Analysis of N-nitrosames in water using micellar electrokinetic chromatography (MEKC) and development of online and offline methods of extraction and enrichment of neutral polar analytes

Evaluation of the quality of the environment is essential for human wellness as pollutants in trace amounts can cause serious health problem. Nitrosamines are a group of compounds that are considered potential carcinogens and can be found in drinking water (as disinfection byproducts), foods, beverages and cosmetics. To monitor the level of these compounds to minimize daily intakes, fast and reliable analytical techniques are required. As these compounds are relatively highly polar, extraction and enrichment from environmental samples (aqueous) are challenging. Also, the trend of analytical techniques toward the reduction of sample size and minimization of organic solvent use demands new methods of analysis. In light of fulfilling these requirements, a new method of online preconcentration tailored to an electrokinetic chromatography is introduced. In this method, electroosmotic flow (EOF) was suppressed to increase the interaction time between analyte and micellar phase, therefore the only force to mobilize the neutral analytes is the interaction of analyte with moving micelles. In absence of EOF, polarity of applied potential was switched (negative or positive) to force (anionic or cationic) micelles to move toward the detector. To avoid the excessive band broadening due to longer analysis time caused by slow moving micelles, auxiliary pressure was introduced to boost the micelle movement toward the detector using an in house designed and built apparatus. Applying the external auxiliary pressure significantly reduced the analysis times without compromising separation efficiency. Parameters, such as type of surfactants, composition of background electrolyte (BGE), type of capillary, matrix effect, organic modifiers, etc., were evaluated in optimization of the method. The enrichment factors for targeted analytes were impressive, particularly; cationic surfactants were shown to be suitable for analysis of nitrosamines due to their ability to act as hydrogen bond donors. Ammonium perfluorooctanoate (APFO) also showed remarkable results in term of peak shapes and number of theoretical plates. It was shown that the separation results were best when a high conductivity sample was paired with a BGE of lower conductivity. Using higher surfactant concentrations (up to 200 mM SDS) than usual (50 mM SDS) for micellar electrokinetic chromatography (MEKC) improved the sweeping. A new method for micro-extraction and enrichment of highly polar neutral analytes (N-Nitrosamines in particular) based on three-phase drop micro-extraction was introduced and its performance studied. In this method, a new device using some easy-to-find components was fabricated and its operation and application demonstrated. Compared to conventional extraction methods (liquid-liquid extraction), consumption of organic solvents and operation times were significantly lower.

Extraction and purification of theobromine using ionic liquids

Theobromine is an alkaloid present in cocoa and it is used in the treatment of atherosclerosis, hypertension, angina, among others. Due to its importance, the aim of this work consists on the development of an efficient and sustainable technology for the extraction of theobromine from cocoa beans. For the development of a purification technique for theobromine extracted from cocoa, aqueous biphasic systems (ABS) composed of ionic liquids (ILs) were initially studied to infer on the most promising systems. Cholinium-based ILs, based on a non-toxic and biocompatible cation, were used combined with two polymers (PPG 400 and PEG 400) and an inorganic salt (K3PO4). The respective phase diagrams at 298 K and atmospheric pressure were determined, as well as their extraction efficiencies for theobromine. The results obtained indicate that K3PO4 has a greater ability to induce the formation of ABS compared to PEG 400 and PPG 400. ABS consisting of K3PO4 also have a high potential for the extraction of theobromine, with extraction efficiencies ranging between 96.4 and 99.9 %. Based on the most promising ILs for the purification step, they were further used in aqueous solution to extract theobromine from cocoa beans, with extraction yields ranging between 4.5% and 6.5 wt%. Finally, ABS were applied to the aqueous solutions containing theobromine from the cocoa extract, with extraction efficiencies ranging between 96.7 and 99.0%.

