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Ejiri usoro sol-gel kwadoro ụmụ irighiri ihe ndị na-adịghị mma nke nwere ụfọdụ mgbanwe iji nweta ihe ndị nwere oghere oghere. Ejiri N-phenylmaleimide-methylvinyl isocyanate (PMI) na styrene wepụta ihe ndị a site na polymerization reverse chain transfer-fragmentation (RAFT) iji mepụta N-phenylmaleimide intercalated polyamides. Styrene (PMP) na-eguzosi ike n'ihe. Ogidi igwe anaghị agba nchara dị warara (dayameta 100 × 1.8 mm n'ime) ka ejiri nkwakọ ngwaahịa slurry juru. A na-enyocha arụmọrụ chromatographic nke kọlụm PMP iji kewaa ngwakọta nke peptides sịntetik mejupụtara peptides ise (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) na tryptic hydrolyzate nke albumin mmadụ. N'okpuru ọnọdụ elution kacha mma, ọnụ ọgụgụ ihe atụ nke efere nwere ngwakọta nke peptides ruru 280,000 plates/sq.m. N'ịtụle arụmọrụ nkewa nke kọlụm mepere emepe na kọlụm Ascentis Express RP-Amide nke azụmahịa, a chọpụtara na arụmọrụ nkewa nke kọlụm PMP dị elu karịa kọlụm azụmahịa n'ihe gbasara arụmọrụ na mkpebi nkewa.
Ụlọ ọrụ biopharmaceutical aghọwo ahịa zuru ụwa ọnụ na-agbasawanye yana mmụba dị ukwuu na òkè ahịa n'afọ ndị na-adịbeghị anya. Site na uto mgbawa nke ụlọ ọrụ biopharmaceutical1,2,3 enwere nnukwu mkpa maka nyocha peptide na protein. Na mgbakwunye na peptide lekwasịrị anya, a na-emepụta adịghị ọcha dị iche iche n'oge njikọ peptide, ya mere a na-achọ ịdị ọcha chromatographic iji nweta ịdị ọcha nke peptide chọrọ. Nyocha na nkọwapụta nke protein na mmiri ahụ, anụ ahụ, na sel bụ ọrụ siri ike nke ukwuu n'ihi ọnụ ọgụgụ buru ibu nke ụdị nwere ike ịchọpụta dị n'otu nlele. Ọ bụ ezie na spectrometry mass bụ ngwá ọrụ dị irè maka ịhazi peptides na protein, ma ọ bụrụ na a na-ewebata ihe nlele ndị dị otú ahụ na spectrometer mass, nkewa ahụ agaghị enwe afọ ojuju. Enwere ike idozi nsogbu a site n'ịrụ chromatography mmiri (LC) tupu nyocha MS, nke ga-ebelata ọnụ ọgụgụ ndị nyocha na-abanye na spectrometer mass na oge enyere4,5,6. Na mgbakwunye, ndị nyocha nwere ike itinye uche na mpaghara dị warara n'oge nkewa mmiri mmiri, si otú a na-etinye uche na nyocha ndị a ma na-abawanye nghọta nke nchọpụta MS. Liquid chromatography (LC) abawanyela nke ukwuu n'ime afọ iri gara aga ma bụrụkwa usoro eji eme ihe maka nyocha proteomic7,8,9,10.
