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Tinthu tating'onoting'ono ta silika tinakonzedwa ndi njira ya sol-gel ndi zosintha zina kuti tipeze tinthu tating'onoting'ono ta pore. Izi particles anali derivatized ndi N-phenylmaleimide-methylvinyl isocyanate (PMI) ndi styrene kudzera n'zosiyana unyolo kusamutsa kugawanika (RAFT) polymerization kubala N-phenylmaleimide intercalated polyamides. Styrene (PMP) stationary gawo. Mizati yopapatiza yokhala ndi zitsulo zosapanga dzimbiri (100 × 1.8 mm m'mimba mwake) inali yodzaza ndi zotengera zotayirira. Maonekedwe a chromatographic pagawo la PMP adawunikidwa kuti alekanitse chisakanizo cha ma peptides 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 humanS hydrolyzate album. Pansi mu mikhalidwe mulingo woyenera elution, ongoyerekeza chiwerengero cha mbale ndi osakaniza peptides anafika 280,000 mbale/sq.m. Poyerekeza ntchito yolekanitsa ya gawo lotukuka ndi gawo lazamalonda la Ascentis Express RP-Amide, zidawoneka kuti kulekanitsa bwino kwa gawo la PMP kunali kopambana kuposa gawo lazamalonda potengera magwiridwe antchito komanso kusamvana.
Bizinesi ya biopharmaceutical yakhala msika womwe ukukula padziko lonse lapansi ndikuwonjezeka kwakukulu kwa msika m'zaka zaposachedwa. Ndi kukula kwamphamvu kwa mafakitale a biopharmaceutical1,2,3 pakufunika kwambiri kusanthula kwa peptide ndi mapuloteni. Kuphatikiza pa peptide yomwe mukufuna, zonyansa zosiyanasiyana zimapangidwa panthawi ya peptide, kotero kuyeretsedwa kwa chromatographic kumafunika kuti mupeze chiyero chomwe mukufuna. Kusanthula ndi mawonekedwe a mapuloteni m'madzi am'thupi, minofu, ndi maselo ndi ntchito yovuta kwambiri chifukwa cha kuchuluka kwa mitundu yomwe ingathe kuzindikirika yomwe ilipo mu chitsanzo chimodzi. Ngakhale misa spectrometry ndi chida chothandiza potsata ma peptides ndi mapuloteni, ngati zitsanzo zotere zimalowetsedwa mwachindunji mu spectrometer yamisala, kulekanitsa sikungakhale kosangalatsa. Vutoli litha kuthetsedwa popanga chromatography yamadzimadzi (LC) pamaso pa kusanthula kwa MS, komwe kungachepetse kuchuluka kwa ma analytes omwe amalowa mu spectrometer ya misa pa nthawi yoperekedwa4,5,6. Kuphatikiza apo, osanthula amatha kuyang'ana kwambiri kudera lopapatiza panthawi yopatukana yamadzimadzi, potero amangoyang'ana zowunikirazi ndikuwonjezera chidwi cha kuzindikira kwa MS. Liquid chromatography (LC) yapita patsogolo kwambiri m'zaka khumi zapitazi ndipo yakhala njira yogwiritsidwa ntchito kwambiri pakuwunika kwa proteinomic7,8,9,10.
