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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 uas muaj qhov dav. Cov khoom me me no tau derivatized nrog N-phenylmaleimide-methylvinyl isocyanate (PMI) thiab styrene ntawm kev hloov pauv ntawm cov saw hlau rov qab (RAFT) polymerization los tsim N-phenylmaleimide intercalated polyamides. Styrene (PMP) theem ruaj khov. Cov kem hlau stainless hlau nqaim (100 × 1.8 hli sab hauv txoj kab uas hla) tau ntim nrog cov slurry packing. Kev ua tau zoo chromatographic ntawm PMP kem tau soj ntsuam kom cais cov khoom sib xyaw ntawm cov peptides tsim los ntawm tsib peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leu amino acid enkephalin) thiab tryptic hydrolyzate ntawm tib neeg cov ntshav albumin (HAS). Nyob rau hauv qhov zoo tshaj plaws elution mob, tus lej theoretical ntawm cov phaj nrog cov sib xyaw ntawm peptides tau txog 280,000 phaj / sq.m. Piv rau qhov kev ua tau zoo sib cais ntawm cov kem tsim nrog cov lag luam Ascentis Express RP-Amide kem, nws tau pom tias qhov kev ua tau zoo sib cais ntawm PMP kem zoo dua li cov lag luam kem hauv cov nqe lus ntawm kev ua tau zoo sib cais thiab kev daws teeb meem.
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 hauv xyoo tas los no. Nrog rau kev loj hlob sai ntawm kev lag luam biopharmaceutical1,2,3 muaj kev xav tau ntau rau peptide thiab protein tsom xam. Ntxiv rau lub hom phiaj peptide, ntau yam tsis huv tau tsim thaum lub sijhawm peptide synthesis, yog li chromatographic purification yog qhov yuav tsum tau ua kom tau txais qhov huv ntawm peptide. Kev tsom xam 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 muaj nyob rau hauv ib qho qauv. Txawm hais tias mass spectrometry yog ib qho cuab yeej zoo rau kev txheeb xyuas cov peptides thiab cov protein, yog tias cov qauv zoo li no tau qhia ncaj qha rau hauv mass spectrometer, kev sib cais yuav tsis txaus siab. Qhov teeb meem no tuaj yeem daws tau los ntawm kev ua kua chromatography (LC) ua ntej MS tsom xam, uas yuav txo qhov ntau ntawm cov analytes nkag mus rau hauv mass spectrometer ntawm lub sijhawm muab4,5,6. Tsis tas li ntawd, analytes tuaj yeem tsom mus rau hauv thaj chaw nqaim thaum lub sijhawm sib cais kua, yog li ua kom cov analytes no tsom mus rau thiab ua rau muaj kev nkag siab ntawm MS. Kev siv kua chromatography (LC) tau nce qib ntau heev nyob rau kaum xyoo dhau los thiab tau dhau los ua txoj hauv kev siv dav rau kev tshuaj xyuas proteomic7,8,9,10.
Cov kua chromatography rov qab-theem (RP-LC) yog siv dav los ntxuav thiab cais cov khoom sib xyaw ntawm peptides siv octadecyl-modified silica (ODS) ua theem ruaj khov11,12,13. Txawm li cas los xij, vim lawv cov qauv nyuaj thiab amphoteric xwm,14,15 RP theem ruaj khov tsis tuaj yeem muab kev sib cais zoo ntawm peptides thiab cov protein. Yog li ntawd, kev tshuaj xyuas ntawm peptides thiab cov protein nrog cov khoom polar thiab tsis-polar xav tau cov theem ruaj khov tsim tshwj xeeb los sib cuam tshuam thiab khaws cov analytes16. Mixed chromatography, uas muaj kev sib cuam tshuam ntau hom, tuaj yeem yog lwm txoj hauv kev rau RP-LC rau kev sib cais peptides, cov protein, thiab lwm yam sib xyaw ua ke. Ntau hom theem ruaj khov sib xyaw tau npaj thiab cov kem puv nrog cov theem ruaj khov no tau siv los sib cais peptides thiab cov protein17,18,19,20,21. Vim muaj cov pab pawg polar thiab tsis-polar, cov theem sib xyaw ua ke (WAX/RPLC, HILIC/RPLC, polar intercalation/RPLC) yog qhov tsim nyog rau kev sib cais ntawm peptides thiab cov protein22,23,24,25,26,27,28., polar intercalated stationary phases nrog cov pab pawg polar sib txuas ua ke qhia txog kev sib cais zoo thiab kev xaiv tshwj xeeb rau polar thiab non-polar analytes vim tias kev sib cais nyob ntawm kev sib cuam tshuam ntawm analyte thiab theem stationary Multimodal kev sib cuam tshuam 29,30,31,32. Tsis ntev los no, Zhang et al. 30 tau txais behenyl-terminated stationary phases ntawm polyamines thiab ua tiav kev sib cais hydrocarbons, antidepressants, flavonoids, nucleosides, estrogens, thiab qee lwm yam analytes. Cov khoom siv polar embedded stationary muaj ob qho tib si polar thiab non-polar pawg, yog li nws tuaj yeem siv los cais peptides thiab cov protein ua hydrophobic thiab hydrophilic qhov chaw. Cov kab polar inline (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 tshuaj xyuas ntawm amine 33.
