Figure 1
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Starch is composed of two macromolecules differing in size and structure. Amylose is a weakly branched polymer and amylopectin
is a highly branched polymer with a higher molecular weight than amylose. Natural starch characterization with SEC/GPC is
a challenge, because of the high number of reactive groups present in the chains, the high molar mass and the structure. Only
a well balanced SEC/GPC method, based on optimized stationary phase and solvent polarity, allows true SEC/GPC separation by
size and not by interaction or by a mixed mode of SEC/GPC and LAC.
Sample preparation
Exact sample preparation is very important for starch characterization. To make sure that the sample is fully dissolved and
not decomposed one of the established procedures has to be strictly adhered to. In this instance the samples were dissolved
in DMSO at 80 °C for 8 hours and then at 120 °C for 4 hours. This procedure ensures that the sample is representative.
Experimental:SEC/GPC analysis was performed on a PSS GPC 1100 System.
The analysis conditions were:
Columns: PSS GRAM, 10 μm, 30000 Å, 8 × 300 mm + precolumn
Solvent: DMSO with 5 g/L LiBr
Flow-rate: 0.5 mL/min
Temperature: 60 °C
Sample: amylopectin, amylose and potato starch
Calibration: PSS pullulan calibration kit
10 calibration standards: 342–710000 Da
Concentration: 0.5 g/L
Inject volume: 100 μL
Data acquisition: PSS WinGPC Unity
Results and Conclusion
Figure 2:
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Three different types of starches were separated on PSS GRAM columns with DMSO, 5 g/L LiBr as mobile phase. Calibration with
polysaccharides (pullulanes) allows the relative determination of the molar mass distributions (Figure 1).
A light-scattering detector was used to check if the sample preparation had been successful and if true SEC/GPC separation
is achieved. Light-scattering detectors are molar mass sensitive detectors. These detectors are ideal for high molecular weight
samples and would detect non-dissolved particles with high sensitivity. Additionally they can measure the molar mass directly,
if the refractive index increment, dn/dc, is known. For high molar masses large light scattering signals are expected, for
low molar masses small signals are expected. Figure 2 shows the chromatogram of potato starch with the light scattering and
refractive index signal. This figure shows that the separation on the GRAM columns is based on true SEC/GPC mode: the large
molecules elute first and show a large light scattering signal, the smaller molecules elute at the end and show a small signal.
Sample preparation has been successful since no non-dissolved particles can be detected by the light scattering detector.
Whereas, sample decomposition can be excluded because the refractive index detector does not show signal in the low molecular
weight region after the main peak.
Dr Günter Reinhold, PSS Polymer Standards Service, Mainz, Germany.
PSS Polymer Standards Service GmbH
In der Dalheimer Wiese 5, Mainz, Germany
tel. +49 6131 96239 0 fax +49 6131 96239 11
E-mail: info@polymer.de
Website: http://www.polymer.de/
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