Product Focus: UHPLC
Method transfer between HPLC and UHPLC has been a standard complaint against high-pressure, sub-two-micron technology. Despite the accumulation of application notes from UHPLC vendors, end users continue to cite method transfer difficulties for not switching to UHPLC.
Scaling methods, changing modes
Regulated methods in environmental, food, and pharmaceutical analysis are particularly prone to not switching below the five-micron particle barrier.
Extending knowledge and practice from UHPLC to preparative LC is similarly difficult.
According to Helmut Schulenberg-Schell, director of business development for liquid phase separations at Agilent Technologies (Waldbronn, Germany), the body of experience for transferring HPLC methods to UHPLC is voluminous compared with transfers from either HPLC or UHPLC to preparative work.
The problem, says Schulenberg-Schell, is purification chemistry’s lack of what he terms industrialization. “Chemists can often reach 80 percent purity or higher, but at that stage they cannot afford the additional time and effort required to optimize purification.” Nor is calling in a chromatography expert for projects at this relatively low level of sophistication warranted.
At issue is not just hardware differences—stationary phases and columns are much larger in prep work than in UHPLC—but in adapting factors such as solvents, gradients, and flow rates to preparative systems.
Agilent has developed an algorithm that with the assistance of “industrialized,” automated injection and fraction collection systems can seamlessly scale from analytical runs to preparative without user interaction. Users simply select the UHPLC peak they would like to isolate, and the system does the rest.
Industrialized scaling from UHPLC to prep LC will replace flash chromatography in labs that need to purify many samples at milligram-to-gram scale. Flash is rapid but requires some level of development and hands-on time. “Flash chromatography’s advantage [relative to other open column methods] is speed, but it does not provide the purity of HPLC,” says Schulenberg-Schell. “Prep LC conveys the same purification efficiency of analytical LC to purification.”
Mixed-mode UHPLC
A presentation earlier this year by Xiaodong Liu, PhD, manager of R&D, chromatography consumables at Thermo Fisher Scientific (Sunnyvale, CA), illustrates one drawback of conventional UHPLC for life science analytes. Reverse-phase has been the go-to separation mode, but it shows poor retention for hydrophilic molecules, is incompatible with aqueous buffers, and has limited selectivity. “Selectivity has the greatest impact on separation,” Liu says.
Hydrophilic interaction chromatography (HILIC) suffers from matrix effects and is unsuitable for hydrophobic analytes. “Plus the method is not rugged,” Liu adds. “It works for some separations, but for many real-world analytes it is not a good choice. And ion exchange works only for charged molecules.”
The answer to the complexity and diversity of life science samples is mixed-mode chromatography. Mixed-mode consisting of hydrophobic or hydrophilic interaction plus ion exchange on a single resin has been around for decades, but users considered it too complex and finicky. Liu and coworkers have been at the forefront of developing and applying mixed mode resins for everyday UHPLC applications where ionex, reverse phase, and HILIC fall short.
Under mixed mode separations, elution order may often be altered—even reversed—by changing mobile phase pH. Additionally, mixed mode provides baseline separation of basic, neutral, and acidic molecules in a single sample within the same run.
“We’re not trying to replace reverse phase but rather focus on specific, significant applications for mixed mode,” Liu says. He is working with the U.S. Environmental Protection Agency, for example, on developing mixed-mode analytics for drinking water.
Similarly, biopharmaceutical labs are beginning to notice mixed-mode columns for glycan analysis. In another Thermo study, mixed-mode UHPLC baseline separated enzymatically released glycans from bovine fetuin, an important serum component. Individual sugar moieties were then identified with mass spectrometry.
Mixed-mode chromatography complements UHPLC and mass detection with novel, sometimes strange separation capabilities. While it is not suitable for every situation, labs with difficult separations not currently served by HILIC, reverse phase, or ion exchange should keep an eye open for mixed-mode protocols for molecules they routinely work with.
For additional resources on UHPLC, including useful articles and a list of manufacturers, visit www.labmanager.com/uhplc