Analytica Chimica Acta 522 (2004) 179187

Ultrasound-assisted extraction procedure for the fast estimation of major, minor and trace elements in lichen and mussel samples by ICP-MS and ICP-AES
M.V. Balarama Krishna, J. Arunachalam
Department of Atomic Energy, National Center for Compositional Characterization of Materials (CCCM), Bhabha Atomic Research Centre, ECIL Post, Hyderabad 500062, India Received 10 March 2004; received in revised form 6 July 2004; accepted 6 July 2004 Available online 9 August 2004

Abstract The application of ultrasound-assisted extraction procedure has been examined for the estimation of major, minor and trace elements in a set of bio-environmental samples (IAEA lichen 336 and mussel tissue NIST 2976) as a possible alternative to conventional digestion methods. Inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma atomic emission spectrometry (ICP-AES) were used for the quantication of the elements. Parameters affecting extraction such as extractant concentration, sonication time and ultrasound amplitude were optimized to get quantitative recovery of elements. These studies indicate that the method is a fast (within 15 min including centrifugation time) and simple method for the estimation of Na, K, Ca, Mg, Cr, Mn, Co, Ni, Cu, Zn, Ge, As, Se, Rb, Sr, Zr, Ag, Cd, In, Sb, Cs, Ba, Pb and Bi. Quantitative recoveries for most of the elements for which certied concentrations available, were obtained using a 1% (v/v) HNO3 as extractant and metal solubilization can be achieved within 4 min sonication time at 40% sonication amplitude and 100 mg sample weight. The supernatant obtained upon centrifugation was used for analysis. Quantitative recovery was achieved for many elements which are well in agreement with certied values. An overall precision of better than 10% could be achieved for many elements in multiple extractions. Similar extraction procedure was tested on two eld lichen samples. Closed-microwave digestion method was also used for the estimation of various elements in lichen and mussel samples for comparison purposes. 2004 Elsevier B.V. All rights reserved.
Keywords: Lichens; Mussel tissue; Ultrasound-assisted extraction; Microwave digestion; ICP-MS; ICP-AES

1. Introduction Human activities often mobilize and redistribute natural substances in the environment so much so that they can cause adverse effects [1]. Trace elements pollutants have received considerable attention since they have a signicant effect on its ecological quality. Various plant species are being extensively used as bio-monitors by many of environmental research groups for monitoring air pollution and to follow changes in pollution patterns [2,3]. Lichens and mosses, which depend on surface absorption of nutrients, have been
Corresponding author. E-mail address: aruncccm@rediffmail.com (J. Arunachalam).

widely used in biomonitoring surveys [3,4]. As a result, the number of environmental samples submitted to analyses in the frame of routine monitoring or risk and sustainability assessment studies is continuously growing. But the multielemental (major, minor and trace) analysis of all environmental matrices requires dissolution of the sample prior to instrumental analysis by highly sensitive spectroscopic techniques such as inductively coupled plasma-optical emission and -mass spectrometry (ICP-AES and ICP-MS) [5]. Most of the sample digestion methods for various environmental samples are largely limited to the conventional acid digestion procedures with different heating systems [612]. But most of these digestion procedures are somewhat limited by various primary factors including long dissolution times

