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  Chroma means Color
 Graphein means Writing
 First chromatography was introduced by Russian
botanist, Mikhael Tswett,
 In 1906 Tswett used chromatography to
separate plant pigments

 He called the new technique

chromatography because the result of
the analysis was 'written in color' along
the length of the adsorbent column
 Chromatography is an analytical method that allow the
separation, identification and determination of components of a
mixture, which is based on the difference in the degree of
interaction of the components of the mixture with two immiscible
phases: a stationary phase and mobile phase.
 In all chromatographic separations the sample is dissolved in a
mobile phase, which may be a gas, a liquid, or a supercritical fluid.
This mobile phase is then forced through an immiscible stationary
phase, which is fixed in place in a column or on a solid surface.
 Upon movement through the stationary phase, each components
will interact with both the stationary phase and mobile phase.
 Since they have different degree of interactions, they will not have
the same speed.
 The one that strongly interacts with the stationary phase will move
slowly while the one that interact weakly will move fast.
 upon this movement they will be separated across the column or
plate.
 Definitions of Terms
Mobile phase: is a phase (substance) that is used to transport the
sample across the stationary phase.
 What kinds of substances can be used as mobile
phase? Which ones can not? Why?
 A single mobile phase or a mixture of solvents
may be used
 Examples, hydrogen, nitrogen, water, methanol
etc…………………………………….
Stationary phase: is a phase (substance), which can be
packed inside a column or coated on inner part of
the column or coated on a surface of a flat plate,
the sample is being carried away by the mobile
phase.
 What kind of substances can be used as stationary
phases? Which ones can not? Why?
 It may be packed or coated by itself or it may be
 In a chromatographic separations the mobile phase and the
stationary phase must have low interaction (theoretically).
 Always when we select a mobile phase we should consider this.
This is because
1. The mobile phase must be the least eluted species.
2. The mobile phase could not wash the stationary phase.

Classification of Chromatography
 Chromatography can be classified into groups based on several
properties. Here in this chapter we will classify chromatography
based on three criteria.
1. based on the physical means by which the stationary phase
and mobile phase are brought together.
2. based on the type of mobile phase and stationary phase
3. based on the type of interaction occur between the
sample and stationary phase.
1. Classification of chromatography based on the physical means by
which the stationary phase and mobile phase are brought together.
 In this classification chromatography is classified into two groups.
A.Column chromatography: in this type of chromatography the
stationary phase is held in a narrow tube (column). The stationary
phase can be
A. packed inside a column (packed column chromatography)
B. coated on the internal surface of a column (open
tubular column chromatography)
 The mobile phase moves in the column either due to gravity
or due to the force of a pump.
B.Planar chromatography: in this type of chromatography the
stationary phase is held in a planar, flat plate or on the interstices
of paper. The stationary phase material is coated on the wall of
a flat plate.
 The mobile phase is transported across the stationary
phase by a capillary action or gravity. Example, thin layer
and paper chromatography.
 Which types of mobile phase can be used for column and planar
chromatography? Why?
 Which types of stationary phases can be used for column and
planar chromatography? Why?

2. Classification of chromatography based on the type of mobile

phase and stationary phase
 In this case the classification is based on the physical state of
the mobile and stationary phase.
 During naming using this classification the first letter
represents the type of mobile phase and the second letter
represents the type of stationary phase used.
Type of chromatography M.P S.P
A.Liquid chromatography (LC)
i. Liquid – liquid chrom. (LLC) Liquid Liquid
ii. Liquid – solid Chrom. (LSC) Liquid Solid
B.Gas chromatography (GC)
i. Gas – liquid chrom. (GLC) Gas Liquid
ii. Gas – solid chrom. (GSC) Gas Solid
C.Supercritical fluid chromatography (SFC)
 We use supercritical fluid as a mobile phase.
 We may use liquid immobilized on support material or solid
stationary phase
Exercise
1. Justify
a. All planar chromatographic techniques are liquid
chromatography.
b. All Gas and supercritical fluid chromatographic techniques
are column chromatography.
3. Classification of chromatography based on the type of
interaction occur between the sample and stationary phase.

