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Uduce twa silica dufite imyenge twateguwe hakoreshejwe uburyo bwa sol-gel hamwe n'impinduka zimwe na zimwe kugira ngo haboneke uduce twa macroporous. Utu duce twakuwemo hakoreshejwe polymerization ya reversible addition fragmentation chain transfer (RAFT) hamwe na N-phenylmaleimide-methylvinylisocyanate (PMI) na styrene kugira ngo hategurwe N-phenylmaleimide intercalation ya polystyrene (PMP) stationary phase. Inkingi z'icyuma kidafunze zifite narrow-bore (100 × 1.8 mm id) zapakiwe hakoreshejwe slurry packing. Isesengura rya PMP column ry'uruvange rwa peptide rugizwe na peptide eshanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) chromatographic performance) hamwe na trypsin digestion ya human serum albumin (HAS). Mu gihe cyo gusesengura neza, umubare wa plate y'uruvange rwa peptide ni hejuru ya plates 280.000 / m². Ugereranije imikorere yo gutandukanya inkingi yakozwe hamwe n'inkingi y'ubucuruzi ya Ascentis Express RP-Amide, byagaragaye ko imikorere yo gutandukanya inkingi ya PMP yari iruta inkingi y'ubucuruzi mu bijyanye n'imikorere myiza no gukemura ikibazo.
Mu myaka ya vuba aha, inganda zikora imiti ya biopharmaceutical zabaye isoko rikura ku isi hose hamwe n’ubwiyongere bugaragara bw’isoko. Bitewe n’iterambere rikomeye ry’inganda zikora imiti ya biopharmaceutical1,2,3, isesengura rya peptide na poroteyine ririfuzwa cyane. Uretse peptide yibandwaho, imyanda myinshi iravuka mu gihe cyo gukora peptide, bityo bigasaba gusukurwa kwa chromatografiya kugira ngo haboneke peptide z’ubuziranenge bwifuzwa. Isesengura n’imiterere ya poroteyine mu mazi yo mu mubiri, mu ngingo no mu turemangingo ni akazi katoroshye cyane bitewe n’umubare munini w’ubwoko bushobora kugaragara mu gipimo kimwe. Nubwo spectrometry y’ubunini ari igikoresho cyiza cyo gukurikirana peptide na poroteyine, niba ingero nk’izo zishyizwe muri spectrometer y’ubunini mu gihe kimwe, gutandukanya ntabwo bizaba byiza. Iki kibazo gishobora kugabanywa hakoreshejwe uburyo bwo gutandukanya chromatography y’amazi (LC) mbere yo gusesengura MS, bizagabanya umubare w’ibisesengura byinjira muri spectrometer y’ubunini mu gihe runaka4,5,6. Byongeye kandi, mu gihe cyo gutandukanya imiterere y’amazi, ibisesengura bishobora kwibandwa mu turere duto, bityo bigashyira hamwe ibi bisesengura kandi bikanoza uburyo bwo kubona MS. Uburyo bwo kumva. Koromatogarafi y'amazi (LC) yateye imbere cyane mu myaka icumi ishize kandi yabaye uburyo buzwi cyane mu isesengura rya proteomiki7,8,9,10.
