Chromatography

 In greek words for “chromo -- colour “
and “graphy – to write “.
 Chromatography is meant for coloured
compounds as well as colourless compounds
.
 In 1942 partition chromatography was
developed by martin and syug.
 In 1961 paper chromatography was
discovered by Sehon bein.

 All chromatographic systems consists of two
phases
STATIONARY PHASES:-
 It may be solid ,gel ,liquid or a solid –liquid
mixture which is immobilised(stable).
MOBILE PHASE :-
 It may be liquid or gaseous in state,which is
mobilised (free moving).
 The principle of separation is mainly partition
rather than adsorption.
 Cellulose layers in filter paper
(watt man No-1)contains moisture which
acts as stationary phase and organic solvents or
buffers are used as mobile phase

 Different components of
The sample mixture
Interacts with two phases
Differently on the basis of
Small difference in their
Physico-chemical properties.
 the compound which interacts more with mobile phase
and least with the stationary phase migrates fast .
 The component showing least interaction with mobile
phase while strongly with stationary
phase migrates slow(retarded).

 Paper commonly used for chromatography is
Wattman no 1 filter paper (highly purified
cellulose)
 In PC the stationary phase is supported by
cellulose fibers of the sheet
 cellulose is a homo ploysaccharide of glucose .
 The paper also contains impurities through
organic substances,salts,minerals which are
deposited on the paper while being processed .
 these impurities will be removed by washing the
paper with 0.1N HCl and dry it before
chromatography is carried out.

solvent system:-
 In PC the stationary phase is water since it is very
well adsorbed by cellulose .
 The mobile phase which flows over the stationary
phase may not be necessarily immiscible with
water if water is used as stationary phase(SP) .
 The mobile phase is usually a mixture of various
solvents such as alcohols, acids ,esters, ketones,
phenols, amines and hydro carbons.
 the solvent system should be chosen that the two
phases are immiscible .
 such a choice would lead to maximum separation,
time required is also be short .
 Cellulose fibres in the paper hold moisture tightly
through formation of hydrogen bonds .

PROCEDURE :-
 The sample is applied to the paper as small
spot with the help of platinum loop ,
capillary tube and micropipette.
 The sample should be placed at equal
distance.
 sample is added to the paper and dry it for
some time and repeat the process for 3 to 4
times .

 Based on the way the development of
chromatogram on paper is done in procedure we
have broadly 4 types of paper chromatography.
 Ascending chromatography
 Descending chromatography
 Radial chromatography
 2-D chromatography

ASCENDING CHROMATOGRAPHY
 Solvent is placed in the base of sealed tank or
glass jar to allow the chamber to be saturated
with the solvent vapours .
 the paper is suspended into the solvent
The sample spot should be
in a Position just above the
surface of the solvent.
So that as the solvent
moves vertically up the
paper by capillary action .
The solvent moves upward
against the gravity .

 Firstly ,the setup required for it is very simple
 Secondly ,the resolution of sample by ascending
technique is better as compared to the descending
technique.
 In ascending chromatography two forces are
acting on the solute : the capillary force which
makes it move up and the gravitational force which
opposes this movement.
 The only disadvantage of the ascending technique
is it is very slow.

 The solvent is placed in the base of a sealed tank
or glass jar to allow
chamber to become saturated
with solvent vapour.
 The paper is allowed to
hang in a manner that the
base of paper is not in
contact with the solvent.
 Development is started by adding the solvent to
the trough .
 Negative capillary action is observed .
 The separation of the sample is acheived as the
solvent moves downward the gravity.

 Firstly setup required for it is very complex.
 Secondly the resolution of sample by descending
technique is not better when compared to the
ascending technique.
 In descending chromatography two forces are
acting on the solute .The capillary force which
makes it down and the gravitational force also
towards downward which decreases it’s accuracy .
 The only advantage of descending paper
chromatography is very fast process .

