Kubata Kuyerera: A Lab Veteran's Guide kune HPLC Mobile Phase

Nhanganyaya

Ndanga ndiri pauri - ndakatarisa pamonitor na2 AM, ndichiona baseline drift sechikepe chakarasika pagungwa, ndichifunga kana iyo "HPLC doo-hickey" yakangogadzirwa kuti ndiedze njere dzangu. Kana iwe urikunzwa kufara kuyedza kuputira musoro wako kutenderedza yakakwira performance liquid chromatography (HPLC), tura mafemo. Haisi bhokisi rezvikamu zvinodhura; kutamba kwakapfava pakati pechemistry nefizikisi.

MuLab, isu tinowanzo taura kuti mobile phase ndiyo "yehupenyu mutsara" weiyo system. Pasina iyo, sampuli yako yakangogara mujekiseni, hapana kwainoenda. Asi iwe unosarudza sei "chitima" chakakodzera kutakura mamorekuru ako kune detector? Ngatizviputse sekunge tiri kutaura pamusoro pekofi muimba yekutandarira.

1. Chii chinonzi HPLC, Chaizvoizvo?

Pakati payo, HPLC ndeyekuparadzana. Funga nezvayo semujaho apo "track" ndiyo nhanho yakamira (iyo column) uye "mhepo" inosundira vamhanyi pamwe chete ndiyo hplc mobile phase.

Mashiripiti anoitika nekuti mamorekuru akasiyana ane "affinities" akasiyana. Mamwe mamorekuru anoda mobile phase uye zoom kuburikidza nekoramu. Vamwe "vanonamira" - vane pfumo padanho rakamira uye vanosara kumashure. Uyu mutsauko mukumhanya ndiwo unogadzira iwo mapikisi pachromatogram yako.

2. Normal Phase vs. Reverse Phase

Kazhinji apa ndipo panotanga munhu wese kudhonza bvudzi rake panze. Mumakore angu ekudzidzira, ndakawana nzira iri nyore yekurangarira iyi ndeyekutarisa polarity.

A. Normal Phase Chromatography

Iyi ndiyo nzira ye "chikoro chekare". Mune yakajairika chikamu chromatography, iyo yakamira chikamu ndeye polar-funga nezvayo semagineti yemamorekuru epolar. Nharembozha yatinoshandisa pano haisi-polar, kazhinji zvinonyungudutsa sehexane kana heptane.

• Iyo Logic: Polar "inonamira" mamorekuru anodzoserwa kumashure neiyo polar column. Non-polar mamorekuru anogeza nekukasira neasiri-polar mobile phase.

• Iyo Pro Tip: Kana iwe ukawedzera polarity yechikamu chako chefoni (kuwedzera zvishoma isopropanol), samples dzako dzinonyatso famba nekukurumidza.

B. Reverse Phase Chromatography

Nhasi, iyi ndiyo chiyero chegoridhe. Pamwe 90% yebasa randinoita murabhoritari rinosanganisira reverse phase chromatography. Pano, isu tinoshandura script. Iyo column haina-polar (hydrophobic, semafuta), uye phase mobile is polar (kazhinji musanganiswa wemvura uye methanol kana acetonitrile).

• The Logic: Mvura-inoda (polar) mamorekuru anoswededza mukati nekuti anovenga mbiru ine mafuta. Mafuta-anofarira (asiri-polar) mamorekuru anonamira pambiru senamo.

• Chikonzero nei tichichida: Zvinogoneka, zvinogoneka, uye zvinoshandisa mvura-based solvents izvo zviri nyore kubata.

3. Iyo "Golden Rule" yeSolvent Quality

Iwe uchaona "HPLC Giredhi" pane mavara nechikonzero. Usatombo funga nezvekushandisa ACS giredhi kana chero zvishoma. Sei? Nekuti izvo "zvidiki" tsvina mune yakaderera-giredhi zvinonyungudutsa zvichaungana pakoramu yako nekufamba kwenguva, zvichiuraya kugadzirisa kwako uye kugadzira zvisinganzwisisike ghost peaks.

