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Basic Principles of HPLC

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Basic Principles of HPLC. Martin R. Hackman. NJ- DEP. Office of Quality ... Separation is based on the analyte's relative solubility between two liquid phases ... – PowerPoint PPT presentation

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Title: Basic Principles of HPLC


1
Basic Principles of HPLC
  • Martin R. Hackman
  • NJ- DEP
  • Office of Quality Assurance

2
H
igh
  • P

erformance
L
iquid
C
hromatography
3
H
igh
  • P

ressure
L
iquid
C
hromatography
4
H
igh
  • P

riced
L
iquid
C
hromatography
5
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
6
Compounds
2,4,5-T
Benzidine
Fluorene
7
Compounds
TNT (2,4,6-Trinitrotoluene)
H2CCH-CN
Acrylonitrile
Carbaryl
8
Partitioning
  • Separation is based on the analytes relative
    solubility between two liquid phases

Stationary Phase
Mobile Phase
Solvent
Bonded Phase
9
HPLC - Modes
  • Normal Phase.- Polar stationary phase and
    non-polar solvent.

Reverse Phase. - Non-polar stationary phase
and a polar solvent.
10
Common Reverse Phase Solvents
  • Methanol

CH3OH
Acetonitrile
CH3CN
Tetrahydrofuran
Water
H2O
11
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.
12
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

13
Instrumentation
Gradient Controller

Column
Pump
Detector
Injector
Mobile Phases
14
Detectors
  • UV
  • Single wavelength (filter) 610, 8330
  • Variable wavelength (monochromator) 8316, 8325
  • Multiple wavelengths (PDA) 555
  • Fluorescence 610
  • Electrochemical 605
  • Mass Spectrometric 8325

15
Chromatograms
Restek ULTRA C-18 and CN Columns (250mm x 4.6mm,
5µ), Mobile Phase (11 MethanolWater), 1.5
mL/min.
16
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
17
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