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Tinthu ta silica tokhala ndi porous tinakonzedwa pogwiritsa ntchito njira ya sol-gel ndi zosintha zina kuti tipeze tinthu tating'onoting'ono tokhala ndi pore. Tinthu tating'onoting'ono timeneti tinapangidwa ndi N-phenylmaleimide-methylvinyl isocyanate (PMI) ndi styrene kudzera mu reverse chain transfer-fragmentation (RAFT) polymerization kuti tipange N-phenylmaleimide intercalated polyamides. Styrene (PMP) stationary phase. Narrow bore stainless columns (100 × 1.8 mm inner diameter) zinadzazidwa ndi slurry packing. Magwiridwe antchito a PMP column adayesedwa kuti alekanitse chisakanizo cha ma peptide opangidwa ndi ma peptide asanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) ndi tryptic hydrolyzate ya human serum albumin (HAS). Pansi pa mikhalidwe yabwino kwambiri yopezera ma peptide, chiwerengero cha ma plates okhala ndi ma peptides chinafika pa ma plates 280,000/sq.m. Poyerekeza momwe chigawo chopangidwacho chinagawidwira ndi chigawo cha Ascentis Express RP-Amide chamalonda chimagawidwira, zinaoneka kuti mphamvu yogawa ya chigawo cha PMP inali yabwino kuposa chigawo chamalonda pankhani ya mphamvu yogawa ndi kutsimikiza.
Makampani opanga mankhwala a biopharmaceutical akhala msika womwe ukukula padziko lonse lapansi ndipo gawo lawo la msika lawonjezeka kwambiri m'zaka zaposachedwa. Chifukwa cha kukula kwakukulu kwa makampani opanga mankhwala a biopharmaceutical1,2,3, pakufunika kwambiri kusanthula kwa peptide ndi mapuloteni. Kuphatikiza pa peptide yofunikira, zonyansa zosiyanasiyana zimapangidwa panthawi yopangira peptide, kotero kuyeretsa kwa chromatographic ndikofunikira kuti peptidi ikhale yoyera. 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 pofufuza ma peptide ndi mapuloteni, ngati zitsanzo zotere zilowetsedwa mwachindunji mu mass spectrometer, kulekanitsidwako sikudzakhala kokhutiritsa. Vutoli likhoza kuthetsedwa pochita chromatography yamadzimadzi (LC) musanayese kusanthula kwa MS, zomwe zimachepetsa kuchuluka kwa ma analytes omwe amalowa mu mass spectrometer panthawi inayake4,5,6. Kuphatikiza apo, ma analytes amatha kuyang'ana m'dera lopapatiza panthawi yolekanitsa gawo lamadzimadzi, potero amaika ma analytes awa ndikuwonjezera chidwi cha kuzindikira kwa MS. Chromatography yamadzimadzi (LC) yapita patsogolo kwambiri m'zaka khumi zapitazi ndipo yakhala njira yogwiritsidwa ntchito kwambiri pofufuza za proteomic7,8,9,10.
