Acceptable volatile buffer system for pH 7-8?

[khimik]

Hi,

I'm trying to develop an MS-friendly RP-LC separation of basic compounds, and it looks like I'll need to park the method at about pH 7.5 to get the best separation. Can anyone tell me a good choice of buffer system for this application?

My MS folks tell me they don't like ammonium citrate or ammonium carbonate. The latter is awkward at this pH anyway - bubble CO2 through an ammonium bicarb solution? Adjusting the pH down with anything other than carbonic acid will reduce the buffering capacity, right? I doubt ammonium acetate will work - almost 2 units away from ammonium's pKa and I think I'll need strong buffering - unless you know something I don't, so chime in.

Any reason not to try N-methyl-morpholine (with acetate I suppose)? Any stability, toxicity, reactivity or purity issues? If you've used it successfully in LCMS, please let me know.

Any other proven volatile systems for this pH range?

Thanks in advance for any info.

[SIELC_Tech]

Khimik,

If you are interested in using lower pH check the following link for retention of basic compounds at lower pH (2-7). You can use ammonium formate, ammonium acetate or formic acid. It shows comparison of analysis at high pH (over 9) and friendlier one (below 7):
http://www.sielc.com/pdf/SIELC_June_2005.pdf

You can also find bunch of methods for basic compounds here:

http://www.sielc.com/Applications_By_Compound.html

Regards,

Vlad
Toze Himik

[Uwe Neue]

khimik,

I still have not yet found a good MS compatible buffer at pH 7. I have played with carbonate only to find that it was not stable enough. I did do some investigations over a prolonged period of time using ammonium phosphate buffer at pH 7 with a Z-spray interface. The MS people declared me to be ready for a mental institution, one could see the buffer build up at the interface, but we were able to run for several weeks with such a set-up. Just had to clean the interface once in a while.

If the MS machine is not your own toy, the MS owner is not likely to let you play with this though.

[MG]

I don't know about the stability or toxicity of N-methyl-morpholine. From a volatility standpoint it looks like it won't clog the MS. But I've observed a problem with the supposed "mass spec friendly" amines, such as triethylamine, that they will suppress your signal in positive electrospray or APCI. Plus, if you are scanning for low masses, you'll have a huge background at 102. If your MS method is negative ion, you might not have these problems. If you try it, I'd be interested to know how it works.

[khimik]

Thanks for the replies.

I've just discovered that N-methyl-morpholine has an auto-ignition temperature not far above its boiling point. I.e., BP ~115, ignition ~165, if I remember correctly. I don't know jack about MS but I'm assuming some similarity to ELSD (which I have used) in how it handles the eluent stream - am I correct in assuming this is a fatal flaw?

Uwe Neue: Yes, I'm not the owner of the MS, so I am likely to be frowned upon if I try ammonium phosphate. Or worse laughed at. Sounds clever though. But this is discouraging - if you haven't found a buffer system for this type of situation, maybe I should stop trying and try PrimeSep...

HeyVlad: Thanks for the links. I've been meaning to educate myself on PrimeSep for some time but time of course time is the problem. Looks like it's time I made the time. The trick is that I work for a conservative pharmaceutical company that doesn't like to take risks on "new" column technologies. But if I continue to be unsuccessful with the tried-and-true, doors may open... On other topics, ya izuchil russkiy yazik v universitete. Vui iz rossii? Wow I sure wish I could figure out how to use a Cyrillic font in here.

If anyone has any further suggestions, please post!

Thanks,
khimik

[khimik]

What about ethylenediamine (pKa ~6.9) adjusted with ammonia to 7.5? Kostas' very elegant study (LC-GC Europe, Feb 2002) suggests this system is MS-friendly up to 25mM. Has anyone tried this system? Feedback? Any MS dudes finding ED to be objectionable?

Thanks!

[Eston]

Hi. Well I'm working with an Integrity Sistem (Waters) and I've got good results with ammonium acetate 10mM / ACN mobile phase working at pH 7 or even pH 9.5

[SIELC_Tech]

Khimik,

I know big pharma approach-I spend 10 years in the biggest pharma in the world.
We will be happy to help you with method development on Primesep column. We are offering free and confidential method development and can do it in our lab in parallel with your efforts:

http://sielc.com/Services_MethodDevelopment.html


We have secrecy agreements with bunch of companies in US and outside. Contact us if you are interested.

Regards,

Vlad

P.S. For Cyrillic typing you can use http://translit.ru/ (для кирилицы можно использовать эту ссылку)-you type with your regular keyboard using Russian transcription).

[MG]

Khimik, on the flammability issue: Your eluent stream is either going to be sprayed through a needle held at ~ 5kV, and the droplets dried with a heated gas with temperatures ranging from 250-500C (this is called electrospray ionization (ESI)); or nebulized then vaporized in a heated tube, with the vaporized analyte + mobile phase passing near a corona-discharge for ionization (this is called atmospheric pressure chemical ionization (APCI)).

It sounds like the sort of thing you wouldn't want to spray flammable solvents into, yet people do it (e.g. methanol). The important thing is the heated gas. If you make sure all gases entering the ion source are nitrogen (or other inert gas), and that the ion source is under positive pressure and free of leaks that might allow room air into the source, then you are safe to spray flammable solvents. If the mass spec uses compressed air for any of the gases in the ion source, then you need to be more careful.

[Kostas Petritis]

I wouldn't worry that much about it personally as it is just a mobile phase additive, which by definition is added in very small amounts....

[alealbor]

Hi Khimik,
You can used Triethylammonium acetate buffer (pH 7.0) 100 mM, but you must to used Divert Valve option in order to avoid to soil the ion source (ESI) too much.

You can try with 20 mM ammonium acetate solution (pH 7.0 adjusted with ammonia-acetonitrile) (2:1, v/v).

Another mobile phase could be: 25 mM of N',N'-dimethylhexylamine, DMHA (pH 7.0) adjusted with acetic acid


Alejandro