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Handbook of Size Exclusion Chromatography and Related ...

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Both absolute approaches are extraordinarily sensitive towards high<br />

molecular components, in particular towards glucan aggregates. Thus, the<br />

molecular weight <strong>of</strong> superstructures <strong>and</strong> not <strong>of</strong> the constituting molecules will<br />

primarilybedeterminedusingthesetechniques.Analternativeapproachtoobtain<br />

information about molecular weight <strong>of</strong> constituting glucans is a combined<br />

chemical/analytical one:<br />

. Quantitative derivatization <strong>of</strong> each glucan molecule combining specific<br />

molar detection (e.g., UV/VIS or fluorescence <strong>of</strong> aunique chromophor<br />

in each molecule) <strong>and</strong> universal mass detection (SEC-mass/molar).<br />

SEC elution pr<strong>of</strong>iles are also affected by variations in the branching pattern <strong>and</strong><br />

the more or less pronounced presence <strong>of</strong> supermolecular structures. However,<br />

these phenomena are superimposed, somewhat secondary influences, which need<br />

complementary analyses to evaluate them from SEC experiments.<br />

5 STARCH GLUCAN ANALYSIS: EXPERIMENTAL<br />

APPROACH<br />

Benefits<strong>and</strong>limitations<strong>of</strong>SECinstarchglucancharacterizationwillbediscussed<br />

<strong>and</strong>illustratedforwheatstarchglucaninthefollowingsections.Wheatstarchisan<br />

important industrial source <strong>of</strong> starch, <strong>and</strong> is amix <strong>of</strong> lcb/scb-glucans <strong>and</strong>, thus,<br />

shows amix <strong>of</strong> characteristics from both “extreme” components. In particular,<br />

results from different detector combinations with SEC providing fraction<br />

informationwillbecomparedtodatafrombulktechniqueswhichprovideintegral<br />

characteristics (Table 7).<br />

5.1 Preparative SEC: Purification <strong>and</strong> Pooling<br />

Mass detection <strong>of</strong> separated glucan fractions in preparative SEC is typically<br />

obtained <strong>of</strong>f-line as total carbohydrates (Fig. 8). Therefore an equivalent <strong>of</strong> 1mL<br />

<strong>of</strong> each fraction is mixed with 2 mL anthron reagent (200 mg crystalline anthron<br />

pA dissolved in 100 mL H2SO4 96% pA) <strong>and</strong> heated for 10 min in a boiling water<br />

bath. After cooling <strong>and</strong> degassing by ultrasound, extinction at 540 nm is<br />

determined. Carbohydrate concentration is obtained from extinction values via<br />

calibration with D-glucose (51,52).<br />

5.2 Semipreparative SEC: Branching Analysis<br />

Whereas the granular consistency <strong>of</strong> starch glucans typically is considered to be<br />

rather relevant for starch material quality, importance <strong>of</strong> molecular level<br />

characteristics such as branching pattern, potential to form supermolecular<br />

© 2004 by Marcel Dekker, Inc.

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