MODULE TWO

TOPIC: Extraction, Isolation and Purification of Organic Compounds

INTRODUCTION: Before the properties and structures of an organic compound can be

completely investigated and put to use the compound must attain a high level of purity which
is determined by certain criteria (for liquids, narrow boiling point range and sharp melting
point for solids). This criterion proves important in the study and investigation of substances that
are suspected to possess organic compounds in them.
STUDY OBJECTIVES: At the end of this module, students should be able to:
(i) Define extraction, isolation and purification as relating to organic compounds
(ii) Identify & familiarize themselves with the various procedures for carrying out the
aforementioned processes
(iii) Differentiate between the techniques for carrying out the aforementioned processes
MODULE CONTENT: In natural product chemistry (this deals with the study of organic
compounds that are synthesized by living organisms), extraction process involves separation,
from a plant or animal material, of desired organic compound, which contains other (undesirable)
compounds. It can be carried out in various ways ranging from:

(i) Soxhlet extraction/Hot continuous extraction

Fig 1: Diagram of a Soxhlet Extractor

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Fig 2: Detailed diagram of a Soxhlet Extractor showing the labelled parts

Principles of Conventional Soxhlet Extraction

Soxhlet extraction is a very useful tool for preparative purposes in which the analyte is
concentrated from the matrix as a whole or separated from particular interfering substances.
Sample preparation of environmental samples has been developed for decades using a wide
variety of techniques. Solvent extraction of solid samples, which is commonly known as solid–
liquid extraction, is one of the oldest methods for solid sample pre-treatment.

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 Conventional Soxhlet extraction remains as one of the most relevant techniques in the
environmental extraction field. This assertion is supported by the double use of conventional
Soxhlet: (1) as an extraction step in a given method, and/or
(2) as a well-established model for comparison of new extraction alternatives.
In this method, the finely ground crude organic material is placed in a porous bag or
“thimble” 3 made of strong filter paper, which is placed in chamber 4 of the Soxhlet
apparatus (Figure 2). The extracting solvent 1 in flask 2 is heated by 8, and its vapours condense
in condenser 5 passing through the side arm 7. The condensed extract drips into the thimble 3
containing the crude organic material through 4, and extracts it by contact. When the level
of liquid in chamber 3 rises to the top of siphon tube 6, the liquid contents of chamber 3
siphon into flask 2. This process is continuous and is carried out until a drop of solvent from the
siphon tube does not leave residue when evaporated. The advantage of this method, compared to
other extraction methods, is that large amounts of the organic material can be extracted with a
much smaller quantity of solvent. This procedure helps to save time, energy and consequently
financial costs. At small scale, it is employed as a batch process only, but it becomes much
more economical and viable when converted into a continuous extraction procedure on medium
or large scale.
(ii) Cold maceration- In this process, the whole or coarsely powdered crude drug is placed in a
stoppered container with the solvent and allowed to stand at room temperature for a period
of at least 3 days (72 hrs) with frequent agitation (shaking) until the soluble matter has dissolved.
The mixture then is strained, the marc (the damp solid material) is pressed, and the combined
liquids are clarified by filtration or decantation after standing. This must be carried out carefully
and accurately.
(iii) Decoction- In this process, the crude plant material is boiled in a specified volume of water for
a defined time. It is then cooled and strained or filtered. This procedure is suitable for extracting
water-soluble, heat-stable constituents. This is undoubtedly the most medieval of all extraction
procedures as its usage is as old as man’s existence.
(iv) Steam distillation (suitable for volatile oils)

Isolation, in organic chemistry, is the removal, in a pure and uncombined state, of a required
compound from a mixture of many other similar compounds which are undesirable and present
in relatively large amount.

Purification of organic compounds involves the process of removing small amounts of

contaminating materials. As the process of isolation and purification are related, similar methods
are employed, they include:
(i) Filtration

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 (ii) Crystallization/ Recrystallization (for solids)
(iii) Solvent extraction
(iv) Distillation (simple, steam, fractional and reduced pressure)
(v) Chromatography (for gases, liquids and solids)

FILTRATION- this is the technique used in the separation of a solid substance (solute) which
is in contact with a liquid or a solution in which it is insoluble, or a solute in contact with a gas
in which it is insoluble. In simple filtration, the mixture is poured slowly into a filter funnel into
which a correctly folded filter paper is inserted and placed on a filtration flask. The solid
substance that remains inside the filter paper is called residue, while the clear liquid is called the
filtrate. For better separation by filtration a Buchner funnel connected to water or vacuum (suction)
pump is used. The suction pump sucks up as much liquid as possible from the solid. Practically it is
easier and faster to filter a solid while it is still hot.

CRYSTALLIZATION- this is a procedure for purification of a solid based on the differences

in solubility in a solvent or mixture of solvents. This method is generally used to obtain crystals
of a substance from a solution. It involves the evaporation of the solvent from the solution until the
solution becomes saturated and as the saturated solution cools, crystals of solids start to separate
out. The crystals are then filtered off by the use of a Buchner funnel and then dried in the open
air or in a desiccator.

