Determination of nickel, calcium and magnesium in xylem sap by flame atomic absorption spectrometry using a microsampling technique

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Phytochem. Anal. 2009, 20, 365–371 Copyright © 2009 John Wiley & Sons, Ltd.

Research Article

Received: 25 September 2008, Revised: 18 December 2009, Accepted: 31 March 2009 Published online in Wiley InterScience: 16 June 2009

(www.interscience.wiley.com) DOI 10.1002/pca.1135

Determination of Nickel, Calcium and Magnesium in Xylem Sap by Flame Atomic Absorption Spectrometry using a Microsampling TechniqueSheila Alves, Maria L. Simões Gonçalves and Margarida M. Correia dos Santos*

ABSTRACT:Introduction – Knowledge of xylem sap chemical composition is important to the understanding of translocation, detoxifi cation and tolerance mechanisms. However, the small amount of sample available often hampers its characterisation. Hence, low volume consumption techniques are needed for xylem sap analysis.Objective – To develop a microsampling technique for the determination of elements in xylem sap from diff erent plants by fl ame atomic absorption spectrometry (FAAS).Methodology – The microsampling device was optimised in terms of sample volume and integration time. The analytical characteristics of the microsampling technique (m-FAAS) were established and compared with those of FAAS with traditional continuous nebulisation. The method was validated by means of an independent technique.Results – Ca, Mg and Ni were determined in a 50 mL aliquot of xylem sap solution/element that was introduced directly into the fl ame via the microsampling accessory. Good precision was obtained with relative standard deviations of 1.1, 0.6 and 2.3% for Ca, Mg and Ni, respectively. Matrix eff ects resulting from the physical characteristics of the samples and possible chemical interferences caused by phosphate and/or sulphate were ruled out.Conclusion – A simple, rapid and reproducible microsampling technique coupled to FAAS was developed and successfully applied in the determination of Ca, Mg and Ni in xylem sap. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: Xylem sap composition; fl ame atomic absorption spectrometry; microsampling technique

* Correspondence to: M. M. C. dos Santos, Centro de Química Estrutural, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001, Lisboa, Portugal. E-mail: [email protected]

Centro de Química Estrutural, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001, Lisboa, Portugal

Contract/grant sponsor: POCI 2010.

Contract/grant sponsor: European Social Fund.

Contract/grant sponsor: FCT; Contract/grant number: SFRH/BD/21597/2005.

IntroductionFlame atomic absorption spectrometry (FAAS) is the technique most commonly applied for the determination of metals at the 1.0 or 0.1 mg/L levels. This method typically depends on the continuous introduction of the sample into the fl ame during the measurement period. Problems might arise when the sample is too viscous, or contains a high concentration of dissolved solids that can cause a rapid blockage of the burner slot, or when the available volume is in the microlitre range since a minimum of 1–2 mL of sample solution is required for classical nebulisation techniques. These problems can be overcome by nebulising discrete sample aliquots and recording the transient signal.

Discrete nebulisation, or microsampling as it is sometimes called, have been employed in several studies. Single pulse nebulisation techniques were reported for the determination of several elements in steel and whole blood using a disposable pipette tip inserted into the end of the silicone rubber nebuliser uptake tube. Discrete volumes, typically 25–250 µL of solution, could be nebulised by dipping the tip in the sample solution (Thompson and Godden, 1976a, b). A similar approach was used in fl ame emission spectrometry in the determination of the alkaline and alkaline earth cations in human blood serum (Fry et al., 1978; Berndt, 1979; Matusiewicz, 1982). More recently, microsampling techniques were reported for the determination of metals in strong brines and serum where the nebuliser uptake

tube was manually introduced into the sample solution (Wai et al., 1996; Ji and Ren, 2002). Microvolume injection techniques have also been described either using hydrodynamic injection (Xu and Fang, 1995) or installing an interface for sample injection in the atomic absorption instrument by removing the capillary tube and the glass impact bead (Shang and Hong, 1997a,b).

Xylem sap represents a single and specifi c compartment in plants and knowledge of its composition is important in the understanding of translocation, detoxifi cation and tolerance mechanisms (Marschner, 1995). Xylem sap comprises an aqueous solution in which constituents such as Ca, Mg, K, nitrate, phos-phates and amino and carboxylic acids are found in milimolar concentration ranges, while other elements such as Ni, Zn, Cu and Mn may occur in µM concentrations. However, since the

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volume of xylem sap that may be extracted is very small (often limited to a few hundred microliters) chemical analysis of the material is somewhat diffi cult (Bhatia et al., 2005; Goodger et al., 2005). It is therefore essential to have low volume consumption techniques for sample analysis.

