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Ua hoʻomākaukau ʻia nā ʻāpana silica porous e ke ʻano sol-gel me kekahi mau hoʻololi e loaʻa ai nā ʻāpana macroporous. Ua hoʻokaʻawale ʻia kēia mau ʻāpana e ka polymerization reversible addition fragmentation chain transfer (RAFT) me N-phenylmaleimide-methylvinylisocyanate (PMI) a me styrene e hoʻomākaukau i ka intercalation N-phenylmaleimide o ka polystyrene (PMP) stationary phase. Ua hoʻopaʻa ʻia nā kolamu kila kila haiki (100 × 1.8 mm id) e ka slurry packing. Ua loiloi ʻia ka hoʻokaʻawale ʻana o ke kolamu PMP o kahi hui peptide i loaʻa i ʻelima mau peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) chromatographic performance) a me ka trypsin digestion o ka human serum albumin (HAS). Ma lalo o nā kūlana elution kūpono, ʻo ka helu papa theoretical o ka hui peptide he kiʻekiʻe e like me 280,000 mau papa / m². Ke hoʻohālikelike nei i ka hana hoʻokaʻawale o ke kolamu i hoʻomohala ʻia me ka Ascentis kalepa. Ma ke kolamu RP-Amide Express, ua ʻike ʻia ʻoi aku ka maikaʻi o ka hana hoʻokaʻawale o ke kolamu PMP ma mua o ke kolamu kalepa ma ke ʻano o ka pono hoʻokaʻawale a me ka hoʻonā.
I nā makahiki i hala iho nei, ua lilo ka ʻoihana biopharmaceutical i mākeke honua e hoʻonui ana me ka piʻi nui ʻana o ka māhele mākeke. Me ka ulu nui ʻana o ka ʻoihana biopharmaceutical1,2,3, makemake nui ʻia ka nānā ʻana o nā peptides a me nā protein. Ma waho aʻe o ka peptide target, ua hana ʻia kekahi mau haumia i ka wā o ka peptide synthesis, no laila e koi ana i ka hoʻomaʻemaʻe chromatographic e loaʻa ai nā peptides o ka maʻemaʻe i makemake ʻia. ʻO ka nānā ʻana a me ke ʻano o nā protein i loko o nā wai kino, nā ʻiʻo a me nā cell he hana paʻakikī loa ia ma muli o ka nui o nā ʻano hiki ke ʻike ʻia i loko o kahi laʻana hoʻokahi. ʻOiai he mea hana pono ka spectrometry mass no ka peptide a me ka protein sequencing, inā e hoʻokomo ʻia kēlā mau laʻana i loko o ka spectrometer mass i hoʻokahi hala, ʻaʻole kūpono ka hoʻokaʻawale ʻana. Hiki ke hoʻēmi ʻia kēia pilikia ma ka hoʻokō ʻana i nā hoʻokaʻawale chromatography wai (LC) ma mua o ka nānā ʻana o MS, kahi e hōʻemi ai i ka helu o nā analytes e komo ana i ka spectrometer mass i kahi manawa i hāʻawi ʻia4,5,6. Eia kekahi, i ka wā o ka hoʻokaʻawale ʻana o ka pae wai, hiki ke hoʻopili ʻia nā analytes ma nā wahi haiki, no laila e hoʻopaʻa ana i kēia mau analytes a hoʻomaikaʻi i ka ʻike ʻana o MS. Loaʻa i ka chromatography wai (LC) ua holomua nui i nā makahiki he ʻumi i hala iho nei a ua lilo i ʻenehana kaulana i ka nānā ʻana i ka proteomic7,8,9,10.
