A Method to Polarize Stored Antiprotons to a High Degree

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One hundred fold increase in current carrying capacity in a carbon nanotube-copper composite.

ARTICLEOPENReceived9Jan2013|Accepted26Jun2013|Published23Jul2013One hundred fold increase in current carrying capacity in a carbon nanotube–copper compositeChandramouli Subramaniam1,T akeo Yamada1,2,Kazufumi Kobashi1,2,Atsuko Sekiguchi2,Don N.Futaba1,2, Motoo Yumura1,2&Kenji Hata1,2,3Increased portability,versatility and ubiquity of electronics devices are a result of theirprogressive miniaturization,requiring currentﬂow through narrow channels.Present-daydevices operate close to the maximum current-carrying-capacity(that is,ampacity)ofconductors(such as copper and gold),leading to decreased lifetime and performance,creating demand for new conductors with higher ampacity.Ampacity represents themaximum current-carrying capacity of the object that depends both on the structure andmaterial.Here we report a carbon nanotube–copper composite exhibiting similar conductivity(2.3–4.7Â105S cmÀ1)as copper(5.8Â105S cmÀ1),but with a100-times higher ampacity(6Â108A cmÀ2).Vacuum experiments demonstrate that carbon nanotubes suppress theprimary failure pathways in copper as observed by the increased copper diffusion activationenergy(B2.0eV)in carbon nanotube–copper composite,explaining its higher ampacity.Thisis the only material with both high conductivity and high ampacity,making it uniquely suitedfor applications in microscale electronics and inverters.1T echnology Research Association for Single Wall Carbon Nanotubes(TASC),Central5,1-1-1Higashi,Tsukuba305-8565,Japan.2National Institute of Advanced Industrial Science and T echnology(AIST),Central5,1-1-1Higashi,Tsukuba305-8565,Japan.3Japan Science and T echnology Agency(JST), Honcho4-1-8,Kawaguchi332-0012,Japan.Correspondence and requests for materials should be addressed to K.H.(email:kenji-hata@aist.go.jp).D evelopment of electronic devices is undergoing a para-digm shift with their continued miniaturization1.Suchminiaturization provides greater portability and versatility to the devices,leading to their ubiquity.Simultaneously,the functionality and performance of these devices have been increasing.Although single-atom transistors2and few-atom memory devices3have been demonstrated to keep pace with the progressive miniaturization,there has been very little progress in conductors(such as Cu and Au),which supply power to these components within devices.With shrinking size of devices, pathways for carrying current to operate these components have signiﬁcantly reduced.This has resulted in higher current density being carried by the conductors,reaching the limit of conventional conductors(such as Cu and Au)in present-day devices.In fact,the International Technology Roadmap for Semiconductors(ITRS)predicts4that the current density in these devices is expected to exceed the breakdown limit of Cu and Au in2015.Therefore,new conductors with higher ampacity are in great demand1.Ampacity represents the maximum current-carrying capacity of the object that depends both on the structure and material.However,high ampacity and high conductivity are mutually exclusive properties.This is because the former requires a strongly bonded system,whereas the latter requires the free electrons from a weakly bonded system5.Therefore,achieving high electrical conductivity and ampacity in the same material has been impossible.Here we report a carbon nanotube–copper(CNT–Cu) composite that overcomes this mutual exclusivity to achieve an ampacity(630Â106A cmÀ2)100times higher than common electrical conductors,such as Cu6–9and Au9,10 (B106A cmÀ2),approaching the theoretical limit for CNTs11–16 (1000Â106A cmÀ2).Compared with copper,the macroscopic CNT–Cu conductivity(4.7Â105S cmÀ1)rivaled,exceeded and doubled at23°C,above80°C and227°C,respectively from a one-order-of-magnitude lower temperature coefﬁcient of resistivity.To understand the high ampacity of CNT–Cu composite,we performed activation energy(E a)analysis to evaluate the energy required for Cu diffusion in CNT–Cu composite.Our analysis demonstrated that the E a for Cu diffusion in the composite was B2.0eV.In comparison,the primary electromigration failure pathways in pure Cu have much lower E a,with surface and grain-boundary Cu diffusion requiring B0.6eV and B1.0eV, respectively17.Thus,the primary electromigration failure pathways(surface and grain-boundary diffusion)are suppressed by the presence of CNTs.Further,Cu diffusion is thus smaller(104 times)in our composite compared with bulk Cu,explaining its high ampacity.ResultsConductivity of CNT–Cu composite.Signiﬁcantly,a macro-scopic(2.5Â3.5cm;Fig.1a)CNT–Cu composite solid exhibited a room temperature conductivity of4.7±0.3Â105S cmÀ1,as measured by the four-probe method,comparable to that of Cu18 (5.8Â105S cmÀ1),three orders higher than pristine CNT19,20 (B102S cmÀ1)and an order higher than pure CNTﬁbre21 (104S cmÀ1).A microscopic CNT–Cu composite fabricated in an identical protocol(Fig.1b)exhibited a room temperature conductivity of 2.1±0.3Â105S cmÀ1.High CNT volume fraction(45vol%)resulted in a42%density reduction (5.2g cmÀ3)compared with Cu18(8.9g cmÀ3).Therefore,the speciﬁc conductivity for our CNT–Cu composite was26%higher than Cu and exceeded most materials(Au,Ag and Cu)with the exception of Al(Fig.1j).The temperature dependence of the conductivity,that is,temperature coefﬁcient of resistivity, of the CNT–Cu composite(7.5Â10À4KÀ1)was one order of magnitude lower than that of Cu22(6.8Â10À3KÀ1)(Fig.1i), highlighting one beneﬁt of our CNT–Cu composite. Consequently,the decrease in conductivity with temperature for the CNT–Cu was far less than Cu,and the conductivity was on par at room temperature,exceeded above80°C,and was double at227°C.This feature is important for heavy load applications,because the operating temperature is often higher than80°C.Ampacity of CNT–Cu composite.Central to this report,we found that our CNT–Cu exhibited a100times higher ampacity than Cu6–9.For this measurement,a CNT–Cu composite line structure(width¼800nm,height¼900nm,length¼50m m)was fabricated by electrodepositing Cu into a CNT line fabricated with conventional electron-beam lithography and reactive ion etching from a thin,closely packed and aligned CNTﬁlm23,24, that is,‘CNT wafer’.Similar structures with Cu and Au were used for comparison.The current was increased while monitoring the resistivity(Fig.1d).Initially,the resistivity unexpectedly decreased with current.Scanning electron microscopy(SEM) images revealed a smoothening of the initially rough CNT–Cu surface,indicating reorganization of the deposited Cu (Supplementary Fig.S1).We believe that this phenomenon is similar toﬁlament aging to increase lifetime,which contributed to the higher ampacity.The resistivity remained unchanged up to 600Â106A cmÀ2(600MA cmÀ2),after which the resistivity exponentially increased leading to failure at B690MA cmÀ2at the central region of the test structure(Fig.1e,f).The ampacity is deﬁned as the maximum current density where the resistivity remains constant and thus was estimated to be600MA cmÀ2from an average ofﬁve measurements.For comparison,we measured the ampacity of Cu and Au(both wire and sputter-deposited)test structures,which showed ampacities of 6.1MA cmÀ2and 6.3MA cmÀ2,respectively,close to literature values6–10 (B1MA cmÀ2)B100times smaller than that of the CNT–Cu composite(inset,Fig.1d).Not only was the ampacity of CNT–Cu composite100times higher than conventional conductors,such as Cu,Al,Au and Ag6–10,25(B1MA cmÀ2)and reports using Pt decorated CNTs26(7.5MA cmÀ2),it approached the highest value(1,000MA cmÀ2)reported from individual CNTs11–16. Further,the composite was stable at a DC current density of 100MA cmÀ2for over1200h(50days)with less than10% variation in resistivity(Supplementary Fig.S2).SEM observation of the failure point revealed thinning at both ends of the rupture (Fig.1e).Energy dispersive X-ray microscopy(EDX)detected only carbon at the ruptured surfaces(Fig.1g,h).These results demonstrate that Cu had diffused away from this region and that the failure mechanism of the CNT–Cu composite was Cu electromigration27–29.The CNT–Cu composite was plotted onto a conductivity-ampacity Ashby map for comparison with other high-perfor-mance materials(Fig.1c).An inverse trend between conductivity and ampacity was clearly apparent with metals possessing higher conductivities and nanocarbons possessing higher ampacities. The CNT–Cu composite did not follow this trend creating a distinct and isolated point in the high-ampacity and high-conductivity domain with conductivity1,000times higher than nanocarbons and ampacity100times higher than metals. Although having been proposed theoretically30,no methods,to date,have achieved simultaneous increase in both properties.For example,alloying of copper decreases conductivity and doping or bundling of CNTs decreases ampacity31.For example,Xu et al.32 have demonstrated a similar conductivity(no estimation on ampacity)for CNTﬁbre–Cu composite,but the Cu deposition was limited to the outer surface of the CNTﬁbre.In this case,thehigh conductivity is derived from the surface Cu,but we expect that it will fail to exhibit high ampacity.Furthermore,Behabtu et al.21reported high conductivity of 2.9Â104S cm À1in pure CNT ﬁbre;however,this report also did not characterize the ampacity.Fabrication and characterization of CNT–Cu composite .To fabricate a CNT–Cu composite (Fig.1a)that synergistically combines the strengths of both materials,we developed a unique fabrication process.In contrast to conventional approaches that use CNT–Cu ion dispersions,we electrodeposited Cu into the pores of premade,macroscopic CNT structures,for example,buckypaper,and CNT solids (bulk,packed,aligned CNT material 33).Long,vertically aligned single-wall CNT forests (diameter ¼3nm,height ¼500–700m m,density ¼0.04g cm À3)were synthesized on a substrate by the water-assisted method 34.The sparse forests were densiﬁed (B 0.5g cm À3)into closely packed and aligned CNT structure by the liquid densiﬁcation technique 33with Cu ions.Then,this CNT structure was transformed into CNT–Cu composite by two-stage nucleation-growth electrodeposition processes (Fig.1b),as described below.Several key processes were developed to achieve this CNT–Cu composite.First,the electrodeposition was separated into two phases:(1)wetting the hydrophobic CNTs with Cu ions in an organic solution to nucleate Cu seeds on CNT surface and (2)grow the Cu seeds in an aqueous solution until all the mesopores were ﬁlled.Second,in the organic electrodeposition phase,homogeneous seeding required that the rate-limiting step be the Cu nucleation on the CNTs rather than the Cu ion diffusion through the CNT structure.Therefore,slow deposition rates (1–5mA cm À2versus conventional rates 35of 50–100mA cm À2)were required to electrodeposit Cu throughout the dense CNT matrix,not only on the surface.At low current densities (1–5mA cm À2),Cu homogeneously nucleated throughout the CNT matrix,resulting in high conductivity,high Cu ﬁlling-ratio and low speciﬁc surface area (an indicator of accessible CNT surface area;Fig.2a).In contrast,at high current densities (5–15mA cm À2)Cu deposited only at the outer surfaces of the CNT matrix,resulting in exactly the opposite effect (Fig.2a).Third,annealing in hydrogen ambient reduced the (111),(200)and (220)Cu x O y phases to a pure (111),(200)and (220)Cu phase as seen from X-ray diffraction (Fig.2b).In addition,the Cu particles sintered resulting in an improvement inelectricalda Vertically aligned CNT forestShear forceHorizontally alignedCNT forestOrganic electroplatingAqueous electroplatingCNT-Cu compositeschematicef jcghbiCu seed particlesTest lineFailure pointCNT-Cu CNT-Cu CopperCopper Gold00408012016040801202468Current density (×106 A cm –2)Current density (×106 A cm –2)C o n d u c t i v i t y (×106 S c m –1)200400600300350400450500Temperature (K)0.20.30.40.50.6R e s i s t i v i t y (μ .c m )R e s i s t i v i t y (μ .c m )4k8k12kWS nF eN i A uZ nA gC u C N T -C u A IS p e c i f i c c o n d u c t i v i t y (c m 2 k g –1)Conductivity (S cm –1)1010109108107106105104106105104103102101A m p a c i t y (A c m –2)Figure 1|Fabrication and ampacity of CNT–Cu composite.(a )Picture of CNT–Cu composite alongside a paperclip for size reference.(b )Schematicrepresentation of various steps for CNT–Cu composite fabrication.Cu seeds nucleate on the CNT surface during the organic electrodeposition and subsequently grow during the aqueous electrodeposition to yield the CNT–Cu composite.(c )Ashby plot of ampacity versus conductivity for various relevant materials,including metals (such as Cu,Au,Ag,Al,etc),alloys (such as Sn-Pb),nanocarbons (such as SWNT,graphene)and composites (such as Pt–CNT).The ITRS recommended level of current density is also shown for comparison,with the performance of CNT–Cu exceeding it.(d )Variation of resistivity with current density for CNT–Cu composite.The electrical conductivity of the wire was 2Â105S cm À1.Similar traces for Cu and Au lines are shown in inset for comparison.(e )SEM image of CNT–Cu test structure after failure.Scale bar,4m m.(f )SEM image of the same CNT–Cu composite test line before failure.Scale bar,4m m.(g ,h )EDX mapping (based on Cu)of the failure points of CNT–Cu composite line presented in Fig.1e.Scale bar,500nm.(i )Variation of conductivity with temperature for CNT–Cu (red)and Cu (black),showing the largely invariant conductivity of CNT–Cucomposite with temperature.In comparison,Cu shows a decreasing conductivity with temperature,as expected for metals.(j )Comparison of conductivity per unit weight (speciﬁc conductivity)of CNT–Cu with different metals.conductivity from 8.2Â103to 9.1Â104S cm À1(Fig.2c).Finally,through aqueous electrodeposition,the Cu volume fraction was increased,as seen by the steep density increase (Fig.2c).Through this scheme,we succeeded in making a CNT–Cu composite with the CNT covering the surfaces and grain boundaries of Cu (inset,Fig.2d,e).No contamination by oxygen was observed by EDX (Fig.2d).Thermo-gravimetry showed that the composite was composed of B 55vol%Cu and 45%CNT (Supplementary Figs S3and S4)(total density ¼5.2g cm À3),indicating a 42%density reduction from pure Cu 18(8.9g cm À3).A control experiment was carried out to verify that the observed conductivity arose from the CNT–Cu bulk as opposed to its outer surface.Hence,the thickness and conductivity were plotted as a function of electrodeposition time (10–840min;Fig.2f).Initially (up to 600min),the electrical conductivity monotonicallyincreased and saturated at 105S cm À1while the thickness remained unchanged.This meant that the Cu deposited within the CNT matrix.Further electrodeposition (beyond 600min)led to a thickness increase,which indicated deposition on the outer surfaces.Importantly,the conductivity did not increase during this phase,meaning that the measured conductivity did not stem from the surface deposition of Cu.DiscussionAs experimental results demonstrate,electromigration of Cu atoms is also the failure mechanism of the CNT–Cu composite (like pure Cu).Within this framework,several experiments and analyses were carried out to understand the mechanism of high ampacity of the CNT–Cu composite.The activation energy fordbace f100k 80k 60k 40k 20k 0246810121416202530354045SSA (m 2 g –1)Conductivity (S cm –1306090120150Electrodeposition current density (mA cm –2)I n t e n s i t y (a .u .)I n t e n s i t y (a .u .)3040506070802 θ°(220)(220)(220)(200)(200)(111)(111)Before heat treatment After heat treatment1k 8006004002000246810Energy (keV)CuK αCuK βCuL αCNT-Cu (org)CNT-Cu (aq)HeatingPure Cu0.00.20.40.60.81.01.2Cu volume %Cu volume fractionLog (conductivity, S cm –1)Density (g cm –3)3456789D e n s i t y a n d l o g (c o n d u c t i v i t y )Cu2,0004,0006,0008,00010,00012,000Thickness (nm)(10)(600)(840)ExternalCopperfilling CNT matrix1.52.02.53.03.54.04.55.05.5l o g (c o n d u c t i v i t y , S c m –1)Figure 2|Characterization of CNT–Cu composite.(a )Variation of CNT–Cu composite properties as a function of organic electrodeposition current density.The direct correlation between conductivity and volume occupancy of Cu is observed.(b )XRD traces of CNT–Cu composite before (red)and after (black)heat treatment.(c )Evolution of the CNT–Cu composite properties (conductivity,density and Cu volume occupancy)with every advancing step in the fabrication process.Similar properties of bulk Cu are also given alongside for comparison.(d )EDX spectrum of CNT–Cu composite showing the absence of any other impurities with the cross-sectional SEM and EDX mapping images.Scale bar,2m m.(e )Cross-sectional SEM images showing the polycrystalline Cu tightly bound with long,intertwined,well-dispersed CNTs.Scale bar,6m m.(f )Evolution of CNT–Cu conductivity with thickness and electrodeposition time for a current density of 5mA cm À2.Values given in brackets pertain to electrodeposition time in minutes.Cu diffusion in CNT–Cu composite was estimated by failure kinetics tests carried out at different temperatures (440K,450K,473K and 498K).Applying a current density (720MA cm À2)higher than the ampacity (600MA cm À2)of CNT–Cu results in accelerated failure (Fig.3a).The ‘time of failure,’t ,was estimated from the point of 40%resistivity increase.The results of several such experiments carried out at different temperatures (constant current density)were assembled into an Arrhenius plot of ln(t )versus 1/kT (Fig.3b).Therefore,the activation energy for Cu diffusion in CNT–Cu composite was estimated from the slope of this plot,based on Black’s equation 36,ln t ¼ln A Àn ln j þE akTð1Þwhere j is the current density,E a is the activation energy,k is the Boltzmann constant,T is the mean temperature of the test structure,n is the current-density exponent and A is the pre-exponential factor.The activation energy for Cu diffusion in CNT–Cu composite was estimated,from slope of Fig.3b to be 2.03eV.We wish to note that our choice of the current-density exponent (n )and pre-exponential factor (A )were taken from similar electromigration studies on Cu under identical measurement conditions,namely in vacuum and with the test structure not surrounded by any dielectric 27,37,38.Further,we note that this estimation of the activation energy (E a )is independent of the choice of pre-exponential factor (A )and current-density exponent (n ).The value of 2.03eV was identical to that of Cu lattice diffusion 17,28,29(B 2.0–2.3eV),which means that the Cu diffusion follows the most difﬁcult pathway.For bulk Cu,diffusion occurs at the surfaces and grain boundaries that possess much lower activation energies 17,28,29(B 0.7eV and B 1.0eV,respectively).Our results showed that for the CNT–Cu composite,the Cu diffusion pathways through surface and grain boundaries were greatly suppressed.The Cu diffusion coefﬁcient (D Ã,related the ease of electromigration)was calculated from the activation energy by:D Ã¼D 0expÀE akT ð2Þwhere D 0is the diffusion coefﬁcient at inﬁnity 17.As a result,the estimated diffusion coefﬁcient of Cu in the CNT–Cu composite by bulk diffusion was 104times lower than that of copper,which should signiﬁcantly contribute to the observed high ampacity.Internal structure observations the CNT–Cu composites by SEM and EDX revealed a ﬁne,continuous,mesh-like CNT network covering the surfaces and grain boundaries of Cu (inset,Fig.2d).Theoretical studies have proposed that the activation energy forcarbon-doped Cu diffusion increased by suppressing surface and grain boundary pathways 39.Although lowering of Cu electromigration by CNT has been reported,the exact mechanism and the 100times ampacity increase,without compromising on electrical conductivity,reported here have never been achieved 40.We draw a distant parallel of this effect with the well-known practice of alloying steel with carbon for strengthening.We believe that the CNT in CNT–Cu composite has a similar role in suppressing Cu diffusion 39.We numerically estimated the ampacity of the CNT–Cu composite by assuming that the material ruptured by current-induced Joule heating,leading to melting.The temperature increase by current is described by 41,j crit ¼2K l ﬃﬃﬃﬃﬃL n p cosÀ1T 0T m;ð3Þwhere j crit is the ampacity,K is the thermal conductivity(B 800Wm À1K À1),l is the length of the test structure (50Â10À6m),and L n is the Lorentz number (B 3.44Â10À8W O K À2).The melting point of the composite was taken as that of Cu 18(as 1357K),because the CNT melting point is signiﬁcantly higher in an inert ambient.The Lorentz number (ratio of thermal and electrical conductivities)was calculated from the experimentally measured temperature-dependent thermal conductivity and were plotted versus temperature (Fig.3c).Interestingly,the Lorentz number for the CNT–Cu composite linearly increased while remaining constant for Cu.Increasing Lorentz number indicates phonon contribution to the thermal conductivity,unlike a constant Lorentz number for a free electron system 42.We extrapolated the Lorentz number at higher temperatures from the slope.The theoretically estimated ampacity was thus estimated as B 1,200MA cm À2,which agrees with our measured ampacity of 600MA cm À2.These analyses and experimental results demonstrate the signiﬁcant role of the CNT phonons in achieving high thermal conductivity and Lorentz number at elevated temperatures for the CNT–Cu composite.This phenomenon was vital for the high ampacity.Furthermore,we quantitatively evaluated the electromigration by estimating the Cu atomic ﬂuxes in bulk Cu and CNT–Cu due to current (mass ﬂux)described by the Nernst–Einstein equation 43:J total ¼J E þJ T ¼CD ÃF E kT þCD ÃF TkT;ð4Þwhere C is the concentration of atoms,k is the Boltzmann constant,T is the temperature,D Ãis the diffusion coefﬁcient,F E and F T are the respective driving forces due to electronwindabc20181614121086100200300400500600Time (s)I n (t i m e -t o -f a i l u r e )109876543232425261/k B T (eV –1)Temperature (K)3003103203303403502.42.62.83.0L o r e n t z n u m b e r (×10–8 W K –2)R e s i s t i v i t y (μ .c m )Figure 3|Mechanistic analysis of CNT–Cu composite.(a)Variation of resistivity with time for CNT–Cu composite at a current density of 720MA cm À2at 473K.(b )Experiment in a repeated at different temperatures to give an Arrhenius plot of ln(time-to-failure)versus 1/k B T .The activation energy for Cu diffusion in CNT–Cu is determined from the slope of this graph.(c )Comparison of Lorentz number for CNT–Cu (red)with Cu (black)as a function of temperature,indicating an increasing phonon contribution of CNT with temperature in CNT–Cu composite.and temperature gradient.Electromigration consists of two components:the massﬂux from electron wind(electron-Cu atom collisions,J E)and from thermal gradient(diffusion caused by Joule heating,J T).For Cu,the electron wind is known to be one order larger than that of the thermal gradient44;thus,thermal gradient was neglected in the following discussion.We calculated the electron wind driving force by,F E¼ze r j where,z is the effective charge number38(¼10),e the electronic charge(1.6Â10À19C),r the material resistivity (4.3Â10À6O cm),and j the current density(600MA cmÀ2). By using experimentally obtained data,the electron-driving force was estimated to be(2,083eV cmÀ1),two orders higher than that of Cu44(40eV cmÀ1);accordingly,the electron wind massﬂux was B100times smaller than that of pure Cu.We think that this 100times difference agrees well with the observed100times difference in ampacity.In summary,we developed a high-performance electrical conductor(CNT–Cu composite)that combines the best electrical properties of CNT(high ampacity)and Cu(high conductivity). Moreover,the CNT–Cu composite is the only material satisfying the ampacity and conductivity levels stipulated by ITRS4for2015.MethodsMaterials.Single-walled CNTs were synthesized through the water-assisted supergrowth technique34.All chemicals were purchased from Wako and used without further puriﬁcation,unless speciﬁed otherwise.Preﬁlling of CNT matrix with Cu electrolyte.A free-standing CNT forest was obtained by carefully removing it from the growth substrate.This was subjected to a shear force between two glass slides to change the alignment direct of the CNT forest from vertical to horizontal(with reference to the glass slides).The arrangement,held in place using clips,was immersed into a densiﬁcation solution of Copper acetate dissolved in acetonitrile(2.75mM)and left undisturbed was 20min.The densiﬁed solid was carefully removed and assembled into the electroplating setup.Electroplating setup.The electroplating setup consists of a cathode of densiﬁed, preﬁlled CNT solid backed onto an stainless steel mesh for providing an equipo-tential surface for electroplating.The cathode was sandwiched between thin strips of pure Cu which served as anodes.Filter paper from Advantech acted as insulating separators between the anodes and cathode.The whole assembly was held in place using a specially designed polyether ether ketone cell.For the organic electro-plating,the electrolyte consists of the same liquid,which was used during forest densiﬁcation.In case of the second step aqueous electrodeposition,the electrolyte used was a commercially available electroplating solution(ATMI,without accel-erators or suppressors).Trapped air and dissolved gases was removed from the setup before electrodeposition.Electrodeposition of Cu was carried out under galvanostatic conditions using a VMP3Electrochemical workstation(Princeton Applied Research).Reductive annealing.The composite after each electrodeposition was washed with pure acetonitrile and dried using a vacuum desiccator at60°C for30min.This was followed by heating at250°C for3h in a tube furnace.The heating and subsequent cooling was carried out under a controlledﬂow of H2gas at150sccm.Conductivity measurement.The electrical conductivity measurements were carried out in two different geometries.The four-probe conductivity was measured using a hand-held four-probe conductivity meterﬁtted with Au-coated electrodes. For the two-probe measurement,the edges of the composite were cut using a CO2 laser to remove the Cu deposited on the edgesﬁrst removed to force the electrical path through the thickness(bulk)of the composite.Thin Cu strips were attached to either faces of the composite and were in turn connected to a digital multimeter. Plastic clips were used to hold the setup in place and ensure good,stable electrical contact between the electrodes and samples.Thus,the resistance measured was from the bulk of the composite,in a direction perpendicular to the thickness of the composite.The resistance of the composite was normalized to the thickness and cross-sectional area of the composite to arrive at its two-probe resistivity.The two-probe and four-probe conductivity values presented here are an average ofﬁve such measurements at different places of the composite across three composites.Microfabrication of CNT–Cu composite.For microfabrication,vertically aligned CNT thinﬁlms were grown from patterned catalyst using water-assisted super-growth chemical vapor deposition method.The average size of theﬁlms used here was700m mÂ700m m with an as-grown thickness of8m m.This was transferred to the desired substrate in the desired orientation using an liquid-induced densiﬁ-cation approach23,24.Thickness of the CNTﬁlms after transfer was measured to be around700nm.Patterning of CNTﬁlm.Ti-Au(3/100nm)electrode lines,required during elec-trodeposition,were deﬁned using conventional E-beam lithography followed by sputtering and lift-off.We used a TiN-coated Si3N4substrate to prevent Cu diffusion into the substrate,and large Ti/Au bottom contacts that ensured breakdown at the centre of the line.After CNT thin-ﬁlm transfer,the substrate was vacuum baked at180°C for15min before resist coating.Positive tone PMMA-495 was spin-coated at4,700r.p.m.for60s on the substrate followed by baking at 180°C for60s.This was followed by spin coating a negative tone FOX16resist at 4,500r.p.m.for60s.The substrate was baked at120°C for8min for slow curing of the resist.E-beam lithography was used to deﬁne the required areas of CNT.The sample was developed using tetramethylammonium hydroxide,rinsed with water and dried under a dry N2ﬂow.Reactive-ion etching using a mixture of O2/Ar/ CHF3was carried out to remove the unwanted CNT areas.The negative resist was stripped by immersing in buffered hydrogenﬂuoride for B10s followed by thorough rinsing and drying.The positive resist was removed using1:1micture of methylisobutyl ketone/isopropyl alcohol followed by rinsing in isopropyl alcohol and drying under N2stream.Current density testing of CNT–Cu composite.A home-built setup was used to perform the current density testing on CNT–Cu composite.It consists of a T-joint ﬁtted with electrical feedthrough on one side.The other two openings were connected to a vacuum pump and a vacuum gauge,respectively.All experiments were carried out at the pressure of1.3Â10À4Pa.The electrical feedthrough was connected to an Agilent U3606A DC power supply-Digital multimeter system capable of supplying up to3A.For higher currents,a Kikusui10–105DC power supply was used.The experiments were carried out by sequentially stepping up the voltage and simultaneously recording the currentﬂowing through the system. Resistance at every voltage step,computed from the I–V correlation,was translated to resistivity with knowledge of length and cross-sectional area of the test structure. The cross-sectional area was also used to compute the applied current density at every stage of experiment.The testing was carried out onﬁve devices with identical results.Sputtered and electroplated Cuﬁlms,Cu and Au wires(25m m diameter), which are conventionally used in wire-bonding were also tested under identical conditions using the same setup.Activation energy analysis.Accelerated lifetime tests were carried out at different temperatures using a modiﬁed setup consisting of a test chamber with temperature control.Electrical feedthrough in the test chamber enabled a constant current density to be applied while the temperature of the experiment could be modiﬁed. The time-to-failure of the sample at each temperature was recorded at a constant current density,providing an Arrhenius plot of ln(time-to-failure)versus1/kT. Activation energy for Cu diffusion in CNT–Cu composite was estimate from the slope of this graph.Lorentz number estimation.Thermal diffusivity(a)of CNT–Cu composite was measured at various temperatures with a Bethel Thermowave Analyser systemﬁtted with a temperature controller.Differential scanning calorimetry was used to estimate the speciﬁc heat capacity(C p)of the material in the same temperature range.Thermal conductivity(k)of the sample at various temperatures was estimated using the relationk¼a:r:C pð5Þwhere r is the density of the material(5.2g cmÀ3).From the knowledge of elec-trical conductivity(s)at various temperatures(Fig.1i),the Lorentz number(L) was estimated using Wiedmann–Franz law asL¼ks Tð6ÞThe Lorentz number estimated for Cu and CNT–Cu composite is plotted in Fig.3c.References1.U.S.Congress,Ofﬁce of Technology Assessment.Miniaturization Technologies.OTA-TCT-514(ernment Printing Ofﬁce,Washington,DC,1991).2.Fuechsle,M.et al.A single atom transistor.Nat.Nanotech.7,242–246(2012).3.Loth,S.,Baumann,S.,Lutz,C.P.,Eigler,D.M.&Heinrich,A.J.Bistability inatomic-scale antiferromagnets.Science335,196–199(2012).4.ITRS International Technology Working Groups.International TechnologyRoadmap for Semiconductors()(2010).5.Pauling,L.The Nature Of The Chemical Bond.3rd edn(Cornell UniversityPress,USA,1960).6.Li,P.-C.&Young,T.K.Electromigration:the time bomb in deep-submicronICs.IEEE Spectrum33,75–78(1996).。

A method for monitoring a dose of penetrating radi

专利名称：A method for monitoring a dose ofpenetrating radiation absorbed by an object 发明人：Struye, Luc,Leblans, Paul,Willems, Peter申请号：EP99203350.6申请日：19991011公开号：EP1001278B1公开日：20050406专利内容由知识产权出版社提供摘要：A method for monitoring a dose of penetrating radiation absorbed by an object, comprising the steps of providing the object with a device for absorbing penetrating radiation, including a storage phosphor for storing energy from the penetrating radiation, at predetermined intervals coupling the storage phosphor to a source of stimulation light, in such a way that the stimulation light impinges on the phosphor, activating the source of stimulation light so as to cause the storage phosphor to emit an amount of fluorescent light in proportion to an amount of stored energy, reading the amount of fluorescent light and converting it in an electric signal value, storing electric signal value(s) obtained at the predetermined intervals and processing them so as to evaluate a total amount of radiation absorbed by the object, comparing the total amount with a pre-defined threshold value for obtaining a difference value, and displaying the difference value on a decentralised display.申请人：AGFA GEVAERT地址：BE国籍：BE更多信息请下载全文后查看。

