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Authors:ID Goljat, Leja (Author)
ID Franko, Mladen (Mentor) More about this mentor... New window
Files:.pdf Dissertation_Leja_Goljat_final.pdf (3,65 MB)
MD5: 544DBC6E1A684C55A0870897F2C6EB51
Work type:Doctoral dissertation
Typology:2.08 - Doctoral Dissertation
Organization:FPŠ - Graduate School
Abstract:Environmental pollution is one of the greatest challenges that the world is facing today. Toxic compounds, such as pesticides, allergens, pharmaceuticals, toxins and heavy metals are widely present in the air, water and soil, and can affect the health of people and animals even in small quantities, as well as they may cause long- or short-term damage in plants [Hill, 1997]. Heavy metals (mercury, arsenic, cadmium…) are widely spread in the environment. They derive from a number of sources, including mining, industrial wastes and vehicle emissions [Tchounwou et al., 2012]. They are easily incorporated into biological molecules and exert their toxic effects by displacing essential metals of a lower binding power in biologically active molecules or by acting as noncompetitive inhibitors of enzymes, affecting neurological, reproductive, renal and hematological systems [Sunil D’Souza et al., 2003; Heavy-Metal Pollution, 2018]. Metals form countless compounds (e.g. metal complexes and organometallic compounds) which are essential for living organisms (vitamin B12, hemoglobin, chlorophyll) and/or have a wide range of applications in industry and other areas, including analytical chemistry. Because of the potential risk which toxic metals represent to the living organisms and also because of the importance of some essential metals, different analytical techniques and detection methods have been developed for studies of their occurrence, fate and concentration in the environment and in organisms. However, providing a required sensitivity for determination and speciation of different metals and their compounds, especially in small- volume samples is still a challenge. Therefore, general objectives of this dissertation were development of novel analytical methods for sensitive, reliable and fast determination of metal species, based on highly sensitive optothermal technique thermal lens spectrometry (TLS), which can be used as detection tool following colorimetric reaction of a selected metal ion or for direct detection of colored organometallic compounds. This dissertation is composed of the following chapters: introduction, research goals, theoretical background, results and discussion, conclusion and references. The core of this dissertation is presented in the fifth chapter (results and discussion), which is divided into three parts. They separately cover development of methods for determination of iron redox species, pyoverdine and Fe-pyoverdine complexes and mercury. Pyoverdine is a siderophore, excreted by a certain bacteria in order to scavenge iron in the environment and is closely related to the chemistry of iron in such biological systems. Therefore, the first two parts are closely related. Procedures for batch mode thermal lens microscopy (TLM), flow-injection thermal lens sprectrometry (FIA-TLS) and µFIA-TLM (flow injection and TLS detection in microspace) were developed for Fe(II) and Fe(III) determination, based on colorimetric reaction of Fe(II) with 1,10-Phenanthroline. All these procedures were focused on cloudwater examination with a tendency to minimize sample consumption but at the same time preserve low limits of detection (LOD) and limits of quantification (LOQ). TLM measurements with highly collimated probe beam were performed in a 100 μm optical path length cell (40 µL volume), which resulted in a considerably smaller sample volume requirement (500 µL in total) and consumption, as compared to UV-Vis spectrophotometry, which required at least 25 mL of sample due to large volume (almost 30 mL) of the 10 cm optical path-length sample cell. LODs for mode-mismatched TLM were 0.16 and 0.14 µM for Fe(II) and Fe(total) (sum of Fe(II) and Fe(III) concentrations), respectively, while LODs for UV-Vis spectrophotometry were 0.01 µM for both Fe(II) and Fe(total). By using the mode mismatched TLM we were able to detect concentrations corresponding to absorbances as low as 1.5 × 10-5, while the lowest absorbance detectable on the UV-Vis spectrophotometer corresponded to 1.1 × 10-3, despite the use of the 10 cm optical path-length cell. Another important step in the development of new methods for Fe(II) and Fe(III) determination was the use of TLS detection in FIA (FIA-TLS). By injecting 50 µL of the sample into the FIA-TLS system, cca. 10 times lower LODs were achieved (1 × 10-3 µM for Fe(II) and 8 × 10-4 µM for Fe(total)), as compared to the UV- Vis spectrophotometry. Nevertheless, the development of μFIA-TLM method, with on-line colorimetric reaction for Fe(II) and Fe(III) determination is considered as the most important achievement of this study. The results show that despite 100 times shorter optical path length and low sample consumption (3 µL of each sample/injection) compared to UV-Vis spectrophotometry, LODs for µFIA-TLM were 0.10 and 0.07 μM for Fe(II) and Fe(total) respectively, which is sufficiently for cloudwater analysis, since concentrations, lower