Problem with HPLC Colum. HELP. Please

[Arkangel5000]

We Cleaned an used HPLC colum with strong solvents (Isopropanol - Chloroform - Hexane), after that we have a really strong big peak (more like a hill) that elute between the 70% and 80% of ACN. We thought it could be an impurity of some of this solvents, but we are not sure of that and we also think it could be an inmiscibility problem. Moreover, we have a random noisy baseline that could not be stabilized at all.
Something like this__---__--__----___-----_____^(Broad Peak)^______
The problem is in the colum and not in the equipment.
It will be very aprecciated your HELP.

[tom jupille]

What did you do in between the hexane and the ACN/water gradient?

[Arkangel5000]

First we don ACN --> Isopropane-ol--> Chloroform-->Hexane, And After Hexane, we do Chloroform, then isoprone-ol, and then ACN, and then ACN/Water.

[Slammy1]

For extreme RP column regeneration we'll run aqueous, ACN, Chloroform, then reverse it out to MP. This is very generic, and may be insufficient depending on the analysis. I remember for one method where we lost columns after 2-3 runs, cleaning with an acidied aqueous portion of the clean out produced S/N in the LOQ solution where the requirement was 10 now typically runs 100-200.

[Bruce Hamilton]

Have you ordered a spare replacement column yet?.

I assume that you used HPLC grade solvents, and also used solvents the manufactuer recommended for cleaning, if not repeat the clean with suitable grades and recommended solvent cleaning procedure.

If you used suitable solvent grades, I would first run the gradient method, and try to measure the approximate size of the contaminant.

I would increase the column temperature by about 10-15C, flush the column with some IPA at low flow rate ( 0.5 ml/min ) for about 20 mins, followed by methanol at 1 ml/min for same time, and acetonitrile at 2 ml/min for about the same time, and then return to your gradient starting conditions.

Then I'd run your gradient, flush with column with acetonitrile at higher temperature for another 20 minutes, rerun the gradient, and see if the peak has decreased in size.

If so, repeat the initial MeOH, CH3CN wash cycle - don't bother with the IPA, see if peak is most reduced by MeOH or CH3CN, if not consider using another cleaning solvent, such as tetrahydrofuran, in place of the methanol, or using the replacement column.

Please keep having fun,

Bruce Hamilton

[mbicking]

You don't mention what equipment you have (manufacturer), so there will be some differences for different instruments, but you could have a residual chloroform problem.

In some instruments, like the older Agilent 1100's, the degasser has a very large internal volume (12 mL). It can take a long time to completely remove all traces of a different solvent. In one laboratory, I was still seeing chloroform residues four hours after switching to a conventional reversed phase solvent. So, you may just have to let the system flow with a new solvent for a longer time.

[cactus]

Hi,

I am currently coming up with a column cleaning method for my column and would like to know, how should I clean my waters XTerra C18 column?

I'm thinking of using water -> 50%water50%ACN -> ACN -> THF -> DCM -> THF -> ACN. Should this be alright?

I couldn't afford to kill my column, so I haven't really experiment this out yet. Would like to know how many tried this method and if this is ok for my column? I heard from a few reports that THF is hard to get rid of once it gets into the column.

Please advise, the flow rate and how long it should be flushed? By the way, I'm flushing the column only, without the detector.

Thank you!

[mbicking]

You might have better response if you posted this as a new topic.

Perhaps Uwe can comment on the particular stability of these columns to these solvents, but in general what you are proposing is not a big problem. It's drastic, but not a problem!

You also should spend some time considering why the column is becoming so contaminated that you need to clean it with methylene chloride to clean it!

A better long-term answer would be a guard column, or better sample preparation to remove those things that are retained on the column.

[Uwe Neue]

The first question about any column cleaning procedure is: what do you want to remove from the column? Once you know this, you can design a very specific cleaning procedure.

For removing hydrophobic materials from a C18, THF is sufficient. You do not need to go to DCM. Also, you can go straight from a salt-free aqueous solution to THF.

[cactus]

Thanks uwe and mbicking, i've reposted this message as a new topic.