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Iingqungquthela ze-silica ezinobuncwane zalungiswa ngendlela ye-sol-gel kunye nokuguqulwa okuthile ukufumana i-macroporous particles.Ezi ngqungquthela zithathwe ngokubuyisela ukuguqulwa kwe-reversible fragmentation chain transfer (RAFT) i-polymerization kunye ne-N-phenylmaleimide-methylvinylisocyanate (PMI) kunye ne-styrene ukulungiselela i-N-phenylmaleide yensimbi ye-polymaleimide ye-polymer I-s (i-100 × 1.8 mm id) yayipakishwe ngokupakisha i-slurry.I-Vavaluated PMP ikholamu ukuhlukana komxube we-peptide oqukethe i-peptide emihlanu (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arnkeg, i-albhamu ye-leucineps ye-leucineps ye-leucineps ye-leucineps ye-albhamu ye-leucineps ye-try ye-leucineps kunye ne-try ye-leucineps ye-try) .Ngaphantsi kweemeko ezifanelekileyo ze-elution, i-theory plate count yomxube we-peptide iphezulu njenge-280,000 plates/m².Ukuthelekisa ukusebenza kokwahlulwa kwekholamu ephuhlisiweyo kunye nekholamu yorhwebo ye-Ascentis Express RP-Amide, kwaphawulwa ukuba ukusebenza kokwahlula kwekholamu ye-PMP kwakungaphezulu kwekholamu yorhwebo ngokwemigaqo yokwahlula kunye nesisombululo.
Kwiminyaka yakutshanje, umzi-mveliso we-biopharmaceutical uye waba lurhwebo olukhulayo lwehlabathi ngokunyuka okukhulu kwisabelo semarike.Ngokukhula okugqabhukileyo kweshishini le-biopharmaceutical1,2,3, uhlalutyo lweepeptides kunye neeproteni lunqweneleka kakhulu. ulwelo lomzimba, izicubu kunye neeseli ngumsebenzi onzima kakhulu ngenxa yenani elikhulu leentlobo ezinokuthi zibonwe kwisampulu enye.Nangona i-mass spectrometry sisixhobo esisebenzayo sokulandelelana kwe-peptide kunye neprotheyini, ukuba iisampuli ezinjalo zifakwe kwi-spectrometer yobunzima kwipasi enye, ukwahlukana akuyi kuba yinto efanelekileyo.Le ngxaki inokuncitshiswa ngokuphumeza uhlalutyo lwe-MSLC ye-mass paraography (ukunciphisa inani le-MS romat), ukuhlalutya kwe-MS romat ngaphambi kokunciphisa ulwelo. i-spectrometer ngexesha elinikiweyo4,5,6.Ukongezelela, ngexesha lokwahlulwa kwesigaba solwelo, abahlalutyi banokujoliswa kwimimandla emxinwa, ngaloo ndlela bagxininise aba bahlalutyi kunye nokuphucula uvakalelo lwe-MS.
I-chromatography yenqanaba le-Promatography (i-RP-LC) isetyenziselwa ukuhlanjululwa kunye nokulwa umxube we-Peptide (i-ODS) njengesakhiwo se-octadecyl esiyi-14,15. Iiproteni ze-petives kunye neeproteni zePolar kunye ne-Poralar Tranch yokusebenzisana kwaye zigcine unxibelelwano lwe-RP-LC ye-Promarides, kwaye i-Promering Peods I-SPRORDE isetyenziselwe ukwahlula-hlulwa kwi-Poptide kunye neProten R77,18, I-19 ,2,21.21 Ngohlalutyo lwePolar kunye no-On-Polilar, njengoko ukwahlulwa kuxhomekeke kwintsebenzo phakathi kwehlalutyo kunye nenqanaba lesitali.Ukusebenzisana kwe-Multimodal 29, 30, 31, 32.Kutshanje, uZhang et al.I-30 ilungiselele i-dodecyl-terinated polyamine isigaba sokumisa kwaye yahlula ngempumelelo i-hydrocarbons, i-antidepressants, i-flavonoids, i-nucleosides, i-estrogens, kunye nezinye i-analyte ezininzi.I-polar intercalator inamaqela amabini angama-polar kunye nangekho-polar, ngoko ingasetyenziselwa ukwahlula i-peptides kunye neeprotheni ezine-hydrophobic kunye ne-hydrophilicembed, i-hydrophilic-e-hydrophilicembed, i-polar-polar, i-hydrophilicembed, i-polar-8. ikholamu) zifumaneka ngokurhweba phantsi kwegama lorhwebo Ascentis Express RP-Amide ikholamu, kodwa ezi kholamu zisetyenziselwa uhlalutyo lwe-amine 33 kuphela.
