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How to test chitosan oligosaccharide's content by chemical methods?

Posted by:bozhihuili    Published:2014-06-08 18:15

1. Elson-Morgan
Elson-Morgan is the classic method of determination of glucosamine. And it has good specificity on glucosamine. Its 
principle is that the acetylacetone alkaline solution makes amino acetylation of glucosamine molecules, generate the 
original 2 - methyl - 3-2 b pyrrole derivatives, And then it looks red when the action between the original and the 
dimethyl amino benzene formaldehyde under acid condition.

There has a good linear relationship between absorbance value (Y) and glucosamine hydrochloride 
content (X, mu g)

Calculation formula of the content of Chitosan oligosaccharide:

m- The weight of glucosamine hydrochloride indicated by the standard curve
M- The weight of samples of chitosan oligosaccharide
0.8309 - coefficient of glucosamine converted by Glucosamine hydrochloride

The content of glucosamine after COS hydrolysis was determined by Elson - Morgan, the recovery rate is only 37.33% ~ 
45.94%, even if the test conditions are optimized, the recovery rate is still less than 50%

Main reasons: The structure of glucosamine is destructed in the process of acid hydrolysis ofCOS, so that the chromogenic 

reaction cannot proceed. And acetylglucosamine still exist afterCOShydrolysis, which makes the chromogenic reaction unable 

to proceed. Therefore Elson - Morgan method, is unsuited to determine the accurate content ofCOS

2. Phenol - sulfuric acid method
Phenol - sulfuric acid method is commonly used in measuring the content of oligosaccharide, polysaccharide. Its principle: 

the polysaccharides or oligosaccharide hydrolyzes into monosaccharide under the action of sulfuric acid, furfural derivatives 

generated quickly, and then orange compound, measured by colorimetric method.

Linear equation between Absorbance (Y) and glucosamine hydrochloride content (X, mu g):
Y = 0.0004X + 0.0724      r = 0.9977

The phenol-sulfuric acid method, low sensitivity, its measuring range is 200 ~ 3000 ug, the calculation formula of COS 

CONTENT is the same as Elson - Morgan 

The recovery rate is 106.7% ~ 106.7% determined by Phenol-sulfuric acid method, the average recovery is 109.4%, 

the RSD is 2.1%, but the phenol - sulfuric acid method is simple, convenient, good repeatability, applied in the measurement 

of COS content.

3. DNS

DNS method is commonly used in measuring the content of reducing sugar; there are a wide range of applications. Its 

principle: reducing sugars containing free aldehyde or ketone group are oxidated into saccharic acid and other products 

under the condition of alkaline and heating, 3, 5 - 2 nitro salicylic acid is reduced into red-brown amino - 5-3 - nitro salicylic 

acid. Within a certain range, the amount of reducing sugars is proportional to the color depth of palm red material.

DNS method, there also has a good linear relationship between absorbance (Y) and glucosamine hydrochloride content 

(X, mu g) , the standard curve equation is:

Y = 0.2192X - 0.0077     r = 0.9948

Calculation formula of the content of Chitosan oligosaccharide:

m- The weight of glucosamine hydrochloride indicated by the standard curve 
f- Dilution ratio of the sample solution in the process of measurement.
M- The weight of the samples of COS
0.994- The coefficient of polysaccharide converted by monosaccharide

For DNS method of measurement of chitosan oligosaccharide, the recovery rate is between 93.4% ~ 100.7%, the RSD 

is 2.9%., DNS method is high recovery rate, good repeatability, which can be applied to the content of COS. But in the 

process of measurement, the hydrolysis of COS is important, especially to the operation of the sulfuric acid. It shall be in

the iced water, during the neutralization process, otherwise the structure of glucosamine will be destructed by the sulfuric 

acid, and disturb the measurement. 

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