CHROMATOGRAPHY

INTRODUCTION

Chromatography is used to separate

molecules based on differences in
size, mass, shape, solubility, charge,
adsorption properties.

Tswett, Russian botanist is a Father

of Chromatography.
 Chromatography is an
important laboratory
technique that enables the
separation, identification,
DEFINITION and purification of the
components of a mixture for
the purpose of qualitative and
quantitative analysis.
Principle

 Sample mixture are

separated as a result of
their differential
distribution between
stationary phase and
mobile phases.

 Partition coefficient
k=Cs/Cm
STATIONARY PHASE

Either a solid or liquid particle attached

to a glass or a metal surface on which
the components of the mixture to be
separated is adsorbed selectively.

Gel beads, thin uniform paper, silica,

glass or even liquid components are
used as a stationary phase.
MOBILE PHASE

Either a liquid or gas that is passed through a

chromatographic system where the components of the
mixture are separated at different rates by adsorbing
them to the stationary phase.

The mobile phase is the solvent that carries the mixture as

it moves down the stationary phase.

Alcohol, water, acetic acid, acetone

TYPES
Types Based on separation on Stationary Phase
 • The stationary phase is held on a solid support of
filter paper (cellulose).
• The mobile phase is a mixture of immiscible solvents
which are mixtures of water, a nonpolar solvent and
an acid or base. e.g. butanol-acetic acid-water or
phenol-water-ammonia.
Paper • Either ascending or descending type of
Chromatography chromatography can be done with the mobile phase
being applied from the bottom (ascending) or at the
top (descending).
• In ascending chromatography, the paper is placed in a
glass trough containing the solvent which ascends up
the solid support medium.
• The components of the mixture to be separated are
carried up with the solvent.
PAPER CHROMATOGRAPHY
  Ascending Chromatography
Principle
 Descending Chromatography
 1. The stationary phase is selected as a fine
quality cellulosic paper.
2. Different combinations of organic and
inorganic solvents are taken as the mobile
phase.
Procedure 3. About 2-200 µl of the sample solution is
injected at the baseline of the paper, and it is
allowed to air dry.
4. The sample loaded paper is then dipped into
the mobile phase not more than the height of
1 cm.
5.After the mobile phase reaches near the edge of the
paper, the paper is taken out.
6.The retention factor is calculated, and the separated
components are detected by different techniques.
 Paper
Chromatogra
phy.
Analysis
• SOLVENT FRONT (SF)- The
distance to which solvent has
moved on the filter paper along
with relative mobility of different
compounds.

• RF(RATIO OF FRONTS)- Ratio of

distance moved by a compound
to the distanced moved by the
solvent front.
 • The spots may be identified by the Rf
value of the unknown substance and
compared with those of pure standards
(Rf = ratio of fronts).
• It is the ratio of the distance travelled by
The Rf Value the substance/compound (solute) to the
distance travelled by the solvent.
• The Rf value of a specific compound is
a constant for a particular solvent
system at a given temperature.
• ADVANTAGES • DISADVANTAGES
1. Very easy, simple , rapid and highly 1. Complex mixture cannot be
efficient method of separation. separated
2. Can be applied even in micrograms 2. Less accurate compared to HPLC.
quantities of the sample.
3. Used for identification of type of
aminoaciduria . Eg- cystinuria,
phenylketonuria.
Thin Layer Chromatography
• This is another version of liquid–liquid
chromatography.
• A thin layer of silica gel (Kieselguhr) is spread on
a glass plate. Biological sample is applied as a
small spot.
• The plate is placed in a trough containing the
solvent.
• The stationary water phase is held on the silica
gel and mobile phase of non-polar solvent moves
upwards.
• In the case of paper chromatography, it takes 14–
16 hours for separation of components to be
separated.
• But in the case of thin layer chromatography
(TLC), it takes only 2–4 hours.
 THIN LAYER
CHROMATOGRAPHY
PRINCIPLE

A Mobile Phase consisting of the

Stationary Phase consists of a thin solution to be separated which is
layer of adsorbent material, dissolved in an appropriate solvent
usually silica gel , aluminium and is drawn up the plate via
oxide, or cellulose immobilized capillary action , separating the
onto a flat carrier sheet. solution based on the polarity of
the compound.
 The stationary phase is uniformly applied on the
solid support (glass, thin plate or aluminum foil)
and dried.

