Determination of Small Amounts of Copper in Spray Residues D. E. H. FREAR Agricultural Experiment Station, State College, Penna.
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URING the past few years it has been necessary for the author to make a large number of copper determina-
The method as finally adopted in this laboratory is as follows:
tions on various surfaces sprayed with copper fungicides. The surfaces upon which the copper was deposited included the leaves and fruits of apples, cherries, and tomatoes, and certain synthetic surfaces,. particularly Pyralin, which is a t present being used in this laboratory for most of the studies on the deposition and retention of sprays. A number of methods have been investigated, and two have been selected as the most satisfactory under the present conditions. Since an increasing number of workers in the fields of insecticides and fungicides are turning their attention to the copper compounds, and since few rapid methods for the determination of the small amounts of copper present upon plant surfaces have been described in the literature, the following two methods are presented briefly. These are modifications of well-known analytical procedures and have been used with complete success in this laboratory for some time,
The solution containing the copper is freed from organic matter, if the latter is present, by digestion with sulfuric and nitric acids. For routine analysis of fruits, it has been customary to wash the surfaces thoroughly in hot 10 per cent nitric acid solution, making the washings to volume and digesting an aliquot portion. The laboratory sprayed plates are washed in 50 per cent nitric acid and the wash solution is concentrated, made to volume, and used without digestion. The solution, free from organic matter, is neutralized with concentrated ammonium hydroxide, and about 10 ml. are added in excess. This mixture is then boiled for a few minutes, allowed to stand for 30 minutes, filtered through a fast filter paper, and washed. The entire filtrate, or an aliquot of it, is transferred t o a Nessler tube, 25 ml. of concentrated ammonium hydroxide are added, and it is made t o a volume of 100 ml. The tube is then placed in a photoelectric colorimeter of the type described by Frear and Haley (5) or Samuel and Shockey (6), the light intensity is adjusted to the maximum, 1 ml. of a 1 per cent solution of sodium diethyl dithiocarbamate is added, the solution is stirred, and a second reading is taken. By calibrating the instrument with known amounts of copper, the reading in microamperes may be converted directly into milligrams of copper. A typical calibration curve is shown in Figure 1.
Method A When copper is present in the sample in amounts greater than 2 mg., the most accurate and convenient method of determination is by direct weighing after electrodeposition on platinum electrodes. The determination of small quantities (under 50 mg.) is considerably more difficult, however, than the ordinary electrodeposition as followed in the case of copper ores or alloys. The procedure found satisfactory for leaf samples which have received one or more applications of copper sprays is as follows:
Accuracy of Method B For the greatest accuracy, a sample should be selected which contains between 0.05 and 0.15 mg. of copper. Numerous recovery tests have been run with apple wash solutions containing no copper, to which have been added known amounts of copper as copper sulfate. A typical set of re-
A sample of from 2 t o 20 grams of the dried material is ashed at a temperature not exceeding 450' C. The ash is dissolved in nitric acid (1 to I) and transferred to a 150-ml. beaker. To this solution are added 10 ml. of a saturated solution of ammonium nitrate and 1 gram of urea, and the volume is made up to about 100 ml. The electrolysis is then carried out in the usual manner, usin a platinum gauze cathode and a rotating platinum loop anofe. The current between the electrodes must be much lower than is usually recommended in the methods for the electrolytic deposition of copper described in the literature, and should not exceed 0.15 ampere. Currents in excess of this amount will cause the deposition of copper oxide. The time required for complete deposition is a function of the quantity present, but for the amounts normally present on leaf samples 15 minutes is usually sufficient.
Typical results on different types of leaf material are shown in Table I.
Method B When the total quantity of copper in the sample is less than 2 mg., it is usually not possible to weigh the metal directly with sufficient accuracy. Samples of fruits and small areas of synthetic surfaces sprayed in the laboratory usually bear less than 1 mg., and hence require a method of analysis sensitive to smaller amounts. After thorough trials of several methods, including the chromotropic reagent method of Ansbacher, Remington, and Culp (I), the xanthate method (7), and the thiocyanate method of Elvehjem and Lindow (Q), the method of Callan and Henderson (2) as modified by Cockburn and Herd (3) was selected as most adaptable to the present use.
0
OJOMG. COPPER a20
FIGURE1. TYPICAL CALIBRATION CURVE 494
SEPTEMBER 15, 1939
ANALYTICAL EDITION
TABLEI. COPPERPRESENT IN APPLE AND CHERRY LEAVES AS DETERMINED BY ELECTROLYTIC METHOD Type
of Leaf
Copper Present A B
Treatment
Mo./sq. m.
