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E bonts'a carousel ea li-slide tse tharo ka nako e le 'ngoe. Sebelisa likonopo tsa E fetileng le tse Latelang ho tsamaea ka har'a li-slide tse tharo ka nako, kapa sebelisa likonopo tsa li-slider qetellong ho tsamaea ka har'a li-slide tse tharo ka nako.
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 N-phenylmaleimide-methylvinyl isocyanate (PMI) le styrene ka polymerization ea reverse chain transfer-fragmentation (RAFT) ho hlahisa li-polyamides tse kopaneng tsa N-phenylmaleimide. Styrene (PMP) e sa sisinyeheng. Litšiea tse tšesaane tsa tšepe e sa hloekang (bophara ba ka hare ba 100 × 1.8 mm) li ne li tletse ka ho paka ha lerōle. Ts'ebetso ea chromatographic ea kholomo ea PMP e ile ea hlahlojoa ho arola motsoako oa li-peptide tsa maiketsetso tse nang le li-peptide tse hlano (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) le tryptic hydrolyzate ea human serum albumin (HAS). Tlas'a maemo a matle a ho hlakisa, palo ea lipoleiti tse nang le motsoako oa li-peptide e fihlile ho lipoleiti tse 280,000/sq.m. Ha ho bapisoa ts'ebetso ea karohano ea kholomo e ntlafalitsoeng le kholomo ea khoebo ea Ascentis Express RP-Amide, ho ile ha hlokomeloa hore katleho ea karohano ea kholomo ea PMP e ne e phahametse kholomo ea khoebo mabapi le katleho ea karohano le qeto.
Indasteri ea meriana ea bio e fetohile 'maraka o ntseng o hola oa lefats'e ka keketseho e kholo ea karolo ea 'maraka lilemong tsa morao tjena. Ka kholo e matla ea indasteri ea meriana ea bio1,2,3 ho na le tlhoko e kholo ea tlhahlobo ea peptide le protheine. Ntle le peptide e reriloeng, litšila tse fapaneng lia thehoa nakong ea tlhahiso ea peptide, kahoo tlhoekiso ea chromatographic ea hlokahala ho fumana bohloeki bo lakatsehang ba peptide. Tlhahlobo le tlhaloso ea liprotheine marong a 'mele, liseleng le liseleng ke mosebetsi o boima haholo ka lebaka la palo e kholo ea mefuta e ka bonoang e teng sampole e le 'ngoe. Le hoja spectrometry ea boima e le sesebelisoa se sebetsang sa ho latellana ha li-peptide le liprotheine, haeba lisampole tse joalo li kenngoa ka ho toba ho spectrometer ea boima, karohano e ke ke ea khotsofatsa. Bothata bona bo ka rarolloa ka ho etsa 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, li-analyte li ka tsepamisa mohopolo sebakeng se moqotetsane nakong ea karohano ea karolo ea metsi, ka hona li tsepamisa maikutlo ho li-analyte tsena le ho eketsa kutloisiso ea ho lemoha MS. Ho sebelisa chromatography ea metsi (LC) ho tsoetse pele haholo lilemong tse leshome tse fetileng 'me e se e le mokhoa o sebelisoang haholo bakeng sa tlhahlobo ea proteomic7,8,9,10.
