I have separated a chiral drug with HPLC via a ChromTech AGP Chiral column. The mobile phase composition is 4% IPA, with 20mM phosphate buffer, pH 7. However, the column is very old (around 7 years), and therefore the sensitivity is only down to 250ng/ml using the LC system. I tried using the same column, and moved it to the Tandem MS sytem. The mobile phase is changed to a MS-compatible combination, and I can see the chiral drug with increase sensitivity; however, there is no enantioselectivity (i.e. I can see only one peak for the chiral drug).

I tried using different organic modifiers (i.e. acetonitrile, IPA, etc.) and change of pH, but there is still no enantioselectivity.

I would appreciate any valuable suggestions.

Thanks in advance.

Do you achieve enantioselectivity when you switch to MS compatible solvents and use whatever your initial detection system?

Do you operate in selected reaction monitoring in MS/MS?

I use everything in the initial detection system, except that I decrease the flow rate, and changed phosphate buffer to ammonium acetate buffer.

Yes, I operated in a SRM mode in MS/MS.

Any suggestions?

How important is the pH of 7 in your analysis? One gets essentially no buffering with NH4Ac at ph = 7, so this may be off. (In other words, I am restating the question of Anon March 15 3:19 pm.)

The chiral column is good from pH 2-7, and I have tried pH4 and pH7 with the MS system. However, I can't see the enantiomers still. I used pH7 initially because the enantiomers can be separated at the LC system at pH7. Will I need to change the pH because I have change the buffer?

I have tried using ammonium formate today, but still nothing. What pH would you recommend? Sorry, can you please elaborate on the "no buffering at pH 7, and this may be off"? I don't quite understand the theory behind.

Thanks!!

This is Anon March 15 3:19 pm,

I will rephrase my questions with some insights. I asked if you achieve enantioselectivity by switching in the volatile additives (ammonium acetate). Maybe by switching to this additive you loose your enantioselectivity that is why you see only one peak (so check with your initial detector if you still retain your enantioselectivity with the new mobile phase).

There is always the possibility that the second peak you saw by using another detector (by the way what was the other detector?), was just an impurity. As now you are selecting only one mass to be detected with the mass spectrometry you do not detect the impurity.

You mentioned that you reduced the flow rate. What is the column i.d., old flow rate, and new flow rate?

Thank you all.

Kostas, I have tried using ammonium acetate with the UV detector. and I did not see enantioselectivity; however, and I saw it when I used phosphate buffer for the UV detector. But since one must use volatile buffers for MS system, and I saw that most papers will switch to ammonium acetate in place of phosphate buffer, so I tried using ammonium acetate and ammonium formate for the MS system.

I have pure standards for the enantiomer, and so it should be the real peaks.

MG, I actually used the same column for the LC system and the MS system. It is a 4x100mm, 5um column. The initial flow rate for the LC system is 0.9ml/min, and I now changed to 0.6ml/min for the MS sytem (however, I did increase the run time relatively to detect for the enantioselectivity)

Thanks.

One uses buffer to impede pH changes (thatīs what buffering means). Without this buffering the pH can change drastically if minute amounts of acid or base are contacting the solution. Obviously, if you had selectivity with phosphate, you changed your conditions drastically by going to the NH4Ac, it could be the pH, among other things.

Thanks Mr. Mueller, I have used different pH with different buffers (formic acid, acetic acid, ammonium formate, ammonium acetate). Do you think it is the problem of the chiral column (i.e. separation can only be achieved by the use of phosphate buffer?) Or what else would you suggest? Thanks!

First I would check whether the phosphate buffered mp still does the trick, then I would try to find a volatile substance which buffers at pH=7, keeping flow rate and ionic strength close to the original, etc, etc.