Nkhani zina zokhudzana ndi mavuto a LC sizimathera nthawi, chifukwa pali mavuto omwe amakumana nawo mu LC, ngakhale ukadaulo wa zida ukusintha pakapita nthawi. Pali njira zambiri zomwe mavuto angayambire mu dongosolo la LC ndikutha kukhala ovuta. Mavuto okhudzana ndi mawonekedwe a peak akabuka, mndandanda waufupi wa zomwe zingayambitse zotsatirazi zimathandiza kuti tichepetse mavuto athu.
Zakhala zosangalatsa kulemba gawo ili la "LC Troubleshooting" ndikuganizira mitu mwezi uliwonse, chifukwa mitu ina siitha. Pamene ndili mu gawo la chromatography, kafukufuku wa mitu kapena malingaliro ena amakhala osagwira ntchito chifukwa amalowedwa m'malo ndi malingaliro atsopano komanso abwino, pankhani yothetsa mavuto, kuyambira pomwe nkhani yoyamba yothetsa mavuto idawonekera mu magazini ino (LC Journal panthawiyo) popeza mitu ina ikadali yofunika) mu 1983 (1). M'zaka zingapo zapitazi, ndayang'ana kwambiri magawo angapo a LC Troubleshooting pazochitika zamakono zomwe zimakhudza madzi chromatography (LC) (mwachitsanzo, kufananiza kwa kumvetsetsa kwathu za momwe kupanikizika kumakhudzira kusunga [2] Zatsopano Zotsogola) Kutanthauzira kwathu zotsatira za LC ndi momwe tingathetsere mavuto ndi zida zamakono za LC. Mu gawo la mwezi uno, ndikupitiliza mndandanda wanga (3), womwe unayamba mu Disembala 2021, womwe unayang'ana kwambiri mitu ina ya "moyo ndi imfa" ya LC troubleshooting - zinthu zomwe zili zabwino kwa wothetsa mavuto aliyense ndizofunikira, mosasamala kanthu za zaka za dongosolo lomwe tikugwiritsa ntchito. Mutu waukulu wa mndandanda uwu ndi wofunikira kwambiri ku LCGC yotchuka Tchati cha pakhoma cha “LC Troubleshooting Guide” (4) chomwe chili m'ma laboratories ambiri. Pa gawo lachitatu la mndandanda uno, ndasankha kuyang'ana kwambiri pa nkhani zokhudzana ndi mawonekedwe a peak kapena makhalidwe a peak. Chodabwitsa n'chakuti, tchati cha pakhoma chili ndi zifukwa 44 zosiyanasiyana zomwe zingayambitse mawonekedwe oipa a peak! Sitingathe kuganizira zonsezi mwatsatanetsatane m'nkhani imodzi, kotero mu gawo loyamba ili la nkhaniyi, ndiyang'ana kwambiri pa zina zomwe ndimawona nthawi zambiri. Ndikukhulupirira kuti ogwiritsa ntchito a LC achinyamata ndi achikulire apeza malangizo othandiza komanso zikumbutso pankhaniyi yofunika.
Ndimadzipeza ndikuyankha mafunso othetsa mavuto ndi mawu akuti "chilichonse n'chotheka". Yankho ili lingawoneke losavuta poganizira zomwe zawonedwa zomwe zimakhala zovuta kuzimvetsa, koma nthawi zambiri ndimapeza kuti ndi loyenera. Popeza pali zifukwa zambiri zomwe zimapangitsa kuti vuto likhale loipa, ndikofunikira kukhala ndi maganizo otseguka poganizira zomwe zingakhalepo, ndikutha kuyika patsogolo zomwe zingayambire mavuto athu, poganizira zomwe zingachitike nthawi zambiri, mfundo iyi ndi yofunika kwambiri.
