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Tinthu ta silika tokhala ndi porous tinakonzedwa pogwiritsa ntchito njira ya sol-gel yokhala ndi zosintha zina kuti tipeze tinthu ta macroporous. Tinthu ta silika timeneti tinapangidwa pogwiritsa ntchito reversible addition fragmentation chain transfer (RAFT) polymerization ndi N-phenylmaleimide-methylvinylisocyanate (PMI) ndi styrene kuti tikonzekere N-phenylmaleimide intercalation ya polystyrene (PMP) stationary phase. Narrow-bore stainless columns (100 × 1.8 mm id) zinadzazidwa ndi slurry packing. Kupatukana kwa PMP column kwa peptide mixture yokhala ndi ma peptide asanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) chromatographic performance) ndi trypsin digestion ya human serum albumin (HAS). Pansi pa mikhalidwe yabwino kwambiri yopezera, chiwerengero cha ma plate cha peptide mixture ndi chokwera kufika pa 280,000 plates/m². Poyerekeza magwiridwe antchito a kupatukana kwa Mzere wopangidwa ndi mzati wamalonda wa Ascentis Express RP-Amide, zinaonedwa kuti magwiridwe antchito a PMP anali apamwamba kuposa mzati wamalonda pankhani ya magwiridwe antchito ndi kutsimikiza kwa kulekanitsa.
M'zaka zaposachedwapa, makampani opanga mankhwala a biopharmaceutical akhala msika womwe ukukula padziko lonse lapansi ndi kuwonjezeka kwakukulu kwa gawo la msika. Ndi kukula kwakukulu kwa makampani opanga mankhwala a biopharmaceutical1,2,3, kusanthula kwa ma peptide ndi mapuloteni kukufunidwa kwambiri. Kuphatikiza pa peptide yofunidwa, zonyansa zingapo zimapangidwa panthawi yopangira peptide, motero zimafuna kuyeretsa kwa chromatographic kuti mupeze ma peptide a chiyero chomwe mukufuna. Kusanthula ndi kufotokozera mapuloteni m'madzi amthupi, minofu ndi maselo ndi ntchito yovuta kwambiri chifukwa cha kuchuluka kwa mitundu yomwe ingapezeke mu chitsanzo chimodzi. Ngakhale kuti mass spectrometry ndi chida chothandiza pakutsata ma peptide ndi mapuloteni, ngati zitsanzo zotere zilowetsedwa mu mass spectrometer kamodzi kokha, kulekanitsa sikungakhale kwabwino. Vutoli likhoza kuchepetsedwa pokhazikitsa kulekanitsa kwa liquid chromatography (LC) musanayese kusanthula kwa MS, komwe kudzachepetsa chiwerengero cha ma analytes omwe amalowa mu mass spectrometer panthawi inayake4,5,6. Kuphatikiza apo, panthawi yolekanitsa gawo lamadzimadzi, ma analytes amatha kuyang'aniridwa m'madera opapatiza, potero kuyang'ana ma analytes awa ndikuwongolera kuzindikira kwa MS. Kuzindikira kwa madzi. Kusanthula kwa madzi (LC) kwapita patsogolo kwambiri m'zaka khumi zapitazi ndipo kwakhala njira yotchuka pakusanthula kwa proteomic7,8,9,10.
