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基于液相31P-NMRNMRNMR技术的沉积物中有机磷分析方法构建及其应用研究
Alternative TitleThe analytical method establishment based on solution 31P-NMR for sediment organic phosphorus analysis
张文强
Subtype博士
Thesis Advisor单保庆
2014-05
Degree Grantor中国科学院研究生院
Place of Conferral北京
Degree Discipline环境工程
Keyword沉积物 有机磷 液相 P-nmr Naoh-edta Sediment Organic Phosphorus 31 P-nmr Naoh-edta
Other Abstract      核磁共振(NMR)技术是现代科学中用途广泛的分析工具,它可以快速准确的用于土壤、动植物组织、沉积物等环境样品的分析。自然界中的磷仅有一种稳定同位素(31P),31P原子核质量数为奇数,原子核的自旋量子数为半整数,在外加磁场件下可以产生核磁共振现象。31P-NMR技术被应用于环境样品Po分析,但是 Po提取效率较低和核磁共振图谱质量不高等问题仍有待于进一步完善。本研究以 Fe/Al型沉积物为研究对象,通过逐步优化的方式,构建了包括样品保存方法、样品前处理方法、提取剂选择与配比、浸提比例与浸提时间优化等内的沉积物中 Po液相 31P-NMR分析方法,并在河流、湖泊等沉积物中   Po分析上进行了应用验证。利用构建的液相  31P-NMR分析方法,不仅揭示了沉积物中 Po形态特征,而且探索了沉积物中    Po的转化规律及驱动因素。期望为深入探讨沉积物中磷生物地球化学循环过程及影响机制提供技术支撑。主要研究内容和结果如下:
      (1)构建了一套适合于  Fe/Al型沉积物的有机磷液相   31P-NMR分析方法,实现了沉积物中有机磷化合物的精确定量分析。
      以 Fe/Al型沉积物为研究对象,分别从沉积物样品保存方式、沉积物的前处理方式、沉积物提取剂的选择和提取剂的配比、沉积物与提取液的浸提比例、浸提时间和提取液的浓缩方式等方面对液相  31P-NMR分析方法进行了优化。研究发现:①样品保存方法:沉积物取样后采用隔氧保护层法进行样品保存可以有效的降低沉积物中 Po的降解和转化;②样品前处理方式:沉积物带回实验室后,采用冷冻干燥的方法对样品进行处理,冷干法不仅可以避免沉积物中 Po的转化,而且可以批量处理样品;③提取剂选择与配比:从提取效率和图谱质量等分析来看,0.25 M NaOH+50  mM EDTA混合溶液可以得到较为理想的提取效果;④浸提时间与提取比例:研究结果表明,浸提时间以 16小时,浸提比例为  1:10(w:v)为最优配比;⑤提取液浓缩:浸提液冷冻干燥后研磨过筛,溶解到 0.6 mL D2O+0.1mL 10 M  NaOH混合溶液中,上机检测;⑥NMR分析与图谱解译:根据沉积物中磷含量,弛豫时间选择 3.6秒,扫描次数  20000次左右。获得图谱后,确定Po化合物峰值,对图谱进行积分计算有机磷化合物浓度。
      (2)利用构建的液相   31P-NMR技术,结合   H-J磷分级方法,在滏阳新河和滏阳河沉积物中检测到 Mono-P、PL-P和  DNA-P等有机磷化合物,并发现沿河流流向,有机磷化合物种类和含量呈衰减趋势。
      利用  31P-NMR在滏阳新河沉积物  NaOH-EDTA提取液中共检测到   6种磷化合物。磷酸单酯(Mono-P:8.96-29.58%)是生物物质磷(Biogenic-P)的主要组成部分。其他 Biogenic-P包括焦磷酸(Pyro-P:0.22-0.86%),DNA-P(0.75-2.03%),膦酸盐(Phon-P:0-1.57%)和磷脂(Lipids-P:0-2.66%)。沿河流流向,TP和Biogenic-P含量降低。磷分级过程中 NaOH-Po的种类和含量低于提取液   31P-NMR分析所得结果。滏阳河沉积物的分析表明:滏阳河沉积物累积了大量磷;不同形态磷含量为:H2SO4-P>NaOH-Pi>Res-P>NaOH-Po>KCl-P;利用  31P-NMR技术在NaOH-EDTA提取液中共检测到七种磷化合物。正磷酸盐(Ortho-P:45.16-92.35%)和磷酸单酯(Mono-P:  6.57-45.70%)是沉积物中磷的主要成分,其次是  Pyro-P(0.13-6.58%),DNA-P(0.33-3.96%),Phon-P(0-3.34%),Lipids-P(0-2.74%)和多聚磷酸盐(Poly-P:0-0.04%);基于磷分级和   31P-NMR分析结果,Pi中的35%为活性 Pi,主要包括 KCl-P和  NaOH-Pi(Fe-P和  Al-P),Biogenic-P占到沉积物中总磷的 24%。
      (3)利用构建的液相 31P-NMR分析方法,在巢湖沉积物中检测到包括Mono-P和  Diesters-P在内的多种有机磷化合物。在沉积物剖面上,有机磷化合物种类和含量呈递减趋势,且磷酸二酯类物质衰减速度要高于磷酸单酯类物质。
       液相 31P-NMR分析发现,巢湖表层沉积物主要包括 Ortho-P、Mono-P、Lipids-P、DNA-P、Pyro-P、Phon-P。Mono-P占  Po总量的  70%以上,为 Po的主要组成部分。巢湖东西部湖区磷形态及含量存在差异:巢湖表层沉积物中 TP平均含量为 686.93±254.94 mg∙kg-1,西部湖区沉积物中  TP高于东湖。沉积物中Po平均占 TP的  27.47±6.05%。与 TP不同,东部湖区的  Po百分含量大于西部湖区,其中 Mono-P和   DNA-P含量明显高于西部湖区。随着沉积柱深度的增加,磷的种类和含量都逐步降低,且西部湖区的降低速率要大于东部湖区。Po化合物的降低速率差异显著,磷酸二酯( Diesters-P)类化合物随深度增加含量和种类降低的速率要高于 Mono-P类化合物。
      (4)液相  31P-NMR方法结合  SMT分级方法揭示了湖泊沉积物中有机磷含量高于湿地和河流沉积物,河流 Mono-P的平均含量要高于湖泊与湿地;对于东部所有水系,引起流域沉积物中磷差别的主要是 Pi;在区域尺度上,沉积物 pH、LOI和  TC/TN都会影响液相      31P-NMR的分析效率。
