Methodology:

REAGENT PREPARATION
Reagent A - 25 milligrams of sulfanilic acid was weighed and dissolved in 375
l concentrated HCl. The resulting mixture was diluted to 25.0 ml with distilled
water in a volumetric flask.
Reagent B (0.5% NaNO2 solution, 25ml) 125 milligrams of sodium nitrate
was weighed. The obtained sodium nitrate was dissolved and diluted to 25.0 ml with
distilled water in a volumetric flask.
Freshly Prepared Diazo Reagent - Inside a sterile falcon tube, 20 ml of reagent
A was mixed with 1.6 ml of reagent B.
Standard Bilirubin Stock Solution 5.0 milligrams of pure bilirubin was
weighed and dissolved in a beaker using 10 ml chloroform. The resulting solution
was transferred into a 50 ml volumetric flask and was diluted to volume with
chloroform. 1.0ml of the stock was transferred into another 50ml volumetric flask
and was diluted to volume with methanol to a final bilirubin concentration of 2
grams/ml.
Generation of bilirubin standard calibration curve and analysis of total serum and
serum-conjugated bilirubin
Different reagents with varying amounts were dispensed in separate 10ml
test tubes consecutively as follows, standard bilirubin, diazo reagent, methanol,
distilled water, and lastly 0.18 M HCl. The solutions were mixed thoroughly and were
incubated for 30 minutes at room temperature. After incubation, respective
absorbance values of each mixture were measured at 540nm.
After measuring the absorbance values, a standard curve was constructed by
plotting the absorbance versus bilirubin concentration. Using the generated linear
equation, the concentration values (in mg/dL) of total,conjugated, and unconjugated
bilirubin were computed in the test serum sample.