(17) Zbid., p. 4139. (18) Yoe, J. H., J. Chem. Educ. 14, 170 (1937). (19) Yoe, J. H., Armstrong, +4.R., IND. Esa. CHEM.,ANAL. ED. 19, 100 (1947). (20) Yoe, J. H., Barton, C. J., I b i d . , 12, 405 (1940). (21) Ibid., p. 456. (22) Yoe, J . H., Boyd, G. R., Jr., J . A m . Chem. SOC.64, 1511 (1942). 123J Toe, J. H., Grob, R. L., ANAL.CHEM. 26, 1465 (1954).
(24) Yoe, J. H., Hall, R. T., J . Am. Chern. SOC.59, 872 (1937). (25) Yoe, J. H., Harvey, A. E., Jr., Zbid., 70, 648 (1948). (26) Yoe, J. H., Hill, W,I,.,Ibid., 49,2395 (1927). (27) Yoe, J. H., Jones, A. L., IND.ESG. CHE?d., AN.4L. ED. 16, 111 (1944). (28) Yoe, J. H., Kirkland, J. J., ANAL. CHEM.26, 1335 (1954). (29) Yoe, J. H., Overholser, L. G., IND. ESG. CHEM..A s . 4 ~ .ED. 14, 148 (1942).
Zbid., 15, 73 (1943). Yoe, J. H., Overholser, L. G., J . A m . Chem. SOC.61, 2058 (1939). Y_ o e.. J. H.. Sarver. L. A.. “Oreanic Aklyticftl Reagents,”’ p. -112,
Wiley, New York, 1941. (33) Yoe, J. H., Will, F. 111,Black, R. A., ANAL.CHEM.25, 1200 (1953). (34) Yoe. J. H.. Wirsina F. H., J . A m . Chem. Soc. 54, lg66 (1932).
RECEIVED for review April 25, 1957. .ICcepted ?\lay29, 1957.
Spectrophotometric Determination of Boron with Diaminochrysazin, Diaminoanthrarufin, and Tribromoanthrarufin Determination in Plant Tissue with Diarninochrysazin EVERETT C. COGBILL’ and JOHN H. YOE
P raft Trace Analysis laboratory, Department o f Chemistry, University o f Virginia, Charloftesville, Va.
b Diaminochrysazin, diaminoanthrarufin, and tribromoanthrarufin give sensitive color reactions with borate in concentrated sulfuric acid solution. These color reactions and the variables that are important to their analytical use were studied, in order to apply them to the determination o f trace amounts of boron. The spectrophotometric sensitivities o f the three reagents in 95.4% sulfuric acid are, respectively: 0.0022, 0.0025, and 0.0009 y of boron per sq. cm. Boron in the range 2 to 7 y may b e determined with a precision within 1%. Titanium is the only common metallic ion likely to interfere; oxidizing anions and fluoride must b e absent. A procedure for the spectrophotometric determination of boron in plant tissue employs wet digestion and separation of the boron b y methyl borate distillation. Using diaminochrysazin, 6 to 12 y o f boron in a 0.1- to 0.3-gram sample of plant tissue may b e determined with a precision within 4%.
T
spectral absorption characteristics of six new organic reagents which give sensitive color reactions with borate ion in concentrated sulfuric acid medium have been described ( 2 ) . These reagents are derivatives of the tlihydroxyanthraquinones: anthrarufin, chrysazin, and quinizarin, having amino, nitro, cyano, or halogen substituents in the molecule. HE
Present address, Research Laboratory, .4merican Tobacco Co., Richmond, Va.
Anthrarufin, chrysazin, and quinizarin are the common names for the isomeric dihydroxyanthraquinones with the hydroxyl groups in the lJ5-, I,%, and 1,4-positionsJ respectively. The six derivatives of these substances investigated as colorimetric reagents for boron were : diaminoanthrarufin (1,5diamino-4,s- dihydroxyanthraquinone), diaminochrysazin (1,8 - diamino - 4,5dihydroxyanthraquinone), dinitroanthrarufin (1,5 dinitro - 4,8 dihydroxyanthraquinone), dinitrochrysazin (1,8dinitro - 4,5 - dihydroxyanthraquinone), tribromoanthrarufin [1,5(?)-tribromo4,8-dihydroxyanthraquinone], and dicyanoquinizarin (2,3 - dicyano - 1,4dihydroxyanthraquinone) . Further examination showed three of these to be the most promising of the group as colorimetric reagents for boron-dinminochrysazin, diaminoanthrarufin, and tribromoanthrarufin-and more detailed investigation was confined t o these. The present paper describes a study of these three compounds as reagents for the determination of trace amounts of boron. It shows the effect on their color reactions of the variables which are important to their analytical use and details a procedure by JThich they may be applied to the determination of boron in plant tissue. From an overall consideration of the characteristics of the three reagents, diaminochrysazin !vas judged to be somewhat superior to the other two, and was used in the analyses of plant material reported. The two anthrarufin derivatives can be substituted for it, with only minor changes in the procedure.
