Product Focus: HPLC Columns

High-performance liquid chromatography (HPLC) columns are rightly considered the “heart” of the instrument because that is where the separations occur. Columns consist of stainless steel tubes with inlet and outlet openings. Plastic or glass may also be used, but steel supplies the highest mechanical strength. Conventional columns are filled with porous particles coated with a polymeric material that interacts with the injected sample. In contrast to gas chromatography columns, HPLC has a true stationary phase: column “chemistries” are bonded tightly to the base material and do not bleed off.

Go with the flow

Understanding trends in HPLC column technology requires some background on liquid flow through tightly packed particles, which is a feature of all HPLC columns. All other things being equal, the smaller the particles, the more efficient the separation. But as particles decrease in size from conventional 10-, 7-, 5-, and 3-micron nominal diameters, the backpressure buildup increases exponentially. Thus, a column using 3-micron particles is about twice as efficient as a 5-micron column, but attendant pressures are three times as high. While additional separation efficiencies are possible by reducing particle size even more, to below 2 microns, more expensive hardware is required to handle the extremely high pressures. Such systems are generically referred to as UHPLC (ultra- HPLC), which represents one of the most significant trends in HPLC column technology. Note that the many vendors of UHPLC columns and systems use unique, proprietary names for their products.

Accompanying UHPLC has been the move from first-generation stationary phases toward ultra-low metal content, high-performance, spherical particles and a general trend toward lower particle size columns, even for instruments operating at conventional pressures. Together, these changes and others related to column dimensions and instrument pressure ratings have brought about tenfold improvements in HPLC performance, according to some experts. As a result, users can expect shorter run times, cleaner separations, sharper/taller peaks, and improved detection limits.

Yet UHPLC is not without its drawbacks. Users bristle at the higher cost, and methods developed with conventional- sized particle columns do not transfer to sub-2-micron or UHPLC format. For these reasons, hints Michael McGinley, bioseparations product manager at Phenomenex (Torrance, CA), pharmaceutical companies with hundreds of instruments running validated methods have been somewhat reluctant to jump onto the sub-2-micron bandwagon.

Biggest thing in column technology?

Recently, several vendors have introduced fused-core column technology that advocates claim provides all the performance of sub-2-micron particles but at normal pressures. Vendors name their fused-core columns uniquely, but the technology remains the same. Instead of a porous particle, fused core employs a solid silica particle covered with a layer of porous silica, which is then infused with the bonded phase. This has the effect of shortening the path length of solutes into and out of the particles and decreasing backpressure relative to UHPLC while providing, according to McGinley (and other vendor firms), performance equivalent in many cases to UHPLC.

Fused core has caused potential purchasers to rethink their need for a new HPLC, McGinley says. “Now they can use the same instrument they’ve had for ten years and not rock the boat.” But users who are planning to purchase a new system anyway are probably better off purchasing one that can handle higher-pressure columns, because “the distinction between UHPLC and HPLC has been blurring.”

Since high-pressure instruments work with both conventional and UHPLC columns, users might prefer the instrument with greater capability even if they don’t yet need its higher-end performance. Some vendors, he adds, have discontinued older HPLC systems in favor of those that can handle both conventional columns and ones that generate very high backpressures. “Everyone will soon have higher backpressure capability,” McGinley says, although not all will absolutely need it at the time of purchase.

Wayne Way, marketing manager at Sigma-Aldrich (Bellefonte, PA), calls fused-core particles the “biggest trend” in HPLC column technology. “Users were skeptical at first, but today they view this technology quite positively.” According to Way (and my own independent research), review articles supporting performance claims for fused core abound in the literature.

Fused-core particles, Way tells Lab Manager Magazine, produce more rugged columns, particularly when compared with sub-2-micron technology. “They’re great for openaccess instruments that take a lot of abuse, and provide an easy transfer of methods.” Way is referring to the fact that methods developed on UHPLC or conventional HPLC cannot be transferred unless the two labs have the same instrument. With fusedcore particle columns, different labs need only have the same column.

Angelo DePalma holds a Ph.D. in organic chemistry and has worked in the pharmaceutical industry. You can reach him at angelo@adepalma.com.

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