published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

We aimed to identify the volatile compounds in inflorescences of Rudbeckia hirta that may be responsible for the olfactory attraction of the crab spider Misumenoides formosipes to this plant. Our approach was to use ultrasonic extraction, separate the extract into fractions using flash chromatography with different solvent systems, and test the attraction of the male spiders to the pooled fractions using a y-tube olfactometer. Ultrasonic extraction is carried out using a mixture of 1:2 hexane/diethyl ether with 10 g of inflorescences for 30 minutes. Bioassay results indicated that male spiders chose the inflorescences, bulk ultrasonic extract, and the pooled 100% dichloromethane fractions over controls. Nuclear magnetic resonance experiments and infrared spectroscopy experiments were carried out on the 100% dichloromethane fractions and these experiments indicated that a long chain hydrocarbon is the main component in the 100% dichloromethane fractions / Chromatographic method and bioassay development method -- M. formosipes olfactory response to R. hirta -- Separation and identification of the possible attractants in the 100% dichloromethane fractions. / Department of Chemistry

Lam, Hon Yiu. / "November 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 122-135). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Contents --- p.vii / List of Abbreviations --- p.xiii / List of Figures --- p.xiv / List of Tables --- p.xvii / Chapter 1 --- Literature review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Flavor of fish --- p.3 / Chapter 1.2.1 --- Carbonyls (aldehydes and ketones) and alcohols --- p.4 / Chapter 1.2.2 --- Sulfur-containing compounds --- p.5 / Chapter 1.2.3 --- Thermally-induced flavor --- p.5 / Chapter 1.2.4 --- Deteriorated fish flavor --- p.6 / Chapter 1.2.5 --- Autoxidation --- p.7 / Chapter 1.2.6 --- Bromophenols --- p.8 / Chapter 1.3 --- Bromophenols in aquaculture --- p.8 / Chapter 1.3.1 --- General properties of bromophenols --- p.9 / Chapter 1.3.2 --- Biosynthetic pathway of bromophenol in marine algae --- p.12 / Chapter 1.3.3 --- Thresholds of bromophenols --- p.14 / Chapter 1.3.4 --- Toxicity of bromophenols --- p.17 / Chapter 1.4 --- Giant Grouper --- p.19 / Chapter 1.4.1 --- Living Habitat of Giant Grouper --- p.19 / Chapter 1.4.2 --- Biological features of Giant Grouper --- p.23 / Chapter 1.4.3 --- Aquaculture of Giant Grouper --- p.23 / Chapter 1.5 --- Flavor analysis and extraction methods --- p.23 / Chapter 1.5.1 --- Solvent extraction --- p.25 / Chapter 1.5.2 --- Simultaneous Steam Distillation/Extraction --- p.25 / Chapter 1.5.3 --- Headspace sampling --- p.27 / Chapter 1.5.4 --- Gas Chromatography/Olfactometry (GCO) --- p.28 / Chapter 1.5.5 --- Food chemistry and Odor Threshold Value --- p.30 / Chapter 2 --- Distribution of bromophenols in selected Hong Kong dried seafood / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and Methods --- p.34 / Chapter 2.2.1 --- Sample preparation --- p.34 / Chapter 2.2.2 --- "Preparation of the internal standard, Pentachloroanisole" --- p.35 / Chapter 2.2.3 --- Simultaneous steam distillation-solvent extraction (SDE) --- p.35 / Chapter 2.2.4 --- Gas chromatography-mass spectrometry (GC-MS) --- p.36 / Chapter 2.2.5 --- Compound identification --- p.37 / Chapter 2.2.6 --- Quantification of compounds --- p.37 / Chapter 2.2.7 --- Recovery --- p.37 / Chapter 2.2.8 --- Odor activity value (OAV) --- p.38 / Chapter 2.2.9 --- Statistical Analysis --- p.38 / Chapter 2.3 --- Results and discussion --- p.39 / Chapter 2.3.1 --- Distribution of bromophenols in dried seafoods --- p.39 / Chapter 2.3.2 --- Bromophenol contents in dried seaweeds --- p.51 / Chapter 2.3.3 --- Bromophenol contents in dried crustacean --- p.52 / Chapter 2.3.4 --- Bromophenol contents in dried mollusks --- p.53 / Chapter 2.3.5 --- Bromophenol contents in dried-salted fishes --- p.54 / Chapter 2.3.6 --- Relationship between living habitat and bromophenol contents --- p.55 / Chapter 2.3.7 --- Flavor impact of bromophenols in dried seafood --- p.57 / Chapter 2.3.8 --- Comparison of bromophenol content in purchased dried laminaria with Qingdao seaweed powder and bloodworms --- p.64 / Chapter 2.4 --- Conclusion --- p.67 / Chapter 3 --- Bromophenol content retention and fish quality in giant grouper / Chapter 3.1 --- Introduction --- p.70 / Chapter 3.2 --- Materials and Methods --- p.71 / Chapter 3.2.1 --- Abbreviation of treatment groups --- p.71 / Chapter 3.2.2 --- Sample preparation --- p.72 / Chapter 3.2.3 --- Ingredients --- p.72 / Chapter 3.2.4 --- Production of fish feed --- p.73 / Chapter 3.2.5 --- Preparation of the internal standard，Pentachloroanisole --- p.73 / Chapter 3.2.6 --- Simultaneous steam distillation-solvent extraction (SDE) --- p.75 / Chapter 3.2.7 --- Gas chromatography-mass spectrometry (GC-MS) --- p.75 / Chapter 3.2.8 --- Bromophenol identification and quantification --- p.76 / Chapter 3.2.9 --- Recovery of bromophenols --- p.76 / Chapter 3.2.10 --- Muscle color determination --- p.76 / Chapter 3.2.11 --- Texture analysis --- p.77 / Chapter 3.2.12 --- Moisture determination --- p.78 / Chapter 3.2.13 --- Ash determination --- p.78 / Chapter 3.2.14 --- Fat determination --- p.78 / Chapter 3.2.15 --- Protein determination --- p.79 / Chapter 3.2.16 --- Statistical Analysis --- p.80 / Chapter 3.3 --- Results and discussion --- p.80 / Chapter 3.3.1 --- Muscle color of giant grouper --- p.81 / Chapter 3.3.2 --- Texture of giant grouper --- p.85 / Chapter 3.3.3 --- Proximate analysis of giant grouper --- p.86 / Chapter 3.3.4 --- Bromophenol depuration of giant grouper --- p.92 / Chapter 3.4 --- Conclusion --- p.101 / Chapter 4 --- Volatile compounds in giant grouper / Chapter 4.1 --- Introduction --- p.102 / Chapter 4.2 --- Materials and Methods --- p.103 / Chapter 4.2.1 --- Sample preparation --- p.103 / Chapter 4.2.2 --- "Preparation of the internal standard, 2,4,6Trimethylpyridine (TMP)" --- p.104 / Chapter 4.2.3 --- Dynamic headspace (purge-and-trap) --- p.104 / Chapter 4.2.4 --- Simultaneous steam distillation-solvent extraction (SDE) --- p.105 / Chapter 4.2.5 --- Gas chromatography-mass spectrometry (GC-MS) --- p.105 / Chapter 4.2.6 --- Compound identification --- p.106 / Chapter 4.2.7 --- Quantification of compounds --- p.106 / Chapter 4.2.8 --- Recovery --- p.107 / Chapter 4.2.9 --- Odor activity value (OAV) --- p.108 / Chapter 4.2.10 --- Statistical analysis --- p.108 / Chapter 4.3 --- Results and discussion --- p.108 / Chapter 4.3.1 --- Comparison of extraction between dynamic headspace and SDE --- p.108 / Chapter 4.3.2 --- Flavor profile of giant grouper --- p.113 / Chapter 4.3.2.1 --- carbonyls and alcohol --- p.113 / Chapter 4.3.2.2 --- Other aroma volatile compounds in giant grouper --- p.116 / Chapter 4.3.3 --- Giant grouper tainted by water contamination --- p.116 / Chapter 4.4 --- Conclusion --- p.118 / Chapter 5 --- General conclusion --- p.119 / References --- p.122 / Appendix --- p.136