Kromatography ea metsi ea mokhahlelo o ka morao (RP-LC) e sebelisoa haholo ho hloekisa le ho arola metsoako ea li-peptide ho sebelisoa octadecyl-modified silica (ODS) e le mokhahlelo o sa fetoheng11,12,13. Leha ho le joalo, ka lebaka la sebopeho sa tsona se rarahaneng le tlhaho ea amphoteric, mekhahlelo e sa fetoheng ea RP e 14,15 e ke ke ea fana ka karohano e khotsofatsang ea li-peptide le liprotheine. Ka hona, tlhahlobo ea li-peptide le liprotheine tse nang le likaroloana tsa polar le tse seng polar e hloka mekhahlelo e sa fetoheng e entsoeng ka ho khetheha ho sebelisana le ho boloka li-analyte tsena16. Kromatography e tsoakiloeng, e fanang ka likamano tse ngata, e ka ba mokhoa o mong oa ho arola li-peptide, liprotheine le metsoako e meng e rarahaneng. Mekhahlelo e 'maloa e sa fetoheng ea mofuta o tsoakiloeng e ile ea lokisoa 'me likholomo tse tletseng mekhahlelo ena e sa fetoheng li ile tsa sebelisoa ho arola li-peptide le liprotheine17,18,19,20,21. Ka lebaka la boteng ba lihlopha tsa polar le tse seng polar, mekhahlelo e tsitsitseng ea mokhoa o tsoakiloeng (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) e loketse karohano ea li-peptide le liprotheine22,23,24,25,26,27,28. , mekhahlelo e tsitsitseng ea polar e nang le lihlopha tsa polar tse kopantsoeng ka covalently e bontša bokhoni bo botle ba karohano le khetho e ikhethang bakeng sa li-analyte tsa polar le tse seng polar hobane karohano e itšetlehile ka tšebelisano pakeng tsa analyte le mohato o tsitsitseng Likamano tse ngata tsa 29,30,31,32. Haufinyane tjena, Zhang et al. 30 ba fumane mekhahlelo e tsitsitseng ea polyamines e felisitsoeng ke behenyl 'me ba arotse ka katleho li-hydrocarbon, li-antidepressants, li-flavonoid, li-nucleoside, li-estrogen, le li-analyte tse ling. Thepa e tsitsitseng e kentsoeng polar e na le lihlopha tsa polar le tse seng polar, kahoo e ka sebelisoa ho arola li-peptide le liprotheine ka likarolo tsa hydrophobic le hydrophilic. Likholomo tse ka hare ho polar (mohlala, likholomo tsa C18 tse nang le amide inline) li fumaneha tlas'a lebitso la khoebo la likholomo tsa Ascentis Express RP-Amide, empa likholomo tsena li sebelisitsoe feela bakeng sa tlhahlobo ea amine 33.
Phuputsong ea hona joale, ho lokisitsoe le ho hlahlojoa karolo e sa fetoheng ea polar embedding (N-phenylmaleimide, embedding polystyrene) bakeng sa karohano ea peptide le karohano ea tryptic HSA. Leano le latelang le sebelisitsoe ho lokisa karolo e sa fetoheng. Likaroloana tsa silica tse nang le masoba li lokisitsoe ho latela mekhoa e hlalositsoeng likhatisong tsa rona tse fetileng, ka liphetoho tse ling mererong ea ho lokisa 31, 34, 35, 36, 37, 38, 39. Litekanyetso tsa urea, polyethylene glycol (PEG), TMOS le aqueous-acetic acid li ile tsa fetoloa ho fumana likaroloana tsa silica tse nang le boholo bo boholo ba masoba. Ea bobeli, phenylmaleimide-methylvinyl isocyanate ligand e ncha e ile ea etsoa 'me likaroloana tsa eona tsa silica tse nkiloeng li ile tsa sebelisoa ho lokisa mekhahlelo e sa fetoheng ea polar embedded. Karolo e sa fetoheng e fumanoeng e ile ea pakoa kholomong ea tšepe e sa hloekang (bophara ba ka hare ba 100 × 1.8 mm) ho latela moralo o ntlafalitsoeng oa ho paka. Ho paka kholomong ho thusoa ke ho thothomela ha mechini ho netefatsa lera le lekanang ka har'a kholomo. Kholomo e pakiloeng e ile ea hlahlojoa bakeng sa karohano ea motsoako oa li-peptide o nang le li-peptide tse hlano (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). le tryptic hydrolysates ea human serum albumin (HSA). Ho ile ha hlokomeloa hore motsoako oa peptide le HSA tryptic digest li arohane ka qeto le bokgabane bo botle. Bokgoni ba karohano ba kholomo ea PMP bo bapisoa le ba kholomo ea Ascentis Express RP-Amide. Ho ile ha hlokomeloa hore li-peptide le liprotheine li na le qeto e ntle le bokgabane bo phahameng ba karohano kholomong ea PMP, 'me bokhoni ba karohano ba kholomo ea PMP bo phahame ho feta ba kholomo ea Ascentis Express RP-Amide.
