BrJAC-2018-V5-N19 by BrJAC

Water Chemical Analyses

April - June 2018

Volume 5

Number 19

BrMASS - 8 to 12 December Windsor Hotel Barra da Tijuca 7th IBERO-AMERICAN CONFERENCE ON MASS SPECTROMETRY Five days of Conferences. More than 20 Plenary Talks. 50 Invited Guests. Your chance to strengthen your networks and deepen your understanding of Mass Spectrometry - all of this in one of Brazil´s most beautiful landscapes.

SPEAKERS MASS 2018 will feature over 48 speakers from all over the world, exposing their newest and brightest ideas on Mass Spectometry.

PROGRAM A CONFERENCE YOU WON´T SOON FORGET Five days of events, full with meetings & greetings, poster presentations, oral presentations, public performances and much more.

LATEST NEWS KEEP UP TO DATE WITH MASS SPECTROMETRY Get ready for BrMASS 2018! Here you will ﬁnd the latest news about the topics and researches featured in our Congress

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About Br. J. Anal. Chem. The Brazilian Journal of Analytical Chemistry (BrJAC) is a peer-reviewed scientiﬁc journal intended for professionals and institutions acting mainly in all branches of analytical chemistry. BrJAC is an open access journal which does not charge authors an article processing fee. Scope BrJAC is dedicated to professionals involved in science, technology and innovation projects in the area of analytical chemistry at universities, research centers and in industry. BrJAC publishes original, unpublished scientiﬁc articles and technical notes that are peer reviewed in the double-blind way. In addition, it publishes reviews, interviews, points of view, letters, sponsor reports, and features related to analytical chemistry. Manuscripts submitted for publication in BrJAC, either from universities, research centers, industry or any other public or private institution, cannot have been previously published or be currently submitted for publication in another journal. For manuscript preparation and submission, please see the Guidelines for the Authors section at the end of this edition. When submitting their manuscript for publication, the authors agree that the copyright will become the property of the Brazilian Journal of Analytical Chemistry, if and when accepted for publication. Published by Visão Fokka Communication Agency Publisher Lilian Freitas MTB: 0076693/ SP lilian.freitas@visaofokka.com.br Advertisement Luciene Campos luciene.campos@visaofokka.com.br ISSN 2179-3425 printed www.brjac.com.br

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Editorial Board Editor-in-Chief Lauro Tatsuo Kubota Full Professor / Institute of Chemistry - University of Campinas - Campinas, SP, BR Associate Editors Cristina Maria Schuch Analytical Department Manager / Solvay Research & Innovation Center - Paris, FR Elcio Cruz de Oliveira Technical Consultant / Technol. Mngmt. - Petrobras Transporte S.A. and Aggregate Professor / Post-graduate Program in Metrology - Pontiﬁcal Catholic University, Rio de Janeiro, RJ, BR Fernando Vitorino da Silva Chemistry Laboratory Manager - Nestle Quality Assurance Center - São Paulo, SP, BR Marco Aurélio Zezzi Arruda Full Professor / Institute of Chemistry - University of Campinas - Campinas, SP, BR Pedro Vitoriano Oliveira Full Professor / Institute of Chemistry - University of São Paulo - São Paulo, SP, BR Renato Zanella Full Professor / Dept. of Chemistry - Federal University of Santa Maria - RS, BR Advisory Board Adriano Otávio Maldaner Criminal Expert / Forensic Chemistry Service - National Institute of Criminalistics - Brazilian Federal Police – Brasília, DF, BR Auro Atsushi Tanaka Full Professor / Dept. of Chemistry - Federal University of Maranhão, São Luís, MA, BR Carlos Roberto dos Santos President of CETESB - Environmental Company of São Paulo State, São Paulo, SP, BR Gisela de Aragão Umbuzeiro Professor / Technology School - University of Campinas - Campinas, SP, BR Isabel Cristina Sales Fontes Jardim Full Professor / Institute of Chemistry, University of Campinas, Campinas, SP, BR Janusz Pawliszyn Professor / Department of Chemistry - University of Waterloo, Ontario, Canada Joaquim de Araújo Nóbrega Full Professor / Dept. of Chemistry - Federal University of São Carlos - São Carlos, SP, BR José Anchieta Gomes Neto Associate Professor / São Paulo State University (UNESP), Institute of Chemistry, Araraquara, SP, BR José Dos Santos Malta Junior Pre-formulation Lab. Manager / EMS / NC Group – Hortolandia, SP, BR Luiz Rogerio M. Silva Quality Assurance Associate Director / EISAI Lab. – São Paulo, SP, BR Márcio das Virgens Rebouças Process & Technology Manager – GranBio Research Center - Campinas, SP, BR Marcos Nogueira Eberlin Full Professor / Institute of Chemistry - University of Campinas - Campinas, SP, BR Maria das Graças Andrade Korn Full Professor / Institute of Chemistry - Federal University of Bahia - Salvador, BA, BR Ricardo Erthal Santelli Full Professor / Institute of Chemistry - Federal University of Rio de Janeiro, RJ, BR

Br. J. Anal. Chem., 2018, 5 (19)

Contents Editorial Water Chemical Analysis Interview Professor Mário César Ugulino de Araújo, who is an active member of the scientiﬁc community, recently spoke with BrJAC

1-2 3-6

Points of View The latest advances in chemical analysis of waters at CETESB

7-7

Water Chemistry: A fundamental knowledge base for aquatic ecosystem management

8-9

Letters The importance of advances in analytical chemistry for the quality control of water and chemicals used in the water treatment process

10-10

In Memoriam Atomic Spectrometry loses one of its fathers: Professor Bernhard Welz

11-11

Articles Feasibility of using Total Reﬂection X-ray Fluorescence Spectrometry for Drinking Water Analysis

12-21

Evaluation of nitrate as internal standard for quantitative determination of urea in urine by Raman spectroscopy

22-28

Prediction of glucose, fructose and sucrose content in cassava (Manihot esculenta Crantz) genotypes from Amazon using PLS models

29-37

Exploratory on-line pyrolysis and thermally assisted hydrolysis and methylation for evaluating non-hydrolyzable organic matter in anthropogenic soil from Central Brazilian Amazon

38-53

Features The 41st Annual Meeting of the Brazilian Chemical Society was marked by discussions on the future of scientiﬁc research in chemistry

54-60

Sponsor Reports Determination of mercury in water by direct mercury analysis: A study to comply with Brazilian water legislation

61-64

Fully automated, intelligent, high-throughput elemental analysis of drinking waters using SQ-ICP-MS

65-69

Fast anion determinations in environmental waters using a high-pressure compact ion chromatography system

70-81

Automated extraction of acrylamide from underground water prior to HPLC-DAD analysis

82-85

Releases CHROMacademy helps increase your knowledge, eﬃciency and productivity in the lab

87-87

Join Pittcon Conference & Expo 2019

89-90

SelectScience® – The Fastest Way to Expert Opinion

92-92

DMA-80 - The most successful Hg analyzer in the market

94-94

Thermo Scientiﬁc Dionex Integrion HPIC System - Advancing Ion Chromatography with High Pressure

96-96

Thermo Scientiﬁc iCAP RQ ICP-MS - Simplicity, productivity and robustness for routine labs

98-98

GX-27X Large-Volume SPE Systems - Versatile SPE workﬂow with ﬂexibility for small or large sample volumes

100-100

Notices of Books

101-101

Periodicals & Websites

102-102

Events

103-103

Author's Guidelines

104-107

Br. J. Anal. Chem., 2018, 5 (19), 1-2 DOI: 10.30744/brjac.2179-3425.2018.5.19.1-2

Editorial

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Water Chemical Analysis

Water Chemical Analysis Renato Zanella Full Professor Laboratory for Pesticide Residue Analysis (LARP) Chromatography and Mass Spectrometry Research Group (CPCEM) Chemistry Department, Federal University of Santa Maria, Santa Maria RS, Brazil renato.zanella@ufsm.br

Considering the current and recent use of a large number of compounds, it is of great importance to have appropriate methods for determining the levels of water pollution and, from the results, to assess the risks of exposure and to examine measures to mitigate the effects on the environment and life. The demands of analysis of environmental samples are very broad in terms of environmental matrices, with emphasis on different types of water, effluents, soil, sediments, air, particulate matter, animals and plants, as well as in relation to the analytes of interest; among them, persistent organic pollutants, endocrine disruptors, pesticides, pharmaceuticals and personal care products (PPCPs), veterinary drugs, flame retardants, metals and organometallic compounds. The proper qualification of analysts to work in this area is of fundamental importance in order to generate reliable results that allow a greater control of the occurrence of pollution. Several parameters of water chemical analysis are well established; however, there is a limited availability of personnel with broad knowledge of sample preparation techniques and wide-scope analyses, with the ability to establish and apply appropriate methods, as in the case of emerging pollutants, and to participate in decision-making in this area. The search for analyses in accredited laboratories is a growing trend and, therefore, the training of analysts in laboratories with implanted quality systems is of fundamental importance. The determination of chemical elements in water is well established, including the speciation of the most important cases from the point of view of environmental and toxicological control. The major challenges are in the analysis of samples such as seawater and ultrapure water, as a function of interferences and low concentrations, respectively. However, the determination of organic compounds, due to the wide range of analytes of interest and their stability, has required a great effort in the establishment of adequate analytical methods. The assessment of the extent of environmental impacts due to the presence of pollutants is generally hampered by the lack of reliable results, as well as of knowledge of the situation prior to the occurrence of the problem. In addition, it is important to highlight that food is produced in the environment and, in general, requires a large amount of water, the pollution of which can cause problems in the final product. Therefore, the risks to human health are much greater than is generally perceived. It is imperative to make continuous investment in the qualification of laboratories in terms of infrastructure and training of analysts in the area. The search for an intensification of cooperation between research groups, as well as with government agencies and the private sector, will allow progress in the area of monitoring and control of water quality, since new pollutants, such as microplastics, are continually being identified. The instrumentation available today has allowed faster and more comprehensive analyses, with detection limits much lower than those obtained in previous decades. Chromatography coupled to mass spectrometry has enabled multi-residue and multi-class analyses of organic compounds with great 1

Editorial efficiency, even in complex samples. The use of detectors with high resolution allows the analysis of target and non-target compounds with high reliability for a large number of analytes, allowing screening analyses that are of great importance, especially in situations when pollutants are unknown, as well for retrospective evaluations of the data. The availability of reference materials for most analytes of interest, as well as a wide variety of isotopically-labeled compounds, provides for greater reliability on the results obtained. Proficiency tests, essential to increase the reliability of the analyses, are becoming more accessible and offer a wider range of analytical parameters, although for organic compounds problems of the stability of most analytes in environmental matrices persist. The determination of metabolites is also important, especially in cases where these are stable and relevant from the toxicological point of view; however, few studies have evaluated these compounds in water samples, due to the analytical difficulties and the limited availability of standards. It is important to highlight that the methods must comply with the limits established by legislation, providing that those limits can be changed. It is also important to analyze properly at levels below the maximum limits allowed, to minimize the risks of possible problems in water, which is essential for terrestrial life.

Br. J. Anal. Chem., 2018, 5 (19), pp 3-6 DOI: 10.30744/brjac.2179-3425.2018.5.19.3-6

Interview

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Professor Mário Ugulino, who is an active member of the scientific community, recently spoke with BrJAC Mário César Ugulino de Araújo Full Professor at the Department of Chemistry, Center of Exact and Nature Sciences, Federal University of Paraíba, PB, BR laqa@quimica.ufpb.br Since 1988, Prof. Mário Ugulino, as he is best known, has been the coordinator of the Laboratory of Automation and Instrumentation in Analytical Chemistry and Chemometrics (LAQA) of the Department of Chemistry, Center of Exact and Nature Sciences (CCEN), at the Federal University of Paraíba, Campus I, João Pessoa, Paraíba, Brazil. The LAQA is a consolidated laboratory in the northeast region of Brazil, specializing in the areas of analytical instrumentation, automation of analytical processes, and chemometrics. Prof. Mário is currently a research productivity fellow of the National Council for Scientific and Technological Development (CNPq) at level 1A. He holds a degree in industrial chemistry from the Federal University of Paraíba (UFPB) and a PhD in chemistry from the University of Campinas (Unicamp), São Paulo, Brazil. The title of his PhD thesis was “Development of automated systems of standard additions and elimination of interferences in spectrophotometric analyses of rocks, minerals, and alloys by applying the generalized standard addition method”. His advisor was Prof. Dr. Roy Edward Bruns, one of the pioneers in the development of computational quantum chemistry and chemometrics in Brazil. Mário Ugulino, Elias Ayres Guidetti Zagatto, and Ieda Spacino Scarminio were the first students taught in Brazil by Prof. Bruns in Chemometrics. Mário Ugulino's doctorate involved the development of automated flow injection analysis (FIA) systems and relied on the collaboration with good friends Boaventura Freire dos Reis, Francisco José Krug, Elias Zagatto, and Henrique Bergamin Filho, from the Center for Nuclear Energy in Agriculture (CENA) at the University of São Paulo, and Celio Pasquini from Unicamp. The chemometrics he used in data processing, especially the generalized standard addition method (GSAM), he developed with the invaluable help of his advisor and friends at Unicamp and CENA. He was also reliant on the expressive scientific cooperation in chemometrics from Prof. Dr. Roberto Kawakami Harrop Galvão, based in the Electronic Engineering Division of the Technological Institute of Aeronautics (ITA), São José dos Campos, São Paulo, Brazil. Since February 1982, Mário Ugulino has been Professor of the Department of Chemistry at the CCEN of the UFPB. He could have retired in 2008 but has no intention of retiring any time soon. He has already supervised 50 master's dissertations, 40 PhD theses, and more than 100 scientific initiation students. In addition, he has worked with 22 postdoctoral researchers and PhD students from other Brazilian and foreign research laboratories. He also acts as an ad-hoc advisor to the main research funding agencies in Brazil and national and international scientific journals in the area of analytical chemistry. Furthermore, he had more than 300 papers presented at national and international scientific events and over 190 published articles; an H index/number of citations of approximately 33/4036 on the Web of Science, 33/4409 on Scopus, 33/4365 on Research Gate, 33/4387 on Mendeley and 41/6013 on Google Scholar. This score is greater than 97.5% of Portal Research Gate members. 3

Interview Mário Ugulino is an active member of the scientific community, having worked as president of the organizing committee of the 14th Brazilian Meeting of Analytical Chemistry, held from October 7 to 11, 2007, in João Pessoa, Paraíba, Brazil; a member of the CNPq Advisory Committee for chemistry in 2008 and from July 2013 to June 2016; a member of the Coordination of Superior Level Staff Improvement (CAPES) Advisory Committee for the evaluation/conceptualization of graduate programs in chemistry from 1999 to 2001 and from 2010 to 2012; a member of the Management Committee of the National Institute of Advanced Analytical Sciences and Technologies (INCTAA/CNPq) since 2009; a coordinator of the postgraduate program in chemistry at the UFPB from 1989 to 1991, from 1997 to 2002, and from 2010 to 2012; and as a coordinator of the CNPq registered research group named “Group of Instrumentation and Automation in Analytical Chemistry / Chemometrics of Paraíba” since 1988. He is currently the coordinator of several research projects and scientific and technological collaborative projects at national and international level, funded by the main Brazilian research funding agencies: CAPES, CNPq, and the Funding Authority for Studies and Projects (FINEP). He works with his students to develop instrumentation and automatic methods of analysis for the determination of different analytes and/or the detection of contaminants in water, beverages, food, medicines, and fuels. When were you first introduced to chemistry? Did you have an influencer, such as a teacher? My first contact with chemistry was during practical classes in the chemical, physical, and biological sciences during the 7th grade at the elementary state school Liceu Paraibano. I owe my great passion for chemistry to what I learned from Professor of General Chemistry, Dr. Josué Eugênio Viana, Professor of Physical Chemistry, Dr. Natarajan Subramanian, and to several other professors of the chemistry undergraduate course of the UFPB. When did you decide to focus on chemistry? What motivated you in this decision? How were the early stages of your career? At the end of high school, I did not have a definite vocation, because I was equally interested in chemistry, physics, and mathematics. I chose by chance to take the entrance exam for the Bachelor's degree in Industrial Chemistry, and in the first semester I fell in love with Chemistry. For this reason, I decided that chemistry teaching/researching would be my profession. In the second semester, I became a teaching assistant in the discipline of general chemistry, and following my graduation I became a statutory teaching assistant in other chemistry disciplines, assisting the teachers in the preparation and follow-up of practical classes, and helping the students with their school work. What are your lines of research? What work are you currently developing? You have published many scientific papers. Are there any to highlight? There are many jobs for which I have passion, I cannot highlight one. I have been working for several years, alongside my students, in the following lines of research: ·Instrumentation and automatic systems for chemical analysis: this involves the development of instruments, methodologies, and automatic systems for flow analysis, such as spectrometers, potentiostats /galvanostats, photometers, luminometers, and FIA and flow-batch systems. ·Chemometric methods in analytical chemistry: this involves the development of algorithms and chemometric methods for: (a) first- and higher-order multivariate classification and calibration; (b) pretreatment of analytical data (filtering, smoothing, compression, baseline correction, and selection of variables and samples, etc); and (c) transfer of multivariate models employing NIR, UV-Vis and molecular fluorescence spectrometry, voltammetry, liquid chromatography and gas chromatography, etc. ·Analytical methods based on digital images: this involves the development of systems for univariate and multivariate analysis using digital images obtained from scanners, digital cameras (webcam), and cell phones. These digital images are treated using RGB (red, green, blue), HSI (hue, saturation, and intensity), CMYK (cyan, magenta, yellow, and black), and grayscale, and chemometric techniques. 4

Interview ·Microfabrication in analytical chemistry: this involves the development of automated microfabricated systems using ultraviolet deep photolithography with commercial resin based on urethane and acrylate oligomers. ·Development of electroanalytical methods: this involves electrochemical oxidation/reduction studies using different types of electrodes, such as vitreous carbon and boron doped diamond, and the development of methods for the voltammetric determination of toxins, pesticides, and polycyclic aromatic hydrocarbons PAHs in food, beverages, and environmental matrices. What is the importance of coordinating the LAQA for you? Being the LAQA coordinator is a huge passion. It was extremely difficult to set up this laboratory due to financing issues in Paraíba. I stay at the LAQA for at least 10 hours every day, except when I am traveling, in meetings, in classes, or resting at weekends and on holidays. Do you keep informed about the progress of chemistry research? What is your opinion of the current progress of chemical research in Brazil? What are the latest advances and challenges in scientific research in Brazil? I always try to stay well informed of chemistry research in my country and in the world. Brazil is very well developed and has made great progress in almost all areas of chemistry compared with any first world country. However, I and several researchers are extremely concerned about the lack of investment in research and education in Brazil, especially in recent years. We urgently need to reverse this situation, otherwise Brazil will see a decline in the advances made in recent decades and there will be a serious risk of losing great researchers to other countries. For you, what have been the most important recent achievements in the world of analytical chemistry research? What are the landmarks? There have been major and important milestones in analytical chemistry in recent decades. I could say that, for example, the coupling of analytical instrumentation to microcomputers in the late 1970s was an important milestone. However, with the generation of enormous amounts of data using these instrumentations, the development of chemometrics for the treatment of these data has been a fundamental framework for analytical chemistry. It is also worth mentioning the resurgence of NIR spectroscopy in the late 1980s, instrumentation hyphenation, and the emergence of laser-induced breakdown spectroscopy (LIBS) more recently. Nowadays, I find the use of nanotechnology, digital and hyperspectral images, and the generation of second-, third-, and fourth-order analytical data treated using higher-order chemometric techniques to be interesting landmarks. There are several scientific meetings in the area of chemistry held in Brazil and worldwide. To you, how important are these meetings? How do you see the development of national meetings for chemistry in Brazil? These scientific meetings are of fundamental importance for the exchange of ideas between researchers and students. Since the early 1980s, I have participated effectively at the Annual Meetings of the Brazilian Chemical Society (RASBQ) and the National Meeting of Analytical Chemistry (ENQA), which are important events for the growth of chemistry and analytical chemistry in Brazil. You have already received a few awards. What is it like to receive this kind of recognition? What is the importance of these awards for the development of science and new technologies? Some papers presented by my students at scientific events have been awarded, and others received an honorable mention at the UFPB. I am extremely happy to have been a professor/researcher honored for relevant contribution to analytical chemistry at the 15th National Meeting on Analytical Chemistry & 3rd Ibero-American Congress on Analytical Chemistry held in Salvador in October 2009. An interesting fact 5

Interview that also honored me was to have been invited and written together with Roberto Kawakami Harrop Galvão the chapter: RKH Galvão and MCU Araújo "Variable Selection" In: Brown SD, Tauler R, Walczak B (eds.) "Comprehensive Chemometrics : Chemical and Biochemical Data analysis", vol. 3, pp. 233-283, Elsevier, Oxford, 2009. For you, what is the importance of the support of research funding agencies (CAPES and São Paulo Research Foundation (FAPESP), among others) for scientific development in Brazil? Allocation of resources to researchers by development agencies is of fundamental importance for the development of science in Brazil. Universities do not have the financial resources to pay for scientific and technological research in the country. Therefore, we researchers rely heavily on these development agencies. How is a career in analytical chemistry? What advice would you give to a newcomer to this area? I am extremely passionate about my career as a professor/researcher in analytical chemistry. I do not have advice specifically for a professional in this area. My advice is for professionals from any area. Do everything in your professional life with passion and dedication. If you do not have passion for your profession, immediately seek another profession that will provide this. Doing things in life without passion can be very sad. Everything you do with passion and dedication brings you good results and happiness.

Br. J. Anal. Chem., 2018, 5 (19), pp 7-7 DOI: 10.30744/brjac.2179-3425.2018.5.19.7-7

Point of View

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The latest advances in Chemical Analysis of Waters at CETESB Carlos Roberto dos Santos President of CETESB - Environmental Agency of São Paulo State, São Paulo, SP, Brazil carlosrs@sp.gov.br

One of the most important topics for the environmental laboratories at CETESB (Environmental Agency of Sao Paulo State) is provide reliable data to support decision makers when it comes to regulations, monitoring programs, responses to chemical emergencies, sources inspections and other applications. All these different goals need high quality procedures in compliance with ISO 17025 for several matrices such as water, sludge, air, soils, solid waste and biological samples. For the Toxicological Analyses Laboratory to achieve excellence in our analytical methods was necessary to improve some steps of water sample preparation for bioassays and chemical analysis. For Ames test (Salmonella typhimurium reverse mutation assay) solid-phase extraction (SPE) was previously carried out manually using open glass column filled with adsorbent resin. At this point, large amount of organic solvent were being used per sample and low analytical frequency of four samples a week during the whole process. Over the last seven years, the interest in endocrine disrupting chemicals (EDC) has been growing worldwide including Sao Paulo State, Brazil and the number of samples increased greatly. It leaded us to research new techniques and find automated instrumentation. According to the advances in analytical chemistry in water analysis an automated extraction method using SPE disk was developed and implemented. This system controlled by specific software, consists of three programmable extraction towers capable of processing aqueous samples directly from their original containers. Furthermore, a combination of prefilter and SPE disk with large surface area provides better throughput even for samples that contain substantial amount of particulate matter. Finally, extract is concentrated using evaporating system for solvent removal that can be used simultaneously for 16 vials of 40 mL and 96 vials of 2 mL. This instrumental innovation increased the capacity of the laboratory up to 700% samples/year and volume reduction of 500% of the total organic solvent volume. The final result was less toxic chemicals in the environment and high productivity. References Thurman, E. M.; Snavely, K. Advances in solid-phase extraction disks for environmental chemistry. TrAC, Trends Anal. Chem., 2000, 19 (1), pp 18-26. (DOI: 10.1016/S0165-9936(99)00175-2) Umbuzeiro, G. A.; Roubicek, D. A.; Sanchez, P. S.; Sato, M. I. Z. The Salmonella mutagenicity assay in a surface water quality monitoring program based on a 20-year survey. Mutat. Res., Genet. Toxicol. Environ. Mutagen., 2001, 491 (1-2), pp 119-126. (DOI: 10.1016/S1383-5718(01)00139-5)

Br. J. Anal. Chem., 2018, 5 (19), pp 8-9 DOI: 10.30744/brjac.2179-3425.2018.5.19.8-9

Point of View

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Water Chemistry: A Fundamental Knowledge Base for Aquatic Ecosystem Management José Galizia Tundisi International Institute of Ecology, São Carlos, SP, Brazil Universidade Feevale, Novo Hamburgo, RS, Brazil tundisi@iie.com.br Water availability, water quality, water management and water governance are today at the center of discussion on water security and sustainability. The chemical composition of pristine inland waters is depending upon the hydrogeochemistry of the watersheds, geological background, soil type and the aquatic biota (this due to metabolism of aquatic organisms). Water is the universal solvent. Dissolved substances in water are ions and anions, gases, nutrients for phytoplankton, macrophyte growth and photosynthetic bacteria. Nutrient such as phosphorus and nitrogen with different chemical species dissolved in water are thus fundamental for the biological productivity of the +2 +2 +6 inland waters. Trace ions such as Cu , Zn , Mo , organic refratory substances, such as humic acids and labile organic substances are also present in almost all inland waters: lakes, rivers, reservoirs, wetlands. High level of dissolved organic substances with complex molecules decreases oxygen concentration in all layers of water affecting the aquatic biota and the underwater radiation climate. Geographic variability of principal dissolved substances and elements in natural pristine waters occurs due to the continental / regional differentiation in hydrogeochemistry. Processes of interest in an environmental context are: · Leaching of ions and organic compounds in soil · Evaporation of organic chemicals from soil and surface water · Sedimentation of heavy metals and organic chemicals in aquatic ecosystems · Hydrolisis of organic chemicals · Dry-humid deposition from the atmosphere · Chemical oxidation · Photochemical processes Biotic processes such as respiration, excretion and decomposition of organisms further complicate this picture. All the processes above described are first order reactions at least in some situations [1]. Human actitvity has resulted in extensive contamination of pristine waters all over Planet Earth. Even when some contaminants are natural components of soils, they can be mobilized and redistributed in potencially toxic forms throughout many industrial activities, or other man made actions. Several contaminants wash into the aquatic environment, dissolve in water or may settle in the sediments. Good water quality, which is the basic chemical composition of water, is essential for water security. The history of degradation of water quality shows a continuous deterioration, with increasing complexity, mainly in the 20th century with cumulative impacts, increasing vulnerability of human populations and economic impacts. There is an increase in complexity of water analysis due to this degradation process [2]. Endocrine disruptors, pesticides, herbicides, cosmetics, antibiotics, and toxins dissolved in water turn the water chemistry a very important knowledge data base for water resources management. Several million of different compounds and 100.000 chemicals have environmental interest because they may threaten the environment. The number of possible reactions among these chemicals is enormous. The solubility products of hydroxides, oxides, and carbonates have particular interest in aquatic chemistry because these anions are present in high concentrations in natural aquatic ecosystems [3]. 8

Point of View Chemical analysis of waters from inland ecosystems is therefore fundamental for their management. This management is an interdisciplinary and complex operation. Chemical analysis of efluents from point sources, identification of non point sources of contamination, complexes formation, interaction sedimentwater, are some of the problems to be solved and they are challenges for an adequate management perspective. Acidification, eutrophication, very high concetration of toxic substances, are some of the problems that have to be solved by the development of advanced technologies and strategies in order to optimize multiple uses of surface waters. Contamination of underground waters is another threat to water quality. Integrated environmental approach means that all biogeophysical, chemical, social and economic processes have to be considered for the overall systemic view necessary to promote water governance and water resources management. In order to follow up water quality problems it is necessary to keep track of all material flows (in the air, water and soils) and their cycles in organic and inorganic compartments. Methodological and technical advances are relevant for the consolidation of chemical information on aquatic ecosystems. Chemical analysis of fresh waters can help with the anticipation of impacts and or in the prediction capacity of future impacts [4]. Solving the analytical complexity and developing an advanced process of predictions is one important task for the future of aquatic chemistry, with a relevant contribution for the integrated management of inland aquatic ecosystems.

