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Zvidimbu zvesilica zvine maporous zvakagadzirwa nenzira ye sol-gel pamwe nekugadziriswa kwezvimwe zvinhu kuti zviwane zvidimbu zvemacroporous. Zvidimbu izvi zvakatorwa kuburikidza ne reversible addition fragmentation chain transfer (RAFT) polymerization ne N-phenylmaleimide-methylvinylisocyanate (PMI) uye styrene kugadzirira N-phenylmaleimide intercalation ye polystyrene (PMP) stationary phase. Narrow-bore stainless steel columns (100 × 1.8 mm id) dzakazadzwa ne slurry packing. Kupatsanurwa kwePMP column musanganiswa wepeptide une mapeptide mashanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) chromatographic performance) uye trypsin digestion ye human serum albumin (HAS). Pasi pemamiriro epamusoro ekugadzirisa, theoretical plate count ye peptide mixture inosvika 280,000 plates/m². Kuenzanisa performance yekupatsanurwa kwe Pakuumbwa kwekoramu yakagadzirwa nekoramu yeAscentis Express RP-Amide yekutengeserana, zvakaonekwa kuti mashandiro ekuparadzanisa kwekoramu yePMP aive akakwirira kupfuura koramu yekutengeserana maererano nekushanda zvakanaka uye kugadziriswa kwekuparadzanisa.
Mumakore achangopfuura, indasitiri yemishonga yemuchadenga yave musika wepasi rose uri kukura nekuwedzera kukuru kwemusika. Nekukura kwakanyanya kweindasitiri yemishonga yemuchadenga1,2,3, kuongororwa kwemapeptide nemapuroteni kunodiwa zvikuru. Pamusoro pepeptide inotarirwa, tsvina yakawanda inogadzirwa panguva yekugadzira peptide, nokudaro ichida kucheneswa kwechromatographic kuti uwane mapeptide ekuchena kwaunoda. Kuongorora uye kutsanangurwa kwemapuroteni mumvura yemuviri, tishu nemasero ibasa rakaoma zvikuru nekuda kwehuwandu hwakawanda hwemarudzi anogona kuonekwa mumuenzaniso mumwe chete. Kunyangwe mass spectrometry iri chishandiso chinoshanda che peptide neprotein sequencing, kana masampuli akadaro akapinzwa mu mass spectrometer kamwe chete, kupatsanurwa hakuzove kwakanaka. Dambudziko iri rinogona kuderedzwa nekushandisa liquid chromatography (LC) separations isati yaitwa MS analysis, iyo ichaderedza huwandu hweanalytes dzinopinda mu mass spectrometer panguva yakatarwa4,5,6. Pamusoro pezvo, panguva yekupatsanurwa kwechikamu chemvura, analytes dzinogona kutariswa munzvimbo dzakamanikana, nokudaro dzichiisa analytes idzi uye dzichivandudza kuonekwa kweMS. Kunzwa kwemvura.Kuongorora kwemvura (LC) kwafambira mberi zvakanyanya mumakore gumi apfuura uye kwave nzira inozivikanwa mukuongorora kweproteomic7,8,9,10.
