Buffered Solutions M HC 2 H 3 O 2 (acid) and 0.10M NaC 2 H 3 O 2 (conjugate base) 0.25 M NH 3 (base) and 0.20 M NH 4 Cl (conjugate acid)

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1 Buffered Solutions Objective: Buffering of weak acid/weak base solutions is very important, especially in biological chemistry. In this experiment you will demonstrate the buffer effect to yourself, and will investigate a situation in which a buffered solution may arise. Introduction: A buffered solution is one that does not change its ph significantly when a strong acid or base is added to it. Buffered solutions typically consist of an approximately equimolar mixture of a conjugate acid-base pair. For example, the following mixtures would be expected to act as buffered solutions: 0.10 M HC 2 H 3 O 2 (acid) and 0.10M NaC 2 H 3 O 2 (conjugate base) 0.25 M NH 3 (base) and 0.20 M NH 4 Cl (conjugate acid) The two components of the buffered solution do not have to be present in exactly equal amounts, but there must be comparable amounts of the components for the buffer to have a significant capacity to resist changes in its ph. A buffered solution is able to resist changes in its ph when strong acids or strong bases are added because the components of the buffered solution are able to react chemically with such added substances. If the added strong acid or strong base is chemically consumed by one of the components of the buffered solution, then the acid or base will not have an effect on the total hydrogen ion concentration (ph) of the solution. For example, for the HC 2 H 3 O 2 / NaC 2 H 3 O 2 buffer above, the weak acid portion of the buffer will react with any added strong base, and the conjugate base portion of the buffer will react with any added strong acid. HC 2 H 3 O 2 + NaOH NaC 2 H 3 O 2 + H 2 O C 2 H 3 O HCl HC 2 H 3 O 2 + Cl - Buffered solutions are vitally important in the physiology of living cells. Many biochemical reactions are extremely sensitive to ph, and will not take place if the acidity of the physiological system is outside a very narrow range. For example, if a few drops of acid are added to whole milk, the milk will almost instantly curdle as the protein in the milk precipitates. The change in the ph of the milk when the acid is added is enough to cause the disruption of the structure of the milk protein so that it is no longer soluble in water. In this experiment you will demonstrate the ability of buffered solutions to resist changes in ph, and will investigate a common situation in which buffered solutions arise. Safety Precautions: Safety eyewear approved by your institution must be worn at all times while you are in the laboratory, whether or not you are working on an experiment. Assume that all the acid and base solutions used in this experiment are corrosive to the eyes, skin, and clothing. Wash immediately if spilled and inform the instructor. Clean up all spills on the bench top. Apparatus/Reagents Required ph 7 buffer concentrate, universal indicator and color chart, 3 M HCl, 3 M NaOH, and dropper bottles of 1.0M HCl, 1.0M acetic acid, and 1.0M NaOH, albumin (egg white)

