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O mea'ai silica Porous na saunia e se metotia sol-gel ma nisi o suiga e maua ai ni vaega macroporous. O nei vaega na maua mai i le toe fa'aopoopoina fa'aopoopo fragmentation filifili fesiitaiga (RAFT) polymerization ma N-phenylmaleimide-methylvinylisocyanate (PMI) ma styrene e saunia N-phenylmaleimide u'amea u'amea (PMP) polystyrene koluma fa'atasi. (100 × 1.8 mm id) sa teuina e slurry packing.Evaluated PMP koluma vavaeesega o se paluga peptide e aofia ai lima peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg) faʻataʻitaʻiga faʻataʻitaʻiga o le tagata (leucine sepsinkephalin) (HAS) .I lalo o tulaga elution sili ona lelei, o le numera plate theoretical o le paluga peptide e maualuga e pei o 280,000 ipu / m². Faatusatusa le faatinoga o le tuueseeseina o le koluma atiina ae ma le koluma pisinisi Ascentis Express RP-Amide, na matauina o le tuueseeseina faatinoga o le koluma PMP na sili atu i le koluma faapisinisi i tulaga o le vavaeeseina lelei ma le faaiuga.
I tausaga talu ai nei, o le biopharmaceutical alamanuia ua avea ma faʻalauteleina maketi i le lalolagi atoa ma le tele o le faʻatupulaia o maketi maketi. Faatasi ai ma le faʻateleina o le tuputupu aʻe o le biopharmaceutical industry1,2,3, o le auiliiliga o peptides ma proteins e matua manaʻomia. o polotini i sua o le tino, tissues ma sela o se galuega sili ona faigata ona o le tele o ituaiga e mafai ona iloa i se faataitaiga e tasi. analytes ulufale atu i le spectrometer tele i se taimi tuuina atu 4,5,6.In le faaopoopo, i le taimi o le vaeluaga vai, e mafai ona taulaʻi analytes i itulagi vaapiapi, o lea e taulai atu nei auʻiliʻili ma le faʻaleleia o le MS detection sensitivity.Liquid chromatography (LC) ua alualu i luma tele i luga o le sefulu tausaga talu ai ma ua avea ma se auala lauiloa i proteomic,917.
Fa'aliliuina-vaega vai chromatography (RP-LC) o loʻo faʻaaogaina lautele mo le faʻamamaina ma le vavaeeseina o paluga peptide e faʻaaoga ai le silica octadecyl-modified (ODS) e pei o le tulaga tumau 11,12,13. Ae peitai, RP tulaga tumau e le maua ai le vavaeeseina faamalieina o peptides ma porotini ona o lo latou fausaga faʻapitoa 14, philore philore ma le natura faʻapitoa. vaega e manaomia e iloilo peptides ma polotini faatasi ma polar ma non-polar moieties e fegalegaleai ma taofi nei analytes16. Mixed-mode chromatography, lea e maua ai fegalegaleaiga multimodal, e mafai ona avea ma se isi mea i le RP-LC mo le tuueseeseina o peptides, polotini, ma isi fefiloi lavelave. E tele fefiloi-mode chromatography ua uma ona saunia mo nei vaega tulaga tumau ma polotini vaega ua saunia ma le polotini vaega. separations17,18,19,20,21.Mixed-mode stationary phases (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) e talafeagai mo le peptide ma le protein separations ona o le i ai o vaega uma e lua polar ma non-polar vaega22,23,24,25,26,27,28 faʻatasi faʻatasi, faʻasolosolo faʻatasi, faʻasolosolo faʻatasi, pola. fa'apipi'i polar vaega fa'aalia lelei le tu'ueseeseina o le mana ma le tulaga ese o le filifilia mo polar ma non-polar analytes, aua o le vavae'esega e fa'alagolago i le fegalegaleaiga i le va o analyte ma vaega tumau. Fegalegaleaiga tele 29, 30, 31, 32. Talu ai nei, Zhang et al. 30 saunia se dodecyl-terminated polyamine tulaga tumau vaega ma manuia vavae hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, ma le tele o isi analytes. O le intercalator polar e lua vaega polar ma non-polar vaega, o lea e mafai ona faaaoga e tuueseese peptides ma porotini o loo i ai uma hydrophobic ma hydrophilic moieties. koluma) o loʻo avanoa faʻapisinisi i lalo o le igoa tau fefaʻatauaʻiga Ascentis Express RP-Amide koluma, ae o nei koluma e faʻaaogaina mo le auiliiliga o amine 33 naʻo.