Reversed-phase liquid chromatography (RP-LC) inoshandiswa zvakanyanya pakuchenesa nekuparadzanisa peptide mixes uchishandisa octadecyl-modified silica (ODS) se stationary phase11,12,13. Zvisinei, RP stationary phases hadzipe kupatsanurwa kunogutsa kwepeptides nemaproteins nekuda kwechimiro chazvo chakaoma uye hunhu hwazvo hwe amphiphilic 14,15 .Naizvozvo, stationary phases dzakagadzirirwa zvakananga dzinodiwa kuti dziongorore peptides nemaproteins ane polar ne non-polar moieties kuti dzibatane nekuchengetedza aya analytes16. Mixed-mode chromatography, iyo inopa multimodal interactions, inogona kuva imwe nzira yeRP-LC yekupatsanurwa kwepeptides, maproteins, uye mimwe complex mixes. Mixed-mode stationary phases dzakagadzirwa, uye makoramu akazara neaya mazinga akashandiswa pakupatsanurwa kwepeptide neprotein17,18,19,20,21. Mixed-mode stationary phases (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) dzakakodzera kupatsanurwa kwepeptide neprotein nekuda kuvapo kwemapoka ese ari maviri epolar neasiri epolar22,23,24,25,26,27,28. Saizvozvowo, mapolar intercalating stationary phases ane mapoka epolar akabatana necovalently anoratidza simba rakanaka rekuparadzanisa uye sarudzo yakasarudzika yeanalytes dzepolar neasiri epolar, sezvo kupatsanurwa kunoenderana nekudyidzana kuripo pakati peanalyte nechikamu chestationary. Kutaurirana kweMultimodal 29, 30, 31, 32. Munguva pfupi yapfuura, Zhang et al. 30 vakagadzira dodecyl-terminated polyamine stationary phase uye vakabudirira kuparadzanisa mahydrocarbon, antidepressants, flavonoids, nucleosides, estrogens, nedzimwe nzira dzakawanda dzekuongorora. Polar intercalator ine mapoka epolar neenon-polar, saka inogona kushandiswa kuparadzanisa mapeptide nemapuroteni ane hydrophobic ne hydrophilic moieties. Polar-embedded columns (semuenzaniso, amide-embedded C18 columns) anowanikwa mumusika pasi pezita rekutengeserana rekuti Ascentis Express RP-Amide columns, asi ma columns aya anoshandiswa kuongorora amine 33 chete.
Muchidzidzo chazvino, polar-embedded stationary phase (N-phenylmaleimide-embedded polystyrene) yakagadzirwa uye yakaongororwa kuti iparadzirwe mapeptides uye trypsin digests eHSA. Stanary phase yakagadzirwa uchishandisa nzira inotevera. Porous silica particles dzakagadzirwa zvichienderana nemaitiro akapihwa muchinyorwa chedu chekare pamwe nekugadziriswa kweprotocol yekugadzirira. Chiyero cheurea, polyethylene glycol (PEG), TMOS, water acetic acid chakagadziriswa kuti chigadzirire silica particles dzine pore yakakura. Chechipiri, ligand itsva, phenylmaleimide-methyl vinyl isocyanate, yakagadzirwa uye yakashandiswa kutora silica particles kuti igadzirire polar embedded stationary phase. Stanary phase yakabuda yakarongedzwa mu stainless steel column (100 × 1.8 mm id) uchishandisa optimized packing scheme. Column packing inobatsirwa ne mechanical vibration kuti ive nechokwadi chekuti homogeneous bed inoumbwa mukati me column. Ongorora packed column separation ye peptide mixtures ine 5 peptides; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) uye Trypsin digest ye human serum albumin (HAS). Musanganiswa wepeptide ne trypsin digest yeHSA zvakaonekwa kuti zvakaparadzana zvine resolution yakanaka uye zvinobudirira. Kushanda kwekupatsanurana kwePMP column kwakaenzaniswa nekwe Ascentis Express RP-Amide column. Ma peptide nemaprotein ese akaonekwa kuti akagadziriswa zvakanaka uye anoshanda zvakanaka paPMP column, iyo yaishanda zvakanyanya kupfuura 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, uye Acetone Zvakatengwa kubva kuSigma-Aldrich (St. Louis, MO, USA).
Musanganiswa weurea (8 g), polyethylene glycol (8 g), uye 8 mL ye 0.01 N acetic acid yakakurungwa kwemaminitsi gumi, uye 24 mL yeTMOS yakazowedzerwa ipapo mumamiriro echando. Musanganiswa we reaction wakapiswa pa40°C kwemaawa matanhatu uye wozopiswa pa120°C kwemaawa masere mu autoclave yesimbi isina ngura. Mvura yakadururwa uye zvasara zvakaomeswa pa70°C kwemaawa gumi nemaviri. Hunyoro hwakaomeswa hwakakuyiwa muovheni ndokupiswa pa550°C kwemaawa gumi nemaviri. Mabheti matatu akagadzirwa uye akatsanangurwa kuti aongorore kugona kudzokororwa kwehukuru hwezvidimbu, saizi yemaburi uye nzvimbo yepamusoro.
