Figure 1.
Calibration curves for Cr standards
These standards contain Ti, Fe, and the following interference suppressors: ( 0 )K H S 0 4 , ( A ) Na2S04, ( X ) aqueous, ( B ) NHICI
Effect of Anions. Some anions interfere with Cr absorption ( 3 ) , but as standards and sample are closely matched and recovery was satisfactory the effect of anions can be practically ignored. Precision and Accuracy. Lack of a suitable analyzed standard ilmenite was a problem. The accuracy of the method was checked against various ilmenites analyzed by conventional techniques (Table IV). Precision was established by replicate analysis of E P 139 over several months. A standard deviation of 0.0036%
was obtained. The relative standard deviation was 3.4%. The method is simple, rapid, and is applicable to a wide range of ilmenite types and alteration products.
ACKNOWLEDGMENT My thanks are due to E. S. Pilkington of CSIRO, Melbourne, for kindly providing the analyzed ilmenite E P 139 and to A. Pepper for critically reading the manuscript. Received for review November 30, 1972. Accepted March 28, 1973.
Determination of Low Concentrations of Cobalt in Plant Material by Atomic Absorption Spectrophotometry W. J. Simmons Department of Soil Science and Plant Nutrition, lnstifute of Agriculture, University of Western Australia, Nedlands, W. A. 6009
Co deficiency develops in animals when the Co concentrations of their pastures and fodders fall below 0.07 to 0.10 pg/g (dry weight) for some time ( I ) . In many areas levels as low as 0.01 pg/g (dry weight) are encountered. There is, therefore, considerable interest in simple, rapid, and accurate procedures for measuring these extremely low concentrations. The most common procedures in current use for determining Co in biological materials are based on the nitroso R salt colorimetric method (2-4). This technique is not ( 1 ) E. J. Underwood, "Trace Elements in Human and Animal Nutrition," 3rd ed. Academic Press, New York, N . Y . , 1971, p 150.
ideal for routine analyses because it is long and great care is required during the color development of the cobalt nitroso R salt complex. Also a t low Co concentrations, a t least 20 g of dry plant material is required for sufficient sensitivity. A more rapid technique using small samples was required for Co assays of a large number of samples collected in a survey of the trace element content of a range of (2) H. R. Marston and D . W. Dewey, Aust. J. Exp. Biol. Med. Sci., 18, 343 (1940). (3) D. W . Dewey and ti. R. Marston, Anal. Chirn. Acta., 57, 45 (1971). (4) B. E. Salzman, Anal. Chern., 27, 284 (1955).
ANALYTlCAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
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~~
~
~
Table I . Effect of Sample Weight on Precision and Result Mean conNo. of centration," Plant material Weight, g analyses p g Co/g RSD"
Wheat Subterranean clover Yellow lupin Lucerne
5.2 10.4 1.9 5.8 2.0 6.0 1.o 5.8
0.0121 0.01 27 0.0448 0.0427 0.0856 0.0834 0.154 0.149
9.6 4.2 7.2 4.1 4.2 1.7 7.9 4.5
~
Table II. Recovery of Co Added to Plant Material cos Weight, Plant material
Wheat Subterranean clover Yellow lupin Lucerne
a Calculated using mean of 12 blanks (mean blank 0.0182 pg of Co, standard deviation 0.0084pg of Co).
pasture species undertaken at this Institute (5). Recently two methods using atomic absorption have been published. Jago, Wilson, and Lee (6) concentrated the Co from 5 g of plant material into 1 ml of methyl ethyl ketone (MEK) which was then sprayed into the flame. Gelman (7) used 10 g of plant material and 5 ml of isobutyl methyl ketone (MIBK). The procedure of Jag0 et al. is superior to Gelman's because it is appreciably more sensitive, thus requiring a smaller amount of plant material. However we have encountered a serious problem in using Jago's method resulting from the MEK extract blocking the nebulizer of our instrument. This paper describes an alternative atomic absorption procedure having the same sensitivity as that of Jag0 et al. but using a different complexing agent, pH, and final solvent for extraction and concentration of the Co. Removal of the excess complexing agent completely prevents nebulizer blockage.
