Chromatography
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
Uses for Chromatography
Real-life examples of uses for
chromatography:
• Pharmaceutical Company – determine amount of
each chemical found in new product
• Hospital – detect blood or alcohol levels in a
patient’s blood stream
• Law Enforcement – to compare a sample found at
a crime scene to samples from suspects
• Environmental Agency – determine the level of
pollutants in the water supply
• Manufacturing Plant – to purify a chemical
needed to make a product
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
 Ex; Column chromatography
(CC) (K.Turus), TLC, HPLC
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
Ion Exchange Chromatography
 Use ionic stationary phase
– ions separated on the basis of their tendency to
displace counter ions adsorbed on stationary phase
(Depends on charge, hydration, “solubility”
– Used for analysis of aminoacids and its base pair.




Anionic stationary phases: used for cation separation
Cationic stationary phases : for anion separation
for ionic compounds
- Ex : CC (K.turus), HPLC
Size Exclusion Chromatography
 Separation is a result of
“trapping” of molecules in the
pores of the packing material
• Very large molecules can’t get
into the pores – unretained
• Very small molecules get hung up
in to pores for a long time - most
retained – longest retention time
• stationary phase is a porous
matrix
• Ex: CC, HPLC
Types of Chromatography
• 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)
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)
Filter paper, cellulose
Silica gel, alumina,
polyamide
Squalene, apezion,
carbowax M
C-8, C-18, Licosorb,
Silicone
MOBILE PHASE
Type of chromatography
Solvent
Paper chromatography
Air, alcohol
(KK = kertas kromatografi)
Thin Layer
Chromatography
(KLN = Kromatografi
lapisan nipis)
Hexane, ether petroleum,
alcohol.
Gas chromatography
(GC)
He, Ar, N2
High Performance Liquid
Chromatography
(KCPT = kromatografi
cecair prestasi tinggi)
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
The moving components depends on :
a. Solubility of solute in solvent
b. Intermolecule forces
c. Pore size of filter paper
d. Size of solute
At the end of process, components that are separated
will emerge to different distance on filter paper.
Rf values are used to identification of each the
component.
• The retention factor, or Rf, is defined as the distance
traveled by the compound divided by the distance
traveled by the solvent
For example, if a compound travels 2.1 cm and the
solvent front travels 2.8 cm, the Rf is 0.75:
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 solvent solution
• Prepare the solvent solution in various concentration:
- 0%, 5%, 10%, 20%, 50%, and 100%
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
USES OF PAPER CHROMATOGRAPHY
1. Separation of ink dyes
- To compare ink dyes use in any
company.
2. Food coloring
- To differentiate coloring agent
used in food product such as :
M&M, Smarties dan Reese
candies.
3. Botanist/herbalist
- To isolate plant pigment from
root and leaves