Determination of Nitrogen
Prepare chemical catalyst
- Salt mixture : K2SO4 : CuSO4. 5H2O
Take 930 g of K2SO4 and mix well with 70g CuSO4.5H2O
- Sulphuric acid for digestion : Concentrated, industrial grade is OK (and is cheaper!!)
- Sodium hydroxide : 40% NaOH in tap water = approximately 10 N
Dissolve 2 kg NaOH in tap water and make up to 5 litres
- Mix indicator : Methylene blue and Methyl red
Dissolve 0.12g Methylene blue in 100 ml Ethanol
Dissolve 0.270g Methyl red in 200 ml Ethanol
Mix the two!
*Boric acid + indicator
2% boric acid in water + mix indicator
Dissolve 100 g of boric acid in water and make up to 5 litres. Add 25 ml of mixed indicator.
* Sulphuric acid (analytical grade) for titration 0.1 N
Prepare a 1.00 N (0.5 M) H2SO4 from "ampoule" if possible.
Dilute it 10 times. Take 100 ml and make up to 1000 ml
Principles
Procedure
Digestion
Weigh accurately the sample to be analysed and put it in a kjeldahl flask (about 1 g for feeds with expected 20% protein in DM, and more or less of the sample according to expected protein content). Add 3 g of the salt mixture and add 10 ml concentrated sulphuric acid (industrial grade) and then put the flask on the heater and boil until the colour of the liquid has turned to green.
Allow the flask to cool down for at least 30 min
Add 250 ml of distilled water
Distillation
Put 50 ml of boric acid + indicator solution in a 250 - 300 ml E-flask (Erlenmeyer flask) and place it below the condenser with the receiver tube dipped in the acid
Pour carefully 90 - 100 ml of NaOH (40% w:v) in the flask
Connect the flask to the distillation system. This has to be done as quickly as possible to avoid loss of nitrogen
When an additional 100 ml distillate have collected in the E-flask (200 ml in total), lower the E-flask so the received tube is not in the acid and continue for 5 more minutes so the last distillate can come down into the E-flask
Titration and calculation
Titrate with 0.1 N H2SO4 until the colour goes from greenish yellow to pink. Correct the titration with the blank (no sample - only water) value (Do the digestion and distillation using all the chemicals but without the sample).
Two blanks should be run every time a new preparation of boric acid is used and also when you start using a new bulk quantity of any of the chemicals.
Calculation
First calculate the N content
% N = (V-B)*14/1000*0.1/S*100
(B= blank titration; V is sample titration; S is weight of sample in g)
1 g of N = 6.25 g "crude" protein
Therefore % crude protein in sample = N * 6.25
* 0.1 is the strength (normality) of the sulphuric acid used for titration
* 14 is equivalent weight of nitrogen
* 6.25 is factor to transfer from nitrogen to protein on most feed samples
* 1000 is 1000 ml N H2SO4 solution = Equivalent weight of H2SO4
* 100 = To get to %
Note : Always use protective clothing like laboratory coats, gloves and spectacles when working with strong acids and alkalis.