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Izinhlayiya ze-silica ezinama-porous zalungiselelwa indlela ye-sol-gel ngokulungiswa okuthile ukuze kutholwe izinhlayiya ezinama-wide-pore. Lezi zinhlayiya zikhishwe nge-N-phenylmaleimide-methylvinyl isocyanate (PMI) kanye ne-styrene nge-reverse chain transfer-fragmentation (RAFT) polymerization ukuze kukhiqizwe i-N-phenylmaleimide intercalated polyamides. I-styrene (PMP) isigaba esimile. Amakholomu ensimbi engagqwali emincane (100 × 1.8 mm ububanzi bangaphakathi) ayegcwele ukupakishwa okunodaka. Ukusebenza kwe-chromatographic yekholomu ye-PMP kwahlolwa ukuze kuhlukaniswe ingxube yama-peptide okwenziwa ahlanganisa 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 humanS hydrolyzation album Ngaphansi kwezimo ezingcono kakhulu ze-lution, inombolo yethiyori yamapuleti anengxube yama-peptide ifinyelele ku-280,000 amapuleti/sq.m. Uma kuqhathaniswa nokusebenza kokuhlukaniswa kwekholomu ethuthukisiwe nekholomu ye-Ascentis Express RP-Amide yezentengiselwano, kubonwe ukuthi ukusebenza kahle kokuhlukaniswa kwekholomu ye-PMP kwakuphakeme kunekholomu yezohwebo mayelana nokusebenza kahle nokuhlukaniswa.
Imboni ye-biopharmaceutical isiphenduke imakethe yomhlaba wonke ekhulayo ngokukhuphuka okukhulu kwesabelo semakethe eminyakeni yamuva nje. Ngokukhula okukhulu kwemboni ye-biopharmaceutical1,2,3 kunesidingo esikhulu sokuhlaziywa kwe-peptide namaprotheni. Ngokungeziwe ku-peptide eqondiwe, kwakheka ukungcola okuhlukahlukene ngesikhathi sokuhlanganiswa kwe-peptide, ngakho-ke ukuhlanzwa kwe-chromatographic kuyadingeka ukuze kutholwe ubumsulwa obufiswayo be-peptide. Ukuhlaziywa nokuhlukaniswa kwamaphrotheni oketshezini lomzimba, izicubu, namaseli kuwumsebenzi oyinselele kakhulu ngenxa yenani elikhulu lezinhlobo ezingase zibonakale ezikhona kusampula eyodwa. Nakuba i-mass spectrometry iyithuluzi elisebenzayo lokulandelanisa ama-peptide namaprotheni, uma amasampula anjalo ethulwa ngokuqondile ku-mass spectrometer, ukuhlukaniswa ngeke kugculise. Le nkinga ingaxazululwa ngokwenza i-liquid chromatography (LC) ngaphambi kokuhlaziywa kwe-MS, okuzonciphisa inani labahlaziyi abangena ku-mass spectrometer ngesikhathi esinikeziwe4,5,6. Ngaphezu kwalokho, abahlaziyi bangagxila endaweni encane phakathi nokuhlukaniswa kwesigaba soketshezi, ngaleyo ndlela bagxilise lezi zihlaziyi futhi bakhulise ukuzwela kokutholwa kwe-MS. I-Liquid chromatography (LC) ithuthuke kakhulu kule minyaka eyishumi edlule futhi isiphenduke indlela esetshenziswa kakhulu yokuhlaziya i-proteomic7,8,9,10.
