Enhanced degradation of gaseous benzene under vacuum ultraviolet

通过胺改性活性炭加强汞离子的吸附作用摘要：汞是在水中和沉淀物中发现的最有毒性的金属之一。

在努力建立一个有效的吸附作用用于水汞去除的过程中，活性炭（AC）与氨基封端的有机硅被修改（3-氨基丙基，APTES）。

氨基丙基与已修改的活性炭（MAC）的表面性质在扫描电子显微镜（SEM-EDS）和能量色散光谱法的结合下具有傅立叶变换红外光谱（FT-IR）和电位的特征。

对溶剂，APTES浓度，反应时间和温度对表面改性的影响进行了评价。

水汞吸附动力学和能力也被确定。

结果证明强汞结合胺配体通过碳表面官能团之间（-COOH，-COH）硅烷醇反应和APTES分子有效地引入到了活性碳表面。

在零电荷（PZC）下，改性将pH从9.6降到4.54，更有利于阳离子吸附。

已修改的活性碳呈现出了Hg（II）吸附作用更快的速率并且与未修改的活性碳相比，已修改活性碳的吸附能力是未修改活性碳的两倍以上。

关键词：活性炭；APTES；表面改性；汞吸附1.介绍汞（Hg）是在环境中的毒性极高的金属之一，它能引起人类不可逆的神经损伤。

世界卫生组织（WHO）建议的最大汞摄入量为0.3微克每周。

在我们的饮用水中可接受的最大混合浓度为1微克每升。

美国环保局允许的废水总汞的排放限值为10毫克每升而饮用水的极限为2毫克每升。

日本环境部建立了更加严格的限制在0.5微克每升到5微克每升之间。

因此，除去汞来控制在一个可接受浓度是饮用水中和废水处理中的一个挑战。

传统的方法已经应用于去除和分离水汞离子，包括硫化物沉淀，离子交换，明矾和铁凝血。

这些方法中，常常具有的去除效率很低。

所以需要一个额外的处理过程，但是拆除过程可能产生更多的污泥。

然而，当汞含量低的时候去除是无效的的。

因此，有一种强烈的动机来开发新的吸附剂能够具有高的效率和选择性的从含水介质中去除汞离子。

活性炭（AC）是一种常见的吸附剂，通常用于水性金属的去除。

因为其优良的多孔结构，特殊的表面特性，可重用性，以及最低的成本和环境友好的性质。

Unit 34 Oil Pollution Responding and Damage Control油污染应急和损害控制1. Fate and behavior of oil in the marine environment石油在海洋环境中的演变和特性Complex processes of oil transformation in the marine environment start developing from the first seconds of oil’s contact with seawater. The progression, duration, and result of these transformations depend on the properties and composition of the oil itself, parameters of the actual oil spill, and environmental conditions. The main characteristics of oil transformations are their dynamism, especially at the first stages, and the close interaction of physical, chemical, and biological mechanisms of dispersion and degradation intoxicated living organism, a marine ecosystem destroys, metabolizes, and deposits the excessive amounts of hydrocarbons, transforming them into more common and safer substances.自从石油及其制品与海洋接触的那一该开始，在海洋环境中转化石油的复杂性就在不断增长。

吹扫捕集GC-MS法测定饮用水中挥发性有机物—苯乙烯摘要：吹扫捕集GC-MS气相色谱-质谱法测定水中苯乙烯,通过对吹扫捕集、GC-MS等条件的优化，分析了吹扫时间、吹扫温度对吹扫捕集效率的影响。

分析结果表明，在此条件下，苯乙烯的线性相关系数为0.9974，加标回收率为78.9%~115.6%，相对标准偏差为2.53~2.58%，该方法具有较好的线性关系，操作简单，灵敏度高，准确性及重现性好；方法检出限为0.5 ug/L，远低于《生活饮用水卫生标准》（GB5749-2006）中限值20ug/L。

关键词：GC-MS；苯乙烯；生活饮用水苯乙烯属于挥发性有机物，地表水和饮用水中的苯乙烯一般来自于化工企业排放的废水、废气。

另外，水中的腐植酸、富里酸及藻类代谢产物等经过加氯消毒后也会产生一些，会经呼吸、皮肤接触和饮水进入人体，如果浓度过大将对人体的健康造成危害，《地表水环境质量标准》和《生活饮用水卫生标准》中均对苯乙烯提出了限值要求[1-2]，目前我国对地表水和生活饮用水中苯乙烯的测定采用的是静态顶空气相色谱法[3]，该法的灵敏度低，精密度差，本文采用的吹扫捕集法主要有无需使用有机溶剂，对环境不造成二次污染，而且取样量少、富集效率高、受机体干扰小、操作简便快捷等优点[4-5]。

质谱的选择离子监测（SIM）可以消除于目标化合物保留时间接近的其他化合物干扰，对于成分较复杂的样品可以得到好的测定结果[6]。

1 材料与方法1.1 仪器与试剂7890A-5975C气相-质谱联用仪；P&T9800和AQUATEK100;苯乙烯标准溶液：中国计量科学研究院，质量浓度为100ug/ml；氯化钠（优级纯），在马弗炉中于450℃条件下烘烤2h，然后置于干燥器中备用；微量注射器，规格为1ul、10ul和100ul等。

