Performing analytical work in the contract pharmaceutical environment presents many challenges. Clients typically want the
best price and the highest quality service provided in the shortest possible time. A contract facility's analytical department
must analyze the samples in a timely fashion with the utmost quality and in a cost-effective manner. Methodology must be precise,
robust, and time efficient. Ultra high performance liquid chromatography (UHPLC) is one of the most recent analytical tools
to help laboratories reach these objectives.
For more than 30 years, high performance liquid chromatography (HPLC) has been used routinely as the primary test for potency
and related substances in the pharmaceutical industry. During this time, there have been many improvements in detectors, column
chemistries, and other hardware. The particle size in columns also has decreased from 10 μm to 2.5 μm. The decrease in particle
size has corresponded to an increase in selectivity and column efficiency. However, there has also been a corresponding increase
in system pressure. Because of the limitations of pressure in HPLC systems to a maximum of approximately 6000 psi, the particle
size has been limited to about 2.5 μm or larger for routine HPLC analysis. Smaller particle sizes have been available but
have been limited in application and needed reduced flow rates to compensate for the pressure limit. The reduced flow rates
has hindered the usefulness of these columns in routine analysis.
Figure 1: Van Deemter plots for several column particle sizes. HPLC is high performance liquid chromatography and HETP is
height equivalent to a theoretical plate.
In 2003, Jerkovich et al. published an article on the advantages of micrometer-sized (1–2 μm) particles in ultrahigh pressure
liquid chromatography. The Van Deemter Plot (see Figure 1) demonstrates that the most efficient particle size is 1.7 μm at
a relatively high linear velocity. The relationship between mobile phase flow velocity (u) and plate height (H or column efficiency) is described by the Van Deemter equation:
in which A, B, and C are the coefficients for eddy diffusion, longitudinal diffusion, and resistance to mass transfer, respectively—the lower
the plate height, the more efficient the column. Smaller particles increase efficiency by providing faster linear velocities
and better sensitivity as well as a significant reductions in analysis time. The slope of the high-velocity side of the curve
decreases with the particle size. This result enables operation at greater flow rates without sacrificing efficiency. Peak
capacity, which is the number of peaks that can be resolved per unit time of chromatography, is also increased significantly
using 1.7-μm particles.
Using small particle packing materials increases the resistance to flow, which increases backpressure. The pressure drop in
a column is inversely proportional to the square of the particle diameter. In addition, to achieve maximum separation efficiency
for a 1.7-μm particle, higher flow rates are required for faster linear velocities, which generate even higher backpressure.
Silica-based particles do not possess the mechanical strength or efficiency needed to meet the demands of UHPLC separations.
A bridged ethylsiloxane–silica (BEH) hybrid particle has been developed to meet the demands of UHPLC. It provides improved
mechanical strength when formed into fully porous particles. The narrow size distribution of the particles facilitates packing
into high-efficiency columns.