SCHOOL OF BIOLOGICAL SCIENCES

UNIVERSITI SAINS MALAYSIA

BOI 206/4
PRINCIPLES OF BIOCHEMISTRY

EXPERIMENT 3
AMINO ACID TITRATION

NAME: NORHIDAYATI BINTI MD GHAZALI

MATRIC NUMBER: 158767
OBJECTIVES
1. Prepare buffer solution and determine its pKa value.
2. Carry out acid-base titration for glycine.
3. Explain that an amino acid consist at least carboxyl group and amino group.
4. Determine pKa1 and pKa2 for glycine and pKa of acetic acid
5. Calculate pI value of glycine.

INTRODUCTION

A titration with an acid and a base is a method of quantitative analysis that is used to
determine the concentration of an acid or base by precisely neutralising it with a standard
solution of base or acid whose concentration is already known. This approach is also known
as an acid–base titration. In order to keep track of how far along the acid–base interaction has
progressed, a pH indicator is used. It is possible to determine the concentration of the analyte
solution by knowing either the acid dissociation constant (pKa) of the acid or the base
dissociation constant (pKb) of the base in the solution. Constructing a titration curve is
another alternative method that may be used to calculate the pKa of an analyte solution if the
concentration of the solution is already known.

MATERIALS

• Three solutions of standard buffer pH 4, pH 7 and pH 9.2.

• 0.1 M acetic acid.
• 0.1 M natrium acetate.
• 0.1 M NaOH.
• 0.1 M HCl.
• 0.1 M glycine solution.
• Biuret reagent.
• 50 ml beaker.
• 10 ml pipette.
• pH meter.
METHODS
A. Preparation of Buffer Solution

1. Seven solutions which contain the following content in the table below was
prepared by using 0.1 M acetic acid and 0.1 M natrium acetate that was given.

Solution 1 2 3 4 5 6 7
Distilled
Water 19 18 15 10 15 18 19
(ml)
Acetic 10 10 10 10 5 2 1
acid (ml)
Natrium 1 2 5 10 10 10 10
acetate
(ml)

2. The pH of each solution was determined and the pK a value for buffer solution was
calculated.

B(1). Determination of pKa1 and pKa2 for Glycine and pKa of Acetic Acid.

1. 20 ml of 0.1 M glycine solution was added to the beaker.

2. pH electrode was placed into the beaker and the pH value of the solution was
recorded.
3. 0.5 ml (each time) of 0.1 M HCL was added until 2 ml.
4. Then, 1 ml (each time) was added until 20 ml.
5. When HCL was added, the mixture was mixed well and the pH of solution was
recorded.
6. The step was repeated until a total of 25 ml 0.1 M HCL is added.
7. Steps 1-6 was repeated using 0.1 M NAOH to titrate with 20 ml of 0.1 M and new
glycine.
8. The pH for each titration was determined until 20 ml of 0.1 M NAOH was added.
9. The titration graph of glycine with HCL and NAOH was plotted on graph paper and
the pKa1, pKa2, pl value of glycine was determined.

B(2): Determination of pKa1 of acetic acid.

1. 25 ml of 0.1 M acetic acid was added into the beaker.

2. The magnetic stirrer was inserted and the beaker was placed on the stirrer.
3. The pH electrode was placed into the beaker and was stirred then the pH value was
recorded.
4. 2 ml of 0.1 M NAOH was added and the pH value of solution was recorded.
 5. The steps was repeated until 24 ml of 0.1 M NAOH is added.
6. The graph was plotted as in B(1) for the titration of acetic acid and NAOH.

RESULTS:

TABLE:
(A): Preparation of Buffer Solution

Solution 1 2 3 4 5 6 7
Distilled
Water (ml) 19 18 15 10 15 18 19
Acetic acid 10 10 10 10 5 2 1
(ml)
Natrium 1 2 5 10 10 10 10
acetate (ml)
Concentration
of conjugate 0.0033 0.0067 0.0167 0.0333 0.0333 0.0333 0.0333
base
Concentration 0.0333 0.0333 0.0333 0.0333 0.0167 0.0067 0.0033
of weak acid
pH 3.91 4.16 4.26 4.38 4.64 5.44 5.83
pKa formula: 3.91 -log10 4.16 -log10 4.26 -log10 4.33 -log10 4.64 -log10 5.44 -log10 5.83 -log10
pH-log10¿ ¿ [ 0.0033 ] [ 0.0067 ] [ 0.0167 ] [ 0.0333 ] [ 0.0333 ] [ 0.0333 ] [ 0.0333 ]
[ 0.0333 ] [ 0.0333 ] [ 0.0333 ] [ 0.0333 ] [ 0.0167 ] [ 0.0067 ] [ 0.0033 ]