References 1. Chapman , D. (Ed.). Water Quality Assessments. UNESCO/ WHO/UNEP, 1995, p 585. 2. Tundisi , J. G.; Matsumu ra-Tundisi, T.; Ciminelli , V. S.; Barbosa , F. A. ‘Water availability, water, quality, water governance: the future ahead’. Hydrological Sciences and Water Security: Past, Present, Future . IAHS Press Publ., 2015, 366, pp 75-79. (Proceedings of the 11 th Kovacs Colloquium, Paris, France, June 2014). 3. Tundisi , J. G.; Matsumura -Tundisi, T. Limnology . CRC Press, Taylor & Francis, London, 2013, p 864. 4. Jorgensen, S. E.; Tundisi , J. G.; Matsumura -Tundisi , T. Handbook of Inland Aquatic Ecosystem Management . CRC Press. Taylor & Francis, London, 2013, p 421.

Br. J. Anal. Chem., 2018, 5 (19), pp 10-10 DOI: 10.30744/brjac.2179-3425.2018.5.19.10-10

Letter

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The Importance of Advances in Analytical Chemistry for the Quality Control of Water and Chemicals used in the Water Treatment Process Danielle Polidorio Intima Laboratory Supervisor Department of Water and Sewage Quality Control Water and Sanitation Company of the State of São Paulo (SABESP) dpolidorio@sabesp.com.br

The analytical chemistry is inserted in all stages of the water production process, that is, in the characterization of the water of the lake from where it will be collected, in the characterization of the chemicals that will be applied in the process, in the quality control laboratory and in the monitoring the quality of the drinking water distributed to the population. There are thousands of analytical tests performed in sanitation companies daily. In order to produce water for human consumption, processes must be used to allow the removal of impurities present in the water to be treated. The conventional process of water treatment is divided into phases, such as: pre-chlorination, pre-alkalinisation, coagulation, flocculation, decantation, filtration, postalkalization, disinfection and fluorination. In each of them there is a strict dosage control of the chemicals, which must be within the technical specifications required by the sanitation companies. Therefore, all technological evolution in chemical analysis is important in order to optimize the processes of monitoring drinking water quality. Whether for field, inline or bench-top equipments. For example, the quantitative elemental measurements in drinking water are a very important task since Brazilian legislation establishes the threshold limit value for each element. Besides Atomic Absorption Spectrometry (AAS) and Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES) have been routinely used for this purpose, Total Reflection X-ray Fluorescence spectrometry (TXRF) shows to be very interesting alternative. It provides a fast and easy sample preparation, low analytical operation and maintenance costs. This technique alows monitoring simultaneously the presence of arsenic, barium, lead, copper, chromium, nickel, selenium, uranium, iron, manganese and zinc in drinking water samples. This technique was so certain that, nowadays, Sabesp performs all the monitoring of the toxicity of the chemicals used in the treatment of water by TXRF. Since any product used in the treatment must promote the potability of water unconditionally, without the risk of transferring any contaminant to the process. All this control is not an easy task, the sanitation company chemists must be always up to date and seeking technical cooperation agreements with universities. This partnership will promote the evolution of the analytical methodologies used to control the quality of the water we drink.

Br. J. Anal. Chem., 2018, 5 (19), pp 11-11

In Memoriam

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Atomic Spectrometry loses one of its fathers: Professor Bernhard Welz On Saturday night (6/2/2018) the death of Professor Bernhard Welz was announced. The German professor, who liked to be called Bernardo, had been a volunteer professor at the Department of Chemistry of the Federal University of Santa Catarina (UFSC) in Florianópolis, Brazil since 1999. Considered one of the fathers of atomic absorption spectrometry, he worked with great prominence in Analytical Chemistry, producing about 300 publications, which yielded an H-index of 55 – one of the largest in Brazil among all scientific areas. Professor Welz has received numerous national and international honors throughout the many decades of his brilliant scientific career. His charisma, sympathy and cordiality will be deeply missed by those who have known him. His path Bernhard Welz received his undergraduate degree in chemistry from the Technical University of Munich in 1960, and his doctorate degree from the University of Stuttgart in 1966. Welz was part of the Perkin Elmer chemical analyst group from 1967 to 1998, where he was also director of research in atomic spectrometry. In 1999, Welz accepted a proposal to act as a guest lecturer at the UFSC, where he taught instrumental analysis and atomic absorption spectrometry. Consequently, he also moved to the city of Florianópolis, Santa Catarina. He also participated in the postgraduate program of UFSC, advising students in scientific initiation, masters and doctorates, and had as research line the development of methods in atomic and molecular absorption spectrometry, focusing on direct analysis of solids. Bernhard Welz was a member of the following associations: the Society for Applied Spectrometry (SAS), the Brazilian Chemical Society (SBQ), and the German Chemical Society (GDCh). He also belonged to the board of editors of the following scientific journals: Spectrochimica Acta Part B, Journal of Trace Elements in Medicine and Biology, and Talanta. Some awards and honors he received 1982 – Senior Scientist (PerkinElmer) 1988 – Jana Marcus Marci Medal (Czechoslovak Spectroscopic Society) 1988 – Pergamon/Spectrochimica Acta Atomic Spectroscopy Award 2006 – Clemens-Winkler-Medaille (GDCh) 2016 – Adilson José Curtius Medal (Brazilian Chemical Society / National Meeting of Analytical Chemistry – ENQA) 2017 – Colloquium Spectroscopicum Internationale Award Bernhard Welz was born on October 5, 1936, in Augsburg, Germany, and died on June 2, 2018, in Florianópolis, Brazil.

Br. J. Anal. Chem., 2018, 5 (19), pp 12-21 DOI: 10.30744/brjac.2179-3425.2018.5.19.12-21

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Feasibility of using Total Reﬂection X-ray Fluorescence Spectrometry for Drinking Water Analysis Isabella O. Silva1, Denise Akemi F. T. Trugillo2, Edson Joanni3, Danielle Polidorio Intima3*, Cassiana Seimi Nomura1 1

Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo 05508-000, São Paulo, SP, Brazil. 2 Departamento de Ciências Exatas e da Terra, Universidade Federal de São Paulo 09972-270, Diadema, SP, Brazil 3 Departamento de Controle de Qualidade, Companhia de Saneamento Básico do Estado de São Paulo 020037-021, São Paulo, SP, Brazil.

The quantitative elemental measurements in drinking water are a very important task since Brazilian legislation establishes the threshold limit value for each element. Besides Atomic Absorption Spectrometry (AAS) and Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES) have been routinely used for this purpose, Total Reflection X-ray Fluorescence spectrometry (TXRF) seems to be a very interesting alternative. It provides a fast and easy sample preparation, low analytical operation and maintenance costs. This study shows the feasibility of using TXRF for monitoring simultaneously the presence of arsenic, barium, lead, copper, chromium, nickel, selenium, uranium, iron, manganese and zinc in drinking water samples. All measurements were performed using TXRF spectrometer, equipped with an air cooled low power X-ray tube (Mo target). For internal calibration, 950 µL of sample was mixed with 50 µL of a standard solution containing 10 mg L-1 of gallium. For most of the elements, the direct quantification by TXRF using Ga as internal standard could be used. For the Cr, Ni and U measurements, analytical calibration curves have to be adopted. The limits of detection for proposed method are appropriate for all elements investigated. In addition, the method showed good precision and accuracy and seems to be a very interesting alternative to be used in the routine analysis involving elemental measurement in drinking water samples analysis. Keywords: drinking water, elemental analysis, TXRF INTRODUCTION Supply of safe drinking water is crucial to human life and safe drinking water should not impose a significant risk to humans [1]. Although a few elements are essential for human health, an excess amount of these metals can have negative effects. The Brazilian Drinking Water legislation provide the reference which defines safe, good quality water, how it can be achieved and how it can be assured. They are concerned with health safety aesthetic quality [2]. Inorganic species in drinking water usually occur as dissolved salts, especially carbonates, chlorides and sulfates. Inorganic compounds are generally present in water in concentrations substantially higher than organic compounds. Taste thresholds for some -1 -1 -1 commonly occurring inorganic ions are about 0.1 mg L for manganese, 0.3 mg L for iron, 2 mg L for copper, 5 mg L-1 for zinc, 250 mg L-1 for chloride, and 250-500 mg L-1 for sulfate. Most of these ions have health guidelines even at concentrations higher than their taste thresholds. However, in most cases the customer would reject the water for aesthetic reasons. Table I presents the Brazilian drinking water guideline value for the inorganic species [2].

*dpolidorio@sabesp.com.br https://orcid.org/0000-0002-9311-4044

Feasibility of using Total Reﬂection X-ray Fluorescence Spectrometry for Drinking Water Analysis

Article Table I. Brazilian drinking water guideline value (DMGV) for the inorganic species

Parameters

Toxic

Organoleptic

Element Antimony Arsenic Barium Cadmium Lead Copper Chromium Nickel Selenium Uranium Aluminum Iron Manganese Zinc

DWGV* (mg L-1) 0.005 0.01 0.7 0.005 0.01 2 0.05 0.07 0.01 0.03 0.2 0.3 0.1 5

Flame atomic absorption spectrometry (FAAS) and Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES) have been routinely used for water quality control. Besides the good performance of those methods and the adequate limits of detection for this purpose, the cost of analysis is high. The requirement of analytical grade gases (acetylene and argon) and high cost of consumables increase the cost of analysis. The total reflection X-ray fluorescence spectrometer used in this study, for example, is a plug and play system, compact with low energy consumption, completely independent of any cooling medium or exhaustion system, without the need for disposables or analytical grade gases, greatly reducing the cost of analysis. In this context, the use of Total Reflection X-ray Fluorescence spectrometry (TXRF) show to be a very interesting alternative. It provides fast and easy sample preparation processes and the costs of analytical operation and maintenance are low [3]. In this technique, X-ray is focused onto the sample and its energy is able to excite the atoms and induce the emission of fluorescence radiation, which is specific for each element. At very shallow angles of incidence of the exciting X-rays with respect to a flat specimen surface, external total reflection of the incident radiation occurs and drastically reduces the penetration depths of the incident radiation into the specimen bulk, thus enhancing the surface sensitivity by decreased substrate fluorescence and scattered excitation radiation intensities. During the last few decades this Total-reflection XRF (TXRF) method became one of the faster and lower cost competitive techniques for trace element analyses [4]. Radiation sources commonly used in TXRF is made with the molybdenum or tungsten. Each of them has a useful range for the determination and quantification of the elements present in the samples. The molybdenum source shows a useful range between 3 and 15 keV. For this reason, elements whose emission of fluorescence is near the extremes of this range cannot be quantified. In this condition, Al can be detected, but its quantification is compromised due to the interference caused by Si and Ar. In another situation, elements like Cd and Sb are subject to spectral interference by molybdenum coming from the tube. To minimize the spectral interference allowing the quantitative measurement of low energy elements, the use of TXRF instrument equipped with an air cooled low power X-ray tube (W target) and monochromator with signal amplifier is recommended [5]. In recent years, TXRF has been used in elemental measurement in water samples. Riaño et al. developed a practical and easy guideline for the correct preparation of aqueous samples, showing the effect of different parameters as measurement time, carrier position, sample volume and sample drying time [6]. Floor et al. assessed the components contributing to the combined uncertainty budget associated 13

Silva, I. O.; Trugillo, D. A. F. T.; Joanni, E.; Intima, D. P.; Nomura C. S.

Article Article with TXRF measurements using Cu and Fe concentrations in different spiked and natural water samples [7]. Romero et al. developed a headspace thin-film microextraction onto graphene membranes for specific detection of methyl(cyclopentadienyl)-tricarbonyl manganese in water samples and the reflectors containing graphene membranes were directly used as sample carriers for TXRF analysis [8,9]. Woelfl et al. determined Cr, Mn, Fe, Ni, Cu, Zn, As and Pb in freshwater rotifers and ciliates by TXRF spectrometry [10]. Bahadir et al. proposed a method to preconcentration and determination of Cr species in water samples, and another methodology based on the combination of dispersive microsolid-phase extraction (DMSPE) with total reflection X-ray fluorescence (TXRF) spectrometry for the determination of hexavalent chromium in drinking waters [11,12]. This work proposes evaluate the feasibility to use the total reflection X-ray fluorescence spectrometry for monitoring the presence of metals presented in Table I, with the exception of Al, Cd and Sb, by multielemental direct determination in drinking water samples, attending the parameters established by Brazilian legislation. MATERIALS AND METHODS Instruments All measurements were performed using a benchtop S2 PICOFOX TM TXRF – spectrometer (Bruker Nano GmbH, Karlsruhe, Germany) equipped with a Mo tube – Kα 17.5 keV excitation source (600 mA, 50 kV, 50 W), a multi-layer monochromator and a silicon drift detector with an active area of 10 mm2. The resolution of the detector was better than 160 eV at 10 kpcs (MnKα). The X-ray tube is provided with a primary radiation protection made of 5 mm-brass, a thin Beryllium window (100 µm) by which the X-rays escape to the outside. In the direction to the monochromator a 3 mm-opening for the emission of the useful beam is provided. Sample carriers made of synthetic quartz glass. The measurement time was 600 s per sample. The processing of the X-ray spectra and the accounting for fluorescence peak overlaps were performed using the software SPECTRA version 7.0 (Bruker Nano GmbH, Karlsruhe, Germany). The analytical results obtained with the TXRF spectrometer were cross-checked with results obtained by ICP OES Thermo Scientific™ iCAP™ 7400 (Thermo Scientific, China) with dual view configuration and multichannel CCD array detector. An analytical balance (Mettler Toledo, Switzerland) was employed for weighing samples and a vortex shaker (Scientific Industries, USA) to homogenize the samples. An electronic pipette (Rainin/Mettler Toledo, USA) was employed to transfer the sample aliquot to quartz disk. A dry oven (Ethik Technology, Brazil) was employed to dry the samples aliquot. Reagents and samples All analytical solutions were prepared with high purity deionized water obtained from a Milli-Q water purification system (Millipore, USA) and stored in decontaminated polypropylene tubes (Aton, USA) and all samples were stored in microtubes of 2.0 mL (Aton, USA). Standards solutions containing 1000 mg L-1, produced by laboratories with ISO 17025, ISO Guide 34 accreditation and NIST traceability (Table II), were added over real water samples and used to check the accuracy of the analytical method developed. The 1000 mg L-1 gallium (Ga) standard solution was obtained from Inorganic Venture (Christiansburg, USA). A silicone solution in isopropanol was obtained from SERVA Electrophoresis Gmbh (Heidelberg, Germany).

Feasibility of using Total Reﬂection X-ray Fluorescence Spectrometry for Drinking Water Analysis

Article Table II. NIST traceability of the standards solutions

Element

Stock standard solutions(1000 mg L-1)

NIST traceability

Arsenic

Inorganic Ventures - K2-AS650402

NIST SRM 3103a

Barium

SPC Science - S161003016

NIST SRM 3104a

Inorganic Ventures - K2-PB03074

NIST SRM 3128

Absolute Standard, Inc–060515

NIST SRM 3114

Chromium

Inorganic Ventures - J2-CR03111

NIST SRM 3112a

Nickel

Inorganic Ventures - J2-NI02103

NIST SRM 3136

Selenium

Inorganic Ventures - J2-SE02058

NIST SRM 3149

Uranium

Inorganic Ventures - J2-U01107R

NIST SRM 3164

Absolute Standard, Inc–102815

NIST SRM 3126a

Ultra Scientific - P01081 Inorganic

NIST SRM 3132

Ventures - J2-ZN

NIST SRM 3103a

Lead Copper

Iron Manganese Zinc

Procedure Sample carriers were pretreated with 5 μL of silicone in isopropanol at room temperature and dried for 15 min in a drying oven at 60 °C. This procedure was followed to make the surface hydrophobic and avoid spreading of the aqueous sample on the carrier. For internal calibration, 950 μL of sample was mixed with 50 μL of a standard solution (Ga; 10 mg L-1). For the measurement, a volume of 5 μL of the sample was added onto the carrier at room temperature and dried at 60 °C in a drying oven for 15 min. This procedure was performed three times to preconcentrate the samples. The gain correction was done before starting the analysis. Samples were measured for 600 s. The optimization of the instrumental parameters was performed according to the preparation of aqueous samples proposed by Riano et al. which involves 15 min for silicone in isopropanol drying time and 600 s for measurement time [6]. Spectra were analyzed with the Bruker Spectra Picofox version 7.5.3.0 software and the limits of detection were calculated by the same software. This software use the 3-sigma criterion to calculate the limits of detection. As the elements are identified and quantified by means of their fluorescence peaks, the definition of the detection limits is based on a statistical inspection of the peak area and the subjacent spectral background. Thereby, it is assumed that an element is considered to be detected if the peak area is three times larger than as the counting statistics of the background (Equation 1).

with the meaning LLDi lowest limit of detection of the element i in milligrams per liter Ci concentration of the element i in milligrams per liter Ni area of the fluorescence peak in counts NBG background area subjacent the fluorescence peak in counts The quantification limits were calculated according to CGCRE 008 of Instituto Nacional de Metrologia, Normalização e Qualidade Industrial (INMETRO) (Equation 2) [13].

Silva, I. O.; Trugillo, D. A. F. T.; Joanni, E.; Intima, D. P.; Nomura C. S.

Article Article with the meaning LQi limit of quantification of the element i LLDi lowest limit of detection of the element i si standard deviation of lowest limit of detection of the element i The results obtained by the proposed method were compared with those obtained by the conventional method using ICP OES in which the sample was analyzed directly without any dilution. The wavelengths (nm) used for each element were: As (189.042), Ba (455.403), Cd (228.802), Pb (220.353), Cu (324.754), Cr (283.563), Fe (259.940), Mn (257.610), Ni (341.476), Se (196.090), U (409.014) e Zn (213.856). The measurements were performed using axial view, 2 L min-1 of plasma gas flow, 1200W RF power and concentric nebulizer. RESULTS AND DISCUSSION All experiments were performed using Ga as internal standard which is the most common element used in TXRF measurements and it is rarely found in drinking water samples. Figure 1 shows the TXRF spectrum of a spiked drinking water sample using Ga as internal standard for quantification of each analyte.

Figure 1. TXRF spectrum of a spiked drinking water sample

Table III shows the results obtained for direct analysis of the drinking water using Ga 0.5 mg L−1 as internal standard. Table III. Limits of quantiﬁcation and results obtained for direct analysis of the drinking water by TXRF using 0.5 mg L−1 of Ga as internal standard

Element

LQ* (mg L -1)

Drinking water samples(mg L -1)

Spike (mg L -1)

Arsenic

0.003

0.007+0.001

0.01

Drinking water spiked samples (mg L -1) 0.035+0.001

Barium

0.002

<0.002

0.7

0.64+0.03

Lead

0.005

<0.005

0.01

0.009+0.001

Copper

0.029

0.006+0.001

2.02+0.14

100

Chromium

0.005

<0.005

0.05

0.044+0.014

Nickel

0.006

<0.006

0.07

0.065+0.005

Selenium

0.003

<0.003

0.01

0.010+0.001

Uranium

0.003

<0.003

0.03

0.025+0.001

Iron

0.02

0.840+0.008

0.3

1.19+0.02

103

Manganese

0.02

0.170+0.009

0.1

0.28+0.02

101

Zinc

0.76

0.120+0.007

2.05+0.14

Recovery (%) 106

*Limit of Quantiﬁcation

Considering the threshold limits established by the legislation (Table I), limits of quantification for the proposed method (Table III) show to be appropriate. As the concentration of As, Ba, Pb, Cu, Cr, Ni, Se, U, Fe, Mn and Zn were lower than the limit of detection of the method, addition and recovery test was performed to check the matrix interference. The concentration used for spiking was based on Brazilian drinking water guideline value (Table I). For most analytes, recoveries between 83 and 106% (Table III) were obtained, indicating the absence of significant matrix interference in the direct analysis of drinking water by TXRF. For the analytes Cr, Ni and U, the recoveries were lower than 90% at different concentrations showed in Figure 2. To improve the analytical results, analyses were performed using analytical calibration curves (Figure 2) instead of using direct methods which involves the use of Ga as internal standard. Addition and recovery tests were also performed and the obtained results were compared to those obtained by using direct methods (Figure 3).

Figure 2. Analytical calibration curves in deionized water for TXRF analysis for Cr (a), Ni (b) and U (c)

Silva, I. O.; Trugillo, D. A. F. T.; Joanni, E.; Intima, D. P.; Nomura C. S.

Article Article

Figure 3. Recoveries values for analysis made using direct method and method using analytical calibration curve for Cr, Ni and U

Figure 3 shows that the recoveries for Cr, Ni and U were improved when analysis were done using calibration methods showing that for these elements the quantiďŹ cation using Ga as internal standard was not enough. For this reason, for those elements, calibration curves were adopted for analysis. The direct quantiďŹ cation of Cr, Ni and U by the internal standard would be possible by altering the constants of these analytes in the equipment software. The constants correspond to the slope (Sli) in a plot of the net counts of the element peak versus the net counts of the Ga peak multiplied by concentration of the element divided by concentration of Ga solution. Sli is determined by the Equation 3 and it is a part of the Equation 4, used to determine the concentration of the analyte. As the software doesn't allow the access to change the constants, the external calibration was performed through the analytical curve, correcting the results mathematically.

with the meaning Sli sensitivity of the element i in milligrams per liter Ni element i peak in net counts CGa concentration of Ga solution in milligrams per liter NGa Ga peak in net counts Ci concentration of the element i in milligrams per liter

with the meaning Ci concentration of the element i in milligrams per liter CGa concentration of Ga in milligrams per liter Ni element i peak in net counts SGa sensitivity of Ga in milligrams per liter NGa Ga peak in net counts Sli sensitivity of the element i in milligrams per liter 18

Feasibility of using Total ReďŹ&#x201A;ection X-ray Fluorescence Spectrometry for Drinking Water Analysis

Article Using the proposed method, drinking water samples were analyzed by TXRF method and the results obtained by the proposed method were compared to those obtained by ICP OES technique (Table IV). Table IV. Results obtained for direct analysis of drinking water samples by ICP OES and by TXRF

Silva, I. O.; Trugillo, D. A. F. T.; Joanni, E.; Intima, D. P.; Nomura C. S.

Article Article Table IV. Results obtained for direct analysis of drinking water samples by ICP OES and by TXRF (cont.) Element

Manganese

Zinc

Sample

Results TXRF (mg L -1)

A – 1* A – 2* A – 3* B – 1** B – 2** B – 3**

0.023 0.031 0.022 0.061 0.033 0.041

+ 0.001 + 0.001 + 0.003 + 0.003 + 0.003 + 0.001

A – 1* A – 2* A – 3* B – 1** B – 2** B – 3**

0.152 0.201 0.269 0.129 0.116 0.154

+ 0.009 + 0.012 + 0.016 + 0.011 + 0.009 + 0.011

Spike (mg L -1)

TXRF (mg L -1)

REC (%)

ICP OES (mg L -1)

REC (%)

0.1

0.134 0.138 0.129 0.166 0.138 0.144

+ 0.021 + 0.015 + 0.013 + 0.014 + 0.011 + 0.008

109 105 106 103 104 102

0.125 0.128 0.123 0.164 0.137 0.142

+ 0.009 + 0.008 + 0.009 + 0.007 + 0.011 + 0.009

102 98 101 102 103 101

2.109 2.267 2.360 2.065 2.010 2.176

+ 0.097 + 0.128 + 0.122 + 0.022 + 0.097 + 0.088

98 103 104 97 95 101

2.238 2.333 2.292 2.172 2.074 2,219

+ 0.022 + 0.087 + 0.023 + 0.031 + 0.042 + 0.061

104 106 101 102 98 103

* A = Drinking water treatment system sample ** B = Drinking water distribution system sample

Results from Table IV show that the values obtained by TXRF are in accordance with the results obtained by ICP OES, according to t-Test (95%) realized using the statistic function of Excel. Limits of quantification for TXRF and ICP OES presented in the Table V show that for the values obtained for Cu, Cr, Ni and Zn were higher for TXRF probably due to the less efficient X-ray fluorescence excitation in those concentrations presents in drinking water samples [14]. Besides this, due to the simplicity and low cost of analysis, TXRF is strongly recommended for routine analysis of drinking water. Table V: Limits of quantification (LQ) for direct analysis of the drinking water samples by TXRF and ICP OES Elem ent

LQ TXRF (m g L-1)

LQ ICP O ES (m g L-1)

Arsenic

0.003

Barium

0.002

0.010

Lead

0.005

0.004

Copper

0.029

0.005

Chrom ium

0.005

0.002

Nickel

0.006

0.002

Selenium

0.003

0.007

Uranium

0.003

0.019

Iron

0.02

0.05

M anganese

0.02

0.01

Zinc

0.76

0.01

CONCLUSIONS This study presented the feasibility of using total reflection X-ray fluorescence spectrometry for multielemental direct determination in drinking water samples. The technique presented a simple, fast and safe sample preparation with optimized measurement time, resulting in a method three times faster, with high analytical frequency for the elemental determination compared to ICP OES, considering sample preparation, measurement time and data analysis. Besides this, the total reflection X-ray fluorescence spectrometer used in this study is a plug and play system, compact with low energy consumption, completely independent of any cooling medium or exhaustion system, without the need for disposables or analytical grade gases, greatly reducing the cost of analysis. For most of the elements investigated, direct 20

Feasibility of using Total Reﬂection X-ray Fluorescence Spectrometry for Drinking Water Analysis

Article quantiﬁcation by TXRF using Ga as internal standard was used. For the Cr, Ni and U elements measurements, the use of analytical calibration curves are recommended. The limits of detection are appropriate for all elements investigated since they are below Brazilian drinking water guideline value. In addition, the method showed good precision and accuracy. Considering the simplicity of the proposed procedure associated to low cost of analysis, TXRF is strongly recommended for routine analysis of drinking water. Manuscript received May 15, 2018; revised manuscript received Aug. 3, 2018; accepted Aug. 6, 2018.

REFERENCES 1. Conselho Nacional do Meio Ambiente – CONAMA, Brazil. Resolution Nº 357, March 17, 2005. 2. Ministério da Saúde, Brazil. Portaria nº 2914, December 12, 2011. 3. Stosnach, H. Trace element analysis of fresh water samples by TXRF spectrometry, Lab Report XRF 425, Bruker Nano GmbH, Berlin, Germany, 2007. 4. Klockenkamper, R.; Bohlen, A. Total-reflection X-ray fluorescence analysis and related methods. Chemical analysis: a series of monographs on analytical chemistry and its applications. John Wiley & Sons, Inc., Hoboken, New Jersey, 2015. 5. Stosnach H.; Gross, A. Tungsten (W) excitation and its application to pharmaceutical and environmental samples. Lab Report XRF 436, Bruker Nano GmbH, Berlin, Germany, 2015. 6. Riaño, S.; Regadío, M.; Binnemans, K.; Hoogerstraete, T. V. Spectrochim. Acta Part B, 2016, 124, pp 109–115. 7. Floor, G. H.; Queralt, I.; Hidalgo, M.; Marguí, E. Spectrochim. Acta Part B, 2015, 111 pp 30–37. 8. Romero, V.; Costas-Mora, I.; Lavilla, I.; Bendicho, C. Spectrochim. Acta Part B, 2016, 126, pp 65–70. 9. Romero, V.; Costas-Mora, I.; Lavilla, I.; Bendicho, C. RSC Adv., 2016, 6, pp 669-676. 10. Woelfl, S.; Óvári, M.; Nimptscha, J.; Neu, T. R.; Mages, M. Spectrochim. Acta Part B, 2016, 116, pp 28–33. 11. Bahadir, Z.; Bulut, V. N.; Hidalgo, M.; Soylak, M.; Marguí, E. Spectrochim. Acta Part B, 2016, 115, pp 46–51. 12. Bahadir, Z.; Bulut, V. N.; Hidalgo, M.; Soylak, M.; Marguí, E. Spectrochim. Acta Part B, 2015, 107, pp 170–177. 13 .Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMETRO). DOQ-CGCRE-008 Orientação sobre validação de métodos analíticos, Revision July 4, 2011. http://www.inmetro.gov.br/sidoq/ arquivos/Cgcre/DOQ/DOQ-Cgcre-8_04.pdf 14. S2 Picofox, User Manual, Bruker AXS Microanalysis, GmbH, Berlin, Germany, 2012.