Reverse-phase liquid chromatography (RP-LC) imagwiritsidwa ntchito kwambiri kuyeretsa ndi kulekanitsa zosakaniza za peptides pogwiritsa ntchito octadecyl-modified silica (ODS) monga stationary phase11,12,13. Komabe, chifukwa cha mawonekedwe awo ovuta komanso chikhalidwe cha amphoteric, magawo oima a 14,15 RP sangathe kupereka kulekanitsa kokwanira kwa ma peptide ndi mapuloteni. Chifukwa chake, kusanthula kwa ma peptides ndi mapuloteni okhala ndi zidutswa za polar komanso zopanda polar kumafuna magawo okhazikika opangidwa mwapadera kuti agwirizane ndikusunga zowunikirazi16. Kuphatikizika kwa chromatography, komwe kumapereka kuyanjana kwamitundu yosiyanasiyana, kumatha kukhala njira ina ya RP-LC yolekanitsa ma peptides, mapuloteni, ndi zosakaniza zina zovuta. Magawo angapo osasunthika osasunthika adakonzedwa ndipo mizati yodzazidwa ndi magawo osasunthikawa idagwiritsidwa ntchito kulekanitsa ma peptides ndi mapuloteni17,18,19,20,21. Chifukwa cha kukhalapo kwa magulu a polar ndi osakhala a polar, magawo osakanikirana (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) ndi oyenera kulekanitsa ma peptides ndi mapuloteni22,23,24,25,26,27,28. , magawo a polar intercalated stationary omwe ali ndi magulu a polar ogwirizana amawonetsa luso lolekanitsa bwino komanso kusankha kwapadera kwa ma analyte a polar ndi omwe si a polar chifukwa kulekana kumadalira kuyanjana pakati pa analyte ndi stationary gawo Multimodal interactions 29,30,31,32. Posachedwapa, Zhang et al. 30 adapeza ma polyamines oimitsidwa a behenyl ndipo adalekanitsa bwino ma hydrocarbon, antidepressants, flavonoids, nucleosides, estrogens, ndi ma analyte ena. Zinthu zokhazikika za polar zili ndi magulu onse a polar komanso omwe si a polar, kotero zitha kugwiritsidwa ntchito kulekanitsa ma peptide ndi mapuloteni kukhala magawo a hydrophobic ndi hydrophilic. Mizati ya polar (mwachitsanzo, mizati ya C18 yokhala ndi amide inline) imapezeka pansi pa dzina la malonda la Ascentis Express RP-Amide mizati, koma mizatiyi yangogwiritsidwa ntchito posanthula amine 33.
Mu kafukufuku wapano, gawo loyika polar (N-phenylmaleimide, embedding polystyrene) idakonzedwa ndikuwunikidwa kuti ilekanitse peptide ndi tryptic HSA cleavage. Njira yotsatirayi idagwiritsidwa ntchito pokonzekera gawo loyima. Tinthu tating'onoting'ono ta silika tinakonzedwa molingana ndi njira zomwe tafotokoza m'mabuku athu apitawa, ndikusintha kwina kokonzekera 31, 34, 35, 36, 37, 38, 39. Magawo a urea, polyethylene glycol (PEG), TMOS ndi aqueous-acetic acid adasinthidwa kuti apeze tinthu tating'onoting'ono ta silika. Kachiwiri, phenylmaleimide-methylvinyl isocyanate ligand yatsopano idapangidwa ndipo tinthu tating'ono ta silika tinagwiritsidwa ntchito pokonzekera magawo okhazikika a polar. Gawo loyima lomwe linapezedwa lidadzazidwa ndi chitsulo chosapanga dzimbiri (m'mimba mwake 100 × 1.8 mm) molingana ndi dongosolo lokonzekera bwino. Kuyika kwa gawoli kumathandizidwa ndi kugwedezeka kwamakina kuti kuwonetsetse kuti yunifolomu yosanjikiza mkati mwa ndimeyo. Mzere wodzazawo udawunikidwa kuti apatulidwe osakaniza a peptides okhala ndi ma peptide 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). Zinawonedwa kuti osakaniza peptide ndi HSA tryptic digest analekanitsidwa ndi kusamvana bwino ndi bwino. Kusiyanitsa bwino kwa gawo la PMP kuyerekezedwa ndi gawo la Ascentis Express RP-Amide. Zinawoneka kuti ma peptides ndi mapuloteni ali ndi chisankho chabwino komanso kupatukana kwakukulu pazitsulo za PMP, ndipo kulekanitsa bwino kwa gawo la PMP ndipamwamba kuposa gawo la Ascentis Express RP-Amide.