Kuphononongo lwangoku, i-polar-embedded stationary phase (i-N-phenylmaleimide-embedded polystyrene) ilungiselelwe kwaye ivavanywe ukuhlukana kwe-peptides kunye ne-trypsin digests ye-HSA.Isigaba sokumisa salungiswa ngokusebenzisa isicwangciso esilandelayo.Iincinci ze-silika ze-silika zilungiselelwe ngokwenkqubo enikwe kwipapasho yethu yangaphambili kunye nolunye uhlengahlengiso. I-asidi ye-cetic yahlengahlengiswa ukulungiselela i-silica particles kunye nobukhulu obukhulu be-pore.Okwesibini, i-ligand entsha, i-phenylmaleimide-methyl vinyl isocyanate, yaxutywa kwaye isetyenziselwa ukukhupha i-silica particles ukulungiselela i-polar embedded stationary phase.Isigaba se-stationary esibangelwayo sifakwe kwikholamu ye-stainless steel packing kunye ne-packing i-100 mm i-packing i-packing i-100 mm. ukungcangcazela koomatshini ukuqinisekisa ukuba ibhedi ene-homogeneous yenziwa ngaphakathi kwikholamu.Vavaluya ukwahlulwa kwekholamu epakishiweyo yemixube yepeptide equka iipeptides ezintlanu;(I-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-Leucine Enkephalin) kunye ne-Trypsin digest ye-albumin ye-serum yabantu (HAS) .Umxube we-peptide kunye ne-trypsin digest ye-HSA yabonwa ukuba yahlula kunye nesisombululo esihle kunye nokusebenza kakuhle kwekholamu ye-AMP. i-peptides kunye neeprotheni zabonwa ukuba zixazululwe kakuhle kwaye zisebenza kakuhle kwikholamu ye-PMP, eyayisebenza ngakumbi 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-Albumin ye-Human Serum (HSA), i-Ammonium Chloride, i-Urea, i-Hexane Methyldisilazane (HMDS), i-Methacryloyl Chloride, i-Benzol-Chloride (MC), i-Perzoydroksidi, i-Mc, i-Benzol, i-Mc. I-HPLC yeBanga le-Acetonitrile (ACN), i-Methanol, i-2-Propanol, kunye ne-Acetone Ethengwe kwi-Sigma-Aldrich (eSt. Louis, MO, USA).
Umxube we-urea (8 g), i-polyethylene glycol (8 g), kunye ne-8 mL ye-0.01 N acetic acid yavuselelwa imizuzu eyi-10, kwaye i-24 mL ye-TMOS yongezwa phantsi kweemeko ezibandayo ze-ice. I-70 ° C kwiiyure ze-12. Ubunzima obomileyo obuthambileyo bungumhlaba ococekileyo kwi-oven kunye ne-calcined kwi-550 ° C kwiiyure ze-12. Iibhakethi ezintathu zilungiselelwe kwaye zibonakaliswe ukuba zihlolisise ukuveliswa kwakhona kwi-particle size, ubukhulu bepore kunye nommandla ophezulu.
Ngokuguqulwa komphezulu we-silica particles kunye ne-ligand yangaphambili ye-phenylmaleimide-methylvinylisocyanate (PCMP) elandelwa yi-polymerization ye-radial kunye ne-styrene, i-polar equkethe iqela eliqulethwe lilungiswe.Isigaba esimileyo se-aggregates kunye ne-polystyrene chain.Inkqubo yokulungiselela ichazwe ngezantsi.
