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, 5), 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