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Laura Bush is the Editor in Chief for BioPharm International
Recently, LCGC asked a panel of experts (listed in the sidebar) to assess the current state of the art of liquid chromatography (LC) columns and methods and to try to predict how the technology will develop in the future.
Recently, LCGC asked a panel of experts (listed in the sidebar) to assess the current state of the art of liquid chromatography (LC) columns and methods and to try to predict how the technology will develop in the future. Here are some highlights from that discussion.
The Liquid Chromatography Expert Panel
The Size of Superficially Porous Particles
The first question we asked our expert panel was whether they foresee any limits to the decrease in size of superficially porous particles (SPPs).
There should be no limits in terms of synthesizing the particles themselves, points out Ron Majors. It has already been demonstrated, he notes, that both smaller and larger SPP particles can be prepared with various shell thicknesses and pore sizes, and that they are robust, can be derivatized with the popular bonded phases, and can be successfully packed into most column configurations (albeit with a bit of work).
The question, then, is whether smaller particle sizes for SPPs are desirable, given the higher pressures generated, the real possibility of frictional heating, and the instrumental considerations of extracolumn effects. "Perhaps a new generation of low dispersion instruments may be required for smaller SPPs," he considers.
And of course, as several experts point out, making these particles smaller would eliminate the advantage of being able to use them on regular LC equipment to achieve efficiencies close to those of ultrahigh-pressure liquid chromatography (UHPLC). Thus, the panel almost unanimously agreed that the current size of SPPs, with most commercial SPPs at 2.7 μm and experimental SPPs between 1 and 2 μm, is likely to remain where it is.
And as Jack Kirkland notes, particles smaller than 1 μm likely are not practical, and particles with diameters of <2 μm already are problematic, requiring very high pressures that tax instruments, decrease reliability, and increase operating costs. "Such small particles are only of value for fast separations of samples with limited components," he says. "How many users require <10-s separations rather than 30 s? Complex samples require larger particles, longer columns."
Joe Glajch also notes, "Additional gain in efficiency is probably not the major direction for column particles in the future; selectivity will become more important."
And there is indeed a lower limit to the size of SPPs, says Georges Guiochon. The pressure required to achieve the optimum flow rate, he says, increases as the square of the inverse of the particle size at constant efficiency, for a given compound on a given system.
This, he says, becomes practically impossible for the analysis of low-molecular-weight compounds (such as pharmaceuticals and peptides) on columns packed with particles finer than 1 μm. However, Guiochon continues, the analysis of large biochemicals on columns packed with 0.5-μm particles would be possible if particles that size (but not smaller), with large mesopores could be manufactured and packed in efficient columns.
David Hage agrees. "Pore sizes of 100 Å or less work well for small molecules, but pore sizes of 300–500 Å or even larger are needed to provide suitable access if the same kinds of supports are to be used for biomacromolecules," he says.
"The limit might also depend on the column diameter desired," adds Guiochon, "The efficiencies of wide-bore columns (>4.6 mm) and of capillary columns (<0.1 mm) tend to exceed that of narrow-bore (0.5–2.1 mm) ones."
Operating at Very High Pressure
In a related question, we then asked our panelists if they foresee a move to high performance liquid chromatography (HPLC) instruments operating at even higher pressure in the future, to increase speed or take advantage of ever-smaller packings.
A few like the pursuit of higher pressure, at least to some extent, noting that higher pressures have provided excellent results so far.
"There are many applications in which UHPLC has been used to achieve better separations," says Hage. "One example has been to combine UHPLC with mass spectrometry (MS) to look at mycotoxins in food."
But most are not convinced that going to much higher pressures makes sense, both in terms of need as well as the costs and complications involved. "I think the current operating pressures are more than sufficient for the vast majority of problems in HPLC today and in the future," says Glajch.
Chris Welch also points out that a lot of the recent improvements in HPLC performance can be attributed to simple adjustments like tuning up internal plumbing connections and reducing the volume devoted to mixing. "If higher pressure means higher complexity and cost, I would rather see simple solutions that improve performance while decreasing cost," he says. "I think vendors should give some thought to selling at the low end of the market, expanding the overall base of HPLC users to include things like microfluidic devices operating in medical and dentist offices, perhaps even in the home."
