How to analysis nitrogen content & protein analysis in food product
Kjeldhal Method
The
method is applicable for determination of nitrogen content and hence proteins
present in the sample. The method is
based on digestion of test sample in sulphuric acid during which organic
compounds are oxidized to carbon dioxide and water and nitrogen of amino groups
of protein is converted to ammonium sulphate.
Further, in the presence of sodium hydroxide, ammonium sulphate is
broken down to form ammonia, which is distilled along with steam into a conical
flask containing acid. The amount of ammonia
is determined by titration.
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APPARATUS
1. Micro kjeldhal assembly (Parnas-Wagner
Assembly)
2. Kjeldhal flask of volume 100 ml for
digestion of sample
3. Heating mantle (for digestion)
REAGENTS
1. Concentrated Sulphuric acid.
2. 50 % w/v solution of sodium hydroxide
3. 0.02 N HCl solution - dilute 1.7 ml of
hydrochloric acid (11.5 N in strength) to 1000 ml with water.
4. 4 % boric acid solution
5. Catalyst (mixture of 95 g K2SO4
+ 5 g C4SO4)
6. Tashiro Indicator (0.2 g Methyl red
dissolved in 50 ml of 96 % ethyl alcohol and 0.1 g Methylene blue dissolved in
150 ml of 50 % ethyl alcohol. Mix both
solutions and filter into a dark bottle.
7. 1 % Phenolphthalein solution.
PROCEDURE
A. Digestion
1.
Take the prescribed weight of the
sample weighed accurately upto 0.0001 g into the Kjeldahl digestion flask.
2.
Add 2 g of the catalyst and some glass
beads.
3.
Add 20 ml of concentrate H2SO4
to this mixture.
4.
A small funnel is placed on the flask.
5.
A blank is carried out simultaneously
using the catalyst mixture and H2SO4 without the sample.
6.
Carry out the digestion, keeping the
Kjeldahl flask slightly inclined, gently at first till foaming is stopped and
later very intensely.
7.
If the H2SO4 used
for the sample is about to dry up, add more H2SO4,
carefully adding 5 ml at a time.
(Precaution should be taken to cool the flask and then add H2SO4)
8.
When the solution in the flask is clear
and having a light green colour, continue digestion for one hour further.
9.
For savory oats colour of the digested
solution may not be light green colour due to seasoning colour interference.
10.
Cool the solution
11.
Transfer this solution to a 50-ml
volumetric flask. Rinse the flask and funnel thoroughly and make up the volume
to 50 ml with distilled water (digest solution).
B. Distillation
1.
Connect the apparatus as shown in the
diagram.
2.
Boil distilled water in the steam
generator A using a Bunsen burner.
3.
Transfer about 25 ml of water into the
distilling flask G.
4.
Close stopcock E and pinch clamp D as
soon as steam starts to emerge from the steam-trap tubing.
5.
Run cold water through the condenser
from which about 5 ml of distillate should collect per minute.
6.
As soon as the distillate starts to
emerge, remove burner.
7.
The water in flask G is back-sucked
into the steam-trap C.
8.
Repeat the procedure twice more to
ensure that the set-up is clean and ready for use.
9.
Pipette out 5 ml of boric acid solution
into a 100 ml conical flask and add 4 drops of mixed indicator solution
(Tashiro’s indicator solution).
10.
Replace the burner under the steam
generator and open the pinch clamp D to remove liquid from the steam-trap C.
11.
Leave the pinch clamp on the glass
tubing through which the steam escapes.
12.
Place the conical flask containing the
boric acid under the condenser F and support the flask in an oblique position,
so that the tip of the condenser is completely immersed in the liquid.
13.
Open the stopcock E with one hand, and
with the other hand, pipette out 5.0 ml of the digest solution into G.
14.
Add 1 ml of Phenolphthalein through
funnel E
15.
Rinse the funnel twice with 2 - 3 ml
portions of distilled water. Then
introduce 25 ml of 50 % NaOH and close stopcock.
16.
Replace the pinchcock D on the rubber
tubing, whereupon steam enters G, stirs up the digestion mixture and sodium
hydroxide, liberator ammonia, which distills along with steam through the
condenser into the boric acid solution.
17.
The boric acid changes from reddish
purple to bluish green as soon as it comes in contact with ammonia.
18.
Continue distillation for 10 minutes
after the boric acid has changed its colour.
19.
Remove the condenser tip from boric
acid solution and rinse the tip with distilled water.
20.
Stop heating only after removing the
flask as vacuum created will pump out the solution into the steam-trap.
21.
Titrate the boric acid and ammonia
complex with 0.02 N HCl solution till colour changes to reddish purple.
22.
Repeat procedure for blank.
CALCULATION:
Nitrogen
% = [Sample titre-Blank titre] x NHCl
x 14 x vol.of digest (50 ml) x 100
Aliquot of
digest (5 ml) x Wt. of sample x 1000
Protein
% =
Nitrogen % x 6.25
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