| Entomology 401/801. Insect Physiology |
| Dr. David W. Stanley |
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Lab 9. Isolation of Natural Product.
Extraction and Purification of Cinnamaldehyde from Cinnamon Sticks.
Objective:
To extract and purify the active ingredient of a spice from its natural product source using a solvent extraction method.
Introduction.
One of the easiest physical methods for isolating a compounds is solvent extraction. In solvent extraction, a compound is separated from inert or impure material using a solvent. The choice of solvent is important since it should preferentially dissolve the desired material. An everyday example of solvent extraction is the preparation of coffee or tea: in this case, a variety of organic compounds is leached out of the coffee grounds using hot water. Many flavors, extracts, and medicines are isolated from plants using simple solvent extraction techniques.
How are coffee, tea and colas decaffeinated?
In this laboratory, you will use very hot water to initially extract the oil of cinnamon from cinnamon sticks.
| Cinnamon is the dried inner bark of a tropical evergreen tree, of which there are about 100 different species, all with similar aromatic properties. The two most commonly available varieties are Ceylonese cinnamon and Chinese cinnamon. Chinese cinnamon, which is actually from the bark of the cassia tree, is not considered a true cinnamon (species Cinnamomum verum). Grown in Southern China, and other parts of Eastern Asia, cassia is a dark reddish color and stronger in flavor than its Ceylonese cousin (Cinnamomum zeylancium). Cassia is less expensive to process than true cinnamons and is the type of "cinnamon" most commonly sold in supermarkets--though it is sometimes blended with Ceylonese cinnamon. |
An organic solvent, ethyl ether, is then used to isolate the major organic component of the oil, cinnamaldehyde. Cinnamaldehyde is the compound that gives cinnamon its unique aroma and taste. As you might guess, cinnamaldehyde is an organic compound containing an aldehyde functional group.
The material that is isolated in this lab will not be pure cinnamaldehyde but rather a mixture of organic compounds, with cinnamaldehyde as a major component. We will further purify this extract using a micro silica chromatographic column. The column will be made from a 1000 mL pipette tip containing a silanized glass-wool plug with 500 mg of acidified silica gel. The cinnamon organic mixture will be eluted with 2x500 mL n-hexane, 2x500 mL, n-hexane/methanol (1:1 v/v) and 2x500 mL methanol. The eluted solution should be collected into tarred eppendorfs, evaporated and assessed for cinnamaldehyde by smell. We will take the elutant that is "purified" cinnamaldehyde and suspend in isooctane to yield a 1.0mg/mL solution. This will be injected into the Gas Chromatograph/Mass Selective detector during our next lab session.
For review of Column Chromatography
We will basically follow this Procedure for Microscale Flash Column Chromatography
Lab Procedure
Extracting Cinnamaldehyde from cinnamon stick
1. Set up a 400-mL beaker as a hot bath using a hot plate
2. Mass out about 1.0 to 1.5 g of slivered cinnamon stick. Place the pieces in a reaction vial.
3. Add ~ 5mL nanopure water to cover the cinnamon stick pieces then seal the vial with a Teflon lined cap.
4. Sit the reaction vial into the hot bath and reflux for 20-25 minutes.
5. After refluxing, remove the vial, allow to cool then place in cold water bath for 3 minutes.
6. Pipet the aqueous fraction from the cinnamon stick into a clean vial. Be sure the pieces of cinnamon stick are not removed with the solution. You should be able to recognize the cinnamon smell.
7. Extract the organic fraction with ethyl ether. Add 2 mL of ethyl ether to the cinnamaldehyde/water mixture, vortex the vial on low speed for 15 seconds, centrifuge using the clinical centrifuge at 1300 xg for 1 minute.
8. Remove the top organic fraction and place into a clean reaction vial.
9. Repeat steps 7 & 8 two more times.
10. Dry the organic fraction with anhydrous sodium sulfate. Add only a small amount (tip of a microspatula). If the sodium sulfate clumps, then add more sodium sulfate.
11. Transfer the dried organic fraction to a clean, tarred reaction vial. Rinse the sodium sulfate with 2 mL of clean ethyl ether and combine with the dried organic fraction.
12. Evaporate the ethyl ether then mass the vial containing the cinnamaldehyde oil. The boiling point of cinnamaldehyde is 248 oC so there is little threat of losing product. Record your results.
13. Notice the smell of the oil that remains.
Purification by flash chromatography
14. Set up a flash chromatography column as directed by your instructor.
15. Dissolve the oil into 100 uL of n-hexane and add to the silica top in the column.
16. Rinse the vial with 50 mL of n-hexane and add to the top of the silica. (X2).
17. Using 2x500 mL n-hexane, 2x500 mL, n-hexane/methanol (1:1 v/v) and 2x500 mL methanol as eluting solvents, collect each fraction into a tarred eppendorf.
18. Evaporate the solvents from the eppendorfs and assay for cinnamaldehyde by smell.
19. Mass the eppendorf with the most distinct cinnamon smell.
20. Suspend the cinnamaldehyde in isooctane to make a 1.0mg/mL solution. Store until next week