A number of our experts see the need for various types of new stationary-phase chemistries.
"When it comes to regular LC columns, I think that there must be more than reversed-phase-LC, hydrophilic interaction chromatography (HILIC) and the various ion exchangers," says Rainer Bischoff. "Orthogonality of separations is a critical attribute to getting a more comprehensive view of very complex samples. There was a time when mixed-mode stationary phases as used in solid-phase extraction were popular. Maybe there should be a revival of such specialty columns for high-efficiency LC."
Other areas for development that were suggested include new phases for chiral and biochemical applications, achiral phases that improve separations for closely related isomers and analogs, improved columns for oligonucleotide separations, and selective adsorbents for on-line extraction.
For supercritical fluid chromatography (SFC), says Welch, the "holy grail" is a column that would give comparable generality to C18 reversed-phase separations. "This would enable a shift to SFC for carrying out the routine mass-directed, small-scale purifications that support high-throughput synthetic chemistry," he says. "Until generality is comparable with reversed phase, it doesn't make sense to switch to SFC."
Majors predicts that for both HILIC and chiral separations, new phases will be developed that are more universal. For intact biological molecules, he feels improvements in biocompatible phases, especially for faster separations, would be of interest. "Non-silica-based superficially porous packings could fill this need," he concludes.
Nevertheless, he doesn't foresee a significant change in the landscape of stationary phases. "For the last three decades, for nonchiral small molecules, reversed-phase chromatography has been dominant as the preferred HPLC mode and I don't see this changing," he says. "Neutral, ionic, and ionizable compounds can all be separated using this mode."
Glajch sees the overall trend moving toward fewer stationary phases, not more, as stationary phases with overlapping selectivity are weeded out. "Some new phases are warranted, to fill in the gaps in current offerings, but many existing ones will likely be removed from use due to duplication," he says.
Kirkland believes that separation scientists should focus more on mobile-phase selection to obtain selectivity differences. "This technology has been exhaustively researched but largely ignored, even though it is much more powerful than stationary-phase selectivity," he says.
New Particle Technology?
We also asked our panelists if they see any new particle technology on the horizon. A number of ideas emerged.
Welch suspects that self-assembled porous coatings within microfluidic capillaries may become important.
Cortes, in turn, is intrigued by various recent developments, particularly shell-on-shell technology being developed by Peter Myers at the University of Liverpool, and monolith modifications, such as addition of particles, pioneered by Emily Hilder at the University of Tasmania.
Majors foresees that comprehensive multidimensional LC — particularly once the method is more widely used outside of academia — may require newer types of column configurations and truly orthogonal stationary phases.
Bischoff believes that the sub-1-μm particles being explored by Mary Wirth at Purdue University could bring about a new era of efficiency in LC. "However, the technical challenges are significant on all levels," he acknowledges. "We'll have to wait and see whether they can be tackled."
New ways of making LC packings may generate improvements in efficiency that were unheard of just 10 years ago, Majors notes. "Don't count out silica-, silica-hybrid-, and polymer-based monolithic columns as academics and industry work to further improve these interesting separation media."
In Guiochon's view, the only progress needed is to develop particles with a higher thermal conductivity, to reduce the heat effects that plague analyses performed with long, wide-bore columns packed with fine core–shell particles. The best candidate would be to use a solid alumina core instead of silica, he believes.
But, he says, the real problem with modern fine particles is their packing procedure. "It has proved to be very difficult for all manufacturers to develop a suitable procedure," he says, and wonders if it could still be improved.
Guiochon would also like to see instrument changes that reduce band broadening. "I suggest that manufacturers integrate the whole LC system — the sample injection device, the column, and the detector — into one subunit," he says. "Or if that is impossible, I suggest they design it in a way that minimizes the extracolumn volumes and reduces the length and diameters of connecting tubes."