APPLICATION REPORT | LABORATORY EQUIPMENT | MICROWAVE DIGESTION S. 1/4 APPLICATION REPORT | LABORATORY EQUIPMENT Microwave digestion Diluted HNO3 as green chemistry reagent for sample preparation Sample preparation Evaluation of different oxidant mixtures for microwave digestion of biological samples Different oxidant mixtures were evaluated for microwave-assisted digestion of biological samples. Parameters as HNO3 concentration (2, 7, and 14 mol/L), use of H2O2 and internal volume of the digestion vessels (30 and 100 mL) were studied. The quality of digestion was evaluated by determining residual carbon content and acidities of the digested solutions. The use of diluted HNO3 (2 mol/L) plus H2O2 is recommended due to its high effectiveness. Quantitative recoveries were reached for Al, B, Ca, Cu, Fe, K, Mg, Mn, P, and Zn. Authors Lucimar L. Fialho, Érica F. Batista, Amália G. G. Pessoa, Edenir R. PereiraFilho, Joaquim A. Nóbrega, Group of Applied Instrumental Analysis, Department of Chemistry, Federal University of São Carlos, São Carlos, SP, Brazil APPLICATION REPORT | LABORATORY EQUIPMENT | MICROWAVE DIGESTION Introduction Analysis of solid samples often requires a pretreatment prior to analyte determination which is usually the most time consuming step of the whole analytical sequence. 1,2 Most of the conventional sample preparation methods for atomic spectrometric techniques involve complete or partial matrix decomposition (digestion) using concentrated acids. The most commonly used oxidizing acid is HNO3. The use of concentrated acids always bears a certain safety and health risk especially for inexperienced user, e.g. chemical burns or adverse health effects upon inhalation. Moreover, it also requires higher dilution of the solutions leading to higher amount of disposal.3 Aiming to circumvent these difficulties, procedures based on diluted solutions of HNO3 were developed for digestion of biological samples.4,5 The efficiency of diluted HNO3 solutions for oxidation of organic matter can be explained by the regeneration of this acid promoted by the oxidation of NO to NO2 and the absorption of this latter compound in the solution followed by its disproportioning reaction.2 Other important aspect for understanding the efficiency of this process is the temperature gradient inside the microwave-assisted heated vessels that plays a fundamental role in the regeneration of nitric acid.2 Therefore, in this study microwave-assisted digestion procedures were investigated using solutions containing 2, 7, or 14 mol/L HNO3. The use of H2O2 and two types of digestion vessels (DAP-30+ and DAP-100+) were also studied. Chicken thigh and forage (Brachiaria brizantha Stapf. cv. Marandu) samples were digested as typical examples of animal and vegetable tissues. Material and Methods | Reagents and Solutions All reagents were of analytical grade and deionized water (18.2 M/cm) produced using a Milli-Q® Plus Total Water System (Millipore Corp., Bedford, MA, USA) was used to prepare all solutions. Prior to use, all glassware and polypropylene flasks were washed with soap, soaked in 10% v/v HNO3 for 24 h, rinsed with deionized water and dried to ensure that no contamination may occur. Concentrated HNO3 was previously purified using a sub-boiling apparatus BSB-939-IR distillacid (Berghof, Eningen, Germany). Carbon reference solutions used for external calibration to determine carbon content in digests (CCD) were prepared by dissolution of urea (Reagen, Rio de Janeiro, RJ, Brazil) in water (15–5000 mg/L of C). Aluminum, B, C, Ca, Cu, Fe, K, Mg, Mn, P, and Zn were determined by ICPOES (iCAP 6000, Thermo Scientific, Waltham, MA, USA) with external calibration using analytical solutions containing from 1.0 to 20 mg/L, prepared in 0.14 mol/L HNO3 by appropriate dilution of the stock solution (1000 mg/L) of each analyte (Qhemis, Jundiaí, SP, Brazil). Standardized NaOH (Qhemis) solution (0.1968 mol/L) was prepared for determination of residual acidity in digests by acid-base titration. S. 2/4 Instrumentation A microwave oven (Speedwave four, Berghof) equipped with twelve digestion vessels completely made of TFMTM-PTFE was used in all experiments (Fig. 1). The internal vessels volume used were 30 mL (DAP-30+) or 100 mL (DAP-100+). The maximum operational pressures of these vessel types are 80 and 40 bar, respectively. The maximum operational temperature of both vessel models is 230°C. Internal