In the present study, we report a simple, rapid and reproduc-ible procedure for the determination of Ca, Mg and Ni in xylem sap by FAAS using a microsampling technique (µ-FAAS). Sap samples from diff erent species growing in non-serpentine (NS) and serpentine (S) soils of the northeast of Portugal were collected. Serpentine soils are particularly interesting since they are char-acterised by elevated concentrations of Ni and Mg combined with low Ca concentrations (Menezes de Sequeira and Pinto da Silva, 1992). The plants analysed were: Alyssum serpyllifolium ssp. Lusitanicum, an Ni hyper-accumulator endemic of the serpentine area, and two tolerant species of serpentine soils Quercus ilex (Holm Oak) and Cistus ladanifer (Brown-Eyed Rock Rose) (Freitas et al., 2004). For xylem sap extraction two diff erent procedures were used. In the case of Q. ilex, sap was collected using an opti-mised version of the vacuum extraction method (Bollard, 1957; Nabais, 2000), while a modifi ed Scholander et al. (1965) proce-dure was used for A. serpyllifolium and C. ladanifer.

The analytical characteristics of the microsampling method for Ca, Mg and Ni were established and compared with those of FAAS applied with traditional continuous nebulisation. The methodology was validated by comparing the results obtained for Ni by µ-FAAS with those of an independent method, namely, square wave voltammetry (SWV), after proper digestion of the xylem sap.

Although elemental determination in low sample amounts can be performed by graphite furnace atomic absorption spectro-metry (GFAAS), many laboratories can only handle elemental determination by FAAS, particularly in nonspecifi c analytical chemistry areas, including plant physiology. Furthermore, for particular applications, GFAAS might not be feasible owing to element concentration, sample manipulation requirements and high risk of contamination. Therefore, some comparison was also carried out with GFAAS in terms of small volume requirements and sample contamination.

ExperimentalReagents and solutions

All reagents were of analytical grade. Deionised water from a Milli-Q 185 Plus water purifi cation system (Millipore, Bedford, MA, USA) was used to prepare all solutions.

Standard stock solutions (1.000 g/L) of Ca, Mg and Ni were prepared by dissolving the corresponding nitrate salts of AnalaR grade (BDH Chemicals, Poole, UK) in 3.5% v/v nitric acid 65% of Puriss. p.a. grade (Riedel-de Haën, Seelze, Germany). Calibration solutions were prepared daily by dilution of the standard stock solutions in nitric acid and used to establish the calibration curves.

Lanthanum stock solution (25.000 g/L) was prepared by dis-solving lanthanum(III) oxide (Riedel-de Haën) in hydrochloride acid 37% PA-ACS-ISO (Panreac, Barcelona, Spain).

Hydrogen peroxide 30% (w/w) solution from Sigma (St. Louis, MO, USA) and nitric acid 65% of Suprapur® grade from Merck (Darmstadt, Germany) were used for wet digestion of xylem sap. For the vacuum extraction of xylem sap, a 1% w/v safranin dye solution (Safranin T, Riedel-de Haën) was prepared.

Sampling

A serpentine area of 70 × 35 m located in Maciço de Morais (Northeast Portugal) was selected for plant collection, and further divided into fi ve sampling sites. At each sampling site, samples of A. serpyllifolium (entire bush) and C. ladanifer (branches) from randomly selected plants (minimum of fi ve individuals/species) were collected. A similar approach was followed for a non-serpentine neighbouring area where branches of C. ladanifer were collected as well as branches from the upper crown of Q. ilex trees.

Immediately after collection the samples were sprinkled with distilled water and kept in plastic bags until required for sap extraction.

Xylem sap extraction

For Q. ilex, a vacuum extraction procedure was used and the xylem sap was extracted from the branches with a diameter greater than 5 mm (Nabais, 2000). The branches were submerged in water, the bark on each end was removed to prevent contam-ination of xylem sap with cellular constituents of phloem and parenchyma cells and the submerged ends were cut back several times to minimise embolisation or cavitation of the vessels within the branches. The branches were then attached with their apical end on top of a vacuum fl ask and exposed to a vacuum of 80 Pa. The safranin dye solution was fed at the basal end of the section to prevent contamination of xylem sap with water during extrac-tion. Both ends were rinsed thoroughly with distilled water before extraction.