Chromatography yamadzimadzi ya reverse-phase (RP-LC) imagwiritsidwa ntchito kwambiri kuyeretsa ndi kulekanitsa zosakaniza za ma peptide pogwiritsa ntchito octadecyl-modified silica (ODS) ngati gawo lokhazikika11,12,13. Komabe, chifukwa cha kapangidwe kawo kovuta komanso mtundu wa amphoteric, magawo 14,15 a RP stationary sangapereke kulekanitsa kokwanira kwa ma peptide ndi mapuloteni. Chifukwa chake, kusanthula ma peptide ndi mapuloteni okhala ndi zidutswa za polar ndi non-polar kumafuna magawo okhazikika opangidwa mwapadera kuti agwirizane ndikusunga 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 okhazikika amitundu yosiyanasiyana adakonzedwa ndipo mizati yodzazidwa ndi magawo okhazikika awa idagwiritsidwa ntchito kulekanitsa ma peptide ndi mapuloteni17,18,19,20,21. Chifukwa cha kukhalapo kwa magulu a polar ndi non-polar, magawo osakanikirana a stationary (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) ndi oyenera kulekanitsa ma peptide ndi mapuloteni22,23,24,25,26,27,28. , magawo okhazikika a polar intercalated okhala ndi magulu a polar ogwirizana bwino amasonyeza kuthekera kolekanitsa bwino komanso kusankha kwapadera kwa analytes a polar ndi non-polar chifukwa kulekanitsa kumadalira kuyanjana pakati pa analyte ndi gawo lokhazikika Multimodal interactions 29,30,31,32. Posachedwapa, Zhang et al. 30 adapeza magawo okhazikika a polyamines okhala ndi behenyl ndipo adalekanitsa bwino ma hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, ndi ma analytes ena. Zinthu zokhazikika zomwe zili mu polar zili ndi magulu a polar ndi non-polar, kotero zimatha kugwiritsidwa ntchito kulekanitsa ma peptides ndi mapuloteni kukhala magawo a hydrophobic ndi hydrophilic. Ma columns okhala ndi polar (monga C18 columns okhala ndi amide inline) amapezeka pansi pa dzina lamalonda lakuti Ascentis Express RP-Amide columns, koma ma columns awa agwiritsidwa ntchito pongofufuza amine 33.
Mu kafukufuku wapano, gawo lokhazikika lokhazikika (N-phenylmaleimide, embedding polystyrene) linakonzedwa ndikuyesedwa kuti liwone kusiyana kwa peptide ndi tryptic HSA cleavage. Njira yotsatirayi idagwiritsidwa ntchito pokonzekera gawo lokhazikika. Tinthu ta silica tokhala ndi porous tinakonzedwa motsatira njira zomwe zafotokozedwa m'mabuku athu am'mbuyomu, ndi kusintha kwina mu njira zokonzekera 31, 34, 35, 36, 37, 38, 39. Ma ratios a urea, polyethylene glycol (PEG), TMOS ndi aqueous-acetic acid adasinthidwa kuti apeze tinthu ta silica tokhala ndi ma pore akuluakulu. Kachiwiri, phenylmaleimide-methylvinyl isocyanate ligand yatsopano idapangidwa ndipo tinthu ta silica tomwe timachokera ku polar tinagwiritsidwa ntchito pokonzekera magawo okhazikika okhazikika. Gawo lokhazikika lomwe linapezedwa linayikidwa mu mzati wachitsulo chosapanga dzimbiri (m'mimba mwake wamkati 100 × 1.8 mm) malinga ndi dongosolo lokonzekera bwino. Kulongedza kwa mzati kumathandizidwa ndi kugwedezeka kwa makina kuti zitsimikizire kuti pali gawo lofanana mkati mwa mzati. Mzere wodzaza unayesedwa kuti ulekanitse chisakanizo cha ma peptides okhala ndi ma peptides asanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). ndi tryptic hydrolysates ya human serum albumin (HSA). Zinaonedwa kuti chisakanizo cha peptide ndi HSA tryptic digest zinalekanitsidwa bwino komanso moyenera. Kutha kwa PMP column kunayerekezeredwa ndi kwa Ascentis Express RP-Amide column. Zinaonedwa kuti ma peptides ndi mapuloteni ali ndi mphamvu yabwino komanso mphamvu yayikulu yolekanitsa pa PMP column, ndipo mphamvu yolekanitsa ya PMP column ndi yapamwamba kuposa ya Ascentis Express RP-Amide column.
PEG (polyethylene glycol), urea, acetic acid, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, human serum albumin (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride (MC), styrene, 4-hydroxy- TEMPO, benzoyl peroxide (BPO), acetonitrile (ACN) ya HPLC, methanol, 2-propanol ndi acetone. Sigma-Aldrich Company (St. Louis, Missouri, USA).
