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Zvidimbu zvesilica zvine maburi zvakagadzirwa nenzira yesol-gel pamwe nekugadziriswa kuti zviwane zvidimbu zvine maburi akawanda. Zvidimbu izvi zvakatorwa neN-phenylmaleimide-methylvinyl isocyanate (PMI) uye styrene kuburikidza ne reverse chain transfer-fragmentation (RAFT) polymerization kuti zvigadzire N-phenylmaleimide intercalated polyamides. Styrene (PMP) stationary phase. Narrow bore stainless steel columns (100 × 1.8 mm inner diameter) dzakazadzwa neslurry packing. Kushanda kwechromatographic kwePMP column kwakaongororwa kuti kupatsanurwe musanganiswa we synthetic peptides une mapeptide mashanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) uye tryptic hydrolyzate ye human serum albumin (HAS). Pasi pemamiriro ekugadzirisa akakodzera, huwandu hwemaplate ane musanganiswa wemapeptide hwakasvika 280,000 plates/sq.m. Zvichienzanisa mashandiro ekuparadzanisa kwekoramu yakagadzirwa nekoramu yeAscentis Express RP-Amide yekutengeserana, zvakaonekwa kuti kushanda kwekuparadzanisa kwekoramu yePMP kwaive kwakakwirira kupfuura koramu yekutengeserana maererano nekushanda kwekuparadzanisa nekugadzirisa.
Indasitiri yemishonga yemuchadenga yave musika wepasi rose uri kukura nekuwedzera kukuru kwemusika mumakore achangopfuura. Nekukura kwakanyanya kweindasitiri yemishonga yemuchadenga1,2,3, pane kudiwa kukuru kwekuongorora peptide neprotein. Pamusoro pepeptide inotarirwa, tsvina dzakasiyana-siyana dzinoumbwa panguva yekugadzira peptide, saka kucheneswa kwechromatographic kunodiwa kuti uwane kuchena kwaunoda kwepeptide. Kuongorora uye kutsanangurwa kwemapuroteni mumvura yemuviri, tishu, nemasero ibasa rakaoma zvikuru nekuda kwehuwandu hwakawanda hwemhando dzinogona kuonekwa dziripo mumuenzaniso mumwe chete. Kunyangwe mass spectrometry iri chishandiso chinoshanda chekutevera mapeptide nemapuroteni, kana masampuli akadaro akapinzwa zvakananga mumass spectrometer, kupatsanurwa kwacho hakuzogutsi. Dambudziko iri rinogona kugadziriswa nekuita liquid chromatography (LC) isati yaongororwa neMS, izvo zvichaderedza huwandu hweanalytes dzinopinda mumass spectrometer panguva yakatarwa4,5,6. Pamusoro pezvo, analytes dzinogona kutarisisa munzvimbo yakamanikana panguva yekupatsanurwa kwechikamu chemvura, nokudaro dzichitarisisa analytes idzi uye dzichiwedzera kunzwa kwekuona kweMS. Kuongororwa kwemvura (Liquid chromatography) (LC) kwafambira mberi zvakanyanya mumakore gumi apfuura uye kwave nzira inoshandiswa zvakanyanya pakuongorora kweproteomic7,8,9,10.
