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Probabilistic fatigue life prediction using AFGROW and accounting for material variabilityWilliam A.Grell,Peter z *Department of Mechanical and Materials Engineering,University of Denver,2390S.York St.,Denver,CO 80208,USAa r t i c l e i n f o Article history:Received 8July 2009Received in revised form 22October 2009Accepted 7December 2009Available online 16December 2009Keywords:Fatigue life prediction ProbabilisticMaterial variability Aluminum 2024AFGROW Lap jointa b s t r a c tThe fatigue performance of components contains signiﬁcant amounts of scatter,and variability has been characterized in initial crack sizes,crack growth rates,and material properties.Probabilistic methods have recently been gaining acceptance as an approach to account for uncertainty in various sources to predict component fatigue life.However,computation time associated with the accepted standard Monte Carlo method can be prohibitive during design phase evaluations.Accordingly,the objectives of the cur-rent study were to develop a probabilistic interface for the AFGROW life prediction software and to dem-onstrate the use of efﬁcient probabilistic methods,as an alternative to Monte Carlo analysis,to accurately predict fatigue lives for three veriﬁcation cases.The veriﬁcation cases were based on experimental data for compact tension,single edge notched tension,and single lap joint specimens from the literature.Based on experimentally determined distributions of crack growth rate,material properties,and initial crack size,predicted distributions of fatigue life agreed closely with replicate experimental test putation time with the Advanced Mean Value (AMV)and FORM methods were reduced by 100-fold compared to Monte Carlo,promoting the notion of utilizing probabilistic assessments within the design process.Ó2009Elsevier Ltd.All rights reserved.1.IntroductionComponent fatigue data contain signiﬁcant amounts of scatter [1,2].Variation in life of less than a factor of 2for high stresses and up to a factor of 100for low,uniform stresses is not uncom-mon [1].Inherent scatter has also been reported in crack nucle-ation sites [3–5]and crack growth rates [6,7].Additional uncertainties arise due to microstructure,processing,in-service loading,and environment.Historically,these uncertainties have been dealt with by applying experience-based safety factors to the fatigue analysis of critical components,driving the likelihood of failure to an acceptable level.In contrast to deterministic analy-ses,probabilistic methods represent the input parameters as distri-butions and predict distributions of performance.This allows assessment of performance at a corresponding probability level,which can aid in risk-related decision making for fatigue critical parts.In addition,similar to parametric studies,sensitivity factors are determined as part of the analysis to identify which input parameters are most critical to the performance distribution.Numerous studies have investigated probabilistic aspects of fa-tigue,including variability in stress-life (S –N )data [2,8],fatigue crack growth rate [6,7,9–13],and life prediction considering micro-structure [3,4,14–20],for structures [19,21–24]and under multi-axial conditions [25,26].In cases where a large amount of S –N data are available,statistical approaches may be used to quantify the scatter in the data [8].In more general approaches,probabilis-tic fracture mechanics based fatigue models are applied.The most straightforward probabilistic fracture mechanics models are ana-lytical and are based on randomizing the Paris relation (e.g.[10,11]).Typically these models describe the C-parameter in the Paris relation as a random variable,then numerically integrate the relation to determine a distribution of life.The limitations of this approach are that only one variable may be modeled as a dis-tribution and that the analysis is restrained to the Paris region of the material.More recently,models have been expanded to account for com-plex fatigue considerations (e.g.crack closure,variable amplitude loading)and multiple stochastic material behaviors (e.g.[14,21]).These models typically evaluate the crack growth process cycle-by-cycle while accounting for variability in crack growth rate.Other models accounted for the fatigue life variability by utilizing a distribution of material features or anomalies as the initial cracks [15,19]or incorporating initiation mechanisms [20].Fatigue life predictions using probabilistic fracture mechanics have typically employed time intensive Monte Carlo analysis,used simpliﬁed fracture mechanics models,or required complicated mathematical manipulation and formulation.Accordingly,the0142-1123/$-see front matter Ó2009Elsevier Ltd.All rights reserved.doi:10.1016/j.ijfatigue.2009.12.001*Corresponding author.Tel.:+13038713614;fax:+13038714450.E-mail address:plaz@ (z).International Journal of Fatigue 32(2010)1042–1049Contents lists available at ScienceDirectInternational Journal of Fatiguejournal homepage:w w w.e l s e v i e r.c o m/l o c a t e /i j f a t i g ueobjectives of the current study were to develop a probabilistic life prediction platform and to demonstrate the use of efﬁcient proba-bilistic methods,as an alternative to Monte Carlo analysis,to pre-dict the distribution of fatigue lives for three veriﬁcation cases.As an additional objective,the platform was to be developed with minimal software development in order to highlight the accessibil-ity of such an approach to engineers in the industry.The platform interfaced a commercially available probabilistic software package,NESSUS (Southwest Research Institute,San Antonio,TX)[27]or UNIPASS (PredictionProbe,Inc.,Irvine,CA),with the AFGROW (Wright-Patterson Air Force Base,OH)fatigue life prediction software.Wieland and Millwater [24]interfaced NESSUS and AFGROW to predict the sensitivity of fatigue life to probabilistic input parameters in T-38aircraft structures under spectrum loading;their study utilized Monte Carlo simulation,but did not consider the use of efﬁcient probabilistic methods.By interfacing with AFGROW,the model leverages all of the capabili-ties of the analysis package,including classical and user-deﬁned K-solutions,advanced weight function techniques,residual stress capabilities,multiple closure models,material databases,and ever improving numerical integration techniques.Further,the probabi-listic packages offer a wide array of numerical probabilistic tech-niques as well as useful tools to summarize results.Two probabilistic softwares were utilized to verify agreement in the probabilistic predictions independent of analysis package.The ver-iﬁcation cases evaluated in this study were based on experimental data from the literature for compact tension CT,single edge notched tension SENT,and single lap joint specimens of common engineering aluminum alloys.2.Methods2.1.Probabilistic analysis platformA probabilistic fatigue life prediction model was developed that interfaced commercially available probabilistic software and the AFGROW life prediction software (Fig.1).The AFGROW softwaremakes efﬁcient life predictions incorporating spectrum loading,crack closure,and inspection and repair features.The probabilistic modeling was demonstrated using commercially available proba-bilistic softwares,either NESSUS or UNIPASS.In the current study,the key input parameters were modeled as distributions,and the perturbed variables for the probabilistic methods were passed to AFGROW using the component object model (COM)capabilities and custom scripting.Life prediction was performed using AF-GROW with results passed back to the probabilistic software.The end result was a predicted distribution of fatigue lives.A variety of probabilistic methods were implemented and eval-uated to consider efﬁciency and accuracy for the various speci-mens.The most common and robust probabilistic method is Monte Carlo simulation,which involves repeated model evalua-tions with input values sampled at random according to their dis-tributions.Monte Carlo does not put restrictions on the number of random variables,nor the behavior of the response (e.g.linearity,continuity).However,Monte Carlo simulation is computationally expensive and may be prohibitive when trying to perform timely analyses within the design cycle.A series of approximate most probable point (MPP)methods have been developed that are more computationally efﬁcient than Monte Carlo simulation.The MPP represents the combination of input parameter values that predict performance,fatigue life in this study,at a speciﬁed probability level.The MPP methods typi-cally determine the most probable point using optimization on a ﬁrst-order Taylor series approximation of the performance func-tion [28].Some implementations map the original random vari-ables into independent standard normal variables to facilitate optimization with variables of similar magnitudes.The various techniques differ in terms of how performance is computed;for example,FORM (ﬁrst-order reliability method)uses a ﬁrst-order approximation.While the MPP methods are approximate,they have been shown in many analyses to be quite accurate in compar-isons with Monte Carlo simulation results,while requiring a small fraction of the number of computations.The low computational cost of the MPP methods comes with a tradeoff;these methods only provide information for a single point (e.g.probability),soResults (e.g. life)Perturbed variablesAFGROWCOMUNIPASSW.A.Grell,z /International Journal of Fatigue 32(2010)1042–10491043in order to construct a full PDF or CDF,the method must be applied repeatedly at each point of interest.The MPP methods work well with well-behaved monotonic systems,like fatigue life predictions.Further detailing the MPP family of methods,the mean-value (MV)method constructs a mean-based response function and computes the MPP for the speciﬁed probability levels.As aﬁrst-or-der method,it provides a good approximation of the solution near the mean.It is suitable for fairly linear problems,but can deviate signiﬁcantly toward the tails for non-linear problems.The MV method requires n+1trials,where n is the number of random vari-ables.The advanced mean-value(AMV)method utilizes the MV as a basis to achieve a better representation of the response.It does this by including corrective terms to approximate higher order ef-fects and requires n+1+m trials,where m is the number of spec-iﬁed probability levels[29].AMV takes the MV prediction and using data from the calculated MPP of interest,corrects this value for a single level of desired probability(or desired output).The higher-order approximation achieved by AMV cannot be applied at any point other than that for which it was derived;getting esti-mates for additional points requires additional applications of the AMV method.The advanced mean-value with iterations(AMV+) method involves the implementation of AMV but also includes iterations on the MPP to ensure that convergence to a speciﬁed le-vel is reached.AMV+has been shown to be very accurate even for non-linear problems,though the number of trials varies with the problem[29].2.2.Probabilistic sensitivity factorsIn addition to predicting the distribution of fatigue lives(e.g. cumulative distribution function),the probabilistic methods can identify the relative importance of each input parameter by com-puting sensitivity factors.Sensitivity factors can assess the change in probability with respect to a change in the mean or standard deviation of the input distribution.Sensitivity factors computedwith a partial differential equation,@p f@l and@p f@r,are calculated as partof the MPP methods.These values are typically nondimensional-ized by multiplying by the respective input standard deviation and probability to facilitate comparison across a variety of proba-bility levels in the CDF.The resulting sensitivity factors,@p f@l rpand@p f r r,provide a relative importance ranking of the inputparameters.3.Probabilistic fatigue life predictionsProbabilistic fatigue life predictions were performed for three veriﬁcation cases developed from the literature:CT specimens of aluminum2024-T351from Wu and Ni[12],SENT specimens of aluminum2024-T3from[15,30],and single lap joint geometries of aluminum2024-T3from Moreira et al.[31].These studies from the literature were selected as they contained a large number of re-peated tests.The parameters modeled as distributions varied for each geometry studied(Table1)and are discussed in the following sections on the respective geometries.The fatigue life predictions were performed in AFGROW utilizing the FASTRAN II plasticity in-duced closure model and the appropriate closed-form K-solutions for the CT and SENT specimens or tabulated solutions for the single lap joint.Parameters for the FASTRAN II[32]plasticity induced clo-sure model for Al2024-T3were based on the work by Newman et al.[33].3.1.Input distributionsIn the probabilistic analyses,input parameter variability was considered for fracture toughness,yield strength,initial crack size, and fatigue crack growth rate.Distributions for the input parame-ters were developed from available literature data for aluminum 2024in the-T3and-T351conditions.The fracture toughness of Al2024-T351was represented by a distribution with a mean of34MPa m0.5and a standard deviation of5.6MPa m0.5[34].The data was from testing on11specimens.It was assumed to be normally distributed based on observations made in the literature[7,21].The yield strength was considered be-cause of its impact in crack closure calculations.Yield strength data for2024-T3and2024-T351were very similar,and accordingly the same distribution was used for both.The yield strength distribu-tion was determined from A-basis and B-basis values of331and 345MPa,respectively[34].The A-and B-basis values represent the levels above which the actual yield strength falls99%and 90%of the time,respectively.The reporting of A-and B-basis im-plies that at least100samples were tested,though the exact num-ber was not reported[34].Here,it was assumed that the yield strength followed a lognormal distribution,and the distribution parameters were calculated from the A-and B-basis values as shape=0.03907and scale=5.8929(in ln(MPa))corresponding to a mean of362.7MPa and standard deviation of14.2MPa.The distribution of crack nucleating particles observed in Al 2024-T3SENT specimens were characterized in Laz et al.[15]. Based on180crack nucleation sites from31SENT and10double edge notch tension specimens[15],the study showed that the par-ticle widths,2a,and depths,c,were described well by lognormal distributions,and that the two exhibited a small degree of correla-tion.The corresponding mean and standard deviations were l a=4.478l m and r a=2.052l m for particle half-width(a)and l c=13.64l m and r c=5.582l m for depth.To model the fatigue crack growth rate(FCGR)relation,the baseline(mean)da/dN versus D K eff curve for Al2024-T3was rep-resented as a piece-wise curve from Newman et al.[33].Utilizing data from multiple sources,the extent of scatter for the material was evaluated by Wang[7,14],and standard deviations of da/dN were listed for ranges of D K eff values[14].The data were shown to be described well by a lognormal ing the infor-mation from these sources,a probabilistic crack growth rate curve was developed(Fig.2)with the mean piece-wise curve bounded by plus or minus three standard deviation scatter bands[14].The amount of scatter follows the expected trend in that the smaller crack region has larger standard deviations.Also included in the ﬁgure is the extrapolation which AFGROW performs when D K eff levels are less than the minimum from Newman et al.