Dinitro salicylic acid (DNSA) method

Protocol

1. Dinitro salicylic acid (DNSA) method

2. Aim: To estimate the amount of carbohydrate present in the given sample (maltose) by DNSA method. Principle: In alkaline solution, reducing sugars form enediols. The enediols are readily oxidized to its respective sugar acids by the oxidizing agent 3, 5 dinitro salicylic acid to form orange red color complex. The absorbance is measured at 540 nm.

3. Reagents: Working standard: 200 mg maltose in 100ml water (Conc. 2 mg/ml). 2 N NaOH: 4 g in 50 ml distilled water. DNSA reagent: Solution A: Dissolve 3 g of Rochelle's salt (sodium potassium tartrate) in 50 ml distilled water Solution B: Dissolve 1 g of 3, 5-dinitrosalicylic acid in 20 ml 2N NaOH Mix solution A and B and make up to 100 ml with distilled water.

4. Procedure: Aliquots of standards were taken in a series of test tubes (0.2 to 1 ml). 1 ml of sample is taken as test. All the tubes were made up to 2 ml with water. A blank was also set. 1 ml of DNSA reagent is added to all tubes and kept in boiling water bath (90˚ C) for not more than 1.5 min. The tubes were cooled and made up to 20 ml with water. The color developed was read at 540 nm.

5. Particulars B S1 S2 S3 S4 S5 Test Working standard (in ml) - 0.2 0.4 0.6 0.8 1.0 1.0 Concentration in std (in μg ) - 400 800 1200 1600 2000 - Water (in ml) 2 1.8 1.6 1.4 1.2 1.0 1.0 DNSA reagent (ml) 1 1 1 1 1 1 1 Boiling water bath At 90˚ C for 1.5 min. Water (ml) 17 17 17 17 17 17 17 OD read at 540 nm