Reverse-phase liquid chromatography (RP-LC) inoshandiswa zvakanyanya kuchenesa nekuparadzanisa musanganiswa wemapeptide uchishandisa octadecyl-modified silica (ODS) sechikamu che stationary phase11,12,13. Zvisinei, nekuda kwechimiro chazvo chakaoma uye hunhu hwe amphoteric, zvikamu zve RP stationary phase14,15 hazvigone kupa kupatsanurwa kunogutsa kwemapeptide nemaprotein. Nokudaro, kuongororwa kwemapeptide nemaprotein ane zvikamu zve polar ne non-polar kunoda zvikamu zve stationary zvakagadzirirwa kuti zvibatane uye zvichengetedze analytes idzi16. Mixed chromatography, iyo inopa multimodal interactions, inogona kuva imwe nzira yeRP-LC yekuparadzanisa mapeptide, maprotein, uye mimwe misanganiswa yakaoma. Zvikamu zvakati wandei zve stationary phases zvakagadzirwa uye makoramu akazadzwa nezvikamu izvi zve stationary akashandiswa kupatsanura mapeptide nemaprotein17,18,19,20,21. Nekuda kwekuvapo kwemapoka epolar neasiri polar, ma mixed mode stationary phases (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) akakodzera kupatsanurwa kwema peptide nema proteins22,23,24,25,26,27,28. , ma polar intercalated stationary phases ane ma covalently bonded polar groups anoratidza kugona kwakanaka kwekupatsanurwa uye kusarudza kwakasiyana kwema polar neasiri polar analytes nekuti kupatsanurwa kunoenderana nekudyidzana kuripo pakati peanalyte ne stationary phase Multimodal interactions 29,30,31,32. Munguva pfupi yapfuura, Zhang et al. 30 vakawana behenyl-terminated stationary phases e polyamines uye vakaparadzanisa zvinobudirira ma hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, nedzimwe analytes. Zvinhu zvepolar embedded stationary zvine mapoka epolar neasiri polar, saka zvinogona kushandiswa kupatsanura ma peptides nemapuroteni kuita zvikamu zve hydrophobic ne hydrophilic. Makoramu ari mumutsara wepolar (semuenzaniso, makoramu eC18 ane amide inline) anowanikwa pasi pezita rekutengeserana rekuti Ascentis Express RP-Amide columns, asi makoramu aya akangoshandiswa chete pakuongorora amine 33.
Muchidzidzo chazvino, polar embedding stationary phase (N-phenylmaleimide, embedding polystyrene) yakagadzirwa uye yakaongororwa kuti ionekwe separation yepeptide uye tryptic HSA cleavage. Nzira inotevera yakashandiswa kugadzirira stationary phase. Porous silica particles dzakagadzirwa zvichienderana nemaitiro akatsanangurwa mumabhuku edu apfuura, pamwe neshanduko muzvirongwa zvekugadzirira 31, 34, 35, 36, 37, 38, 39. Ratio dze urea, polyethylene glycol (PEG), TMOS uye aqueous-acetic acid dzakagadziriswa kuti dziwane silica particles dzine pore sizes hombe. Chechipiri, phenylmaleimide-methylvinyl isocyanate ligand itsva yakagadzirwa uye derivatized silica particles dzayo dzakashandiswa kugadzirira polar embedded stationary phases. Standing phase yakawanikwa yakarongedzwa mu stainless steel column (inner diameter 100 × 1.8 mm) zvichienderana ne optimized packing scheme. Kurongedza kwe column kunobatsirwa ne mechanical vibration kuti ive nechokwadi chekuti layer yakaenzana mukati me column. Koramu yakarongwa yakaongororwa kuti ione kupatsanurwa kwemusanganiswa wemapeptide ane mapeptide mashanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). uye tryptic hydrolysates ye human serum albumin (HSA). Zvakaonekwa kuti musanganiswa wepeptide neHSA tryptic digest zvakapatsanurwa zvine resolution yakanaka uye efficiency. Kugona kwekuparadzanisa kwePMP koramu kwakaenzaniswa nekweAscentis Express RP-Amide column. Zvakaonekwa kuti mapeptide nemaprotein ane resolution yakanaka uye efficiency yakanyanya yekuparadzanisa paPMP column, uye efficiency yekuparadzanisa yePMP column yakakwira kupfuura yeAscentis 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) yeHPLC, methanol, 2-propanol uye acetone. Sigma-Aldrich Company (St. Louis, Missouri, USA).
