2. Chromatography Chromatography is a method of separation in
which the components to be separated are distributed between two
phases, one of these is called a stationary phase and the other is
a mobile phase which moves on stationary phase in a definite
direction. The component of the mixture redistribute themselves
between two phases by a process which may be adsorption, partition,
ion exchange or size exclusion. The stationary phase can be solid
or a liquid and the mobile phase can be liquid, gas or a
supercritical fluid.
3. Illustration of Chromatography Separation Mobile Phase
Mixture Components
4. Examples of 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 aluminium foil, which is coated with the thin
layer of adsorbent material, usually silica gel, aluminium oxide,
or cellulose. Paper Chromatography is an analytical method that is
used to separate coloured chemicals or substances, especially
pigments. This can also be used in ink experiments. Column
Chromatography in Chemistry is a method use to purify individual
chemical compounds from a mixtures of compounds. It is often used
for preparative applications on scale from micrograms up to
kilograms.
5. Examples of Chromatography Ion Exchange Chromatography (Ion
Chromatography) is a process that allows the separation of ions and
polar molecules based on their affinity to the ion exchanger. It
can be used for almost any kind of charged molecules including
large protein, small nucleotide and amino acids. The solution to be
injected is called Sample and individually separated components are
called analytes. Size-Exclusion Chromatography (SEC) is a
chromatographic method in which molecules in a solution are
separated by their size, and in some cases molecular weight. It is
usually applied to large molecules or macromolecular complexes such
as proteins and industrial polymers.
6. Introduction The Term Chromatography (chroma = a colour;
graphein = to write) is the collective term for a set of laboratory
techniques for the separation of mixtures. Chromatography involves
a sample (or sample extract) being dissolved in a mobile phase
(which may be a gas, a liquid or a supercritical fluid). The mobile
phase is then forced through an immobile, immiscible stationary
phase. The phases are chosen such that components of the sample
have differing solubilities in each phase. A component which is
quite soluble in the stationary phase will take longer to travel
through it than a component which is not very soluble in the
stationary phase but very soluble in the mobile phase.
7. As a result of these differences in mobilities, sample
components will become separated from each other as they travel
through the stationary phase. Techniques such as H.P.L.C. (High
Performance Liquid Chromatography) and G.C. (Gas Chromatography)
use columns - narrow tubes packed with stationary phase, through
which the mobile phase is forced. The sample is transported through
the column by continuous addition of mobile phase. This process is
called elution.
8. History The subject of Chromatography was introduced into
scientific world in a very modest way by M. Tswett in 1906. He
employed a technique to separate various pigments such as
chlorophylls and xanthophylls by passing the solution of these
compounds into the glass column which was packed with finely
divided calcium carbonate. After the later, Thompson and Way had
realized the Ion Exchange properties of soils. Almost after three
decades, in 1935 Adams and Holmes observed the Ion Exchange
characteristics in crushed phonograph. This observation opened the
field for preparation of Ion Exchanged resins. The concept of
Gas-Liquid Chromatography was first introduced by Martin and Synge
in 1941.
9. They were also responsible for the development in Liquid-
Liquid chromatography. In 1944, from Martin laboratory, the
separation of amino acid by paper chromatography was reported. In
1952, the importance of the chromatography was observed when both
Synge and Martin were awarded with Nobel Prize. In 1959, a
technique known as Gel Filtration chromatography was observed which
is used to separate low molecular weight substances from high
molecular substances. In 1960, further improvement in liquid
chromatography led to the development of High Performance Liquid
Chromatography. The following decade of 1970s saw an improvement in
the field of adsorption chromatography in the form of Affinity
chromatography which was mainly based on biological
interactions.
10. A new field was originated which was supercritical fluid
chromatography. Supercritical fluid chromatography is a hybrid of
gas and liquid chromatography and combine advantageous feature of
the both gas and liquid chromatography. It will not be wrong to say
that the entire twentieth century can be named as the century of
chromatography.
11. Classification Of Chromatography On the basis of
interaction of solute to the stationary phase On the basis of
chromatographic bed shape On the basis of physical state of mobile
phase Adsorption Chromatography Partition Chromatography Ion
Exchange Chromatography Size Exclusion Chromatography Two
Dimensional Three Dimensional Thin Layer Chromatography Paper
Chromatography Column Chromatography Liquid Chromatography Gas
Chromatography Super Critical Fluid Chromatography
12. On the basis of interaction of solute to the stationary
phase Adsorption Chromatography Partition Chromatography Ion
Exchange Chromatography Size Exclusion Chromatography
13. Adsorption Chromatography Definition: 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.
14. Principle: Principle of Adsorption Chromatography involves
competition of components of sample mixture for active site on
adsorbent. These active sites are formed in molecule due to Cracks
Edges Separation occurs because of the fact that an equilibrium is
established between molecules adsorbed on stationary phase and
those which are flowing freely in mobile phase. The more the
affinity of the molecule of particular component, less will be its
movement.
