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Yerekana karuseli ya sisitemu eshatu icyarimwe. Koresha Utubuto na Ibikurikira kugirango unyuze mumashusho atatu icyarimwe, cyangwa ukoreshe utubuto twa slide kumpera kugirango wimuke unyuze mubice bitatu icyarimwe.
Ibice bya silika binini byateguwe nuburyo bwa sol-gel hamwe na bimwe byahinduwe kugirango ubone ibice binini. Utwo duce twakomotse kuri N-phenylmaleimide-methylvinyl isocyanate (PMI) na styrene binyuze mu guhinduranya urunigi rwimurwa (RAFT) polymerisation kugirango habeho polyamide N-fenylmaleimide. Styrene (PMP) icyiciro gihagaze. Inkingi zifite ibyuma bidafite ingese (100 × 1.8 mm z'imbere z'imbere) zari zipakiye ibintu bipfunyitse. Imikorere ya chromatografique yinkingi ya PMP yasuzumwe kugirango itandukane uruvange rwa peptide yubukorikori igizwe na peptide eshanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acide enkephalin) na hydrolyzate ya tryptic hydrolyzate ya serumu yumuntu (HAS). Mugihe cyiza cyo gukuraho, umubare wibyerekezo byamasahani hamwe nuruvange rwa peptide wageze kuri plaque 280.000 / sq.m. Ugereranije imikorere yo gutandukanya inkingi yateye imbere hamwe nubucuruzi bwa Ascentis Express RP-Amide inkingi, byagaragaye ko uburyo bwo gutandukanya inkingi ya PMP bwarutaga inkingi yubucuruzi muburyo bwo gutandukana no gukemura.
Inganda zikomoka ku binyabuzima zahindutse isoko ry’isi yose ryiyongera cyane ku mugabane w’isoko mu myaka yashize. Hamwe no kwiyongera guturika kwinganda zikomoka ku binyabuzima 1,2,3 harakenewe cyane isesengura rya peptide na proteyine. Usibye intego ya peptide, habaho umwanda utandukanye mugihe cya synthesis ya peptide, bityo rero chromatografique isabwa kugirango ubone peptide yifuzwa. Isesengura no kuranga poroteyine mu mazi yo mu mubiri, mu ngingo, no mu ngirabuzimafatizo ni umurimo utoroshye cyane kubera ubwinshi bw’ibinyabuzima bishobora kumenyekana biboneka mu cyitegererezo kimwe. Nubwo mass spectrometrie nigikoresho cyingirakamaro mugukurikirana peptide na proteyine, niba izo ngero zinjiye muburyo butaziguye, gutandukana ntibizaba bishimishije. Iki kibazo gishobora gukemurwa no gukora chromatografiya yamazi (LC) mbere yisesengura rya MS, bizagabanya umubare wa analyite yinjira muri mass spectrometer mugihe runaka4,5,6. Byongeye kandi, analyse irashobora kwibanda mukarere kagufi mugihe cyo gutandukanya icyiciro cyamazi, bityo igahuza analyse kandi ikongerera sensibilité yo kumenya MS. Amazi ya chromatografiya (LC) yateye imbere cyane mumyaka icumi ishize kandi yabaye uburyo bukoreshwa muburyo bwo gusesengura proteomic 7,8,9,10.
