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Izinhlayiya ze-silica ezinama-porous zalungiselelwa indlela ye-sol-gel enezinguquko ezithile ukuze zithole izinhlayiya ezinkulu.Lezi zinhlayiya zithathwe yi-reversible addition fragmentation chain transfer (RAFT) polymerization ne-N-phenylmaleimide-methylvinylisocyanate (PMI) kanye ne-styrene ukuze kulungiswe i-N-phenylmaleide ye-polyboridi ye-polymaleimide amakholomu ensimbi engagqwali (i-id engu-100 × 1.8 mm) ayepakishwe ngokupakishwa okunodaka.Ukuhlukaniswa kwekholomu ye-PMP ehloliwe yengxube ye-peptide ehlanganisa ama-peptide amahlanu (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Gly-Ser-Arg e-Arg, ukusebenza kwe-Try-Ile-Gly-Ser-Ser-Arg-Arg) ukugaywa kwe-albumin ye-serum yomuntu (HAS).Ngaphansi kwezimo ezingcono kakhulu ze-elution, inani lepuleti lethiyori lengxube ye-peptide liphezulu ngokufika kumapuleti angu-280,000/m². Uma kuqhathaniswa nokusebenza kokuhlukaniswa kwekholomu ethuthukisiwe nekholomu yezohwebo ye-Ascentis Express RP-Amide, kwaphawulwa ukuthi ukusebenza ngokuhlukana kwekholomu yekholomu ye-PMP kwakuphakeme ngokwemibandela yekholomu yokuhlukaniswa kwekholomu ye-PMP kusezingeni eliphezulu ngokwemibandela yokulungiswa kwekholomu ye-PMP.
Eminyakeni yamuva nje, imboni ye-biopharmaceutical isiphenduke imakethe yomhlaba wonke ekhulayo ngokukhula okukhulu kwesabelo semakethe. Ngokukhula okunamandla kwemboni ye-biopharmaceutical1,2,3, ukuhlaziywa kwama-peptides namaprotheni kufiseleka kakhulu. ukuhlukaniswa kwamaprotheni oketshezini lomzimba, izicubu namaseli kuwumsebenzi oyinselele kakhulu ngenxa yenani elikhulu lezinhlobo ezingase zibonakale kusampula eyodwa.Nakuba i-mass spectrometry iyithuluzi elisebenzayo lokulandelana kwe-peptide ne-protein, uma amasampula anjalo ejova ku-spectrometer enkulu ngephasi eyodwa, ukuhlukaniswa ngeke kube okufanelekile. ukunciphisa inani labahlaziyi abangena ku-spectrometer enkulu ngesikhathi esinikeziwe4,5,6.Ngaphezu kwalokho, ngesikhathi sokuhlukaniswa kwesigaba soketshezi, ama-analyte angagxila ezifundeni ezincane, ngaleyo ndlela agxilise lezi zihlaziyi futhi athuthukise ukuzwela kokutholwa kwe-MS.I-Liquid chromatography (LC) ithuthuke kakhulu kule minyaka eyishumi edlule futhi isibe inqubo ethandwayo ekuhlaziyweni kwe-proteomic,9,8,17.
