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Extraction and Determination of Total Wax and Cuticular Wax of Citrus Fruits   

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实验目的:检测柑橘果实表面总蜡及外蜡含量。

关键词: 柑橘, 果实, 蜡质

材料与试剂

  1. 毛刷
  2. 滤纸
  3. 10 ml离心管
  4. 1 ml注射器
  5. 0.22 µm微孔滤膜
  6. 气相质谱上样瓶
  7. 柑橘果实
  8. 蒸馏水
  9. 三氯甲烷
  10. 正二十四烷
  11. 无水硫酸钠
  12. 吡啶
  13. BSTFA: N,O-双 (三甲基硅烷基) 三氟乙酰胺 (N,O-bis(trimethylsilyl) trifluoroacetamid)
  14. 正二十四烷内标溶液 (见溶液配方)

仪器设备

  1. 游标卡尺
  2. 1 L烧杯
  3. 100 ml蓝盖瓶
  4. 分液漏斗
  5. -20°C冰箱
  6. 烘箱
  7. 氮气吹干仪
  8. 玻璃内插管
  9. 气相色谱-质谱联用 (Thermo,DSQⅡ)
  10. TR-1ms毛细管柱
  11. EI离子源

实验步骤

  1. 选取果面光洁,无破损,大小一致的果实用清水洗净,然后在蒸馏水中润洗3次,晾干。 
  2. 果实表面积测定:本方法通过建立果实表面积与横纵径相关模型,以果实横纵径推算果实表面积。温州蜜柑、椪柑、柠檬、纽荷尔脐橙及沙田柚果实表面积与横纵径相关模型参见王金秋 (2015) 博士毕业论文。
  3. 总蜡提取:在1 L烧杯中加入300 ml三氯甲烷,每个果实浸泡2 min,其中轻微振荡烧杯。所有果实脱蜡处理两次,将两次含有蜡液的三氯甲烷溶液混合在一起,加入200 μl(1 μg/μl)正二十四烷内标。
  4. 外蜡提取:阿拉伯胶粉用三氯甲烷去除杂质后,配成浓度为90% (w/v)的乳浊液,用毛刷将其均匀地涂在柑橘果实表面,室温放置5 h左右,剥离干燥的阿拉伯树胶。以上步骤重复一次,并将将两次获取的阿拉伯胶研磨均匀,取其中16 g粉末,溶解于25 ml双蒸水中。按照水:三氯甲烷(v/v)1:3加入三氯甲烷,随后加入100 μl (1 μg/μl) 正二十四烷内标,剧烈振荡,直到阿拉伯树胶完全溶解为止;静置一段时间,待分层后,用分液漏斗取下层三氯甲烷澄清液,并用滤纸过滤。
  5. GC-MS 样品制备:总蜡和外蜡三氯甲烷溶液中加入等量的无水硫酸钠,过滤后用氮气吹干仪在40 °C下吹至剩余15 ml液体左右,平均分到两个干净的10 ml离心管中,氮气下吹干放置入-20 °C冰箱中。留一份备用。每个样品取其中一份加入200 μl吡啶,50 °C于烘箱中孵育30 min,加入等体积的BSTFA,60 °C孵育40 min。氮气吹干多余的衍生化试剂,加入2.5 ml三氯甲烷定容。用孔径0.22 μm的针筒微孔滤膜过滤至玻璃内插管中,用于GC-MS测定。
  6. GC-MS测定条件:气相色谱-质谱联用 (Thermo,DSQⅡ),TR-1ms毛细管柱 (100% 二甲基聚硅氧烷,30 m x 25 μm i.d. x 0.1 μm),EI离子源(70 eV),扫描范围50~650 m/z,进样口温度250 °C,离子源温度300 °C,传输线温度250 °C,氦气为载气,流量为1.2 ml/min;程序升温:70 °C起始,以10 °C/min升至200 °C,保持2 min,2 °C/min升至285 °C,1 °C/min升至290 °C,2 °C/min升至300 °C,保持10 min, 3 °C/min升至320 °C,保持20 min;分流比10:1;自动进样,进样量1 μl。

注意事项

  1. 剥去外蜡时一定小心,防止损失。
  2. 内标一定要在蜡质提取后立即加入。

溶液配方

  1. 正二十四烷内标溶液:浓度为1 μg/μl,溶剂为氯仿
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:汪洋, 杨宏斌, 王金秋, 何义仲, 程运江. (2018). 柑橘果实总蜡和外蜡的提取及测定. Bio-101: e1010213. DOI: 10.21769/BioProtoc.1010213.
How to cite: Wang, Y., Yang, H. B., Wang, J. Q., He, Y. Z. and Cheng, Y. J.  (2018). Extraction and Determination of Total Wax and Cuticular Wax of Citrus Fruits. Bio-101: e1010213. DOI: 10.21769/BioProtoc.1010213.
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