Comparison of slurry preparation and microwave digestion of

View Article Online / Journal Homepage / Table of Contents for this issue

Comparison of slurry preparation and microwave digestion of freshwater algae for multi-element analysis by total reflection X-ray fluorescence spectrometry Imre Varga,* Erika Rierpl and Attila Tusai Department of Inorganic and Analytical Chemistry, L . Eo¨tvo¨s University, P.O. Box 132, H-1518 Budapest, Hungary

Downloaded on 31 January 2013 Published on 01 January 1999 on http://pubs.rsc.org | doi:10.1039/A809028I

Received 18th November 1998, Accepted 10th March 1999

The purpose of this study was to investigate the usefulness of slurry sampling for total reflection X-ray fluorescence ( TXRF ) analysis of freshwater algae. Slurry samples were prepared from freeze-dried algae by sonication. Solution samples were prepared by vapour-phase microwave digestion. TXRF analyses of 20–200 mg of specimens made from freeze-dried algal samples are reported. The concentrations of P, K, Ca, Mn, Fe, Cu, Zn and Pb were determined in the range 5–40 000 mg g−1; similar values have been published by other investigations for marine and freshwater algae samples. Good agreement was found between the results of the TXRF analysis of the slurries and the digested samples, and also the results of an additional inductively coupled plasma atomic emission spectrometric analysis of the digested samples. Student’s t-test was used to demonstrate the applicability of slurry sampling. It is concluded that the slurry preparation simplifies the sample treatment and is suitable for TXRF measurement. The accumulation of elements by plants, animals and microorganisms has been investigated by a wide variety of analytical methods. Biological materials have been analysed by inductively coupled plasma atomic emission spectrometry (ICP-AES) using a microconcentric nebulizer,1 by inductively coupled plasma mass spectrometry (ICP-MS)2 and by total reflection X-ray fluorescence (TXRF ) spectrometry3 after digestion. Sonication and slurry sampling have been combined with electrothermal atomisation atomic absorption spectrometry, flame atomic absorption spectrometry, ICPAES4 and electrothermal vaporisation ICP-MS measurements.5 Algae, as living organisms, can accumulate toxic elements in addition to essential elements. Algae have been used as biomonitors6 since the 1970s. Recently, Schorin et al.7 published a method for accurate trace element determinations in algae using ICP-AES. Schorin et al.’s algae samples were prepared by the Analytical Quality Control Services of the International Atomic Energy Agency. TXRF spectrometry needs only micrograms of sample and features simultaneous multi-element analysis for up to 20 elements in one measuring step. Calibration can easily be accomplished by internal standardisation, because of the absence of absorptionenhancement effects. Pettersson8 described a method for the direct TXRF analysis of marine periphyton communities grown on soda float glass discs. Pettersson and Olsson9 developed a nitric acid–hydrogen peroxide digestion for microalgae and compared the TXRF analytical results of digested and directly measured periphyton samples. Solid sampling can minimise the risk of contamination. In some cases only a limited amount of biological material is available for the analysis. The time consuming digestion procedure can be eliminated if a sufficient amount of sample is available to permit representative solid sampling. In this study, two methods for preparing algae for TXRF analysis were compared, namely slurry sampling and vapour-phase microwave digestion. Using a single species system, each cell of an unicellular algae has a fairly uniform size and almost the same composition, so the sampling must be representative even in the case of several individual cells.

Experimental Instrumentation The study was performed using an EXTRA IIA TXRF spectrometer (Atomika Instruments, Oberschleissheim, Germany), equipped with an energy dispersive detector. ICP-AES analysis was carried out using a Plasmalab 8440 sequential–simultaneous spectrometer (Labtam, Melbourne, Australia). The operating conditions are listed in Table 1. Sample preparation The freshwater algae selected for the study were the following: Chlorella (unicellar, green), Synehococcus (unicellular, blue) and Cylindrospermopsis (nematodeous, blue). Each algal

Table 1 Experimental conditions for TXRF and ICP-AES measurements TXRF— Excitation

Mo tube, excitation energy 17.4 keV, 30 mm Mo filter W tube, excitation energy 35 keV, 100 mm Ni filter Si(Li) detector, 80 mm2 area, 160 keV resolution at 5.9 keV 300 s (Mo tube), 600 s ( W tube)

Detector Measuring time Analytical lines

Ka lines were used to quantify P, K, Ca, Mn, Fe, Cu and Zn and the La line was used for Pb

ICP-AES— Plasma source Ar flow rates Outer Intermediate Aerosol Nebulisation Optics Analytical lines

27.1 MHz crystal controlled r.f. generator 12.5 L min−1 0.6 L min−1 0.8 L min−1 GMK nebuliser, 2.2 mL min−1 sample uptake Paschen–Runge-type vacuum polychromator, 1 m focal length Ca II 393.36, Mn II 257.61, Fe II 259.94, Cu I 324.75, Zn II 213.86 nm

