Comparison of Amylose Determination Methods and the Development of a Dual Wavelength Iodine Binding Technique

It has long been recognized that limitations exist in the analytical methodology for amylose determination. This study was conducted to evaluate various amylose determination methods. Purified amylose and amylopectin fractions were obtained from corn, rice, wheat, and potato and then mixed in proportion to make 10, 20, 30, 50, and 80% amylose content starch samples for each source. These samples, considered amylose standards, were analyzed using differential scanning calorimetry (DSC), high-performance size-exclusion chromatography (HPSEC), and iodine binding procedures to generate standard curves for each of the methods. A single DSC standard equation for cereal starches was developed. The standard curve of potato starch was significantly different. Amylose standard curves prepared using the iodine binding method were also similar for the cereal starches, but different for potato starch. An iodine binding procedure using wavelengths at 620 nm and 510 nm increased the precision of the method. When HPSEC was used to determine % amylose, calculations based on dividing the injected starch mass by amylose peak mass, rather than calculations based on the apparent amylose/amylopectin ratio, decreased the inaccuracies associated with sample dispersion and made the generation of a cereal amylose standard curve possible. Amylose contents of pure starch, starch mixtures from different sources with different amylose ranges, and tortillas were measured using DSC, HPSEC, iodine binding, and the Megazyme amylose/amylopectin kit. All the methods were reproducible (±3.0%). Amylose contents measured by these methods were significantly different (P < 0.05). Amylose measurements using iodine binding, DSC, and Megazyme procedures were highly correlated (correlation coefficient >0.95). DSC and traditional iodine binding procedures likely overestimated true amylose contents as residual butanol in the amylose standards caused interference. The modified two-wavelength iodine binding procedure seemed to be the most precise and generally applicable method. Each amylose determination method has its benefits and limitations.