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Plant volatiles (PVs) mediate manifold interactions between plants and their biotic and abiotic environments (Dicke and Baldwin, 2010; Holopainen and Gershenzon, 2010). An understanding of the physiological and ecological functions of PVs must therefore be based on measurements of PV emissions under natural conditions. Yet sampling PVs in natural environments is difficult, limited by the need to transport, maintain, and power instruments, or else to employ expensive sorbent devices in replicate. Thus PVs are usually measured in the artificial environments of laboratories or climate chambers. However, polydimethysiloxane (PDMS), a sorbent commonly used for PV sampling (Van Pinxteren et al., 2010; Seethapathy and Górecki, 2012), is available as silicone tubing (ST) for as little as 0.60 €/m (versus 100-550 € apiece for standard PDMS sorbent devices). Small (mm-cm) ST pieces can be placed in any experimental setting and used for headspace sampling with little manipulation of the organism or headspace. ST pieces have absorption kinetics and capacities sufficient to sample plant headspaces on a timescale of minutes to hours, producing biologically meaningful “snapshots” of PV blends. When combined with thermal desorption (TD)-GC-MS analysis - a 40-year-old and widely available technology - ST pieces yield reproducible, sensitive, spatiotemporally resolved, quantitative data from headspace samples taken in natural environments (Kallenbach et al., 2014).

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Application of Silicone Tubing for Robust, Simple, High-throughput, and Time-resolved Analysis of Plant Volatiles in Field Experiments
采用硅胶试管法稳固、简单、高效和快速的分析田间试验植物的挥发性物质

植物科学 > 植物生物化学 > 其它化合物
作者: Mario Kallenbach
Mario KallenbachAffiliation: Departments of Molecular Ecology and Technical Services, Max Planck Institute for Chemical Ecology, Jena, Germany
Bio-protocol author page: a1972
Daniel Veit
Daniel VeitAffiliation: Departments of Molecular Ecology and Technical Services, Max Planck Institute for Chemical Ecology, Jena, Germany
Bio-protocol author page: a1973
Elisabeth J. Eilers
Elisabeth J. EilersAffiliation: Institute of Biology, Applied Zoology/Animal Ecology, Freie Universität Berlin, Berlin, Germany
Bio-protocol author page: a1974
 and Meredith C. Schuman
Meredith C. SchumanAffiliation: Departments of Molecular Ecology and Technical Services, Max Planck Institute for Chemical Ecology, Jena, Germany
For correspondence: mschuman@ice.mpg.de
Bio-protocol author page: a1975
Vol 5, Iss 3, 2/5/2015, 3123 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1391

[Abstract] Plant volatiles (PVs) mediate manifold interactions between plants and their biotic and abiotic environments (Dicke and Baldwin, 2010; Holopainen and Gershenzon, 2010). An understanding of the physiological and ecological functions of PVs must therefore be based on measurements of PV emissions under natural conditions. Yet sampling PVs in natural environments is difficult, limited by the need to transport, maintain, and power instruments, or else to employ expensive sorbent devices in replicate. Thus PVs are usually measured in the artificial environments of laboratories or climate chambers. However, polydimethysiloxane (PDMS), a sorbent commonly used for PV sampling (Van Pinxteren et al., 2010; Seethapathy and Górecki, 2012), is available as silicone tubing (ST) for as little as 0.60 €/m (versus 100-550 € apiece for standard PDMS sorbent devices). Small (mm-cm) ST pieces can be placed in any experimental setting and used for headspace sampling with little manipulation of the organism or headspace. ST pieces have absorption kinetics and capacities sufficient to sample plant headspaces on a timescale of minutes to hours, producing biologically meaningful “snapshots” of PV blends. When combined with thermal desorption (TD)-GC-MS analysis - a 40-year-old and widely available technology - ST pieces yield reproducible, sensitive, spatiotemporally resolved, quantitative data from headspace samples taken in natural environments (Kallenbach et al., 2014).
Keywords: Polydimethylsiloxane(聚甲基硅氧烷), Silicone tubing(硅胶管), Headspace analysis(顶空分析), Herbivore-induced plant volatiles(虫害诱导植物挥发物), Nicotiana attenuata(野生烟草)

[Abstract]

Materials and Reagents

  1. Acetonitrile (gradient grade)
  2. Methanol (gradient grade)
  3. Nitrogen (gaseous)
  4. Scentless soap (Labosol S)
  5. Argon (gaseous)
    Note: Argon provides a clean, inert atmosphere which is heavier than air, for long-term storage of conditioned ST pieces. For shorter time periods, storage under nitrogen is adequate.