Chromatography yamadzimadzi yosinthidwa (RP-LC) imagwiritsidwa ntchito kwambiri poyeretsa ndi kulekanitsa zosakaniza za peptide pogwiritsa ntchito octadecyl-modified silica (ODS) ngati gawo lokhazikika11,12,13. Komabe, magawo osakhazikika a RP sapereka kulekanitsa kokwanira kwa ma peptide ndi mapuloteni chifukwa cha kapangidwe kawo kovuta komanso mawonekedwe awo a amphiphilic 14,15. Chifukwa chake, magawo osakhazikika opangidwa mwapadera amafunika kuti afufuze ma peptide ndi mapuloteni okhala ndi magawo a polar ndi non-polar kuti agwirizane ndi kusunga ma analytes awa16. Chromatography yosakanikirana, yomwe imapereka kuyanjana kwamitundu yambiri, ikhoza kukhala njira ina m'malo mwa RP-LC yolekanitsa ma peptide, mapuloteni, ndi zosakaniza zina zovuta. Magawo angapo osakhazikika a mixed-mode akonzedwa, ndipo mizati yodzaza ndi magawo awa yagwiritsidwa ntchito polekanitsa ma peptide ndi mapuloteni17,18,19,20,21. Magawo osakhazikika a mixed-mode (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) ndi oyenera kulekanitsa ma peptide ndi mapuloteni chifukwa kupezeka kwa magulu onse a polar ndi non-polar22,23,24,25,26,27,28. Mofananamo, magawo okhazikika a polar intercalating okhala ndi magulu a polar ogwirizana bwino amasonyeza mphamvu yabwino yolekanitsa komanso kusankha kwapadera kwa ma analyte a polar ndi non-polar, chifukwa kulekanitsa kumadalira kuyanjana pakati pa analyte ndi gawo lokhazikika. Kuyanjana kwa Multimodal 29, 30, 31, 32. Posachedwapa, Zhang et al. 30 adakonza gawo lokhazikika la polyamine lomwe linatha ndi dodecyl ndipo adasiyanitsa bwino ma hydrocarbon, mankhwala oletsa kupsinjika maganizo, ma flavonoid, ma nucleoside, ma estrogen, ndi ma analytes ena angapo. Polar intercalator ili ndi magulu a polar ndi non-polar, kotero ingagwiritsidwe ntchito kulekanitsa ma peptide ndi mapuloteni omwe ali ndi hydrophobic ndi hydrophilic moieties. Polar-embedded columns (monga amide-embedded C18 columns) amapezeka pamsika pansi pa dzina lamalonda la Ascentis Express RP-Amide columns, koma ma columns awa amagwiritsidwa ntchito pofufuza amine 33 yokha.
Mu kafukufuku wapano, gawo lokhazikika lopangidwa ndi polar-embedded (N-phenylmaleimide-embedded polystyrene) linakonzedwa ndikuyesedwa kuti lilekanitse ma peptide ndi trypsin digests a HSA. Gawo lokhazikika linakonzedwa pogwiritsa ntchito njira yotsatirayi. Tinthu ta silica tomwe timakhala ndi porous tinakonzedwa motsatira njira yomwe yaperekedwa m'buku lathu lapitalo ndi kusintha kwina kwa njira yokonzekera. Chiŵerengero cha urea, polyethylene glycol (PEG), TMOS, water acetic acid chinasinthidwa kuti chikonzekere tinthu ta silica tokhala ndi pore yayikulu. Chachiwiri, ligand yatsopano, phenylmaleimide-methyl vinyl isocyanate, idapangidwa ndikugwiritsidwa ntchito kuchotsa tinthu ta silica kuti tikonzekere gawo lokhazikika lopangidwa ndi polar. Gawo lokhazikika lomwe linatsatira linayikidwa mu mzati wachitsulo chosapanga dzimbiri (100 × 1.8 mm id) pogwiritsa ntchito njira yokonzekera bwino. Kuyika mzati kumathandizidwa ndi kugwedezeka kwa makina kuti zitsimikizire kuti bedi lofanana limapangidwa mkati mwa mzati. Yesani kulekanitsa mzati wodzaza wa zosakaniza za peptide zokhala ndi ma peptide asanu; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) ndi Trypsin digest ya human serum albumin (HAS). Kusakaniza kwa peptide ndi trypsin digest ya HSA zinawonedwa kuti zimasiyana ndi resolution yabwino komanso bwino. Kugawa kwa PMP column kunayerekezeredwa ndi kwa Ascentis Express RP-Amide column. Ma peptide ndi mapuloteni onse awiri adawonedwa kuti ali okhazikika bwino komanso ogwira ntchito bwino pa PMP column, yomwe inali yothandiza kwambiri kuposa Ascentis Express RP-Amide column.
PEG (Polyethylene Glycol), Urea, Acetic Acid, Trimethoxy Orthosilicate (TMOS), Trimethyl Chlorosilane (TMCS), Trypsin, Human Serum Albumin (HSA), Ammonium Chloride, Urea, Hexane Methyldisilazane (HMDS), Methacryloyl Chloride (MC), Styrene, 4-Hydroxy-TEMPO, Benzoyl Peroxide (BPO), HPLC Grade Acetonitrile (ACN), Methanol, 2-Propanol, ndi Acetone Zogulidwa ku Sigma-Aldrich (St. Louis, MO, USA).
