Ch 28 - Liquid Chromatography

1. Ch 28 - Liquid Chromatography HPLC = High Performance Liquid Chromatography (lots of theoretical plates) Scope of HPLC - there are many different types of LC; we are going to concentrate on "Partition Chromatography"

3. Adsorption chromatography • stationary phase = solid (e.g. SiO2 particles that analyte adsorbs onto) • mobile phase = gas or liquid • Partition chromatography • stationary phase = thin liquid film coating inside surface of column ("capillary columns") or coats the solid support (SiO2) as with "packed columns" • mobile phase = gas or liquid • Ion-exchange chromatography • stationary phase = charged resin with covalently bound ionic groups such as -SO3- or -N(CH3)3+; electrostatically attracts ionic analytes • mobile phase = liquid • Molecular exclusion chromatography • e.g. gel filtration, gel permeation, molecular sieve • smaller molec ules trapped in pores while the larger ones elute faster • Affinity chromatography • most selective, covalently bonded antibody binds a specific protein

4. High Performance Liquid Chromatography "High-performance" because smaller solid support particle size decreases the Eddy Diffusion contribution to plate height. There's also no Longitudinal Diffusion contribution to plate height except at very low (and impractical) flow rates. Why?

5. Instrumentation

6. Mobile Phase Reservoirs and Solvent Treatment Systems "Sparging" = "isocratic elution = "gradient elution =

7. Polar Stationary Phase gradient elution: initially 40:60 methanol-water, then methanol increased 8%/min isocratic elution: 50:50 methanol-water polarity

8. Reciprocating Pumps • high pressure (1000-5000 psi) • pulse-free (eliminate "ghost peaks") • range of flow rates (0.1 - 10 mL/min) • corrosion resisitant • reproducible

9. Sample Injection Systems

10. Columns for HPLC • stainless steel tubing, 25 cm long, i.d. = 5 mm • 40,000 - 60,000 plates/meter • solid support packing size = 5 m • some systems allow column thermostatting

11. "Bonded Phase" Columns - same as in GC

12. Common Nonpolar Bonded Phases

13. Common Polar Bonded Phases

14. Detectors 1. UV-Vis - the most commonly employed wavelength is 254 nm (most intense peak from a Hg-vapor lamp)

15. 2. Refractive Index Detector - if two solutions (sample and reference) vary in refractive index, the light is bent.

16. 3. LC - MS - another "hyphenated" method similar to GC-MS

17. Normal vs. Reversed Phase Chromatography "Normal" Phase "Reversed" Phase solid support solid support polar stat. phase nonpolar stat. phase nonpolar mobile phase polar mobile phase

18. solute polarities: A > B > C

19. The Mobile Phase • The selection of the proper mobile phase for a given separation is basically a trial-and-error process. • Generally, the polarity of the analyte is matched to the column polarity as in GC. The mobile phase should have the opposite polarity. • Desirable mobile phase properties are - • high purity (HPLC grade) • cheap (some people redistill after use) • boiling point higher than the column temperature • low viscocity • low reactivity • nonflammable and nontoxic

20. Applications of HPLC