Column Chromatography 1

Submitted To.
Ma’am Asma Shaheen

Submitted By. Anosh Asif , Hammad Bubak
M.Adnan , Ameer Muawiya
Registration No: 21-UON-0198,0222,0223,0211
Course Code: CHEM-304
Course Title: Analytical Chemistry 11
Department: Chemistry
Chromatography:
In chemical analysis, chromatography is a laboratory technique for the separation
of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or
liquid) called the mobile phase, which carries it through a system (a column, a
capillary tube, a plate, or a sheet) on which a material called the stationary phase is
fixed.
Column Chromatography
Column chromatography is a separation technique that is used to isolate
compounds from complex mixtures. Either small or large-scale column
chromatography can be carried out to separate and purify the analytes. Column
chromatography has two phases viz a mobile phase and a stationary phase.
Principle of Column Chromatography: Column chromatography process

involves the uniform percolation of a liquid solute through a column packed with
finely divided material. It involves adsorption, partition or ion exchange
phenomenon. The adsorption column chromatography is based on the retention of
solute by surface adsorption. This technique is useful in the separation of nonpolar
substances and constituents of low volatility.
The sample mixture is set on the top of the column and is made to absorb on the
top of the stationary phase. Eventually, the mobile phase which is applied to the
column is used to remove the mixture via the stationary phase. Separation of
compounds in column chromatography by utilizing the molecule’s polarity. The
rate at which molecules move in the column during the separation process depends
on their polarity. As the mobile phase comes out of the column it is collected in
small fractions in the test tube. Then it is allowed to isolate and purify. Later, the
removal of salt is done by a rotary evaporator to obtain the separated compound.
Column chromatography is the most widely used technique for the purification of
compounds due to its versatility. Also, it is a very convenient process.
Column chromatography is a chromatography technique used to separate chemical
substances of a mixture into individual compounds.
This separation method consists of two phases viz: contiguous stationary phase and
mobile phase.
The column is prepared by mixing the silica and a suitable solvent. It is later
poured into a column made of glass.
The mobile phase (suitable solvent) is moved along with a compound mixture
through the Column based on the polarity.
Component Use in column Chromatography:

Various stationary phases, such as silica, alumina, calcium phosphate,
calcium carbonate, starch, and magnesia, and different solvent
compositions based on the nature of compounds to be separated and
isolated, are used in column chromatography.
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.
2-Two types of preparing the column, known as packing techniques
namely:
 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.
 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.
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.
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:
 Isocratic elution technique – Throughout the procedure, a solvent of the

same polarity or same solvent composition is utilized.
 Example: Use of chloroform alone
 Gradient elution technique – Throughout the separation procedure, solvents

of gradually increased polarity or increased elution strength are utilized.
 Example: Benzene → Chloroform → Ethyl acetate → Chlorofor
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 – 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.
Applications:
Column Chromatography is used to isolate active ingredients.

It is very helpful in separating compound mixtures.
It is used to determine drug estimation from drug formulations.
It is used to remove impurities.
Used to isolate metabolites from biological fluid
Advantages and Disadvantages of Column Chromatography
Some advantages and disadvantages of column chromatography are discussed
below. So let us find out its advantages and disadvantages to know more about
column chromatography.
Advantages of Column Chromatography are:

Column chromatography enhances detection of compounds.
Column chromatography enables trace analysis.
Increases signal to noise ratio
It also detects low concentration.
Column chromatography improves analytical accuracy.
It is best suitable various detection methods.
Column chromatography optimizes sensitivity easily.
It separates closely related compounds.
Enables detection of impurities.
Disadvantages:
Disadvantages of Column Chromatography are:
Column chromatography equipment is expensive.
Costly consumables.
The operating cost is high.
Column chromatography requires specialized training.
It is time consuming process.
It is labor intensive technique.
Difficult to automate.
Requires significate sample for

Column Chromatography 1

Uploaded by

Copyright:

Available Formats

Column Chromatography 1

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Column Chromatography 1

Uploaded by

Copyright:

Available Formats

Submitted To.

Ma’am Asma Shaheen

Principle of Column Chromatography: Column chromatography process

Component Use in column Chromatography:

1-Preparation of the column

 Dry packing technique – The amount of absorbent needed is added as a fine

Introduction of the sample

 The process of elution can be carried out by employing two techniques:

 Isocratic elution technique – Throughout the procedure, a solvent of the

 Gradient elution technique – Throughout the separation procedure, solvents

Types of Column Chromatography:

2. Partition column chromatography – The stationary phase, as

3. Gel column chromatography – In this method of

4. Ion exchange column chromatography – A chromatography

Column Chromatography is used to isolate active ingredients.

Advantages of Column Chromatography are:

You might also like