Chisakanizo cha urea (8 g), polyethylene glycol (8 g) ndi 8 ml ya 0.01 N. acetic acid chinasakanizidwa kwa mphindi 10, ndipo 24 ml ya TMOS inawonjezedwa pamenepo pansi pa kuziziritsidwa ndi ayezi. Chisakanizocho chinatenthedwa pa 40°C kwa maola 6 kenako pa 120°C kwa maola 8 mu autoclave yachitsulo chosapanga dzimbiri. Madziwo anachotsedwa ndipo zotsalirazo zinaumitsidwa pa 70°C kwa maola 12. Mabuloko ofewa ouma anaphwanyidwa bwino ndikuyikidwa mu uvuni pa 550°C kwa maola 12. Magulu atatu anakonzedwa ndikuyesedwa kuti ayesere kubwerezabwereza kukula kwa tinthu, kukula kwa ma pore ndi malo a pamwamba.
Gulu la polar ndi gawo lokhazikika la unyolo wa polystyrene. Njira yokonzekera yafotokozedwa pansipa.
N-phenylmaleimide (200 mg) ndi methyl vinyl isocyanate (100 mg) zinasungunuka mu anhydrous toluene, kenako 0.1 ml ya 2,2′-azoisobutyronitrile (AIBN) inawonjezedwa ku reaction flask kuti ipange copolymer ya phenylmaleimide ndi methyl vinyl isocyanate (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), tinasakaniza ndi kusakaniza kwa mphindi 10 mu botolo lozungulira la 500 ml. PMCP (10 mg) inasungunuka mu toluene ndikuwonjezeredwa ku botolo loyankhira kudzera mu funnel yowonjezera. Chosakanizacho chinasungunuka pa 100°C kwa maola 8, chinasefedwa, chinatsukidwa ndi acetone ndikuumitsidwa pa 60°C kwa maola 3. Kenako, tinthu ta silika tomwe timagwirizana ndi PMCP (100 g) tinasungunuka mu toluene (200 ml), ndipo 4-hydroxy-TEMPO (2 ml) inawonjezedwa pamenepo 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 ku 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 usiku wonse pa 60°C. Ndondomeko yonse ya zomwe zachitika ikuwonetsedwa pachithunzi choyamba.
Zitsanzozo zinachotsedwa mpweya pa 393 K kwa ola limodzi mpaka mphamvu yotsalira ya zosakwana 10–3 Torr itapezeka. Kuchuluka kwa N2 komwe kunayamwa pa mphamvu yochepera P/P0 = 0.99 kunagwiritsidwa ntchito kudziwa kuchuluka kwa ma pore onse. Kapangidwe ka tinthu ta silica toyera ndi tomwe tinamangidwa ndi ligand kanayesedwa pogwiritsa ntchito microscope ya scanning electron (Hitachi High Technologies, Tokyo, Japan). Zitsanzo zouma (tinthu ta silica toyera ndi tomwe tinamangidwa ndi ligand) zinayikidwa pa ndodo za aluminiyamu pogwiritsa ntchito tepi ya carbon. Golide anaikidwa pa chitsanzocho pogwiritsa ntchito chipangizo chothira madzi cha Q150T, ndipo Au layer yokhuthala ya 5 nm inayikidwa pa chitsanzocho. Izi zimathandizira kuti njira yochepetsera mphamvu ya magetsi igwire bwino ntchito ndipo zimapereka kupopera kozizira bwino. Kusanthula kwa elemental kunachitika pogwiritsa ntchito Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental composition analyzer. Chowunikira kukula kwa tinthu ta Malvern (Worcestershire, UK) Mastersizer 2000 chinagwiritsidwa ntchito kupeza kugawa kwa kukula kwa tinthu. Tinthu ta silika tosaphimbidwa ndi tinthu ta silika tomwe timakhala 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 kumachitika pa liwiro la 5 °C pamphindi imodzi kutentha kuyambira 30 mpaka 800 °C.
