Torn twixt tradition and innovation
Standards-setting organizations such as the U.S. Environmental Protection Agency and the U.S. Pharmacopoeia (USP) institute change cautiously. For example, USP’s initiative to modernize pharmaceutical monographs, which include many HPLC methods, relies heavily on tried-and-true column and LC platforms, but these don’t work as well as they might with today’s products.
Many monographs specify C18 columns, a highly hydrophobic phase that was the only game in town when the methods were written years ago. C18 still works for older drugs but not with newer molecules, some of which are highly polar. Ion-pairing reagents improve resolution and sensitivity, but they are incompatible with mass detectors. That is why USP has recruited major industrial collaborators to invent newer methods that provide the required degree of sensitivity without compromising robustness.
No individual column technology will emerge victorious. It appears that switching to a more sensitive stationary phase, such as HILIC or monoliths, or a smaller particle size will be sufficient to modernize monograph methods. But routine migration to UHPLC will probably not occur, at least for a while.
Lab Manager will continue to follow this story from the perspectives of both columns and instrumentation.
Transferring methods to prep LC
A good deal of technical writing still centers on transferring analytical methods from conventional HPLC to UHPLC. The sticking points—high pressures and small particle sizes—are very real issues with chromatographers, particularly those who rely on legacy methods.
Today, thanks to the efforts of major instrument companies, a good process development scientist can overcome most of the hurdles and devise a robust UHPLC method. But what about the ultimate column scale-up, from analytical to preparative?
Much less is known about scaling up to prep work, says Helmut Schulenberg-Schell, director of business development, liquid phase separations at Agilent Technologies (Waldbron, Germany).
Despite traditionally long development times, preparative LC is replacing flash chromatography as the go-to method for purifying research-stage materials in milligram to low-gram quantities. Flash columns require a mini development program, which synthetic chemists don’t have the time to carry out, and purity is rarely as high as with HPLC. Moreover, knowledge from dozens of analytical runs does not transfer to flash columns.
“After analyzing samples at the resolution that analytical HPLC provides, you’d like to convey that separation efficiency into purification. Flash does not allow you to do that,” says Schulenberg-Schell.
In December 2013, Agilent introduced the 1260 Infinity Automated Purification System, which automates scaling from analytical to preparative LC columns. The operator need only select a column from the analytical LC/MS run, and software automatically sets prep conditions, including gradients.
The system “industrializes” compound purification, says Schulenberg-Schell. “It’s a more rational approach.” From a management perspective, it also allows specialist chromatography groups to focus on more pressing, scientifically-challenging projects.
Combining stationary phase ideas …
We tend to think of sub-two-micron particles and superficially porous technologies as either-or: the former for high-pressure UHPLC systems, the latter for labs that desire UHPLC performance at HPLC back-pressures. No longer.
Phenomenex (Torrance, CA) has produced superficially porous Kinetix® “core shell” columns incorporating 1.7-micron particles—the best of both worlds. Michael McGinley, senior product manager for bioseparations, explains that for a given particle size, fused core will always provide up to 35 percent higher performance than conventional sub-two-micron columns. A typical UHPLC system, for example, has 250,000 theoretical plates per meter, which is achievable with 2.6-micron core-shell columns. Phenomenex’s 1.3-micron product achieves more than 400,000 plates per meter.
“We’ve continued to take column evolution further, especially as more folks adapt UHPLC in labs as they retire older HPLC systems,” McGinley says. “Coreshell columns are higher-performing than fully porous particles regardless of particle size.”
Innovation is ongoing even within the world of superficially porous materials. Supelco, Advanced Materials Technology (of Fused Core™ fame), and others have introduced stationary phases with varying shell-to- core thicknesses and different pore size diameters for macromolecule separations. “Companies are developing materials that operate across all pressure regiments and in some cases focus more on longer columns to optimize peak capacity and counts instead of speed,” McGinnis says.
…Down to the chemistry
Mixed-mode chromatography, where a single resin incorporates two separation modes, is better known in large-scale preparative chromatography than in HPLC. That is beginning to change as scientists from Thermo Fisher Scientific (Sunnyvale, CA) look more deeply into the technique for analytical LC.
“Mixed mode has been around for thirty years,” says Thermo scientist Xiaodong Liu, PhD. “Only in the past ten years has mixed mode been revived.” Liu attributes slow acceptance to the familiarity and ease of use for reverse phase and the perception that mixed mode is difficult due to the extra separation mode and all its mobile phase implications.
Yet as noted above, modern chemistry increasingly finds reverse-phase C18 (and related) columns to be inadequate. Reverse phase is incompatible with many aqueous mobile phases and has limited selectivity. Similarly, hydrophobic interaction columns suffer from solubility and matrix effects.
Mixed mode allows development scientists to fine-tune affinities based on pH and ionic strength. Changing the aqueous-organic buffer ratio slightly, as little as from 80:20 to 60:40, can reverse the selectivity of certain compounds.
Mixed mode’s lack of familiarity practically guarantees method development times that many companies cannot tolerate. Labs often prefer an adequate, out-of-the-box separation to a superior one that takes weeks to invent. “We realize that reverse phase is the workhorse column in LC today,”Liu admits.
Liu is instead working with the U.S. Environmental Protection Agency on revising methods for drinking water that remain problematic for conventional HPLC—for example, glycans, surfactants, and drinking water analysis.
“We’re not trying to replace reverse phase,” Liu says. “Rather, we are trying to address important applications where mixed mode has something unique to offer.”
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