Post on 06-Aug-2015
Basic Principles of HPLC
Martin R. Hackman
NJ- DEP
Office of Quality Assurance
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Chromatography
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Chromatography
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Chromatography
HPLC Methods• Parameter Group Method Compounds
• SDW05.23000’s EPA 555 Cl-PhenoxyAcids
• WPP05.13000’s EPA 610 PAHs
• WPP05.06000’s EPA 605 Benzidines
• SHW06.26000’s SW-846 8316 Acrylics
• SHW06.28000’s SW-846 8330’s Explosives
• SHW07.06000’s SW-846 8325 Benzidines and N- Pesticides
Compounds
2,4,5-T
Benzidine
Fluorene
Compounds
TNT (2,4,6-Trinitrotoluene)
H2C=CH-CN Acrylonitrile
Carbaryl
Partitioning
• Separation is based on the analyte’s relative solubility between two liquid phases
Stationary PhaseMobile 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
• Analytical - Performs the separation.
• 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
Bonded Phases
• C-2 Ethyl Silyl -Si-CH2-CH3
•CN Cyanopropyl Silyl -Si-(CH2)3-CN
• C-18C-18 Octadecyl SilylOctadecyl Silyl -Si-(CH -Si-(CH22))1717--
CHCH33
•C-8 Octyl Silyl -Si-(CH2)7-CH3
Instrumentation
Pump
Injector
ColumnDetector
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, 5µ),
Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.
Chromatograms
Supelcosil LC-PAH Columns. A BConditions: 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