I developed a method for cetylpyridinium analysis with pentanesolfonate sodium salt as ion pairing agent using a C18 BDS column (hypersil) and a ACN/H2O gradient. The method works fine on my formulation but the tailing factor is above 3.

I have two questions:

1) Changing from the column used to a newer generation column (like XTerra) with lower silanol activity could improve peak tailing?

2) I read about TFA as ion pairing agent (in the forum). Could it be useful for cetylpyridinium chloride analysis (pyridinium salt so positively charged)? This would permit me to switch to HPLC-MS to study the degradation products.

2a) is there any method for pyridine analysis in HPLC? (probable degradation product of cetylpyridinium chloride?)


Thank you in advance for your help!

Hello rp,

Cetylpyridinium chloride, as a quaternary ammonium compound, may be separated using your column with a simple modification of the mobile phase. I would prefer using ACN and 0.1% TFA in WATER as a gradient. Though I have seen some people using 0.01 N sulfuric acid instead of TFA.

Good luck

I would not even use TFA. Formic acid and a high quality modern reversed-phase packing will do fine.

Why deal with troublesome ion-pairing agents. Have you considered HILIC?

Although the aliphatic chain is fairly large I think HILIC is an alternative.

Then you have compatibility with MS and most other detection modes.

For the retention of polar compounds without ion-pairing reagent (including pyridine ) check these two methods for pyridine.

http://allsep.com/makeCmp.php?cmp=Cmp_079

Next week we will try to develop a method for cetylpiridinium chloride.

Wow - I appreciate these commercials from Allseptech: cetylpyridinium as a polar compound...

Dear "Anonymous",
RP, asked for the HPLC method for pyridine and reference was for pyridine with other polar compounds (amino acids, organic acids and benzylamine)
We are working on the cetylpiridinium chloride and other quaternary amines and will update.

There is a related issue posted two posts later. You need an increased ionic strength, not a low pH. You will be better off with a high ionic strength formate or acetate instead of TFA.

Hi rp,

in theory (I don't have any practice with moleculs like that) I guess the tailing is caused partly through the different polarity of the cetylpyridinium and the resulting different kinetic.
With TFA the polar part of the molecule is building a polar ion complex (faster kinetic), but the c16 chain remains very unpolar (slower kinetic). To get more retention with a TFA ion pair complex you have to use a fluorinated phase (but that won't speed up the slow kinetic of the unpolar alkyl chain).
Have you tried to analyse your sample with a suitable column at pH around 9 and higher conc. of methanol?

Dear RP,
You might want to consider the method we have developed for cetylpyridinium chloride. The method is on our Primesep B column with ACN-water-TFA. Very good symmetry with no tailing. We are not going to "bad mouth" other columns but all our attempts with other columns did not produce good peak shape and symmetry - but we don't know other columns as well as ours.
This method is also applicable to other quaternary amines.

http://allsep.com/makeChr.php?chr=Chr_047

P.S. Please do not consider this as a "commercial" we just trying to help chemists with their problematic separations.

Thank you. I have seen your application example and it seems quite nice. We ended the validation of the method with the high Tf. But for sure i will consider this kind of stationary phase if i have problems during analytical transfer. Quite interesting the fact to use a stationary phase with basic groups to increase the peak shape of this kind of compounds.

To andreas: i'm working with a gradient 70-90% ACN. With ion pairing agents like pentansulfonic acid sodium salt i have seen only methods working at acid pH.I think that at higher pH silanol activity is higher and i don't think that this could improve peake shape.

Maybe the lower the ionic interactions the bettere the peak shape.

Hi rp,

you're right, at higher pH the silanol activity increases.
My idea was to counter the ionic interaction of cetylpyridinium with the silanol groups by using a higher pH to analyse the analyt as non-ionised molecule. The use of MeOH istead of ACN based on the assumption that cetylpyridinium is solved better in MeOH.
When this condition proves to be not good enough I would have suggested to use a SiOH-Column instead of RP.
But, as I wrote earlier, my ideas based on theory. If someone out there has some experience with the conditions I would be very thankfully for some feedback.