Title: Basic Principles of HPLC
1Basic Principles of HPLC
- Martin R. Hackman
- NJ- DEP
- Office of Quality Assurance
2H
igh
erformance
L
iquid
C
hromatography
3H
igh
ressure
L
iquid
C
hromatography
4H
igh
riced
L
iquid
C
hromatography
5HPLC Methods
- Parameter Group Method Compounds
- SDW05.23000s EPA 555
Cl-PhenoxyAcids
- WPP05.06000s EPA 605
Benzidines
- WPP05.13000s EPA 610
PAHs
- SHW06.26000s SW-846 8316
Acrylics
- SHW06.28000s SW-846 8330s
Explosives
SHW07.06000s SW-846 8325
Benzidines and
N- Pesticides
6Compounds
2,4,5-T
Benzidine
Fluorene
7Compounds
TNT (2,4,6-Trinitrotoluene)
H2CCH-CN
Acrylonitrile
Carbaryl
8Partitioning
- Separation is based on the analytes relative
solubility between two liquid phases
Stationary Phase
Mobile Phase
Solvent
Bonded Phase
9HPLC - Modes
- Normal Phase.- Polar stationary phase and
non-polar solvent.
Reverse Phase. - Non-polar stationary phase
and a polar solvent.
10Common Reverse Phase Solvents
CH3OH
Acetonitrile
CH3CN
Tetrahydrofuran
Water
H2O
11Columns
- Solid Support - Backbone for bonded phases.
- Usually 10µ, 5µ or 3µ 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.
12Bonded Phases
- C-2 Ethyl Silyl -Si-CH2-CH3
- C-8 Octyl Silyl -Si-(CH2)7-CH3
- C-18 Octadecyl Silyl -Si-(CH2)17-CH3
- CN Cyanopropyl Silyl -Si-(CH2)3-CN
13Instrumentation
Gradient Controller
Column
Pump
Detector
Injector
Mobile Phases
14Detectors
- UV
- Single wavelength (filter) 610, 8330
- Variable wavelength (monochromator) 8316, 8325
- Multiple wavelengths (PDA) 555
- Fluorescence 610
- Electrochemical 605
- Mass Spectrometric 8325
15Chromatograms
Restek ULTRA C-18 and CN Columns (250mm x 4.6mm,
5µ), Mobile Phase (11 MethanolWater), 1.5
mL/min.
16Chromatograms
A
B
Supelcosil LC-PAH Columns.
Conditions A 150mm x 4.6mm, 5µ. Flow Rate 1.5
mL/min
Conditions B 50mm x 4.6mm, 3µ. Flow Rate 3.0
mL/min
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