The most frequently used methods for measuring protein content in foods include the Kjeldahl method, Dumas method, direct measurement methods using UV-spectroscopy and refractive index measurement. Each method has advantages and disadvantages

1. Presented by, vaishnavi janjal and Samiksha wanare
Student of pharm d 2nd year
Goverment college of pharmcy, aurangabad

2. Protein Definition
A protein is a naturally occurring, extremely complex substance
that consists of amino acid residues joined by peptide bonds.

3. kjeldahl apparatus
1.Digestion
The purpose of this step is to break down the bonds that hold
the polypeptides together, and convert them to simpler
chemicals such as water, carbon dioxide andammoni

4. The food sample to be analyzed is weighed into a digestion flask
and then digested by heating it in the presence of sulfuric acid (an
oxidizing agent which digests the food), potassium sulfate (K2SO4)
(to speed up the reaction by raising the boiling point of the
digesting acid) and a catalyst, such as copper, selenium, titanium, or
mercury (to speed up the reaction).
Ammonia gas is not liberated in an acid solution because the
ammonia is in the form of the ammonium ion (NH4 +) which binds
to the sulfate ion (SO4 2-) and thus remains in solution:
The general equation for the digestion of an organic sample is
shown below:
Protein + H2SO4 → (NH4)SO4 + H2O + CO2

5. 2.Distillation
The purpose of the distillation step, is to separate the
ammonia (that is, the nitrogen) from the digestion mixture.
This is done by, raising the pH of the mixture using (NaOH )This
has the effect of changing the ammonium(NH4 +) ions (which
are dissolved in the liquid) to ammonia (NH3), which is a gas as
indicated in the following equation.
The ammonia gas is led into a trapping solution (an acid )
where it dissolves and become an ammonium ion once again
NH3 + HCl (0.1 N) NH4 +Cl- + HCl (left back) (in excess)
(NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O

6. 3. Titration
Left back HCl (0.1 N ) is titrated with standard NaOH (0.1
N).

7. Calculation
• Let the weight of the organic substance be x gm and V ml of (N) HCl is
required for complete neutralization of ammonia evolved.
• V ml (N) HCl = V ml of (N) NH3
• 1000 ml of a N NH3 contain 17 gm of NH3 or 14 gm of Nitrogen
•
• Amount of Nitrogen present in V ml of (N) NH3 = 14/1000 x V x N = y gm
• Percentage of nitrogen = Weight of Nitrogen (y gm) / Weight of
Substance (x gm) ×100
• Where ,
• N = Normality of Acid Used
• V = Volume of Acid used up
• %N× 6.25(Correction Factor) = %protein

8. 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

9. Dumas (Nitrogen Combustion) Method
This method is a more advanced form of the method proposed by
Dumas which was based on the principle of heating nitrogen
containing compounds at high temperatures to yield free nitrogen.
Presently it has transformed into an automated instrumental
technique which helps in rapid determination of proteins in the
sample.
1. Weighed quantity of sample is subjected to very high
temperature (about 900°C) along with oxygen in the heating
chamber. Such combustion yields N₂, CO₂ and H₂O.
2. The gases so liberated are then allowed to pass over
specialized columns which function to absorb CO, and water.
Procedure

10. 3. Remaining gases are passed through a column which is connected
to a thermal conductivity detector. This column
functions in selective absorption of nitrogen gas from the residual
mixture of CO, and water.
4. Instrument is calibrated by utilizing a known pure standard, whose
nitrogen concentration is already known.
5. Sample is also analyzed and nitrogen content is determined by
suitable conversion of the signal from thermal
conductivity detector.
6. Similar to Kjeldahl method, Dumas method also requires
appropriate conversion factors for converting the
concentration of nitrogen in the sample to protein content.

12. Applications
The combustion method is an alternative to the Kjeldahl
method and is suitable for all types of foods.
Advantages
1. Requires no hazardous chemicals.
2. Can be accomplished in 3 min.
Disadvantages
1. Expensive equipment is required.
2. Measures total organic nitrogen, not just protein
nitrogen

13. Ultraviolet 280nm Absorption Method
Principle
• Proteins show strong absorption in the region at ultraviolet
(UV) 280nm, primarily due to tryptophan and tyrosine
residues in the proteins. Because the content of tryptophan
and tyrosine in proteins from each food source is fairly
constant, the absorbance at 280nm could be used to estimate
the concentration of proteins, using Beer’s law.
• Since each protein has a unique aromatic amino acid
composition, the extinction coefficient (E280) or molar
absorptivity (Em) must be determined for individual proteins
for protein content estimation.

14. Procedure
1. Proteins are solubilized in buffer or alkali.
2. Absorbance of protein solution is read at 280nm
against a reagent blank.
3. Protein concentration is calculated according to the
equation
A = abc
where:-
• A = absorbance
• a = absorptivity
• b = cell or cuvette path length
• c = concentration

15. Advantages
1. Rapid and relatively sensitive; At 280 nm, 100 μg or more
protein is required; several times more sensitive than the
biuret method.
2. No interference from ammonium sulfate and other buffer
salts.
Disadvantages
1. Nucleic acids also absorb at 280 nm.
2. Aromatic amino acid contents in the proteins from
various food sources differ considerably.
3. The solution must be clear and colorless. Turbidity due to
particulates in the solution will increase absorbance falsely.

25. Thank you.....