ANALYST, JANUARY 1992, VOL. 117 23 Determination of Lead in Plant Tissues: A Pitfall due to Wet Digestion Procedures in the Presence of Sulfuric Acid Erwin J. M. Temminghoff and Ivo Novozamsky Department of Soil Science and Plant Nutrition, Agricultural University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands Two wet digestion procedures for the determination of lead in plant materials by electrothermal atomic absorption spectrometry have been evaluated. The combination of HN03, H202 and HF, though leading to incomplete digestion, yielded values corresponding well with the reference or indicative values of the vegetal tissues used. The mixture of H2S04, HN03 and HC104 gave results that were too low in some instances, owing t o the formation of a mixed precipitate (Pb,Ba)S04.Keywords: Wet digestion of plant tissue; lead determination; electrothermal atomic absorption spec- trometry; lead coprecipitation; formation of (lead, barium) sulfate Wet digestion methods for organic matter in trace element determinations are well documented. 1-3 Often, H2S04 is used in the acid mixtures with the oxidizing agents: nitric acid, perchloric acid, hydrogen peroxide, dichromate and perman- ganate. There are two reasons for this; hot concentrated H2S04 destroys most organic compounds by charring and the oxidizing power of the other agents present is enhanced. The mixture of H2SO4, HC104 and HN03 is known to be one of the most efficient and fast operating reagents, resulting in far better destruction of organic compounds than a mixture of perchloric and nitric acid only.4 In this laboratory, a modification of the tri-acid (H2S04, HCIO4 and HN03) digestion mixture proposed by Schaum- loffel5 has been used for a long time for the digestion of plant tissues.The mixture of H2S04-HC104-HN03 (1 + 4 + 40) contains a rather small amount of sulfuric acid so that for the conditions used (2 g of dried plant material, 20 ml of digestion mixture, 100 ml final volume), the danger of precipitation of CaS04 is minimized. In addition to the 'classical' analysis for micronutrients, determination of Pb and Cd is increasingly important with an increasing interest in the pollution of the environment. The determination of Cd in the above men- tioned tri-acid digest proved successful, but for Pb, although the majority of analyses were correct, in some samples the values were too low as was made clear by the use of laboratory quality control.In the following study, the reasons for the incomplete recovery of Pb are examined and an alternative digestion procedure using a mixture of HN03-H202-HF is proposed. Experimental Instrumentation All measurements were performed using a Varian SpectrAA- 300 atomic absorption spectrometer equipped with a graphite tube atomizer, an automated sampler and a Zeeman-effect background correction system. The operating parameters and temperature programme for the determination of Pb and Ba are given in Table 1. Pyrolytic graphite coated partition tubes were used throughout the experiment. For Pb, 0.2% palladium chloride was used as a chemical modifier (Varian Part No. 63-100012- 00) and for Ba no chemical modifier was necessary.The sample volume that was injected for both the Pb and Ba determination was 10 pl and the volume of the injected chemical modifier was 5 pl. As the Zeeman-effect system was able to correct properly for the background (there was no change in sensitivity when plant digests with standard additions or synthetic standard solutions were measured), all signals were read against the calibration graph. Samples Analyses of plant materials were carried out on two certified reference material samples produced by the Community Bureau of Reference (BCR)6 in Brussels, two reference