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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
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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
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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 | [email protected]
Alberto Iglesias-Vila | T +49.7121.894-202 | [email protected]
Dr. Kerstin Dreblow | T +49.7121.894-202 | [email protected]
Berghof Products + Instruments GmbH | Harretstrasse 1 | 72800 Eningen | www.berghof.com
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