Coulometer from a Digitally Controlled Galvanostat with Photometric Endpoint Detection

Ci a ion: González-A jona, D.; Roldán González, E.; López-Pé ez, G.; Domínguez Pé ez, M.M.; Cale o-Cas illo, M. Coulome e om a Digi ally Con olled Gal anos a wi h Pho ome ic Endpoin De ec ion. Senso s 2022,22, 7541. h ps:// doi.o g/10.3390/s22197541 Academic Edi o s: Eladio Du án A anda, JoséLuis Ál a ez Macías and Juan Daniel Mozo Llamaza es Recei ed: 6 Sep embe 2022 Accep ed: 1 Oc obe 2022 Published: 5 Oc obe 2022 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2022 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). senso s A icle Coulome e om a Digi ally Con olled Gal anos a wi h Pho ome ic Endpoin De ec ion Domingo González-A jona *, Emilio Roldán González, Ge mán López-Pé ez, Manuel Ma ía Domínguez Pé ez and Ma ina Cale o-Cas illo Depa men o Physical Chemis y, Facul y o Chemis y, Uni e si y o Se illa, 41012 Se illa, Spain *Co espondence: [email p o ec ed] Abs ac : In his wo k, a coulome e was de eloped om a digi ally con olled gal anos a . A simple colo ime e based on a RGB LED was used as a ligh emi e coupled o ligh de ec o s, while ligh dependen esis ance (LDR) and pho odiodes ha e been de eloped as endpoin de ec o s. Bo h ha dwa e and so wa e ha e been adap ed om he o iginal gal anos a design. Rega ding he ha dwa e, new elec ical signal condi ione s ( il e s and ol age di ide s) we e included o op imize he wo king sys em. The so wa e was de eloped based on an open sou ce A duino UNO mic ocon olle . The di e en a iables ha con ol he i a ion p ocess a e managed by an add-in module o Excel da a acquisi ion so wa e ha is eely a ailable. A s udy o he possible a iables ha in luence he i a ion p ocess has been ca ied ou . The sys em was es ed wi h wo classical coulome ic i a ions such as iodome y ( hiosul a e, asco bic acid) and acid/base (po assium acid ph hala e as s anda d). The de eloped sys em is e sa ile as di e en endpoin colo indica o s can be employed (s a ch and phenolph halein o he in es iga ed eac ions). Di e en expe imen al a angemen s ha e been s udied: he na u e o he elec odes (P , Ag), ype o cells ( wo sepa a e compa men s o a single compa men ), and ligh de ec o s (LDR, pho odiode). The in luence o se e al expe imen al pa ame e s (bo h elec ical, ligh , and in eg a ion ime) was s udied and chosen o ob ain he bes pe o mance o he comple e sys em. Rep oducibili y esul s below 1% can be ob ained unde con olled condi ions. In he case o acid/base i a ions, he p esence o a mosphe ic ca bon dioxide was de ec ed, whose in e e ence was mainly a ec ed by he s i ing a e and he i a ion ime. Keywo ds: elec ochemical ins umen a ion; gal anos a ; coulome y; op ical de ec o 1. In oduc ion One o he mos common analy ical echniques o quan i ica ion in a chemical labo- a o y is olume ic i a ion. The unknown concen a ion o a compound in a sample is ob ained by adding an amoun o eac an ha has a as , i e e sible, and quan i a i e eac ion wi h i . The amoun o eac an added is s opped when he compound unde s udy in he sample is consumed. Thus, a eliable me hod o de ec ing he endpoin needs o be p o ided. Mo eo e , he concen a ion o he eac an solu ion o be added has o be known p ecisely and i s concen a ion kep s able, among o he analy ical conside a ions. Elec ochemis y, h ough elec olysis echniques, can be employed o he con olled p oduc ion o nume ous eagen s in si u, which can be used as i an agen s. This is an especially adequa e me hodology o he case o uns able o di icul o manipula e eac- an s due o pa icula eac ion condi ions [ 1 , 2 ]. Measu emen s o he elec ic cu en can be pe o med wi h g ea p ecision, allowing o accu a e de e mina ion a e y low concen a- ion le els. In coulome ic me hods based on Fa aday’s law, he numbe o coulombs ha passed h ough he cell a e quan i a i ely ela ed o he amoun o compound elec olyzed. The cha ge associa ed wi h he elec ons is used as he p ima y s anda d eagen , gene a ing he i an in con olled quan i ies wi hin he elec oly ic cell, which a oids he necessi y o Senso s 2022,22, 7541. h ps://doi.o g/10.3390/s22197541 h ps://www.mdpi.com/jou nal/senso s Senso s 2022,22, 7541 2 o 15 p epa a ion and he s o age eagen s anda d solu ions. In any case, he elec ochemical eac ion has o be a single and as p ocess, wi h a well-de ined s oichiome y. The i s quan i a i e applica ion o elec ic cha ge was pe o med a he beginning o he 20 h cen u y, bu i was no un il 1938 ha a succession o pape s appea ed wi h he e m “coulome ic” in he i le [ 3 ]. These pape s, oge he wi h he ad en o elec- onic ins umen a ion and he nume ous and asso ed elec o-gene a ed chemicals, led o he widesp ead applica ion o he coulome ic analysis. A. J. Ba d published an ex- ensi ely e iew on coulome ic analysis [ 4 ] and oge he wi h chap e s 11 and 15 in his popula ex book, bo h cons i u e an excellen s a ing poin o s udy and a ich sou ce o e e ences [ 5 ]. The coulome ic echnique shows some impo an ad an ages o e he classical i a ion me hods such as high sensi i i y, he usage o aa e y low amoun o eagen equi ed, and/o uns able compounds as i a ion agen s, no p ima y s anda d is needed and he main eac an (elec ons) can be p ecisely con