Youre planning to isolate RNA to load into nanoparticles as part of creating a new therapeutic, and realize that youve run out of the 0.24 M sodium citrate buffer, pH 5.5 that you need. Your lab is well equipped and you have all of the reagents and equipment needed to prepare the buffer on hand, including trisodium citrate dihydrate (Na₃C₆H₅O₇2H₂O MW: 294.1 g/mol), and citric acid monohydrate (C₆H₈O₇H₂O, MW: 210.14 g/mol).

3a. Write out the equilibrium reactions for citric acid [3 points]

3b. Citric acid is a polyprotic weak acid, with pKa values of 3.09, 4.75, and 5.41. Which should be used to do the calculations to make a pH 6.0 buffer. Why? [2 points]

3c. Use the Henderson-Hasselbalch equation to determine what concentration you need of each component in your buffer. Show your work and clearly state the concentration needed of each component in mM. [6 points]

3d. Assuming you want to make 250 mL, calculate the amount of solid trisodium citrate dihydrate and citric acid monohydrate needed to make your buffer. Show your work and clearly indicate how much of each compound is needed. [2 points]

3e. After combining your reagents with the appropriate amount of water and mixing well, you need to check the pH of your buffer. After having just done all the calculations to make sure you made a buffer with the correct pH, why do you need to check the pH (and possibly adjust it)? [1 point]

3f. After you finished making your buffer you realized that the technician just made 1M stock solutions of both trisodium citrate, and citric acid. How much of each liquid stock would you have needed to make your 250 mL of buffer if youd used these stocks instead of the solid powders? Show your work and give the answer in reasonable units for the volume needed (i.e. what units would you want to work in if measuring these chemicals in the lab). [2 points]