RP-HPLC of Oligonucleotides: Regeneration

[GCasi]

Hello everyone,

I have a problem with RP-HPLC purification of oligonucleotides.

I am using a three moths old 250/4.6 Nucleosil 300-5 C18 column to purify oligonucleotides with triethylammonium acetate / Acetonitrile buffer system, and I wanted to regenerate the column.

In a first attempt I reversed the flow on the column and applied a 1 mL/min flow with the following solvents: Acetonitrile, MeOH, THF, MeOH and back to acetonitrile. Every wash lasted 25 minutes. The result was that I eluted a lot of material from the column; the separation quality increased, but the back-pressure raised from 120 bars to 160 bars.

In a second attempt I used 0.4 mL/min and only Acetonitrile,THF and back to acetonitrile, and this time the pressure raised evem more to 180 bars.
I don't know what I am doing wrong.

Does anybody of you know how I could wash the column and still maintain moderatly-high pressure? Do you think I will be able to recover the column, or should I trash it? Would you suggest to use ionexchange instead?

Thank you for your help

Best regards,
Giulio

[Steve]

In cleaning, the “LAST RESORTâ€

[MG]

Now I have a question in response to Steve's post. I have considered in the past injecting a DMSO plug to clean a column, or to clean a dirty injector, but was worried that the polymer in my injection valve would not withstand DMSO. Normally the valve seal is Vespel in my case, although the user can optionally install a Tefzel or PEEK seal. Can any of these withstand DMSO?

[Steve]

Dimethyl sulfoxide (DMSO), a by-product of the wood industry, has been in use as a commercial solvent since 1953. DMSO has Food and Drug Administration (FDA) approval only for use as a preservative of organs for transplant and for interstitial cystitis, a bladder disease. DMSO ability is to pass through cell membranes which allows for a mechanism that can be used as a drug transport system.

There are no problems associated when injecting DSMO into a Vespel seal.

Here is the MSDS for DMSO http://www.dmso.com/ (amazing even chemicals have their own web address)

Dupont does not list non compatible chemicals for Vespal.

[HW Mueller]

Giulio,
your problem appears to be the same we had with proteins. I have not done oligos (might have to, thus my interest in this), so I was waiting for someone experienced in this field to answer. But, it would not be surprising that your organic solvents precipitate oligos on the frit and/or column. It may well be that chaotropic conditions, etc., also enable to dissolve oligos (the example referenced by Steve seems to indicate this also).
Steve,
modern columns take reverse flow very well, I always wash columns, which showed a pressure increase, backwards. A partially plugged frit (I know that for samples where proteins are involved) is extremely difficult to clean in forward flow (flow direction of the analysis). Surprisingly, some people even report that reversing a column with a void can improve performance.

Generally: It would be very much appreciated if somebody in the know would contribute on handling oligonucleotides regarding HPLC.

[Steve]

HW Mueller

I did not know that your an HPLC column cleaning expert and technical rep for a column manufacture that sells voids as an increase in column performance. If a litte void offers better performance that why not just empty your column altogether and get great results. It would be beneficial to this forum to keep your personal comments to yourself and just provide your technical comments however dumb they are.

[MG]

Geeze, Steve, I didn't think HW Mueller's comments were dumb. For years, the instructions provided with Zorbax columns have claimed that reversing flow will not harm their columns, and can be used to unclog the inlet frit. Also I think you misunderstood his comment regarding column voids. What I take from his comment is that in some cases the poor performance resulting from a void can be mitigated by reversing flow, not that voids themselves are a good thing.

[HW Mueller]

Yes MG, obviously!

[Steve]

hehehehe

Just trying to stir the pot.

No, I do think HW Mueller's comments are lame.

In a small percentage of time, reverse flow will be the best way to clean a column, but should not be our first practice on all columns.

Hope the DSMO works for you.

[ananda]

This is an interesting coincidence. I am also developing a RP-HPLC method to resolve ~30 aa peptide from its impurity using asimilar type of RP column and with tetrabutyl ammonium dihydrogen phophate as the IPR. After 15-20 injections which give me very good resolution and data then suddenly column gets bad and shows lots of junk peaks. I also found out the reason is peptide and TBA and C18 somehow together cause this column degeneration.
I also tried the same column regeneration method as MG and I reversed washed the column and actually it helped to clean the column but I am having the same chromatographic problem

So my opinion in reversing the column is It is OK if the manufacturer recommend or/and in general. However, if the manufacturer say not to, you shouldnt.

To remove protien buildup in the column LCGC recommends to inject ~250ul of TFE (trifluoro ethanol) .

My problem is also I cant use the column over and over after 10-15 injections and I have to either give up TBAHP as my IPR and use another IPR.

Regards!

Ananda

[Uwe Neue]

Ananda:

Unless a sample contains mostly junk, such as a plasma sample, or a natural products sample, there is no reason whatsoever for a column to show lousy separations after 15-20 injections. Something else must have gone wrong. Have you considered the quality of the TBA, for example? How clean is your peptide?

[HW Mueller]

Ananda,
after cleaning the column it runs well for about another 15 injections before refouling? If so it would be very interesting to know the exact cleaning protocol. Can you run your peptides with pH control only (no ion pairing)? Rememnber that it can take extremely long to get rid of ion pairing reagents (if at all) from a column.
It would be very surprising to me if you could get rid of proteins with neat trifluoroethanol, that´s the first time I ever hear of its use. Do you have the reference?
The handling of proteins has been discussed several times before.
Steve,
the columns I bought in the last ten years are all reversible, why should one wash the dirt, which usually sits in the frit or the first mm of the column, through the whole column? Incidentally, I am by no means the initiator of washing columns backwards. Lots of lameies out there.

[ananda]

Uwe,

My peptide is very clean and it is actually a drug product (~98%) and I am using TBADHP ampoules. So both are very pure and no contaminants expected. I totally agree with you that there can be another reason for the column to become foul but cant find whats' wrong.

HWM,

This is the reference for TFE method of cleaning. I think it worked to a certain level but my problem is still there with lack of resolution and builudup of peptides with time. We tried Ammonium bicarbonate without the IPR and the peaks were co-eluted. We are using Vydac C18 columns.
Reference for TFE: LC-GC, volume 17, # 4, April 1999, Pages 354-355

Thanks for your comments/suggestions.

Ananda

[HW Mueller]

Ananda,
The LC GC web page did not yield that article, but on the LC GC Europe web I found the following article which mentioned TFE: Majors, LC GC Europr, July 2003, p.2. The TFE is referenced to Bhardwaj, Day, LC GC 17 (#4), 354 (1997). Is that your source? Since I can´t get that here I would appreciate if you could tell me whether these authors promote this as a general method or give a specific example only.

GCasi,
a quick look at my material on oligos showed that MeOH is commonly used to precipitate them. Various aqu. buffers or aqu. buffer - organic mixtures are used to keep them in solution, so maybe a strong buffer (~0.1 molar) at ?pH will clean your columns. Certainly would like to her more on this.

[ananda]

HMW,

Yes, the reference and authors are all the same in the reference you mentioned for this TFE use to clean bound protines. However, the year is 1999 and not 1997. I have the article right here in my hand now. If you want, I can fax it now.
Let me know. It is a general method according to authors. I agree with their justification since I am a protein biochemist. So bascially they beleive TFE can promote alpha helical formation and that helps to remove proteins from the column but as you know it may of course depend on the type of protien who knows?

Regards!

Ananda