A na-eji chromatography mmiri mmiri reverse-phase (RP-LC) mee ka ọ dị ọcha na kewapụ ngwakọta nke peptides site na iji octadecyl-modified silica (ODS) dị ka nkeji oge 11,12,13. Otú ọ dị, n'ihi usoro mgbagwoju anya ha na ọdịdị amphoteric, usoro nkwụsị 14,15 RP enweghị ike inye nkewa na-eju afọ nke peptides na protein. Ya mere, nyocha nke peptides na protein ndị nwere iberibe polar na ndị na-abụghị polar chọrọ usoro ndị a haziri ahazi nke ọma ka ha na-emekọrịta ihe na idowe nyocha ndị a16. Ngwakọta chromatography, nke na-enye mmekọrịta multimodal, nwere ike ịbụ ihe ọzọ na RP-LC maka ikewapụta peptides, protein, na ngwakọta ndị ọzọ dị mgbagwoju anya. A kwadebere ọtụtụ usoro nkwụsị ụdị agwakọtara na kọlụm jupụtara n'usoro ndị a guzoro ọtọ ka ejiri kewaa peptides na protein17,18,19,20,21. N'ihi ọnụnọ nke polar na ndị na-abụghị polar otu, agwakọta mode guzo n'ókè (WAX / RPLC, HILIC / RPLC, polar intercalation / RPLC) kwesịrị ekwesị maka nkewa nke peptides na proteins22,23,24,25,26,27,28. , polar intercalated guzo n'usoro na covalently bonded pola iche iche na-egosi ezi nkewa ike na pụrụ iche selectivity maka pola na-abụghị polar analytes n'ihi na nkewa dabere na mmekọrịta dị n'etiti nyocha na ọdụ ọdụ Multimodal interactions 29,30,31,32. N'oge na-adịbeghị anya, Zhang et al. 30 nwetara usoro nke polyamines kwụsịrị behenyl wee kewaa hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens na ụfọdụ nyocha ndị ọzọ. Polar agbakwunyere ihe na-adịgide adịgide nwere ma polar na ndị na-abụghị polar, yabụ enwere ike iji ya kewaa peptides na protein n'ime akụkụ hydrophobic na hydrophilic. Ogidi inline dị n'okpuru aha ahia Ascentis Express RP-Amide, mana ogidi ndị a ka ejirila naanị nyocha nke amine 33.
N'ime ọmụmụ ihe ugbu a, a kwadebere ma nyochaa usoro ntinye nke polar (N-phenylmaleimide, embeding polystyrene) maka nkewa peptide na nkwụsị nke HSA tryptic. Ejiri atụmatụ a na-esonụ iji kwado oge nkwụsị. A na-akwadebe ihe ndị dị na silica porous dị ka usoro ndị a kọwara na akwụkwọ anyị gara aga, na mgbanwe ụfọdụ na atụmatụ nkwadebe 31, 34, 35, 36, 37, 38, 39. A na-edozi oke nke urea, polyethylene glycol (PEG), TMOS na aqueous-acetic acid iji nweta nnukwu mkpụrụ osisi silica. Nke abuo, ihe ohuru phenylmaleimide-methylvinyl isocyanate ligand bụ synthesized na-ejiri silica ụmụ irighiri ihe na-ewepụtara ya iji kwadebe pola agbakwunyere usoro guzobe. Achịkọbara akụkụ a na-akwụghị ụgwọ enwetara n'ime kọlụm igwe anaghị agba nchara (dayameta dị n'ime 100 × 1.8 mm) dịka atụmatụ mbukota kachasị mma. A na-enyere mkpọkọ nke kọlụm aka site na ịma jijiji n'ibu iji hụ na otu oyi akwa dị n'ime kọlụm. A tụlere kọlụm a juru eju maka nkewa nke ngwakọta peptides nwere peptides ise (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). na tryptic hydrolysates nke mmadụ serum albumin (HSA). Achọpụtara na ngwakọta peptide na HSA tryptic digest kewapụrụ na mkpebi dị mma na arụmọrụ. Atụnyere arụmọrụ nkewa nke kọlụm PMP na nke kọlụm Ascentis Express RP-Amide. Achọpụtara na peptides na ndị na-edozi ahụ nwere mkpebi dị mma na oke nkewa dị elu na kọlụm PMP, na nkewa nkewa nke kọlụm PMP dị elu karịa nke Ascentis Express RP-Amide kọlụm.