1.2试验方法1.2.1 仪器条件谱柱：DB-624毛细管色谱柱（60m*0.250mm*1.40um）。

Zeolite-encapsulated Ru(III)tetrahydro-Schiffbase complex:An efficient heterogeneous catalyst for the hydrogenation ofbenzene under mild conditionsPing Chen,Binbin Fan,Minggang Song,Chun Jin,Jinghong Ma,Ruifeng Li*Key Laboratory of Coal Science and Technology,MOE,Institute of Special Chemicals,Taiyuan University of Technology,79West Yinze Street,Taiyuan 030024,PR ChinaReceived 7January 2006;received in revised form 7April 2006;accepted 7April 2006Available online 18April 2006AbstractA series of Ru(III)tetrahydro-Schiffbase complexes (denoted as Ru[H 4]-Schiffbase with Schiffbase =salen,salpn and salcn,see Scheme 1)were encapsulated in the supercages of zeolite Y by flexible ligand method.The prepared catalysts were characterized by X-ray diffraction,diffuse reflectance UV–Vis spectroscopy,Infrared spectroscopy,elemental analysis,as well as N 2adsorption tech-niques.It was shown that upon encapsulation in zeolite Y,Ru(III)tetrahydro-Schiffbase complexes exhibited higher activity for the hydrogenation of benzene than the corresponding Ru(III)-Schiffbase complexes.This indicates that hydrogenation of the C @N bond of the Schiffbase ligands led to a modification of the coordination environment of the central Ru(III)cations.The stability of the prepared catalysts has also been confirmed against leaching of the complex molecule from the zeolite cavities,as revealed by the result that no loss of catalytic activity was observed within three successive runs with regeneration.Ó2006Elsevier B.V.All rights reserved.Keywords:Benzene hydrogenation;Ru(III)tetrahydro-Schiffbase complex;Encapsulation;Zeolite Y1.IntroductionTransition metal complexes have been extensively used for homogeneous hydrogenation of organic substrates such as benzene due to their high selectivity under mild reaction conditions [1,2].However,the difficult recovery and recy-cling of the catalysts limit their reuse.Therefore,increasing demands have been generated for the preparation of effec-tive heterogeneous catalysts.In this context,immobiliza-tion of metal complex is an attractive strategy.Up to date,several methods,such as covalent bonding of ligands with host materials,entrapment or occlusion of complex molecule in polymer matrices or porous inorganic materi-als by steric hindrance,and adsorption of the complexes on the support via ionic interaction,have been examinedfor the immobilization of transition metal complexes [3].Among them,the physical encapsulation of transition metal complex molecules into the supercages of zeolites gains incomparable advantages for homogeneous and con-ventional heterogeneous catalysts [4–7].Upon encapsula-tion in the cavities of the zeolites with FAU and EMT topological structures,palladium and rhodium salen com-plexes are highly active for the selective hydrogenation of 1,5-cyclooctadiene to cyclooctene [8],particularly chiral palladium salen complex makes it possible of enantioselec-tive hydrogenation of unsaturated organic compounds such as 3-methyl-2-cyclohexenone [9].In addition,palla-dium and nickel salen complexes grafted on mesostruc-tured silicates and delaminated ITQ zeolites also show good catalytic performance in the hydrogenation of imines [10],while Ru(III)-Schiffbase complex anchored on poly-mer exhibits considerably improved catalytic efficiency for the hydrogenation of styrene [11].The catalytic activity1566-7367/$-see front matter Ó2006Elsevier B.V.All rights reserved.doi:10.1016/j.catcom.2006.04.003*Corresponding author.Tel./fax:+863516010112.E-mail address:rfli@ (R.Li)./locate/catcomCatalysis Communications 7(2006)969–973of transition metal Schiffbase complexes in a given process is highly dependent on the environment of metal center and their conformationalflexibility[12].Compared to Schiffbase ligands,the corresponding tetrahydro-Schiffbase ligands possess stronger N-basicity and higherflexibility owing to the hydrogenation of C@N bond[13].It was found that zeolite-encapsulated Cu(II)tetrahydro-salen complex is more active than the encapsulated Cu(II)salen for the oxidation of cycloalkane in the presence of H2O2 [14].To the best of our knowledge,the hydrogenation per-formance of transition metal tetrahydro-salen complex has not been reported yet.Here,we report the preparation of zeolite Y encapsulated Ru(III)tetrahydro-Schiffbase com-plexes by usingflexible ligand method and their catalytic property in an industrially important reaction of benzene hydrogenation(see Scheme1).2.Experimental2.1.Preparation of ligandsSchiffbase L(L=salen,salph and salcn)was prepared by following the procedures reported in Ref.[13,15]. Ligand[H4]L was prepared with the following method [16]:0.01mol Schiffbase L was dissolved in60ml metha-nol,followed by the addition of0.011mol NaBH4at ambi-ent temperature.After2h of stirring,the solvent was removed by distilling under vacuum conditions.The solid product was washed with distilled water and re-crystallized from ethanol.The purity of the ligands was confirmed by IR and1H NMR before coordination to Ru(III)cations.2.2.Preparation of zeolite Y encapsulated Ru(III) complexesZeolite Y was ion-exchanged with RuCl3aqueous solution(0.005mol/l,Liquid/Solid=20ml/g)at room temperature for24h,and then washed with deionized water and dried at100°C overnight.The obtained Ru-Y was mixed with an excessive amount of tetrahydro-Schiffbase ligands(n ligand/n Ru=3:1mol/mol),followed by heat-ing in a sealed glass ampoule for24h under vacuum conditions at temperatures of150,130and130°C for Ru[H4]-salen,Ru[H4]-salpn and Ru[H4]-salcn,respectively. The resultant materials were soxhlet-extracted with acetone to remove the complex molecules on the external surface and the free ligands.The extracted samples were dried at 373K for24h under vacuum conditions.2.3.Characterization of zeolite Y encapsulated Ru(III) complexesRu content was determined by an inductively coupled plasma-atomic emission spectrometer(TJA Atom Scan 16).X-ray power diffraction(XRD)patterns were recorded on a Rigaku Dmax X-ray diffractometer(Ni-filtered, CuK a radiation).Infrared spectra(IR)were measured on a FTIR spectrometer by using the conventional KBr pellet method.Diffuse reflectance(DR)UV–Vis spectra in the range of200–800nm were measured against a halon white reference standard by a Perkin–Elmer Lambda Bio40spec-trophotometer equipped with an integration sphere.The Brunauer–Emmett–Teller(BET)surface area was mea-sured on a NOVA1200instrument.Before the measure-ment,the sample was evacuated at200°C for12h.2.4.Catalytic testHydrogenation of benzene was carried out in a Parr4842 stainless steel batch reactor,which is equipped with a gauge,a thermocouple,an internal mechanic stirring sys-tem,gas inlet valves,liquid sampling valves and a temper-ature-programmed electric heater.In a typical batch,0.36g catalyst and30.0ml(26.4g)benzene were employed.To remove the air,the closed reactor was purged with hydro-gen for three times.When the temperature reached60°C, hydrogen was charged into the reactor.The stirring speed was maintained at a value as high as600rpm in order to eliminate the diffusion problem.The product was analyzed by a GC-9A gas chromatography equipped with aflame ionization detector.3.Results and discussion3.1.Characterization of catalystsNo remarkable difference was observed from the XRD patterns of the zeolitic host before and after complexation as well as the further soxhlet-extraction,indicating that the zeolite framework was not significantly affected by the for-mation of complexes.The BET surface areas of different Ru[H4]L-Y are given in Table1.Clearly,inclusion of Ru(III)Schiffbase com-plex considerably reduced the adsorption capacity and the surface area of the zeolite host.This is indicative of the presence of Ru(III)complexes in the zeolite cavities rather than on the external surface.The surface area,as]970P.Chen et al./Catalysis Communications7(2006)969–973expected,decreased with increasing molecular dimensions of ligands.The surface areas of Ru[H 4]salen-Y,Ru[H 4]-salpn-Y and Ru[H 4]salcn-Y were 500,465and 484m 2/g,respectively,in contrast to 570m 2/g of Ru-Y.Table 1also shows the Ru content in different samples.Although all samples were prepared from the same Ru(III)-exchanged zeolite Y matrix,their Ru content were obviously different,and decreased with the increase in the complex molecule size.Irrespective of its coincidence with our previous results on the encapsulation of Ru(III)-Schiffbase com-plexes in zeolite Y [17],it is different from the inclusion of Mn(III)salen complex in zeolite X and zeolite Y by zeo-lite synthesis method and template synthesis method,respectively [18,19].It was argued that the increase in the complex size could enhance its physical entrapment in zeo-lite supercages,resulting in an increase in the encapsulation efficiency.The contrary results might arise from the differ-ent preparation methods.Concerning the flexible ligand method,an increase in the ligand size would cause its diffu-sion into the zeolite channels difficult,and consequently hindering its access to Ru(III)cations and the formation of complex molecules in the supercages of zeolite Y.IR spectra of Ru-Y and Ru[H 4]L-Y are shown in Fig.1.Although in the low-wavenumber region the bands for the encapsulated complexes were overlapped by the framework vibration of the zeolite matrix,in the range from 1200to 1600cm À1,the bands due to C–N,C @C and atomic ring vibrations could be clearly distinguished for the zeolite-encapsulated Ru(III)tetrohydro-Schiffbase complex sam-ples,while these bands were not observed for the Ru-Y.The encapsulation of Ru(III)tetrohydro-Schiffbase com-plexes in zeolite Y was further confirmed by DR UV–Vis spectroscopy.It is evident in Fig.2that after complexation and further soxhlet-extraction,a relatively intense absorp-tion band could still be seen around 325nm in the DR UV–Vis spectra of all zeolite Y encapsulated Ru(III)tetrohydro-Schiffbase samples.As we know,this band is attributed to ligand-to-metal charge transfer (CT),giving a strong evidence for the formation of Ru(III)tetrahydro-Schiffbase complex molecules inside the cavity of zeolite Y.3.2.Hydrogenation of benzeneTable 2compares the catalytic results for the hydroge-nation of benzene over Ru-Y,RuL-Y and Ru[H 4]L-Y sam-ples.RuL-Y was prepared by the same method as that used for Ru[H 4]L-Y [17].Under the same reaction conditions,RuL-Y and Ru[H 4]L-Y catalysts gave much higher ben-zene conversion than Ru-Y despite that the product was only cyclohexane for all the catalysts.This indicates that the electronic environment of central Ru(III)ions drasti-cally influences the catalytic performance.This also gives another piece of evidence for the successful complexation of Ru(III)ions with the tetrahydro-Schiffbase ligands inside the zeolitic host.Nevertheless,it is worth noting that all Ru[H 4]L-Y catalysts showed higher activity than the corresponding RuL-Y materials,as revealed by the fact that the Ru[H 4]L-Y catalysts gave a TOF being 2–4times as high as that obtained for the RuL-Y samples.At a reac-tion time of 2h,the benzene conversion reached 75.3%with a TOF of 4361over the Ru[H 4]salen-Y catalyst.In contrast,the Rusalen-Y sample only gave a benzene con-version of 19.7%,and thus a TOF of 1155,although it con-tained a comparable