Hauv kev tshawb fawb tam sim no, ib qho polar embedding stationary phase (N-phenylmaleimide, embedding polystyrene) tau npaj thiab soj ntsuam rau kev sib cais peptide thiab tryptic HSA cleavage. Cov tswv yim hauv qab no tau siv los npaj cov theem stationary. Cov khoom me me porous silica tau npaj raws li cov txheej txheem piav qhia hauv peb cov ntawv tshaj tawm yav dhau los, nrog qee qhov kev hloov pauv hauv cov phiaj xwm npaj 31, 34, 35, 36, 37, 38, 39. Cov piv ntawm urea, polyethylene glycol (PEG), TMOS thiab aqueous-acetic acid tau kho kom tau txais cov khoom me me silica nrog cov qhov loj me. Qhov thib ob, ib qho tshiab phenylmaleimide-methylvinyl isocyanate ligand tau synthesized thiab nws cov khoom me me silica tau siv los npaj cov theem polar embedded stationary. Cov theem stationary tau txais tau ntim rau hauv ib lub stainless hlau kem (sab hauv txoj kab uas hla 100 × 1.8 hli) raws li cov phiaj xwm ntim khoom zoo tshaj plaws. Kev ntim ntawm kem yog pab los ntawm kev co tshuab kom ntseeg tau tias muaj ib txheej sib xws hauv kem. Lub kem ntim tau raug soj ntsuam rau kev sib cais ntawm cov peptides sib xyaw 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 tib neeg serum albumin (HSA). Nws tau pom tias cov peptide sib xyaw thiab HSA tryptic digest tau sib cais nrog kev daws teeb meem zoo thiab kev ua haujlwm zoo. Kev ua haujlwm sib cais ntawm PMP kem tau piv rau Ascentis Express RP-Amide kem. Nws tau pom tias peptides thiab cov protein muaj kev daws teeb meem zoo thiab kev ua haujlwm sib cais siab ntawm PMP kem, thiab kev ua haujlwm sib cais ntawm PMP kem siab dua li ntawm 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 chloride (MC), styrene, 4-hydroxy- TEMPO, benzoyl peroxide (BPO), acetonitrile (ACN) rau HPLC, methanol, 2-propanol thiab acetone. Sigma-Aldrich Company (St. Louis, Missouri, USA).
Ib qho sib tov ntawm urea (8 g), polyethylene glycol (8 g) thiab 8 ml ntawm 0.01 N. acetic acid tau do rau 10 feeb, thiab 24 ml ntawm TMOS tau ntxiv rau hauv qab dej khov txias. 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 decanted thiab cov seem tau qhuav ntawm 70 ° C rau 12 teev. Cov blocks qhuav tau zom du thiab calcined hauv qhov cub ntawm 550 ° C rau 12 teev. Peb pawg tau npaj thiab ua cim los sim qhov rov ua dua ntawm cov khoom me me, qhov loj me thiab thaj chaw nto.
Pawg polar thiab theem ruaj khov rau cov saw polystyrene. Cov txheej txheem npaj tau piav qhia hauv qab no.
