Chromatography Forum

I want to try primesep column with a quaternary ammonie compound (has several postive charge on the chain). I tried Primesep A, it seems this compound can not be eluted from the column no-matter how much ammonium salt and ACN I used. I also have primesep C column. But I am not sure whether I get the right column type or not. Or whether I can use this mix mode column for this permanent charged molecule or not.

Thanks a lot!

:lol:

If you have more than 2 quats in your molecule you might have hard time eluting your compound even from Primesep C column (particularly if you have 150 or 250 mm column). In mixed-mode chromatography very often 1+1 is much more than 2 (so binding is much stronger for oppoly charged molecules). You can try to elute quat from Primesep C column at lower pH. Your objective is to partially suppress ion-exchange strength of the column. pKa of Primesep C column is around 3, so you need to go lower than 3. The alternative would be Obelisc R column with pKa around 5. Here is application for separation of paraquat and diquat:
http://www.sielc.com/application_180.html

Please provide me with your email and I will send you few other applications similar to yours.

Kind regards,

Vlad

Welcome to the forum, yangxiaomi.

Been there, done that (i.e. I've had the same problem previously) with the quaternary ammonium on Primesep 100 - yes, it gives far to much retention for this situation.

You are on the right lines with the Primesep C; Primesep B or B2 might also work. Depending on whether the analyte possesses sufficient hydrophobicity, you may even get away with a decent C18 or polar embedded phase. HILIC is another alternative if the analyte is hydrophilic. Plenty of options then

These previous discussion might help to get your method dev moving:
Quaternary ammonium cpd on Primesep 100
Peak tailing for quaternary ammonium compound on C18 column

edit: beaten by Vlad who's got another column technology to offer. Out of interest, Vlad: are the applications you are offering not available on your site? If not, why?

We are way behind in transferring methods to the web. We have around 100 new methods on Primesep and Obelisc columns. It is not direct transfer (posting picture) so it requires some additional manipulations and time (we have PowerPoint/PDF presentation files which I can send).

If your quat is retained on C18 but produces poor peak shape you can use Primesep D columns to obtain perfect peak shape:
http://www.sielc.com/application_151.html

Thank you so much for the suggestion. Yes my compound is polycation with several quaternary pyridine sepeated by carbon chain. We have a regular RP method with TFA adding. If no TFA, my molecule has no retention on RP column. With 0.1% TFA, at 30% ACN it can be eluted from the column with moderate retention time. However, TFA will supress MS signal, so we have some challenge of getting good MS spectrum for this molecule. We want to utilize the mix-mode column to get rid of TFA and develope a method more MS compatible. I tried Primesep A. No matter how high the ammonium formate concentration is or how high ACN is, the recovery is no more than 10%. Once I coat all the stationary phase with my polycation molecule, it may becomes a regular RP column. Right? Now I am planning to try any luck of Primesep C column. Do you have some good idea or suggestion?

Thanks!

One more interesting thing I found for this PrimesepA column and my polycation molecule is the retention time of those tiny peaks. A huge peak is just after the viod and then several small peaks at longer retention time. My counter ion is Cl. At UV 254, there is almost no absorbance of Cl. Does this mean some of my sample has no rention at 30%ACN with 50mM ammonium formate while others have stronger interaction with the stationary phase?

Thanks!

Yangxiaomi,

try Primesep C column with ammonium formate. Adjust pH of ammoniu8m formate to 2.9 (a lot of formic acid) and use 50 mmol with various amount of ACN. Based on your data I suspect that linker on your quat is not long (3-8 carbons?) so you can try 30% ACN first.
Is you compound more like obidoxime or dequalinium chloride? I have methods for these two types (pdf files). One has two pyridinium quats connected by short linker another one has two pyridinium quats connected by C10.

regards,

Vlad

Is there a reason for not using a lower % of ACN in reversed phase conditions? Have a look at the paper below as it discuss in general chromatographic problems with quaternary amines, ion-suppression with TFA etc. The authors suggest the use of 10 mM ammonium acetate with triethylamine as additive and pH of 4. Having said that, I like the idea of using the Primesep C column if you can achieve enough retention...

BIOMEDICAL CHROMATOGRAPHY
Biomed. Chromatogr. 19: 579–585 (2005) Quantitative determination of alkylated quaternary amines and their n-hydroxylated metabolites in an enzyme incubation matrix by liquid chromatography electrospray
ionization mass spectrometry

Thanks for the suggestion, Could you please email me these two methods to yangmimi2008@yahoo.com

yangxiaomi,

I sent you three applications for compounds with two quaternary amines connected by different linkers (hydrophobic and hydrophilic compounds) (check you bulk folder, sometimes yahoo sends it there).

Xiaomi,

Alternatively, you can use FA instead of TFA on a C18 column for better MS compatibility.