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Ubonisa ikharawusi yezilayidi ezithathu ngaxeshanye. Sebenzisa amaqhosha angaphambili nalandelayo ukutyhutyha izilayidi ezithathu ngexesha, okanye sebenzisa amaqhosha esilayidi ekupheleni ukuya kwizilayidi ezithathu ngexesha.
Iincinci ze-silica ze-porous zalungiselelwa yindlela ye-sol-gel kunye nohlengahlengiso oluthile lokufumana amaqhekeza abanzi. La masuntswana athathwe kwi-N-phenylmaleimide-methylvinyl isocyanate (PMI) kunye ne-styrene nge-reverse chain transfer-fragmentation (RAFT) polymerization ukuvelisa i-N-phenylmaleimide intercalated polyamides. I-styrene (PMP) isigaba sokumisa. Iikholamu zetsimbi ezingenasici ezinqamlekileyo (i-100 × 1.8 mm ububanzi bangaphakathi) zazipakishwe ngokupakishwa kwe-slurry. Ukusebenza kwe-chromatographic yekholomu ye-PMP yavavanywa ukuba ihlukanise umxube weepeptide zokwenziwa eziqukethe i-peptides ezintlanu (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-Leu amino acid enkephalin) kunye ne-tryptic ye-albhamu ye-Hydrolyzan yabantu. Ngaphantsi kweemeko ezifanelekileyo ze-lution, inani lethiyori yeepleyiti ezinomxube weepeptide zafikelela kwi-280,000 plates/sq.m. Ukuthelekisa ukusebenza kokuhlukana kwekholamu ephuhlisiwe kunye nekholamu ye-Ascentis Express RP-Amide yorhwebo, kwabonwa ukuba ukusebenza kakuhle kokuhlukana kwekholamu ye-PMP kwakuphezulu kwikholamu yorhwebo ngokwemigaqo yokwahlula kakuhle kunye nesisombululo.
Umzi-mveliso we-biopharmaceutical uye waba yimakethi ekhulayo yehlabathi ngokunyuka okukhulu kwisabelo semarike kwiminyaka yakutshanje. Ngokukhula okugqabhukileyo kweshishini le-biopharmaceutical1,2,3 kukho imfuneko enkulu yohlalutyo lwepeptide kunye neprotheyini. Ukongeza kwi-peptide ekujoliswe kuyo, ukungcola okwahlukeneyo kwenziwa ngexesha le-peptide synthesis, ngoko ke ukucocwa kwechromatographic kuyafuneka ukufumana ukucoceka okufunwayo kwepeptide. Uhlalutyo kunye nokubonakaliswa kweeprotheyini kumanzi omzimba, izicubu kunye neeseli ngumsebenzi onzima kakhulu ngenxa yenani elikhulu leentlobo ezinokuthi zibonwe kwisampulu enye. Nangona i-mass spectrometry sisixhobo esisebenzayo sokulandelelanisa i-peptides kunye neeprotheni, ukuba iisampulu ezinjalo zingeniswa ngokuthe ngqo kwi-spectrometer yobunzima, ukwahlukana kuya kunganelisi. Le ngxaki ingasombululwa ngokwenza i-chromatography ye-liquid (LC) phambi kohlalutyo lwe-MS, oluya kunciphisa inani labahlalutyi abangena kwi-spectrometer yobunzima ngexesha elithile4,5,6. Ukongeza, abahlalutyi banokugxila kwindawo emxinwa ngexesha lokwahlulwa kwesigaba solwelo, ngaloo ndlela kugxininiswe kwaba bahlalutyi kunye nokwandisa uvakalelo lokufunyanwa kwe-MS. I-Liquid chromatography (LC) iqhubele phambili kakhulu kule minyaka ilishumi idlulileyo kwaye ibe yindlela esetyenziswa ngokubanzi yohlalutyo lweproteomic7,8,9,10.
