Transcript Lecture Notes 13

High Performance
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: 5mL 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
“A sampling loop for liquid chromatography”
Components
http://www.youtube.com/watch?v=mPO7Tv2mIJU
Columns
– straight, 15 to 150 cm in length; 2 to 3 mm i.d.
– packing - silica gel, alumina, Celite
Normal phase – Polar Stationary Phase and Nonpolar Solvent
Reverse phase – Nonpolar Stationary Phase and Polar Solvent
Size exclusion (gel permeation) - Small molecules penetrate
into the pores within the packing while larger molecules only
partially penetrate the pores. The large molecules elute before
the smaller molecules.
Effect of Particle Size
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.”