Chromatography Forum

Hi all,
I got a old Knauer c18 HPLC column from my colleague ... I could not ask him how and what for he has used the column
The CoA of the column says @1ml/min 80/20 MeOH/water the back pressure would be around 170bar ..
but I get ~240bar ... I first want to try cleaning the column ...
Can some one give me a step by step method of reviving the column ... a nice link might also suffice ...

This is the first time I am trying to do something of this sort ...


with best wishes

First of all take the column out of your system, open the column top and replace the frit!
Than built in the column in reversed flow but don't connect it to the detector.
Reduce flow and flush first with water, than with organic solvents like Isopropanol, Methanol and/or Acetonitrile.
You can also flush with "hot" water.
You can use the column after this cleaning procedure in reversed flow direction. Backpressure should be lower than 240bar but will not go down to 170bar. But you can work with the column
Good luck

thanks dear Kratz ..
Actually I just ran benzene sample .. as it was used in the column CoA ...
The retention time given in CoA is 4.8min and I get 4.5 min ... of couse as i said the back pressure is 240 bar ..
but the peak has a bit of tailing ...
so what should i do .. can i use the column .. or i should still go for cleaning it ..?

I would do the cleaning following this protocol:
Regeneration of RP packings
C18, C8, C4, C1, C30, CN and Phenyl stationary phases:
• Flush the column with 20 column volumes of water
• Flush the column with 20 column volumes of acetonitrile
• Flush the column with 5 column volumes of isopropanol
• Flush the column with 20 column volumes of heptane
• Flush the column with 5 column volumes of isopropanol
• Flush the column with 20 column volumes of acetonitrile

In addition I would run the column in reversed flow direction.
Good luck.

.... but don't spend an awful lot of time and solvent on an old column. If you look at what your time is worth, and the amount you have spent on your samples already, and balance this against the price of a new column, you may find that columns are more consumable than they appear at first sight!

If you can get 500 injections out of a column, it has almost certainly cost you less than the plastic-ware and vial you used to make the samples (a spin-filter is nearly a pound in the UK, good quality vial, insert and cap is getting on for a pound, and a typical hplc column maybe 500 pounds).