Sept., 19581 TITRATION CNIT FOR PROCESS USE 503 A System for the Determination of Certain Trace Metals in Crops* BY W. D. DUFFIELD (26 Pavk Crescent, Povtland Place, Londoia, W.1) The system described has been developed to meet the need for a rapid routine evaluation of trace-element levels in crops and foodstuffs. Copper, cobalt, nickel, molybdenum and boron are determined by paper chromato- graphy. Zinc, manganese, and, in certain contingencies, molybdenum are determined colorimetrically. Is the course of a comprehensire long-term agricultural investigation, I have examined large numbers of assorted crops, fodders and pastures with a view to establishing concentration levels of trace metals and detecting significant fluctuations in these levels. Because of the number of elements of established importance to plant growth that had to be considered and the variety of samples involved, it was obvious that classical methods of separation and determination of these metals would be excessively laborious and time-consuming.A rapid method for routine use must satisfy the following criteria: (a) simplicity and rapidity of technique, ( b ) use of the minimum of apparatus, which should be unspecialised in nature and economical of bench space, and (c) suitability to the examination of large batches of samples in routine series. The methods chosen are either paper chromatographic or colorimetric, and in every instance the final determination is effected by visual comparison against standards. This technique has been found to give accuracies adequate to the scope of the system and within the limits of experimental error of the methods as a whole.DISCUSSION OF METHODS The methods available for determining boron are primarily based on distillation as methyl borate and subsequent titration of boric acid after the addition of glycerol or mannitol. * Presented a t the meeting of the Society on Wednesday, October 2nd, 1957.504 DUFFIELD A SYSTEM FOR THE DETERMINATION [Vol. 83 Alternatively, a colorimetric end-point can be achieved with curcumin. It was thought that this separation would be unsuitable for the routine examinations envisaged and that it should be avoided if possible. The possibility of a paper-chromatographic separation was considered, and the work of Lacourt, Sommereyns, Claret and W’antier was investigated.l? It was found that reproducible results were only obtained under rigidly controlled conditions of temperature and humidity, and frequent unexplained inconsistencies were encountered.The reaction of boron with curcumin was next investigated. Boron, in company with iron, molybdenum, vanadium and titanium, forms a pink colour with curcumin in faintly acid solutions. When the solution is made alkaline, however, only boron produces a blue-green complex. This reaction was utilised in developing the method described later. The term paper chromatography is loosely applied to the technique, which is, in fact, a controlled spot test and then a paper-chromatographic procedure, i.e., separation of excess of reagent from the stationary test spot.The colour produced is fugitive and must be compared with simultaneously prepared standards within 20 minutes. Copper, cobalt and nickel are determined simultaneously by paper-chromatographic separation in an ethyl methyl ketone - hydrochloric acid - water system and then colour development with rubeanic acid. These metals lend themselves readily to paper chromatography and many successful methods with ketone - acid solvents have been d e s ~ r i b e d . ~ 2 ~ 1 ~ 9 ~ The most consistent of those investigated was that of Hunt, North and Wells,6 which was adopted in toto with satisfactory results. During the investigation, it was found that zinc also was separated in this solvent system and that its presence a t R, 0.9 (approximately) could be detected by spraying with a dilute solution of dithizone in chloroform.Unfortunately, the stains produced were not quantita- tively consistent, partly owing to the extraordinary sensitivity of the reaction. The method finally adopted for the determination of zinc was colorimetric, based on the reaction of zinc with dithizone at pH 5.0 after interfering metals had been complexed with sodium thio- sulphate. Visual comparison of the “mixed-colour” zinc dithizonate was found to be satis- factory, and the use of sodium thiosulphate avoided interference, particularly by copper, in the ranges normally encountered in crops.’ In this respect, the almost invariable heavy preponderance of zinc over copper, cobalt and nickel is advantageous, since the method as applied is only valid in the presence of up to a twofold excess of copper or a tenfold excess of cobalt or nickel.Molybdenum is separated by paper chromatography in a a-butyl alcohol - hydrochloric acid - water system. Several solvent mixtures have been recommended4 ~8 and investigation led to the adoption of rt-butyl alcohol saturaled with 10 per cent. v/v hydrochloric acid, which gave the most consistent results with the materials under examination. It was also established that separation, particularly from iron, was improved on pre-conditioning the paper by suspending it in an atmosphere saturated with water vapour at room temperature. Under these conditions, molybdenum was satisfactorily separated from up to one hundred times its concentration of iron. Toluene-3 : 4-dithiol is used for colour development, a strong apple-green band being formed: amounts from 0.5 to 5.0 pg can be compared directly with standards. This is insufficiently sensitive for the amounts of molybdenum normally en- countered in crops.Sensitivity is increased by standing the papers in a-butyl acetate, which diffuses up the strips and collects the dithiol cohur into a thin concentrated line, thus permit- ting the detection of 0.005pg of molybdenum and visual colour comparison in the range When the concentration of iron exceeds that of molybdenum by more than one hundred times, an adaptation of the well known potassium thiocyanate - stannous chloride method is used with solvent extraction of the thiocyanate colour in order to concentrate it for comparison. It has been establishedg that, for optimum colour development, the concentration of hydro- chloric acid should be between 4 and 6 per cent ; the concentration of potassium thiocyanate should be not less than 0.6 per cent.The amount of stannous chloride present is not critical, and no differences have been observed in colour developed at a range between 0.05 and 1.0 per cent. Of necessity, there must be sufficient present to furnish a slight excess over what is needed to decolorise the ferric thiocyanate. Some iron must be present to ensure complete development of the molybdenum c o l x r , particularly if the molybdenum concen- tration is above 4pg. The amount of iron needed at this concentration is 0.5mg.10 If these not particularly stringent conditions are observed, excellent results can be obtained 0.02 to 0.2 pg.Sept., 19583 OF CERTAIN TRACE METALS IIY CROPS 505 in the presence of normally occurring acid radicles, such as sulphate and perchlorate, and of more than thousandfold excesses of such metals as titanium, vanadium, chromium and uranium.The technique adopted specifies extraction of the molybdenum thiocyanate complex in 1 ml of n-butyl acetate. This permits the detection of 0.1 pg of molybdenum and visual colour comparison in the range 0 to 4.0 pg in 0.2-pg stages. Manganese is determined by the familiar potassium periodate oxidation to permanganate. The statement by Koroleffll that silver nitrate enhances colour development with microgram amounts of manganese has been in the main confirmed. It was also confirmed that, at low concentrations, full colour development could only be ensured by heating for 2 hours on a water bath after the initial boiling with periodate.12 METHOD PREPARATION OF THE SAMPLE- At this temperature the alkaline nature of cereal and vegetable ashes is sufficient to prevent serious losses of the more volatile metals, such as zinc, molybdenum and boron.Triturate the ash, in the crucible, with 0.5 ml of water, and set aside for 2 hours. From the resulting solution remove 0.02 ml for the determination of bcron. To the remainder of the aqueous solution add a few millilitres of 50 per cent. hydrochloric acid, evaporate to dryness