number of complex molecules.A further increase in the reaction time to 3h could result in a benzene conversion of 98.3%over the Ru[H 4]salen-YTable 1Ru content and surface area of Ru-Y and Ru[H 4]L-Y samples Catalyst Ru content (wt%)Surface area (m 2/g)Ru-Y1.00571Ru[H 4]salen-Y 0.82500Ru[H 4]salpn-Y 0.57465Ru[H 4]salcn-Y0.44484P.Chen et al./Catalysis Communications 7(2006)969–973971catalyst.Homogeneous catalysis and Ru(III)cations play a minor role since at the same reaction conditions neat Rusalen and Ru-Y with a comparable Ru content to Rusalen-Y both showed a conversion of about 2.5%.It was suggested that hydride complexes as intermediate species or starting materials play a key role in most hydro-genation reactions [20].The required dihydrogen molecule cleavage is proposed to occur as a result of the interaction of a filled metal d orbit with the empty sigma antibonding molecular orbit of H 2,which could weaken the H–H bond.In addition,an electron-rich atmosphere around the metal atom may also facilitate the breaking of the H–H bond [20].Compared to the Schiffbase ligands,the hydrogenated tetrahydro-Schiffbase ligands have stronger N-basicity and more flexibility because of the hydrogenation of C @N bond [13].This makes the metal center electron-rich,pro-moting the appropriate overlap of the filled metal d orbit with the empty sigma antibonding molecular orbit of H 2,and hence favoring the cleavage of H–H bond,which is a crucial step in the hydrogenation reaction.It is noteworthy that although three types of Ru[H 4]L-Y catalysts all showed higher activity than the corresponding RuL-Y ana-logues,the degree substantially decreased with increasing ligand size.The conversion obtained over the Ru[H 4]sa-len-Y at the reaction time of 2h was about 3.8times as high as that obtained on the Rusalen-Y.In contrast,for the Ru[H 4]salcn-Y catalyst,it was only about 1.7times ashigh as that of its corresponding Schiffbase analogue.This indicates that the effect of the C @N bond hydrogenation on the metal center weakens with the ligand size.Effect of the reaction temperature on the hydrogenation of benzene over the Ru[H 4]salen-Y catalyst is illustrated in Fig.3.As expected,the benzene conversion was highly dependent on the reaction temperature.It sharply increased with increasing reaction temperature when the temperature was lower than 60°C.When the reaction was carried out at 60°C for 2h,the benzene conversion was 75.3%in contrast to 19.4%at 50°C.Regardless of this,a further increase in the reaction temperature to 70°C had no such an apparent effect.The influence of hydrogen pressure was investigated at 60°C between 2.0and 4.0MPa.The relationship between the hydrogen pressure and the benzene conversion is shown in Fig.4.Obviously,the benzene conversion obtained at 3.0MPa was higher than those obtained at 2.0andTable 2Catalytic results of different catalysts in benzene hydrogenation a Catalyst Ru content (wt%)Reaction time (h)Conversion (%)TOF b(mol/(mol Æh À1))Ru-Y0.872 2.614748.7611.5Rusalen c 22.5141Rusalen-Y0.81219.71115448.5675.2Ru[H 4]salen-Y 0.82275.34361398.3Rusalpn-Y0.5528.3717420.9644.9Ru[H 4]salpn-Y 0.57223.61967452.0672.9Rusalcn-Y 0.432 6.0663416.3632.3Ru[H 4]salcn-Y 0.44210.21101423.5648.1aReaction conditions:0.36g catalyst,26.4g benzene,60°C,3.0MPaH 2.bThe amount of the converted benzene per mole of Ru(III)complexes per hour.c0.0135g Rusalen,26.4g benzene,60°C,3.0MPa H 2.972P.Chen et al./Catalysis Communications 7(2006)969–9734.0MPa,showing that the optimum hydrogen pressure was 3.0MPa.This is in accordance with the results reported by Wang and co-workers for the partial hydrogenation of benzene to cyclohexene on a Ru–Zn/m-ZrO2catalyst[21]. This was accounted for by assuming a competitive adsorp-tion between hydrogen and benzene on the same active sites on the basis of the slow adsorption theory and the existence of a stagnant waterfilm around the catalysts. However,the effect of hydrogen pressure depends on the reaction system and the catalytic mechanism.Therefore, the reason is not unambiguous yet for the moment,and further studies are in progress.The stability and recycling possibility of the prepared Ru[H4]salen-Y catalyst in benzene hydrogenation were fur-ther investigated.After one reaction run,the catalyst was recovered by the centrifugation of hot reaction mixture so as to avoid the readsorption of possibly leached complex molecules,and further washed with ethanol and dried at 90°C under vacuum conditions.The dried sample was then used for the next run under the same reaction conditions. Fig.5shows the catalytic results of three successive recy-cles.It is clear that the obtained benzene conversion was almost the same within three reaction runs,verifying that Ru[H4]salen-Y is highly stable and can be reused.This is further confirmed by the following experiment.When the reaction was performed for2h,the catalyst was separated from the reaction mixture,and left the reaction for another 2h.It was found that the benzene conversion remained at 75%without a further increase.In contrast,if the catalyst was not separated,the benzene in the reaction mixture was completely converted.This proves that the reaction is cat-alyzed heterogeneously since homogeneous catalysis,as above discussed,plays a negligible role.This is also sup-ported by the ICP analysis that a trace amount of Ru was observed in the reaction liquid.Thus,the possibility of leaching of the complex molecules from the zeolite cav-ities could be excluded.Otherwise,the benzene conversion would decrease because of a reduction in active sites of the encapsulated complex molecules.4.ConclusionsA series of Ru(III)tetrahydro-Schiffbase complexes have been encapsulated in zeolite Y,and show much higher activity than the corresponding Ru(III)Schiffbase ana-logues for the hydrogenation of benzene under mild condi-tions.This results from a modification of the coordination environment of central Ru(III)cations by the hydrogena-tion of C@N bond,and consequently a more H2coordina-tion activation.The prepared catalysts are also highly stable and reusable,as verified by the unchanged activity within three successive reaction runs and no further increase in the benzene conversion with increasing reaction time after removing the catalyst.AcknowledgementThis work is supported by the National Science Founda-tion of China(Nos.20443004and50472083). 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Published Ahead of Print 5 January 2007. 10.1128/AEM.02236-06.2007, 73(5):1474. DOI:Appl. Environ. Microbiol. Henriksen, Carsten S. Jacobsen and Ole Andersen Anders R. Johnsen, Stine Schmidt, Trine K. Hybholt, SidselMycobacteriaBioremediated Soil Dominated by on PAH Degradation when Priming with of a PAH-Polluted Soil but Marginal Effect Hydrocarbon (PAH)-Degrading Community Strong Impact on the Polycyclic Aromatic /content/73/5/1474Updated information and services can be found at: These include:REFERENCES/content/73/5/1474#ref-list-1This article cites 27 articles, 9 of which can be accessed free at:CONTENT ALERTSmore»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new /site/misc/reprints.xhtml Information about commercial reprint orders: /site/subscriptions/To subscribe to to another ASM Journal go to: on May 8, 2012 by SOUTH CHINA UNIVERSITY OF TECHNOLOGY/Downloaded fromA PPLIED AND E NVIRONMENTAL M ICROBIOLOGY,Mar.2007,p.1474–1480Vol.73,No.5 0099-2240/07/$08.00ϩ0doi:10.1128/AEM.02236-06Copyright©2007,American Society for Microbiology.All Rights Reserved.Strong Impact on the Polycyclic Aromatic Hydrocarbon(PAH)-Degrading Community of a PAH-Polluted Soil but Marginal Effect on PAH Degradation when Priming with Bioremediated SoilDominated by MycobacteriaᰔAnders R.Johnsen,1*Stine Schmidt,1,2Trine K.Hybholt,1,2Sidsel Henriksen,1Carsten S.Jacobsen,1and Ole Andersen2Geological Survey of Denmark and Greenland,Department of Geochemistry,Øster Voldgade10,DK-1350Copenhagen K,Denmark,1and Department of Life Sciences and Chemistry,Roskilde University,Postboks260,DK-4000Roskilde,Denmark2Received22September2006/Accepted21December2006Bioaugmentation of soil polluted with polycyclic aromatic hydrocarbons(PAHs)is often disappointingbecause of the low survival rate and low activity of the introduced degrader bacteria.We therefore investigatedthe possibility of priming PAH degradation in soil by adding2%of bioremediated soil with a high capacity forPAH degradation.The culturable PAH-degrading community of the bioremediated primer soil was dominatedby Mycobacterium spp.A microcosm containing pristine soil artificially polluted with PAHs and primed withbioremediated soil showed a fast,100-to1,000-fold increase in numbers of culturable phenanthrene-,pyrene-,andfluoranthene degraders and a160-fold increase in copy numbers of the mycobacterial PAH dioxygenasegene pdo1.A nonpolluted microcosm primed with bioremediated soil showed a high rate of survival of theintroduced degrader community during the112days of incubation.A nonprimed control microcosm containingpristine soil artificially polluted with PAHs showed only small increases in the numbers of culturable PAHdegraders and no pdo1genes.Initial PAH degradation rates were highest in the primed microcosm,but later,the degradation rates were comparable in primed and nonprimed soil.Thus,the proliferation and persistenceof the introduced,soil-adapted degraders had only a marginal effect on PAH degradation.Given the small effectof priming with bioremediated soil and the likely presence of PAH degraders in almost all PAH-contaminatedsoils,it seems questionable to prime PAH-contaminated soil with bioremediated soil as a means of large-scalesoil bioremediation.Various studies have investigated the possibility of bioaug-mentation of polycyclic aromatic hydrocarbon(PAH)-polluted soil with PAH-degrading strains or consortia.In vitro experi-ments often show improved degradation of soil PAHs when PAH-degrading lab strains or consortia are added.However, scaling up of this type of experiment is often highly disappoint-ing.For instance,a recent microcosm study of bacterial com-munity dynamics and PAH degradation during bioremediation and bioaugmentation of creosote-contaminated soil did not detect any effect of the introduced degrader consortium either on community profiles or on PAH degradation(26).Likewise, a pilot-scale study using a PAH-degrading Mycobacterium strain and an enrichment culture did not result in increased numbers of pyrene degraders or increased PAH degradation compared to creosote-polluted soil simply treated with N,P, and K(15).The poor results may be caused by the focus on the meta-bolic capacity of the introduced strains.However,bioaugmen-tation should be more than only the addition of a metabolic function;the introduced degrader cells should also show high persistence and be able to compete with the indigenous bac-teria for space and resources(13).The natural PAH-degrading communities of bioremediated soil with high PAH degradation potential may have these prop-erties.These degrader cells have proven to be efficient in surviving and obtaining PAHs under natural conditions and may therefore constitute an appropriate inoculum for bioaug-mentation.The aim of our study was to use such degrader-enriched, bioremediated soil to prime“freshly polluted”pristine soil simply by adding bioremediated clay soil suspended in water and then to follow PAH degradation and the degrader com-munities in microcosms over time.MATERIALS AND METHODSChemicals and reagents.All chemicals were of analytical grade.