A current "hot topic" in high performance liquid chromatography (HPLC) column technology (1) is the alternative approaches
for developing faster separations and generating more column efficiency at lower pressure drop. The big question: Which approach
will be favored in the long run? These approaches include
packed columns with small porous particles (sub-2 μm);
packed columns with small porous particles (2–3 μm);
monoliths (silica and polymeric);
superficially porous packings (~2.7 μm).
The purpose of this installment of "Column Watch" will be to compare and contrast these various approaches for readers to
assess which one might be the best solution to his or her particular separations problem.
Table I: Commercial 2-mm and sub-2-mm totally porous HPLC columns at Pittcon 2007*
In general, all these approaches work in balancing column efficiency, phase ratios and column permeability. The first two
approaches are variations of a theme of the increased efficiency that results when the average particle size of a packed bed
is reduced. As for the third approach, monoliths have been around for a decade now but have not yet achieved dominance in
the applications arena. The oldest approach, superficially porous packings, dates back to the beginnings of HPLC in the late
1960s when chromatographers left the open gravity-fed columns to achieve faster more efficient separations. But the most recent
versions have a new twist.
Small Porous Particles
Figure 1
Because the particles smaller than today's more common 3.0–3.5 μm sizes have the same properties, the first two categories
will be combined in this discussion. Most recently, the introduction of numerous commercial HPLC columns with particle sizes
in the range of 2 μm and under (Table I) has brought about a new area of controversy. As one can see in Table I, 1.5–2.0 μm
particles are now available on the market. This packed-column approach uses conventional spherical silica particles but with
sub-2-μm particles sizes being favored, each having various particle size distributions (2). Short columns, usually less than
50 mm in length, are run at high linear velocities giving high sample throughput. Flatter van Deemter curves for these particle
sizes compared with larger sizes allow these higher flow rates without major losses in efficiency. On the other hand, if more
theoretical plates are required, these small particles are packed into longer columns (up to 15 cm) but when these columns
are run at these higher flow velocities, more back pressure is generated. Much has been written on this topic so I will not
elaborate further. A new nomenclature has come about with terms such as ultrahigh-pressure liquid chromatography (UHPLC) has
arisen to describe the higher back pressure requirement. An illustrative chromatogram in Figure 1 depicts the essence of the
story. One can achieve faster separations by shortening the column and can maintain the resolution by decreasing the particle
size concurrently. The evolution of HPLC is further shown in Figure 2 where plate count versus run time is plotted for various
particle size columns at various lengths. The HPLC pressure limit shown on the graph is indicative of conventional pumps that
can achieve pressures up to 6000 psi. New generation instrumentation allows for greatly increased pressure capability allowing
the use of longer columns packed with sub-2-μm particles. In addition, high temperatures are now fashionable in HPLC to lower
backpressure as well as solute improve mass transfer. In some cases with changes in temperature, chromatographic selectivity
is affected in both positive and negative ways.
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