Tris Buffer Calculations

Showing three methods to make a Tris buffer.
1) Tris base + Tris•HCl
2) Tris base + 1 M HCl
3) Tris•HCl + 1 M NaOH

[@e.z.4692]

It is 1:18 AM and I have a lab tomorrow, and you sir have just saved my sleep schedule. Thank you!!!

[@MichaelGaroutte]

Glad that it helped!

[@Belenbelenn]

Thank you so much I was looking for a good explanation

[@Cabbage22927]

Thanks for the video. Very helpful

[@yimingxu1913]

Thank you for making the video and very clear , thorough explanation . But if you are a compulsory math fanatic, you would realize that the mathematics behind the reasoning for all three ways of marking the Tris buffer is an approximation, although an extremely close approximation with errors truly negligible. The approximation occurs due to the fact that contributions from hydrogen and hydrogen ions are completely ignored or assumed to be absolutely zero.

[@MichaelGaroutte]

Yes, the Henderson-Hasselbalch equation gives only an approximation due to the assumptions made in deriving it. This is why the best practice is to use a meter to check the actual pH and use acid or base to adjust the pH before making the final dilution.

[@ajike]

thank you!!!

[@АнастасияКаргапольцева-ъ4щ]

OMG thank you SO MUCH

[@jessicaaldayjlpa7544]

thanks

[@lavenderwilliams1147]

Hello, say I was looking to make my own buffer, would I randomly choose the desired concentration of the buffer? In this case the 50mM number? Are there any rules that govern choosing this number when trying to make your own buffer

[@MichaelGaroutte]

Good question! We use a buffer to keep the pH constant, often when studying a biochemical reaction. We need enough buffer capacity to neutralize any added acid or base, for example, if the reaction generates protons (acid). In other words, the concentrations of the reactants should be much less than the concentration of the buffer species. Since most biochemical reactions with enzymes use concentrations of 1mM or less, a 50 mM or 100 mM buffer is usually sufficient. We don't want to add more ions than necessary, both because there is no need, and also to avoid the ionic strength changing the reaction we are studying, so we don't want to use higher concentrations of buffer than necessary (say, 1 M).

[@alejandralarasosa7187]

how did you get 1.269

[@MichaelGaroutte]

Hello, Alejandra! Please check to see if the other comments on the video answer your question. 😊

[@MailessTembo-we7yi]

I'm looking for the correction factor when not at 25 degrees 😢. Please explain further sir.

[@MichaelGaroutte]

The pKa values of some buffers change with temperature more than others. I found this link to a PDF that lists correction factors (dpKa/dT) for many buffers. www.itwreagents.com/download_file/info_point/IP-022/en/IP-022_en.pdf

[@MailessTembo-we7yi]

@@MichaelGaroutte thank you so much Sir

[@pasinduwimalasiri1835]

How I prepare 10 liters of 0.5% Tris buffer with 1% NaCl solution

[@jpedroam1692]

Why is the number of Tris-HCl moles equal to the sum of acid and base moles? Since each HCl molecule joins a Tris molecule, shouldn't it be n(Tris-HCl) = n(Tris) = n(HCl)? I think I'm missing something, I just don't know what it is.

[@jpedroam1692]

Nevermind, I was thinking the conjugate acid was HCl instead of TrisHCl. So it makes sense, since there is still some Tris left, besides the amount that reacted with HCl to form Tris-HCl, hence n(Tris) + n(TrisHCl) = total amount of moles in the buffer. Just leaving this here in case anyone has the same problem. Thank you for the video!

[@jpedroam1692]

but shouldn't 50mM be the concentration for Tris-Hcl only, and not Tris-HCl +Tris?

[@MichaelGaroutte]

@@jpedroam1692 By definition, the concentration of a buffer is the *total moles of buffer* (conjugate acid + conjugate base) per liter. There are three ways to make a buffer. Since you need to add enough Tris acid and/or Tris base to equal the total number of moles, you can add (1) all acid form, (2) all base form, or (3) some of each. It is often easiest to just add one form and then use either a strong acid or base to adjust the pH to the desired value. I show all three methods in this video.

[@jpedroam1692]

@@MichaelGaroutte alright, got it. I thought the concentration refered only to Tris-HCl and not Tris. Thanks again!

[@i.k3650]

where you get the 0.269? i tried log 0.57 but i get -0.244 instead. please help me

[@MichaelGaroutte]

You have to do the *inverse* log (antilog) of -0.57. This is likely a button labeled 10^x on your calculator.

[@i.k3650]

@@MichaelGaroutte thank you so much for your help

[@video_camera]

Really helpful! One thing I don't understand is why you choose to put all the Tris base at 11:47. Is it to make sure it's all going to react?

[@MichaelGaroutte]

You need to add enough Tris acid and/or Tris base to equal the total moles of buffer required. You can add all acid form, all base form, or some of each. It is often easiest to just add one form and then use a strong acid or base to adjust the pH to the desired value. I show all three methods in this video.

[@heytheredelilah4174]

hello sir, where did you get the 1that you added to 0.269?

[@jomarienarag435]

the 1 added to the 0.269 was from the CA

[@jomarienarag435]

in math, for example x +x = 2x, same with the 1 of CA present in the solution

[@heytheredelilah4174]

@@jomarienarag435 pagpalain ka sana ni maam roxanne hahahahahahahhaha

[@jomarienarag435]

@@heytheredelilah4174 omg HAHAHAHAHA amen

[@MichaelGaroutte]

@@jomarienarag435 Perfect explanation. Thanks!