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Iinxalenye ze-silica ezinemibhobho zalungiswa ngendlela ye-sol-gel kunye notshintsho oluthile ukuze kufunyanwe iinxalenye ezinemibhobho ebanzi. Ezi nxalenye zahluzwa nge-N-phenylmaleimide-methylvinyl isocyanate (PMI) kunye ne-styrene nge-reverse chain transfer-fragmentation (RAFT) polymerization ukuvelisa ii-N-phenylmaleimide intercalated polyamides. I-Styrene (PMP) stationary phase. Iikholamu zentsimbi engagqwaliyo ene-narrow bore (100 × 1.8 mm inner diameter) zazigcwele i-slurry packing. Ukusebenza kwe-chromatographic kwekholamu ye-PMP kwavavanywa ukuze kwahlulwe umxube wee-peptides zokwenziwa eziquka ii-peptides ezintlanu (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-Leu amino acid enkephalin) kunye ne-tryptic hydrolyzate ye-human serum albumin (HAS). Phantsi kweemeko ezifanelekileyo zokucoca, inani leepleyiti ezinomxube weepeptides lifikelele kwiipleyiti ezingama-280,000/sq.m. Xa kuthelekiswa ukusebenza kokwahlula kwekholamu ephuhlisiweyo kunye nekholamu ye-Ascentis Express RP-Amide yorhwebo, kwabonwa ukuba ukusebenza kokwahlula kwekholamu ye-PMP kwakungcono kunekholamu yorhwebo ngokubhekiselele ekusebenzeni kakuhle kokwahlula kunye nesisombululo.
Ishishini le-biopharmaceutical liye laba yimarike yehlabathi ekhulayo ngokwanda okukhulu kwesabelo semarike kwiminyaka yakutshanje. Ngokukhula okukhulu kweshishini le-biopharmaceutical1,2,3 kukho isidingo esikhulu sohlalutyo lwe-peptide kunye neproteni. Ukongeza kwi-peptide ekujoliswe kuyo, ukungcola okwahlukeneyo kwenziwa ngexesha lokwenziwa kwe-peptide, ngoko ke ukucocwa kwe-chromatographic kuyadingeka ukuze kufunyanwe ubunyulu obufunekayo be-peptide. Uhlalutyo kunye nokuchazwa kweeproteni kulwelo lomzimba, izicubu, kunye neeseli ngumsebenzi onzima kakhulu ngenxa yenani elikhulu leentlobo ezinokubonwa ezikhoyo kwisampulu enye. Nangona i-mass spectrometry sisixhobo esisebenzayo sokulandelelanisa ii-peptide kunye neeproteni, ukuba ezo sampuli zingeniswa ngqo kwi-mass spectrometer, ukwahlukana akuyi kuba kuhle. Le ngxaki ingasombululwa ngokwenza i-liquid chromatography (LC) ngaphambi kohlalutyo lwe-MS, oluya kunciphisa inani lee-analytes ezingena kwi-mass spectrometer ngexesha elithile4,5,6. Ukongeza, ii-analytes zinokugxila kwindawo emxinwa ngexesha lokwahlulwa kwesigaba solwelo, ngaloo ndlela zigxila ezi analytes kwaye zonyusa uvakalelo lokufunyanwa kwe-MS. I-chromatography yolwelo (LC) ihambele phambili kakhulu kwiminyaka elishumi edlulileyo kwaye iye yaba yindlela esetyenziswa kakhulu kuhlalutyo lweproteomic7,8,9,10.
