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Appendix III

Appendix III

Appendix III PURPOSE 1.1 To determine the degree of deacetylation of chitosan using uv-vis spectrophotometry. 1.1.1. 1.1.2. 2. The degree of deacet...

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Appendix III

PURPOSE 1.1 To determine the degree of deacetylation of chitosan using uv-vis spectrophotometry. 1.1.1. 1.1.2.


The degree of deacetylation of chitosan defines many properties of chitosan including the reactivity, processing and biodegradability. This protocol is intended as a simple, rapid, reliable and cost efficient method to determine the degree of deacetylation of chitosan.

SCOPE 2.1 All shellfish derived sources of chitosan from commercial vendors or from other shellfish processors and fungal chitosan derived in-house.


RESPONSIBILITY 3.1 All students and staff of Dr. E. Khor' s research laboratory.


SAFETY 4.1 All acids and bases must be handled in the fume-hood. 4.2 Safety glasses and gloves must be worn at all times.


EXPERIMENTAL 5.1 Equipment 5.1.1.

Uv-vis spectrophotometer: The wavelength range should cover the entire visible and near uv with a lower wavelength cutoff of at least 150nm. The slit width should be l nm or better and the path-length of 10mm as standard for the sample cuvette. The spectrophotometer should have software capable of converting the absorption spectrum into the first derivative spectrum and superimposing spectra.


Appendix III


5.2 5.2.1 5.2.2

Reagents and Materials

5.2.3 5.2.4 5.2.5 5.2.6 5.2.7 5.2.8

Acetic acid (Spectra grade). Only high purity chitosan that is readily soluble in dilute acetic acid shall be used. 99% minimum N-acetyl-D-glucosamine. 99% D-glucosamine hydrochloride. Distilled water. Glass cuvette for uv-vis spectrophotometers with a pathlength of 10mm. 100 ml volumetric flask. 10 ml volumetric flask.




A 100ml (volumetric) sample solution is obtained by dissolving l mg of freeze-dried chitosan sample in 10ml of 0.1 M acetic acid and subsequently topped up to 100ml with distilled water.

Zero Point Crossing (ZPC)


The first derivative spectra of 0.010, 0.020 and 0.030 M of acetic acid solutions in the range of 190nm to 250nm. Fix the zero crossing point (ZPC) at 203 nm.

Calibration curve 5.3.3


Prepare reference solutions of 0.005, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.045 and 0.050 mg of GIcNAc per ml of 0.01 M acetic acid. Obtain the first derivative spectra of these solutions in the range of 190nm to 250nm.

Appendix III

5.3.5 5.3.6


Superimpose all 10 spectra and determine the vertical distance from the ZPC to each reference solution, H~ to Hi0 in mm. Plot the H1 to H~0 values against their corresponding concentrations to obtain a linear calibration curve.

Sample solutions 5.3.7

Obtain the first derivative spectra of sample solutions in the range of 190nm to 250nm. Determine the H value for each sample as outlined in Calibration Curve. This is the contribution of GlcNAc to the chitosan sample. Perform five replicates and calculate the average for each sample.

Determination of the DD of chitosan 5.3.8

Determine the DD of the samples by the formula, DD = 100 - [A/(W 204 A)/161 + A] x 100), Where: A is the amount of GlcNAc determined/204, and W is the mass of chitosan sample used.

Correction of the effect of GlcN on H values 5.3.9

5.3.10 5.3.11

5.3.12 5.3.13 5.3.14 5.3.15

The presence of GIcN will give rise to a larger H value for GlcNAc than expected. Therefore, obtain a reference curve for correcting this discrepancy, as follows: Prepare a stock 0.10 mg GlcNAc/ml of 0.01 M acetic acid solution. To 5 x 10 ml of the stock solution, add separately 99, 19, 9, 5.67 and 4 mg of GIcN to give a 1%, 5%, 10%, 15% and 20% GIcNAc solutions (w/w), respectively. Make similarly other solutions of intermediate GIcNAc (%) concentrations, if deemed necessary Obtain the first derivative spectra for all these solutions. Measure the H values of the pure GIcNAc solution, H~, and the H values of the solutions of different percentages of GIcNAc, H2. Plot the reference curve H~/H2 versus the corresponding GIcNAc percentage Apply the correction to the H value obtained in 5.3.7 to obtain the final value for GIcNAc for the DD calculation in 5.3.8.

Appendix III



6.1 The make of the uv-vis spectrophotometer. 6.2 The source of the N-acetyl-D-glucosamine and D-glucosamine hydrochloride references. 6.3 The first derivative spectra of acetic acid (5.3.2), references for calibration and correction and samples. 6.4 Calibration and correction curves. 6.5 The DD of the sample. [Note: enter directly into laboratory notebook; spectra stored in data file properly referenced to laboratory notebook.] 7.


7.1 RAA Muzzarelli, R Rochetti, Determination of the degree of acetylation of chitosan by first derivative ultraviolet spectrophotometry. Carbohydrate Polymers 5 (1985) 461-472. 7.2 SC Tan, E. Khor, TK Tan, SM Wong, The degree of deacetylation of chitosan: Advocating the first derivative UV-spectrophotometry method of determination. Yalanta 45 (1998) 713-719.