0003-2670/$ see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.07.006

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(hours to days), the potential loss of volatile elements, contamination of the sample by excessive amounts of reagents, high process blanks due to air born contaminants, etc. [13]. In view of these aspects, microwave energy has gained importance in the analysis of various environmental samples including biological and geological samples [14]. Although the introduction of microwave ovens allows shorted digestion times, the main limitation of these procedures is the large cooling times required before opening the low or highpressure bombs. In contrast to intensive sample preparation methods involving high temperatures and pressures, acid extraction (solidliquid extraction) under mild conditions appears promising with the use of ultrasonication [15]. Ultrasonic extractions have proved to be successful for achieving quantitative recoveries from various environmental matrices [1619] and various parameters associated with the ultrasonic solidliquid extraction for elemental analysis (total content) has been reviewed [20]. Various digestion methods have been reported for the estimation of trace elements in lichens and mussel samples [2125]. Only about four to ve elements have been estimated in most of the reported ultrasound-assisted extraction procedures. But in the case of environmental assessment programs, it very essential to estimate as many elements as possible to assess the trace element pollution levels. Hence, methods capable of extracting as many major, minor and trace elements possible are appropriate to match the capabilities of ICP-MS such as extreme sensitivity and high sample throughput. Ultrasound-assisted extraction of various elements from lichen and mussel tissue samples, although not yet sufciently exploited, could be an attractive alternative to microwave-assisted digestion methods. In contrast to ultrasonic baths, the use of high intensity ultrasonic probes can drastically accelerate extraction of analytes from solid samples [26]. The sonication time required with ultrasonic probe is less (up to 100 times) than that needed when using ultrasonic baths. Sonication time of 5 min or less is usually reported when ultrasonic probes are used for solid liquid extraction [27,28]. In this work, the capability of probe sonicator for the quantitative leaching of as many elements as possible using common reagents such as HNO3 and HCl, was evaluated in a set of bio-environmental samples (IAEA lichen 336 and mussel tissue NIST 2976). The different parameters such as composition of the extractant solution, sonication amplitude and sonication time, etc. associated with extraction process have been optimized in order to obtain the maximum extraction efciency for as many elements as possible. The results obtained from ultrasound-assisted extraction procedure were compared with the values obtained by microwave-assisted extraction. Similar extraction procedure was applied on two eld lichen samples collected in Kodaikanal to see the efcacy of same ultrasound extraction procedure for the estimation of various elements.

2. Experimental 2.1. Instrumentation The determination of elements in the sample after extraction was carried out using VG Plasma Quad 3 inductively coupled plasma mass spectrometry (ICP-MS) (VG elemental, Winsford, Cheshire, UK) located in class 200 area. The system is equipped with Meinhard concentric nebuliser and Scott-type double pass cooled spray chamber. The ion lens voltage settings and other parameters of the instrument was tuned everyday with 10 ng ml1 solution containing Be, Co, In, Pb and Bi. 103 Rh was used as an internal standard in all the sample solutions to correct for instrumental drift and matrix effects. All the measurements were performed in peak jump mode of data acquisition. The determination of Na, K, Ca, Mg and Fe was carried by inductively coupled plasma atomic emission spectrometry (ICP-AES) (JY-2000, France). 2.2. High intensity probe sonicator A 130 W power and 20 kHz frequency (Cole Parmer instruments, Illinois, USA, Model: CP 130PB-1) high intensity probe sonicator equipped with a Ti probe was used for ultrasound-assisted extraction. The amplitude control of the ultrasonic processor allowed the ultrasonic vibrations at the probe to be set any desired level in the 10100% range of the nominal power. After sonication, all the extracts were centrifuged at 4000 rpm for about 10 min for the rapid separation from the solidliquid mixture after sonication. A domestic microwave oven, programmable for the time and microwave power, was used for the total digestion of the sample. 2.3. Reagents and standards Sub-boiled HCl and HNO3 were prepared by sub-boiling distillation in quartz stills. Ultra-pure water with >18 M resistivity, obtained using a Milli-Q high purity water system, located in class 200 area, was used for dilution of standards, for preparing samples and for nal rinsing of the acid cleaned vessels. All containers were decontaminated in 20% HNO3 prior to use. All stock standards have been prepared by dissolving 99.999% metal in sub-boiled acids. Working standards at ng ml1 level were prepared daily in 1% sub-boiled HNO3 from stock standards by sequential dilutions. Polypropylene centrifuge tubes of 50 ml capacity were used for sonication experiments. Elemental concentrations of the laboratory standard solutions used in this work were cross-validated using NIST multi-element standards 3171A (mix A1) and 3172 (mix B1). 2.4. Lichen sample collection and preparation The lichen Parmelia sulcata is found abundantly on tree barks, on the surfaces of rocks or any wood posts throughout

