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Porous silica hais tau npaj los ntawm txoj kev sol-gel nrog qee qhov kev hloov kho kom tau txais qhov dav-pore hais. Cov khoom no tau derivatized nrog N-phenylmaleimide-methylvinyl isocyanate (PMI) thiab styrene ntawm rov qab saw hloov-fragmentation (RAFT) polymerization los tsim N-phenylmaleimide intercalated polyamides. Styrene (PMP) nyob ruaj khov theem. Nqaim bore stainless hlau kab (100 × 1.8 mm puab txoj kab uas hla) tau ntim nrog cov slurry packing. Lub chromatographic kev ua tau zoo ntawm PMP kem tau soj ntsuam kom cais cov khoom sib xyaw ntawm cov peptides uas muaj tsib peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) thiab tryptic hydrolyzate ( human albumS ). Raws li kev pom zoo elution, tus lej theoretical ntawm daim hlau nrog sib xyaw ntawm peptides mus txog 280,000 daim hlau / sq.m. Sib piv qhov kev sib cais ntawm cov kab ke tsim nrog kev lag luam Ascentis Express RP-Amide kem, nws tau pom tias kev sib cais ntawm PMP kem yog qhov zoo tshaj rau kab kev lag luam hauv cov ntsiab lus ntawm kev sib cais thiab kev daws teeb meem.
Kev lag luam biopharmaceutical tau dhau los ua kev lag luam thoob ntiaj teb nrog kev nce qib hauv kev lag luam hauv xyoo tas los. Nrog rau kev loj hlob tawg ntawm kev lag luam biopharmaceutical1,2,3 muaj qhov xav tau zoo rau peptide thiab cov tshuaj ntsuam xyuas protein. Ntxiv nrog rau lub hom phiaj peptide, ntau yam impurities yog tsim thaum lub sij hawm peptide synthesis, yog li chromatographic purification yuav tsum tau kom tau qhov xav tau purity ntawm peptide. Kev tsom xam thiab kev ua tus yam ntxwv ntawm cov protein nyob hauv lub cev, cov ntaub so ntswg, thiab cov hlwb yog ib txoj haujlwm nyuaj heev vim muaj coob tus neeg uas muaj peev xwm kuaj tau cov tsiaj nyob hauv ib qho qauv. Txawm hais tias huab hwm coj spectrometry yog ib qho cuab yeej zoo rau kev sib luag peptides thiab cov proteins, yog tias cov qauv no ncaj qha nkag mus rau hauv qhov loj spectrometer, kev sib cais yuav tsis txaus siab. Qhov teeb meem no tuaj yeem daws tau los ntawm kev ua cov kua chromatography (LC) ua ntej MS tsom xam, uas yuav txo tau cov analytes nkag mus rau hauv huab hwm coj spectrometer ntawm ib lub sijhawm 4,5,6. Tsis tas li ntawd, cov analytes tuaj yeem mloog zoo rau hauv thaj av nqaim thaum lub sij hawm ua kua theem sib cais, yog li tsom rau cov analytes no thiab ua rau muaj kev nkag siab zoo ntawm MS nrhiav pom. Liquid chromatography (LC) tau nce siab heev nyob rau kaum xyoo dhau los thiab tau dhau los ua ib txoj hauv kev siv rau kev soj ntsuam proteomic7,8,9,10.
