Given their widespread effects and distribution in both natural and industrial environments, the monitoring of phenolic compounds is of considerable analytical interest. Electrochemical biosensor technologies, in particular those comprising laccase enzymes, afford many potential benefits to address this analytical need. However, several key factors affecting sensor response currently limit their applicability. This Thesis reports on the fabrication and optimisation of an electrochemical laccase-based biosensor towards the application of the monitoring of phenolic compounds. Selected factors considered to affect sensor response were investigated using the optimised biosensor. These included: electrochemical, biochemical and substrate-dependent factors, which were found to intersect in modulating biosensor response signals. Through the application of transducer-dependent and substrate-dependent parameters, the selective and simultaneous detection of a mixture of different phenolic analytes is successfully demonstrated. This Thesis also investigates the use of Quartz-Crystal Microbalance with Dissipation (QCM-D) technology, an analytical technique that measures physical parameters of thin-film structures, towards the successful monitoring of enzyme immobilisation strategies. These strategies are fundamental to the successful fabrication of biosensors, and the real-time monitoring of immobilised film formations is of considerable research interest. In the studies reported on in this Thesis, QCM-D technology was demonstrated to be an effective complementary technology in the prediction of film immobilisation techniques on the resultant biochemical kinetics of immobilised enzymes.

The chemical shift of protons in intramolecular hydrogen bonds has been measured in 41 ortho substituted phenol type compounds. The change in chemical shift " ΔσOH " on formation of these hydrogen bonds is taken as the difference between the infinite dilution chemical shift of the parent phenol compound in CCl₄ solution and the chemical shift measured for the proton in the intramolecular hydrogen bond. This change in chemical shift is correlated with the corresponding frequency shift " ΔνOH " in the -OH stretching region of the infra-red spectrum. The dilution chemical shift for the -OH proton in the o-halophenols has been investigated over a concentration range 1-5 mole % in CS₂ and a temperature region -53 to 107°C Using the infinite dilution shift values, the equilibrium constants of the cis-trans conversion were obtained. Values for δCIS, the chemical shift of the completely hydrogen bonded form; δ TRANS, the chemical shift of the unbonded form, and ΔH , the enthalpy of formation of the hydrogen bond, were also calculated. From these results a value for ΔH, the enthalpy of formation for the dimer was calculated, assuming that the major dimer species in solution was formed from the combination of a cis and trans bonded form. Temperature studies of the change in chemical shift of the -OH proton for 2,1,6-trihalosubstituted phenols is also reported. The temperature range in this case was 0°C to 111°0. / Science, Faculty of / Chemistry, Department of / Graduate

The possibility of separating homologous phenols by liquid-liquid extraction with aqueous sodium hydroxide has been considered. In particular, phenol-o-cresol mixtures have received attention and the system phenol, o-cresol, water, and sodium hydroxide has been defined both with respect to the boundary of the two-phase region and the phase equilibria involved. Some qualitative and quantitative considerations have been given to possible extraction operations. It is concluded that extraction in the quaternary system is likely to be attractive only in the case of particular alkaline phenolic feeds. / Applied Science, Faculty of / Chemical and Biological Engineering, Department of / Graduate

I. Previous studies on biogenesis of simple phenols in plants have been restricted to hydroquinone. Among the other simple phenols, catechol is of particular interest because of its potential role as a ring-cleavage substrate. Tracer studies on the biogenesis of catechol in Gaultheria leaf discs showed that it was formed from salicylic acid by oxidative decarboxylation. Salicylate decarboxylating activity could be detected in buffered extracts of very young leaves. II. Among the numerous caffeic acid esters presently known in plants, only 3-0-caffeoylquinic acid (chlorogenic acid) has been studied in detail. Rosmarinic acid (alpha-O-caffeoyl-3, 4-dihydroxyphenyllactic acid) has been reported to occur in a number of plants but nothing was known of its biosynthesis or metabolic role. Tracer studies demonstrated that in Mentha the caffeic acid moiety was formed from phenylalanine via cinnamic and para-coumaric acids. In contrast, the structurally similar 3, 4-dihydroxyphenyllactic acid moiety was formed from tyrosine and 3, 4-dihydroxyphenylalanine. There was no evidence of the participation of a para-coumaroyl ester intermediate. Time-course studies and use of labelled rosmarinic acid showed that endogenous rosmarinic acid was turning over slowly. The caffeoyl moiety, however, does not appear to be contributing to the formation of insoluble polymers, as has been suggested for chlorogenic acid in other plants. III. Bacteria and fungi readily degrade aromatic compounds to carbon dioxide. Despite the large quantities of aromatic compounds formed in plants, little attention has been paid to the ability of plant tissues to degrade aromatic rings. No reported studies have used completely sterile plants and techniques. This has left open the possibility that the microflora associated with the plant might be carrying out the observed reactions. The ability of sterile plant tissue cultures to degrade aromatic ring-¹⁴C compounds to carbon dioxide was studied. It was established that a number of tissues (Ruta, Triticum, Phaseolus, Melilotus) have the ability to cleave the aromatic ring of phenylalanine. Melilotus tissue could also degrade cinnamic acid-ring-¹⁴C suggesting that a dihydroxy phenolic acid may be the ring-cleavage substrate. Neither Ruta nor Melilotus tissues were able to degrade benzoic acid or salicylic acid-ring-¹⁴C. Tryptophan benzene ring-¹⁴C was shown to be degraded to carbon dioxide by both Ruta and Melilotus. In summary, the ability of plants to cleave the benzene ring of aromatic compounds when free of micro-organisms was thus established. / Science, Faculty of / Botany, Department of / Graduate

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