Chisakanizo cha urea (8 g), polyethylene glycol (8 g), ndi 8 mL ya 0.01 N acetic acid chinasakanizidwa kwa mphindi 10, kenako 24 mL ya TMOS inawonjezedwa pamenepo pansi pa kuzizira kwambiri. Chisakanizocho chinatenthedwa pa 40°C kwa maola 6 kenako pa 120°C kwa maola 8 mu autoclave yachitsulo chosapanga dzimbiri. Madzi anathiridwa ndipo zinthu zotsalazo zinaumitsidwa pa 70°C kwa maola 12. Chidutswa chofewa chouma chinaphwanyidwa bwino mu uvuni ndikusungunuka pa 550°C kwa maola 12. Magulu atatu anakonzedwa ndikuyesedwa kuti awone momwe tinthu tating'onoting'ono tingaberekere, kukula kwa ma pore ndi malo ake.
Mwa kusintha pamwamba pa tinthu ta silica ndi pre-synthesized ligand phenylmaleimide-methylvinylisocyanate (PCMP) kutsatiridwa ndi radial polymerization ndi styrene, polar group-containing compound inakonzedwa. Gawo lokhazikika la ma aggregates ndi ma polystyrene chains. Njira yokonzekera yafotokozedwa pansipa.
N-phenylmaleimide (200 mg) ndi methyl vinyl isocyanate (100 mg) zinasungunuka mu toluene youma, ndipo 0.1 mL ya 2,2′-azoisobutyronitrile (AIBN) inawonjezedwa ku flask yopangira phenylmaleimide-methyl vinyl isocyanate copolymer (PMCP).. Chosakanizacho chinatenthedwa pa 60°C kwa maola atatu, chinasefedwa ndikuumitsidwa mu uvuni pa 40°C kwa maola atatu.
Tinthu ta silika touma (2 g) tinamwazidwa mu toluene youma (100 mL), tinasonkhezeredwa ndi kulowetsedwa mu botolo lozungulira la 500 mL kwa mphindi 10. PMCP (10 mg) inasungunuka mu toluene ndikuwonjezeredwa ku botolo loyankhira kudzera mu funnel yodontha. Chosakanizacho chinasungunuka pa 100°C kwa maola 8, chinasefedwa ndikutsukidwa ndi acetone ndikuumitsidwa pa 60°C kwa maola 3. Kenako, tinthu ta silika tomwe tinalumikizidwa ndi PMCP (100 g) tinasungunuka mu toluene (200 ml) ndipo 4-hydroxy-TEMPO (2 mL) inawonjezedwa pamaso pa 100 µL ya dibutyltin dilaurate ngati chothandizira. Chosakanizacho chinasunthidwa pa 50°C kwa maola 8, chinasefedwa ndikuumitsidwa pa 50°C kwa maola 3.
Styrene (1 mL), benzoyl peroxide BPO (0.5 mL), ndi tinthu ta silica tomwe timalumikizidwa ndi TEMPO-PMCP (1.5 g) tinamwazidwa mu toluene ndikutsukidwa ndi nayitrogeni. Kupopera kwa styrene kunachitika pa 100°C kwa maola 12. Chotsatiracho chinatsukidwa ndi methanol ndikuumitsidwa pa 60°C usiku wonse. Ndondomeko yonse ya reaction ikuwonetsedwa mu Chithunzi 1.
Zitsanzozo zinachotsedwa mpweya pa 393 K kwa ola limodzi kuti zipeze mphamvu yotsalira yosakwana 10-3 Torr. Kuchuluka kwa N2 komwe kunayamwa pa mphamvu ya P/P0 = 0.99 kunagwiritsidwa ntchito kudziwa kuchuluka kwa pore. Kapangidwe ka tinthu ta silica topanda kanthu ndi topanda kanthu kanayesedwa ndi scanning electron microscopy (Hitachi High Technologies, Tokyo, Japan). Zitsanzo zouma (tinthu ta silica topanda kanthu ndi tinthu ta silica topanda kanthu) zinayikidwa pa aluminiyamu pogwiritsa ntchito tepi yomatira ya carbon. Golide anaikidwa pa zitsanzozo pogwiritsa ntchito Q150T sputter coater, ndipo 5 nm Au layer inayikidwa pa zitsanzozo. Izi zimapangitsa kuti ntchito igwire bwino ntchito pogwiritsa ntchito ma voltage otsika ndipo zimapereka timadzi tochepa, timadontho tozizira. Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental analyzer inagwiritsidwa ntchito pofufuza zinthu. Malvern (Worcestershire, UK) Mastersizer 2000 particle size analyzer inagwiritsidwa ntchito kuti ipeze kufalikira kwa tinthu. Naked silica Tinthu tating'onoting'ono ndi tinthu ta silica tomwe timagwirizana ndi ligand (5 mg iliyonse) tinamwazidwa mu 5 mL ya isopropanol, tinayikidwa mu sonic kwa mphindi 10, tinasungunuka kwa mphindi 5, ndipo tinayikidwa pa benchi yowunikira ya Mastersizer. Kusanthula kwa thermogravimetric kunachitika pamlingo wa 5 °C pamphindi pa kutentha kwa 30 mpaka 800 °C.