Reverse-theem kua chromatography (RP-LC) yog dav siv los ntxuav thiab cais cov sib tov ntawm peptides siv octadecyl-hloov silica (ODS) raws li nyob rau theem 11,12,13. Txawm li cas los xij, vim lawv cov qauv nyuaj thiab cov xwm txheej amphoteric, 14,15 RP nyob ruaj khov theem tsis tuaj yeem muab kev sib cais txaus siab ntawm peptides thiab cov proteins. Yog li ntawd, kev tsom xam ntawm peptides thiab cov proteins nrog lub ncov qaumteb qabteb thiab tsis-polar fragments yuav tsum tau tsim tshwj xeeb tsim nyob rau theem nyob rau hauv kev sib cuam tshuam thiab khaws cov analytes16. Mixed chromatography, uas muaj kev sib cuam tshuam ntau yam, tuaj yeem yog lwm txoj hauv kev rau RP-LC rau kev sib cais peptides, proteins, thiab lwm yam sib xyaw ua ke. Ob peb theem sib xyaw ua ke tau npaj thiab cov kab uas muaj cov theem nyob ruaj khov no tau siv los cais cov peptides thiab cov proteins17,18,19,20,21. Vim muaj cov pawg polar thiab tsis-polar, sib xyaw hom nyob ruaj khov theem (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) yog tsim rau kev sib cais ntawm peptides thiab proteins22,23,24,25,26,27,28. , polar intercalated nyob ruaj ruaj theem nrog covalently bonded polar pawg qhia zoo sib cais muaj peev xwm thiab tshwj xeeb xaiv rau polar thiab non-polar analytes vim hais tias kev sib cais nyob ntawm kev sib cuam tshuam ntawm cov analyte thiab nyob ruaj ruaj theem Multimodal kev sib tshuam 29,30,31,32. Tsis ntev los no, Zhang et al. 30 tau txais behenyl-terminated nyob ruaj ruaj theem ntawm polyamines thiab ntse cais hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, thiab ib co lwm yam analytes. Lub ncov qaumteb qabteb embedded khoom nyob ruaj ruaj muaj ob pawg polar thiab non-polar, yog li nws tuaj yeem siv los cais cov peptides thiab cov proteins rau hauv qhov chaw hydrophobic thiab hydrophilic. Polar inline kab (piv txwv li, C18 kab nrog amide inline) muaj nyob rau hauv lub npe lag luam Ascentis Express RP-Amide kab, tab sis cov kab no tsuas yog siv rau kev tsom xam ntawm amine 33.
Nyob rau hauv txoj kev tshawb no tam sim no, ib tug polar embedding nyob ruaj ruaj theem (N-phenylmaleimide, embedding polystyrene) tau npaj thiab soj ntsuam rau peptide sib cais thiab tryptic HSA cleavage. Cov tswv yim hauv qab no tau siv los npaj cov theem nyob ruaj khov. Porous silica hais tau npaj raws li cov txheej txheem uas tau piav qhia hauv peb cov ntawv tshaj tawm yav dhau los, nrog qee qhov kev hloov pauv hauv kev npaj 31, 34, 35, 36, 37, 38, 39. Qhov sib piv ntawm urea, polyethylene glycol (PEG), TMOS thiab aqueous-acetic acid tau hloov kho qhov loj me ntawm poov tshuaj. Thib ob, ib qho tshiab phenylmaleimide-methylvinyl isocyanate ligand tau ua ke thiab nws cov khoom siv silica derivatized tau siv los npaj cov polar embedded nyob ruaj khov theem. Cov theem tau nyob ruaj khov tau ntim rau hauv cov kab hlau tsis muaj steel (sab hauv txoj kab uas hla 100 × 1.8 hli) raws li cov txheej txheem ntim khoom zoo. Kev ntim ntawm kem yog pab los ntawm cov neeg kho tshuab kev co los xyuas kom meej ib txheej txheej nyob rau hauv kem. Cov kab ntawv ntim tau raug soj ntsuam rau kev sib cais ntawm kev sib xyaw ntawm peptides uas muaj tsib peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin peptide). thiab tryptic hydrolysates ntawm human serum albumin (HSA). Nws tau pom tias peptide sib tov thiab HSA tryptic zom tau sib cais nrog kev daws teeb meem zoo thiab ua haujlwm zoo. Kev sib cais ua haujlwm ntawm PMP kem tau muab piv rau Ascentis Express RP-Amide kem. Nws tau pom tias peptides thiab cov proteins muaj kev daws teeb meem zoo thiab kev sib cais siab ntawm PMP kem, thiab kev sib cais ntawm PMP kem siab dua li Ascentis Express RP-Amide kem.
PEG (polyethylene glycol), urea, acetic acid, trimethoxyorthosilicate (TMOS), trimethylchlorosilane (TMCS), trypsin, human serum albumin (HSA), ammonium chloride, urea, hexamethylmethacryloyldisilazane (HMDS), methacryloyl EMPO chloride (MC), peroxide (BPO), acetonitrile (ACN) rau HPLC, methanol, 2-propanol thiab acetone. Sigma-Aldrich Company (St. Louis, Missouri, USA).