Extraction and purification of immunoglobulin G with aqueous biphasic systems

The immune system is able to produce antibodies, which have the capacity to recognize and to bind to foreign molecules or pathogenic organisms. Currently, there are a diversity of diseases that can be treated with antibodies, like immunoglobulins G (IgG). Thereby, the development of cost-efficient processes for their extraction and purification is an area of main interest in biotechnology. Aqueous biphasic systems (ABS) have been investigated for this purpose, once they allow the reduction of costs and the number of steps involved in the process, when compared with conventional methods. Nevertheless, typical ABS have not showed to be selective, resulting in low purification factors and yields. In this context, the addition of ionic liquids (ILs) as adjuvants can be a viable and potential alternative to tailor the selectivity of these systems. In this work, ABS composed of polyethylene glycol (PEG) of different molecular weight, and a biodegradable salt (potassium citrate) using ILs as adjuvants (5 wt%), were studied for the extraction and purification of IgG from a rabbit source. Initially, it was tested the extraction time, the effect on the molecular weight of PEG in a buffer solution of K3C6H5O7/C6H8O7 at pH≈7, and the effect of pH (59) on the yield (YIgG) and extraction efficiency (EEIgG%) of IgG. The best results regarding EEIgG% were achieved with a centrifugation step at 1000 rpm, during 10 min, in order to promote the separation of phases followed by 120 min of equilibrium. This procedure was then applied to the remaining experiments. The results obtained in the study of PEGs with different molecular weights, revealed a high affinity of IgG for the PEG-rich phase, and particularly for PEGs of lower molecular weight (EEIgG% of 96 % with PEG 400). On the other hand, the variation of pH in the buffer solution did not show a significant effect on the EEIgG%. Finally, it was evaluated the influence of the addition of different ILs (5% wt) on the IgG extraction in ABS composed of PEG 400 at pH≈7. In these studies, it was possible to obtain EEIgG% of 100% with the ILs composed of the anions [TOS]-, [CH3CO2]-and Cl-, although the obtained YIgG% were lower than 40%. On the other hand, the ILs composed of the anions Br-, as well as of the cation [C10mim]+, although not leading to EEIgG% of 100%, provide an increase in the YIgG%. ABS composed of PEG, a biodegradable organic salt and ILs as adjuvants, revealed to be an alternative and promising method to purify IgG. However, additional studies are still required in order to reduce the loss of IgG.

Extraction and detection of mycotoxins in medicinal and aromatic plants: a case study with Aloysia citrodora P.

Plants frequently suffer contaminations by toxigenic fungi, and their mycotoxins can be produced throughout growth, harvest, drying and storage periods. The objective of this work was to validate a method for detection of toxins in medicinal and aromatic plants, through a fast and highly sensitive method, optimizing the joint co-extraction of aflatoxins (AF: AFB1, AFB2, AFG1 and AFG2) and ochratoxin A (OTA) by using Aloysia citrodora P. (lemon verbena) as a case study. For optimization purposes, samples were spiked (n=3) with standard solutions of a mix of the four AFs and OTA at 10 ng/g for AFB1, AFG1 and OTA, and at 6 ng/g of AFB2 and AFG2. Several extraction procedures were tested: i) ultrasound-assisted extraction in sodium chloride and methanol/water (80:20, v/v) [(OTA+AFs)1]; ii) maceration in methanol/1% NaHCO3 (70:30, v/v) [(OTA+AFs)2]; iii) maceration in methanol/1% NaHCO3 (70:30, v/v) (OTA1); and iv) maceration in sodium chloride and methanol/water (80:20, v/v) (AF1). AF and OTA were purified using the mycotoxin-specific immunoaffinity columns AflaTest WB and OchraTest WB (VICAM), respectively. Separation was performed with a Merck Chromolith Performance C18 column (100 x 4.6 mm) by reverse-phase HPLC coupled to a fluorescence detector (FLD) and a photochemical derivatization system (for AF). The recoveries obtained from the spiked samples showed that the single-extraction methods (OTA1 and AF1) performed better than co-extraction methods. For in-house validation of the selected methods OTA1 and AF1, recovery and precision were determined (n=6). The recovery of OTA for method OTA1 was 81%, and intermediate precision (RSDint) was 1.1%. The recoveries of AFB1, AFB2, AFG1 and AFG2 ranged from 64% to 110% for method AF1, with RSDint lower than 5%. Methods OTA1 and AF1 showed precision and recoveries within the legislated values and were found to be suitable for the extraction of OTA and AF for the matrix under study.

