1. Chapter 22 GC & LC

2. 22.1 Gas Chromatography 1.Schematic diagram

3. 22.1 Gas Chromatography 2.Columns : open tubular columns

4. 22.1 Gas Chromatography A)m.p.(gas) - s.p. 1)s.p.: solid （ using adsorption ） ex: SiO 2 column ages: Si-O-H cause tailing peak. 2) s.p.: liquid （ GLC, using partition ） a range of polarities (Table 22-1), “like dissolves like” Decrease thickness of stationary phase leads to a)Resolution  (H  ) b) t r  c)Sample capacity 

6. 22.1 Gas Chromatography B) The effects of column polarity on separation Like dissolves like (a) S.P: nonpolar, b.p. dependent (b) S.P: polar

7. Figure 22-4 Resolution of trans fatty acids in hydrogenated food oil improves when the stationary phase is changed from DB-23 to HP-88 (aryl group) P.484 How changing the S.P. can affect separation

8. 22.1 Gas Chromatography C) Common solid s.p. : a) Porous carbon : larger molecules bind more tightly than small ones, flexible molecules bind more than rigid ones b) Molecular sieves : inorganic materials with nanometer-size cavities that retain & separate small molecules : H 2, O 2, N 2, CO 2, CH 4. (Fig. 22-5) c) Guard column Collect nonvolatile components that would otherwise be injected into a column and never be eluted.

9. 22.1 Gas Chromatography packed column vs. open tubular column higher resolution lower sample capacity

10. 22.1 Gas Chromatography 3.Temperature programming  temp of column   v.p. solute,   t r  sharpens peaks isothermal : constant temp. temp. programming (gradient) : raise the column temp. during the separation.

11. 22.1 Gas Chromatography -9 Figure 22-6 (a) Isothermal and (b) programmed temperature chromatography of linear alkanes through a packed column with a nonpolar stationary phase.

12. 4. Carrier Gas 22.1 Gas Chromatography

13. 5. Sample Injection 1) gasses, liquids, or solids  vaporized, not decomposition 2) injection time   bands broader 3) injected by syringe (manual or automatic injection)

14. 22.1 Gas Chromatography Figure 22-7 Injection port operation for (a) split, (b) splitless, and (c) on-column injection into an open tubular column.

15. 22.1 Gas Chromatography split injection (350 ℃ ) (only 0.1-10% sample) Routine method concentrated sample high resolution dirty samples could cause thermal decomposition splitless injection (220 ℃ ) (80%) For quantitative analysis and for analysis of trace components of mixture high resolution solvent trapping (T solvent < 40 ℃ ) for dilute sample cold trapping (T solute < 150 ℃ ) for high-boiling solutes on-column injection (50 ℃ ) (100%) best for thermally unstable solutes.

16. 22.1 Gas Chromatography 5. Detectors Qualitative analysis : mass spectrometer, IR Quantitative analysis : area of a chromatographic peak.

17. 22.1 Gas Chromatography a) Thermal conductivity detector: - most general way -responds to everything -not sensitive enough for high resolution. b) Flame ionization detector : - most popular -mainly responds hydrocarbons (C-H) c) Electron capture detector : -for compounds containing atoms with high electron affinities. -sensitive for halogen, C=O, NOx, & orgaometallic compounds.

19. 22.1 Gas Chromatography d) Mass Spectrometric Detection and Selected Reaction Monitoring : - A mass spectrometer is the single most versatile detector. - Total Ion Chromatogram (TIC) - selected ion monitoring (SIM) at on value of m/z - selected reaction monitoring (SRM) = tandem mass = MS/MS - Multiple reaction monitoring (MRM)

20. QQQ Mass Spectrometer Precursor ion (parent ion) vs. Product ions (daughter ion) Solid phase extraction (SPE)

21. Caffeine as example

22. Caffeine ( 13 C) as an internal standard

23. 22.2 Liquid Chromatography 1. open, gravity-feed column 2. closed column (under high pressure) packed with micron-size particles. (HPLC) 3. stationary phase : a. adsorption : silica (SiO 2  xH 2 O), alumina (Al 2 O 3  xH 2 O), b. molecular exclusion, c. ion-exchange,  affinity

24. 22.2 Liquid Chromatography compete with ▲ for binding on s.p. the more strongly bind to s.p.   eluent strength 

25. 22.2 Liquid Chromatography 4. Eluent strength : Table 22.2 The more polar solvent   eluent strength   t r 5. Gradient elution : increased the eluent strength during the separation in liquid chromatography.

27. 22.3 High-Performance Liquid Chromatography (HPLC)

28. 1. Through a closed column, and needs high pressure. 2. s.p. particles size  microporous particles of silica with diameters of 1.5-10 um s.p. m.p. faster, i.e. C  in van Deemter eqn.  resolution  22.3 High-Performance Liquid Chromatography (HPLC)

29. 22.3 HPLC

30. 3. Stationary phase a) Normal-phase chromatography : polar s.p. and less polar solvent. Eluent strength is increased by adding a more polar solvent. b) Reversed-phase chromatography : low- polarity s.p. and polar solvent. Eluent strength is increased by adding a less polar solvent.

31. 22.3 HPLC c) Bonded stationary phase. polar vs. nonpolar d) Optical isomers D- & L-amino acids for drug industry see p.494 for R = polar or nonpolar

32. 22.3 HPLC d) Optical isomers separation ex: for ant-inflammatory drug Naproxen

33. 4. Column a)Guard column b)Injection valve 22.3 HPLC

34. 5. Solvents a) Isocratic elution : elution with single solvent or a constant solvent mixture b) Gradient elution : solvent is changed continuously from a weak eluent strength to a strong eluent strength by mixing more and more of a strong solvent to a weak solvent during the chromatography.

35. 22.3 HPLC A : KH 2 PO 4 (aq) B: CH 3 CN (l) Figure 22-20 Isocratic HPLC separation of a mixture of aromatic compounds at 1.0 mL/min on a 0.46×25 cm Hypersil ODS column (C 18 on 5-μm silica) at ambient temperature (~22 ℃ ) ： (1)benzyl alcohol; (2)phenol; (3)3’, 4’-dimethoxyacetopheneone; (4)benzoin; (5)ethyl benzoate; (6)toluene; (7)2,6-dimethoxytoluene; (8)o-methoxybiphenyl.

36. 22.3 HPLC The gradient can be used to resolve all peaks by reducing the time from 2 h to 38 min.

37. Detectors - Ultraviolet detector - Electrochemical detector redox reaction - Fluorescence detector LC-MS - ESI (Electrospray ionization) - APCI (atmospheric pressure chemical ionization)

38. P.500 Figure 22-23 Atmospheric pressure chemical ionization interface between liquid chromatography column and mass spectrometer.