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柑橘挥发性物质提取与鉴定   

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Original research article

A brief version of this protocol appeared in:
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实验原理:柑橘挥发性物质属于有机化合物,利用有机试剂MTBE萃取,超声波清洗仪超声加速萃取,利用气相色谱-质谱联用仪 (GC-MS) 分析鉴定挥发性物质。
实验目的:有机试剂萃取结合GC-MS分析鉴定柑橘中挥发性物质。

关键词: 柑橘, 挥发性物质, GC-MS

材料和试剂

  1. 2 ml离心管
  2. 1 ml注射器
  3. 0.22 μm滤膜 (SCAA-104)
  4. 2 ml进样瓶
  5. 200 μl内插管
  6. 柑橘组织
  7. 液氮
  8. Methyl tert-butyl ether (MTBE)
  9. 壬酸甲酯 (Sigma-Aldrich)
  10. 提取液 (见溶液配方)

仪器设备

  1. 电子秤
  2. 涡旋仪
  3. GC-MS (TRACE GC Ultra GC, DSQ II mass spectrometer, Thermo Fisher Scientific, Waltham, MA, USA)
  4. TRACE TR-5 MS column (30 m x 0.25 mm x 0.25 μl, Thermo Scientific,Bellefonte, PA, USA)
  5. Eppendrof centrifuge 5424R
  6. 超声波清洗仪 (DTG-15J)
  7. 研钵

实验步骤

  1. 柑橘组织在研钵中液氮下研磨成粉。
  2. 称取柑橘组织 (黄皮层0.2 g,汁胞1 g, 叶片0.3 g) 装入2 ml离心管中,加入500 μl超纯水,涡旋混合均匀。
  3. 再加入500 μl提取液,涡旋混合均匀。
  4. 4 °C冰浴,超声波萃取40 min。
  5. 4 °C,10,000 x g离心10 min,分层。
  6. 用1 ml注射器吸取上清液,用0.22 μm滤膜过滤进上样瓶 (内插管)。
  7. GC-MS上机检测。

结果与分析

  1. GC-MS检测条件
    GC-MS (TRACE GC Ultra GC结合DSQ II mass spectrometer) 的进样量为1 μl,色谱柱为TRACE TR-5 MS柱 (30 m x 0.25 mm x 0.25 μl)。载气为高纯氦气(99.999%),分流比50:1,恒流模式,流速为1 ml/min。进样口,离子源和传输线的温度分别为250 °C、260 °C和280 °C。GC的升温程序如下:40 °C保持3 min,然后以3 °C/min的速度升温至160 °C并保留1 min,然后以5 °C/min的速度升温至200 °C并保留1 min,最后以8 °C/min的速度升温至240 °C并保留3 min。MS的条件如下:EI (电子轰击) 离子源,电子轰击能量70 eV,正离子扫描模式,质量扫描范围m/z 45-400 amu。
  2. 检测结果如图1所示


    图1. 柑橘挥发性物质GC-MS检测色谱图 (部分). A:β-月桂烯

注意事项

  1. 叶片分流比20:1;汁胞:不分流

溶液配方

  1. 提取液 (20ml)
    试剂
    含量
    壬酸甲酯
    1 μl
    MTBE
    20 ml

参考文献

  1. Liu, C., Cheng, Y., Zhang, H., Deng, X., Chen, F. and Xu, J. (2012). Volatile constituents of wild citrus Mangshanyegan (Citrus nobilis Lauriro) peel oil. J Agric Food Chem 60(10): 2617-2628.
  2. Zhang, H., Xie, Y., Liu, C., Chen, S., Hu, S., Xie, Z., Deng, X. and Xu, J. (2017). Comprehensive comparative analysis of volatile compounds in citrus fruits of different species. Food Chem 230: 316-326.
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:张海朋 , 刘翠华 , 徐娟 . (2018). 柑橘挥发性物质提取与鉴定. Bio-101: e1010207. DOI: 10.21769/BioProtoc.1010207.
How to cite: Zhang, H. P., Liu, C. H. and Xu, J. (2018). Extraction and Identification of Volatile Substances from Citrus. Bio-101: e1010207. DOI: 10.21769/BioProtoc.1010207.
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