Musanganiswa weurea (8 g), polyethylene glycol (8 g) uye 8 ml ye 0.01 N. acetic acid wakakurungwa kwemaminitsi gumi, uye 24 ml yeTMOS yakawedzerwa ipapo pasi pekutonhodza nechando. Musanganiswa we reaction wakapiswa pa40°C kwemaawa matanhatu uye wozopiswa pa120°C kwemaawa masere mu autoclave yesimbi isina ngura. Mvura yakabviswa mvura uye zvakasara zvakaomeswa pa70°C kwemaawa gumi nemaviri. Mabhuroko akapfava akaomeswa akakuyiwa zvakanaka ndokuiswa mu calcined muovheni pa550°C kwemaawa gumi nemaviri. Mabheti matatu akagadzirwa uye akatsanangurwa kuti ayedze kugona kudzokororwa kwehukuru hwezvimedu, saizi yemaburi uye nzvimbo yepamusoro.
Boka rePolar uye chikamu chisingachinji che polystyrene chains. Maitiro ekugadzirira anotsanangurwa pazasi.
N-phenylmaleimide (200 mg) uye methyl vinyl isocyanate (100 mg) zvakanyungudutswa mu anhydrous toluene, uye 0.1 ml ye2,2′-azoisobutyronitrile (AIBN) yakawedzerwa mu reaction flask kuti pave ne copolymer yephenylmaleimide uye methyl vinyl isocyanate (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 sonic kwemaminitsi gumi mu 500 ml round bowl. PMCP (10 mg) yakanyungudutswa mu toluene ndokuwedzerwa dropwise ku reaction flask kuburikidza ne addition funnel. Musanganiswa wacho wakadzoserwa pa 100°C kwemaawa masere, wakaseferwa, wakasukwa ne acetone ndokuomeswa pa 60°C kwemaawa matatu. Zvadaro, zvidimbu zve silica zvine chekuita ne PMCP (100 g) zvakanyungudutswa mu toluene (200 ml), uye 4-hydroxy-TEMPO (2 ml) yakawedzerwa ipapo paine 100 μl ye dibutyltin dilaurate se catalyst. Musanganiswa wacho wakakurunga pa 50°C kwemaawa masere, wakaseferwa uye waomeswa pa 50°C kwemaawa matatu.
Styrene (1 ml), benzoyl peroxide BPO (0.5 ml) uye silica particles zvakabatanidzwa kuTEMPO-PMCP (1.5 g) zvakaparadzirwa mu toluene ndokucheneswa ne nitrogen. Kugadziriswa kwe styrene kwakaitwa pa 100°C kwemaawa gumi nemaviri. Mushonga wakabuda wakagezwa ne methanol ndokuomeswa husiku hwese pa 60°C. Hurongwa hwese hwe reaction hunoratidzwa mumufananidzo wekutanga.
Masampuleti akabviswa mweya pa393 K kweawa imwe chete kusvika pawanikwa kumanikidzwa kwasara kuri pasi pe10–3 Torr. Huwandu hweN2 hwakanyungudutswa papressure P/P0 = 0.99 hwakashandiswa kuona huwandu hwese hwepore. Maumbirwo ezvikamu zvesilica zvakachena uye zvakasungwa neligand akaongororwa uchishandisa scanning electron microscope (Hitachi High Technologies, Tokyo, Japan). Masampuleti akaoma (zvikamu zvesilica zvakachena uye zvikamu zvesilica zvakasungwa neligand) akaiswa pamatanda ealuminium uchishandisa carbon tape. Goridhe rakaiswa pasampuli uchishandisa Q150T sputtering device, uye Au layer ine ukobvu hwe5 nm yakaiswa pasampuli. Izvi zvinovandudza kushanda kwe low voltage process uye zvinopa spraying inotonhora zvakanaka. Elemental analyzer yakaitwa uchishandisa Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental composition analyzer. Malvern particle size analyzer (Worcestershire, UK) Mastersizer 2000 yakashandiswa kuwana kugoverwa kwehukuru hwezvikamu. Zvidimbu zvesilica zvisina kuputirwa uye zvidimbu zvesilica zvakasungwa ne ligand (5 mg chimwe nechimwe) zvakapararira mu 5 ml ye isopropanol, zvikaiswa sonic kwemaminitsi gumi, zvikazungunuswa kwemaminitsi mashanu, uye zvikaiswa pabhenji reMastersizer optical. Kuongororwa kweThermogravimetric kunoitwa nechikero che 5 °C paminiti muhurefu hwekupisa kubva pa 30 kusvika 800 °C.