Zipilala zachitsulo zosapanga dzimbiri zokhala ndi ulusi wagalasi zokhala ndi miyeso yopapatiza (ID 100 × 1.8 mm) zinapakidwa ndi njira yodzaza ndi slurry motsatira njira yomweyi monga momwe zilili mu bukhu 31. Chipilala chachitsulo chosapanga dzimbiri (chokhala ndi galasi, ID 100 × 1 .8 mm) ndi chotulutsira chokhala ndi 1 µm frit chinalumikizidwa ku makina opakira slurry (Alltech Deerfield, IL, USA). Konzani kuyimitsidwa kwa gawo losapanga dzimbiri poyimitsa 150 mg ya gawo losapanga dzimbiri mu 1.2 ml ya methanol ndikuyiyika mu mzati wosungiramo madzi. Methanol idagwiritsidwa ntchito ngati slurry solvent ndi control solvent. Ikani mzatiwo poyika njira yokakamiza ya 100 MP kwa mphindi 10, 80 MP kwa mphindi 15, ndi 60 MP kwa mphindi 30. Njira yopakira idagwiritsa ntchito ma vibrator awiri a gasi chromatography column (Alltech, Deerfield, IL, USA) kuti zigwedezeke kuti zitsimikizire kuti zipilalazo zikufanana. Tsekani chopakira slurry ndikutulutsa kuthamanga pang'onopang'ono kuti chingwe chisawonongeke. Mzatiwo unachotsedwa pa nozzle ya slurry ndipo cholumikizira china chinalumikizidwa ku cholowera ndipo chinalumikizidwa ku makina a LC kuti ayesere momwe amagwirira ntchito.
MLC yapadera inapangidwa pogwiritsa ntchito pampu ya LC (10AD Shimadzu, Japan), sampler yokhala ndi lupu ya 50 nL injection (Valco (USA) C14 W.05), membrane degasser (Shimadzu DGU-14A), ndi UV-VIS capillary window. Detector device (UV-2075) ndi enamelled microcolumn. Gwiritsani ntchito machubu olumikizana opapatiza komanso afupiafupi kuti muchepetse zotsatira za kukulitsa kwina kwa mizati. Mukadzaza mizati, ikani capillary (50 µm id 365) pamalo otulukira 1/16″ reducing junction ndikuyika capillary (50 µm) ya reducing junction. Kusonkhanitsa deta ndi kukonza chromatogram kumachitika pogwiritsa ntchito pulogalamu ya Multichro 2000. Pa 254 nm, UV absorbance ya subjects analytes inayang'aniridwa pa 0. Deta ya Chromatographic inasanthulidwa pogwiritsa ntchito OriginPro8 (Northampton, MA).
Albumin ya seramu ya munthu, ufa wophilized, ≥ 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 linasakanizidwa kwa mphindi 10 ndipo linasungidwa m'madzi osamba pa 37°C kwa maola 6, kenako linazimitsidwa ndi 1 ml ya 0.1% TFA. Sefa yankholo ndikusunga pansi pa 4°C.
Kulekanitsa kwa chisakanizo cha ma peptide ndi tryptic digest HSA pa chigawo cha PMP kunayesedwa padera. Yang'anani tryptic hydrolysis ya chisakanizo cha ma peptide ndi HSA cholekanitsidwa ndi chigawo cha PMP ndikuyerekeza zotsatira ndi chigawo cha Ascentis Express RP-Amide. Chiwerengero cha ma plate a theoretical chimawerengedwa pogwiritsa ntchito equation yotsatirayi:
Zithunzi za SEM za tinthu ta silika toyera ndi tinthu ta silika tomangidwa ndi ligand zikuwonetsedwa mu Chithunzi 2. Zithunzi za SEM za tinthu ta silika toyera (A, B) zikuwonetsa mawonekedwe ozungulira momwe tinthu ta silika timatalikira kapena tili ndi kusinthasintha kosasinthasintha poyerekeza ndi maphunziro athu am'mbuyomu. Pamwamba pa tinthu ta silika tomangidwa ndi ligand (C, D) ndi yosalala kuposa ya tinthu ta silika toyera, zomwe zitha kukhala chifukwa cha unyolo wa polystyrene womwe umaphimba pamwamba pa tinthu ta silika.
