Isolation and Characterization of Eugenol from Cloves

Alyssa Lynn P. Emata De La Salle University Dasmarinas Dasmarinas City, Cavite, Philippines

ABSTRACT The isolation of essential oil (eugenol) from botanical sources such as cloves undergoes a classic method of steam distillation. Eugenol is an phenylpropanoid, made up of C=C and an aromatic hydroxyl group, phenol. This experiment undergoes in 2 part procedure to isolate eugenol from the powdered cloves. First part was steam distillation of clove oil. Eugenol, essential oil from o cloves, is 80 90% found in cloves. In nature, eugenol needs 254 C to boil but because of coo distill with water (100 C), it boils lower than the boiling point of the 2 pure substances because of o o Ptot = PA + PB leads having higher total vapor pressure that also leads to lower boiling point than their natures boiling point. Second part was active solvent extraction by hexane extraction where the eugenol isolated from other constituents pertaining to eugenol acetate and beta caryophyllene. The extract from the done active solvent extraction was test and characterizedvthrough 2 test Bayers test and Ferric chloride (FeCl3). Bayers reagents only works for unsaturated hydrocarbon, specifically in Alkene group resulting for oxidizing the C=C and gave a positive result due of reaction, purple color turns to brown precipitate. Ferric chloride works for phenol (Ar-OH) and for positive results, it gave purplish color from the original color, yellow.

INTRODUCTION Eugenol, an phenylpropanoid which synthesized from amino acid and phenylalanine in may aromatic plants like, cloves, Syzygium aromaticum, the best source of eugenol, where 80-90% of its component is eugenol while the remaining 10% is the eugenol acetate. It is made up of C=C and an aromatic hydroxyl group, phenol. (1)(2) It is widely used for its anesthetic and analgesic action in dentistry, posseses significant antioxidant, anti-inflammatory and cardiovascular properties, also act as penetration enhancer. Steam distillation is performing a co-distillation with water where oil can be isolated with lower temperature than the actual boiling point of the two components. (3,4) Active solvent extraction is the process by which the oil (eugenol) isolated from the other constituents of cloves oil.

MATERIALS AND METHOD 5.00 g of powdered clove was undergoing to steam distillation, see figure 1.

Figure 1. Steam Distillation with Powdered Cloves and Distillate (cloudy) After collecting the distillate, the boiling point was recorded. 3.0 g of NaCl was added to the distillate and let to cool down. The organic compound drives into organic layer and transferred to separatory funnel (see figure 2) where 25.0 ml of C6H14 (Hexane) was added and shaken to extract the organic clove oils from the aqueous layer and shaken and vent to release pressure. After 5 minutes, 2 layers was formed, the bottom aqueous layer was drained and discarded. 25.0 ml of 10% NaOH was added , same procedure after adding hexane, shaking, venting, discard aqueous layer, this procedure repeats also after adding 6.0 M HCl to the remaining organic layer to acidify, and by the used of red litmus paper, the acidity was tested and repeat again the shaking, venting steps after adding the reagents. Meanwhile, anhydrous Na2SO4 was added and swirled. The purified extract were undergo two test, Bayers test and Ferric Chloride (FeCl3) to check the presence of eugenol. The % yield was computed.

Figure 2. Separatory funnel and Erlenmeyer flask

RESULTS AND DISCUSSION A. Yield of Eugenol and Chemical Reactions MASS OF GROUND CLOVE: 4.8 g MASS OF EUGENOL OBTAINED:0.3 g % YIELD:..5% COLOR REACTION OF EUGENOL WITH 1% FeCl3:.Yellow Purplish COLOR REACTION OF EUGENOL WITH BAYERS REAGENT:.Purple Brown B. DISTILLATION CURVE

Temperature (Degrees Celcuis)

120 Temperature oC 100 80 60 40 20 0 0 20 40 60 Volume (ml) Temperature (Degrees Celcuis)

Solution: Mass of eugenol obtained: 99.74 g (Mass of Erlenmeyer flask with eugenol) 91.16 g (Mass of Erlenmeyer flask) 8. 58 g

13. 8 ml ( eugenol) x 0.60 g/ml (hexane) = 8.28 g 8.58 g 8.28 g 0. 30 g % yield: 0.3 g ( mass of eugenol) / 4.8 (mass of cloves) x 100 = 6%

H3C

CH2

HO
Eugenol

Eugenol acetate

Figure 4. Structure of EUGENOL in Skeletal structure (top), EUGENOL ACETATE (left), BETA CARYOPHYLLENE (right) (6)

H2O-soluble sodium eugenate (sodium 4-(2-propenyl)-2- methoxyphenoxide) was formed after the eugenol reacts with NaOH . The beta-caryophyllene and acetyleugenol remain in the organic layer and the eugenol was protonized . The polar(eugenol) and non- polar layers (beta-caryophyllene and acetyleugenol) are separated, the solution was acidified with HCl to regenerate eugenol and recharged. Because of organic solvent was added each time, the eugenol was extracted from its other minor consistuents. Non polar compound dissolves in organic solvents. (5)

In nature, eugenol needs 254 C to boil but because of co- distill with water (100 C), it boils lower o o than the boiling point of the 2 pure substances because of Ptot = PA + PB leads having higher total vapor pressure that also leads to lower boiling point than their natures boiling point. Ptot = PA + PB
o o o o

(254 C)PA + (100 C)PB = 354 C The vapor pressure and the external pressure is equal (VP=AP), also leads to lower boiling point o o than their natural boiling point. Lower than 254 C, and also must lower than 100 C.(2) It is widely used for its anesthetic and analgesic action in dentistry, posseses significant antioxidant, anti-inflammatory and cardiovascular properties, also act as penetration enhancer. Steam distillation is performing a co-distillation with water where oil can be isolated with lower temperature than the actual boiling point of the two components. (3,4)

Figure 5. Reaction between Eugenol and FeCl3 (7) Ferric chloride works for phenol (Ar-OH) and for positive results, it gave purplish color from the original color, yellow. It shows the characteristic of Eugenol having an aromatic compound.

Figure 6. Reaction between Eugenol and Bayers reagent (7) Bayers reagent known to test the C=C in your solution. In nature, Bayers reagents only works for unsaturated hydrocarbon, specifically in Alkene group resulting for oxidizing the C=C and gave a positive result due of reaction, purple color turns to brown precipitate. (2,7)

REFERENCE: (1) manual (2) Distillation Introduction Retrived 3 January 2013 from http://classes.kvcc.edu/chm220/Clove_Oil_Extraction/prelab/introduction.htm (3) Paramod, K., Ansari, SH., Ali J., Eugenol: A natural compound with versatile pharmacological actions December 2010 Retrieved 3 January 2013 from www.ncbi.nlm.nih.giv/pubmed/21299140 (4) Steam Distillation: The isolation of eugenol from clove Retrieved 3 January 2013 from www. Westfield.ma.edu/cmasi/organic_lab/organic_lab/isolation_of_eugenol.pdf. (5) Retrieved 3 January 2013 from http://www.sonic.net/miyasaki/SRJC/Exam%20solutions/12B%20Exam%20I%20Key%20S04. pdf (6) Chapter 6 - Clove Oil (eugenol) Marin Municipal Water District. 2013. Retrieved 3 January 2013 from http://www.marinwater.org/documents/Chap6_CloveOil_8_28_08.pdf (7) Eugenol reaction between, FeCl3 and Bayers reagent Retrived 3 January 2013 from http://www.seattlecentral.edu/faculty/ptran/bastyr/summer%2007/organic%20lecture/Lab%20P acketfall07.pdf