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Qaybaha silica ee daloolka leh ayaa lagu diyaariyey habka sol-gel iyadoo wax laga beddelayo si loo helo qaybo dalool ballaadhan. Qaybahaan waxaa lagu soo saaray N-phenylmaleimide-methylvinyl isocyanate (PMI) iyo styrene iyada oo la adeegsanayo polymerization-ka wareejinta silsiladda roga ah (RAFT) si loo soo saaro N-phenylmaleimide polyamide-ka is-dhex-galka ah. Styrene (PMP) marxalad taagan. Tiirar bir ah oo cidhiidhi ah oo cidhiidhi ah (100 × 1.8 mm dhexroorka gudaha ah) ayaa lagu soo xidhay baakad dhoobo ah. Waxqabadka chromatographic ee tiirka PMP ayaa la qiimeeyay si loo kala saaro isku dhafka peptides synthetic oo ka kooban shan peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) iyo tryptic hydrolyzate ee serum aadanaha. Marka la eego xaaladaha ugu fiicnaanta, tirada aragtida ee taarikada leh isku dhafka peptides waxay gaareen 280,000 taariko/sq.m. Isbarbardhigga waxqabadka kala-soocidda tiirka horumaray iyo tiirka ganacsiga ee Ascentis Express RP-Amide, waxaa la arkay in hufnaanta kala-soocidda tiirka PMP uu ka sarreeyay tiirka ganacsiga marka loo eego hufnaanta iyo xallinta kala-soocidda.
Warshadaha biopharmaceutical waxay noqdeen suuq caalami ah oo sii fidaya iyadoo koror weyn ku yimid saamiga suuqa sanadihii ugu dambeeyay. Iyada oo koritaanka qarxa ee warshadaha biopharmaceutical1,2,3 waxaa jira baahi weyn oo loo qabo falanqaynta peptide iyo borotiinka. Marka lagu daro peptide bartilmaameedka ah, wasakh kala duwan ayaa la sameeyaa inta lagu jiro isku-dhafka peptide, sidaas darteed nadiifinta chromatographic ayaa loo baahan yahay si loo helo nadiifinta la rabo ee peptide. Falanqaynta iyo sifaynta borotiinnada ku jira dareeraha jidhka, unugyada, iyo unugyada waa hawl aad u adag sababtoo ah tirada badan ee noocyada suurtagalka ah ee la ogaan karo ee ku jira hal muunad. Inkasta oo spectrometry mass ay tahay qalab wax ku ool ah oo loogu talagalay isku xigxiga peptides iyo borotiinada, haddii shaybaarradan oo kale si toos ah loogu soo bandhigo spectrometer-ka mass, kala-soociddu waxay noqon doontaa mid aan fiicnayn. Dhibaatadan waxaa lagu xalin karaa iyadoo la sameeyo chromatography dareere ah (LC) ka hor falanqaynta MS, taas oo yareyn doonta qadarka falanqeeyayaasha gelaya spectrometer mass spectrometer waqti la siiyay4,5,6. Intaa waxaa dheer, falanqeeyayaashu waxay ku soo ururin karaan gobol cidhiidhi ah inta lagu jiro kala-soocidda wajiga dareeraha, taas oo xoogga saaraysa falanqaynta iyo kordhinta dareenka ogaanshaha MS. chromatography dareere ah (LC) ayaa si aad ah u horumaray tobankii sano ee la soo dhaafay wuxuuna noqday hab si weyn loo isticmaalo falanqaynta borotiinka7,8,9,10.