olled. Ne e heless, some d awbacks ha can be o e come a e also p esen such as a mo e complex ins umen a ion, he non-comme cial a ailabili y o speci ic expe imen al de ices, speci ic aining o he labo a o y specialis , and no mally, a highe budge ha dwa e [6–8]. A li e a u e e iew om 2000 o he p esen on he aspec s o he “coulome y a cons an cu en ” was ca ied ou . A ound hi y ela ed a icles we e e e enced as an indica ion o he use ulness o he cons an -cu en coulome y. In he las six yea s, some a icles based on he use o coulome y, om he analy ical poin o iew, we e published desc ibing he applica ion o he de ec ion and quan i ica ion o a a ie y o compounds om me als, pha maceu icals, wa e , an ioxidan s, gases, and ce i ied ma e ials [ 9 – 18 ]. Fu he mo e, nume ous pape s on coulome y ha e been published in educa ion jou - nals due o hei ins uc ional cha ac e (see ecen pape s on coulome y o chemical educa ion [19–21] and he e e ences he ein). Two me hods can be dis inguished based on he elec ical a iable con olled du ing he elec oly ic p ocess, wi h ei he he elec ical po en ial o cu en being kep cons an . In cons an -po en ial elec olysis, he edox eac ion o in e es is con olled by he po en ial alue, a oiding o he possible elec ochemical side eac ions. When no compound is le o be consumed by he elec olysis, he p ocess ceases. Thus, no sepa a e signaling/endpoin de ec ion me hod is necessa y, as he cu en dec eases exponen ially o a ze o alue. The ime o comple e eac ion is la ge and i is usually di icul o disce n he endpoin om he esidual cu en [ 5 ]. Mo eo e , he ins umen a ion is mo e complica ed, mo e ex- pensi e, and can be applied o a limi ed numbe o subs ances [ 22 ]. On he o he hand, cons an -cu en elec olysis allows o a g ea e numbe o subs ances o be con e ed and i p o ides a s aigh o wa d ela ionship be ween he elec ic cha ge passed and he elec olysis ime. Thus, he elec olysis ime is di ec ly p opo ional o he amoun o com- pound gene a ed by he edox eac ion. Mo eo e , he necessa y elec onic ins umen a ion is simple (a gal anos a ). Thus, a cons an cu en and when he edox eac ion o in e es eaches comple ion, he gal anos a modi ies he applied po en ial o keep he cu en cons an , which ine i ably leads o side eac ions. The e o e, a sys em indica ing he end o he elec olysis ime o he s udied edox p ocess is necessa y. In any case, o analy ical pu poses, a 100% cu en e iciency is necessa y wi h bo h me hodologies. One way o p o iding an e ec i e means o con ol, minimizing he side eac ions, is by moni o ing he e olu ion o he elec ical a iable ha is no kep cons an du ing he elec olysis. Thus, exponen ial changes in he e olu ion o he cu en when ope a ing in po en ios a ic mode o sudden changes in he po en ial in gal anos a ic mode can be indica i e o side eac ions, which lowe he p ocess e iciency. An addi ional complica ion in designing an elec olysis cell a ises when he chemical componen s o med a bo h elec odes can eac each o he in he bulk solu ion. In his case, he solu ion a ound each elec ode has o be sepa a ed by an elec ically conduc ing memb ane. The coulome ic sys em de eloped in his p ojec was based on an adap a ion o he o iginal p o o ype o a digi al gal anos a [ 23 ], used in he cha ac e iza ion o p ima y and seconda y ba e ies. Ini ially, he same digi al mic ocon olle (A duino UNO) [ 24 ] was Senso s 2022,22, 7541 3 o 15 u ilized due o i s low cos , use - iendly p og amming en i onmen , and open so wa e– ha dwa e en i onmen ha uns on Windows, Mac OsX, and Linux ope a ing sys ems [ 25 ]. The inpu /ou pu da a by se ial communica ion we e de ec ed/p ocessed by using he ee Da a Acquisi ion ool PLX-DAQ [ 26 ] embedded as a con olle in an Excel ype sp eadshee , as p e iously desc ibed [ 27 ]. Thus, he inpu pa ame e s o he ins umen con ol and he da a acqui ed can be pe o med ia sp eadshee , a oiding he need o upload he so wa e o he mic ocon olle e e y ime an expe imen al un is s a ed. Mo eo e , he ange o di e en inpu pa ame e s is alida ed in he sp eadshee , while he signal o he op ical de ec o is also g aphically moni o ed du ing he ini ial elec olysis p ocess. Ne e heless, some adap a ions o he o iginal gal anos a ha dwa e/so wa e a e equi ed o pe o m coulome ic i a ions. Thus, he ba e y connec o s a e now elec ical e minals o he elec olysis cell. The e minal used o ead he ba e y po en ial du ing i s cha ac e iza ion is now employed o moni o he s a us o he elec olysis cell o he de ec ion o inco ec elec ode connec ions, he po en ial obs uc ion o he anode/ca hode sepa a ion memb ane, and e en o eco ding possible changes ha may be due o he exis ence o side eac ions. Di e en elec ochemical echniques can be used as endpoin de ec o s. These a e based on checking he ab up change o an elec ical a iable a he endpoin . An addi ional elec ic ci cui wi h a pai o elec odes isola ed om he elec oly ic gene a o sys em is used: po en iome y (elec ic po en ial agains a e e ence elec ode), ampe ome y, and dead-s op [ 28 ]. The use o an independen elec ic ci cui , combined wi h he mic ocon- olle ’s di icul y in eading di ec ly nega i e alues o ol ages, complica es he endpoin de ec o design. On one hand, he spec ome ic de ec o s a e based