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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 pore ākea. Ua hoʻokaʻawale ʻia kēia mau ʻāpana me ka N-phenylmaleimide-methylvinyl isocyanate (PMI) a me ka styrene ma o ka polymerization reverse chain transfer-fragmentation (RAFT) e hana i nā polyamides intercalated N-phenylmaleimide. ʻO ka pae paʻa Styrene (PMP). Ua hoʻopaʻa ʻia nā kolamu kila kila haiki (100 × 1.8 mm ke anawaena o loko) me kahi slurry packing. Ua loiloi ʻia ka hana chromatographic o ke kolamu PMP e hoʻokaʻawale i kahi hui ʻana o nā peptides synthetic i haku ʻia me ʻelima mau peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) a me ka tryptic hydrolyzate o ka human serum albumin (HAS). Ma lalo o nā kūlana elution kūpono, ua hiki ka helu theoretical o nā papa me ka hui ʻana o nā peptides i 280,000 mau papa/sq.m. I ka hoʻohālikelike ʻana i ka hana hoʻokaʻawale o ke kolamu i hoʻomohala ʻia me ke kolamu Ascentis Express RP-Amide kalepa, ua ʻike ʻia ʻoi aku ka maikaʻi o ka pono o ka hoʻokaʻawale ʻana o ke kolamu PMP ma mua o ke kolamu kalepa ma ke ʻano o ka pono a me ka hoʻonā ʻana o ka hoʻokaʻawale ʻana.
Ua lilo ka ʻoihana biopharmaceutical i mākeke honua e hoʻonui ana me ka piʻi nui ʻana o ka māhele mākeke i nā makahiki i hala iho nei. Me ka ulu nui ʻana o ka ʻoihana biopharmaceutical1,2,3 aia kahi pono nui no ka nānā ʻana o ka peptide a me ka protein. Ma waho aʻe o ka peptide i manaʻo ʻia, ua hoʻokumu ʻia nā ʻano haumia like ʻole i ka wā o ka peptide synthesis, no laila pono ka hoʻomaʻemaʻe chromatographic e loaʻa ai ka maʻemaʻe i makemake ʻia o ka peptide. ʻO ka nānā ʻana a me ke ʻano o nā protein i loko o nā wai o ke 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 hoʻonohonoho ʻana i nā peptides a me nā protein, inā hoʻokomo pololei ʻia kēlā mau laʻana i loko o ka spectrometer mass, ʻaʻole e ʻoluʻolu ka hoʻokaʻawale ʻana. Hiki ke hoʻoponopono ʻia kēia pilikia ma ka hana ʻana i ka chromatography wai (LC) ma mua o ka nānā ʻana o MS, kahi e hōʻemi ai i ka nui o nā analytes e komo ana i ka spectrometer mass i kahi manawa i hāʻawi ʻia4,5,6. Eia kekahi, hiki i nā analytes ke kau i kahi ʻāpana haiki i ka wā o ka hoʻokaʻawale ʻana o ka pae wai, no laila e kau ana i kēia mau analytes a hoʻonui i ka ʻike o ka ʻike ʻana o MS. Ua holomua nui ka chromatography wai (LC) i nā makahiki he ʻumi i hala iho nei a ua lilo ia i ʻano hana i hoʻohana nui ʻia no ka nānā ʻana i ka proteomic7,8,9,10.