N-phenylmaleimide (200 mg) thiab methyl vinyl isocyanate (100 mg) tau yaj rau hauv anhydrous toluene, thiab tom qab ntawd 0.1 ml ntawm 2,2′-azoisobutyronitrile (AIBN) tau ntxiv rau hauv lub raj tshuaj kom tau txais cov copolymer ntawm phenylmaleimide thiab methyl vinyl isocyanate (PMCP). ) Cov 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 rau 10 feeb hauv lub raj mis 500 ml puag ncig hauv qab. PMCP (10 mg) tau yaj rau hauv toluene thiab ntxiv rau hauv lub raj mis tshuaj tiv thaiv los ntawm lub raj mis ntxiv. Cov khoom sib tov tau rov qab los ntawm 100 ° C rau 8 teev, lim, ntxuav nrog acetone thiab ziab ntawm 60 ° C rau 3 teev. Tom qab ntawd, cov khoom me me silica uas cuam tshuam nrog PMCP (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 tus catalyst. Cov khoom sib tov 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 cov khoom me me silica uas txuas nrog TEMPO-PMCP (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 ib hmos ntawm 60 ° C. Cov qauv dav dav ntawm cov tshuaj tiv thaiv tau qhia hauv daim duab ib.
Cov qauv kuaj tau degassed ntawm 393 K rau 1 teev kom txog thaum muaj qhov siab seem tsawg dua 10-3 Torr tau txais. Tus nqi ntawm N2 adsorbed ntawm qhov siab sib piv P / P0 = 0.99 tau siv los txiav txim siab tag nrho cov qhov ntim. Cov qauv ntawm cov khoom silica ntshiab thiab ligand-bound tau kuaj xyuas siv lub tshuab ntsuas hluav taws xob scanning (Hitachi High Technologies, Tokyo, Nyiv). Cov qauv qhuav (silica ntshiab thiab ligand bound silica particles) tau muab tso rau ntawm cov pas nrig txhuas siv cov kab xev carbon. Kub tau tso rau ntawm cov qauv siv lub cuab yeej Q150T sputtering, thiab 5 nm tuab Au txheej tau tso rau ntawm cov qauv. Qhov no txhim kho kev ua haujlwm ntawm cov txheej txheem qis voltage thiab muab cov tshuaj tsuag txias zoo. Kev tshuaj xyuas cov khoom siv tau ua tiav siv Thermo Electron (Waltham, MA, USA) Flash EA1112 elemental composition analyzer. Lub Malvern particle size analyzer (Worcestershire, UK) Mastersizer 2000 tau siv los txais cov khoom faib tawm ntawm cov khoom siv. Cov khoom me me silica uas tsis tau coated thiab cov khoom me me ligand-bound silica (5 mg txhua) tau dispersed hauv 5 ml ntawm isopropanol, sonicated rau 10 feeb, agitated rau 5 feeb, thiab muab tso rau ntawm Mastersizer optical bench. Kev tshuaj xyuas thermogravimetric yog nqa tawm ntawm tus nqi ntawm 5 ° C ib feeb hauv qhov kub thiab txias ntawm 30 txog 800 ° C.
Cov kem hlau stainless hlau uas muaj iav fiber ntau thiab nqaim nrog qhov loj (ID 100 × 1.8 hli) tau ntim los ntawm txoj kev sau slurry ua raws li cov txheej txheem zoo ib yam li hauv kev siv 31. Cov kem hlau stainless hlau (iav fiber, ID 100 × 1 .8 hli) thiab qhov hluav taws xob uas muaj 1 µm frit tau txuas nrog lub tshuab ntim slurry (Alltech Deerfield, IL, USA). Npaj ib qho kev ncua ntawm theem ruaj khov los ntawm kev ncua 150 mg ntawm theem ruaj khov hauv 1.2 ml ntawm methanol thiab pub nws rau hauv kem reservoir. Methanol tau siv ua cov tshuaj slurry thiab cov tshuaj tswj. Ntim kem los ntawm kev siv cov txheej txheem siab ntawm 100 MP rau 10 feeb, 80 MP rau 15 feeb, thiab 60 MP rau 30 feeb. Cov txheej txheem ntim khoom siv ob lub tshuab vibrators roj chromatography (Alltech, Deerfield, IL, USA) rau kev co tshuab kom ntseeg tau tias cov kem ntim sib xws. Kaw lub slurry packer thiab tso siab qeeb qeeb kom tiv thaiv kev puas tsuaj rau txoj hlua. Lub kem tau raug txiav tawm ntawm lub tshuab nqus dej thiab lwm qhov khoom siv tau txuas rau qhov nkag thiab txuas nrog lub kaw lus LC los sim nws txoj haujlwm.
