High-performance liquid chromatography (HPLC) columns are 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. 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. Systems employing particle sizes below 2 microns are referred to as UHPLC (ultra-HPLC), one of the most significant trends in HPLC column technology.
UHPLC has prompted a move toward ultra-low metal content, highperformance, spherical particles and a general trend toward lower particle size columns, as well as significant 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. In addition to the higher cost, methods developed with conventional-sized particle columns do not transfer to sub-2-micron or UHPLC format. For these reasons, hints Michael McGinley, bio-separations product manager at Phenomenex (Torrance, CA), pharmaceutical companies with hundreds of instruments running validated methods have been reluctant to jump onto the sub-2- micron bandwagon.
Advocates of new fused-core column technology claim it provides all the performance of sub-2-micron particles but at normal pressures. 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 performance equivalent in many cases to UHPLC, McGinley says. Fused core has caused potential purchasers to rethink their need for a new HPLC, McGinley adds. “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 have replaced older HPLC systems with those that can handle both conventional columns and ones that generate high backpressures. “Everyone will soon have higher backpressure capability,” McGinley says.
Fused-core particles, says Wayne Way, marketing manager at Sigma- Aldrich (Bellefonte, PA), produce more rugged columns, particularly when compared with sub-2-micron technology. “They’re great for openaccess instruments... and provide an easy transfer of methods.” Methods developed on UHPLC or conventional HPLC cannot be transferred unless the two labs have the same instrument. With fused-core particle columns, different labs need only have the same column.
• Aids in the analysis of compounds that are usually difficult to retain using conventional C18 columns
• Available in 1.9 μm, 3 μm and 5 μm particle sizes
• Highly organic mobile phase contains low salt levels, making them ideal for electrospray mass spectroscopy
• Ideal for applications within drug discovery, food testing and environmental analysis
Thermo Fisher Scientific
• Provide extra resolving power for a wide variety of sample mixtures
• Exceptional bonded phase stability and ultra-low bleed characteristics make them suitable for LC/MS
• Achieves baseline resolution of all compounds in a sample when other C18 columns fail
• Bonded to the silica is a proprietary phase consisting of a C18 chain with integral phenyl functionality
• Lux Cellulose-4 is a novel cellulose tris (4-chloro-3-methylephenylcarbamate) phase
• Lux Cellulose-3 uses cellulose tris (4-methylbenzoate) as the selector
• Deliver high loading capacity and are pressure-stable up to 300 bar
• Offered in 3 µm and 5 µm particle sizes
• Operate at high pH, as well as low and mid pH
• For use with Jasco X-LC™, Agilent 1290® or Waters Acquity UPLC® systems
•Small particles will not lose efficiency even at higher linear velocity
• Fully scalable with proprietary C18 analytical 3 µm, 5 µm and 10 µm particles