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E ji usoro sol-gel kwadebe obere silica ndị nwere oghere dị n'ime ya, ha nwekwara mgbanwe ụfọdụ iji nweta obere oghere dị n'ime ya. E jiri N-phenylmaleemide-methylvinyl isocyanate (PMI) na styrene mepụta obere ihe ndị a site na iji reverse chain transfer-fragmentation (RAFT) polymerization mepụta polyamides ndị a na-akpọ N-phenylmaleemide. Styrene (PMP) standalone phase. E ji ngwugwu slurry jupụta ogidi ígwè anaghị agba nchara dị warara (100 × 1.8 mm dayameta dị n'ime). A tụlere arụmọrụ chromatographic nke kọlụm PMP iji kewaa ngwakọta nke peptides sịntetik nke nwere peptides ise (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) na tryptic hydrolyzate nke mmadụ serum albumin (HAS). N'okpuru ọnọdụ nhichapụ kacha mma, ọnụọgụ efere ndị nwere ngwakọta peptides ruru efere 280,000/sq.m. Site na iji arụmọrụ nkewa nke kọlụm emepụtara tụnyere kọlụm Ascentis Express RP-Amide azụmaahịa, a hụrụ na arụmọrụ nkewa nke kọlụm PMP dị elu karịa kọlụm azụmaahịa n'ihe gbasara arụmọrụ nkewa na mkpebi.
Ụlọ ọrụ ọgwụ biopharmaceutical aghọọla ahịa zuru ụwa ọnụ na-abawanye ụba na oke ahịa n'afọ ndị na-adịbeghị anya. Site na mmụba dị egwu nke ụlọ ọrụ ọgwụ biopharmaceutical1,2,3, enwere nnukwu mkpa maka nyocha peptide na protein. Na mgbakwunye na peptide a na-elekwasị anya, a na-emepụta ọtụtụ ihe na-adịghị ọcha n'oge mmepụta peptide, yabụ ọ dị mkpa ka a sachaa chromatographic iji nweta ịdị ọcha achọrọ nke peptide ahụ. Nyocha na njirimara nke protein na mmiri ahụ, anụ ahụ, na mkpụrụ ndụ bụ ọrụ siri ike nke ukwuu n'ihi ọnụ ọgụgụ dị ukwuu nke ụdị nwere ike ịchọpụta dị na otu ihe nlele. Ọ bụ ezie na spectrometry mass bụ ngwaọrụ dị irè maka usoro peptides na protein, ọ bụrụ na etinyere ihe nlele ndị dị otú ahụ ozugbo na spectrometer mass, nkewa ahụ agaghị eju afọ. Enwere ike idozi nsogbu a site na ime chromatography mmiri mmiri (LC) tupu nyocha MS, nke ga-ebelata ọnụọgụ nke ndị nyocha na-abanye na spectrometer mass n'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 ndị nyocha ndị a n'ọrụ ma na-eme ka mmetụta nke nchọpụta MS dịkwuo elu. Nnwale chromatography mmiri (LC) emeela nke ọma n'ime afọ iri gara aga ma aghọọla ụzọ a na-ejikarị eme ihe maka nyocha proteomic7,8,9,10.