Ib qho MLC kev cai tau tsim los ntawm kev siv lub twj tso kua mis LC (10AD Shimadzu, Nyiv), ib lub sampler nrog 50 nL txhaj tshuaj voj (Valco (Tebchaws USA) C14 W.05), lub membrane degasser (Shimadzu DGU-14A), thiab lub qhov rais capillary UV-VIS. Lub cuab yeej ntes (UV-2075) thiab enamelled microcolumn. Siv cov raj txuas nqaim thiab luv luv kom txo qhov cuam tshuam ntawm kev nthuav dav ntxiv. Tom qab sau lub kem, teeb tsa capillary (50 µm id 365) ntawm qhov tawm ntawm 1/16″ txo qhov sib koom ua ke thiab teeb tsa capillary (50 µm) ntawm qhov sib koom ua ke. Kev sau cov ntaub ntawv thiab kev ua cov chromatogram tau ua tiav siv Multichro 2000 software. Ntawm 254 nm, UV absorbance ntawm cov ntsiab lus analytes tau saib xyuas ntawm 0. Cov ntaub ntawv Chromatographic tau soj ntsuam siv OriginPro8 (Northampton, MA).
Cov albumin hauv cov ntshav ntawm tib neeg, hmoov lyophilized, ≥ 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 rau hauv qab 4°C.
Kev sib cais ntawm cov peptides sib xyaw thiab tryptic digest HSA ntawm PMP kem tau raug soj ntsuam cais. Tshawb xyuas qhov tryptic hydrolysis ntawm cov peptides sib xyaw thiab HSA sib cais los ntawm PMP kem thiab piv cov txiaj ntsig nrog Ascentis Express RP-Amide kem. Tus lej ntawm cov phaj theoretical yog xam siv cov qauv hauv qab no:
Cov duab SEM ntawm cov khoom silica ntshiab thiab cov khoom silica ligand khi tau pom hauv Daim Duab 2. Cov duab SEM ntawm cov khoom silica ntshiab (A, B) qhia txog lub voj voog uas cov khoom me me ntev lossis muaj qhov sib npaug tsis sib xws piv rau peb cov kev tshawb fawb yav dhau los. Qhov chaw ntawm cov khoom silica khi los ntawm ligand (C, D) yog du dua li ntawm cov khoom silica ntshiab, uas tej zaum yuav yog vim cov saw polystyrene npog qhov chaw ntawm cov khoom silica.
Cov duab thaij hluav taws xob ntawm cov khoom me me ntawm silica (A, B) thiab cov khoom me me ntawm ligand bound silica (C, D).
Qhov kev faib tawm ntawm cov khoom me me ntawm cov khoom silica ntshiab thiab cov khoom silica ligand-bound tau qhia nyob rau hauv daim duab 2. 3 (A). Cov kab sib faib tawm ntawm cov khoom me me volumetric tau qhia tias qhov loj me ntawm cov khoom silica tau nce tom qab kev hloov kho tshuaj (Daim duab 3A). Cov ntaub ntawv faib tawm 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 me ntawm cov khoom me me d (0.5) ntawm PMP yog 3.36 µm, piv rau tus nqi ad (0.5) ntawm 3.05 µm hauv peb txoj kev tshawb fawb yav dhau los (polystyrene bonded silica particles)34. Vim yog qhov hloov pauv ntawm qhov sib piv ntawm PEG, urea, TMOS thiab acetic acid hauv cov tshuaj sib xyaw, qhov kev faib tawm ntawm cov khoom me me ntawm cov khoom no tau nqaim dua piv rau peb txoj kev tshawb fawb yav dhau los. Qhov loj me ntawm cov khoom me me ntawm theem PMP yog me ntsis loj dua li ntawm theem polystyrene bound silica particle uas peb tau kawm ua ntej. Qhov no txhais tau hais tias qhov kev ua haujlwm ntawm cov khoom silica nrog styrene tsuas yog tso ib txheej polystyrene (0.97 µm) rau ntawm qhov chaw silica, thaum nyob rau 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 silica ntshiab thiab cov khoom me me silica ligand bound.
