Methylene blue da leucomethylene blue: ma'auratan redox guda ɗaya, launuka biyu

Wata ɗaliba ta ƙara 'yan ɗigon rini shuɗi a cikin flask, ta juya shi a hankali, sannan ta kalli launin yana ɓacewa. Ta girgiza flask ɗin, sai shuɗin ya dawo. Babu abin da aka ƙara ko aka cire tsakanin yanayin biyu sai electron da haɗuwa da iska. Wannan flask ya ƙunshi duk abin da wannan maƙala ke magana a kai: methylene blue da abokin ma'auratarsa da aka samu ta ridaksiyon, leucomethylene blue, suna sauyawa zuwa juna a matsayin ma'auratan redox masu amsawa ta alkiblu biyu, inda launi yake nuna sakamakon.

Muhimmin abin amfani shi ne na farko. Sauyin launi ba wani tasiri ne na gefe a nan ba. Shi ne siginar nazari. Duk wanda yake haɗa samfur da methylene blue, yin titration da shi, ko koyarwa da shi ya kamata ya iya rubuta rabin amsawar sinadaran ba tare da dubawa ba, ya bayyana yanayin da ke tura ta hagu ko dama, sannan ya ambaci ƙazantattun sinadaran da ke sa sakamakon ya zama marar bayyana.

Ma'auratan a cikin lissafi guda ɗaya

Methylene blue da aka yi wa oksidawa shi ne katayon na methylthioninium: tsarin phenothiazinium mai shimfiɗa, wanda yake da sulfur da nitrogen a zoben tsakiya da kuma rukunin dimethylamino a kowane ƙarshensa, ana samar da shi a matsayin gishirin chloride. PubChem ya rubuta shi a matsayin methylthioninium chloride, C.I. Basic Blue 9. A cikin ruwa, monomer ɗin yana shan haske kusa da 664 nm, wanda shi ne shuɗin da ido yake gani.

Leucomethylene blue (LMB) da aka samu ta ridaksiyon yana da irin wannan kwarangwal ɗin tsarin, amma nitrogen na zoben tsakiya ya karɓi proton kuma an katse chromophore ɗin. Ba shi da cajin lantarki a pH kusa da tsaka-tsaki, kuma kusan ba shi da launi; shan hasken da ido ke gani bai kai a lissafta shi ba, amma yana da ƙaƙƙarfan band ɗin shan hasken ultraviolet kusa da 258 nm (bayanan PubChem na leucomethylene blue, CAS 613-11-6). Yawan proton a cikin rabin amsawar ya dogara da pH, saboda nau'in da aka samu ta ridaksiyon yana karɓar proton a cikin acid (ƙimomin pKa na kusan 4.5 da 5.8). Yanayi uku sun rufe zangon da ake amfani da shi a aikace:

MB(+) + 2 e(-) + H(+) <=> LMB, shuɗi <=> marar launi (pH kusa da tsaka-tsaki, proton 1)

MB(+) + 2 e(-) + 2 H(+) <=> LMBH(+), shuɗi <=> marar launi (mai matsakaicin acidity, proton 2)

MB(+) + 2 e(-) + 3 H(+) <=> nau'in leuco da ya karɓi proton biyu (mai tsananin acidity, proton 3)

Duk waɗannan rubuce-rubucen uku suna bayyana ma'auratan guda ɗaya a ƙarƙashin yanayi daban-daban na karɓar proton, batun da wallafe-wallafen halayen redox na rinayen thiazine suka tattauna cikin tsari. Dogaro da pH yana bin lissafin Nernst na Ox + mH(+) + 2e(-) <=> Red, yana ba da gangara ta kusan 29.6, 59.2, ko 88.7 mV ga kowace raka'ar pH idan m = 1, 2, ko 3. Gangarorin da aka auna kusa da 28, 58, da 87 mV ga kowace raka'ar pH suna tabbatar da wannan dangantaka, don haka potenshiyal da aka bayar ba tare da pH ɗinsa ba ba shi da ma'ana.

