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E ji usoro sol-gel kwadebe obere silica ndị nwere oghere, ya na mgbanwe ụfọdụ iji nweta obere ihe ndị dị n'ime ya. E si na N-phenylmaleemide-methylvinylisocyanate (PMI) na styrene mepụta obere ihe ndị a iji kwadebe N-phenylmaleemide intercalation nke polystyrene (PMP) stainless phase. E ji ngwugwu slurry jupụta kọlụm ígwè anaghị agba nchara dị warara (100 × 1.8 mm id). E lere anya na nkewa kọlụm PMP nke ngwakọta peptide nke nwere peptides ise (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) na mgbaze trypsin nke albumin serum mmadụ (HAS). N'okpuru ọnọdụ elu kacha mma, ọnụọgụ efere echiche nke ngwakọta peptide dị elu dị ka efere 280,000/m². Na-atụnyere arụmọrụ nkewa nke kọlụm emepụtara. Site na kọlụm Ascentis Express RP-Amide nke azụmahịa, a hụrụ na arụmọrụ nkewa nke kọlụm PMP dị elu karịa kọlụm azụmaahịa n'ihe gbasara arụmọrụ nkewa na mkpebi.
N'afọ ndị na-adịbeghị anya, ụlọ ọrụ biopharmaceutical aghọọla ahịa zuru ụwa ọnụ na-abawanye ụba na oke ahịa. Site na mmụba dị egwu nke ụlọ ọrụ biopharmaceutical1,2,3, a na-achọsi ike inyocha peptides na protein. Na mgbakwunye na peptide a na-elekwasị anya, a na-emepụta ọtụtụ ihe na-adịghị ọcha n'oge mmepụta peptide, si otú a chọrọ nhicha chromatographic iji nweta peptides nke ịdị ọcha achọrọ. 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 na otu ihe atụ. Ọ bụ ezie na spectrometry mass bụ ngwaọrụ dị irè maka peptide na usoro protein, ọ bụrụ na etinyere ihe atụ ndị dị otú ahụ n'ime spectrometer mass n'otu ngafe, nkewa agaghị adị mma. Enwere ike ibelata nsogbu a site na itinye nkewa chromatography mmiri mmiri (LC) tupu nyocha MS, nke ga-ebelata ọnụọgụgụ nke ndị nyocha na-abanye na spectrometer mass n'oge enyere4,5,6. Na mgbakwunye, n'oge nkewa nkewa nke mmiri mmiri, enwere ike ilekwasị anya na ndị nyocha na mpaghara dị warara, si otú a na-etinye ndị nyocha ndị a n'ọrụ ma melite nchọpụta MS. mmetụta uche. Mmiri chromatography (LC) emeela nke ọma n'ime afọ iri gara aga ma aghọọla usoro ama ama na nyocha proteomic7,8,9,10.
A na-eji chromatography mmiri mmiri nke a gbanwere agbanwe (RP-LC) eme ihe nke ukwuu maka nhicha na nkewa nke ngwakọta peptide site na iji octadecyl-modified silica (ODS) dị ka usoro kwụ ọtọ11,12,13. Agbanyeghị, usoro RP anaghị enye nkewa zuru oke nke peptides na protein n'ihi nhazi ha dị mgbagwoju anya na ọdịdị amphiphilic 14,15. Ya mere, a chọrọ usoro kwụ ọtọ pụrụ iche iji nyochaa peptides na protein nwere polar na non-polar moieties iji mekọrịta ma jide ndị nyocha a16. Chromatography ụdị agwakọtara agwakọta, nke na-enye mmekọrịta multimodal, nwere ike ịbụ ihe ọzọ na RP-LC maka nkewa nke peptides, protein, na ngwakọta ndị ọzọ dị mgbagwoju anya. A kwadebela ọtụtụ usoro agwakọtara agwakọta, ejiriwokwa ogidi ndị a jupụta na usoro ndị a maka nkewa peptide na protein17,18,19,20,21. Usoro agwakọtara agwakọta (WAX/RPLC, HILIC/RPLC, njikọ polar/RPLC) dabara adaba maka peptide na protein Nkewa n'ihi ọnụnọ nke ma otu polar na nke na-abụghị polar22,23,24,25,26,27,28. N'otu aka ahụ, usoro polar na-anọpụ iche na otu polar jikọtara ọnụ na-egosi ike nkewa dị mma na nhọrọ pụrụ iche maka ndị na-enyocha polar na ndị na-abụghị polar, ebe nkewa dabere na mmekọrịta dị n'etiti usoro analyte na nke na-anaghị anọgide. Mmekọrịta multimodal 29, 30, 31, 32. N'oge na-adịbeghị anya, Zhang et al. 30 kwadebere usoro polyamine nke dodecyl kwụsịrị ma kewaa hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, na ọtụtụ ndị ọzọ na-enyocha ihe nke ọma. Onye na-eme ihe na polar intercalator nwere ma otu polar na nke na-abụghị polar, yabụ enwere ike iji ya kewaa peptides na protein ndị nwere ma hydrophobic na hydrophilic moieties. Ogidi ndị e tinyere na polar (dịka ọmụmaatụ, kọlụm C18 nke amide) dị n'ahịa n'okpuru aha azụmaahịa Ascentis Express RP-Amide, mana ejiri kọlụm ndị a maka nyocha nke amine 33 naanị.
