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Porous silica particles anakonzedwa ndi Sol-gel osakaniza njira ndi zosintha zina kupeza macroporous particles.These particles anali derivatized ndi reversible Kuwonjezera kugawika unyolo kutengerapo (RAFT) polymerization ndi N-phenylmaleimide-methylvinylisocyanate (PMI) ndi styrene kukonzekera N-phenylmaleide polyboride polyboride (MPI) polymerization (N-phenylmaleimide). zitsulo zosapanga dzimbiri (100 × 1.8 mm id) zinali zodzaza ndi slurry kulongedza.Kuyesedwa kwa PMP ndime kulekanitsidwa kwa peptide osakaniza opangidwa ndi ma peptides asanu (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Gly-Ser-Ser-Arg-Ser-Arg) ntchito digestion of human serum albumin (HAS) .Pansi pazikhalidwe zabwino kwambiri za elution, chiwerengero cha mbale zowerengera za osakaniza a peptide ndizokwera kwambiri mpaka 280,000 mbale / m².
M'zaka zaposachedwa, msika wa biopharmaceutical wakhala msika womwe ukukula padziko lonse lapansi ndikuwonjezeka kwakukulu pamsika. Makhalidwe a mapuloteni mumadzimadzi a m'thupi, minofu ndi maselo ndi ntchito yovuta kwambiri chifukwa cha kuchuluka kwa mitundu yomwe ingathe kuzindikirika mu chitsanzo chimodzi.Ngakhale kuti misa spectrometry ndi chida chothandiza pa peptide ndi mapuloteni sequencing, ngati zitsanzo zoterezi jekeseni mu spectrometer misa mu chiphaso chimodzi, kulekana sikungakhale kwabwino. kuchepetsa chiwerengero cha analytes kulowa spectrometer misa pa nthawi anapatsidwa4,5,6. Komanso, pa madzi gawo kulekana, analytes akhoza lolunjika pa zigawo yopapatiza, potero tcheru kusanthula izi ndi kusintha MS kuzindikira sensitivity.Liquid chromatography (LC) wapita patsogolo kwambiri pa zaka khumi zapitazi ndipo wakhala njira wotchuka mu proteomic,8,17,17.
Reversed-phase liquid chromatography (RP-LC) amagwiritsidwa ntchito kwambiri kuyeretsa ndi kulekanitsa zosakaniza za peptide pogwiritsa ntchito octadecyl-modified silica (ODS) monga gawo lokhazikika11,12,13. magawo osasunthika amafunikira kusanthula ma peptides ndi mapuloteni okhala ndi polar ndi omwe si a polar moieties kuti agwirizane ndi kusunga ma analytes awa16.Mixed-mode chromatography, yomwe imapereka ma multimodal interactions, ikhoza kukhala njira ina ya RP-LC yolekanitsa ma peptides, mapuloteni, ndi zosakaniza zina zovuta. Kupatukana kwa peptide ndi mapuloteni17,18,19,20,21.Mixed-mode stationary phases (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) ndi yoyenera kupatukana kwa peptide ndi mapuloteni chifukwa cha kukhalapo kwa magulu onse a polar ndi omwe si a polar22,23,24,25,26,26,26,26,28 Magulu ogwirizana kwambiri a polar amawonetsa mphamvu zolekanitsa zabwino komanso kusankha kwapadera kwa ma analyte a polar ndi omwe si a polar, popeza kulekana kumadalira kuyanjana pakati pa analyte ndi gawo loyima. Kuyanjana kwa Multimodal 29, 30, 31, 32.Posachedwapa, Zhang et al. 30 anakonza dodecyl-zitha polyamine stationary gawo ndi bwinobwino analekanitsa hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, ndi angapo analytes.The polar intercalator ali onse polar ndi sanali polar magulu, kotero angagwiritsidwe ntchito kulekanitsa peptides ndi mapuloteni kuti onse hydrophobic ndi hydrophilic-monga hydrophilic mouembed (polar-polar-polar). mizati ya C18 yophatikizidwa ndi amide) imapezeka pamalonda pansi pa dzina la malonda la Ascentis Express RP-Amide, koma mizatiyi imagwiritsidwa ntchito pofufuza amine 33 okha.