Chromatography y'amazi ihinduka (RP-LC) ikoreshwa cyane mu gutunganya no gutandukanya uruvange rwa peptide hakoreshejwe octadecyl-modified silica (ODS) nk'urwego ruhoraho11,12,13. Ariko, urwego ruhoraho rwa RP ntirutanga gutandukanya ku buryo bushimishije peptide na poroteyine bitewe n'imiterere yazo ikomeye n'imiterere yazo ihindagurika 14,15. Kubwibyo, urwego ruhoraho rwagenewe gusesengura peptide na poroteyine zifite ibice bya polar na non-polar kugira ngo zifatanye kandi zigumane izi analytes16. Chromatography y'uburyo buvanze, itanga imikoranire myinshi, ishobora kuba ubundi buryo bwa RP-LC bwo gutandukanya peptide, poroteyine, n'izindi mvange zikomeye. Hateguwe amashami menshi ahuza uburyo buvanze, kandi inkingi zipakiye izi mvange zakoreshejwe mu gutandukanya peptide na poroteyine17,18,19,20,21. Urwego ruhoraho rwa Mixed-mode (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) zikwiriye gutandukanya peptide na poroteyine bitewe ku kuba hari amatsinda yombi ya polar na non-polar22,23,24,25,26,27,28. Mu buryo nk'ubwo, ibice bihoraho bya polar intercalating hamwe n'amatsinda ya polar afatanye neza bigaragaza imbaraga nziza zo gutandukanya hamwe n'uburyo bwihariye bwo guhitamo ku basesengura ba polar na non-polar, kuko gutandukanya biterwa n'imikoranire iri hagati ya analyte na stationary phase. Interaction multimodal 29, 30, 31, 32. Vuba aha, Zhang et al. 30 bateguye uburyo bwa polyamine budahinduka bwa dodecyl kandi batandukanya neza hydrocarbons, imiti igabanya ubukana bw'indwara zo mu mutwe, flavonoids, nucleosides, estrogens, n'izindi analytes nyinshi. Polar intercalator ifite amatsinda yombi ya polar na non-polar, bityo ishobora gukoreshwa mu gutandukanya peptides na poroteyine zifite moieties zo mu mazi na hydrophilic. Polar-embedded columns (urugero, amide-embedded C18 columns) ziboneka ku isoko ku izina ry'ubucuruzi Ascentis Express RP-Amide columns, ariko izi columns zikoreshwa mu gusesengura amine 33 gusa.
Mu bushakashatsi buriho, hateguwe kandi hasuzumwa uburyo bwo gutandukanya peptides na trypsin digests bya HSA. Uburyo bwo gutandukanya imiterere bwateguwe hakoreshejwe ingamba zikurikira. Uduce twa silica dufite imyenge twateguwe hakurikijwe uburyo bwatanzwe mu nyandiko yacu yabanje hamwe n'impinduka zimwe na zimwe ku buryo bwo gutegura. Igipimo cya urea, polyethylene glycol (PEG), TMOS, aside asetiki y'amazi cyahinduwe kugira ngo hategurwe utuntu twa silica dufite imyenge minini. Icya kabiri, ligand nshya, phenylmaleimide-methyl vinyl isocyanate, yakozwe kandi ikoreshwa mu gukura utuntu twa silica kugira ngo hategurwe uburyo bwo gutandukanya imiterere. Uburyo bwo gutandukanya imiterere bwashyizwe mu cyuma (100 × 1.8 mm id) hakoreshejwe uburyo bwo gupakira bwakozwe neza. Gupakira inkingi bifashwa no guhindagura ibyuma kugira ngo harebwe ko inkingi ihuye neza. Suzuma uburyo bwo gutandukanya inkingi za peptide zigizwe na peptides eshanu; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) na Trypsin digest ya human serum albumin (HAS). Uruvange rwa peptide na trypsin digest ya HSA byagaragaye ko bitandukanye neza kandi bifite ubushobozi. Imikorere yo gutandukanya PMP column yagereranyijwe n'iya Ascentis Express RP-Amide column. Peptides na poroteyine byombi byagaragaye ko bifite ubushobozi kandi bitunganye ku gice cya PMP, cyari gifite ubushobozi kurusha inkingi ya Ascentis Express RP-Amide.
PEG (Polyethylene Glycol), Urea, aside asetiki, Trimethoxy Orthosilicate (TMOS), Trimethyl Chlorosilane (TMCS), Trypsin, Serum Albumin y'umuntu (HSA), Ammonium Chloride, Urea, Hexane Methyldisilazane (HMDS), Methacryloyl Chloride (MC), Styrene, 4-Hydroxy-TEMPO, Benzoyl Peroxide (BPO), HPLC Grade Acetonitrile (ACN), Methanol, 2-Propanol, na Acetone Byaguzwe muri Sigma-Aldrich (St. Louis, MO, USA).
