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Liquid Chromatography
HPLC:
High Pressure Liquid Chromatography
High Performance Liquid Chromatography
? Which Species
Liquid Chromatography
Stationary Phase
Sample injection:
liquid
signal
Compound Separation:
time
Sample is always
equilibria
Repeated repartition
Use of
HPLC:
- Separation of various
compounds dissolved in liquid,
- Great variety of analytes: non-polar
(organic), polar (fatty
acids,..), anions,
cations..
- Separation of thermally labile
compounds
Chromatogram
Detection:
15/04/2012
Chromatography
set-up for
HPLC
HPLC columns
expensive !
15/04/2012
Chromatography
set-up for
HPLC
Injection of
Sample:
6-wayvalve
15/04/2012
http://www.restek.com/info_sixport.asp
http://www.vici.com/support/app/app11j
.php
Stationary phases of LC
Polarity
of
compound
s!
Normal-phase chromatography:
Polar stationary phase and less polar solvent
e.g., R = (CH2)3CN, mobile phase hexane
Reverse-phase chromatography
Low polarity stationary phase and polar solvent
e.g, R = (CH2)17-CH3 (C18), mobile phase water/acetonitrile
15/04/2012
Stationary phases of LC
Stationary Phase
mainly
RP (reverse
phase)
SiO2 backbone (polar)
R = C18:
C CH
reversed
polar
phase
(nonpolar),
most common
CH3
H2
H2O/MeOH
3
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Column: Infuence of
Particle Size
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40%
ACN
30%
ACN
35%
ACN
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Mixing Pump
Gradient
Pump
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Ionisation of a liquid:
Electrospray Ionisation (ESI)
drying gas
+++
Information: pseudo
molecular mass (M+H+)
droplets
15/04/2012
John Fenn
Nobel P. 2002
Determination of
biomolecules
such as proteins
and peptides
Electrospray : wings
for molecular
elephants
15/04/2012
Take Home
Message
Chromatography = Separation
Sample preparation injection
separation
detection