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GC Advantages Very Large N (Very Long Columns) No Packing Material (A=0) PowerPoint PPT Presentation


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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

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GC Advantages Very Large N (Very Long Columns) No Packing Material (A=0)

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Gc advantages very large n very long columns no packing material a 0

  • 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 advantages very large n very long columns no packing material a 0

  • GC Limitations

  • Analytes must be Thermally Stable

  • Analytes must be Relatively Volatile

  • MW < 400

  • 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 advantages very large n very long columns no packing material a 0

  • GC Limitations

  • Analytes must be Thermally Stable

  • Analytes must be Relatively Volatile

  • MW < 400

  • Not possible to operate at biological

  • conditions

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


Gc advantages very large n very long columns no packing material a 0

Modern Liquid Chromatography

(post 1969)

HPLC

High Performance Liquid Chromatography

(originally High Pressure Liquid Chromatography)


Gc advantages very large n very long columns no packing material a 0

LIQUID CHROMATOGRAPHY


Gc advantages very large n very long columns no packing material a 0

HPLC INSTRUMENTATION

1. Mobile Phase Supply

2. Sample Injector

3. Column (Stationary Phase)

4. Detector


Gc advantages very large n very long columns no packing material a 0

HPLC Stack Configuration


Gc advantages very large n very long columns no packing material a 0

Mobile Phase Supply: Solvent Reservoirs


Gc advantages very large n very long columns no packing material a 0

Mobile Phase Supply: Solvent Reservoirs

To Pump


Gc advantages very large n very long columns no packing material a 0

Mobile Phase Supply: Syringe Pump

Provides a constant, smooth, high pressure flow.

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


Gc advantages very large n very long columns no packing material a 0

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)


Gc advantages very large n very long columns no packing material a 0

Mobile Phase Composition

Reversed Phase ≡Retention decreases as mobile phase

polarity decreases

Aqueous Mobile Phases

Normal Phase ≡Retention decreases as mobile phase

polarity increases

Organic Mobile Phases


Gc advantages very large n very long columns no packing material a 0

  • Advantages of Reversed Phase HPLC

  • Weak Attractive Forces

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

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

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


Gc advantages very large n very long columns no packing material a 0

Mobile phase: Water + % MeOH + 0.5% H3PO4

Effect of Organic Modifiers

(separation of common analgesics)


Gc advantages very large n very long columns no packing material a 0

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

Effect of pH

(separation of sulfa drugs)


Gc advantages very large n very long columns no packing material a 0

Isocratic:

constant 0.055 M sodium nitrate

Gradient:

0.01 to 0.1 M sodium nitrate in 25 min

Separation of aromatic carboxylic acids


Gc advantages very large n very long columns no packing material a 0

Separation of amino acids with

pH Gradient Elution


Gc advantages very large n very long columns no packing material a 0

Sample Injection


Gc advantages very large n very long columns no packing material a 0

Sample Injection


Gc advantages very large n very long columns no packing material a 0

Auto-injection


Gc advantages very large n very long columns no packing material a 0

Stop Here for now!


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