High-performance liquid chromatography (HPLC) columns are considered the “heart” of the instrument used to transport the analyte and the mobile phase and provide the environment in which separation is achieved.
HPLC columns are stainless steel tubes generally 30 to 300 mm in length with internal diameters of 2 to 5 mm, internally coated with a stationary phase. 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.
Reverse-phase and normal-phase chromatography separation methods are based on polarity. Reversephase columns separate analytes based on their hydrophobicity, with the more hydrophobic compounds being retained longer on the column. Separations based on charge utilize ion-exchange chromatography.
More recently, many companies have introduced hydrophilic interaction chromatography (HILIC) columns for analysis of polar analytes. Reverse-phase is the most popular method among survey respondents. Reverse phase
|Hydrophilic interaction (HILIC)||7%|
|Gel permeation (GPC)||7%|
|Gel filtration (GFC)||5%|
As particles decrease in size from conventional 10-, 7-, 5-, and 3-micron diameters, back pressure buildup increases exponentially. Thus, a 3-micron column 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 further reducing particle size (to below 2 μ), more expensive hardware is required to handle extremely high pressures. Such systems are referred to as UHPLC, a significant trend in LC column technology.
|Narrow-bore (1 to 2 mm diameter)||20%|
|Large ID (>10 mm diameter)||11%|
|Capillary (<0.3 mm diameter)||11%|
Of the three common chain lengths, C4 is generally used for proteins and C18 is used to capture peptides or small molecules. Peptides are smaller and need longer chain lengths to be captured, so C8 and C18 are appropriate.
In reverse-phase HPLC, the stationary phase is often a silica-based packing covalently bonded with hydrophobic alkyl chains of C8 (octyl group) or C18 (octadecyl group), though there are many variations on this theme.
|C18 (polar end-capped)||10%|
It’s a challenge is to pick the right column to analyze the right sample correctly. Several factors, including particle and pore sizes, can affect separation efficiency, inertness, resolution, solvent usage and more.
Lab professionals need to know that the column will elute the analyte peaks at the same time, every time. Along with elution times, getting good peak shapes—sharp, narrow, symmetrical peaks—is important for various applications.
|Technical performance of HPLC columns||98%|
|Shorter run times/Increased throughput||94%|
|Lot-to-lot reproducibility of HPLC columns||93%|
|Ruggedness/durability of HPLC columns||92%|
|Lower operating costs (reduce solvent use and waste)||87%|
|Reputation of column manufacturer||75%|
|Purchase price of column||72%|
|Breadth of HPLC column offering (selectivity)||70%|
|Method validation/compliance support||57%|
|Safety and health features||70%|
In an effort to be economical, many users are moving to smaller columns, packed with smaller particles (sub-2 μ) because they use less solvent. However, slower, longer columns that offer better resolution are sometimes preferred to separate sample components in extremely complex samples.
Since high-pressure instruments work with both conventional and UHPLC columns, users might prefer an instrument with greater capability even if they don’t yet need its higher-end performance. Some vendors have discontinued older HPLC systems in favor of those that can handle both conventional columns and ones that generate very high back pressures.
|What can we do to reduce analysis time and increase resolution?||21%|
|How do I determine which column(s) makes the most sense for my lab?||19%|
|What should I consider when selecting a column(s) for faster throughput and higher resolution?||18%|
|What are the considerations for selecting a column(s) to achieve longer column life/retention?||18%|
|Are newer models of LC columns significantly better for developing faster LC methods?||15%|
|What type(s) of column(s) could be used to reduce solvent use and waste?||12%|
Completed Surveys: 311
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