Laser ablation inductively coupled plasma optical emission spectrometry for analysis of pellets of plant materials

Spectrochimica Acta Part B 94–95 (2014) 27–33

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Laser ablation inductively coupled plasma optical emission spectrometry for analysis of pellets of plant materials Marcos S. Gomes a,b, Emily R. Schenk c,e, Dário Santos Jr. d, Francisco José Krug b, José R. Almirall c,e,⁎ a

Departamento de Química, Universidade Federal de São Carlos, Rod. Washington Luís, km 235, 13565-905 São Carlos, SP, Brazil Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av. Centenário 303, 13416-000 Piracicaba, SP, Brazil Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA d Departamento de Ciências Exatas e da Terra, Universidade Federal de São Paulo, Rua Professor Arthur Riedel 275, Diadema, SP, Brazil e International Forensic Research Institute, Florida International University, Miami, FL, USA b c

a r t i c l e

i n f o

Article history: Received 30 January 2014 Accepted 7 March 2014 Available online 18 March 2014 Keywords: LA-ICP OES Laser ablation Optical emission spectroscopy Plant material Macronutrient Micronutrient

a b s t r a c t An evaluation of laser ablation inductively coupled plasma optical emission spectroscopy (LAICP OES) for the direct analysis of pelleted plant material is reported. Ground leaves of orange citrus, soy and sugarcane were comminuted using a high-speed ball mill, pressed into pellets and sampled directly with laser ablation and analyzed by ICP OES. The limits of detection (LODs) for the method ranged from as low as 0.1 mg kg−1 for Zn to as high as 94 mg kg−1 for K but were generally below 6 mg kg−1 for most of the elements of interest. A certified reference material consisting of a similar matrix (NIST SRM 1547 peach leaves) was used to check the accuracy of the calibration and the reported method resulted in an average bias of ~5% for all the elements of interest. The precision for the reported method ranged from as low as 4% relative standard deviation (RSD) for Mn to as high as 17% RSD for Zn but averaged ~6.5% RSD for all the elements (n = 10). The proposed method was tested for the determination of Ca, Mg, P, K, Fe, Mn, Zn and B, and the results were in good agreement with those obtained for the corresponding acid digests by ICP-OES, no differences being observed by applying a paired t-test at the 95% confidence level. The reported direct solid sampling method provides a fast alternative to acid digestion that results in similar and appropriate analytical figures of merit with regard to sensitivity, accuracy and precision for plant material analysis. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Essential elements are classified as macronutrients (N, P, K, Ca, Mg, S) and micronutrients (Fe, Cu, Mn, Zn, B, Mo, Ni and Cl) and the classification is based on the relative abundance in plants. From the knowledge of the concentration of the most important nutrients, it is possible to define a strategy to correct for deficiencies, if present, that will limit the production and/or the quality of plant materials. Under limited conditions, the plant may exhibit visual symptom(s) indicating the deficiency for a specific nutrient, which normally can be corrected or prevented by supplying the most appropriate fertilizer. Foliar nutrient analysis is a useful diagnostic tool to complement soil testing as a best-management practice with plants [1]. In general, the determination of nutrients in plant materials is carried out in leaves properly collected, washed, dried and homogenized [2]. The homogenized samples are frequently acid digested [3] for further analysis by inductively coupled plasma optical emission spectroscopy (ICP OES),

⁎ Corresponding author at: 11200 SW 8th St, OE116, Miami, FL 33199, USA. Tel.: +1 305 348 3917; fax: +1 305 348 4485. E-mail address: almirall@fiu.edu (J.R. Almirall).

http://dx.doi.org/10.1016/j.sab.2014.03.005 0584-8547/© 2014 Elsevier B.V. All rights reserved.

