Of course as always you must also prepare your procedure and your notebook as indicated on the notebooks page.

Reading
1. There are several good web sites which provide useful information about amino acids. There are also some excellent study guides about properties and behavior. Not all web sites are good.  Many of them have the Fischer formulas drawn as D instead of L.  I have reviewed the information contained in the following sites for accuracy.

a. Acid-base chemistry and structure of amino acids
http://www.biology.arizona.edu/biochemistry/problem_sets/aa/aa.html
It is recommended that you draw the structures of all 20 AA in your pre lab for the laboratory. 
Amino acid chirality: http://chemistry.about.com/library/weekly/blaminochiral.htm
The chemical properties of amino acid side chains:
http://en.wikipedia.org/wiki/Amino_acid#Chemical_properties
b. Titrations general information:
http://www.math.duke.edu/education/ccp/materials/calculus_projects/TitrationProj/Titration1.html
Titrations of amino acids - take some time experimenting with the plot options on this site.  See if you can correlate structure with the titration plots.
http://cti.itc.virginia.edu/~cmg/Demo/markPka/markPkaInstr.html

2. Garrett and Grisham Chapter 4.

Questions (completed in notebook prior to class):
1. Sketch a titration curve for the amino acid glycine.
2. Sketch a titration curve for the amino acid aspartic acid.
3. Explain the differences in the curves for 1 and 2 (don't describe, explain).
4. Discuss potential sources of error in this experiment, more than one please. (Hopefully this will help you avoid them!)
5.  Sketch a titration curve for the standardization.  Explain differences between your curve and the curve in question 1.
6.  For the standardization of NaOH you will use both indicator and the pH meter to detect your endpoint.  Which is more reliable?  Do you think you will be able to titrate easily to perfect neutrality?

1. Acid/base chemistry of amino acids
2. Titrations
3. Experimental determination of pKa values
4. Identification of an unknown amino acid

Introduction:
Amino acids are the building blocks of proteins. Proteins are biomolecules essential to the function of living systems. The amino acids also serve as the monomers used to create polymeric proteins. Amino acids all have the same basic backbone which contains a carboxylic acid and an amine attached by a tetrahedral methylene carbon. Each amino acid is structurally defined by the side chain or R group pendant to the methylene carbon. Twenty different R groups occur naturally which provide twenty different natural amino acids. Individually, amino acids play important roles in metabolism and signaling (especially as neurotransmitters). A key chemical property of every amino acids is that it has at least one acidic (carboxylic acid) and one basic (amine) functional group (some amino acids have more than one titratable group, if the R group exhibits acidic or basic properties). Amino acids are ampholytes.

Figure 1. The ionization of a generic amino acid.

Each titratable group on an amino acid molecule has a characteristic pKa value. Thus, by titrating an unknown amino acid sample, one can often identify the compound from the shape of the titration curve and the experimentally determined pKa values.

Experimental Procedure:

You will be working in groups for this experiment. Each group will get one unknown amino acid solution to titrate both with acid and with base. The HCl is a standard solution purchased from Fisher Scientific Company. The NaOH solution must be standardized on the day of the experiment (see below). In addition to titrating the amino acid solution, each group must also titrate the blank (water). Each person in the group should perform at least one part of the experiment; all data obtained by the group needs to be recorded by each member in the laboratory notebook. Remember to record the unknown number and the names of all group members.

Methods

(Note:  Pay attention to the laboratory implement you chose to use to measure volume - make sure you chose an appropriate vessel which will not sacrifice your accuracy)

I. Standardize pH meters: Standardize (calibrate) the pH meter with the pH 4.0 and pH 7.0 standard buffer solutions.

II. Calibrate the NaOH stock solution:
Because the precise concentration of a NaOH solution can change with time due to Na₂CO₃ formation, the solution should be titrated with the standard HCl solution to determine the exact concentration of NaOH on the day of the experiment.

To standardize the base add 5.00 ml of the NaOH solution to 50 ml of water and titrate with 1.000 M HCl standard (use the 5 ml pipettor). Include a few (2 -3) drops of phenolpthalein -- a pH indicator dye. Determine the volume of HCl required to neutralize the base. Compare the volume of acid required to reach the endpoint (neutralization) as indicated by the dye color change with that required to bring the solution to pH 7.00.

The volume of acid required to neutralize the NaOH solution is used to determine the concentration of the NaOH solution. For example, if it takes 4.81 ml of the HCl to reach a pH of 7.00, you should be able to use this information to determine the exact concentration of the NaOH via introductory chemistry principles and rather simple calculations.

HINT: Remember to convert data to moles and that, for neutralization, there will be equivalent moles of acid (H+) and base (OH-).

III. Amino acid titration
1. Place 25 ml of the unknown amino acid solution into an appropriate (use the 25 ml volumetric pipet) beaker. Dilute by adding 50 ml of distilled water.

2. Place a stir bar in the beaker, and mix solution on the stir plate.

3. Standardize the pH meter with pH 4 standard buffer.

4.  Carefully place the pH probe into the amino acid solution, so that the probe is far enough into the solution, but not touching the stir bar or beaker.

5. Begin titrating the solution with the standard HCl solution by adding 0.1 ml aliquots.  It is essential to know the exact volume of acid added, so pipet carefully, noting the number of significant figures that will be dictated by the precision of the pipet used. After each addition of 0.1 ml, record the volume added and measure and record the pH of the solution, after it stabilizes.

6. Continue titrating until the pH drops to 1.5.

7. Repeat steps 1-6, only this time, calibrate the pH meter with the pH 10 standard, and titrate with the calibrated NaOH solution until the pH is approximately 11.5. Be sure to start with a fresh 25 ml of amino acid added to 50 ml of distilled water.

Questions: These questions (as all post lab questions) should be answered in your laboratory notebook
1. For each functional group of your deduced amino acid, write the ionization reaction; indicate the pH where conjugate acid and base pairs are equal in concentration.
2.  If a more dilute acid or base had been used for the titration it would be necessary to titrate a water blank (titrate water with acid and with base and subtract the titrant volume from the curve). Why?

Report:

The following are points that should be addressed in the Introduction of your laboratory report:
1. The general structure of amino acids.
2. The acid/base chemistry of amino acids.
3. The objective of the experiment in more than a single sentence statement. (i.e. "The objective of this experiment was to identify an unknown amino acid" is not an acceptable statement of the objective.)

Results/Discussion - Include:
1. Prepare a table of moles of HCl and NaOH added during titration and the observed pH value for the unknown amino acid and the water blank.
2. Plot moles HCl or NaOH added vs. pH for the amino acid (titration curve).
3. From the plot, prepare a table of calculated moles HCl and NaOH vs. pH for the amino acid.

Address the following:
1. From the  titration curve, what can you tell about the number and types of titratable groups on the amino acid?
2. What were the experimentally determined pKa values?
3. Possible unknown identities were noted in laboratory. Discuss the identity of your unknown. Explain your decision using your data to support your conclusion; you may also include other references or source(s) of information used to verify your conclusion. These resources must be cited.
4. From the data obtained, calculate the concentration of the stock solution of the unknown amino acid.

Misc:
1. Make sure you include your unknown number.
2. Be sure to include titles, numbers and legends for your figures and tables.
3. Be sure to include a bibliography.
4. Write in full sentences and paragraph format, third person passive voice. Outlines are not acceptable.