- Estimation of total bilirubin in serum

* Principle :

- Bilirubin is coupled with diazotized sulfanilic acid in the presence of caffeine to give azodye .

- Normal saline is used instead of caffeine when direct bilirubin is determined.

diazotization

1- Sulphanilic acid + HNO2 ( NaNo2 + dil. HCl )

diazotized sulphanilic acid 2- Bilirubin + diazo reagent + ( caffeine and sod. benzoate )Acid azo bilirubin ( pink color ) 3- Acid azo bilirubin + alkaline tartrate soln. Alkaline azo bilirubin (blue green color )

* Reagents :

1- Sulphanilic acid :

0.054 gm sulphanilic acid in 10 ml ( 0.2 M ) HCl .

2- Sodium nitrite :

0.02 gm in 10 ml H2O

3- Caffiene and sodium benzoate :

0.0054 gm of caffeine + 0.0079 gm of sod. benzoate in 100 ml H2O

4- Alkaline tartrate :

0.8 gm NaOH + 0.002 gm Na,k tartrate in 10 ml H2O

5- Saline soln. :

0.9 gm of NaCl in 100 ml H2O

* Procedure :

1- To determine total biliribin :

Blank Test Reagents

0.2 ml ------1 ml 0.2 ml 1 ml 0.2 ml 50l1 ml 0.2 ml1 ml 1- Sulphanilic acid2- NaNO2

3- caffeine and sod.

benzoate

4- Serum

Mix , wait for 10 mins.

5- Tartrate soln.

- Mix , wait for 5 mins. , then read at 578 nm .

2- To determine direct bilirubin:

Blank Test Reagents

0.2 ml------2 ml 200l

0.2 ml 50l2 ml 200l

1- Sulphanilic acid2- NaNO2

3- Saline soln.

4- Serum

- Mix , incubate for exactly 5 mins. At 20 25 c .

- Read at 546 nm .

* Calculations :

- Calculate the concentration of bilirubin in serum in mg % . ** Total bilirubin = A sample X 10.8

** Direct bilirubin = ( A sample A blank ) X 14.4

** Free bilirubin = Total direct

* Results :

Absorbance Tube

Test

Blank

Test

Blank