Kusanthula ma electron micrograph a tinthu ta silika tokha (A, B) ndi tinthu ta silika tomwe timalumikizidwa ndi ligand (C, D).
Kufalikira kwa tinthu tating'onoting'ono ta tinthu ta silika tokha ndi tinthu ta silica tomwe timamangidwa ndi ligand kukuwonetsedwa mu Chithunzi 2. 3(A). Ma curve a kukula kwa tinthu ta volumetric adawonetsa kuti kukula kwa tinthu ta silika kudakwera pambuyo pa kusintha kwa mankhwala (Chithunzi 3A). Deta yogawa kukula kwa tinthu ta silika kuchokera ku kafukufuku wapano ndi kafukufuku wam'mbuyomu ikuyerekezeredwa mu Table 1(A). Kukula kwa tinthu ta volumetric d(0.5) kwa PMP kunali 3.36 µm, poyerekeza ndi mtengo wa ad(0.5) wa 3.05 µm mu kafukufuku wathu wam'mbuyomu (tinthu ta silica tomangidwa ndi polystyrene)34. Chifukwa cha kusintha kwa chiŵerengero cha PEG, urea, TMOS ndi acetic acid mu reaction mix, kufalikira kwa tinthu ta gulu ili kunali kocheperako poyerekeza ndi kafukufuku wathu wam'mbuyomu. Kukula kwa tinthu ta PMP ndi kwakukulu pang'ono kuposa kwa gawo la tinthu ta silica tomangidwa ndi polystyrene lomwe tidaphunzira kale. Izi zikutanthauza kuti kugwira ntchito kwa pamwamba pa tinthu ta silika ndi styrene kunangoyika polystyrene wosanjikiza (0.97 µm) 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 timagwirizana ndi ligand.
Kukula kwa ma pore, kuchuluka kwa ma pore, ndi malo a pamwamba pa tinthu ta silica tomwe tagwiritsidwa ntchito mu kafukufukuyu zikuwonetsedwa mu Gome 1 (B). Ma profiles a PSD a tinthu ta silica tokha ndi tinthu ta silica tomwe tamangidwa ndi ligand akuwonetsedwa mu Zithunzi 3 (B). Zotsatira zake zinali zofanana ndi kafukufuku wathu wakale34. Kukula kwa ma pore a tinthu ta silica tokha ndi tomwe tamangidwa ndi ligand kunali 310 Å ndi 241 Å, motsatana, zomwe zikusonyeza kuti pambuyo pa kusintha kwa mankhwala, kukula kwa ma pore kunachepa ndi 69 Å, monga momwe zasonyezedwera mu Gome 1 (B), ndipo shift curve ikuwonetsedwa mu Chithunzi. Malo enieni a pamwamba pa tinthu ta silica mu kafukufukuyu ndi 116 m2/g, zomwe zikufanana ndi kafukufuku wathu wakale (124 m2/g). Monga momwe zasonyezedwera mu Gome 1 (B), malo a pamwamba (m2/g) a tinthu ta silica pambuyo pa kusintha kwa mankhwala nawonso anachepa kuchoka pa 116 m2/g kufika pa 105 m2/g.
