Determination of the pattern of acetylation of chitosan samples: Comparison of evaluation methods and some
9.3 Results and discussion
9.3.2 Determination of PA by 1H-NMR spectroscopy
In the1H-NMR spectrum of chitosan, the anomeric signals of GlcNAc (A) and GlcN (D) are sensitive to their nearest neighbor126. The signal of GlcNAc is relatively broad due to its proximity to the rapidly relaxing nitrogen atom, which is in a more asymmetric environment than in the GlcN residue. The experimental intensities IAD and IAA of the sequences AD and AA, respectively, were determined from the H1 signal intensities of GlcNAc and the frequencies IDD and IDA based on the H1 signal intensities of GlcN.
Unfortunately, the resonances of the H-1 of GlcN were not well resolved, so establishing the frequencies IDD and IDA was a complicated matter.
Figure 9.2: Comparison native vs. de-graded chitosan- Sections of the13C-NMR spec-tra of native (a) and degraded (b) samples of chi-tosan B (FA 0.02).
IDD and IDA frequencies can be cal-culated with the help of the intensity of the H1 signals of GlcN and the fol-lowing relationships: IAD = IDA and ID = IDA + IDD, where ID indicates the intensity of the H1 signals of GlcN
126. The signals of the anomeric pro-tons of the GlcNAc and GlcN residues were not sufficiently well resolved in the
1H-NMR spectra of chitosan samples recorded on a 400MHz NMR spectrom-eter. Their shapes did not enable the experimental frequencies IAA, IDD, IAD and IDA to be determined (Fig.9.1);
this step is essential for calculating the parameters FAA, FDD and FAD, and then transforming them into one sin-gle parameter PA. The correct determi-nation of PA for chitosan samples was impossible, so the 1H-NMR technique using a 400MHz spectrometer is not a suitable method for determining PA.
9.3.3 Determination of PA by 13C-NMR spectroscopy using carbon C6 signals
According to the literature127, the intensities of C6 carbon signals can be used to de-termine the experimental sequences AD, DD, AA and DA along the chitosan chain.
Unfortunately, the only limited depolymerization of chitosan by nitrous acid, a pro-cedure recommended to enhance NMR spectral resolution, may impair the correct analysis of the intensities of the C6 signals, and artifact peaks may appear in this part of the 13C-NMR spectrum209. However, these disadvantages did not prevent the use of the experimental intensities of C6 signals to determine PA.
9.3.4 Determination of PA by 13C-NMR spectroscopy using carbon C5 signals
In the13C-NMR spectrum of chitosan, the carbon C5 signals also have different chem-ical shifts, depending on the nature of the neighboring units. As shown in Fig.9.2a, limited depolymerization is essential to obtain a good quality spectrum; this permits further analysis and does not result in the appearance of artifact signals in that part of the13C-NMR spectrum containing the carbon C5 signals (Fig.9.2b). Analysis of C5 signal intensities is specific and enables PAto be estimated accurately. In addition, the C5 signals representing the sequences AD, DD, AA and DA in the 13C-NMR spectra were much better resolved than the H1 signals of GlcNAc and GlcN representing the same sequences in the1H-NMR spectra (Fig.9.1). Thus,13C-NMR spectroscopy based on the analysis of carbon C5 signals is clearly the most suitable method for determining PA. During a typical PAdetermination by13C-NMR spectroscopy the carbon spectrum of the chitosan sample is recorded only once, because these analyses are very costly.
Knowledge of the sensitivity and precision (repeatability and reproducibility) of this method is very important.
9.3.5 Sensitivity
The method’s sensitivity was tested on chitosan samples with a degree of acetylation from 0.02 to 0.48. The intensities of the frequencies DA, AA and AD of carbon C5 increase, whereas the intensity of the DD sequence decreases for chitosan samples with higher values of FA (Fig.9.3). The small signal between DD and AA peaks visible in the carbon spectrum is probably an effect of the resonance due to one of the following sequences: ADDD, DADD, DDDA or DDAD. The assumption was made because the intensity of this signal decreases for chitosan samples of high FA127. This signal also appears in the carbon spectra of native chitosans209. It means that it is not an artefact after degradation of chitosan with NaNO2.
Table 9.2: Intra-day (n = 6) and inter-day (n = 18) precision test expressed as RSD%. The analyses of the carbon spectra were performed by the same analyst (analyst A).
