PROGRAMME: EurpeanMasterinQualityinAnalyticalLaboratories(EMQAL)2008Ͳ2010 RESEARCHWORK: Manufacture,characterisationandanalyticalapplicationofnanomaterialsin sensordevices. Department:AnalyticalChemistry. Student:ChukwuemekaAjaeroSupervisor:JoséMaríaPalaciosSantander Signature:Signature:
TABLEOFCONTENTS LISTOFTABLES. ... 4 LISTOFFIGURES ... 5 ACKNOWLEDGEMENTS ... 7 ABSTRACT... 9 1. StateͲofͲtheart ... 10 1.1. Objectives... 10 1.2. Nanomaterials... 10 1.3. SolͲgelcompositeelectrodes ... 11 1.4. GeneralprocedureformanufacturingSonogel ... 12 1.5. FactorsaffectingthesolͲgelprocess... 14 1.6. Ultrasoundsynthesisofsonogel ... 14 1.7. Applicationofultrasoundinthesynthesisofgoldnanoparticles... 16 1.8. Comparisonbetweenultrasonicandthermalmethodsofsynthesisofgold nanoparticles... 18 1.9. Backgroundandcurrentstatus... 18 1.10. Voltammetrictechniques... 21 1.10.1. Cyclicvoltammetry... 23 1.10.2. Differentialpulsevoltammetry ... 25 1.11. UVͲvisiblespectroscopy ... 27 1.12. Electronmicroscopy:theoryandfundamentals... 28 1.12.1. ScanningElectronMicroscopy(SEM)... 30 1.12.2. ScanningElectronMicroscopy(SEM)... 30 1.12.3. SEMinstrumentation ... 31 1.12.4. XͲrayEnergyDispersiveSpectroscopy(EDS)... 32 1.12.5. TransmissionElectronMicroscopy(TEM)... 34 2. MaterialsandMethods... 36
Reagents... 37 Preparationofsolutions... 38 Instruments ... 39 2.2. Methods ... 41 Classicalsynthesisofgoldnanoparticles(SN1)... 41 Ultrasonicsynthesisofgoldnanoparticles(SN2)... 41 PreparationoftheSonogelͲCarbonelectrodes ... 41 ElectrochemicalpreparationofSonogelͲCarbonelectrodes... 43 Preparationofthegoldnanoparticlesmodifiedsensors... 43 3. Resultsanddiscussion ... 44 3.1. UVͲvisiblespectraofgoldnanoparticles:stabilitystudies. ... 44 3.2. Electrochemicalcharacterization... 46 3.2.1. Cyclicvoltammetry(CV). ... 46 3.2.2. Differentialpulsevoltammetry(DPV)... 55 3.3. Structuralcharacterization... 63 3.3.1. Opticalmicroscopy(OM). ... 64 3.3.2.Scanningelectronmicroscopy(SEM)andXͲrayenergydispersivespectroscopy (EDS). ... 65 3.3.3.Transmissionelectronmicroscopy(TEM). ... 82 3.4. RealSampleanalysis ... 83 4. Conclusions. ... 86 5. Futurework... 88 6. References... 89
LISTOFTABLES. Table3.1.:ElectrochemicaldataforPBS0.2M(pH=6.90),inthepresenceof K4Fe(CN)61.00mM ... 48 Table3.2.:Peakintensityvaluesfortheanodicandthecathodicscans correspondingtoaAuNPs(SN2Ͳ5ʅL)ͲmodifiedSonogelͲCarbon(SNGC)electrode andaAuNPs(SN1Ͳ5ʅL)ͲmodifiedSonogelͲCarbonelectrode ... 52 Table3.3.:Experimentalvaluesoftheobservedcapacity(Cobs)at100mV.sͲ1and thedoubleͲlayercapacity(Cdl)forthedifferentconfigurationsoftheSonogelͲ Carbonelectrodes... 53 Table3.4:ResultsoftheoptimizationoftheDPVparameterswhenapplyingthree factorsandthreelevelBoxͲBehnkenexperimentaldesign... 56 Table3.5:ResultsofthebestcalibrationcurvesobtainedforAAwhenusingthe differenttypesofSonogelͲCarbonelectrodesemployed. ... 59 Table3.6:Resultsobtainedforthereproducibilitystudiesbyusingthreedifferent SNGCelectrodesforeachoneoftheconfigurationstested. ... 60 Table3.7:Resultsobtainedfortherepeatabilitystudiesbyusingindependently threedifferentSNGCelectrodesforeachoneoftheconfigurationstested.The numberofcalibrationcurvesobtainedforeachelectrodewastwoatleast. ... 62 Table3.8:BestdetectionandquantificationlimitsobtainedfordifferentSNGC electrodesforeachoneoftheconfigurationstested... 63 Table3.9:ExperimentalresultsforthedeterminationofAAinapplejuiceforbabies byusingthestandardadditionmethod ... 84
LISTOFFIGURES
Figure1.1:Typicalexcitationsignalforcyclicvoltammetry... 23
Figure1.2.CyclicvoltammogramoftheredoxcoupleFe2+/Fe3+inH2SO41M. ... 24
Figure1.3.Excitationsignalindifferentialpulsevoltammetry... 26 Figure1.4.Schemeofascanningelectronmicroscope. ... 32 Figure1.5.ExampleofanEDXspectrum... 33 Figure1.6.Electronictransitionsandspectrallines... 34 Figure1.7.Schemeofatransmissionelectronmicroscope... 35 Figure2.1:SchemeofthestepsfollowedforthefabricationoftheSonogelͲCarbon compositeelectrodes. ... 42 Figure3.1.UVͲvisspectrarecordedforgoldnanoparticlesfromsynthesis1 ... 44 Figure3.2.UVͲvisspectrarecordedforgoldnanoparticlesfromsynthesis2 ... 45 Figure3.3:Cyclicvoltamogrammsinphosphatebuffersolution0.2M(pH6.9)at variousscanratesfortheAuNPs(SN2Ͳ3ʅL)ͲmodifiedSonogelͲCarbonelectrode... 47 Figure3.4:Plotsofanodic(a)andcathodic(b)peakcurrentsofK4Fe(CN)6vsʆ1/2 (squarerootofthescanrate)fromthecyclicvoltammogramsofFigure3.3for AuNPs(SN2Ͳ3ʅL)ͲmodifiedSonogelͲCarbonelectrode... 49 Figure3.5:Cyclicvoltamogrammsinphosphatebuffersolution0.2M(pH6.9)at differentscanrates(10,25,50,75and100mV.sͲ1),correspondingtoabare SonogelͲCarbonelectrode(red)andaAuNPs(SN1Ͳ3ʅL)ͲmodifiedSonogelͲCarbon electrode(black). ... 50 Figure3.6:Cyclicvoltamogrammsinphosphatebuffersolution0.2M(pH6.9)at differentscanrates(10,25,50,75and100mV.sͲ1),correspondingtoaAuNPs(SN1Ͳ3 ʅL)ͲmodifiedSonogelͲCarbonelectrode(red)andaAuNPs(SN1Ͳ5ʅL)Ͳmodified SonogelͲCarbon ... 51 Figure3.7:Cyclicvoltamogrammsinphosphatebuffersolution0.2M(pH6.9)at differentscanrates(10,25,50,75and100mV.sͲ1),correspondingtoaAuNPs(SN2Ͳ5 ʅL)ͲmodifiedSonogelͲCarbonelectrode(red)andaAuNPs(SN1Ͳ5ʅL)Ͳmodified SonogelͲCarbon ... 52 Figure3.8:Representationoftheaverage(absolute)valuesoftheanodicand cathodiccurrentdensitiesatdifferentscanratesversusthescanratevaluesinthe rangefrom10to200mV.sͲ1fortheAuNPs(SN2Ͳ5µL)ͲmodifiedSNGCelectrode. .. 54
Figure3.9.BoxͲBehnkendesignandsampledpoints... 55 Figure3.10:CalibrationcurveforAAusinganAuNPs(SN1Ͳ5ʅL)ͲmodifiedSNGC electrode... 58 Figure3.11:DPVvoltammogramscorrespondingtothecalibrationcurvereportedin Figure3.10forAA;theelectrodeusedwastheAuNPs(SN1Ͳ5ʅL)ͲmodifiedSNGC electrode... 59 Figure3.12:OpticalmicroscopyphotographsofSonogelͲCarbonelectrodesatthe magnification10x:A)AuNPs(SN1Ͳ5ʅL)ͲmodifiedSNGCelectrode;B)AuNPs(SN2Ͳ5 ʅL)ͲmodifiedSNGCelectrode;C)bareSNGCelectrode. ... 65 Figure3.13:SEMmicrographsandtheEDSspectracorrespondingtothebareSNGC electrode:A)usedandB)notused. ... 67 Figure3.14:SEMmicrographsandtheEDSspectracorrespondingtothe AuNPs(SN1Ͳ3ʅL)ͲmodifiedSNGCelectrode:A)usedandB)notused. ... 69 Figure3.15:SEMmicrographsandtheEDSspectracorrespondingtothe AuNPs(SN1Ͳ5ʅL)ͲmodifiedSNGCelectrode:A)usedandB)notused. ... 71 Figure3.16:SEMmicrographsandtheEDSspectracorrespondingtothe AuNPs(SN2Ͳ3ʅL)ͲmodifiedSNGCelectrode:A)usedandB)notused. ... 73 Figure3.17:SEMmicrographsandtheEDSspectracorrespondingtothe AuNPs(SN2Ͳ5ʅL)ͲmodifiedSNGCelectrode:A)usedandB)notused. ... 75 Figure3.18:SEMmicrographsandtheEDSspectracorrespondingtothebare SonogelͲCarbonelectrodepolished:polarisedA)andnotpolarisedB). ... 78 Figure3.19:SEMmicrographsandtheEDSspectracorrespondingtothebare SonogelͲCarbonelectrodepolished,polarised,andcoatedwith5ʅLofAuNPsfrom synthesis1:usedA)andnotusedB). ... 80 Figure3.20:SEMmicrographsandtheEDSspectracorrespondingtothebare SonogelͲCarbonelectrodepolished,polarised,andcoatedwith5ʅLofAuNPsfrom synthesis2:usedA)andnotusedB). ... 81 Figure3.21:TEMmicrographscorrespondingtothegoldnanoparticlessynthesized bytheultrasonicmethod(SN2)... 82 Figure3.22:Sizedistributionofthegoldnanoparticlessynthesizedbytheultrasonic method(SN2)... 83
