The development and application of green analytical techniques aiming at the sample preparation of complex matrices for the study of organic compounds have been growing considerably over the last 15 years. Miniaturisation, automation and solventless techniques are gaining importance in this field, associated to others, as is the case of metrics. However, the unreflected use of the so-called green analytical techniques “might lead to doing the same things better, rather than rethinking solutions altogether”. Some limits and potentialities of the green sample preparation towards sustainable separations of organic compounds using the biorefinery concept will be also discussed in this paper, a promising biobased route that can integrate sustainable extraction and purification processes in a whole complete circular unity.
Especially after the industrial revolution, analytical chemistry has been considered central in all research areas, manufacture sectors, regulatory agencies and human activities, enabling the qualitative and quantitative understanding of natural and artificial complex systems. For such challenging matrices, not only the amount and identity of the components, but also how they are distributed in space and time as well as the best ways to measure them are fundamental data to be known, which include the design of the necessary analytical instruments and approaches [1, 2]. In spite of the present concern about the socio-environmental impacts caused by the chemical practices from lab scale to production processes over the last decades, most of the conventional analytical methods employed for the determination of organic compounds in several matrices show negative effects, mainly due to the high quantities of hazardous substances involved in multiple steps of the whole procedure. In many contexts, these procedures can generate a larger amount and variety of toxic residues which can be more deleterious than the studied analytes .
From the 1960s onwards, when processes and chemicals showed adverse effects well beyond their intended use, the society began to realise that catastrophic implications could result from conventional chemicals and practices, which, among other initiatives, contribute to generate laws and regulations to address the problems derived from chemistry in diverse contexts. As a result, a philosophy known as green chemistry started and got much attention in the 1990s worldwide and is defined as the utilisation of a “set of principles that reduces or eliminates the use or generation of hazardous substances in design, production and application of chemicals” .
According to US Environmental Protection Agency (EPA), the efforts to speed the adoption of this philosophy have led to significant environmental benefits, innovation and a strengthened economy, emphasising that green chemicals will save industry $65.5 billion by 2020 . The RD&I in green chemistry have grown significantly over the last 15 years and, as can be seen in Fig. 1, a total of 9774 papers have been published during this period, among which 1330 manuscripts focus on green analytical chemistry (ISIS Web of Knowledge; Thomson Reuters) (Fig. 1). Not surprisingly, one of the papers that has the highest number of citations was written by Anastas and Kirchhoff (>880), pointing out that among the main topics studied in green chemistry, analytical method development has always occupied a primordial place in this wide research field [6, 7].
Green analytical chemistry can be understood as the development and application of safer techniques and methods that generate less hazardous waste. As expected, some principles are more directly related to analytical chemistry, as the prevention of waste, the use of ideally innocuous reagents and renewable materials, omission of derivatisation steps, real-time analysis, the application of practices that minimise accidents and design for energy efficiency. In fact, no analytical procedure is completely benign, but it is necessary to follow this ideal to make a method as environmentally friendly as possible. Therefore, operating conditions associated with adequate quantity and low toxicity of reactants, products and by-products are considered as valuable as other parameters of critical analytical quality, such as selectivity, sensitivity, accuracy, precision, linearity and robustness . Additionally, another important dimension that should be taken into account is the cost of a method. Nowadays, increased greenness and efficiency do not always equate with overall reduced cost as a number of factors need to be considered, e.g., price of reagents, energy used in the system, disposal of by-products, etc. . However, a more comprehensive economical understanding of the greening efforts in method development will be one of the most attractive aspects of green analytical chemistry in the near future. New devices and systems are available in the market at a competitive cost, providing analytical determinations as fast-as-possible where, in many cases, time is also a matter of business (Fig. 2) .