Zipilala zosapanga dzimbiri zopangidwa ndi chitsulo chosapanga dzimbiri chokhala ndi galasi zokhala ndi miyeso yopapatiza (100 × 1.8 mm id) zinapakidwa pogwiritsa ntchito njira yopakira matope, pogwiritsa ntchito njira yomweyi yomwe inagwiritsidwa ntchito mu Ref. 31. Chipilala chachitsulo chosapanga dzimbiri (chokhala ndi galasi, 100 × 1.8 mm id) chokhala ndi cholumikizira chotulutsira mpweya chokhala ndi 1 µm frit chinalumikizidwa ku chopakira madzi (Alltech Deerfield, IL, USA). Konzani slurry ya gawo lokhazikika poyimitsa 150 mg ya gawo lokhazikika mu 1.2 mL ya methanol ndikuitumiza ku column yosungiramo. Methanol idagwiritsidwa ntchito ngati chosungunulira madzi komanso chosungunulira madzi. Dzazani column motsatizana poika mphamvu ya 100 MP kwa mphindi 10, 80 MP kwa mphindi 15, ndi 60 MP kwa mphindi 30. Pakulongedza, kugwedezeka kwa makina kunagwiritsidwa ntchito ndi ma GC column shakers awiri (Alltech, Deerfield, IL, USA) kuti muwonetsetse kuti column ikuyenda bwino. Tsekani chopakira madzi ndikutulutsa kupanikizika pang'onopang'ono kuti mupewe kuwonongeka kulikonse mkati mwa column. Chotsani column kuchokera ku slurry packing unit ndikulumikiza china cholumikizira ku inlet ndi ku LC system kuti muwone ngati chili magwiridwe antchito.
Pampu ya LC (10AD Shimadzu, Japan), injector (Valco (USA) C14 W.05) yokhala ndi 50nL injection loop, membrane degasser (Shimadzu DGU-14A), UV-VIS capillary window idapangidwa. Chowunikira chipangizo chapadera cha µLC (UV-2075) ndi ma microcolumns okhala ndi galasi. Gwiritsani ntchito chubu cholumikizira chopapatiza kwambiri komanso chachifupi kuti muchepetse zotsatira za kukulitsa kwa mzere wowonjezera. Pambuyo poyika, ma capillaries (50 μm id 365 ndi ma reducing union capillaries (50 μm) adayikidwa pamalo otulukira a 1/16″ a reducing union. Kusonkhanitsa deta ndi kukonza chromatographic kunachitika pogwiritsa ntchito pulogalamu ya Multichro 2000. Kuwunika pa 254 nm Analytes kunayesedwa kuti awone ngati UV imayamwa. Deta ya chromatographic idasanthulidwa ndi OriginPro8 (Northampton, MA).
Albumin yochokera mu seramu ya anthu, ufa wophilizidwa, ≥ 96% (agarose gel electrophoresis) 3 mg wosakaniza ndi trypsin (1.5 mg), 4.0 M urea (1 mL), ndi 0.2 M ammonium bicarbonate (1 mL). Yankho linasunthidwa kwa mphindi 10 ndikusungidwa m'madzi osambira pa 37°C kwa maola 6, kenako limazimitsidwa ndi 1 mL ya 0.1% TFA. Sefa yankholo ndikusunga pansi pa 4 °C.