-
-
APPARATUS
Spectrophotometer. Transmittance measurements were made with a Beckman Model DU quartz spectrophotometer, using matched 1-cm. Corex or silica cells. Glassware. Glassware employed for t h e storage and ordinary handling of cold acid solutions was either of t h e “soft-glass” t y p e or made of Corning alkali-resistant (“boron-free”) glass No. 7280. Pipets and volumetric flasks were of Kimble Brand glass. The No. 7280 glassware was employed exclusively for operations requiring heating or evaporation of solutions, and for the storage of alkaline solutions. Distillation Apparatus. T h e apparatus used for t h e separation of boron by distillation as methyl borate was similar in design t o t h a t described by Yoe and Grob (6, I S ) . The exit tube of the receiver flask mas an Nshaped tube with funnel top on its outlet arm. A few drops of water placed in the lower loop of this guard tube during the distillation serve as a trap for any escaping vapors of methanol or borate ester. REAGENTS
Sulfuric Acid, 960j0. Except where otherwise indicated, t h e reagent grade concentrated sulfuric acid of commerce (assay 95 t o 96%) was used, and all solutions were made u p in this medium. Care should be t a k e n t o select a n acid which is water-white and has a low boron blank. B and A Brand reagent grade sulfuric acid, manufactured by General Chemical Division, New York, N. Y., is satisfactory. The lot of acid used for the work reported was analyzed 95.4% sulfuric acid, by alkimetric titration. VOL. 29, NO. 9, SEPTEMBER 1957
1251
Table
I.
Color Reactions of Reagents with Borate
Reagent Xaximum, mfi
Reagent Diaminochrysazin
44u
Diaminoanthrarufin
435
Tribromoanthrarufin
560
Table II.
Boron Complex Maxima, Color mfi Reagent soln. complex soh. 495 Lemon yellow Orange 525 570 Brownish yellow Deep blue 620 590 Reddish violet Blue 635
Sensitivities of Color Reactions in
95.4y0Sulfuric Acid
Optimum Analytical Spectrophotometric Wave Length, Sensitivity, Reagent mt.i T j S q . Cm. Diaminochrysazin 525 0.0022 Diaminoanthrarufin 605 0 0025 Tribromoanthrarufin 625 0 0009
Sulfuric Acid, 99.5%. One pound of B and A reagent grade fuming sulfuric acid (manufacturer's assay, 30.0 t o 33.0% free sulfur trioxide) \vas added to 440 ml. of concentrated sulfuric acid (assay 9 6 . 9 ~ o ) . T h e resulting product. mas analyzed b y alkalimetric titration and found t o have a sulfuric acid content of 99.6%. By mixing this acid with 99.47, acid in calculated proportions by weight, acids of intermediate concentrations were prepared a,? needed. Organic Reagents. T h e hydroxyanthraquinone derivatives n-ere of commercial purity and were used without further purification. except t h a t they were vacuum dried a t 96' C. Solutions of t h e reagents in concentrated sulfuric acid containing 0.300 mg. of the reagent per ml. viere prepared h!- dissolving weighed amounts of the compounds in appropriat'evolumes of the solvent. All dissolre readily in the sulfuric acid to a concentration of 0.300 mg. per ml., except the tribromoanthrarufin, which leaves a sinal1 amount of undiesolred residue. I n this case, the saturated solution is slloived to st'aiid for several days in a stoppered cylinder until t'he residue has settled out, and the clear supernatant liquid is removed by pipetting. Standard Borate Solutions. Stock standard solutions, containing 1 mg. of boron per ml., may be prepared b y dissolving the calculated quantity of pure boric acid or borax (sodium tetraborate decahydrate) in concentrated sulfuric acid. Reagent grade crystalline boric acid may be vacuum-dried at about 60" C. before use, b u t should not be dried a t elevated temperatures (above 100') because this may convert i t partially t o metaboric acid. Reagent grade borax is oft.en part,ly effloresced. It may be reconverted completely to the decahydrate b y placing it in a hygrostat over a saturated solution of sodium tetrabromate until it has ceased to gain weight (several days), and then transferring it to a second
1252
ANALYTICAL CHEMISTRY
Limits of Detection B in 2 ml. Dilution 0.08 1 :25,000,000 0.12 1:15,000,000 0.06 1 :35,000,000
hygrostat over some of the original partially effloresced material, where it is allowed to remain until its weight becomes constant. Working standard solutions having concentrations in the range 0.5 to 8.0 y of boron per ml. are prepared from the stock standard solution by appropriate dilution. Hydrogen peroxide, 30%, analytical reagent grade. with stabilizer. Hydrogen peroxide, 90%, Becco C.P. reagent, manufactured by Buffalo Electro-Chemical Co., Inc., Buffalo, N. Y. Calcium hydroxide solution, a saturated aqueous solution (approximately O.O4S), prepared from thoroughly washed reagent grade calcium hydroxide. Ammonium Hydroxide, 3 N . Prepare ammonium hydroxide solution either from t a n k ammonia gas, or b y distilling commercial concentrated ammonium hydroxide in a "boron-free" glass still. Determine the concentration of the product by acidimetric titration, and dilute to 3.V. The solution is best preserved in a polyethylene bottle. Ferrous sulfate solution, a saturated
Table
111.
