You need to carry out an assay reaction using reagent (Rg) that dissolves in wat

Question

You need to carry out an assay reaction using reagent (Rg) that dissolves in water according to the reaction:

Rg(aq)+ H2O(l)--> RgH+(aq) +OH-(aq)

Concentration of Rg is 25 mM and you need to add 5ml of Rg solution to your sample. Sample must be dissolved in 20 ml of one of the buffers listed here, but it must meet two requirements: i) before Rg is added buffer must have pH=7.3 and ii) after the reaction pH must remain between pH 6.9 and 8.4, where sample is stable. Which buffer will you choose? Justify quantitatively.

Assume:

Explanation / Answer

A buffer system will allow small additions of acid/base without dramatic changes in pH. If you want the final pH to be in a particular range, then it is best to choose a buffer whose optimal pH ranges fall within that ideal value. This automatically leads to MOPS, HEPES, or TAPS.

Then, you need to consider the buffer capacity, and we can do that in terms of absolute number of moles, as follows:

For the Rg, the total number of moles =

25mM = .0025moles / L x .005L = 1.25 x 10^-5 moles Rg that need to be neutralized by the buffer.

What about the absolute moles of the buffer? Let's try HEPES, because it is right in the middle of the optimal pH range. The total number of moles =

25 mM = .0025moles / L x .020L = 5 x 10^-5. So clearly this buffer (as well as MOPS, which is same concentration) will have the buffering capability to neutralize the Rg.

However, neither buffer has an initial pH of 7.3. Because of this, in looking at their pKa values, it would be wise to use 10mL of each of the two (a combination of 10mL HEPES and 10mL MOPS). This will satisfy all requirements.

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