The last two years have seen a continuous introduction of new high performance liquid chromatography (HPLC) and ultrahigh-pressure liquid chromatography (UHPLC) packing materials, including a range of sub-2-µm particles, superficially porous particles and second-generation monoliths. Here, we look at these introductions and their impact on instrumentation and laboratory productivity. In addition, we discuss future directions in column technology.
High-Throughput and Productivity: Still a Driving Force in Liquid-Phase Separations
When sub-2-µm porous particles are packed into short columns, separations can be performed faster, sometimes in just a minute or two, than longer columns packed with larger particles (~5 µm) without sacrificing chromatographic resolution. The flat van Deemter curves noted for sub2µm columns allow (and perhaps demand) relatively high flow rates to be used, in the range of 1.5–2 mL/min, if necessary. Even though the column pressure increases with the inverse square of the average particle diameter, these shorter columns (usually 50 mm and under) can be used with most conventional HPLC pumping systems, even at these increased flow rates.
For more demanding separations, longer columns of 100- and 150-mm lengths may be required. With such lengths, column back pressures may increase beyond the capabilities of conventional pumping systems (~400 bar upper limit). Thus, in recent years, pumping systems capable of operation at pressures as high as 20000 psi (1330 bar) have come onto the market. The advent of the term ultrahigh-pressure liquid chromatography (UHPLC) has somewhat mesmerized the chromatography world into thinking that an entirely new technology has arisen. However, it is rare to see a separation run at such high pressures, even at 1000 bar. User concerns about stress on instrument hardware and on the columns themselves have limited widespread applications at these high pressures. The demands of sample cleanliness and easier "pluggability" of the small porosity frits terminating the ends of sub-2-µm columns also have made some users cautious about jumping into their routine application. Nevertheless, these columns have proven to be rugged, even at extremely high pressures, if used properly by ensuring particle-free samples are injected. With the introductions of zero- or low-dead-volume, high-pressure guard columns and zero-dead-volume in-line filters to help protect the analytical column, liquid chromatographers are becoming more comfortable using these columns and many publications are now seen employing sub-2-µm columns, even in routine environments.
What the advent of UHPLC has done is make instrument manufacturers more aware of the need to provide systems with minimal extra column effects (that is, band broadening outside of the column itself). These small-particle columns are so efficient (~250000 plates/m) that any unswept and unnecessary volumes and connections in the flow path have to be minimized. In addition to the high-pressure capability required for the pump and other hardware components (such as injector valves, mixers, and fittings), attention also is paid to the dwell volume now (also referred to as the gradient delay volume — the volume from the point of mixing of the solvents to the head of the column). When attempting to develop 1–2 min separations, liquid chromatographers can no longer wait for a gradient that takes several minutes to reach the column because ballistic gradients are now the norm.
Some column and instrument suppliers have held off on joining the sub-2-µm bandwagon and have made more moderate reductions in the particle size from the popular 3–3.5 µm columns. The particle diameter is larger for packed columns in the range of 2–3 µm than the sub-2-µm particles, so the pressure drop is lower but efficiency is better than the more popular 3–3.5 µm particles. The arguments for using packings in the 2–3 µm range revolve around considering the entire separation cycle time: Higher temperatures improve efficiency and reduce back pressure, improved LC system hydraulics decrease band dispersion and gradient re-equilibration time, and faster autosamplers, detectors and data systems increase overall system efficiency. The use of lower pressures also places less stress on the instrumentation and column materials.
Interestingly, I have not seen even smaller totally porous particle columns coming to the market in the last four years. Currently, the smallest totally porous particle size that is commercially available is 1.5 µm and this diameter has been available for many years. However, there are other competing technologies now on the market (and coming onto the market) that do not expose the column and instrumentation to such dramatic pressure conditions: SPP columns and monoliths.