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Extraction of Nonstructural Carbon and Cellulose from Wood for Radiocarbon Analysis
提取木材中的非结构性碳和纤维素用于放射性碳素分析   

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Abstract

This method aims at isolating nonstructural organic carbon (NSC) pools, i.e. soluble sugars and starch, from wood for radiocarbon (14C) analysis at natural abundance levels (≤1 ppt).
Pools are operationally defined to 1) physically isolate pools - prohibiting the use of destructive methods, such as compound-specific enzyme digestion, and 2) minimize possible contamination with extraneous carbon form organic solvents.

Materials and Reagents

  1. Nonstructural carbon and cellulose
    1. MQ H2O
    2. Laboratory grade pure methanol for cleaning

  2. Nonstructural carbon
    1. 14C standards [international secondary standards: ANU sucrose (IAEA-C6), in-house laboratory standards, e.g. wheat flour, rye flour]
    2. Laboratory-grade ethanol
    3. HPLC-grade ethanol
    4. Analytical-grade 20% HCl [≈ 6 M HCl, 50:50 (v: v) conc. HCl]

  3. Cellulose
    1. 14C standards (international secondary wood standards: e.g. IAEA-C4, -C5)
    2. Laboratory-grade toluene
    3. Laboratory-grade glacial acetic acid (CH3COOH)
    4. Technical grade sodium chlorite (NaClO2)

Equipment

  1. Nonstructural carbon and cellulose
    1. Aluminum (Al) foil (cover surfaces and prevent dust contaminating equipment and samples with extraneous carbon)
    2. Marker pen
    3. Spatula
    4. Wipes for cleaning (e.g. KCWW)
    5. Duster for cleaning (I L) (e.g. InnoveraTM)
    6. Pyrex beakers to hold reagents
    7. Graduated volumetric cylinder to prepare reagents
      Note: All glassware is baked and stored covered with Al foil to remove extraneous sources of carbon. Quartz is pre-combusted at 900 °C for 2 h, Pyrex at 550 °C for 3 h.

  2. Nonstructural carbon
    1. Mill with mesh 10 or scalpel
    2. Graduated, adjustable volume, 5 ml pipette (e.g. VWR International, Eppendorf) or cylinder
    3. Disposable borosilicate glass culture tubes (13 x 100 mm) with plastic vented caps and snap caps (VWR International)
    4. Disposable, plastic (extended fine tip) transfer pipette (e.g. Thermo Fisher Scientific, Samco Scientific)
    5. Quartz combustion tubes (120 mm length, 9 mm O.D.)
    6. 50 ml reusable glass centrifugation tube with screw cap or plastic (e.g. PP) centrifuge tubes with screw cap
    7. Speedvac-dryer (Thermo Fisher Scientific, Savant, model: SC200 speedvac with RT400 cold trap) (operated at room temperature)
    8. Block heater with thermometer for 10 or 20 (13 x 100 mm) disposable borosilicate glass culture tubes

  3. Cellulose
    1. General-purpose extra-long, stainless steel forceps
    2. Quartz combustion tubes (150 mm length, 6 mm O.D.)
    3. Heat sealer
    4. 1,000 ml pyrex beaker
    5. 1,000 ml pyrex round bottom flask (still pot)
    6. Soxhlet extraction apparatus (e.g. VWR International)
    7. Heated magnetic stirrer & stir bar
    8. Drying oven
    9. Cork ring
    10. Boiling chips
    11. Fiber filter bags for sample digestion (ANKOM Technology, catalog number: F57 )

Procedure

Part I: Extraction of nonstructural carbon
The sequential extraction protocol was developed for Reference 2, based on References 3 and 4. Following extractions, samples are dried in 9 mm O. D. quartz combustion tubes and combusted to CO2 at 900 °C for 2 h with CuO (80 mg CuO for 1 mg C); the CO2 is isolated cryogenically and converted to graphite (Xu et al. 2007). Ideally, sugar and starch concentrations should be known prior to this extraction to guide how much material should be extracted and combusted. Concentrations can be quantified as described in References 3 and 4. The target amount for a radiocarbon sample analyzed with accelerator mass spectrometry is typically 0.3-1.2 mg C.


