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Izinhlayiya ze-silica ezinezimbobo zalungiswa ngendlela ye-sol-gel kanye nokuguqulwa okuthile ukuthola izinhlayiya ezinezimbobo ezinkulu. Lezi zinhlayiya zathathwa yi-reversible addition fragmentation chain transfer (RAFT) polymerization nge-N-phenylmaleimide-methylvinylisocyanate (PMI) kanye ne-styrene ukuze kulungiselelwe i-N-phenylmaleimide intercalation ye-polystyrene (PMP) stationary phase. Amakholomu ensimbi engagqwali anezimbobo ezincane (100 × 1.8 mm id) apakishwe nge-slurry packing. Ukuhlukaniswa kwekholomu ye-PMP okuhloliwe kwengxube ye-peptide equkethe ama-peptide amahlanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) chromatographic performance) kanye nokugaya kwe-trypsin kwe-human serum albumin (HAS). Ngaphansi kwezimo ezifanele zokuxazulula, inani lamapuleti eliyinkolelo-mbono lengxube ye-peptide lifinyelela kuma-plate angu-280,000/m². Uma kuqhathaniswa ukusebenza kokuhlukaniswa kwe- Ikholomu ethuthukisiwe enekholomu ye-Ascentis Express RP-Amide yezentengiselwano, kwaphawulwa ukuthi ukusebenza kokuhlukaniswa kwekholomu ye-PMP kwakungcono kunekholomu yezentengiselwano ngokusebenza kahle kokuhlukaniswa kanye nesisombululo.
Eminyakeni yamuva nje, imboni ye-biopharmaceutical isibe yimakethe yomhlaba wonke ekhulayo ngokukhula okukhulu kwesabelo semakethe. Ngokukhula okukhulu kwemboni ye-biopharmaceutical1,2,3, ukuhlaziywa kwama-peptide namaprotheni kuyadingeka kakhulu. Ngaphezu kwe-peptide eqondiwe, ukungcola okuningana kukhiqizwa ngesikhathi sokwenziwa kwe-peptide, ngaleyo ndlela kudinga ukuhlanzwa kwe-chromatographic ukuze kutholakale ama-peptide ahlanzekile afunwayo. Ukuhlaziywa kanye nokuhlukaniswa kwamaprotheni oketshezini lomzimba, izicubu kanye namaseli kuwumsebenzi onzima kakhulu ngenxa yenani elikhulu lezinhlobo ezingatholakala kusampula eyodwa. Nakuba i-mass spectrometry iyithuluzi eliphumelelayo lokulandelana kwe-peptide kanye namaprotheni, uma amasampula anjalo efakwa ku-mass spectrometer ngesikhathi esisodwa, ukuhlukaniswa ngeke kube kuhle. Le nkinga ingancishiswa ngokusebenzisa ukuhlukaniswa kwe-liquid chromatography (LC) ngaphambi kokuhlaziywa kwe-MS, okuzonciphisa inani lama-analytes angena ku-mass spectrometer ngesikhathi esithile4,5,6. Ngaphezu kwalokho, ngesikhathi sokuhlukaniswa kwesigaba soketshezi, ama-analytes angagxila ezindaweni ezincane, ngaleyo ndlela agxile kulawa ma-analytes futhi athuthukise ukutholwa kwe-MS. Ukuzwela. I-chromatography yoketshezi (LC) ithuthuke kakhulu eminyakeni eyishumi edlule futhi isibe yindlela ethandwayo ekuhlaziyweni kwe-proteomic7,8,9,10.
