HPLC Columns: Where the Action Is
Now that sub-2-micron columns have become the stationary phase technology of choice for research labs and have made substantial inroads in quality, food, and pharmaceutical settings, another breakthrough in column technology looms.
Now that sub-2-micron columns have become the stationary phase technology of choice for research labs and have made substantial inroads in quality, food, and pharmaceutical settings, another breakthrough in column technology looms. Superficially porous particles (SPPs), also known as fused-core, shell, porous- shell, and Poroshell (Agilent’s trademark) particles, have been around since the 1960s. SPPs were used extensively until the 1980s, when fully porous particles began their long domination.
SPPs employ a porous “shell” of silica wrapped around a solid silica core. Molecules diffuse into and out of the particles much more rapidly and efficiently than through fully porous media, which results in faster, sharper separations.
Most vendors claim that SPPs result in separations that are close, and in some cases superior, to those of UHPLC, but at back pressures typical of conventional LC.
Jason Link, Ph.D., LC columns product manager at Agilent (Wilmington, DE), believes that SPPs have revitalized HPLC for aging instruments. “Users with older chromatographs, or that are cash-crunched, can now get many of the benefits of UHPLC without having to purchase a new instrument.”
Dr. Link estimates that SPPs provide “80 to 90 percent of the efficiency of sub-2-micron columns, at 40 to 50 percent of the back pressure.” The performance boost, he says, results from improved mass transfer and the greater particle size uniformity.
Most SPPs are in the high-2- micron size range. Phenomenex (Torrance, CA) has recently introduced a sub-2- micron porous shell particle that combines the advantages of very small particles and SPP technology. These 1.7-micron SPPs are solvent-sparing, as one would expect, and result in back pressures comparable to those of UHPLC, about 1,000 bar.
Dafydd Milton, Ph.D., product manager for LC and LC/MS columns at Thermo Fisher Scientific (Runcorn, UK), says trends in column technology are all about increasing speed.
High-temperature operation is an often overlooked approach to fast LC without encountering backpressure woes. As temperature rises, solvent viscosity decreases, mass transfer increases, and analyte partitioning into the stationary phase is enhanced, resulting in shorter runs and sharper peaks.
Most stationary phases fall apart at a temperature approaching 250°C, and systems are generally not equipped for heating. “Conventional C18 bonded phases are simply not stable at high temperatures,” Dr. Milton says.
Users considering high-temperature operation will need to invest in third-party column ovens and systems for preheating the mobile phase. They will also require rugged columns.
Novel column technologies such as SPPs, hydrophilic interaction chromatography (HILIC), and monoliths provide more options, but they also resurrect sticky questions about method transfer. Labs that submit instrument results to legal and regulatory authorities live by standardized, validated methods, as do QC and other high-volume functions. And while some complaints are no doubt exaggerated, organizations can rarely justify revalidating all their analytic methods.
There is no simple answer, as labs must justify everything economically. Solvent savings and higher throughput alone might justify method transfer for a large pharmaceutical organization, but not perhaps for a forensics or environmental laboratory that makes 20 or 30 injections per week.
Recently Released HPLC Columns
ZORBAX UHPLC RRHD 300SB-C18
1.8 μm columns
- Provide stability at low pH and symmetrical peaks for protein and peptide separations with trifluoroacetic acid or formic acid mobile phase modifiers
- Increased efficiency for improved resolution and more robust quantitation of post translational modifications
- Bring UHPLC performance to protein analysis with pressure stability to 1200 bar
Agilent
www.agilent.com
Kinetex® XB-C18 and C8
- Offer advantages of UHPLC within standard HPLC operating pressures
- XB-c18 incorporates two iso-butyl side chains at the base of the c18 ligand for reduced secondary interactions with residual silanols
- C8 meets United States pharmacopeia (USP) specifications for classification code l7
Phenomenex
www.phenomenex.com
AccucoreTM
- Provide a unique chromatography solution to enhance laboratory workflow and efficiency
- Available in a wide range of stationary phase selectivities and compatible with almost any instrument, these columns give increased sensitivity and peak resolution
- Allow high-speed, high-resolution separation, with backpressures significantly lower than those columns associated with UHPLC
Thermo Fisher Scientific
www.thermo.com
XSelect
- HSS Cyano and HSS PFP columns offer scientists an alternative to traditional C18 column chemistries
- Give scientists more control over the resolving power of their HPLC separations
- Reduce time and method development costs
Waters
www.waters.com