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: 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

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.