I-Reverse-phase liquid chromatography (RP-LC) isetyenziswa kakhulu ukucoca nokwahlula imixube yeepeptides kusetyenziswa i-octadecyl-modified silica (ODS) njenge-stationary phase11,12,13. Nangona kunjalo, ngenxa yesakhiwo sazo esintsonkothileyo kunye nendalo ye-amphoteric, ii-14,15 RP stationary phases azinakubonelela ngokwahlula okwaneleyo kweepeptides kunye neeproteins. Ke ngoko, uhlalutyo lweepeptides kunye neeproteins ezinee-polar fragments kunye nee-non-polar fragments lufuna ii-stationary phases ezenzelwe ngokukodwa ukusebenzisana nokugcina ezi analytes16. I-Mixed chromatography, enika ukusebenzisana kwe-multimodal, inokuba yindlela eyahlukileyo ye-RP-LC yokwahlula iipeptides, iiproteins, kunye neminye imixube enzima. Ii-mixed-type stationary phases ezininzi zalungiswa kwaye iikholamu ezizaliswe zezi stationary phases zasetyenziswa ukwahlula iipeptides kunye neeproteins17,18,19,20,21. Ngenxa yokubakho kwamaqela e-polar kunye namaqela angengawo e-polar, ii-mixed mode stationary phases (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) zifanelekile ukwahlulahlula ii-peptides kunye neeproteni22,23,24,25,26,27,28. , ii-polar intercalated stationary phases ezinamaqela e-polar adibeneyo kunye ne-covalently zibonisa amandla okwahlulahlula amahle kunye nokukhetha okukhethekileyo kwii-polar kunye nee-non-polar analytes kuba ukwahlukana kuxhomekeke kunxibelelwano phakathi kwe-analyte kunye ne-stationary phase Multimodal interactions 29,30,31,32. Kutshanje, uZhang et al. 30 wafumana ii-behenyl-terminated stationary phases ze-polyamines kwaye zahlula ngempumelelo ii-hydrocarbons, ii-antidepressants, ii-flavonoids, ii-nucleosides, ii-estrogens, kunye nezinye ii-analytes. Izinto ezi-polar embedded stationary zinamaqela e-polar kunye namaqela angengawo e-polar, ngoko ke zinokusetyenziselwa ukwahlula ii-peptides kunye neeproteni zibe ngamacandelo e-hydrophobic kunye ne-hydrophilic. Iikholamu ezikwi-polar inline (umz., iikholamu ze-C18 ezine-amide inline) ziyafumaneka phantsi kwegama lorhwebo elithi Ascentis Express RP-Amide columns, kodwa ezi kholamu zisetyenziselwe kuphela uhlalutyo lwe-amine 33.
Kolu phononongo lwangoku, i-polar embedding stationary phase (i-N-phenylmaleimide, embedding polystyrene) yalungiselelwa kwaye yavavanywa ukwahlukana kwe-peptide kunye ne-tryptic HSA cleavage. Eli cebo lilandelayo lasetyenziswa ukulungiselela i-stationary phase. Ii-porous silica particles zalungiselelwa ngokweenkqubo ezichazwe kwiimpapasho zethu zangaphambili, kunye notshintsho oluthile kwizicwangciso zokulungiselela 31, 34, 35, 36, 37, 38, 39. Ii-ratios ze-urea, i-polyethylene glycol (PEG), i-TMOS kunye ne-aqueous-acetic acid zalungiswa ukuze kufunyanwe ii-silica particles ezinobukhulu obukhulu bee-pore. Okwesibini, i-phenylmaleimide-methylvinyl isocyanate ligand entsha yenziwe kwaye ii-silica particles zayo ezithathwe kwi-derivatized zasetyenziselwa ukulungiselela ii-polar embedded stationary phases. I-stationary phase efunyenweyo yapakishwa kwikholamu yentsimbi engagqwaliyo (ububanzi bangaphakathi 100 × 1.8 mm) ngokweskimu sokupakisha esilungisiweyo. Ukupakisha kwekholamu kuncediswa kukungcangcazela koomatshini ukuqinisekisa umaleko ofanayo ngaphakathi kwikholamu. Ikholamu epakishiweyo ihlolwe ukwahlulahlula umxube weepeptides oquka iipeptides ezintlanu (iGly-Tyr, iGly-Leu-Tyr, iGly-Gly-Tyr-Arg, iTyr-Ile-Gly-Ser-Arg, iLeucine-enkephalin peptide). kunye nee-tryptic hydrolysates ze-human serum albumin (HSA). Kubonwe ukuba umxube wepeptide kunye ne-HSA tryptic digest zahlukene ngesisombululo esihle kunye nokusebenza kakuhle. Ukusebenza kakuhle kokwahlulwa kwekholamu ye-PMP kuthelekiswa noko kwekholamu ye-Ascentis Express RP-Amide. Kubonwe ukuba iipeptides kunye neeproteni zinesisombululo esihle kunye nokusebenza kakuhle kokwahlulwa okuphezulu kwikholamu ye-PMP, kwaye ukusebenza kakuhle kokwahlulwa kwekholamu ye-PMP kuphezulu kunokwekholamu ye-Ascentis Express RP-Amide.
I-PEG (i-polyethylene glycol), i-urea, i-acetic acid, i-trimethoxyorthosilicate (TMOS), i-trimethylchlorosilane (TMCS), i-trypsin, i-human serum albumin (HSA), i-ammonium chloride, i-urea, i-hexamethylmethacryloyldisilazane (HMDS), i-methacryloyl chloride (MC), i-styrene, i-4-hydroxy-TEMPO, i-benzoyl peroxide (BPO), i-acetonitrile (ACN) ye-HPLC, i-methanol, i-2-propanol kunye ne-acetone. I-Sigma-Aldrich Company (eSt. Louis, eMissouri, e-USA).
