proceedings of the 16th international students conference

ISBN 978-80-7444-079-3

Pro

ceedin

gs of th

e 16

th In

ternatio

nal Stu

den

ts Co

nferen

ce ldquoMo

dern

An

alytical Ch

emistryrdquo P

rague 2

02

0

788074 440793

Prague 17mdash18 September 2020

Edited by Karel Nesměraacutek

Prague 2020

Proceedings of the

16th International Students Conference

ldquoModern Analytical Chemistryrdquo

Proceedingsofthe

16thInternationalStudentsConference

ldquoModernAnalyticalChemistryrdquo



Prague 2020

Proceedings of the



CATALOGUING-IN-PUBLICATIONndashNATIONALLIBRARYOFTHECZECHREPUBLIC

KATALOGIZACEVKNIZEndashNA RODNIKNIHOVNACR

ModernAnalyticalChemistry(konference)(162020PrahaCesko)

Proceedingsofthe16thInternationalStudentsConferenceldquoModernAnalyticalChemistryrdquo

Prague17ndash18September2020editedbyKarelNesmerak--1stedition--PragueFacultyof

ScienceCharlesUniversity2020--vi154stran

Obsahujebibliografiearejstrıky

ISBN978-80-7444-079-3(brozovano)

543(062534)

analyticalchemistry

proceedingsofconferences

543ndashAnalyticalchemistry[10]

TheelectronicversionoftheProceedingsisavailableattheconferencewebpage

httpwwwnaturcuniczisc-mac

copyCharlesUniversityFacultyofScience2020

ISBN978-80-7444-079-3

Preface

Despitethefactthattheyear2020ismarkedbyCOVID-19morethan40young

analytical chemists gathered in Prague for the 16th annual international

conferenceldquoModernAnalyticalChemistryrdquoTheymeettopresenttheresultsof

theirresearchtomastertheirpresentationandlanguageskillsandtoexchange

anddiscussideasandexperiencesofanalyticalchemistry

Thisvolumeofconferenceproceedingsbringsyouatotalof25papersfrom

thisconferenceAsinpreviousyearsthecontributionspresentedareassortedby

the sequence of their delivery supplemented by indexes at the end of the

proceedingsallowingeasynavigationthroughthepagesYouwillseethattopics

of contributions cover all the aspects of modern analytical chemistry from

theoretical problems through development of new analytical methods and

improvementofanalyticaltechniquestotheapplicationsinvolvingthesolutionof

medicinaltechnicalorenvironmentalproblemsLetushopethatlikeprocee-

dingsofpreviousyearsofourconference thisonewillalsobean interesting

beneficialandenjoyablereading

Itseemstousthattheauthorsofthecontributionsareaguaranteeofthatanew

generationofanalyticalchemistswillprotectbrightandthrillingfutureofour

science

We are very grateful to the Division of

AnalyticalChemistryofEuChemSforitslong-

lasting auspices of our conference Also we

arethankfultooursponsorsnotonlyfortheir

kind sponsorship making the conference

possiblebutalsoforalltheircooperationand

supportinmanyofourotheractivities

Enjoyreadingtheseproceedings

docRNDrKarelNesmerakPhD

editor

Proceedingsofthe16thISCModernAnalyticalChemistryPrague2020 iii

Sponsors

The organizersof16th International Students Conference ldquoModernAnalytical

Chemistryrdquo gratefully acknowledge the generous sponsorship of following

companies

wwwecomsrocom

wwwlach-nercom

iv Proceedingsofthe16thISCModernAnalyticalChemistryPrague2020

wwwthermofishercz

www2thetacz

wwwzentivacz wwwquintacz

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wwwwaterscom

Contents

AlikovaVChernovaAShormanovVKorotkovaEDeterminationof2-methoxyphenol inmodelsolutionsbyspectrophotometry 1

BohmDMatysikF-MTheeffectsoflinearlyassembledcapillarieswithvariousinnerdiametersoncapillaryelectrophoresis 6

PoradaRBasBVoltammetricdeterminationofvitamins 13Baluchova S Klouda J Barek J Schwarzova-Peckova K Wong DKY Dopamine detection at

antifoulingconical-tipcarbonelectrodes 19Tvorynska SBarek J JosypcukBA comparative study of covalentglucose oxidaseand laccase

immobilizationtechniquesatpowderedsupportsforbiosensorsfabrication 25Heigl N Matysik F-M Capillary flow injection analysis with electrochemical detection for

carbohydrateanalysis 31KravchenkoAVKolobovaEAKartsovaLAApplicationofcovalentcoatingsbasedonimidazolium

cations for separationandon-linepreconcentrationof basicandneutralanalytes in capillaryelectrophoresis35

Efremenko E Chernova A Bastrygina O Determination of vanillin in smoking mixtures byspectrophotometry41

PietrzakKWardakCUranylion-selectiveelectrodewithsolidcontact 45Plotnikova K Dubenska L Zeleny I Polarographic determination of metronidazole and

oxytetracyclinehydrochlorideinveterinarydrugforhoneybees 51Deev V Bessonova E Kartsova L Application of microextraction techniques combined with

chromatographicmethodsfortheanalysisofcomplexobjects 57KralMDendisovaMMatejkaPThedevelopmentofreferenceprobesystemfortip-enhancedRaman

spectroscopy 63ChoinskaMHrdlickaVRedondoBRBarekJNavratilTDeterminationofheavymetalpoisoning

antidote23-dimercapto-1-propanesulfonicacidusingsilversolidamalgamelectrode70VymyslickyFKrızekTHert JCanagliflozinoxidation studyusingelectrochemical flowcelland

comparisonwithhydrogenperoxideoxidation 76AugustınMVyskocilVNovelhybridelectrochemicalDNAbiosensorformonitoringoxidativeDNA

damageviaoxidationreductionsignalsoflowmolecularweightdouble-strandedDNA 83SagapovaLKodrık ovaBSvobodaMMusilSKratzerJChemicalvaporgenerationofcadmiumfor

analyticalatomicspectrometry 90S tadlerova B Vyhnanovsky J Dedina J Musil S Photochemical vapour generation of bismuth

coupledwithatomicfluorescencespectrometry 97Cokrtova K Krızek T Separation of liquid crystals using non-aqueous capillary electrokinetic

chromatography104OndrackovaAStiborovaMHavranLSchwarzova-PeckovaKFojtaMElectrochemistryofSudanI

anditsderivatesinaqueousmedia 110BurkinKGalvidisIBurkinMGroupdetectionofaminoglycosidesusingELISAforcontroloffood

contamination 116Vyhnanovsky J Musil S Photochemical vapor generation of cobalt for detection by inductively

coupledplasmamassspectrometry 123LipinskaJMadejMBasBTyczkowskiJOptimizationofconditionforcoldplasmadepositionofthin

layersforsurfacemodificationofworkingelectrodes 129KorbanAAdvancedGC-MSmethodforqualityandsafetycontrolofalcoholicbeverages 135Baroch M Dejmkova H Sladkova S Utilization of a carbon felt as a material for working

electrodes 141Benesova L Zarybnicka A Klouda J Schwarzova-Peckova K Electroanalytical methods for

determinationof7-dehydrocholesterolinartificialserum146

Authorindex 151Keywordindex 152

Proceedingsofthe16thISCModernAnalyticalChemistryPrague2020 v

1Introduction

2-Methoxyphenol(guaiacol)isusedinmedicineasanexpectorantThestructuralformulaisshowninFig1Itiswidelyusedinthepharmaceuticalindustry[1]forthesynthesisofantituberculosisexpectorantdrugs(Kas-nol Sudafed Ascoril Prothiazine Expectorant Guai-phenesinum) Moreover 2-methoxyphenol is often used as anaromaticsubstance [2] in the food industry Inparti-cular itwaswidelyusedintheproductionofsmokedfish and meat products using smokeless smokingtechnologyusingflavourings Ontheotherhand2-methoxyphenolhasthesymbolGHS07andhasahazardcodeXnTXi[2]accordingtotheGHSsystemItisverytoxicbyinhalationitcanirritatethemucousmembraneoftherespiratorytractandtheconjunctivaofthe

Determination of 2-methoxyphenol in model solutions by spectrophotometry

a a b aVALERIYAALIKOVA ANNACHERNOVA VLADIMIRSHORMANOV ELENAKOROTKOVA

a DepartmentofChemicalEngineeringEngineeringSchoolofNationalResourcesNationalResearchTomskPolytechnicUniversityLeninavenue30634050TomskRussiaalikovaleramailru

b DepartmentofPharmaceuticalToxicologicalandAnalyticalChemistryKurskStateMedicalUniversitystKarlaMarks3305000KurskRussia

Proceedingsofthe16thISCModernAnalyticalChemistryPrague2020 1

AbstractA spectrophotometric approach for determination of 2-methoxy-phenol in model solutions has been developed The absorptionspectra of 2-methoxyphenol were determined in the wavelengthrangefrom200to400nminsolutionsof95ethanolacetonitrile01Msodiumhydroxideandethylacetatewithaconcentrationofthe

minus3analyte of 005mgdm For the quantitative determination of2-methoxyphenol a seriesof solutionswaspreparedwithvarious

minus3 minus3concentrationsfrom0001mgdm to005mgdm in95ethanolacetonitrile01MsodiumhydroxideTheopticaldensityof2-meth-oxyphenolinsolventswasmeasuredatawavelengthof276nmand289nmThedevelopedmethodwastestedusingthemethodanalysisofspikedsamples

Keywordsquantitation2-methoxyphenolUVVISspectrophoto-

metry

Fig 1Structuralformulaof2-methoxyphenol

eyeballinhighconcentrationswhenitpenetratestheskincanleadtoneurosiswhenadministeredorallycanstimulatetheesophagusandstomachresultinginheartfailurecollapseanddeathNowadaystherearepublisheddataoncasesofasystemicallergicreaction[3]causedby2-methoxyphenolderivativesandthereisa fatal case known [4] for oral administration of guaifenesin (3-(2-methoxy-phenoxy)propane-12-diol)oneofthecomponentsofcommonlyavailablecoughmedications Thedeterminationof2-methoxyphenolinenvironmentalobjectsaswellasinthefoodindustryiscarriedoutusinggaschromatographymethodswithsolid-phasemicroextraction[5]Inordertocontrol2-methoxyphenolinnaturaldrink-ing and treatedwastewater gas chromatography is used followed by opticaldetectionoftheeluate[6]Themaindisadvantagesofthismethodofanalysisarethelowselectivityanddurationofdetermination(about3hours)AlsoaccordingtoRussianStateStandartGOST33312-2015themethodofgaschromatographyisusedforthequalitativeandquantitativedeterminationof2-metoxyphenolinjuiceproducts Commonlyfortheanalysisoftoxicsubstancesinvariousbiologicalsamples(bloodplasmaurinesalivasweathair)bygaschromatographyitisnecessarytocarryoutmultistagesamplepreparationwhichcomplicatesandslowsdownthecourseofthestudy[7]Atthesametimeitisimportantthatduringtheprepa-rationofsamplesintheanalyzedcompoundstheirstructureisnotviolatedasthiswillleadtothedifficultyoftheiridentification Spectrophotometryintheultravioletregionhaslowersensitivitycomparedtothe abovemethods however thismethod does not require such complicatedpreparation of the analyzed samples it is a relatively affordable simple andinexpensive analysis method In addition its sensitivity can be significantlyimprovedbyapplyinganappropriateseparationprocedureandpreconcentrationbeforedetection[8]MethodUVspectrophotometryisusedtoassessthequalityof both medicinal substances and preparations made from them in terms ofauthenticitygoodqualityandquantitativecontentInadditionitisarelativelyaffordablesimpleandlow-costanalysismethod Ananalysisoftheliteraturedatashowedthattodaytherearefastandsensitivespectrophotometricmethodsforthedeterminationofpyrocatecholderivativesinmedicines[9]vanillininfoodproducts[10]andotherphenolsinwastewaterandwineproducts[1112]Howeverasfarasweknowinformationonthedetermi-nationof2-methoxyphenolfromtheabsorptionspectraintheultravioletregionisabsent Theaimofthisstudyistodevelopmethodsforthequalitativeandquantitativeof2-methoxyphenolinmodelsolutionsusingUVspectrophotometry

2 Proceedingsofthe16thISCModernAnalyticalChemistryPrague2020

2Experimental

21Reagentsandchemicals

Asampleof2-methoxyphenolfromFlukawithabasicsubstancecontentofge98wastakenastheobjectofstudyAssolventsweusedacetonitrile(ChP)95ethanolethylacetateand01MsodiumhydroxidesolutionAllotherchemicalsusedwereofanalyticalreagentgrade

22Instrumentation

Theopticaldensitywasmeasuredincuvetteewithanabsorbinglayerthicknessof10mmusingaCary60spectrophotometer(AgilentUSA)Allmeasurementswerecarriedoutatroomtemperature

3Resultsanddiscussion

Thechangeinthebehavioroftheabsorptionspectrumwasinvestigatedinthewavelengthrangeof200ndash400nmFigure2isshowedthatwithanincreaseinthepolarityofthesolventtheabsorptionmaximumshiftstowardthevisiblepartofthe spectrum The wavelength of absorption maxima of 2-methoxyphenol ispresentedinTable1 A studyof thephotometricbehaviorof2-metoxyphenolinvarious solventsshowedthatacetonitrile95ethanoland01Msodiumhydroxidearethemostsuitablesolventsforthequalitativedeterminationofthetestsubstance

ndash3Fig 1Thespectraof2-methoxyphenolofconcentration005mgdm inthemediumofsolvents(anabsorbinglayerthicknessof10mm)


Forthequantitativedeterminationof2-methoxyphenolaseriesofsolutionsndash3 ndash3withaconcentrationragefrom0001mgdm to005mgdm werepreparedin

acetonitrileand95ethanolTheopticaldensityof2-methoxyphenolinsolventswasmeasuredbywavelengthof276nmThedependenceoftheintensityoftheoptical density on the concentration of 2-methoxyphenol in 01 M sodium

ndash3hydroxide was plotted in the concentration range from 0005mgdm tondash3003mgdm Themeasurementwerecarriedoutbywavelengthof276nmThe

obtainedregressionequationsarepresentedinTable2Dataanalysisobtainedwasperformedusingleast-squaresmethodThedevelopedmethodwastestedusingthemethodanalysisofspikedsamplesTheresultsarepresentedinTable2

4Conclusions

Studieshaveshownthepossibilityofusingspectrophotometricanalysisforthequalitativeandquantitativedeterminationof2-methoxyphenolTheabsorptionmaximaofweredeterminedinsolutionsofethanolandacetonitrile(276nm)inasolutionofethylacetate(277nm)and01Msodiumhydroxide(289nm)Theconstructed calibration curves of thepure substance of 2-methoxyphenol hasshownagoodregressioncoefficient(Rgt099)andcanbeusedforquantitativedeterminationof2-metoxyphenolinbiologicalobjectsInthefutureitisplannedtoapplythistechniquetodetermine2-metoxyphenolinincadavericmaterial

Solvent Regressionequation Found S RSD Δх δ 2-metoxy- phenolg

ndash3 ndash6 ndash5Acetonitrile y=18294C+01130 499times10 50times10 028 2times10 044 Rsup2=09985

ndash3 ndash6 ndash595ethanol y=35131C+00269 503times10 03times10 021 7times10 137 Rsup2=09956

ndash3 ndash6 ndash501Msodium y=31196C+01101 495times10 01times10 018 1times10 028hydroxide Rsup2=09997

Table 2Results of the determination of 2-methoxyphenol (average of three measurements) in modelsolutionsbythemethodanalysisofspikedsamplestheconcentrationofintroduced2-methoxy-

ndash3phenolwas500times10 g(SndashstandarddeviationRSDndashrelativestandarddeviationΔхndashabsoluteerrorδndashrelativeerror)

Table 1Valuesofopticaldensityandwavelengthsinappropriatesolventswith2-methoxyphenol

minus3 minus1Solvent λnm εgdm cm

Acetonitrile 276 0082895ethanol 276 00853Ethylacetate 277 0083101Msodiumhydroxide 289 00744


References

[1] МельниковаИММизерницкии ЮЛКомбинированныеотхаркивающиепрепаратырастительного происхождения в педиатрическои практикеМедицинский совет 2(2018)93ndash97

[2] httpwwwthegoodscentscompanycomdatarw1032272html(accessed27stFebruary2020)

[3] RayMFaltayBHallerNACasereportanaphylacticreactiontoguaifenesinHospPract37(2009)60ndash63

[4] OkicMJohnsonTCrifasiJALongCMitchellEKSwiftonsetofcentralnervoussystemdepressionandasystolefollowinganoverdoseofguaifenesinJAnalToxicol37(2013)318ndash319

[5] ВолковCМЧерновецАНОпределениеконцентрациифеноловвгазовыхвыбросахпромышленных предприятии методом газовои хроматографии с твердофазнои микроэкстракциеи Сорбционныеихроматографическиепроцессы10(2010)723ndash728

[6] ШачневаЕЮОньковаДВСерековаСМСпособыопределенияфеноловвобъектахокружающеи среды Астраханский вестник экологического образования 4 (2013)138ndash142

[7] ГладиловичВДПодольскаяЕПВозможностипримененияметодаГХ-МС(Обзор)Научноеприборостроение4(2010)36ndash49

[8] Pena-PereiraFLavillaIBendichoCHeadspacesingle-dropmicroextractioncoupledtomicrovolumeUVndashVis spectrophotometry for iodine determinationAnal Chim Acta631(2009)223ndash228

[9] NagarajaPMurthyKCSRangappaKSGowdaNMMSpectrophotometricmethodsforthe determination of certain catecholamine derivatives in pharmaceutical preparationsTalanta46(1998)39ndash44

[10] Altunay N Development of vortex-assisted ionic liquid-dispersive microextractionmethodology for vanillin monitoring in food products using ultraviolet-visible spectro-photometryLWT93(2018)9ndash15

[11] Lupetti KO Rocha FRP Fatibello-Filho O An improved flow system for phenolsdetermination exploiting multicommutation and long pathlength spectrophotometryTalanta62(2004)463ndash467

[12] Figueiredo-Gonzalez M Cancho-Grande B Simal-Gandara J Garnacha tintorera-basedsweetwineschromaticpropertiesandglobalphenoliccompositionbymeansofUVndashVisspectrophotometryFoodChem140(2013)217ndash224


1Introduction

Thenumberofsamplesthesamplecomplexityandalsothenumberofsubstanceswhich need to be analysed simultaneously is increasing steadily ThereforepowerfulseparationanddetectionmethodsarerequiredOnewaytoachievethisisthecouplingofaseparationsystemwithmorethanonedetector[12] In recent years capillary electrophoresis (CE)was established as a potentseparation system due to its high separation efficiency and the low sampleconsumption [3] To generate more information numerous dual detectionconceptsforCEweredevelopedwhicharesummarisedelsewhere[12]Acom-binationofamperometricdetectionandmassspectrometry(MS)isaninterestingdual detection concept for CE because both detectors supply complementaryinformationForelectroactivespeciesamperometricdetectionisarobustandoneof the most sensitive detection method [4] Thus it is well suited for the

The effects of linearly assembled capillaries with various inner diameters on capillary electrophoresis

DANIELBO HMFRANK-MICHAELMATYSIK

InstituteofAnalyticalChemistryChemo-andBiosensorsFacultyofChemistryandPharmacyUniversityofRegensburgUniversitaumltsstraszlige3193053RegensburgGermanydanielboehmchemieuni-regensburgde

AbstractDuetotheincreasingneedofpowerfulanalyticalmethodsanewdualdetection concept for capillary electrophoresis (CE) with parallelamperometricdetectionandmassspectrometryshallbedevelopedFor this concept the CE flow has to be divided into two streamsutilizinga flowsplitter In thiswork theeffectsof combinedcapi-llarieswithvarious innerdiameterswerestudiedForpreliminaryinvestigationsthecapillarieswereconnectedinaserialconfigurationwithoutdeadvolumeUsingcapillaryflowinjectionanalysishyphe-natedtocontactlessconductivitydetectionitcouldbeshownthatthecouplingofidenticalcapillariesleadstoaslightdecreaseoftheflowratesWithCEhyphenatedtoUVdetection itcouldbeshownthatthecouplingofcapillarieswithdifferentinnerdiameterhasamuchstrongereffectontheelectroosmoticflowthanthecombinationwiththesameinnerdiameterFurthermorenosignificantchangeinpeakshapewasobserved

Keywordsassembledcapillariescapillaryelectrophoresiscapillaryflowinjection

analysisdualdetectionconceptnon-aqueoussystem


quantificationofsubstanceswhereasMSiswellsuitedfortheidentificationofunknown substances [3] In most dual detection concepts the detectors arearrangedinaserialconfigurationwhichisnotpossibleincaseofamperometricdetection-mass spectrometry [1] The instrumental implementation is morecomplicatedwithbothdetectorsbeingdestructiveFurthermoretheymustbedecoupledfromthehighvoltagefieldoftheCEThereforetheCEflowmustbedividedintotwostreamswithaflowsplitterAsimplifiedsketchofthepossiblenewdualdetectionconceptisshowninFig1 ForthedevelopmentofthenewdualdetectionconceptthreecapillarieswithpotentiallydifferentinnerdiametersmustbecoupledForthisreasonthedeadvolume-freecouplingofcapillarieswithdifferent innerdiameterswas investi-gatedinafirststepTokeepthesetupsimplewefocusedonthelinearcouplingofcapillariesandtheresultingeffectsNon-fragmentedcapillarieswerecomparedwithfragmentedcapillariesofthesameordifferentinnerdiametersEffectsonthe flow rate were investigated with capillary flow injection analysis (CFIA)

4hyphenatedtocontactlessconductivitydetection(C D)EffectslikechangesinthemigrationbehaviourorpeakshapesoccurringinCEwereinvestigatedwithCEhyphenatedtoUVdetection(CE-UV)


Fig 1SchematicillustrationofthenewdualdetectionconceptwithparallelamperometricdetectionandmassspectrometricdetectionforCEAfterinjectionfrom(a)thesamplevialthecomponentsareseparatedbyCEnext(b)theflowsplitterdivides(c)thecapillaryintotwopartsandleadstheCEflowtowards(d)themassspectrometerand(e)theamperometricdetector

2Experimental


Thefollowingchemicalswereusedallofanalyticalgradeferrocenemethanoldecamethylferrocene(ABCRGermany)acetonitrileammoniumacetate01Msodiumhydroxidesolutionultra-purewaterprovidedbyaMilliQAdvantageA10system(MerckGermany)aceticacid(RothGermany)

22Instrumentation

221Capillaries

4Forbothexperiments(CFIA-C DandCE-UV)capillarieswithinnerdiametersof2550and75micromanouterdiameterof360micromandatotallengthof70cmwereusedTheywerepurchasedfromPolymicroTechnologies(USA)Measurementswere carried out with fragmented and non-fragmented capillaries For themeasurementswiththefragmentedcapillariestheoriginalcapillarieswerecutintotwopiecesyieldingatotalof9capillarycombinationswithlengthsof70cm(20cmfirstcapillarypieceand50cmsecondcapillarypiece)ThesecombinationsaresummarizedinTab1(section31)Atbothendsofthecapillariesabout02cmof thepolyimidecoatingwasremovedBothsidesof thecapillarypieceswerepolishedtoreceiveplanarcapillarytipsForthelinearassemblingofthecapillarypiecesMicroTightSleevesF185Xanda capillary connectorUnionAssemblyMicroTightP720fromIDEXHealthampScience(USA)wereusedPriortothefirstCEmeasurementsthecapillarieswereconditionedbyflushingthemfor10minwith01Msodiumhydroxidesolution5minwithultra-purewaterand30minwithseparationbuffer

222Capillaryflowinjectionanalysishyphenatedtocontactlessconductivitydetectionsetup

The flow rates for the fragmented and non-fragmented capillaries were4determinedwithaCFIA-C DsetupschematicallydepictedinFig2ATheflowin

thecapillarywasgravitationdrivenbyaheightdifferencebetweentheinletandoutletcarriersolutionvialTheconceptofCFIAwithgravitationdrivenflowwasfirstdescribedbyMatysiketal[5]AlaboratoryconstructedautosamplerofaCEdevicewasusedforthehydrodynamicinjectionThesamplesolutionconsistedof10mMdecamethylferrocene incarrier solution (10mMCH COONH and1M3 4

4CH COOH in acetonitrile) A high resolution C D was placed after 40 cm for3

detectionThedetectordescribedelsewhere[6]wasconstructedinthedoLagogroup(Brazil)Adoubledeterminationattwodifferentheightswasdoneforthedeterminationoftheflowrates


223Capillaryelectrophoresis-UVdetectionsetup

Fig2BshowsasketchoftheCE-UVsetupItconsistedofalab-builtCEdevicewhichwasconnectedtoahighvoltagepowersupplyfromISEG(Germany)Theseparationswerecarriedoutwithanon-fragmented50micromcapillaryandwithcapillary combinations implementing a 50micromdownstream capillary segment(25+5050+50and75+50microm)ALambda1010UV-VISdetectorfromBischoff(Germany)wasusedfordetectionat210nmThedetectorwasplacedafter40cm


Fig 2Schemeof(A)thecapillaryflowinjectionanalysis(CFIA)hyphenatedtocontactlessconduc-4tivity detection (C D) setup and (B) the capillary electrophoresis hyphenated to UV detection

4(CE-UV) setup Components of the CFIA-C D setup (a) sample (b) inlet and (c)outlet carrier4solution vial (d) fused silica capillary (e) linear capillary connector (f) C D and (g) stand

ComponentsoftheCE-UVsetup(h)sample(i)inletand(j)outletbuffervialand(k)UVdetector4therestofthecomponentswereidenticaltotheCFIA-C DsetupTheoutletbuffervialwaslowered

forthehydrodynamicinjection(j)Theenlargedview(k)depictsthecouplingoftwocapillarieswithdifferentinnerdiametersintheconnectionsidewithoutdeadvolume

Asamplesolutioncontaining1mMferrocenemethanolanddecamethylferroceneinseparationbuffer(10mMCH COONH and1MCH COOHinacetonitrile)was3 4 3

utilizedTheinjectionwasperformedhydrodynamicallybyloweringtheoutletbuffer vial by 20 cm A uniform sample plug was injected to compare bandbroadeningeffectsTheinjectionsegmenthadalengthof035cm(05ofthetotalcapillarylength)andtherespectiveinjectiontimewasdeterminedbasedontheflowratesofthecorrespondingcapillarycombinationFortheelectrophoreticseparationaseparationvoltageof25kVwasappliedandtheinletandtheoutletbuffervialswereplacedatthesameheightsothattherewasnogravityflowwhichaffectedthemigrationbehaviour


31Capillaryflowinjectionanalysishyphenatedtocontactlessconductivitydetectionexperiments

AsshowninTab1theflowratesforaheightdifferenceof20cmwerecalculated4basedontheCFIA-C DmeasurementsItwasobservablethattheflowratesare

slightlylowerforfragmentedcapillariesthanfornon-fragmentedcapillariesofthe same dimension This indicates that a flow resistance arises when twocapillaries are combined Furthermore it was observed that the flow ratedecreasesforupstreamcapillarieswithlowerinnerdiametersandviceversaTheflow rate for the combination 25+75microm could not be determineddue to theformationofairbubblesattheconnectionside


Table 1Flowratesandthecorrespondingstandarddeviations(SD4measurements)ofdifferentcapillarycombinationsforaheightdifferencebetweeninletandoutletvialof20cmbymeansofcapillaryflowinjectionanalysishyphenatedtocontactlessconductivitydetection

ndash1capillary flowratenLs plusmnSDflowndash1combinationmicrom ratenLs

25 00577 0000525+25 0057 000150+25 00775 0000775+25 ndash ndash25+50 0137 000550 0865 000250+50 080 00575+50 105 00625+75 0187 000250+75 1941 000375 456 00275+75 42 01

32Capillaryelectrophoresis-UVdetectionexperiments

Theelectropherogramsforthenon-fragmented50micromcapillaryandthecapillarycombinationswithdownstream50micromcapillaryarepresentedinFig3ThetwoferrocenederivatesdecamethylferroceneandferrocenemethanolwereusedasmodelanalytesDecamethylferrocenewasonlydetectedascationicspeciesasitiseasilyoxidizedbydissolvedoxygeninsolution Forthecombination50+50micromslightlyhighermigrationtimesforthecationic(decamethylferrocene) and neutral species (ferrocenemethanol) were obser-vable compared to thenon-fragmented50micromcapillaryThis indicateda flow

4resistanceattheconnectionwhichwasalsoobservedfortheCFIA-C Dexperi-mentsinsection31Incontrasttothecombinationwiththesameinnerdiameterastrongshift in themigration times for theneuralspecieswasvisible for thecombinationswithdifferentinnerdiametersThisshowedthatthecouplinghadaneffectontheelectroosmoticflow LookingatthepeakshapeitwasfoundthatallpeaksshowednearlyGaussianshape for all combinations Furthermore there was no tailing visible Theferrocenemethanolpeaks for thecombination25+50micromand75+50micromwere


Fig 3Electropherograms of themodelmixture ferrocenemethanol (FcMeOH) and decamethyl-ferrocene(DeMeFc)measuredwithanon-fragmentedcapillary(50microm)andfragmentedcapillaries(25+5050+50and75+50microm)Experimentalparameters1mMFcMeOHandDeMeFcinseparationbuffer(10mMCH COONH and1MCH COOHinacetonitrile)injectionsegment035cmseparation3 4 3

voltage25kVcapillarylength70cm(40cmtothedetectorfragmentedcapillaries20cmfirstpartand50cmsecondpart)UVdetectionat210nm

slightlybroaderthanthepeaksforthenon-fragmented50micromcapillaryorforthe50+50 microm combination But this probably results from longitudinal diffusioneffectsduetothelongerresidencetimes

4Conclusions

FromtheCFIAmeasurementsitcanbeconcludedthattherewasamechanicaldisturbanceoftheflowduetothecouplingFurthermoreitcouldbeshownthatCEmeasurementswithlinearcoupledcapillariesofvariousinnerdiameterwerepossible Unlike to the capillary combinationwith the same inner diameter astrongshiftoftheelectroosmoticflowtowardshighermigrationtimeswasfoundfor capillary combinations with different inner diameters In this work thecapillaries were coupled with almost no dead volume which resulted in nosignificantchangesofthepeakshapeorpeaktailingContrarytoexpectationsthecouplingofcapillarieswithvariousinnerdiametershadnosignificantimpactonthepeakwidth TheknowledgegainedfromthelinearcouplingofcapillariesisagoodbasisforthedevelopmentofthenewdualdetectionconceptInanextstepthreecapillariesshouldbecoupledwitheachother

Acknowledgments

WethanktheGermanResearchFoundation(DFG)forfinancialsupport

References

[1] OpekarFS tulıkKSomeimportantcombinationsofdetectiontechniquesforelectrophoresisincapillariesandonchipswithemphasisonelectrochemicalprinciplesElectrophoresis32(2011)795ndash810

[2] BeutnerAHerlTMatysikF-MSelectivityenhancement incapillaryelectrophoresisbymeans of two-dimensional separation or dual detection conceptsAnal Chim Acta1057(2018)18ndash35

4[3] BeutnerACunhaRRRichterEMMatysikF-MCombiningC DandMSasadualdetectionapproachforcapillaryelectrophoresisElectrophoresis37(2016)931ndash935

[4] MatysikF-MEnd-columnelectrochemicaldetectionforcapillaryelectrophoresisElectro-analysis12(2000)1349ndash1355

[5] MatysikF-MWernerGTracemetaldeterminationintearsbyanodicstrippingvoltammetryinacapillaryflowinjectionsystemAnalyst118(1993)1523ndash1526

[6] FranciscoKJMdoLagoCLAcompactandhigh-resolutionversionofacapacitivelycoupledcontactlessconductivitydetectorElectrophoresis30(2009)3458ndash3464


1Introduction

Thetermldquovitaminsrdquodescribestheheterogeneousgroupofchemicalcompoundswhich are important for the proper functioning of the human body [1 2] Bydefinitionvitaminsarenotsynthesizedbythehumanbodyorthesynthesizedamount is not sufficient to cover the demand That is why they have to besupplementedfromtheexternalsourceslikefoodproductsorpharmaceuticals[1ndash3]Basedontheirsolubilityvitaminsaredividedintowater-soluble(B-groupandvitaminC)andfat-solublevitamins(ADEandK)[3]VitaminC(ascorbicacid) is the most important antioxidant and participates in the activation ofenzymes[4]VitaminB1(thiamine)facilitateswoundhealingandiscrucialforthehumannervoussystem[15]VitaminB2(riboflavin)participatesintheenzy-maticreactionsandthebiotransformationofglucoseandaminoacids[6]Vitamin

+B3(niacin)isthemainconstituentoftheNAD andNADHcoenzymeswhichareresponsible for the transfer of electrons and hydrogen ions in the cellularrespiration [1ndash3] Vitamin B6 possesses six related structures (vitamers) thateasilyinterconvertThemostimportantoneispyridoxinewhichhelpstopreventtongue inflammation and microcytic anemia [2] For the production of well-functioningredbloodcellsandtheavoidanceofmegaloblasticanemiaandfetusdefectsvitaminB9(folicacid)hastobesupplementedintheproperamount[12]

Voltammetric determination of vitamins

RADOSŁAWPORADABOGUSŁAWBAS

DepartmentofAnalyticalChemistryFacultyofMaterialsScienceandCeramicsAGHUniversityofScienceandTechnologyMickiewicza3030-059KrakoacutewPolandrporadaaghedupl


AbstractVitaminsbelongtothegroupofchemicalcompoundsessentialfortheproperfunctioningofthebodySinceboththeirdeficiencyandexcessmay result in serious health problems the amount of vitaminssupplementedinthedietaswellasvitamincontentintheirsourceshavetobestrictlycontrolledInthisworkthepossibilityofsimulta-neous determination of vitamins by means of differential pulseadsorptive stripping voltammetry is discussed The research hasshownthatthedeterminationofsingularvitaminatthemicromolarlevel isrelativelyfastandstraightforwardandthemostimportanthindranceisrelatedtotheanalyteadsorptionattheelectrodesurfaceInthecaseofvitaminswithdifferentredoxpotentialstheycanbeanalyzedsimultaneouslywithouttheneedtoreachfortheadvancedmethodsforsignalprocessing

Keywordsmercuryelectrodevitaminsvoltammetry

VitaminK3(menadione)doesnotoccurnaturallybutitservesasaprecursorforthesynthesisofotherK-groupvitaminsandcanbeusedtotreathypoprothrom-binemiaVitaminK3ispartiallysolubleinwater[17] All of the vitamins are electrochemically active [3] therefore the electro-chemicalmethodscanbeappliedforthedeterminationofvitamincontentinfoodproductspharmaceuticalsandbodyfluidsVoltammetrictechniquesarecharac-terizedbyhighsensitivityandselectivityandtheydonotrequiretime-consumingsamplepreparationMoreovertheelectrochemicalinstrumentationisrelativelyinexpensiveandcanbeappliedintheon-siteconditionsfortheonlineanalyseseginqualitycontrolMostofthepapersreporttheconstructiondevelopmentand characterization of a novel modified working electrodes for quantitativeanalyses of a singular vitamin in the variety of matrices Unfortunately only alimitednumberofpapersdescribethesimultaneousdeterminationofmultiplevitaminsinasinglerun[23] The preliminary research devoted to the simultaneous determination ofB-groupCandK3vitaminswiththeuseofthecontrolledgrowthmercurydropworking electrode in aqueous solutions is presented in this work Particularattentionhasbeenpaidtotheredoxpotentialsofthestudiedcompoundstheshape of the calibration curves and adsorption phenomena As an attempt toovercomethelattertheneutralsurfactantTritonX-100hasbeenintroducedintothestudiedsystem

2Experimental


TheappliedreagentswereofanalyticalgradeandusedassuppliedPhosphateand McIlvaine buffers were obtained by mixing the appropriate amount of

ndash1 ndash1 ndash102molL Na HPO with 02molL NaH PO and 01molL citric acid2 4 2 4

respectively (all reagents purchased from Avantor Performance MaterialsPoland)ThestandardsolutionsofvitaminB1B2B3B9andCwerepreparedbydissolving the corresponding amount of the standard (all Sigma-Aldrich) in

ndash1distilledwater In thecaseofB2andB9 theadditionof02molL NaOHwasinevitabletoobtainaclearsolutionVitaminK3standard(Sigma-Aldrich)was

ndash1dissolvedinthemixtureofmethanoland1molL phosphatebuffer(pH=82)(vv=25)LaboratorygradeTritonX-100(Sigma-Aldrich)wasusedinthestudyoftheadsorptionprocesses22Instrumentation

All the electrochemical measurements were conducted in the three-electrodesystem composed of the Pt auxiliary electrode double-junction silversilverchloride reference electrode and controlled-growth mercury drop electrode


actingastheworkingelectrodeTheusedmeasurementequipmentinvolvedtheM164electrodestandandM161multipurposeelectrochemicalanalyzer(bothmtm-anko Krakow) To control the buffer pH-value the SevenCompact S210laboratorypH-meter(MettlerToledoSwitzerland)wasemployed

23Voltammetricmeasurements

Throughout the course of the study differential pulse adsorptive strippingvoltammetry has been used for recording the current-potential curves BothcathodicandanodicscanswererecordedinapotentialrangeadjustedforthestudiedvitaminsTheinfluenceofvariousmeasurementconditionsontheregis-teredsignalshasbeeninvestigatedFinallythepossibilityofthesimultaneousdeterminationofmultiplevitaminsinonescanhasbeenverified


Figure 1 depicts the redox potentials of the studied vitamins in the aqueoussolutions for the mercury electrode The only exception is vitamin B6 whoseredox potential is higher than the potential of mercury oxidation (ca +02 V)Therefore the given value refers to the glassy carbon electrode The redoxpotentialvalueofstudiedvitamins isnotasingularvaluebut it fallswithinacertainrangeThiscanbeascribedtothedependenceofredoxpotentialonthesolutionpHvaluewhichresultsfromtheparticipationofprotonsintheredoxreactions of vitamins Moreover the potentials for individual vitamins arerelatively well separated indicating that the simultaneous determination ofseveralvitaminsintheonerunmaybepossibleTheonlyencounteredproblems


Fig 1Redoxpotentialsofchosenvitamins

regardthevitaminsB1B3andB12whoseredoxpotentialsoscillatebetweenndash15andndash17VandtheK-groupvitamins(K1K2K3)inwhichonlythecommonstructuralmotifndashthequinoneringndashiselectrochemicallyactiveresultinginthevalueoftheredoxpotentialofcandash02V[8] AtypicalcalibrationcurveisdepictedinFig2Abasedonthedifferentialpulsevoltammograms of the vitamin B9 reduction in the concentration range from

ndash1blank to 01 mg L recorded in the McIlvaine buffer of pH = 52 using thecontrolledgrowthmercurydropelectrodeworkingelectrodeTherelationshipbetweenthepeakcurrentandtheconcentrationofVB9 is linear in thewholetestedrange(r=09999)Basedontheparametersoftheregressioncurvethelimit of detection and limit of quantitation were estimated to 42 and

ndash1142nmolL respectively Similar dependencies and figures of merit can beobtainedforothervitamins QuitedifferentbehaviorwasobservedinthecaseofthevitaminK3forwhichthe increase in current was not strictly proportional to the increase in theconcentrationandthecalibrationplotresemblesanS-shapecurve(Fig2B)ThelatterindicatesthatvitaminK3adsorbsatthesurfaceoftheworkingelectrodeHoweverasnopre-orpost-peakwereobservedwearedealingherewiththeweakadsorption[9]AdsorptionalsoplaysasignificantroleinthecaseofvitaminB2forwhichboththepre-andpost-peakswereobservedindicatingitsstrongaffinitytothemercuryelectrodes To overcome this issue attempts with Triton X-100 were performed TritonX-100isaneutralsurfactant thateasilyadsorbsat thesurfaceof themercury


Fig 2Differentialpulseadsorptivestrippingvoltammogramsof(A)vitaminB9and(B)vitaminK3ndash1reduction recorded in the concentration range from blank to 01 and from blank to 04 mg L

respectivelyInsetcorrespondingcalibrationcurvesSupportingelectrolyte(A)McIlvainebufferndash1(pH=52)(B)04molL phosphatebuffer(pH=82)

electrode partially blocking its surface Doing so it prevents the undesiredadsorption of other molecules and thus allows to obtain a linear relationshipbetweenthepeakcurrentandthevitaminconcentration(Fig3)UnfortunatelyduetotheblockingoftheelectrodesurfacetheslopesofthecalibrationlinesaresmallerincomparisontotheonesobtainedintheabsenceofanysurfactantsThismeans that the sensitivity defined as the increase in current caused by a unitincrease in concentration and the resolution understood as the possibility todistinguishsmallvariationinconcentrationarecorrespondinglydecreased Figure3alsopresentsthepossibilitytodeterminemultiplevitaminsinasinglerunChosenvitaminshavewell-separatedpotentialsandtheydonotinterferewitheachotherthereforenoadvancedmultivariatecalibrationstrategiesareneededTheproblemsinthesimultaneousanalysisincludevarioussensitivitieswith respect to the studied analytes and differences in the influence of themeasurementconditionsontherecordedsignalsDuetothattheexperimentalconditionswillneverensurethehighestpossiblesignalvaluesforallanalyzedcompounds

4Conclusions

Differential pulse voltammetry in conjunction with the controlled growthmercurydropelectrodeisaperfecttoolforquantitativeanalysesofvitaminsTheadsorptionofvitaminB2andK3canbepreventedbytheadditionoftheneutralsurfactantTritonX-100whichselectivelyblockstheworkingelectrodesurface


Fig 3Cathodic voltammograms for the simulatanous determination of vitamin B2 B3 and K3Depictedintheinsetsarethevoltammogramsafterbackgroundsubtractionwiththecorresponding

ndash1callibrationplotsSupportingelectrolye04molL phosphatebuffer(pH=82)with40ppmTritonX-100AccumulationconditionsE =ndash005Vt =20sacc acc

The proposed methodology allows for the simultaneous determination ofmicromolaramountsofvitaminB2B3andK3Suchaprocedurewillhelptoreduce the time and costs of analyses of multivitamin formulations and foodproducts

Acknowledgments

RPhasbeenpartlysupportedbytheEUProjectPOWR0302-00-00-I00416

References

[1] Combs GF JrTheVitaminsFundamentalAspects inNutritionandHealth 3rd ed IthacaElsevierAcademicPress2008

[2] Lovander MD Lyon JD Parr DL Wang J Parke B Leddy J Review Electrochemicalpropertiesof13vitaminsAcriticalreviewandassessmentJElectrochmSoc165(2018)G18ndashG49

[3] Brunetti B Recent advances in electroanalysis of vitamins Electroanalysis 28 (2016)1930ndash1942

[4] BrubacherGMuller-MulotWSouthgateDATMethods forDeterminationofVitamins inFoodNewYorkElsevier1985

[5] Szpikowska-Sroka B A simple and sensitive analytical method for the determination ofthiamineinpharmaceuticalpreparationsJAnalChem68(2013)218ndash222

[6] PetteysBJFrankELRapiddeterminationofvitaminB (riboflavin)inplasmabyHPLCClin2

ChimActa412(2011)38ndash43[7] ZhangZXuJWenYZhangJDingWTheelectro-syntesizedimprintedPEDOTfilmasa

simple voltammetric sensor for highly sensitive and selective detection of vitamin K in3

poultrydrugsamplesSynthMet230(2017)79ndash88[8] JedlinskaKStrusMBasBAnewelectrochemicalsensorwiththeRefreshableSilverLiquid

Amalgam Film multi-Electrode for sensitive voltammetric determination of vitamin K2(menaquinone)ElectrochimActa265(2018)355ndash363

[9] SouthamptonElectrochemistryGroupInstrumentalMethodsinElectrochemistryChichesterHorwood1985


1Introduction

Carbonelectrodesarecommonlyappliedtosensitiveelectrochemicaldetectionofneurotransmittersegdopamine (nor)epinephrineandserotonin in-vivoandin-vitro[12]Neverthelessadsorptionofhigh-molecularweightbiomoleculesinthe matrix on a sensing electrode which then hinders the electron transferreactionofneurotransmitterswillresultinbiofoulingofelectrodesThisremainsachallengingproblemasbiofoulingwillcompromiseelectrochemicalmeasure-mentsThusseveralstrategies foraddressingbiofoulinghavepreviouslybeenreported[34] Thisworkreportsonaneffectiveapproachforminimisingbiofoulingbasedonthehypothesisthatahydrophobicelectrodesurfacewillrepelagainstadsorptionof amphiphilic biomolecules Briefly structurally small conical-tip electrodes

Dopamine detection at antifouling conical-tip carbon electrodes

a a a aSIMONABALUCHOVA JANKLOUDA JIR IBAREK KAROLINASCHWARZOVA -PECKOVA bDANNYKYWONG

a UNESCOLaboratoryofEnvironmentalElectrochemistryDepartmentofAnalyticalChemistryFacultyofScienceCharlesUniversityHlavova812800PragueCzechRepublicsimonabaluchovanaturcunicz

b DepartmentofMolecularSciencesMacquarieUniversitySydneyNSW2109Australia


AbstractA significant achievement in this work is the development ofantifoulingconical-tipcarbonelectrodes(~27micromtipdiameterand~165micromaxiallength)suitablefordetectionoftheneurotransmitterdopamine in-vivo These electrodes were hydrogenated using adiphenylsilanereductionmethodtoyieldahydrophobicsurfacetodeteradsorptionofamphiphilicbiomoleculesInitiallyhydrogenatedcarbonelectrodeswereelectrochemicallycharacterisedusingseveralredoxmarkersThedegreeofantifoulingwasthenassessedbythevoltammetricsignalchangeofdopamineattheseelectrodesbeforeand after being incubated in a fouling solution containing bovineserumalbumincytochromeC(bothareproteins)andcaproicacid(alipid) In our work we have obtained only a 69 (standarddeviation35N=40)decreaseindopaminesignalsatthehydro-genated carbon electrodes These results strongly support thediphenylsilanereductionstrategyforthedevelopmentofantifoulingbiosensorsfordopaminedetectioninbiologicalmatrices

