6Experiment164Experiment8319-Experiment-3.ppt
- 1. LOGO Do you google? Experiment 2 Titration of Amino Acids Sundusit Yangvisit Edwin Hojillla Jayson Doria Renan Difuntorum Dandelo De Guzman Group 4
- 2. Sorensen’s Formol Titration In view of the presence of the amino group, it is not possible to titrate and estimate the total acidity of an amino acid solution, since the H+ ion formed by the ionisation of –COOH will be taken up by –NH2 and be present as –NH3 +. However, an amino acid solution could be titrated to the complete neutralisation point after the addition of excess of formalin (solution of formaldehyde in water).
- 3. Formaldehyde converts the basic –NH2 group of the amino acid forming a neutral dimethylol derivative thus overcoming the interference by –NH2 in the titration. After the addition of formaldehyde, an amino acid behaves as any ordinary organic acid. Thus, formalin makes available the entire H+ ions during titration against alkali.
- 5. Purpose to determine the acid base behavior of select amino acids during titration with an alkali and acid. The effect of formaldehyde on the titration curve of amino acids will also be determined.
- 6. (1) titration of the amino acid with HCl, (2) titration of the amino acid with NaOH, (3) titration of the formaldehyde-treated amino acid with HCl and (4) titration of the formaldehyde-treated amino acid with NaOH. The experiment is divided into 4 parts:
- 7. Materials/Reagents 0.1N NaOH 0.1N HCl 0.1M glycine solution 0.1M lysine solution 0.1M aspartic acid solution Neutralized formaldehyde
- 8. Procedure 1. Take two pipettes and fill the first with 0.1N HCl and the second with 0.1N NaOH. 2. Into each of the two beakers, introduce 10.0 mL of the amino acid solution and measure the resulting pH of the solution. 3. Titrate the first solution with 0.1N HCl adding 2.0 mL at a time and determining the pH after each addition, until a total of 10.0 mL is reached (for glycine and aspartic acid) or 20.0 mL (for lysine). In addition, measure the pH at 5 mL and 15 mL volumes.
- 9. 4. Titrate the second solution in the same manner as the 1st using instead 0.1N NaOH, until 10.0 mL is reached (for glycine and lysine) or 20.0 mL is reached (for aspartic acid). 5. Plot the pH (ordinate) vs. the equivalent acid/base (abscissa). One mL of acid/base=0.1 mEq of acid/base. (Show how this value was obtained). 6. Repeat the above procedure, but add 5.0 mL of neutralized formaldehyde solution into each of the amino acid solutions before determining the pH of the solution. Titrate the solutions. Procedure
- 10. 7. Plot pH vs. the equivalent acid/base on the same graph as above. Construct your titration curves on graphing paper. Solve for the pI and pKa values of your amino acid. www.themegallery.com Procedure
- 11. 1 mL of acid/base = 0.1 mEq of acid/base 0.1 N HCl = 0.1 M HCl = (0.1 mol/L)(36.5 g/mol) = 3.65 g/L 0.1 mEq = 0.1 x (36.5 g / (1 x 1000) = 0.1 x 0.0365 g = 0.00365 g mEq = atomic weight (g) valence x 1000
- 12. Glycine HCl NaOH mL mEq w/o HCHO w/ HCHO w/o HCHO w/ HCHO 0 0.0 5.1 5 6.9 6.5 2 0.2 3 3 9.3 9.4 4 0.4 2.6 3.5 9.7 9.5 5 0.5 2.5 2.4 9.8 9.6 6 0.6 2.4 2.3 10 9.9 8 0.8 2.1 2.1 10.2 10.1 10 1.0 2 2 10.6 10.3
