Protein analysis

1. Proximate analysis
Dr. Arfaa Sajid
Assistant Professor
Department of Chemistry, UOL.

2. Protein analysis:
 Proteins are macromolecules formed by amino
acids. Proteins are large size molecules (macromolecules),
polymers of structural units called amino acids.
 Protein is a very abundant component of all cells. It is very
important for biological functions and cell structure.
 It is composed of 20 different kinds of amino acids. 11 none
essential amino acids and 9 are essential.
 Food proteins are very complex.
 Proteins vary in molecular mass, ranging from approximately
5000 to more than a million Daltons.
 Nitrogen is the most distinguish element present in proteins.
 Its content in various food proteins ranges from 13.4 to 19.1%
due to the variation in the specific amino acid composition of
proteins.
 Generally, proteins rich in basic amino acids contain more
nitrogen.

3. Asparagine 1806
Cystine 1810
Glycine 1820
Essential AA Non Essential AA
Histidine Alanine,
isoleucine Arginine
Leucine Asparagine
Lysine Aspartic acid
Methionine Cysteine
Phenylalanine Glutamic acid
Threonine Glutamine
Tryptophan Glycine
Valine Proline
Serine
Tyrosine

4. Numerous methods have been developed to measure
protein content. The basic principles of these methods
include:
a) Determinations of nitrogen,
b) Peptide bonds,
c) Aromatic amino acids,
d) Dye-binding capacity,
e) Ultraviolet absorptivity of proteins,
f) Light scattering properties

5. There are several methods of protein analysis
a) Kjeldhal method
b) Dumas method
c) Infrared spectroscopy
d) Biuret method
e) Lowry method
f) Bradford method
g) Bicinchoninic acid (BCA)
h) Ultraviolet absorptivity of proteins,

6. Kjeldhal Method:
In 1883, March 7th John Kjeldhal , A Danish Chemist developed this
method.
Objective:
Qualitative determination of organic as well as inorganic nitrogen in
the substances.
Sample preparation:
1. Solid food grind and pass through 20 mesh size screen.
2. Sample should be homogenous.
Procedure:
Digestion Distillation Titration

7. Digestion Neutralization and distillation

8. DIGESTION:
Aim: To convert all nitrogen into ammonium ions.
 For decomposition of nitrogen sample the sample (0.01-5g) is
mixed with sulfuric acid at temperatures between 350 and 380ºC.
 The higher the temperature used, the faster digestion can be
obtained.
 The speed of the digestion can be greatly improved by the addition
of salt and catalysts.
 Potassium sulfate is added in order to increase the boiling point
of sulfuric acid and catalysts are added in order to increase the speed
and efficiency of the digestion procedure.
 Oxidizing agents (Hydrogen peroxide) can also be added to
improve the speed even further.
Nitrogenous Compound + H2SO4 +CuSO4 + K2SO4 = (NH4)2SO4 + H2O + CO2 +
Other by-products

9. After digestion is completed the sample is allowed to cool to room temperature,
then diluted with water and transferred to the distillation unit.
(Catalyst + salt) Kjeldahl tablets / Missouri catalyst.
The catalysts are composed of more than 97% of a salt which increases the
boiling temperature of the sulfuric acid and 1 - 3% of one type of catalyst or a
mixture of catalysts in order to increase the speed and efficiency of the digestion
procedure.
Typical catalysts are selenium or metal salts of copper , titanium and murcury.
The selection of a particular catalyst depends on
 Ecological and toxic aspects
 Practically, reaction time or foaming and sputtering.
Selenium-containing catalyst reacts fastest but it is toxic.
Copper containing catalyst is considerably safer for both humans and the
environment but gives a slower digestion process.
An ideal compromise is the mixed catalyst consisting of copper and titanium
sulfate.
In water containg samples there is a problem of foaming and sputtering.
If foaming is the only problem it is better to use 1-3 drops of a proprietary
antifoam emulsion (Silicone antifoaming liquid).

10. DISTILLATION/ Neutralization:
During the distillation step the ammonium ions (NH4+) are converted into ammonia (NH3)
by adding alkali (NaOH). The ammonia (NH3) is transferred into the receiver vessel by
means of steam distillation.
The receiving vessel for the distillate is filled with an absorbing solution in order to capture
the dissolved ammonia gas.
Aqueous boric acid [B(OH)3] of 2-4% concentration. The ammonia is quantitatively
captured by the boric acid solution forming solvated ammonium ions.
Sulfuric acid or hydrochloric acid that captures the ammonia forming solvated ammonium
ions.