PEG (polyethylene glycol), urea, acetic acid, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, mmadụ serum albumin (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl chloride (MC), POZ-hydrone, styrene. acetonitrile (ACN) maka HPLC, methanol, 2-propanol na acetone. Ụlọ ọrụ Sigma-Aldrich (St. Louis, Missouri, USA).
Ngwakọta urea (8 g), polyethylene glycol (8 g) na 8 ml nke 0.01 N. acetic acid kpalitere maka nkeji 10, na 24 ml nke TMOS tinye ya n'okpuru mmiri oyi. Ngwakọta mmeghachi omume na-ekpo ọkụ na 40 ° C maka awa 6 wee mee ya na 120 Celsius maka awa 8 na igwe anaghị agba nchara autoclave. A na-ewepụ mmiri ahụ ma kpochaa ihe fọdụrụ na 70 Celsius maka awa 12. A na-egweri ihe mgbochi ndị ahụ dị nro nke ọma ma gbanye ya na oven na 550 Celsius maka awa 12. A kwadebere batches atọ ma mara iji nwalee reproducibility nke nha nha, nha pore na mpaghara elu.
Otu polar na oge guzoro maka agbụ polystyrene. A kọwara usoro nkwadebe n'okpuru.
N-phenylmaleimide (200 mg) na methyl vinyl isocyanate (100 mg) gbazere na toluene anhydrous, wee tinye 0.1 ml nke 2,2′-azoisobutyronitrile (AIBN) na flask mmeghachi omume iji nweta copolymer nke phenylmaleimCPide na methylate vinyl (PM). ) A na-eme ka ngwakọta ahụ dị ọkụ na 60 ° C maka awa 3, kpochaa ma kpoo ya na oven na 40 ° C maka awa 3.
A na-achụsasị silica mịrị amị (2 g) na toluene akọrọ (100 ml), kpalitere na sonicated maka 10 min na 500 ml gburugburu flask ala. PMCP (10 mg) gbazere na toluene wee gbakwunye ya na flask mmeghachi omume site na oghere mgbakwunye. A na-emegharị ngwakọta ahụ na 100 ° C maka awa 8, kpochaa, saa ya na acetone ma kpoo ya na 60 ° C maka awa 3. Mgbe ahụ, a na-agbaze akụkụ silica nke metụtara PMCP (100 g) na toluene (200 ml), na 4-hydroxy-TEMPO (2 ml) na-agbakwunye ya na ọnụnọ 100 μl nke dibutyltin dilaurarate dị ka ihe na-eme ka ọ bụrụ ihe na-akpali akpali. A na-emegharị ngwakọta ahụ na 50 ° C maka awa 8, kpochaa ma kpoo ya na 50 Celsius maka awa 3.
Styrene (1 ml), benzoyl peroxide BPO (0.5 ml) na silica particles jikọtara na TEMPO-PMCP (1.5 g) na-agbasasị na toluene ma kpochaa ya na nitrogen. A na-eme polymerization nke styrene na 100 Celsius C maka awa 12. A na-eji methanol sachaa ngwaahịa a rụpụtara ma kpọọ nkụ n'abali na 60 Celsius. E gosipụtara atụmatụ izugbe nke mmeghachi omume na fig. otu .