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) bakeng sa HPLC, methanol, 2-propanol le acetone. Sigma-Aldrich Company (St. Louis, Missouri, USA).
Motsoako oa urea (8 g), polyethylene glycol (8 g) le 8 ml ea 0.01 N. acetic acid o ile oa hlohlelletsoa metsotso e 10, 'me 24 ml ea TMOS ea eketsoa ho eona tlas'a ho pholisa leqhoa. Motsoako oa karabelo o ile oa futhumatsoa ho 40°C ka lihora tse 6 ebe o chesoa ho 120°C ka lihora tse 8 ka har'a autoclave ea tšepe e sa hloekang. Metsi a ile a tlosoa 'me masala a omisoa ho 70°C ka lihora tse 12. Li-block tse bonolo tse omisitsoeng li ile tsa siloa hantle 'me tsa kenngoa ka ontong ho 550°C ka lihora tse 12. Ho ile ha lokisoa lihlopha tse tharo 'me tsa hlalosoa ho leka ho ikatisa hape ha boholo ba likaroloana, boholo ba li-pore le sebaka sa bokaholimo.
Sehlopha sa polar le karolo e sa sisinyeheng bakeng sa liketane tsa polystyrene. Mokhoa oa ho lokisa o hlalositsoe ka tlase.
N-phenylmaleimide (200 mg) le methyl vinyl isocyanate (100 mg) li ile tsa qhibilihisoa ka har'a toluene e sa hloekang, 'me 0.1 ml ea 2,2′-azoisobutyronitrile (AIBN) e ile ea eketsoa ka har'a flask ea karabelo ho fumana copolymer ea phenylmaleimide le methyl vinyl isocyanate (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 metsotso e 10 ka har'a flask e chitja e ka tlase ea 500 ml. PMCP (10 mg) e ile ea qhaloa ka har'a toluene 'me ea eketsoa ka ho theohela ka har'a flask ea karabelo ka funnel e eketsehileng. Motsoako o ile oa khutlisetsoa mochesong oa 100°C ka lihora tse 8, oa sefshoa, oa hlatsuoa ka acetone 'me oa omisoa ka har'a 60°C ka lihora tse 3. Ebe, likaroloana tsa silica tse amanang le PMCP (100 g) li ile tsa qhaloa ka har'a toluene (200 ml), 'me 4-hydroxy-TEMPO (2 ml) ea eketsoa ho eona moo ho nang le 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 hoketsweng ho TEMPO-PMCP (1.5 g) di ile tsa hasana ka toluene mme tsa hlwekiswa ka naetrojene. Polymerization ya styrene e ile ya etswa ka 100°C ka dihora tse 12. Sehlahiswa se hlatswitsweng se ile sa hlatsuwa ka methanol mme sa omiswa bosiu bohle ho 60°C. Moralo o akaretsang wa karabelo o bontshitswe ho setshwantsho sa pele.
Disampole di ile tsa ntshuwa ka mosi ka 393 K ka hora e le nngwe ho fihlela kgatello e setseng ya ka tlase ho 10–3 Torr e fumanwa. Palo ya N2 e amohuweng ka kgatello e lekanyeditsweng P/P0 = 0.99 e sebedisitswe ho fumana bophahamo ba pore kaofela. Sebopeho sa dikarolwana tsa silica tse hlwekileng le tse tlamilweng ke ligand se ile sa hlahlojwa ho sebediswa microscope ya elektrone e skena (Hitachi High Technologies, Tokyo, Japane). Disampole tse omileng (dikarolwana tsa silica tse hlwekileng le tse tlamilweng ke ligand) di ile tsa bewa hodima dithupa tsa aluminium ho sebediswa theipi ya khabone. Kgauta e ile ya bewa hodima sampole ho sebediswa sesebediswa sa ho phunya sa Q150T, mme lera la Au le teteaneng la 5 nm le ile la bewa hodima sampole. Sena se ntlafatsa bokgoni ba tshebetso ya motlakase o tlase mme se fana ka ho fafatsa ka serame se batang. Tlhahlobo ya elemental e entswe ho sebediswa Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental composition analyzer. Analyzer ya boholo ba dikarolwana tsa Malvern (Worcestershire, UK) Mastersizer 2000 e ile ya sebediswa ho fumana kabo ya boholo ba dikarolwana. Likaroloana tsa silica tse sa koaheloang le likaroloana tsa silica tse tlamiloeng ka ligand (5 mg ka 'ngoe) li ile tsa hasana ka har'a 5 ml ea isopropanol, tsa kenngoa ka sonic metsotso e 10, tsa sisinyeha metsotso e 5, 'me tsa beoa bencheng ea optical ea Mastersizer. Tlhahlobo ea Thermogravimetric e etsoa ka sekhahla sa 5 °C ka motsotso mochesong o pakeng tsa 30 le 800 °C.