The Properties of Chromatography

The Properties of ChromatographyChromatography is a widely used technique in the field of analytical chemistry. It separates and identifies the different components of a mixture by using the properties of the mobile and stationary phases of the chromatographic system. The technique is based on the principle that different compounds have different affinities for the stationary and mobile phases of the system, leading to their separation from each other.In this article, we will discuss the properties of chromatography and how they contribute to the separation of different compounds. We will explore the various parameters that affect the chromatographic process and how they can be optimized for better results.1. Mobile phaseThe mobile phase is the solvent used to carry the sample through the chromatographic system. It can be either a liquid or a gas, depending on the type of chromatography being used. In liquid chromatography, the most commonly used solvents are water and organic solvents such as methanol, acetonitrile, and chloroform. In gas chromatography, helium or nitrogen is used as the carrier gas.The choice of mobile phase depends on the polarity of the compounds being separated. For example, polar compounds can be separated using a polar mobile phase such as water, while nonpolar compounds require a nonpolar solvent such as hexane. The composition of the mobile phase can also be varied to optimize the separation of the sample.2. Stationary phaseThe stationary phase is the material through which the sample passes, and it has a high affinity for certain compounds. There are several types of stationary phases, including solid-phase, liquid-phase, and gas-phase materials.The choice of stationary phase depends on the nature of the sample being analyzed. For example, if the sample contains polar compounds, a polar stationary phase such as silica gel will be used. If nonpolar compounds are present, a nonpolar stationary phase such as a C18 resin is used.3. Retention timeRetention time is the time taken for a particular compound to pass through the chromatographic system. It is affected by several factors, including the size and shape of the compound, the polarity of the mobile and stationary phases, and the temperature of the system.The retention time of a compound can be used to identify it, since each compound has a unique retention time. Furthermore, retention time can be used to quantify the amount of a particular compound in a sample by comparing its retention time to that of a known standard.4. ResolutionResolution is the ability of the chromatographic system to separate and resolve different peaks in the chromatogram. It is affected by several factors, including the stationary and mobile phases, the sample size and preparation, and the temperature of the system.High resolution is important in chromatography, since it allows for better identification and quantification of the different compounds in a sample.5. DetectionThe final step in the chromatographic process is detection, where the separated compounds are identified and quantified. There are several methods of detection, including ultraviolet (UV) detector, fluorescence detector, mass spectrometer, and refractive index detector.The choice of detection method depends on the nature of the sample being analyzed, as well as the properties of the compounds being detected. For example, UV detection iscommonly used for detecting aromatic compounds, while mass spectrometry is useful for identifying and quantifying small molecules.ConclusionIn conclusion, chromatography is a versatile and widely used technique in analytical chemistry. Its ability to separate different compounds based on their properties makes it an essential tool in many fields, including pharmaceuticals, food science, and environmental science.By understanding the various properties of chromatography, we can optimize the process for better separation and identification of different compounds. By choosing the right mobile and stationary phases, adjusting the retention time and resolution, and selecting the appropriate detection method, we can achieve accurate and reliable results in our analyses.。

阅读欧洲药典时需要注意的问题

阅读欧洲药典时需要注意的问题阅读欧洲药典时需要注意的问题1. ContentFor a non-specific assay (for example, titrimetry) the assay limits are usually 99.0-101.0% percent (unless otherwise justified). For a specific assay using a separation technique (for example, liquid or gas chromatography), the upper assay limit is normally 102.0 percent; the lower assay limit will take any necessary account of the impurities present and may therefore be lower than 98.0 percent.2. Characteristic / AppearanceThe term “white” is not used without qualification since, if viewed against a standard white material, very few pharmaceutical materials will appear truly white. It is, of course, not intended that such a comparison be made but experience shows that certain users of the Pharmacopoeia may insist on doing so as part of a purchasing contract. The term “white or almost white” is used instead.In English, the dominant is placed second, whereas in French, it is place first. Expressions such as lemon-yellow, buff, salmon-pink are to be avoided.3. IdentificationNormally, a single set of tests for identification is given. Where justified, in order to give users of the Pharmacopoeia a choice between methods requiring complex instrumentation and other methods, two sets of identification tests may be included.This is usually the case when the substance is used in hospital and/or community pharmacies. Monographs then have subdivisions entitled “First identification” and “Second identification”. The test or tests that constitute the “Second identification” may be used instead of the test or tests of the “Fist identification” provided it can be demonstrated that the substance or medicinal product is fully traceable to a batch certified to comply with all the requirements of the monograph.3.1 PolymorphismThe general chapter allows for recrystallisation before recording of the spectrum. Where a monograph mentions polymorphism then a method for recrystallisation is described unless it is the intention to limit the scope of the monograph to the crystalline form represented by the chemical reference substance in the latter case the monograph indicates that the spectrum is recorded “without recrystallisation”.3.2 Ultraviolet and Visible Absorption SpectrophotometryThe concentration of the solution to be examined is such that the absorbance preferably lies between 0.5 and 1.5 measured in a 1 cm cell.3.3 Specific Optical RotationWhen an enantiomer is described in a monograph, a test for optical rotation is given in the IDENTIFICATION section or a cross-reference is made to the test for enantiometic purity in the TESTS section. When both the racemate (or the racemic mixture) and the enantiomer are available then, in the monograph of the racemate, an angle of rotation will be given in the TESTS sectionand will be referred to in the IDENTIFICATION section. When only the racemate is available the angle of rotation will be given in the TESTS section, provided the specific optical rotation of the chiral form is known and is of sufficient magnitude to provide a meaningful test for racemic character.4. Test4.1 Appearance of SolutionThis test makes it possible to ascertain the general purity of a substance by the detection of impurities insoluble in the solvent selected, or of colored impurities.The “Appearance of solution” test is practically always prescribed for substances intended for preparations for parenteral use. Apart from this it is to be applied only if it yields useful information concerning the general purity of the substance.It can comprise both tests or one only, namely:l Clarity and degree of opalescence of liquids (2.2.1);l Degree of coloration of liquids (2.2.2).4.1.1 Clarity and degree of opalescenceThis test is mainly performed on colorless substances or those which give only slightly colored solutions in order to permit valid comparison with reference suspensions.4.1.2 Degree of colorationThe test applies to essentially colorless substances that contain, or may degrade to form, colored impurities that can be controlled by limiting the color of solution of the substance.4.2 Related SubstancesMonographs should include acceptance criteria for:l Each specified impurity;l Unspecified impurities (previously referred to as “any other impurities”), normally set at the identification threshold;l Total impurities.Impurities to be controlled include: intermediates and by-products of synthesis; co-extracted substances in products of natural origin; degradation products. Monographs on organic chemicals usually have a test entitled “Related substances” (or a test with equivalent purpose under a different title), designed for control of organic impurities. Inorganic impurities are usually covered, where applicable, by other tests. Residual solvents are covered by specific provisions.The acceptance criterion for specified impurities may be set at the identification threshold for the substance.The acceptance criteria for specified impurities take account of both:1. qualification data, where applicable, the limit being set at a level not greater than that at which the impurity is qualified; the information on qualification is provided by the producer and the compatibility of the limit with the qualification data and approved specifications is checked by the competent authorities during elaboration of the monograph and/or during the Pharmeuropa comment phase; and2. batch analysis data, the acceptance criteria being set to take account of normal production; data is provided by the producer for typical batches and checked during elaboration of the monograph on not fewer than 3 batches.4.3 Heavy MetalsThe heavy metals detected by the general methods are those that precipitate at pH 3.5 in the form of dark-colored sulphides through the action of sulphide ions or reagents capable of producing them: lead, copper, silver, mercury, cadmium, bismuth, ruthenium, gold, platinum, palladium, vanadium, arsenic, antimony, tin and molybdenum.In routine practice, Methods A, B, C, D, F and G are unsuitable for establishing limits below 5 ppm, unless filtration is prescribed. For lower limits, Method E can be used, which makes it possible to go down to 0.5 ppm. In order to ensure limit contents of less than 0.5 ppm, it is necessary to resort to tests specific for each metal which are frequently based on atomic spectrophotometry.Tests for individual metal catalysts are included where it is known that they are used in the synthesis of an active substance or excipient. Limits take account of toxicity, the route of administration and the manufacturing process.4.4 Loss on DryingGenerally an upper limit for loss on drying is given. If the substance is a hydrate (or solvate) upper and lower limits are indicated. Drying is carried out to constant mass, unless a drying time is specified in the monograph. When a drying time is prescribed, adequate validation data must be provided. Where the drying temperature is indicated using a single value, a tolerance of ±2°C is understood. For temperatures higher than 105 °C, a larger tolerance should be indicated in the monograph, if necessary.Limits less than 10 percent should be given with 2 significantfigures and limits of 10 percent or greater should be given with 3 significant figures. The sample size is chosen to give a difference of 5-50mg before/after drying and is indicated with 4 significant figures.4.5 ThermogravimetryLoss on drying can be determined by this method when the amount of substance has to be restricted, for example to reduce exposure for the analyst or if the substance is very expensive.4.6 Sulphated AshThis test is usually intended for the global determination of foreign cations present in organic substances and in those inorganic substances which themselves are volatilized under the conditions of the test. Thus the test will be of little value as a purity requirement for the majority of inorganic salts of organic substances, due to the resulting high bias.The limit in a test for Sulphated ash is usually set at 0.1 percent, unless otherwise justified. The amount of substance prescribed for the test must be such that a residue corresponding to the limit will weigh not less than 1.0mg and the prescribed mass of substance is then given with the appropriate precision (1.0g).5. AssayWhen the identification and purity tests are sufficiently characteristic and searching, a non-specific but precise assay may be used rather than a specific and less precise assay.。

trypsinpromega

trypsinpromegaI.Description ............................................................................................................1II.Product Components ...........................................................................................2III.Trypsin Protocols ..................................................................................................3A.In-Gel Protein Digestion ..................................................................................3B.Digestion of Proteins in Solution .....................................................................4IV.Troubleshooting ....................................................................................................5V.Appendix ...............................................................................................................5A.Trypsin Inhibitors..............................................................................................5B.ZipTip ? Protocol..............................................................................................6C.References......................................................................................................6I.DescriptionT rypsin is a serine protease that specifically cleaves at the carboxylic side of lysine and arginine residues.The distribution of Lys and Arg residues in proteins is such that trypsin digestion yields peptides of molecular weights that can be analyzed by mass spectrometry.The pattern of peptides obtained is used to identify the protein.The stringent specificity of trypsin is essential for protein identification.Native trypsin is subject to autolysis, generating pseudotrypsin, which exhibits a broadened speci-ficity including a chymotrypsin-like activity (1).Such autolysis products, present in a trypsin preparation, would result in additional peptide fragments that could interfere with database analysis of the mass of fragments detected by mass spectrometry.T rypsin Gold, Mass Spectrometry Grade, has been manufactured to providemaximum specificity.Lysine residues in the porcine trypsin have been modified by reductive methylation, yielding a highly active and stable molecule that is extremely resistant to autolytic digestion (2).The specificity of the purified trypsin is further improved by TPCK treatment, which inactivates chymotrypsin.The treated trypsin is then purified by affinity chromatography and lyophilized to yield Trypsin Gold, Mass Spectrometry Grade.T rypsin Gold, Mass Spectrometry Grade, has extremely high specific activity.Modified trypsin is maximally active in the range of pH 7–9 and is reversiblyinactivated at pH <4.It is resistant to mild denaturing conditions such as 0.1% SDS, 1M urea or 10% acetonitrile (3) and retains 50% of its activity in 2M guanidine HCl (4).The activity of trypsin is decreased when acidic residues are present on either side of a susceptible bond.If proline is at the carboxylic side of lysine or arginine, thebond is almost completely resistant to cleavage by trypsin.Trypsin Gold,Mass Spectrometry GradeINSTRUCTIONS FOR USE OF PRODUCT V5280.PLEASE DISCARD PREVIOUS VERSIONS.All technical literature is available on the Internet at /doc/5c11981137.htmlPlease visit the web site to verify that you are using the most current version of this Technical Bulletin.Technical Bulletin No.309A F 9TB 309 0504T B 309T rypsin is often used for in-gel digestion.In this procedure, complex protein mixtures such as cell extracts are resolved by gel electrophoresis, and the band or spot of interest is excised from the gel and digested with trypsin.The digestion products are purified and concentrated, then analyzed by mass spectrometry to determine their molecular weights.Database searches can then be performed, using the mass of the peptides to identify the protein(s) resolved on the gel (5).Figure 1 is a spectrogram generated using this protocol.When the masses observed on this spectrogram were entered into a database, the protein was identified as carbonic anhydrase II.Each lot of quality-tested T rypsin Gold, Mass Spectrometry Grade, is qualified for use with in-gel digestion and mass spectrometric analysis.A copy of the spectro-gram generated with carbonic anhydrase II and any lot of T rypsin Gold, MassSpectrometry Grade, may be obtained from Promega Technical Services at 1-800-356-9526.Figure 1.Spectrogram of bovine carbonic anhydrase II digested by Trypsin Gold,Mass Spectrometry Grade.A 500ng sample of carbonic anhydrase II was separated by gel elec-trophoresis and digested with 500ng Trypsin Gold, Mass Spectrometry Grade, overnight at 37°C.The peptides generated were purified as described in Sections III and V and analyzed using a PerSeptive BioSystems Voyager-DE? MALDI-TOF system.The peak at 842.51 is due to autolysis of trypsin.II.Product ComponentsProductSize Cat.#T rypsin Gold, Mass Spectrometry Grade100µgV5280Storage Conditions:Store the lyophilized powder at –20°C.Reconstitute the powder in 50mM acetic acid and store at –20°C for up to one month.For long-term storage, freeze reconstituted trypsin at –70°C.Thaw the reconstituted trypsin atroom tem-perature, placing on ice immediately after thawing.Remove the amount of trypsin needed, then refreeze the unused portion.To maintain maximum productactivity, limit the number of freeze-thaw cycles to 5.3868T A 11_2AMass (m/z)C o u n t s1,5001,0002,0002,5003,0005,000979.368973.71013.71018.551102.091346.741582.062099.492197.572253.742511.072583.662851.3710,00015,00020,000III.Trypsin ProtocolsA.In-Gel Protein DigestionNumerous protocols for in-gel protein digestion have been described (6–8).The following procedure has been used successfully by Promega scientists.Materials To Be Supplied by the User 1.Separate protein samples by electrophoresis on an SDS-Tris-Glycine gel (see Note 1 at the end of this section).2.Rinse the gel 3 times, for 5 minutes each rinse, in NANOpure ?water.Stain for 1 hour in SimplyBlue? SafeStain (Invitrogen Corporation) at room tem-perature with gentle agitation (see Note 1 at the end of this section).When staining is complete, discard the staining solution.3.Destain the gel for 1 hour in NANOpure ?water at room temperature with gentle agitation.When destaining is complete, discard the solution.4.Using a clean razor blade, cut the protein bands of interest from the gel,eliminating as much polyacrylamide as possible.Place the gel slices into a 0.5ml microcentrifuge tube that has been prewashed twice with 50% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA).5.Destain the gel slices twice, with 0.2ml of 100mM NH 4HCO 3/50% ACN for 45 minutes each treatment, at 37°C to remove the SimplyBlue? SafeStain.6.Dehydrate the gel slices for 5 minutes at room temperature in 100µl 100%ACN.At this point the gel slices will be much smaller than their original size and will be whitish or opaque in appearance.7.Dry the gel slices in a Speed Vac ?for 10–15 minutes at room temperature to remove the ACN.8.Resuspend the T rypsin Gold at 1µg/µl in 50mM acetic acid, then dilute in 40mM NH 4HCO 3/10% ACN to 20µg/ml.Preincubate the gel slices in a mini-mal volume (10–20µl) of the trypsin solution at room temperature (do not exceed 30°C) for 1 hour.The slices will rehydrate during this time.If the gel slices appear white or opaque after one hour, add an additional 10–20µl of trypsin and incubate for another hour at room temperature.9.Add enough digestion buffer (40mM NH 4HCO 3/10% ACN) to completely cover the gel slices.Cap the tubes tightly to avoid evaporation.Incubate overnight at 37°C.10.Incubate the gel slice digests with 150µl of NANOpure ?water for10 minutes, with frequent vortex mixing.Remove and save the liquid in a new microcentrifuge tube (see Note 2 at the end of this section).11.Extract the gel slice digests twice, with 50µl of 50% ACN/5% TFA (withmixing) for 60 minutes each time, at room temperature (see Note 2).SimplyBlue SafeStain(Invitrogen Cat.# LC6060)?trifluoroacetic acid (TFA)?acetonitrile (ACN)200mM NH 4HCO 3buffer (pH 7.8)NANOpure water ZipTip scx pipette tips (Millipore Cat.# ZTSCXS096)α-cyano-4-hydroxycinnamic acid (CHCA)MALDI targetZipTip ?C18pipette tips (Millipore Cat.# ZTC18S096)We recommendwearing gloves throughout the in-gel digestion proce-dure to avoid contamina-tion of samples by proteinpresent on hands.Note:T o avoid contamina-tion, razor blades, staining containers and all other equipment that comes into contact with the gel or gel slices should be cleaned with laboratory detergent (e.g., Alconox) and rinsed well prior to use.Note:T rypsin specific activities can vary widely,depending on the manu-facturer.Procedures describing the use of trypsin by weight may need to be optimizeddepending on the specific activity of the trypsin being used.12.Pool all extracts (Steps 10 and 11) and dry in a Speed Vac?at roomtemperature for 2–4 hours (do not exceed 30°C).13.Purify and concentrate the extracted peptides using ZipTip?pipette tips(Millipore Corporation) following the manufacturer’s directions or the ZipTip?protocol recommended by Promega scientists (see Section V.B).14.The peptides eluted from the ZipTip?tips are now ready for massspectrometric analysis.Notes:1.Other gel systems and staining reagents can be used for in-gel digestions(6–8) but should be tested to ensure compatibility with the protein of interestand detection system being used.2.Alternatively, the buffer solution may be removed from the in-gel digestionimmediately following the overnight incubation step, dried with aSpeed Vac?, then prepared for mass spectrometry.The effectiveness andutility of the aqueous and organic extractions following in-gel digestsdepends on the target protein and should be determined by the user.Ingeneral, the aqueous and organic extractions are recommended for initialdigestions or for previously uncharacterized samples.B.Digestion of Proteins in SolutionIn general, proteins require denaturation and disulfide bond cleavage in order for enzymatic digestion to reach completion (4).If partial digestion of a nativeprotein is desired, begin this protocol at Step 3.1.Dissolve the target protein in 6M guanidine HCl (or 6–8M urea or 0.1%SDS), 50mM T ris-HCl (pH 8), 2–5mM DTT (or β-mercaptoethanol).2.Heat at 95°C for 15–20 minutes or at 60°C for 45–60 minutes.Allow thereaction to cool.3.For denatured proteins, add 50mM NH4HCO3(pH 7.8) or 50mM T ris-HCl,1mM CaCl2(pH 7.6), until the guanidine HCl or urea concentration is lessthan 1M.If SDS is used, dilution is not necessary.For digestion of nativeproteins, dissolve in buffer with a pH between 7 and 9.4.Add T rypsin Gold to a final protease:protein ratio of 1:100 to 1:20 (w/w).Incubate at 37°C for at least 1 hour.Remove an aliquot and chill theremainder of the reaction on ice or freeze at –20°C.5.T erminate the protease activity in the aliquot from Step 4 by adding aninhibitor (Section V.A).Alternatively, precipitate the aliquot by adding TCA toa 10% final concentration.The reaction can also be terminated by freezingat –20°C.T rypsin can also be inactivated by lowering the pH of the reactionbelow pH 4.T rypsin will regain activity as the pH is raised to above pH 4 (4).6.Determine the extent of digestion by subjecting the aliquot in Steps 4 and 5to reverse phase HPLC or SDS-PAGE.7.If no inhibitors were added to the remainder of the reaction and further pro-teolysis is required, incubate at 37°C until the desired digestion is obtained(9).Reducing the temperature will decrease the digestion rate.Incubationsof up to 24 hours may be required, depending on the nature of the protein.With long incubations, take precautions to avoid bacterial contamination.IV.TroubleshootingSymptoms Possible Causes CommentsNo or insufficient Different target proteins Determine optimum conditionsin-gel digestion may require different for each target protein.Thisdigestion conditions includes optimizing trypsin:proteinratios and incubation times.Peptides not eluting Optimize the ZipTip?elution condi-from the ZipTip?tips tions.Follow the manufacturer’sdirections for use.Incompatible reconstitution Reconstitution of T rypsin Gold inbuffer buffers other than 50mM acetic acidis not recommended.Loss of trypsin activity Reconstituted trypsin is stable for updue to multiple freeze-thaw to 5 freeze-thaw cycles.cyclesThe spectrogram These peaks are due to These peaks are often used to contains peaks autocatalytic trypsin calibrate the mass spectrometer. at 842 and 2211cleavage at arginineresidues, which arenot protected byreductive methylationApparent chymotrypsin The presence of chymo-T ry using lower trypsin:protein peaks on mass trypsin is very unlikely, ratios and/or shorter incubation spectrogram because it is inhibited times.Reconstitution of T rypsinby TPCK.These peaks Gold, Mass Spectrometry Grade,are most likely due to in buffers other than 50mMpseudotrypsin activity acetic acid is not recommended.(1)Reconstituted trypsin subjectedto more than 5 freeze-thaw cyclesmay contain products of autolysis. Reaction Keratin is a contaminant Wear gloves.Work in a hood. contamination from skin and hair Thoroughly clean all equipmentcommonly introduced and use disposable componentsduring sample handling when possible.V.AppendixA.Trypsin InhibitorsThe following are general trypsin inhibitors:Antipain (50µg/ml), antithrombin (1unit/ml), APMSF (0.01–0.04mg/ml), aprotinin(0.06–2µg/ml), leupeptin (0.5µg/ml), PMSF (17–170µg/ml), TLCK (37–50µg/ml),trypsin inhibitors (10–100µg/ml).B.ZipTip?ProtocolPromega scientists have successfully used the following ZipTip?(ZipTip?scx or ZipTip?C18) pipette tip protocol for purification and sample concentration.These conditions may not be optimal for all proteins/peptides.1.Prepare ZipTip?tips by washing with 10µl of 100% ACN, then washing2–3 times with 10µl of 0.1% TFA.2.Reconstitute the samples with 10µl of 0.1% TFA.3.Draw the sample into ZipTip?tips by pipetting fully into and out of tips4–5 times.Expel liquid.4.Wash ZipTip?tips 2–3 times with 10µl of 0.1% TFA to removecontaminants.5.Elute peptides in 2.5µl of 70% ACN/0.1% TFA containing 10mg/ml α-cyano-4-hydroxycinnamic acid (CHCA) and spot directly onto MALDI target.C.References1.Keil-Dlouha, V.et al.(1971) Proteolytic activity of pseudotrypsin.FEBS Lett.16, 291–5.2.Rice, R.H., Means, G.E.and Brown, W.D.(1977) Stabilization of bovinetrypsin by reductive methylation.Biochem.Biophys.Acta492, 316–21.3.Bond, J.S.(1989) “Commercially Available Proteases”, Appendix II.In:Proteolytic Enzymes, A Practical Approach,R.J.Beynon and J.S.Bond,eds., IRL Press, Oxford, 240.4.Wilkinson, J.M.(1986) “Fragmentation of Polypeptides by EnzymicMethods”.In:Practical Protein Chemistry:A Handbook, A.Darbre, ed., JohnWiley and Sons, New Y ork, 122.5.Mann, M., Hendrickson, R.C.and Pandey, A.(2001) Analysis of proteins andproteomes by mass spectrometry.Ann.Rev.Biochem.70, 437–73.6.Flannery, A.V., Beynon, R.J.and Bond, J.S.(1989) “Proteolysis of Proteinsfor Sequencing Analysis and Peptide Mapping”.In:Proteolytic Enzymes:APractical Approach, R.J.Beynon and J.S.Bond, eds., IRL Press, Oxford, 45.7.Shevchenko, A.et al.(1996) Mass spectrometric sequencing of proteinssilver-stained polyacrylamide gels.Anal.Chem.68, 850–8.8.Rosenfeld, J.et al.(1992) In-gel digestion of proteins for internal sequenceanalysis after one- or two-dimensional gel electrophoresis.Anal.Biochem.203, 173–9.9.Sheer, D.G.(1994) Techniques in Protein Chemistry, J.W.Crabb ed., Academic, San Diego Volume V, 243.。

methods to control harmful algal blooms a review

methods to control harmful algal blooms a review Harmful algal blooms (HABs) are a global phenomenon, causing ecological and human health problems. HABs are primarily caused by the excessive growth of certain species of algae that are toxic to humans and marine life. Various methods are used to control HABs, ranging from physical and chemical methods to the use of biological agents. 。

1. Physical control methods: Physical methods are aimed at reducing the exposure of people and marine organisms to HABs. These methods include the use of barriers, such as nets or floating booms, to prevent the spread of the bloom. Physical methods also include the use of pumps and skimmers to remove the HABs from the water.2. Chemical control methods: Chemical methods are used to reduce the growth of HABs by killing or suppressing the growthof the algae. Chemical control methods include the use of herbicides, pesticides, and algaecides. However, the use of chemicals can have unintended consequences, such as the killing of non-targeted organisms or the accumulation of toxins in food webs.4. Nutrient management: Nutrient management aims to reduce the availability of nutrients, such as nitrogen and phosphorus, that promote the growth of HABs. Strategies include reducingfertilizer use and controlling wastewater discharge in coastal areas.5. Genetic control methods: Genetic methods are used to modify the genes of harmful algae or to introduce genes from other organisms to control the growth of HABs. However, genetic methods are still in the experimental stage and are not currently used to control HABs.。