PEG (polyethylene glycol), urea, acetic acid, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, human serum albumin (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride, TPOxyrene, MC4 (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 inalimbikitsidwa kwa mphindi 10, ndipo 24 ml ya TMOS inawonjezeredwa pamenepo pansi pa kuzizira kwa ayezi. Zomwe osakaniza anali usavutike mtima pa 40 ° C kwa maola 6 ndiyeno pa 120 ° C kwa maola 8 mu zosapanga dzimbiri zitsulo autoclave. Madziwo adachotsedwa ndipo zotsalirazo zidawuma pa 70 ° C kwa maola 12. Zowuma zofewa zimaphwanyidwa bwino ndikuwotchedwa mu uvuni wa 550 ° C kwa maola 12. Magulu atatu adakonzedwa ndikuzindikiridwa kuti ayese kuchulukitsa kwa tinthu tating'onoting'ono, kukula kwa pore ndi kumtunda.
Gulu la polar ndi gawo loyima la maunyolo a polystyrene. Njira yokonzekera ikufotokozedwa pansipa.
N-phenylmaleimide (200 mg) ndi methyl vinyl isocyanate (100 mg) zinasungunuka mu anhydrous toluene, ndiyeno 0.1 ml ya 2,2'-azoisobutyronitrile (AIBN) anawonjezedwa ku botolo la zomwe zimachitika kuti apeze copolymer ya phenylmaleimide ndi methylcyanate methylCP (PMM vinyl isocyana). ) The osakaniza anali usavutike mtima pa 60 ° C kwa 3 hours, osasankhidwa ndi zouma mu uvuni pa 40 ° C kwa 3 hours.
Zouma silika particles (2 g) anali omwazika mu youma toluene (100 ml), analimbikitsa ndi sonicated kwa mphindi 10 mu 500 ml yozungulira pansi botolo. PMCP (10 mg) idasungunuka mu toluene ndikuwonjezedwa pang'onopang'ono ku botolo lochitirapo kanthu pogwiritsa ntchito fupa lowonjezera. The osakaniza anali refluxed pa 100 ° C kwa 8 hours, wosefedwa, kutsukidwa ndi acetone ndi zouma pa 60 ° C kwa 3 hours. Kenaka, particles za silika zogwirizana ndi PMCP (100 g) zinasungunuka mu toluene (200 ml), ndipo 4-hydroxy-TEMPO (2 ml) inawonjezeredwa pamenepo pamaso pa 100 μl ya dibutyltin dilaurate monga chothandizira. The osakaniza analimbikitsa pa 50 ° C kwa 8 hours, osasankhidwa ndi zouma pa 50 ° C kwa 3 hours.
Styrene (1 ml), benzoyl peroxide BPO (0.5 ml) ndi tinthu ta silika tophatikizidwa ku TEMPO-PMCP (1.5 g) zidamwazikana mu toluene ndikutsukidwa ndi nayitrogeni. The polymerization wa styrene inachitika pa 100 ° C kwa maola 12. Chotsatiracho chinatsukidwa ndi methanol ndikuwumitsa usiku wonse pa 60 ° C. Ambiri chiwembu cha anachita zikusonyezedwa mkuyu. imodzi .
Zitsanzozo zidachotsedwa pa 393 K kwa 1 h mpaka kukakamizidwa kotsalira kochepera 10-3 Torr kunapezedwa. Kuchuluka kwa N2 adsorbed pa kuthamanga wachibale P/P0 = 0.99 anagwiritsidwa ntchito kudziwa okwana pore voliyumu. Mapangidwe a tinthu tating'onoting'ono ta silika tokhala ndi ligand adawunikidwa pogwiritsa ntchito maikulosikopu ya electron (Hitachi High Technologies, Tokyo, Japan). Zitsanzo zouma (silica woyera ndi ligand womangidwa silika particles) anaikidwa pa ndodo zotayidwa ntchito carbon tepi. Golide adayikidwa pachitsanzocho pogwiritsa ntchito chipangizo cha Q150T, ndipo 5 nm wandiweyani wa Au wosanjikiza adayikidwa pachitsanzocho. Izi bwino dzuwa ndondomeko otsika voteji ndi amapereka zabwino ozizira kupopera mbewu mankhwalawa. Kusanthula kwazinthu kudachitika pogwiritsa ntchito Thermo Electron (Waltham, MA, USA) Flash EA1112 yowunikira yoyambira. Malvern particle size analyzer (Worcestershire, UK) Mastersizer 2000 idagwiritsidwa ntchito kupeza kugawa kwa tinthu. Uncoated silika particles ndi ligand-womangidwa silika particles (5 mg aliyense) anali omwazika mu 5 ml ya isopropanol, sonicated kwa mphindi 10, kukwiya kwa mphindi 5, ndipo anaikidwa pa Mastersizer kuwala benchi. Kusanthula kwa Thermogravimetric kumachitika pamlingo wa 5 ° C pamphindi pa kutentha kuyambira 30 mpaka 800 ° C.
