Liquid chromatography shine babbar hanyar gwada abun ciki na kowane bangare da ƙazanta a cikin albarkatun ƙasa, tsaka-tsaki, shirye-shirye da kayan tattarawa, amma yawancin abubuwa ba su da daidaitattun hanyoyin dogaro da su, don haka ba makawa don haɓaka sabbin hanyoyin. A cikin haɓaka hanyoyin matakan ruwa, ginshiƙi na chromatographic shine ainihin tushen chromatography na ruwa, don haka yadda za a zaɓi ginshiƙin chromatographic mai dacewa yana da mahimmanci. A cikin wannan labarin, marubucin zai yi bayanin yadda za a zaɓi shafi na chromatography na ruwa daga bangarori uku: ra'ayoyi gabaɗaya, la'akari da iyakokin aikace-aikace.
A.Babban ra'ayoyin don zaɓar ginshiƙan chromatography na ruwa
1. Yi la'akari da halayen jiki da sinadarai na mai bincike: kamar tsarin sinadarai, solubility, kwanciyar hankali (kamar ko yana da sauƙi don zama oxidized / rage / ruwa), acidity da alkalinity, da dai sauransu, musamman ma tsarin sinadaran shine mabuɗin. factor a cikin ƙayyadaddun kaddarorin, kamar ƙungiyar haɗin gwiwar tana da ƙarfi mai ƙarfi na ultraviolet da haske mai ƙarfi;
2. Ƙayyade maƙasudin bincike: ko babban rabuwa, babban tasiri na shafi, gajeren lokaci na bincike, babban hankali, tsayin daka mai tsayi, tsawon rayuwar shafi, ƙananan farashi, da dai sauransu.
- Zaɓi ginshiƙin chromatographic mai dacewa: fahimtar abun da ke ciki, kaddarorin jiki da sinadarai na filler chromatographic, kamar girman barbashi, girman pore, haƙurin zafin jiki, haƙurin pH, adsorption na analyte, da sauransu.
- Shawarwari don zaɓar ginshiƙan chromatography na ruwa
Wannan babin zai tattauna abubuwan da za a yi la'akari da su lokacin zabar ginshiƙi na chromatography daga hangen nesa na zahiri da sinadarai na ginshiƙin chromatography da kansa. 2.1 Filler matrix
2.1.1 Silica gel matrix Matrix filler na mafi yawan ginshiƙan chromatography na ruwa shine gel silica. Wannan nau'in filler yana da tsafta mai yawa, ƙarancin farashi, ƙarfin injina, kuma yana da sauƙin gyara ƙungiyoyi (kamar phenyl bonding, amino bonding, cyano bonding, da dai sauransu), amma ƙimar pH da kewayon zafin jiki da yake jurewa suna iyakance: pH kewayon mafi yawan silica gel matrix filler shine 2 zuwa 8, amma kewayon pH na musamman gyaran silica gel bonded phases na iya zama mai faɗi kamar 1.5 zuwa 10, kuma akwai kuma gyare-gyare na musamman na silica gel bonded phases waɗanda ke barga a ƙananan pH, irin su Agilent ZORBAX RRHD stablebond-C18, wanda ya tsaya a pH 1 zuwa 8; Matsakaicin zafin jiki na silica gel matrix yawanci shine 60 ℃, kuma wasu ginshiƙan chromatography na iya jure yanayin zafin jiki na 40 ℃ a babban pH.