I-reverse-phase liquid chromatography (RP-LC) isetyenziselwa ngokubanzi ukuhlambulula kunye nokwahlula imixube yeepeptides usebenzisa i-octadecyl-modified silica (ODS) njengesigaba esimileyo11,12,13. Nangona kunjalo, ngenxa yesakhiwo sabo esiyinkimbinkimbi kunye nendalo ye-amphoteric, i-14,15 izigaba ze-RP ezimileyo azikwazi ukunika ulwahlulo olwanelisayo lweepeptides kunye neeprotheni. Ngoko ke, uhlalutyo lweepeptides kunye neeprotheni ezineengqungquthela ze-polar kunye ne-non-polar zifuna izigaba zokumisa ezilungiselelwe ngokukodwa ukusebenzisana kunye nokugcina aba bahlalutyi16. I-Chromatography edibeneyo, enika intsebenziswano ye-multimodal, inokuba yenye ye-RP-LC yokwahlula iipeptides, iiprotheni, kunye neminye imixube enzima. Izigaba ezininzi zokumisa ezixubeneyo zalungiswa kwaye iikholomu ezizaliswe ngezi zigaba zokumisa zasetyenziselwa ukwahlula iipeptides kunye neeprotheni17,18,19,20,21. Ngenxa yobukho bamaqela e-polar kunye ne-non-polar, i-mixed mode modes stationary phases (WAX / RPLC, HILIC / RPLC, i-polar intercalation / RPLC) ifanelekile ukuhlukana kweepeptides kunye neeprotheni22,23,24,25,26,27,28. , izigaba ze-polar ezidibeneyo ezimileyo kunye namaqela e-polar adibeneyo abonisa amandla okuhlukana okulungileyo kunye nokukhethwa okukhethekileyo kwi-polar kunye ne-non-polar analytes kuba ukwahlukana kuxhomekeke ekusebenzisaneni phakathi kwe-analyte kunye nesigaba esimileyo Ukusebenzisana kwe-Multimodal 29,30,31,32. Kutshanje, uZhang et al. I-30 ifumene izigaba ezimileyo ze-behenyl eziphelisiwe ze-polyamines kwaye zahlula ngempumelelo ii-hydrocarbons, i-antidepressants, i-flavonoids, i-nucleosides, i-estrogens, kunye nolunye uhlalutyi. Izinto ezimileyo ezifakwe kwi-polar zinamaqela amabini amhlophe kunye ne-non-polar, ngoko ke ingasetyenziselwa ukwahlula iipeptides kunye neeprotheni zibe yi-hydrophobic kunye ne-hydrophilic parts. Iikholomu ze-polar inline (umz., iikholomu ze-C18 ezine-amide inline) zifumaneka phantsi kwegama lezorhwebo elithi Ascentis Express RP-Amide iikholomu, kodwa ezi zikholomu zisetyenziselwa uhlalutyo lwe-amine 33.
Kuphononongo lwangoku, i-polar embeding stationary phase (N-phenylmaleimide, embedding polystyrene) yalungiswa kwaye yavavanyelwa ukwahlulwa kwe-peptide kunye ne-tryptic HSA cleavage. Esi sicwangciso-qhinga silandelayo sisetyenziselwe ukulungiselela isigaba sokusebenza. Iinqununu ze-silica ezinobuncwane zalungiswa ngokwemigaqo echazwe kwiincwadi zethu zangaphambili, kunye nolunye utshintsho kwiiskimu zokulungiselela i-31, 34, 35, 36, 37, 38, 39. Imilinganiselo ye-urea, i-polyethylene glycol (PEG), i-TMOS kunye ne-aqueous-acetic acid yalungiswa ukuze ifumane iinqununu ezinkulu ze-silica. Okwesibini, i-phenylmaleimide-methylvinyl isocyanate ligand entsha yenziwe kwaye iinqununu zayo ze-silica ezikhutshwayo zisetyenziselwa ukulungiselela izigaba ezimileyo ezifakwe kwi-polar. Isigaba sokumisa esifunyenweyo sifakwe kwikholomu yensimbi engenasici (ububanzi bangaphakathi 100 × 1.8 mm) ngokweskimu sokupakisha esilungisiweyo. Ukupakishwa kwekholomu kuncediswa yi-vibration yomatshini ukuze kuqinisekiswe uluhlu olufanayo ngaphakathi kwekholomu. Ikholamu epakishweyo yavavanywa ukwahlula umxube we-peptides oquka iipeptides ezintlanu (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, iTyr-Ile-Gly-Ser-Arg, i-leucine-enkephalin peptide). kunye ne-tryptic hydrolysates ye-albumin yabantu ye-serum (HSA). Kwaye kwaphawulwa ukuba umxube we-peptide kunye ne-HSA tryptic digest yahlulwe ngesisombululo esihle kunye nokusebenza kakuhle. Ukusebenza kokuhlukana kwekholamu ye-PMP kwafaniswa nekholamu ye-Ascentis Express RP-Amide. Kwaphawulwa ukuba i-peptides kunye neeprotheni zinesisombululo esihle kunye nokusebenza kakuhle kokuhlukana kwikholamu ye-PMP, kunye nokusebenza kakuhle kokuhlukana kwekholamu ye-PMP iphezulu kunekholamu ye-Ascentis Express RP-Amide.