on a signi ican change in colo (UV–Vis), ei he by he addi ion o an indica o o by he colo change in he elec oly ic so- lu ion i sel . No wi hs anding i s e sa ili y, he implemen a ion o he spec opho ome ic echnique is gene ally mo e expensi e. A possible way a ound such budge a y cons ain s esul ing om using spec ome y wi hou a big loss in lexibili y can be esol ed by he use o a colo ime e o he isible de ec ion [ 10 , 20 , 25 ]. In his wo k, a simple colo ime e was de eloped based on a RGB LED as a ligh emi e , coupled o a isible ligh de ec o as a pho o esis o (LDR/pho odiode). The comple e sys em was es ed o classical iodome ic and acid/base i a ions ( hiosul a e, asco bic acid, pe chlo ic acid, and po assium hyd ogen ph hala e). S a ch and phenolph halein we e used as he isible endpoin indica o s, espec i ely. Mul iple a angemen s o elec ode ma e ials (P , Ag), elec oly ic cells ( wo sepa a ed hal -cells o a single compa men ), ligh de ec o s (LDR, pho odiode), and se e al combina ions o expe imen al pa ame e s (bo h elec ical, ligh and in eg a ion ime) ha e been explo ed o ob ain he bes pe o mance om he de eloped coulome ic sys em. Some impo an ad an ages can be achie ed wi h he p oposed design. On one hand, he usage o a mic ocon olle allows o e y p ecise con ol o he elec olysis ime, and on he o he hand, he open sou ce sys em educes he ha dwa e budge equi ed o he i a ion de ice. The e sa ili y o he sys em allows o he de elopmen o o he ypes o applica- ions using gal anos a ic me hods [ 3 ], o example, he measu emen o he hickness o me allic coa ings. The p oposed sys em allows he po en ial di e ence applied be ween he elec odes o be moni o ed simul aneously wi h he gal anos a ic p ocess. An app eciable change in he po en ial alue will indica e ha he elec olysis o he me allic coa ing has been comple ed. The e o e, aking in o accoun Fa aday’s laws, he densi y o he me al and he su ace a ea exposed, he elapsed ime will be p opo ional o he hickness o he me al coa ing. This would equi e a comple e change in he design o he gal anos a ic cell, he so wa e, and he s udy o he possible a iables in ol ed. Senso s 2022,22, 7541 4 o 15 2. Ma e ials and Me hods 2.1. Ha dwa e (a) Gal anos a : The coulome e ha dwa e se up mainly cons i u es he adap a ion o he o iginal gal anos a p o o ype [ 23 , 27 ], a lexible op ical endpoin de ec ion sys em, and an A duino UNO employed as a mic ocon olle o handling he en i e sys em. The o iginal gal anos a design was based on a scale/in e e ope a ional ampli ie . The cu en anges we e manually selec ed (jumpe s) by a combina ion o he esis o s. Swi ching on/o o he gal anos a ope a ion was digi ally con olled. A di e en ial ope a ional ampli ie (OA) was used o ead he ba e y’s ac ual ol age. The ac ual cu en alue wi hin he ull scale selec ed was con olled by applying a DC bias ol age. This DC po en ial was digi ally gene a ed by using a pulse wid h modula ed (PWM) signal [ 29 ], which was p e iously passed h ough an ac i e second o de 1 Hz low-pass il e . Thus, 256 di e en cu en alues could be gene a ed o he ull scale selec ed. The pola i y o he cu en could be con olled digi ally by ac i a ing o deac i a ing i s passage h ough a ol age in e e . The schema ic design o he o iginal gal anos a p o o ype can be ound in he Supplemen a y Ma e ials in [23]. Thus, he adap a ion o he gal anos a desc ibed abo e mainly consis ed o he subs i u ion o he BAT connec ions o he elec odes o he elec olysis cell. The o he digi al con ols we e kep o adjus he cu en passing h ough he elec olysis cell. Wi h his con igu a ion, he selec ed cu en , which is passed h ough he elec oly ic cell, was kep cons an by con inuously adjus ing he ol age applied o he ba e y unde s udy. Thus, i he elec ic esis ance in he elec oly ic cell inc eases du ing he p ocess, o main ain he cu en alue, he applied ol age will also inc ease. Mo eo e , in he case ha du ing he se up o he elec oly ic cell, he esis ance be ween he elec odes is abno mally high, o example, due o a connec ion ailu e, he ol age measu ed by he di e en ial OA will inc ease o i s maximum alue o 15 V ( he alue o he powe sou ce). The e o e, by moni o ing his ol age du ing he elec olysis p ocess, i is possible o de ec changes in he e iciency o he elec olysis p ocess and/o a ailu e in he elec ical connec ion be ween he elec odes. In any case, his ol age should be a posi i e alue and i has o be scaled o he adequa e ope a ion o he mic ocon olle . The co ec pola i y is imposed by using he digi al con ol o cha ging/discha ging, ensu ing ha a posi i e ol age is ob ained. To scale his elec ical signal o he s anda d alue o 5 V ( he maximum alue suppo ed by he mic ocon olle ), a simple ol age di ide was used, gua an eeing a maximum ou pu ol age o 4.7 V o a 15 V powe sou ce. The ol age di ide ou pu was ead by one analog inpu in he mic ocon olle . (b) Elec olysis cell: Elec odes we e placed in a s anda d 150 mL beake . A pla inum coil wi h an a ea o 1.25 cm 2 was employed as he indica o elec ode. A sil e squa e shee wi h an a ea o 3 cm 2 was employed as a coun e elec ode o single compa men cell expe imen s. A cylind ical ube (15 mm diame e ) wi h a i ed glass bo om co e ed wi h aga –aga gel sa