Umxube we-urea (8 g), i-polyethylene glycol (8 g) kunye ne-8 ml ye-0.01 N. acetic acid yavuselelwa imizuzu eli-10, kwaye i-24 ml ye-TMOS yongezwa kuyo phantsi kokupholisa umkhenkce. Umxube we-reaction wafudunyezwa kwi-40°C kangangeeyure ezi-6 waza emva koko kwi-120°C kangangeeyure ezi-8 kwi-autoclave yentsimbi engatyiwayo. Amanzi asuswa kwaye intsalela yomiswa kwi-70°C kangangeeyure ezili-12. Iibhloko ezithambileyo ezomileyo zacolwa kakuhle zaza zafakwa kwi-oven kwi-550°C kangangeeyure ezili-12. Kwalungiswa iibhetshi ezintathu zaza zachazwa ukuvavanya ukuphinda-phinda kobukhulu beesuntswana, ubungakanani beembobo kunye nommandla womphezulu.
Iqela le-polar kunye nesigaba esimiyo seentambo ze-polystyrene. Inkqubo yokulungiselela ichazwe ngezantsi.
I-N-phenylmaleimide (200 mg) kunye ne-methyl vinyl isocyanate (100 mg) zanyibilikiswa kwi-toluene engenamanzi, kwaza emva koko kongezwa i-0.1 ml ye-2,2′-azoisobutyronitrile (AIBN) kwiflaski ye-reaction ukuze kufunyanwe i-copolymer ye-phenylmaleimide kunye ne-methyl vinyl isocyanate (PMCP). ) Umxube ufudunyezwe kwi-60°C iiyure ezi-3, wahluzwa waza womiswa kwi-oven kwi-40°C iiyure ezi-3.
Iisuntswana ze-silica ezomileyo (2 g) zasasazwa kwi-toluene eyomileyo (100 ml), zaxutywa zaza zafakwa kwi-sonic imizuzu eli-10 kwi-flask esezantsi eyi-500 ml. I-PMCP (10 mg) yanyibilikiswa kwi-toluene yaza yongezwa kwi-flask ye-reaction nge-addition funnel. Umxube wahluzwa kwi-100°C iiyure ezi-8, wahluzwa, wahlanjwa nge-acetone waza womiswa kwi-60°C iiyure ezi-3. Emva koko, iisuntswana ze-silica ezinxulumene ne-PMCP (100 g) zanyibilikiswa kwi-toluene (200 ml), kwaye i-4-hydroxy-TEMPO (2 ml) yongezwa kuyo xa kukho i-100 μl ye-dibutyltin dilaurate njenge-catalyst. Umxube washukunyiswa kwi-50°C iiyure ezi-8, wahluzwa waza womiswa kwi-50°C iiyure ezi-3.
I-Styrene (1 ml), i-benzoyl peroxide BPO (0.5 ml) kunye namasuntswana e-silica anamathele kwi-TEMPO-PMCP (1.5 g) asasazwa kwi-toluene aze acocwe nge-nitrogen. I-polymerization ye-styrene yenziwe kwi-100°C kangangeeyure ezili-12. Imveliso ephumayo yahlanjwa nge-methanol yaza yomiswa ubusuku bonke kwi-60°C. Inkqubo iyonke yempendulo iboniswe kumzobo wokuqala.
Iisampulu zasuswa igesi kwi-393 K kangangeyure e-1 de kwafunyanwa uxinzelelo olushiyekileyo olungaphantsi kwe-10–3 Torr. Ubungakanani be-N2 obufakwe kwixinzelelo elilinganiselweyo P/P0 = 0.99 busetyenzisiwe ukumisela umthamo opheleleyo weembobo. Imo yeenxalenye ze-silica ezicocekileyo nezibotshelelwe kwi-ligand zihlolwe kusetyenziswa i-scanning electron microscope (Hitachi High Technologies, Tokyo, Japan). Iisampulu ezomileyo (iinxalenye ze-silica ezicocekileyo nezibotshelelwe kwi-ligand) zibekwe kwiintonga ze-aluminium kusetyenziswa iteyiphu yekhabhoni. Igolide ibekwe kwisampulu kusetyenziswa isixhobo sokutshiza se-Q150T, kwaye umaleko we-Au onobukhulu obuyi-5 nm ubekwe kwisampulu. Oku kuphucula ukusebenza kakuhle kwenkqubo ye-voltage ephantsi kwaye kubonelela ngokutshiza okubandayo okucolileyo. Uhlalutyo lwe-elemental lwenziwe kusetyenziswa i-Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental composition analyzer. I-Malvern particle size analyzer (Worcestershire, UK) Mastersizer 2000 isetyenzisiwe ukufumana usasazo lobungakanani beenxalenye. Iisuntswana ze-silica ezingagqunywanga kunye neesuntswana ze-silica ezibotshelelwe kwi-ligand (5 mg nganye) zasasazwa kwi-5 ml ye-isopropanol, zafakwa kwi-sonicated imizuzu eli-10, zavuthuzwa imizuzu emi-5, zaza zabekwa kwibhentshi ye-optical ye-Mastersizer. Uhlalutyo lwe-thermogravimetric lwenziwa ngesantya se-5 °C ngomzuzu kububanzi bobushushu obuqala kwi-30 ukuya kwi-800 °C.
