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Analysis of Total Se Content in Rice
水稻中总硒含量的分析   

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New Phytologist
Mar 2014

Abstract

Total Se content in rice is normally low and it is difficult to determine it exactly because of Se volatilization and pollution during the digestion process. In this method, rice sample is digested thoroughly and Se volatilization is reduced greatly by designing a specific digestion tube, increasing digestion temperature by three steps, controlling the amount of mixed acid and adjusting the location of digestion tube in the digestion furnace. Se pollution is also reduced greatly by specific cleaning treatments.

Materials and Reagents

  1. Rice sample: Polished rice or brown rice
  2. 10% HCl (Sigma-Aldrich, catalog number: 7647-01-0 )
  3. 65% HNO3 (Sigma-Aldrich, catalog number: 7697-37-2 )
  4. 72% HClO4 (Sigma-Aldrich, catalog number: 7601-90-3 )
  5. 6 mM HCl (Sigma-Aldrich, catalog number: 7647-01-0) (used for the reduction of SeO42- to SeO32-)
  6. Selenium standard solution (Sigma-Aldrich, catalog number: 7782-49-2 ) (1, 5, 10, 20, 50, and 100 μg/L)
  7. Acid mixture: HNO3: HClO4 (V/V) = 4:1

Equipment

  1. Digestion tube (see Design digestion tube)
  2. 40 mesh nylon sieve (15 cm diameter)
  3. A paper bag (8 x 10 cm)
  4. Parafilm (Parafilm, catalog number: PM-996 )
  5. Volumetric flask (25 ml)
  6. Glass stopper (match 25 ml volumetric flask)
  7. Atomic fluorescence spectrometer (AFS) (model number: BRAIC AFS 610A )
  8. Inductively coupled plasma mass spectrometry (ICP-MS) (PerkinElmer, model number: ELAN-DRCe )

Procedure

  1. Design digestion tube
    1. The digestion tube is designed 300 mm in length. The edge of tube mouth is thickened.
    2. The outer- and inner-diameter of tube mouth is 28 mm and 21 mm, respectively.
    3. The thickness of digestion tube mouth is 3.5 mm. The outer diameter of digestion tube is 26 mm, the thickness is 2 mm.
    4. The digestion tube neck is at 25 mm from tube mouth. The outer diameter of tube neck is 18.5 mm, the length is 15 mm. The thickness of digestion tube at the bottom is 3.5 mm.


      Figure 1. The structure of digestion tube

  2. Clean digestion tube
    1. Digestion tubes are initially soaked in running water added a small amount of cleaning solvent.
    2. Put the tube brush into the bottom of digestion tube, scrub rotatablely the inwall up and down. Funnel is rotatablely scrubed with tube brush. Add diluted cleaning solvent into volumetric flask, then clean ultrasonically for 30 min.
    3. After cleaning, the digestion tubes, funnels and volumetric flasks are rinsed with deionized water (>18 MΩ) for 2-3 times, then put into 10% HCl and soak for more than 24 h, respectively.
    4. After being taken out, digestion tubes and the funnels are scrubed with brush again and rinsed with deionized water for 2-3 times, respectively. Volumetric flasks are directly rinsed with deionized water.
    5. Then, digestion tubes and volumetric flasks are inverted on special shelf until no drop of water appear inwall. At last, digestion tubes and volumetric flasks are oven-dried at 60 °C.

