Chromatography

Importance

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.
 Chromatography
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.
 Thin layer chromatography (TLC)
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.
Thin Layer Chromatography (TLC)
TLC
 Preparing the Chamber

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.
 Preparing the Plates for
Development
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.
 Developing the Plates

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.
Identifying the Spots (visualization)
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.
 Visualizing Agents
Alkaloids: Dragendorff’s reagent

Cardiac glycosides: Antimony trichloride

Sugar: Aniline phthalate

Amino acids: Ninhydrin

 Interpreting the Data

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.
 Summary
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.
 Paper Chromatography
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)
 General Procedure
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.
 Techniques of development with various flow
directions
Radial development Ascending development

Descending development
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.
 Column Chromatography (CC)

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 Chromatography

Column
chromatography
Stationary phase is
held in a narrow
tube through
which the mobile
phase is forced
under pressure or
under the effect
of gravity
 Open Column Chromatography
(Traditional column chromatography)

Traditional column chromatography is

characterized by addition of mobile phase
under atmospheric pressure and the
stationary phase is packed in a glass
column.
 Packing & operating the column
1- Packing

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
 2- Sample Application

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.
 4- Detection

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…
Applications in separation of natural products
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.

 Selection of the solid support
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.
 Selection of the mobile phase

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).
Affinity Chromatography
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
 Ion Exchange Chromatography
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".
 Cation Exchangers
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-
 Anion Exchangers

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
They are supplied as the chloride rather than the
hydroxide as the chloride form is a more
stable. Represented as: X. NR3+Cl -

-
or Cl (where, is the matrix)
e.g. exchange with Na2SO4 solution
- +2 -2 -2 + -
2 Cl + Na2 SO4 SO4 + 2 Na Cl
 Regeneration of the resin

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.
 Applications of Ion Exchange
Chromatography
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.
4- Separation of electrolytes from non-
electrolytes.

5- Separation of carbohydrates & their

derivatives:
Uronic acids separated on anion exchanger.
Sugars converted into ionized form by using
borate& separated on strong anion exchanger.
Hexosamines separated on strong cation
exchanger.
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