I le suʻesuʻega o loʻo i ai nei, o se vaega faʻapipiʻi faʻapipiʻi polar (N-phenylmaleimide-embedded polystyrene) na saunia ma iloiloina mo le vavaeeseina o peptides ma trypsin digests o le HSA. O le vaega faʻamau na saunia e faʻaaoga ai le taʻiala o loʻo i lalo. Sa saunia meaʻai silica Porous e tusa ai ma le faʻagasologa na tuʻuina atu i la tatou lomiga talu ai ma nisi o suiga i le faʻasologa o le vai, polyceleneure a le PPE. acid na fetuutuunai e saunia vaega silica ma le tele pore size.Lua, o se ligan fou, phenylmaleimide-methyl vinyl isocyanate, sa synthesized ma faaaoga e derivatize vaega silica e saunia ai se polar embedded stationary phase.The taunuuga tulaga tumau vaega na teuina i totonu o se koluma uamea stainless (100 × 1.8 mm id faakomepiuta o loo fesoasoani i le packing koluma id). fa'amautinoa o lo'o faia se moega tutusa i totonu o le koluma. Iloilo le vaeluaga o koluma fa'apipi'i o fa'afefiloi peptide e aofia ai peptide e lima; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) ma Trypsin digest o serum albumin tagata (HAS) .O le paluga peptide ma trypsin digest o le HSA sa matauina e vavae ese ma le lelei o le iugafono ma le lelei o le faatinoga o le Express PMPpacent ma le faatinoga o lena serum PMPpacent. RP-Amide koluma.O peptides ma polotini uma na matauina e lelei ona foia ma lelei ile koluma PMP, lea e sili atu le lelei nai lo le koluma 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 (MCTEMP-Hyrene, Benzoxyide), Styrene. HPLC Grade Acetonitrile (ACN), Methanol, 2-Propanol, ma Acetone Faʻatau mai Sigma-Aldrich (St. Louis, MO, ISA).
O se faʻafefiloi o le urea (8 g), polyethylene glycol (8 g), ma le 8 mL o le 0.01 N acetic acid sa faʻafefeteina mo le 10 minute, ona faʻaopoopoina lea o le 24 mL o le TMOS i lalo o le aisa-malili. na faʻagogo i le 70 ° C mo itula 12. O le masini vaivai mamago sa faʻafefeteina i totonu o se ogaumu ma calcined i le 550 ° C mo itula 12. E tolu vaega na saunia ma faʻamaonia e suʻesuʻe ai le toe gaosia i le lapopoa, pore lapoa ma luga o le eleele.
E ala i le suia o luga o vaega silica ma muai synthesized ligand phenylmaleimide-methylvinylisocyanate (PCMP) sosoo ai ma le radial polymerization ma styrene, sa saunia se vaega polar-o loʻo i ai le tuufaatasiga. Vaega tumau mo agregates ma filifili polystyrene. O le faagasologa o sauniuniga o loʻo faʻamatalaina i lalo.
N-phenylmaleimide (200 mg) ma le methyl vinyl isocyanate (100 mg) na faʻamavaeina i toluene mago, ma 0.1 mL o le 2,2′-azoisobutyronitrile (AIBN) na faʻapipiʻiina i le fagu tali e saunia ai le phenylmaleimide-methyl isocyanate copolymer (PMC) 3 itula na faʻafefiloi i le 3°C . fa'amago ile ogaumu ile 40°C mo le 3 itula.