 This classification is based on the types of interactions that

occurs between the components of the mixture to be separated
with the stationary phase or mobile phase.
A. Adsorption chromatography: it is a type of chromatography in
which solutes are separated because they have different degree
of adsorption on a solid stationary phases.
 The type of interaction is adsorptions
 Solid stationary phases are used such as silica and
alumina.
B. Partition chromatography: it is a type of chromatography in
which separation takes place because solutes have different
degree of solubility into two immiscible liquid phases (the
m.p and s.p)
 We usually use liquid stationary phase immobilized on a
solid support materials.
C. Ion exchange chromatography: it is a type of chromatography in
which ions are separated due to their ability to exchange H+ or
OH- found on the stationary phase.
 It is used to analyze ions.
 We usually use ion exchange resins as a stationary phase.
D. Size exclusion chromatography: it is a type of chromatography
in which separation takes place because solutes have different
size to pass through a porous stationary phase differently.
 Solutes are separated based on their size.
 Only used to analyze molecules that have very large
(>10000) molecular weight.
E. Affinity chromatography: it is another type of chromatography
in which separation takes place based on the difference in
affinity of components of a sample for affinity ligand attached
on support material.
 Most selective interactions
 Mostly used to separate biomolecules
 Most affinity ligands are enzymes, inhibitors, antibodies, etc
Note that:
Separation in chromatography is based on the elution of the sample
across the stationary phase by the mobile phase. Across the movement
the components of the sample interacts with the stationary phase
differently.
 The solute, which interacts strongly with the stationary will be
strongly retained. So the speed of this solute in the column or
plane is very slow and takes more time to be eluted. So, they
are the last to be eluted.
 The solute, which interacts with the stationary phase
weakly, will be weakly retained. So the solute will be easily
eluted by the mobile phase. So, they are the first to be eluted.
 The Chromatogram
 If a detector that responds to the solute concentration is placed at
the end of the column and its signal is plotted as a function of
time, a series of peak are observed.

 A chromatogram is a graph showing the detector response as a

function of elution time (rarely volume of mobile phase)
 It is the representation of the variation, with time (rarely volume),
of the amount of the analyte in the mobile phase exiting the
chromatographic column.

 The position of peaks on the time axis (retention time) may

serve to identify the components of the sample.

 The area under each peak provides the quantitative measure of

the amount of each component.
 Resolution Between Two Peaks
 Resolution (Rs): is the separation of two chromatographic
peaks or bands.
 The goal of chromatography is to separate a sample into a
series of chromatographic peaks, each representing a single
component of the sample.
 Resolution is a quantitative measure of the degree of
separation between two chromatographic peaks, A and B, and is
defined as

Where WA and WB are base

line width of peak of 1 and 2
respectively
DZ > 1
 Retention time (tr): It is the time taken for a given substance for
its migration throughout the column from the moment of entrance
to the moment in which it leaves the column(enters into the
detector).
 Retention volume (Vr): It represents the volume of mobile phase
necessary to enable its migration throughout the column from the
moment of entrance to the moment in which it leaves the column
(enters into the detector)
Based on the above equation to increase the resolution,
1. We have to increase the difference in the retention time of
the strongly retained species B and the weakly retained
species, A, i.e. ∆Z and
2. We have to decrease the baseline width of species A and B.

Increasing ∆Z is done by adjusting the migration rate of the two

species. That is by increasing the retention time of the strongly
retained species, solute B and by decreasing the retention time of
the weakly retained species, solute A.
Decreasing the sum of W1 and W2 is done by decreasing the line
broadening.
 The first option of increasing the efficiency of separation
 in GC is
 changing the stationary phase type
 changing the temperature (Temperature
programming)
 In LC
 changing the mobile phase type
 changing the percentage composition of the mobile
phase
 changing the stationary phase type
 changing the pH
Band Broadening and Column Efficiency
 Multiple Paths
 This occurs when different particles of the solutes takes different
path that differs in length as the solute migrates through the
column.
 This multiple path effect sometimes known as Eddy diffusion
 Longitudinal Diffusion
 Diffusion is a process in which species migrate from a more
concentrated part of a medium to a more dilute region.
 The rate of migration is proportional to the concentration
difference between the regions and to the diffusion coefficient D M
of the species. The latter, which is a measure of the mobility of a
substance in a given medium, is a constant for a given species
equal to the velocity of migration under a unit concentration
gradient.
 In chromatography, longitudinal diffusion results in the migration
of a solute from the concentrated center of a band to the more
dilute regions on either side (that is, toward and opposed to the
direction of flow).
 Longitudinal diffusion is a common source of band broadening in
gas chromatography, where the rate at which molecules diffuse is
high. The phenomenon is of little significance in liquid
chromatography, where diffusion rates are much smaller.
 Mass Transfer
 In chromatographic separation, solute particles move from mobile
phase into the stationary phase or vice versa.
 For a solute to move from one phase to the other, it must first
diffuse to the interface between the two phases—a process called
mass transfer.
 A contribution to band broadening occurs whenever the
solute’s movement to the interface is not fast enough to
maintain a true equilibrium distribution of solute between the
two phases.
 This will create unequal mass transfer between the two phases that
could result into band broadening.
 Using Column Efficiency to Optimize Resolution