flame atomic absorption spectrometry (FAAS), inductively coupled plasma mass spectrometry (ICP-MS) [4–7] or by using other analytical methods [8]. The procedures utilized for the acid digestion of plant material are relatively simple, but can be time-consuming compared to the procedures for analysis of solids using atomic spectrometry techniques. Direct solid sampling analysis of plant material offers a practical advantage over wet digestion methods, including time-saving, lower risks of contamination, improved laboratory safety (no reagent manipulation, no chemical residues), and minimal number of uncertainty sources. Solid sampling analysis of plant materials by different analytical techniques have been reported [9], such as micro-energy dispersive X-ray fluorescence spectrometry (μEDX) [4], laser ablation ICP-MS (LA-ICPMS) [10,11], electrothermal vaporization inductively coupled plasma optical emission spectrometry (ETV-ICP OES) [12,13], secondary ion mass spectrometry (SIMS) [9], proton/particle induced X-ray emission (PIXE) [9], X-ray and synchrotron techniques [9,14] and laser-induced breakdown spectroscopy (LIBS) [15,16]. In the case of plant leaves, a grinding step, usually referred to as comminution [2], is necessary prior to the micro-analysis due to the inherent heterogeneity of the elemental concentration distribution within the leaf. In general, pellets of plant materials have been prepared prior

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to analyte determination [15,17]. The test portion usually analyzed by microanalytical techniques varies from 0.1 to 10 mg [18], and thus the comminution procedure should be effective for obtaining appropriate homogeneity. The cohesion of the resulting pellet has also been reported to improve the measurement precision [19], and enhances the analyte emission line intensity by reducing the particle size distribution and pellet porosity [20]. The first attempt for coupling a laser ablation (LA) unit to the inductively coupled plasma of an optical emission spectrometer was made by Thompson et al. [21] for the analysis of steel and silicate rocks. To obtain good results, authors recommended that analytes should be homogeneously distributed in the matrix and that calibration curves should be linear. There are many contributions in the literature dealing with laserbased microsampling coupled to ICP-MS which have been successfully employed in spatially-resolved spectrochemical analysis of biological samples [9,11,22]. Regarding LA-ICP OES, to our knowledge, this method has not yet been used for quantitative analyses of plant material. The reader will find very useful information on fundamentals of laser–sample interactions, including the laser parameters, plasma conditions and sample surface [23–25], and applications such as shell analysis in geochemistry [26,27], analysis of heterogeneous catalysts in industry [28] and analysis of glass in forensic chemistry [29]. More information on laser ablation in analytical chemistry can be found in excellent reviews from Russo et al. [30,31], and in contributions from Gooijer and Mank [32] and Smith [33]. The main objective of this work is to demonstrate that LA-ICP OES can be used for quantitative analysis of pelleted plant materials. In the present study, the evaluation was based on Ca, Mg, P, K, B, Fe, Mn and Zn measurements in test samples of citrus, soy and sugarcane leaves. Sample preparation was also evaluated with particular attention to the optimization of solid sampling by laser ablation, including the use of Sc as an internal standard for the correction of possible variations in the ablated mass. LA-ICP OES can also be viewed as an analogous technique to the previously reported LIBS [15] method and compared within the same context with regard to the analytical figures of merits.

2. Experimental

2.1.2. Internal standard Scandium (Ricca Chemical Company, Arlington, TX, USA) was used as an internal standard by pipetting 160 μL of a solution containing 400 mg kg−1 Sc over the comminuted material prior to pressing into a pellet as described elsewhere [35]. Thereafter, the resulting mixture was homogenized, and dried at 55 °C for 24 h. 2.2. Instrumentation An ICP OES (PerkinElmer, model Optima 7300 DV, Waltham, MA, USA) equipped with an Echelle-based polychromator and two segmented-array charge-coupled device detectors for ultraviolet (UV) and visible (VIS) range was used. Laser ablation analyses were performed using a 266 nm Nd:YAG laser ablation system (CETAC Technologies, model LSX-500, Omaha, NE, USA). The LSX-500 sample cell was mounted on an X–Y–Z translation stage, with a step size increment of 0.25 mm. To achieve optimal reproducibility, highest signal-to-noise ratio (SNR), precision and accuracy, the effect of laser energy per pulse at 10 Hz was also evaluated. The laser pulse was focused on the surface of the test sample using a depth profiling ablation mode and a 200 μm spot size as previously reported for elemental analysis of cotton and glass [29,36]. In addition, the signal integration was accomplished in transient mode [29] with a 20 s argon blank followed by a 50 s ablation of the test sample. As the initial laser coupling can cause signal instability [37], the first 20 s of the ablation signal was ignored for analytical signal integration. The cell was swept for an additional 30 s post-ablation to remove material from the cell and tubing to avoid carryover between replicates. Argon has been utilized as plasma and auxiliary gas as well as the entire makeup gas (0.5 L min−1) for transport of ablated particles [29]. All analyses were conducted with ten replicates for every pellet with distances between spots of at least 1.5 mm. The laser fluence for all studies was approximately 9.2 J cm−2. 2.2.1. Limits of detection The limits of detection for LA-ICP OES analyses were estimated using CRMs with the lowest mass fraction of the analyte in the calibration curve [38]. The standard deviation of the background (s) was measured during the first 20 s gas blank. The LODs were calculated as 3.3 s / b [39, 40], where b is the slope of the calibration curve and s is the estimated standard deviation of the blank signal measurements.

