Kev ua haujlwm siab ua kua chromatography feem ntau suav nrog kev txhaj tshuaj, qhov system infusion, kev sib cais, kev kuaj pom, thiab cov ntaub ntawv ua haujlwm. Cov hauv qab no yuav qhia txog lawv cov kev sib xyaw thiab cov yam ntxwv.
1. Lub tshuab txhaj tshuaj feem ntau yog siv lub diaphragm injector lossis lub tshuab nqus tsev txhaj tshuaj kom ua tiav qhov kev txhaj tshuaj, nrog rau qhov ntim tas li. Qhov no muaj txiaj ntsig zoo rau kev txhim kho kev rov ua dua ntawm cov qauv ntsuas.
2. Lub infusion system muaj peb ntu: lub twj tso kua mis siab, lub twj tso kua mis theem mobile, thiab lub ntsuas gradient. Qhov dav dav ntawm lub twj tso kua mis siab yog l. 47-4.4X107Pa, adjustable thiab ruaj khov txaus tus nqi. Thaum lub sij hawm high-pressure mobile dhau los ntawm chromatographic kem, nws tuaj yeem txo cov nyhuv diffusion ntawm cov qauv hauv kem, ua kom lub zog ceev hauv kem, txhim kho qhov kev sib cais, thiab rov qab cov qauv. Muaj txiaj ntsig zoo rau kev tswj hwm kev ua haujlwm lom neeg ntawm cov qauv. Lub mobile theem cia yuam kev thiab gradient analyzer tuaj yeem hloov pauv theem ntawm lub xov tooj ntawm tes raws li cov khoom ntawm cov theem nyob ruaj khov thiab cov qauv, suav nrog kev hloov pauv polarity, ion zog, thiab pH tus nqi ntawm eluent, los yog siv cov kev sib tw inhibitors lossis denaturing agents. Qhov no ua rau muaj kev sib cais zoo ntawm ntau yam tshuaj, txawm tias tsuas muaj ib qho sib txawv lossis isomers.
3. Cov txheej txheem sib cais muaj xws li chromatography kab, txuas cov raj, ntsuas kub, thiab lwm yam. Qhov ntev ntawm chromatographic kem feem ntau yog 10-50cm (yog tias xav tau ob qho, ib lub raj txuas tuaj yeem ntxiv ntawm lawv), nrog rau txoj kab uas hla. ntawm 2-5mm, ua los ntawm cov hlau tsis zoo los yog tuab phab ntsa iav raj lossis titanium alloy. Muaj ib theem taag nrog ib tug particle loj ntawm 5-10 μ m (sau ntawm ib tug matrix thiab ib tug fixative). Lub matrix nyob rau hauv cov theem nyob ruaj khov yog ua los ntawm cov neeg kho tshuab siab zog resin los yog silica gel, uas muaj inertness (xws li alkaline silica gel tau raug tshem tawm), porosity (pore loj txog li 1000?), thiab loj tshwj xeeb thaj chaw. Lawv yog mechanically coated rau coupling (zoo ib yam li kev npaj cov roj chromatography nyob twj ywm theem), los yog tshuaj ua ke nrog ntau yam noob (xws li phosphate, quaternary amino, hydroxymethyl, phenyl, amino, los yog ntau yam ntev ntawm carbon saw alkyl) los yog ligand organic compounds. . Yog li, qhov kev txheeb ze ruaj khov no muaj kev xaiv zoo. Piv txwv li, tom qab coupling pea agglutinin (PSA) nyob rau saum npoo ntawm silica gel ntxeem tau, glycoproteins hauv fibroblasts tuaj yeem raug cais tawm. Tsis tas li ntawd, cov theem nyob ruaj khov raws li plasmid yog me me thiab cov kab hauv tsev yog ib qho yooj yim kom ua tiav lub xeev zoo li qub thiab ntom. Nws yog ib qho yooj yim los txo cov eddy tam sim no diffusion nyhuv. Lub matrix particle loj me me, cov micropores yog ntiav, thiab qhov loj hloov ntawm cov qauv hauv cheeb tsam microporous yog luv. Cov no muaj txiaj ntsig zoo rau kev txo qis bandwidth thiab txhim kho kev daws teeb meem. Raws li kev tsom xam cov nyhuv ntawm kab ke, qhov me me ntawm cov matrix particle loj thiab qhov loj dua qhov theoretical tooj N ntawm phaj. Qhov no ntxiv ua pov thawj tias qhov me me ntawm cov matrix particle loj, qhov siab dua qhov sib cais degree. Tsis tas li ntawd, kev ua haujlwm siab ua kua chromatography thermostats tuaj yeem kho qhov kub ntawm chav tsev kub mus rau 60 degree, txo lub sij hawm tsom xam los ntawm kev nce tus nqi hloov pauv loj., Nws tuaj yeem txhim kho qhov ua tau zoo ntawm cov kab chromatographic.
4. Cov khoom siv feem ntau siv rau HPLC cov cuab yeej kuaj xyuas muaj xws li cov khoom siv ultraviolet, cov ntsuas ntsuas qhov ntsuas tsis pom, thiab cov khoom siv fluorescence detectors.
(1) UV detector yog tsim los kuaj xyuas cov qauv nrog cov yam ntxwv haum nyob rau hauv ultraviolet (los yog pom) lub teeb. Nws cov yam ntxwv: dav siv ntau yam (xws li cov proteins, nucleic acids, amino acids, nucleotides, peptides, cov tshuaj hormones, thiab lwm yam); rhiab heev (nrhiav txwv ntawm 10-10g/ml); Wide linear ntau; Tsis rhiab rau qhov kub thiab txias hloov pauv; Nws tuaj yeem pom cov qauv eluted nrog cov kev daws teeb meem gradient.
(2) Qhov sib txawv refractive index detector tuaj yeem siv los txheeb xyuas tag nrho cov qauv piv txwv nrog cov ntsuas ntsuas qhov sib txawv ntawm cov khoom kuaj pom los ntawm lub xov tooj ntawm tes. Tam sim no, feem ntau cov carbohydrate tebchaw raug kuaj pom siv qhov kev kuaj xyuas no. Lub kaw lus muaj zog universality thiab yooj yim ua hauj lwm, tab sis tsis tshua muaj rhiab heev (nrhiav txwv ntawm 10-7g/ml), thiab cov kev hloov nyob rau hauv lub mobile theem yuav ua rau kev hloov nyob rau hauv refractive index. Yog li ntawd, nws tsis tsim nyog rau kev soj ntsuam kab mob lossis kev tshawb nrhiav cov qauv gradient elution.
(3) Lub fluorescence detector tsuas yog tsim nyog rau kev txiav txim siab ntawm fluorescent organic compounds (xws li polycyclic aromatic hydrocarbons, amino acids, amines, vitamins, thiab qee cov proteins) nyob rau hauv tej yam kev mob, raws li fluorescence siv thiab concentration ntawm lub teeb emission. Nws qhov rhiab heev yog qhov siab heev (kev kuaj pom qhov txwv yog 10-12~10-14}g / ml), thiab nws tuaj yeem siv rau kev tsom xam thiab tshawb pom hauv gradient elution ua haujlwm.
5. Cov txheej txheem ua cov ntaub ntawv tuaj yeem sau, khaws cia, tso saib, luam tawm, thiab cov txheej txheem kuaj cov ntaub ntawv kom cais cov qauv, thiab tuaj yeem ua tau raws li kev npaj lossis kev txheeb xyuas.