N'ọmụmụ ihe a dị ugbu a, a kwadebere ma nyochaa usoro nkewa nke polar-embedded static phase (N-phenylmaleimide-embedded polystyrene) maka nkewa nke peptides na trypsin digests nke HSA. A kwadebere usoro nkewa nke na-anaghị agbanwe agbanwe site na iji usoro a. A kwadebere mkpụrụ silica porous dịka usoro enyere na mbipụta anyị gara aga yana mgbanwe ụfọdụ na usoro nkwadebe. A gbanwere oke nke urea, polyethylene glycol (PEG), TMOS, na mmiri acetic acid iji kwadebe mkpụrụ silica nwere nnukwu nha oghere. Nke abụọ, a na-emepụta ligand ọhụrụ, phenylmaleimide-methyl vinyl isocyanate, ma jiri ya wepụta mkpụrụ silica iji kwadebe usoro nkewa nke polar. A na-etinye usoro nkewa nke na-anaghị agbanwe agbanwe n'ime kọlụm ígwè anaghị agba nchara (100 × 1.8 mm id) site na iji usoro nkewa nke kachasị mma. A na-enyere nkwakọ kọlụm aka site na iji igwe na-ama jijiji iji hụ na e mepụtara akwa yiri ibe ya n'ime kọlụm ahụ. Nyochaa nkewa kọlụm nke ngwakọta peptide nke nwere peptide ise; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) na Trypsin digest nke mmadụ serum albumin (HAS). A hụrụ ngwakọta peptide na trypsin digest nke HSA ka ha kewapụrụ onwe ha 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 abụọ ahụ edoziri nke ọma ma rụọ ọrụ nke ọma na kọlụm PMP, nke dị irè karịa kọlụm Ascentis Express RP-Amide.
PEG (Polyethylene Glycol), Urea, Acetic Acid, Trimethoxy Orthosilicate (TMOS), Trimethyl Chlorosilane (TMCS), Trypsin, Human Serum Albumin (HSA), Ammonium Chloride, Urea, Hexane Methyldisilazane (HMDS), Methacryloyl Chloride (MC), Styrene, 4-Hydroxy-TEMPO, Benzoyl Peroxide (BPO), HPLC Grade Acetonitrile (ACN), Methanol, 2-Propanol, na Acetone Zụrụ na Sigma-Aldrich (St. Louis, MO, USA).
A gwakọtara ngwakọta urea (8 g), polyethylene glycol (8 g), na 8 mL nke 0.01 N acetic acid ruo nkeji iri, wee tinye 24 mL nke TMOS na ya n'ọnọdụ oyi na-atụ. A kpoo ngwakọta mmeghachi omume ahụ ọkụ na 40°C ruo awa isii wee tinye ya na 120°C ruo awa asatọ na autoclave ígwè anaghị agba nchara. A wụsara mmiri ahụ ma kpọọ nkụ ihe fọdụrụ na 70°C ruo awa iri na abụọ. A gwere ntụ ntụ dị nro nke kpọrọ nkụ ahụ nke ọma n'ime oven ma kpoo ya na 550°C ruo awa iri na abụọ. A kwadebere ma kọwaa otu atọ iji lelee ike imepụtaghachi ya na nha irighiri ihe, nha oghere na mpaghara elu.