Supramolecular interactions between losartan and hydroxypropyl

International Journal of Pharmaceutics 404 (2011) 116–123Contents lists available at ScienceDirectInternational Journal ofPharmaceuticsj 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 /i j p h a rmSupramolecular interactions between losartan and hydroxypropyl-␤-CD:ESI mass-spectrometry,NMR techniques,phase solubility,isothermal titration calorimetry and anti-hypertensive studiesWashington X.de Paula a ,Ângelo M.L.Denadai b ,Marcelo M.Santoro c ,Aline N.G.Braga d ,Robson A.S.Santos d ,Rubén D.Sinisterra a ,∗aLaboratório de Encapsulamento Molecular e Biomateriais (LEMB),Departamento de Química,Instituto de Ciências Exatas,Universidade Federal de Minas Gerais (UFMG),Av.Antônio Carlos 6627,Belo Horizonte 31270-901,MG,Brazil bCentro Federal de Educac ¸ão Tecnológica de Minas Gerais (CEFET-MG),Campus VII,Timóteo 35183-006,MG,Brazil cDepartamento de Bioquímica e Imunologia,Instituto de Ciências Biológicas (ICB),Universidade Federal de Minas Gerais (UFMG),Belo Horizonte 31270-901,MG,Brazil dDepartamento de Fisiologia-Biofísica,Instituto de Ciências Biológicas (ICB),Universidade Federal de Minas Gerais (UFMG),Belo Horizonte 31270-901,MG,Brazila r t i c l e i n f o Article history:Received 3March 2010Received in revised form 3November 2010Accepted 10November 2010Available online 17 November 2010Keywords:LosartanAngiotensin II (AT 1)Receptor antagonist Inclusion compounds Cyclodextrinsa b s t r a c tIn this work,low soluble supramolecular complex between the losartan potassium (Los)and hydroxypropil-␤-cyclodextrin (HP ␤CD)were characterized throughout phase-solubility,NMR tech-niques (1H and 2D-ROESY)and isothermal titration calorimetry (ITC)in order to attain physical–chemical knowledge of the system.In addition,the hypertensive effect of composition Los/HP ␤CD was evaluated aiming to obtain a more efﬁcient oral pharmaceutical composition.ESI mass spectrometry and ITC blank experiment demonstrate the presence of Los clusters at 30mM pure solution.Phase-solubility experi-ments showed a “Bs ”type system,due to the formation of a less soluble complex than pure Los.NMR demonstrated the short distance interactions between the Los and the cyclodextrin,where several pos-sibilities of interactions were observed.ITC data suggest an average 1:1stoichiometry of Los and the cyclodextrin.The complex demonstrated efﬁciency in hypertension control,presenting antagonist action on the pressure effect of angiotensin II within 30h,as compared to Los alone,6h,indicating that inclu-sion of Los in HP ␤CD enhanced the extent and duration of its antagonistic action.In this work,a model of interaction between Los and HP ␤CD was proposed based on dissociation of self-assembled Los followed by complexation with HP ␤CD.© 2010 Elsevier B.V. All rights reserved.1.IntroductionLosartan potassium (Los –Fig.1)is a higher soluble orally active,non-peptide drug used in the treatment of hypertension and related disorders (Kaplan,1999;Lambot et al.,2001;Mcintyre et al.,1997;Oparil et al.,2001).It has been demonstrated to have a greater effect than previous peptide receptor antagonists and angiotensin converting enzyme (ACE)inhibitors because of its enhanced speci-ﬁcity,selectivity,and tolerability.Los is metabolized in the body,forming a pharmacologically active carboxylic acid metabolite EXP-3174that presents low bio-availability of 33%(19–62%)with an approximate one-hour post-administration plasma peak concen-tration and a mean half-life of 2h (Mcintyre et al.,1997).∗Corresponding author at:Departamento de Química,Universidade Federal de Minas Gerais.CEP:31270-901Avenida Pres.,Antônio Carlos 6627,Belo Horizonte,Minas Gerais,Brazil.Tel.:+553134995778;fax:+553134995700.E-mail address:sinisterra@ufmg.br (R.D.Sinisterra).Thus we hypothesized that if sequestering a highly water soluble drug,such as Los,through supramolecular non-covalent host–guest complexes formation using modiﬁed cyclodex-trin,such as hydroxypropil-␤-cyclodextrin (HP ␤CD),we can obtain lower water Los solubility and a more efﬁcient angiotensin II AT1receptor antagonist oral pharmaceutical composition.This host–guest strategy is well-established in literature where cyclodextrins complexes with hydrophobic guests in order to increase water solubility and bioavailability (De Sousa et al.,2008;Denadai et al.,2006a,2007a,b;Irie and Uekama,1997;Loftsson and Brewster,1996;Lula et al.,2007;Sousa et al.,2008;Uekama et al.,1998).However,works using this strategy when water-soluble molecules as Los are used as guest molecules are scarcely found in literature.Cyclodextrins are very important for supramolecular chem-istry,since they form non-covalent complexes with a wide range of host molecules,which can be used as models for studying weak interaction.Their cavity offers a suitable hydrophobic envi-ronment to the guest molecule (Fig.2),allowing the formation0378-5173/$–see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.ijpharm.2010.11.008W.X.de Paula et al./International Journal of Pharmaceutics 404 (2011) 116–123117Fig.1.Losartan potassium structure.of inclusion compounds.Hence cyclodextrins are used for the solubilization and encapsulation of drugs,perfumes and ﬂavor-ings forming supramolecular structures (De Sousa et al.,2008;Denadai et al.,2006a,2007a,b;Irie and Uekama,1997;Loftsson and Brewster,1996;Sousa et al.,2008).In this work,the supramolecular complexes between the Los and HP ␤CD were characterized throughout electrospray mass spectrometry,phase-solubility,NMR techniques (1H and 2D-ROESY)and isothermal titration calorimetry (ITC)in order to look further into the physical–chemical characteristics of the system.Furthermore,in vivo anti-hypertensive tests were carried out aim-ing to assess higher efﬁcient Los oral pharmaceutical composition.2.Materials and methods 2.1.ReagentsLos was purchased from Galena Química e Farmacêutica Ltda,Brazil;and HP ␤CD (substitution degree 5–8and M w ≈1400g/mol)was obtained from Cerestar Company,USA.All the othermate-Fig.3.Electrospray mass spectroscopy of Losartan potassium 5mM.rials and solvents were of analytical reagent grade and used as received.2.2.Inclusion compound preparationFor NMR and ESI-MS analysis and anti-hypertensive evaluation,a 1:1Los/HP ␤CD inclusion compound was prepared by freeze-dry method.In brieﬂy,the Los salt and HP ␤CD were dissolved in milli-Q water at 1:1molar ratio.This mixture was submitted to stirring during 48h.Next,the solution was freeze-dried by 48h.2.3.ESI-MS measurementsMass spectrometry experiments were performed using an ESI Micromass Q-TOF instrument in order to check the previous aggre-gation state of the Los and the stoichiometry of the complex in solution.The data were collected from a 5mM sample of Los and Los/HP ␤CD 1:1aqueous solution by using the electrospray ioniza-tion mode.The standard conditions employed were:vaporization temperature of 120◦C,capillary voltage of 2000V,sample cone 40V,extraction cone 2.0V.Fig.2.Hydroxipropil-␤-cyclodextrin structure.118W.X.de Paula et al./International Journal of Pharmaceutics404 (2011) 116–123Fig.4.(a)Electrospray mass spectrum of 5mM Los/HP ␤CD 1:1water solution.(b)Expansion of the region between 1800and 2000,referent to inclusion compounds.2.4.NMR experiments1HNMR chemical shifts (ı)and 2D 1H-1H-ROESY experimentswere obtained using a Brucker DPX-400Avance (400MHz)spec-trometer,at 300K.The solutions used were 2.0mM of Los and 2.0mM (1:1)of Los/HP ␤CD,both dissolved in D 2O (Cambridge Isotope Laborato-ries,Inc –99.9%of isotopic purity).The HOD signal at ı=4.80was used as reference.No solid formation was observed in the solution during analysis.The 2D-ROESY experiments were recorded at spin lock of 600ms,which was previously calculated throughout the inversion-recovery sequence (Rahman,1989;Werner,1994).The sample for this experiment was prepared by utilizing the freezing-drying method in the molar ratio of 1:1Los/HP ␤CD.In this method,the aqueous solution,which contains the dissolved materials (Los and HP ␤CD),was stirred for 2h.After,the solu-tion was frozen in liquid nitrogen and freeze-dried for 24h before dissolution in D 2O.2.5.Solubility studiesThe phase-solubility diagrams were made according to the Higuchi and Connors method (Higuchi and Connors,1969).For this purpose,aqueous solutions of HP ␤CD with concentrations of 0–2.6mM with a known concentration of Los at 30mM were pre-pared.These were placed in a thermostatic bath at 298K by 48h.The samples were centrifuged at 15rpm for 10min and ﬁltered with an ultra-ﬁltration membrane of 0.22␮m from Millipore ®.Quantiﬁ-cation was performed in triplicate on a UV–vis equipment HP-8453at 248nm and a one-centimeter cell.2.6.Microcalorimetric measurementsCalorimetric titrations were carried out in duplicate with a VP-ITC Microcalorimeter (Microcal Company,Northampton,MA,USA)at 298.15;303.15and 308.15K next the electrical and chemical calibration.Each titration experiment consisted of 41successive injections of Los aqueous solution (100mM)into the reaction cell charged with 1.6mL of HP ␤CD aqueous solution (4.0mM),with time inter-vals of 540s.The ﬁrst injection of 1.0␮L was discarded to eliminate diffusion effects of material from syringe to cell calorimetric.The subsequent injections were used at constant volume of 5.0␮L of Los.The time of injection was 2.0s.The HP ␤CD concentration in the calorimeter cell varied from 4.0to 3.6mM and the concentration of the Los from 0.0to 11.1mM.The raw data were analyzed using the “one site model”set forth by Microcal Origin 5.0for ITC after the subtraction of the blank experiment (dilution of Los in water).The enthalpy values obtained at each temperature were plot-ted against temperature values in order to determinate the heat capacity at pressure constant C ◦p ,according to Eq.(1),where the angular coefﬁcient of the curve ∂ H ◦/∂T ,was obtained by linear regression by using of Microcal Origin 5.0.C ◦p =∂ H ◦∂Tp(1)2.7.Dissolution testsThe dissolution tests were performed in an incubator model Q120A3Qualitas according to the method described by Oskan et al.(2000).Initially were weighed in triplicate 100mg of LosW.X.de Paula et al./International Journal of Pharmaceutics 404 (2011) 116–123119Fig.5.Solubility diagram of [Losartan]vs.[HP ␤CD]at 25◦C.and 300mg of the freeze dried Los/HP ␤CD,after being passed by a separation particle size of 50,100and 200mesh.They were then transferred to tubes eppendorfs and added 2.0mL of phosphate buffer pH 7.4and undergoing agitation 110rpm at 37◦C for 5min.Then 1.0mL was collected from each tube and added more 1.0mL of buffer.This procedure was performed at various time intervals (0.25–24h)after the beginning of the experiment,reaching 48h.The solutions collected from each tube at different time intervals were stored for quantiﬁcation on a UV–vis equipment HP-8453at 248nm and a one-centimeter cell.2.8.Antagonist action of Los and Los/HP ˇCD complex on the pressor effect of angiotensin II in Wistar ratsThe antagonist action of Los and Los/HP ␤CD complex on angiotensin pressor effect was evaluated in Wistar rats (male,weighting 300–350g)obtained from “CEBIO –Centro de Bioterismo de Ciências Biológicas–UFMG”.Before and after surgery,the animals were kept in a temperature-controlled room using a 14/10-light/dark cycle.One day before the experiment,a polyethylene catheter (PE-10con-nected to PE-50)was inserted into the abdominal aorta through the femoral artery for blood pressure measurements.For intravenousinjections and infusions,polyethylene cannulas were implanted into the veins.The cannulas,closed by a metallic pin and ﬁlled with isotonic saline,were driven subcutaneously to the interdescapular region of the back of the animals.After recovery from anesthesia,the rats were kept in individual cages with free access to water and chow until the end of the experiment.A data acquisition system (Biopac,USA)was used to measure systolic,diastolic and mean pressure.In experiments,Los/HP ␤CD solution was prepared by directly dissolution of complex in mili-Q water,which was fol-lowed by further dilutions.Los and Los/HP ␤CD were administered by oral route,and the hypertensive effect produced by i.v.infusion of angiotensin II (Ang II)(0.10mL 20ng in bolus ).The pressure data were collected 2,6,12,24,48and 72h after Los or Los/HP ␤CD com-plex administration.All experimental protocols were performed in compliance with the guidelines on laboratory animals from our institute and approved by local authorities.The basal values mean arterial pressure were (98±0.5)parisons between the control period and the experimental and recovery periods were made by one-way ANOVA,followed by the Dunnet’s test.For this,the control values were obtained by averag-ing all values obtained in the last 3days of the pre-infusion parison between changes in mean arterial pressure produced by Ang II infusion was made using t -test.A value of P <0.05was considered statistically signiﬁcant.3.Results and discussion 3.1.ESI-MS measurementsLos is an amphiphilic substance and shows an intense and endothermic heat of dilution (see below ITC data)suggesting dis-aggregating phenomenon upon dilution.Hence,in order to check the possibility of self-assembly of the Los under the experimen-tal conditions,ESI mass-spectrometry measurements were used once the ESI experiments were able to evaluate weak non-covalent complexes (Fig.3)(Rekharsky et al.,2002;Toma et al.,2004).The data presented in Fig.3show the presence of homologue series of small K n −1Los n clusters,not excluding necessarily the presence of high order self-assembly complexes in water solu-tions,as described in the literature for other amphiphilic systems (Rekharsky et al.,2002;Toma et al.,2004).Fig.6.2D-ROESY spectrum of the inclusion compound between Losartan and HP ␤CD.120W.X.de Paula et al./International Journal of Pharmaceutics404 (2011) 116–123ESI mass-spectroscopy was also used to assess qualitatively the species present in the Los/HP␤CD solution at a1:1molar ratio (Fig.4).The ESI technique is not precise enough to monitor weak non-covalent interactions because this type of experiment may disturb the chemical equilibrium of the system due to the drastic conditions(high electricalﬁeld and high temperature).Fig.4shows parts of the ESI experiment where the m/z distri-bution of peaks between1285.45and1713.90,with maximum at 1537.64,is observed.Such distribution is due the fact that HP␤CD sample is a mixture of several cyclodextrins with different substi-tution degree.At m/z values localized between1800and2000is observed three peaks,1846.13,1905.28and1963.45,relating to three types of inclusion compounds with stoichiometry1:1.The correspondent peaks of free cyclodextrins are those localized at m/z 1423.54,1479.58and1537.64(considering the m/z of Los−anion of423.32).3.2.Phase solubility studiesThermodynamic solubility experiments allow to demonstrate the hydrotrope effect on the solubility of a substance and evaluates the interactions between the species(Higuchi and Connors,1969).Fig.5shows the solubility of Los against HP␤CD concentra-tion where an initial Los constant concentration of up to0.25mM of cyclodextrin is observed.After achieving this value,a strong reduction of Los solubility upon increase of HP␤CD concentration is observed.This diagram corresponds to the formation of a com-plex less soluble than the pure substrate,named Higuchi’s B s type (Higuchi and Connors,1969).Los and HP␤CD are very soluble molecules presenting high aqueous solubility at298K(both species have solubility greater than300mM).However,as demonstrated in Fig.5,a lower soluble than pure Los species is formed upon complexation, showing stronger host–guest interaction than host–solvent and guest–solvent.The Los/HP␤CD complex is a very small and quasi invisible pre-cipitate which was separated from solution only by centrifugation and ultra-ﬁltration.According to the solubility data,the precipi-tate formation is dependent on the molar ratio and it starts to form above0.25mM of HP␤CD.The Los solubility reduction after the HP␤CD interaction could be explained by the ESI results for pure Los.The ESI results showed the presence of Los self-assemblies,which could be an intrinsic sol-ubilization mechanism of the drug.Thus,complexation of Los with HP␤CD could break down the self-assemblies,leading to reduction of Los solubility.3.3.NMR experimentsNMR experiments allowed an investigation of the short dis-tance interaction of supramolecular complexes between Los and HP␤CD.1H NMR chemical shift changes are related to the distur-bance caused by unpaired C1-O-C4electrons of the HP␤CD cavity on electronic density of the Los hydrogen’s(De Sousa et al.,2008; Denadai et al.,2007b;Loftsson et al.,1993;Schneider et al.,1998; Sousa et al.,2008).Changes in chemical environment and confor-mation upon break down of self-assembly also contribute to the changes in chemical shift.The Table1shows the chemical shift of 1H NMR values for free and complexated Los.Possible architectures of the Los complexes with cyclodextrins were evaluated by the2D-ROESY experiments,which are able to detect dipolar coupling in solution through space upon5˚A distance (Denadai et al.,2007a,b;Lula et al.,2007;Rahman,1989;Schneider et al.,1998;Werner,1994).Cross peaks correlations were found between aromatic Los hydrogens(region atı≈6.5–7.6)with H2,H3,H4,H5and H6Table11H NMR(at400MHz)chemical shifts of Los and Los/HP␤CD in D2O at27◦C. HydrogenıLosıLos/HP␤CD ıaH6 2.49 2.51+0.02 H7 1.48 1.40−0.08 H8 1.25 1.20−0.05 H90.810.79−0.02 H10 5.21 5.24+0.03 H12 6.90 6.97+0.05 H137.107.16+0.04 H167.297.28−0.01 H177.347.36+0.02 H187.367.37+0.01 H197.527.54+0.02 H22 4.31 4.30−0.01a ı=ıLos−ıLos/HP␤CD.HP␤CD hydrogens.There were also cross peaks found between H12,H13and H16of the Los with H9hydrogens of HP␤CD hydrox-ypropyl group.These data are indicating correlations of aromatic and aliphatic moieties of Los with external hydrogens H9of HP␤CD (Fig.6).These results suggest very complex supramolecular architecture between Los and HP␤CD which could be explained in terms of three plausible hypotheses:(1)dynamic equilibrium between the species,where several com-plexes stabilized by different non-covalent interactions can exist within the time scale of NMR detection,(2)conformational equilibrium of Los molecule,which could letseveral kinds of interactions with cyclodextrins within the time scale of NMR detection,(3)self-assembly of inclusion compounds,where the complex willbe packaged in different way,stabilized by hydrogenbonds.Fig.7.Dilution curves obtained from41successive5␮L injections of an aqueous solution of100mM Losartan potassium into a1.4mL cell containing water at298, 308and318K.W.X.de Paula et al./International Journal of Pharmaceutics404 (2011) 116–123121Fig.8.Titration curves obtained from41successive5␮L injections of an aqueous solution of100mM Losartan potassium into a1.4mL cell initially containing4mM HP␤CD at298,308and318K,after subtracted from blank data at each temperature.3.4.Microcalorimetric measurementsIn order to know the average stoichiometry of the complex in solution as well as its thermodynamic parameters,isothermal titrations calorimetry(ITC)of Los100mM into the4mM HP␤CD solution,at298,308and318K were performed(Figs.7and8). ITC experiment allows for the simultaneous determination of the enthalpy H◦,equilibrium constant K eq and stoichiometry N from a single titration curve,using the least square non-linear adjustment (MicroCal,1998a,b;Turnbull and Daranas,2003).Through the use of classical thermodynamic equations(Levine,1995),is possible calculate the changes on free energy G◦,entropy energy T S◦and heat capacity at pressure constant C◦p.For all temperatures,the sigmoid adjust showed an equivalence point at molar ratio of approximately0.8(Table2).These data and the relative size between the molecules suggested a1:1minimal stoichiometry not necessarily excluding the existence of higher order complex.It is well accepted in the literature that the cyclodextrin system there is a distribution of equilibrium species,named Los n/HP␤CD m, wich has an average stoichiometry(Denadai et al.,2007b;Loftsson and Brewster,1996;Sousa et al.,2008).Thus,the equilibrium constant calculated on the basis of ITC experiment is a global equi-librium constant K eq,g,referring to some process which leads to the formation of a Los n/HP␤CD m(Eqs.(2)and(3)).nS+mCD K eq,gS m CD n(2)Fig.9. C◦p calculus from Eq.(1).K eq,g=[S mCD n][S]n[CD]m(3)As depicted in Fig.7,the Los dilution experiment showed endothermic signals in an overall range of analyzed concentration, indicating that dilution is entropy-driven.Considering that ESI mass spectroscopy experiments showed the presence of clusters at30mM solutions,the endothermic dilu-tion of Los could be understood in terms of disaggregation of self-assemblies,which requires energy to separate monomers from the clusters.Analyzing the data from Table2,one can observe that the overall interaction between Los and HP␤CD is exothermic and accompa-nied by entropy increase.However,the global equilibrium constant is relatively low and similar to other cyclodextrins/guest systems (Rekharsky and Inoue,1998).Enthalpy changes may well be attributed to the binding of enthalpy-rich water molecules released from the cyclodextrin cav-ity together with bulk water molecules(De Sousa et al.,2008; Denadai et al.,2006b,2007a,b;Loftsson and Brewster,1996; Rekharsky and Inoue,1998;Sousa et al.,2008),by the formation of cooperative Van der Waals interactions between guest and the cyclodextrin cavity through the hydrophobic Los moiety and by the formation of ion-dipole interaction between tetrazolic ring and cyclodextrin OH groups.By performing ITC experiments,positive entropy changes were found,although the Los/HP␤CD complex had been precipitated. For an increase of entropy in the precipitation process,high des-solvation of molecules is needed to compensate the formation of solid-state phase,and the water molecules must gain translational and rotational freedom degree(Rekharsky et al.,2002).The deepest understanding of the host–guest interactions was achieved by considering the C p changes during the process,cal-culated by Eq.(1)through linear regression in a“ H◦versus T”graphic(Fig.9).It is well known that C p data is related to changes in hydropho-bic interactions during binding process.According to the literature (Rekharsky and Inoue,1998),if C p is negative(−0.15kJ/mol K for the Los/HP␤CD system),hydrophobic bonds are formed as con-Table2Thermodynamic parameters obtained by ITC experiments.T/K N K H◦/kJ mol−1 G◦/kJ mol−1T S◦/kJ mol−1 C p/kJ mol−1K−1298.150.741147.0−11.8−17.5 5.8308.150.79739.2−13.4−16.9 3.5−0.15318.150.83496.8−14.7−16.4 1.7122W.X.de Paula et al./International Journal of Pharmaceutics404 (2011) 116–123Fig.10.Dissolution proﬁle of Losartan and Los/HP␤CD complex.Mean±SE(vertical bar),n=3.sequence of breakdown water clatrates around apolar molecules, corroborating the dessolvation hypothesis mentioned above.Thus,in this work it was suggested that the interaction between Los and HP␤CD involves an initial disaggregation of Los self-assemblies,followed by complexation with cyclodextrins.The formed complex presents low solubility and it is stabilized by favor-able interactions such as Van der Waals,hydrogen bonding and ion-dipole interactions.The interaction is increased by the release of the enthalpy-rich water molecules from cyclodextrins cavity and hydration shells of cyclodextrins and Los,which gain rotational and translational freedom degree in order to overcome the entropy reduction due to precipitation.3.5.Dissolution testsFig.10shows the in vitro dissolution proﬁles for Losartan and Los/HP␤CD.The curve of Losartan showed a rapid dissolution,with approximately100%dissolved in theﬁrst minutes,as compared to 65%dissolution of Los/HP␤CD at the same time interval.Moreover, the dissolution of the complex was observed until20h after the start of testing,conﬁrming that inclusion leads to a decrease in solubility of Losartan.3.6.Antagonist action of Los and Los/HPˇCD complex on the pressor effect of angiotensin II in Wistar ratsFig.11shows the antihypertensive effect of Los and Los/HP␤CD in Wistar rats.The Los/HP␤CD complex,when administered orally as a single dose of0.7mg/kg,blocked in approximately75% (p<0.01,n=4)the pressor effect of Ang-II for approximately30h. In contrast,the free Los given at the same dose blocked the effect of Ang II for a period of only6h(p<0.01,n=4).The increase in pres-sure due to angiotensin II administration in the control period was 20.2mmHg and22.3mmHg for Los and Los/HP␤CD,respectively. After3h,13mmHg and9mmHg,reaching around a variation of 20.5mmHg and17.1mmHg after30h.These data indicate that inclusion compound Los/HP␤CD enhanced the extent and duration of its Los antagonist action.The higher Los bioavailability upon inclusion could be due to the lower Los solubility and solubilization rate as veriﬁed by phase solubility studies and dissolution tests.To the extent of our knowledge,such an approach to alter the pharmacological proﬁle of highly water-soluble drug when admin-istered orally has not yet been explored.Thus,the formulation using the host–guest strategy could be improved by increasing the efﬁcacy and reducing the dose or spacing with each doseintake.Fig.11.Effect of Losartan and Los/HP␤CD complex administered by oral route at 0.7mg/kg to Wistar rats,on the mean arterial pressure changes induced by Ang II (20ng i.v.).*p<0.05compared with the average of the3last day of the period before infusion(98±0.5mmHg)(ANOVA followed by Dunnett’s test).Mean±SE(vertical bar),n=4.4.ConclusionA model based on dissociation of the Losartan potassium clus-ters,followed by complexation with HP␤CD forming a lower soluble1:1complex than their precursor has been proposed. This approach offers an alternative means of improving the bio-availability of water-soluble drugs and represents a signiﬁcant step towards the development of sustained release of oral dosage forms. The Los formulation accompanied with this strategy could be useful in reducing costs and increasing compliance with the antihyperten-sive treatment.AcknowledgmentsThe authors acknowledgeﬁnancial support by Brazilian agen-cies FAPEMIG,CAPES and CNPq and Biolab-Sanus pharmaceutical company for the technology transfer contract assigned with the UFMG to scale up this technology.ReferencesDe Sousa,F.B.,Denadai,A.M.,Lula,I.S.,Lopes,J.F.,Dos Santos,H.F.,De Almeida, W.B.,Sinisterra,R.D.,2008.Supramolecular complex ofﬂuoxetine with beta-cyclodextrin:an experimental theoretical study.Int.J.Pharm.353,160–169. Denadai,A.M.,Ianzer,D.,Alcantara,A.F.,Santoro,M.M.,Santos,C.F.,Lula,I.S.,de Camargo,A.C.,Faljoni-Alario,A.,dos Santos,R.A.,Sinisterra,R.D.,2007a.Novel pharmaceutical composition of bradykinin potentiating penta peptide with beta-cyclodextrin:physical–chemical characterization and anti-hypertensive evaluation.Int.J.Pharm.336,90–98.Denadai,A.M.,Teixeira,K.I.,Santoro,M.M.,Pimenta,A.M.,Cortes,M.E.,Sinisterra, R.D.,2007b.Supramolecular self-assembly of beta-cyclodextrin:an effec-tive carrier of the antimicrobial agent chlorhexidine.Carbohydr.Res.342, 2286–2296.Denadai,A.M.L.,Santoro,M.M.,Da Silva,L.H.,Viana,A.T.,Santos,R.A.S.,Sinis-terra,R.D.,2006a.Self-assembly characterization of the beta-cyclodextrin and hydrochlorothiazide system:NMR,phase solubility,ITC and QELS.J.Incl.Phe-nom.Macro.55,41–49.Denadai,A.M.L.,Santoro,M.M.,Lopes,M.T.P.,Chenna,A.,de Sousa,F.B.,Avelar,G.M., Gomes,M.R.T.,Guzman,F.,Salas,C.E.,Sinisterra,R.D.,2006b.A supramolecular complex between proteinases and beta-cyclodextrin that preserves enzymatic activity–physicochemical characterization.Biodrugs20,283–291.Higuchi,T.,Connors,K.,1969.Phase-Solubility Techniques.Advances in Analytical Chemistry and Instrumentation,4,pp.117–212.Irie,T.,Uekama,K.,1997.Pharmaceutical applications of cyclodextrins3.Toxicolog-ical issues and safety evaluation.J.Pharm.Sci.86,147–162.Kaplan,N.M.,1999.Angiotensin II receptor antagonists in the treatment of hyper-tension.Am.Fam.Physician60,1185–1190.Lambot,M.A.,Vermeylen,D.,Noel,J.C.,2001.Angiotensin-II-receptor inhibitors in ncet357,1619–1620.Levine,I.N.,1995.Physical Chemistry,fourth edition.McGRAW-HILL INC.,New York. Loftsson,T.,Brewster,M.E.,1996.Pharmaceutical applications of cyclodextrins1.Drug solubilization and stabilization.J.Pharm.Sci.85,1017–1025.。

光谱法研究药物小分子与蛋白质大分子的相互作用的英文

Spectroscopic Study of the Interaction between Small Molecules and Large Proteins1. IntroductionThe study of drug-protein interactions is of great importance in drug discovery and development. Understanding how small molecules interact with proteins at the molecular level is crucial for the design of new and more effective drugs. Spectroscopic techniques have proven to be valuable tools in the investigation of these interactions, providing det本人led information about the binding affinity, mode of binding, and structural changes that occur upon binding.2. Spectroscopic Techniques2.1. Fluorescence SpectroscopyFluorescence spectroscopy is widely used in the study of drug-protein interactions due to its high sensitivity and selectivity. By monitoring the changes in the fluorescence emission of either the drug or the protein upon binding, valuable information about the binding affinity and the binding site can be obt本人ned. Additionally, fluorescence quenching studies can provide insights into the proximity and accessibility of specific amino acid residues in the protein's binding site.2.2. UV-Visible SpectroscopyUV-Visible spectroscopy is another powerful tool for the investigation of drug-protein interactions. This technique can be used to monitor changes in the absorption spectra of either the drug or the protein upon binding, providing information about the binding affinity and the stoichiometry of the interaction. Moreover, UV-Visible spectroscopy can be used to study the conformational changes that occur in the protein upon binding to the drug.2.3. Circular Dichroism SpectroscopyCircular dichroism spectroscopy is widely used to investigate the secondary structure of proteins and to monitor conformational changes upon ligand binding. By analyzing the changes in the CD spectra of the protein in the presence of the drug, valuable information about the structural changes induced by the binding can be obt本人ned.2.4. Nuclear Magnetic Resonance SpectroscopyNMR spectroscopy is a powerful technique for the investigation of drug-protein interactions at the atomic level. By analyzing the chemical shifts and the NOE signals of the protein in thepresence of the drug, det本人led information about the binding site and the mode of binding can be obt本人ned. Additionally, NMR can provide insights into the dynamics of the protein upon binding to the drug.3. Applications3.1. Drug DiscoverySpectroscopic studies of drug-protein interactions play a crucial role in drug discovery, providing valuable information about the binding affinity, selectivity, and mode of action of potential drug candidates. By understanding how small molecules interact with their target proteins, researchers can design more potent and specific drugs with fewer side effects.3.2. Protein EngineeringSpectroscopic techniques can also be used to study the effects of mutations and modifications on the binding affinity and specificity of proteins. By analyzing the binding of small molecules to wild-type and mutant proteins, valuable insights into the structure-function relationship of proteins can be obt本人ned.3.3. Biophysical StudiesSpectroscopic studies of drug-protein interactions are also valuable for the characterization of protein-ligandplexes, providing insights into the thermodynamics and kinetics of the binding process. Additionally, these studies can be used to investigate the effects of environmental factors, such as pH, temperature, and ionic strength, on the stability and binding affinity of theplexes.4. Challenges and Future DirectionsWhile spectroscopic techniques have greatly contributed to our understanding of drug-protein interactions, there are still challenges that need to be addressed. For instance, the study of membrane proteins and protein-protein interactions using spectroscopic techniques rem本人ns challenging due to theplexity and heterogeneity of these systems. Additionally, the development of new spectroscopic methods and the integration of spectroscopy with other biophysical andputational approaches will further advance our understanding of drug-protein interactions.In conclusion, spectroscopic studies of drug-protein interactions have greatly contributed to our understanding of how small molecules interact with proteins at the molecular level. Byproviding det本人led information about the binding affinity, mode of binding, and structural changes that occur upon binding, spectroscopic techniques have be valuable tools in drug discovery, protein engineering, and biophysical studies. As technology continues to advance, spectroscopy will play an increasingly important role in the study of drug-protein interactions, leading to the development of more effective and targeted therapeutics.。

