Natural Products / Secondary Plant Metabolites / Terpenes
Essential Oils / Steam Distillation / Extraction
Introduction * Procedure

(Adapted from University of Colorado, Boulder; Department of Chemistry and Biochemistry)

Introduction:

A natural product is a chemical compound that is produced by a living organism. Natural products are found as mixtures of many compounds, which have to be separated and purified in order to be studied chemically. The ease in which a molecule of interest can be isolated and purified depends on its molecular structure, stability, and the amount of the compound available in the organism. For example, Alexander Fleming recognized the antibiotic qualities of penicillin and its remarkable non-toxic nature to humans, but he was unable to purify it. Many natural products like pencillin have been used as drugs to treat many different ailments from headache to cancer. Salicylic acid, which is found in willow bark, was used by several native cultures as an analgesic. Taxol, which was isolated from the Pacific yew, Taxus brevifolia, led to the semi-synthetic anti-cancer chemotherapeutic, Paxitel. And, the list goes on and keeps growing.

Plants are rich with a widely diverse array of molecules that are not involved with primary metabolism as proteins and carbohydrates are. The presence of these "secondary" metabolites is not fully understood, but many, if not all are believed to play some role in chemical ecology, such as pheromones and allomones: attracting, repelling, alarming, defending. A number of the plant natural products have been categorized as "essential oils", which produce a distinct scent such as clove oil.

Essential oils are generally isolated by distillation or solvent extraction. They are used in perfumes, cosmetics and bath products, for flavoring foods and beverages, and for aerosol sprays and household cleaning products. Various essential oils have been used medicinally in history. Medicinal interest in essential oils has been revived in recent years with the arrival of aromatherapy, a branch of alternative medicine.

Clove oil is an essential oil from the clove plant, Syzygium aromaticum, CAS: [8015-98-2]. It is a natural analgesic and antiseptic used primarily in dentistry for its main ingredient eugenol. It can also be purchased over the counter, as a home remedy for dental pain relief, and is also found in the aromatherapy section of health food stores. The main clove oil producing countries are Madagascar and Indonesia.

"Essential oils" generally do not have a specific property in common, beyond all of them having characteristic fragrances. But, many of them have chemical structures with two or more repeating units of a five carbon building block, isoprene, . These are referred to as terpenes, which are responsible for the odor of eucalyptus, pine, mint, lavender, rose, and others. Terpenes are often referred to as isoprenoids. They are classified according to the number of carbon atoms that they contain: 10 carbons is a monoterpene, 15 is a sesquiterpene, 20 is a diterpene, 25 is a sesterpene, 30 is a triterpene, and 40 is a tetraterpene. They respectively have 2, 3, 4, 5, 6, and 8 isoprene units in their structures.

Limonene, a monoterpene that is the chief component of orange oil, is widely used as a fragrance and flavoring, as well as a cleaning solvent. Organic chemists use terpenes and other natural products as chiral starting materials for complex chemical syntheses or as inspirations for pharmaceuticals. Some natural products are attractive synthetic targets because of interesting or unusual structural features or medicinal applications.

Isolation of natural products typically involves multiple extractions and chromatographic steps, but certain organic oils can be freed of contamination by subjecting them to a process known as steam distillation. In this lab, you will perform a steam distillation to isolate a natural product. You will then use IR spectroscopy to analyze your isolated essential oil.

Normally, a liquid boils when its vapor pressure is equal to the surrounding pressure (generally, this is the same as the atmospheric pressure, 1 atm. or 760 mm Hg). A solution that is a homogeneous mixture of two or more miscible liquids will boil when the combined vapor pressures of its dissolved components is equal to the sur-rounding pressure. The pressure of each component in a solution is related to its concentration in the mixture, and so the boiling point of a solution, or homogeneous mixture, is normally between the boiling points of the individual components.

A heterogeneous mixture of two immiscible liquids will also boil when the combined vapor pressures of its components is equal to the surrounding pressure, however, because the liquids are immiscible, the vapor pressures of the individual components are independent of one another and not related to their concentrations. The two liquids independently exert vapor pressures against the external pressure, and when the sum of the partial pressures is equal to the external pressure, boiling occurs. Thus, the total vapor pressure of a heterogeneous mixture is given by the following equation:

Ptotal is the total pressure of a system at a given temperature, and P°A, and P°B are the individual pressures of components A and B at the given temperature. Generally, a heterogeneous mixture will boil at some temperature below the boiling point of either component. In the figure below, individual vapor pressures are plotted against temperature. Pure compound B, which has the lower boiling point, will boil at a temperature slightly above 125 °C. But when compound A is also present, the combined pressures add up to 760 mm—and the mixture boils—when the temperature is about 85 °C. This boiling temperature is lower than the boiling point of either compound alone, and it is the result of the combined effects of both compounds.


