Translator for HPLC HINTS and TIPS for Chromatographers

Friday, July 20, 2012

Column Temperature in HPLC / UHPLC / LC-MS

Let us not forget the role of temperature in liquid chromatography. Just as mobile phase composition changes are used to develop better methods, column temperature is an important chromatography variable which must be addressed. I would like to call to your attention to a few different ways temperature can change your chromatography in this "hint and tip".

(1) Stability & Reproducibility of the Method: 
Maintaining a stable column temperature during a separation is important. Excellent temperature stability can lead to a high degree of reproducibility (*Their are of course many other factors to consider as well). For a typical analysis, temperature stability of 1.0 °C / hour (over the course of the analysis) is usually enough. If you are not using a thermostatted column compartment to perform your chromatography you may have already noticed the hour-to-hour or day-to-day fluctuations which can result from running samples under ambient temperature conditions. The normal changes in room temperature can be several degrees C over an eight hour period. These types of temperatures changes can make it impossible to achieve reproducible results for some samples. It is for this reason that it is critical that you include some type of temperature control as part of your method. Always record the temperature at the start and end of each run and include this data with your report. Most of the automated chromatography data systems provide this data as standard today and it is very valuable in reproducing the data as well as for troubleshooting, if needed.

(2) Back Pressure:
Column back pressure is directly changed by temperature. As the temperature rises, the column back pressure decreases. As the temperature decreases, the back pressure increases. This can be a useful variable when working with some of the newest sub-two micron particles on the market. The very high back pressures produced by these particles can be significantly reduced by increasing the column temperature [See "Pressure Drop Across an HPLC Column"]. 

When practical, try experimenting with your method by increasing the temperature, in increments of 5°C, to measure the change. You may discover an improved method with lower back pressures, a shorter run time and sharper peaks.

(3) Viscosity: 
Viscous mobile phase systems can take advantage of using higher temperatures to reduce the overall system back pressure. Since efficiency often improves with higher temperatures a double bonus of higher efficiency (sharper peaks) and lower back pressure can be achieved just by increasing the column temperature (peaks sometimes change elution order too so use standards to check this). 

(4) Practical Considerations:
Their are limits to using higher temperatures in chromatography which must be respected. The stability and solubility of your sample, the boiling point of your solvent, the maximum temperature setting of your column heater (mobile phase, flow cell and the rest of the HPLC system) and the stability of your column over time will determine how far you can safely push this.

(5) Specifications: 
One other issue worth mentioning here is that many traditional silica columns can loose their bonded phase at temperatures above 60°C. Some specialty silica phases (i.e. Waters XBridge & Zorbax StableBond) have temperature ratings to ~ 90°C. The more exotic non-silica based supports (e.g. Zirconium, graphitized carbon and/or PSDVB) often provide poor efficiency compared to the silica based products, but can handle temperatures in excess of 100°C

*Always consult with the column and/or instrument manufacturer to determine what the correct and safe operating conditions are before using any instrument, column or chemical.