[33].This extrapolation is conservative because this region is below the threshold of crack growth for the material.Because of the similar crack growth rates for aluminum2024-T351[12],the probabilistic crack growth rate curve for2024-T3was used for both materials.To simulate specimen to specimen material property variability, each probabilistic variable was perturbed from its mean according to its distribution.This is a straightforward task for yield strength, fracture toughness,and initial crack size but is more complex forTable1Probabilistic inputs(U=active)for various specimens analyzed with the life prediction model.Material of specimens and input data source indicated in parenthesis.Probabilistic input CT(2024-T351)SENT(2024-T3)Single lap joint(2024-T3)Yield strength2024-T3/T351)U U–Fracture toughness(2024-T351)U––Fatigue crack growth rateoffset(2024-T3)U U UInitial crack size(2024-T3)–U U1044W.A.Grell,z/International Journal of Fatigue32(2010)1042–1049the crack growth rate variability.To set crack growth behavior for agiven specimen,the entire piece-wise curve is offset based on a single scale factor,‘‘FCGR Offset”.The piece-wise curve is deﬁned with mean and standard deviations for da/dN at each D K eff point. The FCGR Offset is a standard normal variate that is used to offset each point according to the mean and standard deviation.This pro-cess will offset the entire curve from the baseline and simulate overall variability in the crack growth rate rather than cycle-to-cy-cle variability.The overall crack growth rate variability contributes much more to scatter in fatigue life,as the cycle-to-cycle variabil-ity is averaged out over the entire fatigue life[11].Also,it has been observed that,when tested under identical conditions,a specimen with faster crack growth rates generally remained faster than other specimens over its life[6].pact tension(CT)specimen predictionsConstant amplitude fatigue experimental data on30identical CT specimens of Al2024-T351plate were taken from the work by Wu and Ni[12].Commonly used for material characterization, the CT specimens were50.0mm wide and12.0mm thick.Pre-cracking started at c=15mm and extended to c=18mm.Each specimen was tested under the same maximum and minimum loads of P=4.5and0.9kN at a frequency of15Hz for pre-cracking and the remainder of testing.Specimen failure occurred at56,314 cycles on average with a standard deviation of10,231cycles[12].The data from the work of Wu and Ni[12]are plotted in Fig.3as cumulative probability(CDF)versus cycles to failure.A lognormal distribution wasﬁt to the experimental data with the following parameters:shape=0.1802and scale=10.922.A Kolmogorov–Smirnov(K–S)test was conducted to show that the lognormal dis-tribution was acceptable(level of signiﬁcance a=0.05,D=0.170 was less than D0:0530=0.242).In addition to plotting the distribution ﬁt,Fig.3also includes the5%and95%conﬁdence intervals on the mean.The fatigue life data appears bimodal indicating the poten-tial for a different mechanism or growth rates in the short and long life specimens.In the current work,probabilistic life prediction analysis was performed for the CT geometry with dimensions and loading con-ditions from Wu and Ni[12].Variability in fracture toughness,yield strength,and fatigue crack growth rate was considered.The initial crack size wasﬁxed at c=18mm.The Monte Carlo(MC) method was implemented with NESSUS and UNIPASS for1000iter-ations each.More efﬁcient techniques were also considered, namely the MV and AMV methods with NESSUS and FORM with UNIPASS.Experimental and predicted fatigue life distributions are plotted as cumulative distribution functions(Fig.3),obtained by ordering the data from smallest to largest and uniformly distrib-uting them along the y-axis.The predictions compared reasonably well and captured the variability in the shortest fatigue lives(CDF values<0.5),but failed to predict the overall amount of scatter and the longest fatigue lives.The amount of variability in the crack growth rate curve must not have accounted for the amount of scat-ter present in the experimental data.The AMV and FORM results followed the MC predictions very closely,but required only a small fraction of the iterations.To evaluate the accuracy of the different probabilistic methods,their predictions of fatigue life for aﬁxed probability of failure,P f,of0.01were compared.This low probabil-ity was chosen on the grounds that conservatism dictates the shortest fatigue lives be considered in design to ensure a low P f.A summary of the predicted lives using the different methods is shown in Table2.A comparison with the AMV+method applied at this probability level was also performed to conﬁrm conver-gence and agreed well with results from the other methods.The analyses indicate that similar results can be obtained with AMV or FORM in less than one percent of the time required for MC.Nor-Table2Comparison of CT life prediction and efﬁciency for different probabilistic methods.(N=NESSUS,U=UNIPASS).The shortest experimental life was43,172cycles(P f=0.03)[12].Method Life(cycles)P f=0.01#of Trials Analysis timeN-MC-1k42,697100017hU-MC-1k43,671100016hN-MV43,29244minN-AMV43,49555minN-AMV+43,56555minU-FORM43,91187minW.A.Grell,z/International Journal of Fatigue32(2010)1042–10491045malized sensitivity factors at P f=0.01from the AMV analysis iden-tiﬁed the most important parameters(Fig.4).Yield strength and fracture toughness variability are not contributing signiﬁcantly to the scatter in fatigue life,and thus may be modeled as determinis-tic variables in order to expedite future predictions.3.3.Single edge notch tension(SENT)specimen predictionsData from constant amplitude fatigue experiments on similar single edge notch tension(SENT)specimens were taken from [15,30].Designed to simulate a rivet hole in an aircraft structure, the SENT specimens were machined from Al2024-T3sheet and had nominal dimensions of203mm long,45mm wide,and 2.54mm thick with a notch radius of r=2.813mm.The corre-sponding elastic stress concentration factor at the notch root was 3.165[15].Between the two studies,a total of24fatigue tests were conducted at the120MPa stress range with an R ratio(minimum-to-maximum stress)of0.01.While the tests were carried through to failure,the number of cycles to crack breakthrough,deﬁned as crack transitions from a nominally semi-elliptical crack to a through-thickness crack,was reported[15,30].Considering results from both studies,the life-to-breakthrough averaged198,515cycles with a standard devia-tion of146,200cycles.The results of the combined dataset are plotted as cumulative probabilities(CDF)versus the number of cy-cles to breakthrough in Fig.5.A K–S test(a=0.05)was again used to show that the lognormal distributionﬁt the SENT data accept-ably,resulting in the following distribution parameters: shape=0.6583and scale=11.982.The distribution along with the5%and95%bounds on the mean are also shown in Fig.5.In addition,tests were stopped periodically to create replicas of the notch root surface in order to observe and determine the details of crack formation at constituent particles[15].Crack nucleating particles were identiﬁed and their dimensions measured digitally.Probabilistic life predictions for the SENT specimens were con-ducted considering variability in yield strength,initialﬂaw size, and fatigue crack growth rate.As the simulations predicted fatigue life-to-breakthrough,fracture toughness was not a factor.The ini-tialﬂaw was assumed to be semi-elliptical with input distributions for the half-width,a,and depth,c.Again,the MC,MV,AMV,and FORM methods were implemented in NESSUS and UNIPASS.The predicted distributions of life-to-breakthrough showed good agreement with the experimental data over the entire CDF (Fig.5),with accurate predictions of the mean and important shortest lives.Furthermore,the AMV and FORM methods gave very similar results to the MC simulations in small fractions of the time. The MV predictions differed considerably from the experimental data for probabilities away from0.50;this result is somewhat ex-pected as the MV method assumes linear behavior which causes inaccuracies in non-linear systems.Each method’s predictions are compared for P f=0.01in Table3;again,the AMV+method was ap-plied at this probability level to conﬁrm convergence and agreed well with the AMV and FORM methods.This illustrates another case where AMV and FORM may be used to give similar predictions in small fractions of the time of MC.The sensitivity factors from the AMV analysis(Fig.6for P f=0.01)indicated that the initial crack size was most important,while the yield strength contributed neg-ligibly to fatigue life scatter.3.4.Single lap joint predictionsConstant amplitude fatigue experimental data were taken from the work by Moreira et al.[31].They conducted tests on45identi-cal single lap joint specimens at maximum remote stress of 160MPa and stress ratio of0.05[31].The single lap joint speci-mens consisted of a single column and three rows of rivets joining two160mmÂ20mmÂ1.2mm thick Al2024-T3sheets.TheTable3Comparison of SENT life prediction and efﬁciency for different probabilistic methods.(N=NESSUS,U=UNIPASS).The shortest experimental life was68,525cycles(P f=0.04)[15,30].Method Life to breakthrough(cycles)P f=0.01#of Trials Analysis timeN-MC-1k68,4651000 1.3hU-MC-1k72,8641000 1.3hN-MV24,035517sN-AMV75,327620sN-AMV+72,850937sU-FORM73,5051040s1046W.A.Grell,z/International Journal of Fatigue32(2010)1042–1049overlap was 60mm and the rivets rows were spaced 20mm apart.The specimen was designed to represent a normal rivet pitch of an aircraft fuselage.Fatigue cracks were observed to initiate at and grow from the ﬁrst (or third)rivet hole on the faying surface side [31].The faying surface is the surface of contact between the two sheets.In the work by Moreira et al.[31],the average fatigue life to fail-ure was 77,688cycles with a standard deviation of 18,320cycles.Their results are shown as cumulative probabilities (CDF)versus fatigue life in Fig.7.Based on a K–S test (a =0.05),a lognormal dis-tribution acceptably ﬁt the single lap joint life data,resulting in the following distribution parameters:shape =0.2348and scale =11.235.The distribution,along with the 5%and 95%bounds on the mean,is shown in Fig.7.The single lap joint specimen of Moriera et al.[31,35]was sim-ulated using the probabilistic AFGROW model incorporating vari-ability in initial crack size and crack growth rate.The yield strength and fracture toughness values were modeled as determin-istic as they did not affect the results in CT and SENT specimen pre-dictions.The crack was observed to be quarter elliptical in the lap joint study [31]and in typical observations for similar geometries [36].Three loading conditions were considered together:the by-pass stress from the remotely applied load,the bearing stress from the load transfer through the rivet,and the bending stresses due to the eccentricity of the joint.The stress intensity factor solutions for these cases have been developed by ﬁnite element techniques for the quarter elliptical crack [37,38]and the oblique through crack [39]and are implemented in AFGROW.Each loading condition is dealt with separately,and they are superposed to determine the stress intensity factor and crack growth rate.The solutions given by Newman and Raju [37],Zhao et al.[38],and Fawaz [39]are for any location along the elliptical crack front,denoted by u .;however,AFGROW uses values for u of 10°and 80°for the c and a solutions,respectively,to improve computational efﬁciency [40].The stress levels for the cases of tension,bending,and bearing loads were determined based on the geometry of the joint and a re-mote stress of 160MPa.At the ﬁrst (or third)rivet where failure occurs,the remote tensile load is divided into bypass and pin com-ponents.The bypass load is that which is transferred around the rivet hole through the top sheet.The pin load is that which is trans-ferred though the rivet to the bottom sheet.In this geometry,37.5%of the remote load is transferred through the ﬁrst rivet,leaving 62.5%as bypass load [35].In the analysis [35],friction between the sheets was neglected.Due to the eccentricity of the joint,a bending moment is produced inducing a bending stress in theExp. Moriera et al.Exp. log-normal fit Monte Carlo NESSUS MV NESSUS AMV NESSUS AMV+UNIPASS FORMSingle Lap JointTable 4Comparison of single lap joint life prediction and efﬁciency for different probabilistic methods.(N =NESSUS,U =UNIPASS).The shortest experimental life was 41,486cycles (P f =0.02)[31].Method Life (cycles)P f =0.01#of Trials Analysis time N-MC-1k 45,168100040min N-MV 26,40649s N-AMV 49,228512s N-AMV+46,0111229s U-FORM46,893821sW.A.Grell,z /International Journal of Fatigue 32(2010)1042–10491047。

江苏省苏州市2023-2024学年高一下学期期中阳光指标调研英语试卷

江苏省苏州市2023-2024学年高一下学期期中阳光指标调研英语试卷一、阅读理解Explore more in Thailand’s parts unknownThailand’s southern beaches attract travelers worldwide, but there’s more to explore in the Southeast Asian country.Ban Krut for beach fansIn Ban Krut, travelers will find one of the cleanest and quietest white sandy beaches within driving distance of the capital, Bangkok. This sleepy seaside community, known mostly by locals, is a five-hour drive or six-hour train trip down the Gulf of Thailand. Don’t miss the great Wat Tang Sai, a Buddhist temple located on Thong Chai Mountain.Lopburi for history loversLopburi, one of Thailand’s oldest cities, is home to Khmer-era temples and the uncrowded ruins of King Narai’s Palace, which was built in the 1600s. It’s also known for the monkeys that gather at Phra Prang Sam Yot temple in the center of town. A tip: Keep a safe distance from the monkeys and hide anything you don’t want them to steal.Cave (洞穴) for holiday hikersHiking trails, wetlands and forests make Khao Sam Roi Yot National Park a weekend adventure worthy of topping your Thailand to-do list. The park’s crown jewel is the amazing Phraya Nakhon Cave. Outdoorsy travelers can camp in a park or choose better rooms in the nearby tourist town of Hua Hin.Car rental costs about $20 per day, and an international driver’s permit is required. You can also hire a driver at most major car rental companies, book a taxi or explore by train.1．What do we know about Ban Krut?A．It is a crowded tourist destination.B．It is where Wat Tang Sai is located.C．It is the cleanest and quietest beach.D．It is known to the people nationwide.2．What will happen if you get close to monkeys in Lopburi?A．They might stare at you curiously.B．They might suddenly hide themselves.C．They might offer you a friendly wave.D．They might take away your belongings.3．Which of the following best suits people who enjoy adventures?A．The Thong Chai Mountain.B．The tourist town of Hua Hin.C．The Phra Prang Sam Yot temple.D．Khao Sam Roi Yot National Park.From the deserts of Southern California to the forests of Alaska, the U. S. possesses a diversity (多样性) of nature. For most of her life, Joy Ryan never saw the scenery beyond her home state of Ohio. Then, when she was in her mid-eighties, her grandson Brad Ryan decided to take her on a journey to all of the U. S. National Parks, which took eight years to complete. At the age of 93, Grandma Joy has finally seen all 63 parks, ending her journey in the National Park of American Samoa.“When I learned she had never seen the great wildernesses of America-deserts, mountains, oceans, I decided to intervene (干预) in some way,” Brad says. This idea inspired their first trip to the Great Smoky Mountains. However, once they were on the road, Brad realized that they could visit more parks that were nearby. Over the years, they managed to see all of the famous sights at Yosemite, the Grand Canyon, the Redwoods, and so on. But Brad values the time spent with his grandmother just as much as the amazing places they were able to visit.Despite Grandma Joy’s older age, she was able to hike mountains. Brad regards her love for life as the key to her good health. She is grateful for each new day and is always excited for the next journey. Finally, their long journey across the U.S. came to an end with American Samoa.“I am thankful every morning for giving me another day,” Grandma Joy shares. “And you’re your choice whether you’re going to have a rainy day or a sunny day. So, smile at everybody, and let everybody know you’re having a whale of a time.” While her tour of the U. S. has come to a close, she and her grandson are not done traveling. Next up, they have their sights set on Kenya.4．Why did Brad decide to intervene in some way?A．To celebrate Grandma Joy’s 93rd birthday.B．To complete a task he promised Grandma Joy.C．To enrich Grandma Joy’s travelling experiences.D．To realize Grandma Joy’s dream of visiting parks.5．Which of the following best describes their tour in Brad’s eyes?A．Tough and challenging.B．Tiring but educational.C．Stressful but eye-opening.D．Amazing and worthwhile.6．Which factor mainly contributes to Grandma Joy’s good health?A．Her grandson’s inspiration.B．Her positive attitude.C．Her regular exercise.D．Her adventurous spirit.7．What can be a suitable title for the text?A．Stay Curious, Stay Young B．Never too Old to Hit the RoadC．Laughter, the Best Medicine D．Travelling Contributes to Long LifeSome of us like to keep some things private — maybe we don’t want people knowing about our relationship, our age or where we live — because these details are personal. The problem is, everywhere we go online, we seem to leave a digital footprint.On our social media accounts, we often present things like our opinions, connections and holiday photos. The issue is, how do we stop everybody seeing everything? Maybe we don’t want a stranger knowing our favourite type of coffee. And cookies — there was a time they were just something nice to eat. Now they seem to appear unexpectedly on websites. So how can you stay more private?There are some steps you can take. First, on your social media accounts, check your privacy settings. Many social media sites allow everything to be seen by default (默认), and the responsibility is on you to set your boundaries. And limiting the amount you share isn’t only confined (局限于) to social media. There are many sites out there that help you to share documents — so a good tip may be to not put anything too private, like passwords, on there. One careless click may send it to the wrong person.Finally, cookies are designed to improve your surfing experience by tracking and savinginformation about your visits to a site, helping to personalise your sessions. Deleting (删除) your using history may help, but there are certain apps which can block them if you don’t feel comfortable.So, if you want to maintain your privacy online, maybe only share things you’d be happy with a stranger seeing — and if you don’t feel comfortable with cookies, you might decide to apply some tech to block them. If that’s not enough, just don’t accept them and visit another site — and why not have a yummy cookie to eat instead?8．What is the article aimed at?A．Reminding you to update information.B．Advising you to maintain privacy online.C．Encouraging you to live a simple digital life.D．Warning you to delete social media accounts.9．Why is it important to check privacy settings on social media accounts?A．To increase the number of followers.B．To block all the cookies from tracking.C．To limit the amount of information shared.D．To prevent strangers from viewing using history.10．Which step does the author suggest readers take to stay more private?A．Disable some social media sites.B．Share passwords with familiar ones.C．Store documents on public sites.D．Use suitable apps to block cookies.11．What is the last paragraph mainly about?A．Concerns for personal privacy.B．Recommendation of private apps.C．Suggestions for protecting privacy.D．