Dzimwe nyaya dzekugadzirisa matambudziko eLC hadzimbofi dzakapera, sezvo paine matambudziko muLC practice, kunyangwe tekinoroji yemidziyo ichivandudzika nekufamba kwenguva. Kune nzira dzakawanda idzo matambudziko anogona kumuka muLC system uye oguma asina kunaka. Kana matambudziko ane chekuita nechimiro chepamusoro amuka, runyorwa rupfupi rwezvikonzero zvinogona kukonzera matambudziko aya zvinobatsira kurerutsa ruzivo rwedu rwekugadzirisa matambudziko.
Zvave zvichinakidza kunyora chikamu ichi che "LC Troubleshooting" uye kufunga nezvenyaya mwedzi wega wega, nekuti dzimwe nyaya hadzimboperi. Ndiri mubasa rekutsvaga chromatography, dzimwe nyaya kana pfungwa dzinopera basa sezvo dzichitsiviwa nepfungwa itsva uye dziri nani, mubasa rekugadzirisa matambudziko, kubvira pakabuda chinyorwa chekutanga chekugadzirisa matambudziko mumagazini ino (LC Journal panguva iyoyo) sezvo dzimwe nyaya dzichiri kukosha) muna 1983 (1). Mumakore mashoma apfuura, ndakatarisana nezvikamu zvakati wandei zveLC Troubleshooting pamaitiro emazuva ano anokanganisa liquid chromatography (LC) (semuenzaniso, kuenzanisa kwekunzwisisa kwedu mhedzisiro yekumanikidzwa pakuchengetedza [2] New Advances) Dudziro yedu yemhedzisiro yeLC uye maitiro ekugadzirisa matambudziko nemidziyo yemazuva ano yeLC. Muchikamu chemwedzi uno, ndiri kuenderera mberi nenhevedzano yangu (3), yakatanga muna Zvita 2021, iyo yakatarisana nedzimwe dzenyaya dze "hupenyu nerufu" dzekugadzirisa matambudziko eLC - zvinhu zvakanaka kune chero mugadziri wematambudziko zvakakosha, zvisinei nezera rehurongwa hwatiri kushandisa. Musoro wenyaya iyi wakakosha zvikuru kune yakakurumbira yeLCGC Chati yemadziro ye "LC Troubleshooting Guide" (4) yakaturikwa muma laboratories mazhinji. Kwechikamu chechitatu chenyaya iyi, ndakasarudza kutarisa nyaya dzine chekuita nechimiro chepamusoro kana hunhu hwepamusoro. Zvinoshamisa kuti chati yemadziro inonyora zvikonzero makumi mana nezvina zvakasiyana zvinogona kukonzera chimiro chisina kunaka chekumusoro! Hatigone kufunga nezvenyaya idzi dzese zvakadzama mune imwe nyaya, saka muchikamu chekutanga ichi pamusoro penyaya iyi, ndichatarisa pane dzimwe dzandinowanzoona. Ndinovimba kuti vashandisi veLC vechidiki nevakuru vachawana mazano anobatsira nezviyeuchidzo panyaya iyi inokosha.
Ndinowana ndichipindura mibvunzo yekugadzirisa matambudziko ndichiti "chero chinhu chinogoneka". Mhinduro iyi ingaita seiri nyore kana ndichifunga nezvezvakaonekwa zvakaoma kududzira, asi ndinoona zvichiwanzo kukodzera. Nezvikonzero zvakawanda zvinogona kukonzera kusakwana kwechimiro chepamusoro, zvakakosha kuti munhu agare akasununguka pakufunga nezvedambudziko, uye kuti akwanise kuisa zvinhu zvakakosha pakutanga kuedza kwedu kugadzirisa matambudziko, achitarisa pane zvingangoitika, pfungwa iyi inokosha zvikuru. Zvinogoneka.
