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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 the diffusion of significant and original knowledge in all branches of Analytical Chemistry. BrJAC is addressed to professionals involved in science, technology and innovation projects in 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 BrJAC publishes original, unpublished scientific articles and technical notes that are peer reviewed in the double-blind way.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 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. BrJAC is Published by: VisĂŁo 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

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Editorial Board Editor-in-Chief

Marco Aurélio Zezzi Arruda 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. at Petrobras Transporte S.A. and Aggregate Professor at the 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 Mauro Bertotti Full Professor / Institute of Chemistry, University of São Paulo, São Paulo, 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, 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 Director of Engineering and Environmental Quality of CETESB, São Paulo, SP, BR Gisela de Aragão Umbuzeiro Professor / Technology School, 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), Inst. of Chemistry, Araraquara, SP, BR José Dos Santos Malta Junior Pre-formulation Lab. Manager / EMS / NC Group, Hortolandia, SP, BR Lauro Tatsuo Kubota Full Professor / Institute of Chemistry, University of Campinas, Campinas, SP, BR Luiz Rogerio M. Silva Quality Assurance Associate Director / EISAI Lab., São Paulo, SP, BR Márcio das Virgens Rebouças Global Process Technology / Specialty Chemicals Manager - Braskem S.A., Campinas, SP, BR Marcos Nogueira Eberlin Full Professor / School of Engineering, Mackenzie Presbyterian University, São Paulo, SP, BR Maria das Graças Andrade Korn Full Professor / Institute of Chemistry, Federal University of Bahia, Salvador, BA, BR Ricardo Erthal Santelli Full Professor / Analytical Chemistry, Federal University of Rio de Janeiro, RJ, BR

Contents

Br. J. Anal. Chem., 2019, 6 (24)

Editorial Chromatographic Challenges............................................................................................................2-3 Renato Zanella

Interview Professor Fabio Augusto, a pioneer researcher in Brazil in the development of modern analytical separation techniques, discussed with BrJAC his memories and lucid ideas about the situation of science in the country...................................................................................................................... 4-11 Point of View Trends and Perspectives on Future Developments in Liquid Chromatography............................12-13 Carla Beatriz Grespan Bottoli

Letter Is Comprehensive Two-Dimensional Gas Chromatography Here to Stay?...................................14-15 Carin von Mühlen

Articles Harvest Influence in Volatile Composition of Chocolates Produced with Hybrid Varieties of Bahia’s Cocoa was Investigated using GC×GC-QMS and Chemometrics................................................16-26 Soraia Cristina Gonzaga Neves Braga, Luciana Fontes Oliveira, Adriana Reis de Andrade Silva, Priscilla Efraim, Ronei J. Poppi, Fabio Augusto

Selective Extraction of Manganese using Moringa oleifera Seeds as Bioadsorbent....................27-37 Vanessa Nunes Alves, Sângela Nascimento do Carmo, José Alistor de Sousa Neto, Luciana Melo Coelho, Nívia Maria Melo Coelho

Evaluation of the Quality of Formulations Containing Lactase (β-galactosidase) Employing Gel Electrophoresis and Cell Phone....................................................................................................38-46 Bruna Soares Dionizio, Diego Victor Babos, Dulce Helena Ferreira de Souza, Edenir Rodrigues Pereira-Filho

Micelar-Electrokinetic Chromatography Separation of Nitrogen-Containing Aromatic Compounds in Diesel Prepared as Microemulsion............................................................................................47-62 Anastácia Sá Pinto da Silva, Alessandra Licursi Maia Cerqueira da Cunha, Selma Cunha Mello, Ricardo Queiroz Aucelio

Features The 42nd Annual Meeting of the Brazilian Chemical Society Discussed the Importance of Current Decisions for a Better Future of Chemistry in Brazil......................................................................63-68 COLACRO 2019 Discussed the Importance of Chromatography in the Development of Latin America.. 69-73 Sponsor Reports Flow Modulated Comprehensive Two-Dimensional Gas Chromatography. Part I - Low Duty Cycle Modulation of Hidroprocessed Vegetable Oil................................................................................74-81 Rodrigo Passini, Danilo Pierone, Angelo L. Gobbi, Leandro W. Hantao

Determination of Organic Acids in Fruit Juices and Wines by High-Pressure IC..........................82-93 Thermo Scientific

Contents

Sponsor Reports Overcome the Memory Effect in Dioxins Extraction with Ethos X Powered by fastEX-24............94-97 Milestone

Releases Thermo Scientific TRACE 1300 Series GC — Productivity Solution for Your Needs......................... 98 Thermo Scientific Dionex ICS-6000 HPIC System — A new highly-configurable ion chromatography system.............................................................................................................................................. 100 Advanced Microwave Extraction System for Environmental Laboratories....................................... 102 Pittcon Conference & Expo — Be Amongst the best in PITTCON 2020... The Future of Laboratory Sciences........................................................................................................................................... 104 SelectScience® Pioneers Online Communication and Promotes Scientific Success since 1998................................................................................................................................................. 106 CHROMacademy Helps Increase your Knowledge, Efficiency and Productivity in the Lab............. 108 Notices of Books

.....................................................................................................110-111

Periodicals & Websites ........................................................................................................... 112 Events

........................................................................................................... 113

Guidelines for the Authors ............................................................................................... 114-116

Editorial

Br. J. Anal. Chem., 2019, 6 (24) pp 2-3 DOI: 10.30744/brjac.2179-3425.editorial.rzanella.N24

Chromatographic Challenges

Renato Zanella Full Professor Laboratory for Pesticide Residue Analysis (LARP) Chromatography and Mass Spectrometry Research Group (CPCEM) Department of Chemistry, Federal University of Santa Maria Santa Maria, RS, Brazil Chromatography, with its varied analytical possibilities, plays a prominent role in the various analyses, especially of organic compounds, and it has applications in several application areas, such as environmental, forensics, metabolomics, clinical, and industrial. Coupling chromatography with mass spectrometry has provided broad, fast, selective, and sensitive methods. Chromatography coupled to high-resolution or tandem mass spectrometry has enabled multiresidue and multi-class analyses with great performance for different complex matrices. The use of high-resolution mass spectrometry allows target, non-target, and suspect screening analyses of a large number of compounds, which is of great importance today. Comprehensive chromatography technologies are adequate tools for complex samples, such as petrochemicals, natural products, foods, and clinical applications. Innovations in chromatography columns and the higher performance of new equipment systems in recent decades have allowed for faster analyses without sacrificing the reliability of the results. Chromatography allows for the quality control of food and beverages through the analysis of pesticide multiresidue, veterinary drugs, mycotoxins, dyes, preservatives, and contaminants contained in the packaging. This technique is also important in determining functional compounds, vitamins, and antioxidants, among other parameters, present in foods. These analyses ensure the authenticity of the products tested. In clinical analysis, chromatography has played a fundamental role in disease diagnosis and prevention, as well as in supporting clinical evaluations during and after complex surgeries. It can also be used to assess the exposure of workers to pollution by determining the pollutants found in ambient air or in biological fluids, such as blood and urine. In the environmental area, chromatography allows to evaluate the occurrence of persistent organic pollutants, as well as emerging pollutants in different matrices, such as in water, effluents, air, soil, and sediments. The analysis of contaminants in wild animals is of great importance and can be used to evaluate the exposure of animals to contaminants and to establish measures to mitigate contamination problems. Multi-residue pesticide determinations are required today, but analyses of endocrine disruptors, pharmaceuticals, personal care products (PPCPs), and flame retardants have been increasingly frequent. The determination of a wide range of organic compounds of interest has required a great effort from the instrumentation companies in the development of sensitive and selective equipment in order to achieve the required levels. Despite this effort, the training of new analysts prepared to develop new analytical methods is a very important challenge. Continuous investment in the qualification of laboratories in terms of infrastructure and training of analysts is very important, including collaboration of the instrumentation companies and the laboratories that require these analysts. Cooperation between research groups can provide each with access to new technologies for more complex applications.

Editorial

The establishment and application of faster and more efficient analytical methods for a large number of compounds makes the implementation of a quality system, such as ISO 17025, even more important in enabling more efficient control of working conditions and traceability of results. The qualification of personnel with experience in sample preparation and analysis techniques, as well as in quality system, should be encouraged since the demand for professionals with these skills is increasing due to the need for reliable results in the most diverse sectors. In chromatography, speed and accuracy are always being pushed to the limits by the analytical demands of real samples.

Interview

Br. J. Anal. Chem., 2019, 6 (24) pp 4-11 DOI: 10.30744/brjac.2179-3425.interview.faugusto

Professor Fabio Augusto, a pioneer researcher in Brazil in the development of modern analytical separation techniques, discussed with BrJAC his memories and lucid ideas about the situation of science in the country

Fabio Augusto

Full Professor Department of Analytical Chemistry, Institute of Chemistry University of Campinas, Campinas, SP, Brazil Prof. Dr. Fabio Augusto graduated from the Institute of Chemistry at the University of Campinas (IQ-Unicamp) in 1986 with a degree in chemistry. Before that, in 1982, he was a trainee at Rhodia S.A. Chemical Unit in Santo André, SP, Brazil, where he took the first steps in the area of chromatography. In 1990, he became a Master in Chemistry, and in 1996 a Doctor in Sciences, both from IQ-Unicamp. In 1996, Fabio Augusto, along with Antonio Luiz Pires Valente, professor at IQ-Unicamp, founded the “Laboratório de Cromatografia Gasosa-LCG,” which was the pioneer chromatography laboratory in important fields of analytical chemistry in Latin America, developing methods and technologies for solid-phase microextraction (SPME) and comprehensive two-dimensional gas chromatography (GCxGC). In 2005, Fabio Augusto presented his free-docency thesis with the monograph entitled “Solid Phase Microextraction SPME” at IQ-Unicamp. It was Fabio Augusto’s experience at Rhodia as a trainee that gave him direction for his professional career. At Rhodia, he worked with chromatographs made in Brazil by “CG Instrumentos Científicos”, a company founded by Remolo Ciola, a professor at the Institute of ChemistryUniversity of Sao Paulo, SP, Brazil, who died in 2010. After 37 years of work in chromatography, Fabio Augusto received the “Ciola Medal” in recognition of his dedication to the development and dissemination of chromatography in Brazil and for his excellent contributions to science. The Ciola Medal was awarded to Fabio Augusto at the XVII Latin American Symposium on Chromatography and Related Techniques (COLACRO) in 2019. Fabio Augusto has previously received the following awards and recognitions: • “Genzo Shimadzu” in 2018 for the best poster at the 42nd International Symposium on Capillary Chromatography/15th GCxGC Symposium; • Dr. Janusz Pawliszyn Medal 2016 for his contribution to the sample preparation area in Latin America, awarded at the Workshop on Recent Advances on Sample Preparation (WARPA);

Interview

• Unicamp Inventors Award 2013 for the granted patent “Device for Solid Phase Micro-Extraction combined with dynamic headspace analysis (DHS-SPME)”, granted by the Innovation Agency Inova Unicamp; • Top Ten Chromatography, Mass Spectrometry and Lab Automation Papers Analytical Chemistry 2014, Chemical & Engineering News American Chemical Society; • Capes Thesis Award 2009 (area of Food Science) as coadvisor to Cláudia H. Kowalski doctoral thesis (advisor: Profa. Dra. Helena T. Godoy), award granted by the Brazilian Coordination for the Improvement of Higher Education Personnel (CAPES). From 1999 to 2000, Fabio Augusto was a postdoc at the University of Waterloo (Canada) with the research group of Prof. Dr. Janusz Pawliszyn. In 2015, he held the Joliot Chair for invited foreign researchers from ESPCI (Ecole Supérieure de Physique et de Chimie Industrielles, Paris, Fabio Augusto and Cláudia Kowalski at the France). 14th International Symposium on Advances in Fabio Augusto is currently full professor of the Department Extraction Technologies (ExTech) in Alesund, of Analytical Chemistry at IQ-Unicamp. To date, he has Norway (2007). mentored over 60 postgraduate students, participated in over 40 thesis examining boards, and published over 200 papers. He works in the area of analytical separations, and his research is based on the development of comprehensive two-dimensional gas chromatography (GCxGC) instrumentation and methodologies combining this tool with extraction microtechniques and chemometrics for bioanalytical chemistry, and metabolomic, petroleomic, forensic, and food & beverage analysis applications. What early influences encouraged you to study science? Did you have any influencers, such as a teacher? In fact, I don’t remember any time in my life when I wanted to be something other than a “scientist”. I’m from the first generation who saw man landing on the Moon, and I think that this left an impression on many kids and teens from the late 60s through the mid-70s. To be a scientist was to participate in the future that we saw in movies and television series. It was a very different mood from what we have today, in which science is purposely discredited and undervalued by some groups and sectors of society. So, for me and for many people who are researchers or who work in the industrial sector today, the great incentive to become a scientist was this: being part of something that, in our minds, would be a great adventure and change the world. When did you decide to go into the field of chemistry? What motivated you? How was the beginning of your career in chemistry? Like many of my colleagues, my first contact with chemistry was through children’s games, such as “The Little Chemist”, which were very popular at that time. They were not cheap, and my family wasn’t exactly wealthy (quite the opposite), but during my childhood I had a couple of these games. To the dismay of my parents, I started to say to everyone that I wanted to study chemistry. This seemed odd because being a chemist was not considered a “successful profession”. But, eventually they accepted, and when I finished eighth grade in elementary school at the neighborhood school in São Bernardo do Campo, SP, I got a scholarship to attend a technical course in Industrial Chemistry from the former “Instituto de Ensino de São Caetano do Sul”. This course was deficient in disciplines such as Portuguese and mathematics; however, we had some teachers with a good chemistry background and industry 5

Interview

experience. In mid-1981, the Rhodia Chemical Unit in Santo André opened an internship position for a technical trainee, lasting until December 1982. More than 60 people applied for this position, and after a selection process that lasted almost a month, I was selected. I started the internship in August 1981 at the Central Laboratory Service. This laboratory centralized all quality control of raw plant materials and finished products and was in charge of monitoring boiler water quality and many other functions. The manager of the Central Laboratory Service was Dr. Claudio Puschel, who had a solid background in chemistry and was one of the best laboratory administrators I had ever met. He led me to work in the quality control of all available production lines, as well as assisting the senior laboratory chemist (Dejair Soria) in solving problems related to batches returned by customers for errors in analysis or developing methodologies for analysis of new products. That’s when I came into contact with something very close to what would be Fabio Augusto at Rhodia S.A. Gas Chromatography Lab analytical chemistry research, and I was sure that I in Santo André, SP, working on a chromatograph from had chosen the right career for myself. At the end of “CG Instrumentos Científicos” (1982). the internship, I was transferred to the Gas Chromatography Sector. The senior technician, João Luiz de Souza Carvalho, was a great teacher and encourager. This chemical unit of Rhodia had a very good instrument park at the time, and it was there that I started learning chromatography, working on the now legendary chromatographs of “CG Instrumentos Científicos.” In early 1982, I decided to take the university entrance exam only as an experience since there were still 6 months left for me to complete the technical course and one year left to finish my internship at Rhodia. I decided to take the entrance exam at Unicamp, in Campinas, SP, so that I would have a greater chance to pass to the second phase of the exam. To my astonishment, I was ranked sixth among the participants. This created a problem for my parents because they could not afford to support me outside the family home and without working. I decided to take time off from the university in 1982 to finish my internship and to save money. I started the chemistry course at Unicamp in 1983 and relied on financial help from family members. At the end of the first year of the chemistry course at Unicamp, I sought out Prof. Antonio Luiz Pires Valente from the Analytical Chemistry Department at IQ-Unicamp. He was still doing his doctorate under Prof. Carol Collin’s guidance, and he was a specialist in gas chromatography. Prof. Pires guided me through almost every undergraduate degree as a student of the Scientific Initiation program. He was a great connoisseur of analytical instrumentation and greatly appreciated teaching. When I graduated, I entered the master’s degree and then the doctorate, always under the guidance of Prof. Pires. In 1992, I was hired at IQ-Unicamp. What has changed in the student profile, ambitions, and performance since the beginning of your career? There have been so many changes that it doesn’t even seem like another generation; it’s another world. The generation that is entering universities today is the first generation that has lived their entire lives with the ease of access to information provided by the Internet and smartphones. This profoundly influences their form of reasoning and world perspective. Our teaching uses models that worked for us and those who came before us, but they are no longer good for today’s students. So, their performance is often poor, and I don’t think it is entirely due to deficiency in their education (although this certainly plays a significant role). We need to change the way we teach science: I do not have the recipe for what 6

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must be done, but I am fully convinced that the current form of teaching has no future. The profile of the newly graduated professionals is very different as well. Twenty or thirty years ago, it was very common for the highest achieving chemistry graduates to be strongly interested in working in the field of analytical chemistry, either in academic research or in private laboratories. Today, topics such as material science, energy, and biochemistry are much more attractive to recent graduates, both for job market opportunities and for being areas universally considered state-of-the-art in science. Could you briefly comment on recent developments in analytical chemistry, considering your contributions? As I mentioned above, the most active areas in cutting-edge developments are material science, new energy matrices, and biological chemistry. I believe this has changed the focus of modern analytical chemistry. For example, metabolomics, proteomics, and related areas demand analytical methods that are especially fast, automated and highly reliable, as well as able to handle the typical chemical species in samples from these fields, which include analytes traditionally regarded as problematic (large and highly polar molecules, labile substances, etc.). Thus, the various modalities of mass spectrometry, as well as their hyphenations with chromatographic systems and other techniques that allow ultrafast and minimal sample preparation, have been the target of many interesting developments. The availability of new materials has also been widely exploited by analytical chemists, such as in the increasing use of nanomaterials in sensors, electrodes, and as sorbent materials in various extraction formats. Another field that has developed quite sharply is that of portable and simplified instrumentation (often based on microfabricated components), notably for field analysis. In addition, many of these new analytical techniques and approaches result in large volumes of data that must be properly interpreted and manipulated. Increasingly, analytical chemistry is an information science, so the importance of using chemometric tools has become more and more pronounced. Many of these chemometric tools have become routine for modern analytical chemists. For some time, my point of view has been that the future of analytical chemistry research lies in the interfacing of these areas. Therefore, over the past few years, I have been working hard on combining multidimensional chromatography, fast extraction techniques such as SPME, and chemometric tools for multivariate analysis applied to highly complex samples. We have had very interesting results, with a reasonable impact on the literature and in the development of this area. What are your lines of research? You have published many scientific papers. Would you highlight any? Essentially, we operate in two major areas: development of systems and methodologies in comprehensive two-dimensional gas chromatography (GC×GC), for applications in petroleomics, food analysis, and plant and microbiological metabolomics, and microextraction techniques for chromatographic analysis. To date, we have published close to 120 articles in indexed journals. Although, in recent years, we have devoted ourselves much more to GCxGC. From the papers we have published, I find two of them more significant. As soon as I returned from my postdoctoral degree in Professor Janusz Pawliszyn’s group at the University of Waterloo, Canada, I got financial resources and started my work with solid phase microextraction (SPME), which, at the time, was a relatively new technique, with many aspects to Poster presentation at the Congress of the be explored. One interesting application of SPME is its use in the Latin American Geochemistry Association, analysis of volatile food and beverage constituents associated Búzios, RJ. From left: Noroska Mogollon, with the aroma and quality of these products. At the time, I did Fabio Augusto, and Paloma Prata (2015). 7

Interview

not have my own GC-MS system, but I got Agilent to temporarily provide us with a GC-MS system for evaluation. We had this system in the lab for about a month. With the participation of a scientific initiation student and a master student, Eduardo Tada and Sandra Rivellino, respectively, we screened the composition of the volatile pulp fraction from a series of native fruits from Brazil. The chromatograph was used almost non-stop during the month while working on these analyses. Afterwards, we spent a few months processing the files acquired in the analyses, systematically identifying the detected compounds using public domain software since we did not have our own software. The results of this work were published in the Journal of Chromatography (J. Chromatogr. A, 2000, v. 873, p 117), and this was one of the first descriptions of a systematic application of SPME in the study of flavorings. For this reason, it has been (and still is) cited often in the literature. Another paper that I consider equally significant was from our collaboration with my colleague, Prof. Ronei Poppi, who is one of the leading chemometrics specialists in Brazil. We developed a strategy to detect any kind of adulteration in gasoline using GCxGC and multivariate chromatogram processing. Our approach was to determine the purity of the samples by quantifying the base gasoline content. In adulterated samples, regardless of the nature of the adulterant, this content will be lower than that specified in the legislation. At the time, GCxGC was still a seldom explored tool, and this work (J. Chromatogr. A, 2008, v. 1201, p 176) was one of the first to demonstrate the enormous potential of combining this technique with multivariate data analysis strategies. The GCxGC system we employed was fully designed and built in our laboratory as part of the doctoral theses of Márcio Pozzobon Pedroso (now professor at the Federal University of Lavras, Lavras, MG) and Ernesto Correa Ferreira (who is a professor at the Federal Institute of Espírito Santo, Vitória, ES). Do you keep yourself informed about the progress of research in chemistry? What is your opinion about the current progress of chemistry research in Brazil? What are the recent advances and challenges in scientific research in Brazil? Overall, I believe there are some centers in Brazil where cutting-edge chemistry takes place, comparable with good research centers abroad. Approximately 10 chemistry postgraduate programs are rated 7 by CAPES, which means they are at an international level. Overall, research in Brazil, at least in universities and public institutions, has made a huge qualitative and quantitative leap in recent years. However, the current economic and financial situation of the country is calamitous, and, therefore, we are living something new: the National Council for Scientific and Technological Development (CNPq) will stop paying scholarships starting next September due to the exhaustion of its budget (unless new events occurs). This has been happening for some time with research project funding. Unfortunately, I have the feeling that this situation will continue for a long time, both because of the economic environment and also due to the unscientific environment that has established itself in important sectors of Brazilian society. The biggest scientific challenge in Brazil today is to survive this storm. For you, what have been the most important recent achievements in the analytical chemistry research? What are the landmarks? I believe that in the last 10 years, three important events have occurred in analytical chemistry: the “revival” of Raman spectroscopy as a routine analytical tool; the development of handheld, pocket-based analytical platforms recently implemented on smartphones; and the popularization of the combination between liquid chromatography and mass spectrometry to unveil the chemistry of biological systems. The first two were the result of the collaboration of many researchers, and I don’t think it is possible to point out just one of them as being the most responsible. However, in the latter case, I think the big milestone was the 2002 Nobel Prize award to the inventor of the electrospray interface, John Fenn. There are in Brazil and in the world several meetings on chemistry. To you, how important are these meetings to the scientific community? How do you see the development of national 8

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chemistry meetings in Brazil? They are essential for science anywhere in the world, and especially in Brazil. Even today, with instant access to journals and the proliferation of alternative channels for scientific dissemination, personal interaction is indispensable for the emergence of new ideas and the discussion of trends and advances. In Brazil, this is the way researchers from smaller universities and research institutes can interact with scientists from the most advanced and up-to-date international centers. In addition, as the number of researchers increases, the importance of regionalized or specific meetings grows. The National Analytical Chemistry Meetings (ENQA) are increasingly large events, attended by almost the entire analytical chemistry community in Brazil. Other meetings specializing in specific topics, such as COLACRO, have also grown in importance since they allow for direct contact of Brazilian students and researchers with colleagues from abroad; in these events, there is increasing participation of invited foreign researchers.

15th International Symposium on Advances in Extraction Technologies, ExTech 2013 in João Pessoa, PB, Brazil. Former members of Prof. Janusz Pawliszyn’s group at the University of Waterloo, Canada. From left to right: Eduardo Carasek (Federal University of Santa Catarina, Brazil); Érica Silva (Federal University of São Paulo, Brazil); Elena Stashenko (Industrial University of Santander, Colombia); Janusz Pawliszyn; Zoltan Mester (National Research Council, Canada); Cláudia Zini (Federal University of Rio Grande do Sul, Brazil); Fabio Augusto; Maria Eugênia Queiroz (University of Sao Paulo, Brazil); and Tadeusz Goreki (University of Waterloo, Canada).

You have already received some awards. What is it like to receive this kind of recognition? How did you feel about winning the “Ciola Medal”? For me, being chosen to receive the Ciola Medal was an honor and a pleasant surprise. I have been working with chromatography for 38 years, as a researcher, teacher, and technician, and this award represented to me the knowledge I was able to contribute to this area in Brazil. Obviously, nobody achieves anything on their own, and I think much of the merit comes from my students, both those I mentored in the masters and doctorate programs and those who attended undergraduate and postgraduate subjects I taught. They gave me the opportunity to grow professionally. I am absolutely sure that a good teacher learns more from his students than he teaches them.

Interview

Luiz Bravo, director of the company Nova AnalĂtica (left), handing the Ciola Medal to Fabio Augusto (right) at the opening ceremony of the XVII COLACRO in Aracaju, SE, Brazil (2019).

What is the importance of these awards in the development of science and new technologies? I believe awards such as the Ciola Medal, regardless of its role in recognizing the importance of the recipient to the area, are also important to the new generation of scientists. They indicate researchers and scientists who have achieved successful careers, contributed to science, and are examples to follow. For you, what is the importance of the funding support for the scientific development of Brazil? Quality scientific research in Brazil is mainly done at public universities and federal and state research institutes, such as the National Institute of Pure and Applied Mathematics (IMPA), the National Institute of Amazonian Research (INPA), the National Institute of Space Research (INPE), Campinas Agronomic Institute (IAC), and the National Center for Research in Energy and Materials (CNPEM). Everywhere in the world, public funding is an essential source of research resources. However, in our country, for a variety of reasons, research funding depends almost exclusively on public funding agencies. The basis of our economy is the production of commodities, such as soybean, iron ore, and oil. Except for the oil industry, these are not sectors that create a high demand for new technologies, cutting edge knowledge, or innovative processes. Local companies in areas that might be interested in investing heavily in science and innovation are, largely, multinationals based abroad. Thus, unlike most of Europe and the United States, I do not see much potential for heavy private sector investment in research. One of the few exceptions has been the Brazilian Petroleum Corporation Petrobras (in any case, a state-owned company), which, both by necessity and legal demands, has invested heavily in research both internally and in universities. While the matrix of our industrial production and wealth generation is based on agricultural and mineral products, we will naturally depend on public investments for scientific development. At the moment, the situation for scientific research in Brazil is one of decreasing investment. How do you see this situation, and what would you say to young researchers? It seems to me that the present situation is a little worse than a simple occasional reduction in investments because of the unfavorable economic environment in the country. In recent years, in addition to experiencing a long-running financial crisis, which does not seem to be ending in the short term, (as I already mentioned, but not only in Brazil but worldwide), anti-scientificism has grown at an alarming rate. What was simply a manifestation of eccentric and localized niches is, in some cases, 10

Interview

becoming a major trend, and even a determining factor in shaping environmental, scientific, and public health policies in countries of Brazil’s size. Who would have thought 30 years ago that things like the belief that the earth is flat and intelligent design would be taken seriously by so many people? What I can say to those starting out in science today is this: As bad as the current picture may seem, don’t give up. Although this crisis is the most serious I’ve ever seen, economically, morally, and ethically, it is certainly also part of a cyclical movement that has always existed and will exist. Crises are not eternal, and when the crisis is over we will need scientists and researchers more than ever. What advice would you give to a young scientist who wants to pursue a career in Chromatography? One should always keep in mind that analytical chemistry is essentially an applied science. It exists to solve real problems. Anyone wishing to make a significant contribution to the development of chromatography must be aware of the demands of industry, academia, and society. An analytical method or a new technique or device is only justified if it addresses a real problem. A new analytical concept or device developed and tested, if it generates only a scientific paper, later forgotten in a drawer, would have been nonsense and a waste of resources and time. To know where the relevant problems lie, we should always listen and interact with professionals in fields such as medicine, agronomy, food science, and environmental science, even though, in many cases, this is a naturally difficult dialogue. How would you like to be remembered? As relevant as a scientific paper or academic research may be, it will one day become obsolete. If I am remembered in the future, I hope it would be for having formed new professionals and researchers, whether they are chromatographers or not, and for having a positive impact on their careers.

Fabio Augusto’s research group at IQ-Unicamp in 2015. From left to right: Noroska Mogollon (now professor at the Amazon Regional University - Ikiam, Ecuador); Lucília Vilela (lab. technician); Mayra Fontes Furlan (senior chemist at “Kerry Aromas” and still in the group as a doctoral student); Jadson Reis (currently a PhD student at the Federal University of Espírito Santo); Bruna Sampaio (today, at Rhodia Solvay Group, Paulínia Industrial Unit, SP); Fabio Augusto and his wife, Rachel; Paloma Prata (postdoctoral student at the Federal University of São Carlos); Paula Lima (chemical analyst at “Valer Laboratórios”); and Sandra Rivellino (technical high school teacher).

Point of View

Br. J. Anal. Chem., 2019, 6 (24) pp 12-13 DOI: 10.30744/brjac.2179-3425.pointofview.cbgbottoli.N24

Trends and Perspectives on Future Developments in Liquid Chromatography

Carla Beatriz Grespan Bottoli Associate Professor Institute of Chemistry, University of Campinas — Unicamp Campinas, Brazil Since the early 1970s, there has been a tremendous surge in the development and refinement of high performance liquid chromatography (HPLC) instrumentation as well as in HPLC applications. The molecules can be very small or their molar mass may range from thousands to hundreds of thousands. In a short time, vast amounts of highly reproducible data can be generated. The use of HPLC has not just been confined to analytical chemistry or biological sciences. HPLC is frequently used as a powerful separation technique in all branches of science or industry that require the separation of components in a complex mixture. The characterization of different molecules requires a variety of chromatographic approaches, including reversed-phase, ion exchange, and size exclusion. Reversed-phase has been responsible for almost all applications in chromatographic separations because a majority of analytes can be separated by this technique. Hydrophilic interaction liquid chromatography (HILIC) has emerged as an alternative for the separation of polar analytes and hydrophilic compounds that are problematic in other separation modes. It has become more routine because of the reliable and stable results it produces. Innovations in stationary phases have focused on the development of more stable supports, new bonded phases, and novel materials. These innovative materials must be capable of improving specific selectivity and providing faster analysis times. There is still potential for further improvements and new materials. The availability of reliable and sensitive detectors underscores the success of HPLC as a multipurpose analytical technique for miscellaneous applications. Three categories of HPLC detectors have featured in recent years: ultraviolet (UV) detectors for molecules with chromophores, evaporative light scattering detectors (ELSD) or charged aerosol detectors (CAD) for non-chromophoric compounds, and mass spectrometers (MS) for general use. There is continued growth in the use of liquid chromatography with tandem mass spectrometry (LC-MS/MS). This use is because the improvement in MS technology — both hardware and software — allows highly sensitive and selective methods to be developed for diverse applications, including clinical research, food safety, biopharmaceutical research and development, environmental protection, and “omics”-related research. Several areas are growing fast with the use of LC-MS because MS can now provide a level of sensitivity and selectivity that opens doors to replace other technologies, for example, costly immunoassays in clinical laboratories. However, this development requires very easy-to-use, robust instruments and the production of reliable, high-quality data, regardless of the user’s experience. Advances in column technology (smaller particles, superficially porous particles) and instrumentation 12

Point of View

(pressure capabilities, detector cell size) have provided the means to increase peak capacity. Hyphenating comprehensive two-dimensional liquid chromatography (LC×LC) with high-resolution MS is also an effective way to significantly increase resolving power and peak capacity in LC. In the near future, if more tools are developed to make this powerful technology simpler, LC×LC will also be used for routine analysis. Software developments to improve “feature recognition” should be investigated further. Although much effort has been made in simplifying data processing, it remains far from fully automated and consumes marked amounts of time and analytical expertise. The implementation of artificial intelligence to assist data processing, self-diagnosis, and self-maintenance — in order to reduce human intervention in daily laboratory routine — should be developed to improve workflow solutions. In the future, LC will continue to be the separation technique of choice, and nano-LC and capillary LC are the way forward for faster LC and greener technology. The demand for increased sensitivity, throughput, and robustness has made capillary LC a potential technique because of its ability to provide increased MS sensitivity compared to typical analytical flow LC-MS. An additional advantage includes lower solvent consumption and sample size. However, improvements in micro-LC instruments are required, for example, decreasing dead volumes to make them more efficient.

