preparing ACN with 0.1% ammonium Acetate

[Elham]

Hello,

I am going to prepare the mobile phase for HPLC (Gradient elution). According to articles, I need Acetonitrile with 0.1% aqueous Ammonium acetate. In one article they write w/w and in another they didn't indicate w/w or w/v. So I get confused how I could prepare this mobile phase. It seems that w/v is more logical and I have to prepare 0.1% ammonium acetate straight in ACN (0.1 g Ammonium Acetate in 100 ml ACN). But I have realised that as soon as adding Ammonium Acetate in ACN, It is going to precipitate. So what should I do to dissolve ammonium acetate in ACN.

On the other hand, I don't know if I have to prepare the ammonium acetate solution in water initially (to make aqueous ammonium acetate) and then use this solution in ACN? So in which molarity or percentage should I prepare this aqueous stock solution to make ACN with 0.1% ammonium acetate?

Do you have any experience in this regard?
Kind regards,
Elham

[tom jupille]

If both atticles were by the same authors, then they were probably consistent (i.e., if one was w/w, so was the other).

I don't know for sure, but I suspect that ammonium acetate is reasonably soluble in acetonitrile.

[Elham]

If both atticles were by the same authors, then they were probably consistent (i.e., if one was w/w, so was the other).

I don't know for sure, but I suspect that ammonium acetate is reasonably soluble in acetonitrile.

Hi Tom,
No, The authors are different. My main concern is which concentration needs to prepare the aqueous ammonium acetate in order to make ACN with 0.1% Ammonium acetate?

[Andy Alpert]

If the article calls for aqueous ammonium acetate to be mixed with acetonitrile, then it must specify how much water will be in the final mixture overall. Do they provide a figure for the percentage?

This sounds like the sort of solvent one might prepare for an application in HILIC. HILIC requires at least 2% water in the starting mobile phase in order to be able to form the immobilized layer of water on the surface of the stationary phase.

[Elham]

If the article calls for aqueous ammonium acetate to be mixed with acetonitrile, then it must specify how much water will be in the final mixture overall. Do they provide a figure for the percentage?

This sounds like the sort of solvent one might prepare for an application in HILIC. HILIC requires at least 2% water in the starting mobile phase in order to be able to form the immobilized layer of water on the surface of the stationary phase.

Thanks for your comment,
Unfortunately they did not provide any figure for percentage and they just mention ACN with 0.1% aqueous ammonium acetate.
Meanwhile, I agree that water is essential for ammonium acetate solution but how much?

[Andy Alpert]

The total amount of water present is as important a variable as the amount of ammonium acetate.

Please cite the reference that you got this method from. I'll look it up if possible and see if I can figure this out.

[Elham]

The total amount of water present is as important a variable as the amount of ammonium acetate.

Please cite the reference that you got this method from. I'll look it up if possible and see if I can figure this out.

Hi,

This is a link for supplementary Methods of the article (Okada, et al., 2005, Structure of the Bacillus subtilis
quorum-sensing peptide pheromone ComX)

http://www.nature.com/nchembio/journal/ ... 709-S8.pdf

Thanks a lot

[Andy Alpert]

This helps. OK, your original posting asked about the possibility of dissolving ammonium acetate in pure acetonitrile to prepare a mobile phase. That's not what this article is promoting, though, which is why we were all confused. The article describes a gradient of 20-55% ACN in 0.1% ammonium acetate with a reversed-phase (ODS) column or a 20-32% ACN gradient in 0.1% ammonium acetate with a cyanopropyl column ("reversed-phase lite"). I suspect you assumed that you'd have to start with one mobile phase consisting of purely aqueous 0.1% ammonium acetate, a second mobile phase consisting of pure ACN with 0.1% ammonium acetate, and that you'd generate the gradient by blending them online. Not so! If I were you, I'd do the following:
1) Prepare a stock solution (say, 500 ml) of 1% (w/v) of ammonium acetate in water. Do this by weighing 5 g. of the salt into a 500-ml volumetric flask. Add about 250-300 ml of water and swirl until the salt is dissolved. Add enough water to reach the calibration mark and invert 8-9x. Use an HPLC grade of the salt if available, and filter the stock solution (0.45 um nylon filter) if the water wasn't already filtered and if you aren't using HPLC-grade ammonium acetate.
2) To prepare 1 L. of a mobile phase containing 20% ACN with 0.1% ammonium acetate, put 100 ml. of that stock solution into a 1-L. volumetric flask. Add 700 ml. water. Now add enough ACN so the volume is about 1-2 ml below the calibration mark. Invert 8-9x. The solution will cool. Let it warm up to room temperature (hint: I like to sonicate the solutions in a sonicator bath filled with warm or hot water for 1-2 minutes). The solution will expand (which is why you don't add ACN to the calibration mark at the beginning). Now add enough ACN to reach the calibration mark. Invert 8-9x.
3) Use the same method to prepare the other mobile phases with different % ACN, keeping the volume of added stock solution the same but varying the volume of additional water and ACN.

Hope this helps.