I-reverse-phase liquid chromatography (RP-LC) isetshenziswa kakhulu ukuhlanza nokuhlukanisa izingxube zama-peptide kusetshenziswa i-octadecyl-modified silica (ODS) njengesigaba esimile11,12,13. Kodwa-ke, ngenxa yesakhiwo sabo esiyinkimbinkimbi kanye nemvelo ye-amphoteric, izigaba ezimile ze-14,15 RP azikwazi ukuhlinzeka ngokuhlukaniswa okwanelisayo kwama-peptide namaprotheni. Ngakho-ke, ukuhlaziya ama-peptide namaprotheni anezingcezu ze-polar nezingezona ze-polar kudinga izigaba ezimile eziklanywe ngokukhethekile ukuze zihlanganyele futhi zigcine lezi zihlaziyi16. I-chromatography ehlanganisiwe, enikeza ukusebenzisana kwe-multimodal, ingaba enye ye-RP-LC yokuhlukanisa ama-peptide, amaprotheni, nezinye izingxube eziyinkimbinkimbi. Kwalungiswa izigaba zokuma ezixubile eziningana futhi amakholomu agcwaliswe ngalezi zigaba ezimile asetshenziswa ukuhlukanisa ama-peptide namaprotheni17,18,19,20,21. Ngenxa yokuba khona kwamaqembu e-polar ne-non-polar, izigaba ezimile zemodi exubile (i-WAX/RPLC, i-HILIC/RPLC, i-polar intercalation/RPLC) ifanele ukuhlukaniswa kwama-peptide namaprotheni22,23,24,25,26,27,28. , izigaba ze-polar intercalated ezimile ezinamaqembu e-polar ahlanganiswe ngokubambisana zibonisa amakhono amahle okuhlukanisa kanye nokukhetha okuyingqayizivele kwabahlaziyi be-polar nabangewona we-polar ngoba ukuhlukana kuncike ekusebenzelaneni phakathi kwe-analyte nesigaba esimile Ukusebenzisana kwe-Multimodal 29,30,31,32. Muva nje, uZhang et al. 30 bathole izigaba ezimile ze-behenyl-term of polyamines futhi bahlukanise ngempumelelo ama-hydrocarbon, ama-antidepressants, ama-flavonoid, ama-nucleosides, ama-estrogens, nezinye izihlaziyi. I-polar embedded stationary material inawo womabili amaqembu e-polar kanye ne-non-polar, ngakho-ke ingasetshenziswa ukuhlukanisa ama-peptide namaprotheni abe izingxenye ze-hydrophobic kanye ne-hydrophilic. Amakholomu asemgqeni we-Polar (isb, amakholomu e-C18 ane-amide esemgqeni) atholakala ngaphansi kwegama lokuhweba elithi Ascentis Express RP-Amide amakholomu, kodwa lawa makholomu asetshenziswe kuphela ekuhlaziyeni i-amine 33.
Ocwaningweni lwamanje, isigaba sokushumeka se-polar (N-phenylmaleimide, embedding polystyrene) salungiselelwa futhi sahlolwa ukuhlukaniswa kwe-peptide kanye ne-tryptic HSA cleavage. Kusetshenziswe isu elilandelayo ukulungisa isigaba sokunganyakazi. Izinhlayiya ze-silica ezinama-porous zalungiswa ngokuvumelana nezinqubo ezichazwe ezincwadini zethu zangaphambilini, ngezinguquko ezithile ezinhlelweni zokulungiselela i-31, 34, 35, 36, 37, 38, 39. Izilinganiso ze-urea, i-polyethylene glycol (PEG), i-TMOS kanye ne-aqueous-acetic acid yalungiswa ukuze kutholwe izinhlayiya ezinkulu ze-silica nge-poly particles. Okwesibili, i-phenylmaleimide-methylvinyl isocyanate ligand entsha yahlanganiswa futhi izinhlayiya zayo ze-silica ezikhishwe zasetshenziswa ukuze kulungiswe izigaba ezimile ze-polar. Isigaba esimisiwe esitholiwe sipakishwe kukholamu yensimbi engagqwali (ububanzi bangaphakathi obuyi-100 × 1.8 mm) ngokohlelo lokupakisha olulungiselelwe kahle. Ukupakishwa kwekholomu kusizwa ukudlidliza komshini ukuze kuqinisekiswe ungqimba olufanayo phakathi kwekholomu. Ikholomu epakishiwe yahlolelwa ukuhlukaniswa kwengxube yama-peptide ahlanganisa 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 ne-tryptic hydrolysates ye-albhamuin ye-human serum (HSA). Kwaqashelwa ukuthi ingxube ye-peptide kanye ne-HSA tryptic digest ihlukaniswe ngokulungiswa okuhle nokusebenza kahle. Ukusebenza kahle kokuhlukanisa kwekholomu ye-PMP kwaqhathaniswa nalokho kwekholomu ye-Ascentis Express RP-Amide. Kwaphawulwa ukuthi ama-peptide namaprotheni anokulungiswa okuhle nokusebenza kahle kokuhlukaniswa kukholomu ye-PMP, futhi ukusebenza kahle kokuhlukaniswa kwekholomu ye-PMP kuphakeme kunekholomu 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, i-hydroxyrene, i-MC4 (BPO), i-acetonitrile (ACN) ye-HPLC, i-methanol, i-2-propanol ne-acetone. I-Sigma-Aldrich Company (St. Louis, Missouri, USA).
