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 Introduction
 Basic terminologies
 History
 Principles
 Applications
 Types
 It is a separation technique based on the different interactions
of compounds with two phases a mobile phase and a
stationary phase, as the compounds travels through a
supporting medium
 Chromatography is non destructive procedure for resolving a
multi-component mixture of trace, minor, or major
constituents into its individual fraction. While
chromatography may be applied both quantitatively, it is
primarily a separation too
 The word chromatography is derived from two Greek words
1) Chroma ……… color
2) Graphos ……… writing
 Chromatograph: Instrument employed for a
chromatography.
 Stationary phase: Phase that stays in place inside the
column. Can be a particular solid or gel-based packing (LC)
or a highly viscous liquid coated on the inside of the column
(GC).
 Mobile phase: Solvent moving through the column, either a
liquid in LC or gas in GC..
 Elution: The process of passing the mobile phase through the
column.
 Chromatogram: Graph showing detector response as a
function of a time.

 Linear velocity: Distance passed by mobile phase per 1 min

in the column.
 In 1903 Tswett a Russian Botanist coined the term
chromatography . He passed plant tissue extracts through
a chalk column to separate pigments by differential
adsorption chromatography 1915 R.M Willstattar ,
German Chemist with Nobel prize for similar experiment
 In 1922 L.S Palmer, American scientist used tswett’s
techniques on various natural products
 In 1931 Richered Kuhn used chromatography to separate
isomers of polyene pigments; this is the first known
acceptance of chromatographic methods
 Chromatography usually consist of mobile phase and
stationary phase.
 The mobile phase refers to the mixture of substances to
be separated dissolved in a liquid or gas.
 The stationary phase is a porous solid matrix through
which the sample contained in the mobile phase
percolates.
 The interaction b/w the mobile phase and stationary
phase results in the separation of the compound from the
mixture
 Chromatography plays an important role in
many pharmaceutical industries and also in the chemical
and food industry
 Pharmaceutical industries use this method both to prepare
huge quantities of extremely pure materials, and also to
analyze the purified compounds for trace contaminants
 The other applications of chromatography especially HPLC is
used in Protein Separation like Insulin Purification, Plasma
Fractionation and Enzyme Purificanation
 Chromatography is used for quality analyses and checker
in the food industry, by identifying and separating,
analyzing additives, vitamins, preservatives, proteins,
and amino acids
 Chromatography like HPLC is used in DNA
fingerprinting and bioinformatics.
 Assay & Content Uniformity
 HPLC in fingerprinting and Bioinformatics
 Petrochemicals and Catalysis
 Polymer Synthesis
 Clinical diagnosis of diseases and disorder
 LC-NMR
The twelve types are
1) Column Chromatography
2) Paper Chromatography
3) Thin Layer Chromatography
4) Gas Chromatography
5) High Performance Liquid Chromatography
6) Fast Protein Liquid Chromatography
7) Supercritical Fluid Chromatography
8) Affinity Chromatography
9) Reversed Phase Chromatography
10) Two Dimensional Chromatography
11) Pyrolysis Gas Chromatography and
12) Counter Current Chromatography.
 Paper chromatography is one of the types of
chromatography procedures which runs on a piece of specialized
paper.
 It is a planar chromatography system wherein a cellulose filter
paper acts as a stationary phase on which the separation of
compounds occurs
 Principle of paper chromatography:
 The principle involved is partition chromatography wherein the substances
are distributed or partitioned between liquid phases. One phase is the
water, which is held in the pores of the filter paper used; and other is the
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
 Paper chromatography works in few steps:
Step 1: A horizontal line is drawn near one end (about 1.5 cm
from the bottom edge) of the paper. In figure below 6 is
the horizontal line
 Step 2: The sample needs to be separated is placed as
a small drop or line on to the paper using capillary
tube. Labelling the drop by a pencil with an alphabet
or number help to identify the compound later. In
figure above 3 and 4 are the drops labelled. The
drops are then soaked on the paper and dried.
 Step 3: The paper is then placed into a sealed
container with a swallow layer of suitable solvent.
The solvent level must be lower than the pencil line
or drop on it. The container need to be covered to
stop the solvent to evaporate
 Step 4: The solvent rises up the paper chromatography taking
each component of the sample with it. The components travel
with the solvent depends on three things:
 The polarity of the sample molecule. The non polar
components travel faster than the polar component.
 The attraction between the sample molecule and the solvent
or solvent mixture.
 The attraction between the sample and the silica.
 Suppose any sample compound mixture contains three
colored molecules green, blue and red. According to their
polarity, the order of these compounds is green<blue<red.
