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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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Presentation Transcript
slide1

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)
slide2

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
slide3

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.
slide4

Modern Liquid Chromatography

(post 1969)

HPLC

High Performance Liquid Chromatography

(originally High Pressure Liquid Chromatography)

slide6

HPLC INSTRUMENTATION

1. Mobile Phase Supply

2. Sample Injector

3. Column (Stationary Phase)

4. Detector

slide10

Mobile Phase Supply: Syringe Pump

Provides a constant, smooth, high pressure flow.

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

slide11

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)

slide12

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

slide13

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).
slide15

Mobile phase: Water + % MeOH + 0.5% H3PO4

Effect of Organic Modifiers

(separation of common analgesics)

slide16

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

Effect of pH

(separation of sulfa drugs)

slide17

Isocratic:

constant 0.055 M sodium nitrate

Gradient:

0.01 to 0.1 M sodium nitrate in 25 min

Separation of aromatic carboxylic acids

slide18

Separation of amino acids with

pH Gradient Elution

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