CPB 30103 Biochemical Engineering

EXPERIMENT 1:

PREPARATION OF BUFFER SOLUTIONS

OBJECTIVES To prepare buffer solutions and test the effects of added acid or base upon the pH. To review acid-base chemistry and calculation. INTRODUCTION Buffers are very important to chemical and biological systems. Biochemical reactions are catalyzed by enzymes that have very narrow ranges of optimum pH values. The pH of a living system is maintained with buffers. In humans, the pH of blood is maintained between 7.35 and 7.45. Typically 5 liters of blood contains enough buffering capacity to absorb 150 mL of 1M acid. The principal buffers in blood are bicarbonate, proteins (including hemoglobin and oxyhemoglobin), and phosphates. A buffer is a mixture of a weak acid and its conjugate base. Because the solute can either absorb protons or release protons, the buffer solution can maintain the pH at a constant value. THEORY Buffer solutions are solutions that resist change in hydronium ion and the hydroxide ion concentration (and consequently pH) upon addition of small amounts of either acid or base, or upon dilution. Buffer solutions consist of a weak acid and its conjugate base (more common) or a weak base and its conjugate acid (less common). The resistive action is the result of the equilibrium between the weak acid (HA) and its conjugate base (A): HA (aq) H+ (aq) + A- (aq) (1)

The ionization constant for the acid is given by: Ka = [H+][A-] / [HA] (2)

Any base (OH-) added to the solution is consumed by the hydrogen ions to form water, thus increasing the pH. However, the equilibrium reaction will shift to the right as H+ ions are removed and result in the generation of hydrogen ion. The [H+] will therefore remain fairly constant, as will the pH. If a strong acid is added, the equilibrium reaction will shift to the left. This will cause the [H+] to decrease, to close to what it was before, and thus the pH will stay fairly constant.

CPB 30103 Biochemical Engineering

This is an example of Le Chatelier's principle and the common ion effect. The most effective buffering solutions are those which have similar concentrations of HA and A-, because then the buffer has the capacity to absorb both acid and base, with the same effectiveness in either direction. Since pH = -log [H+], we can rewrite the above equation (2) to obtain (3) Equation (3) is the Henderson-Hasselbach equation. LABORATORY PROCEDURE Apparatus 1. pH meter 2. Pipettes and pipette bulbs 3. Volumetric flask 4. Beakers 5. Graduated cylinder 6. Buret 7. Funnel 8. Spatula 9. Stirring hot plate and stir bars 10. Weighing paper Procedure Wastes and Spills: All of the chemicals in this experiment can be put in the trash or down the drain. Be sure the water is running. Use wet paper towels to wipe up small spills. Thoroughly rinse the pipet, buret and other glassware with water before returning them to the stockroom. You will want to clean all of your glassware before beginning this experiment. Be sure to triple rinse with distilled water and then triple rinse with the solution you are using before filling burets, beakers or pipets. pH electrode: The pH electrode is very fragile. It must be kept immersed in water at all times. Rinse it with deionized water from your wash bottle when moving it from one solution to another.

CPB 30103 Biochemical Engineering

A. Preparation of the Buffer Solution Prepare 500 mL of a 0.1 M buffer solution with a pH of 5.0 using acetic acid, HC2H3O2 and sodium acetate, NaC2H3O2. You are required to do your own calculations. There are several methods that can be used to make buffers, two of these methods are described below. Be sure to carefully describe the procedure that you used in the method section of your lab report. Combining volumes method: Since the total molarity of the solution must be 0.1 M, you can make up a 0.1 M solution of the acetic acid and a 0.1 M solution of the conjugate base (sodium acetate), and then combine the appropriate volumes of the two solutions to get the correct ratio of base/ acetic acid. (Use the Henderson-Hasselbalch equation to calculate the ratio, and remember that the total volume is 500 mL) A modification of this method that is used frequently, is to start with the appropriate volume of either solution (you must first calculate the appropriate volume) and add the other solution until the correct pH is obtained. For this you will need to monitor the pH of the solution as you are adding the second solution. You will need a pH meter, a stirring plate and a stir bar. You may end up with more or less than 500 mL, do not add water to make up the volume. Mass method: Calculate the mass of the acetic acid and the mass of the acetate ion that are required to make a pH 5.0 solution that has a combined molarity of 0.1M. 1. Use the Henderson-Hasselbalch equation to determine the molar ratio of acetic acid to acetate ion. 2. (moles of acetic acid + moles acetate ion)/0.5 L = 0.1M 3. moles x MW = grams The molar masses of acetic acid and sodium acetate are 60.05 g mol-1 and 82.03 g mol-1, respectively. Weigh out each substance and add distilled water to make a 0.5L solution. B. Testing your Buffer Solution 1. Measure the pH of your buffer: Place 25.00 ml of your buffer in a clean 100 ml beaker. Determine the pH of the solution using the pH electrode. 2. How effective is your buffer in resisting pH change? To each of two clean, dry 100 mL beakers add 50 mL of deionized water. Measure and record the pH of each one. To one beaker, add 1.0 mL of 1 M HCl and to the other add 1.0 mL of 1 M NaOH. Measure the pH of each solution. What is the pH change caused by the addition of the acid and base, respectively?

CPB 30103 Biochemical Engineering

3. Repeat the procedure in the above paragraph using your buffer solution instead of the deionized water. Results/Discussion Clearly show your calculations for making a buffer. Report the initial pH of your buffer. Show your complete calculations for pH of your buffer after adding acid or base. (2 calculations: acid + buffer, base + buffer) Make a table showing the calculated and measured pH values for adding base or acid for adding base or acid to buffer. The pH obtained in practice may differ from that calculated for a number of reasons. Discuss the relative error of your pH measurements and postulate reasons for the error. For example if the pH electrode was not calibrated correctly, and the actual pH was lower than the measured pH. Make a table and compare the pH changes resulting from the addition of 1 M HCl and 1 M NaOH, respectively, to pure water and to the buffer solution. Is your buffer solution resistant to pH change? Please explain. Tutorial: 1. Describe how would you prepare a (CH3CO2H / CH3CO2-) buffer with a pH of 5.2 given that the pKa (CH3CO2H / CH3CO2-) is 4.76. 2. What is buffer capacity? What determines the capacity of a buffer?