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Likaroloana tsa silica tse nang le masoba li lokisitsoe ka mokhoa oa sol-gel ka liphetoho tse ling ho fumana likaroloana tse nang le masoba a maholo. Likaroloana tsena li nkiloe ka polymerization ea phetisetso ea ketane e ka fetoloang hape (RAFT) ka N-phenylmaleimide-methylvinylisocyanate (PMI) le styrene ho lokisa N-phenylmaleimide intercalation ea polystyrene (PMP) stationary phase. Litšiea tsa tšepe e sa ruseng tse nang le moqotetsane (100 × 1.8 mm id) li ne li pakiloe ka ho paka ka slurry. Karohano ea kholomo ea PMP e hlahlobiloeng ea motsoako oa peptide o nang le li-peptide tse hlano (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) chromatographic performance) le trypsin ea human serum albumin (HAS). Tlas'a maemo a matle a ho hlakisa, palo ea lipoleiti tsa khopolo-taba ea motsoako oa peptide e holimo ho fihla ho lipoleiti tse 280,000/m². Ha ho bapisoa ts'ebetso ea karohano ea kholomo e ntshetsweng pele e nang le kholomo ya kgwebo ya Ascentis Express RP-Amide, ho ile ha bonwa hore tshebetso ya karohano ya kholomo ya PMP e ne e phahametse kholomo ya kgwebo mabapi le bokgoni ba karohano le tharollo.
Lilemong tsa morao tjena, indasteri ea meriana ea bio e fetohile 'maraka o ntseng o hola oa lefats'e ka keketseho e kholo ea karolo ea' maraka. Ka kholo e matla ea indasteri ea meriana ea bio1,2,3, tlhahlobo ea li-peptide le liprotheine e lakatseha haholo. Ntle le peptide e reriloeng, litšila tse 'maloa li hlahisoa nakong ea tlhahiso ea peptide, kahoo ho hlokahala tlhoekiso ea chromatographic ho fumana li-peptide tsa bohloeki bo lakatsehang. Tlhahlobo le tlhaloso ea liprotheine marong a' mele, lisele le lisele ke mosebetsi o boima haholo ka lebaka la palo e kholo ea mefuta e ka bonoang sampole e le 'ngoe. Le hoja spectrometry ea boima e le sesebelisoa se sebetsang bakeng sa tatellano ea peptide le protheine, haeba lisampole tse joalo li kenngoa ka har'a spectrometer ea boima ka nako e le 'ngoe, karohano e ke ke ea ba ntle. Bothata bona bo ka fokotsoa ka ho kenya tšebetsong karohano ea chromatography ea metsi (LC) pele ho tlhahlobo ea MS, e tla fokotsa palo ea li-analyte tse kenang ho spectrometer ea boima ka nako e itseng 4,5,6. Ho phaella moo, nakong ea karohano ea karolo ea metsi, li-analyte li ka tsepamisoa libakeng tse moqotetsane, ka hona li tsepamisa li-analyte tsena le ho ntlafatsa ho fumanoa ha MS kutloelo-bohloko.Chromatography ea metsi (LC) e tsoetse pele haholo lilemong tse leshome tse fetileng 'me e se e le mokhoa o tsebahalang oa tlhahlobo ea proteomic7,8,9,10.