than 0.1 μM are not expected [Parazols et al., 2006; Deguillaume et al., 2014]. Linear range for Fe(II) and Fe(III) determination by μFIA-TLM was between 0.1 and 70 µM. To test the accuracy of this method, artificial cloudwater was prepared, spiked with different amounts of Fe(II) and Fe(III) and analyzed for iron content by µFIA-TLM and UV-Vis spectrophotometry. Good agreement was observed between the two methods. To ascertain the ruggedness of the method 7 (or more) replicate determinations at two different concentrations for both, Fe(II) and Fe(total) in artificial cloudwater were carried out on day 1 (replicates were measured instantly after fortification), day 2 and day 5. A student’s t-test (p=0.05) was applied to compare 3 sets of obtained data (day 1, day 2 and day 5) and showed that sets are not significantly different from each other. Considering very low sample volume requirement of µFIA-TLM, this should be the method of choice for determination of Fe(II) and Fe(III) in investigations of processes in cloudwater, where multiparameter analysis is desired (determination of other ions, ligands, microbial counts, etc.). When larger sample volumes are available, FIA-TLS can be used for accurate determination of iron species at lowest concentration levels. High performance liquid chromatography (HPLC) was applied for separation and detection of pyoverdine (PVD), produced by Pseudomonas fluorescens 36b5, a bacterial strain isolated from the aqueous phase of clouds at the Puy de Dôme station (1465 m, France). Reversed-phase (RP) chromatography (RP-18 chromatographic column Hypersil gold), hydrophilic interaction liquid chromatography (HILIC) (ZIC®-Hilic column) and three different detection systems (diode-array (DAD), spectrofluorimetry (FLD) and TLS) were tested for their performance in separation and determination of pyoverdines and corresponding complexes of pyoverdine with iron (Fe(III)-PVDs). PVDs and Fe(III)-PVD complexes could not be separated and quantified by applying HILIC technique, therefore it was concluded, that HILIC is not suitable for HPLC-DAD and also not for HPLC-TLS, since the method should offer a simultaneous sensitive detection of free PVDs as well as Fe(III)-PVD complexes in a single chromatographic run. Since pyoverdine standards were available only as a mixture of several different forms of PVDs, whereby the exact composition was unknown, the quantification of each of the four major specie (two fluorescent PVDs and two nonfluorescent Fe(III)-PVDs) in the standard, which was obtained from Université Clermont Auvergne, Institut de Chimie de Clermont-Ferrand, was performed. When applying Hypersil gold column, a linear correlation between fluorescence intensity and absorbance of each component was observed in a concentration range 3–24 µg/mL, whereby LODs were estimated to be 0.03–0.04 µg/mL for each of the major PVD species (HPLC-DAD). Even though HPLC-FLD method provided cca. 100 times lower LODs, it is not the method of choice for determination of PVD species in cloudwater, because it does not allow detection of PVD complexes with Fe(III). When comparing HPLC-TLS and HPLC-DAD, LODs were 5 to 8 times lower in case of HPLC-TLS, which was a significant improvement. Furthermore, recoveries (89–111 %) at two concentration levels of four PVD species in two independent samples, showed good reliability of the method. Almost all mercury in uncontaminated drinking-water is thought to be in the form of Hg2+ [WHO, 2010]. Therefore, the method for Hg2+determination based on colorimetric reaction with triamterene, described originally by Al-Kady and Abdelmonem was further investigated in this study, as well as the possibilities of application of this reaction for Hg2+ determination by TLS. The stoichiometry of the complex formation was determined by the method of continuous variations and saturation experiment, suggesting formation of the complex with the formula Hg2-triamterene. The obtained value of the molar absorption coefficient was 9988 Lmol-1cm-1 at 403 nm, which significantly contradicts the existing data in literature, which reports the molar absorption coefficient of 5.32 × 104 Lmol-1cm-1 [Al-Kady and Abdelmonem, 2013]. Even though the spectrophotometric results were not encouraging for triamterene as colorimetric reagent for Hg2+ determination, it was further investigated for its performance in TLS system. Fe(II)-1,10-phenanthroline (ferroin) was used for comparison, because it was well studied for TLS applications previously. The results showed that Hg2-triamterene in solutions was degraded when it was exposed to the light of the excitation beam. Due to the lower molar absorptivity than reported in literature, fotodegradation and unfavorable complex stoichiometry, triamterene was not confirmed as a suitable colorimetric reagent for highly sensitive Hg2+ determination by TLS. In summary, this dissertation investigates alternative approaches for analysis of metal complexes and organometallic compounds in small-volume environmental water samples. Methods, which were developed in this research, could potentially serve as improvements of existing technologies, to facilitate analysis of such samples, by offering simple handling of samples and superior sensitivity over the UV-Vis spectrophotometry.