Danho guru mune chero chiito chekugadzirisa matambudziko - asi chandinofunga kuti hachina kukosheswa - kuziva kuti pane dambudziko rinofanira kugadziriswa. Kuziva kuti pane dambudziko kunowanzoreva kuziva kuti zvinoitika kuchishandiso zvakasiyana nezvatinotarisira, izvo zvinoumbwa nedzidziso, ruzivo rwekuongorora, uye ruzivo (5). "Chimiro chepamusoro" chinotaurwa pano hachirevi chete chimiro chekumusoro (chakaenzana, chisina kuenzana, chakapfava, chakapfava, chinotungamirwa, muswe, nezvimwewo), asiwo kuhupamhi. Zvatinotarisira zvechimiro chekumusoro chaiko zviri nyore. Dzidziso (6) inotsigira zvakanaka tarisiro yebhuku rechikoro yekuti, kazhinji, makomo echromatographic anofanira kunge akafanana uye achienderana nechimiro chekugoverwa kweGaussian, sezvakaratidzwa muMufananidzo 1a. Zvatinotarisira kubva kuhupamhi hwepamusoro inyaya yakaoma, uye tichakurukura nyaya iyi mune imwe nyaya inotevera. Mamwe maumbirwo ekumusoro muMufananidzo 1 anoratidza mamwe emamwe mikana anogona kuonekwa - nemamwe mazwi, mamwe enzira dzinogona kukanganisa zvinhu. Muchikamu chasara chechikamu chino, tichapedza nguva tichikurukura mimwe mienzaniso chaiyo yemamiriro ezvinhu ayo zvinogona kutungamira kumarudzi aya echimiro.
Dzimwe nguva makomo haaonekwe zvachose mu chromatogram kwaanotarisirwa kuti asaonekwe. Chati iri pamusoro apa inoratidza kuti kusavapo kwegomo (tichifunga kuti muenzaniso wacho une analyte inotarirwa pahuwandu hunofanira kuita kuti mhinduro ye detector ikwane kuiona pamusoro peruzha) kazhinji ine chekuita nedambudziko remidziyo kana mamiriro asina kunaka e mobile phase (kana akaonekwa zvachose). makomo, kazhinji "asina simba"). Rondedzero pfupi yematambudziko anogona kuitika uye mhinduro muchikamu ichi inogona kuwanikwa muTable I.
Sezvambotaurwa pamusoro apa, mubvunzo wekuti kuwedzera kwenhamba yemakomo kunofanira kubvumidzwa sei tisati tanyatsoteerera uye kuedza kugadzirisa inyaya yakaoma yandichakurukura mune imwe nyaya inotevera. Zvandakaona ndezvekuti kuwedzera kwenhamba yemakomo kunowanzo perekedzwa nekuchinja kukuru kwechimiro chenhamba yemakomo, uye kuwedzera kwenhamba yemakomo kwakajairika kupfuura kupatsanurwa kwenhamba isati yasvika. Zvisinei, nhamba dzemakomo dzakafanana dzakatambanudzwawo, izvo zvinogona kukonzerwa nezvikonzero zvakasiyana:
Nyaya imwe neimwe iyi yakakurukurwa zvakadzama mumagazini ekare eTroubleshooting LC, uye vaverengi vanofarira nyaya idzi vanogona kutarisa nyaya idzi dzekare kuti vawane ruzivo nezvezvikonzero zvikuru uye mhinduro dzinogona kuitika kumatambudziko aya.
Kureba kwemusoro, kumberi kwemusoro, uye kupatsanurana zvinogona kukonzerwa nezviitiko zvemakemikari kana zvemuviri, uye runyorwa rwemhinduro dzinogona kugadzirisika kumatambudziko aya runosiyana zvakanyanya, zvichienderana nekuti tiri kubata nedambudziko remakemikari kana remuviri. Kazhinji, nekuenzanisa misoro yakasiyana muchromatogram, unogona kuwana zviratidzo zvakakosha zvekuti chii chinokonzera. Kana misoro yese muchromatogram ichiratidza maumbirwo akafanana, chikonzero chingangove chisiri chemuviri. Kana misoro imwe chete kana mishomanana ikakanganiswa, asi yasara ichitaridzika zvakanaka, chikonzero chingangove chemakemikari.
