ANALELE UNIVERSITĂŢII BUCUREŞTI 2004

ANALELE UNIVERSITII

BUCURETI CHIMIE

2004

EDITORIAL BOARD

Editor-in-chief: Prof. Dr. Doc. Rodica VLCU

Executive Editor: Prof. Dr. Cristina MANDRAVEL

Members: Prof. Dr.

Prof. Dr.

Prof. Dr.

Prof. Dr.

Prof. Dr.

Elena CRISTUREAN

Cornelia CERCASOV

Vasile MAGEARU

Dumitru OANCEA

Ioan SNDULESCU

Editorial Scientific Secretary: Prof. Dr. Niculae POPA

Electronic Submission Secretary: Lect. Dr. Valentina CHIOSA

Techno-Redacting Reviser: Lect. Dr. Valentin MUNTEANU

EDITURA UNIVERSITII BUCURETI

os. Panduri nr. 90 Bucureti

Telefon 410.31.78/122

Copyright Analele Universitii din Bucureti 2004

Conducerea Universitatii din Bucuresti Omagiaza memoria Profesorului

Grigore Popa Umanist desavrsit

Om al scolii, devotat profesiunii sale si Creatorul primei catedre de Chimie Analitica

din Romnia

Rectorul Universitii din Bucureti Profesor Dr. Ioan Mihilescu

CUPRINS SOMMAIRE TABLE OF CONTENTS

ADEVRUL DESPRE UN MARE OM .............................................................................XIG.-E. Baiulescu.

OMAGIU LAUREATULUI NOBEL ILYA PRIGOGINE.............................................. XIII Rodica Vlcu.

ANALYTICAL CHEMISTRY 1. DEVELOPMENT OF A NEW ETHANOL BIOSENSOR WITH

ELECTROPOLYMERISED MELDOLA BLUE AS MEDIATOR .............................. 19 Camelia Bala, L. Rotariu, Alina Vasilescu and V. Magearu....................................19

2. DETERMINATION OF TRACE AMOUNTS OF MOLYBDENUM BY ADSORPTIVE STRIPPING VOLTAMMETRY USING MORIN AS CHELATING AGENT ................................................................. 27

Mihaela Buleandr, I.G. Tnase, M. Ionic and M.M. Buleandr ...........................27 3. DETERMINATION OF SULPHIDE BY POTENTIOMETRIC TITRATION............. 33

V. Dumitrescu, Nina Dumitrescu and Dana Anghel ................................................33 4. PARTICLE-INDUCED X-RAY EMISSION (PIXE): A TOOL FOR

MULTIELEMENT ANALYSIS IN THE YEAST CELLS ........................................... 39 Ileana Cornelia Farcasanu, F. Nishiyama and T. Miyakawa....................................39

5. ENZYMATIC MARKER FOR TOXICITY OF CONTAMINATED SOILS............... 45 S. Fleschin, A. Bunaciu and Mihaela Scripcariu......................................................45

6. ANIONS COMPLEXES DU PT(IV) EN ANALYSE ET CONTROLE DES MEDICAMENTS. DETERMINATION ANALYTIQUE DE LA PRENYLAMINE A LAIDE DE K2[PT (SCN)6] ....................................................................................... 49

I. Gnescu, G. Brtulescu, V. Magearu and Anca Gnescu.....................................49 7. ANIONS COMPLEXES DU PLATINE EN ANALYSE ET LE CONTROLE

DES MEDICAMENTS. DETERMINATION DE L'ANAFRANILE ........................... 57 I. Gnescu, G. Brtulescu, V. Magearu, I. Papa and Anca Gnescu........................57

8. DETERMINATION OF VITAMIN C FROM SOME NATURAL PRODUCTS PRESERVED UNDER DIFFERENT STORAGE CONDITIONS ............................... 65

Nicoleta Matei and V. Magearu ...............................................................................65

VI CUPRINS SOMMAIRE TABLE OF CONTENTS

9. PARAMETERS INFLUENCING THE RETENTION MECHANISM OF RALOXIFEN AND ITS RELATED IMPURITIES IN HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ................................... 69

A. Medvedovici, F. Albu and V. David ...................................................................69 10. FIRST DERIVATIVE SPECTROMETRY FOR DETERMINATION

OF A RUBBER ANTIOXIDANT ................................................................................. 79 Zenovia Moldovan and Laurenia Alexandrescu .....................................................79

11. DNA-BASED BIOSENSOR FOR DETECTION OF GENETICALLY-MODIFIED ORGANISMS......................................................... 85

A.G. Nica, M. Mascini and A.A. Ciucu ...................................................................85 12. THE SEPARATION AND PURIFICATION OF NEW COMPACT CONDENSED

HETEROCYCLIC SYSTEMS WITH THIAZOLIC RING BY HPLC......................... 95 Cristina Rdulescu, C.M. Trbanu, Ana Maria-Hossu, Ionica Ionit and V. Magearu...................................................................................95

13. FAST ENZYMATIC METHOD FOR ACETALDEHYDE DETERMINATION IN WINE QUALITY CONTROL................................................................................ 105

L. Rotariu, Adina Arvinte, Simona-Carmen Litescu and Camelia Bala ................105 14. ANALYTICAL CHARACTERIZATION OF STABILITY FOR SOME

FAT-SOLUBLE VITAMINS IN PHARMACEUTICAL PRODUCTS ...................... 111 Mihaela Scripcariu, Ana-Maria Hossu and V. Magearu ........................................111

15. OPTIMIZED MICROWAVE DIGESTION METHOD FOR IRON AND ZINC DETERMINATION BY FLAME ABSORPTION SPECTROMETRY IN FODDER YEASTS OBTAINED FROM PARAFFIN, METHANOL AND ETHANOL ........... 117

A. Tanase, A. Vamanu, Cornelia Niculae and C. Patroescu ..................................117 16. VOLTAMMETRIC BEHAVIOR OF ETHYLMERCURY

USING PLATINUM AND GLASSY CARBON ELECTRODES .............................. 125 I.G. Tnase, Florentina Panru (Soare), A. Ciucu .................................................125

17. DETERMINATION OF ARYLSULPHONYLBENZAMIDES RELATED PRODUCTS OF ARYLSULPHONYLTHIO-BENZAMIDES BY DERIVATIVE SPECTROMETRY....................................................................... 133

Luminia Vldescu, Florentina Bdu, Mariana Surmeian and Irinel Adriana Badea.........................................................133

INORGANIC CHEMISTRY 18. COMPLEXES WITH TRIAZOLE DERIVATIVES II.

MONONUCLEAR COMPLEXES OF Co(II) WITH 1-[METHYLAMINO-N-(O-TOLYL)]-1H-BENZOTRIAZOLE......... 139

Mihaela Badea, Rodica Olar, Dana Marinescu, Elena Cristurean, Lucia Ivan and Ana Emandi .....................................................139

CUPRINS SOMMAIRE TABLE OF CONTENTS VII

19. COMBINAISONS COMPLEXES DU NICKEL(II) AVEC SALICYLEALDHYDE BENZOTHIAZOLYL HYDRAZONE. SYNTHSE, CARACTRISATION ET PARAMTRISATION DU CHAMP DES LIGANDS.......................................... 147

Mirela Clinescu, Emilia Ion, D. Negoiu et Ana Emandi ......................................147 20. CONTROLLED SYNTHESIS II. REACTION OF Sn(IV) AND Zr(IV)

WITH ISATIN-3-TIOSEMICARBAZONE AND n-METIL ISATIN-3-TIOSEMICARBAZONE ........................................................... 155

Elena Cristurean, Carmen Prnu, Mihaela Badea and Rodica Olar .....................155 21. SYNTHESIS AND CHARACTERISATION OF SOME NEW METAL COMPLEXES

OF 3-NITROZO-4-HYDROXY-COUMARIN AND ANTRANILIC ACID.............. 161 Ana Emandi, Mirela Calinescu, Mihaela Badea and Rodica Olar .........................161

22. SYNTHESIS AND CHARACTERISATION OF SOME NEW COMPLEXES OF Ni(II), Cu(II), Zn(II), Cd(II) AND Hg(II) WITH MIXED LIGANDS (NITROGEN, OXIGEN) DONOR GROUPS.................. 167

Ana Emandi, Anca Nicolae, Mihaela Badea and Rodica Olar ...............................167 23. UREA AND N-SUBSTITUTED UREA DERIVATIVES COMPOUNDS OF IRON (II)

ARE SUITABLE INTERMEDIATES IN THE SYNTHESIS OF COMPLEXES MIMIKING THE NONHEME IRON-CONTAINING ENZIMES ............................. 173

Monica Ilis, Angela Kriza, Irinel Badea and Veronica Pop ...................................173 24. CONTROLLED SYNTHESIS I. REACTION OF Sn(IV) AND Zr(IV)

WITH SCHIFF BASES DERIVED FROM 1-H-INDOL-2,3-DIONE AND DIAMINES................................................................ 179

Angela Kriza, Carmen Prnu, N. Popa and T. Rou ............................................179 25. SYNTHESIS, SPECTROSCOPIC AND MAGNETIC CHARACTERISATION

OF SOME NICKEL(II) COMPLEXES WITH 2-AMINO-1,3,4-THIADIAZOLE-5-THIOL ............................................................... 185

Rodica Olar, Mihaela Badea, Nicolae Stanica, Elena Cristurean, Dana Marinescu and Ana Emandi .............................................185

ORGANIC CHEMISTRY 26. NEW PYRROLO[1,2-A][1,10]PHENANTHROLINE DERIVATIVES..................... 191

F. Dumitracu, C. Drghici, Loredana Barbu and Christina Zlaru.......................191 27. NEW SUBSTITUTED 2-(3,5-DIMETHYL-PYRAZOL-1-YL)- ACETANILIDES

WITH PHARMACOLOGICAL ACTIVITY............................................................... 197 Christina Zlaru, M. Iovu, F. Dumitracu, Elena Cristea and Isabela Trcomnicu197

PHYSICAL CHEMISTRY 28. VAPOUR PRESSURE EQUATIONS FOR 2-PHENOXYETHANOL

AND ACETYL- PHENOXYETHANOL .................................................................... 201 Zoica Cenuse and Cornelia Cercasov.....................................................................201

VIII CUPRINS SOMMAIRE TABLE OF CONTENTS

29. RESEARCH OVER SOME PHYSICAL-CHEMICAL PROCESSES TAKING PLACE IN THE SULPHOUROUS MINERAL WATERS FOR THE KNOWLEDGE OF THEIR QUALITIES ALTERATIONS OCCURRED IN TIME ........................... 207

Madalina Ciocan and Rodica Vlcu .......................................................................207 30. THE SOLUBILITY OF CO2 IN SOME C4 ALCOHOLS AT HIGH PRESSURES ... 213