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Kodaikanal, Tamilnadu, a southern state of India, because of cool climatic conditions. Two lichen samples were collected in Kodaikanal and near Berijam, a pristine area which is about 20 km away from main city of Kodaikanal (L1 and L2, respectively). Soil and bark particles adhering to the lichens were carefully removed after wetting with a ne brush and the samples stored in the refrigerator till analysis. At the laboratory lichen samples were further cleaned of the adhering soil and washed thoroughly with de-ionized water with agitation in an ultra sonic bath for 30 s. After draining, fresh water added and washed once again. The washed samples were transferred to a plastic sieve and water was completely drained off. The samples were then taken in a wide mouthed beaker and dried in an oven for 24 h at 4050 C. The dried samples were ground to a ne powder in an agate planetary ball-mill (Fritsch, Germany) and sieved to get 200300 mesh particles. 2.5. Analytical procedure 2.5.1. Ultrasound-assisted extraction procedure Accurately weighed amount (0.1 g) of different aliquots of IAEA lichen 336 and mussel tissue NIST SRM 2976 (taken as representative of biological tissue sample) was placed in to polypropylene centrifuge tubes (50 ml volume) containing 5 ml of corresponding extractant (HNO3 /HCl) solution. Then the sampleextractant mixture was sonicated at room temperature by varying sonication time and sonication amplitude settings. After sonication, the supernatant liquid was separated from the solid phase by centrifugation for about 10 min at 4000 rpm and then transferred the known volume of clear supernatant to another pre-cleaned centrifuge tube for the analysis by ICP-MS and ICP-AES using external calibration method. Corresponding process blanks were also prepared in the same way without any sample material. Three aliquots of each sample were used for each extraction procedures. With each series of extractions a blank was also measured. All the analytical measurements were run in triplicate for the sample solutions. Calculations of the metal contents in samples are based on a calibration graph obtained from aqueous standards. The percentage recovery of all the metals after ultrasoundassisted extraction were calculated using the following equation: value obtained recovery (%) = 100 value reported 2.5.2. Optimization of extractant concentration Among the acids employed as extractants, HNO3 is reported to have an enhanced performance due to its oxidants properties. The typical acid concentrations used in probe sonication are generally less than 1 M [29]. In the present work, HNO3 used as extractant and the concentration of HNO3 was varied from 0.5 to 5% (v/v). With the aim of improving extraction efciency conditions for the maximum number of el-

ements, the following acid concentration mixtures were also used HNO3 (1%) + HCl (1%), HNO3 (1%) + HCl (2.5%) and HNO3 (1%) + HCl (5%) (v/v). Corresponding extractant solutions were employed as blanks. The extractant volume of 5 ml was kept constant in all the ultrasound extraction studies. 2.5.3. Optimization of sonication time The extraction time necessary for achieving total solidliquid extraction depends on analytematrix interaction, composition of the liquid media and ultrasonic device being used [26]. In the present study, sonication time varied between 1 and 6 min was attempted by keeping ultrasound amplitude (40%), extractant concentration (1% HNO3 , v/v), extractant volume (5 ml) and sample weight (100 mg) constant. 2.5.4. Optimization of ultrasound amplitude Different authors have extensively investigated the effect of sonication amplitude in the extraction efciency, since the ultrasonic power delivered by probe is directly related to the amplitude [20,32]. In this work, to study the inuence of the sonication power in the ultrasonic extraction efciency, a solution of 1% HNO3 (v/v) was used as an extractant agent. The sonication amplitude studied were 2080% where as extractant concentration (1% HNO3 ), extractant volume (5 ml), sonication time (4 min) and sample weight (100 mg) was kept constant. 2.6. Microwave-assisted procedure for total digestion of the samples Accurately weighed amount (100 mg) of lichen 336, two eld lichen samples and mussel tissue (NIST 2976) were placed in a PTFE vessel containing 2 ml of sub-boiled HNO3 and closed. Then the vessel was put in to a Parr digestion vessel and closed. The closed vessels were placed inside the microwave oven (650 W) where they were irradiated for total time of 4 min at maximum power in two 2 min steps using cooling period of around 5 min after rst step to avoid an excess of pressure. On cooling at room temperature the vessels were opened and the sample was diluted to required volume with high purity water. The digests were analysed by ICPMS and ICP-AES using an external calibration method. For ICP-MS, Rh was added as internal standard in the all the sample digests. Appropriate blanks prepared similarly were also analysed.