A na-ejikarị chromatography mmiri mmiri nke na-agbanwe agbanwe (RP-LC) eme ihe iji sachapụ ma kewaa ngwakọta nke peptides site na iji octadecyl-modified silica (ODS) dị ka usoro kwụ ọtọ11,12,13. Agbanyeghị, n'ihi nhazi ha dị mgbagwoju anya na ọdịdị amphoteric ha, usoro RP 14,15 enweghị ike inye nkewa zuru oke nke peptides na protein. Ya mere, nyocha nke peptides na protein nwere iberibe polar na nke na-abụghị polar chọrọ usoro kwụ ọtọ pụrụ iche iji mekọrịta ma jigide ndị nyocha a16. Chromatography agwakọtara agwakọta, nke na-enye mmekọrịta multimodal, nwere ike ịbụ ihe ọzọ na RP-LC maka ikewapụ peptides, protein, na ngwakọta ndị ọzọ dị mgbagwoju anya. E kwadebere ọtụtụ usoro agwakọta nke na-adịghị agbanwe agbanwe ma jiri kọlụm jupụtara na usoro ndị a na-adịghị agbanwe agbanwe kewapụ peptides na protein17,18,19,20,21. N'ihi ọnụnọ nke otu polar na nke na-abụghị polar, usoro nhazi nke ụdị agwakọta (WAX/RPLC, HILIC/RPLC, njikọ polar/RPLC) dabara adaba maka nkewa nke peptides na protein22,23,24,25,26,27,28., usoro nhazi nke polar na-ejikọta ọnụ yana otu polar jikọtara ọnụ na-egosi ikike nkewa dị mma na nhọrọ pụrụ iche maka ndị nyocha polar na ndị na-abụghị polar n'ihi na nkewa dabere na mmekọrịta dị n'etiti onye nyocha na usoro nhazi nke Multimodal mmekọrịta 29,30,31,32. N'oge na-adịbeghị anya, Zhang et al. 30 nwetara usoro nhazi nke behenyl nke polyamines na-akwụsị ma kewaa hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, na ụfọdụ ndị nyocha ndị ọzọ nke ọma. Ihe a na-etinye na polar nwere otu polar na nke na-abụghị polar, yabụ enwere ike iji ya kewaa peptides na protein n'ime akụkụ hydrophobic na hydrophilic. Ogidi dị n'ime polar (dịka ọmụmaatụ, ogidi C18 nwere amide inline) dị n'okpuru aha azụmaahịa Ascentis Express RP-Amide columns, mana ejiri ogidi ndị a naanị maka nyocha nke amine 33.
N'ọmụmụ ihe a dị ugbu a, a kwadebere ma nyochaa usoro nhazi nke polar (N-phenylmaleemide, embedding polystyrene) maka nkewa peptide na tryptic HSA cleavage. E jiri usoro a kwadebe usoro nhazi nke stainless. A kwadebere mkpụrụ silica porous dịka usoro akọwara na mbipụta anyị gara aga si dị, yana mgbanwe ụfọdụ na atụmatụ nkwadebe 31, 34, 35, 36, 37, 38, 39. A gbanwere oke nke urea, polyethylene glycol (PEG), TMOS na aqueous-acetic acid iji nweta mkpụrụ silica nwere nnukwu nha pore. Nke abụọ, a rụpụtara ligand phenylmaleemide-methylvinyl isocyanate ọhụrụ ma jiri mkpụrụ silica ya nke a rụpụtara iji kwadebe usoro nhazi nke polar. A kpọchiri usoro nhazi nke enwetara n'ime kọlụm ígwè anaghị agba nchara (dayameta dị n'ime 100 × 1.8 mm) dịka atụmatụ nhazi kachasị mma si dị. Nkwakọba kọlụm ahụ na-enyere aka site na iji igwe na-eme ka ọ dị otu oyi akwa n'ime kọlụm ahụ. A tụlere kọlụm a kpọchiri akpọchi 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 albumin serum mmadụ (HSA). A hụrụ na ngwakọta peptide na HSA tryptic digest kewara na mkpebi na arụmọrụ dị mma. A tụnyere arụmọrụ nkewa nke kọlụm PMP na nke kọlụm Ascentis Express RP-Amide. A hụrụ na peptides na protein nwere mkpebi dị mma na arụmọrụ nkewa dị elu na kọlụm PMP, arụmọrụ nkewa nke kọlụm PMP dị elu karịa nke kọlụm 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) maka HPLC, methanol, 2-propanol na acetone. Ụlọ ọrụ Sigma-Aldrich (St. Louis, Missouri, USA).