Br. J. Anal. Chem., 2018, 5 (19), pp 22-28 DOI: 10.30744/brjac.2179-3425.2018.5.19.22-28

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Evaluation of Nitrate as Internal Standard for Quantitative Determination of Urea in Urine by Raman Spectroscopy Felipe Manfroi Fortunato1, Marcos André Bechlin1, Edilene Cristina Ferreira1, Silvana Ruella Oliveira2 and José Anchieta Gomes Neto1* 1 São Paulo State University - UNESP, Analytical Chemistry Department, Rua Prof. Francisco Degni 55, CEP 14800-060, Araraquara, SP, Brazil 2 University of São Paulo - USP, Faculty of Pharmaceutical Sciences of Ribeirão Preto – FCFRP Avenida do Café s/n, CEP 14040-903, Ribeirão Preto, SP, Brazil Graphical Abstract Nitrate was tested as internal standard Nitrate testedmethod as internalfor standard a inwas a new urea indetermination new method for urea in urine in urine by determination Raman spectroscopy. The

proposed method by Raman spectroscopy. The furnished proposed better p rfurrnished e c i s i o better n i nprecision c o m inp a r i s o n w i t h method conventional methods. Besides, it is comparison with conventional methods. simple, robust and provides faster results, minimum sample preparation faster results, minimum sample preparation and lower reagent consumption and and lower reagent consumption and minimum waste generated than the minimum waste generated than the reference spectrophotometric method. Besides, it is simple, robust and provides

reference spectrop hotometric method.

Nitrate was evaluated as internal standard (IS) for urea determination in urine by Raman spectroscopy. The influence of main operating parameters (laser power and integration time) on the Raman scattering of urea (N-C-N stretching at 1003 cm-1) and nitrate (N-O stretching at 1045 cm-1) was evaluated and the results showed good correlation between analyte and IS signals. The method was then developed and applied in five urine samples which presented urea concentrations in the 7.3 - 18.3 g L-1 range. For comparative purposes, samples were also analyzed by the reference method based on the enzymatic hydrolysis of urea followed by the spectrophotometric determination of ammonium ions. Results obtained by the proposed IS method were in agreement with the reference method at the 95% confidence level (paired t-test). Relative standard deviations (n = 12) of a sample analyzed by the reference method, proposed IS method and conventional external calibration method were in the ranges of 3.8 - 6.3%, 1.4 3.9% (IS) and 1.8 - 7.5%, respectively. Recoveries improved from 85 - 108% to 97% - 103% intervals when samples were analyzed by the conventional external calibration and IS proposed method, respectively. Keywords: Urea; urine; nitrate; internal standard; Raman spectroscopy. INTRODUCTION Analysis of biological fluids provides information about the health of a patient [1,2]. For example, the urea contents in blood or urine furnish an indicative for hepatic and kidney diseases diagnosis [3]. The normal levels of urea concentration in blood and urine are in the 0.15 - 0.4 g L-1 and 9.3 - 23.3 g L-1 ranges, respectively [4]. That determination is commonly carried out by the enzymatic hydrolysis of urea to produce ammonium ions, which are quantified by spectrophotometric methods based on Berthelot [5] and Nessler 22

*anchieta@iq.unesp.br https://orcid.org/0000-0002-8388-9866

Evaluation of Nitrate as Internal Standard for Quantitative Determination of Urea in Urine by Raman Spectroscopy

Article [6] reactions. However, these spectrophotometric methods require sample pretreatments and consume a large amount of reagents, which generates substantial amounts of residues and are time-consuming. Therefore, some electrochemical [7,8] and chromatographic [9,10] methods were proposed as alternatives. The electrochemical methods are based on the use of selective electrodes or biosensors with immobilized enzymes on the electrode surface [7,8]. Despite the low cost of these methods, they require rigid temperature and pH control, and are deeply susceptible to matrix interferences. Regarding chromatographic methods, clean up steps (sample pretreatments) are usually required. Furthermore, the high costs for equipment acquisition and maintenance restrict their use in small laboratories. Hence, the development of alternative methods of quantitative analysis that operate in adherence to the principles of Green Chemistry is attractive [11]. In this sense, laser-based techniques have showed potential for using in assorted chemical analyses [12]. The Raman vibrational spectroscopy [13] has been presented noteworthy advances in the last decades due to the possibility of direct and non-destructive sample analysis, instrumental simplicity, high analytical throughput and portability [14], and reduced interference caused by the sample fluorescence by using diode lasers (780 – 830 nm) [15]. Despite the technical improvements in Raman instrumentation in the last years, systematic and random errors are often observed and the choice of a proper calibration method is crucial to reduce/eliminate fluctuations caused by instrumental oscillations or matrix interferences [16]. The internal standardization calibration method is simple, fast, of easing construction and has been employed to minimize random/systematic errors [17]. The use of internal standard (IS) in Raman is reported in the literature with good results for different analytes (A) and samples (S). Among the main uses are (IS/A/S): 4-mercaptopyridine/3,4-methylenedioxymethamphetamine and α-methyltryptamine hydrochloride/drugs [18]; d-nicotine/nicotine/liquids for electronic cigarettes [19], amide/poly(3-hydroxybutyrate)/bacteria [20]; OH stretching band of water/sulfate/aqueous solution [21]; 1-propanethiol/polycyclic aromatic hydrocarbons/food contact materials [22]. However, the selection of a suitable IS for a specific problem is still a challenge. Despite of potentialities of both Raman and internal standardization for analytical purposes, little attention has been given for the use of internal standardization for urine quantitative analysis. Also, the evaluation of nitrate as IS for quantitative determination of urea in urine is not found in the literature. Thus, the goal of this work is the development of a simple, fast and green method for urea determination in urine by Raman spectroscopy and the evaluation of nitrate as a possible IS. MATERIALS AND METHODS Instrumentation Raman spectra were obtained using a B&W Tek iRaman BWS415-785H spectrometer with a 785 nm laser (power < 350 mW) and 3.5 cm-1 spectral resolution. The excitation and acquisition spectra of blanks, standards and urine samples were carried out by means of an optical fiber coupled to a B&W Tek BCR100A cuvette support. -1 All the Raman shifting spectra (n = 5) were obtained in the 150 - 2700 cm range employing the following instrumental parameters: 50% of laser power and 20 s integration time. The background of all spectra was corrected by using the Background Removal tool from the software BWSpec (B&W Tek). A Bel Photonics UV-M51 spectrophotometer was used for urea determination by the reference method. All measurements were carried out at 700 nm, and at temperate controlled of 37 °C using a SOLAB SL 150/10 thermostatic bath. Reagents, analytical solutions and samples High-purity deionized water obtained using a Millipore Rios 5TM reverse osmosis and a Millipore Milli-QTM Academic® deionizer system (resistivity 18.2 MΩ.cm) was used throughout for solutions preparation.

Fortunato, F. M.; Bechlin, M. A.; Ferreira, E. C.; Oliveira, S. R.; Neto, J. A. G.

Article Article A 100 g L-1 urea standard stock solution was prepared by dissolving 50 g of urea (Sigma Aldrich) in 500 mL of deionized water. Analytical solutions in the 100 - 700 mg L-1 interval were daily prepared by appropriate dilution of the stock solution. A 100 g L-1 nitrate standard stock solution was prepared by dissolving 67.2 g of NaNO3 (Merck) in 500 mL of deionized water. The reference method for urea determination was carried out by using the commercial Labtest kit containing the following solutions: solution 1 (phosphate buffer 10 mmol L-1 + urease: ≥ 268 kU L-1); solution -1 -1 -1 2 (phosphate buffer 100 mmol L + C7H5O3Na 312 mmol L + Na2[Fe(CN)5NO] 16.8 mmol L ); solution 3 (NaOH 2.8 mol L-1 + NaClO 121 mmol L-1); solution 4 (700 mg L-1 urea). These solutions were prepared and used according to the manufacturer instructions, as follow: 100 mL of solution 2 + 25 mL of solution 3 were diluted to 500 mL with deionized water; the solution 1 was mixed with the solution 2 and 20-fold diluted. All urine samples were analyzed by Raman spectroscopy without any previous sample preparation. For the reference method, samples were diluted 50-fold with deionized water. Analytical procedure The behaviors of nitrate and urea were evaluated at different laser power and integration time conditions. -1 -1 Raman scattering of a solution containing 40 g L urea + 10 g L nitrate were measured fixing the integration time at 10 s and varying the laser power from 10 to 90% maximum power. Thereafter, the laser power was fixed at 50% (optimized condition) and the integration time varied from 1 to 40 s. -1 -1 The linear working ranges for nitrate and urea were evaluated in the 0.5 g L - 50 g L intervals using 50% laser power and 20 s integration time. Urea was determined in five urine samples employing the proposed IS method. All blanks, standard solutions and samples were spiked with 10 g L-1 of nitrate as internal standard. Raman scatterings (Relative intensities) of blanks, standard solutions and samples were measured in triplicate using operating parameters fixed at 50% of laser power and 20 s integration time. The calibration curve was built up by plotting on the y-axis scattering measurements of urea/scattering measurements of nitrate (Iurea/Initrate) and on the x-axis the urea concentration. The contents of urea in samples were obtained by interpolation of measured scatterings of samples on that curve. For comparative purposes, all samples were also analyzed by a spectrophotometric reference method -1 and Raman spectroscopy with conventional external calibration (0 – 40 g L of urea). For the reference method, measurements of blanks, standards and diluted samples were carried out according to the following steps: in a test tube, it was added 20 µL of blanks, standards or diluted samples + 2 mL of the mixture 1:20 of solution 1 and solution 2 (buffered urease + salicylate + sodium nitroprusside) + 2 mL of the diluted solution 3 (NaOH + NaClO). After homogenizing, the tubes were immersed in a 37 °C bath for 5 minutes, cooled and measured at 700 nm.

Accuracy was also by means of addition and recovery tests for ﬁve urine samples spiked with 15 g L of urea and analyzed by the proposed IS method and conventional external calibration.

-1

RESULTS AND DISCUSSION Evaluation of nitrate as internal standard Urea is a nitrogen compound that presents the chemical formula (NH2)2CO and a Raman spectrum with a characteristic intense peak at nearly 1003 cm-1 referring to the N-C-N stretching [17]. The nitrate (NO3-) -1 presents an intense peak at nearly 1045 cm due to the N-O stretching [23]. Shown in Figure 1 is the -1 -1 Raman spectrum of 50 g L of urea and 5 g L of nitrate using 20 s integration time and laser ﬁxed at 50% of maximum power. Figure 1 shows that the nitrate and urea peaks are well resolved (do not occur overlaps) and appear in the same spectral region, which allows the simultaneous monitoring. These ﬁndings make nitrate a possible candidate as IS for urea determination.

Evaluation of Nitrate as Internal Standard for Quantitative Determination of Urea in Urine by Raman Spectroscopy

Article

Figure 1. Raman spectrum of 50 g L-1 of urea and 5 g L-1 of nitrate using laser power ﬁxed at 50% and integration time ﬁxed at 20 s.

Considering the requisite of internal standardization method by which the behavior of the IS should mimic that of the analyte, the performance of nitrate as IS for urea was evaluated by studying the behavior of nitrate and urea standards at different integration times and excitation conditions. First, Raman scattering of nitrate and urea were measured by fixing the integration time at 10 s and varying the laser power from 10 to 90% of maximum power. It was found an increase in the signals of both nitrate and urea with increasing laser power (Figure 2). This figure shows that both species presented similar behaviors since the ratio (Iurea/Initrate) remains almost constant in the 20 - 90% laser power interval. Thereby, eventual fluctuations in the power laser during the measurements do not reflect in significant errors if the internal standardization is used.

Figure 2. Inﬂuence of laser power on Raman scattering of urea (-■-), nitrate (-●-) and ratio urea/nitrate (-▲-). Signals obtained with integration time ﬁxed at 10 s. Relative intensity means the Raman scattering measurements. Iurea/Initrate means the ratio scattering measurements of urea/scattering measurements of nitrate.

Fortunato, F. M.; Bechlin, M. A.; Ferreira, E. C.; Oliveira, S. R.; Neto, J. A. G.

Article The behavior of the urea and nitrate was also evaluated by changing the integration time (1 - 40 s) but ﬁxing the laser power at 50% of the maximum. Figure 3 shows that signals of both urea and nitrate increased with increasing integration time. These ﬁndings show that nitrate and urea present similar behavior since the ratio (Iurea/Initrate) remains almost constant in the monitored interval. Considering nitrate and urea showed similar behaviors through variations in the main instrumental conditions of Raman technique, the nitrate was considered a potential IS for urea determination in urine samples, so it was further evaluated in real samples.

Figure 3. Inﬂuence of integration time on Raman scattering of urea (-■-), nitrate (-●-) and ratio urea/nitrate (-▲-). Signals obtained with laser power ﬁxed at 50%. Relative intensity means the Raman scattering measurements. Iurea/Initrate means the ratio scattering measurements of urea/ scattering measurements of nitrate.

Optimization of the method The generation of intense background fluorescence for some workable samples and spectrometers may hinder the observation of the target Raman spectrum [24]. Preliminary tests carried out in urine samples showed noteworthy background fluorescence. Considering the signal is proportional to the power of the laser exciting the sample, the laser power was elected as 50% of maximum as a compromise between required sensitivity and sample preservation (possibility of burning when exposed to full laser power). When the integration time was evaluated (10, 15, 20, 25 and 30 s) in a urine sample, an increase in the Raman signal of urea and the shot noise (noise proportional to the intensity of the either Raman or fluorescence) was observed (Figure 4). Integration times 25 s resulted in lower signal-to-noise (SNR) ratios. Longer exposure time did not improve SNR. The shot noise caused by the fluorescence signal contributes more to the spectrum noise level than other sources of noise. So, the optimized parameters for further experiments were laser power at 50% of maximum and 20 s integration time.

Figure 4. Inﬂuence of integration time (10 – 30 s) on Raman shift corresponding to an urine sample using laser power at 50% of maximum. 26

Evaluation of Nitrate as Internal Standard for Quantitative Determination of Urea in Urine by Raman Spectroscopy

Article Afterwards, the linear working ranges for nitrate and urea were evaluated by measuring the Raman scatterings for nitrate and urea standards. Curves with good correlation coefficients (r ≥ 0.9996) for nitrate -1 -1 and urea in the 0.5 - 50 g L and 1 - 50 g L , respectively, were found. The concentration of a given candidate as IS should be within the limit of quantification and the upper limit of linear response, and the optimum concentration of it depends on its usual concentration in urine samples and the required precision. The use of 10 g L-1 of nitrate as IS was chosen taking into consideration the linear working range for nitrate and one of the requisites of internal standardization by which the IS concentration must be fixed and constant in blanks, standards and samples [25]. The use of high concentration of IS may be an effective strategy to make negligible signal fluctuations that may occur due to variations of analyte in workable -1 -1 samples like human urinary nitrate. Analytical curves in the range of 1 – 50 g L plus 10 g L of nitrate was adopted for further studies. Determination of urea in urine After method optimization, five urine samples were analyzed by the IS proposed method. Urea -1 concentrations ranged from 7.6 to 16.8 g L (Table I). For comparative purposes, the samples were also analyzed by a reference method based on spectrophotometry. Analysis of Table I reveals that values obtained by both methods are in agreement at 95% confidence level (paired-t-test). Indeed, it shows that the use of IS improved precision. The relative standard deviations (RSD) associate to the urine sample #4 analyzed successively (n=12) by the proposed IS method, reference method and conventional external calibration were in the ranges of 1.4 - 3.9% (IS), 3.8 - 6.3% (Reference method), and 1.8 - 7.5% (Conventional external calibration). Table I. Results (g L-1) of the determination (n=3) of urea in urine samples by the proposed (IS), reference and conventional external calibration methods.

The performance of the proposed method was also evaluated by addition and recovery tests. Recoveries were in the 97 - 103% interval (Table II). The application of internal standardization improved the recoveries in comparison with those obtained by conventional external calibration (87 - 108%). Also, the IS furnished better precision: RSD improved from 1.8 – 7.5% (without IS) to 1.4 – 3.9% (with IS). Table II. Recoveries (%) for 15 g L-1 urea added to urine samples determined by the proposed method (IS) and conventional external calibration.

Fortunato, F. M.; Bechlin, M. A.; Ferreira, E. C.; Oliveira, S. R.; Neto, J. A. G.

Article CONCLUSION Nitrate was an IS eﬀective to the direct determination of urea in urine samples by Raman spectroscopy. The proposed method furnished better precision in comparison with the conventional external calibration and reference spectrophotometric method. In general, the proposed IS method here is simple, robust and may be considered environmentally friendly since it provides faster results, minimum sample preparation and lower reagent consumption and minimum waste generated than the reference method.

ACKNOWLEDGMENTS The authors thank the Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico - CNPq for ﬁnancially supporting this work (Project 471453/2013-7) and the fellowship granted to J.A.G.N. (303255/2013-7). São Paulo Research Foundation - FAPESP is also thanks for the fellowship to F.M.F. (2012/23323-7). Manuscript received Nov. 14, 2017; revised manuscript received Feb. 20, 2018; accepted March 27, 2018

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Br. J. Anal. Chem., 2018, 5 (19), pp 29-37 DOI: 10.30744/brjac.2179-3425.2018.5.19.29-37

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Prediction of Glucose, Fructose and Sucrose content in Cassava (Manihot esculenta Crantz) genotypes from Amazon using PLS models Arthur Abinader Vasconcelos1*, Roberto Lisboa Cunha2, Elisa Ferreira Moura Cunha2, Willison Eduardo Oliveira Campos3, Paulo Sérgio Taube1, Heronides Adonias Dantas Filho3 1

Universidade Federal do Oeste do Pará, Rua Vera Paz s/n, CEP: 68035-110, Santarém, PA, Brazil 2 Embrapa Amazônia Oriental, Trav. Dr. Enéas Pinheiro, s/n , CEP: 66095-903, Belém, PA, Brazil 3 Universidade Federal do Pará, Rua Augusto Corrêa, 01, CEP: 66075-110, Belém, PA, Brazil

Graphical Abstract

The mid infrared spectrum shows a facile method to quantify glucose, fructose and sucrose obtaining PLS models with considerable robustness.

The chemical characterization by classical methods requires a long time of analysis and the use of expensive and environmentally aggressive reagents. The use of the partial least squares (PLS) tool applied to FT-MIR data represents a reduction of these considered variables. The relative contributions of glucose, fructose, and sucrose obtained for the 26 cassava samples varied between 0.111-0.383 g/100g; 0.0317-0.256 g/100g and 0.286-0.775 g/100g, respectively. For ﬁve latent variables the mean of predicted glucose content in external samples was 0.220 g/100g and had the RMSEP value of 0.00590 g/100g; The best number of LVs for the prediction of the fructose content for new samples were ﬁve, where the mean of the predicted value was 0.0994 g/100g against the mean fructose reference value 0.0879 g/100g, with a 0.0115 g/100g RMSEP; The mean sucrose content in the external samples was 0.451 g/100g, compared with the reference value 0.515 g/100g, with a RMSEP 0.138 g/100g. The use of the PLS1 algorithm generated two good models with the second derivative in spectral data and one with the raw data in spectral data using four samples external to the prediction step.

Keywords: Partial least squares, PLS, cassava, spectroscopy, Amazon region. INTRODUCTION Cassava root has great importance both in economic and nutritional aspects, since the genus has approximately 98 species, with Manihot esculenta being the most cultivated in tropical regions [1]. The consumption of cassava in Africa is great, and in Mozambique its marketing can reach more than 240 million dollars. In other regions of the world, it is consumed by approximately 700 million people [2,3]. The content of glycines in the cassava root is closely related to the starch content present because this plant structure is essentially starchy with low protein, lipid and ash contents. Other carbohydrates of diﬀerent molecular weights may be present, from sugars to glycosides and cellulosic material. Therefore, the root is a fundamental raw material for obtaining sugars by hydrolysis process, which has been used in recent years by the sugar industry, mainly in enzymatic processes to obtain glucose and fructose syrups, used in the pharmaceutical and food industry [4]. *arthurnadervas@yahoo.com.br https://orcid.org/0000-0003-4496-6475 29

Article

Prediction of Glucose, Fructose and Sucrose content in Cassava (Manihot esculenta Crantz) genotypes from Amazon using PLS models

Sucrose levels in vitro culture influence diverse metabolic processes in cassava such as tissue differentiation and growth. The trend of studies involving sugars content in amylaceous sources is reflected in the ethanol obtained from the hydrolysis of cassava starch from syrups. The improvement of instrumental methodologies with few uses of the high amount of chemicals is a good environmental alternative. Among these techniques, the Fourier transform medium infrared spectroscopy (FT-MIR) stands out because it is a simple, fast and inexpensive technique that has been used with chemometric tools such as Partial Least Squares Regression (PLS) to quantify some classic parameters of local models. The medium infrared (MIR) region shows promise since foods are mainly composed of water, carbohydrates, proteins, lipids and other minor components such as vitamins and minerals, all of these compounds have the ability to absorb radiation. Thus, the quantification of the compounds is possible from the evaluation of the intensity and region characteristic of the absorption bands of the main functional groups present. If signals are intercepted, it is often possible to extract spectral data more efficiently using multivariate mathematical methods [5]. Many authors report the importance of using chemometric tools coupled to infrared spectroscopy to evaluate some chemical properties of food, where PLS modeling has been the most widely used chemometric tool in recent years [6-15]. The multivariate calibration models generated for a universe of samples allow the creation of a local model in which new samples with similar spectral characteristics can be inserted and the parameter is quickly determined. Thus, it is increasingly necessary to explore this procedure, as shown by some recently published studies [16,17]. The present work aims to use data from a reference method for quantification of glucose, fructose, and sucrose to create PLS models by FT-MIR spectral data. MATERIALS AND METHODS Sample preparation A total of 30 cassava genotypes were planted in the municipality of Igarapé Açu, Pará state, Brazil (01°07'33"S and 47°37'27"W) in an active germplasm bank of an experimental area of the Brazilian Company of Agricultural Research (Embrapa). The 26 samples were used for the construction of calibration models and 4 samples were used as external samples for prediction. The general characteristics of the planting site are medium-textured yellow latosol, "Am" type in the Köppen classification, with high annual precipitation (over 2,350 mm). The average annual temperature is around 25 ºC and relative humidity around 85%. After one year of planting, the samples were harvested, taken with running water, packed in plastic bags and stored under refrigeration between -18 and -4 ºC. They were peeled and cut into plastic planks, forming disks with approximately 2 cm, and separated in triplicates. After cutting, samples were ground and transferred to 50 mL Falcon tubes previously decontaminated with 10% (v/v) HCl aqueous solution and taken to the freezer where they remained at -18 °C for 24 h. The frozen samples were then lyophilized for 96 h to ensure removal of as much water as possible. The lyophilized samples were then crushed in an agate mortar and transferred to 80 mL plastic vials previously decontaminated with 10% (v/v) HCl aqueous solution and stored in dissent at room temperature until further analysis. Determination of glucose, fructose, and sucrose by Elisa method A mass of 0.2 g of fresh root was macerated and the sugars were extracted with 80% (v/v) of ethanol. An aliquot of 5 μL of ethanolic extract and 5 μL of hexokinase, phosphoglucoisomerase and β-fructosidase enzymes were used to determine the different sugars. Absorbance reading was performed on an ELISA plate reader (Thermo Scientific Multiskan® FC Microplate Photometer) using a wavelength of 340 nm [18].

Vasconcelos, A. A.; Cunha, R. L.; Cunha, E. F. M.; Campos, W. E. O.; Taube, P. S.; Dantas Filho, H. A.

Article FT-MIR analysis Analysis by medium infrared spectroscopy was performed by the ATR method. Samples were used in triplicates using 1 mg of each lyophilized sample. The background of each sample was obtained by the environment spectral record. The samples were analyzed on infrared spectrometer as Fourier transform (Thermo Fisher Scientific Inc., model Nicolet) with a spectral range between 4000 and 500 cm-1. The instrumental analysis parameters were: spectral -1 resolution 4 cm , 32 scans per spectrum, correcting the bands of water and carbon dioxide. Multivariate analysis The PLS models were created from FT-MIR data, which were processed by the software Unscrambler 9.1© (CAMO Software AS). The spectral data were converted to the matrix form using mean absorbance -1 values obtained in 600-1450, 2900-3000 and 3300 cm . The validation method adopted was cross-validation of the complete segment of a sample. The elimination of anomalous samples was done according to the leverage values and the residual variance of matrix Y, in addition to the automatic detection by the program. The data of the matrices X and Y were not weighted, initially being constructed models for 10 PCs (principal components), observing the precision of each model with the elimination of abnormal samples and PC reduction. The data from both matrices were centered on the mean, the models were constructed by performing pre-processing of the spectral data to verify the optimization of the spectral responses in the determination of each parameter, obtaining a total of 9 models constructed for each parameter. The spectral preprocesses used to construct the models were: raw data, normalized data, normalized first derivative data, normalized second derivative data, first derivative MSC, second derivative MSC, MSC with normalization, first derivative and second derivative. For the prediction step the spectra of 4 external cassava samples, in the same wavenumbers of calibration samples were used, using the average content of each parameter as a reference value. RESULTS AND DISCUSSION Sugars contents by ELISA analysis The analytical parameters of validation to Elisa analysis are shown in Table I. Table I. Validation parameters for sugars quantification

Parameter

Glucose

Fructose

Sucrose

LOD ( g/100 g)

0.0105

0.0120

0.0160

LOQ ( g/100 g)

0.0315

0.0300

0.0480

Recovery (%)

120

115

105

Article

Prediction of Glucose, Fructose and Sucrose content in Cassava (Manihot esculenta Crantz) genotypes from Amazon using PLS models

The glucose, fructose and sucrose contents obtained by Elisa analysis are shown in Table II. Table II. Contents of sugars in cassava samples

Sam ples ID

Glucose (g/100g)

Fructose (g/100g)

Sucrose (g/100g)

0.258 ± 0,0712

0.103 ± 0.00230

0.775 ± 0.124

0.201 ± 0.0320

0.0507 ± 0.00193

0.382 ± 0.00291

0.185 ± 0.0173

0.0391 ± 0.00278

0.464 ± 0.0545

0.173 ± 0.0477

0.0521 ± 0.0364

0.616 ± 0.0309

CP1

0.288 ± 0.114

0.0375 ± 0.0165

0.502 ± 0.001

0.282 ± 0.0808

0.182 ± 0.0514

0.428 ± 0.0557

CP2

0.217 ± 0.0721

CP3

0.297 ± 0.0703

0.0751 ± 0.00096 9

0.439 ± 0.0616

0.236 ± 0.0302

0.0704 ± 0.0063 0

0.508 ± 0.0889

0.216 ± 0.0875

0.0356 ± 0.0037 5

0.479 ± 0.0406

0.347 ± 0.156

0.196 ± 0.071 7

0.337 ± 0.00551

0.227 ± 0.0596

0.0826 ± 0.093 1

0.394 ± 0.0754

0.383 ± 0.125

0.104 ± 0.0032 7

0.471 ± 0.0457

0.244 ± 0.0766

0.0697 ± 0.0075 1

0.736 ± 0.0137

0.324 ± 0.0748

0.141 ± 0.031 7

0.616 ± 0.0125

0.343 ± 0.0640

0.256 ± 0.043 5

0.529 ± 0.0234

0.189 ± 0.0436

0.0697 ± 0.0049 7

0.759 ± 0.0267

AMR

0.164 ± 0.0708

0.0591 ± 0.0033 9

0.624 ± 0.0772

P24

0.190 ± 0.0496

0.0612 ± 0.0056 9

0.637 ± 0.0469

BMG

0.124 ± 0.00497

0.0911 ± 0.0016 9

0.387 ± 0.00254

0.115 ± 0.0342

0.0567 ± 0.016 8

0.303 ± 0.0209

SAP

0.166 ± 0.0172

0.122 ± 0.0077 5

0.452 ± 0.0963

0.0966 ± 0.0304

0.0317 ± 0.0057 5

0.286 ± 0.0136

0.194 ± 0.0234

0.0779 ± 0.0030 3

0.767 ± 0.00887

0.111 ± 0.0132

0.0662 ± 0.012 6

0.610 ± 0.0704

CPATU

0.172 ± 0.0127

0.0411 ± 0.0032 7

0.473 ± 0.0203

0.0881 ± 0.0093

0.489 ± 0.0182

FT-MIR and multivariate calibration results A total of 26 spectra were generated for each sample in triplicate (N=3), after this, the mean of spectra is calculated. The spectrum of the 26 samples is shown in Figure 1.

