Thaum kawm txog cov tshuaj hauv cov organic chemistry, ntau tshaj li kaum ob qhov kev ua tau zoo sib txawv yog siv los txiav txim siab cov ntsiab lus ntawm qee cov tebchaw. Xws li kev tsom xam pom tso cai rau koj kom nkag siab tam sim ntawd seb puas muaj cov khoom tsim nyog tam sim no, thiab yog tias lawv tsis nyob, koj tuaj yeem txo qhov kev sim ntxiv los txheeb xyuas lawv. Cov tshuaj tiv thaiv no suav nrog ninhydrin, uas yog qhov tseem ceeb hauv kev txiav txim siab pom cov amino compounds.
Qhov no yog dab tsi?
Ninhydrin yog ib qho dicarbonyl compound uas muaj ib lub nplhaib uas muaj ntxhiab nrog lub heterocycle txuas rau nws, lub atom thib ob uas muaj 2 pawg hydroxyl (OH-). Cov tshuaj no tau txais los ntawm oxidation ncaj qha ntawm inandione - 1, 3, thiab, yog li ntawd, raws li lub thoob ntiaj teb nomenclature, nws muaj cov nram qab no lub npe: 2, 2 - dihydroxyinandione -1, 3 (Fig. 1).
Ntshiab ninhydrin yog cov xim daj lossis dawbxim uas, thaum rhuab, yaj zoo nyob rau hauv dej thiab lwm yam polar organic solvents, xws li acetone. Qhov no yog ib qho teeb meem zoo nkauj, yog tias nws nkag mus rau hauv daim tawv nqaij hauv qhov ntau lossis cov mucous membranes, nws ua rau khaus, suav nrog thaum nqus tau. Ua hauj lwm nrog cov tshuaj no yuav tsum tau ceev faj thiab tsuas yog nrog hnab looj tes, raws li thaum nws los rau hauv kev sib cuag nrog daim tawv nqaij, nws reacts nrog daim tawv nqaij cell proteins thiab stains nws liab doog.
Reactive Substances
Raws li tau hais los saum no, cov tshuaj ninhydrin yog siv feem ntau rau kev txiav txim siab pom ntawm cov ntsiab lus ntawm cov amino compounds:
- α-amino acids (nrog rau cov proteins);
- amino qab zib;
- alkaloids uas muaj -NH2 thiab -NH pawg;
- ntau yam amines.
Nws yuav tsum tau muab sau tseg tias theem nrab thiab theem nrab amines qee zaum hnov mob tsis muaj zog, yog li xav tau kev tshawb fawb ntxiv kom paub meej tias lawv muaj.
Ntau txoj hauv kev ntawm chromatography yog siv rau kev txiav txim siab ntau, piv txwv li, ntawv chromatography (BC), nyias txheej chromatography (TLC) lossis nrog kev ntxuav cov khoom nqa nrog cov tshuaj ninhydrin hauv ntau yam xov xwm.
Cov tshuaj tiv thaiv no tsis yog tshwj xeeb rau cov amino compounds, txij li cov reagent tuaj yeem nkag mus rau hauv nws nrog tag nrho ib zaug. Txawm li cas los xij, ntawm ib feem ntawm cov khoom siv tshuaj tiv thaiv, nws muaj qhov tshwj xeeb hauv daim ntawv tso tawm cov pa roj carbon dioxide npuas (CO 2), thiab qhov no yog ib txwm tsuas yog thaum cuam tshuam nrog α-amino. acids.