Hoʻohana nui ʻia ka chromatography wai hoʻohuli ʻia (RP-LC) no ka hoʻomaʻemaʻe ʻana a me ka hoʻokaʻawale ʻana o nā hui peptide me ka hoʻohana ʻana i ka silica i hoʻololi ʻia e ka octadecyl (ODS) ma ke ʻano he pae paʻa11,12,13. Eia nō naʻe, ʻaʻole hāʻawi nā pae paʻa RP i ka hoʻokaʻawale kūpono o nā peptides a me nā protein ma muli o ko lākou ʻano paʻakikī a me ke ʻano amphiphilic 14,15. No laila, pono nā pae paʻa i hoʻolālā kūikawā ʻia e kālailai i nā peptides a me nā protein me nā ʻāpana polar a me nā non-polar e launa pū me a mālama i kēia mau analytes16. ʻO ka chromatography mode-mixed, kahi e hāʻawi ai i nā pilina multimodal, hiki ke lilo i mea ʻē aʻe i ka RP-LC no ka hoʻokaʻawale ʻana o nā peptides, nā protein, a me nā hui paʻakikī ʻē aʻe. Ua hoʻomākaukau ʻia kekahi mau pae paʻa mixed-mode, a ua hoʻohana ʻia nā kolamu i hoʻopaʻa ʻia me kēia mau pae no ka hoʻokaʻawale ʻana o ka peptide a me ka protein17,18,19,20,21. ʻO nā pae paʻa mixed-mode (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) he kūpono no ka hoʻokaʻawale ʻana o ka peptide a me ka protein ma muli o ke alo o nā polar a me nā hui non-polar22,23,24,25,26,27,28. Pēlā nō, hōʻike nā pae paʻa intercalating polar me nā hui polar i hoʻopaʻa ʻia me ka covalently i ka mana hoʻokaʻawale maikaʻi a me ke koho kūikawā no nā analytes polar a me non-polar, ʻoiai ke hilinaʻi nei ka hoʻokaʻawale ʻana i ka pilina ma waena o ka analyte a me ka pae paʻa. Nā pilina multimodal 29, 30, 31, 32. I kēia manawa, ua hoʻomākaukau ʻo Zhang et al. 30 i kahi pae paʻa polyamine dodecyl-terminated a hoʻokaʻawale pono i nā hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, a me kekahi mau analytes ʻē aʻe. Loaʻa i ka intercalator polar nā hui polar a me non-polar, no laila hiki ke hoʻohana ʻia e hoʻokaʻawale i nā peptides a me nā protein i loaʻa nā moieties hydrophobic a me hydrophilic. Loaʻa nā kolamu polar-embedded (e.g., nā kolamu C18 amide-embedded) ma ke kālepa ma lalo o ka inoa kālepa ʻo Ascentis Express RP-Amide columns, akā hoʻohana ʻia kēia mau kolamu no ka nānā ʻana i ka amine 33 wale nō.
Ma ke aʻo ʻana o kēia manawa, ua hoʻomākaukau ʻia a loiloi ʻia kahi pae paʻa polar-embedded (N-phenylmaleimide-embedded polystyrene) no ka hoʻokaʻawale ʻana o nā peptides a me nā trypsin digests o HSA. Ua hoʻomākaukau ʻia ke kahua paʻa me ka hoʻohana ʻana i kēia hoʻolālā. Ua hoʻomākaukau ʻia nā ʻāpana silica porous e like me ke kaʻina hana i hāʻawi ʻia i kā mākou puke mua me kekahi mau hoʻololi i ka protocol hoʻomākaukau. Ua hoʻoponopono ʻia ka lakio o ka urea, polyethylene glycol (PEG), TMOS, ka wai acetic acid e hoʻomākaukau i nā ʻāpana silica me ka nui o ka pore. ʻO ka lua, ua synthesized ʻia kahi ligand hou, phenylmaleimide-methyl vinyl isocyanate, a hoʻohana ʻia e hoʻopuka i nā ʻāpana silica e hoʻomākaukau i kahi pae paʻa polar i hoʻokomo ʻia. Ua hoʻopaʻa ʻia ke kahua paʻa hopena i loko o kahi kolamu kila kila (100 × 1.8 mm id) me ka hoʻohana ʻana i ka hoʻolālā hoʻopaʻa i hoʻonui ʻia. Kōkua ʻia ka hoʻopaʻa ʻana o ke kolamu me ka haʻalulu mechanical e hōʻoia i ka hoʻokumu ʻia ʻana o kahi moena homogeneous i loko o ke kolamu. E loiloi i ka hoʻokaʻawale kolamu i hoʻopaʻa ʻia o nā hui peptide i loaʻa i ʻelima mau peptides; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) a me ka Trypsin digest o ka human serum albumin (HAS). Ua ʻike ʻia ka hui peptide a me ka trypsin digest o HSA e hoʻokaʻawale me ka hoʻonā maikaʻi a me ka pono. Ua hoʻohālikelike ʻia ka hana hoʻokaʻawale o ke kolamu PMP me ko ke kolamu Ascentis Express RP-Amide. Ua ʻike ʻia ua hoʻonā maikaʻi ʻia a kūpono hoʻi nā peptides a me nā protein ma ke kolamu PMP, ʻoi aku ka maikaʻi ma mua o ke kolamu Ascentis Express RP-Amide.
ʻO PEG (Polyethylene Glycol), Urea, Acetic Acid, Trimethoxy Orthosilicate (TMOS), Trimethyl Chlorosilane (TMCS), Trypsin, Human Serum Albumin (HSA), Ammonium Chloride, Urea, Hexane Methyldisilazane (HMDS), Methacryloyl Chloride (MC), Styrene, 4-Hydroxy-TEMPO, Benzoyl Peroxide (BPO), HPLC Grade Acetonitrile (ACN), Methanol, 2-Propanol, a me Acetone i kūʻai ʻia mai Sigma-Aldrich (St. Louis, MO, USA).
