1. TLS Detection in Liquid Chromatography, Flow Injection Analysis and Microfluidic Systems for Food Quality Control and Biomedical DiagnosticsMladen Franko, 2023, invited lecture at foreign university Abstract: The basics of thermal lens spectrometry (TLS) and TLS microscopy (TLM) will be explained considering in particular the dependence of the thermal lens signal on the excitation power, thermo-optical properties of the sample, as well as sample flow and photostability of the analyte, with particular focus on the TLS enhancement factor, sensitivity and sample throughput of TLS and TLM measurements.
Examples of applications will include most notable achievements of TLS and TLM measurements in liquid chromatography (detection of bilirubin and biliverdin in blood plasma, bilirubin in vascular endothelial cells), flow injection analysis (organophosphate insecticides and allergens in foodstuffs) and microfluidics (high throughput determination of Cr(VI), microcystin, HPV virus antibodies, and biomarkers of acute kidney injury).
Before the concluding remarks, recent progress in multi-pass mode-mismatched TLS instrumentation and applications of incoherent light sources will be presented s part of the future outlook for TLS and TLM techniques. Keywords: Spektrometrija TLS, TLM, chromatography, microfluidics, FIA Published in RUNG: 14.11.2023; Views: 1857; Downloads: 8 Full text (21,62 MB) |
2. Macroporous epoxy-based monoliths functionalized with anti-CD63 nanobodies for effective isolation of extracellular vesicles in urineJulia Neumair, Claudia D'Ercole, Matteo De March, Martin Elsner, Michael Seidel, Ario De Marco, 2023, original scientific article Keywords: extracellular vesicles, monolith chromatography, nanobodies, functionalitazion strategies Published in RUNG: 27.03.2023; Views: 2273; Downloads: 14 Full text (2,20 MB) |
3. Novel analytical approaches in quality and safety control in production of fermented beverages : dissertationJelena Topić, 2022, doctoral dissertation Abstract: The exploitation of microorganisms for fermentation goes back centuries. Two types of fermentation are usually used in the winemaking process – alcoholic fermentation and malolactic fermentation. Nowadays, inoculated fermentations with the use of starter cultures are commonly used in order to produce wine with more consistent quality. However, wines can lack in flavour complexity, so scientists and the industry are constantly looking for new and improved starters that can be adapted to different types of wine.
In this work we focused on the development and implementation of novel analytical methods for wine quality control. In the course of method development native yeasts and lactic acid bacteria isolates were characterized for wine starter properties. We focused on the determination of biologically active compounds that determine wine quality and safety. Yeasts can influence wine colour through their adsorption capacity and synthesis of stable colour pigments pyranoanthocyanins and lactic acid bacteria can produce biogenic amines which can have adverse detrimental health effects on sensitive consumers when they are present in wines. Keywords: Saccharomyces yeasts, non-Saccharomyces yeasts, pyranoanthocyanins, thermal-lens spectrometry, lactic acid bacteria, biogenic amines, thin layer chromatography, dissertations Published in RUNG: 18.02.2022; Views: 3864; Downloads: 157 Link to full text This document has many files! More... |
4. Implementation of high performance liquid chromatography coupled to thermal lens spectrometry (HPLC‑TLS) for quantification of pyranoanthocyanins during fermentation of Pinot Noir grapesJelena Topič Božič, Lorena Butinar, Natka Ćurko, Karin Kovačević Ganić, Branka Mozetič Vodopivec, Dorota Korte, Mladen Franko, 2020, original scientific article Abstract: In this work high performance liquid chromatography coupled to thermal lens spectrometry (HPLC-TLS) was applied for monitoring of vinylphenolic pyranoanthocyanins formation during the fermentation of Pinot Noir wines. Vinylphenolic pyranoanthocyanins are wine pigments, present in low concentrations, but very important for wine colour stability.