Ingxube ye-urea (8 g), i-polyethylene glycol (8 g) kanye no-8 ml we-0.01 N. acetic acid yanyakaziswa imizuzu engu-10, futhi u-24 ml we-TMOS wengezwa lapho ngaphansi kokupholisa iqhwa. Ingxube yokusabela ishisiswe ku-40 ° C amahora angu-6 bese ifika ku-120 ° C amahora angu-8 ku-autoclave yensimbi engagqwali. Amanzi ayencibilikisiwe futhi insalela yomiswa ku-70°C amahora angu-12. Amabhulokhi athambile omisiwe agaywa kahle futhi afakwa kuhhavini ongu-550°C amahora angu-12. Amaqoqo amathathu alungiswa futhi ahlukaniswa ukuze kuhlolwe ukukhiqizwa kabusha kosayizi bezinhlayiyana, usayizi wembotshana 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) zincibilikiswe ku-toluene anhydrous, kwase kuthi u-0.1 ml we-2,2′-azoisobutyronitrile (AIBN) yengezwe ku-reaction flask ukuze kutholwe i-copolymer ye-phenylmaleimide ne-vinyl methylCP (vinyl isoso). ) Ingxube yashiswa ku-60 ° C amahora angu-3, ​​ihlungiwe futhi yomiswa kuhhavini engu-40 ° C amahora angu-3.
Izinhlayiya ze-silica ezomile (2 g) zihlakazwe ku-toluene eyomile (100 ml), zinyakaziswa futhi zenziwe nge-sonicated imizuzu engu-10 ku-flask engaphansi eyindilinga engu-500 ml. I-PMCP (10 mg) yancibilika ku-toluene futhi yengezwa ngokudonsela phansi kufulaski wokusabela ngefaneli yokwengeza. Ingxube ihlanjululwe ku-100 ° C amahora angu-8, ihlungiwe, igezwe nge-acetone futhi yomiswe ku-60 ° C amahora angu-3. Khona-ke, izinhlayiya ze-silica ezihlotshaniswa ne-PMCP (100 g) zachithwa ku-toluene (200 ml), futhi i-4-hydroxy-TEMPO (2 ml) yengezwe lapho phambi kwe-100 μl ye-dibutyltin dilaurate njenge-catalyst. Ingxube yashukunyiswa ku-50 ° C amahora angu-8, ihlungiwe futhi yomiswe ku-50 ° C amahora amathathu.
I-styrene (1 ml), i-benzoyl peroxide BPO (0.5 ml) kanye nezinhlayiya ze-silica ezinamathiselwe ku-TEMPO-PMCP (1.5 g) zihlakazwe ku-toluene futhi zahlanzwa nge-nitrogen. I-polymerization ye-styrene yenziwa ku-100 ° C amahora angu-12. Umkhiqizo ovelayo wagezwa nge-methanol futhi womiswa ngobusuku obungama-60°C. Uhlelo olujwayelekile lokusabela luboniswa ku-fig. eyodwa .
Amasampula akhishwe ku-393 K ngehora elingu-1 kuze kube yilapho kutholakala ingcindezi esele engaphansi kuka-10–3 Torr. Inani le-N2 elikhangisiwe ekucindezelweni okuhlobene P/P0 = 0.99 lasetshenziselwa ukunquma ingqikithi yevolumu yembotshana. I-morphology yezinhlayiya ze-silica ezimsulwa neziboshwe nge-ligand yahlolwa kusetshenziswa isibonakhulu se-electron yokuskena (Hitachi High Technologies, Tokyo, Japan). Amasampula omile (i-silica ehlanzekile kanye nezinhlayiya ze-silica eziboshwe nge-ligand) abekwe ezindongeni ze-aluminium kusetshenziswa i-carbon tape. Igolide lafakwa kusampula kusetshenziswa idivayisi yokuphalaza ye-Q150T, futhi ungqimba lwe-Au olungu-5 nm lwafakwa kusampula. Lokhu kuthuthukisa ukusebenza kahle kwenqubo ye-voltage ephansi futhi kunikeza isifutho esihle esibandayo. Ukuhlaziywa kwe-Elemental kwenziwe kusetshenziswa i-Thermo Electron (Waltham, MA, USA) Flash EA1112 eyisisekelo yokuhlaziya ukwakheka. I-Malvern particle size analyzer (Worcestershire, UK) Mastersizer 2000 isetshenziselwe ukuthola ukusatshalaliswa kosayizi wezinhlayiyana. Izinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-silica eziboshwe nge-ligand (5 mg ngayinye) zihlakazwe ku-5 ml ye-isopropanol, i-sonicated imizuzu ye-10, iphazamise imizuzu engu-5, futhi ibekwe ebhentshini le-Mastersizer optical. Ukuhlaziywa kwe-Thermogravimetric kwenziwa ngesilinganiso esingu-5 °C ngomzuzu ebangeni lokushisa elisuka ku-30 ​​kuya ku-800 °C.