u a ed wi h KCl was used as a sal b idge when wo king wi h a wo-compa men elec olysis cell. In he la e case, ano he P coil was used as he coun e elec ode. (c) Endpoin de ec o sys em: A simple colo ime e was de eloped based on a RGB LED ligh sou ce [ 30 ], coupled wi h wo kinds o ligh de ec o s: a pho o esis o LDR [ 31 ] and a pho odiode OPT101 [ 32 ]. The endpoin de ec o sys em allows o a limi ed selec ion o wa eleng hs, cen e ed close o he h ee maxima o he RGB ligh (460 nm o blue, 520 nm o g een, and 630 nm o ed) o ob ain he maximum sensi i i y depending on he colo de eloped in he solu ion. Be e sensi i i y is achie ed when he colo o he RGB illumina ion and he indica o colo a e nea ly complemen a y. The in ensi y o he ligh emi e colo is con olled by using h ee mic ocon olle PWM digi al pins. In ou case, a single colo o he RGB LED was u ned on, aking in o accoun he colo emi ed by he chemical indica o used. The elec ical signal ac i a ing he emi e can be se easily, and he change in he ligh in ensi y eaching he de ec o has o be scaled and adjus ed o maximize he ol age change. Senso s 2022,22, 7541 5 o 15 (d) LDR de ec o con igu a ion: A LDR changes he esis ance om ens o k Ω unde b igh ligh (1000 Lux) o app oxima ely 1 M Ω in he da k (<0.1 Lux). They a e eadily a ailable a low cos bu ha e a d awback because i s elec ical esponse is slow and no linea . Ne e heless, cheap LDRs possess su icien sensi i i y and can be easily implemen ed. The pho o conduc i e cell is elec onically a anged in a ol age di ide con igu a ion in which he ini ial ou pu alue can be p ecisely selec ed by using a couple o mul i u n po en iome e s: one o ough selec ion and ano he o ine- uning he ou pu ol age adjus men . The ou pu ol age dec eases as ligh in ensi y inc eases nonlinea ly. Figu e 1schema ically shows he connec ions acco ding o [33]. Senso s 2022, 22, x FOR PEER REVIEW 5 o 16 he change in he ligh in ensi y eaching he de ec o has o be scaled and adjus ed o maximize he ol age change. (d) LDR de ec o con igu a ion: A LDR changes he esis ance om ens o kΩ unde b igh ligh (1000 Lux) o app oxima ely 1 MΩ in he da k (<0.1 Lux). They a e eadily a ailable a low cos bu ha e a d awback because i s elec ical esponse is slow and no linea . Ne e heless, cheap LDRs possess su icien sensi i i y and can be easily imple- men ed. The pho o conduc i e cell is elec onically a anged in a ol age di ide con ig- u a ion in which he ini ial ou pu alue can be p ecisely selec ed by using a couple o mul i u n po en iome e s: one o ough selec ion and ano he o ine- uning he ou pu ol age adjus men . The ou pu ol age dec eases as ligh in ensi y inc eases nonlinea ly. Figu e 1 schema ically shows he connec ions acco ding o [33]. Figu e 1. A schema ic o he ol age di ide LDR connec ion. R1: LDR (pho o- esis o ); R2: 100 kΩ, R3: 1 MΩ, R4 and R5: 10 kΩ mul i- u n o he ough and ine- uned ol age ou pu adjus men , espec i ely. By using his ci cui and employing wo en- u n po en iome e s, he ou pu ol age can be adjus ed be ween 0 V and +5 V wi h a esolu ion (R4, R5) o abou one milli ol . This p o ides g ea e sa ili y o he selec ion o he esponse ange o he pho o esis o elemen . (e) Pho odiode (OPT101) de ec o con igu a ion: Ligh de ec o de ices and elec- onic ensembles o g ea e sensi i i y, which exhibi linea esponses, can be employed, o example, hose based on pho odiodes, pho o ansis o s, and e en a simple LED [20]. Based on he adequacy o i s cha ac e is ics o his applica ion, a pho odiode ( ype OPT101) was selec ed [32]. Basic connec ions o his de ice we e pe o med ollowing he schema ics depic ed in igu e 21 in [32] minimizing he numbe o elec onic compo- nen s equi ed. Minimizing he high- equency noise ha no mally occu s in da a acqui- si ion when in e acing he digi al mic ocon olle o an analog ligh de ec o o senso is impe a i e. Fo bo h de ec o s, a simple RC low pass il e wi h a cu o equency o 15Hz was inse ed be ween he de ec o ou pu and he analog inpu o he A duino [28]. ( ) Colo ime ic de ec o a angemen : The emi e and de ec o we e placed a he bo om o wo s anda d plas ic spec opho ome ic cu e es (1 cm o pa h ligh ). Bo h elec- onic de ices we e kep in place using a non-conduc ing black oam. The emi e and he Figu e 1. A schema ic o he ol age di ide LDR connec ion. R1: LDR (pho o- esis o ); R2: 100 k Ω , R3: 1 M Ω , R4 and R5: 10 k Ω mul i- u n o he ough and ine- uned ol age ou pu adjus men , espec i ely. By using his ci cui and employing wo en- u n po en iome e s, he ou pu ol age can be adjus ed be ween 0 V and +5 V wi h a esolu ion (R4, R5) o abou one milli ol . This p o ides g ea e sa ili y o he selec ion o he esponse ange o he pho o esis o elemen . (e) Pho odiode (OPT101) de ec o con igu a ion: Ligh de ec o de ices and elec onic ensembles o g ea e sensi i i y, which exhibi linea esponses, can be employed, o exam- ple, hose based on pho odiodes, pho o ansis o s, and e en a simple LED [ 20 ]. Based on he adequacy o i s cha ac e is ics o his applica ion, a pho odiode ( ype OPT101) was selec ed [ 32 ]. Basic connec ions o his de ice we e pe o med ollowing he schema ics depic ed in Figu e 2in [ 32 ] minimizing he numbe o elec onic componen s equi ed. Minimizing he high- equency