Chromatography ea metsi e fetotsoeng mohato (RP-LC) e sebelisoa haholo bakeng sa ho hloekisa le ho arola metsoako ea peptide ho sebelisoa octadecyl-modified silica (ODS) e le mohato o sa fetoheng11,12,13. Leha ho le joalo, mekhahlelo e sa fetoheng ea RP ha e fane ka karohano e khotsofatsang ea li-peptide le liprotheine ka lebaka la sebopeho sa tsona se rarahaneng le tlhaho ea tsona e sa fetoheng 14,15. Ka hona, mekhahlelo e sa fetoheng e entsoeng ka ho khetheha ea hlokahala ho sekaseka li-peptide le liprotheine tse nang le likarolo tsa polar le tse seng tsa polar ho sebelisana le ho boloka li-analyte tsena16. Chromatography ea mokhoa o tsoakiloeng, e fanang ka likamano tse ngata, e ka ba mokhoa o mong oa RP-LC bakeng sa karohano ea li-peptide, liprotheine le metsoako e meng e rarahaneng. Mekhahlelo e 'maloa e sa fetoheng ea mokhoa o tsoakiloeng e se e lokisitsoe, 'me likholomo tse tletseng mekhahlelo ena li sebelisitsoe bakeng sa karohano ea peptide le protheine17,18,19,20,21. Mekhahlelo e sa fetoheng ea mokhoa o tsoakiloeng (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) li loketse karohano ea peptide le protheine ka lebaka la ho ba teng ha lihlopha tsa polar le tse seng polar22,23,24,25,26,27,28. Ka ho tšoanang, mekhahlelo e sa fetoheng ea polar e nang le lihlopha tsa polar tse kopantsoeng ka covalently e bontša matla a karohano a matle le khetho e ikhethang bakeng sa li-analyte tsa polar le tse seng polar, kaha karohano e itšetlehile ka tšebelisano pakeng tsa analyte le mohato o sa fetoheng. Likamano tsa Multimodal 29, 30, 31, 32. Haufinyane tjena, Zhang et al. 30 ba lokisitse karolo e sa fetoheng ea polyamine e felisitsoeng ke dodecyl mme ba arotse ka katleho li-hydrocarbon, li-antidepressants, li-flavonoid, li-nucleoside, li-estrogen, le li-analyte tse ling tse 'maloa. Sehokelo sa polar se na le lihlopha tsa polar le tse seng tsa polar, kahoo se ka sebelisoa ho arola li-peptide le liprotheine tse nang le likarolo tse sa rateheng le tse phelang. Likholomo tse kentsoeng ka polar (mohlala, likholomo tsa C18 tse kentsoeng ka amide) li fumaneha khoebong tlas'a lebitso la khoebo la Ascentis Express RP-Amide columns, empa likholomo tsena li sebelisetsoa ho sekaseka amine 33 feela.
Phuputsong ea hona joale, ho lokisitsoe le ho hlahlojoa karolo e sa fetoheng e kentsoeng polar (polystyrene e kopantsoeng ea N-phenylmaleimide) bakeng sa karohano ea li-peptide le trypsin digests tsa HSA. Karolo e sa fetoheng e lokisitsoe ho sebelisoa leano le latelang. Likaroloana tsa silica tse nang le masoba li lokisitsoe ho latela mokhoa o fanoeng khatisong ea rona e fetileng ka liphetoho tse ling ho protocol ea ho lokisa. Karolelano ea urea, polyethylene glycol (PEG), TMOS, asiti ea metsi e ile ea fetoloa ho lokisa likaroloana tsa silica tse nang le boholo bo boholo ba pore. Ea bobeli, ligand e ncha, phenylmaleimide-methyl vinyl isocyanate, e ile ea etsoa 'me ea sebelisoa ho ntša likaroloana tsa silica ho lokisa karolo e sa fetoheng e kentsoeng polar. Karolo e sa fetoheng e ileng ea hlahisoa e ile ea kenngoa kholomong ea tšepe e sa hloekang (100 × 1.8 mm id) ho sebelisoa mokhoa o ntlafalitsoeng oa ho paka. Ho paka kholomo ho thusoa ka ho thothomela ha mechini ho netefatsa hore bethe e ts'oanang e thehoa ka har'a kholomo. Lekola karohano ea kholomo e pakiloeng ea metsoako ea peptide e nang le li-peptide tse hlano; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) le Trypsin digest ea human serum albumin (HAS). Motsoako oa peptide le trypsin digest ea HSA li ile tsa bonoa li arohane ka qeto e ntle le katleho. Tshebetso ea karohano ea kholomo ea PMP e ile ea bapisoa le ea kholomo ea Ascentis Express RP-Amide. Li-peptide le liprotheine ka bobeli li ile tsa bonoa li rarollotsoe hantle ebile li sebetsa hantle kholomong ea PMP, e neng e sebetsa hantle ho feta kholomo ea Ascentis Express RP-Amide.
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, le Acetone E rekiloe ho Sigma-Aldrich (St. Louis, MO, USA).
Motsoako oa urea (8 g), polyethylene glycol (8 g), le 8 mL ea 0.01 N acetic acid e ile ea hlohlelletsoa metsotso e 10, eaba 24 mL ea TMOS e eketsoa ho eona tlas'a maemo a batang joaloka leqhoa. Motsoako oa karabelo o ile oa futhumatsoa ho 40°C ka lihora tse 6 ebe ho 120°C ka lihora tse 8 ka har'a autoclave ea tšepe e sa hloekang. Metsi a ile a tšolloa 'me lintho tse setseng tsa omisoa ho 70°C ka lihora tse 12. Motsoako o bonolo o omisitsoeng o ile oa siloa ka ontong 'me oa chesoa ho 550°C ka lihora tse 12. Lihlopha tse tharo li ile tsa lokisoa 'me tsa khetholloa ho hlahloba ho ikatisa hape ka boholo ba likaroloana, boholo ba li-pore le sebaka sa bokaholimo.