Xylem sap from A. serpyllifolium and C. ladanifer was obtained by application of pneumatic pressure using a Scholander pressure chamber from Manofrígido (Amadora, Portugal) pressurised with nitrogen. Sap was extracted from branches and twigs, cut from the shrubs, with 10–30 cm in length and less then 1 cm in diameter. The cut end was cleaned with distilled water and the bark removed. The branch was placed in the pressure chamber with the cut end on the outside through a rubber stopper fi tted into the chamber cover. After pressure application the xylem sap was removed from the cut end using Pasteur pipettes.

Xylem sap samples were pooled in Eppendorf caps in order to obtain one unique composite sample/species and soil type, and stored at −20°C.

Xylem sap digestion

For total nickel determination by SWV, the xylem sap of A. serpyl-lifolium was digested using a mixture of nitric acid and hydrogen peroxide (Huang and Schulte, 1985). Briefl y, a 50 µL aliquot of xylem sap was transferred to a 10 mL Pyrex cup, 1 mL of Suprapur nitric acid (65%) was added and the mixture heated for 30 min on a hot plate at 80–90°C. Then, 1 mL of hydrogen peroxide 30% (w/w) solution was carefully added and the mixture was evapo-rated to dryness. After cooling, the dry residue was dissolved to a fi nal volume of 10 mL in 0.010 M nitric acid.

Instrumentation

Flame atomic absorption spectrometry. A Unicam M Series atomic absorption spectrometer from Thermo Electron Corpora-tion (Cambridge, UK) equipped with a 50 mm slot universal Ti burner and Ca, Mg and Ni hollow cathode lamps from Photron

Determination of Nickel, Calcium and Magnesium in Xylem Sap

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(Victoria, Australia), and a stoichiometric air–C2H2 fl ame was used for the determinations. Optimum instrumental and opera-tion conditions are given in Table 1.

The manual aliquot microsampling accessory, consisting of a Tefl on block with a funnel shaped hole and a stainless steel clip is shown in Fig. 1, and was built following the general idea presented in the spectrometer manual (Thermo Electron Corporation, 2004). The Tefl on block was fi tted to the fl ame compartment door via the stainless steel clip. One end of the sample uptake capillary tube (250 × 0.5 mm i.d.) was pushed into the hole of the Tefl on block base and the other end was fi tted to the nebuliser.

Graphite furnace atomic absorption spectrometry. A Unicam M Series atomic absorption spectrometer (Thermo Electron Corporation) equipped with a GF95Z Zeeman graphite furnace and a FS95 furnace auto-sampler was used for Ni determination in xylem sap samples after appropriate dilution. Absorbance measurements were made at 232.0 nm, with a spectral band-pass of 0.1 nm, using a Ni hollow cathode lamp from Photron operated at 5 mA and partition extended lifetime cuvettes from Thermo Electron Corporation.

The electrothermal program of 104.4 s included two drying stages together with a pyrolysis, an atomisation and a cleaning stage with temperatures of 95, 150, 400, 2250 and 2500°C, ramps of 8, 3, 13°C/s (for the fi rst three temperatures) and times of 10, 20, 20, 3 and 2 s, respectively.

Square wave voltammetry. Voltammetric measurements were performed using an Autolab/PGSTAT10 potentiostat/galvanostat (Eco-Chimie, Utrecht, Netherlands) connected to a Metrohm Stand 663 (Herisau, Switzerland) featuring a conventional three-electrode

confi guration, namely, a static mercury drop electrode (SMDE) as the working electrode, an Ag–AgCl–KCl(sat) as the reference electrode and a carbon rod auxiliary electrode.

SWV was used for Ni determinations with a square wave amplitude (Esw) of 25 mV, a step height (∆E) of 5 mV and a frequency (f) of 50 Hz. In all experiments, the initial potential was −0.75 V and the fi nal potential was −1.25 V vs Ag–AgCl. All measurements were performed in deaerated solutions with high purity nitrogen.

Procedure

Depending on the plant species, the element to be determined and the technique employed, xylem sap samples were diff erently diluted with acidifi ed aqueous solution. All samples were analysed at least in duplicate sets. In all cases, calibration against standard solutions was done. In the microsampling technique an adjustable volume manual pipette was used for standards and xylem solutions injection. A transient signal was recorded and the absorbance area was selected for all analytical measurements. In GFAAS the analytical measurements were also based on absorbance peak area.

