3.2 DESCRIPCIÓN DE LA SOLUCIÓN AAA
3.2.2 REQUERIMIENTOS DEL SISTEMA
Eight modem vegetation reference specimens from the Lochan Uaine catchment were selected for lipid analysis. These were selected to represent the major plant groups in the catchment rather than the most abundant species. Lipid composition is more likely to vary between different groups than between different species of the same group, and the intention was to use lipid analysis to determine the broad inputs of organic matter to the lake sediment. The reference specimens chosen included a lichen, a moss, a liverwort, a grass, a fem, a dwarf shrub, and an aquatic bryophyte. An algal scrape from submerged rocks in Lochan Uaine did not provide sufficient material for analysis, and a scrape from a lowland lake in southem England was used as a substitute.
Due to the length of time required for lipid extraction, sample preparation and analysis it was not possible to analyse lipid contents contiguously through core UACT6. Twenty-eight samples were chosen from UACT6. While these were approximately evenly spaced throughout the core, a conscious effort was made to choose samples from maxima and minima in profiles of LOI and bulk organic ô^^C. This was to allow the lipid record to be used to investigate the variations in bulk parameters, and to see whether similar variations are observed in Hpid concentrations. O f the twenty-eight samples used for Hpid analysis, sixteen samples were selected for compound-specific
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isotope analysis. This analysis was performed on the hydrocarbon, acid, and alcohol/sterol fractions only. The sixteen samples analysed were from the same depths for each fraction, and additionally two further hydrocarbon samples were analysed. As for the hpid analysis, samples for compound-specific isotope analysis were chosen to coincide with maxima and minima in bulk parameters, while also being roughly evenly spread downcore.
2.2.7.1 Extraction
All samples were freeze-dried, crushed and passed through a 500 |xm sieve. Due to the high resolution 2 mm sampling interval it was necessary to combine core samples across three contiguous levels to provide a sufficient quantity of extract for hpid analysis. This was achieved by taking 0.5 g dry sediment from each sample to give a total sample mass of 1.5 g, representing a sediment depth of 6 mm. An attempt was made to combine only contiguous samples having similar organic content, so that the contribution of organic matter from each 2 mm sample to the combined sample would be similar (Appendix B). Smaller amounts of modem plant material were used due to their higher hpid content.
Samples were placed in a 12 m l glass centrifuge tube and 100 |Lil internal standard was
added to each. This contained five components relating to different hpid classes, each with a concentration of 200 |Xg ml'^ (Table 2.1).
Table 2.1 Internal standard mixture used in hpid analysis
Component Lipid class
5a-cholestane Hydrocarbon
Hexadecyl octadecanolate Ketones and wax esters 2-hexadecanol Alcohols and sterols 5P-pregnan-3a-ol Alcohols and sterols
Heptadecanoic acid Acids
The recovery of these standards was assessed by comparing the peak areas of the two standards added to the alcohol and sterol fraction, 2-hexadecanol and 5P-pregnan-3a- ol. A very close correlation was observed, with an R^-value of 0.969 (Figure 2.3). This shows that the recovery of standards added to samples is highly reproducible.
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and it can thus be assumed that GC peak areas of components found in the sediment have equally high reproducibility.
250000 I 200000 y 150000 I00000 -
§
c cd c &o 2 c£ 50000 y = 1.0784X- 1198 R" = 0.9686 T--- 1---r 50000 100000 150000 200000 250000 2-hexadecanol, GC peak areasFigure 2.3 Comparison of standards added to the alcohol/sterol fraction of twenty-eight samples from core UACT6. The slope of the regression line (y = 1.0784x) indicates that the standard mixture
appeared to contain roughly 8% more 5P-pregnan-3a-ol than 2-hexadecanol. Arbitrary scale.
Lipids were extracted by sequential sonication and centrifugation (10 min sonication followed by 10 min centrifugation at 3000 rpm). Three extractions were carried out with 100% methanol (MeOH), three with 1:1 v/v MeOH/dichloromethane (DCM), and five with 100% DCM, in that order. Seven millilitres of solvent were used at each stage. After each centrifugation the supernatant was decanted into a round-bottomed flask, and the solvents were removed by evaporation under vacuum. A short silica column was made by adding 1 cm^ silica gel (stored at 130°C) to a glass pipette plugged with solvent-extracted cotton wool. DCM/isopropanol (2:1 v/v) was used to rinse the column, remove the lipids from the round-bottomed flask, and elute them through the column. This stage removes sugars and any particulate matter. Anything that eluted through the column was collected in a pre-weighed glass vial and blown
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down under a gentle stream of nitrogen. Samples were immediately removed from nitrogen blow-down when dry to prevent the loss of more volatile components. The vial was reweighed to allow the mass of total lipid extract (TLE) to be calculated.
The TLE was separated into neutral and acid fractions by solid phase extraction using an aminopropyl Bond Elut column (Figure 2.4). The column was prewashed with 2:1 v/v DCM/isopropanol, and the TLE was dissolved in a small quantity of the same solvent and added to the column. Care was taken not to allow the activated aminopropyl surface of the column to dry out. The neutral fraction was eluted from the column into a round-bottomed flask with 12 ml 2:1 v/v DCM/isopropanol, and the acid fraction was similarly eluted with 12 ml 2% acetic acid in diethylether.
The neutral fraction was further separated into hydrocarbon, aromatic, ketone and wax ester, alcohol and sterol, and polar fractions by ‘flash’ column chromatography. Silica gel (0.6 g) was added to the ‘flash’ column and the column eluted with hexane. Again without allowing the silica to dry, the neutral lipid extract was added and the fractions were eluted from the column by addition of solvents in the order and quantities given in Table 2.2. The first 1 cm^ of hexane was used to dissolve the neutral lipid extract.
Table 2.2 Sequential fractionation of neutral lipid extract by ‘flash’ column chromatography Fraction Solvent added Quantity / cm^
Hydrocarbons Hexane 3.0
Aromatics 9:1 v/v Hexane/DCM 1.5
Ketones and wax esters DCM 5.5
Alcohols and sterols 1:1 v/v DCM/MeOH 3.0
Polar fraction MeOH 2.5
The five fractions were blown down with nitrogen to remove the solvents. Hydrocarbons required no further preparation prior to GC analysis. The other four fractions, and the acid fraction, needed to be derivatised. For those lipids extracted from sediment samples, each fraction was dissolved in 300 |xl DCM. Twenty microlitres of this were placed in a vial and blown down with nitrogen, and 30 fil of the derivatising agent BSTFA [A/0-bis(trimethylsilyl)trifluoroacetamide] were
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