I-Reversed-phase liquid chromatography (RP-LC) isetshenziswa kakhulu ekuhlanzeni nasekuhlukaniseni izingxube ze-peptide kusetshenziswa i-octadecyl-modified silica (ODS) njengesigaba esimile11,12,13.Nokho, izigaba ezimile ze-RP azihlinzeki ngokuhlukana okwanelisayo kwama-peptide namaprotheni ngenxa ye-amphiphilic eklanyelwe imvelo,15 isakhiwo sazo esikhethekile. izigaba ezimile ziyadingeka ukuze kuhlaziywe ama-peptide namaprotheni anezingxenye ezincanyana nezingezona ze-polar ukuze kuhlangatshezwane nazo futhi kugcinwe lezi analytes16.I-Mixed-mode chromatography, ehlinzeka ngokusebenzisana kwe-multimodal, ingaba enye indlela ye-RP-LC yokuhlukaniswa kwama-peptide, amaprotheni, nezinye izingxube eziyinkimbinkimbi.Amakholomu amaningana ahlanganisiwe asetshenzisiwe, lezi zigaba ezixubile ziye zapakishwa ngezigaba ezimile, lezi zikholomu ezixubile ziye zapakishwa ngezigaba ezimile, lezi zikholomu ezihlanganisiwe ziye zapakishwa ngezigaba ezimile. ukuhlukaniswa kwe-peptide namaprotheni17,18,19,20,21.Izigaba ezimile zemodi exubile (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) zifanele ukuhlukaniswa kwe-peptide kanye namaprotheni ngenxa yokuba khona kokubili kwamaqembu e-polar kanye ne-non-polar22,23,24,25,25,26,26,26,26. Amaqembu e-polar ahlanganiswe ngokuhlanganyela abonisa amandla amahle okuhlukanisa nokukhetha okuyingqayizivele kuma-polar analyte angewona ama-polar, njengoba ukuhlukana kuncike ekusebenzelaneni phakathi kwe-analyte nesigaba esimile. Ukusebenzisana kwe-Multimodal 29, 30, 31, 32.Muva nje, u-Zhang et al. I-30 ilungiselele isigaba sokumisa se-dodecyl-terinated polyamine futhi ihlukanise ngempumelelo ama-hydrocarbon, ama-antidepressants, ama-flavonoid, ama-nucleosides, ama-estrogens, nezinye izihlaziyo ezimbalwa.I-polar intercalator inamaqembu e-polar kanye ne-non-polar, ngakho-ke ingasetshenziswa ukuhlukanisa ama-peptide namaphrotheni anakho kokubili i-hydrophobic ne-hydrophilic moiembed. Amakholomu e-C18 ashumekwe i-amide) atholakala ngokwentengiso ngaphansi kwegama lokuhweba elithi Ascentis Express RP-Amide amakholomu, kodwa lawa makholomu asetshenziselwa ukuhlaziywa kwe-amine 33 kuphela.
Ocwaningweni lwamanje, isigaba sokumisa esishunyekiwe se-polar (i-N-phenylmaleimide-embedded polystyrene) salungiswa futhi sahlolwa ukuze kuhlukaniswe ama-peptides kanye ne-trypsin digests ye-HSA.Isigaba esimile salungiswa kusetshenziswa isu elilandelayo.Izinhlayiya ze-silica ezinama-porous zalungiswa ngokuvumelana nenqubo enikezwe ekushicilelweni kwethu kwangaphambilini ngokunye ukuguqulwa, i-polylethy ratio ye-uprotocol ye-u. I-TMOS, i-acetic acid yamanzi yalungiswa ukuze kulungiswe izinhlayiya ze-silica ezinosayizi omkhulu wembotshana.Okwesibili, i-ligand entsha, i-phenylmaleimide-methyl vinyl isocyanate, yahlanganiswa futhi yasetshenziselwa ukukhipha izinhlayiya ze-silica ukuze kulungiswe isigaba esimile esishunyekiwe se-polar. isikimu.Ukupakishwa kwekholomu kusizwa ngokudlidliza kwemishini ukuze kuqinisekiswe umbhede olinganayo kwakheka ngaphakathi kwekholomu.Hlola ukuhlukaniswa kwekholomu epakishiwe yezingxube ze-peptide ezihlanganisa ama-peptide amahlanu; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) kanye ne-Trypsin digest ye-human serum albumin (HAS).Ingxube ye-peptide kanye ne-trypsin digest ye-HSA yaqashelwa ukuze ihlukaniswe ngokulungiswa okuhle nokusebenza kahle kwe-Ps ye-AMP ye-Express yaqhathaniswa nokusebenza kokuhlukaniswa kwekholomu ye-AMP. Ikholomu ye-RP-Amide.Kokubili ama-peptide namaprotheni abonwe exazululwe kahle futhi esebenza kahle kukholomu ye-PMP, eyayisebenza kahle kakhulu kunekholomu ye-Ascentis Express RP-Amide.