and heat on a high-temperature electric hot-plate for 30 minutes. Twice repeat this treatment with hydro- chloric acid to ensure precipitation of silica in pure white crystals.Finally, moisten the residue with 50 per cent. hydrochloric acid, and evaporate to dryness on a steam-bath. An alternative procedure consists in transferring the ash solution to a platinum crucible and removing the silica by evaporation to dryness with hydrofluoric acid. To the cooled crucible add accurately 0.5 ml or, if necessary, 1.0 ml of 50 per cent. hydrochloric acid, cover with a watch-glass, gently warm, and set aside for a few hours. CHROMATOGRAPHIC PROCEDURE- In the paper-chromatographic procedures, the papers used are Whatman No. 1 (Chemical Research Laboratory pattern). This is a slotted paper that permits the simultaneous treatment of ten separate specimens. The 0.01 or 0.02-ml aliquots are applied to the paper by means of capillary pipettes prepared in the laboratory and calibrated by weight with distilled water.These pipettes can be made and calibrated rapidly and have a reproducibility of k0.2 mg per delivery. A satisfactory pipette contains 0.01 ml in a length of 3 to 4 cm, fills to the mark by capillary action and delivers steadily and evenly on to the chromatographic strip by absorption of the liquid on to the paper. The test aliquot is applied as an even band across the whole width of the strip, about 1 6 c m from the bottom. DETERMINATION OF COPPER, COBALT AND KICKEL- Ash 10 g of finely ground sample at a temperature not exceeding 550" C. Reagents- Solvent mixture-Mix ethyl methyl ketone, concentrated hydrochloric acid and water, Ammonia solution, sp.gr. 0.880-Analytical-reagent grade.Rubeanic acid reagent solution-Dissolve 0.1 g of rubeanic acid (dithio-oxamide) in 60 ml Pyocedure- Apply 0.01 ml of the hydrochloric acid solution of the sample to the chromatographic strip as previously described. Form the paper into a cylinder by clipping together the two ends with a paper clip at the top so that the separate strips stand in the vertical plane. Dry the paper by standing it for 5 minutes in a 600-ml beaker that is floating in a 2-litre beaker of boiling water. Remove the paper and stand it with the test spot downward in 20 ml of solvent mixture contained in a 600-ml tall beaker. Cover with a watch-glass and set aside until the solvent front has diffused to the top of the strips. This should take about 30 minutes. Remove the paper, allow to dry in air for 5 minutes and stand it in a 600-ml beaker that has a 50-ml beaker of ammonia solution, sp.gr.0.880, in the centre. Cover with a clock-glass and set aside for 5 minutes to neutralise. in the volumetric ratio of 15 : 3 : 2 , respectively. of ethanol. Filter the solution, and dilute the filtrate to 100ml with water.506 DCFFIELD : A SYSTEM FOR THE DETERMINATION [Vol. 83 When the small beaker of ammonia solution is placed inside the 600-ml beaker before the introduction of the paper cylinder, take care to spill no liquid into the larger vessel, as any moisture will diffuse up the paper and distort or completely destroy the separated zones. Remove the paper and spray it on both sides with rubeanic acid reagent solution. Separate colour bands will appear, denoting nickel (purple-blue, RF about O - l ) , cobalt (yellow, RF about 0.4) and copper (olive-green, RF about 0.6).Compare these bands with similarly prepared standards covering the range 0.2 to 2.Opg. The limit of visual detection is of the order of 0.05 to 0.1 pg. In this solvent system, iron travels in the solvent front (RF 1.0) and is visible as a band of brown ferric hydroxide. On counter-spraying with a dilute solution of dithizone in chloroform, zinc is detectable as a pink band a t R, about 0.9. DETERMINATION OF ZINC- Reagents- Hydrochloric acid, 1 per cent. v / v . Bufer solution, pH 5--Mix equal volumes of 2 S sodium acetate solution and 2 AT Sodium thiosulphate solution, 25 per cent. w / v . Dithizone reagent solution-Prepare a saturated solution of dithizone in 0.05 X sodium Carbon tetrachloride-Analytical-reagent grade. Procedure- By pipette, place 0.01 ml of sample solution in 1 ml of 1 per