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Lab 3: Acid-Base Properties of

Amino Acids
Titration of Glycine

Justine Chirichella
Lab Partner: Jarin Tasnim

Purpose
The purpose of this lab is to use standard titration methodology to calculate the pKa
values of the amino group (-NH3) and the carboxylic acid group (-COOH) as well as the pI value
(isoelectric point) of the amino acid glycine. These values will be determined using the generated
titration curve.

Introduction
Amino acids are the building blocks of proteins. There are 20 amino acids in total, 9 of
which are considered essential because humans must obtain them from food. The remaining 11
are considered inessential and are synthesized in vivo. All amino acids have the same general
structure:

H3N

O-

R
In this general scheme, the first carbon is the alpha carbon, the + H3N represents an amino
group, the COO- portion represents a carboxylic acidic group, and the R group is the functional
group which differentiates each amino acid from each other. No two amino acids share the same
R group, and this experiment analyzes the effect of changing pH on the amino acid glycine. In
glycine, the R group is represented by a single hydrogen atom, making it the simplest amino acid
structurally.
In the above amino acid structure, it is critical to note that this molecule is in zwitterion
form. Zwitterion form refers to a charged molecule (contains both negative and positive charges)
which exists as neutral because the charges on both groups cancel each other out. This occurs
most commonly around a neutral pH value above the pKa of the carboxylic acid group, but
below the pKa of the amino group. This point, the pH at which a molecule has no net electrical
charge, is called the isoelectric point (pI).
Furthermore, amino acids are also examples of amphoteric molecules. An amphoteric
species can serve as both an acid (proton donor) and a base (proton acceptor). The ionizable
amino and carboxylic groups on all amino acids provide these molecules with their acid-base
properties.
The mechanism for deriving the pKa1, pKa2, and pI values of glycine is to construct a
titration curve. A titration curve is a type of plot which measures pH vs. mL of added base. By
adding base to a glycine solution, the pH will change considerably and follow a specific pattern
dictated by the acid-base properties of the amino acid. The curve will consist of two legs (areas

where the trendline flattens) where pH does not change much with added base. The area where
pH changes dramatically indicates that the isoelectric point is being reached. Titration curves can
be incredibly useful in determining pKa1, pKa2, and pI values because they act as a visual aid of
acid-base activity.
As would be expected from general acid-base chemistry, the behaviors of the amino and
carboxylic groups are very important in titration studies. Regarding the carboxylic group, at pH
values below the pKa protonation occurs (-COOH) giving a neutral charge and at pH values
above the pKa deprotonation (-COO-) occurs and it remains negatively charged. Regarding the
amino group, at pH values below the pKa protonation occurs (+ H3N) remains positively charged
whereas at pH values above the pKa, deprotonation occurs (H2N) and it is neutral. At pH values
near the pKa, both protonated and deprotonated forms exist and this region is most clearly
illustrated by the titration curve. pI will be generated using the lowest basic and highest acidic
values provided by the titration curve. In this experiment, pKa1 will refer to the point at which
the acidic side chain (carboxylic group) becomes deprotonated whereas pKa2 will refer to the
deprotonation of the basic side chain (amino group).

Methods
In this experiment, a PASCO Spark unit with a pH electrode attachment was used to
measure the pH of 0.1 M-glycine solution with varying levels of 0.25 M NaOH. The pH of the
glycine solution was adjusted to 1.5 by the addition of HCl prior to titration. The pH sensor unit
was calibrated using standard buffers of pH 3 and pH 7. 0.25 M NaOH was added in increments
of 0.5 mL using a calibrated disposable pipette. A pH reading was recorded after each addition of
base until a pH of 11 was reached.

