Titrimetric determination of ascorbic acid using chloranil
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Updated: Oct 20, 2026

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
Abstract:
Ascorbic acid is oxidized and quantitatively titrated with chloranil (2,3,5,6-tetrachloro-1,4-benzoquinone) in the presence of hexamethylenetetramine in acetone-water; the end point is determined visually by the appearance of a golden yellow color. Colored solutions are assayed by setting the initial absorbance at 451 nm to zero or the minimum, titrating with chloranil solution, and measuring absorbance after each increment of titrant. A plot of the volume of chloranil added against the absorbance gives a straight line with the volume intercept as the end point. Interference by the thiol group of cysteine, glutathione, etc., is avoided by masking with acrylamide; interference by iron(II) is masked with ammonium thiocyanate and sodium potassium tartrate. Hydrogen sulfite and thiourea (which do not interfere) are added as antioxidants during extraction of ascorbic acid from drugs and fruits.
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