Hoʻohana nui ʻia ka chromatography wai hoʻohuli-phase (RP-LC) e hoʻomaʻemaʻe a hoʻokaʻawale i nā hui ʻana o nā peptides 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, ma muli o ko lākou ʻano paʻakikī a me ke ʻano amphoteric,14,15 ʻaʻole hiki i nā pae paʻa RP ke hāʻawi i kahi hoʻokaʻawale kūpono o nā peptides a me nā protein. No laila, ʻo ka nānā ʻana i nā peptides a me nā protein me nā ʻāpana polar a me nā non-polar e pono ai nā pae paʻa i hoʻolālā kūikawā ʻia e launa pū a mālama i kēia mau analytes16. ʻO ka chromatography hui ʻia, e hāʻawi ana i nā pilina multimodal, hiki ke lilo i mea ʻē aʻe i ka RP-LC no ka hoʻokaʻawale ʻana i nā peptides, nā protein, a me nā hui paʻakikī ʻē aʻe. Ua hoʻomākaukau ʻia kekahi mau pae paʻa ʻano hui ʻia a ua hoʻohana ʻia nā kolamu i hoʻopiha ʻia me kēia mau pae paʻa e hoʻokaʻawale i nā peptides a me nā protein17,18,19,20,21. Ma muli o ke alo o nā hui polar a me nā hui non-polar, kūpono nā pae paʻa ʻano hui (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) no ka hoʻokaʻawale ʻana o nā peptides a me nā protein22,23,24,25,26,27,28. , hōʻike nā pae paʻa polar intercalated me nā hui polar i hoʻopaʻa ʻia me ka covalently i nā hiki ke hoʻokaʻawale maikaʻi a me ke koho kūikawā no nā analytes polar a me non-polar no ka mea pili 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 loaʻa iā Zhang et al. 30 nā pae paʻa behenyl-terminated o nā polyamines a ua hoʻokaʻawale pono i nā hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, a me kekahi mau analytes ʻē aʻe. Loaʻa i ka mea paʻa i hoʻokomo ʻia i loko o ka polar nā hui polar a me non-polar, no laila hiki ke hoʻohana ʻia e hoʻokaʻawale i nā peptides a me nā protein i nā ʻāpana hydrophobic a me hydrophilic. Loaʻa nā kolamu inline polar (e laʻa, nā kolamu C18 me ka amide inline) ma lalo o ka inoa kālepa ʻo Ascentis Express RP-Amide kolamu, akā ua hoʻohana wale ʻia kēia mau kolamu no ka nānā ʻana i ka amine 33.
Ma ke aʻo ʻana o kēia manawa, ua hoʻomākaukau ʻia a loiloi ʻia kahi pae paʻa polar embedding (N-phenylmaleimide, embedding polystyrene) no ka hoʻokaʻawale ʻana o ka peptide a me ka cleavage tryptic HSA. Ua hoʻohana ʻia ke ʻano hana ma lalo nei e hoʻomākaukau i ka pae paʻa. Ua hoʻomākaukau ʻia nā ʻāpana silica porous e like me nā kaʻina hana i wehewehe ʻia ma kā mākou mau puke i hala, me kekahi mau hoʻololi i nā papahana hoʻomākaukau 31, 34, 35, 36, 37, 38, 39. Ua hoʻoponopono ʻia nā lakio o ka urea, polyethylene glycol (PEG), TMOS a me ka aqueous-acetic acid e loaʻa nā ʻāpana silica me nā nui pore nui. ʻO ka lua, ua synthesized ʻia kahi ligand phenylmaleimide-methylvinyl isocyanate hou a ua hoʻohana ʻia kāna mau ʻāpana silica derivatized e hoʻomākaukau i nā pae paʻa polar embedded. Ua hoʻopaʻa ʻia ka pae paʻa i loaʻa i loko o kahi kolamu kila kila (ke anawaena o loko 100 × 1.8 mm) e like me kahi papahana hoʻopaʻa i hoʻonohonoho pono ʻia. Kōkua ʻia ka hoʻopaʻa ʻana o ke kolamu e ka haʻalulu mechanical e hōʻoia i kahi papa like i loko o ke kolamu. Ua loiloi ʻia ke kolamu i hoʻopaʻa ʻia no ka hoʻokaʻawale ʻana i kahi hui ʻana o nā peptides i loaʻa i ʻelima mau peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). a me nā hydrolysates tryptic o ka albumin serum kanaka (HSA). Ua ʻike ʻia ua hoʻokaʻawale ʻia ka hui peptide a me ka HSA tryptic digest me ka hoʻonā maikaʻi a me ka pono. Ua hoʻohālikelike ʻia ka pono hoʻokaʻawale o ke kolamu PMP me ko ke kolamu Ascentis Express RP-Amide. Ua ʻike ʻia he hoʻonā maikaʻi ko nā peptides a me nā protein a me ka pono hoʻokaʻawale kiʻekiʻe ma ke kolamu PMP, a ʻoi aku ka kiʻekiʻe o ka pono hoʻokaʻawale o ke kolamu PMP ma mua o ko ke kolamu Ascentis Express RP-Amide.