Keywordsantifoulingelectrodesdiphenylsilanereduction

methodhydrogenatedconical-tip

carbonelectrodesvoltammetricdopamine

detection

(denoted as CTEs) are fabricated by thermally pyrolysing acetylene gas in anitrogenatmospheretodepositcarbonatthetipandontheshankofpulledquartzcapillaries [5] Spectroscopic studies confirmed that the electrode surface

2 3consistsofsp -likegraphiticcarbonandsp -hybridiseddiamond-likecarbon[6]In addition there is also a range of carbon-oxygen functionalities includingcarbonylquinonecarboxylphenolsalcoholsandethergroupsontheelectrodesurface[6]whichcaninteractwithspectatorbiomoleculesthroughdipole-dipoleorion-dipoleinteractionleadingtotheirirreversibleadsorptionontheelectrodesurface[7]HoweverbysubjectingthesecarbonelectrodestosilanereductionCndashObondsareconvertedtoCndashHbondsandphenolicgroupsaretransformedtosiloxane dendrimers [6] to yield a more hydrophobic carbon surface that isexpectedtobesimilarlylesssusceptibletobiofoulingcomparedtoboron-dopeddiamondelectrodes[78] In this work we will present a methodology involving diphenylsilanereduction to fabricate physically small hydrogenated conical-tip carbonelectrodes(denotedasHCTEs)withanti-foulingcapabilityBothCTEsandHCTEswere electrochemically characterised using several redox probes to elucidatetheirsurfacepropertiesbeforeevaluatingtheirresistancetobiofoulingduringdopaminedetectionin-vitro

2Experimental


Analyticalgradereagents(Sigma-AldrichAustralia)including4-methylcatecholhexaammineruthenium(III) chloride potassium hexacyanoferrate(III) dop-amine hydrochloride sodium phosphate dibasic citric acid perchloric acidpotassiumchloride sodiumhydroxide anhydrousdichloromethanediphenyl-silanetris-(pentafluorophenyl)boraneandgraphitepowderwereusedas-recei-ved Ultra-high purity gases acetylene and nitrogenwere obtained from BOCGases (Australia) All aqueous solutionswere preparedwith deionisedwater(MilliporeMiliplusQsystemUSA)witharesistivityof182MΩcm

22Instrumentation

Chronoamperometric and voltammetric experiments were carried out usingalow-currentpicostateDAQoperatedbyanEChemversion212softwareviaanE-corderinterface(eDAQPtyLtdAustralia)Athree-electrodeset-upinvolving

minus1eitheraCTEorHCTEasaworkingelectrodeaAgAgCl(3molL KCl)referenceelectrode (Bioanalytical SystemsUSA)andaplatinumwire counterelectrode

minus1(CypressSystemsUSA)wasusedCyclicvoltammetryatascanrateof100mVs anddifferential pulse voltammetry (pulse height +25mV pulsewidth 50ms

minus1sampling time 20 ms and scan rate 20mVs ) were used in this work All


electroanalyticalexperimentswereperformedinanaluminiumFaradaycageatanambienttemperature(23plusmn1degC)

23Preparationofhydrogenatedconical-tipcarbonelectrodes

Asreportedpreviously[5]structurallysmallCTEswerefabricatedbythermallypyrolysing C H (a pressure of 50 kPa) in a pulled quartz capillary (Sutter2 2

minus1InstrumentUSA)housedinaN atmosphere(counterflowof60mLmin )Prior2

to hydrogenation the catalyst tris(pentafluorophenyl) borane (100 mg) wasdissolvedinanhydrousCH Cl (50mL)bystirringfor5minbeforethehydroge-2 2

natingagentdiphenylsilane(25μL)wasaddedCTEswerethenplacedinthereagentmixturefor2hThepreparedHCTEsweredriedovernightbeforeuse

24Biofoulingexperiments

A laboratory synthetic fouling solution consisting of 4 (wv) bovine serumalbumin001(wv)cytochromeC(bothareproteins)and10(vv)caproicacid(alipid)waspreparedbyhomogenisingtheminapH=74citrate-phosphate

minus1buffer(01molL )AllfoulingcompoundswereacquiredfromSigmaAldrichAustralia


31Electrochemicalcharacterisation

minus1InthisworkallCTEswerecharacterisedbycyclicvoltammetryof10mmolL 3+ minus1[Ru(NH ) ] in10molL KClAsdisplayedinFig1(A)onlyCTEsthatshow3 6

asigmoidal-shapedvoltammogramwithasmallchargingcurrentwereemployedinfurtherexperimentsUsingchronoamperometry[5]ameantipdiameterof27μm(standarddeviation(SD)28μmN=142)ameanaxiallengthof165μm(SD=114μmN=142)wereestimatedfortheseCTEs TocomparesurfacecharacteristicsofbothCTEsandHCTEs cyclicvoltam-

minus1 3+2+ minus1 minus1metryof (1)10mmolL [Ru(NH ) ] in10molL KCl (2)10mmolL 3 63minus4minus minus1 minus1[Fe(CN) ] in 10 mol L KCl and (3) 10 mmol L 4-methylcatechol in6minus101molL HClO wasconductedatthesameelectrodesbeforeandafterhydroge-4

nation The results obtained are shown in Fig 1(A-C)We observed a ~20(SD=5N=10)decreaseinthelimitingcurrentofallthreeredoxmarkersafterdiphenylsilane reduction most likely attributable to the hindrance to theirelectron transfer reactions by the phenylsiloxane group formed on HCTEs

3minus4minusMoreover as an inner-sphere redox probe both [Fe(CN) ] and 4-methyl-6

catechol reactionsare sensitive to thepresenceofoxygen functionalitiesonacarbonsurface [7]Accordingly theconversionof these functionalities toCndashHbondsbydiphenylsilanereductionwasexpectedtoyieldmoresluggishelectron


transfer kinetics at HCTEs as supported by a negative potential shift (from3minus4minus+75mVtominus10mV)inthecyclicvoltammogramof[Fe(CN) ] andapositive6

potentialshift(from+580mVto+675mV)inthecorrespondingcyclicvoltammo-2gram of 4-methylcatechol In addition the conversion of sp -carbon to

3sp -diamond-likecarbon[6] isalsoexpectedtoreducetheconductivityof thecarbonelectrodesurface

32Dopaminedetectionduringbiofoulingexperiments

minus1Theelectrochemicalbehaviourof1mmolL dopamine inapH=74citrate-minus1phosphate buffer (01 mol L ) at CTEs and HCTEs was studied by cyclic

voltammetryTheresultsobtainedareshown inFig1(D)Acomparable12decrease(SD=6N=10)inthedopamineoxidationlimitingcurrenttothatof4-methylcatechol was observed A positive potential shift from +285 mV to+305mVinthevoltammogramsisalsoaccountedforasdescribedabove


minus1 3+ minus1 minus1Fig 1Cyclicvoltammetryof(A)10mmolL [Ru(NH ) ] in10molL KCl (B)10mmolL 3 63minus4minus minus1 minus1 minus1[Fe(CN) ] in10molL KCland(C)10mmolL 4-methylcatechol in01molL HClO at3 4

minus1(a)aCTEand(b)aHCTE(D)10mmolL dopamineinapH=74citrate-phosphatebufferrecordedminus1at(a)aCTEandaHCTE(b)beforeand(c)afterbiofoulingScanrate100mVs

Next HCTEs were incubated in a synthetic fouling solution containing4(wv)bovineserumalbumin001(wv)cytochromeC(bothareproteins)and 10 (vv) caproic acid (a lipid) for 30 min Cyclic voltammetry of

minus110mmolL dopamineattheseHCTEswasthenconductedinapH=74citrate-phosphatebuffertoevaluatetheirantifoulingpropertyNotablyaconsiderable515(SD=183N=6)decreaseindopaminesignalwasobservedatCTEsIncontrastonlyacorresponding69decrease(SD=35N=40)wasestimatedatHCTEsasshowninFig1(D)Clearlythisrepresentsamajorimprovementinthe antifouling capability of HCTEs obtained using diphenylsilane reductioncomparedtoCTEsandotherpreviouslytestedhydrogenatingagents[9]includingn-butylsilane (35decrease) triethylsilane (23decrease) andphenylsilane(18decrease)Thereforethisworkhassuccessfullydemonstratedtheeffecti-venessofdiphenylsilanereductionmethodindevelopingantifoulingelectrodesfordopaminedetection


Smallconical-tipelectrode

As-prepared Hydrogenated

minus1LinearrangemicromolL 1ndash10 1ndash20minus2 minus2Intercept10 pAmicrom 108plusmn004 273plusmn011

minus3 minus2 minus1Slope10 pALmicrom micromol 128plusmn007 443plusmn012R 0993 0998

minus1LimitofdetectionmicromolL 100 077

Table 1Analyticalparametersofconcentrationdependencesofdopamineobtainedbydifferentialpulse

minus1voltammetryinapH=74citrate-phosphtebuffer(01molL )Allquoteduncertaintiesrepresentthe95confidenceintervalandthecorrelationcoefficient(R)wasfoundtobestatisticallysigni-ficantatthe95usingStudentrsquost-test

Fig 2DifferentialpulsevoltammetryofdopamineataHCTEinapH=74citrate-phosphatebufferminus1 minus1(01molL )atconcentrations(a)1(b)2(c)4(d)6(e)8(f)10and(g)20μmolL

WehavealsostudiedtheconcentrationdependenceofdopamineinapH=74citrate-phosphatebufferbydifferentialpulsevoltammetryAtypicalcalibrationplotobtainedisshowninFig2Theanalyticalparametersandestimatedlimitsofdetection are summarised inTable 1 These results show thatHCTEs outper-formed CTEs because they exhibit a ~35times higher sensitivity a 23 lowerdetectionlimitandawiderlinearrange

4Conclusions

InthisstudyphysicallysmallHCTEswithanti-foulingcharacteristicsachievedbyhydrogenationusingdiphenylsilanereductionweresuccessfullyfabricatedandelectrochemically characterised using several redox probes Next dopaminedetectionwasperformedbeforeandafterincubationofelectrodesinasyntheticfoulingsolutioncontainingahighconcentrationofbiomoleculesOnlyalow69(SD=35)decreaseindopaminelimitingcurrentwasachievedatHCTEsobtain-edbydiphenylsilanereductionindicatingtheirsignificantlylesssusceptibilitytobiofoulingthanCTEsThesepromisingresultsindicatethatantifoulingHCTEswillpotentiallybenefitthedevelopmentofbiosensorsfordopaminedetectionin-vivoinbiologicalmedia

Acknowledgments

ThisresearchwasperformedwithintheframeworkofSpecificUniversityResearch(SVV260560)FinancialsupportsprovidedbytheGrantAgencyofCharlesUniversity(project390119)andbytheCzechScienceFoundation(project20-03187S)aregratefullyacknowledgedSBandJKalsothankthe Mobility Fund of Charles University and Hlavkova nadace for providing funding for theirresearchinternshipsatMacquarieUniversitySydneyAustralia

References[1] BaranwalAChandraPClinicalimplicationsandelectrochemicalbiosensingofmonoamine

neurotransmittersinbodyfluidsinvitroinvivoandexvivomodelsBiosensBioelectron121(2018)137ndash152

[2] CaoQPuthongkhamPJillVentonBReviewnewinsightsintooptimizingchemicaland3Dsurface structuresof carbonelectrodes forneurotransmitterdetectionAnalMethods11(2019)247ndash261

[3] LinP-HLinB-RAntifoulingstrategiesinadvancedelectrochemicalsensorsandbiosensorsAnalyst145(2020)1110ndash1120

[4] HanssenBLSirajSWongDKYRecentStrategiestoMinimiseFoulinginElectrochemicalDetectionSystemsRevAnalChem35(2016)1ndash28

[5] McNallyMWongDKYAnin-vivoprobebasedonmechanicallystrongbutstructurallysmallcarbonelectrodeswithanappreciablesurfaceareaAnalChem73(2001)4793ndash4800

[6] SirajSMcRaeCRWongDKYEffectiveactivationofphysicallysmallcarbonelectrodesbyn-butylsilanereductionElectrochemCommun64(2016)35ndash41

[7] ParkJShowYQuaiserovaVGalliganJFinkGDSwainGMDiamondmicroelectrodesforuseinbiologicalenvironmentsJElectroanalChem583(2005)56ndash68

[8] ShinDTrykDAFujishimaAMerkociAWang JResistance to surfactantandproteinfoulingeffectsatconductingdiamondelectrodesElectroanalysis17(2005)305ndash311

[9] Roshni RAnAntifouling Structurally Small Carbon Electrode forDetectionof theNeuro-transmitterDopaminePhDThesisMacquarieUniversitySydney2019


1Introduction

Theanalyticalperformancesofenzymaticbiosensorsarestronglyaffectedbytheenzyme immobilization process There is no universal technique for enzymesattachmentThereforespecialattentionshouldbepaid to theselectionof theappropriatesupportandthedevelopmentoftheoptimalbindingstrategyinordertoensure thebestcharacteristicsof immobilizedenzymeDespiteavarietyofpreviouslyreportedcovalentimmobilizationmethodsfordifferentenzymesthepresentedprocedurescanbehardlycomparedtofindtheoptimalonesbecauseofdifferentanalyticalmethodsandexperimentalconditionsusedUptodatethere

A comparative study of covalent glucose oxidase and laccase immobilization techniques at powdered supports for biosensors fabrication

ab a bSOFIIATVORYNSKA JIR IBAREK BOHDANJOSYPCUK

a UNESCOLaboratoryofEnvironmentalElectrochemistryDepartmentofAnalyticalChemistryFacultyofScienceCharlesUniversityHlavova2030812843Prague2CzechRepublicsofiiatvorynskajh-instcascz

b JHeyrovskyacuteInstituteofPhysicalChemistryoftheCzechAcademyofSciences Dolejškova318223Prague8CzechRepublic


AbstractInordertodeveloptheoptimalstrategyandtodeepentheknowledgeinthefieldofenzymeimmobilizationthreedifferenttechniquesofcovalentbindingfortwoenzymes(glucoseoxidaseandlaccase)atpowdered surfaces were compared Immobilization protocol wasoptimized by changing supports (twomesoporous silica powders(SBAminus15 MCMminus41) and a cellulose powder) the functionalizedgroupsintroducedatsupportsurfaces(minusNH andminusCOOH)andthe2

methodsofactivation(glutaraldehydeandcarbodiimide)Aminoandcarboxyl functionalized mesoporous silica and cellulose powderswerepreparedbysilanizationusing(3-aminopropyl)triethoxysilaneandcarboxyethylsilanetriolrespectivelyItwasfoundthatcouplingof both enzymes by their ndashNH groups through glutaraldehyde to2

ndashNH functionalized supports in particular SBA15minusNH and2 2

celluloseminusNH forglucoseoxidaseMCM41minusNH forlaccaseshowed2 2

thehighestactivityandthebeststability

Keywordsbiosensorcovalentimmobilizationenzymaticreactorglucoseoxidaselaccase

is still a lack of the comparative systematic studies focusing on the enzymesimmobilizationonthevarioussupportsusingdifferenttechniques The aim of this work is the systematic comparative study of the differenttechniques for covalent coupling of the enzymeswhich ensures not only thedevelopmentoftheoptimalimmobilizationstrategyfortheselectedenzymesbutalsoenablestofindoutsometendenciesinenzymeattachmentprocessgenerallyThusthisworkisfocusedonadetailedanalysisoftheeffectofthekindofsupportits anchor groups and the activation methods on activity and stability ofimmobilizedenzymesTwoenzymeswithdifferentnature(glucoseoxidase(GOx)andlaccase(Lac))werechosenasthetestingbioreceptors

2Experimental


AllchemicalswereofpaorbettergradeGlucoseoxidasefromAspergillusnigerminus1(GOxEC11341452Umg )laccasefromTrametesversicolor(LacEC11032

minus1129Umg )D-(+)-glucosedopamineglutaraldehyde(GAgradeII25aqueoussolution) N-(3-dimethylaminopropyl)-Nʹ-ethylcarbodiimide hydrochloride(EDC ge980) N-hydroxysuccinimide (NHS ge970) (3-aminopropyl)-triethoxysilane (APTES) mesoporous silica powder SBAminus15 (particle size

2 minus12ndash6μmporesizeasymp7nmsurfaceareaasymp600m g )mesoporoussilicapowder2 minus1MCMminus41 (pore size 21ndash27 nm surface area asymp 1000 m g ) cellulose (Cell

microcrystalline powder particle size 20 microm) were purchased from SigmaAldrichCarboxyethylsilanetriol(CEST25aqueoussolution)waspurchased

regfromabcr (Germany)

22Instrumentation

Amperometric measurements were carried out at room temperature usingcomputer-controlled electrochemical stand (Polaro-Sensors Czech Republic)withMultiElchemv31software(JHeyrovskyInstituteofPhysicalChemistryoftheCAS)Flowinjectionanalysis(FIA)withthethree-electrodelaboratory-madeflow-through cellwas usedworking electrode minus tubular detector of polishedsilversolidamalgam(TD-p-AgSAlaboratory-madeinnerdiameter05mmtheamalgamtube length60mm) referenceelectrodeminusaminiaturizedsaturatedcalomelelectrodebasedonsilverpasteamalgam[1](laboratory-madeithasthesamepotential as classical saturated calomel electrode) auxiliary electrodeminusplatinum wire (diameter 10 mm length 10 mm) The system for FIA withelectrochemicaldetectioncomprisedofalinearsyringepumpa2-position6-port

regsampleinjectorvalveaninjectionlooplaboratory-madeofTeflon (PTFE)tubing(100μL) a solenoid operatedmicro-pumpan enzymatic reactor and a flow-through cell for TD The enzymatic reactor consists of a tube filled by theenzymaticpowder



Basedonthedatareportedintheliterature[23]andontheresultsofourpreviousworks[4ndash8]forthisstudyCellandmesoporoussilicapowders(namelySBAminus15andMCMminus41)havebeenselectedas thepotentialpromisingsupports for thecovalent enzyme immobilizationBecauseof thehigh contentof surficial ndashOHgroupswhich are capable of chemical reactions these supports can be easilyfunctionalizedThewell-knownandfrequentlyutilizedtechniqueofsilanizationhasbeenusedtomodifythesurfacesofSBA15MCMminus41andCellbythedesiredfunctionalizedgroupsAminosilaneAPTESwasappliedtoformminusNH groupson2

thematrixsurfaceswhereascarboxylsilaneCESTwasusedtointroducendashCOOHgroups Generallytheprocedureofthecovalentimmobilizationofenzyme(eitherLacorGOx)onthefunctionalizedsupportconsistsofthreestepsI Synthesisofthefunctionalizedsupportwhichmeansthemodificationofthematrix(MCMminus41SBAminus15andCell)withsuitableanchoredgroups(minusNH or2

minusCOOH)II Activationstepofthefunctionalizedsupportwithspecificactivatingagents(glutaraldehydeorEDCNHS)tomakeitreactivetowardsenzyme

IIIEnzyme(LacorGOx)couplingtotheactivatedsupport

To investigate the effect of support its surface functionalizedgroups and themethodsofactivationontheefficiencyofthecovalentenzymeimmobilizationthreedifferentstrategies(ABandC)forLacandGOxattachmenthavebeenusedThedetailsoftheusedtechniquesandthedenotationsofthepreparedenzymaticpowdersaresummarizedinTable1(nextpage)ToexaminetheefficiencyofLacandGOximmobilizationtheenzymaticreactors(filledbytheenzymaticpowderspreparedwithdifferenttechniques)coupledwithTDwereusedforamperometricdetermination of dopamine and glucose respectively in flow systems Theprincipleofglucosedetection isbasedonamperometricmeasurementsof theenzymatically consumed oxygen whereas dopamine was detected by thereductionoftheenzymaticallyoxidiseddopamine Asdepicted inFig1 thebiosensors responsesare stronglyaffectedby thestrategyusedforLacorGOximmobilizationAsshowntheresponsesofLacandGOx biosensors decrease in the order strategy A gt strategy B gt strategy Cirrespectiveofthetypeofsupport It isclearlyseenthataminofunctionalizedsupports(SBA15minusNH MCM41minusNH andCellminusNH )providehigheractivitiesof2 2 2

the immobilized Lac andGOx than these supports functionalized by carboxylgroups(SBA15minusCOOHMCM41minusCOOHandCellminusCOOH)BycomparingactivitiesofimmobilizedenzymesusingstrategiesAandBtheinfluenceoftheactivationagenthasbeenevaluatedThebestresultsforbothenzymeswereobtainedforndashNH functionalizedsupportsactivatedbyGAItcouldbeexplainedbythefact2

thatGAcontrary tocarbodiimidewithnonemolecularspaceprovidesa long


spacerarmensuringminimalsterichindrancesforenzymesbindingItcanbeconcludedthatthecovalentimmobilizationofbothenzymesbytheirndashNH groups2

viaGAtondashNH functionalizedmesoporoussilicapowders(strategyA)provided2

the highest activities Interestingly in the similar comparative studies it isreportedthatamongndashOHminusCOOHandndashNH functionalizedsupportsactivatedby2

divinylsulfonecarbodiimideandGArespectivelythelastonewasfoundasthemostsuitabletechniqueforthecovalentbindingofLac[9]invertase[10]andpepsin[11]


Tab

le 1

Theprinciplesofthecovalentimmobilizationmethodsofenzymesusedinthisstudy

StrategyA

StrategyB

StrategyC

Support

SBAminus15MCMminus41Cell



Supportfunctio-

minusNH

minusNH

minusCOOH

22

nalizedgroup

Activationagent

Glutaraldehyde(GA)

Carbodiimide

Carbodiimide

(EDCNHS)

(EDCNHS)

Enzymereactive

minusNH

minusCOOH

minusNH

22

group

Typeofbond

secondaryamine

amide

amide

Denotationsofthe

GOxminusNHminusCHminus(CH)minusCHminusNHminusSBA15

GOxminusC(=O)minusNHminusSBA15

GOxminusNHminusC(=O)minusSBA15

23

preparedenzy-

GOxminusNHminusCHminus(CH)minusCHminusNHminusMCM41

GOxminusC(=O)minusNHminusMCM41

GOxminusNHminusC(=O)minusMCM41

23

maticpowders

GOxminusNHminusCHminus(CH)minusCHminusNHminusCell

GOxminusC(=O)minusNHminusCell

GOxminusNHminusC(=O)minusCell

23

LacminusNHminusCHminus(CH)minusCHminusNHminusSBA15

LaxminusC(=O)minusNHminusSBA15

LacminusNHminusC(=O)minusSBA15

23

LacminusNHminusCHminus(CH)minusCHminusNHminusMCM41

LacminusC(=O)minusNHminusMCM41

LacminusNHminusC(=O)minusMCM41

23

LacminusNHminusCHminus(CH)minusCHminusNHminusCell

LacminusC(=O)minusNHminusCell

LacminusNHminusC(=O)minusCell

23

When the effect of the method of the covalent enzyme coupling on thebiosensorstabilitywasevaluateditwasfoundthatLacboundedtondashNH functio-2

nalizedsupportsviaGA(strategyA)hasshownthehigheststability(gt65oftheinitial responses after 1 month) compared to other strategies whereas GOximmobilizedwithtwostrategies(AandB)possessedapproximatelysimilarhighstability(gt80oftheinitialresponsesin1month)BothenzymesboundedviandashNH groupstondashCOOHfunctionalizedsupportsthroughEDCNHS(strategyC)2

showedquitelowstability

4Conclusions

Threedifferent strategies including the support selection the anchor surfacegroups and the activationmethod havebeen compared for efficient covalentimmobilization of Lac and GOx The results showed that ndashNH functionalized2

supports(SBA15minusNH CelluloseminusNH forGOxandMCMminusNH forLac)activatedby2 2 2

GAmaybeusedtoeffectivelybindenzymesintermsofhighactivityandstability

Acknowledgments

ThisworkwasfinanciallysupportedbytheGrantAgencyofCharlesUniversityinPrague(Project1356120)theGrantAgencyoftheCzechRepublic(Project20-07350S)anditwascarriedoutwithintheframeworkofSpecificCharlesUniversityResearch(SVV260440)

References

[1] YosypchukBBarekJYosypchukOPreparationandpropertiesofreferenceelectrodesbasedonsilverpasteamalgamElectroanalysis23(2011)2226minus2231


Fig 1Effectof the covalent attachment techniqueson (A) laccaseand (B) andglucoseoxidaseminus1biosensor responses Experimental conditions (A) c = 500 micromol L E = minus50 mVDOP det

minus1 minus1v = 01 mL min V = 40 microL carrier solution 01 mol L acetate buffer pH = 48flow DOPminus1 minus1(B)c =500micromol L E =minus1100mVv ==01mLmin V =40microL carrier solutionGlu det flow Glu

minus1 minus101molL acetatebuffer0001molL Na EDTApH=652

[2] LiuYChenJYEnzymeimmobilizationoncellulosematrixesJBioactCompactPolym31(2016)553ndash567

[3] Hartmann M Kostrov X Immobilization of enzymes on porous silicas ndash benefits andchallengesChemSocRev42(2013)6277minus6289

[4] JosypcukOBarekJJosypcukBElectrochemicalbiosensorsbasedonenzymaticreactorsfilledbyvarioustypesofsilicaandamalgampowdersformeasurements inflowsystemsElectroanalysis28(2016)3028minus3038

[5] Josypcuk O Barek J Josypcuk B Amperometric determination of catecholamines byenzymaticbiosensorsinflowsystemsElectroanalysis30(2018)1163minus1171

[6] TvorynskaSBarekJJosypcukBAmperometricbiosensorbasedonenzymaticreactorforcholinedeterminationinflowsystemsElectroanalysis31(2019)1901minus1912

[7] TvorynskaSBarekJJosypcukBFlowamperometricbiosensorbasedontwoenzymaticreactors (acetylcholinesterase-choline oxidase) for the detection of neurotransmitteracetylcholine In Proceedings of the 15th International Students Conference ldquoModernAnalyticalChemistryrdquoKNesmerak(ed)PragueFacultyofScienceCharlesUniversity2019p61minus66

[8] TvorynskaSBarekJJosypcukBAcetylcholinesterase-cholineoxidase-basedmini-reactorscoupledwithsilversolidamalgamelectrodeforamperometricdetectionofacetylcholineinflowinjectionanalysisJElectroanalChem860(2020)113883

[9] RekucAKruczkiewiczPJastrzembskaBLiesieneJPeczynska-CzochWBryjakJLaccaseimmobilizationonthetailoredcellulose-basedGranocelcarriersIntJBiolMacromol42(2008)208minus215

[10] Bryjak J Liesiene J S tefuca V Man-tailored cellulose-based carriers for invertaseimmobilizationCellulose15(2008)631minus640

[11] SzałapataKOsinska-JaroszukMBryjakJJaszekMJarosz-WilkołazkaANovelapplicationofporousandcellularmaterialsforcovalentimmobilizationofpepsinBrazJChemEng33(2016)251minus260


1Introduction

Carbohydrates are crucial for energy structure and signaling in the humanbody[1]Thereisavarietyofcarbohydratesbutthemostimportantoneforlifeisglucoseasitisfundamentalinthemetabolismandphotosynthesis[2]GlucoseisclassifiedashexoseThesemonosaccharidesonlydifferinthepositionofhydroxylsubstituentsinsomecasesInadditiontothestructuralsimilaritiesthesemole-culeslackachromophoreandarenoteasilyionizable(pK ~12)Thusdetectiona

intheUVregionandseparationofanalytesbycapillaryelectrophoresis(CE)arechallenging [1 3] At the moment there are many different techniques for theanalysis of carbohydrates commonly including time-consuming derivatizationstepsoreluentswithhighpHvalues(pHgt12)inionchromatographyAwell-esta-blishedtechniquefortheanalysisofcarbohydratesishigh-performanceanion-exchangechromatographywithpulsedamperometricdetection(HPAE-PAD)[4]Electrochemical detection like AD is matching miniaturization simple instru-mentationlowcostandrobustnessandthusisoftenusedforflow-basedsystemssuchasCEandflowinjectionanalysis(FIA)[5]

Capillary flow injection analysis with electrochemical detection for carbohydrate analysis

NICOLEHEIGLFRANK-MICHAELMATYSIK

InstituteofAnalyticalChemistryChemo-andBiosensorsFacultyofChemistryandPharmacyUniversityofRegensburgUniversitaumltsstraszlige3193053RegensburgGermanynicoleheiglchemieuni-regensburgde


AbstractAsimplecapillaryflowinjectionanalysissystemwithamperometricdetection was arranged for the development of a method for fastoptimization of detection conditions in the context of thedetermination of carbohydrates by means of electrochemistry-capillary electrophoresis-mass spectrometry This setup is free ofelectricalinterferencebyhighvoltageandisperfectforstudyingtheoxidationofvariousanalytesFurthermoreitassureseasycouplingtoMS and thus is an useful tool to investigate the correspondingoxidationproductsofananalyte

Keywordscapillaryflowinjection

analysiscarbohydratesmassspectrometrydisposableelectrodespulsedamperometric

detection

Inthiscontributioncapillaryflowinjectionanalysis(CFIA)withADwillbepresentedasamethodtoapplyandtestADforthedetectionofmonosaccharidesondifferentdisposablethin-filmorscreen-printedelectrodesCFIAwaschosenoverconventionalFIAforthispurposeasthegravityflowinCFIAisstableforalongertimeandverylowsampleconsumptioncanbeachieved[6]TheCFIAsystemwasarrangedassimpleaspossibleandperformedhydrodynamicallytoavoidanyinterferencesFurthermoreitassuresthecouplingoftheflowsystemtoamassspectrometerThusthesamesetupasusedforCFIAcanbeutilizedforcapillaryelectrophoresis-massspectrometry(CE-MS)experimentsbychangingthe flow through the electrochemical flow cell in opposite direction In futureexperimentstheexperienceintermsofADonthoseelectrodeswillbeusedtodevelopelectrochemicalpretreatmentprotocolsforcarbohydratedeterminationbyCE-MS

2Experimental


The following chemicals were used for this study all of analytical gradeAmmoniumacetate(NH OAc)wasobtainedfromMerck(DarmstadtGermany)4

and ferrocene methanol (FcMeOH) from ABCR (Karlsruhe Germany) Milli-Qregwater(182MΩcm)wasgeneratedbyaMilli-QAdvantageA10 system(Merck

Millipore Darmstadt Germany) Carrier solution was prepared by dissolvingndash1NH OAc (50mmolL ) in Milli-Q water FcMeOH solution was prepared by4

dissolvingFcMeOHincarriersolution

22Instrumentation

Electrochemical measurements were performed using a microAutolab Type IIIpotentiostatgalvanostat (Metrohm Autolab B V Utrecht Netherlands)controlledbyNOVA20softwareforexperimentalcontrolanddataacquisitionCFIAwasperformedusingthesetupillustratedinFig1(A)consistingofacarrierreservoirsamplevialandtwofusedsilicacapillaries(PolymicroTechnologiesPhoenix AZ USA inner diameter 100 microm length inlet 40 cm length outlet10cm)connectedtoacommerciallyavailableflowcellfromMicruxTechnologies(model ED-FLOW-CELL Oviedo Spain) Inside of the flow cell the fused silicacapillary was placed in a so-called wall-jet configuration above the workingelectrode of a disposable thin-film gold electrode (model ED-SE1-Au MicruxTechnologiesOviedoSpain)ascanbeseeninFig1(B)Thethin-filmelectrodeswerebasedonathree-electrodesystemwithagoldworkingauxiliaryandquasi-referenceelectrodeThehydrostaticpressurewasachievedbyaheightdifferencebetweeninletandoutletreservoirof30cmresultinginagravityflowofthecarriersolutionthroughafusedsilicacapillaryandsubsequentlythroughtheflowcell


Theinjectionwascarriedoutbyloweringthevialcontainingthecarriersolutiontotheleveloftheoutletexchangingthecarrierreservoirwiththesamplevialandliftingthesamplevialto20cmforadefinedperiodoftimeRe-establishingthecarrierreservoirtookplacethesameway


AsimpleCFIA-ADsystemwasarrangedwherehydrostaticpressurebyaheightdifferencebetweeninletandoutletreservoirresultedinagravitationalflowToobtain general information about the behavior of the assembled CFIA systempreliminaryexperimentswithFcMeOHwereperformedToassurecompatibilitywithMSlateronNH OAcwaschosenastheelectrolyteVariousheightdifferences4

andinjectiontimesweretestedandtheinjectionataheightdifferenceof20cmlasting for 10 s was found to be the optimum concerning feasibility and peakshapes Injections of several solutions of FcMeOH of different concentrationsshowed that the concentration dependence of FcMeOH was linear in theinvestigatedrange(Fig2)Furthermoreexperimentsrevealedthattheinjectionprocedure was established with reasonable precision When repeating the

ndash1injectionof05mmolL FcMeOHincarriersolutionfortentimestherelativestandarddeviationwasfoundtobe3forthemanualinjectionprotocol


Fig 1(A)Schemeoftheusedcapillaryflowinjectionanalysissetup(1)carrierreservoir(2)samplevial(3)inletcapillarywithalengthof40cmandaninnerdiameterof100microm(4)outletcapillarywithalengthof10cmandaninnerdiameterof100micrombothcapillariesconnectedto(5)acommerciallyavailableflowcellfromMicruxand(6)awastevial(B)Configurationinsidetheflowcell(7)thefusedsilicacapillarywasplacedinaso-calledwall-jetconfigurationabovetheworkingelectrodeof(8)adisposablethin-filmgoldelectrode

4Conclusions

The presented system for CFIA-AD was arranged as simple as possible andrepresents a useful approach for the development of a method for fastoptimization of detection conditions in the context of the determination ofcarbohydratesbymeansofelectrochemistry-CE-MSThesetupisfreeofelectricalinterferencebyhighvoltagecompatiblewithMSandthuspromisingforstudyingtheoxidationofvariousanalytes

References

[1] LuGCrihfieldCLGattuSVeltriLMHollandLACapillaryelectrophoresisseparationsofglycansChemRev118(2018)7867ndash7858

[2] GalantALKaufman RCWilson JDGlucoseDetectionandanalysisFoodChem188(2015)149ndash160

[3] Sarazin C Delaunay N Costanza C Eudes V Gareil P Application of a new capillaryelectrophoreticmethodforthedeterminationofcarbohydratesinforensicpharmaceuticalandbeveragesamplesTalanta99(2012)202ndash206

[4] Rohrer JS Basumallick L Hurum D High-performance anion-exchange chromatographywithpulsedamperometricdetectionforcarbohydrateanalysisofglycoproteinsBiochem78(2013)697ndash709

[5] IslamMAMahbubPNesterenkoPNPaullBMackaMProspectsofpulsedamperometricdetectioninflow-basedanalyticalsystemsndashAreviewAnalChimActa1052(2019)10ndash26



Fig 2ndash1 ndash1(A) CFIA-AD recordings of three consecutive injections of (1) 025mmolL (2) 05mmolL

ndash1 ndash1 ndash1(3) 075mmolL and (4) 1mmolL FcMeOH in 50 mmolL NH OAc detection at a Micrux4

thin-filmAuelectrodeataconstantpotentialof03VinaflowcellHydrodynamicinjectionlasted10sataheightdifferenceof20cm(B) Calibration dependence of FcMeOH for CFIA-AD determination and detection at a Micruxthin-filmAuelectrodeataconstantpotentialof03VinaflowcellThestandarddeviationsofpeakheights(n=3)areindicatedbyerrorbars

1Introduction

Topreventsorptiononthecapillarysurfaceandimproveseparationefficiencyand selectivity of determined analytes coatings are formed on the capillarysurface There are two types of ones namelydynamic and covalently bondedcoatings Despite the simplicity of creation dynamic coatings cannot providerequiredreproducibilitywhilecovalentcoatingscontributethestableelectro-osmoticflow(EOF)andhighreproducibilityoftheanalysisInmostcasesthe

Application of covalent coatings based on imidazolium cations for separation and on-line preconcentration of basic and neutral analytes in capillary electrophoresis

a ab aANASTASIAVKRAVCHENKO EKATERINAAKOLOBOVA LIUDMILAAKARTSOVA

a DepartmentofOrganicChemistryInstituteofChemistrySaintPetersburgStateUniversity 26Universitetskiiprospect198504StPetersburgPeterhofRussiakravchenko161216gmailcom

b TheFederalStateInstituteofPublicHealthldquoTheNikiforovRussianCenterofEmergencyandRadiationMedicinerdquoTheMinistryofRussianFederationforCivilDefenceEmergenciesandEliminationofConsequencesofNaturalDisasters54Optikovst197082StPetersburgRussia


AbstractThemethodofcapillaryelectrophoresis (CE) isactivelydevelopedandmoreandmoreattractsscientistsattentioneveryyearHoweverthesorptionofanalytesonsurfaceoffused-silicacapillarywallsisoneof thesignificantdisadvantagesof thisapproachThe formationofcoatings on the inner capillary surface is typical way to preventsorption and to increase separation efficiency and selectivity ofdetermined analytes Coatings that covalently bonded to capillarywalls is more suitable because they are stable and provides highreproducibilityofanalysisThepresentworkisfocusedonthedevelo-pmentofthemethodofelectrophoreticdeterminationofbiologicalactiveanalytesusingacovalentcoatingbasedonimidazoliumcationsTheeffectofsubstituentinimidazoliumringonmainelectrophoreticparameters was examined It was shown that alkylimidazoliumcoatingscontributetosignificantreducingofbiogenicamineslimitsof detection while β-cyclodextrinimidazolium covalent coatingallowstoseparatebothofhydrophobicandhydrophilicanalytesinonerun

Keywordsbiologicalactiveanalytescapillarycoatingcapillaryelectrophoresisimidazoliumionicliquids

analytes nature determines type of usedmodifiers because suitable ones canprovideaccessorial interactionbetweentheanalytesandthestationaryphaseimproving separation selectivity and efficiency [1 2] Ionic liquidshave beenwidelyusedinanalyticalchemistry[3]andseparationtechniquesparticularlyincapillaryelectrophoresis[4]Earlyresearches[5ndash9]haveshownopportunityofcovalently bonded imidazolium ionic liquids for electrophoretic separationHowever the effect of various substituents in imidazolium ring hasnot beendescribed previously Thus the purpose of this study was to create covalentcoatingsbasedon ionic liquidwithvarioussubstituentsand tocompare theiranalytical capabilities in the electrophoretic separation of biologically activecompounds

2Experimental


(3-Glycidyloxypropyl)trimethoxysilane (GPTMS) hydrochloric acid sodiumdodecyl sulfate (SDS) imidazole 22-diphenyl-1-picrylhydrazyl (DPPH)p-toluen-sulfonylchlorideβ-cyclodextrinhydrocortisone(F)11-deoxycortisol(S)Corticosterone(B)rac-ketoprofen(ndash)-adrenaline(A)L-(ndash)-norepinephrine(NE) DL-normetanephrine (NMN) dopamine (DA) DL-metanephrine hydro-chloride(Met)serotoninhydrochloride(Ser)homovanillicacid(HVA)24-di-hydroxy-benzoic acid (24-DHBA) 34-dihydroxy-L-phenylalanine (DOPA)L-tryptophan(Trp)L-tyrosine(Tyr)werepurchasedfromSigma-Aldrich(USA)1-Bromo-butane1-bromooctanewerepurchasedfromReagentPlus(Ukraine)Sodium dihydrogenphosphate dihydrate acetone NN-dimethylformamide(DMF)wereobtainedfromMerck(Germany)AllreagentsusedwereanalyticalgradeAllsolutionswerepreparedusingdeionizedwater

22Instrumentation

Capillary electrophoresis experiments were carried out using the system ofcapillaryelectrophoresisCAPEL-105M(LumexRussia)withUV-spectrophoto-metricdetector(wavelengthrange190ndash360nm)Separationswereperformedusing 58times49 cm (9 cm to the detector outside diameter 360 microm and innerdiameter50microm) coated silica capillaries (LumexRussia) Thebuffer pHwasmeasuredwithapH-meterHI2210ndash2216(Hanna)

23Capillarycoatingsynthesis

Earlier our research team has proposed the synthesis route for the covalentcoatingsbasedonimidazoliumcationfunctionalizedwithalkylgroup[10]andβ-cyclodextrin [11] All capillarieswere prepared according above-mentioned


manuscriptsandcharacterizedbytheEOFmobilitymeasurementandscanningelectronmicroscopyThesynthesisconsistedoffollowingstepspreparationofacapillarytocreateacovalentcoating(heatingcapillaryfilledwith2MNaOHat90degCfor1handdryingfollowed)silylationwithGPTMSandfunctionalizationwiththeimidazolesolutionfollowedmodificationbybutyl-andoctylbromideortosyl-β-cyclodextrin(seedetailsin[10]and[11])

24Solutions

A stock buffer solution in concentration 50mM was prepared by dissolvingappropriate amount of sodium dihydrogenphosphate dihydrate in deionizedwateradjustingpHto20with1MhydrochloricacidThisbuffersolutionwasthendilutedwithdeionizedwater

ndash1 Allthesamplestocksolutionswerepreparedwithconcentration10mgmL Thestocksolutionsoftheneurotransmittersandtheirmetabolites(adrenalinenoradrenaline dopamine normetanephrine metanephrine serotonin homo-vanillicacid)and24-dihydroxybenzoicacidasinnerstandardandaminoacids(tryptophan34-dihydroxy-L-phenylalanine tyrosine)wereprepared in01MhydrochloricacidThestocksolutionsofsteroids(hydrocortisone11-deoxycor-tisolandcorticosterone)wereprepared inacetonitrileThestocksolutionsofketoprofen racemate and S-ketoprofen were prepared in acetonitrilewatersolution(1090vv) Untilelectrophoreticanalysisthestocksolutionswerestoredatndash16degCTheworkingsolutionswerepreparedbydilutingtheinitialsolutionswithwaterjustbeforetheexperiments


CovalentcoatingsbasedonN-alkylimidazoliumcationwereespeciallysuitableforseparationofneurotransmittersandtheirmetabolites(Fig1)Inadditionthecombinationofcovalentcoatingwithon-linepreconcentrationtechniquesallowstothesignificantdecreaseoftheseanalyteslimitsofdetection(LOD)Accessorialinteractions positively charged analytes with positively charged imidazoleimproveseparationselectivity(viaπ-πinteraction)andefficiency(concentrationintightzonesviaelectrostaticrepulsion)Sodiumdodecylsulfate(SDS)addedintobackgroundelectrolyte(inconcentrationabovecriticalmicelleconcentra-tion) strongly interacts with hydrophobic alkyl groups in covalent coatingstructureThenegativelychargedSDSlayerisformedoninnercapillarysurfaceThedoublereversingEOFallowsustocarryoutelectrokineticinjectionofsampleandon-linepreconcentrationbysweepingsimultaneouslyLODweredeclineto

ndash108ndash20ngmL ThelengthofalkylsubstituentalsoaffectsthestackingefficiencyfactorandLODMorehydrophobicoctylgroupscomparetobutylprovidemoreeffectiveinteractionwithSDSandasresultlowerLOD


Covalentcoatingmodifiedβ-CDhasnotshownsharpreducingofLODbyon-linepreconcentrationStacking sweeping (SDSasmicelle reagent) field-enhancedsample injection were examined using different model mixtures of analytesNeverthelessthiscoatingallowssimultaneousseparationofbothofhydrophobicsteroidhormonesandhydrophilicbiogenicaminesinasinglerun(Fig2) The guest-host interaction hydrophobic cavity of β-cyclodextrin with thehydrophobicsteroids leadsto the formationofcomplexwhichaffectssteroidselectrophoretic mobility At the same time β-cyclodextrin can act as a chiralselector and baseline separation of ketoprofen enantiomers has also beenachieved


Fig 1Electropherogramofmixtureof neurotransmitters and theirmetabolitesadrenaline (A)norepinephrine (NE)normetanephrine (NMN)dopamine (DA)metanephrine (Met) serotonin(SER)homovanillicacid(HVA)andtheinnerstandard24-dihydroxybenzoicacid(24-DHBA)oncovalentlymodifiedwithN-buthylimidazoliumionicliquidscapillaryConditions10mMNaH PO 2 4

(adjusted to pH= 20 by 1MHCl) injection 50 stimes30mbar ndash20 kV 220 nmmodelmixurendash1 ndash1 ndash110microgmL (METADNMNNADA24-DHBA)5microgmL (SER)and20microgmL (HVA)

4Conclusions

ItwasshownthatstructurecovalentcoatingaffectsitsanalyticalcharacteristicsWecomparedtwotypesofcovalentcoatingdifferingsubstituentinimidazoliumringnamelyalkylgroupandβ-cyclodextrinThefirsttypeisgreatcoupledwithon-line preconcentration technic but it is limited to effectively determine ofbiogenicaminesonlywhilethesecondtype(withβ-cyclodextrin)showedthepossibilitiestoseparatevariousanalytesbutsuitableon-linemodehasnotbeenfoundThemainpointsaresummarizedinTable1


Covalentcoatingtype Electrophoreticseparationof On-lineprecon- centration biogenicamines amino steroid ketoprofen andtheirmeta- acids hormones enantiomers bolites N-β-cyclodextrinimida- yes yes yes yes thesuitableapproachzoliumcovalentcoatings wasnotfoundedN-alkylimidazolium yes yes nonsepa- nonsepa- thesignificantreducingcovalentcoatings rated rated forbiogenicaminesLOD

