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Izinhlayiya ze-silica ezinezimbobo zalungiswa ngendlela ye-sol-gel kanye nokuguqulwa okuthile ukuthola izinhlayiya ezinezimbobo ezibanzi. Lezi zinhlayiya zathathwa nge-N-phenylmaleimide-methylvinyl isocyanate (PMI) kanye ne-styrene nge-reverse chain transfer-fragmentation (RAFT) polymerization ukuze kukhiqizwe ama-polyamides ahlanganisiwe e-N-phenylmaleimide. Isigaba esimile se-Styrene (PMP). Amakholomu ensimbi engagqwali ane-narrow bore (ububanzi obuyi-100 × 1.8 mm ngaphakathi) agcwele i-slurry packing. Ukusebenza kwe-chromatographic kwekholomu ye-PMP kwahlolwa ukuze kuhlukaniswe ingxube yama-peptide okwenziwa aqukethe ama-peptide amahlanu (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-Leu amino acid enkephalin) kanye ne-tryptic hydrolyzate ye-human serum albumin (HAS). Ngaphansi kwezimo ezifanele zokuxazulula inkinga, inani lamapuleti anengxube yama-peptide lafinyelela amapuleti angu-280,000/sq.m. Uma kuqhathaniswa ukusebenza kokuhlukaniswa kwekholomu ethuthukisiwe nekholomu ye-Ascentis Express RP-Amide yezentengiselwano, kwabonwa ukuthi ukusebenza kahle kokuhlukaniswa kwekholomu ye-PMP kwakungcono kunekholomu yezentengiselwano ngokusebenza kahle kokuhlukaniswa kanye nesisombululo.
Imboni ye-biopharmaceutical isibe yimakethe yomhlaba wonke ekhulayo ngokukhula okukhulu kwesabelo semakethe eminyakeni yamuva nje. Ngokukhula okukhulu kwemboni ye-biopharmaceutical1,2,3 kunesidingo esikhulu sokuhlaziywa kwe-peptide kanye ne-protein. Ngaphezu kwe-peptide eqondiwe, ukungcola okuhlukahlukene kwakheka ngesikhathi sokwenziwa kwe-peptide, ngakho-ke ukuhlanzwa kwe-chromatographic kuyadingeka ukuze kutholakale ubumsulwa obufunwayo be-peptide. Ukuhlaziywa kanye nokuhlukaniswa kwamaprotheni oketshezini lomzimba, izicubu, kanye namaseli kuwumsebenzi onzima kakhulu ngenxa yenani elikhulu lezinhlobo ezingatholakala ezikhona kusampula eyodwa. Nakuba i-mass spectrometry iyithuluzi eliphumelelayo lokulandelanisa ama-peptide namaprotheni, uma amasampula anjalo efakwa ngqo ku-mass spectrometer, ukuhlukaniswa ngeke kunelise. Le nkinga ingaxazululwa ngokwenza i-liquid chromatography (LC) ngaphambi kokuhlaziywa kwe-MS, okuzonciphisa inani lama-analytes angena ku-mass spectrometer ngesikhathi esithile4,5,6. Ngaphezu kwalokho, ama-analytes angagxila endaweni encane ngesikhathi sokuhlukaniswa kwesigaba soketshezi, ngaleyo ndlela agxile kula ma-analytes futhi andise ukuzwela kokutholwa kwe-MS. I-chromatography yoketshezi (LC) ithuthuke kakhulu eminyakeni eyishumi edlule futhi isiyindlela esetshenziswa kabanzi yokuhlaziya i-proteomic7,8,9,10.
