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Real-Time Reaction Monitoring and Flash Separation As a Process Seton Hall University eRepository @ Seton Hall Seton Hall University Dissertations and Theses Seton Hall University Dissertations and Theses (ETDs) Fall 2012 Real-time Reaction Monitoring and Flash Separation as a Process Analytical Technology Tool and HPLC Method Development for Melamine in Infant Formula Gopalakrishnan Venkatasami Seton Hall University Follow this and additional works at: https://scholarship.shu.edu/dissertations Part of the Chemistry Commons Recommended Citation Venkatasami, Gopalakrishnan, "Real-time Reaction Monitoring and Flash Separation as a Process Analytical Technology Tool and HPLC Method Development for Melamine in Infant Formula" (2012). Seton Hall University Dissertations and Theses (ETDs). 1819. https://scholarship.shu.edu/dissertations/1819 Real-time Reaction Monitoring and Flash Separation as a Process Analytical Technology Tool and HPLC Method Development for Melamine in Infant Formula by Gopalakrishnan Venkatasami Ph.D. DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry and Biochemistry of Seton Hall University December, 2012 South Orange, New Jersey We certify that we have read this thesis and that in our opinion it is sufficient in scientific scope and quality as a dissertation for the degree of Doctor of Philosophy APPROVED >¥ft><il~ JeKn R. Sowa, Jr., Ph.D. Mentor, Member of Dissertation Committee, Seton Hall University Cecilia Marzabadi, Ph.D. Member of Dissertation Committee, Seton Hall University David Sabatino, Ph.D. Member of Dissertation Committee, Seton Hall University c5Y~tb~ Stephen P. Kelty, Ph.D. Chair, Department of Chemistry and Biochemistry, Seton Hall University ii Dedicated to my Parents, Wife and my Kids iii Table of Contents Abstract ............................................................................................................................ xi Motivation ...................................................................................................................... xiii Aim of the Thesis ........................................................................................................... xiv 1. Introduction and Literature Overview ............................................................................ 1 1.1. Process analytical technology (PAT) ........................................................................... 1 1.2. Attentuated Total Reflectance-Infrared (ATR-IR) spectroscopy ................................. 2 1.3. The optical fiber probes used in mid-infrared analysis ................................................. 6 1.4. Advantage of mid-IR over near-IR .............................................................................. 8 1.5. ReactIR techniques ................................................................................................... 11 1.6. Flash chromatography ............................................................................................... 15 1.7. Detectors used in flash chromatography .................................................................... 16 1.8. Reaction carried out by on column synthesis ............................................................. 18 1.9. Flow chemistry ......................................................................................................... 20 1.10. Reactions in flow chemistry .................................................................................... 24 1.11. Conclusion.............................................................................................................. 27 2. Experimental Section ................................................................................................... 35 2.1. Instrumentation for real-time reaction monitoring ..................................................... 35 2.1.2. Instrumentation for Separation ............................................................................... 37 2.2. Synthesis of benzyl tert-butyl ether (Williamson ether synthesis) .............................. 41 2.3. Reaction monitoring of conversion of cholesterol to cholesteryl acetate (acetylation).43 2.4. Isomerization of cyclohexen-I-ol to cyc1ohexanone .................................................. 46 3.0. Results and Discussion ............................................................................................. 48 3.1. Synthesis of benzyl tert-butyl ether (Williamson ether synthesis) ........................41 3.2. Reaction monitoring and separation of conversion of cholesterol to cholesteryl acetate (acetylation) ................,', .................................................................................................. 60 3.3. Reaction monitoring and separation of cyclohexene-l-ol into cyclohexanone (Isomerization) ................................................................................................................ 70 3.6. Conclusion................................................................................................................ 85 4.0.0n-column Synthesis of Cyclohexanone by Isomerization of Cyclohexen-l-ol. ......... 86 4.1. Introduction .. , ........................................................................................................... 86 4.2. Experimental section ................................................................................................. 87 iv 4.3. Results and discussion.......................................... ......................... ..... 83 4.3.1. First Pass ........................................................'................................83 4.3.2. Second Pass .....................................................................................85 4.3.3. Third Pass ....................................................................................... 89 4.4. Conclusion........................................................................................ 91 5.0 A Rapid, acetonitrile-free method for determination of melamine in infant formula by HPLC............................................................................................................................ 100 5.1. Introduction ............................................................................................................ 100 5.1. Experimental Section .............................................................................................. 103 5.1.1. Chemicals and reagents ........................................................................................ 103 5. ] .2. Chromatographic equipment ................................................................................ 103 5.1.3. Chromatographic conditions ............................................................................... 103 5.2. Standards preparation ............................................................................................. 104 5.2.1. Diluent................................................................................................................. 104 5.2.2. Melamine stock solution ...................................................................................... 104 5.2.3. Intermediate melamine stock solution .................................................................. 104 5.2.4. Working melamine solution ................................................................................. 104 5.3. Sample preparation ................................................................................................. ]04 5.3.1. Preparation of stock infant formula samples ......................................................... 104 5.3.2. Preparation of working infant formula samples .................................................... 105 5.4. Method validation ................................................................................................... 105 5.4.1. Linearity .............................................................................................................. lOS 5.4.2. Accuracy.............................................................................................................. 106 5.4.3. Precision .............................................................................................................. 106 5.5. Results and discussion ............................................................................................ 107 5.5.1. Linearity .............................................................................................................. ]08 5.5.2. Limit of detection (LOD) and limit of quantification (LOQ) ................................ 108 5.5.3. Accuracy.............................................................................................................. 109 5.5.4. Precision .............................................................................................................. 109 6. Conclusion................................................................................................................. ] 10 7. Research conclusions ................................................................................................. I ] 2 8.0. Appendix ................................................................................................................ ] 13 Raw data and model calculation for Williamson ether synthesis and Separation ............. 113 v List of Figures Figure 1-1. Steps for PAT implementation ........................................................... 1 Figure 1-2. A schematic representation of a multiple reflection
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