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Preparation of ammonium acetate buffer for HPLC
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Larry Tracy
Preparation of ammonium acetate buffer for HPLC
This is not correct for a $\ce{100 mM}$ ammonia buffer. When you add more ammonia to adjust the pH to the desire value, you increase the molarity of the buffer so that the total amount of $\ce{NH4+/NH3}$ is more than $\pu{100 mol}$. Here the critical buffer ion is the ammonium/ammonia couple, not the acetate which has no role as a buffer in this case.
To prepare an exact ammonia buffer at $\ce{100 mM}$:
pour the quantity (volume) of ammonium necessary for $\pu{100 mM}$ in water (less than the final volume)
adjust to the desired pH with acetic acid
complete with water to full volume.
NB: regardless of the pH, this will be a $\pu{100 mM}$ buffer
This is not correct for a $\ce{100 mM}$ ammonia buffer. When you add more ammonia to adjust the pH to the desire value, you increase the molarity of the buffer so that the total amount of $\ce{NH4+/NH3}$ is more than $\pu{100 mol}$. Here the critical buffer ion is the ammonium/ammonia couple, not the acetate which has no role as a buffer in this case.To prepare an exact ammonia buffer at $\ce{100 mM}$:
pour the quantity (volume) of ammonium necessary for $\pu{100 mM}$ in water (less than the final volume)
adjust to the desired pH with acetic acid
complete with water to full volume.
NB: regardless of the pH, this will be a $\pu{100 mM}$ buffer
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.
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.
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.
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.
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 ?More
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 youMore
@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.More
This is not correct for a $\ce{100 mM}$ ammonia buffer. When you add more ammonia to adjust the pH to the desire value, you increase the molarity of the buffer so that the total amount of $\ce{NH4+/NH3}$ is more than $\pu{100 mol}$. Here the critical buffer ion is the ammonium/ammonia couple, not the acetate which has no role as a buffer in this case. To prepare an exact ammonia buffer at $\ce{100 mM}$:
NB: regardless of the pH, this will be a $\pu{100 mM}$ buffer
This is not correct for a $\ce{100 mM}$ ammonia buffer. When you add more ammonia to adjust the pH to the desire value, you increase the molarity of the buffer so that the total amount of $\ce{NH4+/NH3}$ is more than $\pu{100 mol}$. Here the critical buffer ion is the ammonium/ammonia couple, not the acetate which has no role as a buffer in this case.To prepare an exact ammonia buffer at $\ce{100 mM}$:
NB: regardless of the pH, this will be a $\pu{100 mM}$ buffer
More
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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.
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.
More
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