Abin da lissafin yake gaya wa mai haɗa samfur

Abubuwa uku suna fitowa daga stoichiometry. Na farko, electron biyu ga kowane molecule yana nufin cewa methylene blue mai nuna alama ne kuma mai shiga tsakani na electron biyu, ba mai jigilar electron ɗaya ba, idan aka duba amsawarsa gaba ɗaya. Na biyu, proton suna shiga cikin amsawar, don haka potenshiyal na formal yana sauyawa da pH kamar yadda aka bayyana a sama. Na uku, potenshiyal na formal a pH 7 yana kusa da +0.011 V idan aka kwatanta da daidaitaccen electrode na hydrogen (kusan +0.53 V a pH 0), wanda yake sanya ma'auratan cikin sinadaran oksidawa marasa tsanani: suna da ƙarfin karɓar electron daga masu bayarwa masu kyau kamar NAD(P)H ko ascorbate, amma ba su da ƙarfin da zai hana su miƙa electron ga oxygen da ya narke ko cibiyoyin ferric. Wannan matsayi na tsakiya shi ne ainihin dalilin da ya sa rinin yake zagayawa maimakon ya makale a gefe ɗaya.

Taro yana ƙara wani abin lura. Idan ya haura kusan micromolar ɗari a cikin ruwa, daidai da ƙimar da aka bayar ta ma'aunin rabuwar monomer da dimer kusa da 170 zuwa 250 uM a pH 7, katayon ɗin da aka yi wa oksidawa yana jeruwa zuwa dimer da manyan taruka, tare da band ɗin shan haske da ya matsa zuwa kusa da 610 zuwa 615 nm. Saboda haka, bakan haske da kololuwarsa ba ta bayyana a wurin da ake tsammani ba na iya nufin taruwar molecule, ba ƙazanta ba. A fara tsarma shi, sannan a yanke hukunci.

Yanayin da ke tura kowace alkibla

Teburin da ke ƙasa abin dubawa ne yayin aiki a dakin gwaje-gwaje. Ridaksiyon yana buƙatar mai bayar da electron; sake oksidawa yana buƙatar mai karɓar electron, kuma yawanci iska ta wadatar.

Kowane abin da ke cikin teburin yana bin lissafi guda ɗaya. Ƙarfin da yake tura amsawar ne kawai yake sauyawa. Wannan shi ne ma'anar cewa ma'auratan suna iya komawa baya: hanya ɗaya ce, ana binta ta alkiblu masu kishiyantar juna a ƙarƙashin yanayi masu kishiyantar juna.

Bayanin kwalbar shuɗi

Ya dace a bayyana tsarin wannan gwajin a sarari domin shi ne ma'auratan a ƙaramin tsari. A cikin maganin glucose na alkaline, base yana taimaka wa glucose yin enolization, kuma enediolate da ya samu yana yin ridaksiyon ga methylene blue zuwa leucomethylene blue, don haka flask ɗin ya zama marar launi. Girgizawa yana narkar da oxygen daga sararin da ke saman ruwan; oxygen yana sake yin oksidawa ga nau'in leuco, sai shuɗin ya dawo. Barin shi tsaye yana rage oxygen da ya narke kusa da yankin amsawar a saman ruwan, kuma glucose ya sake yin rinjaye. Zagayowar tana maimaituwa har sai an cinye glucose, kuma kowace girgizawa lissafin Nernst ne da ake gani: ƙara aikin sinadarin oksidawa, sai daidaiton ya karkata zuwa shuɗi.

Radikal ɗin da ke tsakiya

Amsawar gaba ɗaya tana motsa electron biyu, amma matakan asali suna motsa ɗaya a lokaci guda. Tsakanin katayon shuɗi da nau'in leuco marar launi akwai tsaka-tsakin radikal na semiquinone wanda yawanci yake yin disproportionation, ya koma ma'auratan biyu maimakon ya taru. Potenshiyal ɗinsa na electron ɗaya kusa da minus 0.23 V a pH 7 yana ƙasa sosai da tsakiyar potenshiyal na amsawar electron biyu gaba ɗaya, shi ya sa radikal ɗin yake ci gaba da kasancewa nau'i mai ƙaramin adadi a yanayin da ya daidaita. Yana da muhimmanci saboda dalilai biyu. Idan aka haska rinin, yana kaiwa yanayin kuzari mai ƙaruwa da ke buɗe hanyoyin radikal; wannan shi ya sa magungunan ruwa na methylene blue suke da saurin sauyawa saboda haske, kuma shi ya sa sinadaran haske suke cikin duk wata ƙa'idar gwajin kwanciyar hankali. Haka kuma, idan taro ya yi yawa, nau'in da aka yi wa oksidawa yana haɗuwa ya samar da dimer, wanda yake sauya bakan haske da saurin amsawar da ake gani ba tare da sauya ainihin ma'auratan ba. Babu ɗayan waɗannan abubuwan da yake hana amsawar komawa baya. Dukansu suna bayyana abubuwan da ake gani waɗanda, in ba haka ba, za su yi kama da rashin iya komawa baya.