Application of high technologies.It is a well-known fact that not all animals have the ability to instantly recognize themselves in the mirror. However, a few animals can recognize themselves, such as dolphins, Asian elephants, and of course humans! A species of fish with the ability to recognize itself in the mirror is commonly known as the cleaner fish.Found in the Indian Ocean and the Pacific Ocean, cleaner fish exist as “cleaning stations” for other fish (known as hosts). They “clean” the dead skin and parasites (寄生虫) from other fish. This is of benefit for cleaner fish, which get a meal, as well as for the other fish species, which are freed from parasites. Such a type of relationship is known as mutualism.Recently, a group of scientists in Japan found that cleaner fish might have the ability to identify (识别) themselves in photographs! It was already known that cleaner fish could recognize themselves in mirrors and would attack unknown cleaner fish. So, the team wanted to test whether the same was seen in the case of photos as well.Thus, the team of scientists showed four types of photos to the cleaner fish. The first type was a photo of themselves, while the second involved an unknown cleaner fish. The third type of picture was a photo of an unknown cleaner fish with the fish’s own face edited onto it. The fourth type of photo included its own body but with the face of an unknown cleaner fish.They found that cleaner fish attacked only those photographs which did not have their own faces. This result was similar to the ability of humans to recognize each other based on their faces and not by their bodies.This study suggests that self-awareness may be far more widespread among animals than scientists once thought. People shouldn’t be surprised that fish could have a sense of self given that they have already been shown to have complex behavior including tool use and planning. 12．What does the underlined word “mutualism” probably mean in paragraph 2?A．A host-parasite relationship.B．A master-servant relationship.C．Dependence on each other.D．Beneficial cleaning behavior.13．What is the new finding about cleaner fish?A．They can identify themselves in the mirror.B．They can recognize themselves in the images.C．They have the same sense of self as humans.D．They have a better sense of self than some animals.14．Which types of photos would cleaner fish most probably attack?A．The first and second types.B．The second and fourth types.C．The first and third types.D．The second and third types.15．What is the author’s attitude toward the study?A．Unclear.B．Doubtful.C．Supportive.D．Unconcerned.Humiliation (蒙羞). Many of us have experienced that fearful feeling of just wanting the ground to open up and get down, right? 16 But what is humiliation, and how can we move on from it?Humiliation is quite often compared to the feeling of shame. Shame is more of a private feeling of regret and dishonor, which results from having said or done something wrong or stupid. For example, a good person will feel ‘shameful’ for speaking or doing something mean to a friend.17 However, the case of humiliation differs. It is described as a strong feeling that comes with loss of self-respect. Imagine your workmates shock you by unfairly calling you out on your work during a meeting with your boss. Or your friend may look down upon you by questioning your knowledge in a hobby or interest in front of your other friends. 18So, how can you deal with it? According to psychologist Robert J. Sternberg, there are a few steps you can take. First, you must accept the humiliation and realize you’re not the only one who has felt like this. 19 Also, don’t immediately try and fight back for ‘round 2’ —often humiliation isn’t personal.Lastly, resilience (恢复力) seems to be extremely important. Some people say: “What doesn’t kill you only makes you stronger”. 20 Why not use the disadvantage as motivation (动力) to become a better worker or learn more about that hobby or interest? It could be an opportunity to start spending more time in doing something you love. That way it may have an unexpected benefit.A．While humiliation probably won’t kill you, it can hurt.B．It occurs when someone decides to make us feel small.C．It makes that person feel bad and regretful of his behavior.D．Therefore, talk to others who have experienced it and move on.E．Thus, this article helps to understand this painful private feeling.F．The presence of others is the key factor—a public dressing-down.G．So, take the challenge to make it known to the public and fight back.二、完形填空I have always enjoyed cold climates and connecting to the environment.In November 2023, I joined the UK Antarctic Heritage Trust (英国南极遗产信托基金会), the charity that looks after British heritage in Antarctica, as their 21 . I’m responsible for sorting the letters that are sent here, the southernmost post office in the world. 22 ,I observe the environment and 23 my neighbours, the penguins(企鹅).Five of us now share the island with 1,000 penguins, who of course are given 24 . They have their own paths to walk down to the shore(岸边)and we get used to slowing down, always keeping our 25 , and waiting for them to pass. Every two days we 26 the nests and eggs, and send that information to British Antarctic Survey.When we arrived here in November, the base had been shut for winter, 27 when we got off the ship, we dug 28 through snow for hours before we could open the front door. 29 our lives here takes a lot of work, as we’ve got no running water, no showers, and there is a lot of snow 30 .As postmaster, I’ve 31 20,000 letters so far, including postcards from stamp 32 . They request particular postmarks or stamps, and can be very 33 about where they want them placed.On our days off, we enjoy going on long walks around the island, taking more time for all the 34 things, such as slight changes in the weather and tiny green plants. Sometimes I feel 35 simply by looking at the sun reflected in the water.21．A．reporter B．postmaster C．researcher D．explorer 22．A．In other words B．In turn C．In addition D．In summary 23．A．keep track of B．guard against C．take control of D．get hold of 24．A．duties B．supplies C．preference D．treatment 25．A．secrets B．balance C．word D．distance 26．A．clean B．remove C．count D．replace 27．A．so B．but C．because D．or 28．A．easily B．breathlessly C．unwillingly D．separately 29．A．Discovering B．Maintaining C．Escaping D．Enriching30．A．fighting B．remaining C．disappearing D．digging 31．A．processed B．written C．delivered D．collected 32．A．collectors B．sellers C．buyers D．makers 33．A．hesitant B．curious C．anxious D．specific 34．A．small B．obvious C．powerful D．important 35．A．worried B．touched C．confused D．surprised三、语法填空阅读下面材料，在空白处填入适当的内容(1个单词)或括号内单词的正确形式。

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冠词 Article 的用法

in hospital in the hospital
在住院在医院里
in school in the school
在学习；在学校上课在学校里
out of question 不成问题；豪无疑问 out of the question 不可能；办不到
go to school
上学
go to the school 到学校去
6）在称呼或表示官衔，职位的名词前不加冠词： The guards took the American to General Lee. 士兵们把这个美国人送到李将军那里。
7）在三餐、球类运动和娱乐运动的名称前，不加冠词如：
have breakfast，play chess 8）当两个或两个以上名词并用时，常省去冠词：
go to hospital go to the hospital
go to bed go to the bed
去看病到医院去
去睡觉向那张床走去
5
冠词用法歌诀
名词是秃子，常要戴帽子。可数名词单，须用a或an。辅音前用a，an用元音前。若为特指时，则须用定冠。复数不可数，泛指the不见。碰到代词时，冠词均不现。
12）如果名词前已有this, that, these, those, whose, which, my, your, their, his, her, it词，意义不同
试比较：
at table at the table
在进餐在餐桌旁
Where do you live? I live on the second floor. 你住在哪？我住在二层。
That‘s the very thing I’ve been looking for. 那正是我要找的东西。

sdarticle3

Analysis of Homoisoflavonoids in Ophiopogon japonicus by HPLC-DAD-ESI-MS nMin Ye and Dean GuoThe State Key Laboratory of Natural and Biomimetic Drugs,School of Pharmaceutical Sciences,Peking University,Beijing,People’s Republic of ChinaGuan Ye and Chenggang HuangShanghai Institute of Materia Medica,Chinese Academy of Sciences,Shanghai,People’s Republic of ChinaThe homoisoflavonoids in Ophiopogon japonicus(Thunb.)Ker-Gawler were analyzed by high-performance liquid chromatography-diode array detection-electrospray ion trap tandem mass spectrometry(HPLC-DAD-ESI-MS n).Homoisoflavonoids gave prominent[MϪH]Ϫions by electrospray ionization monitored in the negative ion mode.They could be classified into two types depending on the fragmentation behavior of their[MϪH]Ϫions in the ion trap mass analyzer.The[MϪH]Ϫions of homoisoflavonoids with a saturated C2-3bond underwent C3-9bond cleavage to lose the B-ring,which was followed by the loss of a molecule of CO.The[MϪH]Ϫions of homoisoflavonoids with a C2-3double bond usually eliminated a CO molecule first,and then underwent the cleavage of C3-9or C9-1=bonds.For homoisofla-vonoids with a C-6formyl group,however,the neutral loss of CO was the first fragmentation step;the presence of a methoxyl group at C-8could lead to the cleavage of C-ring.No retro Diels-Alder(RDA)fragmentation characteristic for normal flavonoids was observed.The above fragmentation rules were reported for the first time,and were implemented for the analysis of homoisoflavonoids in O.japonicus.The CHCl3-MeOH extract was separated on a Zorbax Extend-C18column,eluting with a acetonitrile-0.3%acetic acid gradient.A total of18 homoisoflavonoids,including seven new minor constituents,were identified or tentatively characterized based on the UV spectra and tandem mass spectra of the HPLC peaks.(J Am Soc Mass Spectrom2005,16,234–243)©2004American Society for Mass SpectrometryF lavonoids are a class of natural products with aC6-C3-C6skeleton widely distributed in theplant kingdom.They have been reported to be the major bioactive constituents of medicinal herbs because of their antioxidant,anti-inflammatory,and anti-carcinogenic activities [1, 2]. The development of simple and rapid methods for the analysis of fla-vonoids is therefore of great significance in the qual-ity control of herbal medicine.The plant extract, however,is a very complex mixture,and contains a variety of secondary metabolites.A potent and cur-rently widely used technique to analyze plant ex-tracts is fingerprinting,especially by high-perfor-mance liquid chromatography-diode array detection (HPLC-DAD) [3]. An informative and convincing fingerprint should have most of the diagnostic peaks elucidated.Unfortunately,such routine techniques as HPLC-DAD could only provide very limited struc-tural information like UV spectrum;standard com-pounds,which are commercially unavailable in most cases,are usually necessary for the characterization of individual constituents.As a result,the isolation and purification from crude plant extracts of ade-quate amounts(at least5–10mg)of pure compounds (Ͼ90%purity)for nuclear magnetic resonance(NMR) identification was needed before they could serve as reference compounds.The whole process is tedious, laborious,and expensive.Moreover,some constitu-ents are only present in raw plant materials in very low amounts,and their enrichment and purification are extremely difficult.High-performance liquid chromatog-raphy-mass spectrometry(HPLC-MS)combines the effi-cient separation capabilities of HPLC and the great power in structural characterization of MS,and pro-vides a new powerful approach to identify the constit-uents in plant extracts rapidly and accurately [4]. In addition,mass spectrometer is a very sensitive detector. In many cases,minor constituents in plant extracts that are difficult to obtain by conventional phytochemical means could be detected,especially when selected ion monitoring(SIM)technique was used.Thus,HPLC-MS could also be used to discover new constituents from medicinal herbs.Presently,HPLC-MS has been used for the analysis of a variety of natural products.Flavonoids,especiallyPublished online December21,2004Address reprint requests to Professor D.Guo,The State Key Laboratory ofNatural and Biomimetic Drugs,School of Pharmaceutical Sciences,PekingUniversity,Xueyuan Road38,Beijing100083,People’s Republic of China.E-mail:gda@©2004American Society for Mass Spectrometry.Published by Elsevier Inc.Received September13,2004 1044-0305/05/$30.00Revised November4,2004 doi:10.1016/j.jasms.2004.11.007Accepted November8,2004flavonols,flavones,and their glycosides,are the most frequently reported [5–12]. Electrospray ionization (ESI)and atmospheric pressure chemical ionization (APCI)sources are usually used as the interface between HPLC and a mass spectrometer.Homoisoflavonoids are a type of special flavonoids,with their B-and C-rings connected by an additional CH 2group.These compounds are rare in plants and few reports can be found on their HPLC-MS analysis.Ophio-pogon species (Liliaceae)are abundant in homoisofla-vonoids.Up to now,at least 30homoisoflavonoids have been isolated from these plants [13–19] (Figure 1). Re-cently,we isolated five pure homoisoflavonoids from O.japonicus ,a traditional Chinese medicine (Mai-Dong)fre-quently used as a tonic drug in the clinic.These com-pounds have been reported to be important active constit-uents for its therapeutic effects [15]. In this study, we investigated the fragmentation behaviors of homoisofla-vonoids in an electrospray ion trap mass spectrometer,and analyzed the homoisoflavonoids in O.japonicus by high-performance liquid chromatography-diode array de-tection-electrospray ionization-tandem mass spectrometry (HPLC-DAD-ESI-MS n ).ExperimentalStandards and ReagentsPure homoisoflavonoids 1–5were isolated from the tubers of Ophiopogon japonicus (Thunb.)Ker-Gawler by the authors.Their structures were unambiguously iden-tified by NMR techniques,and their purities were above 98%as determined by HPLC.HPLC grade acetonitrile (Fisher,Loughborough,UK)and ultra-pure water were used for all analyses.The chloroform and methanol for plant extraction and acetic acid used in mobile phase was of ARgrade,Figure 1.Chemical structures of homoisoflavonoids identified from Ophiopogon japonicus .235J Am Soc Mass Spectrom 2005,16,234–243ANALYSIS OF HOMOISOFLAVONOIDS BY LC/MSpurchased from Beijing Chemical Corporation(Beijing, China).Plant Materials and Sample PreparationThe tubers of O.japonicus were purchased from a drug store in Beijing.The materials were pulverized and dried to constant weight before use.An aliquot of1g of the sample was extracted with50ml of chloroform-methanol(1:1)in an ultrasonic water bath for30min and then filtered.The filtrate was evaporated to dryness and the residue was dissolved in1ml of acetonitrile. The solution was filtered through a0.45-␮m micropore membrane prior to use.A volume of10␮l was injected into the HPLC instrument for analysis.HPLC ConditionsThe analyses were performed on an Agilent series1100 HPLC instrument(Agilent,Waldbronn,Germany) equipped with a quaternary pump,a diode-array de-tector,an autosampler,and a column compartment.The sample was separated on a Zorbax Extend-C18column (5␮m,␸4.6ϫ250mm,Agilent).The mobile phase consisted of acetonitrile(A)and water containing0.3% acetic acid(B).A gradient program was used as follows: 40%A in the first10min,linearly gradient to65%A over25min,then hold for10min.The mobile phase flow rate was1.0ml/min;the detector was monitored at285nm;column temperature was set at25°C. Mass SpectrometryFor HPLC-MS analysis,a Finnigan LCQ Advantage ion trap mass spectrometer(ThermoFinnigan,San Jose,CA) was connected to the Agilent1100HPLC instrument via an ESI interface.The LC effluent was introduced into the ESI source in a post-column splitting ratio of2:1. Ultrahigh pure helium(He)was used as the collision gas and high purity nitrogen(N2)as the nebulizing gas. To tune the mass spectrometer,a0.1mg/ml solution of methylophiopogonanone A(1)in acetonitrile(MeCN) was pumped into a0.5ml/min MeCN-0.3%HOAc(1:1, vol/vol)LC eluate at a steady rate of2.5␮l/min by using a TEE union.The combined flow was allowed to enter the ESI source for ionization.The mass spectrom-eter parameters were adjusted to obtain maximum response of the[MϪH]Ϫion at m/z341.The optimized parameters in the negative ion mode were as follows: ion spray voltage,4.5kV;sheath gas(N2),45arbitrary units;auxiliary gas(N2),10units;capillary temperature, 320°C;capillary voltage,Ϫ15V.For full scan MS analysis,the spectra were recorded in the range of m/z 120–800.A data-dependent program was used in the liquid chromatography-tandem mass spectrometry analysis so that the two most abundant ions in each scan were selected and subjected to MS/MS and MS3 analyses.The collision-induced dissociation(CID)en-ergy was adjusted to45%.The isolation width of precursor ions was3.0mass units.Results and DiscussionTandem Mass Spectrometry of Pure Standards APCI and ESI sources were tried for the ionization of homoisoflavonoids both in positive and negative ion modes.Either source gave[MϪH]Ϫions as the base peak in the negative mode,while[MϩH]ϩions with remarkably lower intensity were obtained in the posi-tive mode.APCI and ESI produced very similar ions. Therefore,ESI in the negative ion mode was selected as the ion source for follow-up analyses.Pure Compounds1–5(0.1mg/ml in MeCN)were respectively injected into the ESI source by continuous infusion.The[MϪH]Ϫions were selected for CID fragmentation to produce MS/MS spectra.The promi-nent MS/MS ions were then selected for further MS n analysis(nϭ3up to5).The collision energy ranged from30to50%.Unexpectedly,the homoisoflavonoid[MϪH]Ϫions underwent new and unique fragmentations,which were completely different from the retro Diels-Alder(RDA) cleavage characteristic for flavones and flavonols [20].The five analyzed homoisoflavonoids could be clearly classified into two groups according to their fragmentation behavior.Interestingly,the classification was well correlated with the saturation status of their C2-3bond.For Compounds1,2,and3,which contain saturated C2-3bonds,the predominant fragmentation of their[M ϪH]Ϫions was the cleavage of C3-9bond to lose theB-ring,followed by the neutral loss of a molecule of CO (Scheme1).This class of homoisoflavonoids would be cited as Type I in this paper for convenience.For Compounds4and5,where the C-2and C-3are connected by a double bond(Type II),however,the[M ϪH]Ϫions appeared to be more stable.They produced a major product ion by losing a CO molecule first,and then underwent the cleavage of C3-9or C9-1=bonds to lose the B-ring(Scheme2).For any homoisoflavonoid with a formyl group at C-6,the fragmentation was triggered by the initial loss of a molecule of CO.Fragmentation of Standards1,2,and3When applied to an collision energy of50%,the[MϪH]Ϫion at m/z341of methylophiopogonanone A(1) produced a prominent ion at m/z206,which should result from the cleavage of C3-9bond.Similar ions could be found in the electron impact(EI)mass spectra of homoisoflavonoids.The major difference was that the B-ring fragments were usually base peaks in the EI