Ho Tseba Phallo: Tataiso ea Lab Veteran ho HPLC Mobile Phase

Selelekela

Ke bile moo u leng teng — ke shebile sebali ka 2 AM, ke shebile seketsoana se hoholehang joalo ka seketsoana se lahlehileng leoatleng, ke ntse ke ipotsa hore na "HPLC doo-hickey" e qapiloe ho leka bohlanya ba ka. Haeba u ikutloa u tsielehile ha u leka ho koahela hlooho ea hau ka har'a high performance liquid chromatography (HPLC), nka moea. Hase feela lebokose la likarolo tse theko e boima; ke motjeko o bobebe lipakeng tsa k'hemistri le fisiks.

Ka laboratoring, hangata re re mobile phase ke "mohala oa bophelo" oa sistimi. Ntle le eona, sampole ea hau e lutse feela ka enteng, ha e ea kae kapa kae. Empa u khetha "terene" e nepahetseng joang ho isa limolek'hule tsa hau ho ea ho detector? Ha re e qhekelle joalokaha eka re ntse re qoqa re noa kofi ka kamoreng ea ho phomola.

1. HPLC ke eng, Ha e le hantle?

Motheong oa eona, HPLC e mabapi le karohano. E nahane joalo ka peiso eo "pina" e leng karolo ea ho ema (kholomo) 'me "moea" o sutumetsang limathi ke karolo ea hplc mobile.

Boselamose bo etsahala hobane limolek'hule tse fapaneng li na le "likamano" tse fapaneng. Limolek'hule tse ling li rata mohato oa selefouno 'me li atometsa kholomo. Ba bang ba "khomarela" - ba na le khatello ea maikutlo 'me ba salla morao. Phapang ena ea lebelo ke eona e etsang litlhoro tseo ho chromatogram ea hau.

2. Normal Phase vs. Reverse Phase

Hangata mona ke moo bohle ba qalang ho hula moriri oa bona. Lilemong tsa ka tsa boikoetliso, ke fumane tsela e bonolo ka ho fetisisa ea ho hopola sena ke ho sheba polarity.

A. Chromatography ea Phase e Tloaelehileng

Ena ke mokhoa oa "sekolo sa khale". Ka mokhoa o tloaelehileng oa chromatography, karolo e emeng ke polar - nahana ka eona joalo ka makenete bakeng sa limolek'hule tsa polar. Mohato oa selefouno oo re o sebelisang mona ha o polar, hangata lihlapolli tse kang hexane kapa heptane.

• The Logic: Limolek'hule "tse khomarelang" tsa polar li khutlisetsoa morao ke karolo ea polar. Limolek'hule tse se nang polar li hlatsuoa kapele ka karolo ea selefouno e seng polar.

• The Pro Tip: Haeba u eketsa polarity ea karolo ea hau ea selefouno (ho eketsa isopropanol), lisampole tsa hau li tla tsamaea kapele.

B. Reverse Phase Chromatography

Kajeno, sena ke tekanyetso ea khauta. Mohlomong 90% ea mosebetsi oo ke o etsang lab o kenyelletsa reverse phase chromatography. Mona, re phetla script. Kholomo ha e polar (hydrophobic, joalo ka oli), 'me phase mobile ke polar (hangata motsoako oa metsi le methanol kapa acetonitrile).

• The Logic: Limolek'hule tse ratang metsi (polar) lia feta hobane li hloile kholomo e mafura. Limolek'hule tse ratang mafura (tse seng polar) li khomarela kholomong joalo ka sekhomaretsi.

• Lebaka leo re e ratang ka eona: E ka lebelloa esale pele, e sebetsa ka mefuta-futa, 'me e sebelisa lihlapolli tse thehiloeng metsing tseo ho leng bonolo ho li laola.

3. "Molao oa Khauta" oa Boleng ba Solvent

U tla bona "HPLC Grade" ho li-label ka lebaka. Se ke oa nahana ka ho sebelisa sehlopha sa ACS kapa eng kapa eng e tlase. Hobaneng? Hobane litšila tse "nyenyane" tse hlapollang tsa maemo a tlase li tla bokella kholomong ea hau ha nako e ntse e ea, li bolaee qeto ea hau le ho theha litlhoro tse makatsang tsa ghost.