Vasconcelos, A. A.; Cunha, R. L.; Cunha, E. F. M.; Campos, W. E. O.; Taube, P. S.; Dantas Filho, H. A.

Article

Figure 1. Mean FT-MIR spectrum of each sample.

The spectral data shown in Figure 1 allows extracting the main region responsible to inform carbohydrate fingerprints to generate models based on nine pre-treatments. The regions that were extracted varied from 1000 to 1400 cm-1, which includes the fingerprint region (900-1200 cm-1), useful in the analysis of -1 carbohydrates [19]. In addition to the region from 3229.61 to 3301.53 cm , which contains more relevant information about the OH group from the hydrocarbon chains. The correlation coefficient in the calibration step reached the ideal value (≈ 1.0) with a lower number of PCs. Table III shows the diagnostic parameters of the calibration and validation steps for the glucose prediction model. Table III. Calibration and validation parameters for each glucose model

Model

RMSEC

Y- explained variance/%

RMSECV

Crude dataa

0.0513 (3 b)

56.00 (3)

0.7483 (3)

0.0608 (2)

MSC/1st Derivativea

0.0359 (6)

69.57 (6)

0.8341 (6)

0.0509 (2)

MSC/2nd Derivative

0.0156 (6)

95.89 (6)

0.9792 (6)

0.0888 (2)

MSC/Normalization

0.0065 (6)

98.98 (6)

0.9949 (6)

0.0830 (2)

Normalization/1st Derivative

0.0167 (6)

94.74 (6)

0.9733 (6)

0.0822 (3)

Normalization/2nd Derivative

0.0226 (6)

90.90 (6)

0.9534 (6)

0.0895 (1)

Normalization

0.0086 (6)

98.02 (6)

0.9900 (6)

0.0459 (8)

1st Derivative

0.0229 (6)

89.83 (6)

0.9478 (6)

0.0933 (4)

2nd Derivative

0.0078 (6)

98.86 (6)

0.9943 (6)

0.0926 (3)

Models submitted to Marten's uncertainty test; Number of PCs.

According to Table II, the calibration models with the best diagnostic parameters were those with MSC/ Normalization and second derivative, so the next step was to verify the predictive power of the glucose content of each one for the external samples. Figure 2 shows the prediction of the model with an only second derivative of the spectral data, proving that this better predicts the glucose content in new samples compared to the average of glucose level.

Figure 2. Prediction of glucose content. 33

Article

Prediction of Glucose, Fructose and Sucrose content in Cassava (Manihot esculenta Crantz) genotypes from Amazon using PLS models

For four latent variables, the predicted mean glucose content had the RMSEP value of 0.0059 g/100g (second derivative) versus 1.9266 g/100g (MSC/normalization) for the same number of latent variables. The mean value predicted for glucose was 0.220 g/100g versus the mean value of 0.226 g/100g determined by Elisa method. For six latent variables, the second derivative model had RMSEP and SEP equal to 0.0063 g/100g and 0.0011 g/100g respectively, while the other model had RMSEP and SEP equal to 2.1686 g/100g and 0.2079 g/100g respectively. The high SEP value indicated that although the calibration set was adequate, the model has probably high residues as the result of pre-processing of the spectral data. Authors evaluating cassava biomass parameters by PLS-DA regression found higher prediction errors for 4 latent variables in the prediction of ash, potassium and chlorine contents [20]. The errors had a factor of 10 relative to the errors found, showing the robustness of the obtained model. Other authors report the use of PLS to Lotus seed infrared data, generating models with a maximum of 4 latent variables for parameter prediction, however, the authors performed few pre-treatments [21]. Some authors obtained diďŹ&#x20AC;erences in glucose prediction parameters by the diďŹ&#x20AC;erential use of accessories in the spectrometer. Reporting SEP value between 0.46 and 0.38 mg/g for the use of ATR mode and dial-path respectively [22]. Thus, not only logical data processing is fundamental in a PLS analysis, but the proper use of accessories in the equipment as well. Table IV shows the performance parameters of the models created for the prediction of fructose content. Table IV. Calibration and validation parameters for each fructose model

number of PCs

The best models obtained with better parameters of calibration and validation were second derivative, MSC/Normalization, Normalization/1st Derivative and Normalization / 2nd Derivative. The models that had the best RMSECV value with 1 principal component were inadequate, due to the increase of error. Among the best models, the one with the second derivative was the most robust, because the validation error reached the lowest value with only 2 LVs in comparison to the others. To prove that this model was able to predict better than the other three that are shown in Table V. Table V. RMSEP values to predict fructose contents in external samples

Vasconcelos, A. A.; Cunha, R. L.; Cunha, E. F. M.; Campos, W. E. O.; Taube, P. S.; Dantas Filho, H. A.

Article The RMSEP values clearly show the good ability of the second derivative model to predict fructose levels in new samples with the decrease of the error associated. The model was the only one that had a good decrease of prediction error with the increase of latent variables. The best number of LVs for the prediction of the fructose content for new samples were four, where the mean of the predicted value is 0.0994 g/100g versus the average of the reference 0.0879 g/100g. Figure 3 shows the prediction of fructose in external samples.

Figure 3. Prediction of fructose content.

Authors performed the coupling of Raman spectra obtained from prebiotic sugars with PLS analysis, obtaining weak correlations of the calibration and validation stages of the models, respectively, in the values of 0.989 and 0.984 [19]. Contrasting the best model obtained for fructose in which correlation assumed the value of 0.9941. In the determination of functional compounds in baby food, authors report the creation of prediction models using NIRS data. These models presented high values of prediction error even after performing st spectral pre-treatments, the 1 derivative, SNV and centering in the mean [23]. These results conďŹ rm the robustness of the model for prediction of fructose in relation to the most recent published works. Table VI shows the calibration and validation parameters for each ideal model considered for sucrose prediction. Table VI. Calibration and validation parameters for each sucrose model

Models submitted to Marten's uncertainty test; bNumber of PCs

The elucidation of the predictive capacity of the models is shown in Table VII, considering only three latent variables. Table VII. RMSEP values to predict sucrose contents in external samples

Article

Prediction of Glucose, Fructose and Sucrose content in Cassava (Manihot esculenta Crantz) genotypes from Amazon using PLS models

These prediction error values have shown that the crude data model has the minimum RMSEP value for only two LVs, this error value being smaller than others. This model was the only one that had the decrease of prediction error with the increase of latent variables. The elimination of some wavenumbers improved the model, predicting a mean sucrose content in external samples of 0.4510 g/100g, compared with the reference value 0.5150 g/100g. Figure 4 shows the prediction in the external samples for this model.

Figure 4. Prediction of sucrose content.

Experiments involving chemical characterization of cassava with FT-MIR and construction of prediction models with PLS are not mentioned in the literature. Some authors use PLS together with infrared spectroscopy data to predict physicochemical properties. These authors had ﬁgures of merit and number of latent variables higher compared to models obtained using medium infrared spectroscopy and Raman spectroscopy [24,25]. In spite of this, some authors report that FT-MIR spectroscopy is a technique with great advantage in showing a spectrum directly related to chemical information, as it was observed in the case of glucose, fructose and sucrose which have been quantiﬁed in many agricultural matrices by many methods among which infrared spectroscopy has been highlighted [26,13]. Some authors have used FT-MIR spectroscopy coupled to PLS regression to quantify many properties, generating models with RMSEP in the range of 4.3000-5.9000 g/100g to quantify phenolic compounds in red grape [27,28]. The use of this coupling was reported involving Raman spectroscopy, showing a great possibility of applying the PLS regression with spectroscopy techniques [29]. In the determination of phenolic compounds in wine, some authors have found prediction error values ranging from 0.99 to 3.24 g/100g. The use of the PLS1 algorithm is also reported as more advantageous in the creation of models for the prediction of sucrose, presenting RMSEP reduction of 13.6 to 10.0 g/L in the prediction of the reference average content in direct determination of fructooligosaccharides treated by β-fructofuranosidases enzymes [30]. CONCLUSIONS The pre-processing of spectral data was used to stabilize the constructed models. At this point, the second derivative was the best pre-processing for the prediction of glucose and fructose and the crude data explained better sucrose contents. This work has shown that it is possible to do less use of classical analysis techniques in the repeatability of the determination of essential compounds in food matrices, from the creation of local models of multivariate regression by partial least squares. It is always important to expand the sampling to create ideal models of the studied properties, a factor that must be taken into account by future work. Manuscript received Jan. 26, 2018; revised manuscript received April 23, 2018; accepted May 2, 2018.

Vasconcelos, A. A.; Cunha, R. L.; Cunha, E. F. M.; Campos, W. E. O.; Taube, P. S.; Dantas Filho, H. A.

Article REFERENCES 1. Bayoumi, S. A. L.; Rowan, M. G.; Beeching, J. R.; Blagbrough, I. S. Phytochemistry, 2010, 71, pp 598-604. 2. http://www.embamoc.co.za/trade-industry/ [Accessed 23 January 2018]. 3. Albuquerque, J. A. A.; Sediyama, T.; Silva, A. A.; Sediyama, C. S.; Alves, J. M. A.; Neto, F. A. Brazilian Journal of Agricultural Sciences, 2009, 4, pp 388-394. 4. Silva, R. N.; Quintino, F. P.; Monteiro, V. N.; Asquieri, E. R. Ciênc. Tecnol. Aliment., 2010, 30, pp 213-217. 5. Karoui, R.; Downey, G.; Blecker, C. Chemical Reviews, 2010, 110, pp 6144-6168. 6. Souza, A. M. de; Poppi, R. J. Química Nova, 2012, 1, pp 223-229. 7. Pierce, K. M.; Kehimkar, B.; Marney, L. C.; Hoggard, J. C.; Synovec, R. E. J. Chromatogr. A, 2012, 3, p 1255. 8. Gy, B.; Ovádi, Z.; Salgó, A. J. Near Infrared Spectrosc., 1998, 6, p 341. 9. Ferreira, M. M. C.; Antunes, A. M.; Melgo, M. S.; Volpe, P. L. O. Química Nova, 1999, 22, p 724. 10. Rebouças, M. V.; Barros Neto, B. J. Near Infrared Spectrosc., 2001, 9, p 263. 11. Sun, D. W. Infrared Spectroscopy for Food Quality Analysis and Control, Netherlands, 2009, chapter 1, p 275. 12. Scibisz, I.; Reich, M.; Bureau, S.; Gouble, B.; Causse, M.; Bertrand, D.; Renard, C. M. G. C. Food Chem., 2011, 125, p 1390. 13. Magwaza, L. S.; Opara, U. L.; Cronje, P. J. R.; Landahl, S.; Nieuwoudt, H. H.; Mouazen, A. M.; Nicolaï, B. M.; Terry, L. A. Scientia Horticurae, 2014, 165, p 421. 14. Sánchez, T.; Ceballos, H.; Dufour, D.; Ortiz, D.; Morante, N.; Calle, F.; Felde, T. Z.; Domínguez, M.; Davrieux, F. Food Chem., 2014, 151, p 444. 15. Alander, J. T.; Bochko, V.; Martinkauppi, B.; Saranwong, S.; Mantere, T. Int. J. Spectrosc., 2013, 36, p 2013. 16. Wei, M.; Geladi, P.; Lestander, T. A.; Xie, G.; Xiong, S. Anal Bioanal Chem, 2015, 407, p 5443. 17. Xu, L.; Chai, C. B.; She, Y. B.; Chen, L. J. J. Spectrosc., 2015, 8, p 2015. 18. Stitt, M.; Lilley R. M.; Gerhardt, R.; Heldt, H. W. Methods in Enzymol., 1986, 174, p 518. 19. Santos, M. I.; Andrade, C. A.; Tymczyszyn, E. E.; Zvaglia, A. G. Food Res. Int., 2014, 64, pp 514-519. 20. Wei, M.; Geladi, P.; Lestander, T. A.; Xie, G.; Xiong, S. Anal Bioanal Chem, 2015, 407, p 5443. 21. Xu, L.; Chai, C. B.; She, Y. B.; Chen, L. J. J. Spectroscopy, 2015, 8, p 2015. 22. Ayvaz, H.; Santos, A. M.; Moyseenko, J.; Kleinhenz, M.; Saona, L. E. R. Plant Foods Hum Nutr, 2015, 70, p 215. 23. Cascant, M. M.; Garrigues, S.; de la Guardia, M. Anal Bioanal Chem, 2015, 407, p 1961. 24. Henrique, C. M.; Teóﬁlo, R. F.; Sabino, L.; Ferreira, M. M. C.; Cereda, M. P. J. Food Sci., 2007, 72, p E184. 25. Almeida, M. R.; Alves, R. S.; Nascimbem, L. B. L. R.; Stephani, R.; Poppi, R. J.; de Oliveira, L. F. C. Anal Bioanal Chem, 2010, 397, p 2693. 26. Ruiz-Altisent, M.; Ruiz-Garcia, L; Moreda, G.P.; Lu, R.; Hernandez-Sanchez, N.; Correa, E.C.; Diezma, B.; Nicolai, B.; García-Ramos, J. Computer and Electronics in Agriculture, 2010, 74, p 176. 27. Fragoso, S.; Aceña, L.; Guasch, J.; Mestres, M.; Busto, O. J. Agric. Food Chem., 2011, 59, p 10795. 28. Fragoso, S.; Aceña, L.; Guasch, J.; Busto, O.; Mestres, M. J. Agric. Food Chem., 2011, 59, p 2175. 29. Meza-Márquez, O. G.; Gallardo-Velázquez, T.; Dorantes-Álvarez, L.; Osorio-Revilla, G.; Arana, J. L. de La R. Analyst, 2011, 136, p 3355. 30. Trollope, K. M.; Volschenk, H.; Görgens, J. F.; Bro, R.; Nieuwoudt, H. H. Anal Bioanal Chem, 2015, 407, p 1661.

Br. J. Anal. Chem., 2018, 5 (19), pp 38-53 DOI: 10.30744/brjac.2179-3425.2018.5.19.38-53

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Exploratory on-line Pyrolysis and Thermally Assisted Hydrolysis and Methylation for evaluating NonHydrolyzable Organic Matter in Anthropogenic Soil from Central Brazilian Amazon Paulo S. Taube1*, Douglas S. Silva1, Arthur A. Vasconcelos1, Lilian Rebellato2, 3 4 Luiz A. dos Santos Madureira , Fabricio A. Hansel 1

Instituto de Biodiversidade e Florestas, Universidade Federal do Oeste do Pará, Rua Vera Paz, s/n, Santarém – PA, 68005-100, Brazil. 2Instituto de Ciências da Sociedade, Universidade Federal do Oeste do Pará, Rua Mendonça Furtado, 2946, Santarém – PA, 68040-079, Brazil. 3Departamento de Química, Universidade Federal de Santa Catarina, Campus Universitário Trindade, CP 476, Florianópolis – SC, 88040-900, Brazil. 4Embrapa Florestas, Estrada da Ribeira, km 111, cx 319, Colombo – PR, 88411-000, Brazil.

Graphical Abstract

Amazonian dark earths (ADE) are characterized by high fertility and organic carbon content, the latter being associated with black carbon (BC). BC is recalcitrant in nature due to its aromatic building blocks and it is expected to remain in the non-hydrolyzable fraction after the sequential extraction of soil organic matter (SOM). In this context, the aim of this study was to compare the composition of non-hydrolyzable SOM samples from an ADE and an adjacent soil using pyrolysis - gas chromatography - mass spectrometry and thermally-assisted hydrolysis and methylation with tetramethylammonium hydroxide. We tested two hypotheses: (i) non-hydrolyzable organic matter preserves BC in ADE and adjacent soils; and (ii) pyrolysis products are also produced thermally and are different for ADE and adjacent soils. The results of this study showed that polycyclic aromatic hydrocarbons were the main pyrolysis products for both soils. In addition, the benzene / toluene, naphthalene / methylnaphthalene and benzofuran / methylbenzofuran ratios of the pyrolysis products observed in the case of ADE were around two times higher than the corresponding values for the adjacent soil, except in the surface horizon, which indicates the presence of a higher recalcitrant BC in the ADE. The specific organic matter sources for the ADE and adjacent soil could not be differentiated. Keywords: black carbon; charcoal; carbonization; Py-GC-MS; THM-GC-MS. *pstjunior@yahoo.com.br https://orcid.org/0000-0001-5786-7615 38

Taube, P. S.; Silva, D. S.; Vasconcelos, A. A.; Rebellato, L.; Madureira, L. A. S.; Hansel, F. A.

Article INTRODUCTION Anthropogenic soils called Amazonian dark earths (ADE) can be found in the Amazon region. They have surface horizons with a dark color and contain ceramic material, lithic artifacts, charcoal, fish and mammal bones, human burial remains and detritus [1]. ADE are fertile soils with high contents of Ca, Mg, Zn, Mn, P (total and available) and carbon in comparison with adjacent soils [2-9]. The dark color is related to the presence of large amounts of pyrogenic carbon, i.e., black carbon (BC) [10-11]. The high concentration of BC in ADE results from incomplete combustion of organic matter (OM) during low intensity burning [5,12], slash-and-char is another potential explanation [6,13-14]. Biochar plays a significant role in the carbon cycle and it can be used in sustainable agricultural practices [15]. This pyrogenic carbon may be modified by different processes after deposition and incorporation into soil [16]. The degradation rate of BC is dependent on the temperature of the burning, intensity of carbonization, fuel source and soil conditions [17]. Laboratory incubation experiments have shown that ADE under cultivation has both rapidly mineralizable in very stable types of soil organic matter (SOM) [18]. The recalcitrance of the stable SOM is due to physical or chemical interaction with soil minerals and the intrinsic stability of the condensed aromatic structures of BC [11,18]. The incomplete combustion of wood or other fuel material generates a wide range of products with diverse sources and recalcitrant BC formed anthropogenically or through natural fires can be preserved in the non-hydrolyzable fraction [19]. A previous study of extractable and hydrolysable SOM revealed some differences between anthropogenic and natural soils [9]. Pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) and thermally-assisted hydrolysis and methylation (THM-GC-MS), using tetramethylammonium hydroxide (TMAH), can provide information on the macromolecular structure of SOM, including BC [20-24]. Nevertheless, some limitations are associated with the use of Py-GC-MS for the characterization of charred OM in soils, including: (i) only a semi-quantitative response is possible [24-27]; (ii) carboxylic groups are often not detected [24-26]; (iii) aromatic compounds are formed in situ at high pyrolysis temperatures [24,28-30]; and (iv) highly condensed BC is stable under pyrolysis conditions [24,30-32]. Furthermore, THM-GC-MS can provide additional information on SOM with respect to carboxyl, hydroxyl and phenolic groups, since they are transformed into methyl esters and methyl ethers, which are more easily identified and separated by GC-MS than their underivatized counterparts [26,33-34]. In this context, the aim of this study was to evaluate the presence of BC in the non-hydrolyzable SOM fraction and to compare the composition of samples of this fraction obtained from ADE and an adjacent soil using Py-GC-MS and THMGC-MS. The hypotheses considered were: (i) non-hydrolyzable SOM preserves BC in the case of ADE; and (ii) Py-GC-MS and THM-GC-MS products show differences between the ADE and the adjacent soil. MATERIALS AND METHODS Sample location The ADE and adjacent soil (AS) samples were collected from the Caldeirão Experimental Station (Embrapa, Iranduba - AM, Brazil; 03º14'22”-03º15'47”S and 60º13'02”-60º13'50”W). Samples from two soil profiles, one ADE and one non-anthropogenic adjacent soil, were collected at four different depths for each profile. The soil with an anthropogenic horizon was classified as Pretic Anthrosol (Orthodystric, Clayic) and the non-anthropogenic soil as Haplic Xanthic Acrisol (Hyperdystric, Clayic) [35]. These soils were formed in the Tertiary period during the Alter do Chão formation. Both soils were covered by tropical rainforest. The ADE profile contained a significant quantity of archaeological pottery fragments whilst in the adjacent soil no pottery or charcoal fragments were visually observed. Other general information regarding the ADE and adjacent soil can be found in Table I.

Cation exchange capacity. 2Base saturation. 3Aluminium saturation. 4Total organic carbon (mg kg-1).

Table I. Chemical characteristics of the studied soils obtained according to the methodology used by Moreira et al. (2009) [36]

Taube, P. S.; Silva, D. S.; Vasconcelos, A. A.; Rebellato, L.; Madureira, L. A. S.; Hansel, F. A.

Article

Exploratory on-line pyrolysis and thermally assisted hydrolysis and methylation for evaluating non-hydrolyzable organic matter in anthropogenic soil from Central Brazilian Amazon

Article

Sample preparation The samples were dried in an oven with air circulation at 60 ºC for 24 h. They were then macerated in a porcelain mortar and sieved (250 µm). Free lipids were removed by extraction with a mixture of CHCl3: (CH3)2CO (9:1 v/v, 10 mL in an ultrasound system for 30 min) and ester-bound lipids were eliminated by alkaline hydrolysis with a 1.0 mol L-1 KOH solution in MeOH (96%, 10 mL, 70 °C, 30 min) and then extracted with MeOH:CHCl3 (1:1, v/v, 1 x 10 mL) and CHCl3 (2 x 10 mL). After alkaline extraction the residue was dried (60 ºC) and crushed to obtain fine particles for the Py and THM analysis. The carbon content (%) was measured using an elemental analyzer (Vario Macro cube, Elementar, Hanau, Germany) [37]. Py-GC-MS and THM-GC-MS Dried soil (ca. 500 µg) was placed in a quartz tube and the tube was inserted into a Pyroprobe inlet. For the thermally-assisted hydrolysis and methylation, 5 µL of TMAH (0.25 mol L-1 in MeOH) was added and then the MeOH was evaporated by pre-heating the sample at 110 °C for 60 s. Pyrolysis and THM were then carried out at 700 ºC for 10 s, and 600 °C for 5 s, respectively, both with heating at 10 ºC/ms, using a CDS 5000 pyrolyzer (CDS analytical, Oxford, UK). The Pyroprobe interface and the oven were at a temperature of 290 °C. Both the Py and THM products were analyzed on-line (inlet 290 °C with split mode 1:10) using GC-MS (Focus GC instrument coupled to a Polaris ion trap mass spectrometer; Thermo, Waltham, USA) and a DB5MS column (60 m x 0.25 mm, 0.25 µm film thickness). The GC oven programs were: (i) 40 ºC (held 5 min) to 300 ºC (held 5 min) at 7 ºC/min for Py-GC-MS; and (ii) 40 ºC (held 5 min) to 150 ºC at 10 ºC/min and then to 300 °C (held 5 min) at 4 °C/min for THM-GC-MS. Helium was used as the carrier gas at a constant flow of 1.0 mL/min. The GC-MS interface and ion source temperatures were 290 °C and 200 °C, respectively. The ion trap mass spectrometer was operated in electron ionization mode at 70 eV, scanning at m/z 50 - 650 (0.58 total scan time), with an emission current of 250 mA. All compounds were identified with the support of an interactive chemical information structure (ICIS) and the NIST/EPA/NIH mass spectral database version 4.5 1994, as well as from the interpretation of the mass spectra. The relative contribution (RC) of products was quantified as the ratio between the peak area for each compound and the total area of identified peaks, according to the formula:

Where xij is the total integrated area of compound “j” in sample “i”, “∑xi” the sum of all integrated peak areas of identiﬁed compounds and “RCij” the relative contribution of compound “j” in sample “i”. RESULTS AND DISCUSSION General distribution of Py-GC-MS and THM-GC-MS The major Py-GC-MS products were monoaromatic compounds (benzene, toluene, etc.), polycyclic aromatic hydrocarbons (PAHs) (naphthalene, phenanthrene, etc.), phenols, N-compounds and polysaccharide pyrolysis products (Table II). The same compound classes were detected with THM-GCMS, but with additional information on lignin-derived products. Derivatized alkanoic and phosphoric acids were the major products observed by THM-GC-MS. Benzene was not detected with the THM-GC-MS technique, because it eluted within the solvent delay period programmed in the software.

Taube, P. S.; Silva, D. S.; Vasconcelos, A. A.; Rebellato, L.; Madureira, L. A. S.; Hansel, F. A.

Article Table II. Relative contribution (%) of compounds obtained in Py-GC-MS

Ion fragment used to quantify the relative contribution. 2n-alkane. 3n-alkene.