Letter

Br. J. Anal. Chem., 2019, 6 (24) pp 14-15 DOI: 10.30744/brjac.2179-3425.letter.cvmuhlen.N24

Is Comprehensive Two-Dimensional Gas Chromatography Here to Stay?

Carin von Mühlen Associate Professor Chemistry and Environmental Department Faculty of Technology at Rio de Janeiro State University Resende Regional Campus, Resende, RJ, Brazil Gas chromatography is the most established analytical technique to investigate volatile and semivolatile organic compounds in real life samples. Since its onset, one of the most pursued challenges of this technique has been to work with very complex samples associated with high throughput analysis. In 1991, one important analytical tool was added to this family: comprehensive two-dimensional gas chromatography (GC×GC). With this technique, in its variable configurations, it is possible to separate thousands of substances in one unique chromatographic run. The association of this technique with fast data acquisition rate detectors, such as time-of-flight mass spectrometers, and, recently, highresolution mass spectrometers, has added the analytical power needed to elegantly identify organic compounds in very complex samples. There are more than 1.4 thousand papers published in Science Direct records in 2019 alone with GC×GC, which is close to 6% of publications in gas chromatography. Twenty years ago, GC×GC was responsible for less than 2% of publications. The question that arises is why this technique is not being used everywhere. Why it is growing so slowly? What are people afraid of? Are there specific difficulties associated with it? We can think of possible answers to these questions. The first resistance of chromatographers to jumping to GC×GC may be unfamiliarity with a different way of performing chromatography and analyzing data. When dealing with two columns with different internal diameters and stationary phases coupled together, you need to revisit the optimization of parameters such as the injection speed in your method, flow rates, column temperatures and other parameters that improve your separation. Chromatograms are now tridimensional, and how to interpret and group thousands of peaks is another challenge. Once you get used to it, you become familiar with it, but this knowledge is not straightforward. The second resistance is related to instrumentation costs and operation. While commercial GC×GC equipment is considerably more expensive than traditional chromatographs, the home-made systems are limited by the data processing systems available. Some modulators need cryogenic liquids, such as liquid nitrogen or CO2, for their operation, which increases the costs and logistics for operation. The third resistance is related to the way the technique has been presented in the literature. As a frontier technique with high resolving power, most publications presented only qualitative results, leading the reader to conclude that this technique is not recommended for quantitative work or for fast throughput analysis. On top of that, different column configurations have been tested in several publications directing the chromatographers to consider these studies as a mandatory step for method optimization. This could be true in the earlier publications, but once the technique was established, method optimization became as easy as one-dimensional chromatographic methods. 14

Letter

As a rising technology, authors are starting to push the technique to its limit, demonstrating high throughput quantitative work and the limits of its resolution power. Instrumentation is becoming less expensive, and liquid cryogenic-free modulators are growing in importance. Software is becoming user friendly, with data set volumes easier to handle. Consequently, I believe that in the near future the GCĂ&#x2014;GC will occupy the prominent position it deserves in the history of chromatography. In my opinion, this technique is here to stay. REFERENCE Liu, Z. Y.; Phillips, J. B. J. Chromatogr. Sci., 1991, 29, pp 227â&#x20AC;&#x201C;234 (https://doi.org/10.1093/ chromsci/29.6.227).

Article

Br. J. Anal. Chem., 2019, 6 (24) pp 16-26 DOI: 10.30744/brjac.2179-3425.AR-02-2019

Harvest Influence in Volatile Composition of Chocolates Produced with Hybrid Varieties of Bahia’s Cocoa was Investigated using GC×GC-QMS and Chemometrics Soraia Cristina Gonzaga Neves Braga1 Luciana Fontes Oliveira2, Adriana Reis de Andrade 3 3 Silva , Priscilla Efraim , Ronei de Jesus Poppi2, Fabio Augusto2 Instituto Federal do Espírito Santo, Campus Viana, Rodovia BR-262, Universal, 29134-400 - Viana, ES - Brazil Instituto de Química, Universidade Estadual de Campinas (Unicamp), Cidade Universitária Zeferino Vaz s/n, Barão Geraldo, 13084971 - Campinas, SP, Brazil 3 Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas (Unicamp), Rua Monteiro Lobato, 80, Cidade Universitária Zeferino Vaz, Barão Geraldo, 13083-862 - Campinas, SP, Brazil 1 2

Graphical Abstract

Cocoa from eleven different varieties harvested from two different harvests was used to produce chocolate that had its volatile composition analyzed by GCxGC and interpreted by multivariate statistical procedure.

Cocoa varieties are important in Brazil due to their resistance to “witch’s broom” disease. In this work, the chromatographic profiles of chocolates produced from eleven cocoa varieties harvested from two different harvests were analyzed. A total of 22 samples were analyzed by comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GC×GC-MS), using headspace solid phase microextration (HS-SPME). Multiway principal component analysis (MPCA) were conducted to study the differences between the two harvests. Two principal components were responsible for the separation of the profiles between the harvests and their respective loadings were evaluated. Hydrocarbons were the principal group responsible for this separation, as some pyrazines and aldehydes. Keywords: comprehensive two-dimensional gas chromatography, volatile organic compounds, cocoa varieties, multiway principal component analysis. INTRODUCTION Until the 1980s, Brazil had a large cocoa production, but a disease known as witch’s broom, caused by the fungus Moniliophthora perniciosa [1], reached plantations in Bahia, a state located in the Brazilian 16

Harvest Influence in Volatile Compounds of Chocolates Produced with Hybrid Varieties of Bahia’s Cocoa using GC×GC-QMS and Chemometrics

Article

Northeast Region, dramatically reducing the production of cocoa [1]. Two ways were adopted to combat this disease in Brazil: the selection of clones and the selection of hybrid varieties [2]. Currently, new works have been conducted using biological control to reduce witch’s broom infestation [3]. The first cocoa clones were selected in the 1930s and a series called ICS were originated from these clones [4]. Some genotypes of this ICS series were selected and crossed with resistant clones, generating the series TSH [4]. A cross of Scavinas with Amazonian clones generated the TSA series [4]. In Ecuador, CCN 51 clones have high productivity and are resistant to witch’s broom. Researches developed in Brazil by CEPLAC — The Executive Commission for Cocoa Cultivation Planning, an agency of the Brazilian Ministry of Agriculture, Livestock and Food Supply — obtained hybrid cocoas of the types Forastero x Forastero and Forastero x Trinitario. These cocoas’ hybrid have been used by producers in the Bahia region since 1995 [4]. In 2009, Efraim [4] characterized hybrid cocoas of a CEPLAC experimental farm, studding the varieties: CEPEC 42 (hybrid of TSA 644 x SIC 19), EET 397 (coming from SCA), TSA 654 (hybrid of SCA6 x IMC 67), TSA 656 (hybrid of SCA6 x IMC 67), TSAN 792 (hybrid of TSA 641 and unknown genotype), TSH 516 (hybrid of SCA6 x ICS1), TSH 565 (hybrid of SCA6 x ICS1), TSH 774 (unknown origin) e TSH 1188 (unknown origin) [4]. Ramos et al. [5] evaluated, in 2014, the fermentation of four cocoa varieties through the volatile composition in the fermentation. In the new hybrids developed, it is necessary to characterize the volatile profile in chocolates obtained with these cocoas. Cocoa and chocolate are complex samples, and analysis of volatile compounds in such samples usually requires complex analytical methods. Analysis of volatile compounds is usually conducted by gas chromatography; another technique that has been used is the comprehensive two-dimensional gas chromatography (GC×GC), which is widely used in the characterization of complex biological [6-8] and food [9-11] samples. It is also used to obtain chromatographic profiles with less sample preparation. GC×GC has a higher analyte resolution power, besides facilitating the identification of analyzed components by the chromatographic structuring that can occur with some sets of chromatographic columns. In this work, eleven varieties of cocoa developed by CEPLAC were studied. The differences in the volatile composition of the chocolates obtained from two harvests of cocoa clones were investigated using HS-SPME and GCxGC-MS, analyzed with MPCA as chemometric tool. With this work it was possible to characterize the chocolate obtained by the new cocoa varieties, showing that the cocoa harvest influences the volatile composition in the final product, not only depending on the cocoa variety. MATERIALS AND METHODS Chocolate Samples Cocoa was obtained from an experimental farm in Bahia, in a CEPLAC study. The produced nibs belonged to different cocoa varieties, and the samples were collected from two different crops, at harvests with a difference of six months between them. The varieties studied were: TSH 1188, TSH 565, LP 06, EET 397, OS 13.19, CCN 10, CA 1.4, CCN 51, PH 129, FM 31 and COMUM. For the chocolate production, the nibs were processed under the same conditions used at the School of Food Engineering, University of Campinas (FEA-Unicamp). Dark chocolate with 65% cocoa and 35% sugar was produced according to Reis [12]. After production, the chocolate was stored in a cold chamber at -18 ºC until analysis. SPME Extraction and Chromatographic Conditions Collection of the volatile fraction of the samples was performed by HS-SPME using a previously optimized procedure [13]. Aliquots of 1.000 ± 0.005 g of the chocolate samples were weighed into 15 mL vials. After a 5 min period for sample/headspace equilibration at 60 °C, a 50/30 μm divinylbenzene/ carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber was exposed to the sample headspace for 50 min; the fiber was then immediately inserted into the GC×GC system injector and held there for 5 min. Before 17

Article

Braga, S. C. G. N; Oliveira, L. F.; Silva, A. R. A.; Efraim, P.; Poppi, R. J.; Augusto, F.

the extractions, the fiber was conditioned for 2 h at 250 ºC according to the manufacturer’s instructions. The method optimization, including the fiber selection, was the same used by Oliveira et al. [14]. GC×GC-QMS GC×GC-QMS analyses were carried out on a prototype based on a Shimadzu QP2010 + fast GC-QMS gas chromatograph fitted with a lab-made 4-jet cryogenic modulator, already described in the literature [7]. The column set consisted of a 30 m × 0.25 mm × 0.25 μm HP-5 column (Agilent Technologies, Wilmington, DE) connected to a 0.80 m × 0.1 mm × 0.1 μm Solgel Wax column (SGE Analytical Science, Ringwood - Victoria, Australia). The split-splitless injector was operated in splitless mode at 260 ºC. The column oven temperature was programmed as follows: 60 ºC (3 min) to 70 ºC (7 min) at 3 ºC min−1; 70 ºC to 110 ºC at 3 ºC min−1; and 110 ºC to 240 ºC at 10 ºC min−1. The carrier gas used was high purity (99.9999%) hydrogen at 0.6 mL min−1, and the modulation period was 6 s. The interface temperature was 260 ºC and the photomultiplier power was programmed to 0.8 kV up to 10 min of the chromatographic run, and then increased to 0.9 kV until the end of the analysis. The scanned mass range was set from m/z = 40 D to 340 D, resulting in a data collection frequency of 25 spectra s−1. Data Processing Peaks in chromatograms were identified by NIST 2010 mass spectral library and 1st dimension linear temperature programming retention indexes (LTPRI) performed using GC Image software (Zoex Corp., Houston – TX, USA), and samples spiked with n-alkanes mixture (C8-C20). Chemometric analyzes were performed using the softwares: MATLab environment (Mathworks, EUA) version R2011b, and PLS_toolbox (Mathworks, EUA) version 654. Differences between chromatographic profiles of chocolates were evaluated by MPCA. Before the use of MPCA, the unfolded chromatograms were aligned using the COW (Correlation Optimizing Warping) algorithm [15,16]. RESULTS AND DISCUSSION Chocolate samples were produced with cocoa of 11 varieties and from two harvests, named harvest 1 and harvest 2, totaling 22 samples. After obtaining the chromatographic profiles by GC×GC-QMS, variations in these samples related to harvests were investigated. Chromatograms of harvest 1 and harvest 2 presented differences in a specific region of the chromatogram near to the retention time of 1.0 second in the 2D dimension. MPCA was developed with the entire chromatograms. Chromatographic profiles obtained were exported as TIC (total ion current) in .txt format and were investigated with MATLab. Alignment was conducted with the unfolded chromatogram and in small segments independently, using the COW algorithm [15,16]. After the alignment, a MPCA model was developed using the PLS_toolbox. Three principal components were required and they explained 83,47% of data variance (PC1 69,70%, PC2 10,36% and PC3 3,41%). Hotelling (T2) versus Q residual (Q) showed no outliers. Two principal components were responsible for separation of chromatographic profiles in harvest 1 and harvest 2: PC1 and PC2. A score graphic between these principal components is shown in Figure 1.

Harvest Influence in Volatile Compounds of Chocolates Produced with Hybrid Varieties of Bahiaâ&#x20AC;&#x2122;s Cocoa using GCĂ&#x2014;GC-QMS and Chemometrics

Article

Figure 1. Graphic of scores of principal components 1 and 2, showing the separation between harvest 1 (triangles) and harvest 2 (asterisks).

To identify the compounds responsible for the class separation, the loadings graphics of PC1 and PC2 were evaluated and the compounds found were identified by matching their mass spectra with the NIST 10 library and confirmed by comparing the LTPRI with the standard. Table I and Table II present the chromatographic peaks identified in loadings of PC1 and PC2. Table I. Compounds corresponding to loadings of PC1 in chocolate samples of two different harvests tR 1D (min)

tR 2D (s)

Retention index calculated

Identified compounds

10.5

1.84

961

Benzaldehyde

12.9

1.52

1000

Decane

13.9

1.48

1015

14.6

1.48

1025

15.7

1.44

1042

3-methyl-decane

16.7

1.52

1056

2-methyl-decane

17.3

1.52

1065

(Z)-5-methyl-dec-2-ene

18.2

1.52

1078

2,5-dimethyl-decane

19.7

1.44

1105

20.4

1.44

1112

2,3,5-trimethyl decane

21.9

1.44

1138

4-methyl-undec-1-ene

22.8

1.44

1154

3-methyl-undecane

24.3

1.44

1180

3,8-dimethyil-undecane

13.2

4.20

1006

Trimethylpyrazine

18.9

3.36

1088

Tetramethylpyrazine

20.0

2.44

1105

Nonanal

28.5

1.36

1285

2,3-dimethyldodecane

NI: Not identified 19

Article

Braga, S. C. G. N; Oliveira, L. F.; Silva, A. R. A.; Efraim, P.; Poppi, R. J.; Augusto, F.

Table II. Compounds corresponding to loadings of PC2 in chocolate samples of two different harvests tR 1D (min)

tR 2D (s)

Retention index calculated

Identified compounds

5.1

1.44

843

5.8

1.40

863

4-methyl-octane

8.2

1.68

903

Dihydro-2(3H)-furanone

10.1

3.32

952

(E)-hept-2-enal

12.1

2.60

975

Octan-3-one

12.5

3.48

994

(Z)-hex-2-enyl acetate

17.4

4.04

1066

3-methoxybutanoic acid

18.1

5.04

1089

Octan-1-ol

19.0

4.24

1090

19.7

4.04

1099

Linalool

19.9

3.56

1101

Nonan-2-ol

21.7

3.28

1129

Dihydrolinalol

27.1

1.84

1245

NI: Not identified

Analyzing the identified compounds by loadings of PC1, it was possible to verify that hydrocarbons were responsible for the separation between the chocolate samples produced firstly (harvest 1), and those harvest later (harvest 2). In addition to this group of compounds, other compounds such as benzaldehyde, trimethylpyrazine, tetramethylpyrazine and nonanal were identified as the major compounds (after the hydrocarbons) in the chromatograms of chocolate samples, also being known as important contributors to chocolate aroma [2]. In PC2 there is a predominance of alcohols (4 compounds) and 1 compound of each class: furanone, aldehyde, ester, ketone, acid and alkane. Figure 2 shows the characteristic chromatograms of the COMUM cocoa variety from harvest 1 (A) and harvest 2 (B). The red overlapping rectangle in chromatogram (A) highlight some analytes not solved. All the components in chromatograms were identified and separated in chemical classes. In Figure 3, the graphics show this classification for harvest 1 (A) and harvest 2 (B). Table III shows all the compounds identified in harvest 1 and harvest 2. The developed MPCA showed that hydrocarbons are the compounds with the greatest difference between the two harvests, being this showed by the chemical class studies and presented in Figure 3. There was no change in the number of compounds in the classes of acids (1), aldehydes (8), ketones (11) and compounds containing nitrogen (6) between the two harvests. A decrease of 3 alcohols and an increase of 2 esters from harvest 1 to 2 were also observed.

Harvest Influence in Volatile Compounds of Chocolates Produced with Hybrid Varieties of Bahiaâ&#x20AC;&#x2122;s Cocoa using GCĂ&#x2014;GC-QMS and Chemometrics

Article

Figure 2. Chromatograms obtained for the COMUM cacao variety from harvest 1 (A) and harvest 2 (B). The region in (A) surrounded by red corresponds to some analytes not resolved (splitless mode).

Figure 3. Classes of compounds found in chocolates from harvest 1 (A) and harvest 2 (B) and the number of compounds in each class.

Regarding the distribution of compounds in Table III, in the alcohol class, hept-4-en-1-ol, 3-methylheptan-2-ol and triethylene glycol were observed in harvest 1 and not observed in harvest 2. Among the esters, in harvest 2 there were 2-pentyl acetate, prop-2-ene-1,1-diol diacetate, isoamyl pyruvate and 3,4-dihydroxy-3-methyl-butyl acetate, while harvest 1 presented ethyl octanoate and ethyl benzene acetate. Regarding the aldehydes and N-compounds there were no variation in number and distribution of them. For the other classes, there was no variation in the number of compounds, but their distribution was different. In the ketone class, harvest 1 had undecan-2-one, pentan-2-one and 2-(formyloxy)-1phenyl-etanone, while harvest 2 had heptan-2-one, decan-2-one and 4-hidroxy-3-methyl-butanone. The compound octan-1-ol was present only in FM 31 and LP 06, while dihydrolinalool and undecan2-one were present in EET 397 and PS 13.19, both in harvest 1; o-cimene was present in the varieties EET397 and PH 129 - in the latter, triethylene glycol was identified. Furthermore, in harvest 1 variety PS13.19, only the ethyl octanoate compound was found. In harvest 2, the varieties FM31, CCN551, PH129, TSH and PS13.19 contained single compounds, with the last two varieties presenting N,N,Otriacylhydroyilamine, the first containing prop-2-en-1,1-diol acetate, the second having 1-methyl-4-(1methylethyl)-benzene and the third containing tetrahydrolinalol.

9.3

8.8

8.7

8.3

8.2

7.9

7.6

7.3

7.2

7.0

6.8

6.1

5.9

5.8

5.4

4.7

4.2

3.8

tR 1 D (min)

3.28

2.24

0.88

5.92

1.48

3.52

1.64

1.40

3.24

4.60

3.28

2.12

1.40

2.12

2.60

5.84

2.32

1.88

4.00

1.40

1.76

2.96

3.40

5.76

1.84

tR 2 D (s)

Benzaldehyde

1-ethyl-3-methyl-benzene

(E)-hept-2-enal

Benzeneacetaldehyde

5-hydroxy-2-methyl-hexan-3-one

4-Hydroxy-butan-2-one acetate

Non-2-ene

2,3-dimethyl-pyrazine

Dihydro-5-methyl-2 (3H) -furanone

2,4,6-trimethyl-octane

2,6-dimethyl-pyrazine

Butyl propanoate

3-methyl -heptan-2-ol

Heptan-2-ol

Nonane

Heptan-2-one

Ethenyl benzene

1,2-Diacetoxyethane

2-hydroxy-3-methyl-2-cyclopenten1-one

Ethyl benzene

Hept-4-en-1-ol

4-methyl-octane

2-pentyl acetate

Methyl-pyrazine

2,3-Butanediol

Acetic acid

Hexanal

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1,2))

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1,2)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1,2)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

EET 397

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1,2)

PS 13.19

X(1,2)

X(2)

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1,2)

CCN10

X(1,2)

X(2)

X(1,2)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1,2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(2)

X(1,2)

CA 1.4

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1,2)

CCN551

X(1,2)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1,2)

PH129

Table III. Compounds identified in all the chromatograms obtained in the two harvests

10.0

2.32

X(1,2)

COMUM

10.1

1.84

X(2)

X(1,2)

LP06

10.4

Prop-2-ene-1,1-diol diacetate

X(1,2)

TSH565

10.5

3.12

Octan-3-one

X(1,2)

TSH1188

11.3

2.56

5-hepten-2-one

FM 31

12.0

3.48

Compound

12.1

Braga, S. C. G. N; Oliveira, L. F.; Silva, A. R. A.; Efraim, P.; Poppi, R. J.; Augusto, F.

Article

tR 2 D (s)

2.36

2.76

3.44

5.20

1.52

2.72

4.20

1.48

2.84

2.32

2.38

1.92

5.12

3.32

2.16

1.44

2.68

1.48

4.00

5.04

4.24

3.36

2.48

1.48

4.04

2.76

3.56

2.44

tR1D (min)

12.4

12.5

12.7

12.9

13.2

13.7

14.6

14.7

14.8

15.0

15.2

15.8

16.0

16.1

16.2

16.7

17.3

17.4

18.0

18.3

18.9

19.3

19.6

19.7

19.8

19.9

20.0

Nonanal

Nonan-2-ol

Tetrahydro-linalool

Linalool

Undecane

Nonan-2-one

Tetramethyl-pyrazine

1,3-Butanediol diacetate

Octan-1-ol

3-Methoxybutanoic acid

5-methyl-dec-2-ene

2-methyl-decane

Phenylacetaldehyde

5-methyl-decane

Isoamyl Pyruvate

oct-3-en-2-one

2-ethyl-hexan-1-ol

Limonene

1-methyl-4-(1-methylethyl)-benzene

o-Cimene

1,2,4-trimethyl-benzene

3-ethyl-2,7-dimethyloctane

Trimethylpyrazine

Octanal

Decane

4-hydroxy-3-methyl-butan-2-one

(Z) -2-hex-2-enyl acetate

Octan-2-one

2-pentyl-furan

Compound

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

FM 31

X(1,2)

X(2)

X(1,2)

X(2)

X(1,2)

X(1)

X(2)

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

TSH1188

X(1,2)

X(2)

X(1,2)

X(1)

X(2)

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

TSH565

X(1,2)

X(1)

X(2)

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

LP06

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

COMUM

X(1,2)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

EET 397

X(1,2)

X(2)

X(1,2)

X(1)

X(2)

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

PS 13.19

X(1,2)

X(2)

X(1,2)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

CCN10

X(1,2)

X(2)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

CA 1.4

Table III. Compounds identified in all the chromatograms obtained in the two harvests (Cont.)

X(1,2)

X(2)

X(1,2)

X(2)

X(1,2)

X(1)

X(2)

X(1,2)

X(2)

X(1)

X(1,2)

X(2)

X(1,2)

CCN551

X(1,2)

X(2)

X(1,2)

X(2)

X(1,2)

X(1)

X(2)

X(1,2)

X(1)

X(1,2)

X(2)

X(1,2)

PH129

Harvest Influence in Volatile Compounds of Chocolates Produced with Hybrid Varieties of Bahia’s Cocoa using GC×GC-QMS and Chemometrics

Article

26.5

25.7

25.4

25.3

25.1

24.3

23.7

23.5

23.0

22.8

21.9

21.7

21.3

20.9

20.7

20.4

20.3

tR 1 D (min)

3.16

1.24

3.36

1.40

3.24

2.24

1.48

2.04

2.36

1.44

1.48

1.44

1.52

1.48

1.44

3.32

1.44

1.48

4.72

1.48

1.44

5.84

tR 2 D (s)

2,3-dimethyldodecane

2-Phenylethyl acetate

Triethylene glycol

3,4-Dihydroxy-3-methyl-butyl acetate

5,5-dimethyl-undecane

Benzene Ethyl acetate

Decanal

Dodecane

Ethyl octanoate

Decan-2-one

3,8-dimethyl-undecane

2,2,6-trimethyl-decane

5-methyl-undec-2-ene

5-methyl-undecane

3-methyl-undecane

4-methyl-undec-1-ene

Dihydrolinalol

Tetradecane

2,6,7-trimethyl- decane

Benzeneethanol

2,5,6-trimethyl-decane

2,3,5-trimethyl-decane

N, N, O-Triacetylhydroxylamine

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

EET 397

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

PS 13.19

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(2)

X(1)

X(1,2)

X(1)

CCN10

X(1)

X(1,2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

CA 1.4

X(1)

X(1,2)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1)

X(1,2)

X(1)

CCN551

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)

X(1)

X(1,2)

X(1)

X(1,2)

X(1)

X(2)*

X(2)

X(1)

X(1,2)

X(1)

PH129

Table III. Compounds identified in all the chromatograms obtained in the two harvests (Cont.)

27.4

1.38

Tridecane

Undecan-2-one

X(1)

COMUM

27.9

1.84

X(1)

LP06

28.5

1.36

X(1)

TSH565

28.9

2- (formyloxy) -1-phenyl-ethanone

X(1)

TSH1188

29.1

2.00

Tetradecane

FM 31

31.2

1.40

Compound

31.3

X: compound present in the sample; *Compound missing in the sample; (1): Harvest 1; (2): Harvest 2; (1,2): Harvest 1 and 2.

Braga, S. C. G. N; Oliveira, L. F.; Silva, A. R. A.; Efraim, P.; Poppi, R. J.; Augusto, F.

Article

Harvest Influence in Volatile Compounds of Chocolates Produced with Hybrid Varieties of Bahia’s Cocoa using GC×GC-QMS and Chemometrics

Article

Regarding the quantity of volatile compounds in each variety, in harvest 1 the order was as follows: FM31 = PS13.19 > TSH565 = EET397 > LP06 = PH129 > COMUM > TSH 1188 > CA 1.4 > CCN10> CCN 51. In harvest 2, the order was PS13.19 > TSH565 > TSH1188 > EET397 > LP06 = CCN10> FM31 = PH129 = CCN551 > COMUM > CA1.4. It is possible to verify that in the two harvests the richest sample in volatiles compounds was PS13.19, followed by TSH565, being these two samples the ones that had smallest variation between the harvests. There was no pattern among the other varieties, so the distribution was random. CONCLUSIONS Applying the MPCA to the chocolate chromatographic profiles obtained from cocoa beans of 11 varieties and two harvests, it was possible to verify the differences in these profiles according to the harvest — the largest difference observed was in the hydrocarbon class found in higher quantity in harvest 1. PS13.19 was found to be the richest variety in volatile compounds, regardless of the original harvest. Furthermore, it was found that even within the same variety, the volatile composition of chocolate may change according to the cocoa harvesting period. Manuscript submitted: February 14, 2019; revised manuscript submitted: May 28, 2019; manuscript accepted: August 19, 2019; published online: September 27, 2019. REFERENCES 1. Pereira, J. L.; de Almeida, L. C. C.; Santos, S. M. Crop Prot., 1996, 15, pp 743–752 (https://doi. org/10.1016/S0261-2194(96)00049-X). 2. Pires, J. L. Avaliação Quantitativa e Molecular de Germoplasma para o Melhoramento do Cacaueiro com Enfase na Produtividade, Qualidade de Frutos e Resistencia a Doenças. Doctoral Thesis, 2003, Federal University of Viçosa, Viçosa, MG, Brazil. 3. Medeiros, F. H. V; Pomella, A. W. V; de Souza, J. T.; Niella, G. R.; Valle, R.; Bateman, R. P.; Fravel, D.; Vinyard, B.; Hebbar, P. K. Crop Prot., 2010, 29, pp 704–711 (htpps://doi.org/10.1016/j. cropro.2010.02.006). 4. Efraim, P. Contribuição à melhoria de qualidade de produtos de cacau no Brasil, por meio da caracterização de derivados de cultivares resistentes à vassoura-de-bruxa e de sementes danificadas pelo fungo. Doctoral thesis, 2009, Faculty of Food Engineering, University of Campinas, Campinas, SP, Brazil. 5. Ramos, C. L.; Dias, D. R.; Miguel, M. G. C. P.; Schwan, R. F. Food Res. Int., 2014, 64, pp 908– 918 (https://doi.org/ 10.1016/j.foodres.2014.08.033). 6. Hantao, L. W.; Toledo, B. R.; de Lima Ribeiro, F. A.; Pizetta, M.; Pierozzi, C. G.; Furtado, E. L.; Augusto, F. Talanta, 2013, 116, pp 1079–1084 (https://doi.org/10.1016/j.talanta.2013.08.033). 7. Hantao, L. W.; Aleme, H. G.; Passador, M. M.; Furtado, E. L.; Ribeiro, F. A. L.; Poppi, R. J.; Augusto, F. J. Chromatogr. A, 2013, 1279, pp 86–91 (https://doi.org/10.1016/j. chroma.2013.01.013). 8. de Lima, P. F.; Furlan, M. F.; de Lima Ribeiro, F. A.; Pascholati, S. F.; Augusto, F. J. Sep. Sci., 2015, 38, pp 1924–1932 (https://doi.org/10.1002/jssc.201401404). 9. Humston, E. M.; Knowles, J. D.; Mcshea, A.; Synovec, R. E. J. Chromatogr. A, 2010, 1217, pp 1963–1970 ( https://doi.org/10.1016/j.chroma.2010.01.069). 10. Cordero, C.; Bicchi, C.; Rubiolo, P. J. Agric. Food Chem., 2008, 56, pp 7655–7666 (https://doi.org/ 10.1021/jf801001z). 11. Souza-Silva, E. A.; Gionfriddo, E.; Pawliszyn, J. TrAC Trends Anal. Chem. 2015, 71, pp 236-248 (https://doi.org/10.1016/j.trac.2015.04.018).

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Braga, S. C. G. N; Oliveira, L. F.; Silva, A. R. A.; Efraim, P.; Poppi, R. J.; Augusto, F.

