Construction And Application Of Green Iminazole-ionic Liquid Aqueous Two-phase System Based On The Separation And Analysis Of Antibiotic Residues

The abuse of antibiotics in clinical medicine, aquaculture and food processing industry has been posed a serious threat to the ecological environment and human health. Therefore, the establishment of the method for separation and enrichment of antibiotic residues in the environment and food becomes the focal point of attention. Due to the composition of antibiotics matrix is complicated and usually trace or ultra-trace exists, we must find a reasonable method for separation and enrichment of antibiotics in the environment and food. It will achieve the separation of interfering substances, lower detection limits and improve the measurement accuracy of the purpose.This thesis deeply researches into the behavior of liquid-liquid equilibrium (LLE) of iminazole-ionic liquid aqueous two-phase system (ILATPS) and its phase-separation abilities, so this provides sufficient theoretical foundation for its application. On the basis of which, an ionic liquid aqueous two-phase extraction (ILATPE) system and an ionic liquid aqueous two-phase flotation (ILATPF) system were established, they can be applied to separate and concentrate antibiotic residues in the environment and food. Various factors influencing the extraction and flotation of antibiotics were optimized; the mechanisms of extraction and flotation were discussed. These studies provide a feasibility evidence on separation and concentration of other biological small molecules by ILATPE and ILATPF. The main results are as follows:1. Construction of ILATPS and the research of LLE(1) Through the fitting of a series of iminazole ILATPS of binodal data, the optimum equation was selected, and it was w,= exp(a+bw20.5+cw2+dw22). Based on the titration method, the densimeter method and "lever rule" calculation, the LLE compositions were obtained. The equations of Othmer-Tobias and Bancroft were used to fitting the LLE data, and the result was satisfactory.(2) The salting-out abilities of salt ions were obtained by calculating the effective excluded volume (EEV) of different species of phase-separation salts in aqueous l-butyl-3-methylimidazolium tetrafluoroborate ([C4mimJBF4) solutions, and drawing binodal curves in molar mass concentration. The phase-separation abilities of cation are in the order Na+>NH4+, and the phase-separation abilities of anion are PO43->C6H5O73->CO32->SO42-≈C4H4O62->C4H4O42->H2PO4->C2H3O2->Cl- Similarly, the phase-separation abilities of ionic liquids were obtained by calculating the EEV of six species of phase-separation salts in aqueous 1-ethyl-3-methylimidazolium tetrafluoroborate ([C2mim]BF4), 1-propyl-3-methylimi-dazolium tetrafluoroborate ([C3mim]BF4) or [C4mim]BF4, and drawing binodal curves in molar mass concentration. The phase-separation abilities of ionic liquids are in the order [C4mim]BF4> [C3mim]BF4> [C2mim]BF4.(3) The effect of temperature on ILATPSs was investigated. When the temperature decreased, the binodal curves shifted to the left, then the area of two-phase region and the slope of tie-line simultaneously increased, so the ability of forming ILATPSs was increased gradually.2. Separation and concentration of antibiotic residues using ILATPS(1) An ILATPS consisting of [C4mim]BF4 and Na2CO3, which was a sample pretreatment technique coupled with molecular fluorescence spectrophotomery, was developed for simultaneous separation and enrichment of roxithromycin. The extraction yield of roxithromycin in [C4mim]BF4-Na2CO3 ILATPS was influenced by the types of salts, the concentrations of Na2CO3 and [C4mim]BF4, as well as the extracting temperature. Under the optimum conditions, this method was practical when applied to the analysis of roxithromycin in real water samples with the detection limit of 0.03μ.g/mL, relative standard deviation (RSD) of 1.9%, and the average extraction efficiency was up to 90.7%. The mechanism of ILATPS formation was discussed by hydration theory, and the extraction mechanism of the [C4mim]BF4-salt ILATPS was investigated by FT-IR spectroscopy and UV-vis spectroscopy. The results demonstrated that no chemical (bonding) interactions were observed between ionic liquid and roxithromycin, while the nature properties of the roxithromycin are not altered, the ionic liquid just as the role of the solvent.