RADIAL CHROMATOGRAPHY
 In this method the sample is spotted at the center
of a circularly cut disc of paper which is placed
horizontally.
 The center of paper is connected to a WICK of the
solvent, which is placed at base of a jar .
 The solvent rises up the wick and then on to the
paper through
capillary action.
 The sample components
Move outward radially
Forming concentric circle
Of increasing diameter.
 The sample components to be separated are
coloured , the chromatogram developed by this
method looks please to the eye.

 The resolution of components by this technique is
sharper , the apparatus also simple.
 One way is to press the paper between two glass plates
with a hole in the center through which the wick can be
connected for solvent supply.
 A larger circular glass jar covered with a glass plate
serves as a very good chamber

 A plane surface is developed in two directions
using two different solvents.
 The paper, with sample applied as a spot close to
corner, is developed in the normal by ascending or
descending procedure.
 The development is continued until the faster
moving component front approaches the end of the
paper .

 The paper is then removed and the solvent
is allowed to evaporate . This paper is
turned to 90° or right angle and develop it
for second time with another solvent having
totally different eluting properties.
 Since, the two solvents used have different
eluting properties, the distribution
coefficients of individual components in
them will also differ.

 Therefore components which could not be
separated using one solvent can be easily
separated by this procedure .
 This technique is known as
TWO –DIMENSIONAL PAPER CHROMATOGRAPHY

 There are various methods for detection .
 If the sample components are colourless they can
be imparted colour by spraying the paper with
colour producing reagents .
EX:- For amino acid detection ninhydrin reagent
spreads on the paper reacts with amines and amino
acids to form a blue or purple colour.
 If not sprayed the reagent may be applied to the
paper by either immersing the paper in the
reagent or by treating the paper to the reagent
vapours .

 Various methods of detection are ;-
I. Ultra violet and Infrared absorption.
II. Fluorescence
III.Radio active

 It is also known as Relative Flow .
 It is the ratio which reflects the
distribution coefficient of the given solute .
 The compound can be identified on the basis
of the distance transveresed by the solute
relative to the distance moved by the
solvent front .
 Rf = The distance moved by the sample
The distance moved by the solvent
front

 For carbohydrates the term Rf is replaced
by RG where G stands for Glucose
 RG = The distance moved by a
carbohydrate
The distance moved by the glucose
Since, each compound has specific Rf(RG)
value,this value can be used to detect the
unknown compounds.

 A qualitative method for identifying
components of mixture .
 Identification of drugs and impurities .
 Unknown organic and in organic compounds .
 Identification of biomolecules.
 Its an inexpensive and rapid method for
separation.

 This technique is more or less similar to
paper chromatography .
 The technique , a thin layer of a finely
divided substances is deposited on to a flat
glass plate.
 The sample to be separated is spotted at
one end . This plate is dipped into the
solvent in a glass jar and the development
carried out by asscending technique.

 After the development ,the layer can be
dried and the components are detected .
 TLC may be either carried out by the
Adsorption principle (thin layer is prepared
by adsorbent _ kiselghur).
 Or by partition principle (layer is prepared
by a substance called _silica gel which hols
water)

o The glass plate on which the thin layer is
prepared should be even and is thoroughly
washed and dried before layer application .
o The thin layer is to be made is usually
mixed with water in such a proportion that a
thick suspension known as “SLURRY”.
o This slurry is applied to a plate surface as a
uniform thin layer .

o The thickness of slurry layer used for used
for analytical separation is 0.25mm, while
preparative separation the thickness f layer
is about0.5mm.
o Stationary phase for adsorption
chromatography is to be performed , The TL
is activated by heating at 110°c for several
hours .
o PRECAUTION – Not to scrape the thin layer
while applying the sample.

 The choice of solvents and the methods of
elution are much the same as for paper
chromatography .
 This procedure must be conducted in a
closed chamber to prevent evaporation of
the solvent and this technique is carried out
in ascending or 2 dimensional methods.
 One of the greatest advantage of TLC is the
speed of separation.
 Generally 10 to 30 minutes are sufficient
and for certain compounds about 90
minutes may be required.

