Chromatography is a method used to separate mixtures by distributing components between a stationary and mobile phase. There are several types including thin layer chromatography (TLC), which separates compounds on plates coated with adsorbents, and column chromatography, where the stationary phase is packed in a tube. High performance liquid chromatography (HPLC) uses pumps to force liquid mobile phases through columns at high pressures for improved separation.
2. Chromatography is a method of
physically separating mixtures of
gases, liquids, or dissolved
substances.
Chromatography can be used to identify
drugs, poisons and many other substances.
Separation is determined by the molecular
size and/or charge.
Chromatography
3. General Principles of Chromatography
• Separation of molecules by distribution between a
stationary phase and a mobile phase.
– A stationary phase (absorbent) phase the material
on which the separation takes place. can be solid, gel, or
liquid. Also called matrix, resin, or beads.
– The mobile phase is the solvent transports the
sample and it is usually a liquid, but may also be a gas.
Also called eluting buffer
• The compounds to be separated are
considered solutes
4. The mechanism that causes the stationary
phase to retard the movement of
molecules:
1. Sieve mechanism separation according to
size or MW.
(molecular sieve = gel filtration = size exclusion = gel
permeation).
2. Charge interaction separation based on
net charge.
Classification by the Separation mode
5. 3. Solubility characteristics separation
based on polarity
Hydrophobic chromatography, reverse-phase
chromatography, Adsorption or normal-phase
chromatography
4. Biological or Specific interaction capture
any molecule that exhibit such property
affinity chromatography, dye-chromatography
Antibody-antigen: (Immuno precipitations and
other forms)
Classification by the Separation mode
6. Classification of Chromatography
Gas Chromatography
Gas - solid Gas - liquid
Liquid
chromatography
High
performance
(pressure flow)
Thin layer
(adsorption)
Column
(gravity
flow)
By mobile phase:
1. Liquid chromatography.
2. Gas chromatography.
7. Type of chromatography Material
Paper chromatography Filter paper, cellulose
Thin Layer Chromatography Silica gel, alumina,
polyamide
Gas chromatography Squalene, apezion,
carbowax M
High Performance Liquid
Chromatography
C-8, C-18, Licosorb,
Silicone
8. Type of chromatography Solvent
Paper chromatography Air, alcohol
Thin Layer
Chromatography
Hexane, ether petroleum,
alcohol.
Gas chromatography He, Ar, N2
High Performance Liquid
Chromatography
Cyclohexane, n-hexane,
carbon tetrachloride, ethanol,
methanol, air
10. (A) Thin Layer Chromatography:
Is a method for identifying substances
and testing the purity of compounds.
TLC is a useful technique because it is
relatively quick and requires small
quantities of material
11. Separations in TLC involve distributing a mixture of two
or more substances between a stationary phase and a
mobile phase.
The stationary phase:
is a thin layer of adsorbent (usually silica gel or
alumina) coated on a plate.
The mobile phase:
is a developing liquid which travels up the stationary
phase, carrying the samples with it.
Components of the samples will separate on the
stationary phase according to
how much they adsorb on the stationary phase versus
how much they dissolve in the mobile phase.
A. Thin Layer Chromatography:
13. If no spots are obvious, the
most common visualization
technique is to hold the
plate under a UV lamp.
Many organic compounds can
be seen using this
technique, and many
commercially made plates
often contain a substance
which aids in the
visualization of compounds.
Identifying the Spots (visualization)
15. The Rf (retention factor) value for each
spot should be calculated.
It is characteristic for any given
compound on the same stationary phase
using the same mobile phase for
development of the plates.
Hence, known Rf values can be compared
to those of unknown substances to aid in
their identifications.
Interpreting the Data
16. Rf values often depend on the temperature and the
solvent used in the TLC experiment.
The most effective way to identify a compound is
to spot known substances next to unknown
substances on the same plate.
In addition, the purity of a sample may be
estimated from the chromatogram.
An impure sample will often develop as two or more
spots, while a pure sample will show only one spot
Interpreting the Data
18. A method of partition chromatography using filter
paper strips as carrier or inert support.
The factor governing separation of mixtures of
solutes on filter paper is the partition between
two immiscible phases.
One is usually water adsorbed on cellulose fibers in
the paper.
The second is the organic solvent flows past the
sample on the paper (stationary phase).
B. Paper Chromatography
19. Definition:
A chromatographic analytical separation
technique for complex mixtures involving
the progressive adsorption of the
dissolved component onto a special grade
of paper.
B. Paper Chromatography
20. Principle:
• The certain solvent are used to separate a mixture
ex: water, alcohol.
• With capillary action the solvent will move up to filter
paper.
• Movement of a solvent will bring together component
that are separated from the mixture.
• Every component that are separated will move to
several velocity
B. Paper Chromatography
21. The moving components are depend on :
a. Solubility solute in solvent
b. Intermolecule forces
c. Pore size of filter paper
d. Size of solute
At the end of process, components that are separated
will emerge to different distance on filter paper.
Rf values are used to identification of each the
component.
