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World Journal of Chemical Education, 2014, Vol. 2, No. 4, 59-61
Available online at http://pubs.sciepub.com/wjce/2/4/3
© Science and Education Publishing
The Identification of Amino Acids by Interpretation of
Titration Curves: An Undergraduate Experiment for
Cassidy M. Dobson, Nathan S. Winter*
Department of Chemistry and Biochemistry, St. Cloud State University, St. Cloud, Minnesota, United States
*Corresponding author: nswinter@stcloudstate.edu
Received November 08, 2014; Revised December 08, 2014; Accepted December 25, 2014
Abstract Undergraduate biochemistry students should have great familiarity with titration curves. These curves
allow the prediction of protonation states, charges, and isoelectric points. Here we describe an experiment in which
students identify four amino acids based on their titration behavior. Students make solutions of each unknown amino
acid and monitor the change in pH upon adding aliquots of a strong base. They identify the amino acids based on the
shapes of the curves. They annotate the plots with isoelectric points, pKas, buffering regions and the structures of the
amino acids.
Keywords: titration curve, amino acids, pH, biochemistry, pKa, isoelectric point
Cite This Article: Cassidy M. Dobson, and Nathan S. Winter, “The Identification of Amino Acids by
Interpretation of Titration Curves: An Undergraduate Experiment for Biochemistry.” World Journal of Chemical
Education, vol. 2, no. 4 (2014): 59-61. doi: 10.12691/wjce-2-4-3.
1. Introduction
Familiarity with amino acid chemistry including pKa
values, pI values and protonation states is important for
every biochemist. The protonation states of amino acids
are important for understanding enzymatic catalysis, pH
induced conformational changes, and the intermolecular
interactions which stabilize tertiary and quaternary
structure of proteins.
Creating a titration curve for a weak acid, such as
phosphoric acid or an amino acid [1,2,3,4,6] is a typical
undergraduate laboratory experiment. Indeed dozens of
examples of titration curve exercises can be found by
running a simple internet search. The amino acid titration
described here is unique in that the students do not know
which amino acid they are titrating. Instead students know
they have one of four amino acids which they have to
identify based on the shape of their titration curves. This
experiment is valuable because students must fully
exercise their understanding of amino acid chemistry.
This experiment is a beneficial introductory lab for
students in biochemistry. It familiarizes them with how
functional groups contribute to form a complex titration
curve as well as how pH can influence protonation state
[2,4,6,7,8,9]. This experiment requires only minimal
laboratory equipment including pH meters and micropipettes.
Other reagents include concentrated solutions of hydrochloric
acid and sodium hydroxide and the four amino acids.
Students analyze lysine, glutamine, glutamic acid, and
histidine (CAS numbers L5501, G3203, G1251 and
H8000). These amino acids were selected because they
have quite similar molar masses (155.1 for histidine, 146.2
for lysine, 146.1 for glutamine and 147.1 for glutamic acid
[7]), so that equal masses of the amino acids will make
solutions of approximately the same concentration. This is
important so that the titration curves can be superimposed
to assist with the correct identification of the amino acids.
Each of these amino acids has a different side chain
chemistry (Figure 1). Lysine and histidine are basic amino
acids, glutamic acid is acidic and glutamine is a neutral,
polar amino acid. The differences in the type of amino
acid (Table 1) produce different titration curve signatures.
Figure 1. The predominate forms of glutamine, glutamic acid, lysine and
histidine as they occur at physiological pH.
Table 1. pKa and pI values of the amino acids utilized in this
Amino Acid
pKa –NH3+
pKa -R
Glutamic acid
World Journal of Chemical Education
Each amino acid utilized in this experiment has a
different side chain chemistry. Glutamine does not have a
titratable R-group and therefore has a distinct titration
curve signature from all of the other amino acids who
have three titratable groups. Histidine contains an
additional pKa value at around a pH of 6 and is thus able
to be differentiated from the acidic residue Glutamic acid
and the basic residue Lysine.
2. Experimental Procedure
Amino acid solids labeled A, B, C and D are provided
and each student is instructed to make 25 mLs of a 20 mM
solution of each amino acid. The amino acids in this
experiment have comparable molar masses (155.1 for
histidine, 146.2 for lysine, 146.1 for glutamine and 147.1
for glutamic acid [7]) and therefore the same mass can be
used to prepare each amino acid solution irrespective of
the identity. For consistency all students are directed to
use the molar mass of lysine.
After calibrating the pH meter, a 60 mM HCl solution
is used to bring the pH of each amino acid solution to 2 to
ensure all the unknowns have the same starting point.