Qhov loj ntawm cov pore, qhov ntim ntawm cov pore, thiab thaj chaw ntawm cov khoom silica siv hauv txoj kev tshawb fawb no tau qhia hauv Rooj 1 (B). Cov qauv PSD ntawm cov khoom silica ntshiab thiab cov khoom silica ligand-bound tau qhia hauv Figs. 3 (B). Cov txiaj ntsig tau sib piv rau peb txoj kev tshawb fawb yav dhau los 34. Qhov loj ntawm cov khoom silica ntshiab thiab ligand-bound yog 310 Å thiab 241 Å, feem, qhia tias tom qab kev hloov kho tshuaj lom neeg, qhov loj ntawm cov pore txo qis los ntawm 69 Å, raws li tau qhia hauv Rooj 1 (B), thiab cov kab hloov pauv tau qhia hauv Daim Duab. Thaj chaw tshwj xeeb ntawm cov khoom silica hauv txoj kev tshawb fawb tam sim no yog 116 m2 / g, uas sib piv rau peb txoj kev tshawb fawb yav dhau los (124 m2 / g). Raws li tau qhia hauv Rooj 1 (B), thaj chaw ntawm cov khoom silica tom qab kev hloov kho tshuaj lom neeg kuj txo qis los ntawm 116 m2 / g mus rau 105 m2 / g.
Cov txiaj ntsig ntawm kev tshuaj xyuas cov ntsiab lus ntawm theem ruaj khov tau nthuav tawm hauv Rooj. 2. Cov ntsiab lus carbon ntawm theem ruaj khov tam sim no yog 6.35%, uas qis dua li peb txoj kev tshawb fawb yav dhau los (silica particles cuam tshuam nrog polystyrene, 7.93%35 thiab 10.21%, feem) 42. Cov ntsiab lus carbon ntawm theem ruaj khov tam sim no hauv qab no, txij li qee cov polar ligands xws li phenylmaleimide methyl vinyl isocyanate (PCMP) thiab 4-hydroxy-TEMPO tau siv ntxiv rau styrene hauv kev npaj SP. Qhov feem pua ​​​​​​ntawm nitrogen hauv theem ruaj khov tam sim no yog 2.21% piv rau 0.1735 thiab 0.85% hauv kev tshawb fawb yav dhau los42. Qhov no txhais tau tias theem ruaj khov tam sim no muaj feem pua ​​​​​​ntawm nitrogen vim yog phenylmaleimide. Ib yam li ntawd, cov khoom (4) thiab (5) muaj cov pa roj carbon ntawm 2.7% thiab 2.9%, raws li, thaum cov khoom kawg (6) muaj cov pa roj carbon ntawm 6.35%, raws li pom hauv Rooj 2. Kev tshuaj xyuas Thermogravimetric (TGA) tau siv rau ntawm theem ruaj khov ntawm PMP los sim rau qhov poob phaus, thiab cov kab TGA tau pom hauv Daim Duab 4. Cov kab TGA qhia txog qhov poob phaus ntawm 8.6%, uas yog qhov sib haum xeeb zoo nrog cov pa roj carbon (6.35%), txij li cov ligands tsis yog tsuas yog C, tab sis kuj muaj N, O thiab H.