Equipment

  1. Silicone laboratory tubing (ST, 1 mm i.d. x 1.8 mm o.d.) (Carl Roth, catalog number: 9555.1 )
  2. 4 ml screw-neck glass vials (brown) (Macherey-Nagel, catalog number: 702973 ) and screw caps (N13 PP) (Macherey-Nagel, catalog number: 702051 )
  3. 1.5 ml screw-neck glass vials (Macherey-Nagel, clear, catalog number: 702282 ; brown, catalog number: 702293 ) and screw caps (N9 polypropylene [PP], Macherey-Nagel, catalog number: 702028)
  4. Polytetrafluoroethylene (PTFE) seal tape (RS, catalog number: 231-964 )
  5. Paddle wire
  6. Retractable-blade utility knife or single-edged razor blade
  7. Cutting mask (custom-made) for uniformly cutting 5 mm PDMS pieces, made out of polyoxymethylene (POM) (Figure 1A)
    Note: PDMS pieces can also be cut manually, but a cutting mask increases uniformity and saves time when cutting large numbers of PDMS pieces.
  8. Laboratory borosilicate glass bottle (volume: 100-250 ml) (Carl Roth, catalog numbers: A358.1 or A359.1 )
  9. Stainless steel spatula (Carl Roth, catalog number: 1902.1 )
  10. Stainless steel forceps (Carl Roth, catalog number: 2687.1 ) and aluminum foil (Carl Roth, catalog number: 0192.1 )
  11. Glass column (50 ml) with screw-cap ends, containing a size P0 glass frit (Figure 1C)
    Note: The glass frit should be sufficiently small to cause a turbulent nitrogen gas flow.
  12. Polyether ether ketone (PEEK) screw-caps with PTFE lining and stainless steel hose connections, for glass column (Figure 1D)
  13. Flow meter (5-10 L volume)
  14. PTFE gas hoses
  15. Heating oven (max. temperature at least 210 °C)
  16. Polyethylene tetraphthalate (PET) trapping containers (30-600 ml, Solo or Iso-pack)
    Note: The trapping container volume should be as small as possible without significantly changing environmental conditions (e.g. humidity, temperature). This can be tested by enclosing a test sample for the planned trapping period and checking for any visual change (e.g. wilting, condensation) as well as by placing environmental sensors (e.g. temperature, humidity) inside and outside of the trapping container.
  17. Thermo desorption (TD) unit (Shimadzu, model: TD-20 )
  18. 89 mm TD sampler tubes (Supelco, catalog number: 28714-U ) and caps with O-rings (Shimadzu, caps, catalog number: 2235461791 and O-rings, catalog number: 2235716691 )
    Note: Caps can also be milled from brass as long as the exact dimensions are kept.
  19. GC-MS (Shimadzu, model: GC-MS-QP2010Ultra )
  20. 5MS GC column (30 m long, 0.25 mm i.d., 0.25 µm film thickness) (Restek Rtx-5MS)
  21. WAX column (30 m long, 0.25 mm i.d., 0.25 µm film thickness) (Phenomenex ZB-Wax Plus)

Procedure

  1. Preparation of PET trapping containers (please see Figure 2 for examples)
    1. Make a hole of a standard size in the lid of each trapping container (if necessary; 600 ml Iso-pack cups have lids with holes), so that the sample can be easily inserted without plant damage.
    2. In each cup, make a hole in the bottom of a standard size, to allow movement of air through the container and to enable exchange of ST pieces without opening trapping containers during sampling.
    3. Thread paddle wire through small holes in each cup to use for fixing trapping containers to supports.
      Note that this will block the inner surface of the ST pieces from exposure to the headspace and could thus potentially increase equilibration time, although we have not found such use of wire to significantly change required exposure times.
    4. Wash cups with scentless soap (Neolab Labosol S) and water and air-dry before and after use.
      Note: Paddle wire may be removed before washing to avoid rusting; otherwise, drying should be accelerated by shaking or patting dry, heating at a low temperature (30-50 °C), or airflow.

