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HPLC Techniques and Methods Overview

This document discusses the basic principles of high performance liquid chromatography (HPLC). HPLC is used to separate and analyze compounds based on their relative solubility between a mobile liquid phase that carries the sample through a stationary phase within the column. There are two main modes - normal phase uses a polar stationary phase and non-polar mobile phase, while reverse phase uses a non-polar stationary phase like C18 bonded silica and a polar mobile phase like water or acetonitrile. Key instrumentation includes the pump, injector, column, detector, and gradient controller. Common detectors are UV, fluorescence, electrochemical and mass spectrometry. EPA and SW-846 methods are listed that use HPLC to analyze compounds like acids, explosives

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127 views14 pages

HPLC Techniques and Methods Overview

This document discusses the basic principles of high performance liquid chromatography (HPLC). HPLC is used to separate and analyze compounds based on their relative solubility between a mobile liquid phase that carries the sample through a stationary phase within the column. There are two main modes - normal phase uses a polar stationary phase and non-polar mobile phase, while reverse phase uses a non-polar stationary phase like C18 bonded silica and a polar mobile phase like water or acetonitrile. Key instrumentation includes the pump, injector, column, detector, and gradient controller. Common detectors are UV, fluorescence, electrochemical and mass spectrometry. EPA and SW-846 methods are listed that use HPLC to analyze compounds like acids, explosives

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 PPT, PDF, TXT or read online on Scribd

Basic Principles of HPLC

High

Performance
L iquid

C hromatography

High

Pressure
L iquid

C hromatography

High

Priced

L iquid

C hromatography

HPLC Methods
Parameter Group Method

SDW05.23000s

EPA 555

Compounds
Cl-PhenoxyAcids

WPP05.06000s

EPA 605

Benzidines

WPP05.13000s

EPA 610

PAHs

SHW06.26000s

SW-846 8316

Acrylics

SHW06.28000s

SW-846 8330s

Explosives

SW-846 8325

Benzidines and
N- Pesticides

SHW07.06000s

Compounds
2,4,5-T

Benzidine

Fluorene

Compounds
TNT (2,4,6-Trinitrotoluene)

H2C=CH-CN

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
Analytical
- Performs
theof
separation.
Prolongs
the life
the analytical column

Bonded Phases
C-2
C-8
C-18
CN

Ethyl Silyl

-Si-CH2-CH3

Octyl Silyl

-Si-(CH2)7-CH3

Octadecyl Silyl

-Si-(CH2)17-CH3

Cyanopropyl Silyl

-Si-(CH2)3-CN

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]

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