Pakafukufuku wamakono, polar-embedded stationary phase (N-phenylmaleimide-embedded polystyrene) inakonzedwa ndikuwunikiridwa kuti ikhale yolekanitsa ma peptides ndi trypsin digests ya HSA. Gawo lokhazikika linakonzedwa pogwiritsa ntchito njira yotsatirayi.Tinthu tating'onoting'ono ta silika tinakonzedwa molingana ndi ndondomeko yomwe inaperekedwa m'buku lathu lapitalo ndi zosintha zina za glycerea, polyethylene protocol, TMOS, madzi acetic acid anasinthidwa kukonzekera silika particles ndi lalikulu pore size.Chachiwiri, latsopano ligand, phenylmaleimide-methyl vinyl isocyanate, anali apanga ndi ntchito derivatize silika particles kukonzekera polar ophatikizidwa stationary gawo. scheme.The mzati kulongedza amathandizidwa ndi makina kugwedera kuonetsetsa homogeneous bedi aumbike mkati mwa column.Valuate odzaza ndime kulekana kwa peptide osakaniza wopangidwa asanu peptides; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) ndi Trypsin digest ya human serum albumin (HAS) .Kusakaniza kwa peptide ndi trypsin digest ya HSA inawonedwa kuti ikulekanitsidwa ndi kusamvana kwabwino komanso kothandiza. RP-Amide column.Ma peptide onse ndi mapuloteni adawonedwa kuti amathetsedwa bwino komanso akugwira ntchito bwino pazamba za PMP, zomwe zinali zogwira mtima kwambiri kuposa gawo la 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, xyPOYL, MC-TEM, Perzol oxide, MCTEM (BPO), HPLC Grade Acetonitrile (ACN), Methanol, 2-Propanol, ndi Acetone Ogula kuchokera ku Sigma-Aldrich (St. Louis, MO, USA).
Chisakanizo cha urea (8 g), polyethylene glycol (8 g), ndi 8 mL wa 0.01 N acetic acid analimbikitsidwa kwa mphindi 10, ndiyeno 24 mL wa TMOS anawonjezedwa pamenepo pansi pa ayezi-ozizira mikhalidwe. The osakaniza anachita unatenthedwa pa 40 ° C kwa maola 6 ndiyeno pa 120 ° C kwa maola 8 anathira ndi zitsulo zosapanga dzimbiri. zinthu zotsalira zinauma pa 70 ° C kwa maola 12. Zouma zofewa zouma zinali zosalala pansi mu ng'anjo ndi calcined pa 550 ° C kwa maola 12. Magulu atatu adakonzedwa ndipo amadziwika kuti ayang'ane kuberekana mu kukula kwa tinthu, kukula kwa pore ndi pamwamba.
Pogwiritsa ntchito kusinthidwa kwa silika particles ndi pre-synthesized ligand phenylmaleimide-methylvinylisocyanate (PCMP) yotsatiridwa ndi ma polima a radial ndi styrene, gulu la polar lomwe lili ndi gulu linakonzedwa. Zosasunthika gawo la ma aggregates ndi unyolo wa polystyrene. Njira yokonzekera ikufotokozedwa pansipa.
N-phenylmaleimide (200 mg) ndi methyl vinyl isocyanate (100 mg) anasungunuka mu toluene youma, ndipo 0.1 mL ya 2,2'-azoisobutyronitrile (AIBN) anawonjezedwa mu botolo la reaction kuti akonze phenylmaleimide-methyl vinyl isocyanate copolymer pa maola 6 osakaniza, 6PM ° C. zosefedwa ndi zouma mu uvuni pa 40 ° C kwa 3 hours.