In general, analytical processes encompass sample collection, transportation, storage, followed by sample preparation and final determination. Overall, sample preparation is considered the most demanding stage, consuming around 60–80% of the analysis time and reagents for the treatment and separation of the components or fractions of interest. This step is the primary source of error, and is most often a necessity since the simplest samples are frequently inadequate for direct determination, which arise from excessive dilution of the analytes, the high number of interferents or from the incompatibility with the standard operational requirements for some equipment .
In order to make sample preparation greener, a number of approaches for the determination of organic compounds have been described in the literature, e.g., techniques based on miniaturised liquid-liquid extraction (LLE), solid-phase extraction (SPE), solid-phase microextraction (SPME), headspace (HS) analysis, membrane techniques or the use of alternative solvents .
It is noteworthy that the increasing amount of data obtained with the use of instrumentation for high throughput screening has also driven the development of tools for data processing, with special attention to chemometric methods. Also, another important and relatively incipient approach is the use of metrics, which enable a meaningful assessment of green processes corresponding to the principles of green chemistry. In real-world scenarios, these metrics can provide part of the story of a process, i.e., encompassing “shades of green” [10, 11].
Green metrics cover topics as resources, processing, materials, cleaning and renewability. Amongst them, life cycle assessment (LCA) is a methodology that explores the environmental impact assessment known as a cradle-to-grave evaluation, in which the resource consumption, pollutants generated, and their environmental impacts are taken into account and assessed at each stage .
As can be noted, the trends in analysis and monitoring indicate that progress has been particularly prominent in the development of those methods and techniques ensuring compliance with green analytical chemistry principles, being solvent-free sample preparation techniques of particular interest . Based on current achievements , and on the personal author’s experience and views on present and future issues related to sample preparation of complex matrices, a new perspective about sustainable separations of organic compounds related to the biorefinery concept will be discussed.
Trends and challenges in green sample preparation: demands for plural point of view
Ideally, the best sample preparation is “no sample preparation”, but this can be a far-away dream mainly because of the complexity of the samples and the concentration of the analytes . However, depending on the case, some approaches can be of great interest. For instance, the direct determination of rutin in green tea infusions was successfully performed using square-wave voltammetry with a rigid carbon-polyurethane composite electrode. It was observed that for tea infusions the electroanalytical method with deconvolution procedure could be applied without previous separation, showing that from 75.4 to 91.3×10–6 mol L–1 of this flavonoid was present in the commercial teas investigated. In addition, very satisfactory reproducibility (3.5%) and LOD values for rutin determinations were found, similar to the chromatographic ones (2.5×10–6 mol L–1) .
However, despite the current degree of development of the instrumentation used for final determinations, this ideal approach cannot be fully accomplished for the majority of real samples. Concepts like miniaturisation, hyphenation, integration and simplification became key ones that have already been shown to effectively contribute to face a number of drawbacks of conventional sample preparation methods and that, in some studies involving size-limited samples, can probably be considered the best, if not the only, analytical alternatives [3, 8, 17, 18]. Aiming at illustrating some of the sample preparations to determine organic compounds proposed by the author, examples of those will be presented, which can reduce, replace or eliminate the use of hazardous solvents, as well as minimise the number of steps of the whole method, which guarantee all the necessary analytical quality parameters for the given cases. It is important to highlight that these case studies can be considered greener and simpler alternatives to solve (or contribute to face) real problems related to socio-environmental studies, quality control of phytomedicines, healthy food and food security, among other topics of ultimate importance all over the world.
Reduction, replacement and solvent elimination: the next step forward?
Over recent years, a number of techniques that make it possible to reduce, replace or avoid solvents derived from nonrenewable resources have been developed or improved for the extraction and concentration of analytes in a fast, efficient and reproducible way. In this group accelerated solvent extraction (ASE), supercritical fluid extraction (SFE), membrane extraction, ultrasonic extraction and microwave extraction may be included. While ASE usually employs pressure and temperature above 200°C, the other techniques use lower temperatures, allowing the extraction of thermolabile analytes, which is especially interesting for polyphenols or essencial oils in food, phytocosmetics and phytomedicines [3, 19].
