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Cov khoom me me silica uas muaj qhov porous tau npaj los ntawm txoj kev sol-gel nrog qee qhov kev hloov kho kom tau txais cov khoom me me macroporous. Cov khoom me me no tau derivatized los ntawm kev hloov pauv rov qab ntxiv fragmentation saw hlau hloov pauv (RAFT) polymerization nrog N-phenylmaleimide-methylvinylisocyanate (PMI) thiab styrene los npaj N-phenylmaleimide intercalation ntawm polystyrene (PMP) theem ruaj khov. Cov kab hlau stainless hlau nqaim (100 × 1.8 hli id) tau ntim los ntawm slurry packing. Kev soj ntsuam PMP kem sib cais ntawm cov peptide sib xyaw uas muaj tsib peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) chromatographic kev ua tau zoo) thiab trypsin zom ntawm tib neeg serum albumin (HAS). Nyob rau hauv qhov zoo tshaj plaws elution cov xwm txheej, cov phaj theoretical suav ntawm cov peptide sib xyaw yog siab txog 280,000 phaj / m². Sib piv qhov kev ua tau zoo sib cais ntawm cov kem tsim nrog Hauv kev lag luam Ascentis Express RP-Amide kem, nws tau pom tias qhov kev ua tau zoo ntawm kev sib cais ntawm PMP kem zoo dua li kem lag luam hauv kev ua haujlwm sib cais thiab kev daws teeb meem.
Nyob rau xyoo tas los no, kev lag luam biopharmaceutical tau dhau los ua kev lag luam thoob ntiaj teb uas nthuav dav nrog kev nce ntxiv ntawm kev lag luam. Nrog rau kev loj hlob sai ntawm kev lag luam biopharmaceutical1,2,3, kev tshuaj xyuas cov peptides thiab cov protein yog qhov xav tau heev. Ntxiv rau lub hom phiaj peptide, ntau yam tsis huv tau tsim thaum lub sijhawm peptide synthesis, yog li xav tau chromatographic purification kom tau txais peptides ntawm qhov purity xav tau. Kev tshuaj xyuas thiab kev piav qhia ntawm cov protein hauv cov kua dej hauv lub cev, cov ntaub so ntswg thiab cov hlwb yog ib txoj haujlwm nyuaj heev vim muaj ntau hom tsiaj uas tuaj yeem pom hauv ib qho qauv. Txawm hais tias mass spectrometry yog ib qho cuab yeej zoo rau peptide thiab protein sequencing, yog tias cov qauv zoo li no raug txhaj rau hauv mass spectrometer hauv ib qho dhau, qhov kev sib cais yuav tsis zoo tagnrho. Qhov teeb meem no tuaj yeem txo qis los ntawm kev siv cov kua chromatography (LC) kev sib cais ua ntej MS kev tshuaj xyuas, uas yuav txo cov lej ntawm cov analytes nkag mus rau hauv mass spectrometer ntawm lub sijhawm muab4,5,6. Tsis tas li ntawd, thaum lub sijhawm kua sib cais, analytes tuaj yeem tsom mus rau thaj chaw nqaim, yog li ua kom cov analytes no thiab txhim kho MS kev kuaj pom. kev rhiab heev. Cov kua chromatography (LC) tau nce qib ntau heev hauv kaum xyoo dhau los thiab tau dhau los ua ib txoj hauv kev nrov hauv kev tshuaj xyuas proteomic7,8,9,10.