chromatography dareeraha wejiga-celinta (RP-LC) ayaa si ballaaran loo isticmaalaa si loo nadiifiyo oo loo kala saaro isku dhafka peptides iyadoo la adeegsanayo octadecyl-modified silica (ODS) sida marxaladda taagan11,12,13. Si kastaba ha ahaatee, iyada oo ay ugu wacan tahay qaab-dhismeedkooda adag iyo dabeecadda amphoteric, 14,15 RP wejiyada taagan ma bixin karaan kala soocitaan lagu qanco ee peptides iyo borotiinada. Sidaa darteed, falanqaynta peptides iyo borotiinada leh jajabyada polar iyo non-polar waxay u baahan yihiin wejiyaal taagan oo si gaar ah loo habeeyey si ay ula falgalaan una xajistaan ​​falanqayntan16. chromatography isku dhafan, kaas oo bixiya isdhexgalka hababka kala duwan, waxay noqon kartaa beddelka RP-LC ee kala soocida peptides, borotiinnada, iyo isku dhafka kale ee adag. Waxaa la diyaariyey dhowr weji oo isku dhafan oo isku dhafan waxaana tiirar ay ka buuxaan wejiyadan taagan la isticmaalay si loo kala saaro peptides iyo borotiinada17,18,19,20,21. Sababtoo ah joogitaanka kooxaha polar iyo non-polar, wejiyada isku dhafan ee isku dhafan (WAX / RPLC, HILIC / RPLC, polar intercalation / RPLC) waxay ku habboon yihiin kala-soocidda peptides iyo borotiinka22,23,24,25,26,27,28. , Polar wejiyada taagan ee intercalated oo leh kooxo dabaylaha covalently bond waxay muujinayaan awoodaha kala soocida wanaagsan iyo xulashada gaarka ah ee dabaylaha iyo falanqaynta aan dabaylaha ahayn sababtoo ah kala soociddu waxay ku xiran tahay isdhexgalka u dhexeeya falanqaynta iyo marxaladda taagan ee isdhexgalka Multimodal 29,30,31,32. Dhawaan, Zhang et al. 30 waxay heleen wejiyada joogtada ah ee behenyl-ka ee polyamines waxayna si guul leh u kala sooceen hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, iyo qaar ka mid ah falanqaynta kale. Maaddada taagan ee darafku waxay leedahay labada koox ee polar iyo non-polar, sidaas darteed waxaa loo isticmaali karaa in lagu kala saaro peptides iyo borotiinada qaybaha hydrophobic iyo hydrophilic. Tiirarka khadadka dalalka ah (tusaale, tiirarka C18 oo leh inline amide) ayaa laga heli karaa hoosta magaca ganacsiga Ascentis Express RP-Amide columns, laakiin tiirarkan waxa kaliya loo isticmaalay falanqaynta amine 33.
Daraasadda hadda jirta, wejiga ku dhejinta dabaylaha (N-phenylmaleimide, dhejinta polystyrene) ayaa la diyaariyey oo lagu qiimeeyay kala-soocidda peptide iyo kala-baxa HSA-ga tryptic. Istaraatiijiyadan soo socota ayaa loo adeegsaday diyaarinta marxaladda taagan. Qaybaha silica ee daloola ayaa loo diyaariyey iyadoo loo eegayo habraacyada lagu sharraxay daabacaadii hore, iyadoo isbeddellada qaarkood lagu sameeyay qorshayaasha diyaarinta 31, 34, 35, 36, 37, 38, 39. Saamiga HOL, polyetylen glycol (PEG), TMOS iyo aqueous-acetic acid ayaa la hagaajiyay si loo helo qaybo silica ah oo waaweyn. Marka labaad, ligand cusub ee phenylmaleimide-methylvinyl isocyanate ligand ayaa la soo saaray oo qaybo silica ah oo laga soo dejiyey ayaa loo isticmaalay si loogu diyaariyo wejiyada taagan ee cirifka leh. Marxaladda taagan ee la helay waxaa lagu soo guray tiir bir ah (dhexroorka 100 × 1.8 mm) iyadoo loo eegayo nidaamka xirxiran ee la hagaajiyay. Xiritaanka tiirka waxaa caawiya gariirka farsamada si loo hubiyo in lakabka lebbiska ah ee tiirka dhexdiisa. Tiirka la soo buuxiyay ayaa lagu qiimeeyay kala soocida isku dhafka peptides oo ka kooban shan peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). iyo tryptic hydrolysates ee serum albumin (HSA). Waxaa la arkay in isku darka peptide-ka iyo HSA tryptic dheefshiidku ay ku kala tageen xalin wanaagsan iyo hufnaan. Waxtarka kala soocida tiirka PMP ayaa la barbardhigay tiirka Ascentis Express RP-Amide. Waxaa la arkay in peptides iyo borotiinada ay leeyihiin xallin wanaagsan iyo hufnaanta kala-soocidda sare ee tiirka PMP, iyo waxtarka kala-soocidda tiirka PMP ayaa ka sarreeya tiirka Ascentis Express RP-Amide.
