Column Temperature – How to Maintain Accuracy and Stability

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 benchtop! 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 for both the method’s reproducibility and the column’s overall lifetime. But why? I know you are asking yourself that very question right now, so let’s talk about it!


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 the 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 of kinetic energy for both the mobile phase and the analytes. The increase in the kinetic energy of the analytes will cause them to move and vibrate more intensively and will disrupt the intermolecular binding that is the separation and retention mechanism between the analytes and the stationary phase.  Many times this causes all of 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, as in the example below with temperature increases in 20 °C intervals.

column temperature increases in 20C intervals

Changes in temperature of only a few degrees Celsius can affect only a few specific analytes within a chromatogram. Small temperature differences are enough to encourage a favorable interaction of an analyte with the stationary phase or with the mobile phase. Such specific interactions are seen in sample chromatograms as only one analyte shifting in retention compared to the others, which otherwise maintain their retention times. As it is hard to predict which analytes, if any, could exhibit shifts in retention due to the small temperature fluctuations, the best practice is to maintain a consistent temperature by turning on the column heater and attaching the cover onto the column heater.

Thermodynamics continues to impact chromatography by affecting the viscosity of the mobile phase as a function of temperature. Many chromotographers have caught on and harness this relationship for their own benefit. Generally speaking, as the viscosity of a mobile phase decreases, the backpressure on the column will directly decrease, allowing the method to flow at higher flow rates without reaching the max pressure of the column or instrument.

One of the reasons that Acetonitrile is favored over Methanol would be the lower viscosity of acetonitrile  (0.37 cPoise compared to methanol’s 0.60 cPoise). When methanol is used as the organic solvent during reverse-phase LC, methods tend to have temperatures of 40 – 60 °C to help reduce the backpressure by reducing the viscosity.

The unfolding of large biological analytes can vary causing their 3D structure to change. Denaturing, or unfolding, can happen due to a number of factors, one of which is temperature. The partial unfolding could expose hydrophobic or hydrophilic groups that may have been previously buried within the folded structure, which could then impact the overall retention time of the analytes. Oligonucleotides provide an example of higher temperatures causing an overall reduction in the retention time. 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 column’s overall lifetime as well. The media/phase of the column is what drives its specification for max temperature.

A column’s temperature stability can differ depending on what solvent and buffer are used. An 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 columns, 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 that shift earlier due to the loss of stationary-phase that provides retentive interactions for the analytes. A loss of resolution between peaks will occur sooner than the overall shift in retention times.

It is common to see either a loss of baseline resolution or peaks merging together when compared to a new column with the same phase. The 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 when the column is stored improperly.

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!

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