Chapter 22

GC & LC

22-1 Gas Chromatography1. Schematic diagram

22-1 Gas Chromatography2. Columns : open tubular columns

22-1 Gas Chromatography

A) m.p.(gas) - s.p. 1) 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 a) Resolution (H)b) tr

c) Sample capacity

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

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

22-1 Gas ChromatographyC) 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.

22-1 Gas Chromatographypacked column vs. open tubular column

higher resolutionlower sample capacity

22-1 Gas Chromatography

3. Temperature programming

temp of column v.p. solute,

tr

sharpens peaksisothermal : constant temp.temp. programming (gradient) : raise the column temp. during the

separation.

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.

4. Carrier Gas

22-1 Gas Chromatography

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)

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.

22-1 Gas Chromatographysplit 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.

22-1 Gas Chromatography

5. Detectors

Qualitative analysis :mass spectrometer, IR

Quantitative analysis :area of a chromatographic peak.

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.

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)

QQQ Mass Spectrometer

Precursor ion (parent ion) vs. Product ions (daughter ion)

Solid phase extraction (SPE)

Caffeine as example

Caffeine (13C) as an internal standard

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 (SiO2xH2O), alumina

(Al2O3xH2O),b. molecular exclusion,

c. ion-exchange, affinity

22-2 Liquid Chromatographycompete with ▲ for binding on s.p.

the more strongly bind to s.p.eluent strength

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.

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

22-3 High-Performance Liquid Chromatography (HPLC)

22-3 HPLC

22-3 HPLC

3. 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.

22-3 HPLCc) Bonded stationary phase.

polar vs. nonpolar

d) Optical isomersD- & L-amino acidsfor drug industry

see p.494 for R = polar or nonpolar

22-3 HPLCd) Optical isomers separation

ex: for ant-inflammatory drug Naproxen

4. Columna) Guard columnb) Injection valve

22-3 HPLC

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.

22-3 HPLC

A : KH2PO4(aq)

B: CH3CN(l)

Figure 22-20 Isocratic HPLC separation of a mixture of aromaticcompounds at 1.0 mL/min on a 0.46×25 cm Hypersil ODS column (C18 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.

22-3 HPLC

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

Detectors- Ultraviolet detector

- Electrochemical detector

redox reaction

- Fluorescence detector

LC-MS

- ESI (Electrospray ionization)

- APCI (atmospheric pressure chemical ionization)

P.500

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