Column Chromatography – Instrumental Methods of Analysis B. Pharma 7th Semester

Column Chromatography

Contents

•       Column
chromatography

•       Principle
involved

•       Practical
requirements of column chromatography

•       Stationary
phase (adsorbent)

•       Mobile
phase

•       Column
characteristics

•       Preparation
of column

•       Introduction
of sample

•       Development
technique (elution)

•       Detection
of components

•       Recovery
of components

•       Factors
effecting column efficiency

•       Applications

Objectives

By the end of this session, students will be able to:

Ø  Define
Column Chromatography

Ø  Explain
the principle involved in the column chromatography

Ø  Explain
the practical requirements of column chromatography

Ø  Explain
the procedure involved in quantitative estimation of compound by Column
Chromatography 

Ø  Discuss
the components of column chromatography

Ø  Discuss
the factors effecting column efficiency

Ø  Explain
the pros and cons of column chromatography

Column
Chromatography 

•       Column
of stationary phase is used

•       Stationary
phase is solid- Column adsorption chromatography

•       Stationary
phase is liquid- Column partition chromatography

•       Column
partition chromatography is not widely used

Principle

•       Solid
stationary phase and liquid mobile phase is used

•       Principle
of separation is adsorption

•       Mixture
of components dissolved in mobile phase is introduced in the column

•       Individual
components move with different rates depending upon their relative affinities

•       Compounds
with lesser affinity towards stationary phase moves faster and eluted out first

•       Component
with greater affinity towards stationary phase moves slower and eluted later

•       Compounds
are separated

•       Type
of interaction between stationary phase and solute is reversible in nature

•       Rate
of movement of component is given as

•       R
= rate of movement of component /rate of movement of mobile phase

•       Can
be simplified as

•       R
= distance moved by the solute /distance moved by the solvent 

Practical
Requirements

•       Stationary
phase (adsorbent)

•       Mobile
phase

•       Column
characteristics

•       Preparation
of column

•       Introduction
of sample

•       Development
technique (elution)

•       Detection
of components

•       Recovery
of components

Stationary
Phase

•       Adsorbent
used in column chromatography

Should meet the following criteria

•       Particle
size and geometry

•       Particles
should have uniform size distribution and have spherical shape

•       Particle
size- 60-200 µ

•       Should
have high mechanical stability

•       Should
be inert and should not react with solute or other components

•       Insoluble
in the solvents or mobile phase used

•       Should
be colorless to facilitate observation of zones and recovery of components

•       Should
allow free flow of mobile phase

Should meet the following criteria

•       Should
be useful for separating wide variety of compounds

•       Should
be freely available, inexpensive, etc

Types of adsorbents

Selection
of Stationary Phase

•       Success
of chromatography depends upon the proper selection of stationary phase

•       Selection
of stationary phase depends upon

Removal of impurities

•       Small
quantity of impurity is present and there is a difference in affinity

•       Weak
adsorbent is used

No. of components to be separated

•       Few
components are to be separated- weak adsorbent is used

•       More
components are to separated- strong adsorbent is used

Affinity difference between components

•       Components
with similar affinities- strong adsorbent

•       More
difference in affinities- weak adsorbent

Length of the column used

•       Shorter
column- strong adsorbent

•       Longer
column- weak adsorbent

Quantity of adsorbent

•       20
or 30 times the weight of adsorbent is used for effective separation

•       Adsorbate
: adsorbent ratio = 1:20 or 1:30

Mobile
Phase

•       Very
important and serve several functions

•       Act
as solvent, developer and as eluent

Functions of a mobile phase

•       To
introduce the mixture into column- as solvent

•       To
develop the zones for separation- as developing agent

•       To
remove pure compound out of the column- as eluent

•       Different
mobile phases used- in increasing order of polarity or elution strength

•       Petroleum
ether, carbon tetrachloride, cyclohexane, carbondisulphide, ether, acetone,
benzene, toluene, ethylacetate, chloroform, alcohols (methanol, ethanol, etc),
water, pyridine, organic acids (acetic acid, etc)

•       Can
be used in either pure form or as mixture of solvents of varying compositions

Column
Characteristics

•       Material
of column is mostly good quality neutral glass

•       Should
not be effected by solvents, acids or alkalies

•       Ordinary
burette can also be used as column for separation

•       Column
dimensions are important for effective separation

•       Length:diameter
ranges from 10:1 to 30:1

•       For
more efficiency 100:1 can also be used

Length of column depends upon

•       Affinity
of compounds towards adsorbent

•       Number
of compounds to be separated

•       Type
of adsorbent used

•       Quantity
of the sample

Preparation
of the Column

•       Bottom
portion of column is packed with cotton wool or glass wool or may contain
asbestos pad

•       Above
it column of adsorbent is packed

•       Whatmann
filter paper disc is also used

•       After
packing the column, similar paper disc is kept on the top

•       Adsorbent
layer is not disturbed during introduction of sample or mobile phase

•       Disturbance
in the layer of adsorbent will leads to irregular bands in separation

