The document provides an overview of column chromatography, including its history, key components, principles, procedures, applications, and advantages/limitations. Column chromatography is a separation technique that uses a stationary phase (often silica gel or alumina) packed into a column and a liquid or gas mobile phase to separate mixtures based on how strongly components interact with the stationary phase. It was developed in 1901 and is widely used today to purify compounds and remove impurities from mixtures.

1. Assignment Topic Column Chromatography
Contents
⮚ Introduction
⮚ Column Chromatography
⮚ History
⮚ Forms of Column Chromatography

2. ⮚ Types of Column Chromatography
⮚ Principle of Column Chromatography
⮚ Components of Column Chromatography
⮚ Procedure of Column Chromatography
⮚ Column Chromatography works in few steps
⮚ Instrumentation in Column Chromatography
⮚ Steps in Column Chromatography
⮚ References
Introduction
Chromatography is an important biophysical technique that enables the separation, identification, and
purification of the components of a mixture for qualitative and quantitative analysis.
It is a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a
selectively absorbent stationary phase.

3. There are a number of different kinds of chromatography, which differ in the mobile and the stationary phase used.
COLUMN CHROMATOGRAPHY
• Column chromatography is a technique in which the substances to be separated are introduced onto the top
of a column packed with an adsorbent, passed through the column at different rates that depend on the
affinity of each substance for the adsorbent and for the solvent or solvent mixture, and are usually collected
in solution as they pass from the column at different times.
• It is a solid – liquid technique in which the stationary phase is a solid & mobile phase is a liquid or gas.
History
• The first column chromatography was developed by the Russian botanist Mikhail Tsvet in 1901.
• Who washed an organic solution of plants pigments through a vertical glass column packed with an
adsorptive material.
• He discovered that the pigments separated into a series of discrete colored bands on the column, divided by
regions entirely free of color.

4. Forms of Column Chromatography
There are two forms of column chromatography.
1. Liquid chromatography (LC)
2. Gas chromatography (GC)
Types of Column Chromatography:
1. Adsorption column chromatography – Adsorption chromatography is a technique of separation, in
which the components of the mixture are adsorbed on the surface of the adsorbent.
2. Partition column chromatography – The stationary phase, as well as mobile phase, are liquid in.
partition chromatography.
3. Gel column chromatography – In this method of chromatography, the separation takes place through a
column packed with gel. The stationary phase is a solvent held in the gap of a solvent.
4. Ion exchange column chromatography – A chromatography technique in which the stationary phase is
always ion exchange resin.
Column Chromatography Principle
• When the mobile phase along with the mixture that needs to be separated is introduced from the top of the
column, the movement of the individual components of the mixture is at different rates.
• The components with lower adsorption and affinity to stationary phase travel faster when compared to the
greater adsorption and affinity with the stationary phase.
• The components that move fast are removed first whereas the components that move slow are eluted out
last.
• The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the movement
of the components is expressed as:
• Rf = the distance travelled by solute/ the distance travelled by solvent
Rf is the retardation factor.

5. Components of C.C.
Adsorbent
• A variety of adsorbents can be used as the stationary phase;
silica gel ,caco3,cellulose,starch etc. is most commonly used in organic chemistry
Mobile phase
• Solvent also play important role in this c.c.
Many types of solvents are available like cyclo hexane, benzene, chloroform, etc
Column
• The column dimensions are imp. for the effective column separation.

6. • Ranges from 1:10 to 1:100
Column Chromatography Procedure
Before starting with the Column Chromatography Experiment let us understand the different phases involved.
Mobile phase – This phase is made up of solvents and it performs the following functions:
1. It acts as a solvent – sample mixture can be introduced in the column.
2. It acts as a developing agent – helps in the separation of components in the sample to form bands.
3. It acts as an eluting agent – the components that are separated during the experiment are removed from the
column
4. Some examples of solvents used as mobile phase based on their polarity are – ethanol, acetone,
water, acetic acid, pyridine, etc.
Stationary phase – It is a solid material which should have good adsorption property and meet the
conditions given below:
1. Shape and size of particle: Particles should have uniform shape and size in the range of 60 – 200μ in
diameter.
2. Stability and inertness of particles: high mechanical stability and chemically inert. Also, no reaction with
acids or bases or any other solvents used during the experiment.
3. It should be colourless, inexpensive and readily available.
4. Should allow free flow of mobile phase
It should be suitable for the separation of mixtures of various compounds
Column chromatography works in few steps:
Step 1: The mobile phase or eluent is either solvent or solvent mixture. The upper level of mobile
phase should be same as the stationery phase. That means the stationery phase should be wet with the solvent. On
this stage the compound mixture what need to be separated, are added from the top of the column in such a way
that the top level of it is not disturbed. By turning on the tap below it is allowed to adsorbed on the surface of the
silica.

