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ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978
centration of DGA used in the reaction cell was 1.1 mM in all cases. Small variations (&lo%)about this value had little effect on results. Though doubling the DGA concentration in pH 8.0 synthetic seawater did increase signals slightly at 25 "C and significantly a t 51 "C, it did little to reduce the x-intercept; more curvature of the response curve was also evident under these conditions. Effect of Fluorogenic Reagent. Only homovanillic acid (HVA) and p-HPA ( 5 , 14) were employed as fluorescence reagents in this work. It was found that HVA exhibited a smaller linear region in the analytical curve for Mn(I1) than did p-HPA and was thus unsuitable. The effect of concentration of p-HPA on the observed signal was not large, but a small increase in signal was observed when the p-HPA concentration was reduced to one-half that used by Guilbault e t al. (14). This concentration (59 pg/mL in the reaction cell) was thus used for all reported studies. Effect of Buffers. A comparison of two supposedly "non-complexing" buffers was made in synthetic seawater, both a t 0.05 M: T E S and BES (19). Though the larger pK, of TES was more desirable for use at higher pH, the response curve with TES a t pH 8.5 showed a much smaller slope than that obtained in BES a t pH 8.0. Hence, BES was chosen for use in this work. Effect of Temperature. Temperature often has dramatic effects on reaction rates in enzyme analyses since enzymes usually exhibit a temperature coefficient of about 10% per degree ( 1 4 ) . In synthetic seawater a t pH 8.0, increasing the bath temperature from 25 to 51 "C did generally effect increases in fluorescence signals; however, the data for 25 "C were superior in terms of x-intercept and linearity of the response curve. A bath temperature of 25 "C was thus used for all reported studies. C 0NCLUS IO N The major attractiveness of this Mn(I1) assay procedure is its ability to function where other methods fail or are unusually difficult to execute. For example, the Ag(1)-catalyzed persulfate reaction to produce Mn0,- ( 4 ) would be virtually inapplicable in high halide concentrations (such as those encountered in seawater). Also, analysis by atomic
absorption in seawater samples could be expected to exhibit difficulties with salt build-up in the burner (20). Since the seawater analysis is performed using a modified standard addition method, matrix effects can be expected to be minimal. The fact that the range of salinity in the Earth's oceans is only 33.8-36.4%0(21) indicates that a seawater matrix is relatively constant in nature. A 20% dilution of synthetic seawater produced a signal increase of only 7 % relative to the undiluted matrix a t the same [Mn(II)]. Assay of Mn(I1) in seawater by the method reported in this paper could thus be performed in a reasonable length of time and without the need to refer to calibration plots.
LITERATURE CITED M. M. Fishman and H. F. Schiff, Anal. Chem., 48, 322R (1976). H. U. Bergmeyer, Ed., "Methods of Enzymatic Analysis", Vol. 1, 2, 3, 4, Academic Press, New York, N.Y., 1974. G. G. Guilbault, "Enzvmatic Methods of Analvsis", Peraamon Press, Elmsford, N Y., 1970.. B. K. Pal and D. E. Ryan, Anal. Chim. Acta, 47, 35 (1969). G. G. Guilbault, P. J. Brignac. Jr., and M. Zimrner, Anal. Chem., 40, 190 11968). b. N. Lisitsyna and D. P. Shcherbov, Zh. Anal. Khim., 28, 1203 (1973). I. E. Kalinichenko, Ukr. Khim. Zh., 35, 755 (1969). L. I. Dubovenko and A. P. Tovmasyan, Zh. Anal. Khim., 25, 904 (1970). A. K. Babko, L. I . Dubovenko, and L. S.Mikhailova, Ukr. Khim. Zh., 32. 614 (1966). H. Habermann and H. Gaffron. Photochem. fhotobiol.. 1. 159 (1962). P. Homann and H. Gaffron, Science, 141, 905 (1963). P. Homann and H. Gaffron, Photochem. Photobiol., 3, 499 (1964). P. Homann, Biochemistry, 4, 1902 (1965). G. G. Guilbaul, P. J. Brignac, Jr., and M. Juneau, Anal. Chem., 40, 1256 (1968). C. 0". Curtin and C. G. King, J . Bioi. Chem., 216, 539 (1955). J. P. Riley and G. Skinow, Ed., "Chemical Oceanography", Vol. 2, Academic Press, New York, N.Y., 1965, pp 362-384. R. H. Fairbridge, Ed., "The Encyclopedia of Oceanography", Reinhold Publishing Company, New York, N.Y., 1966, p 758. I.B. Berlmn, "Handbook of Fluorescence Spectra of Aromtic Molecules", Academic Press, New York. N.Y., 1965, p 87. N. E. Good, G. D. Winget, W. Winter, T. N. Connolly, S.Izawa, and R. M. M. Singh, Blochemistry5, 467 (1966). H. H. Willard, L. L. Merritt. Jr., and J. A. Dean, "Instrumental Methods of Analysis", 5th ed., D. Van Nostrand Company, New York, N.Y., 1974, p 374. D. K. Todd, Ed., "The Water Encyclopedia", Water Information Center, Inc., Port Washington, N.Y., 1970, p 184.
