ISOLATION OF LIMONENE AND IDENTIFICATION OF THE ANTIMICROBIAL

PROPERTIES OF POMELO (Citrus maxima) EXTRACT

INTRODUCTION
Citrus oils are mixtures of very volatile components as terpenes and oxygenated compounds.
Limonene, a monoterpene and most common terpenes in nature, is the major constituent in several citrus
essential oils (orange, lemon, mandarin, lime, and grapefruit). These oils are used in the pharmaceutical,
perfumery and food industries and the quality of the oils is related to the value of total aldehydes,
basically citral content, which is between 4-5%.
Pomelo (Citrus maxima)
In this experiment we try to isolate Limonene from Pomelo peel it said that limonene can be
found in all most all citrus fruits in various degrees. The main concentration lies in the zest of the skin of
the fruit. And (+)-Limonene can be found in all most all cases of Citrus family and since it is not soluble
in water hence it is like more soluble in organic solvent one way to do it is by using the method steam
distillation it will appear as an oily layer on top of the water layer.
It is found that most of the Citrus family exhibits extraordinary antimicrobial properties
in this experiment we also want to investigate and identify the antimicrobial properties POMELO (Citrus
maxima)

OBJECTIVES
1. To isolate limonene from pomelo peels through steam distillation
2. To analyze the essential oil obtained through GC-MS
3. To test the microbial properties of the essential oil in a gram positive and gram negative bacteria

MATERIALS

Apparatus
500mL round bottom flask

Chemicals
ether

250 ml volumetric flask

Anhydrous magnesium sulfate

Pipette
Aspirator
Separatory funnel

Agar
Bromine water
E. coli

Tripod
Condenser
Iron ring, iron clamp, iron stand
Rubber tubbing
Graduated cylinder
Autoclave

Staph. aureus

Petri Dishes
250mL Erlenmeyer Flask
wire loop
Autoclavable plastic bags
Alcohol lamp
Vernier caliper / ruler
Aluminum foil
Test tubes
Test tube rack
50mL beaker
microspatula
PROCEDURES
A. STEAM DISTILLATION OF POMELO PEELS
1. Wash and slice the pomelo peels into small pieces and weigh about 100 g of the cut pomelo peels.
2. Put the pomelo peel in the 500 ml round bottom flask and add about 250 ml of distilled water.
Caution: The peel will swell during the distillation! If pieces are too large, it will impossible to get
the peels out of the flask when the distillation is complete. Clamp the flask securely in the
distillation set up as shown in Figure 1.
3. Heat the flask with stirring and vigorously distill the mixture until you have collected about 50 to 70
mL of distillate. Note: You can observe an oily suspension in the distillate, it is the formation of
limonene.
4. Transfer about half of the distillate in a clean vial in preparation for the GC-MS analysis and
antimicrobial properties test of essential oil.
B.
1.
2.
3.

ISOLATION OF LIMONENE
Transfer the distillate into a separatory funnel.
Extract the distillate with 20 mL of ether. The lower layer is the remaining aqueous distillate.
To finalize the extraction, the ether layer (b.pt. 37C) will be evaporated on a water bath to leave
the limonene (b.pt. 176C).
4. Transfer the limonene extracted in a clean vial or beaker in preparation for test for limonene.

Figure 1
C. TEST FOR LIMONENE
1. Take about 0.5ml of limonene distillate and put it in a test tube.
2. Add few drops of bromine water. Note: Decolorization of bromine water means the
distillate contains limonene.

D. ANALYSIS OF ESSENTIAL OIL THROUGH GC-MS

E. PREPARATION OF MEDIA AND CULTURES
1. Before preparing the media assure first that all equipment is sterilize. It is important to follow
the proper rules in aseptic technique. Clean first your working area use alcohol then spray it
all over to surface are you will be working on.
2. Wash all the equipment to be use with dishwashing liquid and run with plentiful of water
after that use deionized water run it through and use soft cloth or tissue for drying.
3. Put the petri dish in an autoclavable bags sealed it properly then put it in the autoclave
machine. Set it for 30 minutes for maximum sterilization. wait until the pressure gauge is
zero then at that time when the gauge is zero it is safe to open.
4. Weigh around 15g then dissolve this in 85mL of distilled water in a 250mL E-Flask. Cover
the flask with foil. Cook the agar through boiling until clear solution is observed.
5. After cooling for some time pour the agar solution the sterilized petri dishes then let it stand
until solidified.

F.TEST FOR ANTIMICROBIAL PROPERTIES

1. A gram positive, Staphylococcus aureus, and gram negative bacteria, E. coli will be used in
the test for antimicrobial properties of the essential oil. And disk diffusion will be the method
to be used.
2. Obtain a plate culture of one of the organisms to be tested.
3. Using a sterile loop, emulsify a colony from the plate in the sterile saline solution. Mix
thoroughly making sure that no solid material from the colony is visible.
4. Repeat this procedure until the turbidity of the saline solution matches that of the standard
available for your class.
5. Dip the swab into the broth culture of the organism.
6. Gently squeeze the swab against the inside of the tube to remove excess fluid. Use the swab
to streak a Mueller-Hinton agar plate or a nutrient agar plate for a lawn of growth. This is best
accomplished by streaking the plate in one direction, then streaking at right angles to the first
streaking, and finally streaking diagonally.
7. End by using the swab to streak the outside diameter of the agar. Allow the plates to dry for
about 5 minutes.
8. Wrap the plates with foil and invert it and incubate for 24 hours at 37 C.
9. Measure the diameter of the zone of inhibition. Record and report this observation.