PEG (polyethylene glycol), urea, acetic acid, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, human serum albumin (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride (MC), styrene, 4-hydroxy-TEMPO, benzoyl peroxide (BPO), acetonitrile (ACN) no HPLC, methanol, 2-propanol a me acetone. ʻO Sigma-Aldrich Company (St. Louis, Missouri, 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 ua hoʻohui ʻia he 24 ml o TMOS i laila ma lalo o ka hoʻomaʻalili ʻana o ka 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 hoʻokahe ʻia ka wai a ua hoʻomaloʻo ʻia ke koena ma 70°C no 12 mau hola. Ua wili mālie ʻia nā poloka palupalu maloʻo a ua calcined ʻia i loko o ka umu ma 550°C no 12 mau hola. Ua hoʻomākaukau ʻia ʻekolu mau ʻāpana a ua wehewehe ʻia e hoʻāʻo i ka hana hou ʻana o nā nui o nā ʻāpana, ka nui o ka pore a me ka ʻili.
Hui polar a me ka pae paʻa no 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 anhydrous, a laila ua hoʻohui ʻia ʻo 0.1 ml o 2,2′-azoisobutyronitrile (AIBN) i ka ʻōmole hana e loaʻa ai kahi copolymer o phenylmaleimide a me methyl vinyl isocyanate (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 no 10 min i loko o kahi ʻōmole lalo poepoe 500 ml. Ua hoʻoheheʻe ʻia ka PMCP (10 mg) i loko o ka toluene a hoʻohui ʻia i loko o ka ʻōmole hopena ma o kahi funnel hoʻohui. Ua refluxed ʻia ka hui ʻana ma 100°C no 8 mau hola, kānana ʻia, holoi ʻia me ka acetone a hoʻomaloʻo ʻia ma 60°C no 3 mau hola. A laila, ua hoʻoheheʻe ʻia nā ʻāpana silica e pili ana me PMCP (100 g) i loko o ka toluene (200 ml), a ua hoʻohui ʻia ʻo 4-hydroxy-TEMPO (2 ml) i laila 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 hoʻomaloʻo 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 iā TEMPO-PMCP (1.5 g) i loko o toluene a 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 i ka pō ma 60°C. Hōʻike ʻia ke ʻano maʻamau o ka hopena ma ke kiʻi. ʻekahi.
Ua hoʻokaʻawale ʻia nā laʻana i ke kinoea ma 393 K no 1 h a hiki i ka loaʻa ʻana o ke kaomi koena ma lalo o 10-3 Torr. Ua hoʻohana ʻia ka nui o N2 i hoʻopili ʻia ma ke kaomi pili P/P0 = 0.99 e hoʻoholo i ka nui o ka pore holoʻokoʻa. Ua nānā ʻia ke ʻano o nā ʻāpana silica maʻemaʻe a me nā ligand-bound me ka hoʻohana ʻana i kahi microscope electron scanning (Hitachi High Technologies, Tokyo, Iapana). Ua kau ʻia nā laʻana maloʻo (silica maʻemaʻe a me nā ʻāpana silica i hoʻopaʻa ʻia me ka ligand) ma nā koʻokoʻo alumini me ka hoʻohana ʻana i ka lipine kalapona. Ua waiho ʻia ke gula ma luna o ka laʻana me ka hoʻohana ʻana i kahi mea sputtering Q150T, a ua waiho ʻia kahi papa Au 5 nm mānoanoa ma luna o ka laʻana. Hoʻomaikaʻi kēia i ka pono o ke kaʻina hana haʻahaʻa haʻahaʻa a hāʻawi i ka pīpī anu maikaʻi. Ua hana ʻia ka loiloi elemental me ka hoʻohana ʻana i kahi mea kālailai haku mele elemental Thermo Electron (Waltham, MA, USA) Flash EA1112. Ua hoʻohana ʻia kahi mea kālailai nui ʻāpana Malvern (Worcestershire, UK) Mastersizer 2000 e loaʻa ai ka hoʻolaha nui ʻāpana. Ua hoʻopuehu ʻia nā ʻāpana silica i uhi ʻole ʻia a me nā ʻāpana silica i hoʻopaʻa ʻia e ka ligand (5 mg kēlā me kēia) i loko o 5 ml o isopropanol, sonicated no 10 mau minuke, hoʻoluliluli ʻia no 5 mau minuke, a kau ʻia ma luna o kahi noho optical Mastersizer. Hana ʻia ka nānā ʻana o Thermogravimetric ma ka wikiwiki o 5 °C i kēlā me kēia minuke ma ka pae mahana mai 30 a 800 °C.