2.1.2 Polymer matrix Polymer fillers yawanci polystyrene-divinylbenzene ko polymethacrylate. Amfanin su shine cewa zasu iya jure wa fa'idar pH - ana iya amfani da su a cikin kewayon 1 zuwa 14, kuma sun fi tsayayya da yanayin zafi (zai iya kaiwa sama da 80 ° C). Idan aka kwatanta da silica na tushen C18 filler, irin wannan nau'in filler yana da ƙarfi hydrophobicity, kuma macroporous polymer yana da matukar tasiri wajen rarraba samfuran kamar sunadaran. Lalacewar sa shine cewa ingancin ginshiƙi ya yi ƙasa da ƙarfi kuma ƙarfin injin ya yi rauni fiye da na filaye na tushen silica. 2.2 Siffar barbashi
Yawancin filaye na zamani na HPLC ɓangarorin sikeli ne, amma wani lokacin ɓarna ce marasa daidaituwa. Siffar nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i)) wanda zai iya samar da ƙananan ginshiƙan ginshiƙan, mafi girman tasiri, kwanciyar hankali da tsawon rai; lokacin amfani da matakai na wayar hannu mai girma (kamar phosphoric acid) ko kuma lokacin da samfurin samfurin ya kasance danko, ƙwayoyin da ba a saba da su ba suna da wani yanki na musamman na musamman, wanda ya fi dacewa da cikakken aikin sassan biyu, kuma farashin yana da ƙananan ƙananan. 2.3 Girman barbashi
Karamin girman barbashi, mafi girman ingancin ginshiƙi kuma mafi girman rabuwa, amma mafi muni da juriya mai ƙarfi. Shagon da aka fi amfani da shi shine ginshiƙin girman barbashi 5 μm; idan buƙatun rabuwa yana da girma, za'a iya zaɓar filler 1.5-3 μm, wanda zai dace don magance matsalar rabuwa na wasu matrix mai rikitarwa da samfurori masu yawa. UPLC iya amfani da 1.5 μm fillers; 10 μm ko mafi girman girman girman barbashi ana amfani dashi akai-akai don shirye-shirye na rabin-shirya ko ginshiƙan shirye-shirye. 2.4 Abubuwan Carbon
Abubuwan da ke cikin carbon yana nufin rabon lokacin haɗin gwiwa akan saman silica gel, wanda ke da alaƙa da takamaiman yanki na ƙasa da ɗaukar lokaci mai alaƙa. Babban abun ciki na carbon yana ba da ƙarfin ginshiƙi da babban ƙuduri, kuma ana amfani dashi sau da yawa don samfurori masu rikitarwa waɗanda ke buƙatar babban rabuwa, amma saboda tsawon lokacin hulɗar tsakanin sassan biyu, lokacin bincike yana da tsawo; ƙananan ginshiƙan chromatographic ƙananan abun ciki na carbon suna da ɗan gajeren lokaci bincike kuma suna iya nuna zaɓuɓɓuka daban-daban, kuma ana amfani da su sau da yawa don samfurori masu sauƙi waɗanda ke buƙatar bincike mai sauri da samfurori waɗanda ke buƙatar yanayin yanayin ruwa mai zurfi. Gabaɗaya, abun cikin carbon na C18 ya tashi daga 7% zuwa 19%. 2.5 Girman Pore da takamaiman yanki na fili
Kafofin watsa labarai na adsorption na HPLC ɓangarorin ɓarke ne, kuma yawancin hulɗar suna faruwa a cikin pores. Don haka, dole ne kwayoyin halitta su shiga ramukan don a datse su kuma a raba su.
Girman pore da ƙayyadaddun farfajiyar fage biyu ne masu dacewa. Ƙananan girman pore yana nufin babban yanki na musamman, kuma akasin haka. Babban ƙayyadaddun yanki na musamman na iya haɓaka hulɗar tsakanin ƙwayoyin samfuri da matakan haɗin gwiwa, haɓaka riƙewa, haɓaka ɗaukar samfuri da ƙarfin shafi, da rabuwar hadaddun abubuwan. Cikakkun filaye masu ƙyalƙyali suna cikin irin wannan nau'in filaye. Ga waɗanda ke da manyan buƙatun rabuwa, ana ba da shawarar zaɓin filaye tare da babban yanki na musamman; ƙananan ƙayyadaddun yanki na musamman na iya rage matsa lamba na baya, inganta ingantaccen shafi, da rage lokacin daidaito, wanda ya dace da nazarin gradient. Abubuwan filaye na Core-shell suna cikin irin wannan nau'in fillers. A kan yanayin tabbatar da rabuwa, ana bada shawara don zaɓar filaye tare da ƙananan yanki na musamman don waɗanda ke da buƙatun ingantaccen bincike. 2.6 Ƙarfin ƙura da ƙarfin inji
Ƙarfin ƙura, wanda kuma aka sani da "ƙarar pore", yana nufin girman ƙarar ƙarar kowane barbashi. Yana iya da kyau nuna ƙarfin injin filler. Ƙarfin injina na filaye tare da babban ƙarar pore ya ɗan yi rauni fiye da na filaye tare da ƙaramin ƙarar pore. Fillers tare da ƙarar pore ƙasa da ko daidai da 1.5 mL / g ana amfani da su galibi don rabuwar HPLC, yayin da masu filaye tare da ƙarar pore sama da 1.5 mL/g ana amfani da su galibi don chromatography keɓance kwayoyin halitta da ƙananan chromatography. 2.7 Tattalin Arziki
Capping zai iya rage kololuwar wutsiya da ke haifar da hulɗar tsakanin mahadi da ƙungiyoyin silanol da aka fallasa (kamar haɗin gwiwar ionic tsakanin mahaɗan alkaline da ƙungiyoyin silanol, sojojin van der Waals da haɗin gwiwar hydrogen tsakanin mahadi acidic da ƙungiyoyin silanol), don haka inganta ingantaccen shafi da siffar kololuwa. . Matakan da ba a rufe ba za su samar da zaɓuɓɓuka daban-daban dangane da matakan da aka rufe, musamman don samfuran polar.