EPA 3510C-1996 分液漏斗的液体提取

METHOD 3510CSEPARATORY FUNNEL LIQUID-LIQUID EXTRACTION1.0SCOPE AND APPLICATION1.1This method describes a procedure for isolating organic compounds from aqueous samples. The method also describes concentration techniques suitable for preparing the extract for the appropriate determinative methods described in Section 4.3 of Chapter Four.1.2This method is applicable to the isolation and concentration of water-insoluble and slightly water-soluble organics in preparation for a variety of chromatographic procedures.1.3This method is restricted to use by or under the supervision of trained analysts. Each analyst must demonstrate the ability to generate acceptable results with this method.2.0SUMMARY OF METHOD2.1 A measured volume of sample, usually 1 liter, at a specified pH (see Table 1), is serially extracted with methylene chloride using a separatory funnel.2.2The extract is dried, concentrated (if necessary), and, as necessary, exchanged into a solvent compatible with the cleanup or determinative method to be used (see Table 1 for appropriate exchange solvents).3.0INTERFERENCES3.1Refer to Method 3500.3.2The decomposition of some analytes has been demonstrated under basic extraction conditions. Organochlorine pesticides may dechlorinate, phthalate esters may exchange, and phenols may react to form tannates. These reactions increase with increasing pH, and are decreased by the shorter reaction times available in Method 3510. Method 3510 is preferred over Method 3520 for the analysis of these classes of compounds. However, the recovery of phenols may be optimized by using Method 3520, and performing the initial extraction at the acid pH.4.0APPARATUS AND MATERIALS4.1Separatory funnel - 2-liter, with polytetrafluoroethylene (PTFE) stopcock.4.2Drying column - 20 mm ID Pyrex® chromatographic column with Pyrex® glass wool at bottom and a PTFE stopcock.NOTE:Fritted glass discs are difficult to decontaminate after highly contaminated extracts have been passed through. Columns without frits may be purchased.Use a small pad of Pyrex® glass wool to retain the adsorbent. Prewash theglass wool pad with 50 mL of acetone followed by 50 mL of elution solvent priorto packing the column with adsorbent.CD-ROM3510C - 1Revision 3December 19964.3Kuderna-Danish (K-D) apparatus.4.3.1Concentrator tube - 10-mL, graduated (Kontes K-570050-1025 or equivalent).A ground-glass stopper is used to prevent evaporation of extracts.4.3.2Evaporation flask - 500-mL (Kontes K-570001-500 or equivalent). Attach toconcentrator tube with springs, clamps, or equivalent.4.3.3Snyder column - Three-ball macro (Kontes K-503000-0121 or equivalent).4.3.4Snyder column - Two-ball micro (Kontes K-569001-0219 or equivalent).4.3.5Springs - 1/2 inch (Kontes K-662750 or equivalent).NOTE:The following glassware is recommended for the purpose of solvent recovery during the concentration procedures requiring the use of Kuderna-Danishevaporative concentrators. Incorporation of this apparatus may be requiredby State or local municipality regulations that govern air emissions of volatileorganics. EPA recommends the incorporation of this type of reclamationsystem as a method to implement an emissions reduction program. Solventrecovery is a means to conform with waste minimization and pollutionprevention initiatives.4.4Solvent vapor recovery system (Kontes K-545000-1006 or K-547300-0000, Ace Glass 6614-30, or equivalent).4.5Boiling chips - Solvent-extracted, approximately 10/40 mesh (silicon carbide or equivalent).4.6Water bath - Heated, with concentric ring cover, capable of temperature control (± 5E C). The bath should be used in a hood.4.7Vials - 2-mL, glass with PTFE-lined screw-caps or crimp tops.4.8pH indicator paper - pH range including the desired extraction pH.4.9Erlenmeyer flask - 250-mL.4.10Syringe - 5-mL.4.11Graduated cylinder - 1-liter.5.0REAGENTS5.1Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity to permit its use without lessening the accuracy of the determination. Reagents should be stored in glass to prevent the leaching of contaminants from plastic containers.CD-ROM3510C - 2Revision 3December 1996CD-ROM 3510C - 3Revision 3December 19965.2Organic-free reagent water - All references to water in this method refer to organic-freereagent water, as defined in Chapter One.5.3Sodium hydroxide solution (10 N), NaOH. Dissolve 40 g NaOH in organic-free reagentwater and dilute to 100 mL. Other concentrations of hydroxide solutions may be used to adjustsample pH, provided that the volume added does not appreciably change (e.g., <1%) the totalsample volume.5.4Sodium sulfate (granular, anhydrous), Na SO . Purify by heating to 400E C for 4 hours24in a shallow tray, or by precleaning the sodium sulfate with methylene chloride. If the sodium sulfateis precleaned with methylene chloride, a method blank must be analyzed, demonstrating that thereis no interference from the sodium sulfate. Other concentrations of acid solutions may be used toadjust sample pH, provided that the volume added does not appreciably change (e.g., <1%) the totalsample volume.5.5Sulfuric acid solution (1:1 v/v), H SO . Slowly add 50 mL of H SO (sp. gr. 1.84) to 5024 24mL of organic-free reagent water.5.6Extraction/exchange solvents - All solvents must be pesticide quality or equivalent.5.6.1Methylene chloride, CH Cl , boiling point 39E C. 225.6.2Hexane, C H , boiling point 68.7E C.6145.6.32-Propanol, CH CH(OH)CH , boiling point 82.3E C. 335.6.4Cyclohexane, C H , boiling point 80.7E C. 6125.6.5Acetonitrile, CH CN, boiling point 81.6E C.36.0SAMPLE COLLECTION, PRESERVATION, AND HANDLINGSee the introductory material to this chapter, Organic Analytes, Sect. 4.1.7.0PROCEDURE7.1Using a 1-liter graduated cylinder, measure 1 liter (nominal) of sample. Alternatively, ifthe entire contents of the sample bottle are to be extracted, mark the level of sample on the outsideof the bottle. If high analyte concentrations are anticipated, a smaller sample volume may be takenand diluted to 1-L with organic-free reagent water, or samples may be collected in smaller samplebottles and the whole sample used.7.2Pipet 1.0 mL of the surrogate spiking solution into each sample in the graduated cylinder(or sample bottle) and mix well. (See Method 3500 and the determinative method to be used fordetails on the surrogate standard solution and matrix spiking solution).7.2.1For the sample in each batch (see Chapter One) selected for use as a matrixspike sample, add 1.0 mL of the matrix spiking standard.7.2.2If Method 3640, Gel-Permeation Cleanup, is to be employed, add twice thevolume of the surrogate spiking solution and the matrix spiking standard, since half of the extract is not recovered from the GPC apparatus. (Alternatively, use 1.0 mL of the spiking solutions and concentrate the final extract to half the normal volume, e.g., 0.5 mL instead of1.0 mL).7.3Check the pH of the sample with wide-range pH paper and adjust the pH, if necessary, to the pH indicated in Table 1, using 1:1 (v/v) sulfuric acid or 10 N sodium hydroxide. Lesser strengths of acid or base solution may be employed, provided that they do not result in a significant change (<1%) in the volume of sample extracted (see Secs. 5.3 and 5.5).7.4Quantitatively transfer the sample from the graduated cylinder (or sample bottle) to the separatory funnel. Use 60 mL of methylene chloride to rinse the cylinder (or bottle) and transfer this rinse solvent to the separatory funnel. If the sample was transferred directly from the sample bottle, refill the bottle to the mark made in Sec. 7.1 with water and then measure the volume of sample that was in the bottle.7.5Seal and shake the separatory funnel vigorously for 1 - 2 minutes with periodic venting to release excess pressure. Alternatively, pour the exchange solvent into the top of the Snyder column while the concentrator remains on the water bath in Sec. 7.11.4.NOTE:Methylene chloride creates excessive pressure very rapidly; therefore, initial venting should be done immediately after the separatory funnel has been sealedand shaken once. The separatory funnel should be vented into a hood to avoidexposure of the analyst to solvent vapors.7.6Allow the organic layer to separate from the water phase for a minimum of 10 minutes. If the emulsion interface between layers is more than one-third the size of the solvent layer, the analyst must employ mechanical techniques to complete the phase separation. The optimum technique depends upon the sample and may include stirring, filtration of the emulsion through glass wool, centrifugation, or other physical methods. Collect the solvent extract in an Erlenmeyer flask. If the emulsion cannot be broken (recovery of < 80% of the methylene chloride, corrected for the water solubility of methylene chloride), transfer the sample, solvent, and emulsion into the extraction chamber of a continuous extractor and proceed as described in Method 3520, Continuous Liquid-Liquid Extraction.7.7Repeat the extraction two more times using fresh portions of solvent (Secs. 7.2 through 7.5). Combine the three solvent extracts.7.8If further pH adjustment and extraction is required, adjust the pH of the aqueous phase to the desired pH indicated in Table 1. Serially extract three times with 60 mL of methylene chloride, as outlined in Secs. 7.2 through 7.5. Collect and combine the extracts and label the combined extract appropriately.7.9If performing GC/MS analysis (Method 8270), the acid/neutral and base extracts may be combined prior to concentration. However, in some situations, separate concentration and analysis of the acid/neutral and base extracts may be preferable (e.g. if for regulatory purposes the presence or absence of specific acid/neutral or base compounds at low concentrations must be determined, separate extract analyses may be warranted).7.10Perform the concentration (if necessary) using the Kuderna-Danish Technique (Secs.7.11.1 through 7.11.6).CD-ROM3510C - 4Revision 3December 19967.11K-D technique7.11.1Assemble a Kuderna-Danish (K-D) concentrator (Sec. 4.3) by attaching a 10-mLconcentrator tube to a 500-mL evaporation flask.7.11.2Attach the solvent vapor recovery glassware (condenser and collection device)(Sec. 4.4) to the Snyder column of the K-D apparatus following manufacturer's instructions.7.11.3Dry the extract by passing it through a drying column containing about 10 cm ofanhydrous sodium sulfate. Collect the dried extract in a K-D concentrator. Rinse the Erlenmeyer flask, which contained the solvent extract, with 20 - 30 mL of methylene chloride and add it to the column to complete the quantitative transfer.7.11.4Add one or two clean boiling chips to the flask and attach a three-ball Snydercolumn. Prewet the Snyder column by adding about 1 mL of methylene chloride to the top of the column. Place the K-D apparatus on a hot water bath (15 - 20E C above the boiling point of the solvent) so that the concentrator tube is partially immersed in the hot water and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of the apparatus and the water temperature as required to complete the concentration in 10 -20 minutes. At the proper rate of distillation the balls of the column will actively chatter, butthe chambers will not flood. When the apparent volume of liquid reaches 1 mL, remove the K-D apparatus from the water bath and allow it to drain and cool for at least 10 minutes.7.11.5If a solvent exchange is required (as indicated in Table 1), momentarily removethe Snyder column, add 50 mL of the exchange solvent, a new boiling chip, and reattach the Snyder column. Alternatively, pour the exchange solvent into the top of the Snyder column while the concentrator remains on the water bath in Sec. 7.11.4. Concentrate the extract, as described in Sec. 7.11.4, raising the temperature of the water bath, if necessary, to maintain proper distillation.7.11.6Remove the Snyder column and rinse the flask and its lower joints into theconcentrator tube with 1 - 2 mL of methylene chloride or exchange solvent. If sulfur crystals are a problem, proceed to Method 3660 for cleanup. The extract may be further concentrated by using the technique outlined in Sec. 7.12 or adjusted to 10.0 mL with the solvent last used.7.12If further concentration is indicated in Table 1, either the micro-Snyder column technique (7.12.1) or nitrogen blowdown technique (7.12.2) is used to adjust the extract to the final volume required.7.12.1Micro-Snyder column techniqueIf further concentration is indicated in Table 1, add another clean boiling chip to the concentrator tube and attach a two-ball micro-Snyder column. Prewet the column by adding0.5 mL of methylene chloride or exchange solvent to the top of the column. Place the K-Dapparatus in a hot water bath so that the concentrator tube is partially immersed in the hot water. Adjust the vertical position of the apparatus and the water temperature, as required, to complete the concentration in 5 - 10 minutes. At the proper rate of distillation the balls of the column will actively chatter, but the chambers will not flood. When the apparent volume of liquid reaches 0.5 mL, remove the K-D apparatus from the water bath and allow it to drain and cool for at least 10 minutes. Remove the Snyder column, rinse the flask and its lower joints into the concentrator tube with 0.2 mL of methylene chloride or the exchange solvent, and adjust the final volume as indicated in Table 1, with solvent.CD-ROM3510C - 5Revision 3December 19967.12.2Nitrogen blowdown technique7.12.2.1Place the concentrator tube in a warm bath (35E C) and evaporate thesolvent to the final volume indicated in Table 1, using a gentle stream of clean, drynitrogen (filtered through a column of activated carbon).CAUTION:New plastic tubing must not be used between the carbon trap andthe sample, since it may introduce contaminants.7.12.2.2The internal wall of the tube must be rinsed several times withmethylene chloride or appropriate solvent during the operation. During evaporation, thetube must be positioned to avoid water condensation (i.e., the solvent level should bebelow the level of the water bath). Under normal procedures, the extract must not beallowed to become dry.CAUTION:When the volume of solvent is reduced below 1 mL, semivolatileanalytes may be lost.7.13The extract may now be analyzed for the target analytes using the appropriate determinative technique(s) (see Sec. 4.3 of this Chapter). If analysis of the extract will not be performed immediately, stopper the concentrator tube and store refrigerated. If the extract will be stored longer than 2 days it should be transferred to a vial with a PTFE-lined screw-cap or crimp top, and labeled appropriately.8.0QUALITY CONTROL8.1Any reagent blanks, matrix spikes, or replicate samples should be subjected to exactly the same analytical procedures as those used on actual samples.8.2Refer to Chapter One for specific quality control procedures and Method 3500 for extraction and sample preparation procedures.9.0METHOD PERFORMANCERefer to the determinative methods for performance data.10.0REFERENCESNone.CD-ROM3510C - 6Revision 3December 1996CD-ROM 3510C - 8Revision 3December 1996METHOD 3510CSEPARATORY FUNNEL LIQUID-LIQUID EXTRACTION。

冲激响应不变法极点

冲激响应不变法极点英文回答：The impulse response invariance method, also known as the zero-pole matching method, is a technique for designing digital filters by transforming an analog filter designinto a digital filter design. This method is based on the principle that the impulse response of an analog filter is invariant under a transformation from the analog domain to the digital domain.The impulse response invariance method is a two-step process. In the first step, an analog filter is designed using traditional analog filter design techniques. In the second step, the analog filter design is transformed into a digital filter design using a bilinear transformation or a matched z-transform.The bilinear transformation is a simple transformation that maps the s-plane (the complex plane used to representanalog filters) to the z-plane (the complex plane used to represent digital filters). The matched z-transform is a more complex transformation that takes into account the sampling rate of the digital filter.Once the analog filter design has been transformed into a digital filter design, the impulse response of thedigital filter will be identical to the impulse response of the analog filter. This means that the digital filter will have the same frequency response and phase response as the analog filter.The impulse response invariance method is a simple and effective technique for designing digital filters. However, this method can only be used to design digital filters that have a finite impulse response (FIR).中文回答：冲激响应不变法，也称为零极点匹配法，是一种通过将模拟滤波器设计转换为数字滤波器设计来设计数字滤波器的方法。

Antiprotonic hydrogen in static electric field

a rXiv:n ucl-t h /5132v112Ja n25Antiprotonic hydrogen in static electric ﬁeld G.Ya.Korenman and S.N.Yudin Skobeltsyn Institute of Nuclear Physics,Moscow State University,Moscow,Russia e-mail:korenman@anna19.sinp.msu.ru Abstract Eﬀects of the static electric ﬁeld on the splitting and annihilation widths of the levels of antiprotonic hydrogen with a large principal quantum number (n =30)are studied.Non-trivial aspects of the consideration is related with instability of (p ¯p )∗-atom in ns and np -states due to coupling of these states with the annihila-tion channels.Properties of the mixed nl -levels are investigated depending on the value of external static electric ﬁeld.Speciﬁc resonance-like dependence of eﬀective annihilation widths on the strength of the ﬁeld is revealed.1Introduction When antiproton enters a matter it is slowed down and captured eventually by atom forming an antiprotonic atom.From the general point of view this new physical object is of the great interest.For the long time the experimental methods to investigate an-tiprotonic atoms have been limited by studying their X-ray radiation and the products of antiproton annihilation at the end of cascade transitions.However,about 15years agothe investigations of these atoms have got on the new qualitative level especially due to the new experimental methods (see [1,2]and references therein)that used antiprotonic beam from storing ring of LEAR operated in CERN up to 1996.New setup AD (”an-tiproton decelerator”)that has been operating since the end of 2001opens,in deﬁnite aspects,even more possibilities for experimental study of antiprotonic atoms.In particu-lar,in the frame of ASACUSA project [3]it is supposed,among other experiments,direct observation and investigation of antiprotonic atoms formation in very rareﬁed gases.The new experimental possibilities pose various problems of the theory of antiprotonic atoms that were not considered early because they seemed to be of little importance in the former experimental conditions.One of such problem could be an inﬂuence of ex-ternal static electric ﬁeld on the properties of antiprotonic atoms,especially antiprotonic hydrogen.Static electric and magnetic ﬁelds can be generated by some parts of an exper-imental setup,therefore they have to be taken into account in precision measurements of the properties of excited (p ¯p )atom.On other hand,a special choice of the ﬁelds could be used in order to obtain an additional information on the properties of antiprotonic atoms.According to current concepts of the theory of exotic atom formation (see,e.g.,[4,5]),the antiprotonic atoms are formed initially in highly exited states with principal quantum number n n 0≃√(1≪l n−1),whereµis a reduced mass of the¯p-nucleus system in the units of electron mass(so,for the formation in atomic hydrogenµ=918,n0=30).When (p¯p)nl atom is formed in molecular hydrogen,the distribution over quantum number is more complicated,however the probability of population of the states with large n and l remains rather appreciable.Among the highly excited states,speciﬁc interest can present circular orbits with l=n−1and nearly-circular orbits.In isolated antiprotonic hydrogen atom these states can decay only by radiative E1-transition.Radiative life time of hydrogen-like atom at large n,l can be estimated by the equation[6]τγ(nl)≃(n3l2/µZ4)·84.5ps(1) that givesτγ=2.09µs for the circular orbit with n=30of antiprotonic hydrogen. This life time is of the same order of value as for antiprotonic helium metastable states [1],therefore the same high-precision method of laser-induced transition could be,in principle,applied for the study of energy structure and dynamics of the excited states of antiprotonic hydrogen.However,the(p¯p)atom,as compare with(¯p He+),may be less stable against atomic collisions and inﬂuence of externalﬁelds,because a most part of the(p¯p)nl states with a deﬁnite n is degenerated in l,contrary to antiprotonic helium. Therefore theoretical study of the quenching eﬀects on the long-lived states of antiprotonic hydrogen is important for the possible future experiments.One of the most important factors to quench long-lived states is the electricﬁeld arising in the processes of atomic collisions or as a results of the external conditions.The dynamical(collisional)Stark eﬀect in hadronic hydrogen atoms was discussed in the literature for a long time[6,7]. However the inﬂuence of the external static electricalﬁeld,as far as we know,was not discussed in the literature,in spite of the seeming simplicity of this eﬀect.In this paper we discuss the inﬂuence of electricﬁeld on the splitting and decay rates of highly-excited (n=30)states of antiprotonic hydrogen atoms.2Formulation of the problemThe states with large quantum numbers n,l of isolated antiprotonic hydrogen(p¯p)nl are long-lived,because annihilation in the states with l≫1is practically absent and,on other hand,these states have large radiative life times.However,if some external action forces transitions of the(p¯p)system to the states with low orbital angular momenta,then antiproton will annihilate quickly.In the problem under consideration an external action is produced by the static external electricﬁeld.Formally this problem is formulated as follows.If the nuclear(p¯p)-interaction is disregarded,all the states nl of antiprotonic hydrogen atom will be degenerated in l.In the real system nuclear interaction is important in the states with low angular momenta and it produces the both shifts and annihilation widths of the levels,or,in other words,complex shifts∆E nl=−ǫnl−iΓnl/2.(2) The estimations show that these values at n∼30are important for l=0,1and can be neglected for l≥2.So,the complex shifts remove the degeneration of the ns and np−levels,whereas other levels remain to be degenerated.A dependence of the complexshifts on the principal quantum number can be obtained taking into account that the radius of annihilation region and of nuclear interaction is small as compare with a radius of the antiprotonic hydrogen atom.Radial wave function of antiprotonic atom this region should have the simple form R nl(r)≃C nl·r l.It allows to express the dependence the complex shifts∆E nl on n in terms of the coeﬃcients C nl:∆E nl=∆E l+1,l|C nl/C l+1,l|2(3) The coeﬃcients C nl are estimated,as a rule,with the hydrogen-like wave functions that givesΓns=Γ1s/n3,(4)Γnp=Γ2p·32(n2−1)/(3n5),(5) and similarly forǫnl.As the input data we have taken the following values for the energy shifts and widths[8]:Γ1s/2=561eV,ǫ1s=691eV,Γ2p/2=17meV,ǫ2p=0.(6)With these values we obtain from the Eqs.(4),(5)for the states with n=30:Γns/2=20.78meV,ǫns=25.59meV,Γnp/2=0.00671meV,ǫnp=0.(7)Total hamiltonian H of the antiprotonic hydrogen atom in the external electricﬁeld can be written as a sum of two terms,H=H0+V,(8)where H0is a diagonal matrix with elementsnl|H0|nl′ =δll′(δl0∆E ns+δl1∆E np).(9) The matrix of interaction of the atom with the electricﬁeld E directed along Oz axis has the form:nlm|V|nl′m′ =eEr0(3/2)ntheﬁeld less than a critical value,at which non-diagonal matrix element nl|V|n′l′ is comparable with the distance between the levels with diﬀerent n.It is easy to estimate that for the levels with n≃30this critical value of theﬁeld is E a∼109V/cm,that is far from real laboratoryﬁelds.It should be noted that in the problem under consideration there are also two other critical values of theﬁeld.Theﬁrst one is theﬁeld that mixes eﬀectively ns and np states, and other one is that mixed np and nd states,the latter being degenerated with all other nl states for l>2.With the above-mentioned values of strong interaction shifts and annihilation widths of the ns and np states,we estimate the corresponding criticalﬁelds as E b∼3.3·106V/cm for a mixing of ns and np at n=30,and E c∼2000V/cm for a mixing of np and nd states.We restrict our consideration by theﬁelds up to5000V/cm, therefore only third critical value E c of theﬁeld is covered by these calculations.For the further discussion of the obtained results let us remember also,that the prob-lem of Stark mixing in the degenerated(without initial shifts and widths)hydrogen-like system at theﬁxed n has the exact solution in the parabolic coordinates[9].Eigenstates of a such system are labeled by parabolic quantum numbers n1,n2,m(n1+n2+|m|+1=n), the splitting of the levels is linear in electricﬁeld,whereas the coeﬃcients nlm|n1n2m of mixing of the states with diﬀerent l do not depend on the value of theﬁeld and can be expressed in terms of Clebsh-Gordan coeﬃcients,nlm|n1n2m 2= kν1kν2|lm 2,(11) where k=(n−1)/2,ν1=(n1−n2+m)/2,ν2=m−ν1.Therefore the admixture of ns-state to all parabolic states with m=0should be ns|nn1 2=1/n,and the weights of the np-state in the parabolic states are np,m|nn1,m 2=0.019÷0.025at n=30.These relationships could describe the eﬀect of externalﬁeld on antiprotonic hydrogen at very high values of the electricﬁeld(E b<E<E a).However for the realistic values of E<E b the inﬂuence of theﬁeld can not be predicted without calculations.The results of our calculations are shown on Figs.1-4.On theseﬁgures the shifts W(E)and widthsΓ(E)of the states are presented in the relative units of|W max|andΓmax, which are the maximum values of these quantities in the considered interval of electric ﬁelds(0≤E≤5000V/cm).Diﬀerent types of dependencies of the shifts on the value E of electricﬁeld are shown on Figs.1-2for several levels with m=0.The levels are labeled by the indexes l that correspond to the quantum numbers of the states in the limit E=0.With respect to the dependence of the shifts on E,the levels can be divided into two main types.The shifts are negative for l≤14and positive for l≥16,and in the both cases they rise in absolute value with increasing of the electricﬁeld.Within the intermediate interval l∈13−16 the shifts are very small and change the signs.It is seen from Fig.2that in this region the shifts are nonlinear in E and,moreover,they behave themselves for theﬁrst sight unusually,showing a resonance-like behaviour at E∼E c.However,the nature of a such behaviour is quite clear-this is a result of some crossing of the levels that takes place with a change of the values of electricﬁeld.For the levels with|m|=1we observe a similar behaviour with E.The widths of the levels with m=0are shown on Figs.3-4depending on the value of electricﬁeld.For the states with l≤13and l≥16the widths grow with increasing of the electricﬁeld,however in the aforementioned interval13≥l≤16the widths depend10002000300040005000E,V cm -1-0.8-0.6-0.4-0.2W W max(a)10002000300040005000E,V cm 0.20.40.60.81W W max (b)Figure 1:The dependence of the shifts of (p ¯p )states on electric ﬁeld.Part (a):l =1,W max =3.7·10−5eV;part (b):l =28,W max =3.43·10−5eV.10002000300040005000E,V cm -1-0.8-0.6-0.4-0.2W W max (a)10002000300040005000E,V cm 0.10.20.30.40.5W W max (b)Figure 2:Resonance-like dependence of the shifts of (p ¯p )states on electric ﬁeld.Part (a):l =14,W max =2.62·10−6eV;part (b):l =15,W max =1.95·10−9eVon the ﬁeld in a resonance-like manner.The reason of this phenomenon is the same as for the shifts,i.e.it is connected with the crossing of levels in the static electric ﬁeld.The shift and width of the ns state are practically unchanged at the considered values of E .It is related with the large values of the ’self’shift and width,or,in other words,with the fact that the value of E is small as compare with critical ﬁeld E b as estimated above.Thus the obtained values of ’induced’widths of all the states with l =0are due to the inﬂuence of np-states in the considered interval of electric ﬁelds.At larger electric ﬁelds the contribution of ns states would be a more essential.4ConclusionIn this paper we have made the analysis of the splitting and annihilation widths of the levels of antiprotonic hydrogen atom in the static external electric ﬁeld.As compare with the theoretical case when all the levels are degenerated and the exact analytical solution of this problem exists,we meet here a more complicated situation.This complication10002000300040005000E,V cm 0.20.40.60.81max(a)10002000300040005000E,V cm0.20.40.60.81 max (b)Figure 3:Dependence of the widths on the value of electric ﬁeld.Part (a):l =1;Γmax =3.42·1081/s;part (b):l =28,Γmax =4.52·1081/s.10002000300040005000E,V cm 0.20.40.60.81max(a)10002000300040005000E,V cm 0.20.40.60.81 max (b)Figure 4:Resonance-like dependence of the widths on the value of electric ﬁeld.Part (a):l =14,Γmax =1.51·1091/s;part (b):l =16,Γmax =1.52·1091/s.is related with the strong interaction shifts and annihilation widths of ns and np states participated in the mixing of nl levels of the antiprotonic atom.The results of the paper can be formulated as follows:a)due to mixing of diﬀerent nl states (l >1)with np states at the value of electric ﬁeld E ∼1000V/cm,all the states acquire the widths of order 108÷1091/s.The relevant shifts for the most part of the states are linear in E and have the order of value 10−5eV.b)Contribution of the ns state into the widths of other levels is negligible at the considered values of electric ﬁeld (E ≤5000V/cm),because the ns state is very distant in complex energy from all other levels and can be admixed to these states at E ∼3·106V/cm.As a result,the main contribution to the widths of the mixed levels gives np state.c)For the states that are close to the point,where the sign of the shift is changed,the shifts and widths of exact states have a resonance-like dependence on the value of electric ﬁeld.The ’resonance’value of electric ﬁeld is of the same order as critical value E c ,which provides strong mixing of the np state with other states (l ≥2).This phenomenon is due to a crossing of the levels at some value of the electric ﬁeld.AcknowledgementsThis investigation was supported by Russian Foundation for Basic Research as a part of the project03-02-16616.Authors thank to N.P.Yudin for the useful discussions.One of the authors(G.K.)thanks to Y.Yamazaki for attracting our interest to the considered problem.References[1]T.Yamazaki,N.Morita,R.S.Hayano,E.Widmann and J.Eades,Physics Reports366(2002)183.[2]A.Bertin,M.Bruschi,M.Capponi et al.,Phys.Rev.A54(1996)5441;E.Lodi Rizzini,A.Bianconi,G.Bonomi et al.,Phys.Lett.B507(2001)19.[3]ASACUSA collaboration.Status report CERN/SPSC2002-002,SPSC/M-674,Jan.2002.[4]G.Ya.Korenman,Hyperﬁne Interaction,101/102(1996)81.[5]J.S.Cohen,Phys.Rev.A56(1997)3583.[6]K.Omidvar,Atomic Data and Nucl.Data Tables28(1983)1.[7]M.Leon and H.A.Bethe,Phys.Rev.127(1962)636.[8]T.P.Terada and R.S.Hayano,Phys.Rev.C55(1996)73.[9]ndau and E.M.Lifshits,Quantum Mechanics,Fizmatgiz,Moscow,1963,§77.。

LS-dyna接触设置

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Introduction

Purpose of Contact
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Surface_to_Surface Contacts

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Master
Slave
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+
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Search Methods
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共聚物和乙烯,丙烯,和更高的α烯烃与使用金属茂催化剂的高丙烯含量的三聚