Ua hoʻopaʻa ʻia nā kolamu kila kila haiki i hoʻopaʻa ʻia me ke aniani me nā ana (ID 100 × 1.8 mm) e ke ʻano hoʻopiha slurry ma muli o ke kaʻina hana like me ka kuhikuhi 31. Ua hoʻopili ʻia ke kolamu kila kila (i hoʻopaʻa ʻia me ke aniani, ID 100 × 1 .8 mm) a me kahi puka e loaʻa ana kahi frit 1 µm i kahi mīkini hoʻopili slurry (Alltech Deerfield, IL, USA). E hoʻomākaukau i kahi hoʻokuʻu o ka pae paʻa ma ke kau ʻana i 150 mg o ka pae paʻa i loko o 1.2 ml o ka methanol a hānai iā ia i loko o kahi kolamu waihona. Ua hoʻohana ʻia ka Methanol ma ke ʻano he mea hoʻoheheʻe slurry a me ka mea hoʻoheheʻe kaohi. E hoʻopaʻa i ke kolamu ma ka hoʻopili ʻana i kahi kaʻina kaomi o 100 MP no 10 min, 80 MP no 15 min, a me 60 MP no 30 min. Ua hoʻohana ke kaʻina hana hoʻopili i ʻelua mau vibrators kolamu chromatography gas (Alltech, Deerfield, IL, USA) no ka haʻalulu mechanical e hōʻoia i ka hoʻopili like ʻana o ke kolamu. E pani i ka mea hoʻopaʻa slurry a hoʻokuʻu mālie i ke kaomi e pale i ka hōʻino ʻana i ke kaula. Ua hoʻokaʻawale ʻia ke kolamu mai ka nozzle slurry a ua hoʻopili ʻia kekahi mea kūpono i ka puka komo a ua hoʻopili ʻia i ka ʻōnaehana LC e hoʻāʻo i kāna hana.
Ua kūkulu ʻia kahi MLC maʻamau me ka hoʻohana ʻana i kahi pamu LC (10AD Shimadzu, Iapana), kahi sampler me kahi loop injection 50 nL (Valco (USA) C14 W.05), kahi degasser membrane (Shimadzu DGU-14A), a me kahi puka makani capillary UV-VIS. Mea ʻike (UV-2075) a me ka microcolumn enamelled. E hoʻohana i nā paipu hoʻohui haiki loa a pōkole e hōʻemi i ka hopena o ka hoʻonui ʻana o ke kolamu. Ma hope o ka hoʻopiha ʻana i ke kolamu, e hoʻokomo i kahi capillary (50 µm id 365) ma ka puka o ka hui hoʻemi 1/16″ a hoʻokomo i kahi capillary (50 µm) o ka hui hoʻemi. Hana ʻia ka hōʻiliʻili ʻikepili a me ka hana ʻana i ka chromatogram me ka hoʻohana ʻana i ka polokalamu Multichro 2000. Ma 254 nm, ua nānā ʻia ka absorbance UV o nā analytes kumuhana ma 0. Ua kālailai ʻia ka ʻikepili Chromatographic me ka hoʻohana ʻana iā OriginPro8 (Northampton, MA).
