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Chromatography

Pn. Suryati Bt. Syafri


2008

LEARNING OUTCOME
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

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


patients 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)

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 cant 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
strip (stationary phase)

paper

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
Hexane, ether petroleum,
Chromatography
alcohol.
(KLN = Kromatografi
lapisan nipis)
Gas chromatography
(GC)
High Performance Liquid
Chromatography
(KCPT = kromatografi

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

The moving components are depend on :


a. Solubility 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