The downside to using a sub-2-μm-particle column is increased system pressure, which requires a limited column length to remain
within the capabilities of the available hardware in the laboratory. This restriction occurs because the pressure arising
from the column is in an inverse-square relationship to the particle diameter. Reducing the particle diameter by half results
in a fourfold increase in observed pressure. This pressure relationship makes it difficult to use small-particle columns under
a high (i.e., optimum) flow-rate range or choose a column long enough to achieve the desired resolution.
In response to this difficulty, some companies have developed specialized instruments with improved pressure tolerances. These
instruments, however, have their own limitations when it comes to the accepted levels of performance expected from HPLC analysis
with respect to injection reproducibility and gradient accuracy. In addition, these systems may have a higher cost of ownership
because of the extreme conditions under which they operate and the resulting wear and tear on consumable components such as
pump seals and injection-valve rotors. The question then arises: Is it really necessary or beneficial to use a sub 2-μm particle
to increase the speed when the back pressure increases so much?
The authors suggest that using a well-packed column with uniform silica gel-based materials of a mid-size diameter is the
proper solution (1). Mid-size particles are between 2 and 3 μm. Sub 2-μm particles are defined as small. This may seem like
an academic difference, but recall the inverse-square relationship of particle size to pressure. Mid-size particles allow
users to achieve high speed and high resolution simultaneously on existing equipment. Consider that the performance difference
between a 2.2-μm column, for instance, and a 1.8-μm column is minimal, but the expected back pressure with the 2.2-μm particle
column is less than two-thirds that of the sub-2-μm particle, meaning the highest performance level is achieved without sacrificing
Furthermore, in some cases, small-particle columns, owing to their shorter length, may not be suitable for separating closely
eluting peaks. If resolution is critical, the natural strategy is to use a longer column. With a small-particle column, it
may be impossible to use a long column because the back pressure exceeds the allowed operating pressure, regardless of the
system capability because every system has a limit. As a result, it is necessary to choose either a lower flow rate (thus
eliminating the advantage of going to a smaller particle) or to switch to a larger particle size and longer column to reduce
the back pressure.
The time needed to perform the separation is longer, so a balance must be struck between time and resolution. This problem
demonstrates that one cannot necessarily have an ideal separation. The separation must be optimized for each individual's
needs—the highest speed and the ultimate resolution may not always be possible in the same run. Columns with mid-size particles
have proven to be optimal for balancing the time reduction and high resolution required at the highest levels. It is more
useful if the same or better separation with a shorter run time can be achieved with lower back pressure.
Separation at elevated temperature
Interest in elevated-temperature liquid chromatography (LC) is increasing because better and faster separation can be obtained
simultaneously by using higher temperatures. Separation performed at increased temperature has an effect similar to that of
reducing particle size. Both techniques accelerate the mass diffusion of the analyte between the stationary phase and mobile
phase, thus resulting in a "flatter" van Deemter curve.
Both factor A and factor C of the van Deemter equation decrease as temperature increases and result in a lower HETP. High-temperature HPLC also reduces
the viscosity of the mobile phase, thus lowering the resistance to flow through the column and allowing greater flow rates
compared with ambient conditions. The resistance to column flow is inversely proportional to temperature, such that the column
pressure at 80 °C is about 40% lower than that at 40 °C. This relationship highlights the importance of temperature as a factor
in accelerating separation. Of course, increasing the temperature of the column raises questions of itself.