A gwakọtara ngwakọta urea (8 g), polyethylene glycol (8 g) na 8 ml nke 0.01 N. acetic acid ruo nkeji iri, a gbakwụnyekwara 24 ml nke TMOS na ya mgbe a na-eme ka ọ dị jụụ. A kpoo ngwakọta mmeghachi omume ahụ ọkụ na 40°C ruo awa isii, wee kpoo ya na 120°C ruo awa asatọ n'ime autoclave ígwè anaghị agba nchara. A kpochapụrụ mmiri ahụ ma kpọọ ihe fọdụrụ na 70°C ruo awa iri na abụọ. A gwere blọk ndị ahụ dị nro nke ọma ma kpoo ha n'ime oven na 550°C ruo awa iri na abụọ. A kwadebere ma kọwaa ha n'ime otu atọ iji nwalee nha mkpụrụ, nha oghere na mpaghara elu.
Otu polar na usoro kwụ ọtọ maka agbụ polystyrene. A kọwara usoro nkwadebe n'okpuru.
A gbazere N-phenylmaleimide (200 mg) na methyl vinyl isocyanate (100 mg) na toluene anhydrous, wee tinye 0.1 ml nke 2,2′-azoisobutyronitrile (AIBN) na karama mmeghachi omume iji nweta copolymer nke phenylmaleimide na methyl vinyl isocyanate (PMCP). ) A na-ekpo ọkụ ngwakọta ahụ na 60°C ruo awa atọ, a na-asacha ya ma kpọọ ya nkụ n'ime oven na 40°C ruo awa atọ.
A gbasasịrị mkpụrụ silica kpọrọ nkụ (2 g) n'ime toluene kpọrọ nkụ (100 ml), gwakọta ma gbanye ya ruo nkeji iri n'ime karama gburugburu ala 500 ml. A gbazere PMCP (10 mg) na toluene ma tinye ya na dropwise na karama mmeghachi omume site na funnel mgbakwunye. A gbazere ngwakọta ahụ na 100°C ruo awa 8, a gbazere ya, jiri acetone saa ya ma kpọọ ya nkụ na 60°C ruo awa 3. Mgbe ahụ, a gbazere mkpụrụ silica ndị metụtara PMCP (100 g) na toluene (200 ml), a gbazerekwa 4-hydroxy-TEMPO (2 ml) na ya n'ihu 100 μl nke dibutyltin dilaurate dị ka ihe na-akpali akpali. A gbazere ngwakọta ahụ na 50°C ruo awa 8, a gbazere ya ma kpọọ ya nkụ na 50°C ruo awa 3.
A gbasasịrị Styrene (1 ml), benzoyl peroxide BPO (0.5 ml) na ihe ndị silica dị na TEMPO-PMCP (1.5 g) na toluene ma jiri nitrogen sachaa ha. Emere polymerization nke styrene na 100°C ruo awa iri na abụọ. A sachara ngwaahịa ahụ na methanol ma kpọọ nkụ n'abalị na 60°C. E gosiri usoro izugbe nke mmeghachi omume ahụ na fig. 1.
