Ukulawula ukuHamba: IsiKhokelo seLab Veteran kwiZigaba eziSelfowuni zeHPLC

Intshayelelo

Ndikhe ndaya apho ukhoyo-ndijonge esweni nge-2 AM, ndibukele isiseko sokuhamba njengephenyane elilahlekileyo elwandle, ndizibuza ukuba "i-HPLC doo-hickey" yasungulwa nje ukuvavanya ubukrelekrele bam. Ukuba uziva unobudenge uzama ukusonga intloko yakho kwi-high performance liquid chromatography (HPLC), thatha umoya. Ayisiyobhokisi nje yamalungu exabiso eliphezulu; ngumdaniso obuthathaka phakathi kwekhemistri kunye nefiziksi.

Kwilebhu, sihlala sisithi isigaba esiphathwayo "ngumgca wobomi" wenkqubo. Ngaphandle kwayo, isampuli yakho ihleli nje kwi-injector, ayiyi ndawo. Kodwa ukhetha njani "uloliwe" ofanelekileyo wokuthwala iimolekyuli zakho ukuya kwi-detector? Masiyiqhekeze ngathi sincokola phezu kwekofu kwigumbi lokuphumla.

1. Yintoni i-HPLC, Ngokwenene?

Kwisiseko sayo, i-HPLC imalunga nokwahlukana. Cinga nje njengogqatso apho "umkhondo" sisigaba esimileyo (ikholamu) kunye "nomoya" otyhala abagijimi kunye nesigaba seselula se-hplc.

Umlingo uyenzeka ngenxa yokuba iimolekyuli ezahlukeneyo zine "affinities" ezahlukeneyo. Ezinye iimolekyuli zithanda isigaba esiphathwayo kwaye sondeza kwikholamu. Abanye "bancamathele" -banokuphazamiseka kwinqanaba lokumisa kwaye bashiye ngasemva. Lo mahluko kwisantya yeyona nto idala ezo ncochoyi kwichromatogram yakho.

2. ISigaba esiQhelekileyo vs

Oku kuqhelekile apho wonke umntu eqala ukutsala iinwele zakhe. Kwiminyaka yam yokuziqhelanisa, ndifumene eyona ndlela ilula yokukhumbula oku kukujonga i-polarity.

A. ISigaba esiQhelekileyo seChromatography

Le yindlela "yesikolo esidala". Kwi-chromatography yesigaba esiqhelekileyo, isigaba sokumisa sikwipolar-cinga ngayo njengemagnethi yeemolekyuli zepolar. Isigaba esihambayo esisisebenzisayo apha asiyiyo-polar, ngokuqhelekileyo izinyibilikisi ezifana ne-hexane okanye i-heptane.

• Ingqiqo: Iimolekyuli zepolar "ezincangathi" zibanjelwa umva yikholomu yepolar. Iimolekyuli ezingezizo ipolar zihlamba ngokukhawuleza kunye nesigaba se-non-polar mobile.

• Ingcebiso yePro: Ukuba unyusa i-polarity yesigaba sakho esiphathwayo (ukongeza isuntswana le-isopropanol), iisampulu zakho ziya kuhamba ngokukhawuleza.

B. ISigaba sokuReverse Chromatography

Namhlanje, lo ngumgangatho wegolide. Mhlawumbi i-90% yomsebenzi endiwenzayo kwilebhu ubandakanya i-chromatography yesigaba sokubuyela emuva. Apha, siflip iskripthi. Ikholomu ayinayo i-polar (i-hydrophobic, njengeoli), kwaye i-mobile yesigaba i-polar (ngokuqhelekileyo umxube wamanzi kunye ne-methanol okanye i-acetonitrile).

• Ingqiqo: Iimolekyuli ezithanda amanzi (ipolar) ziyasondeza kuba ziyithiyile ikholamu enamafutha. Iimolekyuli ezithanda amanqatha (ezingezizo ipolar) zinamathela kwikholamu njengeglu.

• Kutheni siyithanda: Inokucingeleka, iyaguquguquka, kwaye isebenzisa izinyibilikisi ezisekwe emanzini ekulula ukuzilawula.

3. "Umthetho weGolide" woMgangatho weSolvent

Uya kubona "iBanga le-HPLC" kwiilebhile ngesizathu. Ungaze ucinge ngokusebenzisa ibakala le-ACS okanye nantoni na engaphantsi. Ngoba? Kuba ezo zinto zimdaka “zincinci” kwizinyibilikisi zomgangatho ophantsi ziya kuqokelelana kwikholamu yakho ngokuhamba kwexesha, zibulala isisombululo sakho kwaye zidale iincopho zesiporho ezingaqhelekanga.