Makoramu esimbi isina tsvina akaputirwa negirazi ane saizi (ID 100 × 1.8 mm) akaputirwa nenzira yekuzadza slurry achitevedzera maitiro akafanana neari mu reference 31. Koramu yesimbi isina tsvina (yakapetwa negirazi, ID 100 × 1 .8 mm) uye nzvimbo yekubuda ine 1 µm frit zvakabatanidzwa kumuchina wekupaka slurry (Alltech Deerfield, IL, USA). Gadzirira suspension yechikamu chisina tsvina nekumisa 150 mg yechikamu chisina tsvina mu 1.2 ml ye methanol woiisa mukoramu yedhaivha. Methanol yakashandiswa seslurry solvent uye control solvent. Rongedza koramu nekushandisa pressure sequence ye 100 MP kwemaminitsi gumi, 80 MP kwemaminitsi gumi nemashanu, uye 60 MP kwemaminitsi makumi matatu. Maitiro ekurongedza akashandisa ma vibrators maviri e gas chromatography column vibrators (Alltech, Deerfield, IL, USA) kuti zvidengenyeke kuti zvive nechokwadi chekuti column packing yakafanana. Vhara slurry packer uye sunungura pressure zvishoma nezvishoma kudzivirira kukuvara kwetambo. Chikwangwani chakabviswa kubva pamuromo wemvura uye chimwe chinhu chakasungirirwa pakupinda kwemvura chikabatanidzwa kune sisitimu yeLC kuti chiedze kushanda kwayo.
MLC yakagadzirwa uchishandisa pombi yeLC (10AD Shimadzu, Japan), sampler ine 50 nL injection loop (Valco (USA) C14 W.05), membrane degasser (Shimadzu DGU-14A), uye UV-VIS capillary window. Device detector (UV-2075) uye enamelled microcolumn. Shandisa machubhu akamanikana uye mapfupi ekubatanidza kuti uderedze mhedzisiro yekuwedzera kwekoramu. Mushure mekuzadza koramu, isa capillary (50 µm id 365) panzvimbo yekubuda ye1/16″ reducing junction uye isa capillary (50 µm) ye reducing junction. Kuunganidzwa kwedata uye chromatogram processing zvinoitwa uchishandisa Multichro 2000 software. Pa254 nm, UV absorbance yesubjects analytes yakatariswa pa0. Chromatographic data yakaongororwa uchishandisa OriginPro8 (Northampton, MA).
Serum albumin yemunhu, 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). Mhinduro yacho yakakurungwa kwemaminitsi gumi ndokuchengetwa mumvura yakashambidzwa pa37°C kwemaawa matanhatu, yozodzimwa ne1 ml ye0.1% TFA. Sefa mhinduro yacho woichengeta pasi pe4°C.
Kupatsanurwa kwemusanganiswa wemapeptides ne tryptic digest HSA pa PMP column kwakaongororwa zvakasiyana. Tarisa tryptic hydrolysis yemusanganiswa wemapeptides ne HSA wakapatsanurwa ne PMP column woenzanisa mhedzisiro ne Ascentis Express RP-Amide column. Huwandu hwemaplates edzidziso hunoverengerwa uchishandisa equation inotevera:
Mifananidzo yeSEM ​​yezvimedu zvesilica zvakachena uye zvimedu zvesilica zvakasungwa neligand zvinoratidzwa muMufananidzo 2. Mifananidzo yeSEM ​​yezvimedu zvesilica zvakachena (A, B) zvinoratidza chimiro chakatenderera umo zvimedu zvakareba kana kuti zvine symmetry isina kurongeka zvichienzaniswa nezvidzidzo zvedu zvekare. Pamusoro pezvimedu zvesilica zvakasungwa neligand (C, D) zvakapfava kupfuura zvezvimedu zvesilica zvakachena, izvo zvinogona kunge zvichikonzerwa necheni dzepolystyrene dzakafukidza pamusoro pezvimedu zvesilica.
