# Preparation of ammonium acetate buffer for HPLC

Buffer solutions are typically prepared by dissolving an appropriate salt in water and then adjusting the $$\mathrm{pH}$$ to the desired position by addition of the conjugate acid or base.

For example, to prepare a $$\pu{100 mM}$$ ammonium acetate solution at $$\mathrm{pH}~9,$$ you’d first prepare a $$\pu{100 mM}$$ ammonium acetate, and then add ammonium hydroxide dropwise until the desired $$\mathrm{pH}$$ was achieved (a titration, using a pH meter to determine endpoint).

I’d like to work out (roughly—I understand that what I’m about to describe is nothing more than an approximation, but it’s a reproducible approximation at least which is good enough for our needs) directly how much ammonium hydroxide and acetic acid I would need to make up the same solution.

I understand the fundamental Henderson–Hasselbalch equation:

$$\mathrm{pH} = \mathrm{p}K_\mathrm{a} + \log\frac{[\ce{A}]}{[\ce{HA}]}$$

But as I see $$\ce{A}$$ is ammonium and $$\ce{HA}$$ is ammonium acetate. Thus, in my example I don’t really simplify things that much.

Additionally the majority of examples I’ve found look at acidic buffers rather than alkali ones. Does my equation therefore need to be rearranged further to take account of this?

• HA is ammonium NH4+ and A is ammonia NH3, with pKa approx. 9.25. – Poutnik Jan 9 at 19:39
• I know the pKa of ammonium, my issue is piecing everything together to work out the acetic acid amount. All of the examples on the internet seem to be for acid buffers – user103231 Jan 9 at 19:46
• Eg. Many examples of preparing acidic ammonium acetate solutions calculated from a HH approximation – user103231 Jan 9 at 19:47
• pKa of ammonia is very high, you mean pKa of ammonium. – Poutnik Jan 9 at 19:48

to prepare a 100 mM ammonium acetate solution at pH 9, you’d first prepare a 100 mM ammonium acetate, and then add ammonium hydroxide dropwise until the desired pH was achieved.

Well, this procedure is good enough for a rough screening solution- call it a quick and dirty method. The main issue is that one would not prepare a solution of 100 mM ammonium acetate beforehand and add drops of ammonia to that. Whenever you need to prepare a buffer solution, you should know what volume is desired and what concentration is needed.

For HPLC, let us say you need 1000 mL of buffer containing 100 mM ammonium acetate. One would dissolve the required amount of ammonium acetate in about 900 mL water in a 1 L beaker, add ammonium hydroxide dropwise till you reach the desired pH, add more water till you are close to 950 mL. Check the pH again, and adjust as desired, and then quantitatively transfer the solution to a 1 L volumetric flask and then make up the volume to 1 L. This will be your analytically correct 100 mM ammonium acetate buffer.

Now, coming to the buffer equation. Again, for that you need to define the concentration and volume. What would ask yourself what should be concentration of ammonia should be present in 1 L of 0.1 M ammonium acetate solution such that the final pH is 9 solution. You would use the pK$$_a$$ of ammonium ion and from the equation you would figure out the concentration of ammonia.

See example no. 2 here: Buffer calculation

And also study example 3. Ammonia buffers

Now the problem is that conc. ammonia is sold as w/w % solution. Using the density and percentage, you need to determine the molarity of ammonia solution as well to actually prepare a buffer.

• Thanks for your answer. The main purpose of my problem is that we don’t have a reliable way of measuring pH and such so I was trying to estimate how much of A and B I’d need to mix to theoretical have the solution at the right pH. I’ll work it through and see where I get. Thank you – user103231 Jan 9 at 19:54
• I’m still lost about the ammonium acetate since I don’t want to use the salt but rather just mix HPLC grade ammonium hydroxide and acetic acid - would they just be mixed 1:1 to make the 100mM solution and then work out how much overall ammonia is needed based on your links ? – user103231 Jan 9 at 19:57
• @user103231, You have an HPLC but not a pH meter? – M. Farooq Jan 9 at 21:14
• @user103231 For given pH, you need to fit pH=9.25 + log (( c1.V1 - c2.V2)/(c2.V2)), where c1,V1 are molar concentration and volume of ammonia and c2, V2 the same for acetic acid. Nothing beats pH meter, but if this is good enough for you... At this pH, acetic acid can be considered like if it were a strong acid. – Poutnik Jan 10 at 14:23