Paragraph 7 PROCEDURE

Transfer 2, 5, 10, 20, 25 and 50 ml aliquot portions of biuret standard solution (4.6) into a series of six 100 ml graduated flasks. Make up the volumes to about 50 ml with water, add one drop of indicator solution (4.7) and neutralise, if necessary, with 0.05 M sulfuric acid (4.2). Add with swirling 20.0 ml of the alkaline tartrate solution (4.4) and then 20.0 ml copper sulfate solution (4.5). Make up to the mark with water, mix and allow to stand at 30 ± 2°C for fifteen minutes.

• At the same time prepare a reagent blank as follows. Place 50 ml water in a 100 ml graduated flask and proceed as described above from ‘… add one drop of indicator solution …'

• Measure the absorbance of each solution at 546 nm against the reagent blank as reference, using cells of suitable path length. Plot the calibration curve, using the absorbances as the ordinates and the corresponding quantities of biuret in milligrams, as the abscissae.

Weigh to the nearest 0.001 g, 10 g of the prepared sample; dissolve in about 150 ml of water in a 250 ml graduated flask and make up to the mark and mix. Filter if necessary.

Note 1:

If the sample for analysis contains more than 0.015 g of ammoniacal nitrogen, dissolve in 50 ml methanol (4.1) in a 250 ml beaker. Reduce by evaporation to a volume of about 25 ml. Transfer quantitatively to a graduated 250 ml flask. Make up to the mark with water. Filter, if necessary, through a dry fluted paper into a dry receiver.

Note 2:

Elimination of the opalescence: if any colloidal substance is present difficulties may arise during filtration. In that case the solution for analysis is prepared as follows:

Dissolve the sample in 150 ml of water, add 2 ml 1 M hydrochloric acid, and filter the solution into a 250 ml graduated flask. Wash the filters with water and make up to volume. Continue the process according to the method described in 7.3.

According to the presumed biuret content, transfer with a pipette 25 or 50 ml from the solution prepared in 7.2, to a 100 ml graduated flask and neutralise if necessary with 0.05 M sulfuric acid or sodium hydroxide solution (4.2 or 4.3) as required, using methyl red indicator (4.7). Add 20.0 ml of the alkaline solution of potassium sodium tartrate (4.4) and 20.0 ml of the copper solution (4.5). Make up to volume, mix thoroughly and leave standing for 15 minutes at 30°C ± 2. Measure the absorbance of the solution as described in 7.1.