GC Advantages Very Large N (Very Long Columns) No Packing Material (A=0) Simple Mobile Phase (Compressed Gas) Universal Detectors (FID) Easy to Change k’ (Temperature Program). GC Limitations Analytes must be Thermally Stable Analytes must be Relatively Volatile MW < 400
High Performance Liquid Chromatography
(originally High Pressure Liquid Chromatography)
1. Mobile Phase Supply
2. Sample Injector
3. Column (Stationary Phase)
Provides a constant, smooth, high pressure flow.
Difficult to mix or change solvent (reservoir is inside the pump)
Draws solvent(s) from an external reservoir.
Flow is not as uniform, dissolved gas can be troublesome.
Pump only works if liquid is in chamber (must be primed)
Reversed Phase ≡ Retention decreases as mobile phase
Aqueous Mobile Phases
Normal Phase ≡ Retention decreases as mobile phase
Organic Mobile Phases
Effect of Organic Modifiers
(separation of common analgesics)
Mobile phase: 20 mM KH2PO4 : acetonitrile (95:5)
Effect of pH
(separation of sulfa drugs)
constant 0.055 M sodium nitrate
0.01 to 0.1 M sodium nitrate in 25 min
Separation of aromatic carboxylic acids
pH Gradient Elution