Chapter 2
DETERMINATION OF THE DEACETYLATION DEGREE OF
CHITOSAN BY MEANS OF POTENTIOMETRY AND
CONDUCTOMETRY TITRATION
2.1 OBJECTIVES
1. Developing an understanding of the principles and practical operation of conductometric and potentio-
metric titration
2. To measure the deacetylation degree of chitosan using potentiometry and conductometry titration
3. Analyze, interpret, and report the results of both experiments
4. Comparison of Experimental mean of both methods to evaluate the possible difference between these
methods
5. Data treatment and reported the obtained experimental data in a publication style report.
2.2 OVERVIEW
Chitosan is a biodegradable biopolymer with unique biological and physicochemical properties.
Lysozymes in the body degrade it, is biocompatible and enhancer of humoral and cell-mediated immune
responses, and present immune-stimulating, suppressing tumor growth, wound-healing, mucoadhesive, anti-
fungal, and antibacterial properties, among others. Due to this wide range of favorable features and being an
inexpensive material, chitosan has become a relevant compound in several fields closely related to pharmacies,
such as cosmetics, ophthalmology, dietetics, and biomedicine. Nevertheless, despite all its valuable character-
istics, chitosan is restricted due to its poor solubility in physiological conditions. Chitosan solubility is derived
from its chemical structure and composition (Figure 2.1).
Figure 2.1: Chemical structure of (A) chitin and (B) chitosan
2.3 EXPERIMENTAL
Chitosan, poly[β-(1-4)-linked-2-amino-2-deoxy-D-glucose], is usually obtained by alkaline partial deacety-
lation reaction of chitin. Figure 1 shows the chemical structures of fully N-acetylated chitin and completely
N-deacetylated chitosan. The degree of deacetylation, DD, is the parameter that indicates the molar percent-
age of glucosamine monomeric units and varies from 0 (chitin) to 100 (fully deacetylated chitin). DD of
chitin/chitosan is the most critical parameter that influences their biological, physicochemical, and mechanical
properties and, subsequently, the effectiveness of chitosan and its derivatives. Therefore, determining the degree
of deacetylation of chitosan is essential to predict its properties and validate it for specific applications. But
how can we determine the deacetylation degree of chitosan?
U.S. Pharmacopoeia has selected the 1 H NMR high resolution as the standard method for determining the
DD in chitosan samples.[1] However, this technique is expensive and dependent on a technician, while others
are simple, inexpensive, and suitable available in most laboratories. These techniques include conductometric
titration, potentiometric titration, IR spectroscopy, UV–vis spectroscopy, and CHN elemental analysis. This
project deals with determining the deacetylation degree of chitosan using different experimental techniques
available in the lab, such as potentiometry titration and conductometry titration.[2, 3]
2.3 EXPERIMENTAL
2.3.1 Reagents
Hydrochloric acid (37%, 1.19 g mL−1 at 25◦ C), sodium hydroxide (≥98%, anhydrous, pellets), commercial
Chitosan (food grade), water type I (< 0.056 mS cm−1 ) and type II (< 1.0 mS cm−1 ).
2.3.2 Preparation of stock solutions
2.3.2.1 Secondary standard solution of sodium hydroxide (approximately 0.15 M)
Alkali hydroxides such as sodium hydroxide are NOT suitable for use as primary standards because
they absorb moisture and carbon dioxide from the atmosphere. However, you can prepare a sodium hydroxide
solution with an approximate concentration by weighing out some NaOH pellets and dissolving them in distilled
water in a volumetric flask. This solution can then be titrated with an acidic primary standard solution, such
as potassium hydrogen phthalate, KH(C8 H4 O4 ), to determine the concentration of the basic solution more
accurately. Because the accurate concentration of the base has been derived using a primary standard, the base
is referred to as a secondary standard.
Preparation of sodium hydroxide (NaOH) solution approx. 0.15 M
a Calculate the mass of NaOH needed to prepare 500 mL of a solution approx. 0.15 M. Record the mass
on your lab notebook.
b Weigh the amount needed of NaOH in a weigh boat and transfer it to a 250 mL beaker. Dissolve all the
NaOH in water by adding distilled water to the beaker. Mix and allow to cool to room temperature and
transfer to a 500 mL volumetric flask. (Remember, the dissolution process of NaOH is exothermic).
c Fill the flask with distilled water to about half. Swirl the flask to mix the contents thoroughly.
d Top up the flask with distilled water to just below the ring mark.
e Using a wash bottle or pipette, top up the remaining volume until the meniscus is precisely at the ring
mark. Important: meniscus must be read at eye level. The wall of the flask must not be wetted above the
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2.3 EXPERIMENTAL
mark.
f Close the flask and shake it upside down to mix the contents.
g Transfer the solution to an appropriate container. Label your solution with the date and the contents.
Standardization of sodium hydroxide NaOH solution
a Clean a burette by rinsing it with several portions of tap water. (The burette is clean enough when water
droplets do not cling to the inner surface.) This rinse water can be poured down the drain.
b Obtain about 75 mL of the NaOH solution in a clean, dry 100 mL beaker. Cover with a watch glass.
c Rinse the burette with three portions (about 5 mL) of the NaOH solution. Drain each NaOH rinse and
discard into the waste container located under the hood.
d Fill the burette with NaOH slightly above the zero mark and clamp the burette up vertically.
e Remove the air bubbles from the tip of the burette by draining the NaOH into a small beaker. Bring the
NaOH level to precisely the 0.00 mL mark.
f Calculate the mass of KHP (potassium acid phthalate; HKC8 H4 O4 ) needed to react with approximately
25 mL de NaOH in the titration. Record the mass on your lab notebook.
g Weigh the amount needed of KHP in a weigh boat and transfer quantitatively into a clean 250 mL
Erlenmeyer flask with the help of a wash bottle. Dissolve all the KHP in water by adding distilled water
to the Erlenmeyer (If necessary, you can warm the solution to dissolve all the solid acid.)
h Add 2 to 3 drops of phenolphthalein indicator to the KHP solution in the Erlenmeyer flask.
i Add NaOH from the burette to the flask by swirling until the color of the solution is a faint pink. This
light pink color should last only 45 to 60 seconds. There should be a one-drop difference between when
the solution is colorless and when it is pink. If too much base is added (if you "over-shoot" the endpoint),
discard the solution and repeat the titration. A white piece of paper placed under the flask will aid color
detection.
