SPDS Aloe Vera Method Book

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Procedure Title: Determination of Absolute Molecular Weight and Content of Polysaccharides in Aloe vera Fresh Leaf and Concentrated Juice Powder by SEC-MALS-RI

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Prepared By: Yuehong Zhang, Zhichao Bao Department: R&D Applies To: Herbalife International

CONTENTS Procedures Definitions References Forms/Chromatograms Ownership/Management Sign-off Revision History

SECTION 1.0 2.0 3.0 4.0 5.0 6.0

PURPOSE The purpose of the test method is to determine the absolute molecular weight, the molecular weight distribution and the content of polysaccharides in Aloe ver a fresh leaf and concentrated juice powder by Size Exclusion Chromatography Coupling with Multi-Angle Laser Light Scattering and Refractive Index Detectors (SEC-MALS-RI).

REAGENTS 

Bovine serum albumin standard solution (5mg/mL), China National Institute of Metrology, CAS#BW3627-10 or equivalent  Sodium chloride, Sinopharm Reagents, Catalog# 10019318 or equivalent  Methanol, HPLC grade  Ultrapure water or equivalent EQUIPMENT  Analytical Balance  Nitrocellulose Membrane, 0.22μm GSWP or equivalent  Disposable Syringe filters, 25mm, 0.45 μm Hydrophilic PTFE membrane filter or equivalent  Disposable Syringes (1mL)  Disposable Plastic Pipette  Vortex mixer  SCIENTZ-12N Lyophilizer  25 mL Volumetric Flask  5 mL Volumetric Flask  1.5 mL Glass HPLC Vials HPLC  HPLC System: Waters 2695 Alliance Separations Module consisting of pump and auto sampler and an inline 0.10 μm x 25 mm Durapore membrane filter or equivalent  Waters 2414 refractive index detector or equivalent  Wyatt Technology DAWN-HELEOS-II multi-angle laser light scattering (MALS) detector equipped with 100 mW 662nm Ga/As laser and 18 angle research grade light scattering photometer  Size Exclusion Chromatographic (SEC) Column: Phenomenex PolySep™-SEC GFC-P Linear, LC Column 300 x 7.8 mm, Product No. CHO-9232 or equivalent  Guard Column: Phenomenex PolySep™-SEC GFC-P Guard, LC Guard Column 35 x 7.8 mm, Product No. CHO-9225 or equivalent  HPLC chromatographic conditions: - Mobile phase: 100mM Sodium chloride solution - Flow rate: 0.7 mL/min

- Column temperature: 35 o C - Sample temperature: 20 o C - Refractive index detector temperature: 35 o C - Injection volume: 100 μL - Run time: 30 minutes

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