Chromatography

Manzara Arshad
0151-BH-CHEM-11
Government college
university, Lahore

 History
 Introduction
 Principle
 Theory
 Classification
 Mobile-Stationary Phase
 Shape of support
 Mechanism Based
 Purpose

 Russian scientist Tswett in 1906 used
a glass columns packed with finely
divided CaCO3 to separate plant
pigments extracted by hexane. The
pigments after separation appeared as colour
bands that can come out of the column
one by one.
 Tswett was the first to use the term "chromatography"
derived from two Greek words "Chroma" meaning color
and "graphein" meaning to write.

 1931 Lederer & Kuhn - LC of carotenoids
 1938 TLC and ion exchange
 1950 reverse phase LC
 1954 Martin & Synge (Nobel Prize)
 1959 Gel permeation
 1965 instrumental LC (Waters)

What is chromatography?
Tswett (1906) stated that
 mobile phase = solvent
 stationary phase = column packing material

International Union of pure and
applied Chemistry (1993):

Chromatogram - Detector signal
vs. retention time or volume
time or volume
DetectorSignal 1 2

 Typical Response obtained by
chromatography (Chromatogram)
 Chromatogram= Concentration term vs Elution
Time
Where:
tR = retention time
tM = void time
Wb = baseline width of
the peak in time units
Wh = half-height width
of the peak in time units

 The separation of solutes in chromatography depends on two factors:
(a) a difference in the retention of solutes (i.e., a difference
in their time or volume of elution.)
(b) a sufficiently narrow width of the solute peaks (i.e,
good efficiency for the separation system.)

Peak width & peak position
determine separation of peaks
A similar plot can be made in terms of elution volume
instead of elution time. If volumes are used, the volume of
the mobile phase that it takes to elute a peak off of the
column is referred to as the retention volume (VR) and the
amount of mobile phase that it takes to elute a non-
retained component is referred to as the void volume (VM).

Solute Retention:
A solute’s retention time or retention volume in
chromatography is directly related to the strength of
the solute’s interaction with the mobile and stationary
phases.
Retention on a given column pertain to the
particulars of that system:
 - size of the column
 - flow rate of the mobile phase

Capacity factor (k’):
more universal measure of retention, determined from tR
or VR.
k’ = (tR –tM)/tM or
k’ = (VR –VM)/VM
capacity factor is useful for comparing results
obtained on different systems since it is independent
on column length and flow-rate.
When k' is # 1.0, separation is poor
When k' is > 30, separation is slow
When k' is = 2-10, separation is optimum

3.) Efficiency:
Efficiency is related experimentally to
a solute’s peak width.
 an efficient system will produce narrow
peaks
 narrow peaks  smaller difference in
interactions in order to separate two
solutes

Different methods were attempted for
classification of chromatography.
Chromatography is classified according to:
 1-Mobile-Stationary Phase
 2-Mechanism of Separation

According to mobile-stationary phase
chromatography is classified into:
Liquid chromatography
Gas chromatography
1-Classification according
to mobile-stationary phase

NORMAL PHASE CHROMATOGRAPHY:
In normal phase chromatography, the
mobile phase is non-polar and stationary phase
is polar.
REVERSE PHASECHROMATOGRAPHY:
In reverse phase, the mobile phase is polar
and stationary phase is non-polar.

Present day liquid chromatography that
generally utilizes very small packing
particles and a relatively high pressure is
referred to as high performance liquid
chromatography (HPLC).
In HPLC the sample is forced by a liquid at
high pressure (the mobile phase) through a
column that is packed with a stationary
phase composed of irregularly or
spherically shaped particles, a porous
monolithic layer, or a porous membrane
High Performance Liquid
chromatography

High Performance Liquid
chromatography

Supercritical Fluid Chromatography (SFC) is a form
of normal phase chromatography first used in
1962, that is used for the analysis and purification
of low to moderate molecular weight, thermally
labile molecules. It can also be used for the
separation of chiral compounds. Principles are
similar to those of high performance liquid
chromatography(HPLC), however SFC typically
utilizes carbon dioxide as the mobile phase;
therefore the entire chromatographic flow path
must be pressurized. Because the supercritical phase
represents a state in which liquid and gas properties
converge, supercritical fluid chromatography is
sometimes called "convergence chromatography."

