Mastering the Flow: Lab Veteran's Guide to HPLC Mobile Phases

Taw qhia

Kuv tau nyob qhov twg koj nyob - ntsia ntawm lub saib thaum 2 teev sawv ntxov, saib lub hauv paus drift zoo li lub nkoj ploj hauv hiav txwv, xav tsis thoob yog tias "HPLC doo-hickey" tau tsim los sim kuv lub siab. Yog tias koj xav tias xav tau sim qhwv koj lub taub hau nyob ib puag ncig kev ua haujlwm siab ua kua chromatography (HPLC), ua pa. Nws tsis yog ib lub thawv uas kim kim; nws yog ib qho kev seev cev zoo nkauj ntawm chemistry thiab physics.

Hauv chav kuaj, peb feem ntau hais tias lub sijhawm txawb yog "lub neej kab" ntawm qhov system. Yog tsis muaj nws, koj tus qauv tsuas yog zaum hauv ib qho tshuaj txhaj, mus tsis pom qhov twg. Tab sis ua li cas koj xaiv txoj cai "tsheb ciav hlau" los nqa koj cov molecules mus rau lub ntes? Cia peb tawg nws zoo li peb tab tom tham hauv kas fes hauv chav so.

1. HPLC yog dab tsi, Tiag?

Ntawm nws lub hauv paus, HPLC yog hais txog kev sib cais. Xav txog nws zoo li kev sib tw uas "khiav" yog theem nyob ruaj khov (kem) thiab "cua" thawb cov neeg sib tw nrog yog hplc mobile theem.

Cov khawv koob tshwm sim vim hais tias sib txawv molecules muaj sib txawv "affinities." Qee cov molecules nyiam lub xov tooj ntawm tes thiab zoom los ntawm kab. Lwm tus yog "nqa" - lawv muaj kev sib tsoo ntawm lub sijhawm nyob ruaj khov thiab poob qab. Qhov sib txawv ntawm qhov ceev no yog dab tsi tsim cov peaks ntawm koj chromatogram.

2. Theem ib txwm vs. Rov qab Theem

Qhov no feem ntau yog txhua tus neeg pib rub lawv cov plaub hau tawm. Hauv kuv lub xyoo ntawm kev xyaum, kuv tau pom txoj hauv kev yooj yim tshaj plaws kom nco ntsoov qhov no yog los ntawm kev saib cov polarity.

A. Nquag Theem Chromatography

Qhov no yog txoj kev "qub tsev kawm ntawv". Nyob rau hauv ib txwm theem chromatography, cov theem nyob ruaj ruaj yog polar-xav txog nws zoo li cov hlau nplaum rau polar molecules. Lub sijhawm txawb peb siv ntawm no tsis yog-polar, feem ntau cov kuab tshuaj zoo li hexane lossis heptane.

• Lub Logic: Polar "nplaum" molecules tau tuav rov qab los ntawm tus ncej polar. Cov tsis-polar molecules ntxuav tawm sai sai nrog cov tsis-polar mobile theem.

• Lub tswv yim Pro: Yog tias koj nce polarity ntawm koj lub sijhawm txawb (ntxiv me ntsis ntawm isopropanol), koj cov qauv yuav txav nrawm dua.

B. Rov qab Theem Chromatography

Niaj hnub no, qhov no yog tus qauv kub. Tej zaum 90% ntawm cov hauj lwm kuv ua nyob rau hauv lub chav kuaj nrog reverse theem chromatography. Ntawm no, peb tig tsab ntawv. Cov kem tsis yog-polar (hydrophobic, zoo li roj), thiab theem mobile yog polar (feem ntau yog sib xyaw ntawm dej thiab methanol lossis acetonitrile).

• Lub Logic: Dej-hlub (polar) molecules zoom los ntawm vim lawv ntxub cov oily kem. Fat-hlub (tsis-polar) molecules lo rau hauv kem zoo li cov kua nplaum.

• Yog vim li cas peb hlub nws: Nws yog kwv yees, ntau yam, thiab siv cov kuab tshuaj raws li dej uas yooj yim los tswj.

3. "Golden Rule" ntawm Solvent Quality

Koj yuav pom "HPLC Qib" ntawm cov ntawv lo vim yog vim li cas. Tsis txhob xav txog kev siv ACS qib lossis tsawg dua. Vim li cas? Vim tias cov "me me" impurities hauv cov kuab tshuaj qis dua yuav sau rau ntawm koj kem lub sijhawm, tua koj qhov kev daws teeb meem thiab tsim cov dab txawv txawv.