Our research involves the isolation and identification of the possible chemical compounds in black-eyed Susans that may be responsible for the olfactory attraction of the crab spider Misumenoides formosipes to the inflorescences of these plants. In olfactometric bioassays, 80% of 30 male spiders moved towards olfactory-only cues from R. hirta inflorescences over a water control (P = 0.0014). The bulk extract was separated using flash column chromatography (silica column) with a series of solvents. Spiders in olfactometer bioassays showed a significant preference for the fractions collected using 100% dichloromethane over the solvent-only control (P=0.039). The 100% dichloromethane pooled fractions were separated using solid phase extraction (SPE). Three compounds were isolated and identified using TLC, infrared and NMR spectroscopy. Two compounds were identified as contaminants, di(2-ethylhexyl) phthalate and erucamide, found in the flash column chromatography apparatus and SPE apparatus, respectively. A long-chain crystalline hydrocarbon wax was extracted from R. hirta inflorescences. Research shows that several insects use the lipids of the wax layer, specifically various long-chain alkanes and alcohols, as cues in host plant selection or as kairomones, chemical cues used in communication from one organism to another [3]. It also shows that the waxes can act as absorbents or release agents for biologically active material. Thus, the long-chain hydrocarbon wax interacting with the volatile components could play a major role in attracting the male crab spiders to the R. hirta inflorescences / Introduction and background -- Olfactory bioassay studies of M. formosipes -- Chromatographic separation of components in the 100% dichloromethane fractions -- Identification of the possible attractants in the 100% dichloromethane fractions using spectroscopic methods. / Department of Chemistry

The determination of carbon monoxide is also possible by trapping CO on preconditioned molecular sieve and thermal desorption. Analysis in this case is performed by gas chromatography/mass spectroscopy, although the trapping technique is applicable to other suitable GC techniques.