I-PEG (i-polyethylene glycol), i-urea, i-acetic acid, i-trimethoxyorthosilicate (TMOS), i-trimethylchlorosilane (TMCS), i-trypsin, i-albumin ye-human serum (HSA), i-ammonium chloride, urea, i-hexamethylmethacryloyldisilazane (HMDS), i-methacryloyl chloride, i-Hydroxyrene (MC4), i-methacryloyl chloride, i-MC4 (BPO), i-acetonitrile (ACN) ye-HPLC, i-methanol, i-2-propanol kunye ne-acetone. Inkampani yaseSigma-Aldrich (eSt. Louis, eMissouri, eU.SA).
Umxube we-urea (8 g), i-polyethylene glycol (8 g) kunye ne-8 ml ye-0.01 N. i-acetic acid yaxutywa imizuzu eyi-10, kwaye i-24 ml ye-TMOS yongezwa apho phantsi kwe-ice-cooling. Umxube wokuphendula ushushu kwi-40 ° C kwiiyure ze-6 kwaye emva koko kwi-120 ° C kwiiyure ze-8 kwi-autoclave yensimbi engenasici. Amanzi ahlanjululwa kwaye intsalela yomiswa kwi-70 ° C kwiiyure ze-12. Iibhloko ezithambileyo ezomisiweyo zacolwa kakuhle kwaye zafakwa kwi-oven engu-550 ° C iiyure ezili-12. Iibhetshi ezintathu zalungiswa kwaye zibonakaliswe ukuvavanya ukuphindaphinda kobungakanani be-particle size, ubukhulu bepore kunye nommandla wendawo.
Iqela le-polar kunye nesigaba esimileyo samakhonkco e-polystyrene. Inkqubo yokulungiselela ichazwe ngezantsi.
I-N-phenylmaleimide (200 mg) kunye ne-methyl vinyl isocyanate (i-100 mg) yachithwa kwi-toluene e-anhydrous, kwaye emva koko i-0.1 ml ye-2,2'-azoisobutyronitrile (AIBN) yongezwa kwi-flask yokuphendula ukufumana i-copolymer ye-phenylmaleimide kunye ne-vinyl methylCP (vinyl isoso). ) Umxube wawushushu kwi-60 ° C kwiiyure ze-3, ucofe kwaye womiswe kwi-oven kwi-40 ° C ngeeyure ze-3.
Iinqununu ze-silica ezomileyo (i-2 g) zahlakazwa kwi-toluene eyomileyo (i-100 ml), ixutywe kwaye i-sonicated i-10 min kwi-flask ephantsi ye-500 ml. I-PMCP (10 mg) yachithwa kwi-toluene kwaye yongezwa i-dropwise kwi-reaction flask nge-funnel yokongeza. Umxube uhlanjululwe kwi-100 ° C kwiiyure ze-8, uhlanjululwe, uhlanjwe nge-acetone kwaye womiswe kwi-60 ° C ngeeyure ze-3. Emva koko, i-silica particles ehambelana ne-PMCP (100 g) yachithwa kwi-toluene (200 ml), kunye ne-4-hydroxy-TEMPO (2 ml) yongezwa apho phambi kwe-100 μl ye-dibutyltin dilaurate njenge-catalyst. Umxube wawuxutywe kwi-50 ° C kwiiyure ze-8, ucofe kwaye womiswe kwi-50 ° C ngeeyure ze-3.
I-Styrene (1 ml), i-benzoyl peroxide BPO (0.5 ml) kunye ne-silica particles efakwe kwi-TEMPO-PMCP (1.5 g) yachithwa kwi-toluene kwaye yahlanjululwa nge-nitrogen. I-polymerization ye-styrene yenziwa kwi-100 ° C kwiiyure ze-12. Imveliso efunyenweyo ihlanjwe nge-methanol kwaye yomiswa ngobusuku kwi-60 ° C. Iskimu esiqhelekileyo sokuphendula siboniswa kumfanekiso. Nye .