Magalasi okhala ndi mizere yopapatiza yokhala ndi zitsulo zosapanga dzimbiri zokhala ndi miyeso (ID 100 × 1.8 mm) anali odzaza ndi njira yodzaza ndi slurry motsatira njira yofananira ndi 31. Chitsulo chachitsulo chosapanga dzimbiri (galasi lokhala ndi ID 100 × 1 .8 mm) ndi chotulukira chokhala ndi 1 µm fritter fritter, zonse zidalumikizidwa ndi makina a tech delurry. IL, USA). Konzani kuyimitsidwa kwa gawo loyima poyimitsa 150 mg ya gawo loyima mu 1.2 ml ya methanol ndikuyipatsa mzati yosungiramo madzi. Methanol imagwiritsidwa ntchito ngati slurry solvent ndi control solvent. Ikani gawoli pogwiritsa ntchito kukakamiza kwa 100 MP kwa mphindi 10, 80 MP kwa 15 min, ndi 60 MP kwa 30 min. Njira yolongedza idagwiritsa ntchito ma vibrators awiri a gasi a chromatography (Alltech, Deerfield, IL, USA) pakugwedezeka kwamakina kuti atsimikizire kulongedza kofananako. Tsekani slurry packer ndi kumasula kuthamanga pang'onopang'ono kuti muteteze kuwonongeka kwa chingwe. Mzerewu udachotsedwa pamphuno ya slurry ndipo cholumikizira china chidalumikizidwa ndi cholowera ndikulumikizidwa ku LC system kuyesa momwe amagwirira ntchito.
MLC yachizolowezi inamangidwa pogwiritsa ntchito LC pump (10AD Shimadzu, Japan), sampler yokhala ndi 50 nL jekeseni loop (Valco (USA) C14 W.05), membrane degasser (Shimadzu DGU-14A), ndi UV-VIS capillary window. Chipangizo chojambulira (UV-2075) ndi microcolumn enamelled. Gwiritsani ntchito timachubu tating'ono kwambiri komanso tating'onoting'ono tolumikizira kuti muchepetse zotsatira zakukulitsa ndime zina. Mukadzaza gawoli, ikani capillary (50 µm id 365) potuluka pa 1/16 ″ podumphadumpha ndikuyika capillary (50 µm) ya mphambano yochepetsera. Kusonkhanitsa deta ndi kukonza chromatogram kumachitika pogwiritsa ntchito pulogalamu ya Multichro 2000. Pa 254 nm, kuyamwa kwa UV kwa osanthula maphunzirowo kunayang'aniridwa pa 0. Chromatographic data inayesedwa pogwiritsa ntchito OriginPro8 (Northampton, MA).
Albumin ya seramu yamunthu, ufa wa lyophilized, ≥ 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) . Njira yothetsera vutoli idalimbikitsidwa kwa mphindi 10 ndikusungidwa m'madzi osamba pa 37 ° C kwa maola 6, kenako imazimitsidwa ndi 1 ml ya 0,1% TFA. Sefa yankho ndikusunga pansi pa 4°C.