spectra,whilst they could not be observed in the ESI spectra.The m/z206ion was further subjected to MS3 analysis to produce a signal at m/z178,which should be attributed to the loss of the carbonyl group(CO,⌬mϭ236YE ET AL.J Am Soc Mass Spectrom2005,16,234–24328)at C-4.The m/z 178ion then underwent successive losses of CH 3(⌬m ϭ15)or CO to generate ions at m/z 163, 150, and 135 (Figure 2). The above product ions allowed us to propose a fragmentation pathway for Compound 1,as depicted in Scheme 1.The fragmentation behaviors of 1could also be applied to Compounds 2and 3.The [M ϪH]Ϫion of Compound 2at m/z 327gave a prominent product ion at m/z 206,which further yielded ions at m/z 178,163,150,and 135in the MS 3spectra by the same pathway as Compound 1.The fragmentation of Compound 3was somewhat different from 1and 2.The MS/MS spectrum of its [M ϪH]Ϫion at m/z 355gave a base peak at m/z 327,due to the neutral loss of a CO molecule.We assumed this fragmentation should involve the loss of the formyl group at C-6,since free carbonyl group is easy to lose by ␣-cleavage.The obtained product ion at m/z 327then undertook similar fragmentations as quasi-molecular ions of 1and 2(Scheme 1).Fragmentation of Standards 4and 5The [M ϪH]Ϫion (m/z 339)of methylophiopogonone A (4)fragmented into m/z 311at a 50%energy collision.There could be two possible ways to form this ion.One was to lose the carbonyl group (CO)at C-4,and the other involved the conversion of methylenedioxyl group at B-ring into a hydroxyl group (Scheme 2).The first pathway was normal for the loss of 28mass units,and was described above for Type I homoisoflavonoids.Unfortunately,no supporting product ions could be observed in the MS 3spectrum of Compound 4.Thus,C 2-3double bond seemed to inhibit the cleavage of C-4carbonyl group.The second fragmentation pathway had rarely been reported before,but the MS 3ions at m/z 205and m/z 218strongly suggested the presence of a carbonyl group in their parent m/z 311ion.Very similar fragmentations had been reported recently for apor-phine alkaloids with a methylenedioxyl group [21].With its extended ␲-conjugation,the ion at m/z 311was relatively stable and fragmented only upon 50%energy collision to form the ions at m/z 205and 218,presum-ably due to the cleavages of C 3-9and C 9-1=bonds,respectively.Also with a formyl group at C-6,the [M ϪH]Ϫion at m/z 353of 5first underwent the loss of CO like Compound 3.The obtained m/z 325ion thenproducedm/z 136m/z 192m/z 164m/z 327Scheme 1.Proposed MS fragmentation pathway for the [M ϪH]Ϫions of Compounds 1,2,and 3.237J Am Soc Mass Spectrom 2005,16,234–243ANALYSIS OF HOMOISOFLAVONOIDS BY LC/MSions at m/z 297,204,and 191in the same pathways as Compound 4.HPLC-DAD-ESI-MS n Analysis of the Plant ExtractFigure 3 shows the HPLC-UV and TIC profiles of the extract of Ophiopogon japonicus .A variety of solvents were tried for the extraction of homoisoflavonoids from Ophiopogon tubers.Pure meth-anol (MeOH)could extract homoisoflavonoids effec-tively,but suffered from the concomitant extraction of large amounts of saponins and saccharides,which remarkably affected the HPLC-MS chromatogram.Chloroform (CHCl 3)and acetonitrile extracts gave good HPLC profiles,while the more polar homoisofla-vonoids could not be readily extracted.Finally,a mix-ture of MeOH-CHCl 3(1:1,vol/vol)was used.The homoisoflavonoids,including the minor ones,could be effectively extracted,and most HPLC peaks were base-line resolved in a 45-min run.Acetic acid was added in the mobile phase to im-prove the ionization of homoisoflavonoids,though they did not alter their chromatographic retention signifi-cantly [22].The UV detector was monitored at 285nm to make sure all homoisoflavonoids gave good responses.The HPLC peaks were preliminary identified as homoisoflavonoids according to their on-line UV spec-tra.Because of different ␲-conjugation extensions,Type I homoisoflavonoids show a maximum absorption band at 294–298nm,while Type II at 262–266nm.When a C-6formyl group is present,however,the band shifts to 274–276nm.The structures were further elu-cidated based on their tandem mass spectra.Fragmen-tation rules described above for standards 1–5were extensively used for the elucidation.At least 25homoisoflavonoids were detected from the extract of O.japonicus ,and 18of them were charac-terized (Table 1). Compounds 1–5 were identified by comparing their HPLC retention times and UV spectra with the standards.In the HPLC-MS analysis,they gave MS and MS n spectra almost identical with those ob-tained by continuous infusion.Identification of Type I HomoisoflavonoidsIn addition to 1–3,eleven C 2-3saturated homoisofla-vonoids (Type I)were plausibly identified.Five of them were known constituents previously reported from Ophiopogon species.The other six compounds were reported for the first time and were tentatively charac-terized on the basis of their tandem mass spectra.The peak at 11.23min (no.4)gave a [M ϪH]Ϫionat2m/z 204m/z 297m/z 191..Scheme 2.Proposed MS fragmentation pathway for the [M ϪH]Ϫions of Compounds 4and 5.238YE ET AL.J Am Soc Mass Spectrom 2005,16,234–243Figure2.Full scan MS and MS n spectra for Compound1.(a)full scan MS spectrum;(b)MS2 spectrum of the[MϪH]Ϫion at m/z341;(c)MS3spectrum of the m/z206ion(341Ͼ206);(d)MS4 spectrum of the m/z178ion(341Ͼ206Ͼ178).239J Am Soc Mass Spectrom2005,16,234–243ANALYSIS OF HOMOISOFLAVONOIDS BY LC/MSm/z373.Its MS/MS and MS3spectra yielded prominent ions at m/z207and m/z179,respectively.These ions should result from C3-9bond cleavage,followed by the neutral loss of CO,which were characteristic for Type I homoisoflavonoids.The above fragments were consis-tent with the known5,7,4=-trihydroxy-3=,5=-dimethoxy-6,8-dimethyl homoisoflavanone(10),and allowed the identification of Peak4.The pseudo-molecular ion(m/z343)of Peak6(t Rϭ13.35min)yielded very similar MS/MS and MS3spec-tra as Peak4,with m/z207and m/z179ions as the base peak,respectively.However,ions at m/z192and163 resulting from the neutral loss of15mass units were also observed as abundant fragments in the MS3spec-trum of Peak6,suggesting the presence of a methoxyl group in A-ring [8, 11]. Although a Type I homoisofla-vonoid(5,7,4=-trihydroxy-3=-methoxy-6,8-dimethyl ho-moisoflavanone,without a methoxyl group at A-ring) with molecular weight of344Da had been isolated from O.japonicus,Peak6was tentatively identified as the new5,4=-dihydroxy-7,3=-dimethoxy-8-methyl homoiso-flavanone.Peak9(t Rϭ21.22min)and Peak14(t Rϭ27.42min, 2)both gave a[MϪH]Ϫion at m/z327.This pair of isomers could be distinguished by their MS/MS spec-tra,which yielded m/z192and206ions as the base peak,respectively.They suggested different substitu-tion patterns at the A-ring.By examining known ho-moisoflavonoids,Peak9was characterized as ophio-pogonanone A(6).Peak1(t Rϭ7.23min)gave a[MϪH]Ϫion at m/z 329.Its MS/MS spectrum gave a base peak at m/z207, consistent with it being a Type I homoisoflavonoid.In the MS3spectrum,an abundant demethylated product at m/z192(75%relative intensity,⌬mϭ15)was observed besides the expected m/z179ion(⌬mϭ28), suggesting the presence of a methoxyl group in A-ring. The neutral loss of a methyl radical was common for methoxylated flavonoids [8, 11]. Thus, Peak 1 was plausibly identified as5,3=,4=-trihydroxy-7-methoxy-8-methyl homoisoflavanone.Peak8(t Rϭ17.69min)gave a[MϪH]Ϫion at m/z355. Its MS/MS spectrum yielded prominent ions at m/z340 and205,and excluded the possibility of the known ophiopogonanone C(5,7-dihydroxy-3=,4=-methylenedi-oxy-8-aldehydo-6-methyl homoisoflavanone,MWϭ356). These two ions should result from the loss of a methyl radical and the cleavage of C3-9bond,respectively.Thus, Peak8was tentatively identified as5-hydroxy-7-methoxy-3=,4=-methylenedioxy-6,8-dimethyl homoisoflavanone. Peak10(t Rϭ22.35min)gave a[MϪH]Ϫion at m/z 313.No homoisoflavonoid with molecular weight of 314Da had been reported from Ophiopogon species, hence it should be a new constituent.The MS/MS spectrum gave ions at m/z192and164.Based on the general fragmentation rules of Type I homoisofla-vonoids,Peak10was preliminarily characterized as 5,7-dihydroxy-4=-methoxy-6-methyl homoisoflavanone.Three peaks in the HPLC chromatogram gave a quasi-molecular ion at m/z341,including CompoundRT:0.00 - 45.01SM:3GFigure3.HPLC-DAD-ESI-MS n analysis of the CHCl3-MeOH(1:1)extract of Ophiopogon japonicus.(a)HPLC-UV chromatogram monitored at285nm.(b)LC-negative ion ESI-MS total ion current(TIC)profile.240YE ET AL.J Am Soc Mass Spectrom2005,16,234–2431.Peak5(t Rϭ12.20min)gave prominent MS/MS ions at m/z326and191,suggesting the presence of a methoxyl group.Its structure was plausibly estab-lished as5-hydroxy-3=,4=-dimethoxy-6,8-dimethyl ho-moisoflavanone.The third peak appeared at37.23 min(no.18).Its MS/MS spectrum gave a base peak at m/z313(⌬mϭ28).The preferential loss of CO was characteristic for homoisoflavonoids with a formyl group.The MS2and MS3spectra were consistent with the known5,7-dihydroxy-4=-methoxy-6-aldehydo-8-methyl-homoisoflavanone(9),and allowed the par-tial characterization of Peak18.Peak7(t Rϭ14.37min)represented a main constitu-ent of O.japonicus.It gave a[MϪH]Ϫion at m/z359.A moderate intensity ion at m/z223was observed in the MS/MS spectrum,attributed to C3-9bond fragmenta-Table1.Characterization of homoisoflavonoids by HPLC-DAD-tandem mass spectrometry from Ophiopogon japonicusPeak no.Retention time(tR,min)UV␭max(nm)[M-H]Ϫm/z HPLC-ESI-MS a m/z(%base peak)Identification17.23296329MS2[329]:207(100)MS3[207]:192(75),179(100),165(25),163(40)5,3=,4=-trihydroxy-7-methoxy-8-methyl homoisoflavanone28.18296373MS2[373]:358(20),355(30),222(5),183(100),168(35),153(20)MS3[183]:168(100)5,2=-dihydroxy-7,8,4=-trimethoxy-6-methyl homoisoflavanone39.92–339MS2[339]:324(100),311(5)MS3[324]:296(30),295(100),237(5)5-hydroxy-7,4=-dimethoxy-6, 8-dimethyl homoisoflavone411.23–373MS2[373]:207(100)MS3[207]:179(100)5,7,4=-trihydroxy-3=,5=-dimethoxy-6,8-dimethyl homoisoflavanone(10)512.20–341MS2[341]:326(50),191(100)MS3[326]:191(100)5-hydroxy-3=,4=-dimethoxy-6,8-dimethyl homoisoflavanone613.35296343MS2[343]:207(100)MS3[207]:192(45),179(100)163(15)5,4=-dihydroxy-7,3=-dimethoxy-8-methyl homoisoflavanone714.37296359MS2[359]:344(100),223(15),Ophiopogonanone E(7)208(12),169(85),154(43)MS3[344]:326(20),208(100),154(15)MS3[169]:154(100)817.69296355MS2[355]:340(40),205(100)MS3[340]:205(100)MS3[205]:190(35)177(100),161(60)5-hydroxy-7-methoxy-3=,4=-methylenedioxy-6,8-dimethyl homoisoflavanone921.22294327MS2[327]:205(20),192(100),MS3[192]:164(40)Ophiopogonanone A(6) 1022.35294313MS2[313]:192(100),164(15)5,7-dihydroxy-4=-methoxy-6-methyl homoisoflavanone 1124.23–339MS2[339]:311(100),219(25)Methylophiopogonone A(4) 1225.67296341MS2[341]:206(100),178(40)MS3[206]:178(100)MS3[178]:150(100)Methylophiopogonanone A(1)1325.71–373MS2[373]:358(30),237(100),222(20),183(55),168(20),153(5)MS3[237]:222(100)Ophiopogonanone F(8)1427.42296327MS2[327]:206(100),178(15)MS3[206]:178(100)Methylophiopogonanone B(2)MS3[178]:150(100)1534.79276353MS2[353]:325(100)MS3[325]:297(80),204(20)6-aldehydo-isoophiopogonone A(5)1635.53274355MS2[355]:337(30),327(100),307(65),193(45)MS3[327]:192(100),164(30)MS3[307]:279(100)6-formyl-isoophiopogonanone A(3)1736.03–339MS2[339]:324(8),311(100),296(5)MS3[324]:296(100)MS3[311]:296(100)6-aldehydo-isoophiopogonone B(11)1837.23274341MS2[341]:323(50),313(100),220(50),193(45)MS3[313]:192(100),164(10)MS3[220]:192(100),178(10)5,7-dihydroxy-4=-methoxy-6-aldehydo-8-methyl homoisoflavonanone(9)–UV absorption maximum unavailable because of low amounts.241J Am Soc Mass Spectrom2005,16,234–243ANALYSIS OF HOMOISOFLAVONOIDS BY LC/MStion.The base peak at m/z 344suggested the presence of a methoxyl group.Thus,Peak 7was identified as the known ophiopogonanone E (7).The m/z 344ion was further subjected to MS 3analysis,and a prominent ion at m/z 208resulting from C 3-9bond cleavage was produced,while no signal at m/z 223was observed.Hence,the m/z 344ion should be attributed to the loss of CH 3at A-ring.Another abundant ion at m/z 169in the MS/MS spectrum could not be explained by previ-ously known fragmentation pathways.We assumed it should result from the simultaneous cleavage of C 2O O and C 4-11bonds.This ion could further fragment into m/z 154by CID.The fragmentation pathway for Com-pound 7is given in Scheme 3.The new fragmentation behavior for Compound 7was also observed in Peak 2(t R ϭ8.18min)and Peak 13(t R ϭ25.71min).They represented a pair of isomers,both giving a [M ϪH]Ϫion at m/z 373.Their MS/MS spectra gave ions at m/z 358,237,and 222,generated by the loss of a methyl radical and C 3-9bond cleavage.They were 14units greater than the corresponding ions at m/z 344,223,and 208observed in Compound 7,suggesting the presence of an addi-tional methoxyl group in the A-ring,presumably at C-5or C-7.The above fragments were consistent with the known ophiopogonanone F (8),containing a C-5methoxyl group.Coincidentally,Compounds 7and 8both had a C-8methoxyl group.This structural characteristic might be the reason for the new frag-mentation pathway.Indeed,an abundant ion at m/z 183was observed in the MS/MS spectra for both Peak 2and Peak 13,which corresponded to the m/z 169ion in Compound 7.The next work was to differentiate the two isomers.Their MS/MS spectra were ex-tremely similar,except that an ion at m/z 237,due to the direct C 3-9bond cleavage from [M ϪH]Ϫions,was observed as the base peak in the spectrum for Peak 13,while it was not observed in Peak 2.We assumed it depended on whether or not the [M ϪH]Ϫion was easy to lose a methyl radical.When a methoxyl group was present at C-7,the [M ϪH]Ϫionfavored to eliminate a methyl group to form a more extended and stable ␲-conjugation structure.All fur-ther product ions would derive from the resulting m/z 358ion.On the other hand,the loss of a methyl radical at C-5was not likely to produce a more stable structure.Therefore,Peak 13(with the m/z 237ion)was identified as the known ophiopogonanone F (8),and Peak 2was tentatively characterized as a C-7methoxylated isomer,5,2=-dihydroxy-7,8,4=-trime-thoxy-6-methyl homoisoflavanone.Identification of Type II HomoisoflavonoidsRelatively fewer Type II homoisoflavonoids were de-tected from O.japonicus .Besides standards 4and 5,only one known compound was identified.It appeared at 36.03min in the HPLC chromatogram (Peak 17)and gave a [M ϪH]Ϫion at m/z 339.In addition to the ion at m/z 311attributed to the initial loss of CO,an ion at m/z 324also was observed in the MS/MS spectrum,indicating the presence of a methoxyl group.Based upon the above evidences,Peak 17was identified as the known 6-aldehydo-isoophiopogonone B (11).In its MS/MS spectrum,the m/z 311ion was much more abundant than m/z 324,since the elimination of formyl group was easier than methyl radical.No C 3-9or C 9-1=bond fragmentation characteristic for Type II ho-moisoflavonoids was observed in the HPLC-MS analy-sis because of the low amounts of precursor ions and their relatively stable structures.Another peak at t R 9.92min (Peak 3)gave almost identical MS and MS/MS spectra to Compound 11,except that the m/z 324ion was more abundant than m/z 311.This result excluded the presence of a formyl group in the molecule,and the m/z 311ion should result from the cleavage of C-4carbonyl group.Peak 3was thus tentatively character-ized as 5-hydroxy-7,4=-dimethoxy-6,8-dimethyl ho-moisoflavone,which was reported from Ophiopogon species for the first time.This structure was consistent with our assumption that C-7methoxyl group could trigger the loss of 15mass units,as observed for Peak2.3m /z 223m/z 208m/z 154Scheme 3.Proposed MS fragmentation pathway for the [M ϪH]Ϫions of Compounds 7.242YE ET AL.J Am Soc Mass Spectrom 2005,16,234–243ConclusionsIn this paper,the fragmentation behavior of ho-moisoflavonoids in electrospray ion trap mass spec-trometer was studied.The observed fragmentation pathway was very different from those of flavones and flavonols.The major fragmentation of C2-3saturated homoisoflavonoids(Type I)was C3-9bond cleavage, which was followed by the loss of carbonyl group(CO). Homoisoflavonoids with a C2-3double bond(Type II) usually eliminated a CO molecule first,and then under-went the cleavages of C3-9or C9-1=bonds.The pathway for CO elimination in Type II homoisoflavonoids was still unclear,and needs further investigation with com-pounds devoid of a methylenedioxyl group.When a C-6formyl group was present,the homoisoflavonoids readily lost one molecule of CO to form a base peak. The unique MS fragmentation of homoisoflavonoids should be due to the additional CH2group(C-9),which divided the structure into two isolated conjugation parts.Like other flavonoids,methoxylated homoisofla-vonoids could lead to the loss of15mass units.This fragmentation appeared to take place more easily for C-7or C-8methoxyl groups than that at C-4=.As a result,demethylated ions were low or could not be observed in compounds with only a C-4=methoxyl group,such as Compounds2and9,and Peak10.In addition,the presence of a C-8methoxyl group ap-peared to trigger the cleavage of C-ring,which might be characteristic for this substitution.However,this frag-mentation should be further confirmed with standard compounds.Based on the above MS fragmentation rules,18 homoisoflavonoids were identified or tentatively char-acterized from Ophiopogon japonicus in one LC-MS n run. Seven of them were reported from Ophiopogon species for the first time.Isomers and close analoges could be distinguished from each other by comparing their MS/MS and MS3spectra.This research set a good example for the rapid identification of bioactive constit-uents in plant extracts and their products. 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annual volatilities of spot prices were 163.91% for copper, 119.05% for chromium, 168.24% for uranium, 153.87% for zinc, and 44.46% for lead. These data show that increasingly more companies are being exposed to high risks in natural resources prices, implying that the stable supply of natural resources is very important for the national economy and for the survival of a company.