Uruvange rwa urea (8 g), polyethylene glycol (8 g), na mL 8 za aside asetiki ya 0.01 N byakanzwe mu minota 10, hanyuma hongerwamo mL 24 za TMOS mu gihe cy'ubukonje bukabije. Uruvange rw'ibisubizo rwashyuhijwe kuri 40 ° C mu gihe cy'amasaha 6, hanyuma kuri 120 ° C mu gihe cy'amasaha 8 muri autoclave y'icyuma kitagira umugese. Amazi yarasutswe maze ibisigaye byumishwa kuri 70 ° C mu gihe cy'amasaha 12. Ingano yumye yoroshye yasibwe neza mu itanura maze ishyirwa kuri 550 ° C mu gihe cy'amasaha 12. Hateguwe uduce dutatu kandi twagaragajwe kugira ngo harebwe uburyo bwo kongera kubyara mu bunini bw'uduce, ingano y'imyenge n'ubuso.
Hakoreshejwe guhindura ubuso bw'uduce twa silica hakoreshejwe ligand ya phenylmaleimide-methylvinylisocyanate (PCMP) yakozwe mbere, hagakurikiraho polymerization ya radial hamwe na styrene, hateguwe imvange irimo polar group. Icyiciro gihoraho cy'ibice by'ibumba n'iminyururu ya polystyrene. Uburyo bwo gutegura burasobanurwa hano hepfo.
N-phenylmaleimide (200 mg) na methyl vinyl isocyanate (100 mg) byashongeshejwe muri toluene yumye, maze 0.1 mL ya 2,2′-azoisobutyronitrile (AIBN) yongerwa mu icupa rya reaction kugira ngo hategurwe phenylmaleimide-methyl vinyl isocyanate copolymer (PMCP).. Uruvange rwashyuhijwe kuri 60°C mu gihe cy'amasaha 3, ruyungururwa kandi rwumishwa mu itanura kuri 40°C mu gihe cy'amasaha 3.
Uduce twa silica twumye (2 g) twajugunywe muri toluene yumye (100 mL), tuvangwa hanyuma dushyirwa mu icupa ryo hasi rya mL 500 mu gihe cy'iminota 10. PMCP (10 mg) yashongeshejwe muri toluene hanyuma yongerwamo agace gato k'icupa ry'ikirahure binyuze mu muyoboro utonyanga. Uruvange rwasubiwemo kuri 100 °C mu gihe cy'amasaha 8, ruyungururwa kandi rwozwa na acetone hanyuma rwumishwa kuri 60 °C mu gihe cy'amasaha 3. Hanyuma, uduce twa silica twa PMCP-bonded (100 g) twashongeshejwe muri toluene (200 ml) hanyuma hongerwamo 4-hydroxy-TEMPO (2 mL) imbere ya 100 µL ya dibutyltin dilaurate nk'umuvuduko. Uruvange rwashongeshejwe kuri 50 °C mu gihe cy'amasaha 8, ruyungururwa kandi rwumishwa kuri 50 °C mu gihe cy'amasaha 3.
Styrene (1 mL), benzoyl peroxide BPO (0.5 mL), na TEMPO-PMCP-attached silica particles (1.5 g) byashyizwe muri toluene hanyuma bisukurwa na azote. Polymeralization ya styrene yakozwe kuri 100°C mu gihe cy'amasaha 12. Umusaruro wavuyemo wogejwe na methanol hanyuma wumishwa kuri 60°C ijoro ryose. Gahunda rusange y'ibizamini igaragazwa ku Ishusho ya 1.