Litšiea tsa tšepe e sa hloekang tse nang le lesoba le lesesaane tse nang le fiber ea khalase tse nang le litekanyo (ID 100 × 1.8 mm) li ne li pakiloe ka mokhoa oa ho tlatsa slurry ho latela mokhoa o tšoanang le o boletsoeng ho referense ea 31. Kholomo ea tšepe e sa hloekang (e nang le khalase, ID 100 × 1 .8 mm) le sehokelo se nang le frit ea 1 µm li ile tsa hokahanngoa le mochini oa ho paka slurry (Alltech Deerfield, IL, USA). Lokisetsa ho emisoa ha karolo e sa sisinyeheng ka ho emisa 150 mg ea karolo e sa sisinyeheng ka 1.2 ml ea methanol le ho e fepa kholomong ea polokelo. Methanol e sebelisitsoe e le tharollo ea slurry le tharollo ea taolo. Paka kholomo ka ho sebelisa tatellano ea khatello ea 100 MP metsotso e 10, 80 MP metsotso e 15, le 60 MP metsotso e 30. Ts'ebetso ea ho paka e sebelisitse li-vibrator tse peli tsa kholomo ea chromatography ea khase (Alltech, Deerfield, IL, USA) bakeng sa ho thothomela ha mechini ho netefatsa ho paka kholomo ka mokhoa o ts'oanang. Koala sephutheloana sa slurry 'me u lokolle khatello butle ho thibela tšenyo khoeleng. Kholomo e ile ea khaoloa ho tloha nozzle ea lerōle 'me sehokelo se seng sa hokeloa ho pompo 'me sa hokeloa ho sistimi ea LC ho leka ts'ebetso ea eona.
Ho hahiloe MLC e ikhethileng ho sebelisoa pompo ea LC (10AD Shimadzu, Japane), sampole e nang le loop ea ente ea 50 nL (Valco (USA) C14 W.05), degasser ea membrane (Shimadzu DGU-14A), le fensetere ea capillary ea UV-VIS. Sesebelisoa sa ho lemoha (UV-2075) le microcolumn e nang le enamel. Sebelisa lipeipi tse moqotetsane haholo le tse khutšoane tsa ho hokahanya ho fokotsa phello ea katoloso e eketsehileng ea kholomo. Kamora ho tlatsa kholomo, kenya capillary (50 µm id 365) sebakeng sa ho tsoa sa 1/16″ reduction junction 'me u kenye capillary (50 µm) ea reduction junction. Pokello ea data le ts'ebetso ea chromatogram li etsoa ho sebelisoa software ea Multichro 2000. Ho 254 nm, monyako oa UV oa bahlahlobi ba lihlooho o ile oa hlahlojoa ho 0. Lintlha tsa Chromatographic li ile tsa hlahlojoa ho sebelisoa OriginPro8 (Northampton, MA).