Ka ho fetola bokaholimo ba dikarolwana tsa silica ka ligand phenylmaleimide-methylvinylisocyanate (PCMP) e seng e entsoe pele e latelwa ke radial polymerization ka styrene, ho ile ha lokiswa motsoako o nang le sehlopha sa polar. Mokgahlelo o sa sisinyeheng bakeng sa di-aggregates le diketane tsa polystyrene. Mokgwa wa ho lokisa o hlaloswa ka tlase.
N-phenylmaleimide (200 mg) le methyl vinyl isocyanate (100 mg) li ile tsa qhibilihisoa ka har'a toluene e omileng, 'me 0.1 mL ea 2,2′-azoisobutyronitrile (AIBN) e ile ea eketsoa ka har'a flask ea karabelo ho lokisa phenylmaleimide-methyl vinyl isocyanate copolymer (PMCP).. Motsoako o ile oa futhumatsoa ho 60°C ka lihora tse 3, oa sefshoa 'me oa omisoa ka ontong ho 40°C ka lihora tse 3.
Dikaroloana tsa silica tse ommeng (2 g) di ile tsa hasana ka har'a toluene e omileng (100 mL), tsa hlohlelletsoa le ho hlatsuoa ka har'a botlolo e chitja ea 500 mL ka metsotso e 10. PMCP (10 mg) e ile ea qhalana ka har'a toluene 'me ea eketsoa ka ho theohela ka har'a botlolo ea karabelo ka funnel e theohelang. Motsoako o ile oa khutlisetsoa mochesong oa 100°C ka lihora tse 8, oa sefshoa 'me oa hlatsuoa ka acetone 'me oa omisoa ka har'a 60°C ka lihora tse 3. Eaba, likaroloana tsa silica tse kopantsoeng le PMCP (100 g) li ile tsa qhalana ka har'a toluene (200 ml) 'me 4-hydroxy-TEMPO (2 mL) ea eketsoa boteng ba 100 µL ea dibutyltin dilaurate e le catalyst. Motsoako o ile oa hlohlelletsoa ka har'a 50°C ka lihora tse 8, oa sefshoa 'me oa omisoa ka har'a 50°C ka lihora tse 3.
Styrene (1 mL), benzoyl peroxide BPO (0.5 mL), le dikarolwana tsa silica tse kgomaretsweng ke TEMPO-PMCP (1.5 g) di ile tsa hasana ka toluene mme tsa hlwekiswa ka naetrojene. Ho polymerization ha styrene ho ile ha etswa ka 100°C ka dihora tse 12. Sehlahiswa se hlatswitsweng se ile sa hlatsuwa ka methanol mme sa omiswa ka 60°C bosiu bohle. Moralo wa karabelo ka kakaretso o bontshitswe ho Setšoantšo sa 1.
Disampole di ile tsa ntshuwa ka mosi ho 393 K ka hora e le nngwe ho fumana kgatello e setseng ya ka tlase ho 10-3 Torr. Bongata ba N2 bo amohuweng ka kgatello e lekanyelitsoeng ya P/P0 = 0.99 bo sebedisitswe ho fumana bophahamo ba pore kaofela. Sebopeho sa dikarolwana tsa silica tse se nang letho le tse nang le ligand se ile sa hlahlojwa ka microscopy ya elektrone e skenwang (Hitachi High Technologies, Tokyo, Japane). Disampole tse omisitsweng (dikarolwana tsa silica tse se nang letho le tse nang le ligand) di ile tsa bewa hodima kholomo ya aluminium ho sebediswa theipi ya khabone e kgomaretsang. Kgauta e ile ya manehwa hodima disampole ho sebediswa sekwahelo sa Q150T sputter, mme lera la 5 nm Au la bewa hodima disampole. Sena se ntlafatsa bokgoni ba tshebetso ho sebediswa di-voltage tse tlase mme se fana ka makumane a matle, a batang. Sesebelisoa sa Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental analyzer se ile sa sebediswa bakeng sa tlhahlobo ya elemental. Sesebelisoa sa Malvern (Worcestershire, UK) Mastersizer 2000 particle size analyzer se ile sa sebediswa ho fumana kabo ya boholo ba dikarolwana. Silica e hlobotseng Likaroloana le likaroloana tsa silica tse kopantsoeng le ligand (5 mg ka 'ngoe) li ile tsa hasana ka har'a 5 mL ea isopropanol, tsa kenngoa ka sonic metsotso e 10, tsa koaheloa ka vortex metsotso e 5, 'me tsa beoa bencheng ea optical ea Mastersizer. Tlhahlobo ea Thermogravimetric e entsoe ka sekhahla sa 5 °C ka motsotso holim'a mocheso oa 30 ho isa ho 800 °C.