Mechanism nta
BMuaj kev txhais lus sib txawv ntawm qhov sib npaug ninhydrin tshuaj tiv thaiv hauv cov ntaub ntawv. Qee cov kws tshawb fawb tshem tawm qhov tsim ntawm hydrindanthine los ntawm 2-aminoinandione, uas, nrog kev koom tes ntawm ammonia thiab ninhydrin, kuj tsim cov xim xim hu ua "Rueman's ntshav" (lossis "Rueman's xiav"), thaum lwm tus, ntawm qhov tsis sib xws, xav tias tsuas yog nws cov xim liab. kev koom tes tsis muaj cov khoom nruab nrab ntawm cov amino acids. Tseem muaj qee cov ntsiab lus nthuav dav hauv cov ntaub ntawv ntawm cov tshuaj tiv thaiv nws tus kheej, tshwj xeeb, qhov no cuam tshuam cov txheej txheem ntawm kev txuas cov amino derivative ntawm ninhydrin rau nws lub ntsiab molecule los tsim cov xim. Qhov taw qhia ntawm qhov chaw ntawm "taug kev hydrogen" tau txais los ntawm qhov nruab nrab amine los ntawm qhov nruab nrab aqueous tseem muaj lus nug: nws tuaj yeem yog nyob rau hauv pawg ketone lossis ib sab ntawm -NH 2.
Hauv kev muaj tiag, qhov nuance nrog H atom yog qhov tsis tseem ceeb, txij li nws txoj haujlwm hauv cov khoom sib xyaw tsis ua lub luag haujlwm tshwj xeeb hauv cov tshuaj tiv thaiv, yog li nws yuav tsum tsis txhob saib xyuas. Raws li rau qhov kev tshem tawm ntawm ib qho ntawm cov theem ua tau, vim li cas ntawm no nyob rau hauv qhov kev xav: txog tam sim no, lub caij nyoog mechanism rau kev tsim ntawm Rueman's ntshav tsis tau txiav txim siab, yog li cov tswv yim sib txawv ntawm cov tshuaj tiv thaiv ninhydrin tuaj yeem pom.
Qhov ua tiav qhov ua tiav ntawm kev sib cuam tshuam ntawm cov reagent nrog cov amino tebchaw yuav raug qhia hauv qab no.
Reaction mechanism
Ua ntej, ninhydrin cuam tshuam nrog α-amino acid, txuas rau ntawm qhov chaw ntawm kev sib cais ntawm pawg hydroxy thiab tsim cov khoom condensation (Daim duab 2a). Tom qab ntawd cov tom kawg raug rhuav tshem, tso cov amine nruab nrab, aldehyde, thiab carbon dioxide (Fig. 2b). Los ntawm cov khoom kawg thaum koom nrogninhydrin, Rueman ntshav qauv (diketonhydrindenketohydrinamine, Fig. 2c) yog synthesized. Tsis tas li ntawd qhia tau hais tias muaj peev xwm tsim hydrindanthine (txo ninhydrin) los ntawm nruab nrab amine, uas kuj hloov mus rau hauv cov xim sib xyaw nrog ammonia (ntau dua, ammonium hydroxide) nrog ntau tshaj ntawm cov reagent nws tus kheej (Fig. 2d).
Kev tsim ntawm hydrindantine tau ua pov thawj los ntawm Rueman nws tus kheej thaum hydrogen sulfide ua rau ninhydrin molecule. Cov tshuaj no muaj peev xwm yaj hauv sodium carbonate Na 2CO3, pleev xim rau cov tshuaj tsaus liab. Thiab thaum dilute hydrochloric acid ntxiv, hydrindanthine precipitates.
Feem ntau yuav, qhov nruab nrab amine, hydridanthin, ninhydrin thiab cov qauv ntawm cov xim, vim lawv tsis ruaj khov thaum rhuab, nyob rau hauv qee qhov sib npaug, uas tso cai rau muaj ntau theem ntxiv.
Cov txheej txheem no yog tsim los piav qhia cov tshuaj ninhydrin nrog rau lwm cov amino compounds, tshwj tsis yog cov khoom lag luam uas tshwm sim los ntawm kev tshem tawm cov seem ntawm cov qauv los ntawm -NH 2, -NH or -N.