RECEIVED for review January 26, 1978. Accepted March 13, 1978.
Background Absorption Error in Determination of Copper in Plants by Flame Atomic Absorption Spectrometry W. J. Simmons Department of Soil Science and Plant Nutrition, Institute of Agriculture, University of Western Australia, Nedlands, Australia 6009
Background absorptlon is shown to be a potentially serlous source of error when analyzlng perchlorlc acld digests of plant material for Cu by flame atomlc absorptlon spectrometry. The effect was malnly due to the presence of Ca; the magnltude of the background absorptlon error belng modlfled by flame condltlons, Ca concentratlon, and the levels of other major elements present In plant digests. A lean flame, a low total gas flow rate, and an observatlon helght of 11 mm mlnlmlzed the effect of hlgh Ca levels. Background correctlon or chemlcal separatlon of the Cu from the matrlx Is recommended when very low concentratlons of Cu accompany hlgh Ca levels.
Bowen ( 1 ) has recently drawn attention to the discrepancy 0003-2700/78/0350-0870$01 .OO/O
between the Cu results obtained for his kale when measured by flame atomic absorption spectrometry (AAS), neutron activation analysis (NAA), and colorimetry. The AAS value of 5.23 wg/g was biased positively with respect to the results from either of the other techniques (4.68 and 4.77 pg/g for NAA and colorimetry, respectively). These results suggest that the AAS method may be subject to serious error, particularly when extremely low concentrations of Cu are to be measured. Work done in this laboratory has indicated that the Cu readings of plant digests analyzed by AAS may contain an appreciable background absorption component. Measurements made a t the 333.8 nm spectral line of the Cu hollow cathode lamp (2), which does not exhibit absorption by Cu atoms and is quite close to the commonly used 324.7 and 327.4 nm Cu lines, have sometimes given readings whose magnitude 1978 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978
was 30% of the 324.7 nm reading when low Cu concentrations were being determined in perchloric acid digests of plant material. Against this background, it seemed timely to evaluate the extent to which background absorption affects the accuracy of the direct determination of Cu in plant digests. T h e effects of flame conditions and various levels of different elements on the magnitude of background absorption a t 324.7 nm are examined in this paper. Ca is identified as the element mainly responsible for the presence of background absorption in perchloric acid digests of plant material. Additionally, several ways of making corrections for background absorption occurring a t 324.7 are evaluated.