ACKNOWLEDGEMENTS
IwouldliketoexpressmysinceregratitudetomyresearchsupervisorJoséMaría Palacios Santander, for his continuous support, motivation and outstanding scientific guidance and patience throughout this research work, supporting me in thisworkandgivingmehisopinionanddirectiveseverytimeIneededsuch.Iwould alsoliketothankLauraCubillanaAguileraandProf.IgnacioNaranjoRodríguezfor theirkindassistance.Finally,IalsothankProf.JoséLuisHidalgoHidalgodeCisneros aswell,responsibleoftheresearchgroupinwhichIhavecarriedoutmyresearch work.
Special thanks also go to the European Commission Education and Culture DG for supporting my graduate research and studies through the Erasmus Mundus studentship. I also want to thank the European Master in Quality in Analytical LaboratoriesProjectManagementTeam(PMT)fortheopportunitytoparticipatein thisErasmusMundusprogramme,theEMQALCoͲordinatorProf.IsabelCavacoand the CoͲordinator of EMQAL in University of Cadiz Prof. Miguel Palma Lovillo and those who taught me in the Erasmus Mundus programme for the knowledge and skillsgainedthroughthisprogramme.
IalsowanttothankmycolleaguesintheEMQAL2008programme,CharlesKosore, SamsonKilaza,Antenehforbeingtheregivingmesupport.Mentionalsoshouldbe made of Leo, Belachew, Fasil, Teeka, Kumar, Moosa, Jelena, Rufy, Liping, Yingxu, Leire, Joana, Marta, Anu, Rami, Christy, Sintayehu, Saif, Glauce and Phoebe for those enjoyable moments and for being such good friends and coursemates. Worthy of thanks are my friends Joaquín Crespo Rosa, María Franco, Roberto, Laura, Miguel, Sandra who at one time or the other were my Spanish language teachershelpingmetolearnthelanguagewhereIleftoffintheintensivelanguage courseandallwhohavesharedsomehoursinthelabwithme.Iwouldalsoliketo thanktheotherstaffsandpeopleintheDepartmentofAnalyticalChemistry,more thanoncehavegivenmetheirhelpandsupport.
I also want to thank my friends Ugochukwu and his Family in Denmark, Mrs.Rita Oze, Chike Nweze, Amara Egwim, Ndui Aikayo, Henrik, Olawumi Makinde, Ana Paula, Ana Laura, Roger, Ley, little Kamil, Yahil (yahaya), Sergio and others numeroustomention.
Iwanttothankmywonderfulparents,Chief(Sir)andLadyE.U.Ajaero,forgiving myeducationandlifeafacelift,andmysiblingsVera,Anita,Uche,Chidumemand Ikenna for being behind me all this time. I can not forget my uncles and their familiesnamelyDr.B.N.AjaeroKSMandChief(Sir).F.C.NwokoroKSJ,thanksfor yourcontributioninhelpingmetoattainthislevel.
I also want to thank those people I have known, for giving me all their support, share their opinions, help in the work we have done since the beginning of my programmerightfromUniversityofAlgarve,Faro,Portugalandtheentirestaffof the Department of Chemistry at the Federal University of Technology, Owerri, Nigeria, Prof.E.N.Ejike, and Dr.E.E.Oguzie for being there and sharing my life both personallyandprofessionally.WorthyofthanksalsoisMrs.AnnUdunwa.
Finally, I thank God for his divine mercy throughout my study period; my sincere gratitudegoestomywonderfulfriendWahzforhisguidance.
ABSTRACT
The main objective of this study consisted of developing a new type of amperometric sensors based on depositing gold nanoparticles obtained from two different synthetic routes (thermal and ultrasonic methods) on the surface of a SonogelͲCarbonmatrix,inordertobuildelectrochemicaldevices.
UVͲvisible spectroscopy was employed to study the time stability of the gold nanoparticles,withtheaimofcomparingbetweenthetwosyntheses.Asexpected fromtheoreticalconsiderationsbothtypesofgoldnanoparticlesshowedonepeak at 525 nm corresponding to the surface plasmon resonance effect typical of this kindofnanomaterials.
Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to investigate electrochemical behaviour of AuNPsͲmodified SonogelͲCarbon electrodesatdifferentconfigurationswiththeaimofcomparingtheirperformance. The AuNPsͲmodified SonogelͲCarbon electrodes showed significantly enhanced peakswithrespecttotheunmodifiedSonogelͲCarbonelectrodes.Furthermore,the peakcurrentsincreasedlinearlywiththesquarerootofthescanrateindicatingthat electrochemicalreactionswerecontrolledbydiffusionprocess.
Differential pulse voltammetry under optimized conditions showed good reproducibility and repeatability towards ascorbic acid. The detection and quantificationlimitswerealsodetermined.
The AuNPsͲmodified SonogelͲCarbon electrodes and unmodified SonogelͲCarbon electrodeswerestructurallycharacterizedwithOpticalMicroscopy(OM),Scanning Electron Microscopy (SEM) and XͲray Energy Dispersive Spectroscopy (EDS). Information about the size distribution and shape of the gold nanoparticles was obtainedthankstoTransmissionElectronMicroscopy(TEM)technique.
Thenewamperometricsensorsdevelopedthroughoutthisresearchworkwerealso usedforthedeterminationofascorbicacidinapplejuiceforbabies,givingregular results.
1. StateͲofͲtheart 1.1. Objectives
The main objective of this research work consists of learning to manufacture carbonͲbasedelectrochemicalsensordevices,modifiedwithmetalnanoparticlesin order to characterize the sensor devices structurally (on its surface) and electrochemically,usingdifferenttechniquesforeachpurpose.Theresearchstudy includes the first attempts for the application of the amperometric sensors developedthroughoutthisresearchworktorealsamples.
Thegoalstobeachievedwiththislineofresearchare:
Ͳ First of all, to compare two different syntheses of gold nanoparticles in terms of simplicity and fastness of synthesis, time stability and size of the nanoparticles.
Ͳ Secondly,toevaluatethepossibilityofmodifyingSonogelͲCarbonelectrodes withthesegoldnanoparticles.