Nekuchinja pamusoro pezvinhu zvakaita se silica particles ne pre-synthesized ligand phenylmaleimide-methylvinylisocyanate (PCMP) zvichiteverwa ne radial polymerization ne styrene, polar group-containing compound yakagadzirwa. Standary phase ye aggregates ne polystyrene chains. Maitiro ekugadzirira anotsanangurwa pazasi.
N-phenylmaleimide (200 mg) uye methyl vinyl isocyanate (100 mg) zvakanyungudutswa mu dry toluene, uye 0.1 mL ye2,2′-azoisobutyronitrile (AIBN) yakawedzerwa mu reaction flask kugadzira phenylmaleimide-methyl vinyl isocyanate copolymer (PMCP).. Musanganiswa wacho wakapiswa pa60°C kwemaawa matatu, wakaseferwa uye wakaomeswa muovheni pa40°C kwemaawa matatu.
Zvidimbu zvesilica zvakaomeswa (2 g) zvakaparadzirwa mu toluene yakaoma (100 mL), zvakasanganiswa uye zvakaiswa mu flask yepasi ye500 mL kwemaminitsi gumi. PMCP (10 mg) yakanyungudutswa mu toluene ndokuwedzerwa mu flask ye reaction kuburikidza ne dropping funnel. Musanganiswa wacho wakadzoserwa pa 100°C kwemaawa masere, wakaseferwa ndokusukwa ne acetone ndokuomeswa pa 60°C kwemaawa matatu. Zvadaro, zvidimbu zve silica zvakasungwa ne PMCP (100 g) zvakanyungudutswa mu toluene (200 ml) uye 4-hydroxy-TEMPO (2 mL) yakawedzerwa paine 100 µL ye dibutyltin dilaurate se catalyst. Musanganiswa wacho wakamutswa pa 50°C kwemaawa masere, wakaseferwa uye waomeswa pa 50°C kwemaawa matatu.
Styrene (1 mL), benzoyl peroxide BPO (0.5 mL), uye TEMPO-PMCP-attached silica particles (1.5 g) zvakaparadzirwa mu toluene ndokucheneswa ne nitrogen. Kugadziriswa kwe styrene kwakaitwa pa 100°C kwemaawa gumi nemaviri. Chigadzirwa chakabuda chakagezwa ne methanol ndokuomeswa pa 60°C husiku hwese. Maitiro ese ekuita anoratidzwa muMufananidzo 1.
Masampuleti akabviswa mweya pa393 K kweawa imwe chete kuti awane kumanikidzwa kwakasara kuri pasi pe10-3 Torr. Huwandu hweN2 hwakanyudzwa pakumanikidzwa kweP/P0 = 0.99 hwakashandiswa kuona huwandu hwese hwepore. Maumbirwo ezvimedu zvesilica zvisina simbi uye ligand-bonded akaongororwa ne scanning electron microscopy (Hitachi High Technologies, Tokyo, Japan). Masampuleti akaomeswa (zvimedu zvesilica zvisina simbi uye ligand-bonded silica) akaiswa pakoramu yearuminiyamu uchishandisa adhesive carbon tape. Goridhe rakaputirwa pazvimedu uchishandisa Q150T sputter coater, uye 5 nm Au layer yakaiswa pazvimedu. Izvi zvinovandudza kushanda zvakanaka kwemaitiro uchishandisa low voltages uye zvinopa fine grain, cold sputtering. Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental analyzer yakashandiswa pakuongorora elemental. Malvern (Worcestershire, UK) Mastersizer 2000 particle size analyzer yakashandiswa kuwana kugoverwa kwehukuru hwezvimedu. Naked silica Zvidimbu nezvidimbu zve silica zvakasungwa ne ligand (5 mg chimwe nechimwe) zvakapararira mu 5 mL ye isopropanol, zvikaiswa sonic kwemaminitsi gumi, zvikabviswa kwemaminetsi mashanu, zvikaiswa pabhenji re optical reMasterizer. Kuongororwa kweThermogravimetric kwakaitwa nechiyero che 5 °C paminiti pamusoro pekupisa kwe 30 kusvika 800 °C.