Amazi ya chromatografiya yinyuma (RP-LC) akoreshwa cyane mugusukura no gutandukanya imvange ya peptide ukoresheje silika ya octadecyl yahinduwe (ODS) nkicyiciro gihagaze11,12,13. Nyamara, bitewe nuburyo bugoye hamwe na amphoteric, ibyiciro 14,15 bya RP ntibishobora gutanga itandukaniro rishimishije rya peptide na proteyine. Kubwibyo, isesengura rya peptide na proteyine hamwe nuduce twa polar na non-polar bisaba ibyiciro byabigenewe byabugenewe kugirango bihuze kandi bigumane analyite16. Chromatografi ivanze, itanga imikoranire myinshi, irashobora kuba inzira ya RP-LC yo gutandukanya peptide, proteyine, nibindi bivangavanze. Hateguwe ibyiciro byinshi bivanze-bihagaze kandi inkingi zuzuyemo ibyiciro bihagaze zikoreshwa mugutandukanya peptide na proteyine 17,18,19,20,21. Bitewe nuko hariho amatsinda ya polar na non-polar, ivanze ryuburyo buhagaze (WAX / RPLC, HILIC / RPLC, polar intercalation / RPLC) birakwiriye gutandukanya peptide na proteyine 22,23,24,25,26,27,28. , polar ihujwe nicyiciro gihagaze hamwe na colar ihuriweho na polar matsinda yerekana ubushobozi bwiza bwo gutandukana hamwe no guhitamo bidasanzwe kuri polar na non-polarite kuko gutandukana biterwa nimikoranire hagati ya analyte nicyiciro gihagaze Imikoranire ya Multimodal 29,30,31,32. Vuba aha, Zhang n'abandi. 30 yabonye ibyiciro bya behenyl byarangiye icyiciro cya polyamine kandi bitandukanya neza hydrocarbone, antidepressants, flavonoide, nucleoside, estrogene, hamwe nizindi analyite. Ibikoresho bya polar byashizwemo bifite amatsinda yombi ya polar na non-polar, bityo irashobora gukoreshwa mugutandukanya peptide na proteyine mubice bya hydrophobique na hydrophilique. Inkingi ya inline (urugero, C18 inkingi hamwe na amide inline) iraboneka mwizina ryubucuruzi Ascentis Express RP-Amide inkingi, ariko izi nkingi zakoreshejwe gusa mu gusesengura amine 33.
Mu bushakashatsi burimo gukorwa, hateguwe icyiciro cya polarike (N-phenylmaleimide, gushyiramo polystirene) kandi hasuzumwa uburyo bwo gutandukanya peptide hamwe na tryptic ya HSA. Ingamba zikurikira zakoreshejwe mugutegura icyiciro gihagaze. Uduce duto twa silika twateguwe dukurikije inzira zasobanuwe mu bitabo byabanjirije iki, hamwe n’impinduka zimwe na zimwe muri gahunda yo gutegura 31, 34, 35, 36, 37, 38, 39. Ibipimo bya urea, polyethylene glycol (PEG), TMOS na acide aque-acetike byahinduwe kugirango babone uduce duto twa silika nini nini. Icya kabiri, ligand nshya ya fenylmaleimide-methylvinyl isocyanate ligand yarashizwe hamwe hanyuma uduce duto twa silika tuvamo twakoreshejwe mugutegura polar yashizwemo ibyiciro bihagaze. Icyiciro cyahagaritswe cyapakiwe mu nkingi zidafite ingese (diameter y'imbere 100 × 1.8 mm) ukurikije gahunda yo gupakira neza. Gupakira inkingi bifashwa no kunyeganyega kugirango hamenyekane urwego rumwe murinkingi. Inkingi yapakiwe yasuzumwe kugirango itandukane ivanze rya peptide igizwe na peptide eshanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, peptide ya leucine-enkephalin). na tryptic hydrolysates ya serumu yumuntu albumin (HSA). Byagaragaye ko imvange ya peptide hamwe na HSA tryptic digest yatandukanijwe no gukemura neza no gukora neza. Gutandukanya imikorere yinkingi ya PMP yagereranijwe nubwa Ascentis Express RP-Amide. Byagaragaye ko peptide na proteyine bifite imiterere myiza kandi itandukanya cyane kurwego rwa PMP, kandi uburyo bwo gutandukanya inkingi ya PMP burenze ubw'inkingi ya Ascentis Express RP-Amide.
PEG. peroxide (BPO), acetonitrile (ACN) kuri HPLC, methanol, 2-propanol na acetone. Isosiyete ya Sigma-Aldrich (St. Louis, Missouri, Amerika).
Uruvange rwa urea (8 g), polyethylene glycol (8 g) na ml 8 ya 0.01 N. acide acetike yashizwemo iminota 10, hanyuma hongerwaho ml 24 za TMOS munsi yo gukonjesha urubura. Uruvangitirane rwa reaction rwashyutswe kuri 40 ° C mumasaha 6 hanyuma kuri 120 ° C mumasaha 8 muri autoclave idafite ibyuma. Amazi yarashizwemo hanyuma ibisigara byumishwa kuri 70 ° C mumasaha 12. Ibice byumye byumye byashizwe hasi kandi bibarwa mu ziko kuri 550 ° C mumasaha 12. Ibice bitatu byateguwe kandi birangwa kugirango bigerageze kubyara ingano yubunini, ubunini bwa pore nubuso bwubuso.