2.1. Description of samples and sample pre-treatment Leaves of orange citrus (Citrus sinensis), soy (Glycine max) and sugarcane (Saccharum officinarum) were used in this work. The leaf samples were collected from plants and washed with tap water, rinsed twice with distilled water and three times with high purity water to remove contaminants [20]. For sugarcane leaves, the central vein was removed as recommended [34]. For soy and orange citrus leaves, whole leaves were used. After washing, samples were dried, chopped, and ovendried to constant mass at 60 °C.

2.1.1. Grinding procedures and pellet preparation Samples were initially ground using a cutting mill (Marconi LTDA, model MA680, Piracicaba, SP, Brazil) with an outlet aperture of 600 μm. Thereafter, samples were comminuted in a mixer mill (Retsch, model MM 200, Haan, NW, Germany), with a tungsten carbide (WC) container and one 9 mm WC ball. Each sample was homogenized for 5–120 min, with a frequency of 25 Hz, in order to investigate the influence of particle size distributions on the quality of pellet formation for appropriate sampling in the test samples. Pellets were prepared by using a manual press (Carver Bench top Pellet Press, model 4350L, Wabash, IN, USA) by transferring approximately 0.4 g of comminuted material to a 15 mm die set under vacuum at 8.0 t cm−2 for 5 min. Resulting pellets were approximately 2 mm thick and 15 mm diameter.

2.2.2. Certified reference materials Calibrations were carried out with the following CRMs from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA): apple leaves (SRM 1515), peach leaves (SRM 1547), spinach leaves (SRM 1570a), tomato leaves (SRM 1573a), and pine needles (SRM 1575a). 2.2.3. Acid digestion and ICP OES The CRMs and plant materials were microwave-assisted acid digested in triplicate. A closed vessel microwave oven (ETHOS 1600, Milestone, Italy) was used according to the following procedure: 250 mg of ground material was accurately weighed in the TFM® vessels and then 6.0 mL of 65% v v−1 HNO3 and 1.0 mL of 30% v v−1 H2O2 were added. Thereafter, the residual solutions were transferred to 25 mL volumetric flasks and the volume was made up with high purity deionized water (resistivity 18.2 MΩ cm). The final solutions were analyzed by a radially viewed ICP OES (Vista RL, Varian, Australia) [41]. 2.2.4. Sample characterization A digital microscope (Keyence, model VHX-1000, Osaka, Japan) was used for particle size inspection and imaging of the morphology and volume of the resulting craters. A small portion of milled sample was fixed onto a double-sided carbon conductive tape and observed with 200× magnification. The density of pelleted material was determined taking into account the width and thickness of the pellet.

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29

2.0x104

3. Results and discussion

(a)

3.1. Working parameters The LA-ICP OES operating parameters have been listed in Table 1. The emission lines were selected taking into account the concentration ranges of the elements in the test samples. The laser fluence of 9.2 J cm−2 was chosen after testing between 70 and 90% of the maximum laser power. This range was defined based on preliminary experiments, where, in principle, both conditions were appropriate for analysis. Notwithstanding, with 90% of laser power, the corresponding fluence of 9.2 J cm−2 provided the highest signal emission intensities for all tested analytes, as a consequence of the higher ablated mass, and this condition was chosen for further experiments. Under this condition the highest signal-to-noise ratios were also obtained. Examples of typical P and B transient signals under working conditions for the analysis of a pellet of orange citrus leaves are shown in Fig. 1a and b, respectively.

Emission intensity (a. u.)