Ua hoʻoulu ʻia kahi hui ʻana o ka urea (8 g), polyethylene glycol (8 g), a me 8 mL o 0.01 N acetic acid no 10 mau minuke, a laila ua hoʻohui ʻia he 24 mL o TMOS i laila ma lalo o nā kūlana hau. Ua hoʻomehana ʻia ka hui hopena ma 40°C no 6 mau hola a laila ma 120°C no 8 mau hola i loko o kahi autoclave kila kila. Ua ninini ʻia ka wai a ua hoʻomaloʻo ʻia ke koena i koe ma 70°C no 12 mau hola. Ua wili ʻia ka nui palupalu maloʻo i loko o ka umu a ua calcined ma 550°C no 12 mau hola. Ua hoʻomākaukau ʻia ʻekolu mau ʻāpana a ua wehewehe ʻia e nānā i ka hana hou ʻana i ka nui o ka ʻāpana, ka nui o ka pore a me ka ʻili.
Ma ka hoʻololi ʻana i ka ʻili o nā ʻāpana silica me ka ligand phenylmaleimide-methylvinylisocyanate (PCMP) i hana mua ʻia a ukali ʻia e ka polymerization radial me ka styrene, ua hoʻomākaukau ʻia kahi hui polar i loaʻa ka hui. Pae paʻa no nā aggregates a me nā kaulahao polystyrene. Ua wehewehe ʻia ke kaʻina hana hoʻomākaukau ma lalo nei.
Ua hoʻoheheʻe ʻia ʻo N-phenylmaleimide (200 mg) a me ka methyl vinyl isocyanate (100 mg) i loko o ka toluene maloʻo, a ua hoʻohui ʻia he 0.1 mL o 2,2′-azoisobutyronitrile (AIBN) i loko o ka ʻōmole hana e hoʻomākaukau i ka phenylmaleimide-methyl vinyl isocyanate copolymer (PMCP). Ua hoʻomehana ʻia ka hui ʻana ma 60°C no 3 mau hola, kānana ʻia a hoʻomaloʻo ʻia i loko o ka umu ma 40°C no 3 mau hola.
Ua hoʻopuehu ʻia nā ʻāpana silica maloʻo (2 g) i loko o ka toluene maloʻo (100 mL), hoʻoulu ʻia a sonicated i loko o kahi ʻōmole lalo poepoe 500 mL no 10 min. Ua hoʻoheheʻe ʻia ka PMCP (10 mg) i loko o ka toluene a hoʻohui ʻia i ka kulu i ka ʻōmole hopena ma o kahi funnel hāʻule. Ua refluxed ʻia ka hui ʻana ma 100 ° C no 8 mau hola, kānana ʻia a holoi ʻia me ka acetone a maloʻo ʻia ma 60 ° C no 3 mau hola. A laila, ua hoʻoheheʻe ʻia nā ʻāpana silica i hoʻopaʻa ʻia e PMCP (100 g) i loko o ka toluene (200 ml) a ua hoʻohui ʻia ka 4-hydroxy-TEMPO (2 mL) i ke alo o 100 µL o dibutyltin dilaurate ma ke ʻano he catalyst. Ua hoʻoulu ʻia ka hui ʻana ma 50 ° C no 8 mau hola, kānana ʻia a maloʻo ʻia ma 50 ° C no 3 mau hola.
Ua hoʻopuehu ʻia ʻo Styrene (1 mL), benzoyl peroxide BPO (0.5 mL), a me nā ʻāpana silica i hoʻopili ʻia ʻo TEMPO-PMCP (1.5 g) i loko o toluene a ua hoʻomaʻemaʻe ʻia me ka nitrogen. Ua hana ʻia ka polymerization o ka styrene ma 100°C no 12 mau hola. Ua holoi ʻia ka huahana hopena me ka methanol a hoʻomaloʻo ʻia ma 60°C i ka pō. Ua hōʻike ʻia ke ʻano hana holoʻokoʻa ma ke Kiʻi 1.
Ua hoʻokaʻawale ʻia nā laʻana ma 393 K no 1 hola e loaʻa ai ke kaomi koena ma lalo o 10-3 Torr. Ua hoʻohana ʻia ka nui o N2 i hoʻopili ʻia ma ke kaomi pili o P/P0 = 0.99 e hoʻoholo i ka nui o ka pore. Ua nānā ʻia ke ʻano o nā ʻāpana silica ʻōlohelohe a me nā ligand-bonded me ka microscopy electron scanning (Hitachi High Technologies, Tokyo, Iapana). Ua kau ʻia nā laʻana maloʻo (silica ʻōlohelohe a me nā ʻāpana silica i hoʻopaʻa ʻia me ka ligand) ma luna o kahi kolamu alumini me ka hoʻohana ʻana i ka lipine kalapona hoʻopili. Ua uhi ʻia ke gula ma luna o nā laʻana me ka hoʻohana ʻana i kahi Q150T sputter coater, a ua waiho ʻia kahi papa Au 5 nm ma luna o nā laʻana. Hoʻomaikaʻi kēia i ka pono o ke kaʻina hana me ka hoʻohana ʻana i nā voltages haʻahaʻa a hāʻawi i ka palaoa maikaʻi, ka sputtering anuanu. Ua hoʻohana ʻia kahi mea kālailai elemental Thermo Electron (Waltham, MA, USA) Flash EA1112 no ka nānā ʻana i nā elemental. Ua hoʻohana ʻia kahi mea kālailai nui ʻāpana Malvern (Worcestershire, UK) Mastersizer 2000 e loaʻa ai ka hoʻolaha nui ʻāpana. Nā ʻāpana silica ʻōlohelohe a me ka silica i hoʻopaʻa ʻia me ka ligand Ua hoʻopuehu ʻia nā ʻāpana (5 mg kēlā me kēia) i loko o 5 mL o isopropanol, sonicated no 10 min, vortexed no 5 min, a kau ʻia ma ka noho optical o ka Mastersizer. Ua hana ʻia ka nānā ʻana o Thermogravimetric ma ka wikiwiki o 5 °C i kēlā me kēia minuke ma luna o kahi pae mahana o 30 a 800 °C.