Itsinda rya polar hamwe nicyiciro gihagaze kumurongo wa polystirene. Uburyo bwo gutegura bwasobanuwe hano hepfo.
N. ) Uruvange rwashyutswe kuri 60 ° C mumasaha 3, rwungururwa hanyuma rwumishwa mu ziko kuri 40 ° C mumasaha 3.
Ibice bya silika byumye (2 g) byakwirakwijwe muri toluene yumye (ml 100), bikaranga hanyuma bigashyirwa muminota 10 mumashanyarazi ya ml 500. PMCP (10 mg) yashongeshejwe muri toluene hanyuma yongerwaho ibitonyanga kuri flask ya reaction ikoresheje umuyoboro wongeyeho. Uruvange rwongeye kugaruka kuri 100 ° C mu masaha 8, rwungururwa, rwozwa na acetone hanyuma rwumishwa kuri 60 ° C mu masaha 3. Noneho, ibice bya silika bifitanye isano na PMCP (100 g) byashongeshejwe muri toluene (200 ml), hanyuma 4-hydroxy-TEMPO (2 ml) byongerwaho imbere ya 100 μl ya dibutyltin dilaurate nka catalizator. Uruvange rwavanze kuri 50 ° C mumasaha 8, rwungururwa hanyuma rwumishwa kuri 50 ° C mumasaha 3.
Styrene (ml 1), benzoyl peroxide BPO (0,5 ml) hamwe na silika ya selile ifatanye na TEMPO-PMCP (1.5 g) yakwirakwijwe muri toluene hanyuma isukurwa na azote. Polymerisation ya styrene yakozwe kuri 100 ° C mumasaha 12. Ibicuruzwa bivamo byogejwe na methanol hanyuma byumishwa ijoro ryose kuri 60 ° C. Gahunda rusange ya reaction irerekanwa mugitabo. imwe.
Ingero zamanuwe kuri 393 K kuri 1 h kugeza habonetse umuvuduko usigaye uri munsi ya 10-3 Torr. Ingano ya N2 yamamajwe kumuvuduko ugereranije P / P0 = 0,99 yakoreshejwe kugirango hamenyekane ingano ya pore yose. Imiterere y’imiterere ya silika itunganijwe neza na ligande yasuzumwe hifashishijwe microscope ya elegitoronike (Hitachi High Technologies, Tokiyo, Ubuyapani). Ingero zumye (silika itunganijwe neza na ligand iboshye bya silika) yashyizwe kumukono wa aluminium ukoresheje kaseti. Zahabu yashyizwe kuri sample ikoresheje Q150T igikoresho cyo gusohora, hanyuma igipimo cya nm 5 nm cyashyizwe kuri sample. Ibi bitezimbere imikorere yumubyigano muke kandi bitanga imiti ikonje. Isesengura ryibanze ryakozwe hakoreshejwe Thermo Electron (Waltham, MA, USA) Flash EA1112 isesengura ibice. Isesengura ry'ubunini bwa Malvern (Worcestershire, UK) Mastersizer 2000 yakoreshejwe kugirango ibone ingano yo kugabana. Uduce duto twa silika idatwikiriwe hamwe na silika ihujwe na ligande (mg 5 buri umwe) yakwirakwijwe muri ml 5 ya isopropanol, ikorwa muminota 10, ihagarika iminota 5, ishyirwa ku ntebe ya optique ya Mastersizer. Isesengura rya Thermogravimetric rikorwa ku gipimo cya 5 ° C ku munota mu bushyuhe buri hagati ya 30 na 800 ° C.
