Don’t Overlook LC Column Temperature!
11 Dec 2022Many things can affect peak shape and retention times, such as column size, mobile phase additives, and analyte characteristics. Column temperature is an important and often overlooked factor when it comes to LC method development. This is because column temperature can impact peak shape, retention, and backpressure. Regarding retention time, as the temperature of the column is increased, the retention time is decreased. This is because, the higher the temperature of the column, the faster the analytes are exchanged between the stationary phase of the column and the mobile phase. As this process becomes faster, analytes are not retained as long, resulting in shorter retention times, and often more efficient peak shapes. However, keep in mind that increasing temperature does not affect all analytes equally, meaning that the impact of retention factor may be different for each analyte. For example, in Figure 1 and Figure 2 below the same method and same column were used but the methods were run using two different column temperatures, 30oC and 55oC. In this example, shorter retention times are noted for all of the analytes up to a difference of 0.19 minutes.
Figure 1: Fatty acid ethyl esters on Force Biphenyl 50 x 3mm, 3 µm at 30oC.
Figure 2: Fatty acid ethyl esters on Force Biphenyl 50 x 3mm, 3 µm at 55oC.
However, if you are having trouble separating peaks, or want to get better resolution between peaks, it could help to try a lower column temperature. Just as increasing the column temperature can cause the retention times to be shorter because the speed of interactions on the column has increased, lowering the temperature will do the opposite, which in turn can help with the resolution of analytes. There are also some analytes that show better separation with a lower column temperature. For example, some cannabinoids such as CBDA, CBGA, CBC, and THCA show best resolution at a lower temperature. In the example below, the first chromatogram the compounds were analyzed with a column temperature at 40oC and there is no baseline resolution between CBDA and CBGA or CBC and THCA however, when run with a column temperature of 30oC, the compounds are better resolved. This can be seen in Figure 3 and Figure 4 below.
Figure 3: Chromatogram of cannabinoids at 40oC column temperature.
Figure 4: Chromatogram of cannabinoids at 30oC column temperature.
While the above is true in most cases, there are certain instances where a higher temperature works better for both shorter retention times and analyte resolution. Ergot alkaloid epimers and PFAS compounds show best results with a higher column temperature. In the example below (Figure 5), ergosine and ergosinine were analyzed using a Raptor Biphenyl 100 x 2.1mm, 2.7µm column at 50oC and 60oC. At 50oC there is no separation of the two analytes, but at a higher column temperature, 60oC, the two analytes begin to separate.
Figure 5: Effects of column temperature on ergot alkaloid epimers.
Column temperature can also affect the back pressure of the column. Because high mobile phase viscosity can cause high back pressures, raising the temperature of the column can help to decrease the mobile phase’s viscosity and therefore, lower the back pressure. For example, in Figure 1 and Figure 2 above, the starting conditions, mobile phases, and gradient conditions are the same for both. However, the column temperature was different for both. When the column temperature was set to 30oC, the pressure was 2100 psi. When the column temperature was set to 55oC, the pressure was 1500 psi, yielding a different in psi of approximately 600 psi.
While higher column temperatures can help with peak shape, and shorter retention times, it is important to not go above the recommended temperature for the column. Using too high of a temperature on a column can lead to a drastically shortened column lifetime. You can usually find the maximum temperature for your column in the manufacturer’s column usage guide.
So, remember, though it is often overlooked, column temperature is an important parameter to optimize whenever you are setting up a method. It can take a bit of trial and error to find the right column temperature for the analytes on your method, but considering this parameter in early method development can lead to better resolution and more efficient methods.