Exploratory on-line pyrolysis and thermally assisted hydrolysis and methylation for evaluating non-hydrolyzable organic matter in anthropogenic soil from Central Brazilian Amazon

Article

In relation to the Py-GC-MS products, monoaromatic compounds (benzene, toluene, ethylbenzene, dimethylbenzene, trimethylbenzene and styrene) ranged from 16.7% (15-38 cm) to 18.8% (90-130 cm) in the AS soil, and from 17.9% (0-36 cm) to 22.3% (84-150 cm) in the ADE soil (Table II). Benzene was the main monoaromatic compound, with the exception of the top horizon of the adjacent soil, where toluene was more abundant (Table II). According to the THM-GC-MS results, the relative contribution of monoaromatic compounds in the ADE samples ranged from 3.3 to 6.3% (Table III). Table III. Relative contribution (%) of the compounds detected in THM-GC-MS analysis Samples

m/z1

Depth (cm)

ADE 0-36

36-56

Toluene Ethylbenzene Dimethylbenzene Styrene Trimethylbenzene TOTAL

91+92 91+106 91+106 78+104 105+120

0.7 0.3 0.1 0.5 1.7 3.3

0.8 0.6 0.2 0.7 1.7 4.0

Indene Methylindene Dimethylindene TOTAL

115+116 115+129 115+143

0.7 0.4 0.6 1.7

0.4 0.5 0.5 1.3

Benzofuran Methylbenzofuran Dibenzofuran TOTAL

89+118 131 139+168

0.3 0.5 0.3 1.1

0.7 0.3 0.2 1.2

Dimethylfuran Furfural 5-methyl-2-furaldehyde Trimethylfuran TOTAL

67+95 95 108 109+110

0.3 5.5 1.9 1.5 9.2

0.3 9.2 1.3 0.9 11.7

80+81

2.4

3.2

Methoxybenzene 1-Methoxy-4-methylbenzene Methyl 4-methoxybenzoate Methyl 3-(4-methoxyphenyl)-2-propenoate 1-Methoxy-3,5-dimethylbenzene TOTAL

78+108 91+122 171 161+192 121+136

0.9 4.0 6.1 0.8 1.7 13.6

1.0 3.0 3.1 0.1 7.3 14.5

1,2-dimethoxybenzene 4-methyl-1,2-dimethoxybenzene 4-vinyl-1,2-dimethoxybenzene 4-allyl-1,2-dimethoxybenzene Cis-1,2-dimethoxy-4-propenylbenzene 1,2-dimethoxy-4-propenylbenzene Methyl 3-(3,4-dimethoxyphenyl)propenoate 1,2-Dimethylmethoxybenzene Methyl 3-(3,4-dimethoxyphenyl)propenoate Methyl 3,4-dimethoxybenzoate TOTAL

123+138 137+152 149+164 163+178 163+178 163+178 191+222 121+136 191+222 165+196

1.8 1.4 0.7 0.7 0.3 0.4 0.5 1.7 0.7 2.0 10.2

2.6 1.1 1.5 0.5 0.1 0.1 0.1 7.3 0.3 13.5

1,2,3-Trimethoxybenzene 4-methy-1,2,3-trimethoxybenzene 4-vinyl-1,2,3-trimethoxybenzene 4-allyl-1,2,3-trimethoxybenzene 3,4,5-Trimethoxybenzaldehyde 1,2,3,5-Tetramethoxybenzene* TOTAL

153+168 167+183 193+208 193+208 181+196 183+198

2.0 0.3 0.2 0.1 0.5 1.4 4.5

1.7 0.3 0.1 0.1 0.7 2.9

Dimethyl 1,2-benzenedicarboxylate Trimethyl 1,2,4-benzenetricarboxylate Tetramethyl1,2,4,5-benzenetetracarboxylate Pentamethyl 1,2,3,4,5-benzenepentacarboxylate Hexamethyl 1,2,3,4,5,6-benzenehexacarboxylate TOTAL

163 221+252 279 368 395

0.81 0.19 0.08 0.01 1.09

0.46 0.07 0.02 0.55

1H-methylpyrrole

56-84

AS 84-150

15-38

38-55

55-90

Monoaromatics 4.4 1.3 2.3 7.3 4.5 0.1 0.4 1.7 1.9 2.1 0.6 0.2 2.2 3.6 0.2 0.4 0.6 0.6 2.3 0.7 0.9 1.4 2.5 0.9 2.3 6.3 4.0 9.2 15.8 9.8 Polycyclic aromatic hydrocarbons 0.9 1.1 2.4 4.4 1.5 1.0 0.7 0.8 2.1 2.0 0.3 0.3 1.3 1.0 1.0 2.2 2.0 4.5 7.5 4.6 Oxyaromatics 0.1 1.1 0.7 0.3 0.4 0.2 0.3 1.2 1.2 0.9 0.2 0.2 0.3 0.6 0.4 0.4 1.6 2.2 2.1 1.6 Polysaccharides 0.2 0.3 0.4 0.1 0.4 5.3 8.6 6.2 5.2 6.7 0.9 1.7 1.2 0.4 1.7 1.3 3.6 2.3 1.6 5.1 7.6 14.2 10.0 7.3 13.8 N-compounds 1.5 9.4 3.0 2.9 4.4 Methoxybenzene 2.3 2.4 1.0 1.2 0.7 3.0 7.5 4.7 3.2 6.0 4.5 2.2 5.9 3.9 5.5 0.3 0.4 0.3 3.6 7.5 5.6 2.0 8.3 13.7 19.6 17.6 10.6 20.5 Dimethoxybenzene 3.1 3.0 2.7 2.5 4.3 1.0 1.0 1.5 1.8 2.0 0.1 4.5 6.6 0.8 0.5 1.1 1.1 0.8 0.1 0.1 0.4 0.6 0.1 0.2 0.2 0.2 0.1 1.5 0.3 0.1 3.6 7.5 5.6 2.0 8.3 0.4 0.1 0.3 0.2 1.8 0.5 0.4 9.0 11.5 19.7 15.7 16.9 Trimethoxybenzene 1.6 1.1 2.9 1.4 2.5 0.3 0.4 0.3 0.4 0.2 0.4 0.2 0.4 0.2 0.2 0.6 0.4 0.2 0.4 0.2 2.0 1.4 1.2 2.4 1.3 6.1 4.4 4.5 Dicarboxylic acid 0.76 0.62 0.60 0.42 0.50 0.13 0.03 0.20 0.06 0.03 0.08 0.07 0.02 0.01 0.02 0.01 0.01 0.99 0.65 0.88 0.51 0.54

90-130 3.0 0.7 1.1 0.8 5.7 0.6 0.4 1.1 1.1 0.9 2.8 4.8 7.1 1.0 7.7 1.6 10.2 3.5 1.6 5.1 1.6 0.5 2.1 0.82 0.82

*Tannin derivative [60]. 1Ion fragment used to quantify the relative contribution. 43

Taube, P. S.; Silva, D. S.; Vasconcelos, A. A.; Rebellato, L.; Madureira, L. A. S.; Hansel, F. A.

Article The PAH in the samples detected by Py-GC-MS were dominated by naphthalene and its methylderivatives. The PAH values ranged from 9.4% (0-36 cm) to 22.7% (84-150 cm) in the ADE profile, and from 11.0% (15-38 cm) to 18.6% (90-130 cm) in the AS profile (Table II). Higher molecular weight PAHs, such as anthracene, phenanthrene, fluoranthene, fluorene and pyrene, were observed for both soils. The relative contribution of PAH increased with depth for both profiles, probably due to the presence of BC, since it does not degrade easily and is thus relatively resistant to decomposition (Table II). The relative distributions of oxygen-containing PAHs (e.g., benzofuran and dibenzofuran) in the two profiles were found to be similar in the Py-GC-MS analysis. In contrast, according to the THM-GC-MS results, a minor number of PAHs and oxygen-containing PAHs were detected, that is, only three compounds for each class in each of the soils (Table III). The most abundant polysaccharide products observed by Py-GC-MS were furan, methylfuran, furfural and 5-methyl-2-furaldehyde. Their presence decreased with depth and ranged from 23.0% (0-36 cm) to 8.0% (84-150 cm) for ADE, and from 22.6% (15-38 cm) to 11.1% for AS (Table II). In the THM-GC-MS analysis, the relative contributions of these compounds fluctuated according to depth in the ADE and AS profiles (Table III), with values ranging from 7.6% (56-84 cm) to 14.2% (84-150 cm) and from 4.8% (90130 cm) to 13.8% (55-90 cm), respectively. The N-compounds in the Py-GC-MS products were abundant for both soils, with ranges of 15.7-19.7% in the ADE profile and 14.8-19.2% in the AS profile. Seven Py-GC-MS components were assigned, of which pyridine and benzonitrile were the most abundant. Only one N-compound (1-H-methylpyrrole) was detected in the THM-GC-MS analysis (Tables II and III). Phenol derivatives also contributed significantly to the Py-GC-MS pyrolyzates. Phenol, methylphenol, dimethylphenol, trimethylphenol and 4-vinylphenol were the most abundant (Table II, Figure 1A). The methylation of hydroxyl and carboxyl groups in lignin allowed a series of unambiguous lignin products to be detected with THM-GC-MS, based on 1-methoxy (from p-hydroxyphenyl, H), 1,2-dimethoxy (from guaiacyl, G) and 1,2,3-trimethoxy (from syringyl, S) moieties (Table III, Figure 1B). Of the lignin THM-GCMS-derived compounds, H units were the major type detected, followed by G and S units, with the exception of the 38-55 cm horizon in the AS profile, for which guaiacyl monomers were the most abundant (Table III). Note that the dominance of H over G and S moieties is consistent with the detection of 4vinyphenol using Py-GC-MS (Figure 1A). The presence of 1,2,3,4-tetramethoxybenzene also was observed in both soils (Table III). Benzene polycarboxylic acids (BPCA) were detected in the upperregions in both soils, with similar relative contributions. Dimethyl 1,2-benzenedicarboxylate (B2CA) and trimethyl 1,2,4benzenetricarboxylate (B3CA) were the most abundant BPCAs present (Table III). Aliphatic compounds (RC<1.5%) were also detected among the Py-GC-MS products for both soils and comprised a homologous series of n-alkenes and n-alkanes ranging from C11 to C29, with a predominance of medium-chain length (C11-C19) and a similar contribution of even and odd-numbered chain lengths.

Exploratory on-line pyrolysis and thermally assisted hydrolysis and methylation for evaluating non-hydrolyzable organic matter in anthropogenic soil from Central Brazilian Amazon

Article

Figure 1. Partial total ion current (TIC) showing lignin-derived Py (A) and THM (B) products of non-hydrolisable SOM in top horizon of ADE. H, p-hydroxyphenyl (phenol or methoxybenzene); G, guaiacyl (guaicol or dimethoxybenzene); S, syringyl (syringol or trimethoxybenzene); T, tannin (1,2,3,4-tetramethoxybenzene), and * contamination.

Taube, P. S.; Silva, D. S.; Vasconcelos, A. A.; Rebellato, L.; Madureira, L. A. S.; Hansel, F. A.

Article Dealkylation ratios of pyrolysis products Figure 2 shows a series of ratios of pyrolysis products that reflect the difference between the ADE and adjacent samples. The ratios can be separated into three groups according to the difference in the profiles: (i) benzene/toluene and phenanthrene/methylphenanthrene ratios were higher in the ADE considering the whole profile (p<0.05); (ii) naphthalene/methylnaphthalene and benzofuran/methylbenzofuran were similar in both samples at the three lowest depths (p>0.05); and (iii) dibenzofuran/metlhyldibenzofuran and biphenyl/methylbiphenyl were higher in the ADE in the three deepest horizons (p<0.01, Table II, Figure 2).

Figure. 2. Ratios of Py products for ADE and adjacent soil proﬁles.

Characterization of non-hydrolyzable SOM The non-hydrolyzable SOM was marked by a similar distribution of Py-GC-MS and THM-GC-MS products for the ADE and adjacent soil profiles (Tables II and III), with only small differences in the relative abundances, which was mainly demonstrated by the dealkylation ratios of the pyrolysis product (Figure 2). The pyrolysis of SOM obtained from mineral and organic soils generally leads to a high relative contribution of PAH and benzene derivatives [24,30,38-39] of which alkyl benzenes and PAH have been attributed to the burning of biomass (BC) [24,26-27,30], methoxyphenols from lignin and carbohydrates and – containing compounds from microbes [40]. In this study, benzene was a prominent component in the ADE products, mainly at lower depths (36-150 cm), with a lower abundance being observed for the AS profile (Table II, p<0.05). In fact, the distribution and high abundance of benzene might be indicative of BC, since this is abundant in the Py-GC-MS derived products of charcoal [23] and natural soil affected by fire [39]. It has been reported that alkylbenzenes (e.g., toluene) are pyrolysis products of microbial tissues [41], but a predominance of the pyrolysis product of peptidoglycan (i.e., acetamide), a primary component of bacteria cell walls, was not detected [42]. THM is a suitable technique for obtaining the bacterial alkanoic acid profile [43], but the amount of bacterial biomarkers (saturated branched C15 and C17 and monounsaturated C16 and C18 alkanoic acids) [44] was relatively low. However, it is possible that after the extraction of non-hydrolyzable OM most of the alkanoic acids have been removed. Thus, the alkyl benzenes (including toluene) and PAH detected can be assigned to BC, and this is indicative of the presence of BC in the ADE and adjacent soils. PAHs were important components of the products obtained from the soils, particularly naphthalene and its methylated counterparts, with a less significant contribution from the higher molecular weight PAHs (Table II). PAH and BPCA sources in soils are associated with the products of the incomplete combustion of OM (e.g. BC) incorporated through the sorption of pyrogenic products [22,23,32,45-48]. Some PAH results indicated that the dealkylation ratios of pyrolysis products were different when the profiles were compared, reflecting the relative accumulation of more condensed BC in the ADE profiles (Figure 2) [49]. In the thermochemolysis the PAHs showed the same tendency as the monoaromatic components (see above). 46

Exploratory on-line pyrolysis and thermally assisted hydrolysis and methylation for evaluating non-hydrolyzable organic matter in anthropogenic soil from Central Brazilian Amazon

Article

As seen for the PAH components, no difference in the oxygen-containing PAHs that could differentiate the studied soils was observed (Tables II and III). Benzofuran and its counterparts are products of charred cellulose formed above 350 °C [50], and may indicate the presence of charred cellulose material in the non-hydrolyzable SOM in the two soils. Furans and anhydrosugars are typically pyrolysis products of cellulose and are commonly obtained in the Py-GC-MS of polysaccharide-rich SOM [51]. In this study, only furan-like compounds were detected in the products obtained from the non-hydrolyzable fractions of the ADE and adjacent soils (Tables II and III). The presence of levoglucosan (a product of the pyrolysis of intact polysaccharides) was not observed [50-51]. Its absence and the presence of furans in abundance may be indicative of microorganism activity, related to carbohydrate degradation in the soil, this being more pronounced with depth [52], which is in agreement with tropical soil conditions. The possibility of the presence of microbial carbohydrates cannot be totally excluded, but given the low abundance of the pyrolysis products of microbial biomarkers (e.g., acetamide) [42-44] they would be expected to be present in low amounts in the non-hydrolyzable fraction of soils. Although the Py-GC-MS technique tends to underestimate the values for N-containing biomolecules [53-54], the N-containing pyrolysis products from the non-hydrolyzable fraction of the two soils were abundant. The source of these compounds is related to proteins originating from plant and microbial biomass [53]. The presence of methylated pyridines and pyrroles suggests that these products may be, in part, derived from microbial biomass [55], but this is not reinforced by other biomarkers (e.g., acetamide) [42-44]. With respect to benzonitrile, its presence may be indicative of the presence of BC in the soils (Table II) [26]. A large contribution from N compounds in the pyrolysis products of SOM generally corresponds to a contribution from microbial material [40,56-58]. Moreover, the N compounds in soils are usually considered to be pyrolysis products of residues from burned non-woody material (e.g., leaves and bones) [19,59]. The presence of phenolic compounds may be indicative of a lignin component in the non-hydrolyzable SOM. It is known that lignin components are not degraded during the alkaline hydrolysis of soils [60]. The presence of 1,2,3,4-tetramethoxybenzene indicates the incorporation of tannin derivatives into soils (Table III) [61]. The presence of 4-vinylphenol may be indicative of decarboxylation of lignin during thermal decomposition [22-23,27]. The p-coumaric acid moiety is released from suberin as a primary source of 4-vinylphenol during pyrolysis, since the suberin tissue is broken down and is freely extracted with organic solvents after alkaline hydrolysis, it seems not be the source of such pyrolysis product [9,60]. In the THMGC-MS analysis, some additional lignin units that were not detected in the Py-GC-MS analysis appeared, such as: H, p-hydroxyphenyl (phenol or methoxybenzene); G, guaiacyl (guaicol or dimethoxybenzene); S, syringyl (syringol or trimethoxybenzene) and T, tannin (1,2,3,4-tetramethoxybenzene) (Table III, Figure 1B). The absence of these lignin derivatives during the Py-GC-MS analysis may, in part, be due to demethoxylation and dihydroxylation in the pyrolysis analysis at temperature higher than 600 ºC [22-23,27]. The ADE and adjacent soil profiles showed a high abundance of p-hydroxyphenyl units for all samples, with the exception of the 38-55 cm adjacent soil horizon, which could reflect the incorporation of an abundance of non-woody angiosperms (e.g., grasses - Poaceas and Cyperaceaes) into this horizons [35,62]. The presence of preserved propyl and ester groups in the side chain of the methoxybenzene and dimethoxybenzene derivatives and the absence of 1-(3,4-dimethoxyphenyl)-1,2,3-trimethoxypropane and 1-(3,4,5-trimethoxyphenyl)-1,2,3-trimethoxypropane), which indicates intact propyl site (C3) chains during THM, are not identified, implying that fresh lignin is not present in any of the samples (Table III) [63], but the relatively high abundance of C-1 non-substituted and 1-methyl substituted compounds may indicate a mixture of degraded/charred and fresh lignin in the non-hydrolyzable SOM (Table III). An input of cutan/suberin to the soil profiles is detected by the presence of a homologous series of alkene/alkanes (Py-GC-MS), with a major contribution in the subsoil [64]. The presence of similar contributions of even short-chain alkanes in the two soils may be a result of the “in situ” thermal degradation of biomass during the pyrolysis [65]. The presence of alkanoic acids (THM-GC-MS) could be associated with the residues surviving after the alkaline hydrolysis of plant-derived ester-bound lipids, thus corresponding to roots [64]. 47

Taube, P. S.; Silva, D. S.; Vasconcelos, A. A.; Rebellato, L.; Madureira, L. A. S.; Hansel, F. A.

Article It seems that both the ADE and adjacent soil profiles have BC in their non-hydrolyzable SOM, though it is important to note that the Py-GC-MS and THM-GC-MS data are qualitative and do not reflect the amount of BC in the soils, which, in general, is greater in the ADE compared with the adjacent soil profile [10,12]. This possibility is reinforced by the residual carbon content (%) after alkaline hydrolysis, which decreases with depth (3.3, 2.2, 1.5, and 0.8 for ADE and 2.3, 1.5, 1.2, and 0.7 for AS), but for the same horizon the values were always higher for the ADE. The greater contribution of BC in the ADE is in agreement with the values obtained by Macedo [35], who reported amounts of charcoal of less than 2% in an adjacent soil and ranging from 5-10% in an ADE samples collected from the same site investigated in this study. Therefore, the BC formed from wildfires could be responsible for the BC signals in the AS profile. Forest fires are widespread in the Amazon region nowadays, and it can be assumed that these also occurred in the past, that is, during the ADE formation [1]. The relative increase in the PAHs with depth may indicate a high degree of relative recalcitrance in the deepest horizons of the soils [24]. In theory, adjacent soil has a non-anthropogenic and reflects the natural processes involved in SOM formation. However, the data suggest the presence of BC in the non-hydrolyzable SOM in the AS profile and its preservation in this soil fraction [24]. Special care needs to be taken when differentiating the OM residues from charred (i.e., fuel) and degraded biomass sources. In other words, it is difficult, at this stage, to identify a specific source for OM that could differentiated the ADE and adjacent soils, since it appears that many pyrolysis-derived products from the two processes (i.e., natural OM decay and charred OM) are comprised of similar compounds [20, 23,60,63,66]. Thus, a distinction based solely on Py or THM investigations seems unable to detect which process had the greatest influence (i.e., degradation x charring) on the OM to yield the pyrolysis products. In this case, it is known that an anthropogenic process (e.g., the production of biochar for home gardens [68]) may be responsible for a remarkable difference in the ADE profile, and thus it was expected that a difference would be apparent when the two soil profiles were compared. The presence of BC in soils was characterized by an increase in the relative contributions of benzene, PAH, benzofuran and benzonitrile [23-24,26-27]. Non-hydrolyzable charred OM consists mainly of highly condensed aromatic compounds regardless of the source [67]. In this context, the ADE and adjacent samples displayed practically the same products derived from Py and THM, the differences being, mainly the relative contribution of some compounds and some of the dealkylation ratios (Figure 2). ADE BC features Kaal et al. [23] proposed the use of ratios between aromatics and their methylated counterparts to measure the charring intensity of BC. Figure 2 shows that the ADE sample has a higher benzene/toluene ratio of pyrolysis products in all horizons, and the same result was observed for phenanthrene/ methyphenanthrene (p<0.01, Table II, Figure 2). This trend was less evident for dibenzofuran/ metlhyldibenzofuran and biphenyl/methylbiphenyl, these ratios being higher in the ADE sample considering only the three lowest horizons (p<0.01, Table II, Figure 2). This major relative contribution of aromatic compounds vs. their methylated counterparts in the ADE is a distinct feature of BC, in agreement with the greater contribution from PAHs without alkyl side chains in a TPI (Terra Preta de Índio) soil samples reported by Schellekens et al. [30] and Justi et al. [49]. These results may indicate a more recalcitrant BC with a highly condensed aromatic structure and, consequently, a higher dealkylation ratio of pyrolysis product in anthropogenic soils [26-27,49]. These differences may also be the result of different inputs and/or decomposition processes for the two soils [30]. Toluene from a microbial source may contribute to the lower value for the benzene/toluene ratio in the top horizon of the ADE, which could reflect a higher degree of microbial activity. However, considering the weak evidence of biomarkers from microbial cells in the pyrolysis products [42,44], it can be assumed that most of the toluene thermally-produced during Py-GC-MS has a BC source. Basically, fire needs three elements for ignition: heat, fuel and an oxidizing agent (O2). Natural fuel elements in the forest are wood and leaves, and in anthropogenic soils, besides the natural elements, other materials brought to the location from elsewhere are present (e.g., food residues). Considering the 48

Exploratory on-line pyrolysis and thermally assisted hydrolysis and methylation for evaluating non-hydrolyzable organic matter in anthropogenic soil from Central Brazilian Amazon

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plausible sources for combustion in the Amazon basin (wood and leaves), the more recalcitrant BC in the ADE may be associated with the heating mode (i.e., anthropogenic fire). For example, Wolf et al. reported that plant material burns at a low median temperature (503 °C for shrubs) [69]. In contrast, a domestic fire using wood stacked around pottery reaches a maximum of 962 °C [70] and a domestic fire reportedly has a median temperature of 797 °C [69]. In this context, a more recalcitrant BC in the ADE may be due to human interference in the heating mode (e.g., slash and burn, campfire, bonfire and earth kiln). The burning and charring of debris (e.g., bones) could take place during the clearing of an area for habitat or the production of biochar for home gardens, as currently practiced by the Caboclos people in the Amazon basin [68]. In addition, the use of bone as a fuel source associated with other fuel elements (e.g., grass and wood) in the archaeological context is well reported in the literature [71]. As the burning of this material occurs at lower temperatures and for longer periods than the burning of the wood alone, this prolonged heating could produce a more recalcitrant BC in the ADE [71]. Proteinaceous biomass was detected in the Py-GC-MS but no marked difference was detected in the distribution of N-compounds in the soils (ADE x AS, Table II). Although, the indirect presence of burned bone was suggested by Taube et al. [9], due to the high concentration of short chain (<C20) alkanoic acids in the extracted lipids, the data reported herein does not reinforce this conclusion. However, evidence of fish bones has been observed in ADE profiles of the same region of this study, using scanning electron microscopy in combination with energy-dispersive-X-ray spectroscopy, which revealed the presence of Ca and P-derived from bones, besides if the presence of charred bones exist it could give pyrolysis products which are similar to those of any other charred proteinaceous material (e.g., leaves) [72]. CONCLUSIONS The BC present in an Amazonian dark earth (ADE) sample was produced by high-intensity fire, in contrast to the wildfires that influenced an adjacent soil sample. This afforded more recalcitrant BC with a highly condensed aromatic structure in the ADE. The intense fire associated with the ADE could have occurred as a result of anthropogenic activity (e.g., slash and burn, campfire, bonfire and earth kiln). The dealkylation ratios obtained from the Py-GC-MS analysis suggest a more condensed structure for the BC in the ADE, which may be associated with anthropogenic fires. Thus, there is a need for future investigations using Py-GC-MS and THM-GC-MS analysis to study bone collagen alterations with respect to incomplete combustion. ACKNOWLEDGEMENTS The study was supported by the Brazilian CNPq (477676/2009-0) and EMBRAPA (02.09.01.014.00.00). We also thank the Universidade Federal de Santa Catarina (UFSC) and Universidade Federal do Oeste do Pará (UFOPA) for laboratory and field facilities. The authors would like to express their deepest gratitude to the unknown reviewers, without whom this work would be impossible in this format. Manuscript received Feb. 21, 2018; revised manuscript received June 10, 2018; accepted June 15, 2018.

Taube, P. S.; Silva, D. S.; Vasconcelos, A. A.; Rebellato, L.; Madureira, L. A. S.; Hansel, F. A.

Article REFERENCES 1. Glaser, B.; Birk, J. J. Geochim. Cosmochim. Ac. 2012, 82, pp 39-51 (DOI: 10.1016/j.gca.2010.11.029). 2. Sombroek, W. G. Amazon soil: A reconnaissance of the soils of the Brazilian Amazon region. Centre for Agricultural Publications and Documentation, Wageningen, 1966. 3. Lehmann, J.; Kern, D. C.; Glaser, B.; Woods, W. I. Amazonian Dark Earths. Origin, properties and management. Kluwer Academic Publishers, Dordrecht, 2003. 4. Glaser, B.; Woods, W. Amazonian Dark Earths: Explorations in Space and Time. Springer, Berlin, 2004. 5. Glaser, B. Philos. T. Roy. Soc. B. 2007, 362, pp 187–196 (DOI: 10.1098/rstb.2006.1978). 6. Woods, W. I.; Teixeira, W. G.; Lehmann, J.; Steiner, C.; Winklerprins, A.; Rebellato, L. Amazonian Dark Earths: The First Century of Reports. In: Woods, W. I.; Teixeira, W.G.; Lehmann, J.; Steiner, C.; Winklerprins, A. M. G. A.; Rebellatto L. (Eds.). Amazonian Dark Earths: Wim Sombroeks Vision, Springer, Berlin, 2009, pp 1-14. 7. Falcão, N. P. S.; Clement, C. R.; Tsai, S. M.; Comerford, N. B. Pedology, fertility, and biology of central Amazonian Dark Earths. In: Woods, W. I.; Teixeira, W. G.; Lehmann, J.; Steiner, C.; Winklerprins, A. M. G. A.; Rebellatto, L. (Eds.). Amazonian Dark Earths: Wim Sombroeks Vision. Springer, Berlin, 2009, pp 213-228. 8. Birk, J. J.; Teixeira, W. G.; Neves, E. G.; Glaser, B. J. Archaeol. Sci. 2011, 38, pp 1209-1220 (DOI: 10.1016/j.jas.2010.12.015). 9. Taube, P. S.; Hansel, F. A.; Madureira, L. A. S.; Teixeira, W. G. Org. Geochem. 2013, 58, pp 96–106 (DOI: 10.1016/j.orggeochem.2013.02.004). 10. Glaser, B.; Balashov, E.; Haumaier, L.; Guggenberger, G.; Zech, W. Org. Geochem. 2000, 31, pp 669678 (DOI: 10.1016/S0146-6380(00)00044-9). 11. Novotny, E. H.; de Azevedo, E. R.; Bonagamba, T. J.; Cunha, T. J. F.; Madari, B. E.; Benites, V. M. Environ. Sci. Technol. 2007, 41, pp 400-405 (DOI: 10.102/es060941x). 12. Glaser, B.; Haumaier, L.; Guggenberger, G.; Zech, W. Naturwissenschaften. 2001, 88, pp 37-41 (DOI: 10.1007/s001140000193). 13. Steiner, C.; Teixeira, W. G.; Zecha, W. Slash and char: an alternative to slash and burn practiced in the Amazon Basin. In: Glaser, B.; Woods, B. W. (Eds.). Amazonian Dark Earths. Springer, Berlin and Heidelberg, 2004, pp 183-193. 14. Steiner, C. Slash and char as alternative to slash and burn: soil charcoal amendments maintain soil fertility and stablish a carbon sink. Cuvillier Verlag, Göttingen, 2007. 15. Lehmann, J.; Liang, B.; Solomon, D.; Lerotic, M.; Luizão, F.; Kinyanagi, J.; Schäfer, T.; Wirick, S.; Jacobsen, C. Global Biogeochem. Cy. 2005, 19, pp 1013–1025 (DOI: 10.1029/2004GB002435). 16. Forbes, M. S.; Raison, R. J.; Skjemstad, J. O. Sci. Total Environ. 2006, 370, pp 190–206 (DOI: 10.1016/j.scitotenv.2006.06.007). 17. Czimczik, C. I.; Masiello, C. A. Global Biogeochem. Cy. 2007, 21, GB3005 (DOI: 10.1029/2006GB002798). 18. Glaser, B.; Guggenberger, G.; Zech, W. Past anthropogenic inﬂuence on the present soil properties of anthropogenic dark earths (Terra Preta) in Amazonia (Brazil). In: Glaser, B.; Woods, B. W. (Eds). Amazonian Dark Earths. Springer, Berlin and Heidelberg, 2003, pp 227-242. 19. Knicker, H.; Totsche, K. U.; Almendros, G.; González-Vila, F. J. Org. Geochem. 2005, 36, pp 13591377 (DOI: 10.1016/j.orggeochem.2005.06.006). 20. Nierop, K. G. J.; Verstraten, J. M. Rapid. Commun. Mass Sp. 2004, 18, pp 1081-1088 (DOI: 10.1002/rcm.1449). 21. Nierop, K. G. J.; Filley, T. R. J. Anal. Appl. Pyrol. 2008, 83, pp 227-231 (DOI: 10.1016/j.jaap.2008.07.004). 50

Exploratory on-line pyrolysis and thermally assisted hydrolysis and methylation for evaluating non-hydrolyzable organic matter in anthropogenic soil from Central Brazilian Amazon

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Exploratory on-line pyrolysis and thermally assisted hydrolysis and methylation for evaluating non-hydrolyzable organic matter in anthropogenic soil from Central Brazilian Amazon

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Br. J. Anal. Chem., 2018, 5 (19), pp 54-60

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The 41 Annual Meeting of the Brazilian Chemical Society was marked by discussions on the future of scientific research in chemistry The Brazilian Chemical Society (SBQ) held its 41st Annual Meeting in Foz do Iguaçu (PR, Brazil), from May 21 to 24, 2018. More than 1,500 attendees, including students, professors and researchers from throughout the country, as well as international guests, gathered to discuss the future of scientific research in chemistry. "We knew that it would be challenging to hold this SBQ Annual Meeting (RASBQ) after the success of the IUPAC 2017 World Congress, and we are very pleased with the result, both from the point of view of the high scientific level and the intense political discussion that permeated the event. After all, this is a time of many uncertainties," said Professor Aldo Zarbin (Federal University of Paraná, Brazil), who presided over the SBQ in its last two years and joins its Advisory Board. st With the theme 'Building Tomorrow', the 41 RASBQ proposed an important reflection on the creation of new conditions for the Brazilian scientific and technological environment to grow again and to establish itself as a fundamental axis of the country's economic development. The RASBQ program included 7 workshops, 12 short-courses, 3 poster sessions, 18 coordinated sessions, regional and divisional assemblies, an honor session, a book-launch session, and the traditional exhibition by supporting companies. Major names in contemporary chemistry presented recent results of their researches, including Francisco José Krug, Carlos Moyses Graça Araujo, Claudio D. Borsarelli, Fabrizio Adani, Frank Glorius, Galo Soler Illia, Hamilton Brandão Varela de Albuquerque, Joaquim de Araújo Nóbrega, Jonathan S. Lindsey, Joost N. H. Reek, Luiz Carlos Dias, Mônica Tallarico Pupo, Richard G. Weiss, Teodorico de Castro Ramalho and Wilmo Ernesto Francisco Junior.