Table 1Thesummationofpossibilitiesofcovalentcoatingsbasedonimidazoliumcation

Fig 2Electropherogramofsimultaneousseparationofhydrophobic(steroidhormones)andhydro-philicanalytes (aminoacidsandbiogenicamines) insinglerunwithcovalentcoatingbasedonimidazoleandβ-CDConditions10mMNaH PO (adjusted topH = 20by1MHCl) injection2 4 20 stimes30mbarndash20kV254nm(1ndash8min)and220nm(8ndash15min)0mbar(1ndash10min)and40mbar(10ndash15 min) Model mixture corticosterone (B) hydrocortisone (F) 11-deoxycortisole (S)

ndash1 ndash15μgmL L-tryptophan(Trp)34-dihydroxy-L-phenylalanine(DOPA)10μgmL L-tyrosine(Tyr)ndash15 μg mL noradrenaline (NA) normetanephrine (NMN) adrenaline (AD) dopamine (DA)

ndash120μgmL

Acknowledgments

This work was supported by Russian Science Foundation (grant numbers 19-13-00370) Theauthors are also grateful to the Chemistry Education Centre and Nanothechnologies Centre ofResearchParkSaintPetersburgStateUniversityfortechnicalsupport

References

[1] HuLFYinSJZhangHYangFQRecentdevelopmentsofmonolithicandopen-tubularcapillaryelectrochromatography(2017ndash2019)JSepSci43(2020)1942ndash1966

[2] KartsovaLAKravchenkoAVKolobovaEACovalentcoatingsofquartzcapillariesfortheelectrophoretic determination of biologically active analytes J Anal Chem 74 (2019)729ndash737

[3] HoTDZhangCHantaoLWAndersonJLIonicliquidsinanalyticalchemistryFundamen-talsadvancesandperspectivesAnalChem86(2014)262minus285

[4] TangSLiuSGuoYLiuXJiangSRecentadvancesofionicliquidsandpolymericionicliquids incapillaryelectrophoresisandcapillaryelectrochromatography JChromatogrA1357(2014)147ndash157

[5] QinWLiSFYElectrophoresisofDNAinionicliquidcoatedcapillaryAnalyst128(2003)37ndash41

[6] QinWWeiH Li SFY 13-Dialkylimidazolium-based room-temperature ionic liquids asbackgroundelectrolyteand coatingmaterial in aqueous capillaryelectrophoresis JChro-matogrA985(2003)447ndash454

[7] QinW Fong S Li Y Determination of ammonium andmetal ions by capillary electro-phoresisndashpotential gradient detection using ionic liquid as background electrolyte andcovalentcoatingreagentJChromatogrA1048(2004)253ndash256

[8] QinWLiSFYAn ionic liquidcoating fordeterminationofsildenafilandUK-103320 inhumanserumbycapillaryzoneelectrophoresis-iontrapmassspectrometryElectrophoresis23(2002)4110ndash4116

[9] BorissovaMVaherMKoelMKaljurandMCapillaryzoneelectrophoresisonchemicallybondedimidazoliumbasedsaltsJChromatogrA1160(2007)320ndash332

[10] KolobovaEKartsovaLKravchenkoABessonovaEImidazoliumionicliquidsasdynamicand covalent modifiers of electrophoretic systems for determination of catecholaminesTalanta188(2018)183ndash191

[11] KravchenkoAKolobovaEKartsovaLMultifunctioncovalentcoatingsforseparationofaminoacidsbiogenicaminessteroidhormonesandketoprofenenantiomersbycapillaryelectrophoresisandcapillaryelectrochromatographySepSciplus3(2020)102ndash111


1Introduction

Synthetic 4-hydroxy-3-methoxybenzaldehyde (vanillin) is used as a flavoringagent in foodsdrinksperfumesandpharmaceuticals [1]However at certainconcentrationsthesubstancemayaccumulateinthebodyhaveatoxiceffectand

ndash1at high concentrations may be fatal (lethal dose LD (oral rat) =2gkg 50ndash1 ndash1LD (oral guinea pig) = 14gkg LD (intravenous dog) = 132gkg lethal50 50 ndash1concentrationLC (inhalationmouse)=417gkg )[2]AccordingtoRussianState

StandartGOST121005-88thetoxiceffectsofvanillinintheworkplaceinthendash3formofvapoursoraerosolsareobservedatconcentrationsabove15mgm

Chromatography[3]spectrophotometry[4]capillaryelectrophoresisareusedforvanillindeterminationindifferentobjects CurrentlysmokingmixturesforhookahsandelectroniccigarettesarewidelyusedamongyoungpeopleThesemixturesarenotcontrolledforthecontentofsubstancesandarefreelyavailableconsideringthemmoreharmlesswithrespecttoordinarycigarettesThusthedevelopmentofamethodforthedeterminationof4-hydroxy-3-methoxybenzaldehydeinsmokingmixturesisrelevant

Determination of vanillin in smoking mixtures by spectrophotometry

ELIZAVETAEFREMENKOANNACHERNOVAOLGABASTRYGINA

DepartmentofChemicalEngineeringNationalResearchTomskPolytechnicUniversityLeninavenue30634050TomskRussiaeaetpuru


AbstractTheresearchdealswithdeterminationofvanillin insmokingmix-turesbyultraviolet-visiblespectrophotometryThemethodshowed

ndash1goodlinearityintherangeof005ndash012gL withalimitofdetectionndash1005gL After validation studies the method was successfully

applied to thedeterminationof vanillin in smokingmixtureswithsatisfactoryresultsItwasshownthattheerrorofthismethoddoesnot exceed 1 The developed spectrophotometric procedure fordeterminingvanillininsmokingmixturescanbeusedasacontrol

Keywordssmokingmixturesspectrophotometryvanillin

2Experimental


Asampleofvanillin(purity98)wastakenastheobjectofstudyAssolventsweused95ethanolAllchemicalsusedwereofanalyticalreagentgrade

22Instrumentation

Theopticaldensityofsampleswasmeasuredincuvettewithanabsorbinglayerthickness of 10 mm using a Cary 60 spectrophotometer (Agilent USA) Allmeasurementswerecarriedoutatroomtemperature

23Samplepreparation

Sample preparation of the investigated objects consisted of the preliminarydissolutionofthesamplein95ethanolThesample10mgoftobaccoldquoAdalyandashVanillardquo(Turkey)wasdiluted in10μLof95ethanol to theconcentrationof

ndash11gL Thesample10μLofldquoFlavoringTPAndashVanillaCustardrdquo(USA)wasdilutedin10 μL of 95 ethanol The resulting solution was diluted six times to the

ndash3concentrationof017μLcm


TodeterminevanillininthesamplestheopticalpropertiesofvanillininvarioussolventsweredeterminedAsaresultthe95ethanolwaschosenastheoptimalsolvent[4] IthasbeenestablishedthatintheUVspectraoftheanalyteabsorptionbondsareobservedwithmaximumvaluesat23002800and3100nmwhichcorres-pondstopublisheddata[45](Fig1) To quantify vanillin the calibration curve of the optical density on theconcentrationofvanillin in95ethanolwasobtainedatconcentrations005

ndash1006007008010and012gL Calibrationcurveofvanillinin95ethanolatawavelengthof280nmis

ndash1 A =81914c[gL ]+00357 (1)2802 R =1

Calibrationcurveofvanillinin95ethanolat310nmis

ndash1 A =73824c[gL ]+00301 (2)3102 R =1


Inthespectraoftheanalyzedsamplesolutionsabsorptionmaxima(2800nmand3100nm)characteristicforvanillinwereobservedTheamountofvanillininthesamplewasdeterminedusingcalibrationcurvesat280and310nmWeightedleast square regressionwas applied to the calibration curves to improve theaccuracyespeciallyatinlowconcentrationlevelrangeGoodlinearitywasfound

ndash1 ndash1intherangeof005ndash012gL withadetectionlimitof005gL TheresultsarepresentedintheTable1

4Conclusions

ThedevelopedmethodcanbeusedasacontrolmethodTheerrorinthemethodfordeterminingvanillininthesampleldquoFlavoringTPAndashVanillaCustardrdquowithaknownconcentrationofvanillinwas0004Accordingtothedataobtainedwerecommendawavelengthof280nmforthedeterminationofvanillininsamples


λ nm

Absorban

ce

ndash1Fig 1 Absorption spectrum of vanilin solution in 95 ethanol at concentration 01 mol L (anabsorbinglayerthicknessof10mm)

Sample λnm Tookmg Foundmg S Sх Δх δ

FlavoringTRA 310 10850 104096 00024 00011 00006 00306ndashVanillaCustardAdalyandashVanilla 310 100000 10162 00019 00009 00002 00025


Table 1Testingmethods introduced foundof vanillin in the samplesat310nmby spectrophotometricmethod(n=5p=099SndashstandarddeviationSxndashrelativestandarddeviationΔхndashabsoluteerrorδndashrelativeerror)

ThenthedevelopedmethodwastastedonthesampleldquoAdalyandashVanillardquosamplewithamorecomplexcompositionandanunknownconcentrationofvanillinwastaken The vanillin content in the sample was determined according to thedevelopedmethoditamountedto10ofthetotalmassStudieshaveshownthepossibility of using spectrophotometric analysis for the qualitative andquantitative determination of vanillin Also based on preliminary studies aspectrophotometricprocedurewasdevelopedforthequantitativedeterminationofvanillinbasedonabsorptioninethanolinthewavelengthrange200ndash400nm

References

[1] httpswwwrusnaukacom43_DWS_2015Chimia6_203179dochtm (accessed 25thFebruary2019)

[2] httpswwwcdcgovnioshrtecsdefaulthtm(accessed11stApril2020)[3] AliLPerfettiGDiachenkoGRapidmethodforthedeterminationof342coumarinvanillin

and ethyl vanillin in vanilla extract by reversed-phase liquid 343 chromatography withultravioletdetectionJAOACInt91(2008)383ndash386

[4] БастрыгинаОАЕфременкоЕАЧерноваАПВыделениеванилинаисследованиеегооптическихсвои ствопределениевбиологическомматериалеВХимияихимическаятехнология в XXI веке Материалы XX Международной научно-практическойконференции имени профессора ЛП Кулёва студентов и молодых ученых ТомскНациональныи исследовательскии Томскии политехническии университет 2019с301ndash302

[5] WeastRCHandbookofChemistryandPhysics60thedBocaRatonCRCPress1979p143


1Introduction

UraniumbelongstothegroupofhazardouselementsItisahighlyharmfulandradioactiveelementtoxictohumansandalllivingorganisms[12]Inhaledwithair it has a particularly destructive effect on the kidneys and as a result ofaccumulationinwhitebloodcellsitcanalsocauseimpairmentoftheimmunesystem[2]Uraniumoccursatseveraldegreesofoxidationhoweverinaqueoussolutionsthemoststableformisuranylion(UO (II))[12]Thepresenceofura-2

niumintheenvironmentiscausedbyamongothersnaturalsoilandrockerosionEnvironmentalpollutionwiththiselementisalsoconstantlyincreasingduetohumanactivitycoalcombustionuraniumoreminingandprocessingthearmsindustryandtheuseofuraniumasnuclearfuelinfissionreactors[3]Itisveryimportanttoconstantlymonitortheconcentrationofuraniumbothinthenaturalenvironment in order to assess its state and safety (especially in the case ofdrinkingwater)aswellasinallstagesofprocessingprocessesassociatedwiththenuclearindustrytoavoidtheoccurrenceofnuclearpollution[13] Scientists have made many attempts to develop research methods todeterminethecontentofuranylcompoundsinliquidsamplesEffortsweremadetousemanyanalyticalmethodsforthispurposeincludingspectrophotometry

Uranyl ion-selective electrode with solid contact

KAROLINAPIETRZAKCECYLIAWARDAK

DepartmentofAnalyticalChemistryInstituteofSciencesFacultyofChemistryMariaCurie-SklodowskaUniversityMariaCurie-SklodowskaSq320-031LublinPolandkarolinapietrzakpocztaumcslublinpl


AbstractNewallsolidstateuranylion-selectiveelectrodeswithlowdetection

ndash7 ndash1limits(71times10 molL )shortresponsetimegoodselectivityandstable and reproducible potential were developed Many types ofelectrodeswith different active ingredient content in ion-selectivemembrane (bis(244-trimethylpentyl)phosphonium acid Cyanex-272)were testedAs an additive an ionic liquid1-octyl-3-methyl-imidazole chloride was used The optimal composition of theion-selective membrane was chosen from all electrodes based onthedeterminationand comparisonof analyticalparametersof thesensors

Keywordsion-selectiveelectrodesolidcontacturanyl

plasma spectrometry luminescence spectroscopy voltammetry or chromato-graphymethods[2] Duetomanyadvantagesofpotentiometricmethods(amongthemlowercostseasieroperationofdevicesquickresponseandtheabilitytoperformmeasure-ments in flowmode) [3] a numberof potentiometric sensorshave alsobeendeveloped that could be successfully used in this type of research Themostpopularpotentiometricsensorsincludeion-selectiveelectrodes(ISEs)whicharecharacterized by low-energy consumption small size and portability and aresuccessfullywidelyusedforthedeterminationofbothinorganicandorganicionsinclinicalanalysisprocesstechnologyaswellasincontrolthestateofthenaturalenvironment[45]Removaloftheinternalsolutioncontainingthesameanalytetowhich theelectrode is sensitiveresulted in theso-calledsolidcontact ISEswhicharemuchsmallerinsizethantheirpredecessorsaremoreconvenienttouse and more mechanically resistant In this type of sensors however it isimportanttoachievesatisfactorypotentialstabilitywhichisnecessarytoobtainsatisfactoryresults[5]AveryimportantpartofISEsistheion-selectivemem-branewhosecompositiondeterminestheanalyticalparametersofthesensorsResearchers are currently focusing on the production and testing of newsubstancesthatcouldbesuccessfullyusedasmembranecomponentsandsolidcontacts thatwould allow to obtain new sensorswith lower detection limitslongerlifetimeandbetterpotentialstabilityandtodeterminenewpreviouslyunattainableanalytes[4] AstheactivecomponentsofthemembranesensitivetouranylionscientistshavealreadyusedKryptofix22DD(413-didecyl-171016-tetraoxa-413-diaza-cyclooctadecane)[2]Cyanexextractants(bis(244-trimethylpentyl)phosphinicacid bis(244-trimethylpentyl)monothiophosphinic acid and bis(244-tri-methylpentyl)dithiophosphinic)acid[3]DBBP(dibutylbutylphosphonate)andDOPP (di-n-octyl phenylphosphonate) [6] DMSO (dimethylsuphoxide) [7]TTPTP (5678-tetrahydro-8-thioxopyrido[4345]thieno[23-d]pyrimidine-4(3H)one)[8]orTEHP(tris(2-ethylhexyl)phosphate)andTPTU(O-(12-dihydro-2-oxo-1-pyridyl)-NNNN-bis(tetra-methylene)uronium hexafluorophos-phate)[9]

2Experimental


This paper presents research on the design and properties of ion-selectiveelectrodes with solid contact for the determination of uranyl ions Bis(244-trimethylpentyl)phosphonium acid (Cyanex-272) was used as the activecomponentof themembranewhichwasdescribed in the literatureasagooduranylextractant[10]Inordertoensureaconstantpotentialofthiselectrodeandreducetheelectroderesistancetheion-sensitivemembranewasenrichedwithafewpercentadditionof1-octyl-3-methylimidazolechlorideionicliquid


Several types of ion-selective electrodes were prepared using an AgAgClelectrodeasaninternalelectrodewhichdifferinthequantitativeandqualitativecompositionofthemembranesAllcompositionsarelistedinTable1

22Instrumentation

Measurements were made at room temperature using a 16-channel datacollectionsystem(LawsonLabs IncUSA)coupled toa computer in solutionsmixedwithamechanicalstirrerAsilversilverchlorideelectrodewithdoublejunctionwasusedasthereferenceelectrode


The effect of ion-selective membrane composition on the properties of theobtained potentiometric sensors was examined by determining their basicanalyticalparametersincludingslopeoftheelectrodecharacteristicsdetectionlimitmeasuringrange(concentrationrangeinwhichthecourseoftheelectrodecharacteristics isrectilinear)pHrange(inwhich ithasnoeffect forelectrodepotential)andresponsetimeTheobtainedvaluesofthetestedparametersareshowninTable2 Figure1showsthecalibrationcurvesofthetestedelectrodesdeterminedin

ndash7 ndash1 ndash1UO (NO ) solutionsintheconcentrationrange1times10 ndash1times10 molL Asitcan2 3 2

beseeninFig1andTable2allelectrodesweresensitivetouranylionsbutindifferent extend The best response exhibited ISE-3 containing 1 (ww) ofionophore Increasing the ionophore content in themembrane shortened thelinearityrangeofthecalibrationcurveanditssupernenstianslope Theselectivityofthetestedelectrodeswasestimatedbydeterminingtheselec-tivitycoefficients inrelationto interfering ionsForthispurpose theseparate

ndash1solutionmethodwasused(extrapolatingresponsecurves toa =a =1molL )i j

ComparisonofISE-1andISE-3electrodeselectivityisshowninFig2


Table 1Quantitative and qualitative composition of electrode membranes Cyanex-272 (bis(244-tri-methylpentyl)phosphoric acid) TBP (tri-n-butyl phosphate) and OMImCl (1-octyl-3-methyl-imidazolechloride)

Abbreviation Membranecomposition(ww)ofelectrode Cyanex-272 PVC TBP OMImCL

ISE-1 00 33 620 5ISE-2 05 33 615 5ISE-3 10 33 610 5ISE-4 30 33 590 5ISE-5 50 33 570 5ISE-6 100 33 520 5


Abbreviation Slope Detectionlimit Linearrange Response pHrange2+ ndash1 ndash1ofelectrode mVpa(UO ) molL molL times2

ndash5 ndash5 ndash1ISE-1 297 25times10 5times10 ndash1times10 5ndash8 28ndash42ndash6 ndash5 ndash1ISE-2 292 65times10 1times10 ndash1times10 5ndash8 25ndash60ndash7 ndash5 ndash1ISE-3 298 71times10 1times10 ndash1times10 5ndash8 24ndash60

ndash6 ndash4 ndash1ISE-4(I) 357 31times10 5times10 ndash1times10 5ndash8 ndndash6 ndash6 ndash4ISE-4(II) 242 31times10 5times10 ndash5times10 5ndash8 nd

ndash3 ndash1ISE-5(I) 638 nd 1times10 ndash1times10 5ndash10 ndndash5 ndash3ISE-5(II) 234 nd 5times10 ndash1times10 5ndash10 ndndash3 ndash1ISE-6(I) 733 nd 1times10 ndash1times10 5ndash10 ndndash5 ndash3ISE-6(II) 222 nd 5times10 ndash1times10 5ndash10 nd

Table 2Selectedparametersandtheirdeterminedvaluesoftestedionselectiveelectrodes

Fig 1 Calibration curves of the testedelectrodesobtainedinUO (NO ) solutionsin2 3 2 ndash7the concentration range from 1times10 to

ndash1 ndash11times10 molL

Fig 2Comparisonofselectivitycoefficientspot(log K (UO (II))M) for electrodes ISE-12

(1stcolumn)andISE-3(2ndcolumn)

Inordertoexaminethereversibilityofthepotentialofthetestedelectrodesndash4 ndash1potentialmeasurementsweremadealternatelyinsolutions1times10 molL and

ndash5 ndash11times10 molL ofUO (NO ) TherecordedpotentialreadingsareshowninFig32 3 2

Long-term potential stability and sensor reproducibility were evaluated byndash1determiningtheaveragevalueoftheelectrodepotentialina01molL UO (II)ion2

solutionovertimeforthreeidenticalISE-3Thesemeasurementsweremadetoobservechangesinthepotentialofelectrodeswiththesameconcentrationoveralongperiodoftime(30days)Figure3showsthelong-termpotentialstabilityandreproducibilitydeterminedforthreeidenticalsensors

4Conclusions

Asaresultofthetestsion-selectiveelectrodeforthedeterminationofuranylionswasobtainedwhich iseasy todesignanduseThebestanalyticalparametersexhibitedISE-3containing1ionophoreintheion-selectivemembraneForthis

ndash7 ndash1typeofelectrodesthedetectionlimitof71times10 molL linearityoftheelectrodendash6 ndash1 ndash1calibrationcurve in the range1times10 ndash1times10 molL andresponse time5ndash8s

were obtained In addition the manufactured sensors also showed stablereproducibleandreversiblepotentialandverygoodselectivityinrelationtothetestedinterferents

References

[1] AnsariRMosayebzadehZConstructionofanewsolid-stateU(VI)ion-selectiveelectrodebasedonpolypyrroleconductingpolymerJRadioanalNuclChem299(2014)1597ndash1605

[2] GhanbariMRounaghiGHAshrafNAnuranylsolidstatePVCmembranepotentiometricsensor based on 413-didecyl-171016-tetraoxa-413-diazacyclooctadecane and itsapplicationforenvironmentalsamplesIntJEnvironAnalChem97(2017)189ndash200


Fig 3 Stability () reproducibility andreversibility () of the potential of ISE-3Standard deviations given on the plot aredeterminedforthesamethreeISE-3

[3] Badr IHA Zidan WI Akl ZF Cyanex based uranyl sensitive polymeric membraneelectrodesTalanta118(2014)147ndash155

[4] BiegCFuchsbergerKStelzleMIntroductiontopolymer-basedsolid-contaction-selectiveelectrodes basic concepts practical considerations and current research topics AnalBioanalChem409(2017)45ndash61

[5] Bobacka J IvaskaA LewenstamA Potentiometric ion sensorsChem Rev108 (2008)329ndash351

[6] ZidanWI Badr IHA Akl ZF Development of potentiometric sensors for the selective2+determinationofUO ionsJRadioanalNuclChem303(2015)469ndash4772

[7] SalehMBSolimanEMGaberAAAAhmedSANovelPVCmembraneuranylion-selectivesensorSensActuatorsB114(2006)199ndash205

[8] SalehMBHassanSSMAbdelAAAbdelNAAnoveluranylion-selectivePVCmembranesensor based on 5678-tetrahydro-8-thioxopyrido[4345]thieno[23-d]pyrimidine-4(3H)oneSensActuatorsB94(2003)140ndash144

[9] HassanSSMAliMMAttawiyaAMYPVCmembranebasedpotentiometricsensorsforuraniumdeterminationTalanta54(2001)1153ndash1161

[10] Prabhu DR Ansari SA Raut DR Murali MS Mohapatra PK Extraction behaviour ofdioxouranium(VI) cation by two phosphorous-based liquid cation-exchangers in room-temperatureionicliquidsSepSciTechnol52(2017)2328ndash2337


1Introduction

Metronidazole(2-methyl-5-nitroimidazole-1-ethanol)isoneofthemostwidelyused nitroimidazole antibiotics Metronidazole is used for the treatment ofinflammatorydiseasescausedbyanaerobicorganismsandsomeprotozoaandforpreventionofdysenterycolibacillosiseimeriosisbalantidiasissalmonellosisenteritissepticemiapost-surgicalcomplications[1ndash3]Oxytetracyclinehydro-chlorideisanantibioticofthetetracyclinefamilyItisoneofthemostcommonlyused antibiotics in poultry because of its low cost and effective [4] Thesecompounds are intensively used in poultry breeding and stockbreedingUnreasonableuseofthesedrugscancauseseriousfoodsafetyissues[5] The veterinary drug Nozemat which includemetronidazole and oxytetra-cyclinehydrochloridewaschosenfortheexperimentsNozematisusedtotreat

Polarographic determination of metronidazole and oxytetracycline hydrochloride in veterinary drug for honey bees

a a bKATERYNAPLOTNIKOVA LILIYADUBENSKA IVANZELENYI

a AnalyticalChemistryDepartmentIvanFrankoNationalUniversityofLvivKyrylaiMefodiaStr879005LvivUkrainekaterina27plgmailcom

b DrohobychPedagogicalLyceumIvanaFrankaStr3682100DrohobychUkraine


AbstractWehavedevelopedanewpolarographicmethodforthedetermin-ation of metronidazole and oxytetracycline hydrochloride in theveterinarydrugNozematforhoneybeesThetechniqueisbasedonthereductionofpolarographicallyactivecompoundsonamercurydropletelectrodeTheinfluenceofthecomponentsoftheveterinarydrugNozematonthepolarographicdeterminationofmetronidazolewasstudiedItwasfoundthatthereductionofmetronidazoleisnotaffected by glucose and ascorbic acid but is affected by oxytetra-cyclinehydrochloridewhichisreducedtomercurydropletelectrodeatapotentialofndash145VThedevelopedtechniqueischaracterizedbyeaseofsamplepreparationandcost-effectivenessThistechniquehastheabilitytoidentifysimultaneouslyanddeterminatemetronidazoleand oxytetracycline hydrochloride in solution without the use ofseparationandconcentrationmethods

Keywordselectrochemistrymetronidazoleoxytetracycline

hydrochloridepolarographyveterinarydrug

bees and it can be given in unregulated doses Because of this an unknownamountofmetronidazolecangetintothehoneyanditsometimescausessideeffectsofthehumanbodyanditcouldbeofgreatconcernforpublichealth[56]MedicinesforpeoplearemorestringentandbettertestedthanveterinarydrugsTheproblemofthecontroloftheveterinarydrugsisurgentnowadaysVeterinarymedicinescouldbeunauthorizedandtheuncontrolleduseofmedicinesexistsinretailpharmaciesofmedicineorimportedascontrabandfromothercountries The most widespreadof these classes in thequality controlare chromato-graphic [6ndash9] spectrophotometric [10ndash13] and electrochemical methods[14ndash17]Manyoftheknownmethodsforthedeterminationofmetronidazoleandoxytetracyclinehydrochloridehaveanumberofdisadvantagestime-consumingtheuseoforganicsolventsandexpensivereagentsthesideeffectsofexcipientsandotheractivesubstancesElectrochemicalmethodsarepromisingalternativefor the determination of the electroactive substances Their advantages aresimplicityminiaturizationhighsensitivityandrelativelylowcostThereforethesearch for simple express and affordable methods for the determination ofmetronidazoleremainsrelevantOneofthepromisingmethodsofdeterminationisvoltammetry

2Experimental


VeterinarydrugNozemat (manufacturerAPI-SANRussia) is a yellowpowderwithaslighttypicalodorAvailableinlaminatedbagsof25gCompositionper1gof the drug metronidazole 400 mg oxytetracycline hydrochloride 400 mgglucoseascorbicacid MetronidazoleandoxytetracyclinehydrochloridewerepurchasedfromSigmaAldrich(USA)Stockstandardsolutionofmetronidazolefordeterminationwaspreparedbydissolvingtheexactamountofstandardin7mLof2Mhydrochloricin 500 mL volumetric flask Stock standard solution of oxytetracyclinehydrochloride was prepared by dissolving the exact amount of standard indistilledwaterin500mLvolumetricflaskAfterthatthesolutionswereadjustedtothemarkwithdistilledwaterandmixedthoroughly The Britton-Robinson buffer preparationwas as follows 202 g of sodiumtetraboratedecahydrate287mLofglacialaceticacidand176mLofconcen-tratedorthophosphoricacidweredissolvedin10Lvolumetricflask Working solution preparation was as follows an aliquot of stock standardsolutionwasaddedintoa25mLvolumetricflasktoobtainasolutionwiththenecessaryconcentrationthen2mLofBritton-RobinsonbufferwithnecessarypHwasaddedtotheflaskanddistilledwaterwasaddedtothemark AqueoussolutionofNozematwaspreparedasfollowstheexactportionofthetestveterinarydrugwasdissolved ina250mlvolumetric flaskAnaliquotof


Fig 1Polarogramsof(A)metronidazoleand(B)metronidazolewithoxytetracyclinehydrochloridendash1solutionsat02MBritton-RobinsonbufferbackgroundatpH=96(υ=05Vs c(metronidazole)=

ndash5 ndash5=45times10 Мc(oxytetracyclinehydrochloride)=50times10 М

100mloftheresultingsolutionwasaddedtoa250mlvolumetricflaskandmadeuptothemarkwithwaterAnaliquotof100mloftheresultingsolutionwasaddedtoa250mlvolumetricflask2mlofBritton-RobinsonbufferwithapHof96wasaddedandthevolumewasadjustedtothemarkwithdistilledwater

22Instrumentation

ForpolarographicmeasurementsweuseddigitaldeviceMTechOVA-410 [18]temperature-controlledthree-electrodeamercurydropletindicatorelectrodeasaturatedcalomelreferenceelectrodeandplatinumwireauxiliaryelectrodeTheaccuracyofthepotentialmeasurementis1mVTheuncertaintyofcurrentmeasu-rement is 01 The employed mercury droplet electrode had the following

ndash4 ndash1characteristicsm=594times10 gs τ=10 min in 02 M NH Cl We used cyclic4

voltammetryforthestudyoftheelectrochemicalprocess WeusedMV870DIGITAL-pH-MESSERATpH-meterformeasuringpHofthesolutions Theobtainedworkingsolutionswereintroducedintothecellanddeoxyge-natedwithargonfor10minPolarogramswererecordedintherangeofpotentialsfrom00tondash16V


Previously it was found that using the Britton-Robinson buffer with pH = 96metronidazoleisreducedwiththeformationofacharacteristiconeirreversiblepeakatndash064V(Fig1A)UsingpolarographywithfastpotentialscanitwasfoundthatmetronidazoleisreducedonmercurydropletelectrodeintherangeofpH20to105ThepeakrecoverycurrentofthemetronidazolereachesthemaximumvalueatpH9ndash10againstthebackgroundofatheBritton-Robinsonbuffer


Underpre-selected conditions theeffectof some foreign substanceson thepolarographicdeterminationofmetronidazolewasinvestigatedSubstancesthatare componentsofdrugswere studiedglucose ascorbicacidoxytetracyclinehydrochlorideGlucose and ascorbic acid are not reduced atmercury dropletelectrodeanddonotchangetheappearanceofthepolarogramandpolarographiccharacteristics of the recovery of metronidazole As can be seen from Fig 1oxytetracycline hydrochloride is reduced to mercury droplet electrode andchangestheappearanceofthepolarogramandpolarographiccharacteristicsofthe recovery of metronidazole With the addition of oxytetracycline hydro-chloridetherecoverypeakofmetronidazoledecreasesandslightlyshiftstomorenegativepotentials The composition of the drug is relatively complex excipients affect theanalyticalsignaloftherecoveryofcompoundssototakeintoaccountthematrixeffectusedthemethodofmanyadditives QuantitativelytransferredthesolutionofNozemattothecell(exactvolume)removed dissolved oxygen for 10 min and took polarograms in the range ofpotentialsfrom00tondash16VAliquotsofstandardmetronidazolesolutionwereintroducedintothecelltoobtainasolutionwithagivenconcentrationofadditive

ndash5 ndash5metronidazole 10times10 M to 70times10 M As with the determination of themetronidazole aliquots of a standard oxytetracycline hydrochloride solutionwereaddedtothecellwithsolutionofNozemattoobtainasolutionwithagiven

ndash6 ndash6additiveconcentrationfrom70times10 Mto50times10 M(Fig2) In Table 1 are shown metrological characteristics of the determination ofmetronidazoleandoxytetracyclinehydrochloride inveterinarydrugRecoverywascalculatedFormetronidazoletherecoveryis97andforoxytetracyclinehydrochloridetherecoveryis103Analyticalperformanceofthetechniqueisgoodfordeterminationveterinarydrugs


Fig 2Polarogramsof(A)metronidazolereductionatdifferentmetronidazoleconcentrationsand(B)oxytetracyclinehydrochloridereductionatdifferentoxytetracyclinehydrochlorideconcentra-tionsandtheircorrespondingcalibrationgraphs

Theaccuracywasverifiedbytheldquoadded-foundrdquomethodAliquotsofstandardsolutionofmetronidazoleweremadeina250mlvolumetricflasktoobtaina

ndash5solutionofagivenconcentrationof33times10 Mandthesolutionofoxytetracyclinendash5hydrochloridetoobtainasolutionofagivenconcentrationof15times10 M2mlof

Britton-RobinsonbufferwithpH96wasaddedtoflaskwithstirringandadjustedtothemarkwithwaterTheanalysisprocedureofmodelsolutionissimilartoanalysis procedure of the solution of Nozemat The calculated amount ofmetronidazolebythemethodofmultipleadditivesinthetestedmodelsolutionisinagreementwiththeamountthatwasintroducedintothesample

4Conclusions

The new polarographic method for the determination of metronidazole andoxytetracyclinehydrochlorideintheveterinarydrugNozematforhoneybeeswasdeveloped We conducted principal component analysis of veterinary drugNozemattoassesstheoveralleffectforthedeterminationofmetronidazoleWefoundthatoxytetracyclinehydrochlorideisreducedtomercurydropletelectrodeThismethodhastheabilitytoidentifysimultaneouslyanddeterminatemetro-nidazole and oxytetracycline hydrochloride in solution without the use ofseparationandconcentrationmethodsOnemoreofadvantagesoftechniquearefastprocedureofanalysissimplesamplepreparationlowcostthepossibilityofminiaturization

References

[1] Antibiotic and Chemotherapy Finch R Greenwood D Whitley R (edits) AmsterdamElsevier2006p292ndash299

[2] MitrowskaKPrzyczynyiskutkizakazustosowania5-nitroimidazoliuzwierzątktorychtkanki lub produkty przeznaczone są do spozycia przez ludziMed Weter 71 (2015)736ndash742


Metronidazole Oxytetracycline hydrochloride

PeakspotentialV ndash065 ndash144CorelationcoefficientR 099892 099922

ndash1 4 4SlopebmicroАM 656times10 176times10Δb 1763 400InterceptamicroА 2238 0219Δa 0075 0009

ndash1 ndash5 ndash5cmolL 342times10 125times10ndash1cmgg 389 413

Recovery 97 103

Table 1Validationparametersofthemethodofmetronidazoleandoxytetracyclinehydrochloridedetermi-nationinsolutionsofNozematbythemethodofmanyadditives

[3] VermaPNamboodiryVMishraSBhagwatABhoirSAstabilityindicatingHPLCmethodfor the determination of Metronidazole using Ecofriendly solvent as mobile phasecomponentIntJPharmPharmSci5(2013)496ndash501

[4] Cervini P Ambrozini B Machado LCM Ferreira Garcia AP Cavalheiro Gomes ETThermal behavior and decomposition of oxytetracycline hydrochloride J Therm AnalCalorim121(2015)347ndash352

[5] DangBNAnhNTKKyLXThaiPKAntibioticsintheaquaticenvironmentofVietnamsourcesconcentrationsriskandcontrolstrategyChemosphere197(2018)438ndash450

[6] QuintanillaPHettingaKABeltranMCEscricheIMolinaMPVolatileprofileofmaturedTronchon cheese affected by oxytetracycline in raw goat milk J Dairy Sci 103 (2020)6015ndash6021

[7] Chen F Yu L Jingdong P Xiang W Huanjun P Yu C Yan H Study on simultaneousdetermination of three nitroimidazole residues in honey by high performance liquidchromatographyndashresonanceRayleighscatteringspectraMicrochemJ141(2018)423ndash430

[8] Hernandez-MesaM Cruces-Blanco C Campana GA Simple and rapid determination of5-nitroimidazolesandmetabolitesinfishroesamplesbysalting-outassistedliquid-liquidextractionandUHPLC-MSMSFoodChem252(2018)294ndash302

[9] Xiu-ChunGZhao-YangXHai-HuiWWen-YiKLi-MingLWen-QingCHong-WeiZWen-HuiZMolecularlyimprintedsolidphaseextractionmethodforsimultaneousdeterminationofsevennitroimidazolesfromhoneybyHPLC-MSMSTalanta166(2017)101ndash108

[10] ТеплыхАНИлларионоваЕАКоличественноеопределениеметронидазоласпектро-фотометрическимметодомСибирскиймедицинскийжурнал5(2009)48ndash50

[11] ZheltvayOIZheltvayIISpinulVVAntonovichVPSpectrophotometricdeterminationofmetronidazoleandtinidazoleusingcopper(II)complexesJAnalChem68(2013)663ndash668

[12] Youssef AK Saleh MS Abdel-Kader DA Hashem Facile DY SpectrophotometricdeterminationofmetronidazoleandsecnidazoleinpharmaceuticalpreparationsbasedontheformatioonofdyesIntJPharmPharmSci6(2015)103ndash110

[13] Sversut RA Vieira JC Rosa AM Amaral MS Kassab NM Salgado H ValidatedspectrophotometricmethodsforsimultaneousdeterminationofoxytetracyclineassociatedwithdiclofenacsodiumorwithpiroxicaminveterinarypharmaceuticaldosageformArabianJChem13(2020)3159ndash3171

[14] Nikodimos Y Electrochemical determination of metronidazole in tablet samples usingcarbonpasteelectrodeJAnalMethodsChem(2016)361294

[15] Srivastava AK Upadhyay SS Rawool CR Punde NS Rajpurohit AS Voltammetrictechniques for the analysis of drugs using nanomaterials based chemically modifiedelectrodesCurrAnalChem15(2019)249ndash276

[16] Sahu G Voltammetric behaviour of metronidazole at a composite polymer membraneelectrodeOrienJChem26(2010)81ndash86

[17] Yang Y YanW Guo YWang X Zhang F Yu L Guo C Fang G Sensitive and selectiveelectrochemicalaptasensorviadiazonium-couplingreactionforlabel-freedeterminationofoxytetracyclineinmilksamplesSensorsandActuatorsReports2(2020)1ndash7

[18] httpchemlnueduuamtechdeviceshtml(accesed21stJune2020)


1Introduction

AnimportantpartofanyanalysisthatsignificantlyaffectsthefinalresultsisthesamplepreparationThelowconcentrationofbiologicallyactivecompoundsandthepresenceofaccompanyingcomponentspreventdirectanalysisofthesamplewithcomplexmatrixcompositionTraditionalmethodsofliquidandsolid-phaseextractionhaveaplentyoflimitationssuchashighlytime-consumingprocedureslarge volume of samples expensive cartridges toxic organic solvents andchallenges in automating the process Therefore the application of extractiontechniquesemployinglowamountofsolvents(microextractionmethods)andthelow toxicity extractantes has become the main research direction in recentyears[12] Solid-phasemicroextraction(SPME)wasproposedbyPavlishinin1989[3]Onevariantofthismethodistousethinrodswithvariouspolymercoatingssuch

Application of microextraction techniques combined with chromatographic methods for the analysis of complex objects

VLADISLAVDEEVELENABESSONOVALIUDMILAKARTSOVA

InstituteofChemistrySaint-PetersburgStateUniversityUniversitetskyprospect26198504PeterhofSaint-PetersburgRussiahitchervmailru


AbstractThelowconcentrationofanalytesandthepreventionofthematrixinfluence requires a stage for extraction and concentration of thestudiedcompoundsTheclassicalmethodsofliquidandsolid-phaseextractionhavemanylimitationsthatpreventtheiruseinsomecasesMicroextraction techniques are becoming more widespread WestudiedthepossibilityofusingionicliquidstoextractpesticidesfromwatersampleswiththeirsubsequentHPLS-MSdeterminationTheinfluenceonthedegreeofextractionofsuchparametersasanatureofionicliquidsanddispersersolventtheiramountssaltconcentrationvolumeratioofionicliquidsandwatersampledilutionoftheionicliquidsextractwithmethanolwasperformedBesidesconditionsofsolid-phase microextraction of volatile organic compounds fromurine samples obtained from healthy donors and donors withprostate cancer have been found The analysis of volatile organiccompoundsbyGC-MSfollowedbychemometricprocessingallowedachievingahighvalueofbinaryclassificationaccuracy(91)

Keywordschemometricsdispersiveliquid-liquid

microextractionsolid-phase

microextraction

asdivinylbenzenepolydimethylsiloxanepolyacrylateandpolyethyleneglycolwhichappliedtothesurface[4]Thepolymersorbentisplacedintheequilibriumheadspaceaboveacondensedphaseofthesampleandthevolatilecompoundsareextracted Liquidmicroextractionconsistsofusingsmallamountsofliquid(extractant)inequilibriumwiththegasorliquidphaseofthesampleDispersiveliquid-liquidmicroextraction(DLLME)isavariantofliquidmicroextractionTheessenceofthemethodisasfollowsextractantisdissolvedinthephaseofadispersingsolventandthemixtureisrapidlyinjectedintothesamplevolume[5]Inthiscasethedispersing solvent is dissolved and a ldquocloudrdquo of extractant is formed A largesurfaceareacontributestomasstransferprocessesThecombinationofDLLMEwiththeuseof ionic liquids(ionic liquids)asextractantsreducestheharmfulimpactontheenvironment[6] Sothegoalofthisstudywastheapplicationofmicroextractionmethodsfortheanalysisofrealsamples

2Experimental

21Reagents

Deionizedwaterwas obtained at the AQUILON D 301 deionizer (Russia) Allchemicals and reagents (the highest commercially available purity) werepurchasedfromReachimBakerAcrosorganicsandSigmaAldrich

22Instrumentation

HPLCanalysiswascarriedoutusinganHPLCLCMS-8030(Shimadzu)withatriplequadrupole mass-selective detector with electrospray ionization Analysis ofvolatileorganiccomponents(volatileorganiccompounds)ofurinesampleswasmade by GCMS-QP2010 SE (Shimadzu) Chemometric data processing wasperformedusingRStudio

23DeterminationofvolatileorganiccompoundsinurinesamplesbyGC-MSmethod

ForSPMEofvolatileorganiccompounds inurinesamplewasusedfibercoatedwith a polydimethylsiloxane (PDMS) The volatile organic compounds wereextractedontofibercoatingfor20minat50degCThentheanalytesweredesorbedinto the gas chromatography for 4 minutes at a temperature of 250degCChromatographic separation was carried out on a HP-5 capillary column(30mtimes250μmtimes025μm)usingtemperatureprogrammingmodeThetempera-

ndash1tureofovenwasincreasedfrom50degCupto250degCatarateof10degCmin Tionsourcewas200degCMassspectrometrywasusedinSIMmode(mz=35ndash900)


24ConditionsforLCMSMSdeterminationofpesticides

Separation of pesticides was performed by HPLCMSMS with positiveelectrosprayionizationoncolumnZorbaxBonusRP35μm(21times100mm)with40mMammoniumacetateandmethanolasmobilephaseAandBrespectivelyThefollowinggradientelutionwasapplied20ndash85B(8min)85B(8ndash15min)85ndash95B(150ndash155min)95B(155minus180min)95ndash20B(180minus185min)

ndash1Thevelocityof themobilephasewas03mlmin Thevolumeof the injectedsamplewas 20 microlMS detection capillary voltage +45 kV spray gas velocity

3 ndash1 3 ndash13dm min flow rate and drying gas temperature 15 dm min and 250 degCrespectively

25Selectionofconditionsfordispersiveliquid-liquidmicroextractionofpesticides

The influence of the natures of ionic liquids ([C MIM][PF ] [C MIM][NTf ]4 6 6 2

[C MIM][BF ])andthedispersingsolvent(methanolacetonitrileacetone) the6 4

weightoftheionicliquids(0060ndash0200g)thevolumeofthedispersingsolvent(02ndash10ml)onthedegreeofpesticidesextractionwerestudiedTheinfluenceofthepH(5422)theconcentrationofNaCl(0040ndash0200g)andextractiontime(1-6min)wereinvestigated The effects of different ionic liquids and disperser solvents on DLLMEprocedures were investigated and optimized by using standard solutions ofpesticidesIndetailasolutionofionicliquidsinadispersingsolventwaspreparedand rapidly injected into the aqueous sample solution (2ml) followed bytreatment for 2 min in an ultrasonic bath cooling at ndash4degC for 10 mincentrifugationfor10minat3500rpmandcollectionofionicliquidsThewaterphasewas separatedandanalyzedbyHPLC-MSThe ionic liquidsextractwasdilutedinmethanolandanalyzedbyHPLC-MS


31Microextractionofpesticides

One of the important tasks of environmental monitoring is to control traceconcentrationsofpesticidesinwatersamplesTechniqueofcombiningseveralpesticideshasbecomemorewidespread inagriculture It allowsreducing thetotalconcentrationoftheappliedcompoundsandtodecreasetheadaptabilityofpathogens and insects Therefore the analysis of real samples requires apreliminarystageofselectiveanalytesextractionandconcentration ExtractinganddispersingsolventsarebothimportantinDLLMEofanalytesTheinfluenceofthenatureofthedispersingsolvent(methanolacetonitrileandacetone) and the extractant (imidazolium ionic liquids [C MIM][PF ]4 6

[C MIM][BF ]and[C MIM][NTf ])onthedegreeofextractionofpesticideswas6 4 6 2


studied This parameter was controlled by the residual concentration ofpesticidesinthewaterphaseafterextractionThebestresultswereobtainedforionic liquids [C MIM][PF ] as an extractant and acetonitrile as a dispersing4 6

solvent Thenextstepwastoselecttheamountofionicliquids(0060ndash0200g)andthevolume of acetonitrile (02ndash10 ml) It was found that the highest degree ofextractionofanalyteswasachievedbyusing020gofionicliquidsand03mlofacetonitrile It was shown that the degree of extraction of selected pesticides does notdependonpHofthewatersamplewhichconfirmsthepartitionmechanismofextractionThedegreeofextractionofcarbofosincreasedslightlywithanincreaseinthesaltconcentrationandreachmaximumbyweightto4(008g) ItisknownthatthehighviscosityofionicliquidshavehinderedtheprocessesofelectrosprayionizationDilutionofthesamplewithmethanolby3timesgivesthebestresult(thesignalintensitywas373to845ofthesignalwithoutionicliquids) ThustheconditionsofDLLME-ionicliquidsextractionofpesticidesfromwatersampleswere found The limits of detection for pesticideswere from007 to

ndash1019ngml thereproducibilityofpeakareaswerefrom3to5theextractionrecoverywascloseto100

