Vietnam National University -International University

School of Biotechnology -Department of Food Technology

Food Analysis
Lab report
Instructor: Assoc. Prof. Pham Van Hung
Teaching Assistant: BEng. Tran Thi Yen Nhi

Group 1:
Phm Gia Huy - BTFTIU14132
Nguyn Th Ho - BTBTIU13068
Trn Th Phng ng - BTBTIU14040
 Session 4:
DETERMINATION OF PROTEIN CONTENT
QUESTIONS
1. Calculate the percent nitrogen and the percent protein (wet weight basis (wwb) and
dry weight basis (dwb).
Assume moisture content of 10%. Use 6.25 for the nitrogen to protein conversion
factor.

Tube Sample Volume (mL)

1 Blank 0.500
2 Glutinous rice flour 8.70
Table 4.1 The volume of titrated 2 4 of sample and blank.
On wet weight basis:
() 14
%N = Normality 2 4 100%

0.1 (8.700.500) 14
= 1000 100% 1.16%
0.99

% Protein = % N protein factor = 1.16%6.25 7.25%

On dry weight basis:
() 14
%N = Normality 2 4 100%

() 14
= Normality 2 4 100%
(1)

0.1 (8.700.500) 14
= 1000 0.99(110%) 100% 1.29%

% Protein = % N protein factor = 1.29%6.25 8.06%

 Comparison with food label:
5.95
Protein content in Glutinous rice flour: = 5.95%
100

Experiment result: %Protein (wb) 7.25%

o Discussion: Protein contents of glutinous rice flour sample in food label is lower than
our results. The large error can be explained because of using protein conversion
1
factor 6.25 (= 16% with 16% is the average value of nitrogen content in protein)
Percentage of protein is calculated by using the formula:
% Protein = % N protein factor
However, nitrogen in foods is not only found in protein but also contained in other
compounds, such as free amino acids, nucleotides, creatine and choline (non-protein
nitrogen). Moreover, the nitrogen content of specific amino acids also depends on the
molecular weight of the amino acid and the number of nitrogen atoms it contains.
Therefore, each protein has a different conversion factor depending on its amino acid
composition.
Other factors are proposed to be the reasons for this significant deviation:
- Titration skill.
- Tashiro indicator has a transition range. We need to titrate until having the same
color to color of solution in initial receiving flask which was not easy to be
realized.

2. Could phenolphthalein be used as an indicator in the Kjeldahl titration? Why or why

not?
Phenolphthalein has a transition range (the range of changing the color) from 8.2 -10. If
pH < 8.0, it will change into colourless. If pH > 10, it will change into pink color.
Kjeldahl titration method consists of three main steps: digestion, neutralization and
distillation, titration.
After neutralization and distillation step, only NH4+ and H2BO3- are in receiving flask:
(NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O
NH3 + H3BO3 NH4+ + H2BO3-
pH of the solution at that time was less than or around 7. If phenolphthalein was used, the
solution would be colourless and no sign of colour change occurred after titration because
the solution cannot reach pH 8.0 (pH of boric acid is about 5.12-6.12):
H2BO3- + H+ H3BO3
The phenolphthalein cannot be used as an indicator in the Kjeldahl titration

3. Describe the function of the following chemicals used in this determination

a) Catalyst pellet
In the experiment, catalysts (K2SO4 and CuSO4) was used to speed up the reaction in
digestion step. The amount of K2SO4 added raised boiling point of the digesting acid,
 reduced the digestion time for organic compounds, and facilitated the decomposition of
substances which were hard to be digested.
On the other hand, other substances can be used instead of CuSO4 for increasing speed of
digestion, such as Hg, Cu, Se; oxides: CuO, HgO, P2O5, TiO2; peroxides: H2O2; and salt:
Hg2SO4. However, the most efficient and environmentally neutral catalyst is CuSO4.
b) Borate
Borate ion (BO32-) traped the NH3 produced after neutralizing the solution in digestion
tubes.
(NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O
NH3 + H3BO3 (boric acid) NH4+ + H2BO3-
It also played an intermediate role in quantifying nitrogen contained in the samples by
back titration against H2SO4 solution.
H2BO3- + H+ H3BO3
c) H2SO4
H2SO4 acted as an oxidizing agent which digests the organic compounds to carbon
dioxide and water. It also helped breaking down nitrogencontaining substances, which
produce ammonia in form of ammonium sulfate.
Moreover, sulfuric acid was utilized as a titrant in back titration, which acidify the
solution back to the point at which pH value is the same as that of the initial boric acid.
The volume of used acid are used for determining nitrogen content.
d) NaOH
In the experiment, NaOH neutralized the excess sulfuric acid remaining after digestion
and reacted with ammonium ions (NH4+) which were dissolved in the liquid to form free
ammonia (NH3).
2NaOH + H2SO4 Na2SO4 +2H2O
(NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O

4. Why was it not necessary to standardize the boric acid solution?

The aim of standardization in titration is determining the exact concentration of titrant,
which was used to calculate the concentration of an unknown solution.
In this experiment, boric acid was used to trap NH3 in neutralization and distillation
step. Boric acid solution containing ammonia was then titrated against standard H2SO4
solution to determine the amount of nitrogen.
Therefore, boric acid was not a titrant which need to be standardized. The titrant was
H2SO4.
 5. For each of the disadvantages of the Kjeldahl method, give another protein analysis
method that overcomes (at least partially) that disadvantage.
Disadvantages of the Kjeldahl method Other protein analysis method
Measures nitrogen from both protein and Biuret Method
non-protein sources Does not give true Lowry Method
protein content. Ultraviolet Absorption at 280 nm
Time-consuming (at least 2hrs to complete) Dumas (Nitrogen Combustion) Method
Infrared Spectroscopy
Biuret Method
Ultraviolet Absorption at 280 nm
Dye-Binding Methods
Lowry Method
Kjeldahl method is not sensitive to low Ultraviolet Absorption at 280 nm
protein concentration sample.
Using toxic and corrosive reagents Dye-Binding Methods
(concentrated sulfuric acid H2SO4 at high Dumas (Nitrogen Combustion) Method
temperature, sodium hydroxide) Biuret method
Table 4.2: The disadvantages of the Kjeldahl method and other protein analysis methods that
overcomes (at least partially) that disadvantage.
6. What do we need to pay attention in this experiment in term of lab safety
The sulfuric acid and sodium hydroxide used in the experiment is toxic, corrosive chemicals.
They can burn our skin, eyes and harm our organs if breathed. Therefore, we must:
- Wear lab coat, glass wear, mask, gloves when work with these chemicals.
- These chemicals must be transferred in the fume hood.
- The sulfuric acid must be dispensed with automatic pipette.
Digestion must be conducted in the fume hood We need to leave the sample to cool down
before taking out. If we add NaOH immediately into digestion tubes, violent boiling can
occur.
The chemical waste after doing experiment should be neutralized before discharging to
outside.
 REFERENCES
1. Wrolstad RE, Smith DE. (2010). Food Analysis (4th ed.). S.S. Nielsen (Ed.). Springer,
New York.
2. J. Blarmire. (2003). Kjeldahl Method. In Science @ a Distance. Retrieved March 19,
2017, from
http://www.brooklyn.cuny.edu/bc/ahp/SDKC/Chem/SD_KjeldahlMethod.html