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Biochemistry Laboratory Manual  
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Titration of Amino Acids - Procedure

A. Get your assignment of which unknown you will be working with from the teaching assistant and make sure to record its label in your notebook.

  1. Weigh out ~0.20-g of your unknown sample making sure to record its mass to 4 significant figures.

  2. Add the unknown to enough distilled, deionized water (ddH2O) to make a total volume of 50-mL and mix your solution until the sample is completely dissolved.

  3. After collecting a buret, a buret clamp, stand, pH meter, stir plate, and stir bar set up the titration apparatus.

  4. With the titration apparatus set up, collect 250-mL of 0.1M standardized NaOH. You should pour a little bit of NaOH in your buret and swirl it around to rinse the instrument before filling it completely. For detailed instructions please click here.

  5. Place your 50-mL of unknown amino acid solution in a 250-mL beaker and carefully place the stir bar inside while placing it upon the stir plate. After obtaining a gentle swirl, adjust the probe of the pH meter in the solution so that the stir bar nor glass sides of the beaker are in contact.

  6. Carefully add small drops of 12.0 M HCl to your beaker until the pH is ~1.5, be careful not to overshoot too much as it will only lengthen the amount of time required for your titration.

  7. Once the appropriate pH has been reached, AND RECORDED, the titration can start. Use these guidelines to help with the process: (a) Add base in increments of 1.5-mL until a pH of ~2.0 is reached recording the volume of NaOH added and pH after each addition; (b) Add base in increments of 0.5-mL until reaching a pH of ~12.5 again recording the volume and pH each time.

  8. After reaching the end of your titration, create a rough graph of your results to isolate regions of interest (plateaus, points of inflection, etc.)

  9. Repeat the titration, reducing the amount of base added when necessary to better define the region of interest identified by the first titration. An accurate titration curve here will immensely help you discuss (and prove) the identity of the amino acid contained within your sample!

 

Click here for a printable PDF file of the procedure.