Zouma silika particles (2 g) anali omwazika mu youma toluene (100 mL), analimbikitsa ndi sonicated mu 500 mL wozungulira pansi botolo kwa 10 min.PMCP (10 mg) anali kusungunuka toluene ndipo anawonjezera dropwise kuti anachita botolo kudzera dontho funnel.The osakaniza anali refluxed pa 100 osakaniza ndi zouma ndi zouma maola 100 ° C zouma ndi zouma 100 ° C wosambitsidwa. 60 ° C kwa maola 3. Kenaka, particles za silika za PMCP (100 g) zinasungunuka mu toluene (200 ml) ndi 4-hydroxy-TEMPO (2 mL) zinawonjezeredwa pamaso pa 100 µL ya dibutyltin dilaurate monga chothandizira.Kusakaniza kunasunthidwa kwa maola 50 ° C ndi 50 ° C kwa maola 50, 3 ° C inasinthidwa ndi 50 ° C. maola.
Styrene (1 mL), benzoyl peroxide BPO (0.5 mL), ndi TEMPO-PMCP-attached silica particles (1.5 g) anamwazikana mu toluene ndi kutsukidwa ndi nayitrogeni.The polymerization wa styrene unkachitika pa 100 ° C kwa maola 12. The chifukwa mankhwala anatsukidwa ndi methanol 60 ° C usiku zonse zowuma chiwembu ndi zouma. 1.
Zitsanzozo zinachotsedwa pa 393 K kwa ola la 1 kuti zipeze kupanikizika kotsalira kwa 10-3 Torr. Kuchuluka kwa N2 kudsorbed pamtundu wa P / P0 = 0.99 kunagwiritsidwa ntchito kuti mudziwe kuchuluka kwa pore. Japan) .Zitsanzo zouma (zopanda silika ndi ligand-bonded silica particles) zinayikidwa pa aluminiyamu ndime pogwiritsa ntchito zomatira mpweya tepi.Gold anali yokutidwa pa zitsanzo pogwiritsa ntchito sputter coater Q150T, ndi 5 nm Au wosanjikiza anaikidwa pa zitsanzo.Izi bwino ndondomeko Mwachangu ntchito otsika voteji ndi kupereka mbewu zabwino, USA Wamphamvu ndi Eleron, USA. Kung'anima EA1112 elemental analyzer ankagwiritsidwa ntchito pofufuza zinthu. A Malvern (Worcestershire, UK) Mastersizer 2000 tinthu kukula analyzer ntchito kupeza tinthu kukula kugawa. Naked silika particles ndi ligand-boma silika particles (5 mg aliyense) anali omwazika mu 5 mL wa isopropanol, anaika pa 5 mL wa isopropanol, sonicated pa 5 min, sonicated pa optical, sonicated, sonicated, 5 mg wa silika. benchi ya Mastersizer.Kusanthula kwa Thermogravimetric kunachitidwa pa mlingo wa 5 °C pamphindi pa kutentha kwa 30 mpaka 800 °C.
Zitsulo zosapanga dzimbiri zokhala ndi mizere yagalasi zokhala ndi mizere yopapatiza (id 100 × 1.8 mm) zidapakidwa pogwiritsa ntchito njira yolongedza slurry, kugwiritsa ntchito njira yomweyi yomwe imagwiritsidwa ntchito mu Ref. 31.Chigawo chachitsulo chosapanga dzimbiri (chokhala ndi magalasi, 100 × 1.8 mm id) chokhala ndi chotuluka chokhala ndi 1 µm frit chinalumikizidwa ndi paketi ya slurry (Alltech Deerfield, IL, USA). Konzani slurry ya gawo lokhazikika poyimitsa 150 mg ya gawo lokhazikika la mL ndi kutumiza gawo lokhazikika la menotha pa menotha 1. ankagwiritsidwa ntchito ngati zosungunulira slurry komanso propelling zosungunulira.Tsitsani ndime sequentially ntchito mavuto a MP 100 kwa mphindi 10, 80 MP kwa mphindi 15, ndi 60 MP kwa mphindi 30. Pakulongedza katundu, makina kugwedera anagwiritsidwa ntchito ndi awiri GC ndime shakers (Alltech, Deerfield) kuonetsetsa yunifolomu IL, Deerfield kuonetsetsa yunifolomu yunifolomu. ndikumasulani kupanikizika pang'onopang'ono kuti muteteze kuwonongeka kulikonse mkati mwa chigawocho.Chotsani mzati kuchokera ku slurry packing unit ndikugwirizanitsa kuyenerera kwina kolowera ndi ku LC dongosolo kuti muwone momwe ntchito yake ikuyendera.