Site na mgbanwe elu nke ihe ndị silica nwere ligand phenylmaleemide-methylvinylisocyanate (PCMP) nke e tinyere na radial polymerization na styrene, e mepụtara ihe mejupụtara otu polar. Usoro nkwụsị maka aggregates na polystyrene chain. A kọwara usoro nkwadebe n'okpuru.
A gbazere N-phenylmaleimide (200 mg) na methyl vinyl isocyanate (100 mg) na toluene akọrọ, e tinyekwara 0.1 mL nke 2,2′-azoisobutyronitrile (AIBN) na karama mmeghachi omume iji kwadebe phenylmaleimide-methyl vinyl isocyanate copolymer (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 ihe ndị silica kpọrọ nkụ (2 g) n'ime toluene akọrọ (100 mL), kpalite ma tinye ya na karama gburugburu ala 500 mL maka nkeji iri. A gbazere PMCP (10 mg) na toluene ma tinye ya na karama mmeghachi omume site na funnel na-ada ada. A gbazere ngwakọta ahụ na 100°C ruo awa 8, a sachaa ya ma saa ya na acetone ma kpọọ ya nkụ na 60°C ruo awa 3. Mgbe ahụ, a gbazere ihe ndị silica ejikọtara na PMCP (100 g) na toluene (200 ml) ma tinye 4-hydroxy-TEMPO (2 mL) 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 mkpụrụ silica nke TEMPO-PMCP jikọtara (1.5 g) na toluene ma jiri nitrogen sachaa ya. Emere polymerization nke styrene na 100°C ruo awa 12. A sachara ngwaahịa ahụ na methanol ma kpọọ nkụ na 60°C n'abalị. E gosiri atụmatụ mmeghachi omume zuru oke na Foto 1.
E wepụrụ ihe nlele ndị a na 393 K ruo otu awa iji nweta nrụgide fọdụrụnụ nke na-erughị 10-3 Torr. E jiri ọnụọgụ N2 nke a na-etinye na nrụgide dị n'etiti P/P0 = 0.99 chọpụta oke oghere niile. E jiri igwe nyocha eletrọn (Hitachi High Technologies, Tokyo, Japan) nyochaa ọdịdị nke ihe ndị silica gba ọtọ na nke ligand jikọtara ọnụ. E tinyere ihe nlele kpọrọ nkụ (ihe ndị silica gba ọtọ na ihe ndị silica jikọtara ọnụ) na kọlụm aluminom site na iji teepu carbon gluu. E ji ihe mkpuchi Q150T sputter kpuchie ihe nlele ndị ahụ, e tinyekwara oyi akwa Au 5 nm na ihe nlele ndị ahụ. Nke a na-eme ka arụmọrụ usoro dịkwuo mma site na iji obere voltaji ma na-enye obere ọka, sputtering oyi. E jiri ihe nyocha eletrọnịk Thermo Electron (Waltham, MA, USA) Flash EA1112 eletrọnịk maka nyocha eletrọnịk. E jiri ihe nyocha nha nha Malvern (Worcestershire, UK) Mastersizer 2000 nweta nkesa nha nha. Silica gba ọtọ A gbasasịrị ihe ndị dị na mkpụrụ silica e ji ligand jikọta (5 mg nke ọ bụla) na 5 mL nke isopropanol, gbanye ya ọkụ ruo nkeji iri, gbanye ya ọkụ ruo nkeji ise, ma tinye ya na oche anya nke Mastersizer. E mere nyocha thermogravimetric na ọnụego nke 5 °C kwa nkeji n'elu oke okpomọkụ nke 30 ruo 800 °C.
E ji usoro nkwakọ ngwaahịa slurry jupụta ogidi ígwè anaghị agba nchara nke nwere iko, nke nha ya dị warara (100 × 1.8 mm id), na-etinye otu usoro e ji mee ihe na Ref. 31. E jikọtara ogidi ígwè anaghị agba nchara (nke nwere iko, 100 × 1.8 mm id) nke nwere ihe nkwụnye mmiri nwere 1 µm frit na ihe nkwụnye mmiri slurry (Alltech Deerfield, IL, USA). Kwadebe ihe nkwụnye mmiri stage site na ịkwụsịtụ 150 mg nke usoro stage na 1.2 mL nke methanol wee ziga ya na kọlụm nchekwa. E jiri methanol mee ihe dị ka ihe mgbaze slurry yana ihe mgbaze propelling. Jupụta kọlụm ahụ n'usoro site na itinye nrụgide nke 100 MP maka nkeji 10, 80 MP maka nkeji 15, na 60 MP maka nkeji 30. N'oge nkwakọ ngwaahịa, e jiri igwe jijiji mekaniki mee ihe na ihe ndọba GC abụọ (Alltech, Deerfield, IL, USA) iji hụ na ngwugwu ahụ dị otu. Mechie ihe nkwụnye mmiri slurry ma hapụ nrụgide ahụ nwayọ iji gbochie mmebi ọ bụla n'ime kọlụm ahụ. Kwụpụ kọlụm ahụ na ngalaba nkwakọ ngwaahịa slurry ma jikọọ ihe mgbakwunye ọzọ na ntinye na sistemụ LC iji lelee arụmọrụ ya.