A fast multiresidue method to determine organochlorine and organophosphorus pesticides in Passiflora alata Dryander and Passiflora edulis Sims. f. flavicarpa Deg. leaves by SFE and high-resolution gas chromatography with electron-capture and flame photometric detection (HRGC-ECD/FPD) was successfully proposed. The mild extraction conditions [pure CO2; 100 bar (1 bar=105 Pa) and 40°C; 5 min static plus 10 min dynamic extraction time; C18 trap and elution with 1 mL n-hexane at 2 mL min–1] allow for direct analysis by HRGC-ECD/FPD with no additional clean up. The method provided analytical results that are similar to or even better than the official pharmacopoeia procedures, once it is simpler, cheaper and greener. Mean recoveries of 69.8–107.1% were obtained, with 1.4–14.7% of reproducibility. Commercial samples of Passiflora L. from Brazil were analysed and 23% of them showed the presence of the organochlorine or organophosphorus pesticides investigated, most with dieldrin, lindane, tetradifon, chlorothalonil and α-endosulfan at 21–71.4 ng g–1 level). Although the levels of contamination found were lower than the maximum residue limit stipulated by the European pharmacopeia, due to their high persistence in body tissues, even small amounts of such compounds may represent a serious risk to human health .
In the same direction, methods based on adaptations of polypropylene membranes filled with a small amount of solvents have been proposed (MASE). One of these methods was compared to the solventless stir bar sorptive extraction (SBSE) for the determination of 18 organic contaminant residues in Brazilian sugarcane juice. Stir bar sorptive extraction and thermal desorption coupled to capillary gas chromatography-mass spectrometry using the selected ion monitoring mode [SBSE-TD-GC-MS(SIM)] and membrane-assisted solvent extraction combined with large volume injection [MASE-LVI-GC-MS(SIM)] methods were assessed taking into account the time of extraction [SBSE (3 h) and MASE (30 min)], the recoveries [SBSE (0.2–55.3%) and MASE (13.6–103.1%)], the repeatability [SBSE (0.3–19.2%) and MASE (2.6–18.4%)] and the limits of detection [SBSE (0.002–0.71 μg L–1) and MASE (0.004–0.56 μg L–1)] of the selected triazine, organochlorine and organophosphorus pesticides as well as benzo[a]pyrene in sugarcane juice. In general, faster analyses and much better analyte recovery results were achieved with MASE, whereas greater sensitivity and repeatability were obtained with SBSE. SBSE and MASE procedures were applied to the analysis of six sugarcane juice samples from São Carlos city, in the state of São Paulo, Brazil. A comparison of the results of the pesticide and benzo[a]pyrene levels obtained by the two methods demonstrated good agreement .
As can be noted, SBSE has many similarities to the solid phase microextraction (SPME), as it is also a solventless sample preparation technique, and it uses similar sorbents (based on PDMS). In SBSE, the analytes present are aqueous samples and are extracted by stirring for a certain time with a PDMS-coated stir-bar. The stir-bar is then removed from the sample, and the absorbed compounds are either thermally desorbed and analysed by GC, or desorbed by means of a liquid for interfacing to a LC system. Usually, heat-desorption provides higher sensitivity while liquid desorption provides higher selectivity .
