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HPLC
HPLC
Liquid
Chromatography
HPLC
originally refered to:
High Pressure Liquid Chromatography
currently refers to:
High Precision Liquid Chromatography
high pressure to be able to use small particle size to
allow proper separation at reasonable flow rates
Applications of
Liquid
Chromatography
Partition chromatography
Adsorption, or liquid-solid
chromatography
Ion exchange chromatography
Size exclusion, or gel,
chromatography
Components
Solvent Reservoir and Degassing
System
Pumps
Precolumns
Sample Injection System
Columns
Temperature Control
Detectors
Schematic
of an
apparatus
for
HPLC
Components
Solvent Reservoir and Degassing
System
isocratic elution - single solvent separation
technique
gradiant elution - 2 or more solvents, varied
during separation
Isocratic mixture of
A (aqueous buffer)
and B (acetontrile)
Isocratic elution:
performed with a
single solvent (or
constant solvent
mixture)
Gradient Elution
Gradient elution:
continuous
change of solvent
composition to
increase eluent
strength
Improvement in separation
efficiency by gradient
elution. Column: 1m x
2.1mm id, precision-bore
stainless; packing: 1%
PermaphaseR ODS.
Sample: 5L of chlorinated
benzenes in isopropanol.
Detector: UV photometer
(254 nm). Conditions:
temperature 60oC,
pressure, 1200 psi.
Components
Pumps
1000 - 6000 psi at rate of @ 3 mL/min
reproducible to 2%
screw driven syringe
reciprocating pump
A reciprocating
pump for HPLC.
Components
Precolumns
remove impurities from solvent
saturates mobile phase with liquid of
stationary phase before the analytical column
Components
Sample Injection System
sample valve
syringe
Components
http://www.youtube.com/watch?v=mPO7Tv2mIJU
Columns
Columns Cont.
Components
Temperature Control
Components
Detectors
single wavelength UV
tuneable UV
diode-array
refractive index
MS
Performances of LC Detectors
Ultraviolet
detector cell for
HPLC
Diode-Array
Absorption
Spectra
Absorption
spectra of the
eluent from a
mixture of
three steroids
taken at 5-second
intervals.