Iisampuli zachithwa kwi-393 K kwi-1 h kwaze kwaba yilapho kufunyenwe uxinzelelo olushiyekileyo olungaphantsi kwe-10-3 Torr. Isixa se-N2 esibhengezwe kuxinzelelo olunxulumene ne-P / P0 = 0.99 yasetyenziselwa ukumisela umthamo opheleleyo we-pore. I-morphology ye-silica ecocekileyo kunye ne-ligand-bound-bound particles yahlolwa kusetyenziswa i-electron microscope yokuskena (i-Hitachi High Technologies, e-Tokyo, e-Japan). Iisampulu ezomileyo (i-silica ecocekileyo kunye ne-ligand eboshwe i-silica particles) zifakwe kwiintonga ze-aluminium usebenzisa i-carbon tape. Igolide yafakwa kwisampulu kusetyenziswa isixhobo sputtering Q150T, kunye 5 nm obukhulu Au umaleko wafakwa kwisampulu. Oku kuphucula ukusebenza kakuhle kwenkqubo yombane ophantsi kwaye ibonelela ngokutshiza okubandayo okucolekileyo. Uhlalutyo lwe-Elemental lwenziwa kusetyenziswa i-Thermo Electron (Waltham, MA, USA) I-Flash EA1112 i-elemental composition analyzer. I-Malvern particle size analyzer (Worcestershire, UK) Mastersizer 2000 isetyenziswe ukufumana ukusabalalisa ubungakanani be-particle. Iinqununu ze-silica ezingaxutywanga kunye ne-ligand-bound-bound silica particles (5 mg nganye) zachithwa kwi-5 ml ye-isopropanol, i-sonicated imizuzu ye-10, iphazamise imizuzu ye-5, kwaye ibekwe kwi-bench ye-Mastersizer optical. Uhlalutyo lwe-Thermogravimetric lwenziwa ngesantya se-5 °C ngomzuzu kwiqondo lokushisa ukusuka kwi-30 ukuya kwi-800 °C.
Ifiber yeglasi enemigca emxinwa yazalisa iintsika zetsimbi ezingenasici ezinemilinganiselo (ID 100 × 1.8 mm) zipakishwe ngendlela yokuzalisa uludaka ngokulandela inkqubo efanayo nereferensi 31. Ikholomu yentsimbi engenanto (iglasi elayitiweyo, i-ID 100 × 1 .8 mm) kunye ne-outlet equkethe i-1 µm i-friedfield machine, yonke i-packfield ye-tech iqhagamshelwe. IL, USA). Lungiselela ukunqunyanyiswa kwesigaba sokumisa ngokumisa i-150 mg yesigaba sokumisa kwi-1.2 ml ye-methanol kwaye uyondle kwikholamu yokugcina amanzi. I-Methanol yayisetyenziswa njenge-solvent ye-slurry kunye ne-solvent yokulawula. Pakisha ikholamu ngokusebenzisa ulandelelwano loxinzelelo lwe-100 MP kwi-10 min, i-80 MP ye-15 min, kunye ne-60 MP ye-30 min. Inkqubo yokupakisha isetyenziselwe i-gas chromatography column vibrators ezimbini (Alltech, Deerfield, IL, USA) ukwenzela ukungcangcazela komatshini ukuqinisekisa ukupakishwa kwekholamu efanayo. Vala i-slurry packer kwaye ukhulule uxinzelelo ngokukhawuleza ukukhusela umonakalo kumtya. Ikholomu ikhutshwe kwi-slurry nozzle kwaye enye i-fitting ifakwe kwi-inlet kwaye ixhunywe kwinkqubo ye-LC ukuvavanya ukusebenza kwayo.
I-MLC yesiko yakhiwe kusetyenziswa i-LC pump (10AD Shimadzu, Japan), isampuli ene-50 nL injection loop (Valco (USA) C14 W.05), i-membrane degasser (Shimadzu DGU-14A), kunye nefestile ye-UV-VIS ye-capillary. Isixhobo somtshini (UV-2075) kunye ne-enamelled microcolumn. Sebenzisa imibhobho yokudibanisa emxinwa kakhulu kwaye imfutshane ukunciphisa umphumo wokwandiswa kwekholomu eyongezelelweyo. Emva kokuzalisa ikholamu, fakela i-capillary (50 µm id 365) kwindawo yokuphuma ye-1/16″ yokunciphisa isiphambuka kwaye ufake i-capillary (50 µm) yesiphambuka sokunciphisa. Ukuqokelelwa kwedatha kunye nokulungiswa kwechromatogram kwenziwa kusetyenziswa isoftware ye-Multichro 2000. Kwi-254 nm, i-UV absorbance of the subjects analytes ibekwe esweni kwi-0. Idatha ye-Chromatographic yahlaziywa ngokusebenzisa i-OriginPro8 (Northampton, MA).
I-albumin ye-serum yabantu, i-lyophilized powder, ≥ 96% (i-agarose gel electrophoresis) 3 mg exutywe ne-trypsin (1.5 mg), 4.0 M urea (1 ml) kunye ne-0.2 M ye-ammonium bicarbonate (1 ml) . Isisombululo sixutywe nge-10 min kwaye sigcinwe kwindawo yokuhlambela amanzi kwi-37 ° C kwi-6 h, emva koko sicinywe nge-1 ml ye-0.1% TFA. Hlunga isisombululo kwaye ugcine ngaphantsi kwe-4°C.