Kulekanitsa kwa zosakaniza za peptide ndi HSA trypsin digests kunayesedwa padera pa mizati ya PMP. Yang'anani kulekanitsa kwa peptide mixture ndi trypsin digest ya HSA ndi mizati ya PMP ndikuyerekeza zotsatira zake ndi mizati ya Ascentis Express RP-Amide. Nambala ya plate ya theoretical imawerengedwa motere:
Zithunzi za SEM za tinthu ta silica topanda kanthu ndi tinthu ta silica tolumikizidwa ndi ligand zikuwonetsedwa mu Chithunzi 2. Zithunzi za SEM za tinthu ta silica topanda kanthu (A, B) zikusonyeza kuti, mosiyana ndi maphunziro athu am'mbuyomu, tinthu tating'onoting'ono timeneti ndi tozungulira momwe tinthu tating'onoting'ono timakhala tatalika kapena tokhala ndi mawonekedwe osasinthasintha. Pamwamba pa tinthu ta silica tolumikizidwa ndi ligand (C, D) ndi posalala kuposa pa tinthu ta silica topanda kanthu, zomwe zitha kukhala chifukwa cha kuphimba kwa unyolo wa polystyrene pamwamba pa tinthu ta silica.
Kujambula zithunzi za maikulosikopu ya ma elekitironi za tinthu ta silika topanda kanthu (A, B) ndi tinthu ta silika tolumikizidwa ndi ligand (C, D).
Kugawa kwa tinthu tating'onoting'ono ta tinthu ta silika topanda kanthu ndi tinthu ta silica tolumikizidwa ndi ligand kukuwonetsedwa mu Chithunzi 3(A). Ma curve ogawa tinthu ta silika tochokera ku voliyumu awonetsa kuti kukula kwa tinthu ta silika kunawonjezeka pambuyo pa kusintha kwa mankhwala (Chithunzi 3A). Deta yogawa tinthu ta silika kuchokera ku kafukufuku wapano ndi kafukufuku wam'mbuyomu ikuyerekezeredwa mu Table 1(A). Kukula kwa tinthu ta silika tochokera ku voliyumu, d(0.5), ya PMP ndi 3.36 μm, poyerekeza ndi kafukufuku wathu wam'mbuyomu wokhala ndi mtengo wa ad(0.5) wa 3.05 μm (tinthu ta silika tomangidwa ndi polystyrene)34. Gulu ili linali ndi kugawa kwa tinthu tating'onoting'ono poyerekeza ndi kafukufuku wathu wam'mbuyomu chifukwa cha kusiyana kwa ma PEG, urea, TMOS, ndi acetic acid mu chisakanizo cha reaction. Kukula kwa tinthu ta PMP ndi kwakukulu pang'ono kuposa kwa gawo la tinthu ta silika tomangidwa ndi polystyrene lomwe tidaphunzira kale. Izi zikutanthauza kuti magwiridwe antchito a pamwamba pa tinthu ta silika ndi styrene amangoyika polystyrene wosanjikiza (0.97 µm) pa pamwamba pa silika, pomwe mu gawo la PMP makulidwe a wosanjikiza anali 1.38 µm.
Kugawa kukula kwa tinthu tating'onoting'ono (A) ndi kugawa kukula kwa tinthu tating'onoting'ono ta silika (B) ta tinthu tating'onoting'ono ta silika tomwe tili ndi ligand.
Kukula kwa ma pore, kuchuluka kwa ma pore ndi malo a pamwamba pa tinthu ta silica ta kafukufukuyu aperekedwa mu Gome 1(B). Ma profiles a PSD a tinthu ta silica topanda kanthu ndi tinthu ta silica tolumikizidwa ndi ligand akuwonetsedwa mu Chithunzi 3(B). Zotsatira zake zikufanana ndi kafukufuku wathu wakale. Kukula kwa ma pore a tinthu ta silica topanda kanthu ndi tolumikizidwa ndi ligand ndi 310 ndi 241, motsatana, zomwe zikusonyeza kuti kukula kwa ma pore kumachepa ndi 69 pambuyo pa kusintha kwa mankhwala, monga momwe zasonyezedwera mu Gome 1(B), ndipo kusintha kwa curve kukuwonetsedwa mu Chithunzi 3(B). Mofananamo, kuchuluka kwa ma pore a tinthu ta silica kunachepa kuchoka pa 0.67 kufika pa 0.58 cm3/g pambuyo pa kusintha kwa mankhwala. Malo enieni a tinthu ta silica omwe akuphunziridwa pano ndi 116 m2/g, omwe akufanana ndi kafukufuku wathu wakale (124 m2/g). Monga momwe zasonyezedwera mu Gome 1(B), malo a pamwamba (m2/g) a tinthu ta silica nawonso anachepa kuchoka pa 116 m2/g kufika pa 105 m2/g mutasintha mankhwala.