O fasimea silica mago (2 g) na salalau i toluene mago (100 mL), faʻaoso ma sonicated i se 500 mL lapotopoto pito i lalo fagu mo le 10 min. PMCP (10 mg) na solo i toluene ma faaopoopo dropwise i le fagu tali e ala i se faʻafefe paʻu. O le paluga sa refluxed i le 100 ° C ma faamamago itula i le 100 ° C ma faamamago. 60 ° C mo le 3 itula. Ona, PMCP-faʻapipiʻiina silica particles (100 g) na faʻamavaeina i toluene (200 ml) ma 4-hydroxy-TEMPO (2 mL) faʻaopoopoina i le i ai o le 100 μL o le dibutyltin dilaurate e fai ma faʻamalosi. O le paluga na faʻaosoina i le 50 ° C ma faʻamalo itula 50 ° C mo itula faʻamago.
Styrene (1 mL), benzoyl peroxide BPO (0.5 mL), ma le TEMPO-PMCP-faʻapipiʻi silica particles (1.5 g) na faʻasalalau i toluene ma faʻamamaina i le nitrogen. O le polymerization o le styrene na faia i le 100 ° C mo itula 12. O le mea na maua mai na fufuluina i le methanol ma faʻagogo atoa i le po i le 60 ° C.
O faʻataʻitaʻiga na faʻamalo i le 393 K mo le 1 itula e maua ai le mamafa o totoe o le itiiti ifo i le 10-3 Torr.O le aofaʻi o le N2 adsorbed i se faʻalavelave vavalalata o P / P0 = 0.99 na faʻaaogaina e fuafua ai le aofaʻi o le pore. O le morphology o le silica silica ma ligand-bonded particles na suʻesuʻeina i le suʻesuʻeina o masini eletise eletise, Japan samples, Japan samples. ma ligand-bonded silica particles) na tuʻuina i luga o se koluma alumini e faʻaaoga ai le kaponi faʻapipiʻi. Na faʻapipiʻiina le auro i luga o faʻataʻitaʻiga e faʻaaoga ai se Q150T sputter coater, ma se 5 nm Au layer na teuina i luga o faʻataʻitaʻiga. E faʻaleleia atili ai le faʻaogaina o le eletise maualalo ma maua ai saito lelei, malulu malulu. Malvern (Worcestershire, UK) Mastersizer 2000 na faʻaaogaina e maua ai le tufatufaina o le tele o le vaega. O le silica natia ma le ligand-bonded silica particles (5 mg taʻitasi) na faʻasalalau i le 5 mL o le isopropanol, sonicated mo le 10 min, vortexed mo le 5 min, ma tuʻu i luga o le laulau faʻapipiʻi o le Masters 5 faʻataʻitaʻiga i luga ole fua faatatau o le Masters. va'aiga vevela o le 30 i le 800 °C.
Ua fa'apipi'i i tioata uamea u'amea vaapiapi koluma o fua (100 × 1.8 mm id) sa fa'apipi'iina i le fa'aogaina o le auala e teu ai, fa'atatau i le faiga lava e tasi sa fa'aogaina i le Ref. 31.A koluma uʻamea uʻamea (faataata-laina, 100 × 1.8 mm id) ma se faʻapipiʻi faʻapipiʻi o loʻo i ai se 1 µm frit na faʻafesoʻotaʻi i se packer slurry (Alltech Deerfield, IL, ISA). Saunia se slurry vaega tumau e ala i le taofia o le 150 mg o le vaega tumau i le 1.2 mL faʻaaogaina le methanol o loʻo faʻaaogaina e Methanol. slurry solvent faapea foi ma le solvent propelling. Faatumu le koluma sequentially e ala i le faaaogaina o le mamafa o le 100 MP mo le 10 minute, 80 MP mo 15 minute, ma le 60 MP mo minute 30. I le taimi o le teuina, sa faʻaaogaina le vibration faʻainisinia ma lua GC koluma shakers (Alltech, Deerfield, IL, IL, ISA) e mautinoa le faʻamavaeina o le toniga o le koluma. e puipuia ai so'o se mea leaga i totonu o le koluma. Aveese le koluma mai le vaega fa'apipi'i slurry ma fa'afeso'ota'i se isi mea fa'apipi'i i le inlet ma le LC system e siaki ai lona fa'atinoga.