12. Silva, A. R. de A. Caracterização de amêndoas e chocolate de diferentes variedades de cacau visando a melhoria da qualidade tecnológica. Master’s thesis, 2013, Faculty of Food Engineering, University of Campinas, Campinas, SP, Brazil. 13. Oliveira, L. F.; Braga, S. C. G. N.; Augusto, F.; Hashimoto, J. C.; Efraim, P.; Poppi, R. J. Food Res. Int., 2016, 90, pp 133–138 (https://doi.org/ 10.1016/j.foodres.2016.10.047). 14. Oliveira, L. F.; Braga, S. C. G. N.; Filgueiras, P. R.; Augusto, F.; Poppi, R. J. Talanta, 2014, 129, pp 303-308 (https://doi.org/10.1016/j.talanta.2014.05.038). 15. Nielsen, N. P. V; Carstensen, J. M.; Smedsgaard, J. J. Chromatogr. A, 1998, 805, pp 17–35 (https://doi.org/10.1016/s0021-9673(98)00021-1). 16. Tomasi, G.; Van Den Berg, F.; Andersson, C. J. Chemom., 2004, 18, pp 231–241 (https://doi. org/10.1002/cem.859).

Br. J. Anal. Chem., 2019, 6 (24) pp 27-37

Article

DOI: 10.30744/brjac.2179-3425.AR-10-2019

Selective Extraction of Manganese using Moringa oleifera Seeds as Bioadsorbent Sângela Nascimento do Carmo1, José Alistor de Sousa Neto2, Vanessa Nunes Alves2 Luciana Melo Coelho2, Nívia Maria Melo Coelho1 Instituto de Química, Universidade de Uberlândia. Av. João Naves de Ávila, 2121, 38400-902, Uberlândia, MG, Brazil Departamento de Química, Universidade de Goiás. Av. Dr. Lamartine Pinto de Avelar, 1120, Setor Universitário, 75704-020, Catalão, GO, Brazil

1 2

Graphical Abstract

Moringa oleifera seeds used for selective extraction of manganese in aqueous samples. Around 60% of the Mn(II) was removed at an initial pH of 3.0. The maximum adsorption capacity for Mn(II) was 10.35 mg g-1.

The adsorption of manganese onto Moringa oleifera seeds was optimized as a function of pH, adsorbent mass, particle size and contact time in aqueous solutions in batch tests. The results show that the optimized conditions for manganese adsorption were: pH 3.0, particle size ≤ 180 µm, adsorbent mass 1.0 g and contact time 15 min. Varying the pH allowed the separation of the manganese species, the seeds selectively retained Mn(II) while Mn(VII) remained free in solution. Around 60% of the Mn(II) was removed at an initial pH of 3.0 with a manganese concentration of 4 mg L-1. The adsorption process was evaluated through adsorption isotherms and kinetics studies. The maximum adsorption capacity for Mn(II) was 10.35 mg g-1. The isotherm followed the Langmuir model and the adsorption kinetics followed a pseudo-second-order kinetic model. Keywords: manganese, selective extraction, Moringa oleifera. INTRODUCTION Manganese (Mn) is the tenth element in order of abundance in the earth’s crust and it is mainly used in metallurgical processes, approximately 90% being used in the manufacture of steel, and as a component in metallic alloys. Other uses of Mn include the manufacture of ceramics, dry cell batteries and pigments. These activities constitute the major anthropogenic sources of this metal to the aquatic environment [1]. Manganese is an essential micronutrient in the human diet. It activates many enzymes used in metabolic processes and is also required for protein and fat metabolism. Mn helps to maintain 27

Article

Selective Extraction of Manganese using Moringa oleifera Seeds as Bioadsorbent

healthy nervous and immune systems and to regulate blood sugar levels [2-4]. However, high doses of manganese cause DNA mutations, neurological disorders (e.g., manganism), overflow of the liver, hallucinations, depression and excessive sleep [5,6]. Therefore, the study of trace elements is gaining importance, especially when these elements are present in the environment in different oxidation states [7]. There are 11 oxidation states of manganese, the most prevalent being Mn(II), Mn(IV) and Mn(VII), and their order of toxicity is Mn(II) > Mn(VII) > Mn(IV) [8]. In this context, it is essential to establish methods for the removal of this metal, especially Mn(II), which is the most toxic species in aqueous or solid effluents. Precipitation as a hydroxide is one of the most commonly used techniques for metal removal, through its reaction with a base added to the effluent; however, this procedure is not appropriate for low concentrations. The use of solid phase extraction (SPE) has been frequently proposed as a technique for metal removal and it is suitable for use in batch or flow experiments. The choice of the adsorption phase is dependent on the adsorption capacity associated with each species considered [9]. The use of natural adsorbents has been successfully employed in metal ion adsorption. The significant benefits of adsorption processes include effective and economical contaminant removal, recovery of the adsorbent metals from the adsorbent and its recycling, low sludge production, simple process procedures and high removal efficiencies. In this context, the Moringa oleifera seeds have been gaining attention. Moringa seeds have been most widely applied as a coagulant agent, but many studies have been performed in order to explore other potential applications of this material, especially in the removal of metals from aqueous systems. This material is of low cost and is easy to obtain, and it has good potential for application in procedures for the removal and selective extraction of metal ions [10]. The aim of this study was to describe the use of M. oleifera seeds for the extraction of Mn(II) from natural water samples, since the physiological and toxicological effects of manganese are dependent on its chemical form [11], the bivalent species being the most toxic. MATERIALS AND METHODS Adsorbent Preparation The Moringa oleifera seeds were obtained from trees which were cultivated in the city of Uberlândia (Minas Gerais, Brazil). The seeds were separated from the pods, crushed in a household blender (Black & Decker, São Paulo, Brazil) and sieved at 500, 300 and 180 µm. They were then washed in deionized water and dried at ambient temperature. The functional groups present in the seeds were characterized using a Fourier transform infrared (FT-IR) spectrometer (Shimadzu, IR Prestige-21, Tokyo, Japan). Manganese Determination A Varian Model SpectrAA 220 (Victoria, Australia) flame atomic absorption spectrometer, with airacetylene flame, was used for the manganese determination. A manganese hollow cathode lamp was run under the conditions recommended by the manufacturer. The wavelength used was 279.5 nm and conventional values were applied for the slit width and burner height. Standard Solutions and Reagents All solutions were prepared with analytical grade reagents and high purity deionized water produced by a Milli-Q® system (Millipore, Bedford, MA, USA). The glassware and containers for the storage of the solutions were immersed in 10% (v/v) nitric acid and rinsed with deionized water before use. The working solutions of Mn(II) were prepared through dilution of a 1000 mg L-1 stock solution (Carlo Erba, Val de Ruil, France) in deionized water. Since a stock solution of Mn(VII) was not available, the 1000 mg L-1 solution of this species was prepared by dissolving 0.034 g of KMnO4 (Vetec, Rio de Janeiro) in 100 mL of deionized water. Solutions of 0.1 mol L-1 HNO3 and 0.1 mol L-1 NaOH were used to adjust the pH. 28

do Carmo, S. N.; de Sousa Neto, J. A.; Alves, V. N.; Coelho, L. M.; Coelho, N. M. M.

Article

Adsorption Studies The solution pH is a critical variable that directly affects the ion adsorption. Moringa oleifera seeds can adsorb cations or anions depending on the pH solution. The effect of the pH solution on the Mn(II) and Mn(VII) separation was investigated by varying the pH from 1 to 8. In this procedure, 0.5 g of seeds were shaken for 40 minutes with 25 mL of a manganese solution containing one of the Mn species (4.0 mg L-1). The suspension was then filtered, and the supernatant was directly analyzed by flame atomic absorption spectrophotometry (F AAS). The amount of manganese retained in the biosorbent was calculated by the difference between the initial and final manganese concentrations in the solution. The pH 3.0 is the pH at which separation of the manganese species occurs and this value was used in order to obtain the optimum conditions for Mn(II) adsorption. The following variables were studied: particle size (180 to 500 µm), adsorbent mass (0.05 to 4.0 g) and stirring time (5 to 120 min). Studies on Interference from Ions in Mn(II) Adsorption The Mn(II) adsorption in the presence of Na+, Ca2+, Mg2+ and Fe3+, considered as concomitant ions, in various proportions, was evaluated. The experimental conditions were: pH 3.0, particle size 500 µm, adsorption mass 2 g and stirring time 15 min. Isotherm Adsorption The experiments for the isotherm tests were carried out at a temperature range of 25 to 28 ºC using 50.0 mg of moringa seeds (500 µm) and 50 mL of Mn(II) solution in a concentration of 0.2 to 100 mg L-1. The pH of the mixture was adjusted to 3.0 and the stirring time was 60 min. The mixture was filtered and the Mn(II) was quantified using flame atomic absorption spectrometry (FAAS). Kinetic Studies The kinetics of the Mn(II) adsorption onto moringa seeds in the batch tests was investigated. In this study, 0.05 g of moringa seeds were added to 50 mL of the solution containing manganese ions in the concentration of 4 mg L-1 in polyethylene jars. The mixtures were agitated at 180 rpm for intervals between 5 and 120 min. After agitation, the mixture was filtered and the manganese concentration determined by F AAS. The adsorption capacity of the adsorbent q (mg g-1) in relation to manganese ions was calculated by the Equation 1: q = [(CO-Cf)/m]*VS (1) where CO is the initial concentration in mg L-1, Cf is the final concentration in mg L-1, m (g) is the adsorbent mass and VS (L) is the solution volume. The kinetic parameters of the manganese adsorption process by moringa seeds were determined through linear regression of the graphs for pseudo-first-order, pseudo-second-order model and intraparticle diffusion using the respective equations given below [12,13]. log10(qe – q) = log10(qe – k)*t

(2)

where qe and q are the amounts adsorbed from the solution (mg g-1) at equilibrium and at time (t), respectively, and k is the adsorption rate constant (min-1). The constant k is calculated from the slope of the line in the graph log (qe - q) x t. This model considers that the sorption rate is dependent on the number of active sites available.

(3)

Article

Selective Extraction of Manganese using Moringa oleifera Seeds as Bioadsorbent

where k2 is the pseudo-second-order constant (g mg-1 min-1), t is the time (min) and qe and q are the amounts of solute adsorbed (mg g-1) at equilibrium and at time t, respectively. From the plot of t/q x t, the values of the constants k2 and q can be calculated. The k2 constant is used to calculate the adsorption rate “h” (mg g-1 min-1) for t0, as follows: h = k2 qe2. For the intra-particle diffusion model this equation is: qt = kdif t½ + C

(4)

where qt is the amount of adsorbed solute (mg g-1), t is the agitation time (min) and C (mg g-1) is the constant related to the resistance to the diffusion. The kdif (mg g-1 min-0.5) can be obtained from the slope and the C value from the intersection of the curve for qt versus t0.5 [14]. According to this model, when the intraparticle diffusion is involved in the sorption process, the curve defined by this equation must be linear. Accuracy Tests To verify the selective adsorption of Mn(II), standard addition and recovery tests were carried out with samples containing Mn(II) and Mn(VII). The samples were: tap water, river water and mineral water. The river water samples were collected from Uberabinha River, which runs through an urban zone in the city of Uberlândia, and mineral water samples were acquired from a local store in the city of Uberlândia. Since the analyte concentration was below the detection limit, samples were spiked with the same concentrations of Mn(II) and Mn(VII) (4 and 50 mg L-1). Also, samples of a drinking water certified reference material (APS 1075) was used in the accuracy tests. RESULTS AND DISCUSSION Optimization for the Selective Adsorption of Manganese Metals in aqueous systems are dissolved in different forms, some are present as simple hydrated ions, or ion complexes bound to organic ligands such as amines, humic and fulvic acids and proteins. Thus, the adsorption of the metals can occur through ion exchange mechanisms and complexation, which can even occur simultaneously [15]. In this context, the pH is a critical variable since it directly affects the manganese adsorption. Since the seeds can adsorb cation and anions depending on the electrical charge on the surface, the initial pH of the solution is the determining factor in the separation of inorganic manganese species. Thus, controlling the initial pH of the solution may be sufficient to ensure a significantly different percentage of the adsorption of the Mn(II) or Mn(VII) species, thus promoting their selective extraction. The influence of the pH on the adsorption of the Mn(II) and Mn(VII) species is shown in Figure 1.

do Carmo, S. N.; de Sousa Neto, J. A.; Alves, V. N.; Coelho, L. M.; Coelho, N. M. M.

Article

Figure 1. Evaluation of Mn species retention at different pH values. Experimental conditions: Adsorbent mass = 0.5 g, stirring time = 40 min.

The isoelectric point determined for Moringa oleifera seeds is between 5-6 and for pH values under 6.0 the surface of moringa seeds has a positive charge [10]. In Figure 1 it can be observed that at pH values under 4.0, only the Mn(II) species is adsorbed, the highest adsorption occurring at pH 3.0. Thus, at pH 3.0 it is assumed that the material is positively charged and can thus adsorb negative species, but, in acidic medium, Mn is predominantly in the Mn2+ form. The better adsorption observed can be attributed to the fact that the mechanism responsible for the metal retention by lignocellulosic adsorbents is based not only on ion exchange mechanisms but also complexation. So, at pH 3.0, the Mn(VII) species is predominantly in the uncharged form (MnO4H) and cannot electrostatically interacts with the adsorbent. Another process that may be involved is the oxidation reactions of organic matter present in the seeds, promoted by the permanganate ion in alkaline medium (pH > 6). Permanganate ion is a strong oxidizing agent. So, can be a conversion of MnO4- to the insoluble MnO2, which cannot be quantified by F AAS in the supernatants [16]. Therefore, at pH 3.0, Mn(II) can be adsorbed with no adsorption of Mn(VII), allowing the selective separation of Mn(II) and Mn(VII) with pH control. Optimization Strategy for Mn(II) Adsorption Several variables can interfere with the adsorption process and in the next part of this study the variables evaluated were particle size, adsorbent mass and contact time. The influence of varying the particle size of the Moringa oleifera seeds (≤ 500, ≤ 300 and ≤ 180 µm) on the Mn(II) adsorption was evaluated and the results are shown in Figure 2(a). A decrease in the particle size had a favorable effect on the metal sorption, possibly due to the increased contact surface, suggesting an increase in the number of reactive sites, thus favoring ion adsorption by the adsorbent [17]. The experimental conditions for the study were: pH 3.0, adsorbent mass 0.5 g, contact time 40 min and Mn(II) solution concentration 4 mg L-1. The influence of the adsorbent mass on the Mn(II) adsorption was studied by varying the mass (0.05, 0.5, 1.0, 1.5, 2.0 and 4.0 g) of adsorbent. The percentage of manganese ions adsorbed increased with increasing mass up to 1.0 g of seeds, and then remained relatively constant (Figure 2(b)). This probably occurred because the system reaches saturation. Thus, a mass of 1.0 g is sufficient to ensure the 31

Article

Selective Extraction of Manganese using Moringa oleifera Seeds as Bioadsorbent

adsorption of Mn(II) ions. The effect of the contact time on the adsorption of Mn(II) was studied in the range of 5 to 120 min (Figure 2(c)). The amount of Mn(II) retained by the adsorbent decreased as the contact time was increased to 60 min and after this time there was no significant variation in the percentage of metal adsorbed, possibly due to the system reaching equilibrium. Since 15 min was sufficient time to ensure good adsorption, this time was selected for the subsequent studies, in order to obtain a simple and fast methodology.

Figure 2. Percentage adsorption of Mn(II) onto Moringa oleifera seeds on varying the particle size (a), adsorbent mass (b) and contact time (c).

Adsorption Isotherm The mechanisms associated with metal adsorption by biomass are still not clear; however, it is important to note that this process is not based on a single mechanism. Metal sequestration occurs through complex mechanisms, including ion-exchange and complexation, and it is quite possible that at least some of these mechanisms act simultaneously in various degrees depending on the biomass, the metal ion and the solution environment. The isotherm models can be used to describe this process. Figure 3 shows the sorption isotherm for Mn(II) adsorption onto Moringa oleifera seeds.

Article

do Carmo, S. N.; de Sousa Neto, J. A.; Alves, V. N.; Coelho, L. M.; Coelho, N. M. M.

Figure 3. Adsorption isotherm for Mn(II) adsorbed onto Moringa oleifera seeds. Experimental conditions: pH = 3.0, adsorbent mass = 0.5 g, stirring time = 15 min.

Figure 3 demonstrates graphically the isotherm obtained, which can be compared with the class L proposed by Oscik [18]. The isotherms of this class are nonlinear and the slope is concave in relation to the abscissa. Since the sites on the adsorbent are all occupied, further adsorption of the adsorbate molecules is hindered. This behavior is expected in chemical adsorption and occurs for ion exchange or complexation [19]. The experimental data was analyzed using the Langmuir and Freundlich models, and the associated linearized equations are: Langmuir equation: 1/q = 1/Qmax Ce b + 1/Qmax Freundlich equation: logQe = log(Kf) + 1/n log Ce where: Qe is the amount of species adsorbed in the solid phase at equilibrium, Ce is the species concentration in the liquid phase at equilibrium, Qmax is the Langmuir parameter related to the maximum adsorption capacity, b is the constant related to adsorbent/analyte interaction forces, Kf is the Freundlich constant and is indicative of the degree of adsorption and the constant n is indicative of the heterogeneity of the surface of the solution. The relative values of Qmax, Kf and n calculated from the Langmuir and Freundlich models for the Mn(II) in the adsorbent are listed in Table I. Table I. Parameters of the adsorption isotherm models Langmuir

Freundlich

Qmax (mg g-1)

Kf (mg g-1)

10.35

0.24

0.9943

1.19

9.3

0.9651

As shown in Figure 4, when the Langmuir model was used to described the Mn(II) adsorption onto the adsorbent, the linear plot of Ce vs Ce/Qe, with a coefficient of determination of R2 = 0.9943, was obtained. When the Freundlich model was used, for the linear plot of log q vs log Ce the coefficient of determination was R2 = 0.9651.

Article

Selective Extraction of Manganese using Moringa oleifera Seeds as Bioadsorbent

Figure 4. Plots of the Langmuir isotherm for the sorption of Mn(II) onto Moringa oleifera seeds. Experimental conditions: pH = 3.0, adsorbent mass = 0.5 g, stirring time = 15 min.

The results showed that the value for the coefficient of determination (R2) of the Langmuir isotherm model was higher than that of the Freundlich model. Thus, the experimental data are well fitted to the linear Langmuir isotherm, agreeing with the finding when compared to the system proposed by Oscik [18]. In the Langmuir model it is assumed that the adsorption is limited to the monolayer and thus maximum adsorption indicates the saturation of this monolayer. The process probably occurs through chemical adsorption, basically by ion exchange and complexation, and the maximum adsorption capacity of the adsorbent for Mn(II) was calculated to be 10.35 mg g-1. The RL factor was calculated and its value indicates favorable adsorption (RL > 1). The good adsorption capacity of Moringa oleifera seeds for manganese is shown in Table II, where higher Qmax values can be observed when compared to results reported for the other natural adsorbents. Table II. Comparison of methods for the manganese ion removal Maximum Capacity (mg g-1)

Reference

Chitin + associated protein

5.44

[17]

Black carrot (Daucus carota)

3.87

[20]

Activated carbon (AC) derived from coconut shell

2.54

[21]

Tannic acid immobilized on AC

1.13

[22]

Moring oleifera seeds

10.35

This study

Adsorbent

Kinetic Studies Based on an analysis of Figure 2(b), it can be observed that manganese adsorption increases with increased contact time, reaching equilibrium. The main models used to evaluate the kinetic profile were the pseudo-first-order, pseudo-secondorder, and intraparticle diffusion kinetic models. For the fitting of these models, two criteria must be satisfied. The first is that the linearity must be acceptably high (R2). The second is that the calculated qe 34

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do Carmo, S. N.; de Sousa Neto, J. A.; Alves, V. N.; Coelho, L. M.; Coelho, N. M. M.

values must be close to the experimental qe values. Table III. Kinetic parameters for Mn(II) adsorption onto moringa seeds Pseudo-first-order model K1 (min-1)

qe exp. (mg g-1)

0.2218

1.075

0.637

Pseudo-second-order model K2 (g mg-1 m-1)

qe exp. (mg g-1)

0.27

4.05

0.999

Intraparticle diffusion model C

Kdif

2.58

0.073

0.994

The kinetic parameters for the adsorption of Mn(II) ions onto the Moringa oleifera seeds (Table III) were obtained by linear regression of the graphic models of pseudo-first-order, pseudo-second-order and intraparticle diffusion. The calculated qe was 0.42 and the q values determined experimentally are included in the table for comparison. It can be noted that the kinetics of the manganese ion adsorption onto the seeds could be fitted with a pseudo-second-order kinetic model because it fulfills the two criteria mentioned above. The C value (2.58) being different from zero indicates that the plot of qt vs t0.5 does not pass through the origin and therefore the intraparticle diffusion mechanism is not the rate determining step of the transfer process, and other bulk mechanisms must act simultaneously in the control of the adsorption process. Accuracy Tests The accuracy of the proposed method was evaluated by recovery tests applying the method of standard addition using water samples (tap water, mineral water and river water). By placing the samples in contact with moringa seeds under shaking, Mn(II) ions will be selectively retained and Mn(VII) will be free in the supernatant allowing its determination by F AAS. The Mn total concentration can be determined by direct analysis of the samples with the adsorbent without agitation, and thus the Mn(II) concentration can be calculated as the difference between the total Mn concentration and Mn(VII). Since the concentration of analyte was below the detection limit, samples were spiked with Mn(II) in concentrations of 4 and 50 mg L-1 and the results for the recovery, shown in Table IV, are within the acceptable range of 80 to 120%.

Article

Selective Extraction of Manganese using Moringa oleifera Seeds as Bioadsorbent

Table IV. Determination of Mn(II) in water samples and experimental recovery in water samples spiked with 4.0 and 50.0 mg L-1 Mn(II) Samples Mineral Water

Tap water

River water

Mn(II) spiked (mg L-1)

Mn(II) found (mg L-1)

Recovery (%)

N.D.

4.0

3.6 ± 0.2

50.0

52.1 ± 0.5

104

N.D.

4.0

3.7 ± 0.1

50.0

48.1 ± 0.3

N.D.

4.0

3.9 ± 0.2

50.0

51.0 ± 0.2

102

N.D.: not detectable. N=3.

The accuracy of the method was further evaluated by analysis of the drinking water certified reference material APS 1075. Table V shows the results obtained. These results are consistent with the reference value, confirming the reliability of the method. Table V. Determination of manganese in drinking water certified reference material Sample APS 1075

Proposed method

Certified Value

98.3 ± 1.0 mg L-1

99.4 ± 0.3 mg L-1

CONCLUSIONS The central point of the method developed in this study is the variation in the percentage adsorption of the inorganic species of Mn according to the pH, since the pH changes the surface charge of the adsorbent, influencing its ability to adsorb cations and anions. It was observed that at pH 3.0 only Mn(II) is retained, confirming the possibility of an extraction method with high selectivity. The good accuracy of the method was observed through recovery tests and analysis of reference material. It was verified that the method can be applied in the selective extraction of Mn(II) from water samples using Moringa oleifera seeds as a bioadsorbent. Manuscript submitted: March 25, 2019; revised manuscript submitted: June 24, 2019; revised for the 2nd time submitted: August 16, 2019; manuscript accepted: August 27, 2019; published online: September 27, 2019. REFERENCES 1. Clarke, C.; Upson, S. Neutoxicology, 2017, 58, pp 173-179. 2. Brown, S.; Taylor, N. L. Environ. Toxicol. Pharmacol, 1999, 7, pp 49-57. 3. Michalke, B. J. Chromatogr. A, 2004, 1050, pp 69-76. 4. Ozdemir, S.; Kilinc, E.; Poli, A.; Nicolaus, B.; Guven, K. Chem. Eng. J, 2009, 152, pp 195-206. 36

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5. Crossgrove, J. S.; Allen, D. D.; Bukaveckas, B. L.; Rhineheimer, S. S.; Yokel, R. A. NeuroToxicology, 2003, 24, pp 3-13. 6. Kazi, T. G.; Afridi, H. I.; Nazi, N.; Jamali, M. K.; Arain, M. B.; Jalbani, N.; Kandhro, G. A. Biol.Trace Elem. Res, 2008, 122, pp 1-18. 7. Gerber, G. B.; Léonard, A.; Hantson, P. Crit. Rev. Oncol. Hematol., 2002, 42, pp 25-34. 8. Tobiasz, A.; Soltys, M.; Kurys, E.; Domagala, K.; Dudek-Adamska, D.; Walas, S. Spectrochim Acta B, 2017, 143, pp 11-16. 9. Silva, C. A.; Silva, R. L. S.; Figueiredo, A. T.; Alves, V. N. J. Braz. Chem. Soc., 2019. In Press. 10. Alves, V. N.; Mosquetta, R.; Coelho, N. M. M.; Bianchin, J. N.; Roux, K. C. P.; Martendal, E.; Carasek, E. Talanta, 2010, 80, pp 1133-1138. 11. Martins, I.; Martins, I. V. Cadernos de Referência Ambiental, 2001, 57, pp 1-121. 12. Barka, N.; Abdennouri, M.; Boussaoud, A.; Makhfouk, M. Desalination, 2010, 258, pp 66-71. 13. Bhatti, H. N.; Mumtaz, B.; Hanif, M. A.; Nadeem, R. Process Biochem, 2007, 42, pp 547-553. 14. Marques, T. L.; Alves, V. N.; Coelho, L. M.; Coelho, N. M. M. Bioresource, 2013, 8, pp 2738-2751. 15. Tarley, C. R. T.; Andrade, F. N.; Santana, H.; Zaia, D. A. M.; Biejo, L. A.; Segatelli, M. G. React Func Polym, 2012, 72, pp 83-91. 16. Burriel, F.; Lucena, F.; Arribas, S.; Hernández, J. Química Analítica Cualitativa, 1985. 17. Robinson-Lora, M. A.; Brennan, R. A. Chem. Eng. J, 2010, 162, pp 565-572. 18. Ościk, J. Adsorption. John Wiley & Sons, Toronto, 1982. 19. Ndabigengesere, A.; Narasiah, K. S.; Talbot, B. G. Water Res.,1995, 29, pp 703-710. 20. Guzel, F.; Yakut H.; Topal G. J. Hazard. Mat., 2008, 153, pp 1275-1287. 21. Jusoh, A.; Cheng, W. M.; Low, N. A.; Megat, M. J.; Noor, M. Desealination, 2005, 182, pp 347-353. 22. Üçer, A.; Uyanik, A.; Aygün, S. F. Sep. Purif. Technol., 2006. 47, pp 113-118.

Article

Br. J. Anal. Chem., 2019, 6 (24) pp 38-46 DOI: 10.30744/brjac.2179-3425.AR-13-2019

Evaluation of the Quality of Formulations Containing Lactase (β-galactosidase) Employing Gel Electrophoresis and Cell Phone Bruna Soares Dionizio1, Diego Victor Babos2, Dulce Helena Ferreira de Souza1 Rodrigues Pereira-Filho2 1

, Edenir

Laboratório de Bioquímica Funcional e Estrutural, Departamento de Química, Universidade Federal de São Carlos — P. O. Box 676, São Carlos, SP, 13565-905, Brazil 2 Grupo de Análise Instrumental Aplicada, Departamento de Química, Universidade Federal de São Carlos P. O. Box 676, São Carlos, SP, 13565-905, Brazil

Graphical Abstract

A quantification method of the lactase enzyme in pharmaceutical formulations is proposed employing digital images obtained from a cell phone camera and processed using the colorimetric application. The use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) made it possible to identify the lactase enzyme, to fractionate possible protein impurities and to evaluate the degradation of lactase present in the analyzed formulations. In addition, gel electrophoresis made it possible to determine lactase free of matrix interference. The proposed method presented limit of quantification of 0.002 mg lactase (0.03 FCC), using external calibration. Recoveries of lactase added to the samples were in the range 78-104%. The concentrations found in different pharmaceutical formulations samples containing lactose were in the range 7-621 mg (104-9319 FCC). The relative standard deviations (RSD) were in the range of 3-13%. Of the six samples analyzed, only two (#S2 and #S5) were in agreement with the sample label description. Keywords: lactase, digital image, PhotoMetrix, lactose intolerance, gel electrophoresis

Evaluation of the Quality of Formulations Containing Lactase (β-galactosidase) Employing Gel Electrophoresis and Cell Phone

Article

INTRODUCTION Lactase (β-galactosidase, Enzyme Commission number 3.2.1.23) belongs to the class of hydrolytic enzymes which catalyze the conversion of lactose to glucose and galactose [1]. Several people cannot or inefficiently metabolize lactose, developing lactose intolerance. This syndrome affects approximately 70% of the world’s population, mainly American Indians and Asians [2,3]. The main symptoms resulting from lactose intolerance are gastrointestinal clinical manifestations, such as abdominal distension and pain, diarrhea, nausea, flatulence and abdominal discomfort [3,4]. Thus, the food and pharmaceutical industries are very interested in the production of the lactase enzyme, since it can be used in the production of lactose free food, such as milk and dairy products, and also as a food supplement, used to prevent unwanted symptoms caused by intolerance to lactose. Lactase is usually industrially produced, using the fungi Aspergillus niger, Aspergillus oryzae, Kluyveromyces lactis and Kluyveromyces marxianus, because they can have acceptable productivities and yields of lactase. However, lactase can also be obtained by different microorganisms such as bacteria and yeasts [1,5,6]. Pharmaceutical formulations containing lactase are classified as food supplements according to the Food and Drugs Administration (FDA), so can be submitted to quality, efficacy and stability testing [7,8]. Thus, it is possible that some commercially available formulations may contain irregularities as to their content and enzymatic activity. The pharmaceutical formulations express the lactase content in lactase FCC unit (FCC - Food Chemical Codex Units), and are usually in the range from 3,000 to 10,000 FCC [9]. The lactase-containing formulations present some limitations, since it is reported in the literature that the enzyme has low physicochemical stability and reduced shelf life, although many technologies are proposed to overcome these drawbacks [4,10]. Its exposure to drastic conditions such as extremes pH and temperature can lead to enzymatic inactivation due to the formation of insoluble particles and aggregates, as well as protein unfolding and degradation [11,12]. Thus, it is important to monitor these products for the quality control and efficacy of commercially available supplements containing lactase. Analytical methods for the determination of proteins usually require sample preparation and quantification. The most used methods for the determination of proteins employ molecular absorption spectrophotometry in the UV-Vis region (for example Bradford method) [13,14], liquid chromatography coupled with mass spectrometer [15,16,17], electrochemical methods [18], densitometry [19,20], fluorescence spectroscopy [21], among others. These techniques present certain difficulties in their implementation, such as lack of selectivity, sensitivity, and possible matrix effects. The polyacrylamide gel electrophoresis (PAGE) or Agarose gels are widely used in microbiological assays for separation and identification of proteins and DNA, respectively; besides being simple, effective and low-cost methods. SDS-PAGE processing method consists in the separation of proteins under denaturing sodium dodecyl sulfate (SDS), reducing with β-mercaptoethanol or even heating conditions. The proteins are heated with SDS before electrophoresis so the charge-density of all is made roughly equal. Consequently, when these samples undergo electrophoresis, proteins separate according to their molecular mass [22-25]. The development of methods employing colorimetric detection with unusual devices as cell phones, digital cameras, computers, scanners, webcams, among others is a reality that is increasingly arousing the interest of the scientific community [26-32]. Simplicity in the acquisition of analytical signals (image), and the possibility to perform the data processing in the device is one of the great advantages of employment [27,28,33,34]. In this context, the goal of this study is to develop a procedure for the analysis of pharmaceutical formulations containing lactase, using gel electrophoresis as a method of protein fractionation and processing digital images, obtained from a cell phone through the application PhotoMetrix. In addition, the proposed analytical method could be implemented as a quality control tool in pharmaceutical formulations containing lactase, being an affordable and inexpensive alternative.

