Ka Manaʻo i ka Kahe: He alakaʻi a Lab Veteran i nā HPLC Mobile Phases

Hoʻolauna

Aia wau i kahi āu e noho nei—e nānā ana i kahi mākaʻikaʻi i ka hola 2 AM, e nānā ana i ka neʻe ʻana o ka baseline e like me ka waʻa nalowale i ke kai, me ka noʻonoʻo inā ua hana ʻia ka "HPLC doo-hickey" e hoʻāʻo i koʻu noʻonoʻo. Inā makemake ʻoe e hoʻāʻo e ʻōwili i kou poʻo a puni ka high performance liquid chromatography (HPLC), e hanu. ʻAʻole ia he pahu o nā ʻāpana pipiʻi; he hulahula maalea ia ma waena o ke kemika a me ka physics.

I loko o ka lab, ʻōlelo pinepine mākou ʻo ka pae kelepona ka "laina ola" o ka ʻōnaehana. Inā ʻaʻole ia, ke noho wale nei kāu hāpana i loko o kahi injector, ʻaʻohe hele i kahi. Akā, pehea ʻoe e koho ai i ka "kaʻaahi" kūpono e lawe i kāu mau mole i ka mea ʻike? E wāwahi kākou e like me ke kamaʻilio ʻana ma luna o ke kofe i loko o ka lumi hoʻomaha.

1. He aha ka HPLC, ʻOiaʻiʻo?

Ma kāna kumu, pili ka HPLC i ka hoʻokaʻawale. E noʻonoʻo ʻoe e like me ka heihei kahi o ka "track" ka pae paʻa (ke kolamu) a ʻo ka "makani" e hoʻokuke ana i ka poʻe kukini ma ka pae hplc mobile.

Hana ʻia ka hana kilokilo no ka mea he ʻokoʻa nā "affinities" o nā molekala like ʻole. Makemake kekahi mau mole i ka pae paʻa a hoʻonui i ke kolamu. ʻO nā mea ʻē aʻe he "paʻa" - ua paʻa lākou i ka pae paʻa a lohi ma hope. ʻO kēia ʻokoʻa i ka wikiwiki ka mea e hana ai i kēlā mau piko ma kāu chromatogram.

2. Māhele maʻamau vs. Mahele Hoʻohuli

ʻO kēia ka mea maʻamau kahi e hoʻomaka ai nā mea a pau e huki i ko lākou lauoho. I koʻu mau makahiki o ka hoʻomaʻamaʻa, ua loaʻa iaʻu ke ala maʻalahi loa e hoʻomanaʻo i kēia ma ka nānā ʻana i ka polarity.

A. Kromatography Māhele maʻamau

ʻO kēia ke ala "kula kahiko". I ka chromatography pae maʻamau, polar ka pae kuʻuna—e noʻonoʻo e like me ka magnet no nā molekole polar. ʻO ka pae kelepona a mākou e hoʻohana ai ma ʻaneʻi he polar ʻole, maʻamau nā solvents e like me hexane a i ʻole heptane.

• ʻO ka Logic: Paʻa ʻia nā molekala "sticky" polar e ke kolamu polar. Holoi ʻia nā molekala non-polar me ka pae kelepona ʻole polar.

• ʻO ka Manaʻo Pro: Inā hoʻonui ʻoe i ka polarity o kāu pae kelepona (hoʻohui i kahi isopropanol), e neʻe wikiwiki kāu mau laʻana.

B. Hōʻalo ʻia ʻo Chromatography

I kēia lā, ʻo ia ke kūlana gula. ʻO 90% paha o kaʻu hana i loko o ka lab e pili ana i ka reverse phase chromatography. Eia, hoʻohuli mākou i ka palapala. ʻAʻole polar ke kolamu (hydrophobic, e like me ka ʻaila), a ʻo ka pae mobile he polar (maʻa mau ka hui ʻana o ka wai a me ka methanol a i ʻole acetonitrile).

• ʻO ka Logic: ʻO nā molekole aloha wai (polar) e hoʻonui i loko no ka mea ua inaina lākou i ke kolamu ʻaila. ʻO nā molekala momona (non-polar) pili i ke kolamu e like me ke kāpili.

• No ke aha mākou e makemake ai: He mea wānana, maʻalahi, a hoʻohana i nā mea hoʻoheheʻe wai i maʻalahi ke mālama.

3. Ka "Golden Rula" o ka Solvent Quality

E ʻike ʻoe i ka "HPLC Grade" ma nā lepili no kekahi kumu. Mai noʻonoʻo e pili ana i ka hoʻohana ʻana i ka māka ACS a i ʻole kekahi mea liʻiliʻi. No ke aha mai? No ka mea, e hōʻiliʻili ʻia kēlā mau mea "ʻuʻuku" i loko o nā mea hoʻoheheʻe haʻahaʻa ma kāu kolamu i ka wā lōʻihi, e pepehi ana i kāu hoʻoholo ʻana a me ka hoʻokumu ʻana i nā piko ʻuhane ʻē.