  1. Sample preparation
    Samples are processed in batches of a maximum of 17 samples plus 3 standards.
    1.  Manually chop each dried wood core into small slices or pieces [or grind samples using a mill to mesh 10 (0.8 mm)]. Avoid producing powder to achieve clear separation into supernatant and precipitate. Clean all tools with laboratory grade methanol, then blow dry with an air duster. Cover all surfaces with Al foil.
    2. Assign each sample and standard 2 different ID numbers (1 for soluble sugars, 1 for starch; except ANU which requires only 1 for soluble sugars).
    3. Weigh each sample (entire core section) and standard [ANU (1.5-2 mg), flours (30 mg)] into a culture tube labeled with the ID numbers of both the sugar and the starch fraction. Each sample should not take up more than about 1/3 of the tube as larger samples will not easily absorb solutions or might float out of the tube.
    4. Label combustion tubes with the ID numbers of either the sugar or the starch fraction and record the weight of the empty tubes.
    5. Label centrifuge vials with the starch-fraction ID numbers.

  2. Extraction of soluble sugars
    1. At least 1 h before usage, switch on the speedvac drier to cool the cold-trap to its operating temperature (see operating manual for details).
    2. Pre-heat block heater to 100 °C.
    3. Add 5 ml MQ H2O to each tube, close with a ventilated cap and place in block heater. Bring to boil and gently boil for 15 min. If sample floats, gently tap the tube.
    4. Let solution cool, and then transfer the supernatant with a disposable pipette into a 50 ml glass centrifuge tube. If any sample is floating on the supernatant, centrifuge samples before transfer.
      Note: The disposable culture tubes break easily. They require specific adapters when used in a centrifuge.
    5. Repeat boiling the sample in 5 ml MQ H2O for 15 min TWICE and combine the supernatants in the same 50 ml centrifuge tube. This will yield ≤15 ml of liquid.
    6. In batches of 1-2 ml, transfer 2 ml  of supernatant to a combustion tube (labeled with sugar-fraction ID number) and speedvac-dry.
      Notes:
      1. Ideally, sugar and starch contents should be known prior to 14C analysis. Depending on the sugar yield and on the amount of other soluble compounds recovered with the sugars, samples may explode during combustion or not yield sufficient C for 14C analysis. The target amount is 0.3-1.2 mg C. Samples may have to be combusted multiple times (trial and error) using 0.5-5 ml of supernatant.
      2. This should be done on the same day as the extraction, since samples are prone to growing mold.
      While the sugar samples are drying over several hours, begin starch extraction. Alternatively, cover the solid precipitate remaining in the extraction tube with a snap cap and refrigerate.
    7. Refrigerate remaining liquid sugar fraction.
    8. After speedvac-drying, record the weight of the combustion tubes.

  3. Starch extraction

    Removal of nonpolar compounds, including lipids and pigments
    1. Pre-heat block heater to 70 °C.
    2. Add 5 ml ethanol to the sample in the culture tube, close with vented cap and gently boil for 30 min at about 70 °C.
    3. Let solution cool and then discard the supernatant.
    4. Repeat boiling the sample in fresh ethanol until the supernatant is clear and colorless.
      Note: It is critical that all of the ethanol is removed before proceeding to the starch extraction. If the sample size is very small and sample cannot be sucked very dry, sample might have to be washed with MQ H2O to remove the ethanol.

      Extraction of starch
    5. Lower block heater temperature to 40 °C.
    6. Add 1.5 ml 20% HCl to the sample in the culture tube, close with vented cap and keep at 40 °C for 30 min.
      Note: Make sure the sample is well suspended; samples of finer texture might require more liquid; keep ratios of acid and ethanol (see Part I, step C11) constant.
    7. Switch off the block heater, remove samples and let them stand at room temperature for 8-12 h (over night). Pre-heat block heater to 40 °C.
    8. Transfer supernatant from the culture tubes to a 50 ml centrifuge tube (labeled with the starch-fraction ID number). If any sample is floating in the supernatant, centrifuge samples before transfer.
    9. Again, add 1.5 ml 20% HCl to wood sample in the culture tube (not to the supernatant) and keep at 40 °C for 30 min.
    10. Transfer supernatant to the same 50 ml centrifuge tube - there should be about 3 ml of liquid in the tube. If any sample is floating, centrifuge samples and transfer the supernatant to a fresh tube.
    11. Add 4.5 ml ethanol to the supernatant, vortex, close with a screw cap and let stand over night in the fume hood at room temperature. Starch will precipitate.
    12. The wood sample remaining in the culture tube may be used for subsequent cellulose analysis. If this is required, wash the sample with MQ water until the pH is neutral and dry samples in the block heater or oven at <60 °C.