Ͳ In third place, to characterize these new gold nanoparticlesͲmodified amperometricsensorsbothelectrochemicallyandstructurally.
Ͳ And finally, to test the performance of these electrodes at different configurations when determining certain analyte, namely ascorbic acid. Some tests have been done to determine this analyte in apple juices for babies.
1.2. Nanomaterials
NanomaterialsarematerialsstructuredonthesubͲhundrednanometerscale,which may be assembled and organised to yield nanodevices and nanosystems that possessneworimprovedproperties.
Because of their small size, nanomaterials have unique properties. Their physical, chemical, and mechanical properties are different from the properties of ordinary size materials. For example, they may have difference in structures, surface
compositions, shapes, and optical and electrical properties. Nanomaterials are exceptionally strong, hard, malleable at high temperatures, and chemically active. All physical and chemical properties are size dependent, and the properties of materials on the nanosize scale have important consequences in wide ranging fields.Exploitingnanoscalebehaviorwilleventuallyleadtodevelopmentofdevices that can selectively attack diseased cells, increase computer speed or improve chemical and biological sensors. The composition, size, shape and environment of nanosizedparticlesstronglyinfluencetheirultimateutility.[1,2].
1.3. SolͲgelcompositeelectrodes
Nowadays, the manufacture and application of graphite based solͲgel electrodes have attracted significant interest and importance in the bid to establish alternatives to other solid electrodes [3Ͳ7]. Meritorious features include high conductivity, relative chemical inertness, good mechanical properties, physical rigidity, renewable surface, amenable chemical or biological modification, and stabilityinvarioussolvents[8,9].Theyhaveparticularlyfavourableelectrochemical properties,asawideoperatingrangeofvoltagewindowandverylowloadcapacity observed(Cobs),comparedwithothercarbonelectrodes.Inaddition,theelectrodes respond directly to a broad group of organic compounds and various metal ions which, along with the properties mentioned, makes them very suitable for use as electrochemicalsensors.
Inaddition,theproceduredevelopedforthefabricationoftheelectrodesiseasy; bysimplyaddinggraphitetotheprecursorssolution,conductivityisconferredtoit and the mixture is readily malleable, so that any configuration can be virtually obtained. Furthermore, there is also the opportunity to tailor the structures by controlling the chemistry of the solͲgel process, thus yielding materials with very different properties [10]. The solͲgel process is a chemical synthesis technique. SeveralresearchworkshaveaccountedfortheapplicationofsolͲgeltechnique[11Ͳ 18].
1.4. GeneralprocedureformanufacturingSonogel.
The solͲgel process is a wetͲchemical technique starting from a chemical solution which acts as the precursor for an integrated network (or gel) of either discrete particles or network polymers. Typical precursors are metal alkoxides and metal chlorides, which undergo various forms of hydrolysis and polycondensation reactions. The formation of a metal oxide involves connecting the metal centers withoxo(MͲOͲM)orhydroxo(MͲOHͲM)bridges,thereforegeneratingmetalͲoxoor metalͲhydroxopolymersinsolution.Thus,thesolevolvestowardstheformationof a gelͲlike diphasic system containing both a liquid phase and solid phase whose morphologiesrangefromdiscreteparticlestocontinuouspolymernetworks.Most times,acidorbasecatalystsareused,whichacceleratestheonsetofhydrolysis,and toachievehomogeneousaqueousandorganicphases.
Theinitialhydrolysisofthealkoxide,followedbycondensationreactionsinlocalized regions, results in a suspension of colloidal particles called sol. The polycondensationoftheparticlesincreasestheinterͲconnectionbetweenthem,so that the viscosity increases rapidly and leads to the formation of a rigid network, calledwetpolymergel,consistingessentiallyofsiloxaneunits(SiO );inmostcases,2 unitsareconnectedbyoxygenbridges.
The early stages of hydrolysis and polymerization produces alcohol and water, respectively, which together with the excess of water that we added in the precursormixtureandhumidityoftheexternalenvironment,arehousedinsidethe networkthathasformed.Thereactionproductsarereleasedastheprocessmoves forward,thoughnotcompletelyfree.Evenafterthepointofgelation,inwhichthe entire volume of the solid is interconnected, the gel structure and properties continue to evolve as there is solvent in the pores and the gel remains wet. In addition, polycondensation reactions continue occurring, thereby extending the crossover.
Asaresult,dynamicway,thegelundergoesaprogressivespontaneouscontraction, resulting in the expulsion of pore fluid, and there is an increase in the internal
generated during the process are retained on the network during gelation. Therefore, a gel aging can be defined as a vitreous material with an internal aqueoussolidͲphase.
Oncethisgelgetaging,theyaresubjectedtoamildheattreatment(generallyless than 100oC), obtaining a dry gel, called xerogel which undergoes no further fluid lossandcanbeconsideredstableovertime.Ifinsteadofsubjectingthegeltothis smoothprocess,itisfollowedbythermaltreatmentathightemperatures,thereisa densificationofthematerial,leadingtoatotallycompactcrystal.
The solͲgel process can be classified in three main stages from chemical point of view:
1.Preparationofthesol:colloidaldispersionofparticlesinaliquid.
2. Gelification of the sol: this step results in a rigid network of interͲconnected microscopicporesandpolymerchains.
3.Removalofsolventinthenetwork:atthisstage,asolidcondensateisobtained.
All the above process takes place at room temperature, which is one of the main characteristicsofthistypeofsyntheticroute.
As a result, different species can be introduced during the first stage, even in the case of labile species. Certain solids synthesized via solͲgel have capacity for ionic andelectronicconductionandthispropertymakesthemsuitableforbeingusedin conventionalelectrochemicaltechniquessuchasvoltammetry.
Thefillingofcapillariesisperformedbyinsertingthemwithintheheartofthesolin thecontainer,rotatinginbothdirectionswithrespecttotheverticalaxis,andtrying to avoid the gaps that may remain, what is relatively complicated, taking into account the high viscosity of the sol and the small diameter of the capillary; however,properdistributionofthesolͲgelalongtheglasstubeisensured.
Oncefilled,acopperwireisintroducedineachcapillarytubeinordertocompact thematerialinside.Thepressureismaintainedwiththethreaduntilasmallamount ofsolfallsoutsidethecapillary,thusfacilitatingsubsequentgrindingandpolishing.
Inthemomentsbeforethemeasurethematerialthatprotrudesfromthebaseof the electrode are removed using fine grit sandpaper. If the excess is large, first coarsesandpapercanbeusedtoacceleratetheprocessandsubsequentlythefine grain one. Then the resulting surface is rubbed with a glossy paper placed on a smooth surface. Thus, the surface is polished (shiny and smooth). With regard to electrical contact (Cu wire), it is set once the material is hardened, ensuring its perfectimmobilization.
1.5. FactorsaffectingthesolͲgelprocess.
The microstructure of a material obtained by solͲgel process depends on the interaction of a large number of process parameters, and variation of more than one simultaneously can lead to results quite complex. In general, the most importantfactorsarethosethatinfluencetheratesofhydrolysisandcondensation, sinceduringthesestages,whichoriginatesthesol,areestablished,somehow,the final factors that will influence the gel. These factors have been studied in earlier worksandarelistedbelow.
The factors that rely primarily on the kinetics of hydrolysis and condensation reactionsare: ͲTypeofalkoxideprecursor. ͲInitialmolarratioalkoxideprecursor:water. ͲType(acidorbasic)andconcentrationofthecatalyst. ͲTypeofsolvent. ͲTemperature. 1.6. Ultrasoundsynthesisofsonogel.
Depending on the effect that is being studied, applications of ultrasound can be classifiedintwotypes.
Thefirstareaisrelatedtotheeffectofenvironmentonthewave[19,20],whilethe secondareafocusesonstudyingtheeffectofthewaveontheenvironment,suchas
emulsion formation, based on the use of high power ultrasounds and/or low frequencies. Studies conducted on this last area [21,22] agree that it must take placethephenomenonofultrasoniccavitationforwaveeffectsontheenvironment tobecomeapparent.
Ageneralformofultrasoniccavitationandthe formationofanewsurfacewithin theliquidbytheirradiationofultrasoundwavescanbedefined.
