This research project is an ongoing project in collaboration with colleagues in the Department of Biology at the University of the Pacific. The main purpose of this project was to separate and identify the female-attracting substance secreted by male frog by developing chromatography methods for the male and female aqueous extracts. Three methods were used to concentrate the samples: sample lyophilization,..solid phase extraction (SPE), and direct sample loading. Different parameters such as the type and concentration of the phase modifier, wavelength for UV-detection, composition of the mobile phase, flow rate, and gradient elution were studied to achieve the required separation. The optimal method was determined as: direct sample loading, 0.01% (v v) formic acid in in mobile phase A (water) and mobile phase B (acctonitrile), 200 nm, 0.60 mL/min, and gradient method. The chromatograms of male and female skin gland water samples were compared and the fractions specific to male frog were collected and lyophilized for bio-activity testing and mass spectrometry analysis. Three different mass spectrometer systems, JEOL LCMate (ES+), Micromass Q-Tof Ultima™ Global (ES-). Voyager-DE™ STR Biospectromelry™ Workstation (MALDI-TOF), and Varian Mercury 300 MHz FT-NMR were utilized to investigate the structure of the fractions collected through HPLC. Only Micromass Q-Tof Ultima™ Global (ES+) gave some potential results. After analysis, the proposed protonated molecular ion peak was determined to be at m/z 779 by analyzing the abundance and relationship among the peaks at higher m/z values. The detail structure was inconclusive. vi

Choklad erhålls från kakaobönan som är frukten på kakaoträdet (Theobroma cacao). För att skapa choklad bearbetas kakaobönan vilket påverkar halten av bioaktiva ämnen i produkten. Choklad innehåller flera typer av bioaktiva ämnen där kakaoflavanolerna epikatechin och katechin är de mest framträdande. De hör till klassen flavanoider som i tidigare forskning har kopplats till hälsosamma effekter som sänkt blodtryck, minskad risk för hjärt- och kärlsjukdomar och ökad antioxidantkapacitet. I förordningen nr 432/2012 över godkända hälsopåståenden för livsmedel som EU-kommissionen fastställt framgår att kakaoflavanoler kan bidra till en normal blodcirkulation genom att blodkärlen hålls elastiska. Påståendet får användas för mörk choklad men konsumenten måste upplysas om att ett dagligt intag av 200 mg kakaoflavanoler krävs för att uppnå den gynnsamma effekten. I aktuell studie har kakaobönor från Madagaskar och 70% mörk choklad producerad på kakaobönor från Peru, Mexiko, Filippinerna och Ecuador analyserats i samarbete med Johannas Choklad i Kalmar. Syftet med studien var att undersöka hur halten flavanoider och främst kakaoflavanolerna epikatechin och katechin påverkas vid hantverksmässig chokladtillverkning. Resultatet skulle kunna ge information åt Johannas Choklad vilka bearbetningssteg i processen från kakaoböna till färdig chokladkaka som påverkar halterna mest. Det skulle även kunna ge indikationer på om någon av deras chokladprodukter uppfyller kraven för att erhålla EU:s hälsopåstående angående mörk choklad. Ett antal analyser genomfördes genom att bioaktiva ämnen extraherades med metanol och den slutgiltiga supernatanten indunstades och återstoden löstes i MQ-vatten (v/w =10). Lösningen filtrerades och analyserades därefter med RP-HPLC med UV-detektor (Reversed phase - High Performance Liquid Chromatography). Resultatet från analyserna gav indikationer på att variationen av rostningsgrad (110-135 °C) och processningsförfarandet vid chokladtillverkningen inte påverkade halten av flavanoider nämnvärt. Analysen av den 70% mörka chokladen med bönor av olika ursprung visade att kakaobönor från Mexiko och Peru innehöll högst halt flavanoider och kakaoflavanoler (261 respektive 163 mg kakaoflavanoler/100 g) medans choklad gjord på bönor från Ecuador och Filippinerna innehöll lägre halter (60-70 mg kakaoflavanoler/100 g). Utifrån resultaten av aktuell studie dras slutsatsen att bearbetning av kakaobönor industriellt inte påverkar halterna flavanoider nämnvärt så länge rostnings-temperaturer  135 °C används. Halten flavanoider i choklad tenderar istället att främst bero på kakaobönans ursprung och fermenteringen som sker redan på kakaoplantagen. Mörk choklad (70%) producerat på kakaobönan från Mexiko innehöll högst halter flavanoler i aktuell studie. För att uppnå 200 mg kakaoflavanoler vid daglig konsumtion av denna choklad krävs ett dagligt intag på cirka 77 g. Om detta berättigar Johannas Choklad i Kalmar att utnyttja hälsopåståendet för kakaoflavanoler är oklart. Vid en eventuell ansökan är det europeiska myndigheten för livsmedelssäkerhet, Efsa som genomför bedömningen. Beräkningen av 77 g grundar sig dock på värden utanför kalibreringskurvan vilket gör bestämningen osäker. För att fastställa halterna mer exakt bör ytterligare analyser genomföras. / Chocolate is obtained from the cocoa bean which is the fruit of the cocoa tree (Theobroma cacao). To create chocolate, the cocoa bean is processed, which affects the content of bioactive substances in the product. Chocolate contains several types of bioactive substances, of which the cocoa flavanols epicatechin and catechin are the most prominent. They belong to the class of flavonoids which in previous research have been linked to health effects such as lowered blood pressure, reduced risk of cardiovascular disease, and increased antioxidant capacity. Regulation No. 432/2012 on approved health claims for food, established by the European Commission, states that cocoa flavanols can contribute to the normal blood circulation by keeping the blood vessels elastic. The claim may be used for dark chocolate, but the consumer must be informed that a daily intake of 200 mg of cocoa flavanols is required to achieve the beneficial effect. In the current study, cocoa beans from Madagascar and 70% dark chocolate produced on cocoa beans from Peru, Mexico, the Philippines, and Ecuador have been analyzed in collaboration with Johanna's Chocolate in Kalmar. The purpose of the study was to investigate how the content of flavonoids and mainly the cocoa flavanols epicatechin and catechin are affected by artisanal chocolate production. The result could provide information to Johanna's Chocolate which processing steps in the process from cocoa bean to finished chocolate cake that affects the levels the most. It could also provide indications as to whether any of their chocolate products meet the requirements for obtaining the EU dark chocolate health claim. A number of assays were performed by extracting bioactive substances with methanol and evaporating the final supernatant and dissolving the residue in MQ water (v/w = 10). The solution was filtered and then analyzed by RP-HPLC with a UV detector (Reversed phase - High Performance Liquid Chromatography). The results of the analyzes gave indications that the variation of the degree of roasting (110-135 ° C) and the processing procedure in the chocolate production did not significantly affect the content of flavonoids. The analysis of the 70% dark chocolate with beans of different origins showed that cocoa beans from Mexico and Peru contained the highest levels of flavonoids and cocoa flavanols (208 and 125 mg cocoa flavanols/100 g respectively) while chocolate made of beans from Ecuador and the Philippines contained lower levels (60-70 mg cocoa flavanols/100 g). Based on the results of the current study, it is concluded that the processing of cocoa beans industrially does not significantly affect the levels of flavonoids as long as roasting temperatures ≤135 ° C are used. The content of flavonoids in chocolate instead tends to depend mainly on the origin of the cocoa bean and the fermentation that already takes place on the cocoa plantations. Dark chocolate (70%) produced on the cocoa bean from Mexico contained the highest levels of flavanols in the current study. In order to achieve 200 mg of cocoa flavanols with daily consumption of this chocolate, a daily intake of approximately 77 g is required. Whether this justifies Johanna's Chocolate in Kalmar to use the health claim for cocoa flavanols is unclear. In the event of an application, the European Food Safety Authority, EFSA, will carry out the assessment. The calculation of 77 g is based on values outside the calibration curve, which makes the determination uncertain. To determine the levels more precisely, further analyzes should be carried out.

