3.2 Determination of Amino Acids Using Formal Titration.

PROCEDURE : Refer to FAR 142 Pharmacology and Basic Biochemistry Practical Manual
RESULTS:
Molarity of standardized NaOH : 0.1M
Initial Reading (mL)
Final Reading (mL)
Total Volume (mL)

I
0.00
25.00
25.00

II
0.00
24.00
24.00

Average titer value : 24.50mL

CALCULATION
1) Concentration of amino nitrogen
1 mole of amino acid reacts with 1 mole of formaldehyde.
1 mole of NaOH reacts with 1 mole of H+ produced by 1 mole of formaldehyde.
M1 = Molarity of amino acid

M2 = Molarity of NaOH

V1 = Volume of amino acid

V2 = Volume of NaOH

M 1V 1 = M 2V 2
M1 25 = 0.1 24.50
M1 = 0.1 24.50
25
=0.098M
Atomic weight of N = 14.0
Concentration of amino nitrogen
= 0.098 14.0
= 1.372 g/L
2) Concentration of amino acid

Molecular weight of glycine = 75.00g/mol

Molarity of glycine

= Molarity of amino nitrogen

= 0.098 M

Concentration of glycine = 0.098 75.00

= 7.35 g/L
DISCUSSION:
Concentration of an amino acid can be estimated by using formal titration method. A
direct titration method cannot be used because the alkali added will neutralize the hydrogen ions
from both the protonated amino group and the carboxyl group. Amino acid exists in zwitterionic
form at its isoelectric point, where the H+ ion formed from the ionization of COOH(carboxyl
group) is taken up by NH2(amino group). It will then be represented as NH 3+. This dipolar
character of amino acid makes it difficult to titrate amino group quantitatively. However, in the
presence of a large excess formaldehyde, amino acid can be titrated to a phenolphthalein end
point. When amino acid reacts with formaldehyde, -NH 2 (amino group) in the amino acid will
react with formaldehyde to form methylol and dimethylol derivatives, which in turn will prevent
the amino group from participating in the neutralization reaction of the amino acid with alkali.
Before adding to amino acid, formaldehyde needs to be neutralized first as it may contain
small amounts of free acid from the oxidation of formaldehyde. The amino acids needs to be
reacted with sodium hydroxide solution first. This is to convert the amino acid to its anionic
form, NH2CHRCOO-.
When the formaldehyde has reacted completely with the amino group in the amino acid,
the amino acid can be titrated directly with an NaOH to an phenolphthalein end point. Only the
carboxyl group in the amino acid will react with the alkali, therefore allowing us to determine the
concentration of the amino acid used in the experiment. The second chemical reaction is, the
proton that release through the chemical reaction above is neutralized by 0.1M NaOH during
titration procedure is done.
+

NaOH

Na

H 2O

Based on this experiment, 1 mol of amino acid (glysine) produce 1 mol H + and 1 mol of H+ react
with 1 mol of NaOH.
The volume of NaOH used for this titration is 24.50ml. By using formula M1V1 =
M2V2, the concentration of amino nitrogen is 1.372 g/L and the molarity of glycine used is
0.098mol/L. The concentration of glycine used in g/L is 7.35 g/L.
Theoretical concentration of amino acid is 7.50g/L (0.1mol/L x 75.0 g/mol). The
difference is caused by some errors in the experiment. This may be caused by excess
neutralization of formaldehyde and amino acid by NaOH. Besides, excess of phenolphthalein
which is used as indicator during titration will reduce the volume of NaOH, resulting lesser as
compared with the actual value. Besides, excess of phenolphthalein which is used as indicator
during titration may alter the pH of the initial solution. It also can be caused by the error of
different perception of the end point, which is the pink colour produced during the titration.

3.3 TITRATION OF GLYCINE IN THE PRESENCE AND

ABSENCE OF FORMALDEHYDE
Volume(mL)
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4

pH (Without formaldehyde)
3.20
3.00
2.88
2.72
2.65
2.58
2.55
2.50
2.45
2.40
2.31
2.20

pH (With Formaldehyde)
-

PROCEDURE : Refer to FAR 142 Pharmacology and Basic Biochemistry Practical Manual Pg
7

a) After adding 0.5M HCL(ml)

b) After adding 0.5M NaOH(ml)

Volume(mL)

pH (Without formaldehyde)

pH (With Formaldehyde)

0.2

7.89

5.64

0.4

8.65

6.12

0.6

9.05

6.34

0.8

9.12

6.42

1.0

9.23

6.69

1.2

9.30

6.94

Glycine
1.4
1.6
pK1
1.8pK
2

Without9.50
Formaldehyde
9.67
2.65
9.80
9.23

With Formaldehyde
7.30
7.74
2.65
8.50
6.69

2.0
pI
2.2

10.28
5.94
10.80

10.17
4.67
10.50

2.4

11.17

11.00

CALCULATION:

Ma = Concentration of HCl

Va = Volume of HCl

Mb = Concentration of NaOH

Vb = Volume of NaOH

Mc = Concentration of glycine

Vc = Volume of glycine

MaVa = McVc

MbVb = McVc

(0.5)(Va) = (0.1)(10)

(0.5)(Vb) = (0.1)(10)

Va = 0.1 10

Vb = 0.1 10

0.5
= 2mL

0.5
= 2mL

Refer to the graph 3.3 on how pK1 and pK2 was obtained.