Ua hoʻopaʻa ʻia nā kolamu kila kila haiki i uhi ʻia i ke aniani me nā ana (100 × 1.8 mm id) me ka hoʻohana ʻana i ke ʻano hoʻopaʻa slurry, e hoʻopili ana i ke kaʻina hana like i hoʻohana ʻia ma Ref. 31. Ua hoʻopili ʻia kahi kolamu kila kila (i uhi ʻia me ke aniani, 100 × 1.8 mm id) me kahi mea hoʻokomo puka e loaʻa ana kahi frit 1 µm i kahi mea hoʻopaʻa slurry (Alltech Deerfield, IL, USA). E hoʻomākaukau i kahi slurry pae paʻa ma ke kau ʻana i 150 mg o ka pae paʻa i loko o 1.2 mL o ka methanol a hoʻouna iā ia i ke kolamu mālama. Ua hoʻohana ʻia ka Methanol ma ke ʻano he mea hoʻoheheʻe slurry a me ka mea hoʻoheheʻe propelling. E hoʻopiha i ke kolamu ma ke ʻano maʻamau ma ka hoʻopili ʻana i nā kaomi o 100 MP no 10 mau minuke, 80 MP no 15 mau minuke, a me 60 MP no 30 mau minuke. I ka wā o ka hoʻopaʻa ʻana, ua hoʻopili ʻia ka haʻalulu mechanical me ʻelua mau mea hoʻoluliluli kolamu GC (Alltech, Deerfield, IL, USA) e hōʻoia i ka hoʻopaʻa like ʻana o ke kolamu. E pani i ka mea hoʻopaʻa slurry a hoʻokuʻu mālie i ke kaomi e pale aku i kekahi pōʻino i loko o ke kolamu. E hoʻokaʻawale i ke kolamu mai ka ʻāpana hoʻopaʻa slurry a hoʻohui i kahi mea hoʻokomo ʻē aʻe i ka inlet a i ka ʻōnaehana LC e nānā i kāna hana.
Ua kūkulu ʻia kahi pamu LC (10AD Shimadzu, Iapana), injector (Valco (USA) C14 W.05) me ka loop injection 50nL, membrane degasser (Shimadzu DGU-14A), ka puka makani capillary UV-VIS. Ua kūkulu ʻia kahi mea ʻike µLC kūikawā (UV-2075) a me nā microcolumns i uhi ʻia i ke aniani. E hoʻohana i nā paipu hoʻohui haiki loa a pōkole e hōʻemi i ka hopena o ka hoʻonui ʻana o ke kaula kolamu keu. Ma hope o ka hoʻopili ʻana, ua hoʻokomo ʻia nā capillaries (50 μm id 365 a me nā capillaries union reducing (50 μm) ma ka puka 1/16″ o ka union reducing. Ua hana ʻia ka hōʻiliʻili ʻikepili a me ka hana chromatographic me ka hoʻohana ʻana i ka polokalamu Multichro 2000. Ua hoʻāʻo ʻia ka nānā ʻana ma 254 nm Analytes no ka absorbance UV. Ua kālailai ʻia nā ʻikepili chromatographic e OriginPro8 (Northampton, MA).
ʻO ka albumin mai ka serum kanaka, ka pauka lyophilized, ≥ 96% (agarose gel electrophoresis) 3 mg i hui pū ʻia me ka trypsin (1.5 mg), 4.0 M urea (1 mL), a me 0.2 M ammonium bicarbonate (1 mL). Ua hoʻoulu ʻia ka hopena no 10 mau minuke a mālama ʻia i loko o ka ʻauʻau wai ma 37°C no 6 mau hola, a laila hoʻopau ʻia me 1 mL o 0.1% TFA. E kānana i ka hopena a mālama ma lalo o 4°C.