Cov kua chromatography rov qab (RP-LC) yog siv dav rau kev ntxuav thiab sib cais cov peptide sib xyaw siv octadecyl-modified silica (ODS) ua theem ruaj khov11,12,13. Txawm li cas los xij, RP theem ruaj khov tsis muab kev sib cais zoo ntawm peptides thiab cov protein vim lawv cov qauv nyuaj thiab amphiphilic xwm 14,15. Yog li ntawd, cov theem ruaj khov tsim tshwj xeeb yog qhov yuav tsum tau los tshuaj xyuas peptides thiab cov protein nrog polar thiab non-polar moieties los cuam tshuam nrog thiab khaws cov analytes16. Mixed-mode chromatography, uas muab kev sib cuam tshuam multimodal, tuaj yeem yog lwm txoj hauv kev rau RP-LC rau kev sib cais ntawm peptides, cov protein, thiab lwm yam sib xyaw ua ke. Ntau cov theem ruaj khov sib xyaw tau npaj, thiab cov kem ntim nrog cov theem no tau siv rau kev sib cais peptide thiab protein17,18,19,20,21. Mixed-mode stationary phases (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) yog qhov tsim nyog rau kev sib cais peptide thiab protein vim yog qhov muaj ob pawg polar thiab tsis yog polar22,23,24,25,26,27,28. Ib yam li ntawd, cov theem ruaj khov polar intercalating nrog cov pawg polar sib txuas qhia tau tias muaj zog sib cais zoo thiab kev xaiv tshwj xeeb rau cov analytes polar thiab tsis yog polar, vim tias kev sib cais nyob ntawm kev sib cuam tshuam ntawm analyte thiab theem ruaj khov. Kev sib cuam tshuam ntau hom 29, 30, 31, 32. Tsis ntev los no, Zhang et al. 30 tau npaj ib theem ruaj khov dodecyl-terminated polyamine thiab sib cais cov hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, thiab ntau lwm yam analytes. Lub polar intercalator muaj ob pawg polar thiab tsis yog polar, yog li nws tuaj yeem siv los cais cov peptides thiab cov protein uas muaj ob qho tib si hydrophobic thiab hydrophilic moieties. Cov kab polar-embedded (piv txwv li, amide-embedded C18 kab) muaj nyob rau hauv lub npe lag luam Ascentis Express RP-Amide kab, tab sis cov kab no tsuas yog siv rau kev tshuaj xyuas ntawm amine 33 xwb.
Hauv kev tshawb fawb tam sim no, ib qho polar-embedded stationary phase (N-phenylmaleimide-embedded polystyrene) tau npaj thiab soj ntsuam rau kev sib cais ntawm peptides thiab trypsin digests ntawm HSA. Lub stationary phase tau npaj siv cov tswv yim hauv qab no. Cov khoom me me porous silica tau npaj raws li cov txheej txheem tau muab rau hauv peb cov ntawv tshaj tawm dhau los nrog qee qhov kev hloov kho rau cov txheej txheem npaj. Qhov piv ntawm urea, polyethylene glycol (PEG), TMOS, dej acetic acid tau hloov kho kom npaj cov khoom me me silica nrog qhov loj me pore. Qhov thib ob, ib qho ligand tshiab, phenylmaleimide-methyl vinyl isocyanate, tau synthesized thiab siv los derivatize cov khoom me me silica los npaj ib qho polar embedded stationary phase. Lub stationary phase tshwm sim tau ntim rau hauv ib lub stainless hlau kem (100 × 1.8 mm id) siv cov txheej txheem ntim khoom zoo tshaj plaws. Lub kem ntim khoom tau pab nrog kev co tshuab kom ntseeg tau tias lub txaj homogeneous tau tsim nyob rau hauv kem. Soj ntsuam qhov sib cais kem ntim ntawm cov khoom sib xyaw peptide uas muaj tsib peptides; (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) thiab Trypsin zom cov albumin hauv tib neeg (HAS). Cov peptide sib xyaw thiab trypsin zom HSA tau pom tias sib cais nrog kev daws teeb meem zoo thiab ua haujlwm tau zoo. Kev ua haujlwm sib cais ntawm PMP kem tau piv rau Ascentis Express RP-Amide kem. Ob qho peptides thiab cov protein tau pom tias daws tau zoo thiab ua haujlwm tau zoo ntawm PMP kem, uas ua haujlwm tau zoo dua li Ascentis Express RP-Amide kem.
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 Qib Acetonitrile (ACN), Methanol, 2-Propanol, thiab Acetone Yuav los ntawm Sigma-Aldrich (St. Louis, MO, USA).
Ib qho sib tov ntawm urea (8 g), polyethylene glycol (8 g), thiab 8 mL ntawm 0.01 N acetic acid tau raug do rau 10 feeb, thiab tom qab ntawd 24 mL ntawm TMOS tau ntxiv rau hauv qab dej khov. Cov tshuaj sib tov tau raug cua sov ntawm 40 ° C rau 6 teev thiab tom qab ntawd ntawm 120 ° C rau 8 teev hauv lub tshuab autoclave stainless hlau. Cov dej tau nchuav tawm thiab cov khoom seem tau qhuav ntawm 70 ° C rau 12 teev. Cov khoom mos qhuav tau raug zom du hauv qhov cub thiab calcined ntawm 550 ° C rau 12 teev. Peb pawg tau npaj thiab piav qhia txog kev tshuaj xyuas qhov ua tau rov ua dua hauv qhov loj me ntawm cov khoom me me, qhov loj me ntawm qhov hws thiab thaj chaw ntawm qhov chaw.