32SPMEofvolatileorganiccompoundsfromurinesamples

Oneof the importantdirections is the search for criteriaofearlydiagnosisofcancer Obtaining characteristic profiles of volatile organic compounds fromurinesamplescanhelpdevelopanon-invasivemethodforearlydiagnosisofthedisease ForthiswestudiedtheinfluenceofanumberoffactorsonthetotalnumberofpeaksandthetotalpeakareaTheywerethetemperatureatwhichthevaporandcondensedphasesofurinewerebalanced(30ndash60degC)thepreheatingtimeofthesample(10ndash40min)NaClconcentration(30ndash133wv)andthesorptiontimeonthePDMScoating(5ndash30min) Anincreaseinthepreheatingtemperatureofthesampleto500degCledtoanincreaseinthenumberofsignalswhichdidnotchangewithafurthertempera-tureincreasingNextparameterwasthetimeofachievementequilibriumofthevapor and condensed phases The largest number of peaks was observed at40minbutthisgreatlyincreasedthetimeofanalysisandsowechose20minAlsowestudiedthedesaltingeffectofsodiumchlorideonefficiencyofextractionThebestvolatileorganiccompounds sorptionwasachievedbyadding133saltingagent It is also shown that thenumberofpeaksdidnot changeafter20minutesofsorption Thus to obtain the characteristic profiles of urine samples the followingconditionswereselected1333NaClwasaddedtotheurinesample(3ml)


heatingfor20minat50degCthensorptionofvolatileorganiccompoundsonPDMSfibercoatingat50degCfor20min Undertheselectedconditionsweobtainedvapor-phaseprofilesof52urinesamples (32 normal and 20 pathology) Prior to performing chemometricprocessing of chromatographic profiles of urine samples preliminary datapreparation is necessary [7] The baseline was removed and the peaks werealignedusingdynamictimewarpingwithcontrollingbymassspectra ThePCAmodelwasbasedon52aligned characteristicprofilesThere is asatisfactoryseparationofdataintotwoclustersinthescoresplotrelativetothefirstandsecondprincipalcomponent(Fig1) The original data set (52 samples) was randomly divided into calibration(13pathology21normal)andtest(7pathology11normal)setsThenthePLS-DAmodelwasbuiltusingthecalibrationsetanditspredictiveabilitywasevaluatedusingthetestsetTheprocedurewasrepeated100timesTheaveragevaluesofsensitivityspecificityandaccuracyinthiscasewere9594and91

4Conclusions

Thepossibilityofusingimidazoliumionicliquids([C MIM][PF ])asextractants4 6

forquantitativeextractionandconcentrationofpesticidesundertheconditionsofDLLMEisshownThedegreeofconcentrationwas28ndash33whichallowedreaching

ndash1thedetectionlimits(006ndash019ngml )belowthemaximumpermissibleconcen-trationThepossibilityofnon-invasivediagnosisofprostatecancerbySPMEofvolatileorganiccompounds inurine isshownChemometricprocessingofgaschromatographic profiles using PLS-DA and PCA methods allowed achievingclassificationaccuracyvaluesmorethan90

Acknowledgments

ThisworkwassupportedbytheRussianFoundationforBasicResearchprojectno18-53-80010BRICS_t and the Russian Science Foundations (Projects 19-13-00370) We are grateful to


Fig 1Scoresplotrelativetothefirstandsecondprincipalcomponent

Resource Education Center in Chemistry of St Petersburg State University for the providedequipment

References

[1] Rutkowska M Płotka-Wasylka J Sajid M Andruch V Liquidndashphase microextractionAreviewofreviewsMicrochemJ149(2019)103989

[2] JaliliVBarkhordariAGhiasvandAAcomprehensivelookatsolid-phasemicroextractiontechniqueAreviewofreviewsMicrochemJ152(2020)104319

[3] ArthurCLPawliszynJSolidphasemicroextractionwiththermaldesorptionusingfusedsilicaopticalfibersAnalChem62(1990)2145ndash2148

[4] SchmidtKPodmoreISolidphasemicroextraction(SPME)methoddevelopmentinanalysisof volatile organic compounds (VOCs) as potential biomarkers of cancer JMol BiomarkDiagn6(2015)1000253

[5] Mousavi L Tamiji Z Khoshayand MR Applications and opportunities of experimentaldesign for the dispersive liquidndashliquidmicroextractionmethod ndash A review Talanta190(2018)225ndash356

[6] MarcinkowskaRKoniecznaKMarcinkowskiLNamiesnikJKloskowskiAApplicationofionic liquids inmicroextractiontechniquesCurrent trendsandfutureperspectivesTrACTrendsAnalChem119(2019)115614

[7] WehrensRChemometricswithRMultivariateDataAnalysisintheNaturalSciencesandLifeSciencesBerlinSpringer2011


1Introduction

Theself-assembledtwo-dimensionalmonolayers(2DSAMs)ofvariousmolecules(eg graphene [1] MoS [2] rubrene [3]) offer beneficial properties for the2

constructionofnano-electronic andnano-opticaldevicesThe topological andchemicalcharacterizationof2DSAMsiscrucialtogatherinformationaboutthearrangementofdepositedmoleculesandtheirinteractionwiththesubstrateThistaskrequiresanalyticaltechniqueswith(sub)nanometerspatialresolutionandup to single-molecular detection sensitivity Only few techniques meet therequirements and one of them is tip-enhanced Raman spectroscopy (TERS)whichcombinestheexcellentspatialresolutionofscanningprobemicroscopy(SPM) and chemical sensitivity of surface-enhanced Raman scattering (SERS)spectroscopy[45] TheSERS spectroscopyutilizesplasmonicmetal nanostructures to cause ahighlocalenhancementoftheelectricfieldintheirclosevicinityviathesurfaceplasmonresonance(SPR)effectThelocalelectricfieldcausesanincreaseofthe

The development of reference probe system for tip-enhanced Raman spectroscopy

MARTINKRA LMARCELADENDISOVA PAVELMATE JKA

DepartmentofPhysicalChemistryFacultyofChemicalEngineeringUniversityofChemistryandTechnologyPragueTechnickaacute516628Prague6CzechRepublicMartinKralvschtcz


AbstractThetip-enhancedRamanspectroscopy(TERS)isamodernanalyticaltechniquewithanoutstandingspatialresolutionandchemicalsensi-tivityTheseparametersmainlydependon the structural integrityand chemical purity of employed plasmonic scanning probe tipsUsuallyeachtipistestedbeforeTERSmeasurementsusingcommer-ciallyavailablereferencesamplesHowevertheirpriceandrelativelyshortexpirationdatemustbeconsideredwhenplanningaresearchbudgetWedevelopedaproceduretoproduceself-madereferenceprobesamplesfortestingTERStipsusingcopper(II)phthalocyanineonaAunanolayerwhichispreparedbythermalvacuumevaporationofAuonaSiwaferOurresultsshowthatthepreparedsystemenablesrepeateddetectionofwell-resolvedTERSspectraThecollectedTERSspectraandspectralmapsexhibitsomedegreeofvariabilitywhichmaybeduetovariousphoto-inducedprocessesanditmustbeconsi-deredwhileperformingTERSmeasurements

Keywordscopper(II)

phthalocyaninescanningtunnelling

microscopyresonanceRaman

spectroscopysurface-enhancedRaman

spectroscopytip-enhancedRaman

spectroscopy

Ramanscatteringfrommoleculesboundtothemetalby6ndash8ordersofmagnitude[6]TheenhancementallowsSERSspectroscopytobeusedforsingle-moleculardetectionHoweverRamanmicroscopeshave limitedspatialresolutionbythelightdiffractionwiththeachievableresolutionbeingaroundhalfoftheexcitationwavelengthOntheotherhandthespatialresolutionofSPMtechniquesislimitedonlybythedimensionsoftheapexofthescanningprobetipwhichmayevenbeatomicallysharpByutilizingSPMtipandsubstratemadefromplasmonicmetalsan artificial ldquohotspotrdquomay be createdwith its position and dimensions beingdefinedbythetipItopensthepossibilitytocollectstronglyenhancedRamanspectrafromtheareapreciselylocalizedbellowthetipandthusovercometheopticaldiffraction limitTheartificialhotspotmayberelocatedbymovingthesamplebelowthetipwhichisthefoundationofTERSmapping[578] A successful TERS experiment requires an optimal combination of variousparameters the most important of which are tip sharpness and purity [7]Areferencesampleconsistingofaflatplasmonicnanolayerwithattachedprobemolecules is frequently used to check the state of the tip before using it forexperiments Unfortunately commercially available TERS standards areexpensiveandhaveanexpirationdateofseveralmonths Thegoalofthisstudywastofindapreparationprocedurewhichwouldbeableto produce cheap reference samples for repeated detection of intense TERSspectraAcombinationofaAunanolayeronaSisubstratepreparedbythermalvacuumevaporationwithadsorbedcopper(II)phthalocyanine(CuPc)whichisamoleculewithhighRamancross-sectionwastested[910]Copper(II)phthalo-cyanine known as phthalocyanine blue is a synthetic blue pigment and isfrequently used in paints It has been studied as a potentialmaterial for theconstruction of organic solar cells and other photoelectronic devices [11] AsindicatedbyitscolourCuPcexhibitsseveralabsorptionbandswithinthevisibleregion The effects of a transition to excited electronic states and subsequentluminescencemaybeobservable inRamanmeasurements[12]Au isahighlysuitablemetalforthesampleasitisboththermallyandelectricallyconductivewhichlimitsthelocalheatingofthesampleduringthemeasurementsandenablesthe use of scanning tunnelling microscopy (STM) for tip-surface interactionfeedback[13]

2Experimental


Thesubstrateforthesamplewaspreparedbythermalvacuumevaporationofgoldonsilicon(100)waferFirsta5nmthickCradhesionlayerwasdepositedon

ndash1thewaferfollowedby100nmofAuThedepositionratewas4nmmin forCrand ndash18nmmin for Au The base pressure of the evaporation system was below

ndash65times10 mbarFollowingthepreparationproceduredescribedbyJiangetal[14]


thecleansubstratewasimmersedintoasaturatedsolutionofCuPc(˃99SigmaAldrich USA) in dimethylformamide (˃98 Lach-ner CZ) for at least 12h atambienttemperatureSubsequentlythesamplewasremovedfromthesolutionrinsedwithMilli-Qwaterandmethanol(paPentaCZ)anddriedwithair

22Instrumentation

TheRamanSERSandTERSspectrawererecordedusingRamanspectrometerInVia Reflex (Renishaw UK) equipped with lasers emitting at two differentexcitation wavelengths 633nm (136mW max power output) and 785nm(204mWmaxpoweroutput)Thespectrometerhasathermoelectricallycooled

ndash1CCDdetectorwithaspectralresolutionof2cm and4microscopeobjectiveswith5times20times50timesand100timesmagnitudeForTERSexperiments the laserbeamwasredirectedtotheSPMplatformInnova-IRIS(BrukerUSA)viaasystemoflightguidesElectrochemicallyetchedAuTERS-STMtips(BrukerUSA)wereusedforallTERSmeasurements The spectra were processed using the Spectragryph software (F MengesldquoSpectragryph - optical spectroscopy softwarerdquo Version 1214 2020httpwwweffemm2despectragryph) Using this software all collectedspectra were treated by a Savitzky-Golay noise filter automatic baselinecorrectionspikeremovalandpeaknormalization


31Ramanmeasurementsofcrystallinecopper(II)phthalocyanine

AtfirsttheRamanspectraofpureCuPcwerecollectedtoprovidereferencedatawhileusingboth633and785-nmexcitationlasers(Fig1)Bothspectraexhibit


Fig 1RamanspectraofCu(II)phthalocyanineincrystallineformmeasuredat633(top)and785-nm(bottom)excitationThespectraareoffset

ndash1vibration bands in the 500ndash1600 cm region with slight differences in theirintensityratiosThe633-nmexcitationallowstheobservationofadditionalbands

ndash1inthe2000ndash3000cm region(onaluminescencebackground)whichoriginatefrom the resonance Raman effect as the excitation energy overlaps with theQ-bandofCuPc[12]MoreovertheprocessofelectronicexcitationmaylowertheD symmetry of CuPc during resonance Raman scattering and previously4h

forbiddenbandsmaybecomeobservable[15]Theluminescentbackgroundhasandash1maximumaround2200cm whichcorrespondstoamolecularemissionbandat

735nmEventhoughthespectrameasuredwiththe785-nmlaserlinedonotexhibitapparentresonanceenhancementapre-resonanceRamanenhancementmayoccur

32Surface-enhancedRamanmeasurementsofcopper(II)phthalocyaninelayeronagoldsubstrate

The prepared sample of CuPc on a Au layer was analysed using the Ramanmicroscope Both excitation laserswere used to obtain SERS spectra (Fig 2)whichwerecomparedtothespectraofapurecrystallineCuPc Thepositionsof bands in SERS spectra closelymatch their positions in thespectraofbulkCuPcHoweveraslightshiftofsomespectralbandsisobservable

ndash1(eg1528rarr1532cm )whichmaybeattributedtotheinteractionbetweenCuPcand the Au substrate The disappearance of luminescence background andresonance-enhancedbandsinthespectrumat633-nmexcitationalsosuggeststhemolecule-metalinteractionandthetransferofenergyfromCuPcmoleculestothesubstrateMoreovertherearevariationsintherelativeintensitiesofbands

ndash1whichdependontheexcitationenergyegthebandat1309cm isenhancedinSERSspectraatthe785-nmexcitationwhencomparedtothespectraofpureCuPcorevenSERSspectraat633-nmexcitation


Fig 2SERS spectra of Cu(II) phthalocyanineon aAu layermeasured at 633 (top) and785-nm(bottom)excitationThespectraareoffset

33Tip-enhancedRamanmeasurementsofcopper(II)phthalocyaninelayeronagoldsubstrate

SERS microspectroscopy is a diffraction-limited technique as it provides anaveragedinformationaboutmoleculesintheilluminatedareaofseveralsquaremicrometers Meanwhile TERS spectra are collected from an area of tens ofnanometers and they contain specific information about the local moleculararrangement topography of the underlyingmetal and properties of the localelectric field between the tip and the substrate Therefore a higher spectralvariabilityshouldbeexpected Several TERS mapping experiments were carried out using both 633 and785-nmexcitationwithvaryingexperimentalparameterssuchasthenumberanddistance betweenmeasured points acquisition time number of acquisitionslaserpoweretc TheTERSspectrameasuredat633-nmexcitationexhibited lowersignal tonoiseratioandreproducibilityAsaconsequenceTERSmappingwasimpossibleandonlyafewone-pointTERSspectrawereobtained(Fig3) TheTERSspectracollectedat785-nmexcitationcontainedahighernumberofwell-resolvedbandsMoreoverthespectrawerestableintimeandsotheTERSmappingwaspossibleThe twopresentedTERSspectraareaveragesofTERSmaps which contained 16 and 80 points with 600 and 300-nm spacingrespectively(Fig4) Thespectracollectedusingbothexcitationwavelengthsexhibitavariabilityinrelative intensities and positions of bands between themeasured points ThevariabilitymaybeattributedtothelocalorientationofCuPcmoleculesbetweenthe tip and the Au surface and the properties of strongly enhanced andnon-homogeneous electromagnetic field which depend on the tip-surfacedistancetheirmorphologyandrelativepositionMoreovertheusedexcitation


Fig 3TwoexamplesofTERSspectraofCu(II)phthalocyaninemeasuredat633-nmexcitationThespectraareoffset

wavelengthsareclosetoabsorptionbandsofCuPcandthestrongelectricfieldmay give rise to photo-induced effects These effects include the electronicexcitationofCuPctohigherstateschargetransferbetweentheCuatomandthephthalocyaninering ionizationof themoleculeand formationofradicalsThephoto-inducedprocessesarelikelytoplayabiggerroleinTERSspectradetectedat633-nmexcitationduetotheoverlapwithQ-bandofCuPcwhichmaybethecauseoftheirlowersignaltonoiseratioandreproducibility

4Conclusions

Thedeveloped referenceprobe systemofCuPc adsorbedon aAu surfacehasprovedtobesuitablefortheintendeduseasitenabledthedetectionofintenseandwell-resolvedSERSandTERSspectraTheAulayerpreventsoverheatingofthesampleandallowsfortheuseofSTMTheSERSspectrawereinagoodmatchwith thespectraofpureCuPcAslight shiftof somebandsandchange in theluminescent background indicated the interaction between CuPc and the AusurfaceTheTERSexperimentsresultedinspectralmapswithhighintensitiesofindividualspectraIncreasedvariabilitybetweenmeasuredpointswasobservedPossiblesourcesofthevariabilityarephoto-inducedprocessesthatmayoccurinthestronglyenhancedelectricfieldTheseeffectsareaknownfeatureofTERSmeasurements and they offer valuable insight into the photophysics andphotochemistryofCuPcinteractingwiththeAusurfaceThedependenceofTERSspectra on experimental parameters and the preparation procedure of thereferencesampleshouldbefurtherstudied

Acknowledgments

ThisworkwassupportedfromthegrantofSpecificuniversityresearchndashA2_FCHI_2020_039


Fig 4TwoexamplesofaveragedTERSspectraofCu(II)phthalocyaninemeasuredwith785-nmexcitationThespectraareaveragesfromTERSmapsincluding16(top)and80(bottom)measuredpointsThespectraareoffset

References

[1] Mas-BallesteRGomez-NavarroCGomez-HerreroJZamoraF2DmaterialstographeneandbeyondNanoscale3(2011)20ndash30

[2] ZengHCuiXAnopticalspectroscopicstudyontwo-dimensionalgroup-VItransitionmetaldichalcogenidesChemSocRev44(2015)2629ndash2642

[3] SchultzJFLiLMahapatraSShawCZhangXJiangNDefiningmultipleconfigurationsofrubreneonaAg(100)surfacewith5A spatialresolutionviaultrahighvacuumtip-enhancedRamanspectroscopyJPhysChemC124(2020)2420ndash2426

[4] WhitemanPJSchultzJFPorachZDChenHNJiangNDualbindingconfigurationsofsubphthalocyanineonAg(100)substratecharacterizedbyscanningtunnelingmicroscopytip-enhanced Raman spectroscopy and density functional theory J Phys Chem C 122(2018)5489ndash5495

[5] ShaoFZenobiRTip-enhancedRamanspectroscopyprinciplespracticeandapplicationstonanospectroscopicimagingof2DmaterialsAnalBioanalChem411(2019)37ndash61

[6] ArocaRSurface-EnhancedVibrationalSpectroscopyHobokenWiley2006[7] KumarNMignuzziS SuWRoyDTip-enhancedRamanspectroscopyprinciplesand

applicationsEPJTechInstrum2(2015)9[8] BailoEDeckertVTip-enhancedRamanscatteringChemSocRev37(2008)921ndash930[9] BovillAJMcConnellAANimmoJASmithWEResonanceRamanspectraofα-copper

phthalocyanineJPhysChem90(1986)569ndash575[10] Shaibat MA Casabianca LB Siberio-Perez DY Matzger AJ Ishii Y Distinguishing

polymorphsofthesemiconductingpigmentcopperphthalocyaninebysolid-stateNMRandRamanspectroscopyJPhysChemB114(2010)4400ndash4406

[11] SzybowiczMRunkaTDrozdowskiMBałaWGrodzickiAPiszczekPBratkowskiAHightemperaturestudyofFT-IRandRamanscatteringspectraofvacuumdepositedCuPcthinfilmsJMolStruct704(2004)107ndash113

[12] CaplinsBWMullenbachTKHolmesRJBlankDAFemtosecondtonanosecondexcitedstatedynamicsofvapordepositedcopperphthalocyaninethinfilmsPhysChemChemPhys18(2016)11454ndash11459

[13] SaccoAImbraguglioDGiovannozziAndreaMPortesiCRossiAMDevelopmentofacandidatereferencesampleforthecharacterizationoftip-enhancedRamanspectroscopyspatialresolutionRSCAdv8(2018)27863ndash27869

[14] JiangSChenZChenXNguyenDMatteiMGoubertGVanDuyneRPInvestigationofcobaltphthalocyanineatthesolidliquidinterfacebyelectrochemicaltip-enhancedRamanspectroscopyJPhysChemC123(2019)9852ndash9859

[15] MelendresCAMaroniVARamanspectraandnormalcoordinateanalysisoftheplanarvibrationsofironphthalocyanineJRamanSpectrosc15(1984)319ndash326


1Introduction

The aim of this work has been develo-pmentofanewvoltammetricmethodforthe determination of 23-dimercapto-1-propane-sulfonic acid (DMPS) Fig 1Investigationwasdonetoobtainrelevantinformation about complexingbehaviorofDMPStowardsleadions Lead is one of heavy metals which can cause irreversible neurologicalproblems [1 2]DMPS is a synthetic antidotewith two thiol groups used fortreatmentofpoisoningbyheavymetals[3ndash6]StrongcomplexingpropertieshighwatersolubilityandnegligiblesideeffectsarethemostimportantadvantagesofDMPS[47]

Fig 1 Chemical structure of 23-dimercap-to-1-propane-sulfonicacid

Determination of heavy metal poisoning antidote 23-dimercapto-1-propanesulfonic acid using silver solid amalgam electrode

ab ab bc bMARTACHOIN SKA VOJTE CHHRDLICKA BEATRIZRUIZREDONDO JIR IBAREK aTOMA S NAVRATIL

a JHeyrovskyacuteInstituteofPhysicalChemistryoftheCzechAcademyofSciences Dolejškova21553182thinsp23Prague8CzechRepublicmartachoinskagmailcomb UNESCOLaboratoryofEnvironmentalElectrochemistryDepartmentofAnalyticalChemistryFacultyofScienceCharlesUniversityHlavova20308128thinsp43Prague2CzechRepublic

c UniversityofValladolidPlazadeSantaCruz847002ValladolidSpain

Abstract23-Dimercapto-1-propane-sulfonic acid (DMPS) was investigatedusingdirectcurrentvoltammetry(DCV)differentialpulsecathodicstrippingvoltammetry(DPCSV)differentialpulseanodicstrippingvoltammetry(DPASV)andeliminationvoltammetrywithlinearscan(EVLS)atapolished(p-AgSAE)andatameniscusmodifiedsilversolidamalgam electrode (m-AgSAE) EVLS confirmed two consecutivereductions with coupled protonelectron transfer VoltammetrictitrationsofDMPSwithPb2+provedcomplexformationwithlimits

minus1ofquantification(LOQs)anddetection(LODs)03and01micromolL atminus1m-AgSAE and 08 and 03micromolL at p-AgSAE respectively

Determination of DMPS in commercial drug Dimaval and humanurine samples confirmed practical applicability of the developedmethod

Keywordscathodicstripping

voltammetry23-dimercapto-1-pro-

panesulfonicacideliminationvoltammetry

withlinearscansilversolidamalgam

electrodeunithiol


Voltammetry was chosen as a determination technique due to its highsensitivity and selectivity speed low costs [8] Thiol groups in DMPS can beoxidatively chemisorbed on solid amalgam electrode It can be used as anaccumulation step for cathodic strippingvoltammetry [9ndash11]Moreover solidamalgamelectrode (SAE)was chosen as theworking electrodebecauseof itspropertiesashighsignaltonoiseratiowidepotentialwindowandabilitytoreachlowlimitsofdetection(LOD)[12ndash13]

2Experimental


Allsolutionswerepreparedusingdeionizedwater(Milli-Q-GradientMilliporendash1PragueCzechRepublic)withconductivitylt005microScm Britton-Robinsonbuffer

solutionspHrangefrom2to12werepreparedbymixingtheproperamountsof02MNaOH(alkalinesolution)andof004MH BO 004MH PO and004M3 3 3 4

CH COOH(allLachemaCzechRepublic)acidicsolutionTheacidicsolutionwas3

preparedbydissolutionof1235gofH BO pa088mLofH PO (85)paand3 3 3 4

1435mLofCH COOH(99)pa in500mLofdeionizedwaterThealkaline3

solution was prepared by dissolution of 3995 g of NaOH pa in 500mL ofdeionizedwater(allLachemaCzechRepublic) StocksolutionofDMPSwaspreparedbydissolving10mgofsolid23-dimer-capto-1-propanesulfonic acid monohydrate pa (Merck Czech Republic) in100mLofdeionizedwaterForthepreparationofthemodelsamplesolutiononecapsuleofdrugDimaval(HeylGermany)contains100mgofDMPSwasdissolved

ndash1in10LofdeionizedwatertotheDMPSconcentrationof0531mmolL Twomodel samplesofDimavalwerepreparedbydilutionof theabove-mentioned

ndash1solutionwithBritton-Robinsonbuffersolutiontoconcentrations10micromolL andndash1of 10micromolL respectively Urine model samples were prepared by mixing

Britton-Robinsonbuffer solutionwithurine samples obtained fromvolunteer(manhealthy30yearsold)inratio11SamplepHwasadjustedbyadditionof

ndash1proper amount of 02molL NaOH Before each measurement oxygen wasremoved for 5minbynitrogenbubbling (purity class 46MesserTechnogasPragueCzechRepublic)

22Instrumentation

Measurementswereperformedusingtwotypesofworkingelectrodesmeniscusmodified silver solid amalgam electrode (m-AgSAE working surface of

20382plusmn0025mm α lt 005) and polished silver solid amalgam electrode2(p-AgSAEworkingsurfaceof0196plusmn0015mm αlt005)Ag|AgCl|3MKClwas

used as the reference electrode andplatinumwire (Oslash1mm)wasused as theauxiliary electrode (both from Elektrochemicke detektory Czech Republic)Measurementswereperformedatlaboratorytemperature(25plusmn2degC)


The pH was measured using pH-meter Jenway 3505 with combined glasselectrodetype924001(BibbyScientificLimitedUK)Voltammetricmeasure-ments were performed using the computer-controlled Eco-Tribo Polarograph(Polaro-Sensors Czech Republic) Software used for measurements wasMultiElChem 33 forWindows XP7810 (J Heyrovsky Institute of PhysicalChemistryoftheCzechAcademyofSciencesCzechRepublic)


OptimumconditionsformeasurementswereobtainedbyseriesofmeasurementinwiderangeofpHvaluesandtestingvariouscleaningproceduresOptimumpotentialof accumulation (E ) and timeof accumulation (t )ofDMPSwereacc acc

adjusted for differential pulse cathodic stripping voltammetry (DPCSV) atp-AgSAEandm-AgSAE ThedependencebetweenpeakheightandconcentrationofDMPSatp-AgSAEhasalogarithmicshapewhichcorrespondstotheaccumulationprocessattheelectrodesurfaceThelineardependencewasobservedintheDMPSconcentra-

ndash1 ndash1tionsfrom03micromolL to20micromolL Peakshifttowardsnegativepotentialwithincreasing concentration of DMPS corresponds to the metal-thiol bond andinfluenceofelectrodesurfacestructureonthisbondOnthecontrarysignalsonm-AgSAEweremorestableandthedependencebetweenconcentrationofDMPSandsignalwasalmostlinearinwholetestedrangeofconcentrations The developedmethodwas tested inmodel samples of Dimaval and urineFoundamountsofDMPSwereingoodagreementwithdeclaredcontentsusingbothelectrodesHowevertherepeatabilityofsignalsregisteredusingp-AgSAEinurinesamplesweresignificantlyworsethanthoseinDimavalsamplesItcanbecausedbycomplicatedbiologicalmatricesandfoulingeffectsofurine ELSV measurements confirmed two consecutive reductions of DMPS inadsorbed state At m-AgSAE signal were at about minus415 mV and minus440 mVrespectively and at p-AgSAE at about minus790 mV and minus830 mV respectively

ndash1 ndash1Reductionsatm-AgSAEatthescanratesfrom80mVs to640mVs havebeencontrolledbyakineticprocessatminus400mV Inanodicscansonm-AgSAEonlyonepeakwasvisibleatabout‒390mVItcorrespondswiththeoxidationofmercuryelectrodesurfaceontheelectrodeinthepresenceofDMPSandwithdiffusionfromthebulksolutionofproductsAtp-AgSAEnosignificantanodicsignalwasfound ThelastpartoftheresearchrevealedvoltammetricbehaviorofDMPSinthe

2+presence of Pb Voltammetric titration was investigated by DPCSV anddifferential pulse anodic stripping voltammetry (DPASV) during consecutive

ndash1 ndash1additionsof1mmolL ofPb(NO ) into100micromolL DMPSsolutioninacetate3 2

bufferofpH50InabsenceofPb2+ontheelectrodesurfaceHg(DMPS)complexisformedduringtheaccumulationstepDuringtheanodicscanthereisonlyonewelldevelopedreductionpeak(Fig2A)


ndash1Fig 2DPCSandDPASvoltammogramsof10micromolL ofDMPSinacetatebufferpH=5correspond-2+ingto[Pb DMPS]ratiosof(A)01(B)11and(C)21Uppercurvecorrespondstothecathodic

scanE =0mVt =15sLowercurvecorrespondstoreverseanodicscanwitht =15satacc acc accndash1E =minus1000mVν=20mVs (Ref[16])acc


2+ WhenPb DMPSratioisequalto11twooxidationandthreereductionpeakswereregistered(Fig2B)PeakA ataboutndash300mVcorrespondstoformationofred

0Pb(DMPS)complexThiscomplexwasfurtherreducedtothePb (Hg)atabout2+ndash500mV(C )ReductionpeakoffreePb wasalsoregistered(B )Oxidationred red

peaksA andB correspondtoreverseprocessesandC isnotpresentbecauseox ox ox

nofreeDMPSispresentinthesolutionAtratio21thereisnofreeDMPSinthesolution however excess of lead ions As a consequence A and B peakred ox

2+increasedIncreaseofB correspondstothedepositionofPb duringtheaccu-ox

mulationstepPeaksBredandCredremainedpracticallyunchanged(Fig2C) Voltammetric titrationconfirmed themechanismof formationcomplexesofPb(DMPS)Hg(DMPS)andPb(Hg)Italsoconfirmedpossibilityofdetermination

2+Pb andDMPSinthesamesolution

4Conclusions

ValidationinmodelsampleofdrugDimavalandhumanurinespikedwithDMPSconfirmed that this method can be used for clinical purposes Voltammetric

2+titration of DMPS by Pb ions proved that it can be used for simultaneousdetermination of the drug and heavy metal ions in human urine Moreoverobtained LODs were two orders lower than those in the previously reportedvoltammetricmethod[14](Table1)

Acknowledgments

ResearchwascarriedoutwithintheframeworkofSpecificUniversityResearch(SVV260560)TheauthorsthanktheCzechScienceFoundation(GACRprojectNo20-01589S)

References

[1] AnHHLuchakMCopesRLeadtoxicityAsystematicreviewofrecentlypublishedcasesClinToxicol53(2015)757ndash758

[2] KimYLustMRKreimerbirnbaumM23-Dimercaptopropane-1-sulfonate(DMPS)inthetreatmentoflead-poisoningFasebJ2(1988)A1820ndashA1820

[3] AposhianHVDMSAandDMPS ndashwater-solubleantidotesforheavy-metalpoisoningAnnuRevPharmacol23(1983)193ndash215


Table 1ComparisonofvoltammetricmethodsforDMPSdetermination(LDR

Method Workingelectrode Lineardynamic LOQ LOD Refminus1 minus1 minus1 rangemicromolL micromolL micromolL

LSV glassy-carbonelectrode 18ndash140 41 14 [14] modifiedwithmulti-walled 260ndash690 carbonnanotubes DPCSV p-AgSAE 03ndash20 08 03 thisworkDPCSV m-AgSAE 01ndash10 03 01 thiswork 10ndash100

[4] BjorklundG Crisponi G Nurchi VM Cappai R Djordjevic AB Aaseth J A review oncoordinationpropertiesof thiol-containingchelatingagents towardsmercury cadmiumandleadMolecules24(2019)3247

[5] DonnerAHrubyKDMPSinthetreatmentofacuteandchronicheavy-metalpoisoningActaMedAust14(1987)10ndash10

[6] DonnerAHrubyKPirichKKahlsPSchwarzacherKMeisingerVDimercaptopropan-sulfonate(DMPS) inthetreatmentofacute lead-poisoningVetHumToxicol29 (1987)37ndash37

[7] Blanusa M Varnai VM Piasek M Kostial K Chelators as antidotes of metal toxicityTherapeuticandexperimentalaspectsCurrMedChem12(2005)2771ndash2794

[8] BarekJMoreiraJCZimaJModernelectrochemicalmethodsformonitoringofchemicalcarcinogensSensors-Basel5(2005)148ndash158

[9] Josypcuk B FojtaM Yosypchuk O Thiolatemonolayers formed on different amalgamelectrodesPartIIPropertiesandapplicationJElectroanalChem694(2013)84ndash93

[10] YosypchukBMarecekVPropertiesofthiolatemonolayersformedondifferentamalgamelectrodesJElectroanalChem653(2011)7ndash13

[11] Alvarez JMF SmythMRCathodic strippingvoltammetryofpyridine-2-thiolandsomerelated-compoundsAnalyst114(1989)1603ndash1605

[12] DanhelABarekJAmalgamelectrodesinorganicelectrochemistryCurrOrgChem15(2011)2957ndash2969

[13] Fadrna R Polished silver solid amalgam electrode Further characterization and appli-cationsinvoltammetricmeasurementsAnalLett37(2004)3255ndash3270

[14] ZiyatdinovaGKGrigorevaLVBudnikovGKElectrochemicaldeterminationofunithioland lipoic acid at electrodesmodifiedwith carbonnanotubes J Anal Chem64 (2009)185ndash188

[15] HrdlickaVChoinskaMRedondoBRBarekJNavratilTDeterminationofheavymetalpoisoning antidote 23-dimercapto-1-propanesulfonic acid using silver solid amalgamelectrodeElectrochimActadoiorg101016jelectacta2020136623


Fig 1Structureofcanagliflozin

1Introduction

Canagliflozin is a selective sodium-glucosecotransportertype2inhibitorused for the treatment of type 2 dia-betes mellitus Canagliflozin inhibitssodium-glucose cotransporter type 2present in proximal tubules of the

Canagliflozin oxidation study using electrochemical flow cell and comparison with hydrogen peroxide oxidation

a a bFILIPVYMYSLICKY TOMA S KR IZ EK JAKUBHER T

a DepartmentofAnalyticalChemistryFacultyofScienceCharlesUniversity Hlavova8203012843Prague2CzechRepublicfvymyslickygmailcomb ZentivaGroupasUKabelovny13010237Prague10CzechRepublic

AbstractBystandardstheeffectonoxidationofanactivesubstanceistestedusinghydrogenperoxidesolutionatelevatedtemperatureinastresschamberfor1ndash7daysAnalternativewaytostudytheeffectofoxida-tion on an active substance is to use an electrochemical flow cellSolutionwith active substance flows at low flow rate into a smallreactorwheretheactivesubstanceisoxidizedonworkingelectrodesurfaceTheelectrolytestreamwiththeoxidizedactivesubstanceisthen directed to the sample collector Products of electrochemicaloxidationareanalyzedbyhighperformanceliquidchromatographywithultravioletndashvisiblespectrophotometrydetectionCanagliflozinhasbeenusedbecauseitsmaindegradationpathwayisoxidationThedesign of experiments approach was used to explore the experi-mentalspaceandoptimizeexperimentalconditionsofoxidationTheresultsoftheoxidationstudyperformedintheelectrochemicalflowcellwerestatisticallycomparedwiththeresultsofastandardstudyusinghydrogenperoxidesolutionThemostsuitableconditionsforelectrochemical oxidation were found Electrochemical oxidationproducedcomparableamountsofimpuritiesaschemicaloxidationwithhydrogenperoxide

KeywordscanagliflozindesignofexperimentselectrochemicalflowcellHPLCoxidation


kidneywhichrestrictsglucoseabsorptioninthekidneytherebyincreasingtheurinaryexcretionofglucoseandloweringthelevelofglucoseintheblood[1]TheformulaofcanagliflozinisC H FO SthestructureofcanagliflozinisinFig1The24 25 5

IUPAC name of canagliflozin is (2S3R4R5S6R)-2-[3-[5-(4-fluoro-phenyl)-thiophen-2-ylmethyl]-4-methyl-phenyl]-6-hydroxymethyltetrahydro-pyran-345-triol[2]CanagliflozinisawhitepowderinsolubleinwaterbutverysolubleinorganicsolventslikemethanolordimethylsulfoxideCanagliflozinissoldundertradenameINVOKANA Manyauthorshavestudiedtheelectrochemicalpropertiesofactivesubstancesin the literature One example is the study of electrochemical behaviour andoxidationofbromhexineThesepropertieswerestudiedusingdifferentialpulsevoltammetryandcyclicvoltammetryonacarbonelectrodeTheresultsofelectro-chemicalmethodswerecomparedwithhighperformanceliquidchromatography(HPLC)analysis[3]Anotherexampleisthestudyofelectrochemicalbehaviourand degradation study performed on the active substance atomoxetineDegradation was studied using differential pulse voltammetry and cyclicvoltammetryonacarbonelectrodeTheresultswerealsocomparedwithHPLCanalysis[4]Electrochemicalmethodsareusedmainlytostudythemechanismofoxidationbutinthisworktheelectrochemicalmethodwasusedtodegradetheactivepharmaceuticalingredient In the stability studies of active pharmaceutical ingredient properties theinfluence of temperature pH light and oxidation is studied [5] By TheInternationalCouncilforHarmonisationofTechnicalRequirementsforPharma-ceuticals forHumanUse (ICH) standards the influenceof oxidationon activepharmaceutical ingredient is studied using hydrogen peroxide at roomtemperatureorincreasedtemperatureinthestresschamberduring1ndash7days[6]Analternativewaytostudytheinfluenceofoxidationonactivepharmaceuticalingredient is using electrochemical flow cellwhere an electrolytewith activepharmaceuticalingredientisdrivenbylowflowrateintothesmallreactorInthesmallreactortheactivepharmaceuticalingredientisoxidizedonthesurfaceoftheworkingelectrodeThestreamofelectrolytewithoxidizedactivepharma-ceuticalingredientisdriventothesamplecollectorProductsofelectrochemicaloxidationareanalysedbyHPLCUVVISThedesignofexperimentsapproachwasusedfordevelopmentofanalternativemethodofoxidationofcanagliflozinusinganelectrochemical flowcellThedesignofexperimentsapproachwasusedtoexplore the experimental space of the method and to find the optimalexperimentalconditionsofelectrochemicaloxidationofcanagliflozin

2 Experimental

21Materialandreagents

Canagliflozin(ZentivaCzechRepublic)999methanol(HoneywellGermany)98ammoniumdihydrogenphosphate(Sigma-AldrichJapan)35ortho-phos-phoricacid(PentaCzechRepublic)25ammonia(LachnerCzechRepublic)30hydrogenperoxide (LachnerCzechRepublic)water forchromatography


Fig 2Schemeoftheelectrochemicalflowcell(1)input(2)workingelectrode(3)gasket(4)refe-renceelectrode(5)counterelectrode

wasobtainedbypurifyingdemineralisedwaterusingMilliporetypeSynergyUVpurificationinstrument

22Instruments

An Agilent 1290 HPLC system (Agilent Technologies Germany) with highpressure pump autosampler thermostat and DAD detector was used for allexperimentsThePinnacleDBbiphenylcolumn(100times21mm19micromRestekUSA)wasusedforseparationIntheHPLCmethod10mMammoniumdihydrogenphosphatebufferpH=25wasusedascomponentAandmethanolascomponentBofthemobilephaseThegradientprogramwassetasfollowst(min)B01555010551790229023152515Theflowrateofthemobilephasewas

ndash104mlmin and the injection volume was 2μl The detector operated at awavelengthof220nmTheautosamplertemperaturewassetat20degCandthecolumntemperatureat60degCTheEmpowersoftwarewasusedforevaluationForelectrochemicaloxidationelectrochemicalflowcellfromALS(Japan)wasusedGlassycarbonelectrode(=6mm)andsilversilverchlorideelectrodewereusedasworkingandreferenceelectroderespectivelyTheschemeofelectrochemicalflowcellisinFig2ElectrodeswereconnectedwithpotentiostatPalmSens3fromPalmsens (Netherlands) AnElmasonic S15Hultrasonic bath fromElma (Ger-many)wasusedforsamplepreparationForpHmeasurementspHmeterJenway3540fromJenway(UnitedKingdom)wasused


AtthedevelopmentofthemethodforthestudyofcanagliflozinoxidationusingelectrochemicalflowcellitwasfirstnecessarytofindtheoptimalconditionsofelectrochemicaloxidationThedesignofexperimentsapproachwasusedChosen


ndash1Fig 3Cyclicvoltammogramofcanagliflozin(concentrationofcanagliflozin11mgml electrolyte300 mM ammonium dihydrogen phosphate pH = 40 and methanol (11 vv) and scan rate

ndash1001Vs )

independentvariablesandtheirlevelswereconcentrationofelectrolyte(100200300mM)pHofelectrolyte(406080)cellsize(50100200500microm)and

ndash1flowrate(0102504mlh )ThereducedcombinatorialdesignwasusedIntheModde12 software aworksheet containing 11 experimentswas created Theworkingpotentialof12Vwasselectedbasedoncyclicvoltammetryofcanagli-flozininFig3Fromthisfigureitcanbeseenthattheoxidationofcanagliflozinoccursintheregionfrom11Vto14VAllexperimentswereperformedwith

ndash111mgml canagliflozin samples The glassy carbon electrodewas used as aworkingelectrodeandthesilversilverchlorideelectrodewasusedasareferentelectrode The canagliflozin samples oxidized in the electrochemical flow cellunder theexperimentalconditionsgivenby theworksheetweremeasuredbyHPLCwithUVVISdetectionDependentvariablespeakareasofimpuritiesandpercentage of peak areas of impurities obtained from chromatograms wereevaluated by the partial least squaremethod in theModde 12 software Thevariableimportanceintheprojectionplottoolwasusedforinterpretationofthedata as a whole The significance values of the independent variables were

ndash1evaluatedbufferpH=137flowrateof125mlh bufferconcentration061mMandcell size04micromFromthis tool itwasconcluded that theelectrochemicaloxidationofcanagliflozinisthemostaffectedbythepHoftheelectrolyteandflowrateoftheelectrolyteUsingtheoptimizertoolthemostsuitableconditionsfor

ndash1the oxidation of canagliflozin were evaluated flow rate 01 ml h 300 mMammoniumdihydrogenphosphate pHof electrolyte40 and cell size500micromUsingonefactoratthetimeapproachthedependenceofthecellsizeonthetotalsumofimpuritieswastested(Fig4A) Itisvisiblefromthegraphthatthesmallerthecellweusethemoreoxidationproducts are formed Based on the graph the most suitable conditions were


Fig 4(A)Optimizationofelectrochemicaloxidationconditionsdependenceofsumofimpuritiesoncellsize(B)Chromatogramofasampleoxidizedundermostsuitableconditions

ndash1adjustedtoflowrate01mlh 300mMammoniumdihydrogenphosphatepHofelectrolyte40andcellsize12micromUnderthemostsuitableconditionsarepeat-ability test was performed by ten independent oxidation experiments Therelativestandarddeviationofthepercentageareaofcanagliflozinwas164atasignificant level of 095 The chromatogram of sample oxidized under mostsuitableconditionsisinFig4BThestandardstudyoftheeffectofoxidationoncanagliflozinusinghydrogenperoxideaccordingtoICHguidelineswasperfor-medThestudywasperformedundertwosetsofexperimentalconditionsInthefirstcaseasolutionof50methanolwiththeadditionof3H O wasusedIn2 2

thesecondcasetheconditionsintheelectrochemicalflowcellweresimulatedA300mMammoniumdihydrogenphosphate pH40 andmethanol in a ratio11(vv)withtheadditionof3H O wasusedSamplesfortheoxidationstudy2 2

werestressedinastabilitychamberfor13and7daysattheconstanttempe-ratureof50degC ThetotalsumsofimpuritiesformedduringchemicaloxidationusinghydrogenperoxideinbothmediawerecomparedasisshowninFig5AItisobviousthat


Fig 5(A)Acomparisonofastandardoxidationstudyusinghydrogenperoxidein50methanolwith added buffer andwithout them (B) Chromatogramof sample oxidized electrochemically(C)Chromatogramofsampleoxidizedchemically


ammoniumphosphatesuppressesoxidationofcanagliflozinThereasonofthisphenomenonisunknownFig5BandFig5CshowchromatogramsofsamplesoxidizedelectrochemicallyandchemicallyrespectivelyItcanbeseenthatfiveimpuritieswereformedbybothtypesofoxidationhoweverindifferentamounts

4Conclusion

AnalternativemethodfortheoxidativestudyofcanagliflozinwasdevelopedThedesign of experiments approach was used in the method development ThedevelopedmethodworkswithRSDof165(α=095)Oxidationofcanagliflozinbythedevelopedmethodproducedfiveimpuritiesthatareidenticalwiththoseproducedusingthestandardoxidationstudywithhydrogenperoxide

Acknowledgments

ThisworkhasbeensupportedbyCharlesUniversityResearchCentreprogramNoUNCESCI014SVV260560projectandpharmaceuticalappliedresearchcenter(TheParc)

References

[1] ChaoECCanagliflozinDrugsFuture36(2011)351ndash357[2] NislySAKolanczykDMWaltonAMCanagliflozinanewsodium-glucosecotransporter2

inhibitorinthetreatmentofdiabetesAmJHealthSystPharm70(2013)311ndash319[3] Turchan M Jara-Ulloa P Bollo S Nunez-Vergara LJ Squella JA Alvarez-Lueje A

VoltammetricbehaviourofbromhexineanditsdeterminationinpharmaceuticalsTalanta73(2007)913ndash919

[4] Perez-OrtizMMunoz C Zapata-Urzua C Alvarez-Lueje A Electrochemical behavior ofatomoxetineanditsvoltametricdeterminationincapsulesTalanta82(2010)398ndash403

[5] Baertschi SW Alsante KM Reed RA Pharmaceutical Stress Testing Predicting DrugDegradationLondonInformaHealthcare2011

[6] RignallA ICHQ1A(R2) stability testing of newdrug substance andproduct and ICHQ1CstabilitytestingofnewdosageformsInICH Quality Guidelines An Implementation GuideATeasdaleDElderRWNims(Eds)HobokenWiley2017p3ndash44