materials evaluated by the Comitee Inter-Instituts (CII) d'Etudes des Techniques Analytique (Theiller et a1.7) and nine samples with known Pb contents originating from the Inter- national Plant-analytical Exchange Program (IPE) in Wageningen8.9 (a continuous proficiency control scheme, organized by this laboratory).The Pb contents of all the plant materials used, together with the major matrix components, are given in Table 2. Reagents Standard solutions of Pb and Ba were prepared from Titrisol ampoules (Merck, Darmstadt, Germany; Nos. 9969 and 9968, Table 1 Operating parameters and temperature programmes for the determination of Pb and Ba by electrothermal atomic absorption spectrometry using Zeeman-effect background correction Parameters Pb Ra Wavelength/nm 283.3 553.6 Slit- wid th/nm 0.5 0.5 Measurement mode Integrated Integrated Lamp current/mA 5 10 absorbance absorbance Replicates 3 3 Temperature programmes for the graphite furnuce- Pb Ba Tempera- Tempera- t u r d Sheath t u r d Sheath Step "C Time/s gas "C Tim& gas 1 2 3 4 5 6 7 8 9 10 11 95 5 .o 130 35.0 1000 25.0 1000 15.0 1000 5 .0 1000 2.5 2500 0.8 2500 3.0 2700 0.3 2700 5.0 40 13.3 Ar-H2* Ar-H2 Ar-H2 Ar-H2 Ar - - - Ar Ar Ar 95 5.0 Ar 130 35.0 Ar 1000 25.0 Ar 1000 15.0 Ar 40 5.8 Ar 40 2.0 Ar 40 2.5 - 2700 1.4 - 2700 4.0 - 2700 4.0 Ar 40 13.3 Ar * Ar-H2 = 95% argon and 5% hydrogen.24 ANALYST, JANUARY 1992, VOL.117 Table 2 Lead and major matrix components in 13 different plant samples (Pb in mg per kg of dry matter; average k standard deviation and the major matrix components in g per kg of dry matter; average) Sample BCR No. 61 Aquatic Moss (Platihypnidium riparioides) BCR No.62 Olive Leaves (Olea europaea) CII Hay IPE No. 757 Spinach (Spinacia oleracea) 1PE No. 864 Grass (Ruakura) (Poaceae) CII Lettuce (1) (Lactuca sativa) IPE No. 844 Lettuce (2) (Lactuca sativa) IPE No. 846 Carrots (shoot) (Daucus carota L.) IPE No. 838 Onion (Allium cepa L.) IPE No. 857 Cow Parsley (shoot) (A nth viscus sy fvesfris) IPE No. 847 Carrots (root) (Daucus carota L.) IPE No. 761 Pine (needles) (Pinus radiata) IPE No. 813 Sorghem (shoot) (Sorgh urn vulgare) Major matrix components/g kg- * mgkg-l K Na Ca Mg Mn Fe A1 S Si P N Reference Pb/ 64.4k3.5 12.5 2.97 16.9 3.9 3.77 9.30 10.7 2.3 75.3 9.21 33.5 25.0t-0.7 3.1 0.06 17.5 1.2 0.06 0.28 0.45 1.6 1.26 1.05 19.5 4.70k0.40 18.5 0.91 0.5 1.4 0.24 0.19 0.37 2.3 11.6 2.12 16.7 3.24-1-0.72 68.6 4.69 16.0 5.91 0.09 1.09 1.30 4.17 13.4 7.00 44.4 1.36f0.55 25.5 2.94 6.81 1.85 0.07 0.21 0.29 2.60 6.19 3.65 29.8 6.70k 1.20 70.6 4.90 14.0 4.6 0.05 0.49 0.49 2.7 - 7.13 44.4 2.62+-0.41 55.5 1.10 9.14 2.19 0.04 0.55 0.41 3.56 23.4 6.04 33.6 6.19-tO.82 24.4 0.87 24.2 4.59 0.42 0.49 0.33 3.75 7.83 3.78 30.8 1.05k0.28 14.5 0.60 11.8 1.05 0.02 0.80 0.95 2.85 10.3 2.48 18.2 5.8710.77 34.5 0.32 12.4 1.75 0.03 0.26 0.09 1.60 0.77 3.28 18.9 0.76t0.30 48.6 4.02 3.73 1.80 0.04 0.18 0.12 2.21 1.89 5.48 18.8 1.07-CO.25 8.4 0.57 2.44 1.24 0.32 0.08 0.45 0.99 0.22 1.21 22.3 2.5110.71 23.6 0.16 4.09 2.50 0.03 0.16 0.07 1.60 8.85 3.81 17.1 50-52 mm 0.d.8-9 mm 0.d. IS0 19/26 joint Fig. 1 with HN03, HC104 and H2S04 Construction of the digestion apparatus used for the digestion respectively) and diluted appropriately.The Pd chemical modifier solution was prepared by dissolving 0.20 g of PdC12 in 2 ml of concentrated HCl (p = 1.19 g cm-3) transferring into a 100 ml calibrated flask and diluting to the mark with doubly distilled water. Butanol (0.1 ml) was added, to 2 ml of the Pd solution, before use, in order to achieve more reproducible drying conditions in the graphite atomizer. 