  2. Preparation of silicone tubing (ST)
    1. Cut ST into uniform 5 mm pieces using the knife or scalpel and handmade cutting mask (Figure 1A).
    2. Collect ST pieces in a laboratory glass bottle and add 4/1 (v/v) acetonitrile/methanol until the ST pieces are fully covered (Figure 1B).
    3. Soak for approx. 3 h at room temperature.
    4. Decant the solvent and with the spatula, pour the ST pieces into the glass column with glass frit (Figure 1C).
      Note: Dry ST pieces are electrostatic and will stick to glass walls. Pouring with some solvent facilitates the transfer. This should be done over a glass beaker to collect solvent.
    5. Connect the glass columns to nitrogen gas flow at 5 L/min to flush solvent and contaminants.
    6. Transfer the closed glass columns into a modified heating oven (Figure 1D).
      Note: We laid PTFE gas hose through the ventilation of the heating oven to have a nitrogen connection inside the oven.
    7. Heat under nitrogen flow (5 L/min) for 1.5 h at 210 °C.
      Note: The temperature should be held for 1.5 h. Determine how long your oven requires to reach 210 °C and add this pre-heating time to the total time, e.g. 3 h: 1.5 h heating and 1.5 h at 210 °C. You may, at the same time, place the forceps and spatula on a piece of aluminum foil in the oven, for cleaning.
    8. Switch off the heating and cool to room temperature under nitrogen flow (5 L/min).
    9. Transfer the ST pieces into brown 4 ml glass vials.
      Note: The dry ST pieces are electrostatic. Small numbers are best transferred by using forceps; larger numbers of ST pieces are best transferred by first pouring them on aluminum foil and sliding them from the foil into the vial, assisted with forceps or gloved fingers.
    10. Flush the vials with argon, screw closed, and seal the closed vials with PTFE tape (Figure 1E).


      Figure 1. Working steps for preparation and conditioning of silicone tubing (ST). Photographs by Danny Kessler and Mario Kallenbach.

  3. Sampling of PVs from leaf and flower headspaces with ST pieces
    1. Place the lids on the leaf or flower assigned for trapping as shown in Figure 2A-B.
    2. Place an appropriate number of ST pieces into the trapping container (Figure 2A-B).
      Note: Each ST piece is analyzed only once. We use two to five ST pieces as technical replicates for each sample. It is essential to use the same number of technical replicates for every sample; the total volume of ST in the sample headspace will determine the amount of PVs collected on each individual ST piece.
    3. Close the trapping containers and collect the volatile headspace (Figure 2C).
      Note: In field experiments we stabilize the trapping containers with sticks, tent herrings, stones, etc.
    4. After the trapping, store the ST pieces in tightly-closed 1.5 ml glass vials.
      Note: Technical replicates may be stored in a common vial. This will increase their homogeneity. The storage temperature should be adapted to the compounds of interest. For short term storage (up to several weeks, see Kallenbach et al., 2014) and/or stable substances, storage at room temperature is sufficient; otherwise, storage at -20 °C is recommended.
    5. Store the ST pieces in vials at room temperature for at least 1 day prior to analysis to allow equilibration between ST pieces and the surrounding atmosphere in the glass vial.
      Note: Trapping on ST is an absorptive method with an enrichment of headspace volatiles into the polydimethylsiloxane (PDMS). Therefore, an equilibration between PDMS and the surrounding atmosphere always occurs. Thus for samples which should be directly comparable, we always store similar numbers of technical replicates in similar vial sizes to have an equal ratio of PDMS volume to atmosphere volume.


