3.4. Chemical identification of electrophysiologically-active components
See section 2.2.4 on In-situ volatile collection for details of how volatile components can be identified. Briefly,
- Collect volatiles on adsorbent and wash them-off the filters with an appropriate solvent (hexane, dichloromethane or redistilled ether).
- Analyse aliquots of the volatile extracts using coupled GC-EAD and coupled GC-MS (linked gas chromatography mass spectrometry) using identical GC columns and oven conditions in both equipment. Add a specific amount of Internal Standard (highly recommended to facilitate matching and identification of peaks when comparing traces obtained from the GC-EAD and GC-MS) (see section 2.2.4. on In-situ volatile collection) for qualitative and quantitative comparisons.
- Compare retention times of separated components from both GC-EAD and GC-MS traces and identify peaks representing EAD-active components in the mixture on the GC-MS trace.
- Tentatively identify chemical structures of EAD-active components (based on their representative peaks) using their fragmentation patterns (mass spectral data) while comparing it with those already identified and stored in a mass spectra database (e.g. NIST, ADAMS) (see section 2.2.4. on In situ volatile analysis).
- Individually prepare 50 – 100 ng/μl solutions of the tentatively identified components in an appropriate solvent from authentic compounds obtained from commercial sources or synthesized by a chemist and analyse via linked GC-EAD and GC-MS to confirm identities of EAD-active components. Repeat the same procedure using a mixture of the tentatively identified components from premix authentic compounds obtained from commercial sources or synthesized and constituted by a chemist. Using a mixture of compounds is advantageous in that it saves time and resources in cases where many EAG-active components (>20) are present in the natural extract. You may also analyse each component separately.