Cov ligand phenylmaleimide-methylvinyl isocyanate tau raug xaiv los hloov kho qhov chaw ntawm cov khoom silica vim nws cov pawg polar phenylmaleimide thiab 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 cov dawb radical polymerization. Kauj ruam kawg ntawm kev teb (dawb radical polymerization) yog qhov tseem ceeb vim nws hloov pauv lub polarity ntawm theem ruaj khov. Kev tshuaj xyuas Elemental tau ua tiav los xyuas cov ntsiab lus carbon hauv cov theem ruaj khov no. Nws tau pom tias kev nce ntxiv ntawm styrene thiab lub sijhawm teb ua rau cov ntsiab lus 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. Ib yam li ntawd, cov theem ruaj khov no tau muab tso rau ntawm cov hlau tsis xeb thiab lawv cov yam ntxwv chromatographic (kev xaiv, kev daws teeb meem, N tus nqi, thiab lwm yam) tau raug kuaj xyuas. Raws li cov kev sim no, cov khoom sib xyaw ua ke zoo tshaj plaws rau kev npaj ntawm PMP theem ruaj khov tau raug xaiv los muab kev tswj hwm polarity thiab kev khaws cia zoo ntawm cov analyte.
Lub kem PMP kuj tau raug soj ntsuam rau kev tshuaj xyuas 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 theem 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 mob (200,000 phaj/m), tus naj npawb ntawm cov phaj theoretical (N) ib kem (100 × 1.8 hli) yog 20,000 ± 100. Cov nqi N rau peb kem PMP tau qhia hauv Rooj 3 thiab cov chromatograms tau qhia hauv Daim Duab 5A. Kev tshuaj xyuas sai ntawm qhov nrawm siab (700 µl/min) ntawm ib kab PMP, tsib peptides eluted hauv ib feeb, tus nqi N zoo heev ntawm 13,500 ± 330 ib kab (100 x 1.8 hli txoj kab uas hla), sib npaug rau 135,000 phaj/m2 (Daim duab 5B). Peb kab ntawm tib qhov loj me (txoj kab uas hla sab hauv 100 x 1.8 hli) tau sau nrog peb pawg sib txawv ntawm PMP theem ruaj khov los sim kev rov ua dua. Cov analytes tau kaw rau txhua kab los ntawm kev sib cais tib qhov sib xyaw ntawm txhua kab siv cov xwm txheej zoo tshaj plaws elution, tus lej ntawm cov phaj theoretical N, thiab lub sijhawm khaws cia. Cov ntaub ntawv rov ua dua rau cov kab PMP tau qhia hauv Rooj 4. Kev rov ua dua ntawm kab PMP sib raug zoo nrog cov nqi %RSD qis heev raws li tau qhia hauv Rooj 3.
Kev sib cais 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%), PMP kem qhov ntev (100 x 1.8 hli id), kev tshuaj xyuas Elution order 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 (leucic acid enkephalin).
Ib kem PMP (lub cheeb sab hauv 100 x 1.8 hli) tau raug soj ntsuam rau kev sib cais ntawm tryptic hydrolyzate ntawm tib neeg cov ntshav albumin los ntawm HPLC. Lub chromatogram hauv Daim Duab 6 qhia tau hais tias cov qauv tau sib cais zoo nrog kev daws teeb meem zoo heev. Cov tshuaj HSA tau soj ntsuam siv tus nqi ntws ntawm 100 μl / min, theem txawb ntawm 70/30 acetonitrile / dej thiab 0.1% TFA. Kev sib cais ntawm HSA tau muab faib ua 17 lub ncov, raws li tau pom hauv chromatogram (Daim Duab 6), sib raug rau 17 peptides. Kev sib cais zoo ntawm cov ncov ib leeg los ntawm HSA hydrolyzate tau suav thiab cov nqi tau pom hauv Rooj 5.
Cov HSA tryptic hydrolysates tau sib cais rau ntawm ib kem PMP (lub cheeb sab hauv 100 x 1.8 hli), tus nqi ntws (100 μl/min), theem txawb 60/40 acetonitrile/dej, thiab 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, 60/40 v/v tau siv. ACN/dej. Qhov permeability ntawm kem PMP (ID 100 × 1.8 hli) zoo ib yam li peb txoj kev tshawb fawb Ref.34 yav dhau los. Qhov permeability ntawm ib kem puv nrog cov khoom me me superficially porous yog 1.7 × 10 .6 µm, 2.5 × 10-14 m2 rau 5 µm khoom me me43. Yog li ntawd, qhov permeability ntawm theem PMP zoo ib yam li qhov permeability ntawm cov khoom me me-plhaub nrog qhov loj ntawm 5 μm.