A simple and alternative green methodology for headspace (HS) SPME using a new fibre coated with polydimethylsiloxane-poly(vinyl alcohol) (PDMS/PVA) was reported for the trace determination of organochlorine and organophosphorus pesticides in herbal infusions of Passiflora L. by GC-ECD. The capacity of the PDMS/PVA coating for the pesticides was compared to that of commercial PDMS fibres, with advantageous results. The effects of parameters such as the sample ionic strength, dilution of the infusion, extraction temperature (T) and time (text) were investigated. In order to shorten and make the method even greener, the last two parameters were studied simultaneously through a multivariate approach (22 factorial designs). The upper and lower levels for the variables in the initial experiments were 50°C and 70°C (T) and 20 min and 40 min (text). After the initial factorial design, five additional experiments using T ranging from 57°C to 78°C and text ranging from 17 min to 38 min were performed. The results were fitted to a proper response surface, which was employed to find values of T and text where the extraction efficiency was maximised. The optimised conditions for the determination of the selected pesticides in Passiflora L. infusions were extraction time and temperature, respectively, of 38 min and 67.5°C, with 5 min of sample/headspace equilibration time. The detection limits for the analytes varied from 0.01 ng mL–1 (α-endosulfan) to 1.5 ng mL–1 (malathion). The sensitivity of studied methodology was adequate, as well as its accuracy (78.7–91.5%) and precision (1.2–14.2%) .
Additionally, the application of the direct coupling of SPME with mass spectrometry (MS), a technique known as fibre introduction mass spectrometry (FIMS), to determine organochlorine and organophosphorus pesticides in herbal infusions of Passiflora L. was described . The new fibre coated with a composite of PDMS/PVA was used, resulting in sensitive, selective, simple and simultaneous quantification of several pesticides by monitoring diagnostic fragment ions of m/z 266 (chlorothalonil), m/z 195 (a-endosulfan), m/z 278 (fenthion), m/z 263 (methyl parathion) and m/z 173 (malathion). Simple HS-SPME extraction (25 min) and fast FIMS detection (less than 40 s) of pesticide residues from a highly complex herbal matrix provided good linearity with correlation coefficients of 0.991–0.999 for concentrations ranging from 10 to 140 ng mL–1 of each compound. Good accuracy (80–110%), precision (0.6–14.9%) and low limits of detection (0.3–3.9 ng mL–1) were also obtained. Even after 400 desorption cycles inside the ionisation source of the mass spectrometer, no visible degradation of the novel PDMS/PVA fibre was detected, confirming its suitability for FIMS. Fast (ca 20 s) pesticide desorption occurs for the PDMS/PVA fibre due to the small thickness of the film and its reduced water sorption.
As can be seen briefly, there has been a crescent demand for the establishment and application of green techniques, materials and methods in analytical chemistry, especially for sample preparation of complex matrices containing a high variety and quantity of organic compounds . However, together with these instrumental aspects, the proposal of innovative approaches in this field encompassing a deeper and holistic multidimensional understanding of our natural and constructed world is mandatory. This means that we need to avoid thinking and acting in the same way “just doing the same things better”, without modifying the core of the unsustainable thoughts and practices .
Sustainable separations: biorefinery concept as a starting point
First of all, it is essential to decide what we want and/or need to separate in a complex system. This is also about our capacity of discrimination, i.e., the establishment of the difference between one thing and another and the best way to materialise it. Separation is one of the fundamental processes in chemistry and chemical engineering, involving multiphase and/or multicomponent distinctions. Nearly all reactions generate co-products or residues that usually require extraction and/or purification steps; the same occurs with natural products or raw materials before their use, transformation or removal. For instance, a bottleneck for the lignocelluloses industrialisation is the lack of an efficient and robust separation technique . If the components of the lignocellulosic feedstock cannot be separated adequately, the control of the quality and characteristics of the final products can be impossible. Besides not playing an important role in improving economic efficiency, the lignocellulose industry has also become a source of waste. It is estimated that 350 million tons of lignocellulose waste is available per year only in Brazil, showing that the country has not reached its full potential yet, especially important for the sugarcane bagasse .
In this context, sustainable separations can be understood in the scope of biorefinery concept, conceived as an integrated unity for the conversion of biomass into multiple value-added products including energy, chemicals and materials. According to this model, the circular system can create flexible, zero waste networks, which are applicable to a variety of low value local feedstocks or, ideally, biomass residues (such as forest and agro-industrial residues, food waste, etc.) . Based on our experience, biorefineries are more than promising approaches that are able to generate energy, solvents and biobased chemicals, but also unities that can either produce and/or separate (extracted/purified) high-value compounds using the energy and materials manufactured in the same system. These are the cases of CO2, solvents or biofuels (bioethanol), catalysts or even carbonaceous mesoporous material employed as sorbents [17, 28] (Fig. 3).
