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
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.
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
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)
26.
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