- Ƙimar aikace-aikacen ginshiƙan chromatography na ruwa daban-daban
Wannan babin zai bayyana iyakokin aikace-aikace na nau'ikan ginshiƙan chromatography na ruwa daban-daban ta wasu lokuta.
3.1 Juya-lokaci C18 shafi na chromatographic
Rukunin C18 shine ginshiƙin juzu'i da aka fi amfani da shi, wanda zai iya saduwa da abubuwan ciki da gwaje-gwaje na ƙazanta na mafi yawan sinadarai, kuma yana amfani da matsakaicin iyakacin duniya, ƙarancin iyakacin duniya da abubuwa marasa ƙarfi. Ya kamata a zaɓi nau'in da ƙayyadaddun shafi na chromatographic C18 bisa ga takamaiman buƙatun rabuwa. Misali, don abubuwan da ke da manyan buƙatun rabuwa, ana amfani da ƙayyadaddun ƙayyadaddun 5 μm * 4.6 * 250 mm sau da yawa; don abubuwa tare da hadaddun matrices rabuwa da irin wannan polarity, 4 μm * 4.6 mm * 250 mm ƙayyadaddun ƙayyadaddun ƙayyadaddun abubuwa ko ƙananan ƙananan ƙwayoyin cuta za a iya amfani da su. Misali, marubucin ya yi amfani da ginshiƙi na 3 μm*4.6*250 mm don gano ƙazantattun abubuwa biyu na genotoxic a cikin celecoxib API. Rabuwar abubuwa biyu na iya kaiwa 2.9, wanda yake da kyau. Bugu da ƙari, a ƙarƙashin yanayin tabbatar da rabuwa, idan ana buƙatar bincike mai sauri, ana zaɓar ɗan gajeren ginshiƙi na 10 mm ko 15 mm sau da yawa. Misali, lokacin da marubucin ya yi amfani da LC-MS/MS don gano ƙazantar genotoxic a cikin piperaquine phosphate API, an yi amfani da ginshiƙin 3 μm * 2.1 mm * 100 mm. Rabuwa tsakanin rashin tsarki da babban sashi shine 2.0, kuma ana iya kammala gano samfurin a cikin minti 5. 3.2 Rukunin phenyl Juya-lokaci
Rukunin Phenyl kuma nau'in ginshiƙi ne na jujjuyawar lokaci. Wannan nau'in ginshiƙi yana da zaɓi mai ƙarfi don mahaɗan aromatic. Idan amsawar mahaɗan aromatic da aka auna ta hanyar ginshiƙi na C18 na yau da kullun yana da rauni, zaku iya la'akari da maye gurbin ginshiƙin phenyl. Misali, lokacin da nake yin celecoxib API, babban martanin bangaren da aka auna ta hanyar ginshiƙin phenyl na masana'anta guda da ƙayyadaddun bayanai guda ɗaya (duk 5 μm*4.6 mm*250 mm) ya kusan sau 7 fiye da na ginshiƙin C18. 3.3 Al'ada-lokaci shafi
A matsayin ingantaccen kari zuwa juzu'in juzu'i, ginshiƙi na al'ada ya dace da mahaɗan polar sosai. Idan kololuwar har yanzu yana da sauri sosai lokacin da ake yin sama da kashi 90% na ruwa mai ruwa a cikin juzu'in juzu'i, har ma kusa da mamayewa tare da kololuwar ƙarfi, zaku iya la'akari da maye gurbin ginshiƙi na al'ada. Irin wannan ginshiƙi ya haɗa da ginshiƙin hilic, ginshiƙin amino, ginshiƙin cyano, da sauransu.