Ua loiloi ʻokoʻa ʻia ka hoʻokaʻawale ʻana o nā hui peptide a me nā ʻeli trypsin HSA ma nā kolamu PMP. E nānā i ka hoʻokaʻawale ʻana o ka hui peptide a me ka ʻeli trypsin o HSA e ke kolamu PMP a hoʻohālikelike i nā hopena i ke kolamu Ascentis Express RP-Amide. Ua helu ʻia ka helu pā theoretical penei:
Ua hōʻike ʻia nā kiʻi SEM o nā ʻāpana silica ʻōlohelohe a me nā ʻāpana silica i hoʻopaʻa ʻia e ka ligand ma ke Kiʻi 2. Hōʻike nā kiʻi SEM o nā ʻāpana silica ʻōlohelohe (A, B), i ka hoʻohālikelike ʻana me kā mākou mau haʻawina mua, he poepoe kēia mau ʻāpana kahi i hoʻolōʻihi ʻia ai nā ʻāpana a i ʻole he symmetry ʻano ʻole. ʻOi aku ka laumania o ka ʻili o nā ʻāpana silica i hoʻopaʻa ʻia e ka ligand (C, D) ma mua o nā ʻāpana silica ʻōlohelohe, ma muli paha o ka uhi ʻana o nā kaulahao polystyrene ma ka ʻili o nā ʻāpana silica.
Ke nānā nei i nā kiʻi microscope electron o nā ʻāpana silica ʻōlohelohe (A, B) a me nā ʻāpana silica i hoʻopaʻa ʻia me ka ligand (C, D).
Hōʻike ʻia nā hoʻolaha nui o nā ʻāpana o nā ʻāpana silica ʻōlohelohe a me nā ʻāpana silica i hoʻopaʻa ʻia e ka ligand ma ke Kiʻi 3(A). Ua hōʻike nā piʻo hoʻolaha nui o nā ʻāpana e pili ana i ka nui i hoʻonui ʻia ka nui o nā ʻāpana silica ma hope o ka hoʻololi kemika (Kiʻi 3A). Hoʻohālikelike ʻia ka ʻikepili hoʻolaha nui o nā ʻāpana o nā ʻāpana silica mai ke aʻo ʻana o kēia manawa a me ke aʻo ʻana ma mua ma ka Papa 1(A). ʻO ka nui o nā ʻāpana e pili ana i ka nui, d(0.5), o PMP he 3.36 μm, i hoʻohālikelike ʻia me kā mākou aʻo ʻana ma mua me ka waiwai ad(0.5) o 3.05 μm (nā ʻāpana silica i hoʻopaʻa ʻia e ka polystyrene)34. He ʻoi aku ka liʻiliʻi o ka hoʻolaha nui o kēia pūʻulu i hoʻohālikelike ʻia me kā mākou aʻo ʻana ma mua ma muli o nā lakio like ʻole o PEG, urea, TMOS, a me ka waikawa acetic i loko o ka hui hopena. ʻOi aku ka nui o ka nui o nā ʻāpana o ka pae PMP ma mua o ka pae ʻāpana silica i hoʻopaʻa ʻia e ka polystyrene a mākou i aʻo ai ma mua. ʻO ke ʻano o kēia, ʻo ka hana ʻana o ka ʻili o nā ʻāpana silica me ka styrene i waiho wale i kahi papa polystyrene (0.97 µm) ma ka ʻili silica, ʻoiai ma ka pae PMP ua ʻoi aku ka mānoanoa o ka papa. 1.38 µm.
Ka hoʻolaha ʻana o ka nui o nā ʻāpana (A) a me ka hoʻolaha ʻana o ka nui o nā pore (B) o nā ʻāpana silica ʻōlohelohe a me nā ʻāpana silica i hoʻopaʻa ʻia e ka ligand.
ʻO ka nui o ka pore, ka nui o ka pore a me ka ʻili o nā ʻāpana silica o ke aʻo ʻana o kēia manawa ua hāʻawi ʻia ma ka Papa 1(B). Hōʻike ʻia nā ʻikepili PSD o nā ʻāpana silica ʻōlohelohe a me nā ʻāpana silica i hoʻopaʻa ʻia e ka ligand ma ke Kiʻi 3(B). Hoʻohālikelike ʻia nā hopena me kā mākou aʻo mua. ʻO ka nui o ka pore o nā ʻāpana silica ʻōlohelohe a me ka ligand-bound he 310 a me 241, kēlā me kēia, e hōʻike ana ua emi ka nui o ka pore ma 69 ma hope o ka hoʻololi kemika, e like me ka mea i hōʻike ʻia ma ka Papa 1(B), a ua hōʻike ʻia ka hoʻololi o ke kūlou ma ke Kiʻi 3(B). Pēlā nō, ua emi ka nui o ka pore o nā ʻāpana silica mai 0.67 a i 0.58 cm3/g ma hope o ka hoʻololi kemika. ʻO ka ʻili kikoʻī o nā ʻāpana silica i aʻo ʻia i kēia manawa he 116 m2/g, kahi e hoʻohālikelike ʻia me kā mākou aʻo mua (124 m2/g). E like me ka mea i hōʻike ʻia ma ka Papa 1(B), ua emi pū ka ʻili (m2/g) o nā ʻāpana silica mai 116 m2/g a i 105 m2/g ma hope o ke kemika. hoʻololi.