I. Ginar and Daniela Bala .....................................................................................213 31. INVESTIGATION ON THE REAL STATE OF SOME RADIONUCLIDES

IN VERY DILUTED WATERY SOLUTIONS IN THE PRESENCE OF SOME COMPLEXING AGENTS......................................................................... 219

M. Iliescu, C. Jujea, C. Podina, P. Balea................................................................219 32. ACTUAL TRENDS IN COMPUTATIONAL TOXICOLOGY

I. USING PHYSICAL-CHEMICAL DATA TO EVALUATE THE ACUTE TOXICITY (LD50) .................................................... 229

Cristina Mandravel, Valentina Chiosa and Ioana Stanculescu...............................229 33. SYNTHESIS AND THERMODYNAMIC CHARACTERIZATION OF 4-(P-ETHYL-

N-PHENYL-ACETAMIDOXY)- 4-[P-METHYL-PHENYLAZO]BIPHENYL ....... 233 Viorica Meltzer, Gabriela Rau, Gabriela Iacobescu and Elena Pincu....................233

34. DI-ELECTRODE OF TRANSFER AFFECTED BY DIFFUSION AT ITIES EXPRESSIONS FOR mITIESg AN D

mi

IN POTENTIAL-DEPENDENT KINETIC CONSTANTS I. TRANSFER OF TWO IONS OF SAME CHARGE ACROSS ITIES ..................... 239

C. Mihailciuc and S. Lupu .....................................................................................239 35. CO2 EFFECT ON THE FLAME VELOCITY

IN GASEOUS FLAMMABLE MIXTURES. PRELIMINARY TESTS ON STOICHIOMETRIC ETHYLENE-AIR MIXTURE ............................................ 249

Codina Movileanu, Maria Mu, V. Munteanu, Domnina Rzu and D. Oancea...249 36. CONDITIONING OF ZEOLITE STONE FROM ZALAU.

PHYSICAL-CHEMICAL STUDIES........................................................................... 255 S. Neagoe, O. Pntea, Eva Trmbiau, Daniela Popovici, D. Bombo, Fnica Bacalum, Dorina Matei, C. Neagoe, T. Jugnaru and M. Hotinceanu .......255

37. COPPER (II) CATALYZED OXIDATION OF 2-AMINOPHENOL IN AQUEOUS MEDIUM............................................................................................ 263

Mihaela Puiu and D. Oancea..................................................................................263 38. NEW THERMODYNAMIC STATE FUNCTIONS ................................................... 269

Andrei Rotaru.........................................................................................................269 39. MODELS OF OSCILLATING CHEMICAL REACTIONS.

PARTICULARITIES OF SOME PROPOSED MODELS FOR THE CHARACTERIZATION OF CHEMICAL OSCILLATIONS................... 277

Rodica Vlcu and Daniela Bala ..............................................................................277

CUPRINS SOMMAIRE TABLE OF CONTENTS IX

TECHNOLOGICAL CHEMISTRY AND CATALYSIS 40. ETUDE DE LA DESHYDROGENATION OXYDANTE DU n-BUTANE

SUR LE PYROPHOSPHATE DE TITANYLE, (TIO)2P2O7 .......................................... 287 I.-C. Marcu et I. Sndulescu ..................................................................................287

41. HYDROGENATION REVIVAL OF THE PAREX REAGENT ................................ 293 G. Brebeanu, S. Neagoe and C. Savu.....................................................................293

42. INVESTIGATION OF THE ADSORPTION CAPACITY OF THE ZEOLITE STONE FROM ZALAU .............................................................. 297

S. Neagoe, O. Pntea, Eva Trmbiau, Fnica Bacalum, Mihaela Bombo, V. Dumitrescu, D. Bombo, Lucia Antonescu, Alexandra Schiopescu, G. Brebeanu,C. Neagoe and I. Clp .......................................................................297

43. THE SIMULATION OF MULTIPLE EXTRACTION IN COUNTER CURRENT... 303 T.-M. Sturzu, and I.-C. Marcu ...............................................................................303

PhD THESIS IN CHEMISTIY PRESENTED IN 2003 AT THE BUCHAREST UNIVERSITY............................................................................ 309

SCIENTIFIC EVENTS OF THE YEAR 2003 .................................................................. 311

TEACHING NEWS........................................................................................................... 315

NOTE TO THE AUTHORS.............................................................................................. 317

ADEVRUL DESPRE UN MARE OM

Prin amabilitatea doamnei profesor Rodica Vlcu am fost invitat s prefaez volumul omagial dedicat profesorului Grigore Popa.

Profesorul Grigore Popa sa nscut n 1913 ntrun sat din preajma Cheilor Turzii, fapt care ia inoculat ntreaga via pasiunea pentru munte. A obinut titlul de doctor la Universitatea din Cluj n domeniul combinaiilor complexe cu urotropin, sub conducerea academicienei Raluca Ripan.

Lam cunoscut pe profesorul Popa n anul 1950, fiind student n anul I i asistnd la cursul de chimie anorganic predat de dnsul. n acea perioad a fost i secretarul general al Academiei. Din 1958 am nceput s colaborm n cercetarea tiinific formnd un trio mpreun cu profesorul Negoiu. Profesorul Popa ma ajutat s m transfer din funcia de cercettor n cadrul Academiei la Universitatea din Bucureti, la Catedra de Chimie Anorganic i Analitic.

Grigore Popa profesor

(1913-1990)

n 1961 a nfiinat Catedra de Chimie Analitic, a Facultii de Chimie, prima Catedr de acest fel din nvmntul superior de chimie. Ulterior, a fost creat Catedra de Chimie Analitic a Universitii din Cluj, precum i cea de la Universitatea din Iai. Ca iniiator al catedrei de Chimie Analitic a reuit s conduc o echip de cercetare i didactic de mare valoare, care a contribuit, n mare msur, la dezvoltarea acestei discipline n Romnia. A avut o serie de funcii de conducere, cum ar fi cea de decan al Facultii de Chimie, precum i director general al Institutului de Cercetri Chimice. A fcut tot posibilul s doteze Catedra de Chimie Analitic cu aparatur de cercetare de bun performan, fapt care i-a ridicat prestigiul, dar totodat sau creat o serie de invidii. Fiind n primul rnd un om cu o inut moral deosebit sa nconjurat de o serie de

XII G.-E. BAIULESCU

prieteni i colaboratori de diverse vrste, dar de foarte bun calitate. Este necesar s menionm prietenia dnsului cu Constantin Teodosiu, unul din cei mai buni colaboratori ai regretatului profesor Longinescu, precum i cu toi membrii echipei, printre care menionm pe: Croitoru Vasilica, Ptroescu Constantin, Paralescu Ion, Cruceru Dumitru, Dimonie Elena, Lerch Rudolf, Enea Octavian, Ciurea Constantin, Moldoveanu erban, Magearu Vasile, Dumitrescu Valeric, Luminia Vldescu, dar i cu cercettori din afara catedrei, Todor Dumitru, Tiberiu Ncuiu, i muli alii.

Odat cu unirea Facultii de Chimie cu Facultatea de Chimie Industrial n aa-numitul Institutul Naional de Chimie, sa pensionat nainte de vreme i dei a dorit s lase la conducerea catedrei de Chimie Analitic pe profesor Croitoru Vasilica, conducerea Ministerului nvaamntului din acea perioad, influenat de doi carieriti, din pcate din Catedra de Chimie Analitic, a fost mpotriv, iar din acel moment profesorului Popa, dei a fost iniatorul Catedrei de Chimie Analitic nu i sa asigurat nici un spaiu pentru scopuri didactice i de cercetare.

Profesorul Grigore Popa a fost n primul rnd un mare om i iubitor de munte. Dup o munc n laborator, la care dnsul participa n mod substanial, ne invita pe toi n excursii, plcndu-i n mod deosebit Munii Ciuca.

Dei a crezut ntro societate mai bun, evoluia evenimentelor din Romnia l-a decepionat i la vrsta de 77 de ani a decedat lsnd n urm amintiri frumoase. Cu ocazia a 10 ani de la decesul profesorului Popa, Societatea de Chimie Analitic din Romnia a inut o edin festiv la care au participat cadre didactice de la diverse catedre, iar n final, i s-au dedicat o serie de proiecii cu imagini din munii lumii, prezentate de cel mai mare alpinist romn al tuturor timpurilor, Constantin Lctuu.

Facultatea de Chimie recunoscnd meritele de OM n primul rnd ale profesorului Grigore Popa, i-a dedicat un amfiteatru, care-i poart numele si care pentru chimitii analiti, membrii ai Societii de Chimie Analitic, reprezint un sanctuar al activitii didactice i tiinifice.

George-Emil Baiulescu

OMAGIU LAUREATULUI NOBEL ILYA PRIGOGINE

Dup o via consacrat unor realizri de nalt profesionalism Ilya Prigogine, ilustrul profesor i creatorul unor instituii i coli de cercetare durabile, a trecut n eternitate la 28 mai 2003. Viaa sa s-a stins, dar aureola operei sale monumentale nu se va risipi n neant: rmn posteritii creaii cu rezultate surprinztoare, idei de valoare, deschiztoare de orizonturi noi.

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Ilya Prigogine Ilya Prigogine s-a nscut la Moscova, cu cteva luni naintea revoluiei din ctombrie 1917, urmat de proclamarea Republicii Socialiste Federative Sovietice. a nceputul vieii sale a avut un destin neobinuit: viaa familiei Prigogine a fost arcat de naionalizarea fabricii tatlui su, inginer chimist, nevoit s emigreze n ermania. Copilul Ilya avea patru ani, iar fratele su Alexandru era mai mare. up civa ani de cutri, familia s-a mutat n Belgia. In 1929 Ilya i-a finalizat

tudiile secundare, dar dei avea nclinaii spre domeniul umanist i reale aptitudini uzicale, la sfatul tatlui su i a fratelui mai mare, devenit chimist, s-a nscris la acultatea de Stiine, profilat pe chimie i fizic, din Universitatea Liber din ruxelles. In 1939 era liceniat, iar doi ani mai trziu a obinut titlul de Doctor n tiinele Chimice.

Dup al doilea rzboi mondial, s-a ndreptat spre cariera universitar, la niversitatea Liber din Bruxelles (ULB) i a optat pentru studiul termodinamicii

himice, domeniu pe ct de atractiv, pe att de deschis nnoirilor. In anul 1949 a devenit cetean belgian. Dup o perioad de cca. 10 ani,

nterval n care s-a impus n lumea tiinific prin valoarea contribuiilor sale i a nui temperament activ, I. Prigogine nu a ncetat s-i extind sfera cunoaterii n omeniul tiinelor exacte, manifestnd i caliti manageriale. In anul 1959 a reluat conducerea Institutului Internaional de Fizic i Chimie Solvay care usese fondat n 1911, de acest mare industria, mpreun cu profesorii Nernst i orenty. Prigogine a rmas directorul acestui institut pn la sfritul vieii.

