TBAHP as Ion Pairing Regaent

[ananda]

Hello All,

I need your advice and comments on a problem I am having lately. I am using Tetrabutyl Ammonium Dihydrogen Phosphate (TBAHP) as an IPR for one of my HPLC methods to separate an impurity of ~30 aa peptide from its main peak of 33 aa. A colleague from the lab developed this method and now she is on her maternity leave and now I am struggling.

The problem is, after 10-15 injections, the column ;Vydac C18 and even C8 (4.6X250mm)-300A, 5um ; lose resolution and it seems column become bad. Pressure doesnt change but profile changes completely. We cant figure it out whats wrong but very likely something bound onto the column and changes the resolution. We cleaned the column and there were some improvements but not 100%
MPhae is 1mm TBA in water, pH 6.7 (A) and 1 mM TBA (pH 6.7) in 80%ACN

Anyone of you have any experience working with this TBAHP IPR??Please comment!

Thanks in advance!

Ananda

[Chris Pohl]

Ananda,

There is a good chance that the problem stems from poor quality TBA being used as the reagent source. To get the best quality, it's better to start with highest quality TBA hydroxide from Fluka or Sachem and the adjust the pH with HPLC grade phosphoric acid.

FYI, the most likely culprit is tributylamine, a common impurity in TBA. To completely remove it from a silica based RP column you should use 80% ACN, pH2 with phosphoric acid (the acid source doesn't really matter but since you are using a phophate eluent system this should present the fewest problems).

[ananda]

Hi Chris,

Thank you very much for your comments. In fact right now we are using TBA-OH instead of TBAHP at pH 6.7 adjusted with HCl and no phosphates in the mphase. We are getting the resolution back but have to inject 10-15 injections and see whether we can get this retention continuously. We were using highest purity TBAHP ampoules from Fluka. LIke you said if trimethyl amine is the culprit, we can avoid it by using TBA-OH (hydroxide) instead of TBAHP assuming this trimethyl amine conamination is originated by the phosphoric acid reaction in the TBAHP sysnthesis? What do you think?

Thanks!

Ananda

[Uwe Neue]

It could also be simply a question of slow equilibration with the ion-pair reagent. 1 mM TBA takes along time to equilibrate the column. Only after this equilibration will you get reproducible results. If your separation works only before the complete equilibration and not after this, you do not need the TBA in your mobile phase.

[Chris Pohl]

Ananda,

Now I'm really confused! Are you saying that you've been using the Fluka TBA hydroxide all along or is this a recent adaptation of the procedure? A couple of points relative to your last post: the contaminant I was referring to was tributylamine not trimethylamine and it arises not from the phosphoric acid but from the TBA. It is a degradation product which will accumulate over time in the reagent and the best way to avoid if it is to use fresh good-quality reagents. Perhaps your problem was caused by using an old ampule. Another point is that neutralizing TBA hydroxide with hydrochloric acid and adding a sodium phosphate buffer will not give you the same result as neutralizing the hydroxide with phosphoric acid. The counterion frequently makes a significant difference in the chromatography although this is somewhat dependent upon operating conditions in ion pair chromatography.

[ananda]

Hi Uwe and Chris,

Uwe, usually we equilibrate the column over night before we do any injections but your point is well taken. If it takes more than 24 hours to equilibrate, then TBA may not be required. The optimum pH is 6.7 in the presence of this IPR and no extra phosphate were added other than the counter ioni from the TBA.

Chris, sorry for the mistakes. I did mean to say we were using TBAHP but now we are trying TBA-OH. I thought this tributyl amine contamination comes from the synthesis but if it comes from the TBA group it doesn matter whether it is TBAHP or TBA-OH it may present as a contaminant. We have seen the problem even with fresh and new ampoules as well. Other than the IPR no additional phopshates present in the mobile phase and only the pH brings to 6.7 by the IPR and adjusting it either with HCl or NH4OH. Buffering is an issue too I think?

I have another copncern now. Since my MPhase B has 80% ACN, I dont know whether this problem is due to TBAHP precipitate after sometimes in the presence of ACn like all other phosphates. Thats is why I think TBA-OH may be better than TBAHP. (rememeber i need to pH my Mphase to 6.7) . So I dont know how far I can maintain the pH at 6.7 without any buffers but just with the IPR.

Thanks for your replies.

Ananda

[Chris Pohl]

Ananda,

First off, I'm not sure I understand why you're adjudicating the pH using hydrochloric acid or ammonium hydroxide. Why aren't you adjusting the pH using phosphoric acid or TBA hydroxide? As I said, introduction of other ions especially those which are opposite in charge to TBA can affect the chromatography and because they are used in low concentrations (I assume) it can take a long time for the system to reach equilibrium since the affinity of TBA for chloride is higher than for phosphate.

Regarding your second question, if you think the problem is due to precipitation, you should be able to see this by simply manually preparing your mobile phase as is when it contains 80% acetonitrile and give it some time to see whether any precipitate forms. If after a few days you don't see any precipitate, try adding a few crystals of TBA dihydrogen phosphate to see if this nucleates precipitation. If this fails, precipitation is probably not your problem. Alternatively, you could use MOPS or BES as your counter ion as both of these species buffer in the range you are interested in.

[ananda]

Chris,

When we use TBAHP, we had do adjust (increase) the pH to 6.7 using NH4OH because our optimum pH for this method is 6.7. If mistakenly pH goes up then then we use either phosphoric acid or HCl to bring it down to 6.7. Now i understand your point of adding diffrent counter ions and the effct. I never thought it is going to affect that much at these low levels.

I think now since we are using TBA-OH instead of TBAHP, so fgar it seems better and not loosing the resolution but have to wait and see.

Thanks for your comments and suggestions.

Ananda