Ikirahure cya fibre cyometse kumurongo wibyuma bidafite ibyuma (ID 100 × 1.8 mm) byapakishijwe uburyo bwo kuzuza ibicuruzwa bikurikiza uburyo bumwe nkubwo bwerekanwe 31. Inkingi yicyuma itagira umuyonga (ikirahuri cyanditswemo, ID 100 × 1 .8 mm) hamwe n’isoko ririmo frit 1 µm ryahujwe n’imashini ipakira ibintu (Alltech Deerfield, IL, USA). Tegura ihagarikwa ryicyiciro gihagaze uhagarika mg 150 yicyiciro gihagaze muri ml 1,2 ya methanol hanyuma ukayigaburira mu nkingi yikigega. Methanol yakoreshwaga nkibishishwa byoroshye kandi bigenzura. Gapakira inkingi ukoresheje umuvuduko ukabije wa MP 100 kuminota 10, 80 MP kuri 15 min, na MP 60 kuri 30 min. Uburyo bwo gupakira bwakoresheje gazi ebyiri za chromatografiya ya vibatori (Alltech, Deerfield, IL, USA) kugirango ihindagurika ryimashini kugirango harebwe inkingi imwe. Funga ibipfunyika hanyuma urekure buhoro buhoro kugirango wirinde kwangirika kumurongo. Inkingi yaciwe kuva nozzle ya slurry kandi ikindi gikwiye cyometse kumurongo hanyuma gihuza sisitemu ya LC kugirango igerageze imikorere yacyo.
MLC yabugenewe yubatswe hifashishijwe pompe ya LC (10AD Shimadzu, Ubuyapani), sampler ifite 50 nL yo gutera inshinge (Valco (USA) C14 W.05), degasser ya membrane (Shimadzu DGU-14A), hamwe nidirishya rya capillary UV-VIS. Igikoresho cyo gushakisha (UV-2075) hamwe na microcolumn. Koresha cyane kandi bigufi bihuza imiyoboro kugirango ugabanye ingaruka zo kwagura inkingi. Nyuma yo kuzuza inkingi, shyiramo capillary (50 µm id 365) kumasoko ya 1/16 ″ kugabanya aho uhurira hanyuma ushyire capillary (50 µm) yo kugabanya. Gukusanya amakuru no gutunganya chromatogramu bikorwa hakoreshejwe software ya Multichro 2000. Kuri 254 nm, UV yakira amasomo analytes yakurikiranwe kuri 0. Amakuru ya Chromatografique yasesenguwe hakoreshejwe OriginPro8 (Northampton, MA).
Serumu yumuntu, ifu ya lyofilize, ≥ 96% (agarose gel electrophoreis) mg 3 ivanze na trypsin (1.5 mg), 4.0 M urea (1 ml) na 0.2 M amonium bicarbonate (ml 1). Umuti washyizwe muminota 10 hanyuma ubikwa mu bwogero bwamazi kuri 37 ° C kuri 6 h, hanyuma uzimya ml 1 ya 0.1% TFA. Shungura igisubizo hanyuma ubike munsi ya 4 ° C.
Gutandukanya imvange ya peptide na tryptic digest HSA kumurongo wa PMP byasuzumwe ukundi. Reba hydrolysis ya tryptic ivanze ya peptide na HSA itandukanijwe ninkingi ya PMP hanyuma ugereranye ibisubizo hamwe na Ascentis Express RP-Amide. Umubare wibyapa byerekana ubarwa ukoresheje ikigereranyo gikurikira:
SEM amashusho yibice bya silika itunganijwe hamwe na ligande ihujwe na silika ya silika irerekanwa mumashusho 2. Ishusho ya SEM yibice bya silika yera (A, B) yerekana imiterere ya serefegitire aho ibice birebire cyangwa bifite uburinganire budasanzwe ugereranije nubushakashatsi twabanje. Ubuso bwibice bya silika bihujwe na ligand (C, D) byoroshye kuruta ibya selile silika nziza, bishobora guterwa numunyururu wa polystirene utwikiriye ubuso bwa silika.
Gusikana micrografi ya elegitoronike ya silika yuzuye (A, B) hamwe na ligande iboshye bya silika (C, D).
Ingano yubunini bwikwirakwizwa rya silika yuzuye hamwe na silika ihujwe na ligande irerekanwa mumashusho 2. 3 (A). Ingano yubunini bwikwirakwizwa ryerekanwe ko ingano ya silika yiyongereye nyuma yo guhindura imiti (Ishusho 3A). Ingano ya silika ingano yo gukwirakwiza amakuru avuye mubyigisho byubu hamwe nubushakashatsi bwabanje ugereranije mu mbonerahamwe ya 1 (A). Ingano ya volumetricike d (0.5) ya PMP yari 3,36 µm, ugereranije na ad (0.5) agaciro ka 3.05 µm mubushakashatsi bwacu bwibanze (polystirene ihujwe na silika ibice) 34. Bitewe nihinduka ryikigereranyo cya PEG, urea, TMOS na acide acetike mu ruvangitirane rwa reaction, ingano yubunini bwagabanijwe muri iki cyiciro yari mike ugereranije nubushakashatsi twabanje. Ingano yingingo yicyiciro cya PMP nini cyane ugereranije niy'icyiciro cya polyisirene ihujwe na silika icyiciro twize kare. Ibi bivuze ko ubuso bukora ibice bya silika hamwe na styrene byashyizwe gusa kuri polystirene (0,97 µm) hejuru ya silika, mugihe mugice cya PMP uburebure bwa 1.38 µm.
