Basic Principles of HPLC

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Basic Principles of HPLC

• Martin R. Hackman
• NJ- DEP
• Office of Quality Assurance

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H
igh

• P

erformance
L
iquid
C
hromatography
3
H
igh

• P

ressure
L
iquid
C
hromatography
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H
igh

• P

riced
L
iquid
C
hromatography
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HPLC Methods

• Parameter Group Method Compounds

• SDW05.23000s EPA 555
  Cl-PhenoxyAcids

• WPP05.06000s EPA 605
  Benzidines

• WPP05.13000s EPA 610
  PAHs

• SHW06.26000s SW-846 8316
  Acrylics

• SHW06.28000s SW-846 8330s
  Explosives

SHW07.06000s SW-846 8325
Benzidines and
N- Pesticides
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Compounds
2,4,5-T
Benzidine
Fluorene
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Compounds
TNT (2,4,6-Trinitrotoluene)
H2CCH-CN
Acrylonitrile
Carbaryl
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Partitioning

• Separation is based on the analytes relative
  solubility between two liquid phases

Stationary Phase
Mobile Phase
Solvent
Bonded Phase
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HPLC - Modes

• Normal Phase.- Polar stationary phase and
  non-polar solvent.

Reverse Phase. - Non-polar stationary phase
and a polar solvent.
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Common Reverse Phase Solvents

• Methanol

CH3OH
Acetonitrile
CH3CN
Tetrahydrofuran
Water
H2O
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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.
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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

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Instrumentation
Gradient Controller

Column
Pump
Detector
Injector
Mobile Phases
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Detectors

• UV
• Single wavelength (filter) 610, 8330
• Variable wavelength (monochromator) 8316, 8325
• Multiple wavelengths (PDA) 555
• Fluorescence 610
• Electrochemical 605
• Mass Spectrometric 8325

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Chromatograms
Restek ULTRA C-18 and CN Columns (250mm x 4.6mm,
5µ), Mobile Phase (11 MethanolWater), 1.5
mL/min.
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Chromatograms
A
B
Supelcosil LC-PAH Columns.
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
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