O se pamu LC (10AD Shimadzu, Iapani), injector (Valco (USA) C14 W.05) ma 50nL tui matasele, membrane degasser (Shimadzu DGU-14A), UV-VIS faamalama capillary na fausia Faapitoa μLC masini detector (UV-2075) ma tioata-laina microcolumns faʻapipiʻi faʻaitiitiga puʻupuʻu ma koluma faʻapipiʻi faʻapipiʻi. broadening.A maeʻa le afifiina, capillaries (50 μm id 365 ma faʻaitiitia le faʻapipiʻi faʻapipiʻi (50 μm) na faʻapipiʻiina i le 1 / 16 ″ outlet o le faʻaitiitiga faʻatasi. O le aoina o faʻamaumauga ma le faʻaogaina o le chromatographic na faia i le faʻaogaina o le Multichro 2000 software. Mataʻituina i le 254 nmtographic Analytes na faʻataʻitaʻiina mo le suʻesuʻeina o le 254 nm. (Northampton, MA).
Albumin mai serum tagata, lyophilized paʻu, ≥ 96% (agarose gel electrophoresis) 3 mg faʻafefiloi ma trypsin (1.5 mg), 4.0 M urea (1 mL), ma le 0.2 M ammonium bicarbonate (1 mL). O le vaifofo na faʻaosoina mo le 10 minute ma teuina i totonu o se vai taele i le 67 ° C ma le 67 ° C. 0.1% TFA. Filifili le vaifofo ma teu i lalo ole 4 °C.
O le vavaeeseina o faʻafefiloi peptide ma HSA trypsin digests na iloiloina eseese i luga o koluma PMP. Siaki le vavaeeseina o le paluga peptide ma le trypsin digest o le HSA e le PMP koluma ma faʻatusatusa iʻuga i le koluma Ascentis Express RP-Amide. O le numera o le plate theoretical e fuafuaina e pei ona taua i lalo:
O ata SEM o mea'ai silika e leai ni mea'ai ma fasimea silika fa'apipi'i ligand o lo'o fa'aalia i le ATA. 2 .SEM ata o fasi silica e leai ni mea (A, B) o loʻo faʻaalia ai, e ese mai i a tatou suʻesuʻega talu ai, o nei vaega o loʻo faʻataʻamilomilo lea e faʻaumiumi ai meaʻai poʻo le i ai o se faʻataʻitaʻiga e le masani ai.
Fa'ata'ita'iina o ata fa'aeletonika fa'aeletonika o ni vaega o le silica (A, B) ma vaega o le silica fa'atasi (C, D).
O le tufatufaina o le tele o vaega o le silica e leai ni mea ma ligand-bonded silica particles o loʻo faʻaalia i le Ata 3 (A) . Volume-based particle size distribution curves na faʻaalia ai le tele o le silica particles na faʻateleina ina ua maeʻa le fesuiaiga o vailaʻau (Fig. 3A). μm, faʻatusatusa i la matou suʻesuʻega talu ai ma le ad (0.5) taua o le 3.05 μm (polystyrene-bound silica particles) 34. O lenei vaega sa i ai se vaapiapi lapopoa o le tufatufaina atu i le matou suʻesuʻega talu ai ona o le fesuiaiga o fua faatatau o le PEG, urea, TMOS, ma le acetic acid i le faʻafefiloi o tali. O le fa'aogaina o luga ole vaega ole silica ile styrene na'o le tu'uina o se polystyrene layer (0.97 µm) i luga o le silica surface, ae o le PMP ole vaega ole mafiafia ole 1.38 μm.
