Topics to be studied in this semester:

1. Chromatography
2. Mass Spectrometry
3. Neutron Analysis Activity
 Scintillators
 Gas Filled Detectors
4. Coulometry and Electrogravimetry
5. Advanced Techniques of Voltammetry
6. XRD
 Basics
of Chromatography
Tswett, Russian
botanist (referred
to as Father of
Chromatography)
is credited for the
development of
chromatography.
through
CLASSIFICATION OF CHROMATOGRAPHY
 On the basis of Mechanism

Types of
Chromatography • Adsorption Chromatography
If a solution of a mixture (in a suitable solvent) is
allowed to flow down a column filled with highly
adsorbing material (like alumina and silica gel), the
most readily adsorbed substance tend to be retained
near the top and the other penetrate different
distances down the column, depending upon the
degree of attraction with which they are adsorbed.
 This type of interaction (adsorption) is a result of inter molecular forces between
surface atoms of solid (stationary phase) and molecules of the external solute
(components present in solution) and involve one or some combination of the
following forces

a) London forces between all surfaces and adsorbed molecule.

b) Electrostatic forces between polar surfaces and any absorbed molecule or between
non-polar surface and polar absorbed molecule.
c) Charge transfer forces between strong electron donor and acceptor.
d) Formation of hydrogen bonds.
These forces which lead to physical adsorption are weaker than those found for
covalently or ionically adsorbed species which lead to chemisorption and are not
suitable for chromatographic applications.
Adsorption chromatography (contd.):
Carotenoids, proteins, porphyrins, etc can be isolated from a mixed extract by
adsorption on solid particle of insoluble adsorbents like silica gel, alumina,
calcium hydroxyapatite and charcoal powder depending on the difference between
the component in their affinities for the adsorbent.
The adsorbent should be very finely divided and porous so as to offer a vast
surface area in proportion to the particle size.
On passing the mixed solution through the adsorbent column the solutes are
selectively adsorbed on the adsorbent particles with the help of ionic or hydrogen
bond in case of polar adsorbent and by hydrophobic bonds van der walls forces in
case of non-polar adsorbent.
The different components of the mixture are held at different height of the
adsorption column in the form of separate layers or bonds.
Components A and B form separate layers of
adsorbed particles.
Elution
• In order to collect separately the adsorbed components, a proper solvent (or
buffer) is passed through the column so that the individual component of the
mixture begin to emerge from the column which is collected. The process is called
elution. This process is continued till all the components of the mixture have been
collected in separate flask.

• Since the rate at which a particular component travels down the column depends
on its solubility in the given solvent and the extent of its adsorption by the column
the component that comes out first has maximum solubility in solvent and least
affinity for adsorption by column.
Partition Chromatography
It is based on the use of difference in the coefficient of distribution of different
components of the mixture being analysed between two immiscible liquids or a
liquid and a gas, one of the liquid (stationary phase, is distributed on the porous
substance and the other liquid (mobile phase) is a solvent (or gas) which is
immiscible with the stationary phase. The mobile phase is passed through the
column slowly.

Different value of distribution or partition coefficient ensure different rate of

motion and the separation of the components of the mixture.
• A cylindrical column of an insoluble, inert and hydrophilic solid like starch
or silica gel granules is saturated with water which remains immobilised
around the granules to form a stationary liquid phase.
• The mixture of solute is dissolved in a second immiscible solvent and the
solution is passed through the column. The solvent of this solution form the
mobile liquid phase.
• During the flow of mobile phase alongside the stationary phase, the solute
molecule get partitioned between the two liquid phases according to their
partition coefficients, which depends on their relative dissolution in the two
phases. This produces the separation of the solutes from each other as they
move along the column.
 𝐾𝐷

𝐾𝐷
Synthetic ion-exchange resins are also produced which are high-molecular weights
polymers that contain large number of ionic functional group per molecule. Cation-
exchange resins contain acidic groups and anions exchange resins contain basic
groups.
Cation exchanger

(solid) ( solution) (solid) ( solution)

Where Mx+ represent the cation and R represent that part of Resin molecule that
contains one sulphonic acid group.
Anion exchanger
[ RN [ + x
(solid) ( solution) (solid) ( solution)
Ion- Exchanger exchange ion on stoichiometric basis and tend to show a
well-defined selectivity. Ion-exchange chromatography uses ion-exchange
materials to separate mixtures depending on their acid-base properties
and electric charge. They are water insoluble polymers.

E.g. Cation exchangers- carboxy methyl cellulose and sulphonated

polystyrene
anion exchangers- Dimethyl aminoethyl cellulose and triethyl amino
polystyrene.
Its most important application is in separation of the lanthanides that form +3
ions. A solution containing +3 lanthanide ions is placed at the top of a cation-
exchange column and the lanthanide ions release and equivalent amount of
ions from the column. A solution of ammonium 1- hydroxy -1 methyl
propanate COO is slowly run down the column and the the cations partition
themselves between the ion- exchanger and the eluting solution (with which
they form complex).

