Determination of the Molar Mass as Quality Control Parameter

of Chitosan
Relevant for: Biopolymers, Polyelectrolytes, Chitosan

The wide scale of applications of this natural polymer ranges from agriculture, food industry to high-end
pharmaceutical and medical products. For all of them the molar mass is a significant parameter. The
Lovis 2000 M/ME microviscometer combines precise and fast viscosity measurements for subsequent
molar mass calculations.

The most interesting one is the intrinsic viscosity

which is related to the molar mass of a polymer as
formulated in the Mark-Houwink equation:

[𝜂] = 𝐾 · 𝑀𝑎

In this equation [𝜂] is the intrinsic viscosity and "M"

the molar mass. The Mark-Houwink constants "K" and
"a" are literature values and describe the polymer-
solvent combination at a given temperature. Since
knowledge of the constant values is needed for the
molar mass calculation, it is important to examine and
evaluate the literature source critically.

Tip: A single [𝜂] ~ M relationship is not valid over the

1 Biopolymer - Chitosan
Chitosan is an amino sugar-containing polysaccharide
obtained from crab and shrimp shells. Applications of
the polysaccharide require knowledge of its main
physicochemical properties, especially molar mass,
molecular weight distribution and degree of
deacetylation (DD). These parameters influence the
physical, chemical, and biological properties, such as
solubility, viscosity of solutions, and its biological
activity.
1.1 Dilute solution viscometry using a rolling
ball viscometer
One technique which can be used for quality control is
dilute solution viscometry. Polymers are diluted in
appropriate solvents and the viscosity of the pure
solvent is set in relation to the viscosity of the polymer
solution. This procedure yields the relative viscosity, a
basic parameter from which many others can be
calculated.
whole range of molar masses. The values of the
Mark-Houwink constants need to be specified for a
defined range of molar masses.
1.2 Viscometric determination of the intrinsic
viscosity
Intrinsic viscosity can be measured due to the ability
of polymers to increase the viscosity of a solvent in
the absence of intermolecular interactions between
the polymer molecules. Multipoint measurements
allow for following the evolution of relative viscosity as
a function of increasing concentration.
The absence of intermolecular interactions of polymer
solutions can only be assumed for a relative viscosity
range of 1.2 to 2.5. Within this limit, the trend of the
concentration-dependent viscosity is linear and the
intrinsic viscosity can be extrapolated with a
regression method according to Huggins or Kraemer.
Tip: [𝜂] can only be calculated for low concentration
polymer solutions.

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1.3 Solvent parameter affecting solubility 2.1 Sample preparation

Chitosan is a polyelectrolyte with two functional
groups, namely N-acetyl and hydrogen groups. The
solubility of the solvent can be assessed by caused
chain repulsion or the disruption of the intermolecular
hydrogen bonds.
Various aqueous acidic solvents for chitosan are
described in literature, such as acetic acid, or mixtures
of acetic acid and sodium chloride with or without
urea.
These solvents cause solubility through the
protonation of the amine groups (NH3+) and
consequent coulombic repulsions. Due to the
advantage of complete dissolution without polymer
chain degradations, a buffer of acetic acid or a mixture
of acetic acid and sodium chloride with ionic strength
between 0.05 mol/L and 0.2 mol/L are favorable.
0.01 g of the chitosan sample was dispersed in 50 mL
of a 0.3 M HAc/0.2 M NaAc solution (Table 2). For
dissolving, the samples were stirred with a magnetic
stirrer at room temperature until a homogeneous
solution was formed. The resulting solution was
filtered through a nylon membrane with a pore size of
0.2 µm to get rid of possibly remaining insoluble
material. The concentration series was produced by
further diluting the stock solution. All samples, were
immediately measured to avoid chain scission. If
needed, the samples can be stored at 4 °C for short
time periods without changes in the polymer structure.
Concentration series Concentration [mg/mL]
Stock solution 0.20
Concentration 1 0.08
Concentration 2 0.12

Influencing parameters of [𝜂]: Concentration 3 0.16

Concentration 4 0.20

1. Molecular weight Table 2: Concentration series for both chitosan samples.

2. DD degree
3. pH value 3 Measurement
4. Ionic strength
5. Temperature This section describes the instrument set-up and the
used method settings.
2 Sample and sample preparation
3.1 Instrumental set-up
For both chitosan samples, a stock solution with a For measurements, a Lovis 2000 ME in flow-through
concentration of 0.2 mg/mL was produced (Table 1). mode and additional pH ME for simultaneous pH
The identification of the ideal concentration range for control were used.
the intrinsic viscosity calculation was performed by
previous experiments with a wider concentration For more information about the installation and
range. The final concentration series was determined calibration of the pH module, please check the
according to the relative viscosity limitation for instruction manual pH ME Beverage/ pH ME
polymer solutions. (C88IB001EN).

▪ Capillary: Ø 1.59 mm glass

Sample and chemicals Chemical Description
▪ Ball material: Ø 1.5 mm steel
0.3 M Acetic acid solution (HAc) +
Solvent 0.2 M Ammonium acetate puffer ▪ DMA 4500 M
(NaAc) ▪ pH ME
pH 4.6
Ionic strength 0.2
Chitosan 90/4000/A1
Sample 1 (Sept. 2020)
batch: 200420-S626
Chitosan 90/4000/A1
Sample 2 (Dec. 2020)
batch: 180820-S632
Table 1: Test samples and chemical used. Both chitosan samples
have a DD value of 88 % to 95 %.