Los ntawm kev hloov kho ntawm cov khoom silica nrog cov ligand phenylmaleimide-methylvinylisocyanate (PCMP) ua ntej ua raws li radial polymerization nrog styrene, ib qho polar pawg-muaj compound tau npaj. Theem ruaj khov rau cov aggregates thiab polystyrene saw. Cov txheej txheem npaj tau piav qhia hauv qab no.
N-phenylmaleimide (200 mg) thiab methyl vinyl isocyanate (100 mg) tau yaj rau hauv toluene qhuav, thiab 0.1 mL ntawm 2,2′-azoisobutyronitrile (AIBN) tau ntxiv rau hauv lub raj tshuaj tiv thaiv los npaj phenylmaleimide-methyl vinyl isocyanate copolymer (PMCP).. Cov khoom sib tov tau raug cua sov ntawm 60 ° C rau 3 teev, lim thiab ziab hauv qhov cub ntawm 40 ° C rau 3 teev.
Cov khoom me me silica qhuav (2 g) tau muab faib rau hauv toluene qhuav (100 mL), do thiab sonicated hauv lub raj mis puag ncig 500 mL rau 10 feeb. PMCP (10 mg) tau yaj rau hauv toluene thiab ntxiv rau hauv lub raj mis tshuaj tiv thaiv los ntawm lub raj mis poob. Cov khoom sib xyaw tau rov qab los ntawm 100 ° C rau 8 teev, lim thiab ntxuav nrog acetone thiab ziab ntawm 60 ° C rau 3 teev. Tom qab ntawd, cov khoom me me silica PMCP-bonded (100 g) tau yaj rau hauv toluene (200 ml) thiab 4-hydroxy-TEMPO (2 mL) tau ntxiv rau hauv qhov muaj 100 µL ntawm dibutyltin dilaurate ua catalyst. Cov khoom sib xyaw tau do ntawm 50 ° C rau 8 teev, lim thiab ziab ntawm 50 ° C rau 3 teev.
Styrene (1 mL), benzoyl peroxide BPO (0.5 mL), thiab TEMPO-PMCP-attached silica particles (1.5 g) tau raug faib rau hauv toluene thiab purged nrog nitrogen. Kev polymerization ntawm styrene tau ua tiav ntawm 100 ° C rau 12 teev. Cov khoom tiav lawm tau ntxuav nrog methanol thiab ziab ntawm 60 ° C ib hmos. Cov txheej txheem tshuaj tiv thaiv tag nrho tau qhia nyob rau hauv Daim Duab 1.
Cov qauv kuaj tau degassed ntawm 393 K rau 1 teev kom tau txais qhov siab seem tsawg dua 10-3 Torr. Tus nqi ntawm N2 adsorbed ntawm qhov siab sib piv ntawm P / P0 = 0.99 tau siv los txiav txim siab tag nrho cov qhov ntim. Cov qauv ntawm cov khoom me me thiab ligand-bonded silica tau kuaj xyuas nrog scanning electron microscopy (Hitachi High Technologies, Tokyo, Nyiv). Cov qauv qhuav (cov khoom me me thiab cov khoom me me silica ligand-bonded) tau muab tso rau ntawm ib lub kem txhuas siv cov ntaub nplaum carbon. Kub tau plated rau ntawm cov qauv siv Q150T sputter coater, thiab 5 nm Au txheej tau tso rau ntawm cov qauv. Qhov no txhim kho cov txheej txheem ua haujlwm siv cov hluav taws xob qis thiab muab cov noob zoo, txias sputtering. Ib lub Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental analyzer tau siv rau kev tshuaj xyuas elemental. Ib lub Malvern (Worcestershire, UK) Mastersizer 2000 particle size analyzer tau siv los txais cov khoom me me faib tawm. Silica liab qab cov khoom me me thiab cov khoom me me silica ligand-bonded (5 mg txhua) tau tawg ua 5 mL ntawm isopropanol, sonicated rau 10 feeb, vortexed rau 5 feeb, thiab muab tso rau ntawm lub rooj zaum optical ntawm Mastersizer. Kev tshuaj xyuas Thermogravimetric tau ua tiav ntawm tus nqi ntawm 5 ° C ib feeb dhau ntawm qhov kub thiab txias ntawm 30 txog 800 ° C.