Keywords:thermal lens spectrometry, thermal lens microscopy, high performance liquid chromatography, microfluidics, metal complexes, organometallic compounds, iron, pyoverdine, mercury
Place of publishing:Nova Gorica
Year of publishing:2019
PID:20.500.12556/RUNG-4706-244b28cf-b4f8-5aba-7575-c62cf7dcbf5f New window
COBISS.SI-ID:5447931 New window
Publication date in RUNG:05.09.2019
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Secondary language

Abstract:Onesnaževanje okolja je eden izmed največjih izzivov, s katerimi se dandanes soočamo. Strupene spojine, kot so pesticidi, alergeni, farmacevtski izdelki, toksini in težke kovine, so lahko prisotne v zraku, vodi in tleh ter že v majhnih količinah vplivajo na zdravje ljudi in živali, prav tako pa lahko povzročijo bolezni in poškodbe rastlin [Hill, 1997]. Težke kovine (živo srebro, arzen, kadmij itd.) so prisotne v zemeljski skorji, vodi in atmosferi. Poleg naravnih virov so glavni vir onesnaženja s kovinami rudarstvo, industrija in emisije iz prevoznih sredstev [Tchounwou et al., 2012]. Nekatere kovine so v majhnih količinah esencialne za rast organizmov (kobalt – vitamin B12, železo – hemoglobin, mangan – klorofil), v prevelikih količinah pa so lahko tudi zelo toksične. Nekatere težke kovine se lahko vgradijo v biološke molekule, pri čemer nadomestijo esencialne kovine ali delujejo kot inhibitorji encimov ter pri tem vplivajo na nevrološke, reproduktivne, ledvične in hematološke sisteme [Sunil D'Souza et al., 2003; Heavy-Metal Pollution, 2018]. Kovine tvorijo številne spojine, npr. koordinacijske spojine in organokovinske spojine. Zaradi potencialnega tveganja, ki ga živim organizmom predstavljajo toksične kovine, ter zaradi pomena nekaterih esencialnih kovin za žive organizme so bile v preteklosti razvite številne analizne in detekcijske metode za spremljanje njihove prisotnosti, njihovih transformacij in koncentracije v okolju in v organizmih. Kljub temu je zagotavljanje potrebne občutljivosti za določanje različnih kovin in njihovih spojin v okoljskih vzorcih še vedno izziv, še posebej, ko imamo opravka z majhnimi količinami vzorcev. Cilj te disertacije je bil zato razvoj novih analitskih metod, temelječih na občutljivi spektroskopiji toplotnih leč (TLS), za občutljivo, zanesljivo in hitro določanje kovinskih zvrsti. TLS se lahko uporablja kot detekcijsko orodje po kolorimetrični reakciji izbranega kovinskega iona ali za neposredno detekcijo barvnih organokovinskih spojin. Po začetnem uvodu so predstavljeni cilji, teoretični del in eksperimentalni del. Sledi poglavje »Rezultati in razprava«. Slednje je razdeljeno na tri dele, v katerih je opisan razvoj novih metod za detekcijo železa, pioverdina in živega srebra. Pioverdin je siderofor, ki ga izločajo nekatere bakterije, da bi pridobile železo iz okolja, in je tesno povezan s kemijo železa v takšnih bioloških sistemih. Poglavji o pioverdinu in železu sta zato tesno povezani. Sledita še zaključek in literatura. V sklopu te disertacije smo razvili nove metode za določanje Fe(II) in Fe(III), ki temeljijo na detekciji oranžno-rdeče obarvanega produkta reakcije Fe(II) z 1,10-fenantrolinom, ki ima absorpcijski maksimum pri 508 nm (λmax = 508 nm). Za detekcijo tega produkta smo uporabili spektrometrijo toplotnih leč (TLS), povezano s pretočno injekcijsko analizo (FIA-TLS), njeno miniaturno verzijo – mikroskopijo toplotnih leč (TLM) ter TLM, povezan s pretočno injekcijsko analizo v mikrofluidnih