urea for cleaning hplc system for protein analysis?

[car66]

Hi

Someone I talked to suggested using urea to flush out tubings that may be clogged up in the hplc system due to long term protein separation. I am wondering if anyone has any experience with this? 2M too weak or too strong?

The guard column, a. column, UV detector are detached. This is just for cleaning out the system in hope of relieving the high backpressure I am experiencing, so it shouldn't hurt the hplc?

Thanks!!

[danko]

Hi car 66,

2M urea will be far from effective in your quest. If you’re insisting on using urea you should go for 6 – 7 M.
And even then you’re not guaranteed a successful removal of protein residues.
I will suggest you to try sodium hydroxide (0.02 - 0.04 M) maybe combined with some isopropanol (10 – 20 %).
If the pressure is too high at the present (e.g. > 1000 – 2000 psi at 1mL/min flow rate) then please start with a low flow rate (e.g. 0.2 mL/min) and increase it over time. The procedure shouldn’t take more than a couple of hours.
If it doesn’t work then protein is not the problem.

Good luck.

P.S. As you mentioned your self, remember disconnecting column and detector.

[Kostas Petritis]

It would be best to identify where the high back pressure is coming from by connecting only parts of your system, starting with the last module. If it is problems related to protein analysis, the problem should come from the injector and afterwards. Investigate systematically (i.e. do you have backpressure when nothing is connected from injector and afterwards? if not how about when you connect the injector, if not how about when you connect the tubing that leads to the detector etc...).

Once you identify the plugged parts of your system you can clean them or replace (i.e. in the case of PEEK tubing) them accordingly...

Cleaning with chaotropes or ammonium hydroxide can help.

[HW Mueller]

We have been quite successful in removing proteins from columns with aqu. solutions of Li dodecylsulfate containing dithiothreitol (for instance: 970mL H2O + 30g LDS + 3.9g 1,4-dithiothreitol). It takes a long time to get rid of this "cleaning agent", though.