| (-)-Shikimic acid and(-)-Quinic acid are one of the most important fine chemical products and intermediates of drug synthesis, which has the widespread application in medicine, food, chemical industry, agriculture aspects and so on. Therefore, it have important significance to research how to increase the productivity of(-)-Shikimic acid and(-)-Quinic acid and the related products separation and purification. The objective of this work is to establish a method for the simultaneous determination of(-)-Shikimic acid and(-)-Quinic acid in fermented products, separation and purification of the related products also was studied.A screening method based on LC-MS for the simultaneous determination the content of(-)-Shikimic acid and(-)-Quinic acid in fermented products was developed. The analytical method involves the employment of a Waters Synergy C18 column(150 mm×2.0 mm I.D.) by using the following mobile phase: consisted of methanol-2 m M/L ammonium acetateate solution(1:99, v/v) and adjusted p H = 2.8 with acetic acid, at the flow rate of 0.8 m L/min. The UV detector was used to quantitative analysis, which was set to measure a specific wavelength of 216 nm. The temperature was conditioned at 30℃. The injection volume was 20 μL using partial loop mode for sample injection. The linear regressions of the peak area ratios versus concentrations were fitted over the concentration ranges of 36-504 μg/m L for(-)-Shikimic acid and 25.25-353.5μg/m L for(-)-Quinic acid, respectively. The correlation coefficient(R2) for(-)-Shikimic acid and(-)-Quinic acid were 0.9980 and 0.9996. The LOD was 0.68μg/m L for(-)-Shikimic acid and 5.74μg/m L for(-)-Quinic acid, respectively. The LOQ was 53.26μg/m L for(-)-Shikimic acid and 26.09μg/m L for(-)-Quinic acid, respectively. The intra-day and inter-day precision(relative standard deviation, RSD) values were below 10% and the accuracy(relative error, RE) was within ±5% at three quality control levels. The MS detector was used qualitative detection to confirm the(-)-Shikimic acid and(-)-Quinic acid. The MS was operated in negative ion mode with the declustering potential and source temperature set at 135.0 V and 300 ℃, respectively. Quantification was performed using total ion current(TIC) modality from 100 to 500 m/z, and ion single ion monitoring(SIM) on the ESI generated most abundant ion m/z 172.9 for(-)-Shikimic acid, m/z 191.1 for(-)-Quinic acid. The auxiliary gas(nitrogen) flow rate was set to 6L/min. The result indicates that this method could be well applied to the identification and quantification of(-)-Shikimic acid and(-)-Quinic acid in fermented products. It simultaneously provides analytical methods for the optimization of acid production condition and acid separation experiments.By research the 717 anion exchange resin on the fermentation products(-) shikimic acid adsorption kinetics experiment, breakthrough curve experiment, adsorption thermodynamics experiment investigated the adsorption characteristics of 717 anion exchange resin adsorption(-)-Shikimic acid, established the operating conditions of separation of(-)-Shikimic acid in fermented products by ion-exchange uses a strong-base anion resin 717. The rate Control model of resin adsorption(-)-Shikimic acid were analyzed though the adsorption kinetics experiment. The continuous fixed-bed column studies were carried out by breakthrough curve experiment. Effects of operating parameters such as flow rate, feed(-)-Shikimic acid concentration, operation temperature, and p H solution were studied. Adams-Bohart model, Thomas model and Yoon-Nelson model were takes into account to simulate the experimental breakthrough curves and obtained results showed that the data were correlated well with the Thomas model and Yoon-Nelson model. The optimum operating conditions for separation(-)-Shikimic acid were achieved by 717 resin at a flow rate of 1 m L/min, room temperature and p H solution is 7.5. The filtrate solution was obtained by centrifugation, filtration, adjusting the p H of the fermentation broth. The(-)-Shikimic acid was separation from filtrate solution by 717 anion exchange resin. The loaded(-)-Shikimic acid was eluted with 95% ethonal, thereby the eluent solution was obtained. Finally, the(-) shikimate crude was obtained by concentration crystallization.The obtained crude product contained(-)-Shikimic acid and(-)-Quinic acid two important chemicals. phase equilibria and phase diagram are important tools to use for separation and purification of substances. In order to isolate the pure(-)-Shikimic acid and(-)-Quinic