2.3.2.2 Preparation of solution of hydrochloric acid (approximately 0.10 M)
Hydrochloric acid, HCl, is NOT suitable for use as a primary standard. Although they are commercially
available as concentrated solutions that are easily diluted, the concentration of the "concentrated" solution is
NOT accurately known. This solution can then be titrated with an essential secondary standard solution, such
as sodium hydroxide, to determine the concentration of the acid solution more accurately.
a Preparation of hydrochloric acid solution
b Calculate the volume of HCl concentrated needed to prepare 250 mL of a solution approx. 0.10 M.
Record the volume on your lab notebook.
c Take the among needed HCl in a graduated cylinder and transfer it to 100 mL of water in a 250 mL beaker.
Mix and allow to cool to room temperature and transfer to a 250 mL volumetric flask. (Remember, HCl
produces toxic gases, work in a fume hood).
d Fill the flask with distilled water to about half. Twirl the flask to mix the contents thoroughly.
e Top up the flask with distilled water to just below the ring mark.
f Using a wash bottle or pipette, top up the remaining volume until the meniscus is precisely at the ring
mark. Important: meniscus must be read at eye level. The wall of the flask must not be wetted above the
mark.
g Close the flask and shake it upside down to mix the contents.
h Transfer the solution to an appropriate container. Label your solution with the date and the contents.
8
2.3 EXPERIMENTAL
Standardization of hydrochloric acid HCl solution
a Draw 25.00 mL of the acid solution into the volumetric pipette and transfer this solution into an Erlenmeyer
flask.
b Add 2-3 drops of phenolphthalein to the acid solution in the flask.
c Place the flask under the burette and add the base solution to the Erlenmeyer flask. When pink starts to
develop, add the solution more slowly. You should add one drop at a time, followed by swirling until a
very light pink color persists for at least 45-60 seconds. Remember, the softer the Pink, the better!
d Record the final reading of the burette. Wash the contents of the flask down the drain with water.
e Calculate the molarity of the HCl solution.
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2.3 EXPERIMENTAL
2.3.3 Experimental Methods
Notes on good practice 31
During chitosan titrations with NaOH solution (both potentiometric and conductometric), chitosan pre-
cipitation and viscose solutions formation are observed at pH > 5.5. Thus, heterogeneous titrations will be
performed, resulting in large standard deviations. Vigorous stirring and a long time for equilibrium must
be necessary, which means that potentiometry and conductometry techniques require a long time.
2.3.3.1 Potentiometric Titration
(a) (b)
Figure 2.2: General scheme of the experimental procedure used in the potentiometric titration
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2.4 Results
A given chitosan solution was prepared by dissolution of a known mass of chitosan ( 0.2g) in 40 mL of
a 0.10 mol L−1 HCl solution, which remained under stirring until the sample was dissolved entirely at room
temperature. This solution was then titrated with NaOH solution (approx. 0.15 mol/L), measuring pH with
a pH meter, using a general scheme of the experimental procedure shown in Figure 2.2. pH meter should be
calibrated with standard solutions before use it.
Use the tables in Appendix B to collect your experimental data in your laboratory notebook and technical
report.
2.3.3.2 Conductometric Titration
A given chitosan solution was prepared by dissolution of a known mass of chitosan ( 0.2g) in 40 mL of
a 0.10 mol L−1 HCl solution, which remained under stirring until the sample was dissolved entirely at room
temperature. After adding 100 mL of water type I, conductometric titration was carried out with NaOH solution
(approx. 0.15 mol L−1 ), measuring conductivity, using a general scheme of the experimental procedure shown
in Figure 2.3.
(a) (b)
Figure 2.3: General scheme of the experimental procedure used in the conductometric titration
Notes on good practice 32
Conductimeter should be calibrated with a KCl standard solution (1413 µS cm−1 ± 12µS cm−1 at 25◦ C,
as Potassium Chloride (KCl) (0.01M).) before using it.
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2.4 Results
2.4 Results
a Use the tables B.1 and B.2 in Appendix B to collect your experimental data in your laboratory notebook
and technical report.
b Calculate DD using equations B.4 and B.5
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Bibliography
[1] American Pharmacopoeia. The United States Pharmacopeial Convention. Vol. 36. International Journal
of Pharmaceutical Compounding, 2012, pp. 1754–1756.
[2] Leyre Pérez-Álvarez, Leire Ruiz-Rubio, and Jose Luis Vilas-Vilela. “Determining the deacetylation degree
of chitosan: Opportunities to learn instrumental techniques”. In: Journal of Chemical Education 95.6
(2018), pp. 1022–1028.
[3] ZM Dos Santos et al. “Determination of deacetylation degree of chitosan: a comparison between con-
ductometric titration and CHN elemental analysis”. In: Carbohydrate research 344.18 (2009), pp. 2591–
2595.