Zotsatira za kusanthula kwa elemental kwa gawo lokhazikika zaperekedwa mu Gome. 2. Kuchuluka kwa kaboni mu gawo lokhazikika la pano ndi 6.35%, komwe kuli kotsika kuposa mu kafukufuku wathu wakale (tinthu ta silika tomwe timagwirizanitsidwa ndi polystyrene, 7.93%35 ndi 10.21%, motsatana) 42. Kuchuluka kwa kaboni mu gawo lokhazikika la pano pansipa, popeza ma polar ligands ena monga phenylmaleimide methyl vinyl isocyanate (PCMP) ndi 4-hydroxy-TEMPO agwiritsidwa ntchito kuwonjezera pa styrene pokonzekera SP. Kulemera kwa nayitrogeni mu gawo lokhazikika la pano ndi 2.21% poyerekeza ndi 0.1735 ndi 0.85% mu maphunziro am'mbuyomu42. Izi zikutanthauza kuti gawo lokhazikika la pano lili ndi kuchuluka kwakukulu kwa nayitrogeni chifukwa cha phenylmaleimide. Mofananamo, zinthu (4) ndi (5) zili ndi mpweya wa 2.7% ndi 2.9%, motsatana, pomwe chinthu chomaliza (6) chili ndi mpweya wa 6.35%, monga momwe zasonyezedwera mu Table 2. Kusanthula kwa Thermogravimetric (TGA) kunagwiritsidwa ntchito pa gawo lokhazikika la PMP kuyesa kuchepetsa thupi, ndipo mzere wa TGA wawonetsedwa mu Chithunzi 4. Mzere wa TGA ukuwonetsa kuchepa thupi kwa 8.6%, komwe kukugwirizana bwino ndi mpweya wa carbon (6.35%), popeza ma ligands alibe C yokha, komanso N, O ndi H.
Ligand phenylmaleimide-methylvinyl isocyanate idasankhidwa kuti isinthe pamwamba pa tinthu ta silica chifukwa cha 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 phenylmaleimide moiety ilibe 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 chifukwa limasintha polarity ya gawo lokhazikika. Kusanthula kwa elemental kunachitika kuti muwone kuchuluka kwa kaboni m'magawo awa okhazikika. Zawonedwa kuti kuwonjezera kuchuluka kwa styrene ndi nthawi yochitapo kanthu kumawonjezera kuchuluka kwa kaboni mu gawo lokhazikika komanso mosemphanitsa. Ma SP okonzedwa ndi kuchuluka kosiyana kwa styrene ali ndi mitundu yosiyanasiyana ya carbon loads. Mofananamo, magawo osasinthasintha awa adayikidwa pazitsulo zosapanga dzimbiri ndipo mawonekedwe awo a chromatographic (kusankha, kutsimikiza, mtengo wa N, ndi zina zotero) adawunikidwa. Kutengera ndi zoyeserera izi, kapangidwe kabwino kokonzekera gawo losasinthika la PMP kanasankhidwa kuti kapereke polarity yolamulidwa komanso kusunga bwino kwa analyte.
Gawo la PMP linayesedwanso kuti lifufuze mitundu isanu ya ma peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) pogwiritsa ntchito mphamvu ya gawo loyenda. 60/40 (v/v) ACN/madzi (0.1% TFA) pamlingo woyenda wa 80 µl/min. Pansi pa mikhalidwe yabwino kwambiri yoyeretsera (ma plate 200,000/m2), chiwerengero cha ma plate a theoretical (N) pa chigawo chilichonse (100 × 1.8 mm) ndi 20,000 ± 100. Ma N values ​​​​a ma colon atatu a PMP akuwonetsedwa mu Table 3 ndipo ma chromatogram akuwonetsedwa mu Chithunzi 5A. Kusanthula mwachangu pa liwiro lalikulu la kuyenda (700 µl/min) pa kolamu ya PMP, ma peptide asanu adatuluka mkati mwa mphindi imodzi, mtengo wabwino kwambiri wa N wa 13,500 ± 330 pa kolamu iliyonse (100 x 1.8 mm m'mimba mwake), wofanana ndi mbale 135,000/m (Chithunzi 5B). Zipilala zitatu za kukula kofanana (m'mimba mwake wamkati 100 x 1.8 mm) zidadzazidwa ndi magulu atatu osiyanasiyana a gawo lokhazikika la PMP kuti ayesere kuberekanso. Ma analytes adalembedwa pa kolamu iliyonse polekanitsa kusakaniza komweko kwa mayeso pa kolamu iliyonse pogwiritsa ntchito mikhalidwe yabwino kwambiri yolumikizira, chiwerengero cha mbale zamalingaliro N, ndi nthawi yosungira. Deta yobwerezabwereza ya zipilala za PMP ikuwonetsedwa mu Gome 4. Kubwerezabwereza kwa kolamu ya PMP kunagwirizana bwino ndi ma %RSD values ​​​​otsika kwambiri monga momwe zasonyezedwera mu Gome 3.