Ua hōʻike ʻia nā hopena o ka loiloi elemental o ke kahua paʻa ma ka Papa 2. ʻO ke kaumaha kalapona o ke kahua paʻa o kēia manawa he 6.35%, ʻoi aku ka haʻahaʻa ma mua o ke kaumaha kalapona o kā mākou noiʻi mua (nā ʻāpana silica i hoʻopaʻa ʻia e ka polystyrene, 7.93%35 a me 10.21%, kēlā me kēia) 42. Haʻahaʻa ke kaumaha kalapona o ke kahua paʻa o kēia manawa, No ka mea, i ka hoʻomākaukau ʻana o ka SP o kēia manawa, me ka styrene, ua hoʻohana ʻia kekahi mau ligands polar e like me phenylmaleimide-methylvinylisocyanate (PCMP) a me 4-hydroxy-TEMPO. ʻO ka pakeneka kaumaha nitrogen o ke kahua paʻa o kēia manawa he 2.21%, i hoʻohālikelike ʻia me 0.1735 a me 0.85% ma ke kaumaha o ka nitrogen i nā noiʻi mua, kēlā me kēia. ʻO ke ʻano kēia, ʻoi aku ke kiʻekiʻe o ka wt % o ka nitrogen i ke kahua paʻa o kēia manawa ma muli o phenylmaleimide. Pēlā nō, ʻo nā ukana kalapona o nā huahana (4) a me (5) he 2.7% a me 2.9%, kēlā me kēia, ʻoiai ʻo ke kaumaha kalapona o ka hope loa ʻO ka huahana (6) he 6.35%, e like me ka mea i hōʻike ʻia ma ka Papa 2. Ua nānā ʻia ka pohō kaumaha me ka pae paʻa PMP, a ua hōʻike ʻia ka piʻo TGA ma ke Kiʻi 4. Hōʻike ka piʻo TGA i ka pohō kaumaha o 8.6%, kahi e kūlike maikaʻi me ka hoʻouka kalapona (6.35%) no ka mea ʻaʻole wale nā ​​​​ligands C akā ʻo N, O, a me H.
Ua koho ʻia ka ligand phenylmaleimide-methylvinylisocyanate no ka hoʻololi ʻana i ka ʻili o nā ʻāpana silica no ka mea he mau hui phenylmaleimide polar a me nā hui vinylisocyanate. Hiki i nā hui vinyl isocyanate ke hana hou me ka styrene ma o ka polymerization radical ola. ʻO ke kumu ʻelua, ʻo ia ke hoʻokomo i kahi hui i loaʻa ka pilina kūpono me ka analyte a ʻaʻohe pilina electrostatic ikaika ma waena o ka analyte a me ke kahua paʻa, ʻoiai ʻaʻohe uku virtual o ka phenylmaleimide moiety ma ka pH maʻamau. Hiki ke hoʻomalu ʻia ka polarity o ke kahua paʻa e ka nui kūpono o ka styrene a me ka manawa pane o ka polymerization radical manuahi. ʻO ka hana hope loa o ka hopena (polymerization radical manuahi) he mea koʻikoʻi a hiki ke hoʻololi i ka polarity o ke kahua paʻa. Ua hana ʻia ka loiloi Elemental e nānā i ka hoʻouka kalapona o kēia mau pae paʻa. Ua ʻike ʻia ʻo ka hoʻonui ʻana i ka nui o ka styrene a me ka manawa pane i hoʻonui i ka hoʻouka kalapona o ke kahua paʻa a me ka hope. ʻO nā SP i hoʻomākaukau ʻia me nā ʻano like ʻole o ka styrene he mau hoʻouka kalapona like ʻole. Eia hou, e hoʻouka i kēia mau pae paʻa i loko o nā kolamu kila kila a nānā i kā lākou hana chromatographic. (selectivity, resolution, N value, etc.). Ma muli o kēia mau hoʻokolohua, ua koho ʻia kahi ʻano hana i hoʻonohonoho pono ʻia e hoʻomākaukau i ka pae paʻa PMP e hōʻoia i ka polarity i kāohi ʻia a me ka paʻa ʻana o ka analyte maikaʻi.