      Figure 2. Experimental setup for headspace volatile sampling from leaves and flowers. Red arrows highlight the positions of ST tubing. Photographs by Danny Kessler and Mario Kallenbach.

  4. TD-GC-MS analysis
    1. Place an individual ST piece into a TD sampler tube (Figure 3A).
      Note: To increase sensitivity, multiple ST pieces may be desorbed in the same TD sampler tube. This will lead to larger silicone peaks in GC-MS chromatograms, and thus it may be necessary to modify the MS method to either switch off the ionizing filament during the elution of known silicone peaks, or else reduce the voltage of the detector during these elution times in order to avoid saturating the detector.
    2. Metal springs, supplied with the TD sampler tubes, prevent ST pieces from being flushed out of the sampler tube (red arrow in Figure 3B).
    3. Desorb and analyze the headspace volatile samples by TD-GC-MS (Figure 3C) using the settings described in Table 1 and accompanying method optimization notes.
      Note: Blank tubes should be desorbed periodically, e.g. every 10 samples, to check for contamination.
    4. If required necessary (i.e. in case of detected contamination is detected in blank tubes), clean TD sampler tubes and lids (containing O-rings). Tubes can be cleaned by soaking in acetone for a short period (<1 h is sufficient) – tubes in acetone may also be placed in a sonic bath (15-20 min at room temperature is sufficient) - draining, and heating to 210 °C. Lids can be cleaned by soaking in methanol under the same conditions as tubes, draining, and air-drying under a cover of lint-free tissue paper to avoid dust.
      Note: More aggressive solvents and higher temperatures will damage O-rings, while lids will oxidize if cleaned with water.
    5. Standard solutions can be analyzed by injecting 1 µl of standard at an appropriate concentration (usually ca. 0.1-10 ng/µl, but the range will depend on sensitivity settings of the MS, split setting of the injector, and compound), dissolved in dichloromethane or hexane, onto the inner wall of a clean ST placed in a TD sampler tube. The solvent and standard will be quickly absorbed by the ST.
      Note: This method of analyzing standards may not work well for low-volatility compounds with high affinity for PDMS, e.g., long linear alkanes. For such standards, it may be preferable to inject the 1 µl of standard solution directly onto the inner glass wall of the TD sampler tube and quickly close the tube.


      Figure 3. Loading of ST pieces into TD sampler tubes and TD-GC-MS-equipment. Photographs by Danny Kessler and Mario Kallenbach.

      Table 1. TD-GC-MS settings for leaf and flower headspace analysis
      A. TD sampling and injection
      TD block
      200 °C
      TD line
      230 °C
      GC interface
      230 °C
      Sampling
      N2 flow at 60 ml/min for 8 min at 230 °C
      Cryotrap cold temp.
      -20 °C
      Cryotrap heat temp.
      230 °C for 10 min
      B. GC separation
      Carrier gas
      He at 40 cm/sec
      Split ratio
      1 to 20
      Leaf headspace

      Column
      Rtx-5MS, 30 m long, 0.25 mm i.d., 0.25 µm film thickness
      Oven program
      40 °C for 5 min, ramp to 185 °C with 5 °C/min,
      ramp to 280 °C with 30 °C/min, hold for 0.83 min
      Total run time
      38 min
      Flower headspace

      Column
      ZB-Wax Plus, 30 m long, 0.25 mm i.d., 0.25 µm film thickness
      Oven program
      60 °C for 1 min, ramp to 150 °C at 30 °C/min, ramp to 200 °C
      at 10 °C/min, ramp to 230 °C at 30 °C/min, hold for 1 min
      Total run time
      11 min
      C. MS detection
      Transfer line temperature
      240 °C
      Ion source temperature
      220 °C
      Scan range
      Full scan from 33 to 400 m/z
      Scan speed
      0.3 sec/full scan