Zotsatira za kusanthula kwa elemental kwa gawo lokhazikika zikuwonetsedwa mu Gome 2. Kuchuluka kwa mpweya m'gawo lokhazikika la pano ndi 6.35%, komwe ndi kotsika kuposa kuchuluka kwa mpweya m'kafukufuku wathu wakale (tinthu ta silica tolumikizidwa ndi polystyrene, 7.93%35 ndi 10.21%, motsatana) 42. Kuchuluka kwa mpweya m'gawo lokhazikika la pano ndi kotsika, Chifukwa pokonzekera SP yapano, kuwonjezera pa styrene, ma polar ligands ena monga phenylmaleimide-methylvinylisocyanate (PCMP) ndi 4-hydroxy-TEMPO adagwiritsidwa ntchito. Peresenti ya kulemera kwa nayitrogeni m'gawo lokhazikika la pano ndi 2.21%, poyerekeza ndi 0.1735 ndi 0.85% polemera kwa nayitrogeni m'maphunziro am'mbuyomu, motsatana. Izi zikutanthauza kuti % ya nayitrogeni ndi yokwera m'gawo lokhazikika la pano chifukwa cha phenylmaleimide. Mofananamo, kuchuluka kwa mpweya m'zinthu (4) ndi (5) kunali 2.7% ndi 2.9%, motsatana, pomwe Kuchuluka kwa mpweya m'thupi (6) kunali 6.35%, monga momwe zasonyezedwera mu Table 2. Kuchepa kwa thupi kunayang'aniridwa ndi gawo lokhazikika la PMP, ndipo curve ya TGA ikuwonetsedwa mu Chithunzi 4. Mzere wa TGA ukuwonetsa kuchepa kwa thupi kwa 8.6%, zomwe zikugwirizana bwino ndi carbon loading (6.35%) chifukwa ma ligands alibe C yokha komanso N, O, ndi H.
Phenylmaleimide-methylvinylisocyanate ligand idasankhidwa kuti isinthe pamwamba pa tinthu ta silica chifukwa ili ndi magulu a polar phenylmaleimide ndi magulu a vinylisocyanate. Magulu a Vinyl isocyanate amatha kuchitapo kanthu ndi styrene mwa kukhala ndi radical polymerization. Chifukwa chachiwiri ndikuyika gulu lomwe lili ndi kuyanjana pang'ono ndi analyte ndipo palibe kuyanjana kwamphamvu kwa electrostatic pakati pa analyte ndi gawo lokhazikika, popeza gawo la phenylmaleimide lilibe mphamvu yeniyeni pa pH yachibadwa. Polarity ya gawo lokhazikika ikhoza kulamulidwa ndi kuchuluka kwabwino kwa styrene ndi nthawi yochitapo kanthu ya free radical polymerization. Gawo lomaliza la reaction (free-radical polymerization) ndilofunika kwambiri ndipo likhoza kusintha polarity ya gawo lokhazikika. Kusanthula kwa elemental kunachitika kuti muwone kuchuluka kwa carbon mu magawo okhazikika awa. Zinawonedwa kuti kuwonjezera kuchuluka kwa styrene ndi nthawi yochitapo kanthu kunawonjezera carbon loading ya gawo lokhazikika ndipo mosemphanitsa. Ma SP okonzedwa ndi kuchuluka kosiyana kwa styrene ali ndi carbon loadings zosiyanasiyana. Apanso, ikani magawo okhazikika awa mu chitsulo chosapanga dzimbiri. Ma columns ndikuyang'ana momwe amagwirira ntchito pa chromatographic (kusankha, kutsimikiza, kuchuluka kwa N, ndi zina zotero). Kutengera ndi zoyeserera izi, njira yabwino kwambiri idasankhidwa kuti ikonze gawo lokhazikika la PMP kuti litsimikizire polarity yolamulidwa komanso kusungidwa bwino kwa analyte.
