2. Tahreem Khawar
Afifa Khizar
Arfa Khizar
Farah Masood
Amna Shehzadi

4. It is a physical separation method of separation in
which the components of a mixture are separated
by differences in their distribution between two
phases, one of which is stationary (stationary
phase) while the other (mobile phase) moves
through it in a definite direction. The substances
must interact with the stationary phase to be
retained and separated by it.
It is used for extraction and purification of molecules
in downstreaming processing

5. Mixture
Separate
 Analyze
• Identify
• Purify
• Quantify
Components
CHROMATOGRAPHY

6. Chromatograph: Instrument employed for a chromatography.
Eluent: Fluid entering a column.
Eluate: Fluid exiting the column.
Elution: The process of passing the mobile phase through the
column.
Flow rate: How much mobile phase passed / minute (ml/min).

8. Real-life examples of uses for chromatography:
Pharmaceutical Company
Hospital
Environmental Agency
Manufacturing Plant

9. •Liquid Chromatography – separates liquid samples with a liquid
solvent (mobile phase) and a column composed of solid beads
(stationary phase)
• Gas Chromatography – separates vaporized samples with a
carrier gas (mobile phase) and a column composed of a liquid or
of solid beads (stationary phase)
•Paper Chromatography – separates dried liquid samples with a
liquid solvent (mobile phase) and a paper strip (stationary phase)
•Thin-Layer Chromatography – separates dried liquid samples with
a liquid solvent (mobile phase) and a glass plate covered with a
thin layer of alumina or silica gel (stationary phase)

10. Size
Charge
Hydrophobicity
Charge

12. Invented by grant henry and colin.
does not require an “interaction” to achieve separation.
It is a chromatographic method in which molecules in solution
are separated by their size.
applied to large molecules or macromolecular complexes
such as proteins and industrial polymers.
Also known as “gel filtration chromatography” or “gel
permeation chromatography”.

13. The sponge-like gel beads with pores serve as molecular
sieves for separation of smaller and bigger molecules.
A solution mixture containing molecules of different sizes
(e.g. different proteins) is applied to the column and eluted.
The smaller molecules enter the gel beads through their
pores and get trapped. On the other hand, the larger
molecules cannot pass through the pores and therefore
come out first with the mobile liquid

16. advantages
 Short analysis time.
 Well defined separation.
 Narrow bands and good sensitivity.
 There is no sample loss.
 Small amount of mobile phase required.
 The flow rate can be set.
disadvantages
 Limited number of peaks that can be
resolved within the short time scale of the
GPC run.
 Filtrations must be performed before using
the instrument to prevent dust and other
particulates from ruining the columns and
interfering with the detectors.
 The molecular masses of most of the
chains will be too close for the GPC
separation to show anything more than
broad peaks

19. TLC is one of the simplest, fastest, easiest and least
expensive of several chromatographic techniques
used in qualitative and quantitative analysis to
separate organic compounds and to test the purity of
compounds.

20.  TLC is a form of liquid chromatography consisting of: A
mobile phase (developing solvent) and A stationary phase (a
plate or strip coated with a form of silica gel) Analysis is
performed on a flat surface under atmospheric pressure and
room temperature

21.  It is based on the principle of adsorption chromatography or partition
chromatography or combination of both, depending on adsorbent, its
treatment and nature of solvents employed The components with more
affinity towards stationary phase travels slower. Components with less
affinity towards stationary phase travels faster

23. TLC consists of three steps - spotting, development, and
visualization.
Spotting consists of using a micro pipet to transfer a small
amount of this dilute solution to one end of a TLC plate.
Development consists of placing the bottom of the TLC plate
into a shallow pool of a development solvent

24. Visualization The silica gel on the TLC plate is impregnated
with a fluorescent material that glows under ultraviolet (UV)
light.

25.  Separation of mixtures in microgram quantities by movement
of a solvent across a flat surface; components migrate at
different rates due to differences in solubility, adsorption, size
or charge.

27. Pharmaceutical and drugs
Clinical chemistry, forensic chemistry, and biochemistry
Cosmetology
Food analysis
Environmental analysis
Analysis of inorganic substances

28. Short analysis time
Spot can easily visualized
Adoptable the most pharmaceuticals
Low cost
Reliable and quick

30. Initally it is called salting out
bead polymers were created that are suitable for HIC
chromatographic purposes.
 The surface of the beads was modified by hydrophobic alkyl
or aryl groups.
Such media include the polysaccharide-based Butyl-
Sepharose, Octyl-Sepharose and Phenyl-Sepharose
materials that are derived from polymers that had proven to
be suitable for chromatographic separation of proteins.

32. Different basis of separation
Weaker interactions
 Less structural damage
Maintain high activity

33. Separation of substances is based on their varying strength
of interaction with hydrophobic groups attached to an
uncharged gel matrix
Hydrophobic groups on proteins are sufficiently exposed to
bind to the hydrophobic groups on the matrix.
High salt concentration promote binding of Proteins with
Ligand
Low salt concentration promotes unbinding of protein with
ligand

36. Equilibrium
Sample application and wash
Elution
Regeneration

37. HIC Resin
Protein

38. The type and density of the hydrophobic ligand on the matrix
surface
The quality of the salt used and its concentration
pH
Temperature
Additives

39. Purification of a monoclonal antibody for clinical studies of
passive immunotherapy of HIV- 1.
Purification of recombinant human Epidermal Growth Factor
(h-EGF) from yeast.
Assessment of purity and quantification of plasmid DNA in
process solutions using high-performance hydrophobic
interaction chromatograph
Separation of biomolecules
Economical