Title: Determination of Protein in Bread by Back Titration

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

Apparatus: Kjeldahl flask, electronic scale, Bunsen burner, tripod stand, clamp
stand, burette, retort stand, conical flask, receiving flask, condenser, glass tube,
rubber tube, measuring cylinder, distillation, flask

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.
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

Add seven grams of potassium sulfate and a catalyst, mercury (II) oxide.

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.

Cool the flask to room temperature by placing it in some distilled water.

Sample + H2SO4 (NH4)2SO4(aq) + CO2(g) + SO2(g) + H2O(g)
1]

[equation

Distillation
The purpose of the distillation step is to separate the ammonia (that is, the
nitrogen) from the digestion mixture.

Raise the pH of the mixture using sodium hydroxide (45% NaOH solution).
This has the effect of changing the ammonium (NH 4+) ions (which are
dissolved in the liquid) to ammonia (NH3), which is a gas.

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.

(NH4)2SO4(aq) + 2NaOH Na2SO4(aq) + 2H2O(l) + 2NH3(g)

[equation 2]

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.

Add methyl red indicator to the acid/ammonia trapping solution. This dye
should turn a strong color, indicating that a significant amount of the
original trapping acid is still present.

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.

Record the volume of the neutralizing base (sodium hydroxide solution)

that was necessary to reach the endpoint.

Perform a calculation to find the amount of ammonia, and thus nitrogen

that came from the original sample.

Titrate reagent blank similarly.

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
of NaOH
Volume of
Xb
yb
Zb
Xs
Ys
titre
The table above is displaying the expected results for the back titration of
ammonia/acid trapping solution, and reagent blank with NaOH.

Fs
Zs

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.

All the nitrogen present in the sample is in the form of ammonia.

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

to calculate the mass of N in the ammonia, divide atomic mass of N by the

molar mass of ammonia. Whatever value is obtained, multiply it by the
mass of ammonia in the sample.
g nitrogen = (14.01/17.04 )*(gNH3)
= 0.822*gNH3

The percentage of nitrogen found in the original sample can now be

calculated by:
%nitrogen = (g nitrogen / g sample) x 100
%N = (g nitrogen / 1g) x 100

To calculate the amount of protein in the sample, multiply the percent

Nitrogen by a factor 5.7.

%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.

When transferring solutions from one container to another for example,

from the Kjeldahls flask to distillation apparatus, rinse container three
times with distilled water to ensure all contents are transferred.

Misreading the volume - at any moment, and due to whatever reason. This
can be for example a parallax problem (when someone reads the volume
looking at an angle), or error in counting unmarked graduation marks.

When reading the volume on the burette scale it is not uncommon to read
both upper and lower value in different lighting conditions, which can
make a difference. Therefore it should be ensured that the volume is
rightfully read at all times.

Using solutions of wrong concentration - titrant we use may have different

concentration than expected. This can be due to incorrect standardization,
contamination of the bottle content, titrant decomposition, and solution
being kept in open bottle and partially evaporated and so on. These errors
can be prevented or minimized, if the experiment is cautiously carried out.

Using wrong amount of indicator this can shift end-point. Add no more
than five drops of the methyl red indicator.

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.

The use of concentrated sulfuric acid at high temperatures poses a

considerable hazard, as does the use of some of the possible catalysts.

Precautions

All acids and bases must be handled carefully.

Titrations must be done cautiously in order to not miss the end-point of

the reaction.

Personal protective equipment must be worn during the execution of this

experiment.