Multiple Themes: The Amino Acid Project

• To provide a long-term research project involving a number of aspects of chemical identification, structure, and reactivity
• To enhance observation skills
• To learn and reinforce techniques of synthesis, separation, and characterization
• To enhance skills of experimental design and strategy

The purpose of this project is to give you a long-term, research-like laboratory experience in chemistry. Unlike the 1-period experiments that you have performed during previous terms, this project will occupy the entire term, and will be conducted at your pace and according to your experimental decisions. Instructors will be on hand to provide help and guidance; however, the responsibility for advancing the project to a meaningful conclusion will be yours.

Before coming to lab each week, you should complete the appropriate set of Preparation Questions and hand them in at the beginning of the period, as usual. We expect that you will maintain your laboratory notebook in the same manner as in previous terms; however, the notebook will not be turned in for grading. Instead, you will be expected to submit a formal research report on your findings at the end of the term. Your laboratory grade will be based on the quality of this report, and on the Preparation Questions that you answer each week.

Description of Project You will work with one or more partners throughout the term. At the first lab period, you and your partners will be given a vial containing one of the 20 common amino acids. Your overall goals are to identify and characterize your amino acid. You can accomplish this by addressing, during the successive weeks of the term, the following project objectives:

• Determine the physical properties of your amino acid. These include
  • Solubility in water (quantitative)
  • Solubility in other solvents, such as acetone, ethanol, dichloromethane, dilute HCl (qualitative)
  • Density
  • Melting point
  • R_f value under specified thin-layer chromatographic conditions (Thin-Layer Chromatography (TLC) is a technique for the separation of substances with similar molecular structures). Please save ALL of your TLC plates, even those that you think "didn't work".
  • UV-visible spectrum (Can be done no earlier than the second week of the term). You will run your own UV-visible spectrum, and will compare the results with a table containing l_max(e) values for the 20 amino acids. The spectrum must be included in your final report, so don't abuse it!.
  • Infrared spectrum (Can be done no earlier than the third week of the term). You will run your own IR spectrum, and will compare it with the IR spectra for the 20 amino acids that we will provide. You are looking for a match. Your spectrum must be included in your final report.
  • NMR spectrum (Can be done no earlier than the third week of the term). We will provide you with the NMR spectrum for your amino acid. Please study it and decide whether the number and nature of the signals in the spectrum is consistent with what you suspect your amino acid to be. The spectrum must be included in your final report.
• Determine the Bronsted-Lowry acid-base properties of your amino acid. These include
  • pH of a 0.1 M aqueous solution
  • pK_a values for the carboxylate proton and the ammonium proton
  • Molar mass of your amino acid, using the titration data from the previous experiment
  • pH of isoelectric point (you will have to read about what the isoelectric point is)
  • Synthesis of the adduct of your amino acid with HCl (NOTE: check with the instructor before proceeding). You should
    • Make this and isolate it from aqueous solution
    • Obtain the IR spectrum of the adduct
    • Determine the melting point of the adduct
    • Determine whether the adduct is ionic or covalent (check with the instructor as to whether or not you have to do this)
    • The word, "adduct", is a condensation of the phrase, addition product. Here the word means that HCl somehow adds to the amino acid. Can you describe how?
• Determination of the ligating properties (i.e., Lewis basicity) of your amino acid. This includes
  • Synthesis of the complex, Cu(aa)₂ (aa = amino acid anion)
  • Verification of the stoichiometry of the copper complex using Job's Method. For a detailed Job's Method procedure on a similar system, check here.
  • Characterization of the copper complex. This includes
    • Color
    • melting point
    • density
    • Solubility in common solvents
    • infrared spectrum. The spectrum must be included in your final report.
    • UV-visible spectrum. The spectrum must be included in your final report.
    • Spectrometric Analysis of copper content
  • Structure of the copper complex. Obtain two molecular model kits, and review their use.
    • Assemble two identical models of your amino acid.
    • Determine and write down the stereochemistry at each "central" atom of the amino acid.
    • Build a model of your copper complex. Your model should be consistent with the fact that 2 isomers are known for the complex, even though your synthesis only produces one of them. WHEN FINISHED WITH THE MODELS, SORT THE PIECES BACK IN TO THE KITS SO THAT EACH KIT HAS THE PROPER NUMBER OF EACH PIECE. Then return the kits.
• Analysis of an oligopeptide (a molecule containing two or more amino acids linked together in a chain). This includes
  • melting point
  • infrared spectrum. The spectrum must be included in your final report.
  • Hydrolysis of the oligopeptide.
  • Determination of the amino acid sequence of your oligopeptide. (NOTE: this is a sophisticated and challenging experiment; check with the instructor.)
• Synthesis of polypeptides of your amino acid. (NOTE: this is a sophisticated and challenging experiment; you can try it if you have time at the end.)

By the end of the term, you should be an expert in the physical and chemical properties of your amino acid.