Kupatukana kwa chisakanizo cha peptides ndi tryptic digest HSA pagawo la PMP kudawunikidwa padera. Onani tryptic hydrolysis ya osakaniza a peptides ndi HSA olekanitsidwa ndi gawo la PMP ndikuyerekeza zotsatira ndi gawo la Ascentis Express RP-Amide. Chiwerengero cha mbale za theoretical chimawerengedwa pogwiritsa ntchito equation iyi:
Zithunzi za SEM za tinthu tating'ono ting'onoting'ono ta silika ndi tinthu tating'onoting'ono ta silika tikuwonetsedwa mu Chithunzi 2. Zithunzi za SEM za tinthu tating'onoting'ono ta silika (A, B) zimasonyeza mawonekedwe ozungulira omwe tinthu tating'onoting'ono tating'onoting'ono tating'onoting'ono tating'ono tating'onoting'ono tating'ono tating'ono tating'ono tating'onoting'ono poyerekeza ndi maphunziro athu akale. Pamwamba pa particles silika womangidwa ndi ligand (C, D) ndi yosalala kuposa particles koyera silika, amene mwina chifukwa cha unyolo polystyrene kuphimba pamwamba pa silika particles.
Kusanthula ma electron micrographs a tinthu tating'ono ta silika (A, B) ndi tinthu tating'ono ta silika (C, D).
The tinthu kukula kugawa koyera silika particles ndi ligand-womangidwa silika particles chikuwonetsedwa mkuyu. 2. 3 (A). Volumetric tinthu kukula kugawa zokhotakhota anasonyeza kuti silika tinthu kukula chinawonjezeka pambuyo mankhwala kusinthidwa (mkuyu. 3A). Deta ya silika yogawa kukula kwa tinthu tating'ono kuchokera ku phunziro lamakono ndi phunziro lapitalo likufanizidwa mu Table 1 (A). Volumetric particle size d (0.5) ya PMP inali 3.36 µm, poyerekeza ndi ad (0.5) mtengo wa 3.05 µm mu phunziro lathu lapitalo (polystyrene bonded silica particles)34. Chifukwa cha kusintha kwa chiŵerengero cha PEG, urea, TMOS ndi asidi asidi mu zimene osakaniza, ndi tinthu kukula kugawa mtanda anali yopapatiza poyerekeza ndi phunziro lathu lapita. Kukula kwa tinthu ta gawo la PMP ndikokulirapo pang'ono kuposa gawo la tinthu tating'ono ta polystyrene lomwe tidaphunzira kale. Izi zikutanthauza kuti pamwamba functionalization wa silika particles ndi styrene waikamo kokha polystyrene wosanjikiza (0,97 µm) pa silika pamwamba, pamene PMP gawo wosanjikiza makulidwe anali 1.38 µm.
Tinthu kukula kugawa (A) ndi pore kukula kugawa (B) koyera silika particles ndi ligand womangidwa silika particles.
Kukula kwa pore, voliyumu ya pore, ndi malo a silika omwe amagwiritsidwa ntchito mu phunziroli akuwonetsedwa mu Gulu 1 (B). Mbiri ya PSD ya tinthu tating'ono ting'onoting'ono ta silika ndi tinthu tating'onoting'ono ta silika tikuwonetsedwa mu Mkuyu. 3 (B). Zotsatirazo zinali zofanana ndi phunziro lathu lapitalo34. Kukula kwa pore kwa tinthu tating'onoting'ono ta silika tating'onoting'ono tomwe timakhala ndi 310 Å ndi 241 Å, motsatana, kusonyeza kuti pambuyo pa kusintha kwa mankhwala, kukula kwa pore kunatsika ndi 69 Å, monga momwe tawonetsera mu Table 1 (B), ndi kusintha kosinthika kukuwonetsedwa mkuyu. (124 m2/g). Monga momwe tawonetsera mu Table 1 (B), pamtunda (m2 / g) wa silika pambuyo pa kusintha kwa mankhwala kunatsikanso kuchokera ku 116 m2 / g mpaka 105 m2 / g.