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C. Won / Energy 34 (2009) 1215–1224

pricing model which is both easy to apply and realistic in order to help ﬁrms’ investment decision-making.
There are two major competing procedures for evaluating risky projects in natural resources developments: one is decision analytic, based on traditional discounted cash ﬂow and stochastic dynamic programming (Fleten et al. [11], Smith and McCardle [27], Szklo et al. [28]) and the other is option pricing model (OPM) or contingent claims analysis, based on the no-arbitrage theory of ﬁnancial markets (Brennan and Schwartz [3], Ekern [9], Emhjellen and Alaouze [10], Laughton [18], Paddock et al. [22], Schwartz [25], Sezgen et al. [26]). In this paper, we develop a new pricing model of natural resources projects by applying contingent claim analysis in order to help ﬁrms evaluate the projects more accurately and easily. Our model is different from previous models in several respects. First, our model uses different assumptions about underlying variables and different methodology. For example, we treat both output prices and development costs as stochastic and derive a closed-form solution through Martingale framework. While most of the previous literature treats only output prices as stochastic (Brennan and Schwartz [3], Dixit [6], Gibson and Schwartz [14], Laughton [18], Paddock et al. [22], Pindyck [23], Schwartz [24, 25], Sezgen et al. [26]) and does costs as uncertain, we make our model more realistic by treating both output prices and development costs as stochastic processes, because development costs change over time depending upon the ﬂuctuations in materials and labor costs necessary for the developments. In addition, whereas we utilize Martingale principle to derive the pricing equation, other authors use different methods, such as an arbitrage argument through self-ﬁnancing (Brennan and Schwartz [3]), a dynamic decision tree (Smith and McCardle [27]), and a real option framework by considering various managerial ﬂexibility in the process of developments (Cortazar and Casassus [5], Fleten et al. [11], Laughton [18], Olsen and Stensland [21], Paddock et al. [22], Sezgen et al. [26], Szklo et al. [28]).

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Current Biology19,909–914,June9,2009ª2009Elsevier Ltd All rights reserved DOI10.1016/j.cub.2009.03.060Report A Signaling Module Controllingthe Stem Cell Nichein Arabidopsis Root MeristemsYvonne Stahl,1Rene´H.Wink,1Gwyneth C.Ingram,2and Ru¨diger Simon1,*1Institut fu¨r GenetikHeinrich-Heine Universita¨tUniversita¨tsstrasse1D-40225Du¨sseldorfGermany2Institute of Molecular Plant SciencesUniversity of EdinburghMayﬁeld RoadEdinburgh EH93JRUKSummaryThe niches of the Arabidopsis shoot and root meristems,the organizing center(OC)and the quiescent center(QC), orchestrate theﬁne balance of stem cell maintenance and the provision of differentiating descendants.They express the functionally related homeobox genes WUSCHEL(WUS) and WOX5,respectively,that promote stem cell fate in adja-cent cells[1].Shoot stem cells signal back to the OC by secreting the CLAVATA3(CLV3)dodecapeptide[2],which represses WUS expression[3].However,the signals controlling homeostasis of the root stem cell system are not identiﬁed to date.Here we show that the differentiating descendants of distal root stem cells express CLE40, a peptide closely related to CLV3.Reducing CLE40levels delays differentiation and allows stem cell proliferation. Conversely,increased CLE40levels drastically alter the expression domain of WOX5and promote stem cell differen-tiation.We report that the receptor kinase ACR4,previously shown to control cell proliferation[4],is an essential compo-nent,and also a target,of CLE40signaling.Our results reveal how,in contrast to the shoot system,signals origi-nating from differentiated cells,but not the stem cells,deter-mine the size and position of the root niche.Results and DiscussionCLE40Expression Pattern and Mutant PhenotypesCLE40,the closest homolog of the stem cell restricting signal CLV3,is expressed in speciﬁc root tissues,indicating a role in controlling root cell fates.Stem cells on the proximal(toward the shoot)site of the quiescent center(QC)generate vascula-ture and pericycle(Figure1A),lateral stem cells give rise to endodermis,cortex,epidermis,and lateral root cap,and distal columella stem cells(CSC)generate the protective cap of columella cells(CC)with distinct starch granules that sense the gravitationalﬁeld.After expression in the entire globular stage embryo,CLE40becomes successively restricted to the basal regions of the embryo that form the root meristem and the vasculature.After germination, CLE40remains expressed only in the differentiation zone of the stele that forms the inner layers of the root and in differ-entiating CCs(Figures1B–1G).We identiﬁed two loss-of-function alleles of CLE40to investigate the role of CLE40as a potential signal for intercellular communication in the root (see Figure S1available online).cle40mutant roots are shorter[5]and root tips appeared irregularly shaped,indi-cating that CLE40function is required for organized cell divisions in the root meristem.Because CLE40is normally expressed in CCs,we analyzed development of the distal meristem in detail(Figure2).Wild-type roots carry mostly one(at D1position)or,after a recent cell division,two layers of CSCs distal to the QC(at D1and D2positions)which lack stainable starch granules(Figure2A;Table S1).By day5, additional CSCs in more distal positions(D2)were found in 58%of the cle40roots,but in only33%of the wild-type meri-stems,suggesting that differentiation of CSC daughters into CCs was signiﬁcantly delayed when CLE40was lacking (Figure2B;Table S1).CLE40Peptide Promotes Differentiationin the Distal Root Meristem in a Dose-Dependent Manner We asked whether differentiation toward CC fate depends on the dosage of CLE40peptide(CLE40p).Previous studies have shown that synthetic CLE peptides can activate CLE-dependent signaling pathways in shoot and root development [6–8].Growing cle40mutant roots on medium containing1m M synthetic CLE40p largely suppressed the formation of extra CSCs and restored organized cellﬁle formation.However, further increasing the CLE40dosage by CLE40p treatment of wild-type roots,carrying two functional copies of the CLE40 gene,resulted in ectopic starch granule accumulation also in the D1layer,indicating loss of CSC identity(Figures2C and 2D).Differentiation of D1cells was also triggered by CLV3p, which is closely related to CLE40p,but not by the less similar TDIFp,which controls differentiation of xylem cells[7] (Table S1).Together,this indicated that cell identities in the distal meristem are regulated by a signaling pathway that is governed by the dosage of CLE40p;reduction of CLE40 activity permits stem cell proliferation,whereas increased CLE40levels promote differentiation of distal cells.CLE40Regulates WOX5Expression and Distal Cell Fates WOX5acts from the QC to maintain the distal stem cell popu-lation[1],and can functionally replace WUS.Similarly,CLE40 can replace CLV3if expressed from the shoot stem cell domain[5].Together,this suggests that pathways controlling stem cell fate in shoot and root are at least partially conserved at the molecular level.In wox5mutants the distal root meri-stem appears disorganized,and D1cells lose CSC identity and differentiate as CCs[1](Figure3A).Ectopic expression of WOX5inhibits the differentiation of CSC daughters,result-ing in ampliﬁcation of distal cell layers that maintain CSC iden-tity[1].During wild-type development,WOX5activity in the QC may generate a short-ranging signal that sufﬁces to confer CSC identity to D1,but not to D2,cells.Because CLE40 appears to regulate the distal stem cell domain antagonisti-cally to WOX5,we tested whether WOX5expression is subject to regulation by CLE40.In67%of cle40mutant roots,the*Correspondence:ruediger.simon@uni-duesseldorf.deWOX5expression domain expanded from the QC into the adjacent lateral stem cells (Figures 2F,2G,and 2M;Figures S2and S3;Tables S2and S3),suggesting that CLE40is required to spatially conﬁne WOX5during normal develop-ment.Treatment of cle40roots with CLE40p caused a near total restoration of the wild-type expression pattern of WOX5(Figures 2I and 2M).The presence of supernumerary CSCs at the D2position in cle40mutants may thus be caused by increased or ectopic WOX5expression.When wild-type roots were treated with CLE40p,WOX5expression was reduced in the QC and shifted to a more prox-imal position,indicating that the position of the WOX5expres-sion domain along the proximo-distal axis of the root is controlled by CLE40p levels (Figure 2H).To elucidate whether WOX5is functional at the new,more apical position,we used the enhancer trap line QC184that is normally expressed in the QC in a WOX5-dependent manner [1](Figures 2E and 2J;Table S4).In root meristems growing on CLE40p,the QC184expression domain was similarly displaced from the QC and coincided with the new proximal WOX5domain,showing that WOX5expression,but not WOX5function,is regulated by CLE40(Figures 2E and 2J).Cells at the D1position differen-tiate toward CC in these plants,indicating that WOX5signaling from the more proximal location is insufﬁcient for stem cell maintenance at D1.Phenotypically,CLE40p-treated roots strongly resemble wox5mutants,suggesting that WOX5is a major target for repression by CLE40.However,when wox5mutant roots were grown on CLE40p medium,we observed a further prox-imal shift of CC identity,so that cells at the QC position accumulated starch granules (Figures 3A,3D,and 3G).This indicated that CLE40signaling also interferes with the activity of another,WOX5-independent pathway that acts in parallel to WOX5and promotes distal stem cell maintenance.CLV2Is Not Required for Distal Stem Cell Regulation by CLE40The observation that exogenous application of CLE40p repressed WOX5expression in the QC,but still permitted de novo WOX5expression in a proximal region,reveals that CLE40p is differentially perceived along the root axis,which may reﬂect the differential expression of the corresponding receptor protein(s).In several shoot tissues,CLE peptides have been found to signal via transmembrane receptors carrying extracellular LRR domains [9–11].Shoot stem cells of Arabidopsis secrete the CLV3peptide,which is perceived by the CLV1and CLV2/CRN receptors on subjacent cells [12]and downregulates expression of WUS ,a transcription factor that in turn non-cell-autonomously promotes stem cell fate [13].In root tissues,external application of different CLE peptides,including CLE40p,showed that the LRR receptor protein CLV2is required for overall growth restriction and meristem arrest [6].However,the role of CLV2in root develop-ment has remained unclear,because clv2mutant roots appear aphenotypic and show a normal pattern of cell differentiation in the distal meristem (Figure 3B).Interestingly,clv2roots grow to normal length in the presence of CLE40p,but still show differentiation of D1cells toward CC (Figures 3E and 3G).This indicates that exogenous CLE peptide applica-tions require CLV2function in the proximal meristem to repress overall root growth,but not to perceive the CLE40signal in the QC or the distal meristem.ACR4Perceives the CLE40SignalRoots mutant for ACR4,encoding a receptor-like kinase of the CRINKLY4family,carry additional CSCs at the D2position,revealing that ACR4,like CLE40,controls cell fate in the columella lineage [4](Figures 3C and 3G).In acr4mutant roots,the number of WOX5-expressing cells increased,andtheFigure 1.CLE40Is Expressed in the Embryo and Differentiated Root Cells(A)Diagram illustrating root cell positions from proximal (P1)to distal (D1–D4).Color codes indicate cell fates.Stem cells surrounding the QC are outlined in gray.SI,stele initials;QC,quiescent center;CSC,columella stem cell;CC,columella cell;DM,distal meristem;PM,proximal meristem;LRC,lateral root cap;EP,epidermis;C,cortex;EN,endodermis.Gray dots,starch granules.(B–F)CLE40expression (purple-blue)in embryos.Arrowheads,root progenitor cells (blue)and stele (yellow).High probe concentrations (F)detect CLE40expression also in the stele.(G)pCLE40:CLE40-GFP expression (green)in the stele and CC of a lateral root.The scale bars represent 25m m in (B)–(D)and 50m m in (E)–(G).Current Biology Vol 19No 11910Figure 2.CLE40Regulates Cell Fates in the Distal Meristem(A–D)Cell fates in wild-type (Col-0),cle40-2mutant,or CLE40peptide (CLE40p)-treated root meristems.(A)Wild-type root;arrowheads:blue,QC position;yellow,CSC;red,CC.(B)cle40-2root lacking starch in D1and D2positions,indicating CSC fate in D2.(C)Col-0root treated with CLE40p shows starch in D1,indicating CC fate.(D)CLE40p treatment of cle40-2restores wild-type pattern.(F–I)WOX5expression;white asterisks mark WOX5-expressing cells at QC position.(F)WOX5RNA is conﬁned to QC position.(G)In cle40-2,WOX5expression expands laterally.(H)CLE40p induces proximal shift of WOX5domain.(I)CLE40p treatment of cle40-2restores WOX5in QC.(E and J)Expression of WOX5-dependent QC184reporter (light blue)and starch granules stained by Lugol’s (violet).(J)CLE40p triggers proximal shift of both reporter expression and CC identity.The scale bars represent 20m m.(K)Frequency of roots carrying starch granules in the designated domains.(L)Frequency of WOX5expression in the designated domains.(M)Changes in number of WOX5-expressing cells at QC position.In Col-0,WOX5expression is detected in two cells.Error bars represent the standard error means.Asterisks mark statistically signiﬁcant changes compared to wild-type analyzed via Student’s t test (p <0.001).CLE40Signaling Adjusts Distal Root Stem Cell Fate 911expression domain expanded into lateral positions(Table S2). Thus,WOX5expression was similarly affected in both cle40 and acr4mutants.Treatment of acr4mutants with CLE40p caused only a minor increase in the differentiation of D1cells toward CC(Figures3F and3G),indicating that ACR4acts to perceive the CLE40signal.ACR4Is Positively Regulated by CLE40Expression of ACR4reporters in root meristems is detected mainly in the D1–D3layer,the adjacent lateral root cap,and epidermis initial,but only occasionally,and at a low level,in the QC itself[4,14,15](Figures4A and4D;Table S5).Wild-type roots grown in the presence of CLE40p showed strongly increased ACR4expression that systematically incorporated the QC position(Figures4B and4E).This upregulation was speciﬁc for CLE40p and the closely related CLV3p,and not observed when the less similar TDIFp was used(Figures4C and4F).This alteration in ACR4expression paralleled the proximal displacement of the WOX5expression domain, indicating that CLE40could act via ACR4to regulate WOX5, and that ACR4is also a target gene regulated by CLE40signaling.Figure 3.Targets and Receptors Involved inCLE40Signaling(A and D)In wox5-1roots,D1cells acquire CCfate;CLE40p induces CC fate also in QC position.(B and E)clv2-3mutants are not affected in distalmeristem development and respond to CLE40pwith a proximal shift of CC fate like wild-type.(C and F)acr4-2mutants form supernumeraryCSCs and respond only weakly to CLE40p treat-ment,indicating a central role of ACR4in CLE40signaling.