Dear Jihui

Would you please explain more about your method. it seems that you have a second solution which will be mixed with aetonitril. what is that solution. about 10 mM ammonium acetate, are you sure that it should be added to acetonitril? if yes whay you need such a solution?

Dear Somaieh,

To make up the solution, I have done the following steps:

1. Dissolve solid ammonium acetate into water at 1M;

2. Add 10ul of the above 1M Stock solution to 1L Acetonitrile so the concentration of ammonium acetate would be be 10mM

The acetonitrile containing 10mM Ammonium acetate is a mobile phase B of LC-MS-MS used for gradient elution in compound analysis. The method was published. So I think this kind of solution should be able to be made up.

Thanks a lot

Jihui

10 ul of a 1M solution in 1 L will give you a 10um (microM) solution, not 10mM.

There was an interesting discussion about the solubility of ammonium acetate in acetonitrile: http://www.chromforum.org/viewtopic.php?p=34523&sid=7d47aa3192c7fd38ee046eeed3e12f49

We have had issues with the solubility of ammonium acetate in acetonitrile. I have dropped the concentration down to 5 mM, and gone to slowly (SLOWLY!) adding the acetonitrile to the buffer with stirring and post-filtration. I run 10 mM in the aqueous eluant, and 5 mM in the acetonitrile, and still get good ionization. Note that most of the LC-MS methods use water/methanol rather than water/acetonitrile, especially with ammonium acetate buffers. There is no solubility issue with the methanol.

Thanks a lot, Gaetan. It should be 10ml to 1L. Sorry for the mistake. Thanks very much for the suggestion and I will have a look the discussion.

Hi, Mark,

good suggestion. I may consider to reduce the concentration in acetonitrile or use methanol instead. I have actually run with 10uM and sensitization was good too.

Jihui,

You got ionization with 10 uM? That is huge - we would LOVE to drop the concentration of ammonium acetate, since it just eats up everything in your system. I'll try it and let you know what we see.

Hi Jihui,

You will have problems dissolving ammonium acetate in acetonitrile. Sigm-Aldrich addresses this with 'acid stabilized' acetonitrile containing 0.1% ammoniumn acetate. The attachment is from Sigma-Aldrich and describes using this.

Here is the attachment

Hi, do not trust to everything what is published. Ammonium acetate can´t be disolved in acetonitrile. If you like to have 10mM amonium acetate in ACN in the eluent B, do not use pure ACN but it's mixture with water. Let's say that you will have 85% ACN containing 10mM ammonium acetate. Disolve the salt first in the water. Don't worry sensitivity will be the same. What publication you follow?

Sorry, I did a mistake in the text. I meant to prepare not 85% ACN but 95% one.

Hi,

If you really need 10 mM ammonium acetate then a minimum of 10% water is required to achieve a stable solution. If you insist on using 100% acetonitriel as one of two mobile phase liquids for a binary gradient, you should use either pure acetonitrile or, possibly, acetonitrile with acetic acid (or formic acid), as one, and have an ammonium acetate buffer in the other. Note that ammonium acetate alone has no buffering capacity buffer, you need to add either some acetic acid or some ammonia to achieve a controlled pH (for example at pH 4.8 or pH 9.3 respectively)

Hi Jihui:

You may use 20ml of 0.5M ammonium acetate instead.

Secondly, you may try to warm the acetonitriel in water bathe to about 30c and keep the solution warm. It will help to higher the salt dissolution rate.

Try to optimise your salt solution with some water

Thanks very much for all your contributions to this question. I really appreciate. I agree that the water content has to be >10% in acetonitrile to achieve 10mM ammonium acetate. However, using the acid stabilized ACN as Ken suggested from SigmaAldrich would be helpful.

Mark, lower the concentration to 10uM was OK to my experiments as sufficient signal and sensitivity were achieved. Based on the Sigma document, the influence of salt on ionization is various on different compounds . It would be wise to try a preliminary run on your samples.

Fred, rising temperature to 30C is very interesting. Cheers

Thanks, Kasia. I will be cautious on these issues.

Usually, I prepare a solution of ammonium acetate in water and in a separate bottle acetonitrile. You can use these solutions in the gradient elution for LC/MS/MS.

You can acidified the ammonium acetate solution with concentrated acetic acid at 1% v/v.

Usually the concentration used are ammonium acetate 5 mM or 20 mM with 1% of glacial acetic acid.

Attention, when you have finished to use this solution, wash the column with water and after with acetonitrile.

thanks a lot, Angela.Did you mean that ACN is used without any salt?