Makoramu esimbi isina ngura akaputirwa negirazi ane saizi (100 × 1.8 mm id) akaiswa pasi achishandisa nzira yekurongedza slurry, achishandisa nzira imwecheteyo yakashandiswa muRef. 31. Chivharo chesimbi isina ngura (chakagadzirwa negirazi, 100 × 1.8 mm id) chine chinhu chinobuda chine 1 µm frit chakabatanidzwa kune slurry packer (Alltech Deerfield, IL, USA). Gadzirira slurry yechikamu chisina ngura nekumisa 150 mg yechikamu chisina ngura mu 1.2 mL yemethanol woitumira kukoramu yekuchengetera. Methanol yakashandiswa seslurry solvent pamwe ne propelling solvent. Zadza koramu zvakatevedzana nekushandisa pressures ye100 MP kwemaminitsi gumi, 80 MP kwemaminitsi gumi nemashanu, uye 60 MP kwemaminitsi makumi matatu. Munguva yekurongedza, mechanical vibration yakashandiswa nema GC column shakers maviri (Alltech, Deerfield, IL, USA) kuti ive nechokwadi chekuti koramu yakarongedzwa zvakaenzana. Vhara slurry packer wosunungura pressure zvishoma nezvishoma kudzivirira chero kukuvara mukati mekoramu. Bvisa koramu kubva ku slurry packing unit uye batanidza imwe fitting kune inlet uye kune LC system kuti uone kuti iri mashandiro.
Pombi yeLC (10AD Shimadzu, Japan), injector (Valco (USA) C14 W.05) ine 50nL injection loop, membrane degasser (Shimadzu DGU-14A), UV-VIS capillary window yakagadzirwa. Special µLC device device device device (UV-2075) uye glass-lined microcolumns. Shandisai connecting tube yakamanikana uye pfupi kuderedza mhedzisiro yekuwedzera column band. Mushure mekurongedza, capillaries (50 μm id 365 uye reducing union capillaries (50 μm) zvakaiswa panzvimbo yekubuda ye1/16″ ye reducing union. Kuunganidzwa kwedata uye chromatographic processing zvakaitwa uchishandisa Multichro 2000 software. Kuongorora pa254 nm Analytes kwakaedzwa kuti UV absorbance ionekwe. Chromatographic data yakaongororwa neOriginPro8 (Northampton, MA).
Albumin kubva muropa revanhu, upfu hwakasanganiswa, ≥ 96% (agarose gel electrophoresis) 3 mg yakasanganiswa ne trypsin (1.5 mg), 4.0 M urea (1 mL), uye 0.2 M ammonium bicarbonate (1 mL). Mushonga wacho wakakurungwa kwemaminitsi gumi ndokuchengetwa mumvura inodziya pa37°C kwemaawa matanhatu, wozodzimwa ne 1 mL ye 0.1% TFA. Sefa mushonga wacho wouchengeta pasi pe 4°C.
Kupatsanurwa kwemisanganiswa yepeptide uye HSA trypsin digests kwakaongororwa zvakasiyana paPMP columns. Tarisa kupatsanurwa kwepeptide musanganiswa uye trypsin digest yeHSA nePMP column uye enzanisa mhedzisiro neAscentis Express RP-Amide column. Nhamba yeplate yedzidziso inoverengerwa seinotevera:
Mifananidzo yeSEM ​​yezvimedu zvesilica zvisina chinhu uye zvimedu zvesilica zvakasungwa neligand zvinoratidzwa muMufananidzo 2. Mifananidzo yeSEM ​​yezvimedu zvesilica zvisina chinhu (A, B) inoratidza kuti, kusiyana nezvidzidzo zvedu zvekare, zvimedu izvi zvakatenderera umo zvimedu zvakareba kana kuti zvine symmetry isina kurongeka. Pamusoro pezvimedu zvesilica zvakasungwa neligand (C, D) zvakapfava kupfuura zvezvimedu zvesilica zvisina chinhu, izvo zvinogona kunge zvichikonzerwa nekuputirwa kwecheni dzepolystyrene pamusoro pezvimedu zvesilica.
