Notoriously difficult use of small-particle stationary phases in HPLC gets easier

Researchers from the University at Buffalo, State University of New York, and Johnson and Johnson Pharmacuetical Research and Development, US, have shown that very high pressure HPLC can be performed using commercially available equipment and does not need technically difficult capillary columns, as had been assumed previously.

Traditionally, HPLC is performed using columns 2-5mm in diameter and up to 250mm long. These columns are packed with beads of a stationary phase that are between 3 and 5μm in diameter. Reducing the size of these particles gives more efficient separations, but there is a problem: the extra pressure needed to force the mobile phase through the smaller particles causes ’frictional heating’ and can lead to non-uniform changes in temperature, adversely affecting the separation.

To combat this, analysts usually decrease the sample flow rate by using tiny capillary columns, just fractions of a millimetre thick. But Luis Colón and his colleagues think that this may be unnecessary.

Col?n was motivated to explore how materials behave in HPLC columns after he developed new micron- and sub-micron sized silica particles for chromatographic use. Going back to the equations that govern chromatographic behaviour, he calculates that reducing the particle size of the stationary phase to 1μm will only generate as much frictional heat as routine separations carried out successfully all over the world.

A reduction in column diameter is needed, but not to the extent of using a capillary. ’Our analysis shows that one can use columns of 1 mm internal diameter packed with small, 1μm size materials with power generation similar to that found with current HPLC practices’, Colón says.

The routine use of small particle stationary phases and high mobile-phase pressures is good news for pharmaceutical companies, who rely on HPLC heavily. And analytical instrument manufacturers are already on the case. The Waters Corporation recently announced their UPLC (ultra high pressure liquid chromatography) system, which uses mobile-phase pressures of up to 15,000psi.

But Colón thinks this is only the beginning. ’Our calculations show that an instrument capable of 30 000 psi may show better potential. I think Waters will now push the envelope even further in terms of the actual limitations. In two years there will be instruments out there by other companies’, he predicts.