Gawo lofunika kwambiri pa ntchito iliyonse yothetsa mavuto — koma yomwe ndikuganiza kuti siili yocheperako — ndikuzindikira kuti pali vuto lomwe liyenera kuthetsedwa. Kuzindikira kuti pali vuto nthawi zambiri kumatanthauza kuzindikira kuti zomwe zimachitika ku chidachi ndizosiyana ndi zomwe timayembekezera, zomwe zimapangidwa ndi chiphunzitso, chidziwitso champhamvu, ndi zomwe takumana nazo (5). "Mawonekedwe apamwamba" omwe akutchulidwa pano kwenikweni sakutanthauza mawonekedwe a nsonga yokha (yofanana, yosafanana, yosalala, yofewa, m'mphepete motsogozedwa, mchira, ndi zina zotero), komanso m'lifupi. Zoyembekeza zathu za mawonekedwe enieni a nsonga ndizosavuta. Chiphunzitso (6) chimathandizira bwino chiyembekezo cha buku la maphunziro kuti, nthawi zambiri, nsonga za chromatographic ziyenera kukhala zofanana ndikugwirizana ndi mawonekedwe a kugawa kwa Gaussian, monga momwe zasonyezedwera pa Chithunzi 1a. Zomwe tikuyembekezera kuchokera ku m'lifupi mwa nsonga ndi nkhani yovuta kwambiri, ndipo tidzakambirana nkhaniyi m'nkhani ina yamtsogolo. Mawonekedwe ena a nsonga mu Chithunzi 1 akuwonetsa zina mwazotheka zomwe zingawonekere — mwanjira ina, njira zina zomwe zinthu zingayende molakwika. Mu gawo lotsalali, tidzakhala ndi nthawi yokambirana zitsanzo zina za mikhalidwe yomwe zingayambitse mitundu iyi ya mawonekedwe.
Nthawi zina nsonga sizimaoneka konse mu chromatogram komwe zimayembekezeredwa kuti sizingaoneke. Tchati cha khoma pamwambapa chikuwonetsa kuti kusakhalapo kwa nsonga (poganiza kuti chitsanzocho chili ndi analyte yowunikira yomwe iyenera kupangitsa kuti yankho la detector likhale lokwanira kuti liwone pamwamba pa phokoso) nthawi zambiri limakhudzana ndi vuto linalake la chipangizo kapena mikhalidwe yolakwika ya gawo loyenda (ngati lawonedwa konse). nsonga, nthawi zambiri "zofooka kwambiri"). Mndandanda waufupi wa mavuto ndi mayankho omwe angakhalepo mu gululi ungapezeke mu Table I.
Monga tafotokozera pamwambapa, funso loti kukulitsa peak kuyenera kuloledwa bwanji musanaganizire ndikuyesera kukonza ndi nkhani yovuta yomwe ndidzakambirana m'nkhani ina mtsogolo. Zomwe ndakumana nazo ndikuti kukulitsa peak kofunika nthawi zambiri kumayenderana ndi kusintha kwakukulu kwa mawonekedwe a peak, ndipo kukwera kwa peak kumakhala kofala kwambiri kuposa kugawanika kwa peak kapena pre-peak. Komabe, peaks zomwe zimafanana nazo zimakulitsidwanso, zomwe zingayambitsidwe ndi zifukwa zingapo zosiyana:
Nkhani iliyonse mwa izi yakambidwa mwatsatanetsatane m'magazini am'mbuyomu a Troubleshooting LC, ndipo owerenga omwe ali ndi chidwi ndi mitu iyi akhoza kuwona nkhani zam'mbuyomu kuti adziwe zomwe zimayambitsa komanso njira zothetsera mavutowa. Zambiri.
Kukwera kwa nsonga, kutsogolo kwa nsonga, ndi kugawanikana kungayambitsidwe ndi zochitika za mankhwala kapena zakuthupi, ndipo mndandanda wa mayankho omwe angatheke pamavutowa umasiyana kwambiri, kutengera ngati tikukumana ndi vuto la mankhwala kapena zakuthupi. Nthawi zambiri, poyerekeza nsonga zosiyanasiyana mu chromatogram, mutha kupeza zizindikiro zofunika zokhudza chomwe chikuyambitsa vutoli. Ngati nsonga zonse mu chromatogram zikuwonetsa mawonekedwe ofanana, chifukwa chake sichingakhale chathupi. Ngati nsonga imodzi kapena zingapo zokha zakhudzidwa, koma zina zonse zikuwoneka bwino, chifukwa chake mwina ndi chamankhwala.