J. Anal. At. Spectrom., 1999, 14, 881–883

881

View Article Online


species was cultivated separately in Allen solution under controlled conditions. The Allen solution was prepared from analytical-reagent grade reagents, containing the following constituents: Cl, K, S, Ca, Mg, P and Si (17–6 mg dL−1, in order of decreasing concentration) and Fe, B, Mn, Mo, Co, Zn and Cu (1–0.02 mg dL−1) in 1.5% NaNO solution. 3 After a nutrition period of 4 weeks, the washed samples were freeze-dried for conservation. Vapour-phase microwave digestion was applied to amounts of 6–21 mg of the freezedried sample according to a method described for the digestion of dust-laden cellulose nitrate filters.10 A temperature- and pressure-controlled microwave digestion system (MDS 2100, CEM, Matthews, NC, USA) was used. Open microvials made of quartz glass (volume 2 mL) was placed in PTFE-lined microwave digestion vessels (120 mL) using laboratory-made

Fig. 1 Comparison of slurry sampling and vapour-phase microwave digestion. Mean concentrations (mg g−1) of P, K, Ca, Mn, Fe, Cu, Zn and Pb for n independent sample analyses of ($) Chlorella (n = digested 3, n =7), (#) Synehococcus (n =3, n =4) and (×) slurry digested slurry Cylindrospermopsis (n =2, n =2). digested slurry

glass holders. The samples were wetted with 75 mL of 30% m/v nitric acid in the microvials. The digestion vessels contained a mixture of 9 mL of concentrated nitric acid and 1 mL of 30% v/v hydrogen peroxide. The vessels were heated for 90 min, applying a five-step digestion program. At a maximum power of 270 W the final temperature and pressure were 180 °C and 5.5 bar, respectively. The volumes of the digested samples were adjusted to 1 mL after cooling. Slurry samples were prepared from 5–25 mg of freeze-dried algae by sonication in 0.25–1 mL of high purity water (from a Milli-Q system, Millipore, Bedford, MA, USA) using polypropylene micro test-tubes (1.5 mL). The closed micro testtubes were immersed in the water tank of a laboratory ultrasonic bath for 5 min. Gallium was used as an internal standard at a concentration of 75–100 mg g−1 related to the dry mass of the algae. Gallium nitrate standard solution (1000 mg dL−1) was prepared by dissolving high purity Ga (99.9995%) in nitric acid (Suprapur, Merck, Darmstadt, Germany) in our laboratory. Aliquots of 10 mL were taken from the spiked solutions and from the spiked slurries to prepare specimens for TXRF measurements. The slurries were pipetted on to siliconised quartz glass plates promptly after sonication. The specimens were then allowed to dry in a clean-bench at 60 °C for 20 min. To separate the algae from the nutrition solution they were washed four times. Pettersson8 found a single wash to be effective for marine microalgae before analysis. In contrast, the freshwater algae in this study were not affected by osmotic shock, so repeated washing can be applied. The effect of the washing on the elemental composition was investigated, and it was found that four or five wash steps were sufficient for a complete separation. Washing the sample more than five times might lead to loss of elements to be determined. Prior to the TXRF measurements, the proper distribution of the sample on the quartz glass carriers were determined by diluting the slurries. Visual and microscopic observations showed that at least a 50-fold dilution (2% m/m slurry) was necessary to obtain a homogeneous and continuous layer on the carrier plates. The residual layer of slurries containing more than 2% algae became blistered and peeled off even during drying at ambient temperature in a clean-bench.

Table 2 Calculated concentrations for Chlorella (mg g−1) obtained after a rotation test Rotating angle (°)

Sample Digested sample

Mean SD Slurry sample

Mean SD

0 120 180 240 0 120 180 240

P

K

Ca

Mn

Fe

Cu

Zn

Pb

20869 25052 19018 20321 21315 2609 29414 31587 31728 29513 30561 1269

3313 3386 3360 3106 3291 127 3946 4254 4091 4315 4152 166

29205 30703 29067 28146 29280 1059 33130 36492 34195 37315 35283 1950

4538 4648 4408 4404 4500 117 4156 4591 4491 4784 4505 263

13162 13641 13519 13552 13469 211 14246 15682 15278 16211 15354 832

126 129 118 121 123 5 111 124 110 120 116 7

724 740 713 719 724 12 594 655 650 687 647 39

13 15 22 14 16 4.1 15 12 7 14 12 3.6

Table 3 Comparison of digestion and slurry preparation of Synehococcus samples (concentrations in mg g−1) Sample

P

K

Ca

Mn

Fe

Cu

Zn

Pb

Slurry samples (n=4)