A na-ekpochapụ ihe nlele ahụ na 393 K maka 1 h ruo mgbe a nwetara nrụgide fọdụrụnụ nke na-erughị 10-3 Torr. Ọnụ ego nke N2 adsorbed na nrụgide ikwu P/P0 = 0.99 ka ejiri chọpụta mkpokọta pore. A na-enyocha morphology nke ụmụ irighiri silica dị ọcha na njikọ ligand site na iji microscope eletrọn na-enyocha (Hitachi High Teknụzụ, Tokyo, Japan). A na-etinye ihe nlele akọrọ (silica dị ọcha na ligand bound silica particles) na mkpanaka aluminum site na iji teepu carbon. A na-etinye ọla edo na nlele ahụ site na iji ngwa sputter Q150T, na akwa akwa Au nm 5 nm ka etinyere na nlele ahụ. Nke a na-eme ka arụmọrụ nke usoro voltaji dị ala ma na-enye ezigbo spraying oyi. Emere nyocha nke ihe site na iji Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental compound analyzer. A na-eji ihe nyocha nke Malvern (Worcestershire, UK) Mastersizer 2000 nweta nkesa nha nha. Achịcha silica na-enweghị mkpuchi na ligand-bound silica particles (5 mg nke ọ bụla) gbasasịa na 5 ml nke isopropanol, sonicated maka nkeji 10, kpasuru iwe maka nkeji 5, ma tinye ya na bench anya Mastersizer. A na-eme nyocha nke Thermogravimetric na ọnụego 5 Celsius C kwa nkeji na ọnọdụ okpomọkụ sitere na 30 ruo 800 Celsius.
Glass fiber lined warara gbawara igwe anaghị agba nchara kọlụm nwere akụkụ (ID 100 × 1.8 mm) juru n'ọnụ site slurry ndochi usoro na-eso otu usoro dị ka e kwuru 31. Igwe anaghị agba nchara kọlụm (glass lined, ID 100 × 1 .8 mm) na mwepu nwere 1 µm frit ejikọrọ na a ubi slurry USA. Kwadebe nkwusioru nke oge nkwụsị site na ịkwụsịtụ 150 mg nke oge nkwụsị na 1.2 ml nke methanol ma nye ya nri n'ime kọlụm nchekwa mmiri. A na-eji methanol mee ihe dị ka ihe mgbaze na-achịkwa. Kpochie kọlụm ahụ site n'itinye usoro nrụgide nke 100 MP maka nkeji iri, 80 MP maka nkeji 15 na 60 MP maka nkeji 30. Usoro mbukota jiri kọlụm chromatography gas abụọ (Alltech, Deerfield, IL, USA) maka ịma jijiji n'ọrụ iji hụ na nkwakọ kọlụm otu. Mechie ngwugwu slurry wee hapụ nrụgide nwayọ ka ọ ghara imebi eriri. Akwụsịla kọlụm ahụ na nozzle slurry na mgbakwunye ọzọ dabara adaba na ntinye wee jikọọ na sistemụ LC iji nwalee ọrụ ya.
A na-arụ MLC omenala site na iji mgbapụta LC (10AD Shimadzu, Japan), onye nlele nwere 50 nL injection loop (Valco (USA) C14 W.05), membrane degasser (Shimadzu DGU-14A), na windo capillary UV-VIS. Ngwaọrụ nchọpụta (UV-2075) yana microcolumn enamelled. Jiri tube njikọ dị warara na nke dị mkpụmkpụ iji belata mmetụta nke mgbasawanye kọlụm ọzọ. Mgbe emejuchara kọlụm ahụ, wụnye capillary (50 µm id 365) na mwepu nke 1/16 ″ mbenata njikọ wee wụnye capillary (50 µm) nke nkwụsị mbelata. A na-eme nchịkọta data na nhazi chromatogram site na iji ngwanrọ Multichro 2000. Na 254 nm, a na-enyocha nnabata UV nke ndị na-enyocha ihe na 0. A na-enyocha data Chromatographic site na iji OriginPro8 (Northampton, MA).
Serum albumin mmadụ, lyophilized ntụ ntụ, ≥ 96% (agarose gel electrophoresis) 3 mg agwakọta na trypsin (1.5 mg), 4.0 M urea (1 ml) na 0.2 M ammonium bicarbonate (1 ml) . A na-emegharị ihe ngwọta maka 10 min ma debe ya na mmiri ịsa ahụ na 37 ° C maka 6 h, wee mebie ya na 1 ml nke 0.1% TFA. Wepu ihe ngwọta ma chekwaa n'okpuru 4 Celsius.