Ingero zakuwemo umwuka kuri 393 K mu gihe cy'isaha imwe kugira ngo haboneke igitutu gisigaye kiri munsi ya 10-3 Torr. Ingano ya N2 yashyizwe ku gitutu cya P/P0 = 0.99 yakoreshejwe kugira ngo hamenyekane ingano y'imyenge yose. Imiterere y'uduce twa silica twambaye ubusa na ligand yasuzumwe hakoreshejwe mikorosikope ya electron yo gupima (Hitachi High Technologies, Tokyo, Ubuyapani). Ingero zumye (uduce twa silica twambaye ubusa na ligand-bonded silica) zashyizwe ku nkingi ya aluminiyumu hakoreshejwe kaseti ya karuboni ifatana. Zahabu yashyizwe ku ngero hakoreshejwe Q150T sputter coater, maze urwego rwa 5 nm Au rushyirwa ku ngero. Ibi byongera imikorere myiza hakoreshejwe voltage nke kandi bitanga ingano nziza, ikonje. Isesengura rya Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental yakoreshejwe mu gusesengura elemental. Isesengura rya Malvern (Worcestershire, UK) Mastersizer 2000 particles ryakoreshejwe kugira ngo haboneke ikwirakwizwa ry'ingano y'uduce. Silika yambaye ubusa Uduce duto n'uduce twa silica twa ligand-bonded (5 mg buri kimwe) byashyizwe muri 5 mL ya isopropanol, bishyirwa mu cyuma mu gihe cy'iminota 10, bishyirwa mu cyuma mu gihe cy'iminota 5, hanyuma bishyirwa ku ntebe y'ijisho ya Mastersizer. Isesengura rya Thermogravimetric ryakozwe ku gipimo cya 5 °C ku munota ku bushyuhe buri hagati ya 30 na 800 °C.
Inkingi z'icyuma kidafunze zifite uburebure buto (100 × 1.8 mm id) zapakiwe hakoreshejwe uburyo bwo gupakira ibintu, hakoreshejwe uburyo bumwe bwakoreshejwe muri Ref. 31. Inkingi y'icyuma kitagira umugese (ifite ikirahure, 100 × 1.8 mm id) ifite aho isohoka irimo 1 µm yahujwe n'icyuma gipakira ibintu (Alltech Deerfield, IL, USA). Tegura icyuma gifata ibintu kitagira umugese ushyira 150 mg y'icyuma gifata ibintu kitagira umugese muri 1.2 mL ya methanol hanyuma wohereze ku nkingi yo kubikamo. Methanol yakoreshejwe nk'icyuma gifata ibintu kitagira umugese ndetse n'icyuma gitanga umwuka. Wuzuze inkingi uko bikurikirana ushyiramo imbaraga za 100 MP mu minota 10, 80 MP mu minota 15, na 60 MP mu minota 30. Mu gihe cyo gupakira, hakoreshejwe vibration ya mechanical hamwe na GC column shakers ebyiri (Alltech, Deerfield, IL, USA) kugira ngo inkingi ipakire neza. Funga icyuma gipakira ibintu kitagira umugese hanyuma urekure umugese buhoro buhoro kugira ngo wirinde kwangirika mu nkingi. Kuramo inkingi mu gice gipakira ibintu kitagira umugese hanyuma uhuze ikindi gipakira ku gice cyo kwinjira no kuri sisitemu ya LC kugira ngo urebe ko imikorere.
Pompe ya LC (10AD Shimadzu, mu Buyapani), injektori (Valco (USA) C14 W.05) ifite umuyoboro wa 50nL, imashini ikuraho gaze (Shimadzu DGU-14A), idirishya rya capillary rya UV-VIS ryakozwe. Hakozwe imashini yihariye ipima µLC (UV-2075) n'inkingi nto z'ikirahure. Koresha umuyoboro muto cyane kandi mugufi uhuza kugira ngo ugabanye ingaruka zo kwaguka kw'inkingi. Nyuma yo gupakira, capillaries (50 μm id 365 na capillaries igabanya union (50 μm) byashyizwe ku gice cyo hanze cya 1/16″ cy'umuyoboro ugabanya union. Gukusanya amakuru no gutunganya chromatografiya byakozwe hakoreshejwe porogaramu ya Multichro 2000. Igenzura rya 254 nm Analytes ryapimwe kugira ngo harebwe niba UV ifata. Amakuru ya chromatografiya yasesenguwe na OriginPro8 (Northampton, MA).
Albumine iva mu maraso y'umuntu, ifu ya lyophilized, ≥ 96% (agarose gel electrophoresis) 3 mg ivanze na trypsin (1.5 mg), 4.0 M urea (1 mL), na 0.2 M ammonium bicarbonate (1 mL). Umuti waranzwe mu minota 10 maze ubikwa mu bwogero bw'amazi kuri 37°C mu gihe cy'amasaha 6, hanyuma ukazimya na 1 mL ya 0.1% TFA. Kungura umuti hanyuma ubibike munsi ya 4°C.