Fermentation process was conducted with four different yeast strains, used as starters, either in sequential fermentation of non-Saccharomyces with S. cerevisiae yeast or as single fermentation with S. cerevisiae yeasts in order to test the applicability of developed method for monitoring of selected compounds in real wine fermentation experiments. The
developed HPLC-TLS method showed higher sensitivity compared to HPLC coupled to diode array detection (DAD) technique for particular wine colour compounds. Obtained limits of detection (LODs), were 6- and 22-times lower in comparison to HPLC–DAD in gradient and isocratic elution mode, respectively, whereas limits of quantification (LOQs)
5 and 18-times lower. Lower LODs enabled earlier observation of vinylphenolic pyranoanthocyanins formation during fermentation (already at day 7) in the case of HPLC-TLS method in gradient mode, while by using HPLC–DAD in gradient elution mode the formation of vinylphenolic pyranoanthocyanins was noticed only after 12 days of fermentation. Keywords: Thermal lens spectrometry (TLS), High performance liquid chromatography (HPLC), Pyranoanthocyanins, Wine, Yeasts Published in RUNG: 18.06.2020; Views: 4288; Downloads: 0 This document has many files! More... |
5. DETEKCIJA KOVINSKIH KOMPLEKSOV IN ORGANOKOVINSKIH SPOJIN V VZORCIH IZ OKOLJA S SPEKTROMETRIJO TERMIČNIH LEČLeja Goljat, 2019, doctoral dissertation 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 Published in RUNG: 05.09.2019; Views: 5672; Downloads: 167 Full text (3,65 MB) |
6. Capturing and Storing Exhaled Breath for Offline AnalysisIain R. White, Stephen J. Fowler, 2019, independent scientific component part or a chapter in a monograph Abstract: In this chapter we will summarize and discuss methods for the capture and storage of exhaled breath, prior to offline (and indirect online) analysis. We will detail and compare methods currently in use, including their applications, key strengths, and limitations. In synthesizing the best features of each technique, we will propose an ideal standardized breath sampling solution, and give a personal vision on the next steps to be taken in this exciting area of breath research. Keywords: Breath analysis, Breath sampling, Offline analysis, Thermal desorption, Gas chromatography-mass spectrometry Published in RUNG: 22.07.2019; Views: 4430; Downloads: 0 This document has many files! More... |
7. TD/GC–MS analysis of volatile markers emitted from mono- and co-cultures of Enterobacter cloacae and Pseudomonas aeruginosa in artificial sputumIain R. White, Oluwasola Lawal, Hugo Knobel, Weda Hans, Tamara M E Nijsen, Royston Goodacre, Stephen J. Fowler, Waqar M Ahmed, Antonio Artigas, Jonathan Barnard-Smith, Lieuwe D Bos, Marta Camprubi, Luis Coelho, Paul Dark, Alan Davie, Emili Diaz, Gemma Goma, Timothy Felton, Jan H Leopold, Pouline M P van Oort, Pedro Póvoa, Craig Portsmouth, 2018, original scientific article Abstract: Introduction: Infections such as ventilator-associated pneumonia (VAP) can be caused by one or more pathogens. Current methods for identifying these pathogenic microbes often require invasive sampling, and can be time consuming, due to the requirement for prolonged cultural enrichment along with selective and differential plating steps. This results in delays in diagnosis which in such critically ill patients can have potentially life-threatening consequences. Therefore, a non-invasive and timely diagnostic method is required. Detection of microbial volatile organic compounds (VOCs) in exhaled breath is proposed as an alternative method for identifying these pathogens and may distinguish between mono- and poly-microbial infections. Objectives: To investigate volatile metabolites that discriminate between bacterial mono- and co-cultures. Methods: VAP-associated pathogens Enterobacter cloacae and Pseudomonas aeruginosa were cultured individually and together in artificial sputum medium for 24 h and their headspace was analysed for potential discriminatory VOCs by thermal desorption gas chromatography–mass spectrometry. Results: Of the 70 VOCs putatively identified, 23 were found to significantly increase during bacterial culture (i.e. likely to be released during metabolism) and 13 decreased (i.e. likely consumed during metabolism). The other VOCs showed no transformation (similar concentrations observed as in the medium). Bacteria-specific VOCs including 2-methyl-1-propanol, 2-phenylethanol, and 3-methyl-1-butanol were observed in the headspace of axenic cultures of E. cloacae, and methyl 2-ethylhexanoate in the headspace of P. aeruginosa cultures which is novel to this investigation. Previously reported VOCs 1-undecene and pyrrole were also detected. The metabolites 2-methylbutyl acetate and methyl 2-methylbutyrate, which are reported to exhibit antimicrobial activity, were elevated in co-culture only. Conclusion: The observed VOCs were able to differentiate axenic and co-cultures. Validation of these markers in exhaled breath specimens could prove useful for timely pathogen identification and infection type diagnosis. Keywords: Bacteria, Enterobacter cloacae, Gas Chromatography-Mass Spectrometry, Infection, Pseudomonas aeruginosa, Volatile organic compounds Published in RUNG: 18.07.2019; Views: 5383; Downloads: 118 Full text (1,29 MB) |
