I.

INTRODUCTION

Eugenia aromatica is a tropical evergreen tree that can grow up to 30-40 feet high, has extensive
branching, yellowish bark, rose-colored flowers, and berry-like fruit (Christopher 1953). Its flower buds,
best used when dried and unexpanded, are known as “cloves”, which are the natural resource wherein
the natural product eugenol resides. Eugenol is a member of the allylbenzene class of chemical
compounds; it is used in perfumeries, flavorings, essential oils and in medicine as a local antiseptic and
anesthetic (Broadhurst and Duke 1997). It is also used as a means to preserve food through use of clove
oil wherein eugenol inhibits putrefaction, especially that of meat when cooked.

Similar to the previous exercise wherein piperine was isolated from black peppercorns, eugenol will be
isolated from cloves. The isolation process of eugenol will include steam distillation and liquid-liquid
extraction, followed by characterization through a standard color reaction. Distillation is a separation
process that can be used to separate a pure liquid from a mixture of liquids. Using simple distillation
with immiscible liquids would require high temperatures that could possibly cause the breaking apart of
important molecules; thus, distilling them in the presence of water can avoid this as the temperature is
kept low, which is what steam distillation achieves (Clark 2005). Liquid-liquid extraction, on the other
hand, is a batch-type phase-distribution technique that makes use of immiscible layers (organic and
aqueous) in order to separate mixtures (Cantwell and Losier 2002).

The cloves will undergo steam distillation in order to collect “oil of cloves” or clove oil, which is
comprised of around 85% eugenol, along with around 10% of acetyleugenol and caryophyllene (see
Figure 3.1.). However, the isolation of eugenol from oil of cloves depends largely on the chemically
active proton of its phenolic group.

FIGURE 3.1. Skeletal structures of the known constituents of “oil of cloves”.

Therefore, eugenol may be deprotonated by an alkaline solution to give a phenoxide ion that may be
extracted with water; this acidification, done by extraction of the organic layer with NaOH and addition
of HCl to the aqueous layer, reverts the phenoxide group to its original nonpolar form.

FIGURE 3.2. Acidification of the phenoxide group.

II. Objectives

III. Materials and Methods

A. Schematic Diagram of Procedures
B. Set-Ups
FIGURE 3.3. Steam distillation setup.
C. List of Necessary Chemicals
Dichloromethane
Ethanol
Eugenol
Ferric chloride
Hydrochloric acid
Magnesium sulfate
Sodium chloride
Sodium hydroxide
Sodium sulfate

IV. DATA

VI. Discussion

Eugenol, as was previously mentioned, is a natural product that can be isolated from cloves, which are
dried flower buds of the tropical evergreen tree Eugenia aromatica. It is a phenylpropanoid, is an allyl
chain-substituted guaiacol (as seen from its structure in Figure 3.1.), which is weakly acidic, slightly
soluble in water and soluble in organic solvents; it is a clear to pale yellow liquid with a characteristic
and pleasant odor of cloves and a spicy pungent taste (Kamatou, Vermaak and Viljoen 2012). Through
clove oil, eugenol is able to inhibit the putrefaction of meat when cooked, and can be used in a range of
different ways alluding to food preservation. However, clove oil also has antimicrobial, antifungal,
antiseptic, antiviral, aphrodisiac and stimulating properties (Organic Facts 2015); along with superior
anti-oxidant activity due the presence of its phenolic group (Kamatou et al. 2012), it is also often used
for treating a variety of health issues as well.

This aforementioned “clove oil”, which is comprised of mostly eugenol (approximately 60-90%) is while
it also contains traces of caryophyllene and acetyleugenol (around 10%) along with other minor
constituents such as gallic acid, sesquiterpenes, furfural, vanillin, and methyl-n-amyl ketone (Berestecky
2008), can be obtained by steam distillation of a clove-water mixture.

Steam distillation is used for most water-insoluble substances with very high boiling points (above
100degC so that water boils first and subsequently boils the substance with it), so that they may be
distilled at temperatures below their boiling points without the risk of bond-breaking that can occur at
very high temperatures. As such, with eugenol having a boiling point of approximately 253degC, it was
appropriate to use steam distillation so that the clove-water mixture may boil together at a lower
temperature (though slightly higher than 100degC due to the principle of boiling point elevation). The
duration of time required to perform the steam distillation is affected by the degree of comminution, or
the smallness of pieces produced by cutting the original sample, in a directly proportional relationship;
the greater the degree of comminution (the smaller the pieces), the smaller the essential oil yield
(Mejdoub and Katsiotis 1998), which thus requires a longer period of time to undergo distillation in
order to ensure a maximum amount of oil has been collected from the original sample. The main reason
of essential oil losses can either be due to the hardness of the sample to be used, as with comminution
these tissues keep the minuscule droplets of oil that should be liberated from the inner tissues, thus
avoiding the extraction (Mejdoub and Katsiotis 1998).

