Methodology:

Before the experiment proper, nine clean test tubes were prepared and were rinsed with acetone.
In this experiment, extraction of serum cholesterol, preparation of standard cholesterol solutions and
cholesterol analysis were done. For the extraction of serum cholesterol, three 10 mL blood samples were
transferred in to a dry test tube, the blood samples were chilled in a refrigerator for about 5 minutes, then
were centrifuge. The supernatant of about 0.5 mL was pipetted out to another test tube and then was added
0.5 mL glacial acetic acid.

For the preparation of standard cholesterol solutions, five standard cholesterol solutions with the
following contents indicated in the table below were prepared.

Table 1. Set-up for standard cholesterol solutions

Standard Cholesterol (mg) Glacial CH3COOH (mL)
1 10 10
2 15 10
3 20 10
4 25 10
5 30 10

For the cholesterol analysis, Color Development Mixture was prepared by adding 5 mL
concentrated H2SO4 to 30 mL acetic anhydride in a dry beaker that is placed in an ice bath under the fume
hood. The mixture was then added 15 mL glacial CH3COOH and 0.3 g anhydrous Na2SO4.

The solutions for spectrophotometric analysis were prepared as indicated in the table below. The
absorbance readings of each solutions were taken at 640 nm.

Table 2. Set-up for spectrophotometric analysis

Blank Standard 1 Standard Standard Standard 4 Standard 5 Serum
2 3 Sample
Glacial 0.5 mL - - - - - -
Ch3COOH
Cholesterol - 0.5 mL 0.5 mL 0.5 mL 0.5 mL 0.5 mL 0.5 mL
solution
CDM 2.50 mL 2.50 mL 2.50 mL 2.50 mL 2.50 mL 2.50 mL 2.50
mL

Absorbance readings versus concentration of the standard solutions were plotted and unknown
cholesterol concentration of the three samples were calculated.