Working standards, calibration blank and diluted sample solu-tions were prepared in nitric acid: 3.5% v/v for FAAS and µ-FAAS, and 0.1% v/v for GFAAS.

Nickel determination by SWV was carried out on digested xylem sap after proper dilution (1:200) in 0.010 M nitric acid. Calibration of the voltammetric response to Ni was performed in the absence of xylem sap in the same medium by plotting the SWV peak currents as a function of total nickel added in the range of 0.1–1.0 mg/L.

Method validation

The performance parameters evaluated were: calibration charac-teristics, precision, matrix eff ects caused by the physical charac-teristics of the samples, and trueness. The calibration characteristics were studied according to ISO 8466-1 (International Organiza-tion for Standardization, 1990). The precision of the method was evaluated in terms of repeatability (within run precision) accord-ing to ISO 5725-1 and ISO 5725-2 (International Organization for Standardization, 1994a, b). Matrix eff ects were determined by spiking analysis (with diff erent addition levels) of diluted xylem sap samples (Eurochem, 1998). The recovery percentage and the slope of standard addition methods were critically evaluated (Miller and Miller, 2000). Trueness was established by Ni deter-mination in Alyssum xylem sap using an independent analytical method (Eurochem, 1998). The mean results obtained by the two methods were statistically compared by signifi cance tests (Miller and Miller, 2000).

Results and DiscussionOptimisation of the microsampling technique

A typical transient signal is presented in Fig. 2(a) from which absorbance height or absorbance area could be estimated. Although similar standard deviations were obtained for both types of measurements, the absorbance area was selected since the sensitivity was slightly higher [Fig. 2(b)].

In Fig. 3 the infl uences of the injection volume (a) and of the integration time (b) on the analytical signal peak are shown. The absorbance increases with increasing injection volume within

Table 1. Instrumental and operational conditions for FAAS

Parameter Ca Mg Ni

Wavelength (nm) 422.7 285.2 232.0Spectral bandpass (nm) 0.5 0.5 0.2Lamp current (mA) 5 4 5Acetylene fl ow rate (L/min) 1.1 1.1 1.1Air fl ow rate (L/min) 7 7 7Burner height (mm) 10.2 9.4 7.0

Figure 1. The manual aliquot microsampling accessory showing: (a) transverse section; (b) operational position in the atomic absorption spectrometer.

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the analysed range for all elements [Fig. 3(a)]. A value of 50 µL was chosen as adequate for the required sensitivity.

The sample uptake rate was 4 mL/min, and this was controlled and kept constant by the gas fl ow rate. For an injection volume of 50 µL, it was observed that the greatest value of absorbance was reached in 8 s and therefore the integration time was set at 8 s [Fig. 3(b)].

Calibration function and analytical characteristics

Calibration graphs were constructed under the optimal conditions chosen with standard solutions of Ca, Mg and Ni. A set of six

levels of concentration in the analytical ranges presented in Table 2 was used (International Organization for Standardization, 1990).

The concentration range was established by homogeneity variance tests. Homogeneity of variances was verifi ed using the F-test. The testing value, FExp, was calculated as the quotient of the variance of 10 absorbance measurements of the standards with the highest and lowest concentrations. The calculated FExp values are presented in Table 2 as well as the critical value, F (f1 = 9, f2 = 9, P = 95%). Since FExp < F, no signifi cant diff erences between the variances were obtained and so the unweighted least squares method could be used (International Organization for Standardization, 1990; Huber, 1997; Trancoso et al., 2003).

The linearity of the calibration curve was verifi ed by Mandel’s fi tting test. Testing value, TV, was calculated through equations (1) and (2) (International Organization for Standardization, 1990; Funk et al., 1995):

TVDSsy

=2

22

(1)

DS N s N sy y2

12

222 3= − − −( ) ( ) (2)

where sy1 and sy2 are the residual standard deviations of fi rst- and second-order calibration functions, respectively (calculated from the calibration data), and N is the number of calibration data pairs. Since TV values are lower than the critical Fisher values F (f1 = 1, f2 = N − 3, P = 95%; Table 2), the linear calibration function provides the best adjustment in the selected working range (International Organization for Standardization, 1990; Funk et al., 1995; Huber, 1997; Trancoso et al., 2003). The linear regression equations and the corresponding correlation coeffi cients, r, are presented in Table 2. The calibration was accepted when r > 0.995.