P I 213.619 nm

1.5x104

1.0x104

5.0x103

0.0 0

25

50

75

100

Time (s) 8.0x103

(b)

Plant leaves are complex matrices in that the mineral composition of macro- and micronutrients varies widely between samples. Therefore, it is essential to achieve sample homogeneity on a microscale (analyte microhomogeneity) by using effective milling methods. Depending on the milling step or the type of sample, which includes its physical and chemical characteristics [20], higher milling times may be necessary for obtaining appropriate analytical figures of merit, such as precision, repeatability and reproducibility. It must be stressed that the knowledge of particle size for sample presentation to microanalytical techniques is recommended and the effects of these variables on the quality of measurements and analytical results can be found elsewhere [42,43]. In the present work, the mean particle size diameter was approximately 20 μm for citrus and soy leaves after 10 min milling, and 21 μm for sugarcane leaves after 40 min milling. In all cases the particle size distributions presented at least 90% of particles lower than 100 μm. These values are in good agreement with data found in the literature [20]. The mass removed during ablation was estimated using the density of the material and the volume of the craters and differences in the ablated masses within and between pellets, prepared after different comminution times, were observed particularly for sugarcane. For this species of plant material the ablated masses varied indirectly with respect to comminution times, i.e. 25 ± 1 μg for 120 min and 75 ± 6 μg for 5 min following 500 laser shots (n = 3) at 9.2 J cm−2. For citrus and soy leaves, the ablated masses were practically constant for all comminution times, with a variation of 22 ± 2 and 28 ± 3 μg, respectively,

Emission intensity (a. u.)

B I 249.772 nm

3.2. Test sample presentation

6.0x103

4.0x103

2.0x103

0.0 0

25

50

75

100

Time (s) Fig. 1. Examples of characteristic emission signal intensities in pellets of orange citrus leaves after 20 min milling obtained by LA-ICP OES. (a) 1.8 g kg−1 P and (b) 48 mg kg−1 B (n = 10 replicates, 500 pulses/replicate).

under the same experimental conditions described above. The difficulties in milling sugarcane leaves have been attributed to the different contents of fiber, lignin and cellulose [20] and the variation in the ablated masses can be attributed to differences in particle cohesion and pellet porosity due to the differences in particle size distributions. Fig. 2 shows a typical image of a crater formed by UV laser ablation (500 laser pulses) in a pellet of citrus leaves, prepared after 10 min comminution by ball milling. Fig. 3 shows the crater profiles formed in pellets of citrus, soy

Table 1 Instrumental parameters for the analysis of plant materials by LA-ICP OES. Parameter

LA-ICP OES

ICP OES Power Air flow Auxiliary gas flow Nebulization gas flow Laser Laser fluence (J cm−2) Pulse duration (ns) Spot size (μm) Ablation mode Ablation cell volume (cm3) Number of laser pulses Repetition rate (Hz) Laser focus Emission lines (in nm)

Optima 7300 DV 1.5 kW 15 L min−1 0.5 L min−1 0.5 L min−1 266 nm, Nd:YAG (3 mJ) 9.2 b6 200 Depth profile 56 500 10 On pellet surface Ca II 315.887, K I 404.721, Mg I 285.213, P I 213.618, B I 249.772, Fe I 259.939, Mn II 257.610, Zn I 213.857, Sc II 361.383 nm

600 400 200

Crater depth (µm)

800

0

0

275

550

825

1100

(µm) Fig. 2. Characteristic crater formed by UV laser ablation after 500 laser pulses in pellet of citrus leaves, prepared after 10 min comminution by ball milling.

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distributions could be analyzed without any additional treatment; otherwise the addition of an internal standard prior to pellet preparation should be used in order to correct for differences in element emission intensities due to variations in the ablated masses. The first strategy was used after defining the most appropriate comminution conditions for citrus, soy and sugarcane leaves (similar mean particle size diameters and crater profiles). The calibration curves were obtained with pellets of CRMs, such as those for Mg I 285.213, P I 213.618 and Mn II 257.610 nm as depicted in Fig. 4. The horizontal

Citrus leaves 5 min 20 min 40 min

1125

750

375

(a) 0

8 Mg I 285.213 nm

175

350

525

700

Crater profile (µm)

Soy leaves 5 min 20 min 40 min

1125

750

NIST 1570

4

2

NIST 1547

NIST 1515

(a)

NIST 1575a

0

375

3500

7000

10500

14000

Certified mass fraction (mg kg-1) 0.032

(b)

0

P I 213.618 nm

0

175

350

525

700

Crater profile (µm) 1500

Sugarcane leaves Crater depth (µm)

NIST 1573a

6

0

1125

750

5 min 20 min 40 min

375

Normalized intensity (a.u.)