Pampu ya LC (10AD Shimadzu, Japan), injector (Valco (USA) C14 W.05) yokhala ndi 50nL jakisoni loop, membrane degasser (Shimadzu DGU-14A), UV-VIS capillary window inamangidwa Special µLC device detector (UV-2075) ndi magalasi olumikiza ma microcolum ofupikitsa kwambiri ndi magalasi ang'onoang'ono. Pambuyo pakulongedza, ma capillaries (50 μm id 365 ndi kuchepetsa ma capillaries (50 μm) anaikidwa pa 1/16 ″ potulutsira mgwirizano wochepetsera. kufufuzidwa ndi OriginPro8 (Northampton, MA).
Albumin yochokera ku seramu yaumunthu, ufa wa lyophilized, ≥ 96% (agarose gel electrophoresis) 3 mg wothira trypsin (1.5 mg), 4.0 M urea (1 mL), ndi 0.2 M ammonium bicarbonate (1 mL). Njira yothetserayi inalimbikitsidwa kwa mphindi 10 ndikusungidwa m'madzi osamba kwa maola 6 ndi 67 ° C, kenako 67 m'madzi osamba, ° C 0.1% TFA.Sefani yankho ndi kusunga pansi pa 4 °C.
Kupatukana kwa zosakaniza za peptide ndi ma HSA trypsin digests adawunikidwa padera pazaza za PMP. Onani kulekanitsidwa kwa peptide osakaniza ndi trypsin digest ya HSA ndi ndime ya PMP ndikuyerekeza zotsatira ndi gawo la Ascentis Express RP-Amide.
Zithunzi za SEM za tinthu ting'onoting'ono ta silika ndi tinthu tating'ono ta silika timawonetsedwa mu FIG. 2 .SEM zithunzi za bare silica particles (A, B) zimasonyeza kuti, mosiyana ndi maphunziro athu apitalo, tinthu tating'onoting'ono tating'onoting'ono tating'onoting'ono tating'onoting'ono tating'onoting'ono tating'onoting'ono tating'onoting'ono tating'onoting'ono tating'ono tating'ono tating'ono tating'ono tating'onoting'ono ta silika. silika particles.
Kusanthula zithunzi za maikulosikopu za ma elekitironi za tinthu tating'ono ta silika (A, B) ndi tinthu tating'ono ta silika (C, D).
The tinthu kukula magawidwe anabala silika particles ndi ligand-boma silika particles akuwonetsedwa Chithunzi 3 (A) Volume-based particle size distribution curves anasonyeza kuti kukula kwa silika particles kunawonjezeka pambuyo pa kusinthidwa kwa mankhwala (mkuyu 3A) . d (0.5), wa PMP ndi 3.36 μm, poyerekeza ndi phunziro lathu lapitalo ndi malonda (0.5) mtengo wa 3.05 μm (polystyrene-womangidwa silika particles) 34. Gululi linali ndi gawo lochepa la tinthu tating'onoting'ono poyerekezera ndi phunziro lathu lapitalo chifukwa cha kusiyana kwa PEG, urea, TMOS mu tinthu tating'onoting'ono tomwe timapanga tinthu tating'onoting'ono. kuti wa polystyrene-womangidwa silika tinthu gawo tidaphunzira kale.Izi zikutanthauza kuti pamwamba functionalization wa silika particles ndi styrene yekha waikamo polystyrene wosanjikiza (0.97 µm) pa silika pamwamba, pamene mu gawo PMP wosanjikiza makulidwe anali 1.38 µm.