Litšiea tse tšesaane tsa tšepe e sa ruseng e manehiloeng ka khalase tse nang le mokoti o moqotetsane oa boholo (100 × 1.8 mm id) li ile tsa pakoa ho sebelisoa mokhoa oa ho paka ka lerōle, ho sebelisoa mokhoa o tšoanang o sebelisitsoeng ho Ref. 31. Kholomo ea tšepe e sa hloekang (e manehiloeng ka khalase, 100 × 1.8 mm id) e nang le sehokelo sa ho ntša metsi se nang le 1 µm frit e hokahantsoe le sephutheloana sa slurry (Alltech Deerfield, IL, USA). Lokisetsa sehokelo sa mohato o sa sisinyeheng ka ho fanyeha 150 mg ea mohato o sa sisinyeheng ka 1.2 mL ea methanol 'me u e romele kholomong ea polokelo. Methanol e sebelisitsoe e le tharollo ea slurry hammoho le tharollo ea propelling. Tlatsa kholomo ka tatellano ka ho sebelisa likhatello tsa 100 MP metsotso e 10, 80 MP metsotso e 15, le 60 MP metsotso e 30. Nakong ea ho paka, ho ile ha sebelisoa ho thothomela ha mechini ka li-shaker tse peli tsa GC column (Alltech, Deerfield, IL, USA) ho netefatsa hore kholomo e paka ka ho lekana. Koala sephutheloana sa slurry 'me u lokolle khatello butle ho thibela tšenyo efe kapa efe ka har'a kholomo. Tlosa kholomo ho yuniti ea ho paka ea slurry 'me u hokahanye e' ngoe e kenang le tsamaisong ea LC ho hlahloba hore na e tshebetso.
Pompo ea LC (10AD Shimadzu, Japane), enjector (Valco (USA) C14 W.05) e nang le loop ea ente ea 50nL, membrane degasser (Shimadzu DGU-14A), fensetere ea capillary ea UV-VIS e hahiloe. Sesebelisoa sa ho lemoha sesebelisoa sa µLC (UV-2075) se khethehileng le li-microcolumn tse nang le khalase. Sebelisa tube e moqotetsane haholo le e khutšoane ea ho hokahanya ho fokotsa phello ea ho atoloha ha lebanta la kholomo e eketsehileng. Kamora ho paka, li-capillaries (50 μm id 365 le li-capillaries tse fokotsang (50 μm) li kentsoe sebakeng sa 1/16″ sa mokhatlo o fokotsang. Pokello ea data le ts'ebetso ea chromatographic li entsoe ho sebelisoa software ea Multichro 2000. Tlhokomelo ho 254 nm Analytes e ile ea lekoa bakeng sa ho monya UV. Lintlha tsa chromatographic li ile tsa hlahlojoa ke OriginPro8 (Northampton, MA).
Albumin e tsoang serum ea motho, phofo e lyophilized, ≥ 96% (agarose gel electrophoresis) 3 mg e tsoakiloeng le trypsin (1.5 mg), 4.0 M urea (1 mL), le 0.2 M ammonium bicarbonate (1 mL). Tharollo e ile ea hlohlelletsoa metsotso e 10 'me ea bolokoa ka bateng ea metsi ho 37°C ka lihora tse 6, ebe e tima ka 1 mL ea 0.1% TFA. Sefa tharollo 'me u e boloke ka tlase ho 4 °C.
Karohano ea metsoako ea peptide le HSA trypsin digests e hlahlobiloe ka thoko likholomong tsa PMP. Hlahloba karohano ea motsoako oa peptide le trypsin digest ea HSA ka kholomo ea PMP 'me u bapise liphetho le kholomo ea Ascentis Express RP-Amide. Nomoro ea poleiti ea khopolo-taba e baloa ka tsela e latelang:
Litšoantšo tsa SEM tsa likaroloana tsa silica tse se nang letho le likaroloana tsa silica tse kopantsoeng le ligand li bontšitsoe ho Setšoantšo sa 2. Litšoantšo tsa SEM tsa likaroloana tsa silica tse se nang letho (A, B) li bontša hore, ho fapana le lithuto tsa rona tsa pele, likaroloana tsena li chitja moo likaroloana li leng telele kapa li nang le ho se leka-lekane ho sa tloaelehang. Bokaholimo ba likaroloana tsa silica tse kopantsoeng le ligand (C, D) bo boreleli ho feta ba likaroloana tsa silica tse se nang letho, e leng se ka bakoang ke ho koaheloa ha liketane tsa polystyrene holim'a likaroloana tsa silica.
