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Column Chromatography – Principle,

procedure, Applications

Madhu Kumari
March 18, 2021 % 0 Comments

What is column chromatography?

Column chromatography is described as the useful

technique in which the substances to be isolated
are presented onto the highest point of a column
loaded with an adsorbent (stationary phase), go
through the column at various rates that rely upon
the affinity of every substance for the adsorbent
and the solvent or solvent mixture, and are
typically gathered in solution as they pass from the
column at various time.

The two most common examples of stationary

phases for column chromatography are silica gel
and alumina while organic solvents are regarded as
the most common mobile phases.

Column Chromatography
principle

The main principle involved in column

chromatography is the adsorption of the solutes of
the solution with the help of a stationary phase and
afterward separates the mixture into independent
components.

At the point when the mobile phase together with

the mixture that requires to be isolated is brought
in from the top of the column, the movement of the
individual components of the mixture is at various
rates.

The components with lower adsorption and affinity

to the stationary phase head out quicker when
contrasted with the greater adsorption and affinity
with the stationary phase. The components that
move rapidly are taken out first through the
components that move slowly are eluted out last.

The adsorption of solute molecules to the column

happens reversibly. The pace of the movement of
the components is communicated as:

Rf = the distance traveled by solute/ the distance

traveled by the solvent

Where Rf is called retardation factor

Column Chromatography Diagram (Image

modified from
https://bitesizebio.com/29947/basics-
chromatography-column/)

Column
Chromatography
components
Components of a typical chromatographic system
using a gas or liquid mobile phase include:

Stationary phase – Generally it is a solid

material having a good adsorption property
and should be suitable for the analytes to be
separate. It should not cause any hindrance in
the flow of the mobile phase.

Mobile phase and delivery system – This

phase is made up of solvents that complement
the stationary phase.

The mobile phase acts as a solvent, a developing

agent (promotes separation of components in the
sample to form bands), and an eluting agent (to
remove the components from the column that are
separated during the experiment).

Column –

For liquid chromatography: 2-50cm long and

4mm internal diameter, fabricated with stainless
steel

For gas chromatography: 1-3m long and 2-4mm

internal diameter, fabricated either with glass or
stainless steel

A column’s material and its dimension are very

crucial to support the stationary phase and
promote effective separations.

Injector system – Responsible for delivering

test samples to the column’s top in a
reproducible pattern.

Detector and Chart Recorder – This gives a

continuous record of the presence of the
analytes in the eluate as they come out from
the column.
Detection relies on the measurement of a
physical parameter (like visible or UV
adsorption).
On the chart recorder, each separated analyte
is represented by a peak.

A collector at the bottom is placed at the bottom

end of the column set up to collect the separated
analytes.

Column
chromatography
Procedure
The steps included in the column chromatography
are:

1. Preparation of the column

Mostly the column is comprised of a glass tube

with an appropriate stationary phase
The bottom end of the column is packed with a
glass wool/cotton wool or an asbestos pad
after which the stationary phase is packed.
After packing the column, a paper disc is
placed on the top to avoid the disturbance of
the stationary phase during the introduction of
the sample or mobile phase.
The disturbance in the stationary phase
(adsorbent layer) leads to the irregular bands
of separation.

Two types of preparing the column, known as

packing techniques namely:

1. Dry packing technique – The amount of

absorbent needed is added as a fine dry
powder in the column and the solvent flows
freely through the column until equilibrium is
achieved.
2. Wet packing technique – The slurry of
adsorbent is prepared along with the mobile
phase and is poured into the column.
It is regarded as the ideal technique for
packaging.

The column should be properly washed and

completely dried before in-use.

2. Introduction of the sample

The sample (a mixture of components) is

dissolved in the minimum amount of the mobile
phase.
At one instant, the sample is introduced into
the column and on the top portion of the
column, it is absorbed.
Through the elution process, the individual
sample can be isolated from this zone.

3. Elution technique

Through this technique, the individual components

are separated completely from the column.

The process of elution can be carried out by

employing two techniques:

1. Isocratic elution technique – Throughout the

procedure, a solvent of the same polarity or
same solvent composition is utilized.

Example: Use of chloroform alone

2. Gradient elution technique – Throughout the

separation procedure, solvents of gradually
increased polarity or increased elution strength
are utilized.

Example: Benzene Chloroform Ethyl acetate

Chloroform

4. Detection of Components

In case the mixture separated in a column

chromatography procedure are colored
compounds, then monitoring the separation
progress is simple.
In case the compounds undergoing separation
are colorless, then small fractions of the eluent
are sequentially collected in tubes that are
labeled. Thorugh TLC, the composition of each
fraction is determined.

Types of Column chromatography

1. Adsorption column chromatography –

Technique of separation in which compounds
to be separated (solute) is retained or
adsorbed on the surface of the adsorbent
(stationary phase).
2. Partition column chromatography – It is based
on the variance in partition coefficient of the
individual components of the mixture, where
the stationary phase and the mobile phase
both are in the liquid state.
3. Gel column chromatography – Here, the
separation is carried out through a column
packed with gel and possesses a porous
stationary phase. It is also referred to as size
exclusion chromatography
4. Ion exchange column chromatography – The
basis relies on the charge of the molecules.
The separation is done when molecules get
attracted to the oppositely charged stationary
phase.
5. Gas Chromatography (GC)
6. High-Performance Liquid Chromatography
(HPLC)

Column chromatography uses

Column chromatography is one of the versatile

methods for purifying and separating both solids
and liquids.

Major applications:

1. To isolate active constituents

2. To separate compound mixtures
3. To remove impurities or carry purification
process
4. To isolate metabolites from biological fluids
5. To estimate drugs in drug formulations or
crude extracts

Sources:

1. http://www.rnlkwc.ac.in/pdf/study-
material/physiology/column%20chromatography.pd
2. https://www.biomadam.com/types-of-column-
chromatography
3. https://microbenotes.com/column-
chromatography/

! Categories: General Topics

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