(G)Frequency of roots carrying starch granulesin the designated domains.A CLE40-Dependent SignalingModule Controls Distal Stem Cell FateStem cell proliferation needs to bedynamically adjusted in line with chang-ing requirements for differentiating cellsduring different growth phases.In lightof our results,we propose a new sig-naling pathway providing a mechanismfor homeostasis of the stem cell domaindistal to the QC(Figures4G and4H).WOX5signaling from the QC promotesCSC fate in the distal stem cell domain;this signal diminishes toward the rootcap,permitting differentiation of CSCdaughters into CCs that expressCLE40.Increased CLE40levels in turncreate a negative feedback regulatorthat readjusts the WOX5expressiondomain via ACR4.The CLE40-depen-dent regulation of ACR4expressionlevels is intriguing and could supporta robust signal transmission byincreasing receptor levels upon ligandavailability.Alternatively,ligand seques-tering by ACR4could protect proximalcell layers from CLE40signals[16].Because the overall expression pattern of ACR4is not changed,WOX5remains expressed in a proximal domain where ACR4is not present,even when CLE40levels are high.The identiﬁcation of the CLE40/ACR4/WOX5module reveals further molecular parallels between shoot and root stem cell regulation.However,at the operational level,there are impor-tant differences.In the shoot system,the overall size of the stem cell population is variable,and only loosely conﬁned by CLV3-dependent negative feedback regulation to the organizing center[3,17,18].In the root,proximity to the WOX5-expressing QC,the root niche,is essential for stem cell maintenance.CLE40expression from differentiated cells provides the negative feedback signal that balances stem cell proliferation,acting via ACR4to regulate WOX5expres-sion and the position of the niche.How spatiotemporal communication integrates stem cell proliferation with differen-tiation in the shoot system remains to be discovered. Experimental ProceduresPlant AccessionsArabidopsis thaliana ecotype Columbia(Col-0)was used as wild-type. QC184(in the Wassilevskija background)promoter trap line was originallyCurrent Biology Vol19No11 912from the Institut National de Recherche Agronomique T-DNA collection [19].All mutant seeds analyzed were in the Col-0background.wox5-1mutant seeds (SALK_038262)were obtained from the Nottingham Arabidopsis Stock Centre (NASC,UK).acr4-2,pACR4:H2B-YFP ,and pACR4:ACR4-GFP seeds were previously described [14].clv2-3mutants were previously described [13],and seeds were obtained from Kristen Shepard.Mutants cle40-2and cle40-3were identiﬁed as described in Supplemental Data (Figure S1).Plant Growth ConditionsSeeds were surface sterilized with chlorine gas and imbibed in 0.1%(w/v)agarose for 2days at 4 C before being plated onto 0.53Murashige and Skoog (MS)medium with Gamborgs no.5vitamins (Duchefa),0.5g/l 2-(N-morpholino)ethanesulfonic acid (MES),1%(w/v)sucrose ,and 1.2%(w/v)plant agar.Plates were incubated vertically in a growth chamber with constant light at 21 C for 4–5days.For peptide-containing plates,synthetic dodecapeptides were added to a ﬁnal concentration of 1m M.Construction of pCLE40:CLE40-GFP Reporter LineThe 1061bp CLE40promoter plus coding region without stop codon was ampliﬁed from Col-0genomic DNA with primers CT506(50-AAA AAG CAG GCT TAG TTG AAG ACC TCC ATT GGT C-30)and CLEGATE3(50-AGA AAG CTG GGT ATG GAG TAA AAG GAA TGT GTT TAT-30)and cloned via the GATEWAY system (Invitrogen)into the binary plant transformation vector pMDC107containing GFP in-frame.Subsequent transformation of Col-0plants was carried out with the ﬂoral dip method [20].Expression AnalysesWhole-mount in situ hybridizations on 4–5day roots were carried out according to [21]with an automated system (InSituPro liquid-handling robot;Intavis AG).For CLE40and WOX5probe preparations,the complete cDNA sequences were used and prepared as described in [21].Histochem-ical analysis of b -glucuronidase (GUS)activity in enhancer trap line QC184was carried out by incubation of seedling roots in GUS staining solution [0.05M NaPO 4buffer (pH 7.0),5mM K 3Fe(CN)6,5mM K 4Fe(CN)6,10mM X-glucuronide]at 37 C until blue staining was visible,followed by two washes in distilled water and mounting in 70%(w/v)chloral hydrate,10%(v/v)glycerol for microscopy.Starch StainingsStarch granules and cell walls in root tips were stained with the mPSPI method and imaged with a confocal microscope as described in [22].Lugol’s staining of the GUS-stained enhancer trap line QC184was carried out by mounting root tips in a 1:6dilution of Lugol’s solution in 70%(w/v)chloral hydrate,10%(v/v)glycerol and analyzing them after 15min incubation.MicroscopyImage acquisition was carried out with an Axiocam HR camera attached to a Zeiss Axioscope II microscope.Confocal images were taken with a Zeiss LSM 510Meta laser scanning microscope.Counterstaining of cell walls was achieved by mounting seedling roots in 10m M propidium iodide.Supplemental DataSupplemental Data include three ﬁgures and seven tables and can be found with this article online at /current-biology/supplemental/S0960-9822(09)00914-2.AcknowledgmentsWe thank Carin Theres and Cornelia Gieseler for technical assistance,Andrea Bleckmann,Helge Pallakies,and Adrian Hu ¨lsewede for help with expression analyses,Sandra Komnacki for establishing CLE40reporter lines,the NASC and Arabidopsis Biological Resource Center (Ohio State University)for seeds,and D.Schubert,P.Welters,and T.Klein for critical discussion of the manuscript.Y.S.and R.S.designed the experiments,and Y.S.and R.H.W.carried them out.G.C.I.contributed materials and inter-preted data.Y.S.and R.S.wrote the manuscript.All authors discussed the results and commented on the manuscript.This work was supported by a grant of the Deutsche Forschungsgemeinschaft to R.S.through SFB590.Received:March 9,2009Revised:March 26,2009Accepted:March 27,2009Published online:April 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Chemical Engineering and Processing 49 (2010) 396–401Contents lists available at ScienceDirectChemical Engineering and Processing:ProcessIntensiﬁcationj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c epInternally generated seeding policy in anti-solvent crystallization of ceftriaxone sodiumChun-tao Zhang a ,∗,Hai-rong Wang a ,Yong-li Wang ba College of Chemical Engineering and Technology,Wuhan University of Science &Technology,Wuhan,430081,Hubei Province,China bSchool of Chemical Engineering and Technology,Tianjin University,Tianjin,300072,Chinaa r t i c l e i n f o Article history:Received 16December 2008Received in revised form 20October 2009Accepted 5March 2010Available online 12 March 2010Keywords:Crystal size distribution (CSD)Anti-solvent (or dilution)crystallization Ceftriaxone sodium Seeding policya b s t r a c tA promising seeding strategy of internally generated seeds in producing ceftriaxone sodium with a uni-modal crystal size distribution (CSD)was experimentally investigated in batch anti-solvent crys-tallization.Effects of different seeding policies on product CSDs,both external seeding and internally generated seeding,were experimentally determined.A uni-modal product CSD was obtained without obvious nucleation at high external-seed loadings,while a bi-modal CSD was obtained with plenty of secondary nuclei at low external-seed loadings.The internally generated seeds,prepared by adding pure acetone into the non-seeded saturated solution of ceftriaxone sodium,were ﬁne crystals with a uni-modal,narrow CSD,irrespective of the amount of the added diluent.These ﬁne crystals were grown continuously as seeds by slowly adding a relatively low concentration diluent.The product CSD was uni-modal with relatively large mass-weighted mean size,and the product quality was as good as the one obtained by external seeding.Therefore,such an internally generated seeding policy is expected to be a favorable seeding technology to hopefully substitute for the external seeding policy in the pharmaceuti-cal industry,where external seeding is not welcome because the external seeding policy would destroy the axenic conditions in crystallizer and lead to unstable quality of different batches.© 2010 Elsevier B.V. All rights reserved.1.IntroductionBatch anti-solvent (or dilution)crystallization is widely used in the chemical and pharmaceutical industries,where the demand is increasing for product crystals with precisely controlled sizes and shapes,in addition to high purities.Generally,large crystals of uni-modal (or mono-dispersed)size distribution are desirable to improve downstream processes (such as centrifugal separation and drying after crystallization),product effectiveness (e.g.,bioavail-ability and tablet stability)and product yield [1].Kim [2]concluded that the primary bottleneck to the operation of production-scale drug manufacturing facilities was associated with difﬁculties in controlling the size and shape distribution of crystals produced by complex crystallization processes.Therefore,the control of the product CSD can be of paramount importance in industrial batch crystallization.General reviews on the control of product CSD can be referred to recent papers [3–7],books [8,9],and the Proceed-ings of 2nd International Workshop on Industrial Crystallization of China,2004(Tianjin,September 21–25).Recently,considerable academic efforts have been put to study the seeding policy as an effective and necessary technology for∗Corresponding author.Tel.:+862786559634;fax:+862786559634.E-mail address:zct w@ (C.-t.Zhang).the control of product CSD.What is surprising,however,is that most of these emphases are often laid on the external seeding strategy [3–7,10–12].In the pharmaceutical industries,however,the traditional external seeding strategy had several disadvan-tages while the glovebox was opened for seeding:(1)the axenic conditions in crystallizer would be destroyed and the probability of introducing impurities and bacteria into crystallizer increased accordingly,(2)the volatilized poisonous organic solvents would affect the health of the workers and lead to serious environment pollution,and (3)the dispersancy of the external seeds in the industrial crystallizer was seriously bad,lots of seeds formed into many large conglomerations because the mother liquid was sat-urated and the wetting quality between the seeds and mother liquid was very bad,which would seriously affect the efﬁciency of seeding.In order to overcome the above disadvantages of exter-nal seeding strategy,several novel internally generated seeding policies were developed.Gao et al.[13]introduced a coopera-tion nucleation by adding the organic solvent and the ultrasonic wave to prepare internally generated seeds.However,it is difﬁ-cult and high cost to introduce the device of ultrasonic wave into the industrial crystallizer.Doki et al.[14]developed the internal seeding policy in sodium chloride anti-solvent crystallization and predicted such seeding method would be a promising technology in the pharmaceutical industry.However,it was a pity that little attention had been paid to the down-to-earth application of the0255-2701/$–see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.cep.2010.03.001C.-t.Zhang et al./Chemical Engineering and Processing49 (2010) 396–401397internally generated seeding in the cephalosporin industry up till now.Ceftriaxone sodium is a third generation,semisynthetic,broad-spectrum cephalosporin antibiotic and has a high degree of stability in the presence ofˇ-lactamases,both penicillinases and cephalosporinases of gram-negative and gram-positive bacteria. For its excellent properties,such as long elimination half-life, metabolic stability,low nephrotoxic potential,stability toˇ-lactamases,and relatively high intrinsic activity,ceftriaxone sodium has been greatly saleable all around the world and one of the most important parenterally applied antibiotics since it ﬁrst came into the market in1982[15].In the pharmaceutical industry,ceftriaxone sodium is generally puriﬁed by anti-solvent crystallization with ethanol[16]or acetone[17]as diluent(or anti-solvent),because it is heat-sensitive and has weak solubility temperature dependence[18].According to the previous investi-gations by the group of the present authors,product crystals with serious agglomeration,small mean size and sometimes bi-modal size distribution challenged the home industrial manufacturing of ceftriaxone sodium.A survey of the literature indicated that very little work has been carried out on the control of the product CSD of ceftriaxone sodium.The aim of this paper is to introduce a promis-ing seeding policy into the cephalosporin antibiotic industries for preparing product crystals of a desired mean size with uni-modal distribution by batch anti-solvent crystallization,which might be hopefully substitute for the customary external seeding policy. However,the present study intends neither to explore the mech-anisms of crystal growth and nucleation,nor to analyze the batch process itself.In this study,ceftriaxone sodium was crystallized from a saturated solution by adding pure acetone(99vol.%)or an acetone–water mixture(80vol.%acetone)as a diluent.Effects of external seeding and internally generated seeding on product CSD of ceftriaxone sodium were experimentally analyzed.2.Experimental apparatus and methods2.1.MaterialsA white crystalline powder of ceftriaxone sodium was supplied by Shandong Ruiyang Pharmaceutical co.Ltd.and recrystallized in the State Research Center of Industrial Crystallization and Technol-ogy with purity(>99.5%).The product crystals were vacuum-dried at ambient temperature for24h and then stored in a desiccator.Seeds were prepared by grinding and sieving the above recrys-tallized crystals and its mass-weighted mean size was18.23␮m with a uni-modal CSD.The acetone(purchased from Tianjin Chemical Reagent Comp.,China)used for experiments was of analytical reagent grade,and their mass fraction purities were better than99.5%. Distilled–deionized water was used in all experiments.2.2.Experimental apparatusAn800-ml jacketed glass crystallizer,ﬁtted with a thermometer, a marine type impeller,and a glass nozzle with an inner diameter of1mm,was used to perform the experimental study(see Fig.1). All the experiments were conducted at15◦C with a stirrer speed of 300rpm.At the end of each run,all product crystals were vacuum-ﬁltered and washed by pure acetone alternately,and then dried in a vacuum oven at room temperature.All the product CSDs and mass-weighted mean sizes were deter-mined by Malvern Mastersizer S(Malvern Instruments Ltd.,British) in the State Research Center of Industrial Crystallization and Tech-nology,Tianjin.Fig.1.2.3.External seeding policyA200ml acetone aqueous solution(50vol.%),saturated with ceftriaxone sodium at15◦C,was prepared in the crystallizer,into which seed crystals were added from outside.Then,the pure ace-tone(99vol.%)or the acetone aqueous solution(80vol.%)began to be introduced continuously at a constant rate through the glass nozzle.The seed loading was changed over a wide range,and the adding rate of the diluent was controlled to be1,2and15ml/min for the80vol.%acetone and2ml/min for the pure acetone.In order to keep the same theoretical product yield(10g),the total amount of the added diluent was300ml for the80vol.%,and56ml for the pure acetone,respectively.Afterﬁnishing the addition of diluent, batch operation would last for another10min in order to con-sume the residual supersaturation in crystallizer.The experimental conditions of external seeding were listed in Table1.2.4.Fine crystal generation experimentsFine crystals were generated by adding pure acetone into a 200ml,non-seeded acetone aqueous solution(50vol.%)that was saturated with ceftriaxone sodium at15◦C.The amount of the added pure acetone was changed over a range(10–56ml)at a feed rate of2ml/min.When the amount of the added pure acetone was 56ml,two levels of feed rate(1and2ml/min)were performed.The moment that the pure acetone addition wasﬁnished,the crystal-lization operation was stopped and theﬁne crystals were separated from the suspension immediately byﬁltering and washing.2.5.Internally generated seeding policyFine crystals,generated by the introducing10ml pure acetone into the non-seeded saturated ceftriaxone sodium solution(200ml, 50vol.%acetone)at a feed rate of2ml/min,were grown inter-nally(without removal)to product crystals by continuously adding 150ml diluent(80vol.%acetone)at a constant rate of2ml/min.Table1Feeding conditions in external seeding experiments.Diluent80vol.%acetone Pure acetone(99vol.%) Amounts of seeds[g]0.1120.112Feed rate[ml/min]12152Added amount[ml]30030030056Feed time[min]3001502028Operation time[min]3101603038Theoretical crystal yield=10g.398 C.-t.Zhang et al./Chemical Engineering and Processing49 (2010) 396–401Fig.2.3.Results and discussion3.1.Effects ofﬁltration and washing on product CSDsAdditional experiments were performed to study the effects of ﬁltration and washing operations on product CSDs before the seed-ing experiments.The results,shown in Fig.2,distinctly indicated the effects ofﬁltration and washing operations on product CSDs could be ignored.So,the product CSDs of all the seeding experi-ments could be compared with each other.3.2.Effects of external-seed loadings on product CSDsThe product CSDs,obtained by adding80vol.%acetone solution at a feed rate of2ml/min,varied with the different seed load-ings as shown in Fig.3.The product CSD was widespread and bi-modal for a low external-seed loading of0.1g.The right peak of the CSD was considered to be the grown external seeds and agglomerates,while the left could be assigned to the grown new nuclei which was generated by secondary nucleation.Owing to low seed loading,the supersaturation-generating rate was higher than the supersaturation-consuming rate by seed growth,so the residual supersaturation near the nozzle,through which the dilu-ent was injected,was much high,so plenty of new nuclei occurred near the nozzle,accordingly.On the other hand,for a relatively high external-seed loading of1g,a uni-modal CSD was observedand the mass-weighted mean size of the product crystalswasFig.3.Fig.4.38.00␮m,which was closely in agreement with the theoreticalvalue(40.54␮m)calculated from Eq.(5)(shown later in Sec-tion3.4),assuming no changes in the crystal number and shape.This agreement meant that the crystallization process was domi-nated by the growth of external seeds with suppressing nucleation.What’s more,no obvious nucleation was also observed at an evenhigher external-seed loading of2g.However,the peak of CSDshifted to the small size direction because of the material balanceconstraint that an increase in the number of individual crystalswould decrease the size of the grown part of each crystal.Therefore,the seeding effect on suppressing nucleation was also effective inbatch anti-solvent crystallization processes,and the same conclu-sion had been drawn by Doki et al.[14]and Chivate et al.[19]inearly studies.3.3.Effects of feed rate and diluent concentration on productCSDs in external seeding policyThe product CSDs,obtained at various feed rates and diluentconcentrations with an external-seed loading of1g,were shownin Fig.4.In case of the addition of80vol.