Chromatography Forum

Hi,
I run a method with 95% acetonitrile 5% water with 10mM ammonium acetate modifier. I find it difficult to get the ammonium acetate dissolved and was looking for advice how to make this quicker. Currently I make 200 mM ammonium acetate(aq) and add the acetonitrile to this in steps with mixing in between to get prevent phase separation. Despite this I sometimes get micelles with water at the bottom that takes a very long time to dissolve and I have to heat and sonicate to do it. I do take care to weigh in and measure volumes accurately.

Should I do it in another way, any advise?
Thanks!

Have you tried to make 100 mM in ACN/H2O 50/50 and dilute further with acetonitrile?
If your method is not (yet) validated, try to reduce the amount of salt to 5 mM. Buffer capacity is limited anyway at 10 mM, and for ionisation purposes in ESI-MS 5 mM should be more than enough.

Regardless of the methodology, is there a way to switch your mobile phase component? I'd be very worried that precipitation will occur in the bottle (especially if the temperature fluctuates in your lab), or onto the column you are using. I assume this is apart of a binary phase where this is one of your reservoirs, while the other is the aqueous portion. Could the phases be juggled a little better by any chance? For example, if you're using a 100% aqueous MPA (mobile-phase A), and this mixture as your MPB, then could it be possible to get at least a similar separation with a MPA that includes 10% ACN and a MPB with a concentration of acetonitrile less than 95, maybe like 75-85%?

Perhaps I'm just being pedantic but this would have me very worried when it comes to potential precipitation.

Regardless of the methodology, is there a way to switch your mobile phase component? I'd be very worried that precipitation will occur in the bottle (especially if the temperature fluctuates in your lab), or onto the column you are using. I assume this is apart of a binary phase where this is one of your reservoirs, while the other is the aqueous portion. Could the phases be juggled a little better by any chance? For example, if you're using a 100% aqueous MPA (mobile-phase A), and this mixture as your MPB, then could it be possible to get at least a similar separation with a MPA that includes 10% ACN and a MPB with a concentration of acetonitrile less than 95, maybe like 75-85%?

Perhaps I'm just being pedantic but this would have me very worried when it comes to potential precipitation.

Thanks for advice I think it will be good to make sure to get some ACN mixed into the water phase really well as you suggest. I was using a 2 L bottle that did not fit into the sonication bath so I probably added to much ACN before getting a the first addition mixed enough. This resulted in a bottom layer with H20 and AmAc but it dissolved over night. The method is quite complicated running like 200 MRMs so I have speed a lot of time with validation which is really time consuming to get all compounds right. I am very careful with the ammonium acetate and never leave it the system without flow and have not had any problems so far.

Thanks for the advice and some options to test if problem persists.
Per

Can you use triethylammonium acetate instead of ammonium acetate? It would have less tendency to promote the phase separation that you're observing.

Can you use triethylammonium acetate instead of ammonium acetate? It would have less tendency to promote the phase separation that you're observing.

That is an interesting option as a buffer I will keep this in mind as an option. Never heard that this was an alternative before.
Thanks!