Reduction and Analysis Methods of Indigo

Throughout history indigo was derived from various plants for example Dyer’s Woad (Isatis tinctoria L.) in Europe. In the 19th century were the synthetic dyes developed and nowadays indigo is mainly synthesized from by-products of fossil fuels. Indigo is a so-called vat dye, which means that it needs to be reduced to its water soluble leucoform before dyeing. Nowadays, most of the industrial reduction is performed chemically by sodium dithionite. However, this is considered environmentally unfavourable because of waste waters contaminating degradation products. Therefore there has been interest to find new possibilities to reduce indigo. Possible alternatives for the application of dithionite as the reducing agent are biologically induced reduction and electrochemical reduction. Glucose and other reducing sugars have recently been suggested as possible environmentally friendly alternatives as reducing agents for sulphur dyes and there have also been interest in using glucose to reduce indigo. In spite of the development of several types of processes, very little is known about the mechanism and kinetics associated with the reduction of indigo. This study aims at investigating the reduction and electrochemical analysis methods of indigo and give insight on the reduction mechanism of indigo. Anthraquinone as well as it’s derivative 1,8-dihydroxyanthraquinone were discovered to act as catalysts for the glucose induced reduction of indigo. Anthraquinone introduces a strong catalytic effect which is explained by invoking a molecular “wedge effect” during co-intercalation of Na+ and anthraquinone into the layered indigo crystal. The study includes also research on the extraction of plant-derived indigo from woad and the examination of the effect of this method to the yield and purity of indigo. The purity has been conventionally studied spectrophotometrically and a new hydrodynamic electrode system is introduced in this study. A vibrating probe is used in following electrochemically the leuco-indigo formation with glucose as a reducing agent.

Inversion and assimilation methods with applications in geophysical remote sensing

Stratospheric ozone can be measured accurately using a limb scatter remote sensing technique at the UV-visible spectral region of solar light. The advantages of this technique includes a good vertical resolution and a good daytime coverage of the measurements. In addition to ozone, UV-visible limb scatter measurements contain information about NO2, NO3, OClO, BrO and aerosols. There are currently several satellite instruments continuously scanning the atmosphere and measuring the UVvisible region of the spectrum, e.g., the Optical Spectrograph and Infrared Imager System (OSIRIS) launched on the Odin satellite in February 2001, and the Scanning Imaging Absorption SpectroMeter for Atmospheric CartograpHY (SCIAMACHY) launched on Envisat in March 2002. Envisat also carries the Global Ozone Monitoring by Occultation of Stars (GOMOS) instrument, which also measures limb-scattered sunlight under bright limb occultation conditions. These conditions occur during daytime occultation measurements. The global coverage of the satellite measurements is far better than any other ozone measurement technique, but still the measurements are sparse in the spatial domain. Measurements are also repeated relatively rarely over a certain area, and the composition of the Earth’s atmosphere changes dynamically. Assimilation methods are therefore needed in order to combine the information of the measurements with the atmospheric model. In recent years, the focus of assimilation algorithm research has turned towards filtering methods. The traditional Extended Kalman filter (EKF) method takes into account not only the uncertainty of the measurements, but also the uncertainty of the evolution model of the system. However, the computational cost of full blown EKF increases rapidly as the number of the model parameters increases. Therefore the EKF method cannot be applied directly to the stratospheric ozone assimilation problem. The work in this thesis is devoted to the development of inversion methods for satellite instruments and the development of assimilation methods used with atmospheric models.

Comparison of gas chromatographic and gravimetric methods for quantization of total fat and fatty acids in foodstuffs

Different methods to determine total fat (TF) and fatty acids (FA), including trans fatty acids (TFA), in diverse foodstuffs were evaluated, incorporating gravimetric methods and gas chromatography with flame ionization detector (GC/FID), in accordance with a modified AOAC 996.06 method. Concentrations of TF and FA obtained through these different procedures diverged (p< 0.05) and TFA concentrations varied beyond 20 % of the reference values. The modified AOAC 996.06 method satisfied both accuracy and precision, was fast and employed small amounts of low toxicity solvents. Therefore, the results showed that this methodology is viable to be adopted in Brazil for nutritional labeling purposes.

SIMULTANEOUS DETERMINATION OF PROTOCATECHUIC ALDEHYDE AND PROTOCATECHUIC ACID USING THE LOCALIZED SURFACE PLASMON RESONANCE PEAK OF SILVER NANOPARTICLES AND CHEMOMETRIC METHODS

A simple and sensitive spectrophotometric method is proposed for the simultaneous determination of protocatechuic acid and protocatechuic aldehyde. The method is based on the difference in the kinetic rates of the reactions of analytes with [Ag(NH3)2]+ in the presence of polyvinylpyrrolidone to produce silver nanoparticles. The data obtained were processed by chemometric methods using principal component analysis artificial neural network and partial least squares. Excellent linearity was obtained in the concentration ranges of 1.23-58.56 µg mL-1 and 0.08-30.39 µg mL-1 for PAC and PAH, respectively. The limits of detection for PAC and PAH were 0.039 and 0.025 µg mL-1, respectively.