XIV R. VLCU

Din iniiativa sa a fost nfiinat n anul 1967, un centru de studii, prin colaborarea facultilor profilate pe disciplinele fizico-chimice din ULB i Universitatea Austin din Texas. Prigogine a predat consecvent, o perioad ndelungat studenilor din noua instituie, denumit Ilya Prigogine Center for Studies in Statistical Mechanics and Complex Systems. Institutul a devenit Centru de excelen i principalul nod dintr-o reea de institute similare, infiinate n mai multe orae din lume.

I. Prigogine a lsat o imens oper tiinific de valoare, mbogind tiina cu realizri care i vor reprezenta geniul, peste generaii. Din coninutul ei nu lipsesc tratate de termodinamic chimic de echilibru i ireversibil, mecanica statistic n formulri clasice i de neechilibru, numeroase monografii, unele cu implicaii din filozofia tiinelor, cca.1000 de articole tiinifice, la care se adaug variate conferine prezentate ocazional, adesea n medii academice sau la reuniuni sociale. Chiar din interviurile acordate, adesea cu privire la perspectivele tiinei, reiese gndirea sa spontan i flexibil. De la nceputul carierei sale, maiestrul a manifestat o deosebit predilecie pentru termodinamic, ramur fundamental a tiinelor naturii, cu rezultate concludente, dar limitate un timp la studiul sistemelor n echilibru, sau cel mult, n vecintatea echilibrului.

Primele noiuni de ireversibilitate, s-au introdus prin ingeniozitatea i intuiia lui Clausius care a definit entropia ca funcie de stare, stabilind printr-o teorem o nou form de enun al principiului II, din care decurg, n anumite condiii, criteriile de echilibru sau de evoluie ale sistemelor. Studii ulterioare asupra variaiei locale de entropie cu timpul, n sisteme neuniforme, au condus la corelarea cldurii necompensate cu afinitatea chimic. Pe aceast direcie s-au nscris lucrrile ilustrului fizician Thophile de Donder, autorul definirii termodinamice a afinitii chimice. Prigogine i colaboratorii au iniiat i au desvoltat termodinamica neliniar, departe de echilibru, un principiu general de evoluie adaptat studiului reaciilor chimice.

Inainte de contribuia capital a carierei sale tiinifice, maiestrul Prigogine s-a preocupat intens, mpreun cu un grup de elit dintre colaboratori, de fundamentarea mecanicii statistice n condiii de neechilibru, formulnd previziuni asupra comportrii unor corpuri departe de starea de echilibru. Au urmat, ulterior, verificrile practice ale teoriei propuse. Experimentele au fost descrise pentru starea de gaz, solide i plasm, departe de echilibru, cnd s-a constatat apariia unui fenomen necunoscut, ieit din comun, la creterea etapizat a distanelor fa de starea de echilibru. Fenomenul s-a reprodus la repetarea experimentelor. La creterea distanei fa de poziia de echilibru peste un anumit prag critic, a aprut o bifurcaie, iar prin depirea pragului, sistemul i-a regsito nou ordine stabilizat de fluxul disipativ de energie,care l-a meninut n noua stare. In acea stare au aprut structuri cristaline sau complexe denumite structuri disipative. Prin aceast confirmare experimental a teoriei s-a putut constata c un sistem care ar

OMAGIU LAUREATULUI NOBEL ILYA PRIGOGINE XV

trebui s se degradeze n haos, conform legilor termodinamicii, poate deveni mai complex prin schimb de energie cu mediul. Prigogine a explicat prin modele matematice, modul de producere al structurilor complexe n sistemele departe de echilibru, care au fost obinute experimental. Aceste cercetri au deschis calea unui nou domeniu tiinific denumit Complexitate, aplicabil n descifrarea celor mai variate fenomene dinamice, aparent fr nicio legtur ntre ele. Asemenea sructuri pot fi reprezentate de aproapre tot ce ne nconjoar: o celul vie, creierul, traficul automobilelor pe o autostrad, desvoltarea unui ora, o colonie de furnici, reeaua de internet etc. Toate aceste exemplificri au totui un suport comun: se pot reduce la o multitudine de elemente heterogene, care interacioneaz dup anumite reguli.

In 1977, Comitetul Nobel a distins importantele realizri ale savantrului I. Prigogine din termodinamica de neechilibru cu Premiul Nobel n Chimie, cu meniunea special, pentru teoria structurilor disipative. Aceast distincie a marcat apogeul creaiilor sale tiinifice.

Preocuprile sale tiinifice au avut impact i n domeniul reaciilor oscilante produse n zonele proceselor irevesibile neliniare. Nu a prelucrat ns acest domeniu.

Intr-o perioad care a anticipat celebra sa descoperire, unul din domeniile reprezentative de cercetare din catedra profesorului I. Prigogine (Service de Chimie physique II), era consacrat tratrilor statistice ale soluiilor de neelectrolii. Impreun cu doi dintre principalii si colaboratori, A. Bellemans i V. Mathot, specializai n acest domeniu, a publicat n anul 1957 tratatul The thermodynamic theory of solutions. In aceast conjunctur am nceput stagiul post-doctoral de specializare, n anul universitar 1966/1967, la catedra Profesorului I.Prigogine. Datorez iniiativa realizrii acestei specializri, Academicianului I.G. Murgulescu, eful meu de catedr, recunoscut ca un Mecena al timpurilor noi.

Am ajuns cu bine la destinaie, la catedra Profesorului I. Prigogone, o unitate de elit, de nalt nivel profesional, cu o orientare teoretic, ncadrat cu un numr mare de tineri, chimiti i fizico-chimiti, atent selecionai, dup pregtirea profesional, potenialul lor intelectual i atitudinea etic. Personalul calificat al catedrei nu depea, n medie 35 de ani, iar eful departamentului se apropia de vrsta de 50 de ani. Climatul de lucru dominat de concentrare i seriozitate, asigura fiecrui membru al colectivului linitea i echilibrul att de necesare creaiilor tiinifice.

Cu entuziasmul i energia vrstei m-am ncadrat, n scurt timp, n marele colectiv, unde am fost ntmpinat cu colegialitate i simpatie. La ntoarcerea Profesorului din Statele Unite, la scurt timp dup sosirea mea, i-am fost prezentat de A. Bellemans. Profesorul mi-a demonstrat solicitudine i dup o ntrevedere deosebit de util pentru activitatea mea de cercetare, a expus principalele orientri ale studiilor din catedr, informndu-se asupra propriilor mele preocupri n

XVI R. VLCU

cercetare. I-am prezentat extrase publicate n Z.phys.Chem.(Leipzig) i n J.Chem.Phys.(Chicago), toate din temodinamica soluiilor, obinute experimental sau prin modelarea lor pe bazele termodinamicii statistice.

Mulumit de relatarea mea, m-a ncadrat n sectorul de cercetare teoretic al soluiilor.

Catedra nu dispunea de domenii experimentale, n general, i cu att mai puin pentru soluii. Am fost mulumit de aceast decizie, tiind ce nseamn o cercetare practic i am nceput a doua zi lucrul, n primul rnd documentarea. Omul cel mai orientat n domeniu era A.Bellemans care m-a gsit a doua zi la bibleotec, s-a bucurat c vom colabora i mi-a spus cam ce este util s fac: un program de cercetare, pe care s-l aprobe le Patron adic Prigogine. Peste un timp de documentare, am conceput acest program, care a fost aprobat. Partea grea ncepea de abia atunci! Mi-am impus un program de lucru concentrat, de apte ore zilnic, ntrerupt de cteva ore de asisten la cursul de Termodinamica proceselor ireversibile, pn cnd Profesorul a decis continuarea lui n fiecare sptmn, de cte un alt colaborator.

Colectivele de catedr se ineau cu regularitate de dou ori pe lun. Programul obinuit consta din cte-o conferin cu profesori invitai din ri limitrofe, urmate de 2-3 comunicri ale membrilor din colectiv.Aceste ntlniri se numeau ceaiuri (servite la propriu), fiind organizate dup programul zilnic, ncepnd cu ora 17, iar sala avea aspectul unui salon de recepie. Amfitrionul nu lipsea niciodat: era inima acestor ntlniri i orienta discuiile.

Ajunsesem n faza de a comunica, periodic, etape ncheiate din lucrarea mea, la nceput cu emoie evident. De fiecare dat Profesorul era mulumit iar eu cu o piatr mai puin pe inim. Dup ultima comunicare, Profesorul mi-a sugerat ca din referatele comunicate, s constitui dou lucrri avnd n vedere c din coninut reiese o problem cu particulariti diferite, ceeace era exact. Conlucrnd cu Bellemans, am organizat coninutul textelor celor dou lucrri independente, care au fost prezentate la Societatea de Chimie din Belgia i publicate, ulterior, n dou numere succesive ale periodicului Bull.Soc.Chim.Belges, aprute n sistem de urgen n 1967:

Application of the average potential model to simple liquid mixtures (autori A.Bellemans i R.Vlcu);

Extension of the average potential model of solutions to systems submitted to a moderate pressure (autori R.Vlcu i A.Bellemans). Am privit aceste lucrri ca un trofeu olimpic, tiind c Profesorul Murgulescu se va bucura, la ntoarcerea mea acas, de rezultatul obinut n timpul specializrii.

In ultimele dou sptmni ale stagiului, am rspuns, cu plcere, dezideratului lui A. Bellemans de a aduce la zi bibliografia teoriilor despre soluiile

OMAGIU LAUREATULUI NOBEL ILYA PRIGOGINE XVII

de neelectrolii, ncepnd din 1957, data apariiei primei cri, n vederea reeditrii ei actulizate.

In ajunul plecrii n ar Profesorul a organizat o cin n familia sa, la care am fost invitat mpreun cu soul meu, venit la Bruxelles ca s ne ntoarcem n ar cu cteva escale, n trecere, prin orae europene. Primirea i ntreaga sear, ne-a lsat o amintire deosebit de plcut. Inainte de plecare, Profesorul mi-a adresat invitaia de a reveni la catedr i chiar de a rmne sine die. Pe atunci aceast posibilitate era exclus, i nici eu n-a fi fcut acest pas. Legtura puternic cu familia mea i nostalgia pmntului natal au anihilat cele mai tentante oferte! Am pstrat mult timp legtura, n scris, cu colegii care mi-au organizat la desprire n sala de ceai o sear de au revoir!.