Article

Dionizio, B. S.; Babos, D. V.; de Souza, D. H. F.; Pereira-Filho, E. R.

MATERIALS AND METHODS Instrumentation Bio-Rad Mini-Protean® Tetra system (Biorad, California, USA) were employing for the SDS-PAGE electrophoresis. The system can operate up 500 W and 600 V. The image data were captured by the camera of a cell phone SM-J500M Samsung (Samsung, Seoul, South Korea), camera with 13 MP (megapixels), 5 MP frontal (flash frontal LED). The image data were processed using PhotoMetrix software, freely available in http://www.photometrix.com.br/ [35]. Reagents, Analytical Solutions and Samples All solutions were prepared using high purity water (18.2 MΩ cm resistivity) obtained from a Milli-Q® Plus Total Water System (Millipore Corp., Bedford, MA, USA). All glassware and polypropylene vessels were decontaminated prior to use by detergent washing and then thorough rinsing with distilleddeionized water. Lactase 15,000 FCC g-1 (Fagron, São Paulo, Brazil) was used to prepare standard solutions for calibration curve and addition recovery test. Each 1 g of the lactase standard is equivalent to approximately 15,000 lactase units FCC. The sample buffer was prepared with Tris-HCl 0.188 mol L-1, pH 6.8 (tris(hydroxymethyl) aminomethane, ≥ 99.0%, Sigma-Aldrich, St. Louis, USA), glycerol 30% v/v (≥ 99.0%, Sigma-Aldrich, St. Louis, USA), β-mercaptoethanol 15% v/v (98.0%, Sigma-Aldrich), Sodium dodecyl sulfate – SDS 6% w/v (C12H25NaO4S, ≥ 98.5%, Sigma-Aldrich), and bromophenol blue 0.01% w/v (C19H10Br4O5S, ≥ 99.0%, J.T. BAKER, Phillipsburg, USA). The protein molecular weight standard employed was in the range of 116 - 14.4 kDa (Thermo Scientific, Lithuania). The SDS-PAGE were composed of two parts called concentrator or stacking gel (upper gel, pH 6.8) and gel separator or running (lower gel, pH 8.8). These gels were prepared using bis-acrylamide (bis (N,N’-Methylene-bis-acrylamide) 0.8% w/v, Bio-Rad and acrylamide 30% w/v, ≥ 99.0%, Sigma-Aldrich), Tris-HCl, ammonium persulphate 10% w/v ((NH4)2S2O8, ≥ 99.0%, Synth, Diadema, Brazil), Sodium dodecyl sulfate – SDS 10% w/v (C12H25NaO4S, ≥ 98.5%, Sigma-Aldrich) and TEMED® (N,N,N’,N’tetramethylethyl-enediamine, 99.0%, Sigma-Aldrich) [22]. The SDS-PAGE buffer Tris Base 25 mmol L-1, Glycine 192 mmol L-1, (C2H5NO2, ≥ 99.0%, SigmaAldrich) and SDS 0.1% w/v, was prepared for running the gel [22]. The solution used for staining the gel was Coomassie Brilliant Blue R 250 0.15% w/v (C45H44N3O7S2Na, Sigma-Aldrich), methanol 50% v/v (CH4O, ≥ 99.8%, Synth), and acetic acid 10% v ̸ v (C2H4O2, ≥ 99.7%, Synth). The gel was discolored by diffusion in a solution of acetic acid 7% v/v. Six samples of pharmaceutical formulations of lactase were analyzed using the proposed method. These samples were donated by patients from São Carlos city (São Paulo, Brazil), who use these supplements daily to control lactose intolerance. Analytical Procedure Sample and Standards Preparation The tablets of samples containing lactase were homogenized in mortar and pistil. Subsequently, approximated 50 mg of the sample were transferred to a flask and solubilized in 10 mL high purity water. The solutions obtained were refrigerated at 4 °C until analysis. Before the analysis, the samples were mixed with the buffer in the proportion of 3:1 (sample:sample buffer), and heated to 100 °C in a dry bath for 5 min, for denaturation. A standard solution containing 10 mg mL-1 lactase (150 FCC mL-1 lactase) was prepared using 100 mg lactase solubilized in 10 mL high purity water. Aliquot of standard solution was diluted in sample buffer at the ratio 3:1 (80 µL standard solution:26.6 μL sample buffer) resulting in a 7.5 mg mL-1 lactase solution which was heated to 100 °C in a dry bath for 5 min, for denaturation and analysis. The standard 40

Evaluation of the Quality of Formulations Containing Lactase (β-galactosidase) Employing Gel Electrophoresis and Cell Phone

Article

curve was prepared with aliquots of this solution, in the 0-0.38 mg lactase range. The first standard solution of the calibration curve, corresponding to a concentration of 0 mg, refers to the blank. Gel Preparation The comb was carefully removed from the gel, wells were rinsed with deionized water and the gel mounted in the apparatus Bio-Rad Mini-Protean® Tetra system. The SDS-PAGE buffer was added to the bottom and the top reservoirs. Samples aliquots were placed in the bottom of the wells in the sampling the gel using pipette. For qualitative analysis and verification of impurities in the pharmaceutical formulations containing lactase, 20 µL of all samples were placed in the bottom of wells of sampling the gel. For quantitative analysis, aliquots of 10 µL of the samples #S1, #S2 and #S3, and 5 µL of the samples #S4, #S5 and #S6, were employed. Different aliquots (2.5, 5, 10, 15, 20, 30, 40 and 50 µL) of a standard solution containing 7.5 mg mL−1 lactase were used as standards for analytical curve. For to stack all the samples on the gel were used 80 V. The run was carried out at 120 V for 120 min until the bromophenol blue had reached the lower edges of the gel. Staining the Gel with Coomassie Blue The gel was stained with solution of coomassie blue 0.15% w/v for 5 min and under stirring. The gels were destained by diffusion a solution of acetic acid 7% v/v for 12 h. The blue staining remains only in the protein bands because this is the result of the reaction between the sulfonic groups of the dye and the amine groups of the proteins. Image Data Collection and Quantification of Lactase by Colorimetry on Cell Phone Devices After the discoloration of the gels, samples and standard images were acquired using PhotoMetrix software, according to Figure 1. The images (n = 5) for the lactase protein band on the 90 kDa gel were focused using the camera of the cell, at a distance of 18 cm, and acquired using a 32x32 pixel spot. The camera flash and 640x480 pixels resolution were used.

Figure 1. Acquisition of image data for determination of lactase by proposed method.

Univariate calibration employing multichannel red (R), green (G), blue (B), hue (H), intensity (I), value (V), saturation (S) and lightness (L) was evaluated for the quantification of lactase. The best calibration obtained, with a better linear correlation coefficient and better trueness obtained from the 41

Article

Dionizio, B. S.; Babos, D. V.; de Souza, D. H. F.; Pereira-Filho, E. R.

addition/recovery tests, was chosen for the quantification of lactase in commercial formulations. Figures of Merit The limits of detection (LOD) and quantification (LOQ) of the method proposed were calculated according to IUPAC recommendations: 3 x SDblank/b (LOD), and 10 x SDblank/b (LOQ), where SD is the standard deviation for ten blank measurements (using bleached electrophoresis gel with coomassie blue) and b is the angular coefficient of the calibration curve [36]. Addition / Recovery Tests Accuracy of the proposed method was also evaluated by means of addition / recovery tests. A spike with 0.113 mg lactase was added in all samples analyzed. The trueness was calculated from equation 1:

Eq. 1

where j is the mass determined by the proposed method and jref is the reference mass of the lactase added (spike). RESULTS AND DISCUSSION Evaluation of Impurities and Degradation in Formulations Containing Lactase Employing SDSPAGE Different microorganisms (fungi, bacteria and yeasts) and industrial purifications routes can be used to obtain lactases that may present significant differences in their structures and physicochemical properties resulting from different mechanisms of production. For example, lactase from Aspergillus oryzae is a monomeric enzyme having a molecular mass of 90 kDa, does not require cofactors and has an optimal action pH of 4.5 [10]; but the lactase from Lactobacillus acidophilus has a molecular mass of 540 kDa, needs magnesium as cofactor and presents a optimum pH in 6.2-6.6 [37]. All samples analyzed in this study state in their labels that the lactase used in the formulations originated from the fungus A. oryzae. The lactase produced by this fungus requires relatively inexpensive industrial procedures, but they are more unstable enzymes [11,12,38]. To analyze the purity of the lactase in the formulations, electrophoresis was used in denaturing gel (SDS-PAGE) (Figure 2).

Figure 2. Evaluation of impurities and possible degradation of lactase in pharmaceuticals formulations by SDS-PAGE. Molecular Weight Standard (MWS) and Sample (S). 42

Evaluation of the Quality of Formulations Containing Lactase (β-galactosidase) Employing Gel Electrophoresis and Cell Phone

Article

It is possible to observe in the gel different protein bands, ranging from 18 to 116 kDa, thus indicating that the formulations do not present only the lactase, as expected. The various bands observed on the gel demonstrate that different proteins are present, even after purification steps employed in obtaining lactase. Samples #S3, #S4, #S5 and #S6 show an intense band at about 90 kDa, molecular mass expected for lactase from A. oryzae, being in agreement with the descriptions on the labels. Sample #S2, although slightly above 90 kDa, also can be inferred to lactase from A. oryzae. This difference may be related to the mode of expression of the protein, since the manufacturers do not give details of how these proteins were expressed and purified. If the proteins are recombinant, these can be expressed fused with tags like GST (Glutathione S-transferase), MPB (Maltose-binding protein), His-Tag (Poly-histidine-tagged) [1,6,39], among others and thus the proteins may have molecular mass just above 90 kDa. However, the sample #S1 showed two strong protein bands in the range of 50 to 70 kDa. This may indicate the degradation of the enzyme lactase in this analyzed formulation, since a protein band around 90 kDa was expected, molecular weight of the lactase expressed by A. oryzae. The instability and enzymatic degradation of lactase can be due to several factors as inadequate exposure of the formulations, by the users and or manufacturers, high temperature that can lead to the enzymatic inactivation due to the formation of insoluble particles and aggregates, besides unfolding and protein degradation [4,10-12]. Therefore, these factors may justify what was observed in the analysis of sample #S1. Figures of Merit The main analytical performance parameters (linear range, linear correlation coefficient, LOD, LOQ and precision) calculated for the method proposed are shown in Table I. Among the eight evaluated channels (R, G, B, H, I, V, S and L channel) in the univariate mode, the best calibration curve was obtained using the channel B (blue) with a linear correlation coefficient 0.993, for this reason it was used for lactase determinations, Figure 3. The precision (n=5) was calculated using all samples and RSD varied from 3 to 13%. Overall, the values obtained were considered satisfactory for further development of the analytical methods for determination of lactase in pharmaceuticals formulations employing colorimetry and mobile device. Table I. Parameters of analytical performance for the determination of lactase employing colorimetric on mobile devices proposed method Parameters

Found

Channel

B (blue)

Linearity range (mg ̸ FCC) Calibration curve equation (linear model) Linear correlation coefficient (R) Limit of detection (mg ̸ FCC) Limit of quantification (mg ̸ FCC) RSD (%)

0 – 0.38 ̸ 0 – 5.6 y = 119.82x + 91.67 0.993 0.0006 ̸ 0.009 0.002 ̸ 0.03 3 – 13

Article

Dionizio, B. S.; Babos, D. V.; de Souza, D. H. F.; Pereira-Filho, E. R.

Figure 3. Fragmented chromatograms of different mass (0 - 0.38 mg) of standard lactase (90 kDa) on gel electrophoresis along with molecular weight marker proteins and calibration curve for lactase.

The LOD value calculated for the method proposed in the determination of β-galactosidase (LOD = 0.6 μg), was similar to the values reported in the study developed by Jesus et al. (2019) [26] for the quantification of the albumin (LOD = 0.6 μg), trypsin inhibitor (LOD = 0.7 μg) and carbonic anhydrase (LOD = 0.7μg) proteins in human serum sample, both using SDS-PAGE and PhotoMetrix. These values demonstrate the potential of using these tools in the proteomics field. Addition / Recovery Tests Further tests were performed to verify the accuracy of the analytical method developed for the determination of the lactase. The tests were made for all lactase samples analyzed spiked with 0.113 mg enzyme. The trueness of lactase added to the samples varied within the range 78-104% (Table II), demonstrating the accuracy in the measurements for all samples. Determination of Lactase in Pharmaceutical Formulations The proposed method was applied in the analysis of six formulations containing lactase in the range 4,000-10,000 FCC, and the results are shown in Table II. The measured values were in the range 7-621 mg lactase or 104-9319 FCC. The specifications of most tested pharmaceutical formulations samples were not consistent with the information described on the labels. The determined values for the lactase are not in agreement with the label specifications of formulations #S1, #S3, #S4 and #S6. Only formulations #S2 (3875 ± 168 FCC) and #S5 (9319 ± 316 FCC) are in concordance with the information described on the labels (#S2 – 4,000 FCC and #S5 – 10,000 FCC). The lack of agreement of the lactase values described in the commercial formulations with those determined by the proposed method may be associated with several factors mentioned above, but mainly protein degradation.

Evaluation of the Quality of Formulations Containing Lactase (β-galactosidase) Employing Gel Electrophoresis and Cell Phone

Article

Table II. Determination of lactase (mean ± standard deviation, n = 5) in commercial pharmaceutical formulations by proposed method. Trueness (%) of lactase compared to sample label description and spiked, for all samples. Trueness (%)

Addition / recovery test

7 ± 0.23

104

3875 ± 168

258 ± 11

4500

1692 ± 227

113 ± 15

9000

5140 ± 319

343 ± 21

10000

9319 ± 316

621 ± 21

10000

7635 ± 187

509 ± 11

Sample

Sample label description (FCC)

FCC

4000

104 ± 3

4000

Method proposed

Trueness (%)

In this way the proposed method proves to be an excellent tool to evaluate the quality of the commercial formulations containing lactase, using a method that employs as the analytical technique a mobile device - cell phone. The use of SDS-PAGE and the processing of the digital images acquired with the camera of cell phone by PhotoMetrix software, allow to quantify proteins with sensitivity (microgram levels), accuracy and precision satisfactory. A difficulty encountered in the development of this method was in the acquisition of the calibration standard for β-galactosidase enzyme with adequate specifications (e.g. purity) to be employed. CONCLUSIONS The proposed method based on colorimetric devices and electrophoresis was effective for the determination of lactase in different supplements containing this enzyme. The electrophoresis under denaturing conditions allowed to identify the lactase-related band and the separation of the analyte from possible interferences present in the sample matrix. SDS-PAGE proved to be an excellent analytical tool in the identification of protein impurities and making possible the determination of the concentration of lactase in the formulations using the mobile application. The use of digital images obtained by a cell camera together with the use of Photometrix software, allowed to develop a simple and inexpensive method for determination of lactase. The analytical method should be useful as a tool for assessing the quality of supplements marketed containing lactase, since the monitoring of the enzyme in these formulations may contribute to the efficacy of the supplements consumed. Manuscript submitted: April 18, 2019; revised manuscript submitted: July 8, 2019; manuscript accepted: August 8, 2019; published online: September 27, 2019. REFERENCES 1. Katrolia, P.; Yan, Q.; Jia, H.; Li, Y.; Jiang, Z.; Song, C. J Mol Catal B Enzym., 2011, 69, pp 112-119. 2. Leonardi, M.; Gerbault, P.; Thomas, M. G.; Burger, J. Int Dairy J., 2012, 22, pp 88-97. 3. Swagerty, D. L. Jr.; Walling, A. D.; Klein, R. M. Am Fam Physician., 2002, 65, pp 1845-1850. 4. Traffano-Schiffo, M. V.; Castro-Giraldez, M.; Fito, P. J.; Santagapita, P. R. Food Res Int., 2017, 100, pp 296-303. 5. Khaled, B.; Fethia, F. N.; Kemal, G.; Matpan, B. F.; Omer, A.; Amina, H. Res J Biotechnol., 2016, 11, pp 40-48. 45

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6. Ren, Z. Y.; Liu, G. L.; Chi, Z.; Han, Y. Z.; Hu, Z.; Chi, Z. M. Process Biochem., 2017, 61, pp 38-46. 7. Blendon, R. J.; DesRoches, C. M.; Benson, J. M.; Brodie, M.; Altman, D. E. Arch Intern Med., 2001, 161, pp 805-810. 8. https://www.fda.gov/consumers/consumer-updates/fda-101-dietary-supplements [Assessed on 27 August, 2019]. 9. https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2009.1236 [Assessed on 22 April, 2018]. 10. Gennari, A.; Mobayed, F. H.; Volpato, G.; de Souza, C. F. V. Int J Biol Macromol., 2018, 109, pp 303-310. 11. Nichele, V.; Signoretto, M.; Ghedini, E. J Mol Catal B Enzym., 2011, 71, pp 10-15. 12. Ohtake, S.; Kita, Y.; Arakawa, T. Adv Drug Deliv Rev., 2011, 63, pp 1053-1073. 13. Ku, H. K.; Lim, H. M.; Oh, K. H.; Yang, H. J.; Jeong, J. S.; Kim, S. K. Anal Biochem., 2013, 434, pp 178-180. 14. Zhang, K.; Cai, R.; Chen, D.; Mao, L.; Anal Chim Acta., 2000, 413, pp 109-113. 15. Halvorsen, T. G.; Reubsaet, L. Trends Analyt Chem., 2017, 95, pp 132-139. 16. Vilà-Rico, M.; Colomé-Calls, N.; Martín-Castel, L.; Gay, M.; Azorín, S.; Vilaseca, M.; Planas, A.; Canals, F. J Proteom., 2015, 127, pp 234-246. 17. Magri, A.; Soler, M. F.; Lopes, A. M.; Cilli, E. M.; Barber, P. S.; Pessoa-Junior, A.; Perreira, J. F. B. Anal Bioanal Chem., 2018, 410, pp 6985-6990. 18. Shahmiri, M. R.; Bahari, A.; Karimi-Maleh, H.; Hosseinzadeh, R.; Mirnia, N. Sens Actuators B Chem., 2013, 177, pp 70-77. 19. Muharram, M. M.; Abdel-Kader, M. S. Saudi Pharm J., 2017, 25, pp 359–364. 20. Ahmadifar, S.; Le, T. C.; Marcocci, L.; Pietrangeli, P.; Mateescu, M. A. Anal Chim Acta., 2017, 975, pp 78-85. 21. Wang, K.; Donnarumma, F.; Baldone, M. D.; Murray, K. K. Anal Chim Acta., 2018, 1027, pp 41-46. 22. Laemmli, U. K. Nature 1970, 227, pp 680-685. 23. Reddy, P. R.; Raju, N. Gel-electrophoresis and its applications. In: Magdeldin, S. (Ed.). Gel Electrophoresis - Principles and Basics. Rijeka: InTech. 2012. 24. Sim, J. Z.; Nguyen, P. V.; Lee, H. K.; Gan, S. K. E. Nat. Methods Appl Not., 2015, pp 1-2. 25. Nguyen, P. V.; Ghezal, A.; Hsueh, Y.; Boudier, T.; Gan, S. K.; Lee, H. K. Electrophoresis, 2016, 37, pp 2208-2216. 26. Jesus, J. R.; Guimarães, I. C.; Arruda, M. A. Z. J Proteom., 2019, 198, pp 45-49. 27. Grudpan, K.; Kolev, S. D.; Lapanantnopakhun, S.; McKelvie, I. D.; Wongwilai, W. Talanta, 2015, 136, pp 84-94. 28. Kwon, L.; Long, K. D.; Wanc, Y.; Yua, H.; Cunninghama, B. T. Biotechnol Adv., 2016, 34, pp 291304. 29. Wang, H.; Sun, Y.; Yue, W.; Kang, Q.; Li, H.; Shen, D. Analyst, 2018, 143, pp 1670-1678. 30. Bao, X.; Jiang, S.; Wang, Y.; Yu, M.; Han, J. Analyst, 2018, 143, pp 1387-1395. 31. Malhotra, K.; Noor, M. O.; Krull, U. J. Analyst, 2018, 143, pp 3049-3058. 32. Morbioli, G. G.; Mazzu-Nascimento, T.; Stockton, A. M.; Carrilho, E. Anal Chim Acta., 2017, 970, pp 1-22. 33. Helfer, G. A.; Magnus, V. S.; Böck, F. C.; Teichmann, A.; Ferrão, M. F.; da Costa, A. B. J Braz Chem Soc., 2017, 28, pp 328-335. 34. Shen, H.; Khan, R.; Wang, X.; Li, Z.; Qu, F. Anal Bioanal Chem., 2018, 410, pp 7177-7183. 35. http://www.photometrix.com.br [Assessed on 10 February, 2018]. 36. Currie, L. A.; Anal Chim Acta, 1999, 391, pp 105-126. 37. Harju, M.; Kallioinen, H.; Tossavainen, O. Int Dairy J., 2012, 22, pp 104-109. 38. Yang, S.; Okos, M. R. O. Biotechnol Bioeng., 1989, 33, pp 873-885. 39. Paraskevopoulou, V.; Falcone, F. H. Microorganisms, 2018, 6, pp 47-63. 46

Br. J. Anal. Chem., 2019, 6 (24) pp 47-62

Article

DOI: 10.30744/brjac.2179-3425.AR-14-2019

Micelar-Electrokinetic Chromatography Separation of Nitrogen-Containing Aromatic Compounds in Diesel Prepared as Microemulsion Anastácia Silva Canto1,2 Alessandra Licursi Maia Cerqueira da Cunha3* Selma Cunha Mello4, Ricardo Queiroz Aucelio2 Instituto de Química, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Rodovia BR 465 Km 07, Zona Rural – Seropédica, Rio de Janeiro, Brazil 2 Departamento de Química, Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Rua Marquês de São Vicente, 225 - Gávea, Rio de Janeiro, Brazil 3 Departamento de Química, Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro (IFRJ), Rua Senador Furtado, 121/125 – Maracanã, Rio de Janeiro, Brazil 4 Centro de Pesquisas Leopoldo Américo Miguez de Mello – Petrobras, Gerência de Química, (CENPES/PDISO), Av. Horácio Macedo, 950 - Cidade Universitária, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil 1

Graphical Abstract

Steps for identification and quantification of nitrogen-containing aromatic compounds by capillary electrophoresis in diesel samples, using a stable and homogeneous microemulsion without detergent as a tool.

Nitrogen-containing aromatic compounds (NCACs) are present in petroleum fractions such as diesel. An exploratory study aiming the separation of twelve NCACs of different types (carbazoles, indoles, quinolines, acridine and aniline) by micellar-electrokinetic chromatography (MEKC) was made using micro-emulsified diesel sample and pyrrole as the internal standard. Diesel (previously dissolved in isooctane) was prepared as detergentless microemulsion and then mixed with a surfactant-containing electrolyte solution in order to prepare the microemulsion to be introduced into the capillary. The system BGE was composed by sodium dodecyl sulfate in a borate buffer (10 mmol L-1; pH 9.50), containing acetonitrile and urea as chemical modifiers. Separation of the NCACs was achieved using a gradient of applied voltages (10 kV up to 20 min and then 30 kV up to the end of the run) at 15 ºC. Instrumental limits of detection (LOD) varied from 0.7 to 4.9 mg L-1 depending upon the analyte. LOD in diesel samples depend upon the dilution factor. Diesel samples (fortified with 12 NCACs) were analyzed, 47

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Micelar-Electrokinetic Chromatography Separation of Nitrogen-Containing Aromatic Compounds in Diesel Prepared as Microemulsion

enabling recoveries varying from 85 to 106%. Commercial diesel samples were analyzed and some NCACs were detected in some of the samples and effectively quantified (most carbazoles) in two of them at levels varying from 2.4 to 11.8 g L-1 level. The study has shown the possibility of separating NCACs directly in complex diesel samples providing a way to screening samples for the ones presented at g L-1 levels. Keywords: Diesel; Microemulsion; Nitrogen-containing aromatic compounds; Micellar-electrokinetic chromatography. INTRODUCTION Petroleum is the major energy source in the world and it is the prime raw material for a wide range of products, including fuels such as gasoline and diesel. Among the most relevant contaminants in petroleum are sulfur, oxygen and nitrogen containing compounds [1-3]. In most advanced countries, environmental law severely restric the sulphur content in petroleum derivatives, but for nitrogencontaining compounds, the critical limit is mostly based on information about the long-term stability of the final products rather than based on concerns about emission of pollutants. Little is availabe about the nitrogen-containing aromatic compounds (NCACs). Nitrogen-compounds in crude oil accounts for 0.1 to 2% (w/w) of whole composition. Even at such concentrations they poison cracking catalysts, induce the formation of gum in fuels and produces nitrocompounds and NOX due to incomplete combustion [4]. A fraction of the nitrogen-compounds that compose petroleum are NCACs that concentrate in the higher boiling fractions of petroleum distillates. These substances are generally classified as basic (pyridine, quinoline, indoline and benzoquinoline) and non-basic (pyrrole, indole, carbazole and benzocarbazole) [5,6]. Scientific efforts have been made to screen, identify, quantify and remove NCACs from petroleum derivatives. There are several methods developed for the qualitative detection of NCACs, also for their quantitative determination, varying from those used for screening samples, for identification or quantification of NCACs grouped into classes, to those more sophisticated able to quantify individual isomers. The determination of non-basic nitrogen compounds in gasoline and in diesel was accomplished by differential pulse voltammetry, using a glassy carbon electrode and after solid phase extraction (SPE) allowing limits of detection (LOD) in the Âľg L-1 [7,8]. Quinolines, carbazoles, indoles and anilines along other NCACs were identified by mass spectrometry (MS) in extracts of residual fluid catalytic cracking diesel after column chromatographic separation of the sample fractions [9]. A qualitative evaluation using gas chromatography (GC) coupled to MS finding quinoline, indole and carbazole derivatives in atmospheric gas oil while aniline, indole and carbazole derivatives were found in light cycle oil [10]. GC determination of methylbenzo[c]acridines were achieved after oxidation to formylbenzo[c]acridines followed by reaction with p-fluoroaniline to form Schiff bases that were detected (down to 20 pg) by the electron-capture detector [11]. However, fuel samples were not analyzed. A fingerprinting study, involving diesel samples from three different countries, was made using sample extraction procedures and GC-MS [12]. Pyridine, pyrrole, indoles, quinolones and carbazoles were found. GC after sample extraction procedures was used with chemometrics to characterize nitrogen compounds in crude oils [13]. Two-dimensional GC with nitrogen specific detector [14-16] and with time-of-flight or quadrupole separation and MS detection [17,18] was used to characterize and quantify nitrogen compounds in diesel, heavy gas oil and middle distillate fractions using different sample extraction procedures. Sulfur and nitrogen containing aromatic compounds in shale oil were determined using two dimensional GC with a combination of four different detection systems [19]. A complex protocol was established using different sample preparation procedures depending on the target analytes (with discriminating capability for homologues). Among the NCACs found in shale oil were: indoles (1.11%), quinolines (0.60%), anilines (0.47%), acridines (0.03%) and carbazoles (0.47%). Laser desorption ionization Fourier-transform ionic resonance cyclotron coupled with fast quadrupole 48

Canto, A. S.; da Cunha, A. L. M. C.; Mello, S. C.; Aucelio, R. Q.

Article

MS detector has also been used to determine nitrogen compounds in diesel fuel and in seventy Brazilian crude oil samples [20]. The basic nitrogen content varied from 0.016 to 0.151%. Alkyl indoles, alkyl benzoquinolines and alkyl carbazoles (in the mg kg-1 levels) have been found in acid extracts of Brazilian diesel oil using high performance liquid chromatography (HPLC) with both MS and molecular absorption photometric detection [21,22] and by particle beam LC-MS [23]. Benzoquinolines were found to be the main NCAC group in the basic fraction, with homologues being further separated using a neutral mobile phase. HPLC with fluorimetric detection was used for the determination of six basic azaarenes in jet fuel, reaching absolute LOD values between 2 and 21 pg [24]. Neutral and basic fractions were extracted by SPE with a strong cation exchange sorbent. Oxygen and nitrogen containing aromatic compounds were determined in asphalt mixtures using HPLC-MS after sample fractionation into asphaltenes and maltenes and further liquid extraction [25]. Quinoline was quantified in the acid extract (102.2 mg kg-1) and in the basic extract (63.6 mg kg-1). There is little information on the analysis of petroleum samples using capillary electrophoresis (CE) aiming NCACs. The only reported quantitative work was the one of Luz et al. (2014) [26] concerning the determination five acridines in diesel after SPE, using cationic solid phase, to get the analytes in acidic aqueous phase before separation by capillary zone electrophoresis, using an acidic background electrolyte, and detection by molecular absorption photometry. The concentration of the analytes into the capillary was achieved through analyte stacking, enabling LOD values at the mg L-1 level. For NCACs in other types of samples, two works can be identified. Guarguilo et al. (2000) [27] have separated indoles, carbazoles, acridines and benzoquinolines, along with 16 polycyclic aromatic hydrocarbons, in soil extracts using a capillary packed with octyldecylsilica and laser-induced fluorescence detection (LOD down to 0.4 nmol L-1). Fomin et al. (2010) [28] identified basic NCACs in urine and blood using CE with UV absorption photometric detection but no quantitative application was accomplished. The analysis of fuels usually requires tedious and labor-intensive procedures to separate aromatic compounds from other matrix components. Therefore, any effort to minimize complex protocols is relevant even if it involves some degradation of detection power. In this direction, the dispersion of liquid fuel samples into microemulsions brings advantages for the determination of analytes directly in samples that are immiscible with water. Due to the thermodynamic stability and homogeneity of microemulsions, prepared by mixing oily liquid and water with the aid of a surfactant and/or a co-solvent, the introduction of the oily liquid samples is facilitated and the formation of sample zones, for instance in reverse liquid chromatography, becomes feasible [29]. Sample dispersing into microemulsion reduces the organic load of the analyzed material, avoids analyte losses due to mass transferring of thick samples, minimizes sample contamination and improves the reproducibility of results since variations associated to extraction procedures are eliminated. In recent work, the analysis of diesel was accomplished by HPLC by introducing the whole diesel sample prepared as detergentless microemulsion (or DME) into the aqueous mobile phase. Ten basic and neutral NCACs were determined (fluorescence detection), at the mg L-1 level, in one single chromatographic run [29]. Segura-Carretero et al. (2000) [30] have taken advantage of microemulsions to disperse water insoluble analytes into an aqueous system with composition adjusted to enable phosphorescence measurements. A small amount of samples was prepared as microemulsions composed by a nonpolar solvent, alcohol, water and a detergent (sodium dodecyl sulfate or SDS) [30]. A few reports indicate the use of detergentless microemulsion to perform the analysis of petroleum derivatives aiming the quantification of metals and metalloids by optical emission spectrometry and voltammetry [31,32]. In the present work, a microscale study was made aiming the electrophoretic separation of 12 NCACs in diesel samples. The successful separation of carbazole (CBZ), 9-methylcarbazole (9MC), 3-ethylcarbazole (3EC), 9-ethylcarbazole (9EC), quinoline (QNL), benzo[h]quinoline (BhQ), indole (I), acridine (ACR), 2-methylindole (2MI), 3-methylindole (3MI), 7-methylindole (7MI) and N,N-dimethylaniline (A) (see NCACs structures in Figure 1) was accomplished using micellar-electrokinetic chromatography (MEKC) and sample directly introduced as a microemulsion to guarantee compatibility of the fuel and 49

Article

Micelar-Electrokinetic Chromatography Separation of Nitrogen-Containing Aromatic Compounds in Diesel Prepared as Microemulsion

the aqueous background electrolyte. No need for prior separation of basic and neutral fractions of NCACs from the sample was necessary. The screening character of the method was demonstrated in the analysis of real samples.