      Removal of extraneous carbon (ethanol)
      Note: Failure to remove (traces of) extraneous C will result in a lower 14C content (unrealistically old 14C ages). This removal process is validated by analyzing lab internal 14C standards (flours) of known 14C content alongside each batch of samples.
    13. Switch on the speedvac-drier to cool the cold-trap to its operating temperature.
    14. Centrifuge samples and discard the supernatant with a pipette.
    15. Dissolve starch in 1.5 ml MQ H2O (using vortex mixer) and then speedvac-dry sample within the centrifuge tube; this process will take several hours. Avoid completely drying the sample, as the starch will stick to the tube’s walls and cannot be easily redissolved.
    16. Repeat dissolution in 1.5 ml MQ H2O and speedvac-drying.
    17. Again, dissolve starch in 1.5 ml MQ H2O, but transfer all of this liquid to a combustion tube (labeled with the starch fraction ID number) and dry. To ensure complete transfer, work with 0.5 ml MQ H2O at a time and, or use vortex mixer. The target amount is 0.3-1.2 mg C.
    18. After speedvac-drying, record the weight of the combustion tubes.

Part II: Extraction of holocellulose
The holocellulose extraction protocol is based on Reference 1. Following extraction, samples are combusted to CO2 at 900 °C for 2 h with CuO (80 mg CuO for 1 mg C); the CO2 is isolated cryogenically and converted to graphite (Xu et al., 2007).


  1. Sample preparation
    Samples are processed in batches of up to 38 samples plus 2 standards (one fossil wood and one modern wood, with known 14C age).
    1. Assign each sample and standard a laboratory ID number.
    2. After cleaning scalpel with methanol and blowing it dry with an air duster, manually slice or chop samples of dried wood into pieces. Cover all surfaces with Al foil.
    3. Weigh ~100 mg of homogenized sample into a bag. Avoid producing very thin slices or powder, as this material cannot be recovered from the digestion bags or may pass through the bags. Record the initial sample weight. Each bag can hold up to four samples or standards. Up to 10 bags can be extracted in parallel.
    4. Using a heat sealer, seal bags in unique patterns to allow sample identification following the extraction. Record sealing patterns and sample weight and ID in a notebook.

  2. Lipid extraction
    Note: Since the procedure takes about one week, it is best to start this extraction early Monday morning.
    1. In a fume hood, assemble the soxhlet apparatus.
      Note: Make sure the soxhlet has a small enough thimble; otherwise more solvent is needed in a bigger still pot. In other words, make sure enough solvent is left in the still pot when the thimble volume is filled.
    2. Using forceps, place (up to 10) digestion bags into the apparatus’ thimble.
    3. Fill the still pot (1,000 ml round bottom flask) with 600 ml of 2:1 (v: v) toluene:ethanol mixture; add boiling chips.
      Note: This solution may be used for up to three soxhlet extractions; discard earlier if mixture is discolored.
    4. Turn the hot plate on and bring the solvent to a gentle boil. Wrap the still pot and its neck with Al foil loosely to keep it from cooling. Run the extraction for 24 h.
    5. Turn the hot plate off; let apparatus cool. Remove the bags from the thimble and let dry on Al foil in the fume hood for 2 h.
    6. Repeat the extraction with 600 ml of ethanol for 24 h.
      Note: This ethanol may be used for up to three soxhlet extractions; discard earlier if mixture is discolored.
    7. Dry the bags again for 2 h.

