Molecular Preservation of Eurypterids - Biopolymers: A Molecular Paleontology Approach - page 122

solvent-extracted scorpion or eurypterid cuticle were weighed into quartz tubes
using a microanalytical balance and pyrolysed at 610 °C. No absolute quantitation
was attempted. Separation of pyrolysis products was achieved using a DB-1 fused
silica capillary column (30 m, 0.25 mm i.d., 0.1

m fi lm thickness). The GC oven
was programmed from 40-50 °C (held at 4 min) to 320 °C at 5 °C min −1 and held
at that temperature for 15 min. A thermal hold of 3-5 min was applied. Helium
was the carrier gas. The MS was operated at 70 eV scanning over the range m/z
45-600 at 1 scan/s with an emission current of 300 micro-amperes (full scan
mode). Compounds were identifi ed using the NIST mass spectral library and
spectra reported in the literature (Stankiewicz et al. 1996 ). Modern Limulus cuti-
cle was pyrolysed using a CDS 5150 Pyroprobe by heating at 650 °C for 20 s to
fragment macromolecular organic components. Compound detection and identifi -
cation was performed by on-line GC-MS in full-scan mode on a Hewlett Packard
HP6890 gas chromatograph interfaced to a MicromassAutoSpecUltima magnetic
sector mass spectrometer. GC separation was performed on a J&W Scientifi c
DB-1MS capillary column (60 m length, 0.25 mm internal diameter, 0.25

μ

m fi lm
thickness) using He as the carrier gas. Samples were injected in splitless mode at
300 °C. The oven was programmed from 60 (held for 2 min) to 150 °C at
10 °C min −1 , then at 3 °C min −1 to 315 °C and held isothermal for 24 min. The
source was operated in electron ionization (EI) mode at 70 eV ionization energy
at 250 °C. The AutoSpec full-scan rate was 0.80 s/decade over a mass range of
50-600 Da and a delay of 0.20 s/decade.
For TMAH (tetramethylammonium hydroxide) assisted pyrolysis, an aliquot
of the lipid-extracted residue was transferred to a fresh vial and 1 ml TMAH solu-
tion (25 wt %) was added to the sample. The sample was soaked in TMAH solu-
tion for 3-4 h prior to analysis to ensure that suffi cient TMAH was available
during on-line pyrolysis, also conducted at 610 °C. Blanks with and without
TMAH were run before analysis of all samples as a control to ensure that there
was no contamination.
Raman scattering provides a nondestructive, noninvasive method for microscale
characterization of carbonaceous material. It can be applied to samples ranging
from megascopic to as small as 1

μ

m (Kudryavtsev et al. 2001 ), which is espe-
cially useful when the amount of cuticle is minimal and not amenable to other
techniques. In situ Raman microspectroscopy was carried out with a LABRAM
spectrometer (JobinYvon) with a Nd-YAG 532 nm laser source and a Peltier-
cooled CCD detector. Cuticle samples were mounted on standard glass slides. The
laser was focused on the sample with a 500 nm confocal hole using the 50x objec-
tive under refl ected light. The spot on the sample was ~1.5

μ

m in diameter and had
a power ~1 mW at the sample surface. Jobin Yvon's LabSpec program was used
for data acquisition and estimation of Raman peaks. A minimum of 10 indepen-
dent spots was analyzed and data were collected for 10-20 s per spot depending
upon the Raman intensity. The spot was measured over a spectral window of
1,000-2,000 cm −1 . The spectra were deconvoluted into bands (Rahl et al. 2005 ),
pertaining to ordered and disordered carbonaceous matter. Carbonaceous material
is best characterized by fi rst-order Raman peaks or bands, which occur with

μ