There are a number areas to look at in order to ensure your analysis performs well with reproducible results.
Firstly, review your standards. The absorbance of the standards should be ~ 1, this ensures that the colour development step was performed correctly.
The method should be run as detailed in the data booklet so any modifications that may have been introduced should be also reviewed.
We recommend following method (A) for most samples (those not containing resistant starch), and method (B) for any samples containing resistant starch.
The samples should be weighed directly into the assay tube, do not use a weigh boat to weigh and then transfer as this could cause discrepancy with the sample weight. Ensure to record the exact weight.
The step with NaOH incubation must be carried out carefully so that the sample is homogeneous and gelling of the sample does not occur. If the sample is not homogeneous and lumps occur or the sample is sticking to the side of the tube, this will lead to unreliable results.
The samples should be weighed directly into the assay tube, do not use a weigh boat to weigh and then transfer as this could cause discrepancy with the sample weight. Ensure to record the exact weight.
The step with NaOH incubation must be carried out carefully so that the sample is homogeneous and gelling of the sample does not occur. If the sample is not homogeneous and lumps occur or the sample is sticking to the side of the tube, this will lead to unreliable results.
We recommend vortexing the sample immediately upon addition of the NaOH to aid in the dissolution and ensure that the magnetic stir bars are able to stir in the tube during the 15min incubation in the ice water bath.
The addition of ethanol is also a very important step as it aids in wetting/dispersing the sample. This is particularly important for samples with a high starch content like regular maize starch (kit control).
The addition of ethanol is also a very important step as it aids in wetting/dispersing the sample. This is particularly important for samples with a high starch content like regular maize starch (kit control).
We would recommend to watch the K-TSTA training video (Total Starch Video Method with K-TSTA) and to repeat the analysis exactly as described in the assay protocol.