Dup o via activ n cercetarea de vrf, I. Prigogine a remarcat multiplele necunoscute i aproximri din tiinele fundamentale, recomandnd s se aib n vedere reevaluarea i/sau actualizarea unor asemenea situaii.Pe lng bogata sa contribuie la deschiderea i consolidarea unor ci noi de studiu n tiinele fizico-chimice, Prigogine a avut tentaia, fireasc pentru un savant, de a investiga fenomene intangibile, situate dincolo de limitele cunoaterii contemporane. Colaborarea sa cu Isabelle Stengers, specialist n domeniul filozofiei tiinelor, i-a favorizat publicarea unor reflexii asupra crora planau incertitudini Monografiile publicate, au aprut n intervalul dintre anii 1979 i 1996, cu urmtoarele titluri: Physique, temps et devenir; La nouvelle alliance; Les lois du chaos; La fin des certitudes;Lhomme devant lincertain. Fiecare dintre aceste monografii, conine idei pertinente, cu tent filozofic.Voi cita cteva dintre ele, urmate de unele puncte de vedere ale autorului i al unor deziderate de perspectiv, precum:

Universul este guvernat de legi statistice; Stiina este un dialog cu natura; Timpul este o dimensiune fundamental a universului nostru; Timpul precede existena; Dac universul a avut un nceput timpul nu l-a avut; Dac timpul intervine n mod dinamic n evoluie, viitorul nu este dat de prezent i el nu este determinat; Viaa a aprut printr-o succesiune de instabiliti etc.

In fiecare dintre monografiile publicate apare o bogie de date din sfera tiinelor fundamentale clasice, pe lng meniuni asupra nnoirilor necesare, cu un accent special pe tendinele de trecere de la legile deterministe spre un univers nou, deschis fluctuaiilor i inovaiilor: Muli dintre marii fondatori ai tiinelor clasice ar trebue s ne permit s depim lumea aparenelor pentru a atinge lumea raional - scria Prigogine. Secolul nostru este o perioad de experimentri: au aprut forme noi de manifestare n art, muzic, literatur i n tiin. Acum, la sfritul secolului XX, nu putem prezice nc, cu certitudine, ncotro ne va conduce acest capitol nou al istoriei omenirii. Dar ceea ce este sigur, ncepnd de astzi, este instaurarea unui nou dialog ntre natur i om! Aceleai idei, dar mai complet conturate, se desprind i din prefaa scris de maiestrul I. Prigogine, la cartea Termodinamica proceselor ireversibile, pe care am publicat-o n colaborare cu

XVIII R. VLCU

A. Dobrescu, n Editura Tehnic, Bucureti, n 1982. Citez din expunerea maiestrului un scurt pasaj: Schimbarea de concepie n orientarea cercetrilor spre procesele ireversibile i experienele cu rezultate inedite n acest domeniu, explic interesul unei pri considerabile a lumii tiinifice; printre acestea. termodinamica, un domeniu clasic pn nu demult, este aplicat astzi ntr-un mod nou.Ideile de neliniaritate, instabilitate i fluctuaii, ptrund ntr-o zon din ce n ce mai extins a tiinei

Opera tiinific a maiestrului I. Prigogine caracterizat prin profunzime i diversitate, nu l-a consacrat numai ca un ilustru chimist; remarcabila sa competen n fizic, chimia fizic, n biochimie i chiar n filozofia tiintelor, este nc un mod de afirmare a personalitii sale! Membru al Academiei Regale de Stiine, Litere i Arte frumoase din Belgia, precum i din alte ri i spre satisfacia rii noastre, Membru al Academiei Romne (din 1965 n calitatea de m.c. iar din 1992, membru de onoare), Ilya Prigogine a vizitat n mai multe rnduri ara n scopuri tiinifice.

In afara Premiului Nobel (1977) i s-au atribuit premii n Belgia (1955 i 1965) i din partea unor fundaii din Tokio (1973), Quebec (1993), Rusia (1996) .a. Peste 50 de Universiti din lume i-au decernat titlul de Doctor Honoris Causa, dar lista distinciilor obinute nu se oprete aici.

In anul 1989, Regele Belgiei i-a acordat titlul nobiliar de Viconte, demonstrndu-i deplina sa apreciere pentru onoarea pe care a adus-o rii.

I. Prigogine a vizitat n 1994 Romnia satisfcndu-i pe lng programul tiinific o veche pasiune pentru istorie i arheologie, ajungnd de la Universitatea din Iai, unde fusese invitat, la situl arheologic din Cucuteni. Intors la Bucureti, a expus o conferin la Academia Romn din consideraiile sale vizionare ale perspectivelor n tiin.

Enormul regret al pierderii omului i savantului Ilya Prigogine a marcat generaiile care l-au urmat i care i-au cunoscut opera. Inconjurat cu stim i preuire n nobila sa misiune, Ilya Prigogine va rmne profesorul care a creiat tiin i care a deschis larg orizotul spre tiina viitorului. Alturi de elevii i colaboratorii si, mi exprim gratitudinea pentru condiiile de studiu pe care mi le-a asigurat cu generozitate n departamentul su, ntr-un climat de nalt nivel tiinific.

Rodica Vlcu

Analele Universitii din Bucureti Chimie, Anul XIII (serie nou), vol. I-II, pag. 1925

Copyright 2004 Analele Universitii din Bucureti

DEVELOPMENT OF A NEW ETHANOL BIOSENSOR WITH ELECTROPOLYMERISED MELDOLA BLUE AS MEDIATOR

Camelia Bala , L. Rotariu, Alina Vasilescu and V. Magearu abstract: An amperometric biosensor for ethanol was developed based on immobilization of alcohol dehydrogenase on screen-printed electrodes modified with electropolymerised Meldola Blue carried out by cyclic voltammetry in the range from 0.6 to +1.4 V vs. Ag/AgCl. The calibration plots for ethanol in 0.1 mol.L-1 phosphate buffer pH 8.5 containing 0.1 M KCl shows linearity in the range from 0.1x10-3 to 25x10-3 mol.L-1 ethanol The biosensor showed no decrease in sensitivity after 5 hours of continuous use..

Introduction

The measurement of alcoholic compounds, particularly of ethanol, plays an important role in the quality control of alcoholic beverages such as beer, wines and spirits. A variety of methods had been reported for the determination of this analyte, such as spectrophotometry [1], gas chromatography [2], liquid chromatography with amperometric detection [3,4]. These methods are expensive, slow, need well trained operators and in some cases, require steps of extraction or sample pretreatment, increasing the time of analysis. The food and drink industries need rapid methods to determine compounds of interest [5].

An alternative to facilitate the analysis in routine of industrial products is the biosensors development. Some advantages of this promising tool for food analysis are high selectivity and specificity, relative low cost of construction and storage, potential for miniaturization, facility of automation and simple and portable equipment construction for a fast analysis and monitoring. [6].

In the case of ethanol a number of enzymes based electrochemical devices have been developed either with alcohol oxidase [4,7-9] or alcohol dehydrogenase [10,11]. In the case of biosensors for ethanol based on alcohol oxidase the oxygen consumption or hydrogen peroxide production is monitored [8,12,13]. The alcohol dehydrogenase has the advantage to be more stable and more specific for ethanol but also the disadvantage to be dependent on the coenzyme NAD+ which has to be added to the assay. Additionally, electrochemical detection of NADH requires overpotential of about 1 V for oxidation and at this potential a number of other substances present in food samples, is also oxidized and can interfere in

Laboratory of Quality Control & Process Monitoring, Department of Analytical Chemistry,

Faculty of Chemistry, University of Bucharest, 90 Sos. Panduri, 050663 Bucharest-5, Romania; e-mail: [email protected]; fax: +40 21 4104888

20 C. BALA L. ROTARIU A. VASILESCU V. MAGEARU

the measurement. This disadvantage may be overcome by adding a mediator in order to improve the electron transfer kinetics and lower the applied electrode potential and effectively regenerate NAD+ [14]. Biosensors based on quinones [15], oil-soluble mediators based on phenothiazinium chloride [16], phenoxazines [17], organic dyes [18-21]

In last years the NADH detection based on screen-printed electrodes has attracted growing interest of researchers as these electrodes can be mass-produced at low cost making them suitable for commercial purposes. Immobilization of mediators on screen-printed electrodes was successfully conducted by different way: inclusion in different matrices at the electrode surface [12,22], inclusion of the mediator in the screen-printing ink [20,21] or by electrochemical polymerisation [23-25].

We have chosen as electron transfer mediator, Meldola Blue (7-dimethylamino-1,2-benzophenoxazine), having a fast rate of electron transfer with NADH. This mediator allows to achieve high sensitivity for the amperometric determination of NADH and to detect as low as 2x10-6 mol.L1 [25], with good selectivity since the measurements could be made in an ideal potential window (from 0 V to 0.200 V vs. SCE) where electrochemical interferences are minimal.

In order to improve the stability of the mediator modified electrodes and to avoid the mediator leakage, different strategies were reported [26-28]. The characteristics of poly(MB) screen printed sensors are superior to those previously reported for detectors based on the same screen-printed graphite electrodes containing Meldola Blue-Reinecke salt [28]. In terms of sensitivity, the poly(MB) sensors [31] are superior to the other detectors already reported based on the same mediator [32]. The NADH detection limit attained by screen-printed electrodes modified by the electropolymerisation of Meldola Blue is close to the best value reported for screen-printed sensors (2.0x106 mol.L1) [21].

In this paper an amperometric biosensor for ethanol was developed based on immobilization of alcohol dehydrogenase on screen-printed electrodes with electropolymerised Meldola Blue carried out by cyclic voltammetry in the range from 0.6 to +1.4 V vs. Ag/AgCl. The characteristics of screen-printed electrodes with poly Meldola Blue were already reported. [25,29].

Experimental

The enzyme alcohol dehydrogenase ADH (EC 1.1.1.1.) from bakers yeast 264 U/mg solid, -nicotinamide adenine dinucleotide, in its oxidized form of NAD+ and reduced form NADH, ethanol were purchased from Sigma Chemical Co. Meldola Blue was from Aldrich.

The supporting electrolytes used in this work were Srensen 0.1 mol.L1 phosphate buffers pH 8.5. In amperometry determination the buffer contained also 0.1 mol/L KCl in order to insure proper functioning of the screen-printing pseudo Ag/AgCl reference. All NADH solutions were prepared right before use. Screen-printed graphite electrodes with a geometric area of 0.17 cm2 were fabricated at University of Perpignan, France according to a previously described procedure [28] and were kindly provided by Prof. Jean-Louis Marty.

All the experiments were carried out with a BAS 100B/W Electrochemical Workstation (BioAnalytical System Inc., West Lafayette, USA). Data display and recording were

DEVELOPMENT OF A NEW ETHANOL BIOSENSOR 21

supported by BAS electrochemical software version 3.2. In CV experiments the reference was an Ag/AgCl (3M KCl, BAS) electrode while a platinum wire was used as counter electrode, all BAS, USA.