Ifayibha yengilazi enamakholomu ensimbi engenasici ewumngcingo enobukhulu (ID 100 × 1.8 mm) yayipakishwe ngendlela yokugcwalisa udaka elandela inqubo efanayo neyereferensi engu-31. Ikholomu yensimbi engagqwali (ingilazi enomugqa, i-ID 100 × 1 .8 mm) kanye ne-outlet equkethe i-1 µm gingert yomshini we-packfield, yonke i-packfield ye-tech yayixhumeke IL, USA). Lungiselela ukumiswa kwesigaba esimile ngokumisa u-150 mg wesigaba esimile ku-1.2 ml we-methanol bese uphakela kukholamu yechibi. I-Methanol yayisetshenziswa njengesixazululi se-slurry kanye ne-solvent yokulawula. Pakisha ikholomu ngokusebenzisa ukucindezela kokulandelana kwe-100 MP imizuzu engu-10, 80 MP imizuzu engu-15, kanye no-60 MP imizuzu engu-30. Inqubo yokupakisha isebenzise izidlidlizi zekholomu yegesi yechromatography ezimbili (Alltech, Deerfield, IL, USA) ukuze kudlidlize ngomshini ukuze kuqinisekiswe ukupakishwa kwekholomu efanayo. Vala i-slurry packer bese ukhulula ukucindezela kancane ukuze uvimbele ukulimala kwentambo. Ikholomu inqanyuliwe ku-slurry nozzle futhi okunye ukulingana kwanamathiselwa endaweni yokungena futhi yaxhunywa ohlelweni lwe-LC ukuze kuhlolwe ukusebenza kwayo.
I-MLC yangokwezifiso yakhiwa kusetshenziswa iphampu ye-LC (10AD Shimadzu, Japan), isampuli ene-50 nL injection loop (Valco (USA) C14 W.05), i-membrane degasser (Shimadzu DGU-14A), kanye newindi le-UV-VIS le-capillary. Idivayisi yomtshina (UV-2075) kanye nekholomu encane enamelelwe. Sebenzisa amashubhu axhumayo amancane kakhulu futhi amafushane ukuze unciphise umphumela wokunwetshwa kwekholomu eyengeziwe. Ngemva kokugcwalisa ikholomu, faka i-capillary (50 µm id 365) endaweni yokuphuma ye-1/16″ yokunciphisa futhi ufake i-capillary (50 µm) yokuhlangana kokunciphisa. Ukuqoqwa kwedatha nokucubungula kwechromatogram kwenziwa kusetshenziswa isofthiwe ye-Multichro 2000. Ku-254 nm, ukumunca kwe-UV kwezihlaziyo zezifundo kwaqashelwa ku-0. Idatha ye-Chromatographic yahlaziywa kusetshenziswa i-OriginPro8 (Northampton, MA).
I-albumin ye-serum yomuntu, impushana e-lyophilized, ≥ 96% (i-agarose gel electrophoresis) 3 mg exutshwe ne-trypsin (1.5 mg), 4.0 M urea (1 ml) kanye no-0.2 M ammonium bicarbonate (1 ml) . Isixazululo sixutshwe imizuzu engu-10 futhi sigcinwe emanzini okugeza ku-37 ° C amahora angu-6, bese sicinywa nge-1 ml ye-0.1% TFA. Hlunga isixazululo bese ugcine ngaphansi kuka-4°C.