 Spraying the plate with 25-50% sulphuric
acid in ethanol and heating results in
charring most of the compounds , which
show up as brown spots.
 Iodine along with alcohol vapours is used
extensively a universal reagent for organic
compounds.
 Iodine spot disappears rapidly but can be
made more permanent by spraying with 0.5%
benzidine solution in absolute ethanol.

Applications:
1)The constituents of the mixture of amino acids, and the constituents of
natural lipids and phospholipids
are separated and estimated in a short time.
2)Enzymes, nucleic acids, pigments, sugars can also be
separated by using this technique.
3) TLC has often been used to identify drugs, contaminants &
adulterants.2

 Column chromatography is an often used as
routinely carried out technique which is adaptable
to all the major types of chromatography.
 Column chromatography is of various types
I. Gel filteration chromatography
II. Ion exchange chromatography
III. Affinity chromatography
IV. High performance liquid chromatography(HPLC)

COLUMNS :-
• The columns are usually made up of glass or
polyacrylate plastic .
• Different columns differ in their dimensions.
• Laboratory columns have a diameter of 2-70mm
and length of 15-150 cm ,length shorter than 15cm.
• The commonly used glass columns have a sintered
glass disc at the bottom to support the stationary
phase.
• Certain columns are supported under a replaceable
nylon mesh or glass wool.
• The columns are provided with the inlets and
outlets

• Certain columns are supported under a
replaceable nylon mesh or glass wool.
• The columns are provided with the inlets and
outlets .

 The column is fitted in upright position and
its bottom is sealed with glass wool or nylon
mesh .
 Gel is used as stationary phase in all types of
chromatography .
 We use Agarose , Sephadex , Sephadose
etc…
 They are generally hard in nature .But when
they are soaked in water or buffer and
heated if needed they converts into jelly
substances .
 Some substances will have high porosity while
some have low porosity .

 They are jelly and are spread like beads .
 They are kept in column above the glass wool ,
mobile phase is added to input and released
through out put.
 This process is to remove all the air bubbles in
stationary phase . Buffer is used as mobile phase
in column chromatography
 When all air bubbles are removed stationary phase
is packed in it the level of mobile phase have to be
maintained at a constant level.

column chromatography
Gel Ion - Affinity HPLC
Filteration exchange

It is also known as Molecular sieve chromatography
or Molecular exclusion or Size exclusion and
permeation chromatography.
PRINCIPAL :- In exclusion chromatography the
separation of molecules is based up on the size and
shape . The stationary phase is porous bead
material and the mobile phase is solvent system.
• The apparatus consists of a column packed with
sponge like gel beads containing pores.

• The gels serves as molecular sieves for the
separation of smaller and bigger molecules.
• The large molecules cannot enter the pores
of the beads so they are excluded out and
come down rapidly .
• Small sized molecules enters to the pores of
the beads so that their speed is retarded
and come down slowly .
• There are four types of gels, they are :-
A. Sephadex B. Agarose
C. Polyacrylamide D. Styragel

Sephadex:
1. It is most popular gel for proteins & most of
the biomolecules separation.
2. Sephadex gels are insoluble in water, and
are stable in bases, weak acids and mild
reducing and oxidizing agents.
3. It is cross-linked polymer. .
4. Sephadex, which cannot be used to separate
biopolymers larger than 300,000 Daltons.

Agarose:
1. Agarose gels are produced from AGAR.
2. They are linear polysaccharides alternating
residues of D-Galactose and 3,6-anhydro-L-
galactose units.
3. These gels are hydrophilic and are almost
completely free of charged groups.
4. Agarose gels, due to their greater porosity,
it may be used to separate molecules and
participates up to a molecular weight of
several million Daltons.