Biuret test thiab lwm yam kev cuam tshuam rau cov proteins
Kev tshuaj xyuas zoo rau peptide daim ntawv cog lus txawm tias cov qauv tsis muaj protein ntau tuaj yeem tshwm sim tsis yog nrog kev koom tes ntawm cov reagent saum toj no. Txawm li cas los xij, nyob rau hauv cov ntaub ntawv ntawm ninhydrin cov tshuaj tiv thaiv rau cov protein, kev sib cuam tshuam tsis tshwm sim nrog -CO-NH‒ pawg, tab sis nrog pawg amine. Muaj ib qho kev hu ua "biuret cov tshuaj tiv thaiv", uas yog tus cwj pwm los ntawm kev sib ntxiv ntawm ions rau cov tshuaj nrog cov amino compounds.bivalent tooj liab los ntawm CuSO4 los yog Cu(OH)2 hauv nruab nrab alkaline (Fig. 3).
Thaum kev tshuaj xyuas, nyob rau hauv lub xub ntiag ntawm cov qauv tsim nyog, kev daws teeb meem tig xiav tsaus vim yog kev khi ntawm peptide bonds rau hauv cov xim complex, uas txawv ib qho reagent los ntawm lwm tus. Tias yog vim li cas biuret thiab ninhydrin cov tshuaj tiv thaiv yog universal nyob rau hauv kev sib raug zoo rau cov protein thiab tsis-protein qauv nrog -CO-NH‒ pab pawg.
Thaum txiav txim siab cyclic amino acids, xantoprotein cov tshuaj tiv thaiv nrog cov tshuaj muaj zog ntawm nitric acid HNO3 yog siv, uas muab cov xim daj thaum nitrated. Ib qho poob ntawm reagent rau ntawm daim tawv nqaij kuj pom cov xim daj los ntawm reacting nrog cov amino acids nyob rau hauv daim tawv nqaij hlwb. Nitric acid tuaj yeem ua rau kub hnyiab tau thiab yuav tsum tau siv hnab looj tes.
Piv txwv ntawm kev cuam tshuam nrog cov amino compounds
Ninhydrin cov tshuaj tiv thaiv rau α-amino acids muab qhov pom kev zoo, tshwj tsis yog cov xim proline thiab hydroxyproline cov qauv, uas cuam tshuam nrog kev tsim cov xim daj. Kev piav qhia txog qhov tshwm sim no tau pom nyob rau hauv lwm qhov chaw ib puag ncig ntawm kev sib cuam tshuam ntawm ninhydrin nrog cov qauv no.
Reaction nrog amino pawg
Vim qhov kev sim tsis yog qhov tshwj xeeb, kev pom kev pom ntawm alanine siv cov tshuaj ninhydrin hauv kev sib xyaw ua tsis tau. Txawm li cas los xij, los ntawm daim ntawv chromatography, thaum siv cov qauv ntawm ntau yam α-amino acids, txau nrog cov tshuaj aqueous ntawm ninhydrin thiab txhim kho hauv qhov nruab nrab tshwj xeeb, ib tus tuaj yeem ua tau.xam qhov muaj pes tsawg leeg ntawm tsis yog tsuas yog thov cov compound, tab sis kuj muaj ntau lwm tus.
Schematically, kev sib cuam tshuam ntawm alanine nrog ninhydrin ua raws tib txoj cai. Nws txuas rau cov reagent ntawm pawg amine, thiab nyob rau hauv qhov kev txiav txim ntawm active hydronium ions (H3O+) raug cais tawm ntawm cov pa roj carbon. -nitrogen daim ntawv cog lus, decomposing rau hauv acetaldehyde (CH 3 COH) thiab carbon dioxide (CO2). Lwm cov ninhydrin molecule txuas rau nitrogen, tshem tawm cov dej molecules, thiab cov qauv xim yog tsim (Fig. 4).