Ukuhlukaniswa kwengxube yama-peptide kanye ne-tryptic digest HSA kukholamu ye-PMP kwahlaziywa ngokwehlukana. Hlola i-tryptic hydrolysis yengxube yama-peptide ne-HSA ehlukaniswa yikholomu ye-PMP bese uqhathanisa imiphumela nekholomu ye-Ascentis Express RP-Amide. Inombolo yamapuleti etiyetha ibalwa kusetshenziswa isibalo esilandelayo:
Izithombe ze-SEM zezinhlayiya ze-silica ezihlanzekile kanye nezinhlayiya ze-silica eziboshwe nge-ligand ziboniswa ku-Figure 2. Izithombe ze-SEM zezinhlayiya ze-silica ezihlanzekile (A, B) zibonisa isimo esiyindilinga lapho izinhlayiya zenziwa zinde noma zine-symmetry engavamile uma kuqhathaniswa nezifundo zethu zangaphambilini. Ubuso bezinhlayiya ze-silica eziboshwe i-ligand (C, D) bushelelezi kunezinhlayiya ze-silica ezihlanzekile, okungenzeka kube ngenxa yamaketanga e-polystyrene amboze ubuso bezinhlayiya ze-silica.
Ukuskena ama-electron micrographs wezinhlayiya ze-silica (A, B) kanye nezinhlayiya ze-silica eziboshwe nge-ligand (C, D).
Ukusabalalisa usayizi wezinhlayiyana zezinhlayiya ze-silica ezihlanzekile kanye nezinhlayiya ze-silica eziboshwe nge-ligand kuboniswe ku-Fig. 2. 3 (A). I-Volumetric particle size distribution curves ibonise ukuthi ubukhulu bezinhlayiyana ze-silica buye banda ngemva kokuguqulwa kwamakhemikhali (Fig. 3A). Idatha yokusabalalisa usayizi wezinhlayiyana ze-silica kusukela ocwaningweni lwamanje kanye nocwaningo lwangaphambilini luqhathaniswa kuThebula 1 (A). Usayizi wezinhlayiyana zevolumu d(0.5) we-PMP ubungu-3.36 µm, uma kuqhathaniswa nenani lesikhangiso(0.5) lika-3.05 µm ocwaningweni lwethu lwangaphambilini (izinhlayiya ze-silica eziboshwe nge-polystyrene)34. Ngenxa yoshintsho esilinganisweni se-PEG, i-urea, i-TMOS ne-acetic acid kungxube yokusabela, ukusatshalaliswa kosayizi wezinhlayiyana zaleli qoqo bekuncane uma kuqhathaniswa nocwaningo lwethu lwangaphambilini. Usayizi wezinhlayiyana zesigaba se-PMP mkhulu kancane kunesigaba sezinhlayiyana ze-silica eboshwe nge-polystyrene esifunde ngaphambili. Lokhu kusho ukuthi ukusebenza okungaphezulu kwezinhlayiya ze-silica ezine-styrene kufake kuphela isendlalelo se-polystyrene (0.97 µm) endaweni ye-silica, kuyilapho esigabeni se-PMP ukujiya kwesendlalelo kwakungu-1.38 µm.
Ukusatshalaliswa kosayizi we-particle (A) nokusabalalisa usayizi wembotshana (B) wezinhlayiya ze-silica ezihlanzekile kanye nezinhlayiya ze-silica eziboshwe nge-ligand.
Usayizi wezimbotshana, ivolumu yembotshana, nendawo engaphezulu yezinhlayiya ze-silica ezisetshenziswe kulolu cwaningo kuboniswa kuThebula 1 (B). Amaphrofayili we-PSD wezinhlayiya ze-silica ezihlanzekile kanye nezinhlayiya ze-silica eziboshwe nge-ligand ziboniswa ku-Fig. 3(B). Imiphumela iqhathaniswe nesifundo sethu sangaphambilini34. Amasayizi we-pore wezinhlayiya ze-silica ezihlanzekile futhi eziboshwe nge-ligand zaziyi-310 Å kanye ne-241 Å, ngokulandelana, okubonisa ukuthi ngemva kokuguqulwa kwamakhemikhali, usayizi we-pore wehla ngo-69 Å, njengoba kuboniswe kuThebula 1 (B), futhi ijika lokushintshwa liboniswa ku-Fig. (124 m2/g). Njengoba kuboniswe kuThebula 1(B), indawo engaphezulu (m2/g) yezinhlayiya ze-silica ngemva kokuguqulwa kwamakhemikhali nayo yehla isuka ku-116 m2/g yaya ku-105 m2/g.