Iikholamu zentsimbi engagqwaliyo ezifakwe ifayibha yeglasi ezinobukhulu obuncinci (ID 100 × 1.8 mm) zapakishwa ngendlela yokuzalisa i-slurry ngokulandela inkqubo efanayo naleyo ikwisalathiso 31. Ikholamu yentsimbi engagqwaliyo (efakwe iglasi, ID 100 × 1 .8 mm) kunye nendawo yokuphuma equlethe i-1 µm frit yaqhagamshelwa kumatshini wokupakisha i-slurry (Alltech Deerfield, IL, USA). Lungisa ukumiswa kwesigaba esingagqwaliyo ngokuxhoma i-150 mg yesigaba esingagqwaliyo kwi-1.2 ml ye-methanol kwaye uyifake kwikholamu yokugcina amanzi. I-Methanol yasetyenziswa njenge-slurry solvent kunye ne-control solvent. Paka ikholamu ngokusebenzisa ulandelelwano loxinzelelo lwe-100 MP imizuzu eli-10, i-80 MP imizuzu eli-15, kunye ne-60 MP imizuzu engama-30. Inkqubo yokupakisha isebenzise ii-vibrators ezimbini zekholamu ye-gas chromatography (Alltech, Deerfield, IL, USA) ukuze kungcangcazeliswe ngoomatshini ukuqinisekisa ukupakisha ikholamu ngokulinganayo. Vala i-slurry packer kwaye ukhulule uxinzelelo kancinci ukuthintela umonakalo kwintambo. Ikholamu yasuswa kwi-slurry nozzle kwaza kwaqhotyoshelwa enye i-fitting kwi-inlet yaza yaqhagamshelwa kwinkqubo ye-LC ukuze kuvavanywe ukusebenza kwayo.
I-MLC eyenziwe ngokwezifiso yakhiwe kusetyenziswa ipompo ye-LC (10AD Shimadzu, eJapan), isampulu ene-50 nL injection loop (Valco (USA) C14 W.05), i-membrane degasser (Shimadzu DGU-14A), kunye ne-UV-VIS capillary window. Isixhobo se-detector (UV-2075) kunye ne-enameled microcolumn. Sebenzisa iityhubhu ezixinanisiweyo nezimfutshane kakhulu ukunciphisa isiphumo sokwandiswa kwekholamu eyongezelelweyo. Emva kokugcwalisa ikholamu, faka i-capillary (50 µm id 365) kwindawo yokuphuma ye-1/16″ reducing junction kwaye ufake i-capillary (50 µm) ye-reducing junction. Ukuqokelelwa kwedatha kunye nokucubungula i-chromatogram kwenziwa kusetyenziswa isoftware ye-Multichro 2000. Kwi-254 nm, ukufunxwa kwe-UV kwabahlalutyi bezifundo kujongwe kwi-0. Idatha ye-Chromatographic ihlalutywe kusetyenziswa i-OriginPro8 (Northampton, MA).
I-albumin ye-serum yomntu, umgubo we-lyophilized, ≥ 96% (i-agarose gel electrophoresis) 3 mg ixutywe ne-trypsin (1.5 mg), i-4.0 M urea (1 ml) kunye ne-0.2 M ammonium bicarbonate (1 ml). Isisombululo sivuselelwe imizuzu eli-10 saza sagcinwa emanzini kwi-37°C iiyure ezi-6, emva koko sacinywa nge-1 ml ye-0.1% TFA. Hluza isisombululo kwaye ugcine ngaphantsi kwe-4°C.