%acetone solution,theproduct crystals,obtained at a feed rate of15ml/min,includedmuch moreﬁne crystals than that of a feed rate of2ml/min.In addition,the product CSD became bi-modal with many smallcrystals in the pure acetone case even at a low feed rate of2ml/min.Thus,the effects of external seeding on product CSDdepended not only on feed rate but also on diluent concentration,which were two commonly used operation parameters to con-trol the average(or bulk)supersaturation-generating rate in batchanti-solvent crystallization.The above results suggested that theaverage supersaturation-generating rate played an important rolein producingﬁne crystals.However,the amount of theﬁne crystals generated inanti-solvent crystallization was not governed by the averagesupersaturation-generating rate only,which was contrary to ourexpectations.As shown in Table1,all the external seeding experi-ments were performed under the condition of the same theoreticalcrystal yield of10g,which exactly corresponded to the super-saturation created by the diluent addition.Therefore,the averagesupersaturation-generating rate is inversely proportional to thefeed time.The fastest average supersaturation-generating rate wasrealized at a feed rate of15ml/min(feed time=20min)for theaddition of80vol.%acetone,but the amount of the generatedﬁnecrystals was less than that at a rate of2ml/min(feed time=28min)for the addition of pure acetone.Accordingly,in addition to theaverage(bulk)supersaturation,the local high supersaturation cre-ated near the nozzle,through which the different concentrationC.-t.Zhang et al./Chemical Engineering and Processing 49 (2010) 396–401399Fig.5.diluent was injected,also had a signiﬁcant effect on the generation of ﬁne crystals.It could be concluded that secondary nucleation caused by the local high supersaturation occurred easily in the anti-solvent crystallization under the operation conditions of relatively low seed loading and high diluent concentration.Despite the fact that either a slow feeding rate or low acetone concentration is favorable to obtain good product CSD and quality of ceftriaxone sodium,as seen from Fig.4,the slow feeding rate and low diluent concentration are,however,not recommended from an industrial point of view of production rate or crystal yield,either of which is associated with economic beneﬁts.Assuming no residual supersaturation remained at the end of a batch,the theoretical crystal yield can be calculated by using the solubility model of ceftriaxone sodium in water-acetone system which was expressed as the h equation [20]:ln 1+(1−x 2)x 2= h1T −1T m(1) =11.77exp(−8.6448w )(2)h =121.36exp(8.5231w )(3)where w is the per cent weight concentration of acetone in acetone–water.As seen in Fig.5,the theoretical crystal yield normalized with a total initially dissolved amount of ceftriaxone sodium varied with the acetone concentration in the crystallizer,in addition to the dilu-ent (acetone)concentration added.As the acetone–water (diluent)was added,the acetone concentration in the crystallizer increased,whereas the crystal yield did not increase monotonically,i.e.it increased at ﬁrst,then passed through a maximum,and ﬁnally decreased.This was because that ceftriaxone sodium was very sol-uble in water and the absolute amount of water in the crystallizer increased simultaneously with an increase of the acetone concen-tration in the crystallizer by the addition of diluent.In the early stages of the introduction of the diluent (acetone–water),the effect of acetone concentration (solubility effect)dominated,leading to an increase in the deposit of crystals,while in the ﬁnal stage after the maximum,the effect of the absolute amount of the mixture,which included much water,prevailed.Owing to much addition of diluted anti-solvent at the end of a batch,small crystals,which had been crystallized out,began to dissolve,which would lead to a lower product yield in addition to a smaller coefﬁcient of varia-tion of the CSD.However,the increase of product period and the decrease of product yield implied a loss of economic considerations especially in the high value-added manufactures of ﬁne chemicals and pharmaceuticals,a much slow feeding rate and low diluent concentration were not favorable in the practical manufacturesaccordingly.Fig.6.3.4.Seed chartThe mass-weighted mean particle size,¯L 4,3,can be given as fol-lows [9]:¯L4,3= ∞0L 3n (L )dL ∞n (L )dL1/3=m 3m 01/3=W k v N1/3(4)where L is the characteristic size of particles,n (L )is the populationdensity distribution function,m j is the j -th moment of CSD,W is the crystal mass, is the particle density,k v is the volumetric shape factor,and N is the total number of particles.Assuming no generation of new nuclei during the crystallization process after seeding and no residual supersaturation at the end of the batch,the total particle number of the seeds should equal to the value of the product crystals.Thus L PL S= 1+C SC S1/3(5)where L P ,L S are the mass-weighted mean size of product crystals and seeds,respectively,and the seed loading ratio C S ,C S =W S /W th ,is deﬁned as the ratio of the added seed amount (W S )to the maxi-mum theoretical yield (W th )calculated from solubility.The ideal mass-weighted mean size of products normalized with that of the seeds L p /L S ,calculated from Eq.(5),was plotted in Fig.6as a function of the seed loading ratio C S .This diagram was named as the seed chart by Kubota for the ﬁrst time and more details could be referenced to the recent literatures of the Kubota group [6,7,10–12,14].The mass-weighted mean size of products increased with the seed loading ratio,and then approached the ideal growth line,and eventually coincided with the ideal growthline at a critical seed loading ratio C ∗S,except in the case of adding pure acetone.The value of C ∗Svaried as the supersaturation-generating rate (the feed rate or the diluent concentration)in batchanti-solvent crystallization,while the C ∗Swas always constant in cooling crystallization for a given seed size,irrespective of the aver-age supersaturation-generating rate,i.e.cooling mode [11,12].Such behavior in the anti-solvent crystallization could be considered mainly by having plenty of new nuclei near the glass nozzle,where local high supersaturation was generated by insufﬁcient mixing of the diluent with the bulk solution.The Kubota group [11,14]had demonstrated that the criticalseed loading ratio C ∗S,determined by the seed chart in a laboratory crystallizer,could be directly applied for the design of an industrial batch cooling crystallizer.However,the determination of a critical seed loading in industrial batch dilution crystallizer by using seed400 C.-t.Zhang et al./Chemical Engineering and Processing49 (2010) 396–401Fig.7.a chart experimentally obtained in a laboratory has received little attention.3.5.Effects of feed rate and diluent concentration on productCSDs of internally generated seedsThe CSDs of the internally generatedﬁne crystals of ceftriaxone sodium,obtained by introducing diluent into a non-seeded satu-rated solution of ceftriaxone sodium at15◦C with different feed rates and concentrations,were shown in Fig.7.In the case of the addition of80vol.%acetone solution,the product CSDs were wide-spread,irrespective of the feed rate,while in the case of the addition of pure acetone,the product CSDs were relatively narrow.The effect of the amounts of pure diluent added at a rate of2ml/min on prod-uct CSD was shown in Fig.8.The results indicated the CSDs were almost the same,irrespective of the added amount of the diluent. What’s more,theﬁne crystals yield agreed with the theoretical out-put calculated from solubility data[20]by assuming no residual supersaturation.Therefore,the above results meant that it was pos-sible to generate a desired amount ofﬁne crystals with an identical narrow CSD as seeds in batch anti-solvent crystallization.3.6.Product CSD of the internally generated seeding policyThe CSDs of the external seeds,the internally generated seeds, and the grown internally generated seeds were shown in Fig.9.The internally generated seeds,obtained by adding10ml pureace-Fig.8.Fig.9.tone into a200ml non-seeded solution(50vol.%acetone)saturatedby ceftriaxone sodium at a feed rate of2ml/min,had a uni-modaldistribution with a mass-weighted mean size of10.39␮m and atheoretical mass was1.35g,calculated from a mass balance byassuming no residual supersaturation.The generatedﬁne crystalshad not been removed,and were kept to subsequently grow by con-tinuously adding an80vol.%acetone at a feed rate of2ml/min.Thenormalized mass-weighted mean size was larger than the valueof the ideal growth line(seen in Fig.6),which clearly indicateda decrease in number of the internal seeds during the subsequentgrowth time and the overestimation of the seed mass.The Environ-mental Scanning Electron Microscope(Philips XL30ESEM)analysisof the product crystals of ceftriaxone sodium,seen in Fig.10,revealed that the agglomeration of internalﬁne seeds occurred.Thesame agglomeration behaviour was observed in both batch anti-solvent crystallization and batch cooling crystallization by Doki etal.[12,14].As well known,serious agglomeration is usually considered asigniﬁcant problem for pharmaceutical crystallization because ofsolvent and impurity inclusions,and hence,the pharmaceuticalcrystallization had to avoid the agglomeration to the best of itsabilities.As a result of the lamellar habit,it was hard to completelyavoid agglomeration in the anti-solvent crystallization of ceftriax-one sodium,especially in industrial manufacturing of high initialconcentration and fast adding rate of anti-solvent.So,agglomera-tion was found to occur in nearly all ceftriaxone sodium purchasedfrom home and abroad markets through ESEM.The feedback of theproduct quality reports,determined mainly by high-performanceliquid chromatography(HPLC),showed that the productqualityFig.10.C.-t.Zhang et al./Chemical Engineering and Processing49 (2010) 396–401401would not violate the requirements of the pharmacopoeia until that the extent of agglomeration was serious.Although a lower initial concentration in crystallizer and a slower addition rate of anti-solvent might lower the extent of agglomeration,neither of these conditions is feasible from the point of view of industrial manufacturing because a lower initial concentration in crystallizer means smaller throughput and lower yield,while a slower addition rate of anti-solvent means longer product period needed for each batch.In addition,the polymorphism of ceftriaxone sodium has not been reported so far,what’s more,the polymorph transient in the anti-solvent crystallization of ceftriaxone sodium has not been observed yet.So the internally generated seeds could achieve the same polymorph as the external seeding procedure.The feedback of the product quality reports had shown that the agglomeration phenomenon,occurred in internally generated seeding experiments,did not result in any serious problem in prod-uct CSD and quality of ceftriaxone sodium.Therefore,the above strategy,ﬁrstly generating internal seeds by the introduction of a relatively high concentration of diluent,and then followed by slow addition of another diluent of relatively low concentration,is expected to be a useful technique to industrially manufacture cef-triaxone sodium crystals of a uni-modal size distribution in batch anti-solvent crystallization without external seeding.4.ConclusionsBatch anti-solvent crystallization of ceftriaxone sodium was conducted under two seeding policies,external seeding and inter-nally generated seeding.A promising seeding strategy of internally generated seeds in producing ceftriaxone sodium with uni-modal CSD was experimentally validated to hopefully substitute for exter-nal seeding policy.The following conclusions could be drawn: (1)At high external-seed loadings,uni-modal product crystals ofthe grown seeds with no virtually nucleation were obtained in a batch dilution crystallizer,while at low seed loadings,bi-modal product CSD with plenty of new nuclei was obtained.Because the supersaturation-generated rate was higher than the supersaturation-consuming rate by seeds’growth in the case of low external-seed loading,the residual supersaturation in crystallizer was high,and the local supersaturation was much higher near the nozzle through which the diluent was injected, so plenty of new nuclei occurred near the nozzle.What’s more, too much seed loading would low the product size.Accordingly, the above result meant an existence of a critical seed loading in theory.(2)At the same external-seed loading,both feeding rate andconcentration of diluent could inﬂuence the product CSD of cef-triaxone sodium in anti-solvent crystallization.Although a slow feeding rate and a low concentration were desired to obtain good product CSDs in addition to crystal shape,a slow feeding rate and low concentration were still not recommended from the industrial point of view of production rate or crystal yield, which was usually associated with the economic beneﬁts. (3)The seed chart,which can be used to quantitatively determinethe critical seed loading in industrial manufacturing,settled the challenge that the determination of the seed loading was usually depended on experiences.(4)By adding pure acetone(99vol.%)at a relatively high feed-ing rate into a non-seeded solution saturated by ceftriaxone sodium,ﬁne crystals with a narrow CSD were obtained,irre-spective of the amount of the added diluent.(5)A promising seeding strategy of using internally generatedseeds,ﬁrstly by generating internal seeds through introducinga relatively high concentration of diluent and then followed bythe slow addition of another diluent of relatively low concen-tration,in producing ceftriaxone sodium with a uni-modal CSD was experimentally performed in batch anti-solvent crystal-lization.The product mass-weighted mean size was relatively larger,and the product quality was as good as the one obtained by external seeding.Therefore,such an internal seeding policy is expected to be a favorable seeding technology to hopefully substitute for external seeding policy in the pharmaceutical industry,where external seeding is not welcome because exter-nal seeding would destroy the axenic conditions in crystallizer and lead to unstable quality of different batches.AcknowledgmentsThe authors gratefully acknowledge the Shandong Ruiyang Pharmaceutical co.Ltd.for theﬁnancial supports and raw mate-rial supplies,and staff in the State Research Center of Industrial Crystallization and Technology for technical supports. 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Effect of shearing on crystallization behavior ofpoly(ethylene naphthalate)W.J.Yoon,H.S.Myung,B.C.Kim,S.S.Im *Department of Textile Engineering,Hanyang University,Haengdang,Seongdong,Seoul 133-791,South KoreaReceived 11August 1999;received in revised form 24September 1999;accepted 30September 1999AbstractThe effect of shear history on the isothermal crystallization behavior of poly(ethylene naphthalate)(PEN)was investigated by rheological and morphological measurements.Time sweep measurements of storage modulus (G H )and dynamic viscosity (h H )were carried out on the molten PEN by Advanced Rheometric Expansion System (ARES)in the parallel-plate geometry at several different temperatures and frequencies,followed by structural analysis by differential scanning calorimeter (DSC),X-ray diffractometer,and polarizing microscopy for the shear-induced crystallized PEN specimens in the ARES measurements.The rate of isothermal crystallization of PEN was notably affected by temperature,while the shear rate has an important effect on the structures of the resultant crystals.At a constant shear rate,the rate of crystallization by shear-induced structuring mechanism was increased with lowering temperature over the temperature range 230–250ЊC.The rate of crystallization was increased with increasing shear rate at a given temperature.An increase in shear rate increased both nucleation and number of crystallites.Further,it increased the content of the a -form crystal in the specimen.On the other hand,lower shear rate offered more favorable conditions for forming the b -form crystal.DSC analysis exhibited that the b -form crystal had higher melting temperature (T m )than the a -form crystal.The wide angle X-ray diffraction (WAXD)patterns also ascertained that higher content of the a -form crystal was produced in the PEN specimen crystallized at higher frequency.