Quantitative Chemical Microdetermination of Twelve Elements in Plant Tissue A Systematic Procedure R. Q. PARKS, S. L. HOOD, CHARLES HURWITZ, AND G. H. ELLIS U. S. Plant, Soil, and Nutrition Laboratory, Agricultural Research Administration, Ithaca, N. Y.
A systematic procedure of analysis is presented for the quantitative chemical microdetermination of twelve nutritionally important elements on single samples of plant tissue. The elements determined are calcium, magnesium, potassium, sodium,
I
W E S T I G A T I O S S being carried out a t this laboratory on the relationships of soil fertility to the production of food crops of high nutritive value are concerned with both the macro- and the micronutrient elements. A fairly complete Btudy of these interrelationships necessitates an extensive analytical program. It mould be desirable to have analyses for all the mineral elements commonly accepted as being essential for the growth of either plants or animals. The procedure presented here is for the analysis of calcium, magnesium, potassium, sodium, phosphorus, sulfur, iron, manganese, zinc, molybdenum, copper, and cobalt. It is hoped that future nork will make possible the inclusion of other nutritionally important elements in some such system of analysis. An examination of published analytical procedures reveals a number of excellent methods for the individual determination of these elements. The adoption of these methods in the routine determination of such a list of elements on each of hundreds of samples of plant material is impracticable. Many of the proposed analytical methods are designed for the determination of but one or two elements on the ash of a sample of plant material; hence the determination of a dozen elements would require a t least two or three separate ashings of each sample. This involves not only considerable time and labor, but also an amount of sample which is often not available. This is particularly true in certain greenhouse pot studies, where a n extensive mineral analysis is desired on a sample consisting of only a few plants or plant parts. This situation has been confronted in the majority of the soil and sand pot studies a t this laboratory, in which it was desired to make determinations on the individual replicates of a given treatment. The scheme of analysis reported in this paper was developed to make possible quantitative analyses for twelve nutritionally important elements on single samples of only 5 to 10 grams of dried plant material. The procedure developed is shown schematically in Figure 1. In place of several dry ashings a t different temperatures, and with additions of various chemicals (as recommended in many individual analytical methods), a single 5- to IO-gram sample of dried plant material is digested with nitric and perchloric acids, and then treated with hydrofluoric acid to remove silica. The residue is dried and dissolved in dilute hydrochloric acid. The resultant solution (solution A ) contains all the elements to be subsequently determined.
527
phosphorus, sulfur, iron, zinc, copper, manganese, molybdenum, and cobalt. Data are presented supporting the validity of the separatory procedures and analytical methods used, and their arrangement into a scheme of systematic analysis.
Phosphorus, iron, manganese, and molybdenum are run directly on solution -4. The remainder of this solution is made alkaline and extracted with dithizone in carbon tetrachloride. The aqueous phase (solution C) contains the alkali metals, alkaline earths, and sulfur. The carbon tetrachloride extract (solution B) contains cobalt, copper, and zinc. Zinc is separated from cobalt and copper by an acid dithizone extraction of solution B. Copper and cobalt are determined on the carbon tetrachloride phase of this extraction. Sulfur is determined gravimetrically on an aliquot of solution C. A second aliquot of solution C is digested with acids to remove ammonia and citrates, giving solution F. Sodium and potassium determinations are made on two separate portions of this solution. A third aliquot of solution F is treated with ferric chloride and ammonium hydroxide to remove aluminum, phosphorus, and iron. Calcium is determined on the resultant solution, and magnesium is run on the solution remaining after the calcium precipitation. I n the selection of the individual procedures used, a number of published methods for each element were critically examined under the conditions of the proposed systematic procedure. Colorimetric methods were favored because of their sensitivity and speed. The final selections of methods were made for accuracy in the range desired, size of sample required, speed, and adaptability to systematic analysis. This last requirement is a particularly difficult and exacting one, since each individual determination must be so constituted or so placed in the scheme that elements to be determined subsequently are neither added nor removed. Also, reagents added in the determination must introduce no significant impurities or compounds which interfere with subsequent determinations. All the methods used are adaptations or modifications of published methods. Most of the changes were designed to make the methods applicable to different apparatus or to concentration ranges other than those for which they were originally proposed. The analytical scheme and methods used are described in the following section without comment, a discussion of the developments and details of the scheme is given later, and a separate section is devoted to special precautions and techniques.
