Table 8-2 in your text gives structures and names of several common buffers. You
ID: 930659 • Letter: T
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Table 8-2 in your text gives structures and names of several common buffers. You decide to make a HEPES pH 7.4 buffer and you are lazy and you look up a protocol on -line and it goes like this; it was posted in 2013 by Mr Youssef Farhat. 0.10 MHEPES, pH =7.40 - In this protocol , we will use a solid powder of pure HEPES (MW 238.3 g/mol) to make 100 mL of 0.1 M HEPES, pH 7.4 - Add 2.38g of HEPES to an appropriate beaker(100-200 mL beaker in this case). -Add about 80 mL of deionized water to the beaker. -Add a stir bar to the beaker and leave it on a stir plate until completely dissolved(~1 min). Begin monitoring pH of the solution. It should be acidic(pH ~5). - Add one NaOH pellet to raise the pH towards 7.4 It should reach about pH of 7 -Once the first pellet is fully dissolved, add a second NaOH pellet if necessary to raise the pH to 7.4 Monitor carefully, and if the pH approaches 7.3/7.4 before the pellet is fully dissolved, stop the stir plate from spinning the rod and carefully remove the NaOH pellet with a clean spatla. you may assume the pKa of the sulfonic acid group (on HEPES) is ~ 3.00; get other pKa information from the Table 8.2 Harris . Caluclate the equilibrium molar concentrations of all the protonated and unprotonated forms of HEPS (there are 3) in the above prepared buffer solution. Note that the total formal concentration (according to the method )is 0.1 it would be a good idea to write out all the forms (of HEPS) and their dissociation equilibria. You may also assume that Mr. Youssef's final solution is measured to have a pH of 7.4 exactly. Ignore activity coefficients(of course!)Explanation / Answer
The concentration 0.1 M is too high for biochemical applications (cell cultures). The recommended values are 0.010 M….0.040 M. HEPES is toxic at concentrations greater than 0.040 M.
The amount of NaOH that has to be added, must be a calculated amount, to preserve the buffer capacity if the pH measurement is not very precise (or the pH meter is not exactly calibrated, or the temperature is not known).
The pellets of NaOH will introduce some amount of Na2CO3.
The first pKa (pKa1 = 3) is not relevant for the buffer equilibrium. But the second one pKa2 = 7.55 at 20ºC and 7.31 at 35 ºC is relevant and is used for the calculation of buffer pH.
For a good buffer preparation:
-choose lower concentrations
-calculate the amount of NaOH needed to correctly neutralize HEPES and add this quantity
-or mix equal quantities of HEPES in basic (anionic A-) and acid (zwitterionic –HEPES-H+ ) forms.
-final pH measurement is only for verification
-find a good protocol
Using Henderson-Hasselbalch equation
Log ([A-]/[HA]= pH –pKa2
[A-]/[HA] = 10pH - pKa2
Assume that [A-] + [HA] = Cbuffer
The value depend on pKa2 value at working temperature.
At pH=7.4 , [+H2A] is present in an insignificant proportion (it can be calculated using HH equation [HA]/ [+H2A] = 10pH – pKa1 )
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