HPLC

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