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An shirya ɓangarorin silica mai ƙyalƙyali ta hanyar sol-gel tare da wasu gyare-gyare don samun barbashi mai fadi. An fitar da waɗannan barbashi tare da N-phenylmaleimide-methylvinyl isocyanate (PMI) da styrene ta hanyar juyar da sarkar canja wuri-fragmentation (RAFT) polymerization don samar da N-phenylmaleimide intercalated polyamides. Styrene (PMP) lokacin tsayawa. Ƙunƙarar ginshiƙan bakin karfe (diamita na ciki 100 × 1.8 mm) an cika su tare da fakitin slurry. An kimanta aikin chromatographic na ginshiƙin PMP don raba cakuda peptides na roba wanda ya ƙunshi peptides biyar (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) da tryptic hydrolyzate na albumin ɗan adam. A ƙarƙashin ingantattun yanayin haɓakawa, adadin ka'idar faranti tare da cakuda peptides ya kai faranti 280,000/sq.m. Idan aka kwatanta aikin rabuwa na ginshiƙin da aka haɓaka tare da shafi na Ascentis Express RP-Amide na kasuwanci, an lura cewa ƙimar rabuwa na ginshiƙi na PMP ya fi ginshiƙi na kasuwanci dangane da iyawar rabuwa da ƙuduri.
Masana'antar biopharmaceutical ta zama kasuwa mai faɗaɗawa ta duniya tare da ƙaruwa mai yawa a cikin kason kasuwa a cikin 'yan shekarun nan. Tare da haɓakar fashewar masana'antar biopharmaceutical1,2,3 akwai babban buƙatar peptide da nazarin furotin. Baya ga peptide da aka yi niyya, ana samun ƙazanta daban-daban a yayin haɗin peptide, don haka ana buƙatar tsarkakewar chromatographic don samun tsarkakakken peptide da ake so. Bincike da halayyar sunadaran sunadaran a cikin ruwan jiki, kyallen takarda, da sel aiki ne mai ƙalubale sosai saboda yawancin nau'ikan da ake iya ganowa a cikin samfuri ɗaya. Kodayake ma'aunin spectrometry shine kayan aiki mai tasiri don tsara peptides da sunadarai, idan an gabatar da irin waɗannan samfuran kai tsaye a cikin ma'aunin ma'aunin nauyi, rabuwar ba zata gamsu ba. Ana iya magance wannan matsala ta hanyar yin chromatography na ruwa (LC) kafin nazarin MS, wanda zai rage adadin masu nazarin da ke shiga cikin ma'auni mai yawa a wani lokaci4,5,6. Bugu da kari, manazarta na iya maida hankali a cikin kunkuntar yanki yayin rabuwar lokaci na ruwa, ta haka ne ke ba da hankali ga waɗannan nazarce-nazarcen da kuma haɓaka ƙwarewar gano MS. Liquid chromatography (LC) ya ci gaba sosai a cikin shekaru goma da suka gabata kuma ya zama hanyar da aka yi amfani da ita sosai don nazarin ƙwayoyin cuta7,8,9,10.
Reverse-phase liquid chromatography (RP-LC) ana amfani dashi sosai don tsarkakewa da raba gaurayawan peptides ta amfani da silica-modified octadecyl (ODS) azaman tsayayyen lokaci11,12,13. Koyaya, saboda tsarinsu mai rikitarwa da yanayin amphoteric, matakan tsayawa na 14,15 RP ba zasu iya samar da gamsasshen rabuwa na peptides da sunadarai ba. Don haka, nazarin peptides da furotin tare da ɓangarorin polar da waɗanda ba na polar ba suna buƙatar tsayayyen matakai na musamman don yin hulɗa tare da riƙe waɗannan nazarin16. Haɗaɗɗen chromatography, wanda ke ba da hulɗar multimodal, na iya zama madadin RP-LC don raba peptides, sunadarai, da sauran hadaddun gaurayawan. An shirya nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan tsayi da yawa kuma an yi amfani da ginshiƙan da ke cike da waɗannan matakan tsaye don ware peptides da sunadarai17,18,19,20,21. Saboda kasancewar polar da ƙungiyoyi masu zaman kansu, gauraye matakan matakan tsaye (WAX / RPLC, HILIC / RPLC, polar intercalation / RPLC) sun dace da rabuwa da peptides da sunadarai22,23,24,25,26,27,28. , iyakacin duniya intercalated tsaye bulan tare da covalently bonded iyakacin duniya kungiyoyin nuna mai kyau rabuwa damar da musamman selectivity ga iyakacin duniya da kuma wadanda ba iyakacin duniya analytes domin rabuwa dogara a kan hulda tsakanin Analyte da kuma m lokaci Multimodal hulda 29,30,31,32. Kwanan nan, Zhang et al. 30 sun sami matakan tsayayye na behenyl na polyamines kuma sun sami nasarar raba hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, da wasu masu nazari. Kayan da aka saka na iyakacin duniya yana da ƙungiyoyi biyu na polar da marasa iyaka, don haka ana iya amfani da shi don raba peptides da sunadarai zuwa sassan hydrophobic da hydrophilic. ginshiƙan layi na Polar (misali, ginshiƙan C18 tare da layin amide) suna samuwa a ƙarƙashin sunan kasuwanci Ascentis Express RP-Amide ginshiƙan, amma waɗannan ginshiƙan an yi amfani da su ne kawai don nazarin amine 33.
