Classification of chromatographic techniques

Chromatography:
Chromatography is a set of techniques in which separation of chemical
substances takes place quantitatively as well as quantitatively.
Terminologies in chromatography:
Mobile phase:
In chromatography the substance which is introduced with or along with the
sample and causes the elution of the content of the sample.
Stationary phase:
It refers to that part which is present before the introduction of the sample or
solute in the column(as in column chromatography) or on a solid support.
Eluent:
The substance which separates the component of the mixture in
chromatographic techniques. Eluent is that part which brings separation when
the solution is passed either from the column or from solid support.
Eluate:
The substance which is separated as an individual component of the mixture is
eluate.
Classification of chromatography:
 On the basis of the interaction of solute to the stationary phase
 On the basis of chromatographic bed shape:
 On the basis of physical state of mobile phase
On the basis of interaction of solute to the stationary phase:
Adsorption chromatography:
Adsorption chromatography is one of the oldest type of chromatography. 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.
Principle :
Principle of adsorption chromatography involves competition of components of
sample mixture for active site on absorbent.
These active sites are formed on molecule due to cracks or edges.
Separation occur because of the fact that equilibrium is established between
molecules adsorbed on stationary phase and those are flowing freely in mobile
phase.
The more the affinity of the molecule of particular component, less will be its
movement.
Types:
Column chromatography
Thin layer chromatography
Gas solid chromatography
Partition chromatography:
This form of chromatography is based on a thin film formed on the surface of
solid support by a liquid stationary phase. Solute equilibrates between mobile
phase and the stationary liquid.

Principle:
Separation of components of sample mixture occurs because of partition.
Stationary phase is coated with a liquid which is immiscible in mobile phase.
Partition of components of sample between sample and liquid\gas stationary
phase retard some components of sample more as compared to others.
This gives basis for separation.
Types:
Liquid-liquid chromatography
Gas-liquid chromatography
Ion exchange chromatography:
It is a process that allows the separation of ions and polar molecules based on
affinity to the ion exchanger. It can be used for almost any kind of charged
molecules including large proteins , small nucleotides 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.

Principle:
The ion exchange chromatography is based on 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 the column.
Types:
Cation exchange chromatography
Anion exchange chromatography
Size exclusion chromatography:
It 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 molecular complexes such as proteins and industrial polymers.
Typically an aqueous solution is used to transport the sample through column,
technique is known as gel filtration chromatography, verses the name gel-
permeation chromatography, which is used when an organic solvent is used as
mobile phase.

Principle:
A mixture of molecule dissolved in liquid( the mobile phase) is applied to a
chromatographic column which contains a solid support in the form of
microscopic spheres or beads
The mass of beads is referred to as column bed.
The beads act as traps or sieves and function to filter small molecules which
become temporarily n trapped within pores.
Larger molecules are excluded from beads.
Large molecule samples cannot or can only partially penetrate the pores ,
whereas smaller molecules can access most or all pores.
Thus larger molecules elute first, smaller molecules elute later, while molecules
that can access all the pores elute last from the column.
On the basis of chromatographic bed shape:
Two dimensional:
Thin layer chromatography:
It is useful for separating organic compounds. Because of simplicity and
rapidity of TLC, its often used to monitor the progress of organic reactions and
to check purity of products.
Principle:
It 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 travel over the surface of
stationary phase. During this moment, the compounds with higher affinity to
stationary phase travel slowly while others travel faster. Thus separation of
components in the mixture is achieved.
Once separation occurs individual components are visualized as spots at
respective level on the plate.
Paper chromatography:
paper chromatography method 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
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 90⁰ in between. This is useful for
separating complex mixtures of compounds having similar polarity, for
example, amino acids. The mobile phase is developing solutions that can travel
up to the stationary phase carrying the sample along with it.

On the basis of physical state of mobile phase

  Liquid chromatography
 Gas chromatography
 Super critical fluid chromatography
Liquid chromatography
It 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 phase. Because
There are many mobile\stationary combinations that can be employed when
separating a mixture, there are several types of chromatography that are based
on physical state 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.

Gas chromatography
 Gas chromatography 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 includes testing the purity of a particular substance,
or separating the different components of a mixture.
 In some situations, GC may help in identifying a compound. In
preparative chromatography, GC can be used to prepare pure compounds
from a mixture.
Supercritical fluid chromatography
 It is a form of normal phase chromatography, that is used for the analysis
and purification of low to moderate molecular weight, thermally labile
molecule.
 It can also be used for the separation of chiral compounds.
 Principles are same to HPLC, however, SFC typically uses carbon
dioxide as the mobile phase; therefore the entire chromatographic flow
path must be pressurized.
 The super critical phase represents a state in which liquid and gas
properties converge, supercritical fluid chromatography is sometimes
called “convergent chromatography”.

High performance liquid chromatography

 It is formally called high-pressure liquid chromatography. It is 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 absorbent
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.