Hi everyone,
I want to prepare following two different buffer with different pH stock (20 ml each).
1. Citrate buffer (pH: 3, 4, 5, and 6)
2. Phosphate buffer (pH: 7, 8)
I would be grateful if someone provides me the direct protocol and chemical name to be used in order to prepare these mentioned buffers.
N.B: I am not expert in chemistry, the theory/equations behind the buffer chemistry would be much appreciated.
Thank you in advance.
I want to give basic information about buffer solutions:
Buffer solutions are used to prevent medium for changing pH against to adding a small amount of acid/base or dilution. Buffers can be prepared by weak acid and its salts (for example acetic acid and sodium acetate) or weak base and its salt (ammonia/ammonium). stronge acid and base can never be used.
HA =H+ + A-
There is an equbilirium reaction between weak base and its salt. when strong acid is added into buffer, A- reacts and neutralizes it. After that HA concentration increases. And when a strong base is adde into buffer, the concentration of HA decreases and The concentration of A- increases. But the amount of A- should be as high as to compensate the acid adding. So the most efficient buffer solution contains same amount of HA and A-. Henderson-Haselbach equation is used
pH=pKa+log [A-/HA]
Because pKa value is constant, pH can vary only by [A-/HA] ratio.
For preparing efficient buffer solution, [A-/HA] ratio should be between 0.1 and 10.
Thus; pH=pKa(+/-)1
So, for example if we prepare acetate buffer
pKa=4.74 for acetic acid, We can prepare buffers at pH between 3.74 and 5.74
When we added a strong acid or strong base at small amount only log value of [A-/HA] ratio affects the pH. It is clear that log value of a number is very lower than it.
So when buffer solution is diluted, [A-/HA] does not change, pH value dos not change, either.
The concentration of the buffer is also important. Buffer capacity increases by increasing A- and HA concentration keep same [A-/HA] ratio.
Generally the concentration of the buffer= [A-] + [HA]
If you want to prepare pH 7 phosphate buffer
H3PO4=H+ + H2PO4- pKa1= 2.15 .......................... (1)
H2PO4- = H+ + HPO42- pKa2= 7.2 ...................... (2)
HPO42- = H+ + PO43- pKa3=12.3 .......................(3)
We can use (2) system for pH 7 buffer solution (buffer range 6.2-8.2)
pH=pKa+log [A-/HA]
7=7.2+log[ HPO42-/H2PO4- ]
--------------------------
0.2
HPO42-/H2PO4- =1.58 (100.2)
You can prepare pH 7 buffer which mol ratio of hydrogenphosphate/dihydrogenphosphate at 1.58
Check the Sigma-Aldrich web site for theory and practice:
http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-calculator.html
Or see useful direct protocols of European Pharmacopoeia (attached).
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