3. Results and discussion IAEA lichen 336 and mussel tissue NIST 2976 was used for optimization experiments. Variables inuencing the extraction process were optimized within the intervals shown in Table 1.

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M.V.B. Krishna, J. Arunachalam / Analytica Chimica Acta 522 (2004) 179187 Table 2 Average process blank values (n = 3) obtained after sonication of 5 ml extractant with ultrasonic probe Element Process blank values (ng/ml) 1% HNO3 Naa Ka Caa Mga Fea Al Cr Mn Co Ni Cu Zn Ge As Se Rb Sr Zr Ag Cd In Sb Cs Ba Pb Bi 118 6 210 15 17 1 5.0 0.4 18 1 1.1 0.1 0.27 0.02 0.024 0.003 0.032 0.002 0.45 0.03 0.12 0.01 0.28 0.01 0.25 0.02 0.22 0.01 1.4 0.1 0.26 0.02 0.14 0.01 0.013 0.001 0.061 0.005 0.019 0.001 0.033 0.003 0.031 0.004 0.014 0.001 0.33 0.02 0.22 0.01 0.12 0.01 1% HNO3 HCl 124 5 232 17 22 2 8.3 0.5 23 1 6.9 0.5 0.63 0.06 0.55 0.04 0.031 0.002 4.87 0.24 0.91 0.07 5.8 0.5 0.32 0.02 9.3 0.6 1.6 0.2 0.25 0.02 0.28 0.03 0.028 0.003 0.22 0.02 0.061 0.002 0.071 0.007 0.053 0.004 0.013 0.002 0.35 0.04 2.32 0.15 0.22 0.02 Microwave digestion 170 8 280 16 16 1 11.2 0.8 26 2 2.1 0.2 0.36 0.02 0.28 0.03 0.19 0.01 0.72 0.04 3.03 0.21 3.7 0.8 0.33 0.03 0.11 0.01 1.7 0.2 0.24 0.01 0.25 0.03 0.018 0.002 0.21 0.03 0.059 0.002 0.079 0.005 0.035 0.002 0.011 0.001 0.17 0.02 0.43 0.03 0.18 0.01

Table 1 Experimental conditions for the ultrasound extraction of various elements from lichens Variable parameter Sonication amplitude Sonication time (min) HNO3 (extractant) (%, v/v) Sample amount (g) Studied interval 2080% 16 05 0.050.2 g Optimum extraction conditions obtained 40% 4 min 1 0.1 g

3.1. Effect of acid extractant The composition of the liquid extractant was an important parameter as it effects ultrasound-assisted extraction of metals from the plant materials. In both the samples, extraction efciency with water (in the absence of acidity) was very low (1020%) for most of the elements except for Na, K (6070%). An extractant volume of 5 ml as has been employed by various authors for solidliquid extraction of metals [3031] was chosen in the present study so that the required number of replicated could be performed without exhaustion of the sample solution. The extraction efciency for most of the analytes (major, minor and trace) in both lichen 336 and mussel tissue samples increased with increasing acid concentration; a steady extraction efciency being reached for an acid concentration higher than 1% (v/v). For example, recoveries obtained for the elements Na, Mn, Cd (taken as representatives of major, minor and trace elements, respectively) and Al (representative of low recovery group of elements) with different acid concentrations of HNO3 and under most favourable sonication parameters (sonication amplitude/time) are shown in Fig. 1. When the extraction carried out in 1% HNO3 or 1% HCl or 1% HNO3 + 1% HCl medium, most of the analytes show similar extraction behaviour with quantitative recoveries between 90 and 100%. The analyte content of Ti could not

a Analysed by ICP-AES, sonication time: 4 min, extractant volume: 5 ml (1% HNO3 ), sonication amplitude: 40%.

be determined in this extraction method due to high process blanks obtained after sonication with titanium probe. The process blank values obtained with different acid extractants used for sonication are given in Table 2.

Fig. 1. Effect of extractant concentration on the extraction efciency of Na, Mn, Cd in 1% HNO3 .

