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Chapter 22

Chapter 22. GC & LC. 22.1 Gas Chromatography. Schematic diagram. 22.1 Gas Chromatography. Columns : open tubular columns. 22.1 Gas Chromatography. m.p.(gas) - s.p. s.p.: solid （ using adsorption ） ex: SiO 2 column ages: Si-O-H cause tailing peak.

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Chapter 22

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  1. Chapter 22 GC & LC

  2. 22.1 Gas Chromatography • Schematic diagram

  3. 22.1 Gas Chromatography • Columns : open tubular columns

  4. 22.1 Gas Chromatography • m.p.(gas) - s.p. • s.p.: solid（using adsorption）ex: SiO2 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 • Resolution (H) • tr • Sample capacity

  5. 22.1 Gas Chromatography Like dissolves like (a) S.P: nonpolar, b.p. dependent (b) S.P: polar B) The effects of column polarity on separation

  6. How changing the S.P. can affect separation 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

  7. 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 : H2, O2, N2, CO2, CH4. (Fig. 22-5) c) Guard column Collect nonvolatile components that would otherwise be injected into a column and never be eluted.

  8. 22.1 Gas Chromatography packed column vs. open tubular column higher resolution lower sample capacity

  9. 22.1 Gas Chromatography • Temperature programming  temp of column  v.p. solute,  tr  sharpens peaks isothermal :constant temp. temp. programming (gradient) : raise the column temp. during the separation.

  10. 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.

  11. 22.1 Gas Chromatography 4. Carrier Gas

  12. 22.1 Gas Chromatography • 5. Sample Injection • 1) gasses, liquids, or solids •  vaporized, not decomposition • 2) injection time   bands broader • 3) injected by syringe (manual or automatic injection)

  13. 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.

  14. 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 (Tsolvent < 40℃) for dilute sample cold trapping (Tsolute < 150℃) for high-boiling solutes on-column injection(50℃) (100%) best for thermally unstable solutes.

  15. 22.1 Gas Chromatography 5. Detectors Qualitative analysis :mass spectrometer, IR Quantitative analysis :area of a chromatographic peak.

  16. 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.

  17. 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)

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

  19. Caffeine as example

  20. Caffeine (13C) as an internal standard

  21. 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 (SiO2xH2O), alumina (Al2O3xH2O), b. molecular exclusion, c. ion-exchange, affinity

  22. 22.2 Liquid Chromatography compete with▲for binding on s.p. the more strongly bind to s.p.eluent strength 

  23. 22.2 Liquid Chromatography 4. Eluent strength : Table 22.2 The more polar solvent  eluent strength  tr 5. Gradient elution : increased the eluent strength during the separation in liquid chromatography.

  24. 22.3 High-Performance Liquid Chromatography (HPLC)

  25. 22.3 High-Performance Liquid Chromatography (HPLC) 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 

  26. 22.3 HPLC

  27. 22.3 HPLC 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.

  28. 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

  29. 22.3 HPLC d) Optical isomers separation ex: for ant-inflammatory drug Naproxen

  30. 22.3 HPLC • 4. Column • Guard column • Injection valve

  31. 22.3 HPLC 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.

  32. 22.3 HPLC • 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 • (C18 on 5-μm silica) at ambient • temperature (~22℃)： • benzyl alcohol; • phenol; • 3’, 4’-dimethoxyacetopheneone; • benzoin; • ethyl benzoate; • toluene; • 2,6-dimethoxytoluene; • o-methoxybiphenyl. A : KH2PO4(aq) B: CH3CN(l)

  33. 22.3 HPLC The gradient can be used to resolve all peaks by reducing the time from 2 h to 38 min.

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

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

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