In Table 3, the analytical characteristics of the method are listed. Limits of detection, LOD (mg/L) and quantifi cation, LOQ (mg/L), were determined from equation (3) with k = 3 and k = 10, respec-tively, and b is the slope of the calibration function (IUPAC, 1978):

LDks

by= 1 (3)

Figure 2. (a) Transient signal obtained for a 0.1 mg/L Mg standard solution; (b) signal mea-surement as peak area (white) and peak height (grey) of a 0.8 mg/L Mg standard solution and diluted xylem sap of Q. ilex (1:10); n = 5, tIntegration = 8 s, VInjection = 50 µL.

Figure 3. Relation between absorbance and: (a) injection volume (µL): tIntegration = 8 s, n = 5; (b) integration time (s): VInjection = 50 µL, n = 5.



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Characteristic concentrations, c0 (mg/L), were determined as the concentration of analyte that provided an absorbance signal of 0.0044 (International Organization for Standardization, 1990; Funk et al., 1995; Huber, 1997; Trancoso et al., 2003).

Precision, taken as repeatability (International Organization for Standardization, 1994a, b), was evaluated from the standard deviation of 10 replicate determinations using 10.0 mg/L Ca standard and 1.00 mg/L Mg and Ni standards. The corresponding percentage relative standard deviations, RSD (%), are also shown in Table 3. The results obtained with the conventional sample introduction, where the measurements were carried out in the continuous mode (classical nebulisation system) are presented in the same table. Similar LOD, LOQ and c0 were obtained for both modes of sample delivery. Higher RSD values were found in the case of the microsampling technique, but the values are acceptable for FAAS (Welz and Sperling, 1999).

Matrix eff ects due to the physical characteristics of the samples were accessed from the recoveries of Ca, Mg and Ni added to xylem sap solutions. At least three addition levels were prepared in the working concentration range previous selected (Table 2). Recoveries in the range of 95–105% were obtained for the three elements. The slopes of the calibration graphs and those of the standard additions method were compared statistically using

Student’s t-test. For the three elements, the theoretical t-value was always higher than the experimental value, tExp, at the 95% confi dence level. As an example, t(16,0.95) = 2.12 > tExp = 0.3 was obtained for Ca determined in Q. ilex. These results ruled out matrix interferences and so the determination can be performed by direct calibration.

In order to assess the trueness of the method and since neither matrix nor concentration matching reference materials were available, Ni determination in the xylem sap of A. serpyllifolium was performed by an independent method (Eurochem, 1998). An electroanalytical technique, square wave voltammetry, was chosen, after proper digestion of the sample, in order to deter-mine total Ni in solution (Mota and Correia dos Santos, 1995). The value obtained for Ni concentration in the xylem sap of A. serpyllifolium was 54 ± 5mg/L (n = 3). This concentration was statistically compared with that obtained by m-FAAS (Table 4) through signifi cance tests. Both the F-test, for comparing standard deviations (F(2,4,0.95) = 19.2 > FExp = 1.79), and the t-test, for comparison of means (t(6,0.95) = 2.49 > tExp = 1.00), showed no signifi cant diff erences, thus proving the trueness of the m-FAAS.

As is well known, phosphate as well as sulphate can interfere in the determination of both Ca and Mg due to the formation of compounds of low volatility. Since both anions exist in xylem

Table 2. Characterisation of the linear calibration functions

Parameter Ca Mg Ni

Analytical range (mg/L) 1.0–10 0.1–1.0 0.2–1.0Concentration levels (mg/L) 1.0, 2.0, 4.0, 6.0, 8.0, 10 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 0.2, 0.3, 0.4, 0.6, 0.8, 1.0Homogeneity of variances F(9,9,0.95) = 4.03 > FExp = 3.38 F(9,9,0.95) = 4.03 > FExp = 1.76 F(9,9,0.95) = 4.03 > FExp = 3.07Mandel test F(1,3,0.95) = 10.1 > FExp = 1.54 F(1,3,0.95) = 10.1 > FExp = 3.53 F(1,3,0.95) = 10.1 > FExp = 1.31Linear expression y = (1 ± 3)10−3 + (4.2 ± 0.1)10−2x y = (−1 ± 3)10−3 + (67.6 ± 0.4)10−2x y = (3 ± 2)10−3 + (61 ± 3)10−3xr 0.9996 0.9999 0.9996

Table 3. Analytical characteristics of the method

Parameter Ca Mg Ni

Transient Continuous Transient Continuous Transient Continuous

LOD (mg/L) 0.4 0.4 0.02 0.01 0.07 0.04LOQ (mg/L) 1 1 0.06 0.04 0.2 0.1c0 (mg/L) 0.1 0.1 0.007 0.004 0.07 0.04RSD (%) 1.1 0.1 0.6 0.1 2.3 0.5