  3. Bleaching
    1. Measure out 4 g sodium chlorite.
    2. Place digestion bags into a 1,000 ml beaker.
    3. Add 600 ml MQ H2O, partially cover it with a watch glass and boil the samples on a hot plate for 2 h.
    4. Switch off the hot plate; let the solution cool and discard it.
    5. Add 600 ml MQ H2O, add a stir bar and stir the solution while heating it to 70 °C. Cover it with a watch glass.
    6. Add 4 g sodium chlorite and 2 ml glacial acetic acid to the water at about the same time, and continue stirring this solution at 70 °C.
    7. Change this solution every 3-4 h by adding additional 2 ml glacial acetic acid and 4 g sodium chlorite to the MQ H2O until the samples are white. This process can take 1-2 days.
    8. When the samples are white (like paper), rinse samples at least 4 times with 600 ml MQ H2O over a 3-4 h period. Continue stirring this solution at 70 °C. Make sure no acetic acid smell remains.
    9. Dry samples in a drying oven at 60 °C to constant weight, typically over night.
    10. Record final sample weight.
    11. Transfer sample to combustion tube, labeled with sample ID.

Acknowledgments

We do not wish to add acknowledgments in addition to what was written in the research paper.

References

  1. Leavitt, S. W. and Danzer, S. R. (1993). Method for batch processing small wood samples to holocellulose for stable-carbon isotope analysis. Anal Chem 65(1): 87-89.
  2. Richardson, A. D., Carbone, M. S., Keenan, T. F., Czimczik, C. I., Hollinger, D. Y., Murakami, P., Schaberg, P. G. and Xu, X. (2013). Seasonal dynamics and age of stemwood nonstructural carbohydrates in temperate forest trees. New Phytol 197(3): 850-861.
  3. Richter, A., Wanek, W., Werner, R. A., Ghashghaie, J., Jaggi, M., Gessler, A., Brugnoli, E., Hettmann, E., Gottlicher, S. G., Salmon, Y., Bathellier, C., Kodama, N., Nogues, S., Soe, A., Volders, F., Sorgel, K., Blochl, A., Siegwolf, R. T., Buchmann, N. and Gleixner, G. (2009). Preparation of starch and soluble sugars of plant material for the analysis of carbon isotope composition: a comparison of methods. Rapid Commun Mass Spectrom 23(16): 2476-2488.
  4. Wong, B., Baggett, K. and Rye, A. (2003). Seasonal patterns of reserve and soluble carbohydrates in mature sugar maple (Acer saccharum). Can J Bot 81(8): 780-788.
  5. Xu, X., Trumbore, S. E., Zheng, S., Southon, J. R., McDuffee, K. E., Luttgen, M. and Liu, J. C. (2007). Modifying a sealed tube zinc reduction method for preparation of AMS graphite targets: Reducing background and attaining high precision. Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms 259(1): 320-329.

简介

该方法旨在从木材中分离天然丰度水平的非结构有机碳(NSC)池(即可溶性糖和淀粉)用于放射性碳( 14 C)分析 )。
池的操作定义为:1)物理隔离池 - 禁止使用破坏性方法,如化合物特异性酶消化,以及2)尽量减少可能被外部碳形成的有机溶剂污染。

材料和试剂

  1. 非结构碳和纤维素
    1. MQ H <2> O
    2. 实验室级纯甲醇清洗

  2. 非结构碳
    1. 14标准[国际二级标准:ANU蔗糖(IAEA-C6),   内部实验室标准,例如小麦粉,黑麦粉]
    2. 实验室级乙醇
    3. HPLC级乙醇
    4. 分析级20%HCl [≈6MHCl,50:50(v:v) HCl]

  3. 纤维素
    1. 14 C标准(国际仲裁木材标准:如 -EA,-C5)
    2. 实验室级甲苯
    3. 实验室级冰醋酸(CH 3 COOH)
    4. 工业级亚氯酸钠(NaClO <2>)

设备

  1. 非结构碳和纤维素
    1. 铝(铝)箔(覆盖表面,防止灰尘污染设备和样品与外来碳)
    2. 记号笔
    3. 小铲
    4. 擦拭巾(如 KCWW)
    5. 清洁用除尘器(I L)(如 Innovera TM
    6. Pyrex烧杯装试剂
    7. 刻度量筒,准备试剂
      注意:   所有的玻璃器皿烘烤和存储覆盖着铝箔去除 外来碳源。 将石英在900℃下预燃烧2小时,   Pyrex在550℃下3小时