Ua loiloi ʻia hoʻi nā hui peptide ʻelima (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) me ka hoʻohana ʻana i kahi kolamu PMP me ka hoʻohana ʻana i kahi pae kelepona; 60/40 (v/v) acetonitrile/wai ​​(0.1% TFA) ma ka nui o ke kahe ʻana o 80 μL/min. Ma lalo o nā kūlana elution kūpono, ʻo ka helu pā theoretical (N) no kēlā me kēia kolamu (100 × 1.8 mm id) he 20,000 ± 100 (200,000 mau pā/m²). Hāʻawi ka Papa 3 i nā waiwai N no nā kolamu ʻekolu PMP a ua hōʻike ʻia nā chromatograms ma ke Kiʻi 5A. Ka nānā wikiwiki ʻana ma kahi kolamu PMP ma ka nui o ke kahe ʻana (700 μL/min), ua eluted ʻia ʻelima mau peptides i loko o hoʻokahi minuke, ua maikaʻi loa nā waiwai N, 13,500 ± 330 no kēlā me kēia kolamu (100 × 1.8 mm id), Pili i 135,000 mau pā/m (Kiʻi 5B). ʻEkolu mau kolamu like ka nui (100 × 1.8 mm id) i hoʻopaʻa ʻia me ʻekolu mau ʻāpana like ʻole o ka pae paʻa PMP e nānā i ka hana hou ʻana. ʻO ka Ua hoʻopaʻa ʻia ka nui o ka analyte no kēlā me kēia kolamu me ka hoʻohana ʻana i nā kūlana elution kūpono a me ka helu o nā papa theoretical N a me ka manawa paʻa e hoʻokaʻawale i ka hui hoʻāʻo like ma kēlā me kēia kolamu. Hōʻike ʻia ka ʻikepili reproducibility no nā kolamu PMP ma ka Papa 4. Hoʻopili maikaʻi ka reproducibility o ke kolamu PMP me nā waiwai %RSD haʻahaʻa loa, e like me ka mea i hōʻike ʻia ma ka Papa 3.
Ka hoʻokaʻawale ʻana o ka hui peptide ma ke kolamu PMP (B) a me ke kolamu Ascentis Express RP-Amide (A); pae kelepona 60/40 ACN/H2O (TFA 0.1%), nā ana o ke kolamu PMP (100 × 1.8 mm id); analytical Ke kauoha elution o nā hui: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) a me 5 (leucine) acid enkephalin)).
Ua loiloi ʻia kahi kolamu PMP (100 × 1.8 mm id) no ka hoʻokaʻawale ʻana i nā digests tryptic o ka albumin serum kanaka ma ka chromatography wai hana kiʻekiʻe. Hōʻike ka chromatogram ma ke Kiʻi 6 ua hoʻokaʻawale maikaʻi ʻia ka laʻana a maikaʻi loa ka hoʻonā. Ua kālailai ʻia nā digests HSA me ka hoʻohana ʻana i ka nui o ke kahe o 100 µL/min, ka pae kelepona 70/30 acetonitrile/wai ​​a me 0.1% TFA. E like me ka mea i hōʻike ʻia ma ka chromatogram (Kiʻi 6), ua māhele ʻia ka digestion HSA i 17 mau piko e pili ana i 17 peptides. Ua helu ʻia ka pono o ka hoʻokaʻawale ʻana o kēlā me kēia piko i ka digest HSA a ua hāʻawi ʻia nā waiwai ma ka Papa 5.
Ua hoʻokaʻawale ʻia kahi digest tryptic o HSA (100 × 1.8 mm id) ma kahi kolamu PMP; ka nui o ke kahe (100 µL/min), pae mobile 60/40 acetonitrile/wai ​​me 0.1% TFA.
kahi ʻo L ka lōʻihi o ke kolamu, ʻo η ka viscosity o ka pae mobile, ʻo ΔP ke kaomi hope o ke kolamu, a ʻo u ka wikiwiki linear o ka pae mobile. ʻO ka permeability o ke kolamu PMP he 2.5 × 10-14 m2, ʻo ka nui o ke kahe he 25 μL/min, a ua hoʻohana ʻia he 60/40 v/v ACN/wai. Ua like ka permeability o ke kolamu PMP (100 × 1.8 mm id) me ko kā mākou noiʻi mua Ref.34. ʻO ka permeability o ke kolamu i hoʻopaʻa ʻia me nā ʻāpana porous superficially: 1.7 × 10-15 no nā ʻāpana 1.3 μm, 3.1 × 10-15 no nā ʻāpana 1.7 μm, 5.2 × 10-15 a me 2.5 × 10-14 m2 no nā ʻāpana 2.6 μm No nā ʻāpana 5 μm 43. No laila, ua like ka permeability o ka pae PMP me ko nā ʻāpana core-shell 5 μm.