E wepụrụ ihe nlele ndị a na 393 K ruo awa 1 ruo mgbe e nwetara nrụgide fọdụrụ nke na-erughị 10-3 Torr. E jiri ọnụọgụ N2 a na-etinye na nrụgide dị n'otu P/P0 = 0.99 chọpụta olu oghere niile. E jiri igwe nyocha elektrọn (Hitachi High Technologies, Tokyo, Japan) nyochaa ọdịdị nke ihe ndị dị n'ime silica dị ọcha na nke ligand-bound. E jiri teepu carbon tinye ihe nlele kpọrọ nkụ (ihe ndị dị n'ime silica dị ọcha na ihe ndị dị n'ime silica bound) na mkpanaka aluminom. E jiri ngwaọrụ sputtering Q150T tinye ọlaedo na ihe nlele ahụ, e tinyekwara oyi akwa Au dị arọ 5 nm na ihe nlele ahụ. Nke a na-eme ka arụmọrụ nke usoro voltaji dị ala ka mma ma na-enye obere ịgba mmiri oyi. E jiri ihe nyocha ihe mejupụtara Thermo Electron (Waltham, MA, USA) Flash EA1112 mee nyocha elemental. E jiri ihe nyocha nha nha Malvern (Worcestershire, UK) Mastersizer 2000 nweta nkesa nha ihe. A gbasasịrị ihe ndị e ji silica kpuchie na ihe ndị e ji silica kpuchie (5 mg nke ọ bụla) n'ime 5 ml nke isopropanol, gbanye ya ọkụ ruo nkeji iri, gbanye ya ọkụ ruo nkeji ise, ma tinye ya na oche optical Mastersizer. A na-eme nyocha Thermogravimetric na ọnụego nke 5 °C kwa nkeji n'ọnọdụ okpomọkụ site na 30 ruo 800 °C.
E ji usoro ihe eji emeju ihe mkpuchi iko kpuchie ogidi ígwè anaghị agba nchara nke nwere nha (ID 100 × 1.8 mm) site na iji usoro ihe eji emeju ihe mkpuchi slurry dịka e si kwuo ya. E jikọtara ogidi ígwè anaghị agba nchara (nke nwere iko, ID 100 × 1 .8 mm) na oghere nwere frit 1 µm na igwe nkwakọ ngwaahịa slurry (Alltech Deerfield, IL, USA). Kwadebe nkwụsịtụ nke usoro ihe anaghị agba nchara site n'ịkwụsịtụ 150 mg nke usoro ihe anaghị agba nchara na 1.2 ml nke methanol ma tinye ya na ogidi nchekwa. E jiri methanol mee ihe dị ka ihe mgbaze slurry na ihe mgbaze njikwa. Kpọchie ogidi ahụ site n'itinye usoro nrụgide nke 100 MP maka nkeji 10, 80 MP maka nkeji 15, na 60 MP maka nkeji 30. Usoro nkwakọ ngwaahịa ahụ jiri ihe na-eme mkpọtụ kọlụm gas chromatography abụọ (Alltech, Deerfield, IL, USA) maka ịma jijiji igwe iji hụ na ngwugwu kọlụm ahụ dị otu. Mechie ihe mkpuchi slurry ahụ ma hapụ nrụgide nwayọ iji gbochie mmebi na eriri ahụ. E wepụrụ kọlụm ahụ na ihe mkpuchi slurry ahụ, e tinyekwara ihe ọzọ e ji etinye ya na ntinye ahụ ma jikọọ ya na sistemụ LC iji nwalee ọrụ ya.
E ji ọkpọkọ LC (10AD Shimadzu, Japan) rụọ MLC omenala, ihe nlele nwere aka ntụtụ 50 nL (Valco (USA) C14 W.05), ihe na-eme ka ihe dị n'ime akpụkpọ ahụ dị ọcha (Shimadzu DGU-14A), na windo capillary UV-VIS. Ngwaọrụ nchọpụta (UV-2075) na microcolumn enamel. Jiri ọkpọkọ njikọ dị warara ma dị mkpụmkpụ iji belata mmetụta nke mgbasawanye kọlụm ọzọ. Mgbe ijupụtachara kọlụm ahụ, wụnye capillary (50 µm id 365) na mpụta nke njikọ mbelata 1/16″ ma wụnye capillary (50 µm) nke njikọ mbelata. A na-eji ngwanrọ Multichro 2000 eme nchịkọta data na nhazi chromatogram. Na 254 nm, a na-enyocha nnabata UV nke ndị nyocha isiokwu na 0. E jiri OriginPro8 (Northampton, MA) nyochaa data Chromatographic.
