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. Easy to Change k’ (Temperature Program)

2. GC Limitations 1. Analytes must be Thermally Stable 2. Analytes must be Relatively Volatile 3. MW < 400 4. 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

3. GC Limitations 1. Analytes must be Thermally Stable 2. Analytes must be Relatively Volatile 3. MW < 400 4. Not possible to operate at biological conditions If at least one of these is a serious concern, then another technique must be employed.

4. Modern Liquid Chromatography (post 1969) HPLC High Performance Liquid Chromatography (originally High Pressure Liquid Chromatography)

5. LIQUID CHROMATOGRAPHY

6. HPLC INSTRUMENTATION 1. Mobile Phase Supply 2. Sample Injector 3. Column (Stationary Phase) 4. Detector

7. HPLC Stack Configuration

8. Mobile Phase Supply: Solvent Reservoirs

9. To Pump

10. Mobile Phase Supply: Syringe Pump Provides a constant, smooth, high pressure flow. Difficult to mix or change solvent (reservoir is inside the pump)

11. 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)

12. 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

13. 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).

15. Effect of Organic Modifiers (separation of common analgesics) Mobile phase: Water + % MeOH + 0.5% H 3 PO 4

16. Effect of pH (separation of sulfa drugs) Mobile phase: 20 mM KH 2 PO 4 : acetonitrile (95:5)

17. Separation of aromatic carboxylic acids Isocratic: constant 0.055 M sodium nitrate Gradient: 0.01 to 0.1 M sodium nitrate in 25 min

18. Separation of amino acids with pH Gradient Elution

19. Sample Injection

21. Auto-injection

22. Stop Here for now!