BME 310 Lab 17 pH Measurement, John G. Webster 3/16/99
Introduction Biochemical reactions occur in aqueous solutions maintained closed to neutrality by the presence of buffer. The neutrality can be represented by the pH value, which is the negative logarithm of the hydrogen ion activity. Inside the human body, cellular fluids and tissues are buffered by the presence of bicarbonate and phosphate salts.
Definitions
1 mole (M) = mass (g) numerically equivalent to molecular weight
molarity = concentration [ ] in moles/L.
equivalent weight = molecular weight divided by its valence (1 L of 1 M solution with valence equal to 1 is 1 equivalent weight).
Before the lab: Read material about pH from J. G. Webster (ed.), Bioinstrumentation, Section 3.9 at the coursepage http://www.engr.wisc.edu/interd/bme/courses/bme310.html
Read John W. Suttie, Introduction to Biochemistry, Holt Rinehart and Winston, New York, 1977, Chapter 1 (attached). We will run titration curves, as shown on pg. 6, to discover the effects of buffer systems in the animal body. See pg. 8.
Lab Equipment
1. Accumet Basic AB15 pH meter and instruction book.
2. pH electrode
3. Buffer solutions, chemicals (note that some are caustic, use care), glassware, stirrers. (All lab members should wear safety goggles when handling acids and bases to prevent them from splashing in the eyes.)
Procedure
1. Remove protective cover from pH electrode.
2. Make sure the hole at the top of the electrode is in the open position.
3. Turn on the pH meter. Standardize with pH standard buffers pH 7.05 and pH 4.01 by following the instructions on pages 14 to 16 of the instruction manual. Rinse pH electrode between each new buffer with distilled water in beaker so old buffer solution remaining on electrode does not contaminate new solution.
4. Clean the tall glass beaker with water, then distilled water. Using the graduated cylinder, measure 100 mL of 0.2 M NaAcetate into the beaker. Measure pH. Using a pipette, add concentrated HCl as required to adjust pH to about 2. You have just made acetic acid (vinegar, CH3COOH). NaCl is also present, but does not affect the experiment.
5. Using a funnel, fill a clean burette with 30 mL 1.0 M NaOH. Arrange burette, pH electrode, beaker and stirrer so NaOH can be delivered to beaker while pH is being measured. Prepare labeled titration curve and immediately plot the data of pH vs millequivalents of NaOH added on the plot during the experiment. Add NaOH 1 mL at a time and plot pH on titration curve. Continue until solution becomes very basic (pH = 9 to 11). Discard solution.
6. Rinse beaker and electrode with distilled H2O. Fill with about 100 mL of distilled H2O. Read pH. It should be slightly acidic. If not you have small amounts of impurities (1 part in 106). Add drops of 1.0 M NaOH from the burette into the graduated cylinder to figure out the number of drops/mL. Add drops of NaOH to the H2O in the beaker to raise the pH to between 8 and 10. Plot these points on the titration curve and compare the slope with that of the buffer solution.
7. Using a small amount of dilute acid or base, adjust pH to about 6.5. For 1 min blow expired air containing CO2 through disposable pipette. Note change, if different from that with buffer solution. Discard solutions. Clean and rinse all equipment.
Results
1. Explain what a buffer does.
2. State how many milliequivalents of base were added to the acetate buffer to make the solution basic. Explain this value.
3. Quantitatively, compare buffering action of water and buffer in the pH region of 4 to 6.
4. Explain what happened, including reactions, when blowing CO2 through the solution in each case.
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