Chromatography Column Selection Guide

With thousands of chromatography columns to choose from, selecting the right one for your needs can be a challenging process, especially as there are many stationary phases and mobile phases available. The C18 column (a HPLC column) is the most common, but even it can be customized in a number of ways to fit individual needs.

There is a lot to consider when buying a column, not only are the type and the phases important (including the packing density of the stationary phase), but you also need to consider the application, your analytes, and the column hardware that you want. For this article, we spoke with chromatography column experts from Shimadzu, MilliporeSigma, and Waters Corporation to discuss some of the main factors that guide the decision-making process when purchasing a new column.

Challenges of purchasing a column

There are many things to consider, and the needs vary from person to person and application to application. For example, Sue Steinike, Consumables and Aftermarket Product Manager at Shimadzu Scientific Instruments, believes that “there are too many choices and it’s like picking out paint. It can be overwhelming to decide which C18 column you want when there are different carbon loads, pore sizes, and surface areas to consider.”

On the other hand, Tom Elich, Manager of MSAT Purification Process Engineering at MilliporeSigma, believes that finding the right column dimension can be tricky and “the desired column may not be offered. This usually requires the process developer to round-up their column dimension to the nearest size offering, resulting in an over-sized column for their specific needs.”

Finally, Ken Berthelette, Senior Scientist at Waters Corporation, explains that “the commercial availability of so many columns can make it confusing, and each one will have a different selectivity based on its metal content, ligand density, pore volume, and phase ratio.”

Given that there are several challenges for scientists, we look below at how the selection process can be navigated and what you need to primarily consider when choosing a column.

Choosing the right column length

The column length determines how quickly the analytes pass through and determines the separation and resolution of analytes in a sample. So, it’s an important factor and the decision to choose a long or short column length is mostly guided by resolution requirements.

If an assay has only one peak, a longer column may not be necessary. Conversely, a complex sample with a lot of analytes may need that longer column to improve the resolution of the separation. “However, longer columns do come at a cost of longer run time and more solvent usage,” Berthelette notes. Longer columns are also useful for analyses when you have a larger sample because you can fit more compounds into the elution channels and still get a good enough resolution.

A shorter column is better for a fast analysis and for when you’re using a smaller number of analytes. Additionally, using a shorter column with small particle size stationary phase is a way of achieving high performance without sacrificing resolution but it’s not a suitable approach for all analytes, as some will only work with longer columns.

Even if you want a shorter column, you need to make sure that it’s not too small otherwise you will get an incomplete resolution that will affect the detection and quantitation of each analyte, and you might not be able to differentiate the different elution peaks. It’s therefore critical to choose the right column size for your need because if you choose the wrong column length, you could end up with a back pressure that is too large for your column to handle, an unnecessarily long analysis time, or poor resolution due to the broadening of elution peaks.

Mobile phase vs stationary phase

The mobile phase and stationary phase are two of the most important considerations when it comes to choosing a column, because the mobile phase (most commonly comprised of different ratios of water and organic solvents) is responsible for transporting the analytes through the column and the stationary phase determines how big the pores of the column are and to what degree the analytes interact with the column. Both the mobile and stationary phases are key for separating out different analytes as different molecules will interact differently with both the mobile and stationary phase.

Choosing a stationary phase

Stationary phases come in all forms and are composed of a packaging material with organic functional groups on their surface. The base packaging material are usually made of silica, silane, polymers, glass, or a resin and form the pores of the column. The organic ligands on the surface typically consist of alkyl chains that interact with the analytes. While C18 alkyl chains are the most common, the choice extends from C1 to C30 ligands, as well as aromatic phases, chiral, achiral, and ion exchange ligands.

When choosing a stationary phase, you need to consider the binder capacity of the ligand, but also the required pore size (based on the molecular size of your analytes) of the stationary phase. For analytes, you need to consider their hydrophobicity, isoelectric point, and compatibility with different ligands.

For matching ligands with analyte, “The best thing to do is match the phase with the compound type,” explains Steinike. “Nonpolar, non-aromatic compounds work well on a C18. Compounds with rings work well on a phenyl phase. Polar compounds work with polar phases like amino, cyano, or hydrophilic interaction liquid chromatography (HILIC).”

When it comes to choosing the pores, the size of the molecules being analyzed is the key driver. Antibodies will diffuse through resin structures that have 100 nm pores, but viral vectors, peptides, proteins, and plasmid DNA will require much larger pores (often 400 nm or greater) so will need wide pore columns.

Choosing a mobile phase

“The mobile-phase selection is dictated by stationary-phase selection,” Elich says. “If you are using ion exchange or hydrophobic interaction chromatography, mobile-phase characteristics such as pH and conductivity are critical, and a molecule’s hydrophobicity and isoelectric point are useful for choosing a mobile phase. One example is a monoclonal antibody with an isoelectric point of 8, the mobile phase must be less than pH 8 but higher than pH 6 to maximize binding, so having the right pH in your mobile phase is key.”

You also need to be aware if your analytes are soluble at low organic concentrations (5–10%) for the mobile phase you’re considering. Another aspect is cost. Methanol is the preferred choice for phenyl and biphenyl stationary phases, but it generates more backpressure in the column. Acetonitrile is an alternative to methanol and is a stronger solvent but is more expensive. Both methanol and acetonitrile are common solvents for reverse-phase chromatography.

In the end, the key factors are cost, pH, the elution strength of the solvent (for promoting selectivity), and the solubility of the analytes in the mobile phase because you want them to move effectively through the column.

Picking a column based on the application

While most columns are general purpose, and the choice is directed by the column size, mobile phase, and stationary phase, there are also columns that are geared toward specific applications. Both gel permeation chromatography (GPC) and size exclusion chromatography (SEC) columns are used for the size exclusion of certain molecules and not the interaction between the analyte and the stationary phase. Ion chromatography columns are also specifically designed for cations and ions, whereas chiral columns are used when you’re trying to separate out chiral compounds.

Conclusion

There are a range of columns available to scientists, and many can be customized with different column sizes, stationary phases, and mobile phases. Many of these are suitable for general use but other columns are available when analytes are trickier to elute than normal (ions or chiral compounds for example). While there’s a lot to think about, each of these aspects need to be considered, otherwise it could end up a costly endeavor (in both time and money) if the wrong type of column is selected. The buying process can be a tricky landscape to navigate, and even if you have all the answers to the factors mentioned here, working with column suppliers to communicate your needs is often the most effective approach—but it always helps to have a good idea of what your specific requirements are.

Top Column Selection Tips

• Consider resolution requirements when selecting column length, as longer columns can offer better resolution but also come with longer run times and more solvent usage.
• When choosing a stationary phase, consider the binder capacity of the ligand, the required pore size based on the molecular size of your analytes, and the hydrophobicity, isoelectric point, and compatibility of your analytes with different ligands.
• Compatibility can be matched with the compound type, for instance, C18 is suitable for nonpolar, non-aromatic compounds, and polar compounds work with polar phases like amino, cyano, or HILIC.
• Whenever possible, try several different stationary phases like a C8, C18, and Phenyl or Bipehnyl with the same mobile phase to see how the selectivity, resolution, and run time are affected
• Take into account the needs of your individual application when choosing a column, as different types of columns may be better suited for different types of assays.
• Don't be afraid to seek out expert advice from professionals who work with chromatography columns, as they can help guide you toward the best column for your specific needs.