Real-time Reaction Monitoring and Flash Separation as a Process Analytical Technology Tool and HPLC Method Development for Melamine in Infant Formula

Process analytical technology (PAT) is a technique for the analysis and control of chemical processes to assure the production of acceptable quality of the end-product. In this thesis, the application of mid-IR (1900 -- 650 cm-1) spectroscopy as a real-time monitoring technique for reaction and purification is illustrated. A technique using in-situ ATR-IR spectroscopy to monitor a real-time reaction followed by purification of the product by flash chromatography is developed in this study. The application of mid-IR spectroscopy was focused in etherification, acetylation and isomerization reactions which demonstrate the value of PAT. This study also focusses on the development of flash chromatographic purification conducted with in-situ ATR-IR spectroscopy detection. The results obtained indicated that this technique can be successfully used as an alternative to the traditional UV-Vis detection with the advantage of being able to characterize the eluent via in-situ ATR-IR as a real-time technique. In addition to the methods described above (synthesis and purification), a real-time on-column synthesis is successfully described.;Overall, this work presents the use of ATR-IR as a technique for monitoring chemical reactions and purification in real-time. The use of ATR-IR as a detection technique for flash chromatography is demonstrated to be an orthogonal detection technique to off-line HPLCUV analysis.;Another project within this thesis is development of a simple, precise, accurate and validated, acetonitrile-free, reverse phase high performance liquid chromatography (HPLC) method for the determination of melamine in dry and liquid infant formula. The separation was performed on a Kromasil C18 column (150 mmx3.2 mm I.D., 5 mum particle size) at 25° C. The mobile phase (0.1 % TFA/methanol 90:10) was pumped at a flow rate of 0.3 mLmin -1 with detection at 240 nm. Melamine elutes at 3.7 min. A linear response (r > 0.999) is observed for samples ranging from 1.0 to 80 mugmL -1. The method provides recoveries of 97.2-101. 2% in the concentration range of 5-40 mugmL-1, intra- and inter-day variation in <1.0 % R.S.D. The limit of detection (LOD) and limit of quantification (LOQ) values are 0.1 RgmL-1 and 0.2 [tgmL-1, respectively.