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Russian botanist
Mikhail Tsvet
Chromatography
may be
preparative or
analytical
THEORY
Chromatographic techniques involve the interaction of a mixture between a
stationary and a mobile phase. The stationary phase may be a solid, oil, or gel
while the mobile phase may be a liquid or a gas. The substances to be separated
move with the flow of the mobile phase. Depending upon their relative affinities
for each phase, the components of the mixture may move at different rates.
CHROMATOGRAPHY
OH
OH
OH
OH
OH Si
Si O
Si O
Si O O
Si O O
O O
O O
O Si Si
O O
Si
Si O O O
Si O O
O
O O
O
Si
Si O
O
O O
O
Distribution Coefficient (Equilibrium Distribution )
Definition:
• Paper chromatography
• Thin layer
chromatography
• Column chromatography
• Gas chromatography
• Chromatography is the process use to separate
molecules based on SOME physical property of
the molecule:
1.Adsorption Chromatography
2.Partition Chromatography
3. Ion Exchange Chromatography
4. Molecular Exclusion Chromatography
5. Affinity Chromatography
Types of Chromatography
Adsorption Chromatography
• In this type of
chromatography, the
use of a resin (the
stationary solid phase)
is used to covalently
attach anions or
cations onto it.
Molecular Exclusion Chromatography
• Also known as gel
permeation or gel
filtration.
• No attractive
interaction between the
stationary phase and
solute.
• The liquid or gaseous (Smaller
phase passes through a Mols
porous gel which Trapped
separates the molecules By gel)
according to its size.
Affinity Chromatography
• This is the most
selective type of
chromatography.
• It utilizes the specific
interaction between
one kind of solute
molecule and a second
molecule that is
immobilized on a
stationary phase.
• For example, the
immobilized molecule
may be an antibody to
some specific protein.
Classification of chromatographic processes
Chromatography
Gas C
Liquid C
• Similar to PC.
• Stationary phase of a thin layer of adsorbent like
silica gel, alumina, or cellulose on a flat, inert
substrate.
• Compared to paper, it has the advantage of faster
runs, better separations, and the choice between
different adsorbents.
• For even better resolution and to allow for
quantification, HPTLC can be used.
• TLC is a simple, quick, and inexpensive procedure
Principle of TLC
solvent front
component B
originmixture
solvent front
origin origin
• Simple
• Better separation than PC
• Less time
• Wide choice of adsorbent
Applications of TLC
Solvent(mobile or
moving phase)
OOOOOOOOOOO
A+ B+ C Sample OOOOOOOOOOO
(A+B+C)
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOA OOOO
OOOOOOOOOO Column OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO Solid Particles OOOOOOOOOO
OOOOOOOOOOO (packing material- OOOOOB OOOO
OOOOOOOOOO stationary phase) OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOC OOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
Eluant (eluate)
Reverse Phase
Column Chromatography
• The stationary phase (column packing) is now NON-
POLAR
• Non-polar compounds will move more slowly
because they are attracted to the column packing.
• The more polar component moves more quickly
down the column.
• Polar solvents, such as water and methanol are
used in reverse phase chromatography
• Used mainly in columns, such as HPLC
Reverse phase chromatography
Silica is alkylated with long chain hydrocarbon
groups, using 18
carbons long. This is usually referred to as C-18
silica. CH 3
CH2
CH3 17 CH3
Si
CH2 SiCH3)3 CH3
17 CH3
SiCH3)3
SiCH3)3
O
Si O
CH3 O
O
O Si
Si O
Si O
Si O O
Si O O
O O
O O
O Si
Si O
Si O
Si O O
Si O O
O O
O O
O
Si
Si O
O
O O
O
Types of CC
•These adsorbents are sold in different mesh sizes, “silica gel 60” or
“silica gel 230-400” .
• This number refers to the mesh of the sieve used to size the silica,
specifically, the number of holes in the mesh or sieve through which the
crude silica particle mixture is passed in the manufacturing process.
•If there are more holes per unit area, those holes are smaller, thus
allowing only smaller silica particles go through the sieve.
Polar Solvents
Non-polar Solvents
• Qualitative analysis
• Separation of tautomers
• geometrical isomers
• Diastereomers
• racemates
• In 1978, W. C. Still introduced a modified version
of column chromatography called flash column
chromatography
• the solvent is driven through the column by
applying positive pressure.
• This allowed most separations to be performed in
less than 20 minutes, with improved separations
compared to the old method.
• Modern flash chromatography systems are sold
as pre-packed plastic cartridges, and the solvent
is pumped through the cartridge.
