Understanding the intricate structure of starch involves a crucial step: the fractionation of amylose and amylopectin. This process unveils the distinct characteristics of these macromolecules, shedding light on the composition of starch. In this exploration, we delve into the various techniques employed for the precise fractionation of amylose and amylopectin.
Traditional Amylose and Amylopectin Fractionation
The Schoch traditional method, devised in 1964, remains a cornerstone in starch fractionation. This technique, with slight modifications based on amylose/amylopectin ratios, involves solubilizing samples in a 90% DMSO solution, precipitating in methyl alcohol, and redissolving in distilled water. Adjusting the pH with phosphate buffer, autoclaving, and stirring disperse starch molecules. The formation of an amylose–butyl–alcohol complex, recoverable by centrifugation, separates amylose from amylopectin. The residual amylopectin is then concentrated and treated to eliminate amylose residues.
Thymol and Butanol Method
The method proposed by Banks and Greenwood in 1967, utilizing thymol and butanol for amylose complexation, is widely applied on diverse starches. Starch varieties such as cassava, amaranth, potato, wheat, normal maize, and smooth-seeded peas undergo this method. While effective, evaluation using HPSEC-MALLS-RI has revealed potential challenges, particularly the presence of large amylopectin aggregates in purified amylose solutions. Additional steps, such as ultracentrifugation, have been suggested to enhance purity.
Ultracentrifugation and Prewarming Modifications
For cereal starches like wheat and maize, a modification of the thymol and butanol method has been proposed. This involves additional purification steps employing ultracentrifugation at 10,000 g for 1 hour and prewarming at 40°C. These adjustments enhance the precision of amylose and amylopectin fractionation.
Gel Filtration Chromatography
Gel filtration chromatography emerges as a modern technique for amylose and amylopectin fractionation. This method, compatible with the Schoch (1942) and Banks and Greenwood (1967) techniques, evaluates the purity of both macromolecules post-fractionation. The fractions containing amylopectin and amylose can be pooled, concentrated, and freeze-dried for comprehensive analysis.
Conclusion: Navigating the Landscape of Starch Structure
In the realm of starch structure elucidation, the fractionation of amylose and amylopectin stands as a pivotal chapter. Traditional methods like the Schoch approach and innovative techniques such as thymol and butanol complexation, ultracentrifugation modifications, and gel filtration chromatography offer diverse avenues for isolating these macromolecules. Depending on the source and desired purity, a judicious combination of these techniques opens doors to highly purified samples, paving the way for nuanced starch analysis.