Extraction of limonene using dry ice

Experiment: Extraction of limonene from orange or lemon peel using liquid carbon dioxide.

Theory: Essential oils are organic compounds that are extracted from natural sources and used in flavorings, fragrances and cleaning products. Limonene an essential oil is a chiral molecule. Citrus fruits contain D-limonene ((+)-limonene), which is the (R)-enantiomer. D-limonene give a lemon or orange fragrance and L-limonene has turpentine-like odor. Limonene takes its name from the peel of the lemon. Racemic limonene is known as dipentene.

Traditionally essential oils have been extracted through the use of steam distillation or organic solvent (pentane or DCM) extraction. During the past two decades, great strides have been made in technology that uses supercritical or liquid carbon dioxide in place of organic solvents. The tunable solubility properties, low toxicity, and ease of removal of CO₂ have led to well established CO₂ technology for the extraction of various food products, including essential oils and hops, and for the decaffeination of coffee and tea.

Phase diagram of CO₂

Due to the accessible phase change, carbon dioxide can be used for bench top extraction processes. Unlike other gases, relatively low temperatures and pressures can be used to form liquid and supercritical CO₂. Dry ice (solid CO₂) sublimes (goes directly from solid to gas) at normal atmospheric pressure of ~1atm. The triple point of CO₂, where solid, liquid, and gas phases coexist in equilibrium, is achieved at 5.1atm and -56.6°C. At or near this point, dry ice melts, forming liquid carbon dioxide. If the temperature and pressure are increased to the critical point (72.8atm and 31.0°C), the CO₂ exists as a supercritical fluid and has no distinct liquid or vapor phase, but properties that are similar to both. Supercritical carbon dioxide (ScCO₂) is a fluid state of carbon dioxide where it is held at or above its critical temperature and critical pressure.

Dry ice sublimes at atmospheric pressure and temperatures above -78°C. If the CO₂ is sealed in a vessel during sublimation, the internal pressure in the vessel increases. After the temperature and pressure have increased sufficiently, liquid CO₂ forms.

Although CO₂ is a greenhouse gas, when used as a solvent it is captured from the atmosphere, not generated, resulting in no net environmental harm. The greenness of the CO2 extraction process can be accessed through the evaluation of waste, purity, energy use, yield, and safety.

ScCO₂ is a very good solvent to extract various natural products, but it requires specially designed equipment and is expensive. An effective alternative is use of liquid CO₂.

Procedure:

1. Grate only the colored part of the orange peel.

2. Record the mass of a 15 mL polypropylene centrifuge tube.

3. A solid trap is constructed by bending copper wire into coils and a handle.

4. The grated orange peel (2 g) is placed in the tube, and the tube is filled with dry ice and sealed with a cap.

5. The prepared centrifuge tube is placed in warm (40-50º C) tap water inside a plastic cylinder.

(Any sudden increase in temperature of surrounding water when the centrifuge tube is under pressure can cause the cap to blow off suddenly and violently.)

6. Pressure will begin to build in the tube and gas will escape slowly from the region where the tube and the cap meet.

7. After few seconds, liquid CO₂appear.

8. As the liquid boils, it should pass through the peel and move to the bottom of the tube.

(If it cannot reach the bottom of the tube, the oil will deposit in the region of the tube containing the orange peel. This does not allow for isolation of the product.)

9. After the liquid has evaporated and gas is no longer escaping, remove the tube from the cylinder with tweezers and open the cap. Open centrifuge tubes slowly and only after the gas has escaped. Opening tubes that are under pressure could result in the cap shooting a great distance.

10. Repeat the extraction by refilling the tube with dry ice, resealing the cap, and putting the tube back in the water. A third extraction can be performed in the same manner if desired.

11. Pale yellow oil is obtained in the tip of the tube when the extraction is complete.

12. Carefully remove the solid and the trap by pulling the wire handle with tweezers. If any solid remains in the tube, remove it with a spatula or wire.

13. Dry the outside of the tube, weigh the tube, and determine the mass of the product.

Observations and calculations

• Calculated the mass of the product:

(mass of tube and product)-(mass of empty tube) =

• Calculated percent recovery: (mass of product)/(mass of orange rind) x 100 =

Confirmation test for Limonene

Bromine-water test for unsaturation: A dilute Bromine-water solution is prepared and taken in a test tube. To Bromine-water solution citrus oil extracted from Lemon peels is added. If limonene is present in the oil extracted, the colour of the Bromine - water gets changed from red brown to pale yellow. This is because of the fact that the Bromine present in the Bromine – water solution occupies the space between the two double bonds present in limonene.

Safety and precautions

· Use gloves when working with dry ice.

· Contact with dry ice can damage skin tissues.

· Do not liquefy CO₂ more than 5 times in the same centrifuge tube.

· After repeated liquefaction, the tube may become brittle and rupture.

· The plastic cylinder functions as a secondary container and protects you from possible injury. If the tube shatters or the cap shoots off, any projectiles will be directed straight up. Do not place anything (including your face) above the cylinder.

· Watch the extraction from the side, not the top, of the cylinder.

· It is sometimes helpful to slowly rotate the cylinder on its base to prevent the centrifuge tube from freezing to the side of the cylinder.

· Never remove the tube from the plastic cylinder when the CO₂ is liquid. Tubes may rupture due to pressure and therefore must always remain in secondary containment.

Result:

Mass of the product:

Percent recovery:

Confirmatory test for limonene