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After studying this topic student should
be able to :
1. Define chromatography
2. Explain classification of chromatography
technique
3. Explain type of phase in chromatography
4. Describe about paper chromatography
(definition, principle, method & their
application) in pharmaceutical industry.
DEFINITION
CHROMATOGRAPHY
The 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
HISTORY
Chromatography
(from Greek :chromatos -- color ,
"graphein" -- to write)
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)
Purpose of Chromatography
Analytical - determine
chemical composition of a
sample
Preparative - purify and
collect one or more
components of a sample
Classification of Methods
There are two classification
schemes:
mobile phase
attractive forces
Mobile Phase
Gas (GC)
Water (LC)
Organic solvent (LC)
Supercritical fluid
(SCFC)
Classification based on
Mobile Phase
Gas Chromatography
Gas - solid
Gas - liquid
Stationary Phase
Classification based on
Mobile Phase
Liquid chromatography (LC)
Column
(gravity flow)
High performance
(pressure flow)
Thin layer
(adsorption)
Classification based on
Attractive Forces
1.
2.
3.
4.
Adsorption
Ion Exchange
Partition
Size Exclusion
Adsorption Chromatography
Separation based on their
adsorption onto the surface of
solid (stationary phase).
Normal phase-like separation
Nonpolar mobile phase
for polar non-ionic compounds
Partition Chromatography
solute are separated based on their partition
between a liquid mobile phase and a liquid
stationary phase coated on a solid support.
Normal analyte is nonpolar organic;
stationary phase MORE polar than the
mobile phase
Reverse analyte is polar organic; stationary
phase LESS polar than the mobile phase
Ex : TLC, Paper Chromatography
Phase 2
Phase 2
Phase 1
Phase 1
Types of Chromatography
Liquid Chromatography separates liquid samples
with a liquid solvent (mobile phase) and a column
composed of solid beads (stationary phase)
phase)
paper
STATIONARY PHASE
Type of chromatography Material
Paper chromatography
(KK = kertas kromatografi)
Thin Layer Chromatography
(KLN = Kromatografi lapisan
nipis)
Gas chromatography
(GC)
High Performance Liquid
Chromatography
(KCPT = kromatografi cecair
prestasi tinggi)
MOBILE PHASE
Type of chromatography
Solvent
Paper chromatography
Air, alcohol
He, Ar, N2
Cyclohexane, n-hexane,
carbon tetrachloride, ethanol,
methanol, air
PAPER CHROMATOGRAPHY
DEFINITION
A chromatographic analytical
separation technique for
complex mixtures involving
the progressive adsorption
of the dissolved component
onto a special grade of
paper.
PRINCIPLE
The certain solvent are used to separate a
mixture ex: water, alcohol.
With capillary action the solvent will move up
to filter paper.
Movement of a solvent will bring together
component that are separated from the
mixture.
Every component that are separated will move
to several velocity
Materials List
Beakers or jars
Covers or lids
Solvent (Distilled H2O,
Isopropanol)
Graduated cylinder
Filter paper
Sampel (Different colors
of pens, plant extract)
Pencil
Ruler
Scissors
Tape
Preparing the
Chromatography Strips
1. Cut filter paper
2. Draw a line 1 cm above
the bottom edge of the
strip with the pencil
3. Label each strip with
its corresponding
solution
4. Place a spot from each
pen on your starting
line
Developing the
Chromatograms
1. Place the strips in the
beakers
2. Make sure the solution does
not come above your start
line
3. Keep the beakers covered
4. Let strips develop until the
ascending solution front is
about 2 cm from the top of
the strip
5. Remove the strips and let
them dry
Developing the
Chromatograms
Spot Detection
- Color spot observed by naked eye
- Non color spot color reagent will give
specific colors for different compound.
Example :
Ninhydrin a.amino
Iodin dalam etanol bes (termasuk
alkaloid)
AgNO3 berammonia - Karbohidrat
Botanist/herbalist
- To isolate plant pigment from
root and leaves