Opening Lecture The opening conference of the 41st RASBQ was presented by Professor Francisco José Krug, from the Center for Nuclear Energy in Agriculture, University of São Paulo (CENA-USP). According to Professor Rossimiram Freitas (Federal University of Minas Gerais, Brazil), vice-president st of SBQ and president of the organizing committee for the 41 RASBQ, the choice of the opening lecturer is a very important and diﬃcult choice for the SBQ Board, since it is the recognition of a person who has stood out for their contributions relevant to the development of chemistry in the country and in the world, and is important for strengthening the SBQ'. In addition, the opening lecturer receives the 'Simão Mathias Medal', the highest honor granted by the SBQ. “Among many names deserving of this recognition, it was with great enthusiasm that we came to the name of Chico (the nickname of Professor Krug), a person who combines absolutely admirable qualities as a scientist and a human being,” said Professor Freitas.

Professor Francisco José Krug (CENA-USP) presenting the 41th RASBQ opening conference – Photo: SBQ.

Feature In his conference 'A Critical View on Laser-Induced Plasma Optical Spectroscopy (LIBS)', Professor Krug spoke about this technique, which has been used with great success for the direct analysis of materials of industrial, environmental, pharmaceutical, agronomic and geological interest, among others, and which has aroused increasing interest in the scientiﬁc community. "I also took advantage to tell some facts that contributed to the success of the pioneering work on Flow Injection Analysis (FIA), before addressing the main theme that focused on the direct analysis of solids by LIBS for the determination of elements," explained Professor Krug.

Workshops On the first day of the 41st RASBQ, some workshops were held; one of them was the XVI Post-Graduation in Chemistry Workshop. The postgraduate programs in chemistry have attracted increased attention since the cut in federal funding that took place in mid-2014. "Although it is a very difficult situation, if we can see a positive side to this, the scarcity of resources takes us out of the comfort zone and forces us to seek sources of funds that we had not explored before," said Professor Rochel Montero Lago (Federal University of Minas Gerais), who organized one of the symposia in this workshop. This workshop had two more symposia: 'Perspectives of the postgraduate programs in chemistry and the insertion of the future professionals / Future scenarios for the formation of the postgraduate student' coordinated by Professor Maria Domingues Vargas (Fluminense Federal University), with the participation of Professors Adriano Monteiro (Federal University of Rio Grande do Sul) and Norberto Peporine Lopes (University of São Paulo); and 'Evaluation of the Brazilian postgraduate programs – good practices in the conduction of the postgraduate programs' coordinated by Professor Marília Goulart (Federal University of Alagoas), with the participation of Professors Maysa Furlan (São Paulo State University), Hélio Duarte (Federal University of Minas Gerais), Jaisa Fernandes Soares (Federal University of Paraná), Josué Carinhanha Santos (Federal University of Alagoas) and Wendell Coltro (Federal University of Goiás), among others. Another highlight of the workshop sessions was the 'Young SBQ', which involved a room full of undergraduate and young postgraduate students interested in listening to lectures on topics that are not usually addressed in the classroom. "It was an option we made to give a new meaning to the 'Young SBQ', and I believe that from this workshop we will have an even greater growth of the participation of young people in SBQ," said Professor Fernando Carvalho (Fluminense Federal University), general secretary of SBQ and one of the organizers of the workshop. "Our goal is to create new leaderships, new chemists to join the SBQ and continue our work. We still have very active pioneers, but we must always renew the SBQ," concluded Professor Carvalho. The topics dealt with were entrepreneurship, the chemist's career, scientific publications, and research project funding. "Since they were created, the workshops have been constituted of moments of deep reflection and debate, fundamental for self-knowledge, comparative evaluation and improvement of the programs," said Professor Marília Goulart (Federal University of Alagoas), co-coordinator of the event.

International Partnership The internationalization process of Brazilian chemistry has grown signiﬁcantly in recent years and was evidenced at the RASBQ by the renewal of the cooperation agreement between the SBQ and the Royal Society of Chemistry (RSC). With this agreement renewal, the SBQ and the RSC extended the partnership started in 2007. At the 41st RASBQ, Professor Aldo Zarbin, President of the SBQ, and Professor Dominic Tildesley, Immediate Past President of the RSC, signed a memorandum of intent as one of the highlights of the 'Year Brazil-UK of Science and Innovation 2018–19'. There were also scientiﬁc conferences by researchers from both countries and the participation of diplomatic authorities. The Brazilian lecturer was Professor Carlos Roque Duarte Correia of Unicamp, a 55

Feature specialist in organic synthesis and a Fellow of the RSC since 2017. The speaker representing the United Kingdom was Mirella Di Lorenzo, who coordinates the biosensors and biofuels group at the University of Bath, UK. "We at SBQ believe that representative societies should work together, and that is one of the reasons why we have established a partnership with RSC since 2007. We need to have more and more partnerships with respectable people and entities that can contribute to our history," said Professor Zarbin.

Professors Dominic Tildesley (RSC) and Aldo Zarbin (SBQ) renew the partnership between the societies – Photo: 'Boletim SBQ'.

Professor Zarbin also recalled that the SBQ has established partnerships with the American Chemical Society, and a partnership with the German Chemical Society is in an advanced stage of negotiation, the protocol of which will be signed during the IUPAC World Chemistry Congress 2019 in Paris.

SBQ at Secondary School As every year, the 41th RASBQ also received the 'SBQ at Secondary School', which is a set of highly visual and humorous experiments, designed to bring children and young people closer to chemistry.

Young people delighted with the 'SBQ at Secondary School' – Photo: 'Boletim SBQ'.

This year, 'SBQ at Secondary School' focused on the interactivity of the students who, in the company of instructors, performed the experiments themselves. They were able to build molecules with straws and pieces produced using laser cutting machines. Professors Alfredo Mateus (Federal University of Minas Gerais), José Ricardo Salgado (Federal University of Latin American Integration) and Renata Mello Giona (Federal University of Technology, Paraná), who conducted the 'SBQ at Secondary School' presentation, received more than 500 secondary school teenagers from Foz do Iguaçu and region. 56

Feature Homage Session At the Homage Session, the pioneering spirit of Professor Jaime Rabi was recognized with the 'SBQ Innovation Award Fernando Galembeck'. This award recognizes the contributions made by researchers in the ﬁeld of chemistry that have resulted in innovation; that is, new knowledge that produces economic, strategic or social results. Jaime Rabi was a professor at the Nucleus of Research on Natural Product (now Institute) of the Federal University of Rio de Janeiro for 20 years. In 1985, still at the academy, he was invited to join Microbiológica Química e Farmacêutica Ltda. as a partner responsible for creating and developing a chemical approach within the pharmaceutical company. “Jaime Rabi has a remarkable trajectory. He was a researcher and university professor with active participation in the ﬁrst years of the SBQ and created, developed and produced some important drugs that are in the market. He is an example of innovation that has been made in Brazil for many years, and that many insist on ignoring,” said Professor Fernando Galembeck, of the University of Campinas.

The honorees at the 41th RASBQ: Professors Jaime Rabi, Fábio Minoru Yamaji, Francisco José Krug and Richard Weiss – Photo: 'Boletim SBQ'.

“The SBQ Innovation Award, which honors me, symbolizes the recognition of the Brazilian chemical academy that Brazil's prosperity depends on the generation of good quality science, the training of highlevel human resources and the incorporation of the benefits of science by society,” said Professor Rabi. In addition to the SBQ Innovation Award, four other honors were awarded during the RASBQ: Professor Fabio Minoru Yamaji (Federal University of São Carlos, Sorocaba) received the 'Virtual Journal of Chemistry Prize'. He leads the research group Biomass and Bioenergy, which works with several sources of biomass, such as agricultural residues and materials from forest-based industries. The group seeks to contribute with research into the better use and production of solid fuels such as coal, briquettes and pellets. The research is carried out in partnership with industry, mainly in the agroforestry sector. In addition to the research, the group has organized events to discuss and disseminate research, such as the Congress of Renewable Energies – ConER and the BBC Brazil – Biomass and Bioenergy Conference. "I was extremely honored with the 'Virtual Journal of Chemistry Prize'. It was a real surprise! In fact, this award is for the students of the Biomass and Bioenergy research group who are the authors of the articles. Certainly, an award from an entity so recognized as the SBQ is an incentive for every researcher,” Professor Yamaji acknowledged. The 'Journal of the Brazilian Chemical Society Medal of Honor' was given to Professor Richard Weiss (Georgetown University, USA), who also held a conference entitled 'Molecular and polymeric hydro- and organo-gels. What are they and why are they important?', and the 'Simão Mathias Medal' was given to the opening lecturer, Professor Francisco José Krug (CENA-USP). In order to complete the Homage Session, the 'Angelo da Cunha Pinto Award' was given to the Scientific Electronic Library Online (Scielo), an electronic library and cooperative model of the digital publication of Brazilian open-access scientific journals, for its strong contribution in the preparation, storage, dissemination and evaluation of scientific production. Thematic Sections: Women in Science The 41st RASBQ was remarkable for women scientists. The importance of the debate on women in science has been increasingly evident and has been addressed in many national and international scientific events. 57

Feature At the symposium named 'They, the women scientists!' three women at diﬀerent stages of their careers – the beginning, middle and top – presented their own point of view. For women, this symposium was not only about citing numbers, but also about the challenges and initiatives that must be taken to improve this scenario in the future for Brazilian and world science. It was a start to demythologize stereotypes and to give the opportunity for new generations of women to consider the career of scientist. At the end of this discussion, the names of 40 women who had contributed to the strengthening of the SBQ in its 40 years of existence were mentioned, and those who were present went on stage.

Women scientists who held positions in the SBQ were honored in a thematic session coordinated by Professors Rossimiriam Pereira de Freitas, president of the 41st RASBQ organizing committee, and Elisa Orth – Photo: 'Boletim SBQ'.

In addition to the symposium 'They, the women scientists!', RASBQ crowned the unique work of Professor Rossimiriam Pereira de Freitas, the ﬁrst person responsible for the organization of three consecutive RASBQs. Professor Freitas is the second woman to assume the position of general secretary of the SBQ (Professor Vanderlan Bolzani was secretary general in the period 2004–2006).

Conferences The 41st RASBQ also featured conference sessions in which renowned scientists addressed the recent advances made in academia in several areas of chemistry. The theme of one of these conferences was 'Complex Dynamics in Electrocatalysis', given by Professor Hamilton Varela (University of São Paulo, São Carlos). In his conference, Professor Varela presented results obtained by his research group and discussed some perspectives and practical applications of the electrochemical oscillations. "The study of electrocatalytic reactions in oscillatory regime has opened excellent perspectives in the study of reaction mechanism and obtaining relevant parameters as velocity constants," said Professor Varela to the SBQ Bulletin.

Attendees during one of the conferences held during the 41st RASBQ – Photo: SBQ. 58

Feature Posters and Book Launch The traditional poster session also took place at this RASBQ edition. There were two days in which all participants had the opportunity to present and discuss themes from diﬀerent areas in chemistry.

Poster session – Photo: SBQ.

In parallel, as is traditional, the SBQ opened space for the launch of books by its associates. In total, there were seven new books, all in the Portuguese language: QUÍMICA – matéria, energia e transformações. José Carlos de Azambuja Bianchi, Carlos Henrique Albrecht, and Daltamir Justino Maia - Authors Fundamentos de Espectrometria e Aplicações. Valdemar Lacerda Júnior - Editor Química Orgânica – Estrutura e Propriedades de Compostos Orgânicos. Paulo Marcos Donate - Editor Estudos de Caso para o Ensino de Química 1. Salete Linhares Queiroz and Erasmo Moisés dos Santos Silva - Authors nd

Comunicação e Linguagem Cientíﬁca: Guia para Estudantes de Química (2 Ed.). Salete Linhares Queiroz and Jane Raquel Silva de Oliveira - Authors Introdução à Modelagem Molecular para Química, Engenharia e Biomédicas: Fundamentos e Exercícios. André Mauricio de Oliveira - Author Grandezas, Unidades e Símbolos em Físico-Química (Livro Verde). Translation updated to Portuguese of the 3rd edition in English, coordinated by Romeu C. Rocha-Filho and Rui Fausto For more information access: h p://www.sbq.org.br/41ra/pagina/lancamento-livros.php

Exhibition Area The exhibition area is a unique space for networking and a great opportunity for professors, students of all levels (undergraduate, masters and doctorate), scientists and chemistry sympathizers to know what chemistry does best.

Feature

Participants circulating around the exhibition area of the 41th RASBQ – Photo: SBQ.

The sponsoring companies of RASBQ take advantage of this space to present their launches and innovations. Companies that were present included Agilent, Allcrom, Bruker, Büchi, Horiba, Metrohm, Nova Analítica, Shimadzu, Sigma-Aldrich Brasil, Thermo Scientific, Peak, Waters, and others. On the closing night, the ceremony to announce the new board of directors, advisory board, fiscal council, scientific divisions and regional secretariats took place in the General Assembly of the Associates. The new boards have a mandate until May 2020. (see full report at: http://boletim.sbq.org.br/noticias/ 2018/n3193.php) "We have a lot of work ahead of us. Fortunately, we have had a slight financial recovery at SBQ in recent years, but we know that this is a difficult time for the science in Brazil," said Professor Norberto Peporine Lopes (University of São Paulo, Ribeirão Preto, SP, Brazil), president of the SBQ for the next two years, to the SBQ's weekly Electronic Bulletin.

This feature contains information from the SBQ Electronic Bulletin.

Br. J. Anal. Chem., 2018, 5 (19), pp 61-64

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This section is dedicated for sponsor responsibility articles.

Determination of Mercury in Water by Direct Mercury Analysis: A Study to Comply with Brazilian Water Legislation Bruno M. Siqueira1*, Valerio Rindone2 1

Nova Analítica Importação e Exportação, São Paulo, SP, Brazil 2 Milestone SRL, Bergamo, Italy

In this study, application of the DMA-80 (Milestone) instrument for the determination of mercury in water samples according to Brazilian legislation is demonstrated. The determination of mercury did not require any previous sample treatment, and the limits of detection achieved allowed for exact quantifications, as demonstrated for reference materials DORM-4 and NIST 1568a. Keywords: water and waste water, mercury determination, thermal decomposition. INTRODUCTION According to the World Health Organization (WHO), millions of people die each year from diseases caused by water contamination. Overcoming these tragedies is possible with an increase in the quality of the sanitation system [1]. On December 12, 2011, The Ministry of Health of Brazil promulgated Ordinance 2914, which establishes procedures and parameters for the quality control of water intended for human consumption and its standards of potability and organoleptic content [2]. On March 17, 2005, the Regional Environment Council (CONAMA) promulgated Resolution 357, which provides environmental guidelines for the classification of surface water (seawater, wastewater, etc.), as well as establishes the conditions and standards for effluent release [3]. Table I shows the maximum mercury limits permitted for each legislation. Table I. Maximum mercury limits permitted in water samples by Brazilian legislation.

Ordinance 2914 (µg L -1)

CONAMA 357 (µg L -1)

0.2 *

*Lowest value according to the water classiﬁcation.

Many laboratories use cold vapor with atomic absorption or Inductively Coupled Plasma - Atomic Emission Spectrometry (ICP OES) to analyze mercury. However, this kind of method requires sample preparation, dilutions, and reagents. In this work, we propose a method of thermal decomposition followed by atomic absorption determination to analyze mercury without sample preparation or the need of reagents. MATERIALS AND METHODS This study was conducted in the Analysis Laboratory at Milestone (Bergamo, Italy). All materials used in the analysis were decontaminated by HNO3 steam cleaning using Traceclean System (Milestone, Bergamo, Italy). After this, materials were intensively rinsed with ultrapure deionized water with a resistivity ≥18.2 MΩ cm and then dried. All solutions were prepared using ultrapure deionized water in a Veolia™ Element system. High purity 65% HCl (v/v) was obtained in a DuoPur Acid Puriﬁcation System (Milestone, Bergamo, Italy). Mercury *bruno.menezes@novanalitica.com.br

Bruno M. Siqueira, Valerio Rindone

Sponsor Report −1

(Hg) stock solution (999 ± 3 mg L ) from SpecSol (São Paulo, SP, Brazil) was used to prepare the standard solutions. Quartz sample boats (Milestone, Bergamo, Italy) were used for the calibration curves. The certified reference materials NIST DORM-4 (fish protein) and NIST 1568a (rice flour) were used to check the accuracy of the method. The samples were weighed on a digital analytical balance with four decimal places to the nearest 0.1 mg (Mettler Toledo, Columbus, Ohio, USA). A direct mercury analyzer (DMA-80, Milestone, Bergamo, Italy) was used for total Hg determination in animal feed. DMA-80 technology is based on drying a sample and subsequent thermal decomposition, followed by electrothermal atomization of Hg. Two sample boats are available to use, quartz and nickel. Quartz sample boats were used in this study. An internal thermocouple (ATC) sensor controlled the drying/decomposition temperature. The responsible of Hg reduction is a catalyst system (Milestone). After this, Hg vapor is trapped by a gold amalgamator system (Milestone, DMA 8134). A gold amalgamator is used to selectively trap and pre-concentrate the mercury from the flow of decomposition products. The determination is carried out by atomic absorption spectrometry at 253.65 nm after the thermally desorbed Hg is trapped in the amalgamator. This system has a tricell spectrophotometer that allows the instrument to work at low concentrations. A peak height was used for the signal evaluation. The working parameters used to determine Hg in animal feed are described in Table II. The precision of the method was verified by the analysis of certified reference materials. Table II. Working parameters for mercury determination in animal feed.

Parameter

Setting

Maximum start temperature/°C

Drying temperature/°C

200

Drying time/s

Decomposition temperature/°C

650

Decomposition time/s

Amalgamator heating time/s

Signal recording time/s

Purge time/s

Cuvette temperature/°C

120

Cuvette type

quartz

Air compressed pressure/bar Air compressed flow rate/mL Sample mass/mg

3 min-1

100 200 for water, 35 for Dorm-4, 50 for NIST 1568a

RESULTS AND DISCUSSION Figure 1 describes the calibration curve, with the ﬁrst point of calibration at 0.01 ng. The linearity was more than 0.9978, which was satisfactory for this analysis. After this, a reproducibility test (5 replicates) -1 was performed at 0.2 µg Kg . The certiﬁed reference materials NIST 1568a and DORM-4 were also analyzed, and the results are given in Table III.

Bruno M. Siqueira, Valerio Rindone

Sponsor Report

Figure 1. Calibration curve for low mercury concentrations.

Table III. Mercury concentration results.

Sample

Mercury Concentration Found (µg kg-1)

Expected Mercury Concentration (µg kg-1)

Replicate 1

0.195

0.200

Replicate 2

0.203

0.200

Replicate 3

0.195

0.200

Replicate 4

0.201

0.200

Replicate 5

0.198

0.200

σabs=0.003 µg kg-1, σrel=1.74% NIST 1568 (5 replicates) DORM-4 (5 replicates)

5.89 ± 0.19

5.8 ± 0.5

390.06 ± 8.97

410 ± 55

Figure 2 provides an example of the 0.2 µg kg-1 signal. The sensitivity of the DMA-80 allows for working with a very signiﬁcant signal. The gold amalgamator can collect the Hg and release it during recording, giving a symmetrical peak.

Figure 2. Example of a 0.2 µg kg-1 signal.

Bruno M. Siqueira, Valerio Rindone

Sponsor Report CONCLUSIONS The DMA tricell can work at 0.2 ppb with a very high sensitivity. It is important to perform an accurate calibration at low levels and to use clean quartz boats. The accuracy tests with certified reference materials matched the expected values, confirming the accuracy and reliability of the DMA-80. The DMA-80 is very easy to use and gives results in about 5 min with no sample preparation required for all sample matrices. The method is flexible in terms of matrices and working range when switching from low to high and high to low quickly, avoiding any carry over.

REFERENCES 1. http://www.who.int/topics/drinking_water/en/ [Accessed July 2013]. 2. The Ministry of Health of Brazil promulgated on December 12, 2011, the Ordinance 2914. 3. http://www.mma.gov.br/port/conama/res/res05/res35705.pdf [Accessed 11 January 2018]. This sponsor report is the responsibility of Nova Analítica and Milestone SRL.

Br. J. Anal. Chem., 2018, 5 (19), pp 65-69

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Fully Automated, Intelligent, High-Throughput Elemental Analysis of Drinking Waters using SQ-ICP-MS Marcus Manecki, Daniel Kutscher, Christoph Wehe, Robert Henry, Julian Wills and Shona McSheehy Ducos Thermo Fisher Scientiﬁc, Bremen, Germany

The goal of this work is to demonstrate robust high-throughput analysis of environmental samples using SQ-ICP-MS in He-KED mode, in accordance with the requirements of U.S. EPA method 200.8 Revision 5.5 and to demonstrate the performance of the Thermo Scientific™ iCAP™ RQ ICP-MS coupled to the ESI prepFAST Autodilution system. Keywords: Autodilution, Drinking water, EPA method 200.8 revision 5.5, He KED, ICP-MS, Sample preparation, SQ-ICP-MS. INTRODUCTION EPA Method 200.8 analyses for the quantification of trace metals in drinking and waste waters are performed routinely in many laboratories. Thousands of analyses are performed per week to support the monitoring and control of drinking water contaminants and water quality. Due to the complexity of the standard operating procedure (SOP), skilled technicians are required to setup and prepare the daily analysis, as well as actively monitor the results and perform further sample manipulation as required throughout the analytical run. The need for technical staff is a factor that keeps the overall expense of routinely running the 200.8 method relatively high. Recent advances in autodilution offer the potential to automate much of the sample preparation and data review with automated re-runs of any samples that do not meet predefined limits. By automatically creating a calibration set of standards from one stock standard and then diluting each sample to a predefined dilution level, an autodilution system can save valuable analysts' time and reduce costs overall through the lowered consumption of utilities and lab supplies. Fast sample throughput is another driving factor when implementing routine SOPs. Throughput in the method described herein is improved by the discrete sampling of the autodilution system, dramatically reducing uptake and washout time, as well as the use of a single measurement mode for the analysis of all the analytes in the method. The use of kinetic energy discrimination with helium as a reaction cell gas (He KED) ensures comprehensive interference removal and confidence in the accuracy of the analytical results. Whereas other single quadrupole (SQ) ICP-MS systems require multiple methods for the analysis of drinking water, the iCAP RQ ICP-MS collision/reaction cell (QCell) has a high ion transmission across the mass range so that all of the analytes in the method, including low mass analytes such as Li and Be, can be measured in He KED mode. This eliminates the extra overheads of switching times between different modes and simplifies method development. This report describes the fully automated, intelligent, high throughput EPA 200.8 analysis of environmental samples using a prepFAST Autodilution system (Elemental Scientific Inc., Omaha, NE, USA) integrated with the iCAP RQ ICP-MS. MATERIALS AND METHODS Sample Preparation for U.S. EPA 200.8 Rev 5.5 All samples were prepared according to the EPA 200.8 method. For the determination of dissolved analytes in drinking water, tap water was collected in an HDPE tank and acidified to 1% v/v HNO3 (Optima™ 65

Fully automated, intelligent, high-throughput elemental analysis of drinking waters using SQ-ICP-MS

Sponsor Report grade acid, Fisher Chemicals). Aliquots (20 mL) from the tank were ﬁlled into 50 mL polypropylene centrifuge tubes for analysis. The standards and quality control (QC) solutions were prepared according to the protocol outlined in Figure 1.

AUSS: Gold Standard Solution, CCV: Continuous Calibration Verification, CS-1 to 4: Calibration Standards, HG-50: Mercury Standard (50 ppb), LFB: Laboratory Fortified Blank, LFM-W: Laboratory Fortified Matrix, MDL-1 to 3: Solutions to determine Method Detection Limit, SQC-1 to 4: Standards for Quality control.

Figure 1. Scheme of (a) standard and (b) QC solutions required for EPA 200.8.

Mass Spectrometry The iCAP RQ ICP-MS coupled to the prepFAST Autodilution system with an SC-2DX Autosampler (Figure 2) was used for acquisition of all data. The iCAP RQ ICP-MS was operated in He KED mode for all analytes. Instrumental parameters are listed in Table I. Table I. Instrument conditions Parameter iCAP RQ ICP-MS Nebulizer Nebulizer Gas Flow Interface Setup Cell Gas Flow KED Voltage prepFAST Sample Loop Time per Analysis

Value PFA-ST 1.02 L min-1 Ni Cones, High Matrix Skimmer insert 4.8 mL min-1 He 3V 1.5 mL 66 s

Figure 2. prepFAST Autodilution system connected to the iCAP RQ ICP-MS (left). ESI SC-2DX Autosampler (right). 66

Manecki, M.; Kutscher, D.; Wehe, C.; Henry, R.; Wills, J.; Ducos, S. McS.

