Hii...Amit..

You must get higher absorbance at higher concentration of glucose than low concentration..

I think problem is with manual error while preparing glucose standards. If your highest glucose concentration is 600mg/ml then dilute it serially for subsequent dilutions by its half, i mean add equal vol buffer to yield 300 mg/ml, then 150, 75, etc.) . 

Also you can try the DNS reagent, containing 1% dinitrosalicylic acid, 0.2% phenol, 0.05% sodium sulfite, and 1% sodium hydroxide (Miller, 1959). 

Hope this will solve your problem.

For detail information about DNS reagent, go through attachment.

Thanks 

6/18/15

Dear Amit,

I would suggest taking a look at this reference (attached file):

Hindawi Publishing Corporation

International Journal of Analytical Chemistry

Volume 2011, Article ID 283658, 4 pages

doi:10.1155/2011/283658

Research Article

Comparison of TwoMethods for Assaying Reducing Sugars in

the Determination of Carbohydrase Activities

Alexander V. Gusakov,1 Elena G. Kondratyeva,2 and Arkady P. Sinitsyn1, 2

1Department of Chemistry, M. V. Lomonosov Moscow State University, Moscow 119991, Russia

2 A. N. Bach Institute of Biochemistry, Russian Academy of Sciences,Moscow 119071, Russia

Correspondence should be addressed to Alexander V. Gusakov, [email protected]

Received 17 January 2011; Accepted 6 April 2011

Academic Editor: P. Haglund

Copyright © 2011 Alexander V. Gusakov et al. This is an open access article distributed under the Creative Commons Attribution

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly

cited.

The Nelson-Somogyi (NS) and 3,5-dinitrosalicylic acid (DNS) assays for reducing sugars are widely used in measurements of

carbohydrase activities against different polysaccharides. Using twelve commercial enzyme preparations, the comparison of the NS

and DNS assays in determination of cellulase, β-glucanase, xylanase, and β-mannanase activities was carried out. When cellulase

activities against CMC were measured, the DNS assay gave activity values, which were typically 40–50% higher than those obtained

with the NS assay. In the analysis of the xylanase, β-mannanase, and β-glucanase activities, the overestimations by the DNS assay

were much more pronounced (the observed differences in the activities were 3- to 13-fold). Reasons for preferential use of the NS

assay for measuring activities of carbohydrases other than cellulases are discussed.

The paper discusses differences between the DNS and Nelson-Somogyi (NS) assays for reducing sugars. DNS assay has some issues that can compromise accurate quantitation. It might be a good idea to try the NS method instead. It is an old procedure (So is the DNS assay), but it is also more sensitive and less prone to the problems sometimes encountered w/ DNS.

I hope this will help you.

Bill Colonna

Iowa State University, Ames, IA, USA

Thanks sir for valuable suggestions

Take care in preparing the standard. This reagent is used in my lab for long time and we have not faced such problem. It is better you used DNS of Merck.

Kindly send me the method by which you prepare DNS solution

send me your e.mail

Respected Dariush Norouzian

Pls find  my email

[email protected]

please also email me [email protected] Thank you

I think the problem here is too high concentrate of glucose. You should use concentrate of glucose standard curve lower than 2mg/mL.

Thanks .....

Any way I was having some reagent issue and I solved that.