Low Molecular Weight Compounds SE Chemical Methods WS

Low Molecular Weight Compounds SE Chemical Methods WS 2009/10 Group A: Angelika Hofer Tshering Doma Grace Djoufack

LMWC - Assay Can Low Molecular Compounds detected in Litter be refound in Soil? !

Molecular Weight ß-D-Glucose approx. 100 Dalton LOW Humic substance approx. 300. 000 Dalton HIGH

Litter Decomposition

Translocation Process – e. g. Hemicellulose Mannose Xylose Galactose Glucose Rhamnose Arabinose LEACHING Mineralisation Immobilisation Humification

Sample Preparation - Dilution (in Chloroform – Methanol – Water) - Filtration (paper filter) - Partition (Amberlite Column)

Adsorption Chromatography 1) Stationary Phase: Amberlite-Resin 2) Mobile Phases: Different polarity 1. Water 2. Ethanol

Elution: Water - Ethanol Amberlite XAD 1180: Water - Ethanol Adsorbed c.

Fractions of different polarity - Waterfraction: polar Sugars polar compounds → GC - MS - Ethanolfraction: nonpolar „Polymers“ aromatic compounds → HPLC - UV

HPLC (High Performance Liquid Chromatography) Amberlite fractionation: • Ethanol phase (phenolic compounds) • Me. OH+Water • different silica gel types (particle size 3 -5 μm) • 100 -300 bar pressure • conjugated double bonds

HPLC Result Oak litter Beech litter

HPLC Result Oak soil Beech soil Hydrolysis !

Expected compounds Dihydrokaempferol Kaempferol

Oak litter Beech litter Oak soil Beech soil

Benzoic acid

Gas chromatography-mass spectroscopy Schematic of a GC-MS instrument Zebron 5, 30 x 0, 25 -Sample is introduced into GC inlet vaporized at 250 °C -Swept onto the column by the carrier gas & separated within the column. -The MS is linked to a computer programme, which records the data. -Identification of a compound based on it's mass spectrum -Every compound has a unique fragmentation pattern. -A large library of known mass spectra is stored on the computer - This is searched using computer algorithms to assist the analyst in identifying the unknown.

Gas chromatography-mass spectroscopy II-2 -Preparation of GC-MS samples - Water phase contains generally very polar compounds such as sugars - This phase was frozen and sublimated to obtain a dry sample - Due to their high polarity, hydrophicity and low volatility, sugars have to be converted into volatilizable and stable derivatives before the GC analysis by silylation with n-methyl-ntrimethylsilyltrifluoroacetamide (MSTFA) with pyridine as solvent.

Gas chromatography-mass spectroscopy Results and discussion of our analysis Identification of sugar of oak litter (EL) EL 1 EL 2 Trehalose EL 3 - Erythritol Xylose methoxamine Quercitol Galactose methoxamine Mannitol

Gas chromatography-mass spectroscopy Identification of sugars of oak soil (ES) ES 1 ES 2 ES 3 Xylose methoxamine Mannose methoxamine Galactose methoxamine Trehalose

Gas chromatography-mass spectroscopy Identification of sugars of Beech (EL) BL 1 BL 2 BL 3 - Xylose 1+2 Galactose 1 Glucose 1+2 Trehalose

Gas chromatography-mass spectroscopy Identification of sugars of beech soil (BS) BS 1 BS 2 BS 3 Xylose methoxamine 1+2 Galactose methoxamine Trehalose

Comparison of the composition of soil and litter oak and beech Oak Beech sugars Litter Soil Erythritol p a a a xylose p p Quercitol p a a a Galactose p p Mannitol p a a a Trehalose p p Mannose a p a a Glucose a a p a

Conclusions: Can Low Molecular Compounds detected in Litter be refound in Soil? ! - Our objective was to compare the presence of Low Molecular Weight Compounds in litter and soil of different sites (oak, beech). - Xylose methoxamine, galactose methoxamine, trehalose are present in the litter and soil extract of oak and beech (Hemicellulose sugars). - Quercitol and erythritol, sugar alcohol are present in the litter sample and not in the soil of oak (specific sugar alcohols in oak). They were completely absent in beech litter and soil. -Mannose methoxamine was detected in soil but not in the litter of oak , but we could not detect it in beech. - Glucose was only detected in the beech litter (cellulose monomer).

Mass spectra of different sugars present in sample quercitol erythritol xylose methoxamine mannitol galactose methoxamine trehalose