I-N-phenylmaleimide (200 mg) kunye ne-methyl vinyl isocyanate (i-100 mg) yachithwa kwi-toluene eyomileyo, kwaye i-0.1 mL ye-2,2'-azoisobutyronitrile (AIBN) yongezwa kwi-flask yokuphendula ukulungiselela i-phenylmaleimide-methyl vinyl isocyanate copolymer eyomileyo kwiiyure ze-3 ze-CP ° kunye ne-3 PM ° yokucoca i-copolymer. i-oven kwi-40 ° C ngeeyure ezi-3.
Iincinci ze-silica ezomileyo (i-2 g) zihlakazwe kwi-toluene eyomileyo (i-100 mL), ixutywe kwaye i-sonicated kwi-flask ye-500 mL ejikelezayo ephantsi kwe-10 min.PMCP (10 mg) yachithwa kwi-toluene kwaye yongezwa i-dropwise kwi-flask yokuphendula nge-funnel yokulahla.Umxube wawuxutywe kunye ne-refluxed kwi-800 ° C kunye ne-100 ° C yokucoca kunye ne-100 ° C yokucoca ithoni kunye ne-6 ° C. Emva koko, iinqununu ze-silica ezidibeneyo ze-PMCP (i-100 g) zachithwa kwi-toluene (200 ml) kunye ne-4-hydroxy-TEMPO (2 mL) yongezwa phambi kwe-100 µL ye-dibutyltin dilaurate njenge-catalyst.Umxube waxutywa kwi-50 ° C kwaye womiswa kwiiyure ze-3 °, i-5 yeeyure ze-5.
I-Styrene (1 mL), i-benzoyl peroxide BPO (0.5 mL), kunye ne-TEMPO-PMCP-eqhotyoshelweyo i-silica particles (1.5 g) yachithwa kwi-toluene kwaye yahlanjululwa nge-nitrogen.I-polymerization ye-styrene yenziwa kwi-100 ° C kwiiyure ze-12. Imveliso efunyenweyo ihlanjwe nge-methanol ye-methanol kunye ne-reactivity yomiswe ngobusuku bonke.
Iisampuli zachithwa kwi-393 K ngeyure ye-1 ukufumana uxinzelelo olushiyekileyo olungaphantsi kwe-10-3 Torr. Isixa se-N2 esibhengezwe kuxinzelelo olusondeleyo lwe-P / P0 = 0.99 yayisetyenziselwa ukugqiba umthamo we-pore. (i-silica engenanto kunye ne-ligand-bonded silica particles) yafakwa kwikholamu ye-aluminium usebenzisa i-adhesive carbon tape.Igolide yafakwa kwiisampuli usebenzisa i-Q150T i-sputter coater, kunye ne-5 nm Au layer yafakwa kwiisampuli.Oku kuphucula ukusebenza kwenkqubo ngokusebenzisa i-voltages ephantsi kwaye inikezela ngengqolowa ecocekileyo, i-Flash sputtering, i-USA1 i-Eleham1 i-Eleham1 i-USA1 i-element ebandayo ye-Eleham1 i-USA1 i-Eleham1 i-element ye-Flash. isetyenziselwa uhlalutyo lwe-elemental.I-Malvern (i-Worcestershire, e-UK) I-Mastersizer i-2000 ye-particle size analyzer isetyenziselwe ukufumana ukusabalalisa ubungakanani be-particle.I-Silica particles kunye ne-ligand-bonded silica particles (5 mg nganye) yahlakazwa kwi-5 mL ye-isopropanol, i-sonicated ye-10 min, i-vortexed, i-benchuptical benched kwi-5 ye-master bench efakwe kwi-bench ye-master kwenziwa ngesantya se-5 °C ngomzuzu kwiqondo lobushushu elingama-30 ukuya kuma-800 °C.