ʻO ka albumin serum kanaka, 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 min a mālama ʻia i loko o ka ʻauʻau wai ma 37°C no 6 h, 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 kahi hui ʻana o nā peptides a me ka tryptic digest HSA ma kahi kolamu PMP. E nānā i ka hydrolysis tryptic o kahi hui ʻana o nā peptides a me HSA i hoʻokaʻawale ʻia e kahi kolamu PMP a hoʻohālikelike i nā hopena me kahi kolamu Ascentis Express RP-Amide. Ua helu ʻia ka helu o nā papa theoretical me ka hoʻohana ʻana i ka hoohalike penei:
Ua hōʻike ʻia nā kiʻi SEM o nā ʻāpana silica maʻemaʻe a me nā ʻāpana silica i hoʻopaʻa ʻia e ka ligand ma ke Kiʻi 2. Hōʻike nā kiʻi SEM o nā ʻāpana silica maʻemaʻe (A, B) i kahi ʻano poepoe kahi i hoʻolōʻihi ʻia ai nā ʻāpana a i ʻole he symmetry ʻano ʻole i hoʻohālikelike ʻia me kā mākou mau noiʻi mua. 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 maʻemaʻe, ma muli paha o nā kaulahao polystyrene e uhi ana i ka ʻili o nā ʻāpana silica.
Nā kiʻi uila scanning o nā ʻāpana silica maʻemaʻe (A, B) a me nā ʻāpana silica i hoʻopaʻa ʻia e ka ligand (C, D).
Ua hōʻike ʻia ka hoʻolaha ʻana o ka nui o nā ʻāpana o nā ʻāpana silica maʻemaʻe a me nā ʻāpana silica i hoʻopaʻa ʻia e ka ligand ma ke Kiʻi 2. 3(A). Ua hōʻike nā kiʻikuhi hoʻolaha nui o nā ʻāpana volumetric ua piʻi ka nui o nā ʻāpana silica ma hope o ka hoʻololi kemika (Kiʻi 3A). Ua hoʻohālikelike ʻia ka ʻikepili hoʻolaha nui 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 volumetric d(0.5) o PMP he 3.36 µm, i hoʻohālikelike ʻia me ka waiwai ad(0.5) o 3.05 µm i kā mākou aʻo ʻana ma mua (nā ʻāpana silica i hoʻopaʻa ʻia me ka polystyrene)34. Ma muli o ka loli o ka lakio o PEG, urea, TMOS a me ka waikawa acetic i loko o ka hui hopena, ua ʻoi aku ka haiki o ka hoʻolaha ʻana o ka nui o nā ʻāpana o kēia pūʻulu i hoʻohālikelike ʻia me kā mākou aʻo ʻana ma mua. 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 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 luna o ka ʻili silica, ʻoiai i ka pae PMP, ʻo ka mānoanoa o ka papa he 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 maʻemaʻe a me nā ʻāpana silica i hoʻopaʻa ʻia e ka ligand.
Ua hōʻike ʻia ka nui o ka pore, ka nui o ka pore, a me ka ʻili o nā ʻāpana silica i hoʻohana ʻia ma kēia haʻawina ma ka Papa 1 (B). Ua hōʻike ʻia nā ʻikepili PSD o nā ʻāpana silica maʻemaʻe a me nā ʻāpana silica i hoʻopaʻa ʻia e ka ligand ma nā Kiʻi 3(B). Ua like nā hopena me kā mākou haʻawina mua34. ʻO ka nui o ka pore o nā ʻāpana silica maʻemaʻe a me ka ligand-bound he 310 Å a me 241 Å, kēlā me kēia, e hōʻike ana ma hope o ka hoʻololi kemika, ua emi ka nui o ka pore ma 69 Å, e like me ka mea i hōʻike ʻia ma ka Papa 1 (B), a ua hōʻike ʻia ke kiʻikuhi neʻe ma ke Kiʻi. ʻO ka ʻili kikoʻī o nā ʻāpana silica i ke aʻo ʻana o kēia manawa he 116 m2/g, kahi e like me kā mākou haʻawina 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 ma hope o ka hoʻololi kemika mai 116 m2/g a i 105 m2/g.