noise ha no mally occu s in da a acquisi ion when in e - acing he digi al mic ocon olle o an analog ligh de ec o o senso is impe a i e. Fo bo h de ec o s, a simple RC low pass il e wi h a cu o equency o 15Hz was inse ed be ween he de ec o ou pu and he analog inpu o he A duino [28]. ( ) Colo ime ic de ec o a angemen : The emi e and de ec o we e placed a he bo om o wo s anda d plas ic spec opho ome ic cu e es (1 cm o pa h ligh ). Bo h elec onic de ices we e kep in place using a non-conduc ing black oam. The emi e and he de ec o cu e es we e ixed acing one ano he and sepa a ed by a hi d one, aised wi h espec o he o he wo, and kep in place wi h glue. The expe imen al assembly is depic ed in Figu e 2. Senso s 2022,22, 7541 6 o 15 Senso s 2022, 22, x FOR PEER REVIEW 6 o 16 de ec o cu e es we e ixed acing one ano he and sepa a ed by a hi d one, aised wi h espec o he o he wo, and kep in place wi h glue. The expe imen al assembly is de- pic ed in Figu e 2. Figu e 2. Colo ime ic endpoin de ec o ensemble (LDR/OPT101_RGB_LED) showing he emi e , he ligh de ec o s, and he spa ial a angemen . In his way, he colo ime e de ec o assembly can be easily imme sed in he cell so- lu ion, keeping he dis ance and posi ion o bo h he de ec o and emi e ixed. When he solu ion unde analysis s a ed o de elop a change in colo due o he excess elec ochem- ically-gene a ed eac an in e ac ing wi h he chemical indica o , he in ensi y o ligh eaching he ligh senso dec eased and he ou pu ol age changed. When he LDR de- ec o was employed, he ou pu ol age inc eased, while i dec eased when he pho odi- ode (OPT101) was used. The elec olysis p ocess con inued un il he ou pu ol age eached a p ese alue. The colo o he solu ion was kep uni o m by using a magne ic s i e (S ua CB162). 2.2. So wa e The gal anos a was con olled by an A duino UNO mic ocon olle , whose desc ip- ion and calib a ion is desc ibed in he li e a u e [23,27,34]. Howe e , he de elopmen o he ch ono-coulome y expe imen s no only equi ed he a o emen ioned ha dwa e ad- ap a ion, bu also he implemen a ion o so wa e adap ed o his ype o expe imen . In o de o pe o m a p ope coulome e ope a ion, he so wa e has o be uploaded in o he mic ocon olle and, in addi ion, an Excel shee in which he add-in is embedded has o be used [26,27]. A simpli ied desc ip ion o he ope a ing so wa e o he coulome e will be gi en in he ollowing de elopmen , while u he de ails a e gi en in he Supplemen- a y Ma e ials. Th ee undamen al s ages can be dis inguished wi hin he p og am, acco ding o he gene al scheme o he mic ocon olle : he ini ializa ion, se up, and loop p ocedu es. The i s s age co esponds o he ini ializa ion and assignmen o he di e en pins o he mi- c ocon olle . In his same s age, he ypes o a iables ha will be used in he expe imen a e de ined: accumula ed ime, po en ial, and cu en in ensi y. The co esponding alues o he o dina e and slope co esponding o he linea calib a ion we e also se . The nex s ep, which is also pe o med only once, ini ially con igu es he de aul al- ues o he a iables and es ablishes he se ial po communica ion. The esis ance be ween he elec odes is checked o e i y ha i s alue is low. Thus, he gal anos a is momen- a ily swi ched ON using a e y low cu en alue. Simul aneously, he ol age de eloped be ween he elec odes is ead, and i he alue is highe han he one p e iously speci ied, he p og am ends, indica ing ha he e is an e o in he elec ical connec ion. The expe - imen al a iables a e hen ead om he Excel shee using he PLX_DAQ se ial commu- nica ion add-on and a e checked o compliance wi h he app op ia e anges se p e i- ously. Thus, he cu en inpu alue en e ed in he sp eadshee has o be a alue wi hin he selec ed sensi i i y ange manually selec ed using he PCB p o o ype gal anos a [23]. Figu e 2. Colo ime ic endpoin de ec o ensemble (LDR/OPT101_RGB_LED) showing he emi e , he ligh de ec o s, and he spa ial a angemen . In his way, he colo ime e de ec o assembly can be easily imme sed in he cell solu- ion, keeping he dis ance and posi ion o bo h he de ec o and emi e ixed. When he solu- ion unde analysis s a ed o de elop a change in colo due o he excess elec ochemically- gene a ed eac an in e ac ing wi h he chemical indica o , he in ensi y o ligh eaching he ligh senso dec eased and he ou pu ol age changed. When he LDR de ec o was employed, he ou pu ol age inc eased, while i dec eased when he pho odiode (OPT101) was used. The elec olysis p ocess con inued un il he ou pu ol age eached a p ese alue. The colo o he solu ion was kep uni o m by using a magne ic s i e (S ua CB162). 