Polyacrylamide:
1. This is very popular medium is produced by
polymerizing acrylamide into bead form.
2. polyacrylamide gels can be used to separate
molecules of up to 300,000 daltons.
3. This gel is insoluble in water and common
organic solvents may be used in the pH
range of 2 to 11.
4. Some common gels are,
Bio-gel P 10, Bio-gel P60, Bio-gel P100,

Styragel:
1. For completely non-aqueous separations, a
gel that will swell in an organic solvent is
required. Styragel provides this option.
2. It is a rigid cross-linked polystyrene gel.
3. The gel structure is unaffected by
temperatures as high as 1500 C

Mathematical relation ship about solute
behaviour on molecular sieve .
 This distribution of a solute particle between
the inner and outer solvent is defined as
“DISTRIBUTION COEFFICIENT” (Kd) .
 If Kd = 0 then the solute molecule is large and
excluded out completely .
 If Kd = 1 Then the solute molecule is small
,retarded it in inner solvents .
 The volume outer solvent .i.e, the solvent
surrounding the gel beads is indicated as vo.

 The technical term for this is void volume.
 The volume of the solvent inside gel bead –
inner solvent =Vi
 The distribution coefficient =Kd
 The effluent volume =Ve
Ve = Vo + KdVi
Vi = awr
Where , a= dry weight of the gel
wr =water regain value

 The main application of gel-filtration is the
purification of molecules, viruses, nucleic acids,
hormones, enzymes, proteins, and antibodies and
can be separated and purified by this technique.
 It is also used for the separation of vitamins,
steroids, neuropeptides and drugs.
 Separations are achieved very quickly by this
technique.
 The molecular weight of the molecule can also be
determined by this technique.
 Protein receptor binding can be understood by this
technique

 Separations are achieved very quickly by
this technique.
 The molecular weight of the molecule can
also be determined by this technique.
 Protein receptor binding can be
understood by this technique

 W.Cohn first developed this procedure .
 The reversible exchange of ions in solution with ions
electrostatically bound to some sort of insoluble
medium.
 Ion exchange chromatography is a process that allows
the separation of ions and polar molecules based on the
charge properties of the molecules .
PRINCIPLE
Exchange of ions is the basic principle in this type of
chromatography.
 In this process two types of exchangers can be used
1. CATIONIC EXCHANGER
2. ANIONIC EXCHANGER

PRINCIPLE
Exchange of ions is the basic principle in this
type of chromatography.
 In this process two types of exchangers can
be used
1. CATIONIC EXCHANGER
2. ANIONIC EXCHANGER

CATION – EXCHANGER
 Cationic exchangers possess negatively
charged group, and these will attract
positively charged cations. These
exchangers are also called “Acidic ion
exchange materials”, because their negative
charges result from the ionization of acidic
group .
.

ANIONIC – EXCHANGER
 Anionic exchangers have positively charged
groups that will attract negatively charged
anions. These are also called “Basic ion
exchange” materials.
Types of ion exchange resins:
 Two main groups of materials are used to
prepare ion exchange resins: Polystyrene
and Cellulose.
 Resins made from both of these materials
differ in their flow properties, ion
accessibility and chemicals and mechanical
stability.

Polystyrene resins
 These are proposed by polymerization reaction of
styrene and divinly benzene.
 A higher concentration of divinyl benzene
produces higher cross linkages.
 Polystyrene resins are very useful for separating
small molecular weight compounds
 Resins substituted with sulfonic acid groups are
strong cationic exchangers.
 To prepare weekly acidic exchanger, carbohydrate
groups can be attached to the aromatic rings
instead of sulfonic acid group.

Cellulose resins
 These have much greater permeability to
macromolecular polyelectrolytes and possess
a much lower charge density as compared to
polystyrene exchangers.
 Carboxymethyl cellulose (CM-cellulose) –
Cationic exchanger
 DEAE cellulose - Anionic exchanger
Choice of Buffers:-
 Anionic exchange Chromatography should be
carried out with cationic buffers.

 Cationic exchange Chromatography should
be carried out with anionic buffers.
 The pK of the buffer should be as near as
possible to the pH at which the system is
buffered

Applications:
 It is extremely used in the analysis of amino acids.
The amino acid “Autoanalyzer” is based on in
exchange principle.
 To determine the base composition of nucleic
acids. Chargaff used this technique for
established the equivalence of Adenine and
Thymine; Guanine and Cytosine.
 This is most effective method for water
purification. Complete deionization of water (or) a
non-electrolyte solution is performed by
exchanging solute cations for hydrogen ions and
solute anions for hydroxyl ions.