I-reversed-phase liquid chromatography (RP-LC) isetshenziswa kabanzi ekuhlanzeni nasekuhlukaniseni izingxube ze-peptide kusetshenziswa i-octadecyl-modified silica (ODS) njengesigaba esimile11,12,13. Kodwa-ke, izigaba ezimile ze-RP azihlinzeki ngokuhlukaniswa okwanelisayo kwama-peptide namaprotheni ngenxa yesakhiwo sawo esiyinkimbinkimbi kanye nesimo sawo esiguquguqukayo 14,15. Ngakho-ke, izigaba ezimile eziklanywe ngokukhethekile ziyadingeka ukuhlaziya ama-peptide namaprotheni anezingxenye ze-polar kanye nezingezona ze-polar ukuze zisebenzisane futhi zigcine lezi zihlaziyi16. I-Mixed-mode chromatography, enikeza ukusebenzisana kwe-multimodal, ingaba yindlela ehlukile ye-RP-LC yokuhlukaniswa kwama-peptide, amaprotheni, kanye nezinye izingxube eziyinkimbinkimbi. Izigaba eziningana ezimile zemodi ehlanganisiwe sezilungisiwe, futhi amakholomu agcwele lezi zigaba asetshenziselwe ukuhlukaniswa kwama-peptide namaprotheni17,18,19,20,21. Izigaba ezimile zemodi ehlanganisiwe (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) zifanele ukuhlukaniswa kwama-peptide namaprotheni ngenxa ukuba khona kwamaqembu e-polar kanye namaqembu angewona ama-polar22,23,24,25,26,27,28. Ngokufanayo, izigaba ezimile ezihlanganayo ze-polar ezinamaqembu e-polar ahlanganiswe ndawonye zibonisa amandla amahle okuhlukanisa kanye nokukhetha okuyingqayizivele kwabahlaziyi be-polar kanye nabangawona ama-polar, njengoba ukuhlukana kuncike ekusebenzisaneni phakathi kwe-analyte kanye nesigaba esimile. Ukusebenzisana kwe-Multimodal 29, 30, 31, 32. Muva nje, uZhang et al. 30 balungiselele isigaba se-polyamine esingapheli esiqediwe yi-dodecyl futhi bahlukanisa ngempumelelo ama-hydrocarbon, ama-antidepressants, ama-flavonoid, ama-nucleoside, ama-estrogen, kanye nezinye i-analytes eziningana. I-polar intercalator inamaqembu e-polar kanye nama-non-polar, ngakho-ke ingasetshenziswa ukuhlukanisa ama-peptide namaprotheni ane-hydrophobic kanye ne-hydrophilic moieties. Amakholomu afakwe nge-polar (isb., amakholomu e-C18 afakwe nge-amide) atholakala kwezentengiselwano ngaphansi kwegama lokuhweba elithi Ascentis Express RP-Amide columns, kodwa lawa makholomu asetshenziselwa ukuhlaziya i-amine 33 kuphela.
Esifundweni samanje, kwalungiselelwa futhi kwahlolwa isigaba esimile esihlanganisiwe (i-N-phenylmaleimide-embedded polystyrene) ukuze kuhlukaniswe ama-peptide kanye nokugaya kwe-trypsin kwe-HSA. Isigaba esimile salungiselelwa kusetshenziswa isu elilandelayo. Izinhlayiya ze-silica ezinezimbobo zalungiselelwa ngokwenqubo enikezwe encwadini yethu yangaphambilini kanye nokuguqulwa okuthile kwiphrothokholi yokulungiselela. Isilinganiso se-urea, i-polyethylene glycol (PEG), i-TMOS, i-acetic acid yamanzi salungiswa ukuze kulungiselelwe izinhlayiya ze-silica ezinobukhulu obukhulu bezimbobo. Okwesibili, i-ligand entsha, i-phenylmaleimide-methyl vinyl isocyanate, yakhiqizwa futhi yasetshenziswa ukuthola izinhlayiya ze-silica ukuze kulungiselelwe isigaba esimile esihlanganisiwe se-polar. Isigaba esimile esiphumelayo sapakishwa kukholomu yensimbi engagqwali (100 × 1.8 mm id) kusetshenziswa uhlelo lokupakisha olulungiselelwe kahle. Ukupakisha ikholomu kusizakala ngokudlidliza komshini ukuqinisekisa ukuthi umbhede ofanayo uyakhiwa ngaphakathi kwekholomu. Hlola ukuhlukaniswa kwekholomu okupakishiwe kwezingxube ze-peptide eziqukethe ama-peptide amahlanu; (I-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-Leucine Enkephalin) kanye ne-Trypsin digest ye-albumin ye-serum yomuntu (HAS). Ingxube ye-peptide kanye ne-trypsin digest ye-HSA kwabonwa kuhlukaniswa ngesisombululo esihle kanye nokusebenza kahle. Ukusebenza kokuhlukaniswa kwekholomu ye-PMP kwaqhathaniswa nalokho kwekholomu ye-Ascentis Express RP-Amide. Womabili ama-peptide namaprotheni kwabonwa kuxazululwe kahle futhi kusebenza 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 (MC), i-Styrene, i-4-Hydroxy-TEMPO, i-Benzoyl Peroxide (BPO), i-HPLC Grade Acetonitrile (ACN), i-Methanol, i-2-Propanol, kanye ne-Acetone Kuthengwe kwa-Sigma-Aldrich (St. Louis, MO, 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, kwabe sekufakwa i-24 mL ye-TMOS ngaphansi kwezimo ezibandayo. Ingxube yokusabela yashiswa ku-40°C amahora ayi-6 kwabe sekuba ku-120°C amahora ayi-8 ku-autoclave yensimbi engagqwali. Amanzi athululwa kwathi okusele komiswa ku-70°C amahora ayi-12. Isisindo esithambile esomile sagaywa kahle kuhhavini futhi safakwa ku-calcium ku-550°C amahora ayi-12. Kwalungiswa amaqoqo amathathu futhi kwachazwa ukuhlola ukuphindaphindeka ngobukhulu bezinhlayiya, usayizi wembobo kanye nendawo engaphezulu.
