Diluted HNO₃ as Green Chemistry Reagent for Sample Preparation

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Vessels completely made of TFM-PTFE

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. 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 WM/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 ICP-OES (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.

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Speedwave Microwave digestion system

Instrumentation
A Speedwave microwave oven equipped with twelve digestion vessels completely made of TFM™-PTFE was used in all experiments (Fig. 1). The internal vessels volume used was 100 ml. The maximum operational pressures of these vessel type is 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.

For the determination of CCD, digested solutions were analyzed by ICP-OES (Thermo Fisher Scientific, Cam-bridge, 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 Martins-Praxair, Sertãozinho, SP, Brazil) was used in all ICP-OES measurements.

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 massive TFM™-PTFE were used for evaluation of different digestion conditions in the Speedwave microwave digestion system. 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 500 mg 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.

(*) Pressure limit for operation: 30 bar in vessels of 100 ml.

Results and Discussion
The extent of digestion in all experiments was evaluated by measuring CCD and residual acidities (Table 3). In all conditions, low values of CCD from 0.21 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 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.

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.

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.

Conclusions
The results prove that microwave-assisted digestion using Speedwave microwave oven is efficient. The digestion was promoted with efficiency and without any safety risks. An 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 Nova Analitica and Berghof.

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. astro, 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.