I-reverse-phase liquid chromatography (RP-LC) isetshenziswa kabanzi ukuhlanza nokuhlukanisa izingxube zama-peptide kusetshenziswa i-octadecyl-modified silica (ODS) njengesigaba esimile11,12,13. Kodwa-ke, ngenxa yesakhiwo sazo esiyinkimbinkimbi kanye nesimo sazo se-amphoteric, izigaba ezimile ezingu-14,15 ze-RP azikwazi ukunikeza ukuhlukaniswa okwanelisayo kwama-peptide namaprotheni. Ngakho-ke, ukuhlaziywa kwama-peptide namaprotheni anezingcezu ze-polar nezingezona ze-polar kudinga izigaba ezimile eziklanywe ngokukhethekile ukuze zisebenzisane futhi zigcine lawa ma-analytes16. I-Mixed chromatography, enikeza ukusebenzisana kwe-multimodal, ingaba yindlela ehlukile ye-RP-LC yokuhlukanisa ama-peptide, amaprotheni, nezinye izingxube eziyinkimbinkimbi. Izigaba eziningana ezimile ezihlanganisiwe zalungiswa futhi amakholomu agcwaliswe ngalezi zigaba ezimile asetshenziswa ukuhlukanisa ama-peptide namaprotheni17,18,19,20,21. Ngenxa yokuba khona kwamaqembu e-polar kanye namaqembu angewona ama-polar, izigaba ezimile zemodi exubile (i-WAX/RPLC, i-HILIC/RPLC, i-polar intercalation/RPLC) zifanelekela ukuhlukaniswa kwama-peptide kanye namaprotheni22,23,24,25,26,27,28. , izigaba ezimile ze-polar intercalated ezinamaqembu e-polar ahlanganiswe ndawonye zibonisa amakhono amahle okuhlukana kanye nokukhetha okuhlukile kwama-analyte e-polar kanye namaqembu angewona ama-polar ngoba ukuhlukana kuncike ekusebenzisaneni phakathi kwe-analyte kanye nesigaba esimile Ukusebenzisana kwe-Multimodal 29,30,31,32. Muva nje, uZhang et al. 30 bathole izigaba ezimile ze-polyamines eziqediwe yi-behenyl futhi bahlukanisa ngempumelelo ama-hydrocarbon, ama-antidepressants, ama-flavonoid, ama-nucleoside, ama-estrogen, kanye namanye ama-analyte. Izinto ezimile ze-polar ezifakwe ngaphakathi zinamaqembu e-polar kanye namaqembu angewona ama-polar, ngakho-ke zingasetshenziswa ukuhlukanisa ama-peptide namaprotheni abe izingxenye ze-hydrophobic kanye ne-hydrophilic. Amakholomu angaphakathi kwe-polar (isb., amakholomu e-C18 ane-amide inline) atholakala ngaphansi kwegama lokuhweba elithi amakholomu e-Ascentis Express RP-Amide, kodwa la makholomu asetshenziswe kuphela ekuhlaziyweni kwe-amine 33.
Esifundweni samanje, kwalungiselelwa futhi kwahlolwa isigaba sokuma esimile se-polar (i-N-phenylmaleimide, i-embedding polystyrene) ukuze kuhlukaniswe i-peptide kanye nokuqhekeka kwe-tryptic HSA. Isu elilandelayo lasetshenziswa ukulungiselela isigaba sokuma. Izinhlayiya ze-silica ezinezimbobo zalungiswa ngokwezinqubo ezichazwe ezincwadini zethu zangaphambilini, kanye nezinguquko ezithile ezinhlelweni zokulungiselela 31, 34, 35, 36, 37, 38, 39. Izilinganiso ze-urea, i-polyethylene glycol (PEG), i-TMOS kanye ne-aqueous-acetic acid zalungiswa ukuze kutholakale izinhlayiya ze-silica ezinobukhulu obukhulu be-pore. Okwesibili, kwenziwa i-ligand entsha ye-phenylmaleimide-methylvinyl isocyanate futhi izinhlayiya zayo ze-silica ezithathwe e-polar zasetshenziswa ukulungiselela izigaba zokuma ezimile ze-polar. Isigaba sokuma esitholiwe sapakishwa kukholomu yensimbi engagqwali (ububanzi bangaphakathi obungu-100 × 1.8 mm) ngokwesimiso sokupakisha esilungiselelwe kahle. Ukupakisha kwekholomu kusizwa ukudlidliza komshini ukuqinisekisa ungqimba olufanayo ngaphakathi kwekholomu. Ikholomu epakishiwe yahlolwa ukuze kuhlukaniswe ingxube yama-peptide aqukethe ama-peptide amahlanu (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-leucine-enkephalin peptide). kanye nama-hydrolysates e-tryptic e-human serum albumin (HSA). Kwabonwa ukuthi ingxube ye-peptide kanye ne-HSA tryptic digest kwahlukaniswa ngesisombululo esihle nokusebenza kahle. Ukusebenza kahle kokuhlukaniswa kwekholomu ye-PMP kwaqhathaniswa nalokho kwekholomu ye-Ascentis Express RP-Amide. Kwabonwa ukuthi ama-peptide namaprotheni anesisombululo esihle nokusebenza kahle kokuhlukaniswa okuphezulu kwikholomu ye-PMP, futhi ukusebenza kahle kokuhlukaniswa kwekholomu ye-PMP kuphakeme kunokwekholomu 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, i-urea, i-hexamethylmethacryloyldisilazane (HMDS), i-methacryloyl chloride (MC), i-styrene, i-4-hydroxy-TEMPO, i-benzoyl peroxide (BPO), i-acetonitrile (ACN) ye-HPLC, i-methanol, i-2-propanol kanye ne-acetone. I-Sigma-Aldrich Company (St. Louis, Missouri, USA).
