Cromatografia

8 SEPARATION SCIENCE REDEFINED MAY 2005

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Ultra Performance Liquid Chromatography (UPLC): An Introduction
ACQUITY Ultra Performance LC™ Systems take advantage of technological strides made in particle chemistry performance, system optimization, detector design, and data processing and control. When taken together, these achievements have created a step-functionimprovement in chromatographic performance. Defined as UPLC™, this new category of analytical separation science retains the practicality and principles of HPLC while increasing the overall interlaced attributes of speed, sensitivity, and resolution.

Michael E. Swartz, Ph.D.
Principal Scientist, Waters Corporation, Milford, Massachusetts, e-mail Michael_Swartz@waters.com.

igh performanceliquid chromatography (HPLC) is a proven technique that has been used in laboratories worldwide over the past 30-plus years. One of the primary drivers for the growth of this technique has been the evolution of packing materials used to effect the separation. The underlying principles of this evolution are governed by the van Deemter equation, which is an empirical formula that describes therelationship between linear velocity (flow rate) and plate height (HETP or column efficiency). Since particle size is one of the variables, a van Deemter curve can be used to investigate chromatographic performance. According to the van Deemter equation, as the particle size decreases to less than 2.5 m, not only is there a significant gain in efficiency, but the efficiency does not diminish at increasedflow rates or linear velocities. By using smaller particles, speed and peak capacity (number of peaks resolved per unit time in gradient separations) can be extended to new limits, termed Ultra Performance Liquid Chromatography, or UPLC. The technology takes full advantage of chromatographic principles to run separations using columns packed with smaller particles and/or higher flow rates forincreased speed, with superior resolution and sensitivity. Figure 1 shows a stability indicating assay of five related substances accomplished in under one minute, proving that the resolving power of UPLC is not compromised even at high speed. The current USP lists

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multiple HPLC methods for the analysis of these same compounds with run times approaching 20 min, with broad, tailed peaks.Chemistry of Small Particles

As shown in Figure 1, smaller particles provide not only increased efficiency, but also the ability to work at increased linear velocity without a loss of efficiency, providing both resolution and speed. Efficiency is the primary separation parameter behind UPLC since it relies on the same selectivity and retentivity as HPLC. In the fundamental resolution (Rs) equation:resolution is proportional to the square root of N. But since N is inversely proportional to particle size (dp):

as the particle size is lowered by a factor of three, from, for example, 5 m (HPLCscale) to 1.7 m (UPLC-scale), N is increased by three and resolution by the square root of three or 1.7. N is also inversely proportional to the square of the peak width:www.chromatographyonline.com

MAY 2005 SEPARATION SCIENCE REDEFINED

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Figure 1: UPLC stability indicating assay. UPLC conditions: Column: 2.1 30 mm 1.7µm ACQUITY BEH C18 at 30 °C. A 45 s, 5–85%B linear gradient, at a flow rate of 0.8 mL/min was used. Mobile phase A was 10 mm ammonium formate, pH 4.0, B was acetonitrile. UV detection at 273 nm and 40 pts/s. Peaks are in order: 5-nitroso-2,4,6-triaminopyrimidine,4-amino-6-chloro-1,3-benzenesulfanamide, hydrochlorthiazide, triamterine, and methylbenzenesulfanamide; 5 L injection, 0.1 mg/mL each.

This illustrates that the narrower the peaks are, the easier they are to separate from each other. Also, peak height is inversely proportional to the peak width:

So as the particle size decreases to increase N and subsequently Rs, an increase in sensitivity...