TITRATION OF

AMINO ACIDS
Hapan | Hernandez M | Hilario | Icaro | Jacinto | Janier
C1-2019

INTRODUCTION

TITRATION

Etymology: Greek Titulus (Title);

French Titre (Rank)
Used to determine the unknown
concentration of a known reactant.
Process of analyzing composition by
measuring the volume of one solution
needed to completely react with another
solution

DEFINITION OF TERMS

Titrant
standardized substance reacted with analyte to determine
analyte concentration
Analyte
substance being analyzed
Indicator
used to mark end point ; dye or pH meter
Equivalence Point
point when amount of added standard reagent is exactly
equivalent to amount of analyte
End point
point in titration when physical change associated with
condition of chemical equivalence occurs

TYPES OF TITRATION

Acid-Base

neutralization

Complexometric / Chelatometric
volumetric analysis
colored complex as endpoint

Oxidation-reduction
redox reactions

Precipitation

ionic compounds of limited solubility

silver nitrate

TITRATION CURVE

A plot of pH vs. the amount of titrant

added
Typically the titrant is a strong
(completely) dissociated acid or base
Such curves are useful for determining
endpoints and dissociation constants of
weak acids or bases.

ACID-BASE TITRATION

neutralization reaction
acid/base of concentration
(titrant) reacts with acid/base of
unknown concentration (analyte)
Analyte + Titrant Products

IONIC PROPERTIES OF AMINO ACID

Components:

amine group
carboxyl group
R group

in aqueous solution

side chain which ionize depending on the pH

can behave as acid & base

IONIC PROPERTIES OF AMINO ACID

Isoelectric Point (pI)

pH at which net charge on a molecule is zero

(zwitterions)
average of two pK values
pK = pH

TITRATION OF AMINO ACIDS

When an amino acid is dissolved in water it

exists predominantly in the isoelectric form.
Upon titration with acid: acts as a base
Upon titration with base: acts as an acid
Amphoteric compound can act as either an
acid or a base is known as an

GLYCINE

smallest of the amino acids

Ambivalent can be inside or outside of
the protein molecule
Exist predominantly as the zwitterion in
aqueous solution at or near neutral pH
Chemical property: Aliphatic
Physical property: Non-polar

LYSINE

Essential amino acid; has a positively

charged -amino group
Lysine is basically alanine with a propylamine
substituent on the carbon.
-amino group has a significantly higher
pKa (about 10.5 in polypeptides) than does the
-amino group.
Chemical property: Basic
Physical property: Polar
(positively charged)

ASPARTIC ACID

Alanine with one of the hydrogens replaced

by a carboxylic acid group
pKa of the carboxyl group of aspartic acid in
a polypeptide is about 4.0
Has -keto homolog (oxaloacetate)
Chemical property: Acidic
Physical property: Polar (charged)

OBJECTIVES
To determine the acid base behavior of the amino
acid during titration with an alkali and acid
To determine the effect of formaldehyde on the
titration curve of the amino acid
To plot the titration curve using the pH values
obtained from the experiment

Materials/Reagents Needed

0.1 N NaOH
0.1N HCl
0.1M glycine solution
0.1M lysine solution
0.1M aspartic acid solution
Neutralized formaldehyde

Materials/Reagents Needed

0.1 N NaOH

0.1M glycine solution

Materials/Reagents Needed

0.1 N Aspartic acid

0.1M lysine solution

Materials/Reagents Needed

0.1 N Aspartic acid

0.1M lysine solution

Materials/Reagents Needed

Neutralized Formaldehyde
0.1N HCl
Distilled water

PROCEDURE

PROCEDURE
0.1N HCl

10 mL of the amino
acid solution

Measure the
resulting pH

Titrate with Hcl or

NaOH, adding 2.0 mL
at a time

Determine pH after
each addition until
10 mL or 20 mL is
reached

Plot the pH vs. the

equivalent acid/base

One mL of
acid/base=0.1 mEq
of acid/base

Repeat, but add first

5.0 mL of neutralized
formaldehyde to the
amino acid solution

Plot on the same

graph and solve for Pi
and pK values of the

0.1N NaOH

Results and Discussion

(GLYCINE)

mL
0
2
4
5
6
8

10

w/o HCHO

Glycine

In 1 M HCl

In 1 M NaOH

2.93

6.23

4.77
2.53
2.39
2.28
2.11
1.96

4.88
6.69
6.88
7.12
7.53

8.19

w/ HCHO

In 1 M HCl

In 1 M NaOH

6.01

6.36

2.55

9.61

2.96
2.40
2.29
2.10

1.96

mEq
0

9.18

.2

9.74

.5

9.88

10.17

10.51

.4
.6
.8
1

Glycine

12

pK2 9.85

10

pH

pI 6.12

pK1 2.37

4
2
0

w/ HCHO

pK2 7.11

pI 4.74

w/o HCHO

pK1 2.38
1

0.8

0.6

0.5

0.4

0.2

mEq

0.2

0.4

0.5

0.6

0.8

Results and Discussion

(ASPARTIC ACID)