EXPERIMENTAL Reagents. All reagents were tested for Co content and none required purification. Apparatus. The atomic absorption spectrophotometer used for the absorption measurements was basically a Techtron AA5 with a Zeiss M4QIII monochromator and a Techtron AA4 lamp supply. A Perkin-Elmer DCR 2B concentration readout connected to the amplifier readout module was used to obtain the readings. Procedure. The procedure used was that which is described below. The extraction step is based on the general colorimetric method for Co using 2-nitroso-1-naphthol described by Sandell (8).
Reference Solutions. Add 2.5 ml of 1000 pg/ml aqueous solution of Co (CoSO4) t o 25 ml of 0.5% w/v ethanolic solution of 2nitroso-1-naphthol. Mix and allow to stand for 1 hr. Dilute to 250 ml with MIBK. Prepare 0, 0.3, 0.6, 1.0, 1.5, and 2.0 pg of Co per ml reference solutions by diluting aliquots of the above stock solution with MIBK. Before making to volume, add sufficient 2-nitroso-1-naphthol solution so that each reference solution contains 0.01% wjv 2-nitroso-1-naphthol.Use these solutions for setting up the instrument and for correcting instrument drift. Digestion. For Co concentrations greater than 0.07 pg/g dry weight, weigh 2 g of dry plant material into a tared 100-ml conical flask. (At lower levels of Co take 5 g but divide the plant material evenly into two conical flasks so that the digestion may be carried out safely and quickly.) Digest the contents of each flask with 30 ml of concentrated "03 and 5 ml of concentrated HC104 (9) by heating the flasks on an electric frypan. At the appearance of white HClOl fumes, continue to reflux for 1 hr a t 190 "C to destroy any refractory organic matter and to dehydrate the Si&. J. S. Gladstones and J . F . Loneragen, Aust. J. Agr. Res., 18, 427 (1 967). (6) J. Jago, P. E. Wilson, and B. M. Lee, Analyst (London), 96, 349 (1971). (7) A. L. Gelman, J. Sci. F o o d A g r . , 23, 299 (1972). ( 8 ) E. B. Sandell, "Colorimetric Determination of Traces of Metals," 3rd ed. Interscience, New York, N . Y . , 1959, p 4 2 0 . (9) C. M. Johnson and A . Ulrich, "Analytical Methods for Use in Plant Analysis," Buli. Calif. Agr. Exp. Stn., 766, 32 (1959). (5)
1948
9
4.954 5.295 4.773 4.675 5.059 4.737 4.605 4.671 4.955 4.842
In
sample 0.061 0.065 0.209 0.205 0.427 0.400 0.700 0.710 0.753 0.736
Added 0.060 0.060 0.300 0.300 0.600 0.600 0.600 0.600 0.600 0.600
Found 0.119 0.141 0.514 0.499 1.013 1.029 1.281 1.300 1.366 1.281
Recovery, %
96.7 126.7 101.7 98.0 97.7 104.8 96.8 98.3 102.2 90.8
Extraction. Dilute the single or combined digests to about 40 ml. Heat to dissolve any KC104 precipitate. Allow the solution to cool to room temperature. Add 10 ml of 40% w/v sodium citrate solution. Adjust the p H within the range 6.0 to 6.4 ( p H meter) with 10M N K O H and use 7M HCl to correct any overshoot. Add 5 ml of 6% v/v hydrogen peroxide solution to reduce any F d + to Fe2+ (8).Decant the solution from any KC104 or Si& precipitate into a 100-ml separating funnel. Add 2 ml of 0.5% w/v ethanolic solution of