METHOD 8:0
Printed with effect from 1st January 2003
AMMONIA AND VOLATILE NITROGENOUS BASES
1:
Scope and Field of Application
This method is to be used for those feeding stuffs for which a declaration of protein
content is required and which are believed to contain ammoniacal nitrogen.
2:
Principle
Ammonia and other nitrogenous bases are liberated from an aqueous extract of the sample
by alkaline hydrolysis, displaced from solution by a stream of air at room temperature and
collected in sulphuric acid solution, excess of which is titrated with sodium hydroxide.
3:
3:1
3:2
3:3
3:4
Reagents
Sulphuric acid solution, O.1N.
Sodium hydroxide solution, O.1N.
Potassium carbonate, saturated solution.
Screened methyl red indicator solution:
(a) dissolve 0.2g methyl red in 100ml of ethanol (95-96% V/V).
(b) dissolve 0.1g methylene blue in 100ml of ethanol (95-96% V/V).
Mix one volume of (a) with one volume of (b).
Octan -1-ol.
3:5
4:
4:1
Apparatus
An example of the recommended apparatus is shown in fig 1. (overleaf). The apparatus is
made up of a specially shaped reaction vessel (A) with a ground-glass neck, a side neck,
connecting tube (C) with a splash head leading into a conical flask (D) and a perpendicular
tube (B) for the introduction of air. Alternatively the tubes can be connected to the
reaction vessel by means of a simple perforated rubber bung. It is important to give a
suitable shape to the end of the tubes introducing air, since the bubbles of gas must be
evenly distributed throughout the solutions contained in the vessel and the absorber. The
best arrangement consists of small mushroom-shaped pieces with an external diameter of
20mm and six openings of 1mm around the periphery.
4:2
Rotary shaker, 35-40 turns per minute.
5:
Procedure
Weigh to the nearest 0.001g, approximately 5g of the prepared sample and transfer to a
250ml graduated flask. Add water to the mark and shake the flask and contents for 10
minutes on the rotary shaker (4.2) to dissolve the soluble salts. Filter the aqueous extract
collecting the filtrate in a clean flask. Transfer by pipette to the dry reaction flask a portion
of the filtrate containing a maximum of 20mg ammoniacal nitrogen. Connect up the
apparatus. Place 50.0ml standard sulphuric acid (3.1) in the 500ml conical flask (D), add a
few drops of indicator and sufficient distilled water so that the level of the liquid is 50mm
above the opening of the delivery tube. Introduce through the side neck of the reaction
vessel distilled water to being the volume up to 50ml; add a few drops of octan-1-ol (3.5)
8:0/1
to prevent foaming during aeration. Add 50ml saturated potassium carbonate solution
(3.3), close the side tube to prevent loss of ammonia. Pass a current of air through the
system (3,000ml per minute). The air should be purified by passing it through washing
flasks containing dilute sulphuric acid and dilute sodium hydroxide. Ammonia should be
completely liberated in 3 hours. Disconnect the conical flask (D) and rinse the delivery
end of the apparatus and the sides of the conical flask (D) with distilled water. Titrate the
excess acid with standard 0.1N sodium hydroxide (3.2). Carry out a blank determination
omitting only the sample.
6:
Expression of the Results
The amount of ammoniacal nitrogen, as a percentage of the sample, is given by:
(a -A) x 0.14
W
where:
a = blank titre, ml;
A= sample titre, ml; and
W= weight of sample extract in grams, present in the aliquot part taken for analysis.
8:0/2
Fig 1
A = Reaction vessel, 350-400ml capacity.
B = Tube for introduction of air.
C = Delivery tube with splash head.
D = Conical Flask, 500ml capacity.
8:0/3