Ngokushintsha ubuso bezinhlayiya ze-silica nge-ligand phenylmaleimide-methylvinylisocyanate (PCMP) ehlanganiswe ngaphambilini elandelwa yi-radial polymerization ene-styrene, kwalungiswa i-compound equkethe iqembu le-polar. Isigaba sokuma sama-aggregates namaketanga e-polystyrene. Inqubo yokulungiselela ichazwe ngezansi.
I-N-phenylmaleimide (200 mg) kanye ne-methyl vinyl isocyanate (100 mg) zancibilikiswa ku-toluene eyomile, kwathi i-0.1 mL ye-2,2′-azoisobutyronitrile (AIBN) yanezelwa eflaskini yokusabela ukuze kulungiselelwe i-phenylmaleimide-methyl vinyl isocyanate copolymer (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 ku-sonic ebhodleleni elingezansi eliyindilinga elingu-500 mL imizuzu eyi-10. I-PMCP (10 mg) yancibilikiswa ku-toluene futhi yanezelwa ngokuwa phansi ebhodleleni lokusabela nge-funnel ewayo. Ingxube yahlungwa kabusha ku-100°C amahora ayi-8, yahlungwa futhi yagezwa nge-acetone futhi yomiswa ku-60°C amahora ama-3. Ngemuva kwalokho, izinhlayiya ze-silica eziboshwe yi-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 ezinamathiselwe ku-TEMPO-PMCP (1.5 g) zahlakazeka ku-toluene futhi zahlanzwa nge-nitrogen. Ukupholishwa kwe-styrene kwenziwa ku-100°C amahora ayi-12. Umkhiqizo ophumayo wagezwa nge-methanol futhi womiswa ku-60°C ubusuku bonke. Uhlelo lokusabela oluphelele luboniswe kuMfanekiso 1.
Amasampula akhishwa ugesi ku-393 K ihora eli-1 ukuze kutholakale ingcindezi eseleyo engaphansi kuka-10-3 Torr. Inani le-N2 elifakwe kungcindezi elinganiselwe ye-P/P0 = 0.99 lasetshenziswa ukuthola ivolumu ephelele yembobo. Isimo sezinhlayiya ze-silica ezingenalutho nezihlanganisiwe zahlolwa nge-scanning electron microscopy (Hitachi High Technologies, Tokyo, Japan). Amasampula omisiwe (izinhlayiya ze-silica ezingenalutho nezihlanganisiwe ze-ligand) abekwa kukholomu ye-aluminium kusetshenziswa i-adhesive carbon tape. Igolide labekwa kumasampula kusetshenziswa i-Q150T sputter coater, kwathi ungqimba lwe-5 nm Au lwafakwa kumasampula. Lokhu kuthuthukisa ukusebenza kahle kwenqubo kusetshenziswa ama-voltage aphansi futhi kunikeza ukusanhlamvu okuhle, okubandayo. I-Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental analyzer yasetshenziswa ekuhlaziyeni kwezinto. I-Malvern (Worcestershire, UK) Mastersizer 2000 particle size analyzer yasetshenziswa ukuthola ukusatshalaliswa kosayizi wezinhlayiya. I-Naked silica Izinhlayiya kanye nezinhlayiya ze-ligand-bonded silica (5 mg ngayinye) zahlakazeka ku-5 mL ye-isopropanol, zafakwa i-sonicated imizuzu eyi-10, zavuthuzwa imizuzu emi-5, zabekwa ebhentshini elibonakalayo le-Masterizer. Ukuhlaziywa kwe-thermogravimetric kwenziwa ngesilinganiso esingu-5 °C ngomzuzu ngaphezu kobubanzi bokushisa obungu-30 kuya ku-800 °C.