Z-nitroso-l-naphthol, mix, and allow the solution to stand for 1 hr. Extract the cobalt 2-nitroso-1-naphtholateserially with 10, 5, and 5 ml of chloroform by shaking the mixture for 1 min. Collect the extracts in a 50-ml weighing bottle. When extraction is completed, remove the aqueous phase by inverting the funnel. Rinse the funnel with deionized water. During these steps, take care that no aqueous phase enters the stopcock or the stem of the funnel. Return the organic phase to the separating funnel uia a filter funnel. Rinse any extract remaining in the filter funnel into the separating funnel with 5 ml of chloroform. Add 10 ml of 2N NaOH to the extract and shake the mixture for 15 sec to remove the excess 2-nitroso-1-naphthol. After phase separation, transfer the organic phase back to the weighing bottle. Remove the chloroform by allowing it to evaporate overnight in a fume cupboard or by heating the bottle over a water bath. When dried and cooled, add 1 ml of MIBK, insert the stopper into the bottle to prevent evaporation, and carefully rotate the bottle to dissolve the residue. Run six standards (0, 0.3, 0.6, 1.0, 1.5, and 2.0 pg of Co), each in 40 ml of 6% v/v HC104, through the procedure commencing with the addition of 10 ml of sodium citrate solution. A digestion blank should also be run through the whole procedure. Standards and the blank are extracted and analyzed with each group of samples. Batches of 20 plant samples a t a time may be handled conveniently. Measurement. Use a spectral band pass of 0.12 nm a t the 240.7-nm Co line. Set the burner height to 6 mm and optimize the air/GH* flame by changing the G& flow rate so that when MIBK is sprayed maximum transmission of the 240.7-nm line is obtained without flame lift-off occurring. Use 3 X scale expansion for measurements.
RESULTS AND DISCUSSION Preliminary Experiments. Attempts to extract the Co in the digest directly into 1.5 ml of isoamyl acetate (less soluble in water than MIBK) using sodium diethyl dithiocarbamate or ammonium 1-pyrrolidinecarbodithioate (APDC) failed because of the formation of a precipitate of the Fe complex. Extracting the Co with 2-nitroso-1-naphthol was successful, provided that the excess reagent, which caused the nebulizer to block, was removed from the isoamyl acetate by an additional extraction with 2h' NaOH. However, this technique was rejected because of the difficulty of handling such a small volume of solvent. Extraction pH. The extraction pH of 6.0 f 0.2 was chosen after preliminary experiments had confirmed Boyland's (10) finding that the reaction between 2-nitroso-lnaphthol and Co proceeds rapidly to completion in the cold in the pH range 4-9. Dewey and Marston ( 3 ) , using (10) E . Boyland, Anaiyst iiondorr). 7 1 , 230 (1946).
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
Table Ill. Recovery of Co Added to High Concentrations of Other Elements co. Pg Element
K P
Fe
a
Form added
+
KOH KHzPO4 KH~POA
Amount of element, m g
225 50 150 41.5 12.5 25 0.1 0.025
Equivalent concentration for a 5-9 plant sample.