      Method optimization notes: For humid or wet samples, or samples containing large amounts of low-volatility compounds, the TD program can be made more stringent by increasing the N2 flow to 100-150 ml/min and by increasing the sampling time. GC methods can be adjusted as appropriate for specific analytes and columns by altering split ratio, ramping rates, temperatures, and holding times. As described under TD-GC-MS analysis, a timed MS program may be used to reduce or eliminate signal from very abundant peaks or contaminants. The Shimadzu QP2010Ultra MS can be tuned to three different sensitivity ranges for less or more dilute samples. For a few PVs which degrade or re-arrange at high temperatures, it may be desirable to reduce desorption, interface, transfer line, and ion source temperatures to 150-200 °C. All such method adjustments should only be undertaken with the assistance of an experienced TD-GC-MS user.

Representative data

  1. Chromatograms of Nicotiana attenuata leaf and flower headspace collections.


    Figure 4. Example chromatograms of a headspace volatile collection from a N. attenuata leaf A and a flower B. The total ion chromatogram (TIC) and representative ion traces (A. m/z 55 and 67 to represent green leaf volatiles; B. m/z 105 and 148 to represent benzyl acetone) are shown. Peak identities can be found in Kallenbach et al. (2014).

Acknowledgments

The presented protocol was adopted from Kallenbach et al. (2014). This work was funded by the Max-Planck-Society and by Advanced Grant no. 293926 of the European Research Council.

References

  1. Dicke, M. and Baldwin, I. T. (2010). The evolutionary context for herbivore-induced plant volatiles: beyond the ‘cry for help’. Trends Plant Sci 15(3): 167-175.
  2. Holopainen, J. K. and Gershenzon, J. (2010). Multiple stress factors and the emission of plant VOCs. Trends Plant Sci 15(3): 176-184.
  3. Kallenbach, M., Oh, Y., Eilers, E. J., Veit, D., Baldwin, I. T. and Schuman, M. C. (2014). A robust, simple, high-throughput technique for time-resolved plant volatile analysis in field experiments. Plant J 78(6): 1060-1072.
  4. Seethapathy, S. and Gorecki, T. (2012). Applications of polydimethylsiloxane in analytical chemistry: a review. Anal Chim Acta 750: 48-62.
  5. van Pinxteren, M., Paschke, A. and Popp, P. (2010). Silicone rod and silicone tube sorptive extraction. J Chromatogr A 1217(16): 2589-2598.

材料和试剂

  1. 乙腈(梯度级)
  2. 甲醇(梯度级)
  3. 氮气(气体)
  4. 无皂香皂(Labosol S)
  5. 氩气(气态)
    注意:氩气提供清洁,惰性的气体,比空气重,用于长期储存条件下的ST片。 对于较短的时间,在氮气下储存就足够了。