Zomwe zimayambitsa kukwera kwa peak tailing ndizovuta kwambiri kuti tikambirane mwachidule pano. Wowerenga yemwe akufuna kudziwa zambiri akutumizidwa ku nkhani yaposachedwa ya "LC Troubleshooting" kuti akambirane mozama (10). Komabe, chinthu chosavuta kuyesa ndikuchepetsa kuchuluka kwa analyte yolowetsedwa ndikuwona ngati mawonekedwe a peak akukwera. Ngati ndi choncho, ndiye kuti ichi ndi chizindikiro chabwino kuti vuto ndi "kuchuluka kwa mass". Pankhaniyi, njirayo iyenera kukhala yongolowetsa ma analyte ang'onoang'ono, kapena mikhalidwe ya chromatographic iyenera kusinthidwa kuti mawonekedwe abwino a peak athe kupezeka ngakhale ndi ma mass akuluakulu olowetsedwa.
Palinso zifukwa zambiri zomwe zingachitike chifukwa cha kutsika kwa mtunda. Owerenga omwe akufuna kudziwa zambiri za kuthekera kwawo akutumizidwa ku nkhani ina yaposachedwa ya "LC Troubleshooting" (11). Chimodzi mwa zifukwa zomwe zimafala kwambiri chifukwa cha kutsika kwa mtunda ndi kulumikizana koyipa pakati pa injector ndi detector (12). Chitsanzo choopsa chikuwonetsedwa mu Chithunzi 1d, chomwe chidapezeka mu labu yanga masabata angapo apitawo. Pankhaniyi, tidapanga makina okhala ndi valavu yatsopano yopangira jekeseni yomwe sitinagwiritse ntchito kale, ndipo tidayika lupu yaying'ono yopangira jekeseni yokhala ndi ferrule yomwe idapangidwa pa capillary yachitsulo chosapanga dzimbiri. Pambuyo poyesa koyamba kuthetsa mavuto, tidazindikira kuti kuya kwa doko mu stator ya valavu yopangira jekeseni kunali kozama kwambiri kuposa momwe tinkazolowera, zomwe zimapangitsa kuti voliyumu yayikulu ikhale pansi pa doko. Vutoli limathetsedwa mosavuta posintha lupu yopangira jekeseni ndi chubu china, titha kusintha ferrule pamalo oyenera kuti tichotse voliyumu yakufa pansi pa doko.
Ma peak front ngati omwe akuwonetsedwa pa Chithunzi 1e amathanso kuyambitsidwa ndi mavuto akuthupi kapena a mankhwala. Chifukwa chofala cha m'mphepete mwa kutsogolo ndichakuti tinthu tating'onoting'ono ta m'mbali mwa mzere sitinapake bwino, kapena kuti tinthu tating'onoting'ono takonzedwanso pakapita nthawi. Monga momwe zimakhalira ndi peak tailing yomwe imachitika chifukwa cha izi, njira yabwino yokonzera izi ndikusintha m'mbali mwa mzere ndikupitilira. Mwachidule, mawonekedwe a peak peak a m'mphepete mwa mzere omwe amachokera ku mankhwala nthawi zambiri amachokera ku zomwe timatcha "zosakhala za mzere". Pansi pa mikhalidwe yabwino (yolunjika), kuchuluka kwa analyte komwe kumasungidwa ndi gawo lokhazikika (motero, retention factor) kumagwirizana molunjika ndi kuchuluka kwa analyte mu m'mbali mwa mzere. Mogwirizana ndi chromatographic, izi zikutanthauza kuti pamene kuchuluka kwa analyte komwe kumalowetsedwa mu m'mbali kumawonjezeka, nsonga imakhala yayitali, koma osati yokulirapo. Ubalewu umasweka pamene khalidwe losunga silili lolunjika, ndipo nsonga sizimangokhala zazitali komanso zokulirapo pamene kulemera kwakukulu kumalowetsedwa. Kuphatikiza apo, mawonekedwe osakhala a mzere amatsimikiza mawonekedwe a nsonga za chromatographic, zomwe zimapangitsa kuti m'mphepete zitsogolere kapena zotsatizana. Monga momwe zimakhalira ndi kuchuluka kwa misa komwe kumayambitsa peak tailing (10), nsonga Kutsogolera komwe kumachitika chifukwa cha kusungidwa kosalunjika kumathanso kupezeka mwa kuchepetsa kuchuluka kwa analyte komwe kwalowetsedwa. Ngati mawonekedwe a peak asintha, njirayo iyenera kusinthidwa kuti isapitirire mtundu wa jakisoni womwe umayambitsa m'mphepete wotsogola, kapena mikhalidwe ya chromatographic iyenera kusinthidwa kuti ichepetse khalidweli.