Ukwahlulwa komxube weepeptides kunye ne-tryptic digest HSA kwikholamu ye-PMP kuhlolwe ngokwahlukeneyo. Jonga i-tryptic hydrolysis yomxube weepeptides kunye ne-HSA ezahlulwe yikholamu ye-PMP kwaye uthelekise iziphumo nekholamu ye-Ascentis Express RP-Amide. Inani leeplate zethiyori libalwa kusetyenziswa i-equation elandelayo:
Imifanekiso ye-SEM yee-particles ze-silica ezicocekileyo kunye nee-particles ze-silica ezibotshelelwe kwi-ligand ziboniswe kuMfanekiso 2. Imifanekiso ye-SEM yee-particles ze-silica ezicocekileyo (A, B) zibonisa imilo engqukuva apho ii-particles zinde okanye zinokulingana okungacwangciswanga xa kuthelekiswa nezifundo zethu zangaphambili. Umphezulu wee-particles ze-silica ezibotshelelwe yi-ligand (C, D) uthambile kunowee-particles ze-silica ezicocekileyo, oku kusenokuba kungenxa yemixokelelwane ye-polystyrene egubungela umphezulu wee-particles ze-silica.
Ukuskena ii-electron micrographs zee-particles ze-silica ezicocekileyo (A, B) kunye nee-particles ze-silica ezibotshelelwe kwi-ligand (C, D).
Ukusasazwa kobungakanani be-particle yee-particle ze-silica ezicocekileyo kunye nee-particle ze-silica ezibotshelelwe kwi-ligand kuboniswe kuMfanekiso 2. 3(A). Ii-curves zokusasazwa kobungakanani be-particle ze-volumetric zibonise ukuba ubungakanani be-particle ye-silica buyanda emva kokuguqulwa kweekhemikhali (Umfanekiso 3A). Idatha yokusasazwa kobungakanani be-particle ye-silica evela kuphononongo lwangoku kunye nophando lwangaphambili ithelekiswa kwiTheyibhile 1(A). Ubungakanani be-particle ye-volumetric d(0.5) ye-PMP yayiyi-3.36 µm, xa kuthelekiswa nexabiso le-ad(0.5) le-3.05 µm kuphononongo lwethu lwangaphambili (ii-particles ze-silica ezibotshelelweyo ze-polystyrene)34. Ngenxa yotshintsho kumlinganiselo we-PEG, urea, TMOS kunye ne-acetic acid kumxube we-reaction, ukusasazwa kobungakanani be-particle kwale batch bekuncinci xa kuthelekiswa nophando lwethu lwangaphambili. Ubungakanani be-particle be-PMP phase bukhulu kancinci kunobo be-particles ze-silica ezibotshelelweyo ze-polystyrene esizifundileyo ngaphambili. Oku kuthetha ukuba ukusebenza komphezulu wee-silica particles ezine-styrene kubeke kuphela umaleko we-polystyrene (0.97 µm) kumphezulu we-silica, ngelixa kwisigaba se-PMP ubukhulu bomaleko babuyi-1.38 µm.
Ukusasazwa kobungakanani bamasuntswana (A) kunye nokusasazwa kobungakanani bemingxuma (B) yamasuntswana e-silica acocekileyo kunye namasuntswana e-silica aboshwe yi-ligand.
Ubungakanani bembobo, umthamo wembobo, kunye nommandla womphezulu wee-silica particles ezisetyenzisiweyo kolu phononongo ziboniswe kwiTheyibhile 1 (B). Iiprofayili zePSD zee-silica particles ezicocekileyo kunye nee-ligand-bound silica particles ziboniswe kwiMifanekiso 3(B). Iziphumo bezifana nophando lwethu lwangaphambili34. Ubungakanani bee-pore zee-silica particles ezicocekileyo kunye nee-ligand-bound silica particles beziyi-310 Å kunye ne-241 Å, ngokulandelanayo, nto leyo ebonisa ukuba emva kokuguqulwa kweekhemikhali, ubungakanani bembobo behle ngo-69 Å, njengoko kuboniswe kwiTheyibhile 1 (B), kwaye i-shift curve iboniswe kwiFig. Indawo ethile yomphezulu wee-silica particles kolu phononongo lwangoku yi-116 m2/g, elingana nophando lwethu lwangaphambili (124 m2/g). Njengoko kuboniswe kwiTheyibhile 1(B), indawo yomphezulu (m2/g) wee-silica particles emva kokuguqulwa kweekhemikhali nayo yehle ukusuka kwi-116 m2/g ukuya kwi-105 m2/g.