Once a pH of 2 is achieved 1.000 mL aliquots of 50 mM
sodium hydroxide are added and the resulting pH is
recorded after each addition after each addition is recorded.
This process is repeated for each amino acid. All amino
acid titrations can be completed in a three hour laboratory
Titration curves (pH vs. equivalents of base) are
graphed, and used to determine the identity of each amino
acid. By overlaying at least three curves the identity of
each amino acid can be determined. The resultant titration
curves are analyzed for pKa values which assists in
determination of the identity of each amino acid. In the
final lab report students annotate each curve with the
experimentally determined pKa values, any buffering
regions present, and the structures of the amino acids at
the different protonation states. Finally, based on the
structures the experimental isoelectric point is determined.
A graph generated from student data showing the
superimposition of all four titration curves is shown in
Figure 2.
2.1. Hazards
Hydrochloric acid and sodium hydroxide may be fatal if
swallowed or inhaled, are extremely corrosive, and skin or
eye contract may cause severe burns and permanent harm.
The amino acids present minimal hazards.
3. Results
As can be seen in Figure 2, each amino acid has a
distinct titration curve. The shapes of these curves allow
the students to properly identify the amino acids. In
addition, this experiment provides a solid foundation of
characteristics of amino acids and how their protonation
states change at different pH values. Amino acid titration
curves are vital to understanding not only how pH
influences amino acid chemistry, but are integral to
understand protein structure.
Figure 2. Titration curves of the amino acids histidine, glutamine,
glutamic acid and lysine. Based on the shape of the titration curves
students are able to differentiate the four amino acids
Each amino acid curve has distinct characteristics that
make them easily interpretable. Histidine shows a distinct
plateau near pH 6 due to the imidazole side chain.
Glutamic acid is easily identified as it contains two acidic
groups. The curves between lysine and glutamine are
harder to distinguish. The glutamine has an additional
inflection point at about pH 11, when the amino group
becomes fully deprotonated, whereas the two amino
groups of lysine blend together for a steady rise for two
equivalents of base. Despite the challenges in
identification, students are able to appropriately identify
the amino acids based on the superimposition of the
4. Conclusion
This experiment successfully demonstrates the ability to
generate and analyze a titration curve to distinguish
unknown amino acids. Titration curves are important for
students to understand. This experiment takes an abstract
concept presented in most General Chemistry and
Biochemistry textbooks and makes it tactile. By matching
the titration curves with the four amino acids students are
required to demonstrate their understanding of side chain
amino acid chemistry. By annotating their plots, students
demonstrate their understanding of protonation states,
buffering regions, and isoelectric points. In addition this
experiment gives students practical skills such as making
solutions and using pH meters.
We would like to thank Nicole Holmberg and our
Biochemistry students for participating in this laboratory
and providing feedback.
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without Calculations. Journal of Chemical Education 76(7): 938942.
World Journal of Chemical Education
Bodner, G. (1986). Assigning the pKa’s of Polyprotic Acids.
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Heimer, E. (1972). Quick Paper Chromatography of Amino Acids.
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Kraft, A. (2003). The Determination of the pKa of Multiprotic,
Weak Acids by Analyzing Potentiometri Acid-Base Titration Data
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Meister, A. (1965). Biochemistry of the Amino Acids. New York,
Academic Press Inc.
Sae, S. W. and B. A. Cunningham (1971). Chromatographic
separation and identification of amino acids. Journal of Chemical
Education 48(4): 275.
Weiss, H. (2007). The Roles of Acids and Bases in Enzyme
Catalysis. Journal of Chemical Education 84(3): 440-442.
Lab 1: Acids, Bases, and Buffers
Learning goals:
Students should:
1. Predict and generate titration curves for common amino acids. (Module A)
2. Identify an unknown amino acid based on the experimentally determined pKa and pI
values. (Module A)
3. Employ proper techniques for making solutions. (Module B)
4. Understand how buffers resist changes in pH. (Module B)
5. Understand the effects of molar concentration on buffering capacity. (Module B)
Pre-lab Reading and Questions:
Read the following article regarding:
Dobson, C. and Winter, N. (2014) The Identification of Amino Acids by Interpretation of Titration
Curves: An Undergraduate Experiment for Biochemistry. World Journal of Chemical Education. 2 p.
Answer the following questions before coming to lab:
What are the components of a buffer in a biological system? What are the proportions of these
components in a buffer working at its maximum capacity?
What chemical changes take place in a system when pH changes? How do buffers deal with these
chemical changes?
Define zwitterion.
What is the net charge on an amino acid at its isoelectric point? Explain.
What proportions of protonated and deprotonated forms of an amino acid are present with pH is equal
to pKa?
Why is it important to understand titration curves?