16. Partition Chromatography Definition: 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.
17. Principle: Separation of components of a sample mixture
occurs because of partition. Stationary phase is coated with a
liquid which is immiscible in mobile phase. Partition of component
of sample between sample and liquid/ gas stationary phase retard
some components of sample more as compared to others. This gives
basis for separation. The stationary phase immobilizes the liquid
surface layer, which becomes stationary phase. Mobile phase passes
over the coated adsorbent and depending upon relative solubility in
the coated liquid, separation occurs. The component of sample
mixture appear separated because of differences in their partition
coefficient.
19. Ion Exchange Chromatography Definition: Ion Exchange
Chromatography (Ion Chromatography) is a process that allows the
separation of ions and polar molecules based on their affinity to
the ion exchanger. It can be used for almost any kind of charged
molecules including large protein, small nucleotide and amino
acids. The solution to be injected is called Sample and
individually separated components are called analytes. It is often
used in protein purification, water analysis, and quality
control.
20. Principle: Ion Exchange Chromatography is based on the
relative retention of the ions during their progress through an ion
exchange column which has functional group of opposite charge
attached to its surface. The stronger the charge on the ion, the
greater is the retention time in the column. Ion chromatography is
used to separate organic or inorganic charged substances. The
stationary phases used are based on typical ion exchange
resins.
21. Ion Exchange Chromatography Cation Exchange Chromatography
Anion Exchange Chromatography Solute cations are attached to the
negatively charged sites covalently bond to the stationary phase
Solute anions are attached to the positively charged sites
covalently bond to the stationary phase Types:
22. Size Exclusion Chromatography Definition: Size-Exclusion
Chromatography (SEC) is a chromatographic method in which molecules
in a solution are separated by their size, and in some cases
molecular weight. It is usually applied to large molecules or
macromolecular complexes such as proteins and industrial polymers.
Typically, when an aqueous solution is used to transport the sample
through the column, the technique is known as gel- filtration
chromatography, versus the name gel- permeation chromatography, ,
which is used when an organic solvent is used as a mobile
phase.
23. Size Exclusion Chromatography
24. Principle: A mixture of molecules dissolved in liquid (the
mobile phase) is applied to a chromatography column which contains
a solid support in the form of microscopic spheres, or beads (the
stationary phase). The mass of beads within the column is often
referred to as the column bed. The beads act as traps or sieves and
function to filter small molecules which become temporarily trapped
within the pores. Larger molecules are excluded from the beads .
Large sample molecules cannot or can only partially penetrate the
pores, whereas smaller molecules can access most or all pores.
Thus, large molecules elute first, smaller molecules elute later,
while molecules that can access all the pores elute last from the
column. Particles of different sizes will elute(filter) through a
stationary phase at different rates.
25. On the basis of chromatographic bed shape Two dimensional
i. Thin Layer Chromatography ii. Paper Chromatography Three
dimensional i. Column Chromatography
26. Thin Layer Chromatography Definition: Thin-layer
chromatography (TLC) is a chromatographic technique that is useful
for separating organic compounds. Because of the simplicity and
rapidity of TLC, it is often used to monitor the progress of
organic reactions and to check the purity of products.
27. Principle: Similar to other chromatographic methods TLC is
also based on the principle of separation. The separation depends
on the relative affinity of compounds towards stationary and mobile
phase. The compounds under the influence of mobile phase (driven by
capillary action) travel over the surface of stationary phase.
During this movement the compounds with higher affinity to
stationary phase travel slowly while the others travel faster. Thus
separation of components in the mixture is achieved. Once
separation occurs individual components are visualized as spots at
respective level of travel on the plate. Their nature or character
are identified by means of suitable detection techniques.
28. Paper Chromatography Definition: Paper chromatography is an
analytical method that is used to separate coloured chemicals or
substances, especially pigments. This can also be used in secondary
or primary colours 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
two-dimensional 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. If a filter paper is used, it should be of a high
quality paper. The mobile phase is developing solutions that can
travel up to the stationary phase carrying the sample along with
it.
