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Chromatography

Chromatography is a separation process that involves the interaction of solutes with a mobile phase and a stationary phase, utilizing methods like frontal analysis, displacement analysis, and elution. Key concepts include retention time, capacity factor, and selectivity factor, which help describe the migration rate of solutes and their separation efficiency. Ideal chromatography results in sharp, distinct peaks, while non-ideal conditions lead to broader, diffuse zones.

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4 views

Chromatography

Chromatography is a separation process that involves the interaction of solutes with a mobile phase and a stationary phase, utilizing methods like frontal analysis, displacement analysis, and elution. Key concepts include retention time, capacity factor, and selectivity factor, which help describe the migration rate of solutes and their separation efficiency. Ideal chromatography results in sharp, distinct peaks, while non-ideal conditions lead to broader, diffuse zones.

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ashlandrph
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© © All Rights Reserved
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© Rehan Gul

Chromatography
• A separation process involving the
interaction of one or more solutes with 2
phases;
1- Mobile phase (gas, liquid)
2- Stationary phase (solid, liquid).
– It does not move and retards the individual
species based on following interactions
 Surface adsorption
 Relative solubility or
 Charge
Types of separation in chromatography
• Frontal analysis
Continuously add sample on the start of column and
monitor components as they evolve e.g. charcoal
filtration
– Not useful as a method of separation
• Displacement analysis
Materials move down a column by being displaced by a
more highly retained solute e.g. ion exchangers/water
softeners
• Elution
A solute partitions between 2 phases where separation is
based on relative retention
Theoretical considerations
• Chromatographic column is imagined to be static system in
equilibrium between mobile and stationary phase,
• If A is an analyte, m is mobile phase and s is stationary phase, then
equilibrium may be expressed as
Am ↔ As
Based on this equilibrium, partition coefficient K is give as
K = Cs/Cm
As K increases, solute A will take a longer time to pass through a
column
The average linear rate V of solute migration is give as

V = L/tR
Where L is column length and tR is retention time of the solute
• Similarly the average linear velocity u of
the mobile phase is
u = L/tM
where tM is a dead time, and is the time
required for a non-retarded substance to
pass through the column, and is given as:
V=uXf
where f= fraction of time solute
is in the mobile phase
Relationship of migration rate and partition
coefficient
• To relate the rate of migration of a solute to its
partition coefficient, we express the rate as
fraction of the velocity of the mobile phase
V = u X fraction of time solute in mobile phase

fraction of time solute in mobile phase = No. of


molecules in mobile phase/total number of
molecules

CM VM
fraction of time solute =
in mobile phase Dividing this
CM VM + CS VS relation by CM VM
1 1
= =
1 + K VS / VM 1 + K/

Where K/ is capacity factor and equal to K VS/VM


1
V =u Putting this value
1 + K/ in rate equation

Thus the rate of the travel of an average molecule, V, is


determined by
1- Carrier velocity
2- VS/Vm ratio
3- Distribution coefficient K or K/
Retention time
• Time required for a component to travel the length of
the column, L.
tR= length / rate of solute migration = L/V
tM= L/u
tR= L (1 + K/) = tM (1 + K/)
U

K/ = tR – tM / tM
Where tM is the time required for carrier to traverse
column and provide a measure of average rate of
migration of the mobile phase
Capacity factor k /

• Important experimental parameter used widely


to describe the migration rate of a solute in a
column
k / = K V S / Vm
Where K is partition coefficient
Vs volume of stationary phase
Vm volume of mobile phase
k/ = tR - tM / tM
k/ = 1-5 ideal
k/ < 1 elution occur too rapid
k/ > 20-30 elution become extremely long
Retention volume (VR)
• Volume of mobile phase required to elute the
sample component
VR = tR F where F is flow rate of mobile phase
Substituting equation
VR/F = tR = L/u (1 + K/)
VR = VM (1 + K/) = Vm + K VS (basic
retention equation)
Where Vm is void volume and is the
volume of the mobile phase within the
column
Selectivity factor or relative retention α
• Selectivity factor of a column for 2 species A and B is
defined as
α= KB /KA
KB = partition coefficient for more strongly retained species B
KA= partition coefficient for less strongly retained species A

we know that k/ = K VS / Vm (k/A = KA VS / Vm)


the α= K/ B /K/ A
α= (tR)B - tM / (tR)A – tM
This equation permits the determination of α from an
experimental chromatogram and it is used to compare the
resolving power of a column
Ideal and non ideal chromatography
• Ideal situation
• All molecules behave identically to the
average molecule
• The component zone also called band or peaks
have no tendency to spread and would elute
out of the column in the same volume or time
interval
• Chromatogram would appear as a series of
very sharp, completely separated peaks
• Non ideal situation
• Random behavior of molecules
• Zones become diffuse and broad as
they pass through the column

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