PEG (polyethylene glycol), urea, acetic acid, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, serum albumin (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride (MC), POZ-hydrone, styrenzolyde, styrezone. acetonitrile (ACN) ee HPLC, methanol, 2-propanol iyo acetone. Shirkadda Sigma-Aldrich (St. Louis, Missouri, USA).
Isku dar ah HOL (8 g), polyetylen glycol (8 g) iyo 8 ml 0.01 N. acetic acid ayaa la walaaqay 10 daqiiqo, 24 ml oo TMOS ah ayaa lagu daray hoos qaboojinta barafka. Isku darka falcelinta waxaa lagu kululeeyay 40 ° C ilaa 6 saacadood ka dibna 120 ° C ilaa 8 saacadood oo ah autoclave birta aan lahayn. Biyihii waa la gooyey, hadhaaga waxaa lagu qallajiyey 70 ° C ilaa 12 saacadood. Baloogyada jilicsan ee la qalajiyey ayaa si habsami leh loo dajiyay waxaana lagu dhex dajiyay foorno heerkul ah 550 ° C ilaa 12 saacadood. Saddex qaybood ayaa la diyaariyey oo lagu sifeeyay in lagu tijaabiyo soo saaridda cabbirrada walxaha, cabbirka daloolka iyo bedka dusha sare.
Kooxda Polar iyo wejiga taagan ee silsiladaha polystyrene. Habka diyaarinta ayaa lagu qeexay hoos.
N-phenylmaleimide (200 mg) iyo methyl vinyl isocyanate (100 mg) ayaa lagu milmay toluene anhydrous, ka dibna 0.1 ml oo ah 2,2'-azoisobutyronitrile (AIBN) ayaa lagu daray weelka falcelinta si loo helo copolymer ee phenylmaleimCPide iyo methyl vinyl methyl (PM). ) Isku darka waxaa lagu kululeeyay 60 ° C ilaa 3 saacadood, la sifeeyay oo la qalajiyey foornada 40 ° C 3 saacadood.
Qaybaha silica ee la qallajiyey (2 g) ayaa lagu kala firdhiyey toluene qalalan (100 ml), walaaqay oo la sonicated 10 min in 500 ml wareega hoose ee baaquli. PMCP (10 mg) ayaa lagu milmay toluene waxaana lagu daray hoos udhaca weelka falcelinta iyada oo loo sii marayo marin dheeri ah. Isku darka waxaa lagu soo celiyay 100 ° C ilaa 8 saacadood, la sifeeyay, lagu dhaqay acetone oo la qalajiyey 60 ° C 3 saacadood. Ka dib, qaybaha silica ee la xidhiidha PMCP (100 g) ayaa lagu milmay toluene (200 ml), iyo 4-hydroxy-TEMPO (2 ml) ayaa lagu daray iyada oo ay jirto 100 μl ee dibutyltin dilaure sida kicinta. Isku darka waxaa lagu walaaqay 50 ° C ilaa 8 saacadood, la sifeeyay oo la qalajiyey 50 ° C 3 saacadood.
Styrene (1 ml), benzoyl peroxide BPO (0.5 ml) iyo walxaha silica ee ku xiran TEMPO-PMCP (1.5 g) ayaa lagu kala firdhiyey toluene waxaana lagu nadiifiyaa nitrogen. polymerization of styrene waxaa lagu fuliyay at 100 ° C 12 saacadood. Alaabta ka soo baxday waxaa lagu dhaqay methanol waxaana lagu qallajiyey habeenkii 60°C. Habka guud ee falcelinta waxa lagu muujiyay berdaha. mid .
