TECHNIQUES USED IN MEDICINAL CHEMISTRY TO

ISOLATE & PURIFY

Contents

Thin-layer chromatography (TLC)

Column chromatography (CC)Fractional distillation (FD)Fractional crystallization (FC)

Chromatography

Chromatography is a physical method of separation in

which the components to be separated are distributed

between two phases. One of which is stationary phase and

the other is mobile phase that moves in a definite direction.

Types of Chromatographic Techniques

■ Thin Layer Chromatography(TLC)■ Column Chromatography■ Partition Chromatography■ Paper Chromatography■ Gas – Liquid Chromatography(GLC)■ Gas – Solid Chromatography(GSC)■ Ion Exchange Chromatography

(i)Thin Layer Chromatography

■ TLC is one of the simplest, fastest, easiest and least expensive chromatographic techniques use to separate organic compounds.

■ TLC is a form of chromatography consisting of: 1. A mobile phase. 2. A stationary phase. 3. Analysis should be performed in

atmospheric pressure and room temperature.

Principle of TLC

It is based on the principle of adsorption chromatography or

partition chromatography or combination of both, depending

on adsorbent and also the nature of solvents employed.

The components with more affinity towards stationary phase

travels slower. Components with less affinity towards stationary phase travels faster.

Principle of TLC

Separations in TLC involve distributing a mixture of two or

more substances between a stationary phase and a mobile

phase.

Stationary phase: It is a thin layer of adsorbent (usually silica or alumina) coated on a plane.Mobile phase: It is a developing liquid which travels up

the stationary phase, carrying the sample with it.

Advantages of TLC

■Short analysis time■All spots can be visualized■Low cost■Uses small quantities of solvent■Reliable and quick■Minimum amount of equipment is needed■Requires minimal training■Adoptable to most pharmaceuticals

Adsorbents for TLC

Inorganic:

1. Silica Gel2. Aluminium Silicate3. Bentonite

Organic:

1. Cellulose2. Charcoal

&activated C3. Polyamides

(ii)Column Chromatography

It is a method that has been used to purify individual chemical compounds from mixtures of compounds. Generally it is used for preparative applications.

PurposeThe stationary phase may be a column of adsorbent,

a paper and a thin layer of adsorbent on a glass plate

through which the mobile phase moves on.

Principle of Column Chromatography

The rate of the movement of the component is given as

follows :R= rate of movement of a compound/rate of

movement of mobile phaseIf the mobile is liquid then,

R= Am/Am+P.AsWhere,

P= Partition coefficientAm= Average cross section of mobile phaseAs= Average cross section of stationary phase

Advantages & Disadvantages

Advantages ■ Any type of mixture

can be separated■ Any quantity of the

mixture can be separated

■ Wider choice of mobile phase

■ Automation is possible

Disadvantages■ Time consuming

method■ More amount of

solvents are required which are expensive.

■ Automation technique makes complication.

Instrumentation

■ Stationary Phase■ Mobile Phase■ Column Characteristics■ Preparation of Column■ Introduction of the Sample■ Development Technique■ Detection of Compounds■ Recovery of Compounds

Step 1 : Prepare the Column

Step 2 : Pre elute the Column

Step 3: Load the sample onto the silica gel column

Step 4 : Elute the Column

Step 5 (Optional): Elute the column with the second elution

Solvent.Step 6: Analyze the fractions.

Precaution

■ Keep the column in a clean and dust free place■ Do not disturb the column till the separation is

completed.■ Avoid the gaps within the stationary phase

peeking

Applications

■Separation of different mixture of compounds

■Removal of impurities■Isolation of active constituents■Isolation of metabolite from biological

fluids■Estimation of drugs in formulations.

(iii)Distillation & Fractional distillation

■ Distillation :

Separating substance from liquid mixture. Selective evaporation and condensation. (Using

Boiling point)

Fractional distillation :

Special type of distillation. Substance’s boiling point difference should be less

than 25°C.

History

Concept was discovered by “Alexander of Aphrodisiacs”.

Most important thing of FD is column, which is packed, was developed later in 1935.

Method was getting better after 16th century.

differences between modern fractionating tower and past is that they collected steam.

Apparatuso Heat source.o Distilling flask.o Receiving flask.o Fractionating column.o Distillation head.o Thermometer(if needed).o Condenser, liebig condenser /

graham condenser.o Boiling chips, CaSO4 / CaCO3

How it works ?

Heating of the mixture with different bp at a high temp. & Pressure.

Starts to boil and forming vapour.

Vapours rise to the each column and filled with trays or plates.

Cools down the vapours, which turns into the liquid again.

In different levels of the column, we can collect different liquids (vapours)

Application

Separating Crude Oil Separating Chemicals in the lab Alcoholic Drinks – Spirits Creating Perfume Alcoholic beverages

Crude oil separation

Top of the fractionating column have lower boiling points than the fractions at the bottom.

All of the fractions are processed further in other refining units.

(iv)Fractional Crystallization Purpose:■ Use the technique of vacuum filtration to separate

a heterogeneous mixture into its components.■ Use the technique of fractional crystallization to

separate a homogeneous mixture into its components.

Safety Considerations:■ Exercise care not to burn yourself on hot

ringstands, rings, and beakers.■ Dispose of all waste materials in the appropriately

labeled containers in the hood.

Procedure

■ Add about 25 g of one of the unknown mixtures to a pre-weighed 250 mL beaker. Weigh the beaker and mixture to determine the mass of the mixture.

■ Add 60 mL of DI water to the beaker and heat the mixture to 85 ºC (do not allow it to boil) while stirring. Keep the mixture near 85 ºC for 5 minutes with stirring.

■ Place a piece of filter paper in the Büchner funnel with the curved edge down. Moisten the paper with a small amount of DI water.

■ Turn on the water to the aspirator and pour the hot solution into the funnel. Use the rubber policeman to transfer all solid material to the funnel.

Procedure

■ Pour the filtrate into a clean 250 mL beaker and save it for the recrystallization.

■ While applying suction, wash the sand twice with 25 mL portions of DI water. Continue to apply suction for 3-4 minutes to help dry the sand. Discard the wash water that collects in the flask.

■ Transfer the sand and filter paper to a pre-weighed watch glass and allow the sand to finish drying.

Applications of crystallization

■ There are two major groups of applications for the artificial crystallization process: crystal production and purification.

■ Powder salt for food" industry.■ Silicon crystal wafer production.■ Production of sucrose from sugar beet, where the

sucrose is crystallized out from an aqueous solution.

■ Used to improve (obtaining very pure substance) and/or verify their purity.

References

Wikipedia (https://en.wikipedia.org) A Powerpoint presentation on “Fractional

Distillation” by “Hae Na Lee”. “The Elements of Fractional Distillation” by “Clark

Shove Robinson” “Handbook of Thin-Layer Chromatography” by

“Joseph Sherma, Bernard Fried”