A study of combustion phenomena in circulating fluidized beds by developing and applying experimental and modelling methods for laboratory-scale reactors

The results shown in this thesis are based on selected publications of the 2000s decade. The work was carried out in several national and EC funded public research projects and in close cooperation with industrial partners. The main objective of the thesis was to study and quantify the most important phenomena of circulating fluidized bed combustors by developing and applying proper experimental and modelling methods using laboratory scale equipments. An understanding of the phenomena plays an essential role in the development of combustion and emission performance, and the availability and controls of CFB boilers. Experimental procedures to study fuel combustion behaviour under CFB conditions are presented in the thesis. Steady state and dynamic measurements under well controlled conditions were carried out to produce the data needed for the development of high efficiency, utility scale CFB technology. The importance of combustion control and furnace dynamics is emphasized when CFB boilers are scaled up with a once through steam cycle. Qualitative information on fuel combustion characteristics was obtained directly by comparing flue gas oxygen responses during the impulse change experiments with fuel feed. A one-dimensional, time dependent model was developed to analyse the measurement data Emission formation was studied combined with fuel combustion behaviour. Correlations were developed for NO, N2O, CO and char loading, as a function of temperature and oxygen concentration in the bed area. An online method to characterize char loading under CFB conditions was developed and validated with the pilot scale CFB tests. Finally, a new method to control air and fuel feeds in CFB combustion was introduced. The method is based on models and an analysis of the fluctuation of the flue gas oxygen concentration. The effect of high oxygen concentrations on fuel combustion behaviour was also studied to evaluate the potential of CFB boilers to apply oxygenfiring technology to CCS. In future studies, it will be necessary to go through the whole scale up chain from laboratory phenomena devices through pilot scale test rigs to large scale, commercial boilers in order to validate the applicability and scalability of the, results. This thesis shows the chain between the laboratory scale phenomena test rig (bench scale) and the CFB process test rig (pilot). CFB technology has been scaled up successfully from an industrial scale to a utility scale during the last decade. The work shown in the thesis, for its part, has supported the development by producing new detailed information on combustion under CFB conditions.

Nutritional status of Eucalyptus grandis clones evaluated by critical level and DRIS methods

Nutritional status of eight 1.0 and 4.7 years old clones of Eucalyptus grandis, cultivated in a medium textured Ustults - US - and a Quartzipsamments - PS - soils, in Lençóis Paulista, São Paulo, were evaluated by the Diagnosis and Recommendation Integrated System (DRIS) and Critical Level (CL) methods. Based on multivariate discriminant analysis, the DRIS indices described the nutritional status of trees better in relation to tree age and soil type than in relation to nutrient composition. Spearman's correlation coefficients showed statistically significant relationships between volumetric tree growth and nutrients when applying DRIS indices or foliar nutrient concentrations. However, the DRIS indices indicated a lower number of trees with nutritional deficiencies, in relation to the CL method. According to the CL method, P, S, and Ca were deficient in the majority of the soils and tree age categories. By the DRIS method, Ca was the only deficient nutrient in PS soils, and appeared to be particularly limited in one-year-old trees. In conclusion, the DRIS method was more efficient than the CL method in evaluating the nutritional status of eucalyptus trees.

Soil penetration resistance analysis by multivariate and geostatistical methods

The penetration resistance (PR) is a soil attribute that allows identifies areas with restrictions due to compaction, which results in mechanical impedance for root growth and reduced crop yield. The aim of this study was to characterize the PR of an agricultural soil by geostatistical and multivariate analysis. Sampling was done randomly in 90 points up to 0.60 m depth. It was determined spatial distribution models of PR, and defined areas with mechanical impedance for roots growth. The PR showed a random distribution to 0.55 and 0.60 m depth. PR in other depths analyzed showed spatial dependence, with adjustments to exponential and spherical models. The cluster analysis that considered sampling points allowed establishing areas with compaction problem identified in the maps by kriging interpolation. The analysis with main components identified three soil layers, where the middle layer showed the highest values of PR.