Albumin ea 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 motsoako oa li-peptide le tryptic digest HSA kholomong ea PMP e hlahlobiloe ka thoko. Hlahloba tryptic hydrolysis ea motsoako oa li-peptide le HSA o arotsoeng ke kholomo ea PMP 'me u bapise liphetho le kholomo ea Ascentis Express RP-Amide. Palo ea lipoleiti tsa khopolo-taba e baloa ho sebelisoa equation e latelang:
Litšoantšo tsa SEM tsa likaroloana tsa silica tse hloekileng le likaroloana tsa silica tse tlamiloeng ka ligand li bontšoa ho Setšoantšo sa 2. Litšoantšo tsa SEM tsa likaroloana tsa silica tse hloekileng (A, B) li bontša sebopeho se chitja seo likaroloana li leng telele kapa li nang le ho se leka-lekane ho sa tloaelehang ha li bapisoa le lithuto tsa rona tsa pele. Bokaholimo ba likaroloana tsa silica tse tlamiloeng ke ligand (C, D) bo boreleli ho feta ba likaroloana tsa silica tse hloekileng, e leng se ka bakoang ke liketane tsa polystyrene tse koahelang bokaholimo ba likaroloana tsa silica.
Ho skena di-micrograph tsa dielektrone tsa dikarolwana tse hlwekileng tsa silica (A, B) le dikarolwana tsa silica tse tlamilweng ke ligand (C, D).
Kabo ea boholo ba likaroloana tsa silika e hloekileng le likaroloana tsa silika tse tlamiloeng ka ligand e bontšoa ho Setšoantšo sa 2. 3(A). Li-curve tsa kabo ea boholo ba likaroloana tsa volumetric li bontšitse hore boholo ba likaroloana tsa silika bo eketsehile ka mor'a phetoho ea lik'hemik'hale (Setšoantšo sa 3A). Lintlha tsa kabo ea boholo ba likaroloana tsa silika ho tsoa thutong ea hona joale le thutong e fetileng li bapisoa ho Tafole ea 1(A). Boholo ba likaroloana tsa volumetric d(0.5) ea PMP e ne e le 3.36 µm, ha bo bapisoa le boleng ba ad(0.5) ba 3.05 µm thutong ea rona e fetileng (likaroloana tsa silika tse tlamiloeng ka polystyrene)34. Ka lebaka la phetoho ea karolelano ea PEG, urea, TMOS le acetic acid motswakong oa karabelo, kabo ea boholo ba likaroloana tsa sehlopha sena e ne e le nyane ha e bapisoa le thuto ea rona e fetileng. Boholo ba likaroloana ba karolo ea PMP bo boholo hanyane ho feta ba karoloana ea silika e tlamiloeng ka polystyrene eo re ithutileng eona pejana. Sena se bolela hore ts'ebetso ea bokaholimo ba likaroloana tsa silika tse nang le styrene e behile feela lera la polystyrene (0.97 µm) holim'a bokaholimo ba silika, ha 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 tse hloekileng tsa silica le likaroloana tsa silica tse tlamiloeng ka ligand.
Boholo ba pore, bophahamo ba pore, le sebaka sa bokaholimo ba likaroloana tsa silica tse sebelisitsoeng thutong ena li bontšitsoe ho Tafole ea 1 (B). Liprofaele tsa PSD tsa likaroloana tsa silica tse hloekileng le likaroloana tsa silica tse tlamiloeng ka ligand li bontšitsoe ho Figs. 3(B). Liphetho li ne li tšoana le thuto ea rona ea pejana34. Boholo ba pore ba likaroloana tsa silica tse hloekileng le tse tlamiloeng ka ligand e ne e le 310 Å le 241 Å, ka ho latellana, ho bontšang hore kamora phetoho ea lik'hemik'hale, boholo ba pore bo fokotsehile ka 69 Å, joalo ka ha ho bontšitsoe ho Tafole ea 1 (B), 'me shift curve e bontšoa ho Setšoantšo. Sebaka se ikhethileng sa bokaholimo ba likaroloana tsa silica thutong ea hona joale ke 116 m2/g, e leng 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 kamora phetoho ea lik'hemik'hale le sona se fokotsehile ho tloha ho 116 m2/g ho ea ho 105 m2/g.