Abọmịn serum mmadụ, ntụ ntụ lyophilized, ≥ 96% (electrophoresis gel agarose) 3 mg agwakọtara ya na trypsin (1.5 mg), urea 4.0 M (1 ml) na 0.2 M ammonium bicarbonate (1 ml). A gwakọtara mmiri ọgwụ ahụ ruo nkeji iri ma debe ya n'ime mmiri ịsa ahụ na 37°C maka awa isii, wee jiri 1 ml nke 0.1% TFA mechie ya. Hichaa mmiri ọgwụ ahụ ma chekwaa ya n'okpuru 4°C.
A tụlere nkewa nke ngwakọta peptides na tryptic digest HSA na kọlụm PMP iche iche. Lelee hydrolysis nke tryptic nke ngwakọta nke peptides na HSA nke kọlụm PMP kewara wee tụnyere nsonaazụ ya na kọlụm Ascentis Express RP-Amide. A na-agbakọ ọnụọgụ nke efere echiche site na iji usoro a:
E gosiri foto SEM nke mkpụrụ silica dị ọcha na mkpụrụ silica ejiri ligand mee na Foto nke 2. Foto SEM nke mkpụrụ silica dị ọcha (A, B) na-egosi ọdịdị gburugburu ebe mkpụrụ silica ahụ dị ogologo ma ọ bụ nwee nhazi na-adịghị mma ma e jiri ya tụnyere ọmụmụ ihe anyị gara aga. Elu nke mkpụrụ silica nke ligand (C, D) kekọtara dị nro karịa nke mkpụrụ silica dị ọcha, nke nwere ike ịbụ n'ihi agbụ polystyrene nke kpuchiri elu mkpụrụ silica ahụ.
Nnyocha elektrọn micrographs nke mkpụrụ silica dị ọcha (A, B) na mkpụrụ silica ligand (C, D).
A na-egosi nkesa nha nke ihe ndị dị ọcha na ihe ndị dị na silica nke ejiri ligand mee na Fig. 2. 3(A). Usoro nkesa nha nke ihe ndị dị na volumetric gosiri na nha nke ihe ndị dị na silica mụbara mgbe mgbanwe kemịkalụ gasịrị (Fig. 3A). A tụnyere data nkesa nha nke silica site na ọmụmụ ihe ugbu a na ọmụmụ ihe gara aga na Tebụl 1(A). Nha nha nke ihe ndị dị na volumetric d(0.5) nke PMP bụ 3.36 µm, ma e jiri ya tụnyere uru ad(0.5) nke 3.05 µm na ọmụmụ ihe anyị gara aga (ihe ndị dị na silica nke ejiri polystyrene mee)34. N'ihi mgbanwe na oke nke PEG, urea, TMOS na acetic acid na ngwakọta mmeghachi omume, nkesa nha nke ihe ndị dị na ngwugwu a dị warara karịa na ọmụmụ ihe anyị gara aga. Nha nha nke ihe ndị dị na PMP buru ibu karịa nke nke polystyrene nke ejiri polystyrene mee nke anyị mụrụ na mbụ. Nke a pụtara na ọrụ elu nke ihe ndị silica nwere styrene na-arụ ọrụ naanị oyi akwa polystyrene (0.97 µm) n'elu silica, ebe n'oge PMP, ọkpụrụkpụ oyi akwa ahụ bụ 1.38 µm.
Nkesa nha nke ihe ndị dị ndụ (A) na nkesa nha nke ihe ndị dị ndụ (B) nke ihe ndị dị ndụ dị ọcha na ihe ndị dị ndụ dị ka silica e ji ligand kee.