Kuskena mifananidzo ye electron microscope yezvimedu zve silica (A, B) uye zvimedu zve silica zvakasungirirwa ne ligand (C, D).
Kugoverwa kwehukuru hwezvikamu zvesilica isina chinhu uye ligand-bonded silica particles kunoratidzwa muMufananidzo 3(A). Makori ekugoverwa kwehukuru hwezvikamu zvevolume-based particles akaratidza kuti hukuru hwezvikamu zvesilica hwakawedzera mushure mekushandurwa kwemakemikari (Mufananidzo 3A). Data rekugoverwa kwehukuru hwezvikamu zvesilica kubva mukudzidza kwazvino uye kudzidza kwapfuura rinoenzaniswa muTafura 1(A). Hukuru hwezvikamu zvevolume-based, d(0.5), hwePMP i3.36 μm, zvichienzaniswa nekudzidza kwedu kwapfuura ne ad(0.5) kukosha kwe3.05 μm (polystyrene-bound silica particles)34. Batch iyi yaive nekugoverwa kwehukuru hwezvikamu zvidiki zvichienzaniswa nekudzidza kwedu kwapfuura nekuda kwehuwandu hwakasiyana hwePEG, urea, TMOS, uye acetic acid mumusanganiswa we reaction. Hukuru hwezvikamu zvePMP hwakakura zvishoma pane hwe polystyrene-bound silica particle phase yatakadzidza kare. Izvi zvinoreva kuti kushanda kwepamusoro kwezvikamu zvesilica nestyrene kwakaisa polystyrene layer (0.97 µm) chete pa pamusoro pesilica, nepo muchikamu chePMP ukobvu hwechikamu hwaive 1.38 µm.
Kugoverwa kwehukuru hwezvikamu (A) uye kugoverwa kwehukuru hwemaburi (B) ezvikamu zvesilica zvisina chinhu uye zvikamu zvesilica zvakasungwa neligand.
Saizi yemapore, vhoriyamu yemapore uye nzvimbo yepamusoro yezvikamu zve silica zvechidzidzo chazvino zvinopihwa muTafura 1(B). Maprofile ePSD ezvikamu zve silica zvisina muripo uye zvikamu zve silica zvakasungwa ne ligand zvinoratidzwa muMufananidzo 3(B). Mhedzisiro yacho yakafanana neyechidzidzo chedu chekare. Saizi yemapore ezvikamu zve silica zvakasungwa ne ligand i310 ne241, zvichiteerana, izvo zvinoratidza kuti saizi yemapore inoderera ne69 mushure mekuchinja kwemakemikari, sezvakaratidzwa muTafura 1(B), uye shanduko ye curve inoratidzwa muMufananidzo 3(B). Saizvozvowo, vhoriyamu yemapore ezvikamu zve silica yakaderera kubva pa0.67 kusvika pa0.58 cm3/g mushure mekuchinja kwemakemikari. Nzvimbo chaiyo yezvikamu zve silica zvakadzidzwa parizvino i116 m2/g, iyo yakafanana neyechidzidzo chedu chekare (124 m2/g). Sezvakaratidzwa muTafura 1(B), nzvimbo yepamusoro (m2/g) yezvikamu zve silica yakadererawo kubva pa116 m2/g kusvika pa105 m2/g mushure mekugadziriswa kwemakemikari.