Liphetho tsa tlhahlobo ea elemental ea mohato o sa sisinyeheng li hlahisoa ho Tafole. 2. Sekhahla sa khabone sa mohato o sa sisinyeheng oa hona joale ke 6.35%, e leng se tlase ho feta thutong ea rona e fetileng (likaroloana tsa silica tse amanang le polystyrene, 7.93%35 le 10.21%, ka ho latellana) 42. Sekhahla sa khabone sa mohato o sa sisinyeheng oa hona joale ka tlase, kaha li-ligand tse ling tsa polar tse kang phenylmaleimide methyl vinyl isocyanate (PCMP) le 4-hydroxy-TEMPO li sebelisitsoe ho phaella ho styrene ho lokiseng SP. Peresente ea boima ba naetrojene mohatong o sa sisinyeheng oa hona joale ke 2.21% ha e bapisoa le 0.1735 le 0.85% lithutong tse fetileng42. Sena se bolela hore mohato o sa sisinyeheng oa hona joale o na le peresente e phahameng ea boima ba naetrojene ka lebaka la phenylmaleimide. Ka ho tšoanang, lihlahisoa (4) le (5) li na le khabone ea 2.7% le 2.9%, ka ho latellana, ha sehlahisoa sa ho qetela (6) se na le khabone ea 6.35%, joalo ka ha ho bontšitsoe ho Tafole ea 2. Tlhahlobo ea Thermogravimetric (TGA) e sebelisitsoe mohatong o sa sisinyeheng oa PMP ho leka tahlehelo ea boima ba 'mele,' me moqolo oa TGA o bontšitsoe ho Setšoantšo sa 4. Moqolo oa TGA o bontša tahlehelo ea boima ba 'mele ea 8.6%, e leng se lumellanang hantle le se nang le khabone (6.35%), kaha ligand ha li na C feela, empa hape le N, O le H.
Isocyanate ea ligand phenylmaleimide-methylvinyl e khethiloe ho fetola bokaholimo ba likaroloana tsa silica ka lebaka la lihlopha tsa eona tsa polar phenylmaleimide le vinylisocyanate. Lihlopha tsa vinyl isocyanate li ka arabela haholoanyane le styrene ka ho phela ka radical polymerization. Lebaka la bobeli ke ho kenya sehlopha se nang le likamano tse itekanetseng le analyte le se nang likamano tse matla tsa electrostatic pakeng tsa analyte le mohato o sa sisinyeheng, kaha karolo ea phenylmaleimide ha e na tefiso ea sebele ho pH e tloaelehileng. Polarity ea mohato o sa sisinyeheng e ka laoloa ke bongata bo nepahetseng ba styrene le nako ea karabelo ea free radical polymerization. Mohato oa ho qetela oa karabelo (free radical polymerization) o bohlokoa kaha o fetola polarity ea mohato o sa sisinyeheng. Tlhahlobo ea elemental e entsoe ho hlahloba litaba tsa carbon mekhahlelong ena e sa sisinyeheng. Ho hlokometsoe hore ho eketsa bongata ba styrene le nako ea karabelo ho eketsa litaba tsa carbon tsa mohato o sa sisinyeheng le ka tsela e fapaneng. Li-SP tse lokisitsoeng ka mahloriso a fapaneng a styrene li na le meroalo e fapaneng ea carbon. Ka ho tšoanang, mekhahlelo ena e sa sisinyeheng e ile ea beoa holim'a litšiea tsa tšepe e sa hloekang 'me litšobotsi tsa tsona tsa chromatographic (khetho, qeto, boleng ba N, jj.) li ile tsa hlahlojoa. Ho latela liteko tsena, motsoako o ntlafalitsoeng bakeng sa ho lokisa mohato o sa sisinyeheng oa PMP o ile oa khethoa ho fana ka polarity e laoloang le ho boloka hantle ha analyte.
