Department of Pure and Applied Chemistry

College of Arts and Sciences

Visayas State University
Visca, Baybay City, Leyte

DETERMINATION OF MOLECULAR WEIGHT OF CHITOSAN USING

INTRINSIC VISCOSITY MEASUREMENT

A Scientific Report
Submitted to Prof. Jacob Glenn Jansalin
In Partial Fulfilment
of the Requirements for
Chem 151 – Physical Chemistry I Laboratory

GIZEL MARIE CUIZON

MARY JANE MANINGO GONATO

DANICA SHAHARA DUPAL ROA

THEORETICAL FRAMEWORK

Molar mass determination is characterized into primary and secondary techniques based

on whether or not standards are needed to calibrate the analytic instrument. Primary or absolute

techniques are based on colligative properties of a compound, which depend only on the number

and not on the nature of the particles of a compound. Examples of this technique include melting

point depression, vapor pressure and osmotic pressure. Secondary or relative techniques are

much simpler and more reducible with the use instrumentation such as gel permeation

chromatography (GPC) or high pressure liquid chromatography (HPLC).

Theoretically, intrinsic viscosity is a primary technique because it does not require

secondary standards in solvent conditions. It only relies on knowledge of the Mark-Houwink

coefficients for the thermal and solvent conditions of measurement. Some handbooks, such as

The Polymer Handbook, 3rd edition by Brandrup and Immergut, provide useful reference for the

values for the Mark-Houwink equation as well as a concise description of intrinsic viscosity.

To describe the relationship between intrinsic viscosity and molecular weight, the Mark-

Houwink-Sakurada equation is used: [η] = K Ma, where a and K are constants for a specific

polymer-solvent-temperature system. The viscometer used to measure dilute solution viscosity is

usually a capillary viscometer. The viscometer uses the Poiseuille equation for laminar pressure

flow in a capillary tube. The volumetric flow rate, Q = dV/dt, under gravity, following the

Poiseuille’s law is given by, Q = rgpR4/(8h). For constant volume, dV, the time for flow is

proportional to the viscosity, t =kh.

Since time is proportional to viscosity, the intrinsic velocity can be calculated from a

capillary viscometer substituting time for viscosity. Intrinsic viscosity method is usually applied

in determination of molar masses of polymers.

I. INTRODUCTION

Nature and Importance of the Study

Chitosan (Figure 1) is a cationic linear polysaccharide composed essentially of (1 → 4)-

linked β-D-glucosamine units together with some proportion of N-acetyl-β-D-glucosamine units

(Peniche et al., 2003). It is a linear polysaccharide obtained by alkaline deacetylation of chitin.

The very high molecular weight and a very high viscosity of chitosan precluded its use in several

biological applications. The efficacy of chitosan depends on its molar mass and polydispersion.

If the molar mass is known and there is good understanding of the conformation of the polymer

in the solvent medium, the rheological and mechanical properties can be estimated (Kassai,

2007).

Figure 1. Structure of Chitosan.

Molecular weight (MW) is one of the most fundamental parameters in characterizing a

polymer. The molecular weight of chitosan can be determined by various techniques. One of the

simplest and rapid methods for determining the polymer is through viscometry. It is the method

used to determine the viscosity of a given polymer solution by means of comparing the flow

times of two liquids of equal volumes.

 The molecular weight of the polymer is measured by using viscometer and the molecular

weight obtained by this technique is called viscosity average molecular weight. The molecular

weight of the polymer solution is very high so the viscosity of polymer solution is very high

compared to that of pure solvent. The intrinsic viscosity reflects the average interactions of

single polymer molecules with the solvent and, if the molecule is considered spherical, intrinsic

viscosity is proportional to the volume of the molecule (Neira-Velasquez, et. al., 2013). The

relationship between intrinsic viscosity and molar mass is given by the Mark-Houwink-Sakurada

equation. The intrinsic viscosity of a polymer in a given solvent increases with the polymer’s

molar mass. This relation is the base for the viscometric method to assess the molar mass of a

polymer from the equation of Mark-Houwink-Sakurada:

[η] = KMα [Equation 1]

where K and α are constants for a given polymer–solvent–temperature system. These constants

are determined by evaluating a plot of log [η] versus log molecular weight.

This study is an application of another method to determine molar masses of compounds

where in a Cannon-Fenske viscometer is used. This is an opportunity for the students to explore

other methods aside from the commonly known, Dumas method, in molar mass determination.

Objectives of the Study

This study generally aimed to determine the molecular weight of chitosan by measuring

its viscosity. The specific objectives are as follows:

i. measure the corresponding average viscosity of low-molecular weight

chitosan sample;
 ii. apply Mark-Houwink-Sakurada equation to compute for the molecular weight

from the viscosity results;

iii. show the relationship between intrinsic viscosity and molecular weight

through a plot.

