Lab #1: Preparing Buffers and Amino Acid Titration

Background 

Application of the Henderson-Hasselbalch equation requires "hands-on" laboratory experience in the development of buffer solutions. In our laboratory, we have sodium and potassium salts of base and acid forms of phosphate. The first part of this laboratory is to prepare phosphate buffer solutions of different pH values.

The second part of this laboratory involves amino acid titration. Alpha-amino acids are the building blocks of polymeric protein molecules, and are ampholytes in that they contain at least one acidic (carboxyl) and one basic (alpha-amino) functional group. Some alpha-amino acids also contain side chains that have additional ionizable groups: carboxyl, amino, sulfyhdryl, etc. The ionic behavior of amino acids is largely due to the ionizable side chain groups, since the alpha-carboxyl and alpha-amino groups of amino acids are connected by peptide linkages in polymeric proteins.

Amino acids are also found in free forms in blood and body tissues, and exist in concentrations up to several millimoles per kilogram (mmol/kg) of tissue. This pool of free amino acids is the direct source of amino acids for protein synthesis, and also transamination and deamination reactions that may be involved in energy metabolism.

This experiment involves the study of the reaction of alpha-amino acids with hydrogen ions. As an acid or base is added to a solution of amino acids, a pH change is observed. Since alpha-carboxyl and alpha-amino groups are weak acids and bases, respectively, buffering action by these groups will occur.

1. Develop 50 mL phosphate buffer (pK=6.86) solutions at the following pH values, a) 7.0, b) 7.2, and c) 6.9
2. Test the pH of the solutions using the pH meter. Perform a two-point calibration of the pH meter prior to use (see below).
3. Report the calculated and measured pH of each solution.
4. Explain any discrepancies

Complete these exercises by using the Henderson-Hasselbalch equation. Show all calculations.

Acid Titration

1. Perform a two point calibration of the pH meter (4.0 and 10.0 pH, and then check the 7.0 pH standard). If values are within 0.01 pH units, proceed with the lab. If values are not accurate, consult Dr. Robergs.

2. Use the electronic magnetic stirrer device. Place a magnetic bar into your 100 ml beaker, and turn on the stirrer so that the bar turns at a moderate speed. Turn off the stirrer.

3. Place approximately 50 ml (know and record the actual volume) of your selected amino acid solution into the beaker. Assemble the titration stand, graduated burette, and pH and temperature electrodes so that both electrodes are submerged into the amino acid solution and the burette is positioned to drop solution into the beaker. If your solutions are at room temperature, you can leave the temperature electrode out of the solution.

4. Record the initial pH of the amino acid solution.

5. Turn on the stirrer, check that the magnetic bar does not collide with the electrodes, and add sufficient 1 N HCl by dropwise addition from a pasteur pipette to lower the pH to below 1.5. Record the volume of added HCl as best as possible (think !!), and record the final pH.

6. Add enough 1 N NaOH solution to the titration burette to read at least 20 ml. It is recommended that you allow the first portion of NaOH solution to run through the burette to clear the bottom constriction from residual distilled water (discard this volume).

7. Record the starting volume reading on the burette, and then carefully titrate the amino acid solution by dropwise addition of the 1 N NaOH. I recommend that you determine the volume of one drop !!!!!

8. Record the volume addition of NaOH (think !!) and pH of the solution throughout the titration until a pH > 12 is reached.

Use care in adding the 1N NaOH, as the pH may change slowly or rapidly depending on the pH and buffering range of the ionizable group(s). Have patience !!!

*Plot pH versus milli-equivalents of NaOH added for the amino acid solutions.

*Determine the pKa for each of the detected ionizable groups of your amino acid.

*Determine the pI of your amino acid.

*Identify the unknowns from text data of amino acid and common acid ionizable group pKa values.

References: use any biochemistry text