Basic Principles of HPLC
Martin R. Hackman
NJ- DEP
Office of Quality Assurance
High
Performance
L iquid
C hromatography
High
Pressure
L iquid
C hromatography
High
Priced
L iquid
C hromatography
HPLC Methods
Parameter Group
SDW05.23000s
Method
EPA 555
Compounds
Cl-PhenoxyAcids
WPP05.06000s
EPA 605
Benzidines
WPP05.13000s
EPA 610
PAHs
SHW06.26000s
SW-846 8316
Acrylics
SHW06.28000s
SW-846 8330s
Explosives
SW-846 8325
Benzidines and
N- Pesticides
SHW07.06000s
Compounds
2,4,5-T
Benzidine
Fluorene
Compounds
TNT (2,4,6-Trinitrotoluene)
H2C=CH-CN
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.
Common Reverse Phase Solvents
Methanol
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
Ethyl Silyl
-Si-CH2-CH3
C-8
Octyl Silyl
-Si-(CH2)7-CH3
Octadecyl Silyl
-Si-(CH2)17-CH3
C-18
CN
Cyanopropyl Silyl
-Si-(CH2)3-CN
Instrumentation
Gradient
Controller
Column
Pump
Mobile Phases
Injector
Detector
Detectors
UV
Single wavelength (filter) [610, 8330]
Variable wavelength (monochromator) [8316, 8325]
Multiple wavelengths (PDA) [555]
Fluorescence [610]
Electrochemical [605]
Mass Spectrometric [8325]
Chromatograms
Restek ULTRA C-18 and CN Columns (250mm x 4.6mm, 5),
Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.
Chromatograms
Supelcosil LC-PAH Columns.
Conditions: A: 150mm x 4.6mm, 5.
Conditions: B: 50mm x 4.6mm, 3.
Flow Rate: 1.5 mL/min
Flow Rate: 3.0 mL/min