E wuru ọkpọkọ LC (10AD Shimadzu, Japan), injector (Valco (USA) C14 W.05) nwere oghere injection 50nL, membrane degasser (Shimadzu DGU-14A), UV-VIS capillary window Special µLC device detector (UV-2075) na microcolumns nwere iko. Jiri ọkpọkọ njikọ dị warara ma dị mkpụmkpụ iji belata mmetụta nke ịgbasa band kọlụm ọzọ. Mgbe nkwakọ ngwaahịa gasịrị, etinyere capillaries (50 μm id 365 na reducing union capillaries (50 μm) na 1/16″ nke njikọ ahụ na-ebelata. E jiri ngwanrọ Multichro 2000 mee nchịkọta data na nhazi chromatographic. E nwalere nlekota na 254 nm Analytes maka nnabata UV. OriginPro8 (Northampton, MA) nyochachara data Chromatographic.
Albumin sitere na serum mmadụ, ntụ ntụ lyophilized, ≥ 96% (agarose gel electrophoresis) 3 mg agwakọtara ya na trypsin (1.5 mg), 4.0 M urea (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 ruo awa isii, wee jiri 1 mL nke 0.1% TFA mechie ya. Sachaa mmiri ọgwụ ahụ ma chekwaa ya n'okpuru 4°C.
A tụlere nkewa nke ngwakọta peptide na HSA trypsin digests iche iche na kọlụm PMP. Lelee nkewa nke ngwakọta peptide na trypsin digest nke HSA site na kọlụm PMP wee tụnyere nsonaazụ ya na kọlụm Ascentis Express RP-Amide. A na-agbakọ nọmba efere echiche dị ka ndị a:
E gosiri foto SEM nke mkpụrụ silica gba ọtọ na mkpụrụ silica gbakọtara ligand na FIG. 2. Foto SEM nke mkpụrụ silica gba ọtọ (A, B) na-egosi na, n'adịghị ka ọmụmụ ihe anyị gara aga, mkpụrụ ndị a bụ okirikiri ebe mkpụrụ ndị ahụ dị ogologo ma ọ bụ nwee nhazi na-adịghị mma. Elu nke mkpụrụ silica gbakọtara ligand (C, D) dị nro karịa nke mkpụrụ silica gba ọtọ, nke nwere ike ịbụ n'ihi mkpuchi nke agbụ polystyrene n'elu mkpụrụ silica.
Ịnyocha onyonyo elektrọn microscope nke mkpụrụ silica gba ọtọ (A, B) na mkpụrụ silica ejiri ligand jikọta (C, D).
A na-egosi nkesa nha nke ihe ndị silica gba ọtọ na ihe ndị silica jikọtara na ligand na Foto 3(A). Usoro nkesa nha nke ihe ndị dị na olu gosiri na nha nke ihe ndị silica mụbara mgbe mgbanwe kemịkalụ gasịrị (Fig. 3A). A na-atụnyere data nkesa nha nke ihe ndị silica sitere na ọmụmụ ihe ugbu a na ọmụmụ ihe gara aga na Tebụl 1(A). Nha nha nke ihe ndị dị na olu, d(0.5), nke PMP bụ 3.36 μm, ma e jiri ya tụnyere ọmụmụ ihe anyị gara aga na uru ad(0.5) nke 3.05 μm (ihe ndị silica jikọtara na polystyrene)34. Ogbe a nwere nkesa nha nke ihe ndị dị na ya dị warara ma e jiri ya tụnyere ọmụmụ ihe anyị gara aga n'ihi oke dị iche iche nke PEG, urea, TMOS, na acetic acid na ngwakọta mmeghachi omume. Nha nha nke usoro PMP dị ntakịrị karịa nke usoro nke ihe ndị silica jikọtara na polystyrene anyị mụrụ na mbụ. Nke a pụtara na ọrụ elu nke ihe ndị silica nwere styrene na-etinye naanị oyi akwa polystyrene (0.97 µm) n'elu silica, ebe na Usoro 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 silica gba ọtọ na ihe ndị dị ndụ silica nke ligand jikọtara.
