Peak tailing for quaternary ammonium compound on C18 column

[chhubert]

Dear all,

I have been analysing a quaternary ammonium compound by chromatographic separation using C18 column and MS for detection and observe peak tailing due to probable interaction of residual Si-OH on the column with the positively charged analyte. Mobile phases are 50mM NH4OAc at pH 4.5 and 0.1% formic acid in ACN. Good encapped C18 columns have already been tried but no improvement in peak symmetry. Should there be any alternative approach to solve the tailing problem if I don't want using TFA or ion-pairing reagent? Thanks advance for your suggestion.

Hubert

[Victor]

Please let us know which columns you have tried so far.

[Uwe Neue]

Quaternary amines can be run well with a silica column in HILIC mode. Much less tailing problems than on a C18.

[SIELC_Tech]

Hi Hubert,

How hydrophobic is your amine? Is it retained well on regular reverse phase column?
For hydrophobic amines you can try to use one of Primesep columns. Our columns are not endcapped but stationary phase with a positive charge shields silanol from interacting with your quaternary compound.
Here is application for cetypyridinim chloride. We used TFA in this application but you can use any source of ions (formic acid, acetic acid, ammonium formate or acetate). Applications shows comparison to regular end-capped column:

http://www.sielc.com/compound_114.html
http://www.sielc.com/compound_151.html

If you tell me more about your compound may be we can mimic your separation in our lab. Please contact me by email if you are interested (I might I have CDA with your company)

Kind regards,

Vlad

[chhubert]

Victor, the LC columns that I tried were Luna C18(2), Thermo Gold C18, YMC CN, Phenomenex PolymerX, Symmetryshield C8.

Uwe, I will try your suggestion, but wondering if the analytes are soluble in organic solvents prior to injection.

Vlad, I still have reservation in putting TFA in the mobile phase. I go thru the application of Primsep columns and most applications use TFA for chromatographic separation. Do you have any applications by replacing TFA with, say formic acid, acetic acid etc and get similar results in peak shapes and separation?

Thanks all for your suggestion.

Hubert

[SIELC_Tech]

Hubert,

How many carbons you have in your quaternary amine? I have couple of hydrophobic quats in the lab. We will try to run it this week. I might have some applications already but they are not on the web yet. If you can provide me with your email I can mail you results. What is your objective? How many compounds do you have in your mixture and at what concentrations?

Vlad

[Patrik Appelblad]

HILIC is a suitable alternative, especially as it will allow you to use the requested experimental conditions. In our application example, the overall ionic strength is 5 mM of ammonium acetate, an acceptable level for MS detection.
http://www.sequant.com/sn/ufiles/SeQuan ... 00-14A.pdf

Do a test tube experiment to check the solubility of your analytes.
If it dissolve in 80% organics, you have nothing to worry about.

[Uwe Neue]

Quaternary amines are well retained on silica. You should be able to get away with 70% acetonitrile or so, with a reasonable amount of MS compatible buffer in the mobile phase. Also, you do not need to inject the sample in 100% organic. With 30% water, your quats will be nicely soluble.

[chhubert]

Vlad, the compounds that we are going to analyse are triarylmethane dyes such as crystal violet, CI Basic Green 4. Problem in peak tailing of this type of compounds leads to integration difficulty.

Uwe, thanks for your advice. I'll try and let you know the result. Any suggestion on buffer/organic mobile phases used for this kind of compound when HILIC is used?

Hubert

[SIELC_Tech]

Hubert,

We have method for some dies (with UV detection). It was developed for one of our customers. Let me see if we run these dyes with LC/MS compatible mobile phase:

http://www.sielc.com/pdf/SIELC_November_2006.pdf

Please scroll down to method for quaternary dyes/amines. Problem with dyes is they are not pure compounds, it is a mixture of various isomers.

Vlad

[Uwe Neue]

My colleague who has worked with quats with HILIC says that the higher the ionic concentration is, the better off you are. I would start with 10 mM ammonium acetate pH 4.5 and increase the concentration, if the peak shape is not good enough. You will encounter concentration limits that depend on the amount of acetonitrile that you need for retention. If you continue to have peak shape problems at 70% acetonitrile and the highest concentration of buffer that you can use, you can also try a higher water concentration. HILIC on silica is a mixture of true HILIC and ion exchange.

[chhubert]

Uwe, I got a better peak shape of the quat amine using Atlantis HILIC column with 0.1% formic acid in water and 0.1% formic acid in ACN as the mobile phase. Initial start at 90% organic/10% aqueous, the gradient is slowly ramped to 30% aqueous till the analyte elutes. Notwithstanding this, the peak width is rather broad, at 1-1.5min at base with the retention time of ~ 7 min. Speeding up the gradient may solve this problem but poor separation of interferent is expected. Replacing with 50mM NH4OAc (pH 4.5) for aqueous mobile phase got even worse, both in peak shape and peak width. Any suggestion? Thanks.

Vlad, unfortunately I don't have any Primsep column at hand and can't get any trial run. Anyway, thanks a lot for your comments.

Hubert

[Uwe Neue]

I would increase the formic and flatten the gradient by using a higher flow rate, if possible. Think also if some of the peak width could be due to the sample solvent.

[SIELC_Tech]

Hubert,

I don't have LC/MS method for dyes, but we will run this in the lab today or Monday. I can send you chromatograms by email (posting on the web might take week or so).

regards,

Vlad

[chhubert]

Vlad,

Thanks for your effort. Grateful if you could send the chromatograms to my e-mail a/c at chhubert@hotmail.com. Looking forward to hearing your good news.

Hubert