Tijaabooyinku waxa lagu daadiyey 393 K 1 saac ilaa inta la helay cadaadis hadhaaga ah oo ka yar 10-3 Torr. Qadarka N2 ee lagu dhajiyay cadaadiska qaraabada P/P0 = 0.99 ayaa loo isticmaalay si loo go'aamiyo wadarta mugga daloolka. Qaab-dhismeedka walxaha silica saafiga ah iyo ligand-ku-xidhan ayaa la baadhay iyadoo la adeegsanayo mikroskoob elektaroonig ah (Hitachi High Technologies, Tokyo, Japan). Tijaabooyin qallalan (silica saafi ah iyo qaybaha silica ligand ee ku xidhan) ayaa lagu dhejiyay ulaha aluminiumka iyadoo la isticmaalayo cajalad kaarboon ah. Dahab ayaa lagu shubay muunadda iyadoo la adeegsanayo aaladda xajinta Q150T, iyo lakab 5 nm ah oo qaro ah ayaa lagu shubay muunadda. Tani waxay wanaajisaa waxtarka habka tamarta hooseeya waxayna bixisaa buufin qabow oo wanaagsan. Falanqaynta aasaasiga ah waxaa lagu sameeyay iyadoo la adeegsanayo Thermo Electron (Waltham, MA, USA) Flash EA1112 falanqeeyaha halabuurka aasaasiga ah. Falanqeeyaha cabbirka walxaha Malvern (Worcestershire, UK) Mastersizer 2000 ayaa loo adeegsaday si loo helo qaybinta cabbirka walxaha. Qaybaha silica aan la daboolin iyo qaybaha silica ligand-ku xidhan (5 mg kasta) ayaa lagu kala firdhiyey 5 ml isopropanol, sonicated 10 daqiiqo, kacday 5 daqiiqo, oo la galiyay kursiga indhaha ee Mastersizer. Falanqaynta Thermogravimetric waxaa lagu sameeyaa heerkul ah 5 °C daqiiqadii heerkulka u dhexeeya 30 ilaa 800 ° C.
31. Tiirar bir ah oo dhuuban (galaas safaysan, ID 100 × 1 .8 mm) iyo meel ka kooban 1 µm frit ah ayaa lagu xidhay mashiinka buuxinta slurry ee USA. Diyaarso hakinta marxaladda taagan adiga oo laalya 150 mg ee marxaladda taagan ee 1.2 ml ee methanol oo ku quudin tiirka kaydka. Methanol waxa loo isticmaalay sidii dareere dareere ah iyo dareeraha xakamaynta. Xidho tiirka adiga oo isticmaalaya isku xigxiga cadaadiska 100 MP 10 daqiiqo, 80 MP 15 daqiiqo, iyo 60 MP 30 daqiiqo. Habka xirxiraddu waxay isticmaashay laba gariir oo gaaska koromograafiga ah (Alltech, Deerfield, IL, USA) gariir makaanikada si loo hubiyo xirid lebbiska tiirka. Xir baakadaha slurry oo si tartiib ah u sii daa cadaadiska si aad uga hortagto dhaawac soo gaara xadhigga. Tiirka ayaa ka go'ay sanbabada slurry iyo qalab kale ayaa lagu dhejiyay meesha laga soo galo oo lagu xiray nidaamka LC si loo tijaabiyo hawlgalkiisa.
MLC caado ah ayaa la dhisay iyadoo la adeegsanayo bamka LC (10AD Shimadzu, Japan), muunadeeyaha leh 50 nL duritaanka loop (Valco (USA) C14 W.05), xuubka degasser (Shimadzu DGU-14A), iyo daaqad UV-VIS ah. Qalabka wax soo saara (UV-2075) iyo koodhka yaryar ee dahaadhay. Isticmaal tuubooyinka isku xidha aadka u dhuuban iyo kuwa gaaban si aad u yarayso saamaynta balaadhinta tiirka dheeraadka ah. Kadib buuxinta tiirka, ku rakib xididka (50 µm id 365) halka laga soo baxo 1/16 ″ yaraynta isgoysyada oo ku rakib xididka (50 µm) ee isgoyska dhimista. Xog ururinta iyo habaynta chromatogram-ka waxa lagu sameeyaa iyada oo la isticmaalayo barnaamijka Multichro 2000. At 254 nm, nuugista UV ee maadooyinka falanqaynta ayaa lagu kormeeray 0. Xogta Chromatographic waxaa lagu falanqeeyay iyadoo la adeegsanayo OriginPro8 (Northampton, MA).
Serum albumin aadanaha, budada lyophilized, ≥ 96% (agarose gel electrophoresis) 3 mg oo lagu daray trypsin (1.5 mg), 4.0 M urea (1 ml) iyo 0.2 M ammonium bicarbonate (1 ml) . Xalka ayaa la walaaqay 10 min waxaana lagu hayaa qubeyska biyaha 37 ° C ilaa 6 saacadood, ka dibna la demiyo 1 ml oo ah 0.1% TFA. Sifee xalka oo ku kaydi meel ka hooseysa 4°C.
