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Khedjahl Method Skippy Kidzzzz
Khedjahl Method Skippy Kidzzzz
Theory:
Hypothesis: The quantity of protein in whole wheat bread is greater than
that of white bread.
Materials: whole wheat bread, white bread, reagent blank, distilled water,
NaOH solution, HCl acid, methyl red indicator, potassium sulphate, mercury
(II) oxide, sulphuric acid
Procedure:
Digestion
In this analysis, the digestion process is the most time-consuming step. The
purpose of digestion is to break down the bonds that hold the polypeptides
together, and convert them to simpler chemicals such as water, carbon
dioxide and, definitely, ammonia. In addition, a reagent blank is also
digested, as a measure of correctness of the digestion parameters.
Reactions as these can considerably be sped up by the presence of a catalyst
and by a neutral substance, like potassium sulfate (K 2SO4), which raises the
boiling point of the digesting acid and eventually the temperature of the
reaction.
Catalysts are also used to help in the digestion process; many different one
have been tried including selenium, mercury, copper, or ions of mercury or
copper.
Dry the sample of white bread and grind it into a powdery form.
Weigh 1 gm of the white bread sample and record its weight. Place the
sample into a digestion flask, along with 12-15 ml of concentrated
sulfuric acid (H2SO4). Measure 1g of reagent blank and record it also.
Proceed with reagent blank, using the same procedures to be used for
the white bread sample, unless another step is specified
Bring the digestion flask and mixture to a "rolling boil" (about 370 oC to
400oC) using a Bunsen burner, a tripod stand and clamp stand.
Heat the mixture in the flask until white fumes can be seen, and then
continue the heating for about 60-90 mins.
Separate the nitrogen away from the digestion mixture by distilling the
ammonia (converting it to a volatile gas, by raising the temperature to
boiling point) and then trap the distilled vapors in a special trapping
solution of about 15 ml HCl (hydrochloric acid). For reagent blank,
pipette 1 mL of acid and add approximately 85 mL water.
Remove the trapping flask and rinse the condenser with water for the
purpose of making sure that all the ammonia has been dissolved.
[equation
Titration:
As the ammonia dissolves in the acid trapping solution, it neutralizes
some of the HCl it finds there. What acid is left can then be "back
titrated", that is titrated with a standard, known solution of base (usually
NaOH). In this way the amount of ammonia distilled off from the digestive
solution can be calculated, and hence the amount of nitrogen in the
protein determined.
Put a standard solution of NaOH into the burette, and slowly, slowly
add small amounts of the sodium hydroxide solution to the acid
solution with the dye.
Watch for the point at which the dye turns orange indicating that the
"endpoint" has been reached and that now all the acid has been
neutralized by the base.
Repeat steps 1 -14 using whole wheat bread instead of white bread.
Expected Results:
Mass of bread = 1 g
Back titration of ammonia/acid trapping with NaOH and reagent blank with
NaOH
Reagent Blank
2
3
Cb
Eb
Titration
Ammonia/acid trapping solution
1
2
3
As
Cs
Es
1
Initial
Ab
volume of
NaOH
Final volume Bb
Db
Fb
Bs
Ds
Fs
of NaOH
Volume of
Xb
yb
Zb
Xs
Ys
Zs
titre
The table above is displaying the expected results for the back titration of
ammonia/acid trapping solution, and reagent blank with NaOH.
Assumptions:
One mole of ammonia coming from the digestion mixture (and hence
from the original protein) will neutralize exactly one mole of the acid in
the trapping flask.
Treatment of Results
First, we need to calculate the volume of sodium hydroxide by subtracting the
volume of titre of the ammonia/acid trapping solution from that of reagent
blank.
YB YS = V
Where: YB is the volume from reagent blank, YS is the volume from
ammonia/acid trapping solution, V is the actual volume of NaOH to The next
calculation, therefore, is to find the number of moles of ammonia that have
been produced and then trapped from the sample(s).
g NH3 = moles NH3 x 17.04 g/mol
%P = %N x 5.7
%Protein of white bread = 5.7* %N of white bread
%Protein of whole wheat bread = 5.7* %N of whole wheat bread
Sources of Errors:
If temperature used in the digestion process exceeds 400 degrees
Celsius, amounts of nitrogen in the sample can be lost.
Limitations
This Kjeldahl method does not give a measure of the true protein, since
all nitrogen in food is not in the form of protein.
Precautions