Gutandukanya uruvange rwa peptide na HSA trypsin digests byagenzuwe ukwabyo ku nkingi za PMP. Reba itandukanyirizo ry'uruvange rwa peptide na trypsin digest bya HSA n'inkingi ya PMP hanyuma ugereranye ibyavuye mu bushakashatsi n'inkingi ya Ascentis Express RP-Amide. Umubare wa plate y'ibitekerezo ubarwa ku buryo bukurikira:
Amashusho ya SEM y'uduce twa silica twambaye ubusa n'uduce twa silica twaboheshejwe na ligand agaragara ku ISHUSHO YA 2. Amashusho ya SEM y'uduce twa silica twambaye ubusa (A, B) agaragaza ko, bitandukanye n'ubushakashatsi bwacu bwa mbere, utu duce turimo uruziga aho uduce tuba turekure cyangwa dufite uburinganire budasanzwe. Ubuso bw'uduce twa silica twaboheshejwe na ligand (C, D) bworoshye kurusha ubw'uduce twa silica twambaye ubusa, bishobora guterwa no gutwikirwa kwa polystyrene ku buso bw'uduce twa silica.
Gusuzuma amashusho ya mikorosikopi ya elegitoroniki y'uduce twa silica twambaye ubusa (A, B) n'uduce twa silica twaboheshejwe na ligand (C, D).
Ingano y'uduce tw'uduce twa silica twambaye ubusa n'uduce twa silica twaboheshejwe na ligand igaragara ku Ishusho ya 3 (A). Imirongo y'ingano y'uduce tw ... ubuso bwa silica, mu gihe mu cyiciro cya PMP ubugari bw'urwego bwari 1.38 µm.
Ingano y'uduce duto (A) n'ingano y'imyenge (B) y'uduce twa silica twambaye ubusa n'uduce twa silica dufatanye na ligand.
Ingano y'imyenge, ingano y'imyenge n'ubuso bw'uduce twa silica tw'ubushakashatsi buriho ubu bigaragazwa mu mbonerahamwe ya 1 (B). Imiterere ya PSD y'uduce twa silica twambaye ubusa n'uduce twa silica twaboheshejwe na ligand igaragazwa mu Ishusho ya 3 (B). Ibisubizo birasa n'inyigo yacu yabanje. Ingano y'imyenge y'uduce twa silica twambaye ubusa n'utw'imenge ni 310 na 241, uko bikurikirana, bigaragaza ko ingano y'imyenge igabanukaho 69 nyuma yo guhindura imiti, nk'uko bigaragara mu mbonerahamwe ya 1 (B), kandi impinduka y'umuyoboro igaragazwa mu Ishusho ya 3 (B). Mu buryo nk'ubwo, ingano y'imyenge y'uduce twa silica yagabanutse kuva kuri 0.67 kugera kuri 0.58 cm3/g nyuma yo guhindura imiti. Ubuso bwihariye bw'uduce twa silica twigwa ubu ni 116 m2/g, bugereranywa n'inyigo yacu yabanje (124 m2/g). Nk'uko bigaragara mu mbonerahamwe ya 1 (B), ubuso (m2/g) bw'uduce twa silica nabwo bwagabanutse kuva kuri 116 m2/g kugera kuri 105 m2/g nyuma yo guhindura imiti.
