How to Determine Crude Protein in Kjeldahl Method

There is a very important test in the Feed Mill Quality Control Laboratory – Crude Protein Test or CP Test. So let’s first find out-

What is Crude Protein?

Basically crude protein is in a word crude protein. It is also called total protein. In general, crude protein is all nitrogen content that comes from protein and non-protein nitrogenous substances (NPN means nitrogen sources such as urea, bi-urates, ammonia, etc., which are not essential proteins). Crude protein tests do not actually extract protein directly; Nitrogen content is extracted and this nitrogen content is calculated in crude protein using formulas. True-protein, on the other hand, refers to all nitrogen content from protein sources (NPN means urea, bi-urets, ammonia, etc., except for nitrogen sources). This means that if you subtract the non-protein nitrogenous substance (NPN) from the crude protein, what you have is the true protein. From this analysis it can be said that, “All True Proteins are Crude Protein; But not all crude proteins are true protein! ” Let’s go to the real test without exaggerating.

Lab Test – Crude Protein

Necessary equipment and chemicals

  • Samples
  • Blender machine
  • Analytical balance
  • Geldal flask
  • Digestion and distillation apparatus (protein machine)
  • Conical flakes
  • Pipette
  • Titration apparatus (burette and stand)


  • Catalyst (Copper Sulphate and Potassium Sulphate) – 6 g (1 g Copper Sulphate + 5 g Potassium Sulphate)
  • Sulfuric acid – 15 ml
  • Sodium hydroxide solution (40%)
  • Hydrochloric acid solution (0.1 Normal)
  • Boric acid solution

Preparation of solution

  • Preparation of 40% Sodium Hydroxide solution: Mix 40 g of Sodium Hydroxide well in 1 liter of Distilled Water to make 40% Sodium Hydroxide solution.
  • Boric acid solution: Add 40 g of boric acid in 1 liter of distilled water and mix well. Once the boric acid is mixed, add 10 ml 0.1% Methyl Red solution and 6 ml 0.1% Bromo Crystal Green solution and mix well to make a solution.
  • Preparation of 0.1 N hydrochloric acid solution: Mix 0.3 N 36% hydrochloric acid solution well in 1 liter distilled water to make 0.1 N hydrochloric acid solution. And take out the standard value of the prepared solution.
Standard Determination Method of 0.1 N Hydrochloric Acid Solution:
  • First weigh 0.530 grams of sodium carbonate in a 250 ml beaker.
  • Then add 100 ml of distilled water to the mixture and mix well.
  • From that solution, take a pipette of 10 ml solution to another conical flax and add 30 ml of distilled water to it.
  • Now add 3-4 drops of Methyl Red solution to this solution and the solution will turn yellow.
  • This now available yellow color solution needs to be titrated with the 0.1N hydrochloric acid solution that will determine the standard value of the previously prepared 0.1N hydrochloric acid solution.
  • The standard value of 0.1N hydrochloric acid solution can be obtained by dividing the number 1 by the titration value obtained (Burette reading-BR).
  • Formula 1 / BR = HCl Standard value.

Crude Protein Test Method:

  1. The first sample to be tested is to be grinded in a blender machine and measured with an analytical balance of 0.5 g.
  2. Along with this 6 gm indicator / catalyst (1 g copper sulphate + 5 g potassium sulphate) should be taken. These should be mixed with 15 ml sulfuric acid in a geldal flask and digested in a digest room heater for 2 – 2.5 hours. At this time the color of the solution will turn green.
  3. Then cool for 30 minutes. Then 100 ml of distilled water should be mixed in the geldal flask with the digested sample and the mixture should be heated again. And leave it for another 30 minutes to cool.
  4. When cool, mix well with 75 ml sodium hydroxide solution and set on distiller point heater.
  5. At the other end of the distillation point, set the mouth with 25 ml boric acid solution in a conical flake and close the mouth. And here you have to hit for 2 – 2.5 hours.
  6. The machine should be turned off when the solution of conical flask is close to 100-150 ml. At this time the color of the conical flask solution will change from pink to green.
  7. Leave it for 10 minutes after distillation.
  8. The conical flakes then need to be titrated by hydratechloric acid in the burette. And crude protein is extracted using the formula-
  9. Calculation: CP% = (Burate Reading X 1.4 X HCl Standard X 6.25) / Sample wt.

 (In the next article I will discuss on ” How to do Crude Fiber Test or CF  Test in quality control lab”)

Dr. Srabon Hasan Sajal


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