Subdivision Of Chromatography

The Subdivision is based on changing type of chromatography used,

the first type is one of Column chromatography type it’s thin
layer chromatography (TlC),Column
chromatography in chemistry is a chromatography method used to
isolate a single chemical compound from a mixture. Chromatography
is able to separate substances based on differential adsorption of
compounds to the adsorbent; compounds move through the column
at different rates, allowing them to be separated into fractions. The
technique is widely applicable, as many different adsorbents (normal
phase, reversed phase, or otherwise) can be used with a wide range of
solvents. The technique can be used on scales from micrograms up to
kilograms. The main advantage of column chromatography is the
relatively low cost and disposability of the stationary phase used in
the process. The latter prevents cross-contamination and stationary
phase degradation due to recycling. Column chromatography can be
done using gravity to move the solvent, or using compressed gas to
push the solvent through the column.
A thin-layer chromatograph can show how a mixture of compounds will
behave when purified by column chromatography. The separation is first
optimised using thin-layer chromatography before performing column
chromatography.
It can be did by sequence of steps The most important is Column
preparation.
A column is prepared by packing a solid adsorbent into a cylindrical glass
or plastic tube. The size will depend on the amount of compound being
isolated. The base of the tube contains a filter, either a cotton or glass
wool plug, or glass frit to hold the solid phase in place. A solvent
reservoir may be attached at the top of the column.
Two methods are generally used to prepare a column: the dry method and
the wet method. For the dry method, the column is first filled with dry
stationary phase powder, followed by the addition of mobile phase, which
is flushed through the column until it is completely wet, and from this
point is never allowed to run dry. For the wet method, a slurry is prepared
of the eluent with the stationary phase powder and then carefully poured
into the column. The top of the silica should be flat, and the top of the
silica can be protected by a layer of sand. Eluent is slowly passed through
the column to advance the organic material.
The individual components are retained by the stationary phase
differently and separate from each other while they are running at
different speeds through the column with the eluent. At the end of the
column they elute one at a time. During the entire chromatography
process the eluent is collected in a series of fractions. Fractions can be
collected automatically by means of fraction collectors. The productivity
of chromatography can be increased by running several columns at a
time. In this case multi stream collectors are used. The composition of the
eluent flow can be monitored and each fraction is analyzed for dissolved
compounds, e.g. by analytical chromatography, UV absorption spectra,
or fluorescence. Colored compounds (or fluorescent compounds with the
aid of a UV lamp) can be seen through the glass wall as moving bands.

Tlc Chromatography Use of thin layer chromatography was first reported

by two Russian scientists, N.A Izmailov and M.S Schreiber. Later this
technique was developed further by other scientist. Thin layer
chromatography (TLC) depends on the separation principle. The
separation relies on the relative affinity of compounds towards both the
phases. The compounds in the mobile phase move over the surface of the
stationary phase.The movement occurs in such a way that the compounds
which have a higher affinity to the stationary phase move slowly while
the other compounds travel fast.Therefore, the separation of the mixture
is attained. On completion of the separation process, the individual
components from the mixture appear as spots at respective levels on the
plates. Their character and nature are identified by suitable detection
techniques.The TLC plate is a thin piece of aluminium coated on one side
with the stationary phase (this phase does not move), an inert material
that does not react chemically with the sample components. A mobile
phase, which consists of a solvent that moves through the stationary
phase, is used to move the components of the sample up through the
stationary phase. A drop of the sample is placed near the bottom end of
the TLC plate. The bottom end of the TLC plate is then dipped into the
solvent (mobile phase). The solvent creeps slowly up the TLC plate. The
various components of the sample move along with the solvent at a rate
that depends on how strongly they are bound to the stationary phase.
Strongly bound substances hardly move at all, a very weakly bound
substance may move at almost the same speed as the solvent. Thus the
different components (or fractions) of the sample are separated into bands
(or spots) along the length of the TLC plate. This separation process is the
basis of the chromatography method.
Another type is Paper Chromatography.Paper chromatography is
an analytical method used to separate coloured chemicals or
substances. It is now primarily used as a teaching tool, having been
replaced in the laboratory by other chromatography methods such as thin-
layer chromatography (TLC).
The setup has three components. The mobile phase is a solution that
travels up the stationary phase by capillary action. The mobile phase is
generally a mixture of non-polar organic solvent, while the stationary
phase is polar inorganic solvent water. Here paper is used to support the
stationary phase, water. Polar water molecules are held inside the void
space of the cellulose network of the host paper. The difference
between TLC and paper chromatography is that the stationary phase in
TLC is a layer of adsorbent (usually silica gel, or aluminium oxide), and
the stationary phase in paper chromatography is less absorbent paper.
A paper chromatography variant, 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.
Steps in Paper Chromatography 
Selection of Solid Support
 Selection of Mobile Phase
 Saturation of Tank
 Sample Preparation and Loading 
Development of the Chromatogram
 Drying of Chromatogram 
Detection

And then another Type of Liquid Liquid Chromatography.