Thesis (DTech (Chemistry))--Cape Peninsula University of Technology, 2016. / The use of ozone as an advanced oxidation process is gathering wide spread attention with the major limitation to its application being its cost of operation and design considerations. While the general approach of most researches is to buttress the already known fact of the efficacy of the process, little attention is given to studying the by-products of ozone reactions with organics. The aims of this study were to investigate the efficacy of the ozonation process for removing recalcitrant phenolics: phenol, 2-chlorophenol (2CP), 4-chlorophenol (4CP) and 2,4-dichloropheno (2,4DCP) from aqueous medium with a view of understanding various reaction pathways of the process and identifying possible intermediates and residual compounds using liquid chromatography-mass spectrometry (LC-MS). The choice of the selected chlorophenols would also elucidate the role of the positioning of the chlorine atoms in determining reaction rates, pathways and subsequent mechanisms and by-products. Sequel to this, oxy-hydroxy iron in β-phase (β-FeOOH, akaganite) and various β-FeOOH bonded composites on support metal oxides (Al2O3, NiO and TiO2) were prepared via hetero-junction joining, and explored as a possible promoter to improve the efficiency of the ozonation process. Apparent first order reaction rates constants of tested phenolics was in the order 2,4-DCP > 2-CP > Phenol > 4-CP, irrespective of the tested pH. The individual rates however increased with increasing pH. The position 4 chlorine atom was found to be least susceptible to hydroxylative dechlorination. Catechol intermediate and pathway was identified as the major degradation pathway for phenol and 2-CP, while 4-chlorocatechol pathways were more important for 4-CP and 2,4-DCP. The formation of polymeric dimers and trimers by all compounds was pronounced at alkaline pH. Heterogeneous catalytic ozonation using β-FeOOH reduced ozonation time for 4-CP by 32%. Mechanism for β-FeOOH/ozone catalysis showed that the catalyst suffered reductive dissolution in acidic pH and the kinetics of 4-CP removal using the catalyst was best described using a two stage kinetic model. The first stage was attributed to heterogeneous catalysis of ozone breakdown on β-FeOOH surface generating faster reacting radicals, while the second stage was due to homogeneous catalysis by reduced Fe2+ ions in solution. β-FeOOH stabilized on NiO at a 5% ratio exhibited superior catalytic property compared to the other tested composites. Characterization by high-resolution transmission electron microscopy (HRTEM) affirmed a β-FeOOH-NiO bonded interfaced composite which was stable as a iv catalyst over four (4) recycle runs. The mechanism of operation of the composite was via an increased ozone breakdown to radicals as monitored via photoluminescence experiments. The composite material produced satisfactory results when tested on real wastewater samples. Results from this study contribute to the current understanding on reaction mechanisms for ozone with phenols and chlorophenols, for the first time monitoring time captured intermediates via liquid chromatography-mass spectrometric method, which preserves the integrity of reaction intermediates. Also this study proposes heterogeneous catalysts; β-FeOOH and β-FeOOH bonded composites as possible improvements for simple ozone based water purification systems.

Iron is an essential nutrient for growth of photosynthetic microorganisms such as cyanobacteria and algae. Iron is required for proteins involved in the important processes of carbon and nitrogen assimilation. Low concentrations of iron in cultures or natural waters can lead to iron limitation which affects many aspects of algal metabolism. In natural waters, iron limitation can have effects on the patterns and rates of primary productivity. The cellular content of certain proteins can be affected by media iron concentrations. Methods have been used that assay components of the cell as an indirect measure of iron nutritional status. For example, spectroscopy can be performed to determine the cellular concentration of iron-containing proteins involved in photosynthesis. Organisms grown in media that imitate natural conditions, or organisms collected from their natural habitat are usually dilute. Methods that assay iron nutritional status such as spectroscopy and column chromatography require large sample sizes which are difficult to obtain from natural samples. In addition, methods that utilize techniques such as immunology or radioactive labelling are complex and time-consuming. These considerations led to the necessity of developing a technique that would be simple, rapid and effective on dilute samples. The method developed here utilized fast protein liquid chromatography (FPLC), which fulfilled these requirements. A complete analysis could be done within two to three hours with minimal sample treatment. The FPLC was simple to operate and was effective on a sample containing less than 100 μg of protein. Some photosynthetic organisms, when iron-depleted, can produce the flavin-containing protein flavodoxin (Flv). This protein substitutes for the iron-containing protein ferredoxin (Fd) in Fd-dependent reactions such as the light-induced reduction of NADP. The FPLC technique identified and quantified, in relative terms, Fd and Flv in the cell. Optical spectroscopy was used to verify FPLC retention time assignments. The results illustrated how the FPLC could be used to observe the changes in relative Fd and Flv content as a function of media iron concentration in cultures of the cyanobacterium Anabaena grown in the laboratory. It was found that Fd content decreased and Flv content increased with decreasing media iron concentration. In addition, samples of the cyanobacterium Trichodesmium collected from the ocean near Barbados were analyzed using FPLC to assay relative Fd and Flv content. By analogy with Anabaena, Fd and Flv retention times were identified. Using this technique conclusions could be drawn regarding the changing iron nutritional status of Trichodesmium in its natural habitat .

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Hydroquinone is a most commonly used whitening agent in cosmetics. A high performance liquid chromatography method was developed and validated for the quantitative determination of hydroquinone in creams. Validation parameters such as linearity, precision, accuracy, and limit of detection and limit of quantitation were determined. HPLC was carried by reverses phase technique on a RP-C18 column with a mobile phase of water and methanol (pH 5.0) 70:30. The linearity in the range of 2.0-40.0 μg/mL presents a correlation coefficient of 0.9998. The LOD and LOQ were 0.16 and 0.53 μg/mL, respectively. The precision of the method was found to be satisfactory with a coefficient of variation below 2.2%. The recovery values were in the range of 92.4 to 99.0%. The method is sensitive, fast, and simple. It has been successfully applied to the determination of hydroquinone in cosmetic creams. The results obtained agreed well with the percentages given by the manufacturers.