Crater depth (µm)

1500

Normalized intensity (a. u.)

0

0.016 NIST 1570a

0

NIST 1515

0.008

NIST 1547 NIST 1575a

(b)

0.000 0

1500

3000

Certified mass fraction (mg

4500

6000

kg-1)

0.4

(c)

0

NIST 1573a

0.024

Mn II 257.610 nm

175

350

525

700

Crater profile (µm) Fig. 3. Crater profiles generated from (a) citrus, (b) soy and (c) sugarcane pellets following different milling time preparations and LA-ICP OES analysis (500 pulses/replicate).

and sugarcane leaves prepared after 5, 20 and 40 min milling. In general, similar crater profiles were observed in pellets of citrus and soy leaves, although large differences were found in pellets of sugarcane leaves (Fig. 3c). For the sake of information, pellets of plant materials without milling (particle diameters b 600 μm, obtained by cutting mill) were also prepared, but they became brittle due to the low cohesion between particles.

Normalized intensity (a. u.)

Crater depth (µm)

1500

0.3 NIST 1575a

0.2 NIST 1573a

0.1

NIST 1547

NIST 1570a

(c)

NIST 1515

0.0 0

150

300

450

600

Certified mass fraction (mg kg-1) 3.3. Analysis of plant materials During method development, two strategies were adopted for analysis: pellets prepared from laboratory samples with similar particle size

Fig. 4. LA-ICP OES calibration curves for Mg, P and Mn with pellets of CRMs. Vertical error bars correspond to ±one standard deviation (n = 10 replicates; 500 pulses/replicate, 9.2 J cm−2). Horizontal error bars correspond to the uncertainties of the certified mass fractions at 95% confidence level.

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Table 2 Figures of merit obtained by LA-ICP OES. Emission line (nm)

Ca II 315.887 K I 404.721 Mg I 285.213 P I 213.618 B I 249.772 Fe I 259.939 Mn II 257.610 Zn I 213.857

NIST 1547 certified mass fractions (mg kg−1)

15,600 24,300 4320 1370 29 218 98 17.9

± ± ± ± ± ± ± ±

200 300 80 70 2 14 3 0.4

LA-ICP OES Precision (RSD %)

Measured conc. (mg kg−1)

Biasa (%)

LOD (mg kg−1)

SNR

5.6 6.0 4.3 5.2 4.2 5.2 4.1 17.5

14,700 25,600 4700 1430 30 250 100 18

−6.1 5.2 8.3 4.3 2.2 11.5 2.4 0.6

14 94 5 10 0.2 0.8 2 0.1

226 330 275 443 319 228 257 69

± ± ± ± ± ± ± ±

800 1500 200 70 1 10 4 3

a Bias was calculated based as a percentage error between measured and certified mass fractions of NIST 1547. Uncertainties are represented by ±1 estimated standard deviation (n = 10) for LA-ICP OES.

Table 3 Concentrations of macro- and micronutrients (mg kg−1) determined in citrus, soy and sugarcane leaves by LA-ICP OES and ICP OES after wet decomposition of samples. Element

Ca K Mg P B Fe Mn Zn

Orange citrus leaves

Soy leaves

LA-ICP OESa

ICP OESb

22,700 13,500 3690 1790 43 58 15.7 17.0

21,620 13,200 3510 1810 48 59 15.2 17.3

± ± ± ± ± ± ± ±

600 900 140 80 1 2 1 1

± ± ± ± ± ± ± ±

40 50 40 20 2.5 0.5 0.3 0.5

Sugarcane leaves

LA-ICP OESa

ICP OESb

34,700 8200 7400 1460 50 770 236 58

33,000 8100 7600 1400 49 790 230 55

± ± ± ± ± ± ± ±

1400 500 400 70 2 70 15 6

± ± ± ± ± ± ± ±

400 50 20 20 0.2 10 4 0.4

LA-ICP OESa

ICP OESb

6100 11,800 3750 1720 6.1 177 54 18

5750 12,600 3610 1810 5.4 199 54 19

± ± ± ± ± ± ± ±

280 800 200 80 0.2 21 3 2

± ± ± ± ± ± ± ±

80 160 30 20 0.7 3 0.7 1

Uncertainties are represented by ±1 estimated standard deviation. a 500 pulses per replicate (n = 10). b Digests (n = 3).

