Restore a polymeric RP column?

[miro2009]

I have a new PLRP-S 5um analytical column, (polymeric reverse phase) and we had an established method for analysis of a certain protein in supernatant samples.
Unfortunately, we decided to make a trial to overload the column and evaluate to what extent carryover we have, (a bad decision), anyway, now the performance seemed to be impaired where the peak shape of the standard reference became deformed (high tailing), in addition to loss of resolution in our supernatant samples (in normal load)
we tried so far to clean the column in reverse flow at a slow rate 0.2ml/min with 90% ACN overnight, very slight improvement (by 5%) the next day. Then we tried cleaning with 2 column volume of 0.5M NaOH, much improvement observed, by 40&... but still resolution is lost between very closely related peaks in supernatant samples.

Is the performance permanantly lost? This is a new column only used for 1 month... is there anything else we could do to restore it?

[tom jupille]

If some is good, more is (perhaps) better. So, try more NaOH. PS-DVB columns are stable to alkali.

[DJ]

You should probably check with the manufacturer first. With that said, I've had luck cleaning a PLRP column with CHCl3/MeOH/1% aqueous formic acid (4:4:1; vol/vol). The advantage with this is that protein contaminants not soluble in your 90% MeCN can be often be solubilized (and removed) with CHCL3/MeOH/HCOOH

One thing to consider is that HCL is generated during chloroform degradation, so it wouldn't hurt to extract the CHCL3 with a few portions of water before introducing it to stainless steel surfaces.

[HW Mueller]

If I were to use crappy CHCL3 I would be more concerned about the phosgene than the HCl. Besides, there are all kinds of more harmless and efficient methods to clean out proteins than chloroform (even if a nondegraded material is available). It has been discussed many times here.

[rhaefe]

I have heard people flushing those columns with 1.0 mol/L NaOH (10-15 column volumes), putting the column end plugs back on and leave the column in an oven or incubator at 80°C over night. Followed by flushing with water the next day until effluent is neutral and re-equilibrate with mobile phase.

Now, I don't want to recommend doing this but a 60-90min flush with 0.5mol/L NaOH (disconnect your detector) at 50°C should remove any protein residue that is left on the stationary phase. Choose a flow rate that does not over pressure your column.

Robert

[miro2009]

Thank you all for your replies...
Well before jumping to more aggressive measures I, again, flushed with another 5 CV of 0.5M NaOH, then flushed with water, and performed a blank run, surprisingly I found a very significant peak at retention time of my protein.. as if NaOH has broken down whatever contaminants bound to my resin acting as "2ndry" stationary phase, and now ACN is just needed to elute my protein, another blank and no more peaks appear.. and performance and my sample profiles are back to normal
I guess this is the only logic explanation I have, feel free to share your opinions... and will keep your cleaning techniques in mind in case needed in the future..

[danko]

surprisingly I found a very significant peak at retention time of my protein.. as if NaOH has broken down whatever contaminants bound to my resin acting as "2ndry" stationary phase, and now ACN is just needed to elute my protein, another blank and no more peaks appear..

You’ve observed a carry-over peak there – the stuff that cased the peak has been in the injector.
The protein that was in the column probably eluted already under the NaOH treatment.

Best Regards

P.S. If your mobile phase is just water and ACN you’ll shortly observe the same problems as before the column cleaning.

[miro2009]

surprisingly I found a very significant peak at retention time of my protein.. as if NaOH has broken down whatever contaminants bound to my resin acting as "2ndry" stationary phase, and now ACN is just needed to elute my protein, another blank and no more peaks appear..

You’ve observed a carry-over peak there – the stuff that cased the peak has been in the injector.
The protein that was in the column probably eluted already under the NaOH treatment.

Best Regards

P.S. If your mobile phase is just water and ACN you’ll shortly observe the same problems as before the column cleaning.

Dear Danko,
Actuallly the cleaning process using NaOH was performed on a different HPLC system (actually an AEKTA system), so I don't think this carryover was from the injector. Still, I know I'm dealing with a very hydrophobic protein, and I always perform blank runs between every 3-4 injections to ensure there's no carry-over, and we usually have a 0.7-0.8% carryover which I guess is acceptable so far, given the fact we've tried a lot of method development trials to avoid this (using C4 columns, using ACN/isoprp mixtr...) with no much improvement...

[HW Mueller]

miro2009,
you get this protein to come off with pure ACN, in a step gradient with water? Is this your method for this protein, a step gradient, water to ACN?

[miro2009]

miro2009,
you get this protein to come off with pure ACN, in a step gradient with water? Is this your method for this protein, a step gradient, water to ACN?

No we use a gradient from 0.1%TFA water to acetonitrile, step gradients usually are not applicable for obtaining good separation between closely related components... can I ask why you're asking about this? do you have a similar problem?

[HW Mueller]

I understood your ". . . and now ACN is just needed to elute my protein, . . ." to mean that you washed the column with H2O, then passed ACN into it and out came the protein at the right rt.
So what did you actually do? You washed it with NaOH, then H2O, then equilibrated with mobile phase A, etc.?
If this is what you did, I still wonder how you destroyed and thus removed everything from the column with the NaOH, except the analyte protein, . . . . miracle?

[miro2009]

I understood your ". . . and now ACN is just needed to elute my protein, . . ." to mean that you washed the column with H2O, then passed ACN into it and out came the protein at the right rt.
So what did you actually do? You washed it with NaOH, then H2O, then equilibrated with mobile phase A, etc.?
If this is what you did, I still wonder how you destroyed and thus removed everything from the column with the NaOH, except the analyte protein, . . . . miracle?

yes.. I "washed it with NaOH, then H2O, then equilibrated with mobile phase A," then applied my method "gradient" with no sample injection.. and then a significant peak at my RT... do you have another logic hypothesis, other than "miracle" of course?!
The point is, that the problem is solved, but we're trying to figure out what exactly happened to gain experience when we face similar problems in the future..

[HW Mueller]

Danko gave you a logical possibility, there is a miriad of ways to get carryover, or simply contamination.

[danko]

Miro wrote:

Dear Danko,
Actuallly the cleaning process using NaOH was performed on a different HPLC system (actually an AEKTA system), so I don't think this carryover was from the injector

Actually it’s not that important where the NaOH treatment was performed, but rather where the subsequent tests were carried out. If the system has been used for injecting the protein in question, it could’ve retained some of it in different parts (e.g. injector loop, needle, various filters etc.).

Btw. am I right in understanding your gradient protocol as follows: 0.1% TFA in pure water at start and then more and more ACN with no TFA?

Best Regards

[miro2009]

Miro wrote:

Dear Danko,
Actuallly the cleaning process using NaOH was performed on a different HPLC system (actually an AEKTA system), so I don't think this carryover was from the injector

Actually it’s not that important where the NaOH treatment was performed, but rather where the subsequent tests were carried out. If the system has been used for injecting the protein in question, it could’ve retained some of it in different parts (e.g. injector loop, needle, various filters etc.).

Btw. am I right in understanding your gradient protocol as follows: 0.1% TFA in pure water at start and then more and more ACN with no TFA?

Best Regards

Thanks Danko for trying to help me figure out this case!
In fact, I don't think it was sample retained somewhere in the system, as we're not observing any carry-over now after column cleaning, if this was the case then it should continue to occur, right..?