Estimation of Total Bilirubin and Direct Bilirubin.

Topics: Bilirubin Pages: 3 (568 words) Published: June 24, 2013
Title : Estimation of Total Bilirubin and Direct Bilirubin.
Aim : To determine the concentration of Total Bilirubin, Direct Bilirubin and Indirect Bilirubin in sample X. Principle :
Procedure (A) : Estimation of Total Bilirubin.
Total bilirubin, both conjugated and free, are measured by using a stabilized diazonium salt of 3, 5-dichloroaniline which reacts with bilirubin to form azobilirubin with maximum absorbance at 540nm. Procedure (B) : Estimation of Direct Bilirubin.

This methods are based on the reaction between direct (conjugated) bilirubin and diazotized sulphanilic acid solutions. Direct Bilirubin reagent utilizes an acid diazo method. Conjugated bilirubin will reacts with diazotized sulphanilic acid to produce an acid azobilirubin, the absorbance of which is proportional to the concentration of direct bilirubin in the sample and can be measured at 550nm.

Materials :
1.Total bilirubin reagent.
2.Direct bilirubin reagent.
3.Calibrator.
4.Sample X.
5.Appropriate number of tube(s) for:
a.Blank
b.Calibrator
c.Sample X
6.Cuvettes.
7.Spectrophotometer.

Methods :
Procedure (A) : Estimation of Total bilirubin.
1.All the tubes was labeled accordingly to blank, calibrator and sample X. 2.1500 µL of Total bilirubin was pipettes into each tubes. 3.All tubes was incubated at 37oC for 5 minutes.
4.75 µL Calibrator and Sample X was pipettes into appropriate labeled tubes. The blank tube was leaved without add any sample. 5.All tubes was incubated at 37oC for 5 minutes.
6.The reading of Absorbance of blank tube in a spectrophotometer at 540 nm wavelength was recorded. This was the Blank Absorbance. ‘0 ABS/100%T’ was pressed to set blank. 7.The reading of Absorbance for Calibrator and Sample X in spectrophotometer at 540 nm wavelength was pointed. These absorbance are the Reaction Absorbance for each tube.

Procedure (A) : Estimation of Direct bilirubin.
1.All the tubes was labeled accordingly to blank, calibrator and sample...
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