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7. Add 4 ml acetone, to the pellet. Mix thoroughly.
8. Centrifuge and discard the supernatant.
9. Dry the residue by placing the tube in a boiling water-bath.
10. Then, add 6 ml glacial acetic acid and solubilize the residue by placing the
tube in a boiling water-bath for 5 min.
c. Cholesterol standards & Blank
1. Prepare a series of cholesterol standards at a concentration range of 0 - 0.3
mmol/L.
Notes: Dilute with glacial acetic acid.
2. The final volume should be 6 ml.
FERRIC
GLACIAL TOTAL
CONCENTRATIO GLUCOSE CHLORIDE
TUBES ACETIC mL PER
N (mg/dL) STANDARD (mL) SULPHURIC
(mL) TUBE
(mL)
Glacial
M1V1 : M2V2 Acetic Acid
1 0 (0.3)V1: 0 (6.0 ) = 6.0 ml - 6.0 4.0
V1 : 0 mL 0.0 mL
= 6.0 mL
Glacial
M1V1 : M2V2 Acetic Acid
2 0.05 (0.3)V1: 0.05 (6.0 ) = 6.0 ml - 6.0 4.0
V1 : 1 mL 1.0 mL
= 5.0 mL
Glacial
M1V1 : M2V2 Acetic Acid
3 0.10 (0.3)V1: 0.10 (6.0 ) = 6.0 ml - 6.0 4.0
V1 : 2 mL 2.0 mL
= 4.0 mL
Glacial
M1V1 : M2V2 Acetic Acid
4 0.15 (0.3)V1: 0.15 (6.0 ) = 6.0 ml - .0 6.0 4.0
V1 : 3 mL mL
= 3.0 mL
5 0.20 M1V1 : M2V2 Glacial 6.0 4.0
(0.3)V1: 0.20 (6.0 ) Acetic Acid
V1 : 4 mL = 6.0 ml -
2
4.0 mL
= 2.0 mL
M1V1 : M2V2 Glacial
(0.3)V1: 0.25 (6.0 ) Acetic Acid
6 0.25 V1 : 5 mL = 6.0 ml - 6.0 4.0
1.0 mL
= 5.0 mL
M1V1 : M2V2 Glacial
(0.3)V1: 0.30 (6.0 ) Acetic Acid
7 0.30 V1 : 6 mL = 6.0 ml - 6.0 4.0
6.0 mL
= 0.0 mL
TUBES 1 2 3 4 5 6 7
TEST ABSORBANCE
TOTAL
0.424
CHOLESTROL
FREE
CHOLESTERO 0.021
L
CALCULATION:
TOTAL CHOLESTEROL • From graph: • Total Cholesterol in 10 ml
= 0.080 mmol/L (In 1000ml) solution (0.5 ml + 9.5 ml
• 1 ml solution; Acetone Alcohol).
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o 0.080/1000 = 10 (8.0 X 10-4 ) / 2
• 10 ml solution; = 4.0 X 10-4 mmol Cholesterol
o 0.080/1000 X 10 • This equivalent to amount
= 8.0 X 10-4 mmol Cholesterol of Cholesterol in 0.5 ml
• This equivalent to Total serum.
DISCUSSION:
In this experiment, acetone alcohol is used for extraction process of cholesterol. This done
by separating the cholesterol from serum by centrifugation process. The cholesterol extract can
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used for to determine the esterified and unesterified cholesterol.
Acetone alcohols vaporized and leave behind the cholesterol. Free cholesterol is easily
determined by direct precipitation. Digitonin is a substrate to differentiate free cholesterol from
total cholesterol and esterified cholesterol. In digitonin precipitation method, alcoholic solution of
cholesterol is mixed with an alcoholic solution of digitonin, which formed cholesterol digitonide.
This complex has a low solubility and making the test is more sensitive.
After precipitation is completed, digitonide is separate into its component. Ester and
digitonide give a purple colour of equal intensity. Cholesterol digitonide is insoluble in water but
soluble in glacial acetic acid. Ferric chloride-sulphuric acid is added to the tube when measuring
the density for cholesterol standard as the radiators.
Below are the information ranges for the serum cholesterol:
3.6-5.2 mmol/L (normal)
5.3 -6.2 mmol/L (borderline high)
> 6.2 mmol/L (hypercholesterolemic)
The serum cholesterol that obtained from this experiment is 7.0 mmol/L. This means the
serum sample is hypercholesterolemic. The free cholesterol level is 1.0 mmol/L and indicated low
than normal ranges. This is because of vaporizations process of acetone alcohol, which means by
overheat it.
CONCLUSION:
1. Concentration of the total cholesterol is 7.0 mmol/L.
2. Whereby, the free cholesterol is 1.0 mmol/L.