29. Principle: The principle involved is partition
chromatography where in the substances are distributed or
partitioned between to liquid phases. One phase is the water which
is held in pores of filter paper used and other phase is that of
mobile phase which moves over the paper. The compounds in the
mixture get separated due to differences in their affinity towards
water(in stationary phase) and mobile phase solvents during the
movement of mobile phase under the capillary action of pores in the
paper. The principle can also be adsorption chromatography between
solid and liquid phases, where in the stationary phase is the solid
surface of paper and the liquid phase is of mobile phase. But most
of the applications of paper chromatography work on the principle
of partition chromatography i.e. partitioned between two liquid
phases.
30. On the base of physical state of mobile phase Liquid
Chromatography Gas Chromatography Super Critical Fluid
Chromatography
31. Liquid Chromatography Liquid chromatography is a technique
used to separate a sample into its individual parts. This
separation occurs based on the interactions of the sample with the
mobile and stationary phases. Because there are many
stationary/mobile phase combinations that can be employed when
separating a mixture, there are several different types of
chromatography that are classified based on the physical states of
those phases. Liquid- solid column chromatography, the most popular
chromatography technique, features a liquid mobile phase which
slowly filters down through the solid stationary phase, bringing
the separated components with it.
32. Liquid Chromatography Liquid-Liquid Chromatography
Liquid-Solid Chromatography Stationary Phase Liquid Solid Normal
Phase Reverse Phase Normal Phase Reverse Phase
33. Normal Phase Chromatography: In normal phase
chromatography, the stationary phase is polar, and so the more
polar solutes being separated will adhere more to the stationary
adsorbent phase. When the solvent or gradient of solvents is passed
through the column, the less polar components will be eluted faster
than the more polar ones. The components can then be collected
separately, assuming adequate separation was achieved, in order of
increasing polarity.
34. Reverse Phase Chromatography: In reverse phase
chromatography, the polarities of the mobile and stationary phases
are opposite to what they were when performing normal phase
chromatography. Instead of choosing a non-polar mobile phase
solvent, a polar solvent and non-polar stationary phase will be
chosen.
35. Extraction Chromatography: An important modification in
terms of stationary phase is introduced by loading the extractant
used for solvent extraction on a hydrophobized inert support and
irrigating the support with aqueous solvent. This is known as
extraction chromatography.
36. Affinity Chromatography: Affinity chromatography is a
method of separating biochemical mixtures based on a highly
specific interaction such as that between antigen and antibody,
enzyme and substrate, or receptor and ligand.
37. Principle: The stationary phase is typically a gel matrix,
often of agarose; a linear sugar molecule derived from algae.
Usually the starting point is an undefined heterogeneous group of
molecules in solution, such as a cell lysate, growth medium or
blood serum. The molecule of interest will have a well known and
defined property, and can be exploited during the affinity
purification process. The process itself can be thought of as an
entrapment, with the target molecule becoming trapped on a solid or
stationary phase or medium. The other molecules in the mobile phase
will not become trapped as they do not possess this property. The
stationary phase can then be removed from the mixture, washed and
the target molecule released from the entrapment in a process known
as elution. Possibly the most common use of affinity chromatography
is for the purification of recombinant proteins.
38. High Performance Liquid Chromatography High-performance
liquid chromatography (HPLC; formerly referred to as high-pressure
liquid chromatography), is a technique in analytic chemistry used
to separate the components in a mixture, to identify each
component, and to quantify each component. It relies on pumps to
pass a pressurized liquid solvent containing the sample mixture
through a column filled with a solid adsorbent material. Each
component in the sample interacts slightly differently with the
adsorbent material, causing different flow rates for the different
components and leading to the separation of the components as they
flow out the column.
39. Gas Chromatography Gas chromatography (GC), is a common
type of chromatography used in analytical chemistry for separating
and analyzing compounds that can be vaporized without
decomposition. Typical uses of GC include testing the purity of a
particular substance, or separating the different components of a
mixture (the relative amounts of such components can also be
determined). In some situations, GC may help in identifying a
compound. In preparative chromatography, GC can be used to prepare
pure compounds from a mixture. Two types of gas chromatography are
encountered a. Gas-Solid Chromatography (GSC) b. Gas-Liquid
Chromatography (GLC)
40. Supercritical Fluid Chromatography Supercritical Fluid
Chromatography (SFC) is a form of normal phase chromatography, 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. The
supercritical phase represents a state in which liquid and gas
properties converge, supercritical fluid chromatography is
sometimes called "convergence chromatography."