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Dr. Mohammed N. Sabir, BSc. Pharm, MSc., Ph.D. Pharmacog.

Lecturer, Dept. of Pharmacognosy and Pharmaceutical Chemistry
College of Pharmacy, University of Sulaimani
mohammed.sabir@univsul.edu.iq

Experimental pharmacognosy I
First semester, third-year pharmacy, 2021 – 2022

Experiment No. 3

Liquid-liquid fractionation for the alcoholic crude extract from Silybum marianum
seed.

The Silymarin seed crude extract obtained by soxhlet extractor using ethanol 70% is a mixture that includes
a wide spectrum of polar to medium polar molecules. The targeted metabolites from the mixture are the
flavonolignans which demonstrate the main pharmacological benefits. To separate the flavonolignans, it will
be necessary to remove the other lipophilic ingredients. A sequential partitioning with several organic solvents
that are immiscible with water and ranging from low to high dielectric constants will be very useful as a pre-
step for the chromatographic separation of the desired components. In each partitioning cycle, the crude
mixture components will be fractioned according to their preferences towards the solvents (like-dissolve-like).
The flavonolignans are more soluble in the ethyl acetate solvent.
This experiment aims to obtain a rich extract of flavonolignans from silymarin crude extract using
liquid-liquid fractionation.

Extraction procedures

1. Concentrate the alcoholic crude extract to a 15mL volume under a vacuum at 40°C using a rotary
evaporator.
2. Assemble two clean and dried separartory funnels on a stand.
3. Prepare 45mL of each n-hexane and ethyl acetate solvents.
4. Using a glass funnel, pour the concentrated crude extract into the separatory funnel.
5. Add 15mL n-hexane solvent to the separatory funnel gently.
6. Start the partitioning process by mixing (shaking) the components.
7. Let the separatory funnel stand with the stopper opened until a clear separation of the two immiscible
liquids is seen.
8. Evacuate the funnel (the lower aqueous layer), then the upper lipid layer in separate flasks.
9. Concentrate the lower layer under vacuum and reduce temperature.
10. Perform another two partitioning cycles.
11. Collect the n-hexane layers from the three fractionation processes.
12. Evaporate the n-hexane under vacuum and reduced temperature (25°C).
13. Lable the fixed oils fraction and preserve for further analysis.
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14. Partition the aqueous layer obteined after the previous three successive fractionations with equal volumes
of ethyl acetate solvent. Repeat the same procedure applied above.
15. Collect the ethyl acetate fractions and vacuum concentrate at 45°C.
16. The obtained concentrated portion is the flavonolignan rich extract.
17. Weight the rich extract and label and preserve for further analysis.
18. Make your report.

Materials
1. Rotary evaporator.
2. Glass beaker (200 mL).
3. Glass conical flasks (250 mL).
4. Glass funnel (100 mL).
5. Glass rod.
6. Measuring cylinder.
7. Distilled water.
8. n-hexane.
9. Ethyl acetate.
10. Reagent bottle.
11. Marker and pen.
12. Clamp.
13. Notebook.
14. Nitrile gloves.
15. Mask.
16. Sensitive electric balance.