Chromatography Forum

I am having a significant amount of peak tailing with my Waters Column (uBondapak C18 125A, 3.9 X 300mm). I validated the column against our method last month and the peaks were all clean (absolutely no tailing and resolution was great). After about 12 assays, the column is experiencing tailing and very low resolutions. The mobile phase is 50:50 methanol: water. The solvent for the samples is 60:40 methanol: water. I spoke to someone at waters and they told me to wash the column with methanol for about an hour and then store the column. This did nothing for the tailing problem. I was then instructed by another person at Waters to run the following mobile phases: water followed by methanol followed by THF, then methanol followed by water. She recommended that each mobile phase run for about 20 minutes, which I did. Still, after this cleaning, the column is not any better. I tried reversing the column and still, the same tailing and poor resolutions for the last two peaks is being seen. If anyone has any suggestions it would be very helpful.

Thanks!

There are multiple possibilities, but I do not yet have enough information.
1. What is the nature of your analytes?
2. What else is in your sample (additives etc.?)
3. Was the decline gradual, or sudden?
4. What happens if you dissolve the sample in mobile phase?
5. Are all peaks tailing to the same degree, or is there a trend in the chromatogram?
6. How was the column stored between the time when it looked good and now?
7. Are you running at room temperature, or are you using a column heater?
etc....

The decline was sudden- I perforrmed maybe 6 assays and the peaks were perfect and then on the next assay, there was extreme tailing. We've never tried dissolving our sample in the mobile phase because we have to follow the USP for our sample and it specifies to use the 60:40 methanol water for dissolving the samples- also we have never seen this tailing before in our other columns so were do not think it is a problem with the method. IN our samples, we are measuring the peak areas for three peaks: benzocaine, butamben, and tetracaine (these are our three anesthetics used in our product- the solutions are dilute and contain mainly methanol). The mobile phase also contains 10% acetic acid and 1% heptanesulfonic acid, sodium salt. Our analytes are all similar in nature- benzocaine, also called ethyl 4-aminobenzoate, has a benzene ring; butamben, also called butyl 4-aminobenzoate, also has a benzene ring; and tetracaine is also called benzoic acid, 4(butylamino)-, 2-(dimethylamino) ethyl ester, monohydrochloride. The column was stored in methanol between the time when it looked good and now. We are running the column at 25C in a column heater.

Was the column dropped?

Is your mobile phase filtered?

I'd just order another column (or 2) and see that the above precautions are taken as appropriate. If you have seen no improvement in your problem column after having done all that flushing, I doubt you ever will. 30cm column jackets do make nice pegs for supporting small plants...

The column has never been dropped and the mobile phase is always filtered. Thank you for the help. I'll probably just send the column back to the manufacturer so they can replace it.

I agree with DR.
I have tried regenerating columns according to manufacturer's instructions. I have usually increased the lifetime by a day and it's not been worthwhile.

(Old columns with endfittings probably make good bones for those mechanical robot dogs....)

Did I read this correctly? You are using 10% acetic acid? I'll check on the USP method tomorrow to see what it says.

If I understand you correctly, the column performance dropped after you stored the column for the first time in methanol. Is this correct?

Sorry- I wrote that wrong- 1% acetic acid and 0.1% heptane sulfonic acid, sodium salt. And yes, I stored the column in methanol (this was the manufacturer's recommendation).

Ok - there is no problem with the acid concentration.

In ion-pair separation, I would keep the column in the mobile phase - forever... Unless I need to clean it.

I am not sure if your column has gone dead or not. It may simply require a more lengthy equilibration with the ion-pair reagent. Equilibrate the column for a few hours at your usual flow rate with the mobile phase containing the ion-pair reagent. The peak shape may recover - actually I think it should recover.

If you indeed get good results as suggested, store the column in mobile phase from now on. I believe that the problem is a departure of the equilibrium of the column with the ion-pair reagent due to storage in an organic solvent.

water,s Microbondapak columns are mfg. using irregular 10 micron silica and i have faced similar problem with these columns, they die out quickly with tailing after couple of days use ( depend upon the back pressure, sample composition, use of gaurd column ).

At what pressure u r using this column ?? the best way to solve your problem is to replace it with more robust spherical silica columns of 5 micron .

JM

Back pressure is about 230 bar. I will definitely consider replacing it

230 bar is too much for microbondapak ( irregular silica get compressed during repeated use).

JM

JM: Do you use a filter and/or guard column?

Istyger:
Have you still got high pressure when using a different column?
If so maybe you have particles from the pump/autosampler.
Have you a filter installed prior to the column?
If not you may face the same problem again.

Hope you can sort this problem out soon.

Regards
WK

No, I don't usually have high backpressure problems. Usually the bondapak column runs in the low 200's. I am actually right now looking into different columns for this assay. Thank you to all who have helped.

lstyger

Macherey-Nagel has been producing HPLC columns for over 20 years. Our wide selection will fit all of your needs. I hope you will at least check out the columns selections from a company that cares.

https://www.macherey-nagel.ch/web/MN-WE ... amesE?Open

Thanks