Identification of an Unknown Amino Acid by Titration
Introduction: Alpha amino acids are the building blocks of proteins. Almost all proteins consist of
various combinations of the same 20 amino acids. Amino acids are compounds containing an amine
group, -NH2, and a carboxylic acid group, -COOH. In addition there is an "R" group that differs
for each amino acid. The symbol "R" is used here to represent a generalized abbreviation for an
organic group.
H
H 2N
C
COOH
R
In physiological systems where the pH is near neutrality, the amino group of an amino acid will be
protonated and the carboxylic acid group will be deprotonated. This is called the zwitterion form.
H
H 3N
C
COO
R
In strongly acidic solutions the carboxylic acid group will also be protonated, while in strongly basic
solutions both the carboxylic acid group and the amino group will both be unprotonated.
H
H 3N
C
H
COOH
H 2N
R
C
COO
R
Acidic
Basic
The acid-base behavior of amino acids is best described by the Brønsted-Lowry theory of acids
and bases. A simple amino acid (that does not have an acid or base group in the "R" group) is a
diprotic acid in its fully protonated form; it can donate two protons during its complete titration
with a base. The titration with NaOH is a two-stage titration represented by the reactions below.
+
NH3CH(R)COOH + OH-  +NH3CH(R)COO- + H2O
+
NH3CH(R)COO- + OH-  NH2CH(R)COO- + H2O
The hydrochloride salt of a simple amino acid contains one mole of HCl for each mole of amino acid
such that the amino acid is fully protonated.
Cl- +NH3CH(R)COOH
The titration curve will contain two equivalence points (see diagram below). There will also be two
separate buffer regions on the titration curve. The midpoint of the buffer region (B) is where the
amino acid is half in the acidic form and half in the zwitterion form. The point of inflection (C)
occurs when all of the original amino acid is in the zwitterions form (assuming the "R" group is not
acidic or basic). The actual pH at which this occurs is called the isoelectric pH (or isoelectric
point), and is given the symbol pI. During the pH titration of an amino acid with a non-ionizable "R"
group, the equivalence point occurs at the pI of the amino acid. At the midpoint of the second
buffer region (D), half the amino acid is in the zwitterion form and half is in the basic form. The
apparent pKa values for the two dissociation steps may be extrapolated from the midpoints of each
step. This can be shown by the Henderson-Hasselbach equation:
pH = pKa + log([Base]/[Acid])
The pKacid (pKa for the carboxylic acid group) is point (B) where half the acid group has been
titrated. Therefore the equation becomes:
pH = pKa
In the same way, point (D) gives us the pKamine. In this experiment you will titrate an unknown amino
acid, determine its pI, pKacid and pKamine, and compare your values to literature values.
Objective : Study titration curves and identify unknown amino acid.
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Procedure :
What you are about to do first time around is called a ‘dry run’. It is done quickly to estimate the
amount of NaOH needed to titrate.
(1) Pipet 10.0mL of amino acid into a 150mL beaker.
(2) Add 25.0mL of distilled water into (1).
 Does the amount of water make difference to the titration? Why, why not?
(3) Prepare a buret with NaOH solution for titration. Report the actual concentration .
(4) While a magnetic stirrer is going in a beaker, read the initial pH.
(5) Titrate the amino acid solution with NaOH. Read pH at the interval of your choice.
 What is NaOH reacting with?
(6) Stop the titration if pH reaches 13.0 or if 50.mL of NaOH is added.
(7) Repeat (1) to (6) twice (total of three titrations!) more carefully.
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Prelab Questions :
(PL1) Sketch a theoretical titration curve for HCl vs. NaOH.
(PL2) Sketch a theoretical titration curve for H3PO4.
(PL3) Discuss the titration curves for (PL1) and (PL2). What can you say about strong/weak acid?
What can you say about mono/tri protic acid?
(PL4) What are pKa values for H3PO4?
(PL5) Under what condition is pKa = pH?
(PL6) Cut and paste a (or two) table that has 20 amino acids. It should include names, structures,
pKa’s. Site the source.
Analysis :
(A1) How many moles of unknown amino acid do you have in 10.0mL?
(A2) How many moles of NaOH do you need to add to reach the first equivalent point?
(A3) Draw the titration curves for unknown amino acid. Label pKa’s. Clearly identify all half points
and all equivalent points.
(A4) Identify the unknown.
Questions :
(Q1) How many mol of NaOH were added when you reached the first pKa for your amino acid?
Show your work clearly.
Summary :
Unknown number :
Include titration curves. (Site the page number.)
Theoretical pKa’s
Experimental pKa’s
ID of your unknown