7. Step 2: Then the solvent or a suitable solvent mixture is added at first touching the side of the glass column
slowly and carefully so that the top level of the stationery phase is not disturbed. The solvent is repeatedly added
as many times as needed throughout the process.
Step 3: When the tap, is on the compounds in the compound mixture move along with the eluent depending on
the polarity of the sample molecule. The non polar components travel faster than the polar component.
Suppose if any compound mixture contains three compounds blue, red and green. According to polarity the order
of these compounds are blue>red>green. That means blue is the most polar compound and thus will have less
tendency to move along with the mobile phase.
Step 4: The green colored compound will travel first as it is less polar that other two. When it is near end of the
column a clean test tube is taken to collect the green sample. After this the red and at last the most polar blue
compound is collected, all in separate test tubes.
Thus a compound mixture is separated or purified by using column chromatography.
Instrumentation of Column Chromatography

8. A typical column chromatographic system using a gas or liquid mobile phase consists of the following
components:
A stationary phase:
• Chosen to be appropriate for the analytes to be separated.
A column:
• In liquid chromatography these are generally 25- 50 cm long and 4mm internal diameter and made of
stainless steel whereas in gas chromatography they are 1-3m long and 2- 4mm internal diameter and made
of either glass or stainless steel.
• They may be either of the conventional type filled with the stationary phase, or of the microbore type in
which the stationary phase is coated directly on the inside wall of the column.
A mobile phase and delivery system:
• Chosen to complement the stationary phase and hence to discriminate between the sample analytes and to
deliver a constant rate of flow into the column.
An injector system:
• To deliver test samples to the top of the column in a reproducible manner.
A detector and chart recorder:
• To give a continuous record of the presence of the analytes in the eluate as it emerges from the column.
• Detection is usually based on the measurement of a physical parameter such as visible or ultraviolet
absorption or fluorescence.
• A peak on the chart recorder represents each separated analyte.
A fraction collector: For collecting the separated analytes for further biochemical studies.
Steps in column chromatography
A : Preparation of the Column

9. • The column mostly consists of a glass tube packed with a suitable stationary phase.
• A glass wool/cotton wool or an asbestos pad is placed at the bottom of the column before packing the
stationary phase.
• After packing, a paper disc kept on the top, so that the stationary layer is not disturbed during the
introduction of sample or mobile phase.
• Before using column, it should be washed properly and dried.
• The column should also be free from impurity and uniformly filled with the stationary phase.
There are two methods of preparing the column, they are:
1. Dry packing / dry filling
In this the required quantity of adsorbent is poured as fine dry powder in the column and the solvent is
allowed to flow through the column till equilibrium is reached.
Demerit : air bubbles are entrapped between mobile phase and stationary phase cracks appear in the
adsorbent layer.
2. Wet packing / wet filling
In this, the slurry of adsorbent with the mobile phase is prepared and is poured into the column. It is
considered as the ideal technique for packing.
Stationary phase settles uniformly and no crack in the column of adsorbent.
B. Introduction of the Sample
• The sample which is usually a mixture of components is dissolved in minimum quantity of the mobile
phase.
• The entire sample is introduced into the column at once and get adsorbed on the top portion of the column.
• From this zone, individual sample can be separated by a process of elution.
C. Elution
By elution technique, the individual components are separated out from the column.
It can be achieved by two techniques:
Isocratic elution technique: Same solvent composition or solvent of same polarity is used
throughout the process of separation.

10. Eg. Use of chloroform alone.
Gradient elution technique: Solvents of gradually ↑ polarity or ↑ elution strength are used
during the process of separation.
E.g. initially benzene, then chloroform, then ethyl acetate then chloroform
D. Detection of Components
If the compounds separated in a column chromatography procedure are colored, the progress of the
separation can simply be monitored visually.
If the compounds to be isolated from column chromatography are colorless.
In this case, small fractions of the eluent are collected sequentially in labelled tubes and the composition of
each fraction is analyzed by TLC.
Factors Affecting Column Efficiency
❖ Dimensions of the column
❖ Particle size of the adsorbent
❖ Nature of the solvent
❖ Temperature of the column
❖ Pressure
Applications
• Column chromatography is one of the most useful methods for the separation and purification of both
solids and liquids. Its major application includes:
• Separation of mixture of compounds.
• Removal of impurities or purification process.
• Isolation of active constituents.

11. • Isolation of metabolites from biological fluids.
• Estimation of drugs in formulation or crude extracts
• Separation of diastereomers
• It is used to determine drug estimation from drug formulations
• It is used to remove impurities.
• Used to isolation metabolites from biological fluids.
Advantages
• Any type of mixture can be separated by column chromatography.
• Any quantity of the mixture can also be separated.
• Wider choice of mobile phase.
• In preparative type, the sample can be separated and reused.
• Automation is possible.
Limitatations
• Time consuming method.
• More amounts of solvents are required which may be expensive.
• Automation makes the technique more complicated and costly.
References
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