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Isolation of Natural Products
And
Some Reactions of Alkenes
Natural Products
A natural product is an organic chemical that
has been isolated and purified from a living
organism (plant or animal).
Compounds that are part of common
metabolic pathways (primary metabolism) for
plants and animals are generally excluded.
Natural products are considered products of
secondary metabolism – metabolism not
directly necessary for growth and survival and are often unique to a family of organisms.
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Natural Products
Many natural products are of interest
because of their biological activity in other
organisms, including humans.
Many natural products are quite toxic to
animals and humans. Natural does not
mean ‘safe’.
Natural Products from Plants
Plants provide the largest number of known
natural products. Their utility to the plant
ranges from reproduction (fragrances that
attract bees for pollination) to protection
(substances that repel animals or insects).
Isolation of a specific natural product
requires separation from polymeric
materials (cellulose, DNA, etc.) and the
multitude of primary metabolites present.
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Natural Product Classification
Natural Products can be classified in a
variety of ways. Common classifications
include structural type and biosynthetic
pathways.
Common classes include terpenes, steroids,
acetogenins, glycosides, and alkaloids.
Today’s compounds are classified as a
terpene (limonene) and as an acetogenin
(eugenol).
Terpene Biosynthesis
All terpenes are derived biosynthetically from the
five-carbon precursor isopentenyl diphosphate.
Terpenes are classified by the number of fivecarbon multiples they contain. Structures with
two isopentenyl precursor units, like limonene, are
called monoterpenes.
O
O
PO32-
P
O
O
isopentenyl diphosphate
limonene
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Acetogenin Biosynthesis
Acetogenins are natural products derived from a
two-carbon precursor, acetyl coenzyme A. Fatty
acids and carbohydrates are also derived from this
precursor. Eugenol biosynthesis includes
carbohydrate and amino acid intermediates in a
long, multi-step synthesis.
O
CH3CSCoA
acetyl CoA
HO
eugenol
OCH3
Isolation of Natural Products
Extraction
– Many small organic molecules can be extracted
from plant material (leaves, flowers, roots) by
extraction of the solid material. One example is
extraction of ground coffee beans by water.
Other compounds could be removed by
extraction with acetone or dichloromethane.
– This procedure generally gives a mixture of
compounds requiring significant further
purification.
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Isolation Procedures
Steam Distillation
– In this process, materials that are insoluble in
water can be distilled from plant material at
temperatures that do not lead to decomposition
products (either of the natural product or of the
plant material).
– Organic molecules that are water soluble are
left behind – provided that they don’t distill
below the boiling point of water.
What is Steam Distillation?
Steam distillation occurs when water and an
immiscible (insoluble) liquid are heated to
boiling. The process is different from
distillation of two miscible liquids as in
simple distillation or fractional distillation.
Let’s review a distillation diagram to see the
difference.
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Figure 1. Vapor-Liquid Composition for an Ideal Mixture of Two Liquids
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Vapor
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100
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Temperature
OC
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90
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b
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_
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a
Liquid
x
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80
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I
100% A
0% B
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I
40% A
60% B
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0% A
100% B
Immiscible Liquids
However, if two liquids are immiscible,
their vapor pressures are not affected by the
presence of the other component – there is
no dilution factor.
Therefore, the total vapor pressure is the
sum of the two independent vapor
pressures.
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v
a
p
o
r
p
r
e
s
s
u
r
e
_ Figure 2. Vapor Pressure for Two Immiscible Liquids _
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atmospheric pressure
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composite
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vapor press.
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water
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I
80
I
high boiling oil
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I
I
90
Temperature OC
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I
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100
Steam distillation of natural products
Many commercial ‘oils’ derived from natural
products are obtained by steam distillation.
Examples include fragrance oils (rose oil,
peppermint oil, etc.).
Today we will provide a sample of clove oil
(obtained by steam distillation of clove buds,
leaves, or stem). The major component of this oil
is eugenol, which can be separated from other
ingredients by base extraction (recall extraction of
benzoic acid).
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Steam Distillation of Natural Products
The steam distillate of orange rinds consists of
greater than 90% limonene. The other 10%
contains a small amount of large number of other
terpenes, and some long chain aldehydes and
alcohols.
Limonene is what gives citrus fruits their aroma
and it can be used as an non-toxic insecticide.
The amount of limonene in orange rind is small,
so you will not obtain a lot of material in this
experiment.
Procedure
You will work in pairs today, BUT EACH OF YOU
NEEDS A BIN.
One student will steam distill orange peel to obtain
limonene and the other will extract eugenol from clove
oil. Make sure you observe the procedure and results of
your partner.
NOTE: You will obtain your clove oil sample from a
dispenser at the chemical storeroom door. Wear
goggles, and take a dry graduated cylinder to contain
the sample.
You will not be allowed to start this experiment over if
you botch the separation!
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Procedure (Cont.)
Use the
coarse side
of the
grater to
obtain
your
orange
peel. (See
picture.)
Procedure (Cont.)
IMPORTANT: Make SURE you know, for your
extraction, which layer in the separatory funnel is the
organic layer. They are NOT the same for
dichloromethane and for ethyl acetate.
NOTE: IF YOUR CALCIUM CHLORIDE
DISSOLVES, YOU HAVE A WATER LAYER!
STOP AND GET THE OTHER LAYER. Calcium
chloride will NOT dissolve in ethyl acetate! Dried
ethyl acetate WILL NOT be cloudy!
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Procedure (Cont.)
SOLVENT REMOVAL: In the final step with both
natural products, you will be distilling solvent
AWAY from the natural product. The products will
remain in the distillation pot; the distillate will NOT
contain product.
The dichloromethane distills at just under 40o, while
the ethyl acetate distills at 77o, so use a higher Variac
setting for ethyl acetate.
Procedure (Cont.)
Essentially all solvent must be removed by
distillation from eugenol; with limonene you need
to remove about half of the solvent.
After the products have been isolated, you will
examine their chemistry with classification tests.
BOTH products will be analyzed by IR, and the
limonene will be analyzed by GC also (if the IR
spectrum indicates that water is not present in
your sample).
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Classification Tests
Classification tests are chemical reactions
that are specific to a limited number of
functional groups and give a visual
indicator that reaction has occurred.
HO
limonene
OCH3
eugenol
Classification Tests - Eugenol
Perform ferric chloride, permanganate, and
bromine tests on your eugenol product.
Also perform the permanganate and
bromine tests on the cyclohexene and
cyclohexane standards.
Take an IR spectrum of your product and
compare with the reference spectrum for
eugenol.
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Classification Tests - Limonene
You will conduct permanganate and bromine
tests on the ethyl acetate solution of limonene
after concentration.
Also perform these tests on the cyclohexene
and cyclohexane standards.
You MUST take an IR spectrum of your
limonene.
IF your classification tests are positive AND
the IR does not show primarily water, you
will analyze your limonene by GC.
GC of Limonene
Your limonene sample for GC needs to be
in a sample vial or small beaker, NOT in a
round-bottomed flask.
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Safety
Sodium hydroxide and hydrochloric acid
solutions are corrosive.
The bromine solution can cause serious skin
burns. Wash with copious amounts of water
and rinse with dilute sodium thiosulfite
solution.
Be careful with the grater; we don’t need
skinned knuckles or blood and skin
chemicals in the limonene.
Cleanup
Dispose of citrus peel in the waste basket.
Aqueous residues and washes can be disposed of
in the sink.
All methylene chloride and ethyl acetate solutions
can be combined and disposed of in the labeled
container in the hood.
Limonene and eugenol go in a bottle in the hood.
Calcium chloride must also be disposed of in the
hood.
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