acid, it need to select a suitable solvent to construct the phase diagram. Therefore, the solubility of(-)-Shikimic acid in H2 O, ethanol, n-propanol, isopropanol, n-pentanol, n-heptane and in(H2O + ethanol) binary solvent mixtures and the solubility of(-)-Quinic acid in H2 O, methanol, ethanol and(H2O + methanol),(H2O + ethanol) binary solvent mixtures was measured at different temperatures by using the synthetic method under atmospheric pressure. The ideal model, Apelblat model, λh model, Wilson model and NRTL model were used to correlate the experimental data. Furthermore, thermodynamic properties of the solution process, including the Gibbs free energy, enthalpy, and entropy were also calculated and analyzed. So as to constructed the phase diagram and use it to separation and purification of(-)-Shikimic acid and(-)-Quinic acid provides a theoretical basis.Experimental results indicates that the solubility of(-)-Shikimic acid and(-)-Quinic acid in the solvents with high polarity is higher than that in weakly polar solvents, and the solubility increases with rise of temperature while it decreases with increasing methanol or ethanol content at constant temperature. The enthalpy, entropy and the standard Gibbs free energy of the solution are positive in the studied solvents. Therefore, the solution process is always endothermic. In all cases(-)-Shikimic acid and(-)-Quinic acid solubility apparently increases with decreasing standard Gibbs energy of solution. Moreover, the main contributor to the positive standard molar Gibbs free energy of solution is the positive enthalpy during the dissolution, because all values of %ζH are ≥ 55.02%.The solid-liquid phase equilibrium for the ternary system of(-)-Shikimic acid +(-)-Quinic acid + H2 O and the quaternary system of(-)-Shikimic acid +(-)-Quinic acid + Ethanol(v~50%,v~75%)+ H2 O was measured at T=(298.15, 318.15, 333.15, 348.15) K and the mutual solubility was obtained. Constant-temperature phase diagrams and variable-temperature p Hase diagrams were constructed according to the experimental solubility. In general, the experimental data and phase diagrams can be used as essential theoretical support and serve as a guide for the purification and separation of(-)-Shikimic acid and(-)-Quinic acid in the industrial production and further theoretical studies.It was found that at anyone temperature, ternary and quaternary phase diagram being present in an invariant point, two invariant curves and three crystalline regions:(-)-Shikimic acid crystallization regions,(-)-Quinic acid crystallization regions and mixture solids of(-)-Shikimic acid and(-)-Quinic acid crystallization regions. The solubility values of(-)-Shikimic acid and(-)-Quinic acid increase with the increase in temperature from T = 298.15 K to 348.15 K, and the co-saturated point moves upward.Different amounts of ethanol was added to change the co-saturation point of(-)-Shikimic acid and(-)-Quinic acid at the same temperature. At the same temperature, the equilibrium concentration of(-)-Shikimic acid decreases with the increase of(-)-Quinic acid concentration. At the same(-)-Quinic acid concentration, the equilibrium concentration of(-)-Shikimic acid increase with the increase in temperature. The crystalline fields of two pure solids increase as the temperature decreases. The crystallization field of(-)-Shikimic acid is larger than that of(-)-Quinic acid at the same temperature.The variable-temperature phase diagrams were processed and application. We find that the efficiency was relatively lower and the energy consumption was relatively larger when using the(-)-Shikimic acid +(-)-Quinic acid + H2 O ternary phase diagram to continuous circulation separation of(-)-Shikimic acid and(-)-Quinic acid. The co-saturated point of(-)-Shikimic acid and(-)-Quinic acid almost on a straight line of passing through the origin which indicates that it is not suitable for separation process. By using the quaternary system of(-)-Shikimic acid +(-)-Quinic acid + Ethanol(v~50%) + H2 O or quaternary system of(-)-Shikimic acid +(-)-Quinic acid + Ethanol(v~50%) + H2 O joint the ternary system of(-)-Shikimic acid +(-)-Quinic acid + H2 O we can continue to separation out the pure of(-)-Shikimic acid and(-)-Quinic acid, and the volume of the system will unchanged after a circulation. The efficiency was relatively larger and the energy consumption was relatively lower which indicates that it is suitable for the real industrial separation(-)-Shikimic acid and(-)-Quinic acid process. |
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