Co-and Terpolymerization of Ethylene,Propylene,and Higher␣-Olefins with High Propylene Contents Using Metallocene CatalystsM.A.VILLAR,M.L.FERREIRAPlanta Piloto de Ingenierı´a Quı´mica,PLAPIQUI(UNS-CONICET),Camino La Carrindanga Km7,C.C.717,8000Bahı´a Blanca,ArgentinaReceived7November2000;accepted17January2001ABSTRACT: The copolymerization of propylene/ethylene and terpolymerization of pro-pylene/ethylene/␣-oleﬁns using long-chain␣-oleﬁns such as1-octene and1-decene havebeen carried out using EtInd2ZrCl2//methylaluminoxane.High concentrations of pro-pylene and low concentrations of␣-oleﬁns(near2mol%of the total oleﬁn concentration in the liquid phase)were used.The effect of the ethylene concentration in copolymer-izations of propylene/␣-oleﬁns was studied at medium ethylene contents(12and40mol %in the gas phase).The polymers were molecularly characterized by gel permeation chromatography-multiangle laser light scattering,wide-angle X-ray scattering,Fourier transform infrared spectroscopy,and DSC analyses.The shorter␣-oleﬁn studied(1-octene)produced the highest improvement of activity in terpolymerization at12mol% ethylene in the gas phase.About2mol%of1-octene in the liquid phase increases the activity and decreases the molecular weight of terpolymers with respect to correspond-ing copolymers,whereas the mp is increased by almost30°C.The“termonomer effect”is less evident when higher amounts of ethylene are used.©2001John Wiley&Sons,Inc. J Polym Sci A:Polym Chem39:1136–1148,2001Keywords:propylene copolymerization;propylene terpolymerization;long-chain ␣-oleﬁns copolymerizationINTRODUCTIONThe copolymerization of ethylene with higher ␣-oleﬁns using metallocene catalysts has been in-vestigated.1A small amount of1-butene,1-hex-ene,or1-octene leads to polymers with similar characteristics of linear low-density polyethylene, a product of high industrial interest.Attractive product properties are also stimulating interest in propylene copolymerization.Random copoly-merization of propylene with ethylene and higher ␣-oleﬁns can be a solution for low-temperature applications because the glass-transition temper-ature(T g)of polypropylene(PP)is relatively high(approximately0°C).2Propene and1-octene havebeen copolymerized using different metal-locenes.3The copolymerization of propylene with1-butene,1-hexene,or cyclic oleﬁns using metal-locenes has also been explored.4Recent reportsshow that the crystallization temperatures seemto be independent of the comonomer length.5However,there is little information on terpoly-merizations of ethylene,propylene,and higher ␣-oleﬁns aiming to obtain terpolymers with low contents of higher␣-oleﬁns(less than2–5mol%) and higher propylene contents.In this area,Ka-minsky and Drogemuller6synthesized ethylene/propylene/diene elastomers using Cp2ZrCl2andEtInd2ZrCl2with a maximum diene incorporationCorrespondence to:M.L.Ferreira(E-mail:caferrei@criba. edu.ar)Journal of Polymer Science:Part A:Polymer Chemistry,Vol.39,1136–1148(2001)©2001John Wiley&Sons,Inc.1136of6–7mol%of diene.To obtain functionalizedpolyoleﬁns,Hackmann et al.7examined the re-sults of the copolymerization of propylene withhigher␣-oleﬁns using linear asymetrically substi-tuted dienes.Other researchers studied the copo-lymerization of propylene with1-octene,showingan important increase of activity in the range of2–10mol%of1-octene in the liquid phase.8In the mid-1980s Seppa¨la¨9noticed an en-hanced enrichment of a higher␣-oleﬁn(1-decene or1-dodecene)in its copolymerization with ethyl-ene on a commercial TiCl3/AlEt3catalyst when ashort-chain␣-oleﬁn such as1-butene was added. This phenomenon was called the“synergistic ef-fect.”The copolymerizability of a longer␣-oleﬁn with1-butene or propylene should be higher thanwith ethylene,and1-butene should be more reac-tive than higher␣-oleﬁns in its copolymerization with ethylene.This effect implies that1-butenehas a less sterically hindered structure in thegrowing chain end,allowing the longer␣-oleﬁn to react.However,this synergistic effect can be al-ternatively explained based on unsteady diffusionkinetics.10Owing to the lower diffusion coefﬁcientand copolymerizability of a longer␣-oleﬁn,the attainment of its equilibrium concentration at thecatalyst is more difﬁcult.The presence of a lower ␣-oleﬁn of a higher copolymerizability should lead to a considerable decrease in the crystallinity ofpolyethylene,facilitating the diffusion of thelonger␣-oleﬁn as well as the attainment of its equilibrium concentration.Additionally,the in-corporation of a small amount of ethylene into PPshould destroy the regularity of PP chains andcreate the conditions for improved diffusion of ahigher␣-oleﬁn.The extent of monomer-concen-tration increases at the catalyst surface for sup-ported Ziegler–Natta copolymerization is in the following order:longer␣-oleﬁnϾpropylene Ͼethylene.Other researchers have demonstrated thatunsteady diffusion kinetics can predict that thereactivity of1-hexadecene with propylene in aheterogeneous Ziegler–Natta copolymerizationshould increase by two to three times by adding asmall amount of ethylene.11Those researchershad found that terpolymers with2–10mol%of1-hexadecene as well as small amounts of ethyl-ene incorporated(2–10mol%)show both lowermps and crystallinity than corresponding copoly-mers(up to5–10°C in T m andﬁve to eight timesin crystallinity values).Using Ziegler–Natta cat-alysts,they established that the addition of asmall amount of ethylene in propylene/higher ␣-oleﬁn copolymerization is an excellent way for improving the copolymerizability of the higher ␣-oleﬁn.Seppa¨la¨et al.12studied the synthesis of co and terpolymers of ethylene with1-butene and1-de-cene using Cp2ZrCl2/methylaluminoxane(MAO). Those polymers were totally soluble in n-heptane, the solvent used as a polymerization medium. The enhancement of catalytic activity found can hardly be attributed to monomer diffusion.Fur-thermore,it has been proposed that in the case of this soluble system,virtually all the zirconium atoms form active sites for polymerization espe-cially at high temperatures(70°C).The molecu-lar weight of ethylene/1-butene and ethylene/1-decene copolymers decreased,and the activity of the catalyst simultaneously increased.They con-cluded that the comonomer takes part in the ac-tivation of catalytic centers,decreasing the acti-vation energy necessary for the insertion of mono-mer into an active metal–carbon bond,making propagation and chain-transfer reactions easier. Similar trends have been discovered for terpoly-merizations:higher activity as well as lower mo-lecular weights but no“synergistic effect”for the terpolymerization of ethylene with1-butene and a longer␣-oleﬁn.The synergistic effect has also been found to be absent using bridged and unbridged soluble met-allocene catalysts such as iPrFluoCpZrCl2//MAO, Me2SiInd2ZrCl2//MAO,and Cp2ZrCl2in the ter-polymerization of ethylene with1-butene and 1-octadecene.13,14Besides the lack of the synergistic effect,the following interesting conclusions have been con-veyed:1.iPrFluoCpZrCl2//MAO was more favorablethan Me2SiInd2ZrCl2//MAO for the inser-tion of higher␣-oleﬁns in ethylene/␣-ole-ﬁns copolymerization because of the gapaperture between the␲ligands.2.No signiﬁcant differences were observedbetween the r parameters obtained for thesame catalyst system when1-dodecene or1-octadecene was used as a termonomer.3.A long block of higher␣-oleﬁns at the endof a growing polymer chain forces the li-gands into a more open arrangement,mak-ing the insertion of an␣-oleﬁn easier.Longer␣-oleﬁns open more of the zircono-cene ligands than shorter ones.4.Consumption of ethylene increased contin-uously with the addition of1-butene al-CO-AND TERPOLYMERIZATION USING METALLOCENE CATALYSTS1137though the content of a longer␣-oleﬁn suchas1-octadecene decreased in the terpoly-mer.The ethylene consumption did notpass through a maximum as it does in thecopolymerization of ethylene and1-octade-cene.Previous research states that no comonomereffect can be expected for the ethylene/longer ␣-oleﬁn copolymerization using the homogeneous catalyst EtInd2ZrCl2//MAO.15However,a verywide range was explored for theﬁrst point oflonger␣-oleﬁn incorporation(from0to13mol%).A recent report on the co-and terpolymerizationof ethylene and higher␣-oleﬁns with metal-locenes concluded that the synergistic effect of longer␣-oleﬁns on propene was negative for (nBu)Cp2ZrCl2//MAO and EtInd2ZrCl2//MAO.In this case,the synthesized polymers were ethyl-ene-rich(more than85mol%ethylene).16 Previous studies dealing with the copolymer-ization of ethylene and propylene using metal-locenes have been published in the last de-cade.17–19Recent reports show reactivity ratio values and the effect of the structural fact of met-allocenes on the copolymer structure.20,21The for-mation of sequences of ethylene and propylene could be correlated with the energy involved in the whole process that leads to the insertion of the comonomer,the approach to the catalytic center, and the following insertion into the metal–carbon bond.For those studies,it is evident that ethylene insertion is preferred when ethylene or propylene is the last inserted unit.However,the relative reactivity of propylene versus ethylene increases when propylene is the last inserted unit(k22/k21Ͼk12/k11).It has been pointed out that these results could be explained considering steric ef-fects,emphasizing that a correct interpretation should also take into account the role played by electronic effects.Electronic effects could be re-sponsible for different values of r1and r2in the series of the racemic-substituted metallocenes. Ethylene and␣-oleﬁns differ from both the steric and the electronic point of view.In this sense,the copolymerization of ethylene with1-hexene or 1-octene using EtInd2ZrCl2//MAO produces poly-mers with lower mps and viscosities as the amount of the incorporated higher␣-oleﬁn in-creases(up to27mol%).22Some researchers have proposed that a decrease in the copolymer molec-ular weight could be due to increasing chain-transfer reactions to the longer comonomer.23 Electronic effects of substituents are becoming an important explanation of the results obtainedwith siloxy-substituted EtInd2ZrCl2in the homoand copolymerization of ethylene and higher ␣-oleﬁns.24The importance of electronic effects in metallocene chemistry can be visualized in sev-eral mechanistic problems that remain unsolvedsuch as chain-termination reactions.25The elec-tronic effect is evidently important whenEtInd2ZrCl2and EtInd2HfCl2//MAO are com-pared.The Hf catalyst presents lower activitiesthan the Zr catalyst in ethylene polymerizationand a higher dependence of comonomer concen-tration in the copolymerization with1-butene.However,under the same conditions,the Hf cat-alyst has a higher capability of comonomer incor-poration.Although Zr and Hf compounds showthe same chemical properties,the nuclear chargeis completely different.This is manifested inshorter bond lengths and stronger bondsstrengths in organometallic Hf compounds.How-ever,this fact does not explain why Hf compoundsare more effective in the incorporation of higher ␣-oleﬁns.26An alternative explanation may be the capability of a highly polarizable oleﬁn to increase the positive charge at the Hf atom by coordination even further,as far as insertion is more probable than coordination for Hf with re-spect to Zr.Because both metallocenes show sim-ilar steric hindrances,the coordination of a longer oleﬁn that is more basic and polarizable than ethylene would be preferred on Hf(more electro-ﬁlic)than on Zr.The Hf catalyst shows remark-ably low r1parameters,and this is in agreement with the preceding idea.Another report on oleﬁn polymerization using highly congested ansa-met-allocenes under high pressure demonstrates that although Me2Si(␩5-C5Me4)2ZrCl2and Me2Si(␩5-C5Me4)2HfCl2have almost identical structures, they showed different activities and molecular weights for the polymerization of1-octene and 1-hexene.In the polymerization of1-hexene at 500MPa,the zirconocene showed an activity200 times higher than the value obtained at0.1MPa, whereas the hafnocene shows only a25-fold higher activity.The molecular weight of poly(1-hexene)was20-fold higher for the zirconocene and22-fold higher for the hafnocene when values at500and0.1MPa were compared.This fact emphasizes that electronic effects are important for understanding the behavior in oleﬁn polymer-izations at high pressures.The present article explores the results of co-and terpolymerization of ethylene,propylene,anda higher␣-oleﬁn using EtInd2ZrCl2//MAO.The1138VILLAR AND FERREIRAcopolymers and terpolymers synthesized are rich in propylene(approximately60–90mol%).The effect of low concentrations of higher␣-oleﬁns in the reaction media,such as1-octene and1-de-cene,on product properties is also investigated. EXPERIMENTALMaterialsThe catalyst EtInd2ZrCl2was obtained from Al-drich and manipulated in N2atmosphere,and the co-catalyst MAO(10wt%in toluene)was pro-vided by Witco.The propylene polymerization grade was puriﬁed using MnO/Al2O3and13X mo-lecular sieves to eliminate water and oxygen. 1-Octene(98%chemical purity)and1-decene (94%chemical purity)were obtained from Aldrich and used as received.The impurities were n-al-kanes and substituted␣-oleﬁns in all cases.No internal oleﬁns exist as impurities in longer␣-ole-ﬁns.Toluene was obtained from JT Baker at a high-performance liquid chromatographic grade (water content less than0.006%),and it was de-hydrated with13X molecular sieves before use. Ethylene was used as received from Scott Spe-cialty Gases.Polymerization ConditionsCopolymerization and terpolymerization reac-tions were carried out at40°C at an oleﬁn total pressure of 1.0atm with a continuousﬂow of ethylene and propylene.Propylene and ethylene ﬂows were separately controlled with a totalﬂow of gas monomer to the reactor of0.35L/min.In terpolymerizations the higher␣-oleﬁn(either 1-octene or1-decene)was added to the reactor before feeding the monomer gases batchwise un-der propylene or ethylene pressure.A concentra-tion of2mol%of1-octene or1-decene,based on total amounts of monomers in the liquid phase, was used.Two ethylene/propylene ratios were used for the gas monomers:12/88and40/60(on a molar basis).A zirconium concentration in the range of1–1.310Ϫ5M was used for polymeriza-tions carried out with12mol%of ethylene in the gas phase.The zirconium concentration was re-duced to the range of0.6–0.6410Ϫ5M when a40 mol%of ethylene was present in the gas phase. The Al/Zr molar ratio varies between2000and 2500,and the polymerization time was60min. The conditions used for the reactions were se-lected from the literature to avoid diffusional problems,and they are presented in Table I.The reactor was purged with propylene or a mixture of ethylene/propylene at highﬂow rate for15min to remove the air.Next,dry toluene was added,and the polymerization temperature was achieved maintaining the highest speed of agitation possible.The solvent was then satu-rated with the gas monomers at the polymeriza-tion pressure.For the terpolymerizations the higher␣-oleﬁn(1-octene or1-decene)was added by syringe methods as liquid.Metallocene cata-lysts and MAO were also introduced by syringe techniques—ﬁrst MAO and then the zirconocene. Once the catalyst compounds were injected,the polymerization proceeded under the previously described conditions.On closing the gas monomer feed,the reaction was stopped,and the polymer-ization media were poured into a mixture of eth-anol and hydrochloric acid andﬁltered.Afterﬁl-tration,the polymer was dried at room tempera-ture until constant weight.Table I.Concentration and Composition of Monomers in the Gas and Liquid PhasesPolymerGas-PhaseComposition(mol%)Liquid-Phase Concentrations(mol/L)Liquid-Phase Composition(mol%) Ethylene Propylene Ethylene Propylene Higher␣-Oleﬁn Ethylene Propylene Higher␣-OleﬁnPP—100———100.0—PP12E11.288.80.01470.358— 4.096.0—PP12E2O11.888.20.01510.3560.0085 4.093.8 2.2PP12E2D12.787.30.01670.3520.0085 4.593.3 2.2PP40E40.859.30.05360.239—18.381.7—PP40E2O40.060.00.05270.2420.006817.580.3 2.2PP40E2D40.060.00.05270.2420.007217.580.2 2.3CO-AND TERPOLYMERIZATION USING METALLOCENE CATALYSTS1139The concentration of propylene in toluene dur-ing the polymerization reaction was calculated using the following correlation:͓P͔ϭp P7.6710Ϫ6e3452.3/T(1) where[P]is the concentration of propylene in the liquid phase(toluene)in moles per liter,p P is the partial pressure of propylene in atmospheres,and T is the absolute temperature.Eq1was obtained considering the data collected by Zambelli and Grassi.27In the calculation,we have taken into account the vapor pressure of toluene at40°C.The ratio of the ethylene concentration to the propylene concentration in the liquid phase,at the reaction conditions,was calculated by the fol-lowing equation:͓E͔͓P͔ϭp Ep P16.946eϪ1236.6/T(2)where[E]is the concentration of ethylene in the liquid phase(toluene)in moles per liter,and p E is the partial pressure of ethylene in atmospheres. The gasﬂows were related to the partial pres-sures,and the relation p E/p P was replaced by the gasﬂows.Predictions of the propylene and ethylene con-centration in the liquid phase using eqs1and2 were compared with the experimental values ob-tained from the literature.27,28An error of less than10%was obtained indicating that the pre-dictions are in good agreement with the reported experimental values.The concentration of the higher␣-oleﬁn was calculated based on the added volume,taking into account the density and the purity of the oleﬁn, and the volume of the solvent(toluene). Polymer CharacterizationAverage molecular weights and molecular weight distributions were obtained with a gel permeation chromatograph(GPC)(Waters150C)coupled with multi-angle laser light scattering(MALLS) (Wyatt Dawn DSP).The GPC was equipped with three photoluminescent gel columns of500,104, and106.1,2,4-Trichlorobenzene(TCB)was used as a solvent at aﬂow rate of1mL/min at135°C. The concentrations used were in the order of4to 510Ϫ3g/mL.The TCB refractive index was taken as1.502,and the speciﬁc refractive-index incre-ment(dn/dc)wasϪ0.104.29DSC measurements were performed on a PerkinElmer Pyris1.Sam-ples were cooled atϪ110°C,heated at10°C/min fromϪ110to140°C,cooled at10°C/min from140 toϪ110°C,and then heated again fromϪ110to 140°C at10°C/min.The T g,the onset and peak melting temperatures(T m onset and T m peak,respec-tively),and the heat-of-fusion(⌬H f)values were taken from the second heating curve.Fourier transform infrared(FTIR)spectra of the polymers in solid state were obtained in a Nicolet20DXB.Theﬁlms were prepared with a hydraulic hot press at temperatures lower than 120°C.Solvent evaporation from a concentrated polymer solution on a NaCl window was an alter-native method ofﬁlm preparation.An estimation of the propylene content in the copolymers using the FTIR information can be done using correlations provided in the literature. Two different expressions were used to obtain the molar percentage of propylene30,31mol%P COPϭϪ9.9610Ϫ3ϩ106Xϩ81X2Ϫ166X3ϩ78.8X4(3) mol%P COPϭ1.59YϪ1.59100(4)where X and Y are deﬁned based on the absor-bance values at720–730cmϪ1and1160–1165 cmϪ1by the following equations:XϭA1160A720ϩA1160andYϭA720Ϫ730A1165.FTIR of the terpolymers show the changes pro-duced when a low concentration of a higher␣-ole-ﬁn is incorporated.Differences with the respec-tive copolymers can be observed in the1600–1700 cmϪ1and500–1000cmϪ1zones.Wide-angle X-ray scattering(WAXS)studies of copolymers and terpolymers,in the powder form, were done at room temperature with a diffractom-eter(Phillips,PW1710).A Cu tube anode at45kV and30mA was used.The crystal size was esti-mated using Scherrer’s equation where the re-ported size is the average dimension of the crys-tallites normal to the diffraction planes(hkl).321140VILLAR AND FERREIRAThe rheological characterization of the synthe-sized polymers was carried out in a rotational rheometer(Rheometrics Dynamics Analyzer RDA-II)at temperatures ranging from110to180°C.Shearﬂow was obtained by dynamic tests using25-mm diameter parallel plates.The stor-age(elastic)modulus,GЈ,and the loss(viscous) modulus,GЉ,were measured for frequencies rang-ing from0.05to500rad/s in the linear viscoelas-tic range of strain.The time–temperature super-position principle was used at a reference temper-ature(T0)of120°C.RESULTS AND DISCUSSIONProductivityThe concentration of zirconium used in the poly-merization reactions was similar to those utilized for propylene homopolymerization because the co-and terpolymers are propylene-rich.In all cases, [Zr]was low to avoid diffusional and temperature problems.Productivity values obtained for each reaction are shown in Table I.An increase in productivity is observed when a higher␣-oleﬁn (1-octene or1-decene)is present in the reaction media.The ethylene concentration also affects the productivity.For terpolymers obtained with 40mol%of ethylene in the gas phase,both1-oc-tene and1-decene have almost the same effects. However,for terpolymers synthesized with12 mol%of ethylene in the gas phase,1-octene is more effective than1-decene,taking into account the increment in productivity.GPC-MALLSIn both series,the molecular weight distribution of terpolymers is shifted to lower molecular weights with respect to the corresponding copol-ymer,although the activity increases.The de-crease in molecular weight is higher for terpoly-mers prepared with1-octene compared with those obtained with1-decene.Polymers prepared with higher ethylene contents(40mol%in the gas phase)show higher polydispersity values com-pared with those obtained with12mol%of eth-ylene.A shoulder of molecules with a higher mo-lecular weight(lower elution volume)can be ob-served in the molecular weight distribution of polymers containing higher ethylene contents. Number and weight average molecular weights as well as polydispersity values of synthesized polymers are shown in Table II.DSC AnalysisThe T g value of copolymers decreases as the eth-ylene content increases.The inﬂuence of higher ␣-oleﬁns on T g values is different for both series. Terpolymers prepared with the lower concentra-tion of ethylene show a stronger inﬂuence than those obtained with a higher concentration of eth-ylene.For the PP40E series,all polymers show T g values fromϪ42toϪ44°C.On the other hand, the PP12E series shows a maximum T g value for the terpolymer obtained with1-octene.The melt-ing temperature and melting enthalpy show im-portant differences when copolymers and terpoly-mers are compared.In both series,the terpoly-mers present melting temperatures that are 10–30°C higher than corresponding copolymers. Melting enthalpies also show similar trends in Table II.Terpolymers synthesized with1-octene have a higher melting temperature as well as a higher degree of crystallinity than those obtained with1-decene.Table II.Average Molecular Weights and Thermal Properties of Synthesized PolymersPolymerProductivity(kg Polymer/mol Zr h atm)Mn(Da)Mw(Da)Mw/MnTg(°C)Tm onset(°C)Tm peak(°C)⌬Hf(J/g)PP177013,30022,500 1.69—127.2134.585.4 PP12E260014,60022,400 1.53Ϫ35.257.374.019.9 PP12E2O4500960015,400 1.59Ϫ19.985.0100.444.5 PP12E2D348010,30017,900 1.72Ϫ27.186.6105.436.8 PP40E435016,70027,200 1.64Ϫ42.258.171.114.4 PP40E2O5190900017,600 1.64Ϫ42.666.384.319.8 PP40E2D524013,00023,900 1.84Ϫ43.967.983.613.9 For polypropylene[Zr]ϭ4.4610Ϫ6M,Al/Zrϭ3360,and Tϭ40°C.40CO-AND TERPOLYMERIZATION USING METALLOCENE CATALYSTS1141FTIR SpectraFTIR results are used only in a qualitative way to evaluate major changes in the spectra.Copoly-mers show only one important band in 732cm Ϫ1(PP12E )and 728cm Ϫ1(PP40E )in the 600–800cm Ϫ1zone.Principal differences with respect to the corresponding copolymers can be observed in the 1600–1700and 500–1000cm Ϫ1zones.Ter-polymer PP12E2O (2mol %of 1-octene)presents only one band at 732cm Ϫ1,whereas terpolymer PP12E2D (2mol %of 1-decene)shows two bands:one at 723cm Ϫ1and a second one at 733cm Ϫ1.The IR band for isolated ethylene units contain-ing three CH 2groups appears at 732.5cm Ϫ1.On the other hand,if there are ethylene units with ﬁve or more CH 2groups,a band at 720cm Ϫ1can be found.33Copolymer PP40E shows a band in 732cm Ϫ1.The terpolymer containing 1-octene (PP40E2O )presents two bands at 725and 732cm Ϫ1,and the terpolymer with 1-decene (PP40E2D )also shows two bands located at 723and 733cm Ϫ1.Copolymers of ethylene/propylene show one band in the 1600–1700cm Ϫ1zone.It is located at 1648cm Ϫ1in the copolymer PP12E and at 1651cm Ϫ1for the copolymer PP40E .Terpolymers be-longing to the PP12E series show several addi-tional bands (as shoulders).On the other hand,terpolymers prepared with 40mol %ethylene in the gas phase (PP40E series)present only one band at 1650cm Ϫ1.In the copolymer with 40mol %of ethylene,several bands at 1028and 1078cm Ϫ1appear.The bands of terminal vinyl (910cm Ϫ1)and transvi-nylene (965cm Ϫ1)are present only as shoulders in the spectra.The band at 888cm Ϫ1(vinylidene)is more important than the band at 900cm Ϫ1in PP12E and PP40E as well as the terpolymers with a high ethylene content (PP40E2O and PP40E2D )and decreases in PP12E2O and PP12E2D .Table III summarizes the FTIR results.Al-though several ﬁlms were prepared,differences arise mainly in the samples with higher ethylene contents.This fact can be correlated to some in-homogeneities in the structure of these copoly-mers.A 998/A 973have been included in Table III because this absorbance ratio can be taken as a measure of the regularity of the PP blocks.WAXS DataJones et al.34claimed that the ␥-phase of PP is characterized by ﬁve strong X-ray reﬂections of d -spacing at 6.37(100),5.29(74),4.415(85),4.19(40),and 4.05Å(63%).Four of these reﬂections lie very close to four of the reﬂection characteristics of the ␣-phase with d -spacing at 6.26,5.19,4.77,4.19,and 4.04Å.From those values,the reﬂection at 4.77Åcan be taken as distinctive of the ␣-phase,whereas the peak at 4.415Åis indicative of the ␥-phase.35,36Figures 1and 2show the WAXS proﬁles of PP40E and PP12E2O as well as PP40E2O ,respectively.Table IV shows the relative intensities of dif-ferent reﬂections in the WAXS difractograms.Table III.FTIR Characterization of Synthesized PolymersPolymer A 998/A 973A 720/A 900A 720/A 1160A 720/A 720ϩA 1160mol %P from eq 3mol %P from eq 4PP12E 0.64Ϯ0.020.78Ϯ0.010.35Ϯ0.110.78Ϯ0.0182.2Ϯ0.285.1Ϯ0.3PP12E2O 0.75Ϯ0.040.80Ϯ0.170.40Ϯ0.140.72Ϯ0.0777.4Ϯ 6.080.2Ϯ 5.7PP12E2D 0.71Ϯ0.01 1.01Ϯ0.010.57Ϯ0.010.64Ϯ0.0170.5Ϯ0.273.6Ϯ0.2PP40E 0.47Ϯ0.07 1.37Ϯ0.100.48Ϯ0.030.68Ϯ0.0173.8Ϯ 1.176.8Ϯ 1.0PP40E2O 0.76Ϯ0.260.92Ϯ0.040.52Ϯ0.200.66Ϯ0.0872.6Ϯ7.475.4Ϯ7.2PP40E2D0.94Ϯ0.010.97Ϯ0.010.85Ϯ0.010.54Ϯ0.0161.6Ϯ0.265.1Ϯ0.2Figure 1.WAXS spectra of copolymer PP40E .1142VILLAR AND FERREIRAPolyethylene presents several crystalline forms.In the ␤form,there are two peaks in the zone analyzed in these polymers:at 4.6(30)and 4.1Å(100%).The other reﬂections lie at angles 2␪higher than 35°.Rheological StudiesFigures 3and 4show the melt viscosities of the polymers synthesized with 12mol %(PP12E se-ries)and 40mol %(PP40E series)of ethylene in the gas phase,respectively.All the samples ob-tained behave as thermorheologically simple ma-terials.In both series,terpolymers show viscosity values lower than the corresponding copolymers.This can be attributed to the average molecular weight because all polymers present similar poly-dispersity values.Terpolymers containing 1-oc-tene present the lowest viscosity,whereas those containing 1-decene show values closer to those obtained with the copolymers.When all polymers analyzed obey the time–temperature principle,then curves of G Ј(␻)and G Љ(␻)measured at different temperatures can be superposed by shifting along the frequency axis using a temperature shift factor (a T ).For the set of polymers studied in the present work,a value of 49.0kJ/mol was found for the ﬂow activation energy in an Arrhenius-type dependency of a T with temperature.37The value found in this work is in good agreement with the values reported by Ferry 37and Llinas et al.38for an ethylene/pro-pylene copolymer containing 16mol %of ethylene as well as PP obtained with a metallocene cata-lyst,respectively.General DiscussionIncreasing the ethylene content in the gas phase gives an increment in the activity because of its minor steric hindrance with respect to propylene.A decrease in the melting temperature is alsoob-Figure 2.WAXS spectra of terpolymer containing 1-octene as termonomer:(a)PP12E2O and (b)PP40E2O .Table IV.Relative Intensity of the Main Reﬂections,Crystal Size,and Crystallinity of Synthesized Polymers from WAXS DataPolymer (111)␥-PP (110)␣-PP near 6.3Å(115)␥-PP (040)␣-PP near 5.2Å(130)␣-PP near 4.8Å(120o ´117)␥-PP near 4.4Å(202)␥-PP (111)␣-PP near 4.2Å(026)␥-PP (041)␣-PP near 4.0ÅCrystal-Size 14°(Å)Crystal Size 17°(Å)%X c PP12E 86.5100—36.038.824.09122429.4PP12E2O 90.390–10033.334.352.044.48620032.4PP12E2D 76.310040.641.860.645.19125632.0PP40E 60.6100—34.919.7—12920014.1PP40E2O 94.710087.772.463.1—11320015.8PP40E2D85.0100—22.238.9—5116414.1Figure 3.Master curves of viscosity (␩Ј)as a function of frequency (␻)at 120°C [symbols:(␭)PP12E ,(␴)PP12E2O ,and (␯)PP12E2D ].CO-AND TERPOLYMERIZATION USING METALLOCENE CATALYSTS 1143。

沉淀法的英语

沉淀法的英语The Precipitation Method。

The precipitation method is a widely used technique in various scientific fields, including chemistry, material science, and environmental science. It involves the formation of a solid precipitate from a solution by adding a precipitating agent. This method is valuable for the synthesis of new materials, the purification of substances, and the removal of pollutants from wastewater. In this article, we will explore the principles, applications, and advantages of the precipitation method.The principle behind the precipitation method lies in the solubility of different compounds in a solvent. When two solutions containing ions that can react with each other are mixed, the solubility product is exceeded, resulting in the formation of an insoluble compound or precipitate. The precipitate can then be separated from the solution through filtration or centrifugation.One of the key applications of the precipitation method is in the synthesis of new materials. By carefully controlling the reaction conditions, researchers can produce nanoparticles, nanowires, and other nanostructures with specific properties. For example, by varying the concentration and pH of the solutions, the size and morphology of the precipitate can be controlled, leading to materials with different optical, electrical, or catalytic properties.Another important application of the precipitation method is in the purification of substances. Impurities can be selectively removed by precipitating them out of the solution. This is particularly useful in the pharmaceutical industry, where the purity of drugs is of utmost importance. By adding a suitable precipitating agent, impurities can be removed, resulting in a higher purity product.The precipitation method also finds application in environmental science, particularly in the treatment of wastewater. Many pollutants, such as heavy metals and organic compounds, can be removed from wastewater by precipitating them as insolublecompounds. This is an effective and economical method for the removal of pollutants before the treated water is discharged into the environment.One of the advantages of the precipitation method is its simplicity and cost-effectiveness. The equipment required for the process is relatively simple and inexpensive, making it accessible to researchers and industries with limited resources. Additionally, the precipitation method can be easily scaled up for large-scale production, making it suitable for industrial applications.However, the precipitation method also has its limitations. One challenge is the control of the size and morphology of the precipitate. The formation of unwanted by-products or the aggregation of particles can occur, affecting the desired properties of the final product. Therefore, careful optimization of the reaction conditions is necessary to achieve the desired results.In conclusion, the precipitation method is a valuable technique in various scientific fields. It allows for the synthesis of new materials, the purification of substances, and the removal of pollutants from wastewater. With its simplicity and cost-effectiveness, it is widely used in research and industrial applications. However, careful control of the reaction conditions is crucial to obtain the desired properties of the precipitate. Further research and development in this area will continue to enhance the effectiveness and efficiency of the precipitation method.。