2.2. So wa e The gal anos a was con olled by an A duino UNO mic ocon olle , whose desc ip- ion and calib a ion is desc ibed in he li e a u e [ 23 , 27 , 34 ]. Howe e , he de elopmen o he ch ono-coulome y expe imen s no only equi ed he a o emen ioned ha dwa e adap a ion, bu also he implemen a ion o so wa e adap ed o his ype o expe imen . In o de o pe o m a p ope coulome e ope a ion, he so wa e has o be uploaded in o he mic ocon olle and, in addi ion, an Excel shee in which he add-in is embedded has o be used [ 26 , 27 ]. A simpli ied desc ip ion o he ope a ing so wa e o he coulome- e will be gi en in he ollowing de elopmen , while u he de ails a e gi en in he Supplemen a y Ma e ials. Th ee undamen al s ages can be dis inguished wi hin he p og am, acco ding o he gene al scheme o he mic ocon olle : he ini ializa ion, se up, and loop p ocedu es. The i s s age co esponds o he ini ializa ion and assignmen o he di e en pins o he mic ocon olle . In his same s age, he ypes o a iables ha will be used in he expe imen a e de ined: accumula ed ime, po en ial, and cu en in ensi y. The co esponding alues o he o dina e and slope co esponding o he linea calib a ion we e also se . The nex s ep, which is also pe o med only once, ini ially con igu es he de aul alues o he a iables and es ablishes he se ial po communica ion. The esis ance be ween he elec odes is checked o e i y ha i s alue is low. Thus, he gal anos a is momen a ily swi ched ON using a e y low cu en alue. Simul aneously, he ol age de eloped be ween he elec odes is ead, and i he alue is highe han he one p e iously speci ied, he p og am ends, indica ing ha he e is an e o in he elec ical connec ion. The expe imen al a iables a e hen ead om he Excel shee using he PLX_DAQ se ial communica ion add-on and a e checked o compliance wi h he app op ia e anges se p e iously. Thus, he cu en inpu alue en e ed in he sp eadshee has o be a alue wi hin he selec ed sensi i i y ange manually selec ed using he PCB p o o ype gal anos a [ 23 ]. The p ocess o e i ica ion ends when all equi emen s o he a iables a e sa is ied: sensi i i y, ac ual cu en alue, RGB LED b igh ness, and iming pa ame e s. In coulome y, i is common o pe o m a p e- i a ion o selec he endpoin alue. Thus, a small amoun o he sample is added o he elec oly e solu ion and i a ed o a ce ain de ec o alue. Subsequen ly, when he sample is added, he i a ion will end Senso s 2022,22, 7541 7 o 15 a he p e iously selec ed endpoin [ 35 ]. Thus, his p ese alue o he op ical de ec o co esponding o he endpoin is hen se . P e iously, he colo o he ligh emi e and he mos app op ia e in ensi y would ha e been selec ed. The ac ual de ec o le el alue, gi en by he de ec o , is e i ied be o e he i a ion can be pe o med. Thus, o he LDR de ec o , he ac ual alue should be lowe han he endpoin selec ed, and he opposi e o he pho odiode OPT101. The ligh de ec o ype has o be p e iously selec ed, in o de o choose he adequa e p og am code lines (see Supplemen a y Ma e ials o de ails). Se ing he expe imen al pa ame e s concludes when he e a e no e o messages on he de ec o le el and equi ed pa ame e s. Two check ma ks o hese possible e o s ha e o be e ased on he con olle . Finally, he s a us o he s a /s op swi ch is checked and i s posi ion is held un il he s a o he i a ion. As he communica ion be ween he A duino and he sp eadshee is pe o med ia RS232 se ial, some pa ame e synch oniza ion e o s may occu , bu his can be easily sol ed by ese ing he Excel con olle wi hou losing any da a. In he hi d pa o he p og am, he p ocess o measu emen , endpoin con ol, and he es ima ion o esul s akes place. Figu e 3shows a low diag am o hese p ocesses. Senso s 2022, 22, x FOR PEER REVIEW 7 o 16 The p ocess o e i ica ion ends when all equi emen s o he a iables a e sa is ied: sen- si i i y, ac ual cu en alue, RGB LED b igh ness, and iming pa ame e s. In coulome y, i is common o pe o m a p e- i a ion o selec he endpoin alue. Thus, a small amoun o he sample is added o he elec oly e solu ion and i a ed o a ce ain de ec o alue. Subsequen ly, when he sample is added, he i a ion will end a he p e iously selec ed endpoin [35]. Thus, his p ese alue o he op ical de ec o co - esponding o he endpoin is hen se . P e iously, he colo o he ligh emi e and he mos app op ia e in ensi y would ha e been selec ed. The ac ual de ec o le el alue, gi en by he de ec o , is e i ied be o e he i a ion can be pe o med. Thus, o he LDR de ec o , he ac ual alue should be lowe han he endpoin selec ed, and he opposi e o he pho odiode OPT101. The ligh de ec o ype has o be p e iously selec ed, in o de o choose he adequa e p og am code lines (see Supplemen a y Ma e ials o de ails). Se ing he expe imen al pa ame e s con- cludes when he e a e no e o messages on he de ec o le el and equi ed pa ame e s. Two check ma ks o hese possible e o s ha e o be e ased on he con olle . Finally, he s a us o he s a /s op swi ch is checked and i s posi ion is held un il he s a o he i a- ion. As he communica ion be ween he A duino and he sp eadshee is pe o med ia RS232 se ial, some pa ame e synch oniza ion e o s may occu , bu his can be easily sol ed by ese ing he Excel con olle wi hou losing any da a. In he hi d pa o he p og am, he p ocess o measu emen , endpoin con ol, and he es ima ion o esul s akes place. Figu e 3 shows a low diag am o hese p ocesses. Figu e 3. Flow diag am o he measu emen and endpoin con ol. The e o condi ions a e also e i ied: high esis ance and eme gency s op. The sample olume is hen added o he solu ion and he p ocess is ini ia ed by ac i- a ing he s a /s op swi ch. Elec olysis a a gi en in ensi y akes place un il he colo o he solu ion eaches he p e iously selec ed de ec o alue. Du ing he i s elec olysis p ocess, he de ec o alue is plo ed on an Excel g aph e sus ime, allowing i o be mon- i o ed. When he selec ed de ec o alue is eached o he i s