 This is usually achieved by method is used for
softening of drinking water.
 Proteins are also successfully separated by this
technique.
 It is also used for the separation of many
vitamins, other biological amines, and organic
acids and bases.

PRINCIPLE:-
 It is based on the property of specific and
non covalent binding of proteins to other
molecules referred to as substrate or co
factors.
PROCEDURE:-
 The technique involves use of ligands
covalently attached to inert and porous
matrix in a column.

 It is mainly based on the biological affinity
(or) biological specificity.
 The materials to be isolated are capable of
binding reversibly to a specific ligand i.e.,
attached to an insoluble matrix.

 The matrix should be inert to other molecules to minimize
non specific adsorption .
 The immobilized ligands acts as molecular hooks to
selectively pickup the desired protein while the remaining
proteins pass through the column.
 Alternatively some reagents that can break protein ligand
interaction can also be employed for the separation .

Types of ligands :-
 The particles, which are uniform, spherical
and rigid, are used. The most commonly used
ones are
A) Agarose
B) Polyacrylamide
C) Controlled glass beads

Agarose:
 The agarose beads have most desired
features as mentioned above. But it has
some advantage when use the denaturant
solution for elution, which have a
susceptibility to contraction .

Polyacrylamide:
 The polyacrylamide bead lacks
porosity. This undesirable trait is
heightened even further when they are
substituted by ligands .

Controlled porosity glass beads:
 This bead provides mechanical rigidity and
chemical inertness in addition to providing very
good flow rates. High degree of nonspecific
protein adsorption is the most serious drawback to
these beads, which avoid to some extent by
treatment with “Hexamethyldisilazane

Ligand selection:
 The selection of ligand should have two most
important requirements:
 Ligand interaction should be less with desired
macromolecules.
 The ligand should possess functional groups that
can be modified to form covalent linkage with the
supporting matrix.

Ligand attachment :-
 Covalent coupling of the ligand to the supporting
matrix involve the following steps:
 Activation of the matrix functional groups
 Covalent attachment of the ligand to the activated
functional groups.

 The technique has been used to purify a large
variety of macromolecule such as enzymes,
Immunoglobulins, membrane receptors, Nucleic
acids and even polysaccharides.
 Immobilized enzymes (Solid-state enzymes) are
also isolated and purified by this method.
 mRNA can be isolated by this technique.
 Native proteins can be separated from
denatured proteins by this technique.
 DNA & RNA can be separated from each other
 Papain and Urease can be separated by this
technique.

 HPLC is also known as high pressure liquid
chromatography.
 HPLC is the optimization of the conventional column
chromatography .
 To reduce the time of experiment one can increase the
flow there by reducing retention time but it also
decrease its accuracy.
 To increase the flow rate pressure has to be increased
.
 This method uses an extremely high pressure upto
8000 psi ,the flow rate therefore is high and the
experimental time is shortened considerably.

 To increase the flow rate pressure has to be
increased .
 This method uses an extremely high
pressure upto 8000 psi ,the flow rate
therefore is high and the experimental time
is shortened considerably
 HPLC requires the use of non compressible
resin materials and strong metal columns.
The eluents of the columns are detected by
methods such as UV absorption and
fluorescence .

 It can be applied in the form of partition
,adsorption ,ion exchange or molecular sieve
chromatography .
THE STATIONARY PHASE :-
 Consists of an immobilized thin layer of
liquid on the micro glass or plastic beads
highly packed into a narrow column

THE MOBILE PHASE :-
 Consists of a buffered solvent system which is
passed under high pressure through the column for
eluting the solutes of the sample.

 The separation of the volatile material based on
the partition of the components between the
mobile phase (gas) ,stationary phase (liquid)
.Hence, is also known as gas liquid chromatography
.
 The father of modern gas chromatography is john
porter martin , who also developed the first liquid
gas chromatogram (1950).

 It gives good percision and accuracy .
 The analysis is completed in a short time .
 The cost of instrument is relatively low and its life
is generally long .
 The technique is relatively suitable for routine
analysis .

Chromatography

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