Ibisubizo by'isesengura ry'ibanze ry'icyiciro gihoraho bigaragazwa mu mbonerahamwe ya 2. Umutwaro wa karuboni mu cyiciro gihoraho ni 6.35%, ari munsi y'umutwaro wa karuboni wo mu bushakashatsi bwacu bwa mbere (uduce twa silica twa polystyrene dufatanye, 7.93%35 na 10.21%, uko bikurikirana) 42. Umutwaro wa karuboni mu cyiciro gihoraho ni muto, Kuko mu gutegura SP y'ubu, uretse styrene, hakoreshejwe ligande zimwe na zimwe za polar nka phenylmaleimide-methylvinylisocyanate (PCMP) na 4-hydroxy-TEMPO. Ijanisha ry'uburemere bwa azote mu cyiciro gihoraho ni 2.21%, ugereranije na 0.1735 na 0.85% by'uburemere bwa azote mu bushakashatsi bwabanje, uko bikurikirana. Ibi bivuze ko % ya azote iri hejuru mu cyiciro gihoraho bitewe na phenylmaleimide. Mu buryo nk'ubwo, umutwaro wa karuboni w'ibicuruzwa (4) na (5) wari 2.7% na 2.9%, uko bikurikirana, mu gihe Umutwaro wa karuboni w’umusaruro wa nyuma (6) wari 6.35%, nk’uko bigaragara mu mbonerahamwe ya 2. Kugabanuka kw’ibiro byagenzuwe hakoreshejwe uburyo bwa PMP stationary phase, kandi umutwaro wa TGA ugaragara ku Ishusho ya 4. Umutwaro wa TGA ugaragaza kugabanuka kw’ibiro bya 8.6%, bihuye neza n’umutwaro wa karuboni (6.35%) kuko ligands zitarimo C gusa ahubwo zirimo N, O, na H.
Ligand ya phenylmaleimide-methylvinylisocyanate yatoranyijwe kugira ngo ihindure ubuso bw'uduce twa silica kuko ifite amatsinda ya phenylmaleimide yo mu bwoko bwa polar n'amatsinda ya vinylisocyanate. Amatsinda ya Vinyl isocyanate ashobora kongera gukorana na styrene binyuze mu gukora polymerization ya radical. Impamvu ya kabiri ni ukwinjizamo itsinda rifite imikoranire iri hagati ya analyte kandi nta imikoranire ikomeye ya electrostatic hagati ya analyte n'igice gihagaze, kubera ko igice cya phenylmaleimide kidafite charge virtual kuri pH isanzwe. Polarity y'igice gihagaze ishobora kugenzurwa n'ingano nziza ya styrene n'igihe cyo gukora polymerization ya free radical. Intambwe ya nyuma y'igikorwa (polymerization ya free radical) ni ingenzi kandi ishobora guhindura polarity y'igice gihagaze. Isesengura ry'ibintu ryakozwe kugira ngo harebwe load ya karuboni y'ibi bice bihagaze. Byagaragaye ko kongera ingano ya styrene n'igihe cyo gukora byongera load ya karuboni yo mu gice gihagaze, naho ibinyuranye nabyo. SP zateguwe zifite ingano zitandukanye za styrene zifite load ya karuboni itandukanye. Nanone, shyira izi stationary phases mu cyuma kitagira umugese. inkingi no kugenzura imikorere yazo ya chromatographic (guhitamo, ubushobozi bwo gusuzuma, agaciro ka N, nibindi). Hashingiwe kuri ubu bushakashatsi, hatoranijwe uburyo bwo gutunganya neza PMP kugira ngo habeho polarity igenzurwa kandi ikanagumana neza isesengura.
Uruvange rw’ibinyabutabire bitanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) nabyo byapimwe hakoreshejwe inkingi ya PMP hakoreshejwe uburyo bwo kugenda; 60/40 (v/v) acetonitrile/amazi (0.1% TFA) ku gipimo cy'amazi cya 80 μL/min. Mu gihe cyo kugabanya neza, umubare wa plate theory (N) kuri buri nkingi (100 × 1.8 mm id) ni 20.000 ± 100 (200.000 plates/m²). Imbonerahamwe ya 3 itanga agaciro ka N kuri inkingi eshatu za PMP kandi chromatograms zigaragara ku Ishusho ya 5A. Isesengura ryihuse ku nkingi ya PMP ku gipimo cyo hejuru cy'amazi (700 μL/min), peptide eshanu zakuweho mu munota umwe, agaciro ka N kari keza cyane, 13.500 ± 330 kuri buri nkingi (100 × 1.8 mm id), Bihuye na plates 135.000/m (Ishusho ya 5B). Inkingi eshatu zingana (100 × 1.8 mm id) zari zipakiye ibice bitatu bitandukanye bya PMP stationary phase kugira ngo bigenzurwe. gusubirwamo. Ingano y'isesengura kuri buri nkingi yanditswe hakoreshejwe uburyo bwiza bwo gusesengura hamwe n'umubare w'amabara ya teorike N n'igihe cyo kubika kugira ngo hatandukanye imvange imwe y'igerageza kuri buri nkingi. Amakuru y'isubiramo ry'itsinda ...
