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Basic Principles of HPLC :Basic Principles of HPLC Martin R. Hackman
NJ- DEP
Office of Quality Assurance
H :H P igh erformance L iquid C hromatography
H :H P igh ressure L iquid C hromatography
H :H P igh riced L iquid C hromatography
HPLC Methods :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 :Compounds 2,4,5-T Benzidine Fluorene
Compounds :Compounds TNT (2,4,6-Trinitrotoluene) H2C=CH-CN Acrylonitrile Carbaryl
Partitioning :Partitioning Separation is based on the analyte’s relative solubility between two liquid phases Stationary Phase Mobile Phase Solvent Bonded Phase
HPLC - Modes :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 :Common Reverse Phase Solvents Methanol CH3OH • Acetonitrile CH3CN • Tetrahydrofuran • Water H2O
Columns :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 :Bonded Phases C-2 Ethyl Silyl -Si-CH2-CH3 CN Cyanopropyl Silyl -Si-(CH2)3-CN C-18 Octadecyl Silyl -Si-(CH2)17-CH3 C-8 Octyl Silyl -Si-(CH2)7-CH3
Instrumentation :Instrumentation Pump Injector Column Detector Mobile Phases Gradient Controller •
Detectors :Detectors UV
Single wavelength (filter) [610, 8330]
Variable wavelength (monochromator) [8316, 8325]
Multiple wavelengths (PDA) [555]
Fluorescence [610]
Electrochemical [605]
Mass Spectrometric [8325]
Chromatograms :Chromatograms Restek® ULTRA C-18 and CN Columns (250mm x 4.6mm, 5µ),
Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.
Chromatograms :Chromatograms Supelcosil LC-PAH Columns. A B 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