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FThe separation of a mixture by
Introduction to distribution of its components between a
mobile and stationary phase over time
–  mobile phase = solvent
Dr. Velazquez
–  stationary phase = column packing

HISTORY Purpose of Chromatography

•  Chromatography
(from Greek :chromatos -- color ,
•  Analytical - determine
"graphein" -- to write) chemical composition of a
•  1903 Tswett - plant pigments separated on sample
chalk columns
•  1931 Lederer & Kuhn - LC of carotenoids
•  1938 TLC and ion exchange •  Preparative - purify and
•  1950 Reverse phase LC collect one or more
•  1954 Martin & Synge (Nobel Prize) components of a sample
•  1959 Gel permeation
•  1965 instrumental LC (Waters)

Uses for Chromatography
Classification of Methods
Real-life examples of uses for
There are two classification
•  Pharmaceutical Company – determine amount of schemes:
each chemical found in new product
•  Hospital – detect blood or alcohol levels in a – mobile phase
patient’s blood stream
– attractive forces
•  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 based on
Mobile Phase
Mobile Phase
•  Gas (GC)
•  Water (LC) Gas Chromatography
•  Organic solvent (LC)
•  Supercritical fluid (SCFC)
Gas - solid Gas - liquid
Stationary Phase

Classification based on Classification based on
Mobile Phase Attractive Forces
1.  Adsorption
Liquid chromatography (LC) 2.  Ion Exchange
3.  Partition
4.  Size Exclusion

Column Thin layer

High performance
(gravity flow) (adsorption)
(pressure flow)

Adsorption Partition Chromatography

Chromatography Ø  solute are separated based on their partition
between a liquid mobile phase and a liquid
Ø  Separation based on their stationary phase coated on a solid support.
adsorption onto the surface of
solid (stationary phase). –  Normal – analyte is nonpolar organic;
Ø  Normal phase-like separation stationary phase MORE polar than the
mobile phase
– Nonpolar mobile phase
Ø  for polar non-ionic compounds –  Reverse – analyte is polar organic;
Ø  Ex; Column chromatography stationary phase LESS polar than the
(CC) (K.Turus), TLC, HPLC mobile phase
–  Ex : TLC, Paper Chromatography

Phase 2 Phase 2

Phase 1 Phase 1

Size Exclusion Chromatography
Ion Exchange Chromatography
Ø  Use ionic stationary phase
Ø  Separation is a result of “trapping”
–  ions separated on the basis of their tendency to of molecules in the pores of the
displace counter ions adsorbed on stationary phase packing material
(Depends on charge, hydration, “solubility”…)
•  Very large molecules can’t get into
the pores – unretained
Ø  Anionic stationary phases: used for cation separation
•  Very small molecules get hung up in
Ø  Cationic stationary phases : for anion separation
to pores for a long time - most
Ø  for ionic compounds retained – longest retention time
Ø  - Ex : CC (K.turus), HPLC
•  stationary phase is a porous matrix
•  Ex: CC, HPLC

Types of Chromatography
•  Liquid Chromatography – separates liquid samples Type of chromatography Material
with a liquid solvent (mobile phase) and a column
composed of solid beads (stationary phase) Paper chromatography Filter paper, cellulose
•  Gas Chromatography – separates vaporized samples (KK = kertas kromatografi)
with a carrier gas (mobile phase) and a column
Thin Layer Chromatography Silica gel, alumina,
composed of a liquid or of solid beads (stationary
phase) (KLN = Kromatografi lapisan polyamide
•  Paper Chromatography – separates dried liquid
samples with a liquid solvent (mobile phase) and a Gas chromatography Squalene, apezion,
paper strip (stationary phase) (GC) carbowax M
•  Thin-Layer Chromatography – separates dried liquid High Performance Liquid C-8, C-18, Licosorb,
samples with a liquid solvent (mobile phase) and a glass Chromatography Silicone
plate covered with a thin layer of alumina or silica gel
(KCPT = kromatografi cecair
(stationary phase)
prestasi tinggi)

Type of chromatography Solvent

Paper chromatography Air, alcohol

Thin Layer
Hexane, ether petroleum,
Gas chromatography He, Ar, N2
High Performance Liquid Cyclohexane, n-hexane,
Chromatography carbon tetrachloride,
(HPLC) ethanol, methanol, air


•  The certain solvent are used to separate a

A chromatographic analytical mixture ex: water, alcohol.
separation technique for
complex mixtures involving •  With capillary action the solvent will move up
the progressive adsorption of to filter paper.
the dissolved component onto
a special grade of paper. •  Movement of a solvent will bring together
component that are separated from the

•  Every component that are separated will move

to several velocity

The moving components are depend on : •  The retention factor, or Rf, is defined as the distance
a. Solubility solute in solvent traveled by the compound divided by the distance
b. Intermolecule forces traveled by the solvent
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 For example, if a compound travels 2.1 cm and the
paper. solvent front travels 2.8 cm, the Rf is 0.75:

Rf values are used to identification of each the


Materials List
Preparing the solvent solution
•  Beakers or jars
•  Covers or lids •  Prepare the solvent solution in various concentration:
•  Solvent (Distilled H2O, - 0%, 5%, 10%, 20%, 50%, and 100%
•  Graduated cylinder
•  Filter paper
•  Sampel (Different colors
of pens, plant extract)
•  Pencil
•  Ruler
•  Scissors
•  Tape

Preparing the Chromatography
Developing the Chromatograms
1.  Cut filter paper 1.  Place the strips in the beakers
2.  Make sure the solution does not
2.  Draw a line 1 cm above come above your start line
the bottom edge of 3.  Keep the beakers covered
the strip with the 4.  Let strips develop until the
pencil ascending solution front is
about 2 cm from the top of the
3.  Label each strip with strip
its corresponding 5.  Remove the strips and let them
solution dry

4.  Place a spot from each

pen on your starting

Developing the Chromatograms

Spot Detection

-  Color spot è observed by naked eye

-  Non – color spot è color reagent will give
specific colors for different compound.
Example :
Ø  Ninhydrin – a.amino
Ø  Iodin dalam etanol –bes (termasuk
Ø  AgNO3 berammonia - Karbohidrat


1.  Separation of ink dyes

- To compare ink dyes use in any
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