Zosakaniza zisanu za peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) zinayesedwanso pogwiritsa ntchito PMP column pogwiritsa ntchito mobile phase; 60/40 (v/v) acetonitrile/madzi (0.1% TFA) pamlingo woyenda wa 80 μL/min. Pansi pa mikhalidwe yabwino kwambiri yoyeretsera, nambala ya plate (N) pa chigawo chilichonse (100 × 1.8 mm id) ndi 20,000 ± 100 (200,000 plates/m²). Gome 3 limapereka ma N values ​​​​a mizati itatu ya PMP ndipo ma chromatogram akuwonetsedwa mu Chithunzi 5A. Kusanthula mwachangu pa mizati ya PMP pamlingo woyenda kwambiri (700 μL/min), ma peptide asanu adachotsedwa mkati mwa mphindi imodzi, ma N values ​​​​anali abwino kwambiri, 13,500 ± 330 pa chigawo chilichonse (100 × 1.8 mm id), Yogwirizana ndi ma plate 135,000/m² (Chithunzi 5B). Ma mizati itatu yofanana (100 × 1.8 mm id) adadzazidwa ndi magawo atatu osiyanasiyana a gawo lokhazikika la PMP kuti awone. kubwerezabwereza. Kuchuluka kwa analyte pa kolamu iliyonse kunalembedwa pogwiritsa ntchito mikhalidwe yabwino kwambiri yolumikizira ndi kuchuluka kwa ma plate a theoretical N ndi nthawi yosungira kuti alekanitse kusakaniza komweko kwa mayeso pa kolamu iliyonse. Deta yobwerezabwereza ya makolamu a PMP ikuwonetsedwa mu Table 4. Kubwerezabwereza kwa kolamu ya PMP kumagwirizana bwino ndi ma values ​​otsika kwambiri a %RSD, monga momwe tawonetsera mu Table 3.
Kulekanitsidwa kwa chisakanizo cha peptide pa PMP column (B) ndi Ascentis Express RP-Amide column (A); gawo loyenda 60/40 ACN/H2O (TFA 0.1%), miyeso ya PMP column (100 × 1.8 mm id); analytical Dongosolo la elution la mankhwala: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) ndi 5 (leucine) acid enkephalin)).
Cholembera cha PMP (100 × 1.8 mm id) chinayesedwa kuti chiwonetse kulekanitsa kwa tryptic digests ya human serum albumin mu high performance liquid chromatography. Chromatogram mu Chithunzi 6 ikuwonetsa kuti chitsanzocho chalekanitsidwa bwino ndipo resolution ndi yabwino kwambiri. HSA digests idasanthulidwa pogwiritsa ntchito flow rate ya 100 µL/min, mobile phase 70/30 acetonitrile/water ndi 0.1% TFA. Monga momwe zasonyezedwera mu chromatogram (Chithunzi 6), HSA digest yagawidwa m'ma peaks 17 ofanana ndi ma peptides 17. Kuchuluka kwa kulekanitsidwa kwa peak iliyonse mu HSA digest kunawerengedwa ndipo miyeso yaperekedwa mu Table 5.
Kuchuluka kwa tryptic digest ya HSA (100 × 1.8 mm id) kunalekanitsidwa pa PMP column; flow rate (100 µL/min), mobile phase 60/40 acetonitrile/madzi ndi 0.1% TFA.
kumene L ndi kutalika kwa mzati, η ndi kukhuthala kwa gawo loyenda, ΔP ndi kupanikizika kwa mzati kumbuyo, ndipo u ndi liwiro la mzere wa gawo loyenda. Kutha kwa mzati wa PMP kunali 2.5 × 10-14 m2, kuchuluka kwa madzi kunali 25 μL/min, ndipo 60/40 v/v ACN/madzi kunagwiritsidwa ntchito. Kutha kwa mzati wa PMP (100 × 1.8 mm id) kunali kofanana ndi kwa kafukufuku wathu wakale Ref.34. Kutha kwa mzati wodzaza ndi tinthu tokhala ndi mapokoso pang'ono ndi: 1.7 × 10-15 pa tinthu ta 1.3 μm, 3.1 × 10-15 pa tinthu ta 1.7 μm, 5.2 × 10-15 ndi 2.5 × 10-14 m2 pa tinthu ta 2.6 μm. Pa tinthu ta 5 μm 43. Chifukwa chake, kutha kwa mzati wa PMP kuli kofanana ndi kwa tinthu ta 5 μm core-shell.
