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Practical Ultra High Performance Liquid Chromatography (UHPLC)

Abstract

The potential to increase chromatographic efficiency and resolution along with significant savings in solvent cost and analysis time have driven the uptake of UHPLC to many application areas. This discussion outlines how instrument and column technologies continually evolve to meet the requirements of UHPLC, providing new options for chromatographers. Example data are provided to show the high speed and high resolution options of UHPLC. Advanced topics such as HPLC→HPLC translations using free downloadable tools are also covered.

Instrument and Column Technology

The drive for increased chromatographic performance has led column manufacturers to continuously develop smaller and smaller particle sizes for liquid chromatography. Column efficiency is inversely proportional to the particle size, hence reducing the particle size increases the number of theoretical plates. Although smaller particles provide higher efficiencies, the trade-off is an increase in the operational back pressure. The introduction of commercially available high pressure capable instrumentation paved the way for the routine use of sub-2 micron particles. These small particles were shown to provide exceptional performance, particularly at flow rates higher than typically used with larger particle sizes, as demonstrated by their respective van Deemter plots (Figure 1, also see AKN0010: Band broadening and the van Deemter Equation). As shown, the optimum flow rate for 1.7 μm particles is greater than that of 3 and 5 μm particles. Additionally, 1.7 μm particles exhibit a flatter curve, meaning that a Figure 1: Van Deemter plots for various particle sizes. The minimum of each curve represents the optimal flow rate (i.e. highest efficiency) for that particle size. they can still deliver high performance at higher flow rates. In contrast, larger particles show a sharper drop-off in performance when the flow rate is elevated beyond the optimum value.

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