DETERMINATION OF VISCOSITY-AVERAGE MOLECULAR WEIGHT OF CHITOSAN USING

INTRINSIC VISCOSITY MEASUREMENT

PENGUKURAN BERAT MOLEKUL KITOSAN MENGGUNAKAN KAEDAH KELIKATAN INTRINSIK

Norzita Yacob, Norhashidah Talip, Maznah Mahmud, Nurul Aizam Idayu Mat Sani, Nor Akma
Samsuddin, Norafifah Ahmad Fabillah
Malaysian Nuclear Agency (Nuclear Malaysia)
Bangi, 43000 KAJANG, MALAYSIA

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Abstract

Molecular weight of chitosan can be determined by different techniques such as Gel Permeation
Chromatography (GPC), Static Light Scattering (SLS) and intrinsic viscosity measurement. Determination of
molecular weight by intrinsic viscosity measurement is a simple method for characterization of chitosan.
Different concentrations of chitosan were prepared and measurement was done at room temperature. The
flow time data was used to calculate the intrinsic viscosity by extrapolating the reduced viscosity to zero
concentration. The value of intrinsic viscosity was then recalculated into the viscosity-average molecular
weight using Mark-Houwink equation.
Abstrak

Berat molekul kitosan dapat ditentukan dengan pelbagai teknik yang berbeza seperti Gel Permeation
Chromatography (GPC), Static Light Scattering (SLS) dan pengukuran kelikatan intrinsik. Penentuan berat
molekul melalui pengukuran kelikatan intrinsik merupakan satu kaedah yang mudah untuk pencirian kitosan.
Kepekatan kitosan yang berbeza disediakan dan pengukuran djalankan pada suhu bilik. Data aliran masa
digunakan untuk mengira kelikatan intrinsik dengan mengekstrapolasi penurunan kelikatan kepada
kepekatan sifar. Nilai kelikatan intrinsik yang diperoleh kemudiannya digunakan dalam persamaan Mark-
Houwink bagi mendapatkan purata kelikatan berat molekul.
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Katakunci/keyword : Ubbelohde Viscometer, viscosity average molecular weight, intrinsic viscosity, chitosan

INTRODUCTION

Chitosan is a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine

(deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). The very high molecular weight and
therefore a very high viscosity of chitosan precluded its use in several biological applications. However, in
some fields (especially in medicine and the food industry) the application of this polysaccharide is limited by
its high molecular weight resulting in its low solubility in aqueous media (Minagawa et al. 2007; Biskup et al.
2005). For some specific applications, its degradation products were found to be much more useful. .A variety
of degradation methods such as chemical (Hasegawa et al. 1993), enzymatic (Ilyina et al. 2000) or radiation
(Won-Seok et al. 2002) degradation of high molecular weight polymer are being worked out to generate these
degradation products. Degradation by radiation is a simple and environmental friendly as it is free of initiators
and side product (Charlesby, 1981).

Molecular weight (MW) is one of the most fundamental parameters in characterizing a polymer. Molecular
weight of chitosan can be determined by different techniques. GPC is the most powerful technique for
characterizing the molecular weight of polymers. However, it is a relative method and needs molecular weight
standards for calibration to obtain the relation between elution volume and molecular weight. One of the
simplest and rapid methods for determining the molecular weight of polymer is viscosimetry, although this is
not an absolute method and requires the determination of constants.
The intrinsic viscosity, η as function of average molecular weight, M is represented by Mark-Houwink-
Sakurada equations
η = KMα (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 which the molecular weight has been
determined by an absolute method such as light scattering method.

The goals of this study are to determine the effect of radiation on viscosity-average molecular weight and
intrinsic viscosity of Ubbelohde viscometer.

EXPERIMENTAL

Materials
Chitosan powder was purchased from Heppe Medical Chitosan GmbH with the following properties, 91.8%
of DDA, 6% water content and the viscosity is 1050 mPas 1% in 1% acetic acid at 20°C. Ammonium acetate
and acetic acid were obtained from Sigma-Aldrich. All reagents were used as received without any
purification.

Sample Preparation And Irradiation

Chitosan powder was weighed about 2g in different plastic bag prior to irradiation process. The irradiation
was carried out using electron beam accelerator at irradiation conditions of 2MeV acceleration energy, 10 mA
beam current and dose exposure of 2, 5, 10, 25 and 35kGy. Samples of unirradiated and irradiated were then
dissolved in 0.15M ammonium acetate and 0.2M acetic acid solvent system for viscosity-average molecular
weight and intrinsic viscosity measurement. This solvent should be used for measurement within 3 days. The
solution was prepared and continuously stirred for 24 hours before doing the measurement to completely
dissolve the chitosan. It can only be used within 24 hours.

