Multi-Element Stable Isotope Analysis of Alkylpyrazines and Pyridine from Roast Coffee

Multi-element Stable Isotope Analysis of Alkylpyrazines and Pyridine from Roast Coffee Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Christina Preston aus Aalen Würzburg 2008 <ul style="display: flex;"><li style="flex:1">Eingereicht am: </li><li style="flex:1">26. September 2008 </li></ul>bei der Fakultät für Chemie und Pharmazie 1. Gutachter: 2. Gutachter: der Dissertation Prof. Dr. P. Schreier Prof. Dr. H.-U. Humpf 1. Prüfer: 2. Prüfer: 3. Prüfer: Prof. Dr. P. Schreier Prof. Dr. H.-U. Humpf Prof. Dr. C. Sotriffer des öffentlichen Promotionskolloquiums Tag des öffentlichen Promotionskolloquiums: 21. Januar 2009 Doktorurkunde ausgehändigt am: ……………………………… Acknowledgements The following research was conducted at the chair of food chemistry, University of Würzburg, from January 2004 to May 2007, under the guidance of Professor Dr. Peter Schreier. Professor Dr. Peter Schreier I thank for the isotopic-topic, the scientific support, the interesting discussions, not always strictly work-related, and the freedom of researching independently. Furthermore, I thank Dr. Thorsten König, from Givaudan (formerly Quest International), for providing samples and Dr. Elke Richling, Dr. Dominique Kavvadias, as well as the student workers Christopher Heppel and Silvia Hummel, for the pre-work conducted on this research topic. Special thanks go to the ‘F-Praktikanten’ Kathrin Kahle, Helena Bader, Stefanie Kohlhepp, Ina Kleinsteuber, Magdalena Müller, Karin Thomas, Christof Madinger and Tanja Welsch as well as to the ‘C-Praktikanten’ Daniela Trost and Fabian Oberle, who all supported me courageously during their advanced practical courses. They all contributed substantially to the success of this work, with their never-tiring energy, their good ideas and friendly relationships. Dr. Frank Heckel and Dr. Sandra Elß divulged their GC-MS knowledge to me and taught me all they could about these machines. I thank them and Michael Kempf, both for the good working relationship between us in the GC-MS department and for their willingness to help out with the ‘special’ problems of the IRMS. Eva-Maria Rumpel successfully gave me a three-month crash course in IRMS measurement, whilst Dr. Markus Appel contributed his extensive knowledge on the IRMS. With Dr. Sandra Elß’s assistance the work with the machine was highly productive and always exciting. I thank the IRMS team for their help in the often eventful and seemingly never-ending work, and without whom stable isotopes would still be a mystery to us all. A very special thank you goes to Theresia Feuerbach, who took loads of work off my shoulders, by conducting the practical work for other projects and helping out, whenever she could. I thank my laboratory colleagues, Dr. Markus Appel, Theresia Feuerbach and Michael Kempf, for sharing rooms and laboratory benches with me, even if it was sometimes hard to find space for working. For interpreting NMR results I had help from Dr. Markus Appel and Sanja Locher and for the work on the HPLC’s I thank the ‘cellar kids’ Kathrin Kahle, Wolfgang Hümmer and Matthias Ackermann. Of the non Food Chemists, Doris Moret gave invaluable pharmaceutical technical help and the workshop crew Matthias, Karl and especially Schorsch, were indispensable for all kinds of repairs and constructions. I will, furthermore, miss the ‘Technischer Betrieb’ with Andi, Pierre, Thomas and of course ‘Herr Fritz’ and thank them, particularly, for the lovely midday breaks we girls were invited to. Otherwise, my never-ending thanks go to our working group, for all the good times, excursions, feasts and friendships we enjoyed together. Here, I would especially like to thank Dr. Sandra Elß and Kathrin Kahle for their friendship and our ‘girls - pizza - ice cream - evenings’, as well as Matthias Ackermann and Wolfgang Hümmer, who both participated in the friendship and sometimes in the evenings, if not always voluntarily for Wolfgang. Thank you also to everyone, who had to endure my questions and corrections to this piece of work, especially my father, who is now probably proficient enough to referate on stable isotope analysis off-by-heart. My thanks also go to the F.R.U.I.T. Foundation, Heidelberg, Unibund and the program ‘Chancengleichheit für Frauen in Forschung und Lehre’ from the University of Würzburg for their financial support. My main thanks go to my parents and my sister, who always let me do what I thought best, and supported me on my way with all means, as well as to my friend, Kathrin Diegelmann, whose friendship always meant a lot to me, and without whom, studying would have only been half as much fun and not as successful. ‘Theory is, when one knows everything, but nothing works, Praxis is, when everything works, but no-one knows why. For us theory and praxis are combined: Nothing works and no-one knows why!’ - Unknown Index IIndex Publications........................................................................................... VI Explanations and Abbreviations ............................................................ IX Summary..............................................................................................XII Zusammenfassung ................................................................................XV 12 Introduction......................................................................................1 State of Knowledge ...........................................................................3 <ul style="display: flex;"><li style="flex:1">2.1 </li><li style="flex:1">Sources, Distribution and Fractionation of Isotopes............................ 3 </li></ul><ul style="display: flex;"><li style="flex:1">2.2 </li><li style="flex:1">Isotope Fractionation of Hydrogen................................................... 6 </li></ul>2.2.1 Thermodynamic Isotope Fractionation.......................................... 6 2.2.2 Isotope fractionation in plants..................................................... 8 <ul style="display: flex;"><li style="flex:1">2.3 </li><li style="flex:1">Isotope Fractionation of Carbon .................................................... 10 </li></ul>2.3.1 Thermodynamic Isotope Fractionation........................................ 10 2.3.2 Isotope Fractionation in Plants .................................................. 11 <ul style="display: flex;"><li style="flex:1">2.4 </li><li style="flex:1">Isotope Fractionation of Nitrogen .................................................. 14 </li></ul>2.4.1 Thermodynamic Isotope Fractionation........................................ 14 2.4.2 Isotope Fractionation in Plants .................................................. 16 <ul style="display: flex;"><li style="flex:1">2.5 </li><li style="flex:1">Methods and Applications of Stable Isotope Analysis in Food Chemistry .. </li></ul>................................................................................................ 19 2.5.1 Site-Specific Natural Isotope Fractionation – Nuclear Magnetic Resonance (SNIF – NMR®) ....................................................... 19 2.5.2 Isotope Ratio Mass Spectrometry (IRMS).................................... 20 2.5.2.1 Elemental Analyser – Isotope Ratio Mass Spectrometer (EA-IRMS) ....................................................................................... 22 2.5.2.1.1 Elemental Analyser – Combustion – Isotope Ratio Mass Spectrometry (EA–C–IRMS)........................................... 22 2.5.2.1.2 Elemental Analyser – Pyrolysis – Isotope Ratio Mass Spectrometry (EA–P–IRMS) ........................................ 23 2.5.2.2 Gas Chromatography – Isotope Ratio Mass Spectrometry (GC- IRMS) .............................................................................. 25 2.5.2.2.1 Gas Chromatography – Combustion – Isotope Ratio Mass Spectrometry (GC – C – IRMS)....................................... 26 II Index 2.5.2.2.2 Gas Chromatography – Pyrolysis – Isotope Ratio Mass Spectrometry (GC – P – IRMS)....................................... 27 2.5.3 Application of Stable Isotope Analysis ........................................ 27 2.5.3.1 Authenticity Assessment of Aroma Compounds....................... 28 2.5.3.2 Further Applications ........................................................... 30 <ul style="display: flex;"><li style="flex:1">2.6 </li><li style="flex:1">Coffee and Coffee Aroma ............................................................. 31 </li></ul>2.6.1 History and Importance of Coffee .............................................. 31 2.6.2 Botanic and Production Types ................................................... 32 2.6.3 Composition of Green Coffee Beans ........................................... 33 2.6.4 Roast Coffee Aroma................................................................. 39 <ul style="display: flex;"><li style="flex:1">2.7 </li><li style="flex:1">Alkylpyrazines ............................................................................ 40 </li></ul>2.7.1 Characteristics of Alkylpyrazines ............................................... 40 2.7.2 Chemistry of formation ............................................................ 41 <ul style="display: flex;"><li style="flex:1">2.8 </li><li style="flex:1">Research Objective ..................................................................... 