Protein Determination in Flour and Egg White using Kjeldahl and Biuret Methods

INTRODUCTION:
PRAC 3:
Why use nitrogen for protein determination? – because the only element not present in
lipids/carbohydrates.
How can nitrogen be used to determine protein?
Determining protein methods: kjedahl & dumas. What’s the difference? How do they work/mechanism
of it/t heoretical aspect?
Expected protein content in flours. Why is there a range of protein content?
Why use biuret test with egg protein? What is the biuret test? How does biuret test work?
Expected protein content of eggs.
Protein is an essential part of life as it is found throughout the body – in the bone, skin,
muscles and every body part or tissue. It also makes up enzymes that are responsible for
chemical reactions and haemoglobin that carries oxygen in the blood. Generally, the body
makes amino acid (AA), which is the building blocks of protein (Wong, Aziz, and
Mohamed, 2008). However, a few AA needs to be obtained from food such as eggs, flour,
milk, yoghurt and fish. The protein content in flour is 10g/100g and in egg white is 11g/100g.
Nitrogen in protein is the most distinguishing element, where it typically ranges from 13.4%
to 19.1%, depending on the variation of specific AA composition in proteins. Proteins that
contain high amounts of basic AA have higher amount of nitrogen (Nielsen, 2010).
Numerous methods have been developed to measure protein content. Kjeldahl and Dumas are
two methods used to measure protein content in food. Kjeldahl method relies on the
conversion of protein nitrogen into ammonium ion by boiling sulphuric acid in the presence
of a catalyst whereas the Dumas method relies on the combustion of samples at a high
temperature and measurement of the resulting elemental nitrogen (Thompson et al., 2002).
Kjeldahl method is used in this experiment to determine the total organic nitrogen in the
sample. The nitrogen concentration of the food can determine the amount of protein present
in the food. In the Kjeldahl method, the total organic nitrogen is converted to ammonium
sulphate, which will then be neutralised with alkali and distilled with boric acid solution. The

borate anions formed are titrated with standardised acid that will be converted into nitrogen in
the sample. The result represents the crude protein content of the food.
The Kjeldahl method is carried out in three different but easy steps. Firstly, digestion takes
place where acid and catalyst is added to allow complete breakdown of all organic matters.
Non-volatile ammonium sulphate is formed from the reaction of nitrogen and sulphuric acid.
During digestion, protein nitrogen is liberated to form ammonium ions. Sulphuric acid then
oxidises organic matter and combine with the ammonium formed while carbon and hydrogen
elements are converted to carbon dioxide and water. Neutralisation and distillation will then
take place where the digest is diluted with water. Alkali containing sodium is added to the
sulphuric acid to allow neutralisation. The ammonia formed is distilled into boric acid
solution containing indicators methylene blue and methyl red. The advantage of using this
method is that its applicable to all types of food with high accuracy, thus being the official
method for crude protein analysis (Nielsen, 2010).
Biuret determination measures the albumin in the egg white, with the addition of copper salt
in an alkaline solution [i.e. sodium hydroxide (NaOH)]. A purple colour complex will be
produced when the cupric ion is complexed with peptide bonds. Steps taken in order to carry
out this procedure is by mixing protein solution with reagents that include copper sulphate,
NaOH and potassium sodium tartrate, which is used to stabilise the cupric ion in the alkaline
solution. The absorbance of the colour is read at 550nm against a reagent blank, where the
intensity is proportional to the protein content in the sample. A standard curve of
concentration versus absorbance is constructed using bovine serum albumin (BSA).
Comparing to Kjeldahl method, the biuret method takes a shorter time and is the simplest
form of protein analysis. Other than that, it doesn’t detect nitrogen from non-peptide and non-
protein sources (Nielsen, 2010).
MATERIALS AND METHODS:
Part A: Macro Kjeldahl – Determination of Protein in Flour
Different kinds of flour and its corresponding blanks were prepared and analysed.
Approximately 1g of flour was weighed out onto two joined Tallyho cigarette papers then
twisted into a ball and added to a digestion tube. Approximately 5g anhydrous sodium
sulphate and two catalyst tablets (each containing 1g sodium sulphate and 100mg copper
sulphate) were added to the same digestion tube. In a fume cupboard and using a face shield,

12mL concentrated sulphuric acid was added to the tube and it was gently shaken to wet the
samples. The digestion tubes were loaded onto a preheated digestion unit (420°C) with the
vacuum manifold attached to it. The samples were digested for approximately an hour until
they were a clear blue/green colour.
After an hour of digestion, the tubes were left to cool down before 50mL of distilled water
(diH2O) was added using a measuring cylinder. For each digestion tube, a 250mL conical
flask containing 30mL of 4% boric acid solution (containing bromocresol green and methyl
red indicators) was prepared. Both the digestion tube and conical flask were placed in the
distillation unit whereby the distillate tube was submerged into the flask. The distillation unit
automatically added 50mL of 40% sodium hydroxide (NaOH) to the distillation tube. It then
boiled the solution in the tube with steam. The distillate from the steam was collected in the
conical flask. The waste liquid in the digestion tube was automatically removed into a waste
bottle. The empty digestion tube was placed in the sink while the solution in the conical flask
was titrated against 0.1M hydrochloric acid (HCl) until the solution turned pink. The nitrogen
content and percentage protein were calculated.
Part B: Biuret Determination of Soluble Protein in Egg White
Approximately 20g of commercially pure liquid egg white was weighed into a beaker.
Approximately equal amount of 0.1M NaOH was added to the beaker to dissolve the egg
white using a magnetic stirrer. The dissolved egg white was washed into a 100mL volumetric
flask using diH2O. The beaker was rinsed several times to ensure all egg white had been
washed into the flask. The solution in the flask was diluted to volume with diH 2O and the
flask was shaken to mix the solution. A standard bovine serum albumin (BSA) protein
solution with 10mg protein/mL was prepared. Twelve test tubes in duplicated pairs were set
up following Table 1 with the biuret reagent added last using a safety dispenser. The test
tubes were vortex and left to stand for 30 minutes. The absorbance of the solution in each test
tubes was read on the spectrophotometer at 550nm. A calibration curve was plotted using the
standards’ average absorbance value against its protein content (mg/mL).
Table 1: Reagents added to each pair of test tubes.
Reagents Blank Standard 1 Standard 2 Standard 3 Standard 4 Egg White
Sample
diH2O 2 mL 1.6 mL 1.2 mL 0.8 mL 0.4 mL 1.8 mL
Egg White
Solution - - - - - 0.2 mL