B. Paper Chromatography
22. 1. Separation of ink dyes
- To compare ink dyes use in any company.
2. Food coloring
- To differentiate coloring agent used in
food product.
3. Botanist/herbalist
- To isolate plant pigment from root and
leaves.
Use of Paper Chromatography
24. This includes chromatographic methods in
which:
The stationary phase is packed into a column.
The mobile phase is a moving liquid.
According to the mechanism of separation of
solutes, five major types of CC are
distinguished. Usually, one mechanism
predominates but does not exclude the
others
C. Column Chromatography
25. Column Chromatography
Stationary phase
is held in a
narrow tube
through which
the mobile
phase is forced
under pressure
or under the
effect of
gravity
C. Column Chromatography
26. The analytes
interacting most
strongly with the
stationary phase
will take longer to
pass through the
system than those
with weaker
interactions.
These interactions
are usually
chemical in nature,
but in some cases
physical
interactions can
also be used.
27. Traditional column chromatography is
characterized by addition of mobile
phase under atmospheric pressure and
the stationary phase is packed in a
glass column.
Open Column Chromatography
(Traditional column chromatography)
28. Detection:
On-column detection for colored or
fluorescent compounds directly after
developing the chromatogram.
Monitoring of eluted fractions (PC or TLC).
Using special detectors connected to the
column such as refractive index, UV
detectors, etc…
C. Column Chromatography
30. • HPLC is a form of liquid chromatography used to
separate compounds that are dissolved in
solution. HPLC instruments consist of a reservoir
of mobile phase, a pump, an injector, a separation
column, and a detector.
• Compounds are separated by injecting a sample
mixture onto the column. The different
component in the mixture pass through the
column at differentiates due to differences in
their partition behavior between the mobile
phase and the stationary phase. The mobile phase
must be degassed to eliminate the formation of
air bubbles.
High-performance liquid
chromatography HPLC
32. This technique is used for chemistry and
biochemistry research analyzing complex
mixtures, purifying chemical compounds,
developing processes for synthesizing
chemical compounds, isolating natural
products, or predicting physical properties.
It is also used in quality control to ensure
the purity of raw materials, to control and
improve process yields, to quantify assays
of final products, or to evaluate product
stability and monitor degradation.
Uses Of HPLC
33. The function of the injector is to place the
sample into the high-pressure flow in as
narrow volume as possible so that the
sample enters the column as a
homogeneous, low-volume plug. To minimize
spreading of the injected volume during
transport to the column, the shortest
possible length of tubing should be used
from the injector to the column.
HPLC Chromatograph injectors
34. Normally, columns are filled with silica gel
because its particle shape, surface properties,
and pore structure help to get a good
separation. Silica is wetted by nearly every
potential mobile phase, is inert to most
compounds and has a high surface activity
which can be modified easily with water and
other agents. Silica can be used to separate a
wide variety of chemical compounds, and its
chromatographic behavior is generally
predictable and reproducible.
HPLC columns
35. The column is one of the most important
components of the HPLC chromatograph
because the separation of the sample
components is achieved when those
components pass through the column. The
High performance liquid chromatography
apparatus is made out of stainless steel
tubes with a diameter of 3 to 5mm and a
length ranging from 10 to 30cm.
HPLC columns
36. Column Parameters
• Column Material
• Deactivation
• Stationary Phase
• Coating Material
Instrument Parameters
• Temperature
• Flow
• Signal
• Sample Sensitivity
• Detector
WHAT AFFECTS SYSTEM
45. 1.Very good separation.
2.Time (analysis is short).
3.Small sample is needed – ml.
4.Good detection system.
5.Quantitatively analyzed.
GLC ADVANTAGES
DISADVANTAGES OF GAS
CHROMATOGRAPHY
• Material has to be volatilized at 250C
without decomposition.
46. • By stationary phase or shape
1.Adsorption.
2.Partition.
3.Ion-Exchange.
4.Molecular Exclusion.
5.Affinity.
Classification of
Chromatography
47. Adsorption chromatography
is probably one of the oldest
types of chromatography
around. It utilizes a mobile
liquid or gaseous phase that
is adsorbed onto the surface
of a stationary solid phase.
The equilibration between
the mobile and stationary
phase accounts for the
separation of different
solutes.
1.Adsorption Chromatography:
48. 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.
2.Partition Chromatography:
49. In this type of
chromatography, the use
of a resin (the
stationary solid phase)
is used to covalently
attach anions or cations
onto it. Solute ions of
the opposite charge in
the mobile liquid phase
are attracted to the
resin by electrostatic
forces.
3.Ion Exchange Chromatography:
50. Also known as gel permeation or gel
filtration, this type of chromatography
lacks an attractive interaction between
the stationary phase and solute. The
liquid or 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, causing them
to flow through a larger volume. This
causes the larger molecules to pass
through the column at a faster rate than
the smaller ones.
4.Molecular Exclusion Chromatography:
52. This is the most selective type of chromatography
employed. 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. When solute
containing a mixture of proteins are passed by 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.
5.Affinity Chromatography:
