Prof.Wagh J.G.

Department of Pharmaceutical Chrmistry.

MES College of pharmacy,
Sonai
Mikhail Tswett, Russian, 1872-1919
Botanist
In 1906 Tswett used to
chromatography to separate plant
pigments
He called the new technique
chromatography because the result
of the analysis was 'written in color'
along the length of the adsorbent
column
Chroma means “color” and graphein means
to “write”
Chromatography has application in every branch of
the physical and biological sciences
12 Nobel prizes were awarded between 1937 and 1972
alone for work in which chromatography played a
vital role
Chromatography is a physical method of
separation in which the components to be
separated are distributed between two phases
one of which is stationary (stationary phase)
while the other (the mobile phase) moves
through it in a definite direction.
The chromatographic process occurs due to
differences in the distribution constant of the
individual sample components.
 Is a technique used to separate
and identify the components of a
mixture.

Works by allowing the molecules present in the

mixture to distribute themselves between a
stationary and a mobile medium.

Molecules that spend most of their

time in the mobile phase are carried
along faster.
Classification of chromatography according to
mobile phase:
1- Liquid chromatography: mobile phase is a
liquid. (LLC, LSC).
2- Gas chromatography : mobile phase is a gas.
(GSC, GLC).
Classification according to the packing of the
stationary phase:

1- Thin layer chromatography (TLC): the

stationary phase is a thin layer supported on
glass, plastic or aluminium plates.

2- Paper chromatography (PC): the stationary

phase is a thin film of liquid supported on an
inert support.

3- Column chromatography (CC): stationary

phase is packed in a glass column.
Classification according to the force of separation:

1- Adsorption chromatography.
2- Partition chromatography.
3- Ion exchange chromatography.
4- Gel filtration chromatography.
5- Affinity chromatography.
is a method for identifying substances and
testing the purity of compounds.

TLC is a useful technique because it is

relatively quick and requires small quantities
of material.
Separations in TLC involve distributing a mixture of
two or more substances between a stationary
phase and a mobile phase.

The stationary phase:

is a thin layer of adsorbent (usually silica gel or
alumina) coated on a plate.

The mobile phase:

is a developing liquid which travels up the
stationary phase, carrying the samples with it.
Components of the samples will separate on the
stationary phase according to
how much they adsorb on the stationary phase
versus how much they dissolve in the mobile
phase.
To a jar with a tight-fitting lid add enough of
the appropriate developing liquid so that it is
0.5 to 1 cm deep in the bottom of the jar.
Close the jar tightly, and let it stand for about 30
minutes so that the atmosphere in the jar
becomes saturated with solvent.
With a pencil, etch two small notches into the adsorbent
about 2 cm from the bottom of the plate.

The notches should be on the edges of the plate, and

each notch should be the same distance up from the
bottom of the plate.

The notches must be farther from the bottom of the

plate than the depth of the solvent in the jar.

Using a drawn-out capillary tube, spot the samples on

the plate so that they line up with the notches you
etched.
After preparing the development chamber and
spotting the samples, the plates are ready for
development.

Be careful to handle the plates only by their edges,

and try to leave the development chamber
uncovered for as little time as possible.

When the plates are removed from the chamber,

quickly trace the solvent front (the highest solvent
level on the plate) with a pencil.
If the spots can be seen, outline
them with a pencil.

If no spots are obvious, the

most common visualization
technique is to hold the plate
under a UV lamp.
Many organic compounds can
be seen using this technique,
and many commercially
made plates often contain a
substance which aids in the
visualization of compounds.
Alkaloids: Dragendorff’s reagent

Cardiac glycosides: Antimony trichloride

Sugar: Aniline phthalate

Amino acids: Ninhydrin

The Rf (retention factor) value for each spot
should be calculated.
It is characteristic for any given compound on
the same stationary phase using the same
mobile phase for development of the plates.
Hence, known Rf values can be compared to
those of unknown substances to aid in their
identifications.
(Note: Rf values often depend on the temperature
and the solvent used in the TLC experiment.

the most effective way to identify a compound is to

spot known substances – authentic - next to
unknown substances on the same plate.)

In addition, the purity of a sample may be

estimated from the chromatogram.

An impure sample will often develop as two or

more spots, while a pure sample will show only
one spot
A TLC plate is a sheet of glass, metal, or plastic which is coated
with a thin layer of a solid adsorbent (usually silica or
alumina).

A small amount of the mixture to be analyzed is spotted near the

bottom of this plate.
The TLC plate is then placed in a shallow pool of a solvent in a
developing chamber so that only the very bottom of the plate
is in the liquid.

This liquid, or the eluent, is the mobile phase, and it slowly rises
up the TLC plate by capillary action.

As the solvent moves past the spot that was applied, an

equilibrium is established for each component of the mixture
between the molecules of that component which are adsorbed
on the solid and the molecules which are in solution.
In principle, the components will differ in solubility
and in the strength of their adsorption to the
adsorbent and some components will be carried
farther up the plate than others.

When the solvent has reached the top of the plate, the
plate is removed from the developing chamber, dried,
and the separated components of the mixture are
visualized.

If the compounds are colored, visualization is

straightforward. Usually the compounds are not
colored, so a UV lamp is used to visualize the plates.
A method of partition chromatography using
filter paper strips as carrier or inert support.

The factor governing separation of mixtures of

solutes on filter paper is the partition between
two immiscible phases.

One is usually water adsorbed on cellulose fibres

in the paper (stationary phase).

The second is the organic solvent flows past the

sample on the paper (stationary phase).
Partition occurs between the mobile phase and
the stationary aqueous phase bound by the
cellulose.

The isolation depends on partition coefficient

of the solute.

c( stationary )
K
c(mobile)
1- Choice of paper and solvent to be used.
2- Desalting of sample.
3- Application of the sample.
4- Equilibration of paper.
5- Development.
6- Detection.
7- Identification of substances.
Radial development Ascending development

Descending development
Multiple chromatography includes all procedures
in which the development is repeated after one
development is completed.

A- multiple development: the chromatogram is

repeatedly developed in the same direction and
thus the complete resolution of two or more
substances which have Rf values close together
can be obtained.
As the mobile phase one can use either the same
solvent system or different solvent systems.
B- two-dimensional chromatography:
When large numbers of substances are to be
separated on a single chromatogram.
Development in a direction perpendicular to the
first, and with a solvent system different from
that used initially is often necessary.

The sample is applied on one corner of a square

piece of paper and after development with the
first solvent, the paper is dried , rotated 90o and
developed in the second direction.
Usually, different types of solvents systems are
used in each direction. It is essential that the
first solvent be completely volatile.
This includes chromatographic methods in
which:
The stationary phase is packed into a column.
The mobile phase is a moving liquid or gas.

According to the mechanism of separation of

solutes, five major types of CC are
ditinguished. Usually, one mechanism
predominates but does not exclude the others
 Different Types of chromatography
Mode or type Stationary phase Mobile phase Mechanism
Adsorption Solid that attracts Liquid or gas Solutes move at different rates
Chromatography the solutes according to the forces of attraction
to the stationary phase.
Partition Thin film of liquid Liquid or gas Solutes equilibrate between the 2
Chromatography formed on the phases according to their partition
surface of a solid coefficients
inert support
Ion Exchange Solid resin that Liquid Solute ions of charge opposite to the
Chromatography carries fixed ions containing fixed ions are attracted to the resin
& mobile electrolytes by electrostatic forces & replace the
couterions of mobile counterions.
opposite charge
attached by
covalent bonds
Molecular Exclusion Porous gel with no Liquid Molecules separate according to
Chromatography attractive action their size:
on solute 1.Smaller molecules enter the pores
molecules of the gel, and need a larger volume
of eluent.
2.Larger molecules pass through the
column at a faster rate.
Affinity Solid on which Liquid or gas Special kind of solute molecules
Chromatography specific molecules interact with those immobilized on
Column
chromatograph
y
Stationary phase is
held in a narrow
tube through
which the
mobile phase is
forced under
pressure or
under the effect
of gravity
 Term Definition

Mobile liquid phase with no affinity to the stationary phase

Solvent
(i.e. inert towards it) & no effect on solutes.

Any liquid with more affinity to the stationary phase than

Developer the solvent but less than solutes and just capable to move
them through the column.

Effluent Any liquid that passes out of the column.

Any liquid that has lesser affinity to the stationary phase

Eluent
than solutes but is capable to move them out of the column.

Eluate Fraction of eluent containing a required specific substance.

