Overloaded Column?

[ZeroAir]

Hello,

I recently acquired a barely used column from another lab group. This column was used a few times with long chain fatty acids by the other group and I think they overloaded the column significantly. When I inject a blank, I observe these types of long chain molecules eluting from the column as the gradient progresses.

Is running a highly organic phase through the column for an extended period of time an effective way of cleaning out whatever they might have left in it? Is there a more efficient way to do this?

Thanks,

Luke

[tom jupille]

If your bonded-phase column is contaminated with very hydrophobic junk, you can back-flush with something like methylene chloride.

Some common-sense precautions apply:
- check with the manufacturer or vendor to confirm that the column can be back-flushed (most can be, but better safe than sorry) and that it is, in fact, compatible with methylene chloride.
- if you were using a guard cartridge, remove it (no sense mobilizing trapped junk into your column)
- don't connect the detector (no sense running junk through there either)
- make sure that when you change solvents, everything is misicible and soluble. A typical sequence would be: mobile phase / buffer-free mobile phase / acetonitrile (or methanol) / methylene chloride / acetonitrile / buffer-free mobile phase / mobile phase.
- a reasonable rule of thumb is that it takes about 10 column volumes of solvent to get complete washout.

[bisnettrj2]

To expand on Tom's advice, you can try using the column cleaning procedure detailed in the following article:

http://chromatographyonline.findanalyti ... rticle.pdf

[ZeroAir]

Thanks for the advice!

Before I left today I ran 100% hexane through the column for about 3 hours. I injected another blank just before leaving and the MS still picks up contaminant. I am going to try the back-flush with methylene chloride on Monday, but before I do, do you think that the 3 hour hexane flush has moved the contaminant down the column, making it equidistant from the top and the bottom? Either way I will check compatibility with the documentation I have.

L

[bisnettrj2]

Just be sure you CAN backflush it - many columns I've seen lately recommend NOT backflushing - granted, these are usually 3 micron or smaller particle size columns, but it does pay to read the column care manual. What column are you using?

[HW Mueller]

If there are nonesterified FA on there I would work with pH as well as with MeOH or CH2Cl2 as Tom suggested. Note that under basic conditions both the acids and the silica gel may be negatively charged. (Obviously I assumed silica gel based stat. phase).

[ZeroAir]

Yeah I will give the CH2Cl2 a shot in reverse if the column is compatible with backwards flow. It is a C18 column.

With regards to the pH: I am concerned with getting all the contaminant out of the column, so then I SHOULD run the column in basic conditions right? Then the packing and the acids will repel each other (at least the charges will, obviously the lipophillic tails will not). How basic do you go? I am at pH = 8.5 or so right now. I think the pKa of the acids is quite low like 4.5 or something, not sure about the pKa of the packing, but seeing as it is C18 does this even apply? I was under the impression that C# columns were neutral at all pH.

Thanks.

Luke

[bisnettrj2]

Exactly what kind of C18 are you running? 8.5 is very high, except for the most rugged of C18s.

[HW Mueller]

I have not encountered a silica based column which is not capable of SiOH >>> SiO-.
A pH of 8.5 should be more than sufficient. I would also check whether something is coming out under acidic conditions, after the basic phase.

[ZeroAir]

I am using a Agilent Eclipse XDB-C18. Reversing the direction of the flow is compatible according to the manufacturer but its not recommended.

pH I guess is closer to 8, our pH meter had not been calibrated recently (out of calibrant buffer).

I will check what comes out under acidic conditions later today, you think I should just add some acid to whatever mobile phase I am using? Is there a particular acid that would be most compatible?

[ZeroAir]

Today I ran a blank on the contaminated column when I arrived at school. I have a gradient that goes from 50:50 DCM:THF to 100% hexanes in 35 minutes. Nothing eluted from the column until 30 minutes when the hexanes are at about 90% (10% THF) and at that time a small amount of my contaminant molecules (30+ carbon fatty acids) eluted.

Following this I ran hexanes through the column for three hours (10 column volumes as you suggested Tom), I did not use DCM because my contaminant molecules are not soluble in 100% DCM. They are barely soluble in 50:50 DCM:THF.

After the hexane wash I ran another blank. Contaminant eluted from the column during the entire gradient. I have no idea what this means. (Suggestions anyone?) Tomorrow I am going to run the gradient in acidic conditions and see what happens. I was thinking to moving on to backflushing the column with hexanes too, but I am somewhat hesitant to do this: it seems a bit extreme.

Open to any and all suggestions. Thanks to you all for your help thus far!

Luke

[Alexandre]

Control pressure/flow. Don’t compress particles too much, the pores should be accessible by solvent for good wash, and heat as high as manufacturers instruction/solvent allow. I prefer to wash col. under gradient condition.

[HW Mueller]

The pH calculator,
http://www.bioinformatics.org/JaMBW/5/4/index.html
is quite helpful in adjusting pH of solutions.
You have lots of "crud" + fatty acids on there? How do you know about the solubility of the crud? Are you actually referring to retention of the crud?

[Peter Apps]

This might be a good time to do a back of envelope calculation on whether a new column might work out cheaper than all the high purity solvents that you are pumping through this one

Peter

[ZeroAir]

Peter - ha, yeah you may be right I will keep that in mind.

HW - Thanks for the calculator link! All the crud (from what the lab who gifted us the column has told me) IS the very-long chain fatty acids. I guess they had been doing single ion monitoring (on molecules other than the FAs) and the FAs creeped into their system as a contaminant, so they had been injecting this contaminant for weeks without eluting it.

Alexandre - Alright, yes the high temp low flow makes sense, I will try that too.


Today I will try some backflushing and pH treatment. Will post results.

Thanks again to all of you.