I-PEG (i-Polyethylene Glycol), i-Urea, i-Acetic Acid, i-Trimethoxy Orthosilicate (TMOS), i-Trimethyl Chlorosilane (TMCS), i-Trypsin, i-Human Serum Albumin (HSA), i-Ammonium Chloride, i-Urea, i-Hexane Methyldisilazane (HMDS), i-Methacryloyl Chloride, i-Benzoyldroksidi, i-Benzoyldroksidi, i-MCTEM (BPO), HPLC Grade Acetonitrile (ACN), Methanol, 2-Propanol, kanye ne-Acetone Kuthengwe kwa-Sigma-Aldrich (St. Louis, MO, USA).
Ingxube ye-urea (8 g), i-polyethylene glycol (8 g), kanye no-8 mL ka-0.01 N acetic acid yanyakaziswa imizuzu engu-10, kwase kwengezwa u-24 mL we-TMOS lapho ngaphansi kwezimo ezibandayo. izinto ezisele zomisiwe ku-70 ° C amahora angu-12. Isisindo esomile esithambile sasiwumhlabathi obushelelezi kuhhavini futhi sibalwa ku-550 ° C amahora angu-12. Amaqoqo amathathu alungiselelwe futhi abonakaliswe ukuhlola ukukhiqizwa kabusha ngobukhulu bezinhlayiyana, ubukhulu be-pore kanye nendawo ephezulu.
Ngokuguqulwa kwendawo yezinhlayiya ze-silica nge-ligand yangaphambili ye-phenylmaleimide-methylvinylisocyanate (PCMP) elandelwa i-polymerization ye-radial nge-styrene, i-polar group-containing compound yalungiswa. Isigaba esimile samaketanga ahlanganisiwe namaketanga e-polystyrene.Inqubo yokulungiselela ichazwe ngezansi.
I-N-phenylmaleimide (200 mg) ne-methyl vinyl isocyanate (100 mg) zincibilikisiwe ku-toluene eyomile, futhi u-0.1 mL we-2,2′-azoisobutyronitrile (AIBN) yengezwe ku-reaction flask ukuze kulungiswe i-phenylmaleimide-methyl vinyl isocyanate copolymer amahora angu-30, i-heart C ° 3 PM. lihlungiwe futhi lomiswe kuhhavini elingu-40°C amahora amathathu.
Izinhlayiya ze-silica ezomile (2 g) zihlakazwe ku-toluene eyomile (100 mL), zinyakaziswa futhi zafakwa ku-sonicated ku-500 mL ye-flask engezansi eyindilinga imizuzu engu-10.PMCP (10 mg) yachithwa ku-toluene futhi yengezwa nge-dropwise ku-flask yokusabela nge-funnel yokudonsa. I-60 ° C amahora angu-3. Khona-ke, izinhlayiya ze-silica eziboshwe nge-PMCP (100 g) zahlakazwa ku-toluene (200 ml) kanye ne-4-hydroxy-TEMPO (2 mL) yengezwe phambi kwe-100 µL ye-dibutyltin dilaurate njenge-catalyst.Ingxube yahlungwa ngo-5 ° C futhi yomiswa ku-5 ° C amahora angu-50. amahora.
I-Styrene (1 mL), i-benzoyl peroxide BPO (0.5 mL), kanye nezinhlayiya ze-silica ezinamathiselwe i-TEMPO-PMCP (1.5 g) zihlakazwe ku-toluene futhi zahlanzwa nge-nitrogen. I-polymerization ye-styrene yenziwa ku-100 ° C amahora angu-12. Umkhiqizo owenziwe wawashwa nge-methanol ebusuku ku-reactivity ye-methanol kanye ne-fire ° C jikelele yomiswa. 1.
