ACID (nmoles) Formic, acetic, oxalic, succinic, citric, Fig. 2. A standard curve for colorimetric deter- . benzoic, and salicylic acids are known not to

COLORIMETRIC DETERMINATION OF GLYCOLIC ACID

interfere with the qualitative test. Glycolic acid may be determined quantitatively in the presence of any of these compounds if due corrections are made for the effect of these compounds on the color yield. Care should also be taken of the fact that some aldehydes such as salicylaldehyde, anisaldehyde, or acetaldehyde react with 2, 7-dihydroxynaphthalene similarly to formaldehyde and that acetic acid, malic acid and certain glycols give different colors (2). Since a number of compounds may give color reactions under the assay condition, this method may be of somewhat limited value for quantitative determination of glycolic acid in

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complex biological fluids. It would be most useful for determination of glycolic acid in samples devoid of interfering substances or for examination of purity of glycolic acid or glycolyl esters. REFERENCES 1. E. Eegriwe, Z. Anal. Chem., 89, 121 (1932). 2. F. Feigl, "Spot tests in organic analysis," American Elsevier Publishing Company, Inc., New York, Ed. 7, p. 465 (1966). 3. K. Takahashi, W.H. Stein and S. Moore, / . Biol. Chem., 242, 4682 (1967). 4. T.H. Plummer, Jr., / . Biol. Chem., 246, 2930 (1971).

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Vol. 71, No. 3, 1972

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