Active Foreground Region Extraction and Tracking for Sports Video Annotation

Automatic video segmentation plays a vital role in sports videos annotation. This paper presents a fully automatic and computationally efficient algorithm for analysis of sports videos. Various methods of automatic shot boundary detection have been proposed to perform automatic video segmentation. These investigations mainly concentrate on detecting fades and dissolves for fast processing of the entire video scene without providing any additional feedback on object relativity within the shots. The goal of the proposed method is to identify regions that perform certain activities in a scene. The model uses some low-level feature video processing algorithms to extract the shot boundaries from a video scene and to identify dominant colours within these boundaries. An object classification method is used for clustering the seed distributions of the dominant colours to homogeneous regions. Using a simple tracking method a classification of these regions to active or static is performed. The efficiency of the proposed framework is demonstrated over a standard video benchmark with numerous types of sport events and the experimental results show that our algorithm can be used with high accuracy for automatic annotation of active regions for sport videos.

DNA extraction and RAPD markers to assess the genetic similarity among Gelidium sesquipedale (Rhodophyta) populations

A simple method developed for genomic DNA isolation from fungus was tested on the red alga, Gelidium sesquipedale (Clem.) Born et Thur., which is commercially exploited for its high sulfated polysaccharide (agar) content. This method is faster, cheaper, and less toxic than conventional phenol/chloroform methods. Random amplified polymorphic DNA (RAPD) amplifications were performed successfully without the necessity of purifying the DNA. RAPD markers were used to investigate the genetic similarity among three natural populations of G. sesquipedale from southern Portugal. Bulked-genomic DNA samples of 15 different individuals were made in each population. These can be conceived of as a sample of the population DNA. Of the 62 primers screened, 41 produced bands and 22 revealed polymorphisms. Genetic similarities among populations were high. Populations that are further away from each other have the lowest similarity coefficients, whereas the intermediate Ingrina population, located on the south coast, showed higher genetic similarity with the Odeceixe population located on the southwest coast, than with the Sao Rafael southern population. This suggests a higher genetic flow between Odeceixe and Ingrina or the result may be a founder effect in the sense that the species has propagated from the east coast to the south coast of Portugal. We conclude that the use of this isolation method with RAPD analysis is appropriate to characterize the genetic variability of this commercial species along its geographical distribution. Large sample sizes can be screened at a relatively low cost. Finding genetic markers for commercial populations of C. sesquipedale may be of industrial interest.

Microwave-assisted solvent extraction and analysis of shikimic acid from plant tissues.

A better method for determination of shikimate in plant tissues is needed to monitor exposure of plants to the herbicide glyphosate [N-(phosphonomethyl)glycine] and to screen the plant kingdom for high levels of this valuable phytochemical precursor to the pharmaceutical oseltamivir. A simple, rapid, and efficient method using microwave-assisted extraction (MWAE) with water as the extraction solvent was developed for the determination of shikimic acid in plant tissues. High performance liquid chromatography was used for the separation of shikimic acid, and chromatographic data were acquired using photodiode array detection. This MWAE technique was successful in recovering shikimic acid from a series of fortified plant tissues at more than 90% efficiency with an interference-free chromatogram. This allowed the use of lower amounts of reagents and organic solvents, reducing the use of toxic and/or hazardous chemicals, as compared to currently used methodologies. The method was used to determine the level of endogenous shikimic acid in several species of Brachiaria and sugarcane (Saccharum officinarum) and on B. decumbens and soybean (Glycine max) after treatment with glyphosate. The method was sensitive, rapid and reliable in all cases.