Applyingultrasoundinimmisciblesystemscanexpandtheinterfacebetweenboth phases significantly, facilitating the exchange of molecules between them, thus obtaining extremely fine emulsions. The application of ultrasonic to precursor alkoxideͲwater mixture that is immiscible in the presence of an acid catalyst is an alternative to synthesize different gels, without solvents, called sonocatalysis. By using this method materials are obtained with different characteristics, called sonogels[23].
The ultrasonic waves will act on the liquidͲliquid interface of the alkoxideͲwater mixturewhichresultsintheformationofbubblesdissolvedintheliquid,reaching extremes of pressures and temperatures (in the bubbles). During the collapse of these bubbles produced by hydrolysis, a homogeneous liquid is yielded which subsequentlypolymerizetoformthegel.
The cavitation induced by the use of ultrasounds, together with the absence of solvent,constitutesanexceptionalsetforachievingthesolͲgelprocess,resultingin the existence of specificity in sonogels such as: high density fine texture, and homogeneousstructure,amongothers.
Forthepreparationofasonogel,hydrolysisoftheprecursormixtureisdone;thenit is subjected to ultrasonic waves in an open glass container. For this purpose, the simplestandmostaccessibleequipmentwouldbeanultrasonicbath.However,it hasthedisadvantagethatthepowerdeliveredbythedevicevariesdependingon the size of the bath, the reaction vessel and its position within the bath, so that energy is dispersed over a larger volume, so ultrasounds levels required for cavitationarenotenoughinthereactionmixture.
Without doubt, the most effective method for transmitting ultrasound energy to themixisthedirectimmersionofaprobeintotheliquidreactant.Thefunctionof this probe is to amplify the vibrational movement originated in the generator (whichisofteninsufficientforpracticalcases).Thematerialforthemanufactureof acousticsensorsisatitaniumalloy.Thankstothisthedirectimmersionoftheprobe into the reaction system can generates ultrasonic power much higher than that reachedinthebathroom,becauseinthissystemtherearenoenergylossesinthe transferofultrasoundsbetweentheprobeandthereactionmixture.Theamountof energysuppliedtothesystemisdeterminedbythetimeofinsonationwhichisan additionalparametertocontrolthepropertiesofsolͲgel.
Compared with traditional methods of obtaining gels, which may include the presence of an alcohol as solvent, the first noticeable effect is the considerable decreaseinthegelificationtime.Thisparameterdecreasesfurtherwhenincreasing the dose of ultrasounds applied to the mixture. When using the classical method, thedilutionexistingatthebeginningcausesalargedecreaseinthedensityofthe gelduetoexcessiveporosityresultingfromtheremovalofthesolvent.Thiseffectis not desirable when you require highͲdensity matrices suffering from low volume contraction.
1.7. Applicationofultrasoundinthesynthesisofgoldnanoparticles.
The major aspect of nanotechnology is the development of synthesis of metal nanoparticles. In this sense, nanosized particles of noble metals, especially gold nanoparticles(AuNPs),havereceivedgreatattentionasaresultoftheirattractive electronic, optical, and thermal properties, as well as catalytic properties and potential applications in the fields of physics, chemistry, biology, medicine, and material science and their different interdisciplinary fields [24]. Therefore, the synthesisandcharacterizationofAuNPshaveattractedsignificantattentionfroma fundamentalandpracticalpointofview.Theparticlesshouldbestablechemically withoutundergoingdegradationsuchaspartialoxidationorundesiredsintering.
The synthesis of gold nanoparticles with diameters ranging from a few to several hundredsofnanometresiswellestablishedinaqueoussolutionaswellasinorganic solvents.Intypicalsynthesis,goldsaltssuchasAuCl3arereducedbytheadditionof a reducing agent which leads to the nucleation of Au ions to nanoparticles. In addition,astabilizingagentisalsorequiredwhichiseitheradsorbedorchemically bound to the surface of the Au nanoparticles. This stabilizing agent (often also called a surfactant) is typically charged, so that the equally charged nanoparticles repeleachothersothattheyarecolloidallystable.Aunanoparticlesprotectedvia selfͲassembly (in two or three dimensional lattices) are also promising for the constructionofnanodevicesandnanocircuits[25,26].
Forthemostcommonsynthesisrouteinaqueoussolution,citricacidservesfirstto reduce the gold salt and thus to trigger nucleation; and secondly, due to its adsorptionontheparticlessurface,itprovidescolloidalstabilitytotheparticlesby itsnegativecharges[27].
Extensivestudies[28Ͳ32]ofaqueoussonochemistryhaveshownthatthechemical effectsofultrasoundsareattributedtocavitation.Thecollapseofthecavitieswhich occursduringultrasonicirradiationleadstothedecompositionofwatermolecules to hydrogen atoms and hydroxyl radicals owing to the production of high temperaturesandpressures.
Overthepastdecade,straightforward,economicallyviable,and‘green’synthesisof nanoparticles has been paid wide attention in the emerging areas of nanoscience andtechnologytoyieldmonoͲdispersedmetalandsemiconductornanoparticlesat ambienttemperatureandwithinashorttime[33].Thesesyntheticrouteshavelead to the application of ultrasounds (sonocatalysis) in the synthesis of nanoparticles. Oneofthegoldnanoparticlesusedinthisresearchworkwassynthesizedfollowing this approach. The application of green synthetic approach in synthesis of gold nanoparticleshasbeenreportedinseveralworks[34Ͳ39].
Other synthetic approach for gold nanoparticles employed in this work was the thermalmethod.
1.8. Comparison between ultrasonic and thermal methods of synthesis of goldnanoparticles.
Ultrasoundssynthesisroutehassomemajoradvantageswhencomparedwiththe thermalmethodofsynthesisofournanoparticles.
The ultrasonic method is associated with local high temperatures and pressures createdduringthebubblecollapsewiththerapidcoolingrate(>109KͼsͲ1),greater thanthoseobtainedbyconventionalquenchingtechnique(105to106KͼsͲ1),which enablestheformationofcolloidsaswellastheformationofnanoparticles[40,41]. The high temperatures, pressures and cooling rate attained during acoustic cavitationleadtomanyuniquepropertiesintheirradiatedsolution[42].
Secondly, kinetics and regiospecificity of ultrasonic degradation are different from those of thermal processes (classical/conventional method). Thermal degradation produces cleavage at random points along the chain of inorganic polymers (e.g. poly(polyorganosiloxanes)andpolyorganosilanes)andvariouspolymersinaqueous media, whereas ultrasonic degradation is much more specific, with cleavage occurringpreferentiallynearthemiddleofthechainofpolymers[43,44].
1.9. Backgroundandcurrentstatus
The solͲgel technology finds numerous applications in the field of chemistry: opticallytransparentmatrices[45,46],electrochromicdevices[47,48],construction ofcathodes[49,50],supportsforsolidphaseextraction(SPE)[51,52]andliquidand gas chromatography [53,54]. Applications of this technology in the area of electroanalysis are relatively recent and require a special type of solͲgel materials whichhastobeconductorsofelectriccurrent[49].Thesubstancesmostcommonly
usedtoprovideelectricalconductivityinsolͲgelmaterialsarefinelydividedmetals suchasgolddust,andgraphite.
A solͲgel electrochemical material can be defined as a mixture in which the inorganic matrix is modified with conductive material, in order to be used as an electrodeinanelectrochemicalsystem.Wecouldalsoconsidertheexistenceofa category within the field of chemically modified electrodes (CMEs), which include electrodes modified with oxides synthesized by solͲgel processes, specifically with siliconoxide(SiO2ͲMonolithic).
Research related to SiO2ͲMonolithic began slowly in the early 90s, despite the appropriatecharacteristicsofthesematerialsforaccumulatingelectroactivespecies priortoelectrochemicaldetection,forinstance,absorptioncapacity,chemicalacid/ baseandthermalstability.Anexampleistheapplicationofelectroactivematerials obtained via solͲgel in the form of thin films, which were developed by Lundgren and Murray [53] in 1987. These investigations showed a rapid rise in 1994 in the field of amperometric biosensors thanks to the discovery of the possibility to incorporateenzymesintheinorganicmatrixkeepingintacttheirbiologicalactivity. Morerecentapplicationsinelectroactivematerialsrelatedtotheworkcarriedout byMaruszewskicanbefoundin[55]andKim[56].
