What Is the Difference Between UHPLC and HPLC?
In short, there are two main differences between UHPLC and HPLC:
- Maximum System Pressure Rating
- Typical Particle Size Used in the HPLC Column Stationary Phase
But first, let’s answer some basic questions.
What is HPLC?
HPLC is an acronym for High-Performance Liquid Chromatography. HPLC is a separation technique that allows analysts to separate a complex mixture of known or unknown compounds and quantify how much of each compound exists in the sample. Separation occurs by introducing a sample into a liquid stream (mobile phase), which moves the sample into the column. The mobile phase moves through the system because of the HPLC pump. HPLC columns are packed with small particles (stationary phase), and separation occurs based on how the different compounds interact with the mobile phase and stationary phase. Some compounds/analytes spend a long time interacting with the stationary phase and are more retentive, while some compounds do not interact with the stationary phase and flow through the column at the same rate as the mobile phase (less retentive or no retention). When compounds exit the column, they flow through a detector, which sends a signal to a data system, and a chromatogram is displayed (graphical plot showing the separation).
Over the past 50 years, significant improvements have been made to the critical components of the HPLC system. HPLC fittings and HPLC connectors have been engineered to withstand higher pressures. HPLC column manufacturers can reproducibly make smaller particles (stationary phase). With the popularity of smaller HPLC column particle sizes due to the decrease in analysis time, HPLC system pressures have increased significantly. These smaller particles led to the development of HPLC systems that can perform at higher pressures.
What Is UHPLC?
UHPLC is an acronym for Ultra High-Performance Liquid Chromatography (or sometimes referred to as Ultra High-Pressure Chromatography). UHPLC systems can withstand pressures up to 20,000 psi or 1,300 bar. Traditional HPLC Pressures are typically limited to 6,000 psi or 400 bar.
In UHPLC, it is common to use HPLC columns with a stationary phase that uses sub 2µm particle size. These smaller particles result in added back pressure to the system. With the added back pressure, it is critical that HPLC connections are made properly and the appropriate fittings and connectors are selected to withstand the higher pressures.
UHPLC and HPLC Fittings
In HPLC and UHPLC, it is critical to make proper HPLC connections to avoid system leaks, which lead to dreaded instrument down time. Prior to the pump, in both HPLC and UHPLC, low-pressure fittings can be used; we recommend Flangeless Fittings for ease of use. These Flangeless fittings are typically ¼-28 threaded flat bottom ferrule connections. After the pump, system pressure dramatically increases due to the back pressure caused by the HPLC column (the smaller the particle size, the higher the system back pressure). It is imperative to make good connections in the high-pressure portion of the system, and we recommend Idex Fittings for HPLC and UHPLC connections. Idex Health and Science is the global authority in fluidics, and we recommend that all new chromatographers (all chromatographers, actually) download the Idex Fittings 101 Guide, A Practical Guide to Connections and Fittings in Your Laboratory. The Fittings 101 Guide was designed to give you the fundamentals of equipment fittings and accessories, as well as the basics about liquid chromatography as an analytical technique.