Zvinokonzera makemikari zvinokonzera peak tailing zvakanyanya kuoma kutaura pano. Muverengi anofarira anotumirwa kumagazini yazvino ye "LC Troubleshooting" kuti akurukure zvakadzama (10). Zvisinei, chinhu chiri nyore kuedza ndechekuderedza huremu hweanalyte yakapinzwa uye kuona kana chimiro chepeak chiri nani. Kana zvakadaro, saka ichi chiratidzo chakanaka chekuti dambudziko nderekuti "huwandu hwakawanda". Muchiitiko ichi, nzira yacho inofanira kuganhurirwa pakupinza mass mass madiki eanalyte, kana kuti mamiriro echromatographic anofanira kuchinjwa kuitira kuti maumbirwo akanaka epeak agone kuwanikwa kunyangwe nemass makuru akapinzwa.
Kunewo zvikonzero zvakawanda zvinogona kukonzera kuti kukwirira kwemuswe kuonekwe. Vaverengi vanofarira kukurukura zvakadzama nezvezvingangoitika vanoendeswa kune imwe nyaya yazvino ye "LC Troubleshooting" (11). Chimwe chezvikonzero zvinowanzoonekwa zvekukwirira kwemuswe kuonekwe kwemuswe inyaya isina kunaka pakati peinjector ne detector (12). Muenzaniso wakanyanya unoratidzwa muMufananidzo 1d, wakawanikwa murabhoritari yangu mavhiki mashoma apfuura. Muchiitiko ichi, takavaka sisitimu ine injection valve itsva yatakanga tisina kushandisa kare, uye takaisa diki injection loop ine ferrule yakanga yakaumbwa pa stainless steel capillary. Mushure mekuedza kwekutanga kugadzirisa matambudziko, takaona kuti kudzika kweport mu injection valve stator kwakadzika zvakanyanya kupfuura zvatakajaira, zvichikonzera dead volume yakakura pasi peport. Dambudziko iri rinogadziriswa nyore nyore nekutsiva injection loop neimwe chubhu, tinogona kugadzirisa ferrule panzvimbo yakakodzera kubvisa dead volume pasi peport.
Mapeji ekumusoro akaita seaya anoratidzwa muMufananidzo 1e anogonawo kukonzerwa nematambudziko emuviri kana emakemikari. Chikonzero chinowanzoitika chemupendero wekumusoro ndechekuti mubhedha wezvikamu zvekoramu hauna kurongeka zvakanaka, kana kuti zvikamu zvakarongekazve nekufamba kwenguva. Sekunge nekukwira kwemusoro kunokonzerwa nechiitiko ichi chemuviri, nzira yakanakisa yekugadzirisa izvi ndeyekutsiva koramu woramba uchienda mberi. Chaizvoizvo, maumbirwo epamusoro emucheto wekumusoro ane mavambo emakemikari anowanzo bva mumamiriro ezvinhu atinodaidza kuti "asiri-linear". Pasi pemamiriro ezvinhu akanaka (akafanana), huwandu hweanalyte hunochengetwa nechikamu chekugara (saka, retention factor) hunoenderana nehukuru hweanalyte mukoramu. Chromatographically, izvi zvinoreva kuti sezvo huwandu hweanalyte hwakapinzwa mukoramu huchiwedzera, peak inova yakareba, asi kwete yakakura. Hukama uhwu hunoputsika kana maitiro ekuchengetedza asina kuenzana, uye peaks hadzingove dzakareba chete asiwo dzakafara sezvo huremu hwakawanda huchipinzwa. Pamusoro pezvo, maumbirwo asina kuenzana anosarudza chimiro che chromatographic peaks, zvichikonzera mipendero inotungamira kana inotevera. Sekunge nekuwanda kwehuwandu hunokonzera kukwirira kwemutsara (10), peak Kutungamira kunokonzerwa nekusachengetedzwa kwemasero kunogonawo kuongororwa nekuderedza huwandu hweanalyte inopinzwa. Kana chimiro chepamusoro chikavandudzika, nzira yacho inofanira kuchinjwa kuti isapfuure mhando yejekiseni inokonzera mucheto unotungamira, kana kuti mamiriro echromatographic anofanira kuchinjwa kuderedza maitiro aya.