1Introduction

AlthoughDNArepresentsarelativelystablecomponentfromthechemicalpointofviewitremainsconstantlyexposedtoalargenumberofchemicalorphysicalagentscausingchemicalchangesinDNAmoleculesthatoccurintheenvironmentoraremajororminorproductsofcellularmetabolism[1] One-electronoxidationoftheDNArepresentsadamagingprocesswheretheloss of an electron (oxidation) fromduplexDNA results in the formationof anucleobase radical cation (electron ldquoholerdquo) that is subsequently consumed inchemicalreactionsthatoftenleadtomutationsAdefiningcharacteristicoftheone-electronoxidationofDNAisthepreferentialreactionattheguaninemoietythatisdetectedasstrandcleavagefollowingchemicalorenzymatictreatmentoftheoxidizedDNA[2ndash3]

Novel hybrid electrochemical DNA biosensor for monitoring oxidative DNA damage via oxidationreduction signals of low molecular weight double-stranded DNA

MICHALAUGUSTINVLASTIMILVYSKOCIL

UNESCOLaboratoryofEnvironmentalElectrochemistryDepartmentofAnalyticalChemistryFacultyofScienceCharlesUniversityHlavova812843Prague2CzechRepublicmichalaugustinnaturcunicz


AbstractDeoxyribonucleicacid(DNA)representsamajortargetmoleculeformanydamagingagentscausingunfavorablechangesinastructureofDNAmoleculethatbindandinteractwithDNAThusahighdemandforreliabletoolsregardingabettercomprehensionofthenatureofDNAdamagingprocessesstillrepresentsoneofthemaingoalsinthisareaHereinwedescribeadevelopmentofanovelhybridelectro-chemicalDNAbiosensorbasedonanldquoedge-planerdquopyrolyticgraphiteelectrode (EPPGE) in connectionwith an elementaryoptimizationprocessprovidingacloserresolutionoftheredoxprocessesoflowmolecularweightdouble-strandedDNA(dsDNA)attheEPPGESub-sequentanalyticalapplicationincorporatinganemploymentofthemodel structure K [IrCl ] (representative of transition metal2 6

complexes)andevaluationofitsdamagingeffectinrelationtoDNAbymeansof linear sweepvoltammetry resp square-wavevoltam-metryarealsopresented

KeywordsbiosensordamageDNAgraphitevoltammetry

DNA-based electrochemical biosensors are successfully used in variousapplicationssuchasmonitoringandevaluatingthemechanismsof interactionbetweenDNAandvariousdrugsordamagingagentsrapidmonitoringoftracemetalsorpollutantspresent in theenvironmentordirectmonitoringofDNAhybridizationprocesses[4] Theelectrochemicalactivityofnucleicacids(boththenativehigh-molecularonesaswellasoligonucleotides)isingeneralreferredtotheelectroactivityofitscomponents ndash nucleobases and sugar residues At mercury-based electrodesadenine and cytosine residues undergo reduction processes close to ndash14 V(againstSCE)inneutralorweaklyacidicmedium(givingrisetothepeakCA)Ontheotherhandallbaseshavebeenreportedtobeelectrochemicallyoxidizedatcarbonelectrodesbutonlyadenineand(particularly)guanineoxidationsignalshavebeenwidelyutilizedinelectrochemicalDNAbiosensors[5] In2017theelectrochemistryofnucleicacidsachievedanimportantmilestoneasthereductionoftheDNAoligonucleotideswasperformedataldquobasal-planerdquopyrolytic graphite electrode which provided wide potential window allowingboththeelectrooxidationaswellastheeletroreductionofthenucleobasesatasingleelectrodefortheveryfirsttimeDespitethesefindingsutilizationoftheaforementionedbiosensorintermsofanalyticalapplicationshasyettobeverifiedandremainsunclearuptothisdate[6] TheaimoftheproposedcontributionisapresentationofthedevelopmentprocessandsubsequenttestingofanoveltypeofhybridelectrochemicalDNAbiosensoranditsverificationasareliableanalyticaltoolintermsofmonitoringDNAdamage

2Experimental


Low molecular weight double-stranded DNA (dsDNA) derived from salmonspermwasobtainedfromSigma-AldrichGermanyStocksolutions(01mgmL)

of dsDNA were prepared in a 01 molL phosphate buffer of pH=74 (PB)Dipotassium hexachloroiridate (K [IrCl ]) was purchased from Sigma-Aldrich2 6

GermanyStocksolutions(0001molL)ofK [IrCl ]werepreparedinthePB2 6

22Apparatus

Voltammetric measurements were performed using the μAutolab IIIFRA2potentiostatgalvanostat(EcoChemieTheNetherlands)drivenbyaNOVA111software(MetrohmAutolabSwitzerland)Allmeasurementswerecarriedoutina three-electrode system using an ldquoedge-planerdquo pyrolytic graphite workingelectrode(EPPGE)withanelectroactivesurfacediameterof3mm(BASJapan)asilver|silver chloride reference electrode (Ag|AgCl|sat KCl) and a platinum


counterelectrode(ElektrochemickeDetektoryCzechRepublic)ina20mLglassvoltammetriccellatambienttemperature

23Preparationofthebiosensor

PriortotheeverymeasurementsurfaceoftheEPPGEwasmechanicallycleanedbygentlewipingoftheelectrodeonthesoftpolishingpadrinsedwithdistilledwaterAfterwardstheelectrodewasrinsedwithdistilledwaterandplacedinthePBforthesubsequentelectrochemicalactivationElectrochemicalactivationwasperformed in thePBbyapplyingpotentialof15V for240swithoutstirringApotentialpulseinworkingrangeofpotentials(00ndash15V)wasthenapplied Additional electrochemical activationwasperformed in the solutionof the

3minus4minusredoxindicator([Fe(CN) ] )byconsecutivecyclingintherangeofpotentials6

from10tondash08V(15scans)andfrom055tondash015V(10scans) The electrochemical DNA biosensor based on the EPPGE (dsDNAEPPGEbiosensor)was prepared by the adsorption of dsDNAon the EPPGEOptimal

parametersofthedsDNAadsorptionwereaconcentrationof01mgmLinthePB

(c )adepositionpotentialof07V(E )andanadsorptiontimeof5ming(dsDNA) dep

(t )withoutstirringthesolutionads

Atlasttheelectrodewasimmersedinthesolutionoftheredoxindicatorandtheconsecutivecyclingintherangeofpotentialsfrom055tondash015V(20scans)wasperformedinordertosecurethestabilityoftheoxidationreductionsignalsofdsDNAattheEPPGE

24Procedures

Theexperimentalparameterswereasfollowssquarewavevoltammetry(SWV)inthePBwithapulseamplitudeof20mVafrequencyof50Hzascanrateof750mVsandapotentialstepof15mVlinearsweepvoltammetry(LSV)inthePBwithscanratesof02ndash10Vandapotentialstepof24mVAllcurveswere

recordedthreetimes(n=3)


Sincetheclosestresolutionoftheprocessesassociatedwiththeelectroreductionof singleDNAcomponents at pyrolytic graphitehasbeenperformedwith theldquobasal-planerdquopyrolyticgraphiteelectrode(BPPGE)wehavedecidedtotakeovercorrespondingexperimentaltechniqueandconditions(LSVscanrateof10Vssteppotentialof24mV)attheverybeginningofouroptimizationprocesswiththeEPPGE[6] Inthisparticularcaseitispossibletonoticetheoccurrenceofthetwomixedvoltammetricpeaksatthedefaultexperimentalconditions(greenlineFig1A)selectedforthereductionofdsDNAattheEPPGEBygraduallydecreasingthe


Fig 1Baseline-correctedLSVrecordingscorrespondingtothereductionofdsDNAattheEPPGEfordifferentvaluesofscanrate(02ndash10VsFig1A)respbaseline-correctedLSVrecordingscorres-pondingtothereductionofdsDNAattheEPPGEandthenegativetestperformedunderthesameexperimentalconditionswithintheblanksolution(phosphatebuffer)atthebareEPPGE(03VsFig1B)

scan rate the optimal conditions (νle03Vs) were found and the mutualseparationof the signalswas allowedndash characterizedby thepresenceof twosinglewell-developedvoltammetricpeaksatpotentialsofndash175Vrespndash190V(03Vs orange line Fig 1A) Taking into account previous work regardingprocessesassociatedwiththereductionofDNAatthemercuryelectrodesrespBPPGE we can assume that the peak appearing at the potential of ndash175 Vcorresponds to themixedpeak for the reductionof the cytosine and adenineresidueswithindsDNA(peakCA)[5ndash6] Closer resolution of the second voltammetric peak appears to be farmoreproblematic Regarding our previous study we have discovered that theutilizationofdifferentE fortheadsorptionofdsDNA(E lt07V)isconnecteddep dep

withanappearanceofthethirdoxidationsignal(besidestheoxidationsignalsofguanine resp adeninemoieties) at apotential of073V corresponding to theoxidationoffreeguaninebases(FGBs)presentwithinthesolutionofdsDNAInthiscasewecanassumethatthepeakappearingatapotentialofndash190VcanpossiblyrepresentthereductioncounterpartofFGBspresentwithinthesolutionofdsDNAThisassumptioncanalsobesupportedbytheaforementionedstudyandbythefactthatthereductionsignalatsuchahighnegativepotentialcanbeobservedfortheoligodeoxynucleotidescontainingguanineresidues[6] AdditionallyinordertoverifythetruenatureofthereductionsignalsdepictedatF ig1Bandtoexcludetheoptionthattherelatedsignalsdonotrepresenttheproductsofpriorelectrochemicalactivationof theEPPGE(variousCndashObasedchemicalspecies)wehavedecidedtoperformanegative(control)testwithinthe


Fig 2Baseline-correctedLSVrecordingscorresponding to thereductionof thedsDNA(02Vs

Fig 2A)respbaseline-correctedSWVrecordingscorrespondingtotheoxidationoftheguanine(098V)respadenine(128V)moieties(075VsFig2B)attheEPPGEafteritsincubationinthephosphatebufferforadefinedtimeperiod(60ndash900s)

blanksolution(PB)employingthesameprotocolasforthedsDNAadsorptionattheEPPGEInthiscaseitispossibletoobservetheabsenceofanypronouncedvoltammetricpeakslinkedtothedsDNAadsorptionandonlythepresenceofoneirreversiblepeakatapotentialofndash153VcorrespondingtotheelectroreductionoftheCndashObasedmoietymoietieswhichdropsafterthedsDNAadsorptiontoonetenthofitsoriginalvalue(approximately) Perhaps the most important parameter regarding further optimizationprocessrepresentedthetime-dependentstabilityofthecorrespondingsignalsofdsDNAwhichcanbespecificallyimportantinrelationtothestudyofthetime-dependentoxidativedamageofdsDNA AsithasalreadybeenprovedasingleelectrochemicalactivationoftheEPPGEinthePBdoesnotrepresentasatisfyingtechniqueregardingstabilityofdsDNAoxidationsignalsat theEPPGEand theadditionalstabilization isachievedbyfurtherelectrochemicalactivationinthesolutionofaredoxindicator(Fig2B)[7]Basedonthisaverificationoftheproposedstabilizationprotocolintermsofthetime-dependent stabilityofdsDNAreduction signals in the solutionof thePBwithinthedefinedtimeperiod(60ndash900s)appearedasareasonablenextstep FromtheresultsdepictedinFig2Aitispossibletonoticethatwithinthefirst300sdsDNAreductionsignalsremainstableinrelationtothecurrentresponseaswellasintermsofthepotentialvalueWithanadditionalincubationtime(t )inc

(900sorangeline)thepeakcurrentofthevoltammetricsignalpresentatmorenegative potentials decreased which can probably be addressed as a slowprogressive elimination of the weak (electro)chemical forces related to theunspecificadsorptionoftheFGBsattheEPPGE


Fig 3Baseline-corrected SWV recordings corresponding to the oxidation of the guanine respadeninemoietiesattheEPPGEafteritsincubationinthesolutionofK [IrCl ](IR)foradefinedtime2 6

period(60ndash3600s)(075VsFig3A)andthecorrespondingrelativebiosensorresponses(ΔI )rel

evaluatedusingtheguanosine(turquoise)andadenosine(red)peaksplottedversustheincubationtime(Fig3C)Baseline-correctedLSVrecordingscorrespondingtothereductionofdsDNAattheEPPGEafteritsincubationinthesolutionofK [IrCl ](IR)fordifferenttimeperiods(60ndash3600s)2 6

(02 VsFig3B)andthecorrespondingrelativebiosensorresponses(ΔI )evaluatedusingtherelpeakCA(darkpink)plottedversustheincubationtime(Fig3-D)

AdditionallywehavedecidedtotesttheapplicabilityofthepresentedhybridbiosensorintermsofmonitoringdsDNAdamagecausedbyarepresentativeofone-electron oxidants ndash K [IrCl ] In this case the prepared dsDNAEPPGE2 6

biosensor was immersed into the solution of K [IrCl ] (0001molL) for the2 6

definedtimeperiod(60ndash3600s) In thecaseof theoxidationpath (SWVrecordingsdepicted inFig3A) it ispossible to observe a time-dependent decrease of the oxidation signal of theguaninemoietieswhereastheoxidationsignaloftheadeninemoietiesremainsunaffectedforthemostof the incubationperiodThisphenomenonis ingoodcorrelationwiththetheoreticalknowledgeregardingoxidativedamageofdsDNAcausedbyone-electronoxidants [3]Simultaneouslywith thisLSVrecordingsdepicted in Fig 3B followed the similar behavior (decrease in relation to the


currentresponseofthedsDNAreductionsignalndashpeakCA)asinthecaseofthesignal regarding oxidation of guanine moieties In addition according totheportionofthepreservedDNA(Fig3C3D)itispossibletoassumethatthepronouncedoxidativedamageofdsDNAcanbemonitoredquitepreciselynotonlydirectlyviathedsDNAoxidationsignaloftheguaninemoietiesbutevenindirectlythroughthedsDNAreductionsignalndashpeakCA

4Conclusions

Inthiscontributionwehavepresenteddevelopmentofanunorthodoxhybridelectrochemical DNA biosensor based on an EPPGE Optimization processconcerning some important parameters was performed as well as closerresolutionofthenatureofthereductionprocessesofdsDNAattheEPPGEwasachievedInordertoconfirmtheresultsoftheoptimizationprocessapplicabilityoftheproposedbiosensorhadbeenprobedintermsofmonitoringDNAdamagecausedbyK [IrCl ]Inthiscasethefinalresultshadprovedthattheprepared2 6

hybridbiosensorcanbeconsideredasaversatileanalyticaltoolformonitoringoxidativeDNAdamage(viaoxidationreductionsignals)andispresentedasafinealternative in comparisonwith conventional electrochemical DNA biosensorsprepared within the group of traditional transducer materials (mercury- orcarbon-based)

Acknowledgments

ThisresearchwassupportedbytheSpecificUniversityResearch(SVV260440)

References

[1] FojtaMDanhelAHavranLVyskocilVRecentprogressinelectrochemicalsensorsandassaysforDNAdamageandrepairTrACTrendsAnalChem79(2016)160ndash167

[2] GieseBSpichtyMWesselySLong-distancechargetransportthroughDNAAnextendedhoppingmodelPureApplChem73(2001)449ndash453

[3] Burrows CJ Muller JG Oxidative nucleobasemodifications leading to strand scissionChemRev98(1998)1109ndash1151

[4] DiculescuVC Chiorcea-PaquimAMOliveira-BrettAMApplications of aDNA-electro-chemicalbiosensorTrACTrendsAnalChem79(2016)23ndash36

[5] PalecekEJelenFElectrochemistryofnucleicacidsInElectrochemistryofNucleicAcidsandProteinsndashTowardsElectrochemicalSensorsforGenomicsandProteomicsPalecekESchellerFWangJ(edits)AmsterdamElsevier2005p74ndash174

[6] SpacekJDanhelAHasonSFojtaMLabel-freedetectionofcanonicalDNAbasesuraciland5-methylcytosineinDNAoligonucleotidesusinglinearsweepvoltammetryatapyrolyticgraphiteelectrodeElectrochemCommun82(2017)34ndash38

[7] AugustınMVyskocilVNovelelectrochemicalDNAbiosensorbasedonedge-planepyrolyticgraphite for DNA interaction studies In Proceedings of the 15th International StudentsConferenceldquoModernAnalyticalChemistryrdquoNesmerakK(edit)PragueFacultyofScienceCharlesUniversity2019p263ndash268


1Introduction

Cadmiumisoneofthemosttoxicmetalsanditswidespreadindustrialusesresultin increased environmental pollution Hence the development of sensitivemethodology for Cd determination is still highly desirable Chemical vaporgeneration(CVG)ofCdbythetetrahydroboratereductioninacidicmediumisasuitable alternative sample introduction technique compatible with atomicspectrometricdetectorsandofferingimproveddetectioncapabilityComparedtocommonliquidnebulizationCVGoffersseveraladvantagessuchassignificantlyhigheranalyteintroductionefficiencyandalsoanalyteseparationfromsamplematrix IncomparisontoCVGofcommonhydrideformingelementsthereisalackofliterature dealing with mechanistic aspects of CVG of Cd [1] as well as withstabilityandidentityofitsvolatilespecies(freeatomshydrideotherspecies)Very little information is also available on achieved generation efficiencyMoreover there are many discrepancies in the literature regarding optimum

Chemical vapor generation of cadmium for analytical atomic spectrometry

a b a b a aLINDASAGAPOVA BARBORAKODRIKOVA MILANSVOBODA STANISLAVMUSIL aJANKRATZER

a InstituteofAnalyticalChemistryoftheCzechAcademyofSciences Veveřiacute9760200BrnoCzechRepublicsagapovaiachczb DepartmentofAnalyticalChemistryFacultyofScienceCharlesUniversity Hlavova8203012843Prague2CzechRepublic

AbstractChemical vapor generation of cadmium volatile compounds wasoptimizedinordertodeterminetraceCdconcentrationsbyatomicabsorptionspectrometry(AAS)Severalreactionmodifiersbasedon

III+ II+ III+ IV+inorganicsaltsandcomplexesofCr Co Ti Ti weretestedtoimproveanalyticalperformanceandgenerationefficiencyTheuseofthese reaction modifiers resulted in 4ndash5 times enhancement insensitivity reflected also in corresponding increase of generationefficiency and better repeatability Generation efficiency wasdeterminedfromacomparisonbetweensensitivitiesobtainedwithchemicalvaporgenerationandconventionalsolutionnebulizationbothsimultaneouslycoupledwithinductivelycoupledplasmamassspectrometryTheidentityofthegeneratedcadmiumcompoundswillbediscussed

Keywordsatomicabsorption

spectrometryatomizationcadmiumchemicalvapor

generation


conditionsforCVGofCdAlthoughstrongacid(HClorHNO )isalwaysemployed3

asacarrierandNaBH asareductantsomeauthorsreportedvariousadditives4III+ III+ IV+(modifiers)basedontransitionmetalions(Cr Ti Ti )inthepresenceof

II+KCN[23]orCo inthepresenceofthioureaandascorbicacid[4]toimproveCdsignalssignificantly TheaimofthisworkwastoinvestigateCVGofCdinacomprehensivewayFirstlyCVGofCdwithoutandwithselectedmodifierswasoptimizedemployingatomicabsorptionspectrometry(AAS)asadetectorandexternallyheatedquartztube(QTA)astheatomizerSecondlytheeffectofatomizationtemperatureonCdsignalwasstudiedallowingthustodeducetheatomicormolecularstructureofgeneratedCd speciesThirdly generation efficiencyofCdvolatile specieswasquantified

2Experimental


minus1Boiled and bubled (Ar per 30min) deionizedwater (lt 01 μScm UltrapurWatrex USA) was used to prepare all solutionsWorking Cd standards were

minus1preparedfrom1000mgL Cdstocksolution(AstasolAnalytikaCzechRepublic)minus1bydilutionin01ndash048molL HCl(basedonthemodifieremployed)from37

HCl(paMerckGermany)Theoptimumgenerationconditionsbeingdifferentforeachmodifiertestedare listedinTable1ThereductantwasasolutionofNaBH (ge 97 Sigma-Aldrich Germany) in 04 (mv) KOH (pa Merck4

Germany) prepared fresh daily The solutions of modifiers were prepared as2+followsCo waspreparedfromCoCl 6H O(ge990PENTACzechRepublic)2 2

3+the solution of Cr from Cr(NO ) 9H O (ge 9999 tracemetal basis Sigma-3 3 23+AldrichGermany)thesolutionofTi fromTiCl solution(about15in10HCl3

4+Sigma-AldrichGermany)andthesolutionofTi fromTiOSO (ge999Sigma-4

AldrichGermany)Tostabilizethelattersolution1H SO wasusedprepared2 4

bydilutionof96H SO (paLach-NerSlovakia)SolutionofKCN(ge9702 43+ 3+FlukaSwitzerland)wasusedasasecondmodifierwhenworkingwithCr Ti or

4+ ndash3Ti asmodifiersitsconcentrationvariedfrom008to016moldm depending


Table 1OptimumconditionsforchemicalvaporgenerationofCdinthepresenceofmodifiersandtheirabsence

onthemetalionThiourea(CH N Sge980LachemaBrno)andascorbicacid4 2

(C H O ge997Riedel-deHaenGermany)wereusedasmodifierscombined6 8 62+withCo

22Instrumentation

221Chemicalvaporgenerationsystems

TwoCVG flow injection systemswere employed either a two channel systemwithoutadditionofamodifier(seeFig1A)orafourchannelsystemallowingadditionofmodifiers(seeFig1B)

ndash1 TheflowratesofHClandNaBH were42and10mLmin respectivelyinatwo4ndash1channelsystem(Fig1A)whiletheywerebothkeptat10mLmin inthefour

channelsystem(Fig1B)Theflowratesofmodifiersinthefourchannelsystemndash1were05mLmin Thevolumeofthesampleloopwas015mLinbothsystems

ndash1Carriergasflowrateof75mLmin Arwascontrolledbyamassflowcontroler(Cole-ParmerUSA)


(A)

(B)

Fig 1Schemesofthechemicalvaporgenerationflowinjectionsystemwith(A)twochannels(nomodifiers)and(B)fourchannels(modifiersemployed)

222Atomicabsorptionspectrometry

ThePerkin-Elmermodel503atomicabsorptionspectrometer(BodenseewerkGermany)wasequippedwithaCdelectrodelessdischargelamp(Perkin-ElmerUSA)operatedat228mAThemeasurementswereperformedat2288nmusinga07nmslitwidthTheShimadzumodelAA-7000atomicabsorptionspectrometer(ShimadzuJapan)wasalsousedACdhollowcathodelamp(PhotronAustralia)operatedat2288nmlinewith07nmspectralbandpassandalampcurrentof12mA Signals were recorded for 2 minutes and peak areas were taken forevaluation The QTA was heated electrically to the temperature required byfurnace(PerkinElmer)andanin-housemadefurnacecontrolledbytheREX-C100controller(SysconIndianaUSA)withtheK-typethermocouplesensor(OmegaEngineeringUSA)223QuantificationofCVGefficiencybyICP-MS

Overall CVG efficiency of Cd was quantified bymeans of inductively coupledplasmamassspectrometry(ICP-MS)fromcomparisonoftheslopesofcalibra-tionsobtainedwithnebulizationliquidCdstandardstothoseobtainedwithCVGThe efficiency of liquid nebulization was quantified using a modified wastecollection method (see reference [5] for details) The Agilent 7700x ICP-MSinstrument(AgilentUSA)wasoperatingat1600WofRFpowerThesignalwas

111 125monitoredat Cdisotopeandcorrectedforthesignalofinternalstandard( Tendash11000ngmL Tein2HNO )NebulizeranddilutionArgasflowrateswere11503

ndash1and0mLmin respectively


31Chemicalvaporgenerationconditions

UnivariateoptimizationswereperformedtofindoptimumconditionsforCVGofCdinpresenceandabsenceofmodifiersTheparameterstobeoptimizedwerecarrieracid(HCl)concentrationreductant(NaBH )concentrationmodifierIand4

modifier II concentrations carrier gas flow rate (Ar) length of reaction coilsRCI-III (see Fig 1B) The optimum conditions for individual modifiers aresummarizedinTable1

32IdentityofCdspecies

ThepeakareasofgeneratedCdspecieswereforagivenmodifiermeasuredintheQTAheatedto900degCandnon-heatedQTAsubsequentlyOptimumCVGconditionswere employed as summarized in Table 1 This simple experiment allowsdistinguishingbetweenatomic(freeatoms)andmolecularformsofgenerated


Fig 2RelativesignalofgeneratedCdspeciesmeasuredintheQTAheatedto900degC(blackbars)andnon-heatedQTA(whitebars)withoutorinthepresenceofmodifiers

speciesOnlyfreeatomscanbedetectedinnon-heatedQTAsimilarlyasincaseofmercurycoldvaporsOn thecontrarymolecularanalytespeciesareatomizedat900degCAsaconsequencethesignalregisteredintheheatedQTAcorrespondstobothatomicandmolecularspeciesgeneratedItmustbehighlightedthattheresidencetimeoffreeatomsintheatomizerisdependentonQTAtemperatureduetogasexpansionAsaconsequencethesignalinQTAheatedto900degCshouldreach25ofthesignalatambienttemperaturetakingintoaccountthatonlyfreeatomsaregeneratedSincethetemperaturealongtheopticalarmofQTAisnotdistributedhomogeneouslydecreasingtobothendtheeffectivetemperatureoftheatomizerislowerOurexperimentswithCVGofHgrevealedsignalinheatedQTAisaround40[6]TheresultsreachedforCVGofCdaredepictedinFig2ThesignalofCdinheatedQTAisaround50ofthesignaldetectedinnon-heatedQTA

4+whennomodifierisemployedorusingTi asthemodifierindicatingclearlyfreeCdatomsarethedominantvolatilespeciesgeneratedOnthecontraryalmostno

2+differenceinpeakareaswasobservedforCo asthemodifierwhilethesignalinheatedQTAwas even 5 times higher in heatedQTA compared to non-heated

3+atomizer with Cr as the modifier suggesting the dominant contribution of3+molecularstructurestoCdsignalespeciallyincaseofCr KCNreactionsystem

33Generationefficiency

TheoverallCVGefficiencywasestimatedfromacomparisonbetweensensitivitiesobtainedwithCVGsampleintroductionandconventionalsolutionnebulizationICP-MSunder the sameexperimental conditionsNebulization efficiency for aMicroMISTnebulizerwasdeterminedas79plusmn01ThegenerationefficiencyofCd was derived from the sensitivity enhancement between CVG and liquidnebulizationTheresultsaresummarizedinTable2indicatingthatCVGwithout


modifiersisonlycatwotimesmoresensitivecomparedtoliquidnebulization3+ 4+Generation efficiency of Cd increases to 60 in the presence of Ti and Ti

modifiers

4Conclusions

CVG of Cd was thoroughly optimized in the presence of selected modifiersreportedpreviouslyintheliteratureGenerationefficiencyofCdintheabsenceofanymodifierswasquantifiedto15whileitcanbeincreasedupto60inthe

3+ 4+presenceofTi KCNorTi KCNasmodifiersFreeCdatomsseemtobe the4+dominantCdformgeneratedintheabsenceofanymodifiersorusingTi KCN

3+modifierwhile rathermolecularCd structuresaregenerated inCr KCNand2+Co thioureaascorbicacidreactionsystems

ExperimentsareinprogresstofinishthiscomprehensivestudyOnlythebestmodifierwillbefurtherusedforCVGofCdtobecoupledwithotherspectrometricdetectorsandappliedtocertifiedreferencematerialsandrealsamples

Acknowledgments

ThisresearchhasbeensupportedbytheCzechScienceFoundationundercontract18-01116SandbytheInstituteofAnalyticalChemistryoftheCzechAcademyofSciences(InstitutionalResearchPlannoRVO68081715)andCharlesUniversity(ProjectnoSVV260440)

References

[1] PitzalisEAngeliniDMascherpaMCDacuteUlivoAInsightintothemechanismscontrollingthechemicalvaporgenerationofcadmiumJAnalAtSpectrom33(2018)2160ndash2171

[2] ArslanZYilmazVRoseLEfficientgenerationofvolatilecadmiumspeciesusingTi(III)andTi(IV)andapplicationtodeterminationofcadmiumbycoldvaporgenerationinductivelycoupledplasmamassspectrometryMicrochemJ123(2015)170ndash178

[3] YilmazVRoseLArslanZLittleMDOn-linechemicalvapourgenerationofcadmiuminthepresenceofhexacyanochromate(III)fordeterminationbyinductivelycoupledplasmamassspectrometryJAnalAtSpectrom27(2012)1895ndash1902

[4] Y Lu SunHW YuanCG YanXP Simultaneous determination of trace cadmiumandarsenic inbiologicalsamplesbyhydridegeneration-doublechannelAFSAnalChem74(2002)1525ndash1529


Table 2GenerationefficiencyofchemicalvaporgenerationofCdasquantifiedbyICP-MS

Modifiers Generationefficiency

Nomodifiers 15plusmn13+Cr KCN ndash2+Co thioureaascorbicacid ndash3+Ti KCN 58plusmn24+Ti KCN 61plusmn2

[5] VyhnanovskyJStrugeonREMusilSCadmiumassistedphotochemicalvaporgenerationoftungsten fordetectionby inductively coupledplasmamass spectrometryAnal Chem91(2019)13306ndash13312

[6] MigasovaMMatousekTSchrenkovaVZ ıdekRPetry-Podgorska IKratzer JMercuryvolatilespeciesgenerationfromHClandTRISbuffermediaAnalChimActa1119(2020)68ndash76


1Introduction

Atomicfluorescencespectrometry(AFS)coupledwithvapourgenerationisanultrasensitive analytical method for determination of various elements ItsanalyticalperformancecanbecomparabletoICP-MSwithliquidnebulizationbutatsubstantiallylowercost[1]SampleintroductiontoAFSisacrucialstepoftheanalyticalproceduresincetheanalytehastobeintroducedtotheatomizerintheformofitsvolatilespecies Hydridegeneration(HG)isamaturetechniqueofsampleintroductionduringwhichvolatileanalytehydridesare formedbyreactionwithareducingagenttypicallysodiumborohydrideAnewemergingtechniquephotochemicalvapourgeneration(PVG)employsUV irradiationof theanalyte in liquidphase in thepresenceofaphotochemicalagent(usuallyalowmolarmassorganicacidformic

minusoraceticacid)Highlyreducingradicalspecies(HbullRbullandCOObull )andaquatedelectronsare formedduring irradiationandreactwith theanalyte to form its

Photochemical vapour generation of bismuth coupled with atomic fluorescence spectrometry

ab ab a aBARBORASTA DLEROVA JAROMIRVYHNANOVSKY JIR IDE DINA STANISLAVMUSIL

a InstituteofAnalyticalChemistryoftheCzechAcademyofSciences Veveřiacute9760200BrnoCzechRepublicstadlerovaiachczb DepartmentofAnalyticalChemistryFacultyofScienceCharlesUniversity Hlavova8203012843Prague2CzechRepublic


AbstractPhotochemical vapour generation of bismuth was successfullycoupledwithnon-dispersiveatomic fluorescencespectrometry forthefirsttimeVolatilespeciesofBiweregeneratedusingastandardmercurylow-pressuretubelampandacoiledreactorfromareaction

2+mediumwhichwas composedof acetic and formic acid Co ionswereusedasasensitizerOptimizationofatomizationconditionsinaflame-in-gas-shieldatomizerwasperformedThismethodologywascomparedtothecommonlyemployedhydridegenerationapproachAbsolutelimitofdetectionof68pgwasachievedwithphotochemicalvapour generation which is still about 7 times worse than withhydride generation The developed methodology was successfullyverifiedbyBideterminationinareferencematerialofwater

Keywordsatomicfluorescence

spectrometrybismuthhydridegenerationphotochemicalvapour

generation

volatilespeciesInbothcasesthegeneratedvolatilespeciesoftheanalytehavetobeseparatedfromtheliquidphaseinthegas-liquidseparatorandarecarriedtotheatomizerbyacarriergas[2] InthisworkanatomizerdesignedspecificallyforAFStheflame-in-gas-shieldatomizerwasused(Fig1) It consistsofaverticalquartz tubesuppliedwithargon and hydrogen together with the analyte volatile species Moreover acapillaryisinsertedintheverticalaxisoftheverticaltubethroughwhichoxygenisintroducedA hydrogen-oxygenmicroflameburns on top of the capillary Themicroflameisshieldedfromtheambientatmospherebyaflowofargonwhichisintroducedthroughashieldingunitfittedaroundtheverticaltube[34] Theaimofthisworkwastooptimizeatomizationconditionsintheflame-in-gas-shieldatomizerusingPVGasasampleintroductiontechniqueandtocomparetheanalyticalcharacteristicsofPVGandHGforultrasensitivedeterminationofbismuthbyAFS

2Experimental


Deionized water (Ultrapur Watrex USA) was used for preparation of all thesolutionsWorkingBisolutionswerepreparedfreshdailybyserialdilutionof

ndash1stock1000mgl BistandardforAAS(Sigma-AldrichGermany)RegardingHG05(mv)NaBH in04(mv)KOHwasusedasa reductantAsolutionof4 ndash11mol l HClwasusedasacarrierandblank


Fig 1Flame-in-gas-shieldatomizerOHndashobser-vationheight

RegardingPVGformicacid(98paLach-NerCzechRepublic)andaceticacid (998 pa Lach-Ner CzechRepublic)were used for preparation of thereaction medium they were purified in a Teflon BSB-939-IR sub-boilingdistillation apparatus (Berghof Germany) The composition of the reactionmedium(40(vv)acetic125(vv)formicacid)wasoptimizedearlier[5]

ndash1The 5000mgl Co stock solution was prepared from cobalt(II) acetatetetrahydrate (pa Lach-Ner Czech Republic) and used as a sensitizer ofphotochemicalreactionTheoptimalconcentrationofCointhestandardsamples

ndash1andblanksolutionscorrespondedto50mgl (ref[5]) Acertifiedreferencematerial(CRM)-1643fTraceElementsinWater(NationalInstituteofStandardsandTechnologyUSA)wasusedtochecktheprecisionofthedevelopedmethodology

22Instrumentation

221Atomicfluorescencespectrometer

An in-house assembled non-dispersive atomic fluorescence spectrometerconstructedatourlaboratorywasusedforBideterminationandisdescribedindetail elsewhere [3] The detector output provided signals in microV Peak areacorrectedtobaselineandmainlysignaltonoiseratioweretheparametersusedtoevaluatethedata

222Hydridegeneratorphotochemicalvapourgeneratorandatomizer

A flow injection hydride generator was employed (Fig 2a) The reductantndash1 ndash1(12mlmin )andthecarrier(4mlmin )werepumpedbyaperistalticpump

Thesamplewasinjectedthrougha1mlsampleloopintotheflowofcarrierAglassgas-liquid separator (5 ml) with forced waste removal was employed forseparatingthegasphasecontainingbismuthanewhichwasthencarriedtotheatomizerbyargon Thephotochemicalvapourgenerator(Fig2b)consistedofthephotoreactorconstructedwitha15Wlow-pressureHggermicidallamp(Cole-ParmerUSA)wrappedaroundwith6mofPTFEtubing(1mmidinternalvolume471ml)The

ndash1reactionmedium(3mlmin )waspumpedbyaperistalticpumpThesamplewasinjected througha056ml sample loopApolypropylenegas-liquid separator

(15ml)withforcedwasteremovalimmersedinanicebath[6]wasemployedforseparatingthegasphasecontainingBivolatilespeciessubsequentlycarriedtotheatomizerbyargon Theflame-in-gas-shieldatomizerisdepictedinFig1detaileddescriptionisgiveninRef[3]Theobservationheight(OH)isdefinedasthedistancefromthetopofthecapillarytothecentreoftheopticalbeam


23Samplepreparation

CRMNIST1643fwasdilutedwith1MHCl80-foldforBideterminationbyHG-AFSRegardingBideterminationbyPVG-AFSthesampleneededtobeevaporatedtodrynessinordertogetridofnitricacidthatseriouslyinterferesatmMlevel[5]Avolume of 3ml of CRMwere pipetted into a 40 ml quartz vial evaporated(temperature asymp100 degC two replicates) and subsequently diluted ca 33-foldAsamplepreparationblank3mlofdeionizedwaterwaspreparedaswell


TheatomizationconditionsforHG-AFSwereoptimizedinourpreviouswork[3]TheseconditionswereusedasinitialtofindtheoptimumconditionsforPVG-AFSwith the flame-in-gas-shieldatomizerwith respect to sensitivityandsignal tonoiseratioFirstlythehydrogenfractionintherange10ndash16wasoptimizedatconstant total gas flow rate (sum of total argon and total hydrogen) of

ndash1500mlmin the lower the hydrogen fraction the better However at 10hydrogenfractiontheflamewasnotstableenoughandwentoftenouthenceitwasoptedfor12hydrogenfraction


Fig 2(a)Hydrideand(b)photochemicalvapourgenerator

The oxygen flow rate through the capillary was optimized in the rangendash15ndash30mlmin Thehighestsignaltonoiseratiowasachievedwiththeflowrateof

ndash120mlmin Thetotalgasflowratewasoptimizedatconstant12hydrogenfractioninthe

ndash1range500ndash800mlmin Theoptimumobservationheightvarieswithtotalgasflow rate so it had to be optimized as well The optimum conditions aresummarizedandcomparedtothoseachievedwithHGinTable1 TheanalyticalfiguresofmeritofPVG-AFSwiththeflame-in-gas-shieldatomi-zerweredeterminedThecalibrationfunctionconstructedwith010025050

ndash1 2100 and 200 microg l Bi standardswas linear (R = 09998) The repeatabilityndash1expressedastherelativestandarddeviation(n=10)was6at1microgl andthe

ndash1relativeandabsolutelimitsofdetection(3σn=10)achievedwere12ngl and68pgrespectively(Table2)TheabsolutelimitofdetectionachievedwithHGwas 76 times lower which can be attributed to several aspects Firstly thegenerationefficiencyforPVGapproachwasaround53while100isexpectedforHG[3]Secondlyafullwidthathalfmaximumofthemeasuredpeakswasca2-foldgreaterwhichnecessitatedlongerintegrationtimeandwasthusreflectedinhighernoiseofthesignalsFinallythelimitofdetectionforPVGapproachwas

ndash1affectedby seriouscontamination (around10ng l )mostprobably from thesensitizersolutionthatcontainedBiasimpurity Tovalidate theproposedmethodologyBi contentwasdetermined inCRMNIST1643f (Table3) and the resultswere compared to thosemeasuredwithHG-AFS[3]Duetosevereinterferencesfrominorganicacidsespeciallynitricacid[5]thesampleneededtobeevaporatedtodrynessandthenfilledupwiththe

2+reactionmediumcontainingCo as thesensitizer(NIST1643f isstabilized in


Parameter HG-AFS(ref[3]) PVG-AFS

ndash1Artotalmlmin 440 528ndash1H totalmlmin 60 722 ndash1O mlmin 7 202

OH mm 6 9ndash1Arshieldlmin 1515 1515

Table 1Atomizationconditionsforflame-in-gas-shieldatomizer


LODpg 09 68ndash1LOQngl 09 12

Repeatability lt1 6

Table 2AnalyticalfiguresofmeritofHG-AFSandPVG-AFS

ndash1032moll nitricacid)Theresultsobtainedbybothmethodologiesareingoodagreementwiththecertifiedvalue

4Conclusion

Photochemical vapour generation of Bi was successfully coupled with non-dispersiveatomicfluorescencespectrometryforthefirsttimeanditsapplicabilitywas verified by determination of Bi in certified reference material of waterComparedtohydridegenerationconditionsofatomizationdifferinanoptimalobservationheightandsupplyofoxygenwhichmaybeneededtoldquoburnoutrdquotheorganicvapoursthatarereleasedfromthereactionmediumtothegasphasehowever this remains tobe verifiedAlthough there are still some limitationsregardingthelimitsofdetectionrepeatabilityandinterferencesthisnewsampleintroductionapproachseemstobepromising

Acknowledgments

The support of the Czech Science Foundation (19-17604Y) Czech Academy of Sciences(Institutional supportRVO68081715)andCharlesUniversity (ProjectSVV260560andProjectGAUK1048120)isgratefullyacknowledged

References

[1] Musil SMatousek T Currier JM StybloM Dedina J Speciation analysis of arsenic byselectivehydridegeneration-cryotrapping-atomicfluorescencespectrometrywithflame-in-gas-shield atomizer achieving extremely low detection limits with inexpensiveinstrumentationAnalChem86(2014)10422ndash10428

[2] SturgeonREPhotochemicalvaporgenerationaradicalapproachtoanalyteintroductionforatomicspectrometryJAnalAtSpectrom32(2017)2319ndash2340

[3] S tadlerova B Kolrosova M Dedina J Musil S Atomic fluorescence spectrometry forultrasensitivedeterminationofbismuthbasedonhydridegenerationndashtheroleofexcitationsourceinterferencefilterandflameatomizersJAnalAtSpectrom35(2020)993ndash1002

[4] DedinaJAtomizationofvolatilecompoundsforatomicabsorptionandatomicfluorescencespectrometryOnthewaytowardstheidealatomizerSpectrochimActaPartB62(2007)846ndash872


Certified HG-AFS PVG-AFSndash1valuemicrogl

ndash1 a ndash1 a valueobtainedmicrogl recovery valueobtainedmicrogl recovery

1262plusmn011 128plusmn01 102plusmn1 121plusmn09 97plusmn5

a Spikedrecovery=slopeofstandardadditions(noadditionandtwospikedconcentrationstoasample)slopeofexternalcalibration

Table 3ThedeterminedcontentofBiinCRMNIST1643fpresentedasmedianvalueplusmncombineduncertainty(n=3)andrecoveries

[5] Vyhnanovsky J Yildiz D Musil S Effect of metal sensitizers on photochemical vaporgeneration of bismuth for analytical atomic spectrometry In Proceedings of the 15thInternationalStudentsConferenceModernAnalyticalChemistryKNesmerak(ed)PragueCharlesUniversity2019p257ndash262

[6] VyhnanovskyJSturgeonREMusilSCadmiumassistedphotochemicalvaporgenerationoftungstenfordetectionbyinductivelycoupledplasmamassspectrometryAnalChem91(2019)13306ndash13312


1Introduction

Liquidcrystalsareorganicsubstancesthatformamesomorphicphaseinsolu-tion[1]TheyareliquidlikeliquidsbuthaveaninternalconfigurationassolidsTheir light transmittance changes in the electric fieldwhich is used in liquidcrystaldisplays(LCDs)CholestericliquidcrystalsaretemperaturesensitiveThecolorofreflectedlightchangeswithsmalltemperaturechangeThisisusedinmedicineasasensitivetemperatureindicatorfordisease-infectedtissues High performance liquid chromatography or supercritical fluid chromato-graphy[23]areusedtoseparateanddetermineliquidcrystalcompoundsAnelectrophoretic method could be complementary to these commonly usedmethods To our best knowledge no study dealing with liquid crystal puritycontrolbyelectrokineticchromatographywaspublisheduntilnow Electroneutralsubstancesmoveincapillaryzoneelectrophoresisatthesamespeedcorrespondingtothespeedoftheelectroosmoticflow(EOF)andtherefore

Separation of liquid crystals using non-aqueous capillary electrokinetic chromatography

KATER INACOKRTOVATOMA S KR IZ EK

DepartmentofAnalyticalChemistryFacultyofScienceCharlesUniversityHlavova8203012843Prague2CzechRepublickaterinacokrtovagmailcom

AbstractLiquid crystals arewidelyused in electronicsmedicine andotherfields Analytical separations are important in the development ofnewliquidcrystalstocontrolthepurityofsynthesizedsubstancesThesampleanalysisisimportantfordetectionofimpuritiesformedduring synthesis Liquid crystal-forming substances cannot beseparated by capillary zone electrophoresis due to the absence ofreadily ionizable groups Therefore electrokinetic chromatographywasused in thisworkAnotherproblemcomplicating theanalysiswastheverylowsolubilityofanalytesinwaterSeparationsinthisworkwere thereforecarriedoutundernon-aqueousconditions inacetonitrilewithaceticacidtoadjustthepHandhexadecyltrimethyl-ammonium chloride as a detergent to mobilize the non-ionizedanalytesUndertheseconditionsitwaspossibletoseparateimpu-ritiesfromsynthesizedanalytesinsamples

Keywordselectrokinetic

chromatographyliquidcrystalsnon-aqueouscapillary

electrophoresis


itisnotpossibletoseparatethemDuetothisanelectrokineticchromatographymethodwasdevelopedInthismethodasurfactantisaddedtothebackgroundelectrolyteMoleculesaggregateandformsphericalformationscalledmicelles[4]ifthesubstanceisaddedinsufficientconcentrationiehigherthanthecriticalmicellar concentration (CMC) Separation is possible due to interactions ofnonpolarmoleculepartswiththenonpolarmicelleinsideAlthoughwateristhemostusedsolventinelectrophoreticmethodsforseparationofwater-insolublesubstances organic solvents are selected However such solvent must meetcertaincriteriatobesuitableforuseincapillaryelectrophoresisAllcomponentsmustbesolubleinthesolvent[5]ItshouldnotbeflammabletoxicorreactiveforpracticalityitshouldbeliquidatroomtemperatureandalsoitspriceistakenintoaccountThevalueof its relativepermittivitywhichdescribes the strengthofinteractionsbetweenionsshouldbearound30Lowdynamicviscosityisalsopreferred to allow faster migration of analytes No organic solvent meets allparameters of the ideal solvent In practice methanol acetonitrile and theirmixturesarethemostusedTheseparationparameterscanbeinfluencedbyusingan organic solvent of the background electrolyte This topic has already beenwidelyexplored[6ndash8] Itwasgenerallyassumedthat inanhydrousconditionsmicellesarenotcreateddespitesufficientsurfactantconcentrationHoweveritwasfoundoutthatdodecylsulfatecanformstablemicelleswhenthebackgroundelectrolyteisdissolvedinformamide[9]Fortheanalysisofactivesubstancesinmedicinal plants Chen et al developed a method in which sodium cholatedissolvedinmethanolisusedasasurfactant[10]Theaddedpseudostationaryphase does not always form micelles but can still affect mobilization andseparationofanalytesiftheanalytesinteractdifferentlywithfreemoleculesofsurfactantInthisstudywater-insolubleliquidcrystalswereseparated(Fig1)Thereforenonaqueouselectrokineticchromatographymethodwasdeveloped