Ingano nini yo gukwirakwiza (A) no gukwirakwiza ingano ya pore (B) ya silika yuzuye ya silika na ligand iboshye silika.
Ingano ya pore, ingano ya pore, hamwe nubuso bwa sisitemu ya silika ikoreshwa muri ubu bushakashatsi irerekanwa mu mbonerahamwe ya 1 (B). Umwirondoro wa PSD wibice bya silika byera hamwe na silika ihujwe na ligande irerekanwa mumashusho. 3 (B). Ibisubizo byagereranijwe nubushakashatsi bwatubanjirije34. Ingano ya pore yibice bya silika byera na ligand byari 310 Å na 241 Å, byerekana ko nyuma yo guhindura imiti, ingano ya pore yagabanutseho 69 Å, nkuko bigaragara mu mbonerahamwe ya 1 (B), naho umurongo uhindagurika werekana ku gishushanyo. Nkuko bigaragara mu mbonerahamwe ya 1 (B), ubuso bwubuso (m2 / g) bwibice bya silika nyuma yo guhindura imiti nabyo byagabanutse kuva kuri 116 m2 / g bigera kuri 105 m2 / g.
Ibisubizo by'isesengura ryibanze ryicyiciro gihagaze bitangwa mumeza. . Uburemere bwa azote mugice cyubu gihagaze ni 2,21% ugereranije na 0.1735 na 0,85% mubushakashatsi bwabanje42. Ibi bivuze ko icyiciro gihagaze gifite uburemere buke bwa azote bitewe na fenylmaleimide. Mu buryo nk'ubwo, ibicuruzwa (4) na (5) bifite karubone ingana na 2.7% na 2,9%, mu gihe ibicuruzwa byanyuma (6) bifite karubone ya 6.35%, nkuko bigaragara mu mbonerahamwe ya 2. Isesengura rya Thermogravimetric (TGA) ryakoreshejwe ku cyiciro gihagaze cya PMP kugira ngo hamenyekane ko hagabanutse ibiro, kandi umurongo wa TGA werekana ku gipimo cya 8.6%, umurongo wa TGA werekana ko ufite uburemere bwa 8.6%. ligande ntabwo zirimo C gusa, ahubwo zirimo N, O na H.
Ligand phenylmaleimide-methylvinyl isocyanate yatoranijwe kugirango ihindure ubuso bwibice bya silika kubera polar ya fenylmaleimide hamwe nitsinda rya vinylisocyanate. Amatsinda ya Vinyl isocyanate arashobora kongera kwitwara hamwe na styrene mubuzima bwa polymerisiyonike. Impamvu ya kabiri ni ugushyiramo itsinda rifite imikoranire iringaniye na analyte kandi nta mikoranire ikomeye ya electrostatike iri hagati ya analyte nicyiciro gihagaze, kubera ko umuyaga wa fenylmaleimide udafite amafaranga yubusa kuri pH isanzwe. Ubuharike bwicyiciro gihagaze burashobora kugenzurwa numubare mwiza wa styrene hamwe nigihe cyo kwitwara kwa polymerisiyasi yubusa. Intambwe yanyuma ya reaction (free radical polymerisation yubusa) irakomeye kuko ihindura polarite yicyiciro gihagaze. Isesengura ryibanze ryakozwe kugirango harebwe ibirimo karubone muri ibi byiciro bihagaze. Byaragaragaye ko kongera umubare wa styrene nigihe cyo kubyitwara byongera karubone yibyiciro bihagaze naho ubundi. SP yateguwe hamwe na styrene itandukanye ifite imitwaro ya karubone itandukanye. Mu buryo nk'ubwo, ibyiciro bihagaze byashyizwe kumurongo wibyuma kandi ibiranga chromatografique (guhitamo, gukemura, N agaciro, nibindi) byagenzuwe. Hashingiwe kuri ubwo bushakashatsi, hatoranijwe uburyo bwiza bwo gutegura icyiciro cya PMP gihagaze kugirango hatangwe polarite igenzurwa no kugumana neza analyte.
