Kudziwa Kuyenda: Malangizo a Lab Veteran ku HPLC Mobile Phases

Mawu Oyamba

Ndakhala komwe muliko, ndikuyang'ana chowunikira ku 2 AM, ndikuwona kusuntha koyambira ngati bwato lotayika panyanja, ndikudabwa ngati "HPLC doo-hickey" idapangidwa kuti ingoyesa misala yanga. Ngati mukumva kutopa poyesa kukulunga mutu pa high performance liquid chromatography (HPLC), pumani mpweya. Si bokosi lokha la magawo okwera mtengo; ndi kuvina kosakhwima pakati pa chemistry ndi physics.

Mu labu, nthawi zambiri timati gawo la mafoni ndi "mzere wamoyo" wadongosolo. Popanda izo, chitsanzo chanu chikungokhala mu jekeseni, osapita kulikonse. Koma kodi mumasankha bwanji "sitima" yoyenera kunyamula mamolekyu anu kupita ku chowunikira? Tiyeni tiphwanye ngati tikucheza pa khofi m'chipinda chochezera.

1. Kodi HPLC ndi chiyani kwenikweni?

Pachimake, HPLC ili pafupi kupatukana. Ganizirani izi ngati mpikisano womwe "njira" ndi gawo loyima (mzere) ndipo "mphepo" ikukankhira othamangawo ndi gawo la mafoni a hplc.

Matsenga amachitika chifukwa mamolekyu osiyanasiyana ali ndi "zogwirizana" zosiyanasiyana. Mamolekyu ena amakonda gawo la foni yam'manja ndikuwonera pagawo. Ena ndi "okanirira" - amakhudzidwa ndi gawo loyima ndikutsalira kumbuyo. Kusiyana kwa liwiro uku ndikomwe kumapanga nsongazo pa chromatogram yanu.

2. Normal Phase vs. Reverse Phase

Apa ndipamene aliyense amayamba kuzula tsitsi. M'zaka zanga zakuchita, ndapeza njira yosavuta kukumbukira izi ndikuyang'ana polarity.

A. Normal Phase Chromatography

Iyi ndi njira ya "sukulu yakale". Mu chromatography yanthawi zonse, gawo loyima ndi polar - ganizirani ngati maginito a mamolekyu a polar. Gawo la mafoni lomwe timagwiritsa ntchito pano silikhala polar, nthawi zambiri zosungunulira monga hexane kapena heptane.

• Zomveka: Mamolekyu "omatira" a polar amabwezeretsedwa ndi gawo la polar. Mamolekyu omwe si a polar amatsuka mwachangu ndi gawo la mafoni a non-polar.

• Malangizo Othandizira: Ngati muwonjezera polarity ya gawo lanu la m'manja (kuwonjezera isopropanol), zitsanzo zanu zidzayenda mofulumira.

B. Reverse Phase Chromatography

Masiku ano, uyu ndiye muyezo wagolide. Mwina 90% ya ntchito yomwe ndimagwira mu labu imakhudza reverse phase chromatography. Apa, tikutembenuza script. Mzerewu ndi wopanda polar (hydrophobic, ngati mafuta), ndipo gawo la mobile ndi polar (nthawi zambiri kusakaniza kwa madzi ndi methanol kapena acetonitrile).

• Zomveka: Mamolekyu okonda madzi (polar) amayandikira chifukwa amadana ndi gawo lamafuta. Mamolekyu okonda mafuta (omwe si a polar) amamatira pamzake ngati guluu.

• Chifukwa chomwe timachikondera: Ndizodziwikiratu, zosunthika, ndipo zimagwiritsa ntchito zosungunulira zochokera m'madzi zomwe ndizosavuta kuziwongolera.

3. The "Golden Rule" ya Zosungunulira Quality

Mudzawona "HPLC Grade" pa zolemba pazifukwa. Musaganize za kugwiritsa ntchito kalasi ya ACS kapena zochepa. Chifukwa chiyani? Chifukwa zonyansa "zing'onozing'ono" zomwe zili muzosungunulira zotsika zimachulukana pamndandanda wanu pakapita nthawi, kupha malingaliro anu ndikupanga nsonga zamatsenga.

