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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.