Inkingi ya PMP yasuzumwe kandi kugira ngo isesengure imvange eshanu za peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) hakoreshejwe ubushobozi bw'icyiciro kigendanwa. 60/40 (v / v) ACN / amazi (0.1% TFA) ku kigero cya 80 µl / min. Mugihe gikwiye cyo gukuraho (200.000 plaque / m), umubare wibyapa (N) kuri buri nkingi (100 × 1.8 mm) ni 20.000 ± 100. Indangagaciro N ku nkingi eshatu za PMP zerekanwa mu mbonerahamwe ya 3 naho chromatogramu zerekanwa ku gishushanyo cya 5A. Isesengura ryihuse ku kigero kinini (700 µl / min) ku nkingi ya PMP, peptide eshanu zavuzwe mu minota umwe, N nziza nziza ya 13.500 ± 330 ku nkingi (100 x 1.8 mm ya diametre), ihwanye n'amasahani 135.000 / m (Ishusho 5B). Inkingi eshatu zingana (diameter y'imbere 100 x 1.8 mm) zuzuyemo ibyiciro bitatu bitandukanye bya PMP icyiciro gihagarara kugirango hamenyekane imyororokere. Isesengura ryanditswe kuri buri nkingi mugutandukanya ikizamini kimwe kivanze kuri buri nkingi ukoresheje uburyo bwiza bwo gukuraho, umubare wibyapa bya N, nigihe cyo kugumana. Imyororokere yamakuru yinkingi ya PMP yerekanwa mu mbonerahamwe ya 4. Imyororokere yinkingi ya PMP ihujwe neza nagaciro gake cyane RSD nkuko bigaragara mu mbonerahamwe ya 3.
Gutandukanya imvange ya peptide kumurongo wa PMP (B) na Ascentis Express RP-Amide inkingi (A), icyiciro cya mobile 60/40 ACN / H2O (TFA 0.1%), ibipimo byinkingi ya PMP (100 x 1.8 mm id), gusesengura gahunda yo gukuraho ibice: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gyr) (Tyr-Ile-Gly-Ser-Arg) na 5 (acide leucic enkephalin).
Inkingi ya PMP (diameter y'imbere 100 x 1.8 mm) yasuzumwe kugirango itandukane rya hydrolyzate ya tryptic hydrolyzate ya serumu yumuntu na HPLC. Chromatogramu mu gishushanyo cya 6 yerekana ko ingero zitandukanijwe neza hamwe no gukemura neza. Ibisubizo bya HSA byasesenguwe hakoreshejwe umuvuduko wa 100 μl / min, icyiciro kigendanwa cya 70/30 acetonitrile / amazi na 0.1% TFA. Cleavage ya HSA yagabanijwemo impinga 17, nkuko bigaragara muri chromatogramu (Ishusho 6), ihwanye na peptide 17. Ingaruka zo gutandukanya impinga ya buri muntu kuri hydrolyzate ya HSA zarabazwe kandi indangagaciro zigaragara mu mbonerahamwe ya 5.
HSA tryptic hydrolysates yatandukanijwe kumurongo wa PMP (diameter y'imbere 100 x 1.8 mm), umuvuduko (100 μl / min), icyiciro kigendanwa 60/40 acetonitrile / amazi, na 0.1% TFA.
aho L nuburebure bwinkingi, η nubukonje bwicyiciro kigendanwa, ΔP nigitutu cyinyuma cyinkingi, na u numuvuduko wumurongo wicyiciro cya mobile. Ubucucike bwinkingi ya PMP bwari 2,5 × 10–14 m2, umuvuduko wa 25 µl / min, 60/40 v / v wakoreshejwe. ACN / amazi. Ubucucike bwinkingi ya PMP (ID 100 × 1.8 mm) bwari busa nubwa mbere ubushakashatsi.34. Ubucucike bwinkingi yuzuyemo ibice bitagaragara ni 1.7 × 10 .6 µm, 2.5 × 10-14 m2 kuri 5 µm ibice43. Kubwibyo, ubwikorezi bwicyiciro cya PMP burasa nubucucike bwibice-shitingi bifite ubunini bwa 5 mm.