Kala saarista isku dhafka peptides iyo dheefshiidka tryptic HSA ee tiirka PMP ayaa si gaar ah loo qiimeeyay. Hubi hydrolysis tryptic ee isku dhafka peptides iyo HSA oo ay kala soocaan tiirka PMP oo barbar dhig natiijooyinka tiirka Ascentis Express RP-Amide. Tirada taarikada aragtida waxaa lagu xisaabiyaa iyadoo la isticmaalayo isla'egta soo socota:
Sawirada SEM ee qaybaha silica saafiga ah iyo qaybaha silica ee ligand ee ku xiran ayaa lagu muujiyay sawirka 2. Sawirada SEM ee qaybaha silica saafiga ah (A, B) waxay muujinayaan qaab wareegsan oo qaybuhu ay fidsan yihiin ama ay leeyihiin simmetry aan caadi ahayn marka loo eego daraasadaheena hore. Dusha sare ee qaybaha silica ee ku xidhan ligand (C, D) ayaa ka jilicsan kuwa silica saafiga ah, taas oo laga yaabo inay sabab u tahay silsiladaha polystyrene ee daboolaya dusha sare ee qaybaha silica.
Baadhitaanka mikrogaraafka elektarooniga ah ee walxaha silica saafiga ah (A, B) iyo qaybaha silica ku xidhan ee ligand (C, D).
Qaybinta xajmiga walxaha ee qaybaha silica saafiga ah iyo qaybaha silica ee ku xiran ligand ayaa lagu muujiyay sawirka 2. 3 (A). Xajmiga qaybinta qaybinta mugga waxay muujisay in cabbirka walxaha silica uu kordhay ka dib beddelka kiimikada (Jaantus. 3A). Xogta qaybinta qaybinta walxaha silica ee daraasadda hadda jirta iyo daraasaddii hore ayaa lagu barbardhigay shaxda 1 (A). Cabbirka walxaha mugga d (0.5) ee PMP wuxuu ahaa 3.36 µm, marka la barbar dhigo ad(0.5) qiimaha 3.05 µm daraasaddeennii hore (qurubyada silica ee polystyrene bonded)34. Sababtoo ah isbeddelka saamiga PEG, HOL, TMOS iyo acetic acid ee isku dhafka falcelinta, qaybinta xajmiga qaybta dufcaddani way ka sii yaraatay marka loo eego daraasaddeennii hore. Baaxadda walxaha wajiga PMP ayaa waxyar ka weyn kan wejiga walxaha silica ee ku xidhan polystyrene ee aan hore u barannay. Tani waxay ka dhigan tahay in shaqeynta dusha sare ee walxaha silica leh styrene ay ku shubtay kaliya lakabka polystyrene (0.97 µm) dusha silica, halka marxaladda PMP ay dhumucdiisu ahayd 1.38 µm.
Qaybinta cabbirka walxaha (A) iyo qaybinta cabbirka daloolka (B) ee qaybaha silica saafiga ah iyo qaybaha silica ee ku xidhan ligand.
Cabbirka daloolka, mugga daloolka, iyo bedka dusha sare ee qaybaha silica ee loo adeegsaday daraasaddan ayaa lagu muujiyey Shaxda 1 (B). Tilmaamaha PSD ee qaybaha silica saafiga ah iyo qaybaha silica ee ku xidhan ligand ayaa lagu muujiyay berdihii. 3(B). Natiijadu waxay la mid ahayd daraasaddeennii hore34. Cabbirrada daloolada ee qaybaha silica ee saafiga ah iyo ligand-ku-xidhan waxay ahaayeen 310 Å iyo 241 Å, siday u kala horreeyaan, taas oo muujinaysa in ka dib markii wax laga beddelo kiimikaad, cabbirka daloolka uu hoos u dhacay 69 Å, sida ku cad Shaxda 1 (B), iyo qalooca isbeddelka ayaa lagu muujiyay sawirka (124 m2/g). Sida ku cad Shaxda 1 (B), aagga dusha sare (m2 / g) ee walxaha silica ka dib isbeddelka kiimikada ayaa sidoo kale hoos u dhacay 116 m2 / g ilaa 105 m2 / g.