cent. hydrochloric acid con- tained in a 10-ml stoppered test-tube.Add 1.0 ml of buffer solution, 1.0 ml of sodium thiosulphate solution and 0.2 ml of dithizone solution. Mix, add 1 ml of carbon tetrachloride, insert the stopper and shake for 1 minute. Allow to separate, withdraw the carbon tetra- chloride layer by means of a capillary pipette and suction bulb and transfer it to a narrow-bore comparison tube. Compare with similarly prepared standards covering the range 0 to 4.0 pg in 0.2-pg stages. acetic acid. hydroxide. PAPER-CHROi'iL4TOGRAPHIC DETERMISATION OF MOLYBDENUM- Reagents- Solvent mixture-Shake n-butyl alcohol with 10 per cent.v/v hydrochloric acid. Allow Dithiol reagent solution-Prepare a solution in i\' sodium hydroxide containing 0.1 per n-Butyl acetate. Procedure- Apply a 0.02-ml test band of sample solm-ion to a paper strip, and dry as previously described. Suspend the paper cylinder for 30 minutes in a closed tank saturated with water vapour a t room temperature. Place the paper with test band downwards in a 600-ml beaker containing 20 ml of solvent mixture, cover with a watch-glass and set aside until the solvent front approaches the top of the strips (1 to 14 hours). Remove the paper, dry for a few minutes a t 100" C and spray with dithiol reagent solution. Molybdenum will appear as an apple-green band at about RF 0.6. Iron can be seen as the purple thioglycollate complex at about RF 0.4.Dry the paper cylinder a t 100" C for a few minutes, and stand it in a few millimetres depth of n-butyl acetate in a covered beaker. Allow the solvent to diffuse just to the top of the original dithiol band, remove immediately and allow to dry. This con- centrates the band and permits comparison with similarly prepared standards covering the range 0 to 0.2 pg in 0.02-pg stages. COLORIMETRIC DETERMINATION OF MOLYBDEXCM- more than one hundred times. the mixture to separate and use the supernatant layer. cent. w/w of toluene-3 : 4-dithiol and 0.1 per cent. w/v of thioglycollic acid. Use this procedure when the concentration of iron exceeds that of molybdenum by Reagents- Potassium thiocyanate solution, 10 per cent.w/w. Stannous chloride solution-Prepare a solut Lon containing 40 per cent, w/v of stannous n-Butyl acetate. chloride in concentrated hydrochloric acid.Sept., 19581 OF CERTAIN TRACE METALS I N CROPS 507 Procedure- By pipette, place 0.2 ml of sample solution in a 10-ml stoppered test-tube. Add 2 ml of potassium thiocyanate solution and 0.1 ml of stannous chloride solution, and mix. Add 1 ml of n-butyl acetate, insert the stopper and shake vigorously for 1 minute. Allow the layers to separate, and withdraw the n-butyl acetate layer. (Use a capillary pipette and bulb to transfer it to a narrow-bore comparison tube,) Compare with similarly prepared standards covering the range 0 to 4.0 pg in 0.2-pg stages. Note that, in the preparation of standards, 0.5 mg of iron must be incorporated to ensure optimum development of the molybdenum colour.DETERMINATION OF BORON- Reagents- Curcumin reagent solzhon-Dissolve 0.1 g of curcumin and 5-0 g of oxalic acid in 100 ml Ethanol. Sodium hydroxide solution, 0.5 per cent. w/w-Freshly prepare this solution before use. Procedure- Lightly mark a chromatographic paper with a pencil line about 1.5 cm from the bottom of the strips to ensure that all test spots are applied at exactly the same level. Apply 0.02 ml of the aqueous extract of the ash (see “Preparation of the Sample,” p. 505) along the pencilled line, and spray with curcumin reagent solution. Dry at 100” C for 1 minute, and stand the paper immediately, test spots downwards, in a Petri dish containing a few millimetres depth of ethanol.Remove from the ethanol, and dry at 100” C for 1 minute. Place immediately in a few millimetres depth of 0.5 per cent. sodium hydroxide solution, and set aside until the purple colour developed by the excess of curcumin has been carried just clear of the stationary blue-green boron band. Compare the band with simultaneously prepared standards covering the range 0 to 2.0 pg in 0.2-pg stages. The colour is fugitive, and comparison must be made immediately. Fading is appreciable