I-glass-lined stainless steel stainless-bore columns of dimensions (100 × 1.8 mm id) yayipakishwe kusetyenziswa indlela yokupakisha i-slurry, isebenzisa inkqubo efanayo esetyenziswe kwi-Ref.I-31.Ikholamu yensimbi engenasici (i-glass-lined, i-100 × 1.8 mm id) kunye ne-outlet fitting ene-1 µm frit yayiqhagamshelwe kwi-slurry packer (Alltech Deerfield, IL, USA) Lungiselela i-slurry yesigaba esimileyo ngokumisa i-150 mg yesigaba esimileyo se-mL esetyenzisiweyo kwi-menotha yokugcina i-menotha kwi-menotha esetyenzisiweyo. I-solvent ye-slurry kunye ne-solvent ejikelezayo.Gcwalisa ikholamu ngokulandelelana ngokufaka uxinzelelo lwe-MP ye-100 imizuzu eyi-10, i-80 MP yemizuzu ye-15, kunye ne-60 MP yemizuzu ye-30. Ngexesha lokupakisha, ukungcangcazela komatshini kwasetyenziswa kunye neengqungquthela ezimbini zekholomu ze-GC (Alltech, Deerfield, IL ukupakisha i-uniform ye-column i-il, i-U. ikholomu.Nqamula ikholamu kwiyunithi yokupakisha i-slurry kwaye udibanise enye i-fitting to inlet kunye ne-LC system ukujonga ukusebenza kwayo.
Impompo yeLC (10AD Shimadzu, Japan), injector (Valco (USA) C14 W.05) ene-50nL injection loop, i-membrane degasser (Shimadzu DGU-14A), i-UV-VIS ifestile yecapillary yakhiwe Special µLC isixhobo somtshina (UV-2075) kunye neglasi-lined yokudibanisa i-microcolbins emfutshane ebanzi kunye ne-mini-lined yokudibanisa i-microcolbins emfutshane ebanzi. Ening.Emva kokupakishwa, ii-capillaries (50 μm id 365 kunye nokunciphisa i-union capillaries (50 μm) zafakelwa kwindawo ye-1/16″ yokuphuma komanyano olunciphisayo.Ukuqokelelwa kwedatha kunye nokusetyenzwa kwechromatographic kwenziwa kusetyenziswa isoftwe ye-Multichro 2000. Ukubeka iliso kwi-254 yedatha ye-Uvromatographic i-Absorption ye-UVtes ye-Chromatographic yovavanyo lwe-Chromatographic ye-Chromatographic vavanyo lwe-UVtes Northampton, MA).
I-Albumin esuka kwiserum yabantu, i-lyophilized powder, ≥ 96% (i-agarose gel electrophoresis) 3 mg exutywe ne-trypsin (1.5 mg), i-4.0 M urea (1 mL), kunye ne-0.2 M ye-ammonium bicarbonate (1 mL) .Isisombululo saxutywa ngemizuzu eyi-10 kwaye sigcinwe kwindawo yokuhlambela amanzi kwi-60% yeeyure ze-1. Hlunga isisombululo kwaye ugcine ngaphantsi kwe-4 °C.
Ukwahlulwa kwemixube ye-peptide kunye ne-HSA ye-trypsin digests yavavanywa ngokwahlukileyo kwiikholamu ze-PMP.Jonga ukuhlukana komxube we-peptide kunye ne-trypsin digest ye-HSA ngekholomu ye-PMP kwaye uthelekise iziphumo kwikholamu ye-Ascentis Express RP-Amide.Inombolo yeplate yethiyori ibalwa ngolu hlobo lulandelayo:
Imifanekiso ye-SEM yeengqungquthela ze-silica ezingenanto kunye ne-ligand-bonded silica particles iboniswe kwi-FIG.I-2 .Semm ye-Bare Sulica amasuntswana (A, B) abonisa ukuba, ngokuchasene nezifundo zethu zangaphambili, amasuntswana aselunxwemeni apho amasuntswana aseLigand-Bold) atsiba kwi-sulticals enobukhulu be-polystyrene kumphezulu wamasuntswana esilika.