Hōʻike ʻia nā hopena o ka loiloi elemental o ke kahua paʻa ma ka Papa. 2. ʻO ka nui o ke kalapona o ke kahua paʻa o kēia manawa he 6.35%, ʻoi aku ka haʻahaʻa ma mua o kā mākou noiʻi mua (nā ʻāpana silica e pili ana me ka polystyrene, 7.93%35 a me 10.21%, kēlā me kēia) 42. ʻO ka nui o ke kalapona o ke kahua paʻa o kēia manawa ma lalo nei, ʻoiai ua hoʻohana ʻia kekahi mau ligands polar e like me ka phenylmaleimide methyl vinyl isocyanate (PCMP) a me 4-hydroxy-TEMPO ma waho aʻe o ka styrene i ka hoʻomākaukau ʻana o SP. ʻO ka pakeneka kaumaha o ka nitrogen i ke kahua paʻa o kēia manawa he 2.21% i hoʻohālikelike ʻia me 0.1735 a me 0.85% i nā noiʻi mua42. ʻO ke ʻano kēia he kiʻekiʻe ka pakeneka kaumaha o ka nitrogen o ke kahua paʻa o kēia manawa ma muli o ka phenylmaleimide. Pēlā nō, ʻo nā huahana (4) a me (5) he 2.7% a me 2.9% ka nui o ke kalapona, ʻoiai ʻo ka huahana hope loa (6) he 6.35% ka nui o ke kalapona, e like me ka mea i hōʻike ʻia ma ka Papa 2. Ua hoʻohana ʻia ka loiloi Thermogravimetric (TGA) ma ka pae paʻa o PMP e hoʻāʻo no ka pohō kaumaha, 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 nui o ke kalapona (6.35%), ʻoiai ʻaʻole wale nā ​​​​ligands i loaʻa iā C, akā ʻo N, O a me H.
Ua koho ʻia ka ligand phenylmaleimide-methylvinyl isocyanate e hoʻololi i ka ʻili o nā ʻāpana silica ma muli o kāna mau hui phenylmaleimide polar a me vinylisocyanate. Hiki i nā hui vinyl isocyanate ke hana hou aku me ka styrene ma o ka polymerization radical ola. ʻO ke kumu ʻelua, ʻo ia ke hoʻokomo i kahi hui i loaʻa nā pilina kūpono me ka analyte a ʻaʻohe pilina electrostatic ikaika ma waena o ka analyte a me ka pae paʻa, ʻoiai ʻaʻohe uku virtual o ka phenylmaleimide moiety ma ka pH maʻamau. Hiki ke hoʻomalu ʻia ka polarity o ka pae paʻa e ka nui kūpono o ka styrene a me ka manawa pane o ka polymerization radical manuahi. He mea koʻikoʻi ka hana hope loa o ka hopena (polymerization radical manuahi) no ka mea e hoʻololi ia i ka polarity o ka pae paʻa. Ua hana ʻia ka loiloi elemental e nānā i ka ʻike kalapona i loko o kēia mau pae paʻa. Ua ʻike ʻia ʻo ka hoʻonui ʻana i ka nui o ka styrene a me ka manawa pane e hoʻonui i ka ʻike kalapona o ka pae paʻa a me ka hope. ʻO nā SP i hoʻomākaukau ʻia me nā ʻano like ʻole o ka styrene he mau ukana kalapona like ʻole. Pēlā nō, ua kau ʻia kēia mau pae paʻa ma luna o nā kolamu kila kila a ua nānā ʻia ko lākou mau ʻano chromatographic (selectivity, resolution, N value, etc.). Ma muli o kēia mau hoʻokolohua, ua koho ʻia kahi haku mele i hoʻonohonoho pono ʻia no ka hoʻomākaukau ʻana o ka pae paʻa PMP e hāʻawi i ka polarity i kāohi ʻia a me ka paʻa maikaʻi ʻana o ka analyte.
