Chromatography
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. Adsorption 2. Ion Exchange 3. Partition 4. 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
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 1
Phase 2
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 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
• 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)
Types of Chromatography
STATIONARY PHASE
Type of chromatography Material
Paper chromatography
(KK = kertas kromatografi)
Filter paper, cellulose
Thin Layer Chromatography
(KLN = Kromatografi lapisan nipis)
Silica gel, alumina, polyamide
Gas chromatography
(GC)
Squalene, apezion, carbowax M
High Performance Liquid Chromatography
(KCPT = kromatografi cecair prestasi tinggi)
C-8, C-18, Licosorb, Silicone
MOBILE PHASE
Type of chromatography Solvent
Paper chromatography
(KK = kertas kromatografi)
Air, alcohol
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