Retention time, the period it takes for a compound to travel through the chromatography column to the detector, is a key metric in liquid chromatography. Obvious factors impacting retention time include column size, column packing, and flow rate, but there are other considerations to take into account.
Here, we delve into column retention time and some of the key factors to keep in mind.
Dead Volume of the System
The question around which factors impact retention time was posed in a Chromatography Forum discussion. In response, forum expert lmh noted that a primary concern is system dead volume.
"Any dead volume between the pump mixer and the column delays the arrival of a gradient at the column, and in isocratic chromatography, any dead volume between the injector and the column delays the arrival of the sample at the column," explains lmh, adding that dead volume at the column's other end delays peak arrival at the detector. "If you really want the same retention time, you need to use not only exactly the same column and solvents, you need to make sure your HPLC is also identical."
Solvent Composition
The solvent used to dissolve the sample can significantly impact retention time. "If you are doing isocratic chromatography, it's probably ideal to dissolve the sample in the mobile phase," says lmh. "If you dissolve the sample in a less-eluting solvent than the mobile phase, there will be little or no change in retention time. If you dissolve the sample in a more eluting phase, the worst-case scenario is that it will elute early (because it is carried into the column in a "bubble" of high-strength solvent before the solvent mixes with the real mobile phase and dilutes it sufficiently for the analyte to bind to the column). This also messes up peak shape."
Column Equilibration
A relatively common occurrence, as highlighted in an HPLC Solutions article by John Dolan, is retention time drift during the first few injections of a new column. This drift usually stabilizes within roughly half a dozen injections. Dolan explains that the issue is linked to column equilibration and isn't typically fixed by extending the initial equilibration period. He notes that in reversed-phase HPLC, samples are separated based on their distribution between a non-polar stationary phase and a polar mobile phase. However, multiple retention mechanisms can be at play, not just the primary hydrophobic retention.
Dolan offers the example of older type-A silica columns with acidic silanol groups acting as cation-exchange sites, causing peak tailing. In contrast, modern type-B silica columns have fewer of these sites, reducing tailing, but retention time drift can still occur initially as active sites get saturated with repeated injections.
To mitigate the issue, Dolan suggests accelerating the equilibration by making several rapid injections or injecting high-concentration samples to quickly saturate active sites. He advises that it's also a good idea to ignore the first injection as it often doesn't interact with the column the same way as subsequent ones.
Temperature
An additional consideration highlighted by Dolan in another troubleshooting article is temperature. "Temperature changes often show up as a diurnal change, particularly if the column is not operated in a column oven," explains Dolan. "As the temperature increases, retention decreases—approximately 2% per 1 ºC temperature increase."
He recalls one experience working in a lab without air conditioning that would heat up an additional 5–10 ºC in the summer. "Retention times decreased when this occurred, but they increased again at night when the lab cooled off," says Dolan. "Even labs with better climate control may have different day and night thermostat settings, and this can cause temperature cycles that correlate with retention changes."
To minimize temperature-related problems, Dolan advises using a column oven and keeping the HPLC system away from drafts.
Conclusion
Column retention time is not without uncertainty. "Generally columns age, life isn't perfect, solvents are never quite the same, and it is really quite hard work to make a method so resilient that it can be transferred from lab to lab without any change in retention time," advises lmh.
While the consistency of retention time is theoretically achievable, practical factors such as system dead volume, solvent composition, and temperature can influence the amount of time it takes for a compound to elute.
By understanding and managing these factors, chromatographers can achieve more reliable and reproducible chromatographic results.
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