EXPERIMENTAL Apparatus. Measurements were made at a scale expansion of about 17 X with a Perkin-Elmer model 403 atomic absorption spectrometer in the 100 average mode (integration time about 11 s), by aspirating the solutions into the air-acetylene flame of the 10-cm single slot burner. The light sources used were a Perkin-Elmer Cu hollow cathode lamp at a current of 20 mA with the spectral band-pass of the instrument set to 0.7 nm and a Varian Techtron hydrogen lamp (current 32 mA, spectral band pass 2 nm). Use of the Cu Hollow Cathode Lamp for Measuring Background Absorption. Because of its excellent stability, the Cu hollow cathode lamp was used to obtain most of the background absorption readings of the synthetic plant digests made from various combinations of reagents. The possibility that Cu contamination may have been responsible for these readings, some of which were equivalent to greater than 1 pg Cu per g of plant material, was eliminated in three different ways. (i) Measurements made on the synthetic solutions using the H lamp gave similar, although less precise results, to those obtained with the Cu lamp. (ii) The reagents contained only extremely low concentrations of Cu. Determination of Cu in the stock solutions by organic extraction with ammonium 1-pyrrolidinecarbodithioate(3)showed that the highest Cu level in any of the synthetic plant digests would have been 0.003 pg/mL. (iii) If Cu was responsible for the readings of the synthetic solutions, then the nearby less sensitive 327.4 nm line should have given a reading which was half that obtained at 324.7 nm. This was not so. The 327.4 nm readings of the synthetic digests, expressed as a percentage of the corresponding 324.7 nm reading, had a mean and 95% confidence interval of the mean of 92.0 f 4.3 (lean flame) and 110 f 2.1 (slightly acetylene rich flame). Further, the nearby 333.8 nm line, at which wavelength no Cu absorption was observed, gave readings similar to or higher than those obtained at 327.4 nm. Such observations were not consistent with absorption due to Cu atoms but were consistent with the presence of background absorption. These results, taken together, showed that the contribution of Cu atoms to the observed hollow cathode lamp readings of the synthetic digests was extremely small. Virtually all the reading was due to background absorption. Composition of Plant Materials. The concentrations of the major elements in Bowen's kale were 4.09% Ca, 2.46% K, 0.157% Mg, 0.251% Na, 1.60% S, and 0.449% P ( 4 ) while the barley straw contained 0.19% Ca, 0.90% K, 0.17% Mg, and 0.046% P. Digestion of Plant Material. This differs from a previous description ( 5 ) in that 1-g samples were digested with 20 mL concentrated HNO, and 2 mL of concentrated HCIOl and that the perchloric acid residue was heated for 15 min to dehydrate the silica after which the residue was quantitatively transferred with deionized water to calibrated polycarbonate vials and made to 20 mL. Terminology. Detection Limit. For uncorrected data, this was defined as the Cu concentration in aqueous solution giving a reading at 324.7 nm equal to twice the standard deviation of ten successive readings of deionized water obtained at 324.7 nm using the 100 average mode of the instrument. For corrected data this was defined as v7D,-"* + Dz~,,,,,tlonwhere Dcu is the detection is twice limit as defined in the previous sentence and Dcarrection the standard deviation of ten successive readings (100 average mode) of deionized water obtained with the instrument set to
871
measure the correction and expressed as a Cu concentration measured at 324.7 nm with the Cu hollow cathode lamp. Height of ObservatLon. This was defined in accordance with the revised Standards Association of the Australian Code of Practice on Atomic Absorption Spectroscopy (6). With the burner completely clear of the light path, the readout was set to zero absorbance. The burner was then raised until the signal fell to an absorbance of 0.30. The distance the burner was racked downwards from this reference point was the height of observation. Because the burner of the instrument began to intercept the light path at a height of 6 mm, all readings were obtained at heights equal to or greater than '7 mm.