Figure 1. Molecular structures of NCACs included in this work.

MATERIALS AND METHODS Instrumentation Micellar electrokinetic chromatography (MEKC) was made on a HP 3DCE capillary electrophoresis system (Agilent, USA) operating in normal polarity mode with a UV-vis diode array type absorption photometric detector. The pH measurements were made on a pHmeter (MS Tecnopon, model MPA-210, Brazil) with a glass membrane electrode conjugated with an Ag/AgCl(KClsat) reference electrode. A highperformance dispersion device - Ultra-Turrax IKA T18 (IKA, Brazil) - was used for the homogenization of the detergentless microemulsion. The determination of the C, H and N content in samples was made on Flash EA 1112 elemental analyzer (Thermo Electron Co). Microscopic pictures of the microemulsion was made on a Zeiss optical microscope Axio Lab.A1 (Zeiss, Germany). Reagents and Materials Quinoline, benzo[h]quinoline, carbazole, 3-ethylcarbazole, 9-ethylcarbazole, 9-methylcarbazole, indole, 2-methylindole, 3-methylindole, 7-methylindole, acridine, N-methylpyrrole and N,N-dimethylaniline were obtained from Sigma-Aldrich (USA). Sodium dodecyl sulfate (SDS), urea, boric acid, ethanol, isopropanol, acetonitrile and iso-octane (HPLC-grade) were obtained from Merck (Germany). Deionized water (resistivity of 18.2 MW cm) was from a Milli-Q gradient A10 ultra-purifier (Milipore, USA). Diesel and gas oil samples were obtained from Petrobras, the Brazilian Energy Company, Brazil. A fused silica capillary column (Agilent, California, USA) of 56.0 cm of length (47.5 cm of effective length) with 50 Âľm of internal diameter. Solutions and Samples Background electrolyte (BGE) solution was composed by 20% v/v of acetonitrile and 80% v/v of an aqueous solution containing H3BO3 (10 mmol L-1), SDS 30 mmol L-1 and urea 2 mol L-1 with pH adjusted to 9.50 (with the addition of aliquots of NaOH 1 mol L-1). NCACs stock solutions (1 Ă&#x2014; 10-3 mol L-1) were 50

Canto, A. S.; da Cunha, A. L. M. C.; Mello, S. C.; Aucelio, R. Q.

Article

prepared in ethanol. Standard detergenless microemulsions were prepared by the dilution of the stock analyte solutions into about 5 mL of a mixture containing isooctane (10% v/v), water (12% v/v), ethanol (55% v/v) and isopropanol (23% v/v). Appropriate aliquots (up to 50 ÂľL) of the standard detergenless microemulsions were added to a solution consisting of 25% (v/v) ethanol and 75% (v/v) of an aqueous solution containing H3BO3 (5 mmol L-1), SDS 20 mmol L-1, urea 1 mol L-1 with pH 9.50 (adjusted by the addition of aliquots of 1 mol L-1 NaOH solution) comprising a final volume of 5.00 or 10.00 mL. This final mixture was the analysis microemulsion. Diesel samples were previously homogenized by mechanical agitation using an Ultra Turrax (7200 rpm) apparatus during 5 min. When analyzing samples, 10 to 200 ÎźL (depending upon the characteristics of the sample in forming stable dispersions) of a homogenized diesel sample was dissolved in isooctane comprising about 1 to 6 mL total volume) then mixed with the appropriate volumes of water, ethanol and isopropanol to form 5.00 mL total volume of detergentless microemulsion. Then, sample detergentless microemulsion was diluted in the appropriate aqueous solution prior to the injection into the capillary. Capillary Electrophoresis Analysis Freshly prepared BGE solutions were employed (changed each 5 to 10 runs). The applied potential was 10 kV from the beginning up to 20 min, increasing to 30 kV up to the end of chromatographic run. Capillary temperature was kept at 15 ÂşC and sample hydrodynamic injection (50 mbar) was made at 50 mbar during 10 s. The diode array UV detector was set at 230 nm. Capillary conditioning was performed in the beginning of each working day and it consisted of three steps (each of 30 min flushing): i) sodium hydroxide 1 mol L-1; ii) ultrapure water and iii) the BGE solution. Before each of the sample or standard injections, a 15 min pre-conditioning procedure was made by flushing water (4 min), sodium hydroxide 1 mol L-1 (4 min), water again (2 min) and BGE solution (5 min). At the end of the working day, the capillary was cleaned by flushing water (5 min), sodium hydroxide 1 mol L-1 (5 min), water again (5 min) then passing a flow of compressed air to dry. Elemental Analysis and Optical Analysis The C, H and N elemental determinations were made under He atmosphere (140 mL min-1) using a thermal conductivity detector and samples placed in tin capsules. For microscopic optical pictures, a droplet of the microemulsion was placed on a glass slide. RESULTS AND DISCUSSION Composition and Stability of Sample Dispersions In order to avoid any labor-intensive treatment aiming analyte separation from the sample matrix, the diesel (or gas oil) sample (typically 50 to 200 mL) was first diluted in isooctane forming a 0.5 mL solution. For standards, an aliquot of the stock standard solution, containing the analytes was directly diluted in isooctane. The dispersion made with the isooctane solution (containing either standards or samples) was stable and homogeneous (for at least 24 h) when prepared in a mixture of solvents based on the one proposed by Cunha et al. [29]: 0.5 mL of the isooctane NCACs solution, 0.6 mL of water, 1.1 mL of propan-2-ol and completing the final 5.00 mL volume with ethanol. This was called sample (or standard) dispersion and although microliters of it can be promptly introduced into an HPLC system [29], it could not be introduced into the capillary to perform electrophoretic analysis. Therefore, a logical strategy was to adjust the final composition of the dispersion to be introduced into the CE system (nominated as analysis dispersion) as close as possible to the BGE. In order to do that, volumes varying from 10 to 50 mL of the sample (or standard) dispersion were further dispersed in a solution containing 25% (in volume) of ethanol and 75% (in volume) of an aqueous solution containing borate buffer, surfactant and the chemical modifiers used in the system BGE (see detailed composition below), forming a final 3.00 mL analysis dispersion that was stable for at least 24 h. 51

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Micelar-Electrokinetic Chromatography Separation of Nitrogen-Containing Aromatic Compounds in Diesel Prepared as Microemulsion

Studies to Establish the Electrophoretic Separation Conditions The starting BGE composition was borate buffer (10 mmol L-1 using H3BO3) at pH 9.50 guaranteeing (based on literature pKa values [33-38]) that QNL (pKa = 4.9), BhQ (pKa = 4.7), ACR (pKa = 5.7) and A (pKa = 7.1) remained non-protonated while the carbazoles (pKa @ 12) the indoles (pKa @ 17) and P (pKa = 14.2) remained protonated, in order to better interact with the pseudostationary phase (SDS organized structures) used for the MEKC separation. The BGE at pH 9.5 also contained SDS (40 mmol L-1) and the chemical modifiers acetonitrile (20% in volume) and urea (2 mol L-1). In these initial separation studies, standard microemulsions, initially containing a mixture of five NCACs (QNL, ACR, CBZ, BhQ and 3MI) in isooctane, were mixed with the BGE (50/50% v/v) and the NCACs final concentrations (in the analysis microemulsion) were in the 10-5 mol L-1 (equivalent to mg L-1 levels) in the final analysis microemulsion introduced into the capillary. Pyrrole was used as an internal standard. The influence of temperature (25 ºC, 30 ºC and 35 ºC) was evaluated using the applied voltage of 30 kV. In all cases, the peaks were separated (baseline separation) and no significant changes in migration times were found. However, peak shape tended to be better at 25 ºC with the splitting of 3MI peak at 30 ºC and at 35 ºC and with large baseline fluctuation at 35 ºC. The electropherogram achieved at 25 ºC can be seen in Figure 2A. The applied voltage was varied from 15 to 30 kV in order to check the effect on the quality of separation at 25 ºC. In all cases, baseline separation of peaks was observed and as expected shorter migration times were found as the applied voltage was increased, with QNL (the NCAC with the faster migration velocity after P) presenting migration time (tm) of 8.1 min with 15 kV (Figure 2B) and 3.6 min with 35 kV. The migration time interval between QNL and CBZ (NCAC with the slower migration velocity) was about 9.2 min with applied voltage of 15 kV and about 3.2 min with 30 kV.

Figure 2. MEKC electropherogram of quinoline (QNL), 3-methylindole (3MI), acridine (ACR), Benzo[h]quinoline (BhQ), carbazole (CBZ) using pyrrole as internal standard (IS), sample introduction for 10 s at 50 mbar, at 25 ºC, detection at 230 nm and BGE consisting of borate buffer (10 mmol L-1; pH 9.5), SDS (40 mmol L-1) acetonitrile (20%) and urea 2 mol L-1). A) 30 kV applied voltage; B)15 kV applied voltage.

Canto, A. S.; da Cunha, A. L. M. C.; Mello, S. C.; Aucelio, R. Q.

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Despite the increasing of analysis time (from 6.9 to 17.2 min, measured using the migration time of CBZ) it was decided to proceed studies using applied voltage of 15 kV as there was larger intervals between the separated peaks, leaving room to accommodate the other eight NPACs to be included in the study. Besides, at this applied voltage, peaks presented better shape and no tendency to split. In order to decrease the somewhat larger analysis time, the concentration of SDS was adjusted from 40 mmol L-1 to 30 mmol L-1, which decreased the migration time for CBZ from 17.2 to 12.1 min still leaving 4.3 min between migration peaks of QNL and CBZ (Figure 3A). The next step of the study was the inclusion of the analyte A in the microemulsion introduced into the capillary. Under the conditions used to separate the first five NCACs, the peak of A appeared baseline separated from the others but with peak symmetry affected. Therefore, a decrease of temperature to 20 ยบC was made in order to improve symmetry but not affecting the peak of 3MI as seen in Figure 3B. The adjustment of temperature also increased the migration time for CBZ from 12.2 to 15.1 min because of the increase of viscosity of the BGE.

Figure 3. MEKC electropherogram of quinoline (QNL), 3-methylindole (3MI), acridine (ACR), Benzo[h]quinoline (BhQ), carbazole (CBZ) using pyrrole as internal standard (IS), sample introduction for 10 s at 50 mbar, applied voltage of 15 kV, detection at 230 nm and BGE consisting of borate buffer (10 mmol L-1; pH 9.5), SDS (30 mmol L-1) acetonitrile (20%) and urea 2 mol L-1). A) at 25 ยบC; B) at 20 ยบC.

The other six NCACs (I, 2MI, 7MI, 9MC, 3EC, 9EC) were included in the micromulsion to be injected into the capillary. In the conditions already adjusted for the six NCACs, baseline separation (Figure 4A) for most of the analytes were achieved and the migration time for 9EC (the NCACs with slower migration velocity) was 23.4 min. Poor separation was achieved for the pairs of NCACs A and I and 7MI and 2MI 53

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Micelar-Electrokinetic Chromatography Separation of Nitrogen-Containing Aromatic Compounds in Diesel Prepared as Microemulsion

(detail of Figure 4), then simple adjustments of conditions were tried to improve resolution. A set of experiments were performed using microemulsion containing only these four NCACs. An improved resolution was achieved by decreasing both the applied voltage to 10 kV and the temperature to 15 ยบC, achieving baseline separation for A and I and good resolution for 7MI and 2MI as can be seen in the electropherogram of Figure 4B. The use of such experimental conditions did affect neither baseline resolution between the other NCACs peaks nor the order of migration but it affected the overall analysis time by delaying the migration of 9EC (over 40 min). In order to improve analysis time, a gradient of applied voltage was used by using 10 kV from 0 to 20 min of analysis time (enough time to get the resolution for A and I and 2MI and 7MI) then increasing the applied voltage to 15 kV for the rest of the analysis. In this way, the peak of EC appeared at 37.3 min as seen in Figure 5. The conditions chosen to perform capillary introduction and electrophoretic separation of the 12 NCACs and the internal standard are indicated in Table I. Peak symmetry and resolution between peaks are presented in Table II.

Figure 4. A) MEKC electropherogram of quinoline (QNL), N,N-dimethylaniline (A), indole (I), 2-methylindole (2MI), 7-methylindole (7MI), 3-methylindole (3MI), acridine (ACR), Benzo[h]quinoline (BhQ), carbazole (CBZ), 9-methylcarbazole (9MC), 3-ethylindole (3EC), 9-ethylcarbazole (9EC) using pyrrole as internal standard (IS), sample introduction for 10 s at 50 mbar, applied voltage of 15 kV at 20 ยบC, detection at 230 nm and BGE consisting of borate buffer (10 mmol L-1; pH 9.5), SDS (30 mmol L-1) acetonitrile (20%) and urea 2 mol L-1). B) Application of 10 kV at 15 ยบC to improve resolution of A-I and 2MI-7MI. 54

Canto, A. S.; da Cunha, A. L. M. C.; Mello, S. C.; Aucelio, R. Q.

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Figure 5. MEKC electropherogram of quinoline (QNL), N,N-dimethylaniline (A), indole (I), 2-methylindole (2MI), 7-methylindole (7MI), 3-methylindole (3MI), acridine (ACR), Benzo[h]quinoline (BhQ), carbazole (CBZ), 9-methylcarbazole (9MC), 3-ethylindole (3EC), 9-ethylcarbazole (9EC) using pyrrole as internal standard (IS), sample introduction for 10 s at 50 mbar, at 15 ºC, detection at 230 nm and BGE consisting of borate buffer (10 mmol L-1; pH 9.5), SDS (30 mmol L-1) acetonitrile (20%) and urea 2 mol L-1) applied voltage of 10 kV from 0 to 20 min and 30 kV from 20 to 40 min.

Table I. Chosen conditions to separation and quantification of 12 NCACs using MECK Parameter

Condition

BGE

10 mmol L-1 H3BO3/SDS 30 mmol L-1

Organic modifier

Acetonitrile (20% in volume) and urea 2 mol L-1

9.5

Applied voltage gradient

5 kV (0 – 20 min) and 30 kV (20 – 40 min)

Temperature

15 ºC

Pressure of sample intoduction

50 mbar

Sample introduction time

10 s

Capillary lenghts: efective/total

47/55 cm

Internal diameter

50 μm

Detection (absorbance)

230 nm

Solution to prepare the analysis microemulsion

Ethanol (25%)/BGE (75%)

Micelar-Electrokinetic Chromatography Separation of Nitrogen-Containing Aromatic Compounds in Diesel Prepared as Microemulsion

Article

Table II. NCACs peak characteristics and resolution Migration time (min)

Peak width (min)

Peak simmetry

Resolution

N-methylpyrrole (P)

11.9

0.15

1.12

baseline

Quinoline (QNL)

14.2

0.15

1.01

baseline

N,N-dimethylaniline (A)

15.4

0.13

1.28

baseline

Indole (I)

15.8

0.13

1.02

baseline

2-methylindole (2MI)

18.6

0.16

1.00

2,31b

7-methylindole (7MI)

19.0

0.17

1.35

baseline

3-methylindole (3MI)

20.3

0.07

1.05

baseline

Acridine (ACR)

22.2

0.07

1.00

baseline

Benzo[h]quinoline (BhQ)

23.8

0,10

0.87

baseline

Carbazole (CBZ)

25.7

0.13

0.82

baseline

9-methylcarbazole (9MC)

30.1

0.20

1.00

baseline

3-ethylcarbazole (3EC)

33.7

0.30

1.18

baseline

9-ethylcarbazole (9EC)

37.3

0.38

1.18

baseline

NCACsa

NCACs = Nitrogen-containing aromatic compounds. Resolution (Rs) calculated using: Rs = 2 x (tm(2MI) â&#x20AC;&#x201C; tm(7MI))/(w2MI + w7MI) where tm is migration time and w is peak width.

a b

Analytical Parameters Instrumental limit of detection (LOD) and limit of quantification (LOQ) were calculated by decreasing the concentration of each of the NCACs until reaching the lowest value that the software was able to perform peak integration. Such concentrations were introduced (in replicates) into the system and interpolated in the respective analytical curves (seven replicates) using six concentration points with equations shown in Table III. Those limits (Table III) were then assumed to be xm + 3s (for LOD) and xm + 10s (for LOQ) with xm as the average recovered concentration in the curve and s the standard deviation of the seven replicates. The repeatability (measured as coefficient of variation or CV) of the method was evaluated at three different concentrations (established for each analyte according to the sensibility of the analytical response) for each of the analytes using five consecutive analyzes of microemulsion containing analyte standards. Best results were achieved for QNL (CV up to 4%) and the poorer precision was for 9MC (CV of 11% in all concentrations) as seen in Table IV. These isooctane microemulsions were stored for 24 h before repeating the analysis again (repeatability) and, for all of the analytes, the results were statistically similar to the ones achieved in the previous day (two-tail Student t-test with 95% confidence level and n=7).

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Canto, A. S.; da Cunha, A. L. M. C.; Mello, S. C.; Aucelio, R. Q.

Table III. Analytical figures of merit to separation and quantification of 12 NCACs using MECK NCACsa

a b

Linear equationb

LOD (mg L-1)

LOQ (mg L-1)

Quinoline (QNL)

y = 0.74x + 0.13

0.78

1.03

N,N-dimethylaniline (A)

y = 0.91x + 0.066

4.86

7.96

Indole (I)

y = 0.32x - 0.087

1.09

1.99

2-methylindole (2MI)

y = 0.35x - 0.027

1.11

2.10

7-methylindole (7MI)

y = 0.30x - 0.063

1.09

1.56

3-methylindole (3MI)

y = 0.32x + 0.15

0.79

1.34

Acridine (ACR)

y = 0.059x + 0.078

3.98

4.62

Benzo[h]quinoline (BhQ)

y = 0.16x + 0.013

1.61

2.26

Carbazole (CBZ)

y = 0.21x + 0.029

1.08

2.31

9-methylcarbazole (9MC)

y = 0.19x + 0.11

0.68

1.15

3-ehtylcarbazole (3EC)

y = 0.19x + 0.13

0.76

1.14

9-ethylcarbazole (9EC)

y = 0.33x - 0.31

0.67

1.53

NCACs = Nitrogen-containing aromatic compounds. On equations y = intensity of analytical response, x = concentration of analyte.

Table IV. Repeatability values NCACsa N,N-dimethylaniline (A)

Indole (I)

2-methylindole (2MI)

7-methylindole (7MI)

3-methylindole (3MI)

Acridine (ACR)

Benzo[h]quinoline (BhQ)

Concentration (mg L-1)

Standard-deviation (mg L-1)

Coeficient of variation (%)

1.7

8.7

0.05

1.2

0.05

0.2

0.06

0.6

0.1

0.9

1.4

0.1

0.9

Micelar-Electrokinetic Chromatography Separation of Nitrogen-Containing Aromatic Compounds in Diesel Prepared as Microemulsion

Article

Table IV. Repeatability values (Cont.) NCACsa Carbazole (CBZ)

9-methylcarbazole (9MC)

3-ethylcarbazole (3EC)

9-ethylcarbazole (9EC)

Quinoline (QNL) a

Concentration (mg L-1)

Standard-deviation (mg L-1)

Coeficient of variation (%)

0.1

1.5

0.2

1.2

0.1

0.7

0.04

1.4

0.03

0.5

NCACs = Nitrogen-containing aromatic compounds

A specific interference study was made only for the pair 2MI and 7MI, by varying their concentrations, from 3 to 30 mg L-1 in mixtures, in order to get a relative concentration proportion between them of 1 to 10 to 10 to 1. Enough resolution was achieved in these extreme proportions (Rs of 1.6 for 2MI:7MI at 1:10 proportion and Rs of 1.8 for 2MI:7MI at 10:1 proportion) which guarantee enough resolution to selectively determine these similar indole derivatives. Finally, an interference test was made by fortifying a diesel sample with a specific concentration of each analyte and performing a recovery test. The diesel sample used was the one that presented the lowest amount of the NCACs (evaluated by the HPLC method reported by da Cunha et al. [29]). The electropherogram of the non-fortified diesel sample was used as blank. The recovered results (Table V) varied between 91 ± 7% (3EC) to 106 ± 4% (CBZ) indicating a good selectivity towards other components present in the diesel matrix. Another set of NCACs fortified diesel sample was analyzed by the proposed MEKC method and by using the HPLC method used to evaluate the baseline concentrations of NCACs at mg L-1 level. In this study, P, 2MI and 7MI were not included since the protocol reported for the HPLC did not include these three NCACs. The results for the quantified analytes were similar as indicated by a two-tailed Student t-test (with 95% confidence level and n=3). Therefore, the proposed method provides reliable information on quantification of NCACs. Table V. Recovery of NCACs in diesel fortified samples Fortification (mg L-1)

Recovered concentration (mg L-1)

Recovery (%)

Quinoline (QNL)

11.6

11.4 ± 0.1

98 ± 1.2

N,N-dimethylaniline (A)

10.9

9.3 ± 0.5

85 ± 4.3

Indole (I)

4.7

4.8 ± 0.2

102 ± 4.2

2-methylindole (2MI)

5.2

5.4 ± 0.9

103 ± 5.0

7-methylindole (7MI)

5.2

4.7 ± 0.01

90 ± 0.2

3-methylindole (3MI)

5.2

4.8 ± 0.2

92 ± 3.7

NCACsa

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Canto, A. S.; da Cunha, A. L. M. C.; Mello, S. C.; Aucelio, R. Q.

Table V. Recovery of NCACs in diesel fortified samples (Cont.) Fortification (mg L-1)

Recovered concentration (mg L-1)

Recovery (%)

Acridine (ACR)

16.1

16.3 ± 1.2

101 ± 7.7

Benzo[h]quinoline (BhQ)

7.2

7.1 ± 0.5

99 ± 7.1

Carbazole (CBZ)

6.7

7,1 ± 0.2

106 ± 3.5

9-methylcarbazole (9MC)

7.2

7,3 ± 0.5

102 ± 7.0

3-ethylcarbazole (3EC)

7.8

7.1 ± 0.5

91 ± 6.8

9-ethylcarbazole (9EC)

7.8

7.5 ± 0.2

96 ± 2.2

NCACsa

NCACs = Nitrogen-containing aromatic compounds

Analysis of real samples Samples (diesel and a mix of diesel and gasoil) were provided by Petrobras. Aliquots of these were diluted in isooctane to be prepared as detergentless microemulsions before final dilution with BGE to form the analysis microemulsion to be introduced into the capillary. Sample amounts dissolved in isooctane varied in function of the sample characteristics (viscosity and density) and after a final volume adjustment, to produce a stable introduction microemulsion, the dilution factors varied from 4.000 to 12.000. Despite the high dilution factor, the presence of NCACs was detected in most samples but in two of them (named I and II), a number of NCACs could be effectively determined. A prior elemental analysis has indicated a total N content of 0.4% in sample I (86.6% of C and 10.2% of H) and of 0.3% in sample II (89.0% of C and 9.2% of H). The N content comprises all of nitrogen compounds present in samples with a fraction being NCACs. In sample I, the analytes 9EC and 9MC were found respectively at 6.5 ± 0.2 g L-1 and 2.4 ± 0.3 g -1 L (values in the original sample after correcting for sample dilution). In Figure 6 the electropherogram of sample I fortified with some NCACs (electropherogram A) and the one of the original sample (electropherogram B).

Figure 6. MEKC electropherogram of sample I with sample introduction for 10 s at 50 mbar, at 15 ºC, detection at 230 nm and BGE consisting of borate buffer (10 mmol L-1; pH 9.5), SDS (30 mmol L-1) acetonitrile (20%) and urea 2 mol L-1) applied voltage of 10 kV from 0 to 20 mim and 30 kV from 20 to 40 min. A) fortified with some NCACs; B) non-fortified sample. 59

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Micelar-Electrokinetic Chromatography Separation of Nitrogen-Containing Aromatic Compounds in Diesel Prepared as Microemulsion

For sample II, concentrations (considering the correction of the dilution factor) of QNL (at 10.8 ± 0.7 g L-1) and 3EC (at 11.8 ± 0.8 g L-1) were found as can be seen in Figure 7. It is important to point out the presence of many unidentified absorption peaks maybe comprising a group of aromatic compounds not included in this work.

Figure 7. MEKC electropherogram of sample II (non-fortified sample) with sample introduction for 10 s at 50 mbar, at 15 ºC, detection at 230 nm and BGE consisting of borate buffer (10 mmol L-1; pH 9.5), SDS (30 mmol L-1) acetonitrile (20%) and urea 2 mol L-1) applied voltage of 10 kV from 0 to 20 mim and 30 kV from 20 to 40 min.

CONCLUSIONS In this exploratory study, the feasibility of separation of NCACs in diesel by MEKC without any sample treatment is demonstrated. The compatibilization of the oily sample and the aqueous BGE was achieved by using microemulsions. The high sample dilution factor was crucial to enable sample stabilization during electrophoretic separation but affected the capability for the detection of NCACs in real samples using absorption photometry. Despite that, NCACs could be detected in many of the samples and quantified in two of them. However, the use of laser-induced fluorescence, even exciting in a wavelength detuned from the maximum excitation, will certainly improve detection capability and also, in some extent, the selectivity because of the choice of excitation/emission pair. When analyzing real samples, migration times of the NCACs may be affected due to the complexity of the matrices, even in diluted conditions, therefore, the use of the internal standard and the NCACs standard addition are important to make the identification of the analytes. Acknowledgements The present study was financed in part by the “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil” (CAPES) - Finance Code 001. Aucelio thanks Brazilian research agencies FAPERJ (E-26/202.912/2017) and CNPq (303866/2017-9) for scholarships. Authors thank Petrobras (The Brazilian Energy Company) for research grant. Anastácia acknowledges VRAC-PUC-Rio for scholarship. Manuscript submitted: April 19, 2019; revised manuscript submitted: July 9, 2019; manuscript accepted: August 26, 2019; published online: September 27, 2019. REFERENCES 1. Snyder, L. R.; Buell, B. E. Anal. Chem. 1968, 40, pp 1295-1302 (http://dx.doi.org/10.1021/ ac60264a004). 2. Snyder, L. R. Acc. Chem. Res. 1970, 3, pp 290-299 (http://dx.doi.org/10.1021/ar50033a002).

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Br. J. Anal. Chem., 2019, 6 (24) pp 63-68

The 42nd Annual Meeting of the Brazilian Chemical Society Discussed the Importance of Current Decisions for a Better Future of Chemistry in Brazil Held from May 27 to 30, 2019, in Joinville, SC, Brazil, the 42nd edition of the Annual Meeting of the Brazilian Chemical Society (RASBQ) received an audience of 1652 people. Of the total participants, 682 were researchers, 432 were graduate students, and 403 were undergraduate students. One thousand one hundred eleven papers were presented, most of them in the areas of organic chemistry (180), materials chemistry (145), analytical chemistry (123), natural products chemistry (107), and inorganic chemistry (102). The distribution of participants from the five regions of Brazil was the following: 855 participants from the Southeast, 395 from the South, 130 from the Northeast, 96 from the Midwest, and 51 from the North. The current president of the Brazilian Chemical Society (SBQ), Prof. Dr. Norberto Peporine Lopes, from the Department of Physics and Chemistry, Faculty of Pharmaceutical Sciences, University of Sao Paulo (FCFRP-USP), Ribeirão Preto, SP, emphasized the results achieved in this edition of RASBQ: “In addition to all the scientific success, SBQ’s partners made a manifestation in defense of teaching and research in Brazil, which was a demonstration that proceeded peacefully and without any mess,” said Prof. Peporine Lopes. This statement by Prof. Peporine Lopes was mainly motivated by the significant reduction in funding for scientific research in Brazil and the recent attacks on Brazilian public universities.

Participants of the 42nd RASBQ in manifestation for the defense of teaching and research in Brazil. Photo: SBQ.

This year, the theme of the RASBQ was “Mobilizing Axes in Chemistry,” which led, after 15 years of the publication of the first document in the Brazilian journal “Química Nova” (ISSN 1678-7064), to a reflection on topics such as chemistry teaching at undergraduate and postgraduate levels and the current research funding model in Brazil.

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Opening Ceremony Participants had the opportunity to attend a performance by the Bolshoi Theater School of Joinville (the only subsidiary of the renowned Bolshoi Theater Ballet Corps in Moscow). This was an unprecedented attraction in the history of SBQ. The show that was presented is called “Gala Bolshoi,” and it is a kind of pot-pourri composed of typical dances and pieces of renowned repertoire ballets, bringing to the public the result of the daily work performed by the Bolshoi School. “For the Bolshoi School, performing at national events such as RASBQ provides our students with experience and contributes to the fulfillment of our mission of training citizen-artists, as well as fostering business tourism in Joinville, making dance art and our city better known,” said Pavel Kazarian, DirectorGeneral of the Bolshoi Theater School in Joinville.

Ballet presentation by students of the Bolshoi Theater School of Joinville, SC, Brazil. Photo: SBQ.

Bolshoi Theater School in Brazil has transformed the lives of children and young people, not only in Joinville and the surrounding area. According to Albenize Ballen Bueno, the school’s communication coordinator, ballet students come from various corners of the country and abroad, mostly from the poorest strata of society. They receive a full scholarship and benefits that guarantee excellent training. Currently, according to Ms. Bueno, the school has students from 22 Brazilian states and 5 countries, with 74% of the graduated dancers performing in the world dance market. After the beautiful performance of the Bolshoi Theater, Prof. Dr. Vítor Francisco Ferreira, from the Fluminense Federal University (UFF), held the opening conference entitled “Naphthoquinones: Occurrence, Medicinal Uses and Biological Importance.” Then, Prof. Ferreira was honored with the Simão Mathias Medal, the highest award from SBQ. “I have dedicated myself to SBQ since 1979, and the awarding of this medal brings me great satisfaction because it is recognition for my dedication to SBQ that I embraced in my academic life, parallel to my work as a chemistry professor and researcher,” said Prof. Ferreira. 64

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Prof. Dr. Vítor Francisco Ferreira, from the Fluminense Federal University and former president of SBQ giving the opening conference. Photo: SBQ.