A na-egosi nha oghere, olu oghere na mpaghara elu nke ihe ndị silica dị na ọmụmụ ihe a na Tebụl 1(B). A na-egosi profaịlụ PSD nke ihe ndị silica gba ọtọ na ihe ndị silica gbakọtara ligand na Foto 3(B). Nsonaazụ ya yiri nke anyị mere na ọmụmụ ihe anyị gara aga. Nha oghere nke ihe ndị silica gba ọtọ na nke ligand gbara bụ 310 na 241, n'otu n'otu, nke na-egosi na nha oghere ahụ na-ebelata site na 69 mgbe mgbanwe kemịkalụ gasịrị, dịka egosiri na Tebụl 1(B), a na-egosikwa mgbanwe nke usoro ahụ na Foto 3(B). N'otu aka ahụ, olu oghere nke ihe ndị silica belatara site na 0.67 ruo 0.58 cm3/g mgbe mgbanwe kemịkalụ gasịrị. Mpaghara elu kpọmkwem nke ihe ndị silica a na-amụ ugbu a bụ 116 m2/g, nke yiri ọmụmụ ihe anyị gara aga (124 m2/g). Dịka egosiri na Tebụl 1(B), mpaghara elu (m2/g) nke ihe ndị silica gbapụrụ agbapụ kwa belatara site na 116 m2/g ruo 105 m2/g mgbe mgbanwe kemịkalụ gasịrị.
A na-egosi nsonaazụ nke nyocha elemental nke usoro kwụ ọtọ na Tebụl 2. Ibu carbon nke usoro kwụ ọtọ ugbu a bụ 6.35%, nke dị ala karịa ibu carbon nke ọmụmụ anyị gara aga (ihe ndị mejupụtara silica polystyrene, 7.93%35 na 10.21%, n'otu n'otu) 42. Ibu carbon nke usoro kwụ ọtọ ugbu a dị obere, N'ihi na na nkwadebe nke SP ugbu a, na mgbakwunye na styrene, ejiri ụfọdụ ligands polar dị ka phenylmaleemide-methylvinylisocyanate (PCMP) na 4-hydroxy-TEMPO. Pasent ibu nitrogen nke usoro kwụ ọtọ ugbu a bụ 2.21%, ma e jiri ya tụnyere 0.1735 na 0.85% site na ibu nitrogen na ọmụmụ ihe gara aga, n'otu n'otu. Nke a pụtara na wt% nke nitrogen dị elu na usoro kwụ ọtọ ugbu a n'ihi phenylmaleemide. N'otu aka ahụ, ibu carbon nke ngwaahịa (4) na (5) bụ 2.7% na 2.9%, n'otu n'otu, ebe ibu carbon nke ngwaahịa ikpeazụ (6) bụ 6.35%, dịka egosiri na Tebụl 2. Ejiri usoro PMP nke na-anaghị agbanwe agbanwe lelee mbelata ibu ahụ, a na-egosikwa usoro TGA na Foto 4. Usoro TGA na-egosi mbelata ibu nke 8.6%, nke kwekọrọ na ibu carbon (6.35%) n'ihi na ligands nwere ọ bụghị naanị C kamakwa N, O, na H.
A họọrọ ligand phenylmaleemide-methylvinylisocyanate maka mgbanwe elu nke ihe ndị silica n'ihi na o nwere otu polar phenylmaleemide na otu vinylisocyanate. Otu Vinyl isocyanate 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 n'efu. Nzọụkwụ ikpeazụ nke mmeghachi omume (polymerization free-radical) dị oke mkpa ma nwee ike ịgbanwe polarity nke usoro kwụ ọtọ. Emere nyocha elemental iji lelee ibu carbon nke usoro ndị a kwụ ọtọ. A hụrụ na ịbawanye ọnụọgụ styrene na oge mmeghachi omume mụbara ibu 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. Ọzọkwa, tinye usoro ndị a kwụ ọtọ n'ime ogidi ígwè anaghị agba nchara ma lelee ha Arụmọrụ chromatographic (nhọrọ, mkpebi, uru N, wdg). Dabere na nnwale ndị a, ahọpụtara usoro kachasị mma iji kwadebe usoro PMP na-anaghị agbanwe agbanwe iji hụ na polarity achịkwa na njide nyocha dị mma.