(2) [C4mim]BF4-(NH4)2SO4 ILATPS coupled with high-performance liquid chromatography with ultraviolet detection (HPLC-UV) was developed for the extraction of sulfonamides. Three sulfonamides, sulfamerazine, sulfamethoxazole and sulfamethizole were selected as model compounds for developing and evaluating the method. The effects of various experimental parameters in extraction step were studied using two optimisation methods, one variable at a time and the response surface methodology (RSM) approach. Under the optimum conditions, this method was practical when applied to the analysis of sulfonamides in the environment and food. The detection limits (S/N=3) and quantification limits (S/N=10) of the proposed method for the target compounds were achieved within the range of 0.15-0.3 ng/mL and 0.5-1.0 ng/mL from spiked samples, respectively, and the average recovery rate was in the range of 83.7～116.5%, which was lower than or comparable with other reported approaches applied to the determination of the same compounds.(3) A sample pretreatment procedure coupled with HPLC was developed for the analysis of chloramphenicol that exploits an aqueous two-phase system based on [C4mim]BF4 and organic salt using a liquid-liquid extraction technique. The influence factors on partition behaviors of chloramphenicol were studied, including the type and amount of salts, the pH value, the volume of [C4mim]BF4, and the extraction temperature. Thermodynamic studies indicated that hydrophobic interactions were the main driving force, and salting-out effects was also important for the transfer of the chloramphenicol. Under the optimal conditions, this method was practical when applied to the analysis of chloramphenicol in feed water, milk, and honey samples with the limit of detection of 0.3 ng/mL and the limit of quantification of 1.0 ng/mL. The recovery of CAP was 90.4-102.7%. This novel process is much simpler and more environmentally friendly and is suggested to have important applications for the separation of antibiotics.3. Separation and enrichment of chloramphenicol using ILATPF and mechanism research(1) ILATPF which combined ILATPS based on [C4mim]Cl and K2HPO4 with solvent sublation was developed for the analysis of chloramphenicol coupled with HPLC. In ILATPF systems, phase behaviors of the ILATPF were studied for different types of ionic liquids and salts. The sublation efficiency of chloramphenicol in [C4mim]C1-K2HPCO4 ILATPF was influenced by the types of salts, the concentration of K2HPO4 in aqueous solution, solution pH, nitrogen flow rate, sublation time and the amount of [C4mim]Cl. Under the optimum conditions, this method was practical when applied to the analysis of chloramphenicol in lake water, feed water, milk, and honey samples with the linear range of 0.5～500 ng/mL. The method yielded limit of detection of 0.1 ng/mL and limit of quantification of 0.3 ng/mL. The recovery of CAP was 97.1-101.9% from aqueous samples of environmental and food samples by the proposed method. Compared with liquid-liquid extraction, solvent sublation and ionic liquid aqueous two-phase extraction, ILATPF can not only separate and concentrate chloramphenicol with high sublation efficiency, but also efficiently reduce the wastage of IL. This novel technique is much simpler and more environmentally friendly and is suggested to have important applications for the concentration and separation of other small biomolecules.(2) An IL-K2HPO4 ILATPF was established to separate chloramphenicol. The response surface experimental design method of four factors and three levels was used to optimize the impact factors of flotation of chloramphenicol, and pick up the best conditions:the ionic liquid was [C6mim]Cl, the concentration of K2HPO4 was 0.74 g/mL, the flotation time was 50 min, and the flow rate was 50 mL/min. Under the best conditions, the partition coefficient of chloramphenicol was 405.71, and the flotation rate was 93.16%. According to the three-dimensional response surface chart, we concluded that the order of influencing factors on the partition coefficient and the flotation rate of chloramphenicol was the time of flotation> flow rate of flotation> the type of IL> the concentration of K2HPO4. The mechanism of ILATPF contained two principal processes. One was the mechanism of IL-salt ILATPS formation, the other was solvent sublation.