设备

  1. 硅胶实验室管道(ST,1mm i.d.×1.8mm o.d.)(Carl Roth,目录号:9555.1)
  2. 将4ml螺旋颈玻璃小瓶(棕色)(Macherey-Nagel,目录号:702973)和螺旋盖(N13 PP)(Macherey-Nagel,目录号:702051)
  3. 将1.5ml螺旋颈玻璃小瓶(Macherey-Nagel,clear,目录号:702282;棕色,目录号:702293)和螺旋盖(N9聚丙烯[PP],Macherey-Nagel,目录号:702028)
  4. 聚四氟乙烯(PTFE)密封胶带(RS,目录号:231-964)
  5. 桨线
  6. 可伸缩刀片实用刀或单刃剃刀刀片
  7. 用于均匀切割由聚甲醛(POM)(图1A)制成的5mm PDMS片的切割面具(定制)
    注意:PDMS切片也可以手动切割,但是当切割大量PDMS切片时,切割面罩可以提高均匀性并节省时间。
  8. 实验室硼硅酸盐玻璃瓶(体积:100-250ml)(Carl Roth,目录号:A358.1或A359.1)
  9. 不锈钢刮刀(Carl Roth,目录号:1902.1)
  10. 不锈钢钳(Carl Roth,目录号:2687.1)和铝箔(Carl Roth,目录号:0192.1)
  11. 玻璃柱(50ml),带有螺旋盖,含有尺寸为P0的玻璃料(图1C) 注意:玻璃料应足够小,以产生湍流的氮气流。
  12. 聚醚醚酮(PEEK)带有PTFE衬里和不锈钢软管接头的螺旋盖,用于玻璃柱(图1D)
  13. 流量计(5-10 L容积)
  14. PTFE气管
  15. 加热炉(最高温度至少210℃)
  16. 聚对苯二甲酸乙二醇酯(PET)捕集容器(30-600ml,Solo或Iso-pack)
    注意:捕集容器体积应尽可能小,而不会显着改变环境条件(例如湿度,温度)。这可以通过围绕计划的捕集周期封闭测试样品并检查任何视觉变化(例如萎凋,冷凝)以及通过在捕集容器内部和外部放置环境传感器(例如温度,湿度)来测试。 >
  17. 热脱附(TD)单元(Shimadzu,型号:TD-20)
  18. 89mm TD取样管(Supelco,目录号:28714-U)和带有O形环(Shimadzu,帽,目录号:2235461791和O型环,目录号:2235716691)的帽子。 注意:帽子也可以从黄铜铣削,只要保持精确的尺寸。
  19. GC-MS(Shimadzu,型号:GC-MS-QP2010Ultra)
  20. 5MS GC柱(30m长,0.25mm i.d.,0.25μm膜厚度)(Restek Rtx-5MS)
  21. WAX柱(30m长,0.25mm i.d.,0.25μm膜厚)(Phenomenex ZB-Wax Plus)

程序

  1. 准备PET捕集容器(请参见图2的示例)
    1. 在每个捕集容器的盖子上制作一个标准尺寸的孔(如果   必要; 600毫升Iso-pack杯子有盖子有孔),所以 样品可以容易地插入而没有植物损伤
    2. 在每个杯中, 在标准尺寸的底部开一个孔,以允许空气移动 通过容器并且使得能够交换ST件 在取样期间打开收集容器
    3. 螺纹桨线穿过每个杯中的小孔,用于将捕集容器固定在支撑件上 请注意,这将阻止ST片的内表面 暴露于顶空并因此可能增加 平衡时间,虽然我们还没有发现这种使用电线 会显着改变所需的曝光时间。
    4. 用无香皂(Neolab Labosol S)洗涤杯子,并在使用前后用水和空气晾干。
      注意:在清洗之前可以取下桨叶丝,以免生锈; 否则,应通过摇动或拍干来加速干燥, 在低温(30-50°C)下加热,或气流。

  2. 硅胶管(ST)的制备
    1. 使用刀或手术刀和手工切割面膜将ST切成均匀的5毫米切片(图1A)
    2. 收集ST片在实验室玻璃瓶中,加入4/1(v/v) 乙腈/甲醇,直到ST块完全被覆盖(图1B)
    3. 浸泡约。 室温下3小时。
    4. 倾析溶剂,用刮刀将玻璃块倒入玻璃柱(图1C)。
      注意:干ST片是静电的,会粘在玻璃墙上。 用一些溶剂倾倒有助于转移。 这应该做 通过玻璃烧杯收集溶剂。
    5. 将玻璃柱以5 L/min的流速连接到氮气流,冲洗溶剂和污染物
    6. 将封闭的玻璃柱转移到改良的加热炉中(图1D) 注意:我们通过加热炉的通风来放置PTFE气体软管,以在烤箱内部形成氮气连接。
    7. 在氮气流(5L/min)下在210℃下加热1.5小时 注意:温度应保持1.5小时。确定你多长时间  烘箱需要达到210°C并将该预加热时间加到 总时间,例如。 3小时:加热1.5小时,210℃1.5小时。你可以在  同时,将钳子和铲子放在一片铝箔上  烤箱,用于清洁。
    8. 关闭加热并在氮气流(5L/min)下冷却至室温
    9. 将ST片转移到棕色4 ml玻璃小瓶中 注意:干燥的ST片是静电的。小数字是最好的 使用镊子转移;更大数量的ST片是最好的 通过首先将它们倒在铝箔上并将它们滑动来转移  将箔片放入小瓶中,用镊子或戴手套的手指辅助。
    10. 用氩气冲洗小瓶,旋紧,用PTFE胶带密封封闭的小瓶(图1E)