Kulekanitsa zosakaniza za peptide pa kolamu ya PMP (B) ndi kolamu ya Ascentis Express RP-Amide (A), gawo loyenda 60/40 ACN/H2O (TFA 0.1%), miyeso ya kolamu ya PMP (100 x 1.8 mm id), kusanthula 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 (leucic acid enkephalin).
Cholembera cha PMP (mkati mwake 100 x 1.8 mm) chinayesedwa kuti chiwonetse kulekanitsidwa kwa tryptic hydrolyzate ya human serum albumin ndi HPLC. Chromatogram yomwe ili pa Chithunzi 6 ikuwonetsa kuti zitsanzozo zalekanitsidwa bwino ndi resolution yabwino kwambiri. Mayankho a HSA adawunikidwa pogwiritsa ntchito flow rate ya 100 μl/min, gawo loyenda la 70/30 acetonitrile/madzi ndi 0.1% TFA. Kugawikana kwa HSA kunagawidwa m'magawo 17, monga momwe zasonyezedwera mu chromatogram (Chithunzi 6), mogwirizana ndi ma peptide 17. Kusiyanitsa bwino kwa ma peaks payokha kuchokera ku HSA hydrolyzate kunawerengedwa ndipo ma values ​​​​akuwonetsedwa mu Table 5.
Ma hydrolysates a HSA tryptic adalekanitsidwa pa column ya PMP (mkati mwake 100 x 1.8 mm), flow rate (100 μl/min), mobile phase 60/40 acetonitrile/water, ndi 0.1% TFA.
kumene L ndi kutalika kwa mzati, η ndi kukhuthala kwa gawo loyenda, ΔP ndi kupsinjika kumbuyo kwa mzati, 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, 60/40 v/v kunagwiritsidwa ntchito. ACN/madzi. Kutha kwa mzati wa PMP (ID 100 × 1.8 mm) kunali kofanana ndi kwa kafukufuku wathu wakale wa Ref.34. Kutha kwa mzati wodzazidwa ndi tinthu tokhala ndi mapokoso pang'ono ndi 1.7×10 .6 µm, 2.5×10-14 m2 pa tinthu ta 5 µm43. Chifukwa chake, kutha kwa mzati wa PMP kuli kofanana ndi kutha kwa tinthu ta core-shell tokhala ndi kukula kwa 5 μm.
