I have tried the iodine-starch method, gained relatively high amylose, about 60% amylose content for nomal starch. Who knowns why?
and the standard amylose and amylopectin must be of the same origin? i can't find the according standard of my variety, as it is a underutilized starch. if i prepared it by myself, purity is also a problem.
I use high performance size exclusion chromatography to determine the amylose content of many types of starches. Using HP-SEC you can also get molecular size information.
The iodine binding method tends to overestimate the amylose content because iodine will also bind to the long chains of amylopectin. The origin of the starch does make a difference when using the iodine binding method.
I recommend amperometric titration with iodine according to B.L. Larson, K.A. Gilles & R. Jenness, Anal. Chem. 25 (1953) 802-804. Advantage over potentiometric titration is the simpler data evaluation, whilst amylose and amylopectin standards as in iodine colorimetric assays are not (strictly) necessary. Some remarks have to be made:
1. It is preferred to use KIO3/KI in acidic medium as a reagent rather than I2/KI because of its much better stability and simpler handling. I2 is generated in situ from KIO3. See Y. Takeda, S. Hizukuri & B.O. Juliano, Carbohydr. Res. 138 (1987) 79-88.
2. Amylose content is calculated by assuming an iodine binding capacity (IBC) of natural amylose of 20 %. This is a reasonable approximation for natural amyloses of high degree of polymerization (DP). For precise results an amylose standard is analysed. Potato amylose is preferred because of its purity. As amylose is insoluble even in hot water, it is recommended to disperse it in water, add the calculated amount of aqueous NaOH, and neutralize after dilution with water. [Aqueous amylose is stable at concentrations < 0.2 %.]
3. The method has to be adjusted for cereal starches with complexed lipids as the latter diminish IBC. Complexed lipids can be traced by DSC (20-33 % starch in water) as these give rise to a (reversible) melting endotherm at 100-110 C. These lipids can be removed with hot 75 % n-propanol as described by W.R. Morrison & A.M. Coventry, Starch 37 (1985) 83-87.
Thanks so much for valuable suggestion!
I have a idea, Idione binding method using a correction index, as idione bingding is much simpler and easy to handle than other methods.
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