Fig 1 Structures of liquid crystals 4-([1-oxo-1-(pentyloxy)propan-2-yl]oxycarbonyl)phenyl4-(octyloxy)-[11-biphenyl]-4-carboxylate (ZL 85) and 4-([1-(decyloxy)-1-oxopropan-2-yl]oxycarbonyl)phenyl 4-(dodecyloxy)-[11- biphenyl]-4-carboxylate (ZL 1210) OpticalisomerismsitesaremarkedwithanasteriskStructurescreatedinMarvinSketch[11]

2Experimental


Acetonitrilege999fromSigma-Aldrich(Germany)aceticacid99fromLach-Ner Neratovice (Czech Republic) and hexadecyltrimethylammonium chloride25(ww)inwaterfromSigma-Aldrich(USA)wereusedforpreparationofback-groundelectrolyteMesyloxidepa(MO)suppliedbyLach-nerNeratovice(CzechRepublic)wasusedasareferencesubstance

22Instrumentation

ForexperimentsG7100ACapillaryElectrophoresisInstrument(AgilentTechno-logiesGermany)wasusedwithUV-VISdetectoroperatingat235nmand254nmwavelengthMeasurementswereconductedinafused-silicacapillaryof50microminner diameter with the total length 500cm and effective length 415cm(PolymicroTechnologiesUSA)

23Method

Capillarywas flushed for3minuteswith1MHCland for2minuteswith thebackgroundelectrolyteBackgroundelectrolytewaspreparedbymixingaceticacid (10mM) and hexadecyltrimethylammonium chloride (40mM) in aceto-nitrile Sampleswere introducedhydrodynamicallybyapressureof5kPa for1secondSampleswerefirstdissolvedinacetonitrileandthendilutedtwotimeswith the background electrolyte A voltage of 20 kV was applied during theseparation


Liquidcrystalsampleswerepractically insoluble inwater theirsolubilitywasndash3testedataconcentrationlevelof1mgcm inmethanolandacetonitrileWhile

samples were not sufficiently soluble in methanol they were successfullydissolvedinacetonitrile Becauseallanalytesaresubstancesthatdonothaveeasilyionizablefunctionalgroups theelectrokineticchromatographymethodwaschosen forseparationAsuitablesurfactantwassoughtCommonlyusedsodiumdodecylsulfate(SDS)isinsoluble in acetonitrile Therefore hexadecyltrimethylammonium chloride(CTAC) which had sufficient solubility for further experiments was chosenAlthoughasuitablebufferwassoughttoensureastablepHduetoproblemswithprecipitationofbuffercomponentsinthenon-aqueousenvironmentaceticacidwasusedtoadjustandmaintainpHofbackgroundelectrolytesolutionAstheadditionofcationicsurfactantsuchasCTACleadstoEOFreversalthedependence


ofEOFmobilityontheconcentrationofCTACinthebackgroundelectrolytewasmeasuredContrarytowhatisobservedinaqueousbackgroundelectrolytesEOFwasnotreversedItsmobilitydecreasedwithincreasingCTACconcentrationbutnomajorchangesoccurredabove40mMconcentrationThecapillarywallwasprobablyalreadysaturatedbyCTACandthefurtherincreaseinconcentrationhadno signifficant effect on the conditionof the capillarywall Therefore a CTAC

ndash3concentrationof40mmoldm was chosenas sufficient for furthermeasure-mentswithrespecttotheincreasingcurrentwithincreasingionicstrengthofthebackgroundelectrolyte The optimized method was used for separation of several liquid crystalsamplesofdifferentpurityInthesampleoftheZL85liquidcrystalwith99purityonezoneoftheanalytewasdetectedImpuritieswereseparatedfromthisanalytewhen the samplewith lower puritywas introduced The peak of theanalytewasidentifiedbasedonrelativemigrationtimerelatedtomesityloxideSeparationoftheanalytefromanimpurityinthesampleZL8576isshowninFig 2The relativemigration timeof the firstpeak is 0834 therefore itwasidentifiedastheZL85analyteThestandarddeviationoftherelativemigrationtimesinfivemeasurementswas0002min(01) Using the available high purity sample it was possible to measure thecalibration line forquantificationof theanalyte in lesspuresamplesLimitof

ndash3detection was determined as 0009mgcm and limit of quantification as ndash30031mgcm FromthecalibrationlineconcentrationofZL85inthesample

with lower purity was calculated The concentration was determined as

48(ww)standarddeviation5(ww)


Fig 2ElectropherogramobtainedwhenasampleofZL85liquidcrystalwithlowerpuritywasndash3introducedSamplewas introduced in01mgcm concentrationandwithaddedmesityloxide

ndash3(10mgcm )Capillarywithinnerdiameterof50micromtotallengthof500cm415cmeffectivelength The background electrolyte was acetonitrile with 10 mM acetic acid and 40 mMhexadecyltrimethylammonium chloride A voltage of 20 kVwith positive polaritywas appliedDetectionat254nm

ForsampleZL121099onlytheanalyteandmesityloxideweredetectedInthe sample ZL 1210 59 several impurities were separated and detected(Fig3)Accordingtotherelativemigrationtimetheanalyteofinterestcorres-pondstothefirstpeakPeakresolutionissufficientTheresolutionoftheanalytepeakandthesecondpeakis284andtheresolutionoftheothertwopeaksis230

4Conclusions

InthisstudyanewmethodforanalysisofnewlysynthesizedliquidcrystalswasdevelopedSomeparametersofthemethodwereoptimizedndashoptimumconcen-tration of hexadecyltrimethylammonium chloride was searched The identifi-cation of analyteswas based on a comparison of relativemigration times InsamplesZL85andZL1210withlowerpuritytheimpuritieswereseparatedfromthepeaksofliquidcrystalsthecontentofanalytewasdeterminedintheZL85sampleaccordingtothecalibrationline

Acknowledgments

IwouldliketothanktheInstituteofPhysicsoftheCzechAcademySciencesforprovidingnewlysynthesized liquid crystals This work has been supported by Specific University Research(SVV260560)andbyCharlesUniversityResearchCentreprogramNoUNCESCI014

References

[1] GennesPGProstJThePhysicsofLiquidCrystals2ndedNewYorkOxfordUniversityPress1993


ndash3Fig 3ElectropherogramofsampleZL121059ataconcentrationof05mgcm withmesitylndash3oxideataconcentrationof10mgcm capillarywith innerdiameterof50microm total lengthof

500 cmeffectivelength415cmThebackgroundelectrolytewasacetonitrilewith10mMaceticacidand40mMCTACAppliedvoltage20kVpositivepolarityDetectionat235nm

[2] Vankatova P KalıkovaK KubıckovaA Ultra-performance supercritical fluid chromato-graphy A powerful tool for the enantioseparation of thermotropic fluorinated liquidcrystalsAnalChimActa1038(2018)191ndash197

[3] Vankatova P Kubıckova A Cigl M Kalıkova K Ultra-performance chromatographicmethodsforenantioseparationofliquidcrystalsbasedonlacticacidJSupercritFluids146(2019)217ndash125

[4] Terabe S Otsuka K Ichikawa K Tsuchiya A Ando T Electrokinetic separations withmicellarsolutionsandopen-tubularcapillariesAnalChem56(1984)111ndash113

[5] RiekkolaMLRecentadvancesinnonaqueouscapillaryelectrophoresisElectrophoresis23(2002)3865ndash3883

[6] Wright PB Lister AS Dorsey JG Behavior and use of nonaqueous media withoutsupporting electrolyte in capillary electrophoresis and capillary electrochromatographyAnalChem69(1997)3251ndash3259

[7] PorrasSPKenndlerECapillaryzoneelectrophoresisinnon-aqueoussolutionspHofthebackgroundelectrolyteJChromatogrA1037(2004)455ndash465

[8] PorrasSPRiekkolaMLKenndlerETheprinciplesofmigrationanddispersionincapillaryzoneelectrophoresisinnonaqueoussolventsElectrophoresis24(2003)1485ndash1498

[9] GuoXWangK ChenGH Shi JWuX Di L LWangY Determination of strobilurinfungicideresiduesinfruitsandvegetablesbynonaqueousmicellarelectrokineticcapillarychromatography with indirect laser-induced fluorescence Electrophoresis 38 (2017)2004ndash2010

[10] Chen AJ Li C Gao WH Hu ZD Chen XG Application of non-aqueous micellarelectrokinetic chromatography to the analysis of active components in radix SalviaemiltiorrhizaeanditsmedicinalpreparationsJPharmBiomedAnal37(2005)811ndash816

[11] MarvinSketch [computer program] version 1990 ChemAxon httpschemaxoncom-productsmarvin


1Introduction

Sudandyesaresyntheticazo-basedaromaticcompoundsTheyaretraditionallyusedinvariousindustriessuchaschemicaltextileandwoodworkingasdyestocolourwaxesplasticsoilspolishesandsoforthTheyhavebeencategorizedasclass3carcinogensbytheInternationalAgencyforResearchonCancerandtheiruseisthereforeforbiddeninthefoodindustryTheyareknownfortheirbrightcolours and easy and cost-effectivemanufactureThey arenearly insoluble inwater but soluble in various organic solvents such asmethanol or trichloro-methane[1] SudanI1-phenylazo-2-naphthol(Fig1A)isadyeusedasanorangecolouringagentItssometimesalsosoldundernamesSolventOrangeRorCISolventYellow14ItisformedasasecondaryproductinthemanufactureoftheSunsetYellowdye

Electrochemistry of Sudan I and its derivates in aqueous media

ad b aANNAONDRA CKOVA MARIESTIBOROVA LUDE KHAVRAN cd adKAROLINASCHWARZOVA -PECKOVA MIROSLAVFOJTA

a CentralEuropeanInstituteofTechnologyMasarykUniversity Kamenice753562500BrnoCzechRepublicannaondrackovaceitecmuniczb DepartmentofBiochemistryFacultyofScienceCharlesUniversity Hlavova8203012843Prague2CzechRepublicc UNESCOLaboratoryofEnvironmentalElectrochemistryDepartmentofAnalyticalChemistry FacultyofScienceCharlesUniversityHlavova8203012843Prague2CzechRepublicd InstituteofBiophysicsCzechAcademyofSciences Kralovopolska13561265BrnoCzechRepublic

AbstractSudanIisanaromaticazo-compoundthathasbeenproventobeacar-cinogenDuringitsmetabolizationbycytochromeP450inliverafewmain derivates can be identified Thiswork sets out to assess themechanismofelectrochemicalreductionandoxidationofSudanIitshydroxylationderivativesfeaturingmetabolitesintheSudanIdetoxi-fication pathway and to introduce their selective voltammetricanalysis on boron-doped diamond electrode We show successfuldifferentiationamongthesecompoundsthankstothedifferencesintheelectrochemicaloxidationoftheirphenolicgroups

Keywordsborondopeddiamond

electrodecytochromeP450electrochemicalanalysisSudanI


InmammalianorganismsSudanIismetabolizedbythemicrosomaldetoxi-fying systemwitha central roleof cytochromeP450hydroxylationactivity inliver[2]DuringtheoxidativeprocessofmetabolizingSudanIseveralmetaboliteswereidentifiedbyprevioustestsThesearegt1-(phenylazo)-naphtalene-26-diol(further abbreviated SI-6OH) 1-(4-hydroxyphenylazo)-2-hydroxynaphtol(furtherabbreviatedSI-4OH)and1-(4-hydroxyphenylazo)-naphtalene-26-diol(furtherabbreviatedSI-46-diOH)thestructuresarepresentedinFig1 ThemainmethodcurrentlyusedtoidentifySudanIamongotherdyeswithsimilar structure is high-performance liquid chromatography (HPLC) It isrecommendedasthestandardmethodtoidentifythelevelofSudanIinfood[3] ComparedtoHPLCelectrochemicalmethodsareprovingtobefastercheaperandcomparablypreciseUnfortunatelyacomprehensiveelectrochemicalstudyofSudanIandparticularlyofitshydroxylatedmetaboliteshasnotbeencompletedyetThedyecanbedetectedthroughelectrochemistryeitherbytheoxidationofitsphenolicgrouporviareductionoftheazogrouppresentinitsmoleculeInbothcasesotherelectrochemicallyactivemoietiesareformedThederivatesofSudanIcanbedetectedandrecognizedfromSudanIthroughanalogousprocesses[4]InthisstudywefocusedoncomparisonofelectrochemicalbehaviourofSudanIanditshydroxylatedmetabolitesonborondopeddiamondelectrodetoaddressthepossibilitiesoftheirrecognitionsinmixturesbasedondifferencesinanodicandcathodicsignals


Fig 1 Chemical structure of (A) Sudan I (B) 1-(phenylazo)-naphtalene-26-diol (SI-6-OH)(C) 1-(4-hydroxyphenylazo)-2-hydroxynaphtol (SI-4-OH) and (D) 1-(4-hydroxyphenylazo)-naphtalene-26-diol(SI-46-diOH)

(A) (B)

(C) (D)

2Experimental


SudanI(Merckanalyticalstandardgrade)wasdissolvedinethanol(Merck)andkept at room temperatureChemicals forBritton-Robinsonbufferpreparation(acetic acid boric acid orthophosphoric acid sodium hydroxide) were fromMerckwithpurityge99pHofthebufferwasadjustedbymixingoftheacidsandsodium hydroxide solution at different ratios The Sudan I metabolites weresynthetized at the Department of Biochemistry Faculty of Science CharlesUniversityandkeptinmethanolattemperature3degC

22Instrumentation

Cyclicvoltammetric (CV)measurementswerecarriedout inBritton-RobinsonbufferofpH=70atroomtemperatureSudanIanditsderivativeswereaddedto

minus1thesolutionofBritton-Robinsonbuffertofinalconcentrationof5micromolL andstirred Before the measurement oxygen was removed from the solution bypurgingwithargonfor3minutesAutolabanalyzerPGSTAT20(EcochemieTheNetherlands)inconnectionwithVA-Stand663(MetrohmSwitzerland)GPES49(MetrohmSwitzerland)andathree-electrodesetup(withborondopeddiamond

2(WindsorScientificUKdiskdiameter3mmA=707mm )asworkingelectrodeminus1AgAgCl3molL KCl as reference electrode and platinum wire as auxiliary

electrode) Five cycles were performed for each measurement at scan rateminus1of1Vs


ForeachcompoundtwoseparateCVmeasurementswereperformedeachwithfive cycles performed in rapid succession For bothmeasurements the initialpotentialwassetat0VTheanodicscancontinuedto+1Vturnedtowardsndash1Vandfinishedat0VIntheothersetupcathodicscanwasperformedfirstfromthestartingpointtondash1Vturnedtowards+1Vandreturnedto0VThiswaywewereabletoobservethebehavioursofSudanIandcompareittothatofitsderivativeswhilefirstbeingreducedandthenoxidisedorviceversa In the anodic scan of Sudan I and its derivatives (Fig 2) differences in thepositionsoftheoxidationpeaksineachcompoundcanbeobservedWhileSudanIwithonlyonephenolgroupisoxidizedat+067VthederivativeswithtwophenolgroupsieSI-4OHandSI-6OHgiveoxidationpeaksatremarkablylesspositivepotentialofca+05VSI-46OHwiththepresenceofoverall3hydroxygroupsyieldstwooxidationpeaksInthereversecathodicscan(vertexpotential+1V)probablythankstothepresencereductionpeaksappearwhichcanbefurtherusedtodifferentiatebetweenSudanIandthederivativesSI-4OHandSI-6OH



Fig 3VoltammetricscanofSudanIanditsderivatesSI-4-OHSI-6-OHandSI-46-diOH1stscanincathodicdirectionfrom0Vvertexpotentialsndash1Vand+1VThemeasurementswereperformedinBritton-Robinsonbuffer(pH=7)withtheconcentrationofeachcompoundat5microMandatscan

ndash1rate1Vs

Fig 2 CyclicvoltammogramofSudanIanditsderivatesSI-4-OHSI-6-OHandSI-46-diOH1stscaninanodicdirectionfrom0Vvertexpotentials+1Vandndash1VThemeasurementswereperformedinBritton-Robinsonbuffer(pH=7)withtheconcentrationofeachcompoundat5microMandatscan

ndash1rate1Vs

possesstwoconjugatedhydroxylgroupswhichcanberegardedashydroquinonestructures and thus undergoing quasireversible redox process due to oxida-tionreductionofthehydroquinonetoquinonemoietyThisiswellvisibleattheCVsastheanodicsignalisfollowedbycathodiconeatthepotentialof+023VforSI-4OHand+03VforSI-6OHTheoxidationofSudanIproceedsbymechanism

minus +typicalforphenoliccompoundsatmorepositivepotentialsleading by1e 1H exchange to naphthoxy-type ndashO radical [5] This species undergoes furtherreactionsleadingtoformationofdimersandpolymersThecathodicpeakinthereversescanatndash02Varisesfromreductionofthesereactionproductsanditsoriginneeds tobe further investigatedSI-46OHwith thepresenceofoverallthree hydroxyl groups yields two oxidation peaks The first one is a result ofoxidationoftwoofthembeinginconjugationandthusbeingoxidizedtoquinonemoietyThesecondsignalatthesamepotentialastheoxidationsignalof2OHonnaphthaleneringofSudan I is consequenceofoxidationof the thirdhydroxylgroupofphenolictypeAsinglewidepeakat0Vinthereversescanispresumablyan overlap of signals arising from reduction of the quinonic moiety and by-products formed during oxidation processes Thanks to differences of theseprocessesspecificforindividualcompoundsitispossibletodifferentiateamongallfourofthemviapropersetting-upoftheinitialandvertexpotentialvalues The cathodic scan of Sudan I and its derivatives (Fig 3) shows a dominantreductionpeakaroundndash08Vwhichisduetoreductionoftheazogroupintheirstructuresaccompaniedbycleavageoftheirmoleculestoseparatethebenzeneandnaphthalene rings [4] The peaks in the subsequent anodic scans (vertexpotentialndash1V)arethereforetheresultoftheelectrochemicalreactionofmoietiesthatareproductsofthedivisionofthearomaticcirclesTheseproductsincludeaniline4-aminophenol1-amino-2-naphtholand1-amino-25-naphthalenediolwith irreversibly oxidizable amino moieties or (quasi)reversibly oxidizableamino-hydroxylsystemonthebenzeneornaphthaleneringandtispossibletodifferentiatebetweenthemTheobtainedoxidationpeaks(+02VforSI-4-OH+073VforSudanIandSI-6-OHand+025Vand+075VforSI-46-diOH)makeitpossibletodifferentiatebetweenallcompoundswiththeexceptionofSudanIandSI-6-OHForthereliablerecognitionofthesetwocompoundsanothermeasure-mentwithdifferentparametersisneeded

4Conclusions

ThestructuresofSudanIanditshydroxyderivatesthatarethemainproductsofthe metabolization of Sudan I by cytochrome P450 are similar and theirrecognitionwhenpresentinmixtureinsolutionisdemandingHereinwepresenta simple approach based on comparison of signals obtained in cathodic andanodic scan in CV measurements without the need of time-demandingchromatographicseparationstepFurtherworkwillbedevotedtoidentificationof observed redoxprocesses and applicationof themethod formonitoringofmetabolictransformationsofSudanIinvitro


Acknowledgments

ThisresearchwassupportedbytheCzechScienceFoundation(projectNo18-01710S)

References

[1] ChailapakulOWonsawatW SiangprohW GrudpanK ZhaoYF Zhu ZW Analysis ofSudanISudanIISudanIIIandSudanIVinfoodbyHPLCwithelectrochemicaldetectionComparison of glassy carbon electrodewith carbon nanotube-ionic liquid gelmodifiedelectrodeFoodChem109(2008)876ndash882

[2] StiborovaMMartinekVRydlovaHHodekPFreiESudanIisapotentialcarcinogenforhumans Evidence for itsmetabolic activation and detoxication by human recombinantcytochromeP4501A1andlivermicrosomesCancerRes62(2002)5678ndash5684

[3] GomezMArancibiaVAliagaMNunezCRojas-RomoCDeterminationofSudan I indrinks containing Sunset yellow by adsorptive stripping voltammetry Food Chem 212(2016)807ndash813

[4] PrabakaranEPandianKAmperometricdetectionofSudanIinredchilipowdersamplesusingAgnanoparticlesdecoratedgrapheneoxidemodifiedglassycarbonelectrodeFoodChem166(2015)198ndash205

[5] Enache T A Oliveira-Brett A M Phenol and para-substituted phenols electrochemicaloxidationpathwaysJElectroanalChem655(2011)9ndash16


1Introduction

Aminoglycosidesarealargegroupofnaturalandsemi-syntheticantibioticswithawidespectrumofantimicrobialactivityagainstmostgram-positiveandgram-negativemicroorganismsCurrentlymultiplerepresentativesofaminoglycosidefamily ndash gentamicin (GM) neomycin B (NM) paromomycin (PM) kanamycin(KM)apramycin(AP) andstreptomycin(STM)Fig1ndashareapproved to treatinfectiousdiseasesinanimalsMaximumresiduelimitsfortheseaminoglycosidesinproductsandtissuesfromedibleanimalsareestablished[1]henceaneffectiveandrobustassayisnecessaryforcontrolofaminoglycosidescontamination Inthisstudyenzyme-linkedimmunosorbentassayforthedetectionofamino-glycosides in foodproductsandenvironmentalobjectswasdevelopedGroup-specificantibodieswereproducedowingtoimmunogenbasedonribostamycin(RS)whichexposedthecommonfragmentofmostaminoglycosides2-deoxy-streptamin (2-DOS) Fig 1 The developed assay was made suitable for thedetectionofresidualaminoglycosidesinhoney[2]

Group detection of aminoglycosides using ELISA for control of food contamination

ab a aKONSTANTINBURKIN INNAGALVIDIS MAXIMBURKIN

a DepartmentofImmunologyIMechnikovResearchInstituteofVaccinesandSera MalyjKazionnyjper5a105064MoscowRussianFederationburkin-kostyandexrub DepartmentofChemicalEnzymologyFacultyofChemistryLomonosovMoscowStateUniversityLeninskieGory1119991MoscowRussianFederation

AbstractThegrowingthreatofglobalantibioticresistanceisforcingtoreducenon-targetconsumptionofantibioticsandtomonitorcontaminationoffoodandenvironmentalobjectsInthisworkELISAwasdevelopedforgroupdetectionofaminoglycosidesToobtaingroup-specificanti-bodies a new immunogen based on ribostamycin was used Thedevelopedindirectcompetitiveformatofassayallowedtherecogni-tionof9aminoglycosidesnamelyneomycinribostamycinneaminparomomycin gentamicin sisomicin kanamycin tobramycin and

ndash1apramycinwithadetectionlimitrangedbetween002ndash020ngmL TheeffectivenessoftheproposedassaywasevaluatedinhoneyasafoodstuffmodelToneutralizea stronghoneymatrixeffect and toavoidalaborioussamplepre-treatmentanewmatriximitatorwassuggested 5 sucrose solution imitated the influence of 50-folddilutedhoneyTheproposedassayallowedustorevealanyofthe9

ndash1mentionedaminoglycosidesinhoneyata10microgkg level

KeywordsaminoglycosidesELISAhoney


2Experimental


NeomycinBribostamycinneamin(NA)paromomycinkanamycintobramycin(TM)amikacin(AM)gentamicinnetilmicin(NTM)sisomicin(SSM)geneticin(GC) apramycin and streptomycin were purchased from Chimmed (MoscowRussia) Bovine serumalbumin (BSA) complete Freund adjuvant 16-hexane-diamine1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)sodiumperio-dateandsodiumborohydrideweretheproductsofSigma-Aldrich(USA)Gelatin(Gel)wasfromBio-Rad(USA)sucrosefromServa(Germany)two-componenttetramethylbenzidine (TMB) substrate solutionwas fromBioservice (Russia)andgoatanti-rabbitIgGantibodiesconjugatedtohorseradishperoxidase(anti-rIgG-HRP)werefromIMTEK(Russia)Honeysampleswerepurchasedfromlocaloutlets


Fig 1 Structuralformulasofstudiedaminoglycosides

22Preparationofconjugatedantigens

TwotypesofconjugateswerepreparedbasedonRSandBSAusingzero-lengthand C6 spacer arm between hapten and protein carrier RSwas treatedwithsodiumperiodatetooxidizehydroxylsofribosefragmenttoreactivealdehydegroupsandthencoupledtoBSAaminesthroughreductiveaminationToremoveuncoupledRSanexhaustingdialysiswascarriedoutusingdialysismembranetubes(MWCO14kDa)UsingthesameprocedureGel-RSconjugatewassynthe-sized ForpreparationofBSA-C6-RSwefirstlymodifiedBSAwith16-hexanediamineThemixtureofBSAandEDCinwaterwerestirredfor30minThen16-hexane-diaminewasaddedandstirredfor2hThemodifiedproteinwasdialyzedfromtheexcessive reagents and resultant BSA-C6-NH2was coupled to RS in reductiveaminationprocessasdescribedabove

23Immunizationandantibodypreparation

BSA-RSandBSA-C6-RSwereusedasimmunogensChinchillarabbits(20ndash25kg)weresubcutaneouslyinjectedat10ndash15pointsonthebackwith01mgofimmuno-gensemulsifiedinthecompleteFreundadjuvantThesamedosesofimmunogensinsalinewereadministeredmonthly forbooster immunizationsAweekaftereachinjectionabloodsamplefromearveinswastakenforthecontrolofimmuneresponseTheantiserainglycerol(11vv)werestoredatndash15degCuntiltestinginELISA

24TheELISAprocedure

AcompetitiveassaywasconductedaccordingtoclassicalprocedureGel-RSwascoatedovernightonpolystyrene96-wellCostarplatesNon-adsorbedconjugatewashedoutusingPBSwith005oftween20(PBS-T)Thenextcompetitivestep

ndash1includedtheadditionof01mLstandardaminoglycosidesolutions(1pgmL to ndash1 ndash11microgmL (B)and0microgmL (B ))inPBS-Tor01mLoftestedsampleand01mLof0

antibodiesinworkingdilution(1h25 degC)Afterwashingtheantibodiesboundtoimmobilized Gel-RS were detected using anti-rIgG-HRP (1h 37degC) Coloredproduct formedasaresultofenzymaticreactionwithTMBsubstratemixture(05h25degC)wasreadat450nmusingaStatFax2100platereader(AwarenessTechnologiesUSA) Relativeantibodybinding(BB )vstheanalyteconcentrationswasplottedas0

standardcurvesfittedtoafour-parameterlogisticfunctionThecross-reactivity(CR) for every aminoglycoside representative was calculated as ratio of half-inhibition concentrations IC NMIC aminoglycoside The dynamic range of50 50

assaywasacceptedasIC ndashIC andthelimitofdetection(LOD)wascalculatedas20 80

B ndash3timesSD0



31Immunogensynthesisandantibodypreparation

Inthemajorityofpublicationsdevotedtoimmunoassayofaminoglycosidestheimmunogenscoatingantigensenzymeconjugatesortracerswerepreparedbycarbodiimide or glutaraldehyde methods involving aminoglycosidesrsquo aminogroups [3ndash7] Due to several amino groups in aminoglycoside molecules theformationofconjugateswithavariableorientationofthehaptenoccurs InpresentstudyRSwaschosenasanimmunizinghaptenduetothefollowingadvantageous features Being a trisaccharide RS has the size of a moleculecomparable to themostof aminoglycosides Ithas three identical ringsA-B-CsimilartothoseinNMUsingaperiodateoxidationwecouldinvolvearibosesiteofRSincouplingtoproteinthatprovidedastrictorientationofhaptenonthecarrier with a favorable presentation of the 2-DOS fragment The resultantimmunogensBSA-RSandBSA-C6-RSwerecomparedtorevealwhichdesignisbetterforpresentationofacommonfragmentofaminoglycosidemoleculeandgenerationofgroup-specificantibody Antibodies to the BSA-RS demonstrated moderate sensitivity (NM

ndash1IC =10ngmL )andhighselectivitytowardsNMwithrelativelylowcross-reac-50

tivity(lt5)forGMKMandAPTheapplicationofthespacerintheimmunogenBSA-C6-RScontributedtoaprominentpresentationofthe2-DOSdeterminantandtheinductionofantibodieswithrecognitionofbroadspectrumofdifferentaminoglycosides In addition anti-BSA-C6-RS exhibited significantly better

ndash1sensitivity(NMIC =02ngmL )Thusallsubsequentstudieswereconducted50

usinganti-BSA-C6-RS

32Examinationofassayspecificityandselectionofimmunoreagents

TheindirectcompetitiveformatofassaywasdevelopedForevaluationofassayspecificity a panel of following aminoglycosideswas studied and their cross-reactivitywasdeterminedNA(625)RS(250)NM(100)KM(475)PM(173)GM(90)TM(78)AP(17)SSM(12)AM(lt01)GC(lt01)STM(lt01)andNTM(lt01)ThemostoftheseanalytesareusedinmedicalandveterinaryareashoweveronlyNMPMGMKMAPandSTMareappliedinanimalhusbandry[1]

33Determinationofaminoglycosidesinhoneyandselectionofthematriximitator

Honey is a complex product consisting of carbohydrates (75ndash80) vitaminsproteinsenzymesorganicacids traceelements inclusionsandothercompo-nentsThesecomponentsmightinterfereimmunochemicalreactionThereforetheisolationofaminoglycosidesfromhoneyisalaboriousandtime-consuming



procedureToavoidthisstephoneymatriximitatorswereappliedtomimictheinfluenceofhoneymatrixonantibodybindingSucrosewaschosenasthehoneyimitatorsinceitexposedastronghoney-matrix-likeeffectonantibodybindingTheadequacywasfoundbetweensolutionsofhoneyandsolutionsofsucroseexpressinganequalmatrixeffectTwopairswithequivalentmatrixeffectwere120honey=20sucroseand150honey=5sucroseThelatterconditionswere chosen asmore preferable due to inconvenience of operatingwith highviscous20sucrosesolution Thedeterminationofaminoglycosidesinhoneycouldbecarriedoutquantita-tivelyiftheanalytetobedetectedisknownForquantificationofaminoglycosideinhoneyasamplewasdiluted50timesinPBS-Tandaminoglycosidestandardcurvewas generated in 5 sucrose-PBST (Fig 2) If analyte is unknown thedevelopedgroup-specificELISAcanbeusedasascreeningtestInthiscasetheanalyzed sample can be considered as contaminated if it caused a relativeantibodybindingbelowthecut-off level (Fig3)Thus thedeveloped testwas

ndash1 ndash1 ndash1Analyte IC ngmL Dynamicrange LODngmL LODinhoneymicrogkg50 ndash1 IC ndashIC ngmL20 80

NM 02 003ndash21 002 10PM 07 008ndash71 005 25GM 15 015ndash133 011 55KM 035 005ndash39 004 20AP 68 05ndash968 023 115

Fig 2StandardcurvesandanalyticalparametersoftheELISA-systemforgroupdeterminationofaminoglycosidesinhoneyInteractionofanti-BSA-RSwithcoatingantigenGel-RSin5sucrosesolutionasthehoneyimitatorThedetectionlimitin5sucrosesolutionwasdeterminedaccordingtoLOD=B ndash3timesSD0

capabletorevealthecontaminationofhoneywith9aminoglycosides5amino-ndash1glycosidesapprovedforveterinary(NMPMGMKMandAP)ata10microgkg level

andalsoNARSSSMandTM

4Conclusions

A novel indirect competitive ELISA for the detection of aminoglycosides wasdevelopedRSwasusedasanewimmunizinghaptentoproducegroup-specificantibodiesagainst2-DOSacommonmoietyofalargenumberofaminoglycosideantibiotics A wide spectrum of aminoglycoside representatives could bedetectedincludingNMRSNAPMGMSSMKMTMandAPThedevelopedassay

ndash1was capable todetect theseanalyteswithaLOD up to002ndash020 ngmL ForanalysisofhoneyamatriximitatorwasdevelopedtoavoidhoneyinterferencesonimmunoassayTheanalysisofthehoneysampleallowedustorevealanyofthe


References

[1] CouncilRegulation(EU)N372010OffJEurCommunitiesInfNotL15(2009)1ndash72[2] GalvidisIABurkinKMEreminSABurkinMA Group-specificdetectionof2-deoxy-

streptamineaminoglycosidesinhoneybasedonantibodiesagainstribostamycinAnalMeth11(2019)4620ndash4628


Fig 3Detectionofaminoglycosidesspikedinhoneysamplesata40ppblevelusinggroup-specificELISAEachsymbolcorrespondstotheaveragerelativebindingandtheerrorisSDobtainedforanindividualhoneysampleanalyzedintriplicateEmptycharactersrepresentindividualblankhoneysamples(limebuckwheatandflower)andfilledsymbolsrepresentthesamesamplesfortifiedwith

ndash1aminoglycosidesata40mgkg (level establishedonlyforSTMinseveralcountries)Thecut-offlevelcorrespondstothelimitofassaydetectionobtainedbythematriximitator(5sucrose-PBS-T)

[3] ThompsonSGBurdJFSubstrate-labeledfluorescentimmunoassayforamikacininhumanserumAntimicrobAgentsChemother18(1980)264ndash268

[4] LiCZhangYEreminSAYakupOYaoGZhangXDetectionofkanamycinandgentamicinresiduesinanimal-derivedfoodusingIgYantibodybasedic-ELISAandFPIAFoodChem227(2017)48ndash54

[5] GalvidisIABurkinMAMonoclonalantibody-basedenzyme-linkedimmunosorbentassayfortheaminoglycosideantibiotickanamycininfoodstuffsRussJBiorganChem36(2010)722ndash729

[6] HaasnootWStoutenPCazemierGLommenANouwsJFKeukensHJImmunochemicaldetectionofaminoglycosidesinmilkandkidneyAnalyst124(1999)301ndash305

[7] Peng JWangYLiuLKuangHLiAXuCMultiplex lateral flowimmunoassayfor fiveantibioticsdetectionbasedongoldnanoparticleaggregationsRSCAdv6(2016)7798ndash7805


1Introduction

PhotochemicalvaporgenerationisanalternativesampleintroductiontechniqueforanalyticalatomicspectrometryThistechniqueisbasedaroundasourceofUV-radiationthatirradiatesalowmolecularweightorganicacidmedium(mostcommonlyformicacidaceticacidortheircombinations)withananalyteHighlyreducingradicalsandaquatedelectronsareproducedandconverttheanalyteintoavolatilespecieswhichisthentransportedintoadetector[1]SofartheuseofPVGhasbeendescribedforhydride-formingelements(AsBiTeSbPbSeSnandTl)andmercury[12]transitionmetals(FeCoNiCuMoWCdAgAuIrPdPtRhandOs)[13ndash6]andevennon-metals(BrIClFandS)[16ndash9] AfirstsuccessfulphotochemicalvaporgenerationofcobaltwasdescribedbyGuoetalin2004[6]whichwasfollowedbymoresystematicstudiesbyGrinbergetalin2008[10]andDengetalin2010[11]LaterworksbydeQuadrosetal[12]anddeJesusetal[13]focusedontheanalysisofrealsamplesInthelatterwork

Photochemical vapor generation of cobalt for detection by inductively coupled plasma mass spectrometry

ab aJAROMIRVYHNANOVSKY STANISLAVMUSIL

a DepartmentofTraceElementAnalysisInstituteofAnalyticalChemistryoftheCzechAcademyofSciencesVeveřiacute9760200BrnoCzechRepublicjaromirvyhnanovskygmailcom

b DepartmentofAnalyticalChemistryFacultyofScienceCharlesUniversity Hlavova8203012843Prague2CzechRepublic


AbstractThisworkfocusedonthephotochemicalvaporgenerationofcobaltVolatilespeciesweregeneratedinaflow-injectionsystememployinga high-efficiency flow-through UV photoreactor and a formic acidbased medium and were introduced by an argon carrier into aninductivelycoupledplasmamassspectrometerfordetectionOptimalgeneration conditions were found as 10 (vv) formic acid and

ndash1 ndash14molL ammonium formate with a 4mLmin flow rate whichcorresponds to irradiation time of around 13 s The influence ofvariousmetalsensitizersofphotochemicalreactionwasinvestigated

2+andonlyCu ionsexhibitedapositiveeffectongenerationefficiencyndash ndash 2ndashInterferencesfromcommoninorganicanions(NO Cl SO ) were3 4

also examined Lastly the limit of detection and repeatability (atndash1 ndash1250ngL )weredeterminedtobe13ngL and41respectively

Keywordscobaltinductivelycoupled

plasmamassspectrometry

photochemicalvaporgeneration

the authors also presented a systematic study on generation conditions andachievedagenerationefficiencyofaround40 Themainaimofthisworkwastooptimizetheconditionsofgenerationwithinductivelycoupledplasmamassspectrometry(ICP-MS)detectionexaminetheeffectofvariousmetal sensitizers toachieve thehighestgenerationefficiencypossibleandreachthelowestlimitofdetectionpossible

2Experimental


minus1Deionizedwater (DIW lt 02μScm UltrapurWatrex USA)was used for thepreparationofallsolutionsFormicacid(98paLach-NerCzechRepublic)andammonium hydroxide (ge25 pa Sigma-Aldrich USA) were used for the

minus1preparationof the reactionmediumA1000mgL Co stock solution (Sigma-AldrichUSA)wasusedforthepreparationofallsamplesolutionsThefollowingcompounds were used as potential metal sensitizers cadmium(II) acetatedihydrate(paLach-NerCzechRepublic)zinc(II)acetatedihydrate(paSigma-AldrichUSA)copper(II)acetatemonohydrate(paMerckGermany)nickel(II)acetatetetrahydrate(paSigma-AldrichUSA)sodiumtungstatedihydrate(paCarlRothGermany)and iron(II) sulphateheptahydrate (pa LachemaCzechRepublic)Nitricacid(65semiconductorgradeSigma-AldrichUSA)hydro-chloricacid(37paMerckGermany)andsulfuricacid(98paLach-NerCzechRepublic)wereusedforaninterferencestudy

22Instrumentation

AschematicdiagramofthePVGsystemcoupledtoICP-MSisshowninFig1andamore detailed description can be found in reference [5] Briefly a singlequadrupole ICP-MS Agilent 7700x (Agilent Technologies USA)was used as a

minus1detector Deionized water was mixed with a 10μgL Rh internal standardsolutionin2HNO andwassubsequentlynebulizedbyaMicroMistnebulizer3

59 103during PVG Isotopes of Co and RhweremonitoredMeasurementswereperformed in time resolved analysis mode and in He collision mode

minus1(41mLmin )AlltubingusedwasmadefromPTFEwiththeexceptionoftygontubing in the peristaltic pump (Reglo ICC Ismatec Switzerland) The high-efficiency flow-through photoreactor was a 19 W low-pressure mercurydischarge lamp (Beijing Titan Instruments Co Beijing China) with a quartzcentralchannel(asymp720μL internalvolume)Samplesolutionswere introducedintoastreamofreactionmediumusinganinjectionvalve(V-451IDEXHealthandScienceUSAsampleloopvolume05ml)Effluentfromthephotoreactorwasmixedwithaflowofargonandcarriedtothechilledgas-liquidseparator(internalvolume15mL)wherethevolatilespecieswereseparatedfromtheliquidwaste


andcarried to the inletofaScott-typespraychamber (originally the inlet formakeupargon)oftheICP-MS


The starting conditionswere adopted fromour earlierwork [14]whichusedatomicabsorptionspectrometerasadetectorandminiaturediffusionflameasanatomizerThefirstparameteroptimizedwasthecompositionofreactionmedium(Fig2)Theadditionofammoniumformate(createdin-situbytheadditionofacalculatedamountofammoniumhydroxidetoformicacid)wasfoundcrucialto

ndash1effectivelygeneratevolatilespeciesofcobalt10(vv)formicacidand4molL ammonium formatewas chosen as the optimumandwasused for all further

experimentsAlthoughhigher concentrations of both components led to even

higher signalstheseconditionswerenotusedfurtherbecauseofthelaboriousprocessofpreparation(mixingofconcentratedacidwithconcentratedbase)andtolimittheconsumptionofchemicals Theinfluenceofirradiationtimewasalsoexaminedandthehighestpeakarea

-ndash1wasobtained for4mLmin corresponding to an irradiation timeof approximately13s ToenhancethegenerationefficiencyadditionofvariousmetalstothereactionmediumwastestedtoldquosensitizerdquothephotochemicalreactionThemetalswerechosenwithrespecttotheirsignificantenhancementeffectdescribedrecentlyforphotochemicalvaporgenerationofotheranalytes[158]Theonlymetalionthat

2+ledtoanenhancementofthesignalwasCu (Fig3a)buteveninthiscasetheeffect was rather negligible reaching only 12-fold enhancement in the range

ndash1 2+ 2+001to01mgL Cu FurtheradditionofmoreCu ledtoadecreaseinthesignal2+TheadditionofZn didnotexhibitanypositiveornegativeeffectacrossthetested ndash1range (01 to 1500mgL not shown in figure) and the addition of higher

ndash1 2+ 2+ 6+concentrations (tens to hundredths ofmg L ) of Cd Fe andW (Fig3b)causedsevereinterferences


ndash ndash 2ndash Interferencescausedbycommoninorganicanions(NO Cl SO addedas3 4ndashtheirrespectiveacids)werealso investigatedOutof theseNO was foundto3

ndash1causethemostsevereinterferencesevenatconcentrationsofsinglemmolL Thendash 2ndashmethodologywasmorerobusttowardstheinterferencesfromCl andSO but4

theystillcausedsignificantdropinsensitivityathigherconcentrationsConsi-deringthewideuseoftheseacidsinanalyticalchemistryforsamplepreparationthisposesabigchallengeintheapplicationofthismethodtorealsamples

ndash1 Usingoptimalconditions(10(vv)formicacid4molL ammoniumformateandirradiationtimeof13s)acalibrationcurvewasmeasuredandevaluatedThelimitofdetectionwasdeterminedas3timesthestandarddeviationof10blank

ndash1measurements and was calculated as 13 ng L The repeatability of 10ndash1consecutivemeasurementsof250ngL was41


Fig 3Effectofvariousmetalionsonthepeakarea(a)metalionswithapositiveeffect(b)metalionsndash1withoutapositiveeffectExperimentalconditions2microgL Coreactionmedium10(vv)formic

ndash1 ndash1acidand4molL ammoniumformateflowrate4mLmin

ndash1Fig 2Effectofthecompositionofreactionmediumonpeakareaexperimentalconditions2microgL ndash1Coreactionmediumflowrate4mLmin (blackdotscorrespondtomeasuredpoints)

4Conclusions

Theconditionsofthephotochemicalvaporgenerationofcobaltwereoptimizedand are in good agreement with previous works [11 13] Copper ions wereidentifiedasapotentialsensitizerincreasingthesignalbyabout12-foldbuttheirpotentialuseisseverelylimitedbythenarrowrangeofconcentrationsinwhichthepositiveeffectisexhibitedSevereinterferencesfrominorganicanionswereobservedwhichisinlinewithotherworksdealingwithphotochemicalgeneration[1358]Furtherexperimentswillfollownamely(i)furtherinvestigationsinnewpotentialsensitizerstoenhancegenerationefficiencyandthusdecreasethe

ndash1limit of detection to sub ng L levels (ii) determination of the generationefficiency(fromcomparisonwithnebulizationandorusingaradioactiveisotope58Co)(iii)verificationoftheaccuracyandpracticalfeasibilityofthismethodologybyanalysisofcertifiedreferencematerials

Acknowledgments

ThesupportofTheCzechScienceFoundation(ProjectNo19-17604Y)CzechAcademyofSciences(Institutional supportRVO68081715)andCharlesUniversity (project SVV260560andprojectGAUK60120)isgratefullyacknowledged

References

[1] SturgeonREPhotochemicalvaporgenerationaradicalapproachtoanalyteintroductionforatomicspectrometryJAnalAtomSpectrom32(2017)2319ndash2340

[2] XuTHuJChenHJTransitionmetalionCo(II)-assistedphotochemicalvaporgenerationofthalliumforitssensitivedeterminationbyinductivelycoupledplasmamassspectrometryMicrochemJ149(2019)103972

[3] SoukalJSturgeonREMusilSEfficientphotochemicalvaporgenerationofmolybdenumforICPMSdetectionAnalChem90(2018)11688ndash11695

[4] deOliveiraRMBorgesDLGUVphotochemicalvaporgenerationofnoblemetals(AuIrPdPtandRh)AfeasibilitystudyusinginductivelycoupledplasmamassspectrometryandseawaterasatestmatrixJAnalAtomSpectrom33(2018)1700ndash1706


[6] GuoXSturgeonREMesterZGardnerGJVaporgenerationbyUVirradiationforsampleintroductionwithatomicspectrometryAnalChem76(2004)2401ndash2405

[7] HuJSturgeonRENadeauKHouXZhengCYangLCopperionassistedphotochemicalvapor generation of chlorine for its sensitive determination by sector field inductivelycoupledplasmamassspectrometryAnalChem90(2018)4112ndash4118

[8] LeonoriDSturgeonREAunifiedapproachtomechanisticaspectsofphotochemicalvaporgenerationJAnalAtomSpectrom34(2019)636ndash654

[9] SturgeonREPaglianoEEvidenceforphotochemicalsynthesisoffluoromethaneJAnalAtomSpectrom(2020)httpsdoiorg101039D0JA00108B

[10] GrinbergPMesterZSturgeonREFerrettiAGenerationofvolatilecobaltspeciesbyUVphotoreduction and their tentative identification J Anal Atom Spectrom 23 (2008)583ndash587

[11] DengHZhengCB LiuLWWuLHouXDLvYPhotochemicalvaporgenerationofcarbonyl for ultrasensitive atomic fluorescence spectrometric determination of cobaltMicrochemJ96(2010)277ndash282


