Basic Principles of HPLC

Martin R. Hackman NJ- DEP Office of Quality Assurance

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Performance
L iquid
C hromatography

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Pressure
L iquid
C hromatography

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Priced
L iquid
C hromatography

HPLC Methods
Parameter Group Method
EPA 555 EPA 605 EPA 610 SW-846 8316 SW-846 8330s SW-846 8325

Compounds
Cl-PhenoxyAcids Benzidines PAHs Acrylics Explosives Benzidines and N- Pesticides

SDW05.23000s WPP05.06000s
WPP05.13000s SHW06.26000s SHW06.28000s

SHW07.06000s

Compounds
2,4,5-T

Benzidine

Fluorene

Compounds
TNT (2,4,6-Trinitrotoluene)

H2C=CH-CN

Acrylonitrile

Carbaryl

Partitioning
Separation is based on the analytes 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 Acetonitrile Tetrahydrofuran Water H2O CH3OH CH3CN

Columns
Solid Support - Backbone for bonded phases. Usually 10, 5 or 3 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 C-8 C-18 CN Ethyl Silyl Octyl Silyl Octadecyl Silyl Cyanopropyl Silyl -Si-CH2-CH3 -Si-(CH2)7-CH3 -Si-(CH2)17-CH3 -Si-(CH2)3-CN

Instrumentation
Gradient Controller

Pump
Mobile Phases

Column 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 (250mm x 4.6mm, 5), Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.

Chromatograms

A
Flow Rate: 1.5 mL/min

Supelcosil LC-PAH Columns.

Conditions: A: 150mm x 4.6mm, 5.

Conditions: B: 50mm x 4.6mm, 3. Flow Rate: 3.0 mL/min