Ukuskena imifanekiso ye-electron microscope ye-silica engenanto (A, B) kunye ne-ligand-bonded silica particles (C, D).
Ukusabalalisa ubungakanani bemilinganiselo ye-silica engenanto kunye ne-ligand-bonded silica particles kuboniswe kwi-Figure 3 (A). I-3.36 μm, xa kuthelekiswa nesifundo sethu sangaphambili kunye nexabiso le-ad (0.5) ye-3.05 μm (i-polystyrene-bound bound silica particles) 34. Le bhetshi yayinokusabalalisa ubungakanani be-particle encinci xa kuthelekiswa nesifundo sethu sangaphambili ngenxa yemilinganiselo eyahlukayo ye-PEG, urea, TMOS, kunye ne-acetic acid kwi-acetic acid kumxube wokuphendula i-particle ye-polysty-esiyifunde ngaphambili. .Oku kuthetha ukuba ukusebenza komphezulu wamasuntswana e-silica kunye ne-styrene kufakwe kuphela umaleko we-polystyrene (0.97 µm) kumphezulu we-silica, ngelixa kwisigaba se-PMP ubukhulu bemaleko yi-1.38 µm.
Ukusabalalisa ubungakanani be-particle (A) kunye nokusabalalisa ubungakanani bepore (B) yeengqungquthela ze-silica ezingenanto kunye ne-ligand-bound bound silica particles.
Ubungakanani be-pore, umthamo we-pore kunye nommandla wendawo yeengqungquthela ze-silica zophando lwangoku zinikezelwa kwiThebhile 1 (B) .Iiprofayili ze-PSD ze-silica ezingenanto kunye ne-ligand-bonded silica particles ziboniswe kuMzobo 3 (B) .Iziphumo zifaniswa nesifundo sethu sangaphambili. Ubungakanani buyancipha nge-69 emva kokuguqulwa kweekhemikhali, njengoko kuboniswe kwiThebhile 1 (B), kwaye ukuguqulwa kwejika kuboniswe kwi-Fig. 3 (B) . Ngokufanayo, umthamo we-pore we-silica particles wehla ukusuka kwi-0.67 ukuya kwi-0.58 cm3 / g emva kokuguqulwa kweekhemikhali. .Njengoko kuboniswe kwiThebhile 1 (B), indawo engaphezulu (m2 / g) ye-silica particles nayo yehla ukusuka kwi-116 m2 / g ukuya kwi-105 m2 / g emva kokuguqulwa kweekhemikhali.
Iziphumo zohlalutyo lwe-elemental yesigaba esimileyo ziboniswe kwiThebhile 2. Ukulayishwa kwekhabhoni yesigaba esimileyo sangoku yi-6.35%, engaphantsi kwekhabhoni yokulayishwa kwesifundo sethu sangaphambili (i-polystyrene edibeneyo ye-silica particles, i-7.93% 35 kunye ne-10.21%, ngokulandelanayo) 42. Ukulayishwa kwekhabhoni ye-polystyrene kwi-stationary yangoku, ukulungiswa kwe-spolar kwisigaba sangoku, i-SP, i-SP, ukulungiswa kwe-lical stationary kwi-polystyrene. I-end ezifana ne-phenylmaleimide-methylvinylisocyanate (PCMP) kunye ne-4-hydroxy-TEMPO zisetyenzisiwe.Ipesenti ye-nitrogen yesisindo senqanaba elikhoyo lokumisa liyi-2.21%, xa kuthelekiswa ne-0.1735 kunye ne-0.85% ngesisindo se-nitrogen kwizifundo zangaphambili, ngokulandelanayo.Oku kuthetha ukuba i-wt% ye-nitrogen i-miximilar imveliso ye-carbonnymilar yangoku, i-carbonnymilar yangoku iphezulu kwimveliso ye-carbonnymile. I-4) kunye ne-5) yayiyi-2.7% kunye ne-2.9%, ngokulandelanayo, ngelixa ukulayishwa kwekhabhoni yemveliso yokugqibela (6) yayingu-6.35%, njengoko kuboniswe kwiThebhile 2. Ukulahlekelwa kwesisindo kuhlolwe kunye nesigaba se-PMP esimileyo, kwaye i-curve ye-TGA iboniswe kwi-Figure 4.I-curve ye-TGA ibonisa ukulahlekelwa kwesisindo se-carbon ephethe i-5. kananjalo N, O, kunye noH.