Dzimwe nguva tinoona zvinoita se "split peak", sezvakaratidzwa muMufananidzo 1f. Danho rekutanga mukugadzirisa dambudziko iri nderekuona kana chimiro che peak chiri nekuda kwepartial co-elution (kureva, kuvapo kwemakemikari maviri akasiyana asi ari pedyo). Kana paine analytes maviri akasiyana ari pedyo, saka inyaya yekuvandudza resolution yavo (semuenzaniso, nekuwedzera selectivity, retention, kana plate count), uye "split" peaks dzinoonekwa dzakabatana ne physical Performance haina chekuita ne column pachayo. Kazhinji, chinhu chakakosha pakusarudza uku ndechekuti peaks dzese dziri mu chromatogram dzinoratidza ma fragment shapes, kana kuti imwe chete kana mbiri. Kana iri imwe chete kana mbiri, pamwe inyaya ye co-elution; kana peaks dzese dzakapatsanurwa, pamwe idambudziko re physical, pamwe rine chekuita ne column pachayo.
Matinji akapatsanurwa ane chekuita nehunhu hwekoramu pachayo anowanzo kukonzerwa nekuvharika kwezvikamu zvinopinda kana kubuda, kana kurongekazve kwezvikamu zviri mukoramu, zvichibvumira chikamu chinofamba kufamba nekukurumidza kupfuura chikamu chinofamba munzvimbo dzakati wandei dzekuumbwa kwenzira yekoramu .mune dzimwe nzvimbo (11). Zvikamu zvakavharika zvishoma dzimwe nguva zvinogona kubviswa nekudzosera kuyerera kuburikidza nekoramu; zvisinei, mune zvakaitika kwandiri, izvi zvinowanzova mhinduro yenguva pfupi pane yenguva refu. Izvi zvinowanzouraya nemakoramu emazuva ano kana zvikamu zvikabatana zvakare mukati mekoramu. Panguva ino, zvakanaka kutsiva koramu woenderera mberi.
Nhamba yepamusoro iri muMufananidzo 1g, zvakare kubva mune imwe nguva yadarika murabhoritari yangu, inowanzo ratidza kuti chiratidzo chakakwira zvekuti chasvika padanho repamusoro remhinduro. Kune ma optical absorbance detectors (UV-vis mune iyi kesi), kana huwandu hweanalyte hwakakwira kwazvo, analyte inotora chiedza chinopfuura nepakati pe detector flow cell, zvichisiya chiedza chidiki kuti chionekwe. Pasi pemamiriro ezvinhu aya, chiratidzo chemagetsi chinobva ku photodetector chinokanganiswa zvakanyanya nemarudzi akasiyana-siyana eruzha, akadai se stray light uye "dark current", zvichiita kuti chiratidzo chive "fuzzy" zvakanyanya muchimiro uye chisina huwandu hweanalyte. Kana izvi zvikaitika, dambudziko rinogona kugadziriswa zviri nyore nekuderedza huwandu hwe injection ye analyte - kuderedza huwandu hwe injection, kuderedza sample, kana zvese zviri zviviri.