A specialist in organic synthesis, Prof. Ferreira graduated in Chemistry from the Federal University of Rio de Janeiro (UFRJ) in 1976. After his master’s degree in Natural Product Chemistry from UFRJ, he earned a doctorate in Organic Chemistry from the University of California, San Diego (1984) and a postdoctoral degree from the University of Oklahoma (1997). He was president of SBQ from 2012 to 2014, is a full member of the Brazilian Academy of Sciences, a Fellow of the Royal Society of Chemistry, and was recently the Pro-Rector of Research, Postgraduate, and Innovation at UFF. The Simão Mathias Medal was established by SBQ in 1997 to recognize personalities who have made significant contributions to chemistry in Brazil and to SBQ. Prof. Dr. Simão Mathias (1908–1991) was a student in the first course of chemistry at the newly opened University of São Paulo in 1935 and the first to obtain a doctorate in science from the faculty. He set up Brazil’s first physicochemical laboratory and helped develop several scientific societies, including SBQ, which he chaired from its founding in 1977 until 1980. A posthumous tribute was paid to Prof. Dr. Octavio Augusto Ceva Antunes, one of the victims of the Air France Flight 447 crash in 2009. Prof. Antunes was a professor of pharmacy and chemistry at UFRJ and a consultant to the World Health Organization for the production of anti-HIV-AIDS drugs. He graduated in pharmacy from UFF in 1974, completed his master’s degree in chemistry in 1977 at the Military Institute of Engineering (IME), and earned the title of doctor, also in chemistry, in 1987 from UFRJ. Shortly thereafter, he did postdoctoral work in organometallics at Pierre and Marie Curie University, Paris, FR. Prof. Antunes has had more than 200 articles published in specialized journals, 5 book chapters, and 1 book. He has 23 patent applications, has supervised 22 postdocs, and has 65

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supervised or co-supervised 46 master’s dissertations and 30 doctoral theses in the areas of chemistry, biochemistry, and chemical engineering, and he was a consultant to the World Health Organization (Geneva) for the production of anti-HIV-AIDS drugs from 2004 to 2008. Important SBQ initiatives were launched in this edition of RASBQ, such as the SBQ Accelerates, the Women SBQ Nucleus, and the Vanderlan Bolzani Award. “SBQ Acelera” The “SBQ Acelera” program, which will foster innovation and entrepreneurship in chemistry, was announced at the opening of RASBQ by SBQ President Dr. Peporine Lopes, it and had a booth in the event exhibition area that attracted the attention of dozens of researchers. Research groups in chemistry and chemical engineering with ongoing projects in technology development may apply for the program. There will be a preselection to choose 10 projects that will be mentored and undergo pre-acceleration work. These selected groups will meet in person and will be accompanied weekly by a mentor. They will then submit Banner in posthumous tribute to Prof. Dr. the projects to an examining panel of potential investors Octavio Augusto Ceva Antunes. Photo: and industry insiders who will select five finalist projects. Lilian Freitas. At the end of the year, “demo day” will be held, when the groups will present the finished projects for investors and companies. Women SBQ Nucleus and Vanderlan Bolzani Award The year 2019 may be remembered in the future as the year of women at SBQ. SBQ’s recent effort to increasingly foster the debate on women’s participation in science has reached a milestone at the 42nd RASBQ, with the thematic symposium entitled “Multiplying Women’s References”, coordinated by Prof. Dr. Rossimiriam Pereira de Freitas (Federal University of Minas Gerais), Prof. Dr. Elisa Orth (Federal University of Paraná), and Prof. Dr. Vanderlan da Silva Bolzani (São Paulo State University, Unesp). The aim of this symposium was to foster and multiply women’s references through the presentation of successful trajectories of women who assumed prominent roles in their professional career. Dr. Alice Rangel de Paiva Abreu, emeritus professor at UFRJ, spoke about how to build a successful trajectory from a similar perspective, Prof. Dr. Nadya Pesce da Silveira (UFRGS) spoke about women’s participation in intense scientific discussions, and Dr. Silmara das Neves, founder of IQX-Inove Qualyx, spoke about the influence of gender on the transition from academia to industrial activity. Also at this symposium, the Women SBQ Nucleus was officially launched and awarded the Vanderlan da Silva Bolzani Award. “It was a milestone in the history of SBQ. The auditorium was crowded, we had forceful lectures, and we were thrilled with the award of the Vanderlan Bolzani Prize to our dear Dirce Campos (a member of SBQ since its foundation and now executive director), and Prof. Dr. Maria Vargas (UFF), for her expressive contribution to Chemistry,” said the vice president of SBQ Prof. Rossimiriam P. de Freitas. The Vanderlan Bolzani Award was created this year by SBQ in recognition of the academic work of Prof. Dr. Vanderlan da Silva Bolzani, who is a full professor at the Institute of Chemistry at Unesp in Araraquara, SP, has worked for four decades in the area of natural product chemistry, is Vice President 66

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of the Brazilian Society for the Progress of Science (SBPC), and is a member of the Superior Council of the State of São Paulo Research Foundation Fapesp. This award is dedicated to recognizing the work of women who excel in chemistry and/or strengthening SBQ. Another highlight of the 42nd RASBQ was the XVII Workshop on Postgraduate in Chemistry, which brings together the coordinators of dozens of postgraduate programs in chemistry in Brazil. This Workshop was also coordinated by Prof. Dr. Rossimiriam Pereira de Freitas, and it had the following guest speakers: Prof. Dr. Aldo Jose Gorgatti Zarbin, from the Federal University of Paraná and former president of SBQ; Prof. Dr. Adriano Lisboa Monteiro, from the Federal University of Rio Grande do Sul and coordinator of the Chemistry area at the Coordination for the Improvement of Higher Education Personnel (CAPES); Prof. Dr. Norberto Peporine Lopes (FCFRP-USP); and Prof. Dr. Carlos Gilberto Carlotti Júnior, from Ribeirão Preto Medical School-USP. Periodic Table The United Nations (UN) and the International Union of Pure and Applied Chemistry (IUPAC) have adopted 2019 as the International Year of the Periodic Table, a significant event for scientific dissemination.

To celebrate the 150th anniversary of the Periodic Table, SBQ has created a locker in the periodic table format. Photo: SBQ.

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SBQ did not miss this milestone and held a Thematic Session entitled “Periodic Table,” which commemorated the 150th anniversary of the Mendeleev Periodic Table and dealt with aspects such as the geopolitics of chemical elements, issues related to rare elements, and educational aspects. The session organizer was Prof. Dr. Claudia Moraes de Rezende (UFRJ), member of the Advisory Board of SBQ. Three guest speakers addressed different aspects of the periodic table: Prof. Dr. Luis Roberto Brudna Hölzle from the Federal University of Pampa, Dr. Robson de Souza Monteiro, a chemical engineer and international market consultant for niobium in the field of technological application, and Prof. Dr. Severino Alves Junior from the Federal University of Pernambuco. Book Release SBQ Annual Meetings traditionally feature books recently released by SBQ members and event attendees. In this edition of RASBQ, the books launched were the following: • “Introdução à Química Experimental” 3rd edition. Authors: Roberto Ribeiro da Silva, Nerilso Bocchi, Romeu C. Rocha-Filho, and Patrícia Fernandes L. Machado. Publisher of the Federal University of São Carlos. • “Potenciometria: aspectos teóricos e práticos”. Authors: Orlando Fatibello-Filho, Tiago Almeida Silva, Fernando Cruz de Moraes, and Bruno Campos Janegitz. Publisher of the Federal University of São Carlos. • “Temáticas para o Ensino de Química: contribuições com atividades experimentais”. Organizers: Mara E. Fortes Braibante and Hugo T. Schmitz Braibante. Publisher CRV. For the full list of books released, go to: http://www.sbq.org.br/42ra/pagina/lancamento-livros.php. A social gathering of the participants of the event and the celebration of the 42 years of SBQ were held in an “Oktoberfest,” with a lot of typical German food, beer, and live music. Members of the Brazilian Journal of Analytical Chemistry were also present at the 42nd RASBQ to cover the event. Print editions of the journal and its mascot were drawn among the participants.

Winners of the draw held by BrJAC. Photo: Luciene Campos.

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Br. J. Anal. Chem., 2019, 6 (24) pp 69-73

COLACRO 2019 Discussed the Importance of Chromatography in the Development of Latin America The XVII Latin American Symposium on Chromatography and Related Techniques (COLACRO XVII) was held from July 14 to 19, 2019 in Aracaju city, SE, Brazil. For the first time in its 34 years of existence, COLACRO was held in the Brazilian Northeast Region and brought together about 400 people from Brazil and abroad. In this edition, COLACRO XVII took place in conjunction with the Brazilian Symposium on Chromatography (SIMCRO) and the Workshop on Recent Advances in Sample Preparation (WARPA).

COLACRO 2019 Opening Ceremony. Photo: COLACRO.

The event brought together students, professors, and researchers in chromatography and related areas, from Latin America and from other continents, under the pleasant Brazilian northeastern climate to celebrate the state of the art of chromatography. The COLACRO opening conference was given by Prof. Dr. Philip Marriott, from the School of Chemistry, Faculty of Science at Monash University, Melbourne, AU, entitled “Is portable GC-MS a suitable technology for bio-prospecting in a natural-product-rich emerging economy?” Subsequently, there was the presentation of two plenary lectures: one by Prof. Dr. Milton Lee, from the Department of Chemistry and Biochemistry at Brigham Young University, Provo, Utah, USA, entitled “Portable GC and LC,” and another by Prof. Dr. Fernando Mauro Lanças, from the Institute of Chemistry, University of São Paulo (IQSC-USP) in São Carlos, SP, BR, entitled “Recent Advances in Miniaturized on-line Sample Preparation-Chromatography-Tandem Mass Spectrometry”. COLACRO XVII was held at the Tiradentes University (Unit) in Aracaju, SE, BR, a young university with excellent infrastructure that offers a large, modern, and multifunctional space for all scheduled events, as well as a comfortable area for exhibiting equipment, which is a strong feature of this event. 69

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COLACRO Exhibition Area. Photo: Lilian Freitas.

The main companies in the sector were present at the event, presenting seminars and new products available in the world market for the Chromatography and related techniques area. Among them, were Agilent, Analitica, Shimadzu, Superlab, Thermo Fisher, and Waters. Event Program Participants had the opportunity to attend plenary conferences and lectures given by national and international experts. In addition to the previously mentioned names, the following professors were also present (in alphabetical order): Dr. Carin von Muhlen, from the Faculty of Technology, State University of Rio de Janeiro, Resende, RJ, BR; Dr. Carlos Bicchi, from the Faculty of Pharmacy, University of Turin, Turin, IT; Dr. Claudia Alcaraz Zini, from the Institute of Chemistry, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, BR; Dr. Eduardo Carasek Da Rocha, from the Department of Chemistry, Federal University of Santa Catarina, Florianopolis, SC, BR; Dr. Elena Stashenko, from the School of Chemistry, Faculty of Sciences, Industrial University of Santander (UIS), Bucaramanga, CO; Dr. Fabio Augusto, from the Institute of Chemistry, University of Campinas (IQ-Unicamp), Campinas, SP, BR; Dr. Francisco Radler de Aquino Neto, Emeritus Professor from the Institute of Chemistry, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, BR; Dr. Jared Anderson, from Iowa State University, Ames, Iowa, USA; Dr. José Manuel Florêncio Nogueira, from the Department of Chemistry and Biochemistry (DQB), Faculty of Sciences, University of Lisbon, Lisbon, PT; Dr. Luigi Mondello, from the Department of Pharmaceutical Chemistry and Health Products, University of Messina, Messina, IT; Dr. Maria Eugenia Queiroz, from the Faculty of Philosophy Sciences and Letters of Ribeirão Preto (FFCLRP), University 70

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of São Paulo, Ribeirão Preto, SP, BR; Dr. Quezia B. Cass, from the Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, BR; Dr. Renato Zanella from the Department of Chemistry, Federal University of Santa Maria (UFSM), Santa Maria, RG, BR; and many others. View the full program of the event at https://colacro2019.com/presentation.

Participants attending one of the event’s lectures. Photo: COLACRO. The program’s guiding line was the foundations, developments, and applications of chromatography and related techniques in areas of recognized importance, notably, environmental, food, forensics, pharmaceuticals, flavorings & fragrances, petrochemicals, and biochemistry. The scientific program of the event also contained satellite symposiums, oral communications and poster discussion. Homage Session During COLACRO, two medals were delivered: CIOLA Medal and COLACRO Medal. The Ciola Medal was awarded to Dr. Fabio Augusto, from IQ-Unicamp, in recognition of his contribution to the development and dissemination of chromatography in Brazil. This Medal was created in 2010 to recognize Dr. Remolo Ciola, who pioneered the design, construction, and commercialization of chromatographs in Latin America. He was also a professor at the former “Escola de Engenharia Mauá,” today, called the “Instituto Mauá de Tecnologia,” in São Caetano do Sul, SP; the Institute of Chemistry at the University of São Paulo (IQ-USP), in São Paulo, SP; and the Technological Institute of Aeronautics (ITA) in São José dos Campos, SP, Brazil. The COLACRO Medal was awarded to Prof. Dr. Philip Marriott (Monash University, MELB, AU) for his significant participation in the development of chromatography in Brazil and Latin America. Prof. Dr. Elina Caramão, a researcher at the Institute of Technology and Research (ITP), in Aracaju, SE, chairman of the COLACRO’s Local Organizing Committee and member of the Local Scientific Committee, delivered this Medal at the event’s Homage Session.

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Ciola Medal Delivery. From left to right: Luiz Bravo, from Nova Analitica company, delivering the medal to Fabio Augusto. Photo: Fabio Augusto.

COLACRO Medal Delivery. From left to right: Claudia Zini, Elina Caramão, delivering the medal to Philip Marriot and Carin von Muhlen. Photo: Divulgação.

About COLACRO It was Prof. Dr. Fernando Mauro Lanças (IQSC-USP) who conceived COLACRO and organized the first event in 1986 in Rio de Janeiro, Brazil. Since then, COLACRO has been occurring biennially in Latin American countries, and it is now the largest event for chromatography and related techniques in Latin America. The event was consolidated as the most important meeting for the exchange of information and ideas in the area of chromatography and separation techniques. In 2016, COLACRO celebrated its 30th anniversary and was organized for the first time outside the American continent by the Chromatography group of the Portuguese Chemical Society, in charming Lisbon, Portugal, with the theme “Building Bridges of Cooperation in Separation Science.”

Poster Discussion Session. Photo: COLACRO. About SIMCRO Founded in 1993 by Prof. Dr. Fernando M. Lanças, SIMCRO quickly became an important scientific meeting in the analytical area of the country. Initially carried out in Águas de São Pedro, SP, it was repeated in 2006 in the same city, with more than 600 participants. In 2008, it was carried out in 72

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partnership with COLACRO in the city of FlorianĂłpolis, SC, where it established its relevance by bringing to COLACRO the Satellite Symposiums. In 2010, SIMCRO was held in Campos do JordĂŁo, SP, with several new features, including the ReCAFluB-Brazilian Workshop on Residues and Contaminants in Food and Biological Fluids, a very specific, and at the same time, comprehensive workshop in the area. About WARPA Beginning in 2012, WARPA aimed to bring together researchers, students, and professors from public and private universities and research centers on different areas of science to discuss recent advances in analytical techniques for sample preparation. Among the topics discussed, we highlight the miniaturization of analytical systems, hyphenation of techniques for automation of analyses, minimization of sample volume, use of selective extractive phases, and the use of environmentally correct technologies.

Members of the Brazilian Journal of Analytical Chemistry were also present at COLACRO to honor and cover the event. During the event, raffles were held for print editions of the journal and its mascot.

Winners of the draw held by BrJAC. Photo: Luciene Campos.

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Br. J. Anal. Chem., 2019, 6 (24) pp 74-81 PDF

This section is dedicated for sponsor responsibility articles.

Flow Modulated Comprehensive Two-Dimensional Gas Chromatography Part I - Low Duty Cycle Modulation of Hidroprocessed Vegetable Oil Rodrigo Passini1, Danilo Pierone2, Angelo L. Gobbi1, and Leandro W. Hantao1,3* Laboratório Nacional de Nanotecnologia (LNNano), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Polo II de Alta Tecnologia, R. Giuseppe Máximo Scolfaro, 10000 - 13083-970 Campinas SP Brazil 2 Nova Analítica, Rua Assungui, 432 - 04131-000 São Paulo SP Brazil 3 Instituto de Química, Universidade Estadual de Campinas, R. Josué de Castro, 126 - Cidade Universitária, 13083-861 Campinas SP Brazil 1

In this paper, we report the quantitation of hydroprocessed vegetable oil (HVO) in petroleum diesel and biodiesel blends. To accomplish this goal, we have developed a low-pressure interface using commercially available components from original equipment manufacturer (OEM) SGE / Trajan Scientific and Medical. The low duty cycle modulation was optimized using a multivariate approach by investigating three important parameters, namely, flush period, auxiliary gas flow, and dimension of restrictor capillary (i.e., bleed). A 5-fold reduction in analysis time was accomplished, while maintaining the group-type separations by exploiting short modulation period. Successful quantitation of HVO was attained due to the inherent low sensitivity of the interface allowing for a wide linear range from 1.0% (w/w) to 100.0% (w/w). The current low pressure and low duty cycle interface exhibits simple and robust operation being particularly suited for fuel analysis. Keywords: renewable fuel, petroleum, green chemistry, esters, fatty acids. INTRODUCTION The demand for cleaner burning and renewable motor fuels, alongside the high cost of petroleum, has drawn great interest in the production and distribution of alternative fuels [1]. Over the past decades, ethanol and biodiesel have been explored as alternative fuels. For instance, biodiesel is produced from the solvolysis of triglycerides using an alcohol and a catalyst [2]. Recently, hydro-processed vegetable oil (HVO) has been considered as a potential candidate for diesel engines, see Table I. Table I. Main characteristics of hydro-processed vegetable oil (HVO)* Renewable fuel generation

2nd generation

Release year

2007 by Neste Oil Porvoo Refinery

Main producers

Neste Oil Porvoo Refinery (Finland), ConocoPhillips (USA), Universal Oil Products-Eni (UK, Italy), Nippon Oil (Japan), SK Energy (Korea), and Syntroleum (USA)

Commercial name and references

NExBTL, hydro-processed vegetable oil (HVO), hydro-processed esters and fatty acids (HEFA), renewable diesel fuel, hydrogenation derived renewable diesel, green diesel, hydrogen treating biodiesel (HBD)

Molecular formula

CnH2n+2

Benefits

High calorific value, high cetane number, oxidation stability, negligible aromatic and sulfur-compounds

*The interested reader is directed to Sonthalia, A.; Kumar, N. J. Energy Inst., 2019, 92 (1), pp 1-17 (https://doi.org/10.1016/j. joei.2017.10.008) for the complete report. 74

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The production of HVO also uses triglycerides as substrates, but it explores elevated temperatures and high hydrogen pressure to promote hydrogenation and cracking producing n-paraffins and isoparaffins in the boiling point range of gasoline and diesel [2]. In Germany, 20% of common diesel fuels are already blended with HVO in percentages of approximately 7.7% (v/v) [3]. However, standard methods have not accompanied the changes in diesel blends using HVO, leading to a debate on the suitability of these analytical methods for quality analysis and quality control (QA/QC). In this context, comprehensive two-dimensional gas chromatography (GC×GC) is the ideal technique for QA/QC of HVO in petroleum diesel / biodiesel blends providing unprecedented peak capacity and shorter chromatographic runs compared to conventional gas chromatography (1D-GC) [4]. The augmented resolving power of GC×GC arises from the sequential combination of two complementary GC separations with distinct selectivity. The two-dimensional separation is accomplished by coupling two GC columns interfaced by the modulator. The modulator is the core of the GC×GC instrument, being responsible for the continuous and periodical transference of solute bands from the first stage (1D) to the second stage (2D) as narrow pulses. Thermal modulation [5,6] is the dominant principle in GC×GC, however it is also the main reason why such technique has not been adopted for routine analysis. For instance, the overhead cost associated with the coolant fluid, e.g. liquid nitrogen, represents a 19-fold increase in operational cost, while the use of working fluid, nitrogen gas, represents an additional 86% to the operating expenses for a GC×GC-FID. Flow modulation, also referred to as flow-switching modulation, uses an auxiliary gas to actuate the vapor stream inside a microfluidic platform to attain modulation during GC×GC analysis [7]. The microfluidic devices are installed inside the GC oven, while an actuating valve is positioned outside the oven. Two alternating stages comprise flow modulation, namely, solute re-injection (flush) and accumulation (fill). Hence, flow modulation encompasses low-duty cycle [8] and full transfer interfaces (forward [9] and reverse fill/flush [10] configurations). A low-duty cycle interface is based on high-speed Deans Switch and typically exhibit much lower sensitivity, compared to full transfer modulators, since it lacks a sampling loop. Despite this drawback, the low-duty cycle flow-switching interface is likely the most robust and easy to operate modulator among all GC×GC solutions. In this paper, we report the development of a low-duty cycle flow modulator using commercially available components from original equipment manufacturer (OEM) SGE / Trajan Scientific and Medical. The flow-switching interface was evaluated using experimental design to account for pure and interaction effects. Such semi-empirical model was used to determine the optimum experimental conditions for GC×GC modulation. Lastly, the quantitation of hydro-processed vegetable oil in petroleum diesel and biodiesel blends was performed. MATERIALS AND METHODS Samples: HVO, Biodiesel, and Diesel Hydro-processed vegetal oil, NExBTL, was purchased from Neste Oil (Finland). Biodiesel and petroleum diesel, S10A, samples were provided by Petrobras SA (Brazil) [11]. The composition of the analytical solutions is described in Table II. Table II. Calibration samples for determination of HVO in petroleum diesel / biodiesel blends Sample

HVO % (v/v)

Diesel % (v/v)

Biodiesel % (v/v)

1.0

73.5

25.5

2.0

73.0

25.0

3.0

72.5

24.5

4.0

72.0

24.0

5.0

71.5

23.5

6.0

71.0

23.0 75

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Table II. Calibration samples for determination of HVO in petroleum diesel / biodiesel blends (Cont.) Sample

HVO % (v/v)

Diesel % (v/v)

Biodiesel % (v/v)

7.0

70.5

22.5

8.0

70.0

22.0

9.0

69.5

22.5

65.5

17.5

63.5

15.5

61.5

13.5

57.5

9.5

53.5

5.5

51.0

3.0

50.0

2.0

49.5

1.5

33.0

7.0

23.0

7.0

13.0

7.0

3.0

7.0

100

0.0

GC×GC-FID analyses The GC×GC-FID system consisted of a TRACE 1310 GC-FID gas chromatograph (Thermo Fisher Scientific – Waltham, MA, USA) equipped with a split/splitless injector and a AS 1300 105-position autosampler. GC×GC separations were performed using a high-speed Deans switch modulator based on SilFlow platform (SGE Analytical– Victoria, Australia) as shown in Figure 1 [12,13].

Figure 1. Low duty cycle flow switching modulator for comprehensive two-dimensional gas chromatography. In (A) the fast Deans Switching device is installed inside a TRACE 1310 GC (Thermo Scientific) highlighting the small footprint of the interface. The small footprint is important to ensure low thermal of the composite system. In (B) we illustrate the simple configuration of the interface for nonexpert users.

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The injector and detector were operated at 250 ÂşC. A split ratio of 100:1 was used in the injection of 1 ÂľL of sample. Modulation period was set to 2.5 s. Column set comprised a 30 m Ă&#x2014; 0.32 mm ID DB-5MS (0.25 Âľm film thickness) primary column and a 2.0 m Ă&#x2014; 0.25 mm MEGAWAX-HT (0.15 Âľm film thickness) secondary column (MEGA srl â&#x20AC;&#x201C; Megano, MI, Italy). The restriction capillary length was evaluated from 15 to 35 cm Ă&#x2014; 0.25 mm ID. The independent variables evaluate in the experimental design were flush duration, auxiliary gas flow, and length of restrictor capillary. RESULTS AND DISCUSSION Flow switching modulation In order to effectively exploit the improved peak capacity of GCĂ&#x2014;GC separations two requisites are mandatory for low-duty cycle FM-GCĂ&#x2014;GC. First, the chromatographic conditions must be optimized to produce a minimum modulation ratio (MR) of 3 to ensure negligible variability from modulation [14]. This condition may be fulfilled by using short modulation periods (PM) or highly retentive primary columns (b < 250), as shown in Equation 1. Secondly, we strongly recommend that the average 2wb not exceed 15% of the value of PM to provide usable separation space in the GCĂ&#x2014;GC chromatograms.

đ?&#x2018;&#x20AC;đ?&#x2018;&#x2026; = 4

1đ?&#x2018;&#x160;

đ?&#x2018;?

đ?&#x2018;&#x192;đ?&#x2018;&#x20AC;

Equation 1

In practice the average peak width in the 2D is directly proportional to the reinjection pulse duration (P), and also to the flow in the 1D (1F) and 2D (2F), as shown in Equation 2. In Figure 2 we illustrate the impact of the injection pulse on the peak width of dodecane.

2đ?&#x2018;¤

đ?&#x2018;?

1đ??š

â&#x2C6;?đ?&#x2018;&#x192;2

đ??š

Equation 2

Figure 2. Impact of the injection pulse to peak profile of dodecane in low duty cycle flow switching modulation for comprehensive two-dimensional gas chromatography.

The efficiency of flow switching modulation is highly dependent on numerous chromatographic conditions. Three independent variables (i.e., factors) are highlighted, namely, bleed capillary, auxiliary 77

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flow, and reinjection pulse. Since the pneumatics of the composite system is easily influenced by each of these variables, a multivariate approach for method optimization is required. This approach, experimental design, is critical for empirical modelling of the modulator behavior because the independent variables may exhibit interaction effects in addition to the pure effects. Hence, a central composite design was assembled using the experiments shown in Table III. Table III. Experimental design used to determine the optimum conditions for modulation of dodecane Experiment

Coded

Original Value

Pulse / ms

Auxiliary gas / mL min-1

Bleed length / cm

-1

100

1.4

-1

100

1.4

-1

100

1.46

-1

100

1.46

-1

140

1.4

-1

140

1.4

-1

140

1.46

140

1.46

-1.68

100

1.43

1.68

140

1.43

-1.68

120

1.4

1.68

120

1.46

-1.68

120

1.43

1.68

120

1.43

120

1.43

120

1.43

120

1.43

A central composite model was built using the asymmetry factor of dodecane. The probe solute, dodecane, was selected because it experiences small retention on the 2D. So, the average peak width may be used to evaluate the quality of the modulation conditions. As previously shown in Figure 2, poor modulation generates asymmetrical peak, so the asymmetry factor is a good parameter to monitor the performance of flow modulation. A response surface was plotted using the statistically valid model (a = 0.05) as illustrated in Figure 3.

Figure 3. Response surface attained for low duty cycle flow switching modulation using the experimental design described in Table III. 78

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Selection of the modulation conditions required that the value of peak asymmetry factor was approximately 1.0. Careful investigation of the response surface indicated that GC×GC analysis attained with 1.46 mL min-1 of auxiliary gas, length of bleed capillary of 35 cm, and injection pulse of 120 ms produced the most symmetrical peak of dodecane. Noteworthy, this multivariate optimization protocol may be easily extended to full transfer interfaces for flow modulation, such as the forward fill/flush [15] and reverse fill/flush interfaces [16]. Quantitation of VGO in diesel/biodiesel blends The optimized GC×GC method was initially used to screen petroleum diesel, biodiesel, and hidroprocessed vegetable oil. The chromatograms are shown in Figure 4. The GC×GC chromatogram of HVO clearly indicates that this particular sample exhibited numerous linear and branched hydrocarbons with a volatility range from n-C6 to n-C17. Also, the chromatographic profile of such sample further supports the absence of aromatic hydrocarbons in this renewable fuel. Inspection of nitrogen- and sulfur-containing compounds would require selective detectors or hyphenation to mass spectrometry, which was not the objective of this case study. In Figure 4 B is shown the GC×GC chromatogram of diesel/biodiesel blend. The cluster of monoaromatic hydrocarbons, which exhibit higher retention in the 2 D, is readily seen in the chromatogram of diesel blend. Also, this diesel sample exhibited a pronounced unresolved complex mixture (UCM) profile in the volatility range from n-C9 to n-C16. Furthermore, the soy biodiesel is easily characterized by the two peaks with van den Dool and Kratz retention indices above 1700.

Figure 4. Flow-modulated comprehensive two-dimensional gas chromatography analysis of hidroprocessed vegetable oil (HVO) (A) and diesel/biodiesel blend (B).

In order to perform quantitation, a suitable analyte must be found that is both present in the HVO sample and absent in the diesel/biodiesel blend. In this case study, we have selected a branched paraffin adjacent to n-heptadecane with retention index of approximately 1770. This marker was ideal for this application as the UCM was pronounced in the volatility range of n-C6 to n-C16 and the two constituents of biodiesel, i.e. C16 and C18 fatty acid methyl esters (FAME), did not coelute with any aliphatic hydrocarbons. The analytical curve is presented in Figure 5. The regression coefficient (r2) of 79

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0.989 was more than suitable for the quantitation of HVO of 1% to 90% (v/v) in diesel/biodiesel blends. Further evidence of adequate calibration was the random distribution of the residues. Noteworthy, the root mean square error of calibration (RMSEC) was 3.2% (v/v) for the broad analytical curve, 1% to 100% (v/v), but a RMSEC value of 1.5% (v/v) may be attained using a narrower analytical curve from 1% to 25% (v/v).

Figure 5. Proof-of-concept analytical curve attained for the quantitation of hidroprocessed vegetable oil (HVO) in HVO/diesel/biodiesel blends. Regression coefficient: r2 = 0.989.

CONCLUSIONS Flow switching modulation is an excellent alternative for QA/QC of fuels, including hidroprocessed vegetable oil, diesel, and biodiesel, as it provides enhanced peak capacity and fast chromatographic analysis. Furthermore, low-duty cycle and full transfer flow modulators are excepted to speed the adoption of GC×GC methods for routine analysis as it offers expectational chromatographic performance and selectivity without increasing the cost of the analysis. Acknowledgments The authors thank São Paulo Research Foundation - FAPESP, (2015/05059-9 and 2017/25490-1) for funding our research. We are indebted to Nova Analítica and for sponsoring our laboratory with Thermo Scientific instruments. Stefano Galli (MEGA srl) is also thanked for providing the GC columns used by our research group. Prof. Ronei Poppi and Dr. Willian Dantas are acknowledged for supplying the samples. REFERENCES 1. Seeley, J. V.; Seeley, S. K.; Libby, E. K.; McCurry, J. D. J. Chromatogr. Sci, 2007, 45, pp 650–656 (http://dx.doi.org/10.1093/chromsci/45.10.650). 2. Sonthalia, A.; Kumar, N. J. Energy Inst., In Press. (http://dx.doi.org/10.1016/j.joei.2017.10.008). 3. Jennerwein, M. K.; Sutherland, A. C.; Eschner, M.; Groger, T.; Wilharm, T.; Zimmermann, R. Fuel, 2017, 187, pp 16–25 (http://dx.doi.org/10.1016/j.fuel.2016.09.033). 4. Pollo, B. J.; Alexandrino, G. L.; Augusto, F.; Hantao, L. W.; Trends Anal. Chem., 2018, 105, pp 202-217 (http://dx.doi.org/10.1016/j.trac.2018.05.007). 5. Pursch, M.; Eckerle, P.; Biel, J.; Streck, R.; Cortes, H.; Sun, K.; Winniford, B. J. Chromatogr. A., 2003, 1019, pp 43–51 (http://dx.doi.org/10.1016/j.chroma.2003.07.016).