Ukwahlulwa komxube we-peptides kunye ne-tryptic digest ye-HSA kwikholamu ye-PMP yavavanywa ngokwahlukeneyo. Jonga i-tryptic hydrolysis yomxube we-peptides kunye ne-HSA ehlulwe yikholomu ye-PMP kwaye uthelekise iziphumo kunye nekholamu ye-Ascentis Express RP-Amide. Inani leepleyiti zethiyori libalwa kusetyenziswa le nxaki ilandelayo:
Imifanekiso ye-SEM ye-silica ecocekileyo kunye ne-ligand eboshiweyo ye-silica iboniswe kwi-Figure 2. Imifanekiso ye-SEM ye-silica ecocekileyo ye-silica (A, B) ibonisa imilo ye-spherical apho iinqununu zande okanye zine-symmetry engavamile xa kuthelekiswa nezifundo zethu zangaphambili. Umphezulu we-silica particles eboshwe yi-ligand (C, D) ilula ngakumbi kuneengqungquthela ze-silica ezicocekileyo, ezinokuthi zibangelwa ngamaketanga e-polystyrene agubungela umphezulu we-silica particles.
Ukuskena i-electron micrographs ye-silica ecocekileyo ye-silica (A, B) kunye ne-ligand eboshwe i-silica particles (C, D).
Ukusabalalisa ubungakanani bemilinganiselo ye-silica ecocekileyo kunye ne-ligand-bound bound silica particles iboniswe kwi-Fig. 2. 3 (A). I-Volumetric particle size distribution curves ibonise ukuba ubukhulu be-silica particle yanda emva kokuguqulwa kweekhemikhali (Umfanekiso 3A). Idatha yokusabalalisa ubungakanani be-silica particle ukusuka kuphononongo lwangoku kunye nophononongo lwangaphambili lufaniswa kwiThebhile 1 (A). I-volumetric particle size d (0.5) ye-PMP yayiyi-3.36 µm, xa kuthelekiswa nexabiso le-ad (0.5) le-3.05 µm kwisifundo sethu sangaphambili (i-polystyrene bonded silica particles)34. Ngenxa yokutshintshwa komlinganiselo we-PEG, urea, TMOS kunye ne-acetic acid kumxube wokuphendula, ukusabalalisa ubungakanani be-particle yale bhetshi yayincinci xa kuthelekiswa nesifundo sethu sangaphambili. Ubungakanani bamasuntswana esigaba se-PMP bukhulu kancinci kuneso se-polystyrene ebotshelelwe i-silica particle phase esiyifunde ngaphambili. Oku kuthetha ukuba ukusebenza komphezulu wamasuntswana e-silica kunye ne-styrene efakwe kuphela umaleko we-polystyrene (0.97 µm) kumphezulu we-silica, ngelixa kwisigaba se-PMP ubukhulu bemaleko yi-1.38 µm.
Ukusabalalisa ubungakanani be-particle (A) kunye nokusabalalisa ubungakanani be-pore (B) ye-silica ecocekileyo ye-silica kunye ne-ligand eboshiweyo ye-silica.
Ubungakanani bepore, umthamo wepore, kunye nommandla womphezulu wamasuntswana e-silica asetyenziswe kolu phononongo iboniswe kwiThebhile 1 (B). Iiprofayili ze-PSD zeengqungquthela ezicocekileyo ze-silica kunye ne-ligand-bound bound silica particles ziboniswe kwiMifanekiso. 3(B). Iziphumo zazithelekiseka nesifundo sethu sangaphambili34. Ubungakanani be-pore ye-silica ecocekileyo kunye ne-ligand-bound-bound particles ye-silica yayiyi-310 Å kunye ne-241 Å, ngokulandelanayo, ebonisa ukuba emva kokuguqulwa kweekhemikhali, ubukhulu be-pore buncitshiswe ngu-69 Å, njengoko kuboniswe kwiThebhile 1 (B), kwaye i-curve shift iboniswe kwi-Fig. (124 m2/g). Njengoko kuboniswe kwiThebhile 1 (B), indawo engaphezulu (m2 / g) ye-silica particles emva kokuguqulwa kweekhemikhali nayo yehla ukusuka kwi-116 m2 / g ukuya kwi-105 m2 / g.