Amasampula ahlanjululwe ku-393 K ngehora le-1 ukuze athole ingcindezi eyinsalela engaphansi kwe-10-3 Torr.Inani le-N2 elikhangisiwe ngengcindezi ehlobene ye-P / P0 = 0.99 yasetshenziselwa ukucacisa ivolumu ye-pore. Japan).Amasampuli omisiwe (i-silica engenalutho kanye nezinhlayiya ze-silica eziboshwe nge-ligand) abekwe kukholomu ye-aluminium esebenzisa i-adhesive carbon tape.Igolide yafakwa kumasampula kusetshenziswa i-Q150T sputter coater, futhi ungqimba we-5 nm Au wafakwa kumasampuli.Lokhu kuthuthukisa ukusebenza kahle kwenqubo usebenzisa ama-voltage aphansi futhi inikeza okusanhlamvu okuhle, i-MAWas ebandayo. I-Flash EA1112 i-elemental analyzer isetshenziselwe ukuhlaziya okuyisisekelo.I-Malvern (Worcestershire, UK) I-Mastersizer 2000 yosayizi wezinhlayiyana analyzer yasetshenziswa ukuze kutholwe ukusatshalaliswa kosayizi wezinhlayiyana.Izinhlayiya ze-silica ezinqunu kanye nezinhlayiya ze-silica eziboshwe nge-ligand (5 mg ngayinye) zahlakazwa ku-5 mL we-isopropanol, yafakwa i-ophthal min, i-sonication emi-5 ibhentshi le-Mastersizer.Ukuhlaziywa kwe-Thermogravimetric kwenziwa ngesilinganiso esingu-5 °C ngomzuzu phezu kwezinga lokushisa elingu-30 kuya ku-800 °C.
Amakholomu ensimbi engenacala anemigqa emincane enobukhulu (i-id engu-100 × 1.8 mm) aye apakishwa kusetshenziswa indlela yokupakisha enodaka, kusetshenziswa inqubo efanayo esetshenziswe ku-Ref. 31.Ikholomu yensimbi engagqwali (efakwe ingilazi, i-id engu-100 × 1.8 mm) ene-outlet fitting equkethe i-1 µm frit yayixhunywe ku-slurry packer (Alltech Deerfield, IL, USA).Lungisa i-slurry yesigaba esimile ngokumisa u-150 mg wesigaba esimile se-mL yesitoreji se-menotha ku-menotha2. yayisetshenziswa njengesinyibilikisi esinodaka kanye ne-solvent edonsayo.Gcwalisa ikholomu ngokulandelana ngokusebenzisa izingcindezi ze-100 MP imizuzu engu-10, i-80 MP imizuzu engu-15, ne-MP engu-60 imizuzu engu-30. Ngesikhathi sokupakisha, ukudlidliza komshini kwasetshenziswa nama-shaker amabili ekholomu ye-GC (Alltech, Deerfield) ukuze kuqinisekiswe ukupakishwa kwekholomu ye-ILrry, i-USA futhi ukhulule ukucindezela kancane ukuze uvimbele noma yimuphi umonakalo ngaphakathi kwekholomu.Nqamula ikholomu kuyunithi yokupakisha enodaka bese uxhuma okunye ukulingana endaweni yokungena kanye nohlelo lwe-LC ukuze uhlole ukusebenza kwayo.
Iphampu ye-LC (10AD Shimadzu, Japan), injector (Valco (USA) C14 W.05) ene-50nL injection loop, i-membrane degasser (Shimadzu DGU-14A), iwindi le-capillary ye-UV-VIS lakhiwe Isitholi sedivayisi esikhethekile se-µLC (UV-2075) kanye nomphumela omncane oxhumanisa ingilazi we-microcolum emifushane kakhulu. ukunwetshwa kwebhande lekholomu eyengeziwe.Ngemva kokupakishwa, ama-capillary (50 μm id 365 kanye nokunciphisa ama-union capillaries (50 μm) afakwe endaweni engu-1/16″ yokuphuma kwenyunyana yokunciphisa. Ukuqoqwa kwedatha nokucutshungulwa kwe-chromatographic kwenziwa kusetshenziswa isofthiwe ye-Multichro 2000. Ukuqapha bekuhlolwe idatha ye-UV ye-UV ye-Chromatographic engu-25. ihlaziywe yi-OriginPro8 (Northampton, MA).
I-Albumin evela ku-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 ne-0.2 M ammonium bicarbonate (1 mL).Isixazululo sanyakaziswa imizuzu engu-10 futhi sigcinwa kubhavu wamanzi amahora angu-67 no-37 inch, ° C. 0.1% TFA.Hlunga ikhambi bese ugcine ngaphansi kuka-4 °C.