[9-14C]Phenan-threne(Ͼ98%purity)and[3-14C]fluoranthene(Ͼ95%purity)were obtained from Sigma-Aldrich(Copenhagen,Denmark).[4,5,9,10-14C]pyrene(Ͼ95%purity)was obtained from Amersham Biosciences(Hillerød,Denmark).The cell proliferation reagent WST-1{4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-ben-zene disulfonate}was obtained from Roche Molecular Biochemicals(Mannheim, Germany).Soils.Sandy,pristine soil was sampled from an A-horizon at Dronningmølle (Sealand,Denmark)in March2004.The soil was sieved to4mm and stored for 2days at15°C.Bioremediated clay soil from a previous project(6)was used as a PAH degrader inoculum.This soil originated from the Ringe Asphalt and Tar Production Plant(Ringe,Denmark)and was excavated from a depth of2to3m, followed by homogenization and fertilization with NPK in piles of170kg for4*Corresponding author.Mailing address:Geological Survey of Denmark and Greenland,Department of Geochemistry,Øster Vold-gade10,DK-1350Copenhagen K,Denmark.Phone:4538142328.E-mail:arj@geus.dk.ᰔPublished ahead of print on5January2007.1474 on May 8, 2012 by SOUTH CHINA UNIVERSITY OF TECHNOLOGY / Downloaded fromyears(6).One sample of the bioremediated soil was used for characterization of the degrader community(14C-PAH mineralization,most probable numbers [MPN],and isolation),and another was used in the microcosms. Microcosms.Phenanthrene,fluoranthene,and pyrene were selected as model compounds because they are found in high concentrations in soil polluted with pyrogenic PAHs.The contents of the microcosms are shown in Table1.Sub-samples of PAH-contaminated soil were prepared by adding phenanthrene,fluoranthene,and pyrene in acetone solution to pristine soil(200mg kgϪ1).The acetone was evaporated at15°C for24h.These subsamples were then mixed with pristine soil to givefinal PAH concentrations of50mg kgϪ1.Control soil contained acetone only.Four different microcosms were prepared in4.0-liter stainless-steel containers containing1.5kg of soil.The water content was ad-justed to21%(wt/wt).The bioremediated clay soil(inoculum)was mixed with this water,whereby the suspendable fraction was added to the microcosms.A control microcosm used to account for abiotic processes was poisoned by the addition of sodium azide to afinal porewater concentration of5g literϪ1.The microcosms were sealed with polypropylenefilm(clingfilm)because thisfilm is permeable to oxygen.Subsamples of30g each for chemical and microbiological analyses were taken at increasing time intervals after homogenization of the microcosms.Soil PAH content.Soil samples(5g)were thoroughly homogenized and were spiked with eight deuterated PAHs as internal standards.The frozen samples (Ϫ18°C)were freeze-dried.All soil samples were then microwave extracted for 25min at110°C in35ml acetone:dichloromethane:water(3:3:1)using a com-mercial microwave system designed for extraction of organic compounds.The extracts were dried with Na2SO4and concentrated by evaporation of the solvent under a stream of nitrogen.The extracts were loaded onto SiO2columns and eluted with dichloromethane.All extracts were spiked with10-[D]-chrysene as a PAH recovery pounds were identified and quantified with a Finni-gan TRACE DSQ single-quadrupole gas chromatographer/mass spectrometer in the selected ion mode.PAH degrader MPN.The MPN of phenanthrene,pyrene,orfluoranthene degraders in the soil was determined for the bioremediated soil used as an inoculum and for subsamples from the microcosms using a microplate method with a fourfold dilution series,four parallel rows,and4weeks of incubation as described previously(7).In this assay,growth is determined by respiratory reduction of the tetrazolium compound WST-1in active cells.The initial micro-cosm MPNs were calculated from the MPNs of the soils used.The optimum incubation time for the MPN plates was determined for the bioremediated soil (inoculum)by initially incubating plates for2,3,4,and5weeks before deter-mination of growth-positive wells.Quantification of PAH dioxygenase genes by real-time q-PCR.Whole-com-munity DNA was extracted from0.5-g samples of soil by application of a bead-beating procedure using the FastDNA spin kit for soil(BIO101,Vista,CA)as described by de Lipthay et al.(5).Conventional PCR detection of nah-like naphthalene dioxygenase genes(e.g.,nahAc and pahA)was performed using the NAH primers described by Baldwin et al.(2).Primers described by Johnsen et al.(9)were used for the detection of pdo1PAH dioxygenase genes(17).Quanti-tative real-time PCR(q-PCR)was performed using a Bio-Rad iCycler(Bio-Rad, Hercules,CA)as described previously(8).The numbers of bacterial cells con-taining pdo1and nah-like genes in the soil samples were determined from standard curves of DNA derived from Pseudomonas putida OUS82(pahAc)(2, 24)and Mycobacterium sp.strain6PY1(pdo1)(17).All standard curves were made from DNA extracts corresponding to3ϫ102to3ϫ107CFU per PCR. Following all q-PCRs,melting-curve analysis and conventional agarose gel elec-trophoresis were done to confirm the validity of the PCR products.To estimate the extraction efficiency of template DNA in soil,10␮l of dilutions of Mycobac-terium sp.strain6PY1cells were mixed with0.5-g samples of the pristine soil or with0.5ml water,followed by DNA extraction and q-PCR detection of pdo1. Potential14C-PAH mineralization of inoculum soil.Ottawa sand was sterilized for1h at200°C.Sterile Ottawa sand(1.5g)was placed in10-ml sterile glass scintillation vials.One hundred microliters of14C-labeled PAH in acetone so-lution(60␮g mlϪ1;Ϸ2,500Bq mlϪ1)was added,and the acetone was evaporated under a stream of nitrogen.Three grams(dry weight)of fresh soil was added to the vials and mixed with the14C-PAH-contaminated sand to give afinal PAH addition of2␮g gϪ1soil.Two subsamples were spiked for each PAH.Filter paper was placed at the bottom of sterile250-ml Blue Capflasks,and2ml of NaH2PO4buffer(0.1M,pHϷ4.8)was added to keep the air water saturated. The vials and test tubes containing2ml NaOH(1M)to trap14CO2were placed in theflasks,and theflasks were closed with Teflon-lined caps and incubated at 15°C in the dark.The NaOH was replaced at increasing time intervals.The trapped amounts of14CO2were quantified by mixing the NaOH with10ml HiSafe3scintillation cocktail(Perkin-Elmer,Boston,MA)and counted on a Wallac1409liquid scintillation counter.Isolation of PAH degraders in the inoculum soil.The numerically dominant phenanthrene-,pyrene-,andfluoranthene-degrading strains were isolated from the inoculum soil(Ringe).Phenanthrene,pyrene,orfluoranthene was dissolved in dimethyl sulfoxide(25mg mlϪ1).Phosphate minimal medium without carbon was prepared as described previously(10)except that20g literϪ1Noble Agar(Difco,Detroit,MI)was added as a solidifying agent. PAH agar was prepared by placing the still-warm medium(1liter,50°C)on a magnetic stirrer(900rpm).Four milliliters of dimethyl sulfoxide-PAH was slowly added using a pipette with the tip placed below the surface of the medium to produce turbid suspensions of PAH microcrystals.The PAH medium was poured into petri dishes.The phenanthrene,fluoranthene,and pyrene degraders were MPN enumerated.Scrapings from the bottom of MPN microplate wells at the highest growth-positive dilutions were streaked on the relevant PAH agar using1-␮l inoculation loops.The inoculated PAH-agar plates were incubated for3to4weeks at20°C in a fume hood.Colonies,often very small,of the two most dominant morphologies were picked and pure streaked two to four times on PAH agar.Liquid cultures were prepared by adding0.4mg PAH in acetone solution(4mg mlϪ1)to20ml sterile glass scintillation vials,followed by evaporation of the acetone in a sterile cabinet. Two milliliters of phosphate minimal medium was added,and the vials were inoculated with colonies from the PAH-agar plates,closed with sterile lids with aluminum foil inserts,and incubated on a rotary shaker for up to5 weeks.When turbid,subsamples of the liquid cultures were stored in glycerol (30%[vol/vol],final concentration)atϪ80°C.The purity of the isolates was confirmed by streaking on20%-strength tryptic soy broth agar.PAH degradation capacity of isolates.The isolates were tested for the ability to mineralize the PAH they were isolated on.Fifty microliters of acetone con-taining0.2mg14C-labeled PAH(Ϸ125Bq)was added to25-ml sterile glass scintillation vials.The acetone was evaporated,and2ml phosphate minimal medium was added to givefinal PAH spikes of100mg literϪ1.14CO2was collected in3-ml sterile glass tubes containing1ml NaOH(1M)placed inside the scintillation vials.Growth on phenanthrene,pyrene,andfluoranthene as sole sources of carbon and energy was tested by a previously published microplate method,based on respiratory reduction of the tetrazolium compound WST-1in growth-positive wells after12to14days of incubation(7).Identification of isolates by partial16S rRNA gene sequencing.Approximately 530bp of the16S rRNA gene was amplified by PCR as described previously(28). The PCR products were sequenced by MWG-Biotech(Ebersberg,Germany). Only results for one isolate were reported when two isolates from the same microplate well gave similar16S sequences.Eight randomly chosen isolates were tested for the presence of mycobacterium-like PAH dioxygenase by q-PCR using the pdo1primers as described above.RESULTS AND DISCUSSION Optimization of PAH degrader MPN enumeration.The op-timum incubation time for PAH MPN counts was determinedposition of microcosmsMicrocosm Amt of pristinesoil(g)Amt of PAH-polluted soil(g)Amt of solventcontrol soil(g)Amt of inoculum(bioremediated soil)(g)Amt of NaN3(g literϪ1)PAH polluted,primed1,1703000300PAH polluted,not primed1,200300000 Nonpolluted,primed1,1700300300PAH polluted,poisoned1,200300005V OL.73,2007PAH BIOREMEDIATION BY SOIL-ADAPTED MYCOBACTERIA1475on May 8, 2012 by SOUTH CHINA UNIVERSITY OF TECHNOLOGY /Downloaded fromby preparing identical MPN plates,containing dilution series of the bioremediated Ringe soil,which were then incubated for 2,3,4,or 5weeks before addition of the respiration indicator WST-1for detecting growth-positive wells.The MPN esti-mates increased during the first 4weeks,and 4weeks was therefore used as the standard incubation time in the rest of the study.Characterization of PAH degraders of the inoculum soil.Bioremediated clay soil from a previous project (6)was used asa PAH degrader inoculum.This soil had been subjected to homogenization and fertilization with NPK for 4years.During the first year,the content of 18selected polycyclic aromatic compounds had dropped from 1,250mg kg Ϫ1to 380mg kg Ϫ1(6),followed by much slower degradation during the following years.The PAH mineralization potentials and the numbers of PAH degraders in the bioremediated Ringe soil were determined be-fore the microcosms were set up.