(or retardation volume): Volume of mobile phase that
Retention
passes out of the column, before elution of a specific
volume (VR)
substance.
Traditional column chromatography is
characterized by addition of mobile phase
under atmospheric pressure and the stationary
phase is packed in a glass column.
The selection of the method of packing depends
mainly on the density of the solid.Techniques
used are the wet, dry & slurry methods.
In all cases avoid inclusion of air bubbles
Apply evenly & in a concentrated solution to the
top of the column which is protected from
disturbance (e.g. add glass wool or filter
paper).
 Elution techniques.1

Technique Procedure

Isocratic elution Addition of solvent mixture of fixed composition

during the whole process.

Gradient elution Continuous or linear elution: in which there is

continuous change in the composition of the
mobile phase over a period of time (e.g. polarity,
pH or ionic strength).

Step wise or fractional elution: in which the

change is not continuous i.e. a sudden change in
the composition of the mobile phase is followed
by a period where the mobile phase is held
constant.
On-column detection for colored or fluorescent
compounds directly after developing the
chromatogram.

Monitoring of eluted fractions (PC or TLC).

Using special detectors connected to the column

such as refractive index, UV detectors, etc…
 Factors affecting solutes separation in CC
( Factors affecting column efficiency)
Factor Effect
Particle size of solid stationary Decrease of size improves separation (but very small
phase (or of support) particles need high pressure).
Column dimensions Efficiency increases as ratio length / width increases.
Non uniform packing results in irregular movement
Uniformity of packing of solutes through column & less uniform zone
formation, (i.e. band broadning or tailing).
Increase in column temperature results in speed of
Column temperature
elution but does not improve separation (tailing).
Solvents should be of low viscosity (to give efficient
Eluting solvent resolution) & h igh volatility (to get rapid recovery of
the substances).
Solvent flow rate Uniform & low flow rate gives better resolution.
Continuity of flow Discontinuous flow disturbs resolution
Condition of adsorbent Deactivation of adsorbent decreases separation.
Concentration of solutes Substances of high concentration move slowly.
H = Theoretical Plate Height
L = Length of the Column.

N=L/H

As HETP decreases efficiency

of the column increases.
Adsorbents:
The most common are Alumina & Silica gel in which the
interactions with solute molecules is due to OH
groups present on their surface.
More polar molecules are adsorbed more strongly &
thus, will elute more slowly
Strength of adsorption of polar groups (solutes) on polar
support is in the following order:
-C=C- < O-CH3 < -COOR < >C = O < -CHO < -NH2 < -
OH < -COOH
Olefins < Ethers < Esters < Lactones < Aldehydes <
Amines < Phenols < Acids.
Alumina: sterols, dyestuffs, vitamins, esters,
alkaloids & inorganic compounds.
Not used for compounds containing phenolic or
carboxylic groups

Silica gel: sterols & amino acids.

Carbon: peptides, carbohydrates & amino acids.

Calcium carbonate: carotenoids & xanthophylls.

In this type, the packing consists of a
theoretically inert support material coated
with a film of the liquid stationary phase.

The division into adsorption & partition is only

of theoretical significance as in partition
chromatography the adsorption effects of the
support can be felt.
The support material should:
adsorb & retain the mobile stationary phase.
expose as large surface as possible to the mobile
phase
be mechanically stable.
be easy to pack.
not retard the solvent flow

Examples of solid supports:

Silica gel, diatomaceous earth (as kieselguhr,
celite etc.) & cellulose.
 The liquid stationary & mobile phases should
have a considerable difference between their
solvent strength parameters.

Pure water > Methanol > Ethanol > Propanol >

Acetone > Ethyl acetate> Ether > Chloroform >
Dichloromethane >Benzene > Toluene >
Carbon tetrachloride > Cyclohexane > Hexane
> Pentane.

e.g. if the stationary phase is water, pentane

would be the eluent of choice.
The mobile phase is usually saturated with the
stationary phase to overcome "stripping"
(washing of the stationary phase from the
column by the mobile phase).
This type is also known as:
Size Exclusion Chromatography (SEC)
Molecular Exclusion Chromatography (MEC)
Molecular Sieve Chromatography (MSC)
Gel Filtration Chromatography (GFC)
Gel Chromatography.
Porous polymeric matrix: formed of spongy
particles, with pores completely filled with the
liquid mobile phase (gel).

The gels (polymers) consist of open, three-

dimensional networks formed by cross-linking
of long polymeric chains.

The pore size varies with the degree of cross-

linking.

The diameter of the pores is critical as separation is

based on that molecules above certain size are
totally excluded from the pores because they
can not enter the gel.

The interior of the pores is reached, partially or

wholly, by smaller molecules.
Mobile phase
Mobile phase is a liquid as water or dilute acohol

Separation mechanism
Based on difference between the solutes molecular
weights.

