Halogenated Molecule Sticking to Column

[hajdaei]

Hey

We currently have a molecule that tends to give a skewed peak shape after a number of injections. I believe that because of the 3 halogens on the molecule it tends to be "sticky" and so once there accumulates some junk on the column it causes a skewed peak shape (there are no basic, or other sites on the molecule that would cause this type of thing).

Any recommendations on a modifier to add to the mobile phase to help counter this problem.

Thank you for any suggestions.

[sassman]

Probably preventing junk accumulating on the column is the best course of action. A suitable sample cleanup to remove the junk prior to running the samples is preferable. Alternatively, you could ramp the mobile phase after your peak elutes to flush the junk out of the column.

[hajdaei]

No one has any suggestions here?

[adam]

I can think of two possible (?) solutions:

- Elevate the temperature of the column somewhat.

- Put some THF in mobile phase B.

[Uwe Neue]

I agree with Sassman: if you have a problem with junk accumulating on the column, you need to do better sample cleanup. The chlorine atoms on your molecule are not likely to do anything, unless they are reactive under your chromatographic conditions. If that is the case, you need to change the chromatographic conditions. However, with the sketchy info that you have provided, it is not possible to make any rational assessment of what your problem might be.

[mohan_2008]

I agree with the validity of the three chlorine atoms "sticking" to the hydrophobic stationaryphase of the column.

This is how, TFA is perceived as an "ion pair reagent" with the "sticky" fluorine atoms.

In addition, your molecule is said to undergo some "localizing interactions" with your stationary phase (worst with silanols).

Check the solubility of your compound in various diluents, Methanol, ACN and THF. Use the best diluent as the organic modifier in your mobile phase. For eg: if solubility is very good in 50% ACN, then use 80% ACN in your mobile phase.

Use an inert column such as Zorbax SB C4.
Use a shorter column 4.6 X 50 mm.
Use high column temperature as 60 C.
Use a higher flow rate, about 1.5 mL/min.

It should get it out completely.

[HW Mueller]

What new chemistry is this "sticking of halogens to" . . . .??
Is everything sticking to teflon as of recently?
If I read hejdaei correctly then there was no appreciable non-Gaussian interaction with the stationary phase at all, the tailing set in later. Some have seen this already and suggested to concentrate on handling the cause of this: gunk.

[mohan_2008]

Yes Mueller,

Initially, I had questions about this concept. But, this is how an ion pair interaction of the TFA is perceived in peptide or protein quantitation.

The Fluorine atoms have an inherent tendency to strongly exert a hydrophobic interaction with the C8 or C18 stationary phase.

[mohan_2008]

As per hajdaei's question:

You haven't given info if you are shooting a neat solution "as is" or a formulation with some excipient such as cellulose.

If this problem is aggravating linearly with the # of injections, obviously it can be the junk (sample matrix) sticking onto your inlet frit.

Try this. Invert the column, and reverse-flush with a strong solvent into a beaker (I am assuming your column i.d. is 4.6 mm).
Re-attach the column in the normal direction and perform about five injections. Keep an eye on the backpressure. If resulting backpressure is low, obviously it is the junk getting onto the head of the column or frit.

If you know it is the junk from the sample matrix, filter your sample solution with a teflon syringe filter prior to injection. In addition add a gradient cleaning step as mentioned earlier to rinse-off any junk sticking onto your column. You may have to re-validate your method to evaluate any sample loss from the filter.

[mohan_2008]

The other possibility would be:

1. Lower your sample inj volume.
2. Make your working concentration level very low, for eg., 1 ug/ml (I hope you have good sensitivity at this level). This way, your sample solution will be heavily diluted before it is injected.

Doing step 1. or 2. may result in revalidating the method with a new linear working range, as essential.

[HW Mueller]

mohan_2008, what would you expect to be retained more, at lets say a mobile phase pH of 2, acetic acid or TFA?
Anyway, what does hydrophobicity have to do with tailing?
Also, why are there these conventional suggestions of improving normal stationary phase interactions when the original post claims that the stat. phase changed? To solve the problem he should try to prevent this change.

[mohan_2008]

Hi Mueller,

The question posted on the original was very sketchy. I am assuming a list of possibilities and gave possible solutions.

The problem is the tailing due to the (?) junk getting on the column with a repeated injection frequency.

I gave conventional means to get rid of the accumulation of the junk on the column (besides the sticky chlorine atoms).

I agree that the original problem is with 'Tailing" and the source of this tailing could (?) be the junk accumulating on the head of the column. I have seen in my work similar thing happened due to the junk loading onto the column head after several injections. Hence, I gave an immediate solution, to get rid of the junk.

I agree there are several solutions, one which to change the stationary phase, to improve tailing. Again, the original said there were no basic sites or acidic sites that could have caused this tailing. In other words, it couldn't be the silanol effect on the column. I presume he was using a base deactivated column such as ACE (?).

I can't be confident though, since the information is very sketchy and a proper analysis cannot be derived at this point.

As far as which elutes faster, acetic acid or TFA at pH = 2.0, I think it should be acetic acid, since both will be protonated at this point, and being neutral stay longer on the column. TFA owing to extra hydrophobic interactions with the stationary phase may stick longer.

If TFA elutes faster than acetic acid, or you have an empirical assessment to prove this, please - I am curious to know.

[HW Mueller]

TFA is protonated like acetic at a pH of 2??

[mohan_2008]

Obviously, Mueller -

I am just trying to compare the relative eluting tendencies of TFA & CH3COOH and not trying to make an argument.

This is a forum trying to help each other and learn more info, but not trying to point at people's mistakes.

TFA has a lower pKa and hence, needs pH lesser than 1.5 to be protonated.

[HW Mueller]

So one should expect acetic to be more strongly retained than TFA.
If we don´t point out each others mistakes it would be far better to stop this forum alltogether.