Analytical Procedure The purification of ail solutions, reagents, and apparatus used in element microdeterminations and in all steps preceding them requires particular care (see section on Special Precautions and
INDUSTRIAL AND ENGINEERING CHEMISTRY
528
Vol. 15, No. 8
Pipet a 1.00-ml. aliquot of solution A into a test tube graduated a t 10.0 ml. Add Tissue Volume Cell Dimensions Wave Length 1 ml. of 10 per cent hydroxylRan e of in of Cqlor Minimum for Color amine hydrochloride solution Methods Used volume Depth Comparisons b Metgod“ Aliquot Solutions ElemerIt Units and 0.5 ml. of o-phenanthroGrama ME. M1. Cm. M P line (1.5 per cent in 95 per .. .., Volumetric Permanganate Mg. 0.02-2.0 0.042 ... Ca cent ethanol). Add a small 1.7 650 Quinolate y 1-50 0.005 50 g g 10 400 Cobaltinitritelo 1.7 Mg. 2-8 0.270 100 square of Congo red indicator dichromate paper, and adjust to the alka10 1.7 440 Zinc uranyl acetate 0.064 50 M g . 0.01-0.36 Na line end point with 1 to 5 10 1.7 600 Molybdate 10 P Mg. 0.001-0.06 0.006 .. . . . Gravimetric Barium chloride 2.700 ... M e-. 2-100 S ammonium hydroxide. Avoid 10 1.7 490 o-Phenanthroiine y 1-60 0.060 10 Fe the formation of a precipi0.2-20 0.162 10 10 1.7 540 Dithisone Zn ~. ~.~ tate which mill result from 10 1.7 430 Diethyldithiocarbamate 1-60 1 080 10 cu 10 1.7 525 Periodate y 5-200 0.600 11 hl n the addition of excess hydrox3 5.0 500 Nitroso R salt y 0.2-10 4.320 5 co ide. Make to volume, mix, 25 5.0 476 Thiocyanate y 0.3-15 1.200 25 M O and read the per cent transa As used in this schematic procedure. mittance, using a reagent blank b Determined b y spectral transmissim curves. as a reference liquid. The working curve remains constant. Phosphorus is determined by an adaptation of the method of Fiske and Subbarow (28) Techniques). A Coleman Model 11 Universal spectrophotomand McCune and Weech (18). eter with several types of cuvettes is used for all colorimetric Pipet a 0.100-ml. aliquot of solution A into a test tube gradumeasurements. The appropriate wave lengths for all color ated a t 10.0 ml., using a 0.100-ml. Mohr pipet. Add 1 ml. of observations were obtained by preparing spectral transmission water to the test tube, and discharge the pipet by blowing, with curves. Table I contains data on wave band, cell dimensions, the tip under water. Add 1.0 ml. of 2.5 per cent ammonium and range for all methods used. The procedure is described in molybdate in 5 AT sulfuric acid. Mix and add 0.4 ml. of 0.25 terms of a single determination for each element on one 7.5per cent 1,2,4-aminonaphtholsulfonic acid (prepared by adding gram sample. 0.125 gram of animonaphtholsulfonic acid to 49 ml. of filtered PREPARATION OF “ASH” SOLUTION. Weigh a 7.50-gram sample 15 per cent sodium bisulfite, and then adding 1.25 ml. of 20 per of olant tissue into a 600-ml. Pvrex beaker. [Pvrex ware is used cent sodium sulfite, l a ) . Make to volume and mix. Prepare thrbughout the procedure, excgpt for the storage of dry samples, a set of standards simultaneously, since the slope of the standard and of concentrated acids.) Add 12.5 ml. of concentrated nitric curve is not constant. Allow color to develop 30 minutes before acid, cover with a watch glass, and evaporate to near dryness reading. Use distilled water as a reference liquid, on a steam plate. One repetition of this digestion is usually DETERMIXATIONS ON THE DITHIZONE SOLUTIONS.Dithizone enough. Add 25 ml. of 1 to 1 nitric acid and 25 ml. of 60 per separations. The dithizone (diphenylthiocarbazone) is purified cent perchloric acid and boil (covered with watch glass) until and the separations are made by a method similar to those nearly dry. Add about 20 ml. of water and transfer quantitadescribed by Bambach and Burkey (3),Cowling and Miller ( 7 ) , tively to a 125-m1. platinum dish. Add 5 ml. of perchloric acid Holland and Ritchie (14, 15), and Sandell (26, 2 7 ) . to the beaker, boil, and add to the solution in the platinum dish. The dithisone reagent used has a concentration of 0.5 gram Add 2 to 8 ml. of 48 per cent hydrofluoric acid, using a paraffinper liter, which is several times more concentrated than is coated dipper. Place the dish in a sand bath on a gas hot plate ordinarily used. To prepare this stock reagent, dissolve 0.5 and heat carefully until fumes are no longer given off. Cool gram of commercial dithizone in about 500 ml. of carbon tetraand transfer to a 400-ml. beaker, using small additions of hot, chloride. Filter into a 5-liter separatory funnel containing 2 to 3 0.6 N hydrochloric acid. Add more hot hydrochloric acid to give liters of 0.02 N ammonium hydroxide. Shake well, extracting the about 100 ml. and dissolve by continued heating and crushing dithieone into the aqueous phase. Separate the carbon tetraof solid material with a flat-ended glass rod. This operation chloride, and use to dissolve the dithizone remaining on the filter is essential to assure complete solution of calcium sulfate and paper. Filter again into the same separatory funnel. Repeat this other salts. If insoluble salts persist, increase the concentration process until little residue remains on the filter paper. After the of hydrochloric acid. Dilute to a 125-ml. volume and label last extraction, discard the carbon tetrachloride phase. Extract solution A. the ammoniacal solution of dithizone lvith fresh 50-ml. portions of DETERMINATIOSS OX SOLUTION A. Molybdenum is determined carbon tetrachloride until traces of pink no longer appear in the by an adaptation of the methods of Marmoy (19) and Rogers carbon tetrarhloride layer. .4dd about 500 ml. of carbon tetra(26). chloride and acidify with hydrochloric acid, 1%ith shaking, until Pipet a 20.0-ml. aliquot of solution A into a 200-ml. srparatory the dithizone is extracted from the aqueous phase into the funnel. Add 30 ml. of 1 to 7 hydrochloric acid, 3.0 ml. of aqueous carbon tetrachloride phase. (If necessary, add more acid and 10 per cent potassium thiocyanate, and 3.0 ml. of fresh 10 per carbon tetrachloride and repeat.) Dilute to 1 liter with carbon cent stannous chloride in 1 to 9 hydrochloric acid. Shake after tetrachloride. Store in a glass-stoppered bottle, in a cold, dark each addition. After 2 minutes add 20 ml. of diethyl ether. place ( 5 ) . This reagent should remain stable for several weeks. (The ether for a