45 </li></ul><ul style="display: flex;"><li style="flex:1">3</li><li style="flex:1">Results and Discussion....................................................................47 </li></ul><ul style="display: flex;"><li style="flex:1">3.1 </li><li style="flex:1">Stable Isotope Stability of Alkylpyrazines in Ethanol and Water.......... 47 </li></ul>3.1.1 Stability of Alkylpyrazines in Ethanol.......................................... 47 3.1.2 Stability of Alkylpyrazines in Water............................................ 49 <ul style="display: flex;"><li style="flex:1">3.2 </li><li style="flex:1">Stable Isotope Analyses, 15N/14N and 2H/1H, of Alkylpyrazines and </li></ul>Pyridine from Roast Coffee Aroma................................................. 50 3.2.1 Roast Coffee Aroma Profile and Sample Preparation..................... 50 3.2.2 15N/14N and 2H/1H Analyses of Volatiles of Self-Roasted Coffee Beans ............................................................................................ 52 3.2.3 15N/14N and 2H/1H Analyses of Volatiles of Commercial Coffee Products ............................................................................................ 60 3.2.4 Aroma Profile and 15N/14N, 2H/1H and 13C/12C Analyses of Roast Coffee Volatiles of Different Roasting Degrees....................................... 69 3.2.5 Conclusion ............................................................................. 74 3.2.5.1 Conclusion to 15N/14N and 2H/1H Analyses of Roasted Coffee Beans and Products..................................................................... 74 3.2.5.2 Conclusion to 15N/14N, 2H/1H and 13C/12C Analyses of Roast Coffee Volatiles of Different Roasting Degrees ................................. 76 <ul style="display: flex;"><li style="flex:1">3.3 </li><li style="flex:1">Stable Isotope Analyses, 15N/14N, 2H/1H and 13C/12C, of Synthesised </li></ul>Alkylpyrazines and Studies to Pyridine Formation............................ 78 3.3.1 Alkylpyrazine Syntheses via Condensation of α-Diketones and α- Diamines ............................................................................... 78 Index III 3.3.1.1 Synthesis and 15N/14N, 2H/1H and 13C/12C Analyses of 2,3- dimethylpyrazine ............................................................... 79 3.3.1.2 Synthesis and 15N/14N, 2H/1H and 13C/12C Analyses of 2-Ethyl-3- methylpyrazine.................................................................. 81 3.3.1.3 Synthesis and 15N/14N, 2H/1H and 13C/12C Analyses of 2,3,5- Trimethylpyrazine.............................................................. 82 3.3.1.4 Synthesis and 15N/14N, 2H/1H and 13C/12C Analyses of 2-Ethyl- 3,5/6-dimethylpyrazine ...................................................... 84 3.3.2 Alkylpyrazine Synthesis via Reduction of Methylpyrazinecarboxylate85 3.3.2.1 Synthesis and 15N/14N, 2H/1H and 13C/12C Analyses of 2-Ethylpyrazine........................................................................... 86 3.3.3 Syntheses of Alkylpyrazines from Amino Acids ............................ 87 3.3.3.1 Synthesis and 15N/14N, 2H/1H and 13C/12C Analyses of Alkylpyrazines from L-Serine...................................................... 87 3.3.3.2 Synthesis and 15N/14N, 2H/1H and 13C/12C Analyses of Alkylpyrazines from L-Threonine................................................. 90 3.3.3.3 Synthesis and 15N/14N, 2H/1H and 13C/12C Analyses of Alkylpyrazines from L-Serine and L-Threonine .............................. 91 3.3.4 Synthesis and 15N/14N, 2H/1H and 13C/12C Analyses of Alkylpyrazines from Diethylentriamine ............................................................ 93 3.3.5 Studies to Pyridine Formation ................................................... 94 3.3.5.1 Synthesis and 15N/14N, 2H/1H and 13C/12C Analyses of Trigonelline ....................................................................................... 94 3.3.5.2 Degradation of Pyridine Precursors ....................................... 95 3.3.6 Conclusion ............................................................................. 95 <ul style="display: flex;"><li style="flex:1">3.4 </li><li style="flex:1">Stable Isotope Analysis of Green Coffee Fractions and Roasting, thereof </li></ul>................................................................................................ 97 3.4.1 Fractionation Scheme .............................................................. 