A cikin binciken na yanzu, an shirya lokacin shigar da igiya mai tsayi (N-phenylmaleimide, saka polystyrene) kuma an kimanta shi don rabuwar peptide da tsagewar HSA. Anyi amfani da dabarar da ke gaba don shirya tsayayyen lokaci. An shirya nau'in siliki mai laushi bisa ga hanyoyin da aka bayyana a cikin wallafe-wallafen da suka gabata, tare da wasu canje-canje a cikin shirye-shiryen shirye-shiryen 31, 34, 35, 36, 37, 38, 39. An daidaita ma'auni na urea, polyethylene glycol (PEG), TMOS da acid-acetic acid don samun manyan silica. Abu na biyu, an haɗa wani sabon phenylmaleimide-methylvinyl isocyanate ligand kuma an yi amfani da barbashi na silica da aka samo asali don shirya matakan da suka dace na polar. Matsayin tsayawar da aka samu an cushe shi cikin ginshiƙin bakin karfe (diamita na ciki 100 × 1.8 mm) bisa ga ingantaccen tsarin tattara kaya. Ana taimakon fakitin ginshiƙi ta hanyar girgizar injin don tabbatar da daidaitaccen Layer a cikin ginshiƙi. An ƙididdige ginshiƙin da aka cika don rabuwa da cakuda peptides wanda ya ƙunshi peptides biyar (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). da kuma tryptic hydrolysates na jikin mutum albumin (HSA). An lura cewa cakuda peptide da HSA tryptic narkewa sun rabu tare da kyakkyawan ƙuduri da inganci. An kwatanta ingancin rabuwa na ginshiƙin PMP da na Ascentis Express RP-Amide shafi. An lura cewa peptides da sunadaran suna da kyakkyawan ƙuduri da babban tasiri na rabuwa akan ginshiƙi na PMP, kuma tasirin rabuwa na ɓangaren PMP ya fi na Ascentis Express RP-Amide shafi.
PEG (polyethylene glycol), urea, acetic acid, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, jini albumin mutum (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride (MC), POZ-hydrone benzo, styrezone, styrene. acetonitrile (ACN) don HPLC, methanol, 2-propanol da acetone. Kamfanin Sigma-Aldrich (St. Louis, Missouri, Amurka).
Cakuda urea (8 g), polyethylene glycol (8 g) da 8 ml na 0.01 N. acetic acid an zuga na minti 10, kuma an ƙara 24 ml na TMOS a ƙarƙashin sanyi-kankara. An gauraya cakudawar da aka yi a 40 ° C na tsawon awanni 6 sannan kuma a 120 ° C na awanni 8 a cikin autoclave bakin karfe. An cire ruwan kuma an bushe ragowar a 70 ° C na tsawon awanni 12. An niƙa busassun busassun busassun lallausan kuma an sanya su a cikin tanda a 550 ° C na awanni 12. Uku batches aka shirya da halin gwada reproducibility na barbashi masu girma dabam, pore size da surface area.
Ƙungiyar Polar da lokacin tsayawa don sarƙoƙin polystyrene. An kwatanta tsarin shirye-shiryen a kasa.
