Basic Principles of HPLC

Basic Principles of HPLC

Shahzad Bashir2009-ag-1231

Center for Agricultural biochemistry and biotechnology

UNIVERSITY OF AGRICULTURE

FAISLABAD

Outline

• What is HPLC?

• Types of HPLC

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Chromatography

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Chromatography

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)

PopularitySensitivityAccurate quantitative determinationsuitability for separating nonvolatile and

thermally sensitive specieswidespread applicability in industry, to many

fields of science, and to the publicIdeally 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 analysisAdaptability 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