Iziphumo zohlalutyo lwezinto ezisisiseko zesigaba esisisigxina ziboniswe kwiTheyibhile. 2. Umxholo wekhabhoni wesigaba esisisigxina sangoku yi-6.35%, esezantsi kunakwisifundo sethu sangaphambili (iisuntswana ze-silica ezinxulumene ne-polystyrene, 7.93%35 kunye ne-10.21%, ngokulandelanayo) 42. Umxholo wekhabhoni wesigaba esisisigxina sangoku ngezantsi, kuba ezinye ii-ligands ze-polar ezifana ne-phenylmaleimide methyl vinyl isocyanate (PCMP) kunye ne-4-hydroxy-TEMPO zisetyenzisiwe ukongeza kwi-styrene ekulungiseleleni i-SP. Ipesenti yobunzima be-nitrogen kwisigaba esisisigxina sangoku yi-2.21% xa kuthelekiswa ne-0.1735 kunye ne-0.85% kwizifundo zangaphambili42. Oku kuthetha ukuba isigaba esisisigxina sangoku sinepesenti yobunzima obuphezulu be-nitrogen ngenxa ye-phenylmaleimide. Ngokufanayo, iimveliso (4) kunye (5) zinomxholo wekhabhoni oyi-2.7% kunye no-2.9%, ngokulandelelana, ngelixa imveliso yokugqibela (6) inomxholo wekhabhoni oyi-6.35%, njengoko kubonisiwe kwiTheyibhile 2. Uhlalutyo lweThermogravimetric (TGA) lusetyenziswe kwisigaba esimiselweyo se-PMP ukuvavanya ukwehla kobunzima, kwaye ijika le-TGA liboniswe kuMfanekiso 4. Ijika le-TGA libonisa ukwehla kobunzima obuyi-8.6%, okuvumelana kakuhle nomxholo wekhabhoni (6.35%), kuba ii-ligands aziqulathanga kuphela i-C , kodwa ne-N, i-O kunye ne-H.
I-ligand phenylmaleimide-methylvinyl isocyanate ikhethwe ukuba itshintshe umphezulu wee-silica particles ngenxa yamaqela ayo e-polar phenylmaleimide kunye ne-vinylisocyanate. Amaqela e-vinyl isocyanate anokusabela ngakumbi kwi-styrene ngokuphinda-phinda okuphilayo. Isizathu sesibini kukufaka iqela elinobudlelwane obuphakathi ne-analyte kwaye lingenazo iintsebenziswano ezinamandla ze-electrostatic phakathi kwe-analyte kunye nesigaba esimileyo, kuba indawo ye-phenylmaleimide ayinayo itshaja ebonakalayo kwi-pH eqhelekileyo. I-polarity yesigaba esimileyo inokulawulwa sisixa esifanelekileyo se-styrene kunye nexesha lokuphendula le-free radical polymerization. Inyathelo lokugqibela le-reaction (free radical polymerization) libalulekile njengoko litshintsha i-polarity yesigaba esimileyo. Uhlalutyo lwe-elemental lwenziwe ukujonga umxholo we-carbon kwezi zigaba ezimileyo. Kuye kwabonwa ukuba ukwandisa ubungakanani be-styrene kunye nexesha lokuphendula kwandisa umxholo we-carbon wesigaba esimileyo kwaye ngokuchaseneyo. Ii-SP ezilungiselelwe ngamanani ahlukeneyo e-styrene zinemithwalo eyahlukeneyo ye-carbon. Ngokufanayo, la manqanaba amileyo abekwe kwiikholamu zentsimbi engagqwaliyo kwaye iimpawu zazo ze-chromatographic (ukukhetha, isisombululo, ixabiso le-N, njl.njl.) zijongiwe. Ngokusekelwe kwezi zilingo, kukhethwe umxube olungiselelweyo wokulungiselela isigaba esimileyo se-PMP ukuze kubonelelwe nge-polarity elawulwayo kunye nokugcinwa kakuhle kwe-analyte.