Nguva dzose shandisa Milli-Q (18.2 MΩ.cm) giredhi remvura. Kana mvura yako "yakasviba," yako yekutanga ichaita senge makomo, uye iwe uchapedza mazuva matatu uchiedza kufunga kuti sei.

4. Degassing uye Filtration: The Performance Killers

Mhepo muvengi wehplc mobile phase. Nekumanikidzwa kwakajairika, mweya unonyungudika muzvinonyungudutsa. Asi kana iyo solvent ikarova pombi, tuturu tudiki twunogona kuvharirwa mumavharuvhu echeki kana sero redetector.

• Mhedzisiro: Kuyerera kweyero kuchinjika, pulsing baselines, uye kufamba kwekuchengetedza nguva.

• Iyo Fix: Shandisa yakavakirwa-mukati degassers, kana sonication\/vacuum filtration kana iwe uchimhanyisa yakakwirira-sevhitivity assays.

Kusefa hakusi Sarudzo. Kunyangwe HPLC giredhi zvinonyungudutsa zvinogona kuva ne microscopic particles kubva mukugadzira maitiro kana bhodhoro cap. Gara uchisefa kuburikidza ne 0.22µm kana 0.45µm membrane isati yabata dura rako. Iri ibasa remaminetsi mashanu iro rinogona kuchengetedza koramu yako kubva pakufa kusati kwasvika.

5. Buffers uye Girazi Musungo

Mureverse phase chromatography, tinoshandisa mabuffers kuchengetedza pH yakagadzikana kune ionzable samples.

• Pro Tip: Kana wakashandisa buffer nhasi, geza sisitimu nemvura yakachena/organic musanganiswa usati waenda kumba. Kana iwe ukasadaro, iwo munyu unopenya uye uchacheka zvisimbiso zvepombi yako.

• Iyo Girazi Musungo: Waizviziva here kuti kana yako hplc nhare mbozha pH iri> 8.0, inogona kunyatso leach simbi ions kubva kumagirodhi egirazi? Chinja kusimbi isina tsvina kana midziyo yepurasitiki yakasarudzika mune idzi kesi.

Ita Kuti Kuparadzana Kwako Kuite Kwakanaka

Kuwana nharembozha yacho i80% yehondo muHPLC. Kana iwe uchinetsekana ne "shag carpet" baselines, pressure spikes, kana zvisingaenderani mhinduro, usatambise rimwe zuva uchifungidzira.

Ndapedza zviuru zvemaawa ndiri pabhenji kugadzirisa nyaya idzi chaidzo. Ini ndinogara ndichifara kugovera zvishoma zve "lab-yakapupurirwa" kuraira kuti purojekiti yako idzoke munzira.

• WhatsApp: 86 18338832256

• Email:boonemi@aijirenvial.com
  

References

• Snyder, L. R., Kirkland, J. J., & Dolan, J. W. (2011). Nhanganyaya kune Yemazuvano Liquid Chromatography. 3rd Edition. John Wiley & Vanakomana. (Iyo "Gold Standard" yekunzwisisa HPLC nzira dzekuparadzanisa).

• Dolan, J. W. (2002). "Mobile Phase Kugadzirira." LCGC North America, Troubleshooting Series. (Yakakosha inoshanda gwara rekubvisa uye kusefa).

• Aparicio, R. (Mupepeti.). (2013). Manual yeHPLC Optimization. Elsevier. (Yakazara data pane solvent polarity uye buffer kusarudzwa mune reverse phase chromatography).

• Agilent Technologies. "HPLC System Troubleshooting Guide: Kudzivirira Common Mobile Phase Errors." Technical Resource Library.

• Iyo kambani Waters Corporation. "Mitemo yeChromatography: Normal Phase vs. Reverse Phase." Dzidzo Series.

• International Conference on Harmonization (ICH). Q2 (R1) Kusimbiswa kweKuongorora Maitiro: Zvinyorwa uye Methodology. (Standard for peak area quantification uye kuchengetedza nguva kugadzikana).