Aspartic acid (HCl)

Aspartic acid (NaOH)

4.64

4.59

4.67

4.62

4.07

3.70

9.27

7.88

mL
2
5
6
8

10
12
14
15
16
18
20

w/o HCHO w/ HCHO

4.30
3.97
3.88
3.71
3.53
3.36
3.18
3.08
2.99
2.83
2.67

4.07
3.53
3.36
3.00
2.68
2.46
2.29
2.23
2.17
2.08
2.00

w/o HCHO w/HCHO

mEq

8.62

.2

9.46
9.63
9.95

10.35
11.07
11.40
11.44
11.53
11.67
11.74

7.21
8.10
8.36
8.70
9.23

10.15
10.67
10.80
10.90
11.05
11.16

.4
.5
.6
.8
1

1.2
1.4
1.5
1.6
1.8
2

Aspartic acid

14

12

10

1.8

1.6

1.5

1.4

1.2

0.8

0.6

0.5

0.4

0.2

w/o HCHO

0.2

0.4

w/ HCHO

0.5

0.6

0.8

1.2

1.4

1.5

1.6

1.8

Results and Discussion

(Lysine)

0.1 N HCl (mL)

0
2
4
5
6
8
10
12
14
15
16
18
20

mEq
0
0.2
0.4
0.5
0.6
0.8
1
1.2
1.4
1.5
1.6
1.8
2

pH
pH
0.1 N NaOH (mL)
w/o HCHO w/ HCHO
w/o HCHO w/ HCHO
9.98
6.52
0
9.41
6.49
8.89
6.37
2
9.73
6.92
8.41
6.23
4
10.04
7.42
8.1
6.13
5
10.18
7.75
7.75
5.98
6
10.32
8.14
3.93
4.01
8
10.61
8.96
3.15
3.18
10
10.93
9.59
2.89
2.94
12
11.2
10.16
2.72
2.77
14
11.4
10.6
2.66
2.71
15
11.47
10.73
2.61
2.66
16
11.53
10.81
2.53
2.58
18
11.61
10.96
2.48
2.51
20
11.67
11.06

Lysine

14

12

10

pH

4
w/o HCHO (Acid)

Milliequivalence (mEq)

w/ HCHO (Acid)

w/o HCHO (Base)

10
w/ HCHO (Base)

11

12

13

Titration of Lysine

14
12

pK3=10.40
pK2=8.80

10

pH

8
pK1=6.5

6
pK2=6.25

pK1=2.75

4
2
0

pK1=2.74
2

1.8 1.6 1.5 1.4 1.2

Milliequivalence (mEq)
0.8 0.6 0.5 0.4 0.2 0
0 0.2 0.4 0.5 0.6 0.8

Milliequivalence of H+(mEq)

Lysine without HCHO

Lysine with HCHO

1.2 1.4 1.5 1.6 1.8

Milliequivalence of OH- (mEq)

Guide Questions

What can account for Sorensens discovery that the

endpoint of titration between an amino acid and a
standard alkali is not reached?

The free amino group at the alpha-carbon acts

as a base and interferes with the end point of
the titration using a standard alkali.
Formaldehyde in excess is needed to modify the
basic free amino group to modify it to a neutral
group, a dimethylol derivative, which allows for
the endpoint to be reached.

Compare the values obtained when the amino acid was

titrated with HCl both in the absence and presence of
formaldeyde. How do you account for this?

- In the presence of formaldehyde, titration

curve is slightly lower
- This is due to the fact that formaldehyde
forms the dimethlol and monomethylol group
increasing the basicity

At which pH will an amino acid exert its maximum

buffering capacity? Why? Where in your graph is the
buffering region for your amino acid?

- Maximum buffering capacity

(pink) when pH = pKa +/- 1
- Nearly equal amounts of proton
donors and acceptors
- Least buffering capacity at the pI
because both the amino and
carboxyl group are protonated
and deprotonated
- Therefore any minute addition
of H+ or OH- will result in a large
change in pH.

From the titration curve of an amino

acid, can you determine the nature of
its R group, i.e., basic, acidic or
neutral?