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Estimation of Bilirubin in Serum

This document describes the principles and procedure for estimating total bilirubin in serum. Total bilirubin is determined by coupling bilirubin with diazotized sulfanilic acid in the presence of caffeine to form an azodye, which is pink in color. Direct bilirubin is determined similarly but using saline instead of caffeine. The absorbance is then read and concentrations are calculated using predetermined factors.

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Mohsen Haleem
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995 views

Estimation of Bilirubin in Serum

This document describes the principles and procedure for estimating total bilirubin in serum. Total bilirubin is determined by coupling bilirubin with diazotized sulfanilic acid in the presence of caffeine to form an azodye, which is pink in color. Direct bilirubin is determined similarly but using saline instead of caffeine. The absorbance is then read and concentrations are calculated using predetermined factors.

Uploaded by

Mohsen Haleem
Copyright
© Attribution Non-Commercial (BY-NC)
Available Formats
Download as DOC, PDF, TXT or read online on Scribd
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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

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