BydopingthesolͲgelsilicaatthebeginningoforduringthepolymerizationprocess, Hsueh and Collinson [57] produced electrodes with a glassy carbon organically modified silica coating withͲNH2 orͲCOOH and voltammetrically characterized by findingaremarkablecapacityforionexchangematerials,attributedtoelectrostatic interactions between the analyte and the acidic or basic function of the organosilane.ThebiggestadvantagethatorganicallymodifiedsolͲgel materialhas consistsofthecombinationofthepropertiesofinorganiccrystalswiththeintrinsic properties of organic molecules. Wang et al. [58] extended this possibility by making composite electrodes and carbon solͲgel modified with dimethylglyoxime andusingvoltammetrytodetecttracesofnickel,thusexploitingthesamedeviceon theadvantagesofsolͲgelchemistryandelectrochemicalpreͲconcentration.Laterhe
used many organic modifiers; two of the most recently employed have been polydimethylsiloxane(PDMS)[59]andtetrapropiortotitanate(TPOT)[60].
Y. Guo and Ana R. Guadalupe [61] developed in 1998 a process for preparing graphite composite electrode/solͲgel precursor mixture applied with silk screenͲ printing techniques. No alcoholic coͲsolvents and catalysts were used, but only a wettingsubstancethatperformsthesefunctions.Themanufactureofthemodified electrodes proceeds in one step by this technique. The reproducibility of results obtained in the applications of these electrodes as sensors and biosensors is not verygood,maybebecauseofthehomemadeprintdevice.
Intheliterature,examplesofspecificuseofelectrochemicalsensorsandbiosensors in the field of applied analysis, such as analysis of food, beverages and drinking watercontrol,showingspecialinterestinspeciesthatestablishpropertiesoffoods andbeveragesthataredirectlyrelatedtoitsflavourand stabilitycanbefoundin [62,63].Thewidevarietyofanalytesandthegreatdiversityofmaterialsisanadded problemtothistypeofapplication,apartfromthoserelatedtothesensoritself,as itsstabilityovertimeandsensitivityoftheirresponse.
With regard to the structural characterization of solͲgel materials, numerous researchgroupshavegoneintothistopic.Outstandingamongallthegroupsisthe researchledbyEsquivias(UniversityofCadiz)[64]andVollet(UniversityofBrasilia) [65].
Awiderangeoftechniquesusedinstructuralcharacterizationexists,whichcanbe used independently to obtain data from a physical characteristic or particular structural material or jointly, with the aim of achieving a more complete characterization. In this case, we can highlight spectroscopy, different types of electronmicroscopy[66]andthetechniquesofXͲraydiffraction[67],amongothers.
The collaboration of various research groups at the University of Cadiz, Spain has resulted in the recent development of a new material and electrodes/sensors, which has been a major development internationally. This has resulted in the publicationofseveralarticlesinreputableinternationaljournals.
The usual procedures to synthesize solͲgel materials for electrodes involve acid catalysis, requiring the precursor mixture in the presence of an alcoholic solvent, times of insonation greater than 10 minutes (at least) in an ultrasonic bath for startingthehydrolysis.Thesubsequentevaporationofthesolventcausesamarked reduction in the volume of material with pore formation, which is completely undesirableforthepreparationofsolidelectrodes.
The most recent proposal done at University of Cadiz is quite different from the manufacturingmethod,andisbasedontheuseofhighͲenergyultrasoundsapplied directly on precursors. In this way, ultrasonic cavitation occurs, which promotes acid hydrolysis in the absence of any additional solvent, yielding a unique phase only in a few seconds. This is known as sonocatalysis. The mixture with spectroscopicgradegraphitehasledtoanewtypeofsolͲgelmaterial:theSonogelͲ Carbonmaterial.
1.10. Voltammetrictechniques.
The experimental part of this work involves the use of two voltammetric techniques. Thus, this section has been included in order to describe the general foundationsofthevoltammetryandthecharacteristicsofbothtechniques.
Voltammetry covers a series of electroanalytical methods developed from polarography.Infact,voltammetryaroseasaresponsetothemultiplechallenges posedbytheuseofpolarographictechniquesinsensitivityandselectivity.
Themaincharacteristicofdcpolarographyisbasedontheuseofamercurydrop electrode as working electrode, so if the working electrode is not this, then the techniqueiscalledvoltammetry.
Thecommoncharacteristicofallvoltammetrictechniquesisthattheyinvolvethe application of a potential (E) to an electrode and the monitoring of the resulting current (i) flowing through the electrochemical cell. In many cases the applied
potential is varied or the current is monitored over a period of time (t). Thus, all voltammetrictechniquescanbedescribedassomefunctionofE,i,andt.Theyare considered active techniques (as opposed to passive techniques such as potentiometry)becausetheappliedpotentialforcesachangeintheconcentration ofanelectroactivespeciesattheelectrodesurfacebyelectrochemicallyreducingor oxidizingit.
The various voltammetric techniques show many analytical advantages: excellent sensitivitywithaverylargeusefullinearconcentrationrangeforbothinorganicand organicspecies(10–12to10–1M),alargenumberofusefulsolventsandelectrolytes, a wide range of temperatures, rapid analysis times (seconds), simultaneous determinationofseveralanalytes,theabilitytodeterminekineticandmechanistic parameters, a wellͲdeveloped theory, and thus, the ability to reasonably estimate the values of unknown parameters, and the ease with which different potential waveformscanbegeneratedandsmallcurrentsmeasured.
Analytical chemists routinely use voltammetric techniques for the quantitative determinationofavarietyofdissolvedinorganicandorganicsubstances.Inorganic, physical, and biological chemists widely use voltammetric techniques for a variety ofpurposes,includingfundamentalstudiesofoxidationandreductionprocessesin various media, adsorption processes on surfaces, electron transfer and reaction mechanisms,kineticsofelectrontransferprocesses,andtransport,speciation,and thermodynamicpropertiesofsolvatedspecies.
The electrochemical cell, where the voltammetric experiment is carried out, consists of a working (indicator) electrode, a reference electrode, and usually a counter(auxiliary)electrode.Ingeneral,anelectrodeprovidestheinterfaceacross whichachargecanbetransferredoritseffectsfelt.Sincethereactionortransferof interesttakesplaceattheworkingelectrode,wheneverwerefertotheelectrode, wealwaysmeantheworkingelectrode.Thereductionoroxidationofasubstance atthesurfaceofaworkingelectrode,attheappropriateappliedpotential,resultsin
current.Eventhoughthevarioustypesofvoltammetrictechniquesmayappearto beverydifferentatfirstglance,theirfundamentalprinciplesandapplicationsderive fromthesameelectrochemicaltheory[68]. 1.10.1. Cyclicvoltammetry.
The cyclic voltammetry can be considered an extension of the linear sweep voltammetry. It is based on making the working potential, (the values oscillating between two limits from a minimum to a maximum in a given time) to grow linearly, and then to descend again to the minimum value in the same minimumͲ maximumperiod.Thesequenceconstitutesacyclefigure1.1.Thepartofthecycle inwhichthepotentialisincreasinginpositivedirectioniscalledpositivesweep;the halfcyclewithdecreasingthepotentialsweepiscallednegative. Figure1.1:Typicalexcitationsignalforcyclicvoltammetry.
The scan profile is usually defined in terms of the redox couple of interest in the experiment. The current intensity measures are carried out throughout the cycle and its representation against the applied potential results in a graph called voltammogram(figure1.1.).Themeasuresofintensityfortheanodicandcathodic peaksareusuallycarriedoutbyextrapolatingthebaselineofeachhalfcyclejust before each peak starts to emerge, so that this is the main drawback of cyclic voltammetry,sincethevaluesofintensityobtainedarenotalwaysreliable.
With regard to the application, it is often the first technique used when an electrochemicalstudyisintended.Themainreasonisthatthisisprobablythemost versatile electrochemical technique to study mechanisms for effective redox systems.Itmakessweepsofpotentialandidentifiesthepresenceofaredoxcouple intheelectrolyticmedium.
Figure1.2.CyclicvoltammogramoftheredoxcoupleFe2+/Fe3+inH2SO41M.
Once found, this redox couple can be characterized by the peak potential of the voltammogram, and the analysis of the possible changes caused by varying the sweep speed of potential as well. It is therefore useful when studying reversible electrochemical systems, i.e. those in which both redox couple species exchange electronswiththeelectroderapidly.