Amakholomu ensimbi engagqwali ambozwe ngengilazi anobukhulu obuncane (100 × 1.8 mm id) apakishwa kusetshenziswa indlela yokupakisha i-slurry, kusetshenziswa inqubo efanayo esetshenziswe ku-Ref. 31. Ikholomu yensimbi engagqwali (efakwe ingilazi, engu-100 × 1.8 mm id) ene-outlet fitting equkethe i-1 µm frit ixhunywe ku-slurry packer (Alltech Deerfield, IL, USA). Lungisa i-slurry yesigaba esingaguquki ngokufaka u-150 mg wesigaba esingaguquki ku-1.2 mL ye-methanol bese uyithumela kukholomu yokugcina. I-Methanol yasetshenziswa njenge-slurry solvent kanye ne-propelling solvent. Gcwalisa ikholomu ngokulandelana ngokusebenzisa ukucindezela okungu-100 MP imizuzu eyi-10, okungu-80 MP imizuzu eyi-15, kanye no-60 MP imizuzu engama-30. Ngesikhathi sokupakisha, ukudlidliza komshini kwasetshenziswa ama-GC column shakers amabili (Alltech, Deerfield, IL, USA) ukuqinisekisa ukupakisha okufanayo kwekholomu. Vala i-slurry packer bese ukhulula ingcindezi kancane ukuze uvimbele noma yimuphi umonakalo ngaphakathi kwekholomu. Nqamula ikholomu kuyunithi yokupakisha i-slurry bese uxhuma okunye ukulingana ku-inlet kanye nohlelo lwe-LC ukuze uhlole ukuthi ukusebenza.
Iphampu ye-LC (10AD Shimadzu, eJapan), i-injector (Valco (USA) C14 W.05) ene-injection loop engu-50nL, i-membrane degasser (Shimadzu DGU-14A), ifasitela le-capillary le-UV-VIS lakhiwe. I-Special µLC device device device (UV-2075) kanye nama-microcolumns agcwele ingilazi. Sebenzisa ipayipi elincane kakhulu nelifushane lokuxhuma ukuze unciphise umphumela wokwandiswa kwebhendi yekholomu eyengeziwe. Ngemva kokupakisha, ama-capillary (50 μm id 365 kanye nama-capillary e-union ancishisiwe (50 μm) afakwe endaweni yokuphuma engu-1/16″ ye-reducing union. Ukuqoqwa kwedatha kanye nokucubungula i-chromatographic kwenziwa kusetshenziswa isofthiwe ye-Multicchro 2000. Ukuqapha kuma-Analytes angu-254 nm kwahlolwa ukumuncwa kwe-UV. Idatha ye-Chromatographic yahlaziywa yi-OriginPro8 (Northampton, MA).
I-Albumin evela ku-serum yomuntu, i-lyophilized powder, ≥ 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 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 kwezingxube ze-peptide kanye nokugaya kwe-trypsin ye-HSA kuhlolwe ngokwehlukana kumakholomu e-PMP.Hlola ukuhlukaniswa kwengxube ye-peptide kanye nokugaya kwe-trypsin kwe-HSA ngekholamu ye-PMP bese uqhathanisa imiphumela nekholomu ye-Ascentis Express RP-Amide. Inombolo yepuleti yethiyori ibalwa kanje:
Izithombe ze-SEM zezinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-ligand-bonded silica ziboniswe ku-FIG. 2. Izithombe ze-SEM zezinhlayiya ze-silica ezingenalutho (A, B) zibonisa ukuthi, ngokungafani nezifundo zethu zangaphambilini, lezi zinhlayiya ziyindilinga lapho izinhlayiya zinde khona noma zinokulingana okungajwayelekile. Ubuso bezinhlayiya ze-silica ezixhunyiwe yi-ligand (C, D) bubushelelezi kunobo bezinhlayiya ze-silica ezingenalutho, okungenzeka ukuthi kungenxa yokumbozwa kwamaketanga e-polystyrene ebusweni bezinhlayiya ze-silica.
