Determination of Protein Content
SMK Negeri 13 Bandung
Protein in Human Body
Protein has many function in human body:
- Energy source
- Tissue and cell reparation and development
- As antibody and hormone and enzym sinthesys
- As leveling substance for acid and base balance
In human body, protein through a cycle in which
protein decomposed to smaller component (amino
acid/peptide). Amino acid and peptide form new
protein molecules to replaced the broken one.
(There is no life long protein used)
Protein in Food
According to the source, 2
kinds of protein known:
- Animal Protein
(fish, meat, egg, milk)
- Plant Protein
(Beans, rice, wheat and
Protein content in food
stuff are various
One of big biomolecules besides
polysacharide, lipid and polynucleotid
which are the main former of live
Protein is a polymer of about 20
amino acid linked by the peptide
Amino acid is the main molecule of protein
Amino acid is an organic substance with
carboxyl (COOH) and amine (NH2) group.
Amino acid structure in general is a C
atom linked to 4 groups/atom: amine
(NH2), gugus carboxcyl (COOH), hydrogen
atom (H), and one rest group (R, residue).
R group distinguish an amino acid with
AMINO ACID STRUCTURE
Peptide bonding is amide bonding which is link 2
amino acids. One peptide has end-N amino acid with
free NH3+ group and end-C amino acid with free COO-
ANALYSIS OF PROTEIN CONTENT
The determination of N developed by
Johan Kjeldahl (Danish chemist), that’s
why it called Kjeldahl’s method
The Kjeldahl method of nitrogen analysis
is the worldwide standard for calculating
the protein content in a wide variety of
materials ranging from human and
animal food, fertilizer, waste water and
Kjeldahl’s Method Step
Diagram of Kjeldahl’s Method
Decomposition Distillation Trapping Titration
Nitrogen calculation Protein calculation
It is possible to calculate
the amount of crude
protein in the sample.
Although there are
different samples, the
amount of "crude protein"
(CP) can be found by
multiplying the percent
Nitrogen by a factor
CP = %N x 6.25
Weighing out approximately 1 gm of the sample
containing protein, and placing the sample into a
digestion flask, along with 12-15 ml of concentrated
sulfuric acid (H2SO4).
Adding seven grams of potassium sulfate and a
catalyst, usually copper.
Bringing the digestion tube/flask and mixture to a
"rolling boil" (about 370oC to 400oC) using a heating
Heating the mixture in the tube/flask until white
fumes can be seen, and then continuing the heating
for about 60-90 mins, the solution should be clear.
Cooling the tube/flask and cautiously adding 250 mls
Raising the pH of the mixture using sodium
hydroxide (45% NaOH solution). This has the effect
of changing the ammonium (NH4+) ions (which are
dissolved in the liquid) to ammonia (NH3), which is a
Separating 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 trapping the distilled vapors in a
special trapping solution of about 15 ml HCl
(hydrochloric acid) in 70 ml of water.
removing the trapping flask and rinsing the
condenser with water so as to make sure that all the
ammonia has been dissolved.
Adding an indicator dye to the
acid/ammonia trapping solution.
Putting a standard solution of NaOH
(sodium hydroxide) into the buret slowly
adding small amounts of the sodium
hydroxide solution to the acid solution with
Watching for "endpoint" has been reached
and that now all the acid has been
neutralized by the base.
Performing a calculation to find the amount
of ammonia, and thus nitrogen.
Moles ammonia represent moles nitrogen
gms nitrogen =
moles nitrogen x atomic mass
(gN = molesN x 14.0067)
(gms nitrogen / gms sample) x 100
%N = (gN / gS) x 100