Mhedzisiro yekuongorora kwechikamu chenguva ino inoratidzwa muTafura 2. Kutakura kabhoni kwechikamu chenguva ino i6.35%, iyo yakaderera pane kutakura kabhoni kwekudzidza kwedu kwekare (polystyrene bonded silica particles, 7.93%35 uye 10.21%, zvichiteerana) 42. Kutakura kabhoni kwechikamu chenguva ino ishoma, Nekuti mukugadzirira kweSP yemazuva ano, kuwedzera kune styrene, mamwe ma polar ligands akadai sephenylmaleimide-methylvinylisocyanate (PCMP) uye 4-hydroxy-TEMPO akashandiswa. Kurema kwenitrogen muzana yechikamu chenguva ino i2.21%, zvichienzaniswa ne0.1735 uye 0.85% nehuremu hwenitrogen muzvidzidzo zvekare, zvichiteerana. Izvi zvinoreva kuti wt % yenitrogen yakakwira muchikamu chenguva ino isingaperi nekuda kwephenylmaleimide. Saizvozvowo, kutakura kabhoni kwezvigadzirwa (4) na (5) kwaive 2.7% na 2.9%, zvichiteerana, nepo Kutakura kabhoni kwechigadzirwa chekupedzisira (6) kwaive 6.35%, sezvakaratidzwa muTafura 2. Kurasikirwa nehuremu kwakaongororwa nePMP stationary phase, uye TGA curve inoratidzwa muMufananidzo 4. TGA curve inoratidza kurasikirwa nehuremu kwe8.6%, izvo zvinoenderana zvakanaka necarbon loading (6.35%) nekuti ligands hadzina C chete asiwo N, O, naH.
Phenylmaleimide-methylvinylisocyanate ligand yakasarudzwa kuti igadzirise pamusoro pezvikamu zvesilica nekuti ine mapoka epolar phenylmaleimide uye mapoka evinylisocyanate. Mapoka eVinyl isocyanate anogona kuita zvakawanda nestyrene nekuita radical polymerization. Chikonzero chechipiri ndechekuisa boka rine hukama huri pakati neanalyte uye risina hukama hwakasimba hwemagetsi pakati peanalyte nechikamu chestationary, sezvo chikamu chephenylmaleimide chisina simba remagetsi pa pH yakajairika. Polarity yechikamu chestationary inogona kudzorwa nehuwandu hwakanaka hwestyrene uye nguva ye reaction yefree radical polymerization. Danho rekupedzisira re reaction (free-radical polymerization) rakakosha uye rinogona kuchinja polarity yechikamu chestationary. Elemental analysis yakaitwa kuti iongorore carbon loading yezvikamu izvi zvestationary. Zvakaonekwa kuti kuwedzera huwandu hwestyrene nenguva ye reaction kwakawedzera carbon loading yechikamu chestationary uye zvinopesana. SP dzakagadzirwa nemazinga akasiyana estyrene dzine carbon loading dzakasiyana. Zvakare, isa zvikamu izvi zvestationary musimbi isina tsvina. makoramu uye kutarisa mashandiro avo echromatographic (selectivity, resolution, N value, nezvimwewo). Zvichibva pakuedza uku, fomura yakagadziriswa yakasarudzwa kugadzirira PMP stationary phase kuve nechokwadi chekuti polarity inodzorwa uye kuchengetedza kwakanaka kweanalyte.