Kamehla sebelisa metsi a kereiti ea Milli-Q (18.2 MΩ.cm). Haeba metsi a hau a "silafetse," motheo oa hau o tla shebahala joaloka thaba, 'me u tla qeta matsatsi a mararo u leka ho fumana hore na ke hobane'ng.

4. Degassing le Filtration: The Performance Killers

Moea ke sera sa hplc mobile phase. Ka tlas'a khatello e tloaelehileng, moea oa qhibiliha ka har'a lihlapolli tsa hau. Empa hang ha solvent eo e otla pompo, li-bubble tse nyane li ka ts'oaroa ka har'a li-valve tsa cheke kapa sele ea detector.

• Sephetho: Ho feto-fetoha ha sekhahla sa phallo, metheo ea ho otla, le linako tsa ho boloka tse tsamaeang.

• Tokiso: Sebelisa li-degassers tse hahelletsoeng ka hare, kapa sonication\/vacuum filtration haeba u ntse u etsa liteko tse matla haholo.

Sefa ha se Taba ea Boikhethelo. Le li-solvents tsa boemo ba HPLC li ka ba le likaroloana tse nyane tse tsoang ts'ebetsong ea tlhahiso kapa sekoaelo sa botlolo. Kamehla sefa ka har'a lera la 0.22µm kapa 0.45µm pele e ama polokelo ea hau. Ke mosebetsi oa metsotso e 5 o ka pholosang kholomo ea hau lefung la pele ho nako.

5. Li-buffers le Sefi sa Khalase

Ho chromatography ea reverse phase, re sebelisa li-buffers ho boloka pH e tsitsitse bakeng sa disampole tse ionisang.

• Malebela a Pro: Haeba u sebelisitse buffer kajeno, hlatsoa sistimi ka metsi a hloekileng/motsoako oa tlhaho pele u ea hae. Haeba u sa etse joalo, matsoai ao a tla khanya le ho senya litiiso tsa hau tsa pompo.

• The Glass Trap: Na u ne u tseba hore haeba hplc mobile phase pH ea hau e le> 8.0, e ka hla ea ntša li-ion tsa tšepe ho tloha matamong a khalase? Fetohela ho tšepe e sa hloekang kapa lijana tsa polasetiki tse khethehileng maemong ana.

Ha re phethe karohano ea hau e phethahetseng

Ho fumana karolo ea selefouno hantle ke 80% ea ntoa ho HPLC. Haeba u ntse u loantšana le "shag carpet" ea motheo, likhatello tsa khatello, kapa liphetho tse sa lumellaneng, u se ke ua senya letsatsi le leng ka ho nahana.

Ke qetile lihora tse likete ke le bencheng ho rarolla mathata ana hantle. Ke lula ke thabela ho fana ka likeletso tse "pakiloe lab" ho khutlisetsa projeke ea hau tseleng.

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Litšupiso

• Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2011). Kenyelletso ho Modern Liquid Chromatography. Khatiso ea 3rd. John Wiley & Bara. (The "Gold Standard" bakeng sa ho utloisisa mekhoa ea karohano ea HPLC).

• Dolan, J. W. (2002). "Mobile Phase Tokisetso." LCGC Amerika Leboea, Letoto la Tharollo ea Mathata. (Tlhahiso ea bohlokoa e sebetsang bakeng sa ho senya le ho sefa).

• Aparicio, R. (Mong.). (2013). Bukana ea HPLC Optimization. Elsevier. (Lintlha tse felletseng mabapi le polarity ea solvent le khetho ea buffer ho reverse phase chromatography).

• Agilent Technologies. "HPLC System Troubleshooting Guide: Ho Qoba Liphoso Tse Tloaelehileng Tsa Phase ea Mohala." Laebrari ea Lisebelisoa tsa Theknoloji.

• Mokhatlo oa Metsi. "Melao-motheo ea Chromatography: Boemo bo Tloaelehileng vs. Reverse Phase." Letoto la Thuto.

• Khonferense ya Boditšhabatšhaba ya Temano (ICH). Q2(R1) Ho netefatsoa ha Mekhoa ea Tlhahlobo: Mongolo le Mokhoa. (E tloaelehileng bakeng sa quantification ea sebaka se phahameng le botsitso ba nako ea ho boloka).