6Gas Chromatography-4 B) The effects of column polarity on separation Like dissolves like (a) S.P: nonpolar, b.p. dependent (b) S.P: polar
7How 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
8Gas Chromatography-5 C) Common solid s.p. : a) Porous carbon : larger molecules bind more tightly than small ones, flexible molecules bind more than rigid onesb) Molecular sieves : inorganic materials with nanometer-size cavities that retain & separate small molecules : H2, O2, N2, CO2, CH4. (Fig. 22-5)c) Guard columnCollect nonvolatile components that would otherwise be injected into a column and never be eluted.
9Gas Chromatography-6 packed column vs. open tubular column higher resolutionlower sample capacity
10Gas Chromatography-7 Temperature programming temp of column v.p. solute, tr sharpens peaksisothermal : constant temp.temp. programming (gradient) :raise the column temp. during the separation.
11Gas Chromatography -8Figure 22-6 (a) Isothermal and (b) programmed temperature chromatography of linear alkanes through a packed column with a nonpolar stationary phase.
13Gas Chromatography-10 5. Sample Injection 1) gasses, liquids, or solids vaporized, not decomposition2) injection time bands broader3) injected by syringe (manual or automatic injection)
14Gas Chromatography-11Figure 22-7 Injection port operation for (a) split, (b) splitless, and (c) on-column injection into an open tubular column.
15-12Gas Chromatography split injection (350℃) (only 0.1-10% sample) Routine methodconcentrated samplehigh resolutiondirty samplescould cause thermal decompositionsplitless injection (220℃) (80%)For quantitative analysis and for analysis of trace components of mixturesolvent trapping (Tsolvent < 40℃) for dilute samplecold trapping (Tsolute < 150℃) for high-boiling soluteson-column injection (50℃) (100%)best for thermally unstable solutes.
16Gas Chromatography-13 5. Detectors Qualitative analysis : mass spectrometer, IRQuantitative analysis : area of a chromatographic peak.
17Gas Chromatography-14d) 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)
18QQQ Mass SpectrometerPrecursor ion (parent ion) vs. Product ions (daughter ion)Solid phase extraction (SPE)
28HPLC-4 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.
29HPLC-5 c) Bonded stationary phase. polar vs. nonpolar d) Optical isomersD- & L-amino acidsfor drug industrysee p.494 for R = polar or nonpolar
30HPLC-6 d) Optical isomers separation ex: for ant-inflammatory drug Naproxen
31HPLC-7 5. Solvents a) Isocratic elution : elution with single solvent or a constant solvent mixtureb) 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.
32HPLC-8 Figure 22-20 Isocratic HPLC separation of a mixture of aromatic compounds at 1.0 mL/min on a0.46×25 cm Hypersil ODS column(C18 on 5-μm silica) at ambienttemperature (~22℃)：benzyl alcohol;phenol;3’, 4’-dimethoxyacetopheneone;benzoin;ethyl benzoate;toluene;2,6-dimethoxytoluene;o-methoxybiphenyl.A : KH2PO4(aq)B: CH3CN(l)
33HPLC-9 The gradient can be used to resolve all peaks by reducing the time from 2 h to 38 min.