column temperature

Guest Author – Genevieve Hodson, Technical Specialist

It is common practice to leave LC columns at ‘room temperature’, or ambient temperature conditions, when running routine analytical methods. So why do chromatography companies tell you not to do this? While putting the column heater cover on and setting the methods column temperature to 25°C, which most scientists agree is room temperature, can seem like more work than leaving the column exposed to the ambient lab conditions. And if you are looking for reproducible results, this is a must.

For example, room temperature in a Quality Lab in Texas during the summer when the AC is on full, can cause the DMSO to freeze solid and chemists to wear sweatshirts under their lab coats, is very different than when the AC goes out and you are now doing stability oven studies on pharmaceuticals sitting in vials on the bench top! That would be a range of 19°C to 37°C, for those of you who do not speak Texan. A change of 15°C can have big implications towards both the methods reproducibility and the columns overall lifetime. But why? I know you are asking yourself that very question right now, so let’s talk about it!

Reproducibility

For those chemists out there reading, we are going back to good old thermodynamics (if you thought you were done with P-Chem, you thought wrong!). As the temperature changes, the thermodynamics inside the column between the stationary phase and the mobile phase is directly affected. Running a method at varying temperatures, may impact analytes capacity factor (k) or how long the analytes are retained on the column. A plot of the apparent capacity factor vs temperature is shown below for some theoretical analytes.

A plot of the apparent capacity factor vs column temperature

Plot of apparent capacity factor (k) vs temperature

At elevated temperatures, there is an increase in the kinetic energy of both the mobile phase and the analytes. The increase in kinetic energy of the analytes will cause them to be heated and could disrupt the intermolecular binding that is the separation mechanism between the analytes and the stationary phase.  Many times this causes all the analytes to come out sooner from the column, causing a reduction in the retention time. Usually this is most noticeable with large temperature jumps, example below with temperature increases in 20°C intervals.

column temperature increases in 20C intervals

Changes in temperature of only a few degrees Celsius could be seen impacting only a specific analytes in a chromatogram. In this case, it is likely that the small temperature difference is enough to push a favorable interaction with the stationary phase or with the mobile phase. It can be seen in samples as only one analyte shifting in retention compared to the others, who maintain their retention times. As it is hard to predict which analyte, if any, could be impacted and shift in retention due to the small temperature fluctuations, best practice is to maintain a consistent temperature by setting the column heater on with the column heater cover on.

Oligonucleotides provide a different. Imagining the 3D structure of this class of compounds, lets thinking about what happens when the temperature changes and they might partially unfold. The partial unfolding could expose hydrophobic groups that may have been previously buried causing the oligo to now retain more on the column. Conversely, the unfolding could expose hydrophilic groups causing the opposite retention to happen. A wonderful Technical Note has been written about this and is referenced here:

Effect of Temperature on Single Stranded Oligonucleotide Analysis

Column Limitations

Temperatures can impact a columns overall lifetime as well. The media/phase of the column are what drives it’s specification for max temperature.

A columns temperature stability can differ depending on what solvent and buffer are used. Example can be found in the table below for our Luna Omega brand column. There is a noted difference in how the C18 compares to the Polar C18 due to slight differences in the bonding of the phases. For most reverse phase column 60°C to 90°C is a common max temperature range.

Luna Omega Stability Column Temperature graph

Elevated temperatures under acidic conditions provide favorable thermodynamic conditions for hydrolysis of the ligand bonded to the silica. Hydrolysis is observed as retention times shifting earlier due to there is less phase to provide interactions in the stationary phase for the analytes. A loss of resolution occurs sooner than the overall shift in retention. It is common to see baseline resolution disappear or peaks merging together, when compared to a new column with the same phase. Best practice is to not leave columns in the oven when turned on and no solvent flow going through the system. Most mobile phases have some organic in them that will evaporate out faster.

Hydrolysis Mechanism


If you have any additional questions regarding column temperature, or other chromatographic inquires, reach out to our Technical Specialists, like Genevieve, through our free online chat service – Chat Now.

You will be able to chat with technical experts nearly 24/7 for any help you might need in the lab from method development, product assistance and recommendations, and so much more. Click the link or image below to start chatting today! www.phenomenex.com/chat

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