Biosensor preparation Sensors were prepared by electrochemical polymerization of Meldola Blue (MB) on screen-printed graphite electrodes. After film deposition, the electrodes were rinsed with distilled water and kept dry at room temperature until use. Immobilization of alcohol dehydrogenase was achieved by cross-linking with glutaraldehyde by depositing 3L of a mixture containing 20 IU/L ADH, 1% (m/v) bovine serum albumin and 0.5% (m/v) glutaraldehyde on the surface of screen-printed electrodes modified with poly(MB). The sensors were left to dry at least 24 h at 4C and kept at this temperature until use.

Results and discussions

Cyclic voltammetry experiments have been performed in the absence and the presence of NADH in order to establish the catalytic ability towards the oxidation of NADH of poly Meldola Blue (Fig. 1).

Fig. 1. Cyclic voltammogramms at poly(MB) electrodes

in the absence () and the presence (----) of 5.103 mol.l1 NADH.

The sensors screen-printed sensors with polyMB show catalytically ability for NADH oxidation with a larger increase in the anodic current in the presence of NADH 5x10-3 mol.L1 ctrodes and a superior sensitivity compare to other modified screen-printed elereported previously [31,32]. The results show that the screen-printed electrodes modified with polyMB can catalyze the oxidation of NADH that is generated from the reaction of NAD+ and ethanol catalyzed by alcohol dehydrogenase, as schematized in Fig. 2.


Fig. 2. Schematic representation of the mechanism of response of mediated ethanol biosensor

An amperometric biosensor for ethanol was developed based on immobilization of alcohol dehydrogenase on screen-printed electrodes with electropolymerised Meldola Blue, Electropolymerisation of MB represents the best approach for obtaining sensors with good operational stability and remark le anab alytical performances for the amperometric detection of NADH [25].

Preliminary experiments with this sensor aimed to the stable immobilization of the enzyme by cross-linking with glutaraldehyde. Several tests were run to illustrate the influence of enzyme loading and the cofactor concentration on the performances of the alcohol detector.

Optimization of cofactor concentration In2.

a first approach, the concentration of NAD+ cofactor was varied from 4.104 to 103 mol.L1 and t ydrogenase was

recorded for et e amperometric determinations were 0.05 V vs. Ag/A phosphate buffer pH 8.5. It was shown that c or concentrations abo did not significantly improve the sensitivity o etection limit but increased itude of the linear range for the detection of ethan ncentration of 1.6.103 AD+ was considered as optimum that allowed a tion limit of 3.104 mol.L1 and a linear range up to 7.5x103 m rized by a ra near range and a low sensitiv

he response of a biosensor with 12 IU alcohol dehhanol concentrations up to 12.103 mol.L1. Th

carried out at gCl in 0.1 mol.L1ofact ve 8.104 mol.L1r the d the magnol. A co mol.L1 N

detec (S/N=3) ther narrow liol.L1. This biosensor was characte

ity (8.54 A.L.mol1).

Effect of enzyme loading on the biosensor sensitivity To emphasize the effect of enzyme loading on the sensitivity of the biosensor, three different volumes (2.5, 3 and 3.5 L) of the same biocatalytic mixture were deposited on the surface of poly (MB) electrodes. Comparison of the resulted biosensors presented in Table 1 revealed a substantial increase in sensitivity. High enzyme loadings thus appear as necessary in order to obtain biosensors with a satisfactory sensitivity.

EL

CT

RO

DE

MBH

ET

HA

NO

L

MB+

2e-

NAD+

NADH

AD

H

AC

ET

AL

DE

HY

DE


Table 1. Effect of enzyme loading on the sensitivity of the biosensor

Enzyme loading (IU)

Biosensor sensitivity (A.L.mol1 SD*)

12.5 4.7 0.6

15.0 11.0 0.9

17.5 31.0 2.5 * standard deviation for five measurements.

Calibration of the biosensors The calibration plots for ethanol in 0.1 mol.L1 phosphate buffer pH 8.5 containing 0.1 M

differences in the responses of the sensors being caused by ed graphite layer.

as the time required reaching 95% of the saturation

KCl shows linearity in the range from 0.1x103 to 25x103 mol.L1 ethanol. the analytical curve is describe by the equation I (nA) = 36.46 (0.02) + 204.3 (0.07) C , Rethanol 2=0.9993 for n=21. Detection limits around 0.008 mM ethanol could be estimated considering Signal/Noise=3. The overall coefficient of variation for the determination of ethanol using described biosensor was 5.4% (n=30). This coefficient was found to be mainly induced by the screenprinted procedure,small differences in area of modifi

The response time was measuredcurrent and is 90 s.

Operational stability The operational stability of alcohol dehydrogenase sensors was tested by performing 10 consecutive series of calibration with the same biosensor. The concentration of cofactor in the electrochemical cell was 1.6.103 mol.L1 and the calibration was performed by adding

Additionall ctrodes as detectors a ted with nt deh that catal s with very low ium const 8.105mol.l ol transfor 7). In these cases is ry, especially if amplification systems or coupled reactions that drive the equilibrium to the product side are not taken i nsideration.

Analysis o holic beverag

different volumes of a 5.102 mol.l1 ethanol solution until reaching saturation. No significant decrease in sensor sensitivity was observed at the end of this 5-hour experiment, which represents the more important result obtained for the ethanol biosensor.

y, the described biosensor shows the potentialities of poly(MB)-elessocia

equilibr NAD+-depende ydrogenases

yse reaction

mation at pHants (Keq= 1 for ethansensitive detection of NADH particularly necessa

nto co

f alco es Th osensor allowed i ion for the di onitoring of et fferent beverages: gin, beer, red wine and Romanian palinca. All the samples were diluted with phosphate buffer solution (1:200 for wine and 1:500 for

order to avoid the interferences effect and to fit the analyte in the linear range of the calibration curve. Table 2 presents the values

e performance of the ethanohanol concentration in di

l bi ts applicat rect m

the other samples), inconcentration withfound and the recoveries obtained with the biosensor.

The quantification method was standard addition. As can be observed the results show excellent agreement between the biosensors results and those certified by the suppliers following the Official Method of the European Community consisting of a distillation.


Table 2. Recovery percentages in different samples obtained with ethanol biosensor

Beverage Added ethanol (m.mol.L1) Found ethanol

(m 1.mol.L ) Recovery* %

Gin 3.0 2.95 0.12 99.5 2 98.7 2

Beer 3.0 2.78 0.11 99.3 3

Wine 3.0 2.87 0.10 99.14 3

Romanian palinca 3.0 2.96 0.12 99.4 2 * Average of five measurements

Conclusions

The biosensor for alcohol has a good operational stability and stands for an example of rogenases, especially

m the t

u nt refers to t

, Research and arty,

1. D alysis: Clinical laboratory aspects, Part II, Laboratory Management.

-3.

rm. Biomed. Anal. 17, 1111-28.

egi, E. (1993) J. of

Acta 327, 223-34.

10. S .C., Wdge, R., Hart, J.P. and Pittson, R. (1996) Anal. Chim. Acta 329, 215-21.

. (1996) Talanta 43,

application of poly (MB) detectors with NAD+-dependent dehydde anding with respect of sensitivity towards NADH. Another common feature for de ection of NADH is the improvement obtained by Meldola Blue polymerization over other procedures for electrode modification with the mediator like incorporation of Meldola Bl e or Meldola Blue-Reinecke salt in the screen-printing ink. This improvemede ection limit, sensitivity and operational stability.

Acknowledgements

We acknowledge the financial support of Romanian Ministry of EducationYouth - CNCSIS grant no. A-273/2003. We are grateful to Prof. Jean-Louis MUniversity of Perpignan for providing the screen-printed graphite electrodes.

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Analele Universitii din Bucureti Chimie, Anul XIII (serie nou), vol. I-II, pag. 2732


DETERMINATION OF TRACE AMOUNTS OF MOLYBDENUM BY ADSORPTIVE STRIPPING VOLTAMMETRY USING MORIN AS CHELATING AGENT

Mihaela Buleandr , I.G. Tnase , M. Ionic and M.M. Buleandrabstract: A highly selective and sensitive procedure for the determination of ultra-trace concentrations of molybdenum by adsorptive stripping voltammetry is presented. The method is based on adsorptive accumulation of the molybdenum - morin complex onto a hanging mercury drop electrode, followed by reduction of the adsorbed species by voltammetric scan using square wave modulation. Optimal analytical conditions were found to be a morin concentration of 5 M, pH = 2.3 (Britton-Robinson buffer), and an adsorption potential at 0.25 V versus SCE. The peak current is proportional to the concentration of molybdenum over the range of 109 106 M with a detection limit of 0.1 nM for an accumulation time of 60 s. The method has been applied to the determination of molybdenum in seawater samples and results were in good agreement with atomic absorption spectrometry determinations.

Introduction

Molybdenum is an essential trace element for all living organisms [1]. Since the concentrations of molybdenum in plants, water and soil are generally at parts per billion levels, selective and sensitive methods for the determination of molybdenum are required. According to literature data methods for determination of molybdenum are mainly spectrophotometric techniques [2,3], spectrofluorimetry [4], atomic absorption spectrometry [5,6], neutron activation analysis [7], plasma emission spectrometry [8], ion chromatography [9] and electroanalytical techniques [10-31].

Many electroanalytical procedures based on the catalytic reduction of some oxianions like nitrate [10,11], bromate [12] and chlorate [13], the adsorptive accumulation of molybdenum complexes onto a HMDE [14-20] or the combination of the adsorptive accumulation with a catalytic current system [21-31]] have been used in the determination of molybdenum in environmental and biological samples. The adsorptive accumulation of molybdenum complexes onto a HMDE has been used by means of chemical interactions of the substrate with specific ligands such as 8-hydroxyquinoline (detection limit of 4 nM for 120 s) [14], phosphate (4 nM for 120 s) [15], tropolone (0.3 nmol L1 for 60s) [16],

University of Bucharest, Faculty of Chemistry, Department of Analytical Chemistry, Panduri 90-92,

Bucharest-5, 76231, Romania Army Center for Medical Research, Toxicology Laboratory, Calea Floreasca 8, Bucharest-2, Romania

28 M. BULEANDR I.G. TNASE M. IONIC M.M. BULEANDR

chloranilic acid (0.3 nmol L1) [17], Eriochrome Blue Black R (5.2 nmol L1 for 30s) [18], phenanthroline (0.6 nM for 20 min) [19], toluidine blue (0.1 nM for 240 s) [20].