“Fresh”[14C]phenanthrene,[14C]pyrene,and [14C]fluoranthene were readily mineralized (Fig.1).Duplicate MPNs of PAH degraders showed 1.5ϫ106and 5.8ϫ106cells g Ϫ1for phenanthrene,1.5ϫ106and 2.6ϫ106cells g Ϫ1for pyrene,and 1.8ϫ106and 2.9ϫ106cells g Ϫ1for fluoranthene.Similar MPNs for the three PAHs suggested that many of the degrader cells could grow on more than one of the PAHs.The numerically dominant PAH degraders in the Ringe soil were isolated from the highest growth-positive dilutions of the MPN plates.The isolates were identified by partial 16S rRNA gene sequencing,and their ability to grow on phenanthrene,pyrene,and fluoranthene was determined (Table 2).Surpris-ingly,all but one isolate were mycobacteria affiliated with the species Mycobacterium frederiksbergense ,Mycobacterium aus-troafricanum ,Mycobacterium gilvum ,Mycobacterium aurum ,Mycobacterium pyrenivorans ,and Mycobacterium vaccae .A pre-vious study showed,on the basis of 13C incorporation of phenanthrene-derived carbon into polar lipid fatty acids,that the phenanthrene degraders of the Ringe soil at that time were dominated by sphingomonads and an unclassifiedbeta-pro-FIG.1.Mineralization of 14C-labeled PAHs (2mg kg Ϫ1)by biore-mediated subsoil used for priming of microcosms.PAH mineralization was estimated in duplicate as the recovery of 14CO 2.TABLE 2.Characterization of numerically dominant,culturable PAH degraders in the bioremediated soil used as an inoculumPAH used for isolationIsolateGenBank accession no.Closest relative based on partial 16S rRNA gene sequence(%homology)Growth on PAHs,determined as reductionof WST-1(A 450-630)a HexPheFlaPyrPhe Ri456a EF012722Arthrobacter oxydans (99.8)0.010.860.010.01Phe Ri463b EF012740M.frederiksbergense (99.8)0.27 1.690.22 1.62Phe Ri452b EF012741M.austroafricanum (97.6)0.07 1.660.060.52Phe Ri465a EF012742M.austroafricanum (100)0.050.790.100.35Phe Ri455EF012743M.gilvum (99.7)0.07 2.040.010.48Phe Ri457EF012744M.gilvum (100)0.030.730.010.31Phe Ri469EF185792M.aurum (99.8)0.24 1.220.240.36Phe Ri464EF012746M.aurum (100)0.17 1.360.430.63Phe Ri458a EF012747M.vaccae (100)0.020.570.110.18Phe Ri460EF012723M.vaccae (100)0.040.610.500.39Phe Ri466EF012725M.vaccae (99.5)0.07 1.360.84 1.18Pyr Ri483a EF012726M.aurum (100)0.090.560.090.26Pyr Ri477EF012727M.frederiksbergense (99.5)0.31 2.070.20 1.67Pyr Ri470a EF012728M.gilvum (99.8)0.03 1.140.020.26Pyr Ri484b EF012729M.gilvum (99.8)0.120.730.100.54Pyr Ri481b EF012730M.gilvum (100)0.03 1.090.020.26Pyr Ri489EF012731M.gilvum (100)0.100.770.120.46Pyr Ri471c EF012724M.vaccae (99.8)0.020.440.180.26Pyr Ri486ba EF012732M.vaccae (99.8)0.030.590.190.29Pyr Ri487EF012733M.vaccae (99.4)0.010.240.070.20Fla Ri505b EF012734M.pyrenivorans (99.8)0.010.010.050.01Fla Ri506EF012735M.pyrenivorans (99.8)0.050.050.340.05Fla Ri488a EF012736M.aurum (100)0.140.070.370.14Fla Ri494EF012737M.aurum (100)0.020.000.490.03Fla Ri495EF012738M.aurum (100)0.070.020.180.03FlaRi496EF012739M.vaccae (100)0.080.740.260.23aNumbers in bold are significantly higher than those for the hexane control (one-sided t test,P Ͻ0.05;n ϭ3).Phe,phenanthrene;Pyr,pyrene;Fla,fluoranthene;Hex,hexane control.1476JOHNSEN ET AL.A PPL .E NVIRON .M ICROBIOL .on May 8, 2012 by SOUTH CHINA UNIVERSITY OF TECHNOLOGY/Downloaded fromteobacterium(14).However,that study was carried out right after the initial,fast PAH degradation.Together,this supports the hypothesis of Leys et al.(18)that initial degradation of PAHs in soil is done by sphingomonads and other fast-growing bacteria(r-strategists),whereas later,the sphingomonads are outcompeted by relatively slow-growing mycobacteria(K-strat-egists).The dominance of mycobacteria in aged,PAH-polluted soil is also in accordance with the results reported by Uytte-broek et al.(25).The Arthrobacter isolate grew only on phenanthrene,whereas the20mycobacteria that grew on phenanthrene could also grow on pyrene.Also,all pyrene-degrading isolates could grow on phenanthrene.If these mycobacteria degrade phenanthrene through the o-phthalate pathway described for mycobacteria(17, 20),then pyrene may be shuttled into the phenanthrene pathway through formation of3,4-dihydroxyphenanthrene from pyrene in six metabolic steps(17).It therefore makes sense that these strains could grow on both phenanthrene and pyrene.The degradation offluoranthene to phthalate by mycobac-teria,on the other hand,proceeds through other pathways,sincefluoranthene contains a C5ring.Mycobacterium sp.strainAP1and strain PYR-1both metabolizefluoranthene to phthal-ate through formation of either9-fluorenone-1-carboxylic acid or acenaphthenone(16,19).This may explain why5fluoran-thene degraders grew neither on phenanthrene nor on pyrene and why12out of20mycobacterial phenanthrene/pyrene iso-lates did not grow onfluoranthene.The nine isolates that grew on all three PAHs presumably have both pyrene andfluoran-thene pathways,like Mycobacterium sp.strain AP1(19). PAH degradation in microcosms.Recently,it was demon-strated that the clay fraction of a long-term PAH-polluted soil had a high potential for PAH degradation,as opposed to the sand and silt fractions(25).We therefore used a PAH-pol-luted,bioremediated clay soil as an inoculum in the primed microcosms.The PAH content of the pristine soil was below the limit of detection.The bioremediated soil contained,at the time of the experiment,9.0mg kgϪ1of phenanthrene,22.1mg kgϪ1of pyrene,and22.4mg kgϪ1offluoranthene and thus constituted less than1percent of the phenanthrene,fluoranthene,and pyrene in the polluted microcosms.The initial PAH concen-trations were not measured but were calculated from the amounts added.Changes in PAH concentrations are depicted in Fig.2.Phenanthrene disappeared rapidly within thefirst2to 4weeks,whereas pyrene andfluoranthene were gradually de-graded so that most had disappeared at week16.The initial degradation rates were higher in the primed microcosm than in the nonprimed microcosm,but at later stages,the degradation rates were comparable.The poisoned control microcosm failed to remain inactive (high numbers of CFU).This probably explains the reductionin PAH concentrations for the poisoned control at the end of the incubation;however,it cannot be excluded that some PAH disappearance might have been due to abiotic processes. PAH-degrading populations.The PAH degraders of the bioremediated inoculum soil showed high survival rates in the control microcosm without PAHs,and34%of phenanthrene degraders,13%of pyrene degraders,and8%offluoranthene degraders were still culturable after16weeks of incubation (Fig.3).The high survival rate demonstrates the greaterfitness of soil-adapted degrader cells compared to the generally fast decline of laboratory strains introduced into soil microcosms. For comparison,a classic survival study showed that eight bacterial strains introduced into unsterile loam soil declined1 to4orders of magnitude within1week due to starvation and protozoan predation(1).The degrader populations of the polluted,primed micro-cosm responded rapidly by increases of3orders of magnitude for the phenanthrene and pyrene degrader MPNs and2orders FIG.2.Degradation of phenanthrene,pyrene,andfluoranthene in soil microcosms.PAH-polluted,polluted with50mg kgϪ1of phenan-threne,pyrene andfluoranthene;primed,primed with2%bioremedi-ated soil.V OL.73,2007PAH BIOREMEDIATION BY SOIL-ADAPTED MYCOBACTERIA1477on May 8, 2012 by SOUTH CHINA UNIVERSITY OF TECHNOLOGY /Downloaded fromof magnitude for the fluoranthene degrader MPNs (Fig.3).For comparison,numbers of CFU of a PAH-degrading Sphin-gomonas lab strain introduced into highly PAH-polluted soil decreased 2to 3orders of magnitude within 20days,depend-ing on pretreatment of the inoculum (4).The MPNs of phenanthrene degraders in the primed,pol-luted microcosm was close to the theoretical growth yield,assuming that the degraded phenanthrene (49.7mg kg Ϫ1)was converted into biomass.The yield of Sphingomonas sp.strain LH128on phenanthrene was 1.3ϫ109Ϯ0.1ϫ109cells mg Ϫ1(9),corresponding to a cell density of 6.5ϫ107Ϯ0.5ϫ107cells g Ϫ1in the microcosm.The maximum MPN of phenan-threne degraders was 4.0ϫ107cells g Ϫ1.This comparison does not take into account that the MPN technique detects only those cells that can grow in mineral medium with PAHs as the sole carbon source,that degraders in the microcosm may have been eaten by protozoa,or that they may have utilized more than one carbon source.Also,the yields of the microcosm strains may be different from those of Sphingomonas .How-ever,the yield of mycobacteria could not be reliably estimated because the hydrophobic cells flocculated when grown on PAHs in mineral medium.Interestingly,the phenanthrene MPN continued to increase after the complete disappearance of phenanthrene in the mi-crocosm (Fig.3,day 28to day 112),suggesting that pyrene degraders and possibly some fluoranthene degraders could also grow on phenanthrene.Alternatively,the delayed growth may be explained by the utilization of temporarily excreted metabolites.The general fast decline of bioaugmentation inocula,compared to the high survival rate for the priming community,may be caused by the genetic changes induced by the liquid techniques used for isolation and production of the inocula.Even a single overnight culture induces profound genetic changes (23).Re-peated growth in liquid batch cultures is highly selective for the planktonic mode of living,and as a result,the bioaugmentation strains may lose the ability to excrete extracellular polymeric sub-stances and to attach and form biofilm (3).However,attached-biofilm growth protects the cells against environmental stress and predation by protozoa (22).Biofilm formation directly on the PAH sources is also an important means for degrader bacteria to reduce the constraints of low PAH bioavailability (11,27).In liquid cultures,the cells also become adapted to growth on or-ganic and mineral nutrients in high concentrations,a situation rarely encountered in soil.PAH degraders in the unpolluted soil were undetectable by MPN enumeration.MPN counts were initially below the limit of detection (Ͻ600cells g Ϫ1)in the nonprimed,polluted mi-crocosm (Fig.3).The number of fluoranthene degraders re-mained rather low,whereas phenanthrene degraders peaked at 2.3ϫ105cells g Ϫ1(day 28)and pyrene degraders peaked at 7.6ϫ104cells g Ϫ1(day 56).These increases explain the PAH degradation that was achieved in this microcosm.The low initial densities,compared to those for primed soils,explain the slower onset of degradation,but once densities of 105cells g Ϫ1were reached,degradation occurred at rates similar to those in the primed soils.The maximum numbers of degraders were lower than in the primed microcosm,which may be ex-plained in three ways.First,the PAH degraders may be less culturable in the nonprimed microcosm.Second,some PAH degradation in the nonprimed microcosm may be cometabolic and therefore not detected in the MPN counts.Third,protozoa may have continuously grazed the degraders.The last expla-nation seems the most likely,as suggested by the declining populations at the end of the experiment.PAH dioxygenase genes.The nah -like genes found in Pseudomonas spp.and related genera encode dioxygenases of the N.2.A subfamily that degrade low-molecular-weight PAHs (2).nah genes were initially present in the two primed micro-cosms at densities of 1.8ϫ104cells g Ϫ1and 2.0ϫ104cells g Ϫ1(day 0).However,they were detected by q-PCR neither inlaterFIG.3.Development in populations of PAH degraders.MPN,most probable number of phenanthrene degraders.1478JOHNSEN ET AL.A PPL .E NVIRON .M ICROBIOL .on May 8, 2012 by SOUTH CHINA UNIVERSITY OF TECHNOLOGY/Downloaded from。