Deprotonation is done to the resulting clove oil by adding portions of a basic solution, in order to give a
phenoxide ion that may be extracted with water; in this exercise 10% sodium hydroxide (NaOH) was
used, though the same result will be reached if another solution is used, such as sodium bicarbonate
(Na2CO3) – this is due to the fact that organic compounds must be modified to become more ionic and
made more water-soluble in order to remove it from the organic layer, solutions with high
concentration are rarely used because of increased evolution of heat during extraction and potential
side reactions with the solvent (Bacher 2013). This portion of separation of eugenol from clove oil is
highly dependent on the chemically active proton of the phenolic group that is removed upon addition
of NaOH as eugenol is converted to an aqueous layer and the organic layer had portions of the other
constituents present in clove oil (see Figure 3.2.). Acidification follows to revert this process by adding
sufficient amounts of hydrochloric acid (HCl) to the combined aqueous layers (where eugenol is
currently present) until the solution reacts and becomes a murky mixture (see Table 3.1.); the addition
of HCl reverts the phenoxide group back to its original nonpolar form through H+ donation and eugenol
becomes part of the organic layer once more (see Figure 3.2.).

A drying agent is used when an organic solution has been in contact solution because of it being “wet”,
or basically containing traces of dissolved water even though the solution is typically immiscible with
water; common drying agents are anhydrous inorganic salts that acquire waters of hydration when
exposed to water-involved substances (University of Calgary 2015). In the exercise, the drying agent
used was magnesium sulfate (MgSO4), a powdery white solid. It was added into the organic layer that
had been washed with distilled water and half-saturated sodium chloride (NaCl), and the crystals settled
at the bottom of the Erlenmeyer flask in large clumps as they absorbed water so that the solution was
readily decanted, becoming relatively free of water, ready for the succeeding evaporation of
dichloromethane (DCM) through a steam bath. Following the evaporation of DCM, the remaining oil was
weighed and, seen in Table 3.2., the eugenol yielded 30.02%.

However, in order to confirm that the obtained liquid was indeed eugenol, or at least contained traces
of it, a standard color reaction was performed for characterization. Ferric chloride (FeCl3) is an
industrial-scale commodity chemical compound; its bond however, can be considered covalent polar as
well as ionic, as all bonds between elements have both ionic and covalent character and can therefore
lean towards both (Neto 2013). In FeCl3, the total electronegativity of their bond landing between the
range of that of covalent polar while also remaining a bond between a metal and a nonmetal.

Eugenol is known to form a blue complex upon contact with FeCl3, due to the phenol group present in
eugenol’s structure (see Figure 3.1.) which reacts with the ferric ion, following the chemical reaction
shown in Equation 3.1. (Harper College 2001). If the solution tested is negative for a phenolic group, the
resulting solution will be yellow or yellow-orange in color, but blue or blue-green for a positive solution,
which was also seen in the data recorded in Table 3.3.

[EQN 3.1.]

VIII. References

A. Journals

CANTWELL F.F., LOSIER M. 2002. Comprehensive Analytical Chemistry 37(1): 297-340.

KAMATOU G.P., VERMAAK I., VILJOEN A.M. 2012. Molecules 2012(17): 6953-6981.

MEJDOUB R., KATSIOTIS S.T. 1998. Scientia Pharmaceutica (Sci. Pharm.) 66: 93-105.

B. Web Resources

BACHER A.D. 2013. Extraction in Theory and Practice (Part I). Retrieved September 24, 2017, from:
http://www.chem.ucla.edu/~bacher/Specialtopics/extraction.html
BERESTECKY J.M. 2008. Some Common Spices and Plants with Antimicrobial and Therapeutic Properties.
Retrieved September 24, 2017, from:
http://www2.hawaii.edu/~johnb/micro/m140/syllabus/week/handouts/m140.8.3.html

BROADHURST C.L., DUKE J.A. 1997. Oil of Cloves: The Benefits of Eugenol. Retrieved September 12, 2017
from http://www.motherearthliving.com/plant-profile/eugenol

CHRISTOPHER J.R. 1953. CLOVES (Caryophyllus aromaticus; Eugenia aromatica; Eugenia caryophyllata).
Retrieved September 12, 2017 from http://online.snh.cc/files/2100/HTML/snh_cloves.htm

CLARK J. 2005. Immiscible liquids and steam distillation. Retrieved September 12, 2017 from
http://www.chemguide.co.uk/physical/phaseeqia/immiscible.html

HARPER COLLEGE. 2001. The FeCl3 Test. Retrieved September 24, 2017, from:
http://www.harpercollege.edu/tm-ps/chm/100/dgodambe/thedisk/qual/fecl3.htm

NETO N.S. 2013. Bonds between metals and non-metals. Retrieved September 24, 2017, from:
https://chemistry.stackexchange.com/questions/6923/bonds-between-metals-and-non-metals

ORGANIC FACTS. 2015. 23 Surprising Benefits of Clove Oil. Retrieved September 24, 2017, from:
https://www.organicfacts.net/health-benefits/essential-oils/health-benefits-of-clove-oil.html

UNIVERSITY OF CALGARY. 2015. Drying agents. Retrieved September 24, 2017, from:
http://www.chem.ucalgary.ca/courses/351/laboratory/drying%20agents.pdf