Zotsatira za kusanthula koyambira kwa gawo loyima zimaperekedwa mu Table. 2. Mpweya wa carbon wa gawo lamakono lokhazikika ndi 6.35%, lomwe ndilotsika kusiyana ndi phunziro lathu lapitalo (tinthu tating'ono ta silika togwirizana ndi polystyrene, 7.93% 35 ndi 10.21%, motero) 42. Zomwe zili mu carbon zomwe zili m'munsimu, popeza ma ligand ena a polar monga phenylmaleimide methyl-POPc hydroxy-cyana (hydroxy-proxy) agwiritsidwa ntchito. kuwonjezera pa styrene pokonzekera SP. Kulemera kwa nayitrogeni mu gawo laposachedwa ndi 2.21% poyerekeza ndi 0.1735 ndi 0.85% m'maphunziro am'mbuyomu42. Izi zikutanthauza kuti gawo lomwe lilipo pano lili ndi kulemera kwakukulu kwa nayitrogeni chifukwa cha phenylmaleimide. Mofananamo, mankhwala (4) ndi (5) ali ndi mpweya wa 2.7% ndi 2.9%, motero, pamene mankhwala omaliza (6) ali ndi mpweya wa 6.35%, monga momwe tawonetsera mu Table 2. Kusanthula kwa Thermogravimetric (TGA) kunagwiritsidwa ntchito pa gawo lokhazikika la PMP kuti ayese kulemera kwake, ndipo TGA curve ya TGA curve ikuwonetsedwa mu TGA. 8.6%, zomwe zimagwirizana bwino ndi mpweya wa carbon (6.35%), popeza ma ligand ali ndi C , komanso N, O ndi H.
Ligand phenylmaleimide-methylvinyl isocyanate inasankhidwa kuti isinthe pamwamba pa particles silika chifukwa cha polar phenylmaleimide ndi vinylisocyanate magulu. Magulu a vinyl isocyanate amathanso kuchitapo kanthu ndi styrene pakukhala ma polymerization. Chifukwa chachiwiri ndikuyika gulu lomwe limalumikizana pang'onopang'ono ndi analyte ndipo palibe kuyanjana kwamphamvu kwa electrostatic pakati pa analyte ndi gawo loyima, popeza phenylmaleimide moiety ilibe ndalama zenizeni pa pH yabwinobwino. The polarity wa stationary gawo akhoza lizilamuliridwa ndi mulingo woyenera kwambiri kuchuluka kwa styrene ndi anachita nthawi ya ufulu ankafuna kusintha polymerization. Gawo lomaliza la zomwe zimachitika (free radical polymerization) ndizovuta chifukwa zikusintha polarity ya gawo loyima. Kusanthula koyambira kunachitika kuti awone zomwe zili mu kaboni mu magawo osasunthika awa. Zawonedwa kuti kuchulukitsa kuchuluka kwa styrene komanso nthawi yochitira kumawonjezera kaboni wagawo loyima komanso mosemphanitsa. Ma SP okonzedwa ndi mitundu yosiyanasiyana ya styrene amakhala ndi katundu wosiyanasiyana wa kaboni. Mofananamo, magawo osayimawa adayikidwa pazitsulo zosapanga dzimbiri ndipo mawonekedwe ake a chromatographic (kusankha, kuthetsa, mtengo wa N, ndi zina zotero) adafufuzidwa. Kutengera zoyesererazi, kapangidwe kokonzedwa bwino kokonzekera gawo loyima la PMP idasankhidwa kuti ipereke polarity yoyendetsedwa ndikusungidwa bwino kwa analyte.
