Quantitative Determination Of Protein In Foods By The Biuret
Substances containing two or more peptide bonds form a purple complex with copper
(2) in alkaline solution. Color development is due to the coordination complex formed
between the copper ion and the nitrogen atoms in the peptide chain. The concentration
of protein in a sample is obtained by reference to a calibration curve prepared with
known concentrations of protein. The objective of this exercise is to quantitate the
amount of protein in a food.
Egg albumin standard: dissolve 100mg protein in 100 ml distilled water.
Biuret solution: Dissolve 1.5g CuSO4.5H2O,6 g of NaKC4H4O6.4H2O in 500ml
distilled water. Add 300ml of 10%NaOH with constant swirling. Dilute to 1L with
distilled water and store in a polyethylene bottle.
1. Pipette a known amount of protein standard and distilled water as shown in
table below into a test tube.
2. Add 4 ml of Biuret solution, mix, and allow to stand 30 min at room
3. Record optical density at 550nm on a spectrophotometer.
4. Plot absorbance at 55nm against the concentration of the protein standard.
5. Prepare an unknown that is neither heavily pigmented nor turbid, such as egg
white, unflavored gelatin, pure protein solutions, or a semi-purified protein
dispersion such as a dialyzate. Estimate the appropriate dilution from a food
composition table so that the final protein concentration will fall within the
concentration used for preparation of the standard curve. Pipette an aliquote
into a test tube and add water if necessary to bring the total volume to
1ml.Continue with step 2 as for the standards.
Concentration and optical density of egg protein.
Tube Volume Volume water Concentration Optical density
standard (ml) (ml) Standard(mg/ml)
1 0 1 0
2 0.1 0.9 1
3 0.25 0.75 2.5
4 0.5 0.5 5
5 0.75 0.25 7.5
6 1 0 10
1. Draw a graph of protein concentration versus optical density using excel, Get the
equation of the graph and using the equation of the graph calculate the
concentration of protein in the unknown solution.
2. Compare the experimental value with the literature value of protein in egg white
and state reasons why your value is high or low.