Thus the most non polar green will travel first along with the
mobile phase. Then blue and at last most polar compound the
red one
 Step 5: When the solvent rises near the end of the paper
then the paper should be taken out from sealed container
and air dried. The paper with separated bands of
components are then observed under UV-light.
 Rf value
 The compounds in the sample travels along with
solvent to give separate bands on the paper. The
distance travelled by same compound with respect to
the solvent is always constant. Thus the ratio of the
distance that the compound travelled and the
distance that the solvent travelled is denoted as Rf.
And mathematically expressed as:

 Column chromatography is a chromatography technique
used to separate mixture of chemical substances into its
individual compounds.
 Column chromatography is a widely used method for the
purification or separation of chemical compound mixture
in lab
 Column chromatography works in few steps:
 Step 1: The mobile phase or eluent is either solvent
or solvent mixture. The upper level of mobile phase
should be same as the stationery phase. That means
the stationery phase should be wet with the solvent.
On this stage the compound mixture what need to be
separated, are added from the top of the column in
such a way that the top level of it is not disturbed. By
turning on the tap below it is allowed to adsorbed on
the surface of the silica.
 Step 2: Then the solvent or a suitable solvent mixture is added
at first touching the side of the glass column slowly and
carefully so that the top level of the stationery phase is not
disturbed. The solvent is repeatedly added as many times as
needed throughout the process.
 Step 3: When the tap, is on the compounds in the compound
mixture move along with the eluent depending on the
polarity of the sample molecule. The non polar components
travel faster than the polar component.
 Suppose if any compound mixture contains three compounds
blue, red and green. According to polarity the order of these
compounds are blue>red>green. That means blue is the most
polar compound and thus will have less tendency to move
along with the mobile phase.
 Step 4: The green colored compound will travel first
as it is less polar that other two. When it is near end
of the column a clean test tube is taken to collect the
green sample. After this the red and at last the most
polar blue compound is collected, all in separate test
tubes
 Column layer chromatography is an chromatography
technique used to separate mixture of chemical substances
into its individual compounds.
 Chromatography consists of two phases: one mobile phase
and one contiguous stationery phase.
 Column is prepared my mixing the silica with suitable solvent
and poured in into a glass column.
 A suitable solvent (mobile phase) is moved along with
compound mixture through the column according to the
polarity.
 Ion exchange chromatography involves the separation of
ionizable molecules based on their total charge.
 This technique enables the separation of similar types of
molecules that would be difficult to separate by other
techniques because the charge carried by the molecule of
interest can be readily manipulated by changing buffer
pH
 It can be used for almost any kind of charged molecule
including large proteins, small nucleotides and amino
acids
 The crude sample containing charged molecules is
used as the liquid phase.
 When it passes through the chromatographic column,
molecules bind to oppositely charged sites in the
stationary phase.
 The molecules separated on the basis of their charge
are eluted using a solution of varying ionic strength.
 By passing such a solution through the column,
highly selective separation of molecules according to
their different charges takes place
Key steps in the ion exchange chromatography procedure
are listed below:
 An impure protein sample is loaded into the ion
exchange chromatography column at a particular pH.
 Charged proteins will bind to the oppositely charged
functional groups in the resin
 A salt gradient is used to elute separated proteins. At low
salt concentrations, proteins having few charged groups
are eluted and at higher salt concentrations, proteins with
several charged groups are eluted.
 Unwanted proteins and impurities are removed by
washing the column
 A pH gradient can also be applied to elute individual
proteins on the basis of their isoelectric point i.e. the
point at which the amino acids in a protein carry neutral
charge and hence do not migrate in an electric field.
 As amino acids are zwitter ionic compounds they
contain groups having both positive and negative
charges.
 Based on the pH of the environment, proteins carry a
positive, negative, or nil charge.
 At their isoelectric point, they will not interact with the
charged moieties in the column resin and hence are
eluted.
 A decreasing pH gradient can be used to elute proteins
using an anion exchange resin and an increasing pH
gradient can be used to elute proteins from cation
exchange resins
 This is because increasing the buffer pH of the mobile
phase causes the protein to become less protonated (less
positively charged) so it cannot form an ionic interaction
with the negatively charged resin, allowing is elution.
Conversely, lowering the pH of the mobile phase will
cause the molecule to become more protonated (less
negatively charged_, allowing its elution.
 Ion exchange resins have positively or negatively
charged functional groups covalently linked to a
solid matrix.