Ikholamu ye-PMP ikwavavanyiwe ukuze kuhlalutywe imixube emihlanu yeepeptides (i-Gly-Tyr, i-Gly-Leu-Tyr, i-Gly-Gly-Tyr-Arg, i-Tyr-Ile-Gly-Ser-Arg, i-leucine-enkephalin) kusetyenziswa amandla esigaba esihambayo. 60/40 (v/v) ACN/amanzi (0.1% TFA) kwisantya sokuhamba se-80 µl/min. Phantsi kweemeko ezifanelekileyo zokululwa (iipleyiti ezingama-200,000/m), inani leepleyiti zethiyori (N) kwikholamu nganye (100 × 1.8 mm) yi-20,000 ± 100. Amaxabiso e-N kwiikholamu ezintathu ze-PMP aboniswe kwiTheyibhile 3 kwaye ii-chromatogram ziboniswe kwiFigure 5A. Uhlalutyo olukhawulezileyo ngesantya esiphezulu sokuhamba kwamanzi (700 µl/min) kwikholamu ye-PMP, iipeptide ezintlanu zaphuma ngaphakathi komzuzu omnye, ixabiso elihle kakhulu le-N eliyi-13,500 ± 330 kwikholamu nganye (ububanzi be-100 x 1.8 mm), elilingana neeplates ze-135,000/m (Umzobo 5B). Iikholamu ezintathu ezinobukhulu obufanayo (ububanzi bangaphakathi be-100 x 1.8 mm) zazaliswa ziibhetshi ezintathu ezahlukeneyo zesigaba se-PMP esimileyo ukuvavanya ukuphinda kuvele. Ii-analytes zarekhodwa kwikholamu nganye ngokwahlula umxube wovavanyo ofanayo kwikholamu nganye kusetyenziswa iimeko ezifanelekileyo zokuphinda kuvele, inani leepleyiti zethiyori u-N, kunye nexesha lokugcina. Idatha yokuphinda kuvele iikholamu ze-PMP iboniswe kwiTheyibhile 4. Ukuphinda kuvele iikholamu ze-PMP kudibene kakuhle namaxabiso aphantsi kakhulu e-%RSD njengoko kubonisiwe kwiTheyibhile 3.
Ukwahlulwa kwemixube ye-peptide kwikholamu ye-PMP (B) kunye nekholamu ye-Ascentis Express RP-Amide (A), i-mobile phase 60/40 ACN/H2O (TFA 0.1%), ubukhulu bekholamu ye-PMP (100 x 1.8 mm id), uhlalutyo Ulandelelwano lwe-Elution lwee-compounds: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) kunye ne-5 (leucic acid enkephalin).
Ikholamu ye-PMP (ububanzi bangaphakathi obuyi-100 x 1.8 mm) ihlolwe ukwahlula i-tryptic hydrolyzate ye-albumin ye-serum yomntu yi-HPLC. I-chromatogram ekuMfanekiso 6 ibonisa ukuba iisampulu zahlulwe kakuhle ngesisombululo esihle kakhulu. Izisombululo ze-HSA zihlalutywe kusetyenziswa isantya sokuhamba se-100 μl/min, isigaba esijikelezayo se-70/30 acetonitrile/amanzi kunye ne-0.1% TFA. Ukuqhekeka kwe-HSA kwahlulwe kwaba ziincopho ezili-17, njengoko kubonisiwe kwi-chromatogram (Umzobo 6), okuhambelana neepeptide ezili-17. Ukusebenza kakuhle kokwahlula iincopho zomntu ngamnye kwi-hydrolyzate ye-HSA kubaliwe kwaye amaxabiso aboniswe kwiTheyibhile 5.
Ii-hydrolysates ze-HSA tryptic zahlulwe kwikholamu ye-PMP (ububanzi bangaphakathi obuyi-100 x 1.8 mm), izinga lokuhamba (100 μl/min), isigaba esishukumayo se-acetonitrile/amanzi esiyi-60/40, kunye ne-0.1% TFA.
apho u-L bubude bekholamu, u-η yi-viscosity yesigaba esihambayo, u-ΔP luxinzelelo lwangasemva lwekholamu, kwaye u yi-linear velocity yesigaba esihambayo. Ukuvuleka kwekholamu ye-PMP yayiyi-2.5 × 10–14 m2, izinga lokuhamba laliyi-25 µl/min, kwasetyenziswa i-60/40 v/v. I-ACN/amanzi. Ukuvuleka kwekholamu ye-PMP (ID 100 × 1.8 mm) kwakufana noko kwakufundwe ngaphambili kweRef.34. Ukuvuleka kwekholamu egcwele iinxalenye ezingenanto yi-1.7×10 .6 µm, 2.5×10-14 m2 kwiinxalenye ze-5 µm43. Ke ngoko, ukuvuleka kwesigaba se-PMP kufana nokuvuleka kweenxalenye ze-core-shell ezinobukhulu be-5 μm.