M.V.B. Krishna, J. Arunachalam / Analytica Chimica Acta 522 (2004) 179187 Table 3 Concentrations of various elements (n = 3) in lichen 336 after sonication with ultrasonic probe Element Lichen 336 (mg/kg) Certied Values Obtained in this work 1% HNO3 Naa Ka Caa Mga Fea Al Cr Mn Co Ni Cu Zn Ge As Se Rb Sr Zr Ag Cd In Sb Cs Ba Pb Bi
a

183

1% HNO3 + 1% HCl 318 16 1874 138 2713 152 532 21 38 1 45 3 1.21 0.08 64 5 0.23 0.02 1.7 0.2 2.2 0.4 37.3 2.2 0.061 0.005 2.1 0.2 0.22 0.02 1.57 0.17 8.8 0.5 0.076 0.006 0.048 0.006 0.205 0.018 0.009 0.002 0.056 0.006 0.046 0.005 4.2 0.4 4.8 0.5 0.021 0.003

Microwave digestion 335 18 1810 128 2282 149 540 14 328 12 374 16 1.33 0.11 59 3 0.35 0.04 1.2 0.1 3.4 0.1 27.8 1.9 0.072 0.007 0.99 0.11 0.60 0.05 1.7 0.15 9.5 0.8 0.064 0.006 0.072 0.005 0.131 0.011 0.011 0.003 0.062 0.006 0.082 0.008 5.2 0.3 4.9 0.8 0.018 0.002

320 40 1840 200 2600 610 430 50 680 110 1.06 0.17 63 7 0.29 0.05 3.6 0.5 30.4 3.4 0.63 0.08 0.22 0.04 1.76 0.22 9.3 1.1 0.117 0.073 0.01 0.1 0.003 6.4 1.1 4.9 0.6

315 13 1764 125 2724 145 537 17 35 2 41 2 0.95 0.1 60 2 0.23 0.02 1.4 0.1 2.6 0.2 27.2 1.8 0.065 0.006 0.22 0.03 0.19 0.02 1.48 0.13 8.2 0.4 0.055 0.004 0.053 0.005 0.119 0.012 0.008 0.001 0.047 0.004 0.063 0.007 5.1 0.2 4.3 0.3 0.013 0.002

Analysed by ICP-AES, sonication time: 4 min, extractant volume: 5 ml (1% HNO3 ), sonication amplitude: 40%.

Different acid mixtures such as 1% HCl + (0.315%) HNO3 , 3% HCl + (0.315%) HNO3 were reported to be used as extractants for solidliquid extraction of heavy metals from an aquatic plant [31]. Recoveries with >90% for many elements from both lichen 336 and NIST 2976 except for Fe and Al was achieved using 1% HNO3 (v/v) as extractant and the results are presented in Tables 3 and 4, respectively. No signicant improvement in the extraction efciency of Fe and Al was achieved even after sonication with either 10% HNO3 or 10% HCl or both as well as with two stage extraction method consecutively on the same sample. The low recoveries (<10%) of Fe and Al might be due to incomplete release of these elements from solid matrix as they normally associated with the aluminosilicate matrix. As it is impossible to dissolve siliceous material completely with only nitric acid, recovery of some elements like Al, Fe that are associated with resistant fraction of sample material can be achieved only with total digestion of the sample. The use of HF is necessary for determining those elements, which are associated with silicious materials [14]. 3.2. Inuence of sonication time In both the lichen and mussel samples, extraction efciency of the most of the elements increased with increasing

sonication time from 1 to 4 min and reached plateau. Choosing sonication amplitude of 40%, a sonication time of 4 min and the sample weight of 100 mg, the best recoveries of all the studied metals can be achieved. The inuence of sonication time for recoveries obtained for the metals Na, Mn, Cd and Al after sonication is shown in Fig. 2. The results clearly indicate that sonication time of 4 min was found to be sufcient for the quantitative extraction of various elements except Fe, Al from both lichen and mussel samples, which is advantageous for high sample through put. 3.3. Inuence of sonication amplitude It is known that intensity of ultrasound transmitted to the medium is directly related to the vibration amplitude of the probe [32]. The inuence of ultrasound amplitude on lichen sample in the range 2080% is shown in Fig. 3 keeping the remaining variables such as extractant concentration, sonication time and sample weight at values xed above. For most of the metals, extraction efciency increased with increasing amplitude from 20 to 40% and remained constant for higher amplitude values. However, at high vibrational amplitude a great number of cavitation bubbles are generated in the solution, which may dampen the passage of ultrasound energy through the liquid [25]. The curve obtained for Na, Mn, Cd

