Question

Here are the directions for making 50X Phosphate Buffer ( 1M; final concentration of 1X=20mM ), which is used to wash Dictyostelium cells.

36g Na₂HPO₄
88g Na₂HPO₄
Bring to 1.0 L in ddH₂O
pH =6.3
​A. Write the chemical equation for the equilibrium that describes this buffer. Identify the weak acid and the conjugate base.
B. Calculate the concentration of the two ingredients?
C. Given these directions, is the final concentration of the 50X solution 1M ?
D. Is the 50X buffer the appropriate pH, given these directions? Use the Henderson-Haselbalch equation to calculate the theoretical pH of the solution made according to the directions.
E. Tina's protocol for washing Dicty cells that have taken up bacteria states that the cells must be washed in 1X phosphate buffer containing 5mM sodium azide. There is a bottle of 1M sodium azide in the chemicals cabinet. How would you use 1M azide and 50X phosphate buffer to make 500ml of the azide wash buffer?

Answer 1

A phosphate buffer is made using Na2HPO4 and NaH2PO4. The weak acid is HPO4^2- and the conjugate base is Na2HPO4. The concentration of the ingredients can be calculated by converting mass to moles and dividing by the volume. The final concentration of the 50X buffer solution is 1.24M. The pH of the solution can be calculated using the Henderson-Hasselbalch equation. To make the azide wash buffer, you need to make a 1X phosphate buffer and add the appropriate amount of 1M sodium azide.

Step-by-step explanation:

A. The chemical equation for the equilibrium that describes the phosphate buffer is:

Na2HPO4 + H2O <-> HPO42- + 2Na+ + OH-

The weak acid in this equation is HPO42- and the conjugate base is Na2HPO4.

B. To calculate the concentration of the two ingredients, you need to convert the mass to moles and divide by the volume in liters. The concentration of Na2HPO4 is 36g/0.1L = 0.36M and the concentration of NaH2PO4 is 88g/0.1L = 0.88M.

C. The final concentration of the 50X solution is 0.36M + 0.88M = 1.24M, which is higher than the desired concentration of 1M.

D. To calculate the theoretical pH of the 50X buffer solution, you can use the Henderson-Hasselbalch equation which is pH = pKa + log ([A-]/[HA]). In this case, the pKa is 6.3 and [A-] is the concentration of the weak base (HPO42-) and [HA] is the concentration of the weak acid (NaH2PO4). Plugging in the values, you can solve for the pH.

E. To make 500ml of the azide wash buffer, you need to make a 1X phosphate buffer and add 5mM sodium azide. First, calculate the amount of 1M sodium azide needed by using the equation: (0.005mol/L) x (0.5L) = 0.0025 mol. Then, calculate the volume of 50X phosphate buffer needed by using the equation: (0.00125mol/L) x (0.5 L) = 0.000625 mol. Finally, add the calculated amounts to the 500ml of 1X phosphate buffer.