E jiri kọlụm PMP jiri usoro mkpanaka nwalee ngwakọta peptide ise (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin); 60/40 (v/v) acetonitrile/mmiri (0.1% TFA) na ọsọ mmiri nke 80 μL/min. N'okpuru ọnọdụ mgbapụta kacha mma, nọmba efere echiche (N) kwa kọlụm (100 × 1.8 mm id) bụ 20,000 ± 100 (200,000 efere/m²). Tebụl 3 na-enye ụkpụrụ N maka kọlụm PMP atọ ahụ na egosiri chromatograms na Foto 5A. Nyocha ngwa ngwa na kọlụm PMP na ọsọ mmiri dị elu (700 μL/min), e wepụrụ peptides ise n'ime otu nkeji, ụkpụrụ N dị mma nke ukwuu, 13,500 ± 330 kwa kọlụm (100 × 1.8 mm id), Kwekọrọ na efere 135,000/m (Foto 5B). E ji ọkwa atọ dị iche iche nke usoro PMP kwụ ọtọ jupụta kọlụm atọ (100 × 1.8 mm id) iji lelee Mmepụtagharị. E dekọrọ ntinye nyocha maka kọlụm ọ bụla site na iji ọnọdụ mmịpụta kachasị mma na ọnụọgụ nke efere echiche N na oge njide iji kewaa otu ngwakọta nnwale ahụ na kọlụm ọ bụla. E gosipụtara data mmepụtagharị maka kọlụm PMP na Tebụl 4. Mmepụtagharị nke kọlụm PMP na-ejikọta nke ọma na ụkpụrụ %RSD dị ala, 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 × 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 (leucine) acid enkephalin)).
A tụlere kọlụm PMP (100 × 1.8 mm id) maka nkewa nke tryptic digests nke albumin serum mmadụ na arụmọrụ dị elu nke mmiri mmiri. Chromatogram dị na Foto 6 na-egosi na ihe nlele ahụ kewapụrụ nke ọma ma mkpebi ya dị mma nke ukwuu. Ejiri ọsọ mmiri nke 100 µL/min, usoro mkpanaka 70/30 acetonitrile/mmiri na 0.1% TFA nyochaa digests HSA. Dịka egosiri na chromatogram (Foto 6), a kewara nri HSA n'ime oke 17 kwekọrọ na peptides 17. A gbakọrọ arụmọrụ nkewa nke oke ọ bụla na nchịkọta HSA ma nye ụkpụrụ ndị ahụ na Tebụl 5.
E kewara tryptic digest nke HSA (100 × 1.8 mm id) na kọlụm PMP; ọnụego mmiri (100 µL/min), usoro mkpanaka 60/40 acetonitrile/mmiri nwere 0.1% TFA.
ebe L bụ ogologo kọlụm, η bụ viscosity nke usoro mkpanaka, ΔP bụ nrụgide azụ kọlụm, na u bụ ọsọ kwụ ọtọ nke usoro mkpanaka. Njupụta nke kọlụm PMP bụ 2.5 × 10-14 m2, ọnụego mmiri ahụ bụ 25 μL/min, e jirikwa 60/40 v/v ACN/mmiri. Njupụta nke kọlụm PMP (100 × 1.8 mm id) yiri nke ọmụmụ anyị gara aga Ref.34. Njupụta nke kọlụm jupụtara na obere oghere dị n'elu bụ: 1.7 × 10-15 maka obere 1.3 μm, 3.1 × 10-15 maka obere 1.7 μm, 5.2 × 10-15 na 2.5 × 10-14 m2 maka obere 2.6 μm Maka obere 5 μm 43. Ya mere, ikike nke usoro PMP yiri nke obere 5 μm.