For instance, from an orange peel based-biorefinery it is possible to produce fibre, essential oil, essences, d-limonene, ethanol, seed oil, limonoids as well as pectin that can be transformed and employed as solid phase to separate endogenous citrus compounds that were extracted in the same processing unity . Based on this circular model, the pectin derived from citrus peel can be used as starting material for the preparation of mesoporous carbon chromatographic columns which, in turn, allows the determination of high-value natural compounds present in the same fruits or their wastes. Pectin aerogel can be prepared in a three-step process consisting of gelation in water, addition of TBA to the desired TBA-water ratio, and freeze-drying, followed by a pre-treatment to be used as a sorbent . In fact, the method based on solid phase extraction (SPE) using residual derived-pectin as sorbent was successfully applied to the determination of nine flavonoids commonly found in citrus waste from Brazil, showing similar accuracy and reproducibility of those determined by conventional C18-SPE, which recoveries from 80.4 to 118.4% (RSD from 0.6 to 10.5%) at 10 ng mL–1 level [17, 30].
The tuneable properties of mesoporous polysaccharide-derived materials from waste can lead to the production of a set of sustainable sorbents that can fit a wide variety of applications. This holistic approach can aggregate analytical methods from lab to large scale, including the biochemical, thermochemical, extraction and purification processes with internal reuse/recycle of materials (as gases, water) and energy. In addition, to guarantee as much as possible sustainable processes that are not only dependent on the “stuffs”, but mainly responsible decision-makers, it is important to perform interdisciplinary studies involving contents from chemistry, engineering, bioeconomy, social science, anthropology fields, etc. [26, 31–34]. However, such interdisciplinary studies associated to the establishment of a biorefinery model that allows to identify possible valuable components derived from biomass waste with a maximum efficiency and the least steps possible is a challenging task yet.
Indubitably, green analytical methods are of fundamental importance to reduce socio-environmental impacts derived from chemical analyses, especially of sample preparation in manufacturing activities, research or regulation, from public or private institutions. Among them, miniaturisation and solvent elimination are of particular interest currently. As has already been stated, extraction of valuable natural products before their transformation during biochemical and thermal treatments can increase the overall financial returns significantly, but it requires a prospective interdisciplinary study involving professionals from other fields. The transition from an instrumental to a more holistic science model that demands a responsible participation to better understand and decide what, how and why to separate the components (or fractions) of a complex mixture is in progress.
Thus, biorefinery is a new perspective beyond the principles of green chemistry so that, based on a comprehensive design, a number of so-called wastes derived from agro-industrial biomass can be employed to prepare solvents and other materials especially useful in making alternative tools, equipment as well as proposing new techniques and methods for the determination of a given compound in different samples, normally of high complexity. As an example, pectin extracted from citrus peel can be used as starting material for the preparation of mesoporous carbon chromatographic columns for SPE, allowing the extraction and purification of high-value natural products present in the same wastes, as is the case of flavonoids. This new circular approach constitutes a real opportunity to bring together sustainable separations and an integrated web of systems that use biological resources, maximise value creation and, as far as possible, work in close conjunction in terms of raw materials, water, by-products, costs and energy supported by interdisciplinary research teams.
A collection of invited papers based on presentations at the 5th international IUPAC Conference on Green Chemistry (ICGC-5), Durban (South Africa), 17–21 August 2014.
The author thanks her colleagues form the Department of Chemistry at UFSCar and in the Green Chemistry Centre at the University of York for their support for the work described here as well as our sponsors including Fapesp (2013/12052-5 and 2014/50.827-1) and Capes (002032/2014-07).
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