Optimization of time for the fixation of a sugar-oxime derivative reaction used for the GC-MS analysis of citrus waste
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Research at Texas A&M University in Kingsville (TAMUK) is being conducted to convert citrus waste biomass feedstock to value-added products. Enzymatic hydrolysis of the citrus waste generates organic molecules that cannot be identified by gas chromatography-mass spectrometry (GC-MS) methods since their functional groups react and form decomposition products. A two-step derivatization analytical method was previously developed at TAMUK that converts any aldehyde, carboxylic acid, alcohol, or ketone functional group in these organic molecules to their corresponding trimethylsilyl-oxime derivatives (TMSO). The resulting derivatives have the required thermal stability to be analyzed using GC-MS. However, this derivatization method was not optimized in terms of reaction time, temperature, and reagent concentrations so that additional investigation is needed to identify the optimal derivatization reaction conditions. The primary focus of this work was to perform a systematic analysis of the derivatization reaction time and substrate concentration on the resulting rate of formation of the TMSO derivatives. The second step in the two-step derivatization procedure was studied owing to its relative importance compared to the first step. As a first level of investigation, the effect of substrate concentration and reaction time were varied over several levels. An Agilent Model 6890 GC connected to a Model 7592 Mass Selective Detector was used to collect the required analytical data. Glucose was used as a model compound since this species was previously identified in various reaction products generated from the enzymatic hydrolysis of citrus waste. The experimental data showed that the second derivatization reaction was complete in less than a minute whereas 30 minutes had been previously allotted for complete conversion. Exposure of the derivatized product from the first step to air during an intermediate quenching step resulted in predicted glucose concentrations that were higher than actual values by a factor of about 20. After applying a suitable correction factor and using an internal standard response factor, reasonable agreement was obtained between the measured versus actual values for glucose. Additional studies to identify the optimal derivatization conditions are required before a detailed model of the reaction kinetics for substrate conversion to the TMSO reaction products can be developed.