葡萄糖在稀硫酸催化下的降解反应动力学1彭新文，吕秀阳浙江大学化工系，杭州（310027）E-mail：luxiuyang@摘要：葡萄糖是纤维素的组成单体。

葡萄糖在酸催化下的降解是从生物质资源出发制备乙酰丙酸过程中重要步骤。

从生物质资源出发制备乙酰丙酸通常是采用1.5%以上的硫酸作为催化剂，既造成严重设备腐蚀，又给环境保护带来很大压力。

为了探索在稀硫酸浓度下水解生物质制备乙酰丙酸工艺的可行性，本文系统地测定了压力5MPa，初始浓度5～20 mg·mL-1、温度160～190℃、硫酸浓度0.05%～0.4wt%范围内葡萄糖的降解反应动力学数据，并以带有平行反应的一阶连串反应动力学模型对数据进行了拟合。

拟合结果表明在实验范围内，葡萄糖降解的主、副反应对葡萄糖均为一级反应；葡萄糖降解的主反应对H+为0.716级，反应的活化能129 kJ·mol-1；副反应对H+为1.06级，反应的活化能为154 kJ·mol-1。

通过对动力学方程进行分析，发现在硫酸浓度到达一定量后（0.3wt%左右），若再增加硫酸浓度，对乙酰丙酸收率影响较少。

因此在综合考虑收率、硫酸用量以及污染等因素的前提下，稀浓度硫酸(0.3%左右)催化降解生物质制备乙酰丙酸工艺是有发展前景的。

关键词：葡萄糖；乙酰丙酸；稀硫酸；降解；动力学中图分类号：TQ 032；O 643.121．引言纤维素含量约占50%的生物质资源，是一种有广阔应用前景的可再生资源，它是由许多D-吡喃葡萄糖彼此以β-1-4糖苷键连接起来的线性高分子化合物，葡萄糖是其组成单体。

乙酰丙酸（levulinic acid，LA）是一种能从纤维素出发，低成本、大规模制备的新平台化合物[1-2]。

从纤维素出发制备LA一般采用1.5～30wt%硫酸做催化剂[3-5]。

但是以高浓度硫酸作催化剂，对反应设备有很大的腐蚀性，且反应后会产生大量的酸性废渣和废液，给环境保护带来严重的问题。

掺碳纳米TiO2光催化降解空气中苯的实验研究
掺碳纳米TiO2光催化降解空气中苯的实验研究
采用火焰化学气相沉积法法制备了掺碳纳米TiO2光催化剂,对催化剂进行了表征.利用自制的连续管式光催化氧化装置研究了掺碳纳米TiO2薄膜对苯气体的光催化降解规律,探讨了相对湿度、初始浓度及苯气体流速等因素对降解率的影响,并与P25粉的光催化性能进行了比较.实验结果表明:在催化剂负载量约为4.7 mg,254 nm和365 nm的8W 紫外灯各一盏,相对湿度为80%、苯的初始浓度约为120 mg/m3、苯气体流量为400 mL/min(苯在光催化器中反应时间约为3.5 s)的条件下,苯的降解率可达到15%,高于P25粉的降解效果.
作者：张亚宁谢洪勇徐巧莲 ZHANG Ya-ning XIE Hong-yong XU Qiao-lian 作者单位：张亚宁,徐巧莲,ZHANG Ya-ning,XU Qiao-lian(大连理工大学,化工学院,辽宁,大连,116012)
谢洪勇,XIE Hong-yong(上海第二工业大学,环境工程系,上海,201209)
刊名：中国粉体技术ISTIC PKU英文刊名：CHINA POWDER SCIENCE AND TECHNOLOGY 年，卷(期)：2007 13(6) 分类号：O643.36 关键词：掺碳纳米TiO2 薄膜光催化降解苯。

