Isolation, Purification, and Characterization of Organic Compounds

Most organic reactions are relatively slow and are not usually quantitative. As a result, the reaction mixture at
the end of the reaction usually consist of unreacted starting material, the desired product, and sometimes
undesired products resulting from side reactions, and solvent used during the reaction.

To obtain a pure compound, three major steps are required:

 Isolation – separating the desired compound from the mixture
 Purification – removing impurities from the desired compound
 Characterization – to confirm the identity and structure of the desired compound
The isolation and purification method adopted will depend on the nature of the desired product relative to that
of the contaminant.

1. ISOLATION OF ORGANIC COMPOUNDS

Isolation is the process of separating a desired compound from a mixture.

METHODS OF ISOLATING SOLID PRODUCTS

(a) Filtration: A product which precipitates out of solution may be separated from soluble contaminants by
filtration method. Simple gravity filtration (a) may be accelerated by applying pressure (vacuum filtration) using a
Buchner funnel (b). It is usually necessary to wash the residue with a very small amount of cold solvent on
completion of filtration to quicken its drying process.

(b) Solvent Extraction (liquid -liquid extraction) Technique

This technique is used to isolate organic compound from aqueous solution (layer). The solution of the
compound in water transfers to the organic solvent (e.g. ether), because organic compounds are more soluble
in organic solvents than water.
 Solvent (Liquid-Liquid) Extraction Apparatus

Procedure:
1. Saturate the liquid to be extracted with common salt: this is to reduce the solubility of the compound in
water (salting out) and to further enhance its transfer to the organic layer.

2. Place the aqueous solution in a separatory funnel and shake vigorously in the desired volume of the ethereal
layer in one, two. or more batches.

3. Separate the aqueous layer from the ether layer using the tap.

4. Dry the ethereal layer over desiccant (drying agents) such as anhydrous MgSO4, CaCl2, anhydrous Na2SO4
etc

Solvent extraction is concerned with the distribution law or partition law which states that when a solute X
distributes itself between 2 immiscible liquids, the ratio of the concentration of X in solvent A and the
concentration of X in solvent B is constant (at constant temperature).

[ ]
=
[ ]

It is assumed that the solute X is in the same molecular state in the 2 solvents.

Note:

ℎ ( )
[ ] ℎ =
ℎ
 ( )
[ ] =

Example
50 cm3 of water containing 2 g of caffeine was extracted with 20 cm3 of ether. Given that the partition
coefficient between ether and water is 8, calculate the mass of caffeine extracted.
Solution:

(c) Steam Distillation

A solid reaction product which is steam volatile (e.g. phenol, benzaldehyde, aniline) can be separated from
non-steam volatile components by steam distillation. To facilitate the isolation of a steam volatile compound
from water, the compound should be either immiscible with or at least easily extractable from water.
The technique of steam distillation has the advantage that distillation occurs at a lower temperature than the
normal boiling point of the compound because some compounds tend to decompose at their normal boiling
point at ordinary atmospheric pressure. The general procedure consists of adding steam from a steam jacket
to the reaction mixture to facilitate distillation. As water distils over (assisted by the steam), it carries the
reaction product with it into the distillate and may be separated either by filtration or solvent extraction. An
organic compound to be suitable for steam distillation, it must possess the following characteristics:
 It should have considerable vapour pressure (25-40 mmHg) at the boiling point of water

 It should have a fairly high molecular weight (90-140)

 It should only be slightly soluble in water but readily soluble in ether to facilitate its extraction with
ether after steam distillation.
 A Steam Distillation Apparatus

Steam distillation method can also be used to estimate the vapour pressure or the molar mass of the unknown
steam volatile organic compound using the following expression:

Where = ℎ ; = vapour pressure of H2O

= mass of H2O in distillate; = vapour pressure of A;

