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


Particle size and efficiency


Figure 3: Efficiency, N, is directly proportional to the column length L; therefore, efficiency increases as column length increases.
Columns packed with sub-2-μm particle sizes can be used to increase resolution and shorten analysis times. How is this done? To better understand the effect of particle size, consider two more concepts in separation science—the theoretical plate and the van Deemter equation.


Figure 4: The van Deemter equation, an empirical equation describing the relationship between efficiency, as measured by theoretical plate height H, and linear velocity is governed by three cumulative terms: A eddy diffusion, B longitudinal diffusion, and C mass transfer.
As previously mentioned, particle size affects the efficiency term of the resolution equation. Efficiency is ultimately derived from the theoretical plate model of chromatography. Conceptually, a plate refers to one complete equilibrated transfer, or partition, of a solute between the mobile and stationary phases. N, therefore, is a qualitative term used to measure the number of theoretical plates in a given column or the extent to which a solute partitions between the mobile and stationary phases. In relation to particle size, N is inversely proportional to the particle diameter (see Figure 2). Put simply, as particle size decreases, efficiency increases, and more resolution is possible with the same length of column.


Figure 5: An empirically determined van Deemter plot for a biphenyl test probe demonstrates that sub-2-μm packings are minimally affected by flow rate, thus allowing higher flow rates to be used and resulting in faster analysis times.
Practically, this means that shorter analysis times through the use of smaller particle-size packings can be achieved by two means. First, N is directly proportional to the column length L (see Figure 3). Therefore, an analyst can decrease the length of the column as the particle size decreases, still maintaining resolution and shortening the analysis time. Second, and probably more significant, higher flow rates can be used without a substantial loss in resolution. The van Deemter equation is an empirical formula describing the relationship between plate height H (the height of one theoretical plate) and linear velocity μ (see Figure 4). This equation explains why higher flow rates can be used with small-particle columns without compromising resolution. Smaller plate-height values correspond to greater peak efficiencies because more plates or analyte partitions can occur over a fixed length of column. The van Deemter equation is governed by three cumulative terms: eddy diffusion A, longitudinal diffusion B, and mass transfer C. Eddy diffusion is caused by disturbances in the solute flow path and is largely unaffected by flow rate. Smaller particles give rise to less interstitial space and therefore, through a decreased A term, have higher overall efficiencies. Mass transfer is the movement of the analyte in and out of the stationary phase. Through this term, higher flow rates typically result in poorer efficiencies.


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