Ua loiloi ʻia hoʻi ke kolamu PMP no ka nānā ʻana i nā hui ʻelima o nā peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) me ka hoʻohana ʻana i ka hiki o ka pae mobile. 60/40 (v/v) ACN/wai (0.1% TFA) ma kahi kahe o 80 µl/min. Ma lalo o nā kūlana elution kūpono (200,000 mau pā/m), ʻo ka helu o nā pā theoretical (N) no kēlā me kēia kolamu (100 × 1.8 mm) he 20,000 ± 100. Hōʻike ʻia nā waiwai N no nā kolamu PMP ʻekolu ma ka Papa 3 a hōʻike ʻia nā chromatograms ma ke Kiʻi 5A. ʻO ka loiloi wikiwiki ma ke kahe kiʻekiʻe (700 µl/min) ma kahi kolamu PMP, ʻelima mau peptides i hoʻoheheʻe ʻia i loko o hoʻokahi minuke, ka waiwai N maikaʻi loa o 13,500 ± 330 no kēlā me kēia kolamu (100 x 1.8 mm ke anawaena), like me 135,000 mau pā/m (Kiʻi 5B). ʻEkolu mau kolamu o ka nui like (ke anawaena o loko 100 x 1.8 mm) i hoʻopiha ʻia me ʻekolu mau pūʻulu like ʻole o ka pae paʻa PMP e hoʻāʻo i ka hana hou ʻana. Ua hoʻopaʻa ʻia nā Analytes no kēlā me kēia kolamu ma ka hoʻokaʻawale ʻana i ka hui hoʻāʻo like ma kēlā me kēia kolamu me ka hoʻohana ʻana i nā kūlana elution kūpono, ka helu o nā pā theoretical N, a me ka manawa paʻa. Hōʻike ʻia ka ʻikepili hana hou no nā kolamu PMP ma ka Papa 4. Ua pili pono ka hana hou ʻana 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 nā hui peptide ma kahi kolamu PMP (B) a me kahi kolamu Ascentis Express RP-Amide (A), pae kelepona 60/40 ACN/H2O (TFA 0.1%), nā ana o ke kolamu PMP (100 x 1.8 mm id), ka nānā ʻana i 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 (leucic acid enkephalin).
Ua loiloi ʻia kahi kolamu PMP (ke anawaena o loko 100 x 1.8 mm) no ka hoʻokaʻawale ʻana o ka hydrolyzate tryptic o ka albumin serum kanaka e HPLC. Hōʻike ka chromatogram ma ke Kiʻi 6 ua hoʻokaʻawale maikaʻi ʻia nā laʻana me ka hoʻonā maikaʻi loa. Ua kālailai ʻia nā hopena HSA me ka hoʻohana ʻana i ka nui o ke kahe o 100 μl/min, kahi pae mobile o 70/30 acetonitrile/wai ​​a me 0.1% TFA. Ua māhele ʻia ka ʻoki ʻana o HSA i 17 mau piko, e like me ka mea i hōʻike ʻia ma ka chromatogram (Kiʻi 6), e pili ana i 17 peptides. Ua helu ʻia nā pono hoʻokaʻawale o nā piko pākahi mai ka hydrolyzate HSA a ua hōʻike ʻia nā waiwai ma ka Papa 5.
Ua hoʻokaʻawale ʻia nā hydrolysates tryptic HSA ma kahi kolamu PMP (ke anawaena o loko 100 x 1.8 mm), ka nui o ke kahe (100 μl/min), ka pae lawe lima 60/40 acetonitrile/wai, a me 0.1% TFA.
kahi ʻo L ka lōʻihi o ke kolamu, ʻo η ka mānoanoa o ka pae lawe lima, ʻo ΔP ke kaomi hope o ke kolamu, a ʻo u ka wikiwiki linear o ka pae lawe lima. ʻO ka permeability o ke kolamu PMP he 2.5 × 10–14 m2, ʻo ka nui o ke kahe he 25 µl/min, ua hoʻohana ʻia he 60/40 v/v. ACN/wai. Ua like ka permeability o ke kolamu PMP (ID 100 × 1.8 mm) me ko kā mākou noiʻi Ref.34 ma mua. ʻO ka permeability o kahi kolamu i piha i nā ʻāpana porous superficially he 1.7 × 10 .6 µm, 2.5 × 10-14 m2 no nā ʻāpana 5 µm43. No laila, ua like ka permeability o ka pae PMP me ka permeability o nā ʻāpana core-shell me ka nui o 5 μm.