A tụlere nkewa nke ngwakọta peptides na tryptic digest HSA na kọlụm PMP iche iche. Lelee tryptic hydrolysis nke ngwakọta nke peptides na HSA kewara site na kọlụm PMP wee tụnyere nsonaazụ ya na kọlụm Ascentis Express RP-Amide. A na-agbakọ ọnụ ọgụgụ nke epekele usoro ihe omume site na iji nhata ndị a:
Ihe oyiyi SEM nke silica silica dị ọcha na ligand bound silica particles na-egosi na eserese 2. Ihe oyiyi SEM nke ihe oyiyi silica dị ọcha (A, B) na-egosi ọdịdị okirikiri nke akụkụ ahụ na-agbatị ma ọ bụ nwee ihe ngosi na-adịghị mma ma e jiri ya tụnyere ọmụmụ ihe anyị gara aga. Elu nke silica na-ejikọta ya na ligand (C, D) dị nro karịa nke silica dị ọcha, nke nwere ike ịbụ n'ihi eriri polystyrene na-ekpuchi elu nke silica.
Na-enyocha micrograph elektrọn nke irighiri silica dị ọcha (A, B) na ligand bounded silica particles (C, D).
The urughuru size nkesa nke dị ọcha silica ahụ na ligand-ekekọta silica ahụ na-egosi na fig. 2. 3 (A). Volumetric urughuru size nkesa akụkụ gosiri na silica urughuru size mụbara mgbe chemical mgbanwe (Fig. 3A). A na-atụnyere data nkesa nke silica site na ọmụmụ ihe ugbu a na ọmụmụ ihe gara aga na tebụl 1 (A). Ogo urughuru d (0.5) nke PMP bụ 3.36 µm, ma e jiri ya tụnyere mgbasa ozi (0.5) uru nke 3.05 µm na ọmụmụ anyị gara aga (paịlị polystyrene bonded silica)34. N'ihi mgbanwe dị na nha nke PEG, urea, TMOS na acetic acid na ngwakọta mmeghachi omume, nkesa nha nke batch a dị warara ma e jiri ya tụnyere ọmụmụ anyị gara aga. The urughuru size nke PMP agba bụ ubé ibu karịa nke polystyrene bound silica akụkụ nke anyị mụrụ na mbụ. Nke a pụtara na arụ ọrụ elu nke silica ụmụ irighiri ihe na styrene na-edobere naanị akwa polystyrene (0.97 µm) n'elu silica, ebe n'oge PMP, ọkpụrụkpụ oyi akwa bụ 1.38 µm.
Nkesa nha nha (A) na nkesa nha pore (B) nke ihe dị ọcha silica na ligand bound silica.
A na-egosi nha pore, olu pore, na elu elu nke silica ụmụ irighiri ihe eji eme ihe n'ọmụmụ ihe a na Tebụl 1 (B). A na-egosi profaịlụ PSD nke silica dị ọcha na ihe silica na-ejikọta ligand na fig. 3 (B). Nsonaazụ dị ka ihe ọmụmụ anyị gara aga34. The pore sizes nke dị ọcha na ligand-bound silica particles bụ 310 Å na 241 Å, n'otu n'otu, na-egosi na mgbe mgbanwe kemịkalụ, pore size belatara site 69 Å, dị ka e gosiri na Table 1 (B), na ngbanwe curve na-egosi na fig. The kpọmkwem elu ebe nke silica particles na 6 gara aga ọmụmụ ihe / 11 nke gara aga ọmụmụ bụ m2 compable 1. (124m2/g). Dị ka e gosiri na Tebụl 1 (B), ebe elu (m2 / g) nke silica particles mgbe mgbanwe kemịkalụ belatara site na 116 m2 / g ruo 105 m2 / g.
