The demand for increased efficiency and speed in pharmaceutical analysis extends from drug discovery and development to clinical screening and trials. The need to meet these throughput requirements without compromising the data required in quality assurance–quality control (QA–QC) applications has highlighted the important technical challenge of providing faster separation in high-performance liquid chromatography (HPLC). This article will focus on the challenges in serial chromatography (i.e., one run at a time) on a single HPLC column. Alternatives such as parallel chromatography and advanced routines using multiple columns with switching valves are best addressed separately.
The direct approach to increasing throughput is to raise the mobile-phase flow rate (linear velocity) to drive the peaks from the separation column faster. Increasing the mobile-phase flow rate in the widely used 5-μm particle-packed column, however, diminishes column efficiency. To counteract this loss in performance, one can consider two approaches:
The common goal of the two approaches is to reduce the height equivalent to theoretical plate (HETP) and, consequently, to increase the column performance per a given column length in an extended flow-rate region. One must consider several factors in both approaches to achieving ultrafast liquid chromatography (UFLC).
- Using smaller particle-size packing material
- Performing separations under elevated temperature.
Smaller particle-size packing material
Generally, columns using smaller particles provide good separation performance, are shorter, and also help to maintain column efficiency as the flow rate is increased. The columns' efficiency is highest at a particular mobile-phase flow rate, but at greater and lesser flow rates, efficiency decreases (HETP increases). This relationship is expressed using the following van Deemter equation:
H is the HETP (column length required to obtain one theoretical plate—the smaller the H value, the greater the efficiency), A is eddy diffusion, dP is the packing-particle diameter, B is longitudinal diffusion, v is the mobile-phase linear velocity, and C is coefficient of mass transfer. The usefulness of smaller packing material with a microsphere-packed column for performing high-speed separation is attributed to the column's efficiency at high flow rates. This effect is demonstrated by the van Deemter curves for a compound that is run on a series of HPLC columns packed with particles of various sizes. As shown in Figure 1, the HETP remains small over a wider flow-rate range if smaller particle-size packing material is used.
Figure 1: Van Deemter plots of different particle-size packing materials.
The plots indicate that smaller particle-size packing material makes it possible to achieve great column efficiency at a high flow rate, thus enabling good, quick separations. One can increase the flow rate of a method developed to meet certain resolution criteria without losing resolution, thereby decreasing the run time.
It is worth making a few observations about the van Deemter plot. First, the plot only applies to isocratic separations because no term in the equation takes changing mobile-phase strength into consideration. Second, the size, shape, and polarity of the analyte influence the observed van Deemter plot. Small, nonpolar molecules behave as shown in Figure 1. As more complexity and polarity are added to the molecule, however, a reduced region of minimized HETP may be observed. This effect results from the behavior of an analyte on the column, which depends on many different interactions that the van Deemter equation does not consider.