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and abundant cuticle of these organisms provide additional advantages for handling
and experimentation. Specimens were obtained live from Bristol Zoo and killed by
freezing at −20 °C for 24 h. They were dissected with a scalpel to remove the
internal tissues and the cuticle was recovered. The cuticle was frozen in liquid nitro-
gen and crushed with a mortar and pestle. It was then boiled in double distilled
water at 100 °C, 3 times for 1 h each, to remove any remaining internal tissue, and
then washed thoroughly with double distilled water. The recovered cuticles were
subjected to two different preparations prior to artifi cial maturation. Cuticle from
scorpion and cockroach was: (1) untreated; or (2) solvent extracted in 2:1 dichloro-
methane/methanol (
v/v
, 5 times, sonication for 30 min each) to remove extractable
lipids, and then saponifi ed (i.e., subjected to base hydrolysis) in 95 % 1 M methano-
lic NaOH for 24 h to remove hydrolysable lipids. Shrimp cuticle was also untreated
prior to maturation. Commercially prepared pure chitin (derived from crab, Sigma
Aldrich) was solvent extracted in the same way as the scorpion and cockroach cuti-
cle to remove soluble impurities.
The cuticles and commercial chitin (0.1-0.15 g) were artifi cially matured at
350 °C, 700 Bars for 24 h in gold cell reactors (Monthioux et al.
1985
; Landais
et al.
1989
; Stankiewicz et al.
2000
). A successful experiment was indicated by no
weight loss; i.e. no change before and after the experiment. No other quantitation
was attempted. The volatiles and condensates that were generated were not char-
acterized. The shrimp cuticle was also matured in two separate experiments in the
presence of commercial powder CaCO
3
(1:1
w/w
) and kaolinite (1:1
w/w
) to
explore the effect of different inorganic matrix materials on cuticle transforma-
tion. The shrimp cuticle was not subjected to any further extraction/saponifi cation
prior to maturation. Pure C
16
and C
18
fatty acids (Sigma Aldrich, 1:1
w
/
w
)
were matured to investigate the transformation of pure lipids reaction products
(as detected in the extractable and hydrolysable fractions). Fresh unmatured sam-
ples were analysed without extraction to reveal the entire range of chemical con-
stituents. The matured samples were analysed using thermal desorption-GC/MS
(TD-GC/MS) at 300 °C followed by (Py-GC-MS) at 610 °C to reveal composition
of experimentally heated biopolymers after thermodesorption (see Gupta and
Pancost
2004
for instrument parameters). Thermal desorption was used to remove
volatile compounds (i.e., thermal extraction: Hartgers et al.
1995
) in order to
purify material prior to pyrolysis (i.e., to thermally extract compounds volatile at
300 °C). It also provided a basis for direct comparison with the results of
Stankiewicz et al. (
2000
). Aliquots of the artifi cially matured samples were sub-
jected additionally to base hydrolysis by heating in 1 M methanolic NaOH for 1 h
at 70 °C to assess the degree of recalcitrance.
Composition of Samples Used in Experiments
Py-GC/MS of solvent extracted commercial chitin (Fig.
8.1a
) yielded 3-acetamido-
4-pyrone, 3-acetamido-5-methylfuran, acetylpyridone, and 3-acetamidofuran as
the major products. However, protein and fatty acyl moieties were not observed.
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