Imiphumela yokuhlaziya okuyisisekelo kwesigaba esimile yethulwe kuThebula. 2. Okuqukethwe kwekhabhoni yesigaba esimile samanje yi-6.35%, ephansi kunesifundo sethu sangaphambilini (izinhlayiya ze-silica ezihlotshaniswa ne-polystyrene, 7.93%35 kanye no-10.21%, ngokulandelana) 42. Okuqukethwe kwekhabhoni yesigaba samanje esimile ngezansi, njengoba amanye ama-polar ligands afana ne-phenylmaleimide methyl-POPC esetshenziswa i-hydroxytecyteMyl-4 i-hydroxyMP. ngaphezu kwe-styrene ekulungiseleleni i-SP. Iphesenti lesisindo se-nitrogen esigabeni samanje esimile singama-2.21% uma kuqhathaniswa no-0.1735 kanye no-0.85% ezifundweni ezedlule42. Lokhu kusho ukuthi isigaba samanje sokuma sinephesenti lesisindo esikhulu se-nitrogen ngenxa ye-phenylmaleimide. Ngokufanayo, imikhiqizo (4) kanye (5) inokuqukethwe kwekhabhoni okungu-2.7% no-2.9%, ngokulandelana, kuyilapho umkhiqizo wokugcina (6) unokuqukethwe kwekhabhoni engu-6.35%, njengoba kuboniswe kuThebula 2. Ukuhlaziywa kwe-Thermogravimetric (TGA) kusetshenziswe esigabeni esimile se-PMP ukuze kuhlolwe ukulahlekelwa isisindo, futhi i-TGA. I-8.6%, evumelana kahle ne-carbon content (6.35%), njengoba ama-ligands aqukethe u-C kuphela, kodwa futhi N, O no-H.
I-ligand phenylmaleimide-methylvinyl isocyanate yakhethwa ukuze iguqule ubuso bezinhlayiya ze-silica ngenxa ye-polar phenylmaleimide namaqembu e-vinylisocyanate. Amaqembu e-vinyl isocyanate angaphinde asabele nge-styrene ngokuphila kwe-radical polymerization. Isizathu sesibili siwukufaka iqembu elinokusebenzelana okumaphakathi ne-analyte futhi elingenakho ukusebenzisana okunamandla kwe-electrostatic phakathi kwe-analyte nesigaba esimile, njengoba i-phenylmaleimide moiety ingenakho ukushajwa okubonakalayo ku-pH evamile. I-polarity yesigaba esimile ingalawulwa inani eliphelele le-styrene kanye nesikhathi sokusabela se-polymerization yamahhala ye-radical. Isinyathelo sokugcina sokusabela (i-free radical polymerization) sibalulekile njengoba sishintsha i-polarity yesigaba esimile. Ukuhlaziywa okuyisisekelo kwenziwa ukuze kuhlolwe okuqukethwe kwekhabhoni kulezi zigaba ezimile. Kuye kwaphawulwa ukuthi ukwandisa inani le-styrene kanye nesikhathi sokuphendula kwandisa okuqukethwe kwekhabhoni yesigaba esimile futhi ngokuphambene nalokho. Ama-SP alungiselelwe ngokugxila okuhlukene kwe-styrene anemithwalo yekhabhoni ehlukene. Ngokufanayo, lezi zigaba ezimile zafakwa kumakholomu ensimbi engagqwali nezici zazo zechromatographic (ukukhethwa, ukulungiswa, inani elingu-N, njll.) zahlolwa. Ngokusekelwe kulokhu kuhlolwa, ukwakheka okuthuthukisiwe kokulungiselelwa kwesigaba esimile se-PMP kwakhethwa ukuze kuhlinzekwe i-polarity elawulwayo nokugcinwa okuhle kwe-analyte.