  2. 非结构碳
    1. 网眼10或手术刀磨料
    2. 刻度,可调体积,5ml移液管(例如VWR International,Eppendorf)或圆柱体
    3. 一次性硼硅酸盐玻璃培养管(13 x 100 mm),带塑料通风帽和卡扣帽(VWR International)
    4. 一次性塑料(延长细尖)移液管(例如 Thermo Fisher Scientific,Samco Scientific)
    5. 石英燃烧管(长120mm,外径9mm)
    6. 具有螺帽的50ml可重复使用的玻璃离心管(例如 PP)离心管,具有螺帽/
    7. Speedvac干燥器(Thermo Fisher Scientific,Savant,型号:SC200 speedvac with RT400冷阱)(在室温下操作)
    8. 带有用于10或20(13×100mm)一次性硼硅酸盐玻璃培养管的温度计的块加热器

  3. 纤维素
    1. 通用超长,不锈钢镊子
    2. 石英燃烧管(长150mm,外径6mm)
    3. 热封机
    4. 1000毫升pyrex烧杯
    5. 1,000ml Pyrex圆底烧瓶(釜)
    6. 索氏萃取装置(例如VWR International)
    7. 加热磁力搅拌器& 搅拌棒
    8. 干燥炉
    9. 软木塞
    10. 沸腾的芯片
    11. 用于样品消化的纤维滤袋(ANKOM Technology,目录号:F57)

程序

第I部分:非结构碳的提取
对于参考文献2,基于参考文献3和4开发了顺序提取方案。提取后,将样品在9mm OD石英燃烧管中干燥并在900℃下燃烧2小时, CuO(对于1mg C,80mg CuO); CO 2被低温分离并转化成石墨(Xu等人,2007)。 理想地,在该提取之前应该知道糖和淀粉的浓度,以指导应提取和燃烧多少材料。 浓度可以如参考文献3和4中所述定量。用加速器质谱法分析的放射性碳样品的目标量通常为0.3-1.2mg C。


  1. 样品准备
    样品以最多17个样品加3个标准品的批次处理。
    1.  手工将每个干木芯切成小片或小块[或 使用研磨机筛网10(0.8mm)研磨样品]。 避免产生粉末 以实现清澈分离成上清液和沉淀。 清洁所有 工具用实验室级甲醇,然后用空气喷雾器吹干。 用铝箔覆盖所有表面。
    2. 分配每个样品和 标准2个不同的ID号(1个可溶性糖,1个淀粉; 除了ANU,其中只需要1个可溶性糖)
    3. 称重 每个样品(整个芯部)和标准[ANU(1.5-2mg),面粉 (30mg)]加入到用两个ID号标记的培养管中 糖和淀粉部分。 每个样本不应超过 约1/3的管,因为较大的样品不容易吸收溶液   或者可能从管中浮出
    4. 标签燃烧管 记录糖或淀粉部分的ID号   空管重量
    5. 标记具有淀粉级分ID号的离心瓶。

  2. 提取可溶性糖
    1. 使用前至少1小时,打开speedvac干燥机冷却 冷阱到其工作温度(参见操作手册 详细信息)。
    2. 将预热块加热器预热至100°C
    3. 加入5 ml MQ   H 2 O至每个管,用通风盖封闭并置于块中 加热器。 煮沸并轻轻煮沸15分钟。 如果样品漂浮, 轻轻点击管。
    4. 让溶液冷却,然后转移 上清液用一次性移液管倒入50ml玻璃离心机中 管。 如果任何样品漂浮在上清液上,离心样品 转移前。
      注意:一次性文化管容易折断。 在离心机中使用时,它们需要特殊的适配器。
    5. 重复将样品在5ml MQ H 2 O中煮沸15分钟,并混合 上清液在相同的50ml离心管中。 这将产生≤15 ml液体
    6. 在1-2毫升的批次,转移2毫升  的 上清液至燃烧管(用糖部分ID号标记)   和速干型 注意:
      1. 理想情况下,糖和淀粉 内容应在 14 C分析之前已知。 取决于糖 产量和用其回收的其它可溶性化合物的量 糖,样品可能在燃烧期间爆炸或不产生足够的C 用于 14 C分析。 目标量为0.3-1.2mg C.样品可能必须   使用0.5-5ml的燃烧多次(试验和错误) 上清液。
      2. 这应该在提取的同一天完成,因为样品容易生长霉菌。
      当糖样品干燥数小时时,开始淀粉提取。 或者,用盖帽盖住萃取管中残留的固体沉淀物并冷藏
    7. 冷藏剩余的液态糖部分
    8. 速干后,记录燃烧管的重量。

