GC & LC

Gas Chromatography-1Schematic diagram

Gas Chromatography-2Columns : open tubular columns

Gas Chromatography-3m.p.(gas) - s.p. s.p.: solidusing adsorptionex: SiO2column ages: Si-O-H cause tailing peak.2) s.p.: liquidGLC, using partition a range of polarities (Table 22-1), like dissolves likeDecrease thickness of stationary phase leads to Resolution (H) tr Sample capacity

Gas Chromatography-4 B) The effects of column polarity on separationLike dissolves like (a) S.P: nonpolar, b.p. dependent (b) S.P: polar

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.484How changing the S.P. can affect separation

Gas Chromatography-5C) 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.

Gas Chromatography-6packed column vs. open tubular columnhigher resolutionlower sample capacity

Gas Chromatography-7Temperature programming

temp of column v.p. solute, tr sharpens peaksisothermal : constant temp.temp. programming (gradient) : raise the column temp. during the separation.

Gas Chromatography -8Figure 22-6 (a) Isothermal and (b) programmed temperature chromatography of linear alkanes through a packed column with a nonpolar stationary phase.

4. Carrier GasGas Chromatography-9

Gas Chromatography-105. Sample Injection1) gasses, liquids, or solids vaporized, not decomposition2) injection time bands broader3) injected by syringe (manual or automatic injection)

Gas Chromatography-11Figure 22-7 Injection port operation for (a) split, (b) splitless, and (c) on-column injection into an open tubular column.

-12Gas Chromatographysplit 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 mixturehigh resolutionsolvent trapping (Tsolvent < 40) for dilute samplecold trapping (Tsolute < 150) for high-boiling soluteson-column injection (50) (100%)best for thermally unstable solutes.

Gas Chromatography-135. DetectorsQualitative analysis : mass spectrometer, IRQuantitative analysis : area of a chromatographic peak.

Gas Chromatography-14 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)

QQQ Mass SpectrometerPrecursor ion (parent ion) vs. Product ions (daughter ion)Solid phase extraction (SPE)

Caffeine as example

Caffeine (13C) as an internal standard

Liquid Chromatography-11. open, gravity-feed column 2. closed column (under high pressure) packed with micron-size particles. (HPLC) 3. stationary phase : a. adsorption : silica (SiO2xH2O), alumina (Al2O3xH2O),b. molecular exclusion, c. ion-exchange, affinity

Liquid Chromatography-2compete with for binding on s.p. the more strongly bind to s.p. eluent strength

Liquid Chromatography-3 4. Eluent strength : Table 22.2The more polar solvent eluent strength tr5. Gradient elution : increased the eluent strength during the separation in liquid chromatography.

High-Performance Liquid Chromatography (HPLC)-1

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

HPLC-2

HPLC-3

HPLC-43. Stationary phasea) 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.

HPLC-5c) Bonded stationary phase. polar vs. nonpolard) Optical isomersD- & L-amino acidsfor drug industrysee p.494 for R = polar or nonpolar

HPLC-6d) Optical isomers separation ex: for ant-inflammatory drug Naproxen

HPLC-75. 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.

HPLC-8A : KH2PO4(aq) B: CH3CN(l)Figure 22-20 Isocratic HPLC separation of a mixture of aromaticcompounds at 1.0 mL/min on a 0.4625 cm Hypersil ODS column (C18 on 5-m silica) at ambient temperature (~22)benzyl alcohol; phenol; 3, 4-dimethoxyacetopheneone; benzoin; ethyl benzoate;toluene; 2,6-dimethoxytoluene; o-methoxybiphenyl.

HPLC-9The gradient can be used to resolve all peaks by reducing thetime from 2 h to 38 min.