aho Wx ni misa yinkingi yuzuyemo chloroform, Wy ni misa yinkingi yuzuye methanol, na ρ nubucucike bwumuti. Ubucucike bwa methanol (ρ = 0.7866) na chloroform (ρ = 1.484). Ubwinshi bwimbaraga za silika-C18 inkingi (100 × 1.8 mm ID) 34 hamwe na C18-urea31 inkingi twize mbere yari 0,63 na 0.55. Ibi bivuze ko kuba urea ligands igabanya ubwikorezi bwicyiciro gihagaze. Kurundi ruhande, porotike yuzuye yinkingi ya PMP (diameter y'imbere 100 × 1.8 mm) ni 0.60. Inkingi ya PMP ntishobora kwemerwa cyane kuruta inkingi zipakishijwe na silika ya C18 iboshye kubera ko mu cyiciro cya C18 icyiciro gihagaze C18 ligande ifatanye nuduce twa silika muminyururu, naho mugice cya polystirene icyiciro gihagaze gikozwe mubyuma bya polymer. urwego A. Mubigeragezo bisanzwe, inkingi yinkingi ibarwa kuburyo bukurikira:
Ku mutini. 7A, B yerekana Van Deemter ibibanza byinkingi ya PMP (id 100 x 1.8 mm) hamwe na Ascentis Express RP-Amide inkingi (id 100 x 1.8 mm) mubihe bimwe byo gukuraho, 60/40 ACN / H2O na 0 .1% TFA 20 µl / min kugeza 800 µl / min kuri izo nkingi zombi. Indangagaciro ntarengwa ya HETP ku gipimo cyiza (80 µl / min) yari 2,6 µm na 3,9 µm ku nkingi ya PMP hamwe na Ascentis Express RP-Amide inkingi. Indangagaciro za HETP zerekana ko gutandukanya imikorere yinkingi ya PMP (100 x 1.8 mm id) isumba cyane iy'ubucuruzi buboneka Ascentis Express RP-Amide inkingi (100 x 1.8 mm id). Igishushanyo cya van Deemter ku gishushanyo cya 7 (A) cyerekana ko igabanuka rya N agaciro ritari hejuru cyane hamwe no kwiyongera gutembera ugereranije nubushakashatsi bwatubanjirije. Uburyo bwiza bwo gutandukanya inkingi ya PMP (id 100 × 1.8 mm) ugereranije na Ascentis Express RP-Amide inkingi ishingiye kumiterere yimiterere yubunini nubunini hamwe nuburyo bukomeye bwo gupakira inkingi zikoreshwa mubikorwa byubu34.
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Icyiciro cya polar ihagaze ya polystirene ihuza intera yateguwe kandi isuzumwa kugirango hatandukanywe uruvange rwa peptide ya sintetike na hydrolyzate ya tryptic hydrolyzate ya serumu yumuntu (HSA) muri chromatografiya ikora neza. Imikorere ya chromatografique ya PMP yinkingi ya peptide ivanze nibyiza muburyo bwo gutandukana no gukemura. Kunoza imikorere yo gutandukanya inkingi ya PMP biterwa nimpamvu nyinshi nkubunini bwa silika ingano nubunini bwa pore, kugenzura kugenzura ibyiciro bihagaze, hamwe nibikoresho bipakira inkingi. Usibye uburyo bwiza bwo gutandukana, ikindi cyiza cyiki cyiciro gihagaze ni inkingi yo hasi yinyuma yumuvuduko mwinshi. Inkingi za PMP zororoka cyane kandi zirashobora gukoreshwa mu gusesengura imvange ya peptide no gusya kwa tryptic ya poroteyine zitandukanye. Turashaka gukoresha iyi nkingi mugutandukanya ibinyabuzima bioaktike nibicuruzwa bisanzwe, ibimera byibiti bivura imiti nibihumyo muri chromatografiya. Mu bihe biri imbere, inkingi za PMP nazo zizasuzumwa kugirango hatandukanywe poroteyine na antibodiyite za monoclonal.
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