Table of Contents
What They Are
How They Work
Glass vials being placed into the HPLC rack for autoinjection
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What They Are
High performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UHPLC) are two types of commonly used separation methods using liquid chromatography.
HPLC has been around since the 1960s, but UHPLC only first appeared in 2004 when Waters created their own trademarked ultra-performance chromatography (UPLC) systems. Since then, companies such as Thermo and Agilent have developed their own UHPLC systems.
Both HPLC and UHPLC can be used for the various types of liquid chromatography, including the popular normal and reverse phases, and can be paired with mass spectrometry systems, but the equipment needed to run both differs significantly.
How They Work
Liquid chromatography (LC) is a technique in analytical chemistry that is used to separate components in a mixture. The separation occurs due to the selective distribution of analytes between the mobile and stationary phases. In liquid chromatography, the mobile phase is the liquid and stationary phase is the column. Both HPLC and UHPLC use high pressures to pump the mobile phase through the column and the rest of the system. The rate of separation depends on the affinity of the analytes to the stationary phase.
The columns and mobile phases used in LC vary depending on the method needed for analysis, with the most commonly used method of separation for both HPLC and UHPLC being reverse phase chromatography using a C18 or C8 column. The columns are different for HPLC and UHPLC, with UHPLC columns having much smaller particles, and being are shorter and smaller in diameter than HPLC columns.
While a lot of labs have upgraded their systems to UHPLC, many still use the traditional HPLC systems. HPLC systems are cheaper than UHPLC systems, and method transfer over to UHPLC is both timely and costly. Time spent transferring methods could be spent developing assays or extracting samples.
Frictional heating caused by the high back pressures in UHPLC can degrade columns faster than they would with HPLC systems. Multiple UHPLC columns for different phases are now available, but very little has been published yet on their performance compared to HPLC.
Injection precision and increased carryover have also been shown to be problems in UHPLC compared to HPLC due to the different sample loops and injection systems. With HPLC, the sample loop is constantly washed with mobile phase. In UHPLC, the higher pressures mean that a different loop and sampling valves with rotors that directly connect to sample components are needed.
Traditional HPLC columns are typically 4.6 mm and between 150 to 250 mm in length. The sub-micron columns used for UHPLC give a better resolution and efficiency, but the systems run at ultra-high pressures that a HPLC system cannot handle. The development of fused core columns has been a big leap forward for traditional HPLC systems, as they can now also achieve the speed and efficiency of the UHPLC sub-2-µm columns without the higher pressures, offering a cheaper alternative to UHPLC systems.
UHPLC has come a long way since Waters developed their own patented UPLC machines in 2004, with a lot of companies now developing their own UHPLC systems and columns.
UHPLC provides a better resolution than traditional HPLC due to the shorter column length and smaller sub-2-micron particles in the columns, and UHPLC systems have specialized pumps that can deal with the higher pressures. Once the cost of buying a new system is overcome, they are cheap to run due to reduced solvent use and wastage.
Fast analysis is important for companies that have a lot of samples to analyze, and UHPLC offers higher throughput and faster run times than traditional HPLC. LC is commonly combined with mass spectrometry, and the low flow rates and no flow splitting of UHPLC allows for increased source ionization for better results.
Many laboratories now have a combination of both HPLC and UHPLC systems for both their LC and liquid chromatography mass spectrometry needs. With the development of fused core and HILIC columns, traditional HPLC systems are able to achieve the resolutions of UHPLC columns without having to spend the time and money investing in UHPLC systems.