Muchikoro chechromatography, tinoshandisa chiratidzo che detector (kureva, y-axis mu chromatogram) sechiratidzo chekuwanda kwe analyte mumuenzaniso. Saka zvinoita sezvisinganzwisisike kuona chromatogram ine chiratidzo chiri pasi pe zero, sezvo dudziro iri nyore ndeyekuti izvi zvinoratidza kuwanda kwe analyte - izvo zvisingagoneke panyama. Mune ruzivo rwangu, peaks dzisina kunaka dzinowanzoonekwa kana uchishandisa optical absorbance detectors (semuenzaniso, UV-vis).
Muchiitiko ichi, negative peak inongoreva kuti mamorekuru anobuda mukoramu anotora chiedza chidiki pane mobile phase pachayo nguva pfupi isati yasvika uye mushure me peak. Izvi zvinogona kuitika, semuenzaniso, kana uchishandisa ma wavelengths mashoma ekuona (<230 nm) uye mobile phase additives anotora chiedza chakawanda pama wavelengths aya. Zvimwe zvinhu zvakadaro zvinogona kuva mobile phase solvent components dzakadai se methanol kana buffer components dzakadai se acetate kana formate. Munhu anogona kushandisa negative peaks kugadzirira calibration curve uye kuwana ruzivo rwakarurama rwehuwandu, saka hapana chikonzero chikuru chekudzivisa izvi (nzira iyi dzimwe nguva inonzi "indirect UV detection") (13). Zvisinei, kana tichida chaizvo kudzivirira negative peaks zvachose, kana tichida kuona absorbance, mhinduro yakanakisa ndeyekushandisa imwe detection wavelength kuitira kuti analyte itore zvakawanda kupfuura mobile phase, kana kushandura chimiro che mobile phase kuitira kuti itore chiedza chidiki pane analytes.
Mapopoma asina kunaka anogonawo kuoneka kana uchishandisa refractive index (RI) detection apo refractive index yezvikamu zvisiri analyte mumuenzaniso, zvakaita se solvent matrix, yakasiyana ne refractive index ye mobile phase. Izvi zvinoitikawo ne UV-vis detection, asi izvi zvinowanzo derera kana tichienzanisa ne RI detection. Muzviitiko zvese izvi, mapopoma asina kunaka anogona kuderedzwa nekubatanidza zviri mukati me sample matrix neiyo ye mobile phase.
Muchikamu chechitatu pamusoro penyaya huru yekugadzirisa matambudziko eLC, ndakakurukura mamiriro ezvinhu umo chimiro chepeak chakaonekwa chinosiyana nechimiro chepeak chinotarisirwa kana chakajairika. Kugadzirisa matambudziko akadaro kunotanga neruzivo rwemaumbirwo epeak anotarisirwa (zvichibva pane dzidziso kana ruzivo rwekare nenzira dziripo), saka kusiyana kubva pane zvinotarisirwa izvi kuri pachena. Matambudziko epeak ane zvikonzero zvakawanda zvakasiyana (zvakafara, kuseri, kutungamira, nezvimwewo). Muchikamu chino, ndinokurukura zvakadzama zvimwe zvezvikonzero zvandinowanzoona. Kuziva ruzivo urwu kunopa nzvimbo yakanaka yekutanga kugadzirisa matambudziko, asi hakubati zvese zvinobvira. Vaverengi vanofarira runyorwa rwakadzama rwezvikonzero nemhinduro vanogona kutarisa chati yemadziro yeLCGC "LC Troubleshooting Guide".
(4) Chati yemadziro yeLCGC "LC Troubleshooting Guide". https://www.chromatographyonline.com/view/troubleshooting-wallchart (2021).
(6) A. Felinger, Kuongororwa kweData uye Kugadziriswa kweSignali muChromatography (Elsevier, New York, NY, 1998), mapeji 43-96.
(8) Wahab MF, Dasgupta PK, Kadjo AF naArmstrong DW, Anal.Chim.Journal.Rev. 907, 31–44 (2016).https://doi.org/10.1016/j.aca.2015.11.043.