Biochemistry Lab
Buffers & Spec Problem Set
Name_______________________________
Buffers Exercise
1. What trends did you observe with temperature and ionic strength on the pH of the two buffers? How
does this relate to the activity coefficient? Why/how did the effect you observe happen? Be
quantitative and mechanistic as well as descriptive in your answer. (2 points)
2. What is the difference between Tris-Cl, Tris-Base and Trizma? What is a Good’s buffer? Does Tris or
KH2PO4 fit the requirements of a Good’s buffer? (2 points)
3. You will prepare 100 ml of a 50 mM HEPES buffer pH 7.0. You have two practical options to prepare a
buffer: 1) calculate the final concentration of acid and base buffer component and prepare exact
amounts of both, and 2) starting with only the acid form of the buffer and titrating NaOH to reach the
desired pH. Describe and calculate the difference in making a buffer by each method. (3 points)
4. Does the pH of a buffered solution in question 3 change if you dilute the solution 1:5 or 1:10? Support
your answer using the appropriate equation and calculation. (3 points)
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Biochemistry Lab
Buffers & Spec Problem Set
Spectrophotometry Exercise
1. Consider the reaction catalyzed by MDH. Oxaloacetate and malate do not absorb in the visible
ultraviolet range. Explain which wavelength you would use to measure the reaction by MDH
converting oxaloacetate to malate and why you’d use that particular wavelength? Would you
expect the wavelength to increase or decrease over time? Why? (2 points)
2. Recreate the graph and calculation used to determine the extinction coefficient of NADH. Why is
this a better approach than using one concentration of NADH, determining the absorbance and
using Beer’s law to calculate the same value? (3 points)
3. Why is the volume of solution pipetted into a 96 well plate important for determining absorbance
while it is less important when using a cuvette in a spectrophotometer? (2 points)
4. Using the extinction coefficient you calculated in this laboratory, determine the concentration of a
NADH solution with an absorbance of 0.753 when you pipet 250 µl into a 96 well plate (3 points).
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