ime, he elec olysis is s opped o a ce ain pe iod, ixed as one o he ini ial condi ions. This p ocedu e allows he colo in he solu ion o become uni o m. Nex , he elec olysis is esumed and i his nex elec olysis ime is sho e han he alue speci ied unde he ini ial condi ions, he i a ion is conside ed comple ed and he o al elec olysis ime is de e mined. Comple ion Figu e 3. Flow diag am o he measu emen and endpoin con ol. The e o condi ions a e also e i ied: high esis ance and eme gency s op. The sample olume is hen added o he solu ion and he p ocess is ini ia ed by ac i a ing he s a /s op swi ch. Elec olysis a a gi en in ensi y akes place un il he colo o he solu ion eaches he p e iously selec ed de ec o alue. Du ing he i s elec olysis p ocess, he de ec o alue is plo ed on an Excel g aph e sus ime, allowing i o be moni o ed. When he selec ed de ec o alue is eached o he i s ime, he elec olysis is s opped o a ce ain pe iod, ixed as one o he ini ial condi ions. This p ocedu e allows he colo in he solu ion o become uni o m. Nex , he elec olysis is esumed and i his nex elec olysis ime is sho e han he alue speci ied unde he ini ial condi ions, he i a ion is conside ed comple ed and he o al elec olysis ime is de e mined. Comple ion o he i a ion in s ages minimizes he e o due o ins an aneous colo a ion exceeding he p ese alue o he de ec o . The numbe o s ages is limi ed o en cycles. In he second and subsequen elec olysis eac i a ion p ocesses (s ages), communica- ion h ough he in e ace wi h Excel is inhibi ed, s opping he simul aneous plo ing o he g aph o a p ecise con ol o he elapsed ime. Th oughou he elec olysis p ocess, he p og am moni o s he alue o he applied po en ial o de ec possible inc eases in elec ical esis ance be ween he elec odes. I his si ua ion occu s, he p ocess s ops, showing an Senso s 2022,22, 7541 8 o 15 e o message. The manual s op swi ch is also con inuously moni o ed, e mina ing he en i e p ocess when ac i a ed. Figu e 4shows a sc eensho o he sp eadshee in e ace o a simula ed measu emen p ocess using LDR as he ligh de ec o and a esis o as he dummy elec ochemical cell. Ini ially, he pa ame e s o be used in he expe imen a e se in sec ion B, as indica ed in Figu e 4. These a e checked agains he accep ed ange (sec ion A). I he e a e no e o s, he senso le el o he i a ion endpoin is se (sec ion C). Fo he LDR ligh senso case, du ing he i a ion p ocess, he senso alue inc eases. Thus, he endpoin selec ed mus be a g ea e alue han ha o he s a ing poin ( his equi emen is checked by he so wa e). Fo his pu pose, wo po en iome e s (R4 and R5 shown in Figu e 1) we e ope a ed. The plo in he sc eensho (Figu e 4) shows he e olu ion o he de ec o signal du ing he p og ess o he simula ed i a ion. Fo hese dummy es s, se ial communica ion was no inhibi ed, allowing o a ious s ages o be plo ed, which a e labeled by he numbe s 1, 2, and 3 in sec ion D. The ho izon al ed line indica es he limi alue o he endpoin senso . Fu he de ails on he measu ing ope a ion can be ound in he Supplemen a y Ma e ials. Senso s 2022, 22, x FOR PEER REVIEW 8 o 16 o he i a ion in s ages minimizes he e o due o ins an aneous colo a ion exceeding he p ese alue o he de ec o . The numbe o s ages is limi ed o en cycles. In he second and subsequen elec olysis eac i a ion p ocesses (s ages), communi- ca ion h ough he in e ace wi h Excel is inhibi ed, s opping he simul aneous plo ing o he g aph o a p ecise con ol o he elapsed ime. Th oughou he elec olysis p ocess, he p og am moni o s he alue o he applied po en ial o de ec possible inc eases in elec ical esis ance be ween he elec odes. I his si ua ion occu s, he p ocess s ops, showing an e o message. The manual s op swi ch is also con inuously moni o ed, e - mina ing he en i e p ocess when ac i a ed. Figu e 4 shows a sc eensho o he sp eadshee in e ace o a simula ed measu e- men p ocess using LDR as he ligh de ec o and a esis o as he dummy elec ochemical cell. Ini ially, he pa ame e s o be used in he expe imen a e se in sec ion B, as indica ed in Figu e 4. These a e checked agains he accep ed ange (sec ion A). I he e a e no e o s, he senso le el o he i a ion endpoin is se (sec ion C). Fo he LDR ligh senso case, du ing he i a ion p ocess, he senso alue inc eases. Thus, he endpoin selec ed mus be a g ea e alue han ha o he s a ing poin ( his equi emen is checked by he so - wa e). Fo his pu pose, wo po en iome e s (R4 and R5 shown in Figu e 1) we e ope a ed. The plo in he sc eensho (Figu e 4) shows he e olu ion o he de ec o signal du ing he p og ess o he simula ed i a ion. Fo hese dummy es s, se ial communica ion was no inhibi ed, allowing o a ious s ages o be plo ed, which a e labeled by he numbe s 1, 2, and 3 in sec ion D. The ho izon al ed line indica es he limi alue o he endpoin senso . Fu he de ails on he measu ing ope a ion can be ound in he Supplemen a y Ma e ials. Figu e 4. A sc eensho o he sp eadshee in e ace o es ing he en i e coulome ic i a ion p o- g am wi h a dummy cell and using he LDR as a ligh senso . (A) Inpu pa ame e s and e i ica ion anges. (B) Ac ual alue se . (C) De ec o le els se . (D) Measu emen esul s: numbe s 1, 2, 3 e e s o di e en expe imen al s ages. (E) Add-on module o se ial communica ion. (F) Plo scales. The comple e p og am code, wi h ema ks, can be supplied by he au ho s upon e- ques . Figu e 4. A sc eensho o he sp eadshee in e ace o es ing he en i e coulome ic i a ion p og am wi h a dummy cell and using he LDR as a ligh senso . ( A ) Inpu pa ame e s and e i ica ion anges. ( B ) Ac ual alue se . ( C ) De ec o le els se . ( D ) Measu emen esul s: numbe s 1, 2, 3 e e s o di e en expe imen al s ages. (E) Add-on module o se ial communica ion. (F) Plo scales. The comple e p og am code, wi h ema ks, can be supplied by he au ho s upon eques . 