Kev sib xyaw ntawm urea (8 g), polyethylene glycol (8 g) thiab 8 ml ntawm 0.01 N. acetic acid tau nplawm rau 10 feeb, thiab 24 ml ntawm TMOS tau ntxiv rau hauv qab dej khov txias. Cov tshuaj tiv thaiv sib xyaw yog rhuab ntawm 40 ° C rau 6 teev thiab tom qab ntawd ntawm 120 ° C rau 8 teev hauv stainless hlau autoclave. Cov dej tau decanted thiab cov seem tau qhuav ntawm 70 ° C rau 12 teev. Cov pob zeb qhuav qhuav tau hauv av kom du thiab calcined hauv qhov cub ntawm 550 ° C rau 12 teev. Peb batch tau npaj thiab tus yam ntxwv los ntsuam xyuas lub reproducibility ntawm particle qhov ntau thiab tsawg, pore loj thiab cheeb tsam saum npoo.
Polar pawg thiab nyob ruaj ruaj theem rau polystyrene chains. Cov txheej txheem npaj tau piav qhia hauv qab no.
N-phenylmaleimide (200 mg) thiab methyl vinyl isocyanate (100 mg) tau yaj hauv anhydrous toluene, thiab tom qab ntawd 0.1 ml ntawm 2,2′-azoisobutyronitrile (AIBN) tau ntxiv rau cov tshuaj tiv thaiv lub khob kom tau txais ib copolymer ntawm phenylmaleimide thiab CPPM isocyanate. ) Cov sib tov yog rhuab ntawm 60 ° C rau 3 xuab moos, lim thiab qhuav hauv qhov cub ntawm 40 ° C rau 3 teev.
Qhuav silica hais (2 g) tau dispersed nyob rau hauv qhuav toluene (100 ml), stirred thiab sonicated rau 10 min nyob rau hauv ib tug 500 ml puag ncig hauv qab hwj. PMCP (10 mg) tau yaj nyob rau hauv toluene thiab ntxiv dropwise rau cov tshuaj tiv thaiv lub hwj ntawm ib qho ntxiv funnel. Cov sib tov yog refluxed ntawm 100 ° C rau 8 teev, lim, ntxuav nrog acetone thiab qhuav ntawm 60 ° C rau 3 teev. Tom qab ntawd, silica hais nrog PMCP (100 g) tau yaj hauv toluene (200 ml), thiab 4-hydroxy-TEMPO (2 ml) tau ntxiv rau qhov ntawd nyob rau hauv muaj 100 μl ntawm dibutyltin dilaurate ua ib qho catalyst. Qhov sib tov yog nplawm ntawm 50 ° C rau 8 teev, lim thiab qhuav ntawm 50 ° C rau 3 teev.
Styrene (1 ml), benzoyl peroxide BPO (0.5 ml) thiab silica hais uas txuas rau TEMPO-PMCP (1.5 g) tau tawg nyob rau hauv toluene thiab purged nrog nitrogen. Lub polymerization ntawm styrene tau ua tiav ntawm 100 ° C rau 12 teev. Cov khoom tsim tau raug ntxuav nrog methanol thiab qhuav ib hmos ntawm 60 ° C. Lub tswv yim dav dav ntawm cov tshuaj tiv thaiv yog qhia hauv daim duab. ib.
Cov qauv no tau degassed ntawm 393 K rau 1 h kom txog thaum qhov seem seem ntawm tsawg dua 10-3 Torr tau txais. Tus nqi ntawm N2 adsorbed ntawm tus txheeb ze siab P / P0 = 0.99 yog siv los txiav txim tag nrho cov pore ntim. Lub morphology ntawm cov ntshiab thiab ligand-bound silica hais tau tshuaj xyuas siv lub tshuab ntsuas hluav taws xob (Hitachi High Technologies, Tokyo, Nyiv). Cov qauv qhuav (ntshiab silica thiab ligand bound silica hais) tau muab tso rau ntawm txhuas rods siv carbon daim kab xev. Kub tau tso rau hauv cov qauv siv Q150T sputtering ntaus ntawv, thiab 5 nm tuab Au txheej tau muab tso rau ntawm cov qauv. Qhov no txhim kho kev ua haujlwm ntawm cov txheej txheem tsis tshua muaj hluav taws xob thiab muab cov tshuaj txau zoo. Elemental tsom xam tau ua tiav siv lub Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental composition analyzer. Lub Malvern particle loj analyzer (Worcestershire, UK) Mastersizer 2000 tau siv los muab cov particle loj faib. Uncoated silica hais thiab ligand-bound silica hais (5 mg txhua) tau tawg nyob rau hauv 5 ml ntawm isopropanol, sonicated rau 10 feeb, agitated rau 5 feeb, thiab muab tso rau ntawm ib tug Mastersizer kho qhov muag lub rooj ntev zaum. Thermogravimetric tsom xam yog nqa tawm ntawm tus nqi ntawm 5 ° C ib feeb nyob rau hauv qhov kub ntawm 30 mus rau 800 ° C.