Sponsor Report Data Analysis Thermo Scientific Qtegra™ Intelligent Scientific Data Solution™ (ISDS) Software was used for quantitative assessment of the data. Working from a predefined EPA 200.8 template, the only user action needed is to enter the number of samples to be analyzed in the analytical batch. All parameters that must be monitored and achieve certain criteria to comply with EPA 200.8 are automatically checked by the Quality Control feature set included in the default installation of the Qtegra ISDS Software. Samples that do not meet all criteria e.g. Internal Standard (ISTD) recovery rates or over-range analyte concentrations, are automatically diluted to an appropriate level as calculated or defined within the software and the measurement automatically repeated. Intelligent Autodilution with prepFAST Dilution factors of up to 400-fold are performed reliably and accurately, with all flows controlled by high precision syringe pumps. With the intelligent dilution feature, Qtegra ISDS Software registers every analyte that falls outside of the defined quality control requirements. If an analyte exceeds the calibration range (Figure 3) the intelligent autodilution dilutes the sample and re-measures only the affected analytes without manual interaction. The applied dilution factor is recorded in the software for full traceability of all dilution steps executed during data acquisition.

Analytes exceeding the calibration curve trigger the intelligent auto-dilution!

Measured with corrected dilution factor of 2.165

Figure 3. Analyte concentration re-analyzed by intelligent auto-dilution. Original sample (left), reanalyzed analyte with dilution factor 2.165 (right). RESULTS AND DISCUSSION Routine Performance of the iCAP RQ ICP-MS Over 320 tap water samples were analyzed according to method EPA 200.8. The analysis time was, on average, 66 s per sample for the analysis of 21 elements listed in EPA method 200.8 plus 6 diﬀerent internal standards, leading to a total number of 48 individual isotopes being read out per sample. The concentration of all analytes and their ISTD recovery was monitored throughout the whole analysis time. In total, 508 analyses were run in less than 10 h. Internal standard recovery was well within the EPA 200.8 method requirements of 60 to 125% (Figure 4).

Figure 4. Internal standard response of running tap water samples and QCs showing recoveries well within the 60 – 125% range speciﬁed in EPA Method 200.8. 67

Fully automated, intelligent, high-throughput elemental analysis of drinking waters using SQ-ICP-MS

Sponsor Report Quality Control (QC) Samples During the analysis run, a Continuing Calibration Veriﬁcation (CCV) QC sample was analyzed every 10 samples to assess the accuracy of the calibration throughout the entire batch. The EPA 200.8 method requires that the recovery of this QC must be within ± 10%, or within the acceptance limits of the method (EPA 200.8, rev 5.5, Table 8). All elements were found to be accurate to within ± 10% of the known concentration, as well as the acceptance criteria, and were stable over all repeated analyses (Figure 5).

Figure 5. QC recovery and stability of the continuous calibration samples over the entire batch.

Laboratory Fortified Blank and Laboratory Fortified Matrix Recoveries The recovery of a Laboratory Fortified Blank (LFB) with known added amounts of analytes (Figure 1a, solution 3) must be measured at least once per batch of samples. During this assessment, the LFB was analyzed 32 times and the calculated recovery rates are shown in Figure 6. All analytes show recoveries within the limits (85–115%) of EPA 200.8. Similar to the LFB recovery for every batch, one sample must also be spiked with a known amount of analytes, (Laboratory Fortified Matrix sample; LFM). All 32 LFM (Figure 1a, solution 7) samples were within the EPA 200.8 recovery limits (75-130%).

Figure 6. Laboratory Fortiﬁed Blank (LFB) recoveries from measurements. Blue bars show the highest (green lowest) recovery of the analyte measured during the 10 h run. The grey area represents the EPA 200.8 acceptance range (85-115%) for LFB recoveries.

Driven by Qtegra ISDS Software Fully Integrated The Qtegra ISDS Software provides all required features needed for the high throughput analysis of environmental samples. Together with the fully integrated prepFAST Autodilution system, Qtegra ISDS Software oﬀers:

Manecki, M.; Kutscher, D.; Wehe, C.; Henry, R.; Wills, J.; Ducos, S. McS.

Sponsor Report · Prescriptive dilution of samples and calibration standards. · Continuous monitoring of all quality controls (LFB and LFM recoveries or duplicate sample verification) · LabBook feature that starts an intelligent sequence, with full QA/QC protocols, and subsequently processes and reports results. · Comprehensive, user definable reports enabling flexible export to external LIMS software packages. Intelligent autodilution for samples exceeding the calibration range is fully integrated. Samples remeasured by the Qtegra ISDS Software are added automatically to the sample list and clearly identified by a plus sign (Figure 7).

Figure 7. Screenshot of the intelligent auto-dilution process in Qtegra ISDS Software.

CONCLUSION The Thermo Scientiﬁc iCAP RQ ICP-MS equipped with an ESI Autosampler and prepFAST Autodilution System was successfully validated for use with US EPA Method 200.8. With the robust iCAP RQ ICP-MS paired with an ESI prepFAST Autodilution system, it is possible to run the entire analysis (encompassing sample dilution, calibration and measurement) with minimal manual intervention. After optimizing the uptake and washout parameters, the high sensitivity and stability of the iCAP RQ ICP-MS readily achieved the goal of 52 EPA Method 200.8 analyses per hour. Robustness The iCAP RQ ICP-MS delivers reliable analysis of drinking water with minimal drift when equipped with the high matrix insert. For extra robust operation in the face of higher matrix samples, the system can be equipped with the robust plasma interface. Productivity The iCAP RQ ICP-MS in combination with the ESI prepFAST Autodilution System is the ideal system to measure environmental samples in a high throughput laboratory. Simplicity With the prescriptive and intelligent dilution capabilities provided by the system, manual sample preparation and data post-processing is minimized. No Impact on Bench Space The integrated dual valve assembly is mounted directly beneath the sample introduction system, minimizing sample pathways. This sponsor report is the responsibility of Thermo Fisher Scientiﬁc

Br. J. Anal. Chem., 2018, 5 (19), pp 70-81

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Fast Anion Determinations in Environmental Waters Using a High-Pressure Compact Ion Chromatography System Terri Christison, Linda Lopez, and Jeﬀ Rohrer Thermo Fisher Scientiﬁc, Sunnyvale, CA, USA

This report demonstrates the fast analysis of environmental water samples containing anions at ppm concentrations using the latest column, suppressor, and IC instrument technology. The run time was reduced to 12 min, saving four minutes of analysis time for characterizing a sample containing seven anions (including phosphate) per sample. Reliability and accuracy with standards and samples are shown by the retention time and peak area reproducibilities with a RSD of < 0.1 and < 0.2, respectively, improved chromatography and resolution of critical peak pairs, and 89–105% recoveries. Keywords: Standard Bore, Integrion, HPIC, Fast IC, IonPac AS18-4 μm, RFIC, Reagent-Free IC INTRODUCTION Ion chromatography (IC) is a well-established and accepted technique for the monitoring of inorganic anions in environmental waters, such as surface, ground, and drinking waters. In the U.S., water quality is legislated through the Safe Drinking Water Act (SDWA) and the Clean Water Act (CWA). The goal of the CWA is to reduce the discharge of pollutants into waters, whereas the SDWA ensures the integrity and safety of drinking waters [1,2]. Inorganic anions are regulated in drinking water as primary contaminants (fluoride, nitrate, nitrite, and disinfection byproducts) for health reasons, as secondary contaminants affecting taste, color, or odor, or for aesthetic reasons, as in the case of chloride or sulfate contamination. Many of these same inorganic anions are regulated through the CWA industrial permitting process. In the U.S., compliance monitoring of inorganic anions in drinking water and wastewater has been required since 1992 to follow U.S. EPA Method 300.0, updated in 1997 to U.S. EPA Method 300.1 [3,4]. Other industrial countries, such as Germany, France, Italy, Japan, and China, have similar requirements (ASTM, EU International Organization for Standardization (ISO), China EPA). In U.S. EPA Method 300.0 (Part A) and 300.1 (Part A), inorganic anions are separated by anion-exchange chromatography on the Thermo Scientific™ Dionex™ IonPac™ AS4A and Dionex IonPac AS14A anion exchange columns, respectively, using manually prepared carbonate-based eluents and detected by suppressed conductivity detection. However, there have been significant advances in technology since 1997. In accordance with the rapid technology advancements, the standard methods allow for comparable results using alternative columns, eluents, suppression devices, and detectors. As such, this application has been updated numerous times. In the latest iteration, Thermo Fisher Scientific Application Note 154 (AN154) [5], demonstrated increased sensitivity, peak retention time, and peak area precision using hydroxide eluents over the previous application (AN133) [6], which used manually prepared carbonate eluents. In AN154, the effectiveness of electrolytic eluent generation of hydroxide eluent combined with a hydroxide-optimized, high-capacity anion-exchange column (Dionex IonPac AS18) was demonstrated on a Thermo Scientific™ Dionex™ ICS-2000 Integrated IC system. In AN154 (originally published in 2003), seven anions were eluted within 16 min using an electrolytically generated hydroxide gradient and detected by suppressed conductivity detection with the continuously regenerated Thermo Scientific™ Dionex™ ASRS™ ULTRA Anion Self-Regenerating Suppressor. 70

Fast Anion Determinations in Environmental Waters Using a High-Pressure Compact Ion Chromatography System

Sponsor Report This document updates AN154 with fast separations (12 min run time) on the higher pressure Dionex IonPac AS18-4μm column, reducing the run time by 8 min. This column is optimized with the same selectivity using the 4 μm resin particles but with a shorter format (150 mm versus 250 mm length). The Dionex IonPac AS18-4μm allows for faster run times while maintaining highly efficient separations, thereby yielding good quantification accuracy and consistently reliable results. This application is demonstrated on the high-pressure-capable Thermo Scientific™ Dionex™ Integrion™ HPIC™ compact IC system. The Dionex Integrion HPIC system includes the recent advances in IC instrument technology, including high-pressure capabilities for Reagent-Free™ IC (RFIC) (up to 5000 psi), column heater control, and many new features designed to increase customer ease-of-use. These features include: • A compact, fully integrated system design. • Easy access to eluent generator and electrolytic trap column. • Separate compartments for pump, column heater with injection valve, and detection-suppressor to provide separate temperature control and faster equilibration. • Thermo Scientifc™ Dionex™ IC PEEK Viper™ fittings replacing standard fitting connections in specified positions to minimize void volume problems, improve chromatography, and ensure accurate reporting. • Components tracked by consumables device monitoring for GMP compliance tracking, which prompts the user to install compatible devices and reduces the likelihood of an improper set-up. • Independent tablet control for convenient, continuous full-screen monitoring, independent manual control, and the online instrument manual and troubleshooting guides. • New Thermo Scientific™ Dionex™ Chromeleon™ 7 Chromatography Data System (CDS) software features that provide easy instrument configuration, monitoring of consumable devices, and online video instructions for conditioning columns, suppressors, and other electrolytic devices. The Dionex Integrion HPIC system can also be configured for electrochemical detection of electroactive ions and mono- and disaccharides. Another model of the Dionex Integrion HPIC system, without RFIC capabilities, can be configured with or without conductivity detection. MATERIALS AND METHODS Equipment • Thermo Scientific Dionex Integrion HPIC High-Pressure IC system includes: - CD conductivity detector - Column oven temperature control - Detector compartment temperature control - Tablet control - Consumables device monitoring capability - Eluent generation capabilities • Thermo Scientific™ Dionex™ AS-AP Autosampler with 10 mL trays Software Thermo Scientific Dionex Chromeleon CM 7.2 SR4 CDS software was used. Consumables Table I lists the consumable products recommended for the Dionex Integrion HPIC system, configured for suppressed conductivity detection.

Christison, T.; Lopez, L.; Rohrer, J.

Sponsor Report Table I. Consumables list for the Dionex Integrion HPIC System Product Name

Product Details

Dionex IC PEEK Viper Fitting Tubing Assembly Kits

Dionex IC PEEK Viper fitting assembly kit for the Dionex Integrion HPIC system includes one each of P/Ns: 088805-088808, 088810, 088811

088798

Guard to separator column: 0.007 × 4.0 in. (102 mm)

088805

Injection valve, Port C (Port 2) to guard column: 0.007 × 5.5 in. (140 mm)

088806

EGC Eluent Out to CR-TC Eluent In: 0.007 × 6.5 in. (165 mm)

088807

Separator column to Suppressor Eluent In: 0.007 × 7.0 in. (178 mm)

088808

Suppressor Eluent Out to CD In: 0.007 × 9.0 in. (229 mm)

088810

CR-TC Eluent Out to Degasser Eluent In: 0.007 × 9.5 in. (241 mm)

088811

Dionex AS-AP Autosampler Vials

Package of 100, polystyrene vials, caps, blue septa, 10 mL

074228

Thermo Scientific™ Dionex™ EGC™ 500 KOH Eluent Generator Cartridge*

Eluent generator cartridge

075778

Thermo Scientific™ Dionex™ CR-ATC™ 600 Electrolytic Trap Column*

Continuously regenerated trap column used with Dionex EGC KOH 500 cartridge

088662

HP EG Degasser Module*

Degasser installed after Dionex CR -TC trap column and before the injection valve. Used with eluent generation

075522

Thermo Scientific™ Dionex™ AERS™ 500 Suppressor

Suppressor for 4 mm and 5 mm columns, using recycle mode

082540

Dionex IonPac AG18-4μm Column

Anion guard column, 4 × 30 mm

076035

Dionex IonPac AS18-4μm Column

Anion separation column, 4 × 150 mm

076034

Thermo Scientific™ Nalgene™ Syringe Filter

Syringe filters, 25 mm, PES membrane, 0.2 μm. This type is compatible with IC analysis

Dionex IC PEEK Viper Fitting Tubing Assemblies, Included in Kit, P/N 088798

* High-pressure device recommended for 4 μm particle resin columns.

Part Number

Thermo Scientific 7252520 / Fisher Scientific 09-740-113

Fast Anion Determinations in Environmental Waters Using a High-Pressure Compact Ion Chromatography System

Sponsor Report Chromatographic Conditions Columns

Thermo Scientific Dionex IonPac AG18 -4μm guard (4 × 30 mm) and Dionex IonPac AS18 -4μm separation (4 × 150 mm)

Eluent

15–44 mM KOH (0.2 –6 min, 44 mM KOH (6 –9 min, 15 mM KOH (9 –12 min)

Eluent Source

Thermo Scientific Dionex EGC 500 KOH cartridge with Thermo Scientific Dionex CR-ATC 600 trap column and high -pressure EG degasser

Flow Rate

1.0 mL/min

Column Temperature

35 °C

Detector Compartment Temperature

15 °C

Injection Volume

10 μL, in Push -Full mode

Detection

Suppressed conductivity, Thermo Scientific Dionex AERS 500 suppressor, 4 mm, recycle mode

Run Time (min)

12 min

Background Conductance (μS)

Typical Noise (nS)

System Backpressure (psi)

~ 2200

Samples and Sample Preparation Samples were municipal drinking water, surface water, wastewater, and well water. Drinking water samples were analyzed with minimal dilution (with deionized water). All surface, wastewater, and well water samples were ﬁltered (0.2 μm) prior to injection. Instrument Setup and Installation The Dionex Integrion HPIC system is a high-pressure-capable integrated Reagent-Free IC (RFIC) system. The Dionex Integrion HPIC system, Dionex EGC 500 KOH cartridge, and Dionex CR-ATC 600 consumable products are designed for high-pressure conditions up to 5000 psi. To set up this application, connect the Dionex AS-AP autosampler and the Dionex Integrion HPIC system modules according to Figure 1. Note that the injection valve is plumbed through diﬀerent ports than previous Dionex IC systems.

Figure 1. Flow diagram for the Dionex Integrion HPIC system. 73

Christison, T.; Lopez, L.; Rohrer, J.

Sponsor Report Connect the USB cables from the Dionex Integrion HPIC system to the Dionex AS-AP autosampler and to the computer. Connect the power cables and turn on the IC instrument and the autosampler. Configuring the Modules in the Chromeleon CDS Software To configure the IC system, first start the Chromeleon CDS software Instrument Controller program and then select the link Configure Instruments (opens the Chromeleon Instrument Configuration Manager). Right-click on computer name, select Add an Instrument, and enter an appropriate name (for example: Integrion_ EPA300_1). Select Add a Module, IC: Dionex Integrated Modules, and Integrion HPIC System. The instructions to configure each module are summarized at the end of this section in Table II. After the multi-tabbed program opens, select the Model Serial No. The Chromeleon CDS software will automatically detect all Dionex Integrion HPIC system devices: the electrolytic devices, detectors, pump degasser, and seal wash requiring minimal data entry during instrument configuration. The Chromeleon CDS software automates the system configuration process by automatically detecting the installed devices. To add pressure monitoring capabilities in the configuration, right-click and select Add a Module, IC: Dionex Integrated Modules, Integrion HPIC Pump (Wellness) module and then select the USB address to link the module to the configuration. Select the Devices tab and select the Pressure Signal(s) checkbox (Figure 2). Table II. Summary of system configuration for high-pressure Dionex Integrion HPIC system Tab

Action

Result

Dionex Integrion HPIC Module General

Link to USB address

Pump

Flow rate and pressure limitations are displayed

Detectors

Automatically detected

Electrolytics

Automatically detects Dionex eluent generator cartridges and Dionex CR -TC trap columns

Inject Device

Automatically detected

Thermal Controls

Automatically detects thermal control options for column, detector, and suppressor

High-Pressure Valves

Automatically detected

Low-Pressure Valves

Automatically detected

Options

Automatically detects pump degasser and seal wash pump

Pump Wellness Module Devices

Select Pressure Signal checkbox

Activates pressure monitoring feature (Figure 2)

Add Dionex AS -AP Autosampler Add Module

Link to USB address

Sharing

Only if more than one instrument is detected. If this option is present, select Instrument

Segments / Pump Link

Select 10 mL polystyrene vials or 1.5 mL vials for “Red”, “Blue”, and “Green”

Options

Select Push, select syringe size, select 1.2 mL buffer line, enter the loop size

Fast Anion Determinations in Environmental Waters Using a High-Pressure Compact Ion Chromatography System

Sponsor Report

Figure 2. Adding the Dionex Integrion HPIC Pump Wellness module to the instrument conﬁguration.

Add the Dionex AS-AP Autosampler to the Configuration Add the Dionex AS-AP autosampler as a module, and select the USB address. In the Segments/Pump Link tab, select the appropriate vial trays for each color zone. In the Options tab, select Push, installed syringe size, 1.2 mL for buffer line, and enter the sample loop volume. Save the configuration, select Check the Configuration, and then close the Chromeleon CDS software Instrument Configuration program. Plumbing the High-Pressure Dionex Integrion HPIC System Tip: To achieve the best chromatography, it is important to gently tighten the IC PEEK Viper fittings to finger-tight plus 1/8 clockwise turn for the first installation, and 1/16 turn the second use. Use the IC PEEK Viper fitting assemblies: · Dionex EGC 500 KOH eluent generator cartridge - Eluent Out to Eluent In on Dionex CR-ATC 600 trap column · Dionex CR-ATC 600 trap column - Eluent Out to Eluent In on the Dionex Degas Module · Injection Valve - Port 2 (Column) to the guard column · Between the guard and separation columns · Separation column to Eluent In on the Dionex AERS 500 Suppressor · Dionex AERS 500 Suppressor - Eluent Out to Eluent In on CD Conductivity Cell First, loosen the waste lines, including the metal-wrapped waste line in the back of the instrument, and direct the free ends to a waste container. To plumb the system, first connect the pump eluent line to the eluent bottle containing previously degassed (vacuum filtration and ultrasonic agitation) deionized water. Prime the pump by opening the priming knob ¼ turn and press the priming button. Prime the pump until no bubbles are visible and water is flowing at a steady rate out of the pump waste line. Close priming knob to finger-tight. For more information, review the product manual by selecting “?” on the tablet. Conditioning Electrolytic Devices and Columns Tip: Do not remove RFIC tags on the columns and consumable devices. These tags are required for RFID monitoring functionality. 75

Christison, T.; Lopez, L.; Rohrer, J.

Sponsor Report Install the Dionex EGC 500 KOH cartridge and Dionex CR-ATC 600 Continuously Regenerating Anion Trap Column in reservoir tray compartment. Condition the devices according to instructions in the dropdown menu under Consumables > Install (Figure 3). (This information is also available in the product manuals and the system installation manual [7-9].) Install one black PEEK (0.010 in i.d. tubing) backpressure loop (exerting an additional ~40 psi) at the cell outlet. To hydrate the Dionex ERS 500 suppressor, follow the QuickStart Instructions received with the suppressor and in the product manual [10]. Wait for 20 min for the suppressor to fully hydrate before installing the suppressor in the detector compartment. Install the backpressure loop between the CD outlet and the suppressor Regen In port. Condition the columns for 30 min according to the instructions in the Consumables, Install Column section (Figure 3). The general practice is to follow the eluent and ﬂow rate conditions listed in the QAR report while directing the eluent exiting the column to a waste container [11]. Complete the installation according to the Figure 1 ﬂow diagram.

Figure 3. Consumables online installation instructions.

Installing and Optimizing the Dionex AS-AP Autosampler The Dionex AS-AP autosampler needle must be aligned to the injection port. To align the autosampler needle, first select the Sampler tab on the instrument panel and press the Alignment button. Follow the commands to align the autosampler needle to the Injection Port and Wash Port (Section B.12 in the Operator's Manual) [12]. Then, connect the autosampler syringe line to wash container containing degassed water to the syringe. Prime the syringe to flush out any air in the Buffer Wash line and syringe. Initially select a 5000 μL wash volume until a steady flow of water is observed at the Wash Port. Then, calibrate the transfer line volume by following the prompts on the TLV Calibration button. This volume will be recorded automatically. For more information, review Section 5.9 in the Dionex IC Series AS-AP Autosampler Operator's Manual [12]. Starting the Dionex Integrion HPIC System To start the system, turn on the pump and immediately turn on both the Dionex EGC 500 cartridge and the Dionex CR-ATC 600 trap when liquid is flowing through the device. The system backpressure is dependent on the flow rate and type of column, but the system must be above 2000 psi to support the Dionex EGC cartridges. Typically, columns with 4 μm resin particles operate, as is, above 2000 psi and therefore do not require backpressure tubing. However, if additional pressure is needed to achieve system pressures > 2000 psi, install yellow PEEK backpressure tubing (yellow PEEK, 0.076 mm i.d., 0.003 in i.d.) between the Dionex HP EG Degasser module and the injection port (Pump position). Set the eluent concentration, column oven, compartment oven, and cell temperatures as shown in the Conditions section in the application. Allow the system to equilibrate for 30 min. For optimum chromatography equilibrate until the total background is stable, 1–2 μS. 76

Fast Anion Determinations in Environmental Waters Using a High-Pressure Compact Ion Chromatography System

Sponsor Report Creating an Instrument Method To create a new instrument method using the Chromeleon Wizard, select Create, Instrument Method, and select Instrument. Enter the values from the Chromatographic Conditions section. Save the instrument method. Consumables Device Monitoring Tip: An action (approve or correct an incompatibility between devices) is required to start a sequence after installing any new consumable device. A new feature of the Dionex Integrion HPIC system is consumables device monitoring and tracking, which automatically detect the electrolytic devices and the columns. Review and approval of the devices is required to start the ﬁrst sequence on the Dionex Integrion HPIC system and after installing new consumable devices. To access this approval, select Consumables and select Inventory (Figure 4).

Figure 4. Consumables tracking.

Device monitoring shows the device history, tracking, part number, size, chemistry, serial numbers, manufacture lot, installed location (On Device), and best if used by date (Figure 4, top). Additionally, the device monitoring will provide warnings if there is incompatibility in the devices installed (Figure 4, bottom left). To start the sequence, review the list of consumables listed as inventory, correct any errors, approve, and close page (Figure 4, bottom right). Then, select the Instrument Queue tab, and conduct a Ready Check on the sequence and press Start. RESULTS AND DISCUSSION

Seven anions, including phosphate, were separated using an electrolytically generated hydroxide gradient from 14 mM to 44 mM KOH (0.2 to 9 min) on the Dionex IonPac AS18-4μm, 4 × 150 mm, highcapacity, 4 μm resin particle anion-exchange column. All anions were eluted within 9 min and detected by suppressed conductivity with the latest innovation in suppressors (Dionex AERS-500) using the highpressure capable Dionex Integrion HPIC system. This method demonstrates shorter run times than those in AN154 using the 4 x 250 mm, 7.5 μm column. Faster runs are made possible using the shorter, 150 mm length columns, while the 4 μm resin particles provide highly efficient separations that allow for the reduction in column length. The improved efficiency also allows the column temperature to be increased five degrees to help shorten the analysis time. Additionally, the Dionex Integrion HPIC system has a separate suppressor-detector compartment that can be set to 15 °C, which provides increased suppressor efficiency, resulting in improved chromatography. In Figure 5, the same standard is compared under the same analytical conditions run on both resin formats of the Fast (4 × 150 mm), IonPac AS18 chemistry columns: Chromatogram A - Dionex IonPac AS18-Fast column composed of 7.5 μm resin particles versus Chromatogram B - Dionex IonPac AS18- 4μm column composed of 4 μm resin particles. This figure shows the improved peak efficiencies with the 4 μm column, as exhibited by the smaller peak widths and higher peak response, as well as the higher resolution of the carbonate-bromide and sulfate-nitrate critical pairs

Christison, T.; Lopez, L.; Rohrer, J.

Sponsor Report

Figure 5. Comparison of Dionex IonPac AS18-Fast columns.

Method Qualiﬁcation To evaluate the method, the retention time and peak area precisions were determined by running seven replicate injections of a 50 mg/L mixed anion standard. The results, summarized in Table III, show excellent precision, < 0.1 and 0.1–0.2 RSDs for retention time and peak area, respectively. Table III. Method reproducibilities using a 50 mg/L standard

Retention Time

Peak Area

(min)

(RSD)

Fluoride

2.307 ± 0.002

0.09

9.237 ± 0.012

0.13

Chloride

3.714 ± 0.001

0.04

6.056 ± 0.011

0.19

Nitrite (NO2-N)

4.506 ± 0.006

0.06

4.206 ± 0.006

0.13

Bromide

5.838 ± 0.002

0.03

2.370 ± 0.003

0.14

Sulfate

6.341 ± 0.002

0.04

4.105 ± 0.006

0.14

Nitrate (NO3-N)

6.630 ± 0.002

0.03

3.300 ± 0.005

0.14

Phosphate (PO4-P)

8.714 ± 0.007

0.08

2.171 ± 0.004

0.19

(μS-min) (

)

(RSD)

n=7

Replicate injections of seven combined anion standards were used to determine the linear concentration ranges based on peak area. The MDLs were determined using a 20–50x dilution of the lowest calibration standard (3.14 × σ (standard deviation)). The results, summarized in Table IV, show linear responses within the calibration range and MDLs < 2 μg/L for all anions except bromide.

Fast Anion Determinations in Environmental Waters Using a High-Pressure Compact Ion Chromatography System

Sponsor Report Table IV. Linearity and MDL results

Calibration Range (mg/L)

Coefficient of Determination

MDL Standard (μg/L)

MDL (μg/L)

Fluoride

0.04–100

0.99995

0.3

Chloride

0.50–200

0.99999

0.6

Nitrite (NO2-N)

0.97–100

0.99961

0.1 (0.4 as NO2)

Bromide

0.7–100

0.99993

2.6

Sulfate

0.8–200

1.00000

1.4

Nitrate (NO3-N)

0.16–100

0.99999

0.9 (4.1 as NO3)

Phosphate (PO4-P)

0.17–100

0.99997

0.8 (2.6 as PO4)

Sample Analysis This method was applied to municipal drinking and wastewater, surface water, well water, softened well water, inland sea, artiﬁcial lake, and pool water samples. Chromatograms of municipal wastewater and surface water samples are shown in Figures 6 and 7. To determine accuracy, recoveries were calculated after adding concentrated anion standards (0.5 to 2× the concentration present) to selected samples: surface, pool, municipal waste, and drinking water. The results, summarized in Table V, show good recoveries, 89–105%, demonstrating acceptable accuracy.

Figure 6. Determination of anions in a diluted municipal wastewater sample.