8. Year-long measurements of C1-C3 halocarbons at an urban site and their relationship with meteorological parametersM Anwar K Khan, M Iqbal Mead, Iain R. White, Ben Golledge, Graham Nickless, Alan Knights, Damien Martin, Alison C Rivett, Brian G R Greally, Dudley E Shallcross, 2009, original scientific article Abstract: The mixing ratios of 11 C1–C3 halocarbons have been measured using a GC–twin‐linked ECD system over the period from October 2004 to December 2005 at an urban site in Bristol, UK. Time series and seasonal variations of the halocarbons were analysed over the period to determine biogenic and anthropogenic sources and sinks. Correlations between the target halocarbons were also observed, suggesting common sources within the area. Wind rose plots for all halocarbons have been used to assist in the determination of halocarbons sources. Halocarbon concentrations are highest at low‐wind speeds and decrease as wind speed increases, a few species (CCl4 and CH3Cl most notably) rise at very high‐wind speeds suggesting release from the Bristol Channel. Keywords: adsorption–desorption system, anthropogenic halocarbons, biogenic halocarbons, electron capture detector, gas chromatography, methyl bromide Published in RUNG: 18.07.2019; Views: 3729; Downloads: 0 This document has many files! More... |
9. Stable carbon isotope analysis of selected halocarbons at parts per trillion concentration in an urban locationM Iqbal Mead, M Anwar H Khan, Ian D Bull, Iain R. White, Graham Nickless, Dudley E Shallcross, 2008, original scientific article Abstract: ∂13C values of a suite of halocarbons have been determined in an urban background site in Bristol, UK. A novel mobile preconcentration system, based on the use of multi-adsorbent sample tubes, has been developed for trapping relatively large-volume air samples in potentially remote areas. An Adsorption Desorption System-Gas Chromatography-Electron Capture Detector was used to measure the mixing ratios of the selected halocarbon species, while a Gas ChromatographyCombustionIsotope Ratio Mass Spectrometer was used to determine ∂13C values. For the species with strong local sources, the variation of isotope ratios has been observed over the experimental period. Some of the results reported in the present study differ from previously reported values and reasons for this are discussed. The reporting of different ∂13C values for selected halocarbons from different areas in the present study suggests that ∂13C values may be used to determine the relative magnitudes of anthropogenic and biogenic sources. Keywords: Adsorption Desorption System (ADS), Automated Thermal Desorber (ATD), Electron Capture Detector (ECD), Gas Chromatography (GC), Isotope Ratio Mass Spectrometry (IRMS) Published in RUNG: 18.07.2019; Views: 4076; Downloads: 0 This document has many files! More... |
10. The detection and study of biologically active compounds in environmental processes and samplesMojca Žorž Furlan, doctoral dissertation Abstract: Environmental pollution in the 21th century still represents a global problem for human and animal health. Despite general awareness about released substances and their degradation products their fate and possibilities of removal are not well investigated. Even though the chemicals released are dispersed and diluted in water cycles, their poor biodegradability and/or strong accumulation can result in the intoxication of exposed organisms. Similarly, as a part of the environment, food can get contaminated by bioactive substances during different steps of preparation. Not only artificial compounds such as pesticides or pharmaceuticals, but also natural toxins enter the food chain and impact negatively on humans' and animals' health. In addition, the activity of some bacteria can influence the production of amines from amino acids after fermentation, to which the human body responds with several symptoms of intoxication.