Cov iav fiber ntau lined nqaim bore stainless hlau kab nrog qhov ntev (ID 100 × 1.8 mm) tau ntim los ntawm slurry filling txoj kev raws li cov txheej txheem raws li nyob rau hauv 31. Stainless steel kem (iav lined, ID 100 × 1.8 mm) thiab ib lub qhov hluav taws xob uas muaj 1 µm frit tau txuas nrog lub slurry tech packing, USA (A. Npaj ib qho kev ncua ntawm qhov chaw nyob ruaj khov los ntawm kev ncua 150 mg ntawm cov theem nyob ruaj khov hauv 1.2 ml ntawm methanol thiab pub rau hauv ib qho chaw cia khoom. Methanol tau siv los ua cov kuab tshuaj slurry thiab tswj cov kuab tshuaj. Ntim cov kem los ntawm kev siv lub siab ua ntu zus ntawm 100 MP rau 10 min, 80 MP rau 15 min, thiab 60 MP rau 30 min. Cov txheej txheem ntim khoom siv ob lub roj chromatography kem vibrators (Alltech, Deerfield, IL, USA) rau cov neeg kho tshuab kev co kom ntseeg tau tias cov khoom ntim zoo ib yam. Kaw lub slurry packer thiab tso siab maj mam kom tsis txhob muaj kev puas tsuaj rau txoj hlua. Cov kem tau raug txiav tawm ntawm lub slurry nozzle thiab lwm qhov haum tau txuas rau lub inlet thiab txuas nrog LC system los ntsuas nws txoj haujlwm.
Ib qho kev cai MLC tau tsim los siv lub twj tso kua mis LC (10AD Shimadzu, Nyiv), tus qauv nrog 50 nL txhaj tshuaj voj (Valco (USA) C14 W.05), daim nyias nyias degasser (Shimadzu DGU-14A), thiab UV-VIS capillary qhov rais. Cov cuab yeej ntes (UV-2075) thiab enamelled microcolumn. Siv cov hlua txuas nqaim thiab luv luv kom txo tau cov nyhuv ntawm cov kab txuas ntxiv. Tom qab sau lub kem, nruab ib lub capillary (50 µm id 365) ntawm qhov hluav taws xob ntawm 1/16 "txo kev sib tshuam thiab nruab ib lub capillary (50 µm) ntawm qhov kev sib tshuam. Kev sau cov ntaub ntawv thiab kev ua chromatogram yog ua los ntawm Multichro 2000 software. Ntawm 254 nm, UV absorbance ntawm cov ntsiab lus analytes tau soj ntsuam ntawm 0. Chromatographic cov ntaub ntawv tau txheeb xyuas siv OriginPro8 (Northampton, MA).
Tib neeg cov ntshav albumin, lyophilized hmoov, ≥ 96% (agarose gel electrophoresis) 3 mg tov nrog trypsin (1.5 mg), 4.0 M urea (1 ml) thiab 0.2 M ammonium bicarbonate (1 ml). Cov tshuaj tau nplawm rau 10 feeb thiab muab tso rau hauv ib da dej ntawm 37 ° C rau 6 h, ces quenched nrog 1 ml ntawm 0.1% TFA. Lim cov tshuaj thiab khaws cia hauv qab 4 ° C.