I-phenylmaleimide-methylvinylisocyanate ligand yakhethwa ukuguqulwa kwendawo ye-silica particles kuba inamaqela e-polar phenylmaleimide kunye namaqela e-vinylisocyanate.Amaqela e-Vinyl isocyanate angaqhubela phambili asabelane ne-styrene ngokuphila kwe-polymerization e-radical.Isizathu sesibini kukufaka iqela elinobudlelwane obuphakathi kunye ne-analyte ye-analyte ye-analyte kunye ne-analyte ye-analyte ye-analyte kunye ne-analyte phase kunye ne-analyte ye-analyte. I-mide moiety ayinayo intlawulo ebonakalayo kwi-pH yesiqhelo.I-polarity yesigaba esimileyo sinokulawulwa yimali efanelekileyo ye-styrene kunye nexesha lokuphendula le-polymerization ye-radical yamahhala.Isinyathelo sokugqibela sokuphendula (i-polymerization yamahhala) ibaluleke kakhulu kwaye inokutshintsha i-polarity yesigaba esimileyo.Uhlalutyo lwe-Elemental lwenziwa ukujonga ukulayishwa kwekhabhoni yale nqanaba lokunyuka kwekhabhoni kunye nokunyuka kwekhabhoni ukulayishwa kwesigaba sokunyuka kunye nexesha lokunyuka kwekhabhoni yokunyusa izinga le-carbon stationary reaction. vice versa.SPs ezilungiselelwe ngemilinganiselo eyahlukeneyo ye-styrene ine-carbon loadings eyahlukeneyo.Kwakhona, layisha ezi zigaba ezimileyo kwiikholomu zensimbi ezingenasici kwaye ukhangele ukusebenza kwazo kwe-chromatographic (ukukhethwa, isisombululo, ixabiso le-N, njl.) Ngokusekelwe kule mifuniselo, ukuqulunqwa okuphuculweyo kukhethwe ukulungiselela isigaba sokumisa se-PMP ukuqinisekisa ukubuyiswa kwe-polarity kunye nokuhlaziywa kakuhle.
Imixube emihlanu ye-peptide (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-leucine enkephalin) nayo yavavanywa ngokusebenzisa ikholomu ye-PMP usebenzisa isigaba seselula;60/40 (v/v) acetonitrile/amanzi (0.1% TFA) kwisantya sokuqukuqela 80 μL/min.Phantsi kweemeko elution ezigqwesileyo, ipleyiti yethiyori inombolo (N) ngekholamu nganye (100 × 1.8 mm id) ngama-20,000 ± 100 (200,000 iipleyiti ze-MP kunye ne-P 3m inika ixabiso le-Pleti ye-N00). iichromatograms ziboniswe kuMzobo 5A. Uhlalutyo olukhawulezayo kwikholamu ye-PMP ngesantya esiphezulu sokuhamba (700 μL/min), iipeptides ezintlanu zaye zahlanjululwa ngaphakathi komzuzu omnye, amaxabiso e-N ayelungile kakhulu, 13,500 ± 330 ngekholamu nganye (100 × 1.8 mm id), ihambelana ne-135 yeplaterem0, icolumn ye-135 . (I-100 × 1.8 mm id) yayipakishwe ngamaqashiso amathathu ahlukeneyo esigaba esimileyo se-PMP ukujonga ukuveliswa kwakhona.I-analyte concentration yekholamu nganye yarekhodwa kusetyenziswa iimeko ezifanelekileyo ze-lution kunye nenani leepleyiti zethiyori N kunye nexesha lokugcinwa ukwahlula umxube wovavanyo olufanayo kwikholam nganye.Idatha yokuvelisa kwakhona kwikholamu ye-PMPble kakuhle kwikholamu ye-PMP ephantsi kakhulu iboniswe kwikholamu ye-PMPble 4 iboniswe ngokuphindaphindiweyo kwikholamu ye-PMP. Amaxabiso e-RSD, njengoko kubonisiwe kwiThebhile 3.