  3. Digestion process
    1. Take about 15-20 g of rice sample, and grind to a powder in a mortar, then pass through 40 mesh nylon sieve and put in a paper bag. After grinding one sample, please open the mortar and take out the nylon sieve, then clean them throughly with a brush.
    2. About 0.5 g of rice powder sample is weighed, and make the digestion tube lean and add it into the bottom. Then 5 ml of an acid mixture of 4 ml HNO3 and 1 ml HClO4 is added slowly along the tube wall, is shaken gently to mix sample and mixed acid throughly.
    3. Seal the digestion tube mouth with single layer parafilm. Put it in a fume hood for over 24 h. Fume hood wall and the top are washed with a wet cloth before digestion. All the reagents used are high-pure grade.
    4. Place the digestion tubes into the holes of digestion furnace. All the samples are initially digested for 30 min at 60 °C and continue to digest for 60 min at 100 °C.
    5. Then, add 2.5 ml of mixed acid into digestion tubes and raise the temperature to 150 °C. Similarly, at least 3 of blank samples and standard tea material samples (GSV-4, 0.072 mg Se/kg, GBW07605) are simultaneously digested with the test samples for quality control, respectively.
    6. The locations of digestion tubes in the digestion furnace are continually adjusted during the digestion process to keep the digested liquid surface of each tube at the same height.
    7. The digestion process is over until the brown acid fog disappears and the white fog appears in the digestion tubes. Then the digestion tubes are cooled to 100 °C, add 2.5 ml of 6 mM HCl to reduce SeO42- to SeO32-, and continue to heat at 100 °C until brown acid fog disappears. If Se content is determinated by inductively coupled plasma mass spectrometry (ICP-MS), the reduction of Se by hydrochloric acid is not needed. It will take about 6-8 h to finish the whole digestion process according to the amount of sample.


      Figure 2. The digestion state of standard tea material samples

  4. Measure Se content
    1. After the temperature reduce to room temperature, the digestion tubes are gently removed from the holes of digestion furnace and placed in the digestion tube shelf. The digests are transferred into the volumetric flasks, the digestion tubes are rinsed with deionized water for 3-4 times. The rinsing solution is also added into the volumetric flask until a final volume reaches to 25 ml, then cover it closely with glass stopper.
    2. Place the bottom of the volumetric flask on a vortex and rotate for 30 sec and mix throughly. Put aside for over 2 h before Se determination.
    3. Selenium standard solution (10 mg/L) is gradually diluted to concentrations of 1, 5, 10, 20, 50, and 100 μg/L with 1% HNO3, respectively.
    4. Then Se content is determined by atomic fluorescence spectrometer (AFS) or by ICP-MS. The standard curve of Se content is drawn. Se content of the test samples are also determined.


      Figure 3. Se content of standard tea material samples

    5. The parameters for the determination of Se by AFS is as follows: Wavelength 196 nm, PMT voltage 280 v, HCl main cathode current 80 mA, Carrier gas flux 300 ml/min, Sampling volume 1.0 ml, Atomizer heigth 7 mm, Sampling pump rate 100 r/min, Sampling time 18 sec; The parameters for determination of Se by ICP-MS is as follows: Power 1,300 W, Plasma gas flow rate 15 L/min, Auxiliary gas flow rate 0.2 L/min, Atomizer flow rate 0.89 ml/min. Reaction gas methane flow rate 0.6 ml/min.

Notes

  1. The edge of tube mouth and the bottom is thickened to prevent to be broken.
  2. The digestion tube neck is made thin at 25 mm from the tube mouth to increase the condensation of acid fog and reduce the volatilization of mixed acid.
  3. The amount of rice sample is controlled within about 0.5 g to reduce the digestion time.
  4. The total amount of mixed acid added into digestion tube is controlled below 7.5 ml. The digestion temperature is controlled at 150 °C. Thus, the digest solution is avoided to splash on the tube wall.
  5. The location of digestion tube in the digestion furnace is needed to adjust to keep the digest solution at the same height in each tube.

Acknowledgments

This work has been published in Zhang et al. (2014). The work was supported by the 43rd China Postdoctoral Science Foundation (No: 20080430588), the talent foundation (No: 09001107) and research foundation from Henan University of Science and Technology (No: 13560036). We thank Hongzhi Zhang (Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences) for selenium content analysis using ICP-MS.

References

  1. Zhang, L.*, Hu, B.*, Li, W., Che, R., Deng, K., Li, H., Yu, F., Ling, H., Li, Y. and Chu, C. (2014). OsPT2, a phosphate transporter, is involved in the active uptake of selenite in rice. New Phytol 201(4): 1183-1191. (*Co-first authors)
  2. Zhang, L., Shi, W. and Wang, X. (2006). Difference in selenite absorption between high-and low-selenium rice cultivars and its mechanism. Plant and Soil 282(1-2): 183-193.
  3. Zhang, L., Shi, W., Wang, X. and Zhou, X. (2006). Genotypic differences in selenium accumulation in rice seedlings at early growth stage and analysis of dominant factors influencing selenium content in rice seeds. J Plant Nutrition 29(9): 1601-1618.

