Hello everyone,
I need a citrate buffer with an optimal pH of 4.8 for the enzymes I'm working with. I'm using citric acid monohydrate (molecular weight: 210.14 g/mol) and adjusting the pH with NaOH. I'm preparing it as a 10x concentration and diluting it to 1x in the final volume (15-200 µl).
However, I've come across recipes for citrate buffer that use both sodium citrate and citric acid.
My question is whether the buffer I'm making will be strong enough to maintain a pH of 4.8 when I dilute it to 1x in my sample. Is the recipe with sodium citrate and citric acid a better option for buffering at pH 4.8?
What is the concentration? That is the only relevant factor determining strength of your buffer (and how much balast you add to it).
If you prepare buffer by mixing citric acid and citrate, you should calculate it such, that the final concentration will be constant, e.g. 0.2 M. For that reasons you have the tables of how much each of solutions should you mix. It's more convenient, because you (almost) get your desired pH instantaneously and do not need to adjust it (much).
But in the end, if you use the same concentration of [citrate], it's the same.
*by [citrate] I mean the sum of all forms, i.e. citric acid, hydrogencitrate, dihydrogencitrate...
Tomáš Hluska I am preaparing 10x by 1 M citric acid monohydrate and then adjust the pH with NaOH. In 1x I need 0.1 M citrate buffer. Is this in line with your suggestion?
I couldn't fully understand what you mean by "final concentration will be constant"?
Robert Adolf Brinzer thank you for the answer. Do you think it would make a difference to prepare the buffer with citric acid +NaOH or sodium citrate + citric acid + NaOH?
Elif Oztemiz Topcu yes, 0.1 M buffer should be fine.
What I mean by the constant final concentration is, that when you check table for preparation of citrate buffer, e.g. here:
https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/protein-biology/protein-concentration-and-buffer-exchange/buffer-reference-center#citric2
You will see, that with increasing pH, the volume of added citric acid decreases and volume of added citrate increases. If would you calculate the molarity of both compounds and sum it up, you would always get 0.1.
It's nicely visible here:
https://www.aatbio.com/resources/buffer-preparations-and-recipes/citrate-buffer-ph-3-to-6-2
use the lower slider to change desired pH and you can see, how the molarity changes. Just sum it and you will see that it is constant.
BTW it doesn't make sense to prepare buffer by mixing citric acid + citrate + NaOH, unless you mean the NaOH only for pH adjustment.
Tomáš Hluska thanks for the answer! for your last question, yes i will just adjust pH with NaOH. But I still can not decide should I prepare buffer again but this time with sodium citrate and citric acid or the buffer I already prepared with citric acid (adjusted with NaOH) will do the same job?
Elif Oztemiz Topcu yes, the one you already have is fine and you can use it.
It doesn't matter how you get there (as long as you don't start with sodium citrate and titrate down) - the buffering capacity will be the same. There are calculators available to help you figure out the amounts of citrate and citric acid to end up at your desired pH without having to titrate. Like this one: https://www.aatbio.com/resources/buffer-preparations-and-recipes/citrate-buffer-ph-3-to-6-2
We used sodium citrate and citric acid to prepare citrate buffer . There is a Table for buffer preparations in Methods in Enzymology Vol 1 .
Buffering capacity depends of the solution molar concentration . When Buffer pH is ajusted the citrate / citric acid ratio is ready, because pH depends of this ratio. When you dilute a buffer solution, the pH will not be changed, just the buffering capacity.
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