Just one question for your helping . Have you controlled your UV cell and washed this one?

i would wash the whole system during the night . elute it and inject acetonitrile samples (as a blank) run a gradient (water to ACN), maybe it helps

What was the strength of formic acid ( if o.1% then ok or else may be some properties of column have changed) But pl.refer the following article for better understanding to reactivate your column:

"

Ion Exchange Chromatography & Chromatofocusing - Principles and

wolfson.huji.ac.il/.../AMERSHAM_iIEXandChromatofocMan...ShareFile Format: PDF/Adobe Acrobat

Buffer volumes used during sample application, elution, washing and re-equilibration ...... or formic acid, but the separation properties of the media will change."

According to:

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/proteomics-and-protein/proteomics/chromatography_gel_filtration.pdf

it is given that superdex column:

"Can be used with detergents, chaotropic salts, dissociating agents … Compatible with organic solvents"

So, I suggest to flush it with water -> isopropanol->water in gradient. IPA is the best water compatibile solvent allowing to remove oils effectively. At the same time rise temperature as high as it is acceptable for the column. I've used this method in difficult cases to clean up my GPC column without any signs of deterioration of separation.

In case of fail you can apply a detergent (like in washing machine ;-) however I would avoid it - detergents could affect your further separation. Good Luck.

Start fresh by getting new beads and keep track of the reproducibility of the beads over time and under varying conditions and remember that not everything can be explained by science :) Good Luck

Did you try wash it with cyclohexane? I know that this solvent is excellent for oil contamination, of course if it can be used as the mobile phase.

After cyclohexane wash column as Małgorzata Jaworska suggest. It should help.

Best wishes

Hi everyone, I´d like to thank you for all of your replies! Well, the data sheet for the column says that formic acid up to 70% is fine for daily use (yes, 70%!), so I used a 50% solution for a couple of hours.

I washed the whole system, and am now focusing on the column cleaning: I used Acetonirile 30% for a bit (recommended by GE), washed extensively with water, washed with NaOH 0.5 M (recommended for cleaning after a few injections), washed with methanol, and then I did a test injection with a known protein sample and the UV chromatogram was AWFUL, as was only the ICP spectrum.

After this, I washed again with water, methanol again and I´m now washing with water, to see if the water pH is what it is in my original ultrapure water, to test for any acidic remains in the column.

I don´t think the oil would STICK to the column filling, would it, if the stain is gone? Do you think the column is a throw-away scenario?

I´m worried about the formic acid 70 %, since someone said 0.1%!!!.. the pdf for this column with manufacterer instructions are at this address, if anyone would like to take a look...

http://www.smansy.org/lib/exe/fetch.php?media=public:superdex_75_200.pdf

thanks in advance...!!!

I'd do a good old hexane or heptane wash

Flush salt/acid out with high water/organic

Flush the system with 10 column volumes of acetonitrile or methanol

Then 10 column volumes of isopropanol (bridge solvent) turn up the termperature and turn down the flow as it is very viscous

Then 10 column volumes of hexanes or heptanes

Then 10 column volumes of isopropanol again

then you can go back to aqeous running conditions.

BTW unhook the line from the detector and just put a tube coming out of the col compartment into a waste beaker.

Hi Steven, I'm not sure the column is compatible with these solvents...! There's nothing about them in the pdf... I'm afraid to risk it...! :(

You need to wash with water after washing the column with formic acid . This would help to remove excess acidity in the column. You can also flush the column with IPA after water wash tha would remove your oil traces. Hope this would help you.

I tested water pH today, what went in was the same pH as what came out, indicating no more formic acid at least... but my UV chromatogram was AWFUL, as was the ICP spectrum... I think maybe something is in there that is denaturing my proteins and maybe carrying out metal ions even after extensive washing? I've washed the thing for over a week now... does it usually take that long to recondition a column?

Also, I dont think I can get the beads, it's an agarose dextran mixture, I'm not sure they sell them for column packing... would anyone know?

You need to check the compatibility of the column. I am not sure but looks like the packing material is got distorted.

Hexane is the best solvent for fixed oil.You need to wash your column with Hexane it will wash your column completely without any doubt .

you can referesh your HPLC column by washing it with benzene with more than 95 percent mixture

Hi... benzene is not compatible with this column, this is a protein column mainly...

Hmm... a lot of you guys are recommending nonpolar solvents such as hexane, cyclohexane, etc. as a wash solvent. I agree that these solvents can effectively remove the oils from the column, but I am not sure if these solvents are compatible with Rachel's column. In our experience, one also has to keep in mind the HPLC system. In our laboratory, some LC systems are only RP compatible. Nonpolar solvents such as hexane can harm different parts of the LC system (piston seals, check valves) so caution must be taken. I hope that Rachel's system is RP and also NP compatible.

@ Rachel unfortunately I don't have any real idea how to "revive" your column at the moment.

After cleaning with formic acid you must conditioned ur column with appropriate solvent. Which can be found in the column guide, i mean store ur column with the recomended solvent.

Hi everyone, I'll try the column storage solution tomorrow (ethanol 20 %)... I tested the pH, the water went in and out at the same pH, but when I tested the pH of my Tris-HCl pH 7.4, it came out at 6... so maybe it's interacting with some formic acid still in the column...?

Unfortunately I can't reverse the flow in this particular column, there is a filter that doesn't allow this, at the column entry point...

I'll keep trying, but I don't think it's going to get any better :( it's been about a week of washing, eluting, washing, testing... I think it's a goner :(

This type of probelm usually arises when lot of fixed oil is retained in the column. If your column is NP compatible, it would be better to wash it with a non polar solvent which u consider suitable and once you are done, give the column repeated washings with water followed by methanol for at least a week. Then see what is the result.... If it is not positive.,,,,, Then some other measures can be tried but first u should perform this things.

Hello, the pressure is already normal and pH as well, but still the profile sucks... I've stored the column in ethanol 20% as manufacturer instructiosn, and see what happens tomorrow...