Università Degli Studi Di Bologna

Università degli Studi di Bologna Dottorato di Ricerca in Scienze degli Alimenti – XIX Ciclo – Settore Scientifico-Disciplinare CHIM. 06 Coordinatore: Chiar.mo Prof. Claudio Cavani A CASE STUDY DEVELOPMENT OF CHEMICAL INDEXES, ORIGINATED FROM THE APPLICATION OF CHEMOMETRIC METHODS TO THE NUCLEAR MAGNETIC RESONANCE (NMR), IN THE ASSESMENT OF THE QUALITY AND OF THE GEOGRAPHICAL ORIGIN OF VEGETABLE PRODUCTS PhD FINAL DISSERTATION Presentato Relatore: dal Dottore: Chiar.mo Prof. FRANCESCO SAVORANI GIUSEPPE PLACUCCI Correlatore: Chiar.mo Prof. FRANCESCO CAPOZZI A.A. 2006-2007 PREFACE........................................................................................................ i 1 INTRODUCTION ........................................................................................1 1.1 FOOD QUALITY................................................................................................1 1.1.1 Instrumental analyses for the evaluation of food quality......................................1 1.2 NUCLEAR MAGNETIC RESONANCE (NMR) IN FOOD SCIENCE .......................3 1.2.1 Low-Field NMR (LF-NMR) spectroscopy ..............................................................4 1.2.2 NMR-MObile Universal Surface Explorer (NMR -MOUSE).....................................5 1.2.3 SNIF NMR.........................................................................................................6 1.2.4 Magnetic Resonance for Imaging (MRI)..............................................................6 1.2.5 High Resolution Nuclear Magnetic Resonance (HR-NMR) .....................................7 1.3 NOTIONS ON NMR SPECTROSCOPY ...............................................................8 1.3.1 Discovery .........................................................................................................8 1.3.2 Continuous wave (CW) spectroscopy..................................................................9 1.3.3 Pulsed NMR spectroscopy and Fourier’s transform...............................................9 1.3.4 Theoretical principles ...................................................................................... 11 1.3.4.1 Nuclear Magnetic Resonance phenomenon................................................. 11 1.3.4.2 Chemical shift........................................................................................... 17 1.3.4.3 Tuning, Locking and Shimming .................................................................. 18 1.3.4.4 Fine structure in a NMR spectrum .............................................................. 21 1.4 CHEMOMETRICS............................................................................................22 1.4.1 Preprocessing of raw data ............................................................................... 23 1.4.1.1 Data Alignment......................................................................................... 24 1.4.1.2 Data Normalization ................................................................................... 24 1.4.1.3 Data Centering and Scaling ....................................................................... 25 1.4.2 Principal Chemometric Tools ............................................................................ 26 1.4.2.1 Principal Component Analysis (PCA) ........................................................... 27 1.4.2.2 Linear Discriminant Analysis (LDA)............................................................. 31 1.4.2.3 Leave One Out (LOO) cross-validation........................................................ 34 1.4.2.4 Mahalanobis Distances (MD)...................................................................... 35 1.4.3 Chemometrics in food science.......................................................................... 36 1.5 A CASE STUDY OF CHEMOMETRICS APPLIED TO NMR DATA ...................... 37 1.5.1 Collective marks ............................................................................................. 37 1.5.2 The "Pomodoro di Pachino" PGI product. ......................................................... 40 1.6 AIM OF THE RESEARCH ................................................................................ 42 2 MATERIALS............................................................................................. 43 2.1 GLASSWARE AND DISPOSABLE MATERIALS................................................ 43 2.2 REAGENTS .................................................................................................... 43 2.3 SOLUTIONS .................................................................................................. 44 2.4 INSTRUMENTATIONS................................................................................... 45 2.4.1 Classical laboratory instrumentation................................................................. 45 2.4.2 200 MHz NMR spectrometer ............................................................................ 45 2.4.3 400 MHz NMR spectrometer ............................................................................ 46 3 METHODS................................................................................................ 49 3.1 PROTOCOLS.................................................................................................. 49 3.1.1 Cherry tomato fruit extraction in D2O buffer solutions ....................................... 49 3.1.2 NMR samples preparation ............................................................................... 50 3.1.3 Protocol for 200 MHz NMR analysis.................................................................. 51 3.1.4 Protocol for 400 MHz NMR analysis.................................................................. 51 3.1.5 Protocol for the temperature and pH dependence of signals.............................. 52 3.2 INSTRUMENTAL PARAMETERS FOR NMR ACQUISITION............................. 53 3.2.1 1H-NMR spectra of Cherry tomato extracts recorded at 200 MHz ....................... 53 3.2.2 1H-NMR spectra of Cherry tomato extracts recorded at 400 MHz ....................... 54 3.3 NMR DATA HANDLING ................................................................................. 55 3.3.1 FID processing for acquisition at 200 MHz .................................................... 56 3.3.2 FID processing for acquisition at 400 MHz .................................................... 56 3.4 CHEMOMETRIC DATA PROCESSING............................................................. 57 3.4.1 Chemometric data processing of spectra recorded at 200 MHz .......................... 58 3.4.2 Chemometric data processing of spectra recorded at 400 MHz .......................... 61 3.5 FRUITS SAMPLING ....................................................................................... 64 3.5.1 Sampling and cataloguing cherry tomato fruits ................................................. 64 3.5.2 Cherry tomatoes for inter-laboratory check test................................................ 67 4 RESULTS AND DISCUSSION ....................................................................71 4.1 NMR ANALYSIS AT 200 MHZ.........................................................................74 4.1.1 Chemometric data processing .......................................................................... 79 4.1.2 Processing of winter samples........................................................................... 90 4.1.3 Processing of summer samples ........................................................................ 96 4.2 NMR ANALYSIS AT 400 MHZ.......................................................................101 4.2.1 Study on the temperature and on pH dependence of signals. .......................... 102 4.2.2 Chemometric data processing of aqueous extracts .......................................... 113 4.2.3 Processing of winter samples......................................................................... 118 4.2.4 Processing of summer samples ...................................................................... 124 4.2.5 Inter-laboratory check test ............................................................................ 132 4.2.5.1 Winter.................................................................................................... 132 4.2.5.2 Summer ................................................................................................. 134 5 CONCLUSIONS ......................................................................................139 REFERENCES ............................................................................................143 APPENDIX A: ALGORITHMS IN R LANGUAGE .............................................. I 200 MHZ DATA PROCESSING................................................................................ I 400 MHZ DATA PROCESSING.............................................................................VII 400 MHZ TEMPERATURE AND PH DEPENDENCE STUDY.................................. XXV APPENDIX B: CHERRY TOMATO SAMPLES CATALOGUE......................XXVIII PUBLICATIONS AND CONGRESS PARTICIPATIONS.............................. XXXV PREFACE The present thesis work has been developed towards the direction of providing an objective method finalized to the definition of food quality, by identification of suitable chemical parameters among a wide range of choices. Since foodstuffs are complex mixtures of substances with a large dynamic range of concentrations, it is restrictive to give importance to a limited number of nutrients in the definition

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