Tufatufaina o le lapopoa (A) ma le lapopoa o le pore (B) o vaega o le silica e leai ni mea ma vaega o le silica ua fusifusia i ligand.
O le tele o pore, pore voluma ma le vaega o luga o le silica particles o le suʻesuʻega o loʻo tuʻuina atu i le Table 1 (B). e 69 ina ua uma le fesuiaiga o vailaau, e pei ona faaalia i le Table 1 (B), ma le suiga o le pupuni o loʻo faʻaalia i le Ata 3 (B) .Faʻapea foʻi, o le pore voluma o le silica particles na faʻaitiitia mai le 0.67 i le 0.58 cm3 / g ina ua maeʻa le fesuiaiga o vailaʻau. m2 / g) . E pei ona faʻaalia i le Laulau 1 (B), o le vaega o luga (m2 / g) o le silica particles na faʻaititia foi mai le 116 m2 / g i le 105 m2 / g pe a maeʻa le faʻaogaina o vailaʻau.
O faʻaiʻuga o suʻesuʻega elemene o le vaega tumau o loʻo faʻaalia i le Laulau 2. O le utaina o le kaponi o le vaega tumau o loʻo i ai nei o le 6.35%, lea e maualalo ifo nai lo le utaina o le carbon o la tatou suʻesuʻega talu ai (polystyrene bonded silica particles, 7.93%35 ma 10.21%, i le faasologa) 42. O le uta kaponi o le taimi nei o loʻo faʻapipiʻiina i le vaega faʻamautu o le SP e maualalo, o le faʻaopoopoina o le SP i le taimi nei o loʻo faʻaopoopoina i le vaega faʻamautu o le po, o loʻo i ai nei. ligands e pei o le phenylmaleimide-methylvinylisocyanate (PCMP) ma le 4-hydroxy-TEMPO na faʻaaogaina. O le pasene o le mamafa o le nitrogen o le vaega tumau o loʻo i ai nei o le 2.21%, faʻatusatusa i le 0.1735 ma le 0.85% i le mamafa o le nitrogen i suʻesuʻega muamua. utaina o oloa (4) ma le (5) e 2.7% ma 2.9%, i le faasologa, ae o le uta carbon o le oloa mulimuli (6) o le 6.35%, e pei ona faaalia i le Laulau 2. O le paʻu mamafa na siakiina i le PMP tulaga tumau, ma o le TGA curve o loʻo faʻaalia i le Ata 4. O le TGA curve o loʻo faʻaalia ai le gau o le mamafa o le 8.6%, lea o loʻo i ai i le 8.6% le mamafa o le mamafa o le 8.6%. ligands e le gata o le C ae faʻapea foʻi le N, O, ma le H.
O le phenylmaleimide-methylvinylisocyanate ligand na filifilia mo le suia o luga o vaega silica ona o loʻo i ai polar phenylmaleimide vaega ma vinylisocyanate vaega. Vinyl isocyanate vaega e mafai ona toe tali atu i le styrene e ola radical polymerization.O le mafuaaga lona lua o le faaofiina o se vaega o loʻo i ai se fegalegaleaiga feololo ma le analyte ma e leai se vaega malosi o le electrostatic, ma e leai se vaega malosi. phenylmaleimide moiety e leai se totogi mama i pH masani.O le polarity o le vaega tumau e mafai ona pulea e le aofaiga sili ona lelei o styrene ma le taimi tali o free radical polymerization.O le laasaga mulimuli o le tali (free-radical polymerization) e taua tele ma e mafai ona suia le polarity o le vaega tumau. Elemental suʻesuʻega sa faia e siaki le kaponi uta o le taimi faʻanofoina sa matauina le faateleina o le taimi styrene. utaina o le vaega tumau ma vice versa.SPs saunia ma concentrations eseese o styrene e eseese carbon loadings.Toe, uta nei tulaga tulaga i koluma uʻamea stainless ma siaki latou faatinoga chromatographic (selectivity, iugafono, N tau, ma isi.) E faavae i luga o nei suesuega, na filifilia se optimized formulation e saunia le PMP stationary vaega ma faamautinoa lelei pulea analy poteetention.