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6. Harynuk, J.; Gorecki, T. J. Chromatogr. A, 2003, 1019, pp 53–63 (http://dx.doi.org/10.1016/j. chroma.2003.08.097). 7. Seeley, J. V. J. Chromatogr. A, 2012, 1255, pp 24–37 (http://dx.doi.org/10.1016/j. chroma.2012.01.027). 8. Ghosh, A.; Bates, C. T.; Seeley, S. K.; Seeley, J. V. J. Chromatogr. A, 2013, 1291, pp 146–154 (http://dx.doi.org/10.1016/j.chroma.2013.04.003). 9. Seeley, J. V.; Micyus, N. J.; McCurry, J. D.; Seeley, S. K. Am. Lab., 2006, 38, pp 24–26. 10. Griffith, J. F.; Winniford, W. L.; Sun, K.; Edam, R.; Luong, J. C. J Chromatogr. A, 2012, 1226, 116–123. 11. Dantas, W. F. C.; Alves, J. C. L.; Poppi, R. J. Chemom. Intell. Lab. Syst., 2017, 169, pp 116–121. 12. Higa, K. M.; Guilhen, A.; Vieira, L. C. S.; Carvalho, R. M.; Poppi, R. J.; Baptistão, M.; Gobbi, A. L.; Lima, R. S.; Hantao, L. W. Energy Fuels, 2016, 30, pp 4667–4675. 13. Kataoka, E. M.; Murer, R. C.; Santos, J. M.; Carvalho, R. M.; Eberlin, M. N.; Augusto, F.; Poppi, R. J.; Gobbi, A. L.; Hantao, L. W. Anal. Chem., 2017, 89, pp 3460–3467. 14. Seeley, J. V.; Micyus, N. J.; Bandurski, S. V.; Seeley, S. K.; McCurry, J. D. Anal. Chem. 2007, 79, pp 1840-1847. 15. Seeley, J. V.; Micyus, N. J.; McCurry, J. D.; Seeley, S. K. American Laboratory 2006, 38, pp 24-26. 16. Griffith, J. F.; Winniford, W. L.; Sun, K.; Edam, R.; Luong, J. R. J. Chromatogr. A, 2012, 1226, pp 116-123. This sponsor report is the responsibility of Nova Analítica.

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Br. J. Anal. Chem., 2019, 6 (24) pp 82-93 PDF

This section is dedicated for sponsor responsibility articles.

Determination of Organic Acids in Fruit Juices and Wines by High-Pressure IC Lillian Chen, Brian De Borba, and Jeffrey Rohrer Thermo Fisher Scientific, Sunnyvale, CA, USA

GOAL To develop a method to determine organic acids in fruit juices and wines using IC with suppressed conductivity detection. Keywords: Dionex IonPac AS11-HC-4 μm Column, Suppressed Conductivity, Grape Juice, Apple Juice, Pomegranate Juice INTRODUCTION Organic acids play important roles in juices and wines because of their influence on the organoleptic properties (flavor, color, and aroma) as well as the stability and microbiological control of the products [1]. The total content of organic acids in juices and wines affects the drink’s acidity, whereas the levels of a specific organic acid can directly influence the flavor and taste of the drink. Therefore, organic acid profiles are monitored to determine the freshness of certain fruit juices; winemakers also monitor the concentration of various organic acids to ensure the quality of their wines. The determination of organic acids also plays an important role when testing the authenticity of fruit juices and wines [2,3]. Certain fruit juices, such as those obtained from pomegranate and various types of berries, are popular because of their high levels of antioxidants and the resulting putative health benefits. The high economic value and the large market demand for these juices make them a likely target for adulteration. The most frequent profit-driven adulteration procedures include dilution with water, addition of sugars or pulp wash, and blending with cheaper alternatives. Characterizations of the organic acid content of certain juices are therefore required to verify their authenticity. Many analytical methods are available to determine organic acids in juices and wines. However, several organic acids have poor UV absorption and therefore lack sufficient sensitivity for detection. In addition, other components commonly present in these types of samples — such as sugars and phenolic compounds — have a much higher UV absorption, which can interfere with the detection of target analytes. In contrast, virtually all carboxylic acids ionize sufficiently; therefore, ion chromatography (IC) with suppressed conductivity detection is the technique of choice to separate a large variety of organic acids with inorganic anions and detect them with high sensitivity while minimizing the sugar interferences. MATERIAL AND METHODS Equipment • Thermo Scientific™ Dionex™ ICS-6000+ HPIC™ system, including: SP Single Pump; EG Eluent Generator; DC Detector/Chromatography Compartment; AS-AP Autosampler with Sample Syringe, 250 μL (P/N 074306) and Buffer Line, 1.2 mL (P/N 074989). • Thermo Scientific Dionex EGC 500 KOH Eluent Generator Cartridge (P/N 075778). • Thermo Scientific Dionex CR-ATC 500 Continuously Regenerated Anion Trap Column (P/N 075550). • Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System software version 7.2. Reagents and Standards • Deionized (DI) water, Type I reagent grade, 18 MΩ cm resistance or better • D(+)-Galacturonic Acid Monohydrate, 99% (Fisher Scientific P/N AC22782) 82

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• • • •

L(-)-Malic Acid, 99% (Fisher Scientific P/N AC15059) L-(+)-Tartaric Acid, Powder, Certified ACS, 99.0% (Fisher Scientific P/N A315) Citric Acid Anhydrous, Crystalline, USP (Fisher Scientific P/N A95) Methanol (CH3OH), Certified ACS, ≥99.8% (Fisher Scientific P/N A412)

Consumables • Vial Kit, 10 mL, Polystyrene with Caps and Blue Septa (P/N 074228) • Thermo Scientific™ Nalgene™ Syringe Filters, PES, 0.2 µm (Fisher Scientific P/N 09-740-61A) • AirTite All-Plastic Norm-Ject™ Syringes, 5 mL, Sterile (Fisher Scientific P/N 14-817-28) • Thermo Scientific™ Dionex™ OnGuard™ II RP Cartridges, 1 cc (P/N 057083) Samples Apple Juice, Grape Juice, White Grape Juice, Pomegranate Juice, Pomegranate/Blueberry Juice (Pomegranate 85%, Blueberry 15%), Merlot (Red Wine), Chardonnay (White Wine), White Zinfandel (Rosé Wine). Conditions System 1 (9 μm) Columns: Eluent Source: Eluent A: Eluent B: Time (min)

Thermo Scientific™ Dionex™ IonPac™ AS11-HC Guard, 2 × 50 mm (P/N 052963) Dionex IonPac AS11-HC Analytical, 2 × 250 mm (P/N 052961) Dionex EGC 500 KOH Eluent Generator Cartridge with Dionex CR-ATC 500 Continuously Regenerated Anion Trap Column DI Water CH3OH

KOH (mM)

Time (min)

B (%)

-2.000

0.000

10.070

19.000

10.071

20.000

24.000

30.000

24.010

31.000

35.000

33.000

40.000

33.010

44.000

44.010

45.000

45.000 Flow Rate:

0.4 mL min-1

Inj. Volume:

2.5 μL

Detection:

Suppressed Conductivity, Thermo Scientific™ Dionex™ ASRS™ 300 Anion Self-Regenerating Suppressor™ (2 mm)*, 82 mA, external water mode

System Backpressure:

~2100 psi (1 mM KOH/7% CH3OH), ~2500 psi (60 mM KOH/10% CH3OH)

Background Conductance: Noise:

~0.14–0.64 μS ~0.8–1 nS min-1, peak-to-peak 83

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Conditions — System 1 (9 μm) (cont.) Run Time:

47 min

*Equivalent or improved results can be achieved on the Thermo Scientific™ Dionex™ AERS 500 Anion Electrolytically Regenerated Suppressor.

System 2 (4 μm) Columns:

Dionex IonPac AS11-HC-4 μm Guard, 2 × 50 mm (P/N 078036) Dionex IonPac AS11-HC-4 μm Analytical, 2 × 250 mm (P/N 078035)

Eluent Source:

Dionex EGC 500 KOH Eluent Generator Cartridge with Dionex CR-ATC 500 Continuously Regenerated Anion Trap Column

Eluent A:

DI Water

Eluent B:

CH3OH

Time (min)

KOH (mM)

Time (min)

B (%)

-2.000

0.000

10.070

19.000

10.071

20.000

24.000

30.000

24.010

31.000

35.000

33.000

40.000

33.010

44.000

44.010

45.000

Flow Rate:

0.4 mL min

Inj. Volume:

2.5 μL

-1

Detection:

Suppressed Conductivity, Dionex ASRS 300 Anion Self-Regenerating Suppressor (2 mm)*, 82 mA, external water mode

System Backpressure:

~3900 psi (1 mM KOH/8% CH3OH), ~4800 psi (60 mM KOH/11% CH3OH)

Background Conductance:

~0.16–0.7 μS

Noise:

~0.6–0.9 nS min-1, peak-to-peak

Run Time:

47 min

*Equivalent or improved results can be achieved on the Thermo Scientific™ Dionex™ AERS 500 Anion Electrolytically Regenerated Suppressor.

Preparation of Solutions and Reagents Stock Solutions of 29 Anions To prepare 1000 mg L-1 stock solutions of 29 inorganic and organic acid anions, use the compounds and masses listed in Table I. To prepare a standard mixture, mix appropriate volumes of the 1000 mg L-1 stock solutions.

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Table I. Masses of compounds used to prepare 1 L of 1000 mg L-1 anion stock solutions Anion

Compound

Mass (g)

Quinate

Quinic Acid

1.000

Fluoride

Sodium Fluoride

2.210

Lactate

Lactic Acid

1.000

Acetate

Sodium Acetate

1.390

Glycolate

Glycolic Acid

1.000

Propionate

Sodium Propionate

1.315

Formate

Sodium Formate

1.511

Butyrate

Butyric Acid

1.000

Pyruvate

Pyruvic Acid

1.000

Valerate

Valeric Acid

1.000

Galacturonate

Galacturonic Acid

1.098

Bromate

Sodium Bromate

1.179

Chloride

Sodium Chloride

1.648

Bromide

Sodium Bromide

1.288

Nitrate

Sodium Nitrate

1.371

Glutarate

Glutaric Acid

1.000

Succinate

Succinic Acid

1.000

Malate

Malic Acid

1.000

Malonate

Malonic Acid

1.000

Tartrate

Tartaric Acid

1.000

Maleate

Maleic Acid

1.000

Sulfate

Sodium Sulfate

1.479

Fumarate

Fumaric Acid

1.000

Oxalate

Sodium Oxalate

1.522

Phosphate

Potassium Phosphate, Monobasic

1.433

Citrate

Citric Acid

1.000

Isocitrate

DL-Isocitric Acid Trisodium Salt Dihydrate

1.306

cis-Aconitate

cis-Aconitic Acid

1.000

trans-Aconitate

trans-Aconitic Acid

1.000

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Working Standard Solutions Dilute 1000 mg L-1 galacturonate stock solution to prepare 2, 5, 10, 20, 50, 100, and 200 mg L-1 standards. Dilute 1000 mg L-1 malate stock solution to prepare 2, 5, 10, 20, 50, 100, 200, and 500 mg L-1 standards. Dilute 1000 mg L-1 tartrate stock solution to prepare 2, 5, 10, 20, 50, 100, and 200 mg L-1 standards. Dilute 1000 mg L-1 citrate stock solution to prepare 1, 2, 5, 10, 20, 50, 100, and 200 mg L-1 standards. Sample Preparation Dilute fruit juice samples 1:20 and filter through a Nalgene syringe filter prior to analysis. Dilute wine samples 1:20 and filter through a Dionex OnGuard II RP cartridge prior to analysis. Prepare the Dionex OnGuard II RP cartridge before use by flushing it first with 5 mL of methanol and then with 10 mL of DI water with maximum flow rate of 4 mL min-1. After filling a 5 mL syringe with sample, push the first 3 mL through the cartridge into a waste container and collect the next 2 mL for injection. Recovery Study For fruit juice samples, spike the appropriate amount of stock solutions into the samples during the 1:20 dilution before the filtration described above. For wine samples, spike the appropriate amount of stock solutions into the samples during the 1:20 dilution. Then filter the spiked samples through a Dionex OnGuard II RP cartridge before injection. System Preparation and Configuration Install and configure the Dionex AS-AP Autosampler in Push Mode. Follow the instructions in the Dionex AS-AP Autosampler Operator’s Manual (Document No. 065361) to calibrate the sample transfer line to ensure accurate and precise sample injections. Prepare the Dionex ASRS 300 Anion Self-Regenerating Suppressor for use by hydrating the internal membrane. Push 3 mL of DI water through the Eluent Out port and 5 mL of DI water through the Regen In port. Note: Allow the suppressor to sit for 20 min to ensure complete hydration before installing it in the system. Also note that when methanol is added to the eluent stream, the suppressor must be operated in the External Water mode. Configure the pressurized water reservoirs to supply external water for suppressor regeneration. Use at least two 4 L bottles plumbed in tandem to ensure uninterrupted external water delivery. Fill the reservoirs with DI water and apply 5–15 psi to the reservoir to deliver DI water through the regenerant channel. Ensure that the cap of the reservoir is sealed tightly. For more information on installation and operation of the Dionex ASRS 300 Anion Self-Regenerating Suppressor, consult the product manual (Document No. 031956). Condition the Dionex EGC 500 KOH cartridge before first use by running 50 mM KOH at 1 mL min-1 for 45 min. For more information on installation and operation of the Dionex EGC 500 KOH cartridge, consult the product manual (Document No. 065018-04). Install the Dionex IonPac AG11-HC-4 µm Guard (2 × 50 mm) and the Dionex IonPac AS11-HC-4 µm Analytical (2 × 250 mm) columns in the lower compartment of the DC detector. After connecting the inlet of the column, pump 30 mM KOH through the column with the outlet directed to waste for at least 30 min before connecting the column outlet to the suppressor using 0.005 in. i.d. PEEK tubing. Keep the lengths of the connective tubing to a minimum. After configuring the system, pump 8% CH3OH (92% Eluent A, 8% Eluent B) through the Dionex EGC 500 KOH cartridge at 0.4 mL min-1, set the KOH concentration at 1 mM, and set the suppressor current at 82 mA. Allow the system to equilibrate for at least 30 min before injection. 86

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RESULTS AND DISCUSSION Summary In this study, the determination of organic acids in juices and wines was demonstrated using a Dionex ICS-6000+ system. The efficient separation was achieved on a Dionex IonPac AS11-HC-4 µm column set, a high-resolution high-capacity anion-exchange product designed to resolve a large number of organic acids and inorganic anions using hydroxide gradient elution. The Dionex EGC 500 KOH eluent generator cartridge produced high-purity KOH, which ensured the excellent reproducibility of the method. A solvent gradient of 8–11% CH3OH was added to the KOH eluent to improve the resolution of a few close-eluting peaks. The separated analytes were detected using suppressed conductivity detection. Separation The performance of the Dionex IonPac AS11-HC (9 µm) and Dionex IonPac AS11-HC-4 µm column sets were compared for separation of a standard mixture containing 29 inorganic and organic acids anions. The chromatographic conditions were individually optimized for the two column sets. Despite a difference in optimal CH3OH concentration for the two column sets, a similar strategy for the separation was applied to both column sets. A KOH gradient was used to separate anions of different degrees of retention with minimal background shift. The separation was further optimized with CH3OH, because the solvating power and hydrophobicity of the organic solvent can influence the retention mechanism and improve the resolution of coeluting species [4,5]. However, with the addition of CH3OH to the eluent stream, the suppressor had to be operated in the External Water mode. The use of CH3OH caused a small increase in retention time and a certain decrease in peak response. A low eluent concentration (1 mM KOH) was used to separate the weakly retained anions, such as quinate, fluoride, lactate, acetate, and glycolate. Methanol was added to resolve acetate and glycolate, which would otherwise coelute. The eluent concentration was then gradually increased to elute more strongly retained anions. The percentage of CH3OH was increased to 11% at 20 min and remained at that level for 10 min, during which three previously coeluting groups of anions resolved, including nitrate, glutarate, succinate, and malate in the first group; malonate and tartrate in the second group; and fumarate and oxalate in the third group. To expedite the elution of late-eluting peaks, including phosphate, citrate, isocitrate, cis-aconitate, and trans-aconitate, no CH3OH was used from 33–44 min. The eluent condition was restored to the initial condition at 44 min to re-equilibrate the column prior to the next injection. As shown in Figure 1, 30 anions were separated on the Dionex IonPac AS11-HC (9 µm) and Dionex IonPac AS11-HC-4 µm column sets with the same elution order, as both are high-capacity anion-exchange products with a similar selectivity and capacity. The Dionex IonPac Figure 1. The organic and inorganic anion standard on AS11-HC column set is packed with 9 µm particles, (A) the Dionex IonPac AS11-HC (complete conditions as shown for System 1) and (B) the Dionex IonPac whereas the Dionex IonPac AS11-HC-4 µm column AS11-HC-4 μm columns (complete conditions as set is packed with 4 µm particles. Because smaller shown for System 2). particle sizes yield better overall peak efficiencies, the 87

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Dionex IonPac AS11-HC-4 µm column set offers much sharper peaks and thus improved resolution for close-eluting peaks [6]. Significant improvements in resolution were observed among weakly retained monovalent anions, including lactate, acetate, and glycolate, formate and butyrate; as well as more strongly retained divalent anion pairs, such as succinate and malate, malonate and tartrate, and sulfate and fumarate. Therefore, the remainder of this study was conducted using the Dionex IonPac AS11HC-4 µm column set. Calibration, Limit of Detection, and Limit of Quantitation In this study, four representative monovalent, divalent, and trivalent organic acids were selected for the calibration study. Galacturonate, malate, tartrate, and citrate are four of the major organic acids found in fruit juices and wines. Calibration curves with seven concentration levels ranging from 2 mg L-1 to 200 mg L-1 were constructed for galacturonate and tartrate. Calibration curves with eight concentration levels ranging from 2 mg L-1 to 500 mg L-1 and from 1 mg L-1 to 200 mg L-1 were constructed for malate and citrate, respectively. Due to incomplete dissociation of these weak carboxylic acids at high concentrations, the calibration curves show deviation from linearity in the selected calibration ranges [7]. Therefore, the calibration plots of peak area versus concentration were fit using quadratic regression functions with coefficients of determination (r2) >0.999. To determine the limit of detection (LOD) and limit of quantification (LOQ), the baseline noise was first determined by measuring the peak-to-peak noise in a representative 1 min segment of the baseline where no peaks elute but close to the peak of interest. The LOD and LOQ were then calculated from the average peak height of five injections of 0.2 mg L-1 each of the standards. The results of the calibration, LOD, and LOQ are summarized in Table II. Table II. Results of calibration, LOD, and LOQ Range (mg L-1)

Coefficient of Determination (r2)a

LODb (mg L-1)

LOQc (mg L-1)

Galacturonate

2–200

0.9999

0.069

0.23

Malate

2–500

0.9997

0.041

0.14

Tartrate

2–200

0.9998

0.053

0.18

1–200

0.9997

0.036

0.12

Analyte

Citrate a

Quadratic fit; LOD = 3 × S/N; LOQ = 10 × S/N b

Sample Analysis A number of fruit juices and different wine samples were studied. The various organic acids were identified by comparing their retention times with those of the standards. The concentrations of all the anions were estimated using the 29 anion standard mixture, except for galacturonate, malate, tartrate, and citrate, which were accurately quantified from their respective calibration curves. As noted in the chromatograms of the selected samples, dissolved CO2 appeared as the carbonate peak in all samples, but did not interfere with the peaks of interest. Pomegranate juice is gaining great attention for its perceived health benefits [8]. Because it is a high-value product, there is interest in authenticity testing for pomegranate juice. One of the common adulterants of pomegranate juice is grape juice, which can be added as a sweetener and coloring agent substitute for natural pomegranate color. One distinguishing difference is that tartaric acid is present in large amounts in grape juice but is either absent or present only in small quantities in pomegranate juice [9,10]. Citric acid is the predominant organic acid found in large quantity in pomegranate juice, as reported in other studies [11,12]. As shown in Figure 2, Chromatogram A, malic acid and citric acid are the main organic acids in the pomegranate juice sample. In comparison, the amount of tartaric acid is very low, 88

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indicating that this pomegranate juice is not adulterated with grape juice. Figure 2B shows the anionic profile of a pomegranate/blueberry juice sample. Quinic acid is found in blueberry juices and, as shown in Figure 2, quinate is absent in pomegranate juice but is present in the pomegranate/blueberry juice sample [13,14].

Figure 2. (A) Pomegranate juice and (B) pomegranate/blueberry juice with a 5% signal offset applied (complete conditions as shown for System 2).

In grape and white grape juices, malic acid, tartaric acid, and citric acid are the major acids (Figures 3 and 4). Among them, tartaric acid is the most abundant acid and its concentration is an important criterion for grape juice and wine stabilization [15]. Compared to white grape juice, grape juice contains a higher content of galacturonic acid, malic acid, and citric acid.

Figure 3. (A) White grape juice and (B) grape juice with a 5% signal offset applied (complete conditions as shown for System 2).

Figure 4. (A) White grape juice and (B) spiked white grape juice with a 5% signal offset applied (complete conditions as shown for System 2).

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In apple juice, quinic, galacturonic, and malic acids are the major acids, whereas tartaric acid is present in a trace amount. Malic acid is the most abundant acid in authentic apple juice, whereas tartaric acid is absent when quinic acid is present in apples, as shown in Figure 5 [16]. Galacturonic acid originates from pectin contained in the primary cell walls of terrestrial plants and can be used to indicate the pectin content in fruit samples [17]. Compared to other juices in this study, apple juice contains a low content of citric acid.

Figure 5. (A) Apple juice and (B) spiked apple juice with a 5% signal offset applied (complete conditions as shown for System 2).

Figure 6. (A) Merlot wine and (B) spiked Merlot wine with a 5% signal offset applied (complete conditions as shown for System 2).

For wine, a common differentiation is made between acids that originate from the grape (tartaric, malic, and citric acids) and those from the fermentation process (succinic, lactic, and acetic acids) [15,18,19]. Two dominant acids in wines are malic and tartaric acids, which are present in large quantities in ripe grapes and virtually determine the acidity of wines. As noted in Figures 6â&#x20AC;&#x201C;8, the ratio of tartrate to malate in Merlot wine is significantly higher than in Chardonnay and White Zinfandel wines. In addition, the Merlot wine contains the lowest concentration of citric acid among the three wine samples, because citric acid is usually not added to red wines. Although lactic acid was found in relatively small quantities in the selected juice samples, these wine samples contain much larger concentrations of lactic acid, which originated from the microbial fermentation process [20]. Similarly, these wine samples contain a higher amount of succinic acid when compared to the juice samples, again due to fermentation. Note the higher resolving power of the Dionex IonPac AS11-HC-4 Âľm column set over the Dionex IonPac AS11-HC column set in separating 30 anions in the standard mixture (Figure 1). The superior performance of the Dionex IonPac AS11-HC-4 Âľm column set is also shown through the comparison of anion separations in fruit juices and wines described in older Dionex Application Notes (ANs), such as AN 143 and AN 273. Considering the slight variations in composition of the fruit juice and wine samples studied in AN 143 and AN 273, the characteristic profiles of the selected samples (such as apple juice, grape juice, red wine, and white wine) show general similarities between the previous studies and this study.

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Compared to AN 143, better signal-to-noise (S/N) ratios for various organic acids are observed in the chromatograms of the apple and grape juices presented in this work as a result of higher-efficiency peaks [21]. In AN 273, the anions in wine samples were separated on a Thermo Scientific™ Dionex™ OmniPac™ PAX-100 column with slightly different column selectivity. This column was chosen because poor separations were observed among acetate, shikimate, and lactate and between succinate and malate using the Dionex IonPac AS11-HC column set, even with the aid of organic solvent elution [22]. In this study, acetate, lactate, succinate, and malate peaks are nearly baseline resolved and more anions are observed in the chromatograms here, likely the result of higher peak capacity delivered by the Dionex IonPac AS11-HC-4 µm column set.

Figure 7. (A) Chardonnay wine and (B) spiked Chardonnay wine with a 5% signal offset applied (complete conditions as shown for System 2).

Figure 8. (A) White Zinfandel wine and (B) spiked White Zinfandel wine with a 5% signal offset applied (complete conditions as shown for System 2).

Sample Accuracy and Precision To validate the determination of galacturonate, malate, tartrate, and citrate in the juices and wines, the selected samples were spiked with known amounts of standards at ~100% of the native concentrations. The recoveries of galacturonate, malate, tartrate, and citrate were in the range of 97.6–107%, 92.4– 107%, 97.5–103%, and 94.1–109%, respectively. The results obtained from the recovery study are summarized in Table III. Figures 4–8 show an overlay of the spiked and unspiked samples of white grape juice, apple juice, Merlot wine, Chardonnay wine, and White Zinfandel wine, respectively. Precision of the method was evaluated with five injections of a standard mixture containing 0.2 mg L-1 each of galacturonate, malate, tartrate, and citrate. The retention time RSDs and peak area RSDs of the four analytes are within 4% and 0.04% respectively (Table IV), indicating excellent method precision.

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Table III. Recoveries of galacturonate, malate, tartrate, and citrate in selected fruit juices and wines Galacturonate

Malate

Found (mg L-1)

Spiked (mg L-1)

Total (mg L-1)

Recovery (%)

Found (mg L-1)

Spiked (mg L-1)

Total (mg L-1)

Recovery (%)

White Grape Juice

39.5

45.0

87.9

108

86.2

165

92.4

Apple Juice

83.5

83.0

165

98.2

235

461

96.1

Merlot

59.7

60.0

120

100

11.2

43.3

107

Chardonnay

20.7

20.0

107

117

115

232

100

White Zinfandel

31.9

32.0

63.1

97.6

175

338

93.2

Sample

Tartrate

Citrate

Found (mg L-1)

Spiked (mg L-1)

Total (mg L-1)

Recovery (%)

Found (mg L-1)

Spiked (mg L-1)

Total (mg L-1)

Recovery (%)

White Grape

93.5

90.0

186

103

58.4

55.0

110

94.1

Apple Juice

<0.2

—

2.11

2.00

4.28

109

Merlot

99.6

95.0

193

98.4

4.61

4.00

8.76

104

Chardonnay

83.7

80.0

163

99.1

20.5

20.0

41.0

102

White Zinfandel

108

107

212

97.5

29.4

30.0

59.7

101

Sample

Table IV. Precisions of peak area and retention time for galacturonate, malate, tartrate, and citrate Analyte

Peak Area RSD

Retention Time RSD

Galacturonate

3.75

0.04

Malate

1.67

0.01

Tartrate

2.69

0.01

Citrate

4.03

0.01

CONCLUSION This study presents the characterization of ionic composition profiles in fruit juices and wines and the determination of organic acids in a selection of juice and wine samples. The separation of 30 anions on the Dionex IonPac AS11-HC (9 µm) and the Dionex IonPac AS11-HC-4 µm column sets are compared. The Dionex IonPac AS11-HC-4 µm column set offers superior resolving power for separation of the target anions. The suppressed conductivity detection offers high sensitivity for the anions, including various organic acids — even those present at low concentrations. The specificity and sensitivity of this method allow simple sample treatments without complex procedures such as extraction and/or derivatization. In addition, the recovery study shows good accuracy of the method. The electrolytically generated high-purity KOH and precise delivery of CH3OH through the proportioning valve ensure good peak area and retention time precisions. REFERENCES 1. Mato, I.; Suárez-Luque, S.; Huidobro, J. F. Food Res. Int., 2005, 38, pp 1175–1188. 2. Ehling, S.; Cole, S. J. Agric. Food Chem., 2011, 59, pp 2229–2234. 3. Kiss, J.; Sass-Kiss, A. J. Agric. Food Chem., 2005, 53, pp 10042–10050. 4. Hajós, P.; Nagy, L. J. Chromatogr., B: Anal. Technol. Biomed Life Sci., 1998, 717, pp 27–38. 5. Masson, P. J. Chromatogr., A, 2000, 881, pp 387–394. 6. Martin, C.; Coyne, J.; Carta, G. J. Chromatogr. A, 2005, 1069, pp 43–52. 92

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7. Brinkmann, T.; Specht, C. H.; Frimmel, F. H. J. Chromatogr., A, 2002, 957, pp 99–109. 8. Tezcan, F.; Gültekin-Özgüven, M.; Diken, T.; Özçelik, B.; Erim, F. B. Food Chem., 2009, 115, pp 873–877. 9. Krueger, D. A. J. AOAC. Int., 2012, 95, pp 163–168. 10. Poyrazoglu, E.; Gökmen, V.; Artk, N. J. Food Compos. Anal., 2002, 15, pp 567–575. 11. Gil, M. I.; Tomás-Barberán, F. A.; Hess-Pierce, B.; Holcroft, D. M.; Kader, A. A. J. Agric. Food Chem., 2000, 48, pp 4581–4589. 12. Gundogdu, M.; Yilmaz, H. Organic Acid, Phenolic Profile and Antioxidant Capacities of Pomegranate (Punica granatum L.) Cultivars and Selected Genotypes. Scientia Horticulturae (Amsterdam, Netherlands) 2012, 143, pp 38–42. 13. Wallrauch, S.; Greiner, G. Fluessiges Obst., 2005, 72, pp 14–17. 14. Jensen, H. D.; Krogfelt, K. A.; Cornett, C.; Hansen, S. H.; Christensen, S. B. J. Agric. Food Chem., 2002, 50, pp 6871–6874. 15. Soyer, Y.; Koca, N.; Karadeniz, F. J. Food Compos. Anal., 2003, 16, pp 629–636. 16. Fuleki, T.; Pelayo, E.; Palabay, R. B. J. Agric. Food Chem., 1995, 43, pp 598–607. 17. Luzio, G. A. Proc. Fla. State Hort. Soc. 2004, 117, pp 416–421. 18. Abrahamse, C. E.; Bartowsky, E. J. World J. Microbiol. Biotechnol., 2012, 28, pp 255–265. 19. López-Rituerto, E.; Cabredo, S.; López, M.; Avenoza, A.; Busto, J. H.; Peregrina, J. M. J. Agric. Food Chem., 2009, 57, pp 2112–2118. 20. Gupta, R.; Singh, S.; Thakur, A. Application of Pectinases in Apple Juice Clarification. In Biotechnology Applications; Mishra, C. S., Champagne, P., Eds.; I. K. International Publishing House Pvt. Ltd.: New Delhi, 2009; p 57. 21. Dionex (now part of Thermo Scientific) Application Note 143: Determination of Organic Acids in Fruit Juices. Sunnyvale, CA, 2003. 22. Dionex (now part of Thermo Scientific) Application Note 273: Higher Resolution Separation of Organic Acids and Common Inorganic Anions in Wine. Sunnyvale, CA, 2011.

This sponsor report is the responsibility of Thermo Fisher Scientifc.

