For the purposes of the investigation, Eocene amber samples (note: Eocene is the name given to a period in the earth’s history that began about 56 million years ago and ended about 34 million years ago) from the Ameki Formation in Nigeria were used that were made available by the Hunterian Museum at Glasgow University. The sample material was ground up and pyrolysed in portions of 200 µg at 480°C for 20 seconds.
The following equipment combination was used for the pyrolysis GC/MS: Agilent GC 6890N with the GERSTEL Cooled Injection System (CIS), the GERSTEL Thermal Desorption Unit (TDU), the GERSTEL Multi Purpose Sampler (MPS) and the Agilent MS 5795B Inert XL (Triple Axis). The pyrolysis operation itself was carried out using the GERSTEL Pyro, a special pyrolysis module, by which the GERSTEL TDU can be extended quickly without any major modifications.
Further details of technical relevance: the TDU was operated in split mode, in order to remove surplus solvent. The Cooled Injection System (CIS) was set at a temperature of 300°C and used as a hot split interface. The GC was operated in split mode (1:20) and was equipped with a ZB-5MS quartz glass column (30 m x 0.25 µ x 0.25 mm, Phenomenex). The temperature program of the GC oven was as follows: 60°C (2 min.) – 6°C/min. – 300°C (10 min.). Helium was used as the carrier gas with a constant flow of 1 ml/min. The mass spectrometer was operated in Electron Impact (EI) ionisation mode, with the ionisation energy set at 70 eV and the source and quadrupole temperatures set at 230°C and 150°C respectively. Full-scan mass spectra were recorded over a mass range of 50 to 650 Da. ChemStation software was used to collect and process the data. Individual compounds were identified by comparing the mass spectra with MS library data and information from the literature.
Step by step to in-depth information
Zunächst erfolgte die reine Pyrolyse des gemahlenen Bernsteins. Im Pure pyrolysis of the ground amber was carried out first of all. A number of the components described in the literature were identified in the chromatogram, such as a series of fatty acids (FA), i.e. norchrysanthemum acid (FA I), naphthalene-1-carboxylic acid-1,2,3,4,4a,7,8,8a-octahydro-1,4a,6-trimethyl (FA II), naphthalene-1-carboxylic acid-1,2,3,4,4a,7,8,8a-octahydro-1,4a,5,6-tetramethyl (FA III) and naphthalene-1-carboxylic acid-1,2,3,4,4a,5,8,8a-octahydro-1,4a,6-trimethyl-5-methylene (FA V).
In order to obtain more in-depth information, the amber samples were derivatised (methylised) in situ, i.e. during the 20-second pyrolysis operation, after the pyrolysis GC/MS process using tetramethylammonium hydroxide (TMAH); this process is also known as thermochemolysis. The fatty acids were converted into their corresponding fatty acid methyl esters (FAMES) in this context: norchrysanthemum acid methyl ester (FAME I), naphthalene-1-carboxylic acid-1,2,3,4,4a,7,8,8a-octahydro-1,4a,6-trimethyl methyl ester (FAME II), naphthalene-1-carboxylic acid-1,2,3,4,4a,7,8,8a-octahydro-1,4a,5,6-tetramethyl methyl ester (FAME III), methyl-1,2,3,4-tetrahydro-1,5,6-trimethyl-1-naphthalene carboxylate (FAME IV), naphthalene-1-carboxylic acid-1,2,3,4,4a,5,8,8a-octahydro-1,4a,6-trimethyl-5-methylene methyl ester (FAME V) and methyl-16,17-dinorcallitrisate (FAME VI).
Dr Eike Kleine-Benne explains the difference between pure pyrolysis and the thermochemolysis process as follows: “In the pyrolysis chromatogram, the peaks of FA (I), FA (II), FA (III) and FA (V) are wide and in some cases have a sawtooth shape, a typical phenomenon for polar compounds like fatty acids that are separated on a non-polar column. In contrast to this, the corresponding methylised compounds FAME (I), FAME (II), FAME (III) and FAME (V) elute in the thermochemolysis chromatogram as well-defined sharp peaks. It was only possible to identify FAME (IV) and FAME (V) in the thermochemolysis chromatogram. It is very clear that pyrolysis with TMAH generates more detailed information about the sample.”