Ukuskena izithombe ze-electron microscope zezinhlayiya ze-silica ezingenalutho (A, B) kanye nezinhlayiya ze-silica eziboshwe yi-ligand (C, D).
Ukusatshalaliswa kosayizi wezinhlayiya zezinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-ligand-bonded silica kukhonjisiwe ku-Figure 3(A). Ama-curve okusabalalisa ubukhulu bezinhlayiya asekelwe ku-volume abonise ukuthi ubukhulu bezinhlayiya ze-silica bukhuphuke ngemva kokuguqulwa kwamakhemikhali (Isithombe 3A). Idatha yokusatshalaliswa kosayizi wezinhlayiya zezinhlayiya ze-silica kusukela ocwaningweni lwamanje kanye nocwaningo lwangaphambilini iqhathaniswa kuThebula 1(A). Usayizi wezinhlayiya ezisekelwe ku-volume, d(0.5), we-PMP ungu-3.36 μm, uma kuqhathaniswa nocwaningo lwethu lwangaphambilini olunenani le-ad(0.5) elingu-3.05 μm (izinhlayiya ze-silica eziboshwe nge-polystyrene)34. Leli qoqo lalinokusatshalaliswa kosayizi wezinhlayiya okuncane uma kuqhathaniswa nocwaningo lwethu lwangaphambilini ngenxa yezilinganiso ezihlukene ze-PEG, i-urea, i-TMOS, kanye ne-acetic acid engxubeni yokusabela. Usayizi wezinhlayiya zesigaba se-PMP mkhulu kancane kunowesigaba sezinhlayiya ze-silica eziboshwe nge-polystyrene esasifunde ngaphambilini. Lokhu kusho ukuthi ukusebenza kobuso bezinhlayiya ze-silica nge-styrene kubeke kuphela ungqimba lwe-polystyrene (0.97 µm) ku- ubuso be-silica, kanti esigabeni se-PMP ubukhulu besendlalelo babungu-1.38 µm.
Ukusatshalaliswa kosayizi wezinhlayiya (A) kanye nokusatshalaliswa kosayizi wezinhlayiya (B) zezinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-silica eziboshwe yi-ligand.
Usayizi wembobo, ivolumu yembobo kanye nendawo engaphezulu yezinhlayiya ze-silica zocwaningo lwamanje kunikezwe kuThebula 1(B). Amaphrofayili e-PSD ezinhlayiya ze-silica ezingenalutho kanye nezinhlayiya ze-ligand-bonded silica aboniswe kuThebula 3(B). Imiphumela iyafana nocwaningo lwethu lwangaphambilini. Ubukhulu bembobo yezinhlayiya ze-silica ezingenalutho kanye neziboshwe yi-ligand bungu-310 no-241, ngokulandelana, okubonisa ukuthi usayizi wembobo uncipha ngo-69 ngemva kokuguqulwa kwamakhemikhali, njengoba kuboniswe kuThebula 1(B), kanti ushintsho lwejika luboniswe kuThebula 3(B). Ngokufanayo, ivolumu yembobo yezinhlayiya ze-silica yehlile kusuka ku-0.67 kuya ku-0.58 cm3/g ngemva kokuguqulwa kwamakhemikhali. Indawo ethile ebusweni bezinhlayiya ze-silica ezifundwayo njengamanje ingu-116 m2/g, okulingana nocwaningo lwethu lwangaphambilini (124 m2/g). Njengoba kuboniswe kuThebula 1(B), indawo engaphezulu (m2/g) yezinhlayiya ze-silica nayo yehlile kusuka ku-116 m2/g kuya ku-105 m2/g ngemva kokuguqulwa kwamakhemikhali.
