Chromatography Forum

I am trying to use hamilton PRP X-100 anion exchange(just this column) to separate a goup of methylphosphonic acids with pka around 2 to 2.7, what pH I should use? Anyone has experience?

The resin of PRP X-100 is PS-DVB (Polystyrene Divinylbenzene), supposed to work with both low or high pH. But when I tried with a 0.5% acetic acid and ACN (90:10), I couldn't see the separation. Thanks

What kind of methyl phosphonic acids are you looking at?
We do have an application note (organic acids on PRP-X100) that has at least one phosphonic acid in the mixture. I don't think it is available on-line but if you send me an e-mail I will forward you the appnote. The mobile phase is NaOH/DI water plus acetonitrile.

rhaefele@hamiltoncompany.com

Here is separation of methylphosphonic acid and related products. If you detection technique is ELSD then you need to watch for the temperature. Some of phosphonic acids are volatile even at 50*C:

http://www.sielc.com/application_203.html

regards,

Vlad

An alternative is HILIC-MS in negative mode.
I've attached a link to an application note for separation of 8 organophosphonate nerve agent metabolites in 12 minutes. Largest analyte is Pinacolyl-methyl-phosphonic acid and the smallest is Methyl-phosphonic acid

http://www.sequant.com/sn/ufiles/SeQuan ... 00-18A.pdf

Guys, thanks for your inputs.

The acids are a group of five nerve agent metbaolites. We in fact use a Hilic column for a LC/MS/MS detection.

I am just curious to see if PRP X100 can separate them, and then we can use a P specific detector.

Thanks again.

Not sure if the PRP-X100 can separate all of them but you have to use a pH at which these acids are deprotonated since the X100 is an anion exchange column. As I mentioned in my earlier response, we used NaOH/DI water plus ACN as mobile phase.

rhaefe, thanks.

I tried 20mM NH4NO3 with pH~10/ACN, no separation either. I will try NaOH/CN next.

It's interesting that PRP X100 is a Polystyrene Divinylbenzene based anion exhange column, the retention mechanism should be different from traditional anion exchange (no couter anions?). Because PS-DVB's large surface area, it should also be able to retain acids in their netural state, but may not be able to separate them?

yangz00g,

One thing you need to understand about ion exchange selectivity is that it is fundamentally based on the size of the hydrated ion. Generally, a pure ion exchange separation will tend to achieve no separation of a homologous series differing only in terms of hydrophobicity because the retention site is common to the homologous series and since the common element is the basis of retention you're not going to see any separation without a secondary retention mechanism. That's the reason why you can't expect to get a lot of separation of your mixture via a pure ion exchange retention mechanism. Fortunately, it's rare that polymer-based ion exchange media are free from reversed phase retention mechanisms. However, if you add acetonitrile to the eluent you will mask this retention mechanism and minimize any separation based on hydrophobicity. Generally, you will get the the maximum amount of hydrophobicity-based retention if you use a highly hydrated displacing ion. In your case, hydroxide should fit the bill but I'm not sure it will work very well with your detection system unless you're using suppressed conductivity detection. Another alternative would be to use formic acid/ammonium formate if you need to use a volatile eluent and you want to stay under acid conditions.

Thanks.

When I tried sodium hydroxoide, I saw some separation, but not all. There may be some hope. I will keep trying.

yangz00g,

Are you trying to run anion exchange chromatography? And if so, forget about ACN (in large portions anyway) and add some salt in your strong solvent (eluent B).
With the above mentioned analyte pKa you can elegantly utilize 0.02 M ammonium acetate buffer pH 4.8 (eluent A) and 0.5 M ammonium acetate buffer pH 4.8 (eluent B). Then try to find a suitable gradient profile.
If you experience excessive tailing, just add 5 – 10 % ACN to both eluents and it should take kare of the peak shape.


Best Regards