      液相  31P-NMR分析方法可以科学有效的应用于区域尺度的磷化合物分析。结果显示,在区域尺度上,当提取效率低于 60%时,ER与  LOI呈正相关关系,当 ER高于  60%时,ER与  LOI无相关关系;ER与   pH存在负相关关系,即在酸性沉积物中 ER较高,而在碱性沉积物中,ER较低;当   TC/TN比值低于  20时,ER较高,但随着   TC/TN比值升高,ER逐步降低,表明沉积物有机质来源不同会影响  NaOH-EDTA的提取效率。在不同生态系统沉积物中,    Pi中的稳态磷(Ca-P)在河流沉积物中含量最高,而作为磷的重要储库,Po在湖泊沉积物中含量最高,可以占到全部沉积物中磷的 27%。在 Po形态上,稳定性较高的   Mono-P
在河流沉积物中含量高于湖泊和湿地沉积物,而活性较高的 Diesters-P主要存在于湖泊和湿地沉积物中。;       Nuclear    magnetic    resonance   (NMR)    is    a    powerful   analysis    tool    for
environmental  science, and  phosphorus-31  nuclear  magnetic  resonance  (31P-NMR) spectroscopy distinguishes different P groups based on specific resonance frequencies,
reflecting   the   chemical  environment   surrounding   the   P  nuclei.     31P-NMR   is  a
non-destructive,  non-invasive  technique for  identifying chemical  forms  that  has the advantage of enabling  multiple P compounds to be characterized  simultaneously with distinct binding properties. The extractant and extraction procedure  used are critical to
the  P recovery  and  the reliable  determination  of  individual P  components,  but  the physical and chemical properties of  soils and sediments vary widely,  so the analytical
method  has to be adjusted  to suit a particular   set of samples.
      The main  contents  and detail  results  are as follows:
      (1)The analytical  method  establishment based on solution  31P-NMR Fe/Al-rich river sediments  that were highly  polluted with P  were used  in tests to determine  the   optimum  preparation  techniques   for   measuring  Po  using   solution 31P-NMR. The optimum  pre-treatment, extraction time, sediment to  solution ratio and sodium hydroxide-ethylenediaminetetraacetic acid  (NaOH-EDTA) extractant  solution composition  were determined.  The  total  P(TP)  and  Po  recovery rates  were  higher from freeze-  and air-dried samples than  from fresh samples. An extraction time  of 16 h was adequate  for extracting Po, and a  shorter or longer extraction  time led to  lower recoveries of TP and Po,  or led to the degradation of  Po. An ideal P recovery rate  and good-quality  NMR   spectra  were   obtained  at   a  sediment:solution   ratio  of  1:10,showing that  this  ratio is  ideal  for extracting  Po. An  extractant solution  of 0.25  M NaOH and 50  mM EDTA was  found to be  more appropriate than either  NaOH on its own,  or a  more  concentrated  NaOH-EDTA mixture  for   31P-NMR  analysis,  as  this combination minimized  interference from paramagnetic  ions and was  appropriate for the detected range of  Po concentrations. The most appropriate preparation  method for Po analysis,  therefore,  was  to extract  the  freeze-dried and  ground  sediment  sample with a 0.25 M  NaOH and 50 mM  EDTA solution at a sediment:solution ratio of 1:10, for 16  h, by shaking.  As lyophilization  of the NaOH-EDTA extracts proved  to be  an optimal pre-concentration  method  for  Po analysis  in the  river  sediment,  the extract was lyophilized  as soon as possible,  and analyzed   by 31P-NMR.