Kev sib cais ntawm kev sib xyaw ntawm peptides thiab tryptic digest HSA ntawm PMP kem raug soj ntsuam cais. Tshawb xyuas cov tryptic hydrolysis ntawm kev sib xyaw ntawm peptides thiab HSA sib cais los ntawm PMP kem thiab sib piv cov txiaj ntsig nrog Ascentis Express RP-Amide kem. Tus naj npawb ntawm cov phiaj xwm theoretical yog xam los ntawm kev sib npaug hauv qab no:
SEM dluab ntawm ntshiab silica hais thiab ligand bound silica hais yog qhia nyob rau hauv daim duab 2. SEM dluab ntawm ntshiab silica hais (A, B) qhia ib tug spherical zoo uas cov hais yog elongated los yog irregular symmetry piv rau peb yav dhau los kev tshawb fawb. Qhov saum npoo ntawm cov silica uas khi los ntawm ligand (C, D) yog smoother dua li cov ntshiab silica hais, uas tej zaum yuav yog vim lub polystyrene chains npog lub nto ntawm silica hais.
Scanning electron micrographs ntawm ntshiab silica hais (A, B) thiab ligand bound silica hais (C, D).
Qhov loj me me ntawm cov khoom siv dawb huv silica thiab ligand-bound silica hais yog qhia hauv daim duab 2. 3(A). Volumetric particle loj faib curves pom tias silica particle loj tau nce tom qab kev hloov kho tshuaj (Fig. 3A). Cov ntaub ntawv loj ntawm silica particle faib los ntawm txoj kev tshawb fawb tam sim no thiab cov kev tshawb fawb yav dhau los tau muab piv rau hauv Table 1(A). Lub volumetric particle loj d(0.5) ntawm PMP yog 3.36 µm, piv rau ad(0.5) tus nqi ntawm 3.05 µm nyob rau hauv peb yav dhau los kev kawm (polystyrene bonded silica hais) 34. Vim qhov kev hloov pauv ntawm qhov sib piv ntawm PEG, urea, TMOS thiab acetic acid nyob rau hauv cov tshuaj tiv thaiv sib tov, qhov loj me me ntawm cov khoom no tau nqaim dua piv rau peb txoj kev tshawb fawb dhau los. Qhov loj me me ntawm PMP theem yog me ntsis loj dua li ntawm polystyrene khi silica particle theem uas peb tau kawm ua ntej. Qhov no txhais tau hais tias qhov chaw ua haujlwm ntawm silica hais nrog styrene tso tsuas yog ib txheej polystyrene (0.97 µm) ntawm silica nto, thaum nyob rau theem PMP txheej thickness yog 1.38 µm.
Particle loj faib (A) thiab pore loj faib (B) ntawm ntshiab silica hais thiab ligand khi silica hais.
Qhov pore loj, pore ntim, thiab qhov chaw ntawm cov silica hais siv nyob rau hauv txoj kev tshawb no yog qhia nyob rau hauv Table 1 (B). PSD profiles ntawm ntshiab silica hais thiab ligand-bound silica hais yog qhia nyob rau hauv daim duab. 3 (B). Cov txiaj ntsig tau muab piv rau peb txoj kev tshawb fawb yav dhau los34. Cov pore qhov loj me ntawm cov ntshiab thiab ligand-bound silica hais yog 310 Å thiab 241 Å, raws li qhia tau hais tias tom qab kev hloov kho tshuaj, qhov pore loj poob los ntawm 69 Å, raws li qhia hauv Table 1 (B), thiab qhov hloov nkhaus yog qhia hauv daim duab. kev kawm yav dhau los (124 m2 / g). Raws li qhia hauv Table 1(B), thaj chaw saum npoo (m2 / g) ntawm silica hais tom qab kev hloov kho tshuaj kuj txo qis los ntawm 116 m2 / g mus rau 105 m2 / g.