A na-egosi nsonaazụ nyocha nke elementrị nke oge a na-akwụsị na tebụl. 2. The carbon ọdịnaya nke ugbu a guzo na-adọ bụ 6.35%, nke dị ala karịa na anyị gara aga ọmụmụ (silica particles jikọtara polystyrene, 7.93% 35 na 10.21%, karị) 42. The carbon ọdịnaya nke ugbu a guzo na-adọ n'okpuru, ebe ọ bụ na ụfọdụ polar ligands ndị dị ka phenylmaleimide na TEvinyl-PCOxyhydrocyhydracd (Phenylmaleimide) eji na mgbakwunye na styrene na nkwadebe nke SP. Pasent dị arọ nke nitrogen n'oge a na-akwụsị ugbu a bụ 2.21% ma e jiri ya tụnyere 0.1735 na 0.85% na ọmụmụ gara aga42. Nke a pụtara na oge a na-eguzo ugbu a nwere pasentị nitrogen dị elu n'ihi phenylmaleimide. N'otu aka ahụ, ngwaahịa (4) na (5) nwere carbon ọdịnaya nke 2.7% na 2.9%, karị, mgbe ikpeazụ ngwaahịa (6) nwere a carbon ọdịnaya nke 6.35%, dị ka e gosiri na Tebụl 2. Thermogravimetric analysis (TGA) e ji mee ihe na-anọ na-adọ nke PMP iji nwalee n'ihi na arọ ọnwụ, na TGA curve na-e gosiri na ọnụ ọgụgụ nke 4. nkwekọrịta na ọdịnaya carbon (6.35%), ebe ọ bụ na ligands nwere ọ bụghị naanị C, kamakwa N, O na H.
A họọrọ ligand phenylmaleimide-methylvinyl isocyanate iji gbanwee elu nke silica ahụ n'ihi polar phenylmaleimide na vinylisocyanate otu. Otu Vinyl isocyanate nwere ike meghachi omume na styrene site na polymerization dị ndụ. Ihe nke abụọ kpatara ya bụ ịtinye otu nke nwere mmekọrịta agafeghị oke na onye nyocha na enweghị mmekọrịta siri ike nke electrostatic n'etiti onye nyocha na oge nkwụsị, ebe ọ bụ na phenylmaleimide moiety enweghị ụgwọ ọ bụla na pH nkịtị. Enwere ike ijikwa polarity nke oge kwụ ọtọ site na ezigbo ego nke styrene na oge mmeghachi omume nke radical polymerization. Nzọụkwụ ikpeazụ nke mmeghachi omume (polymerization free radical) dị oke egwu ka ọ na-agbanwe polarity nke oge nkwụsị. Emere nyocha nke ihe iji lelee ọdịnaya carbon n'ime usoro ndị a guzoro ọtọ. Achọpụtala na ịba ụba nke styrene na oge mmeghachi omume na-abawanye ọdịnaya carbon nke oge nkwụsị na ntụgharị. SP ndị a kwadebere na nha dị iche iche nke styrene nwere ibu carbon dị iche iche. N'otu aka ahụ, a na-etinye usoro ndị a kwụ ọtọ na kọlụm igwe anaghị agba nchara na njirimara chromatographic ha (nhọrọ, mkpebi, uru N, wdg) ka a na-enyocha. Dabere na nnwale ndị a, a na-ahọrọ ihe kachasị mma maka nkwadebe nke oge nkwụsị PMP iji nye polarity na-achịkwa na njide dị mma nke nyocha.