10 All reagents were of the highest purity and doubly distilled water was used throughout. Digestion Procedures Digestion with H N 0 3 , HC104 and H2W4 A 2 g sample of the plant material (in duplicate) was weighed out precisely into a flat-bottomed 100 ml flask (Fig. l), mixed with 20 ml of the acid mixture [HN03 (p = 1.40 g cm-3) + HC104(p= 1.67gcm-3) +H2S04(p= 1.84gcm-3) = 4 0 + 4 Fig.2 H202 and HF. All dimensions in millimetres Construction of PTFE tubes used for digestion with HN03. + 11 and left to stand overnight (to prevent excessive foaming). The flasks were heated moderately for at least 40 min until most of the nitric acid was distilled off. When the heat was increased, the mixture turned black and a vigorous reaction with HC104 took place. The digestion was complete when the solution cleared and white fumes appeared. There- after the digests were diluted with about 20 ml of doubly distilled water and boiled for about 15 min. After cooling, the solution was transferred into a 100 ml calibrated flask and diluted to the mark with doubly distilled water. The samples were filtered through a fine fluted ash-free filter in a polyethylene bottle.Digestion with H N 0 3 , H202 and HF For the digestion with HN03-H202-HF solution, poly- (tetrafluoroethylene) (PTFE) tubes with a diameter of 36 mm and a height of 102 mm (Fig. 2) and an aluminium heatingANALYST, JANUARY 1992, VOL. 117 25 ~ ~~~~~~ Table 3 Total Pb content (mg per kg of dry matter) in the BCR, CII and IPE plant reference material samples in the two digests studied (average _+ standard deviation) Total Pb/mg kg-1 Digestion Indicative and Plant material reference values HN03-HC104-H2S04 HN03-H202-HF Aquatic Moss Olive Leaves Hay Spinach Grass Lettuce (1) Lettuce(2) Carrots (shoot) Onion Cow Parsley (shoot) Carrots (root) Pine (needles) Sorghem (shoot) 64.4 k 3.5 25.0 k 1.5 4.70 f 0.40 3.46 k 0.37 1.36 k 0.27 6.70 k 1.20 2.74 f 0.31 6.17 k 0.48 1.04 f 0.22 5.78 f 0.42 0.74 k 0.27 1.04 k 0.24 2.51 +.0.48 36.5 k 1.8 19.2 f 0.4 1.59 k 0.20 2.83 k 0.01 0.53 k 0.04 6.00 k 0.07 3.30 k 0.01 6.87 k 0.42 1.40 f 0.06 4.97 k 0.24 0.95 k 0.15 1.43 k 0.02 3.06 k 0.37 Table 4 Equilibrium reactions and constants used for solubility calculations of Pb, Ca and of Ba at a total SO4 concentration of 0.081 mol dm-3, an ionic strength of 0.2 mol dm-3 and pH 1 Theoretical Equilibrium reaction log K" molar solubility H+ + OH- H20 14 H+ + SOj2- % HSOj- 1.98 0.0 Pb2+ + SO42- % PbS04" 2.62 Pb2+ + 2S042- Pb(S04)z2- 3.47 2.0 x (Pb total) 7.79 Ca2+ + SO4*- % CaS04" 2.31 Ca2+ + SO4*- % CaSO,(s) 4.62 18 x (Ca total) Ba*+ + HS04- z Ba(HSO,)+ 1.07 Ba2+ + 2HS04- Ba(HS04)20 1.93 Ba2+ + SO4*- BaS04" 2.3 3.6 x lo-' (Ba total) 2H+ + SO12- z H2SO4" Pb2+ + SO42- % PbS04(s) Ba2+ + SO4'- BaSO,(s) 9.77 block were used.A 2 g sample of the plant material (in duplicate) was weighed into the PTFE tubes, mixed with 10 ml of concentrated HN03 ( p = 1.40 g cm-3) and left to stand overnight. The tubes were covered with lids and placed into a port of the aluminium heating block and the contents were boiled for 4 h at about 120 "C (liquid temperature, just boiling). Then the lids were removed and the liquid was allowed to evaporate until the sample was almost dry. A 1 ml volume of H202 (p = 1.11 g (3117-3) was added three times and the liquid was allowed to evaporate after each addition. Thereafter, 2 ml of concentrated HN03 and 5 ml of concentrated HF ( p = 1.13 g cm-3) were added and the tubes again covered with lids and boiled for 4 h.The lids were removed and the liquid was allowed to evaporate until the sample was almost dry. The residue was taken up in 20 ml of 1 mol dm-3 HN03 and boiled for about 15 min, transferred into a 100 ml calibrated flask and diluted to the mark with doubly distilled water. The samples were filtered through a fine fluted ash-free filter into a polyethylene bottle. Results and Discussion The results of the Pb