The sample is injected as spots on the

stationary phase about 1 cm above the edge of
the plate.
PROCEDURE
The sample loaded plate is then carefully
dipped into the mobile phase not more than the
height of 1 cm.

After the mobile phase reaches near the edge of

the plate, the plate is taken out.
ANALYSIS
 1. Arginine
2. Methionine
3. Cystine
4. Leucine
5. Tyrosine
6. Lysine
7. Alanine
8. Glycine

Thin layer chromatography (TLC) separation of amino acids on silica gel.

Solvent medium: Butanol / Acetic acid / Water; 8/2/2 (v/v)
Location reagent: Ninhydrin
  Paper Chromatography, takes 18-20 hrs
for the separation of the components, but
in TLC , it takes only 3-4 hrs.
 Corrosive reagents like sulphuric acid
Advantages of can also be used which pose a limitation
TLC over Paper for the paper chromatography.
Chromatography  It is easier to separate and visualise the
components.
 Analyse multiple samples in a single run.
 Relatively a Cheaper.
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)

 Instead of a solvent (mobile phase)

being allowed to drip through the
column under gravity, it is forced
through under high pressure.

 Pressure of about 1000-3000 psi are

frequently used in routine separations.
PROCEDURE
• The column is prepared by
taking a glass tube that is dried
and coated with a thin, uniform
layer of stationary phase
(cellulose, silica).

• Then the sample is prepared by

adding the mixture to the mobile
phase. The sample is introduced
into the column from the top,
and a high-pressure pump is
used to pass the sample at a
constant rate.
• The mobile phase then moves down to a detector that
detects molecules at a certain absorbance wavelength.
Types of HPLC

1. NORMAL PHASE
2. REVERSE PHASE
3. ION EXCHANGE
4. SIZE EXCLUSION
Applications

1. HPLC used in protein separation like insulin

purification, plasma fractionation , enzyme
purification.
2. Used to separate and determine haemoglobin
A2, haemoglobin F,D,S,C & E and
glycosylated haemoglobin.
MECHANISM OF
SEPARATION
1. ADSORPTION
CHROMATOGRAPHY
2. PARTITION
CHROMATOGRAPHY
3. ION EXCHANGE
CHROMATOGRAPHY
4. SIZE EXCLUSION
CHROMATOGRAPHY
5. AFFINITY
CHROMATOGRAPHY
They are the physico-chemical
reactions involved in separation
of compounds
 Adsorption
Chromatography
• The separation is based on differences in adsorption at the
surface of a solid stationary medium.
• Commonly adsorbing substances used are alumina, or silica gel.
• These are packed into columns and the mixture of proteins to be separated is
applied in a solvent on the top of the column.
• The components get adsorbed on the column of adsorbent with different
affinity.
• The fractions slowly move down.
• The most weakly held fraction moves the fastest.
• Followed by others, according to the order of tightness in adsorption. The
eluent from the column is collected as small equal fractions and the
concentration of each is measured.
•Adsorption Chromatography.
Ion Exchange Chromatography

• This method of separation is based on electrostatic

attraction between charged biological molecules to
oppositely charged groups on the ion exchange resins.
• The resins are either cation exchange resins or anion
exchange resins.
• The separation is based on the ionic character of
proteins and amino acids(isoelectric point).
The cation exchange particles carry acid groups, e.g.
COO- , Na+.

When cations (C+) are passed through the column, Na+

in the resin is replaced by C+

Thus C+ particles are adhered in the column, while

negatively charged particles are eluted out easily.
  Used in the separation and
purification of proteins,
peptides, nucleic acids,
polynucleotides and other
charged molecules, mainly
because of its high resolving
power and high capacity.
Applications
 Used to separate DNA from
cell extract.