Look up the general structure of lysine. Draw the predominant structure of lysine in (a) acidic
environments, (b) basic environments, and (c) its zwitterion form.
Available Equipment and Reagents:
pH meter
0.25M and 0.5M NaOH
0.5M HCl
0.1M amino acid solution (glycine, alanine, phenylalanine, leucine, or valine)
Sodium acetate
2M acetic acid
Monosodium phosphate
Disodium phosphate
Laboratory Objective:
The objective of this lab is to enhance your understanding of how acids and bases work in biological
systems. This laboratory exercise will consist of two modules.
In module A, you will examine the structures of some common amino acids and explore their function
as buffers. You will generate a titration curve of an unknown amino acid and investigate its potential
to resist change in pH at the pKa’s and the isoelectic point (pI).
In module B, you will complete a virtual lab to make a buffer and test buffing capacity.
Testable Question:
Based on the objectives outlined above, formulate 2-3 testable questions that your group will seek to
answer during this laboratory exercise. Discuss these questions with your group mates and your
instructor before proceeding to experimental design.
Module A:
Module A: Amino Acids as Buffers – In Person
To understand how amino acids function as buffers in the body, you need to review the basic
structure of amino acids. Draw the basic structure below.
It is important to note that most of the structures you will find in your textbook or in reference sites are
drawn at pH 7. As the pH changes, the structure and composition of the amino acid changes. Draw
the predominant structure of a general amino acid in (a) an acidic environment and (b) a basic
environment below.
Remember that while the acidic structure predominates at low pHs, some of the amino acid may still
be in the basic form. The same is true for high pH environments – the basic structure may
predominate but some of the amino acid is still present in the acidic form. Thus, at any given pH there
are mixtures of acidic amino acid forms and basic amino acid forms. Remember from above that
buffers are mixtures of acids and conjugate bases. Based on this, we can surmise that amino acids
might function as buffers in the body.
For this portion of the lab, you will test the buffering capacity of an unknown amino acid by generating
a titration curve. Your instructor will provide you with 0.1M amino acid solution. Based on the
experimentally determined pKa and pI values, you will identify your unknown amino acid. Use the
article from the pre-lab reading assignment to help you formulate an experimental plan.
Planning Questions
1. Review titration curves in your textbook or from a trusted source. What is plotted on the graph
to generate a titration curve?
2. To what pH should you adjust your assigned amino acid solution? How will you adjust the pH
to the desired value?
3. Your instructor will provide 0.25M NaOH. What volume of base will you add for each data
point? Be sure to mix your solution thoroughly after the addition of base prior to measuring
and recording the pH.
4. How will you know when your experiment is complete (when to stop adding base)?
5. How will you organize and record your data?
6. How will you analyze and present your data?
7. Predicting likely outcomes: Based on your research and what you know about amino acid
titration curves, predict the basic shape of your titration curve, indicating general areas of the
curve you expect to see. Consider an alternative curve. What other curve shape(s) might be
observed and think about possible explanations for these observations.
Module B: Create Acetic Acid Solutions and Evaluate Buffer Behavior – Virtual
Since biochemical reactions take place in a narrow range of pH values, it is important to be able to
control pH in the laboratory. Buffers, solutions that resist changes in pH, are often used in this way,
so it’s important to know how to make them. In this virtual lab, you will make a buffer and test
buffering capacity.
Buffer strength is usually reported as a molarity and a pH (e.g., a 1 M tris buffer at pH 7.8). The
concentration is determined by the total amount of the named ion in solution (so, in the example,
there is 1 mole of tris per liter of solution but some of the mole is in the acid form and some in the
base form) and the ratio of weak acid to conjugate base determines the pH. The HendersonHasselbalch equation can be used to predict the pH if you know the ratio of weak acid to conjugate
base, and the equation can be more usefully applied to determine the proper acid/base ratio to
generate a particular pH.
For this module, you will complete the following virtual activities:
Walk though activity on buffer capacity:
Virtual Lab: http://chemcollective.org/vlab/104
Planning questions
1. What equation will you use to carry out your calculations? Is there additional information
needed to solve the problem?
2. What equipment or materials do you need to properly prepare your buffers?
Regarding buffer capacity, refer to the data below and answer the questions:
Consider three buffers:
0.025 M acetate buffer at pH 4.76 (Buffer 1);
0.125 M acetate buffer at pH 4.76 (Buffer 2);
0.025 M acetate buffer at pH 4.25 (Buffer 3).
Predicting likely outcomes: Predict how well each buffer will resist changes in pH when 1 mL of 0.5 M
HCl or 1 mL 0.5 M NaOH is added to each and write an appropriate hypothesis. You may treat Buffer
1 as the baseline buffer and make your predictions about the other two as they will compare to that
The following four tests were performed on the three buffers above and the data is below. Analyze
this data to answer the post-lab questions.