Misanganiswa mishanu yepeptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) yakaongororwawo uchishandisa PMP column uchishandisa mobile phase; 60/40 (v/v) acetonitrile/mvura (0.1% TFA) pachiyero chekuyerera kwe80 μL/min. Pasi pemamiriro ekugadzirisa akanakisa, nhamba yeplate yedzidziso (N) pakoramu (100 × 1.8 mm id) i20,000 ± 100 (200,000 plates/m²). Tafura 3 inopa maN values ​​​​emakoramu matatu ePMP uye machromatograms anoratidzwa muMufananidzo 5A. Kuongorora nekukurumidza pakoramu yePMP pachiyero chekuyerera kwepamusoro (700 μL/min), mapeptide mashanu akabviswa mukati meminiti imwe, maN values ​​​​aive akanaka kwazvo, 13,500 ± 330 pakoramu (100 × 1.8 mm id), Anoenderana nemaplates 135,000/m (Mufananidzo 5B). Makoramu matatu akafanana (100 × 1.8 mm id) akazara nezvikamu zvitatu zvakasiyana zvePMP stationary phase kuti atariswe. kudzokororwa. Kuwanda kweanalyte yekoramu yega yega kwakanyorwa uchishandisa mamiriro ekugadzirisa akanakisa uye huwandu hwemaplate edzidziso N uye nguva yekuchengetedza kuti iparadzanise musanganiswa wekuyedza wakafanana pakoramu yega yega. Data rekudzokororwa kwemakoramu ePMP rinoratidzwa muTafura 4. Kudzokororwa kwekoramu yePMP kunoenderana zvakanaka ne %RSD values ​​yakaderera kwazvo, sezvakaratidzwa muTafura 3.
Kupatsanurwa kwemusanganiswa wepeptide paPMP column (B) neAscentis Express RP-Amide column (A); mobile phase 60/40 ACN/H2O (TFA 0.1%), PMP column dimensions (100 × 1.8 mm id); analytical Kurongeka kwe elution yemakomponi: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) uye 5 (leucine) acid enkephalin)).
Koramu yePMP (100 × 1.8 mm id) yakaongororwa kuti ione kupatsanurwa kwe tryptic digests ye human serum albumin mu high performance liquid chromatography. Chromatogram iri muMufananidzo 6 inoratidza kuti sampuro yakaparadzaniswa zvakanaka uye resolution yakanaka kwazvo. HSA digests yakaongororwa uchishandisa flow rate ye100 µL/min, mobile phase 70/30 acetonitrile/water uye 0.1% TFA. Sezvinoratidzwa muchromatogram (Mufananidzo 6), HSA digest yakakamurwa kuita 17 peaks inoenderana ne17 peptides. Kugona kwekupatsanurana kwe peak yega yega muHSA digest kwakaverengerwa uye kukosha kwacho kwakapihwa muTafura 5.
Kuyerwa kweHSA (100 × 1.8 mm id) kwakaparadzaniswa paPMP column; flow rate (100 µL/min), mobile phase 60/40 acetonitrile/water ne 0.1% TFA.
apo L iri kureba kwekoramu, η ndiyo viscosity yechikamu chinofambiswa, ΔP ndiyo kumanikidzwa kwekoramu kumashure, uye u ndiyo linear velocity yechikamu chinofambiswa. Kupinda kwekoramu yePMP kwaive 2.5 × 10-14 m2, mwero wekuyerera waive 25 μL/min, uye 60/40 v/v ACN/mvura yakashandiswa. Kupinda kwekoramu yePMP (100 × 1.8 mm id) kwakafanana nekwechidzidzo chedu chekare Ref.34. Kupinda kwekoramu yakazara nezvikamu zvisina maburi zvakanyanya ndeizvi: 1.7 × 10-15 yezvikamu zve1.3 μm, 3.1 × 10-15 yezvikamu zve1.7 μm, 5.2 × 10-15 uye 2.5 × 10-14 m2 yezvikamu zve2.6 μm. Kune zvikamu zve5 μm 43. Nokudaro, kupinda kwechikamu chePMP kwakafanana nekwezvikamu zve5 μm zvecore-shell.