Cov kem nqaim uas muaj iav txheej stainless hlau ntawm qhov loj (100 × 1.8 hli id) tau ntim siv txoj kev ntim slurry, siv cov txheej txheem tib yam siv hauv Ref. 31. Ib lub kem hlau tsis xeb (ua los ntawm iav, 100 × 1.8 hli id) nrog rau qhov chaw tawm uas muaj 1 µm frit tau txuas nrog lub tshuab ntim slurry (Alltech Deerfield, IL, USA). Npaj ib lub slurry theem ruaj khov los ntawm kev muab 150 mg ntawm theem ruaj khov rau hauv 1.2 mL ntawm methanol thiab xa mus rau kem cia. Methanol tau siv ua cov tshuaj slurry thiab cov tshuaj propelling. Sau lub kem ib ntus los ntawm kev siv lub zog ntawm 100 MP rau 10 feeb, 80 MP rau 15 feeb, thiab 60 MP rau 30 feeb. Thaum lub sijhawm ntim khoom, kev co tshuab tau siv nrog ob lub GC column shakers (Alltech, Deerfield, IL, USA) kom ntseeg tau tias lub kem ntim sib xws. Kaw lub slurry packer thiab tso lub zog maj mam kom tsis txhob muaj kev puas tsuaj hauv kem. Tshem lub kem ntawm lub tshuab ntim slurry thiab txuas lwm lub fitting rau qhov nkag thiab rau LC system los xyuas nws qhov kev ua haujlwm.
Ib lub twj tso kua mis LC (10AD Shimadzu, Nyiv Pooj), lub tshuab txhaj tshuaj (Valco (Tebchaws USA) C14 W.05) nrog 50nL lub voj txhaj tshuaj, lub tshuab degasser membrane (Shimadzu DGU-14A), lub qhov rais capillary UV-VIS tau tsim. Lub cuab yeej ntes µLC tshwj xeeb (UV-2075) thiab cov kab me me uas muaj iav. Siv cov raj txuas nqaim thiab luv luv kom txo qhov cuam tshuam ntawm kev nthuav dav ntawm cov kab kem ntxiv. Tom qab ntim khoom, cov hlab ntsha capillaries (50 μm id 365 thiab cov hlab ntsha txo qis (50 μm) tau teeb tsa ntawm 1/16″ qhov hluav taws xob ntawm lub tshuab txo qis. Kev sau cov ntaub ntawv thiab kev ua cov txheej txheem chromatographic tau ua tiav siv Multichro 2000 software. Kev soj ntsuam ntawm 254 nm Analytes tau sim rau UV absorbance. Cov ntaub ntawv Chromatographic tau soj ntsuam los ntawm OriginPro8 (Northampton, MA).
Albumin los ntawm tib neeg cov ntshav, lyophilized hmoov, ≥ 96% (agarose gel electrophoresis) 3 mg sib xyaw nrog trypsin (1.5 mg), 4.0 M urea (1 mL), thiab 0.2 M ammonium bicarbonate (1 mL). Cov tshuaj tau do rau 10 feeb thiab khaws cia rau hauv lub dab dej dej ntawm 37°C rau 6 teev, tom qab ntawd quenched nrog 1 mL ntawm 0.1% TFA. Lim cov tshuaj thiab khaws cia hauv qab 4 °C.
Kev sib cais ntawm cov peptide sib xyaw thiab HSA trypsin digests tau raug soj ntsuam cais ntawm PMP kem. Tshawb xyuas qhov kev sib cais ntawm cov peptide sib xyaw thiab trypsin digest ntawm HSA los ntawm PMP kem thiab piv cov txiaj ntsig rau Ascentis Express RP-Amide kem. Tus lej theoretical phaj yog xam raws li nram no:
Cov duab SEM ntawm cov khoom me me silica liab qab thiab cov khoom me me silica ligand-bonded tau pom hauv daim duab 2. Cov duab SEM ntawm cov khoom me me silica liab qab (A, B) qhia tias, sib piv rau peb cov kev tshawb fawb yav dhau los, cov khoom me me no yog spherical uas cov khoom me me ntev lossis muaj qhov sib npaug tsis sib xws. Qhov saum npoo ntawm cov khoom me me silica ligand-bonded (C, D) du dua li cov khoom me me silica liab qab, uas tej zaum yuav yog vim muaj txheej polystyrene saw ntawm qhov saum npoo ntawm cov khoom me me silica.