set of determinations is freshly extracted with Place a 90.0-ml. aliquot of solution A in a 500-ml. separatory about one tenth its volume of 1t6 1stannous chloride-potassium funnel. Dilute with 50 ml. of redistilled water. Add enough thiocyanate mixture and washed until clear with 1 to 9 hydro40 per cent ammonium citrate solution (prepared using lead-free chloric acid.) Shake vigorously for 30 seconds, and drain thc citric acid plus concentrated ammonium hydroxide to pH 8.5, aqueous layer into a similar funnel. Shake again with 15 ml. and extracted with dithizone) to prevent precipitation when the of extracted ether. Rinse the ether extracts into a 25-1111. solution is subsequently made alkaline. Usually 10 ml. are sufvolumetric flask and dilute to volume with extracted ether. ficient. Adjust to pH 8.5 with about 7 K ammonium hydroxide, Sufficient evaporation will have taken place to make this possible. using a few drops of m-cresol purple (1 mg. per ml. in ethanol) Compare colorimetrically at once with a t least two standards as an internal indicator. I t may be necessary to add 15 or 20 prepared simultaneously to establish the slope of the curve. drops of indicator, if the solution turned yellow on the addition Read against extracted ether as a referenceliquid. of citrate. Add an excess of concentrated dithizone reagent Manganese is determined by the simplified periodate method (usually 20 to 25 ml.) as indicated by a strongly orange aqueous described by Peech (22). phase after shaking, Drain the carbon tetrachloride phase into Pipet 10.0 ml. of solution A into a 50-ml. beaker, and evaporate a similar, dry separatory funnel, rinsing the last portion through to dryness on a steam plate to remove hydrochloric acid. Diswith carbon tetrachloride. Extract the aqueous phase with solve in 6 ml. of 1.0 N nitric acid and transfer to a test tube successive 25-m1. portions of carbon tetrachloride (maintaining graduated a t 11.0 ml. Add 1 ml. of 85 per cent orthophosphoric an excess of dithizone in the aqueous layer), until the carbon acid and dilute to volume. Place the tube in a boiling water tetrachloride layer is clear green, and combine all the extracts. bath, add about 50 mg. of solid sodium periodate, mix with a This is solution B. glass rod, and heat in the bath for 1hour. Cool, make to volume, Wash the aqueous phase into a 200-ml. volumetric flask with mix, and compare color intensities against a reagent blank 10 ml. of 1 to 1 hydrochloric acld, dilute to 200 ml., and save reference liquid. The working curve is constant, but may be for subsequent determinations of sulfur, calcium, magnesium, checked by the inclusion of one or two standards with each set potassium, and sodium. Designate as solution C. of samples. Into the combined carbon tetrachloride extracts (solution B) Iron is determined by the method of Saywell and Cunningham in a 500-ml. separatory funnel, pipet 50.0 ml. of 0.02 N hydro(88).
TABLEI. PERTINENT DATAON METHODSUSED
Y
~
-
ANALYTICAL EDITION
August 15, 1943
Copper is determined by a modification of the procedures of Coulson (6), Drabkin ( I O ) , and Marston and Dewey (20). Transfer the remaining cobalt and copper solution (D) into a 60-ml. separatory funnel. Add 2 ml. of 40 per cent ammonium citrate adjusted to pH 8.5, 10 ml. of 0.1 per cent sodium diethyldithiocarbamate, and 10.0 ml. of isoamyl acetate. [Eden and Green (11) reported the necessary presence of ammonium ions to deionize iron. The presence of ammonium ions also seems to be necessary to prevent rapid fading of the copper color complex.] Shake for 5 minutes, separate the acetate layer, and centrifuge it for 5 minutes to remove water droplets. (All centrifuging is done a t 2000 r. p. m., using an International Equipment Company laboratory centrifuge, Size 1, Type SB. The head is 13 cm. from axis to trunnion carrier pivot.) Read against a reference liquid of isoamyl acetat?. A few standards should be included with each run to check the working curve.
chloric acid. Shake vigorously for 2 minutes (within 5 or 10 seconds). Separate the carbon tetrachloride phase into a 250ml. beaker, and rinse the funnel with carbon tetrachloride. Drain the aqueous phase into a dry container and save for the subsequent determination of zinc (solution E). [This solution may also be used for the determinations of cadmium (67) and lead (26).] Add 5 ml. of 60 er cent perchloric acid to the carbon tetrachloride phase and toil over a low flame until fumes of perchloric acid appear. Cover with a watch glass and digest until clear. Remove the watch glass and continue boiling until dry. Cool and dissolve in 12.5 ml. of 0.04 molar citric acid (c: P. lead-free). (Most samples of c. P. citric acid contain appreciable amounts of copper and other metals, and must be purified.) Dilute to 25.0 ml. and use for the determinations of cobalt and copper (solution D).
20ml.
0.1 ml.
I ml.
IOml.
529
9 0 ml.
DITHIZONE
Soln. C 200 ml.
Soln. E CCI,
I
I EXTRACTION^ Soln. E
/I
DITHIZONE
DIGESTION
DIGESTIONS
Soln. F 1 2 5 m l H N 0 ,
NITROSOR-SALT
DIETHYLDITHIO CARBAMATE
N H,OH
--
SULFOSALICYLIC ACID
FIGURE 1. SCHEMATIC DIAGRAMOF PROCEDURE
Cobalt is determined by a modification of the method described by Marston and Dewey(60). Pipet 20.0 ml. of the cobalt and copper solution (D) into a 50-ml. Pvrex beaker and rinse the uiuet back into the remaining solution.- Evaporate to about 5 mi.and add 2.5 ml. of buffered sodasolution (6.184 grams of boric acid, 35.62 grams of disodium phosphate dihydrate, and 500 ml. of 1 N sodium hydroxide, diluted to 1liter). The pH should be between 8.0 and 8.5 and may be checked externally with phenol red and mcresol purple. Add 1 ml. of 0.2 per cent nitroso R salt slowly with mixing. Heat to boiling for 1 to 2 minutes, add 2 ml. of 1 to 1 nitric acid, and boil for another 1to 2 minutes. Add 0.5 to 1 ml. of saturated bromine water and let stand warm for 5 minutes. Cover with a watch glass, and boil for 2 or 3 minutes to remove the excess bromine. Cool, transfer, and dilute to 5.0 ml. Intense colors may be further diluted before reading. Where the cobalt is very low, it may be possible to keep the volume to 4 or even 3 ml. Read color intensity within a few hours against a distilled water reference. A few standards should be included in each run to check the working curve.