Cov txiaj ntsig ntawm kev tsom xam ntawm cov theem nyob ruaj khov yog nthuav tawm hauv Table. 2. Cov ntsiab lus ntawm cov pa roj carbon monoxide tam sim no nyob ruaj khov yog 6.35%, uas yog qis dua hauv peb txoj kev tshawb fawb yav dhau los (silica hais nrog polystyrene, 7.93% 35 thiab 10.21%, feem) 42. Cov ntsiab lus ntawm cov pa roj carbon monoxide tam sim no nyob ruaj khov theem hauv qab no, txij li qee cov polar ligands xws li phenylmaleimide methyanate-POMP (PC) thiab vinyl phenylmaleimide methyanate4MP. siv ntxiv rau styrene hauv kev npaj SP. Qhov hnyav feem pua ​​​​ntawm nitrogen nyob rau theem tam sim no nyob ruaj khov yog 2.21% piv rau 0.1735 thiab 0.85% hauv cov kev tshawb fawb yav dhau los42. Qhov no txhais tau hais tias tam sim no nyob ruaj khov theem muaj qhov hnyav feem pua ​​​​ntawm nitrogen vim yog phenylmaleimide. Ib yam li ntawd, cov khoom (4) thiab (5) muaj cov ntsiab lus carbon ntawm 2.7% thiab 2.9%, feem, thaum cov khoom kawg (6) muaj cov ntsiab lus carbon ntawm 6.35%, raws li qhia nyob rau hauv Table 2. Thermogravimetric tsom xam (TGA) tau siv nyob rau theem nyob ruaj khov ntawm PMP los ntsuas qhov hnyav poob, thiab TGA nkhaus yog qhia nyob rau hauv daim duab 6. yog pom zoo nrog cov ntsiab lus carbon (6.35%), vim tias cov ligands tsis tsuas yog C, tab sis kuj N, O thiab H.
Lub ligand phenylmaleimide-methylvinyl isocyanate raug xaiv los hloov kho qhov chaw ntawm cov silica hais vim nws cov polar phenylmaleimide thiab vinylisocyanate pawg. Vinyl isocyanate pawg tuaj yeem cuam tshuam nrog styrene los ntawm kev ua neej nyob radical polymerization. Qhov laj thawj thib ob yog txhawm rau ntxig ib pab pawg uas muaj kev sib cuam tshuam nrog cov tshuaj ntsuam xyuas thiab tsis muaj kev cuam tshuam ntawm electrostatic sib cuam tshuam ntawm cov tshuaj ntsuam xyuas thiab cov theem nyob ruaj khov, vim tias phenylmaleimide moiety tsis muaj nqi virtual ntawm pH li qub. Lub polarity ntawm cov theem nyob ruaj khov tuaj yeem tswj tau los ntawm qhov zoo tshaj plaws ntawm styrene thiab lub sijhawm tshuaj tiv thaiv ntawm cov dawb radical polymerization. Cov kauj ruam kawg ntawm cov tshuaj tiv thaiv (dawb radical polymerization) yog qhov tseem ceeb vim nws hloov lub polarity ntawm cov theem nyob ruaj khov. Elemental tsom xam tau ua los xyuas cov ntsiab lus ntawm cov pa roj carbon nyob rau hauv cov theem no. Nws tau raug pom tias nce tus nqi ntawm styrene thiab cov tshuaj tiv thaiv lub sij hawm nce cov ntsiab lus carbon ntawm cov theem nyob ruaj khov thiab rov ua dua. SPs npaj nrog ntau qhov sib txawv ntawm styrene muaj cov pa roj carbon sib txawv. Ib yam li ntawd, cov theem nyob ruaj khov no tau muab tso rau ntawm cov kab hlau tsis muaj steel thiab lawv cov yam ntxwv chromatographic (selectivity, daws teeb meem, N tus nqi, thiab lwm yam) raug kuaj xyuas. Raws li cov kev sim no, ib qho kev ua kom zoo tshaj plaws rau kev npaj ntawm PMP nyob ruaj khov theem tau raug xaiv los muab kev tswj cov polarity thiab khaws zoo ntawm cov tshuaj ntsuam xyuas.
