Cholesterol Determination

Words of Caution:
The ferric chloride reagent that you will be using is very acidic and must be handled very carefully. Wash any spills immediately with a bicarbonate solution, followed by copious amounts of water. You do not want to wear fancy clothes for this lab exercise.You will be working with very volatile, flammable organic solvents. Therefore,

  • work in the fume hood,
  • do not work near any open flames and
  • be sure to dispose of the excess solvents in the excess SOLVENT container.

Spectrophotometric determination of cholesterol

Be sure to read everything before doing anything.

You need to set up a standard curve for determination of cholesterol.

Set up seven test tubes and pipet the following amounts of a 0.1 mg/mL standard solution of cholesterol and absolute alcohol into each tube:

0.0 mL cholesterol standard, 2.0 mL absolute ethanol
0.2 mL cholesterol standard, 1.8 mL absolute ethanol
0.4 mL cholesterol standard, 1.6 mL absolute ethanol
0.8 mL cholesterol standard, 1.2 mL absolute ethanol
1.2 mL cholesterol standard, 0.8 mL absolute ethanol
1.6 mL cholesterol standard, 0.4 mL absolute ethanol
2.0 mL cholesterol standard, 0.0 mL absolute ethanol
Add 2.0 mL of the ferric chloride reagent (remember this is very a strongly acidic solution—handle very carefully, as described above).

Incubate the reaction tubes for 30 minutes at room temperature, transfer to cuvettes very carefully, and read at 550 nm.

You will be assaying three fractions for their cholesterol content: Total Lipids, Neutral Lipids and Polar Lipids.

For the Total lipids, you will bring the volume up to 20 mL (in a 25 mL graduated cylinder) using ether, then put 1.0 mL of that in a 13x100mm tube, dry it under nitrogen gas and resuspend it in 1.0 mL of absolute ethanol. Then, add 0.1 mL of this resuspended solution to a test tube with 1.9 mL of absolute ethanol, to another test tube add 0.05 mL of the resuspended solution and 1.95 mL of absolute ethanol, and to a third test tube, add 0.02 mL of the resuspended solution and 1.98 mL of absolute ethanol.

For the Neutral lipids fraction, you will bring the column fraction up to 5 mL (in a 10 mL graduated cylinder)using ether:methanol (100:1 v/v), then put 1.0 mL of that in a 13x100mm tube, dry it under nitrogen gas and resuspend it in 0.2 mL of absolute ethanol. Then, add 0.1 mL of this resuspended solution to a test tube with 1.9 mL of absolute ethanol, to another test tube add 0.05 mL of the resuspended solution and 1.95 mL of absolute ethanol, and to a third test tube, add 0.02 mL of the resuspended solution and 1.98 mL of absolute ethanol.

For the Polar lipids fraction, you will bring the column fraction up to 5 mL (in a 10 mL graduated cylinder)using a solution of ether;methanol (1:3, v/v), then put 2.0 mL of that in a 13x100mm tube, dry it under nitrogen gas and resuspend it in 0.2 mL of absolute ethanol. Then, add 0.1 mL of this resuspended solution to a test tube with 1.9 mL of absolute ethanol, to another test tube add 0.05 mL of the resuspended solution and 1.95 mL of absolute ethanol, and to a third test tube, add 0.02 mL of the resuspended solution and 1.98 mL of absolute ethanol.

To one test tube, add 2 mL of absolute ethanol. This is your blank.
At this point you should have 10 test tubes.
Add the 2.0 mL of the cholesterol reagent to each of the tubes and carefully mix with a vortex mixture. Incubate the reaction tubes for 30 min at room temperature and then determine the absorbance at 550 nm. Carefully transfer the solutions to the cuvettes. Wash any spills immediately with a bicarbonate solution, followed by copious amounts of water.

Using the standard curve for cholesterol, determine how much cholesterol was present in each of your fractions. Report these values as mg/g of egg tissue, and calculate the percent cholesterol for each fraction.

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