Zinc is determined by an adaptation of the method of Cowling and Miller ( 7 ) . Pipet a 1.5-ml. aliquot of solution E into a 60-ml. separatory funnel. Add 23.5ml. of 0.02 N hydrochloric acid. Add by pipet, 25.0 ml. of buffer solution [9 parts of (I),a stable solution containing 500 ml. of 0.5 molar, dithizone-extracted ammonium citrate plus 150 ml. of 1N ammonium hydroxide, diluted to 2250 ml.; and 1 part of (11), freshly prepared 0.25 per cent sodium diethyldithiocarbamate]. Add exactly 5.0 ml. of dithizone reagent (concentrated reagent diluted 1 to 4 with carbon tetrachloride). Shake for 5 minutes. Collect the carbon tetrachloride layer in a 10-ml. beaker, dilute a 2.0-ml aliquot to 10.0 ml. with carbon tetrachloride, and read per cent transmittance against a carbon tetrachloride reference. This zinc procedure should be carried out in a darkened room and the colors read within an hour. A standard curve should be prepared with each set of samples. SULFUR, ALKALIES, AND ALKALINEEARTHS.Sulfur (g), Place a 100.0-ml. aliquot of solution C in a 400-ml. beaker. Add about 100 ml. of water and a few drops of bromocresol green,
530
Vol. 15, No. 8
INDUSTRIAL AND ENGINEERING CHEMISTRY
neutralize with concentrated ammonium hydroxide. Then add 0.5 ml. of concentrated hydrochoric acid. Bring to a boil, add 10 ml. of 10 per cent barium chloride dropmise with constant stirring, and boil for another 5 minutes. Cover with a watch glass, and let stand for a t least 5 hours in a warm place (steam plate). Transfer the precipitate to an asbestos pad in a weighed Gooch crucible and wash with boiling water until free of chlorides. Ignite at 600" C., and weigh as barium sulfate. Removal of Ammonia, Iron, Aluminum, and Phosphorus. These steps are modified from the work of Peech (22). Evaporate 25.0 ml. of solution C to dryness on a steam plate. in a 150-mi. beaker. Digest with 10 ml. of freshly prepared aqua regia (1 part of concentrated nitric acid and 3 parts of concentrated hydrochloric acid) and heat to dryness. Digest with perchloric acid (5 ml. are usually sufficient), and boil to dryness. Take to dryness twice more with 5-ml. portions of aqua regia to remove any residual ammonia. Take up and dilute to a 25-1111. volume with 0.1 N nitric acid. This is solution F. For the determinations of calcium and magnesium transfer a 2.0-ml. aliquot of this solution to a 15-ml. centrifu e tube graduated at 13.0 a l . , and save the remainder for the cfeterminations of sodium and potassium. Add 0.2 ml. of ferric chloride solution (1.22 grams of ferric chloride hexahydrate in 250 ml. of 1 to 250 hydrochloric acid), mix, add 8 ml. of buffer solution (25 grams of sodium acetate, 62.5 grams of ammonium chloride, and 0.5 gram of sodium hydroxide in 1 liter of solution), and mix again. Add 1 drop of methyl red indicator solution (0.02 per cent) and 0.6 Nammonium hydroxide until the color of the solution changes from slightly red to deep yellow, and then add 2 drops in excess. Dilute to about 13.2 ml., mix with a stirring rod, and digest in a water bath at 80' C. for 5 minutes to flocculate the precipitate. Mix thoroughly, and centrifuge while hot for 10 minutes. The solution should have evaporated to 13.0 ml. Calcium is determined by the method of Peech (22). Transfer a 10.0-ml. aliquot of the clear supernatant solution (using inclined mirror to observe liquid level while pipetting, 22) into another calibrated centrifuge tube. Add 1.4 ml. of 0.2 N hydrochloric acid and place in a water bath a t 70" C. Mix the contents, add 2 ml. of 3 per cent ammonium oxalate, mix thoroughly again, and digest for 30 minutes at 70" C. Remove the tube from the bath and let stand for 30 minutes. Centrifuge for 15 minutes. The volume should now be 13.0 ml. Decant the clear supernatant liquid gently into a dry test tube and save foi the magnesium determination. Allow the centrifuge tube to drain for several minutes on paper toweling. Add from a wash bottle, so as to break up the precipitate, about 5 ml. of 2 N ammonium hydroxide saturated with calcium oxalate. Centrifuge for 15 minutes, decant carefully and discard the solution. Drain the tube and save the precipitate. One washing is sufficient unless very large quantities of calcium are present. Sdd about 5 ml. of 10 per cent sulfuric acid, heat to 70" C. in a water bath, and titrate nith standard 0.02 A; potassium permanganate. Magnesium is determined by the method of Peech (28). Pipet a 10.0-ml. aliquot from the solution set aside for the magnesium determination into a 15-ml. centrifuge tube graduated at 13.0 ml. Place the tube in a water bath at 70" * 2" C., add 0.8 ml. of a freshly prepared 2 per cent alcoholic solution of 8hydroxyquinoline. Mix immediately and then add 0.4 ml. of concentrated ammonium hydroxide. Stir vigorously (1 minute or longer) until full turbidity develops. Set aside until all samples of the set are precipitated. Replace the centrifuge tube in a water bath at 70" C. for 10 minutes to flocculate the precipitate. After digestion, cool by immersing the centrifuge tube in a bath a t about 25" C., and allow to stand for 45 minutes to assure complete precipitation of magnesium. Add 0.5 ml. of 95 per cent ethyl alcohol slowly down the sides of the centrifuge tube, rotating the tube a t the same time to wash down the precipitate and t o form a layer of alcohol on the surface of the solution. Centrifuge for 15 minutes and by using gentle suction draw off twice 2 to 3 ml. of the clear liquid to remove the layer of alcohol. Decant carefully and discard the solution, wipe the mouth of the tube with filter paper, add 5 ml. of ammoniacal ammonium acetate (8 ml. of concentrated ammonium hydroxide in 300 ml. of 0.7 N ammonium acetate), wash the solution down the sides of the tube and break up the precipitate. Add 0.5 ml. of ethyl alcohol to prevent creeping and centrifuge again. Repeat the washing once more. Dissolve the precipitate in 4 ml. of 0.5 N hydrochloric acid, dilute, to 13.0 ml. with water, and mix. Place a 2.00-ml. aliquot of this solution in a 50-ml. volumetric flask. .Dilute to about 35 ml. with water and add 5 ml. of 20 per cent sodium carbonate solution and 3 ml. of phenol reagent (750 ml. of water, 100 grams of sodium tungstate, 20 grams of phosphomolybdic acid, and 50 ml. of 85 per cent phosphoric acid, boiled &for2 hours, and diluted to 1 liter). Mix the contents after each addition. Place the flask in boiling water for 1 minute, and cool
for 15 minutes.