欧洲药典2.2.25

2.2.25.Absorption spectrophotometry,ultraviolet and visible EUROPEAN PHARMACOPOEIA8.0minimum at 1589cm −1and the absorption maximum at 1583cm −1is greater than0.08.Figure 2.2.24.-1.–Typical spectrum of polystyrene used toverify the resolution performanceVeriﬁcation of the wave-number scale .The wave-number scale may be veriﬁed using a polystyrene ﬁlm,which has transmission minima (absorption maxima)at the wave numbers (in cm –1)shown in Table 2.2.24.-1.Table 2.2.24.-1.–Transmission minima and acceptabletolerances of a polystyrene filmTransmission minima (cm −1)Acceptable tolerance (cm −1)Monochromator instrumentsFourier-transform instruments3060.0±1.5±1.02849.5±2.0±1.01942.9±1.5±1.01601.2±1.0±1.01583.0±1.0±1.01154.5±1.0±1.01028.3±1.0±1.0Method .Prepare the substance to be examinedaccording to the instructions accompanying the referencespectrum/reference ing the operating conditions that were used to obtain the reference spectrum,which will usually be the same as those for verifying the resolution performance,record the spectrum of the substance to be examined.The positions and the relative sizes of the bands in the spectrum of the substance to be examined and the reference spectrum are concordant in the pensation for water vapour and atmospheric carbondioxide .For Fourier-transform instruments,spectralinterference from water vapour and carbon dioxide iscompensated using suitable algorithms according to themanufacturer’s instructions.Alternatively,spectra can be acquired using suitable purged instruments or ensuring that sample and background single beam spectra are acquired under exactly the same conditions.IMPURITIES IN GASESFor the analysis of impurities,use a cell transparent to infrared radiation and of suitable optical path length (for example,1-20m).Fill the cell as prescribed under Gases.For detection and quantiﬁcation of the impurities,proceed as prescribed in the monograph.01/2008:202252.2.25.ABSORPTION SPECTROPHOTOMETRY,ULTRAVIOLET AND VISIBLEDetermination of absorbance .The absorbance (A )of asolution is deﬁned as the logarithm to base 10of the reciprocal of the transmittance (T )for monochromatic radiation:T =I/I 0;I 0=intensity of incident monochromatic radiation;I=intensity of transmitted monochromatic radiation.In the absence of other physico-chemical factors,theabsorbance (A )is proportional to the path length (b )through which the radiation passes and to the concentration (c )of the substance in solution in accordance with the equation:ε=molar absorptivity,if b is expressed in centimetres and c in moles per litre.The expressionrepresenting the speciﬁc absorbance of a dissolved refers to the absorbance of a 10g/L solution in a 1cm cell and measured at a deﬁned wavelength so that:Unless otherwise prescribed,measure the absorbance at theprescribed wavelength using a path length of 1cm.Unlessotherwise prescribed,the measurements are carried out withreference to the same solvent or the same mixture of solvents.The absorbance of the solvent measured against air and at the prescribed wavelength shall not exceed 0.4and is preferably less than 0.2.Plot the absorption spectrum with absorbance or function of absorbance as ordinate against wavelength or function of wavelength as abscissa.Where a monograph gives a single value for the position of an absorption maximum,it is understood that the value obtained may differ by not more than ±2nm.Apparatus .Spectrophotometers suitable for measuring inthe ultraviolet and visible range of the spectrum consist of an optical system capable of producing monochromatic radiation in the range of 200-800nm and a device suitablefor measuring the absorbance.Control of wavelengths .Verify the wavelength scale using the absorption maxima of holmium perchlorate solution R ,the line of a hydrogen or deuterium discharge lamp or the lines of a mercury vapour arc shown in Table 2.2.25.-1.The permitted 40See the information section on general monographs (cover pages)EUROPEAN PHARMACOPOEIA 8.0 2.2.25.Absorption spectrophotometry,ultraviolet andvisibletolerance is ±1nm for the ultraviolet range and ±3nm forthe visible range.Suitable certiﬁed reference materials may also be used.Table 2.2.25.-1.–Absorption maxima for control of wavelengthscale 241.15nm (Ho)404.66nm (Hg)253.7nm (Hg)435.83nm (Hg)287.15nm (Ho)486.0nm (Dβ)302.25nm (Hg)486.1nm (Hβ)313.16nm (Hg)536.3nm (Ho)334.15nm (Hg)546.07nm (Hg)361.5nm (Ho)576.96nm (Hg)365.48nm (Hg)579.07nm (Hg)Control of absorbance .Check the absorbance usingsuitable ﬁlters or a solution of potassium dichromate R at the wavelengths indicated in Table 2.2.25.-2,which gives for each wavelength the exact value and the permitted limits of the speciﬁc absorbance.The table is based on a tolerance for the absorbance of ±0.01.For the control of absorbance,use solutions of potassium dichromate R that has been previously dried to constant mass at 130°C.For the control of absorbance at 235nm,257nm,313nm and 350nm,dissolve 57.0-63.0mg of potassiumdichromate R in 0.005M sulfuric acid and dilute to 1000.0mL with the same acid.For the control of absorbance at 430nm,dissolve 57.0-63.0mg of potassium dichromate R in 0.005M sulfuric acid and dilute to 100.0mL with the same acid.Suitable certiﬁed reference materials may also be used.Table 2.2.25.-2Wavelength (nm)Speciﬁc absorbanceMaximum tolerance 235124.5122.9to 126.2257144.5142.8to 146.231348.647.0to 50.3350107.3105.6to 109.043015.915.7to 16.1Limit of stray light .Stray light may be detected at a given wavelength with suitable ﬁlters or solutions:for example,the absorbance of a 12g/L solution of potassium chloride R in a 1cm cell increases steeply between 220nm and 200nm and is greater than 2.0at 198nm when compared with water as compensation liquid.Suitable certiﬁed reference materials may also be used.Resolution (for qualitative analysis).When prescribed in a monograph,measure the resolution of the apparatus asfollows:record the spectrum of a 0.02per cent V/V solution of toluene R in hexane R .The minimum ratio of the absorbance at the maximum at 269nm to that at the minimum at 266nm is stated in the monograph.Suitable certiﬁed reference materials may also be used.Spectral slit-width (for quantitative analysis).To avoid errors due to spectral slit-width,when using an instrument onwhich the slit-width is variable at the selected wavelength,the slit-width must be small compared with the half-width of the absorption band but it must be as large as possible to obtain a high value of I 0.Therefore,a slit-width is chosen such that further reduction does not result in a change in absorbance reading.Cells .The tolerance on the path length of the cells used is ±0.005cm.When ﬁlled with the same solvent,the cells intended to contain the solution to be examined and thecompensation liquid must have the same transmittance.If this is not the case,an appropriate correction must be applied.The cells must be cleaned and handled with care.DERIVATIVE SPECTROPHOTOMETRYDerivative spectrophotometry involves the transformation of absorption spectra (zero-order)into ﬁrst-,second-or higher-order-derivative spectra.A first-order-derivative spectrum is a plot of the gradient of the absorption curve (rate of change of the absorbance with wavelength,d A /dλ)against wavelength.A second-order-derivative spectrum is a plot of the curvature of the absorption spectrum against wavelength (d 2A /dλ2).The second-order-derivative spectrum at any wavelength λis related to concentration by the following equation:c ′=concentration of the absorbing solute,in grams per litre.Apparatus .Use a spectrophotometer complying with the requirements prescribed above and equipped with an analogue resistance-capacitance differentiation module or a digital differentiator or other means of producing derivative spectra.Some methods of producing second-order-derivative spectra produce a wavelength shift relative to the zero-order spectrum and this is to be taken into account where applicable.Figure 2.2.25.-1Resolution power .When prescribed in a monograph,record the second-order-derivative spectrum of a 0.02per cent V/V solution of toluene R in methanol R ,using methanol R as the compensation liquid.The spectrum shows a small negative extremum located between 2large negative extrema at 261nmGeneral Notices (1)apply to all monographs and other texts412.2.26.Paper chromatography EUROPEAN PHARMACOPOEIA8.0and268nm,respectively,as shown in Figure2.2.25.-1.Unless otherwise prescribed in the monograph,the ratio A/B(see Figure2.2.25.-1)is not less than0.2.Procedure.Prepare the solution of the substance to be examined,adjust the various instrument settings accordingto the manufacturer’s instructions,and calculate the amount of the substance to be determined as prescribed in the monograph.01/2008:20226 2.2.26.PAPER CHROMATOGRAPHY ASCENDING PAPER CHROMATOGRAPHY Apparatus.The apparatus consists of a glass tank of suitable size for the chromatographic paper used,ground at the topto take a closelyﬁtting lid.In the top of the tank is a device which suspends the chromatographic paper and is capable of being lowered without opening the chamber.In the bottom of the tank is a dish to contain the mobile phase into which the paper may be lowered.The chromatographic paper consists of suitableﬁlter paper,cut into strips of sufﬁcient length and not less than2.5cm wide;the paper is cut so that the mobile phase runs in the direction of the grain of the paper. Method.Place in the dish a layer2.5cm deep of the mobile phase prescribed in the monograph.If prescribed in the monograph,pour the stationary phase between the walls of the tank and the dish.Close the tank and allow to stand for 24h at20°C to25°C.Maintain the tank at this temperature throughout the subsequent procedure.Draw aﬁne pencil line horizontally across the paper3cm from one ing a micro pipette,apply to a spot on the pencil line the volume of the solution prescribed in the monograph.If the total volume to be applied would produce a spot more than10mm in diameter,apply the solution in portions allowing eachto dry before the next application.When more than one chromatogram is to be run on the same strip of paper,space the solutions along the pencil line at points not less than3cm apart.Insert the paper into the tank,close the lid and allow to stand for1h30min.Lower the paper into the mobile phase and allow elution to proceed for the prescribed distance or time.Remove the paper from the tank and allow to dry in air. Protect the paper from bright light during the elution process. DESCENDING PAPER CHROMATOGRAPHY Apparatus.The apparatus consists of a glass tank of suitable size for the chromatographic paper used,ground at the topto take a closelyﬁtting glass lid.The lid has a central hole about1.5cm in diameter closed by a heavy glass plate or a stopper.In the upper part of the tank is suspended a solvent trough with a device for holding the chromatographic paper. On each side of the trough,parallel to and slightly above its upper edges,are two glass guide rods to support the paperin such a manner that no part of it is in contact with the walls of the tank.The chromatographic paper consists of suitableﬁlter paper,cut into strips of sufﬁcient length,and of any convenient width between2.5cm and the length of the trough;the paper is cut so that the mobile phase runs in the direction of the grain of the paper.Method.Place in the bottom of the tank a layer2.5cm deep of the solvent prescribed in the monograph,close the tank and allow to stand for24h at20°C to25°C.Maintain the tank at this temperature throughout the subsequent procedure. Draw aﬁne pencil line horizontally across the paper at such a distance from one end that when this end is secured in the solvent trough and the remainder of the paper is hanging freely over the guide rod,the line is a few centimetres below the guide rod and parallel with ing a micro-pipette,apply on the pencil line the volume of the solution prescribed in the monograph.If the total volume to be applied would produce a spot more than10mm in diameter,apply the solution in portions,allowing each to dry before the next application. When more than one chromatogram is to be run on the same strip of paper,space the solutions along the pencil line at points not less than3cm apart.Insert the paper in the tank, close the lid,and allow to stand for1h30min.Introduce into the solvent trough,through the hole in the lid,a sufﬁcient quantity of the mobile phase,close the tank and allow elution to proceed for the prescribed distance or time.Remove the paper from the tank and allow to dry in air.The paper should be protected from bright light during the elution process.01/2008:20227 2.2.27.THIN-LAYER CHROMATOGRAPHYThin-layer chromatography is a separation technique in which a stationary phase consisting of an appropriate material is spread in a uniform thin layer on a support(plate)of glass,metal or plastic.Solutions of analytes are depositedon the plate prior to development.The separation is based on adsorption,partition,ion-exchange or on combinationsof these mechanisms and is carried out by migration (development)of solutes(solutions of analytes)in a solvent or a suitable mixture of solvents(mobile phase)through the thin-layer(stationary phase).APPARATUSPlates.The chromatography is carried out using pre-coated plates as described under Reagents(4.1.1).Pre-treatment of the plates.It may be necessary to wash the plates prior to separation.This can be done by migration of an appropriate solvent.The plates may also be impregnated by procedures such as development,immersion or spraying. At the time of use,the plates may be activated,if necessary,by heating in an oven at120°C for20min. Chromatographic tank with aﬂat bottom or twin trough,of inert,transparent material,of a size suitable for the plates used and provided with a tightlyﬁtting lid.For horizontal development the tank is provided with a trough for the mobile phase and it additionally contains a device for directing the mobile phase to the stationary phase.Micropipettes,microsyringes,calibrated disposable capillaries or other application devices suitable for the proper application of the solutions.Fluorescence detection device to measure directﬂuorescence or the inhibition ofﬂuorescence.Visualisation devices and reagents.Suitable devices are used for derivatisation to transfer to the plate reagents by spraying, immersion or exposure to vapour and,where applicable,to facilitate heating for visualisation of separated components. Documentation.A device may be used to provide documentation of the visualised chromatogram,for example a photograph or a computerﬁle.METHODSample application.Apply the prescribed volume of the solutions at a suitable distance from the lower edge and from the sides of the plate and on a line parallel to the lower edge;allow an interval of at least10mm(5mm on high-performance plates)between the centres of circular spots and5mm(2mm on high-performance plates)between the edges of bands.Apply the solutions in sufﬁciently small portions to obtain circular spots2-5mm in diameter(1-2mm on high-performance plates)or bands10-20mm(5-10mm on high-performance plates)by1-2mm.In a monograph,where both normal and high-performance plates may be used,the working conditions forhigh-performance plates are given in the brackets[]after those for normal plates.42See the information section on general monographs(cover pages)。

Subthreshold Antiproton Spectra in Relativistic Heavy Ion Collisions

a rXiv:h ep-ph/959328v119Se p1995TPR-95-19Subthreshold Antiproton Spectra in Relativistic Heavy Ion Collisions Richard Wittmann and Ulrich Heinz Institut f¨u r Theoretische Physik,Universit¨a t Regensburg,D-93040Regensburg,Germany (February 1,2008)Abstract We study the structure of antiproton spectra at extreme subthreshold bom-barding energies using a thermodynamic picture.Antiproton production pro-cesses and ﬁnal state interactions are discussed in detail in order to ﬁnd out what can be learned about these processes from the observed spectra.Typeset using REVT E XI.INTRODUCTIONThere exist numerous examples for the production of particles in heavy ion collisions at bombarding energies well below the single nucleon-nucleon threshold[1].This phenomenon indicates collective interactions among the many participating nucleons and thus is expected to give information about the hot and dense matter formed in these collisions.At beam energies around1GeV per nucleon the most extreme of these subthreshold particles is the antiproton.Therefore,it is believed to be a very sensitive probe to collective behaviour in nucleus-nucleus collisions.However,presently neither the production mechanism nor theﬁnal state interactions of antiprotons in dense nucleonic matter are well understood.The antiproton yields measured at GSI and BEVALAC[2,3]seem to be described equally well by various microscopic models using diﬀerent assumptions about the production mechanism and particle properties in dense nuclear matter[4–7].This ambiguity raises the question which kind of information can be really deduced from subthreshold¯p spectra.In this paper we use a simple thermodynamic framework as a background on wich we can systematically study the inﬂuence of diﬀerent assumptions on theﬁnal¯p spectrum.In the next section we will focus on the production process.Following a discussion of theﬁnal state interactions of the antiproton in dense hadronic matter in Section III,we use in Section IV a one-dimensional hydrodynamic model for the explodingﬁreball to clarify which features of the production and reabsorption mechanisms should survive in theﬁnal spectra in a dynamical environment.We summarize our results in Section V.II.PRODUCTION OF ANTIPROTONS IN HEA VY ION COLLISIONSA.The Antiproton Production RateUnfortunately very little is known about the production mechanism for antiprotons in dense nuclear matter.Therefore,we are forced to use intuitive arguments to obtain aplausible expression for the production rate.As commonly done in microscopic models[8] we consider only two body collisions and take the experimentally measured cross sections for ¯p production in free NN collisions as input.The problem can then be split into two parts: the distribution of the two colliding nucleons in momentum space and the elementary cross sections for antiproton production.The procedure is later generalized to collisions among other types of particles(Section II C)using phase space arguments.Formally,the antiproton production rate P,i.e.the number of antiprotons produced in the space-time cell d4x and momentum space element d3p,is given by[9]P= i,j ds2w(s)d3σij→¯pδ(p2i−m2i)Θ(p0i).(2)(2π)3Finally,w(s,m i,m j)=√ofﬁnding two nucleons at a center-of-mass energys−4m)αIn order to obtain from the total cross section(3)a formula for the diﬀerential cross section,we assume like others that the momentum distribution of the produced particles is mainly governed by phase space.This leads to the simple relationshipd3σij→NNN¯pσij→NNN¯p(s).(4)R4(P)Here R n is the volume the n-particle phase space,which can be given analytically in the non-relativistic limit[12].P is the total4-momentum of the4-particleﬁnal state,which√reduces to(s min(p)/T.Hereρi,j are the densities of the incoming particles,that the cross section σij →NNN ¯p (s )is independent of the internal state of excitation of the colliding baryons in our thermal picture.The consequences of this assumption are quite obvious.While the distance to the ¯p -threshold is reduced by the larger rest mass of the resonances,the mean velocity of a heavy resonance state in a thermal system is smaller than that of a nucleon.Both factors counteract each other,and indeed we found that the total rate P is not strongly changed by the inclusion of resonances.The role of pionic intermediate states for ¯p -production in pp -collisions was pointed out by Feldman [15].As mesonsare created numerously in the course of a heavy ion collision,mesonic states gain even more importance in this case.In fact,Ko and Ge [13]claimed that ρρ→p ¯p should be the dominant production channel.Relating the ρρ-production channel to the p ¯p annihilation channel [13]byσρρ→p ¯p (s )= 2s −4m ρm σp ¯p →ρρ(s )(5)where S =1is the spin factor for the ρ,the production rate can be calculated straightfor-wardly from Eq.(1):P =g 2ρ(2π)516T .(6)The spin-isospin factor of the ρis g ρ=9,and E is the energy of the produced antiproton.The modiﬁed Bessel function K 1results from the assumption of local thermal equilibration for the ρ-distribution.Expanding the Bessel function for large values of 2E/T we see that the ”temperature”T ¯p of the ¯p -spectrum is only half the medium temperature:T ¯p =1w 2(s,m i ,m j )(8)Comparing this form with measured data onπ−p→np¯p collisions[16],a value ofσ0ij= 0.35mb is obtained.Due to the threshold behaviour of Eq.(8)and the rather large value ofσ0ij,it turns out[17]that this process is by far the most important one in a chemically equilibrated system.However,this chemical equilibration–if achieved at all–is reached only in theﬁnal stages of the heavy ion collision when cooling has already started.So it is by no means clear whether the dominance of the meson-baryon channel remains valid in a realistic collision scenario.This point will be further discussed in Section IV.III.FINAL STATE INTERACTION OF THE ANTIPROTONOnce an antiproton is created in the hot and dense hadronic medium,its state will be modiﬁed by interactions with the surrounding particles.Two fundamentally diﬀerent cases have to be distinguished:elastic scattering,which leads to a reconﬁguration in phase space, driving the momentum distribution towards a thermal one with the temperature of the surrounding medium,and annihilation.Each process will be considered in turn.A.Elastic ScatteringThe time evolution of the distribution function f(p,t)is generally described by the equation[18]f(p,t2)= w(p,p′;t2,t1)f(p′,t1)dp′(9) where w(p,p′;t2,t1)is the transition probability from momentum state p′at time t1to state p at t2.Because the number density of antiprotons is negligible compared to the total particle density in the system,the evolution of f(p,t)can be viewed as a Markov process.Assuming furthermore that the duration of a single scattering processτand the mean free pathλare small compared to the typical time scaleδt and length scaleδr which measure the variation of the thermodynamic properties of the system,τ≪δt,λ≪δr,Eq.(9)can be transformed into a master equation.Considering the structure of the diﬀer-ential p¯p cross section one notices that in the interesting energy range it is strongly peaked in the forward direction[19–21].Therefore,the master equation can be approximated by a Fokker-Planck equation[22]:∂f(p,t)∂p A(p)f(p,t)+1∂2pD(p)f(p,t).(10)For the evaluation of the friction coeﬃcient A and the diﬀusion coeﬃcient D we follow the treatment described by Svetitsky[23].For the diﬀerential cross section we took a form suggested in[19]:dσ|t|2D/A(e2At−1)+2mT0p2≡exp −p2/2mT eﬀ(t) .(14)This shows that the exponential shape of the distribution function is maintained throughout the time evolution,but that the slope T eﬀ(t)gradually evolves from T0to the value D/mAwhich,according to the Einstein relation(12),is the medium temperature T.Looking at Fig.3it is clear that after about10fm/c the spectrum is practically thermalized.Therefore initial structures of the production spectrum(like the ones seen in Fig.2)are washed out quite rapidely,and their experimental observation will be very diﬃcult.B.AnnihilationThe annihilation of antiprotons with baryons is dominated by multi-mesonﬁnal states X.For the parametrisation of the annihilation cross sectionσann(s)we take the form given in[14]for the process¯p+B−→X,B=N,∆, (15)Using the same philosophy as for the calculation of the production rate,a simple dif-ferential equation for the decrease of the antiproton density in phase space can be written down:dd3xd3p =−d6N(2π)3f i( x, p i,t)v i¯pσanni¯p≡−d6Nbecomes questionable.More reliable results should be based on a quantumﬁeld theoretic calculation which is beyond the scope of this paper.IV.ANTIPROTON SPECTRA FROM AN EXPLODING FIREBALLA.A Model for the Heavy Ion CollisionIn order to compare the results of the two previous sections with experimental data we connect them through a dynamical model for the heavy ion reaction.In the spirit of our thermodynamic approach the so-called hadrochemical model of Montvay and Zimanyi [24]is applied for the simulation of the heavy ion collision.In this picture the reaction is split into two phases,an ignition and an explosion phase,and particles which have at least scattered once are assumed to follow a local Maxwell-Boltzmann distribution.In addition, a spherically symmetric geometry is assumed for the explosion phase.The included particle species are nucleons,∆-resonances,pions andρ-mesons.As initial condition a Fermi-type density distribution is taken for the nucleons of the incoming nuclei,ρ0ρ(r)=collision with momentum p z=±700MeV in the c.m.system.One clearly sees that at the moment of full overlap of the two nuclei a dense zone with hot nucleons,resonances and mesons(not shown)has been formed.Only in the peripheral regions”cold”target and projectile nucleons can still be found.On the other hand,chemical equilibrium of the hot collision zone is not reached in the short available time before the explosion phase sets in; pions and in particularρmesons remain far below their equilibrium abundances[17].It is important to note that due to the arguments given in Section II¯p production is strongly suppressed in this initial stage of the reaction.In our simple model we have in fact neglected this early¯p production completely.The ignition phase is only needed to obtain the chemical composition of the hotﬁreball which is expected to be the dominant source for the creation of antiprotons.For the subsequent expansion of the spherically symmetricﬁreball into the surrounding vacuum analytical solutions can be given if the equation of state of an ideal gas is taken as input[25].If excited states are included in the model,an exact analytical solution is no longer possible.Because a small admixture of resonaces is not expected to fundamentally change the dynamics of the system,we can account for their eﬀect inﬁrst order by adjusting only the thermodynamic parameters of the explodingﬁreball,but not the expansion velocity proﬁles.There is one free parameter in the model[24],α,which controls the density and the temperature proﬁles,respectively.Small valuesα→0representδ-function like density proﬁles whereasα→∞corresponds to a homogeneous density distribution throughout the ﬁreball(square well proﬁle).The time-dependent temperature proﬁles for two representative values ofαare shown in Fig.5for diﬀerent times t starting at the time t m of full overlap of the nuclei.Clearly,a small value ofαleads to an unreasonably high temperature(T∼200MeV) in the core of theﬁreball at the beginning of the explosion phase,and should thus be considered unphysical.B.Antiproton Spectra from an Exploding FireballBased on the time-dependent chemical composition of this hadrochemical model we can calculate the spectrum of the antiprotons created in a heavy ion collision.Let usﬁrst con-centrate on the inﬂuence of the density distribution in theﬁreball characterized by the shape parameterα.Due to diﬀerent temperature proﬁles connected with diﬀerentαvalues(see Fig.5)the absolute normalization varies substantially when the density proﬁle is changed. For moreδ-like shapes an extremely hotﬁreball core is generated,whereas for increasingαboth density and temperature are more and more diﬀuse and spread uniformly over a wider area.Because of the exponential dependence upon temperature a small,but hot core raises the production rate drastically.This fact is illustrated in Fig.6for three values ofα.Not only the total normalization,but also the asymptotic slope of the spectrum is modiﬁed due to the variation of the core temperature withαas indicated in the Figure.Comparing the dotted lines,which give the pure production spectrum,to the solid lines representing the asymptotic spectrum at decoupling,the tremendous eﬀect of antiproton absorption in heavy ion collisions is obvious.As one intuitively expects absorption is more pronounced for low-energetic antiprotons than for the high-energetic ones which have the opportunity to escape the high density zone earlier.Therefore,theﬁnally observed spectrum isﬂatter than the original production spectrum.Interestingly,while the baryon-baryon and theρρchannels are comparable in their con-tribution to¯p production,the pion-baryon channel turned out to be much more eﬀective for all reasonable sets of parameters.This fact is indeed remarkable,because here,contrary to the discussion in Section II,the pions are not in chemical equilibrium;in our hadrochemical model the total time of the ignition phase is too short to saturate the pion channel.The meson-baryon channel is thus crucial for understanding¯p spectra.Only by including all channels reliable predictions about the antiprotons can be drawn.We did not mention so far that in our calculations we followed common practice and assumed aﬁnite¯p formation time ofτ=1fm/c;this means that during this time intervalafter a¯p-producing collision the antiproton is assumed to be not yet fully developed and thus cannot annihilate.However,there are some(although controversial)experimental indications of an extremely long mean free path for antiprotons beforeﬁnal state interactions set in[26].We have tested the inﬂuence of diﬀerent values for the formation timeτon the¯p spectrum.Fig.7shows that this highly phenomenological and poorly established parameter has a very strong inﬂuence in particular on the absolute normalization of the spectra,i.e. the total production yield.In the light of this uncertainty it appears diﬃcult to argue for or against the necessity for medium eﬀects on the antiproton production threshold based on a comparison between theoretical and experimental total yields only.parison with Experimental DataIn all the calculations shown above a bombarding energy of1GeV/A has been assumed. Experimental data are,however,only available at around2GeV/A.At these higher energies thermalization becomes more questionable[27],and our simple model may be stretching its limits.Especially,the temperature in theﬁreball core becomes extremely high.In order to avoid such an unrealistic situation and in recognition of results from kinetic simulations [4,6,7]we thus assume that only part of the incoming energy is thermalized–in the following a fraction of70%was taken.Fig.8shows calculations for the antiproton spectrum from Na-Na and Ni-Ni collisions at a kinetic beam energy of2GeV/nucleon.The calculation assumes a¯p formation time of τ=1fm/c,and takes for the density and temperature proﬁles the parameter valueα=1 which corresponds to an upside-down parabola for the density proﬁparing with the GSI data[2]we see our model features too weak a dependence on the size of the collision system;the absolute order of magnitude of the antiproton spectrum is,however,correctly reproduced by our simple hadrochemical model,without adjusting any other parameters. No exotic processes for¯p production are assumed.As mentioned in the previous subsection, the pion-baryon channel is responsible for getting enough antiprotons in our model,withoutany need for a reduced eﬀective¯p mass in the hot and dense medium[4,5].The existing data do not yet allow for a deﬁnite conclusion about the shape of the spectrum,and we hope that future experiments[28]will provide additional contraints for the model.V.CONCLUSIONSHeavy ion collisions at typical BEVALAC and SIS energies are far below the p¯p-production threshold.As a consequence,pre-equilibrium antiproton production in such collisions is strongly suppressed relative to production from the thermalized medium pro-duced in the later stages of the collision.Therefore,¯p production becomes important only when the heavy ion reaction is suﬃciently far progressed,in accordance with microscopic simulations[4].By assuming a local Maxwell-Boltzmann distribution for the scattered and produced particles forming the medium in the collision zone one maximizes the¯p production rate(see Fig.1).If,contrary to the assumptions made in this work,the extreme states in phase space described by the tails of the thermal Boltzmann distribution are not populated, the antiproton yield could be reduced substantially.We also found that the threshold behaviour of the¯p production cross section is not only crucial for the total¯p yield,but also introduces structures into the initial¯p spectrum.This might give rise to the hope that by measuring the¯p momentum spectrum one may obtain further insight into the¯p production mechanism.On the other hand we saw here,using a Fokker-Plank description for the later evolution of the distribution function f¯p(p,t)in a hot environment,that these structures are largely washed out by subsequent elastic scattering of the¯p with the hadrons in the medium.In addition,the large annihilation rate reduces the number of observable antiprotons by roughly two orders of magnitude relative to the initial production spectrum;the exact magnitude of the absorption eﬀect was found to depend sensitively on the choice of the¯p formation timeτ.We have also shown that meson(in particular pion)induced production channels con-tribute signiﬁcantly to theﬁnal¯p yield and should thus not be neglected.We were thus ableto reproduce the total yield of the measured antiprotons in a simple model for the reaction dynamics without including,for example,medium eﬀects on the hadron masses and cross sections[4,5].However,we must stress the strong sensitivity of the¯p yield on various unknown param-eters(e.g.the¯p formation time)and on poorly controlled approximations(e.g.the degree of population of extreme corners in phase space by the particles in the collision region),and emphasize the rapidly thermalizing eﬀects of elasticﬁnal state interactions on the¯p momen-tum spectrum.We conclude that turning subthreshold antiproton production in heavy ion collisions into a quantitative probe for medium properties and collective dynamics in hot and dense nuclear matter remains a serious challenge.ACKNOWLEDGMENTSThis work was supported by the Gesellschaft f¨u r Schwerionenforschung(GSI)and by the Bundesministerium f¨u r Bildung und Forschung(BMBF).REFERENCES[1]U.Mosel,Annu.Rev.Nucl.Part.Sci.1991,Vol.41,29[2]A.Schr¨o ter et al.,Z.Phys.A350(1994)101[3]A.Shor et al.,Phys.Rev.Lett.63(1989)2192[4]S.Teis,W.Cassing,T.Maruyama,and U.Mosel,Phys.Rev.C50(1994)388[5]G.Q.Li and C.M.Ko,Phys.Rev.C50(1994)1725[6]G.Batko et al.,J.Phys.G20(1994)461[7]C.Spieles et al.,Mod.Phys.Lett.A27(1993)2547[8]G.F.Bertsch and S.Das Gupta,Phys.Rep.160(1988)189[9]P.Koch,B.M¨u ller,and J.Rafelski,Phys.Rep.42(1986)167[10]B.Sch¨u rmann,K.Hartmann,and H.Pirner,Nucl.Phys.A360(1981)435[11]G.Batko et al.,Phys.Lett.B256(1991)331[12]burn,Rev.Mod.Phys.27(1955)1,and references therein[13]C.M.Ko and X.Ge,Phys.Lett.B205(1988)195[14]P.Koch and C.Dover,Phys.Rev.C40(1989)145[15]G.Feldman,Phys.Rev.95(1954)1697[16]Landolt-B¨o rnstein,Numerical Data and Functional Relationships in Science and Tech-nology,Vol.12a und Vol.12b,Springer-Verlag Berlin,1988[17]R.Wittmann,Ph.D.thesis,Univ.Regensburg,Feb.1995[18]N.G.van Kampen,Stochastic Processes in Physics and Chemistry,North-Holland Pub-lishing Company,Amsterdam1983[19]B.Conforto et al.,Nouvo Cim.54A(1968)441[20]W.Br¨u ckner et al.,Phys.Lett.B166(1986)113[21]E.Eisenhandler et al.,Nucl.Phys.B113(1976)1[22]S.Chandrasekhar,Rev.Mod.Phys.15(1943)1[23]B.Svetitsky,Phys.Rev.D37(1988)2484[24]I.Montvay and J.Zimanyi,Nucl.Phys.A316(1979)490[25]J.P.Bondorf,S.I.A.Garpman,and J.Zim´a nyi,Nucl.Phys.A296(1978)320[26]A.O.Vaisenberg et al.,JETP Lett.29(1979)661[27]ng et al.,Phys.Lett.B245(1990)147[28]A.Gillitzer et al.,talk presented at the XXXIII International Winter Meeting on NuclearPhysics,23.-28.January1995,Bormio(Italy)FIGURES468101214-810-710-610-510-410-310-210-110010s in GeVλt=0.2 fm/c t=2 fm/c t=5 fm/c ©©©2FIG.1.λ(s,t )at diﬀerent times t calculated from the model of Ref.[10].The starting point is a δ-function at s =5.5GeV 2.The dashed line is the asymptotic thermal distribution at t =∞),corresponding to a temperature T =133MeV.00.10.20.30.40.50.6-16-171010-1510-1410-1310-1210-1110-1010 E in GeVα = 1/2α = 3/2α = 5/2α = 7/2ÄÄÄÄPFIG.2.Antiproton production spectrum for diﬀerent threshold behaviour of the elementaryproduction process (x =12,52from top to bottom).203040506070 8090t in fm/cT e f f T = 10 MeV 0T = 50 MeV 0T = 70 MeV 00 246810i n M e V 10FIG.3.Eﬀective temperature T eﬀfor three Maxwell distributions with initial temperatures T 0=10MeV,50MeV and 70MeV,respectively.0z in fm12ρ / ρ0FIG.4.Density distributions ρ(0,0,z )along the beam axis of target and projectile nucleons for a 40Ca-40Cacollision,normalized to ρ0=0.15fm −3.The solid lines labelled by “incoming nuclei”show the two nuclei centered at ±5fm at time t 0=0.The two other solid lines denote the cold nuclear remnants at full overlap time t m ,centered at about ±3fm.Also shown are ﬁreball nucleons (long-dashed)and ∆-resonances (short-dashed)at time t m .α=0.20T i n G e V r in fm α=5r in fm024680.050.10.150.20.25T i n G e V t = 9 fm/c630FIG.5.Temperature proﬁles for α=0.2and α=5at four diﬀerent times t =0(=beginning of the explosion phase)and t =3,6,and 9fm/c (from top to bottom).E in GeV33d N / d p i n G e V -3α=0.2α=1α=500.10.20.30.40.50.6-11-10-9-8-7-6-5-4-3101010101010101010FIG.6.¯p -spectra for diﬀerent proﬁle parameters α.The dotted lines mark the initial pro-duction spectra.The asymptotic temperatures at an assumed freeze-out density ρf =ρ0/2corre-sponding to the solid lines are,from top to bottom,105MeV,87MeV and 64MeV,respectively.E in GeVd N / d p i n Ge V 33-10101010101010FIG.7.¯p -spectrum for diﬀerent formation times,for a proﬁle parameter α=1.The dashed line indicates the original production spectrum.E in GeVkin d p 3d σ3i n b /Ge V /10101010FIG.8.Diﬀerential ¯p spectrum for Na-Na and Ni-Ni collisions,for a shape parameter α=1.The data are from GSI experiments [2].。

超声-酶辅助低共熔溶剂提取桑叶总黄酮的工艺优化及其抗氧化活性研究

吴均，吴俊葶，杨碧文，等. 超声-酶辅助低共熔溶剂提取桑叶总黄酮的工艺优化及其抗氧化活性研究[J]. 食品工业科技，2024，45（3）：31−39. doi: 10.13386/j.issn1002-0306.2023070033WU Jun, WU Junting, YANG Biwen, et al. Optimization of Ultrasonic-Enzyme-Assisted Deep Eutectic Solvents Extraction Process of Total Flavonoids from Mulberry Leaves and Its Antioxidant Activity[J]. Science and Technology of Food Industry, 2024, 45(3): 31−39.(in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023070033· 特邀主编专栏—食品中天然产物提取分离、结构表征和生物活性（客座主编：杨栩、彭鑫） ·超声-酶辅助低共熔溶剂提取桑叶总黄酮的工艺优化及其抗氧化活性研究吴　均1，吴俊葶2，杨碧文1，王　梅1，赵　珮1，马婧秋1，黄　越1, *，黄传书1,*（1.重庆市蚕业科学技术研究院，重庆 400700；2.中国人民解放军陆军勤务学院，重庆 401331）摘　要：为建立一种绿色高效的桑叶总黄酮提取方法，本研究采用了超声-酶辅助低共熔溶剂法对桑叶总黄酮进行提取。

在单因素实验的基础上，以桑叶总黄酮提取量为响应值，采用Box-Behnken 响应面法对提取工艺进行优化，并研究桑叶总黄酮对 ABTS +和DPPH 自由基的清除能力。

结果表明：在氯化胆碱/果糖/乙醇摩尔比为1:1:3、含水量为30%、液料比为40 mL/g 、超声功率为360 W 、超声温度为40 ℃、超声时间为40 min 、酶添加量为4%条件下，桑叶总黄酮提取量为46.58 mg/g ；当总黄酮质量浓度为0.08 mg/mL 时，对DPPH 自由基清除率为98.36%，当总黄酮质量浓度为0.2 mg/mL 时，对ABTS +自由基清除率为72.12%。

FluorescencePola...