Toevaluatetheelectrochemicalbehaviorofadevelopedelectrodeisalsocommon to start using the technique discussed, because, through it, it is possible to know the electrocatalytic field range that can cover the electrode and the current intensityornonͲfaradaicchargingthatprovidesmediumelectrolyte(intheabsence of species). Cyclic voltammetry, along with other voltammetric techniques, is an important tool to carry out, for example, the study of a chemically modified electrode[69].
1.10.2. Differentialpulsevoltammetry
In general, pulse voltammetric techniques were devised for the purpose of correctingthedrawbacksofcurrentpolarography.Thefirstoneinbeingdeveloped was differential pulse polarography, and thanks to that, the shortcomings arising from the use of mercury drop electrodes were overcome. Thus, in this technique intensity data are only recorded at the end of the droplife and, by applying pulse potential,theintensityismaximized.Equally,whenperformingthistechniquewith negligible charge current, most of the values found are dismissed. With such advances it is able to reduce significantly the detection limit and improve the presentationofdata.
Moreover,lateritwasdiscoveredthatsuchimprovementswerenotonlyapplicable to mercury drop electrodes, but also to solid electrodes and mercury film electrodes. Thus, differential pulse voltammetry arose, whose characteristics are describedbelow.
Figure1.3.Excitationsignalindifferentialpulsevoltammetry. Afulldescriptionofthistechniquewillbedonetakenintoconsiderationfigure1.3. Thetechniqueinvolvestheapplication(overlay)ofaconstantpotentialpulse(ȴEp) ofdurationt(s)attheendofeachstepȴEsheightanddurationT(s)ofastaircase potential. The fact of applying a potential ladder instead of a ramp is due to the adventofdigitalapplicationtoelectrochemicalexperiments,asitisnotpossibleto generate an ideal ramp of potentials, and therefore to perform a series of approximationtoproducesmallstepsofpotential.However,thisapproachhasthe advantage of providing significant improvements through this sampling of current whichisdoneattheendofeachsteporrung,whenFaradaicchargingisnotmissing (andthereforeitshouldonlybefaradaiccurrent).Valuesofcurrentarerecordedin two points: just before applying the potential pulse (S1) and at the very end of it (S2),asshownintheFigure.
When applying a pulsed technique for analytical purposes, the pulse amplitude often increased until 100 mV. Higher values of this parameter provide higher currents,butalsocauselossofresolutionandincreasetheloadcurrent.Typicallyit ispreferredtoutilizevaluesthatprovidethemaximumcurrentvaluewiththelesser
loss of resolution and increase of load current at the same time, 100 mV already achievingagoodcompromisebetweentheseeffects[70].
1.11. UVͲvisiblespectroscopy
Ultraviolet and visible spectroscopy (UVͲvis) is a reliable and accurate analytical laboratory assessment procedure that allows for the analysis of a substance. Specifically, ultraviolet and visible spectroscopy measures the absorption, transmission and emission of ultraviolet and visible light wavelengths by matter. Ultraviolet and visible spectroscopy (UVͲvis spectroscopy) is used to study moleculesandinorganicionsinsolution.
Ultraviolet and visible light comprise only a small portion of the wide ranging electromagnetic radiation spectrum. Although lower in frequency and therefore lowerinenergythancosmic,gammaorXͲrays,ultravioletandvisiblelightareofa higher frequency and, therefore of higher energy, than infrared, microwave and radiowaves.
In the practical sense, spectroscopy measures the absorption, emission, or scattering of electromagnetic radiation by atoms or molecules. By such measurements,thetypeoftheatomsormoleculespresentinasample,aswellasa measure of their concentration or abundance, can be made to an astonishing degreeofaccuracy. Whenultravioletorvisiblelightstrikesatomsormoleculestheycaneitherbounce offorcauseelectronstojumpbetweenenergylevels.Absorptionofultravioletor visibleelectromagneticradiationcauseselectrontomovesfromlowerenergylevels toahigherenergylevels.UltravioletͲvisibleabsorptionspectroscopymeasuresthe absorptionofultravioletorvisiblelight.Sincethespectrumofanatomormolecule depends on its electron energy levels, UVͲvis absorption spectra are useful for identifyingunknownsubstances.
In UV/visible spectroscopy, a beam of light is split into a sample and reference beams.Asitsnameimplies,thesamplebeamisallowedtopassthroughthetarget
sample. Alternately, the reference beam passes through the control solvent or a portionofthesolventthatdoesnotcontaintheactualtarget.Oncethelightpasses through the target sample of interest it is measured by a special meter termed a spectrometer designed to compare the difference in the transmissions of the sample and reference beams. Double beam UVͲvisible spectroscopy instruments allow for simultaneous measures of transmissions through the target sample and solvent.
Thewaybywhichelectromagneticradiationaffectsatomsandmoleculesdepends ontheenergyofthelight.Theenergyis,inturn,dependentuponthefrequencyof thelight.Ultravioletandvisiblelightpromoteselectronsintohigherenergyorbitals. Special instrumentation is used in UVͲvis spectroscopy. Hydrogen or deuterium lights provide the source of light for ultraviolet measurements. Tungsten lamps provide the light for visible measurements. These light sources generate light at specific wavelengths. Deuterium lamps generate light in the UV range (190 to 380nm). TungstenͲhalogen lamps generate light in the visible spectrum (380 to about800nm).XenonlampswhichcanproducelightintheUVandvisibleportions ofthespectrumareusedtomeasurebothUVandvisiblespectra.
The wavelengths of ultraviolet or visible light absorbed by a substance result in a unique ultravioletͲvisible spectroscopic signature for each substance and can be usedasanalyticaltool.
1.12. Electronmicroscopy:theoryandfundamentals
This section describes the most important aspects of Electron Microscopy (EM), focusing mainly on Scanning Electron Microscopy (SEM), Transmission Electron Microscope(TEM) and the XͲrayEnergy Dispersive Spectroscopy (EDS),relating to the techniques used in this research. Deeper aspects and more detailed descriptionsofthebasisforthemcanbefoundin[71,72].
Electron microscopes were developed due to the limitations of light microscopes which are limited by the physics of light to 500x or 1000x magnification and a resolution of 0.2 micrometers. In the early 1930's this theoretical limit had been reached and there was a scientific desire to see the fine details of the interior structuresoforganiccells(nucleus,mitochondria...etc.).Thisrequired10,000xplus magnificationwhichwasjustnotpossibleusinglightmicroscopes.
Thetransmissionelectronmicroscopewasthefirsttypeofelectronmicroscopeto bedevelopedandispatternedexactlyonthelighttransmissionmicroscopeexcept that a focused beam of electrons is used instead of light to ‘see through’ the specimen.ItwasdevelopedbyMaxKnollandErnstRuskainGermanyin1931.
The first Scanning Electron Microscope (SEM) debuted in 1942 with the first commercial instruments around 1965. Its late development was due to the electronics involved in ‘scanning’ the beam of electrons across the sample. An excellentarticlewasjustpublishedinScanningdetailingthehistoryofSEMs.
Electron microscopes (EMs) function exactly as their optical counterparts except thattheyuseafocusedbeamofelectronsinsteadoflightto‘image’thespecimen andgaininformationastoitsstructureandcomposition.
ThebasicstepsinvolvedinallEMscanbedescribedasfollows:
1. A stream of electrons is formed (by the electron source) and accelerated towardthespecimenusingapositiveelectricalpotential.
