ANN 111: Principles of Animal Nutrition and Feed Technology (2+1)

PROCEDURE

Weighing procedure

• Place the balance on flat surface. Rotate the knob upon which the analytical balance rests to manipulate the mercury movement. Ensure that the mercury drop is within the circle, which indicates that balance is placed at symmetry. view video

Catalyst mixture

• This is used to induce oxidation and also to raise the boiling point without any spurting. This mixture is made up of copper sulphate, potassium (or) sodium, sulphate and selenium. Different proportions are used in practice.
• In this laboratory the catalyst mixture is prepared by mixing 85 parts of CuSO₄ + 14 parts of sodium sulphate and 1 part of selenium.

Preparation of the digest

• Transfer about 2g of feed sample wrapped in a butter paper into a clean dry Kjeldahl flask. view video
• Carefully add through the sides about 20ml of concentrated H₂SO_4. Gently rotate the flask to mix well the sample and the acid. view video
• Add a pinch of catalyst to the content of the flask and then place it in electric digestion unit. The content of the flask is black due to charring of the material by the concentrated acid. view video
• The sample is then digested by the electric heater. At the beginning, allow the digestion to proceed gradually on a low temperature. There will be frothing and fuming in the beginning. Carefully regulate the temperature to avoid frothing and excess fuming.
• The content will be free from blackness, when the initial digestion is over. Now raise the temperature and continue the digestion till the content is either greenish yellow or colourless. There may be some sediment of catalyst and insoluble residue, which may be ignored. view video
• Allow the flask and its contents to cool to room temperature.

Making up the volume

• Transfer the contents of the digest carefully to the volumetric flask (100ml) fitted with a funnel. Take care not to allow any drop of digest outside the flask or spill on the table. view video
• Place cold water or ice cubes around the volumetric flask. This is to cool the volumetric flask when intense heat will be produced during the digest is transferred  view video
• After the transfer of the initial digest, add 5ml of distilled water to the flask. Rotate the flask gently so that the sides may be washed by distilled water. Transfer the washing to the volumetric flask. Repeat the process of washing four or five times. The technique of washing lies in taking small quantity of distilled water but repeating the washing more number of times. At the same time take care you do not fill up to the mark. Complete the washings and fill up the flask far below the mark. view video
• After complete washing allow the volumetric flask to cool to room temperature. Remove the volumetric flask from the ice and place it on the table. Make up the volume to the mark. Then label it and keep it for micro distillation.view video

Distillation

• The distillation of NH₃ from the digest is the next step in this procedure.
• The distillation is done in an apparatus called Micro-Kjeldahl distillation unit. This is an all-glass unit, consisting of steam generating unit, boiling casket and condensing unit. view video
• Place at the distilling end, a beaker (100ml) containing 10ml of 0.1N H₂SO₄ with a drop of methyl red. view video
• Check the apparatus for any leakage. The hot plate or burner is so adjusted that the water boils in the flask and steam is produced. view video 
• Shake and mix the volumetric flask well before making use of the digest for distillation. view video
• Transfer 5ml of the digest through the funnel to the boiling casket. view video
• Now add some distilled water and a drop or two of phenolphthalein. view video
• Add 15 to 20ml of 40% NaOH by measuring cylinder. The colour will be pink indicating the excess of alkali. view video
• Now check all the connections.
• Intensify the steam generation. The steam will pass into the boiling casket and the contents (5ml digest + excess alkali) will boil. This releases the ammonia from the digest, which leaves through the condensing unit and distilled into standard acid kept in the beaker. Generally steam distillation for five minutes will do to release all the bound ammonia. view video
• The volume of the contents in the beaker will increase due to addition of distilling NH₃. view video
• When the volume is nearly doubled, stop the distillation. Before cutting off the steam, wash the tip of the distilling end with distilled water and remove the beaker. (Place a beaker with distilled water and cut off the steam. All the water from the beaker and the content of the boiling casket will be sucked back and cleared through evacuating tube. Repeat this process two or three times to wash the unit before commencing next distillation. view video

Titration

• Keep ready, a burette filled with 0.1N NaOH. view video
• Boil the beaker containing the H₂SO₄ and titrate hot, against standard alkali.
• When the alkali and acid are of the same strength, the titre value gives the amount of acid neutralised by alkali. view video
• If this quantity is subtracted from the total quantity (10ml) of acid, we get the amount of standard acid neutralised by NH₃. view video
• Equivalent: 1 ml of 0.1N NaOH = 1 ml of 0.1N H₂SO₄ = 0.0014 g Nitrogen

Calculation

Here,

• V = Volume of 0.1N H₂SO₄ neutralized by NH_3,
• W = Weight of the sample,
• Z = Volume of aliquot taken for distillation.

Precaution

• Glass beads should be added to avoid bumping during digestion .
• Incomplete digestion should be avoided.
• Set the conical flask containing N/10 H₂SO₄ before adding 40% NaOH.
• The tip of the condenser should be dipped in acid to avoid any loss of ammonia.

Note

• Copper sulphate is added as catalyst to speed up the reaction.
• A blank may also be run using all reagents (without the sample) to make correction for nitrogen present in the reagents used.

Semi automatic equipment for nitrogen analysis

• In Semi automation of Kjeldhal analysis, few steps are automated for easy and speedy analysis. In this procedure instead of using Kjeldhal flask, large glass tubes are used for digesting sample, which are then distilled. Thus the dilution procedure is eliminated and hence saves time.
• Semi automation of Kjeldhal analysis involves five steps instead of six steps. They are: sample and reagent addition, sample digestion, NaOH addition, Steam distillation and titration. Sample is loaded in tubes specially designed for the instrument and reagents are added to the sample in the tube. Samples are then digested in block digester.
• The digested sample is then transferred to the distillation unit in the same tube.
• The tube is fixed in the distillation unit where NaOH is dispensed. Distillation process like alkali addition, steam generation, condensation, etc., are automated and thus saves time.

Automatic equipment for nitrogen analysis

• It is similar to Semiautomatic equipment for nitrogen analysis, but once the tube is fitted for distillation, the instrument takes care to automatically dispense alkali, distill ammonia and transfer the receiver with distilled ammonia to the next chamber for automated titration. Thus once the tube is loaded, the user has to just note down the titer value with rest of the procedures automated.
• Currently an electronic balance and the automated nitrogen analyser are attached to computer, thus the weight of the sample and the corresponding titer value are registered in the computer, which converts the titer value to nitrogen/protein content in the sample.