I agree with what Angela wrote. Another alternative is ammonium formiate, pH can be adjusted by ammonia or formic acid. I've the impression that it is somewhat more volatile than acetate. I usually make up a 10 mM solution in water as solvent A, solvent B is ACN to 70 or 80% in solvent A. This works fine for gradient elution on a ESI-IT system.

Thanks a lot, Didier. Yes. if the ACN concentration can be dropped to 70-80%,higher concentration of salt can be achieved.

Dont introduce ammonium acetate to acetonitrile

I prepare my solution with ammonium formate in this way : ACN/H2O 95/5

NH4COOH 20mM

pH 4.8 (water solution)

Add 5 mL of the stock solution HCOONH4 4M to 40 mL H2O and stir it for 2 h, then adjust the pH to 4.8 with few drops of formic acid.

Make up to volume (50 mL) and added to 950 mL of acetonitrile slowly. Stir it for 2 hour.

when you finish wash the column with water and all connection because it is very easy to have the precipitation of salt.

Many thanks Anna. it sounds that we should allow sufficient time for the salt dissolving in ACN and also the pH looks very important. I will try this method.

I it difficult to do it as you described. I use instead 5% isopropanol, 90% acetonitrile and 5% water (in which you can solve your ammonium acetate).

Many thanks for you all. I will try the various options according to the application.

I used to sonicate my 5mM ammonium acetate in ACN:H2O (95:5) for 5 min before using it. Also using acetic acid may help. Good luck!

For making a buffer in pH range between 3,8 and 5, you can weight (for 10 mM Acetate concentration) and solve in a mixture of acetonitrile and water 70:30 (v/v) in a bottle of 2 L. And with acetic acid adjust to pH you want.

First you can make 10 mM ammonium acetate in water.For pH adjustment in acidic site you can go with acetic acid and in basic side you can go with ammonia. Then you prepare your mobile phase with prepared10 mM ammonium acetate and acetonitrile in different combination and you can try in in reverse phase LC-MS/MS system for method developemnt. Even ACN : Buffer ::65 : 35 combination also would not be a problem. FLow rate is important.

The commercial available solution has max. 2% Water and 98% ACN. NH4Ac is a terrible sticky salt. It's convenient if you prepare a 7,5 molar stock solution in water (5,781 g NH4Ac + 5,3515 g water) and store it cold. AB Sciex has the same solution in the PPG-Box. For 10 mM you take 1,333 ml add 0,666 water and I add it slowly into the hot (60°C) ACN while sonificating under the hood. The ionisation in the MS is better with little water.

...and 2% water in 1 liter is 20 ml - not 2 ml. I forgot.

Firstly, prepare the Buffer solution: 10 mM ammonium acetate in HPLC grade water (~ 0.77g of NH4COOCH3 in 1000 mL of water). If the pH adjustment is required, you can make the adjustment with acetic acid or formic acid for acidic ends or ammonia solution for basic side.

If the system condition is gradient and you are ramping from the initial combination of two Mobile Phase (s) (MP) MP A: MP B, ACN: Buffer solution, v/v, 65: 35, it is better to make MP A with the combinations (95% ACN + 5% buffer) and MP B (95% buffer solution + 5% ACN). For the pre-mixing, the two phases already felt each other, because of the endothermic nature of the mixture. Then ramp accordingly and remember to accommodate the 5% added during the gradient programming.

Caution: As long as the buffer solution is not more that 50mM for this (ammonium acetate) salt there should be no precipitation problem and the mixing will be even.

Hello! I am having this same problem. I am trying to prepare UPLC buffer 90% acetonitrile/10% aqueous, contaning 10mM ammonium acetate and 0,04% ammonium hydroxide at pH 9. First I dissolve the salt in the aqueous phase and then mix with acetonitrile. I have tried adding the aqueous solution into the acetonitrile and the other way round but both result in inmiscible phases, where the aqueous phase forms droplets or buffer becomes hazy. I have tried sonicating and heating but after some time it ends up crashing out. Any help? Thanks a lot

I use ammonium hyhdroxide (28% in H2O from Sigma) and titrate the pH to 9.0 with neat acetic acid. I never have problems with solubility in 80% acetonitrile. My application is high pH reversed phase fractionation of protein digests.

The easiest way to solve 10 mM NH4Ac in 100% organic solvent without water is to use about 10% methanol and solve it first only in methanol. Then you can add 90% ACN or IPA. NH4Ac is easy soluble in methanol.