Ukuhlukaniswa kwezingxube ze-peptide kanye nokugaya kwe-HSA trypsin kwahlaziywa ngokuhlukana kumakholomu e-PMP.Hlola ukuhlukaniswa kwengxube ye-peptide kanye ne-trypsin digest ye-HSA ngekholomu ye-PMP bese uqhathanisa imiphumela nekholomu ye-Ascentis Express RP-Amide.Inombolo yepuleti lethiyori ibalwa kanje:
Izithombe ze-SEM zezinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-silica eziboshwe nge-ligand ziboniswa ku-FIG. I-2 .Izithombe ze-SEM zezinhlayiya ze-silica ezingenalutho (A, B) zibonisa ukuthi, ngokungafani nezifundo zethu zangaphambilini, lezi zinhlayiya ziyi-spherical lapho izinhlayiya zinde noma zine-symmetry engavamile.Ubuso bezinhlayiya ze-silica eziboshwe nge-ligand (C, D) bushelelezi kunaleyo ye-silica engenalutho engaba yizinhlayiya ze-polysty ngenxa yezinhlayiya ze-colyne. izinhlayiya ze-silica.
Iskena izithombe zesibonakhulu se-electron zezinhlayiya ze-silica ezingenalutho (A, B) kanye nezinhlayiya ze-silica eziboshwe nge-ligand (C, D).
Ukusabalalisa usayizi wezinhlayiyana zezinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-silica eziboshwe nge-ligand ziboniswa ku-Figure 3 (A). Amajika okusabalalisa usayizi wezinhlayiyana ezisekelwe kuvolumu abonise ukuthi ubukhulu bezinhlayiya ze-silica banda ngemva kokuguqulwa kwamakhemikhali (Fig. 3A) .Idatha yokusabalalisa usayizi wezinhlayiyana zezinhlayiya ze-silica kusukela ocwaningweni lwamanje kanye nevolumu yezinhlayiyana zangaphambilini ziqhathaniswa (usayizi wezinhlayiyana zethebula1) uma kuqhathaniswa (usayizi wethebula). d(0.5), ye-PMP ingu-3.36 μm, uma kuqhathaniswa nocwaningo lwethu lwangaphambilini ngenani lesikhangiso(0.5) lika-3.05 μm (izinhlayiya ze-silica eziboshwe nge-polystyrene)34.Leli qoqo libe nokusabalalisa usayizi wezinhlayiyana ezincane uma kuqhathaniswa nocwaningo lwethu lwangaphambilini ngenxa yezilinganiso ezihlukene ze-PEG, i-urea, i-TMOS kuyizinhlayiyana zesigaba esikhulu sokusabela. leyo yesigaba sezinhlayiya ze-silica eboshwe nge-polystyrene esike sayifunda.Lokhu kusho ukuthi ukusebenza kwendawo yezinhlayiya ze-silica nge-styrene kubeka kuphela isendlalelo se-polystyrene (0.97 µm) endaweni ye-silica, kanti esigabeni se-PMP ukujiya kwesendlalelo kwakungu-1.38 µm.
Ukusabalalisa usayizi we-particle (A) nokusabalalisa usayizi we-pore (B) wezinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-silica eziboshwe nge-ligand.
Ubukhulu bembotshana, ivolumu yembotshana kanye nendawo engaphezulu yezinhlayiya ze-silica zocwaningo lwamanje zinikezwe kuThebula 1(B).Amaphrofayili e-PSD ezinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-silica eziboshwe nge-ligand ziboniswa kuMfanekiso 3(B). Imiphumela iqhathaniswa nesifundo sethu sangaphambilini.Amasayizi wembotshana we-bare kanye nezinhlayiyana ze-ligand nezinhlayiya eziyi-230 ngokuhlonipha, i-silica eboshwe nge-230 ngokuhlonipha. ibonisa ukuthi usayizi we-pore wehla ngo-69 ngemva kokuguqulwa kwamakhemikhali, njengoba kuboniswe kuThebula 1 (B), futhi ukuguqulwa kwejika kuboniswe ku-Fig. 3 (B) .Ngokufanayo, umthamo we-pore wezinhlayiya ze-silica wehla ukusuka ku-0.67 kuya ku-0.58 cm3 / g ngemva kokuguqulwa kwamakhemikhali.Indawo ethize ye-silica engu-1 kuya ku-6 m2 efundwayo okwamanje i-silica 6 / m2 isifundo sethu sangaphambilini (124 m2 / g) .Njengoba kuboniswe kuThebula 1 (B), indawo engaphezulu (m2 / g) yezinhlayiya ze-silica nayo yehla ukusuka ku-116 m2 / g kuya ku-105 m2 / g ngemva kokuguqulwa kwamakhemikhali.