Two types of packing techniques are available

•       Dry
packing technique

•       Wet
packing technique

Dry Packing

•       Here,
required quantity of adsorbent is packed in the column in dry form

•       Solvent
is allowed to flow through column, until equilibrium is achieved

Drawbacks of this technique 

•       Air
bubbles are entrapped between the solvent and the stationary phase

•       Column
may not be uniformly packed

•       Cracks
appear in the adsorbent present in the column

•       No
uniformity in flow characteristics and

•       Clear
band of the separated component may not be obtained

Wet Packing

•       Ideal
technique

•       Required
quantity of adsorbent is mixed with mobile phase solvent in a beaker and poured
into column

•       Stationary
phase settles uniformly in the column

•       No
entrapment of air bubbles

•       No
cracks in the column of adsorbent

•       Bands
eluted from the column will be uniform

•       Ideal
for separation

Introduction
of Sample

•       Sample
which is usually a mixture of components is dissolved in minimum quantity of
mobile phase

•       Or
a solvent of minimum polarity

•       Entire
sample is introduced into column at once

•       Gets
absorbed on to the top portion of column

•       Individual
compound can be separated by process of elution

Development technique (Elution)

•       After
introduction of sample, by elution techniques individual components are
separated from the column

•       Isocratic
elution technique

•       Gradient
elution technique

Elution
Techniques

Isocratic elution technique

•       Here,
same solvent composition or solvent of same polarity is used throughout the
process of elution

•       For
example chloroform only, petroleum ether:benzene = 1:1 only, etc

Gradient elution technique

•       Here,
solvents of gradually increasing polarity or

•       Increasing
elution strength are used during the process of elution

•       Initially
low polar solvent is used followed by gradually increasing the polarity to a
more polar solvent

•       For
example, initially benzene, then chloroform, then ethylacetate, then to
methanol, etc

Detection
of Components

•       Detection
of colored components can be done visually

•       Different
colored bands are seen moving down the column which can be collected
immediately

•       But
for colorless compounds, technique depends on properties of components

Different properties which can be used are

•       Absorption
of light (UV/Visible) – using UV/Vis detector

•       Fluorescence
or light emission characteristics – using fluorescence detector

•       Using
flame ionization detector

•       Refractive
index detector

•       Evaporation
of solvent and weighing the residue

•       By
monitoring the fractions by thin layer chromatography

Recovery of
Components

•       Earlier
done by cutting the column into several distinct zones

•       Later,
extrusion of column into zones were done by using plunger

•       Best
technique is to recover the components by a process called elution

•       Components
are called eluate

•       Solvent
called eluent

•       Process
of removing the components from the column is called elution

•       Recovery
is done by collecting as different fractions of mobile phase of equal volume
like 10 ml, 20 ml, etc or unequal volume

•       Can
also be collected time wise i.e., fraction every 10 or 20 minutes, etc

•       Recovered
fractions are detected by using techniques discussed in last slide

•       Similar
fractions are mixed to get bulk compound in pure form

Factors
Effecting Column Efficiency

•       For
any separation, efficiency of column is important

•       Unless
factors effecting the column efficiency are known, efficiency cannot be
improved

Dimensions of the column

•       A
length:diameter ratio of 20:1, 30:1 are ideal

•       100:1
may be satisfactory

Particle size of the adsorbent

•       Adsorbent
activity depends on the surface area of adsorbent

•       For
increasing the surface area, particle size can be reduced

•       Adsorbent
activity increases

Nature of solvent

•       Flow
rate of solvent is affected by its viscosity and is inversely proportional

•       Less
viscous solvents are better efficient

Temperature of the column

•       Speed
of elution is increased at higher temperature

•       But
adsorbent power is decreased at higher temperatures

•       Balance
should be made between the speed of elution and adsorbent power

•       Normally
room temperature is used for all samples

•       Difficult
samples are separated at high temperatures

 Pressure

•       High
pressure above the column and

•       Low
pressure below column increases the efficiency of separation

•       High
pressure above column can be achieved by maintaining the mobile phase reservoir
on top of column or

•       By
using pressure devices

•       Pressure
below the column decreased by applying vacuum using vacuum pump

Applications

•       Separation
of mixture of compounds

•       Removal
of impurities or purification process

•       Isolation
of active constituents

•       Isolation
of metabolites from biological fluids

•       Estimation
of drugs in formulations or crude extracts

Column
Chromatography Pros & Cons

Pros

•       Any
type of mixture can be separated

•       Any
quantity of mixture can be separated (μg to mg)

•       Wider
choice of mobile phase

•       In
preparative type sample can be separated and reused

•       Automation
is possible

Cons

•       Time
consuming method

•       More
amount of solvents are required which are expensive

•       Automation
makes the technique more complicated and expensive

Summary

•       Column
partition chromatography is not widely used

•       Individual
components move with different rates depending upon their relative affinities

•       Compounds
with lesser affinity towards stationary phase moves faster and eluted out first

•       Success
of chromatography depends upon the proper selection of stationary phase

•       Adsorbate
: adsorbent ratio = 1:20 or 1:30

•       Material
of column is mostly good quality neutral glass

Different properties for detection which can be used are

•       Absorption
of light (UV/Visible) – using UV/Vis detector

•       Fluorescence
or light emission characteristics – using fluorescence detector

•       Using
flame ionization detector

•       Refractive
index detector

•       Evaporation
of solvent and weighing the residue

•       By
monitoring the fractions by thin layer chromatography

Factors effecting column efficiency

•       Dimensions
of the column

•       Particle
size of adsorbent

•       Nature
of solvent

•       Temperature
of the column

•       Pressure

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