sistemih (μFIA-TLM). Pri razvoju teh metod smo se osredotočili na analizo vode iz oblakov, s težnjo, da bi zmanjšali količino vzorcev za analizo, a hkrati ohranili nizke meje detekcije (LOD) in kvantifikacije (LOQ). TLM-meritve smo izvedli z uporabo TLM-mikroskopa v celici z optično dolžino 100 μm in prostornino 40 µl po kolorimetrični reakciji Fe(II) oz. Fe(III) z 1,10-fenantrolinom. S to metodo smo občutno zmanjšali potrebno količino vzorca (v celoti 500 µl) v primerjavi z UV-Vis-spektrofotometrijo, kjer je bila potrebna količina vzorca najmanj 25 ml zaradi velike prostornine merilne celice z optično potjo 10 cm. Vrednosti LOD pri TLM so znašale 0,16 µM za Fe(II) in 0,14 µM za Fe(total), pri čemer predstavlja Fe(total) vsoto koncentracij Fe(II) in Fe(III). Vrednosti LOD pri UV-Vis-spektrofotometriji so bile za Fe(II) in Fe(total) 0,01 µM. Nadalje smo razvili metodo FIA-TLS za detekcijo Fe(II) in Fe(III). Pri tej metodi smo po kolorimetrični reakciji železa z 1,10-fenantrolinom v mobilno fazo injicirali Fe(II) ali Fe(total) in detektirali kompleks s TLS v celici z optično dolžino 10 mm. Z injiciranjem 50 µl vzorca smo dosegli približno 10-krat nižji LOD (1 × 10-3 µM za Fe(II) in 8 × 10-4 µM za Fe(total)) v primerjavi z UV-Vis-spektrofotometrijo. Najpomembnejši korak pri razvoju metod za detekcijo železa je bil razvoj µFIA-TLM, z on-line reakcijo. Pri tej metodi smo injicirali 3 µl vzorca v mobilno fazo reagenta v kanalu mikročipa, kjer je potekla reakcija. Nato smo produkt vodili v drug mikročip in ga detektirali s sistemom TLM. Kljub 100-krat krajši optični poti (v primerjavi z UV-Vis-spektrofotometrijo) smo dosegli meje detekcije 0,10 µM za Fe(II) in 0,07 µM za Fe(total), kar je zadovoljivo za analizo vode iz oblakov, saj bi naj bile koncentracije Fe(II) in Fe(III) višje od 0,1 μM [Parazols et al., 2006; Deguillaume et al., 2014]. Da bi validirali metodo, smo pripravili umetno vodo iz oblakov, v katero smo dodali znane količine Fe(II) in Fe(III) ter te vzorce analizirali z µFIA-TLM in UV-Vis-spektrofotometrijo. Vrednosti koncentracij so se dobro ujemale. Linearno območje TLM-signala je bilo med 0,1 in 70 µM. Da bi ugotovili robustnost metode, smo opravili sedem (ali več) ponovitev določanja koncentracij na dveh koncentracijskih nivojih Fe(II) in Fe(total) v umetni vodi iz oblakov, in sicer smo opravili meritve v določenih časovnih zaporedjih: na dan 1, dan 2 in dan 5. Studentov t-test (p = 0,05) smo uporabili na treh nizih pridobljenih meritev (dan 1, dan 2 in dan 5), pri čemer smo pokazali, da se ti bistveno ne razlikujejo med seboj. Metoda se odlikuje v nizki porabi vzorca, kar je ključno v primeru analize vode iz oblakov (zaradi težkega vzorčenja in majhnih količin vzorcev). V primerih, ko so na voljo večje količine vzorcev, lahko uporabimo FIA-TLS-metodo, ki je najbolj natančna za določevanje zvrsti železa na najnižjih koncentracijskih nivojih. Ker je železo znano po svoji reaktivnosti in lahko z lahkoto prehaja iz Fe(II) v Fe(III) in obratno (npr. v prisotnosti kisika), smo testirali tudi stabilnost železa pri različnih pogojih (sobna temperatura, hladilnik in zamrzovalnik), pri čemer smo ugotovili, da je najbolj stabilno v hladilniku. Drugi del raziskav smo namenili razvoju novih metod za ločitev in detekcijo fluorescentnega pioverdina in njegovega kompleksa s Fe(III), ki ne fluorescira. V ta namen smo testirali dve kromatografski tehniki, reverzno fazno (RP), pri kateri smo uporabili