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Chromatography
Introduction
HPLC is a form of liquid chromatography used to
separate compounds that are dissolved in solution.
HPLC instruments consist of a reservoir of mobile
phase, a pump, an injector, a separation column, and a
detector.
Adsorption, or liquid-solid
chromatography
Concentration
Matrix
Solvent Effect
Sample Effect
Several column types
(can be classified as )
Normal phase
Reverse phase
Size exclusion
Ion exchange
Normal phase
IR Absorbance Electrochemical
Fluorescence Mass-
Spectrometric
Refractive-Index
Photo-Diode Array
Types of Detectors:
UV – Ultraviolet light
Lamp
Grating/Lens - Wave length
FlowCell
PhotoDiode - Differential Light Output
RI – Refractive Index
Universal analyte detector
Solvent must remain the same throughout separation
VERY temperature sensitive
Sometimes difficult to stabilize baseline
FD – Fluorescence
Excitation wavelength generates
fluorescence emission at a higher
wavelength
Analytes must have fluorophore group
Can react analyte with fluorophore reagent
Very sensitive and selective
More difficult methods transfer
Results very dependent upon separation
conditions
MS – Mass Spec
Mass to charge ratio (m/z)
Allows specific compound ID
Several types of ionization techniques
electrospray, atmospheric pressure
chemical ionization, electron impact