E lima faʻafefiloi peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) na iloiloina foʻi e faʻaaoga ai se koluma PMP e faʻaaoga ai se vaega feaveaʻi; 60/40 (v / v) acetonitrile / vai (0.1% TFA) i le tafe o le 80 μL / min. I lalo o tulaga elution sili ona lelei, o le numera o le ipu (N) i le koluma (100 × 1.8 mm id) o le 20,000 ± 100 (200,000 T 3 / m²). ma le chromatograms o loʻo faʻaalia i le Ata 5A. Faʻamatalaga vave i luga o se koluma PMP i le maualuga o le tafe (700 μL / min), e lima peptides na faʻafefe i totonu o le minute e tasi, N taua tele na lelei, 13,500 ± 330 i le koluma (100 × 1.8 mm id), E fetaui ma le 00000000000000000000000000000000000000000000000000000000000000/2008. koluma tutusa tutusa (100 × 1.8 mm id) na faʻapipiʻiina i ni vaega eseese se tolu o le PMP tulaga tumau e siaki ai le toe faʻaleleia. tau maualalo %RSD, pei ona fa'aalia i le Laulau 3.
Tuueseesega o le paluga peptide i luga ole koluma PMP (B) ma le koluma Ascentis Express RP-Amide (A); vaega feaveai 60/40 ACN/H2O (TFA 0.1%), PMP koluma fua (100 × 1.8 mm id); au'ili'ili Le fa'asologa o le fa'aogaina o mea fa'afefiloi: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) ma le 5 (leucine) acid enkephalin)).
O se koluma PMP (100 × 1.8 mm id) na iloiloina mo le vavaeeseina o tryptic digests o le albumin serum tagata i le maualuga o le faʻaogaina o le vai chromatography. O le chromatogram i le Ata 6 o loʻo faʻaalia ai o le faʻataʻitaʻiga o loʻo vaʻavaʻavaʻavaʻa lelei ma o le fofo e lelei tele. HSA digests sa suʻesuʻeina e faʻaaoga ai le tafe o le 100 µL / min. TFA. E pei ona faʻaalia i le chromatogram (Ata 6), o le HSA digestion ua vaevaeina i 17 pito i luga e fetaui ma 17 peptides. O le vavaeeseina lelei o tumutumu taʻitasi i le HSA digest sa fuafuaina ma o tau o loʻo tuʻuina atu i le Laulau 5.
O se tryptic digest o le HSA (100 × 1.8 mm id) na vavaeeseina i luga o se koluma PMP; fua tafe (100 µL / min), vaega feaveai 60/40 acetonitrile / vai ma 0.1% TFA.
lea L o le umi o le koluma, η o le viscosity o le vaega feaveaʻi, ΔP o le koluma i tua o le mamafa, ma u o le saoasaoa laina o le vaega feaveaʻi. O le permeability o le PMP koluma o 2.5 × 10-14 m2, o le tafe o le tafe o le 25 μL / min, ma le 60/40 v / v / v o le PMP / 1 na faʻaaogaina le PMP. mm id) e tutusa ma le matou suʻesuʻega talu ai Ref.34. O le faʻaogaina o le koluma o loʻo faʻapipiʻiina i mea papaʻu porous o le: 1.7 × 10-15 mo le 1.3 μm particles, 3.1 × 10-15 mo le 1.7 μm particles, 5.2 × 10-15 ma le 2.5 × 2.5 μm. μm vaega 43. O le mea lea, o le permeability o le vaega PMP e tutusa ma le 5 μm vaega autu- atigi.
lea o le Wx o le mamafa o le koluma ua tumu i le chloroform, o le Wy o le mamafa o le koluma ua tumu i le methanol, ma o le ρ o le mamafa o le solvent. Densities o le methanol (ρ = 0.7866) ma le chloroform (ρ = 1.484) C18-Urea koluma 31 lea na matou suʻesuʻeina muamua o le 0.63 ma le 0.55, o lona uiga o le i ai o le urea ligands e faʻaitiitia ai le faʻaogaina o le tulaga faʻamautu. silica particles aua i le C18-ituaiga tulaga tumau o le C18 ligands o loʻo faʻapipiʻi i le silica particles e pei o laina laina, aʻo i polystyrene-type stationary phases, o le A faitele polymer layer ua faia faataamilo i ai.