简介

水稻总硒含量通常较低,很难准确地确定它是由于在蒸煮过程中的Se挥发和污染。 在该方法中,通过设计特定的消化管,提高消化温度三个步骤,控制混合酸的量和调节消化管在消化炉中的位置,大大消化大米样品并且大大减少Se的挥发。 由于特定的清洁处理,Se污染也大大减少

材料和试剂

  1. 水稻样品:糙米或糙米
  2. 10%HCl(Sigma-Aldrich,目录号:7647-01-0)
  3. 65%HNO 3(Sigma-Aldrich,目录号:7697-37-2)
  4. 72%HClO 4(Sigma-Aldrich,目录号:7601-90-3)
  5. 6mM HCl(Sigma-Aldrich,目录号:7647-01-0)(用于将SeO 4+还原成SeO 3) > 2 -
  6. 硒标准溶液(Sigma-Aldrich,目录号:7782-49-2)(1,5,10,20,50和100μg/L)
  7. 酸混合物:HNO 3 :HClO 4(V/V)= 4:1

设备

  1. 消化管(见设计消化管)
  2. 40目尼龙筛(直径15cm)
  3. 纸袋(8 x 10厘米)
  4. 石蜡膜(Parafilm,目录号:PM-996)
  5. 容量瓶(25ml)
  6. 玻璃塞(匹配25ml容量瓶)
  7. 原子荧光光谱仪(AFS)(型号:BRAIC AFS 610A)
  8. 电感耦合等离子体质谱(ICP-MS)(PerkinElmer,型号:ELAN-DRCe)

程序

  1. 设计消化管
    1. 消化管的长度设计为300mm。 管口边缘增厚。
    2. 管口的外径和内径分别为28mm和21mm。
    3. 消化管口的厚度为3.5mm。 消化管的外径为26mm,厚度为2mm。
    4. 消化管颈在离管口25mm处。 外面 管颈直径为18.5mm,长度为15mm。 厚度 消化管底部为3.5 mm。


      图1.消化管的结构

  2. 清洁消化管
    1. 消化管最初浸泡在加入少量清洁溶剂的流水中。
    2. 将管刷放入消解管的底部,擦洗 可旋转地向内和向下。 漏斗被可旋转地擦洗 管刷。 加入稀释的清洗溶剂到容量瓶中,然后 超声清洗30分钟。
    3. 清洗后,消化   管,漏斗和容量瓶用去离子水漂洗 (>18MΩ)2-3次,然后放入10%HCl中并浸泡多于 24小时。
    4. 取出后,消化管和   再次用刷子洗涤漏斗并用去离子水冲洗 水2-3次。 将容量瓶直接漂洗   用去离子水。
    5. 然后,消化管和容积 烧瓶在特殊架子上倒置,直到没有水滴出现 inwall。 最后,消化管和容量瓶在烘箱中干燥   60℃。

  3. 消解过程
    1. 取约15-20g的米样品,并在研钵中研磨成粉末, 然后通过40目尼龙筛并放入纸袋中。 后 研磨一个样品,请打开砂浆,取出尼龙 筛,然后用刷子彻底清洗。
    2. 约0.5g 称取米粉样品,使消化管倾斜并加入 它进入底部。 然后加入5ml 4ml HNO 3和1ml的酸混合物 HClO 4缓慢地沿管壁加入,轻轻摇动混合 样品和混合酸
    3. 密封消化管口 与单层石蜡膜。 将其放在通风橱中超过24小时。 烟 罩壁和顶部在消化前用湿布清洗。 所有 使用的试剂是高纯度的
    4. 放置消化 管进入消化炉的孔中。 所有样品最初   在60℃消化30分钟并在100℃继续消化60分钟 C。
    5. 然后,向消化管中加入2.5ml混合酸 将温度升至150℃。 类似地,至少3个空白样品 和标准茶材料样品(GSV-4,0.072mg Se/kg sub,GBW07605) 同时用测试样品消化用于质量控制, 分别。
    6. 消化管的位置 消化炉在消化过程中不断调整 以保持每个管的消化的液体表面在相同的高度。
    7. 消化过程结束,直到棕色酸雾消失 白色的雾出现在消化管中。 然后消化管 冷却至100℃,加入2.5ml的6mM HCl以还原SeO 4→SeO 3→SeO 3→H 2 O,/sup>,   并在100℃继续加热,直到棕色酸雾消失。 如果Se 含量通过电感耦合等离子体质谱法测定 (ICP-MS),不需要通过盐酸还原Se。 它 将需要大约6-8小时来完成整个消化过程   样品量。