Nthawi zonse gwiritsani ntchito madzi a giredi a Milli-Q (18.2 MΩ.cm). Ngati madzi anu ali "odetsedwa," maziko anu adzawoneka ngati mapiri, ndipo mudzakhala masiku atatu kuyesa kudziwa chifukwa chake.

4. Degassing ndi kusefa: The Performance Killers

Air ndi mdani wa hplc mobile phase. Pansi pa kuthamanga kwabwino, mpweya umasungunuka mu zosungunulira zanu. Koma chosungunuliracho chikagunda pampu, tinthu tating'onoting'ono timeneti titha kutsekeredwa mu ma cheke kapena cell cell.

• Zotsatira zake: kusinthasintha kwa liwiro, kusuntha koyambira, komanso kusuntha kwa nthawi yosungira.

• Konzani: Gwiritsani ntchito zopangira zopangira, kapena kusefera kwa sonication\/vacuum ngati mukuyesa kuyesa kwamphamvu kwambiri.

Kusefera Sikosankha. Ngakhale zosungunulira zamtundu wa HPLC zimatha kukhala ndi tinthu tating'onoting'ono tomwe timapanga popanga kapena kapu ya botolo. Nthawi zonse sefa nembanemba ya 0.22µm kapena 0.45µm isanakhudze nkhokwe yanu. Ndi ntchito ya mphindi 5 yomwe ingapulumutse gawo lanu ku imfa yanthawi yake.

5. Ma Buffers ndi Msampha wa Galasi

Mu reverse phase chromatography, timagwiritsa ntchito ma buffers kuti pH isasunthike pamatsanzo omwe amatha ionzable.

• Malangizo Othandizira: Ngati mudagwiritsa ntchito buffer lero, sambani makinawo ndi madzi oyera/organic mix musanapite kunyumba. Ngati simutero, mcherewo udzanyezimira ndikuphwanya zisindikizo zanu zapampu.

• Msampha wa Galasi: Kodi mumadziwa kuti ngati hplc mobile phase pH ndi> 8.0, imatha kutulutsa ma ion zitsulo kuchokera kumadzi agalasi? Sinthani ku zitsulo zosapanga dzimbiri kapena zotengera zapulasitiki zapadera pazochitika izi.

Tiyeni Tipeze Kupatukana Kwanu Kwangwiro

Kupeza gawo la mafoni kumanja ndi 80% yankhondo mu HPLC. Ngati mukulimbana ndi zoyambira za "shag carpet", ma spikes, kapena zotsatira zosagwirizana, musataye tsiku lina kungoganiza.

Ndakhala maola masauzande ambiri pa benchi ndikuthetsa mavuto awa. Ndimakhala wokondwa nthawi zonse kugawana upangiri "wotsimikiziridwa ndi labu" kuti ntchito yanu ibwererenso.

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Maumboni

• Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2011). Chiyambi cha Modern Liquid Chromatography. 3rd Edition. John Wiley & Ana. ("Gold Standard" yomvetsetsa njira zolekanitsa za HPLC).

• Dolan, J. W. (2002). "Mobile Phase Kukonzekera." LCGC North America, Troubleshooting Series. (Chitsogozo chofunikira chothandizira kuchotsa gasi ndi kusefera).

• Aparicio, R. (Mkonzi.). (2013). Buku la HPLC Optimization. Elsevier. (Deta yokwanira pa zosungunulira polarity ndi buffer kusankha mu reverse phase chromatography).

• Agilent Technologies. "HPLC System Troubleshooting Guide: Kupewa Zolakwa Zodziwika Pagawo la Mafoni." Library Resource Library.

• Malingaliro a kampani Waters Corporation "Mfundo za Chromatography: Normal Phase vs. Reverse Phase." Maphunziro Series.

• International Conference on Harmonization (ICH). Q2 (R1) Kutsimikizika kwa Njira Zowunikira: Zolemba ndi Njira. (Muyezo wa kuchuluka kwa malo apamwamba komanso kukhazikika kwa nthawi yosungira).