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HPLC Principles

This document provides an overview of the basic principles of high performance liquid chromatography (HPLC). It discusses HPLC methods, partitioning, modes, common solvents, columns, bonded phases, instrumentation including detectors and chromatograms. A variety of compounds analyzed by HPLC are also listed.

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xbnisha
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59 views16 pages

HPLC Principles

This document provides an overview of the basic principles of high performance liquid chromatography (HPLC). It discusses HPLC methods, partitioning, modes, common solvents, columns, bonded phases, instrumentation including detectors and chromatograms. A variety of compounds analyzed by HPLC are also listed.

Uploaded by

xbnisha
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd

Basic Principles of HPLC

H igh

Performance

L iquid

C hromatography

H igh

Pressure

L iquid

C hromatography

HPLC Methods
Parameter Group

SDW05.23000s

Method
EPA 555

Compounds
Cl-PhenoxyAcids

WPP05.06000s

EPA 605

Benzidines

EPA 610

PAHs

WPP05.13000s
SHW06.26000s

SW-846 8316

SHW06.28000s

SHW07.06000s

SW-846 8330s

SW-846 8325
N- Pesticides

Acrylics
Explosives
Benzidines and

Compounds
2,4,5-T

Benzidine

Fluorene

Compounds
TNT (2,4,6-Trinitrotoluene)

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
C-8

Ethyl Silyl
Octyl Silyl

C-18

Octadecyl Silyl

CN
CN

Cyanopropyl Silyl

-Si-CH2-CH3
-Si-(CH2)7-CH3
-Si-(CH2)17-CH3
-Si-(CH2)3-

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

Thank you

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