Ata 7A,B o loʻo faʻaalia ai le koluma PMP (100 × 1.8 mm id) ma le koluma Ascentis Express RP-Amide (100 × 1.8 mm id) e faʻaaoga ai tulaga tutusa elution (ie, 60/40 ACN / H2O ma 0.1% TFA). ) o le fanua van Deemter. Fa'afefiloi peptide filifilia (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) na saunia i le 20 µL/ O le fua maualalo o le tafe mo koluma e lua o le 800 µL/min.O le la'ititi o le HETP fa'atatau o le 800L/min. koluma ma le koluma Ascentis Express RP-Amide e 2.6 µm ma 3.9 µm, faasologa. O tau HETP ua faailoa mai ai o le vavaeeseina lelei o le koluma PMP (100 × 1.8 mm id) e sili atu le lelei nai lo le avanoa faapisinisi Ascentis Express RP-Amide koluma (100 i × 1.8). o le faʻaitiitia o le tau o le N ma le faʻateleina o le tafe e le taua pe a faʻatusatusa i la matou suʻesuʻega talu ai. O le maualuga o le vavaeeseina o le PMP koluma (100 × 1.8 mm id) faʻatusatusa i le Ascentis Express RP-Amide koluma e faʻavae i luga o le faʻaleleia atili o foliga, lapoa, ma faiga faʻapipiʻi koluma lavelave o loʻo faʻaaogaina i le galuega o loʻo iai nei34.
(A) van Deemter plot (HETP versus mobile phase linear velocity) maua mai i le PMP column (100 × 1.8 mm id) i le 60/40 ACN/H2O ma le 0.1% TFA.(B) van Deemter plot (HETP versus mobile phase linear velocity) maua mai i le Ascentis Express RP-Amide i × 4 koluma (1000 i × 4) i le Ascentis Express RP-Amide 1. ACN/H2O ma le 0.1% TFA.
Sa saunia ma iloiloina se vaega o le polystyrene faʻapipiʻi polar mo le tuʻueseeseina o faʻafefiloi peptide synthetic ma trypsin digests o le albumin serum tagata (HAS) i le maualuga o le faʻaogaina o le vai chromatography. O le faʻatinoga o le chromatographic o koluma PMP mo paluga peptide e sili ona lelei i le vavaeeseina o le lelei ma le fofo. vaega ninii, faamaopoopoina pulea o le vaega tumau, ma koluma lavelave packing.In faaopoopo atu i le maualuga o le vavaeeseina lelei, maualalo koluma i tua o le mamafa o le tafe maualuga o le isi tulaga lelei o lenei tulaga tumau. PMP koluma faaalia reproducibility lelei ma e mafai ona faaaoga mo le auiliiliga o peptide paluga ma trypsin digestion o proteins eseese. Matou te faamoemoe e faaaoga lenei koluma mo le tuueseeseina o laau faagaioiina vai mai mea tau vailaau fa'alenatura. chromatography.I le lumanaʻi, o le a suʻesuʻeina foi koluma PMP mo le vavaeeseina o polotini ma monoclonal antibodies.
Field, JK, Euerby, MR, Lau, J., Thøgersen, H. & Petersson, P. Research on Peptide Separation Systems by Reversed Phase Chromatography Vaega I: Atinaʻeina o se Koluma Faʻamatalaga Faʻamatalaga.J. Chromatography.1603, 113–129.https://doi.org/10.1016/j.chroma.2019.05.038 (2019).