Therefore, Titration with Formaldehyde

Therefore, Titration Without Formaldehyde

Will give isoelectric point of;

Will give isoelectric point of;

pI = (pK1 + pK2)

pI = (pK1 + pK2)

= (2.65 + 6.69 )

= (2.65+ 9.23)

= 4.67

= 5.94

DISCUSSION:
Glycine exists as zwitterions which can either act as an acid (proton donor) or act as a
base (proton acceptor) at isoelectric point. In the presence of acid, the proton reacts with the
ionized carboxyl group of glycine, leaving the glycine positively charged. In contrast, in the
presence of alkali, the hydroxyl group reacts with the ionized amino group, leaving the glycine
become negatively charged.
Titration curve indicate that an amino acid has two dissociation steps, corresponding to
the dissociable group indicated by pK1 and pK2. Both dissociable group are fully but oppositely
charged.
The formula used to calculate pI is:
pI =

1
2 ( pK1 + pK2)

where pK is the ionization constant.

The titration of glycine with NaOH is a two-stage titration represented by the reactions
below.
NH3CH(R)COOH + OH- +NH3CH(R)COO- + H2O

NH3CH(R)COO- + OH- NH2CH(R)COO- + H2O

The same principle applies to the determination of pKa1.

NH2CH(R)COO- + H++NH3CH(R)COO- + H2O
NH3CH(R)COO- + H++NH3CH(R)COOH+ H2O

In this experiment, the titration curve for glycine with NaOH in the presence of
formaldehyde is lower than that without formaldehyde. This is due to the formaldehyde ties
down the amino groups, causing the carboxyl hydrogen ion more available.
The exact volume of HCl required to titrate 10ml of 0.1M glycerine is 2ml while the
exact volume of NaOH required to titrate 10ml of 0.1M glycerine is 2ml.
Based on the titration curve of glycine with HCl and NaOH in the absence of
formaldehyde, the pK1 and pK2 are 2.65 and 9.23 respectively. At these points, the cations
(+NH3CH2COOH) and anions (NH2CH2COO-) exist in equilibrium with the zwitterions where the
volume of HCl and NaOH added is 1ml respectively.
Hence,
pH = pK1 where [+NH3CH2COOH]
[+NH3CH2COO-]

= 1 ; pH = pK2 where [NH2CH2COO-]

=1

[+NH3CH2COO-]

. Based on the calculation shown, the pKa2 values can be obtained when the volume of
NaOH added is 1ml. By looking to the graph, the pKa2 value for the titration of glycine in
absence of formaldehyde (pKa2 =9.23) is higher than the titration of glycine with formaldehyde
(pKa2 =6.69). This is because, in the presence of formaldehyde, the amino acid loses its proton

from the amino group and will cause the increase of hydrogen ions in the solution. Hence the pH
become lower than the normal pH. Formaldehyde will not affect the pK1 value but will lower the
pK2 value and thus, affect the pI value.
Formaldehyde will react with uncharged amino group that will form
monohydroxylmethyl and dihydroxylmethyl derivatives. These two compound, also known as
methylol derivatives, are actually are secondary and tertiary amines which are weaker base or
stronger acids. Because of that, the pKa2 of titration glycine with formaldehyde gives a lower pH
value.
From the graph, the isoelectric point calculated for the titration of glycine in absence and
presence of formaldehyde is 5.94 and 4.67 respectively. However, the theoretical pI for glycine is
5.97. The difference between these pI was due to several mistakes that happened during
experiment. The electrodes of pH meter was not washed and rinsed with distilled water
thoroughly before and after the reading is taken. The remains of the previous solution at the pH
meter detector will alter the readings obtained. Bubbles were found inside the microburette and
glycine was not mixed well with HCl and NaOH during titration .All these mistakes can cause
the deviation of the experimental isoelectric point of glycine from the theoretical isoelectric point
of glycine. This experiment is not repeated with HCl because formaldehyde will react with the
terminal amino group of glycine to form an amino-methylol derivative. [4] The COO- end is not
affected whereas less free amino groups are available for deprotonating. The tendency of
deprotonation is reduces as well. The formaldehyde will also compete with OH- ions to react with
amino acid.

CONCLUSION:
1

The isoelectric point of casein is at

The concentration of amino nitrogen is 1.372g/L

The concentration of amino acid is 7.36g/L

The isoelectric point for glycine without formaldehyde is 5.94

The isoelectric point for glycine in the presence of formaldehyde is 4.67.

The reaction between alkali and glycine is displaced to lower values of pH in the
presence of formaldehyde.

PRECAUTION:
1. The electrode of pH meter must be rinsed and washed with distilled water so that no residue
left on detector that will cause the readings to be not accurate.
2. When measuring HCl and NaOH by using microburette, air bubbles should not be trapped in
it.
3. Solution should be shaked gently to allow even distribution of HCl and NaOH in glycine
solution.
4. Parallax error should be avoided when measuring solutions.

REFERENCES:
1
2
3
4

Reference Guide for Amino Acids [Internet] Sept 2011 [cited 2011 Dec 2]. Available
from http://www.realtime.net/anr/aminoacd.html.
What is Casein? [Internet] 2011 [cited 2011 Dec 2]. Available from
http://www.wisegeek.com/what-is-casein.htm
http://www.cliffsnotes.com/study_guide/Amino-Acids.topicArticleId-24998,articleId24958.html
FAR 142 Pharmacology and Basic Biochemistry Practical Manual