Soloko usebenzisa iMilli-Q (18.2 MΩ.cm) amanzi ebakala. Ukuba amanzi akho "angcolile," isiseko sakho siya kubonakala njengoluhlu lweentaba, kwaye uya kuchitha iintsuku ezintathu uzama ukuqonda ukuba kutheni.

4. I-Degassing kunye noHlelo: Ababulali boMsebenzi

Umoya lutshaba lwesigaba seselula se-hplc. Phantsi koxinzelelo oluqhelekileyo, umoya uyanyibilika kwizinyibilikisi zakho. Kodwa xa eso sinyibilikisi sibetha impompo, loo maqamza amancinci angabanjwa kwiivalvu zokutshekisha okanye kwiseli yokukhangela.

• Isiphumo: Ukuguquguquka kwesantya sokuhamba, iziseko zokubetha, kunye namaxesha okugcina ashukumayo.

• Ukulungiswa: Sebenzisa i-degassers eyakhelwe-ngaphakathi, okanye i-sonication\/vacuum filtration ukuba usebenzisa ii-assays ezinovakalelo oluphezulu.

Uhluzo aluKhenketho. Nokuba i-HPLC grade solvents ingaba neencinci ezincinci ezivela kwinkqubo yokuvelisa okanye ibhotile yebhotile. Soloko uhluza nge-0.22µm okanye 0.45µm inwebu phambi kokuba ichukumise indawo yokugcina amanzi. Ngumsebenzi wemizuzu emi-5 onokugcina ikholamu yakho ekufeni kwangaphambi kwexesha.

5. Izithinteli kunye neTrap yeglasi

Kwisigaba sokubuyela umva chromatography, sisebenzisa izithinteli ukugcina i-pH izinzile kwiisampulu ezi-ionzable.

• Ingcebiso yePro: Ukuba usebenzise isithinteli namhlanje, hlamba inkqubo ngamanzi acocekileyo\/umxube ophilayo ngaphambi kokuba uye ekhaya. Ukuba awukwenzi oko, ezo tyuwa ziya kucwebezela kwaye zikhuphe izitywina zakho zempompo.

• I-Glass Trap: Ngaba ubusazi ukuba ukuba i-hplc yakho yesigaba seselula i-pH i> 8.0, inokukhupha ii-ion zetsimbi kwii-reservoirs zeglasi? Tshintshela kwintsimbi engatyiwa okanye kwizikhongozeli zeplastiki ezikhethekileyo kwezi meko.

Masifumane ukwahlukana kwakho kugqibelele

Ukufumana inqanaba leselula elichanekileyo yi-80% yedabi kwi-HPLC. Ukuba usokola "kwikhaphethi ye-shag", i-spikes yoxinzelelo, okanye iziphumo ezingaguqukiyo, ungachithi olunye usuku ngokuqikelela.

Ndichithe amawaka eeyure ebhentshini ndilungisa le miba kanye. Ndihlala ndonwabile ukwabelana ngengcebiso "engqinwe elebhu" ukubuyisela iprojekthi yakho kwindlela.

• WhatsApp: 86 18338832256

• I-imeyile:boonemi@aijirenvial.com
  

Iimbekiselo

• Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2011). Intshayelelo kwiChromatography yeLiquid yanamhlanje. Uhlelo lwesi-3. UJohn Wiley kunye noonyana. (I-"Gold Standard" yokuqonda iindlela zokwahlula i-HPLC).

• UDolan, J. W. (2002). "ULungiselelo lweSigaba seMobile." LCGC North America, Troubleshooting Series. (Isikhokelo esisebenzayo esibalulekileyo sokususa umoya kunye nokucoca).

• Aparicio, R. (Mhl.). (2013). Incwadi yoLungiso lweHPLC. Elsevier. (Idatha ebanzi malunga ne-solvent polarity kunye nokukhethwa kwe-buffer kwi-chromatography yesigaba esingasemva).

• Ubuchwephesha be-Agilent. "I-HPLC System Troubleshooting Guide: Ukunqanda iimpazamo zeSigaba esiQhelekileyo." Ithala leencwadi leMithombo yobuGcisa.

• Umbutho wamanzi. "Imigaqo yeChromatography: iSigaba esiQhelekileyo kunye neSigaba esibuyela umva." Uthotho lwezeMfundo.

• INkomfa yaMazwe ngaMazwe yoLungelelaniso (ICH). Q2(R1) Ukuqinisekiswa kweeNkqubo zoHlalutyo: Isicatshulwa kunye neNdlela yoLwazi. (Umgangatho wobungakanani bendawo ephezulu kunye nokuzinza kwexesha lokugcinwa).