Molecules will distribute themselves outside &

inside the pores according to their size.

Larger are excluded, medium sized enter half-way

& smallest permeate all the way.
 The retention volume Vo of a substance is
inversely proportional to the molecular weight
(M. Wt) of the solute.

Vo ~ 1 / M.wt
Vo = retention volume
M.wt = Molecular Weight
Determination of M. wt. of peptides, proteins & polysaccharides.

Desalting of colloids e.g. desalting of albumin prepared with 2%

(NH4)2SO4.

Separation of mixture of mono- & polysaccharides.

Separation of amino acids from peptides & proteins.

Separation of proteins of different molecular weights.

Separation of mucopolysaccharides & soluble RNA.

Separation of myoglobin & haemoglobin.

Separation of alkaloids & purification of enzymes.

Principle
Process by which ions of an electrolyte solution are brought
into contact with an ion exchange resin.

The ion exchange resin is an insoluble polymer consisting of a

"matrix" (Lattice or framework) that carries fixed charges
(not exchangeable) and mobile active ions "counter ions"
which are loosely attached to the matrix.

In water, the counter-ions move more or less freely in the

framework & can be replaced by ions of the same sign
present in the surrounding solution.

The "matrix" (framework) of a "cation exchanger" is considered

as a crystalline non-ionized "polyanion" & the matrix of an
"anion exchanger" as a non-ionized "polycation".
Active ions (counter ions) are cations.
The polar groups attached to the matrix are
acidic (sulphonic acids, carboxylic acids,
phenols, phosphoric acids) e.g. a cation
exchanger in the free carboxylic acid form:

X-COO- H+
X = Frame work (matrix)
-COO- = Fixed charge (anionic),
Non-exchangeable
H+ = Counter ion (cation), Exchangeable
They are usually (but not always) supplied in the
Na+ form: X-COO-Na+

or Na + , Where = Matrix
e.g. exchange with CaCl2 aqueous solution
+ ++
2 Na + Ca Cl2 Ca++ +2 Na+ Cl-
Active ions (counter ions) are anions.
The polar groups attached to the matrix are tertiary
or quaternary ammonium groups (basic).
e.g. Anion exchanger in the quaternary ammonium
form:
X. NR3+OH –
X = Framework (matrix)
-NR3 + = Fixed charge (cationic)
Non exchangeable
-OH– = counter ion (anion), Exchangeable
 -
or Cl (where, is the matrix)
e.g. exchange with Na2SO4 solution

They are supplied

-
as the
2 Cl + Na SO chloride
2
+2
SO + rather
-2
2 Na Cl
4 than the 4
-2 + -

hydroxide as the chloride form is a more stable.

Represented as: X. NR3+Cl -
Ion exchange process is generally reversible e.g in
the following:

2 Na+ + Ca++ 2Cl - → Ca++ + 2 Na+ Cl –

The cation exchanger could be exhausted after

exchanging all Na+ for Ca++, the exchanger could
be regenerated (loaded again with Na+) by
contacting it with excess Na+ ions e.g. a solution
of NaCl.
Ion Exchangers
These are either cation or anion exchangers of either
organic or inorganic nature.

A- Inorganic ion exchangers

Common clays, soils, minerals e.g. zeolites used for
"softening water".
Disadvantage: low ion-exchange capacity.
Advantages:
Great resistance to high temperatures.
High volume capacity.
Great selectivity towards simple inorganic ions.
They may be natural or synthetic.
Preparation of organic synthetic ion exchangers :
Polycondensation of phenols, sulpho- & carboxy-
derivatives with formaldehyde → cationic
exchangers.
Polycondensation of aromatic amines with
formaldehyde → anionic exchangers.
These techniques yield products linear in
structure & relatively soluble in water which
are now replaced by resin materials based on
styrene divinyl benzene copolymers and
polyacrylate.
1- Water softening:
Removal of Ca2+, Mg2+ & other multivalent ions
causing hardness of water by filtration through a
layer of strong cation resin.
2-Water demineralization:
Removal of cations & anions dissolved in water.
Usually carried by the two step technique in
which two columns of strongly acid cation
exchanger in [H+] form & strongly basic anion
exchanger in [OH-] form are used in sequence.
3- Neutralization:
Cationic exchanger in [H+] can be used to neutralize
alkali hydroxide & anionic exchanger in [OH+]
form to neutralize the acidity.