N-phenylmaleimide (200 MG) da methyl vinyl isocyanate (100 MG) an narkar da su a cikin anhydrous toluene, sa'an nan 0.1 ml na 2,2′-azoisobutyronitrile (AIBN) aka kara zuwa dauki flask don samun wani copolymer na phenylmaleimCPide da methyl vinyl vinyl (PM). ) An yi amfani da cakuda a zafin jiki na 60 ° C na tsawon sa'o'i 3, an tace kuma a bushe a cikin tanda a 40 ° C na 3 hours.
Dried silica barbashi (2 g) aka tarwatsa a bushe toluene (100 ml), zuga da sonicated for 10 min a 500 ml zagaye kasa flask. PMCP (10 MG) an narkar da shi a cikin toluene kuma an ƙara dropwise zuwa flask na amsawa ta hanyar ƙarin mazurari. An sake dawo da cakuda a 100 ° C na tsawon sa'o'i 8, tace, wanke da acetone kuma a bushe a 60 ° C na 3 hours. Sa'an nan, silica barbashi hade da PMCP (100 g) aka narkar da a toluene (200 ml), da kuma 4-hydroxy-TEMPO (2 ml) aka kara a ciki a gaban 100 μl na dibutyltin dilaurarate a matsayin mai kara kuzari. An motsa cakuda a 50 ° C na tsawon sa'o'i 8, tace kuma a bushe a 50 ° C na 3 hours.
Styrene (1 ml), benzoyl peroxide BPO (0.5 ml) da silica barbashi da aka haɗe zuwa TEMPO-PMCP (1.5 g) an tarwatsa a cikin toluene da kuma tsarkake da nitrogen. An gudanar da polymerization na styrene a 100 ° C na 12 hours. An wanke samfurin da aka samu tare da methanol kuma an bushe shi cikin dare a 60 ° C. An nuna tsarin gaba ɗaya na amsa a cikin fig. daya .
An lalata samfuran a 393 K don 1 h har sai an sami ragowar matsa lamba na ƙasa da 10-3 Torr. Adadin N2 da aka tallata a matsa lamba P/P0 = 0.99 an yi amfani dashi don ƙayyade jimlar pore. An yi nazarin yanayin halittar silica mai tsafta da ligand mai ɗaure ta amfani da na'urar duban haske ta lantarki (Hitachi High Technologies, Tokyo, Japan). Samfuran busassun (tsaftataccen silica da ligand daure silica barbashi) an sanya su akan sandunan aluminum ta amfani da tef ɗin carbon. An ajiye zinari akan samfurin ta amfani da na'urar sputtering Q150T, kuma an ajiye wani Layer Au mai kauri 5 nm akan samfurin. Wannan yana inganta ingantaccen tsarin ƙarancin ƙarfin lantarki kuma yana ba da feshin sanyi mai kyau. An yi nazarin abubuwan da aka haɗa ta amfani da Thermo Electron (Waltham, MA, Amurka) Flash EA1112 mai nazarin abun da ke ciki. An yi amfani da mai nazarin girman girman barbashi na Malvern (Worcestershire, UK) Mastersizer 2000 don samun rabon girman barbashi. Uncoated silica barbashi da ligand-daure silica barbashi (5 MG kowane) aka tarwatsa a 5 ml na isopropanol, sonicated for 10 minutes, agitated for 5 minutes, kuma sanya a kan Mastersizer Tantancewar benci. Ana gudanar da bincike na Thermogravimetric a cikin adadin 5 ° C a minti daya a cikin kewayon zafin jiki daga 30 zuwa 800 ° C.
Gilashin fiber liyi kunkuntar ginshiƙan ginshiƙan ƙarfe tare da girma (ID 100 × 1.8 mm) an cika su ta hanyar slurry cika hanyar bin hanya ɗaya kamar yadda aka yi nuni da 31. Bakin ƙarfe ginshiƙi (layi na gilashi, ID 100 × 1 .8 mm) da kanti mai ɗauke da 1 µm frit ɗin da aka haɗa zuwa injin fakitin USA, IL mashin. Shirya dakatarwar lokaci mai tsayi ta hanyar dakatar da 150 MG na lokaci a tsaye a cikin 1.2 ml na methanol da ciyar da shi cikin ginshiƙin tafki. An yi amfani da methanol azaman slurry da sauran ƙarfi mai sarrafawa. Shirya ginshiƙi ta amfani da jerin matsa lamba na 100 MP na minti 10, 80 MP na minti 15, da 60 MP na minti 30. Tsarin tattarawa ya yi amfani da firgita ginshiƙan chromatography gas guda biyu (Alltech, Deerfield, IL, Amurka) don girgiza injina don tabbatar da tattarawar ginshiƙi iri ɗaya. Rufe fakitin slurry kuma saki matsa lamba a hankali don hana lalacewa ga kirtani. An cire haɗin ginshiƙi daga bututun slurry kuma an haɗa wani abin dacewa zuwa mashigar kuma an haɗa shi da tsarin LC don gwada aikin sa.
