Basic Principles of HPLC

Martin R. Hackman
NJ- DEP
Office of Quality Assurance

High

Performance
L iquid
C hromatography

High

Pressure
L iquid
C hromatography

High

Priced
L iquid
C hromatography

HPLC Methods
Parameter Group

Method

SDW05.23000s

EPA 555

Compounds
Cl-PhenoxyAcids

WPP05.06000s

EPA 605

Benzidines

WPP05.13000s

EPA 610

PAHs

SHW06.26000s

SW-846 8316

Acrylics

SHW06.28000s

SW-846 8330s

Explosives

SW-846 8325

Benzidines and
N- Pesticides

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

CH3OH

Acetonitrile

CH3CN

Tetrahydrofuran
Water

H2O

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

Ethyl Silyl

-Si-CH2-CH3

C-8

Octyl Silyl

-Si-(CH2)7-CH3

Octadecyl Silyl

-Si-(CH2)17-CH3

C-18
CN

Cyanopropyl Silyl

-Si-(CH2)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 (250mm x 4.6mm, 5),

Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.

Chromatograms

Supelcosil LC-PAH Columns.

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

Conditions: B: 50mm x 4.6mm, 3.

Flow Rate: 1.5 mL/min

Flow Rate: 3.0 mL/min