CD-ROM 8325 - 1Revision 0December 1996METHOD 8325SOLVENT EXTRACTABLE NONVOLATILE COMPOUNDS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY/PARTICLE BEAM/MASS SPECTROMETRY (HPLC/PB/MS)1.0SCOPE AND APPLICATION1.1This method describes the use of high performance liquid chromatography (HPLC),coupled with particle beam (PB) mass spectrometry (MS), for the determination of benzidines and nitrogen-containing pesticides in water and wastewater. The following compounds can be determined by this method:Compound CAS No.aBenzidine 92-87-5Benzoylprop ethyl 33878-50-1Carbaryl 63-25-2o-Chlorophenyl thiourea 5344-82-13,3'-Dichlorobenzidine 91-94-13,3'-Dimethoxybenzidine 119-90-43,3'-Dimethylbenzidine 612-82-8Diuron 330-54-1Linuron (Lorox)330-55-2Monuron 150-68-5Rotenone 83-79-4Siduron1982-49-6Chemical Abstract Service Registry Numbera 1.2The method also may be appropriate for the analysis of benzidines and nitrogen-containing pesticides in non-aqueous matrices. The method may be applicable to other compounds that can be extracted from a sample with methylene chloride and are amenable to separation on a reverse phase liquid chromatography column and transferable to the mass spectrometer with a particle beam interface.1.3Preliminary investigation indicates that the following compounds also may be amenable to this method: Aldicarb sulfone, Carbofuran, Methiocarb, Methomyl (Lannate), Mexacarbate (Zectran), and N-(1-Naphthyl)thiourea. Ethylene thiourea and o-Chlorophenyl thiourea have been successfully analyzed by HPLC/PB/MS, but have not been successfully extracted from a water matrix.1.4Tables 4 - 6 present method detection limits (MDLs) for the target compounds, ranging from 2 to 25 µg/L. The MDLs are compound- and matrix-dependent.1.5This method is restricted to use by, or under the supervision of, analysts experienced in the use of HPLC and skilled in the interpretation of particle beam mass spectrometry. Each analyst must demonstrate the ability to generate acceptable results with this method.2.0SUMMARY OF METHOD2.1The target compounds for this method must be extracted from the sample matrix prior to analysis.2.1.1Benzidines and nitrogen-containing pesticides are extracted from aqueousmatrices at a neutral pH with methylene chloride, using a separatory funnel (Method 3510), a continuous liquid-liquid extractor (Method 3520), or other suitable technique.2.1.2Solid samples are extracted using Methods 3540 (Soxhlet), 3541 (AutomatedSoxhlet), 3550 (Ultrasonic extraction), or other suitable technique.2.2An aliquot of the sample extract is introduced into the HPLC instrument and a gradient elution program is used to chromatographically separate the target analytes, using reverse-phase liquid chromatography.2.3Once separated, the analytes are transferred to the mass spectrometer via a particle beam HPLC/MS interface. Quantitation is performed using an external standard approach.2.4An optional internal standard quantitation procedure is included for samples which contain coeluting compounds or where matrix interferences preclude the use of the external standard procedure.2.5The use of ultraviolet/visible (UV/VIS) detection is an appropriate option for the analysis of routine samples, whose general composition has been previously determined.3.0INTERFERENCES3.1Refer to Methods 3500 and 8000 for general discussions of interferences with the sample extraction and chromatographic separation procedures.3.2Although this method relies on mass spectrometric detection, which can distinguish between chromatographically co-eluting compounds on the basis of their masses, co-elution of two or more compounds will adversely affect method performance. When two compounds coelute, the transport efficiency of both compounds through the particle beam interface generally improves, and the ion abundances observed in the mass spectrometer increase. The degree of signal enhancement by coelution is compound-dependent.3.2.1This coelution effect invalidates the calibration curve and, if not recognized, willresult in incorrect quantitative measurements. Procedures are given in this method to check for co-eluting compounds, and must be followed to preclude inaccurate measurements.3.2.2An optional internal standard calibration procedure has been included for use ininstances of severe co-elution or matrix interferences.3.3 A major source of potential contamination is HPLC columns which may contain silicon compounds and other contaminants that could prevent the determination of method analytes. Generally, contaminants will be leached from the columns into mobile phase and produce a variable background. Figure 1 shows unacceptable background contamination from a column with stationary phase bleed.CD-ROM8325 - 2Revision 0December 1996CD-ROM 8325 - 3Revision 0December 19963.4Contamination may occur when a sample containing low analyte concentrations is analyzed immediately after a sample containing relatively high analyte concentrations. After analysis of a sample containing high analyte concentrations, one or more method blanks should be analyzed.Normally, with HPLC, this is not a problem unless the sample concentrations are at the percent level.4.0APPARATUS AND MATERIALS4.1High performance liquid chromatograph (HPLC) - An analytical system with programmable solvent delivery system and all necessary accessories including 5 µL injection loop,analytical columns, purging gases, etc. The solvent delivery system must be capable, at a minimum,of handling a binary solvent system, and must be able to accurately deliver flow rates between 0.20- 0.40 mL/min. Pulse dampening is recommended, but not required. The chromatographic system must be able to be interfaced with a mass spectrometer (MS). An autoinjector is recommended and should be capable of accurately delivering 1 - 10 µL injections without affecting the chromatography.4.1.1HPLC Columns - An analytical column is needed, and a guard column is highlyrecommended.4.1.1.1Analytical Column - Reverse phase column, C chemically bonded to184-10 µm silica particles, 150 - 200 mm x 2 mm, (Waters C-18 Novapak or equivalent).Residual acidic sites should be blocked (endcapped) with methyl or other non-polargroups and the stationary phase must be bonded to the solid support to minimize columnbleed. Select a column that exhibits minimal bleeding. New columns must beconditioned overnight before use by pumping a 75 - 100% v/v acetonitrile:water solutionthrough the column at a rate of about 0.05 mL/min. Other packings and column sizesmay be used if appropriate performance can be achieved.4.1.1.2Guard Column - Packing similar to that used in analytical column.4.1.2HPLC/MS interface - The particle beam HPLC/MS interface must reduce the ionsource pressure to a level compatible with the generation of classical electron ionization (EI)mass spectra, i.e., about 1 x 10 - 1 x 10Torr, while delivering sufficient quantities of analytes -4 -6to the conventional EI source to meet sensitivity, accuracy, and precision requirements. The concentrations of background components with masses greater than 62 Daltons should be reduced to levels that do not produce ions greater than a relative abundance of 10% in the mass spectra of the analytes.4.2Mass spectrometer system - The mass spectrometer must be capable of electron ionization at a nominal electron energy of 70 eV. The spectrometer should be capable of scanning from 45 to 500 amu in 1.5 seconds or less (including scan overhead). The spectrometer should produce a mass spectrum that meets the criteria in Table 1 when 500 ng or less of DFTPPO are introduced into the HPLC.4.3Data system - A computer system must be interfaced to the mass spectrometer, and must be capable of the continuous acquisition and storage on machine-readable media of all mass spectra obtained throughout the duration of the chromatographic program. The computer software must be capable of searching any HPLC/MS data file for ions of a specified mass and plotting such abundance data versus time or scan number.4.4Volumetric flasks - Class A, in various sizes, for preparation of standards.CD-ROM 8325 - 4Revision 0December 19964.5Vials - 10-mL amber glass vials with polytetrafluororethylene (PTFE)-lined screw caps or crimp tops.4.6 Analytical balance - capable of weighing 0.0001 g.4.7Extract filtration apparatus4.7.1Syringe - 10-mL, with Luer-Lok fitting.4.7.2Syringe filter assembly, disposable - 0.45 µm pore size PTFE filter in filterassembly with Luer-Lok fitting (Gelman Acrodisc, or equivalent).5.0REAGENTS5.1Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity to permit its use without lessening the accuracy of the determination.5.2Organic-free reagent water - All references to water in this method refer to organic-free reagent water, as defined in Chapter One.5.3Solvents - All solvents must be HPLC-grade or equivalent.5.3.1Acetonitrile, CH CN 35.3.2Methanol, CH OH 35.3.3Ammonium acetate, NH OOCCH , (0.01M in water).435.4Mobile phase - Two mobile phase solutions are needed, and are designated Solvent A and Solvent B. Degas both solvents in an ultrasonic bath under reduced pressure and maintain by purging with a low flow of helium.5.4.1Solvent A is a water:acetonitrile solution (75/25, v/v) containing ammoniumacetate at a concentration of 0.01M.5.4.2Solvent B is 100 % acetonitrile.5.5Stock standard solutions - Stock solutions may be prepared from pure standard materials or purchased as certified solutions. Commercially-prepared stock standards may be used at any concentration if they are certified by the manufacturer.5.5.1Prepare stock standard solutions by accurately weighing 0.0100 g of pure materialin a volumetric flask. Dilute to known volume in a volumetric flask. If compound purity is certified at 96% or greater, the weight may be used without correction to calculate the concentration of the stock standard. Commercially-prepared stock standards may be used at any concentration if they are certified by the manufacturer or by an independent source.CD-ROM 8325 - 5Revision 0December 19965.5.1.1Dissolve benzidines and nitrogen-containing pesticides in methanol,acetonitrile, or organic-free reagent water.5.5.1.2Certain analytes, such as 3,3'-dimethoxybenzidine, may require dilutionin 50% (v/v) acetonitrile:water or methanol:water solution.5.5.1.3Benzidines may be used for calibration purposes in the free base or acidchlorides forms. However, the concentration of the standard should be calculated as thefree base.5.5.2Transfer the stock standard solutions into amber bottles with PTFE-linedscrew-caps or crimp tops. Store at -10E C or less and protect from light. Stock standard solutions should be checked frequently for signs of degradation or evaporation, especially just prior to preparing calibration standards from them.5.6Surrogate spiking solution - The recommended surrogates are benzidine-D ,8caffeine-N , 3,3'-dichlorobenzidine-D , and bis(perfluorophenyl)-phenylphosphine oxide. Prepare 152 6a solution of the surrogates in methanol or acetonitrile at a concentration of 5 mg/mL of each. Other surrogates may be included in this solution as needed. (A 10-µL aliquot of this solution added to 1L of water gives a concentration of 50 µg/L of each surrogate). Store the surrogate spiking solution in an amber vial in a freezer at -10E C or less.5.7MS performance check solution - Prepare a 100 ng/µL solution of DFTPPO in acetonitrile.Store this solution in an amber vial in a freezer at -10E C or less.5.8Calibration solutionsThis method describes two types of calibration procedures that may be applied to the target compounds: external standard calibration, and internal standard calibration. Each procedure requires separate calibration standards. In addition, the performance characteristics of the HPLC/PB/MS system indicate that it may be necessary to employ a second order regression for calibration purposes, unless a very narrow calibration range is chosen. See Method 8000 for additional information on non-linear calibration techniques.5.8.1For external standard calibration, prepare calibration standards for all targetcompounds and surrogates in acetonitrile. DFTPPO may be added to one or more calibration solutions to verify MS tune (see Sec. 7.3). Store these solutions in amber vials at -10E C or less. Check these solutions at least quarterly for signs of deterioration.5.8.2Internal standard calibration requires the use of suitable internal standards (seeMethod 8000). Ideally, stable, isotopically-labeled, analogs of the target compounds should be used. These labeled compounds are included in the calibration standards and must also be added to each sample extract immediately prior to analysis. Prepare the calibration standards in a fashion similar to that for external standard calibration, but include each internal standard in each of the calibration standards.The concentration of the internal standards should be 50 - 100 times the lowestconcentration of the unlabeled target compounds. In addition, the concentration of the internal standards does not vary with the concentrations of the target compounds, but is held constant.Store these solutions in amber vials at -10E C or less. Check these solutions at least quarterly for signs of deterioration.5.9Internal standard spiking solution - This solution is required when internal standard quantitation is used. Prepare a solution containing each of the internal standards that will be used for quantitation of target compounds (see Sec. 5.8.2) in methanol. The concentration of this solution must be such that a 1-µL volume of the spiking solution added to a 1-mL final extract will result in a concentration of each internal standard that is equal to the concentration of the internal standard in the calibration standards in Sec. 5.8.2. Store this solution in an amber vial at -10E C or less. Check this solution at least quarterly for signs of deterioration. This solution is not necessary if only