Chromatography Forum

Dear members of this forum,

Yesterday I had a serious problem with the pressure in the column while I was injected. The pressure was 110 Bar and it started to increase until when it was 150 Bar I stopped; the mobile phase was Phosphate Buffer pH 7.2 with triethylamine (0.2 %) and Acetonitrile (50:50).
The column is Lichrospher RP-18 from Agilent. I changed the mobile phase to water and washed during 30 minutes (2 ml/min) at 60ºC (the pressure was below to 200 Bar) when I change to methyl alcohol the pressure increase to high values (upper to 200 bar) and I decided to stop. I do not understand because organic molecules were attached to this kind of column but the change of solvents behave against the polaritiy.
I would appreciate any help in order to restore the column because it is new, not more than two months. Other important point, I had worked with precolumn and I worked to pressure about 400 Bar.
Thanks in advance for your help,

Diego Delmonte

It is normal to see higher backpressures with methanol/water mixtures, than with either solvent alone. Thus if switching from water directly to methanol, you should see a rise in backpressure, then it should fall again as the water is washed out. The pressure you see depends on your exact column dimensions, flow rate, and mobile phase. But pressures above 200 bar are not automatically cause for alarm, especially with water/methanol systems.

Look at your care and use manual! I am sure that it recommends suggestions for washing the column. Also, it is possible that you washed debris onto the column from your instrument. This stuff usually sits at the column top or the inlet frit. Replacing the inlet frit might help.

The real issue comes next: you need to identify the source of the problem. A common and often overlooked cause is bacterial growth in your aqueous solvent. These bugs love phosphate buffers at pH 7, especially if there is something else around that they can eat...

Hello Diego,

in addition to the comment of Uwe. If you are checking your buffers, check also your degasser (if you are using one). I have seen many chromatographers observing sudden lifetime issues with columns they have used "forever", that finally ended cleaning the internal tubing of the degasser. (see manufactures manual)

Dirk

I have seen many chromatographers observing sudden lifetime issues with columns they have used "forever", that finally ended cleaning the internal tubing of the degasser. (see manufactures manual)
Dirk

Hi Dirk,

I just wondered the mechanism that makes column lifetime to be shortened with a dirty degasser and also how the degasser became dirty. I really assumed it to be less vulnerable and also put it to the end of the list. I think i was misinterpreting something. Could you please explain the mechanism a bit?

bulent

Hello bulent,

the mechanism of degasser contamination is mainly a biological one. If you have a buffer system and a source for organic carbon (teflon tubing) you have a nice environment for bacteria and other microbes to grow. We all may have seen algae growth in buffer bottles if the buffer solution was in contact with the air for a while (especially in summertimes when some non air condition labs have nice temperatures.) The same will happen in a degasser, just a little bit slower. So if you have a new degasser and your lab temperature is not too high, you are right you can put him to the back of your list. If you regulary rinse all degasser lines with 100% acetonitrile you are also quite save.

I hope this helps a little bit.

Dirk