Cov duab thaij los ntawm lub tshuab hluav taws xob scanning ntawm cov khoom me me ntawm silica liab qab (A, B) thiab cov khoom me me ntawm ligand-bonded silica (C, D).
Cov kev faib tawm ntawm cov khoom me me ntawm cov khoom me me silica liab qab thiab cov khoom me me silica ligand-bonded tau qhia nyob rau hauv Daim Duab 3 (A). Cov kab sib faib tawm ntawm cov khoom me me raws li qhov ntim tau qhia tias qhov loj ntawm cov khoom me me silica tau nce tom qab kev hloov kho tshuaj (Daim Duab 3A). Cov ntaub ntawv faib tawm ntawm cov khoom me me ntawm cov khoom me me silica los ntawm kev tshawb fawb tam sim no thiab kev tshawb fawb yav dhau los tau muab piv rau hauv Rooj 1 (A). Qhov loj ntawm cov khoom me me raws li qhov ntim, d (0.5), ntawm PMP yog 3.36 μm, piv rau peb txoj kev tshawb fawb yav dhau los nrog tus nqi ad (0.5) ntawm 3.05 μm (polystyrene-bound silica particles)34. Cov pawg no muaj qhov faib tawm ntawm cov khoom me me nqaim dua piv rau peb txoj kev tshawb fawb yav dhau los vim muaj qhov sib txawv ntawm PEG, urea, TMOS, thiab acetic acid hauv cov tshuaj sib xyaw. Qhov loj ntawm cov khoom me me ntawm PMP theem yog me ntsis loj dua li ntawm cov khoom me me polystyrene-bound silica particle theem uas peb tau kawm yav dhau los. Qhov no txhais tau tias kev ua haujlwm ntawm cov khoom me me silica nrog styrene tsuas yog tso ib txheej polystyrene (0.97 µm) rau ntawm qhov chaw silica, thaum nyob rau hauv theem PMP qhov tuab ntawm txheej yog 1.38 µm.
Kev faib tawm ntawm qhov loj ntawm cov khoom me me (A) thiab qhov loj ntawm cov qhov (B) ntawm cov khoom me me uas muaj silica liab qab thiab cov khoom me me uas muaj ligand-bound silica.
Qhov loj ntawm cov pore, qhov ntim ntawm cov pore thiab thaj chaw ntawm cov silica particles ntawm kev tshawb fawb tam sim no tau muab rau hauv Rooj 1 (B). Cov PSD profiles ntawm cov particles silica liab qab thiab cov particles silica ligand-bonded tau qhia hauv Daim Duab 3 (B). Cov txiaj ntsig zoo sib xws rau peb txoj kev tshawb fawb yav dhau los. Qhov loj ntawm cov pore ntawm cov particles silica liab qab thiab ligand-bound yog 310 thiab 241, feem, uas qhia tias qhov loj ntawm cov pore txo qis los ntawm 69 tom qab kev hloov kho tshuaj, raws li tau pom hauv Rooj 1 (B), thiab qhov kev hloov pauv ntawm cov nkhaus tau qhia hauv Daim Duab 3 (B). Ib yam li ntawd, qhov ntim ntawm cov pore ntawm cov particles silica tau txo qis los ntawm 0.67 mus rau 0.58 cm3 / g tom qab kev hloov kho tshuaj. Thaj chaw tshwj xeeb ntawm cov particles silica uas tau kawm tam sim no yog 116 m2 / g, uas yog sib piv rau peb txoj kev tshawb fawb yav dhau los (124 m2 / g). Raws li tau pom hauv Rooj 1 (B), thaj chaw ntawm cov particles silica kuj tau txo qis los ntawm 116 m2 / g mus rau 105 m2/g tom qab kev hloov kho tshuaj lom neeg.
