CHROMATOGRAPHY

What is Chromatography?
Chromatography is the science which is studies the separation of molecules based on differences in
their structure and/or composition. In general, chromatography involves moving a preparation of the
materials to be separated - the "test preparation" - over a stationary support. The molecules in the test
preparation will have different interactions with the stationary support leading to separation of similar
molecules. Test molecules which display tighter interactions with the support will tend to move more
slowly through the support than those molecules with weaker interactions. In this way, different types
of molecules can be separated from each other as they move over the support material.
Chromatographic separations can be carried out using a variety of supports, including immobilized
silica on glass plates (thin layer chromatography), volatile gases (gas chromatography), paper (paper
chromatography), and liquids which may incorporate hydrophilic, insoluble molecules (liquid
chromatography).
Some examples of chromatographic techniques are described below:
Paper Chromatography
This technique involves the presence of a solvent that moves along blotting or filter paper. In this
technique, the cellulose of the filter paper acts as an inert support. When a few drops of the mixture is
applied to this filter paper and dipped into the container with water, the water begins to move upwards
by capillary action. The water then spreads out the final color of the mixture into their constituent
colors. Thus, interactions between the sample or mixture, water or solvent and the filter paper brings
about separation of the components. Paper chromatography is used to separate most colored
compounds and is widely used in artificial and natural pigment research.
Thin Layer Chromatography (TLC)
Principle:
Thin-layer chromatography or TLC, is a solid-liquid form of chromatography where the
stationary phase is normally a polar absorbent and the mobile phase can be a single solvent or
combination of solvents. TLC is a quick, inexpensive microscale technique that can be used to:

determine the number of components in a mixture

verify a substances identity
monitor the progress of a reaction
determine appropriate conditions for column chromatography
analyze the fractions obtained from column chromatogrpahy

In partition chromatography the stationary phase is a non-volatile liquid which is held as a thin
layer (or film) on the surface of an inert solid. The mixture to be separated is carried by a gas or a
liquid as the mobile phase. The solutes distribute themselves between the moving and the stationary
phases, with the more soluble component in the mobile phase reaching the end of the chromatography
column first . Thin layer chromatography is an example of partition chromatography.
Materials - This process involves:

a suitable adsorbent (the stationary phase): adsorbent silica-TLC plate

solvents or solvent mixtures (the mobile phase or eluent)
the sample plant extract
Developing chamber
Spray reagents

Significance of Each Materials:

Stationary phase, a special finely ground matrix (silica gel, alumina, or similar material) is coated on a
glass plate, a metal or a plastic film as a thin layer (~0.25 mm). A fluorescent powder is mixed into the
stationary phase to simplify the visualization later on (e.g. bright green when you expose it to 254 nm
UV light).
Solvent mixtures should show a maximum of selectivity in its ability to dissolve or desorb the
substances being separated. A more important property of the solvent is its ability to be itself adsorbed
on the adsorbent.
Developing chamber is to make sure that the atmosphere is saturated with solvent vapour. So the
chamber is often lined with some filter paper soaked in solvent. Saturating the atmosphere with vapour
stops the solvent from evaporating as it rises up the plate.
Sample Plant Extract that one being analyzed in this experiment.
Spray Reagents is used during the visualization stage where the TLC plate is sprayed with reagents in
order to be able to see it under a flourescent light.
Advantages:
1) Simple and unsophisticated procedure of conducting thin layer chromatography makes iteasy to
perform.
2) Its faster runs make it time saving.
3) It provides better separations than paper chromatography.
4) It allows a choice between different stationary phases
Disadvantages:
1) It is dependent on other analytical methods to determine the quantities of components in
amixture.
2) TLCs from low-temperature reactions may give misleading results, when used to qualitatively
monitor reactions.
Column Chromatography
Principle:
Column chromatography is a technique in which a column of stationary phase (chromatographic
column) is used.based upon the stationary phase nature,this column chromatography is further divided
into 2 types.They are if a column of solid stationary phase is used it is called as column adsorption
chromatography. If a column of liquid stationary phase is used then it is called as column partition
chromatography. Column adsorption chromatography is widely used.