[Elham]

This helps. OK, your original posting asked about the possibility of dissolving ammonium acetate in pure acetonitrile to prepare a mobile phase. That's not what this article is promoting, though, which is why we were all confused. The article describes a gradient of 20-55% ACN in 0.1% ammonium acetate with a reversed-phase (ODS) column or a 20-32% ACN gradient in 0.1% ammonium acetate with a cyanopropyl column ("reversed-phase lite"). I suspect you assumed that you'd have to start with one mobile phase consisting of purely aqueous 0.1% ammonium acetate, a second mobile phase consisting of pure ACN with 0.1% ammonium acetate, and that you'd generate the gradient by blending them online. Not so! If I were you, I'd do the following:
1) Prepare a stock solution (say, 500 ml) of 1% (w/v) of ammonium acetate in water. Do this by weighing 5 g. of the salt into a 500-ml volumetric flask. Add about 250-300 ml of water and swirl until the salt is dissolved. Add enough water to reach the calibration mark and invert 8-9x. Use an HPLC grade of the salt if available, and filter the stock solution (0.45 um nylon filter) if the water wasn't already filtered and if you aren't using HPLC-grade ammonium acetate.
2) To prepare 1 L. of a mobile phase containing 20% ACN with 0.1% ammonium acetate, put 100 ml. of that stock solution into a 1-L. volumetric flask. Add 700 ml. water. Now add enough ACN so the volume is about 1-2 ml below the calibration mark. Invert 8-9x. The solution will cool. Let it warm up to room temperature (hint: I like to sonicate the solutions in a sonicator bath filled with warm or hot water for 1-2 minutes). The solution will expand (which is why you don't add ACN to the calibration mark at the beginning). Now add enough ACN to reach the calibration mark. Invert 8-9x.
3) Use the same method to prepare the other mobile phases with different % ACN, keeping the volume of added stock solution the same but varying the volume of additional water and ACN.

Hope this helps.

Dear Andy,
Thanks a lot for your proper and accurate description. It is really helpful. As I am new in HPLC (specially gradient one), I thought that I have to prepare the pure ACN with 0.1%Ammonium acetate as a source of mobile phase and HPLC machine can make the gradient 20-55% solvent for gradient elution. (Since I have done an isocratic run with pure ACN and 0.1%TFA as a source of the mobile phase (A) and water with 0.1 TFA (B) then machine made the 80% mobile phase (A) by itself during the run).

If I have to prepare the gradient mobile phase before run, I don't know which percentage of mobile phase in the range of 20-55% I should prepare, Just 20% and 55%? If so, Shall I put them as A and B? So Water with 0.1% ammonium acetate would be in C?

[Andy Alpert]

I like to have the starting and final mobile phase solutions actually in the reservoir bottles, rather than generating them by blending two very different solutions online. To do the blending method requires a very good mixer or else you will have oscillations in your baseline (not a problem at low sensitivity settings of your detector but sometimes a problem at more sensitive settings). Also, if you use blending of very different solutions, then you must have an HPLC machine that can reliably pump 1% of the flow rate or else you will not generate the beginning or the end of the gradient precisely (when you're pumping 1% B and 1% A, respectively).

If you're starting your chromatography with 20% ACN, then I don't understand why you would need a purely aqueous solution of 0.1% ammonium acetate in reservoir C.

[Elham]

I like to have the starting and final mobile phase solutions actually in the reservoir bottles, rather than generating them by blending two very different solutions online. To do the blending method requires a very good mixer or else you will have oscillations in your baseline (not a problem at low sensitivity settings of your detector but sometimes a problem at more sensitive settings). Also, if you use blending of very different solutions, then you must have an HPLC machine that can reliably pump 1% of the flow rate or else you will not generate the beginning or the end of the gradient precisely (when you're pumping 1% B and 1% A, respectively).

If you're starting your chromatography with 20% ACN, then I don't understand why you would need a purely aqueous solution of 0.1% ammonium acetate in reservoir C.

Dear Andy,

You are right. I don't need 0.1% ammonium acetate in C. I really appreciate your helpful comments. Now, most of the parts of preparing for HPLC is clear for me so lets run. I hope that I would get good result as I am going to do this run with different column (C18) not the recommended columns in article.

Bests,
Elham

[lmh]

Isn't it strange how everyone has different ways of defining 20%!

The problem I have with starting with 200mL of acetonitrile and adding water/salt until you end up with 1L is that there's no guarantee you'll have to add 800mL to get the right volume. You certainly don't with methanol. So it creates an interesting dilemma: if you want to make 50% MeOH, and start with 500mL methanol and make up to 1L with water, you end up with a different solution to what you get if you start with 500mL of water and make up to 1L with methanol. In this case, I would definitely blend 500mL methanol with 500mL water and tolerate the fact that I end up with less than 1L.

The difficulty for my approach is how to handle the salt? Because if I start with 100mL of 1% salt and don't end up with 1000mL of mixture, I can't claim the product is 0.1%.

The good news is that provided you are consistent, it doesn't really matter what approach you adopt. Your retention times will vary slightly from someone else who has a different policy. I would very much like it if journals would insist on methods sections describing exactly how such mixtures were made.