Extraction and characterization of Raphanus Sativus seed oil obtained by different methods

Purpose: To evaluate the impact of three different extraction methods on yield, physicochemical properties and bioactive ingredients of Raphanus sativus seed oil. Methods: Raphanus Sativus seed oil was prepared by traditional solvent extraction (SE), super-critical carbon dioxide extraction (SCE) and sub-critical propane extraction (SPE). The yield, physicochemical properties, fatty acid composition and oxidative stability of the oil extracts were compared. The contents of tocopherol and sulforaphene in the oils were also determined. Results: The oil yield obtained by SPE, SE and SCE were 33.69, 27.17 and 24.10 %, respectively. There were no significant differences in physicochemical properties and fatty acid compositions of oils extracted by the three methods. However, SCE oil had the best oxidative stability, and highest contents of vitamin E and sulforaphene, followed by oils from SPE and SE. Conclusion: SCE is highly selective for tocopherol and sulforaphene, which could explain its high oil oxidative stability. These results suggest that of the three extraction methods, SCE is best suited for preparing medicinal radish seed oil.

DNA extraction and RAPD markers to assess the genetic similarity among Gelidium sesquipedale (Rhodophyta) populations

A simple method developed for genomic DNA isolation from fungus was tested on the red alga, Gelidium sesquipedale (Clem.) Born et Thur., which is commercially exploited for its high sulfated polysaccharide (agar) content. This method is faster, cheaper, and less toxic than conventional phenol/chloroform methods. Random amplified polymorphic DNA (RAPD) amplifications were performed successfully without the necessity of purifying the DNA. RAPD markers were used to investigate the genetic similarity among three natural populations of G. sesquipedale from southern Portugal. Bulked-genomic DNA samples of 15 different individuals were made in each population. These can be conceived of as a sample of the population DNA. Of the 62 primers screened, 41 produced bands and 22 revealed polymorphisms. Genetic similarities among populations were high. Populations that are further away from each other have the lowest similarity coefficients, whereas the intermediate Ingrina population, located on the south coast, showed higher genetic similarity with the Odeceixe population located on the southwest coast, than with the Sao Rafael southern population. This suggests a higher genetic flow between Odeceixe and Ingrina or the result may be a founder effect in the sense that the species has propagated from the east coast to the south coast of Portugal. We conclude that the use of this isolation method with RAPD analysis is appropriate to characterize the genetic variability of this commercial species along its geographical distribution. Large sample sizes can be screened at a relatively low cost. Finding genetic markers for commercial populations of C. sesquipedale may be of industrial interest.

Technology, mining methods and landscapes of a placer mining district in Fairbanks, Alaska, 1900-1930

Placer miners in Alaska’s interior were part of the last great gold rush in North America. As word of gold in the Fairbanks Mining District traveled down the Yukon River, a wave of miners from the Klondike placer fields in Dawson, along with a assortment of speculators and inexperienced green horns from the Lower 48 converged on the confluence of the Tanana and Chena rivers hoping to strike it rich. The steamers coming from Dawson were integral; they carried miners with experience working the frozen subarctic placer deposits of the Klondike. These miners encountered new environmental challenges that required the development of new technologies and mining methods to efficiently harvest gold. These methods and machines were brought into Fairbanks and further perfected to account for the local conditions. This thesis describes the local mining technologies and methods employed in the Fairbanks district and the landscape patterns created during the placer mining boom years of 1903-1909, decline years of 1910-1923 and recovery of 1923-1930.

Jointly event extraction and visualization on Twitter via probabilistic modelling

Event extraction from texts aims to detect structured information such as what has happened, to whom, where and when. Event extraction and visualization are typically considered as two different tasks. In this paper, we propose a novel approach based on probabilistic modelling to jointly extract and visualize events from tweets where both tasks benefit from each other. We model each event as a joint distribution over named entities, a date, a location and event-related keywords. Moreover, both tweets and event instances are associated with coordinates in the visualization space. The manifold assumption that the intrinsic geometry of tweets is a low-rank, non-linear manifold within the high-dimensional space is incorporated into the learning framework using a regularization. Experimental results show that the proposed approach can effectively deal with both event extraction and visualization and performs remarkably better than both the state-of-the-art event extraction method and a pipeline approach for event extraction and visualization.

Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp

High quality, pure DNA is required for ensuring reliable and reproducible results in molecular diagnosis applications. A number of in-house and commercial methods are available for the extraction and purification of genomic DNA from faecal material, each one offering a specific combination of performance, cost-effectiveness, and easiness of use that should be conveniently evaluated in function of the pathogen of interest. In this comparative study the marketed kits QIAamp DNA stool mini (Qiagen), SpeedTools DNA extraction (Biotools), DNAExtract-VK (Vacunek), PowerFecal DNA isolation (MoBio), and Wizard magnetic DNA purification system (Promega Corporation) were assessed for their efficacy in obtaining DNA of the most relevant enteric protozoan parasites associated to gastrointestinal disease globally. A panel of 113 stool specimens of clinically confirmed patients with cryptosporidiosis (n = 29), giardiasis (n = 47) and amoebiasis by Entamoeba histolytica (n = 3) or E. dispar (n = 10) and apparently healthy subjects (n = 24) were used for this purpose. Stool samples were aliquoted in five sub-samples and individually processed by each extraction method evaluated. Purified DNA samples were subsequently tested in PCR-based assays routinely used in our laboratory. The five compared methods yielded amplifiable amounts of DNA of the pathogens tested, although performance differences were observed among them depending on the parasite and the infection burden. Methods combining chemical, enzymatic and/or mechanical lysis procedures at temperatures of at least 56 °C were proven more efficient for the release of DNA from Cryptosporidium oocysts.

Artisanal extraction and traditional knowledge associated with medicinal use of Crabwood Oil (Carapa guianensis Aublet.) in a peri-urban Várzea environment in the Amazon Estuary.

Varzea forests of the Amazon estuary contain species of importance to riverine communities. For example, the oil extracted from the seeds of crabwood trees is traditionally used to combat various illnesses and as such artisanal extraction processes have been maintained. The objectives of this study were to (1) describe the process involved in artisanal extraction of crabwood oil in the Fazendinha ProtectedArea, in the state ofAmap´a; (2) characterise the processes of knowledge transfer associated with the extraction and use of crabwood oil within a peri-urban riverine community; and (3) discern medicinal uses of the oil.The data were obtained using semistructured interviews with 13 community members involved in crabwood oil extraction and via direct observation.The process of oil extraction is divided into four stages: seed collection; cooking and resting of the seeds; shelling of the seeds and dough preparation; and oil collection. Oil extraction is carried out within the home for personal use, with surplus marketed within the community. More than 90% of the members of the community involved in extraction of crabwood oil highlighted the use of the oil to combat inflammation of the throat. Knowledge transfer occurs via oral transmission and through direct observation.

Calibration of the maximum carboxylation velocity (Vcmax) using data mining techniques and ecophysiological data from the Brazilian Semiarid region, for use in Dynamic Global Vegetation Models.

The semiarid region of northeastern Brazil, the Caatinga, is extremely important due to its biodiversity and endemism. Measurements of plant physiology are crucial to the calibration of Dynamic Global Vegetation Models (DGVMs) that are currently used to simulate the responses of vegetation in face of global changes. In a field work realized in an area of preserved Caatinga forest located in Petrolina, Pernambuco, measurements of carbon assimilation (in response to light and CO2) were performed on 11 individuals of Poincianella microphylla, a native species that is abundant in this region. These data were used to calibrate the maximum carboxylation velocity (Vcmax) used in the INLAND model. The calibration techniques used were Multiple Linear Regression (MLR), and data mining techniques as the Classification And Regression Tree (CART) and K-MEANS. The results were compared to the UNCALIBRATED model. It was found that simulated Gross Primary Productivity (GPP) reached 72% of observed GPP when using the calibrated Vcmax values, whereas the UNCALIBRATED approach accounted for 42% of observed GPP. Thus, this work shows the benefits of calibrating DGVMs using field ecophysiological measurements, especially in areas where field data is scarce or non-existent, such as in the Caatinga

Microwave-assisted solvent extraction and analysis of shikimic acid from plant tissues.

2008