An gina MLC na al'ada ta amfani da famfo na LC (10AD Shimadzu, Japan), samfurin samfurin tare da madauki na allura na 50 nL (Valco (Amurka) C14 W.05), membrane degasser (Shimadzu DGU-14A), da kuma UV-VIS capillary taga. Na'urar ganowa (UV-2075) da ƙaramar ƙararrawa. Yi amfani da kunkuntar bututu masu haɗawa don rage tasirin ƙarin faɗaɗa shafi. Bayan cika ginshiƙi, shigar da capillary (50 µm id 365) a madaidaicin 1/16 ″ rage junction kuma shigar da capillary (50 µm) na mahaɗar ragewa. Ana yin tarin bayanai da sarrafa chromatogram ta amfani da software na Multichro 2000. A 254 nm, ana kula da shayarwar UV na masu nazarin batutuwa a 0. An yi nazarin bayanan Chromatographic ta amfani da OriginPro8 (Northampton, MA).
Serum albumin na mutum, lyophilized foda, ≥ 96% (agarose gel electrophoresis) 3 MG gauraye da trypsin (1.5 MG), 4.0 M urea (1 ml) da 0.2 M ammonium bicarbonate (1 ml) . An zuga maganin don 10 min kuma an ajiye shi a cikin wanka na ruwa a 37 ° C na 6 h, sa'an nan kuma ya ƙare tare da 1 ml na 0.1% TFA. Tace maganin kuma adana a kasa 4°C.
An kimanta rarrabuwar cakuda peptides da narkewar HSA a kan ginshiƙin PMP daban. Duba tryptic hydrolysis na cakuda peptides da HSA rabu da wani PMP shafi kuma kwatanta sakamakon da wani Ascentis Express RP-Amide shafi. Ana ƙididdige adadin faranti na ka'idar ta amfani da ma'auni mai zuwa:
Hotunan SEM na nau'in siliki mai tsabta da ligand da aka ɗaure silica an nuna su a cikin Hoto 2. Hotunan SEM na nau'in siliki mai tsabta (A, B) suna nuna siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar siffar . Fuskar siliki da aka ɗaure da ligand (C, D) ya fi santsi fiye da na siliki mai tsabta, wanda zai iya zama saboda sarƙoƙi na polystyrene da ke rufe saman sassan silica.
Ana duba micrographs na lantarki na tsantsar silica barbashi (A, B) da ligand daure silica barbashi (C, D).
The barbashi size rarraba tsarki silica barbashi da ligand-daure silica barbashi aka nuna a cikin siffa 2. 3 (A). Volumetric barbashi size rarraba masu lankwasa ya nuna cewa silica barbashi size ya karu bayan sinadaran gyara (Fig. 3A). Silica barbashi size rarraba bayanai daga halin yanzu da binciken da aka kwatanta a cikin Table 1 (A). Girman barbashi na d (0.5) na PMP shine 3.36 µm, idan aka kwatanta da ad (0.5) ƙimar 3.05 µm a cikin bincikenmu na baya (polystyrene bonded silica barbashi)34. Saboda canji a cikin rabo na PEG, urea, TMOS da acetic acid a cikin cakudewar dauki, girman rabon wannan batch ya fi kunkuntar idan aka kwatanta da bincikenmu na baya. Girman barbashi na lokacin PMP ya ɗan fi girma fiye da na polystyrene daure silica barbashi lokaci wanda muka yi nazari a baya. Wannan yana nufin cewa aikin shimfidar abubuwan silica tare da styrene an ajiye shi kawai Layer polystyrene (0.97 µm) akan saman silica, yayin da lokacin PMP kauri ya kasance 1.38 µm.
Rarraba girman barbashi (A) da rarraba girman pore (B) na tsantsar silica barbashi da ligand daure silica barbashi.
