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

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Presentation on theme: "Basic Principles of HPLC Martin R. Hackman NJ- DEP Office of Quality Assurance."— Presentation transcript:

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

2 H P igh erformance L iquid C hromatography

3 H P igh ressure L iquid C hromatography

4 H P igh riced L iquid C hromatography

5 HPLC Methods Parameter GroupMethodCompounds SDW05.23000s EPA 555 Cl-PhenoxyAcids WPP05.13000s EPA 610 PAHs WPP05.06000s EPA 605 Benzidines 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) H 2 C=CH-CN Acrylonitrile Carbaryl

8 Partitioning Separation is based on the analytes relative solubility between two liquid phases Stationary PhaseMobile Phase SolventBonded 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 MethanolCH 3 OH AcetonitrileCH 3 CN Tetrahydrofuran Water H2OH2O

11 Columns Analytical - Performs the separation. 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

12 Bonded Phases C-2 Ethyl Silyl -Si-CH 2 -CH 3 CNCyanopropyl Silyl -Si-(CH 2 ) 3 -CN C-18Octadecyl Silyl -Si-(CH 2 ) 17 -CH 3C-18Octadecyl Silyl -Si-(CH 2 ) 17 -CH 3 C-8Octyl Silyl -Si-(CH 2 ) 7 -CH 3

13 Instrumentation Pump Injector Column Detector Mobile Phases Gradient Controller

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

16 Chromatograms Supelcosil LC-PAH Columns. AB 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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