Concn in plant material pg/ga
Stand dev
of % Recovery,b %
I n sampleb
Added
Foundb
45000} 10000
0.014
0.300
0.309
98.3
1.2
30000) 8300 2500 \
0.042
0.300
0.342
100.0
2.2
0.1 02
0.300
0.400
99.2
1.6
recovery
50 Mean of 3 determinations.
1-nitroso-2-naphthol to extract Co from 10 to 50 g of plant material prior to its final colorimetric assay as the nitroso R salt, preferred to use an extraction p H of 4.0 because of the possibility of loss of Co by adsorption onto metal hydroxides a t a higher pH. The results described below indicate that this is not a problem in the proposed method. Precision. For Co concentrations around 0.08 pg/g, determinations using a single sample and blank gave good precision for sample weights of 2 g. The relative standard deviation (RSD) was 6.5%. The precision decreased in plant samples containing Co concentrations as low as 0.044 pg/g so that 2-g samples were less satisfactory for a single sample and blank (RSD 12.0%). But the precision was good for 6-g samples (RSD 5.3%). At extremely low Co concentrations of 0.01 pg/g, the RSD for a single sample and blank was 16.4% when the sample weight was 5 g. While too large for some work, this precision is satisfactory for survey work. Precision may easily be improved by taking more blanks. For example, where the mean of the 12 blanks run during the present experiment was used to calculate the concentration of Co, the RSD for a single sample improved from 16.4 to 9.6%. This is a useful way of improving the precision when only a limited amount of sample is available. The fifth column of Table I shows the precision for plant materials calculated in this way. The precision may also be improved by increasing the number of samples. These results indicate that the precision of the procedure is as good as, and possibly better than, that reported by Jag0 et al. They reported RSD values ranging from 9 to 14% for single determinations of Co in plant materials Zontaining 0.42 to 0.07 pg/g, respectively. Accuracy. Recoceries. Table 11 shows that the recovery of Co added to plant material prior to digestion is perfectly satisfactory. The worst recovery of 127% was obtained on a sample of wheat straw which had an extremely low Co concentration of 0.012 bg/g. All recoveries lay within the limits expected from the errors associated with each determination. When 5 7 c 0 was added to four 5-g samples of lucerne prior to digestion, recoveries of activity in the MIBK solution were quite satisfactory. The mean recovery was 98.8% with a standard deviation of 0.8%. E f f e c t of High Concentrations of Other E l e m e n t s . Because plant materials may contain appreciable levels of other elements ( I I ) , the effect of relatively high concentrations of eight of the most common of these elements on the determination of added Co was examined. The smount of each element taken and their manner of group-
ing is shown in Table 111. The concentration of each element in the simulated digest would be higher than that in the digest of 5 g of most plant materials. None of the salts added interfered seriously with the reP covery of added Co (Table 111). The results for the K group are slightly low and in agreement with a similar experiment using 5 7 C in ~ which about 0.7% of the activity was found in the KC104 precipitate. Therefore, the amount of K contributed by 5 g of plant material containing up to 4.5% K should give no difficulty; K could be a problem in samples containing extremely low concentrations of Co and very high concentrations of K. The results indicate that the various elements should cause no interference a t the concentrations encountered in most plant material. Constant Bias. Youden (12) has shown that the presence of constant bias in an analytical method is not revealed by the standard recovery test but is revealed if very different sample weights of the same material are analyzed. The proposed method was tested for constant bias in this way. At least four replicates a t two different sample weights for each of four plant species were analyzed. The mean of the twelve blanks which were carried through the procedure with the samples was subtracted from the amount of Co found in each sample. This reduced the influence of the variability of the blank in assessing the accuracy. Sample weight had no effect on the Co concentration of any of the plant materials analyzed ( P 5 0.05: Table I) indicating that the method is free of constant bias. The problem of nebulizer blockage encountered with another procedure has been prevented by the removal of excess 2-nitroso-1-naphthol with NaOH. Over 1000 samples have been analyzed using the proposed procedure and not once has the nebulizer blocked. The method is superior to the colorimetric procedure because small sample weights can be used. For Co concentrations as low as 0.08 pg/g, sample weights of as little as 2 g have satisfactory precision and accuracy. At even lower Co concentrations, 5-g samples give adequate precision and accuracy.
(11 ) H. D. Chapman, "Diagnostic Criteria for Plants and Soils," University of California Division of Agricultural Sciences, 1966.
(12) W. J. Youden. "Statistical Methods for Chemists," 1st ed, Wiley, New York, N.Y.. 1957, p 40.
+
ACKNOWLEDGMENT The author thanks J. F. Loneragan for helpful, constructive discussion in both the development of this procedure and the preparation of the manuscript. Received for review July 24, 1972. Accepted January 31, 1973.
ANALYTICAL CHEMISTRY, VOL. 45, NO. 11, SEPTEMBER 1973
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