=3.91+1.00 =4.16+0.70 =4.26+0.30 =4.38+0 =4.64-0.30 =5.44-0.70 =5.83-1.00

=4.91 =4.86 =4.56 =4.38 =4.34 =4.74 =4.82

pKa 4.91 4.86 4.56 4.38 4.34 4.74 4.82

B(1)
0.1 M HCL (ml) pH 0.1 M NAOH (ml) pH
0.0 7.55 0.0 6.00
0.5 4.17 0.5 6.56
1.0 3.74 1.0 6.82
1.5 3.61 1.5 6.85
2.0 3.33 2.0 6.92
3.0 3.00 3.0 7.20
4.0 2.82 4.0 7.35
5.0 2.67 5.0 7.70
 6.0 2.56 6.0 7.80
7.0 2.41 7.0 8.20
8.0 2.34 8.0 8.44
9.0 2.32 9.0 9.59
10.0 2.30 10.0 9.62
11.0 2.16 11.0 9.80
12.0 1.98 12.0 9.90
13.0 1.97 13.0 9.95
14.0 1.95 14.0 9.99
15.0 1.89 15.0 10.20
16.0 1.85 16.0 10.34
17.0 1.83 17.0 10.38
18.0 1.81 18.0 10.45
19.0 1.78 19.0 10.72
20.0 1.71 20.0 11.20

pl value of glycine: pKa1 + pKa2 = 2.16 + 9.62 =5.89

2 2

B(2) :

0.1 M NAOH pH
0.0 3.00
2.0 3.09
4.0 3.96
6.0 4.35
8.0 4.60
10.0 4.65
12.0 4.86
14.0 4.91
16.0 5.04
18.0 5.45
20.0 6.02
22.0 11.12
24.0 11.64
DISCUSSION

1. State the glycine species at pKa1 and pKa2.

- Glycine species at pKa1 is acidic while pKa2 the glycine is basic.

2. Why a pH value is equal to pKa value in the middle of titration?

- At the half-equivalence point, the acid is half neutralized, hence produced salt is half in
amount than that of acid making concentration of salt and acid are same ([A−]=[HA]).This
solution now becomes a buffer solution and applying Henderson-Hasselbalch equation, the
log value becomes zero. So, pH=pKa at half-equivalence point.

3. State the functional group (s) at pKa1 and pKa2.

-Functional group at pKa1 is α-carboxyl (-COOH) while for pKa2 is α-amino (-NH3)

4. If aspartic acid is used, determine how many pKa values will be obtained and give
reasons.
-Aspartic acid will have three pKa values: pKa1 for the carboxyl group, pKa2 for the amino
group, and pKa3 for the side chain group. This is due to the fact that aspartic acid, an amino
acid that is negatively charged and has an acidic side chain that is at neutral pH and consists
of carboxylic acid groups (CH2COOH), will react with other amino acids, enzymes, and
proteins.

5. Compare the pKa of glycine and pKa of acetic acid and state which compound is a
strong acid or weak acid. Explain why.

- The value of pKa1 of glycine is 2.30, pKa2 of glycine is 9.62, and pKa of acetic acid is 4.65.
Thus, pKa2 of glycine is weak acid while pKa 1 of glycine is a strong acid. For the pKa of
glycine and pKa of acetic acid, pKa 2 of glycine is a weak acid, and pKa of acetic acid is a
strong acid. While pKa1 of glycine is strong acid and pKa of acetic acid is a weak acid.
CONCLUSION:

After this experiment was finished, the goal of creating a buffer solution utilising
distilled water and two solutions—0.1 M acetic acid and 0.1 M natrium acetate—was
accomplished. Additionally, the formula pKa = pH - log [salt]/[acid] allowed for the
determination of the pKa value. Then, by titrating glycine with HCl and NaOH, acid base
titration for glycine was effectively completed. The fact that amino acids are organic
molecules consisting of a basic amino group, an acidic carboxyl group, and a side chain
group allows us to understand that they at least contain a carboxyl group and an amino group.
Next, the pKa value was able to determine which the value of pKa1 is 2.3o, pKa2 is 9.62, and
pKa acetic acid is 4.65. Lastly, pI value of glycine was able to calculate using formula pI =
(pKa1 + pKa2) ÷ 2 which value is 5.89.

REFERENCE:

Chemistry. (2020, January 6). Why a pH value is equal to pKa value in the middle of titration? –
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equal-to-pka-value-in-the-middle-of-titration/

Den. (2015, October 19). Titration Curve of Glycine : The zwitter ionic changes. Biochemistry Den.
Retrieved December 25, 2022, from https://biochemden.com/titration-curve-of-glycine/

Kristen. (2022, October 25). Should I Be Taking a Glycine Supplement? Verywell Health. Retrieved
December 25, 2022, from https://www.verywellhealth.com/glycine-overview-4583816

Myhre. (2022, October 25). Should I Be Taking a Glycine Supplement? Verywell Health. Retrieved
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Redlinska. (2018, January 24). Determination of the pKas for Glycine. Determination of the pKas for
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