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Basic Principles of HPLC

This document provides an overview of high performance liquid chromatography (HPLC), including its basic principles, components, types of HPLC, popularity and advantages. It describes what HPLC is, the components used including mobile phases, pumps, injectors, columns and detectors. It also discusses reverse phase, normal phase and ion-exchange chromatography and explains why HPLC is widely used.

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shahzad1840
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2K views18 pages

Basic Principles of HPLC

This document provides an overview of high performance liquid chromatography (HPLC), including its basic principles, components, types of HPLC, popularity and advantages. It describes what HPLC is, the components used including mobile phases, pumps, injectors, columns and detectors. It also discusses reverse phase, normal phase and ion-exchange chromatography and explains why HPLC is widely used.

Uploaded by

shahzad1840
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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Basic Principles of HPLC

Shahzad Bashir
2009-ag-1231
Center for Agricultural biochemistry and biotechnology
UNIVERSITY OF AGRICULTURE
FAISLABAD
Outline
• What is HPLC?

• Types of HPLC
H igh
Performance
L iquid
C hromatography
H igh
Pressure
L iquid
C hromatography
What is HPLC?
• The most widely used analytical
separations technique
• Utilizes a liquid mobile phase to
separate components of mixture
• uses high pressure to push solvent
through the column
Components
• Mobile phase reservoirs
• HPLC Pump(s)
• Sample injector (manual or auto)
• Column
• Detector
• Mobile phase waste container
Mobile phases
• Water
• Methanol
• Acetonitrile
• Additives, salts, acids, bases
HPLC Pump(s)
• A high pressure pump is used to generate a
specific flow rate to mobile phase, typically
millimeter per minute.

Sample injector

• An injector is able to introduce the sample into


the continuously flowing mobile phase stream
that carries the sample into HPLC column.
Columns
• Length (5-15 cm)
• The column contains the chromatographic
packing material needed to effect the
separation.
• This packing material is called the
stationary phase.
Detectors

• UV
• Fluorescence
• Electrochemical
• Mass Spectrometric
Chromatograms

Restek® ULTRA C-18 and CN Columns (250mm x 4.6mm, 5µ),


Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.
Types of HPLC
• Reverse-phase (polar mobile phase/non-polar
stationary phase/somewhat polar analytes)
• Normal Phase (non-polar mobile phase/polar
stationary phase/non-polar analytes)
• Ion-Exchange (salts/ionic stationary phase)
• Size-exclusion (aqueous/gel for large MW
solutes, >104)
Popularity
Sensitivity
Accurate quantitative determination
suitability for separating nonvolatile and
thermally sensitive species
widespread applicability in industry, to many
fields of science, and to the public
 Ideally suited for proteomic analysis.
Advantages to HPLC

Higher resolution and speed of


analysis
HPLC columns can be reused without
repacking or regeneration
Greater reproducibility of results.
Easy to operation and data analysis
Adaptability to large-scale
Advantages of HPLC are result of 2
major advances

Stationary supports with very small


particle sizes and large surface
areas
Appliancation of high pressure to
solvent flow
THANKS

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