Reaction nrog heterocyclic amino compound
Cov tshuaj tiv thaiv ninhydrin nrog proline yog qhov tshwj xeeb, tshwj xeeb tshaj yog nyob rau hauv chromatographic analyses, txij li cov qauv no hauv cov kua qaub nruab nrab ua ntej tig daj, thiab tom qab ntawd tig liab doog hauv qhov nruab nrab. Cov kws tshawb fawb piav qhia qhov no los ntawm qhov tshwj xeeb ntawm lub voj voog rov ua haujlwm hauv qhov nruab nrab nruab nrab, uas cuam tshuam ncaj qha los ntawm qhov muaj ntau ntawm hydrogen protons uas ntxiv rau sab nraud lub zog ntawm nitrogen.
Kev puas tsuaj ntawm heterocycle tsis tshwm sim, thiab lwm cov ninhydrin molecule txuas rau nws ntawm 4 carbon atom. Thaum cua sov ntxiv, cov qauv tsim nyob rau hauv nruab nrab nruab nrab hloov mus rau hauv Rueman ntshav (Fig. 5).
Kev npaj ntawm lub ntsiab reagent
Ninhydrin kuaj yog ua tiav nrog cov kev daws teeb meem sib txawv, nyob ntawm kev sib cais ntawm cov qauv amino hauv qee cov organic thiabinorganic tebchaw.
Lub ntsiab reagent yog kev npaj ntawm 0.2% tov hauv dej. Qhov no yog ntau yam sib tov, txij li feem ntau cov tebchaw dissolve zoo nyob rau hauv H2O. Txhawm rau kom tau txais cov tshuaj reagent tshiab, ib qho piv txwv ntawm 0.2 g ntawm cov tshuaj ntshiab ninhydrin yog diluted hauv 100 ml dej.
Nws yog ib qho tsim nyog sau cia tias rau qee qhov kev tshuaj ntsuam kev daws teeb meem no tsis txaus, yog li 1% lossis 2% kev daws teeb meem tuaj yeem npaj tau. Qhov no yog ib qho tseem ceeb rau kev rho tawm los ntawm cov khoom siv tshuaj, vim tias lawv muaj ntau hom amino compounds.
Thaum ua cov kev tshawb fawb chromatographic, cov kev daws teeb meem, piv txwv li, thaum ntxuav cov khoom sib tov ntawm cov khoom thauj los ntawm ib sab, tuaj yeem npaj tau hauv cawv, dimethyl sulfoxide, acetone thiab lwm cov kuab tshuaj polar - txhua yam yuav nyob ntawm qhov hnyav ntawm qee yam. Cov qauv amino.
Daim ntawv thov
Cov tshuaj tiv thaiv ninhydrin ua rau nws tuaj yeem kuaj pom ntau cov amino tebchaw hauv cov tshuaj, uas ua rau nws yog ib qho ntawm thawj zaug siv rau hauv kev soj ntsuam zoo ntawm cov organic tshuaj. Kev txiav txim siab pom qhov txo qis ntawm cov kev sim, tshwj xeeb tshaj yog thaum txheeb xyuas cov nroj tsuag tsis zoo, tshuaj thiab ntau npaum li cas, nrog rau cov kev daws teeb meem tsis paub thiab sib tov.
Hauv kev tshawb fawb forensic, txoj kev no tau siv dav los txiav txim siab muaj cov cim tawm hws ntawm txhua qhov chaw.
Txawm tias txawm tias tsis muaj qhov tshwj xeeb ntawm cov tshuaj tiv thaiv, kev tshem tawm ntawm ninhydrin cov tshuaj tiv thaiv los ntawm kev siv tshuaj tsis tuaj yeem, txij liHloov cov tshuaj no nrog cov tshuaj lom neeg tsawg dua (piv txwv li, oxolin) ua pov thawj tias lawv muaj qhov tsis zoo rau cov amino pawg thiab tsis muab cov txiaj ntsig zoo hauv kev tshuaj ntsuam photometric.