      (2) Pyro-P, DNA-P,  Phon-P and Lipids-P were detected  by 31P-NMR.The  TP and biogenic-P decreased along  the direction  of flows.
      The  Fuyangxin  river  sediments  accumulated  significant  Pi  and  Po;   in  the  P fractionation, the rank  order of the  P fractions was as  follows: H2SO4-Pi>NaOH-Pi >Res-P>KCl-P>NaOH-Po. Six P compounds were detected in the  NaOH-EDTA extract by  31P-NMR.  Mono-P (8.96  - 29.58%)  was  the dominant  forms  of biogenic-P,  and other  smaller  fractions of  biogenic-P were  also  observed,  including  Pyro-P (0.22  - 0.86%), DNA-P (0.75 - 2.03%), Phon-P (0 - 1.57%), and Lipids-P (0 - 2.66%).  The TP and biogenic-P decreased  along the direction of  flows. The concentration  and species of Po in NaOH-Po  were lower than  found in  31P-NMR  analysis in this research.  Thus the use  of 0.25 M NaOH  and 50  mM EDTA extracts and  solution  31P-NMR analysis was a  more accurate method  for quantifying biogenic-P  in the river sediments  than P fractionation.
In  Fuyang  river  sediment,  the  rank  order  of  the  P  fractions  was  as  follows: H2SO4-P>NaOH-Pi>Res-P>NaOH-Po>KCl-P.  Seven P compounds  were detected  by the  31P-NMR  analysis. Ortho-P  (45.2 -  92.4%) and  Mono-P (6.6  - 45.7%)  were  the dominant  forms.   Smaller  amounts  of  Pyro-P  (0.1   -  6.6%),  DNA-P(0.3   -  3.9%), Phon-P(0 -  3.3%),  Lipids-P(0 -  2.7%) and  Poly-P(0  - 0.04%)  were observed  in  the sediments.  Results of P  fractionation and   31P-NMR  analysis showed  that 35%  of Pi
was labile P,  including KCl-P and NaOH-Pi. Biogenic-P accounted for 24% of P in the sediments.
      (3) Mono-P was  the majority  Po compounds in the  Chaohu Lake  sediments. For sediment  cores, the  contents  of Lipids-P  and DNA-P declined  more  rapidly than  that   of  Mono-P  and   Pyro-P  as   sediment  depth   increased,  with   these compounds  primarily  occurring   in the top 10 cm of sediment.
      For the surface sediments, TP in the  NaOH-EDTA extracts was dominated by Pi, with higher levels  being observed in the heavily eutrophic  western lake (79.1 ± 1.7%)than in the  eastern lake (68.1  ± 2.4%).  These findings were opposite  to those  for Po. Pi  (Ortho-P  and  Pyro-P)   and  Po  (Phon-P,  Mono-P,   Lipids-P  and  DNA-P)  were detected in  the NaOH-EDTA extracts of the  sediments by  31P-NMR.  The majority of Po consisted of  Mono-P (80.2  ± 2.7%). For  sediment cores, the  contents of  Lipids-P and DNA-P declined  more rapidly than that of  Mono-P and Pyro-P as sediment  depth increased,  with these  compounds  primarily  occurring  in the  top 10 cm of sediment.
      (4) The  concentation of Mono-P  was higher in  the  river sediment than lakeand  wetland,  but the  Diesters-P  mainly  existed  in lake  and  wetland sediment.
      LOI, pH  and TC/TN effected the  ER  of  31P-NMR in the surface  sediment. Surface sediments  in different ecosystems (lake, river, wetland)  in Eastern China were  extracted  with NaOH-EDTA,  and  the extracts  were  analyzed  by  31P-NMR  to reveal the P species distribution.  Positive relationship between  ER and LOI under the condition  of  ER  under   60%  (R2=0.10,  p<0.05).  The   ER  and  the   LOI  show  no correlation  when  the  ER  exceed  60%.  Negative  relationship   was  found  between EDTA-NaOH extraction rate  and the  pH of  the sediments  (R2=0.38,  p<0.01), which means the  ER  was increased  with  the decrease  of the  pH of  the  sediment.  The ER show negative exponent with the  TC/TN(R2=0.65, p<0.01), which means the origin of OM affect  the NaOH-EDTA extraction  efficiency  in the sediment.The  concentration of Ca-P  was higher  in  river sediment  than that  in lake  and  wetland sediment.  As P pool, the Po was  dominant in lake sediment, which was accounted  for 27% of TP. The concentation of Mono-P  was higher in  the river  sediment than lake and  wetland, but the Diesters-P  mainly  existed  in lake  and wetland  sediment.
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/15644
Collection环境水质学国家重点实验室
Recommended Citation
GB/T 7714
张文强. 基于液相31P-NMRNMRNMR技术的沉积物中有机磷分析方法构建及其应用研究[D]. 北京. 中国科学院研究生院,2014.
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