Buffers Keep the Balance
Experiment Overview
In Part A, seltzer water is used a source of carbonic acid to prepare a model biological carbonic
acid-bicarbonate buffer. The effects of added acid and base on the pH and the buffer capacity
of this model buffer will be examined. The pH of the buffer solution will be estimated using
bromothymol blue, an acid-base indicator that changes color in the pH range 6.0 to 7.6.
Bromothymol blue is yellow when the pH is less than 6.0, blue when the pH is greater than 7.6,
and green (the intermediate color) in the transition range 6.0-7.6.
In Part B, two different phosphate buffers will be prepared. These buffers reflect the
physiological role of buffers within cells. The pH of buffers and the pH range over which they
are effective will be measured. The pH changes will be followed using universal indicator, an
acid-base indicator that can be used over pH range 4-10. Consult the universal indicator color
chart to determine the pH value corresponding to a given color of solution.
Pre-lab Questions
1.
a. Write an equation for the reaction of acetic acid with water.
b. Identify the conjugate base of acetic acid in the reaction equation.
2. Acetic acid and a salt containing its conjugate base, such as sodium acetate, form buffer
solutions that are effective in the pH range 3.7-5.7.
a. What would be the composition and pH of an ideal buffer prepared from acetic
acid and its salt, sodium acetate?
b. In resisting pH change, which buffer component would react with NaOH?
c. What happens to buffer activity when this component is exhausted?
Safety
Dilute solutions of sodium carbonate, hydrochloric acid, and sodium hydroxide are body tissue
irritants. Avoid exposure to eyes and skin. Wear chemical splash goggles.
Procedure
Part A
1. Setup six medium sized test tubes in a rack. Label them 1-6.
2. With Table 1 as a guide, use a graduated cylinder to measure and add the indicated
volume of the required solution to each test tube.
3. Mix the contents of each test tube thoroughly.
Table 1
Test Tube
No.
Solution
Seltzer
Water
(mL)
Na2CO3
(mL)
Distilled
Water
(mL)
4.
5.
6.
7.
8.
9.
10.
11.
1
2
3
4
Carbonic
Model
Water
Model
Acid
Carbonate (Control) Carbonate
(Reference)
Blood
Blood
Buffer
Buffer
4
2
0
2
5
6
Water
(Control)
Sodium
Bicarbonate
Reference
0
0
0
2
0
2
0
4
0
0
4
0
4
0
Add 5 drops of bromothymol blue to each test tube. Shake to mix. Record the initial
color of each solution.
Measure the initial pH of each solution using a piece of narrow range pH paper, pH 6.08.0. Record the results.
Using a pipet, add 0.1 M HCl dropwise to the model carbonate blood buffer in test tube
2. Be sure to swirl the test tube after each drop to ensure thorough mixing.
Count the number of drops of HCl required to change the color to the same shade as the
carbonic acid reference solution in test tube 1. Record the number of drops.
Using pipet, add 0.1 M HCl solution dropwise to the water in test tube 3. Count the
number of drops of HCl required to change the color to the same shade as the carbonic
acid reference solution in test tube 1. Record the number of drops.
Using a pipet, add 0.1 M NaOH dropwise to the model carbonate blood buffer in test
tube 4. Be sure to swirl the test tube after each drop to ensure thorough mixing.
Count the number of drops of NaOH required to change the color to the same shade as
the carbonic acid reference solution in test tube 6. Record the number of drops.
Using pipet, add 0.1 M NaOH solution dropwise to the water in test tube 5. Count the
number of drops of NaOH required to change the color to the same shade as the
carbonic acid reference solution in test tube 6. Record the number of drops.
Part B
12. Obtain two 50 mL beakers and label them A and B.
13. Use clean graduated cylinders to add 12 mL of NaH2PO4 solution and 3 mL of Na2HPO4
solution to beaker A. (This is Buffer A)
14. Use clean graduated cylinders to add 8 mL of NaH2PO4 solution and 8 mL of Na2HPO4
solution to beaker B. (This Buffer B)
15. Stir each buffer with a stirring rod to ensure thorough mixing.
16. Use the following layout plan to fill each indicated well in a 24 well plate with 1.5 mL of
distilled water (the control), Buffer A, or Buffer B respectively.
Water
(A1)
Water
(B1)
Buffer A
(A2)
Buffer A
(B2)
Buffer A
(C2)
Buffer A
(D2)
Buffer B
(A3)
Buffer B
(B3)
Buffer B
(C3)
Buffer B
(D3)
Buffer A
(A4)
Buffer A
(B4)
Buffer A
(C4)
Buffer A
(D4)
Buffer B
(A5)
Buffer B
(B5)
Buffer B
(C5)
Buffer B
(D5)
Water
(A6)
Water
(B6)
17. Add three drops of universal indicator to each filled well.
18. Record the initial indicator colors for the control (A1), Buffer a (A2), and Buffer B (A3).
19. Estimate the initial pH of wells A1, A2, and A3 using the narrow range pH paper.
Effect of HCl Addition:
20. Using a clean pipet, add I drop of HCl to wells B1, B2, and B3. Record the indicator
colors.
21. Add 5 drops of HCl to wells C2 and C3 and record the indicator colors.
22. Add 10 drops of HCl to wells D2 and D3 and record the indictor color.
Effect of NaOH Addition:
23. Using a clean pipet add 1 drop of NaOH to wells B4, B5, and B6. Record the indictor
colors.
24. Add 5 drops of NaOH to wells C4 and C5 and record the indicator colors.
25. Add 10 drops of NaOH to wells D4 and D5 and record the indictor colors.
26. Wash the contents of the well plate down the drain under running water.
Conclusion Questions
1. Compare the measured pH value of the model carbonate blood buffer to
(a) the expected pH of an ideal carbonic acid-bicarbonate buffer (Show work)
(b) the actual pH of the blood.
2. Based on the pH comparisons in Questions #1, which solution, the model carbonate
blood or an actual blood buffer, is more likely to contain a greater proportion of the
carbonic acid component compared to the bicarbonate component? Explain.
3. What is the effect of adding even 1 drop of HCl or NaOH on the pH value of the
control (water)? Compare this to the effect of adding HCl or NaOH to the model
carbonate blood buffer?
4. Which phosphate buffer in Part B corresponds to the composition of an ideal buffer
solution? Compare its measured pH value and the calculated pH of the ideal buffer. (Show
Work)
5. Use the universal indicator chart to compare the observed pH changes for phosphate
buffers A and`` B and the control (water) upon addition of HCl and NaOH. Were
phosphate buffers A and B equally effective in resisting pH changes upon addition of
either HCl or NaOH.