GC Advantages

1. Very Large N (Very Long Columns)2. No Packing Material (A=0)3. Simple Mobile Phase (Compressed Gas)4. Universal Detectors (FID)5. Easy to Change k’ (Temperature Program)

GC Limitations

1. Analytes must be Thermally Stable2. Analytes must be Relatively Volatile3. MW < 4004. Not possible to operate at biological conditions

If these limitations are not critical, then GC is probably the BEST means for analyzing a complex sample

GC Limitations

1. Analytes must be Thermally Stable2. Analytes must be Relatively Volatile3. MW < 4004. Not possible to operate at biological conditions

If at least one of these is a serious concern, then another technique must be employed.

Modern Liquid Chromatography

(post 1969)

HPLC

High Performance Liquid Chromatography

(originally High Pressure Liquid Chromatography)

LIQUID CHROMATOGRAPHY

HPLC INSTRUMENTATION

1. Mobile Phase Supply2. Sample Injector3. Column (Stationary Phase)4. Detector

HPLC Stack Configuration

Mobile Phase Supply: Solvent Reservoirs

Mobile Phase Supply: Solvent Reservoirs

To Pump

Mobile Phase Supply: Syringe Pump

Provides a constant, smooth, high pressure flow.

Difficult to mix or change solvent (reservoir is inside the pump)

Mobile Phase Supply: Reciprocating 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)

Mobile Phase Composition

Reversed Phase ≡ Retention decreases as mobile phasepolarity decreases

Aqueous Mobile Phases

Normal Phase ≡ Retention decreases as mobile phasepolarity increases

Organic Mobile Phases

Advantages of Reversed Phase HPLC

1. Weak Attractive Forces

2. Aqueous mobile phase, sometimes with added liquid organic modifiers (MeOH, Acetonitrile), dissolved salts, and/or buffers.

3. Wide scope: may separate polar, non-polar, ionizeable, and ionic compounds (perhaps at the same time).

4. Elution occurs in order of decreasing polarity (but not at predictable as GC Retention Index).

Effect of Organic Modifiers(separation of common analgesics)

Mobile phase: Water + % MeOH + 0.5% H3PO4

Effect of pH(separation of sulfa drugs)

Mobile phase: 20 mM KH2PO4 : acetonitrile (95:5)

Separation of aromatic carboxylic acids

Isocratic:constant 0.055 M sodium nitrate

Gradient:0.01 to 0.1 M sodium nitrate in 25 min

Separation of amino acids with pH Gradient Elution

Sample Injection

Sample Injection

Auto-injection

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