Kholomo ea PMP e boetse e hlahlobiloe bakeng sa tlhahlobo ea metsoako e mehlano ea li-peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) ho sebelisoa bokhoni ba mohato o tsamaeang. 60/40 (v/v) ACN/metsi (0.1% TFA) ka sekhahla sa phallo sa 80 µl/min. Tlas'a maemo a matle a ho hlakisa (lipoleiti tse 200,000/m), palo ea lipoleiti tsa theoretical (N) ka kholomo (100 × 1.8 mm) ke 20,000 ± 100. Boleng ba N bakeng sa likholomo tse tharo tsa PMP bo bontšitsoe ho Tafole ea 3 'me li-chromatogram li bontšitsoe ho Setšoantšo sa 5A. Tlhahlobo e potlakileng ka sekhahla se phahameng sa phallo (700 µl/min) kholomong ea PMP, li-peptide tse hlano li ile tsa tsoa ka motsotso o le mong, boleng bo botle ba N ba 13,500 ± 330 kholomong (bophara ba 100 x 1.8 mm), bo lekanang le lipoleiti tse 135,000/m (Setšoantšo sa 5B). Likholomo tse tharo tsa boholo bo tšoanang (bophara ba ka hare ba 100 x 1.8 mm) li ile tsa tlatsoa ka lihlopha tse tharo tse fapaneng tsa mohato o sa fetoheng oa PMP ho leka ho ikatisa hape. Li-analyte li ile tsa tlalehoa bakeng sa kholomo ka 'ngoe ka ho arola motsoako o tšoanang oa teko kholomong ka 'ngoe ho sebelisoa maemo a ho ikatisa a nepahetseng, palo ea lipoleiti tsa khopolo-taba N, le nako ea ho boloka. 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 metsoako ea 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 x 1.8 mm id), tlhahlobo Tatellano ea metsoako ea Elution: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) le 5 (leucic acid enkephalin).
Kholomo ea PMP (bophara ba ka hare ba 100 x 1.8 mm) e ile ea hlahlojoa bakeng sa karohano ea tryptic hydrolyzate ea albumin ea serum ea motho ka HPLC. Chromatogram ho Setšoantšo sa 6 e bontša hore lisampole li arotsoe hantle ka qeto e ntle haholo. Litharollo tsa HSA li ile tsa hlahlojoa ho sebelisoa sekhahla sa phallo sa 100 μl/min, mohato o tsamaeang oa 70/30 acetonitrile/metsi le 0.1% TFA. Ho arohana ha HSA ho ile ha aroloa ka litlhōrō tse 17, joalo ka ha ho bontšitsoe ho chromatogram (Setšoantšo sa 6), se tsamaellanang le li-peptide tse 17. Bokhoni ba karohano ea litlhōrō ka bomong ho tloha ho hydrolyzate ea HSA bo baliloe 'me boleng bo bontšitsoe ho Tafole ea 5.
Li-hydrolysate tsa HSA tryptic li arotsoe kholomong ea PMP (bophara ba ka hare ba 100 x 1.8 mm), sekhahla sa phallo (100 μl/min), mohato o tsamaeang oa 60/40 acetonitrile/metsi, le 0.1% TFA.
moo L e leng bolelele ba kholomo, η ke viscosity ea mohato o tsamaeang, ΔP ke khatello ea morao ea 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, ho sebelisitsoe 60/40 v/v. ACN/metsi. Ho kenella ha kholomo ea PMP (ID 100 × 1.8 mm) ho ne ho tšoana le ha phuputso ea rona ea pele ea Ref.34. Ho kenella ha kholomo e tletseng likaroloana tse nang le masoba a manyenyane ke 1.7×10 .6 µm, 2.5×10-14 m2 bakeng sa likaroloana tsa 5 µm43. Ka hona, ho kenella ha karolo ea PMP ho tšoana le ho kenella ha likaroloana tsa core-shell tse boholo ba 5 μm.
