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Basic Principles of HPLC

Basic Principles of HPLC. Martin R. Hackman NJ- DEP Office of Quality Assurance. igh. H. erformance. L. iquid. P. C. hromatography. igh. H. ressure. L. iquid. P. C. hromatography. igh. H. riced. L. iquid. P. C. hromatography. HPLC Methods.

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Basic Principles of HPLC

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  1. Basic Principles of HPLC Martin R. Hackman NJ- DEP Office of Quality Assurance

  2. igh H erformance L iquid P C hromatography

  3. igh H ressure L iquid P C hromatography

  4. igh H riced L iquid P C hromatography

  5. HPLC Methods • SDW05.23000’s EPA 555 Cl-PhenoxyAcids • Parameter Group Method Compounds • WPP05.06000’s EPA 605 Benzidines • WPP05.13000’s EPA 610 PAHs • 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

  6. Compounds 2,4,5-T Benzidine Fluorene

  7. Compounds TNT (2,4,6-Trinitrotoluene) H2C=CH-CN Acrylonitrile Carbaryl

  8. Partitioning • Separation is based on the analyte’s relative solubility between two liquid phases Mobile Phase Stationary Phase Solvent Bonded Phase

  9. HPLC - Modes • Normal Phase.- Polar stationary phase and non-polar solvent. • Reverse Phase. - Non-polar stationary phase and a polar solvent.

  10. Common Reverse Phase Solvents CH3OH • Methanol • Acetonitrile CH3CN • Tetrahydrofuran • Water H2O

  11. Columns • 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.

  12. Bonded 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

  13. Instrumentation Gradient Controller • Column Pump Detector Injector Mobile Phases

  14. Detectors • UV • Single wavelength (filter) [610, 8330] • Variable wavelength (monochromator) [8316, 8325] • Multiple wavelengths (PDA) [555] • Fluorescence [610] • Electrochemical [605] • Mass Spectrometric [8325]

  15. Chromatograms Restek® ULTRA C-18 and CN Columns (250mm x 4.6mm, 5µ), Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.

  16. Chromatograms 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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