Results and Calculations


Calculating pKa1, pKa2, and pI using the titration curve
pKa1 = highest acidic value + lowest acidic value/2
pKa2 = highest basic value + lowest basic value/2
pI = lowest basic value + highest acidic value /2
pKa1 = 3.8 + 1.7/2 = 2.75
pKa2 = 11.0 + 8.1/2 = 9.55
pI = 8.1 + 3.8/2 = 5.95

Results (continued)
Table of added NaOH to 0.1 M glycine solution with pH changes
mL of 0.25 M NaOH Added
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
10
10.5
11
11.5
12

pH
1.7
1.8
1.9
1.9
2
2.2
2.3
2.4
2.6
2.7
2.9
3.2
3.8
8.1
8.7
9
9.2
9.3
9.5
9.6
9.8
10
10.2
10.5
11

Acid-Base Titration Curve of Glycine


12
10
8

pH

8.1

6
4

3.8

2
0
0

10

12

14

mL of 0.25 M NaOH added

Discussion
In conclusion, the titration of the amino acid glycine generated a predictable titration
curve. It included distinct buffer regions for the carboxylic acid and amino groups (useful in
calculating pKa1 and pKa2) as well as a clear midpoint which corresponds to the isoelectric point
(pI) of the titrated amino acid glycine. The titration curve assisted in determining the following
values: pKa1 as 2.75, pKa2 as 9.55 and pI as 5.95. The standard values for pKa1, pKa2 and pI of
glycine are 2.35, 9.78, and 5.97 respectively. The closeness of the experimental and standard
values indicates successful titration of glycine.

Discussion Questions
1) The carboxylic acid group is an acid (capable of donating a proton).
COOH ---COO- + H+
When pH < pKa ---COOH protonated form dominates
When pH > pKa --- COO- deprotonated form dominates
When pH = pKa COOH ---COO- + H+ (mixture of protonated and deprotonated
forms exist). This is the point at which conjugate acid and base pairs are equal in
concentration.
The amino group is basic (capable of accepting a proton).

NH3+ ---NH2 + H+
When pH < pKa --- NH3+ protonated form dominates
When pH > pKa --- NH2 deprotonated form dominates
When pH = pKa --- NH3+ ---NH2 + H+
(mixture of protonated and deprotonated forms exist). This is the point at which
conjugate acid and base pairs are equal in concentration.
2) The amino acid cysteines R group contains a CH group bonded to a SH (thiol) group.
Cysteines R-group is polar and neutral. Titration of cysteine does show a slightly
different titration curve than that of glycine because it contains 3 ionizable groups: the
carboxyl, amino, and thiol. Glycine, in comparison, has a nonpolar and neutral side chain
and thus only the carboxyl and amino groups are dissociable. A titration curve of cysteine
would include a total of 3 legs, with an additional pKR value.
3) The amino acid valines R group contains a CH group branch bonded to two CH3
groups. This chemical structure is similar to that of glycine and therefore would generate
a similar titration curve with only 2 legs, since only the carboxylic acid and amino groups
are ionizable.
4) pI (isoelectric point) of the basic side chain amino acids Histidine and Lysine can be
calculating a modified version of the formula: pI = (pKa1 + pKa2)/2. It is important to
note that the pKa values used here will be that of the amino group and the ionizable R
group. This is used because both histidine and lysine contain an additional positive
charge (provided by the basic side chain) and therefore the midpoint will be determined
using the two higher pKa values.
a) Histidine. pKa (-NH3) = 9.33, pKa (-COOH)= 1.80, pKa(R group)= 6.04.
pI = (9.33 +6.04) / 2 = 7.69.
b) Lysine. pKa (-NH3) = 9.06, pKa (-COOH) 2.16, pKa(R group)= 10.54.
pI = (9.06 + 10.54) / 2 = 9.8.

References

Ballantine, D.S., Hoeger, C.A., McMurry, J., and Peterson, V.E., Fundamentals of General,
Organic, and Biological Chemistry. 7th Edition. 2013. Pages 549-580.
Binyaminov, O. and Saffran, W. Laboratory Manual- Introduction to Biochemistry. Queens
College of the City University of New York. 3rd Edition. January 2015. Pages 34-42.

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