UPLC vs HPLC: Unraveling the Similarities and Differences

Overview

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• Source: Microbioz India


• Date: 16 Oct,2023

Both ultra-performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC) are advanced analytical procedures used to separate and evaluate the constituent parts of complex mixtures. Despite their many similarities, they have important differences that serve to distinguish them.

Here, we’ll unravel the similarities and differences between UPLC and HPLC:

Similarities:

1. Chromatographic Principle: The same basic idea underlying liquid chromatography underlies both UPLC and HPLC. Through the application of pressure and the use of a mobile phase (solvent), they include the separation of chemicals in a liquid sample by forcing the sample through a stationary phase, which is commonly packed in a column.
2. Both processes may quantify and identify isolated molecules using a broad variety of detection techniques, such as UV-Visible spectroscopy, fluorescence, mass spectrometry, and more.
3. Depending on the particular separation requirements, both UPLC and HPLC use columns with similar stationary phase components, such as silica gel, C18, C8, or other bonded phases.
4. Sample Preparation: To make sure the sample is appropriate for analysis, sample preparation techniques like dilution, filtration, and derivatization can be used equally in UPLC and HPLC.

Differences:

1. Particle Size: The particle size of the stationary phase is one of the most important distinctions between UPLC and HPLC. While HPLC uses larger particles (usually 3 to 5 m), UPLC uses columns that are significantly more densely packed with much tiny particles (about 1.7 m or smaller). In UPLC, smaller particles offer greater efficiency and quicker separations.
2. Operating Pressure: In comparison to HPLC, which typically runs at pressures between 4000 and 6000 psi, UPLC operates at much greater pressures (up to 15,000 psi or more). In UPLC, the higher pressure enables faster flow rates and greater separation effectiveness.
3. Flow Rate: UPLC may employ significantly higher flow rates than HPLC because of the smaller particle size and higher pressure. As a result, UPLC’s analysis times are reduced.
4. Sample Volume: Compared to HPLC, UPLC often requires smaller sample volumes, making it more appropriate for evaluating scarce or priceless substances.
5. Sensitivity and Resolution: Due to the smaller particle size and less band broadening effects, UPLC often offers higher sensitivity and better resolution.
6. Cost: Due to the necessity for specialist equipment that can withstand high pressures and the price of tiny particle columns, UPLC systems often cost more than HPLC systems.
7. Method Transfer: Due to variations in column particle size, operating pressure, and flow rates, methods created for HPLC may not be readily transferable to UPLC. There might be some modifications needed.