PRACTICALS: STEP BY STEP

1. ESTIMATION OF CREATININE
(ALKALINE PICRATE METHOD)
AIM:
To estimate the amount of creatinine in the given sample of serum and urine
PRINCIPLE:
The method makes use of the Jaffe’s reaction,involving the production of red colour with an
alkaline picrate solution. The intensity of the colour developed is measured at 500nm.
Creatinine appears to be absorbed on to the protein precipitate at higher pH.Hence a modified
Folin-Wu tungstic acid protein precipitation is used to give more acid filtrate.
REAGENTS:
1) 10% Sodium Tungstate
2) 2/3 N Sulphuric Acid
3) PICRIC ACID, 0.04 M SOLUTION:
0.916g of crystalline picric acid is dissolved in 100ml of distilled water.
4)0.75 N Sodium Hydroxide(NaOH)
5) STOCK STANDARD CREATININE SOLUTION: (1ml = 1mg)
100mg of pure dry creatinine is dissolved in 0.1 Hydrochloric acid and made upto
100ml with acid.
6) WORKING STANDARD: (1ml = 0.02 mg)
1.0 ml stock standard is diluted to 50ml with water. It contains 20µg of creatinine/ml.
PROCEDURE:
Tubes containing 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 ml of the working standard solution
corresponding to concentrating 10, 20, 30, 40, 50 and 60 µg are taken. The volume is made
up to 3.0 ml with water in all the test tubes, 3.0 ml is taken as blank.
1.0 ml of the given urine sample is made up to 100 ml with distilled water. From this
3.0 ml is taken for the experiment.
In another test tube, 2.0 ml of the serum is diluted with 3.0 ml of the water and the
protein is precipitated by adding 1.0 ml of 10% Sodium Tungstate solution and then 2.0 ml of
2/3 N sulphuric acid with shaking. Kept for 10 minutes and centrifuged. 3.0 l of the
supernatant is taken for the experiment.
To all the test tubes namely the blank, standard and serum. 1.0 ml of the 0.04 M picric
acid solution and 1.0 ml of 0.75 N sodium hydroxide is added. Test tubes are left in the stand
for 15 minutes for the colour to develop. The tubes are shaken well and the colour developed
is read in a calorimeter at 500 nm.
RESULT: