Determination of Selenium in Selenium enriched yeast. Kenneth Michael Kennedy B.Sc (Hons). Submitted for the award of Masters of Science. Galway-Mayo Institute of Technology * Supervisor: Dr. Joseph Scanlan Submitted to the National Council of Educational Awards July 1999. Acknowledgements: In the completion of the enclosed thesis I would like to take time to thank a few people. Firstly to Dr. Joseph Scanlan who initially set up this project. He gave all his technical and academic support at any time. With out his help and expertise, this project would never have been finished. Thanks to the Department of Physical Sciences Galway-Mayo Institute of Technology ( Dr Brian Place Head of School and Dr. Tom Gillen Head of Department.) To the Technical Support Staff who never minded if I borrowed a piece of equipment for an extended period of time. To the secretaries of the department who helped with all enquires mundane or otherwise that I threw at them, they were always willing to help. My thanks to the Administration Staff of the G.M.I.T especially to Phil Lydon who ploughed through red tape to make my life easier. To Tom Conlon in the Industrial Liaison Office for all his work. To Ann Joyce and the gang up in the Library who helped so many times in getting literature and waived a fine or two for over-due books. To all the Academic staff in the Life Sciences and Computers especially to Dr. Myles Keogh who was a great man for a chat and who was always on call to have his brain picked. To my fellow insomniacs and coffee pals Steven, Marrion, Steve and Iridia “ye kept me sane - God bless ye!” To Dr. Ronan Power of Altech for the samples which the thesis was based upon. To Dr. Michael J. Hynes of N.U.I.G. To Forbairt for financial support under the Graduate Training Programme. Thanks to my parents Owen and Teresa, it was your constant encouragement and support that helped me get this far. I will never be able to repay the debt. And finally to Ursula Owens who proof read the thesis, who corrected all my grammar mistakes and made me look good before it went to Joe for the final proof read. You have been a Godsend. Always and ever - and then some. 3.3.7 Transmitting a spectrum to the computer 120 3.3.8 Sample Preparation 121 3.3.10 Blending/Mixing 121 3.3.11 Preparation of selenium standards in non-enriched yeast 122 3.3.12 Preparation of a standard curve 124 3.4 Results and discussion 127 3.4.1 Optimisation of conditions for X-ray analysis of selenium 127 3.4.2 X-ray spectra of enriched and non-enriched yeast 131 3.4.3 Effect of specimen thickness 136 3.4.4 Selenium calibration curves 137 3.4.5 Matrix effects in selenium analysis 141 3.4.6 Validation of linearity 143 3.4.7 The limit of detection 144 3.4.8 Reproducibility of the selenium analysis 144 3.4.9 Analysis of using standard addition 145 3.4.10 Analysis of variance 148 4.1 A brief history 151 4.2 Materials, Instrumentation and solutions 160 4.3 Experimental Procedure 163 4.3.1 Hydride generation procedure 163 4.3.2 Procedure for the reduction of sodium selenate standard to sodium selenite at various temperatures 167 4.3.3 Procedure for the digestion of the selenium yeast sample and reduction of selenate to selenite and the subsequent selenium analysis 168 4.3.4 Procedure for determination of a sample by standard additions 169 4.4 Results and discussion 170 4.4.1 The effect of HC1 concentrations on the Se peak 170 4.4.2 Standard Curve for sodium selenite 171 4.4.3 Rate of reduction of Se042' in 6M HC1 173 4.4.4 Effect of temperature on digestion step 180 4.4.5 Analysis of selenium enriched yeast by Standard Addition 182 4.4.6 Method Validation 184 5 Conclusion 185 Appendix I i Appendix 2 vi Appendix 3 xxviii Appendix 4 xxxii Bibliography xxxix Chapter 1 1.1 Distribution of Selenium in nature. Selenium, although a rare mineral, is widely distributed in minute amounts in virtually all materials of the earth’s crust, its average abundance is approximately ~0.09ppm. Selenium is rarely found in its native state i.e. in elemental form, instead it is usually found as an oxide as selenite and selenate or in sulphur or porphyry copper deposits. The greatest abundance of selenium is found in igneous rocks, in hydrothermal deposits, and in the massive sulphide and porphyry copper deposits that are mentioned above. Known deposits of selenium are insufficient to permit their mining for the element alone and consequently it would be far too expensive to mine for its own commercial benefit. Virtually all production of selenium is via its extraction from copper refinery slimes along with the recovery of precious metals. (National Research Council, 1976b) Selenium is obtained commercially by treatment of anode slimes produced during the electrolytic refining of copper. The principal sources of selenium are the sulphidic copper ores in Canada, the United States and in Russia as a result the majority of literature dealing with selenium is from these non-European countries. This is not to say that there is no selenium outside these countries, some of the soils of Ireland contain reasonable amounts of selenium. High concentrations of selenium are found in sedimentary rocks such as shale, sandstone, limestone and phosphorite rocks. The average concentration in shales has ranged from 0.24ppm for Palaeozoic shales of Japan to 277ppm for black shales of Permian age from Wyoming (Lakin and Davidson, 1967). Shales are also the principal sources of selenium-toxic soils in Ireland, Australia and several other l countries of the world (Johnson 1975). In Ireland these shales can be found around Sligo, Carrick-on-Shannon, and Enniskillen. The soil type is gleys which is Shale-Old Red Sandstone Till and due to the Ice Age, the soils have been sculpted into drumlins by the glaciers: sqq Appendix. 1.1 (Supplied by the Geological Survey of Ireland) In general, the selenium content of limestone derived soils is generally low as found by Lakin and Davidson in 1967. Here in Ireland these limestone derived soils can be found around Limerick, south west of Kilkenny, south of Wexford and a large area west of Roscommon. The parent material is limestone-sandstone -shale till. The soil type is gleys dominantly influenced by surface water impendence see Appendix 1.1 (Geological Survey of Ireland). Phosphate rocks contain, relatively high concentrations of selenium; this could lead to an increase in the Se in soils because of the wide use of phosphate fertilisers made from these deposits. Seleniferous sulphur is a source of selenium in phosphate fertilisers and sulphur containing inorganic salts included in livestock diets. The selenium content of Japanese and Hawaiian volcanic sulphur ranged from 67 to 206ppm and 1,026 to 2,000ppm respectively (Lakin and Davidson, 1967), Ireland is not an island created from volcanic activity, so no comparative study can be done with respect to these islands. Although selenium has also been found to occur in fossil fuels, Irish fossil fuels are relatively low in selenium content. Exact concentrations have not been calculated, as the economic importance of selenium in fossil fuels is negligible. Uses of selenium in history In the early Stone Age and through to the Pre-Iron Age selenium, in the form of sodium selenite crystals (NaaSeOj), was used by the inhabitants of Montana, Oregon and Wyoming (North America) as powerful talismans and in religious ceremonies. 2 This is evidenced from caves recently discovered (1980’s) in these states. The caves are over twelve miles in length and entirely devoid of light. Many questions arise from this, why did people dig out the selenium as selenite crystals? And how did they travel such great lengths in total darkness? Answers to these questions have been given by archaeologists. Shaman or religious leaders used the element in ancient ceremonies because in appearance they are very much like quartz crystals, they refract the light within its many prisms producing beautiful colours within the crystal. These people dug the crystals out with primitive tools such as bone picks. Archaeologists have figured out how these ancient people travelled the great distances in pitch- blackness. Using cane-reed, which were in ample abundance around the cave area, the reeds were gathered into bundles to form rough lamps, which archaeologists used when trying to recreate the method of illumination used by these people. The cane lamps were lit at the entrance to the cave, then a group of archaeologists travelled for over three days in these caves, using the cane lamps as the only source of light. They found charcoal marks on the low roofs of the caves where the cane was scratched to mark the path or to scratch off the spent cane to keep the lamp rejuvenated. Present day uses for selenium Today selenium and its compounds have a multitude of uses such as, in the manufacturing of glass to make ruby coloured glass and enamels or to decolourise glass, in xerography as a photographic toner and as a pigment when mixed with a cadmium compound. It is used as conductors, rectifiers, electron emitters, and insulators, as reagents in remedies for eczema and fungus infections in pets, in antidandruff agents for humans, and in veterinary therapeutic agents. In agriculture, early uses for selenium compounds involved control of mites and insects.