Several analytical methods for the determination of trace levels of broad range contaminants have been developed. Due to the largely robust, selective and sensitive features of the conventional (rearguard) techniques, they represent the first choice for analysing multiple organic compounds in frequently very complex matrices. However, screening (vanguard) methods are paving the way in the chemical analytics as a solution that provides simplicity and rapid analytical responses with binary (yes/no) answers. They require little or no sample treatment as well as more economically-efficient instrumentation. The combination of vanguard-rearguard analytical strategies hence offers a compromise between classical analytical figures of merit and productivity-related characteristics.
In the first part of our research feasibility studies for the application of TLS and/or TLM in novel analytical methods for the determination of lipid-lowering drug atorvastatin and a mycotoxin ochratoxin A . The survey on atorvastatin performed spectrophotometrically has shown a decrease of ATV-sulpho-vanillin product at the wavelength of its maximum absorbance after dilution by organic solvent, which was investigated due to the possible increasing of the method sensitivity. As the predicted LODs that could be obtained by TLM (0.3 mg/L) could not reach the concentration of ATV usually present in the environment (ng/L-g/L) further experiments on this subject were therefore not justified. On the other hand, the ELISA assay for the determination of ochratoxin A was performed. In case of μFIA-TLM, the measurements were influenced by high background signal resulting in high LODs of TLM (470 pg/mL), which is known as a background limited technique. It was estimated that the LODs of standard ELISA assay could not be significantly improved, therefore no further research was conducted in this direction.
In the second part of the dissertation, a sensitive rearguard system by coupling HPLC and TLS for the determination of biogenic amines in wine samples was developed. Putrescine, cadaverine, histamine and tyramine were separated and detected on a HPLC-TLS system after derivatization by dabsyl chloride. The method was optimized in terms of chromatographic conditions and in terms of TLS parameters. Also, the sensitivity of the newly developed method was evaluated by comparing the TLS detection with DAD detection in terms of LOD values, where TLS showed 3.6-fold improvement compared to DAD. Afterwards, the standard addition calibration was performed and evaluated for its recoveries (86−117%) in the determination of the four BAs. The applicability of the novel method was tested by the analysis of real white and red wine samples and by comparing the results to the standard HPLC-FL method and concentrations of BAs in wine samples were in good accordance. In addition, the dabsylated BAs showed better stability compared to the OPA derivatives as they have not lost the peak intensity after 17h of storage.
In the third part, a vanguard system for detection of the overall biogenic amines concentration was developed by employing μFIA-TLM. Initially, NH4Cl standard solutions were applied in the indophenol reaction for batch mode, off-line μFIA-TLM and in an on-line indophenol formation for μFIA-TLM detection. By adding 50 % of EtOH to indophenol we obtained 9-fold improvement. In addition, indophenol showed good stability under TLM conditions. We optimized the microfluidic and TLM parameters in the off-line and on-line indophenol reaction. The addition of 5% ethanol to the reagent in the on-line reaction resulted in the 3-fold improvement of the signal-to-noise ratio. Further on, the overall reaction, including the enzymatic and the following indophenol reaction, was optimized by choosing the optimal buffer (pH=7, 0.5 M) and alkaline conditions (2M NaOH). The influence of interferences from amino compounds was also evaluated and discussed. The off-line and on-line μFIA-TLM were evaluated by their performance characteristics. The LOD for ammonia detection reached 2.3 μM and the applicability in ammonia detection in water samples was discussed. Similar LOD of 3.2 μM was obtained for the overall concentration of BAs and LOD of 3.8 μM for histamine, which is more than 4-folds lower value as the lowest suggested limits of intake for histamine in wine samples (2 mg/L; 18 μM). Finally, an immobilization procedure on magnetic nanoparticles was developed for the possible implementation of the selected enzyme in a miniaturized biosensor. Keywords: thermal lens spectrometry, thermal lens microscopy, high performance liquid chromatography, microfluidics, biogenic amines, microbial transglutaminase, indophenol (Berthelot) reaction Published in RUNG: 04.06.2018; Views: 6956; Downloads: 251 Full text (2,90 MB) This document has many files! More... |