Scope and Limitations of the Study

The study was limited to the measurement of intrinsic viscosity to determine the

molecular weight of the low-molecular chitosan sample. A Cannon-Fenske viscometer was used

for the measurement and the Mark-Houwink-Sakurada equation was used for computing the

molecular weight.

Time and Place of the Study

This study was conducted on the 10th and 23rd of October 2017 in the analytical

laboratory of the Department of Pure and Applied Chemistry, Visayas State University, Baybay

City, Leyte.
II. MATERIALS AND METHOD

Materials

Low-molecular weight chitosan from shrimp shells (Sigma-Aldrich) was obtained

through the courtesy of Sir Candelario L. Calibo. The solvent used to dissolve chitosan was

prepared from Analytical Reagent (AR) grade chemicals - glacial acetic acid and ammonium

acetate – provided by the department stockroom.

Sample Preparation

An acetic acid/sodium acetate buffer solvent was prepared by mixing equal volumes of

0.25 M acetic acid and 0.25 sodium acetate solutions. The pH of the buffer was measured then

readjusted to pH=4.0 using sodium hydroxide solution. One gram of chitosan sample was

dissolved in 125 mL of the buffer solvent system. The resulting solution (0.8g/dl) was

continuously stirred for 3-4 hours until the chitosan has completely dissolved. Two other

concentrations of the chitosan solution were prepared by serial dilution – 10 mL aliquot of the

higher concentration chitosan solution was diluted to 100 mL, making 0.08g/dl and 0.008g/dl

chitosan solutions.

Intrinsic Viscosity Determination

The flow rate of the viscous chitosan solutions were measured using a Cannon-Fenske

viscometer (Figure 1). The experiment was initially performed at room temperature (36oC) but

due to the time-consuming preparation and measurements, a water bath was used to maintain the

temperature in the later parts of the experiment. The flow time of the solution at test was read

with the aid of a stopwatch. Three readings were made for each chitosan solution, starting with

the least to the most viscous.

 Computation

For each chitosan concentration, the following viscosities were determined using the

equation given;

Relative viscosity, ηrel = t/tsolv [Equation 2]

Specific viscosity, ηsp = (t/ts)-1 [Equation 3]

Reduced viscosity, ηred = ηsp /c [Equation 4]

Inherent viscosity, ηinh = ln ηrel/C [Equation 5]

The reduced viscosity and inherent viscosity values were plotted against chitosan

concentration. The value of intrinsic viscosity was obtained by extrapolating the graph of

reduced viscosity and inherent viscosity to zero concentration. The average of the two obtained

intercept values will be calculated.

III. RESULTS AND DISCUSSION

The results of the flow time of each chitosan concentration are shown in Table 1. It is

observable that the flow tine of the chitosan solution decreases along with its concentration.

Lower concentration of the polymer resulted to a less viscous solution; with lower viscosity, a

solution flows through the viscometer faster.

Table 1. Measured flow time of the three chitosan concentrations.

Flow rate (in sec)

Concentration Trial 1 Trial 2 Trial 3 Average

1 (0.8 g/dl) 4522 4560 4468 4516.67

2 (0.08 g/dl) 316 311 312 313

3 (0.008 g/dl) 212 213 213 212.67

Using Equations 2-5 and the flow rates, the viscosity values were calculated. These

dimensionless values are shown in Table 2.

Table 2. Calculated viscosities of the chitosan concentrations

Concentration
1 2 3
Relative viscosity, Nrel 22.07 1.53 1.04
Specific viscosity, Nsp 21.07 0.53 0.04
Reduced viscosity, Nred 26.34 6.62 4.89
Inherent viscosity, Ninh 3.32 2.95 4.87

Both relative and specific viscosities depend on polymer concentration. The reduced

viscosity is a measure of the specific capacity of the polymer to increase the relative viscosity

(Flory, 1953). By extrapolating the reduced viscosity to zero concentration, the inherent
properties of the polymer are measured. The plot (Figure 2) shows the reduced viscosity and

inherent viscosity values against chitosan concentration.

Figure 2. Extrapolated reduced viscosity vs. inherent viscosity plot.

From the equation of the curve of the inherent viscosity, ηinh = -0.0595x + 3.9961, the y-

intercept was known. The value of the reduced viscosity at zero concentration is 4.56 based from

the equation ηred = 27.21x + 4.56. The average of the two extrapolations, 4.56 and 3.9961, was

substituted to the MHS equation to obtain the molecular mass of the chitosan polymer. The

constants for the polymer-solvent system as determined by Kassai, et. al. (2000) system:

K=1.4x10-4 and α=0.83 (Kassai, et. al., 2000).