Dilute to volume, mix, and read, using a resgent blank as reference. The working curve may be checked by including a few standards with each run. Potassium is determined by the method of Wander (32). Place a 10.0-ml. aliquot of solution F in a 25 X 150 mm. centrifuge tube, add 5 ml. of 20 per cent trisodium cobaltinitrite (freshlyprepared and filtered), mix, and keep a t 20" C. for exactly 2 hours. Wash down the walls of the tube with 0.01 ;V nitric acid. Centrifuge for 10 minutes (0.01 N nitric acid is used in balancing the tubes before centrifuging), decant, and allow the tube to drain for several minutes. Wash the precipitate thoroughly with 15 ml. of 0.01 N nitric acid, centrifuge, and drain as before. Add 10.0 ml. of standardized 0.1 N otassium dichromate and 5 ml. of concentrated sulfuric acid. %fix thoroughly, cool to room temperature, and make to 100-ml. volume. Mix and read the color against an 11-mg. potassium standard as reference, qince this gives a completely reduced dichromate solution. The color measured is that of the unreduced dichromate. Several potassium standards are carried throughout the procedure. Sodium is determined by the method of Darnel1 and Walker (8) and Broadfoot and Browning (4). Place a 2.00-ml. aliquot of solution F (for samples with very high sodium content, use 1.0 or 0.5 m l ) in a 15-ml. centrifuge tube. Add 5 ml. of freshly. filtered uranyl zinc acetate reagent. [Prepare by mixing while warm equal parts of (I), a solution of 77 grams of uranyl acetate plus 14 ml. of glacial acetic acid in 400 ml., Nith (11), a solution of 231 grams of zinc acetate plus 7 ml. of glacial acetic acid in 400 ml. Let stand at least 24 hours and filter.] At 5-minute intervals, add seven 0.3-ml. portions of ethyl alcohol. These additions must occupy at least 30 minutes. After each of the first five additions of alcohol, mix the liquid in the tube thoroughly. The last two additions of alcohol serve to wash down the \!ails of the tube and are allowed to remain layered on the solution. After the last addition of alcohol, centrifuge the tube for 10 minutes, decant, invert, and allow to drain for 5 minutes. Wipe the mouth of the tube dry and agitate the precipitate by directing on it a fine stream of about 2 ml. of ethyl acetate-acetic acid wash liquid (300 ml. of ethyl acetate diluted to 1 liter with glacial acetic acid). Wash down the walls of the tube with about 5 ml. of the wash liquid, centrifuge, drain, and wipe as before. Wash the precipitate and the walls of the tube with about 5 ml. of diethyl ether. Centrifuge for 5 minutes, decant, and drain for not more than 1 minute (prolonged drying will result in the dropping of the precipitate from the tube). Wash the precipitate a second time with 5 ml. of ether, centrifuge 5 minutes, decant, and drain as before. Put the tube in a warm place for 5 minutes to evaporate the last traces of ether. Dissolve and transfer with water the washed, dried precipitate to a 50-ml. volumetric flask. Dilute the solution to about 35 ml. and to it add, in order, 2 ml. of 5 per cent sulfosalicylic acid, 2 ml. of 10 per cent sodium acetate trihydrate solution, and water to make 50 ml. Mix the solution and read, using as a reference a solution containing 2 ml. of 5 per cent sulfosalicylic acid and 2 ml. of 10 per cent sodiiim acetate diluted to 50 ml. with water. The working curve should be checked by including three or four sodium standards with each set of determinations.
Special Precautions and Techniques
It is not possible to overemphasize the care which must be exercised in the purification of all apparatus, solutions, and reagents used in all the steps preceding the element microdeterniinations, and in the determinations themselves. Pyrex glassware is used exclusively, and redistilled water is used throughout the procedure for all solutions and for rinsing
DETERMINATIONS ON TURNIP GREENS TABLE 11. RECOVERY Element Ca
kk K Na
Pe S
F
Zn
cu )In
co
If0
Units
Present
Added
40.0 2.54 24.6 5.27 4.65 12.1 147 42.0 7.2 35.4 0.07 4 94
10.0 2.00 10.0 2.00 2.00 2.0 40.0 11.1 2.7 16.7 0.08
5.00
Total Present
Total Found
50.0 4.54 34.6 7.27 6.65 14.1 187 53.1 9.9 52.1 0.15 9.94
51.2 4.62 33.3 7.39 6.71 13.7 193 51.5 10.2 54.9 0.18 9.80
% Differenee
++ 21 .. 48 - 3.7 + +- 021 ... 986 +- 32 .. 29 ++ 35 .. 04 +16.6 -
1.4
August 15, 1943
ANALYTICAL EDITION
all glassware. This water is prepared by use of a continuously operating all-glass still similar to those described by Piper and Oertel (23). This water is stored in and dispensed from an all-glass suction-operated siphon apparatus, in which the siphon dispenser and the vacuum line both end in a plasticcoated two-hole stopper. The vacuum system is closed by placing the receiving flask against this stopper. I n this manner the redistilled water may be stored and dispensed without contact with rubber or cork. All ammonium hydroxide, hydrochloric acid, and nitric acid (except concentrated acids), and all organic solvents not purified by extraction (such as ether in the molybdenum determination) are purified by distillation from glass. All carbon tetrachloride should be distilled over calcium oxide to remove acidifying substances. All solutions used in heavy metal determinations come in contact only with glassware which has been rigorously cleaned with aqua regia or hot nitric acid. All ground-glass joints require particular care to accomplish this cleaning. Glass stopcocks usually require a further extraction with dithizone. It is also imperative t h a t none of the solutions or apparatus come in contact with rubber a t any point during the procedure. Rubber contacts are eliminated from all wash bottles, and only glass-stoppered volumetric flasks, separatory funnels, and reagent bottles are used. A digestion and reagent blank must be carried throughout the entire scheme with each set of samples. Not only does the trace metal content of different lots of chemicals vary markedly, but the content of impurities of the reagent solutions may also change with storage in glass apparatus. Extractions are facilitated by the use of two-shelf separatory funnel racks, in which the upper shelf is adjustable in height and may be removed for separate use. The funnels can be clamped into the racks for shaking, with sponge-rubber padded cross-bars. A standard reciprocal shaking machine has been modified to hold two shelves of these racks of separatory funnels in a horizontal position. Thus, the dithizone, zinc, and copper extractions, which are normally rather time-consuming, can be carried out 24 a t a time with uniform and complete extraction. The centrifuge tube techniques and water bath designed by Peech (22) for microanalytical procedures are used to excellent advantage, The conical 15 X 126 mm. centrifuge tubes are graduated a t 13.0 ml. by circling with a diamond point. Similarly graduated test tubes are extensively used in place of (and are more adaptable than) small volumetric flasks. The two sizes most commonly used are 15 X 125 mm., graduated a t 10.0 and 11.0 ml., and commercially available 10 x 115 mm. tubes, graduated a t each milliliter from 1 to 8.