Tinthu kukula kugawa (A) ndi pore kukula kugawa (B) anabala silika particles ndi ligand-womangidwa silica particles.
Kukula kwa pore, pore volume ndi pamwamba pa silika particles ya kafukufuku wamakono akuperekedwa mu Table 1 (B) .The PSD mbiri za bare silica particles ndi ligand-bonded silica particles akuwonetsedwa mu Chithunzi 3 (B) . imasonyeza kuti kukula kwa pore kumachepa ndi 69 pambuyo pa kusintha kwa mankhwala, monga momwe tawonetsera mu Table 1 (B), ndipo kusintha kwa mphutsi kumasonyezedwa mu Chithunzi 3 (B) . Mofananamo, pore voliyumu ya silika particles inatsika kuchokera ku 0.67 mpaka 0.58 cm3 / g pambuyo pa kusintha kwa mankhwala. phunziro lathu lapitalo (124 m2 / g) . Monga momwe tawonetsera mu Table 1 (B), pamtunda (m2 / g) wa silika particles adatsikanso kuchokera ku 116 m2 / g mpaka 105 m2 / g pambuyo pa kusintha kwa mankhwala.
Zotsatira za kusanthula kwapang'onopang'ono kwa gawo loyima zikuwonetsedwa mu Table 2. The carbon loading of the current stationary phase ndi 6.35%, yomwe ili yotsika kusiyana ndi carbon loading ya phunziro lathu lapitalo (polystyrene bonded silica particles, 7.93% 35 ndi 10.21%, motero) 42. The carbon loading of the current stationary is ma ligands ena a polar monga phenylmaleimide-methylvinylisocyanate (PCMP) ndi 4-hydroxy-TEMPO anagwiritsidwa ntchito.Kulemera kwa nayitrogeni peresenti ya malo omwe alipo panopa ndi 2.21%, poyerekeza ndi 0.1735 ndi 0.85% ndi kulemera kwa nayitrogeni m'maphunziro apitalo, motsatira. phenylmaleimide. Mofananamo, carbon loadings ya mankhwala (4) ndi (5) anali 2.7% ndi 2.9%, motero, pamene carbon loading yomaliza mankhwala (6) anali 6.35%, monga taonera Table 2. Kutaya kulemera kunafufuzidwa ndi PMP stationary phase, ndipo TGA curve ikuwonetsedwa mu chithunzi cha 6. ikugwirizana bwino ndi kukweza mpweya (6.35%) chifukwa ma ligand ali ndi C komanso N, O, ndi H.
The phenylmaleimide-methylvinylisocyanate ligand inasankhidwa kuti ikhale yosinthidwa pamwamba pa silika chifukwa ili ndi magulu a polar phenylmaleimide ndi magulu a vinylisocyanate. phenylmaleimide moiety alibe ndalama pafupifupi pa pH yachibadwa. Polarity wa stationary gawo akhoza kulamulidwa ndi mulingo woyenera kuchuluka kwa styrene ndi nthawi anachita ya free radical polymerization. sitepe yomaliza ya anachita (free-radical polymerization) ndi yofunika kwambiri ndipo akhoza kusintha polarity wa stationary gawo. The carbon loading of the stationary phase and vice versa.SPs okonzedwa ndi magulu osiyanasiyana a styrene ali ndi carbon loadings yosiyana.Apanso, ikani magawo awa osasunthika muzitsulo zosapanga dzimbiri ndikuyang'ana momwe ma chromatographic amachitira (selectivity, resolution, N value, etc.)