after 20 minutes. DETERMINATION OF MANGANESE- of ethanol. Allow the ethanol to diffuse just to the top of the pink test band. Reagents- All reagents should be of recognised analytical grade. Sulphuric acid, concentrated. Nitric acid, concentrated. Orthophosphoric acid.Silver nitrate. Potassium periodate. Procedure- Take 0.1 ml of sample solution in a 6-inch x 1-inch boiling-tube. Add 0.4 ml of concentrated sulphuric acid, 0.5 ml of concentrated nitric acid and 0.1 ml of orthophosphoric acid. Add 1.0 ml of water, and again heat until fumes are evolved. Cool, and add 7.0 ml of water and 0.1 g each of potassium periodate and silver nitrate. Boil for 5 minutes, and place in a boiling-water bath for 2 hours. Cool, and dilute suitably for comparison with standard potassium permanganate solutions covering the range 0 to 1OOpg of manganese in 10-pg stages. Heat until fumes are evolved. RESULTS AND CONCLUSIONS The proposed procedures were tested by means of a series of recovery experiments in which known amounts of various metallic salts were added to the hydrochloric acid solution of the ash.The results, which are shown in Table I, indicate that consistent recoveries can be obtained within the intended scope of the system. It is suggested that this analytical system serves as a rapid sorting test, whereby significant fluctuations in trace-element levels can be detected. Naturally, more accurate results can be obtained by using larger amounts of samples. The paper-chromatographic techniques for copper, cobalt, nickel, zinc and molybdenum are strongly recommended, as they afford rapid and absolute separations for amounts up508 DUFFIELD [Vol. 83 to 100 pg. At these levels, the developed chroniatograms can be cut into appropriate sections and the individual metal complexes extracted and determined by colorimetric procedures.TABLE I RECOVERY OF VARIOUS METALS FROM WHEAT, BARLEY AND OATS Wheat Barley Oats P Amount found in Total original Amount amount Element material, added, found, CLg CLg CLg Copper . . 2.4 0.4 3.0 1.6 1.0 2.5 1.5 1.5 2.8 Cobalt . . . . 0.1 0.1 0.2 0.1 0.2 0.4 0.1 0.3 0.5 Xickel . . . . 0.2 0.1 0.3 0.3 0.2 0.4 0.2 0.3 0.6 Zinc . . . . 5.0 1.0 5.8 4.5 2.0 6.4 4-0 5.0 9.4 lIolybdenum* 0.10 0.05 0.16 0.05 0.10 0.16 0.04 0.15 0.20 Molybdenumt 1.2 0.5 1.7 0.4 1.0 1.5 0.4 1.5 2.0 Boron . . . , 1.0 0.5 1.5 0.8 1.0 2.0 0.8 1.5 2.5 Amount found in Total original Amount amount material, added, found, CLg: Pg CLg 2.0 0.5 2.5 1.7 1.0 3.0 1.7 1.5 3.3 0.05 0.1 0.2 0.1 0.2 0.3 0.1 0.3 0.5 0.23 0.1 0.3 0.2 0.2 0.5 0. L 0.3 0.4 4.11 1.0 6.0 4.6 2.0 6.5 3.11 5.0 9.0 0.06 0.05 0.10 0.06 0.10 0.15 044 0.15 0.21 0.h 0.5 1.0 O.El 1.0 1-5 O*CI 1.5 2.1 0.8 0.5 1.2 0.tl 1.0 1.5 0.4 1.5 2.0 7 - 7 Amount found in Total original Amount amount material, added, found, PLg CLg Pg 1.5 0.5 1.9 1.5 1.0 2.4 1.8 1.5 3.5 0.1 0.1 0.2 0.1 0.2 0.4 0.05 0.3 0.4 0.3 0.1 0.4 0.2 0.2 0.5 0.2 0.3 0.5 4.8 1.0 6.8 4.0 2.0 6.2 3.2 5.0 8.0 0.12 0.05 0.16 0.08 0.10 0.20 0.08 0.15 0.22 1.1 0.5 1.5 0.75 1.0 1.8 0.75 1.5 2.4 0.8 0.5 1.4 0.8 1.0 1.6 0.6 1.5 2.0 Manganese ..60 10 72 50 10 59 80 10 90 45 20 65 50 20 73 75 20 90 40 30 71 30 30 60 i 0 30 102 * Determined by paper-chromatographic procedure. t Determined by colorimetric procedure. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. REFERENCES Lacourt, A,, Sommereyns, Gh., and Claret, M., Mikrochemie, 1951, 38, 444. Lacourt, A., Sommereyns, Gh., and Wantier, G., Analyst, 1952, 77, 943. Burstall, F. H., Davies, G. R., and Wells, R. A,, Disc. Faraday SOC., 1949, 179. Burstall, F. H., Davies, G. R., Linstead, R. P., and Wells, R. A,, J . Chem. Soc., 1950, 516. Arden, T. V., Burstall, F. H., and Linstead, I?. P., Nature, 1948, 162, 691. Hunt, E. C., North, A. A,, and Wells, R. A., /!nalyst, 1955, 80, 172. Fischer, H., and Leopoldi, G., 2. anal. Chem., 1937, 107, 241. Shibata, M., and Uemura, T., J . Chem. SOC. Japan (Pure Chem. Sect.), 1951, 72, 541. Hurd, L. C., and Allen, H. O., Ind. Eng. Chem., Anal. Ed., 1935, 7, 396. Perrin, D. D., N.Z. J . Sci. Tech., 1946, 27A, 396. Koroleff, F., Acta Chem. Scand., 1947, 1, 503. Nydahl, F., Anal. Chim. Ada, 1949, 3, 144. Received January 20th, 1958