Adsorption is the principle of separation and here a solid stationary phase and a liquid mobile
phase is used. It was developed first. It has a solid stationary phase and a liquid or gaseous mobile
phase. (Plant pigments were separated at the turn of the 20th century by using a calcium carbonate
stationary phase and a liquid hydrocarbon mobile phase. The different solutes travelled different
distances through the solid, carried along by the solvent.) Each solute has its own equilibrium between
adsorption onto the surface of the solid and solubility in the solvent, the least soluble or best adsorbed
ones travel more slowly. The result is a separation into bands containing different solutes. Liquid
chromatography using a column containing silica gel or alumina is an example of adsorption
chromatography (Fig. 1). The solvent that is put into a column is called the eluent, and the liquid that
flows out of the end of the column is called the eluate. The solutes extracted from the liquid collected
(the eluate) by evaporating off the solvent can then be identified by running a simple TLC experiment.
Column chromatography is separated into two categories, depending on how the solvent flows
down the column. If the solvent is allowed to flow down the column by gravity, or percolation, it is
called gravity column chromatography. If the solvent is forced down the column by positive air
pressure, it is called flash chromatography, a "state of the art" method.
Materials:

Chromatographic column
Glass wool/ washed cotton
Organic Solvent
Silica (adsorbent)
Plant Extract

Significance of Each Material:

Chromatographic columun must be neutral such that it should not be affected by acids ,alkalies or
solvents.the length: diameter must range from 10:1-30:1. It must be 100:1 for more efficiency.
Glass wool/ washed cotton is used as a stopper at the bottom of the set-up in order for the adsorbent
not to pass through the column. Washed cotton is preferable since it does not cause harmful effet when
inhaled.
Organic solvent is used to introduce the mixture into the column as a solvent, to develop the zones of
separation as developing agent, and to remove pure component out of the column as an eluent. The
polarity of the solvent which is passed through the column affects the relative rates at which
compounds move through the column.
Silica (adsorbent) acts as the adsorbent in the set-up. These adsorbents are sold in different mesh sizes.
Adsorbent particle size affects how the solvent flows through the column. Smaller particles (higher
mesh values) are used for flash chromatography, larger particles (lower mesh values) are used for
gravity chromatography. For example, 70-230 silica gel is used for gravity columns and 230-400 mesh
for flash columns.
Disadvantage:

Plant extract the sample being tested.

Advantage:
1) Any type of mixture can be separated
2) any quantity of the mixture can be separated

1) time consuming method

2) more amount of solvents are required which are
expensive
3) automation technique makes complication

3) wider choice of mobile phase

4) automation is possible
Gas Chromatography

Principle:
This technique uses a gas as the mobile phase, and the stationary phase can either be a solid or a
non-volatile liquid (in which case small inert particles such as diatomaceous earth are coated with the
liquid so that a large surface area exists for the solute to equilibrate with). If a solid stationary phase is
used the technique is described as gas-solid adsorption chromatography, and if the stationary phase is
liquid it is called gas-liquid partition chromatography. The latter is more commonly used, but in both
cases the stationary phase is held in a narrow column in an oven and the stationary phase particles are
coated onto the inside of the column.
Materials - A gas chromatographic system consists of:

A regulated and purified carrier gas source, which moves the sample through the GC
An inlet, which also acts as a vaporizer for liquid samples
A column, in which the time separation occurs
A detector, which responds to the components as they occur by changing its electrical output
Data interpretation of some sort

Significance of Each Material:

Gas source. The carrier gas must be pure. Contaminants may react with the sample or the column,
create spurious peaks, load the detector and raise baselines, and so on. A high-purity gas with traps for
water, hydrocarbons and oxygen is recommended.
The inlet introduces the vaporized sample into the carrier gas stream. The most common inlets are
injection ports and sampling valves.
The column is where the separation happens. Because the column type is selected by the user, many
different analyses can be performed using the same equipment.
The gas stream from the column, which contains the separated components, passes through a detector.
The output from the detector becomes the chromatogram.
Data interpretation: the chromatogram leaves the detector as an electrical signal. It can be:
Recorded on a strip chart recorder
Processed by a digital integrator
Processed by a computer-based data system