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

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

ß-D-Glucose approx. 100 Dalton Humic substance approx Dalton Molecular Weight LOWHIGH

Litter Decomposition

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

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

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

Amberlite XAD1180: Water - Ethanol Elution: Water - Ethanol Adsorbed c.

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

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

Beech litter Oak litter HPLC Result

Oak soil Beech soil Hydrolysis ! HPLC Result

Dihydrokaempferol Kaempferol Expected compounds

Oak litter Beech litter Oak soil Beech soil

Benzoic acid

Gas chromatography-mass spectroscopy Schematic of a GC-MS instrument -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. Zebron 5,30 x 0,25 x 0,25

Gas chromatography-mass spectroscopy II-2-Preparation of GC-MS samples - This phase was frozen and sublimated to obtain a dry sample - Water phase contains generally very polar compounds such as sugars - 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-n- trimethylsilyltrifluoroacetamide (MSTFA) with pyridine as solvent.

Gas chromatography-mass spectroscopy Results and discussion of our analysis Identification of sugar of oak litter (EL) EL1 EL2 EL3 - Erythritol Xylose methoxamine Quercitol Galactose methoxamine Mannitol Trehalose

Gas chromatography-mass spectroscopy Identification of sugars of oak soil (ES) ES1 ES2 ES3 Xylose methoxamine Mannose methoxamine Galactose methoxamine Trehalose

Identification of sugars of Beech (EL) BL1 BL2 BL3 - Xylose 1+2 Galactose 1 Glucose 1+2 Trehalose Gas chromatography-mass spectroscopy

Identification of sugars of beech soil (BS) BS1 BS2 BS3 Xylose methoxamine 1+2 Galactose methoxamineTrehalose Gas chromatography-mass spectroscopy

Comparison of the composition of soil and litter oak and beechOakBeech sugarsLitterSoilLitterSoil Erythritolpaaa xylosepppp Quercitolpaaa Galactosepppp Mannitolpaaa Trehalosepppp Mannoseapaa Glucoseaapa

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).

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