HPLC introduction

Welcome to the
T-level Science
lab training hub

studentInitials

college

Password

Heading 2

Introduction to HPLC

High Performance Liquid Chromatography (HPLC) is one of the most widely used techniques in modern laboratories. It is essential in many sectors — from pharmaceutical research and NHS diagnostics to environmental monitoring, food safety testing, and forensic science.

HPLC is a powerful method for separating, identifying, and measuring chemicals in a mixture. Its uses are incredibly varied: from quality control of medicines, to checking pollutants in water, to isolating natural products from plants. If you work in laboratory science, there’s a very good chance you will use HPLC at some point in your career.

In fact, employers often list HPLC knowledge and experience as one of the top skills they look for when hiring laboratory staff.

At first, the theory and equipment can seem complicated — but don’t worry we’ll take a step-by-step approach to understanding how HPLC works. You’ll get hands-on experience with an HPLC simulator, analyse real data, and prepare professional-style scientific reports. By the end, you’ll not only understand the principles but also be able to apply them like a professional scientist.

Introduction to HPLC

On the face of it, paper chromatography and HPLC seem worlds apart, but actually when it comes to the separation of mixtures of compounds, the theory and principle of separation are essentially the same. Both techniques require a stationary phase (SP) and a mobile phase (MP).

For paper chromatography the SP is filter paper (a porous material of intertwined cellulose fibres) and the MP is typically water. As the MP travels up the SP (by capillary attraction) the compounds partition between the two phases at different rates dependent on their chemical structure and properties.

If a compound is highly soluble in water and only weakly binds to the SP it will move quickly up the paper.

If a compound is poorly soluble in water and strongly binds to the SP it will move slowly.

The Retention Factor (Rf) is used as a measure of degree of separation compared to the solvent front.

In this example the SP is spotted with a black dye and a known red dye, for reference. We can observe that the black dye is made of a mixture of blue, green and red dyes - and the red dye has the same Rf value as the reference spot which suggest that they are the same compound.

Click to play

Water

Introduction to HPLC

For HPLC, the stationary phase (SP) is silica (more information about this later) and the mobile phase (MP) is typically a mixture of water and an organic solvent.

Silica is a very fine powder resin (fig 1), that can be sorted and refined to obtain the required particle size - fig 2 shows a zoomed in image of the spherical porous particles.

To make an HPLC column, the manufacturers pack steel columns (fig 3a) with a slurry of the silica, under very high pressure, Fig 3b shows an enlarged image of the column with the inlet fitting removed showing the compacted silica that looks like a solid white rod.

The separation of mixtures of compounds using an HPLC column works in the same way as paper chromatography. As the compound mixture is pushed through the column in the mobile phase (MP), each compound binds to the solid phase (SP) at different rates and the compounds exit the column as separate peaks (fig 4). Instead of Rf values we measure the retention time (RT) of each peak, which is the time it takes from the point of injection to the time the peak reaches the detector.

Fig 1: silica resin