495
When using solutions in which no organic matter is present, such as the washings from the plates sprayed in the laboratory, the procedure may be simplified for the sake of rapidity by the elimination of the filtration, if the standardization is carried out under the same conditions.
Summary Two methods are presented for the rapid determination of copper on surfaces sprayed with insecticide or fungicide mixTABLE11. RECOVERY OF ADDED COPPERSULFATE TO APPLE WASHSOLUTIONS Copper Added Mo. 0.050 0.075 0.100 0.150 ~~~
Copper Recovered %
Mo.
tures containing this element. The first method, for quantities greater than 2 mg., is a modification of the usual procedure of weighing the metal directly after electrodeposition. The second method, for smaller quantities of copper, is based on the photoelectric measurement of the color produced by sodium diethyl dithiocarbamate.
Literature Cited
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sults is shown in Table 11. Each figure is the average of duplicate determinations. Replicate determinations usually agree within 2 microamperes (about 0.003 mg.). If a photoelectric colorimeter is not available, the unknown solutions may be compared with standards in a colorimeter. In this case the color should be developed in both standards and unknowns a t the same time, since the ability of the colored solution to transmit light decreases slightly on standing.
(1) Ansbaoher, Remington, and Culp, IND.EXG.CHEM.,Anal. Ed., 3,314 (1931). (2) Callan and Henderson, Analyst, 54, 650 (1929). (3) Cookburn and Herd, Ibid., 63, 482 (1938). (4) Elvehjem and Lindow, J. Biol. Chem., 81,435 (1939). (5) Frear and Haley, Penna. Agr. Expt. Sta., Bull. 304 (1934). (6) Samuel and Shockey, J. Assoc. Oficial Agr. Chem., 17,141 (1934). (7) Scott, “Standard Methods of Chemical Analysis,” Vol. 1, p. 197, New York, D.Van Nostrand Go., 1922. AUTHORIZED for publioation as paper No. 899 in the journal series of the Pennsylvania Agrioultural Experiment Station.
Determining Riboflavin A Fluorometric and Biological Method G. C. SUPPLEE, R. C. BENDER, AND 0. G. JENSEN The Borden Company Biological and Chemical Research Laboratories, Bainbridge, N. Y.
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HE establishment of riboflavin as a dietary essential has been the incentive for numerous investigations involving its. biological and physico-chemical properties. Its fluorescence in ultraviolet light has suggested the possibility of utilizing this characteristic as a basis for its quantitative determination; under proper conditions as little as one part in 100,000,000 may be detected by this means. A preliminary outline of a quantitative method applicable to solutions of lactoflavin (riboflavin derived from milk) was published in 1936 (3). Since that time the method has been further perfected and applied to various materials, and also used to determine quantitatively small amounts of riboflavin required for certain precision studies involving the water-soluble vitamins (1, 4). I n order that the usefulness of any physical or chemical method for determining known vitamin entities may be fully appraised, i t is necessary to correlate the results obtained with the biological response from experimental animals. The results from the fluorometric method presented in this paper have been correlated with a biological method based upon the principles of simplification and standardization previously proposed (1, 4). Application of the fluorometric principle for the quantitative determination of riboflavin must be predicated upon the examination of appropriate solutions in which extraneous matter does not significantly interfere with the degree of
fluorescence or its observation. Potentially, each riboflavin bearing material presents a different problem in respect to the treatments required to extract the active material, and to obtain a solution suitable for examination. Obviously solutions of pure material present no such problems; likewise, many impure riboflavin concentrates, especially those obtained from whey or whey derivatives following a preliminary adsorption, frequently require no preliminary treatment other than proper dilution. Adsorbates and miscellaneous products require complete elution or extraction if reliable quantitative results are to be referred to the original carrier. The general procedure followed in the present study involves the use of an 80 per cent acetone-water mixture. Lactoflavin or natural riboflavin as derived from milk was used for the development and study of the methods hereinafter presented.
Extraction and Preparation of Riboflavin Solutions for Fluorometric Examination PUREOR COMPARATIVELY PURESOLUTIONS OF RIBOFLAVIN. Such solutions of unknown concentration are diluted to match the fluorescent color of standard riboflavin solutions of known concentration. ELUTION OF RIBOFLAVIN FROM CLAY ADSORBATES AND ~ I M I L A R RESIDUES. A 2-gram sample is shaken with 400 ml. of an 80 per cent acetone-water (by volume) mixture in the dark at room temperature for 30 to 45 minutes, the eluate is filtered off, and the residue is washed with 10 to 15 ml. of the acetone mixture.