Gawo la PMP lidawunikidwanso kuti liwunike zosakaniza zisanu za peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) pogwiritsa ntchito gawo la mafoni. 60/40 (v/v) ACN/madzi (0.1% TFA) pakuyenda kwa 80 µl/mphindi. Pansi pazikhalidwe zowoneka bwino (200,000 mbale / m), kuchuluka kwa mbale zongoyerekeza (N) pagawo lililonse (100 × 1.8 mm) ndi 20,000 ± 100. Miyezo ya N pamizati itatu ya PMP ikuwonetsedwa mu Gulu 3 ndipo ma chromatogram akuwonetsedwa Chithunzi 5A. Kusanthula kwachangu pamlingo wothamanga kwambiri (700 µl / min) pagawo la PMP, ma peptide asanu otulutsidwa mkati mwa mphindi imodzi, mtengo wabwino kwambiri wa N wa 13,500 ± 330 pagawo (100 x 1.8 mm m'mimba mwake), wofanana ndi mbale za 135,000 / m (mkuyu 5B). Mipingo itatu yofanana (m'mimba mwake 100 x 1.8 mm) inadzazidwa ndi magulu atatu osiyana a gawo loyima la PMP kuti ayese kuberekanso. Zowunikira zidalembedwa pagawo lililonse polekanitsa kusakaniza kofananako pagawo lililonse pogwiritsa ntchito mikhalidwe yabwino kwambiri, kuchuluka kwa mbale zongoyerekeza N, ndi nthawi yosungira. Kuchulukitsa kwa magawo a PMP kukuwonetsedwa mu Gulu 4. Kupangidwanso kwa gawo la PMP kumagwirizana bwino ndi zotsika kwambiri za %RSD monga zikuwonetsedwa mu Gulu 3.
Kupatukana kwa peptide zosakaniza pa PMP ndime (B) ndi Ascentis Express RP-Amide ndime (A), mobile gawo 60/40 ACN/H2O (TFA 0.1%), PMP ndime miyeso (100 x 1.8 mm id) , kusanthula Elution dongosolo la mankhwala: 1 (Gly-Tyr-Tyr), 2 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) ndi 5 (leucic acid enkephalin).
Mzere wa PMP (m'mimba mwake wa 100 x 1.8 mm) adawunikidwa pakulekanitsa tryptic hydrolyzate ya serum albumin yamunthu ndi HPLC. Chromatogram mu Chithunzi 6 ikuwonetsa kuti zitsanzozo zimasiyanitsidwa bwino ndi kusamvana kwabwino kwambiri. Mayankho a HSA adawunikidwa pogwiritsa ntchito kuthamanga kwa 100 μl/min, gawo la mafoni a 70/30 acetonitrile/madzi ndi 0.1% TFA. Cleavage wa HSA linagawidwa nsonga 17, monga momwe chromatogram (mkuyu. 6), lolingana 17 peptides. Kuthekera kolekanitsa kwa nsonga zapayekha kuchokera ku HSA hydrolyzate kunawerengeredwa ndipo zikhalidwe zikuwonetsedwa mu Gulu 5.
Ma HSA tryptic hydrolysates adapatulidwa pagawo la PMP (m'mimba mwake 100 x 1.8 mm), kuthamanga kwamadzi (100 μl/min), gawo la mafoni 60/40 acetonitrile/madzi, ndi 0.1% TFA.
kumene L ndi kutalika kwa mzere, η ndi kukhuthala kwa gawo la mafoni, ΔP ndi kukakamiza kumbuyo kwa gawoli, ndipo u ndi liwiro laling'ono la gawo la mafoni. Kuthekera kwa gawo la PMP kunali 2.5 × 10-14 m2, kuthamanga kwa 25 µl / min, 60/40 v / v kunagwiritsidwa ntchito. ACN/madzi. Kuthekera kwa gawo la PMP (ID 100 × 1.8 mm) kunali kofanana ndi kafukufuku wathu wakale wa Ref.34. The permeability wa mzati wodzazidwa ndi mwachiphamaso porous particles ndi 1.7×10 .6 µm, 2.5×10-14 m2 kwa 5 µm particles43. Choncho, permeability ya gawo la PMP ndi ofanana ndi permeability wa pachimake-chipolopolo particles ndi kukula kwa 5 μm.