Zosakaniza zisanu za peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) adayesedwanso pogwiritsa ntchito gawo la PMP pogwiritsa ntchito gawo la mafoni; 60/40 (v/v) acetonitrile/madzi (0.1% TFA) pakuyenda kwa 80 μL/mphindi. Pansi pa mikhalidwe yabwino kwambiri, theoretical plate number (N) pa ndime (100 × 1.8 mm id) ndi 20,000 ± 100 (mbale zitatu za N200/000000). Mizati ya PMP ndi ma chromatogram akuwonetsedwa mu Chithunzi 5A.Kusanthula mwachangu pagawo la PMP pamlingo wothamanga kwambiri (700 μL/mphindi), ma peptide asanu adachotsedwa mkati mwa mphindi imodzi, ma N anali abwino kwambiri, 13,500 ± 330 pagawo lililonse (100 × 1.8 mm id), 13 ikugwirizana / 0 (050m) 5B) Mizati itatu yofanana (100 × 1.8 mm id) inali yodzaza ndi magawo atatu osiyana a PMP yoyima kuti ayang'ane kuberekana. Kusanthula kwa analyte pa ndime iliyonse kunalembedwa pogwiritsa ntchito mikhalidwe yabwino kwambiri ya elution ndi chiwerengero cha mbale zongopeka N ndi nthawi yosungiramo kuti alekanitse chisakanizo chofanana cha mayeso pazambiri za PMP. kupangidwanso kwa gawo la PMP kumagwirizana bwino ndi zotsika kwambiri za %RSD, monga zikuwonetsedwa mu Gulu 3.
Kupatukana kwa peptide osakaniza pa PMP ndime (B) ndi Ascentis Express RP-Amide ndime (A); mobile phase 60/40 ACN/H2O (TFA 0.1%), PMP mizere mizere (100 × 1.8 mm id); analytical The elution dongosolo la mankhwala: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) ndi 5 (leucine) asidi enkephalin)).
Chidutswa cha PMP (ID 100 × 1.8 mm) chinawunikidwa kuti chilekanitse ma tryptic digests a seramu ya anthu albumin mu chromatography yamadzimadzi yapamwamba kwambiri. Chromatogram mu Chithunzi 6 ikuwonetsa kuti chitsanzocho ndi cholekanitsidwa bwino ndipo chigamulo chake ndichabwino kwambiri. Kugaya kwa HSA kunawunikidwa pogwiritsa ntchito mayendedwe a L/7min/3 µ acetonitrile/madzi ndi 0.1% TFA.Monga momwe ziwonetsedwera mu chromatogram (Chithunzi 6), chimbudzi cha HSA chagawanika kukhala nsonga za 17 zogwirizana ndi ma peptide 17. Kusiyanitsa kwapamwamba pa chiwongoladzanja chilichonse mu HSA digest kunawerengedwa ndipo zikhalidwe zimaperekedwa mu Table 5.
Kugaya kwa tryptic kwa HSA (100 × 1.8 mm id) kunapatulidwa pagawo la PMP; kuthamanga (100 µL/mphindi), gawo la m'manja 60/40 acetonitrile/madzi ndi 0.1% TFA.
kumene L ndi kutalika kwa gawo, η ndi kukhuthala kwa gawo la mafoni, ΔP ndi kukakamiza kumbuyo kwa mzere, ndipo u ndi liwiro laling'ono la gawo la mafoni. 1.8 mm id) inali yofanana ndi ya phunziro lathu lapitalo Ref.34.The permeability ya mzati yodzaza ndi tinthu tating'onoting'ono tating'onoting'ono tating'onoting'ono tating'onoting'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono tating'ono ta 1.7 × 10-15 kwa 1.3 μm particles, 3.1 × 10-15 kwa 1.7 μm tinthu tating'onoting'ono, 5.0-1 × 2 × 2 × 2 5.2 × 1. 2.6 μm particles Kwa 5 μm particles 43.Choncho, kutsekemera kwa gawo la PMP kuli kofanana ndi 5 μm core-shell particles.