determination in both digests are given in Table 3. The values given are the means of two completely independent digestions and each digest was measured three times. Clearly, for Aquatic Moss, Olive Leaves, Spinach, Hay and Grass, lower values were found in HN03-HC104-H2S04 digests compared with the HN03-H202-HF digests. All values for the latter compared well with the certified values or reported concentrations in collaborative studies.Examination 66.2 k 2.4 24.4 f 1.3 4.60 k 0.19 4.57 f 0.90 1.80 5 0.24 6.60 f 0.43 3.11 k 0.14 7.53 k 0.21 1.51 k 0.01 4.60 k 0.29 0.91 f 0.03 1.50 k 0.03 2.96 k 0.04 Table 5 Barium content in the BCR, CII and IPE plant samples in the two studied digests. Results given are for total Ba (pmol dm-3; average k standard deviation) Digestion Plant material HN 03-H ClO4-H2S04 H N 03-H*OZ-H F Aquatic Moss 0.41 k 0.06 21.4 k 1.1 Olive Leaves 0.19 k 0.04 5.0 k 0.1 Hay 0.54 k 0.01 4.1 k 0.3 Spinach 0.51 k 0.09 2.8 f 0.2 Grass 0.34 _+ 0.05 3.1 k 0.3 Lettuce (1) 0.52 k 0.03 1.4 f 0.1 Lettuce (2) 0.50 k 0.02 2.6 k 0.1 Carrots (shoot) 0.66 f 0.12 1.8 k 0.2 Onion 0.71 _+ 0.01 1.5 f 0.1 Cow Parsley (shoot) 0.52 k 0.01 1.5 f 0.1 Carrots (root) 0.26 k 0.01 0.57 k 0.02 Pine (needles) 0.09 f 0.02 0.16 f 0.01 Sorghem (shoot) 0.51 k 0.01 0.63 f 0.01 Table 6 Recoveries of Pb, using the HN03-HC104-H2S04 digest after addition of Ba, for three plant tissues Recovery (% ) Added Ba/ Cow Parsley pmol dm-3 (shoot) Carrots (root) Hay 0 100 100 33 3.6 69 96 29 7.3 32 77 25 14.6 20 39 9 of the signals showed no differences and the background signals were not excessive (the sum of the atomic absorption and background signals never exceeded an absorbance value of 1 .2), so that the Zeeman-effect background correction system was able to work correctly.It was concluded that the cause of the differences should be looked for in the sample preparation stage. Looking at the composition of the digestion mixtures, there are two differences that might be responsible for the behav- iour found: the presence of H2SO4 in the tri-acid and the presence of the HF in the proposed digestion.It is known that when the dry ashing without expulsion of silica by HF is used for digestion of plant tissues, often low values for some elements are found." Examination of Table 2 shows that Aquatic Moss and Spinach are high in silica, but not Olive Leaves, Hay and Grass. Therefore, although the influence of the solubilization of silica by HF cannot be excluded as a possible source of differences in Pb concentrations in the two digests, it certainly does not offer a complete explanation.26 ANALYST, JANUARY 1992, VOL.117 Alternatively, the presence of H2S04 can lead to the formation of slightly soluble sulfates and to losses of Pb. The maximal theoretical solubility in the digest in the presence of solid sulfates of Pb itself, of Ca as a major component of the plant tissues and of Ba as the ion forming the most insoluble sulfate known was calculated. For the species taken into consideration, stability constants published by Smith and Martell12 and Sillkn and Martelll3 were used to calculate the molar solubilities of Pb, Ca and Ba for the existing conditions (total sulfate concentration = 0.081 mol dm-3; ionic strength = 0.2 mol dm-3; and pH = 1). The results are given in Table 4. The results of the calculations suggest that direct losses of Pb, due to the precipitation of PbS04 or sorption of Pb by CaS04, are highly improbable in the materials used because the total Pb and Ca content in the matrices is not high enough to allow precipitation of these sulfates. Little is known about the Ba content of plant tissues, Ba being neither a nutrient nor a pollutant.This may be the reason why Ba analysis is almost never performed. In order