kahi ʻo Wx ka nuipa o ke kolamu i hoʻopiha ʻia me ka chloroform, ʻo Wy ka nuipa o ke kolamu i hoʻopiha ʻia me ka methanol, a ʻo ρ ka nuipa o ka mea hoʻoheheʻe. ʻO ka nuipa o ka methanol (ρ = 0.7866) a me ka chloroform (ρ = 1.484). ʻO ka porosity holoʻokoʻa o ke kolamu ʻāpana silica-C18 (100 × 1.8 mm ID)34 a me kā mākou kolamu C18-urea31 i aʻo mua ʻia 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 (ke anawaena o loko 100 × 1.8 mm) he 0.60. ʻOi aku ka liʻiliʻi 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 loko o nā pae paʻa ʻano C18 ua hoʻopili ʻia nā ligands C18 i nā ʻāpana silica i nā kaulahao linear, ʻoiai i loko o nā pae paʻa ʻano polystyrene ua hoʻokumu ʻia kahi polymer mānoanoa a puni nā ʻāpana. papa A. Ma kahi hoʻokolohua maʻamau, ua helu ʻia ka porosity kolamu penei:
Ma ke kiʻi 7A, hōʻike ʻo B i nā kiʻi Van Deemter no kahi kolamu PMP (id 100 x 1.8 mm) a me kahi kolamu Ascentis Express RP-Amide (id 100 x 1.8 mm) ma lalo o nā kūlana elution like, 60/40 ACN/H2O a me 0 .1% TFA 20 µl/min a i 800 µl/min ma nā kolamu ʻelua. ʻO nā waiwai HETP liʻiliʻi loa ma ka helu kahe kūpono (80 µl/min) he 2.6 µm a me 3.9 µm no ke kolamu PMP a me ke kolamu Ascentis Express RP-Amide, kēlā me kēia. Hōʻike nā waiwai HETP ʻoi aku ka kiʻekiʻe o ka pono hoʻokaʻawale o ke kolamu PMP (100 x 1.8 mm id) ma mua o ke kolamu Ascentis Express RP-Amide i loaʻa ma ke kālepa (100 x 1.8 mm id). Hōʻike ka pakuhi van Deemter ma ke Kiʻi 7(A) ʻaʻole i kiʻekiʻe loa 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 pono hoʻokaʻawale o ke kolamu PMP (id 100 × 1.8 mm) i hoʻohālikelike ʻia me ke kolamu Ascentis Express RP-Amide e pili ana i ke ʻano a me ka nui o ka ʻāpana i hoʻomaikaʻi ʻia a me ke kaʻina hana hoʻopili kolamu paʻakikī i hoʻohana ʻia i ka hana o kēia manawa34.
(A) Kiʻi Van Deemter (HETP vs. ka wikiwiki linear pae mobile) i loaʻa ma kahi kolamu PMP (id 100 x 1.8 mm) ma 60/40 ACN/H2O me 0.1% TFA. (B) Kiʻi Van Deemter (HETP vs. ka wikiwiki linear pae mobile) i loaʻa ma kahi kolamu Ascentis Express RP-Amide (id 100 x 1.8 mm) ma 60/40 ACN/H2O me 0.1% TFA.
Ua hoʻomākaukau ʻia a loiloi ʻia kahi pae paʻa polar o ka polystyrene intercalated no ka hoʻokaʻawale ʻana i kahi hui ʻana o nā peptides synthetic a me ka tryptic hydrolyzate o ka albumin serum kanaka (HSA) ma ka chromatography wai hana kiʻekiʻe. He maikaʻi loa ka hana chromatographic o nā kolamu PMP no nā hui peptide ma ke ʻano o ka pono hoʻokaʻawale a me ka hoʻonā. ʻO ka hoʻomaikaʻi ʻana i ka pono hoʻokaʻawale o nā kolamu PMP ma muli o kekahi mau kumu e like me ka nui o ka ʻāpana silica a me ka nui o ka pore, ka synthesis i kāohi ʻia o nā pae paʻa, a me nā mea hoʻopaʻa kolamu paʻakikī. Ma waho aʻe o ka pono hoʻokaʻawale kiʻekiʻe, ʻo kekahi pono o kēia pae paʻa ʻo ia ke kaomi hope haʻahaʻa o ke kolamu ma nā helu kahe kiʻekiʻe. Hiki ke hana hou ʻia nā kolamu PMP a hiki ke hoʻohana ʻia e kālailai i nā hui ʻana o nā peptides a me ka digestion tryptic o nā protein like ʻole. Manaʻo mākou e hoʻohana i kēia kolamu no ka hoʻokaʻawale ʻana i nā hui bioactive mai nā huahana kūlohelohe, nā extracts o nā mea kanu lāʻau lapaʻau a me nā halo 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 antibodies monoclonal.
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