CHM 260

Chapter 4: An Introduction to
Chromatographic Separations

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• Chromatography is a technique in which the components of a
mixture are separated based on the rates at which they are
carried through a stationary phase (SP) by a gaseous or liquid
mobile phase (MB)

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 (Thin layer
Chromatography)

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o Planar Chromatography - the stationary phase is supported on a
flat plate or in the fibers of a paper. Here the mobile phase
moves through the stationary phase by capillary action.
Examples include:
➢ Paper chromatography
➢ Thin layer chromatography (TLC)

o Column Chromatography - the stationary phase is held in a

narrow tube through which the mobile phase is forced either by
pressure or by gravity. Examples include:
➢ Simple column chromatography
➢ High pressure liquid chromatography (HPLC)
➢ Gas chromatography (GC)

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1. Partition chromatography (GC, LC)
• This form of chromatography is based on a thin film formed
on the surface of a solid support by a liquid stationary
phase.
• Solute equilibrates between the mobile phase and the
stationary liquid.
• In partition chromatography the solute moves to and pro
between the stationary phase and the mobile phase and are
partitioned between them.
• Both stationary and mobile phases are liquid.
• The rate of movement depends on relative solubility of the
solute in the two phases.
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2. Adsorption Chromatography
• In the adsorption chromatography, the solute molecules are
held on or attached to the surface of the stationary phase.
• Because of the different degree of intermolecular
attraction, some components of the mixture will be more
attracted or adsorbed to the stationary phase.
• The stationary phase is a polar solid and the solutes are
polar molecules.
• The separation of the solutes depends on the difference in
their polarity. The more polar solutes are more readily
adsorbed than the less polar solutes.
• The equilibration between the mobile and stationary phase
accounts for the separation of different solutes.

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3. Ion Exchange Chromatography
• In type of chromatography, a resin (stationary solid phase) is
used to covalently attach anions and cations onto it.
• Solute ions of the opposite charge in the mobile liquid phase
are attracted to the resin by electrostatic forces.

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4. Affinity Chromatography
• This is the most selective type of chromatography.
• It utilizes the specific interaction between one kind of
solute molecule and as second molecule that is
immobilized on a stationary phase.
• For example: Immobilized molecule maybe antibody to
some specific protein. When a solute containing a mixture
of proteins are passed this molecule, only the specific
protein is reacted to this antibody ➔ binding it to the
stationary phase
• This protein is later extracted by changing the ionic
strength or pH

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5. Molecular (Size) Exclusion Chromatography
• Also known as gel permeation or gel filtration.
• Lacks an attractive interaction between the stationary phase
and solute.
• The liquid/gaseous phase passes through a porous gel which
separates the molecules according to its size.
• The pores are normally small and exclude the larger solute
molecules, but allows smaller molecules to enter the gel.
• This causes the larger molecules to pass through the column
at a faster rate than the smaller ones.

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• Elution: A process in which solutes are washed through a
stationary phase by the movement of a mobile phase.
• Eluent: A solvent used to carry the components of a mixture
through a stationary phase.

• Elution involves washing a solute

through a column by additions of fresh
solvent.

• A single portion of the sample dissolved

in the mobile phase is introduced at the
head of the column, where components
A and B distribute themselves between
the two phases.

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• Introduction of additional mobile phase
(the eluent) forces the dissolved portion
of the sample down the column, where
further partition between the mobile
phase and fresh portions of the
stationary phase occurs

• Partitioning between the fresh solvent

and the stationary phase takes place
simultaneously at the original site of
the sample.

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• Further additions of solvent carry
solute molecules down the
column in a continuous series of
transfers between the two
phases.
• Because solute movement can
occur only in the mobile phase,
the average rate at which a solute
migrates depends on the fraction
of time it spends in that phase.
• This fraction is small for solutes
that are strongly retained by the
stationary phase (component B)
and large where retention in the
mobile phase is more likely
(component A).
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• Ideally, the resulting differences
in rates cause the components in
a mixture to separate into bands,
or zones, along the length of the
column.

• Isolation of the separated species

is then accomplished by passing a
sufficient quantity of mobile
phase through the column to
cause the individual bands to pass
out the end (to be eluted from
the column), where they can be
detected and collected.

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• A detector that responds to solute concentration is placed at
the end of the column and its signal is plotted as a function of
time (or of volume of added mobile phase), a series of
symmetric peaks is obtained.
• The positions of the peaks on the time axis can be used to
identify the components of the sample; the areas under the
peaks provide a quantitative measure of the amount of each
species.

• Chromatogram is a plot of some function of solute

concentration versus elution time or elution volume.
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• The degree of separation can be improved either by increasing
the degree of band separation OR by decreasing the band
spread
• This can be achieved by changing the experimental conditions.

