Paragraph 6 PROCEDURE

Weigh to the nearest 0.001 g, 2 g of the prepared sample, or a suitable amount expected to contain between 50 and 500 mg of urea and transfer it to a 500 ml graduated flask. Add 150 ml 0.02 M hydrochloric acid solution (3.4), shake for 30 minutes using a mechanical shaker (4.1) then add 10 ml sodium acetate solution (3.5) and mix well. Add 2 g activated charcoal (3.1) to the flask, shake well and allow to stand for a further 15 minutes. Add 5 ml Carrez solution I (3.2), followed by 5 ml Carrez solution II (3.3), mixing well between additions. Dilute to volume with water and mix well. Filter a portion of the solution through a dry filter paper into a clean dry 250 ml beaker.

Transfer 10 ml of the filtrate (6.1) to a 50 ml graduated flask, add 10 ml 4-DMAB solution (3.6), dilute to 50 ml with water, mix well and allow to stand for 10 minutes. Measure the absorbance of the solution at 435 nm, in a 10 mm cell against a reference solution prepared by diluting 10 ml 4-DMAB solution (3.6) to 50 ml with water.

Transfer amounts of standard urea solution (3.7) corresponding to 50, 100, 150 and 250 mg of urea into a series of 250 ml graduated flask; add 75 ml 0.02 M hydrochloric acid solution (3.4) and proceed as described above (6.1) commencing at “. . . shake for 30 minutes . . .”. Measure the absorbance of the solutions and construct a calibration graph relating the absorbances to the amounts of urea present.