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CHROMATOGRAPHY
FThe separation of a mixture by
Introduction to distribution of its components between a
mobile and stationary phase over time
Chromatography
– mobile phase = solvent
Dr. Velazquez
– stationary phase = column packing
material
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Uses for Chromatography
Classification of Methods
Real-life examples of uses for
chromatography:
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
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
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Classification based on Classification based on
Mobile Phase Attractive Forces
1. Adsorption
Liquid chromatography (LC) 2. Ion Exchange
3. Partition
4. Size Exclusion
Phase 2 Phase 2
Phase 1 Phase 1
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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
STATIONARY PHASE
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
nipis)
• 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)
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MOBILE PHASE
Type of chromatography Solvent
DEFINITION PRINCIPLE
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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
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%
Isopropanol)
• Graduated cylinder
• Filter paper
• Sampel (Different colors
of pens, plant extract)
• Pencil
• Ruler
• Scissors
• Tape
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Preparing the Chromatography
Developing the Chromatograms
Strips
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
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USES OF PAPER CHROMATOGRAPHY