Natiijooyinka falanqaynta aasaasiga ah ee marxaladda taagan ayaa lagu soo bandhigay shaxda. 2. Waxyaabaha kaarboon ee marxaladda taagan ee hadda taagan waa 6.35%, taas oo ka hooseysa daraasaddeennii hore (qaybaha silica ee la xidhiidha polystyrene, 7.93% 35 iyo 10.21%, siday u kala horreeyaan) loo isticmaalo marka lagu daro styrene diyaarinta SP. Boqolkiiba miisaanka nitrogen ee marxaladda taagan hadda waa 2.21% marka la barbar dhigo 0.1735 iyo 0.85% daraasadihii hore42. Tani waxay ka dhigan tahay in marxaladda taagan ee hadda jirta ay leedahay miisaan sare oo boqolkiiba nitrogen ah sababtoo ah phenylmaleimide. Sidoo kale, alaabooyinka (4) iyo (5) waxay leeyihiin kaarboonka 2.7% iyo 2.9%, siday u kala horreeyaan, halka badeecada ugu dambeysa (6) ay leedahay kaarboonka 6.35%, sida ku cad Shaxda 2. Falanqaynta Thermogravimetric (TGA) ayaa loo adeegsaday marxaladda taagan ee PMP si loo tijaabiyo miisaanka luminta, iyo qalooca TGA ayaa lagu muujiyay miisaanka 4% ee miisaanka TGA. heshiis lala yeesho kaarboonka (6.35%), maadaama ligands ay ku jiraan kaliya C, laakiin sidoo kale N, O iyo H.
The ligand phenylmaleimide-methylvinyl isocyanate ayaa loo doortay si loo beddelo dusha sare ee qaybaha silica sababtoo ah phenylmaleimide polar iyo kooxaha vinylocyanate. Kooxaha isocyanate Vinyl waxay si dheeraad ah uga falcelin karaan styrene iyagoo isticmaalaya polymerization xagjirka ah. Sababta labaad waa in la geliyo koox leh is-dhexgal dhexdhexaad ah oo leh falanqeeye oo aan lahayn is-dhexgal xooggan oo koronto ah oo u dhexeeya falanqaynta iyo marxaladda taagan, maaddaama unugyada phenylmaleimide aysan lahayn wax lacag ah oo muuqaal ah pH caadiga ah. Polarity ee marxaladda taagan waxaa lagu xakameyn karaa qadarka ugu fiican ee styrene iyo waqtiga falcelinta ee polymerization xagjirka bilaashka ah. Talaabada ugu danbeysa ee falcelinta (polymerization xagjirka xorta ah) waa muhiim maadaama ay bedelayso polarity ee marxaladda taagan. Falanqaynta aasaasiga ah ayaa la sameeyay si loo hubiyo waxa ku jira kaarboonka ee wejiyadan taagan. Waxaa la arkay in kordhinta qadarka styrene iyo waqtiga falcelinta ay kordhiso kaarboonka marxaladda taagan iyo lidkeeda. SP-yada lagu diyaariyo xajmiyada kala duwan ee styrene waxay leeyihiin culeysyo kaarboon oo kala duwan. Sidoo kale, wejiyadan taagan waxaa lagu dhejiyay tiirarka birta ah ee aan-dhalatada lahayn waxaana la hubiyay sifooyinkooda chromatographic (doorashada, xallinta, qiimaha N, iwm.) Iyada oo ku saleysan tijaabooyinkan, halabuurka la wanaajiyey ee diyaarinta marxaladda taagan ee PMP ayaa la doortay si ay u bixiso polarity la kantaroolo iyo haynta wanaagsan ee falanqaynta.