Kuskena maelektron micrographs ezvikamu zvesilica zvakachena (A, B) uye zvikamu zvesilica zvakasungwa neligand (C, D).
Kugoverwa kwehukuru hwezvikamu zvesilica yakachena uye zvikamu zvesilica zvakasungwa neligand kunoratidzwa muMufananidzo 2. 3(A). Makori ehuwandu hwezvikamu zvakagoverwa akaratidza kuti hukuru hwezvikamu zvesilica hwakawedzera mushure mekushandurwa kwemakemikari (Mufananidzo 3A). Data rekugoverwa kwehukuru hwezvikamu zvesilica kubva muchidzidzo chazvino nechidzidzo chekare rinoenzaniswa muTafura 1(A). Hukuru hwezvikamu zvevolumetric d(0.5) zvePMP hwaive 3.36 µm, zvichienzaniswa nead(0.5) kukosha kwe3.05 µm muchidzidzo chedu chekare (polystyrene bonded silica particles)34. Nekuda kwekuchinja kwehuwandu hwePEG, urea, TMOS uye acetic acid mumusanganiswa wereaction, kugoverwa kwehukuru hwezvikamu zveboka iri kwaive kudiki zvichienzaniswa nechidzidzo chedu chekare. Hukuru hwezvikamu zvePMP hwakakura zvishoma pane hwechikamu chepolystyrene bound silica particle chatakadzidza kare. Izvi zvinoreva kuti kushanda kwepamusoro pezvikamu zvesilica nestyrene kwakaisa polystyrene layer (0.97 µm) chete pamusoro pesilica, nepo muchikamu chePMP ukobvu hwelayer hwaive 1.38 µm.
Kugoverwa kwehukuru hwezvikamu (A) uye kugoverwa kwehukuru hwemaburi (B) ezvikamu zvesilica zvakachena uye zvikamu zvesilica zvakasungwa neligand.
Saizi yemapore, vhoriyamu yemapore, uye nzvimbo yepamusoro yezvikamu zvesilica zvakashandiswa muchidzidzo ichi zvinoratidzwa muTafura 1 (B). Maprofile ePSD ezvikamu zvesilica zvakachena uye zvikamu zvesilica zvakasungwa neligand zvinoratidzwa muMifananidzo 3(B). Mhedzisiro yacho yakafanana neyatakadzidza kare34. Saizi dzezvikamu zvesilica zvakachena uye zvakasungwa neligand dzaive 310 Å uye 241 Å, zvichiteerana, zvichiratidza kuti mushure mekuchinja kwemakemikari, saizi yemapore yakaderera ne69 Å, sezvakaratidzwa muTafura 1 (B), uye shift curve inoratidzwa muMufananidzo. Nzvimbo chaiyo yezvikamu zvesilica muchidzidzo chazvino i116 m2/g, iyo yakafanana neyatakadzidza kare (124 m2/g). Sezvakaratidzwa muTafura 1(B), nzvimbo yepamusoro (m2/g) yezvikamu zvesilica mushure mekuchinja kwemakemikari yakadererawo kubva pa116 m2/g kusvika pa105 m2/g.