kahi ʻo Wx ke kaumaha o ke kolamu i hoʻopaʻa ʻia me ka chloroform, ʻo Wy ke kaumaha o ke kolamu i hoʻopaʻa ʻia me ka methanol, a ʻo ρ ka nui o ka mea hoʻoheheʻe. Nā nui o ka methanol (ρ = 0.7866) a me ka chloroform (ρ = 1.484). ʻO ka porosity holoʻokoʻa o nā kolamu SILICA PARTICLES-C18 (100 × 1.8 mm id) 34 a me nā kolamu C18-Urea 31 a mākou i aʻo mua ai he 0.63 a me 0.55, kēlā me kēia. ʻO ke ʻano kēia, ʻo ka loaʻa ʻana o nā ligands urea e hōʻemi ana i ka permeability o ka pae paʻa. Ma ka ʻaoʻao ʻē aʻe, ʻo ka porosity holoʻokoʻa o ke kolamu PMP (100 × 1.8 mm id) he 0.60. ʻOi aku ka haʻahaʻa o ka permeability o nā kolamu PMP ma mua o nā kolamu i hoʻopaʻa ʻia me nā ʻāpana silica i hoʻopaʻa ʻia e C18 no ka mea i nā pae paʻa ʻano C18 ua hoʻopili ʻia nā ligands C18 i nā ʻāpana silica ma ke ʻano he kaulahao linear, ʻoiai i nā pae paʻa ʻano polystyrene, ua hoʻokumu ʻia ka papa polymer A mānoanoa a puni ia.Ma kahi hoʻokolohua maʻamau, ua helu ʻia ka porosity kolamu penei:
Hōʻike ka Kiʻi 7A,B i ke kolamu PMP (100 × 1.8 mm id) a me ke kolamu Ascentis Express RP-Amide (100 × 1.8 mm id) me ka hoʻohana ʻana i nā kūlana elution like (ʻo ia hoʻi, 60/40 ACN/H2O a me 0.1% TFA). ) o ka pakuhi van Deemter. Ua hoʻomākaukau ʻia nā hui peptide i koho ʻia (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) i loko o 20 µL/ ʻO ka nui o ke kahe haʻahaʻa no nā kolamu ʻelua he 800 µL/min. ʻO nā waiwai HETP haʻahaʻa loa ma ka nui o ke kahe kūpono (80 µL/min) no ke kolamu PMP a me ke kolamu Ascentis Express RP-Amide he 2.6 µm a me 3.9 µm, kēlā me kēia. Hōʻike nā waiwai HETP ua ʻoi aku ka maikaʻi o ka hoʻokaʻawale ʻana o ke kolamu PMP (100 × 1.8 mm id) ma mua o ke kolamu Ascentis Express RP-Amide i loaʻa ma ke kālepa (100 × 1.8 mm id). Hōʻike ka palapala van Deemter ma ke Kiʻi 7(A) ʻaʻole koʻikoʻi ka emi ʻana o ka waiwai N me ka piʻi ʻana o ke kahe i hoʻohālikelike ʻia me kā mākou noiʻi mua. ʻO ke kiʻekiʻe o ka hoʻokaʻawale ʻana o ke kolamu PMP (100 × 1.8 mm id) i hoʻohālikelike ʻia me ke kolamu Ascentis Express RP-Amide e pili ana i nā hoʻomaikaʻi ʻana i ke ʻano o nā ʻāpana, ka nui, a me nā kaʻina hana hoʻopili kolamu paʻakikī i hoʻohana ʻia i ka hana o kēia manawa34.
(A) kiʻi van Deemter (HETP versus mobile phase linear velocity) i loaʻa me ka hoʻohana ʻana i kahi kolamu PMP (100 × 1.8 mm id) ma 60/40 ACN/H2O me 0.1% TFA.(B) kiʻi van Deemter (HETP versus mobile phase linear velocity) i loaʻa me ka hoʻohana ʻana i kahi kolamu Ascentis Express RP-Amide (100 × 1.8 mm id) ma 60/40 ACN/H2O me 0.1% TFA.
Ua hoʻomākaukau ʻia a loiloi ʻia kahi pae paʻa polystyrene i hoʻokomo ʻia i ka polar no ka hoʻokaʻawale ʻana i nā hui peptide synthetic a me nā trypsin digests o ka human serum albumin (HAS) i ka chromatography wai hana kiʻekiʻe. ʻOi aku ka maikaʻi o ka hana chromatographic o nā kolamu PMP no nā hui peptide i ka hoʻokaʻawale pono a me ka hoʻonā. ʻO ka hoʻomaikaʻi ʻana i ka hana hoʻokaʻawale o nā kolamu PMP ma muli o nā kumu like ʻole, e like me ka nui o ka ʻāpana a me ka nui o ka pore o nā ʻāpana silica, ka synthesis i kāohi ʻia o ka pae paʻa, a me ka hoʻopili ʻana o ke kolamu paʻakikī. Ma waho aʻe o ka pono hoʻokaʻawale kiʻekiʻe, ʻo ke kaomi hope kolamu haʻahaʻa ma nā kahe kiʻekiʻe kekahi pono o kēia pae paʻa. Hōʻike nā kolamu PMP i ka hana hou maikaʻi a hiki ke hoʻohana ʻia no ka nānā ʻana i nā hui peptide a me ka trypsin digestion o nā protein like ʻole. Manaʻo mākou e hoʻohana i kēia kolamu no ka hoʻokaʻawale ʻana i nā huahana kūlohelohe, nā hui bioactive mai nā mea kanu lāʻau lapaʻau a me nā fungal extracts i ka chromatography wai. I ka wā e hiki mai ana, e loiloi ʻia nā kolamu PMP no ka hoʻokaʻawale ʻana o nā protein a me nā monoclonal antibodies.
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