V O L U M E 2 2 , N O . 2, F E B R U A R Y 1 9 5 0 slide to the next would not alter the radius ratios even though the radius measurements themselves varied by as much as 1 mm. or more. This assumption is validated in the data of columns 4 and 5 , where all duplicate chromatograms were made on separate slides. KO significant increase in deviation between duplicates has been noted. Although a more uniform surface depth would be desirable, i t may be concluded that the present surfaces are adequate for preliminary studies. The material presented herein admittedly leaves many questions unanswered. Perhaps most desirable would be a correlation between the chromatographic behavior curves of single elements and the degree of their separation from mixtures. The effect of varying amounts of an impurity on the curve of a given element would be useful. The use of developing solvents other than water, such as dilute hydrochloric acid, has been examined but
351 no organized study made. A number of other problems suggest themselves. The complexing effect, for example, of various anions on a given cation might be studied. Because complexing reduces the hydrogen ion concentration available to the adsorbent, this effect should be measurable by a shift in the point of inflection of the curve. I t is hoped that the present study will evoke interest in some of these problems. LITERATURE CITED
(1) Anderson, H. H., ANAL.CHEM., 20, 1241 (1948). (2) Consden, R . , and Martin, A. J. P., Biochem. J . , 38, 224 (1944). (3) Meinhard, J. E., and Hall, N. F., ASAL. CHEM.,21, 185 (1949).
(4) Shemyakin, F. M., and Mitselovskii, E. S., Doklady Akad. .Vavk S . S . S . R . , 6 1 ,289 (1948).
RECEIVED .4ugust 21, 1949.
Magnesium Content of Plant Tissue Microchemical Determination through Thiazole Yellow Procedure A. J. STERGES A N D W. H. M A C I N T I R E University of Tennessee Agricultural Experiment Station, Knoxville, Tenn.
.i procedure is proposed for rapid and accurate microdetermination of the magnesium content of plant tissue, without removal of other ions or addition of compensative solutions. The prescribed nitric acid-perchloric acid solution of plant tissue is also utilizable for the determination of the concomitant content of calcium, potassium, sodium, and phosphorus, and also nitrogen content in a solution of plant tissue obtained by means of the alternative sulfuric arid and selenium oxychloride digestion (5).
I
S A comparison of 130 organic compounds as reagents for the determination of the magnesium content of soil extracts, IIikkelsen and Toth (6) found thiazole yellow (sodium 2,2disulfonate of methylhenzothiazole) to be the best by far. In two subsequent investigations a t the Sew Jersey Station, thiazole yellow was utilized in a niacroproredurc for the spectrophotometric determination of the magnesium content of solutions that had been freed of interfering ions by means of sodium tungstate and sodium phosphate (3,7 ) 8) or the removal of calcium and R,08 by the il.0.A.C. method ( I ) . Because it appeared more advantageous than the official method, the cited procedure was studied in an effort to adapt i t t o the microdetermination of the magnesium content of plant tissue. The procedure t,hat has proved acceptable in the use of 2-granl charges of plant tissue ( 3 , 7 , 8) was found infeasible for the microanalysis of a solution derived from n charge of only 0.2 gram of plant tissue. Further study c1emonstr:ited that the thiazole procedure could be simplified and adapted to routine microdeterminations of the magnesium content of plant material and without necessity for the prior removal of calcium, manganese, iron, and aluminum from the solution of a 0.2-gram charge of plant tissue. The resultant microprocedure for magnesium was checked thoroughly against the official method ( I ) and found to give accurate results, while also affording a single solution for determinations of the companion elements. ADAPTATION OF THIAZOLE YELLOW T O PROPOSED MICROCHEMICAL PROCEDURE
Mkkelsen et al. ( 7 ) prescribed that a Zgram charge of plant material be brought into nitric-perchloric acid solution for the de-
termination of magnesium by means of thiazole yellow. Because that charge is too large for use in routine microchemical analysis, m a l l aliquots of the solutions derived through digestions of only 0.2 gram of plant material nere used for the microchemical determination of magnesium. (The remaining portion of this solution can be used for the niicrochemical determination of phosphorus, calcium, and potassium.) These trials proved disappointing, however, because the adjustment of the p H of each solution in order to effect the prior removal of calcium, manganese, iron, and aluminum as tungstates militated against speedy analyses. Further work served to establish the fact that adjustment of the pH values and prior removal of the cited elements are not necessary for accurate and rapid microdeterminations of magnesium through the use of aliquots of the solution derived from a 0.2gram charge. When a small aliquot of such a solution is evaporated to drvness on an electiic plate and then calcined in a muffle furnace, the residue can be dissolved and the color then developed with the addition of the respective reagents, with results concordant to those obtained by means of the official method. TESTING THE RE-IGENTS
In the course of this study, each reagent was tested in aliquots larger or smaller than those of Mikkelsen et al. ( 6 ) to ascertain whether the color intensity and persistence of the solutions could be improved in the analytical procedure. The reagents behaved in the manner noted by the cited investigators, with the exception of the starch solution. When that reagent was compared with a 3y0 solution of gum acacia, and with a 0.5% solution of gum ghatti, the gum ghatti reagent was found more suitable for use
