Basic Principles of HPLC Martin R Hackman NJ

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

H igh Performance L iquid C hromatography

H igh Pressure L iquid C hromatography

H igh Priced L iquid C hromatography

HPLC Methods • Parameter Group • SDW 05. 23000’s • WPP 05. 06000’s Method Compounds EPA 555 Cl-Phenoxy. Acids EPA 605 Benzidines • • WPP 05. 13000’s EPA 610 PAHs SHW 06. 26000’s SW-846 8316 Acrylics • SHW 06. 28000’s SW-846 8330’s Explosives SW-846 8325 Benzidines and N- Pesticides • SHW 07. 06000’s

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 analyte’s relative solubility between two liquid phases Mobile Phase Stationary 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 • 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.

Bonded Phases • C-2 Ethyl Silyl -Si-CH 2 -CH 3 • C-8 Octyl Silyl -Si-(CH 2)7 -CH 3 • C-18 Octadecyl Silyl -Si-(CH 2)17 -CH 3 • CN Cyanopropyl Silyl -Si-(CH 2)3 -CN

Instrumentation Gradient Controller • Column Pump Mobile Phases Injector Detector

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 (250 mm x 4. 6 mm, 5µ), Mobile Phase: (1: 1 Methanol: Water), 1. 5 m. L/min.

Chromatograms A Supelcosil LC-PAH Columns. B Conditions: A: 150 mm x 4. 6 mm, 5µ. Conditions: B: 50 mm x 4. 6 mm, 3µ. Flow Rate: 1. 5 m. L/min Flow Rate: 3. 0 m. L/min