COLOUR REMOVAL FROM SUGAR CANE JUICE Danny M. T. Nguyen Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Chemistry, Physics and Mechanical Engineering Science and Engineering Faculty Queensland University of Technology, Brisbane, Australia June 2013 Supervisor: Professor William O. S. Doherty Sugar Research and Innovation Centre for Tropical Crops and Biocommodities Queensland University of Technology, Brisbane, Australia Associate Supervisor: Adjunct Associate Professor John P. Bartley School of Chemistry, Physics and Mechanical Engineering Science and Engineering Faculty Queensland University of Technology, Brisbane, Australia The research was carried out within the Centre for Tropical Crops and Biocommodities at the Queensland University of Technology. ii IMPORTANT NOTICE The information in this thesis is confidential and should not be disclosed for any reason or relied on for a particular use or application. Any invention or other intellectual property described in this document remains the property of the Queensland University of Technology. iii DECLARATION OF AUTHORSHIP The work contained in this thesis has not been submitted for assessment for any other award. Wherever contributions of others are involved, every effort is made to indicate this clearly with proper reference to the literature and acknowledgement of collaborative research and discussions. Some parts of the research work in this thesis have been published and a list of publications arising from this research has been provided. QUT Verified Signature .. ... Danny M. T. Nguyen, BSc (Hons) Date: .......................................................... iv Abstract One of the most important parameters in raw sugar quality is colour. Australian raw sugars are considered to be of high quality with respect to this parameter. However, some raw sugars produced in both Australia and overseas are relatively difficult to decolourise by sugar refiners, and tend to develop colour during storage. A new approach that has the potential to efficiently and cost-effectively decolourise sugar process streams is through the use of the Fenton oxidation and related processes. The Fenton oxidation process involves the catalytic production of hydroxyl radicals from the decomposition of hydrogen peroxide (H2O2) using iron(II), which has the potential to effectively degrade colour and colour precursors present in aqueous systems. As a first step towards developing this technology, this study determined the colour content and the composition of colour precursors (i.e., phenolic acids), present in sugar cane juices processed by Australian sugar factories. The results showed that caffeic, p–coumaric and ferulic acids (classed as hydroxycinnamic acids) are the main phenolic acids present in sugar cane juice. The study was able to identify flavonoids (e.g., chrysin, morin, quercetin and rutin) because of modifications of the methods used in the evaluation of colourants in sugar cane juice. The results also show that juice expressed partly or solely from whole crop harvested cane, has significantly higher colour (11,400–20,000 IU) than juices expressed from burnt harvested cane (10,400–12,700 IU). However, the concentrations of phenolic acids in burnt cane were twice as much as those obtained in whole crop cane. This is probably due to the thermal decomposition of HMW phenolics (viz., lignin, polyphenols) during cane burning. The Fenton oxidation process was used to study the degradation of these hydroxycinnamic acids (i.e., caffeic, p–coumaric and ferulic) in water and sucrose solutions. Central composite design and response surface methodologies were used to evaluate and optimise the interactive effects of the process parameters. Quadratic polynomial models were developed for the degradation of each of the individual v acids, and the total hydroxycinnamic acid mixtures. The optimum degradation efficiency (77%) in an aqueous solution containing the hydroxycinnamic acids (200 mg/L) was optimum at pH 4.7 and at 25 °C. The efficiency dropped in the presence of sucrose to 57% at pH 5.4 and at 36 °C. In a mixture of these hydroxycinnamic acids, the degradation behaviour of caffeic acid differed from those of p–coumaric and ferulic acids, because unlike the other acids, it forms a complex with iron(III). Iron(III) is produced in situ during the oxidation process. Analysis of the Fenton degradation products showed the presence of low molecular weight phenolics, aliphatic carboxylic acids as well as several oligomer products. The tentative mechanisms of formation of these compounds have been proposed. To improve the effectiveness of the Fenton process, aluminium chloride was added to act as a pro-oxidant. This process was evaluated on a synthetic juice solution consisting of sucrose (15% (w/w)), the hydroxycinnamic acids (200 mg/L) and a synthetic glucose-glycine melanoidin (2,000 mg/L). The modified Fenton process degraded the melanoidin and the hydroxycinnamic acid mixture by approximately 69% and 53% respectively. In the absence of aluminium chloride, the Fenton process on its own resulted in 63% and 47% degradation, respectively but only achieved 24% decolourisation. However, the addition of aluminium chloride played a significant role in the removal of colour with up to 43% decolourisation achieved. The modified Fenton process was then evaluated for the decolourisation of authentic factory juices. There were increases in colour measured at pH 4.0 (≤ 45%) and pH 7.0 (≤ 21%). However, there was decrease for the colour measured at pH 9.0 (≤ 42%). Colour is usually measured at pH 7.0 but additional information about the nature of colourants is obtained at pH 4.0 and pH 9.0. Colour measured at pH 4.0 suggests the presence the presence of high molecular weight colourants, while colour measured at pH 9.0 is due to the presence of natural colourants such as flavonoids and phenolics. The colour at pH 9.0 is more likely to be transferred to the crystal, so there may well be colour reduction if the treated juice is further processed to raw sugar. vi The key contribution contained in this thesis is an understanding of the degradation of colour precursors in sugar solutions. A new direction of research for the removal of colour and colour precursors in sugar process streams has been identified. vii Keywords Colour Colourants Colour precursors Sugar Sugar cane juice Sugar quality Sucrose Decolourisation Degradation Fenton Advanced oxidation process Hydroxycinnamic acids Caffeic acid p–Coumaric acid Ferulic acid Response surface methodology Experimental design UV/Visible spectroscopy High-performance liquid chromatography Reaction pathways Clarification Aluminium chloride Melanoidin Reducing sugars viii Acknowledgements A PhD candidature, by its very own nature, is a very unique endeavour. There is no right way to undertake a PhD project. However, there are many wrong ways that one could take throughout their candidature. I am for one, a very glad person, who has taken the best path possible in order to complete my candidature and hopefully graduate with a doctoral degree. I could have not taken this path without the consistent guidance and advice given from the very kind people that I have met throughout my entire candidature to whom I give thanks to. First and foremost, I would like to sincerely thank my primary supervisor, Prof. William (Bill) Doherty, for his constant patience, guidance, encouragement and commitment to this work. Bill, you have been a great mentor. I have learnt and gained so much from you. Despite our differences and heated discussions on several aspects of this thesis, you have always seen the best in me. Towards the end of writing this thesis, I was asked by many for an inspirational and memorable quote from you. In response to that, that would definitely be, “Danny, could you please come to my office? I need to see you.” I am very glad because every time I walked into your office with the heater running on a warm Brisbane day, I would learn something new, no matter how irrelevant it is to my own work. Thank you. I would also like to thank my associate supervisor, Adj. A/Prof. John Bartley. You have always been prompt whenever I needed you most and you have been a great mentor. I appreciate all the times, especially at the very early stages of my candidature, assisting me with certain aspects of organic chemistry. I always gained something useful each time we met. Up to today, I still have a strong passion for organic chemistry and to me, drawing chemical structures for reaction mechanisms is genuinely a form of art. This project would have not happened, if it was not for the financial support from my scholarship sponsors. To the three main sponsors, the Queensland University of Technology (QUT), the Sugar Research and Development Corporation and Sugar Research Limited, a very big thank you for your generosity. My exposure ix to the Australian sugar industry has been very worthwhile. This was a very rare opportunity and I am grateful that each sponsor accepted me to undertake this project. In addition, I would also like to thank the production staff of Condong Sugar Mill, Tully Sugar Mill and Isis Central Sugar Mill who gave me access to their facilities during the crushing seasons. Many thanks must go to all the academic and technical staff who have contributed to this project throughout my candidature. Prof. Robert (Bob) Gilbert (University of Queensland, UQ) for his expertise on food polymers; Dr. Peter Sopade (UQ) and A/Prof. Geoff Kent (QUT) for introducing me into the world of multivariate statistics; Mr. Hakan Bakir (QUT) for his assistance at the mills during the factory trials; Mr. Tony Raftery (QUT) for his assistance on XRD analysis; Dr. Chris Carvalho and Mrs. Leonora Newby for analytical instrument training; and Ms. Wanda Stolz (QUT) for her endless hospitality in the lab. To my fellow colleagues who work closely with me, thank you for your ongoing support. Chris East, you have been a great mate throughout my candidature and thanks for changing my life that day (you know what happened).