From Chitin to Chitosan - Chitosan Based Hydrogels: Functions and Applications - page 10

The greatest advantage of high-performance liquid chromatography (HPLC) in relative
molecular weight measurement is that it can absolutely measure the relative molecular
weight and determine relative molecular weight distribution. Special instrument and guide
samples for relative molecular weights have been developed; thus, HPLC is now a common
method for measuring relative molecular weight and relative molecular weight distribu-
tion. The light scattering method is another common method, especially the coupled light
scattering-gel permeation chromatography method used for absolutely measuring rela-
tive molecular weight. The end group method does not need a special device and is easy to
operate; hence it is widely used. However, error in the end group method is somewhat large.
Viscosity can be measured by many methods with different physical significance. During
production of chitosan, viscosity is usually measured by a rotational viscometer. The result
is apparent viscosity, which means quantification of the chitosan viscosity property in
using, but the molecular weight cannot be figured out through this value. Inherent viscos-
ity can be measured by a Ubbelohde viscometer, which is the most common method for
measuring the viscosity of chitosan. Inherent viscosity is apparent viscosity when the con-
centration of high polymer is infinitely low. High-performance capillary electrophoresis is
featured with high resolution, high sensitivity, and fast separation rate, and is widely used
for measuring the molecular weight of low-molecular-weight chitosan (chitosan oligosac-
charide). The chain of chitosan oligosaccharides is modified by negative charges, chro-
mophores, and fluorophores. Chitosan oligosaccharides can be separated by electrophoresis
due to molecular size, because each oligosaccharide in a complex of different oligosaccha-
rides has just one charge. The separated oligosaccharides can be identified by laser-induced
fluorescence detection, and then polymerization degrees of the oligosaccharides can be
determined according to peak time. The percentage of oligosaccharides of different polym-
erization degrees in the hydrolysate can be determined by peak areas.
1.3.3 Structure identification
Structure identification and characterization can be carried out by paper chromatography,
thin-layer chromatography (TLC), infrared absorption spectroscopy, UV absorption spec-
troscopy, mass spectrometry, nuclear magnetic resonance, ultimate analysis, x-ray diffrac-
tion, and free amino content (or DD) [50]. Low-molecular-weight chitosans of different
polymerization degrees can be separated by a silica gel thin layer with developing solvent
made of ethyl acetate, ethanol, water, and ammonia water in the ratio 5:9:1:1.5. Chromato-
graphy reproducibility of TLC and the linear relation between number of residues and Rf
are good [51,52].
The configurations of glycoside bonds and the substitution state of hydroxyls and
amino can be determined by the infrared spectrum. The infrared spectrum shows similar
structure characterizations of high-molecular-weight chitosans and low-molecular-weight
chitosans when they are pressed with KBr and scanned at 400-4000 cm −1 . Characteristic
absorption bands such as the O-H stretching vibration absorption band at 3450 cm −1 , the
C-H stretching vibration absorption band at 2867 cm −1 , and the amide absorption bands at
1665 and 1550 cm −1 appear in the spectrum. The low-molecular-weight chitosan shows a
strong -OH absorption band at 3450 cm −1 due to increased hydroxyl [53].
Main peaks such as the N-H stretching vibration at about 3400 cm −1 and the N-H bend-
ing vibration at about 1600 cm −1 do not move before and after chitosan is degraded, but
strengths change due to decreased molecular weight. This further confirms that the
concerted reaction goes by cracking β-(1,4) glycoside bonds of chitosan, and the polysac-
charide ring does not change in structure after degradation.