pressure and sample temperature were real-time controlled and monitored by contact-less optical sensor technique in every single vessel. Fig. 1: Vessels completely made of TFMTM-PTFE For the determination of CCD, digested solutions were analyzed by ICP-OES (Thermo Fisher Scientific, Cambridge, UK), model iCAP 6300 Duo. Plasma operating conditions are listed in Table 1. These parameters were used as recommended by the instrument manufacturer. Argon 99.996% (White MartinsPraxair, Sertãozinho, SP, Brazil) was used in all ICP-OES measurements. Table 1: Operational conditions used in ICP OES Parameter Operating Condition RF applied power (W) 1150 Nebulizer gas flow rate (L/min) 0.7 Coolant gas flow rate (L/min) 12 Auxiliary gas flow rate (L/min) 0.5 Sample flow rate (mL/min) 1.1 Viewing mode Axial and/or radial Integration time (s) 5 Emission lines (nm) Al II 167.079 K I 691.107 B I 249.773 Mg II 279.553 C I 193.091 Mn II 259.373 Ca I 431.865 P I 185.942 Cu I 327.396 Zn I 213.856 Fe II 259.940 I - atomic lines / II - ionic lines L_Brasil_53-0215-97-01-00-005.docx, Subject to changes and errors, Printed in Germany APPLICATION REPORT | LABORATORY EQUIPMENT | MICROWAVE DIGESTION Sample preparation Samples from animals (chicken thigh) and vegetables (forage) were used in this study. The forage sample is an in-house reference material supplied by Embrapa Cattle-Southeast, São Carlos, SP, Brazil. The forage sample was oven-dried at 65 °C for 48 h. Chicken thigh sample was previously lyophilized and homogenized. Afterwards, samples were ground using a cryogenic mill (Model 6750, CertiPrep Spex, Metuchen, NJ, USA). Reaction vessels made of TFMTM-PTFE (DAP-30+ and DAP100+) were used for evaluation of different digestion conditions in the Speedwave four. The parameters evaluated were HNO3 solution at different concentrations (2, 7, or 14 mol/L), with or without adding concentrated H2O2 (30% m/m). Sample aliquots of 200 mg (DAP-30+) or 500 mg (DAP-100+) were accurately weighed using an analytical balance (model AY 220, Shimadzu, Kyoto, Japan). Forage and chicken thigh were microwave digested using 5.0 mL of HNO3 (2, 7, or 14 mol/L) and 2.0 mL of 30% m/m H2O2 or only 7.0 mL of HNO3 (2, 7, or 14 mol/L). The microwave oven heating program was performed as shown in Table 2. Digested solutions were quantitatively transferred to polypropylene flasks and diluted with water up to 25 mL. Further dilution was performed to ensure a maximum residual acidity of 0.7 mol/L HNO3 before ICP-OES measurements. Table 2: Temperature program Step T [°C] p [bar] * Ta [min] Time [min] Power [%] 1 170 30 or 70 5 5 40 2 190 30 or 70 5 30 70 3 50 30 or 70 1 10 0 4 50 0 0 0 0 5 50 0 0 0 0 (*) Pressure limit for operation: 30 bar in vessels of 100 mL and 70 bar in vessels of 30 mL. al. These conditions contribute to minimum dilution of sample solutions and avoid loss of detection power. It is shown in Table 3 that the use of a digestion mixture composed of HNO3 plus H2O2 leads to the highest residual acidities. It may be concluded that at least part of H2O2 is thermally decomposed and the generated O2 promotes the regeneration of HNO3 during the digestion process. Therefore, in all experiments using H2O2 the residual acidities are close to the initial acidity. The effect of vessel volume was not critical, but of course its choice is strongly dependent on sample mass and consequently on the pressure increase during the digestion process. It can be concluded that both vessel types were efficient for the analysis of biological material using diluted acid solution. For evaluation of accuracy of the method apple leaves (SRM 1515, NIST, Gaithersburg, MD, USA) were analyzed. Approximately 200 mg of sample were digested using 5.0 mL of HNO3 (2 mol/L) and 2.0 mL of 30% m/m H2O2 in DAP-30+ vessel. Results are shown in Table 4. Recoveries were generally good for all elements (86 to 118 %), except Fe that was low (68 %). Usually Fe is associated to Si in plant leaves. Since no HF was used for digestion it is likely that low recovery of Fe is explained by refractory compounds in the sample matrix. An unpaired t test was performed in order to compare the determined and certified values and no differences were observed for Al (95 %), B (99 %), Ca (95 %), Cu (95 %), K (95 %), Mg (95 %) and P (95 %). For Fe, Mn and Zn we observed differences either with 95 or 99 % of confidence level, but recoveries were in the range from 68 % (Fe) to 118 % (Zn). The CCD in this digestate was 0.11 ± 0.02 %. Despite these observations by using t-test, it should be mentioned that standard deviations are low and recoveries are in acceptable range. Table 4: Determined and certified values (mean ± standard deviation, n = 3) for apple leaves Analyte Results and Discussion The extent of digestion in all experiments was evaluated by measuring CCD and residual acidities (Table 3). The study with DAP-30+ showed lower CCD values than those observed with DAP-100+. This result was expected because the digestion carried out in DAP-30+ used lower sample mass. In all conditions, low values of CCD from 0.07 to 1.34% proved the proper digestion efficiency for all concentrations of HNO3. Maximum CCD was 1.34% indicating full compatibility for further determinations by ICP OES. According Castro et al.4 efficient digestions should allow a complete decomposition of organic material using minimal amounts of HNO3 and leading to low residual carbon contents and residual acidities. The conditions presented in this paper support the observations made by Castro et S. 3/4 Determined Certified value Recovery value (mg/kg) (mg/kg) (%) 292 ± 33 286 ± 9 102 ± 12 B 26 ± 0.3 27 ± 2 97 ± 1 Ca 16,675 ± 1035 15,260 ± 150 109 ± 7 Cu 4.87 ± 0.58 5.64 ± 0.24 86 ± 10 Fe 56 ± 2 83 ± 5 68 ± 2 K* 18,299 ± 1387 16,100 ± 200 114 ± 9 Mg 2,746 ± 172 2,710 ± 80 101 ± 6 Mn 51.4 ± 0.4 54 ± 3 95 ± 1 P 1,771 ± 113 1,590 ± 110 111 ± 7 Zn 14.8 ± 0.4 12.5 ± 0.3 118 ± 3 *axial mode L_Brasil_53-0215-97-01-00-005.docx, Subject to changes and errors, Printed in Germany APPLICATION REPORT | LABORATORY EQUIPMENT | MICROWAVE DIGESTION S. 4/4 Table 3: Carbon content in digests and residual acidities for digestions in different conditions (mean ± standard deviation, n = 3) Vessel Digestion solution [HNO3]/mol/L CCD (%) Chicken thigh DAP-30+ HNO3 14 0.14 ± 0.01 0.19 ± 0.14 8.0 ± 0.6 9.5 ± 2.7 DAP-30+ HNO3 7 0.30 ± 0.05 0.15 ± 0.05 4.7 ± 1.0 3.4 ± 0.6 DAP-30+ HNO3 2 0.47 ± 0.07 0.09 ± 0.02 1.1 ± 0.0 0.8 ± 0.0 DAP-30+ HNO3:H2O2 14 0.34 ± 0.01 0.11 ± 0.02 13.8 ± 0.1 13.5 ± 1.0 DAP-30+ HNO3:H2O2 7 0.38 ± 0.01 0.07 ± 0.00 6.6 ± 1.0 6.0 ± 0.7 DAP-30+ HNO3:H2O2 2 0.49 ± 0.04 0.07 ± 0.02 1.7 ± 0.0 1.6 ± 0.2 DAP-100+ HNO3 14 0.82 ± 0.05 0.21 ± 0.03 10 ± 0.5 7.5 ± 0.8 DAP-100+ HNO3 7 0.81 ± 0.03 0.28 ± 0.02 4.4 ± 0.1 4.4 ± 0.4 DAP-100+ HNO3 2 1.34 ± 0.02 0.66 ± 0.22 0.6 ± 0.1 0.4 ± 0.2 DAP-100+ HNO3:H2O2 14 0.84 ± 0.04 0.33 ± 0.03 13.3 ± 0.4 12.1 ± 1.7 DAP-100+ HNO3:H2O2 7 0.90 ± 0.02 0.40 ± 0.06 6.5 ± 0.3 6.4 ± 1.2 DAP-100+ HNO3:H2O2 2 1.16 ± 0.08 0.39 ± 0.01 2.2 ± 0.4 1.8 ± 0.5 Conclusions The results prove that microwave-assisted digestion using Speedwave four oven is efficient. The digestion in both vessel types was promoted with efficiency and without any safety risks. Using both vessel types, efficient digestions can be carried out using 2 mol/L of HNO3 plus H2O2. Recoveries were quantitative for nine analytes. It may be concluded that microwave-assisted digestion using diluted acids is a good alternative towards the development of green chemistry procedures for sample preparation. Acknowledgement The authors would like to thank equipments loan provided by Analitica-Thermo and Berghof. Forage Residual acidities (mol/L) Chicken thigh Forage References 1. Krug, F. J., Ed., Métodos de Preparo de Amostras: Fundamentos sobre Preparo de Amostras Orgânicas e Inorgânicas para Análise Elementar. 1st ed., Piracicaba, SP, Brazil, 2008. 2. Bizzi, C. A.; Flores, E. M. M.; Barin, J. S.; Garcia, E. E.; Nóbrega, J. A.; Microchem. J., 99 (2011) 193-196. 3. Arruda, M. A. Z., Ed., Trends in Sample Preparation. 1st ed., New York, Nova Science, 2007, v.1., 99 (2011) 193–196. 4. Castro, J. T.; Santos, E. C.; Santos, W. P. C.; Costa, L. M.; Korn, M., Nóbrega, J. A.; Korn, M. G. A.; Talanta, 78 (2009), 1378-1382. 5. Araújo, G. C. L.; González, M. H.; Ferreira, A. G.; Nogueira, A. R. A.; Nóbrega, J. A.; Spectrochim. Acta B 57 (2002) 2121-2132. Contact: Raquel Rainone | T +55.11.2162-8080 | Nova Analitica Imp. Exp. Ltda, São Paulo, PP, Brazil | raquel.rainone@novanalitica.com.br Alberto Iglesias-Vila | T +49.7121.894-202 | laboratorytechnology@berghof.com Dr. Kerstin Dreblow | T +49.7121.894-202 | laboratorytechnology@berghof.com Berghof Products + Instruments GmbH | Harretstrasse 1 | 72800 Eningen | www.berghof.com L_Brasil_53-0215-97-01-00-005.docx, Subject to changes and errors, Printed in Germany