Imiphumela yokuhlaziywa kwezinto eziyisisekelo zesigaba esimile iboniswe kuThebula 2. Umthwalo wekhabhoni wesigaba esimile samanje ungu-6.35%, okuphansi kunomthwalo wekhabhoni wesifundo sethu sangaphambilini (izinhlayiya ze-silica eziboshwe nge-polystyrene, 7.93%35 kanye no-10.21%, ngokulandelana) 42. Umthwalo wekhabhoni wesigaba esimile samanje uphansi, Ngoba ekulungiseleleni i-SP yamanje, ngaphezu kwe-styrene, kwasetshenziswa amanye ama-ligand e-polar njenge-phenylmaleimide-methylvinylisocyanate (PCMP) kanye ne-4-hydroxy-TEMPO. Iphesenti lesisindo se-nitrogen sesigaba esimile samanje lingu-2.21%, uma kuqhathaniswa no-0.1735 kanye no-0.85% ngesisindo se-nitrogen ezifundweni zangaphambilini, ngokulandelana. Lokhu kusho ukuthi i-wt % ye-nitrogen iphakeme esigabeni esimile samanje ngenxa ye-phenylmaleimide. Ngokufanayo, umthwalo wekhabhoni wemikhiqizo (4) kanye no-(5) wawungu-2.7% kanye no-2.9%, ngokulandelana, kuyilapho ukulayishwa kwekhabhoni komkhiqizo wokugcina (6) kwakungu-6.35%, njengoba kuboniswe kuThebula 2. Ukwehla kwesisindo kuhlolwe ngesigaba se-PMP esingaguquki, kanti ijika le-TGA liboniswe kuMfanekiso 4. Ijika le-TGA libonisa ukwehla kwesisindo okungu-8.6%, okuvumelana kahle nokulayishwa kwekhabhoni (6.35%) ngoba ama-ligand aqukethe hhayi u-C kuphela kodwa ne-N, O, kanye ne-H.
I-ligand ye-phenylmaleimide-methylvinylisocyanate yakhethwa ukuze kuguqulwe ubuso bezinhlayiya ze-silica ngoba inamaqembu e-polar phenylmaleimide namaqembu e-vinylisocyanate. Amaqembu e-Vinyl isocyanate angasabela kakhulu nge-styrene ngokupholisa okunamandla. Isizathu sesibili ukufaka iqembu elinokuxhumana okulinganiselwe ne-analyte futhi elingenakho ukusebenzisana okuqinile kwe-electrostatic phakathi kwe-analyte 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 futhi singashintsha i-polarity yesigaba esimile. Ukuhlaziywa kwezinto kwenziwa ukuhlola umthwalo we-carbon walezi zigaba ezimile. Kwabonwa ukuthi ukwandisa inani le-styrene kanye nesikhathi sokusabela kwandisa umthwalo we-carbon wesigaba esimile kanye nokuphikisana nalokho. Ama-SP alungiselelwe ngamanani ahlukene e-styrene anemithwalo ye-carbon ehlukene. Futhi, faka lezi zigaba ezimile zibe yinsimbi engagqwali. amakholomu bese uhlola ukusebenza kwawo kwe-chromatographic (ukukhetha, isixazululo, inani le-N, njll.). Ngokusekelwe kulezi zivivinyo, kukhethwe indlela elungiselelwe ukulungiselela isigaba se-PMP esimile ukuqinisekisa i-polarity elawulwayo kanye nokugcinwa okuhle kwe-analyte.