Girman pore, ƙarar pore, da farfajiyar ɓangarorin silica da aka yi amfani da su a cikin wannan binciken an nuna su a cikin Table 1 (B). Bayanan martaba na PSD na tsantsar silica barbashi da ligand-daure barbashi ana nuna su a cikin Figs. 3 (B). Sakamakon ya yi daidai da binciken da muka gabata34. Matsakaicin nau'ikan silica mai tsafta da ligand sun kasance 310 Å da 241 Å, bi da bi, yana nuna cewa bayan gyare-gyaren sinadarai, girman pore ya ragu da 69 Å, kamar yadda aka nuna a cikin Table 1 (B), kuma an nuna madaidaicin motsi a cikin Fig. Ƙayyadadden yanki na silica barbashi a cikin binciken da aka yi a baya shine m211111 na baya. (124m2/g). Kamar yadda aka nuna a cikin Table 1 (B), filin sararin samaniya (m2 / g) na silica barbashi bayan gyare-gyaren sinadarai kuma ya ragu daga 116 m2 / g zuwa 105 m2 / g.
An gabatar da sakamakon bincike na farko na lokaci a tsaye a cikin Tebura. 2. Abubuwan da ke cikin carbon na lokaci mai tsayi na yanzu shine 6.35%, wanda ya fi ƙasa da bincikenmu na baya (silica barbashi da ke hade da polystyrene, 7.93% 35 da 10.21%, bi da bi) 42. Abubuwan da ke cikin carbon na halin yanzu na tsaye a kasa, tun da wasu nau'o'in polar ligands irin su phenylmaleimide da kuma methyl-TEvinyl TEvinyl (PCYMP) suna da methyl-TEvinyl oxide. An yi amfani da ban da styrene a cikin shirye-shiryen SP. Adadin nauyin nitrogen a cikin lokacin tsayawa na yanzu shine 2.21% idan aka kwatanta da 0.1735 da 0.85% a cikin binciken da suka gabata42. Wannan yana nufin cewa lokacin tsayawa na yanzu yana da babban nauyin kaso na nitrogen saboda phenylmaleimide. Hakazalika, samfurori (4) da (5) suna da abun ciki na carbon na 2.7% da 2.9%, bi da bi, yayin da samfurin ƙarshe (6) yana da abun ciki na carbon na 6.35%, kamar yadda aka nuna a cikin Table 2. Thermogravimetric analysis (TGA) da aka yi amfani da a kan m lokaci na PMP don gwada nauyi asara, da kuma TGA kwana da aka nuna a cikin adadi mai kyau na 6. yarjejeniya tare da abun ciki na carbon (6.35%), tun da ligands sun ƙunshi ba kawai C ba, har ma N, O da H.
An zaɓi ligand phenylmaleimide-methylvinyl isocyanate don gyara saman silica barbashi saboda polar phenylmaleimide da vinylisocyanate kungiyoyin. Ƙungiyoyin isocyanate na Vinyl na iya ƙara mayar da martani tare da styrene ta hanyar polymerization mai rayayye. Dalili na biyu shi ne saka ƙungiyar da ke da matsakaicin hulɗa tare da mai nazari kuma babu ƙarfin hulɗar electrostatic tsakanin analyte da kuma lokacin tsayawa, tun da phenylmaleimide moiety ba shi da cajin kama-da-wane a al'ada pH. Za a iya sarrafa polarity na lokaci mai tsayi ta mafi kyawun adadin styrene da lokacin amsawar polymerization na radicals kyauta. Mataki na ƙarshe na martani (polymerization na radicals kyauta) yana da mahimmanci yayin da yake canza polarity na lokacin tsaye. An gudanar da bincike na asali don bincika abubuwan da ke cikin carbon a cikin waɗannan matakan tsaye. An lura cewa ƙara yawan adadin styrene da lokacin amsawa yana ƙaruwa da abun ciki na carbon na lokaci mai tsayi kuma akasin haka. SPs da aka shirya tare da nau'i daban-daban na styrene suna da nauyin carbon daban-daban. Hakazalika, waɗannan matakan tsaye an sanya su akan ginshiƙan bakin karfe kuma an duba halayensu na chromatographic (zaɓi, ƙuduri, ƙimar N, da sauransu). Dangane da waɗannan gwaje-gwajen, an zaɓi ingantaccen abun da ke ciki don shirye-shiryen tsayayyen lokaci na PMP don samar da polarity mai sarrafawa da kyakkyawar riƙe mai nazari.
