Chromatography Forum

Hello All, I'm in dire need of some expert advice!!

The following mphase/column is used for an impurity method: 40:30:30 water:acn:methanol on Hpersil 5u ODS 120A

The following mobile phase contaminated the column: 45:30:25 methanol:10mM butylamine buffer:acn

(10mM butylamine buffer adjusted to pH = 6 with acetic acid)

The question is: Is there a known successful procedure to wash/regenerate this column to remove the butylamine?? The chromatography using the original mobile phase has now dramatically changed; and I am presuming it is the butylamine that's causing the trouble... Your thoughts are greatly welcomed!!

Thank you all in advance for your help!!!

Hi! i think 80% H20-20%Meoh for 2 hs is enough to remove this primary-amine and the buffer of course!! (remove a primary amine it´s more easy than a secondary amine and a terciary-am.)

however, for your information i´ve recently read the next text from some manteinance column guidance:

Regeneration of RP packings:
RP- packings are C18, C8, C4, C1, C30, CN or Phenyl stationary phases.
• Flush the column with 20 column volumes Water
• Flush the column with 20 column volumes Acetonitrile
• Flush the column with 5 column volumes Isopropanol
• Flush the column with 20 column volumes Heptane
• Flush the column with 5 column volumes Isopropanol
• Flush the column with 20 column volumes Acetonitrile

I´m sure someone more expertise than me will answer your question with more detail.
Bye!

I did run the original mobile phase 40:30:30 water:acn:methanol over night (1 liter = 300 column volumes) and it did not help that much. Do you still think the 80:20 water:methanol would help?

Thanks again!!

mmm....it´s a great quantity of m.phase, but i think may be with more %of water you coud flush the amine better...here is my humble opinion...
g.luck and let me know...

In order to remove an amine from the column, you need to run a buffer, for example 20 mM ammonium acetate, pH 4.5, with a high concentration of organic solvent, maybe 60% acetonitrile or so.

Flushing a C18 with a bunch of organic solvents is a last resort voodoo, if there is some contamination on the column whose nature you do not know nor understand.

Thanks both of you for the feedback! I will try the 20mM ammonium acetate buffer: acn (40:60). I'll let you know how it turns out.

Can you make certain that remaining butylamine is the culprit? If some other change in the column is responsible for the changed performance you will be trying to get rid of butylamine until doomsday.

All,

I ran ~200mLs of the pH 4.5 amm acetate:acn solution recommended and observed improved resolution (2.0 to 2.5), improved peak shape (T = 1.6 to 1.1).

However the noise is higher than typically observed (Millenium baseline noise calc on PDA = 0.048 vs 0.032): ~1.5x.

Hello HW,

Are you suggesting the butylamine is very difficult to remove or that the butylamine would not typically cause issues? As far as I know, this is the only change the column has seen.

All thoughts are welcomed!

I am going to try again by saying it completely different: Maybe your butylamine was long since gone and the column´s deterioration was caused by something else. With the information given one can only speculate, for instance, you did not say why the butylamine was placed on the column in the first place.

Hello HW,

The mobile phase containing butylamine was inadvertantly used with this column. Afterwards, the original and correct mobile phase was used, the peaks tailed more than previously observed and resolution decreased from what was previously observed and the baseline noise increased from what was previously observed.

I suspected it might be the butylamine that caused these changes by adsorbing to the C18 groups and possibly permanently changing the stationary phase. In your experience, have you observed butylamine to adsorb to hypersil c18s (or any C18 columns) and remain on the stationary phase, changing the chemistry of the stationary phase? or do you believe the butylamine would be easily removed from this stationary phase and should not permanently affect the stationary phase?

Thanks again all of you for your help.

Are you now back to your original mobile phase and the original operating conditions (wavelength etc...)?

Also, a small change in the baseline noise (1.5x) is not that exciting, if everything else is fine. The cause may not be associated with the column at all...

Yes, after cleaning the column with your recommended mixture, I switched back to the original conditions (mphase, flow, wavelenghth (260nm), etc). The tailing and resolution improved back to normal. the only difference is the noise...

Do you believe residual butylamine could cause a slight increase in noise? or do you believe the butylamine is gone and the noise is something else? I have routinely observed 0.032-0.035 on this PDA. now it's around 0.048-0.050. Thanks again!!

I wouldn't worry about the small increase in noise. Plus I think that it may be caused by other things than butylamine. Maybe your lamp is aging... Who knows...

I have used other amines and have never seen them stick so badly that they would drastically change all sep parameters even though washing the column as much as you apparently did (over night). When I have made an "inadvertant" mistake like this I always ask myself: What else did you bodge? (Here I would have certainly checked in what shape the amine was, maybe it was brown already.....). Also, before assigning blame, one can run a spectrum (UV detector), etc. etc.
Now, Uwe´s solution could get rid of a lot of other things besides butylamine.

Thank you to All, your help is much appreciated. Now onto creating a new post regarding typical LOQ peak areas/peak heights. Thanks again!!