introduction to chromatographic techniques

1. Introduction to
C hrom a toąra phic
Techniques
Chromatography is a powerful analytical technique used to separate
and identify the components of complex mixtures. It has a wide range of
applications in fields like chemistry, biology, and pharmaceuticals.
by Muzamil Nazeer

2. Affinity C hrom a toąra phy
Principle
Affinity chromatography
utilizes the highly specific
interactions between a target
molecule and its
complementary ligand
immobilized on a solid
support.
Selectivity
This technique allows for the
selective purification of
biomolecules like proteins,
enzymes, and antibodies
from complex mixtures.
Applications
Affinity chromatography is
widely used in the purification
of recombinant proteins,
isolation of enzymes, and
analysis of protein-protein
interactions.

3. Principles of Affinity
Chromatoąraphy
1 Liąa nd Im m ob iliza tion
The complementary ligand is covalently attached to a solid support, such
as agarose beads or a polymer matrix.
2 Sample Application
The sample containing the target molecule is loaded onto the affinity
column, allowing it to bind to the immobilized ligand.
3 Elution
The target molecule is then selectively eluted by changing the buffer
conditions, breaking the specific interaction with the ligand.

4. Ion Exchanąe Chromatoąraphy
Principle
Ion exchange
chromatography separates
molecules based on their
net charge, using a
charged stationary phase
and an ionic mobile phase.
Cation vs. Anion
Cation exchange resins
have negatively charged
functional groups, while
anion exchange resins
have positively charged
functional groups.
Applications
Ion exchange is used to
purify proteins, nucleic
acids, and other charged
biomolecules, as well as in
water treatment and
industrial separations.

5. Principles of Ion Exchanąe
Chromatoąraphy
1 Charąed Stationary Phase
The stationary phase is a resin with charged functional groups that can
attract and bind oppositely charged sample molecules.
2 Sample Application
The sample is loaded onto the column, and the charged molecules
interact with the stationary phase based on their net charge.
3 Elution
The bound molecules are then selectively eluted by changing the ionic
strength or pH of the mobile phase, which disrupts the ionic interactions.

6. Size Exclusion Chromatoąraphy
Principle
Size exclusion
chromatography, also known
as gel filtration, separates
molecules based on their size
and molecular weight as they
pass through a porous
stationary phase.
Separation
M ec ha nism
Larger molecules are
excluded from the pores and
elute first, while smaller
molecules can enter the
pores and are retained,
resulting in separation.
Applications
Size exclusion is used to
purify proteins, determine
molecular weights, and
remove salts or other small
molecules from samples.

7. Principles of Size Exclusion
Chromatoąraphy
1 Porous
S ta tiona ry
Phase
The stationary phase
consists of porous
beads or a gel matrix
with a defined pore size
range.
2 M olec ula r
Sieviną
Larger molecules are
excluded from the pores
and elute first, while
smaller molecules can
enter the pores and are
retained longer.
3 C a lib ra tion
Curve
By using standards of
known molecular weight,
a calibration curve can
be generated to
estimate the molecular
weights of unknown
samples.

8. Applications a n d Comparison of
C hrom a toąra phic Tec hniq ues
T
echnique Separation Basis Applications
Affinity Chromatography Specific molecular
interactions
Protein purification, enzyme
isolation, analysis of
biomolecular interactions
Ion Exchange
Chromatography
Charge differences Purification of proteins,
nucleic acids, and other
charged molecules, water
treatment
Size Exclusion
Chromatography
Molecular size/weight Protein purification,
molecular weight
determination, desalting
By understanding the principles and applications of these key chromatographic techniques,
researchers can select the most appropriate method to effectively separate and analyze their
samples.