The ionic radii of lanthanides decrease progressively with increasing atomic

number and due to this decrease in ionic radii, their tendency to form complex
ions increases as the smaller ions show a greater preference for the
complexing solution and hence smallest ions are the first to emerge out from
the column.
Exclusion Chromatography:
It is also called molecular chromatography. It includes those
chromatographic processes in which separation of the sample
components is based on their molecular size and is extensively used for
the separation of macromolecules (like proteins nucleic acids and even
viruses & ribosomal particles).
A buffered solution of a mixture is made to flow under gravity through
the column of hydrated beads of an inert and cross-linked polymerized
material like polysaccharides or polyacrylamides. Solute particles move
down the column accordingly to their sizes. Large solutes molecules flow
at a faster rate down the column because they cannot penetrate into the
beads through the small pores of the latter and consequently remain in
the mobile phase outside the beads but small molecules penetrate into
the beads through their pores and thus get considerably retarded in their
movement through the column.
The degree of retardation being inversely proportional to their particle
size. Thus, by passing suitable solvents different components can be
collected in different fraction. Thus, such column serves as molecular
sieves.

The solid supports are three- dimensional network of cross-linked

polymer chains. These gels are able to swell in particular solvent and as
the gel structure swells the spaces between the polymer chain increases in
size. For a particular gel there is a critical size of the molecule that can just
penetrate into it. So larger molecules pass unhindered through the column
while the molecules smaller in size than entitled size will be reloaded
differently according to their size.
 Electrophoresis
• Used to separate substances based on their charge to mass ratio e/m using the
effect of an electric field on the charges of these substances.
• Used for charged colloidal particles or macromolecular ions such as those of
proteins, nucleic acids and polysaccharides. Among different types of
electrophoresis zone- electrophoresis is most common.
Zone Electrophoresis
Separation of electrically charged bio-molecules under the effect of an electric
field of strength E, they will freely move towards the electrodes of opposite
charge.
• Here proteins are supported on a solid so that electric migration forces as well as
conventional chromatographic forces may contribute to the separation efficiency.
• The common supports used are starch gel, polyacrylamide gels which minimize
convection and diffusion effect.
• A block or plate of solid support is prepared, and the sample is applied in a
narrow band (line) across the block about midway between the ends which are
connected with electrodes through a connecting bridge. When current is passed
through the cell, the different components of the mixture move with velocities
that depend on their charge, size and shape.
• As electrophoresis proceeds the negatively charged ions migrates to anode
and positively charged components migrate to cathode and give rise to
series of separated bands or lines for different sample constituents.
• This technique can be used for separation of very complex mixture, for e.g.
starch gel electrophoretic separation of plasma proteins can separate 18
components in 30 minutes.
• A densitometer is used to measure the intensity of colored zones and thus
provide quantitative information about the components.
• This technique is used largely in clinical chemistry and biochemistry for
separating amino acids and proteins. These contain amino acid and
carboxylic acid group which can ionize or protonate depending on pH.
• This is also used for separation of nucleic acid in DNA sequencing.
Affinity chromatography
• It involves the covalent attachment of an immobilized biochemical
(called affinity ligand) to a solid support. When a sample is passed
through the column only the solute that selectively binds to the
ligand are retained and other elute out without retention.
• The separation exploit (lock and key binding) that is prevalent in
biological system. The retained solutes can be eluted out by
changing the mobile phase conditions.
• The affinity ligands can be antibodies, enzyme inhibitor, or other
molecules that reversibly and bioselectively bind to the
complementary analyte molecules in the sample.
• The advantage is that tremendous specificity which permits rapid
isolation giving good yield in single step.
Ligands are either specific or group specific.
Specific- bind only one particular solute. Advantage can be taken of this high selectivity
by using short column (~ 1 cm) to perform separation by step elution in less than 1 min.
Group specific ligands- bind to certain group of solutes which can then be separated by
using gradient elution technique. For this we need longer and more efficient column.
Elution:
Retained solutes are retained either by adding excess ligand to mobile phase or by adding
a compound that interfere with solute ligand interaction and such that ligand has more
affinity for the added compound then it has for the solute and thus solute will be replaced
by the compound and it will elute out.
Free ligand called inhibitor is added that has similar affinity as ligand and it competes
with the ligand for sites on the solutes. For e.g. glucosamine are used to purify a lectin
either glucose or N-acetyl-D-glucosamine might be used as an inhibitor.
Chromatogram:
If the adsorbent is colourless and the components in the solution are coloured and
on adsorption form coloured bands on column, this column with coloured bands
on it is called chromatogram.
For e.g.: If an aqueous solution of copper and cobalt salts is passed through
aluminium oxide column. We observe the formation of two differently coloured
bands.
upperdark blue Cu2+
lower pink Co2+
Thus, the presence of Cu2+ and Co2+
cations can be determined in the solution by these coloured bands.
If the components being analysed are colourless then they are seen after
illumination with U.V. Light if the substance possess the property of Fluorescence
or by treating the column with such reagent with which the adsorbed components
form coloured compound.

For e.g. if the K3[Fe (CN)6] solution is passed through the column containing the
band of Fe 2+ ion the adsorbed layer turns blue black.
Characteristics of an Ideal Detector:
1. Adequate Senstivity: In general, now a day’s detector’s sensitivity
lies in the range of ( to )g of solute.
2. Good stability & reproducibility
3. A linear response of solutes that extends over several orders of
magnitude.
4. A temperature range of room temperature to 400ᵒ C
5. A short response time which should be independent of flow rate.
6. High reliability and ease to use.
7. Similarity in response towards all solutes.
8. Should not destruct or destroy the sample.
Factors affecting the formation of Chromatogram

1. Nature and structure of the adsorbent.

2. The properties of the solvent.
3. The composition and structure of the substances being analysed.
4. The rate of motion of the solution.
5. Temperature