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Liquid Chromatography Comparison: HPLC vs. UPLC vs. FPLC

March 15, 2024

This ALT Knowledge Base Video focuses on High Performance Liquid Chromatography (HPLC) versus Ultra-Performance Liquid Chromatography (UPLC) versus Fast Protein Liquid Chromatography (FPLC). While HPLC, UPLC, and FPLC are all powerful chromatographic techniques, they differ in their target molecules, back pressure, flow rate, stationary phase, and particle size. Selecting the most appropriate technique depends on the nature of the samples being analyzed, the desired outcomes, and the specific experimental requirements. In this video, we delve into a comparative analysis of these three techniques across various parameters. 

In the world of analytical chemistry, the terms HPLC, UPLC, and FPLC are frequently used. These are different techniques used in chromatography, which is a method of separating mixtures. Each of these techniques has its own advantages.

Chromatography 101

Chromatography is a laboratory technique used to separate mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound’s partition coefficient result in differential retention times on the stationary phase thus allowing for separation and characterization.


High-Performance Liquid Chromatography (HPLC) is a method used in biochemistry and analytical chemistry to identify, quantify, and purify the individual components of a mixture. It is recognized for its broad application across industries including pharma, environmental, food & beverage, & more. HPLC typically utilizes small particles and a relatively high back pressure of 2,000 – 4,000 psi. The flow rate typically ranges from 0.1 – 2 mL/min and the stationary phase is usually silica. The particle size used in HPLC is typically around 3 – 5 µm.


Ultra Performance Liquid Chromatography (UPLC) is a relatively recent technique that provides higher efficiency, faster analysis times, and increased sensitivity compared to HPLC, making it ideal for complex samples. It utilizes a smaller particle size of 1.7 – 5 µm and a significantly higher back pressure of 6,000 – 19,000 psi. The flow rate is often less than 1 mL/min and like HPLC, it uses silica as the stationary phase.


Fast Protein Liquid Chromatography (FPLC) is another chromatographic technique that is ideal for the analysis and purification of biomolecules. It offers gentle separation conditions suitable for sensitive samples. The back pressure is much lower (<600 psi) and a wider range of flow rates (1 – 150 mL/min) can be used. In FPLC, the stationary phase is typically agarose and the particle size varies.

Choosing Between HPLC, UPLC, & FPLC

When it comes to choosing between HPLC, UPLC, and FPLC, it all comes down to the nature of the sample and the purpose of the analysis. As a general rule, HPLC is a good choice for a broad range of applications, UPLC is ideal for analyzing complex samples due to its higher efficiency and sensitivity, and FPLC is the go-to method for analyzing and purifying biomolecules.

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We hope this blog post has provided you with a better understanding of HPLC, UPLC, and FPLC. Whether you’re in the pharmaceutical, environmental, food and beverage, or other industries that require chromatographic analysis, understanding these techniques can significantly enhance your work.

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