      图1.硅胶的制备和调理的工作步骤 (ST)。 Danny Kessler和Mario Kallenbach的照片。

  3. 从具有ST块的叶和花头空间取样PVs
    1. 将盖子放在分配用于捕集的叶或花上,如图2A-B所示。
    2. 将适当数量的ST片放入捕集容器(图2A-B)。
      注意:每个ST片只被分析一次。我们使用两到五个ST片  作为每个样品的技术重复。这是必要的使用 每个样品相同数量的技术重复;总体积 的ST在样品顶空中将确定收集的PV的量  对每个ST段。
    3. 关闭捕集容器并收集挥发性顶空(图2C)。
      注意:在实地实验中,我们用棍子,帐篷,石头等来稳定捕集容器。
    4. 收集后,将ST片放入紧密封闭的1.5 ml玻璃小瓶中 注意:技术重复可以存储在常用小瓶中。这会 增加它们的同质性。储存温度应适应  感兴趣的化合物。对于短期存储(长达几周,  见Kallenbach等人,2014)和/或稳定物质,在室内储存 温度足够;否则,建议储存在-20°C。
    5. 将ST片在室温下储存在小瓶中至少1天 在分析之前允许ST片和之间的平衡 玻璃瓶中的周围大气 注意:在ST上捕获是   吸附方法与顶空挥发物的富集 聚二甲基硅氧烷(PDMS)。 因此,PDMS之间的平衡 并且总是发生周围的气氛。 因此对于样品 应该直接比较,我们总是存储类似的数字 类似的小瓶尺寸的技术重复有相同的比例 PDMS体积与大气体积。


      图2.实验设置 从叶和花的顶空挥发性取样。 红色箭头 突出显示ST管的位置。 照片由丹尼·凯斯勒和 马里奥·卡伦巴赫。

  4. TD-GC-MS分析
    1. 将单个ST片放入TD取样管(图3A)。
      注意:为了提高灵敏度,可以在中解冻多个ST片   相同的TD取样管。 这将导致GC-MS中较大的硅酮峰 色谱图,因此可能需要将MS方法修改为 或者在已知的洗脱过程中关闭电离灯丝 硅胶峰,或者在这些过程中降低检测器的电压 洗脱时间,以避免检测器饱和。
    2. 金属 弹簧,随TD取样器管提供,防止ST件 从取样器管中冲出(图3B中的红色箭头)
    3. 解吸并通过TD-GC-MS分析顶空挥发性样品 3C)使用表1所述的设置和伴随的方法 优化注释。
      注意:空白管应定期解吸。每10个样品,检查是否有污染。
    4. 如果检测到污染,则需要时(即) 在空白管中检测),清洁TD取样器管和盖(含有 O形环)。管可以通过在丙酮中浸泡短时间来清洁 (<1h足够) - 丙酮中的管也可以置于声波中  浴(15-20分钟,在室温下是足够的) - 排水,和 加热至210℃。盖子可以通过浸泡在甲醇下清洗 相同的条件,管,排水和风干下盖 不起毛的薄纸,以避免灰尘 注意:更强的溶剂和更高的温度会损坏O型圈,而如果用水清洗,则盖子会氧化。
    5. 标准溶液可通过注射1μl标准品进行分析 适当的浓度(通常约0.1-10ng /μl,但范围 将取决于MS的灵敏度设置,分割设置 注射器和化合物),溶解在二氯甲烷或己烷中 一个干净的ST的内壁放在TD取样管中。溶剂 并且标准将很快被ST吸收。
      注意:此方法 的分析标准可能不适用于低挥发性化合物 对PDMS具有高亲和力,例如长线性烷烃。对于这样 标准,可优选注射1μl标准溶液 直接在TD取样器管的内玻璃壁上并快速 关闭管。