12. Reaction with Boric Acid (H3BO3): [Direct titration]
If Boric Acid is receiving solution then the Ammonia produce in distillation
is react with H3BO3 and produce AMMONIUM BORATE (NH4
+H2BO3
-), it
can directly titrated against sulfuric acid or hydrochloric acid (0.01N to
0.5N) by using indicator.s
Indicator Acidic medium Basic medium
Methyl red red yellow
Tashiro,s indicator Purple green
Tashiro,s indicator is a mixture of methylene blue (0.1) and methyl red
(0.3) in methanol or ethanol.
NH3 + H3BO3 = NH4
+H2BO3
-
TITRATION:
The concentration of the captured ammonium ions can be determined using
two types of titrations:

13. ADVANTAGES:
1) Prevent loss of ammonia by volatilization,
2) No need to back titration, too weak acid to interfere further
titration.

14. Reaction with Hydrochloric Acid (HCl)/ Sulfuric acid: [Back
titration]
The ammonia is captured by a, carefully measured excess of a
standardized acid solution in the receiving flask. The excess of acid in
the receiving solution keeps the pH low, and the indicator does not
change color.
NH3 + HCl (in excess) = NH4
+Cl- + HCl (left back)
The excess acid solution is exactly neutralized by a carefully measured
standardized alkaline base solution such as sodium hydroxide. A color
change is produced at the end point of the titration.
Methyl orange: Acid (red), base (Yellow)
HCl + NaOH = NaCl + H2O

15. V ml of X (N) NaOH = V ml of X (N) HCl = V ml of X (N) NH3

17. Numerical:
In the kjeldhal method for estimation of nitrogen
present in biscuit sample, ammonia evolved from 0.75 g of
sample neutralized with 10 mL of 1M H2SO4. What is the
percentage of nitrogen in the sample.

18. Advantages:
1) Applicable to all types of foods
2) Inexpensive (if not using an automated system)
3) Accurate; an official method for crude protein content
4) Has been modified (micro Kjeldahl method) to measure
microgram quantities of proteins
Disadvantages:
1) Measures total organic nitrogen, not just protein nitrogen
2) Time consuming (at least 2 h to complete)
3) Poorer precision than the biuret method
4) Corrosive reagent

19. Dumas Method:
In 1831 Jean Baptiste Dumas developed this method.
Principle:
A known mass of organic compound is heated with excess of copper oxide
in an atmosphere of CO2. Carbon, hydrogen and sulphur (if present) are
oxidized to CO2, H2O and SO2 respectively while nitrogen gas is set free.
Any oxide of nitrogen that may be formed is reduced back to free nitrogen
by passing over a hot reduced copper gauze.
C + 2CuO = CO2 + 2 Cu
2H + CuO = H2O + Cu
Nitrogen + CuO = N2 + small amount of oxides of Nitrogen
oxides of Nitrogen + Cu = CuO + N2
The Nitrogen thus formed is collected over conc. KOH (40%) solution
which absorb all other gasses i.e. CO2, H2O and SO2. The volume of
nitrogen collected is noted and from this the percentage of nitrogen can
be calculated.

20. CO2 generator Combustion tube Schiff’s nitrometer

21. CO2 generator:
CO2 is needed for this purpose is produced by heating sodium bicarbonate or
magnesium bicarbonate. The gas is perfectly dried by bubbling through conc.
H2SO4 before passing it through the combustion tube.
Combustion tube:
It is a hard glass tube about 90cm long and about 2cm in diameter. It is packed
with
1) A roll of oxidized copper gauze which prevent backward diffusion of gasses
produced during combustion.
2) An accurately weighed quantity of the substance mixed with excess of cupric
oxide
3) Coarse CuO that filled half of the combustion tube and
4) A reduced copper gauze which help to reduce any oxides of nitrogen formed
during combustion back to nitrogen gas.

22. Schiff’s nitro meter:
It consist of a long graduated tube having a reservoir and a tap at the upper end.
It contains about 40% KOH solution. It also has a mercury seal at the bottom
which prevent KOH solution from being sucked back into the combustion tube.
Both CO2 and H2O produced during combustion are absorbed by KOH solution
while N2 is collected over it. The volume of nitrogen is measure after careful
leveling (by making the level of KOH in the nitro meter tube and reservoir same).
If we keep the level of KOH same in Schiff’s nitro meter and reservoir same then
at that time pressure will be 1 atm.

24. Nitrogen analyzer:

25. Nitrogen analyzer:

26. Thermal conductivity detector:

27. Advantages:
1. Requires no hazardous chemicals.
2. Can be accomplished in 3 min.
3. Recent automated instruments can analyze up to 150
samples without attention.
Disadvantages:
1. Expensive equipment is required.
2. Measures total organic and inorganic nitrogen, not just
protein nitrogen.