Ikholomu ye-PMP iphinde yahlaziywa 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 umthamo wesigaba seselula. 60/40 (v/v) ACN/amanzi (0.1% TFA) ngesilinganiso sokugeleza esingu-80 µl/min. Ngaphansi kwezimo ezingcono kakhulu zokucacisa (amapuleti angu-200,000/m), inani lamapuleti etiyori (N) ikholomu ngayinye (100 × 1.8 mm) lingu-20,000 ± 100. Amanani angu-N amakholomu amathathu e-PMP aboniswa kuThebula 3 futhi ama-chromatogram aboniswa kuMfanekiso 5A. Ukuhlaziya okusheshayo ngezinga lokugeleza okuphezulu (700 µl/min) kukholomu ye-PMP, ama-peptide amahlanu akhishwe ngomzuzu owodwa, inani elihle kakhulu le-N elingu-13,500 ± 330 ikholomu ngayinye (ububanzi obungu-100 x 1.8 mm), okulingana namapuleti angu-135,000/m (Fig. 5B). Amakholomu amathathu anosayizi ofanayo (ububanzi obungaphakathi 100 x 1.8 mm) agcwaliswe ngamaqoqo amathathu ahlukene esigaba esimile se-PMP ukuze kuhlolwe ukukhiqizwa kabusha. Ukuhlaziya kwarekhodwa kukholomu ngayinye ngokuhlukanisa ingxube efanayo yokuhlola kukholomu ngayinye kusetshenziswa izimo ezingcono kakhulu ze-elution, inombolo yamapuleti etiyori angu-N, nesikhathi sokugcinwa. Idatha yokukhiqiza kabusha yamakholomu e-PMP iboniswa kuThebula 4. Ukuphindaphindeka kwekholomu ye-PMP kuhlotshaniswa 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 seselula 60/40 ACN/H2O (TFA 0.1%), ubukhulu bekholomu ye-PMP (100 x 1.8 mm id) , ukuhlaziywa kwe-Elution order of compounds: 1 (Gly-Tyr-Leu), 2 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) kanye 5 (leucic acid enkephalin).
Ikholomu ye-PMP (ububanzi bangaphakathi obungu-100 x 1.8 mm) yahlolelwa ukuhlukaniswa kwe-tryptic hydrolyzate ye-albumin ye-serum yomuntu yi-HPLC. Ichromatogram kuFigure 6 ibonisa ukuthi amasampula ahlukaniswe kahle ngokulungiswa okuhle kakhulu. Izixazululo ze-HSA zahlaziywa kusetshenziswa izinga lokugeleza elingu-100 μl/min, isigaba seselula esingu-70/30 acetonitrile/amanzi kanye no-0.1% TFA. Cleavage of HSA kwahlukaniswa 17 iziqongo, njengoba kuboniswe chromatogram (Fig. 6), elihambisana 17 peptides. Ukusebenza kahle kokuhlukaniswa kweziqongo ngazinye kusuka ku-HSA hydrolyzate kubaliwe futhi amanani aboniswa kuThebula 5.
Ama-HSA tryptic hydrolysates ahlukaniswa kukholomu ye-PMP (ububanzi bangaphakathi 100 x 1.8 mm), izinga lokugeleza (100 μl/min), isigaba seselula 60/40 acetonitrile/amanzi, kanye no-0.1% TFA.
lapho u-L engubude bekholomu, u-η yi-viscosity yesigaba esihambayo, i-ΔP iyingcindezi yangemuva yekholomu, futhi u-u yijubane lomugqa lesigaba seselula. Ukungena kwekholomu ye-PMP kwakungu-2.5 × 10–14 m2, izinga lokugeleza lalingu-25 µl/min, 60/40 v/v lisetshenzisiwe. I-ACN/amanzi. Ukungena kwekholomu ye-PMP (ID 100 × 1.8 mm) kwakufana nalokho ocwaningweni lwethu lwangaphambilini lwe-Ref.34. Ukungeneka kwekholomu egcwele izinhlayiya ezimboze kancane ngu-1.7×10 .6 µm, 2.5×10-14 m2 wezinhlayiya ezingu-5 µm43. Ngakho-ke, ukufinyeleleka kwesigaba se-PMP kufana nokungena kwezinhlayiya zegobolondo eziyinhloko ezinobukhulu be-5 μm.