- 13. 0 2 4 6 8 10 12 0.0 0.2 0.4 0.6 0.8 1.0 1.2 pH mEq of acid/base Glycine HCL w/o HCHO HCL w/ HCHO NaOH w/o HCHO NaOH w/ HCHO
- 14. Aspartic Acid HCl NaOH mL mEq w/o HCHO w/ HCHO w/o HCHO w/ HCHO 0 0.0 4.5 4.5 4.5 4.5 2 0.2 4 4 8.6 7.1 4 0.4 3.6 3.6 9.7 8.2 5 0.5 3.5 3.6 10 8.5 6 0.6 3.3 3.47 10.2 8.7 8 0.8 2.9 3.1 10.5 9.2 10 1.0 2.6 2.9 10.9 9.6 12 1.2 11.4 10.3 14 1.4 12.2 11.3 15 1.5 12.4 11.6 16 1.6 12.5 11.8 18 1.8 12.7 12 20 2.0 12.9 12.2
- 15. 0 2 4 6 8 10 12 14 0.0 0.2 0.4 0.5 0.6 0.8 1.0 1.2 1.4 1.5 1.6 1.8 2.0 pH mEq of acid/base Aspartic Acid HCl w/o HCHO HCl w/ HCHO NaOH w/o HCHO NaOH w/ HCHO
- 16. Lysine HCl NaOH mL mEq w/o HCHO w/ HCHO w/o HCHO w/ HCHO 0 0.0 8.8 6.2 9 6.4 2 0.2 7 5.5 9.5 6.7 4 0.4 2.6 3.2 10 7.3 5 0.5 2.2 2.6 10.3 7.8 6 0.6 2.1 2.3 10.5 8 8 0.8 2 2 11 8.4 10 1.0 1.8 1.9 1.4 9.3 12 1.2 1.7 1.7 14 1.4 1.5 1.6 15 1.5 1.5 1.6 16 1.6 1.5 1.4 18 1.8 1.4 1.3 20 2.0 1.4 1.2
- 17. 0 2 4 6 8 10 12 0.0 0.2 0.4 0.5 0.6 0.8 1.0 1.2 1.4 1.5 1.6 1.8 2.0 pH mEq of acid/base Lysine HCl w/o HCHO HCl w/ HCHO NaOH w/o HCHO NaOH w/ HCHO
- 18. QUESTIONS
- 19. 1. What can account for Sorensen’s discovery that the endpoint of titration between an amino acid and a standard alkali (without formaldehyde) is not reached?
- 20. ANSWER The free amino group at the alpha- carbon acts as a base and interferes with the end point of the titration using a standard alkali. Formaldehyde in excess is needed to modify the basic free amino group to modify it to a neutral group, a dimethylol derivative, which allows for the endpoint to be reached.
- 21. 2. When an amino acid is titrated with an acid, example HCl, both with and without formaldehyde. How do you account for this?
- 22. ANSWER In the presence of formaldehyde, tritration curve was slight lower. This is due to the fact that formaldehyde ties down the amino groups, making the carboxyl hydrogen ion more available
- 23. 3. Draw the titration curve of glycine and point the areas of maximum buffering capacity, the point of least buffering capacity.
- 24. ANSWER Maximum buffering capacity (pink) when pH = pKa +/- 1. In this region there is nearly equal amounts of proton donors and acceptors, i.e. weak acid/base and conjugate base/acid. Least buffering capacity at the pI because both the amino and carboxyl group are protonated and deprotonated, respectively. Therefore any minute addition of H+ or OH- will result in a large change in pH.
- 25. 4. From the titration curve of an amino acid, can you determine the nature of its R group, explain why?
- 26. ANSWER Yes, a reactive side group can be determined from the titration curve of an amino acid. When the amino acid is titrated and graphed, three buffering regions will be developed. The extra buffering region aside from the alpha-amino (pKa = approx. 2) and alpha-carboxyl (pKa = approx. 9) can be used to determine the identity of the unknown side chain. If the R group has: pKa < pH, it is basic pKa > pH, it is acidic pKa = pH, neutral because zwitterion forms
- 27. LOGO