In order to develop a new sensitive method for determination of molybdenum, the present work deals with a study of electrochemical behavior of the trace amounts of molybdenum in presence of 3,5,7,2',4'-pentahydroxy-flavone (morin) and determination of this element from seawater samples.

Experimental

All reagents were of analytical grade and were used without further purification (Sigma). Solutions were prepared by dissolving the salt or concentrated solution in three-distilled water. The Mo(VI) solution was prepared by dissolving (NH4)6Mo7O244H2O in three-distilled water to give a 102 M stock solutions. Solutions of lower concentration were prepared daily by appropriate dilution of the stock solution. The 3,5,7,2',4'-pentahydroxy-flavone (morin) stock solution, 103 M, was prepared by dissolving the necessary amount of dye in three-distilled water. A 0.04 M stock Britton-Robinson buffer solution with respect to o-boric, o-phosphoric acid and acetic acid was prepared. From this solution of different pH values were prepared by additions of 0.2 M sodium hydroxide.

The adsorptive voltammetric measurements were performed using a Radiometer POL 150 polarographic analyzer coupled to a Radiometer MDE stand with a hanging mercury drop electrode (HMDE), a saturated calomel electrode (SCE) reference electrode and a platinum wire counter-electrode. The potential was scanned from +0 to -1.2 V for the screening experiments and from 0.25 to 1.00 V for the Mo(VI) calibration and sample determination, at a scan rate of 50 mV s1. The solutions were de-aerated with highly purified argon at the beginning of each experiment and a flow of argon was maintained over the solution during the experiment to prevent oxygen interference. All experiments were performed at room temperature. The data acquisition and the scan recordings were done by means of Trace Master 5.0 software.

A sea water sample was filtered to remove suspended particles. The sample solution (10 mL), containing 5 M morin and Britton-Robinson buffer (pH = 2.3) was pipetted into the voltammetric cell. The stirrer was switched on and the solution was purged with argon gas for 5 min. The accumulation potential (0.25 V) was applied to a fresh HMDE for 60 s. Following the accumulation period, the stirrer was stopped, and, after 10 s, the voltammogram was recorded by applying a negative-going square wave scan from 0.25 to 1.00 V. Each standard addition was 0.1 mL of Mo(VI) solution with a concentration of 10 mg L1.

An atomic absorption spectrometer SpectrAA 880 - Varian with a GTA 100 - Varian graphite furnace was used for the determination of molybdenum. The spectrometer was operated using a hollow cathode lamp for molybdenum ( = 313.3 nm, 0.5R nm spectral slit width). A volume of 20 L of both reference and sample solutions were transferred into graphite tube through the autosampler. Argon 99.999% was used as protective and purge gas.

DETERMINATION OF TRACE AMOUNTS OF MOLYBDENUM 29

High purity standard reference solutions of Mo(VI) (from (NH4)6Mo7O24 4H2O in 0.5 M NH4OH AAS standard solutions) in 0.1% HNO3 (v/v) were used to prepare the analytical solutions (0 200 g/L) for calibration of the graphite furnace atomic absorption spectrometer. All measurements were made with three replicates and based on integrated absorbance.

Results of discussions

SWAdSV response of Mo(VI)-morin complex Molybdenum (VI) forms a soluble complex with morin in aqueous acidic medium [32]. In aqueous acidic medium Mo(VI) additions to a solution containing morin give rise to the formation of a Mo(VI)-morin complex, which is susceptible to be adsorbed onto mercury drop electrode and is, therefore, able to be analytically determined using Square Wave Adsorptive Stripping Voltammetry (SWAdSV). The voltammogram Mo(VI) in presence of morin at pH = 2.3 has showed one cathodic peak at about -0.55 V. This peak is proportional with molybdenum concentration.

In order to obtain the coordination number (m) of the adsorbed complex on the electrode surface, the following equation can be used [33]:

[ ] mp p,max p,max Li i i ,

where i

1 1 1= +

ding to the number of ligand molecules bound to the metal. This shows that the composition of the electroactive complex of Mo(VI) is 1:2, thus it can be written as

p and ip,max are the current and the maximum current of the reduction peak of the complex MoLm, [L] the concentration of the ligand and the stability constant of the complex. For a given concentration of molybdenum, additions of morin in the range of 0.1 - 8 M give rise to a saturation curve, typical of adsorption phenomena. Fig. 1 shows that, for a Mo(VI) concentration of 1 nM, the peak current increases with the morin concentration up to 5 M, and then becomes constant. Analyzing this curve by plotting 1/i as function of 1/[L]m for m = 1, 2, 3, , it results a straight line for m = 2 which correspon

Mo(L)2.

Optimization of experimental conditions The formation of the complex, the stability and the potential of reduction are strongly dependent on the pH value of the solution. The influence of pH on stripping current of molybdenum has been studied in the pH range of 1.8 to 5.0 (Fig. 2). In order to keep constant the composition of the buffer, when studying the effect of pH, Britton-Robinson buffers were used. It is clear that higher current can be obtained when pH is in the range of 1.8 2.8. Therefore, pH 2.3 identified as the optimum pH value, under these conditions the peak potential being -0.55 V. As illustrated in Fig. 2, the peak potential varied with pH and was shifted in the negative direction by 57 mV/pH unit.

The dependence of the reduction peak current of Mo(VI) complex on the accumulation potential in the range from 0 to 0.40 V was studied using an accumulation period of 60 s.


The optimum accumulation potential was 0.25 V. From the variation of the current peak has been observed that the peak height of the molybdenum-morin complex rapidly increases with the accumulation time during the first 100 s and then slows down. This is confirming that an adsorption process is involved.

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8 9cmorin [M]

i p [n

A]

Fig. 1: Effect of the morin concentration on the height of the reduction peak of complex in Britton-Robinson buffer

(pH 2.3) for 1 nM Mo(VI), with an accumulation time of 60 s at -0.25 V; scan rate = 50 mV s1.

Fig. 2: Variation of the SWV peak h potential () as function of the pH in Britton-Robinson buffer using 1 nM Mo(VI) and 5 M morin, with an accumulation time of 60 s at -0.25 V;

scan rate = 50 mV s-1.

1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5

2

34

5

6

7

8

9

i [nA

]

500

550

600

650

700

- E [m

V]

eight () and SWV peak

pH

DETERMINATION OF TRACE AMOUNTS OF MOLYBDENUM 31

In order to examine the effect of different scanning wave-forms on the sensitivity and linear dynamic range for determining molybdenum, the optimum conditions for square-wave adsorptive stripping voltammetry (SWAdSV) were compared to differential pulse adsorptive voltammetry (DPAdSV). The peak heights were greater, and a better resolution of the peak was obtained using SWAdSV.

Calibration graph, precision, detection limit and interferences Under optimized conditions, a linear relationship between the reduction peak current of molybdenum complex and the concentration of Mo(VI) can be obtained in the range of 10-9 - 10-6 M. The detection limit for Mo(VI) was calculated as kSB/b, where SB is the standard deviation of blank signal with current values measured at the same potential as for molybdenum-morin signal, and b is the slope of the calibration plot, and k is a constant equal to 3 as recommended by IUPAC [34]. The standard deviation of ten blank curves was 0.54 nA for an accumulation time of 60 s leading to detection limit of 0.1 nM.

Some trace elements can interfere with the determination of molybdenum adsorbing competitively onto HMDE or producing signals close to that of molybdenum. However, no interferences of common interfering ions such as iron and aluminum were caused by the 100-fold excess of these elements for molybdenum determination with tested reagent.

Application to real samples The proposed method was successfully applied to the determination of molybdenum in three seawater samples (Black Sea) without interference removal (Fig. 3). In order to eliminate matrix effects, the standard addition method was used. Molybdenum concentrations in the g L1 range in seawater samples were found to be in good agreement with values determined by GF-AAS (Table 1).

Fig. 3: SWAdSV voltammogram for Mo(VI) determination in water sample no 2 by addition standard method;

1: water sample; 2: + 5 10-6 M morin; 3: first addition; 4: second addition; 5: third addition of Mo(VI)

Table 1: Results of molybdenum determination in sea water by AdSV method with morin and GF-AAS

Sample AdSV (g/L) GF AAS (g/L)

No. 1 129.33 1.01 129.54 0.4

No. 2 125.95 1.54 127.70 1.6

No. 3 125.20 1.23 125.89 0.8


Conclusions

A method for determination of Mo(VI) with morin as an electroactive ligand by SWAdSV is developed and successfully applied in seawater samples. The proposed method can be a potential candidate for the practical use of molybdenum determination with high sensitivity, simplicity and speed.

REFERENCES

1. Chappel, W.R., Petersen, K.K. (1976), Molybdenum in the Environment, Marcel Dekker, New York.

2. Li, Z., Pan, J., Jan, T. (2001) Analyst 126, 1154. 3. Prasada Rao, T., Reddy, M.L.P., Ramalingom Pillai, A. (1998) Talanta 46, 769. 4. Blanco, C.C., Campana A.G., Barrero, F.A., Ceba, M.R. (1993) Anal. Chim. Acta 283, 213. 5. Barrera, P.B., Calvo, C.P., de Juan, J.A.C. (1990) Anal. Chim. Acta 231, 321, 6. Okutani, T., Noshiro, K., Sakuragawa, A. (1998) Anal. Sci. 14, 621. 7. Hirai, S. (1999) Curr. Adv. Mater. Proc. 12, 614. 8. Marczenko, M. (1991) Pure Appl. Chem. 63, 1629. 9. Zolotov, Yu. A., Shpigun, O.A., Bubchikova, L.A. (1983) Fresenius J. Anal. Chem. 316, 8. 10. Kolthoff, I.M., Hodara, I. (1963) J. Electroanal. Chem. 5, 2. 11. Edmonds, T.E. (1980) Anal. Chim. Acta 116, 323. 12. Barrado, E., Pardo, R., Batanero, P.S. (1998) Anal. Lett. 21, 1221. 13. He, H.B., Siow, K.S, Chi, H., Gao, Z.Q., Hsieh, A.K. (1995) Anal. Chim. Acta 309, 73. 14. van den Berg, C.M.G. (1985) Anal. Chem. 57, 1532. 15. Wang, L., Zhang, X., Cheng, G. (1990) Anal. Lett. 23, 2233. 16. Khan, S.H., van den Berg, C.M.G. (1989) Mar. Chem. 27, 31. 17. Karakaplan, M., Gucer, S., Henze, G. (1992) Fresenius J. Anal. Chem. 342, 186. 18. Willie, S.N., Berman, S.S., Page, J.A., van Loon, G.W. (1987) Can. J. Chem. 65, 957. 19. Quentel, F. (1999) Electroanalysis 11, 1355. 20. Zhao, Z., Pei, J., Zhang, X., Zhou X. (1990) Talanta 37, 1007. 21. Yokoi, K., van den Berg, C.M.G. (1992) Anal. Chim. Acta 257, 293. 22. Gao, Z., Siow, K.S. (1996) Talanta 43, 719. 23. Farias, P.A.M., Ohara, A.K., Nobrega, A.W., Gold, J.S. (1994) Electroanalysis 9, 333. 24. Wang, J., Serra, B., Ly, S.Y., Lu, J., Pingarron, J.M. (2000) Talanta 54, 147. 25. Jiao, K., Jin, W., Metzer, H. (1992) Anal. Chim. Acta 260, 35. 26. Fraga, I.C.S., Farias, P.A.M., Ohara, A.K. (2000) Fresenius J. Anal. Chem. 366, 307. 27. Safavi, E., Shams, E. (1999) Anal. Chim. Acta 396, 215. 28. Li, H., Smart, R.B. (1997) J. Electroanal. Chem. 429, 169. 29. Tnase, I., Buleandr, M., Iorgulescu, E.E., Radu, G.L. (2001) U.P.B. Sci. Bull., Series B 63(3), 375. 30. Tnase, I., Radu, G. L., Buleandra, M., Litescu, S. (1998) Aplicatii ale tehnicilor electroanalitice in

bioanaliza, Protransilvania Publishing House, Bucuresti.