RESULTS AND DISCUSSION Background Absorption of Kale Digests. Evidence for the presence of a serious background absorption component in the reading of kale digests and the identification of Ca as the element mainly responsible was obtained by measuring the absorption produced by a kale digest and a series of seven synthetic kale digests containing all or all but one of each of the six major elements. The concentrations of the elements in the synthetic digests were the same as their levels in the kale digest. Since background absorption has been reported only for high concentrations, it seemed unlikely that the trace elements would be involved, so only the major elements were used in the synthetic digests. T h e measurements were made using the 333.8 nm line of the Cu lamp and radiation from the hydrogen lamp with the spectrometer set a t 324.7 nm. Good agreement between the readings of the kale digest and the complete synthetic digest using the hydrogen lamp a t 324.7 nm and the Cu lamp a t 333.8 nm confirmed the presence of a serious background absorption error as well as verifying the opinion that only the major elements were involved. The means of the duplicate 17 X scale expanded readings obtained using a lean flame were 0.0245 and 0.0225 (hydrogen lamp) for kale and the complete synthetic solution, respectively, and 0.0235 and 0.0225 with the Cu lamp. These readings correspond to an apparent Cu level of about 0.8 p g / g plant material for a digest containing 0.05 g of kale per mL. Only the absence of Ca from the synthetic kale digest had a very marked effect on the magnitude of the background absorption. When it was omitted from the solution, the absorption was reduced by a factor of three. Extent of Background Absorption Error. Flame composition, total gas flow rate, height of observation, and nebulizer uptake rate, all of which might affect the background absorption error, were studied in detail with a CaC1, solution containing 4000 pg Ca per mL. Further, because the background absorption error could be worse at other concentrations and combinations of concentrations of the major elements, the potential magnitude of the error and its dependence on the composition of the digest was evaluated by measuring the absorption of synthetic digests covering a range of combinations of a wide range of concentrations of each major element. Studies were also carried out with real plant digests. E f f e c t o f Flame Conditions, Height of Observation, a n d Nebulizer U p t a k e Rate. Flame conditions and the height of observation had a n appreciable effect on the background absorption error. No setting of either of these variables or combinations of them could be found which would completely eliminate the error. The experiments were carried out a t two different air flow rates (15.4 and 21.2 L/min) and four different heights (7 to 20 mm). The acetylene flow rate was varied to provide flame conditions ranging from a very lean to a rich flame. With the air flow rate maintained a t 21.2 L/min,the error decreased with decreasing acetylene flow rate at all heights, provided the ratio of the acetylene to air flow rate was less than 0.22. The rate of decrease in the error increased with increasing height: the effect of decreasing acetylene flow rate
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 7 , JUNE 1978
Table I. Effect of Height of Light Path on Precision and Detection Limit Height, mm 7 11 16
20
RSD"
Detection limit, ugimL
0.4 0.5 1.1 4.8
0.002 0.002 0.005 0.021
Relative standard deviation. Table 11. Concentrations of Elements Used in Series 1 and 2 Synthetic Plant Digests Concentration, pg/mL Ca 0 250b 500
75OC 1000 1500 2000a 4000
K 0 500 1000b 1260" 2000d
Na Ob
125a
Mg
S
P
0 83a2b 249 498
0 201b 403 806a
0 113b 225a 450
The basic level for series 1. The basic level for series This level was not present in series 2. Higher solution concentrations will not be encountered because of KClO, precipitation. a
2.
was not very marked at a height of 7 mm (when the error decreased from the equivalent of 0.065 pg Cu/mL to 0.055 pg/mL) compared with its effect at 20 mm, when the error decreased to the equivalent of 0.032 pg Cu/mL. A t acetylene to air ratios between 0.22 and 0.36 (maximum studied), there was no appreciable effect of the ratio or the height of observation on the error. With the observation height maintained a t 11 mm, reducing the air flow rate from 21.2 to 15.4 L/min decreased the background absorption error by the equivalent of 0.015 pg Cu/mL a t all acetylene to air ratios. The minimum error, corresponding to 0.03 kg Cu/mL, was obtained with a very lean flame a t the air flow rate of 15.4 L/min. These results lead to the conclusion that the best operating conditions for minimizing the effect of the background absorption should be a low total gas flow rate combined with a lean flame and a large height of observation. However, the last condition was not practical for measuring low concentrations of Cu because of poorer precision and poorer detection limits (Table I). When these additional observations were taken into account, the use of a low total gas flow rate, lean flame, and a height of 11 mm was the best compromise for minimizing background absorption due to CaC1, when measuring low Cu concentrations. Changing the nebulizer uptake rate had no appreciable effect on the size of the relative error due to Ca. Background Absorption of Synthetic Digests. Because of the extremely large number of possible combinations of different concentrations of the different elements, these experiments were restricted to a set of solutions containing
