Research on amylase structure has been carried out using HPSEC coupled with two detectors: multiangle laser light scattering and refractive index. Amylose powders isolated from starches with diverse botanical origins (cassava, potato, wheat, normal maize, and smooth-seeded peas) by the thymol and n-butanol complexation method, dispersed in 1 M KOH and diluted in 0.1 M KOH, have been characterized by combined systems of size-exclusion chromatography and MALLS (Roger and Colonna, 1993). In this method, pullulan and dextran commercial standards are used to assess the validity of the HPSEC-MALLS experiments and a semi-logarithmic plot of Mw versus elution volume at the maximum of the RI peak as the reference. For the same elution volume (i.e., hydrodynamic size), the branched polymer exhibits a higher Mw than the linear one due to the higher density compactness, leading to some conclusions about the sample structure. By this method, it was possible to concludethat amylose from cassava and wheat presented a higher average molar mass (1.2 and 1.1 × 106 g/mol, respectively) than commercial amylose (4 ×105 g/mol) (Roger and Colonna, 1993), whereas Takeda, Shitaozono, and Hizukuri (1988) reported Mw 4 × 105 g/mol for amylose from corn starch. However, decrease of Mw in amylose from cassava starch from 1.2 × 106 g/mol to 1.05 × 106 g/mol after ultracentrifugation allowed Roger and Colonna (1993) to conclude that large aggregates eluted at low elution volume often cause contamination of the amylose solution in the amylopectin fraction, which can be reduced by ultracentrifugation (Roger et al., 1996).
SLS and HPSEC-MALLS-RI systems are also employed in starch structure characterization. Both were used to studied amyloses synthesized by phosphorylase from malto oligomers as the primer and glucose-1-phosphate as the substrate, where the Mw ranged between 1.3 × 105 and 1.0 × 106 g/mol and Rg was between 24 and 71 nm. Synthetic amyloses show a broader unimodal molar mass distribution, with a polydispersity similar to that obtained from native starch (Radosta, Haberer, and Vorwerg, 2001).
However, the parameter most commonly used to evaluate amylose structure is the molecular weight and dynamic behavior in solution. Using SLS and HPSEC-MALLS-RI systems, Mw values of amylose from three cultivars of rice starch ranged from 3.12 and 3.44 × 106 g/mol with Rg between 86 and 103.3 nm (Zhong et al. 2006). These systems have also been employed to study the impact of temperature on the amylose structure during its fractionating. Pea starch, which has a high amylose content, showed that Mw (15.7 and 18.5 × 106 g/mol) and recovery percentage of amylose increase as isolation temperature rises (Vorwerg, Radosta, and Leibnitz, 2002).