apho i-Wx bubunzima bekholam ezaliswe yi-chloroform, i-Wy bubunzima bekholam ezaliswe yi-methanol, kwaye i-ρ bubunzima be-solvent. Ubuninzi be-methanol (ρ = 0.7866) kunye ne-chloroform (ρ = 1.484). I-porosity iyonke yekholam ye-silica-C18 particle (100 × 1.8 mm ID)34 kunye nekholam yethu ye-C18-urea31 efundwe ngaphambili yayiyi-0.63 kunye ne-0.55, ngokulandelanayo. Oku kuthetha ukuba ubukho be-urea ligands kunciphisa ukuvuleka kwesigaba esimileyo. Kwelinye icala, i-porosity iyonke yekholam ye-PMP (ububanzi bangaphakathi 100 × 1.8 mm) yi-0.60. Iikholam ze-PMP azinakungena ngaphantsi kuneekholam ezipakishwe zii-silica particles ezibotshelelwe yi-C18 kuba kwii-stationary phases zohlobo lwe-C18 ii-C18 ligands zinamathele kwii-silica particles kwiityathanga ezithe ngqo, ngelixa kwii-stationary phases zohlobo lwe-polystyrene i-polymer etyebileyo yenziwe ijikeleze ii-particles. umaleko A. Kwisilingo esiqhelekileyo, i-column porosity ibalwa ngolu hlobo lulandelayo:
Kumfanekiso 7A, u-B ubonisa iiploti zikaVan Deemter zekholamu ye-PMP (id 100 x 1.8 mm) kunye nekholamu ye-Ascentis Express RP-Amide (id 100 x 1.8 mm) phantsi kweemeko ezifanayo zokululwa, i-60/40 ACN/H2O kunye ne-0 .1% TFA 20 µl/min ukuya kwi-800 µl/min kuzo zombini iikholamu. Amaxabiso aphantsi e-HETP kwisantya sokuhamba esifanelekileyo (80 µl/min) yayiyi-2.6 µm kunye ne-3.9 µm kwikholamu ye-PMP kunye nekholamu ye-Ascentis Express RP-Amide, ngokwahlukeneyo. Amaxabiso e-HETP abonisa ukuba ukusebenza kakuhle kokwahlulwa kwekholamu ye-PMP (100 x 1.8 mm id) kuphezulu kakhulu kunokwekholamu ye-Ascentis Express RP-Amide ethengiswayo (100 x 1.8 mm id). Igrafu yevan Deemter kuMfanekiso 7(A) ibonisa ukuba ukwehla kwexabiso le-N akuphezulu kakhulu xa kuthelekiswa nokunyuka kokuhamba xa kuthelekiswa nophando lwethu lwangaphambili. Ukusebenza kakuhle kokwahlulwa kwekholamu ye-PMP (id 100 × 1.8 mm) xa kuthelekiswa nekholamu ye-Ascentis Express RP-Amide kusekelwe kwimo kunye nobukhulu obuphuculweyo be-particle kunye nenkqubo yokupakisha ikholamu ephucukileyo esetyenziswa kumsebenzi wangoku34.
(A) Iploti yeVan Deemter (isantya seHETP vs. isantya somgca wesigaba esiphathwayo) efunyenwe kwikholamu yePMP (id 100 x 1.8 mm) kwi-60/40 ACN/H2O ene-0.1% TFA. (B) Iploti yeVan Deemter (isantya seHETP ngokuchasene nesantya somgca wesigaba esiphathwayo) efunyenwe kwikholamu yeAscentis Express RP-Amide (id 100 x 1.8 mm) kwi-60/40 ACN/H2O ene-0.1% TFA.
Isigaba esimileyo se-polar se-polystyrene edibeneyo salungiselelwa kwaye savavanywa ukuze kwahlulwe umxube weepeptides zokwenziwa kunye ne-tryptic hydrolyzate ye-human serum albumin (HSA) kwi-chromatography yolwelo olusebenzayo kakhulu. Ukusebenza kwe-chromatographic kweekholamu ze-PMP kwimixube ye-peptide kugqwesile ngokubhekiselele ekusebenzeni kakuhle kokwahlulwa kunye nesisombululo. Ukusebenza ngcono kokwahlulwa kweekholamu ze-PMP kungenxa yezizathu ezahlukeneyo ezifana nobukhulu bee-particle ze-silica kunye nobukhulu bee-pore, ukuhlanganiswa okulawulwayo kwezigaba ezimileyo, kunye nezinto ezintsonkothileyo zokupakisha iikholamu. Ukongeza ekusebenzeni kakuhle kokwahlulwa okuphezulu, enye inzuzo yale sigaba esimileyo kuxinzelelo olusezantsi lwekholamu engasemva kwizinga eliphezulu lokuhamba. Iikholamu ze-PMP zinokuphinda-phindwa kakhulu kwaye zinokusetyenziselwa ukuhlalutya imixube yee-peptides kunye nokugaya i-tryptic yeeproteni ezahlukeneyo. Siceba ukusebenzisa le kholamu ukwahlula iikhompawundi ze-bioactive kwiimveliso zendalo, izicatshulwa zezityalo zonyango kunye neekhowa kwi-chromatography yolwelo. Kwixesha elizayo, iikholamu ze-PMP ziya kuvavanywa kwakhona ukuze kwahlulwe iiproteni kunye nee-antibodies ze-monoclonal.
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