Isigaba esihambayo silingana nesigaba solwelo segazi, kwaye kukho izinto ezahlukeneyo zokuhoya ingqalelo ngexesha lokusetyenziswa. Phakathi kwazo kukho “imigibe” ekufuneka iqwalaselwe.
01. Ukulinganisa i-pH yesigaba seselula emva kokongeza i-solvent ye-organic
Ukuba ulinganisa i-pH kunye ne-organic additive, i-pH oyifumanayo iya kwahluka kunangaphambi kokongeza i-solvent ye-organic. Nangona kunjalo, eyona nto ibalulekileyo kukuba ungaguquguquki. Ukuba uhlala ulinganisa i-pH emva kokufaka i-solvent ye-organic, qiniseka ukuba uchaza amanyathelo akho kwindlela oyisebenzisayo ukuze abanye balandele indlela efanayo. Le ndlela ayikho i-100% echanekileyo, kodwa ubuncinane iya kugcina indlela ehambelanayo. Oku kunokubaluleka ngakumbi kunokufumana ixabiso elichanekileyo le-pH.
02. Akukho sikhuseli sisetyenzisiweyo
Injongo yesithinteli kukulawula i-pH kwaye iyithintele ekutshintsheni. Ezinye iindlela ezininzi zitshintsha i-pH yesigaba esihambahambayo, esinokubangela utshintsho kwixesha lokugcinwa, ukubunjwa okuphezulu, kunye nokuphendula okuphezulu.
Formic acid, TFA, njalo njalo ayizo buffers
03. Ukungasebenzisi isithinteli kuluhlu lwe-pH oluqhelekileyo
Isithinteli ngasinye sinobubanzi beyunithi ye-pH e-2, ngaphakathi apho sibonelela ngeyona pH yozinzo. Izithinteli ngaphandle kwale festile aziyi kubonelela ngokuxhathisa okusebenzayo kutshintsho lwe-pH. Sebenzisa isithinteli kuluhlu oluchanekileyo, okanye khetha isithinteli esigquma uluhlu lwe-pH oyifunayo.
04. Yongeza isithinteli kwisisombululo esiphilayo
Ukudibanisa isisombululo se-buffer kunye nesigaba se-organic kuya kubangela ukuba isithinteli sibe nemvula. Kwiimeko ezininzi, nokuba kukho imvula, kusenzima ukuyibhaqa. Khumbula ukuhlala usongeza isisombululo se-organic kwisigaba samanzi, esinokunciphisa kakhulu ithuba lemvula ye-buffer.
05. Hlanganisa i-gradient yoxinaniso ukusuka kwi-0% kunye nempompo
Iipompo ezikhoyo namhlanje zinokuxuba ngokufanelekileyo izigaba zeselula kunye ne-degas inline, kodwa ayinguye wonke umntu osebenzisa indlela yakho oya kuba nempompo yomgangatho ophezulu. Xuba u-A kunye no-B kwisisombululo esinye kwaye uyiqhube nge-100% emgceni.
Ngokomzekelo, i-950 ml yomxube wokuqala we-organic inokulungiswa ngokuxuba kunye ne-50 ml yamanzi. Inzuzo yale nto kukuba inokunciphisa ukuhluka phakathi kwe-HPLC kunye nokunciphisa amathuba okuba namaqamza kunye nemvula kwinkqubo. Kuyafaneleka ukuba uqaphele ukuba umlinganiselo womxube wepompo ngu-95: 5, oko akuthethi ukuba ixesha lokugcinwa kwangaphambili elixutywe kwibhotile liyi-95: 5.
06. Ukungasebenzisi iasidi elungisiweyo echanekileyo (isiseko) ukutshintsha isithinteli
Sebenzisa kuphela iasidi okanye isiseko esenza ibuffer yetyuwa oyisebenzisayo. Ngokomzekelo, i-sodium phosphate buffer kufuneka ilungiswe nge-asidi ye-phosphoric okanye i-sodium hydroxide kuphela.
07. Ukungaxeli lonke ulwazi malunga nesithinteli kwindlela, njengokudibanisa i-5g yei-sodium phosphate ukuya kwi-1000ml yamanzi.
Uhlobo lwesithinteli lumisela uluhlu lwe-pH olunokuthi luthintelwe. Ugxininiso olufunekayo lumisela amandla e-buffer. I-5 grams okanye i-anhydrous sodium phosphate kunye ne-5 grams ye-monosodium phosphate monohydrate inamandla ahlukeneyo e-buffer.
08. Ukongeza izinyibilikisi ze-organic phambi kokukhangela
Ukuba indlela yangaphambili isebenzise isisombululo se-buffer kwisiseko se-B, kwaye indlela yakho isebenzisa isisombululo se-organic kwisiseko se-B, ungathemba ukuba uhlalise isithinteli kwi-tubing yempompo kunye nentloko yempompo.
09. Phakamisa ibhotile uze ukhuphe ithontsi lokugqibela
Kukho ithuba elihle lokuba awuyi kuba nesigaba seselula esaneleyo ukugqiba yonke into kwaye isampuli yakho iya kutshaya. Ngaphandle kokukwazi ukutshisa inkqubo yempompo kunye nekholomu, isigaba esihambayo siya kunyuka ngokupheleleyo kwaye isigaba esihambayo phezulu kwibhotile siya kutshintsha.
10. Sebenzisa i-ultrasonic degassing isigaba seselula
Eyona ngongoma ibalulekileyo kukuqinisekisa ukuba zonke iityuwa ze-buffer ziyanyibilika, kodwa le yeyona ndlela imbi kakhulu ye-degas kwaye iya kutshisa ngokukhawuleza isigaba esiphathwayo, sibangele ukuba izinto eziphilayo zibe ngumphunga. Ukugcina ingxaki engeyomfuneko kamva, thatha imizuzu emihlanu ukucoca inqanaba lakho leselula.
Ixesha lokuposa: Aug-27-2024