Results The characteristics and applicability of the proposed scheme of analysis are shown by the data in the following tables. The recoveries of elements added to plant tissue are given in Table 11. Thirteen portions of a well-mixed sample of turnip greens were used in this comparison. An increment of but one of the twelve elements to be determined was added t o each of twelve samples, xvhile the last sample had no additions. All samples were then run completely through the schematic procedure described. The values reported are the average of duplicate analyses. The agreement between the “Total Present” and “Total Found” for the different elements can be considered as very satisfactory for most types of plant tissue analysis. The difference between these two values for any determination vias less than 6 per cent, escept for cobalt. The large percentage difference for cobalt is but a small actual difference and represents approximately the rcli’ability (0.02 microgram per gram) of the method
531
used. These recovery determinations not only indicate the precision of the individual methods, but in addition measure losses or contaminations in the sample digestion and in all separations and manipulations, as well as in the actual determinations of the element. The proposed system of analysis was further checked by comparison with referee samples. The data in Table 111 were obtained by analysis of six referee samples of different types of plant material, all carefully prepared and analyzed in other laboratories. These analyses were obtained by ashing techniques and chemical methods different from those used in this study. These samples were likewise carried through all operations of the analytical scheme, and the average of duplicate determinations was reported. The analysw are in satisfactory agreement with the results of other investigators, since for all elements but cobalt the differences are less than 8.5 per cent.
T.4 BLE
Element Ca g g Na
P
s
Fe
Zn cu hfn co M0
111 DETERMINATIONS ON REFEREE SAMPLES
Units
Analyses and T y es of Metgods of Authors’ Other Analyses Laboratories
2.99 (82) 3.36 (84) 13.86 (84) 3.67 (8) 2.09 (SI) 3.85 (8) 78.4 (2) 2 3 . 8 (7) 11.4 11.8 (6) 32.0 34 7 (84) 0.07 0 0 8 (26) KOsample available 2.97 3.47 15.00 3.76 2.03 4.18 81.2 25.8
Difference
%
Type of Materia1
0.7 3.2 7.6 2.5 3.0 7.8 3.5 8.4 3 4 8.4
Cauliflower Alfalfa Alfalfa Sugar beet t o p Alfalfa Tomato leaflet Turnip t o p Alfalfa Alfalfa Alfalfa Turnip t o p
1 2 ,A
Discussion The analytical methods incorporated into the proposed scheme of analysis have been drawn mainly from published literature pertaining to soil and plant tissue analysis. Some modifications were necessary to make the methods applicable to smaller quantities of material than originally proposed. Other changes were made to adapt the methods to rapid schematic analysis. At the same time it was possible to eliminate some of the preliminary operations necessary for the removal of interfering substances. Some of the colorimetric methods were originally developed for filter colorimeters or visual comparison. I n some cases the sensitivity of these methods has been improved by the use of a spectrophotometer, greater cell depth, and smaller final volumes, The use of dry-ashing techniques in the determination of such a list of elements would necessitate a t least two samples, since the magnesium or calcium salt which must be added to prevent losses of sulfur and phosphorus would interfere with subsequent analyses for these elements, and for the trace metals. This type of replicate ashing consumes extra time, apparatus, and sample. The wet-digestion procedure used (modified from Gieseking, Snider, and Getz, 13) has proved to be both rapid and smooth in operation. The resultant solution from such a single digestion contains, without losses, the twelve elements under consideration. The use of nitric acid in this digestion precludes the determination of nitrogen. The removal of silica has generally been accepted as necessary (9, Zl), and is accomplished by volatilization with hydrofluoric acid. If desired, a measure of crude silica may be obtained by measuring the loss in sample weight due to the hydrofluoric acid treatment. Since boron is also volatilized as the fluoride, boron as well as nitrogen must be determined on a separate sample when such an analysis is desired.
532
INDUSTRIAL AND ENGINEERING CHEMISTRY
The use of perchloric instead of sulfuric acid in the removal of residual fluorides forms more readily soluble salts, and makes possible the subsequent determination of sulfur. An ether extraction of molybdenum and iron is not used on the entire “ash” solution, since the partial extraction of phosphorus which occurs (f7)would make its subsequent analysis impossible. The ether-stannous chloride-potassium thiocyanate extraction used for molybdenum is not made on the solution in which other elements are to be determined, because of the presence of relatively large amounts of trace metal impurities in the stannous chloride. Attempts to determine molybdenum on solution C have not been completely successful. The results shown in the following table tend to indicate that this method can be made to work satisfactorily:
Sample No. 164 165 184
Molybdenum Content Solution C, HNOJ and HClOd treatment, determined b y HzS01 method
Solution A, b y standard method Y/Q.
