Elissia Vecere

CHEM 3560 Section 102

1/27/21
Experiment 3: Part 1 Amino Acid Titration
Introduction
Amino acids are the monomers of proteins and are an important part of our daily lives in
regard to energy metabolites and essential nutrients. Proteins consist of a variety of the 20
amino acids linked via peptide bonds. Throughout this lab, the student looked at the side chains
of amino acids and the uniqueness of their properties, including their pKa values. Within this
lab, the student titrated an amino acid both with a strong base and a strong acid. This allowed
for a complete titration curve formation. The student worked directly with the titration curves
they formed in order to be able to identify buffering regions present in their unknown amino
acid and use this data to identify possible amino acid identifications based on their pKa’s and
titration curves. This lab worked with titration skills of the student and deductive reasoning on
identifying the amino acid based on titration curves.

Objective
The objectives throughout this lab included being able to understand the amino acid
structure and the pKas for each group within an amino acid (-NH3+, COOH, and the side
chain/R). Along with this the student gained the ability to titrate an amino acid using a strong
acid and a strong base, while also being able to take this data and form a titration curve. Within
this graph, the student was able to identify the pKas, the pI, and the species present in each
region of the graph. Finally, the student was able to calculate the pKas for each group of the
amino acid, when given the data to do so.

Procedure
To begin, the student needed to obtain 25 mL of their unknown amino acid that was
assigned and add it to the 250 mL beaker. During this time, the student recorded the letter or
number of their unknown into their lab notebook. Next, 50 mL of deionized water was added to
the 250 mL beaker and the contents were mixed. From there, 2 mL of 0.1 M HCl was added and
mixed, while the pH was recorded. The student continued the addition of 2 mL of 0.1 M HCl
until the pH was recorded at less than 1.5. The student checked the pH after each additional 2
mL of 0.1 M of HCl was added. Afterward, the solution was disposed of in the mixed acid
container and the 250 mL beaker was washed out. The student obtained another 25 mL sample
of the unknown amino acid and added it to the clean 250 mL beaker. 50 mL of deionized water
was added into the beaker, along with 2 mL of 0.1 M of NaOH, and the solution was mixed.
Following the mixing, the student recorded the pH of the solution. The student continued to
add 2 mL of the 0.1 NaOH until the pH was greater than 12. The pH was recorded after each
addition of 2 mL of 0.1 M of NaOH. This solution was put in the mixed base container and the
250 mL container was cleaned out. The student took the data of the pH calculations from each
addition of 2 mL of either a strong base or acid and draw out a plot of the titration on one
graph. The lab was repeated once more in order to obtain two independent sets of data.

Observations
Throughout the lab, the student noticed that the amino acid solution was relatively cold
in comparison to the deionized water it was mixed with. Once the student began the first
 Elissia Vecere
CHEM 3560 Section 102
1/27/21
Experiment 3: Part 1 Amino Acid Titration
titration with the HCl solution, they noticed that with the first 2 mL of HCl added, there was a
significantly large decline in the pH of the solution. However, following the first 2 mL of HCl
added, the changes in the pH were less obscene, with acceptance to the recognizable pka
areas. The same observation as made in regard to the NaOH titration, except this time the pH
increased dramatically with the first 2 mL of NaOH added and afterwards, the pH increases less
drastically. Throughout the lab, the student noticed that on the second rounds of titration for
both the HCl solution and NaOH solution, the pH values derived were slightly different than in
the first round of titration. Also, due to the student suspecting their unknown to be aspartic or
glutamic acid, the student focused in on the pKas of the amino and carboxyl groups of both.
Specifically, when the pH of the solution reared near the pKa values that represented the amino
group protonation or carboxyl group deprotonation, the student began titrating at 1 mL of the
HCl solution or NaOH solution. The student did this in order to focus more carefully on which
pKa values were more likely to be a match to the unknown.
 Elissia Vecere
CHEM 3560 Section 102
1/27/21
Experiment 3: Part 1 Amino Acid Titration
Results
 Elissia Vecere
CHEM 3560 Section 102
1/27/21
Experiment 3: Part 1 Amino Acid Titration

Titration Curve Unknown B Run 1

14

12

10

8
pH Value

0
-60 -40 -20 0 20 40 60
mL Added
Left= HCl Right= NaOH
 Elissia Vecere
CHEM 3560 Section 102
1/27/21
Experiment 3: Part 1 Amino Acid Titration

Titration of Unknown B Run 2

14

12

10

8
pH Value

0
-80 -60 -40 -20 0 20 40 60
mL added
Left= HCl Right=NaOH

Discussion
Question 2
pKa: The pKa is the tendency in which a molecule will dissociate into the conjugate base and
the H+ ion. Specifically, the pKa value on a titration curve will represents where, for example,
an amino acid has been fully protonated (pH<7), at an acidic pH, or fully deprotonated, at a
basic pH (pH>7).
pI: Also known as the isoelectric point, pI is the pH at which the amino acids within a protein
are in zwitterion formation. Specifically meaning, the amino group is fully protonated, and the
carboxyl group is fully deprotonated, leaving them with a +1 and -1 charge respectively. Their
overall cancelation of each other’s charges leaves the amino acid neutral, with acceptance of
certain charged side chains.
Question 4
Glutamate/Glutamic Acid= Glu = E
 Elissia Vecere
CHEM 3560 Section 102
1/27/21
Experiment 3: Part 1 Amino Acid Titration
Question 5

Question 6
The charge of the amino acid lysine at a physiological pH of 7.2 is +1 on the side chain, +1 on
the amino group and -1 on the carboxy group. Therefor the overall net charge of lysine at a pH
of 7.2 is +1. The charge of the amino acid glutamate at a physiological pH of 7.2 is -1 on the side
chain, a +1 for the amino group and a -1 on the carboxy group. Therefore, the overall net
charge of the amino acid glutamate at a pH of 7.1 is -1.

Summary
Throughout the lab, the student titrated an unknown amino acid in hopes to derive its
pKa values as a means of identification. After the student completed the first titration with both
NaOH and HCl, they were able to recognize the curve as either glutamic or aspartic acid. With
this information, the student looked at the pKa values of both acid (Glutamic pKa1=2.10 and
pKa2= 10.70; Aspartic pKa1= 1.99 and pKa2= 9.90) and during the second titration, began
observing the detected pH’s based on the variation between glutamic and aspartic acid. When
the detected pH reared near 9.4-10.3 and 2.10-1.93, the student titrated each solution by 1 mL
of either NaOH or HCl. This allowed for the student to identify their unknown amino acid
(AMINO ACID B) as glutamic acid.
 Elissia Vecere
CHEM 3560 Section 102
1/27/21
Experiment 3: Part 1 Amino Acid Titration
 Elissia Vecere
CHEM 3560 Section 102
1/27/21
Experiment 3: Part 1 Amino Acid Titration