apo Wx huremu hwekoramu yakazara nechloroform, Wy huremu hwekoramu yakazara nemethanol, uye ρ huremu hwe solvent. Huremu hwe methanol (ρ = 0.7866) uye chloroform (ρ = 1.484). Huremu hwese hwe SILICA PARTICLES-C18 columns (100 × 1.8 mm id) 34 uye C18-Urea columns 31 dzatakadzidza kare dzaive 0.63 uye 0.55, zvichiteerana. Izvi zvinoreva kuti kuvapo kwe urea ligands kunoderedza kupenya kwechikamu che stationary. Kune rumwe rutivi, huremu hwese hwekoramu ye PMP (100 × 1.8 mm id) i0.60. Kupenya kwekoramu dze PMP kwakaderera pane kwekoramu dzakazadzwa neC18-bonded silica particles nekuti muzvikamu zve C18-type stationary, C18 ligands dzakanamirwa kune zvikamu zve silica se linear chains, nepo muzvikamu zve polystyrene-type stationary, A yakakora zvishoma. polymer layer inoumbwa ichiipoteredza. Mukuyedza kwakajairika, porosity yekoramu inoverengerwa seizvi:
Mufananidzo 7A,B unoratidza koramu yePMP (100 × 1.8 mm id) uye koramu yeAscentis Express RP-Amide (100 × 1.8 mm id) uchishandisa mamiriro akafanana ekudzikisa (kureva, 60/40 ACN/H2O uye 0.1% TFA). ) yevan Deemter plot. Misanganiswa yepeptide yakasarudzwa (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) yakagadzirwa mu20 µL/ Mwero wekuyerera kwezvikamu zvese zviri zviviri i800 µL/min. MaHETP akaderera pachiyero chekuyerera kwepamusoro (80 µL/min) yekoramu yePMP nekoramu yeAscentis Express RP-Amide aive 2.6 µm uye 3.9 µm, zvichiteerana. MaHETP akakosha anoratidza kuti kushanda zvakanaka kwekoramu yePMP (100 × 1.8 mm id) kuri nani pane koramu yeAscentis Express RP-Amide inowanikwa mumusika (100 × 1.8 mm id). Mufananidzo wevan Deemter uri muMufananidzo 7(A) unoratidza kuti kudzikira kweN value nekuwedzera kwekuyerera hakuna kukosha zvichienzaniswa nekudzidza kwedu kwapfuura. Kushanda zvakanaka kwePMP column (100 × 1.8 mm id) zvichienzaniswa nekoramu yeAscentis Express RP-Amide yakavakirwa pakuvandudzwa kwechimiro chezvikamu, saizi, uye maitiro akaomarara ekurongedza makoramu anoshandiswa mubasa razvino34.
(A) van Deemter plot (HETP vs mobile phase linear velocity) yakawanikwa uchishandisa PMP column (100 × 1.8 mm id) mu 60/40 ACN/H2O ine 0.1% TFA. (B) van Deemter plot (HETP vs mobile phase linear velocity) yakawanikwa uchishandisa Ascentis Express RP-Amide column (100 × 1.8 mm id) mu 60/40 ACN/H2O ine 0.1% TFA.
Chikamu chepolar-embedded polystyrene stationary chakagadzirwa uye chakaongororwa kuti chiparadzanise ma peptide mixes uye trypsin digests ye human serum albumin (HAS) mu high performance liquid chromatography. Kushanda kwePMP columns ye peptide mixes kwakanaka kwazvo mukushanda zvakanaka uye kugadziriswa kwekuparadzanisa. Kushanda kwakanaka kwekuparadzanisa kwePMP columns kunokonzerwa nezvikonzero zvakasiyana-siyana, zvakaita sehukuru hwe particles uye saizi ye pore ye silica particles, controlled synthesis ye stationary phase, uye complex column packing. Pamusoro pekushanda zvakanaka kwekuparadzanisa, low column back pressure pa high flow rates ndeimwe mukana wechikamu ichi che stationary. PMP columns dzinoratidza kugona kudzokororwa zvakanaka uye dzinogona kushandiswa kuongorora ma peptide mixes uye trypsin digesting yemapuroteni akasiyana-siyana. Tinoda kushandisa chikamu ichi kuparadzanisa zvigadzirwa zvechisikigo, bioactive compounds kubva mumiti yemishonga uye fungal extracts mu liquid chromatography. Mune ramangwana, PMP columns dzichaongororwawo kuti mapuroteni nema monoclonal antibodies aparadzaniswe.
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