Naphthoresorcinol test

The method most commonly used with biological fluids is based on the naphthoresorcinol test it is sufficiently important to warrant some discussion. Introduced by C. Tollens29 in 1909 as a means for determining urinary conjugates of D-glucuronic acid, it has been subjected to numer-... 

There was now fairly good agreement between the various laboratories regarding the qualitative composition of heparin. Jorpes and Bergstrom offered an explanation for the failure by Charles and Scott to obtain a positive naphthoresorcinol test for uronic acid when they showed that highly active heparin is resistant to depolymerization by acid. 

Enzymic degradation experiments on the polysaccharide were unsuccessful, Hydrolysis was carried out with 2.5% sulfuric acid, and in the hydrolyzate, D-mannose was identified as the phenylhydrazone, D-arabin-ose as the benzylphenylhydrazone and D-galactose as the methylphenyl-hydrazone.,. The acidic components gave a negative naphthoresorcinol test for hexbronic acids, though indications of the presence of glycolic acid were claimed. 

If the plant compound gives the naphthoresorcinol test for a uronic acid and on hydrolysis with a hot dilute solution of a strong acid gives simple sugars and aldobionic or free uronic acids, it is a polyuronide. " ... 

A test for glucuronides in urine, based on their reaction with naphthoresorcinol to give a red colour. 

Procedure. Urine is diluted so as to contain not more than 0.04 mg./ml. of glucuronic acid. Diluted urine (2 ml.), hydrochloric acid (4 ml.), and naphthoresorcinol (2 ml) in a test tube (13 x 150 mm.) are heated in boiling water for 2 hours. Evaporation is reduced by a "cold finger condenser placed in the top of the tube. The tube and contents are then cooled in cold water. n-Butanol (5 ml.) is added, the tube is corked, and is shaken well and left to stand for 3 minutes for air bubbles to disperse. The absorption value is then read within 5 minutes in a photoelectric absorptiometer using an Ilford Spectrum Orange 607 filter. The calibration curve can be constructed using 0.2 to 2 ml. of glucurone solution made up to 2 ml. with water. 

Dehydration is carried out with sulfuric acid, hydrochloric acid, or phosphoric acid. Among phenols, phenol, thymol, guaiacol, resorcinol, orcinol, catechol, phloroglucinol, a-naphthol, and naphthoresorcinol have been proposed. Among amines, aniline, p-anisidine, benzidine, 3,3 -di-methoxybenzidine, diphenylamine, indole and its derivatives, carbazole, N-ethylcarbazole, a,a-dinaphthylamine, and 1,2,7,8-dibenzocarbazole have been found valuable. In its classical form (Molisch reaction) the test for saccharides is carried out With 1-naphthol and cone, sulfuric acid (70). It is supposed that under the conditions of the reaction corresponding triphenylmethane dyes are formed, although the course of the reaction might be more complicated (71). 

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