High Performance Liquid Chromatography

• Columns

• High performance pumps

• Stable flow rate

• Compounds dissolved in liquid sample

• Quantitative and qualitative analysis

• Mobile phase: solvent

• Stationary phase: separation column

Components separation

• Based on compound's speed through the column

• Based on interaction with the content of the stationary phase

• The greater is the affinity of the component with the mobile phase, the faster the component moves
through the column

• The greater is the affinity of the component with the stationary phase, the slower the component
moves through the column

1) Sample is introduced into column with mobile phase

2) Separation is accomplished by different moving speed of each compound

3) Detector connected to the outlet of column monitors each eluting compound from the column
(Chromatogram)

Types of column

• Normal Phase Column

- NP elute first

- more polar stationary phase

- silica stationary phase and methylene chloride or ether as mobile phase

• Reverse Phase Column

- P elute first

- reverse of normal phase

- nonpolar of less polar stationary phase

- hydrocarbons as stationary phase and organic solutions in mobile phase such as water and methanol

Types of column

• lon Exchange Columns

- stationary phase elute first

- either basic or acidic stationary phase with negative or positive charges

- mobile phase is a polar liquid such as salt in water - separation is based on attractive ionic force

- Ion Exchange Chromatography

• Size Exclusion Columns

- separation is based on size

- combination of polymers as stationary phase

- Size Exclusion Chromatography

• Columns vary in length from 30 mm to 250 mm

• Porosity of columns vary from 3 to 5 microns