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

Weak

Medium

Strong

Sucrose

Calcium carbonate

Activated magnesium silicate (Silica gel)

Starch

Calcium phosphate

Activated alumina

Inulin

Magnesium carbonate

Activated charcoal

Talc

Magnesium oxide

Activated magnesia

Sodium carbonate

Calcium hydroxide

Fuller’s earth

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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