Tiirka PMP ayaa sidoo kale lagu qiimeeyay falanqaynta shan isku-dar ah oo peptides ah (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) iyadoo la adeegsanayo awoodda wajiga wareega. 60/40 (v/v) ACN/biyaha (0.1% TFA) ee heerka socodka 80 µl/daqiiqo. Marka la eego shuruudaha ugu fiicnaanta (200,000 saxan / m), tirada taarikada aragtida (N) ee tiirarka (100 × 1.8 mm) waa 20,000 ± 100. Qiimaha N ee saddexda tiir ee PMP ayaa lagu muujiyay Shaxda 3 iyo chromatogram-yada ayaa lagu muujiyay sawirka 5A. Falanqaynta degdega ah ee heerka socodka sare (700 µl / min) ee tiirka PMP, shan peptides ayaa lagu soo saaray hal daqiiqo gudaheed, qiimaha N aad u fiican ee 13,500 ± 330 halkii tiir (100 x 1.8 mm dhexroor), oo u dhiganta 135,000 taarikada / m (Jaantus. 5B). Saddex tiir oo isku mid ah (dhexroorka gudaha 100 x 1.8 mm) ayaa lagu buuxiyey saddex qaybood oo kala duwan oo ah marxaladda taagan ee PMP si loo tijaabiyo soo saarista. Falanqeeyayaasha waxaa loo duubay tiir kasta iyadoo la kala saarayo isku dhafka tijaabada ah ee tiir kasta iyadoo la adeegsanayo xaaladaha ugu fiicnaanta, tirada taarikada aragtida N, iyo waqtiga haynta. Xogta soo saarista ee tiirarka PMP waxa lagu muujiyay shaxda 4. Soo saarista tiirka PMP waxa ay si fiican ula xidhiidhaa % RSD oo aad u hooseeya sida ku cad shaxda 3.
Kala soocida isku dhafka peptide ee tiirka PMP (B) iyo Ascentis Express RP-Amide column (A), wajiga wareega 60/40 ACN/H2O (TFA 0.1%), cabbirada tiirarka PMP (100 x 1.8 mm id) , falanqaynta nidaamka Elution ee xeryahooda: 1 (Gly-Tyr), - 2-Gyrly (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) iyo 5 (enkephalin leucic acid).
Tiirka PMP (dhexroorka gudaha 100 x 1.8 mm) ayaa lagu qiimeeyay kala soocida hydrolyzate tryptic ee serum albumin aadanaha ee HPLC. chromatogram-ka ku yaal Jaantuska 6 wuxuu muujinayaa in shaybaarada si fiican loo kala saaray oo leh xallin aad u wanaagsan. Xalalka HSA ayaa la falanqeeyay iyadoo la isticmaalayo heerka socodka 100 μl/min, wejiga wareega ee 70/30 acetonitrile/biyo iyo 0.1% TFA. Burburinta HSA waxaa loo kala qaybiyay 17 sare, sida ku cad chromatogramka (Jaantus. 6), oo u dhiganta 17 peptides. Waxtarka kala soocida ugu sarreeya ee shakhsi ahaaneed ee HSA hydrolyzate ayaa la xisaabiyay oo qiyamka ayaa lagu muujiyay shaxda 5.
HSA tryptic hydrolysates ayaa lagu kala soocay tiirka PMP (dhexroorka gudaha 100 x 1.8 mm), heerka socodka (100 μl/min), wajiga wareega 60/40 acetonitrile/biyo, iyo 0.1% TFA.
halka L uu yahay dhererka tiirka, η waa viscosity ee wajiga wareega, ΔP waa cadaadiska dambe ee tiirka, iyo u waa xawaaraha toosan ee wajiga wareega. Joogitaanka tiirka PMP wuxuu ahaa 2.5 × 10-14 m2, heerka socodka wuxuu ahaa 25 µl/min, 60/40 v/v ayaa la isticmaalay. ACN/biyaha. Dareemida tiirka PMP (ID 100 × 1.8 mm) waxay la mid tahay daraasaddeennii hore ee Ref.34. Dareemida tiirka ay ka buuxaan walxaha dusha sare ka daloola waa 1.7×10 .6 µm, 2.5×10-14 m2 ee 5 µm particles43. Sidaa darteed, daah-furnaanta wajiga PMP waxay la mid tahay qulqulka qaybo ka mid ah qolof-core oo leh cabbirka 5 μm.