Christison, T.; Lopez, L.; Rohrer, J.

Sponsor Report

Figure 7. Determination of anions in an undiluted surface water sample. Table V. Recovery results

Municipal Wastewater

Added (mg/L)

Recovery (%)

Surface water

Added (mg/L)

Recovery (%)

Fluoride

102

Fluoride

1.0

100

Chloride

103

Chloride

103

Nitrite-N

103

Nitrite-N

94.9

Bromide

89.5

Bromide

90.2

Sulfate

95.3

Sulfate

104

Nitrate-N

102

Nitrate-N

95.1

Phosphate-P

98.7

Phosphate-P

97.0

Pool water

Added (mg/L)

Recovery (%)

Added (mg/L)

Recovery (%)

Fluoride

104

Fluoride

1.0

101

Chlorite

Detected

Chloride

100

96.7

Chloride

99.6

Nitrite-N

98.8

Nitrite-N

98.0

Bromide

90.5

Bromide

88.7

Sulfate

91.4

Sulfate

104

Nitrate-N

99.6

Nitrate-N

91.2

Phosphate-P

105

Phosphate-P

96.0

Municipal Drinking water

Fast Anion Determinations in Environmental Waters Using a High-Pressure Compact Ion Chromatography System

Sponsor Report CONCLUSIONS Fast analysis of environmental water samples containing anions at ppm concentrations were demonstrated using the high resolution capabilities of the Dionex IonPac AS18-4μm column facilitated by the high-pressure capabilities of the Dionex Integrion HPIC system. In this update of AN154: · The method was updated with the latest column, suppressor, and IC instrument technology. · The method demonstrated reliability and accuracy with standards and samples as shown by the retention time and peak area reproducibilities with a RSD of < 0.1 and < 0.2, respectively, improved chromatography and resolution of critical peak pairs, and 89–105% recoveries. · The run time was reduced to 12 min, saving four minutes of analysis time for characterizing a sample containing seven anions (including phosphate) per sample. REFERENCES

1. Fed. Regist., 1999; Vol. 64, No. 230. 2. Fed. Regist., 1995; Vol. 60, No. 201. 3. U.S. EPA Method 300.0., U.S. Environmental Protection Agency; Cincinnati, Ohio, 4. U.S. EPA Method 300.1, U.S. Environmental Protection Agency, Cincinnati, OH,

1993. 1997.

5. Thermo Scientific Application Note 154: Determination of Inorganic Anions in Environmen tal Waters Using a Hydroxide -Selective Column. Sunnyvale, CA [Online] https://www.thermofisher.com/content/dam/tfs/ATG/CMD/CMD%20Documents/Application%20&%20 Technical%20Notes/Chromatography/GC%20HPLC%20and%20UHPLC%20Columns%20and%20A ccessories/Chromatograp hy%20Column%20Accessories/4117 -AN154_V19releasedJC052703.pdf (accessed Mar. 11, 2016). 6. Thermo Scientific Application Note 133: Determination of Inorganic Anions in Drinking Water by Ion Chromatography. Sunnyvale, CA [Online] http://www.thermoscientific.com /content/dam/tfs/ATG/CMD/cmd -documents/sci res/app/chrom/ic/sys/AN -133-IC-Inorganic-Anions-Drinking-Water-AN71691-EN.pdf (accessed Mar. 11, 2016). 7. Thermo Fisher Scientific. Dionex Integrion HPIC System Installation and Operator’s Manual, P/N 22153-97003, Sunnyvale, CA, 2015. 8. Thermo Fisher Scientific. Dionex Product Manual for Eluent Generator Cartridges, P/N: 065018 -05, Sunnyvale, CA, June 2014. 9. Thermo Fisher Scientific. Dionex Product Manual for the Continuously Regenerated Trap Column (CR-TC). P/N: 065018 -05, Sunnyvale, CA, November 2012. 10. Thermo Fisher Scientific. Dionex ERS 500 Suppressor Product Manual. P/N: 031956 -09, Sunnyvale, CA, November 2013. 11. Thermo Fisher Scientific. Dionex IonPac AS18-4μm Column Product Manual. P/N 065499 -02, Sunnyvale, CA, August 2014. 12. Thermo Fisher Scientific. Dionex AS -AP Operator’s Manual. Document No. 065259, Sunnyvale, CA, 2012. 2012 This sponsor report is the responsibility of Thermo Fisher Scientiﬁc.

Br. J. Anal. Chem., 2018, 5 (19), pp 82-85

Sponsor Report

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Automated Extraction of Acrylamide from Underground Water Prior to HPLC-DAD Analysis Santos, A. L. A., Souza, P. L., and Siqueira, S. Mineral Analysis Laboratory, Rio de Janeiro – Geological Survey of Brazil CPRM This report describes a sample concentration method by solid phase extraction (SPE) using the Gilson ASPEC 274 system. Water contaminants were concentrated 150-fold using SPE cartridges and analyzed by HPLC. An assay with a limit of quantification of 0.3 μg/L was developed with this technology. Keywords: Solid Phase Extraction, SPE, acrylamide, underground water, ANVISA Resolution RDC 274, Gilson ASPEC 274 INTRODUCTION Acrylamide is a synthetic compound widely used in the plastics industry and in water treatment. It is considered by the World Health Organization (WHO) as a potential carcinogen and therefore the maximum level allowed in water for human consumption is limited to 0.5 μg/L [1]. The Brazilian water legislation, regulated by the Brazilian Health Regulatory Agency (ANVISA), in Resolution RDC 274, established the same maximum allowed level for acrylamide defined by WHO. In order to achieve such a low limit of quantification, a sample pre-concentration step is necessary before determination of the acrylamide content. Most of the acrylamide produced is used as a chemical intermediate or as a monomer in the production of polyacrylamide. Both acrylamide and polyacrylamide are used mainly in the production of flocculants for the clarification of potable water and in the treatment of municipal and industrial effluents. They are also used as grouting agents in the construction of drinking-water reservoirs and wells. Acrylamide is highly mobile in aqueous environments and readily leachable in soil. As it has a higher mobility and lower rate of degradation in sandy soils than in clay soils, it may contaminate groundwater. The most important source of drinking-water contamination by acrylamide is the use of polyacrylamide flocculants containing residual levels of acrylamide monomer [1]. This report describes a sample preparation method by solid phase extraction (SPE) using the Gilson ASPEC 274 system. Method validation was done by the evaluation of the following metrics: limit of quantification, repeatability, reproducibility and recovery. A limit of quantification of 0.3 μg/L was achieved. MATERIALS AND METHODS Sample concentration by solid phase extraction Samples were collected in 250 mL amber glass flasks and stored at 2 ºC – 8 ºC. A control sample of 0.3 μg/L acrylamide was prepared in 150 mL of ultrapure water. CHROMABOND SPE cartridges (6 mL, 500 mg porous graphitic carbon phase, 730512) and collection tubes were arranged in a Gilson Code 386 rack. Each collection tube contained 0.5 mL of ultrapure water to avoid recovery loss. The SPE cartridge was conditioned with 10 mL of methanol and 10 mL of ultrapure water at 5 mL/min. A sample volume of 150 mL was loaded at 1 mL/min and the column was eluted with 9.3 mL of methanol at 0.6 mL/min. The eluates were concentrated in a TurboVap LV (Caliper Life Sciences) at 62 ºC with 10 psi N2. Samples were concentrated to less than 1 mL and the volume adjusted to 1.0 mL with ultrapure water and transferred to 1.5 mL vials.

Automated Extraction of Acrylamide from Underground Water Prior to HPLC-DAD Analysis

Sponsor Report Chromatographic analysis by HPLC-DAD Vials containing concentrated sample were transferred to an Ultimate 3000 RS autosampler. A control sample was included for each sample sequence for evaluation of the extraction. HPLC was conducted on an Ultimate 3000 RS UHPLC system (Thermo Fisher) with an Acclaim C18 column (250 mm x 4.6 mm, 5 μm particle size) with water/methanol (60:40 v/v) at a ﬂow rate of 0.8 mL/min at 35 ºC. The run was monitored with a DAD detector (Thermo Fisher) at 202 nm. RESULTS AND DISCUSSION The ASPEC 274 (Figure 1A) system was used to suﬃciently concentrate acrylamide (Figure 1B) to levels detectable by HPLC. A

B Figure 1. A: Gilson GX-274 ASPEC and B: chemical structure of acrylamide.

The positive control HPLC chromatogram is a 0.3 μg/L sample of acrylamide in ultrapure water which was concentrated using an ASPEC 274 system. HPLC analysis of the concentrated sample detected acrylamide at 4.2 min (Figure 2). A negative control consisting of an environmental sample showed that acrylamide was not detected (Figure 3). The highlighted boxes represent the spectra of the peak at: baseline (red), 50% before apex (green), apex (black), and 50% height after apex (purple). Polar organic compound present in the matrix was detected at 3 minutes.

Figure 2. HPLC chromatogram at 202 nm of a control sample of acrylamide at 0.3 μg/L in demineralized water.

Figure 3. Chromatogram of an environmental sample without acrylamide at 202 nm.

Santos, A. L. A.; Souza, P. L.; Siqueira, S.

Sponsor Report The limit of quantification (LOQ), repeatability, reproducibility, and recovery of the developed method were determined in accordance with the guidelines on validation of analytical methods in the document DOQ-CGCRE-008 INMETRO [3]. Each of these parameters was determined experimentally with seven independent acrylamide solutions that were subjected to the described solid phase extraction, volume reduction, resuspension, and chromatographic analysis. The limit of quantification was measured using a solution of 0.3 μg/mL acrylamide. The recovery and the relative standard deviation (RSD) were equal to 93.3% and 5.7% respectively. As the recovery and RSD values met the criteria previously established by the laboratory, the limit of quantification of the method was established as being equal to 0.3 μg/L. The repeatability was evaluated with 0.5 μg/L acrylamide and analyzed by the same analyst. The recovery and the relative standard deviation were 89.5% and 7.0%, respectively. The determination of intra-laboratory reproducibility was performed by different analysts on different days with 0.5 μg/L acrylamide. The recovery and the relative standard deviation were 90% and 6.8%, respectively (Table I). Table I. Summary of LOQ, repeatability, and intra-laboratory reproducibility of the SPE-based acrylamide assay

Figure 4. Calibration curve of acrylamide in water.

CONCLUSIONS AND BENEFITS · The GX-274 ASPEC is ideal for concentration of samples for improved sensitivity. · Accommodates large volumes – up to liters of sample can be processed. · Four probe Z-Arm: Process four SPE cartridges simultaneously. ACKNOWLEDGEMENTS The authors thank Nova Analítica for the technical support provided in the development of this study.

Automated Extraction of Acrylamide from Underground Water Prior to HPLC-DAD Analysis

Sponsor Report REFERENCES 1. World Health Organization. Acrylamide in Drinking-water – Background document for development of WHO Guidelines for Drinking-water Quality, 2003. 2. Kontominas, M. G.; Paleologos, E. K. Determination of acrylamide and methacrylamide by normal phase high performance liquid chromatography and UV detection.J Chromat. A, 2005, 1077, pp 128135. 3. Instituto Nacional de Metrologia INMETRO. DOQ-CGCRE-008 Revisão 4. Orientação sobre validação de métodos analíticos, 2011. ORDERING INFORMATION

Description

Part Number

Quantity

2614010

VALVEMATE II - GSIOC

331052AB

Valve, Prep, Multi Pos, 10 Port, PPS .06

49400006

Rack 338, Aluminum, 64 vials, 12 x 32 mm (2 mL)

260440106

Rack 345, Aluminum, 44 tubes, 16 x 150 mm

260440041

Extraction Rack 386, Aluminum,16 (6 mL) SPE Cartridges and 15 Tubes, Collection block holds 15 x 85 mm (10 mL) Tubes

260440109

TRILUTION LH Software, LIFETIME

21063023

Probe, 221 x 1.5 x 1.1 mm CON BEV .45 ID Tip

27067374

Gilson ASPEC 274 with two VERITY 4260 Dual Syringe Pumps

Trademarks All product and company names are trademarks™ or registered® trademarks of their respective holders. Use of the trademark(s) in this document does not imply any aﬃliation with or endorsements by the trademark holder(s). Notice This report has been produced and edited using information that was available at the time of publication. This report is subject to revision without prior notice.

This sponsor report is the responsibility of Gilson, Inc.

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Notices of Books Hydrogeochemistry Fundamentals and Advances, Volume 3 Viatcheslav V. Tikhomirov, Author February 2018, Publisher: John Wiley & Sons st

This is a three-volume set: the 1 volume lays the foundation of the composition, chemistry, and testing of groundwater; the 2nd volume covers practical applications such as mass transfer and transport; the 3rd volume focuses more deeply on the analysis of groundwater, the practical applications of these analyses, and its implications for the future. Read more…

Theory and Practice of Water and Wastewater Treatment, 2nd Edition Ronald L. Droste, Ronald L. Gehr, Authors August 2018, Publisher: John Wiley & Sons Completely updated and expanded, this is the most current and comprehensive textbook available for the areas of water and wastewater treatment, covering the broad spectrum of technologies used in practice today—ranging from commonly used standards to the latest state of the art innovations. Fully updates chapters on analysis and constituents in water. Read more…

Handbook of Smart Materials in Analytical Chemistry, 2 Volume Set Miguel de la Guardia and Francesc A. Esteve-Turrillas, Editors November, 2018, Publisher: John Wiley & Sons A comprehensive guide to smart materials and how they are used in sample preparation, analytical processes, and applications. Volume 1 covers New Materials for Sample Preparation and Analysis. Volume 2 handles Analytical Processes and Applications. Features applications in key areas including water, air, environment, pharma, food, forensic, and clinical. Read more…

Quality Assurance and Quality Control in the Analytical Chemical Laboratory: A Practical Approach, Second Edition Piotr Konieczka, Jacek Namiesnik, Authors April, 2018, Publisher: CRC Press The second edition defines the tools used in QA/QC, especially the application of statistical tools during analytical data treatment. Clearly written and logically organized, it takes a generic approach applicable to any field of analysis. New chapters cover internal quality control and equivalence method, changes in the regulatory environment are reflected throughout, and many new examples have been added to the second edition. Read more… 101

Periodicals & Websites American Laboratory The American Laboratory ® publication is a platform that provides comprehensive technology coverage for laboratory professionals at all stages of their careers. Unlike single-channel publications, American Laboratory® is a multidisciplinary resource that engages scientists through print, digital, mobile, multimedia, and social channels to provide practical information and solutions for cutting-edge results. Addressing basic research, clinical diagnostics, drug discovery, environmental, food and beverage, forensics, and other markets, American Laboratory combines in-depth articles, news, and video to deliver the latest advances in their fields. Read more… LCGC Chromatographyonline.com is the premier global resource for unbiased, peerreviewed technical information on the field of chromatography and the separation sciences. Combining all of the resources from the regional editions (LCGC North America, LCGC Europe, and LCGC Asia-Pacific) of award winning magazines, Chromatographyonline delivers practical, nuts-and-bolts information to help scientists and lab managers become more proficient in the use of chromatographic techniques and instrumentation, thereby making laboratories more productive and businesses around the world more successful. Read more… Scientia Chromatographica Scientia Chromatographica is the first and to date the only Latin American scientific journal dedicated exclusively to Chromatographic and Related Techniques (Mass Spectrometry, Sample Preparation, Electrophoresis, etc.). With a highly qualified and internationally recognized Editorial Board, it covers all chromatography topics (HPLC, GC, SFC) in all their formats, in addition to discussing many related topics such as "The Pillars of Chromatography", Quality Management, Troubleshooting, Hyphenation (GC-MS, LC-MS, SPE-LC-MS/MS) and others. It also provides columns containing general information for the area, such as: calendar, meeting report, bookstore, etc. Read more… Select Science SelectScience® promotes scientists and their work, accelerating the communication of successful science. SelectScience® informs scientists about the best products and applications through online peer-to-peer information and product reviews. Scientists can make better decisions using independent, expert ® information and gain easy access to manufacturers. SelectScience informs the global community through Editorial Features, Event Coverage, Video and Webinar programs. Read more… Spectroscopy Spectroscopy's mission is to enhance productivity, efficiency, and the overall value of spectroscopic instruments and methods as a practical analytical technology across a variety of fields. Scientists, technicians, and laboratory managers gain proficiency and competitive advantage for the real-world issues they face through unbiased, peer-reviewed technical articles, trusted troubleshooting advice, and best-practice application solutions. Read more… 102

Events 2018 July 24 - 27 XII Workshop on Sample Preparation (XII WPA) Institute of Chemistry, University of São Paulo, São Paulo, SP, Brazil www3.iq.usp.br/ September 10 - 12 3rd International Plant Proteomics Organization World Congress (3rd INPPO) Padova, Italy http://inppo2018.dafnae.unipd.it September 12 - 14 th 10 European Conference on Pesticides and Related Organic Micropollutants in the Environment & 16th Symposium on Chemistry and Fate of Modern Pesticides Joined to 10th MGPR International Symposium of Pesticides in Food and the Environment in Mediterranean Countries Bologna, Italy www.iaeac.com/ September 16 - 19 19th Brazilian Meeting on Analytical Chemistry (19th ENQA) 7th Ibero-American Congress of Analytical Chemistry (7th CIAQA) Complexo Acqua DiRoma, Caldas Novas, GO, Brazil http://enqa2018.com.br/ September 19 - 20 19th World Congress on Analytical & Bioanalytical Techniques Singapore http://analytika.pharmaceuticalconferences.com November 4 - 8 6th Brazilian Meeting on Forensic Chemistry (6th ENQFor) & 3rd Meeting of the Brazilian Society of Forensic Sciences (SBCF) Convention Center of Ribeirão Preto, SP, Brazil www.sbcf.org.br November 12 - 15 XIII Latin American Symposium on Environmental analytical Chemistry (XIII LASEAC) Hotel La Bahía Enjoy - La Serena, Chile www.eventotal.cl/ December 8 - 12 7th Brazilian Conference on Mass Spectrometry (7th BrMASS) Windsor Barra Hotel, Rio de Janeiro, RJ, Brazil http://www.brmass.com 103

Author's Guidelines The Brazilian Journal of Analytical Chemistry (BrJAC) is a peer-reviewed scientific journal intended for professionals and institutions acting mainly in all branches of Analytical Chemistry. BrJAC is an open access journal which does not charge authors an article processing fee. Scope BrJAC is dedicated to professionals involved in science, technology and innovation projects in the area of analytical chemistry at universities, research centers and in industry. About this journal BrJAC publishes original, unpublished scientific articles and technical notes that are peer reviewed in the double-blind way. In addition, it publishes reviews, interviews, points of view, letters, sponsor reports, and features related to analytical chemistry. BrJAC's review process begins with an initial screening of the manuscripts by the editor-in-chief, who evaluates the adequacy of the study to the journal scope. Manuscripts accepted in this screening are then forwarded to at least two referees indicated by the editors. As evaluation criteria, the referees will employ originality, scientific quality and contribution to knowledge in the field of Analytical Chemistry, the theoretical foundation and bibliography, the presentation of relevant and consistent results, compliance to the journal's guidelines, and the clarity of writing and presentation. Brief description of the BrJAC sections · Articles: Full descriptions of an original research finding in Analytical Chemistry. Manuscripts submitted for publication as articles, either from universities, research centers, industry or any other public or private institution, cannot have been previously published or be currently submitted for publication in another journal. Articles undergo double-blind full peer review. · Reviews: Articles on well-established subjects, including a critical analysis of the bibliographic references and conclusions. Manuscripts submitted for publication as reviews must be original and unpublished, and undergo double-blind full peer review. · Technical Notes: Concise descriptions of a development in analytical method, new technique, procedure or equipment falling within the scope of BrJAC. Technical notes also undergo double-blind full peer review. The title of the manuscript submitted for technical note must be preceded by the words "Technical note". · Sponsor Reports: Concise descriptions of technical studies not submitted for review by referees. Sponsor responsibility documents. · Letters: Discussions, comments, suggestions on issues related to Analytical Chemistry, and consultations to authors. Letters are welcome and will be published at the discretion of the editor-in-chief. · Points of view: The expression of a personal opinion on some relevant subject in Analytical Chemistry. · Interviews: Renowned chemist researchers are invited to talk with BrJAC about their expertise and experience in Analytical Chemistry. · Releases: Articles providing new and relevant information for the community involved in analytical chemistry, and companies' announcements on the launch of new products of interest in analytical chemistry. · Features: A feature article gives to the reader a more in-depth view of a topic, a person or opinion of acknowledged interest for Analytical Chemistry. Manuscript preparation (download a template on the BrJAC website) The manuscript submitted to BrJAC must be written in English and should be as clear and succinct as possible. It must include a title, an abstract, a graphical abstract, keywords, and the following sections: Introduction, Methods, Results and Discussion, Conclusion, and References. Because the manuscripts are subjected to double-blind review, they must NOT contain the authors' names, affiliations, or acknowledgments. The manuscript must be typed in Arial font size 11 pt., and the lines numbered consecutively and double-spaced throughout the text, except in the figure captions, titles of tables and references. 104

Author's Guidelines The manuscript title should be short, clear and succinct, and a subtitle may be used, if needed. The abstract should include the objective of the study, essential information about the methods, the main results and conclusions. Then, three to five keywords must be indicated. The section titles should be typed in bold and subsections in italics. Graphics and tables must be numbered according to their citation in the text, and should appear close to the discussion about them. For figures use Arabic numbers, and for tables use Roman numbers. The captions for the figures must appear below the graphic; for the tables, above. The same result should not be presented by more than one illustration. For figures, graphs, diagrams, tables, etc. identical to others previously published in the literature, the author must ask for permission for publication from the company or scientific society holding the copyrights, and send this permission to the BrJAC editor-in-chief with the final version of the manuscript. The chemical nomenclature should conform to the rules of the International Union of Pure and Applied Chemistry (IUPAC) and Chemical Abstracts Service. It is recommended that, whenever possible, authors follow the International System of Units, the International Vocabulary of Metrology (VIM) and the NIST General Table of Units of Measurement. Abbreviations are not recommended except those recognized by the International Bureau of Weights and Measures or those recorded and established in scientific publications. If the abbreviations are numerous and relevant, place their definitions in a separate section (Glossary). The manuscript must include only the consulted references, numbered according to their citation in the text, with numbers in square brackets. It is not recommended to mention several references with identical statements - select the author who demonstrated them. It is recommended that references older than 5 (five) years be avoided, except in relevant cases. Include references that are accessible to readers. References should be thoroughly checked for errors before submission. Manuscripts must be submitted in conjunction with an analysis report of plagiarism obtained through anti-plagiarism software. BrJAC indicates CopySpider© 2013 freeware to support plagiarism checking analyzes. Download the CopySpider freeware: www.copyspider.com.br

Examples of reference formatting Journals 1. Arthur, K. L.; Turner, M. A.; Brailsford, A. D.; Kicman, A. T.; David A. Cowan, D. A.; Reynolds, J. C.; Creaser, C. S. Anal. Chem. 2017, 89, pp 7431- 7437 (DOI: 10.1021/acs.analchem.7b000940). The titles of journals must be abbreviated as defined by the Chemical Abstracts Service Source Index (http://cassi.cas.org/search.jsp). If a paper does not have a full reference, please provide its DOI, if available, or its Chemical Abstracts reference information. Electronic journals 2. Natarajan, S.; Kempegowda, B. K. LCGC North America, 2015, 33 (9), pp 718-726. Available from: http://www.chromatographyonline.com/analyzing-trace-levels-carbontetrachloride-drugsubstanceheadspace-gc-flame-ionization-detection [Accessed 10 November 2015]. Books 3. Burgot, J.-L. Ionic Equilibria in Analytical Chemistry. Springer Science & Business Media, New York, 2012, Chapter 11, p 181. 4. Griffiths, W. J.; Ogundare, M.; Meljon, A.; Wang, Y. Mass Spectrometry for Steroid Analysis. In: Mike, S.L. (Ed.). Mass Spectrometry Handbook, v. 7 of Wiley Series on Pharmaceutical Science and Biotechnology: Practices, Applications and Methods. John Wiley & Sons, Hoboken, N.J., 2012, pp 297-338. Standard methods 5. International Organization for Standardization. ISO 26603. Plastics — Aromatic isocyanates for use in the production of polyurethanes — Determination of total chlorine. Geneva, CH: ISO, 2017.

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Author's Guidelines Master’s and doctoral theses or other academic literature 1. 6. Ek, P. New methods for sensitive analysis with nanoelectrospray ionization mass spectrometry. Doctoral thesis, 2010, School of Chemical Science and Engineering, Royal Institute of Technology, Stockholm, Sweden. Patents 2. 7. Trygve, R.; Perelman, G. US 9053915 B2, June 9 2015, Agilent Technologies Inc., Santa Clara, CA, US. Web pages 3. 8. http://www.chromedia.org/chromedia [Accessed 21 June 2015]. Unpublished source 9. Mendes, B.; Silva, P.; Pereira, J.; Silva, L. C.; Câmara, J. S. Poster presented at: 36th International 4. Symposium on Capillary Chromatography, 2012, Riva del Garda, Trento, IT. 10. Author, A. A. J. Braz. Chem. Soc., in press. 5. 11. Author, B. B., 2015, submitted for publication. 6. 7. 12. Author, C. C., 2011, unpublished manuscript. Note: Unpublished results may be mentioned only with express authorization of the author(s). Personal communications can be accepted exceptionally. Manuscript submission Three different PDF files, as described below, must be sent online through the website www.brjac.com.br I. A cover letter addressed to the editor-in-chief with the full manuscript title, the full names of the authors and their affiliations, the complete contact information of the corresponding author, including the ORCID iD, and the manuscript abstract. This letter must present why the manuscript is appropriate for publication in BrJAC, and contain a statement that the article has not been previously published and is not under consideration for publication elsewhere. The corresponding author must declare on behalf of all the authors of the manuscript any financial conflicts of interest or lack thereof. This statement should include all potential sources of bias such as affiliations, funding sources and financial or management relationships which may constitute a conflict of interest. When the manuscript belongs to more than one author, the corresponding author must also declare that all authors agree with publication in BrJAC. II.The manuscript file that must NOT mention the names of the authors or the place where the work was performed, but must include the title, abstract, keywords, and all sections of the work, including tables and figures, but excluding acknowledgments that will be included in the final paper upon completion of the review process. III. An analysis report of plagiarism on the manuscript. A Sponsor Report should be sent as a Word file attached to a message to the email brjac@brjac.com.br Revised manuscript submission Based on the comments and suggestions of the reviewers and editors a revision of the manuscript may be requested to the authors. The revised manuscript submitted by the authors must contain the changes made in the manuscript clearly highlighted. A letter without any author's information must also be sent with each reviewer's comment items and a response to each item. Copyright When submitting their manuscript for publication, the authors agree that the copyright will become the property of the Brazilian Journal of Analytical Chemistry, if and when accepted for publication. The copyright comprises exclusive rights of reproduction and distribution of the articles, including reprints, photographic reproductions, microfilms or any other reproductions similar in nature, including translations. Final Considerations

Whatever the nature of the submitted manuscript, it must be original in terms of methodology, information, interpretation or criticism. As to the contents of published articles, the sole responsibility belongs to the authors, and Br. J. Anal. Chem. and its editors, editorial board, employees and collaborators are fully 106

Author's Guidelines

exempt from any responsibility for the data, opinions or unfounded statements BrJAC reserves the right to make, whenever necessary, small alterations to the manuscripts in order to adapt them to the journal rules or make them clearer in style, while respecting the original contents. The article will be sent to the authors for approval prior to publication.

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19 National Meeting of Analytical Chemistry

19th ENQA th 7 CIAQA

7 Ibero-American Congress of Analytical Chemistry September 16-19, 2018 Venue: Acqua DiRoma Complex Caldas Novas, GO, Brazil www.enqa2018.com.br

April - June 2018

Volume 5

Number 19