Re-crystallization method is used to purify organic solids. The process involves dissolving the solid
in a minimum amount of a solvent. In which it is readily soluble in when hot but only slightly
soluble in when cold. The hot solution is filtered by the use of Buchner funnel to remove solid
impurities and the filtrate cools slowly to obtain pure crystals leaving behind the impurities in
solution (mother liquor or supernatant solution). The crystals are filtered off and the process may
be repeated if required hence the name re-crystallization. The re-crystallized solids are washed with
a small amount of the cold solvent and then dried in air or in desiccator.

The following factors are to be considered when making choice of solvent for crystallization:

1. Sample should not react chemically with the solvent.

2. A good solvent should have a high solvent power for the substance to be purified at elevated
temperatures and a comparatively low solvent power at room temperature.
3. A good solvent should dissolve the sample readily or to only a very small extent.
4. A good solvent must yield well-formed crystals of purified compound.
5. A good solvent should easily be removed from the crystals of the purified compound.

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SOLVENT OR LIQUID-LIQUID EXTRACTION- organic compounds are generally more soluble
in organic solvents e.g ethoxyethane (Et2O) and tetrachloromethane (CCl4) than in water. Hence, it is
possible to separate an organic compound from its solution in water by shaking it with an organic
solvent. Ethoxyethane is the solvent commonly used because it is volatile (BP 36oc) and it does
not react with organic compounds. The extracted organic solvent is then dried over anhydrous sodium
sulphate (Na2SO4) before proceeding for proper isolation and purification.

Liquid-liquid extraction is a basic technique in chemical laboratories, where it is usually

performed using a separatory funnel. The term solvent extraction can also refer to the separation
of a substance from a mixture by preferentially dissolving that substance in a suitable solvent. In that
case, a soluble compound is separated from an insoluble compound or a complex matrix.

DISTILLATION- This is a process or act of purifying liquids through boiling. A particular

liquid is vapourised from a container and then its vapour is condensed into another container.
Distillation is a common procedure in many laboratories for the purpose of separation and/or
purification of components of a liquid mixture. The criterion for purity of liquid organic
compounds is the possession of narrow range of boiling point. They are various forms of
distillation which include:

a) Simple distillation- It is a technique used for the separation and purification of a liquid
contaminated by non-volatile solid impurities. The mixture of the substances is placed in the flask and
heated after boiling chips (or broken porcelain is added). Ideally, the substance with the lowest
boiling point vaporises first, the temperature remaining constant until that substance has completely
been distilled. The vapour is led into the the condenser where on being cooled reverts to liquid state
(condenses) and so turns off into a receiving vessel. The product so obtained is known as distillate.
Those substances having a higher boiling point remain in the flask and constitute the residue.

b) Fractional distillation- It is the technique used to separate a mixture of two or more miscible liquids
with reasonably close boiling point difference of about 10oc. A good example is the mixture of
liquid A (Water with boiling point of 100oc) and liquid B (Methylbenzene with boiling point of 110oc).
The mixture is placed in a distillation flask into which few pieces of broken porcelain are added. A
fractionating column is then attached to the flask, it consist of small glass in form of rings to increase
the surface which do not obstruct the passage of vapour or liquid downward. As the liquid mixture is
heated, the vapour of A and B rise through the fractionating column. Since the vapour of B is less
volatile (heavier than the vapour of A) it will start condensing back into the flask, while the vapour
of A been more volatile and lighter continues to rise up, concentrating itself near the top of the
flask. With time the vapour at the end of the

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 fractionating column will be that of A without B. The vapour of A then passes through the water
cooled condenser and is first distilled leaving B. Because of the slight difference in boiling point, the
column can be tapped at various levels to draw up different fractions.

Fractional distillation is commonly used in refining petroleum, some of the fractions obtained include:
gasoline, benzene, kerosene, fuel oil, lubricating oil e.g grease and paraffin.

c) Reduced pressure distillation- distillation under reduced pressure is a technique generally used
to make a liquid to boil below its boiling point. Generally liquid with high boiling point cannot
be purified by simple or normal distillation since they may decompose at their boiling point. The
only way to purify such liquids is to carry out reduced pressure distillation. It is a method in
which a suction/vacuum pump is attached to the simple distillation apparatus in other to reduce
the pressure inside the flask and hence make high-boiling liquids to oil at relatively lower
temperature. A good example of a liquid that can be distilled using reduced pressure distillation
is dimethyl sulfoxide ((CH3)2SO). At atmospheric pressure it boils at 189oc but under a vacuum,
it distils off into a connected receiver at only 70oc. Reduced pressure distillation is also known
as vacuum distillation.

d) Steam distillation- This technique is used to purify organic liquids which are steam volatile and with
high boiling points contaminated by known volatile impurities and insoluble in water. Steam is
passed into the impure liquid in a distillation flask and then heated. Both the liquid and the water
start to boil when the sum of their saturated vapour pressure equals the atmospheric pressure. Hence
the vapour pressure at which the liquid boils is less than the atmospheric pressure i.e the liquid
boils at a lower temperature. The distillate is collected and the organic compound is separated using
a separatory funnel being insoluble in water. In steam distillation, the mixture boils at a temperature
lower than the boiling point of the liquid.