 Separation and removal of

inorganic ions from aqueous
mixtures
 Gel Filtration (Size Exclusion)
Chromatography
• It is also called molecular sieving.
• Hydrophilic cross-linked gels like acrylamide (Sephacryl), agarose (Sepharose),
and dextran (Sephadex) are used for separation of molecules based on their size.
The gel is packed in a column. The gel particles are porous in nature.
• These pores will allow small molecules enter the gel.
• But larger molecules could not enter pores of the gel and so are excluded.
• Suppose a mixture of insulin plus immunoglobulin is passed through the
column. The small molecule (insulin) has to travel a long distance inside the gels.
Thus, small molecules are held back.
• But the large immunoglobulin molecules cannot enter the pores; so they move in
the column rapidly.
• To Summarize, larger molecules will come out first, while smaller molecules
are retained in the column.
Sephadex (gel filtration) chromatography. (A) Protein solution is added
on the top of the column. (B) Small proteins get inside the beads, and so
takes a longer time to reach the bottom. (C) Larger molecules cannot
enter into the beads, so travels quickly, and reaches the bottom faster.
Applications
1. Fractionation of molecules and
complexes within a predetermined
size range.
2. Size analysis and determination.
3. Removal of large proteins and
complexes.
4. Buffer exchange.
Applications
• Desalting
• Removal of small molecules
such as nucleotides, primers,
dyes and contaminants
• Assessment of sample purity.
• Separation of bound and
unbound radioisotopes.
 • The technique is based on the high
affinity of specific proteins towards
specific chemical groups.
• Thus, coenzymes can be used to
Affinity purify the enzymes.
• For example, NAD (nicotinamide
Chromatography adenine dinucleotide)is used to purify
dehydrogenase enzymes.
• By using antibodies, antigens could be
easily separated.
• Conversely, antibodies can be purified
by passing through a column
containing the antigen.
 Affinity
Chromatography
Gas–Liquid Chromatography
•GLC is another type of partition chromatography
where the stationary phase is a liquid, and the
mobile phase is gas.
• The stationary liquid phase is supported by a
column of inert material such as silica in a long
narrow column.
•The mixture of substances to be separated is
made volatile at one end of the column and the
vapours are swept over the column by an inert
carrier gas like helium, argon or nitrogen.
•The fractions emerging from the column are
detected and quantitated by a detecting device.
•This is more suitable for compounds (e.g. lipids)
which resist degradation at high temperature.
Gas–Liquid
Chromatography
Applications

GC is very Can measure

picomoles of a Used
accurate. substance in a 1ml extensively in
liquid sample or forensic science
parts per billion to identify and
concentration in quantify crime
gaseous samples. scene evidence.
 Advantages of Chromatography

Components from
Simplest method Can be controlled
a complex mixture
for the separation. by a single person.
can separated.

The small amount It is a rapid and Continuous

of sample can be precise method of operation possible
detected. separation. on a large scale.
 Disadvantages of
Chromatography
• The chromatography equipment can only be
operated by a trained person.
• Chromatography instruments are expensive.
• An error can occur due to the overloading of the
samples.
• Some of the chromatography techniques require
more solvent to separate analytes.
• Periodic maintenance and parts need to be
changed.
• Some of the chromatography methods require high
power consumption.
• High operational pressure may be required to
achieve efficient separation.
 ADVANCES IN  Liquid chromatography (LC) coupled with
LIQUID mass spectrometry (MS) is a powerful bio-
CHROMATOGRAPHY analytical technique for the analysis of nucleic
acids (genomics), amino acids, peptides and
proteins (proteomics), metabolites such as
carbohydrates (metabolomics) and lipids
(lipidomics)
ADVANCES IN HPLC

Recent focus of HPLC developments are column switching, multi-

dimensional chromatography and miniaturized systems.

There are eight advanced modes of HPLC for analysis and

purification of proteins, including HP-SEC, HP-IEX, RP-HPLC,
HPIMAC, etc.
ADVANCES IN HPLC

Currently, several types of mass spectrometry systems are available

in the market, e.g. Q, IT, TOF, Orbitrap, etc.

There are mainly two types of atmospheric pressure ionization

interfaces- ESI and APCI.

MS based quantitative proteomics for biomarker discovery has

seen lot of development in past two decades.
Thank you