Iziphumo zocazululo lokuqala lwesigaba sokusebenza zithiwe thaca kwiThebhile. 2. Umxholo wekhabhoni wesigaba esimileyo sangoku yi-6.35%, ephantsi kunesifundo sethu sangaphambili (i-silica particles ehambelana ne-polystyrene, i-7.93% 35 kunye ne-10.21%, ngokulandelanayo) 42. Umxholo wekhabhoni wesigaba esisezantsi esisezantsi, ekubeni ezinye ii-polar ligands ezifana ne-phenylmaleimide methylcyTEM kunye ne-hydroxyMP isetyenziswe i-hydroxy-hydroxyMP. ngaphezu kwe-styrene ekulungiseleleni i-SP. Ipesenti yobunzima be-nitrogen kwinqanaba langoku lokumisa li-2.21% xa kuthelekiswa ne-0.1735 kunye ne-0.85% kwizifundo zangaphambili42. Oku kuthetha ukuba isigaba esimileyo sangoku sinepesenti yobunzima obukhulu be-nitrogen ngenxa ye-phenylmaleimide. Ngokufanayo, iimveliso (4) kunye (5) zinomxholo wekhabhoni we-2.7% kunye ne-2.9%, ngokulandelanayo, ngelixa imveliso yokugqibela (6) inomxholo wekhabhoni we-6.35%, njengoko kuboniswe kwiThebhile 2. Uhlalutyo lwe-Thermogravimetric (TGA) lusetyenziswe kwisigaba esimileyo se-PMP ukuvavanya ukulahleka kwesisindo, kwaye i-TGA ye-curve ye-TGA iboniswe kwi-curve ye-TGA4 I-8.6%, ehambelana kakuhle nomxholo wekhabhoni (6.35%), ekubeni i-ligands ayinayo kuphela i-C, kodwa i-N, O kunye ne-H.
I-ligand phenylmaleimide-methylvinyl isocyanate yakhethwa ukuba iguqule umphezulu we-silica particles ngenxa ye-polar phenylmaleimide kunye namaqela e-vinylisocyanate. Amaqela eVinyl isocyanate anokusabela ngakumbi nge-styrene ngokuphila kwepolymerization ephilayo. Isizathu sesibini kukufaka iqela elinobudlelwane obuphakathi kunye ne-analyte kwaye akukho ukusebenzisana okunamandla kwe-electrostatic phakathi kwe-analyte kunye nesigaba sokumisa, ekubeni i-phenylmaleimide moiety ayinayo intlawulo ebonakalayo kwi-pH eqhelekileyo. I-polarity yesigaba esimileyo sinokulawulwa ngomlinganiselo ofanelekileyo we-styrene kunye nexesha lokuphendula le-polymerization yamahhala. Inyathelo lokugqibela lokusabela (i-polymerization yasimahla yeradical) ibalulekile njengoko itshintsha i-polarity yenqanaba lokumisa. Uhlalutyo olusisiseko lwenziwa ukujonga umxholo wekhabhoni kwezi zigaba zokumisa. Kuye kwaphawulwa ukuba ukwandisa inani le-styrene kunye nexesha lokuphendula kwandisa umxholo wekhabhoni wesigaba sokumisa kunye nokunye. I-SPs elungiselelwe ngemilinganiselo eyahlukeneyo ye-styrene inemithwalo yekhabhoni eyahlukeneyo. Ngokufanayo, ezi zigaba ezimileyo zifakwe kwiikholomu zensimbi ezingenasici kunye neempawu zazo zechromatographic (ukukhethwa, isisombululo, ixabiso le-N, njl.) zahlolwa. Ngokusekwe kolu vavanyo, ukubunjwa okulungiselelwe ukulungiswa kwesigaba esimileyo se-PMP kwakhethwa ukubonelela ngepolarity elawulwayo kunye nokugcinwa kakuhle kohlalutyi.