[𝜂]
[𝜂] = 𝐾𝑀𝛼 ⇒ log 𝑀 = 𝑙𝑜𝑔 ( )
𝐾

4.27805
𝑙𝑜𝑔 ( )
𝑀 = 𝑎𝑛𝑡𝑖𝑙𝑜𝑔 [ 1.4𝑥10−4 ] = 𝟐𝟓𝟑, 𝟑𝟕𝟎. 𝟕𝟏 𝒈/𝒎𝒐𝒍
0.83
 The determined molecular weight of chitosan sample manufactured by Sigma-Aldrich is

around 50,000-190,000 Da (1 Da=1 g/mol) based on viscosity (www.sigmaaldrich.com). The

calculated value from the experiment which is 253,370.71 g/mol, is off the given range. Thus,

there was an error in the experiment. Moreover, as can observed in Figure 2, the curves of the

plotted viscosities are not completely linear. One can expect that the experimental value will not

be accurate. The basis for the determination of intrinsic viscosity through extrapolation is the

Kraemer-Huggins plot. Figure 3 shows the expected plot which is different from result of this

experiment.

Figure 3. The Kraemer-Huggins plot to obtain the intrinsic viscosity

(Neira-Velasquez, et. al., 2013).

Based on the closeness of the flow rates of the chitosan concentrations, the experiment

gave quite precision. The errors that affected the results may be systematic or random.

Systematic error may come from the uncontrolled temperature throughout the experiment. The

water bath was only used in the later part of the experiment when the room temperature dropped

as the time of the day passed because the lengthy time for the experimentation was overlooked.
Since the viscosity of solutions depend on temperature, any slight change in the temperature of

the environment during the experiment will affect the flow rate measurement. It would have been

more appropriate to have used the water bath for all the measurements. The possible major

source of random error that affected the measurements may be the presence of impurities in the

solutions. Based on the literatures, before subjecting the chitosan solution to the viscometer, it

must be filtered using filters with pore size of 5 to 8 μm. However, this was not applied in the

experiment because of time constraint. The filtration of the viscous chitosan solution would take

a lot of time, even before the arduous flow rate reading. Any impurity in the solution will affect

the flow rate of the polymer solution in the viscometer, most especially within the smallest radius

of the capillary.

For future experiments using the intrinsic viscosity method where the experimenters have

a lot of time in their hands, more dilute solutions of the polymer should be prepared and more

measurements must be repeated to obtain a linear behavior in the plot. This will result a better

intrinsic viscosity to determine the average-molecular weight of the polymer at test.

IV. CONCLUSION AND RECOMMENDATION

Conclusion

Based from the experiment, polymer viscosity measurement using a non-automatic

viscometer is feasible but laborious and time-consuming. It is not advised to use this method

especially when doing analytical measurements because the results are not accurate especially

when the system is not kept at uniform condition throughout. For this reason, the determination

of molecular weight of polymers using intrinsic viscosity method requires patience and time.

Instead, many other more advanced and more reliable methods can be used to determine

molecular weight of substances such as gel permeation chromatography (GPC) or high pressure

liquid chromatography (HPLC).

Recommendation

Better results and conclusion can be obtained from reconducting the experiment with the

following recommendations;

i.) Before testing a polymer, measure the viscosity of a solution, preferably with

known viscosity, and compare results with literature values;

ii.) Use other types of viscometers such as Ostwald and Ubbelohde viscometers;

iii.) Use a water bath all throughout the experiment to maintain the temperature of

the solutions and during the flow rate measurement and ensure the solutions come

in thermal equilibrium with the bath for a longer period of time; and

iv.) Filter the solution before subjecting to the viscometer to lessen impurities

which can affect the reading.

V. LITERATURE CITED

FLORY, P.J. 1953. Principles of Polymer Chemistry. New York: Cornell Univesity Press, p. 309

KASSAI, M.R. 2007. Calculation of Mark-Houwink-Sakurada (MHS) equation viscometric

constants in any solvent-temperature system using experimental reported viscometric
constants data. Carbohydrate Polymers, 68, 477-488.

KASSAI, M.R., ARUL, J., and CHARLET, G. 2000. Intrinsic viscosity-molecular weight
relationship for chitosan. Journal of Polymer Science Part B: Polymer Physics, 3, 19: 2591-
2598

NEIRA-VELASQUEZ, M.G., RODRIGUEZ-HERNANDEZ, M.T., HERNANDEZ-

HERNANDEZ, E., and RUIZ-MARTINEZ, A.R.Y. 2013. Polymer Molecular Weight
Measurement. Handbook of Polymer Synthesis, Characterization, and Processing, 1st ed.
SALDIVAR-GUERRA, E. and E. VIVALDO-LIMA (Ed.). USA: John Wiley & Sons Inc.,
pp. 355-366.

PENICHE, C., ARGUELLES-MONAL, W., PENICHE, H., and ACOSTA, N. 2003. Chitosan:
An attractive biocompatible polymer for microencapsulation. Macromolecular Bioscience, 3,
511-520.

http://www.sigmaaldrich.com/PH/en/product/aldrich/448869 Date Accessed: October 30, 2017