12. HPLC vs UHPLC

With the need for faster analyses (lower retention times), the industry has developed so-called UHPLC systems – Ultra High-performance Liquid Chromatography.

The only practical way to reduce analysis time is to push the analyzed sample through the column faster, which means using higher flow rates. However, this results in significantly higher back-pressure on the pump, hence UHPLC systems operate at up to 15,000 – 20,000 psi, as opposed to traditional HPLC operating at up to 6,000 psi.

This need for faster analysis comes with many challenges – increasing the flow rate and building systems to withstand 20,000 psi pressures is not enough. By increasing flow rates, the compounds are pushed through the column faster and hence do not have time to properly separate. As a result, the industry has developed UHPLC columns with smaller particle size, which have much larger surface area to interact with the analyzed compounds. Typically, UHPLC columns are packed with material with particle sizes lower than 2um, while traditional HPLC columns typically have 5um particles. Again, the smaller particles are more tightly packed inside the column causing significantly higher back-pressures on the pump.

Lastly, because of the much higher surface area of the packing material and more interaction sites, the columns tend to be shorter – 100mm in length as opposed to the typical 150mm in a traditional HPLC system.

UHPLC systems must be able to withstand pressures up to 20,000 psi and therefore the parts, such as pumps and tubing are modified to accommodate these conditions. As a result, the UHPLC systems tend to be more expensive compared to traditional HPLC. UHPLC does not necessarily yield a faster or better analysis compared to HPLC – it is often a matter of the chromatographic conditions design.