Preparation Questions

1. Week 1
2. Week 2
3. Week 3
4. Week 4
5. Week 5

Equipment and Materials

• assorted beakers and Erlenmeyer flasks
• Pasteur pipets/bulbs
• assorted watch glasses
• Product vials (1-dram)
• Stirring rods
• 5- and 10-mL volumetric flasks
• 1-, 2-, 5-, and 10-mL graduated pipets
• Syringe pipet pumps
• spatulas
• thermometers
• balances
• aspirator trap
• filtration equipment
  • sidearm test tubes
  • Buchner funnels
  • filter paper
  • funnel collars
• Conductivity meter components
  • 9-V batteries
  • LEDs
  • perf-board
  • resistors
• amino acids
• Stock NaOH solution (about 6 M)
• Stock HCl solution (about 6 M)
• conc HNO₃
• potassium hydrogen phthalate
• Reagents for flotation
  • carbon tetrachloride
  • chloroform
  • dichloromethane
  • 1,2-dichloroethane
  • 1,1,2,2-tetrachloroethane
• pH meters
• 25-mL burets
• Cu(NO₃)₂.6H₂O
• Ni(C₂H₃O₂)₂.4H₂O
• Melting point capillaries
• Rock salt
• Thin-Layer chromatography equipment
  • Silica gel plates
  • TLC Spotting capillaries
  • Developing chambers (120-mL snaptop vials and/or 100-mL wide-mouth plastic bottles)
  • Ink samples
  • 0.2% ninhydrin in ethanol
  • forceps
• Melting point apparatus
• Infrared spectrometers
• Infrared sample preparation equipment
  • agate mortar and pestle
  • NaCl plates
  • NaCl plate holder
  • Sample mount
  • Nujol
  • flat spatula
• UV-visible spectrometers
• UV-visible sample preparation equipment
  • UV-visible cuvets
  • 5- and 10-mL volumetric flasks
  • 1- and 2-mL graduated pipets
• NMR spectrometer
• NMR sample preparation equipment
  • 1-dram vials
  • NMR tubes with caps
  • D₂O

Safety goggles must be worn at all times in the laboratory. Avoid contact of the reagents with the skin. In the event of skin contact, flush the affected area with copious quantities of cold water.

Please print and read the documents dealing with fundamental laboratory operations and procedures:

• The Laboratory Notebook
• Measuring Liquids
• Recommended Procedures for Cleaning Glassware
• Laboratory Techniques
• Hazardous Waste Disposal
• Laboratory Safety Regulations

Record all data and observations in your notebook.

Design of experiments is up to you. Please keep in mind that we have available supplies of all the amino acids used for the unknowns. You are free to use these for comparison tests if and when you think these would be useful.

Clean-up. When you have finished all of your work each week:

• Clean glassware by recommended procedures, shake off excess water, and place in the drying oven.
• Return all borrowed equipment to the instructor before leaving lab.
• Clean up your work area before leaving lab.

Dispose of all solids, liquids, and solutions in the appropriately marked waste bottles.

Guidelines

1. At the beginning of each laboratory session except the 6th one, you are to turn in your answers to the appropriate set of Preparation Questions. Each set of Preparation Questions is valued at 8 points, for a total of 40 points for the 5 sets.
2. You have six 3-hour lab periods in which to complete the project. This is 18 hours, or just a little more than 2 full working days! You should make an overall plan listing which experiments you will attempt each week. In addition, you should make a detailed plan for each laboratory period, designed to make your use of time as efficient as possible. We will not collect or grade these plans, but unless you formulate them, you will not make much progress.

   We suggest that you plan to complete the experiments up to and including Thin Layer Chromatography by the end of the second lab session.

3. You TA will maintain a time/activity log during the term. For each laboratory session, you should enter
   • your time of arrival
   • your time of departure
   • what you accomplished during the session
   • problems that you encountered, if any
4. Midterm Progress. Using the prescribed format, you must submit a partial to-date preliminary version of your Formal Research Report for review following the third lab session. This preliminary version will not be graded; instead, it will be treated as a draft and will be critically reviewed and critiqued. The preliminary version must be submitted NO LATER THAN NOON on the Friday following your third lab session.
5. Your final Formal Research Report is due NO LATER THAN noon on the Monday of the final week of C term.

Cautions

1. Because you will be doing on-going research this term, rather than a series of 1-week, independent laboratory experiments, it is essential that you be well-prepared for every lab session and that you keep detailed records of experimental procedures, observations, and results in your laboratory notebook. THE NOTEBOOK WILL NOT BE SUBMITTED FOR GRADING. IT IS STRICTLY FOR YOUR RECORDS; IN IT SHOULD BE ALL OF THE INFORMATION REQUIRED FOR THE WRITING OF YOUR FINAL REPORT. BE FASTIDIOUS AND THOROUGH IN MAINTAINING YOUR LAB NOTEBOOK.

2. Because you will be working with the same partner(s) for the entire term, it is important that you not casually miss any laboratory periods. Doing so has a strong impact on your partner(s).