A na-egosi nha oghere, olu oghere, na mpaghara elu nke ihe ndị silica ejiri mee ihe n'ọmụmụ ihe a na Tebụl 1 (B). E gosiri profaịlụ PSD nke ihe ndị silica dị ọcha na ihe ndị silica jikọtara ligand na Fig. 3(B). Nsonaazụ ya yiri nke anyị mere na ọmụmụ ihe gara aga34. Nha oghere nke ihe ndị silica dị ọcha na nke ligand jikọtara bụ 310 Å na 241 Å, n'otu n'otu, na-egosi na mgbe mgbanwe kemịkalụ gasịrị, nha oghere ahụ belatara site na 69 Å, dịka egosiri na Tebụl 1 (B), a na-egosikwa usoro mgbanwe na Fig. Mpaghara elu kpọmkwem nke ihe ndị silica na ọmụmụ ihe ugbu a bụ 116 m2/g, nke yiri ya na ọmụmụ ihe anyị gara aga (124 m2/g). Dịka egosiri na Tebụl 1(B), mpaghara elu (m2/g) nke ihe ndị silica mgbe mgbanwe kemịkalụ gasịrị belatakwara site na 116 m2/g ruo 105 m2/g.
A na-egosi nsonaazụ nke nyocha elemental nke usoro kwụ ọtọ na Tebụl. 2. Ọdịnaya carbon nke usoro kwụ ọtọ ugbu a bụ 6.35%, nke dị ala karịa na ọmụmụ anyị gara aga (ihe ndị metụtara silica na polystyrene, 7.93%35 na 10.21%, n'otu n'otu) 42. Ọdịnaya carbon nke usoro kwụ ọtọ ugbu a dị n'okpuru, ebe ọ bụ na ejirila ụfọdụ ligands polar dị ka phenylmaleemide methyl vinyl isocyanate (PCMP) na 4-hydroxy-TEMPO na mgbakwunye na styrene na nkwadebe nke SP. Pasent ibu nke nitrogen na usoro kwụ ọtọ ugbu a bụ 2.21% ma e jiri ya tụnyere 0.1735 na 0.85% na ọmụmụ ihe gara aga42. Nke a pụtara na usoro kwụ ọtọ ugbu a nwere pasentị ibu dị elu nke nitrogen n'ihi phenylmaleemide. N'otu aka ahụ, ngwaahịa (4) na (5) nwere ọdịnaya carbon nke 2.7% na 2.9%, n'otu n'otu, ebe ngwaahịa ikpeazụ (6) nwere ọdịnaya carbon nke 6.35%, dịka egosiri na Tebụl 2. Ejiri nyocha Thermogravimetric (TGA) mee ihe na usoro PMP na-anaghị agbanwe agbanwe iji nwalee maka mbelata ibu, a na-egosikwa usoro TGA na Foto 4. Usoro TGA na-egosi mbelata ibu nke 8.6%, nke kwekọrọ na ọdịnaya carbon (6.35%), ebe ọ bụ na ligands nwere ọ bụghị naanị C, kamakwa N, O na H.
A họọrọ ligand phenylmaleemide-methylvinyl isocyanate iji gbanwee elu nke ihe ndị silica n'ihi otu polar phenylmaleemide na vinylisocyanate ya. Vinyl isocyanate otu nwere ike imeghachi omume na styrene site na polymerization dị ndụ. Ihe nke abụọ bụ itinye otu nwere mmekọrịta dị n'etiti na analyte na enweghị mmekọrịta electrostatic siri ike n'etiti analyte na usoro kwụ ọtọ, ebe ọ bụ na akụkụ phenylmaleemide enweghị chajị mebere emebere na pH nkịtị. Enwere ike ijikwa polarity nke usoro kwụ ọtọ site na oke styrene kacha mma na oge mmeghachi omume nke polymerization radical free. Nzọụkwụ ikpeazụ nke mmeghachi omume (polymerization radical free) dị oke mkpa ebe ọ na-agbanwe polarity nke usoro kwụ ọtọ. Emere nyocha elemental iji lelee ọdịnaya carbon na usoro ndị a kwụ ọtọ. A hụla na ịbawanye ọnụọgụ styrene na oge mmeghachi omume na-eme ka ọdịnaya carbon nke usoro kwụ ọtọ na nke ọzọ. SP ndị a kwadebere na njupụta dị iche iche nke styrene nwere ibu carbon dị iche iche. N'otu aka ahụ, e tinyere usoro ndị a na-anaghị agbanwe agbanwe na kọlụm ígwè anaghị agba nchara ma lelee njirimara chromatographic ha (nhọrọ, mkpebi, uru N, wdg.). Dabere na nnwale ndị a, ahọpụtara nhazi kachasị mma maka nkwadebe nke usoro PMP na-anaghị agbanwe agbanwe iji nye polarity a na-achịkwa na njigide dị mma nke onye nyocha ahụ.