Ho skena ditshwantsho tsa microscope ya elektrone tsa dikarolwana tse se nang letho tsa silica (A, B) le dikarolwana tsa silica tse tlamilweng ke ligand (C, D).
Kabo ea boholo ba likaroloana tsa likaroloana tsa silica e se nang letho le likaroloana tsa silica tse kopantsoeng le ligand li bontšoa ho Setšoantšo sa 3(A). Methapo ea kabo ea boholo ba likaroloana e thehiloeng holim'a bophahamo e bontšitse hore boholo ba likaroloana tsa silica bo eketsehile kamora phetoho ea lik'hemik'hale (Setšoantšo sa 3A). Lintlha tsa kabo ea boholo ba likaroloana tsa silica ho tsoa thutong ea hona joale le thutong e fetileng li bapisoa ho Tafole ea 1(A). Boholo ba likaroloana bo thehiloeng holim'a bophahamo, d(0.5), ea PMP ke 3.36 μm, ha bo bapisoa le thuto ea rona e fetileng ka boleng ba ad(0.5) ba 3.05 μm (likaroloana tsa silica tse kopantsoeng le polystyrene)34. Sehlopha sena se ne se e-na le kabo e fokolang ea boholo ba likaroloana ha e bapisoa le thuto ea rona e fetileng ka lebaka la likarolelano tse fapaneng tsa PEG, urea, TMOS, le acetic acid motsoakong oa karabelo. Boholo ba likaroloana ba karolo ea PMP bo boholo hanyane ho feta ba karoloana ea silica e kopantsoeng le polystyrene eo re ithutileng eona pele. Sena se bolela hore ts'ebetso ea bokaholimo ba likaroloana tsa silica tse nang le styrene e behile lera la polystyrene feela (0.97 µm) holim'a bokaholimo ba silika, athe mohatong oa PMP botenya ba lera e ne e le 1.38 µm.
Kabo ea boholo ba likaroloana (A) le kabo ea boholo ba masoba (B) ea likaroloana tsa silica tse se nang letho le likaroloana tsa silica tse tlamiloeng ke ligand.
Boholo ba pore, bophahamo ba pore le sebaka sa bokaholimo ba likaroloana tsa silica tsa thuto ea hona joale li fanoe ho Tafole ea 1(B). Liprofaele tsa PSD tsa likaroloana tsa silica tse se nang letho le likaroloana tsa silica tse kopantsoeng le ligand li bontšitsoe ho Setšoantšo sa 3(B). Liphetho li tšoana le thuto ea rona ea pejana. Boholo ba pore ba likaroloana tsa silica tse se nang letho le tse kopantsoeng le ligand ke 310 le 241, ka ho latellana, e leng se bontšang hore boholo ba pore bo fokotseha ka 69 kamora phetoho ea lik'hemik'hale, joalo ka ha ho bontšitsoe ho Tafole ea 1(B), 'me phetoho ea mothapo e bontšoa ho Setšoantšo sa 3(B). Ka mokhoa o ts'oanang, bophahamo ba pore ba likaroloana tsa silica bo fokotsehile ho tloha ho 0.67 ho isa ho 0.58 cm3/g kamora phetoho ea lik'hemik'hale. Sebaka se ikhethileng sa likaroloana tsa silica tse ithutoang hona joale ke 116 m2/g, se tšoanang le thuto ea rona ea pele (124 m2/g). Joalokaha ho bontšitsoe ho Tafole ea 1(B), sebaka sa bokaholimo (m2/g) sa likaroloana tsa silica le sona se fokotsehile ho tloha ho 116 m2/g ho isa ho 105 m2/g kamora phetoho ea lik'hemik'hale.