PMP kem kuj tau soj ntsuam rau kev soj ntsuam ntawm tsib qhov sib xyaw ntawm peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine-enkephalin) siv lub peev xwm ntawm lub sijhawm txawb. 60/40 (v/v) ACN / dej (0.1% TFA) ntawm tus nqi ntws ntawm 80 µl / min. Nyob rau hauv qhov zoo tshaj plaws elution tej yam kev mob (200,000 daim hlau / m), tus naj npawb ntawm theoretical daim hlau (N) ib kem (100 × 1.8 mm) yog 20,000 ± 100. N qhov tseem ceeb rau peb PMP kab yog qhia nyob rau hauv Table 3 thiab cov chromatograms qhia nyob rau hauv daim duab 5A. Kev soj ntsuam ceev ceev ntawm qhov siab txaus (700 µl / min) ntawm PMP kem, tsib peptides eluted nyob rau hauv ib feeb, zoo heev N tus nqi ntawm 13,500 ± 330 ib kem (100 x 1.8 mm inch), sib npaug rau 135,000 daim hlau / m (Fig. 5B). Peb kab ntawm tib qhov loj me (sab hauv txoj kab uas hla 100 x 1.8 mm) tau ntim nrog peb qhov sib txawv ntawm PMP nyob ruaj khov theem los kuaj kev rov tsim dua. Cov tshuaj ntsuam xyuas tau sau tseg rau txhua kem los ntawm kev sib cais qhov sib xyaw ua ke ntawm txhua kab uas siv cov kev xav tau zoo, tus lej ntawm cov phiaj xwm theoretical N, thiab lub sijhawm khaws cia. Cov ntaub ntawv rov tsim dua tshiab rau PMP kab yog qhia nyob rau hauv Table 4. Qhov kev rov ua dua ntawm PMP kem sib raug zoo nrog % RSD tus nqi qis heev raws li qhia hauv Table 3.
Kev sib cais ntawm peptide sib tov ntawm PMP kem (B) thiab Ascentis Express RP-Amide kem (A), mobile theem 60/40 ACN/H2O (TFA 0.1%), PMP kem qhov ntev (100 x 1.8 mm id), tsom xam Elution kev txiav txim ntawm cov ntsiab lus: 1 (Gly-Tyr), 2-Tyr (Gly-Tyr), (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) thiab 5 (leucic acid enkephalin).
PMP kem (sab hauv txoj kab uas hla 100 x 1.8 mm) tau soj ntsuam rau kev sib cais ntawm tryptic hydrolyzate ntawm tib neeg cov ntshav albumin los ntawm HPLC. Cov chromatogram hauv daim duab 6 qhia tau hais tias cov qauv sib cais zoo nrog kev daws teeb meem zoo heev. HSA cov kev daws teeb meem tau txheeb xyuas siv tus nqi ntws ntawm 100 μl / min, theem mobile ntawm 70/30 acetonitrile / dej thiab 0.1% TFA. Cleavage ntawm HSA tau muab faib ua 17 peaks, raws li qhia hauv chromatogram (Fig. 6), sib raug rau 17 peptides. Kev sib cais ua tau zoo ntawm ib tus neeg siab tshaj plaws los ntawm HSA hydrolyzate raug xam thiab cov txiaj ntsig tau qhia hauv Table 5.
HSA tryptic hydrolysates tau sib cais ntawm PMP kem (puab txoj kab uas hla 100 x 1.8 mm), tus nqi ntws (100 μl / min), mobile theem 60/40 acetonitrile / dej, thiab 0.1% TFA.
qhov twg L yog kab ntev, η yog lub viscosity ntawm lub xov tooj ntawm tes, ΔP yog lub nraub qaum ntawm kab, thiab u yog linear tshaj tawm ntawm theem mobile. Qhov permeability ntawm PMP kem yog 2.5 × 10-14 m2, tus nqi ntws yog 25 µl / min, 60/40 v / v tau siv. ACN / dej. Qhov permeability ntawm PMP kem (ID 100 × 1.8 mm) zoo ib yam li peb qhov kev kawm yav dhau los Ref.34. Lub permeability ntawm ib kem uas muaj superficially ntxeem tau hais yog 1.7 × 10 .6 µm, 2.5 × 10-14 m2 rau 5 µm particles43. Yog li ntawd, qhov permeability ntawm PMP theem zoo ib yam li permeability ntawm core-plhaub hais nrog ib tug loj ntawm 5 μm.