increasing amounts of Ca and two series of synthetic digests (all solutions contained 10% v/v concentrated HC104). Series No. 1 was based on Bowen's kale and series No. 2 based on plant material containing much less Ca, P, and S. Because of the previous finding that the major elements accounted for the background absorption observed with Bowen's kale, only these elements were used in the solutions. For each of the series, the concentration of one element was changed from 0 to the maximum likely to be encountered with digests of real plant material, while the other elements were held constant a t the basic levels-see Table 11. The solution concentrations were set a t levels which assumed the digest contained 0.05 g of oven dry plant material per mL. Only the results obtained with compromise operating conditions (see previous section) will be described. These experiments showed that only concentrations of Ca and P had an appreciable effect. No experiments were run in the absence of Ca to identify the residual source(s) of background absorption. E f f e c t of Ca. The presence of Ca had the most marked effect, the magnitude of the background error increasing with increasing Ca Concentration. Increasing the level of the other elements (Le., changing from pure Ca to series 2 to series 1 matrices) markedly increased the error, which changed from the equivalent of 0.32 pg Cu/g of plant material (pure Ca solution) to 1.26 pg/g (series 1) a t the 4000 pg Ca/mL level. While Ca levels as high as this are unusual (equivalent to 8% Ca in plant material) and are unlikely to be encountered for most plant samples, they could occur when plant parts are being analyzed. For instance, Loneragan and Snowball (7) have reported levels of 7 and 8% in the cotyledon and hypocotyl of tomato plants. Background absorption errors due to the presence of such levels of Ca would be extremely serious if Cu concentrations below 2 pg/g were encountered. At more common plant Ca concentrations (below 2 % ) , the magnitude of the error could still be serious, ranging downwards from 0.72 kg c u / g . Effect of P. The effect of P was most marked with series 1, the background absorption error rising with increasing P concentration from the equivalent of 0.54 pg Cu/g a t 0 P to 1.18 pg/g a t 460 pg P/ml. With series 2 the apparent Cu concentration rose from 0.22 to 0.40 pg/g as the P level went from 0 to 460 pglml. Background Absorption of Plant Digests. The effects reported for the synthetic digests were substantiated by the plant digest studies. These experiments were done with barley straw, to which increasing amounts of Ca had been added before digestion, and with Bowen's kale. The results obtained with the compromise flame condition and the worst flame condition (slightly acetylene rich flame-air flow rate 21.2 L/min) will be described in this section. Flame composition had a marked effect on the Cu results only if no correction was made for the presence of background absorption. The richer flame gave a higher uncorrected result than the lean flame for all digests (Tables I11 and IV). The mean difference between the six pairs (lean and rich flames) of uncorrected kale results was highly significant (P < 0.001). For barley straw, the effect of increasing Ca concentration was very marked with the richer flame but less evident for the lean
Table 111. Analysis of Six Kale Samples
Uncorrected Mean Std dev
5.20 0.11
Cu concentration, p g / g Lean flame Stoichiometric flame Corrected Corrected H lamp 333.8 nm Uncorrected H lamp 333.8 nm 4.24 0.11
4.23 0.11
5.56 0.10
4.28 0.13
4.00 0.11
ANALYTICAL CHEMISTRY, VOL. 50, NO. 7 , JUNE 1978
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Table IV. Analysis of Nine Samples of Barley Straw
Added Ca, pg/mL
Uncorrected
0 0
1.86
500 750
1.87 1.94 2.01 1.98 2.01 2.15 2.17
1000 1500
2000 3000 4000
2.01
Cu concentration, pg/g Lean flame Stoichiometric flame Corrected Corrected H lamp 333.8 nm Uncorrected H lamp 333.8 nm 1.71 1.89 1.71 1.83 1.78 1.83 1.70 1.71
1.74
