Biochemistry (Chemistry 330)

This page contains some theory on buffers, a procedure for titrating water and a buffer to show the difference, and a procedure for titrating a zwitterion like an amino acid. An appendix is available with help on pH, logs and the pH meter. You can also leave me a message, go to my home page, or go back to the lab schedule. 

The mechanism by which the pH is maintained in biological systems is through buffers - chemical substances which resist changes in the pH of the solution. They do so by reacting with added acids or bases to remove H⁺ ions or add them to the solution, such that a much smaller change in pH occurs than would have without the buffer's presence. This is possible because buffers are themselves weak acids or weak bases (or both as in amino acids) which ionize or are protonated under these conditions. Human blood contains several buffering systems, among them the bicarbonate/carbonate system and the phosphate system, to maintain the pH around 7.4. If the pH is altered ± 0.4 pH units, death follows rapidly.

Procedures: Titration of H₂O and a buffer with NaOH
 

Standardize the pH meter to read accurately between pH 7.0 and 4.0 (see pH Meter). Rinse and fill a buret with NaOH or KOH. (Record the concentration!) Arrange a beaker with a stir bar and 30 mL of distilled water (dH2O) on a magnetic mixer.  Arrange the pH meter probe in the beaker and clamp a buret containing the strong base over it, as shown here.  Read and record the pH of the water. Will it read 7.0? Why not?

Be sure the beaker is large enough to contain the total volume likely to be generated at the end of base addition.

1. Rapidly rotate the stopcock a half turn, once or twice, to add about 0.1 mL of base to the beaker.
2. Stir, and record the volume of base delivered and the pH again.
3. Repeat until the pH change is minimal (about pH = 11). This process is called titration.
4. If desired, plot the pH values on the ordinate (y-axis) and total mL of base added on the abscissa (x-axis) on graph paper or on a computer.

1. Titrate 30.0 mL of a weak acid like acetic acid (about 0.1 M HAc) with base as above.
2. Start with small (0.1-0.5 mL) additions; use larger (1.0-5.0 mL) additions when the pH changes are minimal (from about 2 to 25 mL, total, if acid and base concentrations are similar), and end with small additions again.
3. The end point should be obvious from the rapid jump in pH readings, but stop when the pH reaches 10 -11.
4. Plot your pH vs. mL NaOH data on the same graph as the NaOH vs. H₂O test.

An amino acid is a molecule with at least two ionizable groups (a zwitterion), one acidic (-CO₂H ---> -CO₂¯ + H⁺) and one basic (-NH₂ + H⁺ ---> -NH₃⁺). To titrate such a solution:

1. Recalibrate the pH meter between 7 and 10, and titrate 30 mL of a 0.1 M amino acid like glycine with 0.1 M NaOH. How much NaOH will be required?
2. Again use small, large, then small additions, and stop after adding at least the same volume of base as you had glycine (pH ~ 11). Why?
3. Recalibrate the pH meter between 7 and 4, and titrate another sample of glycine with 0.1 M HCl, stopping at pH ~ 1.5, if more HCl than glycine was added.
4. Plot both sets of data on the same graph with pH on the ordinate (y-axis) and mL of acid to the left of 0, and mL of base to the right on the abscissa (x-axis) as shown below.

If you have questions or comments, write the:

  Author of this page: Terry Helser - helsertl@oneonta.edu
    Web Coordinator: Philip S. Bidwell - bidwelps@oneonta.edu
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  Last Modified on 7/6/06