Y/Q.
4.4 4.4 0.9
4.5 4.2 1.3
The molybdenum thiocyanate color complex is subject to fading due to air oxidation. This effect may be minimized by the use of glass-stoppered volumetric flasks, and by comparison within a few minutes after extraction. Faded colors may be fairly accurately restored by shaking with stannous chloride-potassium thiocyanate solution. Unsuccessful attempts were made to determine manganese on the aqueous phase of the molybdenum extraction. It was not possible to effect the oxidation of tin salts and removal of chlorides (to prevent reduction of sodium periodate added in the manganese determination) without occlusion of part of the manganese in the precipitate formed. Attempts to determine manganese on solution C were more promising, but not conclusive. It is felt that it may subsequently be found possible to determine manganese on solution C. Although iron and phosphorus may be determined on the aqueous phase of the alkaline dithizone extraction, the very small amount of sample required for these determinations makes it unnecessary to introduce other manipulations. The interference of iron in the remaining heavy metal determinations is eliminated by the dithizone separation, in which ferric iron remains quantitatively in the aqueous phase. Iron removal by precipitation reactions is accompanied by heavy metal occlusion and is, therefore, avoided. Although other reducing agents are often used in the molybdate method for phosphorus ( I , S I ) the one selected (aminonaphtholsulfonic acid) is rapid and is stable in solution for 2 weeks. Many extensive discussions of dithizone techniques and principles are to be found in the literature (3, 7 , 1.4, 16, 26, 87). Both carbon tetrachloride and chloroform are used as solvents for dithizone complexes. The partition characteristics with respect to cobalt (20) and other elements make carbon tetrachloride the more satisfactory solvent. Neutral, metal-free carbon tetrachloride is obtained by distillation over calcium oxide. The number and total volume of dithizone-carbon tetrachloride extractions are kept at a minimum by the use of a more concentrated dithizone reagent than is usually advocated. The ammonium citrate buffer used in the alkaline dithizone extraction is best prepared from commercial lead-free citric acid and concentrated ammonium hydroxide and is further purified by dithizone extraction, The completeness of extraction with dithizone may be tested by transferring a small quantity of the green carbon tetrachloride phase to a separatory funnel containing
Vol. 15, No. 8
0.02 N ammonium hydroxide, and shaking. The excess dithizone will be extracted into the aqueous phase, and any traces of pink metal-dithizone complexes will be visible in the carbon tetrachloride layer. The appearance of a yellow color in this layer is indicative of decomposition products of the dithizone reagent. These decomposition products do not interfere in the group separations, but will prevent accurate colorimetric measurements, if this reagent is used later in the actual colorimetric determinations. When the perchloric acid oxidation of the carbon tetrachloride phase of the acid dithizone extraction is carried out as indicated, undesirable tarry residues are avoided. The amounts of cobalt present in plant tissues are so small that the determination of this element limits the extent to which sample size for the entire procedure can be reduced. If cobalt were not determined, analyses for the remainder of the elements could be made on a much smaller sample of plant material. The Marston and Dewey (20) modification of the nitroso R salt cobalt method (16) is the most sensitive of the published cobalt methods. The sensitivity of this method is further increased by reduction of final volume and by increased cell depth. Studies on the development of an even more sensitive cobalt method are being carried out a t this laboratory. When copper is determined using sodium diethyldithiocarbamate without a preliminary dithizone extraction, sodium pyrophosphate is added t o prevent interference by iron and manganese. The dithizone separation in the present procedure makes this step unnecessary, although ammonium citrate is added to prevent interference by any iron added as an impurity subsequent to the dithizone extraction. The zinc dithizonate is only moderately stable. The determination should be carried out in a darkened room, and the colors read within an hour. The single extraction does not give complete separation of the dithizone complex. This necessitates careful control in reagent concentration and volume, and in time of shaking to maintain a constant partition ratio ( 7 ) . The final dilution of the complex with carbon tetrachloride is necessary because of the intensity of the colors. The carbon tetrachloride used for dilution must be neutral (as described under Special Precautions and Techniques) or the color complex will be destroyed. The determination of lead and cadmium on the zinc solution (E) is possible, if desired. Lead is a general contaminant and is present in reagents in quantities exceeding those found in plant tissue, necessitating even more rigorous techniques than herein described. An attempt was made to develop a titration microprocedure with barium chloride for the determination of sulfur in plant tissues, using tetrahydroxyquinone as an internal indicator. Although this indicator has been used successfully in soil and water analysis, interfering substances were present a t every point in the procedure where the method was tried. I n using the standard gravimetric barium chloride method for sulfur, it has been found that the citrate remaining in solution C does not interfere and that the traces of sulfur added by the dithizone are not detectable. It was reported by Smith (29) that losses of sulfur occur during wet digestions using perchloric acid. A later publication (SO) showed that the losses resulted from too vigorous pretreatment with nitric acid and can be avoided. The wetdigestion procedure used has been checked by comparison (in addition to the recoveries reported in Table 11) with the A. 0. A. C. standard method ( Z ) , using magnesium nitrate dry-ashing. The results of this comparison, shown in Table IV, indicate that losses of sulfur have not occurred. I n the acid digestions in the preparation of solution E’, an aqua regia treatment should precede the perchloric acid digestion to prevent the formation of ammonium salts that
August 15, 1943
ANALYTICAL EDITION
LEAFLETS TABLE IV. SULFURIN TOMATO Sample Preparation Perohlor,io acid wet digestion Mg(NOd2 dry ash, 550’
C.
Referee analyst: Mg(NO3h dry ash, 550’
C.
Sulfur
Average Value
Mdg.
Mo./g.