flame, where sample variation obscured the difference except a t the two highest Ca levels. However, the hydrogen lamp readings showed that the background absorption error, expressed as Cu concentration in plant material, rose from about 0.1 to 0.4 pg/g as the level of Ca increased from the native level of 95 pg/mL of digest to 4095 pg/mL. While the use of a lean flame and a low total gas flow rate reduced the error to almost a negligible quantity for the barley straw with Ca levels up to 1500 pg/mL, the error a t higher Ca levels and for kale was too large to ignore. For such matrices, particularly when they occur with low Cu concentrations, background correction is essential. Background Absorption Correction. The two most common ways of correcting for the presence of background absorption utilize either a continuum light source, or a nearby line in the hollow cathode lamp spectrum which exhibits no atomic absorption ( 3 ) . Good agreement between the means of duplicate readings of the synthetic kale digest obtained a t 324.7 nm with the Cu lamp and the hydrogen lamp indicated t h a t this continuum light source was giving an accurate measure of the background absorption. The readings were 0.0235 (Cu lamp) and 0.0225 (H lamp). The plant digest data supported this finding. In contrast to the marked effect flame conditions had had on the uncorrected results, the hydrogen lamp corrected results were unaffected by flame conditions (Tables I11 and IV). T h e mean difference between results obtained with the different flames was not statistically significant (P > 0.4 for pairs of kale results and P > 0.6 for pairs of barley straw results). The mean corrected concentration of 4.26 pg Cu per gram found for Bowen's kale was also satisfactory because, although it was lower than the values given by colorimetry and neutron activation analysis (4.77 and 4.68 pg/g, respectively) it was still within one standard deviation of them (Bowen (1)). Use of the nearby 333.8 nm line for making background corrections suffered from the disadvantage that either underor overcorrection was produced. The accuracy of the background correction given by this line was evaluated by comparing the absorbance of the 333.8 and 324.7 nm lines of the Cu lamp for the synthetic digests as well as comparing the plant digest results obtained using the 333.8 nm correction with the results obtained using the hydrogen lamp correction. The comparisons were done under both flame conditions. With the lean flame, the 333.8 nm line gave slight undercorrection to good correction for both synthetic digests, kale digests (Table 111) and barley straw digests containing less than 1000 pg Ca/mL (Table IV). Barley straw digests
1.75 1.89 1.67 1.71
1.78 1.67 1.54 1.59 1.50
1.88 2.03 2.04 2.23 2.31 2.41 2.63 2.98 3.24
1.75 1.89 1.81 1.76 1.65 1.52 1.61 1.81 1.89
1.79 1.89 1.62 1.71 1.47 1.33 1.23 0.96 0.77
containing more than 1000 pg Ca/mL suffered from overcorrection (Table IV). The richer flame gave an overcorrected result for all solutions containing more than 350 pg Ca/mL, the overcorrection becoming very serious a t high Ca levels (Table IV). These results indicated that the 333.8 nm line may be employed for making a background correction provided a lean flame is used. The result should be more accurate than if no correction is made, but may not be as accurate as the correction obtained with a continuum lamp source. The detection limits of both correction methods were worse than the uncorrected detection limit (by a factor of 5 for the hydrogen lamp and a factor of 2 for the 333.8 nm line) reading. Therefore, when extremely low Cu concentrations are encountered, use of the 333.8 nm line may be preferable to the continuum for making a background correction, provided the limitations described in the previous three paragraphs are borne in mind. For Cu deficiency studies, very low Cu concentrations may be encountered. Levels less than 0.5 pg/g (oven dry basis) may be found in the growing point of wheat plants suffering from Cu deficiency (8). For samples containing such low concentrations, the relative error could be serious, ranging from +92 to +252% a t a Cu level of 0.5 pg/g as the plant Ca concentration goes from 0.5 to 8% in a series 1 matrix. Under these circumstances, either background correction or chemical separation of the Cu from the interference is essential for accurate analysis.
ACKNOWLEDGMENT I thank A. D. Robson for constructive criticism made during the preparation of this manuscript. LITERATURE CITED (1) H. J. M. Bowen, At. Energy Rev., 13, 451 (1975) ( 2 ) J. B. Willis, Aust. J . Dairy Techno/., 19, 70 (1964). (3) G.D. Christian and F. J. Feldman, "Atomic Absorption Spectroscopy: Applications in Agriculture, Biology and Medicine", Wiley-Interscience, New York, N.Y., 1970. (4) H. J. M. Bowen, J . Radioanal. Chem.. 19, 215 (1974). (5) W. J. Simmons, Anal. Chem., 47, 2015 (1975). (6) Standards Association of Australia, Australian Standard CK18 (revised 1977). Chemical Analysis of Materials by Atomic Absorption Spectroscopy. Not yet published. (7) J. F. Loneragan and K. Snowball, Aust. J . Agric. Res., 20, 465 (1969). (8) J. F. Loneragan, K. Snowball, and A. D. Robson, "Transport and Transfer Processes in Plants", Academic Press, New York, N.Y., 1976, Chap. 39.
RECEIVED for review April 4,1977. Accepted February 8, 1978. This work was supported by the Western Australian State Wheat Industry Research Committee.