      图2.标准茶材料样品的消化状态

  4. 测量Se含量
    1. 温度降至室温后,消解管 从消化炉的孔中轻轻取出并放入   消化管架。 摘要转换为容积 烧瓶中,用去离子水冲洗消化管3-4 次。 也将冲洗溶液加入容量瓶中 直到最终体积达到25ml,然后用玻璃紧密覆盖 塞子。
    2. 将容量瓶底部放在涡旋上 并旋转30秒并混合。 放在一起2小时以上 Se确定。
    3. 硒标准溶液(10mg/L) 分别用1%HNO 3缓慢稀释至浓度为1,5,10,20,50和100μg/L。< br />
    4. 然后通过确定Se内容 原子荧光光谱仪(AFS)或通过ICP-MS。 标准曲线 的Se含量。 Se的含量也是测试样品 决心。


      图3.标准茶叶材料样品的含量

    5. 由AFS确定Se的参数如下: 波长196nm,PMT电压280V,HCl主阴极电流80mA, 载气流量300ml/min,取样体积1.0ml,雾化器高度7   mm,取样泵速率100r/min,取样时间18秒; 参数 用于通过ICP-MS测定Se如下:功率1,300W,等离子体 气体流速15L/min,辅助气体流速0.2L/min,雾化器 流速0.89ml/min。 反应气体甲烷流速0.6ml/min。

笔记

  1. 管口边缘和底部加厚以防止破裂。
  2. 消化管颈在离管口25mm处变薄,以增加酸雾的冷凝,并减少混合酸的挥发。
  3. 将米样品的量控制在约0.5g内以减少消化时间
  4. 将加入到消化管中的混合酸的总量控制在7.5ml以下。消化温度控制在150℃。因此,避免了消化溶液飞溅在管壁上
  5. 需要消化炉中的消化管的位置以进行调节以将消化溶液保持在每个管中的相同高度。

致谢

这项工作已发表在Zhang等人(2014)。该工作得到了第43届中国博士后科学基金(编号:20080430588),人才基金(编号:09001107)和河南理工大学研究基金(编号:13560036)的支持。我们感谢中国科学院地理科学与自然资源研究所研究员,使用ICP-MS进行硒含量分析。

参考文献

  1. Zhang,L.,Hu,B. *,Li,W.,Che,R.,Deng,K.,Li,H.,Yu,F.,Ling,H.,Li,Y.and Chu,C (2014)。 OsPT2,一种磷酸盐转运蛋白,参与 活性摄取水稻中的亚硒酸盐。 New Phytol 201(4):1183-1191。 (* Co-first authors)
  2. Zhang,L.,Shi,W。和Wang,X。(2006)。 高硒和低硒水稻品种之间亚硒酸盐吸收的差异及其机制。 植物与土壤 282(1-2):183-193
  3. Zhang,L.,Shi,W.,Wang,X.and Zhou,X。(2006)。 早期生长阶段水稻幼苗硒积累的基因型差异和影响因素的主要因素分析 水稻种子中的硒含量。植物营养 29(9):1601-1618。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, L., Yu, F., Deng, K., Hu, B. and Chu, C. (2014). Analysis of Total Se Content in Rice. Bio-protocol 4(19): e1248. DOI: 10.21769/BioProtoc.1248.
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