2. This stream is confined and focused using metal apertures and magnetic lensesintoathin,focused,monochromaticbeam.
3. Thisbeamisfocusedontothesampleusingmagneticlens.
These interactions and effects are detected and transformed into an image. The abovestepsarecarriedoutinallEMsregardlessoftype.Amorespecifictreatment ofhowthetwodifferenttypesofEMsoperateisdescribedinthenextsubsections. 1.12.1. ScanningElectronMicroscopy(SEM) ThescanningelectronmicroscopeusesafocusedbeamofhighͲenergyelectronsto generate a variety of signals at the surface of solid specimens. The signals that derive from electronͲsample interactions reveal information about the sample including external morphology (texture), chemical composition, and crystalline structureandorientationofmaterialsmakingupthesample.Inmostapplications, data are collected over a selected area of the surface of the sample, and a 2Ͳ dimensionalimageisgeneratedthatdisplaysspatialvariationsintheseproperties. Areas ranging from approximately 1 cm to 5 microns in width can be imaged in a scanningmodeusingconventionalSEMtechniques(magnificationrangingfrom20x to approximately 30,000x; spatial resolution from 50 to 100 nm). The SEM is also capable of performing analyses of selected point locations on the sample; this approach is especially useful in qualitative or semiͲquantitatively analysis for determiningchemicalcompositions(usingEDS). 1.12.2. ScanningElectronMicroscopy(SEM) AcceleratedelectronsinanSEMcarrysignificantamountsofkineticenergy,andthis energyisdissipatedasavarietyofsignalsproducedbyelectronͲsampleinteractions when the incident electrons are decelerated in the solid sample. These signals include secondary electrons (that produce SEM images), backscattered electrons (BSE), diffracted backscatteredelectrons (EBSD that are used to determine crystal structures and orientations of minerals), photons (characteristic XͲrays that are used for elemental analysis and continuum XͲrays), visible light, and heat. Secondary electrons and backscattered electrons are commonly used for imaging
samples: secondary electrons are most valuable for showing morphology and topography on samples and backscattered electrons are most valuable for illustrating contrasts in composition in multiphase samples (i.e. for rapid phase discrimination). XͲray generation is produced by inelastic collisions of the incident electronswithelectronsindiscreteortitals(shells)ofatomsinthesample.Asthe excitedelectronsreturntolowerenergystates,theyyieldXͲraysthatareofafixed wavelength (that is related to the difference in energy levels of electrons in different shells for a given element). Thus, characteristic XͲrays are produced for each element in a sample that is ‘excited’ by the electron beam. SEM analysis is consideredtobe‘nonͲdestructive’;thatis,xͲraysgeneratedbyelectroninteractions do not lead to volume loss of the sample, so it is possible to analyze the same materialsrepeatedly.
TheSEMisroutinelyusedtogeneratehighͲresolutionimagesofshapesofobjects andtoshowspatialvariationsinchemicalcompositions(usuallyEDS,alsoBSE).
Thisinstrumentisalsowidelyusedtoidentifyphasesbasedonqualitativechemical analysis and/or crystalline structure. Precise measurement of very small features and objects down to 50 nm in size is also accomplished using the SEM. Backscattered electron images can be used for rapid discrimination of phases in multiphase samples. SEMs equipped with diffracted backscattered electron detectors can be used to examine microfabric and crystallographic orientation in manymaterials. 1.12.3. SEMinstrumentation EssentialcomponentsofallSEMsincludethefollowing: x Electronsource(‘gun’). x Magneticlenses. x Samplestage. x Detectorsforallsignalsofinterest. x Display/dataoutputdevices.
SEMsalwayshaveatleastonedetector(usuallyasecondaryelectrondetector),and mosthaveadditionaldetectors.Thespecificcapabilitiesofaparticularinstrument arecriticallydependentonwhichdetectorsitaccommodates. Figure1.4.Schemeofascanningelectronmicroscope. 1.12.4. XͲrayEnergyDispersiveSpectroscopy(EDS).
EDX analysis stands for Energy Dispersive XͲray analysis. Sometimes it is also referredtoasEDSorEDAXanalysis.Itisachemicalmicroanalysistechniqueusedin conjunctionwithscanningelectronmicroscopeinordertoidentifycompositionand
anintegratedfeatureofascanningelectronmicroscope,andcannotoperateonits ownwithoutthelatter. DuringEDXanalysis,thespecimenisbombardedwithanelectronbeaminsidethe scanningelectronmicroscope.Thebombardingelectronscollidewiththespecimen atoms'ownelectrons,knockingsomeofthemoffintheprocess.Apositionvacated by an ejected inner shell electron is eventually occupied by a higherͲenergy electronfromanoutershell.Tobeabletodoso,however,thetransferringouter electronmustgiveupsomeofitsenergybyemittingXͲray,tobalancetheenergy differencebetweenthetwoelectrons'states.
Theamountofenergyreleasedbythetransferringelectrondependsonfromwhich shell it is transferred, as well as to which shell it is transferred. Furthermore, the atomofeveryelementreleasesXͲrayswithuniqueamountsofenergyduringthe transferringprocess.Thus,bymeasuringtheamountsofenergypresentintheXͲ rays being released by a specimen during electron beam bombardment, the identityoftheatomfromwhichtheXͲraywasemittedcanbeestablished.
TheoutputofanEDXanalysisisanEDXspectrum(Figure1.5).TheEDXspectrumis just a plot of how frequently an XͲray is received for each energy level. An EDX spectrumnormallydisplayspeakscorrespondingtotheenergylevelsforwhichthe mostXͲrayshadbeenreceived.Eachoneofthesepeaksisuniquetoanatom,and therefore corresponds to a single element. The higher a peak in a spectrum, the moreconcentratedtheelementisinthespecimen.
Figure1.5.ExampleofanEDXspectrum.
AnEDXspectrumplotnotonlyidentifiestheelementcorrespondingtoeachofits peaks,butthetypeofXͲraytowhichitcorrespondsaswell.Forexample,apeak correspondingtotheamountofenergypossessedbyXͲraysemittedbyanelectron in the LͲshell going down to the KͲshell is identified as a KͲAlpha peak. The peak corresponding to XͲrays emitted by MͲshell electrons going to the KͲshell is identifiedasaKͲBetapeak,andsoonFigure1.6.[73,74] Figure1.6.Electronictransitionsandspectrallines. 1.12.5. TransmissionElectronMicroscopy(TEM)
Transmission Electron Microscopy is used to identify imperfections in the atomicͲ levelstructureofmaterialsbytheanalysisofmicroscopicsurfaces.Averythinslice ofthematerialtobetestedisexposedtoabeamofelectrons.Whentheelectrons interact with consistent material structure, a constant fraction of electrons is transmittedbackfromthesampletoadetector.Onceastructuralimperfectionis encountered,thefractionoftransmittedelectronschanges.
Two common methods of TEM microstructural imaging reveal important information about the material being tested. Diffraction contrast is useful in identifyinglargestructuresandcrystallographicfeatures.Phasecontrastisusedfor highmagnificationimagingofatomiccolumns.
Scanned Transmission Electron Microscopy (STEM) is useful for identifying crystal defects and mapping diffracting domains. Electron diffraction analysis can be performedinavarietyofmodesandprovidescrystalphaseidentification,specimen
preferred orientation information, and the determination of crystal lattice constants. Electron diffraction is an important tool employed in crystal defect identification.EELS(ElectronEnergyͲLossSpectroscopy)utilizeselectronsthathave lost energy and have their paths slightly and randomly deflected. The amount of energylostisafunctionofthespecimen'scomposition.Thetechniqueisespecially suitedtoanalyzinglightelements[73,74]. Figure1.7.Schemeofatransmissionelectronmicroscope.
This introduction has addressed the subject of the general procedure for solͲgel synthesisandfoundationofthetechniquebrieflyandsimplytomakeasummaryof theoriginsandrecentdevelopments.Ithasalsomadereferencetothetheoretical introduction of the analytical techniques used in the development of this experimentalwork.
Fromthereadingofthisintroduction,itiseasytounderstandthatthecouplingof SonogelͲCarbon devices and nanomaterials can be a powerful combination to
obtainnewamperometricsensorsforthedeterminationofchemicalspecies.Atthis point,myresearchworkbegins. 2. MaterialsandMethods 2.1. Materials Thematerialsusedintheexperimentalpartofthisworkare: Forvoltammetricmeasurements,electrochemicalcellsof25mLwereused. BIOHIT (10Ͳ100 ʅL, 20Ͳ200 ʅL and 200Ͳ1000 ʅL) automatic adjustable pipetteswereusedforadditionsofsolutionsinthemeasuringcell;totake thenecessaryquantitiesofprecursorsinthemanufactureofsonosols,and to prepare buffer solutions, 5 ʅL microsyringe was also employed for additions of solutions in the measuring cell and incorporation of nanoparticlesontheSonogelelectrodesurface.
During the experimental work, diverse glass material was used: pipettes, beakers,flasksofdifferentvolumes,testtubes,cuvettes,etc …
Glasscapillarytubeswereusedasthebodiesforthecompositeelectrodes: 1.15mmofinternaldiameter,1.55mmofexternaldiameter,and70mmin length.