RP-18-kromatografsko kolono Hypersil gold in kromatografijo hidrofilne interakcije, pri kateri smo uporabili kolono ZIC®-Hilic. Uporabo slednje smo opustili, saj ni omogočila ločitve čistega pioverdina in kompleksov pioverdina z železom. Preizkusili smo tudi tri različne detekcijske sisteme: diode-array (DAD), spektrofluorimetrijo (FLD) in TLS. Pioverdin (PVD) je bil v času izvajanja raziskav na trgu na voljo le v obliki mešanice različnih pioverdinov, katerih vsebnost ni bila natančno določena. Zaradi tega smo v sklopu disertacije najprej določili vsebnost štirih glavnih komponent v standardu, ki smo ga pridobili s kemijskega inštituta Institut de Chimie de Clermont-Ferrand, Université Clermont Auvergne, tako da smo standardu dodajali znane količine Fe(III) in na podlagi razmerja tvorbe kompleksa Fe(III)-pioverdina določili vsebnost dveh čistih pioverdinov in dveh povezanih kompleksov s Fe(III). Pri uporabi kolone Hypersil gold smo opazili linearno korelacijo med intenziteto fluorescence in absorbance v koncentracijskem območju 3–24 µg/ml, pri čemer smo ocenili LOD-vrednosti posameznih komponent zvrsti v vzorcu med 0,03–0,04 µg/ml (HPLC-DAD). Detekcija PVD s FLD je omogočila približno 100-krat nižji LOD, ni pa omogočila detekcije nefluorescentnega kompleksa Fe(III)-PVD, ki bi se prav tako lahko nahajal v oblakih. Z implementacijo TLS (HPLC-TLS) smo uspeli LOD znižati za od 5- do 8-krat (v primerjavi s HPLC-DAD). Z uporabo HPLC-DAD in HPLC-TLS smo ločili in detektirali dve vrsti pioverdina in njuna kompleksa z železom. Zadnji del disertacije je posvečen določanju Hg2+ v vodi, saj ta predstavlja dominantno obliko živega srebra v pitnih vodah [WHO, 2010]. Nadalje smo raziskali metodo za kolorimetrično določanje Hg2+ po tvorbi kompleksa z ligandom triamterenom [Al-Kady in Abdelmonem, 2013], ki se sicer uporablja v medicini kot diuretik, in možnosti uporabe te metode za detekcijo s TLS. Določili smo razmerje tvorbe kompleksa po Jobovi metodi in metodi nasičenja, ki kažeta na tvorbo kompleksa Hg2+ : triamteren = 2 : 1 in molarni absorpcijski koeficient 9988 Lmol-1cm-1 kompleksa Hg2-triamterena. Ocenili smo konstanto stabilnosti (3 ± 1) × 109. Raziskava je pokazala, da je kompleks Hg2-triamteren fotorazgradljiv. To smo dokazali z eksperimentom, pri katerem smo izpostavili raztopine kompleksa laserskemu žarku (λ = 445 nm, P = 268 mW) za 8 min. Pri tem smo merili absorbanco raztopin pred izpostavitvijo žarku in po njej. Povprečno zmanjšanje absorbance zaradi fotodegradacije je bilo 29 ± 13 %, kar je povzročilo tudi nižjo občutljivost pri meritvah TLS. Zaradi relativno nizkega molarnega absorpcijskega koeficienta, fotodegradacije in tudi razmerja tvorbe kompleksa triamteren ni bil potrjen kot ustrezen kolorimetrični reagent za določanje Hg2+. V sklopu te disertacije smo raziskovali alternativne pristope za analizo kovinskih kompleksov in organokovinskih komponent v volumsko majhnih okoljskih vzorcih. Metode (TLS, HPLC-TLS, FIA-TLS in μFIA-TLS), ki smo jih razvili, bi lahko potencialno služile kot izboljšave že obstoječih metod in bi lahko olajšale analize okoljskih vzorcev, saj so preproste za uporabo in ponujajo visoko občutljivost v primerjavi s konvencionalno UV-Vis-spektrofotometrijo.
Keywords:spektrometrija s toplotnimi lečami, mikroskopija s toplotnimi lečami, tekočinska kromatografija visoke ločljivosti, mikrofluidika, kovinski kompleksi, organokovinske spojine, železo, pioverdin, živo srebro