qhov twg Wx yog qhov hnyav ntawm kem uas muaj chloroform, Wy yog qhov hnyav ntawm kem uas muaj 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 kem silica-C18 particle (100 × 1.8 mm ID)34 thiab peb cov kem C18-urea31 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 kem PMP (lub cheeb sab hauv 100 × 1.8 mm) yog 0.60. PMP kem tsis tshua permeable dua li kem uas muaj C18 bound silica particles vim tias hauv C18 hom stationary phases C18 ligands tau txuas rau cov silica particles hauv cov saw hlau, thaum nyob rau hauv polystyrene hom stationary phases ib qho polymer tuab heev tau tsim nyob ib puag ncig cov khoom me me. txheej A. Hauv kev sim ib txwm muaj, qhov porosity ntawm kem yog xam raws li nram no:
Hauv daim duab 7A, B qhia Van Deemter cov duab rau ib kem PMP (id 100 x 1.8 mm) thiab ib kem Ascentis Express RP-Amide (id 100 x 1.8 mm) nyob rau hauv tib lub elution mob, 60/40 ACN/H2O thiab 0 .1% TFA 20 µl/min txog 800 µl/min ntawm ob kem. Cov nqi HETP tsawg kawg nkaus ntawm qhov ntws zoo tshaj plaws (80 µl/min) yog 2.6 µm thiab 3.9 µm rau kem PMP thiab kem Ascentis Express RP-Amide, raws li. Cov nqi HETP qhia tau hais tias qhov kev ua haujlwm sib cais ntawm kem PMP (100 x 1.8 mm id) siab dua li ntawm kem Ascentis Express RP-Amide (100 x 1.8 mm id) uas muaj nyob hauv khw. Daim duab van Deemter hauv daim duab 7(A) qhia tau hais tias qhov txo qis hauv N tus nqi tsis siab dua nrog kev ntws ntau dua piv rau peb txoj kev tshawb fawb yav dhau los. Qhov ua tau zoo dua ntawm PMP kem (id 100 × 1.8 mm) piv rau Ascentis Express RP-Amide kem yog raws li cov duab thiab qhov loj ntawm cov khoom me me uas zoo dua thiab cov txheej txheem ntim kem uas siv hauv kev ua haujlwm tam sim no34.
(A) Daim duab Van Deemter (HETP vs. mobile phase linear velocity) tau txais ntawm PMP kem (id 100 x 1.8 mm) hauv 60/40 ACN/H2O nrog 0.1% TFA. (B) Daim duab Van Deemter (HETP vs. mobile phase linear velocity) tau txais ntawm Ascentis Express RP-Amide kem (id 100 x 1.8 mm) hauv 60/40 ACN/H2O nrog 0.1% TFA.
Ib qho polar stationary phase ntawm intercalated polystyrene tau npaj thiab soj ntsuam rau kev sib cais ntawm cov khoom sib xyaw ntawm cov peptides tsim thiab tryptic hydrolyzate ntawm tib neeg serum albumin (HSA) hauv kev ua haujlwm siab ua kua chromatography. Kev ua haujlwm chromatographic ntawm PMP kem rau cov khoom sib xyaw peptide yog qhov zoo heev hauv kev sib cais ua haujlwm thiab kev daws teeb meem. Kev sib cais ua haujlwm zoo dua ntawm PMP kem yog vim muaj ntau yam xws li qhov loj me ntawm cov khoom me me silica thiab qhov loj me ntawm qhov pore, kev tswj hwm kev sib xyaw ntawm cov theem ruaj khov, thiab cov ntaub ntawv ntim kem nyuaj. Ntxiv nrog rau kev sib cais ua haujlwm siab, lwm qhov zoo ntawm qhov theem ruaj khov no yog qhov siab qis ntawm kem rov qab ntawm cov nqi ntws siab. PMP kem yog cov khoom rov ua dua tshiab tau zoo heev thiab tuaj yeem siv los soj ntsuam cov khoom sib xyaw ntawm peptides thiab tryptic zom ntawm ntau yam protein. Peb npaj siab siv kem no rau kev sib cais cov bioactive compounds los ntawm cov khoom ntuj, cov tshuaj rho tawm ntawm cov nroj tsuag tshuaj thiab cov nceb 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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