bsc3402l\buffer.html
Preparation of a Buffered Solution
A buffer solution is an aqueous solution that resists changes in pH1 upon the addition of small amouts of acid or base.
The Hendersen-Hasselbalch equation,
pH = pK + log (base/acid)
is used to calculate the ratio of the base concentration to the acid concentration for a desired pH. The absolute concentrations of the base and acid are unimportant, assuming a constant pK.
Example
Tris(base) is supplied as a 2 M "stock" solution. The desired "working" concentration of tris is 100 mM at pH 7.8. Tris has a pK of 8.1. The pH will be adjusted with 1 N HCl. To prepare 25 ml of extraction cocktail:
Step 1: Calculate and make the required dilution from the stock.
|
2 M (stock) |
= |
2000 mM |
= 20x dilution |
Thus, to a 25-ml mixing cylinder, add some H2O, then add 1.25 ml of 2 M stock Tris(base) (1.25 ml = 1/20 x 25 ml). Mix.
Step 2: Calculate the required ratio of base to acid, using the Hendersen-Hasselbalch equation.
7.8 = 8.1 + log (base/acid)
log (base/acid) = -0.3
log (acid/base) = 0.3
acid/base = 2
_________________________
1
pH = -log10[H+]. As a refresher, logbN = L and bL = N, e.g., log10100 = 2 and 102 = 100.As the pH will be adjusted with HCl,
|
tris-Cl |
= |
2 |
For a total tris concentration of 100 mM,
|
[2 parts tris-Cl] |
x |
100 mM |
= |
2/3 |
x |
100 mM |
= 67 mM tris-Cl |
Similarly,
|
[1 part tris(base)] |
x |
100 mM |
= |
1/3 |
x |
100 mM |
= 33 mM tris-Cl |
Step 3: Calculate and make the dilution required to achieve the desired tris-Cl concentration
|
1 N(stock H+) |
= |
1000 mM |
= 15x dilution |
Thus, to the mixing cylinder containing the tris(base), add 1.67 ml HCl (1.67 ml = 1/15 x 25 ml).
Step 4: To the mixing cylinder, add some water and mix. (Do not exceed ca. 20 mL total volume.)
Step 5: Add calculated aliquots of other stock reagents (MgCl2, EDTA).
Although mixing between additions of these particular reagents is not necessary, it is "good lab practice," as it can be important in other cocktails.
Step 6: Bring the final volume to 25 ml with water and mix.
Step 7: Remove a small aliquot and check the pH by electrode.
Typically, the pH estimate obtained on a pH meter for Tris buffer is 0.2 units lower than the actual pH.
Exercises
(a) Complete Table 2.1 (on the following page).
(b) Which is the weakest buffer/pH combination for buffering against acid addition or formation?
Answer: Buffer X, 50 mM, pH 7
(c) Which is the weakest buffer/pH combination for buffering against base addition or formation?
Answer: Buffer Y, 100 mM, pH 8.5.
(d) At pH 8, why would you use Buffer X at 50 mM instead of Buffer Y at 100 mM? To answer this question, consider the effect of adding or forming 5 mM acid or base in each solution.
Answer:
Buffer X + 5 mM acid = 30 mM acid: 20 mM base; pH = 7.8
Buffer X + 5 mM base = 20 mM acid: 30 mM base; pH = 8.2
Buffer Y + 5 mM acid = 29 mM acid: 71 mM base; pH = 7.9
Buffer Y + 5 mM base = 19 mM acid: 81 mM base; pH = 8.1
Thus, the pH change in Buffer X is less than the pH change in Buffer Y
Acid:base ratios for different buffers at a range of pHs.
|
pH |
7.0 |
7.4 |
7.5 |
7.6 |
7.7 |
7.8 |
7.9 |
8.0 |
8.1 |
8.5 |
|
Acid (A) or Base (B) in mM |
A B |
A B |
A B |
A B |
A B |
A B |
A B |
A B |
A B |
A B |
|
Buffer X pK 8.0 100 mM |
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|
Buffer X pK 8.0 50 mM |
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|
Buffer Y pK 7.5 100 mM |