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15/04/2012

Liquid Chromatography
HPLC:
High Pressure Liquid Chromatography
High Performance Liquid Chromatography

As opposed to Gas Chromatography,


the mobile phase is a liquid!

? Which Species

Liquid Chromatography

Stationary Phase
Sample injection:
liquid

signal

Mobile Phase: Liquid

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:

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Chromatography
set-up for
HPLC

HPLC columns

column length 10-30 cm


columns are filled with beads linked
to stationary
phases
mobile phase fow
rate 1 mL/min
pressure 50
2000 bar !!
good fittings,
leaks can occur

expensive !

15/04/2012

Chromatography
set-up for
HPLC

Injection of
Sample:
6-wayvalve

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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

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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

Smaller particles, larger surface better


separation

How can we infuence the Separation in


RP-HPLC?
Mobile phase: A: water, B:
acetonitile

Changing the mobile phase


Increase polarity (water) increases retention

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40%
ACN

30%
ACN

35%
ACN

Concentration gradient with HPLC

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How to infuence HPLC separation?


Use of a gradient eluent
program

Mixing Pump
Gradient
Pump

Detectors for HPLC


UV-absorption
spectrometer as
HPLC detector
Detects all
aromatic
compounds

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Detectors for HPLC: MS

Ionisation of a liquid:
Electrospray Ionisation (ESI)

drying gas

Charge load in droplets


due to evaporation of
solvent.
soft ionization of
molecules by adding H+
++

+++

, MH+, MH2 , MH3

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

Liquid Chromatography Summary


Mobile phase is a solvent
Variation in mobile phase
composition determines
separation
Reversed phase column most
common for separation of
organics
Sample preparation is important

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Sample Preparation for GC and


HPLC
Aim: extract your analytes into a solvent
GC: solvent lower boiling point than analytes
GC: Headspace analysis or gases
HPLC: Best solvent to extract you analytes
Important: Concentration of analyte, if
too low use small column to concentrate
Important in HPLC: Do not dirty your
column! Use guard column for sample
clean-up

Take Home
Message
Chromatography = Separation
Sample preparation injection
separation
detection

GC: capillary columns (varying


polarity) + FID, TCD or MS
detection

HPLC: reversed phase column +


UV or MS detection
Improving separation:
GC: T programming
HPLC: gradient elution