  3. 淀粉提取

    去除非极性化合物,包括脂质和颜料
    1. 将预热块加热器预热至70°C
    2. 在培养管中的样品中加入5 ml乙醇,盖上盖子,在约70°C轻轻煮沸30分钟。
    3. 让溶液冷却,然后弃去上清液。
    4. 重复将样品在新鲜乙醇中煮沸直到上清液澄清无色 注意:   关键的是在进行之前除去所有的乙醇 淀粉提取。 如果样品量非常小和样品 不能吸干非常干燥,样品可能必须用MQ H 2 除去乙醇。

      提取淀粉
    5. 将块式加热器温度降至40°C
    6. 向培养管中的样品中加入1.5ml 20%HCl,用通气盖封闭并在40℃保持30分钟。
      注意:   确保样品充分悬浮; 更细的纹理样本可能 需要更多的液体; 保持酸和乙醇的比率(参见第I部分,步骤 C11)常数。
    7. 关闭块加热器,取出样品和 让它们在室温下静置8-12小时(过夜)。 预热 块加热器至40℃
    8. 转移培养物的上清液 管置于50ml离心管(用淀粉级分ID标记) 数)。 如果任何样品漂浮在上清液中,离心 转移前的样品
    9. 再次,在培养管(不是上清液)中的木材样品中加入1.5ml 20%HCl,并在40℃保持30分钟。
    10. 转移上清液到同一个50ml离心管 - 应该 在管中约3ml液体。 如果任何样品浮动, 离心样品并将上清液转移到新管中
    11. 向上清液中加入4.5ml乙醇,涡旋,用螺旋盖封闭 并在通风橱中在室温下静置过夜。 淀粉 将沉淀。
    12. 木材样品保留在培养物中 管可以用于随后的纤维素分析。 如果需要,   用MQ水洗涤样品直至pH为中性,并干燥样品 在块加热器或烘箱中<60℃
      除去无关碳(乙醇)
      注意:   不去除(痕量)外来的C将导致较低的 14℃ 内容(不切实际地旧的 14 C年龄)。 这个删除过程是 通过分析已知的 14 C的实验室内部 14标准(面粉) 内容与每批样品一致。
    13. 打开快速干燥器,将冷阱冷却至其工作温度。
    14. 离心样品,并用移液管丢弃上清液
    15. 将淀粉溶解在1.5ml MQ H 2 O(使用涡旋混合器)中,然后 在离心管内快速干燥样品; 这个过程将需要 几个小时。 避免完全干燥样品,因为淀粉会 粘在管的壁上,不容易再溶解
    16. 重复溶解在1.5ml MQ H 2 O中并快速干燥。
    17. 再次,将淀粉溶解在1.5ml MQ H 2 O中,但转移所有这些 液体到燃烧管(用淀粉部分ID号标记)   并干燥。 为了确保完全转移,一次使用0.5ml MQ H 2 O   和/或使用涡流混合器。 目标量为0.3-1.2mg C
    18. 速干后,记录燃烧管的重量。

第II部分:提取全纤维素
提取后,样品在900℃下用CuO(80mg CuO,1mg C)燃烧至CO 2 2小时; CO 2被低温分离并转化为石墨(Xu等人,2007)。


  1. 样品准备
    样品以多达38个样品加2个标准品(一种化石木材和一种现代木材,已知 14℃的年龄)的批次进行处理。
    1. 将每个样品和标准品分配一个实验室ID号
    2. 后 清洁手术刀用甲醇和吹干它用空气喷粉器, 手动切片或切碎干木材的样品成片。 覆盖所有 表面具有铝箔
    3. 称量〜100 mg均质样品   一个包。 避免产生非常薄的切片或粉末,因为这种材料 不能从消化袋中回收或可能通过 袋子。 记录初始样品重量。 每个包可以容纳四个 样品或标准。 最多可以并行提取10个包。
    4. 使用热封机,密封袋采用独特的图案,允许样品 鉴定后提取。 记录密封图案和 样品重量和笔记本中的ID