Identification of new flagellin-encoding fliC genes in Escherichia coli isolated from domestic animals using RFLP-PCR and sequencing methods

Identification of Escherichia coli requires knowledge regarding the prevalent serotypes and virulence factors profiles allows the classification in pathogenic/non-pathogenic. However, some of these bacteria do not express flagellar antigen invitro. In this case the PCR-restriction fragment length polymorphism (RFLP-PCR) and sequencing of the fliC may be suitable for the identification of antigens by replacing the traditional serology. We studied 17 samples of E. coli isolated from animals and presenting antigen H nontypeable (HNT). The H antigens were characterized by PCR-RFLP and sequencing of fliC gene. Three new flagellin genes were identified, for which specific antisera were obtained. The PCR-RFLP was shown to be faster than the serotyping H antigen in E. coli, provided information on some characteristics of these antigens and indicated the presence of new genes fliC.

Leaching of Atrazine in Commercial and Xerogel Formulations in Oxisol Using Bioassay and Chromatographic Methods

Mobility of atrazine in soil has contributed to the detection of levels above the legal limit in surface water and groundwater in Europe and the United States. The use of new formulations can reduce or minimize the impacts caused by the intensive use of this herbicide in Brazil, mainly in regions with higher agricultural intensification. The objective of this study was to compare the leaching of a commercial formulation of atrazine (WG) with a controlled release formulation (xerogel) using bioassay and chromatographic methods of analysis. The experiment was a split plot randomized block design with four replications, in a (2 x 6) + 1 arrangement. The main formulations of atrazine (WG and xerogel) were allocated in the plots, and the herbicide concentrations (0, 3200, 3600, 4200, 5400 and 8000 g ha-1), in the subplots. Leaching was determined comparatively by using bioassays with oat and chromatographic analysis. The results showed a greater concentration of the herbicide in the topsoil (0-4 cm) in the treatment with the xerogel formulation in comparison with the commercial formulation, which contradicts the results obtained with bioassays, probably because the amount of herbicide available for uptake by plants in the xerogel formulation is less than that available in the WG formulation.

Phenotyping and genotyping methods applied to investigate the relatedness of Brazilian isolates of Enterobacter cloacae

In order to evaluate the resolving power of several typing methods to identify relatedness among Brazilian strains of Enterobacter cloacae, we selected twenty isolates from different patients on three wards of a University Hospital (Orthopedics, Nephrology, and Hematology). Traditional phenotyping methods applied to isolates included biotyping, antibiotic sensitivity, phage-typing, and O-serotyping. Plasmid profile analysis, ribotyping, and macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) were used as genotyping methods. Sero- and phage-typing were not useful since the majority of isolates could not be subtyped by these methods. Biotyping, antibiogram and plasmid profile permitted us to classify the samples into different groups depending on the method used, and consequently were not reliable. Ribotyping and PFGE were significantly correlated with the clinical epidemiological analysis. PFGE did not type strains containing nonspecific DNase. Ribotyping was the most discriminative method for typing Brazilian isolates of E. cloacae.

The estimation of tuberculosis transmission parameters using ABC and MCMC methods

The aim of this work is to apply approximate Bayesian computation in combination with Marcov chain Monte Carlo methods in order to estimate the parameters of tuberculosis transmission. The methods are applied to San Francisco data and the results are compared with the outcomes of previous works. Moreover, a methodological idea with the aim to reduce computational time is also described. Despite the fact that this approach is proved to work in an appropriate way, further analysis is needed to understand and test its behaviour in different cases. Some related suggestions to its further enhancement are described in the corresponding chapter.

Advanced analysis and design methods for preparative chromatographic separation processes

Preparative liquid chromatography is one of the most selective separation techniques in the fine chemical, pharmaceutical, and food industries. Several process concepts have been developed and applied for improving the performance of classical batch chromatography. The most powerful approaches include various single-column recycling schemes, counter-current and cross-current multi-column setups, and hybrid processes where chromatography is coupled with other unit operations such as crystallization, chemical reactor, and/or solvent removal unit. To fully utilize the potential of stand-alone and integrated chromatographic processes, efficient methods for selecting the best process alternative as well as optimal operating conditions are needed. In this thesis, a unified method is developed for analysis and design of the following singlecolumn fixed bed processes and corresponding cross-current schemes: (1) batch chromatography, (2) batch chromatography with an integrated solvent removal unit, (3) mixed-recycle steady state recycling chromatography (SSR), and (4) mixed-recycle steady state recycling chromatography with solvent removal from fresh feed, recycle fraction, or column feed (SSR–SR). The method is based on the equilibrium theory of chromatography with an assumption of negligible mass transfer resistance and axial dispersion. The design criteria are given in general, dimensionless form that is formally analogous to that applied widely in the so called triangle theory of counter-current multi-column chromatography. Analytical design equations are derived for binary systems that follow competitive Langmuir adsorption isotherm model. For this purpose, the existing analytic solution of the ideal model of chromatography for binary Langmuir mixtures is completed by deriving missing explicit equations for the height and location of the pure first component shock in the case of a small feed pulse. It is thus shown that the entire chromatographic cycle at the column outlet can be expressed in closed-form. The developed design method allows predicting the feasible range of operating parameters that lead to desired product purities. It can be applied for the calculation of first estimates of optimal operating conditions, the analysis of process robustness, and the early-stage evaluation of different process alternatives. The design method is utilized to analyse the possibility to enhance the performance of conventional SSR chromatography by integrating it with a solvent removal unit. It is shown that the amount of fresh feed processed during a chromatographic cycle and thus the productivity of SSR process can be improved by removing solvent. The maximum solvent removal capacity depends on the location of the solvent removal unit and the physical solvent removal constraints, such as solubility, viscosity, and/or osmotic pressure limits. Usually, the most flexible option is to remove solvent from the column feed. Applicability of the equilibrium design for real, non-ideal separation problems is evaluated by means of numerical simulations. Due to assumption of infinite column efficiency, the developed design method is most applicable for high performance systems where thermodynamic effects are predominant, while significant deviations are observed under highly non-ideal conditions. The findings based on the equilibrium theory are applied to develop a shortcut approach for the design of chromatographic separation processes under strongly non-ideal conditions with significant dispersive effects. The method is based on a simple procedure applied to a single conventional chromatogram. Applicability of the approach for the design of batch and counter-current simulated moving bed processes is evaluated with case studies. It is shown that the shortcut approach works the better the higher the column efficiency and the lower the purity constraints are.