moo Wx e leng boima ba kholomo e tletseng chloroform, Wy ke boima ba kholomo e tletseng methanol, 'me ρ ke boima ba solvent. Botenya ba methanol (ρ = 0.7866) le chloroform (ρ = 1.484). Bongata ba porosity ea kholomo ea likaroloana tsa silica-C18 (100 × 1.8 mm ID)34 le kholomo ea rona ea C18-urea31 e ithutiloe 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 (bophara ba ka hare ba 100 × 1.8 mm) ke 0.60. Likholomo tsa PMP ha li kenelle habonolo ho feta likholomo tse tletseng likaroloana tsa silica tse tlamiloeng ka C18 hobane mekhahlelong e sa sisinyeheng ea mofuta oa C18 li-ligand tsa C18 li khomaretsoe likarolong tsa silica ka liketane tse otlolohileng, ha mekhahlelong e sa sisinyeheng ea mofuta oa polystyrene ho thehoa polymer e teteaneng e potolohileng likaroloana. lera A. Tekong e tloaelehileng, masoba a kholomo a baloa ka tsela e latelang:
Setšoantšong sa 7A, B e bonts'a lipolanete tsa Van Deemter bakeng sa kholomo ea PMP (id 100 x 1.8 mm) le kholomo ea Ascentis Express RP-Amide (id 100 x 1.8 mm) tlas'a maemo a tšoanang a ho hlakisa, 60/40 ACN/H2O le 0 .1% TFA 20 µl/min ho isa ho 800 µl/min likholomong ka bobeli. Boleng bo tlase ba HETP ka sekhahla se setle sa phallo (80 µl/min) e ne e le 2.6 µm le 3.9 µm bakeng sa kholomo ea PMP le kholomo ea Ascentis Express RP-Amide, ka ho latellana. Boleng ba HETP bo bontša hore katleho ea karohano ea kholomo ea PMP (id 100 x 1.8 mm) e phahame haholo ho feta ea kholomo ea Ascentis Express RP-Amide e fumanehang khoebong (id 100 x 1.8 mm). Grafo ea van Deemter ho Setšoantšo sa 7(A) e bontša hore phokotso ea boleng ba N ha e phahame haholo ka phallo e ntseng e eketseha ha e bapisoa le thuto ea rona e fetileng. Bokhoni bo phahameng ba karohano ea kholomo ea PMP (id 100 × 1.8 mm) ha e bapisoa le kholomo ea Ascentis Express RP-Amide bo ipapisitse le sebopeho le boholo bo ntlafetseng ba likaroloana le mokhoa o rarahaneng oa ho paka kholomo o sebelisoang mosebetsing oa hona joale34.
(A) Plot ea Van Deemter (lebelo la HETP khahlanong le lebelo la mothapo o tsamayang) e fumanoeng kholomong ea PMP (id 100 x 1.8 mm) ho 60/40 ACN/H2O ka 0.1% TFA. (B) Plot ea Van Deemter (lebelo la HETP khahlanong le lebelo la mothapo o tsamayang) e fumanoeng kholomong ea Ascentis Express RP-Amide (id 100 x 1.8 mm) ho 60/40 ACN/H2O ka 0.1% TFA.
Karolo e sa sisinyeheng ea polar ea polystyrene e kopantsoeng e ile ea lokisoa 'me ea hlahlojoa bakeng sa karohano ea motsoako oa li-peptide tsa maiketsetso le tryptic hydrolyzate ea human serum albumin (HSA) ho chromatography ea metsi e sebetsang hantle. Ts'ebetso ea chromatographic ea likholomo tsa PMP bakeng sa metsoako ea peptide e ntle haholo mabapi le katleho ea karohano le tharollo. Bokhoni bo ntlafetseng ba karohano ea likholomo tsa PMP bo bakoa ke mabaka a 'maloa a kang boholo ba likaroloana tsa silika le boholo ba li-pore, tlhahiso e laoloang ea mekhahlelo e sa sisinyeheng, le thepa e rarahaneng ea ho paka likholomo. Ntle le katleho e phahameng ea karohano, molemo o mong oa mohato ona o sa sisinyeheng ke khatello e tlase ea mokokotlo oa kholomo ka sekhahla se phahameng sa phallo. Likholomo tsa PMP li ka ikatisa hape haholo 'me li ka sebelisoa ho sekaseka metsoako ea li-peptide le tryptic ea tšilo ea lijo ea liprotheine tse fapaneng. Re ikemiselitse ho sebelisa kholomo ena bakeng sa karohano ea metsoako e sebetsang ho tsoa lihlahisoa tsa tlhaho, li-extracts tsa limela tsa meriana le li-mushroom 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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