Gas chromatography can be used for both
qualitative and quantitative analysis.
Comparison of retention times can be used to
identify materials in the sample by
comparing retention times of peaks in a
sample to retention times for standards. .
Quantitative analysis is accomplished by
measurement of either peak height or peak
area
Gas Chromatography

According to mobile phase
chromatography is classified into:
Gas-Solid Chromatography
Gas-liquid Chromatography
Classification according to
phase (GC)

Gas-Solid Chromatography (GSC)
The stationary phase, in this case, is a solid
like silica or alumina. It is the affinity of
solutes towards adsorption onto the
stationary phase which determines, in part,
the retention time. The mobile phase is, of
course, a suitable carrier gas. This gas
chromatographic technique is most useful for
the separation and analysis of gases like CH4,
CO2, CO, ... etc.
phase (GC)

Gas-liquid Chromatography (GLC)
The stationary phase is a liquid with very
low volatility while the mobile phase is a
suitable carrier gas. GLC is the most
widely used technique for separation of
volatile species. The presence of a wide
variety of stationary phases with
contrasting selectivities and easy column
preparation add to the assets of GLC or
simply GC
phase (GC)

 Carrier gas
 N2, He, H2
 Injector
 Column
 Detector
 Computer
oven

This classification consists of
 Thin layer Chromatography
 Column Chromatography
 Paper Chromatography

Thin-layer chromatography (TLC) is
a chromatography technique used to separate non-
volatile mixtures. Thin-layer chromatography is
performed on a sheet of glass, plastic, or aluminum
foil, which is coated with a thin layer
of adsorbent material, usually silica gel, aluminum
oxide or cellulose. This layer of adsorbent is known
as the stationary phase.
After the sample has been applied on the plate,
a solvent or solvent mixture (known as the mobile
phase) is drawn up the plate via capillary action.
Because different analytes ascend the TLC plate at
different rates, separation is achieved.

Column chromatography in chemistry is a
method used to purify individual chemical
compounds from mixtures of compounds. It
is often used for preparative applications on
scales from micrograms up to kilograms. The
main advantage of column chromatography
is the relatively low cost and disposability of
the stationary phase used in the process. It is
3D chromatography.

Paper chromatography is an analytical method
that is used to separate colored chemicals or
substances, especially pigments. This can also be
used in secondary or primary colors in ink
experiments. This method has been largely
replaced by thin layer chromatography, but is
still a powerful teaching tool.
Double-way paper chromatography, also
called 2D chromatography, involves using two
solvents and rotating the paper 90° in between.
This is useful for separating complex mixtures of
compounds having similar polarity, for
example, amino acids.

The mechanism of separation depends
mainly on the nature of the stationary
phase. Based on separation mechanisms
chromatography can be classified into:

 It is the oldest and most common type of
chromatography.
 The stationary phase is a solid with
adsorption power.
 Mixture components will be adsorbed on
the surface of the stationary phase with
different powers and that account for
separation.
 Silica gel is the most common stationary
phase in adsorption chromatography

 The stationary phase is a liquid forming a
thin film on an inert solid acts as support.
 The stationary liquid is usually more polar
than the mobile liquid. The two liquids must
be immiscible with each other.
 Cellulose powder and wet silica gel are
examples of supports in partition
chromatography that carry film of water
act as stationary phase.

This chromatography is preferable over
adsorption when dealing with polar compounds.
Solid Support Film of the liqiud
stationary Phase

 It is used for separation of charged
molecules.
 The stationary phase is an ion
exchange resin to which a cationic or
anionic groups are covalently bonded.
 Ions of opposite charges (counter
ions) in the mobile phase will be
attracted to the resin and compete
with the components of the mixture
for the charged group on the resin.

 Both the mixture components and the mobile
phase must be changed.
 Mixture of Alkaloids (compounds with positive
charges) can be separated on anionic exchanger,
while mixture of organic acids (negative charges)
can be separated using cationic exchanger.
 Both types are used for desalination of water.

very large molecules eluted first without separation
large molecules can enter some pores
very small molecules enter all pores and eluted at last

It uses the affinity of proteins to specific
ligands such as enzymes. The ligand is
attached to suitable polysaccharide polymer
such as cellulose - agarose – dextran
Affinity
Chromatography:

In this type we can separate enantiomers –
we used chiral stationary phase that
react with one enantiomer more then the
other so separation takes place.
Chiral
Chromatography:

 Analytical - determine chemical
composition of a sample
 Preparative - purify and collect one or
more components of a sample
Purpose of
Chromatography

Chromatography

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