Ingxube ye-urea (8 g), i-polyethylene glycol (8 g) kanye ne-8 ml ye-0.01 N. acetic acid yavuselelwa imizuzu eyi-10, kwathi i-24 ml ye-TMOS yafakwa kuyo ngaphansi kokupholisa iqhwa. Ingxube yokusabela yashiswa ku-40°C amahora ayi-6 kwathi ku-120°C amahora ayi-8 ku-autoclave yensimbi engagqwali. Amanzi akhishwa amanzi kwathi insalela yomiswa ku-70°C amahora ayi-12. Amabhulokhi athambile omisiwe agaywa kahle futhi afakwa kuhhavini ku-550°C amahora ayi-12. Kwalungiswa amaqoqo amathathu futhi kwachazwa ukuze kuhlolwe ukuphindaphindeka kobukhulu bezinhlayiya, usayizi we-pore kanye nendawo engaphezulu.
Iqembu le-polar kanye nesigaba esimile samaketanga e-polystyrene. Inqubo yokulungiselela ichazwe ngezansi.
I-N-phenylmaleimide (200 mg) kanye ne-methyl vinyl isocyanate (100 mg) zancibilikiswa ku-toluene engenamanzi, kwabe sekufakwa u-0.1 ml we-2,2′-azoisobutyronitrile (AIBN) ebhodleleni lokusabela ukuze kutholakale i-copolymer ye-phenylmaleimide kanye ne-methyl vinyl isocyanate (PMCP). ) Ingxube yashiswa ku-60°C amahora ama-3, yahlungwa futhi yomiswa kuhhavini ku-40°C amahora ama-3.
Izinhlayiya ze-silica ezomile (2 g) zahlakazwa ku-toluene eyomile (100 ml), zavuselelwa futhi zafakwa i-sonic imizuzu eyi-10 ebhodleleni elingezansi eliyindilinga elingu-500 ml. I-PMCP (10 mg) yancibilikiswa ku-toluene futhi yanezelwa ngokuwa phansi ebhodleleni lokusabela nge-funnel eyengeziwe. Ingxube yahlungwa kabusha ku-100°C amahora ayi-8, yahlungwa, yagezwa nge-acetone futhi yomiswa ku-60°C amahora ama-3. Ngemuva kwalokho, izinhlayiya ze-silica ezihlobene ne-PMCP (100 g) zancibilikiswa ku-toluene (200 ml), kwathi i-4-hydroxy-TEMPO (2 ml) yanezelwa lapho kukhona i-100 μl ye-dibutyltin dilaurate njenge-catalyst. Ingxube yavuselelwa ku-50°C amahora ayi-8, yahlungwa futhi yomiswa ku-50°C amahora ama-3.
I-Styrene (1 ml), i-benzoyl peroxide BPO (0.5 ml) kanye nezinhlayiya ze-silica ezinamathele ku-TEMPO-PMCP (1.5 g) zahlakazwa ku-toluene futhi zahlanzwa nge-nitrogen. Ukwenziwa kwe-polymerization ye-styrene kwenziwa ku-100°C amahora ayi-12. Umkhiqizo ophumayo wagezwa nge-methanol futhi womiswa ubusuku bonke ku-60°C. Uhlelo olujwayelekile lokusabela luboniswe ku-fig. 1.
Amasampula akhishwa ugesi ku-393 K ihora eli-1 kwaze kwaba yilapho kutholakala ingcindezi eseleyo engaphansi kuka-10-3 Torr. Inani le-N2 elifakwe ngaphansi kwengcindezi elinganiselwe P/P0 = 0.99 lasetshenziswa ukunquma ivolumu ephelele yembobo. Ukwakheka kwezinhlayiya ze-silica ezihlanzekile neziboshwe yi-ligand kwahlolwa kusetshenziswa i-scanning electron microscope (Hitachi High Technologies, Tokyo, Japan). Amasampula omile (izinhlayiya ze-silica ezihlanzekile neziboshwe yi-ligand) abekwa ezindongeni ze-aluminium kusetshenziswa i-carbon tape. Igolide lafakwa esampula kusetshenziswa idivayisi yokuphalaza ye-Q150T, kwathi ungqimba lwe-Au oluyi-5 nm ubukhulu lwafakwa esampula. Lokhu kuthuthukisa ukusebenza kahle kwenqubo ye-voltage ephansi futhi kunikeza ukufutha okubandayo okuhle. Ukuhlaziywa kwe-elemental kwenziwa kusetshenziswa i-Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental composition analyzer. I-Malvern particle size analyzer (Worcestershire, UK) Mastersizer 2000 yasetshenziswa ukuthola ukusatshalaliswa kosayizi wezinhlayiya. Izinhlayiya ze-silica ezingagqokwanga kanye nezinhlayiya ze-silica eziboshwe yi-ligand (5 mg ngayinye) zahlakazwa ku-5 ml ye-isopropanol, zafakwa i-sonicated imizuzu eyi-10, zavuselelwa imizuzu emi-5, zabekwa ebhentshini le-Mastersizer optical. Ukuhlaziywa kwe-thermogravimetric kwenziwa ngesivinini esingu-5 °C ngomzuzu ebangeni lokushisa elisukela ku-30 ​​kuya ku-800 °C.