ebe Wx bụ ibu nke kọlụm jupụtara na chloroform, Wy bụ ibu nke kọlụm jupụtara na methanol, na ρ bụ njupụta nke ihe mgbaze. Njupụta nke methanol (ρ = 0.7866) na chloroform (ρ = 1.484). Ngụkọta porosity nke kọlụm SILICA PARTICLES-C18 (100 × 1.8 mm id) 34 na C18-Urea kọlụm 31 anyị mụrụ na mbụ bụ 0.63 na 0.55, n'otu n'otu. Nke a pụtara na ọnụnọ nke urea ligands na-ebelata permeability nke phase kwụ ọtọ. N'aka nke ọzọ, mkpokọta porosity nke kọlụm PMP (100 × 1.8 mm id) bụ 0.60. Permeability nke kọlụm PMP dị ala karịa nke kọlụm ejiri C18-bonded silica particles n'ihi na na standart ụdị C18, a na-ejikọ C18 ligands na silica particles dị ka agbụ linear, ebe na standart ụdị polystyrene usoro, a na-akpụ oyi akwa polima A dị oke arọ gburugburu ya. Na nnwale nkịtị, a na-agbakọ porosity kọlụm dị ka:
Foto nke 7A,B na-egosi kọlụm PMP (100 × 1.8 mm id) na kọlụm Ascentis Express RP-Amide (100 × 1.8 mm id) site na iji otu ọnọdụ mgbapụta ahụ (ya bụ, 60/40 ACN/H2O na 0.1% TFA). ) nke atụmatụ van Deemter. A kwadebere ngwakọta peptide ahọpụtara (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) na 20 µL/. Ọnụego mmiri kacha nta maka kọlụm abụọ ahụ bụ 800 µL/min. Ọnụego HETP kacha nta na ọnụego mmiri kachasị mma (80 µL/min) maka kọlụm PMP na kọlụm Ascentis Express RP-Amide bụ 2.6 µm na 3.9 µm, n'otu n'otu. Ọnụego HETP na-egosi na arụmọrụ nkewa nke kọlụm PMP (100 × 1.8 mm id) ka mma karịa kọlụm Ascentis Express RP-Amide dị n'ahịa (100 × 1.8 mm id). Atụmatụ van Deemter na Fig. 7(A) na-egosi na mbelata na uru N na mmụba mmiri adịghị mkpa ma e jiri ya tụnyere ọmụmụ anyị gara aga. Ọrụ nkewa dị elu nke kọlụm PMP (100 × 1.8 mm id) ma e jiri ya tụnyere kọlụm Ascentis Express RP-Amide dabere na mmezi na ọdịdị, nha, na usoro nchikọta kọlụm dị mgbagwoju anya ejiri na ọrụ dị ugbu a34.
(A) atụmatụ van Deemter (ọsọ HETP megide ọsọ nke usoro mkpanaka) enwetara site na iji kọlụm PMP (100 × 1.8 mm id) na 60/40 ACN/H2O nwere 0.1% TFA.(B) atụmatụ van Deemter (ọsọ HETP megide ọsọ nke usoro mkpanaka) enwetara site na iji kọlụm Ascentis Express RP-Amide (100 × 1.8 mm id) na 60/40 ACN/H2O nwere 0.1% TFA.
E kwadebere ma nyochaa usoro polystyrene nke a na-etinye n'ime polar maka nkewa nke ngwakọta peptide sịntetik na nchịkọta trypsin nke albumin serum mmadụ (HAS) na chromatography mmiri mmiri dị elu. Arụmọrụ chromatographic nke kọlụm PMP maka ngwakọta peptide dị mma na arụmọrụ nkewa na mkpebi nkewa. Arụmọrụ nkewa ka mma nke kọlụm PMP bụ n'ihi ọtụtụ ihe kpatara ya, dịka nha irighiri ihe na nha oghere nke mkpụrụ silica, njikọ a na-achịkwa nke usoro kwụ ọtọ, na ngwugwu kọlụm dị mgbagwoju anya. Na mgbakwunye na arụmọrụ nkewa dị elu, nrụgide azụ kọlụm dị ala na ọnụego mmiri dị elu bụ uru ọzọ nke usoro kwụ ọtọ a. Ogidi PMP na-egosipụta mmepụta dị mma ma enwere ike iji ya maka nyocha nke ngwakọta peptide na mgbaze trypsin nke protein dị iche iche. Anyị bu n'obi iji kọlụm a maka nkewa nke ngwaahịa eke, ogige bioactive sitere na osisi ọgwụ na ihe ndị sitere n'okike na chromatography mmiri mmiri. N'ọdịnihu, a ga-enyocha kọlụm PMP maka nkewa nke protein na mgbochi monoclonal.
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