4.16
4.21 4.04
4.18
3.96
4.00
3.80 3.76 3.85
3.85
4.00
are difficult to decompose. Further aqua regia treatments are necessary to remove traces of residual ammonia, which interfere in the potassium determination. Tests with Kessler’s reagent show complete removal of ammonia a t this point. I n the ammonium precipitation of iron (aliquot from solution F) for the calcium determination, the bromine ovidation of manganese necessary in soil analysis (22) has been omitted, although the quinolate method determines both magnesium and manganese. The traces of manganese present in plant tissue do not ordinarily introduce a detectable error in the magnesium determination. In cases where manganese is exceptionally high, the magnesium values should be corrected. The centrifuge tube methods of microtitration for calcium and quinolate precipitation of magnesium are used as proposed for the determination of exchangeable bases in soils (22) and have been found to be directly applicable. Wander’s recent modification (52) of the cobaltinitrite precipitation method for potassium has been found to be both rapid and sufficiently accurate. Blank values should not be subtracted, since the dichromate reduction is not directly proportional to the potassium content below the lower limit specified (Table I). The upper limit may be extended, when a sample of very high potassium content is encountered, by doubling the amount of dichromate, wlfuric acid, and final volume. The schematic procedure is described (section on Bnalytical Procedure) in terms of a single determination for each element on the one 7.5-granl sample of plant tissue. As the scheme of analysis is used in this laboratory, duplicate determinations are made of each element. This duplication is accomplished either by dividing the solution from a 15-gram sample after the hydrofluoric acid digestion, or if platinum supply is not limiting, by beginning with duplicate 7.5-gram plant samples. \F7hen a 15-gram sample is digested, the specified sizes of apparatus are adequate. The concentration of solution A is kept unchanged by diluting to 250 ml. with 0.6 N acid. All subsequent aliquots and volumes are then as indicated. Each run usually consists of 24 samples in duplicate, or a total of 576 individual determinations in addition to standards for the determinations. This system of analysis has been used successfully with turnip greens, oat straw, oat grain, alfalfa, ladino clover,
533
tomato leaflets, cauliflower, and sugar beet leaves. Although these materials represent a fairly wide range of plant composition, further testing will be required to reveal the full extent of the applicability of the proposed system of analysis. There are obvious advantages in the application of the proposed analytical scheme to both field and greenhouse investigations involving plant tissue analysis. The techniques described make possible an extensive mineral analysis, using samples of limited size. Another advantage of this type of analytical procedure is its flexibility. The nature of the operations used makes relatively simple the addition of still other elements a t appropriate points or the deletion of any determinations not required. Even where different elements are required, and other interfering substances occur, the development of appropriate separations and determinations should be possible.
Literature Cited Allen, R. J. L., Biochem. J., 34, 858 (1940). Assoc. Official Agr. Chem., Official and Tentative Methods of Analysis, 4th ed. (1935). (3) Bambach, K., and Burkey, R. E., IND. ENG.CHEM.,ANAL.ED., (1)
(2)
14. 904 11942).
(4)
Broadfoot,‘W. h., and Browning, G. M., J . Assoc. Oficial Agr.
Chem., 24, 916 (1941). (5) Clifford, P. A,, Ibid., 21, 212 (1938). (6) Coulson, E. J., Ibid., 20, 178 (1937). (7) Cowling, H., and Miller, E. J., IND.ENG.CHEX.,ANAL.ED., 13, 145 (1941).
Darnell, M,C., Jr., and Walker, E. S., Ibid., 12, 242 (1940). Davidson, J., J . Assoc. Oficial Agr. Chem., 14, 551 (1931). Drabkin, D. L., Ibid., 22, 320 (1939). Eden, A . , and Green, H. H., Biochem. J., 34, 1202 (1940). Fiske, C. H., and Subbarow, Y., J . Biol. Chem., 66, 375 (1925). Gieseking, J. E., Snider, H. J., and Getz, C. A., IND. ENG.CHEM., -43.4~.ED., 7, 185 (1935). (14) Holland, E. B., and Ritchie, W.S.,J . Assoc. Oficial Agr. Chem.,
(8) (9) (10) (11) (12) (13)
22. 333 ~~- f1939). (15) I bid., 23, 392 (1940). (16) Kidson, E. B., and Asgew, H. O., N e w Zealand J . Sci. Tech.. 21B. 178 (1940). (17) Lundell, G. E. F., and Hoffman, J. I., “Outlines of Chemical Analyses”, New York, John Wiley & Sons, 1938. (18) McCune, D. J., and Weech, -4.A., Proc. SOC.EzplZ. Biol. Med., 45, 559 (1940). (19) Marmoy, F. B., J . SOC.Chem. Ind., 58, 275 (1939). (20) Marston, H. R., and Dewey, D. W., Australian J . Ezptl. Biol. M e d . Sci., 18, 343 (1940). (21) Morris, H. P., Nelson, J. W.,and Palmer, L. S.,IND.ENG. CHEM.,A N ~ LED., . 3, 164 (1931). (22) Peech, M., Ibid., 13, 436 (1941). (23) Piper, C. S., and Oertel, A. C., Ibid., 13, 191 (1941). (24) Robinson, W. 0.. U.S.Dept. Agr., Circ. 139 (1939). (25) Rogers, L. H., Ph.D. thesis, Cornell University, 1941. (26) Sandell, E. B., IND. ENG.CHEM.,ANAL.ED.,9, 464 (1937). (27) Ibid., 11, 364 (1939). (28) Saywell, L. G., and Cunningham, B. B., Ibid., 9, 67 (1937). (29) Smith, G. F., “Mixed Perchloric, Sulfuric and Phosphoric Acids I
\ - - - - , -
and Their Applications in Analysis”, Columbus, Ohio, G. Frederick Smith Chemical Co., 1935. (30) Smith, G. F., “Perchloric Acid”, Columbus, Ohio, G. Frederick Smith Chemical Co., 1940. (31) Truog, E., and Meyer, A. H., IND.ENQ.CHEM.,ANAL.ED., 1, 136 (1929). (32)
Wander, I. W., Ibid., 14, 471 (1942).