A N A L Y T I C A L CHEMISTRY
352 Table I.
Yoneffect of Calcium on Determination of Magnesium Content of Plants
natural to plant material did not interfere in the rnicrodetermination of magnesium (8).
(Thiazole yellow micromethod) .~$$~~o~s
Total Calcium Present
.9ig.
MQ.
0.5 1.0 1, 3 2.0 2.5
2.92 3.42 3.92 4.42 4.92
ro
%
~11Q.
100 96 106 94 98
with either filter 540 in the Evelyn photoelectric colorimeter or filter 525 in the Fisher electrophotometer. NONEFFECT OF CALCIU.\I IONS IN THE hlICROASALYSES
Evidence that coincidence of calcium solutes does not affect the microdetermination of magnesium by means of the t,hiazole yellow method was noted first in the findings given in Table I, wherein no provision was made for prior removal of calcium content. The possibility of calcium interference in microanalyses \vas tested further by the use of multiplc charges of 0.2 gram of red clover, fortified by calcium additions of 0.5, 1.0, 2.0, and 2.3 mg. in the form of calcium chloride, as stipulated in Table 11. Because the clover had a calcium content of 1.21%, the additions brought the calcium contents in the range between 1.21 and 2.46%.
Table 11. Recovery of Magnesium from Additions to Standard Red Clover by Proposed Microchemical Procedure .\Isgnesium Additions
Total 1Iagnesium Present
Total hfagnesinm Found
Recovery of Additive Magnesium
.\lo.
'110.
70
None 0 . .i
0.96 1.46 1.98 2.46 2.9C 3.46
MQ. 0.96
1 0
I r, 2.0 2 .i
ADAPTABILlTY OF THIAZOLE YELLOW PROCEDURE T O MICROCHEMICAL TECHNIQUE
Magnesium Recovery
1.80
1.98 2.52 R . 02 3.44
...
1 ox 101 10% 102 9!)
The fortified charges were digested as usual and the mayncsiuiii contents of the resultitnt solutions were determinrtl colorimetrically by the thiazole yellow micromethod. The firidingr presented in Table I1 show noninterference from calcium, when pxsent in proportions that might occur in tissue of calciphiles. EFFECT O F RIANGANESE, IRON, AND ALURIINURI IN lZIICROANALYSES OF PLANTS
iilthough the natural occurrences of manganese, iron, and aluminum had given no evidence of interference in tlle microdetermination of magnesium in previous experiments, the possibilit>- of such interference was ~ ~ p l ( ~through red sepawtc :id& tions of those three elenients in 0.2-gram charges of red (-lover,as in the case of additive calcium. The amounts of each clcmerit addedwere: 0.02, 0.04, 0.08, 0.12, 0.16, 0.20, 0.21, nnd 0.30 nig., as manganese sulfate, ferrous sulfate, and aluminum chloride. Those additions n-ere equivalent to 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12. and 0,15yoof each eiement. Total percentage occurrences are not given in the presentation of findings in Table 111, because the amounts of the three clenient~snaturally prewllt in the clover were not determined. The analytical findings presented in Table I1 show that manganese and iron did not interfere in the determination of magnesium, when occurrenccs of each of those two elements in the plant materials were not higher than O.l5%, and aluminum was not higher than 0.0S70 in air-dried material. The experimentally imposed percentages of the three elements are beyond those to be expected in normal calciphiles and, thereforc, the smdl ainounts