kumene Wx ndi kulemera kwa ndime yodzaza ndi chloroform, Wy ndi kulemera kwa mzati wodzaza ndi methanol, ndipo ρ ndi kachulukidwe wa zosungunulira. Kachulukidwe wa methanol (ρ = 0.7866) ndi chloroform (ρ = 1.484).Kuchuluka kwa porosity ya SILICA PARTICLES 108 × 10 × 10 mm i. ndi C18-Urea mizati 31 kuti tinaphunzira kale anali 0.63 ndi 0.55, motero.Izi zikutanthauza kuti kukhalapo kwa urea ligands amachepetsa permeability wa stationary gawo.Kumbali ina, porosity okwana a PMP ndime (100 × 1.8 mm id) ndi 0.60 m'munsi mwa mizati ya PMP ndi m'munsi mwake. C18-bonded silica particles chifukwa mu C18-mtundu woyima magawo C18 ligands amamangiriridwa ku tinthu silika ngati unyolo mzere, pamene polystyrene-mtundu wa stationary magawo, A wokhuthala polima wosanjikiza amapangidwa mozungulira.
Chithunzi 7A,B chikuwonetsa ndime ya PMP (100 × 1.8 mm id) ndi Ascentis Express RP-Amide column (100 × 1.8 mm id) pogwiritsa ntchito mikhalidwe yofananira (ie, 60/40 ACN/H2O ndi 0.1% TFA). ) ya chiwembu cha van Deemter. Zosakaniza zosankhidwa za peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) zinakonzedwa mu 20 µL/ Kutsika kochepa kwa mizati yonseyi ndi 800 µL/min. The osachepera 800 HETP miyeso yothamanga pa HETP 8min ndime ndi gawo la Ascentis Express RP-Amide linali 2.6 µm ndi 3.9 µm, motsatana. Miyezo ya HETP ikuwonetsa kuti kulekanitsa bwino kwa gawo la PMP (100 × 1.8 mm id) ndikwabwinoko kuposa gawo la malonda la Ascentis Express RP-Amide (100 × id. kuti kuchepa kwa mtengo wa N ndi kuwonjezeka kwa kutuluka sikofunikira poyerekeza ndi phunziro lathu lapitalo.Kupambana kwapamwamba kolekanitsa kwa gawo la PMP (100 × 1.8 mm id) poyerekeza ndi gawo la Ascentis Express RP-Amide limachokera ku kusintha kwa tinthu tating'ono, kukula, ndi ndondomeko zovuta zonyamula mizere zomwe zimagwiritsidwa ntchito pakali pano34.
(A) van Deemter plot (HETP versus mobile phase linear velocity) yopezedwa pogwiritsa ntchito ndondomeko ya PMP (100 × 1.8 mm id) mu 60/40 ACN/H2O ndi 0.1% TFA.(B) van Deemter plot (HETP versus mobile phase linear velocity) yopezedwa pogwiritsa ntchito Ascent-Ascentis 1 × 1 RP-Ascentis Express 60/40 ACN/H2O yokhala ndi 0.1% TFA.
Gawo lokhazikika la polar-embedded polystyrene stationary linakonzedwa ndikuwunikidwa kuti lilekanitse zosakaniza za peptide ndi trypsin digests ya human serum albumin (HAS) mu chromatography yamadzimadzi yogwira ntchito kwambiri. Kuwonjezera pa kulekana kwapamwamba, kutsika kwapansi kumbuyo kwazitsulo zothamanga kwambiri ndi mwayi wina wa gawo losasunthika. M'tsogolomu, mizati ya PMP idzawunikidwanso pa kulekanitsa mapuloteni ndi ma antibodies a monoclonal.
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