High-pressure liquid chromatography (HPLC)

high-pressure liquid chromatography, is a technique in analytical
chemistry used to separate, identify, and quantify specific components in
mixtures. The mixtures can originated
from food, chemicals, pharmaceuticals[1], biological, environmental and
agriculture, etc, which have been dissolved into liquid solutions. It relies
on high pressure pumps, which deliver mixtures of various solvents,
called the mobile phase, collecting the sample mixture on the way,
through a column filled with a solid adsorbent material, called the
stationary phase. Each component in the sample interacts slightly
differently with the adsorbent material, causing different migration rates
for the different components, leading to their separation, as they flow out
of the column into a specific detector.
HPLC has been used for manufacturing (e.g., during the production
process of pharmaceutical and biological products)[2][3], legal (e.g.,
detecting performance enhancement drugs in urine)[4], research (e.g.,
separating the components of a complex biological sample, or of similar
synthetic chemicals from each other), and medical (e.g., detecting vitamin
D levels in blood serum) purposes.[5]
Chromatography can be described as a mass transfer process
involving adsorption and/or partition. As mentioned, HPLC relies on
pumps to pass a pressurized liquid and a sample mixture through a
column filled with adsorbent, leading to the separation of the sample
components. The active component of the column, the adsorbent, is
typically a granular material made of solid particles (e.g., silica,
polymers, etc.), 1.5–50 μm in size, on which various reagents can be
bonded. The components of the sample mixture are separated from each
other due to their different degrees of interaction with the adsorbent
particles. The pressurized liquid is typically a mixture of solvents (e.g.,
water, buffers, acetonitrile and/or methanol) and is referred to as a
"mobile phase". Its composition and temperature play a major role in the
separation process by influencing the interactions taking place between
sample components and adsorbent. These interactions are physical in
nature, such as hydrophobic (dispersive), dipole–dipole and ionic, most
often a combination.
HPLC is distinguished from traditional ("low pressure") liquid
chromatography because operational pressures are significantly higher
(50–~1400 bar), while ordinary liquid chromatography typically relies on
the force of gravity to pass the mobile phase through the packed column.
Due to the small sample amount separated in analytical HPLC, typical
column dimensions are 2.1–4.6 mm diameter, and 30–250 mm length.
Also HPLC columns are made with smaller adsorbent particles (1.5–50
μm in average particle size). This gives HPLC superior resolving power
(the ability to distinguish between compounds) when separating mixtures,
which makes it a popular cartographic technique.
The schematic of an HPLC instrument typically includes solvents'
reservoirs, one or more pumps, a solvent-degasser, a sampler, a column,
and a detector. The solvents are prepared in advance according to the
needs of the separation, they pass through the degasser to remove
dissolved gasses, mixed to become the mobile phase, then flow through
the sampler, which brings the sample mixture into the mobile phase
stream, which then carries it into the column. The pumps deliver the
desired flow and composition of the mobile phase through the stationary
phase inside the column, then directly into a flow-cell inside the detector.
The detector generates a signal proportional to the amount of sample
component emerging from the column, hence allowing
for quantitative analysis of the sample components. The detector also
marks the time of emergence, the Retention Time, which serves for initial
identification of the component. More advanced detectors, provide also
additional information, specific to the analyte's characteristics, such
as UV-VIS spectrum or Mass spectrum, which can provide insight on its
structural features. These detectors are in common use, such
as UV/Vis, Photodiode array (PDA) / diode array detector and mass
spectrometry detector.
A digital microprocessor and user software control the HPLC instrument
and provide data analysis. Some models of mechanical pumps in an
HPLC instrument can mix multiple solvents together at a ratios changing
in time, generating a composition gradient in the mobile phase. Most
HPLC instruments also have a column oven that allows for adjusting the
temperature at which the separation is performed.