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Table 4 Concentrations of various elements in mussel tissue obtained with ultrasonic probe and microwave digestion Element Mussel tissue (NIST 2976) (mg/kg) Certied values Obtained in this work Probe (1% HNO3 ) Naa Ka Caa Mga Fea Al Cr Mn Co Ni Cu Zn Ge As Se Rb Sr Zr Ag Cd In Sb Cs Ba Pb Bi
a

Microwave digestion 34000 2000 9800 600 7700 600 5200 400 128 9 118 12 0.58 0.08 32 2 0.56 0.05 1.04 0.09 4.05 0.43 132 11 0.068 0.005 15.4 2.1 2.8 0.3 4.6 0.4 95 6 0.022 0.004 0.009 0.002 0.86 0.08 0.024 0.005 0.062 0.009 0.031 0.003 0.55 0.07 1.22 0.12 <0.02

35000 1000 9700 500 7600 300 5300 500 171 4.9 134 34 0.5 0.16 33 2 0.61 0.02 0.93 0.12 4.02 0.33 137 13 13.3 1.8 1.8 0.15 4.14 0.09 93 2 0.011 0.005 0.82 0.16 0.027 0.001 1.19 0.18

33000 2000 10100 600 7400 500 4900 400 27 2 15 2 0.68 0.12 31 1 0.52 0.04 0.99 0.08 3.95 0.32 129 8 0.066 0.004 14.4 1.3 2.2 0.2 4.4 0.1 96 4 0.019 0.002 0.008 0.001 0.88 0.06 0.016 0.004 0.059 0.008 0.021 0.002 0.49 0.05 1.32 0.11 <0.02

Analysed by ICP-AES, sonication time: 4 min, extractant volume: 5 ml (1% HNO3 ), sonication amplitude: 40%.

and Al indicates that 40% amplitude is required for quantitative recovery for most of the elements after extraction where as the extraction efciency (<10%) of Fe and Al has not improved even at high ultrasound amplitude. Hence sonication amplitude of 40% was used in all the extraction experiments.

3.4. Sample amount Various authors had used a sample mass of up to 0.2 g for extraction with ultrasonic probe [26] and a sample mass of up to 0.5 g for extraction with ultrasonic bath [31]. In this work,

Fig. 2. Effect of time on the extraction efciency of Na, Mn, Cd in 1% HNO3 .

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Fig. 3. Effect of sonication amplitude on the extraction efciency of Na, Mn, Cd in 1% HNO3 .

extraction efciency of trace elements when different sample amounts (0.05, 0.1, 0.15 and 0.2 g) used for sonication, was investigated. Extractant concentration and volume (5 ml of 1% HNO3 ), sonication time (4 min) and sonication amplitude (40%) were kept constant in all these experiments. These studies indicate that the quantitative recovery with good pre-

cision for the most of the elements from both lichen 336 and mussel tissue was achieved when the sample weight is 0.1 g. Similar observations were noticed by Filgueiras et al. with plant tissue after ultrasound extraction with EDTA [16]. Filgueiras et al. had also noticed a signicant decrease in Mg, Mn and Zn recovery from plant samples when the sam-

Table 5 Concentrations of various elements (n = 3) obtained in eld lichen samples after sonication with ultrasonic probe Element Field lichen samples (mg/kg) Probe (1% HNO3 ) L1 Naa Ka Caa Mga Fea Al Cr Mn Co Ni Cu Zn Ge As Se Rb Sr Zr Ag Cd In Sb Cs Ba Pb Bi
a