[12] deQuadrosDPBorgesDLDirectanalysisofalcoholicbeveragesforthedeterminationofcobalt nickel and tellurium by inductively coupled plasmamass spectrometry followingphotochemicalvaporgenerationMicrochemJ116(2014)244ndash248

[13] deJesusHCGrinbergPSturgeonRESystemoptimizationfordeterminationofcobaltinbiologicalsamplesbyICP-OESusingphotochemicalvaporgenerationJAnalAtomSpectrom31(2016)1590ndash1604

[14] VyhnanovskyJFotochemickegenerovanıtekavychspeciı kobaltuproanalytickouatomovouspektrometriiMasterthesisFacultyofScienceCharlesUniversityPrague2018


1Introduction

Oneof themethods thatallowobtainingmaterialswithnewproperties is theplasmaenhancedchemicalvapordepositionmethodInthismethodcompoundscalledprecursorsaresuppliedtotheplasmareactorasagasphaseThankstoplasmaenhancedchemicalvapordeposition it ispossible toobtainmaterialswithuniquepropertiesThisisduetothefactthattheplasmaaffectsthesurfaceinfourdifferentways etching cleaning chemicalmodification and crosslinking

Optimization of condition for cold plasma deposition of thin layers for surface modification of working electrodes

a b a cJUSTYNALIPIN SKA MARIAMADEJ BOGUSŁAWBAS JACEKTYCZKOWSKI

a DepartmentofAnalyticalChemistryFacultyofMaterialsScienceandCeramicsAGHUniversityofScienceandTechnologyAdamaMickiewicza3030-059KrakoacutewPolandjustynalipinskaaghedupl

b DepartmentofAnalyticalChemistryFacultyofChemistryJagiellonianUniversityinKrakoacutewGronostajowa230-387KrakoacutewPoland

c Departmentof MolecularEngineeringFacultyofProcessandEnvironmentalEngineeringLodzUniversityofTechnologyWolczanska21390-924ŁoacutedźPoland


AbstractCurrentlyresearchisfocusedonthesearchfornewphysicallyandchemicallystablematerialsaswellasvolumeorsurfacemodificationOneofthemethodsusedforsurfacemodificationistheapplicationofthin layers from inorganic and organic compounds The plasmaenhancedchemicalvapordepositionisamethodthatallowsmaterialmodificationandalsodepositionofthinlayersThisworkconcernsoptimizationofcoldplasmadepositionparametersandtoachievethebestelectrical conductivitywhilemaintaining thehighmechanicalstrength of the formed layers Preliminary tests were focused onoptimizing the layering parameters such as the deposition timedischargepowerpressureofmonomerandthe flowofargonTheobtainedsamplesweresubjectedtothermaltreatmentafterwhichtheywere coveredwitha layerof aluminumThe thicknessof theobtained layers was determined on the basis of interferencemicroscopymeasurementsAsaresultoftheexperimentslayerswithathicknessof20nmto600nmwereobtainedTheconductivityofthedeposited layers was also determined and values from 003 to

ndash1150Sm wereobtained

Keywordscoldplasmadepositionelectrochemical

applicationssurfacemodificationthinlayers

Thismethodisusedtoproducecatalyticstructuresortomodifythepropertiesofmaterials eg improve hydrophobicity The growing popularity of surfacemodificationmethodsusingcoldplasmaisassociatedwiththefactthatitisanenvironmentallyfriendlyandversatilemethod[12] Workingelectrodesusedinvoltammetryareasubgroupofchemicalsensorswhich are small devices that convert real-time chemical information into ameasurableandanalyticallyusefulmeasurementsignalChemicalinformationrangingfromtheconcentrationofaspecificcomponentofthetestedsampletotheoverallcompositionofthematrixcancomefromboththeinitiatedchemicalreactionandbetheresultofphysico-chemicaltransformationstakingplaceinthetested object Chemical sensors are equipped with two basic elements iereceptor and transducer The receptor is responsible for the conversion ofchemicalinformationfromthetestedobjectintoaspecificformofenergyintheconverterthisenergyistransformedintoausefulanalyticalsignal Parameters characterizing the electrochemical sensor include accuracyprecision selectivity accuracy presentation selectivity sensitivity dynamicrange limit of quantification limit of detection lifetime response time andreliability Themost numerous and the oldest group of chemical sensors areelectrochemicalsensorsCommonlyobservedinterest inthisgroupofsensorsresultsfromthefactthatwithrelativelylowproductionandoperatingcoststheyofferthebestmetrologicalandoperationalparameters[3ndash5]Oneofthemaintrendsofmodernanalyticsisthesearchfornewelectrodematerialsandvariousgeometries of working electrodes One way to improve the performance ofworkingelectrodesistomodifytheirsurfaceforexamplebyapplyingthinlayersIn this work were considered plasma enhanced chemical vapor depositionmethod as the method of surface modification designed to performworkingelectrodeforvoltammetricdeterminationsAspartoftheinitialresearchplasmaprocessing parameters such as discharge power time of treatment andcompositionofgasmixtureinwhichplasmawasgeneratedwereoptimizedThelayers obtained in different conditions have been tested for suitability forelectrochemical applications (layer thicknessmeasurement and themeasure-mentofconductivity)

2Experimental


Theprecursor solutions suchasacrylonitriledietoxydimethylsilane trietoxy-methylsilaneandtetramethyldisiloxanewhicharesuppliedbyABCRwereusedOtherreagentsofanalyticalpuritysuchasn-hexane(SigmaAldrich)andargonwereused


22Instrumentation

The thin layers were deposited in a parallel-plate plasma reactor (frequency1356MHz)ThesamplesobtainedwerecalcinedinatunnelfurnaceunderanargonatmosphereThethicknessofthedepositedlayerswasmeasuredafterthealuminum was sputtered using a Nikon microscope type ECLIPSE LV150NElectrometerhigh resistance system (KEITHLEY) was used to measureconductivity


Eachof themonomerswasdepositedonprepared1times1corningglasssamplesSamples prepared with n-hexane were placed in a plasma reactor andadditionallypartiallycoveredwithamicroscopecoverslipSchematiclayoutofsamplesinthereactorshowninFig1ThefirststepwastoetchthesystemusingargonplasmaThisstageallowedfortheeliminationofimpuritiesthatwerenotremovedbythehelpofn-hexaneandthepreparationofthesurfaceofthesamplesforthedepositionoftheproperlayerTheproperstageistheapplicationofathinlayerwiththeplasmainducedbytheselectedprecursoracrylonitriledietoxydi-methylsilanetrietoxymethylsilaneandtetramethyldisiloxaneThethicknessandpropertiesoftheobtainedlayersdependonthedepositionparameterssuchasdischarge power time of treatment and composition of gasmixture inwhichplasmawasgenerated Table1showsallcombinationsofparameterstestedforallfourprecursorsFour different discharge powers for acrylonitrile and two different dischargepowersfororganosiliconmonomersweretestedwithtwodifferenttreatmenttimesEachtimeandpowercombinationwasperformedinplasmainducedbypuremonomerandmonomerwithargonAfterapplyingthelayersthesampleswereplacedinaquartzboatandcalcinedinatunnelfurnaceAllsampleswerecalcinedat500degCfor2hoursunderargonflowSamplesaftercalcinationwerecoveredwithalayerofaluminum


Fig 1 Scheme of sample distribution in plasma reactor(a)reactorelectrode(b)corningglasssamples(c)micro-scopecoverslip

Thethicknessoftheobtainedlayerswasmeasuredusinganinterferencemicro-scopeMeasurements were carried out at amagnification 10times recording theimageinmonochromelightTheimagewassetsothattheinterferencefringeswere perpendicular to the arc on the sample In order to calculate the layerthicknessD[nm]thefollowingformulawasused

(1)

wheredandLweredeterminedonthebasisoftheregisteredimage(disfringeshiftduetorefractionoflightontheslopeLisdistancebetweenthefringes) Thelaststageofthestudywastodeterminethecurrent-voltagecharacteristicstodeterminetheconductivityoftheobtainedlayersThesamplewasplacedinameasuring cell and attachedwith silver paste to the electrometerwires ThechangeincurrentwasrecordedwiththeapplicationofalternatingvoltageintimeBasedontheresultsobtainedthegraphsofdependenceUndashIwereobtainedfromwhichthevalueofresistance(R)wasdeterminedAnexampleofcurrent-voltagecharacteristicsisshowninFig2


Table 1Conditionsforlayersdeposition-parameterwhichweretested

Monomer DischargepowerW Timeoftreatmentmin

Acrylonitrile 10204080 24Dietoxydimethylsilane 2040 255Trietoxymethylsilane 2040 255Tetramethyldisiloxane 2040 255

Fig 2Current-voltagecharacteristicsdeterminedfortheacrylonitrilelayer(depositionparametersW=10Wt=25mingasmixtureonlyacrylonitrile)

Table 4Theresultsofthicknessmeasurementsandconductivityspecifictothetrietoxymethylsilanelayers

DischargepowerW 20 20 40Timeoftreatmentmin 25 5 5Argonflowsccm 10 10 10Thicknessnm 14686 24282 35311

ndash1SpecificconductivitySm 165 93 67

Knowing the value of the resistance and the geometry of the system thespecificresistancewasdeterminedfollowedbythespecificconductivityofthesamplethefollowingequationwasused

(2)

whereρisspecificresistance[Ωm]Risresistance[Ω]bissamplelength[m]Disdepositedlayerthickness[m]andldistancebetweenelectrodes(wires)[m] Theresultsofthicknessmeasurementsandspecificconductivityarepresentedin the Tables 2ndash5 For electrochemical applications it is important that theobtainedlayerhasthehighestspecificconductivityAnalyzingthedatapresentedin Tables 2ndash5 shows that the thinnest layers have the greatest applicationpotentialastheelectrodematerial


Table 2Theresultsofthicknessmeasurementsandconductivityspecifictotheacrylonitrilelayers

DischargepowerW 40 40 80 80 10 10 20 20Timeoftreatmentmin 2 4 2 4 2 4 2 4Thicknessnm 15845 31167 250 4644 247 67 7531 12236

ndash1SpecificconductivitySm 0002 156 191 0003 1512 454 311 185

Table 3The results of thickness measurements and conductivity specific to the dietoxydimethylsilanelayers

DischargepowerW 20 20 40 40Timeoftreatmentmin 25 5 25 5Argonflowsccm 10 10 10 10Thicknessnm 13998 37519 28431 49556

ndash1SpecificconductivitySm 1640 55 105 40

Table 5The results of thickness measurements and conductivity specific to the tetramethyldisiloxanelayers



4Conclusions

InthisworkplasmaenhancedchemicalvapordepositionmethodwasusedforapplyinglayersoffourdifferentmaterialsacrylonitriledietoxydimethylsilanetrietoxymethylsilaneandtetramethyldisiloxaneBychangingparameterssuchasdischargepowertimeoftreatmentandcompositionofgasmixtureanumberofsampleswereobtainedwithlayersofdifferentthicknessandwhatisassociatedwithotherelectricalproperties TestsperformedaspartofthisworkwereusedtoperforminnovativeworkingelectrodesforvoltammetricdeterminationsThesurfacewasmodifiedusingthecoldplasmaofthreesubstratesgraphiteglassycarbonandgold

Acknowledgments

JLandMMhavebeenpartlysupportedbytheEUProjectPOWR030200-00-I00416

References

[1] KapicaRTyczkowskiJBalcerzakJMakowskiMSielskiJWorwaEEnhancingadhesivejointsbetweencommercialrubber(SBS)andpolyurethanebylow-pressureplasmasurfacemodificationIntJAdhesAdhes95(2019)102415

[2] TyczkowskiJKapicaRŁojewskaJThincobaltoxidefilmsforcatalysisdepositedbyplasma-enhancedmetalndashorganicchemicalvapordepositionThinSolidFilms515(2007)6590ndash6595

[3] HulanickiAGłabSIngmanFChemicalsensorsDefinitionsandclassificationPureApplChem63(1991)1247ndash1250

[4] BrzozkaZWroblewskiWSensorychemiczneWarszawaOficynaWydawniczaPolitechnikiWarszawskiej1999(InPolish)

[5] SkoogDAWestDWHollerFJCrouchSRFundamentalsofAnalyticalChemistry9thEdBostonCengageLearning2013


1Introduction

ConcentrationandcompositionofvolatilecompoundsorcongenersisoneofthemostimportantparametersresponsibleforqualityofproducedalcoholicdrinksandhencefortheirsensorycharacteristicsandconsumeracceptanceTodaygaschromatography (GC) is conventionally used to determine qualitative andquantitativecompositionsofvolatilecompoundswithvariousexternalandorinternalstandardcalibrationprocedures Methodemployingethanol as an internal standard (IS) forGCquantitativedeterminationofvolatilecompoundsinalcoholicbeverageshasbeensuggestedquitelongago[1]andsincethattimegreatresearchworkhasbeencarriedoutRecentlyaninterlaboratorystudyofthemethodinvolving9testinglaboratoriesfrom4countrieswascarriedout[2]TheresultsdemonstratedgreatperspectivesofldquoEthanolasISrdquomethodandproveditsreferencecharacterandeaseofroutineimplementation

Advanced GC-MS method for quality and safety control of alcoholic beverages

abANTONKORBAN

a DepartmentofAnalyticalChemistryFacultyofScienceCharlesUniversity Hlavova2030812840Prague2CzechRepublickarbonat7gmailcomb DepartmentofAnalyticalChemistryChemistryFacultyBelarusianStateUniversityLeningradskaya14220050MinskBelarus


AbstractRecently developed and validated simple and reliable quantitativemethod employing ethanol as an internal standard for GC-MSquantification of volatile compounds in alcoholic products wasapplied to 36 samples including commercially available world-famousbrandspirits from18countriesandhomemadedistillatesThe GC-MS analyses were performed simultaneously by thesuggested approach and official internal standard method that isprescribedinthelegislationofEUandUSATheindependentsamplest-testwasemployedtoevaluatethestatisticaldifferenceofresultsofthesetwomethodsThetestrevealednodifferenceintheresultsandtheirrepeatabilityThemainbenefitsofthesuggestedmethodaretheeliminationofthenecessityofmanualinternalstandardadditionandsamplesdensitymeasurementthusmakingitmoreeconomicalandproductive

Keywordsalcoholicbeveragesgaschromatography-

massspectrometry(GC-MS)

internalstandardmethodvolatilecompounds

quantification

AllpreviousstudieswereutilizingflameionizationdetectorssinceGC-FIDisprescribedinthelegislation[3]wheremass-spectrometrydetectorsarenotyetofficially referred However GC-MS instruments are employed in practice toqualifyandorquantifyvolatilesincommercialspiritsintraditionalhomemadealcoholicdrinksinnewlydevelopedbeveragesinspiritwastesandindistillatesobtainedwithdifferentmanufacturingprocesses Our recent researchwasdirected towardsdevelopment of an algorithmofldquoEthanolasISrdquomethodapplicationonGC-MSinstruments[4]WehaveshowedthattopreventMSdetectorfromsaturationethanolshouldberegisteredinthecorresponding SIM timewindow at characteristicmz of low abundance forinstance bymz of 47 ions This ion corresponds to non-fragmented ethanol

13moleculescontaining1heavyisotope(mainly C)FinallytheresultsofmeasuredstandardsolutionsshowedthatthesuggestedapproachisvalidandldquoEthanolasISrdquomethodmaybesuccessfullyusedonGC-MSinstrumentstoo The objective of this studywas to test and further approve the suggestedapproachonalargersetof36realsamplesofalcoholicdrinkseithercommercialorhomemadeThesamplesweresimultaneouslyanalysedbytwoGC-MSmethodsndashaclassicalISmethodprescribedinthelegislationandthesuggestedldquoEthanolasISrdquomethod

2Experimental


Thefollowingvolatilecompoundsweredeterminedintestedsamplesofalcoholicbeverages 11-diethoxyethane (acetal) acetaldehyde methyl acetate ethylacetate methanol 2 propanol 1-propanol 2 methylpropan-1-ol (isobutanol)1-butanol2-butanoland3methylbutan-1-ol(isoamylalcohol)1-pentanolwasemployedasatraditionalIScompound

22Instrumentation

ShimadzuGCMS-QP2010Ultraequippedwithaquadrupolemassspectrometrydetector was employed for GC-MS measurements Rxi-1301Sil MS capillarycolumn(60mlength025mmid025micromfilmthicknessRestek)wasusedfortheseparationofcompoundsInjectionswereperformedinasplitmode(ratio175)Helium(99999purity)wasusedasacarriergasinjectortemperaturewas170degCTheoventemperaturewasheldat30degCfor5minthenraisedto210degC

ndash1at a rate of 30 degCmin and held isothermally for 4minMeasurementswereperformedinaSIMmodeFortheanalysedcompoundsand1-pentanol2ndash3mostabundantionsinthecorrespondingMSspectrumwereselectedethanolSIMtimewindowcontainedonly47mzionsAllGC-MSmeasurementswerecarriedoutintriplicateunderrepeatabilityconditions


AnalysisofeachalcoholicsamplewasperformedinafollowingwayAliquotof09mL of a tested sample was pipetted into a standard 2mL glass vial and

ndash1weighedAfterthat01mloftheISsolution(2355mgkg of1-pentanolinWES)wasaddedtothetestedsampleandthemasswasrecordedTheobtainedmixturewasmixedthoroughlyand05microlofitwasinjectedintotheGCsystem Theoriginoftestedalcoholicbeverageswaseithercommercialorhomemade33 world-famous spirits manufactured at different parts of the world werepurchasedfromcommerciallyavailablesourcesThelistoftypesofpurchasedandanalysedspiritsincludedbourboncalvadoscognacgingrappaliquormetaxaportwine rumsake tequilavodkawhiskeyandvarious fruitdistillatesThepurchased drinks were produced at the territory of the following countriesBelarus Bermuda Cuba Czech Republic Denmark France Germany GreeceGuatemala Jamaica Japan Mexico Moldova Portugal Slovakia Trinidad andTobago UK (England and Scotland) USA Three homemade fruit distillatesproducedbyfermentationofpulpyfruitsortheirmustswereobtainedfromlocalspiritmakersThedeclaredABVvaluesofalltestedsamplesvariedfrom15to81


To fulfil themaingoalof thiswork ie toevaluate thestatisticaldifferenceofresultsyieldedbythecomparedmethodswehaveemployedStudentst-testforindependentsamplestoverifystatisticaldifferencesonthesignificancelevelofp=005Theobtainedempiricalvaluesforallpairsofcongenersrsquoconcentrationswere lower than critical one in all cases demonstrating that concentrationsobtainedbythetwomethodshavenostatisticaldifferenceandleadtothesameresults InadditionrepeatabilityofthetwomethodswascomparedthereforeallRSDvalues obtained from triplicatemeasurements were split in two groupswith

ndash1respecttothecorrespondingconcentrations(lowerthan50mgL AAandhigherndash1than50mgL AA)Theobtainedresultsarepresentedintheformofboxplotin

Fig1AnalysisofthechartinFig1showedthatboththetestedmethodshaveyieldedstatisticallysimilarrepeatability AllofthetestedalcoholicdrinkssatisfiedtherequirementsofEURegulation(EC)no1102008[5]Theconcentrationsofundesirablecompoundssuchasmethanoldidnotexceedthelevelsspecifiedinthesameregulationforcorres-ponding beverages In Table 1 the description of the used SIM method andsummaryoftheexperimentalresultsarepresented Tocomparethetruenessofthemethodsoneofthespiritsampleswasspikedwithstandardsolutions(ABV40)containingallanalysedvolatilecompoundsat

ndash1concentrationsof50500and5000mgL AATheoriginalsamplewasusedasareferenceEachofthespikedsolutionswasmeasuredintriplicateSelectedspirit(cherry distillate) initially contained all 11 volatile compounds in various



Fig 1BoxchartsofRSDsofusedISmethodsat2concentrationrangesMeanisequaltoarithmeticmeanoraverageInterquartileRange(IQR)meansisthedistancebetweentheupper(themedianoftheupperhalfofthedataset)andlower(themedianofthelowerhalfofthedataset)quartile

ndash1Compound Timemin Registeredmz Numberof ConcentrationmgL AA results Minimal Maximal

Acetaldehyde 0ndash42 314344 36 24 715Methanol 36 13 13600

Ethanol(IS) 42ndash48 47 mdash mdash mdash

2-Propanol 48ndash70 29314345 14 27 199Methylacetate 596174 10 34 3201-Propanol 26 361 12070Ethylacetate 27 166 107002-Butanol 11 18 2080

Isobutanol 70ndash200 3141ndash4345 28 19 20001-Butanol 5556 13 28 155Acetal 26 45 270Isoamylol 31 39 26501-Pentanol(IS) mdash mdash mdash

Table 1DescriptionoftheusedSIMmethodandsomestatisticsconcerningallmeasured36spiritsamplesbothpurchasedandhomemade

concentrationsTheobtainedrecoveriesboxchartsareshowninFig2Compa-risonoftherecoveriesobtainedwithtwomethodsindicatesthattheyhavenosignificantdifferenceintermsoftruenessAverageobtainedrecoverywas981whenusingsuggestedmethodand980whenusingtraditionalISmethod

4Conclusions

InthisworktheresultsoftestingtheadvantageousldquoEthanolasISrdquomethodfortheGC-MS quality control analysis of alcoholic beverages were presented33purchasedsamplesofworld-famousalcoholicbeveragesoriginatingfrom18countriesand3homemadefruitdistillateswereanalysedtomakeathoroughandcomprehensive studyof thedevelopedmethodTheconcentrationsofvolatile

ndash1compoundsinanalysedsamplesvariedfrom1to13500mgL AAtheABVvalueofanalysedsamplesvariedfrom15to81ThesuggestedmethodwascomparedwiththetraditionalISmethodthatiscurrentlystatedinlegislationTheindepen-dentsamplest-testrevealedthatwithaprobabilityof095resultsobtainedwithtwo methods do not differ significantly The results of within-run precision(repeatability)showedrelativestandarddeviationswithin3measurementstobelessthat6indicatingthatthetechniqueisreproducibleThetruenessofthemethodwasevaluatedbyrecoverycalculationAccordingtotheobtainedresultsrecoveryofthesuggestedmethod(981plusmn33)wasslightlybetterthanthatofthetraditionalone(980plusmn58) ThesefactsprovethatdevelopedldquoEthanolasISrdquomethodistruepreciseandreliable when employed on GC-MS instruments At the same time to obtain


Fig 2Boxchartsof recoveriesof thesuggested(dottedpattern)and traditional (brickpattern)ISmethodsatdifferentspikeconcentrationsSymbolsdefinitionsarethesameasinFigure1

concentrationsofvolatilecompoundsintheofficiallyrequiredunitsofmeasurendash1 ndash1(mgL AAgL AAetc)suggestedmethodrequiresnodensitometrymeasure-

mentsofthetestedsampleandnoadditionofIScompoundoranyothersamplepre-treatmentThismethodprovidesaninvaluableanalyticaltoolforthequalitycontrolofalcoholicproductsandshouldbeusedinroutineanalysis

Acknowledgments

ThisworkwasfinanciallysupportedbytheVisegradFund

References

[1] CherepitsaSVBychkovSMKovalenkoANMazanikALSeleminaNMSeredinskayaOBThe use of themajor component (solvent) as an internal standard in the gas-chromato-graphicdeterminationofimpuritiesJAnalChem58(2003)368ndash371

[2] CharapitsaSSytovaSKorbanASobolenkoLEgorovVLeschevSZakharovMCabalaRBusarovaRShestakovichITolstouhovaAOndrousekSVavraJYilmaztekinMCabarogluTInterlaboratorystudyofethanolusageasaninternalstandardindirectdeterminationofvolatile compounds in alcoholic products BIO Web Conf 15 (2019) 02030httpsdoiorg101051bioconf20191502030

[3] CommissionRegulation(EC)No28702000layingdownCommunityreferencemethodsfortheanalysisofspiritsdrinkshttpdataeuropaeuelireg20002870oj

[4] KorbanACharapitsaSCabalaRSobolenkoLSytovaSTheperspectivesofethanolusageasaninternalstandardforthequantificationofvolatilecompoundsinalcoholicproductsbyGC-MSJMassSpectr55(2020)e4493

[5] EuropeanUnion(2008)Regulation(EC)No1102008oftheEuropeanParliamentandoftheCouncilof15January2008ontheDefinitionDescriptionPresentationLabellingandtheProtectionofGeographical IndicationsofSpiritDrinksandRepealingCouncilRegulationhttpdataeuropaeuelireg2008110(1)oj


1Introduction

Carbonorgraphitefeltsareusedaselectrodematerialsincethe1990sandtheirutilizationstillgrowsinanalyticalelectrochemistryaswellasinotherareasThisisduetotheirsuitablepropertiesfromwhichwecannamehighporosityhighspecificsurfaceareagoodelectricconductivityandhighphysicalandchemicalstabilityThefirsttwoparametersaregivenbystructureoffeltwhichconsistsoforderlesscarbonfibreswithabouttentotwentymicrometersindiameter[1]theothersby theadvantageouselectricalpropertiesof carbon fibreOn theotherhand porous flow-through electrodes including carbon felt electrode havedisadvantageinapotentialdropintheelectrodevolumewhichcausesdifficultcontrollingoftheexactpotentialappliedontheelectrodeandthereforeresultsindifferentcurrentefficienciesontheoppositesidesoftheelectrode[2] Carbon felt electrode can be utilized for detection of structurally differentcompoundsatvariousconditionsForexampleoperatingatreductionpotentialofndash08V[3]oxidationatrelativelyhighpotential+15V[4]ormeasuringatlowconcentrationsofelectrolyte[5]canbenamedDevelopedtechniquesalsoshowsthatcarbonfeltcanbeusedfordeterminationatsubmicromolarconcentrationsThis ismainly due to its ability to operate as a high-efficiency amperometricdetector Themainaimofthispaperistooverviewandcompareparametersofseveraldetermination methods of different analytes using carbon felt detector in

Utilization of a carbon felt as a material for working electrodes

MARTINBAROCHHANADEJMKOVA SA RKASLA DKOVA

DepartmentofAnalyticalChemistryFacultyofScienceCharlesUniversityHlavova8203012843Prague2CzechRepublicmartinbarochnaturcunicz


AbstractWorkingelectrodemadeofcarbonfeltwasusedincombinationwithHPLC for verificationofpractical applicabilityof the electrodeAlldeveloped methods confirm advantageous physical and chemicalpropertiesofcarbonfeltForelectrochemicalutilizationitispossibleto operate at higher positive potentials and even in low concen-trationsofelectrolyteinmobilephaseObtainedlimitsofdetectionwere mostly in submicromolar range and standard deviations ofmeasurementrepeatabilitywereunder5

KeywordsamperometrycarbonfeltFIAHPLC

combinationwithHPLC(forexampletheirdetectionpotentialslimitsofdetectionorlinearrange)

2Experimental


Stock solutions of propyl gallate butylhroxyanisole tert-butylhydroquinonebutylhydroxytoluene chlortoluron 2-amino-4-nitrophenol and 4-amino-2-

ndash3 ndash1nitrophenol (all SigmandashAldrich) with concentration of 1times10 molL werepreparedbydissolving theappropriateamountof therespectivesubstance inmethanol(HPLCgradeLach-NerCzechRepublic)Stocksolutionsof indole-3-aceticacidandindole-3-butyricacidwerepreparedinthesamemannerbutindeionized water Mobile phase consisted of methanol and phosphate-acetatebufferpreparedfromphosphoricandaceticacid(bothLach-NerCzechRepublic)andsodiumhydroxide(Fluka)

22Instrumentation

Theelectrochemicalcellconsistedofcarbonfelt(KarbotechnikCzechRepublic)flow-throughelectrodewhichwasplacedincapwithplatinumwireelectricalcontactanddrilledoutletholeononesideandwithflatferrulawithcapillaryonthe other side Schematic picture of the assembly is shown in ref [5] Otherelectrodeswereauxiliaryplatinumwireelectrodeandreferencesilverchloride(3MKCl) electrode (bothMonokrystaly Turnov Czech Republic) Potentiostatused in combination with this cell was Amperometric Detector ADLC 2(LaboratonıprıstrojePrahaCzechRepublic) HPLCapparatusconsistedofBeta10gradientpump(ECOMCzechRepublic)degasser DG 4014 (ECOM Czech Republic) six-way valve with 20microl loop(Rheodyne USA) HPLC column used for separation of propyl gallatebutylhydroxytoluenetert-butylhydroquinoneandbutylhroxyanisolefromtheirmixtureandforindole-3-aceticacidandindole-3-butyricacidfromtheirmixture

regwasLichrospher RP-18(125times4mm5micromMerckGermany)ForseparationofregchlortoluroncolumnPurospher RP-18(125times4mm5micromMerckGermany)was

used In case of mixture 2-amino-4-nitrophenol and 4-amino-2-nitrophenolregcolumnGemini C18110A (150times46mm5micromPhenomenexUSA)wasusedfor

separation Measurements of pHwere carriedout at Conductivity andpH-meter3510usingcombinedglasselectrode(JenwayUK)



Performanceofcarbonfeltwastestedonseveraltypesofanalyteswhichneededdifferent separation conditions namely amount of organic solvent in mobilephase and buffer pH The lowest amount of methanol (30) was used forseparation of 2-amino-4-nitrophenol and 4-amino-2-nitrophenol [3] Higherconcentrations of methanol in mobile phase was used for determination ofchlortoluronandforseparationofindole-3-aceticacidandindole-3-butyricacid[4]namely40and60respectivelyThehighestconcentrationsofmethanoland therefore electrolyte with lowest conductivity was used in separation ofantioxidantsnamelypropylgallatebutylhroxyanisolebutylhydroxytolueneandtert-butylhydroquinonewhereamountofmethanolwasrampingfrom55to95[5]DetectionpotentialsofmentionedanalytesandtheirlimitsofdetectionsareshowninTable1 HPLCseparationofantioxidantswas the firstmethodchosen for testingofcarbonfeltelectrodeperformancewiththistechniqueDuetodifferencesintheirstructure when butylhydroxytoluene has a different oxidation mechanismdetectionwith two appliedpotentialswas necessary According to the hydro-dynamic voltammograms potentials 14V and 08Vwere chosen for determi-nationofbutylhydroxytolueneandfortheotherthreeanalytesrespectively[5]AsshowninTable1whenthehigherpotentialwasappliedlimitsofdetectionforpropyl gallate butylhroxyanisole and tert-butylhydroquinone had increasedContrary determination of butylhydroxytoluene had approximately six timeslowerdetectionlimitathigherpotential


ndash1Compound E VLODmicromolL Refdet

Propylgallate 080 088 [5] 140 186 [5]Butylhroxyanisole 080 144 [5] 140 348 [5]tert-Butylhydroquinone 080 121 [5] 140 266 [5]Butylhydroxytoluene 080 3128 [5] 140 463 [5]Chlortoluron 140 013Indole-3-aceticacid 150 033 [4]Indole-3-butyricacid 150 054 [4]4-Amino-2-nitrophenol 080 016 [3]2-Amino-4-nitrophenol 080 021 [3]4-Amino-2-nitrophenol ndash080 35 [3]2-Amino-4-nitrophenol ndash080 37 [3]

Table 1Parametersofdetectionpotentialsandlimitsofdetectionfordifferentcompoundsusingcarbonfeltdetector

Detectionofauxins(indole-3-aceticacidandindole-3-butyricacid)wascarriedout at potential 15VMeasurements at this potential gives repeatabilitywithstandarddeviation31forindole-3-aceticacidand25forindole-3-butyricacidevenwithexchangingoftheworkingelectrodematerialCalibrationcurves

ndash1forbothanalyteswereobservedfrom04to100μmolL withlinearityinwholeconcentrationrangeLimitsofdetectionforbothanalytesreachedsubmicromolarconcentrationsevenwithrelativelyhighpotential[4] Incaseofdeterminationof2-amino-4-nitrophenoland4-amino-2-nitrophenolcarbon felt electrodewasused inbothoxidationand reductionmodeHydro-dynamicvoltammogramsshowedthatoptimaldetectionpotentialinreductionmodewasndash08VThislowpotentialisclosetotheendofthepotentialwindowandthereforeinterferenceswithremnantsofdissolvedoxygeninmobilephasewereobservedTheseinterferencesresultedinapproximately20timeshigherlimitofdetection for 2-amino-4-nitrophenol or 4-amino-2-nitrophenol obtained inreductionthaninoxidationmodeOntheotherhandmaximumvaluesoflinearrangewerethesameforbothanalytesinbothdetectionmodes[3] ForHPLCofchlortolurontheoptimaldetectionpotentialof14VwasfoundIts

ndash1calibration dependence although observed from 025 to 1000μmolL was ndash1linearonly in therange from025to50μmolL Limitofdetectionbasedon

ndash1standardsolutionswas013μmolL andreproducibilityofmeasurementgivenbytwentyconsecutivemeasurementsgaverelativestandarddeviationof05 ForallthedeterminationmethodsattentionwaspaidtotheapplicabilityofthecarbonfeltindetectionofanalytesincomplexmatricesIncaseofantioxidantsedibleoilswerechosenasrealsamples[5]nitrophenolderivatesweredeter-minedinurinesamples[3]andauxinsinrootingpreparation[4]ChlortolurondeterminationwasperformedinsoilandsurfacewaterThefoundvaluesshowanegligiblematrixinfluenceondetection

4Conclusions

CarbonfeltworkingelectrodewassuccessfullyusedincombinationwithHPLCfordeterminationofdifferenttypesofelectroactivecompoundsegantioxidantsauxinsorpesticidesAllmentionedapplicationsshowgreatperformanceofthecarbonfeltasaflow-throughelectrodematerialinelectroanalyticalchemistryforoxidationandreductionwayofanalytesdeterminationLimitsofdetectionforanalytes are mostly in submicromolar concentrations the exceptions areoxidationof analytes athigherpotentials and their reductionwhere limitsofdetectionsareinmicromolarconcentrationsApplicabilityoftheelectrodeonrealmatriceswas proven on analysis of edible oil samples groundwater soil androotingpreparation

Acknowledgments

ThisworkhasbeensupportedbytheCzechScienceFoundation(projectGACR20-01589S)


References

[1] Gonzalez-Garcia J Bonete P Exposito E Montiel V Aldaz ATorregrosa-Macia RCharacterizationofacarbonfeltelectrodeStructuralandphysicalpropertiesJMaterChem9(1999)419ndash426

[2] NavaJLRecendizAGonzalezLGCarrenoGMartın ezFMassTransportandpotentialstudiesinaflow-throughporouselectrodereactorPortugalElectrochimActa27(2009)381ndash396

[3] Dejmkova H Knaf M Application of carbon felt detector for the determination ofdinitrophenolmetabolitesInXXXIXModernElectrochemicalMethodsFojtaMSchwarzovaKNavratilT(Eds)U stınadLabemBestServis2019p41ndash43

[4] DejmkovaHdeAraujoDanielMElectrochemicaldeterminationofindole-3-aceticacidandindole-3-butyric acid using hplc with carbon felt detectorMonatsh Chem150 (2019)439ndash442

[5] DejmkovaHBarochMKrejcovaMBarekJZimaJCoulometricdetectorbasedoncarbonfeltApplMaterToday9(2017)482ndash486


1Introduction

Smith-Lemli-Opitzsyndrome(SLOS)isan autosomal recessive genetic disor-der firstly described in 1964 [1] It iscaused by the inborn deficiency of7-dehydrocholesterol reductase Thisenzyme transforms 7-dehydrocholes-terol (7-DHC Fig 1) to cholesterolduringthefinalstepofbiosynthesisofcholesterolincells The clinical symptoms of SLOS aredecreasedbloodlevelofcholesteroland

Electroanalytical methods for determination of 7-dehydrocholesterol in artificial serum

LENKABENESOVAADE LAZA RYBNICKA JANKLOUDAKAROLINASCHWARZOVA -PECKOVA

UNESCOLaboratoryofEnvironmentalElectrochemistryDepartmentofAnalyticalChemistryFacultyofScienceCharlesUniversityHlavova8203012843Prague2CzechRepublicbenesolenaturcunicz

Abstract7-DehydrocholesterolisabiomarkerofSmith-Lemli-Opitzsyndromeanautosomalrecessivegeneticdisordercausedbytheinborndefici-encyof7-dehydrocholesterolreductaseInthisstudyproceduresforitsdeterminationinartificialserumusingflowinjectionanalysiswithelectrochemical detection and voltammetric detection on borondoped diamond electrode were optimized The proteins wereprecipitatedbyacetonitrileandaftercentrifugationthesupernatantused for analysis For quantitation of 7-DHC by differential pulsevoltammetrytheoptimalratioacetonitrile-artificialserum91(vv)wasappliedInFIA-EDtheratio31(vv)runelectrolyteconsisting

minus1ofwater-acetonitrilecontaining001molL NaClO inthesameratio4minus1anddetectionpotentialof+13VvsAgAgCl(3molL KCl)were

usedQuantitationof7-DHCwaspossibleusingcalibrationdepen-minus1dencewithlimitdetectionof20micromolL inartificialserumNeverthe-

lessthemethodhaslowrecoveryandforsensitivedeterminationinreal matrices of human serum and amniotic fluid a liquid-liquidextraction needs to be applied to prevent presence of 7-dehydro-cholesterolinthephasewithprecipitatedproteins

Keywordsamperometricdetectionborondopeddiamond

electrode7-dehydrocholesteroldifferentialpulse

voltammetryflowinjectionanalysisSmith-Lemli-Opitz

syndrome


Fig 1Structureof7-dehydrocholesterol

increased concentrationof7-DHC inbloodandnervous system [2] SLOS is acomplexofmultipleanomaliesincludingmentalretardationItismanifestedbyholoprosencephaly(anomaliesinbraindevelopmentwithimprecisedivisionintotherightandlefthemispheres)milddysmorphismscardiacrenalandgastro-intestinalmalformations ThecharacteristicfacialanomaliesofSLOS[2]aremicrocephalybitemporalnarrowing ptosis short nasal root short nose with anteverted nares andmicrognathia epicanthal folds and capillary hemangioma over the nasal rootextendingontotheglabellatheearappearlow-setandareposteriorlyrotatedOral finding includes a high-arched and narrow hard plate broad and ridgealveoralridgesandredundancyofsublinqualtissuesCNSanomaliesareagenesisorhypoplasiaBilateralandunilateralpostaxialpolydactylycanbepresentedinthehandsorfeetorboth Concentrationof7-DHCinbloodiscrucialforclinicaldiagnosticofSLOSinpatients Concentration levels in amniotic fluid are used for fetal diagnosticsTable1 summarizes concentration of 7-DHC in plasma and amniotic fluid ofhealthypersonandSLOSpatientsAnalyticalmethodsusedfordeterminationofconcentrationof7-DHCinthesematricesincludecombinationofGCorHPLCwithMS[3]orGCwithflameionizationdetection(FID)orUVdetection[45] Thepossibilitiesofelectrochemicalmethodsfordetectionof7-DHCarelimitedasgenerallythesteroidcoreisratherredox-inactive(detailinreview[6])undervarietyofconditionsNevertheless7-DHCpossessesconjugateddoublebondsonsteroidcoreanditsoxidationwasreportedinseveralstudies[7ndash9]Itsvoltam-metricsignal+095VvsSCEonglassycarbonelectrodewasfirstlyobservedin

minus1non-aqueousmediaofmethanol-benzene7525(vv)using005molL LiClO as4

supporting electrolyte in a study dealing with electrochemical behaviour ofvitaminAandDandtheirprovitaminsD(7-DHCisprecursorofvitaminD3)[7]Determinationof7-DHCinhumanskinispossiblebyHPLCwithUV(λ=286nm)andamperometricdetection(E +17VvsAgAgCl)onglassycarbonelectrodedet

minus1usingmethanol-tetrahydrofuran175mmolL KH PO (9514vvv)asmobile2 4minus1phase7-DHCwasdetectedintherangefrom12to81microgg dryweightwith

minus1detectionlimitof39pmolL [8]Anotherstudy[9]isdevotedtodeterminationof


minus1Author(s)ref Matrix ConcentrationμmolL Healthy SLOS

Kelley[4] Plasma 03plusmn001 385plusmn309 Amnioticfluid lt02plusmn001 16plusmn9Rossiteretal[5] Plasma lt5 179ndash335 Amnioticfluid lt03 12ndash15

Table 1Concentrationof7-dehydrocholesterolinclinicalmatricesforhealthypersonsandforSmith-Lemli-Opitzsyndrome(SLOS)patientsdetectedbyGC-FID

7-DHCandvitaminD3 in fishusingHPLCwithelectrochemicaldetectionTheanalytical cell was a serial combination of two-flow-through porous graphiteworking electrodes The first standard coulometric electrode was used toeliminatepotentially interfering compounds using the second lineardynamic

minus1rangefrom0013to0312micromolL for7-DHCwasachieved Hereinwestudiedpossibilitiestodetect7-DHCbasedonitsoxidationonborondoped diamond (BDD) electrode using differential pulse voltammetry andelectrochemicaldetectioninflowinjectionanalysis(FIA-ED)inartificialserumandperipherallyinhumanserumandamnioticfluid

2Experimental


7-dehydrocholesterol (purity95)wasobtainedSigmaAldrich (USA)and itsstandard solution was prepared in acetonitrile (Honeywell Germany) Theartificial serum was prepared from KCl (Penta Czech Republic) CaCl 2H O2 2

(PentaCzechRepublic)NaCl(PentaPragueCzechRepublic)urineD-glucoseand01albuminfromSigma-Aldrich(USA)NaClO4(PentaCzechRepublic)wasusedassupportingelectrolyte

22Instrumentation

VoltammetricmeasurementweregovernedbythepotentiostatPalmSensusingworking BDD electrode (Windsor Scientific UK d = 31 mm) AgAgNO 3

minus1 minus1(01molL AgNO 1molL NaClO in acetonitrile) non-aqueous reference3 4electrodeandplatinumwirecounterelectrodeBDDsurfacewaspolishedbeforeeachscanusingsuspensionofAl O (ElektrochemickedetektoryTurnovCzech2 3

Republic) HPLC system (Hitachi Merck) consisting of control unit D-7000gradientpumpL-7100autosamplerL-7200andUVdetectorL-7400wasusedfor

minus1FIA-EDdetectionof7-DHCRunelectrolytewascomposedof001molL NaClO 4inacetonitrileanddeionisedwaterinratio31(vv)Flowrateofmobilephase

minus1was30mlmin injectionvolumewas40microLandλ=280nmwasusedforUVdetectionWall-jet detection cell was employedwith working BDD electrode

minus1AgAgCl (3molL KCl) reference electrode and platinum wire auxiliaryelectrode Optimal detection potential of +13 V was controlled using ADLC2potentiostat(Laboratornı prıstrojePragueCzechrepublic)


In this study electroanalytical methods were developed for determination of7-DHCinartificialserumnamelyFIA-EDandDPVBothmethodsarebasedondirectoxidationof7-DHConborondopeddiamondelectroderesultinginanodic


peak at ca +08 V (vs AgAgNO in acetonitrile) in non-aqueous medium of3

acetonitrile or mixedmedium acetonitrile-water using NaClO as supporting4

electrolyteTheoxidationispresumablyinitiatedbyoneelectronremovalfromtheconjugateddoublebondsonthesteroidcoreof7-DHC Fordeterminationof7-DHCinrealmatricesitisnecessarytoremovepresentproteinsArtificialserumcontainingalbuminwasusedasmodelmatrixtostudythe possibilities Firstly albumin was removed simply by precipitation withacetonitrile(serum-water13(vv))andthesupernatantwasanalysed Differentratioswateracetonitrileweretestedinrunelectrolyte(59510902080 2575 3070 4060 and 5050 (vv)) to evaluate the influence of itscompositionontheFIA-EDsignaloftheblankand7-DHCThesameratio13asusedforprecipitationofalbuminwaschosenasoptimalbecauseofminimalandstablesignaloftheblankinjectedinFIA-EDsystemFurtherdetectionpotentialE in the range from +10 V to +15 V was optimized by evaluation of thedet

hydrodynamicvoltammogramsresultinginE of+13Vsetasoptimalvaluedetminus1 Concentrationdependenceof7-DHCislinearintherangefrom25micromolL to

minus1 minus1300micromolL (concentrationinartificialserum)withdetectionlimitof20micromolL and this concentration dependence can be used for quantitation of 7-DHC inartificial serum Nevertheless determination of 7-DHC in human serum andamnioticserumfailedastheyrepresentmorecomplicatedmatricesand7-DHCispresumablypartiallyadsorbedinthepresentproteinsandcannotbequantifiedinthesupernatant Furtherdifferentialpulsevoltammetrywithoptimizedparameterswasusedfordeterminationof7-DHCInthepresenceofproteinsinartificialserum(human

minus1serum albumin) an unacceptably high detection limit of 178micromolL wasachieved When the proteins were precipitated using acetonitrile (aceto-nitrileartificialserumratio91(vv))thelimitofdetectionof7-DHCwaslowered

minus1to15micromolL inartificialserumNeverthelesstherecoveryofthemethodwasonly43to70dependingontheconcentrationof7-DHCagainreflectingthelossof7-DHCduetoproteinprecipitation Thereforeasecondapproachofsamplepretreatmentbasedonliquid-liquidextractionofalllipidsdescribedin[10]wastested(Bligh-Dyerextraction)TheprocedurehastwopartsFirstlymethanolchloroformandthesampleofartificialserum is mixed and shaken to form a monophasic system After addition ofchloroform and water a biphasic system is formed where chloroform phasecontainsalloflipidcompoundsandmethanol-waterphasecontainsallnon-lipidscompoundsChloroformphaseisthendriedunderN2atmosphereat50degCanddried extract dissolved in acetonitrile Preliminary experiments using DPVresultedinrecoveryof97forBligh-Dyerextractionof7-DHCfrominartificialserum