Mhedzisiro yekuongorora kwechikamu chenguva ino inoratidzwa muTafura. 2. Huremu hwekabhoni muchikamu chenguva ino i6.35%, iyo yakaderera pane muongororo yedu yapfuura (silica particles ine chekuita nepolystyrene, 7.93%35 uye 10.21%, zvichiteerana) 42. Huremu hwekabhoni muchikamu chenguva ino isingaperi pazasi, sezvo mamwe ma polar ligands akadai sephenylmaleimide methyl vinyl isocyanate (PCMP) uye 4-hydroxy-TEMPO akashandiswa pamwe chete nestyrene mukugadzirira SP. Huremu hwenitrogen muchikamu chenguva ino i2.21% zvichienzaniswa ne0.1735 uye 0.85% muzvidzidzo zvekare42. Izvi zvinoreva kuti chikamu chenguva ino isingaperi chine huwandu hwakawanda hwenitrogen nekuda kwephenylmaleimide. Saizvozvowo, zvigadzirwa (4) ne (5) zvine huwandu hwekabhoni hwe2.7% ne2.9%, zvichiteerana, nepo chigadzirwa chekupedzisira (6) chine huwandu hwekabhoni hwe6.35%, sezvakaratidzwa muTafura 2. Kuongororwa kweThermogravimetric (TGA) kwakashandiswa pachikamu chePMP chekuedza kudzikira kwehuremu, uye TGA curve inoratidzwa muMufananidzo 4. TGA curve inoratidza kudzikira kwehuremu kwe8.6%, izvo zvinoenderana zvakanaka nehuwandu hwekabhoni (6.35%), sezvo ligands dzisine C chete, asiwo N, O naH.
Iyo ligand phenylmaleimide-methylvinyl isocyanate yakasarudzwa kuti ichinje pamusoro pezvikamu zvesilica nekuda kwemapoka ayo epolar phenylmaleimide nevinylisocyanate. Mapoka evinyl isocyanate anogona kuita zvakare nestyrene nekuita radical polymerization. Chikonzero chechipiri ndechekuisa boka rine hukama huri pakati neanalyte uye risina hukama hwakasimba hwemagetsi pakati peanalyte nechikamu che stationary, sezvo chikamu chephenylmaleimide chisina simba rechokwadi pa pH yakajairika. Polarity yechikamu che stationary inogona kudzorwa nehuwandu hwakakwana hwestyrene nenguva ye reaction ye free radical polymerization. Danho rekupedzisira re reaction (free radical polymerization) rakakosha sezvo richichinja polarity yechikamu che stationary. Elemental analysis yakaitwa kuti iongorore huwandu hwekabhoni muzvikamu izvi zve stationary. Zvakaonekwa kuti kuwedzera huwandu hwestyrene nenguva ye reaction kunowedzera huwandu hwekabhoni yechikamu che stationary uye zvinopesana. SPs dzakagadzirwa ne concentrations dzakasiyana dzestyrene dzine carbon loads dzakasiyana. Saizvozvowo, zvikamu izvi zvakaiswa pamatanda esimbi isina ngura uye hunhu hwazvo hwechromatographic (selectivity, resolution, N value, nezvimwewo) zvakaongororwa. Zvichibva pakuedza uku, musanganiswa wakagadziridzwa wekugadzirira chikamu chePMP chakamiswa wakasarudzwa kuti upe polarity inodzorwa uye kuchengetedza zvakanaka kweanalyte.