bars in the X-values indicate the uncertainties of the certified mass fractions at 95% confidence level, and are in the range from 1 to 16% for test portions of 150–250 mg. The vertical bars in the Y-values correspond to ± 1 standard deviation (n = 10 sampling sites alongside pellet surface) of the LA-ICP OES determinations. The figures of merit, such as precision, bias and LODs, were obtained following pellet analysis of NIST 1547 and have been presented in Table 2. The LA-ICP OES measurement precision was found to be b 6% for all elements with the exception of Zn. The bias, calculated as the percent difference of the measured concentration from the certified concentration of NIST 1547 ranged from −6.1% to 11.5%. Similar results were observed with other CRMs. Calculations for SNR were based on the average of element emission intensities (n = 10) using standard deviation estimates of the background. The measurements resulted in good signal to noise ratios (SNRs), including for elements present in low concentration in the plant materials, such as B and Zn. The LODs obtained in LA-ICP OES analysis are appropriate for plant diagnosis purposes, taking into account the mass fraction ranges of macro- and micronutrients tested herein and found in plants of agricultural interest under good nutritional status [15]. The results obtained by LA-ICP OES in the analysis of pelleted citrus, soy and sugarcane leaves were compared to those from ICP OES after acid digestion (Table 3). In general, no significant differences were observed when employing the Student's t-test at 95% confidence level. The CV of ICP OES results (n = 3 digests) varied from 1 to 8%, while the CV of LA-ICP OES measurements (n = 10 craters, 500 pulses/crater) varied from 4 to 15%. The figures of merit also indicate that the proposed method can be used for the direct analysis of pelleted plant material for the determination of the selected analytes. For the sake of information, the quality of the ICP OES measurements was verified through the analysis of NIST 1547 digests, and no differences were observed by comparing the results with the certified reference values: bias varied from −3.0 to + 5.2% and precision reproducibility (between digest variations) from 2 to 5% (n = 3 digests).

A second strategy was evaluated for pellets of sugarcane leaves after 5 replicate analyses with and without an internal standard, as well as the CRMs for calibration. The necessity of an internal standard [44] for correcting differences in the ablated masses between replicates was evaluated with scandium, which has been successfully used previously [45,46]. The use of different techniques of analytical signal normalization, such as internal standards, which improve figures-of-merit for atomic spectrometry with laser sampling can be found in a comprehensive review from Zorov et al. [47]. Table 4 shows the benefit of Sc for correcting the calculated mass fractions of Ca, K, Mg, P, B, Fe, Mn and Zn determined by LA-ICP OES in pellets of sugarcane leaves comminuted for 5 min, no statistical differences being observed between the results after normalization of emission intensities (Student's t-test at 95% confidence level) and the corresponding ICP OES results. Without Sc the differences were significant, as expected, due to the differences in (larger) mass removal

Table 4 Effect of Sc as internal standard in pellets of sugarcane leaves on the determination of macro- and micronutrients. Case study with pellets prepared from comminuted laboratory samples for 5 min. Data in mg kg−1. Element

Ca K Mg P B Fe Mn Zn

LA-ICP OESa

ICP OESb

Without Sc

With Sc

7260 14,200 4400 2060 6.8 220 66 21

5900 12,800 3450 1910 5.5 205 52 19

± ± ± ± ± ± ± ±

170 900 350 120 0.5 20 4 2

± ± ± ± ± ± ± ±

100 130 100 90 0.4 4 0.6 0.7

Uncertainties are represented by ±1 estimated standard deviation. a 500 pulses per replicate (n = 10). b Acid digests (n = 3).