To establish electrical contact between electrodes and the potenciostat /galvanostat,weusedcopperwiresofdiameter0.5mm.
In the polishing stage of the electrodes, sandpaper (Waterproof Silicon CarbidePaper)fineͲgrainedP#1200Struerswasused.
Reagents
Species purity Commercial
House Sodiumcitrate 99.5% Scharlau Hydrogentetrachloroaurate(lll) ͲͲͲͲ ACROSORGANICS Ascorbicacid 99% SigmaͲAldrich Pottasiumhexacyanoferrate(ll) 99.5% Fluka Hydrochloricacid 32% MERCK Methyltrimethoxysilane p.s. MERCK Sulphuricacid 96% MERCK DiͲpottasium hydrogenorthophosphate 99% Fluka Pottasiumdihydrogenorthophosphate 99.5% Fluka Nitricacid 65%QP Panreac Graphite spectroscopic grade GmbH
AbsoluteEthanol p.a. MERCK
Thenitrogenusedforgettinginertatmospheresanddeaeratingsolutionsin themeasuringcellwasNͲ55type.
Preparationofsolutions. Thedescriptionoftheproceduresforthepreparationofsolutionsissummarizedas follows: SolutionofHCl Forobtainingdifferentsynthesizedsonosolsitwasnecessarytoprepareasolution of HCl 0.2 M. To prepare 100 ml, take 1.966 ml of HCl 32% and make up to the correspondingvolumeinavolumetricflask.
PhosphateBufferSolution(PBS):
Toprepareonelitre,34.83gofdiͲpottasiumhydrogenorthophosphate,and27.22g of pottasium dihydrogen orthophosphate were dissolved in 1 litre flask with biͲ distilledwater.
SolutionofAquaRegia:
9mLofconcentratedhydrochloricacidwaspouredintoa25mLgraduatedcylinder, carefullyandslowly3mLofconcentratedHNO3(nitricacid),wereaddedtoprepare the aqua regia. This solution were used to clean glasswares, afterwards the glasswareswerewashedthoroughlywithbiͲdistilledwater,anddriedpriortouse.
Solutionofascorbicacid:
1 mM solution of ascorbic acid was prepared by dissolving 2.20 g in 25 mL of biͲ distilledwater. Solutionofpottasiumhexacyanoferrate(ll) 1mMofpottasiumhexacyanoferrate(ll)solutionwaspreparedbydissolving5.30g in25mLofbiͲdistilledwater.
Instruments Forperformingtheexperimentalworkthefollowinginstrumentswereused: ThesynthesisoftheSonogelͲCarbonmaterialfortheelectrodes,aswellas ofonetypeofthegoldnanoparticleswascarriedoutsonicatingwithahigh powerultrasonicgenerator,SONICATOR3000,fromMISONIX(MISONIX,Inc. Farmingdale,NY,USA)(equippedwitha13Ͳmmtitaniumtip),thatprovides amaximumoutputpowerof600W.
UVͲVisible measurements were made using a Jasco VͲ550 (Easton, Maryland,USA)UVͲvisiblespectrophotometer,jasco32softwareconnected toapersonalcomputer.
Transmissionelectronmicroscopy(TEM)studieswereperformedonaJEOL, JEOL JEMͲ2011 (Jeol, Tokyo, Japan) microscope, operating at 200 KV, equipped with a thermoionic gun (LaB6), a fast change and cold doubleͲ gradientsampler. OpticalmicroscopystudieswerecarriedoutusingaNIKONSMZ800(Nikon, Tokyo,Japan)stereozoommicroscopewithtablestandand10xwidefield eyepieces.Themicroscopehasacontinuouszoomrangemagnificationfrom 10xto63x. Scanningelectronmicroscopy(SEM)studieswerecarriedoutonaQUANTA 200 (FEI Company, Hillsboro, Oregon, USA), normally operating at 20 keV and equipped with a Microanalyzer (EDAX) to perform energy dispersive spectroscopy(EDS).
The voltammetric measures were made on a potentiostat/galvanostat Autolab®PGSTAT20ofEcochemieconnectedtoapersonalcomputeranda 663 Metrohm VA Stand module. This module offers Teflon platform with three holes in which the working electrode, reference electrode (Ag/AgCl) andauxiliaryelectrode(counterelectrode)(platinumrod)areinserted.The computercontrolstheoperationcarriedoutinthemodulebyGPES(General Purpose Electrochemical System) program version 4.9. This software performsfunctionsincludingthegenerationofsignals,dataacquisitionand processingandstorageofthedata.
ThepHmeasurementswereperformedonadigitalpHmeterCrisonMicro pH2002withbuiltͲintemperaturesensor.
The agitation of the solutions was carried out using a magnetic stirrer SelectaAgimaticͲE.
The various substances used were weighed on a Mettler AE420 analytical balance model of two fields (40 gr., detection up to 0.01 mg, and 200 gr., detectionupto0.1mg).
MilliͲQsystem(Millipore,Bedford,MA)witharesistanceof18MёͼcmͲ1,was used for obtaining nanopure water by passing twoͲtimes distilled water throughthesystem. AnultrasonicbathfromSelectawasalsoemployedwhennecessary.
2.2. Methods
Classicalsynthesisofgoldnanoparticles(SN1)
In a 25 mL Erlenmeyer flask, 20 mL of HAuCl4 1.31 mM was brought to boil with vigorous stirring on a magnetic stirring hotͲplate. Then 2 mL of Na3C6H5O7.2H2O 38.8mMwasaddedtothesolutionimmediately,withvigorousstirring.Theyellow HAuCl4 solution changed to dark red within few minutes. Stirring and boiling was continuedforabout10minutesaftertheobservationofthecolourchange. Thesolutionwasremovedfromheatandallowedtocoolatroomtemperature.The colloidalsolutionswerestoredindarknessinacleanplasticcontainerinrefrigerator priortouse. Ultrasonicsynthesisofgoldnanoparticles(SN2) Inavial,1.25mLofHAuCl41.31mMsolutionwasvigorousmixedwithahighpower ultrasoundsonicatorfor4minutes.Then250ʅLofNa3C6H5O7.2H2O38.8mMwas addedandtheinsonationprocesswascontinuedtilladarkredcolourisobtained (totaltimeofsynthesis=5.5min)[75,76]Thesolutionwasremovedandallowedto cool at room temperature before being transfered to a covered plastic container andkeptindarknessinrefrigeratorpriortouse.
PreparationoftheSonogelͲCarbonelectrodes
Insummary,theSonogelͲCarbonmaterialforthecompositeelectrodesisprepared in a glass container (5.5 cm high and 1.7 cm in diameter) and the following precursorsareaddedintheordergiven:50ʅLofHClsolutionconcentration0.2M necessary and 250 ʅL of MTMOS. Then, the container is placed under the probe transducer,asshowninFigure2.1,andinsonationiscarriedoutforfiveseconds.A single homogeneous phase which is the sol (start of the hydrolysis) is obtained in thisway.Then,0.25gofgraphitepowderisaddedandhomogenizedinthemix.
Figure2.1:SchemeofthestepsfollowedforthefabricationoftheSonogelͲCarbon compositeelectrodes. Withregardtothefillingofthecapillaries,thisoperationisperformedreproducibly andaccurately,avoidinggapsthatcanremain,whatisrelativelycomplicated,taking intoaccountthehighviscosityandlowexternaldiameterofthecapillary.Thefilling isdonebyinsertingthecapillaryintothebosomofthevialofsynthesiscontaining sonosol.Ithasbeenobservedthatitisveryeffectivetorotatethecapillaryinboth directions with respect to the vertical axis, while the insertion is done into the sonosol,thussimplifyinggreatlytheoperation[12]. Oncethecapillariesarefilled,nextstepconsistsofthecompressionofthematerial insidethecapillarytube,forwhichacopperwireisusedtopressonthefilling,with thehelpofawatchglass,wherethecapillaryissupportedtopreventtheexitofthe sonosolfromit.Oncecompacted,thewatchglassisremovedand,withthehelpof thesamecopperwire,thepressureismaintaineduntilasmallamountofsonosol fallsoutsidethecapillary,thusfacilitatingsubsequentgrindingandpolishing.Finally theyaredriedatlabenvironmentforaperiodrangingbetween24and48hours.