Izingxube ezinhlanu ze-peptide (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-leucine enkephalin) nazo zahlolwa kusetshenziswa ikholomu ye-PMP kusetshenziswa isigaba esihambayo; I-acetonitrile engu-60/40 (v/v)/amanzi (0.1% TFA) ngesilinganiso sokugeleza esingu-80 μL/min. Ngaphansi kwezimo ezifanele zokususa, inombolo yepuleti yethiyori (N) ngekholomu ngayinye (100 × 1.8 mm id) ingu-20,000 ± 100 (amapuleti angu-200,000/m²). Ithebula 3 linikeza amanani e-N amakholomu amathathu e-PMP kanye nama-chromatogram aboniswe kuMfanekiso 5A. Ukuhlaziywa okusheshayo kwikholomu ye-PMP ngesilinganiso sokugeleza okuphezulu (700 μL/min), ama-peptide amahlanu asuswa kungakapheli umzuzu owodwa, amanani e-N ayemahle kakhulu, angu-13,500 ± 330 ngekholomu ngayinye (100 × 1.8 mm id), Ahambisana namapuleti angu-135,000/m (Umfanekiso 5B). Amakholomu amathathu anosayizi ofanayo (100 × 1.8 mm id) agcwele izingxenye ezintathu ezahlukene zesigaba se-PMP esingaguquki ukuze kuhlolwe. ukuphindaphindwa. Ukuhlushwa kwe-analyte kwekholomu ngayinye kuqoshwe kusetshenziswa izimo ezifanele zokuxazulula kanye nenani lamapuleti e-theory N kanye nesikhathi sokugcina ukuze kuhlukaniswe ingxube efanayo yokuhlola kukholamu ngayinye. Idatha yokuphindaphindwa kwamakholomu e-PMP iboniswe kuThebula 4. Ukuphindaphindwa kwekholomu ye-PMP kuhlobene 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 esiphathwayo 60/40 ACN/H2O (TFA 0.1%), ubukhulu bekholomu ye-PMP (100 × 1.8 mm id); ukuhlaziya Ukuhleleka kokulungiswa kwama-compounds: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) kanye ne-5 (leucine) acid enkephalin)).
Ikholomu ye-PMP (100 × 1.8 mm id) ihlolwe ukuhlukaniswa kokugaya kwe-tryptic kwe-albumin ye-serum yomuntu ku-chromatography yoketshezi esebenza kahle kakhulu. I-chromatogram ku-Figure 6 ikhombisa ukuthi isampula ihlukaniswe kahle futhi isinqumo sihle kakhulu. Ukugaya kwe-HSA kuhlaziywe kusetshenziswa izinga lokugeleza elingu-100 µL/min, isigaba esihambayo esingu-70/30 acetonitrile/amanzi kanye no-0.1% TFA. Njengoba kuboniswe ku-chromatogram (Figure 6), ukugaya kwe-HSA kuhlukaniswe ngama-peaks angu-17 ahambisana nama-peptides angu-17. Ukusebenza kahle kokuhlukanisa kwe-peak ngayinye ku-chromatography ye-HSA kubalwe futhi amanani anikezwe kuThebula 5.
Ukwehliswa kwe-tryptic kwe-HSA (100 × 1.8 mm id) kwahlukaniswa kukholomu ye-PMP; 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 yokubuyela emuva kwekholomu, kanti u-u uyijubane eliqondile lesigaba esihambayo. Ukuvuleka kwekholomu ye-PMP kwakungu-2.5 × 10-14 m2, izinga lokugeleza lalingu-25 μL/min, kanti kusetshenziswe i-ACN/amanzi angu-60/40 v/v. Ukuvuleka kwekholomu ye-PMP (100 × 1.8 mm id) kwakufana nokwesifundo sethu sangaphambilini. I-Ref.34. Ukuvuleka kwekholomu egcwele izinhlayiya ezingenazo izimbobo: 1.7 × 10-15 ngezinhlayiya ezingu-1.3 μm, 3.1 × 10-15 ngezinhlayiya ezingu-1.7 μm, 5.2 × 10-15 kanye no-2.5 ​​× 10-14 m2 ngezinhlayiya ezingu-2.6 μm. Kuzinhlayiya ezingu-5 μm 43. Ngakho-ke, ukuvuleka kwesigaba se-PMP kufana nokwezinhlayiya ezingu-5 μm zegobolondo eliyinhloko.
