Geology Reference
In-Depth Information
a
Modern Leaf
Extraction
Saponification
Py-GC-MS
TMAH py-GC-MS
Extract
Residue 1
Saponified Extract
GC-MS
Bond-Elut Column
Saponification
Hydrolysable Lipids
(Cutin)
Neutral Lipids
Fatty Acids
Phospholipids
Residue 2
Acid Treatment
GC-MS
Saponification
GC-FID/ GC-MS
Saponification
GC-FID/GC-MS
GC-MS
Carbohydrates
Residue 3
b
SEM
TEM
Fossil Leaf
Extraction
Py-GC-MS
TMAH py-GC-MS
NMR/FTIR
Extract
Residue 1
Saponification
GC-FID/ GC-MS
Hydrolysable Lipids
(Cutin)
Residue 2
GC-MS
Py-GC-MS
TMAH py-GC-MS
Fig. 3.1 Analytical protocol adopted for analysing the modern ( a ) and fossil leaves ( b )
Insoluble residues of the fossil leaves were saponifi ed in 1 M 95 % methanoic
NaOH at 80 °C for 3 h. The saponifi ed extracts were acidifi ed to pH 2 with HCl and
collected in hexane. The extract was methylated with borontrifl uoride/methanol
(14 % w/v) by heating at 70 °C for 1 h and collected in dichloromethane. The
methylated extracts were derivatised for GC analysis by treating with BSTFA at
70 °C for 1 h. A C 34 n- alkane was added as an internal standard for quantifi cation.
Flash pyrolysis involves the thermal fragmentation of the chemical constituents
of a sample at high temperatures in an inert medium. These fragments are then
separated and identifi ed by GC-MS. Thus fl ash pyrolysis reveals bulk macromo-
lecular information and it has been used extensively in molecular characterisation of
both modern and fossil plant material (see van Bergen 1999 for review). Samples
were analysed using a Perkin Elmer GC-MS. A CDS (Chemical Data System)
AS-2500 Pyroprobe pyrolysis unit was used with both the injector and interface
temperature at 290 °C. Between 100 to 150 micrograms of the solvent-extracted
modern or fossil leaves were weighed using a microanalytical balance into quartz
tubes and pyrolysed at 610 °C. No absolute quantitation was attempted. For instru-
ment parameters see Gupta and Pancost 2004 . Compounds were identifi ed from
spectra reported in the literature (Ralph and Hatfi eld 1991 ; Stankiewicz et al. 1997a ;
Bland et al. 1998 ; Stankiewicz and van Bergen 1998 ; Gupta and Pancost 2004 ). For
TMAH (tetramethylammonium hydroxide) assisted pyrolysis an aliquot of the lipid
extracted residue was transferred to a fresh vial and 1 ml TMAH solution added to
the sample. The sample was soaked in TMAH solution for 3-4 h prior to analysis to
ensure that suffi cient TMAH was available during on line pyrolysis.
GC-MS was conducted on a ThermoQuest Trace 2000 gas chromatograph
fi tted with a CP-Sil 5 fused silica capillary column (60 m, 0.32 mm i.d., 0.1 micron
fi lm thickness) coupled to a ThermoQuest single quadrupole mass spectrometer
 
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