Quantification of protein using the biuret reaction

The biuret reaction can be used to estimate the amount of protein in a sample. Like biuret, proteins contain a
-CONH- group which, when reacted with aqueous alkaline solution of copper(II) sulphate, produces the following complex of a blue-violet colour:

The violet color produced, allows the estimation of protein colorimetrically. The colour intensity is proportional to the amount of peptide bonds participating in the reaction. A series of samples are prepared with known amounts of protein along with a sample with the unknown amount of protein. Samples are then transferred to cuvettes and the absorbances measured. the absobance of the unknown sample should fall between the absorbances of the known sample.The complex has an absorbance maximum of 540 nm. A calibration curve can then be produced and the unknown sample's concentration calculated using Beer's Law.

The Cu(II)SO4 is added to the protein dissolved in sodium hydroxide. The sodium hydroxide breaks up the secondary sructure of the protein, i.e., the alpha and beta pleated sheets so it is easier for the Cu2+ ions to access and form complexes with the nitrogen atoms in the proteins.

Disadvantage

One disadvantage of this reaction, however, is that some of the Cu2+ will react with NaOH to produce the insoluble, Cu(OH)2

Formol titration-Formol titration

Formol titration can be used to estimate the amount of nitrogen/nitrogen containing substance present in a sample. It is frequently used to estimate the amount of protein present in food products but,of course, can be used to detect the amount of other nitrogen containing substance such as ammonia and polypeptides in a sample.

Procedure (from wikipedia.com):

1. Pipette out 10 ml samples of milk into a 50ml erlenmeyer flask
2 Add 0.4ml saturated potassium oxalate solution and 0.5 ml phenolphthalein indicator. set aside for 2 minutes.
3. Neutralize the milk by titrating with 0.1N NaOH to the endpoint
4. Add 2 ml of 40% formaldehyde solution and allow to stand for 2 min.
5. Titrate again with 0.1 N NaOH to the same endpoint.
6.Run a blank by titrating 2 ml of 40% formaldehyde solution plus 10 ml distilled water with 0.1 N NaOH CALCULATION: Va = volume of 0.1 N NaOH used to titrate sample after addition of formaldehyde Vb= volume of 0.1N NaOH used to titrate blank Va-Vb= formaldehyde value %protein= Va-Vb X formol factor FORMOL FACTORS 1.74 for cow's milk 1.91 for carabao's milk

Further notes on formol titration:

*Saturated potassium oxalate is added to the sample. This is necessary because Ca 2+ ions react with NaOH to form Ca(OH)2 which will lead to inaccurate volume of NaOH being recorded. To prevent this, the oxalate is added which will react with the Ca2+ ions to form calcium oxalate.

*Sample is neutralised usually with NaOH with a few drops of phenolphthalein. A colour change in phenolphthalein from colourless to pink should be seen


* Formaldehyde (methanal) is then added to the sample. The formaldedye reacts with amino acids . The H+ ions are liberated so the sample becomes more acidic which allows it to be titrated with NaOH . the reaction has a 1:1 mole ratio. The amount of H+ ions reacted is to represent the amount of amino acid present in the sample since each amino acid will liberate one H+ ion.


*Regarding the use of sodium hydroxide instead of barium hydroxide, sodium hydroxide is used instead of barium hydroxide due to the some amino acids , after reacting with formaldedyde , the derivate will form insoluble precipitate with barium, but this problem does not occur with sodium hydroxide