qhov twg Wx yog qhov loj ntawm kem uas muaj chloroform, Wy yog qhov loj ntawm kem uas muaj methanol, thiab ρ yog qhov ntom ntawm cov kuab tshuaj. Qhov ntom ntawm methanol (ρ = 0.7866) thiab chloroform (ρ = 1.484). Tag nrho cov porosity ntawm silica-C18 particle kem (100 × 1.8 mm ID)34 thiab peb yav tas los kawm C18-urea31 kem yog 0.63 thiab 0.55, raws li. Qhov no txhais tau hais tias lub xub ntiag ntawm urea ligands txo cov permeability ntawm cov theem nyob ruaj ruaj. Ntawm qhov tod tes, tag nrho cov porosity ntawm PMP kem (puab txoj kab uas hla 100 × 1.8 mm) yog 0.60. PMP kem tsis tshua muaj permeable dua cov kab uas ntim nrog C18 khi silica hais vim hais tias nyob rau hauv C18 hom nyob ruaj khov theem C18 ligands txuas mus rau silica hais nyob rau hauv linear chains, thaum nyob rau hauv polystyrene hom nyob ruaj ruaj theem ib tug kuj tuab polymer yog tsim nyob ib ncig ntawm cov hais. txheej A. Hauv kev sim ib txwm, kab porosity yog xam raws li hauv qab no:
Ntawm daim duab. 7A, B qhia Van Deemter cov phiaj xwm rau PMP kem (id 100 x 1.8 mm) thiab Ascentis Express RP-Amide kem (id 100 x 1.8 mm) nyob rau hauv tib txoj kev elution, 60/40 ACN/H2O thiab 0 .1% TFA 20 µl/min ob kem. Qhov tsawg kawg nkaus HETP qhov tseem ceeb ntawm qhov kev pom zoo (80 µl / min) yog 2.6 µm thiab 3.9 µm rau PMP kem thiab Ascentis Express RP-Amide kem, feem. HETP qhov tseem ceeb qhia tau hais tias kev sib cais ua haujlwm ntawm PMP kem (100 x 1.8 mm id) ntau dua li cov khoom lag luam Ascentis Express RP-Amide kem (100 x 1.8 mm id). Daim duab van Deemter nyob rau hauv daim duab 7(A) qhia tau hais tias qhov txo qis ntawm N tus nqi tsis yog siab dua nrog kev nce ntxiv piv rau peb txoj kev kawm dhau los. Kev sib cais ntau dua ntawm PMP kem (id 100 × 1.8 mm) piv rau Ascentis Express RP-Amide kem yog raws li kev txhim kho particle thiab loj thiab cov txheej txheem sophisticated kem packing siv nyob rau hauv tam sim no ua hauj lwm34.
(A) Van Deemter plot (HETP vs. mobile theem linear velocity) tau ntawm PMP kem (id 100 x 1.8 mm) hauv 60/40 ACN/H2O nrog 0.1% TFA. (B) Van Deemter zaj duab xis (HETP piv rau theem xov tooj ntawm tes tawm nrawm) tau txais ntawm Ascentis Express RP-Amide kem (id 100 x 1.8 mm) hauv 60/40 ACN / H2O nrog 0.1% TFA.
Ib tug polar nyob ruaj ruaj theem ntawm intercalated polystyrene tau npaj thiab soj ntsuam rau kev sib cais ntawm ib tug sib tov ntawm hluavtaws peptides thiab tryptic hydrolyzate ntawm tib neeg serum albumin (HSA) nyob rau hauv siab ua kua chromatography. Lub chromatographic kev ua tau zoo ntawm PMP kab rau peptide sib tov yog zoo heev nyob rau hauv cov nqe lus ntawm kev sib cais efficiency thiab kev daws teeb meem. Kev txhim kho kev sib cais ntawm PMP kab yog vim muaj ntau qhov laj thawj xws li silica particle loj thiab qhov pore loj, tswj kev sib txuas ntawm cov theem nyob ruaj khov, thiab cov khoom ntim cov khoom nyuaj. Ntxiv rau qhov kev sib cais siab, lwm qhov txiaj ntsig ntawm qhov chaw nyob ruaj khov no yog qhov qis qis rov qab siab ntawm cov nqi ntws siab. PMP kab yog cov khoom tsim tau zoo heev thiab tuaj yeem siv los txheeb xyuas cov sib xyaw ntawm peptides thiab tryptic zom ntawm ntau cov proteins. Peb npaj siab yuav siv kab ntawv no rau kev sib cais ntawm bioactive compounds los ntawm cov khoom ntuj tsim, rho tawm ntawm cov nroj tsuag tshuaj thiab nceb hauv kua chromatography. Nyob rau hauv lub neej yav tom ntej, PMP kab tseem yuav raug soj ntsuam rau kev sib cais ntawm cov proteins thiab monoclonal antibodies.
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