Colorimetric Analysis of Bilirubin in Serum

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BACKGROUND

A breakdown produce of hemoglobin is bilirubin that is not soluble in water. It gets transported from spleen to liver and then to bile.¹ An analysis of bilirubin in blood plasma helps in diagnosing jaundice in human being.

So, the basic objective of this experiment is to analyze bilirubin in serum by colorimetric method.

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REQUIREMENTS

Reagents:   

Solution A (Van den Bergh A solution) : 1 gm of sulphanilic acid +15ml of conc. HCL and then mix water to make it 1 litre.²

       Solution B (Van den Bergh B sol): 0.5 gm of sodium nitrite + water for dissolving and then make up to 100 ml.²

  Diazo reagent: 0.3 ml of solution B + 10 ml of solution A.

  Diazo blank: 1.5% v/v HCL

  Bilirubin std.: 100 mg per 100 ml chloroform.

         Working Bilirubin std – dil 1:10 with methanol (0.1 mg per ml)

         Absolute methanol

Apparatus:  Pipette

        Test tubes

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PROCEDURE

Total bilirubin = conjugated bilirubin + unconjugated bilirubin

Take five test tubes and prepare as follows:

Total (direct + indirect) =0.2 ml serum+ 0.5 ml of diazo reagent + 1.8 ml of distilled water + 2.5 ml methanol.³

Direct (conjugated) =0.2 ml serum + 0.5 ml Diazo reagent + 1.8 ml distilled water and then 2.5 ml distilled water.³

Blank = 0.2 ml standard bilirubin +1.8 distilled water + 0.5 diazo blank +2.5 distilled water

Standard =0.2 ml standard bilirubin + 0.5 diazo reagent + 4.3 methanol

Standard blank = 0.2 ml standard bilirubin + 0.5 ml diazo blank + 4.3 methanol

After preparing this test tube mix it properly and leave it undisturbed for about 30 min and then take the OD reading at 540 nm.

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CONCLUSION

This experiment is an simple approach for analyzing bilirubin.

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REFERENCES

1. Kaplan A et al. Bilirubin. The C.V. Mosby Co. St Louis. Toronto. Princeton. Clin Chem. 1984; 1238-1241. 436 and 650.
   
2. Malloy HT et al. The determination of bilirubin with the photoelectric colorimeter. J. Biol Chem. 1937; 112, 2; 481-491.
   
3. Martinek R. Improved micro-method for determination of serum bilirubin. Clin Chim. 1966: 13: 61-170.