      图3.将ST片装入TD取样管和TD-GC-MS设备。 Danny Kessler和Mario Kallenbach拍摄的照片。

      表1.叶和花顶空分析的TD-GC-MS设置
      A。 TD取样和注射
      TD块
      200℃
      TD行
      230℃
      GC接口
      230℃
      抽样
      N 2流以60ml/min在230℃下流动8分钟
      低温冷阱。
      -20°C
      低温热温度。
      230℃,10分钟
      B。 GC分离
      载气
      他在40厘米/秒
      分割比例
      1到20
      叶顶空间


      Rtx-5MS,30m长,0.25mm i.d.,0.25μm膜厚度
      烤箱程序
      40℃5分钟,以5℃/分钟升至185℃,
      以30℃/min升温至280℃,保持0.83分钟
      总运行时间
      38分钟
      花顶空间


      ZB-Wax Plus,30m长,0.25mm i.d.,0.25μm膜厚度
      烤箱程序
      60℃1分钟,以30℃/分钟升至150℃,升至200℃
      以10℃/min,以30℃/min升温至230℃,保持1分钟
      总运行时间
      11分钟
      C。 MS检测
      传输线温度
      240℃
      离子源温度
      220℃
      扫描范围
      从33到400 m/z的全扫描
      扫描速度
      0.3秒/全扫描

      方法优化注意事项:适用于潮湿或潮湿样品或样品 含有大量低挥发性化合物,TD程序可以   通过将N 2 流量增加到100-150ml/min,可以更严格, 通过增加采样时间。 GC方法可以调整为 通过改变分流比适合于特定的分析物和色谱柱, 斜率,温度和保持时间。 如下所述 TD-GC-MS分析,定时MS程序可用于减少或消除   来自非常丰富的峰或污染物的信号。 岛津 QP2010Ultra MS可以调谐到三个不同的灵敏度范围 更少或更多的稀释样品。 对于几个PV降级或重新安排 在高温下,可能希望减少解吸, 界面,传输线和离子源温度升至150-200°C。 所有 这种方法调整只应在协助下进行 的经验丰富的TD-GC-MS用户。

代表数据

  1. 烟草叶片和花顶空间收集的色谱图。


    图   4.来自N的顶空挥发性收集的示例色谱图。 衰减叶叶A和花B. 总离子色谱图(TIC)和 代表性离子轨迹(A.m/z 55和67代表绿叶 挥发物; B.m/z 105和148以表示苄基丙酮)。 峰的身份可以在Kallenbach等人(2014)中找到。

致谢

所提出的方案获自Kallenbach等人(2014)。 这项工作由马克斯普朗克学会和高级格兰特学会资助。 欧洲研究委员会的293926。

参考文献

  1. Dicke,M。和Baldwin,I.T。(2010)。 草食动物诱导的植物挥发物的进化背景:超出了"求助"的范围。 Trends Plant Sci 15(3):167-175。
  2. Holopainen,J.K。和Gershenzon,J。(2010)。 多重胁迫因素和植物VOC的排放。趋势植物科学/em> 15(3):176-184。
  3. Kallenbach,M.,Oh,Y.,Eilers,E.J.,Veit,D.,Baldwin,I.T。和Schuman,M.C。(2014)。 在田间实验中用于时间分辨的工厂挥发性分析的强大,简单,高通量的技术。/a> Plant J 78(6):1060-1072。
  4. Seethapathy,S.和Gorecki,T。(2012)。 聚二甲基硅氧烷在分析化学中的应用:综述 Anal Chim Acta 750:48-62
  5. van Pinxteren,M.,Paschke,A。和Popp,P。(2010)。 硅胶棒和硅胶管吸附萃取。 J Chromatogr A 1217(16):2589-2598
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How to cite this protocol: Kallenbach, M., Veit, D., Eilers, E. J. and Schuman, M. C. (2015). Application of Silicone Tubing for Robust, Simple, High-throughput, and Time-resolved Analysis of Plant Volatiles in Field Experiments. Bio-protocol 5(3): e1391. DOI: 10.21769/BioProtoc.1391; Full Text



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