Titration of Amino Acids

Introduction

Materials0.1 N NaOH0.1 N HCl0.1 M glycine solution0.1 M lysine solution0.1 M aspartic acid solutionNeutralized formaldehyde

Procedures1. Take two pipettes and fill the first with 0.1N

HCl and the second with 0.1N NaOH2. Into each of the two beakers, introduce 10.0

mL of the amino acid solution and add 10mL of distilled water 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 5mL and 15mL volumes

Procedures4. 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 5.0mL of neutralized formaldehyde solution into each of the amino acid solutions before determining the pH of the solution. Titrate the solutions.

Procedures

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 pK values of your amino acid.

8. Record your results as follows

Glycine: Acid VS Base Data

HCl Gly w/o HCHO

HCl Gly w/ HCHO

NaOH Gly w/o HCHO

NaOH Gly w/ HCHO

0 5.15 5.02 6.91 4.57

2 2.96 2.97 9.28 6.06

4 2.62 2.56 9.69 6.57

5 2.52 2.43 9.75 6.92

6 2.35 2.33 9.98 7.21

8 2.13 2.12 10.26 7.84

10 1.99 1.99 10.58 8.88

Glycine: Acid VS Base Graph

0 2 4 5 6 8 100

2

4

6

8

10

12

HCl Gly w/o HCHO HCl Gly w/ HCHO NaOH Gly w/o HCHO NaOH Gly w/ HCHO

Aspartic Acid: Acid VS Base DataHCl Asp w/o

HCHOHCl Asp w/

HCHONaOH Asp w/o HCHO

NaOH Asp w/ HCHO

0 4.47 4.49 4.5 4.52 8.62 7.12 4.01 4.014 9.66 8.2 3.64 3.645 9.96 8.53 3.47 3.646 10.18 8.72 3.3 3.478 10.52 9.16 2.95 3.11

10 10.9 9.63 2.59 2.7512 11.43 10.34 2.32 2.4514 12.16 11.32 2.12 2.2215 12.37 11.58 2.04 2.1116 12.51 11.78 1.96 2.0318 12.68 11.99 1.84 1.9120 12.8 12.14 1.76 1.81

Aspartic Acid: Acid VS Base Graph

0 2 4 5 6 8 10 12 14 15 16 18 200

2

4

6

8

10

12

14

HCl Asp w/o HCHO HCl Asp w/ HCHO NaOH Asp w/o HCHO NaOH Asp w/ HCHO

Lycine: Acid VS Base DataHCl Lys w/o

HCHOHCl Lys w/

HCHONaOH Lys w/o HCHO

NaOH Lys w/ HCHO

0 8.79 6.25 9.04 6.362 7 5.5 9.54 6.764 2.55 3 10 7.165 2.3 2.58 10.3 7.386 2.14 2.32 10.58 7.668 1.94 2.04 11.03 8.36

10 1.81 1.87 11.42 9.3512 1.67 1.7414 1.59 1.6515 1.55 1.6116 1.53 1.5718 1.47 1.5220 1.42 1.47

Aspartic Acid: Acid VS Base Graph

0 2 4 5 6 8 10 12 14 15 16 18 200

2

4

6

8

10

12

HCl Lys w/o HCHO HCl Lys w/ HCHO NaOH Lys w/o HCHO NaOH Lys w/ HCHO

Questions

Question1. 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?

AnswerFormaldehyde in excess readily

combines with free unprotonated amino groups of amino acids to give dimethylol derivatives, and the proton titrated can be treated directly. Formaldehyde makes the solution more acidic when titrated with a standard base

Question2. Compare the values obtained

when the amino acid was titrated with HCl both in the absence and presence of formaldehyde. How do you account for this?

AnswerIn the presence of formaldehyde,

titration curve was slightly lower. This is due to the fact that formaldehyde ties down the amino groups, making the carboxyl hydrogen ion more available

Question3. At which pH will an amino acid

exert its maximum buffering capacity? Why? Where in your graph is the buffering region for your amino acid?

AnswerAn amino acid exerts its

maximum buffering capacity at the plateau near or on its pKa values because it is in this area that there is equal concentration of proton donors and acceptors.

Question4. From the titration curve of an

amino acid, can you determine the nature of its R group i.e., basic, acidic or neutral?

AnswerYes, the nature of the R group

can be determined from the titration of an amino acid through the determination of the pKa values for each dissociable group of an amino acid by extrapolating the midpoint of each buffering region or plateau within the curve.

AnswerSince the ionic form of the amino

acid present in an aqueous solution is dependent upon solutions pH, if:pH > pKa: basic (negatively charged)pH < pKa: acidic (positively charged)pH = pKa: zwitterion (no charge)

isoelectric point