Flow-Batch Sample Preparation for Fractionation of the Stress Signaling Phytohormone Salicylic Acid in Fresh Leaves.

Salicylic acid (SA) is an important stress signaling phytohormone and plays an essential role in physiological processes in plants. SA fractionation has been carried out batchwise, which is not compatible with the high analytical demand in agronomical studies and increases susceptibility to analytical errors. In this context, a novel flow-batch sample preparation system for SA fractionation on fresh plant leaves was developed. It was based on microwave-assisted extraction with water and conversion of the conjugated species to free SA by alkaline hydrolysis. Free and total SA were quantified by fluorimetry after separation by sequential injection chromatography in a C18 monolithic column. The proposed procedure is directly applicable to plant leaves containing up 16 mg kg-1 SA, with a limit of detection of 0.1 mg kg-1 of SA, coefficient of variation of 3.0% (n = 10), and sampling rate of 4 samples h-1. The flow-batch sample preparation system was successfully applied to SA fractionation in sugarcane, corn, and soybean leaves without clogging or increasing in backpressure. The proposed approach is simple, less time-consuming, and more environmentally friendly in comparison to batchwise procedures.

Systematic evaluation of sample preparation for fractionation of phytohormone salicylic acid in fresh leaves.

The determination of salicylic acid (SA), an important phytohormone responsible for stress signaling in plants, is of great importance in agricultural studies. However, a critical evaluation of the procedures for the extraction of the analytes and hydrolysis of the conjugated forms of SA is lacking in the literature and the available alternatives are complex, time-consuming, and laborious. In this study, the sample preparation methods for SA fractionation were critically evaluated to develop a simpler and faster alternative procedure. Microwave-assisted extractions were carried out with 2.0 g of fresh leaves and 8.0 mL of a 75% v/v ethanol:water solution at 40 °C for 10 min, followed by alkaline hydrolysis using 100 µL of 0.1 mol L-1 NaOH at 80 °C for 60 min. Free and total SA were determined in crude and hydrolyzed extracts, respectively, by fluorimetry after chromatographic separation of the sample matrix under isocratic elution (25% v/v acetonitrile/phosphate buffer) using a C18 column. Recovery experiments using methyl salicylate and acetylsalicylic acid model compounds demonstrated that the soft microwave-assisted extraction did not decompose the SA derivatives and that alkaline hydrolysis was quantitative. The proposed procedure was successfully applied for fractionation of SA in sugarcane, corn, and soybean leaves with extraction and hydrolysis yields up to 70 and 20% higher than those achieved in previously proposed approaches, respectively. The developed procedure is a simple, fast, and reliable alternative for SA fractionation in crude extracts without sample clean-up, and utilizes dilute reagents and green solvents.

Performance evaluation of a high-pressure microwave-assisted flow digestion system for juice and milk sample preparation.

Acid digestion is usually required for metal determination in food samples. However, this step is usually performed in batch mode which is time consuming, labor intensive, and may lead to sample contamination. Flow digestion can overcome these limitations. In this work, the performance of a high-pressure microwave-assisted flow digestion system with a large volume reactor was evaluated for liquid samples high in sugar and fat (fruit juice and milk). The digestions were carried out in a coiled perfluoroalkoxy (PFA) tube reactor (13.5 mL) installed inside an autoclave pressurized with 40 bar nitrogen. The system was operated at 500 W microwave power and 5.0 mL min-1 carrier flow rate. Digestion conditions were optimized with phenylalanine, as this substance is known to be difficult to digest completely. The combinations of HCl or H2O2 with HNO3 increased the digestion efficiency of phenylalanine, and the residual carbon content (RCC) was around 50% when 6.0% V/V HCl or H2O2 was used in combination with 32% V/V HNO3. Juice samples were digested with 3.7 mol L-1 HNO3 and 0.3 mol L-1 HCl, and the RCC was 16 and 29% for apple and mango juices, respectively. Concentrated HNO3 (10.5 mol L-1) was successfully applied for digesting milk samples, and the RCCs were 23 and 25% for partially skimmed and whole milk, respectively. Accuracy and precision of the flow digestion procedure were compared with reference digestions using batch mode closed vessel microwave-assisted digestion and no statistically significant differences were encountered at the 95% confidence level. Graphical abstract Application of a high-pressure microwave-assisted flow digestion system for fruit juice and milk sample preparation.

Proposed flow system for spectrophotometric determination of fluoride in natural waters.

An environmentally-friendly flow system was developed for the determination of fluoride in natural waters, based on its reaction with zirconium ions and 2-(parasulfophenylazo)-1,8-dihydroxy-3,6-naphthalene-disulfonate (SPADNS). The flow system was designed with solenoid valves in order to increase the versatility and improve the analytical performance, since each device handles the solution independently leading to a more efficient use of reagents. The minimization of reagent consumption and waste generation favors the development of a noteworthy system. Under optimized conditions, a linear response was observed within the range of 0.1-2.2 mg L(-1), with the detection limit, coefficient of variation and sampling rate estimated as 0.02 mg L(-1), 4.1% and 60 determinations per hour, respectively. In order to analyze samples containing high fluoride content a wider linear range (0.3-6.6 mg L(-1)) can be obtained by using a low sample flow rate and low sample volume. In comparison with other methods, the proposed system offers advantages such as wider analytical range (1.7-2.2 times), higher sample throughput (1.5-4.0 times) and lower quantification limit (by a factor of 5-68). Along with the consumption of reagents being 20-55 times lower, the generation of wastes is 1.2-3.0 times lower, which contributes to the development of a 'greener' system. This method is fast, amenable to automation, environmentally-friendly and of low-cost. In addition, it could be successfully applied to the determination of fluoride in water samples, the results obtained being in agreement with those of the ISE method.

Removal of Ni(II) from aqueous solution using Moringa oleifera seeds as a bioadsorbent.

Metal contaminants are generally removed from effluents by chemical and physical processes which are often associated with disadvantages such as the use of toxic reagents, generation of toxic waste and high costs. Hence, new techniques have been developed, among them the study of natural adsorbents, for instance, the use of Moringa oleifera seeds. The potential of M. oleifera seeds for nickel removal in aqueous systems was investigated. The seeds utilized were obtained from plants grown in Uberlândia/Brazil. After being dried and pulverized, the seeds were treated with 0.1 mol/L NaOH. Fourier transform infrared spectroscopy, scanning electron microscopy and thermogravimetric analyses were used for the characterization of the material. Using the optimized methodology (50 mL of 4.0 mg/L Ni(II), pH range of 4.0-6.0, agitation time of 5 min and adsorption mass of 2.0 g) more than 90% of Ni(II) could be removed from water samples. The sorption data were fitted satisfactorily by the Langmuir adsorption model. Evaluation applying the Langmuir equation gave the monolayer sorption capacity as 29.6 mg/g. The results indicate that this material could be employed in the extraction of nickel, considering its ease of use, low cost and environmental viability, which make it highly attractive for application in developing countries.