Basic Principles of HPLC

Martin R. Hackman
NJ- DEP
Office of Quality Assurance
H
P
igh
erformance
L
iquid
C
hromatography
H
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ressure
L
iquid
C
hromatography
H
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riced
L
iquid
C
hromatography
HPLC Methods
Parameter Group Method Compounds
SDW05.23000s EPA 555 Cl-PhenoxyAcids
WPP05.13000s EPA 610 PAHs
WPP05.06000s EPA 605 Benzidines
SHW06.26000s SW-846 8316 Acrylics
SHW06.28000s SW-846 8330s Explosives
SHW07.06000s SW-846 8325 Benzidines and
N- Pesticides
Compounds
2,4,5-T
Benzidine
Fluorene
Compounds
TNT (2,4,6-Trinitrotoluene)
H
2
C=CH-CN
Acrylonitrile
Carbaryl
Partitioning
Separation is based on the analytes relative
solubility between two liquid phases
Stationary Phase Mobile 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

CH
3
OH
Acetonitrile CH
3
CN
Tetrahydrofuran
Water
H
2
O
Columns
Analytical - Performs the separation.
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
Bonded Phases
C-2 Ethyl Silyl -Si-CH
2
-CH
3

CN Cyanopropyl Silyl -Si-(CH
2
)
3
-CN
C-18 Octadecyl Silyl -Si-(CH
2
)
17
-CH
3
C-8 Octyl Silyl -Si-(CH
2
)
7
-CH
3
Instrumentation
Pump
Injector
Column
Detector
Mobile Phases
Gradient
Controller

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.
A B
Conditions: A: 150mm x 4.6mm, 5.
Flow Rate: 1.5 mL/min
Conditions: B: 50mm x 4.6mm, 3.
Flow Rate: 3.0 mL/min