  2. 脂质提取
    注意:由于程序大约需要一周,最好在星期一早上开始提取。
    1. 在通风橱中,组装索格利特设备 注意:请确保 索格利特有一个足够小的顶针; 否则在a中需要更多的溶剂   更大还是锅。 换句话说,确保留下足够的溶剂 当套管体积充满时仍然是壶。
    2. 使用镊子,将(最多10个)消解袋放入设备的套管中
    3. 用600ml 2:1(v:v)甲苯:乙醇混合物填充蒸馏釜(1000ml圆底烧瓶) 添加煮沸的薯条。
      注意:此溶液可用于最多三次索氏提取; 如果混合物变色,则舍弃。
    4. 打开热板,使溶剂温和沸腾。 包装   仍然锅和它的脖子与铝箔松散,防止冷却。 运行提取24小时。
    5. 关闭热板; 让 设备冷却。 从套管上取下袋子,在铝箔上干燥 在通风橱内2小时。
    6. 用600ml乙醇重复萃取24小时 注意:这种乙醇可用于多达三次索氏萃取; 如果混合物变色,则舍弃。
    7. 再干燥袋子2小时。

    1. 测量4g亚氯酸钠。
    2. 将消化袋放入1000ml烧杯中。
    3. 加入600ml MQ H 2 O,用表面玻璃部分覆盖,并将样品在热板上煮沸2小时。
    4. 关闭热板; 让溶液冷却并丢弃它
    5. 加入600ml MQ H 2 O,加入搅拌棒并搅拌溶液,同时将其加热至70℃。 用表玻璃盖住。
    6. 向水中加入4g亚氯酸钠和2ml冰醋酸 大约同时,并在70℃下继续搅拌该溶液
    7. 更改此解决方案每3-4小时通过添加额外2毫升冰川 乙酸和4g亚氯酸钠至MQ H 2 O 2直到样品为止 白色。 此过程可能需要1-2天。
    8. 当样品是 白色(如纸),用600ml MQ H 2 O漂洗样品至少4次 超过3-4小时。 在70℃继续搅拌该溶液。 确保   没有乙酸气味。
    9. 将样品在60℃的干燥箱中干燥至恒重,通常过夜。
    10. 记录最终样品重量
    11. 将样品转移到燃烧管,用样品ID标记。

致谢

除了研究论文中写的内容外,我们不希望添加承认。

参考文献

  1. Leavitt,S.W。和Danzer,S.R。(1993)。 将小木材样品批量处理成全纤维素进行稳定碳同位素分析的方法 < em> Anal Chem 65(1):87-89
  2. Richardson,A.D.,Carbone,M.S.,Keenan,T.F.,Czimczik,C.I.,Hollinger,D.Y.,Murakami,P.,Schaberg,P.G.and Xu,X.(2013)。 温带森林树木的木材非结构碳水化合物的季节性动态和年龄。新的Phytol 197(3):850-861。
  3. Richter,A.,Wanek,W.,Werner,RA,Ghashghaie,J.,Jaggi,M.,Gessler,A.,Brugnoli,E.,Hettmann,E.,Gottlicher,SG,Salmon, C.,Kodama,N.,Nogues,S.,Soe,A.,Volders,F.,Sorgel,K.,Blochl,A.,Siegwolf,RT,Buchmann,N.and Gleixner, 制备用于分析碳同位素组成的植物材料的淀粉和可溶性糖:比较方法。 Rapid Commun Mass Spectrom 23(16):2476-2488。
  4. Wong,B.,Baggett,K。和Rye,A。(2003)。 成熟糖枫中储备和可溶性碳水化合物的季节性模式( Acer 81(8):780-788。
  5. Xu,X.,Trumbore,S.E.,Zheng,S.,Southon,J.R.,McDuffee,K.E.,Luttgen,M。和Liu,J.C。(2007)。 修改密封管 用于制备AMS石墨靶的锌还原方法:降低背景并获得高精度。物理研究中的核仪器和方法部分B:与材料和原子的光束相互作用259(1):320 -329。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Czimczik, C. I., Trumbore, S. E., Xu, X., Carbone, M. S. and Richardson, A. D. (2014). Extraction of Nonstructural Carbon and Cellulose from Wood for Radiocarbon Analysis. Bio-protocol 4(13): e1169. DOI: 10.21769/BioProtoc.1169.
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