摘要
建立了测定花生和大豆根瘤菌生物固氮能力的毛细管顶空气相色谱法,利用固氮酶还原乙炔气体为乙烯的经典方法,实现了高浓度乙炔中痕量乙烯的高灵敏分析.利用顶空进样方式,分离柱采用HP-PLOT Q毛细管色谱柱,程序升温,采用氢离子化检测器,对乙烯标准气体进行了定量分析.乙烯检测范围宽为0.125～30000 µmol/mol,相关系数为0.9996,检测限(LOD)为0.0341 µmol/mol,检出限优于已有报道.该方法检测精密度好,5次检测的相对标准偏差为2.32%.乙烯样品的回收率为95.8%～110%,相对标准偏差为1.54%～3.47%.同时,对乙烯和实际样品进行检测时,与GDX-502填充柱的传统气相色谱法的测定结果一致,表明所建立的乙烯顶空气相色谱法具有较高的可靠性.此外,该方法分析时间缩短至3 min,利用自动进样方式,检测通量大,适用于花生和大豆根瘤菌中固氮能力的快速准确测定,能满足花生和大豆生物固氮研究中乙炔还原法测定乙烯的需求.
Abstract
A efficient detection method for nitrogenase activity in peanut and soybean have successfully devel-oped via acetylene reduction method by using capillary column headspace gas chromatography(GC).This method realized highly sensitivity discrimination and analysis of low concentration of C2H4 from high concentration C2H2 at-mosphere.Headspace injection mode was adopted,and HP-PLOT Q capillary-column chromatography acted as the separation column with temperature programming with flame ionization detector for C2H4 detection.The calibration curve of C2H4 showed wide linear relationship ranging from 0.125 to 30000 µmol/mol,where the correlation coeffi-cient of R2 was 0.9996.The limit of detection(LOD)was 0.0341 µmol/mol,which was better than previous.The pro-posed headspace GC method showed a good precision with the relative standard deviation of 5 tests of 2.32%.The recoveries of C2H4 samples were 95.8-110%with the relative standard deviations of 1.54-3.47%.Meanwhile,the re-sults in real samples were consistent with those of conventional gas chromatography with GDX-502 packed column,revealing that this GC method was high reliability.Additionally,analysis time for C2H4 and C2H2 was only 3 min,re-alizing a rapid analysis and qualitative of actual samples by combing the automatic sample injection device.The aforementioned results demonstrated this proposed method could achieve rapid and accurate determination of nitro-genase ability in peanut and soybean samples,which could meet the demand of C2H4 determination by acetylene re-duction method in peanut and soybean biological nitrogen fixation research.
基金项目
湖北洪山实验室重大项目(2021hszd015)
中央级科研院所基本科研业务费专项(1610172022001)
中央级科研院所基本科研业务费专项(110217160011012)
国家自然科学基金(32202171)
湖北省自然科学基金(2021CFB181)
中国农业科学院创新工程(CAAS-ASTIP2021-OCRI)
维普
万方数据