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Jun 1, 2012 (Vol. 32, No. 11)

Finding the Right Balance in HPLC

  • Click Image To Enlarge +
    Particles packed into Advanced Materials Technology’s HALO® columns are manufactured using Fused-Core® particle technology that was developed to deliver fast chromatographic separations.

    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.

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    Agilent Technologies’ manufacturing process generates both unimers (shown) and also a small percentage of dimers.

    While such an apparatus is now in wide use, it’s “still beneficial to the people who use the columns day in and day out to operate at lower back pressure,” says William Barber, Ph.D., Agilent Technologies’ R&D manager for liquid chromatography columns. There is less stress on the instrument and the column, therefore extending the interval between maintenance of the instrument and lengthening the effective lifetime of the column.

    Agilent has for years offered larger particles (e.g., 3.5 µm and 5 µm) with a 300 Å pore size, designed for biochromatography on standard HPLC columns and recently commercialized a 1.8 µm line of 300 Å pore size columns for use with UHPLC.

    About eight years ago Agilent tested the effect of broadening the particle size distribution on the permeability and efficiency of its small molecule (80–120 Å pore size) UHPLC columns. Its manufacturing process generates both spheres (unimers) and also a small percentage of fused particles (dimers), with the vast majority of dimers removed prior to packing the columns to market.

    Dr. Barber is currently conducting similar experiments on the 300 Å pore particles. “We systematically add dimers back in known quantities, to more systematically study what our process would look like if we left a certain amount in or varied the amount of dimers that were left in the product.”

    The results for the large molecule columns are similar to those seen for the small molecule columns. “We still see the same trend where you can add some of these dimers back and it really doesn’t have any effect on the efficiency up to about 10–20% of the dimers,” Dr. Barber says. “But you do tend to see a consistent change in permeability or back pressure right from the beginning, as soon as you start adding dimer.”

    “We haven’t done all the measurements yet,” so the results are still preliminary, Dr. Barber points out. For example, they still want to do van Deemter plots on all the materials to see how the performance versus flow characteristics change with the different mixtures; to test different size molecules to determine whether molecular weight plays a role; and to query different modes of chromatography.

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