Imiphumela yokuhlaziywa okuyisisekelo kwesigaba esimile ikhonjisiwe kuThebula 2.Ukulayishwa kwekhabhoni kwesigaba esimile samanje kungu-6.35%, okungaphansi kokulayishwa kwekhabhoni ocwaningweni lwethu lwangaphambilini (izinhlayiya ze-silica eziboshwe nge-polystyrene, 7.93%35 kanye no-10.21%, ngokulandelana) 42. Ukulayishwa kwekhabhoni kwesigaba esimile, se-SP esikhona njengamanje, ku-SPrene amanye ama-polar ligands afana ne-phenylmaleimide-methylvinylisocyanate (PCMP) kanye ne-4-hydroxy-TEMPO asetshenzisiwe.Amaphesenti esisindo se-nitrogen wesigaba samanje esimile singu-2.21%, uma kuqhathaniswa ne-0.1735 kanye ne-0.85% ngesisindo se-nitrogen ezifundweni zangaphambilini, ngokulandelana.Lokhu kusho ukuthi isigaba se-nitrogen siphezulu ngenxa yesigaba samanje se-nitrogen i-phenylmaleimide.Ngokufanayo, ukulayishwa kwekhabhoni kwemikhiqizo (4) kanye (5) bekuyi-2.7% ne-2.9%, ngokulandelana, kuyilapho ukulayishwa kwekhabhoni komkhiqizo wokugcina (6) bekuyi-6.35%, njengoba kuboniswe kuThebula 2.Ukuncipha kwesisindo kuhlolwe ngesigaba esimile se-PMP, futhi ijika le-TGA eliboniswa ku-Figure 4 ye-TGA ekhombisa ukulahlekelwa kwesisindo. ivumelana kahle nokulayishwa kwekhabhoni (6.35%) ngoba ama-ligand awaqukethe u-C kuphela kodwa futhi no-N, O, no-H.
I-phenylmaleimide-methylvinylisocyanate ligand ikhethelwe ukuguqulwa kwendawo yezinhlayiya ze-silica ngoba inamaqembu e-polar phenylmaleimide kanye namaqembu e-vinylisocyanate.Amaqembu e-vinylisocyanate angakwazi ukuqhubeka asabele nge-styrene ngokuphila kwe-polymerization e-radical.Isizathu sesibili ukufaka iqembu elinokuxhumana okumaphakathi ne-analyte ye-electrostatic kanye nokuxhumana okuqinile phakathi kwesiteshi se-analyte kanye nokungabikho kwe-analyte I-phenylmaleimide moiety ayinakho ukushaja 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 futhi singashintsha i-polarity yesigaba esimile.Ukuhlaziywa kwe-Elemental kwenziwa ukuze kubhekwe ukuthi ukulayishwa kwesigaba se-carbonate anda ukwanda kwalezi zikhathi zokusabela kwekhabhoni. ukulayishwa kwekhabhoni yesigaba esimile futhi okuphambene nalokho.Ama-SP alungiselelwe ngokugxila okuhlukene kwe-styrene ane-carbon loadings ehlukene.Futhi, layisha lezi zigaba ezimile kumakholomu ensimbi engagqwali futhi uhlole ukusebenza kwazo kwe-chromatographic (ukukhethwa, ukuxazulula, inani le-N, njll.) .Ngokusekelwe kulezi zivivinyo, ukwakheka okulungiselelwe kwakhethwa ukuze kulungiswe isigaba sokulawulwa kokumiswa kwe-PMP ukuze kuqinisekiswe ukubuyiswa kwe-analote yokulawula okuhle.