Liphetho tsa tlhahlobo ea motheo ea mohato o sa sisinyeheng li bontšitsoe ho Tafole ea 2. Khabone e jarollotsoeng ea mohato o sa sisinyeheng oa hona joale ke 6.35%, e leng tlase ho feta khabone e jarollotsoeng ea thuto ea rona e fetileng (likaroloana tsa silica tse kopantsoeng tsa polystyrene, 7.93%35 le 10.21%, ka ho latellana) 42. Khabone e jarollotsoeng ea mohato o sa sisinyeheng oa hona joale e tlase, Hobane ha ho lokisoa SP ea hona joale, ho phaella ho styrene, ho ile ha sebelisoa li-ligand tse ling tsa polar tse kang phenylmaleimide-methylvinylisocyanate (PCMP) le 4-hydroxy-TEMPO. Peresente ea boima ba naetrojene ea mohato o sa sisinyeheng oa hona joale ke 2.21%, ha e bapisoa le 0.1735 le 0.85% ka boima ba naetrojene lithutong tse fetileng, ka ho latellana. Sena se bolela hore % ea boima ba naetrojene e phahame mohatong o sa sisinyeheng oa hona joale ka lebaka la phenylmaleimide. Ka ho tšoanang, khabone e jarollotsoeng ea lihlahisoa (4) le (5) e ne e le 2.7% le 2.9%, ka ho latellana, ha ho jara khabone ha sehlahisoa sa ho qetela (6) e ne e le 6.35%, joalo ka ha ho bontšitsoe ho Tafole ea 2. Ho lahleheloa ke boima ho hlahlobiloe ka karolo e sa fetoheng ea PMP, 'me moqolo oa TGA o bontšitsoe ho Setšoantšo sa 4. Moqolo oa TGA o bontša ho lahleheloa ke boima ba 8.6%, e leng se lumellanang hantle le moqolo oa khabone (6.35%) hobane ligand ha li na C feela empa hape le N, O, le H.
Phenylmaleimide-methylvinylisocyanate ligand e kgethilwe bakeng sa phetoho ya bokahodimo ba dikarolwana tsa silica hobane e na le dihlopha tsa polar phenylmaleimide le dihlopha tsa vinylisocyanate. Dihlopha tsa Vinyl isocyanate di ka arabela ka ho eketsehileng le styrene ka ho phela ka radical polymerization. Lebaka la bobedi ke ho kenya sehlopha se nang le tshebedisano e itekanetseng le analyte mme se se na tshebedisano e matla ya electrostatic pakeng tsa analyte le mohato o sa sisinyeheng, kaha karolo ya phenylmaleimide ha e na tjhaja ya sebele ho pH e tlwaelehileng. Polarity ya mohato o sa sisinyeheng e ka laolwa ke bongata bo nepahetseng ba styrene le nako ya karabelo ya free radical polymerization. Mohato wa ho qetela wa karabelo (free-radical polymerization) o bohlokwa mme o ka fetola polarity ya mohato o sa sisinyeheng. Tlhahlobo ya motheo e entswe ho hlahloba carbon loading ya diphase tsena tse sa sisinyeheng. Ho ile ha bonwa hore ho eketsa bongata ba styrene le nako ya karabelo ho ekeditse carbon loading ya mohato o sa sisinyeheng le ka tsela e fapaneng. Di-SP tse lokisitsweng ka mahloriso a fapaneng a styrene di na le carbon loading e fapaneng. Hape, kenya diphase tsena tse sa sisinyeheng ka tshepe e sa hlwekang. dikholomo le ho hlahloba tshebetso ya tsona ya chromatographic (kgetho, qeto, boleng ba N, jj.). Ho itshetlehile ka diteko tsena, ho kgethilwe mokhoa o ntlafetseng ho lokisa mohato o sa fetoheng wa PMP ho netefatsa polarity e laolwang le ho boloka hantle ha analyte.
Metswako e mehlano ea li-peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) le eona e ile ea hlahlojoa ho sebelisoa kholomo ea PMP ho sebelisoa mohato o tsamaeang; 60/40 (v/v) acetonitrile/metsi (0.1% TFA) ka sekhahla sa phallo sa 80 μL/min. Tlas'a maemo a matle a ho hlakisa, nomoro ea poleiti ea khopolo-taba (N) ka kholomo (100 × 1.8 mm id) ke 20,000 ± 100 (lipoleiti tse 200,000/m²). Tafole ea 3 e fana ka boleng ba N bakeng sa likholomo tse tharo tsa PMP 'me li-chromatogram li bontšitsoe ho Setšoantšo sa 5A. Tlhahlobo e potlakileng kholomong ea PMP ka sekhahla se phahameng sa phallo (700 μL/min), li-peptide tse hlano li ile tsa hlakisa ka motsotso o le mong, boleng ba N bo ne bo le botle haholo, 13,500 ± 330 ka kholomo (100 × 1.8 mm id), E tsamaellana le lipoleiti tse 135,000/m2 (Setšoantšo sa 5B). Likholomo tse tharo tse boholo bo tšoanang (100 × 1.8 mm id) li ne li tletse likarolo tse tharo tse fapaneng tsa mohato oa PMP o sa sisinyeheng ho hlahloba ho ikatisa hape.Bongata ba analyte bakeng sa kholomo ka 'ngoe bo tlalehiloe ho sebelisoa maemo a ho hlakisa a nepahetseng le palo ea lipoleiti tsa khopolo-taba N le nako ea ho boloka ho arola motsoako o tšoanang oa teko kholomong ka 'ngoe. Lintlha tsa ho ikatisa hape bakeng sa likholomo tsa PMP li bontšitsoe ho Tafole ea 4. Ho ikatisa hape ha kholomo ea PMP ho amana hantle le boleng bo tlase haholo ba %RSD, joalo ka ha ho bontšitsoe ho Tafole ea 3.