kumene Wx ndi kulemera kwa chigawo chodzaza ndi chloroform, Wy ndi kulemera kwa chigawo chodzaza ndi methanol, ndipo ρ ndi kuchuluka kwa solvent. Kuchuluka kwa methanol (ρ = 0.7866) ndi chloroform (ρ = 1.484). Kuchuluka kwa porosity ya SILICA PARTICLES-C18 columns (100 × 1.8 mm id) 34 ndi C18-Urea columns 31 zomwe tidaphunzira kale zinali 0.63 ndi 0.55, motsatana. Izi zikutanthauza kuti kukhalapo kwa urea ligands kumachepetsa kulowerera kwa gawo lokhazikika. Kumbali inayi, kulowerera konse kwa chigawo cha PMP (100 × 1.8 mm id) ndi 0.60. Kulowera kwa mizati ya PMP ndi kotsika kuposa kwa mizati yodzaza ndi tinthu ta silica tolumikizidwa ndi C18 chifukwa mu magawo okhazikika a C18, ma ligand a C18 amamangiriridwa ku tinthu ta silica ngati unyolo wolunjika, pomwe mu magawo okhazikika a polystyrene, A yokhuthala kwambiri. Polima wosanjikiza umapangidwa mozungulira. Mu kuyesera kwachizolowezi, porosity ya mzati imawerengedwa motere:
Chithunzi 7A,B chikuwonetsa gawo la PMP (100 × 1.8 mm id) ndi gawo la Ascentis Express RP-Amide (100 × 1.8 mm id) pogwiritsa ntchito mikhalidwe yofanana ya elution (monga, 60/40 ACN/H2O ndi 0.1% TFA). ) la van Deemter plot. Zosakaniza za peptide zosankhidwa (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) zinakonzedwa mu 20 µL/ Kuchuluka kwa kayendedwe ka mizati yonse iwiri ndi 800 µL/min. Ma HETP ocheperako pamlingo woyenera wa kayendedwe (80 µL/min) wa mizati ya PMP ndi mizati ya Ascentis Express RP-Amide anali 2.6 µm ndi 3.9 µm, motsatana. Ma HETP akuwonetsa kuti mphamvu yolekanitsa ya mizati ya PMP (100 × 1.8 mm id) ndi yabwino kwambiri kuposa mizati ya Ascentis Express RP-Amide (100 × 1.8 mm id). Chithunzi cha van Deemter mu Chithunzi 7(A) chikuwonetsa kuti kuchepa kwa mtengo wa N ndi kuchuluka kwa kayendedwe sikofunikira poyerekeza ndi kafukufuku wathu wakale. Kuchuluka kwa mphamvu yolekanitsa ya mizati ya PMP (100 × 1.8 mm id) poyerekeza ndi Ascentis Express RP-Amide column imachokera pa kusintha kwa mawonekedwe a tinthu, kukula, ndi njira zovuta zopakira mizati zomwe zimagwiritsidwa ntchito pantchito yomwe ilipo34.
(A) van Deemter plot (HETP motsutsana ndi liwiro la mzere wa gawo loyenda) yopezeka pogwiritsa ntchito chigawo cha PMP (100 × 1.8 mm id) mu 60/40 ACN/H2O yokhala ndi 0.1% TFA.(B) van Deemter plot (HETP motsutsana ndi liwiro la mzere wa gawo loyenda) yopezeka pogwiritsa ntchito chigawo cha Ascentis Express RP-Amide (100 × 1.8 mm id) mu 60/40 ACN/H2O yokhala ndi 0.1% TFA.
Gawo lokhazikika la polystyrene lopangidwa ndi polar linakonzedwa ndikuyesedwa kuti lilekanitse zosakaniza za peptide zopangidwa ndi trypsin ndi trypsin digests ya human serum albumin (HAS) mu high performance liquid chromatography. Magwiridwe antchito a PMP columns a peptide mixtures ndi abwino kwambiri pakugawa bwino komanso kutsimikiza. Magwiridwe antchito abwino a PMP columns ndi chifukwa cha zifukwa zosiyanasiyana, monga kukula kwa tinthu tating'onoting'ono ndi kukula kwa pore ya tinthu ta silica, kupangika kolamulidwa kwa gawo lokhazikika, komanso kulongedza kovuta kwa column. Kuphatikiza pa magwiridwe antchito apamwamba, kuthamanga kochepa kwa column back pamlingo wothamanga kwambiri ndi ubwino wina wa gawo lokhazikika ili. PMP columns imasonyeza kuberekana bwino ndipo ingagwiritsidwe ntchito pofufuza zosakaniza za peptide ndi trypsin digest ya mapuloteni osiyanasiyana. Tikufuna kugwiritsa ntchito column iyi pogawa zinthu zachilengedwe, mankhwala ophatikizika kuchokera ku zomera zamankhwala ndi zotulutsa za bowa mu liquid chromatography. M'tsogolomu, PMP columns idzawunikidwanso kuti igawanitse mapuloteni ndi ma antibodies a monoclonal.
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