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  • Title: [Development of an asymmetrical flow field-flow fractionation system for the size characterization of starch granules].
    Author: Guo Y, Song T, Sun Y, Yu Q, Dou H.
    Journal: Se Pu; 2021 Nov; 39(11):1247-1254. PubMed ID: 34677020.
    Abstract:
    Starch occurs naturally in the form of semicrystalline granules, and is composed of two types of carbohydrate molecules, amylose (AM) and amylopectin (AP). Starch granules and starch molecules have sizes in the range of 1-100 μm and 20-250 nm, respectively; these size ranges are among the key factors affecting the functional properties of starch. Asymmetrical flow field-flow fractionation (AF4) is a size-based separation technique. The major difference between AF4 and dynamic light scattering or microscopy techniques is that AF4 enables the separation of particles based on their size; consequently, the elution profile can be converted to the size distribution of the samples. In the last two decades, AF4 systems, when coupled online with multiangle light scattering (MALS) and differential refractive index (dRI) detectors (AF4-MALS-dRI), have demonstrated to be applicable for the size characterization of starch at the molecular level. Unlike size exclusion chromatography (SEC), AF4 systems use an open channel that does not require a stationary phase or packing materials. Thus, the shear scission of AP molecules during AF4 separation is minimized. The size detection range of a commercial AF4 system ranges from 1 nm to 10 μm, which is smaller than the size range of starch granules. In this study, a home-made AF4 system was developed, and its capability for the size characterization of starch granules extracted from sweet potato, lotus seed, and rice was investigated. The performance of the developed AF4 system was evaluated by running a mixture of polystyrene (PS) with diameter of 2, 6, 12, and 20 μm, respectively. Baseline separation of four PS samples was achieved, and the resolution for 6 μm PS and 12 μm PS was 1.40. The detection limit of the developed AF4 system was higher than that of commercial AF4 systems. Thus, the developed AF4 system is promising for the separation and characterization of starch granules. The effect of the composition of the carrier liquid on the AF4 separation of starch granules was also studied. Moreover, the accuracy of AF4 in terms of size characterization of the starch granules was evaluated by optical microscopy (OM). The results revealed that the type of dispersant and viscosity of the carrier liquid affect the accuracy of size characterization of the starch granules. The size distribution of rice starch granules obtained using a carrier liquid containing 0.01% (w/v) sodium dodecyl sulfate (used as a dispersant), 0.02% (w/v) NaN3 (used as a bactericide), and 0.001% (w/v) hydroxypropylmethylcellulose (used to adjust the viscosity of the carrier liquid) was in agreement with that obtained from OM. Furthermore, a commercial AF4 system coupled with MALS and dRI detectors was employed for the separation and characterization of starch molecules. A molecularly dispersed solution is necessary for the reliable molecular characterization of starch. The effect of the starch dissolution temperature on the AF4 characterization of starch was also investigated. The optimal dissolution temperature for lotus seed and rice starch granules was 75 ℃, while that for sweet potato starch granules was 78 ℃; this difference is mainly attributed to the different botanical origins of the granules. The results showed that the ratio of the radius of gyration (Rg) to the hydrodynamic radius (Rh) of rice starch and sweet potato starch is in the range of 0.9-1.1 over the molar mass range of 10 6-108 g/mol. For rice starch, the Rg/Rh ratio is between 1.2 and 1.4. Rice starch has the highest apparent density among the three starches, indicating that rice starch molecules have a dense structure. The results demonstrated that the AF4 system developed in this study is rapid and accurate for the size characterization of starch granules. The developed AF4 system, when combined with commercial AF4 systems coupled online with MALS and dRI detectors, can provide technical support to study the relationship between the size from the nanoscale to the microscale and functional properties of starch. 淀粉颗粒粒径与分子尺寸分别在1~100 μm和20~250 nm之间,是影响淀粉功能特性的重要因素之一。非对称场流分离(AF4)是一种基于样品与外力场相互作用机制的分离技术,已应用于表征淀粉分子尺寸分布。商品化的AF4系统的粒径检测范围为1 nm~10 μm,对于淀粉颗粒粒径表征具有一定的局限性。该文研制了AF4分离系统;考察了其在微米尺度下对红薯、莲子和大米淀粉颗粒粒径表征的性能;采用微米尺寸的聚苯乙烯乳化球(PS)标准样品验证了构建的AF4系统的分离性能。实验结果显示,构建的AF4系统对PS混合样品(粒径2、6、12、20 μm)实现了基线分离,同商品化AF4相比提高了检测上线,具有分离表征淀粉颗粒的潜力。此外,该文研究了载液组成对淀粉颗粒分离表征的影响;通过光学显微镜验证了构建的AF4系统在微米尺度上对淀粉颗粒粒径分布的表征能力。最后,采用商品化的AF4系统串联多角度激光光散射检测器和示差折光检测器对3种淀粉分子进行了分离表征,考察了淀粉的溶解温度对其表征结果的影响。在摩尔质量10 6~108 g/mol范围内,红薯和莲子淀粉的回转半径和水合半径的比值(Rg/Rh)在0.9~1.1之间,大米淀粉的Rg/Rh在1.2~1.4之间。实验结果证明构建的AF4系统是一种快速、准确的淀粉颗粒粒径表征方法,与商品化的AF4系统结合可为研究淀粉尺寸分布与其功能性质之间的关系提供技术支持。
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