Ikholomu ye-PMP iphinde ihlolwe ukuhlalutya kwemixube emihlanu ye-peptides (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-leucine-enkephalin) isebenzisa umthamo wesigaba seselula. 60/40 (v/v) ACN/amanzi (0.1% TFA) kwisantya sokuhamba kwe80 µl/min. Ngaphantsi kweemeko ezizezona zilungileyo (iipleyiti ezingama-200,000/m), inani leepleyiti zethiyori (N) kwikholamu nganye (100 × 1.8 mm) yi-20,000 ± 100. Amaxabiso e-N kwiikholamu ezintathu ze-PMP abonisiwe kwiThebhile 3 kunye neechromatogram ziboniswe kuMfanekiso 5A. Uhlalutyo olukhawulezayo kwisantya esiphezulu sokuhamba (700 µl / min) kwikholamu ye-PMP, iipeptide ezintlanu zikhutshwe ngomzuzu omnye, ixabiso elihle kakhulu le-N le-13,500 ± 330 ngekholomu (100 x 1.8 mm ububanzi), elilingana ne-135,000 plates / m (Fig. 5B). Iikholamu ezintathu zobukhulu obufanayo (i-diameter yangaphakathi 100 x 1.8 mm) zazaliswa ngeebhetshi ezintathu ezahlukeneyo zesigaba esimileyo se-PMP ukuvavanya ukuveliswa kwakhona. Abahlalutyi barekhodwa kwikholamu nganye ngokwahlula umxube wovavanyo ofanayo kwikholamu nganye kusetyenziswa iimeko ezifanelekileyo ze-lution, inani leeplati zethiyori N, kunye nexesha lokugcinwa. Idatha yokuvelisa kwakhona kwiikholamu ze-PMP iboniswe kwiThebhile 4. Ukuveliswa kwakhona kwekholamu ye-PMP kuhambelana kakuhle namaxabiso aphantsi kakhulu e-%RSD njengoko kubonisiwe kwiThebhile 3.
Ukwahlulwa kwemixube ye-peptide kwikholamu ye-PMP (B) kunye nekholamu ye-Ascentis Express RP-Amide (A), isigaba se-mobile 60/40 ACN/H2O (TFA 0.1%), imilinganiselo yekholomu ye-PMP (100 x 1.8 mm id) , uhlalutyo Umyalelo we-Elution we-compounds: 1 (Gly-Tyr-Leu-Tyr), 2 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) kunye ne-5 (i-leucic acid enkephalin).
Ikholamu ye-PMP (i-diameter yangaphakathi eyi-100 x 1.8 mm) yavavanyelwa ukwahlulwa kwe-tryptic hydrolyzate ye-albumin ye-serum yabantu yi-HPLC. Ikhromatogram kuMfanekiso 6 ibonisa ukuba iisampuli zihlulwe kakuhle kunye nesisombululo esihle kakhulu. Izisombululo ze-HSA zahlaziywa kusetyenziswa izinga lokuhamba kwe-100 μl / min, isigaba esihambayo se-70/30 acetonitrile / amanzi kunye ne-0.1% TFA. I-Cleavage ye-HSA yahlulwe yaba ziincopho ze-17, njengoko kuboniswe kwichromatogram (umzobo 6), ehambelana ne-17 peptides. Ukwahlulwa kobuchule beencopho zomntu ngamnye kwi-HSA hydrolyzate zibaliwe kwaye amaxabiso abonisiwe kwiThebhile 5.
I-HSA i-tryptic hydrolysates yahlulwa kwikholamu ye-PMP (i-diameter yangaphakathi 100 x 1.8 mm), izinga lokuhamba (100 μl / min), isigaba seselula 60/40 acetonitrile / amanzi, kunye ne-0.1% TFA.
apho L ubude bekholamu, η yi-viscosity yesigaba esihambayo, ΔP luxinzelelo lwangasemva lwekholamu, kwaye u-u yisantya somgca wesigaba esihambayo. I-permeability yekholomu ye-PMP yayiyi-2.5 × 10-14 m2, izinga lokuhamba laliyi-25 µl / min, 60/40 v / v isetyenzisiwe. I-ACN/amanzi. I-permeability yekholomu ye-PMP (i-ID 100 × 1.8 mm) yayifana nelo cwaningo lwethu lwangaphambili lweRef.34. Ukungena kwekholamu ezaliswe ngamasuntswana arhabaxa kakhulu yi-1.7×10 .6 µm, 2.5×10-14 m2 kwi-5 µm amasuntswana43. Ngoko ke, ukunyanzeliswa kwesigaba se-PMP kufana nokunyanzeliswa kweengqungquthela ze-core-shell ezinobungakanani be-5 μm.