Sponsor Report

Br. J. Anal. Chem., 2019, 6 (24) pp 94-97 PDF

This section is dedicated for sponsor responsibility articles.

Overcome the Memory Effect in Dioxins Extraction with Ethos X powered by fastEX-24 This report shows the use of Milestone Ethos X with fastEX-24 technology for extraction of dioxins from environmental matrices, with emphasis to one of the most common challenge of this application: the carryover effect. The unique design of the fastEX-24 rotor with disposable glass vials allow to perform easy and efficient extraction of dioxins and other organic pollutants, avoiding any memory effect. The fastEX24 simplify the routine pollutants extractions process and provide superior productivity at lower costs. INTRODUCTION Dioxin is one of the most toxic chemicals known, a serious public health threat. According to the US Environmental Protection Agency (EPA) reports, dioxin and dioxin-like chemicals have been associated to adverse health effects in the population. Dioxin is formed as an unintentional by-product of many industrial processes involving chlorine such as waste incineration, chemical and pesticide manufacturing and pulp and paper bleaching. Dioxin is a general term that describes a group of hundreds of chemicals that are highly persistent in the environment. Dioxins are classified as polychorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF) and dioxin-like PCBs (DLPCB). The analysis of this class of pollutants is fundamental for the environmental and human health protections, but due to the persistent nature of that compounds, the sample preparation and analysis is challenging for many extraction techniques. Many solutions are available for the extraction of dioxin such as soxhlet, automated soxhlet, sonication, pressurized liquid extraction and closed microwave vessels. A common and very critical issue in all sample preparation techniques is not only the dioxins extraction, but the subsequent memory effect/ carryover in the extraction cells. The stability of the dioxins requires long and tedious cleaning procedures of the extraction vessels. Ethos X equipped with the unique fastEX24 rotor is specifically designed to overcome the memory effect and cleaning by using disposable glass vials as reaction vessels, which are placed into a pressure reactor. This approach leads to ensure great analytical blanks and completely avoid carryover between runs. Milestone’s new Ethos X benchtop microwave extraction system offers the ability to extract up to 24 samples simultaneously in 40 minutes. The Ethos X with the fastEX 24 rotor is fully compliant with US EPA 3546 (100-115 ºC and 50-150 psi). In addition, disposable glass vials can accommodate sample up to 30 grams of sample if needed, thereby improving the limit of quantitation (LOQ) for analysis. This exceeds by far both the throughput and sample size capabilities of all the other automated and not automated techniques, such as pressurized fluid extraction. MATERIALS AND METHODS Instrument • Milestone Ethos X microwave system equipped with fastEX-24 extraction rotor • 100 mL disposable glass vials (PN GB00122) • Gas chromatograph with Mass Spectrometer detector (GC-MS) • Analytical balance • Vials for collection of extracts • Glass funnels for filtration • Glass fiber filters Standard and reagents Pesticide grade or grade solvents and chemicals must be used in all tests. The solvent mixture used was 1:1 acetone–hexane according to the US EPA 3546 method. 94

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Sodium sulfate anhydrous, silica gel and glass wool or paper filter were used in the work up procedure. According to the analytical method surrogate and internal standard could be used. Sample information The sandy soil standard reference material BCR®-529 was used for the recovery study on PCDD and PCDF [1]. Analytical Procedure Samples were weighed directly into the 100 mL extraction disposable glass vials. Solvent and in case, an aliquot of the surrogate solution were added to the glass vials than were closed. According to the moisture content, the best suitable built-in method was chosen. The extraction procedure so described follows the detailed method provided by U.S. EPA SW-846 Method 3546. Table I. Suggested solvent volumes according to the used sample amounts Sample amount (g)

Solvent mixture (mL)

Up to 10

10-20

20-30

50 Microwave Program

Step

Time (min)

Power (W)

Temperature (°C)

up to 1600*

110

up to 1600*

110

*The power applied depends on the moisture content. Dedicated methods are pre-loaded in the ETHOS X software according to the moisture content.

After the extraction, samples were filtered on glass fiber filters and sodium sulfate anhydrous and the vials were rinsed with additional solvent aliquots. Extracts and rinsates were collected together. Quantification Dioxins (PCDD and PCDF) analyses were performed with polar and non-polar columns according to the following method. Polar columns: Injection was through a split-splitless injector in a GC-MS equipped with 50 m x 0.20 mm i.d. capillary column (5% methylphenylsiloxane, 0.25 μm). The injector was maintained at 280 ºC. Interface temperature GC/MS: 300 ºC. The detector worked with EI (28 - 34 eV). Non-polar columns: Injection was through a split-splitless injector in a GC-MS equipped with 50 m x 0.22 mm i.d. capillary column (cyanopropyl siloxane, 0.25 μm). The injector was maintained at 240 ºC. Interface temperature GC/MS: 240 ºC. The detector worked with EI (28 - 34 eV). RESULTS AND DISCUSSION Beside the recovery study on PCDD and PCDF, this work was aimed to proof the efficacy of the disposable glass vial to avoid any carryover between runs. The results reported in table Table II shown recovery of all the molecules in the range of 80-120% 95

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with great reproducibility (RSD%). Table III shows the value of the blanks of the following run with new disposable glass vials (new disposable glass vials were used in order to proof the absence of memory effect). The data proof that the fastEX24 designed with disposable glass vial fully overcome memory effect/carryover from the entire vessel (all the blanks were below 0.065 μg/kg), ensuring accurate and precise results. The results demonstrate the efficiency of the Ethos X as sample preparation method for the determination of contaminants. Ethos X provides extracts with the lowest solvent usage and significant lower time compared to all the other extraction techniques, avoiding any memory effect. Table II. Recovery (n=4) of PCDD and PCDF from sandy soil standard reference material BCR®-529 (2g) Analyte

Certified value (μg/kg)

Ethos X (μg/kg)

Recovery (%)

RSD (%)

4.5±0.6

4.2

3.4

1,2,3,7,8-PeCDD

0.44±0.05

0.52

118

2.8

1,2,3,4,7,8-HxCDD

1.22±0.21

1.3

106

3.1

1,2,3,6,7,8-HxCDD

5.4±0.9

4.6

2.1

1,2,3,7,8,9-HxCDD

3.0±0.4

2.5

1.9

2,3,7,8-TCDF

0.078±0.013

0.075

2.7

1,2,3,7,8-PeCDF

0.145±0.028

0.116

3.5

2,3,4,7,8-PeCDF

0.36±0.07

0.33

2.6

1,2,3,4,7,8-HxCDF

3.4±0.5

3.4

100

1.9

1,2,3,6,7,8-HxCDF

1.09±0.15

1.08

3.8

1,2,3,7,8,9-HxCDF

0.022±0.010

0.018

3.6

2,3,4,6,7,8-HxCDF

0.37±0.05

0.45

122

2.2

2,3,7,8-TCDD

Table III. Blank values after PCDD and PCDF extraction (from Table II) Analyte

Blank value (μg/kg))

2,3,7,8-TCDD

<0.001

1,2,3,7,8-PeCDD

<0.001

1,2,3,4,7,8-HxCDD

<0.001

1,2,3,6,7,8-HxCDD

<0.001

1,2,3,7,8,9-HxCDD

<0.001

2,3,7,8-TCDF

<0.001

1,2,3,7,8-PeCDF

<0.001

2,3,4,7,8-PeCDF

<0.001

1,2,3,4,7,8-HxCDF

<0.001

1,2,3,6,7,8-HxCDF

<0.001

1,2,3,7,8,9-HxCDF

<0.001

2,3,4,6,7,8-HxCDF

<0.001

Sponsor Report

CONCLUSIONS The ETHOS X enables simultaneous dioxins extraction of up to 24 samples (from weighing to filtration steps) in less than 1 hour, offering superior productivity of any other technology in the market. In addition, the use of disposable glass vials fully solves a constant issue in Dioxins determination by avoiding tedious cleaning, memory effect and carryover. The use of contactless temperature control ensures high reproducibility and full recovery of the target analytes in full compliance with EPA 3546. Moreover, the above procedure it applies to a wide variety of samples, ensuring reliable extraction also on difficult samples such as solid waste and other environmental samples. The ETHOS X with all its unique features fully addresses the need of environmental laboratories in terms of productivity, ease of use, running costs, and extraction quality. REFERENCE 1. https://www.labmix24.com/files/info/22705.pdf This sponsor report is the responsibility of Milestone.

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Thermo Scientific TRACE 1300 Series Gas Chromatograph Productivity Solution for Your Needs The TRACE 1300 Series GC consists of two models designed to meet the specific needs of all laboratories. The TRACE 1310 GC features a complete icon-driven touch-screen user interface ideal for direct instrument control in larger routine and method development laboratories. The TRACE 1300 GC is the budget-conscious investment for the basic routine laboratory looking for an intuitive single-button system, that provides ease of use with minimal instrument interaction and full data system control. Both instruments offer the same userexchangeable, instant connect injector and detector modules and fast oven performance with exceptional retention time stability to reach an incredibly high lab productivity at reduced cost of ownership. Instant Connect Injector and Detector Modules User-installable miniaturized, plug-in injectors and detectors redefine usability in routine and high throughput laboratories. In two minutes, without special training or tools, the user can change the instrument configuration to respond to a specific work load by simply swapping injector and detector modules. This unique Instant Connect capability dramatically reduces any maintenance downtime by using back-up modules. Instant Connect Helium Saver Module Drastically reduce helium consumption and extend helium cylinder lifetime from 3 to 14 years per instrument, without any GC or GC-MS method modifications. Previously acquired retention times remain unchanged, and no method revalidation is required. This proprietary patented split/splitless injector module greatly reduces helium carrier gas consumption, using it only to supply the capillary column, while nitrogen is used for all other injection processes: inlet purge and septum, split flow and sample vaporization. Non-Cryogenic GCxGC Modulator based on microfluidic technology is available as Instant Connect Module, plug and play, easy to install. Powerful Breakthroughs for Ultimate Productivity Enjoy the benefits of a one-channel GC with industry-leading performance and increase productivity at any time by upgrading to a dual channel GC. Increased injector robustness enables the GC to handle dirtier matrices and reduce sample preparation, resulting in an increased savings of time and money. A completely new range of micro volume GC detectors guarantees higher sensitivity to limit sample reconcentration requirements or reduce injected sample amount. Fast peak detection and wide response linearity complement sensitivity to further boost laboratory performance. Performance Specifications â&#x20AC;˘ Typical retention time repeatability: <0.0008 min â&#x20AC;˘ Typical peak area repeatability: <0.5 % RSD

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Thermo Scientific Dionex ICS-6000 HPIC System A New Highly-Configurable Ion Chromatography System When addressing ion analysis challenges, there are sometimes more questions than answers. The ability to develop and run different methods for a single sample or for different samples is increasingly important for analytical laboratories. The Thermo Scientific Dionex ICS-6000 HPIC System is a highly flexible IC system that provides you with the freedom to develop, explore, and run different methods simultaneously. The Thermo Scientific™ Dionex™ ICS-6000 HPIC™ system is a truly modular, highly configurable, high-performance system. The robust system design enables operation at up to 5000 psi and produces consistent, reliable results. As a top-of-the line ion chromatography system, it is designed for users who want to push the boundaries of what is possible in ion analysis. The Dionex ICS-6000 HPIC System offers • High-pressure operation for fast analyses and high resolution • Reagent-Free™ system operation for reproducibility and ease-of-use • Modular system for adaptability/upgradability Accelerate Your Productivity • Single- and dual-channel system configuration options • Finger-tight connections that minimize dead volume and make connections easier • Automated tracking of the usage and performance of IC consumables • Automated eluent preparation using Reagent-Free IC-Eluent Generation (RFIC-EG™) technology • Tablet control of the IC system, to easily monitor sample runs • An optional, always ready capillary IC configuration to perform 24/7 sample analysis • IC columns featuring 4 μm particles to optimize chromatographic efficiency with shorter sample run times and/or improved resolution The Dionex ICS-6000 HPIC system is available in a variety of configurations, including • Standard bore, microbore, and capillary formats • Multiple, flexible detector options Discover Ion Chromatography-Mass Spectrometry The Dionex ICS-6000 HPIC system is compatible with single quadrupole, triple quadrupole, and high-resolution accurate-mass (HRAM) Thermo Scientific™ Orbitrap™ mass analyzer-based mass spectrometers for performing powerful ion chromatography-mass spectrometry (IC-MS) analyses. Solve Complex Analysis Challenges The Dionex ICS-6000 HPIC system can address the full range of IC analysis applications. It is suitable for techniques from 2-D ion chromatography for trace-level analysis to high-performance anionexchange chromatography with pulsed amperometric detection (HPAE-PAD) for complex carbohydrate analyses. 100

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Advanced Microwave Extraction System for Environmental Laboratories Microwave-assisted solvent extraction offers superior lab efficiency in the determination of organic pollutants with microwave green extraction technique. Typical applications include chlorinated pesticides, semi-volatile organics, PAHs, PCBs, chlorinated herbicides, phenols, organophosphorus pesticides, dioxins and furans. Determination of organic pollutants in environmental matrices is a common task for thousands of laboratories worldwide, as it leads to controlling and protecting our environment from high levels of contaminants. This analysis is often done to evaluate the effectiveness of a remediation process, to assess the contamination in waste, in waste landfills and for general environmental monitoring. Therefore, every day environmental laboratories deal with several challenges to ensure high quality data and fast turnaround time while maintaining their competitiveness. Extraction of pollutants from solid matrices is often performed with techniques that limit the productivity and have high running costs. Many laboratories still use the Soxhlet method that was developed in 1879! Milestone listened to the needs of environmental laboratory professionals by developing the ETHOS X with the fastEX-24 rotor, which allows for simultaneous extraction of 24 samples in 40 minutes with minimal solvent usage. By using large volume disposable glass vials, the fastEX-24 rotor simplifies handling and allows to achieve lower detection limits. • • • • •

High throughput: 24 samples in 40 minutes. Superior return of investment. Substantial reduction in solvent. Simple handling. Disposable glass vials. Consistency & Reproducibility. Consistent and reproducible results. Safety & Reliability.

Achieving lower detection limits with higher sample amount The ETHOS X with fastEX-24 rotor extracts up to 30 grams of sample with minimal solvent volume, helping analysts to accomplish their tasks. The Milestone fastEX-24 rotor uses disposable glass vials, eliminating the need for cleaning and the possibility of memory effect between different runs. The 100 mL vials can accommodate the extraction of a large sample amount. The easy to handle and affordable cost of the vials leads to high productivity at a very low running cost. ETHOS X system easily adapts to existing extraction chemistry through the use of a unique, patented material, called Weflon. Stir bars of Weflon are heated by microwaves and they subsequently transfer this heat to the non-polar solvent, which is not heated by microwaves. COMPLIANCE Several official methods describe the use of microwave closed-vessel technology to enhance the extraction efficiency of organic pollutants, such as US EPA 3546, ASTM and other national methods. The ETHOS X with fastEX-24 further enhances the performance of microwave technology for the extraction of water-insoluble or slightly water-soluble organic compounds from soils, clays, sediments, sludges, and solid wastes. 102

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PITTCON is the Worldâ&#x20AC;&#x2122;s Leading Annual Conference and Exposition on Laboratory Science Pittcon attracts 16,000 attendees from industry, academia and government from over 90 countries worldwide. From laboratory scientists, academicians to researchers in molecular and biological sciences, the PITTCON, a non-profit organization has been a pioneer in providing educational and scientific assistance to individuals who wish to carve a niche for themselves in this world of constant change to excel and provide best services. PITTCON not only covers analytical chemistry and spectroscopy, but also showcases developments made in the field of food safety, environmental sciences, bioterrorism and pharmaceutical industry. Established since 1950, PITTCON works in collaboration with Spectroscopy Society of Pittsburgh (SSP) and the Society of Analytical Chemists of Pittsburgh (SACP) to help in the development, research and future excellence of science education and its implementation for providing best medical assistance. Pittcon, a vital resource for knowledge, happens yearly to help keep you informed of, connected to and up-to-date on these significant ongoing findings and new instrumentation.

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Videos Developing 3D Separation Systems for Complex Analyses Dr. Peter Schoenmakers, Professor of Analytical Chemistry at the University of Amsterdam, discusses his work on improving existing chromatographic methods and developing new multi-dimensional methods for more complex applications. Schoenmaker explains how his lab is using virtual reality (VR) to translate older techniques, such as single-layer chromatography and 2D gel electrophoresis, in multiple planes, as well as embracing 3D printing to product prototypes. Watch this video here Solvent Extraction and GC Analysis of Pesticides in Cannabis Products The Scientists’ Channel speaks to Dr. Nathaly Reyes Garcés, Colton Myers, and Ashlee Gerardi from Restek Corporation. Here, they explain a simple workflow for the accurate analysis of pesticides in cannabis products via solvent extraction and GC analysis and outline how Restek are focussing on helping customers with bespoke cannabis testing workflows and method development, in the face of ongoing changes within the industry, new regulations coming into place, and state-to-state differences. Watch this video here Webinar How to Optimize Workflows for Sample Preparation and Rapid Analysis of Contaminants in Foods by LC-MS/MS This expert webinar presents insights into recent advances in chromatography technology and how to apply them to food commodity testing workflows to overcome common challenges, simplify sample preparation, speed up analysis and minimize reporting of false negatives and positives in your final results. Attend the webinar here

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CHROMacademy is the world’s largest eLearning website for analytical scientists. With a vast library of high-quality animated and interactive eLearning topics, webcasts, tutorials, practical information and troubleshooting tools CHROMacademy helps you refresh your chromatography skills or learn something completely new. A subscription to CHROMacademy provides you with complete access to all content including: • Thousands of eLearning topics covering HPLC / GC / Sample Prep / Mass Spec / Infrared / Basic Lab Skills / Biochromatography Each channel contains e-Learning modules, webcasts, tutorials, tech tips, quick guides and interactive tools and certified assessments. With over 3,000 pages of content, CHROMacademy has something for everyone. • Video Training Courses Each course contains 4 x 1.5-hour video training sessions, released over 4 weeks, with full tutor support and certification. • Ask the Expert – 24-hour Chromatography Support A team of analytical experts are on hand to help fix your instrument and chromatographic problems, offer advice on method development & validation, column choice, data analysis and much more. • Assessments Test your knowledge, certificates awarded upon completion. • Full archive of Essential Guide Webcasts and Tutorials Over 70 training topics covered by industry experts. • Application Notes and LCGC Articles The latest application notes & LCGC articles. • Troubleshooting and Virtual Lab Tools Become the lab expert with our HPLC and GC Troubleshooters. • User Forum Communicate with others interested in analytical science. Lite members have access to less than 5% of CHROMacademy content. Premier members get so much more! For more information, please visit www.chromacademy.com/subscription.html 108

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Gradient HPLC for Practitioners: RP, LC-MS, Ion Analytics, Biochromatography, SFC, HILIC Stavros Kromidas, Editor April 2019. Publisher: John Wiley & Sons This practical guide for analytical scientists explains the use of gradients in liquid chromatography. The fundamentals of gradient separations, as well as the most common application scenarios are addressed, from LC-MS coupling to biochromatography to the separation of ionic substances. Throughout, this handy volume provides detailed hands-on information for practitioners, enabling them to use gradient separation methods reliably and efficiently. Read more … Software-Assisted Method Development in High Performance Liquid Chromatography Szabolcs Fekete, Imre Molnár, Editors January 2019. Publisher: World Scientific This handbook gives a general overview of many possibilities in recent developments in chromatographic retention modeling. As a result of the latest developments in modeling software, several new features are now accessible, opening a new level in HPLC method development. Several modes of chromatography, including RPLC, IEX, HIC, and HILIC are explained in detail. Beside the industrial and practical benefits of retention modeling, the possibilities in teaching are also illustrated. Finally, numerous representative industrial examples are shown. Read more ... Sample Preparation in LC-MS Bioanalysis Wenkui Li, Wenying Jian, Yunlin Fu, Editors February 2019. Publisher: John Wiley & Sons This book is a thorough and timely guide to all important sample preparation techniques used for quantitative LC-MS bioanalysis of small and large molecules. This text provides researchers in industry, academia, and regulatory agencies with detailed sample preparation techniques and step-by-step protocols on proper extraction of various analyte(s) of interest from biological samples for LC-MS quantification, in accordance with current health authority regulations and industry best practices. Read more … Advances in Chromatography, Volume 56 Nelu Grinberg, Peter W. Carr, Authors July 2019. Publisher: CRC Press The clear presentation of topics and vivid illustrations for which this series has become known makes the material accessible and engaging to analytical, biochemical, organic, polymer, and pharmaceutical chemists at all levels of technical skill. Key Features: includes a chapter dedicated to Izaak Maurits Kolthoff, offering a unique look at his non-professional life as well as his impact and legacy in Analytical Chemistry; discusses recent advances in two-dimensional liquid chromatography for the characterization of monoclonal antibodies and other therapeutic proteins; reviews solvation processes, methodologies of their measurement, and parameters influenced solvation; explores recent advances in TLC analysis of natural colorings, determination of synthetic dyes, and determination of EU-permitted natural colors, in foods; offers comprehensive and critical insights on the key aspects of CE-MS analysis of intact proteins. Read more …

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Notices of Books

Basic Gas Chromatography, 3rd Edition Harold M. McNair, James M. Miller, Nicholas H. Snow, Authors August 2019. Publisher: John Wiley & Sons This book provides a quick need-to-know introduction to GC, still the most widely used instrumental analysis technique, and is intended to assist new users in gaining understanding quickly and as a quick reference for experienced users. The new edition provides updated chapters that reflect changes in technology and methodology, especially sample prep., detectors and multidimensional chromatography. The book also covers new detectors recently introduced and sample preparation methods that have become much more easily accessible since the previous edition. Read more … Hyphenations of Capillary Chromatography with Mass Spectrometry Peter Q. Tranchida, Luigi Mondello, Editors November 2019. Publisher: Elsevier This book provides comprehensive coverage of capillary chromatography with mass spectrometry—both single and multidimensional approaches. The book examines nearly all capillary chromatography approaches, combined with a variety of MS forms, giving readers a wide and detailed view on current-day analytical strategies and applications. Of particular focus are novel developments in the field of MS, such as the Orbitrap, HR ToF, ToF MS with variable electron-impact energy, fast MS-MS and APGC technology. Read more … A Practical Guide to Gas Analysis by Gas Chromatography John Swinley, Piet de Coning, Authors May 2019. Publisher: Elsevier This book provides a detailed overview of the most important aspects of gas analysis by GC for both the novice and expert. Authors provide the necessary information on the selection of columns and components, thus allowing the reader to assemble custom gas analysis systems for specific needs. The book brings together a wide range of disparate literature on this technique that will fill a crucial gap for those who perform different types of research. This highly practical, up-to-date reference can be consulted in the lab to guide key decisions about proper setup, hardware and software selection, calibration, analysis, and more, allowing researchers to avoid the common pitfalls caused by incorrect infrastructure. Read more … Modern Supercritical Fluid Chromatography: Carbon Dioxide Containing Mobile Phases Larry M. Miller, J. David Pinkston, Larry T. Taylor, Authors December 2019. Publisher: Wiley Explains why modern supercritical fluid chromatography (SFC) is the leading “green” analytical and purification separations technology. SFC is the leading method used to analyze and purify chiral and achiral chemical compounds, many of which are pharmaceuticals, pharmaceutical candidates, and natural products including cannabis-related compounds. This book covers current SFC instrumentation as it relates to greater robustness, better reproducibility, and increased analytical sensitivity. The authors provided readers with an overview of analytical and preparative SFC equipment, stationary phases, and mobile phase choices. Read more …

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Periodicals & Websites

Br. J. Anal. Chem., 2019, 6 (24)

American Laboratory The American Laboratory® publication is a platform that provides comprehensive technology coverage for lab professionals at all stages of their careers. Unlike singlechannel 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 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, 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, 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

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Events

Br. J. Anal. Chem., 2019, 6 (24)

September 1 - 5 Brazilian Symposium of Electrochemistry and Electroanalysis - XXII SIBEE Convention Center Ribeirão Preto, Ribeirão Preto, SP, Brazil www.xxiisibee.com.br September 1 - 5 Euroanalysis XX - Europe’s Analytical Chemistry Meeting Istanbul, Turkey http://euroanalysis2019.com/ September 3 - 5 Brazilian Meeting on Chemical Speciation - EspeQBrasil-2019 Universidade Federal da Bahia, Campus de Ondina, Salvador, BA, Brazil http://www.espeqbrasil2019.ufba.br/ September 8 - 11 133rd AOAC Annual Meeting & Exposition Sheraton Denver Downtown Hotel, Denver, CO, USA http://www.aoac.org September 24 - 26 15th Analitica Latin America Expo & 6th Analitica Congress Centro de Exposições São Paulo Expo, São Paulo, SP, Brazil https://www.analiticanet.com.br/en September 25 - 26 Women in Science: The Federal University of Pelotas discusses the Challenges and Perspectives of Including New Talent Pelotense Public Library (opening ceremony) and the Auditorium of the UFPel Arts Center, Pelotas, RS, Brazil https://wp.ufpel.edu.br/elasnaciencia/ October 28 - 31 XXI Brazilian Congress of Toxicology & XV The International Association of Forensic Toxicologists (TIAFT) Latin-American Regional Meeting Águas de Lindóia, SP, Brazil http://www.cbtox-tiaft.org/ November 5 - 8 9th International Symposium on Recent Advances in Food Analysis - RAFA 2019 Prague, Czech Republic http://www.rafa2019.eu November 6 - 8 XIV Latin American Symposium on Environmental Analytical Chemistry (LASEAC) & IX National Meeting on Environmental Chemistry (ENQAmb) Bento Gonçalves, RS, Brazil http://www.laseac2019.furg.br

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Guidelines for Authors

PDF

Scope The Brazilian Journal of Analytical Chemistry (BrJAC) is dedicated to the diffusion of significant and original knowledge in all branches of Analytical Chemistry. BrJAC is addressed to professionals involved in science, technology and innovation projects in Analytical Chemistry, at universities, research centers and in industry. Professional Ethics Manuscripts submitted for publication in BrJAC cannot have been previously published or be currently submitted for publication in another journal. BrJAC publishes original, unpublished scientific articles and technical notes that are peer reviewed in the double-blind way. Review process 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 reviewers who are experts in the related field. As evaluation criteria, the reviewers employ originality, scientific quality, contribution to knowledge in the field of Analytical Chemistry, the theoretical foundation and bibliography, the presentation of relevant and consistent results, compliance to the BrJAC’s guidelines, and the clarity of writing and presentation. BrJAC is a quarterly journal that, in addition to scientific articles and technical notes, also publishes reviews, interviews, points of view, letters, sponsor reports, and features related to analytical chemistry. Brief description of the documents that can be submitted by the authors • Articles: Full descriptions of an original research finding in Analytical Chemistry. 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. Reviews 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 doubleblind full peer review. The title of the manuscript submitted for technical note must be preceded by the words “Technical note”. • 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 BrJAC editor-in-chief. • Points of view: The expression of a personal opinion on some relevant subject in Analytical Chemistry. Points of View are welcome and will be published at the discretion of the BrJAC editor-in-chief. • Releases: Articles providing new and relevant information for the community involved in Analytical Chemistry. Download a template here Path: Log In / Manuscript Submission / Online Submission

Manuscript (MS) preparation • Language: English is the language adopted by BrJAC. • Required items: the MS must include a title, a graphical abstract, an abstract, keywords, and the following sections: Introduction, Materials and Methods, Results and Discussion, Conclusion, and References. • Identification of authors: the MS must NOT contain the authors’ names nor affiliations. This information must be in the cover letter to the editor-in-chief. This rule is necessary because the MS is subjected to double-blind review. • Layout: the lines in the MS must be numbered consecutively and double-spaced throughout the text. 114

Guidelines

• Graphics and Tables: must appear close to the discussion about them in the text. For figures use Arabic numbers, and for tables use Roman numbers. • Permission to use content already published: for figures, graphs, diagrams, tables, etc. identical to others previously published in the literature, the author must ask for publication permission 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. • 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. • References: must be cited by numbers in square brackets. 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 by the authors before submission. See how to format the references in the following item. Examples of reference formatting Journals 1. Orlando, R. M.; Nascentes, C. C.; Botelho, B. G.; Moreira, J. S.; Costa, K. A.; Boratto, V. H. M. Anal. Chem. 2019, 91 (10), pp 6471-6478 (https://doi.org/10.1021/acs.analchem.8b04943).

• Publications with more than 10 authors, list the first 10 authors followed by a semicolon and et al. • Titles of journals must be abbreviated as defined by the Chemical Abstracts Service Source Index (http:// cassi.cas.org/search.jsp).

Electronic journals 2. Sapozhnikova, Y.; Hoh, E. LCGC North Am. 2019, 37 (1), pp 52-65. Available from: http:// www.chromatographyonline.com/suspect-screening-chemicals-food-packaging-plastic-filmcomprehensive-two-dimensional-gas-chromatogr [Accessed 20 January 2019]. 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. Master’s and doctoral theses or other academic literature 6. Dantas, W. F. C. Application of multivariate curve resolution methods and optical spectroscopy in forensic and photochemical analysis. Doctoral thesis, 2019, Institute of Chemistry, University of Campinas, Campinas, SP, Brazil. Patents 7. Trygve, R.; Perelman, G. US 9053915 B2, June 9, 2015, Agilent Technologies Inc., Santa Clara, CA, US. 115

Guidelines

Web pages 8. http://www.chromedia.org/chromedia [Accessed 10 January 2019]. Unpublished source 9. Viner, R.; Horn, D. M.; Damoc, E.; Konijnenberg, A. Integrative Structural Proteomics Analysis of the 20S Proteasome Complex (WP-25). Poster presented at the XXII International Mass Spectrometry Conference (IMSC 2018) / August 26-31, 2018, Florence, IT. 10. Author, A. A. J. Braz. Chem. Soc., in press. 11. Author, B. B., 2017, submitted for publication. 12. Author, C. C., 2018, unpublished manuscript. Note: Unpublished results may be mentioned only with express authorization of the author(s). Personal communications can be accepted exceptionally.

Download templates here Path: Log In / Manuscript Submission / Online Submission Manuscript submission Three different files, as described below, must be sent online through the website www.brjac.com.br 1. A Cover Letter (PDF file): addressed to the editor-in-chief, with the manuscript title, the full names of the authors and their affiliations, and the complete contact information of the corresponding author, including the ORCID iD. This letter should also inform to which section of the BrJAC the manuscript is being submitted (e.g. Article, Review, Technical Note, Point of View or Letter). The cover letter should also contain a statement that the article has not been previously published and is not under consideration for publication elsewhere. 2. The manuscript PDF file that must NOT mention the names of the authors nor their affiliations. 3. A similarities analysis report on the manuscript obtained through an anti-plagiarism software. BrJAC indicates CopySpiderÂŠ freeware to support similarities checking analyzes. Download the CopySpider freeware at: www.copyspider.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. A revised manuscript should be submitted by the authors, containing the changes made in the manuscript clearly highlighted. Letters to the reviewers, one to each reviewer, must also be submitted answering in detail to the questions made by them, and describing the changes made in the manuscript. 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 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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