halka Wx ay tahay cufnaanta tiirka oo ay ka buuxaan chloroform, Wy waa cufka tiirka uu ka buuxo methanol, iyo ρ waa cufnaanta dareeraha. Cufnaanta methanol (ρ = 0.7866) iyo chloroform (ρ = 1.484). Isugeynta porosity ee tiirka walxaha silica-C18 (100 × 1.8 mm ID) 34 iyo tiirkeenii hore ee C18-urea31 ayaa ahaa 0.63 iyo 0.55, siday u kala horreeyaan. Tani waxay ka dhigan tahay in joogitaanka ligands urea ay yareyneyso qulqulka marxaladda taagan. Dhanka kale, wadarta guud ee tiirka PMP (dhexroorka gudaha 100 × 1.8 mm) waa 0.60. Tiirarka PMP way ka yar yihiin tiirarka ay ka buuxaan qaybaha silica ee ku xidhan C18 sababtoo ah nooca C18 wejiyada joogtada ah ee C18 ligands waxay ku xiran yihiin qaybaha silica ee silsiladaha toosan, halka polystyrene nooca wejiyada taagan ay ka samaysan tahay polymer qaro weyn agagaarka qaybaha. lakabka A. Tijaabo caadi ah, porosity column waxa loo xisaabiyaa sida soo socota:
On berde. 7A, B waxay tusinaysaa dhulalka Van Deemter ee tiirka PMP (id 100 x 1.8 mm) iyo tiirka Ascentis Express RP-Amide (id 100 x 1.8 mm) oo hoos imanaya isla xaalado la mid ah, 60/40 ACN/H2O iyo 0 .1% TFA 20 μl/min labada tiir ilaa 8. Qiimaha ugu yar ee HETP ee heerka socodka ugu fican (80 µl/min) waxay ahaayeen 2.6 µm iyo 3.9 µm tiirka PMP iyo tiirka Ascentis Express RP-Amide, siday u kala horreeyaan. Qiimayaasha HETP waxay muujinayaan in hufnaanta kala-soocidda tiirka PMP (100 x 1.8 mm id) uu aad uga sarreeyo kan tiirka Ascentis Express RP-Amide ee ganacsi ahaan loo heli karo (100 x 1.8 mm id). Garaafka van Deemter ee Jaantuska 7 (A) wuxuu muujinayaa in hoos u dhaca qiimaha N uusan si weyn uga sarreyn socodka sii kordhaya marka loo eego daraasaddeena hore. Waxtarka sare ee wax ku oolnimada tiirka PMP (id 100 × 1.8 mm) marka la barbar dhigo tiirka Ascentis Express RP-Amide wuxuu ku salaysan yahay qaabka iyo cabbirka qaybta wanaajiyay iyo habka xirmidda tiirarka casriga ah ee loo isticmaalo shaqada hadda34.
(A) Sheekada Van Deemter (HETP vs. xawaaraha tooska ah ee wareega wareega) oo laga helay tiirka PMP (id 100 x 1.8 mm) gudaha 60/40 ACN/H2O oo leh 0.1% TFA. (B) Qorshaha Van Deemter (HETP oo ka soo horjeeda xawaaraha tooska ah ee wareega wareega) ee laga helay tiirka Ascentis Express RP-Amide (id 100 x 1.8 mm) gudaha 60/40 ACN/H2O oo leh 0.1% TFA.
Marxaladda taagan ee dabaylaha ah ee polystyrene intercalated ayaa la diyaariyey oo lagu qiimeeyay kala soocida isku dhafka peptides synthetic iyo tryptic hydrolyzate ee serum albumin (HSA) ee waxqabadka sare ee chromatography dareere ah. Waxqabadka chromatographic ee tiirarka PMP ee isku dhafka peptide waa mid aad u fiican marka loo eego hufnaanta iyo xallinta kala-soocidda. Waxtarka kala soocida ee la hagaajiyay ee tiirarka PMP waxaa sabab u ah dhowr sababood sida cabbirka walxaha silica iyo cabbirka daloolka, isku-darka kontoroolka ee wejiyada taagan, iyo agabka xirxirida tiirarka adag. Marka lagu daro waxtarka sare ee kala-soocidda, faa'iidada kale ee marxaladdan taagan waa cadaadiska hoose ee dhabarka ee heerarka socodka sare. Tiirarka PMP waa kuwo si heer sare ah loo soo saari karo waxaana loo isticmaali karaa in lagu falanqeeyo isku dhafka peptides iyo dheefshiidka tryptic ee borotiinno kala duwan. Waxaan dooneynaa inaan u isticmaalno tiirkaan kala soocida xeryahooda bioactive ee alaabada dabiiciga ah, soosaarka dhirta daawada iyo boqoshaada ee chromatography dareeraha ah. Mustaqbalka, tiirarka PMP ayaa sidoo kale lagu qiimeyn doonaa kala soocida borotiinnada iyo unugyada difaaca jirka ee monoclonal.
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