31. Tnase, I. (2000) Tehnici i metode electrometrice de analiz, Ars Docendi, Bucureti.

32. Tarek, M., Yaki, M., Abdel-Kader, A.K., Abdalla, M.M. (1991) Fresenius J. Anal. Chem. 339, 197.

33. Wang, L., Ma, C., Zhang, X., Wang J. (1994) Anal. Lett. 27, 1165.

34. IUPAC, (1978) Spectrochim. Acta B 33, 242.

Analele Universitii din Bucureti Chimie, Anul XIII (serie nou), vol. I-II, pag. 33-37


DETERMINATION OF SULPHIDE BY POTENTIOMETRIC TITRATION

V. Dumitrescu , Nina Dumitrescu and Dana Anghel abstract: Sulphide anion was determined indirectly, by potentiometric titration. For this purpose, a known concentration of Ce4+ and sulphide ions was added into the electrochemical cell. Ce4+ remained in the cell after his reduction by the sulphide ions was titrated with a Fe2+ solution having a known concentration. The difference between total Ce4+ and the titrated Ce4+ corresponds to the amount of Ce4+ reduced by the sulphide ion. The proposed method is reproducible, fast and allows the determination of sulphide ion in waters.

keywords: sulphide, potentiometric determination

Introduction

The sulphide ion is found in various waters and often arises the problem of quantitative analysis. Regarding the methods used in the analysis of the sulphide from waters can be applied well-known techniques in analytical chemistry, depending on the variable amount of sulphide. For this purpose classical and instrumental methods of analysis can be used.

The sulphide determination can be made using volumetric methods [1], spectrophotometric methods [2-7] and electrometric methods [9-13]. Among all electrometric methods those frequently used are direct or indirect potentiometric methods.

In this paper indirect determination of sulphide ion was achieved by potentiometric titration.

Experimental

Apparatus and reagents For measuring the electromotive tension an electrochemical cell was used containing a platinum electrode as indicator electrode and a calomel-saturated electrode as the reference electrode. The electrochemical cell was connected to a pH-meter/millivoltmeter MV-84 Clahmann - Grahnet. The solution was stirred with a magnetic stirrer.

For the determinations the following solutions were used:

101N Ce(SO4)2 solution in 101N H2SO4, provided by Merck;

Department of Analytical Chemistry, University of Bucharest, 90 road Panduri, Bucharest, ROMANIA

34 V. DUMITRESCU N. DUMITRESCU D. ANGHEL

102N Na2S solution in 101N H2SO4, provided by Merck; 101N (NH4)2Fe(SO4)2. 6H2O solution, provided by Merck.

All reagents used in our studies were of analytical purity. The solutions were obtained in bidistilled water.

Results and Discussion

Potentiometric determinations were made in an electrochemical cell containing a platinum electrode and a calomel-saturated electrode. The Ce4+ solution was titrated with a Fe2+ solution. The reaction that takes place is:

Ce4+ + Fe2+ Ce3+ + Fe3+

In the electrochemical cell were also added S2 ions. After the reduction of one part of the initial amount of Ce4+, the Ce4+ remained was titrated with Fe2+.

From the titration curve the equivalence volume was calculated. Knowing the Ce4+ initial concentration, the amount of Ce used to oxidize S2 ions can be calculated by difference and the sulphide amount present in solution can be implicitly calculated.

In order to verify of the Ce4+ solutions titre, 5 ml 101N Ce4+ solution was titrated with 101N Fe2+ solution. In the titration cell was also added 20 ml 101N H2SO4 solution. The titration curve is presented in the Fig. 1.

200

400

600

800

1000

1200

0 1 2 3 4 5 6 7 8 9 10

V(ml)

E(m

V)

Fig. 1: Potentiometric titration curve of Ce4+ with Fe2+ ( )4 21 1Ce Fe10 N; 10 Nc c+ + = = .

From this figure 5 ml equivalence volume was obtained.

a) The time influence The time influence on Ce4+ reduction by S2 was studied. For this purpose, 5 ml 101N Ce4+ solution, 10 ml 102N S2 solution and 10 ml 101N H2SO4 solution were added into the electrochemical cell. From the potentiometric titration curves presented in Fig. 2, Fig. 3, Fig. 4 and Fig. 5 the equivalence volumes were determined at 10 minutes, 20 minutes, 25 minutes, 30 minutes after adding S2 solution into the electrochemical cell.

DETERMINATION OF SULPHIDE BY POTENTIOMETRIC TITRATION 35

200

400

600

800

1000

1200

0 1 2 3 4 5 6 7 8 9 10

V(ml)

E(m

V)

Fig. 2: Potentiometric titration curve of Ce4+ and S2- with 10-1N Fe2+ solution at 10 minutes

after adding S- (Ve=4.75 ml)

200

400

600

800

1000

1200

0 1 2 3 4 5 6 7 8 9 10

V(ml)

E(m

V)


after adding S- (Ve=4.125 ml)

200

400

600

800

1000

1200

0 1 2 3 4 5 6 7 8 9 10

V(ml)

E(m

V)


after adding S2 (Ve=4 ml)

36 V. DUMITRESCU N. DUMITRESCU D. ANGHEL

200

400

600

800

1000

1200

0 1 2 3 4 5 6 7 8 9 10

V(ml)

E(m

V)


after adding S2 (Ve=4 ml)

The reactions that take place are:

2Ce4+ + S2- 2Ce3+ + S0

Ce4+ + Fe2+ Ce3+ + Fe3+

In Fig. 6 the equivalence volume is graphically represented as a function of time.

3.9

4.1

4.3

4.5

4.7

5 10 15 20 25 30 35 40

t(min)

V(m

l)

Fig. 6: Graphic representation of equivalence volume as a function of time

From this figure a constant equivalence volume (4 ml) is observed at 25 minutes after adding 102N S2 solution. In the following determinations 30 minutes were allowed to pass before the titrations were done.

b) The influence of sulphide concentration 5 ml 101N Ce4+ solution, 101N H2SO4 solution and variable amounts of 102N S2 solution (10 ml; 5 ml; 2.5 ml; 1 ml) were introduced into the electrochemical cell. The final volume in the cell was 25 ml (101N H2SO4 solution was added).

The variation of equivalence volume with the S2 added amount was followed.

All titration were made at 30 minutes after adding S2 solution.

DETERMINATION OF SULPHIDE BY POTENTIOMETRIC TITRATION 37

In Table 1 the equivalence volumes obtained for variable amounts of 102N S2 solution are presented.

Table 1. The equivalence volumes (ml) of 101N Fe2+ obtained at the titration of variable amounts of S2

102N S2 volume (ml) 10 5 2.5 1

101N Fe2+ equivalence volume (ml) 4 4.5 4.75 4.9

S2 concentration 4.103N 2.103N 1.103N 4.104N

From this study is ascertain that, using the proposed method, S2 concentrations higher than 4.104N can be determined.

Conclusions

In this work the experimental conditions for potentiometric determination of sulphide anion were established. An indirect method of sulphide anion determination was used. Known concentrations of Ce4+ and S2- were added into the electrochemical cell. The Ce4+ was titrated with a Fe2+ solution with known concentration. The difference between total Ce4+ and the resulted Ce4+ at the titration corresponds to the amount of Ce4+ reduced by S2-.

It was observed that the titration can be done at 25-30 minutes after adding S2- to the Ce4+ solution.

This method can be used for determination of S2- concentrations higher than 4.10-4N. The method is reproducible, fast and allows the determination of sulphide ion in waters.

REFERENCES

1. Liteanu, C. and Hoprteanu, L. (1972) Chimie analitic cantitativ. Volumetrie, Ediia a asea, Ed. Didactic i pedagogic, Bucureti, 405, 508, 622.

2. Hofmann, R. and Hamm, Z. (1967) Z. Anal. Chem. 232, 167.

3. Matheson, N.A. (1974) Analyst 99, 577.

4. Rahim, S. and Salim, A.J. (1994) Anal. Chim. Acta 2, 193.

5. Glister, M.G. and Moody, G.J. (1990) Talanta 21, 215.

6. Ensafi, A. (1992) Anal. Lett. 25(8), 1525.

7. Kester, M.D., Schiundu, P.M. and Wade, A.S. (1992) Talanta 39, 299.

8. Hu, X.Z. and Leng, Z. (1996) Anal. Commun. 33(9), 297.

9. Egorova, V.I., Vasilieva S.V., and Stolyarova, V.S. (1991) Zavod. Lab. 57(3), 18.

10. Back, R. and Ruff, H.J. (1995) Anal. Chem. 64, 305.

11. Mascini, M. (1970) Anal. Chim. Acta 51, 231.

12. Tse, Z.H., Janda, P., Herman, L. and Lever, A.B.P. (1995) Anal. Chem. 67(5), 981.

13. Tse, Z.H., Janda, P. and Lever, A.B.P. (1994) Anal. Chem. 66, 384.



PARTICLE-INDUCED X-RAY EMISSION (PIXE): A TOOL FOR MULTIELEMENT ANALYSIS IN THE YEAST CELLSa

Ileana Cornelia Farcasanu , F. Nishiyama and T. Miyakawaabstract: The analytical particle-induced X-ray emission (PIXE) procedure for the multielement analysis offers great potential for trace element analysis in the biological and medical fields. Here we present the PIXE procedure as an accurate tool to detect both the major and the trace chemical elements within the Saccharomyces cerevisiae cells.

