presentation

1. Presentation on
Presented to
Sir M. AAMIR
Presented by
BS-BIOTECHNOLOGY-4th semester
Group # 3

2. Group Members
ʘ ZAHRA NAZ BS-BT-04
ʘ FARAZ AHMAD BS-BT-11
ʘ TOOBA MAQBOOL BS-BT-20
ʘ EJAZ-ul-HASSAN BS-BT-27
ʘ BUSHRA RAFIQUE BS-BT-18
ʘ SHEEBA RAUF BS-BT-19
ʘ FAIZA SHAUKAT BS-BT-23

3. INTRODUCTION TO CHROMATOGRAPHY
by
ZAHRA NAZ BS-BT-04

4. colour
to write

5. CHROMATOGRAPHY
The physical technique for separation of a mixture
by distribution of its components between a
mobile and stationary phase over time
– mobile phase = solvent
– stationary phase = column packing material

6. Invention of chromatography
Micheal Tswett invented
the chromatography in
1901 during his research
on plant pigments.
He used the technique to
separate various plant
pigments such as
chlorophyll, xanthophylls,
Carotenoids.
Micheal Tswett
Russian botanist
1872-1972

7. Milestones in Chromatography
1903 Tswett - plant pigments separated on
chalk columns
1931 Lederer & Kuhn - LC of carotenoids
1938 TLC and ion exchange
1950 reverse phase LC
1954 Martin & Synge (Nobel Prize)
1959 Gel permeation
1965 instrumental LC (Waters)

8. Purpose of Chromatography
 Analytical - determine chemical composition of a
sample
 Preparative - purify and collect one or more
components of a sample

9. Components of Chromatography
Mobile Phase – phase that carries the
mixture of components through the
stationary phase.
E.g. gas, liquid
Stationary Phase –phase that holds the
components as they move through it &
separates them.
E.g. silica gel, aluminium oxide

10. ANALOGY…

11. Important definitions
Analyte- the substance to be separated during
chromatography
Bonded phase-a stationary phase that is
covalently bonded to the support particles
Mobile phase -the phase that moves in a
definite direction.(liquid/gas)
Stationary phase-the substance fixed in place
for the chromatography procedure

12.  Eluate -the mobile phase
leaving the column.
 Eluent -the solvent that
carries the analyte.
 Sample-the matter
analyzed in chromatography.
It may consist of a single
component or it may be a
mixture of components.

13. Chromatograph-equipment that
enables separation, e.g. gas
chromatograph.
Chromatogram-the visual
output of the chromatograph.
Detector-refers to the
instrument used for qualitative and quantitative
detection of analytes after separation.

14. Retention time
The characteristic time it takes for a particular analyte
to pass through the system (from the column inlet to
the detector) under set conditions.
Depends on:
 Length of column
 Packing material
 Type of carrier gas
 Flow rate of carrier gas
 Temperature of column

15. Retention RF VALUE
factor
It is the distance travelled by the sample or analyte
divided by distance travelled by the solvent front in
chromatography.
For substances that are very soluble in the liquid Rf will be close to 1
For substances that are rather insoluble in the liquid
Rf will be close to 0

16. Retardation factor (R)
Fraction of an analyte in the mobile phase of a
chromatographic system.

17. Mechanism of chromatography
CHROMATOGRAPHY
ADSORPTION
ELUTION
PARTITION

18. Adsorption
Surface phenomenon where interaction takes place
only on the surface of one substance.

19. Elution
The process of extracting
a substance that is
adsorbed to another by
washing it with a
solvent.

20. Partition

21. Difference b/w absorbance & adsorbance
ABSORPTION
• DIFFUSION
• One substance
penetrate into the
bulk of another
substance
ADSORPTION
• ADHESION
• Surface
phenomenon where
interaction takes
place only on the
surface of one
substance

22. Classification of Chromatography
22
Paper
HPLC Gas
Thin layer
Column

23. According to separation mode
a) Adsorption chromatography
b) Partition chromatography
c) Ion-exchange chromatography
d) Size exclusion chromatography
e) Affinity chromatography

24. According to mobile phase
a) Gas Chromatography
i- Gas solid chromatography
ii- Gas liquid chromatography
b) Liquid chromatography
i- Paper chromatography
ii- Thin-layer chromatography
iii- Column chromatography

25. According to form of stationary phase
a) Planar chromatography
i- Paper chromatography
ii- Thin-layer chromatography
b) Column chromatography
i- Gas chromatography
ii- Liquid chromatography (LC/HPLC)

27. Common Types of Chromatography
Liquid Chromatography – separates liquid samples
with a liquid solvent and a column composed of solid
beads
 Gas Chromatography – separates vaporized
samples with a carrier gas and a column composed
of a liquid or of solid beads
Paper Chromatography – separates dried liquid
samples with a liquid solvent and a paper strip
Thin-Layer Chromatography – separates dried
liquid samples with a liquid solvent and a glass plate
covered with a thin layer of alumina or silica gel

28. Uses for Chromatography
Pharmaceutical Company
Hospital
Law Enforcement
Environmental Agency
Manufacturing Plant
Separating mixtures of compounds
Identifying unknown compounds
Establishing the purity or concentration of
compounds
Monitoring product formation in the pharmaceutical
and biotechnology industries

29. Forensics
Medicine
Research Pharmaceutical industry