11/30/2012

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 _ _ _ _ Vapor _ _ 100 _ _ _ _

Temperature OC

_ _ _ 90 _ _ _

b

_ _ _ _ _ _

a

Liquid

x

_ 80 _ I 100% A 0% B

I

I

I

I

I 40% A 60% B

I

I

I

_ _ _ 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 _ _ _ _ _ atmospheric pressure _ _ _ _ composite _ _ vapor press. _ _ _ _ _ _ _ _ _ _ water

_ _ I 80

I

high boiling oil I

I I 90 Temperature OC I

I

I

I

_ _ _ 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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