I want to achieve good peak shape for quaternary amines on a reverse phase column. So far I tried two of the most common in my lab reverse phase columns and various combination of ACN-water-TFA (formic and acetic acids as well) including isocratic and gradient elution. Peak symmetry is not acceptable (0.5-0.6) with a very significant tailing. Can somebody advice me on how to improve peak symmetry?
Thank you in advance

PJ

Lower the pH further to reduce silanol ionization with sulfuric acid or better still with perchloric acid. Use an ion pair reagent such as octane sulfonate at around 5mM. Switch to a polymeric reversed phase column such as Dionex's MPIC NS1 column or Hamilton's PRP1 column.

Thank you Chris,
I tried to lower pH to 1-1.5 but still see very asymmetric peak (with TFA and sulfuric acids). I need symmetry of at least 0.85 and I am far away from this number. I am using Xterra 4.6x150 right now and will try polymeric column next week. My quaternary salts are not UV active and I am using ELSD.

I would increase the ionic strength. A volatile acid such as formic acid or acetic acid will do, but use a higher concentration than what you are normally using. It is an ionic strength phenomenon, not a pH or silanol phenomenon.

Anon,

Sorry, I didn't realize you are using ELSD for your detection. Obviously, true ion pair reagents such as octane sulfonate won't work for this application since they aren't volatile enough for use with your detector. I agree with Uwe that your best choice would be to use either formate or acetic acid (I would suggest acetic acid). In my experience, it's always much easier to get reasonable peak symmetry using a polymeric material for this class of compounds.

FYI: the conventional practice is to assign an asymmetry value of greater than 1 for peaks which are tailing. I believe that you are calculating asymmetry backwards from the normal convention.

Chris,
I am using Agilent 1100 and in my report it gives me symmetry below 1 if the peak is tailing and above 1 if there is fronting. How can I change the set up on Chemstation?
I tried to use AcOH and formic acid (up to 1%) and still have asymmetric peak - trianle peak with tailing. My mobile phase is ACN-water-AcOH (HCOOH)=with 20-50% water abd up to 1% of acid. Column Xterra 4.6x150 mm.
What other mobile phase s can I try? (Water's people?)

The ELSD should be able to deal with ammonium formate or acetate instead of just the acids.

Dear Uwe,
I tried both ammonium acetate and ammonium formate (20-60 mmol in ACN-water, pH=3-5) but did not achieve peak symmetry I need. Would you please suggest conditions for the separation of quaternary amines on Xterra column (as Waters main scientist you might know better than anybody else). Thank you in advance.

PJ

Anonymous from Feb 10,

Please give me your email and shipping address and I will send you one of our columns to try (Primesep B, 4.6x150 mm) for your separation. I think that you might achieve good peak shape for you quaternary amines. Here are couple links - just compare these chromatograms with one you already have:

http://allsep.com/makeChr.php?chr=Chr_047
http://allsep.com/makeChr.php?chr=Chr_043

P.S. I have only one column to give away.

PJ,

Our applications to date with quaternary amines use UV detection, and neither MS nor ELSD. All the techniques with UV detection employ ion-pair reagents. One of us is will be working on suitable MS conditions, starting in about 2 weeks.

Uwe