= molecular weight of A; MH2O = molecular weight of H2O

N. B: PA + PH2O = 760 mmHg

Example
An insoluble compound A was steam distilled, the atmospheric pressure being 760 mmHg. Of the 69cm3
distillate obtained, 60 cm3 was water. The mixture boiled at 99.3 oC at which the vapour pressure of water is
740 mmHg. If the density of A and water are 1.22g/cm3 and 1.00g/cm3 respectively, calculate the molar mass
of A.
(d) Chromatography:

This method is used when compounds are present in small amounts. The technique is based on the distribution
of substances between two phases – one phase is stationary and the other is mobile phase. The most common
phases are:
(i) Solid-liquid e.g. column, thin layer chromatography

(ii) Liquid-liquid e.g. paper chromatography, High performance liquid chromatography

(iii) Liquid-gas – Gas chromatography

When stationary phase is a solid like alumina or silica gel, separation is based on adsorption phenomenon, but
when the stationary phase is a liquid or gas, separation is based on partition phenomenon.
Procedure:
A concentrated solution of the mixture of substances is applied to the top of the column of adsorbent which is
solid material [stationary phase e.g. alumina (Al2O3) and silica (SiO2)]. Depending on the polarities, the
components of the mixture are adsorbed to different degrees on the surface of the adsorbent. Therefore,
suitable solvent (mobile phase) is run through the column to allow each component of the mixture distribute
themselves between the solvent and the surface of the adsorbent. Th distribution coefficient will be different
for each compound. The least adsorbed will pass into the solvent more than the others. It will be preferentially
eluted by the solvent and hence be separated from the others more strongly adsorbed. The process of gradual
and differential elution continues until all the components of the mixture have been separated from one
another.
(e) Sublimation:
This is a separation technique for solids that change directly to gas. Examples are iodine and camphor. A
mixture of sand and iodine can be separated by this method. Iodine when heated will change from solid to gas
without passing through a liquid phase. Thus, sand can be isolated from the mixture.

A Sublimation Setup

2. ISOLATION OF LIQUID PRODUCTS

(a) Distillation:
When the desired reaction product is a liquid and if it is dissolved in another liquid, the conventional method
of isolation is distillation. If any of the contaminants also happens to be a liquid, the process of fractional
distillation will have to be adopted.
Distillation process is based on the fact that a pure liquid usually has a definite boiling point within a narrow
temperature range at a given pressure. The desired liquid product should therefore distil off in a pure state at
this temperature and pressure. The boiling point of a pure liquid is the temperature (at a given pressure) at
which the liquid and gaseous are in equilibrium i.e. the temperature at which the free energy of the change
from liquid – gas is zero.

(b) Fractional Distillation:

This method is used for liquids with close boiling points. A fractionating column is inserted vertically between
boiling flask and the condenser. A thermometer is inserted on top of the fractionating column.
 Fractional Distillation Apparatus
2. PURIFICATION TECHNIQUES
Purification is the removal of impurities in a compound to obtain a pure compound with constant properties.
Purification of solid products is done by crystallization which is based on the difference in their solubilities in
the solvent or mixture of solvents at different temperatures.

Sample Recrystallization:
Recrystallization involves repeated crystallization to yield compounds with higher purity. Recrystallization
process involves:

(i) Choosing a suitable solvent after examining the solubility properties of the compound
(ii) Making a saturated solution of the compound in a hot solvent
(iii) Allowing the solution to cool to enable the solid to crystallize out
(iv) Filtering the solid off and washing it with cold solvent

Qualities of a Good Solvent

(i) It must not react chemically with the solid to be purified
(ii) The solid must be relatively insoluble in the solvent at laboratory temperature but soluble in the hot
solvent
(iii) The hot solution of the compound on cooling should yield well defined crystals of pure compound
(iv) It should dissolve the impurities very readily at room temperature or not at all

Removal of Coloured Impurities

Sometimes, a product which should be colourless when pure is isolated in a coloured form due to
contamination by coloured impurities. To remove this, boil the solution of crude product in the crystallization
solvent for 5-10 minutes with about 1-2 % by weight of animal charcoal. Filter the hot solution through a layer
of celite which has been moistened with hot crystallization solvent to give a clear colourless filtrate free of
carbon particles. Purification of liquid products is usually carried out by distillation as earlier described.