Ib txwm siv Milli-Q (18.2 MΩ.cm) qib dej. Yog tias koj cov dej "qhuav", koj lub hauv paus yuav zoo li lub roob, thiab koj yuav siv sijhawm peb hnub los xyuas seb yog vim li cas.

4. Degassing thiab Lim: Cov Kev Ua Haujlwm Killers

Cua yog tus yeeb ncuab ntawm hplc mobile theem. Nyob rau hauv lub siab ib txwm, huab cua dissolves nyob rau hauv koj cov kuab tshuaj. Tab sis thaum cov kuab tshuaj ntawd tsoo lub twj tso kua mis, cov npuas me me tuaj yeem nkag mus rau hauv cov tshev valve lossis lub ntsuas hluav taws xob.

• Qhov tshwm sim: Flow rate fluctuations, pulsing baselines, thiab txav lub sij hawm tuav.

• Kev Kho: Siv cov degassers built-in, lossis sonication\ / lub tshuab nqus tsev yog tias koj tab tom khiav cov kev ntsuas siab.

Kev lim dej tsis yog xaiv tau. Txawm tias HPLC qib cov kuab tshuaj muaj peev xwm muaj cov kab mob me me los ntawm cov txheej txheem tsim khoom lossis lub raj mis cap. Ib txwm lim los ntawm 0.22µm lossis 0.45µm daim nyias nyias ua ntej nws kov koj lub kais dej. Nws yog 5-feeb ua haujlwm uas tuaj yeem cawm koj kab los ntawm kev tuag ntxov ntxov.

5. Buffers thiab iav ntxiab

Nyob rau theem rov qab chromatography, peb siv buffers kom pH ruaj khov rau cov qauv ionizable.

• Tswv yim Pro: Yog tias koj siv lub tshuab tsis siv hnub no, ntxuav lub kaw lus nrog dej ntshiab\ / organic mix ua ntej koj mus tsev. Yog tias koj tsis ua li ntawd, cov ntsev no yuav crystallize thiab shred koj cov twj tso kua mis.

• Lub iav Trap: Koj puas paub tias yog tias koj lub hplc mobile theem pH yog> 8.0, nws tuaj yeem tso cov hlau ions los ntawm iav reservoirs? Hloov mus rau stainless hlau lossis cov thawv yas tshwj xeeb hauv cov rooj plaub no.

Cia Peb Ua Koj Txoj Kev Sib Ncaim Zoo

Tau txais lub sijhawm txawb txoj cai yog 80% ntawm kev sib ntaus sib tua hauv HPLC. Yog tias koj tab tom tawm tsam nrog "daim ntaub pua plag" hauv paus, kev sib tw siab, lossis cov txiaj ntsig tsis sib xws, tsis txhob nkim lwm hnub kwv yees.

Kuv tau siv ntau txhiab teev ntawm lub rooj ntev zaum daws cov teeb meem no. Kuv ib txwm zoo siab los qhia me ntsis ntawm "lab-pov thawj" cov lus qhia kom tau txais koj qhov project rov qab los.

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Cov ntaub ntawv

• Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2011). Taw qhia rau Modern Liquid Chromatography. 3rd Edition. John Wiley & Tub. (Lub "Gold Standard" rau kev nkag siab txog HPLC cov txheej txheem sib cais).

• Dolan, J. W. (2002). "Mobile Phase Npaj." LCGC North America, Kev daws teeb meem Series. (Cov lus qhia tseem ceeb rau kev degassing thiab pom).

• Aparicio, R. (Ed.). (2013). Phau ntawv ntawm HPLC Optimization. Elsevier. (Cov ntaub ntawv nthuav dav ntawm cov kuab tshuaj polarity thiab kev xaiv tsis nyob hauv cov theem rov qab chromatography).

• Agilent Technologies. "HPLC System Troubleshooting Guide: Zam Txim Txiaj Ntsig Feem Ntau." Technical Resource Library.

• Waters Corporation. "Lub hauv paus ntsiab lus ntawm Chromatography: Lub Sij Hawm Ib Nrab vs. Rov Qab Theem." Kev Kawm Series.

• International Conference on Harmonization (ICH). Q2(R1) Validation of Analytical Procedures: Text and Methodology. (Txheej txheem rau thaj chaw ncov kom muaj nuj nqis thiab khaws lub sijhawm ruaj khov).