Table 4. Ca, Mg and Ni concentrationsa (mg/L) in xylem sap of plants growing in serpentine (S) and non-serpentine (NS) soils

Sample Soil Elements

Ca Mg Ni

Alyssum serpyllifolium S 77 ± 3 (n = 4, 1/50) 36 ± 1 (n = 3, 1/100) 56 ± 3 (n = 5, 1/100)Quercus ilex NS 39 ± 3 (n = 5, 1/20) 16 ± 1 (n = 3, 1/20) —Cistus ladanifer S — — 10 ± 1 (n = 3, 1/10)

NS — — 2.8 ± 0.3 (n = 3, 1/4)

a Results are expressed in terms of mean and standard deviation at 95% (x̄ ± smt). Number of replicates and dilution factor are shown in parentheses. m-FAAS: tIntegration = 8 s; VInjection = 50 µL.

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sap, possible chemical interferences were assessed by analysing the eff ect of La added to standard and xylem sap solutions (Welz and Sperling, 1999). For both Ca and Mg, no signal depression was observed in the absence of La, thus ruling out the existence of interferences.

Analytical applications

The microsampling technique (µ-FAAS) was applied to the deter-mination of Ca, Mg and Mg in the xylem saps and the results are listed in Table 4. The determination of each element was per-formed using just 50 µL of sample, which corresponds to about 1/50 of the volume needed with conventional nebulisation.In the case of the hyper-accumulator A. serpyllifolium, owing to the high concentrations of the elements present in the xylem sap, samples were diluted 1:50, 1:100, 1:100, for the determina-tion of Ca, Mg and Ni, respectively. For Ca and Mg in Q. ilex, the dilution factor was 1:20. For Ni in C. ladanifer, xylem sap was diluted 1:4 (non-serpentine) and 1:10 (serpentine). In all cases, no matrix eff ects caused by the physical characteristics of the sap were observed and direct calibration could be used.It should be pointed out that in the case of conventional sampling, a minimum of 6–12 mL solution would be necessary for the determination of the three elements in duplicate sets. In most situations, the amount of xylem sap available (<1 mL) would be insuffi cient even when dilutions were performed.

The Ni concentration in xylem sap of Q. ilex was below the LOQ of the method. This aspect, together with the small amount of sample available, justifi ed the use of GFAAS for its determina-tion. Xylem sap of Q. ilex was diluted 1:4 and total Ni was found to be 24 ± 3 µg/L. In all other situations, however, unreasonable dilutions would be necessary in order to fi t the sample concen-tration to the analytical range of GFAAS. Furthermore, in order to avoid contamination, additional cleaning procedures would be necessary as well. This aspect is especially relevant in Mg and Ca determination owing to ease of contamination by these ubiquitous elements (Powell and Tease, 1982; Kántor et al., 1994; Godlewska-Źylkiewicz et al., 1998). The described technique allows sample volumes of the same order of magnitude as those used in GFAAS to be handled, thus diminishing sample manipulation.

The microsampling technique for fl ame atomic absorption spectrometry proposed in the present work is rapid, simple and involves a low risk of sample contamination. It was successfully applied in a typical situation where the small amount of sample available can prevent its chemical characterisation, as was recently emphasised in studies of xylem saps composition (Bhatia et al., 2005; Goodger et al., 2005).

Compared with previous reports of microsampling techniques coupled to FAAS, a better precision (RSD%) was obtained even when compared with microvolume injection techniques (Xu and Fang, 1995; Shang and Hong, 1997a, b). Other advantages of the microsampling accessory employed include: easy washing and decontamination, simple handling, straightforward construction, low cost and allowing further coupling with other commercially available FAAS accessories such as the slotted tube atom trap (Thermo Electron Corporation, 2003).

The technique might also be applicable to the determination of other elements and/or in other types of matrices, including biological and environmental samples, when only small volumes of sample are available without the need to use sophisticated apparatus.

Acknowledgements

This work was co-fi nanced by POCI 2010 and by the European Social Fund. Sheila Alves acknowledges FCT for Ph.D. grant SFRH/ BD/21597/2005. The authors thank Cristina Nabais (Departamento de Botânica, Universidade de Coimbra) for guidance in sampling.

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Determination of nickel, calcium and magnesium in xylem sap by flame atomic absorption spectrometry using a microsampling technique