Amakholomu ensimbi engagqwali afakwe i-fiber yengilazi enobukhulu obuncane (ID 100 × 1.8 mm) apakishwa ngendlela yokugcwalisa i-slurry ngokulandela inqubo efanayo naleyo esesibukweni 31. Ikholomu yensimbi engagqwali (efakwe ingilazi, ID 100 × 1 .8 mm) kanye nendawo yokuphuma equkethe i-1 µm frit kwaxhunywa emshinini wokupakisha i-slurry (Alltech Deerfield, IL, USA). Lungisa ukumiswa kwesigaba esimile ngokumisa u-150 mg wesigaba esimile ku-1.2 ml ye-methanol bese uyifaka kukholomu yokugcina amanzi. I-Methanol yasetshenziswa njenge-slurry solvent kanye ne-control solvent. Paka ikholomu ngokusebenzisa ukulandelana kwengcindezi kwe-100 MP imizuzu eyi-10, i-80 MP imizuzu eyi-15, kanye ne-60 MP imizuzu engama-30. Inqubo yokupakisha yasebenzisa ama-vibrator amabili ekholomu ye-gas chromatography (Alltech, Deerfield, IL, USA) ukuze kudlidlize ngomshini ukuqinisekisa ukupakisha ikholomu okufanayo. Vala i-slurry packer bese ukhulula ingcindezi kancane ukuze uvimbele umonakalo entanjeni. Ikholomu yanqanyulwa embhobho we-slurry kwathi okunye ukufakelwa kwaxhunywa endaweni yokungena futhi kwaxhunywa ohlelweni lwe-LC ukuze kuhlolwe ukusebenza kwayo.
I-MLC eyenziwe ngokwezifiso yakhiwe kusetshenziswa ipompo ye-LC (10AD Shimadzu, eJapane), isampula ene-loop yomjovo engu-50 nL (i-Valco (USA) C14 W.05), i-membrane degasser (i-Shimadzu DGU-14A), kanye nefasitela le-capillary le-UV-VIS. Idivayisi yokuthola (i-UV-2075) kanye nekholomu encane enamelled. Sebenzisa amashubhu okuxhuma amancane kakhulu futhi amafushane ukuze unciphise umphumela wokwandiswa okwengeziwe kwekholomu. Ngemva kokugcwalisa ikholomu, faka i-capillary (50 µm id 365) endaweni yokuphuma kwe-1/16″ reducing junction bese ufaka i-capillary (50 µm) ye-reducing junction. Ukuqoqwa kwedatha kanye nokucubungula i-chromatogram kwenziwa kusetshenziswa isofthiwe ye-Multichro 2000. Ku-254 nm, ukumuncwa kwe-UV kwabahlaziyi bezihloko kwaqashwa ku-0. Idatha ye-Chromatographic yahlaziywa kusetshenziswa i-OriginPro8 (Northampton, MA).
I-albumin ye-serum yomuntu, i-lyophilized powder, ≥ 96% (i-agarose gel electrophoresis) 3 mg exutshwe ne-trypsin (1.5 mg), i-4.0 M urea (1 ml) kanye ne-0.2 M ammonium bicarbonate (1 ml). Isixazululo sivuselelwe imizuzu eyi-10 futhi sigcinwe emanzini okugeza ku-37°C amahora ayi-6, bese sicishwa nge-1 ml ye-0.1% TFA. Hlunga isixazululo bese ugcina ngaphansi kuka-4°C.