A tụlekwara kọlụm PMP maka nyocha nke ngwakọta ise nke peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) site na iji ikike nke usoro mkpanaka. 60/40 (v/v) ACN/mmiri (0.1% TFA) na ọsọ ọsọ nke 80 µl/min. N'okpuru ọnọdụ elution kachasị mma (200,000 plates / m), ọnụ ọgụgụ nke efere usoro (N) kwa kọlụm (100 × 1.8 mm) bụ 20,000 ± 100. N'ihi na ụkpụrụ N maka ogidi PMP atọ na-egosi na Tebụl 3 na chromatograms na-egosi na Figure 5A. Nyocha ngwa ngwa na ọnụ ọgụgụ dị elu (700 µl / min) na kọlụm PMP, peptides ise gbapụrụ n'ime otu nkeji, ezigbo N uru nke 13,500 ± 330 kwa kọlụm (100 x 1.8 mm dayameta), nke kwekọrọ na 135,000 efere / m (Fig 5B). Ogidi atọ nke otu nha (dayameta dị n'ime 100 x 1.8 mm) jupụtara na batches atọ dị iche iche nke oge PMP iji nwalee mmeghari. Edere ihe nyocha maka kọlụm ọ bụla site na ikewa otu ngwakọta nnwale na kọlụm ọ bụla na-eji ọnọdụ elution kacha mma, ọnụ ọgụgụ nke efere usoro N, na oge njide. E gosipụtara data mmeghari maka kọlụm PMP na Tebụl 4. Mmegharị nke kọlụm PMP jikọtara nke ọma yana ụkpụrụ% RSD dị ala dị obere ka egosiri na Tebụl 3.
Nkewa nke ngwakọta peptide na kọlụm PMP (B) na kọlụm Ascentis Express RP-Amide (A), usoro mkpanaka 60/40 ACN / H2O (TFA 0.1%), akụkụ kọlụm PMP (100 x 1.8 mm id) , nyocha Elution nke ogige: 1 (Gly-Tyr), - 2-Tyr (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) na 5 (leucic acid enkephalin).
A tụlere kọlụm PMP (dayameta dị n'ime 100 x 1.8 mm) maka ikewapụ tryptic hydrolyzate nke serum albumin mmadụ site na HPLC. Chromatogram dị na eserese 6 na-egosi na ihe atụ ndị ahụ kewapụrụ nke ọma na mkpebi dị mma. A na-enyocha ihe ngwọta HSA site na iji ọsọ ọsọ nke 100 μl / min, usoro mkpanaka nke 70/30 acetonitrile / mmiri na 0.1% TFA. E kewara mkpochapụ nke HSA na ọnụ ọgụgụ 17, dị ka egosiri na chromatogram (Fig 6), nke kwekọrọ na 17 peptides. A na-agbakọ arụmọrụ nkewa nke elu onye ọ bụla site na HSA hydrolyzate ma gosipụta ụkpụrụ ya na Tebụl 5.
E kewapụrụ HSA tryptic hydrolysates na kọlụm PMP (dayameta dị n'ime 100 x 1.8 mm), ọnụego ịgba ọsọ (100 μl / min), usoro mkpanaka 60/40 acetonitrile / mmiri, na 0.1% TFA.
ebe L bụ ogologo kọlụm, η bụ viscosity nke usoro mkpanaka, ΔP bụ nrụgide azụ nke kọlụm, na u bụ ọsọ ọsọ nke usoro mkpanaka. Ntughari nke kọlụm PMP bụ 2.5 × 10-14 m2, ọnụego erute bụ 25 µl / min, 60/40 v / v ejiri mee ihe. ACN/mmiri. Mgbanwe nke kọlụm PMP (ID 100 × 1.8 mm) yiri nke ọmụmụ Ref.34 gara aga. The permeability nke kọlụm juputara na ihe ndị na-enweghị isi bụ 1.7 × 10 .6 µm, 2.5 × 10-14 m2 maka 5 µm particles43. Ya mere, ikike nke usoro PMP yiri ihe nkedo nke isi-shell ahụ na nha nke 5 μm.