3.3.1 Rukunin Hilic Shagon Hilic yawanci yana haɗa ƙungiyoyin hydrophilic a cikin sarkar alkyl mai ɗaure don haɓaka martani ga abubuwan polar. Irin wannan ginshiƙi ya dace da nazarin abubuwan sukari. Marubucin ya yi amfani da irin wannan shafi lokacin yin abun ciki da abubuwan da ke da alaƙa na xylose da abubuwan da suka samo asali. Hakanan ana iya raba isomers na abin da aka samu na xylose da kyau;
3.3.2 Amino shafi da cyano shafi Amino shafi da cyano shafi suna nufin gabatar da amino da cyano gyare-gyare a karshen bonded alkyl sarkar, bi da bi, don inganta selectivity ga musamman abubuwa: misali, amino shafi ne mai kyau zabi. don rabuwa da sukari, amino acid, tushe, da amides; ginshiƙin cyano yana da mafi kyawun zaɓi lokacin raba abubuwan da aka haɗa da hydrogenated da unhydrogenated tsarin kamannin abubuwa saboda kasancewar haɗin haɗin gwiwa. Rukunin Amino da ginshiƙin cyano galibi ana iya sauyawa tsakanin ginshiƙin lokaci na al'ada da juzu'in juzu'i, amma ba a ba da shawarar sauyawa akai-akai ba. 3.4 Rukunin Chiral
Rukunin Chiral, kamar yadda sunan ya nuna, ya dace da rabuwa da bincike na mahadi na chiral, musamman a fannin magunguna. Irin wannan ginshiƙi za a iya la'akari da lokacin da al'ada baya lokaci da ginshiƙan lokaci na al'ada ba za su iya cimma rabuwar isomers ba. Misali, marubucin ya yi amfani da 5 μm * 4.6 mm * 250 mm chiral shafi don raba isomers guda biyu na 1,2-diphenylethylenediamine: (1S, 2S) -1, 2-diphenylethylenediamine da (1R, 2R) -1, 2 -diphenylethylenediamine, kuma rabuwa tsakanin su biyun ya kai kusan 2.0. Koyaya, ginshiƙan chiral sun fi sauran nau'ikan ginshiƙai tsada, yawanci 1W+/ yanki. Idan akwai buƙatar irin waɗannan ginshiƙai, sashin yana buƙatar yin isasshen kasafin kuɗi. 3.5 ion musayar shafi
ginshiƙan musayar ion sun dace da rarrabuwa da nazarin ions da aka caje, kamar ions, sunadarai, acid nucleic, da wasu abubuwan sukari. Dangane da nau'in filler, an raba su zuwa ginshiƙan musayar cation, ginshiƙan musayar anion, da ginshiƙan musayar cation mai ƙarfi.
Rukunin musayar cation sun haɗa da ginshiƙan tushen calcium da ginshiƙan hydrogen, waɗanda galibi sun dace da nazarin abubuwan cationic kamar amino acid. Alal misali, marubucin ya yi amfani da ginshiƙan tushen calcium lokacin da yake nazarin calcium gluconate da calcium acetate a cikin maganin ruwa. Duk abubuwan biyu suna da martani mai ƙarfi a λ = 210nm, kuma matakin rabuwa ya kai 3.0; marubucin ya yi amfani da ginshiƙan tushen hydrogen lokacin nazarin abubuwan da ke da alaƙa da glucose. Yawancin manyan abubuwan da ke da alaƙa - maltose, maltotriose da fructose - suna da babban hankali a ƙarƙashin bambance-bambancen ganowa, tare da iyakacin ganowa ƙasa da 0.5 ppm da matakin rabuwa na 2.0-2.5.
ginshiƙan musayar anion sun fi dacewa don nazarin abubuwan anionic kamar kwayoyin acid da halogen ions; ginshiƙan musayar cation mai ƙarfi suna da ƙarfin musayar ion mafi girma da zaɓin zaɓi, kuma sun dace da rarrabuwa da bincike na samfuran hadaddun.
Abin da ke sama gabatarwa ne kawai ga nau'ikan da kewayon aikace-aikace na ginshiƙan chromatography na ruwa gama gari haɗe da ƙwarewar marubucin. Akwai wasu nau'ikan nau'ikan ginshiƙan chromatographic na musamman a cikin ainihin aikace-aikacen, kamar manyan ginshiƙan chromatographic manyan-pore, ginshiƙan ƙananan pore chromatographic, ginshiƙan ginshiƙan chromatography, ginshiƙan ginshiƙan chromatographic multimode, ginshiƙan ginshiƙan ruwa mai ƙarfi (UHPLC), ginshiƙan ginshiƙan ruwa mai ƙarfi (UHPLC), ginshiƙan chromatography na ruwa mai ƙarfi ( SFC), da dai sauransu Suna taka muhimmiyar rawa a fagage daban-daban. Ya kamata a zaɓi takamaiman nau'in ginshiƙi na chromatographic bisa ga tsari da kaddarorin samfurin, buƙatun rabuwa da sauran dalilai.
Lokacin aikawa: Juni-14-2024