In the course of this study, it was observed that the color of the solution changes gradually, although not to the extent of serious vitiation of the results. This disadvantage can be obviated, however, through the preparation of a fresh standard tinted solution for the calibration curve in each set of determinations. The reading obtained from the calibrat,ion curve then will be morv accurate, because the change in the tint of the "unknown" is likely to occur in parallel with the change in.the tint of tlic: standards, from which the calibration curve is constructed. It is advantageous also to have a systematic arrangement 01' thcb steps in the analytical procedure. After the last reagent is sddetl to develop the tint in the unknown solution and in the s t a r u h d s , the solutions should be made to volumo :ind the colorimetric r ~ t d ing should be taken immediately, so thnt any error resultant 1'rom the effect of light upon the solutions is diminished to a minimuni. Presence of perchloric acid in the solutions tendv to induce grxlual fading of the tint or its hilure to develop. Perchloric :tcid fumes are likely to zdherc on the sides of the beaker during evaporation and they should be expcllcd. Their complctc 1.cmoval after evaporation is accomplished by placing the benltci, in muffle furnace for 15 to 20 minutes a t 550" C.
Sitric acid. conrentrated. Perchloric acid, 70 to 72%. Hydrochloric acid, 1 1. Hydroxylamine hydrochloride, 5%. Dissolve 5 glanis l r i L: 100-ml. flask, make to volume, filter, and store in a glass-stoppered
+
bottle.
+
Sodium hydroxide, 3 iV. Transfer 162 ml. of 1 1stock solution of sodium hydroxide into a 1000-ml. volumetric flask, add 600 ml. of nater, shake, and cool. Make to volume, and transfer to a reagent bottle. S o standardization is required. Thiazole yellow solution. Dissolve 0.25 gram of thiazole yellon. in 500 nil. of water and store the solution in an amber glashstoppered bottle. Gum ghatti solution, 0.5%. Put 200 nil. of water in a 400-nil. heakcr and add 1 gram of powered gum ghatti, while stirring to effect complete difisolution without, caking. Centrifuge the solution, filter the supernatant through a fluted filter, transfer thcs tiltrate into a reagent bottle, nnd stow it i n tht: refrigerator.
'I'able 111. Effect of Coincidental Occurrences of' Manganese, Iron, and Aluminum on Determination of Magnesium Content of Plant Solutions (Thiazole yellow inicrometliod J
Elements
-___h d d r d :bfD.
.lIQ.
0.9j o:Oi 0.96 1.02 0.02 0.92 0.04 I) . 0 i 1.04 O.0R 1 .00 0.10 1.02 0.12 0.96 0 . IS 0 . 9(i Basis of air-dry material.
h-orie 1). 02 0.04 0.08 0.12 0.16 0.20 0.24 0.30 Q
%a
J1-i t h .\Ian,vanesc 'kdditionAI?&nOJIUili R,~. forind c o v r y
'L 11)O 100 10, !)'I
108 101 107 100
100
\Vit !I Iroii
.
Additions __ __. .\lagrie-iiiin
Itr-
fourid
co\-c~.v
.\lo.
'.A
\Vir11 .\Iii!niriu,ti .idditirini. 1 1axnesiuiri Ht.foiirid co~.('r.~. .Ilg.
''1
0.97 1 00
100 104 10" 100
0.98 0 . 90 0.91:
0.88 0.84 0.8L 0 98
100 9' 88 88 109
Standard Magnesium Sulfate Solution. Solution A , 1 ml. = 1.0 mg. of magnesium. Dissolve approximately 12 grams of C.P. magnesium sulfate (l\lgS04.713?0)in a 1000-nil. volumetric flask arid make to volume. Introduce 10-ml. aliquots into beakers, determine magnesium by means of the official method ( I ) , and use the results in adjustment of solution A , so that 1 ml. will contnin 1 .O mg. of magnesium. Solution B. Transfer 20 1111. (if soluiir)ii .\ into a 1000-ml.