Introduction

One of the main characteristics of living organisms is their ability to maintain certain parameters within the physiological limits. Out of the basic homeostasis mechanisms, regulation of the ratio between chemical elements must be an important determinant for adaptation to various environments. Many elements, both major and trace elements [1, 2], take part in the complicated biochemical reactions and one cannot say beforehand which one has a crucial role in certain case. It is also known that there exist synergetic effects, such that the concentration of certain element influences the function of another. If one has a complete elemental profile, there is less chance of overlooking valuable information.

Particle-induced X-ray emission (PIXE) analysis is a multielement technique that has been developing worldwide over the past years [3-6]. Certain advantages make it useful for applications in biological sciences: 1) PIXE offers its maximum sensitivity or minimum detection limit in two atomic number regions, 20 < Z < 35 and 75 < Z < 85; the former region happens to contain the majority of trace elements of interest in various areas of biological sciences. 2) PIXE analysis also determines several major elements such as K, Ca, S and P, and it is often of great interest to know their concentrations. 3) Optimum sensitivities are attained at rather low proton energies; this is important since it means that small accelerators are most suitable for PIXE, with consequent benefits in both reliability and economics. 4) Analysis times are typically a few minutes in duration. 5) The

a This work was performed at Hiroshima University, Japan

Bucharest University, Faculty of Chemistry, Department of Organic Chemistry and Biochemistry, Sos. Panduri 90-92, Bucharest, Romania. Tel.: 40-21-4103178/ext.135. E-mail: [email protected]. Corresponding author

Department of Power Engineering and Applied Physics, Graduate School of Engineering, Hiroshima University, Japan

Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Japan

40 I.C. FARCASANU F. NISHIYAMA T. MIYAKAWA

multielement nature of PIXE in combination with high sensitivity permits the determination of many elements in a single run.

Trace metals (Fe, Zn, Cu, Mn, Co) play an important role in cell metabolism, especially as coenzymes and cofactors in various cellular processes. Usually, the environmental concentrations of such elements are low and cells must develop active transport processes to accumulate them. On the other hand, when the internal concentration of these metals gets higher than the physiological levels, they become toxic, mainly by non-specific binding to proteins or by interference to other metals' metabolism. Under such circumstances, cells develop defense strategies, by limiting the influx, by sequestering the excess cations in a non-toxic form, or by activating the export. PIXE was used to monitory the uptake or efflux rates of individual trace elements [7, 8] as well as the intracellular distribution of manganese [9], in the Saccharomyces cerevisiae cells, but no multielement analysis was carried out.

In this article we present PIXE as a way to measure highly encountered elements (K, S, P, Mg) along with trace elements (Fe, Mn, Zn, Ca, Cu, Ni, Co) inside the cells of the yeast Saccharomyces cerevisiae grown under standard conditions.

Experimental

Strains, media, and preparation of the PIXE samples The brewer's yeast Saccharomyces cerevisiae, strain W303 1A (MAT a trp1 leu2 ade2 ura3 his3 can1-100) was used for our experiments. Cells were grown in liquid YPD (yeast extract-polypeptone-dextrose) [10] supplemented with adenine (400 g/ml) and uracil (200 g/ml). A preculture was used to inoculate the culture at a concentration of about 2 x 105 cells/ml. The cell suspensions were incubated with shaking for 8 hours at 28C. Cells were harvested by centrifugation and were washed three times with deionized water. Finally, cells were brought to the desired density by adding an appropriate volume of deionized water. All centrifugation (3 min, 3000 x g) was done at 4C.

PIXE experiments Suspensions with various cell densities were placed in aliquots of approximately 5 l on polycarbonate membranes (Nucleopore Corp., U. S. A., pore size 0.1 m, thickness ~ 6 m) and let dry overnight in a desiccator containing silicagel. The elements in the cellular material were determined using a Van de Graaff accelerator (Nissin High Voltage Co. Ltd., model AN-2500). The proton energy used was 2 MeV for heavy elements (Z > 18), but for detection of light elements (Z < 18), the proton energy was degraded to 0.75 MeV in order to obtain a good signal-to-noise ratio. For Mg, P and S determinations, a Si (Li) X-ray detector (Canberra SL80175) with 25 m beryllium window was used. For K, Ca, Mn, Fe, Ni, Cu, Zn determinations, a Ge (int) X-ray detector (ORTEC GLP-10180/07) with 127 m beryllium window was used. All data were accumulated by a multichannel pulse height analyzer AMS-1000 (Laboratory Equipment Corp.). The elements determined were expressed as g/mg total cellular protein; total cellular protein content was determined by

PARTICLE-INDUCED X-RAY EMISSION: A TOOL FOR MULTIELEMENT ANALYSIS 41

the method described by Bradford [11]. The measurements were carried out in five different experiments, using triplicates for every sample.

Results and discussion

A typical example of a PIXE spectrum of the main elements present in the yeast cell are presented in Fig. 1. The amounts of various elements determined by PIXE inside the S. cerevisiae cells are presented in Table 1. Minimal detection limit within an organic matrix is considered to be 107~106 mM [3]. If the yeast cell is considered to be a sphere of 5 diameter, then for detecting for example Mn2+, whose average concentration in the cell is around 5 mM [12], the minimal cell density in the sample should be of the 106 order. We found for the majority of elements detected that the cell density 105-106 cells/sample gave accurate and reproducible values (Table 1). However, when the cell density was higher than 5 x 106, the reproducibility was poor, probably because of the interference of the cell matrix.

Counts per channel

101

10200400600800

Channel number

K

MnFeZn

Ca

CoNi

V

BrCu

102

103

104

Fig. 1. A PIXE spectrum of elements found in yeast cell.

The yeast cells were grown to the log phase and concentrated by centrifugation. 5 l of cell suspension (106 cells, in this case) were used for PIXE analysis as described in the

Experimental section. One typical PIXE spectrum is presented.

Certain trace elements (Fe, Cu, Zn) were easy to determine by PIXE (Fig. 1), but in order to measure some of the minor trace elements (e. g., Co, Ni,) it was necessary to optimize the sensitivity. This was done by superposing two different spectral data.

The lightest element that could be detected in our experiments was Na, but the cellular content determined increased with sample dilution and reproducibility of results was poor. This observation suggested us that for light elements (Li, Na) other analytical techniques (e. g., atomic spectroscopy) may be more appropriate.

42 I.C. FARCASANU F. NISHIYAMA T. MIYAKAWA

Table 1. The element composition of yeast cells as determined by PIXE. The results are the average of five different measurements.

Element Cellular content (g/mg protein) OCD1

(cells/sample)2

Mg 21.930.57 1x105~1x106

P 167.55.95 1x105~1x106

S 19.560.93 2x105~1x106

K 169.7715.23 1x105~1x106

Ca 2.330.15 2x105~1x106

Mn3 0.0250.015 1x106~5x106

Fe 0.3550.005 2x105~5x106

Co4 1.4x10-2 5x106

Ni4 2.7x10-3 5x106

Cu 0.1220.003 1x106~5x106

Zn 2.260.02 2x105~5x106

Br4 1.3x10-2 5x106

Rb3 0.0330.001 1x106~5x1061OCD = optimal cell density; 2Sample volume = 5l;3 Minimal cell density for detection: 1x 106 cells/ml; 4 Minimal cell density for detection: 5x106 cells/ml.

REFERENCES

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3. Johansson, S. A. E. and Campbell, J. L. (1988) PIXE: a novel technique for elemental analysis, John Wiley and Sons Ltd.

4. Nygren, O. (2002) New approaches for assessment of occupational exposure to metals using on-site measurements, J Environ Monit. 4: 623-7.

5. Hou, X, Peters, HL, Yang, Z, Wagner, KA, Batchelor, JD, Daniel, MM, and Jones, BT. (2003) Determination of trace metals in drinking water using solid-phase extraction disks and X-ray fluorescence spectrometry, Appl Spectrosc. 57: 338-42.

6. Ebara, M, Fukuda, H, Hatano, R, Yoshikawa, M, Sugiura, N, Saisho, H, Kondo, F. and Yukawa, M. (2003) Metal contents in the liver of patients with chronic liver disease caused by hepatitis C virus. Reference to hepatocellular carcinoma, Oncology 65, 323-30.

7. Farcasanu, IC, Hirata, D, Tsuchiya, E, Nishiyama, F. and Miyakawa, T. (1995) Protein phosphatase 2B of Saccharomyces cerevisiae is required for tolerance to manganese, in blocking the entry of ions into the cells, Eur J Biochem. 232, 712-7.

8. Farcasanu, IC, Mizunuma, M, Hirata, D. and Miyakawa T. (1998) Involvement of histidine permease (Hip1p) of Saccharomyces cerevisiae in Mn2+ transport, Mol. Gen. Gen. 259, 541-8.

9. Farcasanu IC, Hirata D, Tsuchiya E, Mizuta K and Miyakawa T. (1999). Involvement of thioredoxin peroxidase type II (Ahp1p) of Saccharomyces cerevisiae in Mn2+ homeostasis, Biosci Biotechnol Biochem. 63, 1871-81.

PARTICLE-INDUCED X-RAY EMISSION: A TOOL FOR MULTIELEMENT ANALYSIS 43

10. Sherman, F., Fink, G. R. and Hicks, J. B. (1986) Methods in yeast genetics, Cold Sprig Harbor Laboratory Press, Cold Spring Harbor, New York

11. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Anal. Biochem. 72, 248-54.

12. Okorokov, L. A., Lichko, L. P., Kadomtseva, V. M., Kholodenko, V. P., Titovski, V. T. and Kulaev, I. S. (1977) Energy-dependent transport of manganese into yeast cells and distribution of accumulated ions. Eur. J. Biochem. 75, 373-7.



ENZYMATIC MARKER FOR TOXICITY OF CONTAMINATED SOILS

S. Fleschin, A. Bunaciu and Mihaela Scripcariuabstract: The treatment of toxic wastewater and remediation of soil is greatly facilitated by oxidizing the chemicals with selected activated sludge or mixed bacterial strains. Many different chemicals are destroyed or degraded to less hazardous products by altering the chemical structure of the molecule through reaction with the powerful hydroxyl radical. This simple to use oxidant power of microorganisms is easy to prepare, is inexpensive and is used at ambient temperatures and pressures..

Introduction

The concept of applications of enzymatic determination in wastewater treatment or soil remediation is reconsidered now in modern way. The treatability of any industrial wastewater, of course, depends upon its composition. A wastewater containing any of the compounds susceptible to enzymatic oxidation is a candidate for treatment [1,2].

Industries produce wastewaters which contain some toxic organics susceptible to oxidation. For example, the petroleum industry produces waste streams containing phenols, benzenes, toluens and xylenes. Plywood and adhesive manufactures frequently have phenol, formaldehyde and resorcinol wa

ANALELE UNIVERSITĂŢII BUCUREŞTI 2004

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