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
