Polar analyte analysis

[carls]

Its posssible the cyano will work but Ive found the sucess rate in HILIC to be fairly low with this column.

You can run the 1100 in normal phase mode for a short period of time without changing seals.

If you have a bare silica column, any bare silica column, its your next best bet.

Any column that has not been previously used in HILIC mode should be conditioned with 50/50 ACN/20mM ammonium acetate (or formate) prior to use to ensure any previously used salts are removed and to aid later equilibration.

[Vlad Orlovsky]

HILIC is not going to work on cyano or diol columns, you can try different columns but bare silica (among traditional columns) will give you more retention than diol or cyano columns, so if you don't have retention attributed to HILIC on bare silica (which we had in case of cyanoguanidine) you are not going to have retention on cyano column. It is less expensive to buy seals for Agilent and try normal phase then spend several days on the method development which has no chance to work. One week of chemist in US cost company at least $3000 in direct and indirect expenses.

[Blaza]

Hello!

I am certainly not an expert, but I am wondering why you didn't ask your Phenomenex dealer or Phenomenex itself yet?! I am a their dealer from Serbia and know that application support at PHE works very well, so you can expect at least some advices about the problem. At least, but becuase the column is new and very important, I believe you will get much more.

Please try via their web-page or, if it doesn't work (I really doubt), I would like to try to handle this.

Best regards,

Blaza

[bisnettrj2]

I have contacted my rep at Phenomenex. I didn't get anything exceptionally helpful, which I found unfortunate. I got an application note about melamine and cyanuric acid, and an article about nitroguanidine, RDX, and HMX that I'd found myself. There really doesn't seem to be a lot out there about this analyte. I'm tempted to take up Vlad's offer to do free method development, but I really want to figure this out for myself as a learning experience. Does anyone think there is any possibility to using ion pairing reagents like HFBA, TFA, or sodium hexanesulfonic acid in 100% water to retain this analyte? I also have a Hamilton PRP-1 column - I think I might see ifthere be a possibility to retain nitroguanidine when using a basic buffered mobie phase around 9 or 10 or so. We'll see.

[Vlad Orlovsky]

In my opinion nitroguanidine does not have strong enough ionic properties to benefit from ion-pairing reagents or high pH buffers. You can try 100 other things but before doing this ask yourself "What mechanisms of retention and properties of the molecule I am exploring in this particular case?" If there are no mechanisms present don't waste your time, machine time and solvents. Although some people will consider this as "job security" and "research"

Also I think that HILIC properties of cyano column and some diol columns come from residual silanols. Look at properties of propylonitrile vs. acetonitrile, first one is much more hydrophobic and does not mix with water well, now you expect to create a polar layer in case of cyano column? I bet you that well covered cyano column with end-capping is nor demonstrating any HILIC behavior.

Happy Holidays to everybody!!

P.S. Hans if you do search on Google for pKa of nitroguanidine you will end up with few references although pKa numbers have a wide reported range Based on the structure I would say that the correct number is between 2.5 and 4.

[HW Mueller]

Hm, if it had a pKa between 2.5 and 4 it could benefit from ionic interactions. Also, I have trouble conceiving of that as an acid, maybe the structure is really undefined?

[Uwe Neue]

It's not an acid. It is a very weak base with a pKa around 3.5 according to Vlad. At pH 2 it should be protonated and positively charged, at pH 5 it is neutral. This makes sense. If the pKa is correct, ion-pairing is likely to work.

[Vlad Orlovsky]

My statement on IP was based on our experience with cyanoguanidine. As you know on our columns IP is attached to the surface. We tried our strongest columns and there was no retention attributed to ion-exchange.

[Hollow]

bisnettrj2:
Why don't you try to get retention on bare silica TLC plates with "HILIC" mobile phases such as ACN/MeOH or IPA etc. (try those solvents which are ok with your HPLC seals, so you won't have to buy special NP components).
Once you get some retention on TLC, maybe you than can convince your boss to buy some bare silica HILIC column and adopt the eluent system to HPLC and fine tune the method?

[Uwe Neue]

So maybe we still do not yet know what its pKa is. Maybe it does not have any at all... Then ion-pairing or mixed mode are not going to work either. Normal phase chromatography sounds better and better...

[Vlad Orlovsky]

My bet is on normal phase, mixed-mode did not work, so RP and ion-exchange is out of question. HILIC did not work, and RP-pi/pi did not work....so what else is out there. I was also concern about absence of similar data on pKa, my guess is people have no idea what is pKa for nitroguanidine.

[bisnettrj2]

OK, here's an update - per the Phenomenex rep, the Kinetex HILIC IS a bare silica column, not a diol phase. I could have sworn that he said diol when we spoke about it, so this is totally my mistake here. What does that change for everyone? He's suggested low pH buffer and ion-pairing reagents. I probably won't have time to try it out until January, though.

[Vlad Orlovsky]

Did Phenomenex representative tell you how ion-pairing reagent works with bare silica column. May be I will learn something too

[bisnettrj2]

Nope. And I did try a pH 2.5 ammonium formate buffer. But, like I said, due to holidays and vacations, this probably will get back-burnered until January, and we'll see how busy I am when I get back to a normal work schedule.

[Vlad Orlovsky]

That was a joke, IP is not working with bare silica. IP is used in combination with reversed-phase chromatography. I am surpised that Phenomenex representative did not know that
if you used bare silica column with 95% ACN and did not have retention then don't try any more HILIC, because you are wasting solvent big time.
Also may be you have Atlantis T3 column in your lab and can use it with 0% ACN in RP mode.


P.S. Uwe, you see I am trying to recommend/sell Atlantis T3 column!