An kuma kimanta ginshiƙin PMP don nazarin gaurayawan peptides guda biyar (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) ta amfani da ƙarfin lokacin wayar hannu. 60/40 (v/v) ACN/ruwa (0.1% TFA) a yawan gudu na 80 µl/min. A karkashin yanayi mafi kyau duka (200,000 faranti / m), adadin faranti na ka'idar (N) a kowane shafi (100 × 1.8 mm) shine 20,000 ± 100. Ana nuna ƙimar N na ginshiƙan PMP guda uku a cikin Table 3 kuma ana nuna chromatograms a cikin Hoto 5A. Bincike mai sauri a babban adadin kwarara (700 µl / min) akan ginshiƙi na PMP, peptides biyar sun ɓace a cikin minti daya, kyakkyawan darajar N na 13,500 ± 330 a kowane shafi (100 x 1.8 mm diamita), daidai da 135,000 faranti / m (Fig. 5B). An cika ginshiƙai uku na girman ɗaya (diamita na ciki 100 x 1.8 mm) da batches uku daban-daban na lokacin tsayawar PMP don gwada sake haifuwa. An yi rikodin ƙididdiga don kowane ginshiƙi ta hanyar rarraba cakuda gwajin guda ɗaya akan kowane shafi ta amfani da mafi kyawun yanayin haɓakawa, adadin faranti N, da lokacin riƙewa. An nuna bayanan sake fasalin ginshiƙan PMP a cikin Tebura 4. Sake fasalin ginshiƙin PMP yana da alaƙa da kyau tare da ƙananan ƙimar % RSD kamar yadda aka nuna a cikin Table 3.
Rabewar cakuda peptide akan shafi na PMP (B) da kuma Ascentis Express RP-Amide shafi (A), tsarin wayar hannu 60/40 ACN/H2O (TFA 0.1%), Ma'aunin shafi na PMP (100 x 1.8 mm id) , Binciken Elution na mahadi: 1 (Gly-Tyr), - 2 (Tyr) (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) da 5 (leucic acid enkephalin).
An kimanta ginshiƙin PMP (diamita na ciki 100 x 1.8 mm) don rabuwa na tryptic hydrolyzate na albinin ɗan adam ta HPLC. chromatogram a cikin hoto na 6 ya nuna cewa samfurori sun rabu da kyau tare da ƙuduri mai kyau. An yi nazarin hanyoyin HSA ta amfani da ƙimar kwarara na 100 μl / min, tsarin wayar hannu na 70/30 acetonitrile / ruwa da 0.1% TFA. An raba raguwa na HSA zuwa kololuwa 17, kamar yadda aka nuna a cikin chromatogram (Fig. 6), daidai da 17 peptides. An ƙididdige ingancin rarrabuwar kawuna na kowane kololuwa daga HSA hydrolyzate kuma ana nuna ƙimar a cikin Tebur 5.
An raba HSA tryptic hydrolysates akan ginshiƙin PMP (diamita na ciki 100 x 1.8 mm), ƙimar kwarara (100 μl / min), lokacin wayar hannu 60/40 acetonitrile / ruwa, da 0.1% TFA.
inda L shine tsayin ginshiƙi, η shine danko na lokaci na wayar hannu, ΔP shine matsa lamba na baya na ginshiƙi, kuma u shine saurin layin wayar hannu. Matsakaicin ginshiƙi na PMP shine 2.5 × 10-14 m2, yawan kwarara ya kasance 25 µl/min, 60/40 v/v an yi amfani da shi. ACN/ruwa. Matsakaicin ginshiƙin PMP (ID 100 × 1.8 mm) yayi kama da na bincikenmu na baya Ref.34. Matsakaicin ginshiƙi da ke cike da ɓangarorin da ba su da ƙarfi shine 1.7 × 10 .6 µm, 2.5 × 10-14 m2 don 5 µm barbashi43. Sabili da haka, ƙaddamarwa na lokaci na PMP yana kama da ƙaddamar da ƙananan ƙananan harsashi tare da girman 5 μm.