Nthawi zina timaona chomwe chikuwoneka ngati "kugawanika", monga momwe zasonyezedwera pa Chithunzi 1f. Gawo loyamba pothetsa vutoli ndikuwona ngati mawonekedwe a peak akuchokera ku mgwirizano wosakwanira (mwachitsanzo, kukhalapo kwa zinthu ziwiri zosiyana koma zosagwirizana kwambiri). Ngati pali ma analyte awiri osiyana omwe akuyandikana kwambiri, ndiye kuti ndi nkhani yokonza kutsimikiza kwawo (mwachitsanzo, mwa kuwonjezera kusankha, kusunga, kapena kuchuluka kwa mbale), ndipo ma peak "ogawanika" omwe akuwoneka akugwirizana ndi physical Performance sikugwirizana ndi colon yokha. Nthawi zambiri, chidziwitso chofunikira kwambiri pa chisankhochi ndichakuti ngati ma peak onse mu chromatogram akuwonetsa mawonekedwe ogawanika, kapena amodzi kapena awiri okha. Ngati ndi amodzi kapena awiri okha, mwina ndi vuto la co-elution; ngati ma peak onse agawanika, mwina ndi vuto la physical, mwina logwirizana ndi colon yokha.
Ma peak ogawanika okhudzana ndi mawonekedwe enieni a chigawocho nthawi zambiri amakhala chifukwa cha ma frits otsekeka pang'ono olowera kapena otulukira, kapena kukonzanso tinthu tating'onoting'ono mu chigawocho, zomwe zimapangitsa kuti gawo loyenda liziyenda mofulumira kuposa gawo loyenda m'malo ena a mapangidwe a njira ya chigawocho. m'madera ena (11). Ma frit otsekeka pang'ono nthawi zina amatha kuchotsedwa pobweza kuyenda kudzera mu chigawocho; komabe, malinga ndi zomwe ndakumana nazo, nthawi zambiri izi zimakhala yankho la kanthawi kochepa osati la nthawi yayitali. Izi nthawi zambiri zimakhala zoopsa ndi mizati yamakono ngati tinthu tating'onoting'ono tiphatikizananso mkati mwa chigawocho. Pakadali pano, ndibwino kusintha chigawocho ndikupitiliza.
Chiwongola dzanja chomwe chili mu Chithunzi 1g, chomwe chachitika posachedwapa mu labu yanga, nthawi zambiri chimasonyeza kuti chizindikirocho chili chokwera kwambiri kotero kuti chafika pachimake pa yankho. Pa zowunikira za kuwala (UV-vis pankhaniyi), pamene kuchuluka kwa analyte kuli kwakukulu kwambiri, analyte imayamwa kuwala kochuluka komwe kumadutsa mu selo yowunikira, zomwe zimasiya kuwala kochepa kwambiri kuti kuzindikirike. Pansi pa izi, chizindikiro chamagetsi kuchokera ku photodetector chimakhudzidwa kwambiri ndi magwero osiyanasiyana a phokoso, monga kuwala kosochera ndi "mdima wamagetsi", zomwe zimapangitsa chizindikirocho kukhala "chosawoneka bwino" komanso chopanda kuchuluka kwa analyte. Izi zikachitika, vutoli nthawi zambiri limatha kuthetsedwa mosavuta pochepetsa kuchuluka kwa jakisoni wa analyte—kuchepetsa kuchuluka kwa jakisoni, kuchepetsa chitsanzo, kapena zonse ziwiri.
