Ukuqonda Ukugeleza: Umhlahlandlela Womakadebona Welebhu weZigaba Zeselula ze-HPLC

Isingeniso

Ngike ngafika lapho ukhona—ngigqolozele imonitha ngo-2 AM, ngibuka isisekelo sokukhukhuleka njengesikebhe esilahlekile olwandle, ngizibuza ukuthi ingabe i-"HPLC doo-hickey" yasungulwa ukuze kuvivinywe ingqondo yami. Uma uzizwa udidekile ngokuzama ukusonga ikhanda lakho ku-high performance liquid chromatography (HPLC), thatha umoya. Akulona nje ibhokisi lezingxenye ezibizayo; kungumdanso obucayi phakathi kwekhemistri nefiziksi.

Kulebhu, sivame ukuthi isigaba seselula "umugqa wempilo" wesistimu. Ngaphandle kwayo, isampula yakho ihlezi nje kumjovo, ayiyi ndawo. Kodwa ungasikhetha kanjani "isitimela" esifanele ukuthwala ama-molecule wakho uwayise kumtshina? Asihlephule sengathi sixoxa siphuza ikhofi endlini yokuphumula.

1. Iyini i-HPLC, Ngempela?

Emgogodleni wayo, i-HPLC imayelana nokuhlukana. Kucabange njengomjaho lapho "ithrekhi" iyisigaba esimile (ikholomu) futhi "umoya" osunduza abagijimi kuyisigaba seselula se-hplc.

Umlingo uyenzeka ngoba ama-molecule ahlukene "anokuhambisana." Amanye ama-molecule athanda isigaba seselula futhi asondeza kukholamu. Abanye "bayanamathela" -banokuchoboza esigabeni sokumisa futhi basalela ngemuva. Lo mehluko ngesivinini yiwo odala lezo ziqongo ku-chromatogram yakho.

2. Isigaba Esijwayelekile vs. Isigaba Esihlehlayo

Lokhu ngokuvamile yilapho wonke umuntu eqala ukudonsa izinwele zakhe. Eminyakeni yami yokuzijwayeza, ngithole indlela elula yokukhumbula lokhu ngokubheka i-polarity.

A. I-Chromatography Yesigaba Esijwayelekile

Lena indlela "yesikole esidala". Ku-chromatography yesigaba esivamile, isigaba esimile siyindawo epholile—cabanga ngaso njengozibuthe wama-molecule asezindaweni ezishisayo. Isigaba seselula esisisebenzisa lapha asisona i-polar, ngokuvamile izincibilikisi njenge-hexane noma i-heptane.

• I-Logic: Ama-molecule "anamathelayo" e-Polar abuyiselwa emuva yikholomu ye-polar. Ama-molecule angewona ama-polar ageza ngokushesha ngesigaba seselula esingesona i-polar.

• Ithiphu ye-Pro: Uma ukhulisa i-polarity yesigaba sakho seselula (ungeza i-isopropanol kancane), amasampula akho azohamba ngokushesha.

B. Reverse Phase Chromatography

Namuhla, leli izinga legolide. Cishe u-90% womsebenzi engiwenzayo elebhu uhlanganisa i-chromatography yesigaba esihlanekezelwe. Lapha, siphenya iskripthi. Ikholomu ayiyona i-polar (i-hydrophobic, njengowoyela), futhi isigaba seselula siyi-polar (imvamisa iyingxube yamanzi ne-methanol noma i-acetonitrile).

• Umqondo: Ama-molecule athanda amanzi (i-polar) asondeza ngoba azonda ikholomu enamafutha. Ama-molecule athanda amafutha (okungewona ama-polar) anamathela kukholamu njengeglue.

• Kungani sikuthanda: Kuyabikezelwa, kuyaguquguquka, futhi kusebenzisa izincibilikisi ezisekelwe emanzini okulula ukukulawula.

3. "Umthetho Wegolide" Wekhwalithi Ye-Solvent

Uzobona "i-HPLC Grade" kumalebula ngesizathu. Ungacabangi ngisho nokusebenzisa ibanga le-ACS noma yini encane. Kungani? Ngoba lokho kungcola "okuncane" kuzincibilikisi zebanga eliphansi kuzonqwabelana kukholamu yakho ngokuhamba kwesikhathi, kubulale ukulungiswa kwakho futhi kudale iziqongo zezipoki eziyinqaba.