Ukwahlula umxube we-peptide kwikholamu ye-PMP (B) kunye ne-Ascentis Express RP-Amide ikholamu (A);isigaba seselula 60/40 ACN / H2O (TFA 0.1%), imilinganiselo yekholomu ye-PMP (100 × 1.8 mm id);uhlalutyo Umyalelo we-lution we-compounds: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) kunye ne-5 (leucine) acid enkephalin)).
Ikholamu ye-PMP (i-ID ye-100 × 1.8 mm) yavavanyelwa ukwahlulwa kwe-tryptic digests ye-albumin ye-serum yabantu kwi-chromatography yolwelo oluphezulu. Ichromatogram kuMfanekiso 6 ibonisa ukuba isampulu yahlulwe kakuhle kwaye isisombululo sihle kakhulu. I-HSA digests yahlalutywa kusetyenziswa umyinge wokuqukuqela we-100/tole3/3/10000 µamanzi ahambahambayo kunye ne-10000000/03/2/20 I-1% TFA.Njengoko kubonisiwe kwi-chromatogram (Umfanekiso 6), ukugaywa kwe-HSA kuye kwahlulahlulwe kwiincopho ze-17 ezihambelana ne-peptides ye-17. Ukwahlula kakuhle kwincopho nganye kwi-digest ye-HSA kubalwa kwaye amaxabiso anikezelwa kwiThebhile 5.
I-tryptic digest ye-HSA (i-100 × 1.8 mm id) yahlulwe kwikholamu ye-PMP;izinga lokuhamba (100 µL/min), isigaba esihambayo 60/40 acetonitrile/amanzi ane-0.1% TFA.
apho uL ubude bekholomu, η yi-viscosity yesigaba esihambahambayo, i-ΔP yi-column yoxinzelelo lwangasemva, kwaye u-uyisantya somgca wesigaba esihambahambayo.I-permeability yekholomu ye-PMP yayiyi-2.5 × 10-14 m2, izinga lokuhamba laliyi-25 μL / min, kunye ne-60/40 v / v. I-ACN ye-1 mm i-ACN isetyenziswe i-1 × i-column ye-1 mm . yayifana naleyo yophononongo lwethu lwangaphambili Ref.34.I-permeability yekholamu epakishwe ngamasuntswana anqabileyo kakhulu: 1.7 × 10-15 ye-1.3 μm amasuntswana, 3.1 × 10-15 kwi-1.7 μm amasuntswana, 5.2 × 10-5 × 2 × 2 × 2 × 2. Iinqununu ze-μm 43. Ngoko ke, ukunyanzeliswa kwesigaba se-PMP kufana ne-5 μm i-core-shell particles.
apho i-Wx bubunzima bekholamu epakishwe ngeklorofomu, iWy bubunzima bekholamu epakishwe ngemethanol, kwaye ρ bubuninzi bento enyibilikayo.Uxinaniso lwemethanol (ρ = 0.7866) kunye nechloroform (ρ = 1.484).Itotali yeporosity yeSILICA PARTICLES 180 × 18 × 18 × 18 × 18 × 18 mm i-porosity 1. Iikholamu ze-Urea 31 esizifunde ngaphambili ziyi-0.63 kunye ne-0.55, ngokulandelanayo.Oku kuthetha ukuba ubukho be-urea ligands kunciphisa ukunyanzeliswa kwesigaba esimileyo.Ngakolunye uhlangothi, i-porosity epheleleyo yekholomu ye-PMP (i-100 × 1.8 mm id) yi-0.60.I-permeability ye-permeability ye-C1 yekholamu ye-silili ye-C1 ye-C1 yekholamu ye-silil ifakwe kwi-column ye-silil kwi-C1. Izigaba ezimileyo ze-8 ze-C18 ligands zifakwe kwi-silica particles njengamatyathanga anqamlekileyo, ngelixa kwizigaba ze-polystyrene-type stationary, i-A polymer layer ye-polymer engqingqwa yenziwa ngeenxa zonke.