kumene Wx ndi unyinji wa mzati wodzazidwa ndi chloroform, Wy ndiye kuchuluka kwa gawo lodzazidwa ndi methanol, ndipo ρ ndiye kuchuluka kwa zosungunulira. Kuchulukana kwa methanol (ρ = 0.7866) ndi chloroform (ρ = 1.484). Chiwerengero chonse cha silika-C18 particle column (100 × 1.8 mm ID) 34 ndi gawo lathu la C18-urea31 lomwe tinaphunzira kale linali 0.63 ndi 0.55, motero. Izi zikutanthauza kuti kupezeka kwa urea ligands kumachepetsa permeability wa stationary gawo. Kumbali ina, porosity yonse ya mzere wa PMP (m'mimba mwake 100 × 1.8 mm) ndi 0.60. Mizati ya PMP ndi yocheperako kuposa mizati yodzaza ndi tinthu ta silika ta C18 chifukwa mu magawo a C18 amtundu wa C18 ligand amamangiriridwa ku tinthu ta silika mu unyolo wozungulira, pomwe m'magawo amtundu wa polystyrene pali polima wokhuthala kwambiri amapangidwa mozungulira tinthu ting'onoting'ono. wosanjikiza A. Pakuyesa wamba, porosity imawerengedwa motere:
Pa mkuyu. 7A, B akuwonetsa ziwembu za Van Deemter za gawo la PMP (id 100 x 1.8 mm) ndi gawo la Ascentis Express RP-Amide (id 100 x 1.8 mm) m'mikhalidwe yomweyi, 60/40 ACN/H2O ndi 0 .1% TFA 20 µl/mphindi µ0 mphindi zonse mpaka 8/min. Miyezo yocheperako ya HETP pamlingo woyenda bwino (80 µl/min) inali 2.6 µm ndi 3.9 µm pagawo la PMP ndi gawo la Ascentis Express RP-Amide, motsatana. Makhalidwe a HETP amawonetsa kuti kulekanitsa kwa gawo la PMP (100 x 1.8 mm id) ndikokwera kwambiri kuposa komwe kumapezeka malonda Ascentis Express RP-Amide id (100 x 1.8 mm id). Chithunzi cha van Deemter mu Fig. 7 (A) chikuwonetsa kuti kuchepa kwa mtengo wa N sikuli kwakukulu kwambiri ndi kuwonjezeka kwa kuyenda poyerekeza ndi phunziro lathu lapitalo. Kusiyanitsa kwakukulu kwa gawo la PMP (id 100 × 1.8 mm) poyerekeza ndi gawo la Ascentis Express RP-Amide kumatengera mawonekedwe a tinthu tating'ono ndi kukula kwake komanso njira yolozera bwino yomwe imagwiritsidwa ntchito pakali pano34.
(A) Van Deemter chiwembu (HETP vs. mobile gawo liniya liwiro) analandira pa PMP ndime (id 100 x 1.8 mm) mu 60/40 ACN/H2O ndi 0.1% TFA. (B) Van Deemter plot (HETP versus mobile phase linear velocity) yopezedwa pa Ascentis Express RP-Amide column (id 100 x 1.8 mm) mu 60/40 ACN/H2O ndi 0.1% TFA.
Gawo la polar stationary la intercalated polystyrene linakonzedwa ndikuwunikidwa kuti lilekanitse osakaniza a peptides opangidwa ndi tryptic hydrolyzate ya human serum albumin (HSA) mu chromatography yamadzimadzi yapamwamba kwambiri. Kuchita kwa chromatographic kwa mizati ya PMP pazosakaniza za peptide ndizabwino kwambiri pakusiyanitsa bwino komanso kusamvana. Kupititsa patsogolo kulekana kwa mizati ya PMP ndichifukwa cha zifukwa zingapo monga kukula kwa tinthu ta silika ndi kukula kwa pore, kaphatikizidwe kagawo kakang'ono, ndi zida zovuta zonyamula. Kuphatikiza pa kulekanitsa kwakukulu, ubwino wina wa gawo loyima ndi kutsika kwapansi kumbuyo kwazitsulo zothamanga kwambiri. Mizati ya PMP ndi yopangika kwambiri ndipo imatha kugwiritsidwa ntchito kusanthula kusakanikirana kwa ma peptide ndi kagayidwe ka tryptic ka mapuloteni osiyanasiyana. Tikufuna kugwiritsa ntchito gawoli polekanitsa zinthu zachilengedwe, zopangira mankhwala ndi bowa mu chromatography yamadzi. M'tsogolomu, mizati ya PMP idzawunikidwanso kuti tisiyanitse mapuloteni ndi ma antibodies a monoclonal.
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