98 3.4.2 15N/14N, 2H/1H and 13C/12C Analyses of Green Coffee Fractions ......101 3.4.3 Aroma Profile of Roasted Green Coffee Fractions ........................106 3.4.4 15N/14N, 2H/1H and 13C/12C Analyses of Roasted Green Coffee Fractions ...........................................................................................110 3.4.5 Conclusion ............................................................................113 <ul style="display: flex;"><li style="flex:1">4</li><li style="flex:1">Materials and Methods ..................................................................115 </li></ul><ul style="display: flex;"><li style="flex:1">4.1 </li><li style="flex:1">Coffee Bean Samples..................................................................115 </li></ul>Chemicals.................................................................................115 Consumables for Isotope Ratio - Mass Spectrometry.......................116 4.2 4.3 IV Index <ul style="display: flex;"><li style="flex:1">4.4 </li><li style="flex:1">Chromatography........................................................................116 </li></ul>4.4.1 Column Chromatography.........................................................116 4.4.1.1 Silica Gel Chromatography .................................................116 4.4.1.2 Cation Exchange Chromatography.......................................117 4.4.1.3 Anion Exchange Chromatography........................................117 4.4.2 Thin Layer Chromatography (TLC)............................................117 4.4.2.1 Detection of Sucrose via TLC ..............................................117 4.4.2.2 Detection of Amino Acids via TLC ........................................118 4.4.2.3 Detection of Trigonelline via TLC .........................................118 4.4.2.4 Detection of Citric Acid via TLC ...........................................118 <ul style="display: flex;"><li style="flex:1">4.5 </li><li style="flex:1">Instruments ..............................................................................119 </li></ul>4.5.1 Capillary Gas Chromatography - Mass Spectrometry (HRGC-MS) ..119 4.5.2 Isotope Ratio Mass Spectrometry (IRMS)...................................119 4.5.2.1 Capillary Gas Chromatography............................................119 4.5.2.2 HRGC-‘Interfaces’ .............................................................120 4.5.2.3 Elemental Analysers ..........................................................121 4.5.2.4 Isotope Ratio – Mass Spectrometer......................................121 4.5.3 Nuclear Magnetic Resonance Spectroscopy (NMR).......................122 4.5.4 Other Instruments .................................................................122 <ul style="display: flex;"><li style="flex:1">4.6 </li><li style="flex:1">Methods ...................................................................................123 </li></ul>4.6.1 Stability of Alkylpyrazines in Water and Ethanol..........................123 4.6.2 Sample Preparation................................................................124 4.6.2.1 References.......................................................................124 4.6.2.2 Coffee Samples.................................................................124 4.6.2.2.1 Roasting of Green Coffee Beans.....................................124 4.6.2.2.2 Simultaneous Distillation Extraction (SDE) ......................124 4.6.2.2.3 Silica Gel Fractionation.................................................124 4.6.2.2.4 Coffee Aroma..............................................................125 4.6.3 Fractionation of Green Coffee Beans .........................................125 4.6.3.1 Grinding of Green Coffee Beans ..........................................125 4.6.3.2 Extraction of Green Coffee Powder with n-Hexane..................125 4.6.3.3 Extraction of n-Hexane Insoluble Fraction with Ethanol/Water (80:20, v/v).....................................................................126 4.6.3.4 Isolation of Amino Acids from the Ethanol/Water Soluble Fraction via Cation Exchange Chromatography..................................127 4.6.3.5 Isolation of Organic Acids from the Anionic/Neutral Fraction via Anion Exchange Chromatography........................................128 4.6.3.6 Extraction of Ethanol/Water Insoluble Fraction with Water ......128 Index V4.6.3.7 Precipitation of Polysaccharides of the Water Soluble Fraction with Ethanol ...........................................................................129 4.6.3.8 Extraction of Pectin of the Water Insoluble Fraction with Ammonium Oxalate ..........................................................129 4.6.3.9 Roasting of Green Coffee Fractions ......................................130 4.6.4 Roasting of Model Mixtures for Pyridine Formation Studies ...........131

Multi-Element Stable Isotope Analysis of Alkylpyrazines and Pyridine from Roast Coffee

Details

Download

Copyright

We respect the copyrights and intellectual property rights of all users. All uploaded documents are either original works of the uploader or authorized works of the rightful owners.

Support