Izingxube ze-peptide ezinhlanu (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-leucine enkephalin) nayo yahlolwa kusetshenziswa ikholomu ye-PMP kusetshenziswa isigaba seselula; 60/40 (v/v) i-acetonitrile/amanzi (0.1% TFA) ngenani lokugeleza elingu-80 μL/min. Ngaphansi kwezimo ezingcono kakhulu zokucacisa, inombolo yepuleti lethiyori (N) ikholomu ngayinye (i-id engu-100 × 1.8 mm) ingu-20,000 ± 100 (amapuleti angu-200/m² anikeza i-0²). Amakholomu e-PMP nama-chromatogram aboniswa kuMfanekiso 5A. Ukuhlaziywa okusheshayo kukholomu ye-PMP ngenani lokugeleza okuphezulu (700 μL/min), ama-peptide amahlanu akhishwa phakathi nomzuzu owodwa, amanani e-N abemahle kakhulu, 13,500 ± 330 ikholomu ngayinye (100 × 1.8 mm id), i-13 ihambisana/i-0 (0 Ihambisana no-0, i-0 ihambisana no-0) 5B).Amakholomu amathathu anosayizi ofanayo (i-id engu-100 × 1.8 mm) apakishwe ngezigaba ezintathu ezihlukene zesigaba esimile se-PMP ukuze kuhlolwe ukukhiqizwa kabusha.Ukugxiliswa kohlaziyo kwekholomu ngayinye kwarekhodwa kusetshenziswa izimo ezingcono kakhulu zokucacisa kanye nenani lamapuleti etiyetha engu-N kanye nesikhathi sokugcinwa ukuze kuhlukaniswe ingxube efanayo yokuhlola kukholomu ye-PMP eboniswa kabusha kukholamu ngayinye eboniswayo. ukukhiqizwa kabusha kwekholomu ye-PMP kuhlotshaniswa kahle namanani aphansi kakhulu e-%RSD, njengoba kuboniswe kuThebula 3.
Ukuhlukaniswa kwengxube ye-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 × 1.8 mm id); i-analytical I-elution order of the compounds: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) kanye 5 (leucine) acid enkephalin)).
Ikholomu ye-PMP (i-id engu-100 × 1.8 mm) yahlolelwa ukuhlukaniswa kokugaya kwe-tryptic kwe-albumin ye-serum yomuntu ekusebenzeni okuphezulu kwe-chromatography ye-liquid. I-chromatogram ekuMdwebo 6 ibonisa ukuthi isampula ihlukaniswe kahle futhi ukulungiswa kuhle kakhulu. Ukugaya kwe-HSA kwahlaziywa kusetshenziswa izinga lokugeleza elingu-100/3 µ i-acetonitrile/amanzi kanye no-0.1% TFA.Njengoba kukhonjisiwe ku-chromatogram (Umfanekiso 6), ukugayeka kokudla kwe-HSA kuhlukaniswe kwaba iziqongo eziyi-17 ezihambisana nama-peptide angu-17. Ukusebenza kahle kokuhlukanisa kwenani eliphakeme ngalinye ku-HSA digest kubaliwe futhi amanani anikezwe kuThebula lesi-5.
I-tryptic digest ye-HSA (100 × 1.8 mm id) yahlukaniswa kukholamu ye-PMP; izinga lokugeleza (100 µL/min), isigaba seselula 60/40 acetonitrile/amanzi ano-0.1% TFA.
lapho u-L engubude bekholomu, η yi-viscosity yesigaba seselula, i-ΔP iyikholomu yokucindezela emuva, futhi u-u isivinini somugqa wesigaba esihambayo.Ukufinyeleleka kwekholomu ye-PMP kwakungu-2.5 × 10-14 m2, izinga lokugeleza lalingu-25 μL/min, futhi 60/40 v/v ikholomu ngayinye ye-ACN/amanzi yasetshenziswa i-ACN/amanzi angu-1 × 1.8 mm id) yayifana naleyo yocwaningo lwethu lwangaphambilini Ref.34.Ukuvunywa kwekholomu egcwele izinhlayiya ezimboze kakhulu yilokhu: 1.7 × 10-15 yezinhlayiya ezingu-1.3 μm, 3.1 × 10-15 ngo-1.7 μm izinhlayiya, 5.0-1 × 2 × 2 × 2 × 2 5.2 × 1. 2.6 μm izinhlayiya Ku-5 μm izinhlayiya 43.Ngakho-ke, ukungena kwesigaba se-PMP kufana nalokho kwezinhlayiya ze-core-shell ezingu-5 μm.