Chitosan Repetition of line-fitting only Repetition of the whole carbon spectrum analysis samples Mean PA Intra-day Mean PA Inter-day Mean PA Intra-day Mean PA Inter-day
precision precision precision precision
(n = 6) (n = 18) (n = 6) (n = 18)
B [0.02] 0.42 3.45 0.41 7.95 0.41 7.95 0.40 12.76
C [0.06] 0.76 2.83 0.77 6.54 0.76 8.36 0.78 7.74
U [0.10] 0.85 2.51 0.86 3.88 0.87 3.98 0.87 2.98
L [0.14] 0.96 1.17 0.96 2.23 0.96 1.72 0.96 2.47
G [0.18] 0.90 1.74 0.91 2.26 0.90 2.22 0.92 2.92
J [0.22] 0.96 0.99 0.97 1.95 0.97 1.20 0.98 2.04
X [0.44] 1.11 0.62 1.12 1.63 1.11 0.73 1.11 1.85
V [0.48] 1.11 0.57 1.11 0.66 1.11 0.57 1.11 0.75
9.3.6 Precision
In the present study, eight chitosan samples with FAfrom 0.02 to 0.48 were used to test the intra-day and inter-day precision of the PA determination. Every day the carbon spectra were analysed by analyst A in two different ways (see Experimental section).
The last step of this analysisthe line-fitting procedureis illustrated in Fig.9.4. Each of these steps can exert a different influence on the PA results, so the intra-day and inter-day precision of the analysis of the 13C-NMR spectra were tested by repeating only the line-fitting procedure in the carbon spectra. Table 9.2 lists the mean values of PA, SD and the RSD% of PA obtained for chitosan samples in the intra-day and the inter-day precision tests. The data obtained clearly indicate that the intra-day and inter-day precision of the PA determination using 13C-NMR spectra depended on the degree of acetylation of the chitosan samples. For example, the RSD% of PA in the interday precision test was the highest for chitosan B (FA 0.02; RSD% of PA <12.75) and the lowest for chitosan V (FA 0.48, RSD% of PA <0.72) (Table 9.2).
Figure 9.3: Extracts of13C-NMR spectra - C5 regions of the13C-NMR spectra of chitosans with different FA. (a) Chitosan C, FA= 0.06; (b) chitosan G, FA= 0.18; (c) chitosan V, FA= 0.48.
This is due to the lower signal-to-noise ratio (S/N) of signals DA, AA and AD for chitosans of low FA. For exam-ple, the S/N ratios of signals DA, AA and AD in the carbon spectrum of chi-tosan B (FA 0.02) were 9.4, 14.2 and 8.4, respectively, whereas for chitosan V (FA 0.48) they were 78.1, 53.5, and 75.3. The lower RSD% of PA for chi-tosan L (Fig.9.4) compared to those ob-tained for chitosan G (Fig.9.3b) were the result of the good-quality carbon spectrum for chitosan L. Comparison of the RSD% of the PA values ob-tained in the intra- and inter-day pre-cision test, when the whole analysis of the carbon spectra was performed, with those when only the line-fitting procedure was repeated indicated that phase adjustment and baseline correc-tion of the carbon spectra strongly in-fluenced the precision of the method, especially for chitosans of low FA (Ta-ble 9.2). The S/N ratios of signals DA, AA and DA probably changed, thereby adversely affecting the correct estima-tion of their intensities. The repro-ducibility of PA determination by 13 C-NMR spectroscopy was also tested by the having the same carbon spectrum investigated by three analysts: A, B and C. Table 9.3 sets out the mean PA, and the RSD% values for chitosan G (FA0.18) obtained by three analysts in the intra-day and inter-intra-day precision tests. The results were very similar, apart from the higher
RSD% obtained by analyst B. The resolution between 1.00 and 2.00 Hz used by ana-lyst B during Fourier transformation of the fid may have been too low, which precluded more precise results. The use of the correct parameters during carbon spectrum anal-ysis allows reproducible results to be obtained. The results of this study show that the method of PA determination using the intensities of signals in the carbon spectrum of chitosan is sensitive, repeatable and reproducible.
Table 9.3: Comparison of mean values of PA and RSD% of PA in intra-day (n = 6) and inter-day (n = 18) precision tests obtained from carbon spectrum analyses of chitosan G (FA = 0.18) performed by three different analysts (A, B and C).
Chitosan G [0.18] Repetition of the whole carbon spectrum analysis Mean PA Intra-day Mean PA Inter-day
precision precision
(n = 6) (n = 18)
Analyst A 0.90 2.22 0.92 2.92
Analyst B 0.91 3.95 0.93 5.37
Analyst C 0.92 1.87 0.92 1.81