Fluorescence Polarization (FP)Ewald TerpetschnigISS Inc.PrinciplesFluorescence polarization (FP) measurements are based on the assessment of the rotational motions of species. FP can be considered a competition between the molecular motion and the lifetime of fluorophores in solution. If linear polarized light is used to excite an ensemble of fluorophores only those fluorophores, aligned with the plane of polarization will be excited. There are 2 scenarios for the emission.Provided the fluorescence lifetime of the excited fluorescent probe is much longer than the rotational correlation time θ of the molecule it is bound to (τfl >> θrot) (θ is a parameter that describes how fast a molecule tumbles in solution), the molecules will randomize in solution during the process of emission, and, as a result, the emitted light of the fluorescent probe will be depolarized. If the fluorescence lifetime of the fluorophore is much shorter than the rotational correlation time θ (τfl << θrot) the excited molecules will stay aligned during the process of emission and as a result the emission will be polarized.Figure 1. Relationship between fluorescence lifetime τ and rotational correlation time θ. Dependence of Fluorescence Polarization on Molecular Mobility [1]The fluorescence polarization (P) of a labeled macromolecule depends on the fluorescence lifetime (τ) and the rotational correlation time (θ)where P0 is the polarization observed in the absence of rotational diffusion. The effect of the molecular weight on the polarization values can be seen from an alternative form of the above equation:where k is the Boltzman constant, T is the absolute temperature, η the viscosity and V the molecular volume [2]. The molecular volume of the protein is related to the molecular weight (MW) and the rotational correlation time as given bywhere R is the ideal gas constant, v is the specific volume of the protein and h is the hydration, typically 0.2 g H2O per gram of protein. Generally, the observed correlation times are about two-fold longer than calculated for an anhydrous sphere due to the effects of hydration and the non-spherical shapes of most proteins. Hence, in aqueous solution at 20°C (η = 1 cP) one can expecta protein such as HSA (MW ~ 65,000, with h = 1.9) to display a rotational correlation time (θ) near50 ns.The measurement of fluorescence polarization is relatively straight-forward (Figure 2). In a typical experiment the sample containing the fluorescent probe is excited with linear polarized light and the vertical and horizontal components of the intensity of the emitted light are measured and the polarization (P) or anisotropy (r) are calculated using the following equations:Polarization (P) = (I v - I h) / (I v+ I h)Anisotropy (r) = (I v - I h) / (I v+ 2I h)where I v is the intensity parallel to the excitation plane and I h is the emission perpendicular to the excitation plane. They are interchangeable quantities and only differ in their normalization. Polarization P ranges from –0.33 to +0.5 while the range for anisotropy r is –0.25 to +0.4.P = 3 r / 2 + rr = 2 P / 3 - PFigure 2. Schematic drawing for the measurement of fluorescence polarization.Tracers for Polarization AssaysTracers used in fluorescence polarization assays include peptides, drugs, antibiotics etc. and they are typically synthesized by the reaction of a fluorescent dye with a reactive derivative of the analyte.Linker chemistries can have an impact on the fluorescence polarization. While tracers with short linkers between the fluorophore and the labeled molecule minimize the “propeller-effect”, a too short linker can affect the binding affinity of the tracer [3].Typical fluorophores used in FP are fluorescein and rhodamines. BODIPY dyes have longer excited-state lifetimes than fluorescein and rhodamine dyes, making their fluorescence polarization sensitive to binding interactions over a larger molecular weight range [4].A limitation of current fluorescence polarization immunoassays (FPIs) is that they are useful only for measurement of low molecular weight antigens. This limitation is the result of the use of fluorophores, such as fluorescein, which display lifetimes near 4 ns. An FPI requires that the emission from the unbound labeled antigen be depolarized, so that an increase in polarization may be observed upon binding to antibody. For depolarization to occur the antigen must display a rotational correlation time much shorter than 4 ns, which limits the FPI to antigens with molecular weight less than several thousand Daltons.A class of dyes that have been shown to combine long lifetime and high polarization are the metal-ligand complexes of Ru, Os and Re. These labels have lifetimes in the range of a few hundred ns to microseconds and would therefore allow measurement of higher molecular weight antigens but the strong propeller effect of the MLC when labeled to proteins other than HSA has limited their use as labels for high molecular weight analytes [2].Figure 3. Excitation polarization spectra of Ru-metal ligand complexes in solution at -55°C.ApplicationsFluorescence polarization measurements have been used in analytical and clinical chemistry [5,6] and as a biophysical research tool for studying membrane lipid mobility [7], domain motions in proteins, and interactions at the molecular level [8]. Fluorescence polarization based immunoassays are also extensively utilized for clinical diagnostics [9-11]. FP has the advantage that it requires only one labeled species for the assay (unlike energy-transfer based read outs that require two labeled species) and thus FP has become a very popular read out format for HTS (12-17). Many of these assays are based on the use of antibodies that provide the specificity needed to selectively detect a wide variety of antigens.An example for a homogeneous binding assay based on FP is shown below. Any material that enables a mass-increase of the labeled species can replace antibodies. In the IMAP assay™ (Molecular Devices) the high affinity of trivalent metal-ions to phosphate is utilized to generate the FP read-out [18].Figure 4. IMAPTM homogeneous binding assay for kinases and phosphatases.Books and Book Chapters related to Fluorescence Polarization1. Schulman, S.G. (Ed.) (1985). Molecular Luminescence Spectroscopy. Methods and Applications: Part1, J. Wiley & Sons, New York.2. Ichinose, N., Schwedt, G., Schnepel, F.M. and Adachi, K. (Eds.) (1987). Fluorometric Analysis inBiomedical Chemistry, J. Wiley & Sons, New York.3. Van Dyke, K. and Van Dyke, R. (Eds.) (1990). Luminescence Immunoassay and MolecularApplications, CRC Press, Boca Raton, FL.4. Lakowicz, J.R. (1999). Principles of Fluorescence Spectroscopy, 2nd Edition, Kluwer Academic/PlenumPublishers, New York.5. Steiner, R. F. (1991). Fluorescence anisotropy: theory and applications. In Topics in FluorescenceSpectroscopy. Vol. 2. Principles. Lakowicz, J.R. (Ed.) Plenum Press, New York.References1. Weber, G. in Hercules, D.M. (1966) Fluorescence and Phosphorescence Analysis. Principles andApplications, Interscience Publishers (J. Wiley & Sons), New York, pp. 217-240.2. Szmacinski, H., Terpetschnig, E. and Lakowicz, J.R. “Synthesis and Evaluation of Ru-complexes asAnisotropy Probes for Protein Hydrodynamics and Immunoassays of High-Molecular-Weight Antigens”.Biophysical Chemistry 62, 109-120 (1996).3. Huang. “Fluorescence Polarization Competition Assay: The Range of Resolvable Inhibitor Potency IsLimited by the Affinity of the Fluorescent Ligand”. J Biomol Screen. 8, 34-38 (2003).4. Schade SZ, Jolley ME, Sarauer BJ, Simonson LG. “BODIPY-alpha-casein, a pH-independent proteinsubstrate for protease assays using fluorescence polarization.” Anal Biochem. 243, 1-7 (1996).5. Jameson, D.M. and Seifried, S.E. “Quantification of Protein-Protein Interactions Using FluorescencePolarization”. Methods 19,222-233 (1999).6. Van Dyke, K. and Van Dyke, R. (Eds.) (1990) Luminescence Immunoassay and Molecular Applications,CRC Press, Boca Raton, FL. b) Jameson D.M. and Croney J.C. “Fluorescence Polarization: Past, Present and Future”. Combinatorial Chemistry & High Throughput Screening 6 (3), 167-176 (2003).7. Yu W., So PTC, French T, and Gratton E. Fluorescence generalized polarization of cell membranes-atwo photon scanning microscopy approach. Biophys. J. 70, 626-636 (1996).8. LeTilly V, and Royer CA. (1993), Fluorescence Anisotropy Assays Implicate Protein-Protein Interactionsin Regulating trp Repressor DNA Binding, Biochemistry 32, 7753-7758.9. Y and Potter JM. “Fluorescence polarization immunoassay and HPLC assays compared for measuringmonoethylglycinexylidide in liver-transplant patients”. Clinical Chemistry 38, 2426-2430 (1992).10. Bittman R and Fischkoff SA. “Fluorescence Studies of the Binding of the Polyene Antibiotics Filipin III,Amphotericin B, Nystatin, and Lagosin to Cholesterol”. PNAS 69 (12), 3795-3799 (1972).11. B, Verbesselt R, Scharpe S, Verkerk R, Lambert WE, Van Liedekerke B, De Leenheer A. “Comparisonof cyclosporin A measurement in whole blood by six different methods”. J. Clin Chem Clin Biochem.28(1):53-7 (1990).12. Parker GJ, Law TL, Lenoch FJ, Bolger RE. “Development of high throughput screening assays usingfluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays”. J Biomol Screen 5, 77-88 (2000).13. Kim et al. “Development of a Fluorescence Polarization Assay for the Molecular Chaperone Hsp90”. JBiomol Screen 9, 375-381 (2004).14. Banks P, Gosselin M, Prystay L. “Fluorescence polarization assays for high throughput Screening of G-protein coupled receptors”. J.Biomol. Screen. 5, 159–167 (2000).15. Turek TC, Small EC, Bryant RW et al. “Development and validation of a competitive AKTserine/threonine kinase fluorescence polarization assay using product-specific antiphospho-serine antibody”. Anal. Biochem. 299, 45–53 (2001).16. Fowler A, Swift D, Longman E et al. “An evaluation of fluorescence polarization and lifetimediscriminated polarization for high-throughput screening of serine/threonine kinases”. Anal.Biochem.308, 223–231 (2002).17. Lu Z, Yin Z, James L et al. “Development of a fluorescence polarization bead-based coupled assay totarget different activity/conformation states of a protein kinase”. J. Biomol. Screen. 9, 309–321 (2004).18. Zaman G.J.R., Garritsen A. de Boer T., van Boeckel C.A.A. “Fluorescence Assays for High-ThroughputScreening of Protein Kinases”. Combinatorial Chemistry & High Throughput Screening 6 (4), 313-320。

