Isolation and Characterization of Eugenol from Cloves

Bella, Rossana Carmela M. Blasurca, Jolyn Naira G. Cruz, Heidi S. De Guzman, Jean Marco T.

Abstract Natural Products are compounds that can be found or extracted in nature and usually produced by plants, animals, and/or fungi. Phenyl propanoid is a class of natural product in many aromatic plants. Eugenol is an example of phenylpropanoid that will be isolated from cloves in this experiment by steam distillation process. The essential oils were co distilled with water at temperature that is below the boiling points of the components. The emulsion of water and the essential oil can be further separated by organic solvent extraction. In this experiment, clove oil was isolated by steam distillation followed by hexane extraction. Eugenol from clove oil was purified by chemically active extraction, wherein the acidic property of the compound was taken advantage of to isolate it from other minor constituents. Introduction Essential oils are volatile, ethereal liquids of oily consistency. They are extracted from plants, usually by steam distillation or extraction. The oils have odors highly characteristic of the original plants. They were thought to be the essence of plants hence the term essential oil. Eugenol is the main ingredient of the essential oil that can be isolated from the spice clove. Cloves are the dried flower buds of the clove tree, Eugenia caryophyllata, found in India and other locations in the Far East. It belongs to a large class of natural products called the essential oils. It is well known that cloves possess a phenolic compound, 4-allyl-2-methoxyphenol, commonly called as eugenol, with a chemical formula of (C10H12O2). Eugenol acts as an antioxidant on olegenous foods, as an anti-carminative, antispasmodic, antiseptic in pharmacy and also as an antimicrobial agent. Steam distillation of freshly ground cloves results in clove oil, which consists of several compounds. It is commonly used to extract chemicals from plant material. In this method, steam is passed through the plant material containing the desired oils. The steam is produced at greater pressure than the atmosphere and therefore boils at above 100 degrees Celsius which facilitates the removal of the essential oil from the plant material at a faster rate and in so doing prevents damage to the oil. Methodology Part 1: Steam Distillation of Clove Oil 5.00 grams of ground cloves was weighed on a triple beam balance and it was transferred quantitatively into a 250-ml distilling flask. 75.0 ml of distilled water and a few boiling chips were added. The contents were swirled gently to mix. The steam distillation set-up was assembled as shown to Figure 1. The water supply and heat source were turned on. The temperature was recorded at the point where the students started to collect the initial distillate.

The heat was adjusted properly to prevent rapid boiling and foaming of the mixture. A total of 50 ml of distillate was collected while recording the temperature for every 5.0 ml of collected liquid. A distillation curve was constructed with the gathered data. The water and heat supply was then turned off when the needed sample have collected. The residue was disposed properly and the set-up was disassembled when it was cooled for handling.

Figure 1. Steam Distillation Set-up Part 2: Active Solvent Extraction and Characterization The distillate was transferred into a 250-ml separatory funnel and 3.0 grams of NaCl was added. It was swirled to dissolve the salt. 7.0 ml of hexane was added, it was closed by a stopper then it was shaken for 5 minutes with occasional venting in order to release the built-up pressure inside. After 5 minutes of shaking, there were two layers formed. The separatory funnel was placed through an iron ring and the layers were allowed to separate. The aqueous layer at the bottom was drained to a 50-ml Graduated cylinder; the organic layer was left inside. A set-up for the separation of layers was prepared and is shown in Figure 2 below. The aqueous layer was discarded to the sink and flushed with copious amount of water. 25.0 ml of 10% NaOH was added to the remaining hexane extract, stopper then shaken for 5 minutes with occasional venting in order to release the built-up pressure inside. The separatory funnel was placed through an iron ring and the layers were allowed to separate. The aqueous layer at the bottom was drained to a 50-ml Graduated cylinder; the organic layer was left inside. The aqueous layer was discarded into an acid waste jar. The acidified aqueous layer was placed back in the separatory funnel. 7.0 ml of Hexane was added, repeating the same shaking and venting process for five minutes. The bottom aqueous layer was discarded, and the upper organic layer was left inside the separatory funnel. A small amount of anhydrous sodium sulphate was added to the obtained upper organic layer then the contents were swirled for a couple of seconds. The hexane extract was filtered into two small glass vials in order to remove the sodium sulphate. About 10 drops of the extract was placed into two separate test tubes. 1.0-ml of FeCl3 was added to one test tube and 1.0-ml of Bayers reagent to the other. Then the ensuing reactions were observed. The extract was placed under the fume hood for 1 day. Afterwards, the weight of the obtained eugenol was determined and the percent yield was calculated.