4Conclusions

FIA-EDandDPVwereoptimizedfordeterminationof7-DHCinartificialserumUsing precipitation of proteins by acetonitrile limit of detection of 7-DHC in

minus1artificialserumusingFIA-EDwas20micromolL andthismethodcanbeusedfortheirquantificationusingcalibrationdependenceNeverthelessdeterminationusingDPVisunreliableduetolowrecoveryoftheprocedureDevelopmentofamethodincludingliquid-liquidextractionstepisinprogresssothat7-DHCcouldbedeterminedinrealmatricesashumanserumandamnioticfluid

Acknowledgments

TheresearchwassupportedbytheCzechScienceFoundation(projectGACR19-11268S)andtheSpecificUniversityResearch(SVV260560)

References

[1] Smith DW Lemli L Opitz JM A newly recognized syndrome of multiple congenitalanomaliesJPediatr64(1964)210minus217

[2] Nowaczyk M Waye J The SmithndashLemlindashOpitz syndrome a novel metabolic way ofunderstandingdevelopmentalbiologyembryogenesisanddysmorphologyClinGenet59(2001)375minus386

[3] BeckerSRohnikeSEmptingSHaasDMohnikeKBebloSMutzeUHusainRAThieryJCeglarekULC-MSMS-basedquantificationofcholesterolandrelatedmetabolitesindriedblood for the screening of inborn errors of sterolmetabolismAnal Bioanal Chem407(2015)5227minus5233

[4] Kelley RI Diagnosis of Smith-Lemli-Opitz syndrome by gas-chromatography mass-spectrometryof7-dehydrocholesterolinplasmaamniotic-fluidandculturedskinfibroblastsClinChimActa236(1995)45minus58

[5] Rossiter JP Hofman KJ Kelley RI Smith-Lemli-Opitz SyndromePrenatal-diagnosis byquantification of cholesterol precursors in amniotic-fluid Am J Med Genet 56 (1995)272minus275

[6] KloudaJBarekJNesmerakKSchwarzova-PeckovaKNon-enzymaticelectrochemistryincharacterization and analysis of steroid compounds Crit Rev Anal Chem 47 (2017)384minus404

[7] AtumaSSLundstromKLindquistJTheelectrochemicaldeterminationofvitaminAPartIIFurthervoltammetricdeterminationofvitaminAandinitialworkonthedeterminationofvitaminDinthepresenceofvitaminAAnalyst100(1975)827minus834

[8] MoodyJPHumphriesCAAllanSMPatersonCRDeterminationof7-dehydrocholesterolinhumanskinbyhigh-performance liquid-chromatography JChromatogrB530 (1990)19minus27

[9] OstermeyerUSchmidtTVitaminDandprovitaminDinfishEurFoodResTechnol222(2005)403minus413

[10] BlighEGDyerWJArapidmethodoftotallipidextractionandpurificationCanJBiochemPhysiol37(1959)911minus917


Author Index

AlikovaV1

AugustınM83

BaluchovaS19

BarekJ192570

BarochM141

BasB13129

BastryginaO41

BenesovaL146

BessonovaE57

BohmD6

BurkinK116

BurkinM116

ChernovaA141

ChoinskaM70

CokrtovaK104

DedinaJ97

DeevV57

DejmkovaH141

DendisovaM63

DubenskaL51

EfremenkoE41

FojtaM110

GalvidisI116

HavranL110

HeiglN31

HertJ76

HrdlickaV70

JosypcukB25

KartsovaLA3557

KloudaJ19146

Kodrık ovaB90

KolobovaEA35

KorbanA135

KorotkovaE1

KralM63

KratzerJ90

KravchenkoAV35

KrızekT76104

LipinskaJ129

MadejM129

MatejkaP63

MatysikF-M631

MusilS9097123

NavratilT70

OndrackovaA110

PietrzakK45

PlotnikovaK51

PoradaR13

RedondoBR70

SagapovaL90

ShormanovV1

Schwarzova-PeckovaK19110146

SladkovaS 141

S tadlerovaB97

StiborovaM110

SvobodaM90

TvorynskaS25

TyczkowskiJ129

VyhnanovskyJ97123

VymyslickyF76

VyskocilV83

WardakC45

WongDKY19

ZarybnickaA146

ZelenyI51

Proceedingsofthe16thISCModernAnalyticalChemistry Prague2020 151


Keyword Index

alcoholicbeverages135

aminoglycosides116

amperometricdetection146

amperometry141

antifoulingelectrodes19

assembledcapillaries6

atomicabsorptionspectrometry90

atomicfluorescencespectro-

metry97

atomization90

biologicalactiveanalytes35

biosensor2583

bismuth97

borondopeddiamond

electrode110146

cadmium90

canagliflozin76

capillarycoating35

capillaryelectrophoresis635

capillaryflowinjectionanalysis631

carbohydrates31

carbonfelt141

cathodicstrippingvoltammetry70

chemicalvaporgeneration90

chemometrics57

cobalt123

coldplasmadeposition129

copper(II)phthalocyanine63

covalentimmobilization25

cytochromeP450110

damage83

7-dehydrocholesterol146

designofexperiments76

differentialpulsevoltammetry146

diphenylsilanereductionmethod19

dispersiveliquid-liquid

microextraction57

disposableelectrodes31

DNA83

dualdetectionconcept6

electrochemicalanalysis110

electrochemicalapplications129

electrochemicalflowcell76

electrochemistry51

electrokineticchromatography104

eliminationvoltammetrywith

linearscan70

ELISA116

enzymaticreactor25

FIA141146

gaschromatography-mass

spectrometry(GC-MS)135

glucoseoxidase25

graphite83

honey116

HPLC76141

hydridegeneration97

hydrogenatedconical-tipcarbon

electrodes19

imidazoliumionicliquids35

inductivelycoupledplasmamass

spectrometry123

internalstandardmethod135

ion-selectiveelectrode45

laccase25

liquidcrystals104

massspectrometry31

mercuryelectrode13

metronidazole51

non-aqueouscapillary

electrophoresis104

non-aqueoussystem6

oxidation76

oxytetracyclinehydrochloride51

phenol2-methoxy1

photochemicalvaporgene-

ration97123

polarography51

1-propanesulfonicacid23-

dimercapto-70

pulsedamperometricdetection31

quantitation1

resonanceRamanspectroscopy63

scanningtunnellingmicroscopy63

silversolidamalgamelectrode70

Smith-Lemli-Opitzsyndrome146

smokingmixtures41

solidcontact45

solid-phasemicroextraction57

spectrophotometry141

SudanI110

surfacemodification129

surface-enhancedRaman

spectroscopy63

thinlayers129

tip-enhancedRamanspectroscopy

63

unithiol70

uranyl45

vanillin41

veterinarydrug51

vitamins13

volatilecompoundsquantifi-

cation135

voltammetricdopaminedetec-

tion19

voltammetry1383


Proceedings of the 16th International Students Conference ldquoModern Analytical Chemistryrdquo

EditedbyKarelNesmerak

PublishedbyCharlesUniversityFacultyofScience

Prague2020

1steditionndashvi154pages

ISBN978-80-7444-079-3

ISBN 978-80-7444-079-3

Pro

ceedin

gs of th

e 16

th In

ternatio

nal Stu

den

ts Co

nferen

ce ldquoMo

dern

An

alytical Ch

emistryrdquo P

rague 2

02

0

788074 440793



Prague 2020

Proceedings of the



Proceedingsofthe

16thInternationalStudentsConference

ldquoModernAnalyticalChemistryrdquo



Prague 2020

Proceedings of the











543(062534)

analyticalchemistry






ISBN978-80-7444-079-3

Preface


















science










editor


Sponsors



companies

wwwecomsrocom

wwwlach-nercom


wwwthermofishercz

www2thetacz


wwwshimadzueucom

wwwwaterscom

Contents



























1Introduction











metry




2Experimental



22Instrumentation










4Conclusions












References














1Introduction













2Experimental



22Instrumentation

221Capillaries
























25 00577 0000525+25 0057 000150+25 00775 0000775+25 ndash ndash25+50 0137 000550 0865 000250+50 080 00575+50 105 00625+75 0187 000250+75 1941 000375 456 00275+75 42 01








4Conclusions


Acknowledgments


References








1Introduction










2Experimental























4Conclusions






Acknowledgments


References













1Introduction











detection



2Experimental



22Instrumentation













































4Conclusions


Acknowledgments













1Introduction














2Experimental







22Instrumentation


















Tab

le 1


StrategyA

StrategyB

StrategyC

Support




Supportfunctio-

minusNH

minusNH

minusCOOH

22

nalizedgroup

Activationagent

Glutaraldehyde(GA)

Carbodiimide

Carbodiimide

(EDCNHS)

(EDCNHS)

Enzymereactive

minusNH

minusCOOH

minusNH

22

group

Typeofbond

secondaryamine

amide

amide

Denotationsofthe




23

preparedenzy-




23

maticpowders




23




23




23




23




4Conclusions




Acknowledgments


References

















1Introduction










detection


2Experimental






22Instrumentation










4Conclusions


References











1Introduction










2Experimental



22Instrumentation






24Solutions











4Conclusions







ndash120μgmL

Acknowledgments


References













1Introduction













2Experimental



22Instrumentation


23Samplepreparation







ndash1 A =81914c[gL ]+00357 (1)2802 R =1


ndash1 A =73824c[gL ]+00301 (2)3102 R =1




4Conclusions



λ nm

Absorban

ce







References







1Introduction











2Experimental





22Instrumentation


























4Conclusions




References














1Introduction











2Experimental







22Instrumentation















4Conclusions


References








Recovery 97 103



















1Introduction









microextraction


2Experimental

21Reagents


22Instrumentation


























4Conclusions




Acknowledgments





References









1Introduction








Keywordscopper(II)





spectroscopy


2Experimental







22Instrumentation























4Conclusions


Acknowledgments




References
















1Introduction














electrodeunithiol



2Experimental












22Instrumentation

























4Conclusions



Acknowledgments


References






















1Introduction










2 Experimental






22Instruments







ndash1001Vs )















4Conclusion


Acknowledgments


References








1Introduction










2Experimental





22Apparatus












24Procedures
























4Conclusions



Acknowledgments


References









1Introduction









generation





2Experimental















22Instrumentation







(A)

(B)























modifiers

4Conclusions





Acknowledgments


References












1Introduction











generation


2Experimental









22Instrumentation









23Samplepreparation





















Repeatability lt1 6



4Conclusion


Acknowledgments


References














1Introduction








electrophoresis





2Experimental



22Instrumentation


23Method















4Conclusions


Acknowledgments


References
















1Introduction












(A) (B)

(C) (D)

2Experimental



22Instrumentation










ndash1rate1Vs


ndash1rate1Vs



4Conclusions



Acknowledgments


References







1Introduction










2Experimental









24TheELISAprocedure






B ndash3timesSD0








usinganti-BSA-C6-RS












andalsoNARSSSMandTM

4Conclusions




References












1Introduction















2Experimental




22Instrumentation


























4Conclusions



Acknowledgments


References

















1Introduction













2Experimental




22Instrumentation







(1)











(2)












4Conclusions


Acknowledgments


References







1Introduction



abANTONKORBAN







quantification



2Experimental



22Instrumentation





















4Conclusions







Acknowledgments


References







1Introduction









2Experimental




22Instrumentation
















4Conclusions


Acknowledgments



References







1Introduction












syndrome














2Experimental




22Instrumentation


















4Conclusions



Acknowledgments


References












Author Index

AlikovaV1

AugustınM83

BaluchovaS19

BarekJ192570

BarochM141

BasB13129

BastryginaO41

BenesovaL146

BessonovaE57

BohmD6

BurkinK116

BurkinM116

ChernovaA141

ChoinskaM70

CokrtovaK104

DedinaJ97

DeevV57

DejmkovaH141

DendisovaM63

DubenskaL51

EfremenkoE41

FojtaM110

GalvidisI116

HavranL110

HeiglN31

HertJ76

HrdlickaV70

JosypcukB25

KartsovaLA3557

KloudaJ19146

Kodrık ovaB90

KolobovaEA35

KorbanA135

KorotkovaE1

KralM63

KratzerJ90

KravchenkoAV35

KrızekT76104

LipinskaJ129

MadejM129

MatejkaP63

MatysikF-M631

MusilS9097123

NavratilT70

OndrackovaA110

PietrzakK45

PlotnikovaK51

PoradaR13

RedondoBR70

SagapovaL90

ShormanovV1


SladkovaS 141

S tadlerovaB97

StiborovaM110

SvobodaM90

TvorynskaS25

TyczkowskiJ129

VyhnanovskyJ97123

VymyslickyF76

VyskocilV83

WardakC45

WongDKY19

ZarybnickaA146

ZelenyI51



Keyword Index


aminoglycosides116


amperometry141





metry97

atomization90


biosensor2583

bismuth97

borondopeddiamond

electrode110146

cadmium90

canagliflozin76

capillarycoating35



carbohydrates31

carbonfelt141



chemometrics57

cobalt123




cytochromeP450110

damage83






microextraction57


DNA83





electrochemistry51



linearscan70

ELISA116

enzymaticreactor25

FIA141146



glucoseoxidase25

graphite83

honey116

HPLC76141

hydridegeneration97


electrodes19



spectrometry123



laccase25

liquidcrystals104

massspectrometry31

mercuryelectrode13

metronidazole51


electrophoresis104

non-aqueoussystem6

oxidation76


phenol2-methoxy1


ration97123

polarography51


dimercapto-70


quantitation1





smokingmixtures41

solidcontact45



SudanI110



spectroscopy63

thinlayers129


63

unithiol70

uranyl45

vanillin41

veterinarydrug51

vitamins13


cation135


tion19

voltammetry1383





Prague2020


ISBN978-80-7444-079-3

ISBN 978-80-7444-079-3

Pro

ceedin

gs of th

e 16

th In

ternatio

nal Stu

den

ts Co

nferen

ce ldquoMo

dern

An

alytical Ch

emistryrdquo P

rague 2

02

0

788074 440793



Prague 2020

Proceedings of the





Prague 2020

Proceedings of the











543(062534)

analyticalchemistry






ISBN978-80-7444-079-3

Preface


















science










editor


Sponsors



companies

wwwecomsrocom

wwwlach-nercom


wwwthermofishercz

www2thetacz


wwwshimadzueucom

wwwwaterscom

Contents



























1Introduction











metry




2Experimental



22Instrumentation










4Conclusions












References














1Introduction













2Experimental



22Instrumentation

221Capillaries
























25 00577 0000525+25 0057 000150+25 00775 0000775+25 ndash ndash25+50 0137 000550 0865 000250+50 080 00575+50 105 00625+75 0187 000250+75 1941 000375 456 00275+75 42 01








4Conclusions


Acknowledgments


References








1Introduction










2Experimental























4Conclusions






Acknowledgments


References













1Introduction











detection



2Experimental



22Instrumentation













































4Conclusions


Acknowledgments













1Introduction














2Experimental







22Instrumentation


















Tab

le 1


StrategyA

StrategyB

StrategyC

Support




Supportfunctio-

minusNH

minusNH

minusCOOH

22

nalizedgroup

Activationagent

Glutaraldehyde(GA)

Carbodiimide

Carbodiimide

(EDCNHS)

(EDCNHS)

Enzymereactive

minusNH

minusCOOH

minusNH

22

group

Typeofbond

secondaryamine

amide

amide

Denotationsofthe




23

preparedenzy-




23

maticpowders




23




23




23




23




4Conclusions




Acknowledgments


References

















1Introduction










detection


2Experimental






22Instrumentation










4Conclusions


References











1Introduction










2Experimental



22Instrumentation






24Solutions











4Conclusions







ndash120μgmL

Acknowledgments


References













1Introduction













2Experimental



22Instrumentation


23Samplepreparation







ndash1 A =81914c[gL ]+00357 (1)2802 R =1


ndash1 A =73824c[gL ]+00301 (2)3102 R =1




4Conclusions



λ nm

Absorban

ce







References







1Introduction











2Experimental





22Instrumentation


























4Conclusions




References














1Introduction











2Experimental







22Instrumentation















4Conclusions


References








Recovery 97 103



















1Introduction









microextraction


2Experimental

21Reagents


22Instrumentation


























4Conclusions




Acknowledgments





References









1Introduction








Keywordscopper(II)





spectroscopy


2Experimental







22Instrumentation























4Conclusions


Acknowledgments




References
















1Introduction














electrodeunithiol



2Experimental












22Instrumentation

























4Conclusions



Acknowledgments


References






















1Introduction










2 Experimental






22Instruments







ndash1001Vs )















4Conclusion


Acknowledgments


References








1Introduction










2Experimental





22Apparatus












24Procedures
























4Conclusions



Acknowledgments


References









1Introduction









generation





2Experimental















22Instrumentation







(A)

(B)























modifiers

4Conclusions





Acknowledgments


References












1Introduction











generation


2Experimental









22Instrumentation









23Samplepreparation





















Repeatability lt1 6



4Conclusion


Acknowledgments


References














1Introduction








electrophoresis





2Experimental



22Instrumentation


23Method















4Conclusions


Acknowledgments


References
















1Introduction












(A) (B)

(C) (D)

2Experimental



22Instrumentation










ndash1rate1Vs


ndash1rate1Vs



4Conclusions



Acknowledgments


References







1Introduction










2Experimental









24TheELISAprocedure






B ndash3timesSD0








usinganti-BSA-C6-RS












andalsoNARSSSMandTM

4Conclusions




References












1Introduction















2Experimental




22Instrumentation


























4Conclusions



Acknowledgments


References

















1Introduction













2Experimental




22Instrumentation







(1)











(2)












4Conclusions


Acknowledgments


References







1Introduction



abANTONKORBAN







quantification



2Experimental



22Instrumentation





















4Conclusions







Acknowledgments


References







1Introduction









2Experimental




22Instrumentation
















4Conclusions


Acknowledgments



References







1Introduction












syndrome














2Experimental




22Instrumentation


















4Conclusions



Acknowledgments


References












Author Index

AlikovaV1

AugustınM83

BaluchovaS19

BarekJ192570

BarochM141

BasB13129

BastryginaO41

BenesovaL146

BessonovaE57

BohmD6

BurkinK116

BurkinM116

ChernovaA141

ChoinskaM70

CokrtovaK104

DedinaJ97

DeevV57

DejmkovaH141

DendisovaM63

DubenskaL51

EfremenkoE41

FojtaM110

GalvidisI116

HavranL110

HeiglN31

HertJ76

HrdlickaV70

JosypcukB25

KartsovaLA3557

KloudaJ19146

Kodrık ovaB90

KolobovaEA35

KorbanA135

KorotkovaE1

KralM63

KratzerJ90

KravchenkoAV35

KrızekT76104

LipinskaJ129

MadejM129

MatejkaP63

MatysikF-M631

MusilS9097123

NavratilT70

OndrackovaA110

PietrzakK45

PlotnikovaK51

PoradaR13

RedondoBR70

SagapovaL90

ShormanovV1


SladkovaS 141

S tadlerovaB97

StiborovaM110

SvobodaM90

TvorynskaS25

TyczkowskiJ129

VyhnanovskyJ97123

VymyslickyF76

VyskocilV83

WardakC45

WongDKY19

ZarybnickaA146

ZelenyI51



Keyword Index


aminoglycosides116


amperometry141





metry97

atomization90


biosensor2583

bismuth97

borondopeddiamond

electrode110146

cadmium90

canagliflozin76

capillarycoating35



carbohydrates31

carbonfelt141



chemometrics57

cobalt123




cytochromeP450110

damage83






microextraction57


DNA83





electrochemistry51



linearscan70

ELISA116

enzymaticreactor25

FIA141146



glucoseoxidase25

graphite83

honey116

HPLC76141

hydridegeneration97


electrodes19



spectrometry123



laccase25

liquidcrystals104

massspectrometry31

mercuryelectrode13

metronidazole51


electrophoresis104

non-aqueoussystem6

oxidation76


phenol2-methoxy1


ration97123

polarography51


dimercapto-70


quantitation1





smokingmixtures41

solidcontact45



SudanI110



spectroscopy63

thinlayers129


63

unithiol70

uranyl45

vanillin41

veterinarydrug51

vitamins13


cation135


tion19

voltammetry1383





Prague2020


ISBN978-80-7444-079-3

ISBN 978-80-7444-079-3

Pro

ceedin

gs of th

e 16

th In

ternatio

nal Stu

den

ts Co

nferen

ce ldquoMo

dern

An

alytical Ch

emistryrdquo P

rague 2

02

0

788074 440793



Prague 2020

Proceedings of the











543(062534)

analyticalchemistry






ISBN978-80-7444-079-3

Preface


















science










editor


Sponsors



companies

wwwecomsrocom

wwwlach-nercom


wwwthermofishercz

www2thetacz


wwwshimadzueucom

wwwwaterscom

Contents



























1Introduction











metry




2Experimental



22Instrumentation










4Conclusions












References














1Introduction













2Experimental



22Instrumentation

221Capillaries
























25 00577 0000525+25 0057 000150+25 00775 0000775+25 ndash ndash25+50 0137 000550 0865 000250+50 080 00575+50 105 00625+75 0187 000250+75 1941 000375 456 00275+75 42 01








4Conclusions


Acknowledgments


References








1Introduction










2Experimental























4Conclusions






Acknowledgments


References













1Introduction











detection



2Experimental



22Instrumentation













































4Conclusions


Acknowledgments













1Introduction














2Experimental







22Instrumentation


















Tab

le 1


StrategyA

StrategyB

StrategyC

Support




Supportfunctio-

minusNH

minusNH

minusCOOH

22

nalizedgroup

Activationagent

Glutaraldehyde(GA)

Carbodiimide

Carbodiimide

(EDCNHS)

(EDCNHS)

Enzymereactive

minusNH

minusCOOH

minusNH

22

group

Typeofbond

secondaryamine

amide

amide

Denotationsofthe




23

preparedenzy-




23

maticpowders




23




23




23




23




4Conclusions




Acknowledgments


References

















1Introduction










detection


2Experimental






22Instrumentation










4Conclusions


References











1Introduction










2Experimental



22Instrumentation






24Solutions











4Conclusions







ndash120μgmL

Acknowledgments


References













1Introduction













2Experimental



22Instrumentation


23Samplepreparation







ndash1 A =81914c[gL ]+00357 (1)2802 R =1


ndash1 A =73824c[gL ]+00301 (2)3102 R =1




4Conclusions



λ nm

Absorban

ce







References







1Introduction











2Experimental





22Instrumentation


























4Conclusions




References














1Introduction











2Experimental







22Instrumentation















4Conclusions


References








Recovery 97 103



















1Introduction









microextraction


2Experimental

21Reagents


22Instrumentation


























4Conclusions




Acknowledgments





References









1Introduction








Keywordscopper(II)





spectroscopy


2Experimental







22Instrumentation























4Conclusions


Acknowledgments




References
















1Introduction














electrodeunithiol



2Experimental












22Instrumentation

























4Conclusions



Acknowledgments


References






















1Introduction










2 Experimental






22Instruments







ndash1001Vs )















4Conclusion


Acknowledgments


References








1Introduction










2Experimental





22Apparatus












24Procedures
























4Conclusions



Acknowledgments


References









1Introduction









generation





2Experimental















22Instrumentation







(A)

(B)























modifiers

4Conclusions





Acknowledgments


References












1Introduction











generation


2Experimental









22Instrumentation









23Samplepreparation





















Repeatability lt1 6



4Conclusion


Acknowledgments


References














1Introduction








electrophoresis





2Experimental



22Instrumentation


23Method















4Conclusions


Acknowledgments


References
















1Introduction












(A) (B)

(C) (D)

2Experimental



22Instrumentation










ndash1rate1Vs


ndash1rate1Vs



4Conclusions



Acknowledgments


References







1Introduction










2Experimental









24TheELISAprocedure






B ndash3timesSD0








usinganti-BSA-C6-RS












andalsoNARSSSMandTM

4Conclusions




References












1Introduction















2Experimental




22Instrumentation


























4Conclusions



Acknowledgments


References

















1Introduction













2Experimental




22Instrumentation







(1)











(2)












4Conclusions


Acknowledgments


References







1Introduction



abANTONKORBAN







quantification



2Experimental



22Instrumentation





















4Conclusions







Acknowledgments


References







1Introduction









2Experimental




22Instrumentation
















4Conclusions


Acknowledgments



References







1Introduction












syndrome














2Experimental




22Instrumentation


















4Conclusions



Acknowledgments


References












Author Index

AlikovaV1

AugustınM83

BaluchovaS19

BarekJ192570

BarochM141

BasB13129

BastryginaO41

BenesovaL146

BessonovaE57

BohmD6

BurkinK116

BurkinM116

ChernovaA141

ChoinskaM70

CokrtovaK104

DedinaJ97

DeevV57

DejmkovaH141

DendisovaM63

DubenskaL51

EfremenkoE41

FojtaM110

GalvidisI116

HavranL110

HeiglN31

HertJ76

HrdlickaV70

JosypcukB25

KartsovaLA3557

KloudaJ19146

Kodrık ovaB90

KolobovaEA35

KorbanA135

KorotkovaE1

KralM63

KratzerJ90

KravchenkoAV35

KrızekT76104

LipinskaJ129

MadejM129

MatejkaP63

MatysikF-M631

MusilS9097123

NavratilT70

OndrackovaA110

PietrzakK45

PlotnikovaK51

PoradaR13

RedondoBR70

SagapovaL90

ShormanovV1


SladkovaS 141

S tadlerovaB97

StiborovaM110

SvobodaM90

TvorynskaS25

TyczkowskiJ129

VyhnanovskyJ97123

VymyslickyF76

VyskocilV83

WardakC45

WongDKY19

ZarybnickaA146

ZelenyI51



Keyword Index


aminoglycosides116


amperometry141





metry97

atomization90


biosensor2583

bismuth97

borondopeddiamond

electrode110146

cadmium90

canagliflozin76

capillarycoating35



carbohydrates31

carbonfelt141



chemometrics57

cobalt123




cytochromeP450110

damage83






microextraction57


DNA83





electrochemistry51



linearscan70

ELISA116

enzymaticreactor25

FIA141146



glucoseoxidase25

graphite83

honey116

HPLC76141

hydridegeneration97


electrodes19



spectrometry123



laccase25

liquidcrystals104

massspectrometry31

mercuryelectrode13

metronidazole51


electrophoresis104

non-aqueoussystem6

oxidation76


phenol2-methoxy1


ration97123

polarography51


dimercapto-70


quantitation1





smokingmixtures41

solidcontact45



SudanI110



spectroscopy63

thinlayers129


63

unithiol70

uranyl45

vanillin41

veterinarydrug51

vitamins13


cation135


tion19

voltammetry1383





Prague2020


ISBN978-80-7444-079-3

ISBN 978-80-7444-079-3

Pro

ceedin

gs of th

e 16

th In

ternatio

nal Stu

den

ts Co

nferen

ce ldquoMo

dern

An

alytical Ch

emistryrdquo P

rague 2

02

0

788074 440793



Prague 2020

Proceedings of the



Preface


















science










editor


Sponsors



companies

wwwecomsrocom

wwwlach-nercom


wwwthermofishercz

www2thetacz


wwwshimadzueucom

wwwwaterscom

Contents



























1Introduction











metry




2Experimental



22Instrumentation










4Conclusions












References














1Introduction













2Experimental



22Instrumentation

221Capillaries
























25 00577 0000525+25 0057 000150+25 00775 0000775+25 ndash ndash25+50 0137 000550 0865 000250+50 080 00575+50 105 00625+75 0187 000250+75 1941 000375 456 00275+75 42 01








4Conclusions


Acknowledgments


References








1Introduction










2Experimental























4Conclusions






Acknowledgments


References













1Introduction











detection



2Experimental



22Instrumentation













































4Conclusions


Acknowledgments













1Introduction














2Experimental







22Instrumentation


















Tab

le 1


StrategyA

StrategyB

StrategyC

Support




Supportfunctio-

minusNH

minusNH

minusCOOH

22

nalizedgroup

Activationagent

Glutaraldehyde(GA)

Carbodiimide

Carbodiimide

(EDCNHS)

(EDCNHS)

Enzymereactive

minusNH

minusCOOH

minusNH

22

group

Typeofbond

secondaryamine

amide

amide

Denotationsofthe




23

preparedenzy-




23

maticpowders




23




23




23




23




4Conclusions




Acknowledgments


References

















1Introduction










detection


2Experimental






22Instrumentation










4Conclusions


References











1Introduction










2Experimental



22Instrumentation






24Solutions











4Conclusions







ndash120μgmL

Acknowledgments


References













1Introduction













2Experimental



22Instrumentation


23Samplepreparation







ndash1 A =81914c[gL ]+00357 (1)2802 R =1


ndash1 A =73824c[gL ]+00301 (2)3102 R =1




4Conclusions



λ nm

Absorban

ce







References







1Introduction











2Experimental





22Instrumentation


























4Conclusions




References














1Introduction











2Experimental







22Instrumentation















4Conclusions


References








Recovery 97 103



















1Introduction









microextraction


2Experimental

21Reagents


22Instrumentation


























4Conclusions




Acknowledgments





References









1Introduction








Keywordscopper(II)





spectroscopy


2Experimental







22Instrumentation























4Conclusions


Acknowledgments




References
















1Introduction














electrodeunithiol



2Experimental












22Instrumentation

























4Conclusions



Acknowledgments


References






















1Introduction










2 Experimental






22Instruments







ndash1001Vs )















4Conclusion


Acknowledgments


References








1Introduction










2Experimental





22Apparatus












24Procedures
























4Conclusions



Acknowledgments


References









1Introduction









generation





2Experimental















22Instrumentation







(A)

(B)























modifiers

4Conclusions





Acknowledgments


References












1Introduction











generation


2Experimental









22Instrumentation









23Samplepreparation





















Repeatability lt1 6



4Conclusion


Acknowledgments


References














1Introduction








electrophoresis





2Experimental



22Instrumentation


23Method















4Conclusions


Acknowledgments


References
















1Introduction












(A) (B)

(C) (D)

2Experimental



22Instrumentation










ndash1rate1Vs


ndash1rate1Vs



4Conclusions



Acknowledgments


References







1Introduction










2Experimental









24TheELISAprocedure






B ndash3timesSD0








usinganti-BSA-C6-RS












andalsoNARSSSMandTM

4Conclusions




References












1Introduction















2Experimental




22Instrumentation


























4Conclusions



Acknowledgments


References

















1Introduction













2Experimental




22Instrumentation







(1)











(2)












4Conclusions


Acknowledgments


References







1Introduction



abANTONKORBAN







quantification



2Experimental



22Instrumentation





















4Conclusions







Acknowledgments


References







1Introduction









2Experimental




22Instrumentation
















4Conclusions


Acknowledgments



References







1Introduction












syndrome














2Experimental




22Instrumentation


















4Conclusions



Acknowledgments


References












Author Index

AlikovaV1

AugustınM83

BaluchovaS19

BarekJ192570

BarochM141

BasB13129

BastryginaO41

BenesovaL146

BessonovaE57

BohmD6

BurkinK116

BurkinM116

ChernovaA141

ChoinskaM70

CokrtovaK104

DedinaJ97

DeevV57

DejmkovaH141

DendisovaM63

DubenskaL51

EfremenkoE41

FojtaM110

GalvidisI116

HavranL110

HeiglN31

HertJ76

HrdlickaV70

JosypcukB25

KartsovaLA3557

KloudaJ19146

Kodrık ovaB90

KolobovaEA35

KorbanA135

KorotkovaE1

KralM63

KratzerJ90

KravchenkoAV35

KrızekT76104

LipinskaJ129

MadejM129

MatejkaP63

MatysikF-M631

MusilS9097123

NavratilT70

OndrackovaA110

PietrzakK45

PlotnikovaK51

PoradaR13

RedondoBR70

SagapovaL90

ShormanovV1


SladkovaS 141

S tadlerovaB97

StiborovaM110

SvobodaM90

TvorynskaS25

TyczkowskiJ129

VyhnanovskyJ97123

VymyslickyF76

VyskocilV83

WardakC45

WongDKY19

ZarybnickaA146

ZelenyI51



Keyword Index


aminoglycosides116


amperometry141





metry97

atomization90


biosensor2583

bismuth97

borondopeddiamond

electrode110146

cadmium90

canagliflozin76

capillarycoating35



carbohydrates31

carbonfelt141



chemometrics57

cobalt123




cytochromeP450110

damage83






microextraction57


DNA83





electrochemistry51



linearscan70

ELISA116

enzymaticreactor25

FIA141146



glucoseoxidase25

graphite83

honey116

HPLC76141

hydridegeneration97


electrodes19



spectrometry123



laccase25

liquidcrystals104

massspectrometry31

mercuryelectrode13

metronidazole51


electrophoresis104

non-aqueoussystem6

oxidation76


phenol2-methoxy1


ration97123

polarography51


dimercapto-70


quantitation1





smokingmixtures41

solidcontact45



SudanI110



spectroscopy63

thinlayers129


63

unithiol70

uranyl45

vanillin41

veterinarydrug51

vitamins13


cation135


tion19

voltammetry1383





Prague2020


ISBN978-80-7444-079-3

ISBN 978-80-7444-079-3

Pro

ceedin

gs of th

e 16

th In

ternatio

nal Stu

den

ts Co

nferen

ce ldquoMo

dern

An

alytical Ch

emistryrdquo P

rague 2

02

0

788074 440793



Prague 2020

Proceedings of the



Sponsors



companies

wwwecomsrocom

wwwlach-nercom


wwwthermofishercz

www2thetacz


wwwshimadzueucom

wwwwaterscom

Contents



























1Introduction











metry




2Experimental



22Instrumentation










4Conclusions












References














1Introduction













2Experimental



22Instrumentation

221Capillaries
























25 00577 0000525+25 0057 000150+25 00775 0000775+25 ndash ndash25+50 0137 000550 0865 000250+50 080 00575+50 105 00625+75 0187 000250+75 1941 000375 456 00275+75 42 01








4Conclusions


Acknowledgments


References








1Introduction










2Experimental























4Conclusions






Acknowledgments


References













1Introduction











detection



2Experimental



22Instrumentation













































4Conclusions


Acknowledgments













1Introduction














2Experimental







22Instrumentation


















Tab

le 1


StrategyA

StrategyB

StrategyC

Support




Supportfunctio-

minusNH

minusNH

minusCOOH

22

nalizedgroup

Activationagent

Glutaraldehyde(GA)

Carbodiimide

Carbodiimide

(EDCNHS)

(EDCNHS)

Enzymereactive

minusNH

minusCOOH

minusNH

22

group

Typeofbond

secondaryamine

amide

amide

Denotationsofthe




23

preparedenzy-




23

maticpowders




23




23




23




23




4Conclusions




Acknowledgments


References

















1Introduction










detection


2Experimental






22Instrumentation










4Conclusions


References











1Introduction










2Experimental



22Instrumentation






24Solutions











4Conclusions







ndash120μgmL

Acknowledgments


References













1Introduction













2Experimental



22Instrumentation


23Samplepreparation







ndash1 A =81914c[gL ]+00357 (1)2802 R =1


ndash1 A =73824c[gL ]+00301 (2)3102 R =1




4Conclusions



λ nm

Absorban

ce







References







1Introduction











2Experimental





22Instrumentation


























4Conclusions




References














1Introduction











2Experimental







22Instrumentation















4Conclusions


References








Recovery 97 103



















1Introduction









microextraction


2Experimental

21Reagents


22Instrumentation


























4Conclusions




Acknowledgments





References









1Introduction








Keywordscopper(II)





spectroscopy


2Experimental







22Instrumentation























4Conclusions


Acknowledgments




References
















1Introduction














electrodeunithiol



2Experimental












22Instrumentation

























4Conclusions



Acknowledgments


References






















1Introduction










2 Experimental






22Instruments







ndash1001Vs )















4Conclusion


Acknowledgments


References








1Introduction










2Experimental





22Apparatus












24Procedures
























4Conclusions



Acknowledgments


References









1Introduction









generation





2Experimental















22Instrumentation







(A)

(B)























modifiers

4Conclusions





Acknowledgments


References












1Introduction











generation


2Experimental









22Instrumentation









23Samplepreparation





















Repeatability lt1 6



4Conclusion


Acknowledgments


References














1Introduction








electrophoresis





2Experimental



22Instrumentation


23Method















4Conclusions


Acknowledgments


References
















1Introduction












(A) (B)

(C) (D)

2Experimental



22Instrumentation










ndash1rate1Vs


ndash1rate1Vs



4Conclusions



Acknowledgments


References







1Introduction










2Experimental









24TheELISAprocedure






B ndash3timesSD0








usinganti-BSA-C6-RS












andalsoNARSSSMandTM

4Conclusions




References












1Introduction















2Experimental




22Instrumentation


























4Conclusions



Acknowledgments


References

















1Introduction













2Experimental




22Instrumentation







(1)











(2)












4Conclusions


Acknowledgments


References







1Introduction



abANTONKORBAN







quantification



2Experimental



22Instrumentation





















4Conclusions







Acknowledgments


References







1Introduction









2Experimental




22Instrumentation
















4Conclusions


Acknowledgments



References







1Introduction












syndrome














2Experimental




22Instrumentation


















4Conclusions



Acknowledgments


References












Author Index

AlikovaV1

AugustınM83

BaluchovaS19

BarekJ192570

BarochM141

BasB13129

BastryginaO41

BenesovaL146

BessonovaE57

BohmD6

BurkinK116

BurkinM116

ChernovaA141

ChoinskaM70

CokrtovaK104

DedinaJ97

DeevV57

DejmkovaH141

DendisovaM63

DubenskaL51

EfremenkoE41

FojtaM110

GalvidisI116

HavranL110

HeiglN31

HertJ76

HrdlickaV70

JosypcukB25

KartsovaLA3557

KloudaJ19146

Kodrık ovaB90

KolobovaEA35

KorbanA135

KorotkovaE1

KralM63

KratzerJ90

KravchenkoAV35

KrızekT76104

LipinskaJ129

MadejM129

MatejkaP63

MatysikF-M631

MusilS9097123

NavratilT70

OndrackovaA110

PietrzakK45

PlotnikovaK51

PoradaR13

RedondoBR70

SagapovaL90

ShormanovV1


SladkovaS 141

S tadlerovaB97

StiborovaM110

SvobodaM90

TvorynskaS25

TyczkowskiJ129

VyhnanovskyJ97123

VymyslickyF76

VyskocilV83

WardakC45

WongDKY19

ZarybnickaA146

ZelenyI51



Keyword Index


aminoglycosides116


amperometry141





metry97

atomization90


biosensor2583

bismuth97

borondopeddiamond

electrode110146

cadmium90

canagliflozin76

capillarycoating35



carbohydrates31

carbonfelt141



chemometrics57

cobalt123




cytochromeP450110

damage83






microextraction57


DNA83





electrochemistry51



linearscan70

ELISA116

enzymaticreactor25

FIA141146



glucoseoxidase25

graphite83

honey116

HPLC76141

hydridegeneration97


electrodes19



spectrometry123



laccase25

liquidcrystals104

massspectrometry31

mercuryelectrode13

metronidazole51


electrophoresis104

non-aqueoussystem6

oxidation76


phenol2-methoxy1


ration97123

polarography51


dimercapto-70


quantitation1





smokingmixtures41

solidcontact45



SudanI110



spectroscopy63

thinlayers129


63

unithiol70

uranyl45

vanillin41

veterinarydrug51

vitamins13


cation135


tion19

voltammetry1383





Prague2020


ISBN978-80-7444-079-3

ISBN 978-80-7444-079-3

Pro

ceedin

gs of th

e 16

th In

ternatio

nal Stu

den

ts Co

nferen

ce ldquoMo

dern

An

alytical Ch

emistryrdquo P

rague 2

02

0

788074 440793



Prague 2020

Proceedings of the



Contents



























1Introduction











metry




2Experimental



22Instrumentation










4Conclusions












References














1Introduction













2Experimental



22Instrumentation

221Capillaries
























25 00577 0000525+25 0057 000150+25 00775 0000775+25 ndash ndash25+50 0137 000550 0865 000250+50 080 00575+50 105 00625+75 0187 000250+75 1941 000375 456 00275+75 42 01








4Conclusions


Acknowledgments


References








1Introduction










2Experimental























4Conclusions






Acknowledgments


References













1Introduction











detection



2Experimental



22Instrumentation













































4Conclusions


Acknowledgments













1Introduction














2Experimental







22Instrumentation


















Tab

le 1


StrategyA

StrategyB

StrategyC

Support




Supportfunctio-

minusNH

minusNH

minusCOOH

22

nalizedgroup

Activationagent

Glutaraldehyde(GA)

Carbodiimide

Carbodiimide

(EDCNHS)

(EDCNHS)

Enzymereactive

minusNH

minusCOOH

minusNH

22

group

Typeofbond

secondaryamine

amide

amide

Denotationsofthe




23

preparedenzy-




23

maticpowders




23




23




23




23




4Conclusions




Acknowledgments


References

















1Introduction










detection


2Experimental






22Instrumentation










4Conclusions


References











1Introduction










2Experimental



22Instrumentation






24Solutions











4Conclusions







ndash120μgmL

Acknowledgments


References













1Introduction













2Experimental



22Instrumentation


23Samplepreparation







ndash1 A =81914c[gL ]+00357 (1)2802 R =1


ndash1 A =73824c[gL ]+00301 (2)3102 R =1




4Conclusions



λ nm

Absorban

ce







References







1Introduction











2Experimental





22Instrumentation


























4Conclusions




References














1Introduction











2Experimental







22Instrumentation















4Conclusions


References








Recovery 97 103



















1Introduction









microextraction


2Experimental

21Reagents


22Instrumentation


























4Conclusions




Acknowledgments





References









1Introduction








Keywordscopper(II)





spectroscopy


2Experimental







22Instrumentation























4Conclusions


Acknowledgments




References
















1Introduction














electrodeunithiol



2Experimental












22Instrumentation

























4Conclusions



Acknowledgments


References






















1Introduction










2 Experimental






22Instruments







ndash1001Vs )















4Conclusion


Acknowledgments


References








1Introduction










2Experimental





22Apparatus












24Procedures
























4Conclusions



Acknowledgments


References









1Introduction









generation





2Experimental















22Instrumentation







(A)

(B)























modifiers

4Conclusions





Acknowledgments


References












1Introduction











generation


2Experimental









22Instrumentation









23Samplepreparation





















Repeatability lt1 6



4Conclusion


Acknowledgments


References














1Introduction








electrophoresis





2Experimental



22Instrumentation


23Method















4Conclusions


Acknowledgments


References
















1Introduction












(A) (B)

(C) (D)

2Experimental



22Instrumentation










ndash1rate1Vs


ndash1rate1Vs



4Conclusions



Acknowledgments


References







1Introduction










2Experimental









24TheELISAprocedure






B ndash3timesSD0








usinganti-BSA-C6-RS












andalsoNARSSSMandTM

4Conclusions




References












1Introduction















2Experimental




22Instrumentation


























4Conclusions



Acknowledgments


References

















1Introduction













2Experimental




22Instrumentation







(1)











(2)












4Conclusions


Acknowledgments


References







1Introduction



abANTONKORBAN







quantification



2Experimental



22Instrumentation





















4Conclusions







Acknowledgments


References







1Introduction









2Experimental




22Instrumentation
















4Conclusions


Acknowledgments



References







1Introduction












syndrome














2Experimental




22Instrumentation


















4Conclusions



Acknowledgments


References












Author Index

AlikovaV1

AugustınM83

BaluchovaS19

BarekJ192570

BarochM141

BasB13129

BastryginaO41

BenesovaL146

BessonovaE57

BohmD6

BurkinK116

BurkinM116

ChernovaA141

ChoinskaM70

CokrtovaK104

DedinaJ97

DeevV57

DejmkovaH141

DendisovaM63

DubenskaL51

EfremenkoE41

FojtaM110

GalvidisI116

HavranL110

HeiglN31

HertJ76

HrdlickaV70

JosypcukB25

KartsovaLA3557

KloudaJ19146

Kodrık ovaB90

KolobovaEA35

KorbanA135

KorotkovaE1

KralM63

KratzerJ90

KravchenkoAV35

KrızekT76104

LipinskaJ129

MadejM129

MatejkaP63

MatysikF-M631

MusilS9097123

NavratilT70

OndrackovaA110

PietrzakK45

PlotnikovaK51

PoradaR13

RedondoBR70

SagapovaL90

ShormanovV1


SladkovaS 141

S tadlerovaB97

StiborovaM110

SvobodaM90

TvorynskaS25

TyczkowskiJ129

VyhnanovskyJ97123

VymyslickyF76

VyskocilV83

WardakC45

WongDKY19

ZarybnickaA146

ZelenyI51



Keyword Index


aminoglycosides116


amperometry141





metry97

atomization90


biosensor2583

bismuth97

borondopeddiamond

electrode110146

cadmium90

canagliflozin76

capillarycoating35



carbohydrates31

carbonfelt141



chemometrics57

cobalt123




cytochromeP450110

damage83






microextraction57


DNA83





electrochemistry51



linearscan70

ELISA116

enzymaticreactor25

FIA141146



glucoseoxidase25

graphite83

honey116

HPLC76141

hydridegeneration97


electrodes19



spectrometry123



laccase25

liquidcrystals104

massspectrometry31

mercuryelectrode13

metronidazole51


electrophoresis104

non-aqueoussystem6

oxidation76


phenol2-methoxy1


ration97123

polarography51


dimercapto-70


quantitation1





smokingmixtures41

solidcontact45



SudanI110



spectroscopy63

thinlayers129


63

unithiol70

uranyl45

vanillin41

veterinarydrug51

vitamins13


cation135


tion19

voltammetry1383





Prague2020


ISBN978-80-7444-079-3

ISBN 978-80-7444-079-3

Pro

ceedin

gs of th

e 16

th In

ternatio

nal Stu

den

ts Co

nferen

ce ldquoMo

dern

An

alytical Ch

emistryrdquo P

rague 2

02

0

788074 440793



Prague 2020

Proceedings of the



proceedings of the 16th international students conference

Documents