Microwave digestion L2 295 23 1576 123 2424 131 435 34 78 4 56 2 0.56 0.04 63 2 0.52 0.02 1.23 0.08 1.6 0.1 7.9 0.7 0.032 0.005 0.12 0.01 0.18 0.02 0.56 0.03 6.9 0.4 0.027 0.003 0.029 0.003 0.08 0.01 0.012 0.002 0.023 0.003 0.11 0.01 23 2 2.5 0.2 <0.01 L1 452 35 2416 143 3508 253 714 51 385 32 316 26 0.98 0.12 117 13 1.31 0.05 8.5 0.6 6.8 0.7 43 4 1.6 0.2 0.22 0.02 0.84 0.03 13.9 2 48 3 0.253 0.017 0.041 0.005 0.16 0.02 0.038 0.003 0.066 0.004 0.23 0.03 171 16 7.3 1.4 0.026 0.004 L2 324 26 1592 128 2572 164 468 36 273 18 178 12 0.82 0.11 68 3 0.11 0.02 1.31 0.13 2.3 0.2 8.3 0.9 0.048 0.007 0.13 0.03 0.21 0.02 0.63 0.05 7.4 0.4 0.038 0.004 0.031 0.003 0.11 0.01 0.017 0.002 0.032 0.002 0.16 0.02 26 2 2.8 0.4 <0.01

434 31 2354 134 3382 225 687 42 97 6 85 4 0.73 0.03 115 9 1.18 0.04 9.1 0.4 5.2 0.4 41 3 1.5 0.1 0.15 0.02 0.78 0.06 11.9 0.8 43 2 0.168 0.011 0.038 0.004 0.13 0.01 0.029 0.003 0.057 0.004 0.16 0.02 162 12 6.8 0.5 0.036 0.004

Analysed by ICP-AES, L1: lichen sample collected near the trafc road, L2: collected at about 20 km away from main city.

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ple mass is larger than 0.10.15 g [25]. Hence sample weight of about 0.1 g is used in all the extraction experiments in the present work. 3.5. Comparison of the proposed leaching method with those obtained with microwave-assisted digestion As seen from Tables 3 and 4, a good agreement between the results obtained by proposed ultrasound-assisted extraction method, microwave-assisted total digestion when a 0.1 g sample (lichen 336 and mussel tissue 2976) used for digestion and certied metal contents could be observed for most of the metals studied. Average recoveries for many metal contents were found to be in the range of 90100%. Good accordance was observed between the values for Zn and Mn, obtained by both the procedures and analysed by both ICP-MS and ICP-AES. But ICP-MS technique allowed enlarging the analytical information on some elements such as Cd, Co, Ni, Ge, Zr, Se, In, Cs, Sb, Bi not observable by ICP-AES due to sensitivity limitation. Similar ultrasound-assisted and microwave-assisted procedures were applied to two real lichens samples collected in Kodaikanal and good agreement was observed between the ultrasound-assisted and microwave-assisted methods and are tabulated in Table 5. 3.6. Sample throughput and reagent consumption In the case of ultrasound-extraction procedure, the time needed for one extraction was only about 15 min (4 min for sonication and 10 min for centrifugation) and hence it is possible to prepare as many as 3035 samples per 8 h working day for analysis. In the case of microwave-assisted extraction procedure also requires 4 min for digestion, but the time required for the reactor to cool before opening is about 90120 min (at room temperature) which restricts the preparation of samples to very few (assuming that the two sets of digestion bombs are used). Hence the sample throughput is limited by digestion time and by the number of digestion vessels that can be used simultaneously. The consumption of reagents is considerably reduced. Extractant concentration of 1% HNO3 is sufcient enough to recover most of the metal contents from plant as well as biological samples. But in the case of microwave-assisted extraction procedure, 2 ml of concentrated HNO3 is required for digestion.

extraction can be used as an alternative to traditional sample preparation methods for the estimation of wide range of major, minor and trace elements in lichens and mussel tissues using the combination of ICP-MS and ICP-AES. The procedure is also safer as it involves neither high pressure nor high temperature; reagent consumption and the acid waste generation are considerably less than microwave digestion method.

Acknowledgement The authors thank Dr. A.C. Sahayam for carrying out ICPAES measurements through out this work.

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4. Conclusion The proposed ultrasound-assisted extraction procedure has been found to prove a faster and simpler approach for the estimation of various elements in lichen and mussel samples. The number of analytical steps involved is minimum and lowers the contamination risks. With 4 min leaching time and 5 ml extractant solution of 1% HNO3 , ultrasound-assisted

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