Im going to test cereal leaf cellulose using updegraff, seaman hydrolysis and anthrone test. But before that, i need to process my samples to obtain alcohol insoluble residue (AIR). I am using fresh leaf. The first step is to incubate the freeze dried powder samples in 70% ethanol at 70 degree celcius for an hour, discard supernatant and repeat again. Then wash with 70% ethanol 3 times. The wash with chloroform:methanol 1:1 twice, followed by acetone 3 times before drying in oven at 37C overnight.
My problem is even after i incubate at 70C twice, the supernatant (70%ethanol) still turn green). Should i repeat the incubation until the solution is colorless or should i just proceed with the next step?
From the look of the photo you posted you need to increase the amount of solvent being used. Generally aim for about 15:1 to 20:1 v/w. The other clue of course is that you are still seeing green (chlorophyll) in your extract. The acetone will also extract residual chlorophyll. A good rule of thumb is to continue to extract with 70% EtOH until there is no colour in the extract. This will not impact the final AIR.
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