Mu sukulu ya chromatography, timagwiritsa ntchito chizindikiro chowunikira (monga y-axis mu chromatogram) ngati chizindikiro cha kuchuluka kwa analyte mu chitsanzo. Chifukwa chake zikuwoneka zachilendo kuwona chromatogram yokhala ndi chizindikiro chochepera zero, chifukwa kutanthauzira kosavuta ndikuti izi zikuwonetsa kuchuluka kwa analyte koyipa - zomwe sizingatheke mwakuthupi. Mu zomwe ndakumana nazo, ma peaks oyipa nthawi zambiri amawonedwa pogwiritsa ntchito zida zowunikira zowunikira (monga UV-vis).
Pankhaniyi, chiwongolero choyipa chimangotanthauza kuti mamolekyu omwe amachoka pa chigawocho amayamwa kuwala kochepa kuposa gawo loyenda nthawi yomweyo asanayambe komanso atamaliza chiwongolerocho. Izi zitha kuchitika, mwachitsanzo, pogwiritsa ntchito mafunde otsika kwambiri (<230 nm) ndi zowonjezera za gawo loyenda zomwe zimayamwa kuwala kwakukulu pamafunde awa. Zowonjezera zotere zitha kukhala zigawo zosungunulira za gawo loyenda monga methanol kapena zigawo za buffer monga acetate kapena formate. Munthu angagwiritse ntchito ma peaks oyipa kuti akonze curve yowunikira ndikupeza zambiri zolondola, kotero palibe chifukwa chofunikira chopewera izi (njira iyi nthawi zina imatchedwa "kuzindikira kwa UV kosalunjika") (13). Komabe, ngati tikufunadi kupewa ma peaks oyipa kwathunthu, pankhani yozindikira kunyamulidwa, yankho labwino kwambiri ndikugwiritsa ntchito mafunde osiyana kuti analyte inyamule zambiri kuposa gawo loyenda, kapena kusintha kapangidwe ka gawo loyenda kuti Anyamule kuwala kochepa kuposa analytes.
Ma peaks oipa amathanso kuwonekera pogwiritsa ntchito refractive index (RI) detection pamene refractive index ya zigawo zina kupatula analyte mu chitsanzo, monga solvent matrix, ndi yosiyana ndi refractive index ya gawo loyenda. Izi zimachitikanso ndi UV-vis detection, koma izi zimakhala zochepa poyerekeza ndi RI detection. M'zochitika zonsezi, ma peaks oipa amatha kuchepetsedwa pofananiza kwambiri kapangidwe ka chitsanzo cha matrix ndi ka gawo loyenda.
Mu gawo lachitatu pa nkhani yoyambira yothetsera mavuto a LC, ndakambirana za zochitika zomwe mawonekedwe a peak omwe awonedwa amasiyana ndi mawonekedwe a peak omwe akuyembekezeka kapena abwinobwino. Kuthetsa mavuto otere kumayamba ndi kudziwa mawonekedwe a peak omwe akuyembekezeka (kutengera chiphunzitso kapena zomwe zidachitika kale ndi njira zomwe zilipo), kotero kusiyana ndi zomwe zikuyembekezeredwazi ndikodziwikiratu. Mavuto a mawonekedwe a peak ali ndi zifukwa zosiyanasiyana zomwe zingachitike (zotakata kwambiri, zopinga, zotsogola, ndi zina zotero). Mu gawoli, ndikukambirana mwatsatanetsatane zina mwa zifukwa zomwe ndimawona nthawi zambiri. Kudziwa tsatanetsatane uwu kumapereka malo abwino oyambira kuthetsa mavuto, koma sikutchula zonse zomwe zingatheke. Owerenga omwe ali ndi chidwi ndi mndandanda wozama wa zifukwa ndi mayankho amatha kuwona tchati cha khoma la LCGC "LC Troubleshooting Guide".
(4) Tchati cha khoma la LCGC “LC Troubleshooting Guide” https://www.chromatographyonline.com/view/troubleshooting-wallchart (2021).
(6) A. Felinger, Kusanthula Deta ndi Kukonza Zizindikiro mu Chromatography (Elsevier, New York, NY, 1998), pp. 43-96.
(8) Wahab MF, Dasgupta PK, Kadjo AF ndi Armstrong DW, Anal.Chim.Journal.Rev. 907, 31–44 (2016).https://doi.org/10.1016/j.aca.2015.11.043.