Protein by Kjeldahl Method

DETERMINATION OF CRUDE PROTEIN (By Kjeldahl Method)
OBJECTIVE:
This method determines total nitrogen content and protein as 6.25 times the nitrogen content
of the RM & feed samples.
Crude protein is ascertained by multiplying the percentage of nitrogen with 6.25.
PRINCIPLE:
The organic matters in the feed samples are digested with concentrated sulphuric acid and in
the presence of catalyst. The free ammonia forms a compound ammonium sulfate with
sulphuric acid. This compound is treated with excess alkali and distilled. Free ammonia is
liberated which is received in known amount of standard acid. After distillation, by back
titration standard acid with standard alkali the amount acid reacted with free ammonia is
found. From this the nitrogen content of the feed is calculated and the crude protein is found
out by conversion factor.
2NH3 + H2SO4 (NH4)2SO.
(NH4)2SO4 + 2 NaOH 2 NH3 + Na2SO4 + 2H2O
Crude protein is ascertained by multiplying the percentage of nitrogen with 6.25.
SCOPE:
Applicable to testing of the RM, animals feeds & related materials.
REFERENCE: AOAC 954.01-1990
BIS 7874 PART-1-1975
AOAC 976.05-2005
IS/ISO/5983-2-2005
AOAC 979.09-2005
GAFTA Method 4:0
MATERIALS REQUIRED:
 Weighing balance (0.0001g)

 Non protein paper/butter sheet
 Spatula
 Digestion and Distillation unit.
 Auto burette-50 cc
 Pipette volume-25ml.
 Conical flask-250/100 ml.
 Kjeldahl tube -500 ml.
 Measuring cylinder - 25ml.
 Volumetric flask-100/1000 ml.
 Beaker-2000/25 ml.
 Reagent bottle - 1000 ml.
 Indicator bottle-150 ml.
 Hydrochloric acid 0.1 N
 Catalyst mixture. (Potassium sulphate: copper sulphate) or Sodium
 sulfate: copper sulphate)
 Sodium Hydroxide solution (40%)
 Hydrochloric solution 0.1 N.
 Conc. Sulphuric acid
 Boric acid Solution (2%)
 Potassium hydrogen phthalate (KHC8H3O4)
 Mixed indicator (methyl red/ bromocresol green Indicator)
By Boric Acid & Hydrochloric acid method:
DIGESTION:
 Take 1 gm of sample (for low protein;CP <30 %) & 0.5 gm sample (for high protein; CP
<30 %)) {Before 2 gram sample was taken} weight in nitrogen free sheet/butter sheet &
pack it properly.
 Then pour it in digestion tube. {As the digestion system is done in old way so the catalyst
is 1:10 otherwise for rapid Kjeldahl test we use ratio 35:4}
 Put 10 gm of catalyst (copper sulfate: potassium sulphate 1:10 ratio) in digestion tube.
And add 20 ml. of conc. sulphuric acid (before 25 ml used) to it.
 Then put the digestion flask in digestion unit deep green colours appear. (Digestion time
differs so we need to check color to finalize whether digestion is over or need to
continue heating)
 Remove the digestion flask & allow cooling for 30 minutes.
 Add 100 ml distilled H₂O (auto unit) & 250 ml distilled H₂O (manual unit) in digestion
flask. Same way goes for a blank without sample. Do not dilute the digested sample
otherwise the data may vary which was being done before.
DISTILLATION:
 Then take 40 ml. of boric acid (2%) solution in a 250 ml. conical flask.
 Add 4-5 drops of mix indicator & keep it in receiver point.
 Then add 80 ml. of sodium hydroxide (40%) in digestion flask & set immediately in
distillation unit.
 Start the distillation process & collect the distillate approximately 50ml.
 Then titration with 0.1N Hydrochloric acid solution.
 Color change from green to pink.
Calculation:
Nitrogen % = (TV-Blank) x N. of HCl x 6.25 x 14.007 X 100
Sample wt. x 1000
Protein %= Nitrogen % x 6.25
PREPARATION OF SOLUTION
Boric Acid 2%:
 Take 20 gm. of boric acid in a 2000 ml beaker.
 Pour 1000 ml. distilled water & stir well till mix proper.
Mix indicator:
 Take 0.1 gm methyl red & 0.1 gm. bromocresol green in a 100 ml. reagent bottle.
 Add 100 ml. of ethyl alcohol & mix well.
Sodium Hydroxide 40%:
 Take 400 gm of sodium hydroxide pellet in a 2000 ml beaker.
 Add 1000 ml. of distill water swirly & mix well.
 Don't stand the solution without mixing.
Hydrochloric acid (0.1 N)
 Take 500 ml. of distilled water in 1000 ml vol. flask.
 Add 9 ml. of conc, hydrochloric acid in it & mix well.
 Then add distilled water fill to mark.
 Cool & stay for overnight.
STANDARDIZATION
Hydrochloric acid (0.1 N)
 Take 0.4 gm of pre dried sodium carbonate (Na2CO3) in to a 100 ml. conical flask.
 Add 25 ml of distilled water in it & dissolved properly.
 Add 3-4 drops of mix indicator (methyl red & bromocresol green) & mix well.
 Titrate with 0.1N hydrochloric acid solution until pink color is occurs.
Calculations:
N of HCL = Wt. of sodium carbonate x 18.868
Titer value
N of HCI = Wt. of Sodium carbonate X 1000
Titer value X 53

Protein by Kjeldahl Method

Uploaded by

Copyright:

Available Formats

Protein by Kjeldahl Method

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Protein by Kjeldahl Method

Uploaded by

Copyright:

Available Formats

DETERMINATION OF CRUDE PROTEIN (By Kjeldahl Method)

 Weighing balance (0.0001g)

By Boric Acid & Hydrochloric acid method:

You might also like