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 ọnụego mmiri nke 80 µl/min. N'okpuru ọnọdụ mmịpụta kacha mma (200,000 efere/m), ọnụọgụ nke efere echiche (N) kwa kọlụm (100 × 1.8 mm) bụ 20,000 ± 100. Egosiri uru N maka kọlụm PMP atọ ahụ na Tebụl 3 na egosiri chromatograms na Foto 5A. Nnyocha ngwa ngwa na oke mmiri dị elu (700 µl/min) na kọlụm PMP, peptide ise ewepụtara n'ime otu nkeji, ezigbo uru N nke 13,500 ± 330 kwa kọlụm (dayameta 100 x 1.8 mm), nke hà nhata efere 135,000/ m (Foto 5B). E ji ogidi atọ nke otu nha (dayameta dị n'ime 100 x 1.8 mm) jupụta na ogidi atọ dị iche iche nke usoro PMP na-anaghị agbanwe agbanwe iji nwalee mmụba. E dekọrọ ndị nyocha maka kọlụm ọ bụla site na ikewapụ otu ngwakọta nnwale ahụ na kọlụm ọ bụla site na iji ọnọdụ mwepụ kacha mma, ọnụọgụ nke efere echiche N, na oge nchekwa. Egosiri data mmụba maka kọlụm PMP na Tebụl 4. Mmegharị nke kọlụm PMP jikọtara nke ọma na obere uru %RSD dịka 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%), nha kọlụm PMP (100 x 1.8 mm id), nyocha Usoro mwepụ nke ihe mejupụtara: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (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 nkewa nke tryptic hydrolyzate nke albumin serum mmadụ site na HPLC. Chromatogram dị na Foto 6 na-egosi na a kewapụrụ ihe nlele ndị ahụ nke ọma na mkpebi dị mma. E jiri ọsọ mmiri nke 100 μl/min, usoro mkpanaka nke 70/30 acetonitrile/mmiri na 0.1% TFA nyochaa ngwọta HSA. E kewara nkewa HSA n'ime oke 17, dịka egosiri na chromatogram (Fig. 6), nke kwekọrọ na peptides 17. A gbakọrọ arụmọrụ nkewa nke oke nke ọ bụla na HSA hydrolyzate ma egosiri uru ndị ahụ na Tebụl 5.
E kewapụrụ HSA tryptic hydrolysates na kọlụm PMP (dayameta dị n'ime 100 x 1.8 mm), ọnụego mmiri (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 ahụ, na u bụ ọsọ kwụ ọtọ nke usoro mkpanaka. Permeability nke kọlụm PMP bụ 2.5 × 10–14 m2, ọnụego mmiri ahụ bụ 25 µl/min, ejiri 60/40 v/v mee ihe. ACN/mmiri. Permeability nke kọlụm PMP (ID 100 × 1.8 mm) yiri nke ọmụmụ Ref.34 anyị gara aga. Permeability nke kọlụm jupụtara na obere oghere dị n'elu bụ 1.7 × 10 .6 µm, 2.5 × 10-14 m2 maka obere 5 µm43. Ya mere, permeability nke usoro PMP yiri permeability nke obere core-shell nwere nha 5 μm.