Sinadarai masu dangantaka da aka cire wa methyl suna sauya ma'aunin

Cire rukunonin methyl daga rassan dimethylamino biyu, kuma ma'auratan suna ci gaba da kasancewa amma halayensu sun sauya. Matsayin mafi yawan shan haske yana matsawa a jere zuwa gajeren tsawon igiyar haske a bangaren shuɗi duk lokacin da aka cire methyl: methylene blue kusa da 664 nm, Azure B kusa da 654, Azure A kusa da 630, Azure C kusa da 615, da thionine kusa da 600 nm. Azure B, wanda ba shi da rukunin methyl guda ɗaya, shi ne mafi kusancin ɗan'uwa: babban sinadarin da metabolism ke samarwa daga methylene blue, kuma ƙazanta da aka rubuta cewa tana cikin kayan methylene blue. Azure A da Azure C suna ci gaba da jerin tare da irin wannan kwarangwal mai shiga amsawar redox. Thionine, wanda aka cire duk methyl daga rukunonin amino biyunsa, yana ƙarshen jerin da potenshiyal na formal kusa da +0.06 V a pH 7, idan aka kwatanta da methylene blue a +0.011 V; ya fi sauƙin yin ridaksiyon sosai. Potenshiyal ɗin redox a cikin jerin ya dogara da matsakaicin amsawar da electrode, don haka a kwatanta ƙimomi ne kawai idan yanayinsu ɗaya ne. Wannan shi ya sa tantance nau'o'in ƙazanta batun redox ne, ba wai kawai wani akwatin tabbatar da tsarki ba. Ƙananan kaso na Azure B yana nufin ƙananan kaso na wasu ma'auratan redox daban a cikin kwalbar, kuma Azure B a matsayin ƙazanta da samfurin metabolism yana bayyana yadda ake sarrafa wannan kaso.

Inda ilimin halittu yake shiga

A cikin ƙwayar jan jini, NADPH yana yin ridaksiyon ga methylene blue zuwa leucomethylene blue, sannan nau'in leuco yana miƙa electron ga ƙarfen ferric na heme, yana mayar da hemoglobin zuwa yanayin ferrous. Wannan miƙa electron daga ɗaya zuwa ɗaya shi ne tushen sinadarai na amfani da methylene blue a methemoglobinemia da aka samu bayan haihuwa. Duk abin da ya biyo bayan wannan miƙawar, wato metabolism na kuzari, kwararar electron a mitochondria, da duk wani sakamakon halittu, yana cikin jigilar electron a mitochondria kuma da gangan aka bar shi a can.

Sayi ma'auratan, ba launin kaɗai ba

Ingancin rinin redox bai wuce abin da ƙa'idodin samfurinsa suka tabbatar ba. Babban tsarki yana da muhimmanci saboda methylene blue da ba na USP ba yawanci na masana'antar yadi ne, yana ɗauke da Azure B, ƙarafa masu nauyi, ko ragowar sinadaran narkarwa waɗanda suke sauya karatun redox da ɓata wuraren ƙarshen amsawa—abubuwan da babu wata ƙa'idar sanya launi da take bincika su. Yawancin masu sayarwa da suke ikirarin bin USP suna gwada ƙarafa masu nauyi kawai, sannan su ce sun cika sharuddan. Cikakken bin ƙa'idar ya haɗa da tabbatar da ainihi, auna yawan sinadarin, ƙazantattun sinadaran organic, ragowar sinadaran narkarwa, ƙazantattun abubuwan sinadarai, ragowar ƙonawa, iyakokin ƙananan halittu, da endotoxin na ƙwayoyin bacteria; kuma yadda za a zaɓi methylene blue na matsayin magunguna yana bayyana yadda ake karanta waɗannan takardu. Blupreme yana aiki tare da mai kera methylene blue na matsayin magunguna, yana gwada cikakkun ƙa'idodin USP, sannan yana wallafa cikakken COA. Idan launi shi ne abin da ake aunawa, waɗannan takardu wani ɓangare ne na kayan aunawa.