pH and Buffers Homework CHEM331 Biochemistry
Name
(_____ / 25 points)
For each question show all calculations and simply describe the process. Use complete sentences, bullets or
outlines will NOT be accepted. For formula weights, pKa, Ka or other information needed for the problem,
start with your book then look for other sources.
1) (1 point) How many grams of citric acid and sodium citrate must be added to 0.5 L of water to produce
a solution with pH 4.5 and a total solute concentration of 100 mM?
2) (3 points) Calculate how to prepare 250 ml of 1.0 M sodium acetate buffer at pH 4.5. Use your textbook
to determine the molecular weight and pKa of the acid and base. Calculate the grams of sodium
acetate and number of milliliters of glacial acetic acid required. THEN using this stock solution, calculate
and describe how you would prepare 250 ml of a 12.5 mM acetate buffer, pH 4.8. By the way, what is
the molarity of glacial acetic acid?
3) (1 point) What is the pH of a solution containing 32 mM of sodium succinate- and 5 mM disodium
succinate? The total volume of the buffer is 750 ml.
4) (2 points) What did Norman Good and his colleagues contribute to buffer development? Be specific
and look for detail in your explanation.
5) (1 point) a) Chemically define Tris and Trizma? Is there a difference? If so what (details please)?
(1 point) b) What is Tris base and Tris HCl?
(1 point) c) Describe how you would make a Tris buffer at pH 7.5 (you pick a volume and molarity).
(3 points) d) Your laboratory assistant Beaker, prepared a 20 mM Tris buffer at room
temp (20oC) from a 1M, pH 7.4 stock solution of Tris buffer. Because the buffer was at
the same pH you needed, Beaker simply diluted the buffer without checking the pH,
capped and stored the solution on ice (1oC). Just before using the buffer, you double
checked the pH. It was not at the right pH! Predict change (increase or decrease)
Lab assistant Beaker
and estimate the magnitude of the pH you found. What was the cause for this?
There are two chemical reasons (there was no error in calculation or pH meter) for the unexpected pH.
6) (2 points) You have made a 20 mM Succinate buffer at pH 5.64 in a 2.0 ml solution to run an enzyme
reaction which generates H+. When you finish the reaction, you find the pH has shifted to a pH of 4.0.
Much lower than you would like. How would you adjust your experiment to avoid such a significant pH
change? Unfortunately you must use Succinate buffer and because of the enzyme’s optimal pH
requirement it must stay be at pH 5.64. No calculations are required for this answer, just identify the
problem and describe how/what you would do differently. There is a key buffer concept for this
question you should identify in your answer.
7) (2 points) Draw the structure and net charge of histidine for the predominant molecular species at each
of the following pHs 2, 7, and 10. Draw a graph of pH versus H+ dissociated/molecule for histidine
(similar to graph from Voet, Voet & Pratt, Fig. 2-17).
8) (4 points) The pKa’s of amino acid functional groups can change depending on microenvironment. If
the side chain of glutamate were in a hydrophobic pocket, what would you predict the shift in pKa to
be and why? If a basic amino acid side chain such as lysine were in close proximity with two carboxyl
groups would you predict the pKa to change? In what direction and why? Answer both with a
predicted value AND the chemical explanation.
9) (1 point) In order to purify a certain protein you require 0.5 liters of a 0.125 M HEPES buffer at pH 7.2. You
have both HEPES and HEPES sodium salt forms. How many grams of each do you need?
10) An enzyme-catalyzed reaction was carried out in 100 ml of a 0.050 M phosphate buffer, pH 6.90. As a
result of the reaction, 0.075 mole/liter of H+ was produced.
a) (1 point) What were the concentrations of each form of the buffer at the start of the reaction?
b) (1 point) What was the pH at the end of the reaction?
c) (1 point) Was this an appropriate buffer for this experiment? Why?