CHARACTERIZATION TECHNIQUES
Characterization is the process of identifying and confirming the structure of a compound.
(a) Physical Characterization Methods
 Melting Point: Solid products are usually characterized by their melting points. The melting point
of a solid is the temperature at which the solid changes into liquid under a pressure of one atmosphere.
For most pure substances the change from the solid to the liquid phase are to be sharp usually within
2oC range, hence melting point is valuable for the purpose of identification and asserting the purity of
a compound. A sharp melting point is usually indicative of the high purity of a single compound.
Impurities lower and broaden the melting point range of a solid.

 Boiling Point: Liquids are generally characterized by their boiling points – which is obtained when the
liquid distils. Pure liquids have constant boiling points. Boiling point of liquids are usually affected by
pressure

 Density and Refractive Index: These methods are used for the identification of compounds

(b) Chemical Characterization Methods

These are carried out through qualitative laboratory analysis. The most deployed method is functional group
tests such as:
 Lucas test for alcohols
 Tollens’ test for aldehydes
 NaHCO3 test for carboxylic acids

(c) Spectroscopic Methods

This is the most modern and reliable techniques that involves the use of equipment. They include:

 Infrared Spectroscopy – identifies functional groups present

  Nuclear Magnetic Resonance – provides the number of hydrogen/carbon atoms present, its
chemical environment, and structure

 Mass Spectrometry – determines the molecular mass and molecular formula of compounds
 UV-Visible Spectroscopy – used for conjugated systems

(d) Chromatographic Characterization – (TLC Method)

This is a simple and useful technique for the identification of organic compounds, progress of a reaction,
examination of reactions for side products, or to follow the elution in column chromatography. It can also
be adapted for the separation of compounds on a small preparative scale. A very small amount of a solution
of the sample is spotted on the adsorbent on a glass plate which is then placed into a glass tank with the solvent
– mobile phase. The developing solvent ascends gradually due to capillary action and carries in the mobile
phase the now separated spots/bands of crude material by the process called elution.
Separation of the components of a mixture is achieved due to their different polarity which determine their
migration along with the ascending liquid. In principle a single compound should indicate a single spot while
a mixture of compounds should have the corresponding number of spots.

Compounds may be identified and characterized by their Retention factor (Rf).

Distance traveled by the spot from the origin
=
Distance traveled by solvent from the origin
Rf values range from 0 to 1.
In using Rf values for characterization, standard conditions like temperature, solvent system, type of adsorbent,
thickness, degree of saturation of chromatographic chamber with solvent vapour should be considered and
maintained for comparative values.
Example 3
(i) Calculate the Rf of a compound which travels 4.5 cm and the solvent travels 9.0 cm.
Answer: 0.50

(ii) If a spot moved 3.0 cm while the solvent front moved 6.0 cm, calculate the Rf of the compound.
Answer: 0.50

(iii) The Rf of a compound is 0.60. if the solvent front is 10 cm, find the distance travelled by the compound.
Answer: 6.0 cm

Visualization
Coloured compounds are easily located on the slide but colourless compounds can be visualized by different
methods namely:
(i) Eradication – with ultra violet light – useful for fluorescent compounds – e.g. aromatic compounds
(ii) Reversible association with iodine
(iii) Spraying with reagent that colour the spots e.g. conc. H2SO4.

Iodine tank is commonly used – resulting in a brown coloration at the location of each component, which
results from a weak complex with iodine. This will slowly evaporate and the colour fades. To make a
permanent record, the location of the sample spots is indicated with pencil marks on the developed plate.