Using Selectivity to Optimize UHPLC Separations - Pharmaceutical Technology

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Using Selectivity to Optimize UHPLC Separations
Ultrahigh pressure liquid chromatography maximizes efficiency, but, as defined by the resolution equation, the stationary phase is still a crucial consideration when attempting to resolve mixtures of compounds.


Pharmaceutical Technology



Figure 6: A biphenyl stationary phase is more selective for aromatic or fused-ring compounds than a conventional C18 phase, providing improved resolution.
If we look at an empirically determined van Deemter plot of efficiency versus flow rate, when using a 1.9-μm particle size (see Figure 5), we can see that, in practice, smaller particle sizes are minimally affected by higher flow rates. Column efficiency does not diminish when flow rate increases, as indicated by the relatively flat slope of the curve. Peak efficiency is comparable even when the flow rate increases to 1 mL/min on a 2.1-mm internal diameter column. This illustrates the most significant influence that small particles have on chromatographic separations—a much wider range of usable flow rates, which translates into significantly faster analysis times. This benefit, and the shorter column lengths needed for a given resolution, result in higher sample throughput without loss of separation quality.

The role of selectivity


Figure 7: A biphenyl stationary phase, combined with a 1.9-μm packing, makes possible a fast and highly selective analysis of steroids.
UHPLC does maximize efficiency (e.g., theoretical plates), but, as defined by the resolution equation, stationary-phase selectivity is still a crucial consideration when attempting to resolve mixtures of compounds. By choosing a stationary phase that produces optimum selectivity for the specific compounds of interest, one can maximize the benefit of UHPLC. For example, the use of a biphenyl stationary phase can greatly enhance a separation. A biphenyl stationary phase differs from that of an alkyl (e.g., C18) phase in that the phenyl rings present can employ pi–pi interactions. This creates a separation mechanism with improved selectivity for aromatic or fused-ring compounds (see Figure 6). When using a biphenyl stationary phase with a highly efficient 1.9-μm particle-size column, one can produce fast, highly selective separations of steroids (see Figure 7). A 1.9-μm biphenyl column can separate a test mix of seven hormones in less than 2 min, a feat not possible through C18 selectivity (1, 2).


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