Ukuhlukaniswa kwengxube yama-peptide kanye ne-tryptic digest HSA kukholamu ye-PMP kuhlolwe ngokwehlukana. Hlola i-tryptic hydrolysis yengxube yama-peptide kanye ne-HSA ehlukaniswe yikholamu ye-PMP bese uqhathanisa imiphumela nekholomu ye-Ascentis Express RP-Amide. Inani lamapuleti e-theoretical libalwa kusetshenziswa i-equation elandelayo:
Izithombe ze-SEM zezinhlayiya ze-silica ezimsulwa kanye nezinhlayiya ze-silica eziboshwe yi-ligand ziboniswe kuMfanekiso 2. Izithombe ze-SEM zezinhlayiya ze-silica ezimsulwa (A, B) zibonisa isimo esiyindilinga lapho izinhlayiya zinde khona noma zinokulingana okungajwayelekile uma kuqhathaniswa nezifundo zethu zangaphambilini. Ubuso bezinhlayiya ze-silica eziboshwe yi-ligand (C, D) bubushelelezi kunobo bezinhlayiya ze-silica ezimsulwa, okungenzeka ukuthi kungenxa yamaketanga e-polystyrene amboze ubuso bezinhlayiya ze-silica.
Ukuskena ama-micrograph e-electron ezinhlayiya ze-silica ezimsulwa (A, B) kanye nezinhlayiya ze-silica eziboshwe yi-ligand (C, D).
Ukusatshalaliswa kosayizi wezinhlayiya zezinhlayiya ze-silica emsulwa kanye nezinhlayiya ze-silica eziboshwe yi-ligand kuboniswe ku-Fig. 2. 3(A). Ama-curve okusabalalisa usayizi wezinhlayiya ze-volumetric abonise ukuthi usayizi wezinhlayiya ze-silica ukhuphukile ngemva kokuguqulwa kwamakhemikhali (Fig. 3A). Idatha yokusatshalaliswa kosayizi wezinhlayiya ze-silica kusukela ocwaningweni lwamanje kanye nocwaningo lwangaphambilini iqhathaniswa kuThebula 1(A). Usayizi wezinhlayiya ze-volumetric d(0.5) we-PMP wawungu-3.36 µm, uma kuqhathaniswa nenani le-ad(0.5) lika-3.05 µm ocwaningweni lwethu lwangaphambilini (izinhlayiya ze-silica eziboshwe yi-polystyrene)34. Ngenxa yokushintsha kwesilinganiso se-PEG, i-urea, i-TMOS kanye ne-acetic acid engxubeni yokusabela, ukusatshalaliswa kosayizi wezinhlayiya kwaleli qoqo kwakuncane uma kuqhathaniswa nocwaningo lwethu lwangaphambilini. Usayizi wezinhlayiya wesigaba se-PMP mkhulu kancane kunowesigaba sezinhlayiya ze-silica eziboshwe yi-polystyrene esisifunde ngaphambilini. Lokhu kusho ukuthi ukusebenza kobuso bezinhlayiya ze-silica nge-styrene kwafaka ungqimba lwe-polystyrene kuphela (0.97 µm) ebusweni be-silica, kuyilapho esigabeni se-PMP ubukhulu bengqimba babungu-1.38 µm.
Ukusatshalaliswa kosayizi wezinhlayiya (A) kanye nokusatshalaliswa kosayizi wezimbobo (B) zezinhlayiya ze-silica ezimsulwa kanye nezinhlayiya ze-silica eziboshwe yi-ligand.
Usayizi wembobo, umthamo wembobo, kanye nendawo engaphezulu yezinhlayiya ze-silica ezisetshenziswe kulolu cwaningo kuboniswe kuThebula 1 (B). Amaphrofayili e-PSD ezinhlayiya ze-silica ezihlanzekile kanye nezinhlayiya ze-silica eziboshwe yi-ligand aboniswe ku-Figs. 3(B). Imiphumela yayifana nocwaningo lwethu lwangaphambilini34. Ubukhulu bembobo yezinhlayiya ze-silica ezihlanzekile kanye neziboshwe yi-ligand babungu-310 Å kanye no-241 Å, ngokulandelana, okubonisa ukuthi ngemva kokuguqulwa kwamakhemikhali, usayizi wembobo wehle ngo-69 Å, njengoba kuboniswe kuThebula 1 (B), kanti ijika lokushintsha liboniswe ku-Fig. Indawo ethile ebusweni bezinhlayiya ze-silica ocwaningweni lwamanje ingu-116 m2/g, okulingana nocwaningo lwethu lwangaphambilini (124 m2/g). Njengoba kuboniswe kuThebula 1(B), indawo engaphezulu (m2/g) yezinhlayiya ze-silica ngemva kokuguqulwa kwamakhemikhali nayo yehle kusukela ku-116 m2/g kuya ku-105 m2/g.