 Matrices are usually made of cellulose, polystyrene,
agarose, and polyacrylamide. Some of the factors
affecting resin choice are anion or cation exchanger,
flow rate, weak or strong ion exchanger, particle size
of the resin, and binding capacity.
 The stability of the protein of interest dictates the
selection of an anion or a cation exchanger – either
exchanger may be used if the stability is of no
concern
 It is the most widely used chromatographic method for the
separation and purification of charged biomolecules such as
polypeptides, proteins, polynucleotides, and nucleic acids. Ion
exchange chromatography is widely used in several industrial
applications some of which are as follows:
1) Separation and Purification of blood components such as
albumin,recombinant growth factors and enzymes.
2) Biotechnology - Analytical applications such as quality
control and process monitoring
3) Food and clinical research - to study wheat varieties and the
correlation of proteinuria with different renal diseases.
4) Fermentation - Cation exchange resins are used to monitor
the fermentation process during ß-galactosidase production
in analytical chemistry, technique for separating
dissolved chemical substances by virtue of their
differential migration over glass plates or plastic
sheets coated with a thin layer of a finely ground
adsorbent, such as silica gel or alumina, that is
mixed with a binder such as starch or plaster of
paris.TLC is a type of planar chromatography..
 It is a semi-quantitative method consisting of analysis.
 High-performance thin-layer chromatography (HPTLC)
is the more sophisticated or more precise quantitative
version
Similar to other chromatographic methods, thin layer chromatography
is also based on the principle of separation.
 The separation depends on the relative affinity of compounds
towards stationary and the mobile phase.
 The compounds under the influence of the mobile phase (driven by
capillary action) travel over the surface of the stationary phase.
During this movement, the compounds with higher affinity to
stationary phase travel slowly while the others travel faster. Thus,
the separation of components in the mixture is achieved.
 Once separation occurs, the individual components are visualized
as spots at a different level of travel on the plate. Their nature or
character are identified using suitable detection techniques.
TLC system components consist of
 TLC plates, preferably ready-made with a stationary phase:
These are stable and chemically inert plates, where a thin layer
of stationary phase is applied on its whole surface layer. The
stationary phase on the plates is of uniform thickness and is in
fine particle size.
 TLC chamber. This is used for the development of the TLC
plate. The chamber maintains a stable environment inside for
proper development of spots. It also prevents the evaporation of
solvents and keeps the process dust-free.
 Mobile phase. This comprises of a solvent or solvent mixture.
The mobile phase used should be particulate-free and of the
highest purity for proper development of TLC spots. The
solvents recommended are chemically inert with the sample, a
stationary phase.
 A filter paper. This is moistened in the mobile phase, to
be placed inside the chamber. This helps develop a
uniform rise in a mobile phase over the length of the
stationary phase.
The stationary phase is applied onto the plate uniformly and
then allowed to dry and stabilize. These days, however,
ready-made plates are preferred.
 With a pencil, a thin mark is made at the bottom of the
plate to apply the sample spots.
 Then, samples solutions are applied on the spots marked
on the line in equal distances.
 The mobile phase is poured into the TLC chamber to a
leveled few centimeters above the chamber bottom. A
moistened filter paper in the mobile phase is placed on
the inner wall of the chamber to maintain equal humidity
(and also thereby avoids edge effect this way).
 Now, the plate prepared with sample spotting is placed in
the TLC chamber so that the side of the plate with the
sample line is facing the mobile phase. Then the chamber
is closed with a lid.
 The plate is then immersed, such that the sample spots
are well above the level of mobile phase (but not
immersed in the solvent — as shown in the picture) for
development.
 Allow sufficient time for the development of spots. Then
remove the plates and allow them to dry. The sample
spots can now be seen in a suitable UV light chamber or
any other methods as recommended for the said sample
 It is a simple process with short development time.
 It helps with the visualization of separated compound
spots easily.
 The method helps to identify the individual compounds.
 It helps in isolating of most of the compounds.
 The separation process is faster and the selectivity for
compounds is higher (even small differences in
chemistry is enough for clear separation).
 The purity standards of the given sample can be assessed
easily.
 It is a cheaper chromatographic technique.
 To check the purity of the given samples.
 Identification of compounds like acids, alcohols, proteins,
alkaloids, amines, antibiotics, and more.
 To evaluate the reaction process by assessment of
intermediates, reaction course, and so forth.
 To purify samples, i.e., for the purification process.
 To keep a check on the performance of other separation
processes.
 Being a semi-quantitative technique, TLC is used more for
rapid qualitative measurements than for quantitative purposes.
But due to its rapidity of results, easy handling, and
inexpensive procedure, it finds its application as one of the
most widely used chromatography techniques