LC at high temperature

[stanlee]

I have developed a method thats seems optimum at 70 C.

Has any one how robust this would be (column lifetime etc).

I've done a web search but can't seem to find any info.

TIA

Stanlee

[avitan]

Few weeks ago I succesfully ruined a couple of columns, with some extreme pH=9, 70C.

Optimal conditions, also supported by some application notes from manufacturer that claimed stability of stationary phase above pH=10 for 200-300 injections. Well, that's just fine ,but it was at room temperature and run time ~10 minutes. My method was a litle bit longer, and after a week of intense work it just developed backpressure above 400bar. I've opened column frit and void volume of ~1cm was discovered.

According literature the combination of phosphate buffers, high temperature (>40C) and high pH increase deterioration of silica bed.

However, contact your column vendor - I know, that Phenomenex, for example, recommends work temperature with there RP columns below 60C.

Good Luck!

[skunked_once]

Most modern HPLC C-18 columns can be used at 70 C if proper precautions are taken. One of the most important is the pH of the mobile phase. pH should be between 2 and 7; pH above 7 will dissolve the silica in the packing material and increasing the temparature speeds up the dissolution. The best practice is to check with the column manufacturer for their column recommendations. For a good explanation of column heating see "Column heating and resolution-A case study" in LC-GC vol 10(5) p. 364, 1992.

[Cliff Mitchell]

I have heard of a company called Selerity that specilizes in high temp LC, and making high temp stable stationary phases.

http://www.selerity.com/main/main_produ ... c_acc.html

I have had no dealings with them and cannot speak to the quality of their products, just passing on a link.

Good Luck
Cliff

[Uwe Neue]

Column stability at elevated temperature depends on the packing as well as the mobile phase conditions. A packing that is stable at room temperaure at pH 8 does not necessarily make it to 70 degrees C. The rule of thumb applies that column lifetime deteriorates by a factor of 3 for every 10 degrees change in temperature. This makes it a factor of 250, going from room temperature to 70 degrees C. If the column last for a year at room temperature, it will make it for 1.5 days at 70 degrees...

We have tested the stability of our XBridge columns under extreme conditions (low pH) at 80 degrees C. You can go to the Waters webside and examine the data for yourself.

For a complete judgement on the stability under your conditions, one would nee to knwo the remainder of your conditions (pH etc...).

[pawan ratra]

I have worked at such high temperatures. I have been using it for the last 6 months without any damage to the column.
But column life depends upon multiple factors and you have to be very cautious about chosing your chromatographic conditions and column before deciding whether you want to use such high temperatures. You should also ensure that only column temperature is the only option for the optimum separation. Otherwise I wll recommend you to improvise your method to keep column temp below 60.

[Rob Burgess]

Stanlee,

I'd be interested to know how you came by the method that is seemingly optimised at 70°C. Did you use DryLab perchance to reach your conclusion. We have used this software and come up with "curious" temperature extremes (65°C and above) to separate a difficult critical pair of peaks. Its not a method that is used routinely so I cannot say what the lifetime of the column is, I'm afraid.

Many column manufacturer's have a purported high temperature stable column, but I suppose in all fairness its still a try it and see approach still!

[tom jupille]

Rob: in the "tG/T" (gradient time/temperature) mode the DryLab software will only let you extrapolate 10% beyond the calibration runs on the temperature axis. In other words, if you ran your calibrations at 35 and 50 degrees, the highest temperature the program would let you model would be 52 degrees. In effect, it's the user's choice regarding maximum acceptable temperature.

As far as that choice is concerned, I think most people would prefer to run at less extreme temperatures (column lifetime is only one of the issues: mobile phase preheating and instrument-to-instrument variations are also problems). However, a method that works but has a short column lifetime is a lot better than a column which fails to work (but fails the same way for a long time).

[stanlee]

Rob, yes it was a drylab run and it also worked in real life.

I agree that its not ideal as we're not preheating eluents etc so there's no idea what temp we're actually running through the column, but it works.
Not sure for how long though !

[Uwe Neue]

If you are not preheating, the column is likely to be much cooler than you think. Of course, this all depends on your other conditions, i.e. how the column itself is heated, the flow rate etc. You could easily be at 50 degrees inside the column...