We can take advantage of the fact that many water-insoluble liquids and solids behave in the manner described above for heterogeneous mixtures, volatilizing at temperatures below their boiling points. The effect described above is exploited in a technique called steam distillation, where an organic compound of moderate volatility and vapor pressure is distilled as part of a heterogeneous mixture with water. The boiling point of the mixture is slightly below 100°C, the boiling point of water. At this temperature, a fraction of the distillate will be the compound of interest. The greater the vapor pressure of the organic compound, the larger the fraction that will co-distill with the water. This technique is considerably more gentle than regular distillation, since some organic compounds can decompose at temperatures approaching their higher boiling points.

Procedure: (Budget 1.5 lab periods)

Clove Oil: (preferred)

Steam Distillation:
Weigh ~ 2 g of ground cloves, record an exact mass; transfer into your largest round bottom flask, add 2-3 boiling stones and enough deionized water to the flask so that it is slightly less than half full. Set up the steam distillation apparatus shown below. Place a heating mantle under the distillation flask, and heat to boiling. Periodically add ~5 mL portions of water from the separatory funnel to the distillation flask to replace the water that has been distilled over. Try to maintain a fairly constant liquid level in the distillation flask. Continue collecting distillate until the distillate is clear without any oil droplets. (Test by collecting a drop or two on a watch glass.) The total volume collected will be about 50-75 mL. Dispose of the spent clove residue in the waste container equipped with a funnel having a glass wool plug.

Isolation of eugenol:
Extract the distillate with 2 x 15 mL portions of dichloromethane. Combine the extracts. Extract the combined volume with 2 x 15mL portions of 1 M NaOH solution. Combine the aqueous layers. Discard the dichloromethane in the organic waste container.

Acidify the basic aqueous layer with 3 M HCl and extract with 2 x 15mL portions of dichloromethane. Combine the dichloromethane layers. Dry over anhydrous magnesium sulfate. Evaporate the dichloromethane.

Transfer the liquid to a clean, tared, labeled vial and record the mass. Obtain an IR spectrum. Turrn in the vial.

Post Lab Questions:

  1. Draw the structure of eugenol. Circle and label the chemical functions.
  2. On your IR: clearly identify and label the peaks that correspond to the functions cited above.
  3. Draw a separation scheme [organic layer (dichloromethane) vs. aqueous layer] that shows how eugenol was recovered from the distillate using NaOH and HCl. Use chemical equations and arrows to indicate electron movement for each step.
  4. Clove oil contains a minor component that has the formula C12H14O3. It produces eugenol and acetic acid upon acid catalyzed hydrolysis similar to methylsalicylate-salicylic acid. Draw a structure for the compound. Analyze your IR for its presence. Explain whether it is there or not from the IR data.
  5. Clove oil contains a structurally interesting isoprenoid in relatively small amounts. Its IUPAC name is (E)-4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene. What is the common name for this compound? Draw a structure for the compound. How many isoprene units are in the compound? Which class of terpene is it in?.... monoterpene, sesquiterpene, diterpene, sesterpene, triterpene, tetraterpene.

Experimental options: Follow the procedure above for anise, caraway, or cumin. For orange peel follow the procedure below.

Orange: (option)

Steam Distillation:
Peel two large oranges with a sharp knife or peeler. Remove just the exterior, brightly colored portion of the peel, called the “zest”, as the white material underneath (the pith) contains little or no oil. Determine the mass of the peelings—you should have about 25 g—then place them in a blender. Add about 30 mL water, put the lid on the blender, and blend the mixture until a smooth puree is obtained. Pour the mixture into a 100 mL round bottom flask equipped with several boiling stones. Attach the flask to the steam distillation apparatus shown for clove oil. Fill the separatory funnel about half-way with water. Place a heating mantle under the distillation flask, and heat to boiling. Periodically add ~5 mL portions of water from the separatory funnel to the distillation flask to replace the water that has been distilled over. Try to maintain a fairly constant liquid level in the distillation flask. Continue collecting distillate until you have about 100 mL. You should notice an upper layer of oil in the receiving flask.

Isolation of limonene:
Transfer the condensate to an empty separatory funnel. Allow the layers to separate and discard the lower, aqueous layer.

Dry the oil over anhydrous sodium sulfate, then transfer it to a clean, tared, labeled vial. Record the mass. Obtain an IR spectrum. Turn in the vial.