Figure 2. Separation Set-up Results and Discussion A. Yield of Eugenol and Chemical reactions Mass of Ground Cloves: 5 grams Mass of Eugenol Obtained: 3 grams Percentage Yield: 60% Color reaction or Eugenol with 1% FeCl3: none Color Reaction of Eugenol with Bayers reagent: brown Eugenool in cloves that was used in grounded form is 5 grams. The amount obtained after the simulation of the laboratory activities is 3 grams. Eugenol when reacted with 1% FeCl3 showed no reaction. Phenols generally change in color when they are complex. The more complex a phenol is, the longer the appearance of color change. Hence, the obtained Eugenol is not that complex. On the other hand, potassium permanganate of the Bayers reagent showed a positive result with the Eugenol since there was MnO2 that was formed after the reaction took place which is a brown precipitate. B. Distillation Curve

Temperature C - Volume
120 100 80 60 40 20 0 0 5 10 15 20 25 30 35 40 45 50 ml ml ml ml ml ml ml ml ml ml ml

Temperature C

The figure above shows the relationship of temperature with the volume of Eugenols initial distillate in the experiment. From the first 5 ml calculation to its last required amount, which is 50 ml, it showed constant temperature which is 100oC.

GUIDE QUESTIONS 1. Draw the structures of Eugenol, Eugenol acetate, and caryopyllene and explain the basis of separation of eugenol from the other components in clove oil.

Eugenol contains a carbon-carbon double bond and an aromatic hydroxyl group called a phenol. It is responsible for the aroma of cloves. It is the main component in the essential oil extracted from cloves, comprising 7290% of the total. These functional groups provide the basis for simple chemical tests used to characterize the clove oil. Eugenol is not soluble in all liquids, so depending on the % of eugenol in a particular preparation, it will separate out of the solution over time. 2. What is the temperature volatilization of Eugenol. Is this higher or lower than the actual boiling point of eugenol and why? The actual boiling point of Eugenol is 256oC and its volatilization is 243oC which is 10oC lower than its boiling point

3. What are the biological and pharmacological properties of eugenol? Eugenol, the major constituent of clove oil, has been widely used for its anesthetic and analgesic action in dentistry. Eugenol exhibits pharmacological effects on almost all systems and our aim is to review the research work that has identified these pharmacological actions. Eugenol possesses significant antioxidant, anti-inflammatory and cardiovascular properties, in addition to analgesic and local anesthetic activity. The metabolism and pharmacokinetics of the compound in humans have been studied. Eugenol has also been used as a penetration enhancer. The compound is a very promising candidate for versatile applications, and the design of new drugs based on the pharmacological effects of eugenol could be beneficial.

4. Illustrate the reaction between eugenol and FeCl3.What is the implication of this reaction?

Reaction of Eugenol to Ferric chloride (FeCl3) The ferric chloride test is used to determine the presence of a phenol structure in a compound. Change incolorations such as turning blue, red, green, or purple shows that phenol is present. [1] 5. Illustrate the reaction between eugenol and Bayers reagent. What is the implication of this reaction?

Reaction of Eugenol to Potassium permanganate (KMnO4) Bayers test using Bayers reagent or potassium permanganate (KMnO4) is used to test unsaturation or the presence of alkyne structures or double bonds. [2] REFERENCES [1]http://seattlecentral.edu/faculty/ptran/bastyr/Summer%2008/organic/packet%20org%20chem. pdf [2]http://van.physics.illinois.edu/qa/listing.php?id=505
http://www.ncbi.nlm.nih.gov/pubmed/21299140

http://www.chemistry.sc.chula.ac.th/bsac/Org%20Chem%20Lab_2012/Exp.7%5B1%5D.pdf