SUBLIMATION- some organic compounds when heated change directly from solid to gas and on
cooling change to solid without forming liquid, this process is known as sublimation. Compounds such
as camphor, ammonium chloride (NH4Cl) and naphthalene will sublime. In other to purify a substance
by sublimation the impure substance is placed in an evaporating dish and then covered with an
inverted filter funnel blocked with cotton wool. As the impure substance is heated it sublimes
leaving the impurities on the evaporating dish. The pure substance form crystals on the side of the
funnel and can be collected when completely dried.

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Fig 3: Setup showing Sublimation procedure

CHROMATOGRAPHY- this is a technique that is used for the separation of complex mixtures
that rely on the differential affinity of substances for a gas or liquid mobile medium and for a stationary
absorbing medium through which they pass, such as paper gelatin, magnesia, silica, alumina or
cellulose. Chromatography offers a means of concentrating a product that occur naturally in great
dilution and it is an extremely valuable method for the separation, purification and identification
of the constituents of a mixture (especially organic compounds). Chromatography depends on the
distribution of the mixture between two phases namely:

a. Fixed/Stationary phase (the solid material that takes up the particle) and

b. Moving/Mobile phase (fluid that carries the mixture of substances in the sample
through the adsorptive material).

The mixture is dissolved in the moving phase and passed over a fixed phase which may be a glass
column (column chromatography) or a paper strip (paper chromatography). The moving phase may be
a liquid or a gas while the fixed phase is a solid e.g alumina, silica gel etc. The process is usually

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called adsorption chromatography. In this process, the solutes are adsorbed on different parts of the
column the adsorbed compounds are then eluted by the passage of a series of eluents (solvents).

Types of chromatographic techniques:

i). Paper chromatography (PC) - a special case of partition chromatography in which a paper
strip is known as the adsorbent.

ii). Thin-layer chromatography (TLC) - this method uses a chromatoplate as the column i.e a
glass or aluminium plate coated with the adsorbent usually silica gel or alumina. Alternatively
the fixed phase may consist of a liquid strongly adsorbed on a solid (as support). This
is also a form of partition chromatography.

iii). Column chromatography- The adsorbent (such as silica gel, alumina or cellulose) is packed
into a column (usually a glass tube of various sizes between 20-60 cm in length and between
1-4 cm in diameter) and a solution of the mixture to be separated is added from the top.

An appropriate solvent is passed through the column washing or eluting the compounds down the
column. A polar substance that is adsorbed very tightly to the surface, will be efficiently retarded
by the column, will a non-polar substance will elute (dissolve in the solvent) very rapidly. By
varying the nature of the solid adsorbent and the eluting solvent, a wide variety of resolutions, even of
very similar substances can be carried out. The different types of column chromatography include:

A) Gas chromatography (could be gas-solid chromatography GSC or gas-liquid chromatography

GLC)
B) Liquid chromatography (LC)
C) Gel-Permeation chromatography
D) Ion-exchange chromatography

In ion-exchange chromatography, the solid phase consists of a synthetic resin (which may be a
cation or anion exchanger). In this way mixture of bases and acids may be separated.

In gel-permeation chromatography, compounds are separated on the basis of their molecular size.
Porous beads of the gel are packed into a column and the mixture is added at the top in an
appropriate solvent. Large molecules move straight down the column, while small molecules stick
in the pores and are retarded.

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The major difference between GSC, GLC and normal LC is that in GSC and GLC a carrier gas has
replaced the solvent in the column. The carrier gas is therefore responsible for the elution.
Examples of carrier gas include nitrogen, hydrogen, helium etc.

Fig 4: Setup showing Liquid chromatography (LC)

NB: the speed at which a substance moves in a chromatographic system is important. A measure of this
speed is called the retention. In paper and thin- layer chromatography, retention is measured as retention
of retardation factor (Rf).
Rf = distance moved by compound = a
distance moved by solvent front b

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STUDY QUESTIONS: Attempt the following study questions:
1. Describe with the aid of a well labelled diagram how continuous extraction of a solid organic plant
sample can be done using a soxhlet extractor.
2. Using a schematic diagram, briefly classify organic compounds based on their structures citing
relevant example(s) for each class.
3. Define the following terms in relation to organic chemistry:
(a) Extraction (b) Isolation (c) Purification

SUMMARY AND CONCLUSION: This topic has definitely introduced you to natural product
chemistry which is an integral part of organic chemistry. The need to carry out extraction,
isolation and purification on organic materials is integral in the study of organic compounds

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