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Inulin Analysis - COSMOSIL Application Note

Due to the lack of retention on ODS columns, oligosaccharides have typically been analyzed on specialty sugar columns. We have successfully analyzed inulins using COSMOSIL Sugar-D, a specialty column for saccharide analysis, and PBr, a unique reversed-phase column for polar and non-polar analytes.

About Inulin

Inulin structure Inulin (G-Fn-F) is a polysaccharide composed of a glucose unit (G) bonded to multiple fructose units (F). It is an indigestible, water-soluble dietary fiber, abundant in the roots of chicory and tubers of Jerusalem artichokes. Recently, oolong tea with inulin has been released as a functional food in Japan (1) on the basis that inulins feed intestinal bacteria by forming short-chain fatty acids and facilitate the peristaltic movement in the bowels. In the literature as well, there are references to inulin's effect on bowel movements. (2),(3)

Sample preparation

Inulin standard
  1. Weigh inulin standard.
  2. Dissolve in Milli-Q water to a concentration of 10 mg/mL.
Oolong tea with inulin
  1. Load 400 µL of oolong tea with inulin into a Cosmospin Filter H (pore size 0.45 µm) and centrifuge.
  2. Use filtrate as sample.

Analysis using Sugar-D

Sugar-D chromatogram
Peaks
  1. 1-Kestose
  2. Nystose

Separation of inulin (G-Fn-F) was accomplished using Sugar-D, a specialty column for sugars. Based on the retention time of the short-chain 1-kestose (G-F-F) and nystose (G-F-F-F), it is thought that the separation is based on sugar chain length.

Conditions
ColumnCOSMOSIL Sugar-D, 4.6mmI.D.-250mm
Mobile phaseMeCN / H2O = 60 / 40
Temperature30°C
Flow rate1.0 mL/min
DetectionRI (cell temp. 40°C)
SampleInulin standard (10 mg/mL)
Inj. vol.10 µL

Reversed-phase analysis

PAQ chromatogram PBr chromatogram
Peaks
  1. 1-Kestose
  2. Nystose
5C18-PAQ
PBr

Inulins are not retained on the ODS column 5C18-PAQ. However, PBr can utilize dispersion force interaction to separate inulin by chain length.

Conditions
ColumnCOSMOSIL ***, 4.6mmI.D.-250mm
Mobile phaseMeOH / H2O = 15 / 85
Temperature30°C
Flow rate1.0 mL/min
DetectionRI (cell temp. 40°C)
SampleInulin standard (10 mg/mL)
Inj. vol.10 µL

Oolong tea with inulin: reversed-phase analysis

PBr chromatogram: oolong tea with inulin

Upon analyzing oolong tea with inulin (inulin conc. 7.3 g / 500 mL), a similar chromatogram to the standard was obtained.

Conditions
ColumnCOSMOSIL PBr, 4.6mmI.D.-250mm
Mobile phaseMeOH / H2O = 15 / 85
Temperature30°C
Flow rate1.0 mL/min
DetectionRI (cell temp. 40°C)
SampleOolong tea with inulin (inulin conc. 14.6 mg/mL)
Inj. vol.10 µL

Product information

References

  1. https://www.fld.caa.go.jp/caaks/cssc02/?recordSeq=41804260390603 (in Japanese)
  2. Kleessen B. et al. (1997) The American Journal of Clinical Nutrition 65(5):1397-1402
  3. Castiglia-Delavaud C. et al. (1998) British Journal of Nutrition 80(4):343-352

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