Intrinsic Viscosity Determination

Intrinsic viscosity was examined using an automatic system Ubbelohde capillary type viscometer which
allows the reading of flow times of the sample taken automatically without using stop watch. The capillary
number is 531/10 I and the measurement was conducted at 25 ± 0.1°C. The measurement was started with
solvent until five consecutive values differ from their mean value by no more than 0.3s. Next, flow times of
five different concentrations of chitosan solution were recorded. Directly before charging the chitosan
solution into the viscometer, the solution should be filtered using hydrophilic membrane filter of 0.45 micron
size.

The corrected average flow time for a solvent and each chitosan concentration, ti(corr was calculated by
subtracting the Hagenbach correction, ∆tHC(i) from the average flow time for that concentration, ti. according
to :
ti(corr = ti - ∆tHC(i) (2)

The Hagenbach correction was provided in the operating instruction for the viscometer. For each chitosan
concentration, the following viscosities were determined using the equation given;

Relative viscosity, ηrel = t/ts (3)

Specific viscosity ηsp = (t/ts)-1 (4)

Reduced viscosity ηred = ηsp/c (5)

Inherent viscosity, ηinh = ln ηred /C (6)

The reduced viscosity and inherent viscosity were plotted against chitosan concentration. The value of
intrinsic viscosity can be calculated by extrapolating graph of reduced viscosity and inherent viscosity to zero
concentration. The average of the two obtained intercept values was calculated.

Viscosity-Average Molecular Weight Determination

Viscosity-average molecular weight was identified by viscometric measurements using an Ubbelohde
Capillary Viscometer type 531/10 I. This value was calculated from [η] = KMα equation, where K= 9.66x10-5
(dm3/g) and a = 0.742 determined in 0.15M Ammonium Acetate and 0.2M Acetic Acid solution at 25°C.

RESULT AND DISCUSSION

Upon radiation, two main reactions which influence the final properties of polymer: a) scission of main chain,
also known as degradation and b) cross-linking, the opposite process to degradation. The former will be
followed by reduction in molecular weight while the later will cause an increase in molecular weight.
Degradation process usually occurs when natural polymer is subjected to ionizing radiation.

Intrinsic viscosity of chitosan which was irradiated at different doses was demonstrated in Figure 1. It is
showed that the intrinsic viscosity dropped dramatically when the chitosan exposed upon radiation at dose of
2kGy. The intrinsic viscosity reduced from 1 353ml/g for unirradiated chitosan to 861.5ml/g of irradiated
chitosan at dose of 2kGy. For chitosan irradiated at 5kGy and 10kGy, the intrinsic viscosities were 704.5ml/g
and 543.5ml/g respectively, whereas at dose of 25kGy the intrinsic viscosity of irradiated chitosan was
330.5ml/g. The intrinsic viscosity was further decreased to 247ml/g of irradiated chitosan at dose of 35kGy. A
reduction of intrinsic viscosity was followed by decreasing in viscosity-average molecular weight of chitosan.
Changes of viscosity-average molecular weight of chitosan with irradiation dose are depicted in figure 2. A
sudden drop of viscosity-average molecular weight was observed at 2kGy where the molecular weight
reduced from 390kDa to 210kDa. After 5kGy, the viscosity-average molecular weight of chitosan fell steadily
from 160kDa to 40kDa at dose of 35kGy. This reduction was due to the chain scission of chitosan backbone,
where degradation process took place.

1600
Intrinsic viscosity (η, ml/g)

1400
1200
1000
800
600
400
200
0
0 10 20 30 40
Irradiation dose (kGy)

Figure 1: Changes of intrinsic viscosity as function of irradiation dose. Chitosan was irradiated in powder
form using electron beam radiation at 2, 5, 10, 25 and 35kGy.
 500000

Viscosity Average Mw (Da)

400000

300000

200000

100000

0
0 5 10 15 20 25 30 35 40
Irradiation Dose (kGy)

Figure 2: Changes of viscosity-average molecular weight and viscosity as function of irradiation dose.
Chitosan was irradiated in powder form using electron beam radiation at 2, 5, 10, 25 and 35kGy.

CONCLUSION

Chain scission or degradation took place in the chitosan when exposed to irradiation at solid state. Both
intrinsic viscosity and viscosity-average molecular weight were reduced when the chitosan was exposed to
irradiation at dose up to 35kGy. Further, it was concluded that the intrinsic viscosity and viscosity-average
molecular weight for chitosan can be determined by Ubbelohde Viscometer.

REFERENCE

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644.
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Physics Research B, 236: 383–390
3. Hasegawa, M., Isogai, A. & Onabe, F., (1993), Preparation of low molecular weight chitosan using
phosphoric acid. Carbohydr. Polym., 20, 279–283.
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