Umzobo 7A, B ubonisa ikholomu ye-PMP (i-100 × 1.8 mm id) kunye nekholamu ye-Ascentis Express RP-Amide (100 × 1.8 mm id) isebenzisa iimeko ezifanayo ze-lution (okt, 60/40 ACN / H2O kunye ne-0.1% TFA).) yeploti yevan Deemter.Imixube yepeptide ekhethiweyo (iGly-Tyr, iGly-Leu-Tyr, iGly-Gly-Tyr-Arg, iTyr-Ile-Gly-Ser-Arg, iLeucine Enkephalin) yalungiswa nge-20 µL/ Ubuncinane besantya sokuhamba kuzo zombini iikholamu yi-800 µL/L Ikholomu ye-Express RP-Amide yayiyi-2.6 µm kunye ne-3.9 µm, ngokulandelanayo. Amaxabiso e-HETP abonisa ukuba ukwahlukana kakuhle kwekholamu ye-PMP (100 × 1.8 mm id) ingcono kakhulu kunokuthengiswa kwe-Ascentis Express RP-Amide ikholamu (100 × 1.8 mm id id. ayibalulekanga xa kuthelekiswa nesifundo sethu sangaphambili.Ukuphumelela okuphezulu kokuhlukana kwekholamu ye-PMP (i-100 × 1.8 mm id) xa kuthelekiswa nekholamu ye-Ascentis Express RP-Amide isekelwe ekuphuculeni kwi-particle shape, ubukhulu, kunye neenkqubo eziyinkimbinkimbi zokupakisha ikholamu ezisetyenziswe kumsebenzi wangoku34.
(A) i-van Deemter plot (i-HETP ngokubhekiselele kwi-mobile phase ye-linear velocity) ifunyenwe ngokusebenzisa ikholomu ye-PMP (100 × 1.8 mm id) kwi-60/40 ACN / H2O kunye ne-0.1% TFA. (B) i-van Deemter plot (HETP versus phase mobile linear velocity) ifunyenwe ngokusebenzisa i-Ascent0 mm 1 ikholomu ye-Ascentis × i-1 RP-Ascentis × i-1 RP-Ascentis Express. I-0 ACN / H2O kunye ne-0.1% TFA.
I-polar-embedded i-polystyrene isigaba esimileyo salungiswa kwaye savavanywa ukwahlula imixube ye-peptide eyenziweyo kunye ne-trypsin digests ye-albumin ye-serum yabantu (HAS) kwi-chromatography yolwelo oluphezulu. amasuntswana, ulungelelwaniso olulawulwayo lwesigaba esimileyo, kunye nokupakishwa kwekholamu enzima.Ukongezelela ekusebenzeni kokuhlukana okuphezulu, uxinzelelo oluphantsi lwekholamu yangasemva kwinqanaba lokuhamba okuphezulu yenye inzuzo yeli nqanaba elimileyo.Iintsika zePMP zibonisa ukuveliswa kakuhle kwaye zingasetyenziselwa ukuhlalutya imixube ye-peptide kunye ne-trypsin digestion yeeprotheyini ezahlukeneyo.Sizimisele ukusebenzisa le kholamu kwimveliso yendalo ye-biomedical liquids ye-separation ye-biomedical liquids, i-biomedical ye-separation yezityalo ze-biomedical zendalo. Kwixesha elizayo, iikholamu ze-PMP ziya kuphinda zivavanywe ukuhlukana kweeprotheni kunye ne-antibodies monoclonal.
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