kumene Wx ndi kulemera kwa chigawo chodzazidwa ndi chloroform, Wy ndi kulemera kwa chigawo chodzazidwa ndi methanol, ndipo ρ ndi kuchuluka kwa solvent. Kuchuluka kwa methanol (ρ = 0.7866) ndi chloroform (ρ = 1.484). Kuchuluka kwa porosity ya chigawo cha tinthu ta silica-C18 (100 × 1.8 mm ID)34 ndi chigawo chathu cha C18-urea31 chomwe chidaphunziridwa kale chinali 0.63 ndi 0.55, motsatana. Izi zikutanthauza kuti kukhalapo kwa urea ligands kumachepetsa kulowerera kwa gawo lokhazikika. Kumbali ina, porosity yonse ya chigawo cha PMP (m'mimba mwake wamkati 100 × 1.8 mm) ndi 0.60. Mizati ya PMP siiloledwa kulowa pansi kuposa mizati yodzaza ndi tinthu ta silica tomangidwa ndi C18 chifukwa mu magawo okhazikika a mtundu wa C18, ma ligand a C18 amamangiriridwa ku tinthu ta silica mu unyolo wolunjika, pomwe mu magawo okhazikika a mtundu wa polystyrene, polima wokhuthala amapangidwa mozungulira tinthu. Gawo A. Mu kuyesera kwachizolowezi, kupendekeka kwa mzati kumawerengedwa motere:
Pa chithunzi 7A, B ikuwonetsa zithunzi za Van Deemter za kolamu ya PMP (id 100 x 1.8 mm) ndi kolamu ya Ascentis Express RP-Amide (id 100 x 1.8 mm) pansi pa mikhalidwe yofanana ya elution, 60/40 ACN/H2O ndi 0 .1% TFA 20 µl/min mpaka 800 µl/min pa makolamu onse awiri. Mitengo yocheperako ya HETP pamlingo woyenera wa kuyenda (80 µl/min) inali 2.6 µm ndi 3.9 µm pa kolamu ya PMP ndi kolamu ya Ascentis Express RP-Amide, motsatana. Mitengo ya HETP ikuwonetsa kuti mphamvu yolekanitsa ya kolamu ya PMP (100 x 1.8 mm id) ndi yayikulu kwambiri kuposa ya kolamu ya Ascentis Express RP-Amide (100 x 1.8 mm id). Chithunzi cha van Deemter chomwe chili mu Chithunzi 7(A) chikuwonetsa kuti kuchepa kwa mtengo wa N sikukwera kwambiri poyerekeza ndi kuchuluka kwa madzi poyerekeza ndi kafukufuku wathu wakale. Kuchuluka kwa mphamvu yolekanitsa kwa PMP column (id 100 × 1.8 mm) poyerekeza ndi Ascentis Express RP-Amide column kumadalira mawonekedwe ndi kukula kwa tinthu tating'onoting'ono komanso njira yodziwika bwino yopakira mizati yomwe imagwiritsidwa ntchito pa ntchito yomwe ilipo34.
(A) Chithunzi cha Van Deemter (HETP vs. mobile phase linear velocity) chopezeka pa PMP column (id 100 x 1.8 mm) mu 60/40 ACN/H2O yokhala ndi 0.1% TFA. (B) Chithunzi cha Van Deemter (HETP vs mobile phase linear velocity) chopezeka pa Ascentis Express RP-Amide column (id 100 x 1.8 mm) mu 60/40 ACN/H2O yokhala ndi 0.1% TFA.
Gawo lokhazikika la polar stationary la polystyrene yolumikizidwa linakonzedwa ndikuyesedwa kuti lilekanitse chisakanizo cha ma peptide opangidwa ndi tryptic hydrolyzate ya human serum albumin (HSA) mu high performance liquid chromatography. Magwiridwe antchito a PMP columns a peptide mixtures ndi abwino kwambiri pankhani ya kulekanitsa bwino komanso kutsimikiza. Kupambana kwa kulekanitsa bwino kwa PMP columns kumachitika chifukwa cha zifukwa zingapo monga kukula kwa tinthu ta silica ndi kukula kwa pore, kulamulira kaphatikizidwe ka magawo okhazikika, ndi zinthu zovuta zonyamula column. Kuphatikiza pa magwiridwe antchito apamwamba, ubwino wina wa gawo lokhazikika ili ndi kupsinjika kochepa kumbuyo kwa column pamlingo wothamanga kwambiri. PMP columns ndi yoberekanso kwambiri ndipo ingagwiritsidwe ntchito kusanthula zosakaniza za ma peptide ndi tryptic digestion ya mapuloteni osiyanasiyana. Tikufuna kugwiritsa ntchito column iyi kuti tilekanitse zinthu zachilengedwe kuchokera ku zinthu zachilengedwe, zotulutsa za zomera zamankhwala ndi bowa mu liquid chromatography. Mtsogolomu, PMP columns idzawunikidwanso kuti ilekanitse mapuloteni ndi ma monoclonal antibodies.
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