Reproducibility Measurement
of
Color
(Boron taken, 5.05 y ; final concentration, 0.505 p.p.m.) Absorbance DiaminoDiaminoTribromochrysazin anthrarufin anthrarufin 0 275 0. 261 0 700 ~.~ 0.271 0,255 0.i06 0.273 0.258 0,708 0.2il 0.258 0 .io4 0.272 0.258 0.703 0,269 0.258 0.705 Av . 0.272 0.258 0 . io4 Std. dev. 0.002 0.002 0.003
solution of ferrous sulfate heptahydrate in 0 . 1 S sulfuric acid. Methanol, absolute, analytical reagent grade; assay, minimum 99.5%. Plant Tissue Samples. T h e samples of alfalfa, ground for analysis, were obtained from W. T. Mathis, Connecticut dgricultural Experiment Station, Xew Haven, Conn. Ground samples of apple and citrus tree leaves mere supplied by A. L. Kenworthy, Nichigan State College, East Lansing, Mich. The spinach sample was prepared from fresh leaves by air-drying and grinding. Before analysis, the samples were either dried for several days in a desiccator over anhydrous magnesium perchlorate or oven-dried a t 60" to 70' C. The desiccated leaf material is hygroscopic, and samples should be neighed by difference from a stoppered weighing bottle. CHARACTERISTICS
OF
COLOR REACTIONS
Transmittance curves for the reagents and their boron complexes have been published ( 2 ) . Table I summarizes the important features of the absorption spectra and the color changes observed when the reagents undergo reaction with borate. Sensitivities of Reagents. The spectrophotometric sensitivities of t h e reagents, in micrograms per square centimeter as defined by Sandell (IO), are given in Table 11. These values are calculated from the absorbances of solutions containing 0.4 p.p.m. of boron, a t a sulfuric acid concentration of 95.4%. Listed also are the minimum quantities of boron (and the corresponding concentrations) which can be detected ~ i t hcertainty when color is developed as in the recommended procedure, JThere a 2-ml. aliquot of sample solution is taken for analysis. Precision of Spectrophotometric Determination. The reproducibility of t h e spectrophotometric measurement of boron \\-ith t h e three reagentfi is s h o n n by the sets of absorbance values listed in Table 111. These were obtained by analyzing separate 2-nil. aliquots of solution containing 5.05 y of boron by the recommended procedure. I n each case, the standard deviation in absorbance values corresponds to less than 1yo of the boron present. Adherence to Beer's Law. T h e colors produced by diaminochrysazin and tribromoanthrarufin shoR a very good linear relation between absorbance and boron concentration u p to approximately 6 y of boron in the 10-ml. final volume (0.5 p.p.m.); from 5 to 7 y, a slight departure from linearity occurs. The Beer's law plot for diaminoanthrarufin is essentially linear up to approximately 0.36 p.p.m. in the final solution. Through the over-all range 0 to 7 y of boron (in 10 ml.) the plot shows a slight upward curvature.
0.500 I
I
0.400
m (?:
0 m m q.200'
0.1 00
94 0
95.0
96.0
97.0
WEIGHT PERCENT
Figure 1.