Cov txiaj ntsig ntawm kev tshuaj xyuas cov ntsiab lus ntawm theem ruaj khov tau qhia hauv Rooj 2. Qhov kev thauj khoom carbon ntawm theem ruaj khov tam sim no yog 6.35%, uas qis dua qhov kev thauj khoom carbon ntawm peb txoj kev tshawb fawb yav dhau los (polystyrene bonded silica particles, 7.93%35 thiab 10.21%, feem) 42. Qhov kev thauj khoom carbon ntawm theem ruaj khov tam sim no yog qis, Vim tias nyob rau hauv kev npaj ntawm SP tam sim no, ntxiv rau styrene, qee cov polar ligands xws li phenylmaleimide-methylvinylisocyanate (PCMP) thiab 4-hydroxy-TEMPO tau siv. Qhov hnyav nitrogen feem pua ​​ntawm theem ruaj khov tam sim no yog 2.21%, piv rau 0.1735 thiab 0.85% los ntawm qhov hnyav ntawm nitrogen hauv kev tshawb fawb yav dhau los, feem. Qhov no txhais tau tias wt% ntawm nitrogen yog siab dua hauv theem ruaj khov tam sim no vim yog phenylmaleimide. Ib yam li ntawd, cov khoom thauj carbon ntawm cov khoom (4) thiab (5) yog 2.7% thiab 2.9%, feem, thaum cov khoom thauj carbon ntawm cov khoom kawg (6) yog 6.35%, raws li tau pom hauv Rooj 2. Qhov hnyav poob tau kuaj xyuas nrog PMP theem ruaj khov, thiab cov kab TGA tau pom hauv Daim Duab 4. Cov kab TGA qhia txog qhov hnyav poob ntawm 8.6%, uas yog qhov sib haum xeeb zoo nrog cov pa roj carbon (6.35%) vim tias cov ligands tsis yog tsuas yog C tab sis kuj muaj N, O, thiab H.
Cov phenylmaleimide-methylvinylisocyanate ligand tau raug xaiv rau kev hloov kho ntawm cov khoom silica vim nws muaj cov pawg polar phenylmaleimide thiab cov pawg vinylisocyanate. Cov pawg vinyl isocyanate tuaj yeem ua rau muaj kev cuam tshuam nrog styrene los ntawm kev ua kom muaj zog radical polymerization. Qhov laj thawj thib ob yog ntxig ib pawg uas muaj kev sib cuam tshuam nruab nrab nrog analyte thiab tsis muaj kev sib cuam tshuam electrostatic muaj zog ntawm analyte thiab theem ruaj khov, txij li thaum phenylmaleimide moiety tsis muaj virtual charge ntawm pH ib txwm muaj. Lub polarity ntawm theem ruaj khov tuaj yeem tswj tau los ntawm qhov ntau ntawm styrene thiab lub sijhawm teb ntawm free radical polymerization. Kauj ruam kawg ntawm kev teb (free-radical polymerization) yog qhov tseem ceeb thiab tuaj yeem hloov pauv polarity ntawm theem ruaj khov. Kev tshuaj xyuas cov khoom siv tau ua los xyuas cov pa roj carbon ntawm cov theem ruaj khov no. Nws tau pom tias kev nce ntxiv ntawm styrene thiab lub sijhawm teb ua rau cov pa roj carbon ntawm theem ruaj khov thiab rov ua dua. SPs npaj nrog cov concentration sib txawv ntawm styrene muaj cov pa roj carbon sib txawv. Dua li, thauj cov theem ruaj khov no rau hauv cov hlau tsis xeb thiab xyuas lawv cov kev ua tau zoo chromatographic. (kev xaiv, kev daws teeb meem, N tus nqi, thiab lwm yam). Raws li cov kev sim no, ib qho kev tsim kho zoo tshaj plaws tau raug xaiv los npaj PMP theem ruaj khov kom ntseeg tau tias muaj kev tswj hwm polarity thiab kev khaws cia analyte zoo.