 One way is by increasing the length of the column. But it may be

disadvantageous. Why?
 Other way is by minimizing factors which result into band
broadening.
 Multiple path can be minimized by
 using a stationary phase with very small particle size
 using a stationary phase with very ordered arrangement.
 Longitudinal diffusion can be minimized by
 using small thickness of stationary phase packing or coating.
 large mobile phase flow rate
 Mass transfer problem can be minimized by
 decreasing the mobile phase flow rate
 using, smaller diameter packing materials, and thinner films of
stationary phase.
 Gas Chromatography (GC)
 It encompasses all chromatographic techniques which use gaseous
mobile phase.
 We can use liquid stationary phase which is held in solid support
material.
 The type of interaction is the partitioning of solute particles between
the liquid stationary phase and gas mobile phase.
 We can use also solid stationary phase.
 Separation takes place due to the difference in the degree of
adsorption of solute particles on the solid stationary phase.
 It is entirely column chromatographic technique.
 We can`t use stationary phase coated in paper or flat plate.
Instrumentation
1. Mobile Phase (Carrier gas supply)
 He, H and N are most common mobile phases in GC
 They are chemically inert toward both the sample and
stationary phase.
 They interact weakly (no) with both the sample and stationary
phase.
 Their only purpose is to transport the sample across the
stationary phase.
 This is why they are usually called carrier gas.
 The choice of mobile phase in GC is based on the type of
detector used, viscosity and purity.
 The best suitable gas for the detector used is selected as mobile
phase.
 Example, when we use thermal detectors, helium is used as
mobile phase because it is more suitable as a mobile phase for
thermal detectors than other gases.
2. Sample Introduction
 Can all samples be analyzed by GC?
 What kinds of samples can be analyzed by GC?
 What should the sample be in order to be transported by
gaseous mobile phase?
 Three considerations determine how samples are
introduced to the gas chromatograph.
a. all constituents injected into the GC must be volatile or
made to be volatile.
 By chemical derivatization or by heating
 For example, amino acids which are not
sufficiently volatile can be made volatile by reacting
it with 1-butanol and acetyl chloride which produces
an esterfied amino acid and subsequent treatment
with trifluoroacetic acid gives the amino acid’s
volatile Ntrifluoroacetyl-n-butyl ester derivative.
  Solutes of low volatility may be retained by the
column and continue to elute during the analysis
of subsequent samples.
 Nonvolatile solutes condense on the column,
degrading the column’s performance.
b. the analytes must be present at an appropriate
concentration.
c. Finally, injecting the sample must not degrade the
separation.

Analytes present at concentrations too small to give an

adequate signal need to be concentrated before analyzing.
When an analyte is too concentrated, it is easy to overload
the column, thereby seriously degrading the separation.
In addition, the analyte may be present at a concentration
level that exceeds the detector’s linear response.
4. Chromatographic columns
 A chromatographic column provides a location for
physically retaining the stationary phase.
 The column’s construction also influences
 the amount of sample that can be handled,
 the efficiency of the separation,
 the number of analytes that can be easily separated,
 the amount of time required for the separation.
 Both packed and capillary columns are used in gas
chromatography.
Stationary phase
 Selectivity in gas chromatography is influenced by the
choice of stationary phase.
 Elution order in GLC is determined primarily by the
solute’s boiling point and, to a lesser degree, by the
solute’s interaction with the stationary phase.
 The following are criteria which are considered while selecting
a stationary phase. The stationary phase
a. must be chemically inert
b. must be thermally inert
c. low volatility (i.e. mostly the boiling point of the
stationary phase must be 100 oC greater than the optimum
temperature)
d. must have an appropriate polarity for the solutes being
separated
 Usually the column is housed in a thermostat, which is
used to adjust the temperature of the column.
 This temperature is used for temperature programming
purpose. ( preheaters – convert sample to vapor form)!
 The optimum temperature for the separation depends
on the boiling point of the stationary phase and
degree of separation.
 Interfacing GC with different techniques

 GC can be coupled to selective techniques of

spectroscopy and electro analytical methods.
 These combination provide a powerful tool for identification
and quantification of complex mixtures.
 In the early time, the eluates from the column were collected
as separate fractions after being detected by non destructive
and non selective methods. Then the composition of each
fraction was identified and investigated by other
techniques.
 This way is not favorable for small amount of each fraction.
 In the hyphenated methods, the eluates from the column
are directly introduced into the other technique.
a. Gas chromatography hyphenated with mass spectrometer
(GC/MS )
 In this technique, gas chromatography is coupled with mass
spectrometer.
 In GC –MS effluent from the column is introduced directly
into the mass spectrometer’s ionization chamber in a
manner that eliminates the majority of the carrier gas.
 In the ionization chamber all molecules (remaining carrier
gas, solvent, and solutes) are ionized, and the ions are
separated by their mass-to-charge ratio.
 Because each solute undergoes a characteristic
fragmentation into smaller ions, its mass spectrum of ion
intensity as a function of mass-to-charge ratio provides
qualitative information that can be used to identify the
solute.