5750 12,600 3610 1810 5.4 199 54 19

± ± ± ± ± ± ± ±

80 160 30 20 0.7 3 0.7 1

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from the sugarcane pellets, when compared to the CRM calibration standard pellets. 4. Conclusions The potential of LA-ICP OES for the analysis of pelleted plant materials was demonstrated. Data for 8 essential elements critical to plant nutrition (Ca, Mg, K, P, B, Fe, Mn and Zn), indicate that the proposed method is a viable alternative for solid sampling of plant materials to the traditional ICP OES analysis using time consuming and cumbersome wet acid digestion procedures. Regarding the ablation process, the quality of the test sample preparation depends on the quality of the particles after comminution, which is a function of the grinding time and on the chemical composition of samples. Scandium can be used as an effective internal standard for correcting emission signal variations due to differences in mass ablated between shots and between samples, particularly in pellets prepared with larger particle size distributions, as from the comminuted sugarcane leaves for 10–20 min. It is important to point out that the comminution step can be easily employed in ball mills where up to 8 samples can be ground simultaneously in 10–20 min providing relatively high sample throughputs for large scale routine analysis. In general, it can be concluded that the direct microsampling using laser ablation provides several advantages in terms of time savings and reduced complexity while the analytical performance is found to be appropriate for the analysis of sugarcane, orange citrus and soy leaves. Acknowledgments Financial support and fellowships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (process: CAPES BEX 8867/11-9) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 140926/2009-7, 305913/2009-3, 309800/2011-0). The authors are thankful to Dr. Tatiana Trejos for the assistance at the Trace Evidence Analysis Facility (TEAF) at Florida International University. References [1] K. Mengel, E.A. Kirkby, Principles of Plant Nutrition, 5 ed. Kluwer Academic Publishers, Dordrecht, 2001. [2] B. Markert, Sample preparation (cleaning, drying, homogenization) for trace element analysis in plant matrices, Sci. Total Environ. 176 (1995) 45–61. [3] F.J. Krug, Métodos de Preparo de Amostras: Fundamentos sobre Preparo de Amostras Orgânicas e Inorgânicas para Análise Elementar, Copiadora Luiz de Queiroz: Piracicaba, SP, Brasil, 2008, chapter 5. [4] M.B.B. Guerra, C.E.G.R. Schaefer, G.G.A. Carvalho, P.F. Souza, D.S. Junior, L.C. Nunes, F. J. Krug, Evaluation of micro-energy dispersive X-ray fluorescence spectrometry for the analysis of plant materials, J. Anal. At. Spectrom. 28 (2013) 1096–1101. [5] G.C.L. Araujo, M.H. Gonzalez, A.G. Ferreira, A.R.A. Nogueira, J.A. Nobrega, Effect of acid concentration on closed-vessel microwave-assisted digestion of plant materials, Spectrochim. Acta Part B 57 (2002) 2121–2132. [6] B. Madeddu, A. Rivoldini, Analysis of plant tissues by ICP-OES and ICP-MS using an improved microwave oven acid digestion, At. Spectrosc. 17 (1996) 148–154. [7] M. Pouzar, T. Cernohorsky, M. Prusova, P. Prokopcakova, A. Krejcova, LIBS analysis of crop plants, J. Anal. At. Spectrom. 24 (2009) 953–957. [8] Y.P. Kalra, Handbook of Reference Methods for Plant Analysis, CRC Press, Boca Raton, 1998. [9] E. Lombi, K.G. Scheckel, I.M. Kempson, In situ analysis of metal(loid)s in plants: state of the art and artefacts, Environ. Exp. Bot. 72 (2011) 3–17. [10] J. Becker, R. Dietrich, A. Matusch, D. Pozebon, V. Dressler, Quantitative images of metals in plant tissues measured by laser ablation inductively coupled plasma mass spectrometry, Spectrochim. Acta Part B 63 (2008) 1248–1252. [11] J. Cizdziel, K.X. Bu, P. Nowinski, Determination of elements in situ in green leaves by laser ablation ICP-MS using pressed reference materials for calibration, Anal. Methods 4 (2012) 564–569. [12] P. Masson, Direct phosphorus determination on solid plant samples by electrothermal vaporization-inductively coupled plasma atomic emission spectrometry, J. Anal. At. Spectrom. 26 (2011) 1290–1293. [13] A. Detcheva, P. Barth, J. Hassler, Calibration possibilities and modifier use in ETV ICP OES determination of trace and minor elements in plant materials, Anal. Bioanal. Chem. 394 (2009) 1485–1495.

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