LCMS newbie

[hopam002]

Hi I am new to LCMS and having trouble with what are probably some basic concepts, which are hindering my ability to develop a new method for a compound called teriflunomide. I have done a fair bit of reading but can’t realy find an answer to some of my questions.



Firstly, I am trying to recreate a previously developed method, which is quite close to working however I am having problems with broad tailing peaks, which on occasions appear to split.
I have tried several variations of the gradient flow methods to try and tighten this up with no luck. With the mobile phase consisting of
A
H20 (95%), Acetonitrile (5%), 0.5mM ammonium acetate and 0.02 formic acid
B
H20 (5%), Acetonitrile (95%), 0.5mM ammonium acetate and 0.02 formic acid

Which leads to my first question of what is the purpose of ammonium acetate in mobile phase?

Dont i just want acid condition to ionize the compound for the MS?

Secondly what does negative ion mode imply? Is this needed because formic acid is responsible for taking the hydrogen of the alcohol group in the structure?

Anyway it appears apparent that I will have to alter the mobile phases, I have tried using methanol, which made little difference to the peak shape but did increase the retention. I have also tried the methanol with no ammonium acetate and 0.1 formic acid which just made the peak split. Should I try next without any formic acid?, just ammonium acetate?

Also is pKa of this compound going to be important for determining the required pH’s of the mobile phases and why are the mobile phase of the two original MP different at 3 and 6.5 for A and B respectively, and can this affect peak shape. Does anyone have any guesses on what the pKa will be because I can’t find anything in the literature and I’m no chemist.

If relevant I am using a C18 column, which has been suggested to me may be binding (retaining) this compound poorly?

Thanks for any help

This is the advice that has been given to me previously

"I think you need to exhaust the possibilities with the formic acid:ammonium acetate ratio - trying just ammonium acetate may work, but my feeling is that a little bit of formic acid (say 0.01%) will be needed. The pH of about 5-6 and 7-8 for mobile phase A and B respectively is what we should be aiming for. This may mean a lower amount of ammonium acetate into mobile phase B (although the acetonitrile will tend to increase the pH) compared to A..."

[michaelbarnes42]

I am afraid to offer too much advice, since I may lead you down the wrong path, but to answer your question as to what the Ammonium Acetate is for.

Ammonium Acetate is the buffer in your mobile phase, there are various different buffers that can be used and buffers aren't always needed.

Buffers can change your retention time, and improve your peak shape.

[lmh]

I feel the same, but a few points:
(1) The ammonium acetate and formic acid together operate as a buffer. It's quite a complicated buffer because ammonium, acetate, and formate all have pKas. I would usually make a buffer with ammonium acetate and acetic acid, or ammonium formate and formic acid, but there's no reason why you shouldn't have everything together, it will still be a buffer.
(2) I wouldn't attempt to measure the pH of any solution containing acetonitrile. Normal practice is to sort out the pH of an aqueous buffer and then mix it with organic solvents to taste. There are two different viewpoints on the pH of buffers. Some people trust pH meters more than their ability to make solutions, and set the pH by pH meter. Others trust their ability to make solutions more than their pH meter, and simply weigh out the appropriate ratio of ingredients. I personally don't think it matters what you do, provided you do the same every time, and anyone else using the method knows what you did!
(3) The buffer is there for the chromatography, not for the mass spec. Electrospray doesn't need acidic conditions to create hydrogen adducts. There's a lot of electrochemistry going on (which I don't understand) and it will give good signals for things that, at the pH of the mobile phase, really shouldn't be ionised significantly in solution.
(4) I didn't understand your bit about negative ion mode. Are you looking for negative ions from this compound, or positive? I would expect it to form positive ions more easily.

And most important:

(5) There are other causes of bad tailing and split peaks. For example, (a) is your column in good condition? Very elderly columns with voids and problems will give bad peak shape; (b) is your sample being injected in a weak solvent (water), or is it in high organic content? If so, you may find bad peak shape if it elutes fairly early in the gradient (because if it's injected in a solvent mix in which it doesn't actually stick to the column, at least a portion of it will migrate a long way into the column before the solvent is diluted out by the gradient buffer).