Monosaccharide profile of the extracts and the residues of extraction, from R.raetam young stems (YS) and adult stems (AS)
Description
Prior to analysis, polysaccharidic fractions were submitted to methanolysis. The methylglucosides released obtained were then derivatized by trimethylsilylation. The analysis was carried out on 200 to 500 μg of freeze-dried polysaccharide powder, added to an internal standard, mesoinositol (MI), at a rate of 10% of the quantity of polysaccharide. Methylglycosides trimethylsylilated have been identified by GLC by comparison with authentic samples using a Perichrom PR 2100 chromatograph equipped with a capillary tube (0.32 mm by 60 m) OPTIMA® 1-Accent 0.25 μM (Macherey-Nagel) and with a flame ionization detector (FID). The chromatograph has been controlled by the software Winilab III (Perichrom). The results were expressed as a molar percentage after correction of the areas of the chromatographic peaks by the response factors.
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Qualitative analysis by gas liquid chromatography (GLC) : Prior to analysis, polysaccharidic fractions were submitted to methanolysis. The methylglucosides released obtained were then derivatized by trimethylsilylation. The analysis was carried out on 200 to 500 μg of freeze-dried polysaccharide powder, added to an internal standard, mesoinositol (MI), at a rate of 10% of the quantity of polysaccharide. This method was used for the direct analysis of crude plant powders (Marga et al. 1995). It can also be used for the study of the monosaccharidic composition of the residues of extraction. Methanolysis Monosaccharides were released as methylglycosides by adding 1 mL of 1 M hydrochloric methanol to the anhydrous polysaccharide sample. After 24 h, at 80°C in a sealed tube, the methanolisis was stopped by evaporation of the hydrolyzate under a nitrogen flux. The residue was dissolved in 1 mL methanol and then delipidated by three successive extraction with heptane (3 x 1 mL). Finally, it was evaporated again under nitrogen flux. Trimethylating The methylglycosides were then trimethylated with 100 μL of N,O-bis-trim ethylsilyl- trifluoroacetamide (BSTFA) with 1% of trimethylchlorosilane (TMCS) in pyridine (100 μL), in the darkness during 2 h at 27°C. These samples were maintained at -20°C during 24 h and then directly injected in GLC. Analysis of the trimethylsilylation derivatives by gas liquid chromatography Methylglycosides trimethylsylilated have been identified by GLC by comparison with authentic samples using a Perichrom PR 2100 chromatograph equipped with a capillary tube (0.32 mm by 60 m) OPTIMA® 1-Accent 0.25 μM (Macherey-Nagel) and with a flame ionization detector (FID). The carrier gas is nitrogen under a pressure of 75 kPa. The temperature of the injector was fixed at 260°C. The rise in the temperature of the furnace has been programmed from 130 to 210°C at a rate of 2°C.min-1, with a 5 min mitigation at 190°C, then from 210 to 260°C at a rate of 5°C.min-1. The chromatograph has been controlled by the software Winilab III (Perichrom). The results were expressed as a molar percentage after correction of the areas of the chromatographic peaks by the response factors.