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6Experiment164Experiment8319-Experiment-3.ppt
1. LOGO
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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.
4.
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:
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.
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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
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
