Water for UHPLC
Application Overview
One of the biggest driving forces in HPLC innovation has been speed - speed without sacrificing the quality of separations. The van Deemter equation predicts that sample throughput and separation efficiency in chromatography are improved by decreasing the size of the particles packed in the column. (The van Deemter equation relates the plate height of a column, which is a measure of its efficiency, to the linear mobile phase velocity by considering physical, kinetic, and thermodynamic properties of a separation.) But smaller particle size also means an increase in system pressure. Since the early 1970s up until 2004, HPLC performance has been constrained by a system pressure limit of 6000 psi in most systems, which limited the particle size to 3µm or larger, thereby limiting the speed and efficiency of separation that could be achieved. The pioneering research by Dr. James Jorgensen and colleagues in the mid-1990’s demonstrated that substantially higher efficiencies and ultrafast separations could be achieved using very high system pressures with capillary columns packed with nonporous silica particles that are less than 2 μm in diameter. This was the beginning of ultrahigh-performance LC (UHPLC). In 2004, Waters introduced the first commercially available UHPLC system, called the Acquity UltraPerformance Liquid Chromatography (UPLC) system capable of running up to 15,000 psi using columns packed with sub-2µm particles.
The benefits of smaller particle size have been briefly mentioned. It makes possible high resolution chromatography by offering better separation efficiency and higher sensitivity. Separations using sub-2µm particles give 70% higher resolution and 3x higher efficiency compared to 5 µm particles, which makes it ideal for fast analysis of complex mixtures. Smaller particles allow faster analyses by using higher flow rates or shorter columns. In fact, 10x faster analyses and increased sample throughput can be achieved by using shorter columns without reducing efficiency. Additional advantages of sub-2 µm particle sizes are:
- Higher sensitivity because the peaks are taller and narrower
- Standard HPLC methods can be easily transferred to UHPLC within the same column family
- Less eluent consumption per sample because equilibration and run times are shorter
More Information
- Chromatography Online: Fast and Ultrafast HPLC on sub-2 μm Porous Particles — Where Do We Go From Here? http://chromatographyonline.findpharma.com/lcgc/article/articleDetail.jsp?id=333246&pageID=1&sk=&date=
- Other articles, user guides, and links for UHPLC: http://www.forumsci.co.il/HPLC/lcms_page.html#UPLC
- Investigating organic contamination in high purity water using UHPLC with ultrasensitive diode array detection and LC/MS, Agilent Technologies: http://www.chem.agilent.com/en-US/Search/Library/_layouts/Agilent/PublicationSummary.aspx?whid=71884&liid=5921
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