Behavioral and physiological methods for early quantitative assessment of spinal cord injury and prognosis in rats

Methods for reliable evaluation of spinal cord (SC) injury in rats at short periods (2 and 24 h) after lesion were tested to characterize the mechanisms implicated in primary SC damage. We measured the physiological changes occurring after several procedures for producing SC injury, with particular emphasis on sensorimotor functions. Segmental and suprasegmental reflexes were tested in 39 male Wistar rats weighing 250-300 g divided into three control groups that were subjected to a) anesthesia, b) dissection of soft prevertebral tissue, and c) laminectomy of the vertebral segments between T10 and L1. In the lesion group the SC was completely transected, hemisected or subjected to vertebral compression. All animals were evaluated 2 and 24 h after the experimental procedure by the hind limb motility index, Bohlman motor score, open-field, hot-plate, tail flick, and paw compression tests. The locomotion scale proved to be less sensitive than the sensorimotor tests. A reduction in exploratory movements was detected in the animals 24 h after the procedures. The hot-plate was the most sensitive test for detecting sensorimotor deficiencies following light, moderate or severe SC injury. The most sensitive and simplest test of reflex function was the hot-plate. The hemisection model promoted reproducible moderate SC injury which allowed us to quantify the resulting behavior and analyze the evolution of the lesion and its consequences during the first 24 h after injury. We conclude that hemisection permitted the quantitation of behavioral responses for evaluation of the development of deficits after lesions. Hind limb evaluation scores and spontaneous exploration events provided a sensitive index of immediate injury effects after SC lesion at 2 and 24 h. Taken together, locomotion scales, open-field, and hot-plate tests represent reproducible, quantitatively sensitive methods for detecting functional deficiencies within short periods of time, indicating their potential for the study of cellular mechanisms of primary injury and repair after traumatic SC injury.

Comparison of anaerobic threshold determined by visual and mathematical methods in healthy women

Several methods are used to estimate anaerobic threshold (AT) during exercise. The aim of the present study was to compare AT obtained by a graphic visual method for the estimate of ventilatory and metabolic variables (gold standard), to a bi-segmental linear regression mathematical model of Hinkley's algorithm applied to heart rate (HR) and carbon dioxide output (VCO2) data. Thirteen young (24 ± 2.63 years old) and 16 postmenopausal (57 ± 4.79 years old) healthy and sedentary women were submitted to a continuous ergospirometric incremental test on an electromagnetic braking cycloergometer with 10 to 20 W/min increases until physical exhaustion. The ventilatory variables were recorded breath-to-breath and HR was obtained beat-to-beat over real time. Data were analyzed by the nonparametric Friedman test and Spearman correlation test with the level of significance set at 5%. Power output (W), HR (bpm), oxygen uptake (VO2; mL kg-1 min-1), VO2 (mL/min), VCO2 (mL/min), and minute ventilation (VE; L/min) data observed at the AT level were similar for both methods and groups studied (P > 0.05). The VO2 (mL kg-1 min-1) data showed significant correlation (P < 0.05) between the gold standard method and the mathematical model when applied to HR (r s = 0.75) and VCO2 (r s = 0.78) data for the subjects as a whole (N = 29). The proposed mathematical method for the detection of changes in response patterns of VCO2 and HR was adequate and promising for AT detection in young and middle-aged women, representing a semi-automatic, non-invasive and objective AT measurement.