Imiphumela yokuhlaziywa kwezinto eziyisisekelo zesigaba esimile ivezwe kuThebula. 2. Okuqukethwe kwekhabhoni kwesigaba esimile samanje kungu-6.35%, okuphansi kunasesifundweni sethu sangaphambilini (izinhlayiya ze-silica ezihlotshaniswa ne-polystyrene, 7.93%35 kanye no-10.21%, ngokulandelana) 42. Okuqukethwe kwekhabhoni kwesigaba esimile samanje ngezansi, njengoba amanye ama-ligand e-polar njenge-phenylmaleimide methyl vinyl isocyanate (PCMP) kanye ne-4-hydroxy-TEMPO asetshenziswe ngaphezu kwe-styrene ekulungiseleleni i-SP. Iphesenti lesisindo se-nitrogen esigabeni esimile samanje lingu-2.21% uma kuqhathaniswa no-0.1735 kanye no-0.85% ezifundweni zangaphambilini42. Lokhu kusho ukuthi isigaba esimile samanje sinephesenti lesisindo esiphezulu se-nitrogen ngenxa ye-phenylmaleimide. Ngokufanayo, imikhiqizo (4) kanye (5) inokuqukethwe kwekhabhoni okungu-2.7% kanye no-2.9%, ngokulandelana, kanti umkhiqizo wokugcina (6) unokuqukethwe kwekhabhoni okungu-6.35%, njengoba kuboniswe kuThebula 2. Ukuhlaziywa kwe-Thermogravimetric (TGA) kwasetshenziswa esigabeni esimiyo se-PMP ukuhlola ukwehla kwesisindo, kanti ijika le-TGA liboniswe kuMfanekiso 4. Ijika le-TGA likhombisa ukwehla kwesisindo okungu-8.6%, okuvumelana kahle nokuqukethwe kwekhabhoni (6.35%), njengoba ama-ligands equkethe hhayi i-C kuphela, kodwa ne-N, O kanye ne-H.
I-ligand phenylmaleimide-methylvinyl isocyanate yakhethwa ukuguqula ubuso bezinhlayiya ze-silica ngenxa yamaqembu ayo e-polar phenylmaleimide kanye ne-vinylisocyanate. Amaqembu e-vinyl isocyanate angasabela ngokwengeziwe ne-styrene ngokupholisa okunamandla. Isizathu sesibili ukufaka iqembu elinokuxhumana okulinganiselwe ne-analyte futhi elingenakho ukusebenzisana okuqinile kwe-electrostatic phakathi kwe-analyte kanye nesigaba esimile, njengoba ingxenye ye-phenylmaleimide ingenayo ishaja ebonakalayo ku-pH evamile. I-polarity yesigaba esimile ingalawulwa yinani elifanele le-styrene kanye nesikhathi sokusabela se-free radical polymerization. Isinyathelo sokugcina sokusabela (i-free radical polymerization) sibalulekile njengoba sishintsha i-polarity yesigaba esimile. Ukuhlaziywa kwe-elemental kwenziwa ukuhlola okuqukethwe kwe-carbon kulezi zigaba ezimile. Kuye kwabonwa ukuthi ukwandisa inani le-styrene kanye nesikhathi sokusabela kwandisa okuqukethwe kwe-carbon kwesigaba esimile kanye nokuphikisana nalokho. Ama-SP alungiselelwe ngamanani ahlukene e-styrene anemithwalo ehlukile yekhabhoni. Ngokufanayo, lezi zigaba ezimile zabekwa ezinsikeni zensimbi engagqwali futhi izici zazo ze-chromatographic (ukukhetha, isixazululo, inani le-N, njll.) zahlolwa. Ngokusekelwe kulezi zivivinyo, kwakhethwa ukwakheka okuhle kokulungiselela isigaba esimile se-PMP ukuze kunikezwe i-polarity elawulwayo kanye nokugcinwa okuhle kwe-analyte.