7491 1147 5047 1667 2.32 0.07

1469 322 1542 110 0.37 0.73

614 88 521 49 1.63 0.16

78 14 78 7 0.07 0.95

1209 161 1321 92 1.07 0.33

14 2.4 15 2 0.87 0.42

55 9 63 6 1.26 0.26

4.8 1.3 4.7 0.8 0.11 0.97

Mean SD Digested samples (n=3) Mean SD Pooled variance t Probability

882


View Article Online


Results and discussion According to the recommendation of Klockenha¨mper,11 the layer thickness of an organic specimen should not exceed ca. 12 mm when TXRF analysis is performed with internal standardisation. An appropriate calculation resulted in an estimated layer thickness of 10–20 mm for a 1% slurry, coinciding with the recommended upper limit. In addition, the layer thickness is restricted by the measure of the cells and could not be infinitely reduced by dilution. As a compromise 0.5, 1 and 2% slurries were chosen for TXRF analysis. In the case of Chlorella more serious limitations had to be considered. Chlorella accumulated larger amounts of the elements than the other two algae investigated, and needed higher dilution prior to slurry sampling. Specimens made from 0.2, 0.5 and 1% Chlorella slurries were successfully analysed. The concentrations of P, K, Ca, Mn, Fe, Cu, Zn and Pb were determined in the range 5–40 000 mg g−1; similar values were found by other investigators for marine and freshwater algae samples7–9 and for TXRF analysis of 13 and 130 mg specimens made from CRM 414 plankton slurries.8 In our work, the mass of the specimens ranged from 20 to 200 mg. The probable precision of the analysis itself was determined by using a rotation test. Standard deviations were calculated from the data obtained by rotating the sample carriers 120°, 180° and 240° clockwise around the normal axis. A typical data set is given in Table 2. The results were similar for Cylindrospermopsis and Synehococcus also. The relative standard deviation (RSD) values were in the range 1–12% (digested samples and 4–13% (slurry samples) for P, K, Ca, Mn, Fe, Cu and Zn. Lead was close to the detection limit in all of the investigated samples. The precision of Pb determination was relatively poor (18–30% RSD) owing to the counting statistical fluctuations.11,12 Slurry sampling was compared with microwave digestion. The concentration means of P, K, Ca, Mn, Fe, Cu, Zn and Pb for Chlorella, Synehococcus and Cylindrospermopsis samples are plotted in Fig. 1. Satisfactory agreement between the two sample preparation methods was observed for all elements investigated. Student’s t-test was used to test the null hypothesis that the two preparation methods gave the same mean element concentrations. Concentration means, standard deviations of four slurries and three digested samples of Synehococcus, pooled variance t-values and the probabilities are listed in Table 3. It can be seen that only the probability

for phosphorus is close to the conventional critical level of our test (0.05). Although the small number of the samples made the test not very robust, the null hypothesis could not be rejected at the 95% confidence level. In the case of phosphorus the concentrations have to be used with particular caution. The concentrations of Ca, Mn, Fe, Cu and Zn were determined additionally by ICP-AES for digested samples. The relative differences between the results of TXRF and ICPAES analysis were below 12%. Accumulation factors were in the range of 50–15 000 related to the nutrient solution for the elements investigated. In this paper, TXRF analyses of 20–200 mg of specimens made from freeze-dried algal samples have been presented. The analytical results for slurry samples were compared with those for digested samples. We can conclude that there is no significant difference between two sampling methods. The slurry preparation of single species algae simplifies the sample treatment and is suitable for TXRF measurement.

Acknowledgement I. Varga thanks K. Barka´cs for her valuable contributions.

References 1 2 3 4 5 6 7 8 9 10 11 12

M. De Wit and R. Blust, J. Anal. At. Spectrom., 1998, 13, 515. A. Marcos, A. Fisher, G. Rea and S. J. Hill, J. Anal. At. Spectrom., 1998, 13, 521. M. Xie, A. von Bohlen, R. Klockenka¨mper, X. Jian and K. Gu¨nther, Z. Lebensm. Unters. Forsch. A, 1998, 207, 31. H. El Azouzi, M. L. Cervera and M. de la Guardia, J. Anal. At. Spectrom., 1998, 13, 533. S. F. Chen and S. J. Jiang, J. Anal. At. Spectrom., 1998, 13, 673. D. J. H. Phillips, Environ. Pollut., 1979, 18, 31. H. Schorin, Z. Benzo, E. Marcano, C. Gomez and F. O. Bamiro, At. Spectrosc., 1998, 19, 129. R. P. Pettersson, Spectrochim. Acta, Part B, 1998, 53, 101. R. P. Pettersson and M. Olsson, J. Anal. At. Spectrom., 1998, 13, 609. ´ va´ri, M. Garcia Tapia and Zs. Cze´ge´ny, B. Berente, M. O Gy. Za´ray, Microchem. J., 1998, 59, 100. R. Klockenka¨mper, Total-Reflection X-Ray Fluorescence Analysis, Wiley, New York, 1997, ch. 4, pp. 156–159 and ch. 5, pp. 177–181. E. P. Bertin, Principles and Practice of X-Ray Spectrometric Analysis, Plenum Press, New York, 2nd edn., 1984, ch. 11, pp. 474–476.

Paper 8/09028I


883