Chromatographic Analysis Of Amine Additives In PBAT Biodegradable Plastic Film And Its Effect On Nitrogen Metabolism In Nicotiana Tabacum L

1.A method for simultaneous qualitative and quantitative analysis determination of 8 different types amine additives in PBAT biodegradable plastic film by ultrasonic extraction-hydrolysis,ethyl chloroformate（ECF）derivatization,liquid-liquid extraction,combined with GC-MS chromatographic separation and identification technology was established.The extraction-hydrolysis of isocyanate and the derivatization of amine were optimized by single-factor control method.The results showed that the optimal extraction-hydrolysis conditions were V（HCl）:V（acetonitrile）of 1:2,HCl concentration of was 3 mol/L,ultrasound time of 60 min and solid-liquid ratio of 1:60（g:m L）.Under optimal conditions,the various isocyanate have a satisfactory hydrolysis rate（77～104%）.Subsequent optimization of the derivatization process of the amine and the most suitable conditions were as follows:adjusting the p H with Na₂CO₃,50μL ECF as the derivatization reagent,ethyl acetate as the extraction solvent and V（ethyl acetate）:V（ECF）=950:50,derivation-extraction time as 5 min and repeated twice extraction.Under optimal conditions,this method showed good linearity（R~2>0.998）,lower limits of detection（0.0096～0.0282μg/g）and limits of quantification（0.0302～0.0885μg/g）.The recoveries of target analytes were between 93.9～101.2%,while the intra-day and inter-day precision ranged from1.22～10.24%and 1.46～15.09%,respectively.Moreover,the final derivative products were stable and less prone to degradation.The newly method was used to analyze amine additives in PBAT biodegradable mulch films from different suppliers.The results showed that all samples contained 2,6-diisopropylaniline（2,6-DIPA）and the content was more than 1000μg/g,a small amount of samples were added with trace amounts of hexamethylenediamine（HDA）,2,6-diaminotoluene（2,6-TDA）,2,4-diaminotoluene（2,4-TDA）.The method can be used to monitor the type and content of amine additives in biodegradable mulch films,so as to evaluate the safety of related products.This method also provides a certain theoretical reference for the subsequent development and application of new biodegradable plastic films.2.Flue-cured Nicotiana tabacum L.K326 was treated with different concentrations（CK,T1/1.77μg/m L,T2/17.7μg/m L,T3/88.5μg/m L）of 2,6-DIPA at the seedling stage in hydroponic experiments.The growth,physiological properties and nitrogen metabolism of Nicotiana tabacum L.seedlings were analyzed and evaluated.The results of this study indicated that as the concentration of 2,6-DIPA increased,the more obvious the inhibition effect on Nicotiana tabacum L.growth was observed.The plant height,stem girth,maximum leaf area,biomass,root activity of Nicotiana tabacum L.seedlings were significantly decreased with the increase of 2,6-DIPA concentration,but the chlorophyll contents was significantly increased with it.With the increase of 2,6-DIPA concentration,malondialdehyde（MDA）content,superoxide dismutase（SOD）,peroxidase（POD）,catalase（CAT）,phenylalanine ammonia-lyase（PAL）activity increased,while glutathione reductase（GR）activity showed a tendency of first increasing and then decreasing.Analysis of nitrogen metabolism showed that 2,6-DIPA significantly changed the nitrogen-related metabolites.The nicotine synthesis was significantly inhibited at low concentration of 2,6-DIPA,and was promoted at high concentration.Most amino acids and related metabolites were significantly down-regulated at high concentrations of 2,6-DIPA and only alanine（Ala）,aspartic acid（Asp）and tryptamine（Try）were drastically up-regulated.In conclusion,2,6-DIPA can significantly change the agronomic traits of Nicotiana tabacum L.seedlings,and increase chlorophyll content and antioxidant enzyme activity,thus affecting the nitrogen absorption and interfering amino acid,alkaloid synthesis.