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 Method Compounds

• SDW05.23000’s EPA 555 Cl-PhenoxyAcids

• 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
Compounds

2,4,5-T

Benzidine

Fluorene
 Compounds

TNT (2,4,6-Trinitrotoluene)

H2C=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
CH3OH
• Acetonitrile CH3CN

• Tetrahydrofuran

• Water H2O
 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-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

 Instrumentation
Gradient
Controller
•
Pump Column
Detector
Injector
Mobile Phases
 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, 5µ),

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

A Supelcosil LC-PAH Columns. B

Conditions: A: 150mm x 4.6mm, 5µ. Conditions: B: 50mm x 4.6mm, 3µ.
Flow Rate: 1.5 mL/min Flow Rate: 3.0 mL/min