• Systems may also be linked with detectors and
fraction collectors providing automation.
• The introduction of gradient pumps resulted in
quicker separations and less solvent usage
HPLC
A
.
Photodiode 2
Conductivity Detector
Applications of HPLC
• Qualitative and quantitative analysis
• Pollutants, drugs, insecticides, organic
compounds,polymers, amino acids-
• Quality control of the industrial products
Gas Chromatography
• The mobile phase is a gas
• Types-GSC and GLC
• Principle-when vapors of sample in gas stream are
allowed to pass through a column containing
stationary liquid or solid phase, components get
separated due to difference in B.P’s, solubility or
adsorption.
GSC and GLC
• GSC- the principle involved is adsorption
chromatography-the rate of movement of solute is
determined by the relative polarities of the solute
and solid SP.
• GLC-The principle involved is partition
chromatography-the relative solubility of solute in
both the phases determines the rate of movement of
the solute.when solute is gas then movement is
determined by volatility.
Some terminologies
• The analyte is the substance that is to be separated
during chromatography
• The retention time is the characteristic time it
takes for a particular analyte to pass through the
system (from the column inlet to the detector)
under set conditions
• A chromatogram is the visual output of the
chromatograph. In the case of an optimal
separation, different peaks or patterns on the
chromatogram correspond to different
components of the separated mixture.
Terminologies
• D = (Solute)sp
(Solute)mp
(D-Distribution ratio)
tR = length of the column
linear flow rate of solute migration
Time
C
B
A
Detector response
Chromatogram
Rf = Distance the solute moves
Distance the solvent front moves
Effect of temperature
• For speed, higher temp may be desired but at
the expense of resolution
• At lower temp resolution may be better, but
time required may be very long.
Instrumentation
Regulator, injection port, glass or fused quartz
column, thermostat detector and recorder.
• Feed injection-The feed is injected into the
mobile phase. Through the pump the mobile
phase flows through the system
• Separation in the column- components having
weaker attraction for the stationary phase move
faster
• Elution from the column- components elute out
at different time
• Detection-measures the concentration and
characteristic chemical composition
Instrumentation
• Chemically inert carrier gas employed is He,
N2, Ar or H2.
• Temperature of the column is equal or slightly
above the B.P of the least volatile component
of the sample.
Recorder
Injector Amplifier
P gauge
Detector
P reg
F Thermostat
M chamber
Carrier gas Capillary
supply column
Instrumentation
Theory
• Thermally stable volatile samples are introduced into
the gas streams via an injection port.
• Different constituents migrate at different rates due
to their different distribution ratios between MP and
SP.
• Components get eluted in the column and then pass
through Detector and reach the recorder
Detectors
• Thermally stable and chemically inert SP
Gas flow
filament
Gas from
column
• The TCD is based on the principle of thermal
conductivity which depends upon the
composition of the gas.
• The sample components in the carrier gas pass
into the measuring channel.
• A second channel serves as a reference channel
where only pure carrier gas flows. Electrically
heated resistance wires are located in both
channels.
• The difference in thermal conductivity between
the column effluent flow (sample components in
carrier gas) and the reference flow of carrier gas
alone, produces a voltage signal proportional to
this difference.
• The signal is proportional to the concentration of
the sample components
TCD
• The Thermal Conductivity Detector (TCD) is truly a
universal detector because it responds to all
compounds and can detect air, hydrogen, CO, Ar, O2,
N2, CO2, sulfur oxide, inorganic gases and many other
compounds.
• The TCD is a non-specific and non-destructive
detector. For most organic molecules, the sensitivity
of the TCD is lower compared to the Flame Ionization
Detector (FID.)
• Chemically active compounds like acids and
halogenated compounds should be avoided when using
TCD since they can attack the filament (wires) and thereby
change the resistance and permanently reduce the
detector sensitivity.
• Oxidizing substances, such as oxygen, can also damage
the filament, and a leak free environment should be
maintained.
• For an optimal and proper response of the TCD, there
are a couple of critical factors:
• temperature of the detector block
• flow rate of the carrier gas and the reference gas
• resistance of the filaments
All these factors must be optimal to obtain a
representative TCD response.
• ECD-The gases from column exit pass
between two electrodes. One of the
electrodes on its surface has a radioisotope
that emits electrons c/a thermal electrons
the compound absorb these electrons and
produce negative ions. Decrease in thermal
electrons shows dec in detector current.
ECD
Radioactive foil
e- e-
Negative
Voltage
Ceramic insulator
pulse
(Pulsed mode or
DC mode) Electron collector