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 between. Out of the runtimes of the pure solvent and
the polymer solution all polymer parameters were
automatically calculated.

According to Huggins, the intrinsic viscosity is

determined from the y-axis intercept of a plot of
reduced viscosity against concentration.
[η] = lim(ηred )
c→0

The Kraemer equation uses the inherent viscosity

instead for an extrapolation of the linear fit of the data.
[η]K = lim(ηinh )
c→0
Figure 1: DMA 4500 M with Lovis 2000 ME
The applicability of the different regression models
depends on the type of polymer used and needs to be
evaluated. The Mark-Houwink constants for the given
polymer-solution combination were taken from
literature sources (Table 3).

Mark-Houwink constants Literature values

K 76,0 · 10-5 dL/g
a 0.76
Table 3: Mark-Houwink constant values for chitosan in 0.3 M acetic
acid + 0.2 M ammonium acetate puffer solution for DD 89.5 %
degree of a molecular weight range of 100 kDa to 600 kDa.

Table 4 displays the results of the triple determination

of both chitosan batches, expressed in intrinsic
viscosity [mL/g] and molar mass [g/mol] according to
Figure 2: DMA M with pH ME module. (The pH ME is inserted in Huggins and Kraemer model.
the sample cycle as last measuring unit)

Intrinsic
Extrapolation Molar mass
Sample type viscosity
method [g/mol]
Tip: The connection of the pH ME module to the [mL/g]
master instrument is described in the Lovis Reference 15.555 1098.7
Guide. Huggins 15.811 1122.5
Sample 1 17.947 1326.2
(Sept. 2020) 15.914 1132.1
3.2 Method settings
Kraemer 16.019 1149.8
▪ Measurement mode: Polymer 18.016 1335.1

▪ Temperature: 25 °C 17.421 1275.0

▪ Measurement cycles: 2 Huggins 19.807 1510.0
Sample 2 20.207 1560.3
▪ Manual angle: 30°
(Dec. 2020) 17.717 1303.9
▪ Measuring distance: long
Kraemer 19.709 1500.1
▪ Variation Coefficient: 0.35 %
20.165 1523.7
Table 4: Calculated intrinsic viscosity and the molar mass according
to Huggins and Kraemer by the triple determination of the run time
of the solution.
3.3 Measurements and results

Measurements were performed as single The mean value of the intrinsic viscosity and the molar
determinations (n = 3). Each biopolymer solution was mass is given in Table 5.
filled and measured separately with cleaning steps in

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 Extrapolation
Sample type [η] [mL/g] ± σ M [g/mol] ± σ
method 5 References
Sample 1 Huggins 16.44 ± 1.31 1182.46 ± 125.1
(Sept. 2020) Kraemer 16.69 ± 1.18 1205.70 ± 112.5 1 Rao, D. G. (1993). Studies on viscosity-molecular
Sample 2 Huggins 19.18 ± 1.54 1448.49 ± 152.1 weight relationship of chitosan solutions. Journal of
(Dec. 2020) Food Science and Technology (India), 30(1), 66-67.
Kraemer 18.71 ± 1.41 1442.56 ± 120.7
2. Kasaai, M. R. (2007). Calculation of Mark–
Table 5: Mean value of the intrinsic viscosity and the molar mass
with their standard deviation. The calculated molar mass of
Houwink–Sakurada (MHS) equation viscometric
C56H103N9O39 by PubChem (release 2021.05.07) is 1526.5 g/mol. constants for chitosan in any solvent–temperature
system using experimental reported viscometric
constants data. Carbohydrate polymers, 68(3), 477-
By additional density measurements, the dynamic 488.
viscosity can be calculated. Table 6 shows the
calculated dynamic viscosity values of the two 3. Norzita, Y., Norhashidah, T., & Maznah, M. (2011).
chitosan stock solutions. Determination of viscosity-average molecular weight
of chitosan using intrinsic viscosity measurement.

Dynamic Viscosity
Sample type pH
[mPa s]
Sample 1 (Sept. 2020) 4.5 1.543
Contact Anton Paar GmbH
Tel: +43 316 257-0
Sample 2 (Dec. 2020) 4.5 1.588
support-visco@anton-paar.com |
Table 6: Dynamic viscosity of the stock solution (0.2 mg/mL) at the
present pH value and a temperature of 25 °C. www.anton-paar.com

4 Conclusion

It could be shown that Lovis 2000 ME can be used for

quality control of chitosan by means of molar mass
determination. While the dynamic viscosity of the two
chitosan samples differs by less than 0.05 mPa·s, the
molar mass values show a significant difference of
more than 200 g/mol. With the help of the molar mass
determination, the quality differences and the different
performance of the final product can be evaluated and
explained.

4.1 Lovis 2000 M – user benefits

The Lovis 2000 M is perfectly suited for determining

the properties of polymers using dilute solution
viscometry as it offers many features making
measurements easy and comfortable:

▪ Polymer software for automatic calculation of

all desired polymer parameters
▪ Two different measuring distances (short and
long) for time saving measurements
▪ Possibility of time and temperature scans
▪ Measurements over a wide viscosity range
▪ (0.3 mPa·s to 10,000 mPa·s)
▪ Peltier thermostatting for quick and precise
temperature control
▪ Optional combination with DMA and pH
measuring cell

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