ebe Wx bụ uka nke kọlụm jupụtara na chloroform, Wy bụ oke kọlụm jupụtara na methanol, na ρ bụ njupụta nke ihe mgbaze. Njupụta nke methanol (ρ = 0.7866) na chloroform (ρ = 1.484). Mkpokọta porosity nke kọlụm silica-C18 (100 × 1.8 mm ID) 34 na kọlụm C18-urea31 anyị mụọburu bụ 0.63 na 0.55, n'otu n'otu. Nke a pụtara na ọnụnọ nke urea ligands na-ebelata ikike nke oge a na-akwụsị. N'aka nke ọzọ, ngụkọta porosity nke kọlụm PMP (dayameta dị n'ime 100 × 1.8 mm) bụ 0.60. Ogidi PMP dị obere karịa ogidi ndị ejiri C18 kechie silica particles n'ihi na n'ụdị C18, a na-ejikọta ligands na silica na eriri linear, ebe n'ụdị polystyrene, a na-emepụta polymer dịtụ ukwuu n'akụkụ akụkụ ahụ. oyi akwa A. Na a-ahụkarị nnwale, kọlụm porosity gbakọọ dị ka ndị a:
Na fig. 7A, B na-egosi atụmatụ Van Deemter maka kọlụm PMP (id 100 x 1.8 mm) na kọlụm Ascentis Express RP-Amide (id 100 x 1.8 mm) n'okpuru otu ọnọdụ elution, 60/40 ACN/H2O na 0 .1% TFA 20 µl/min na kọlụm abụọ 80. Ụkpụrụ HETP kacha nta na ọnụego kacha mma (80 µl / min) bụ 2.6 µm na 3.9 µm maka kọlụm PMP na kọlụm Ascentis Express RP-Amide, n'otu n'otu. Ụkpụrụ HETP gosipụtara na arụmọrụ nkewa nke kọlụm PMP (100 x 1.8 mm id) dị elu karịa nke kọlụm Ascentis Express RP-Amide (100 x 1.8 mm id). Ihe eserese nke van Deemter na Fig. 7 (A) na-egosi na mbelata nke uru N adịghị elu karịa na-abawanye ụba ma e jiri ya tụnyere ọmụmụ ihe anyị gara aga. Ọrụ nkewa dị elu nke kọlụm PMP (id 100 × 1.8 mm) ma e jiri ya tụnyere kọlụm Ascentis Express RP-Amide dabere na ọdịdị dị mma na nha nha yana usoro mbukota kọlụm ọkaibe ejiri na ọrụ ugbu a34.
(A) Van Deemter nkata (HETP vs. mobile phase linear velocity) nwetara na kọlụm PMP (id 100 x 1.8 mm) na 60/40 ACN/H2O nwere 0.1% TFA. (B) Atụmatụ Van Deemter (HETP vesos mobile phase linear velocity) nwetara na kọlụm Ascentis Express RP-Amide (id 100 x 1.8 mm) na 60/40 ACN/H2O nwere 0.1% TFA.
A kwadebere ma nyochaa usoro nkwụsị nke polar nke intercalated polystyrene maka nkewa nke ngwakọta nke peptides sịntetik na tryptic hydrolyzate nke serum albumin mmadụ (HSA) na chromatography mmiri mmiri dị elu. Ọrụ chromatographic nke kọlụm PMP maka ngwakọta peptide dị mma n'ihe gbasara arụmọrụ na mkpebi nkewa. The mma nkewa arụmọrụ nke PMP ogidi bụ n'ihi na ọtụtụ ihe ndị dị otú ahụ dị ka silica urughuru size na pore size, akara njikọ nke guzo n'ụzọ, na mgbagwoju kọlụm mbukota ihe. Na mgbakwunye na arụmọrụ nkewa dị elu, uru ọzọ nke oge a na-eguzo bụ obere kọlụm azụ nrụgide na ọnụ ọgụgụ dị elu. Ogidi PMP nwere ike imepụtagharị nke ukwuu ma enwere ike iji ya nyochaa ngwakọta nke peptides na mgbaze tryptic nke protein dị iche iche. Anyị bu n'obi iji kọlụm a maka nkewa nke ogige bioactive sitere na ngwaahịa eke, mpụta nke osisi ọgwụ na mushrooms na chromatography mmiri mmiri. N'ọdịnihu, a ga-enyocha kọlụm PMP maka nkewa nke protein na ọgwụ mgbochi monoclonal.
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