inda Wx shine yawan ginshiƙin da ke cike da chloroform, Wy shine yawan ginshiƙin da ke cike da methanol, kuma ρ shine yawan ƙarfi. Yawan methanol (ρ = 0.7866) da chloroform (ρ = 1.484). Jimlar porosity na silica-C18 barbashi ginshiƙi (100 × 1.8 mm ID) 34 da kuma binciken da muka yi a baya C18-urea31 shafi ya kasance 0.63 da 0.55, bi da bi. Wannan yana nufin cewa kasancewar urea ligands yana rage haɓakar lokaci na tsaye. A gefe guda, jimlar porosity na ginshiƙin PMP (diamita na ciki 100 × 1.8 mm) shine 0.60. ginshiƙan PMP ba su da ƙarancin lalacewa fiye da ginshiƙan da ke cike da nau'ikan silica da ke daure C18 saboda a cikin nau'ikan nau'ikan nau'ikan nau'ikan silica na C18 ana haɗa haɗin C18 zuwa barbashi na silica a cikin sarƙoƙi na madaidaiciya, yayin da a cikin nau'ikan nau'ikan polystyrene an kafa polymer mai kauri a kusa da barbashi. Layer A. A cikin gwaji na yau da kullun, ana lissafta porosity na shafi kamar haka:
A kan fig. 7A, B yana nuna makircin Van Deemter don ginshiƙin PMP (id 100 x 1.8 mm) da ginshiƙin Ascentis Express RP-Amide (id 100 x 1.8 mm) ƙarƙashin yanayin haɓaka iri ɗaya, 60/40 ACN/H2O da 0 .1% TFA 20 μl/min akan ginshiƙan 8. Matsakaicin ƙimar HETP a mafi kyawun ƙimar kwarara (80 µl/min) sun kasance 2.6 µm da 3.9 µm don ginshiƙin PMP da ginshiƙin Ascentis Express RP-Amide, bi da bi. Ƙimar HETP sun nuna cewa ingancin rabuwa na ginshiƙin PMP (100 x 1.8 mm id) ya fi girma fiye da na kasuwancin Ascentis Express RP-Amide (100 x 1.8 mm id). Hoton van Deemter a cikin hoto na 7 (A) ya nuna cewa raguwar darajar N ba ta da girma tare da karuwa mai yawa idan aka kwatanta da bincikenmu na baya. Mafi girman ingancin rarrabuwa na ginshiƙin PMP (id 100 × 1.8 mm) idan aka kwatanta da ginshiƙin Ascentis Express RP-Amide ya dogara ne akan ingantattun sifofin ɓangarorin da girman da ƙayyadaddun tsarin tattarawa na shafi da aka yi amfani da su a cikin aikin yanzu34.
(A) Makircin Van Deemter (HETP vs. saurin layin layin wayar hannu) da aka samu akan ginshiƙin PMP (id 100 x 1.8 mm) a cikin 60/40 ACN/H2O tare da 0.1% TFA. (B) Makircin Van Deemter (HETP tare da saurin layin layi na wayar hannu) da aka samu akan ginshiƙin Ascentis Express RP-Amide (id 100 x 1.8 mm) a cikin 60/40 ACN/H2O tare da 0.1% TFA.
An shirya wani lokaci mai tsayayye na polystyrene tare da kimantawa don rarrabuwar cakuda peptides na roba da tryptic hydrolyzate na albinin ɗan adam (HSA) a cikin babban aikin chromatography na ruwa. Ayyukan chromatographic na ginshiƙan PMP don gaurayawan peptide yana da kyau kwarai dangane da iyawar rabuwa da ƙuduri. The ingantattun rabuwa yadda ya dace na ginshiƙan PMP shine saboda dalilai da yawa kamar silica barbashi girman da pore size, sarrafawa kira na tsaye bulan, da hadaddun shafi shiryawa kayan. Bugu da ƙari ga babban haɓakar rabuwa, wani fa'ida na wannan lokaci na tsaye shine ƙananan ginshiƙan baya a madaidaicin magudanar ruwa. ginshiƙan PMP suna da haɓakawa sosai kuma ana iya amfani da su don nazarin gaurayawan peptides da narkar da ƙwayoyin sunadarai daban-daban. Mun yi niyyar amfani da wannan shafi don rabuwa da mahaɗan bioactive daga samfuran halitta, tsantsa na tsire-tsire na magani da namomin kaza a cikin chromatography na ruwa. A nan gaba, za a kuma kimanta ginshiƙan PMP don rarrabuwar sunadaran da ƙwayoyin rigakafi na monoclonal.
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