Everything in chromatography is a compromise. It’s like a game, trying to come up with the best system of compromises that gives you the optimum platform for the separation process.
“There are lots of ways you can do it,” explains Jack (J.J.) Kirkland, Ph.D., vp for R&D at Advanced Materials Technology (AMT), who will be among those speaking at CASSS’ “HPLC” conference later this month. “What you try to do is balance all the parameters that are involved and try to come up with the best thing you can."
The theory suggesting that smaller particles in an HPLC column increase the ability to separate components goes back at least to the early 1960s, recalls Dr. Kirkland.
Another critical parameter is that “you want enough surface area so you can load a sample that you need, and particles creating the required surface area to give you the kind of retention that you need.” By the early 1970s, both of these needs were addressed with small—5–7 µm—totally porous particles. Pores wending through the particles increased the surface area.
But the further a molecule has to diffuse into the totally porous shell, the more time it takes. “More time for diffusion means a broader band, and that means less column efficiency,” he notes. This is especially important for larger molecules such as peptides and proteins for which diffusion can be rate-limiting for separation speed.
But what if you could make smaller particles—say, 2.7 µm—with pores that don’t go all the way through, so that the diffusion path is much shorter? Such superficially porous particles (SPPs)—which AMT introduced in 2006—allow “you to run faster mobile phases, increase the efficiency of the system, and reduce the separation time,” Dr. Kirkland says. They have an efficiency equivalent to totally porous particles of 1.8 µm.
The reduction of surface area means that the amount of sample that can be loaded onto SPPs is less than what totally porous particles can handle. But Dr. Kirkland notes that because mass spectrometry (MS)—which many people are doing—doesn’t require a lot of sample, this isn’t a big problem.
AMT recently introduced 2.7 µm SPPs with wider pores to insure unhindered access of larger solutes to the porous shell for rapid, efficient separations. Because the pressure required to drive the mobile phase through the column goes up with the square of the particle size, 2.7 µm is about as small as particles can be and still run on conventional HPLC equipment—sub-2 µm particles require expensive, specialized high-pressure (UHPLC or UPLC) equipment with optimum separation speed, he points out.