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.
I'm certain that you have gotten the answer to your problem by now. For anyone else facing a similar problem, this worked for me:
1) Prepare the 0.3% anthrone solution by dissolving powdered anthrone in concentrated sulfuric acid. The sulfuric acid does not have to be cold prior to this. Tightly cap the mixture in a bottle, vortex to disperse any clumps. Allow the mixture to sit on ice for about 15 minutes. Any remaining precipitates will dissolve.
2) Add 750ul of cold anthrone reagent to 375ul of glucose standard slowly. Do this by touching the wall of the tube with your pipette tip and dispensing the reagent in a slow, steady stream. The reagent will settle and collect at the bottom of your tube and form a layer below your glucose standard. There will be some mixing towards the top. After which, immediately cap and invert twice, before keeping on ice. Keep on ice until you have completed adding the anthrone reagent to all samples. Before boiling in a water bath, invert 3 times again.
- Badges
- Science topic
- Similar topics
- Social Psychology
- Attitude