Koramu yePMP yakaongororwawo kuti iongorore musanganiswa mishanu yemapeptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) uchishandisa kugona kwechikamu chekufamba. 60/40 (v/v) ACN/mvura (0.1% TFA) pachiyero chekuyerera kwe80 µl/min. Pasi pemamiriro epamusoro ekugadzirisa (200,000 plates/m), huwandu hwemaplate edzidziso (N) pakoramu (100 × 1.8 mm) i20,000 ± 100. MaN values ​​​​emakoramu matatu ePMP anoratidzwa muTafura 3 uye machromatogram anoratidzwa muMufananidzo 5A. Kuongorora nekukurumidza pakuyerera kwakanyanya (700 µl/min) pakoramu yePMP, mapeptide mashanu akabviswa mukati meminiti imwe chete, kukosha kwakanaka kweN kwe13,500 ± 330 pakoramu (dhayamita ye100 x 1.8 mm), yakaenzana nemaplates e135,000/m (Mufananidzo 5B). Makoramu matatu ehukuru hwakafanana (dhayamita yemukati ye100 x 1.8 mm) akazadzwa nemabatch matatu akasiyana ePMP stationary phase kuti ayedze kudzokororwa. Analytes dzakanyorwa pakoramu yega yega nekuparadzanisa musanganiswa wekuyedza wakafanana pakoramu yega yega uchishandisa mamiriro ekugadzirisa akanakisa, huwandu hwemaplates edzidziso N, uye nguva yekuchengetedza. Data re kudzokororwa kwemakoramu ePMP rinoratidzwa muTafura 4. Kudzokororwa kwekoramu yePMP kwakabatana zvakanaka ne %RSD values ​​​​yakaderera kwazvo sezvakaratidzwa muTafura 3.
Kupatsanurwa kwemisanganiswa yepeptide pakoramu yePMP (B) nekoramu yeAscentis Express RP-Amide (A), chikamu chemobile phase 60/40 ACN/H2O (TFA 0.1%), saizi yekoramu yePMP (100 x 1.8 mm id), ongororo Kurongeka kwemakomputa: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) uye 5 (leucic acid enkephalin).
Koramu yePMP (dhayamita yemukati 100 x 1.8 mm) yakaongororwa kuti ione kupatsanurwa kwe tryptic hydrolyzate ye human serum albumin neHPLC. Chromatogram iri muMufananidzo 6 inoratidza kuti sampuro dzakaparadzaniswa zvakanaka uye dzakagadziriswa zvakanaka. Mhinduro dzeHSA dzakaongororwa uchishandisa flow rate ye100 μl/min, mobile phase ye70/30 acetonitrile/water uye 0.1% TFA. Kuchekwa kweHSA kwakakamurwa kuita 17 peaks, sezvakaratidzwa mu chromatogram (Mufananidzo 6), zvinoenderana ne17 peptides. Kupatsanurwa kwema peaks ega ega kubva kuHSA hydrolyzate kwakaverengerwa uye kukosha kwacho kunoratidzwa muTafura 5.
HSA tryptic hydrolysates dzakaparadzaniswa paPMP column (dhayamita yemukati 100 x 1.8 mm), flow rate (100 μl/min), mobile phase 60/40 acetonitrile/water, uye 0.1% TFA.
apo L iri kureba kwekoramu, η ndiyo viscosity yechikamu chinofamba, ΔP ndiyo kumanikidzwa kwekumashure kwekoramu, uye u ndiyo linear velocity yechikamu chinofamba. Kupinda kwekoramu yePMP kwaive 2.5 × 10–14 m2, mwero wekuyerera waive 25 µl/min, 60/40 v/v yakashandiswa. ACN/mvura. Kupinda kwekoramu yePMP (ID 100 × 1.8 mm) kwakafanana nekwechidzidzo chedu chekare cheRef.34. Kupinda kwekoramu yakazadzwa nezvikamu zvisina maburi zvakanyanya i1.7×10 .6 µm, 2.5×10-14 m2 yezvikamu zve5 µm43. Nokudaro, kupinda kwechikamu chePMP kwakafanana nekupinda kwezvikamu zvecore-shell zvine saizi ye5 μm.
