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