3. Resul s and Discussion In his sec ion, he main esul s ob ained when using ue elec ochemical cell es s will be p esen ed. The issues encoun e ed and possible imp o emen s a e also analyzed and discussed. Two common coulome ic eagen s we e elec o-gene a ed in a ious com- bina ions, assemblies, and di e en con igu a ions [ 36 ]. All measu emen s we e pe o med a a con olled empe a u e o 25 ±2◦C. Be o e using he gal anos a , i is impo an o ha e he cu en calib a ions o he di e en cu en scales a ailable. P ecision esis o s a e connec ed a he elec ode e minals o he di e en scales. The sys em exhibi s a mono onic and pe ec ly linea ela ionship Senso s 2022,22, 7541 9 o 15 be ween he measu ed cu en and he PWM digi al alue, wi h less han 1% e o , inde- penden ly o he selec ed scale. Conside ing he cu en scale used (sensi i i y), a single linea eg ession (slope, in e cep ) can also be used o all scales (see he Supplemen a y Ma e ials o a de ailed desc ip ion o he calib a ion p ocess). 3.1. Iodome y Analysis The main edox eac ion in iodome ic i a ions is he educ ion o iodine o iodide and, con e sely, he oxida ion o iodide o iodine: I2(aq)+2e−2I−(aq) Thus, iodine is gene a ed a he anode by oxida ion o he iodide, bu he iodine p oduced can be educed again a he ca hode. The e o e, he coulome ic elec oly ic cell needs o ha e sepa a e compa men s, as can be seen in Figu e 5. A glass ube con aining i ed glass a he bo om wi h a laye o aga –aga illed wi h a sa u a ed KCl solu ion, was used as a sal b idge. In his way, con ec i e eac an anspo om one compa men o ano he was hinde ed, keeping he elec ical esis i i y o he memb ane low. Senso s 2022, 22, x FOR PEER REVIEW 9 o 16 3. Resul s and Discussion In his sec ion, he main esul s ob ained when using ue elec ochemical cell es s will be p esen ed. The issues encoun e ed and possible imp o emen s a e also analyzed and discussed. Two common coulome ic eagen s we e elec o-gene a ed in a ious com- bina ions, assemblies, and di e en con igu a ions [36]. All measu emen s we e pe - o med a a con olled empe a u e o 25 ± 2 °C. Be o e using he gal anos a , i is impo an o ha e he cu en calib a ions o he di e en cu en scales a ailable. P ecision esis o s a e connec ed a he elec ode e mi- nals o he di e en scales. The sys em exhibi s a mono onic and pe ec ly linea ela ion- ship be ween he measu ed cu en and he PWM digi al alue, wi h less han 1% e o , independen ly o he selec ed scale. Conside ing he cu en scale used (sensi i i y), a sin- gle linea eg ession (slope, in e cep ) can also be used o all scales (see he Supplemen- a y Ma e ials o a de ailed desc ip ion o he calib a ion p ocess). 3.1. Iodome y Analysis The main edox eac ion in iodome ic i a ions is he educ ion o iodine o iodide and, con e sely, he oxida ion o iodide o iodine: 𝐼(aq) + 2𝑒⇄2𝐼 (aq) Thus, iodine is gene a ed a he anode by oxida ion o he iodide, bu he iodine p o- duced can be educed again a he ca hode. The e o e, he coulome ic elec oly ic cell needs o ha e sepa a e compa men s, as can be seen in Figu e 5. A glass ube con aining i ed glass a he bo om wi h a laye o aga –aga illed wi h a sa u a ed KCl solu ion, was used as a sal b idge. In his way, con ec i e eac an anspo om one compa - men o ano he was hinde ed, keeping he elec ical esis i i y o he memb ane low. Figu e 5. Schema ics o he wo-compa men coulome ic cell employed o iodome ic i a ions. Molecula iodine is no soluble in wa e , bu is solubilized in he p esence o iodide, o ming iodine–iodide complexes such as 𝐼  o 𝐼 . These anions in e cala e in o he s a ch s uc u e, o ming a cha ge ans e complex wi h an in ense blue colo , wi h a b oad abso p ion band in he isible ange be ween 600 nm and 650 nm [37]. A ew d ops Figu e 5. Schema ics o he wo-compa men coulome ic cell employed o iodome ic i a ions. Molecula iodine is no soluble in wa e , bu is solubilized in he p esence o iodide, o ming iodine–iodide complexes such as I− 3 o I− 5 . These anions in e cala e in o he s a ch s uc u e, o ming a cha ge ans e complex wi h an in ense blue colo , wi h a b oad abso p ion band in he isible ange be ween 600 nm and 650 nm [ 37 ]. A ew d ops o colloidal s a ch solu ion added o an iodide solu ion will cause he solu ion o u n a blue colo in he p esence o iodine. The ligh emi e endpoin de ec o is illumina ed by he ed ligh o he RGB LED, hus ob aining op imal sensi i i y. Two ypes o iodome ic i a ions we e pe o med using I 2 as he oxidizing agen : he classical hiosul a e i a ion is acco ding o he eac ion: 2S2O2− 3(aq)+I2→S4O2− 6(aq)+2I−(aq) Subsequen ly, he i a ion o asco bic acid is acco ding o he eac ion: C6H8O6(aq)+I2→C6H6O6(aq)+2I−(aq)+2H+(aq)