2 Procedure: 1. The buffer effect Place approximately 50mL of distilled water into each of two small beakers. To one beaker of distilled water, add 1mL of concentrated ph 7.00 standard reference buffer. Add 5 drops of universal indicator to each of the beakers. Record the color of the liquid in each beaker after the indicator is added. Use the color chart provided for the indicator to record the ph of the water and of the buffered solution. To each beaker, add 1 drop of 3 M HCl solution and stir to mix. Record the color of the solution in each beaker. Use the indicator color chart to record the ph of the two solutions. Which solution underwent the larger change in its ph when the acid was added? To each beaker, add 2 drops of 3 M NaOH solution (one drop of the NaOH is to neutralize the acid which was added previously) and stir to mix. Record the color of the solution in each beaker. Use the color chart provided for the indicator to record the ph of each solution. Which solution underwent the larger change in its ph when the base was added? 2. Buffering During Titrations In Part 1 above, you demonstrated the buffer effect on a buffered solution that was purposely prepared. Buffered solutions may also arise in situ during titrations of weak acids or bases, which may make the endpoint of the titration less sharp. If we were to titrate a solution of the strong acid HCl with a sodium hydroxide solution, the ph would change drastically and suddenly when we had reached the point where sufficient NaOH had been added to react with the HCl present. On the other hand, if we were to titrate a solution of the weak acid acetic acid with sodium hydroxide, the change in ph at the endpoint would be much less sudden or drastic. As sodium hydroxide is added to acetic acid, the acetic acid is converted to sodium acetate, and a buffered solution arises. Set up 2 clean, dry test tubes in a rack. Add 20 drops of 1.0M HCl to one of the test tubes and 20 drops of 1.0M acetic acid to the other. Add 1 drop of universal indicator to each of the test tubes and mix. Use the color chart provided with the indicator to record the ph of each of the solutions before any change is made to them. Obtain a dropper bottle of 1.0M NaOH solution, and prepare to titrate each of the acid samples. Use the indicator color chart to record the ph of the solutions after each drop of NaOH has been added (give your best estimate of the ph if the color seems intermediate to two of the colors on the chart). Titrate the acid samples in parallel so that you can compare them: that is, add a drop of NaOH to the HCl solution and record the ph, then add a drop of NaOH to the acetic acid solution and record its ph. Continue adding NaOH dropwise until the ph has reached a value of 10. Using Graph paper from the back of this manual construct two graphs (one for each titration): plot the ph of the solution at each point in the titration versus the

3 number of drops of NaOH that had been added to reach that point. Draw a smooth curve through your data points (do not connect the dots ). Notice the difference in the shape between the two curves: the strong acid/strong base titration shows a sharp, sudden increase in ph after approximately 20 drops of the NaOH has been added. The weak acid/strong base titration shows a much more gradual change in ph as the NaOH was added, reflecting the fact that the system was buffering. 3. Proteins as Buffers Proteins are constructed of long chains of linked amino acid molecules. An amino acid molecule contains both a weak acid group (the carboxyl group) and a weak base group (the amino group). Since amino acids have both acidic and basic properties, solutions of proteins behave as buffered solutions when strong acids or strong bases are added. H H 2 N C C=O R OH Place approximately 10 ml of water in a small beaker. Add a tiny amount of egg albumin approximately the size of the head of a match. Stir the mixture vigorously to dissolve as much albumin as possible. Decant approximately 5 ml of the albumin solution into a clean test tube (avoid transferring any undissolved albumin from the beaker to the test tube.) Set up the test tube containing the albumin and a second test tube containing an equal amount of water in a test tube rack. Add 1 drop of universal indicator to each of the test tubes and mix. Use the color chart provided with the indicator to record the ph or each of the solutions before any change is made to them. Add 1 drop of 1M HCl to each test tube. Record the color of the indicator. Then add 2 drops of 1M NaOH to each test tube. Record the color of the indicator. Did the albumin solution appear to behave as a buffered solution?

4 Pre-laboratory Questions 1. Use your textbook to write a specific definition for a buffered solution. 2. Give two examples of mixtures that would behave as buffered solutions, and show how (by writing equations) the components of each of your solutions would consume added strong acid (HCl) and added strong base (NaOH). Do not use examples discussed in either this lab manual or in your textbook. 3. Buffered solutions are especially important in biological systems. Why? Give two examples of buffered solutions in biological systems.

5 Results/Observations 1. The Buffer Effect Color of distilled water + indicator Color of buffered solution + indicator ph of distilled water ph of buffered solution ph s after adding HCl: distilled water buffer ph s after adding NaOH: distilled water buffer Which solution underwent the larger changes in ph when either HCl or NaOH was added? Why? 2. Buffering During Titrations Initial ph of HCl solution Drops ph Drops ph Drops ph

6 Initial ph of acetic acid solution Drops ph Drops ph Drops ph Proteins as Buffers Did the albumin solution appear to exhibit the properties of a buffered solution (compared to water) when strong acid and strong base were added? Explain your observations. Question: Describe in your own words how the shapes of your two titration curves differ as determined in Part 2 of the experiment. Why does one curve show a sudden, dramatic increase in ph, whereas the other curve shows a much more gradual increase in ph?