Determination of glycolic acid by the Eegriwe (Calkins) method

ANALYTICAL

BIOCHEMISTRY

83,

785-787

(1977)

Determination of Glycolic Acid by the Eegriwe (Calkins) Method Interference

by Ethylenediaminetetraacetic

Acid and Formaldehyde

Glycolic acid, an intermediate in the photorespiratory carbon oxidation cycle, is frequently determined by the procedure described first by Eegriwe (1) and later by Calkins (2). In this method, solutions of glycolic acid are heated with 2,7-dihydroxynaphthalene in concentrated H,SO,. A violet color (h,,, 535 nm) develops, the absorbance of which is proportional to the quantity of glycolic acid originally present. The nature of the violet reaction product is unknown, but the overall reaction is thought to involve the following sequence (1).

Consistent with this scheme are the observations that (a) the concentration dependency for color development is the same for glycolic acid and formaldehyde (Fig. 1); and (b) the product formed from glycolic acid has a visible absorption spectrum very similar to that formed from formaldehyde (Fig. 2). Thus, it appears that compounds which form formaldehyde in the presence of concentrated H,SO, will also form the violet reaction product with 2,7-dihydroxynaphthalene. During a study of glycolate synthesis by photosynthesizing cells (Chromatium and Chlorella) unacceptably high absorbance values were obtained for time zero controls. Investigation revealed that EDTA, included in the cell suspension medium, was the reagent responsible for the violet color. As Fig. 2 indicates, the absorption maximum (X,,, 535 nm) of the product formed from EDTA was identical to that formed from either formaldehyde or glycolic acid. The absorption sepctra are not quite identical, but the small differences are of little practical value. Treatment of the EDTA with Dowex 1 anion-exchange resin (formate form) showed that no color-positive material was eluted either in the wash through or with 2% (v/v) formic acid, conditions which would have eluted 78.5 Copyright 0 1977 by Academic Ress. Inc. All rights of reproduction in any form reserved.

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I 1.0

0.50

0

~molet

FIG. 1. The concentration dependency of color formation from glycolic acid or formaldehyde (I ‘I) and EDTA (0 0). To the quantities of reagent indicated in 0.2 ml of 2 N H,SO, was added 1.Omt of0.01% (w/v) 2,5-d~hydroxynaphth~ene in concentrated H&‘O,. After mixing with a Vortex mixer, the solutions were heated at 100°C for 20 min. After cooIing, 3 ml of2 N HzS04 were added, and the solutions were again mixed. The absorbance at 535 nm (A J.?J“In1 cm) was then determined.

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6 0.4 T;c

0.2

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600 Alnml

FIG. 2. The absorption spectra of the products formed from the reaction of (a) EDTA, (b) glycotate, and(c) form~dehyde with 2,S~ihydroxynaphth~ene in concentrated H,SO, by the procedure outlined in the legend to Fig. 1. The spectra labeled (a) and (c) have been offset by 0.10 and -0.10 absorbance units, respectively, for clarity.

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formaldehyde and glycolic acid, respectively. Thus, the color reaction obtained with EDTA is not due to contamination of the EDTA by either formaldehyde or glycolic acid. Caution is therefore required in determining glycolate by this procedure, especially if formaldehyde or EDTA are present. Purification of putative glycolate by ion-exchange chromatography would alleviate the problem caused by the presence of either formaldehyde or EDTA. REFERENCES 1. Eegriwe, 2. Calkins,

E. (1932) 2. Anal. V. P. (1943) Anal.

Chem. Chem.

89, 121- 125. 15, 762-763.

G. H. Department of Environmental Biology Research School of Biological Sciences The Australian National University P.O. Box 475, Canberra City, A.C.T., 2601, Australia Received May 16, 1977; accepted July 26, 1977

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