HPLC – High-Performance Liquid Chromatography

This technique is a form of column chromatography, it pumps a mixture in a solvent at high pressure through a column with chromatographic material.

The HPLC can separate and identify different compounds present in any sample that can be dissolved in a liquid.

HPLC is a very sensitive, efficient, and accurate technique.

Types of Chromatography (different column resin compositions):

• Normal Phase: The column is filled with polar silica particles and the buffer is non-polar. The polar particles in the injected sample will stick to the silica more and have a longer retention time.

• Reverse Phase: The column is filled with hydrophobic particles and the running buffer used in the system is polar. Hydrophobic particles will stick to the resin more.

Required Material:

• HPLC autosampler vials

• Centrifugal filters

• Eppendorf vials

• HPLC machine

In a HPLC there are the following variables:

• Flow rate

• Pressure

• Solvent buffers

• Column type

• Detection parameters

Protocol:

Mobile phase preparation:

1. To 1.5 litres of deionized water add 400ml of acetonitrile/ methanol.

2. To this solution add 2.4ml of glacial acetic acid.

3. Using purified deionised water, dilute the solution to make a total of 2 litres.

4. Add 40% sodium hydroxide and read pH - pH should be 4.2.

5. Filter the mobile phase under a vacuum to remove the solids that could clog the chromatographic column.

Sample injection and data collection:

1. Prepare your sample and slowly inject 100µl of the sample into the system.

2. Load the stationary phase to the column.

3. Start the HPLC and wait for the components to develop.

4. On completion the data is collected and saved, and the syringe can be removed.

Results and calculations:

1. Calculate the concentration of all the separated components.

2. The triangular method determines the peak areas for all standards and samples.

3. Calculate the time taken for each component to reach the peak.

4. Draw calibration curves of peak area vs. concentration for all components.

5. Calculate the least-squares fit for calibration curves.