᭧2000Elsevier Science Ltd.All rights reserved.Keywords :Poly(ethylene naphthalate);Rheology;Shear-induced crystallization1.IntroductionShear-induced structural changes in polymeric materials take an increasing interest in the ﬁeld of polymer proces-sing.In real polymer processing very complex deformation histories are involved,which can inﬂuence ultimate proper-ties of plastics.Recent advances in experimental techniques that allow in situ measurements of materials under deforma-tion have escalated research in this subject area.It has been known for a long time that ﬂow stress have accelerating effect on the crystallization of semi-crystalline polymers [1–6].It is supposed that the application of a shear stress to a polymer melt should lead to formation of orientation and reduce the entropy of the melt,which results in a higher melting temperature and,hence,lead to an increased super-cooling [3,7].Several experiments have been described in the literature where attempts were made to quantify the shear stress-induced crystallization in molten semi-crystal-line polymers such as polypropylene [3,8,9],polyethylene oxide [10],polypropylene [11–13],and polybutene-1[3,14].Some investigators used rotational viscometers andmeasured either the volume change [15]or the number of nuclei formed during shearing [11,14].The polymers enum-erated above are apt to process because of low melting point and viscosity.On the other hand,PEN has good thermal and mechanical properties and is being used as engineering plastics.PEN is reported to have two different triclinic crystalline structures,a -form and b -form crystals.Of two crystal forms,the b -form crystal is known to be more stable than the a -form.The effect of crystallization temperature on the resultant crystal structure is well recognized;lower temperature favors formation of the a -form crystal.The critical temperature is reported about 230ЊC.However,the effect of shear history on the crystal structure of PEN has not been reported.In this study,the shear-induced crystallization behavior of PEN was investigated on the rheological basis.The effect of shear history on the crystalline structure was also discussed in terms of thermal and morphological properties.2.Experimental 2.1.MaterialThe PEN tested was a commercially available gradePolymer 41(2000)4933–49420032-3861/00/$-see front matter ᭧2000Elsevier Science Ltd.All rights reserved.PII:S0032-3861(99)00703-X*Corresponding author.Tel.:ϩ82-2-2292-0495;fax:ϩ82-2-2297-5859.E-mail address:imss007@email.hanyang.ac.kr (S.S.Im).supplied by Kolon Group in South Korea.The inherent viscosity,0.344dl/g was determined in a mixture of triﬂuoroacetic acid and chloroform (1/3v/v%)with an Ubbelohde viscometer at 25^0:1ЊC :The polymer was dried in a vacuum oven at 120ЊC for 24h prior to use.2.2.Measurement of physical propertiesThe dynamic rheological properties were measured by ARES (Rheometric Scientiﬁcs)in the parallel plate geome-try.The plate diameter was 12.5mm,strain level was 5%,and gap between the plates was 1mm.The PEN chips were melted at 300ЊC.The initial gap was set to a value equiva-lent to ﬁnal gap plus 50m m.The excess sample squeezed out by reducing the gap was carefully trimmed off.The value was reset to the ﬁnal gap value,1mm.To remove the residual stress the newly set PEN specimen was relaxed for about 5min at the temperature in nitrogen atmosphere,then cooled to the predetermined temperature for rheologi-cal measurements.A time-sweep experiment was continued for the specimen till the G H reached the ceiling value of the apparatus.After ARES measurement,the molten PEN sample was detached from the plates for measuring other properties such as thermal and morphological properties by DSC,X-ray diffractometer and polarizing optical micro-scopy.Thermal properties were measured by Perkin–Elmer DSC-7over the temperature 50–300ЊC at the heating rate of 10ЊC/min under nitrogen purge.The isothermalcrystallization experiment was performed by two different methods.Firstly,the PEN sample was heated to 300ЊC at the heating rate of 200ЊC/min,and held for about 5min,then they were cooled to the preset temperature to bring about the isothermal crystallization for same time required in ARES experiment.Secondly,the PEN chips were melted at 300ЊC between two slide glasses for 5min on the hot stage.They were moved to an oil bath very quickly and isothermally crystallized at 230,240,and 250ЊC for 4,10,and 24h,respectively.Wide angle X-ray diffraction patterns of the isothermally crystallized PEN specimen in the oil bath and ARES were obtained by X-ray diffractometer (Rigaku Denki)with Ni-ﬁltered CuK a radiation at 35kV and 35mA.Morphology of quiescent and shear-induced crystallized PEN specimen was observed by polarized microscopy (Nikon HFX-IIA).The spherulite structure was observed by microtoming the specimen.3.Results and discussionIn the plot of G H and h H versus time at a given frequency for a polymer,the two parameters may give information on the change in physicochemical properties of the polymer.For thermally sensitive polymer melts,an irreversible decrease of viscosity with time at a constant shear rate suggests the possibility of thermal degradation of polymer molecules,whereas an irreversible increase of viscosityW.J.Yoon et al./Polymer 41(2000)4933–49424934Fig.1.Variation of G H with time for PEN melt at 240ЊC at three different frequencies.with time indicates the possibility of chemical crosslinkingbetween polymer molecules.Both thermal degradation andchemical crosslinking show irreversibility in the rheologicalresponses.On the other hand,a reversible change in G H and h H with time at a constant frequency may be caused by changing in the physical state of the polymer melts.A typi-cal example of the physical change is the isothermal crystal-lization.As the crystallites grow to larger sized spheruliteswithin the PEN melt through nucleation and growth,thehomogeneous melt system changes to the heterogeneoussystem.Thus the G H and h H increase with the crystallization time.Figs.1and2show the variation of the G H and h H of PEN melt with time at240ЊC at three different shear rates(1,3, and5rad/s).At the early stage of experiment,both G H and h H are increased slowly,indicating an induction time for crystallization.The induction period is the stage when randomly entangled polymer chains transform to the regular aligned lattice.Because of topological obstruction of such entanglements,the polymer crystallization is extremely slow[16].However,an abrupt increase of both parameters follows in some minutes.This phenomenon can be ascribed to the formation of tiny crystals so-called crystallites prob-ably due to shear-induced crystallization.It can be easily imagined that the homogeneous PEN melt changes to a suspension system with proceeding crystallization,in which numerous crystallites are dispersed in the homo-geneous molten polymer matrix.The viscosity increases due to increasing the volume fraction of dispersed crystal-lites with progressing crystallization,which is also reportedby others[7,9,11,14,17].The ceiling value of G H is the same regardless of frequen-cies and temperatures whenﬁnishing crystallization asshown in Fig.1.On the other hand,the ceiling value of h H is gradually decreased with increasing the applied frequency as shown in Fig.2.This is attributable to pseudo-plasticity.That is,the heterogeneous system is expected toshow yield behavior[18].At low shear rates the hetero-geneous systems exhibit very high viscosity,and almostunbounded viscosity at zero shear rate.The viscosity,however,is rapidly decreased if the shear rate exceeds acritical value.Consequently,the ceiling viscosity at1rad/sis greater than at5rad/s.In addition,the ceiling value of h H shows a gradual decrease with time after having reached maximum as shown in Fig.2,which is more noticeable at the higher frequency.The gradual decrease of h H seems to result from the restructuring of the heterogeneous systems. That is,the viscosity is decreased with shearing on account of destruction of the orderedﬁller particle structure.The destruction of the pseudostructure ofﬁller particles is increased as shear rate is increased.Fig.2reﬂects this.It is also noted in Figs.1and2that the induction time forcrystallization is decreased as frequency is increased.Anapplication of shear stress to a polymer melt would giverise to two characteristic responses,orientation and slippageof polymer molecules.They are associated with theW.J.Yoon et al./Polymer41(2000)4933–49424935Fig.2.Variation of h H with time for PEN melt at240ЊC at three different frequencies.W.J.Yoon et al./Polymer41(2000)4933–49424936Fig.3.Variation of G H(A)and h H(B)for PEN melt at3rad/s at three different temperatures.W.J.Yoon et al./Polymer41(2000)4933–49424937Fig.4.DSC thermograms of PEN isothermally crystallized at(A)230ЊC,(B)240ЊC and(C)250ЊC at various frequencies.macroscopic phenomena of elasticity and ﬂow,respectively.That is,the oriented polymer molecule has fewer possible conformations than the unoriented one,which results in lower entropy.At the melting temperature,the free energy of the crystal equals the free energy of the melt as written by [3]T mD H f D S f H m ϪH cS m ϪS c1Hence,for an oriented melt,the ensuing reduction in entropy raises T m .Further,it increases the degree of super-cooling,accelerating the rate of crystallization.In general,higher shear rate gives better chance for orientation.Con-sequently,the induction time for cystallization is decreased with increasing shear rate.In Fig.3(A)and (B)shows that the annealing temperaturehas a profound effect on the nucleation and crystallization mechanism of PEN melts.The increase of G H and h H with annealing time represents the extent of crystallization of the melts with annealing time.Fig.3suggests that the number and growth rate of the nucleated crystallites is greater at 230ЊC than at 250ЊC.That is,both nucleation density and growth rate of crystallites are diminished with raising the annealing temperature.This stands to reason because the maximum rate of the homogeneous crystallization of PEN melts is observed in the vicinity of 215ЊC.The viscosity behavior of the PEN melt with crystalliza-tion in Fig.3may be accounted for by adopting the Mooney equation in a qualitative manner [19].ln h =h 1K E F 21ϪF 2=F m2W.J.Yoon et al./Polymer 41(2000)4933–49424938Fig.4.(continued )Table 1The values of T m H and T m HH of PEN with frequency (230,240and 250indicate temperature.(a)and (b)indicate v 0 a and v 0 b ;respectively (see Fig.6).1,3and 5indicate frequencyT m HT m HH T m HT m HH T m HT m HH PEN230(a)259.2272.1PEN240(a)266.7PEN250(a)270.1PEN230(b)264.1270.9PEN240(b)272.1PEN250(b)280.1PEN2301255.6270.3PEN2401259.3269.3PEN2501269.1PEN2303256.0270.0PEN2403261.9268.7PEN2503270.4PEN2305256.2269.7PEN2405263.6268.0PEN2505270.7F m true volume of fillerapparent volume occupied by the filler3 in which h is the viscosity of the suspension,h l is the viscosity of the suspending medium,f2is the volume frac-tion of theﬁller,f m is the maximum volume fraction that theﬁller can have,and K E is the Einstein coefﬁcient,whose value is known to be2.5for the dispersed sphericalﬁller.W.J.Yoon et al./Polymer41(2000)4933–49424939Fig.5.WAXD patterns of PEN isothermally crystallized at(A)230ЊC,(B)240ЊC and(C)250ЊC at various frequencies.Referring to the Mooney equation,the crystallization patterns of PEN melts at 230ЊC and at 250ЊC are distinc-tively different from each other.The Mooney equation predicts that the degree of increasing the suspension visc-osity with increasing f 2is greatly increased if the spheres form aggregates because the aggregation of spheres (spheru-litic crystallites or crystals in this study)increases the appar-ent ﬁller volume fraction.That is,the immobile portions (homogeneous molten PEN matrix in this study)caged by aggregated spheres also act as ﬁller portion.On this assump-tion,it may be suggested that an application of higher shear rate during isothermal crystallization tends to increase the heterogeneous crystallization characteristics.Hence,the higher nucleation density and higher growth rate of the nucleated crystallites is obtained at higher frequency,and the resultant is more abundant with less stable a -form crys-tals (this will be discussed later in detail).The melt endotherms of quiescently and shear-induced crystallized PEN were shown in Fig.4and the correspond-ing peak temperatures are listed in Table 1.The double melting endotherm behavior is displayed during heating the PEN sample in the DSC cell.In the melting process of the shear-induced crystallized PEN sample,three endother-mic peaks are identiﬁed;a broad endotherm,a low endotherm (T m H ),and a high endotherm (T m HH )as shown in Fig.4.The broad endotherm might be due to the thermal history during cooling and reheating,and both low and high endotherms are due to the melting of original lamella and recrystallized one,respectively.These results well coincide with the results of Zachman et al.[20]:(1)no change of crystal modiﬁcation is observed during DSC scanning;(2)the double melting behavior of PEN is due to the mechan-ism based on melting and recrystallization;(3)the b -form crystal has the T m higher than the a -form crystal by 2ЊC;and (4)the peaks of two forms of crystal are not separated in DSC thermogramsIn Fig.4(A)–(C)v 0rad =s indicates quiescent crystal-lization.(A)indicates that the PEN sample was crystallized at 230,240,and 250ЊC for the same time that required in the ARES experiments,and (B)expresses the PEN sample crys-tallized in an oil bath at the same temperature as in (A)for the time long enough to fully crystallize.Since the crystal-lization time in (A)is much shorter than in (B),an exother-mic peak is observed in the thermogram (A)at around 205ЊC.The v 0rad =s (b)curves in Fig.4(B)show a single melting peak.The T m shifts to higher temperature and peak width gets narrower as the crystallization time and tempera-ture are increased.This is attributable to the increased perfectness of the resultant crystal structure,which is observed more clearly when the sample is crystallized at higher temperature as can be seen in Fig.4(C).Only the b -form crystal exists when the sample is isothermally crys-tallized at 250ЊC after having melted at 300ЊC.This result matches well with the X-ray data.As mentioned the T m of the b -form crystal is higher than the a -form crystal by 2–4ЊC.It has been known that PEN has two different triclinic crystal structures.Buchner et al.reported that crystalW.J.Yoon et al./Polymer 41(2000)4933–49424940Fig.5.(continued )structures are inﬂuenced by both melting and isothermal crystallization temperature.They observed that the b -form crystal appeared mainly when PEN was isothermally crys-tallized above 230ЊC quiescently and the a -form crystal did below 230ЊC after having melted at 300ЊC [20].Fig.5presents WAXD patterns of PEN specimens shear-inducedcrystallized at (A)230ЊC,(B)240ЊC,(C)250ЊC at several frequencies.In Fig.5(A)the WAXD patterns for v 0rad =s shows diffraction peaks at 15.6and 23.3Њwhich correspond to (010)and (100)plane of the a -form crystal,respectively.The intensity of these peaks has a tendency to increase with increasing frequency.It means that the appli-cation of shear promotes the formation of the a -form crystal and the increase of frequency increases the content of the a -form crystal.In Fig.5(B)the (010)plane peak of the a -form crystal is smaller than that of the sample crystallized at 230ЊC in Fig.5(A)for v 0rad =s :However,the plane peak is increased with increasing frequency.In the case of the (100)plane peak,a shoulder appears at v 0rad =s :As frequency increases,the intensity of the plane peak standing for the a -form crystal shows tendency to increase.Particu-larly,for v 0rad =s in Fig.5(C)any plane peak of the a -form crystal is not observed,which is consistent with the results reported by Buchner et al.[20].The (010)and (100)plane peaks appear simultaneously,and keep on growing with increasing frequency.In addition,all diffraction peaks of Fig.5shift to lower angle when frequency is increased.This suggests that there is deformation in the crystal struc-tures as well.Thus,this X-ray trace of the sample is similar to those of Fig.5(A)and (B),suggesting similarity in the crystallization behavior at 230–250ЊC.In general,the b -form crystal is thermodynamically more stable but more difﬁcult to nucleate than the a -form crystal and the form of the crystal is largely determined by kinetic factors during crystallization such as the rate of nucleation and spherulite growth [21].In the case of the a -form crystal,one chain passes through the unit cell and the chains in the crystal are extended.In the case of the b -form crystal,however,four chains pass through the unit cell and the chains in the crystal are not completely extended.When the polymer is sheared,the number of crystallites increases with shear rate,representing faster nucleation.Wolkowicz [14]mentioned that the number of crystallites increased exponentially with time at all shear rates.Also,this can be conﬁrmed in Fig.6,which indicates that nuclea-tion becomes increasingly profuse with increasing frequency until the crystalline structure formed is no longer distinguishable with a microscope [3,22].Hence,the content of the a -form crystal in the speci-men increases with frequency because the a -form crys-tal is apt to nucleate due to fast nucleation.Consequently,the resultant a -form crystal is thermody-namically less stable than the b -form crystal because of much reduced entropy by molecular orientation under high shear force.References[1]Hill MJ,Keller A.J Macromol Sci (Phys)1969;B3(1):153.[2]Andrews EH.J Polym Sci 1966;A-2(4):663.[3]Haas TW,Maxwell B.Polym Eng Sci 1969;9:226.W.J.Yoon et al./Polymer 41(2000)4933–49424941(B)(A)(C)Fig.6.Polarizing optical micrographs of 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