to discover the Ba status in the plant materials used, the Ba content in both digests (tri-acid and proposed) was determined. The results, shown in Table 5 , strongly suggest that precipitation of BaS04 took place in the digest containing H2S04. The mean concentration of soluble Ba in this digest (0.47 k 0.15 pmol dm-3; Pine Needles excluded) corresponds well with the calculated value from Table 4 (0.36 pmol dm-3).The formation of a mixed precipitate (Pb, Ba)S04 is a possibility; the ionic crystal radii of Pb2+ and Ba2+ are 1.20 and 1.34 A, respectively, which is close enough for the formation of mixed crystals.14 Also, the same crystalline form, rhombic, is possible for both sulfates. Examination of the Ba and Pb concentration in the two digests for samples where losses of Pb took place leads to the conclusion that the precipitate formed has an approximate composition (Pb0.1Ba0,9)S04; about 3 pmol dm-3 of Ba should be present in the digest in order to start coprecipitation. As a further check, two plant samples in which no losses of Pb in the tri-acid digests were observed were chosen and increasing amounts of Ba as BaCI2 added. After digestion with HN03- HC104-H2S04, the Pb content was measured.The results are given in Table 6. It can be seen that in both Cow Parsley, containing a reasonable amount of Pb, and Carrots (root), with very little lead, after addition of about 3-4 pmol dm-3 of Ba the recovery of Pb is not complete any more. Similarly, further addition of Ba to the Hay sample resulted in a further decrease of the recovery of Pb in this sample. From these experiments it can be concluded that when H2S04 is used in the digestion mixture and sufficient Ba is present in the plant tissue itself, coprecipitation of (Pb,Ba)S04 occurs, leading to low results in the determina- tion of Pb. Because the Ba content of plant materials is usually unknown and the danger of coprecipitation cannot be evaluated in time, it is recommended that H2S04 never be used for the digestion of plant samples when Pb is to be determined.1 2 3 4 5 6 7 8 9 10 11 12 13 14 References Gorsuch, T. T., The Destruction of Organic Matter, Pergamon Press, Oxford. 1970. Bock, R., Aufschlussmethoden der Anorganischen und Organ- ischen Chemie, Verlag Chemic, Weinheim. 1972. Sandell, E. B., and Oniski, H., Photometric Determination of Traces of Metals (General Aspects), Wiley, New York, 1978. Martinic, G. D., and Schilt, A. A.. Anal. Chem., 1976, 48, 70. Schaumloffel, E., Landwirtsch. Forsch., 1960, 13, 278. Community Bureau of Reference, Certificates of Analysis Aquatic Moss (BCR No. 61) and Olive Leaves (BCR No. 62), Brussels, 1986. Theiller, G., et Les Membres du Comitke Inter Institut, Rksultats Analytiques sur de Nouveaux Etalons Vigitaux du CII, Proceedings of the VI International Colloquium for the Optimization of Plant Nutrition, AIONP, Montpellier, 1984, vol. 4, p. 1339. Houba, V. J. G., van der Lee, J . J., and Novozamsky, I., Analusis, 1991, 19, 45. Houba, V. J . G., Uittenbogaard, J., and De Lange-Harmse, A.-M., Chemical Composition of Various Plant Species (1980- 1990). Report of the International Plant-analytical Exchange Program, Department of Soil Science and Plant Nutrition, Wageningen Agricultural University, The Netherlands, 1991. Temminghoff, E. J. M., J. Anal. At. Spectrom., 1990, 5 , 273. Ledent. G., dc Borger, R., and van Hentenrijk, S., Analusis. 1984, 12, 393. Smith, R. M., and Martell, A. E . , Critical Stability Constants: Inorganic Complexes, Plenum, New York, vol. 4, 1981. Sillkn, L. G.. and Martell, A. E., Stability Constants of Metal-ion Complexes, The Chemical Society, London, 1964. Kolthoff, I . M., J. Phys. Chem., 1932. 36, 860. Paper 1 I02652 F Received June 4, 1991 Accepted August 19, 1991