apho i-Wx ibubunzima bekholamu ezaliswe yiklorofomu, i-Wy bubunzima bekholamu ezaliswe yimethanol, kwaye u-ρ bubuninzi besinyibilikisi. Ubuninzi be-methanol (ρ = 0.7866) kunye nekloroform (ρ = 1.484). I-porosity epheleleyo yekholamu ye-silica-C18 ye-particle (100 × 1.8 mm ID) i-34 kunye nekholomu yethu yangaphambili ye-C18-urea31 yayiyi-0.63 kunye ne-0.55, ngokulandelanayo. Oku kuthetha ukuba ubukho be-urea ligands kunciphisa ukunyanzeliswa kwesigaba sokumisa. Ngakolunye uhlangothi, i-porosity iyonke yekholomu ye-PMP (ububanzi bangaphakathi 100 × 1.8 mm) ngu-0.60. Iikholamu ze-PMP azikwazi ukungena ngaphantsi kweekholomu ezipakishwe ngamasuntswana e-silica e-C18 ngenxa yokuba kwizigaba ze-C18 zohlobo olumileyo i-C18 ligands idityaniswe kwi-silica particles kumatyathanga ahambelanayo, ngelixa kwi-polystyrene uhlobo lwezigaba zokumisa i-polymer engqingqwa kakhulu yenziwa malunga namasuntswana. umaleko A. Kumfuniselo oqhelekileyo, ikholam porosity ibalwa ngolu hlobo lulandelayo:
Kwikhiwane. I-7A, B ibonisa iiplani zeVan Deemter zekholamu ye-PMP (id 100 x 1.8 mm) kunye nekholomu ye-Ascentis Express RP-Amide (id 100 x 1.8 mm) phantsi kweemeko ezifanayo zokulution, 60/40 ACN/H2O kunye ne-0 .1% TFA 20 µl/min ¼ zombini kwikholamu ye-8/min. Ubuncinci bexabiso le-HETP kwinqanaba lokuhamba kakuhle (80 µl/min) yayiyi-2.6 µm kunye ne-3.9 µm yekholamu ye-PMP kunye nekholamu ye-Ascentis Express RP-Amide, ngokulandelanayo. Amaxabiso e-HETP abonisa ukuba ukusebenza kakuhle kokuhlukana kwekholamu ye-PMP (i-100 x 1.8 mm id) iphezulu kakhulu kunekholamu ye-Ascentis Express RP-Amide ekhoyo ngokurhweba (100 x 1.8 mm id). Igrafu ye-van Deemter kwi-Fig. 7 (A) ibonisa ukuba ukuhla kwexabiso le-N aliphezulu kakhulu kunye nokunyuka kokuhamba xa kuthelekiswa nesifundo sethu sangaphambili. Ukuphumelela okuphezulu kokuhlukana kwekholamu ye-PMP (id 100 × 1.8 mm) xa kuthelekiswa nekholamu ye-Ascentis Express RP-Amide isekelwe kwimilo ephuculweyo ye-particle kunye nobukhulu kunye nenkqubo yokupakisha ikholamu eyinkimbinkimbi esetyenziswe kumsebenzi wangoku34.
(A) icebo leVan Deemter (i-HETP vs. I-mobile phase linear velocity) ifunyenwe kwikholamu ye-PMP (id 100 x 1.8 mm) kwi-60/40 ACN/H2O kunye ne-0.1% TFA. (B) i-Van Deemter plot (i-HETP ngokubhekiselele kwi-mobile phase linear velocity) ifunyenwe kwikholamu ye-Ascentis Express RP-Amide (id 100 x 1.8 mm) kwi-60/40 ACN/H2O kunye ne-0.1% TFA.
Inqanaba elimileyo le-polar le-polystyrene edibeneyo yalungiswa kwaye yavavanyelwa ukwahlula umxube we-peptides eyenziweyo kunye ne-tryptic hydrolyzate ye-albumin ye-serum yabantu (HSA) kulwelo olusebenza kakhulu lwechromatography. Ukusebenza kwechromatographic kwiikholamu ze-PMP zemixube ye-peptide igqwesileyo ngokwemigaqo yokwahlula kakuhle kunye nesisombululo. Ukuphuculwa kokwahlula okuphuculweyo kweekholamu ze-PMP ngenxa yezizathu ezininzi ezifana nobukhulu be-silica particle kunye nobukhulu be-pore, ukulawulwa kwe-synthesis yezigaba ezimileyo, kunye nezinto eziyinkimbinkimbi zokupakisha ikholamu. Ukongeza kwintsebenzo ephezulu yokwahlula, enye inzuzo yesi sigaba sokumisa yikholomu ephantsi yoxinzelelo lwasemva kumazinga aphezulu okuhamba. Iikholamu ze-PMP zikwazi ukuveliswa kwakhona kwaye zingasetyenziselwa ukuhlalutya imixube yeepeptides kunye ne-tryptic digestion yeeprotheni ezahlukeneyo. Sizimisele ukusebenzisa le kholamu ukuhlukana kwee-bioactive compounds kwiimveliso zendalo, izicatshulwa zezityalo zonyango kunye nama-mushroom kwi-chromatography yamanzi. Kwixesha elizayo, iikholamu ze-PMP ziya kuphinda zivavanywe ukuhlukana kweeprotheni kunye ne-antibodies monoclonal.
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