Gutandukanya uruvange rwa peptide ku nkingi ya PMP (B) na Ascentis Express RP-Amide (A); igice cyo kugenda 60/40 ACN/H2O (TFA 0.1%), ingano z'inkingi ya PMP (100 × 1.8 mm id); isesengura Uburyo bwo gusesengura ibintu: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) na 5 (leucine) aside enkephalin)).
Inkingi ya PMP (100 × 1.8 mm id) yapimwe kugira ngo hamenyekane itandukaniro ry’ibinyabutabire bya tryptic bya albumin yo mu maraso y’umuntu muri chromatography y’amazi ifite ubushobozi bwo hejuru. Chromatogram iri ku Ishusho ya 6 igaragaza ko icyitegererezo gitandukanye neza kandi ubushobozi bwo gupima ni bwiza cyane. Ibinyabutabire bya HSA byasesenguwe hakoreshejwe umuvuduko w’amazi wa 100 µL/min, igice cyo kugenda cya 70/30 acetonitrile/amazi na 0.1% TFA. Nkuko bigaragara kuri chromatogram (Ishusho ya 6), igogorwa rya HSA ryagabanijwemo imisozi 17 ijyanye na peptide 17. Ubuhanga bwo gutandukanya buri nshuro muri HSA bwarabazwe kandi agaciro kabyo katanzwe mu Imbonerahamwe ya 5.
Igipimo cya tryptic cya HSA (100 × 1.8 mm id) cyatandukanijwe ku nkingi ya PMP; umuvuduko w'amazi (100 µL/min), igice cyo kugenda cya acetonitrile 60/40/amazi hamwe na 0.1% TFA.
aho L ari uburebure bw'inkingi, η ni ubunini bw'igice cyo kugenda, ΔP ni igitutu cy'inyuma cy'inkingi, naho u ikaba umuvuduko ugororotse w'igice cyo kugenda. Ingufu z'inkingi ya PMP zari 2.5 × 10-14 m2, umuvuduko w'amazi wari 25 μL/min, naho 60/40 v/v ACN/amazi zakoreshejwe. Ingufu z'inkingi ya PMP (100 × 1.8 mm id) zari zisa n'iz'ubushakashatsi bwacu bwa mbere Ref.34. Ingufu z'inkingi zipakiyemo uduce dufite imyenge mike ni: 1.7 × 10-15 ku duce twa 1.3 μm, 3.1 × 10-15 ku duce twa 1.7 μm, 5.2 × 10-15 na 2.5 × 10-14 m2 ku duce twa 2.6 μm. Ku duce twa 5 μm 43. Kubwibyo, ingufu z'igice cya PMP zisa n'iz'uduce twa 5 μm tw'ibanze.