碳包覆的磁性纳米材料萃取酞酸酯

第３２卷㊀第１２期２０１３年㊀㊀１２月环㊀境㊀化㊀学ＥＮＶＩＲＯＮＭＥＮＴＡＬＣＨＥＭＩＳＴＲＹＶｏｌ．３２，Ｎｏ．１２Ｄｅｃｅｍｂｅｒ２０１３㊀２０１３年３月２７日收稿．㊀∗国家自然科学基金（２１２０７０５９，２１１７１０８５）；山东省自然科学基金（ＺＲ２０１０ＢＭ０２７，ＺＲ２０１１ＢＱ０１２）资助．㊀∗∗通讯联系人，Ｔｅｌ：１３８６４５９３６９８；Ｅ⁃ｍａｉｌ：ｌｄｕｚｓｘ＠１２６．ｃｏｍＤＯＩ：１０．７５２４／ｊ．ｉｓｓｎ．０２５４⁃６１０８．２０１３．１２．００３碳包覆的磁性纳米材料萃取酞酸酯∗王颖辉１㊀腾㊀飞２㊀张媛媛３㊀张升晓３∗∗㊀刘军深３㊀吴㊀茜３（１．烟台开发区城市管理环保局，烟台，２６４００６；㊀２．上海通用东岳汽车有限公司，烟台，２６４００６；３．鲁东大学，烟台，２６４０２５）摘㊀要㊀利用水热反应制备碳材料包覆的Ｆｅ３Ｏ４（Ｆｅ３Ｏ４／Ｃ）纳米颗粒，并将这种材料用作固相萃取吸附剂，从环境水样中富集酞酸酯类（ＰＡＥｓ）污染物．由于纳米材料大的比表面积和碳材料强的吸附能力，Ｆｅ３Ｏ４／Ｃ吸附剂拥有较高的萃取效率．从５００ｍＬ的环境水样中定量萃取ＰＡＥｓ目标物，只需５０ｍｇ的吸附剂．ＰＡＥｓ在Ｆｅ３Ｏ４／Ｃ表面的吸附可快速达到平衡，并且用少量的乙腈就能将分析物洗脱下来．在优化的固相萃取条件下，几种ＰＡＥｓ的检出限在１７５８ｎｇ㊃Ｌ－１之间．通过水样的加标回收率来评价该方法，其加标回收率在９４．６％１０６．６％之间，相对标准偏差为２％８％．关键词㊀酞酸酯，磁性固相萃取，碳材料．酞酸酯类化合物（ＰＡＥｓ）作为增塑剂用于塑料制品的生产，来提高塑料强度和增大可塑性．这类物质会从塑料制品中迁移转化进入环境，从而对环境造成污染．ＰＡＥｓ具有三致（致突㊁致畸㊁致癌）作用，属于环境内分泌干扰物，其中有６种化合物已被美国国家环保局列为优先监控污染物［１］．目前测定水体ＰＡＥｓ的前处理方法主要有液液萃取［２］㊁固相萃取［３］㊁固相微萃取［４］等方法．李玫瑰等［５］以甲苯为萃取溶剂，采用微滴液相微萃取方法快速分析水溶性食品中的ＰＡＥｓ．王东新［６］以中空纤维膜液相微萃取方法从天然水体中萃取了４种ＰＡＥｓ，并结合气相色谱进行分析．胡庆兰［７］采用自制的固相微萃取涂层，通过顶空固相微萃取与气相色谱法联用测定水中的ＰＡＥＳ．固相萃取法以有机溶剂消耗少㊁操作简便等优点被广泛采用．王鑫等［８］采用Ｃ１８小柱萃取饮用水和自来水中的４种ＰＡＥｓ类污染物，并用气相色谱法测定．陈永山等［９］研究了流速和除水方式等条件对Ｃ１８固相萃取柱富集ＰＡＥｓ回收率的影响，他们还用罗里硅土净化土壤提取液，测定了土壤中的１１种ＰＡＥｓ类污染物［１０］．纳米材料拥有大的比表面积和短的扩散路径，使其具有很高的萃取效率和较快的吸附速度，近年来被开发研究用作固相萃取吸附剂，从环境或生物样品中富集污染物［１１⁃１２］．然而纳米材料填充的固相萃取小柱具有很大的反压，样品通过小柱需要消耗大量的时间．因此，磁性纳米材料用作固相萃取吸附剂的研究应运而生［１３⁃１５］，张小乐等［１６］合成了Ｃ１８基团修饰的磁性介孔硅胶材料，并利用该材料建立了磁性固相萃取⁃色谱分析方法，测定了环境水样中ＰＡＥｓ污染物的含量．我们以前的研究［１７⁃１９］表明，在基于磁性纳米颗粒的固相萃取方法中，可将经过修饰的磁性纳米材料分散到样品中进行目标物吸附，达到吸附平衡后，利用磁铁快速将吸附了目标物的磁性材料分离出来，然后将目标物进行洗脱㊁测定，该快速磁分离方法避免了传统固相萃取中耗时的过柱操作，能够节省大量时间．本文制备了一种新型的磁性纳米材料（Ｆｅ３Ｏ４／Ｃ），并以该纳米材料为固相萃取吸附剂从环境水样中富集ＰＡＥｓ污染物．优化了吸附剂用量㊁平衡时间㊁盐度㊁ｐＨ㊁解吸附等固相萃取条件，并将这种新型的固相萃取技术与高效液相色谱（ＨＰＬＣ）结合，开发了一种分析环境水体中ＰＡＥｓ的新方法．１㊀实验部分１．１㊀化学试剂和材料㊀㊀酞酸丙酯（ＤＰＰ）㊁酞酸丁酯（ＤＢＰ）㊁酞酸环己酯（ＤＣＰ）和酞酸辛酯（ＤＯＰ）标准样品从美国Ａｃｒｏｓ２２４４㊀环㊀㊀境㊀㊀化㊀㊀学３２卷Ｏｒｇａｎｉｃｓ公司购得．十八烷基三乙氧基硅烷购买自日本东京化学试剂公司．ＦｅＣｌ３㊃４Ｈ２Ｏ和ＦｅＣｌ２㊃６Ｈ２Ｏ从北京化学试剂公司购买．葡萄糖购买自北京红星化学公司．色谱纯的甲醇和乙腈购自美国的ＴｈｅｒｍｏＦｉｓｈｅｒ公司．实验用的纯水来自于美国Ｍｉｌｌｉ⁃Ｑ纯水系统．１．２㊀Ｆｅ３Ｏ４和Ｆｅ３Ｏ４／Ｃ纳米材料的制备和表征采用化学共沉淀的方法制备Ｆｅ３Ｏ４磁性纳米材料．首先将２．０ｇＦｅＣｌ２㊃４Ｈ２Ｏ和５．２ｇＦｅＣｌ３㊃６Ｈ２Ｏ溶解到２５ｍＬ的预先脱氧的去离子水，再加入０．８５ｍＬ浓盐酸．将以上溶液逐滴加到２５０ｍＬ１．５ｍｏｌ㊃Ｌ－１的ＮａＯＨ溶液，边加边机械搅拌并通氮气保护．反应完成后，生成的黑色Ｆｅ３Ｏ４纳米颗粒用２００ｍＬ去离子水洗２次，再分散到１１０ｍＬ的去离子水中，悬浮液中Ｆｅ３Ｏ４纳米颗粒的浓度约为２０ｍｇ㊃ｍＬ－１．采用水热法在纳米Ｆｅ３Ｏ４的表面包覆一层碳材料．将０．４ｇＦｅ３Ｏ４用去离子水冲洗，直至溶液为中性，然后分散到８０ｍＬ０．５ｍｏｌ㊃Ｌ－１的葡萄糖溶液中，将此混合物超声２０ｍｉｎ后转移到聚四氟乙烯内衬的反应釜中．将反应釜在１８０ħ加热４ｈ，反应完成后，用磁铁将Ｆｅ３Ｏ４／Ｃ纳米材料分离，再用去离子水和乙醇冲洗几次除去杂质．最后将反应产物分散到７０ｍＬ的去离子水中得到１０ｍｇ㊃ｍＬ－１的Ｆｅ３Ｏ４／Ｃ．利用透射电镜（ＴＥＭ，Ｈ⁃７５００，Ｈｉｔａｃｈｉ，Ｊａｐａｎ）观察制得的吸附材料的形貌和粒径，工作电压为８０ｋＶ．材料的能谱图（ＥＤＳ）采用Ｓ⁃３０００Ｎ能谱仪（Ｈｉｔａｃｈｉ，Ｊａｐａｎ）测定．材料的红外谱图采用ＫＢｒ压片的方式在ＮＥＸＵＳ６７０傅立叶变换红外光谱仪（ＮｉｃｏｌｅｔＴｈｅｒｍｏ，Ｕ．Ｓ．）采集．１．３㊀固相萃取的程序将５０ｍｇ的Ｆｅ３Ｏ４／Ｃ吸附剂分散到５００ｍＬ水样中，搅拌２０ｓ使吸附剂在溶液中均匀分散．将１个Ｎｄ⁃Ｆｅ⁃Ｂ强力磁铁置于容器底部进行磁分离．经过大约１０ｍｉｎ，溶液变清澈，吸附剂被完全分离到容器底部，将上清液倒掉．在吸附剂中加入２ｍＬ乙腈，超声１０ｓ后置于磁铁上分离，洗脱液转移到离心管中，取２０μＬ进ＨＰＬＣ系统测定．由于塑料制品可能会释放出ＰＡＥｓ，因此在实验过程中使用玻璃容器．１．４㊀ＨＰＬＣ分析利用美国安捷伦公司的高效液相色谱系统对ＰＡＥｓ进行分离和分析，迪马Ｃ１８色谱柱（２５０ˑ４．６ｍｍ，粒径５μｍ），采用梯度淋洗的方法分离，５０％的乙腈水溶液和纯乙腈分别为流动相Ａ和Ｂ，流速为１ｍＬ㊃ｍｉｎ－１．梯度淋洗程序如下：前１５ｍｉｎ内Ｂ保持在４０％，在１０ｍｉｎ内Ｂ由４０％升至１００％，保持１０ｍｉｎ，随后在３ｍｉｎ内回到初始状态．ＰＡＥｓ采用紫外检测器测定，波长设在２２６ｎｍ．２㊀结果与讨论２．１㊀吸附剂的表征图１为Ｆｅ３Ｏ４和Ｆｅ３Ｏ４／Ｃ的透射电镜图片．Ｆｅ３Ｏ４纳米颗粒为近似球形，粒径分布比较均匀，平均直径大约为１０ｎｍ．Ｆｅ３Ｏ４／Ｃ纳米颗粒呈现出明显的核壳结构，内层颜色较深的为Ｆｅ３Ｏ４核，而外层颜色相对较浅的部分是碳层．Ｆｅ３Ｏ４核使得材料具有磁性，而碳包覆层赋予材料强的吸附能力．图１㊀Ｆｅ３Ｏ４（ａ）和Ｆｅ３Ｏ４／Ｃ（ｂ）纳米材料的透射电镜图（放大倍数：２０００００）Ｆｉｇ．１㊀ＴＥＭｉｍａｇｅｓｏｆＦｅ３Ｏ４（ａ），ａｎｄＦｅ３Ｏ４／Ｃ（ｂ）ｎａｎｏｐａｒｔｉｃｌｅ㊀１２期王颖辉等：碳包覆的磁性纳米材料萃取酞酸酯２２４５㊀Ｆｅ３Ｏ４和Ｆｅ３Ｏ４／Ｃ的能谱图见图２．在Ｆｅ３Ｏ４纳米颗粒的能谱图中只有一个很小的碳峰，在其表面包覆一层碳材料后，Ｆｅ３Ｏ４／Ｃ的能谱图中出现了一个较大的碳峰，其表面碳元素的摩尔百分含量达到５４４％，而铁元素的摩尔百分含量降低到１３．８％，说明碳材料被成功的包覆到了Ｆｅ３Ｏ４纳米颗粒表面．图２㊀Ｆｅ３Ｏ４（ａ）和Ｆｅ３Ｏ４／Ｃ（ｂ）纳米材料的能谱图Ｆｉｇ．２㊀ＥＤＳｓｐｅｃｔｒａｏｆＦｅ３Ｏ４（ａ）ａｎｄＦｅ３Ｏ４／Ｃ（ｂ）ｎａｎｏｐａｒｔｉｃｌｅ利用傅立叶变换红外光谱分析Ｆｅ３Ｏ４和Ｆｅ３Ｏ４／Ｃ材料的表面基团．由图３可见，Ｆｅ３Ｏ４和Ｆｅ３Ｏ４／Ｃ的红外谱图上都有１个５８０ｃｍ－１的吸收峰，为Ｆｅ３Ｏ４的特征吸收峰［２０］．在Ｆｅ３Ｏ４／Ｃ的谱图上１７００ｃｍ－１和１６１６ｃｍ－１的峰是ＣＯ和ＣＣ的振动吸收峰［２１］，这表明在水热反应的过程中，葡萄糖碳化包覆到了纳米Ｆｅ３Ｏ４的表面．１０００１３００ｃｍ－１的峰是ＣＯＨ的伸缩振动峰和ＯＨ弯曲振动峰［２２］，３１００３７００ｃｍ－１之间的宽峰是ＯＨ伸缩振动峰［２３］．Ｆｅ３Ｏ４／Ｃ纳米材料的表面上羟基和羧基基团的存在使得该材料具有表面亲水性，能够在水溶液中分散和稳定存在，并且材料的表面亲水性降低了分析物在其表面的不可逆吸附，能够在一定程度上克服碳材料用作固相萃取洗脱困难的缺点．２．２㊀萃取条件的优化２．２．１㊀吸附剂用量的选择为了获得最优的目标物回收率，在０１００ｍｇ的范围改变Ｆｅ３Ｏ４／Ｃ吸附材料的加入量进行萃取，其结果列于图４．随Ｆｅ３Ｏ４／Ｃ吸附剂加入量的增加，ＰＡＥｓ的回收率也逐渐增加，当吸附剂用量为４０ｍｇ时，ＰＡＥｓ的回收率达到最大值，吸附剂的用量继续增加，回收率基本保持平衡，不会再有明显的增加．使用没有修饰的Ｆｅ３Ｏ４作为固相萃取的吸附剂，即使其用量达到１００ｍｇ，对ＰＡＥｓ的回收率也不会超过１０％．图３㊀Ｆｅ３Ｏ４和Ｆｅ３Ｏ４／Ｃ纳米材料的红外谱图Ｆｉｇ．３㊀ＩＲｓｐｅｃｔｒａｏｆＦｅ３Ｏ４ａｎｄＦｅ３Ｏ４／Ｃｎａｎｏｐａｒｔｉｃｌｅ图４㊀吸附剂用量对ＰＡＥｓ回收率的影响Ｆｉｇ．４㊀ＥｆｆｅｃｔｏｆｔｈｅａｍｏｕｎｔｏｆＦｅ３Ｏ４／ＣｓｏｒｂｅｎｔｏｎｔｈｅｅｘｔｒａｃｔｉｏｎｅｆｆｉｃｉｅｎｃｙｏｆＰＡＥｓ㊀㊀结果表明，能够有效吸附ＰＡＥｓ的是Ｆｅ３Ｏ４／Ｃ材料表面的碳层．碳纳米管和有机化合物之间的吸附主要通过疏水作用，π⁃π键㊁氢键作用和静电作用［２４］，Ｆｅ３Ｏ４／Ｃ纳米颗粒表面也是碳材料，因此其相互２２４６㊀环㊀㊀境㊀㊀化㊀㊀学３２卷作用机理相同．要达到满意的回收率只需要Ｆｅ３Ｏ４／Ｃ吸附剂４０ｍｇ，可能是因为Ｆｅ３Ｏ４／Ｃ材料拥有大的比表面积和碳材料的强吸附能力．为保证分析物的完全吸附，在水样中加入５０ｍｇ的Ｆｅ３Ｏ４／Ｃ纳米吸附剂．２．２．２㊀乙腈用量和平衡时间的选择选用乙腈从Ｆｅ３Ｏ４／Ｃ吸附剂上解吸ＰＡＥｓ．为了能够将ＰＡＥｓ充分解吸附，将Ｆｅ３Ｏ４／Ｃ吸附剂在洗脱液中超声２０ｓ再分离．由图５（ａ）可以看出，只需１ｍＬ乙腈就可将８０％以上的目标物解吸下来，增加乙腈的用量，ＰＡＥｓ的回收率略有增加，但影响不大，本实验选用２ｍＬ乙腈作为洗脱剂．传统的碳材料用作固相萃取吸附剂时，常常面临洗脱困难的问题［２５］．而本研究中Ｆｅ３Ｏ４／Ｃ材料较易洗脱，一方面可能是因为包覆在纳米材料上的薄层碳使得分析物的扩散路径较短，便于洗脱，另一方面可能是因为包覆上的碳材料表面是亲水性，有效避免了分析物在其上发生不可逆吸附．Ｆｅ３Ｏ４／Ｃ是超顺磁性并且拥有大的饱和磁强度，但由于其在水溶液中分散良好且表面羧基使颗粒之间存在静电斥力，因此从纯水中用磁铁分离需要较长时间．为了帮助磁分离，在水溶液中加入２ｍＬ１ｍｏｌ㊃Ｌ－１的ＮａＣｌ溶液提供补偿离子．用一个Ｎｄ⁃Ｆｅ⁃Ｂ强力磁铁能在１０ｍｉｎ将吸附剂完全吸附到容器的底部．通常要达到完全吸附目标物和吸附剂要有充分的接触时间．改变平衡时间考察目标物回收率的变化，由图５（ｂ）可以看出，随着平衡时间由０到１２０ｍｉｎ，ＰＡＥｓ的回收率没有明显变化，说明分析物能在很短的时间内完全吸附到Ｆｅ３Ｏ４／Ｃ材料上．这种快的吸附速率应归功于纳米材料短的扩散路径．磁性分离和快速的吸附，使这种新型固相萃取方法能够避免耗时的过柱操作，具有可操作性和实用性．图５㊀洗脱液乙腈用量（ａ）和平衡时间（ｂ）对ＰＡＥｓ回收率的影响Ｆｉｇ．５㊀Ｅｆｆｅｃｔｏｆａｃｅｔｏｎｉｔｒｉｌｅｖｏｌｕｍｅ（ａ）ａｎｄｓｔａｎｄｉｎｇｔｉｍｅ（ｂ）ｏｎｔｈｅｒｅｃｏｖｅｒｙｏｆＰＡＥｓ２．２．３㊀盐度和ｐＨ的选择为了考察盐度对ＰＡＨｓ回收率的影响，在溶液中加入ＮａＣｌ调整盐度．由图６（ａ）可以看出，在盐度为１５００ｍｍｏｌ㊃Ｌ－１的范围内，目标物的回收率基本不受影响，表明Ｆｅ３Ｏ４／Ｃ能从高盐度水样中萃取目标物而不受影响．吸附剂表面电荷的类型和密度通常会随溶液ｐＨ的改变而变化，因此溶液的ｐＨ可能会影响一些分析物在Ｆｅ３Ｏ４／Ｃ纳米材料上的吸附．改变溶液的ｐＨ值在３１０范围内考察其对ＰＡＨｓ萃取回收率的影响．由图６（ｂ）可以看出，在设定的ｐＨ范围内ＰＡＥｓ的回收率没有明显变化，说明Ｆｅ３Ｏ４／Ｃ纳米材料在此ｐＨ范围内比较稳定，不会被破坏，另一方面可能是因为ＰＡＥｓ在设定的条件以中性分子形式存在，材料表面电荷的改变对它们的吸附没有明显的影响．基于Ｆｅ３Ｏ４／Ｃ的磁性固相萃取方法无需严格控制溶液ｐＨ，用于环境水样品的分析更加便利和稳定．２．２．４㊀水样体积选择利用Ｆｅ３Ｏ４／Ｃ作吸附剂进行固相萃取，可以将材料分散在溶液中进行吸附，达到吸附平衡后用磁铁将吸附剂分离出来进行洗脱，该方法免去了耗时的过柱操作，因此非常适用于大体积环境水样的分析．如图７所示，利用５０ｍｇＦｅ３Ｏ４／Ｃ吸附剂，当水样的体积由２００ｍＬ增加到１０００ｍＬ时，ＰＡＨｓ的回收率没有明显下降，说明Ｆｅ３Ｏ４／Ｃ吸附材料具有大的穿透体积．通过从１０００ｍＬ水样中萃取目标物并将洗脱液浓缩到２ｍＬ，ＰＡＥｓ的富集系数可以达到５００倍．㊀㊀１２期王颖辉等：碳包覆的磁性纳米材料萃取酞酸酯２２４７图６㊀盐度（ａ）和溶液ｐＨ值（ｂ）对ＰＡＥｓ回收率的影响Ｆｉｇ．６㊀Ｅｆｆｅｃｔｏｆｓａｌｉｎｉｔｙ（ａ）ａｎｄｓｏｌｕｔｉｏｎｐＨ（ｂ）ｏｎｔｈｅｅｘｔｒａｃｔｉｏｎｅｆｆｉｃｉｅｎｃｙｏｆＰＡＥｓ图７㊀水样体积对ＰＡＥｓ回收率的影响Ｆｉｇ．７㊀ＥｆｆｅｃｔｏｆｗａｔｅｒｓａｍｐｌｅｖｏｌｕｍｅｏｎｔｈｅｒｅｃｏｖｅｙｏｆＰＡＥｓ２．３㊀分析方法相关参数固相萃取程序结合ＨＰＬＣ⁃ＵＶ检测建立了标准曲线，其线性范围为０．１２０ｎｇ㊃ｍＬ－１．从５００ｍＬ纯水样品中萃取目标物并用２ｍＬ溶剂洗脱，ＰＡＥｓ的富集系数为２５０倍．线性范围㊁线性相关系数和检出限等参数列于表１．由表１可以看出，该方法有较高的灵敏度㊁宽的线性范围和良好的精密度，ＰＡＥｓ的线性相关系数Ｒ２＞０．９９９．ＤＰＰ㊁ＤＢＰ㊁ＤＣＰ㊁ＤＯＰ的检出限（信噪比为３）分别为３５㊁５８㊁１７㊁３３ｎｇ㊃Ｌ－１．表１㊀标准曲线的方法参数Ｔａｂｌｅ１㊀Ａｎａｌｙｔｉｃａｌｐａｒａｍｅｔｅｒｓｏｆｔｈｅｐｒｏｐｏｓｅｄｍｅｔｈｏｄ分析物线性范围／（ｎｇ㊃ｍＬ－１）曲线方程线性相关系数（Ｒ２）方法检出限ａ／（ｎｇ㊃Ｌ－１）ＤＰＰ０．１２０ｙ＝０．５５７８ｘ＋０．０５６１０．９９９８３５ＤＢＰ０．１２０ｙ＝０．５４７６ｘ＋０．０６３４０．９９９７５８ＤＣＰ０．１２０ｙ＝０．９８６５ｘ－０．０３２７０．９９９７１７ＤＯＰ０．１２０ｙ＝０．５９４８ｘ＋０．１０３６０．９９９９３３㊀㊀ａ检测限根据信噪比为３确定（Ｓ／Ｎ＝３），ｘ为分析物浓度，ｙ为紫外信号强度．２．４㊀实际水样的测定实际水样选取实验室自来水和校园人工湖的湖水，采用所建立的磁性固相萃取方法测定水样中和加标后的水样中的ＰＡＥｓ，３次平行测定水样和加标水样结果平均值㊁加标水样的回收率平均值及标准偏差列于表２．自来水样中ＤＰＰ浓度为０．１０４ｎｇ㊃ｍＬ－１，其他未检出；湖水中ＤＰＰ和ＤＢＰ浓度分别为０２１５和０．１３２ｎｇ㊃ｍＬ－１，其余未检出．水样中低浓度的ＰＡＥｓ可能来自于塑料管路或者是塑料制品的释放．加标水样中ＰＡＥｓ的平均回收率在９４．６％１０６．６％之间，相对标准偏差为２％８％．图８为湖水样品及其加标色谱图，其峰型良好，基质相对比较干净，没有太多杂峰的干扰．２２４８㊀环㊀㊀境㊀㊀化㊀㊀学３２卷表２㊀实际水样中ＰＡＥｓ的浓度及其加标回收结果Ｔａｂｌｅ２㊀ＣｏｎｃｅｎｔｒａｔｉｏｎｓｏｆＰＡＥｓｉｎｒｅａｌｗａｔｅｒｓａｍｐｌｅｓａｎｄｒｅｃｏｖｅｒｉｅｓｏｆｓａｍｐｌｅｓｓｐｉｋｅｄｗｉｔｈａｎａｌｙｔｅｓ水样加标／（ｎｇ㊃ｍＬ－１）测定值ａ／（ｎｇ㊃ｍＬ－１）ＤＰＰＤＢＰＤＣＰＤＯＰ回收率ʃ相对标准偏差／％ｂＤＰＰＤＢＰＤＣＰＤＯＰ０．０００．１０４ｎｄｃｎｄｎｄ自来水０．５０．６１２０．５０４０．５２４０．５３３１０１．６ʃ３１０１．０ʃ２１０４．８ʃ６１０６．６ʃ８５５．１５５．１０４．８６４．７９１００．９ʃ２１０２．０ʃ４９７．２ʃ４９５．８ʃ７０．０００．２１５０．１３２ｎｄｎｄ湖水０．５０．７４５０．６５３０．５２３０．４９２１０６．０ʃ７１０４．２ʃ６１０４．６ʃ７９８．４ʃ４５４．９５４．９６４．７３４．８４９４．７ʃ６９６．６ʃ５９４．６ʃ７９６．８ʃ６㊀㊀注：ａ３次平行测定结果．ｂ３次测定相对标准偏差．ｃｎｄ未检出．图８㊀湖水样品中ＰＡＥｓ的色谱图．湖水样品（ａ），湖水加标０．５ｎｇ㊃ｍＬ－１（ｂ）和５ｎｇ㊃ｍＬ－１（ｃ）Ｆｉｇ．８㊀ＨＰＬＣ⁃ＵＶｃｈｒｏｍａｔｏｇｒａｍｓｏｆｌａｋｅｗａｔｅｒ（ａ），ａｎｄｉｔｓｓｐｉｋｅｄｓａｍｐｌｅｓｗｉｔｈ０．５ｎｇ㊃ｍＬ－１（ｂ）ａｎｄ５ｎｇ㊃ｍＬ－１（ｃ）ｏｆｅａｃｈａｎａｌｙｔｅ３㊀结论制备了超顺磁性的Ｆｅ３Ｏ４／Ｃ吸附剂，并用其从大体积环境水样中富集痕量的ＰＡＥｓ污染物．与传统的和文献报道的固相萃取方法比较，该方法具有如下优点：（ａ）超顺磁性吸附剂可以直接分散到水样中吸附目标物，达到吸附平衡后用磁铁分离洗脱，磁分离方法避免了耗时的过柱操作．（ｂ）Ｆｅ３Ｏ４／Ｃ具有纳米材料大的比表面积和碳材料强的吸附能力，因此从大体积水样中萃取目标物只需要少量吸附剂就能获得满意的结果．（ｃ）目标物的回收率不受溶液盐度和ｐＨ值的影响，因此在萃取时不需要对水样进行繁琐的调整．（ｄ）Ｆｅ３Ｏ４／Ｃ吸附剂的制备方法简单，所用试剂价格低廉，无毒．在进行萃取的过程中不会在环境水样中引入有毒有害的物质，环境友好．参㊀考㊀文㊀献［１］㊀王红芬，程晗煜，洪坚平．环境中酞酸酯的污染现状及防治措施［Ｊ］．环境科学与管理，２０１０，３５（７）：３３⁃３６［２］㊀ＣａｉＹＱ，ＣａｉＹＥ，ＳｈｉＹＬ，ｅｔａｌ．Ａｌｉｑｕｉｄ⁃ｌｉｑｕｉｄｅｘｔｒａｃｔｉｏｎｔｅｃｈｎｉｑｕｅｆｏｒｐｈｔｈａｌａｔｅｅｓｔｅｒｓｗｉｔｈｗａｔｅｒ⁃ｓｏｌｕｂｌｅｏｒｇａｎｉｃｓｏｌｖｅｎｔｓｂｙａｄｄｉｎｇｉｎｏｒｇａｎｉｃｓａｌｔｓ［Ｊ］．ＭｉｃｒｏｃｈｉｍｉｃａＡｃｔａ，２００７，１５７：７３⁃７９［３］㊀王超英，李碧芳，李攻科．固相微萃取／高效液相色谱联用分析水中邻苯二甲酸酯［Ｊ］．分析测试学报，２００５，２４（５）：３５⁃３８［４］㊀ＰｅñａｌｖｅｒＡ，ＰｏｃｕｒｌｌＥ，ＢｏｒｒｕｌｌＦ，ｅｔａｌ．Ｄｅｔｅｒｍｉｎａｔｉｏｎｏｆｐｈｔｈａｌａｔｅｅｓｔｅｒｓｉｎｗａｔｅｒｓａｍｐｌｅｓｂｙｓｏｌｉｄ⁃ｐｈａｓｅｍｉｃｒｏｅｘｔｒａｃｔｉｏｎａｎｄｇａｓｃｈｒｏｍａｔｏｇｒａｐｈｙｗｉｔｈｍａｓｓｓｐｅｃｔｒｏｍｅｔｒｉｃｄｅｔｅｃｔｉｏｎ［Ｊ］．ＪＣｈｒｏｍａｔｏｇｒＡ，２０００，８７２：１９１⁃２０１［５］㊀李玫瑰，李元星，毛丽秋．微滴液相微萃取技术用于气相色谱⁃质谱法测定食品中的酞酸酯［Ｊ］．色谱，２００７，２５（１）：３５⁃３８［６］㊀王东新．中空纤维液相微萃取⁃气相色谱测定不同天然水体中的酞酸酯类化合物［Ｊ］．南京师范大学学报（工程技术版），２００８，８（３）：４３⁃４６［７］㊀胡庆兰．自制固相微萃取涂层同时测定水中的多环芳烃和酞酸酯［Ｊ］．吉林大学学报（理学版），２０１３，５１（２）：３１７⁃３２０［８］㊀王鑫，许小苗，俞晔，等．固相萃取⁃气相色谱法同时测定水中的酞酸酯类环境激素［Ｊ］．食品工业科技，２００８，２９（４）：２８７⁃２８９［９］㊀陈永山，骆永明，章海波，等．固相萃取法处理环境水样中酞酸酯：流速与除水方式的影响［Ｊ］．环境化学，２０１０，２９（５）：９５４⁃９５９［１０］㊀黄玉娟，陈永山，骆永明，等．气相色谱⁃质谱联用内标法测定土壤中１１种酞酸酯［Ｊ］．环境化学，２０１３，３２（４）：６５８⁃６６５㊀㊀１２期王颖辉等：碳包覆的磁性纳米材料萃取酞酸酯２２４９［１１］㊀ＣａｉＹＱ，ＪｉａｎｇＧＢ，ＬｉｕＪＦ，ｅｔａｌ．Ｍｕｌｔｉｗａｌｌｅｄｃａｒｂｏｎｎａｎｏｔｕｂｅｓａｓａｓｏｌｉｄ⁃ｐｈａｓｅｅｘｔｒａｃｔｉｏｎａｄｓｏｒｂｅｎｔｆｏｒｔｈｅｄｅｔｅｒｍｉｎａｔｉｏｎｏｆｂｉｓｐｈｅｎｏｌＡ，４⁃ｎ⁃Ｎｏｎｙｌｐｈｅｎｏｌ，ａｎｄ４⁃ｔｅｒｔ⁃Ｏｃｔｙｌｐｈｅｎｏｌ［Ｊ］．ＡｎａｌＣｈｅｍ，２００３，７５：２５１７⁃２５２１［１２］㊀ＷａｎｇＨＹ，ＣａｍｐｉｇｌｉａＡＤ．Ｄｅｔｅｒｍｉｎａｔｉｏｎｏｆｐｏｌｙｃｙｃｌｉｃａｒｏｍａｔｉｃｈｙｄｒｏｃａｒｂｏｎｓｉｎｄｒｉｎｋｉｎｇｗａｔｅｒｓａｍｐｌｅｓｂｙｓｏｌｉｄ⁃ｐｈａｓｅｎａｎｏｅｘｔｒａｃｔｉｏｎａｎｄｈｉｇｈ⁃ｐｅｒｆｏｒｍａｎｃｅｌｉｑｕｉｄｃｈｒｏｍａｔｏｇｒａｐｈｙ［Ｊ］．ＡｎａｌＣｈｅｍ，２００８，８０：８２０２⁃８２０９［１３］㊀ＺｈａｏＸＬ，ＳｈｉＹＬ，ＣａｉＹＱ，ｅｔａｌ．Ｃｅｔｙｌｔｒｉｍｅｔｈｙｌａｍｍｏｎｉｕｍｂｒｏｍｉｄｅ⁃ｃｏａｔｅｄｍａｇｎｅｔｉｃｎａｎｏｐａｒｔｉｃｌｅｓｆｏｒｔｈｅｐｒｅｃｏｎｃｅｎｔｒａｔｉｏｎｏｆｐｈｅｎｏｌｉｃｃｏｍｐｏｕｎｄｓｆｒｏｍｅｎｖｉｒｏｎｍｅｎｔａｌｗａｔｅｒｓａｍｐｌｅｓ［Ｊ］．ＥｎｖｉｒｏｎＳｃｉＴｅｃｈｎｏｌ，２００８，４２：１２０１⁃１２０６［１４］㊀ＺｈａｏＸＬ，ＳｈｉＹＬ，ＷａｎｇＴ，ｅｔａｌ．Ｐｒｅｐａｒａｔｉｏｎｏｆｓｉｌｉｃａ⁃ｍａｇｎｅｔｉｔｅｎａｎｏｐａｒｔｉｃｌｅｍｉｘｅｄｈｅｍｉｍｉｃｅｌｌｅｓｏｒｂｅｎｔｓｆｏｒｅｘｔｒａｃｔｉｏｎｏｆｓｅｖｅｒａｌｔｙｐｉｃａｌｐｈｅｎｏｌｉｃｃｏｍｐｏｕｎｄｓｆｒｏｍｅｎｖｉｒｏｎｍｅｎｔａｌｗａｔｅｒｓａｍｐｌｅｓ［Ｊ］．ＪＣｈｒｏｍａｔｏｇｒＡ，２００８，１１８８：１４０⁃１４７［１５］㊀ＬｉＪＤ，ＺｈａｏＸＬ，ＳｈｉＹＬ，ｅｔａｌ．Ｍｉｘｅｄｈｅｍｉｍｉｃｅｌｌｅｓｓｏｌｉｄ⁃ｐｈａｓｅｅｘｔｒａｃｔｉｏｎｂａｓｅｄｏｎｃｅｔｙｌｔｒｉｍｅｔｈｙｌａｍｍｏｎｉｕｍｂｒｏｍｉｄｅ⁃ｃｏａｔｅｄｎａｎｏ⁃ｍａｇｎｅｔｓＦｅ３Ｏ４ｆｏｒｔｈｅｄｅｔｅｒｍｉｎａｔｉｏｎｏｆｃｈｌｏｒｏｐｈｅｎｏｌｓｉｎｅｎｖｉｒｏｎｍｅｎｔａｌｗａｔｅｒｓａｍｐｌｅｓｃｏｕｐｌｅｄｗｉｔｈｌｉｑｕｉｄｃｈｒｏｍａｔｏｇｒａｐｈｙ／ｓｐｅｃｔｒｏｐｈｏｔｏｍｅｔｒｙｄｅｔｅｃｔｉｏｎ［Ｊ］．ＪＣｈｒｏｍａｔｏｇｒＡ，２００８，１１８０：２４⁃３１［１６］㊀张小乐，王巍杰，张一江，等．磁性介孔硅胶萃取剂的制备及萃取性能研究［Ｊ］．环境化学，２０１２，３１（４）：４２２⁃４２８［１７］㊀ＺｈａｎｇＳＸ，ＮｉｕＨＹ，ＣａｉＹＱ，ｅｔａｌ．ＢａｒｉｕｍａｌｇｉｎａｔｅｃａｇｅｄＦｅ３Ｏ４＠Ｃ１８ｍａｇｎｅｔｉｃｎａｎｏｐａｒｔｉｃｌｅｓｆｏｒｔｈｅｐｒｅ⁃ｃｏｎｃｅｎｔｒａｔｉｏｎｏｆｐｏｌｙｃｙｃｌｉｃａｒｏｍａｔｉｃｈｙｄｒｏｃａｒｂｏｎｓａｎｄｐｈｔｈａｌａｔｅｅｓｔｅｒｓｆｒｏｍｅｎｖｉｒｏｎｍｅｎｔａｌｗａｔｅｒｓａｍｐｌｅｓ［Ｊ］．ＡｎａｌＣｈｉｍＡｃｔａ，２０１０，６６５：１６７⁃１７５［１８］㊀ＺｈａｎｇＳＸ，ＮｉｕＨＹ，ＨｕＺＪ，ｅｔａｌ．ＰｒｅｐａｒａｔｉｏｎｏｆｃａｒｂｏｎｃｏａｔｅｄＦｅ３Ｏ４ｎａｎｏｐａｒｔｉｃｌｅｓａｎｄｔｈｅｉｒａｐｐｌｉｃａｔｉｏｎｆｏｒｓｏｌｉｄ⁃ｐｈａｓｅｅｘｔｒａｃｔｉｏｎｏｆｐｏｌｙｃｙｃｌｉｃａｒｏｍａｔｉｃｈｙｄｒｏｃａｒｂｏｎｓｆｒｏｍｅｎｖｉｒｏｎｍｅｎｔａｌｗａｔｅｒｓａｍｐｌｅｓ［Ｊ］．ＪＣｈｒｏｍａｔｏｇｒＡ，２０１０，１２１７：４７５７⁃４７６４［１９］㊀ＺｈａｎｇＳＸ，ＮｉｕＨＹ，ＺｈａｎｇＹＹ，ｅｔａｌ．Ｂｉｏｃｏｍｐａｔｉｂｌｅｐｈｏｓｐｈａｔｉｄｙｌｃｈｏｌｉｎｅｂｉｌａｙｅｒｃｏａｔｅｄｏｎｍａｇｎｅｔｉｃｎａｎｏｐａｒｔｉｃｌｅｓａｎｄｔｈｅｉｒａｐｐｌｉｃａｔｉｏｎｉｎｔｈｅｅｘｔｒａｃｔｉｏｎｏｆｓｅｖｅｒａｌｐｏｌｙｃｙｃｌｉｃａｒｏｍａｔｉｃｈｙｄｒｏｃａｒｂｏｎｓｆｒｏｍｅｎｖｉｒｏｎｍｅｎｔａｌｗａｔｅｒａｎｄｍｉｌｋｓａｍｐｌｅｓ［Ｊ］．ＪＣｈｒｏｍａｔｏｇｒＡ，２０１２，１２３８：３８⁃４５［２０］㊀ＢｒｕｃｅＩＪ，ＳｅｎＴ，Ｓｕｒｆａｃｅｍｏｄｉｆｉｃａｔｉｏｎｏｆｍａｇｎｅｔｉｃｎａｎｏｐａｒｔｉｃｌｅｓｗｉｔｈａｌｋｏｘｙｓｉｌａｎｅｓａｎｄｔｈｅｉｒａｐｐｌｉｃａｔｉｏｎｉｎｍａｇｎｅｔｉｃｂｉｏｓｅｐａｒａｔｉｏｎｓ［Ｊ］．Ｌａｎｇｍｕｉｒ，２００５，２１：７０２９⁃７０３５［２１］㊀ＬｉＹ，ＬｅｎｇＴＨ，ＬｉｎＨＱ，ｅｔａｌ．ＰｒｅｐａｒａｔｉｏｎｏｆＦｅ３Ｏ４＠ＺｒＯ２ｃｏｒｅ⁃ｓｈｅｌｌｍｉｃｒｏｓｐｈｅｒｅｓａｓａｆｆｉｎｉｔｙｐｒｏｂｅｓｆｏｒｓｅｌｅｃｔｉｖｅｅｎｒｉｃｈｍｅｎｔａｎｄｄｉｒｅｃｔｄｅｔｅｒｍｉｎａｔｉｏｎｏｆｐｈｏｓｐｈｏｐｅｐｔｉｄｅｓｕｓｉｎｇｍａｔｒｉｘ⁃ａｓｓｉｓｔｅｄｌａｓｅｒｄｅｓｏｒｐｔｉｏｎｉｏｎｉｚａｔｉｏｎｍａｓｓｓｐｅｃｔｒｏｍｅｔｒｙ［Ｊ］．ＪＰｒｏｔｅｏｍｅＲｅｓ，２００７，６：４４９８⁃４５１０［２２］㊀ＬｉＹ，ＸｕＸＱ，ＱｉＤＷ，ｅｔａｌ．ＮｏｖｅｌＦｅ３Ｏ４＠ＴｉＯ２ｃｏｒｅ⁃ｓｈｅｌｌｍｉｃｒｏｓｐｈｅｒｅｓｆｏｒｓｅｌｅｃｔｉｖｅｅｎｒｉｃｈｍｅｎｔｏｆｐｈｏｓｐｈｏｐｅｐｔｉｄｅｓｉｎｐｈｏｓｐｈｏｐｒｏｔｅｏｍｅａｎａｌｙｓｉｓ［Ｊ］．ＪＰｒｏｔｅｏｍｅＲｅｓ，２００８，７：２５２６⁃２５３８［２３］㊀ＬｉｍＳＦ，ＺｈｅｎｇＹＭ，ＺｏｕＳＷ，ｅｔａｌ．ＣｈａｒａｃｔｅｒｉｚａｔｉｏｎｏｆｃｏｐｐｅｒａｄｓｏｒｐｔｉｏｎｏｎｔｏａｎａｌｇｉｎａｔｅｅｎｃａｐｓｕｌａｔｅｄｍａｇｎｅｔｉｃｓｏｒｂｅｎｔｂｙａｃｏｍｂｉｎｅｄＦＴ⁃ＩＲ，ＸＰＳ，ａｎｄｍａｔｈｅｍａｔｉｃａｌｍｏｄｅｌｉｎｇｓｔｕｄｙ［Ｊ］．ＥｎｖｉｒｏｎＳｃｉＴｅｃｈｎｏｌ，２００８，４２：２５５１⁃２５５６［２４］㊀ＰａｎＢ，ＸｉｎｇＢＳ．Ａｄｓｏｒｐｔｉｏｎｍｅｃｈａｎｉｓｍｓｏｆｏｒｇａｎｉｃｃｈｅｍｉｃａｌｓｏｎｃａｒｂｏｎｎａｎｏｔｕｂｅｓ［Ｊ］．ＥｎｖｉｒｏｎＳｃｉＴｅｃｈｎｏｌ，２００８．４２（２４）：９００５⁃９０１３［２５］㊀ＨｅｎｎｉｏｎＭＣ．Ｇｒａｐｈｉｔｉｚｅｄｃａｒｂｏｎｓｆｏｒｓｏｌｉｄ⁃ｐｈａｓｅｅｘｔｒａｃｔｉｏｎ［Ｊ］．ＪＣｈｒｏｍａｔｏｇｒＡ，２０００，８８５：７３⁃９５Ｃａｒｂｏｎｃｏａｔｅｄｍａｇｎｅｔｉｃｎａｎｏｐａｒｔｉｃｌｅｆｏｒｓｏｌｉｄ⁃ｐｈａｓｅｅｘｔｒａｃｔｉｏｎｏｆｐｈｔｈａｌａｔｅｅｓｔｅｒｓｆｒｏｍｗａｔｅｒｓａｍｐｌｅｓＷＡＮＧＹｉｎｇｈｕｉ１㊀㊀ＴＥＮＧＦｅｉ２㊀㊀ＺＨＡＮＧＹｕａｎｙｕａｎ３㊀㊀ＺＨＡＮＧＳｈｅｎｇｘｉａｏ３∗㊀㊀ＬＩＵＪｕｎｓｈｅｎ３㊀㊀ＷＵＱｉａｎ３（１．ＣｉｔｙＭａｎａｇｅｍｅｎｔＢｕｒｅａｕｏｆＥｎｖｉｒｏｎｍｅｎｔａｌＰｒｏｔｅｃｔｉｏｎｏｆＹａｎｔａｉＤｅｖｅｌｏｐｍｅｎｔＺｏｎｅ，Ｙａｎｔａｉ，２６４００６，Ｃｈｉｎａ；２．ＳｈａｎｇｈａｉＧｅｎｅｒａｌＭｏｔｏｒ（Ｄｏｎｇｙｕｅ）ＡｕｔｏＣｏｍｐａｎｙ，Ｙａｎｔａｉ，２６４００６，Ｃｈｉｎａ；㊀３．ＬｕｄｏｎｇＵｎｉｖｅｒｓｉｔｙ，Ｙａｎｔａｉ，２６４０２５，Ｃｈｉｎａ）ＡＢＳＴＲＡＣＴＣａｒｂｏｎｃｏａｔｅｄＦｅ３Ｏ４ｎａｎｏｐａｒｔｉｃｌｅｓ（Ｆｅ３Ｏ４／Ｃ）ｗｅｒｅｓｙｎｔｈｅｓｉｚｅｄｂｙａｓｉｍｐｌｅｈｙｄｒｏｔｈｅｒｍａｌｒｅａｃｔｉｏｎａｎｄａｐｐｌｉｅｄａｓｓｏｌｉｄ⁃ｐｈａｓｅｅｘｔｒａｃｔｉｏｎ（ＳＰＥ）ｓｏｒｂｅｎｔｓｔｏｅｘｔｒａｃｔｔｒａｃｅｐｈｔｈａｌａｔｅｅｓｔｅｒｓ（ＰＡＥｓ）ｆｒｏｍｅｎｖｉｒｏｎｍｅｎｔａｌｗａｔｅｒｓａｍｐｌｅｓ．ＴｈｅＦｅ３Ｏ４／Ｃｓｏｒｂｅｎｔｓｐｏｓｓｅｓｓｈｉｇｈｅｘｔｒａｃｔｉｏｎｅｆｆｉｃｉｅｎｃｙｄｕｅｔｏｓｔｒｏｎｇａｄｓｏｒｐｔｉｏｎａｂｉｌｉｔｙｏｆｃａｒｂｏｎｍａｔｅｒｉａｌｓａｎｄｌａｒｇｅｓｐｅｃｉｆｉｃｓｕｒｆａｃｅａｒｅａｏｆｔｈｅｎａｎｏｐａｒｔｉｃｌｅｓ．Ｏｎｌｙ５０ｍｇｏｆｓｏｒｂｅｎｔｓｗｅｒｅｎｅｅｄｅｄｔｏｅｘｔｒａｃｔＰＡＥｓｆｒｏｍ５００ｍＬｗａｔｅｒｓａｍｐｌｅｓ．Ｔｈｅａｄｓｏｒｐｔｉｏｎｒｅａｃｈｅｄｅｑｕｉｌｉｂｒｉｕｍｒａｐｉｄｌｙａｎｄｔｈｅａｎａｌｙｔｅｓｗｅｒｅｅｌｕｔｅｄｗｉｔｈａｃｅｔｏｎｉｔｒｉｌｅｒｅａｄｉｌｙ．ＳａｌｉｎｉｔｙａｎｄｓｏｌｕｔｉｏｎｐＨｈａｄｎｏｏｂｖｉｏｕｓｅｆｆｅｃｔｏｎｔｈｅｒｅｃｏｖｅｙｏｆＰＡＨｓ，ｗｈｉｃｈａｖｏｉｄｓｆｕｓｓｙａｄｊｕｓｔｍｅｎｔｔｏｗａｔｅｒｓａｍｐｌｅｂｅｆｏｒｅｅｘｔｒａｃｔｉｏｎ．Ｕｎｄｅｒｏｐｔｉｍｉｚｅｄｃｏｎｄｉｔｉｏｎｓ，ｔｈｅｄｅｔｅｃｔｉｏｎｌｉｍｉｔｏｆＰＡＥｓｗａｓｉｎｔｈｅｒａｎｇｅｏｆ１７５８ｎｇ㊃Ｌ－１．Ｔｈｅａｃｃｕｒａｃｙｏｆｔｈｅｍｅｔｈｏｄｗａｓｅｖａｌｕａｔｅｄｂｙｔｈｅｒｅｃｏｖｅｒｉｅｓｏｆｓｐｉｋｅｄｓａｍｐｌｅｓ．Ｇｏｏｄｒｅｃｏｖｅｒｉｅｓ（９４．６％１０６．６％）ｗｉｔｈｌｏｗｒｅｌａｔｉｖｅｓｔａｎｄａｒｄｄｅｖｉａｔｉｏｎｓｆｒｏｍ２％ｔｏ８％ｗｅｒｅａｃｈｉｅｖｅｄ．ＴｈｉｓｎｅｗＳＰＥｍｅｔｈｏｄｐｒｏｖｉｄｅｓｓｅｖｅｒａｌａｄｖａｎｔａｇｅｓ，ｓｕｃｈａｓｈｉｇｈｅｘｔｒａｃｔｉｏｎｅｆｆｉｃｉｅｎｃｙ，ｈｉｇｈｂｒｅａｋｔｈｒｏｕｇｈｖｏｌｕｍｅ，ｃｏｎｖｅｎｉｅｎｔｅｘｔｒａｃｔｉｏｎｐｒｏｃｅｄｕｒｅ，ａｎｄｓｈｏｒｔａｎａｌｙｓｉｓｔｉｍｅ．Ｋｅｙｗｏｒｄｓ：ＰＡＥｓ，ｍａｇｎｅｔｉｃｓｏｌｉｄ⁃ｐｈａｓｅｅｘｔｒａｃｔｉｏｎ，ｃａｒｂｏｎｍａｔｅｒｉａｌｓ．。

雪地茶甲醇提取物体外抑菌活性及其

任国媛，郭启新，王静，等. 雪地茶甲醇提取物体外抑菌活性及其稳定性研究[J]. 食品工业科技，2022，43（1）：147−154. doi:10.13386/j.issn1002-0306.2021050011REN Guoyuan, GUO Qixin, WANG Jing, et al. Antibacterial Activity and Stability of Methanol Extract from Thamnolia subuliformis in Vitro [J]. Science and Technology of Food Industry, 2022, 43(1): 147−154. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2021050011雪地茶甲醇提取物体外抑菌活性及其稳定性研究任国媛1，郭启新2，王　静1，丁海燕1,*（1.大理大学公共卫生学院, 大理大学预防医学研究所, 云南大理 671000；2.大理州质量技术监督综合检测中心, 云南大理 671000）摘　要：为初步探究雪地茶体外抑菌活性及其稳定性，采用平板打孔法测定雪地茶甲醇提取物对常见致病菌的体外抑菌活性，以金黄色葡萄球菌作为指示菌，测定不同温度、pH 、紫外照射时间等因素对抑菌稳定性的影响，使用石油醚、乙酸乙酯、正丁醇分级萃取甲醇提取物，确定其抑菌活性物质的极性，并测定其松萝酸含量。

结果表明雪地茶甲醇提取物对革兰氏阳性菌如金黄色葡萄球菌、枯草芽孢杆菌、斯氏李斯特菌、表面葡萄球菌、伊氏李斯特氏菌，革兰氏阴性菌如甲型副伤寒杆菌、乙型副伤寒杆菌以及致病真菌白色念珠菌等具有良好的抑菌效果。

其中，对金黄色葡萄球菌的最小抑菌浓度和最小杀菌浓度分别为0.625、10 mg/mL ；抑菌稳定性实验结果表明，温度对雪地茶甲醇提取物的抑菌活性无显著影响（P >0.05），100 ℃处理30 min 后仍具有较高的抑菌活性；紫外光照射40 min 及50 min 的处理组抑菌活性显著下降（P <0.05），但抑菌率仍保持在90%以上；pH 对雪地茶甲醇提取物的抑菌稳定性影响最大，当pH 接近生理中性时（pH=6.0），其抑菌活性与对照组（pH=4.83）无显著差异（P >0.05），表现出较高抑菌活性，当pH 较高（8.0、10.0）或较低（2.0、4.0）时，处理组的抑菌活性极显著下降（P <0.05），且随着pH 的升高或降低而降低；雪地茶甲醇提取物中松萝酸含量为0.8613%，乙酸乙酯萃取物中松萝酸含量为0.9379%。