ebe Wx bụ oke nke kọlụm jupụtara na chloroform, Wy bụ oke nke kọlụm jupụtara na methanol, ρ bụkwa njupụta nke ihe mgbaze. Njupụta nke methanol (ρ = 0.7866) na chloroform (ρ = 1.484). Ngụkọta porosity nke kọlụm silica-C18 (100 × 1.8 mm ID)34 na kọlụm C18-urea31 anyị mụrụ na mbụ bụ 0.63 na 0.55, n'otu n'otu. Nke a pụtara na ọnụnọ nke urea ligands na-ebelata ikike nke usoro kwụ ọtọ. N'aka nke ọzọ, mkpokọta porosity nke kọlụm PMP (dayameta dị n'ime 100 × 1.8 mm) bụ 0.60. Ogidi PMP adịghị agafe agafe karịa kọlụm ejiri C18 kee obere silica n'ihi na na usoro kwụ ọtọ nke ụdị C18, a na-ejikọ ligands C18 na obere silica n'usoro n'usoro, ebe na usoro kwụ ọtọ nke ụdị polystyrene, a na-emepụta polymer dị oke arọ gburugburu obere ihe ndị ahụ. oyi akwa A. N'ime nnwale nkịtị, a na-agbakọ porosity kọlụm 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 ahụ, 60/40 ACN/H2O na 0 .1% TFA 20 µl/min ruo 800 µl/min na kọlụm abụọ ahụ. Ọnụego HETP kacha nta na ọnụego mmiri 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. Ọnụego HETP na-egosi na arụmọrụ nkewa nke kọlụm PMP (100 x 1.8 mm id) dị elu karịa nke kọlụm Ascentis Express RP-Amide dị n'ahịa (100 x 1.8 mm id). Grafụ van Deemter dị na Fig. 7(A) na-egosi na mbelata na uru N adịghị elu nke ukwuu ma ọ bụrụ na mmụba na-arị elu ma e jiri ya tụnyere ọmụmụ anyị gara aga. Ọganihu dị elu nkewa nke kọlụm PMP (id 100 × 1.8 mm) ma e jiri ya tụnyere kọlụm Ascentis Express RP-Amide dabere na ọdịdị na nha nke ihe ndị ahụ emelitere na usoro nchikọta kọlụm dị elu ejiri na ọrụ ugbu a34.
(A) Atụmatụ Van Deemter (ọsọ HETP vs. ọsọ linear nke usoro mkpanaka) enwetara na kọlụm PMP (id 100 x 1.8 mm) na 60/40 ACN/H2O yana 0.1% TFA. (B) Atụmatụ Van Deemter (ọsọ HETP vs ọsọ linear nke usoro mkpanaka) enwetara na kọlụm Ascentis Express RP-Amide (id 100 x 1.8 mm) na 60/40 ACN/H2O yana 0.1% TFA.
E kwadebere ma nyochaa usoro polar nke polystyrene intercalated maka nkewa nke ngwakọta nke peptides sịntetik na tryptic hydrolyzate nke mmadụ serum albumin (HSA) na chromatography mmiri mmiri dị elu. Arụmọrụ chromatographic nke kọlụm PMP maka ngwakọta peptide dị mma n'ihe gbasara arụmọrụ na mkpebi nkewa. Mmezi nkewa ka mma nke kọlụm PMP bụ n'ihi ọtụtụ ihe dịka nha silica na nha pore, njikọ a na-achịkwa nke usoro ndị na-anọpụ iche, na ihe mkpuchi kọlụm dị mgbagwoju anya. Na mgbakwunye na arụmọrụ nkewa dị elu, uru ọzọ nke usoro a na-anọpụ iche bụ nrụgide azụ kọlụm dị ala na ọnụego mmiri dị elu. Ogidi PMP nwere ike imegharị nke ọma 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 site na ngwaahịa eke, ihe ewepụtara nke osisi ọgwụ na ero na chromatography mmiri mmiri. N'ọdịnihu, a ga-enyocha kọlụm PMP maka nkewa nke protein na monoclonal antibodies.
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