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HPLC Principles
HPLC Principles
H igh
Performance
L iquid
C hromatography
H igh
Pressure
L iquid
C hromatography
HPLC Methods
Parameter Group
SDW05.23000s
Method
EPA 555
Compounds
Cl-PhenoxyAcids
WPP05.06000s
EPA 605
Benzidines
EPA 610
PAHs
WPP05.13000s
SHW06.26000s
SW-846 8316
SHW06.28000s
SHW07.06000s
SW-846 8330s
SW-846 8325
N- Pesticides
Acrylics
Explosives
Benzidines and
Compounds
2,4,5-T
Benzidine
Fluorene
Compounds
TNT (2,4,6-Trinitrotoluene)
Acrylonitrile
Carbaryl
Partitioning
Separation is based on the analytes relative
solubility between two liquid phases
Mobile Phase
Stationary Phase
Solvent
Bonded Phase
HPLC - Modes
Normal Phase.
- Polar stationary phase and non-polar
solvent.
Reverse Phase.
- Non-polar stationary phase and a polar
solvent.
CH3OH
Acetonitrile
CH3CN
Tetrahydrofuran
Water
H2O
Columns
Solid Support - Backbone for bonded phases.
Usually 10, 5 or 3 silica or polymeric particles.
Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support.
Extremely stable
Reproducible
Guard - Protects the analytical column:
Particles
Interferences
Prolongs the life of the analytical column
Analytical - Performs the separation.
Bonded Phases
C-2
C-8
Ethyl Silyl
Octyl Silyl
C-18
Octadecyl Silyl
CN
CN
Cyanopropyl Silyl
-Si-CH2-CH3
-Si-(CH2)7-CH3
-Si-(CH2)17-CH3
-Si-(CH2)3-
Instrumentation
Gradient
Controller
Column
Pump
Mobile Phases
Injector
Detector
Detectors
UV
Single wavelength (filter) [610, 8330]
Variable wavelength (monochromator) [8316, 8325]
Chromatograms
Chromatograms
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