apo Wx huremu hwekoramu yakazadzwa nechloroform, Wy huremu hwekoramu yakazadzwa nemethanol, uye ρ huremu hwe solvent. Kuwanda kwe methanol (ρ = 0.7866) uye chloroform (ρ = 1.484). Kuwanda kwe silica-C18 particle column (100 × 1.8 mm ID)34 uye C18-urea31 column yedu yatakambodzidza yaive 0.63 uye 0.55, zvichiteerana. Izvi zvinoreva kuti kuvapo kwe urea ligands kunoderedza kupenya kwe stationary phase. Kune rumwe rutivi, porosity yese ye PMP column (dhayamita yemukati 100 × 1.8 mm) i0.60. PMP columns hadzipindi mvura zvakanyanya kupfuura columns dzakazadzwa ne C18 bound silica particles nekuti mu C18 type stationary phases, C18 ligands dzakanamirwa kune silica particles mumaketani akatsetseka, nepo mu polystyrene type stationary phases, polymer yakakora inoumbwa yakakomberedza particles. layer A. Mukuyedza kwakajairika, kuyerera kwekoramu kunoverengerwa seizvi:
Pamufananidzo 7A, B inoratidza Van Deemter marongero ePMP column (id 100 x 1.8 mm) uye Ascentis Express RP-Amide column (id 100 x 1.8 mm) pasi pemamiriro akafanana ekudzikisa, 60/40 ACN/H2O uye 0 .1% TFA 20 µl/min kusvika 800 µl/min pamakoramu ese ari maviri. Makomu eHETP mashoma pachiyero chakanaka chekuyerera (80 µl/min) aive 2.6 µm uye 3.9 µm pakoramu yePMP nekoramu yeAscentis Express RP-Amide, zvichiteerana. Makomu eHETP anoratidza kuti kushanda zvakanaka kwePMP column (100 x 1.8 mm id) kwakanyanya kupfuura kwekoramu yeAscentis Express RP-Amide (100 x 1.8 mm id). Girafu revan Deemter riri muFig. 7(A) rinoratidza kuti kudzikira kwehuwandu hweN hakuna kukwira zvakanyanya nekuwedzera kwemvura kana tichienzanisa nekudzidza kwedu kwekare. Kubudirira kwakanyanya kwekuparadzanisa kwePMP column (id 100 × 1.8 mm) kana tichienzanisa neAscentis Express RP-Amide column kwakavakirwa pachimiro cheparticle chakavandudzwa uye saizi uye maitiro ekugadzira column anoshandiswa pabasa razvino34.
(A) Chiratidzo cheVan Deemter (HETP vs. mobile phase linear velocity) chakawanikwa paPMP column (id 100 x 1.8 mm) mu60/40 ACN/H2O ine 0.1% TFA. (B) Chiratidzo cheVan Deemter (HETP vs mobile phase linear velocity) chakawanikwa paAscentis Express RP-Amide column (id 100 x 1.8 mm) mu60/40 ACN/H2O ine 0.1% TFA.
Chikamu chepolar stationary chepolystyrene yakabatana chakagadzirwa uye chakaongororwa kuti chiparadzaniswe musanganiswa wepeptides dzakagadzirwa uye tryptic hydrolyzate yehuman serum albumin (HSA) mu high performance liquid chromatography. Kushanda kwechromatographic kwePMP columns ye peptide mixtures kwakanaka kwazvo maererano ne separation efficiency uye resolution. Kubudirira kwekuparadzanisa kwePMP columns kuri nani nekuda kwezvikonzero zvakati wandei zvakaita se silica particle size ne pore size, controlled construction ye stationary phases, uye complex column packing materials. Pamusoro pe high separation efficiency, chimwe chinhu chakanaka che stationary phase iyi i low column back pressure pa high flow rates. PMP columns dzinogona kudzokororwa zvakanyanya uye dzinogona kushandiswa kuongorora musanganiswa wepeptides uye tryptic digestion yemapuroteni akasiyana-siyana. Tinoda kushandisa column iyi kuparadzanisa bioactive compounds kubva kuzvigadzirwa zvechisikigo, extracts yemiti yemishonga uye howa mu liquid chromatography. Mune ramangwana, PMP columns dzichaongororwawo kuti paparadzaniswe mapuroteni ne monoclonal antibodies.
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