Sebenzisa njalo i-Milli-Q (18.2 MΩ.cm) amanzi ebanga. Uma amanzi akho "engcolile," isisekelo sakho sizobukeka njengentaba, futhi uzochitha izinsuku ezintathu uzama ukuthola ukuthi kungani.

4. I-Degassing and Filtration: Ababulali Bokusebenza

Umoya uyisitha sesigaba seselula se-hplc. Ngaphansi kwengcindezi evamile, umoya uyancibilika kuzincibilikisi zakho. Kodwa uma leso sincibilikisi sishaya ipompo, lawo mabhamuza amancane angavaleleka kumavalvu okuhlola noma iseli lomtshina.

• Umphumela: Ukushintshashintsha kwezinga lokugeleza, isisekelo sokushaya, nezikhathi zokugcina ezihambayo.

• Ukulungisa: Sebenzisa ama-degasser akhelwe ngaphakathi, noma i-sonication\/vacuum filtration uma usebenzisa izivivinyo ezizwela kakhulu.

Ukuhlunga akuyona inketho. Ngisho nezinyibilikisi zebanga le-HPLC zingaba nezinhlayiya ezincane ezivela kunqubo yokukhiqiza noma ikepisi yebhodlela. Hlala uhlunga nge-membrane engu-0.22µm noma engu-0.45µm ngaphambi kokuthi ithinte indawo yokugcina amanzi. Kungumsebenzi wemizuzu emi-5 ongasindisa ikholomu yakho ekufeni ngaphambi kwesikhathi.

5. Amabhafa kanye ne-Glass Trap

Ku-chromatography yesigaba esihlanekezelwe, sisebenzisa amabhafa ukuze sigcine i-pH izinzile kumasampuli e-ionzable.

• Ithiphu Yochwepheshe: Uma usebenzise isigcinalwazi namuhla, geza isistimu ngamanzi ahlanzekile\/ingxube yemvelo ngaphambi kokuya ekhaya. Uma ungenjalo, lawo masawoti azocwebezela futhi ahlukanise ama-pump seals akho.

• I-Glass Trap: Ubuwazi ukuthi uma i-hplc yakho yesigaba seselula i-pH ingu> 8.0, ingakhipha ama-ion ensimbi kumareservoirs engilazi? Shintshela kunsimbi engagqwali noma iziqukathi zepulasitiki ezikhethekile kulezi zimo.

Ake senze Ukwehlukana Kwakho Kuphelele

Ukuthola isigaba esifanele samaselula angu-80% wempi ku-HPLC. Uma ulwa nezisekelo "ze-shag carpet", ama-pressure spikes, noma imiphumela engahambisani, ungachithi olunye usuku ngokuqagela.

Ngichithe izinkulungwane zamahora ebhentshini ngixazulula lezi zinkinga ngqo. Ngihlala ngijabule ukwabelana ngeseluleko "esifakazelwe elebhu" ukuze ubuyisele iphrojekthi yakho endleleni efanele.

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Izithenjwa

• Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2011). Isingeniso se-Modern Liquid Chromatography. Uhlelo lwesi-3. UJohn Wiley & Amadodana. (I-"Gold Standard" yokuqonda izindlela zokuhlukanisa ze-HPLC).

• Dolan, J. W. (2002). "Mobile Phase Ukulungiselela." LCGC North America, Troubleshooting Series. (Umhlahlandlela osebenzayo obalulekile wokukhipha umoya nokuhlunga).

• U-Aparicio, R. (Umhl.). (2013). Imanuwali ye-HPLC Optimization. Elsevier. (Idatha ephelele ye-solvent polarity kanye nokukhethwa kwebhafa ku-reverse phase chromatography).

• I-Agilent Technologies. "I-HPLC System Troubleshooting Guide: Ukugwema Amaphutha Ajwayelekile Esigaba Seselula." Ilabhulali Yensiza Yezobuchwepheshe.

• Ukulinganisa kwenkampani Waters Corporation "Izimiso Ze-Chromatography: Isigaba Esivamile vs. Isigaba Esihlehlayo." Uchungechunge Lwezemfundo.

• Inkomfa Yamazwe Ngamazwe Yokuvumelana (ICH). Q2(R1) Ukuqinisekiswa Kwezinqubo Zokuhlaziya: Umbhalo kanye Nendlela yokwenza. (Okuvamile kokulinganisa indawo ephezulu kanye nokuzinza kwesikhathi sokugcina).