lapho i-Wx iyisisindo sekholomu egcwele i-chloroform, i-Wy isisindo sekholomu egcwele i-methanol, futhi u-ρ ukuminyana kwe-solvent.Ukuminyana kwe-methanol (ρ = 0.7866) kanye ne-chloroform (ρ = 1.484).Ingqikithi ye-porosity yekholomu ye-SILICA PARTICLES i-108 × 10 × 10 mm kanye namakholomu e-C18-Urea angu-31 esiwafunde ngaphambilini ayengu-0.63 no-0.55, ngokulandelana.Lokhu kusho ukuthi ukuba khona kwe-urea ligand kunciphisa ukufinyeleleka kwesigaba esimile.Ngakolunye uhlangothi, ingqikithi ye-porosity yekholomu ye-PMP (100 × 1.8 mm id) ingu-0.60 wekholomu ephansi yekholomu ye-MP. Izinhlayiya ze-silica eziboshwe nge-C18 ngoba ezigabeni ezimile zohlobo lwe-C18 ama-ligand e-C18 anamathiselwe ezinhlayiyeni ze-silica njengamaketango aqondile, kuyilapho ezigabeni ezimile zohlobo lwe-polystyrene, kwakheka ungqimba lwe-polymer oluwugqinsi oluyizungezile. Ekuhloleni okuvamile, ikholomu porosity ibalwa kanje:
Umfanekiso 7A,B ubonisa ikholomu ye-PMP (i-id engu-100 × 1.8 mm) kanye nekholomu ye-Ascentis Express RP-Amide (i-id engu-100 × 1.8 mm) kusetshenziswa izimo ezifanayo zokugqama (okungukuthi, 60/40 ACN/H2O kanye no-0.1% TFA). ) yesakhiwo sika-van Deemter. Izingxube ze-peptide ezikhethiwe (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-Leucine Enkephalin) ilungiswe ngo-20 µL/ Izinga lokugeleza eliphansi lawo womabili amakholomu ngu-800 µL/min. Ubuncane besilinganiso se-HETP µmin. ikholomu kanye nekholomu ye-Ascentis Express RP-Amide bekungu-2.6 µm no-3.9 µm, ngokulandelanayo. Amanani e-HETP abonisa ukuthi ukusebenza kahle kokuhlukaniswa kwekholomu ye-PMP (100 × 1.8 mm id) kungcono kakhulu kunekholomu ye-Ascentis Express RP-Amide etholakalayo kwezohwebo (100 × id em 1.8 ikhombisa) Isiqephu esingu-7 ukuthi ukwehla kwenani le-N ngokugeleza okwandayo akubalulekile uma kuqhathaniswa nesifundo sethu sangaphambilini.Ukuphumelela okuphezulu kokuhlukaniswa kwekholomu ye-PMP (100 × 1.8 mm id) uma kuqhathaniswa nekholomu ye-Ascentis Express RP-Amide isekelwe ekuthuthukisweni kokuma kwezinhlayiyana, usayizi, kanye nezinqubo zokupakisha zekholomu eziyinkimbinkimbi ezisetshenziswe emsebenzini wamanje34.
(A) icebo le-van Deemter (i-HETP uma iqhathaniswa nesivinini somugqa wesigaba seselula) itholwe kusetshenziswa ikholomu ye-PMP (100 × 1.8 mm id) ku-60/40 ACN/H2O ne-0.1% TFA.(B) isiqephu se-van Deemter (i-HETP uma iqhathaniswa nesivinini somugqa wesigaba seselula) etholwe kusetshenziswa ikholomu ye-Ascentis × 1 i-Ascentis × 1 RP-Ascentis Express. 60/40 ACN/H2O nge-0.1% TFA.
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