Gomez, B. et al.Improved active peptides fuafuaina mo le togafitia o faʻamaʻi pipisi.Biotechnology.Advanced.36(2), 415-429.https://doi.org/10.1016/j.biotechadv.2018.01.004 (2018).
Vlieghe, P., Lisowski, V., Martinez, J. & Khrestchatisky, M. Synthetic therapeutic peptides: science and the market.drug discovery.15 (1-2) today, 40-56.https://doi.org/10.1016/j.drudis.2009.10.009 (2009.10.009).
Xie, F., Smith, RD & Shen, Y. Advanced Proteomic Liquid Chromatography.J. Chromatography.A 1261, 78–90 (2012).
Liu, W. et al.Advanced liquid chromatography-mass spectrometry e mafai ai ona tu'ufa'atasia lautele fa'atatau metabolomics ma proteomics.anus.Chim.Acta 1069, 89-97 (2019).
Chesnut, SM & Salisbury, JJ O le matafaioi a le UHPLC i le atinaeina o fualaau faasaina.J. Sep. Sci.30(8), 1183-1190 (2007).
Wu, N. & Clausen, AM Vaega fa'avae ma fa'atinoga ole chromatography vai fa'aoso ultrahigh mo le vavae'ese vave.J. Sep. Sci.30(8), 1167-1182.https://doi.org/10.1002/jssc.200700026 (2007).
Wren, SA & Tchelitcheff, P. Fa'aaogāina ole chromatography vai fa'atino ultra-maualuga ile atina'eina o vaila'au.J. Chromatography.1119(1-2), 140-146.https://doi.org/10.1016/j.chroma.2006.02.052 (2006).
Gu, H. et al.Monolithic macroporous hydrogels saunia mai le suauu-i-vai emulsions vaega maualuga totonu mo le faamamaina lelei o enteroviruses.Chemical.Britain.J. 401, 126051 (2020).
Shi, Y., Xiang, R., Horváth, C. & Wilkins, JA O le matafaioi a le chromatography vai i proteomics.J. Chromatography.A 1053(1-2), 27-36 (2004).
Fekete, S., Veuthey, J.-L.& Guillarme, D. Fa'asolo a'e aga'i i le fa'aliliuina-vaega vai chromatography vaeluaga o peptides therapeutic ma polotini: theory and applications.J. Pharmacy.Biomedical Science.anus.69, 9-27 (2012).
Gilar, M., Olivova, P., Daly, AE & Gebler, JC Vaeluaga lua-dimensional o peptides e faʻaaoga ai le RP-RP-HPLC system e faʻaaoga ai tau pH eseese i le vaeluaga muamua ma le lua.J. Sep. Sci.28(14), 1694-1703 (2005).
Feletti, S. et al. Na su'esu'eina le uiga fe'avea'i tele ma le fa'agaioiga o koluma chromatographic maualuga fa'atumuina i le C18 sub-2 μm atoa ma papa'u.J. Sep. Sci.43 (9-10), 1737-1745 (2020).
Piovesana, S. et al.Tula'iga lata mai ma lu'itau au'ili'ili i le tu'uesea, fa'ailoaina ma le fa'amaoniaina o laau toto bioactive peptides.anus.biological anus.Chemical.410(15), 3425-3444.https://doi.org/10.1007/s00216-02018-0 (2).
Mueller, JB et al.O le laufanua proteomic o le malo o le ola.Natura 582(7813), 592-596.https://doi.org/10.1038/s41586-020-2402-x (2020).
DeLuca, C. et al. Downstream process of therapeutic peptides by preparative liquid chromatography.Molecule (Basel, Suiselani) 26(15), 4688(2021).
Yang, Y. & Geng, X. Fa'afefiloi-mode chromatography ma lona fa'aogaina i biopolymers.J. Chromatography.A 1218(49), 8813–8825 (2011).
Zhao, G., Dong, X.-Y.& Sun, Y. Ligands mo fa'afefiloi-mode protein chromatography: mataupu faavae, uiga, ma le mamanu.J. Biotechnology.144(1), 3-11 (2009).