Ikholomu ye-PMP iphinde yahlolwa ukuze kuhlaziywe izingxube ezinhlanu zama-peptide (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-leucine-enkephalin) kusetshenziswa amandla esigaba esihambayo. 60/40 (v/v) ACN/amanzi (0.1% TFA) ngesilinganiso sokugeleza esingu-80 µl/min. Ngaphansi kwezimo ezifanele zokuxazulula (amapuleti angu-200,000/m), inani lamapuleti e-theoretical (N) ngekholomu ngayinye (100 × 1.8 mm) lingu-20,000 ± 100. Amanani e-N amakholomu amathathu e-PMP aboniswe kuThebula 3 kanti ama-chromatogram aboniswe kuMfanekiso 5A. Ukuhlaziywa okusheshayo ngesivinini sokugeleza okuphezulu (700 µl/min) kukholamu ye-PMP, ama-peptide amahlanu aphumile ngaphakathi komzuzu owodwa, inani elihle kakhulu le-N elingu-13,500 ± 330 ngekholomu ngayinye (ububanzi obungu-100 x 1.8 mm), elilingana namapuleti angu-135,000/m (Isithombe 5B). Amakholomu amathathu anobukhulu obufanayo (ububanzi bangaphakathi obungu-100 x 1.8 mm) agcwaliswe ngamaqoqo amathathu ahlukene esigaba sokuma se-PMP ukuhlola ukuphindaphinda. Ama-analyte aqoshwa kukholamu ngayinye ngokuhlukanisa ingxube efanayo yokuhlola kukholamu ngayinye kusetshenziswa izimo ezifanele zokuhlukanisa, inani lamapuleti e-theory u-N, kanye nesikhathi sokugcina. Idatha yokuphindaphinda kwamakholomu e-PMP iboniswe kuThebula 4. Ukuphindaphinda kwekholomu ye-PMP kuhlobene kahle namanani aphansi kakhulu e-%RSD njengoba kuboniswe kuThebula 3.
Ukuhlukaniswa kwezingxube ze-peptide kukholomu ye-PMP (B) kanye nekholomu ye-Ascentis Express RP-Amide (A), isigaba esihambayo 60/40 ACN/H2O (TFA 0.1%), ubukhulu bekholomu ye-PMP (100 x 1.8 mm id), ukuhlaziywa Ukuhleleka kokuhlanganiswa kwama-compounds: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) kanye no-5 (leucic acid enkephalin).
Ikholomu ye-PMP (ububanzi bangaphakathi obuyi-100 x 1.8 mm) ihlolwe ukuhlukaniswa kwe-tryptic hydrolyzate ye-albumin ye-serum yomuntu yi-HPLC. I-chromatogram ku-Figure 6 ikhombisa ukuthi amasampula ahlukaniswe kahle ngesisombululo esihle kakhulu. Izixazululo ze-HSA zihlaziywe kusetshenziswa izinga lokugeleza elingu-100 μl/min, isigaba esihambayo esingu-70/30 acetonitrile/amanzi kanye no-0.1% TFA. Ukuhlukaniswa kwe-HSA kwahlukaniswa kwaba yiziqongo ezingu-17, njengoba kuboniswe ku-chromatogram (Isithombe 6), okuhambisana nama-peptide angu-17. Ukusebenza kahle kokuhlukanisa kweziqongo ngazinye kusuka ku-hydrolyzate ye-HSA kubalwe futhi amanani aboniswe kuThebula 5.
Ama-hydrolysates e-HSA tryptic ahlukaniswe kukholomu ye-PMP (ububanzi bangaphakathi obuyi-100 x 1.8 mm), izinga lokugeleza (100 μl/min), isigaba esihambayo esingu-60/40 acetonitrile/amanzi, kanye ne-0.1% TFA.
lapho u-L ubude bekholomu, u-η uyi-viscosity yesigaba esihambayo, u-ΔP uyingcindezi yangemuva yekholomu, kanti u-u uyijubane eliqondile lesigaba esihambayo. Ukuvuleka kwekholomu ye-PMP kwakungu-2.5 × 10–14 m2, izinga lokugeleza lalingu-25 µl/min, kwasetshenziswa u-60/40 v/v. I-ACN/amanzi. Ukuvuleka kwekholomu ye-PMP (ID 100 × 1.8 mm) kwakufana nokwesifundo sethu sangaphambilini se-Ref.34. Ukuvuleka kwekholomu egcwele izinhlayiya ezingenazo izimbobo kungu-1.7×10 .6 µm, 2.5×10-14 m2 wezinhlayiya ezingu-5 µm43. Ngakho-ke, ukuvuleka kwesigaba se-PMP kufana nokuvuleka kwezinhlayiya zegobolondo eliyinhloko ezinobukhulu obungu-5 μm.