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 yamakholomu e-SILICA PARTICLES-C18 (100 × 1.8 mm id) amakholomu angu-34 kanye ne-C18-Urea 31 esiwafundile ngaphambilini ayengu-0.63 kanye no-0.55, ngokulandelana. Lokhu kusho ukuthi ukuba khona kwama-urea ligands kunciphisa ukuvuleka kwesigaba esimile. Ngakolunye uhlangothi, i-porosity iyonke yekholomu ye-PMP (100 × 1.8 mm id) ingu-0.60. Ukuvuleka kwamakholomu e-PMP kuphansi kunokwamakholomu agcwele izinhlayiya ze-silica eziboshwe yi-C18 ngoba ezigabeni ezimile zohlobo lwe-C18 ama-ligands e-C18 anamathele ezinhlayiyeni ze-silica njengezintambo eziqondile, kuyilapho ezigabeni ezimile zohlobo lwe-polystyrene, i-A iwugqinsi kakhulu. ungqimba lwe-polymer lwakhiwe nxazonke. Ekuhlolweni okuvamile, i-porosity yekholomu ibalwa kanje:
Umfanekiso 7A, B ukhombisa ikholomu ye-PMP (100 × 1.8 mm id) kanye nekholomu ye-Ascentis Express RP-Amide (100 × 1.8 mm id) kusetshenziswa izimo ezifanayo zokuluthwa (okungukuthi, i-60/40 ACN/H2O kanye ne-0.1% TFA). ) yesakhiwo se-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) zalungiswa ngo-20 µL/ Izinga lokugeleza eliphansi lamakholomu womabili lingu-800 µL/min. Amanani aphansi e-HETP ngesilinganiso sokugeleza esifanele (80 µL/min) sekholomu ye-PMP kanye nekholomu ye-Ascentis Express RP-Amide ayengu-2.6 µm kanye no-3.9 µm, ngokulandelana. Amanani e-HETP abonisa ukuthi ukusebenza kahle kokuhlukaniswa kwekholomu ye-PMP (100 × 1.8 mm id) kungcono kakhulu kunekholomu ye-Ascentis Express RP-Amide etholakala kwezentengiselwano (100 × 1.8 mm id). I-van Deemter plot ku-Fig. 7(A) ikhombisa ukuthi ukwehla kwenani le-N ngokugeleza okwandayo akubalulekile uma kuqhathaniswa nesifundo sethu sangaphambilini. Ukusebenza kahle kokuhlukaniswa okuphezulu kwekholomu ye-PMP (100 × 1.8 mm id) uma kuqhathaniswa nekholomu ye-Ascentis Express RP-Amide isekelwe ekuthuthukisweni kwesimo sezinhlayiya, usayizi, kanye nezinqubo eziyinkimbinkimbi zokupakisha amakholomu ezisetshenziswa emsebenzini wamanje34.
(A) i-van Deemter plot (i-HETP vs. ijubane eliqondile lesigaba esiphathwayo) etholwe kusetshenziswa ikholomu ye-PMP (100 × 1.8 mm id) ku-60/40 ACN/H2O ene-0.1% TFA.(B) i-van Deemter plot (ijubane eliqondile lesigaba esiphathwayo) etholwe kusetshenziswa ikholomu ye-Ascentis Express RP-Amide (100 × 1.8 mm id) ku-60/40 ACN/H2O ene-0.1% TFA.
Isigaba esimile se-polystyrene esifakwe nge-polar salungiswa futhi sahlolwa ukuze kuhlukaniswe izingxube ze-peptide zokwenziwa kanye nokugaya kwe-trypsin kwe-albumin ye-serum yomuntu (HAS) ku-chromatography yoketshezi esebenza kahle kakhulu. Ukusebenza kwe-chromatographic kwamakholomu e-PMP ezingxube ze-peptide kuhle kakhulu ekusebenzeni kahle nasekuxazululeni ukuhlukana. Ukusebenza okuthuthukisiwe kokuhlukaniswa kwamakholomu e-PMP kungenxa yezizathu ezahlukahlukene, njengosayizi wezinhlayiya kanye nosayizi we-pore wezinhlayiya ze-silica, ukuhlanganiswa okulawulwayo kwesigaba esimile, kanye nokupakisha okuyinkimbinkimbi kwamakholomu. Ngaphezu kokusebenza kahle kokuhlukana okuphezulu, ingcindezi ephansi emuva kwekholomu ngamazinga aphezulu okugeleza kungenye inzuzo yalesi sigaba esimile. Amakholomu e-PMP abonisa ukuphindaphindeka okuhle futhi angasetshenziswa ekuhlaziyeni izingxube ze-peptide kanye nokugaya kwe-trypsin kwamaprotheni ahlukahlukene. Sihlose ukusebenzisa le kholomu ukuze kuhlukaniswe imikhiqizo yemvelo, amakhemikhali asebenzayo ezitshalweni zokwelapha kanye nezingcezu ze-fungus ku-chromatography yoketshezi. Esikhathini esizayo, amakholomu e-PMP azohlolwa futhi ukuze kuhlukaniswe amaprotheni kanye nama-antibodies e-monoclonal.
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