GC Advantages 1. Very Large N (Very Long Columns) 2. No Packing Material (A=0) 3. Simple Mobile...
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Transcript of GC Advantages 1. Very Large N (Very Long Columns) 2. No Packing Material (A=0) 3. Simple Mobile...
![Page 1: 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.](https://reader035.fdocuments.in/reader035/viewer/2022070409/56649e755503460f94b7633b/html5/thumbnails/1.jpg)
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)
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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
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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.
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Modern Liquid Chromatography
(post 1969)
HPLC
High Performance Liquid Chromatography
(originally High Pressure Liquid Chromatography)
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LIQUID CHROMATOGRAPHY
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HPLC INSTRUMENTATION
1. Mobile Phase Supply2. Sample Injector3. Column (Stationary Phase)4. Detector
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HPLC Stack Configuration
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Mobile Phase Supply: Solvent Reservoirs
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Mobile Phase Supply: Solvent Reservoirs
To Pump
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Mobile Phase Supply: Syringe Pump
Provides a constant, smooth, high pressure flow.
Difficult to mix or change solvent (reservoir is inside the pump)
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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)
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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
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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).
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Effect of Organic Modifiers(separation of common analgesics)
Mobile phase: Water + % MeOH + 0.5% H3PO4
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Effect of pH(separation of sulfa drugs)
Mobile phase: 20 mM KH2PO4 : acetonitrile (95:5)
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Separation of aromatic carboxylic acids
Isocratic:constant 0.055 M sodium nitrate
Gradient:0.01 to 0.1 M sodium nitrate in 25 min
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Separation of amino acids with pH Gradient Elution
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Sample Injection
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Sample Injection
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Auto-injection
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