- 1) Kjeldahl Method for crude protein Estimation
Kjeldahl Method for crude protein Estimation
Crude protein vs true protein
Crude protein– It is also called Total Protein, i.e, it is the total protein content present in the food sample. It is different from the True protein.
We know that True protein is only the actual protein content in a food sample, whereas, Crude protein also contains the NPN (Nonprotein Nitrogens) which come from the UREA that is used in the field as a fertilizer or it may be added as an adulterant in the food sample like milk.
So, crude protein is the summation of all the sources of Nitrogen in the food sample.
So, for Crude protein calculation, we multiply the total Nitrogen content calculated with conversion factor-like 6.25 or 6.38 to express the results on a protein equivalent basis.
In this short article, we will see how to determine the Crude protein in a food sample by the Kjeldahl method.
The Kjeldahl method is used for the determination of the nitrogen content in organic and inorganic samples.
We can take different samples for determining their nitrogen content, for example- meat samples, drinks or beverages, cereals, feeds, etc.
SAMPLE TAKEN- Potato fried food, also called Aloo Bhujia
- Firstly grind the given sample.
- Pass through 1mm sieve.
- Use filtrate as a SAMPLE source
1. Digestion– In this step, we want the sample to be completely digested, so it is treated with boiling concentrated sulphuric acid (H2SO4). And to speed up the reaction, we add a catalyst. Due to H2SO4, the sample gets completely dissolved and oxidized.
Protein (N) + H2SO4 ————→ (NH4)2SO4 + CO2 + H2O
Since (NH)4+ is acidic in nature, so firstly we have to make the solution neutral by adding excess NaOH.
On reaction, NH4 is converted to NH3, then it is distilled in a distillation flask and finally received on a solution of Boric acid or HCl or H2SO4.
(NH4)2SO4 + 2NaOH ————→ 2NH3 (gas) + Na2SO4 + 2H2O
Now, we have got NH3 gas in the previous step. So, the amount of N in NH3 will be proportional to the crude protein content in the sample.
So, titration of the NH3 solution is done with NaOH solution. The result is %N or %NH3.
The protein content will be equal to the product of %N content × factor.
This was a modern automatic setup for the Kjeldahl apparatus. The info is taken from www.applichem.com/fileadmin/images/Broschueren/Nitrogen_Determination_by Kjeldahl_Method_en.pdf
DETAILED PROCEDURE for LAB METHOD-
1. Digestion step– Put 0.2-0.5g sample in te digestion flask, add 3g of Kjeldahl catalyst with 10ml of concentrated H2SO4. Boil the solution in the flask till it gets clean. Frothing should not be there, if yet frothing occurs, then increase the temperature to 420°C to reduce frothing.
2. Distillation step- Firstly add the 60mL of distilled water into the flask. Then immediately connect the flask with the condenser (see above fig.) which is immersed in a standard acid in a flask. Then add 5-7 drops of miscellaneous indicator. Rotate the flask to mix the content thoroughly and heat until all the NH3 is released.
3. Titration step- Remove the condenser from the flask and titrate with standard acid which is already distilled with 0.1N H2SO4.
HYPOTHETICAL CALCULATION FOR ALOO BHUJIA SAMPLE-
14= Atomic weight of Nitrogen
0.1= Strength of acid used for titration
Blank= Volume of 0.1N acid used for blank
Sample= Volume of 0.1N acid used for sample
6.25= Derivative factor ( varies with different amino acids), Here, for most general foods contain 16% N, so 100/16= 6.25 is the conversion factor for converting %N to %Protein.
So, the final crude content in Aloo Bhujia is 8.05% (hypothetical value, only for understanding the procedure).
ADVANTAGES OF KJELDAHL METHOD-
1. It is an accurate method for the best results.
2. Applicable to all types of foods
3. We can reproduce results, so it has good reproducibility.
DISADVANTAGES OF KJELDAHL METHOD-
1. We can’t get the accurate value of protein, as this method is for the determination of crude protein only which contain both the organic protein and the inorganic protein like ammonium ions
2. We have to use different conversion factors for different foods, as shown in the table above
3. Its very time consuming and tedious
4. Here, we have to use H2SO4 that too at a high temperature
Please comment on what you are feeling…
Also see how to calculate the ASH CONTENT IN A FOOD SAMPLE-