Véronique Marceau, product manager for chromatography and purification at SiliCycle (Quebec, PQ), says that trends in HPLC column technology closely follow those in the pharmaceutical industry. Specifically, vendors continue to develop stationary phases that are suitable for analyzing biomolecules— peptides, proteins, and genes—in complex matrices.
“The industry has had to come up with new materials, stationary phases, and formats,” Marceau says. SiliCycle specializes in functionalized silicacore column materials.
For example, pore sizes must reflect the large sizes of proteins and peptide digests. Small molecules can easily fit into conventional pores, whereas proteins do not.
According to literature from Sigma Aldrich (see https://bit.ly/18IyqLT), the HPLC analysis of peptides and proteins differs significantly from that of small molecules. Reverse-phase (RP) columns are the modality of choice, with C18 bonded to silica as the usual starting point. From there, investigators might try C8 or C5 to obtain the desired selectivity.
But large molecules do not exhibit a “finite partitioning equilibrium” between stationary and mobile phases. Instead, analytes adsorb into the stationary phase through differences in the molecules’ hydrophilicity. Elution occurs when the solvent strength is sufficient to overcome the attraction between analyte and column. That is why peptides and proteins are almost always run under gradient conditions. Peptides and proteins will eventually elute under isocratic conditions, but their peaks will be broad.
The other “stealth” trend noted by Ms. Marceau is the use of three- and five-micron columns in UHPLC systems designed for high-pressure, sub-two-micron particle column operation. “People love their UHPLC, the systems, the software, but three-micron columns persist. Not everyone has switched to sub-two-micron technology.”
UHPLC systems are perfectly capable of running columns designed for older systems. In fact, many UHPLC setups are routinely used in both standard and sub-twomicron HPLC. More important, laboratories are reluctant to invest time and resources for redeploying and revalidating legacy methods, particularly in regulated industries.
“UHPLC is popular, but it takes time for an entire group of analysts to switch to new technology,” Marceau observes.
Protect your investment
Stationary phase constituents and columns represent the largest ongoing costs for HPLC. For columns, the operative consideration is usable lifetime. Modern HPLC columns are sturdy, robust, and operate fine as long as users stick with recommended buffers and solvents.
Yet all chromatography columns degrade over time due to buildup of impurities, degradation of column chemistry, and plain old wear and tear. According to J.T. Presley, brand manager for consumables at Phenomenex (Torrance, CA), smaller particle sizes increase the likelihood that sample matrix or other impurities will cause degradation in performance.
“The vast majority of the time, column lifetime is a function of the sample and matrix,” Presley tells Lab Manager. Therefore, the secrets to long column life are sample preparation and column protection.
Sample preparation should be designed to maximize sample recovery while minimizing injection of “gunk.” Filtration is the minimum preparation. Many methods call for solid-phase extraction or other cleanup methods. In some instances, target analytes may be concentrated during this process.
Another strategy involves the use of guard columns, which protect columns from the nastiest contaminants. “The point is to improve the balance of performance, value, and price,” Presley advises.
Article Courtesy of Lab Manager Magazine: HPLC Columns: Following Trends in End-User Markets
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