lapho i-Wx iyisisindo sekholomu egcwele i-chloroform, i-Wy iyisisindo sekholomu egcwele i-methanol, kanti i-ρ iyisisindo se-solvent. Ubuningi be-methanol (ρ = 0.7866) kanye ne-chloroform (ρ = 1.484). I-porosity iyonke yekholomu yezinhlayiya ze-silica-C18 (100 × 1.8 mm ID)34 kanye nekholomu yethu ye-C18-urea31 efundwe ngaphambilini yayingu-0.63 kanye no-0.55, ngokulandelana. Lokhu kusho ukuthi ukuba khona kwama-urea ligands kunciphisa ukugeleza kwesigaba esimile. Ngakolunye uhlangothi, i-porosity iyonke yekholomu ye-PMP (ububanzi bangaphakathi obungu-100 × 1.8 mm) ingu-0.60. Amakholomu e-PMP awangeni kakhulu kunekholomu egcwele izinhlayiya ze-silica eziboshwe yi-C18 ngoba ezigabeni ezimile zohlobo lwe-C18 ama-ligands e-C18 anamathele ezinhlayiyeni ze-silica ngamaketanga aqondile, kuyilapho ezigabeni ezimile zohlobo lwe-polystyrene kwakheka i-polymer ejiyile ezungeze izinhlayiya. ungqimba A. Ekuhlolweni okuvamile, i-porosity yekholomu ibalwa kanje:
Ku-fig. 7A, u-B ukhombisa ama-plot e-Van Deemter ekholomu ye-PMP (id 100 x 1.8 mm) kanye nekholomu ye-Ascentis Express RP-Amide (id 100 x 1.8 mm) ngaphansi kwezimo ezifanayo zokulungisa, i-60/40 ACN/H2O kanye ne-0 .1% TFA 20 µl/min kuya ku-800 µl/min kuzo zombili izinsika. Amanani aphansi e-HETP ngesilinganiso sokugeleza esifanele (80 µl/min) ayengu-2.6 µm kanye no-3.9 µm ekholomu ye-PMP kanye nekholomu ye-Ascentis Express RP-Amide, ngokulandelana. Amanani e-HETP abonisa ukuthi ukusebenza kahle kokuhlukaniswa kwekholomu ye-PMP (100 x 1.8 mm id) kuphakeme kakhulu kunokwekholomu ye-Ascentis Express RP-Amide etholakala kwezentengiselwano (100 x 1.8 mm id). Igrafu ye-van Deemter ku-Fig. 7(A) ikhombisa ukuthi ukwehla kwenani le-N akuphakeme kakhulu ngokugeleza okwandayo uma kuqhathaniswa nesifundo sethu sangaphambilini. Ukusebenza kahle kokuhlukaniswa okuphezulu kwekholomu ye-PMP (id 100 × 1.8 mm) uma kuqhathaniswa nekholomu ye-Ascentis Express RP-Amide kusekelwe ekubunjweni kwezinhlayiya nosayizi kanye nenqubo yokupakisha ikholomu eyinkimbinkimbi esetshenziswa emsebenzini wamanje34.
(A) I-Van Deemter plot (i-HETP vs. ijubane eliqondile lesigaba esiphathwayo) elitholwe kukholamu ye-PMP (id 100 x 1.8 mm) ku-60/40 ACN/H2O ene-0.1% TFA. (B) I-Van Deemter plot (i-HETP vs ijubane eliqondile lesigaba esiphathwayo) elitholwe kukholamu ye-Ascentis Express RP-Amide (id 100 x 1.8 mm) ku-60/40 ACN/H2O ene-0.1% TFA.
Isigaba esimile se-polar se-polystyrene ehlanganisiwe salungiswa futhi sahlolwa ukuze kuhlukaniswe ingxube yama-peptide okwenziwa kanye ne-tryptic hydrolyzate ye-human serum albumin (HSA) ku-chromatography yoketshezi esebenza kahle kakhulu. Ukusebenza kwe-chromatographic kwamakholomu e-PMP ezingxubeni ze-peptide kuhle kakhulu maqondana nokusebenza kahle kokuhlukanisa kanye nesisombululo. Ukusebenza kahle kokuhlukanisa okuthuthukisiwe kwamakholomu e-PMP kungenxa yezizathu eziningana ezifana nosayizi wezinhlayiya ze-silica kanye nosayizi we-pore, ukuhlanganiswa okulawulwayo kwezigaba ezimile, kanye nezinto zokupakisha eziyinkimbinkimbi zekholomu. Ngaphezu kokusebenza kahle kokuhlukanisa okuphezulu, enye inzuzo yalesi sigaba esimile ukucindezela okuphansi kwekholomu emuva ngamazinga aphezulu okugeleza. Amakholomu e-PMP aphindaphindwa kakhulu futhi angasetshenziswa ukuhlaziya izingxube zama-peptide kanye nokugaya kwe-tryptic kwamaprotheni ahlukahlukene. Sihlose ukusebenzisa le kholomu ukuze kuhlukaniswe amakhemikhali asebenzayo emikhiqizweni yemvelo, izingcezu zezitshalo zokwelapha kanye namakhowe ku-chromatography yoketshezi. Esikhathini esizayo, amakholomu e-PMP azohlolwa futhi ukuze kuhlukaniswe amaprotheni kanye nama-antibodies e-monoclonal.
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