Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Separating molecules by liquid chromatography is a tried-and-true method and is most often performed using HPLC systems. But higher-powered ultra-HPLC (UHPLC) is gaining rapid ground.
UHPLC (or as Waters calls it, UPLC) is a specialized chromatographic method that runs faster, resolves better and uses less solvent than its cousin, HPLC. UHPLC accomplishes this by using a smaller column packed with smaller particles (usually less than 2 µm in diameter). Researchers in biochemistry, cell and molecular biology, clinical medicine and many other areas rely on UHPLC to separate different types of molecules from a mixture—whether these molecules are proteins, peptides, metabolites, pharmaceutical compounds or other chemicals.
Despite its apparently better performance, though, UHPLC isn’t always the best option. Here we help you separate what's important from what's not as you consider your switch to UHPLC.
Why UHPLC?
Although UHPLC and HPLC both have advantages in various circumstances, there are times when UHPLC is clearly the best option. In particular, UHPLC provides better resolution because of the smaller column particles. But researchers don’t always need that kind of power. D.J. Tognarelli, liquid-chromatography applications specialist at Jasco Analytical Instruments, recommends considering which separation will provide the best efficiency in a manageable amount of time. “If you are separating two components in five minutes and only running a few analyses per day, UHPLC is unlikely to be necessary,” he says, because HPLC resolution is both sufficient and relatively rapid.
Even if you don’t need more resolving power, UHPLC can still increase your throughput. “The higher chromatographic resolution can be used to increase the separation power for complex samples, or to increase the speed of an analysis, by using a shorter column and maintaining the resolution compared to [an] HPLC column,” says Michael Frank, senior director of global marketing in the liquid-phase separations business at Agilent Technologies. “So, either high-throughput applications or high-resolution analysis will benefit the most from UHPLC.”
Depending on the type of packing material inside the column (also known as resin, or the stationary phase), UHPLC can separate compounds based on their molecular size, polarity or electrical charge.
UHPLC is not without drawbacks, however. Because UHPLC uses smaller particles in the column, it is not suitable for dirty or unfiltered samples. “The smaller-particles columns are an excellent filter,” says Frank, noting that using unfiltered samples might result in more “downtime because of clogged columns or other parts.” Clogs cost time and money, but advance preparation does, too, so if your samples aren’t very pure, you might need to weigh the costs. “Generally, sample and mobile-phase cleanliness needs to be higher for UHPLC, and this can add an additional overhead to the preparation of solvents and samples,” says Tognarelli.
Tognarelli adds that UHPLC is a good choice when it gives better performance than HPLC, for example, “in terms of speed for an individual analysis where a quick result is needed, for dealing with large numbers of samples [or] to improve the efficiency of a long or difficult separation.”
Considerations for choosing UHPLC
Migrating from HPLC to UHPLC isn’t simply a matter of buying a new column; you might also need a hardware upgrade. Because UHPLC uses smaller particles within the column, more force is required to drive the solvent and sample through the column, for instance. Thus, UHPLC requires pumps that can function at higher pressures than those used in HPLC. “Smaller-diameter column particles generate higher back pressures, requiring a stronger pump,” says Tognarelli. This may mean the additional purchase of a new pump, a dedicated UHPLC system or a liquid-chromatography system with both HPLC and UHPLC capabilities.
On the other hand, though the initial cost of a UHPLC system is usually higher than its HPLC counterpart,total cost of ownership may actually be lower. UHPLC systems can produce faster results, with less reagent, compared with HPLC systems, and increased throughput can move productivity forward. In this case, the higher initial cost of a UHPLC system might actually save lab funds over several years.
Adapting protocols
If you are new to UHPLC, be aware that the HPLC protocols you used previously probably cannot be transferred directly to the new system without making at least a few changes. Adapting an old and trusted HPLC protocol for use in a new UHPLC system can be challenging. There are enough differences between the two systems (i.e., they usually differ in column lengths, column diameters, flow rates and gradient times) that your old protocol likely will need some tweaking.
Luckily, there is help available—UHPLC vendors have experience adapting protocols and many offer online tools for translating an HPLC protocol to UHPLC. For example, Agilent offers the “Agilent 1200 Infinity Series Method Translator and Cost Savings Calculator”, which has two helpful components for users of Agilent’s 1200 Infinity Series instruments. The Method Translator can help you adapt your old HPLC protocol into a newer UHPLC protocol. The Cost Savings Calculator estimates the savings you may derive from using an Agilent Infinity instrument vs. a generic “conventional liquid chromatography” system.
Agilent also offers a software tool called Intelligent System Emulation Technology (ISET), “a software setting that allows a 1290 Infinity UHPLC LC to emulate the exact behavior of other LC systems, like a conventional HPLC system,” says Frank. ISET lets users run both new UHPLC protocols and their well-validated HPLC protocols on one system. This can be handy, because “the old HPLC system can be retired without the immediate need to retire or modify all existing methods,” says Frank. ISET also can be valuable for labs that are verifying the work of, or collaborating with, other labs using HPLC/UHPLC.
Future developments
Despite their differences, new types of column-packing materials are beginning to blur the boundaries between HPLC and UHPLC. For instance, new “core-shell” or “porous” particles offer much better performance than conventional column particles for both HPLC and UHPLC. Core-shell particles have more surface area, which means they outperform their conventional particle cousins at lower operating pressures. “These can give comparable efficiencies to sub-2-µm packing materials without some of the headaches of running at higher pressures,” says Tognarelli.
This means that using core-shell particles (such as the Kinetex core-shell particles from Phenomenex), it is now possible to achieve the “ultra” efficiency of UHPLC using the lower pressures—and maybe even the equipment—of HPLC. “For existing users of HPLC who want to improve separation efficiency and are already using 3-µm particles, a switch to [core-shell particles] may yield significant improvement without having to invest in expensive hardware,” suggests Tognarelli.
Balancing equipment costs and performance is becoming more flexible for HPLC and UHPLC. And whether you need straightforward, high-throughput separations or higher resolving power, UHPLC is growing to meet more types of needs than ever before.