aho Wx ari uburemere bw'inkingi ipakiye chloroform, Wy ni uburemere bw'inkingi ipakiye methanol, naho ρ ikaba ubucucike bw'ikintu gishongesha. Ubucucike bwa methanol (ρ = 0.7866) na chloroform (ρ = 1.484). Ubucucike bwose bwa porosity ya SILICA PARTICLES-C18 columns (100 × 1.8 mm id) 34 na C18-Urea columns 31 twabanje kwiga bwari 0.63 na 0.55, uko bikurikirana. Ibi bivuze ko kuba hari urea ligands bigabanya ubucucike bw'igice gihagaze. Ku rundi ruhande, ubucucike bwose bw'inkingi ya PMP (100 × 1.8 mm id) ni 0.60. Ubucucike bw'inkingi ya PMP buri hasi ugereranyije n'inkingi zipakiye uduce twa silica twa C18 kuko mu bice bihagaze bya C18 ligands za C18 zifatanye n'uduce twa silica nk'iminyururu igororotse, mu gihe mu bice bihagaze bya polystyrene, A ifite ubunini bungana. Urukuta rwa polymer rukorwa rukikije. Mu igerageza risanzwe, porosity y'inkingi ibarwa muri ubu buryo:
Ishusho ya 7A, B yerekana inkingi ya PMP (100 × 1.8 mm id) n'inkingi ya Ascentis Express RP-Amide (100 × 1.8 mm id) hakoreshejwe uburyo bumwe bwo kugena (ni ukuvuga 60/40 ACN/H2O na 0.1% TFA).) y'ikibanza cya van Deemter. Uruvange rwa peptide rwatoranijwe (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) rwateguwe muri 20 µL/ Igipimo gito cy'amazi ku nkingi zombi ni 800 µL/min. Agaciro gato ka HETP ku gipimo cyiza cy'amazi (80 µL/min) ku nkingi ya PMP n'inkingi ya Ascentis Express RP-Amide kari 2.6 µm na 3.9 µm, uko bikurikirana. Agaciro ka HETP kerekana ko ubushobozi bwo gutandukanya inkingi ya PMP (100 × 1.8 mm id) ari bwiza cyane kuruta inkingi ya Ascentis Express RP-Amide iboneka ku isoko (100 × 1.8 mm id). Igishushanyo cya van Deemter kiri ku Ishusho ya 7 (A) kigaragaza ko kugabanuka k'agaciro ka N iyo amazi yiyongera atari ingenzi ugereranije n'ubushakashatsi bwacu bwa mbere. Ubuhanga bwo gutandukanya inkingi ya PMP (100 × 1.8 mm id) ugereranije n'inkingi ya Ascentis Express RP-Amide ishingiye ku kunoza imiterere y'uduce, ingano, n'uburyo bugoye bwo gupakira inkingi bukoreshwa muri iki gihe34.
(A) icyerekezo cya van Deemter (umuvuduko wa HETP ugereranije n'umuvuduko wa elegitoroniki) cyabonetse hakoreshejwe inkingi ya PMP (100 × 1.8 mm id) muri 60/40 ACN/H2O hamwe na 0.1% TFA. (B) icyerekezo cya van Deemter (umuvuduko wa HETP ugereranyije n'umuvuduko wa elegitoroniki) cyabonetse hakoreshejwe inkingi ya Ascentis Express RP-Amide (100 × 1.8 mm id) muri 60/40 ACN/H2O hamwe na 0.1% TFA.
Hateguwe kandi hasuzumwa uburyo bwo gutandukanya peptide zikozwe muri polar-embedded polystyrene stationary phase kugira ngo hamenyekane uburyo bwo gutandukanya peptide zivanze na trypsin digest ya human serum albumin (HAS) muri chromatography y'amazi ifite imikorere myiza. Imikorere ya chromatography y'inkingi za PMP ku mvange za peptide ni myiza cyane mu mikorere myiza no mu buryo bwo gutandukanya. Imikorere myiza yo gutandukanya inkingi za PMP iterwa n'impamvu zitandukanye, nko ingano y'uduce n'ingano y'uduce twa silica, ikorwa ry'uduce duto, hamwe no gupakira inkingi zikomeye. Uretse ubushobozi bwo gutandukanya inkingi, umuvuduko muto w'inyuma ku muvuduko mwinshi w'amazi ni ikindi cyiza cy'iki cyiciro gihoraho. Inkingi za PMP zigaragaza ubushobozi bwo kongera kubyara kandi zishobora gukoreshwa mu gusesengura uruvange rwa peptide no gusya proteini zitandukanye za trypsin. Turateganya gukoresha iyi nkingi mu gutandukanya ibikomoka ku bimera karemano, imvange zikora ku bimera n'ibikomoka ku bihumyo muri chromatography y'amazi. Mu gihe kizaza, PMP columns nazo zizasuzumwa mu gutandukanya poroteyine na antibodies za monoclonal.
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