Tsib qhov sib xyaw peptide (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, leucine enkephalin) kuj tau soj ntsuam siv PMP kem siv theem txawb; 60/40 (v/v) acetonitrile/dej (0.1% TFA) ntawm tus nqi ntws ntawm 80 μL/min. Nyob rau hauv qhov zoo tshaj plaws elution mob, tus lej theoretical phaj (N) ib kem (100 × 1.8 hli id) yog 20,000 ± 100 (200,000 phaj/m²). Rooj 3 muab cov nqi N rau peb kab PMP thiab cov chromatograms tau qhia nyob rau hauv Daim Duab 5A. Kev tshuaj xyuas sai ntawm kab PMP ntawm tus nqi ntws siab (700 μL/min), tsib peptides tau eluted hauv ib feeb, N tus nqi zoo heev, 13,500 ± 330 ib kem (100 × 1.8 hli id), Sib raug rau 135,000 phaj/m (Daim Duab 5B). Peb kab sib npaug (100 × 1.8 hli id) tau ntim nrog peb ntau yam ntawm PMP theem ruaj khov los xyuas qhov rov ua dua. Lub analyte Kev ntsuas qhov concentration rau txhua kem tau raug kaw cia siv cov xwm txheej zoo tshaj plaws ntawm kev elution thiab tus lej ntawm cov phaj theoretical N thiab lub sijhawm khaws cia kom cais cov khoom sib xyaw ua ke ntawm txhua kem. Cov ntaub ntawv rov ua dua rau cov kem PMP tau qhia hauv Rooj 4. Kev rov ua dua ntawm cov kem PMP sib raug zoo nrog cov nqi %RSD qis heev, raws li tau qhia hauv Rooj 3.
Kev sib cais ntawm cov peptide sib xyaw rau ntawm PMP kem (B) thiab Ascentis Express RP-Amide kem (A); theem txawb 60/40 ACN/H2O (TFA 0.1%), qhov ntev ntawm PMP kem (100 × 1.8 hli id); analytical Qhov kev txiav txim ntawm cov tshuaj sib xyaw: 1 (Gly-Tyr), 2 (Gly-Leu-Tyr), 3 (Gly-Gly-Tyr-Arg), 4 (Tyr-Ile-Gly-Ser-Arg) thiab 5 (leucine) acid enkephalin)).
Ib kab PMP (100 × 1.8 hli id) tau raug soj ntsuam rau kev sib cais ntawm cov tryptic digests ntawm tib neeg serum albumin hauv high performance liquid chromatography. Lub chromatogram hauv Daim Duab 6 qhia tau hais tias cov qauv tau sib cais zoo thiab qhov kev daws teeb meem zoo heev. HSA digests tau soj ntsuam siv tus nqi ntws ntawm 100 µL / min, mobile theem 70/30 acetonitrile / dej thiab 0.1% TFA. Raws li tau pom hauv chromatogram (Daim Duab 6), HSA digestion tau raug faib ua 17 peaks sib raug rau 17 peptides. Qhov ua tau zoo ntawm kev sib cais ntawm txhua lub peak hauv HSA digest tau suav thiab cov nqi tau muab rau hauv Rooj 5.
Ib qho tryptic digest ntawm HSA (100 × 1.8 mm id) tau sib cais rau ntawm PMP kem; tus nqi ntws (100 µL/min), theem txawb 60/40 acetonitrile/dej nrog 0.1% TFA.
qhov twg L yog qhov ntev ntawm kem, η yog qhov viscosity ntawm theem mobile, ΔP yog lub zog rov qab ntawm kem, thiab u yog qhov ceev ntawm theem mobile. Qhov permeability ntawm kem PMP yog 2.5 × 10-14 m2, qhov ntws yog 25 μL / min, thiab 60/40 v / v ACN / dej tau siv. Qhov permeability ntawm kem PMP (100 × 1.8 hli id) zoo ib yam li peb txoj kev tshawb fawb yav dhau los Ref.34. Qhov permeability ntawm kem ntim nrog cov khoom me me superficially porous yog: 1.7 × 10-15 rau 1.3 μm khoom me me, 3.1 × 10-15 rau 1.7 μm khoom me me, 5.2 × 10-15 thiab 2.5 × 10-14 m2 rau 2.6 μm khoom me me Rau 5 μm khoom me me 43. Yog li ntawd, qhov permeability ntawm theem PMP zoo ib yam li ntawm 5 μm core-shell khoom me me.