lapho i-Wx iyisisindo sekholomu egcwele i-chloroform, i-Wy iyisisindo sekholomu egcwele i-methanol, futhi u-ρ uwubuningi bento encibilikayo. Ukuminyana kwe-methanol (ρ = 0.7866) ne-chloroform (ρ = 1.484). Ingqikithi ye-porosity yekholomu yezinhlayiyana ze-silica-C18 (100 × 1.8 mm ID)34 kanye nekholomu yethu ye-C18-urea31 eyafundwa ngaphambilini yayingu-0.63 no-0.55, ngokulandelana. Lokhu kusho ukuthi ukuba khona kwe-urea ligands kunciphisa ukufinyeleleka kwesigaba esimile. Ngakolunye uhlangothi, ingqikithi ye-porosity yekholomu ye-PMP (ububanzi bangaphakathi 100 × 1.8 mm) ngu-0.60. Amakholomu e-PMP awakwazi ukungena kalula kunamakholomu apakishwe izinhlayiya ze-silica eziboshwe nge-C18 ngoba ezigabeni ezimile zohlobo lwe-C18 ama-C18 ligand anamathiselwe ezinhlayiyeni ze-silica ngamaketanga aqondile, kuyilapho ezigabeni ezimile zohlobo lwe-polystyrene kwakheka i-polymer ewugqinsi uma kuqhathaniswa nezinhlayiya. ungqimba A. Esivivinyweni esijwayelekile, ikholomu porosity ibalwa ngendlela elandelayo:
Emkhiwaneni. I-7A, B ibonisa iziqephu zika-Van Deemter zekholomu ye-PMP (id 100 x 1.8 mm) kanye nekholomu ye-Ascentis Express RP-Amide (id 100 x 1.8 mm) ngaphansi kwezimo ezifanayo zokuchazwa, 60/40 ACN/H2O kanye no-0 .1% TFA 20 µl/min kokubili kukholomu engu-8/min kuya ku-80min. Amanani amancane e-HETP ngezinga lokugeleza elilungile (80 µl/min) abengu-2.6 µm no-3.9 µm kukholomu ye-PMP kanye nekholomu ye-Ascentis Express RP-Amide, ngokulandelanayo. Amanani e-HETP abonisa ukuthi ukusebenza kahle kokuhlukaniswa kwekholomu ye-PMP (i-id engu-100 x 1.8 mm) kuphakeme kakhulu kunaleyo yekholomu ye-Ascentis Express RP-Amide etholakala ngokwentengiso (i-id engu-100 x 1.8 mm). Igrafu ye-van Deemter ku-Fig. 7 (A) ibonisa ukuthi ukwehla kwenani le-N akuphakeme kakhulu ngokugeleza okwandayo uma kuqhathaniswa nesifundo sethu sangaphambilini. Ukusebenza kahle okuphakeme kokuhlukanisa kwekholomu ye-PMP (id 100 × 1.8 mm) uma kuqhathaniswa nekholomu ye-Ascentis Express RP-Amide kusekelwe ekubunjweni kwezinhlayiyana ezithuthukisiwe nosayizi kanye nenqubo yokupakisha yekholomu eyinkimbinkimbi esetshenziswa emsebenzini wamanje34.
(A) Isakhiwo se-Van Deemter (i-HETP iqhathaniswa nesivinini somugqa wesigaba seselula) sitholwe kukholomu ye-PMP (id 100 x 1.8 mm) ngo-60/40 ACN/H2O no-0.1% TFA. (B) Isakhiwo sika-Van Deemter (i-HETP iqhathaniswa nesivinini somugqa wesigaba seselula) sitholwe kukholomu ye-Ascentis Express RP-Amide (id 100 x 1.8 mm) ngo-60/40 ACN/H2O ngo-0.1% TFA.
Isigaba se-polar esimile se-polystyrene ehlanganisiwe salungiselelwa futhi sahlolwa ukuze kuhlukaniswe ingxube yama-peptide okwenziwa kanye ne-tryptic hydrolyzate ye-human serum albumin (HSA) ku-chromatography yoketshezi esebenza kahle. Ukusebenza kwe-chromatographic kwamakholomu e-PMP yezingxube ze-peptide kuhle kakhulu ngokuya ngokusebenza kahle nokuxazulula ukuhlukaniswa. Ukusebenza okuthuthukisiwe kokuhlukaniswa kwamakholomu e-PMP kungenxa yezizathu eziningana ezifana nobukhulu bezinhlayiyana ze-silica nosayizi we-pore, ukuhlanganiswa okulawulwayo kwezigaba ezimile, nezinto zokupakisha zekholomu eziyinkimbinkimbi. Ngaphezu kokusebenza kahle kokuhlukanisa, enye inzuzo yalesi sigaba esimile ingcindezi ephansi yekholomu emuva emazingeni aphezulu okugeleza. Amakholomu e-PMP akhiqizwa kakhulu futhi angasetshenziswa ukuhlaziya izingxube zama-peptide kanye nokugaya kwe-tryptic kwamaphrotheni ahlukahlukene. Sihlose ukusebenzisa le kholomu ukuze sihlukanise izinhlanganisela ze-bioactive emikhiqizweni yemvelo, okukhishwe kwezitshalo zokwelapha namakhowe ku-chromatography ewuketshezi. Ngokuzayo, amakholomu e-PMP azophinde ahlolwe ukuze kuhlukaniswe amaprotheni nama-monoclonal antibodies.
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