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Author Topic: How to Make a HEPES Buffer???  (Read 10995 times)

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How to Make a HEPES Buffer???
« on: September 05, 2013, 06:59:15 AM »

I would REALLY appreciate any assistance or advice on how to solve this question!!!

Show how you would make 500 ml of 20 mM HEPES buffer (pH 7.0) with 100 mM NaCl. You cannot use a pH meter, but pretend you were given the following:

1.) HEPES dry powder (CAS Number 7365-45-9, Empirical Formula C8H18N2O4S, Molecular Weight 238.30; pKa 7.5) (Hint: What form of the compound is this?)

2.) 1.0 M solutions of NaOH and HCl (Hint: You will have to decide to use one of these or both of these, then calculate how much to use.)

3.) 5 M NaCl

Here’s where I started:

I looked up the CAS number for HEPES and it appears to be in the acid (fully-protonated) form, from what I can tell. So I figured I would use only NaOH, not HCl, to make the buffer. Then I plugged the target pH (7) and the pKa of HEPES (7.5) into the Henderson-Hasselbach equation and solved for the ratio of conjugate base to acid (A-/HA), for which I got 0.316. Then I used algebraic substitution with the following two equations:

A-/HA = 0.316 and A- + HA = 20 mM

and then solved for HA, for which I got 15.198 mM. Then I solved for A-, for which I got 4.802 mM. At this point, I am stumped…I don’t know what quantities of HEPES, NaOH, and NaCl I need, nor how to even find them.  :-\   
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Re: How to Make a HEPES Buffer???
« Reply #1 on: September 05, 2013, 08:00:00 AM »

How much NaCl do you need to prepare 500 mL of 100 mM solution?

How much HEPES do you need to prepare 500 mL of 20 mM solution?

How much NaOH do you need to neutralize correct amount of HEPES?
ChemBuddy chemical calculators - stoichiometry, pH, concentration, buffer preparation,,


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Re: How to Make a HEPES Buffer???
« Reply #2 on: May 16, 2017, 03:36:51 PM »

Since HEPES is a sulphonic acid derivative I presume that HEPES solution would be highly acidic. Then the best approach to prepare this buffer is first to prepare a solution of 20mM HEPES. Initially add only 400 ml dH20. Add the NaCl and adjust the pH with 1N NaOH. Adjust the volume to 500 ml. The conjugate base is formed internally. Based on your calculations. You will need 2.4 ml of 1N NaOH to adjust the pH to the desired pH, presuming your calculations are correct. But let us verify:

Total moles = 0.020 * 0.5 = 0.01
7 = 7.5 + Log [Salt]/[Acid]
-0.5 = Log [Salt]/[Acid]
10^ -(0.5) = [Salt]/[Acid]
0.316/1= [Salt]/[Acid]
moles salt = 0.01 * (0.316/1.316) = 0.0024 moles
moles Acid = 0.01 * (1/1.316) = 0.0076 moles
So our calculations seem to be correct. Since the salt is made internally from sodium hydroxide you have to add 0.0024 moles of NaOH. That corresponds to
2.4 ml of 1N NaoH.



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Re: How to Make a HEPES Buffer???
« Reply #3 on: May 29, 2017, 03:54:46 AM »

I only have time for a quick response just at the moment.  However, one aspect of your premise is wrong.  Yes, sulfonic acids are strong, but in a solution of the free acid form of HEPES, the proton transfers to the nitrogen atom.  In other words the free acid form of HEPES is a zwitterion in aqueous solution, conceptually similar to the free acid form of, say, glycine.
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