I'm trying to quantify cellulose in rice leaf. i'm using nitric acetic method, followed by 67% sulfuric acid digestion, then using anthrone reagent to quantify the glucose released.

My sample and filter paper positive control produce clear blue solution but my glucose standard produced whitish precipitate immediately after anthrone reagent was added. What went wrong with my glucose standard? i tried to centrifuge it but the precipitation floats up instead of settling at bottom of tube, so even when i tried to pipette out the solution, a bit of the precipitate still get pipette in, so this affects the absorbance reading. i tried several times but yield same result for standard. The anthrone protocol used is as follows:

1) prepare 0.3% anthrone reagent by adding cold sulfuric acid (i cooled sulfuric acid on ice before use), then put the yellow anthrone reagent on ice until use.

2)add 1ml cold anthrone solution to 1ml glucose standard. I added anthrone reagent slowly. This is done in fumehood and the test tubes for the mixture was arranged on a rack(not on ice). then when finish adding into all standards, i vortexed all tubes and put in boiling water bath for 5min, then back on ice to cool before transferring to cuvette.

What went wrong with my protocol? Please advise, any opinions are welcomed and appreciated.

Thank you.