In the classical test (48 hour), The benzoic acid produced was detected using ferric chloride

indicator, by its precipitation in an excess of ferric chloride. Gas Liquid Chromatography

(GLC) method for detection of benzoic acid is also available nowadays.

Hippurate hydrolysis test-Left (positive tube), Right (Negative tube)

In the rapid test (2 hour) Ninhydrin is used as the indicator to detect Glycine, the second
byproduct of hippurate hydrolysis. Ninhydrin reacts with glycine to form a deep blue or
purple color (Ruhemann’s purple). This rapid test used in most laboratories is one of several
variations of the ninhydrin tube test described by Hwang and Ederer for detection of glycine.
Principle:
Hippuric acid is hydrolyzed to benzoic acid and glycine by the enzymatic action of
hippuricase. The glycine end product is detected by the addition of ninhydrin reagent.