Karohano ea motsoako oa peptide kholomong ea PMP (B) le kholomong ea Ascentis Express RP-Amide (A); karolo e tsamaeang ea 60/40 ACN/H2O (TFA 0.1%), litekanyo tsa kholomo ea PMP (100 × 1.8 mm id); tlhahlobo Tatellano ea ho hlakisa ea metsoako: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) le 5 (leucine) acid enkephalin)).
Kholomo ea PMP (100 × 1.8 mm id) e hlahlobiloe bakeng sa karohano ea tryptic digests ea albumin ea serum ea motho ho chromatography ea metsi e sebetsang hantle. Chromatogram ho Setšoantšo sa 6 e bontša hore sampole e arohane hantle 'me qeto e ntle haholo. Li-digests tsa HSA li hlahlobiloe ho sebelisoa sekhahla sa phallo sa 100 µL/min, karolo e tsamaeang ea 70/30 acetonitrile/metsi le 0.1% TFA. Joalokaha ho bontšitsoe ho chromatogram (Setšoantšo sa 6), tšilo ea HSA e arotsoe ka litlhōrō tse 17 tse tsamaellanang le li-peptide tse 17. Bokhoni ba karohano ea tlhōrō ka 'ngoe ho HSA digest bo baliloe 'me boleng bo fanoe ho Tafole ea 5.
Ho tjheseha ha HSA ka tryptic (100 × 1.8 mm id) ho ile ha arolwa hodima kholomo ya PMP; sekgahla sa phallo (100 µL/min), mokgahlelo wa ho tsamaya wa 60/40 acetonitrile/metsi ka 0.1% TFA.
moo L e leng bolelele ba kholomo, η ke viscosity ea mohato o tsamaeang, ΔP ke khatello ea mokokotlo oa kholomo, 'me u ke lebelo le otlolohileng la mohato o tsamaeang. Ho kenella ha kholomo ea PMP e ne e le 2.5 × 10-14 m2, sekhahla sa phallo e ne e le 25 μL/min, 'me ho sebelisitsoe 60/40 v/v ACN/metsi. Ho kenella ha kholomo ea PMP (100 × 1.8 mm id) ho ne ho tšoana le ha thuto ea rona e fetileng Ref.34. Ho kenella ha kholomo e tletseng likaroloana tse nang le masoba a manyenyane ke: 1.7 × 10-15 bakeng sa likaroloana tsa 1.3 μm, 3.1 × 10-15 bakeng sa likaroloana tsa 1.7 μm, 5.2 × 10-15 le 2.5 × 10-14 m2 bakeng sa likaroloana tsa 2.6 μm Bakeng sa likaroloana tsa 5 μm 43. Ka hona, ho kenella ha karolo ea PMP ho tšoana le ha likaroloana tsa 5 μm core-shell.