Find the pH of each buffer after the addition of 1 mL of 0.5 M NaOH
Find the pH of each buffer after the addition of 1 mL of 0.5 M HCl
Find the number of mL of 0.5 M NaOH it takes to move each buffer to a pH of 5.76
Find the number of mL of 0.5 M HCl it takes to move each buffer to a pH of 3.76.
Table 2. Trials 1 and 2 For Buffer Solutions (Addition of 1mL of HCl and NaOH)
Trial 1 Buffer 1
Trial 1 Buffer 2
Trial 1 Buffer 3
.5M NaOH
.5M HCl
Table 1: Trials 1 and 2 for Buffer Solutions
Table 3. Trial 3 For Buffer Solutions
.5M NaOH
Trial 1 Buffer 1
Trial 1 Buffer 2
Trial 1 Buffer 3
100 μL pH 4.91
200 μL pH 4.74
1mL pH 5.25
100 μL pH. 4.94
200 μL pH 4.82
1.3mL pH 5.98
100 μL pH 5
200 μL pH 4.97
100 μL pH 5.04
200 μL pH 4.93
100 μL pH 5.08
200 μL pH 4.96
100 μL pH 5.12
200 μL pH 5.01
100 μL pH 5.14
200 μL pH 5.06
100 μL pH 5.21
200 μL pH 5.17
100 μL pH 5.28
200 μL pH 5.29
100 μL pH 5.34
200 μL pH 5.43
100 μL pH 5.40
200 μL pH 5.51
100 μL pH 5.46
200 μL pH 5.56
100 μL pH 5.54
200 μL pH 5.61
100 μL pH 5.62
200 μL pH 5.66
100 μL pH 5.72
200 μL pH 5.72
50 μL pH 5.77
200 μL pH 5.74
200 μL pH 5.76
Table 2: Trial 3 for Buffer Solutions (Addition of .5M NaOH)
Table 4. Trial 4 For Buffer Solutions
.5M HCl
Trial 1 Buffer 1
Trial 1 Buffer 2
Trial 1 Buffer 3
100 μL pH 4.54
200 μL pH 4.54
20 μL pH 4.04
100 μL pH 4.62
200 μL pH 4.46
20 μL pH 3.97
100 μL pH 4.65
200 μL pH 4.41
20 μL pH 3.93
100 μL pH 4.60
200 μL pH 4.36
20 μL pH 3.88
100 μL pH 4.57
200 μL pH 4.31
20 μL pH 3.85
100 μL pH 4.50
200 μL pH 4.28
20 μL pH 3.78
100 μL pH 4.44
200 μL pH 4.23
20 μL pH 3.73
100 μL pH 4.38
200 μL pH 4.15
100 μL pH 4.33
200 μL pH 4.12
100 μL pH 4.29
200 μL pH 4.09
100 μL pH 4.24
200 μL pH 4.03
100 μL pH 4.19
200 μL pH 3.97
100 μL pH 4.15
200 μL pH 3.92
100 μL pH 4.09
200 μL pH 3.87
100 μL pH 4.01
200 μL pH 3.83
100 μL pH 3.95
200 μL pH 3.80
100 μL pH 3.87
50 μL pH 3.82
50 μL pH 3.77
Table 3: Trial 4 for Buffer Solutions (Addition of .5M HCl)
Post-lab Questions:
1. Write a summary of the purpose of this lab and describe the most important thing you learned.
2. Compare the isoelectric point value your group calculated for your amino acid titration with the
value listed in your textbook or from a trusted source. How does it compare? If they are not
the same, propose an explanation.
3. Explain the shape of your amino acid titration curve. If there are plateaus, describe the
biochemistry that is occurring. If there are regions of significant slope, describe why pH is
increasing so rapidly.
4. From the pKa values you obtained in your amino acid titration, calculate the equilibrium
constants (Ka) for the carboxylic acid and the amino group.
5. Consider the amino acid lysine. Predict the shape of the titration for this amino acid based on
what you have learned.
6. Based on the virtual lab activity you completed, what concentrations of weak acid and
conjugate base did you use to create your buffer?
7. Based on the results above, what is true about a buffer that has the same pH as another
buffer, but a higher molarity?
8. What experimental evidence can you provide that a buffer solution is most able to maintain its
pH despite addition of acid OR base when it is at its pKa?
9. Take this one step further: What additional questions surfaced during this investigation? What
gaps in your knowledge are left unfilled? How could you extend or alter this experiment to
answer those questions?

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