external standard calibration will be used.5.10Sodium chloride, NaCl - granular, used during sample extraction.6.0SAMPLE COLLECTION, PRESERVATION, AND HANDLING6.1See the introductory material to this chapter, Organic Analytes, Sec. 4.1.6.2Samples should be extracted within 7 days and analyzed within 30 days of extraction. Extracts should be stored in amber vials at -10E C or less.7.0PROCEDURE7.1Samples may be extracted by Method 3510 (separatory funnel), Method 3520 (continuous extractor), Method 3535 (solid-phrase extraction), or other appropriate technique. Prior to extraction, add a 10-µL aliquot of the surrogate spiking solution and 100 g of sodium chloride to the sample, and adjust the pH of the sample to 7.0. Samples of other matrices should be extracted by an appropriate sample preparation technique. The concentration of surrogates in the sample should be 20-50 times the method detection limit. Concentrate the extract to 1 mL, and exchange the solvent to methanol, following the procedures in the extraction method.7.2Establish chromatographic, particle beam interface, and mass spectrometer conditions, using the following conditions as guidance.Mobile phase purge:Helium at 30 mL/min, continuousMobile phase flow rate:0.25 - 0.3 mL/min through the columnGradient elution:Hold for 1 min at 25% acetonitrile (Solvent A), thenprogram linearly to about 70% acetonitrile (60%Solvent B) in 29 min. Start data acquisitionimmediately.Desolvation chamber temperature:45 - 80E CIon source temperature:250 - 290E CElectron energy:70 eVScan range:62 to 465 amu, at #1.5 sec/scan NOTE:Post-column addition is an option that improves system precision and, thereby, may improve sensitivity. Post-column flow rates depend on the requirements ofthe interface and may range from 0.1 to 0.7 mL/min of acetonitrile. Maintain aminimum of 30% acetonitrile in the interface.Analyte-specific chromatographic conditions are also shown in Table 2. (The particle beam interface conditions will depend on the type of nebulizer).CD-ROM8325 - 6Revision 0December 19967.2.1The analyst should follow the manufacturer's recommended conditions for theirinterface's optimum performance. The interface is usually optimized during initial installation by flow injection with caffeine or benzidine, and should utilize a mobile phase of acetonitrile/water (50/50, v/v). Major maintenance may require re-optimization.7.2.2Fine tune the interface by making a series of injections into the HPLC column ofa medium concentration calibration standard and adjusting the operating conditions (Sec. 7.2)until optimum sensitivity and precision are obtained for the maximum number of target compounds.7.3Initial calibration7.3.1Once the operating conditions have been established, calibrate the MS mass andabundance scales using DFTPPO to meet the recommended criteria in Table 1.7.3.2Inject a medium concentration standard containing DFTPPO, or separately injectinto the HPLC a 5-µL aliquot of the 100 ng/µL DFTPPO solution and acquire a mass spectrum.Use HPLC conditions that produce a narrow (at least ten scans per peak) symmetrical peak.If the spectrum does not meet the criteria (Table 1), the MS ion source must be retuned and adjusted to meet all criteria before proceeding with calibration. An average spectrum across the HPLC peak may be used to evaluate the performance of the system.Manual (not automated) ion source tuning procedures specified by the manufacturer should be employed during tuning. Mass calibration should be accomplished while an acetonitrile/water (50/50, v/v) mixture is pumped through the HPLC column and the optimized particle beam interface. For optimum long-term stability and precision, interface and ion source parameters should be set near the center of a broad signal plateau rather than at the peak of a sharp maximum (sharp maxima exhibit short-term variations with particle beam interfaces and gradient elution conditions).7.3.3System performance criteria for the medium concentration standard - Evaluatethe stored HPLC/MS data with the data system software and verify that the HPLC/PB/MS system meets the following performance criteria.7.3.3.1HPLC performance - 3,3'-dimethylbenzidine and3,3'-dimethoxybenzidine should be separated by a valley whose height is less than 25%of the average peak height of these two compounds. If the valley between them exceeds25%, modify the gradient. If this fails, the HPLC column requires maintenance. SeeSec. 7.4.6.7.3.3.2Peak tailing - Examine a total ion chromatogram and examine thedegree of peak tailing. Severe tailing indicates a major problem and systemmaintenance is required to correct the problem. See Sec. 7.4.67.3.3.3MS sensitivity - The signal-to-noise ratio for any compound's spectrumshould be at least 3:1.7.3.3.4Column bleed - Figure 1 shows an unacceptable chromatogram withcolumn bleed. Figure 2 shows an acceptable ion chromatogram. Figure 3 is the massspectrum of dimethyloctadecyl-silanol, a common stationary phase bleed product. Ifunacceptable column bleed is present, the column must be changed or conditioned toproduce an acceptable background.CD-ROM8325 - 7Revision 0December 19967.3.3.5Coeluting compounds - Compounds which coelute cannot be measuredaccurately because of carrier effects in the particle beam interface. Peaks must beexamined carefully for coeluting substances and if coeluting compounds are present atgreater than 10% of the concentration of the target compound, either conditions must beadjusted to resolve the components, or internal standard calibration must be used.7.3.4Once optimized, the same instrument operating conditions must be used for theanalysis of all calibration standards, samples, blanks, etc.7.3.5Once all the performance criteria are met, inject a 5-µL aliquot of each of theother calibration standards using the same HPLC/MS conditions.7.3.5.1The general method of calibration is a second order regression ofintegrated ion abundances of the quantitation ions (Table 3) as a function of amountinjected. For second order regression, a sufficient number of calibration points must beobtained to accurately determine the equation of the curve. (See Method 8000 for theappropriate number of standards to be employed for a non-linear calibration). Non-linearcalibration models can be applied to either the external standard or the internal standardcalibration approaches described here.7.3.5.2For some analytes the instrument response may be linear over a narrowconcentration range. In these instances, an average calibration factor (externalstandard) or average response factor (internal standard) may be employed for samplequantitation (see Method 8000).7.3.6If a linear calibration model is used, calculate the mean calibration factor orresponse factor for each analyte, including the surrogates, as described in Method 8000.Calculate the standard deviation (SD) and the relative standard deviation (RSD) as well. The RSD of an analyte or surrogate must be less than or equal to 20%, if the linear model is to be applied. Otherwise, proceed as described in Method 8000.7.4Calibration verificationPrior to sample analysis, verify the MS tune and initial calibration at the beginning of each 8-hour analysis shift using the following procedure:7.4.1Inject a 5-µL aliquot of the DFTPPO solution or a mid-level calibration standardcontaining 500 ng of DFTPPO, and acquire a mass spectrum that includes data for m/z 62-465. If the spectrum does not meet the criteria in Table 1, the MS must be retuned to meet the criteria before proceeding with the continuing calibration check.7.4.2Inject a 5-µL aliquot of a medium concentration calibration solution and analyzewith the same conditions used during the initial calibration.7.4.3Demonstrate acceptable performance for the criteria shown in Sec. 7.3.3.7.4.4Using the initial calibration (either linear or non-linear, external standard or internalstandard), calculate the concentrations in the medium concentration calibration solution and compare the results to the known values in the calibration solution. If calculated concentrations deviate by more than 20% from known values, adjust the instrument and inject the standard again. If the calibration cannot be verified with the second injection, then a new CD-ROM8325 - 8Revision 0December 1996initial calibration must be performed after taking corrective actions such as those described in Sec. 7.9.7.5Sample Analysis7.5.1The column should be conditioned overnight before each use by pumping aacetonitrile:water (70% v/v) solution through it at a rate of about 0.05 mL/min.7.5.2Filter the extract through a 0.45 µm filter. If internal standard calibration isemployed, add 10 µL of the internal standard spiking solution to the 1-mL final extract immediately before injection.7.5.3Analyze a 5-µL aliquot of the extract, using the operating conditions establishedin Secs. 7.2 and 7.3.7.6Qualitative identificationThe qualitative identification of compounds determined by this method is based on retention time and on comparison of the sample mass spectrum, after background correction, with characteristic ions in a reference mass spectrum. The reference mass spectrum must be generated by the laboratory using the conditions of this method. The characteristic ions from the reference mass spectrum are defined as the three ions of greatest relative intensity, or any ions over 30% relative intensity, if less than three such ions occur in the reference spectrum. Compounds are identified when the following criteria are met.7.6.1The intensities of the characteristic ions of a compound must maximize in thesame scan or within one scan of each other. Selection of a peak by a data system target compound search routine where the search is based on the presence of a target chromatographic peak containing ions specific for the target compound at a compound-specific retention time will be accepted as meeting this criterion.7.6.2The retention time of the sample component is within ± 10% of the retention timeof the standard.7.6.3The relative intensities of the characteristic ions agree within 20% of the relativeintensities of these ions in the reference spectrum. (Example: For an ion with an abundance of 50% in the reference spectrum, the corresponding abundance in a sample spectrum can range between 30% and 70%.)7.6.4Structural isomers that produce very similar mass spectra should be identified asindividual isomers if they have sufficiently different HPLC retention times. Sufficient GC resolution is achieved if the height of the valley between two isomer peaks is less than 25% of the sum of the two peak heights. Otherwise, structural isomers are identified as isomeric pairs.7.6.5Identification is hampered when sample components are not resolvedchromatographically and produce mass spectra containing ions contributed by more than one analyte. When HPLC peaks obviously represent more than one sample component (i.e., a broadened peak with shoulder(s) or a valley between two or more maxima), appropriate selection of analyte spectra and background spectra is important.CD-ROM8325 - 9Revision 0December 19967.6.6Examination of extracted ion current profiles of appropriate ions can aid in theselection of spectra, and in qualitative identification of compounds. When analytes coelute(i.e., only one chromatographic peak is apparent), the identification criteria may be met, buteach analyte spectrum will contain extraneous ions contributed by the coeluting compound.7.7Quantitative Analysis7.7.1Complete chromatographic resolution is necessary for accurate and precisemeasurements of analyte concentrations. Compounds which coelute cannot be measured accurately because of carrier effects in the particle beam interface. Peaks must be examined carefully for coeluting substances and if coeluting compounds are present at greater than 10% of the concentration of the target compound, either conditions must be adjusted to resolve the components, or the results for the target compound must be flagged as potentially positively biased.7.7.2Calculate the concentration of each analyte, using either the external standardor internal standard calibration. See Method 8000 for the specific equations to be employed for either the non-linear or linear calibration models.7.7.3If the response for any quantitation ion exceeds the initial calibration range of theHPLC/PB/MS system, the sample extract must be diluted and reanalyzed. When internal standard calibration is employed, additional internal standard must be added to the diluted extract to maintain the same concentration as in the calibration standards.7.8HPLC-UV/VIS Detection (optional)7.8.1Prepare calibration solutions as outlined in Sec. 5.8.7.8.2Inject 5 µL of each calibration solution onto the HPLC, using the chromatographicconditions outlined in Secs. 7.2.1 and 7.2.2. Integrate the area under the full chromatographic peak at the optimum wavelength (or at 230 nm if that option is not available) for each target compound at each concentration.7.8.3The retention time of the chromatographic peak is an important criterion foranalyte identification. Therefore, the ratio of the retention time of the sample analyte to the standard analyte should be 1.0 ± 0.1.7.8.4Calculate calibration factors or response factors as described in Method 8000,for either external standard or internal standard calibration, and evaluate the calibration linearity as described in Method 8000.7.8.5Verify the calibration at the beginning of each 8-hour analytical shift, as describedabove.7.8.6Once the calibration has been verified, inject a 5-µL aliquot of the sample extract,start the HPLC gradient elution, load and inject the sample aliquot, and begin data acquisition.Refer to Method 8000 for guidance on calculation of concentration.7.9Corrective ActionsWhen the initial calibration cannot be verified, one or more of the following corrective actions may be necessary.CD-ROM8325 - 10Revision 0December 1996。