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 ( Sulphanilic acid + HNO2 ( NaNo2 + dil. HCl -1 diazotized sulphanilic acid ( Bilirubin + diazo reagent + ( caffeine and sod. benzoate -2 Acid azo bilirubin ( pink color ) .Acid azo bilirubin + alkaline tartrate soln -3 Alkaline azo bilirubin ( blue green color)

: Reagents *
: Sulphanilic acid -1
. gm sulphanilic acid in 10 ml ( 0.2 M ) HCl 0.054

: Sodium nitrite -2
gm in 10 ml H2O 0.02

: Caffiene and sodium benzoate -3

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

: Alkaline tartrate -4
gm NaOH + 0.002 gm Na,k tartrate in 10 ml H2O 0.8

: .Saline soln -5
gm of NaCl in 100 ml H2O 0.9

: Procedure *
: To determine total biliribin -1 Reagents
Sulphanilic acid -1 NaNO2 -2 .caffeine and sod -3 benzoate Serum -4 Mix , wait for 10 .mins .Tartrate soln -5 ml 1 ml 1

Test
ml 0.2 50 l ml 1 ml 0.2

Blank
ml 0.2 -----ml 1 ml 0.2

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

:To determine direct bilirubin -2

Reagents
Sulphanilic acid -1 NaNO2 -2 .Saline soln -3 Serum -4

Test
ml 0.2 50 l ml 2 200 l

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

. 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 *

Tube
Test Blank Test Blank

Absorbance