moo Wx e leng boima ba kholomo e tletseng chloroform, Wy ke boima ba kholomo e tletseng methanol, 'me ρ ke boima ba solvent. Bongata ba methanol (ρ = 0.7866) le chloroform (ρ = 1.484). Bongata ba porosity ea likholomo tsa SILICA PARTICLES-C18 (100 × 1.8 mm id) 34 le C18-Urea columns 31 tseo re ithutileng tsona pele e ne e le 0.63 le 0.55, ka ho latellana. Sena se bolela hore boteng ba li-ligand tsa urea bo fokotsa ho kenella ha karolo e sa sisinyeheng. Ka lehlakoreng le leng, porosity eohle ea kholomo ea PMP (100 × 1.8 mm id) ke 0.60. Ho kenella ha likholomo tsa PMP ho tlase ho feta ha likholomo tse tletseng likaroloana tsa silica tse kopantsoeng ke C18 hobane mekhahlelong e sa sisinyeheng ea mofuta oa C18 li-ligand tsa C18 li khomaretsoe likarolong tsa silica e le liketane tse otlolohileng, ha mekhahlelong e sa sisinyeheng ea mofuta oa polystyrene, A e teteaneng haholo. lera la polymer le thehoa ho potoloha lona. Tekong e tloaelehileng, masoba a kholomo a baloa ka tsela ena:
Setšoantšo sa 7A, B se bonts'a kholomo ea PMP (100 × 1.8 mm id) le kholomo ea Ascentis Express RP-Amide (100 × 1.8 mm id) ho sebelisoa maemo a tšoanang a elution (ke hore, 60/40 ACN/H2O le 0.1% TFA). ) ea van Deemter plot. Metswako e khethiloeng ea peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) e lokisitsoe ka 20 µL/ Sekhahla se fokolang sa phallo bakeng sa likholomo ka bobeli ke 800 µL/min. Boleng bo tlase ba HETP ka sekhahla se setle sa phallo (80 µL/min) bakeng sa kholomo ea PMP le kholomo ea Ascentis Express RP-Amide e ne e le 2.6 µm le 3.9 µm, ka ho latellana. Boleng ba HETP bo bontša hore bokhoni ba karohano ba kholomo ea PMP (100 × 1.8 mm id) bo betere haholo ho feta kholomo ea Ascentis Express RP-Amide e fumanehang khoebong (100 × 1.8 mm id). Setšoantšo sa van Deemter ho Setšoantšo sa 7(A) se bontša hore phokotso ea boleng ba N ka phallo e ntseng e eketseha ha e bohlokoa ha e bapisoa le thuto ea rona e fetileng. Bokhoni bo phahameng ba karohano ba kholomo ea PMP (100 × 1.8 mm id) ha e bapisoa le kholomo ea Ascentis Express RP-Amide e ipapisitse le ntlafatso ea sebopeho sa likaroloana, boholo, le mekhoa e rarahaneng ea ho paka kholomo e sebelisoang mosebetsing oa hona joale34.
(A) van Deemter plot (HETP khahlanong le lebelo le otlolohileng la mokgahlelo o tsamayang) e fumanoeng ho sebelisoa kholomo ea PMP (100 × 1.8 mm id) ho 60/40 ACN/H2O e nang le 0.1% TFA.(B) van Deemter plot (HETP khahlanong le lebelo le otlolohileng la mokgahlelo o tsamayang) e fumanoeng ho sebelisoa kholomo ea Ascentis Express RP-Amide (100 × 1.8 mm id) ho 60/40 ACN/H2O e nang le 0.1% TFA.
Mokhahlelo o sa fetoheng oa polystyrene o kentsoeng polar o ile oa lokisoa 'me oa hlahlojoa bakeng sa karohano ea metsoako ea peptide ea maiketsetso le ho siloa ha trypsin ea albumin ea serum ea motho (HAS) ho chromatography ea metsi e sebetsang hantle. Ts'ebetso ea chromatographic ea likholomo tsa PMP bakeng sa metsoako ea peptide e ntle haholo ka katleho ea karohano le tharollo. Ts'ebetso e ntlafetseng ea karohano ea likholomo tsa PMP e bakoa ke mabaka a fapaneng, joalo ka boholo ba likaroloana le boholo ba li-pore tsa likaroloana tsa silika, tlhahiso e laoloang ea mohato o sa fetoheng, le ho paka ho rarahaneng ha likholomo. Ntle le katleho e phahameng ea karohano, khatello e tlase ea mokokotlo oa likholomo ka sekhahla se phahameng sa phallo ke molemo o mong oa mohato ona o sa fetoheng. Likholomo tsa PMP li bontša ho ikatisa ho hotle 'me li ka sebelisoa bakeng sa tlhahlobo ea metsoako ea peptide le tšilo ea trypsin ea liprotheine tse fapaneng. Re ikemiselitse ho sebelisa kholomo ena bakeng sa karohano ea lihlahisoa tsa tlhaho, metsoako e sebetsang ea bio ho tsoa limela tsa meriana le li-fungus tse ntšitsoeng ho chromatography ea metsi. Nakong e tlang, likholomo tsa PMP li tla boela li hlahlojoe bakeng sa karohano ea liprotheine le li-antibodies tsa monoclonal.
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