qhov twg Wx yog qhov hnyav ntawm kem uas ntim nrog chloroform, Wy yog qhov hnyav ntawm kem uas ntim nrog methanol, thiab ρ yog qhov ceev ntawm cov kuab tshuaj. Qhov ceev ntawm methanol (ρ = 0.7866) thiab chloroform (ρ = 1.484). Tag nrho cov porosity ntawm SILICA PARTICLES-C18 kem (100 × 1.8 mm id) 34 thiab C18-Urea kem 31 uas peb tau kawm ua ntej yog 0.63 thiab 0.55, raws li. Qhov no txhais tau tias qhov muaj urea ligands txo qhov permeability ntawm theem ruaj khov. Ntawm qhov tod tes, tag nrho cov porosity ntawm PMP kem (100 × 1.8 mm id) yog 0.60. Qhov permeability ntawm PMP kem qis dua li ntawm cov kem uas ntim nrog C18-bonded silica particles vim tias hauv C18-hom stationary phases C18 ligands txuas rau cov silica particles ua linear chains, thaum nyob rau hauv polystyrene-hom stationary phases, A txheej polymer tuab dua. yog tsim nyob ib puag ncig nws. Hauv kev sim ib txwm muaj, qhov porosity ntawm kem yog xam raws li:
Daim Duab 7A, B qhia PMP kem (100 × 1.8 hli id) thiab Ascentis Express RP-Amide kem (100 × 1.8 hli id) siv tib yam elution conditions (piv txwv li, 60/40 ACN/H2O thiab 0.1% TFA). ) ntawm van Deemter plot. Cov peptide sib xyaw xaiv (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine Enkephalin) tau npaj rau hauv 20 µL/ Tus nqi ntws tsawg kawg nkaus rau ob kab yog 800 µL/min. Tus nqi HETP tsawg kawg nkaus ntawm tus nqi ntws zoo tshaj plaws (80 µL/min) rau kab PMP thiab kab Ascentis Express RP-Amide yog 2.6 µm thiab 3.9 µm, raws li. Cov nqi HETP qhia tias qhov kev sib cais ntawm kab PMP (100 × 1.8 hli id) zoo dua li kab Ascentis Express RP-Amide (100 × 1.8 hli id) muaj nyob hauv khw. Daim duab van Deemter hauv daim duab 7(A) qhia tias qhov txo qis hauv tus nqi N nrog kev ntws nce ntxiv tsis tseem ceeb piv rau peb txoj kev tshawb fawb yav dhau los. Qhov kev sib cais siab dua ntawm kab PMP (100 × 1.8 hli id) piv rau Ascentis Express RP-Amide kem yog raws li kev txhim kho hauv cov duab ntawm cov khoom me me, qhov loj me, thiab cov txheej txheem ntim kem nyuaj uas siv hauv kev ua haujlwm tam sim no34.
(A) daim duab kos van Deemter (HETP piv rau qhov ceev ntawm theem txav tau) tau los ntawm kev siv kab PMP (100 × 1.8 hli id) hauv 60/40 ACN/H2O nrog 0.1% TFA.(B) daim duab kos van Deemter (HETP piv rau qhov ceev ntawm theem txav tau) tau los ntawm kev siv kab Ascentis Express RP-Amide (100 × 1.8 hli id) hauv 60/40 ACN/H2O nrog 0.1% TFA.
Ib qho polar-embedded polystyrene stationary phase tau npaj thiab soj ntsuam rau kev sib cais ntawm cov khoom sib xyaw peptide thiab trypsin digests ntawm tib neeg serum albumin (HAS) hauv high performance liquid chromatography. Kev ua haujlwm chromatographic ntawm PMP kem rau peptide sib xyaw yog qhov zoo heev hauv kev sib cais efficiency thiab kev daws teeb meem. Kev ua haujlwm sib cais zoo dua ntawm PMP kem yog vim ntau yam, xws li qhov loj me ntawm cov khoom me me thiab qhov loj ntawm cov khoom silica, kev tswj hwm kev tsim ntawm theem ruaj khov, thiab kev ntim kem nyuaj. Ntxiv nrog rau kev sib cais zoo, qhov siab qis ntawm kem rov qab ntawm tus nqi ntws siab yog lwm qhov zoo ntawm theem ruaj khov no. PMP kem qhia txog kev rov ua dua zoo thiab tuaj yeem siv rau kev tshuaj xyuas cov khoom sib xyaw peptide thiab trypsin digestion ntawm ntau yam protein. Peb npaj siab siv kem no rau kev sib cais cov khoom ntuj, cov tshuaj bioactive los ntawm cov nroj tsuag tshuaj thiab cov tshuaj fungal extracts hauv kua chromatography. Yav tom ntej, PMP kem kuj tseem yuav raug soj ntsuam rau kev sib cais ntawm cov protein thiab monoclonal antibodies.
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