E hoʻohana mau i ka wai o Milli-Q (18.2 MΩ.cm). Inā he "palapala" kou wai, e like ana kou pae kumu me kahi pae mauna, a e hoʻolimalima ʻoe i ʻekolu lā e hoʻāʻo ai e noʻonoʻo i ke kumu.

4. Degassing and Filtration: The Performance Killers

ʻO ka ea ka ʻenemi o ka hplc mobile phase. Ma lalo o ke kaomi maʻamau, hoʻoheheʻe ka ea i kāu mau mea hoʻoheheʻe. Akā i ka wā e pā ai kēlā mea hoʻoheheʻe i ka pama, hiki i kēlā mau ʻōpū liʻiliʻi ke hoʻopaʻa ʻia i loko o nā kiwi kikoʻī a i ʻole ke kelepona ʻike.

• ʻO ka hopena: ʻO nā loli kahe o ke kahe, nā pulsing baselines, a me ka neʻe ʻana o nā manawa paʻa.

• ʻO ka hoʻoponopono: E hoʻohana i nā degassers i kūkulu ʻia, a i ʻole sonication\/vacuum filtration inā ʻoe e holo ana i nā assays kiʻekiʻe.

ʻAʻole koho ka kānana. Hiki i nā mea hoʻoheheʻe helu HPLC ke loaʻa nā ʻāpana microscopic mai ke kaʻina hana a i ʻole ka pāpale ʻōmole. E kānana mau ma kahi membrane 0.22µm a i ʻole 0.45µm ma mua o ka pā ʻana i kāu waihona. He hana 5 mau minuke hiki ke hoʻopakele i kāu kolamu mai ka make mua.

5. Hoopaa a me ka Pahele Aniani

I ka chromatography hoʻololi, hoʻohana mākou i nā pale e mālama i ka pH paʻa no nā laʻana ionizable.

• Manaʻo Pro: Inā ʻoe i hoʻohana i kahi pale i kēia lā, e holoi i ka ʻōnaehana me ka wai maʻemaʻe / hui kūlohelohe ma mua o kou hoʻi ʻana i ka home. Inā ʻaʻole ʻoe e hana, e hoʻokaʻawale kēlā mau paʻakai a ʻokiʻoki i kāu mau sila pauma.

• ʻO ka Pahele Aniani: Ua ʻike anei ʻoe inā he > 8.0 kāu hplc mobile phase pH, hiki iā ia ke hoʻokahe i nā ion metala mai nā waihona aniani? E hoʻololi i ke kila kila a i ʻole nā ​​ipu plastik kūikawā i kēia mau hihia.

E Hana Pono i Kou Kaawale

ʻO ka loaʻa ʻana o ka pae kelepona ʻo 80% o ke kaua ma HPLC. Inā ʻoe e hakakā nei me ka "shag carpet" baselines, ka piʻi ʻana o ke kaomi, a i ʻole nā ​​hopena like ʻole, mai hoʻopau i kekahi lā e koho ai.

Ua hoʻohana au i mau kaukani mau hola ma ka papa e hoʻoponopono i kēia mau pilikia pololei. Hauʻoli mau wau e kaʻana like i nā ʻōlelo aʻoaʻo "lab-hōʻoia" e hoʻihoʻi hou i kāu papahana.

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Nā kuhikuhi

• Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2011). Introduction to Modern Liquid Chromatography. 3rd Edition. John Wiley & Keikikane. (Ka "Gold Standard" no ka hoʻomaopopo ʻana i nā hana hoʻokaʻawale HPLC).

• Dolan, J. W. (2002). "Hoʻomākaukau ʻia ʻo Mobile Phase." LCGC ʻAmelika ʻĀkau, Pūʻulu Hoʻoponopono. (Alakaʻi hoʻokō pono no ka degassing a me ka kānana).

• Aparicio, R. (Ed.). (2013). Manual o ka HPLC Optimization. Elsevier. (Ka ʻikepili piha e pili ana i ka polarity solvent a me ke koho pale ma ka chromatography hoʻohuli.

• ʻenehana Agilent. "HPLC System Troubleshooting Guide: Ke pale aku nei i nā hewa maʻamau maʻamau." Hale Waihona Puke Punawai.

• Hui Wai. "Principles of Chromatography: Normal Phase vs. Reverse Phase." Papa Hoʻonaʻauao.

• ʻAhaʻi Kūkākūkā o ka Harmonization (ICH). Q2(R1) Hōʻoia o nā Kaʻina Hana Analytical: Text and Methodology. (Ka mea maʻamau no ka helu ʻāina kiʻekiʻe a me ke kūpaʻa manawa paʻa).