Adjust the migration rates for A and Adjust zone broadening (decrease
B (increase band separation) band spread)
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• The effectiveness of a chromatographic column in separating two
solutes depends on the relative rates at which the two species
are eluted.
• The rates are determined by the partition ratios of the solutes
between two phase (mobile & stationary phases)

Partition Ratio, K

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Retention time

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Retention factor or Capacity factor (k’A)
• Describe the migration rates of solutes on a column
• Retention factor relates to how much material
can chromatograph without adversely
affecting peak shape

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Selectivity factor (α)
• The quantity which describes the separation of two species (ie.
A and B) on the column
• The relative selectivity is directly related to how different
analyte molecules interacts with the phase involved in the
column

• When calculating the selectivity factor, species A elutes faster

than species B. The selectivity factor is always greater than one.

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• To obtain optimal separations, sharp, symmetrical
chromatographic peaks must be obtained
• This means that band broadening
must be limited.

Band broadening.. Why??

1. Non-linear distribution coefficient
Cause ➔ Fronting and tailing

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2. Diffusion – molecules tend to diffuse
from more concentrated part of a
solution to a more dilute

3. Individual molecules in the mobile

phase follow path of different
length as they traverse the column

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• Two related terms are widely used as quantitative measures of
the efficiency of chromatographic columns:
(Number of theoretical plates and plate height)
1. Number of theoretical plates, N
(the plate model supposes that the chromatographic column is contains a large
number of separate layers, called theoretical plates. Separate equilibrations of the
sample between SP and MB occur in these ‘plates’.)

N measures the EFFICIENCY of columns

To increase N ➔ decrease H

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 2. Plate Height, H
• An indication of efficiency of column in terms of length of
column required for a degree of separation

where σ2 is variance
unit for L (cm), σ2 (cm2)

where,
Ʈ is standard deviation
W width of peak at its base (in units of
time)
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 Van Deemter plot

1 Effect of Mobile-Phase Flow Rate

• A minimum of H – maximum efficiency
(occurs at low linear flow rates)

2 Effect of Column Lengths

• Longer columns – higher efficiency
• But for liquid chromatography (LC),
column length about 25-50 cm only to prevent high
pressure drops
• In gas chromatography (GC), columns may be 50 m or more
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• Optimize column resolution ➔ separate mixture to individual
components and subsequently identify them
• Column resolution (Rs): A quantitative measure of the ability to
separate two analytes

Enhance retention
Selectivity of the
time difference (Better
column
to optimize)

Decrease band widths Efficiency of the

column

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Effect of Capacity and Selectivity Factor on Resolution

Effect of Resolution on Retention Time

• To double the resolution R ➔ column length must increased by

factor of 4
• To obtain high resolution, MAXIMISED:
1) Increase N (reduce the size of SP particles)
2) Lengthening the column
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• Controlling the capacity factor, k’ ➔ separation can be greatly
improved
• How?? 1) changing the temperature (in GC)
2) changing the composition of the mobile phase (in LC)

• Selectivity factor, α can also be manipulated to improve

separations

• How? First, optimised k’

Then, α is increased by:

1. Changing the mobile phase composition (for HPLC)

2. Changing column temperature (for GC)
3. Changing composition of SP
4. Using special chemical effects

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• For multiple components, conditions rarely optimum for all
components

Solution? Change column conditions during elution

➢ change in liquid mobile phase composition –gradient

elution or solvent programming (in LC)
➢ change in temperature for GC – temp. programming

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• The diameter of a column and the thickness of the stationary
phase should always be considered together because they
interact with regards to column performance. However, the
general effect of decreasing column diameter is to increase
the speed of analysis. This is because the optimal carrier gas
velocity increases as the diameter decreases (assuming that
the retention factor and plate number are held constant).
Smaller diameter columns usually have lower plate height
(higher N) because of improved mass transfer. Increasing
carrier gas velocity results directly in faster analyses. On the
other hand, larger diameter columns have increased sample
capacity which can provide higher detectability with mass
sensitive detectors such as an FID.

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• Another consideration of column diameter is how it affects
the amount of sample that can be injected. Larger samples
require a larger amount of stationary phase to interact with,
otherwise the column will be overloaded, resulting in poor
chromatography. Larger diameter columns have higher
capacity and less problems with overloading.

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Chromatography is a versatile tool for SEPARATING closely
related chemical species. It can be employed for quaLitative
identification and quanTitative determination of separated
species.

Qualitative and quantitative determination (Chapter GC)

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1) Chromatogram :
A graph showing the detectors
response as a function of elution
time :
band’s shapes, position, resolution.
2) For individual band :
a) Retention time (tr) :
The time needed after injection for an
individual solute to reach detector.
b) An ideal chromatographic peak
 Gaussian shape.
w½ = 2.35σ, w = 4σ
3) For pairs of bands
a) Efficiency : two factors contribute to how
well components are separated :
the widths of the peaks :
the wider the peak, the poorer separation.
the spacing in time :
the further apart, the better separation.
b) Theoretical plates (N): (from distillation)
the more plates on a column, the more
equilibration steps, and the better the
separation.
Number of plates on column :
N = 5.55(tr/w½)2
Plate height : H = L/N
The smaller plate height
 narrower peaks  better separation
c) Resolution (Rs)
Rs = Δ
tr
=
(tr − tr )
2 1
 L
w av 1 2 (w 1 + w 2 )

For quantitative analysis, Rs  1.5

2  length of the s.p.   2 Rs