Degree of Deacetylation of Chitosans
The degree of deacetylation (DDA) is a measure for the proportion of deacetylated (glucosamine) units in the chitosan polymer; in Fig.1 deacetylated units are depicted by R = H.
Figure 1: Generic structure of a chitosan.
It is one of the main characterizing features of chitosans, determining the chemical, physical and mechanical properties of the material and therefore the respective applications. Chitosans with high (≥70%) DDA have higher solubility, mechanical strength and viscosity, finding their applications in wound dressings, seed or edible film coatings, while medium DDA (50-70%) materials are used for controlled drug release formulations or as fertilizer carriers. [1,2] The main analytical methods for determining the DDA are potentiometric titration of the free amine groups, IR and NMR spectroscopy or various derivatization techniques. An ideal method would be highly accurate, non-destructive and not overly costly as well as applicable across the whole range of chitosans. 1H NMR spectroscopy, while highly accurate and non-destructive, is often considered as too costly, but with the wide availability of benchtop NMR systems this is not the case any more. Here we show the application of the USP-NF Chitosan Monograph method [3] for determining chitosan samples with medium and high DDAs.
Experimental Setup
Four lots of chitosans with varying degrees of DDA as determined by alternative methods were analysed by 1H NMR spectroscopy. 10 mg of each chitosan were dissolved in 1 mL of formic acid-d2. 1H{13C} spectra to eliminate the 13C satellites of formic acid for the samples were measured on a Spinsolve 80 MHz Multi X system with a repetition time of 4 s and 32 or 128 scans, yielding experiment times of 2.1 and 8.5 min. Each sample was run ten times.
NMR Results & Discussion
Figure 2: 1H{13C} NMR Spectra of four chitosans with varying DDAs, indicating the acetyl group and the DDA provided by alternative methods.
Figure 3: Overlay of ten 1H{13C} NMR spectra of one chitosan with integral regions (32 scans)
Table 1: DDAs for five chitosans, with the relative standard deviation from 10 measurements per sample
The NMR results are in excellent agreement with the given DDA values, with relative standard deviations for ten measurements well below 1%. Acquiring only 32 scans yields acceptable results, allowing the measurement of a large number of samples very rapidly.
Data acquisition, processing, and analysis parameters were implemented in the Spinsolve software (Fig. 4) to create a fully automated qNMR method. The user workflow is reduced to inserting the sample, entering a sample ID, and pressing start, with all subsequent steps—including data processing, analysis, and PDF report generation—performed automatically.
Figure 4: Screenshots of the qNMR method interface in Spinsolve. The interface is organized into distinct areas: (i) the Measurement / Data Area, where the sample ID is entered, the measurement is initiated or data is loaded; (ii) the Configuration / Results Area, where the method can be configured, results are displayed; and (iii) the Spectra Area, where the acquired spectra are shown together with the defined integration ranges and measurement parameters. Upon completion of the measurement, a PDF report with built-in data integrity verification is generated automatically.
Conclusion
[1] Czechowska-Biskup, R., Jarosińska, D., Rokita, B., Ulański, P., & Rosiak, J. M. (2012). Progress on Chemistry and Application
of Chitin and its Derivatives, 17, 5-20.
[2] El-Araby, A., Janati, W., Ullah, R., Ercisli, S. & Errachidi, F. (2024). Frontiers in Chemistry, 11, 1327426.
[3] Lavertu, M., Xia, Z., Serreqi, A. N., Berrada, M., Rodrigues, A., Wang, D. & Gupta, A. (2003). Journal of Pharmaceutical and
Biomedical Analysis, 32(6), 1149-1158.
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