Tribromoanthrarufln Diaminochrysazin Diaminoanthrarufin
The poorer adherence of the diaminoanthrarufin to Beer's lan niaj be due to the much slower reaction of this reagent, compared n itli that of the diaminochrysazin or the trihromoanthrarufin. The 4 o n nes. of reaction of this reagent prolmbly alio accounts for the upnard curvature of the Beer's law plot, relatively greater color intensity being achieled a t the higher conrentrations of horon because equilihrium is appromhed more rapidly. RECOMMENDED PROCEDURE FOR COLOR DEVELOPMENT
Having obtained the horon to be deterniinetl in solution in concentrated sulfuric acid. transfer a 2-ml. aliquot of the solution to a 10-nil. glass-stoppered volumetric flask. (If an aliquot smaller than 2 ml. is desired, add sufficient sulfuric acid to make its volume approxiinat'ely 2 ml.) Wit'h a pipet, add 1.00 ml. of reagent solution (0.3 mg. per ml.). Stopper the flask, and mix the solut'ion thoroughly by swirling and flowing the liquid around the n-alls and neck of the fl:iik. Allow the solution to stand for the prescribed period for color developnient~-for (IiaIninochrysazin,15minutes; ior tribromoanthrarufin~ 30 minutes; for diaminoanthrarufin, 1 hour. Then tlilut'e to the 10-nil. mark with concwit'rated sulfuric acid and mix. After 10 or 15 minutes of standing, transfer the solution to a spect'rophotometer cell :ind measure its absorliance (against a sulfuric acid blank) a t the analytical the reagent used-for 525 mp; for tri625 m p ; for diamino::nthr:irufin, 605 nip. Determine the qu:iiitity of boron present by reference t o :I calibration curve plotting absorh:tiire 2's. micrograms of boron, established l)y running a series of standard solutions covering the range 1 to G y of the element (per 2-ml. aliquot). ~
990
Effect of acid concentration on sensitivity I. II. 111.
~
98.0 HrSO,
FACTORS INFLUENCING COLOR DEVELOPMENT
Concentration of Sulfuric Acid. T h e sensitivity to boron of hydrosganthraquinone reagents of this type depends greatly upon t h e concentration of t h e solvent acid. Belo\y a n acid strength of about 9070, t'he color react'ions do not occur wit'h trace concentrations of boron. The effect of acid concentration on the sensitivity of a g k e n reagent is important, not only from the point of view of obtaining the highest sensitivity from the reagent', but also because it enables an estimate to be macle of the amount of water that can be tolerat,ed without introducing an appreciable error into the analysis. This effect was studied with the three reagents by measuring the intensity of color produced by each wit'h 4 y of boron in 10 ml. of sulfuric acid of concentrations 94.3, 94.7, 94.9, 95.4, 9G.9, and 98.i7c,. The variation of color intensity ~ i t hacid concentration is shown graphically in Figure 1. The concentration range of most importance n-ith tlie ordiiiary concentrated sulfuric acid of commerce is betwren 95.0 and 96.55%. Over this range, the sensitivity of the dianiinochrysazin increases approuimately 44%, that of t'he diaminoanthrarufin 32'3 ; the tribromoanthrarufjn phon-s w r y little change. When 95.5% sulfuric acid is used as solvent, tlie introduction of 0.1% of water causes a decreax of about' 2% in the color intensity il-ith the two diamino reagents. To increase the sensitivity of the dianiinoanthrarufin and dianiinochrysaziii reagents, acid stronger than the ordinary concentrated sulfuric co~.ild be employed; by using 98y0 acid, a sensitivity approximately twice that in the 95% mat'erial could be achieved.
Time of Standing. With t h e two anthranifin derivatives, development of t h e color after mixing t h e reactants is slow. I n 10 ml. of solution containing 0.3 ing. of reagent and 1 to 7 y of boron, approximately t'hree-fourths of the color is developed in the first 15 minutes, but full color intensity is reached only after standing 12 to 15 hours. Tery nearly the niaxiiiiuin color can be achieved more rapidly, however. if the reaction is allowed to take place in a smaller volume, :ind after standing for a short time the solution is diluted to the final 10 1111. K h e n the recommended procedure is folloiT-ed, where a 2-ml. aliquot of solution containing the boron is treatetl XT-ith 1 nil. of reagent solution (0.3 ing. per nil.), approximately 9 i c c of the maximum color is obtained with 30-niinute standing for the tribro1iic-ianthrarufin and 1-hour standing for the t1i:iminoaiit~lirarufin. The color illtensit?. is then coilstarit betn-een I5 minutei and 2 or 3 hours after dilution. The tliaminochrysazin reacts a1nio.t imiiiet1i;itely; even a t a dilution of 10 inl. color development is eqsentially complete in less than 15 minutes. Se\-ertheless,. the technique of developing the color first in a 3-nil. volume ; i n ( \ then diluting has proved convenient in making series analyses with these reagents, :und it was considered desirable to adopt a short standing period even with this reagent, to assure attainment of color ecjuilibrium; hence. a 15-minute standing period for the di:iniinoclirysazin has been specified. The >tanding periods recommendetl for the til-o anthrarufin derivatives are niinimum times for development of zipproximately 9 i % of the equililriuni color n-liich \\-oulcl lie obtained fafter 15 hours) if the reaction were carried out in tlie filial 10-nil. volume. Longer periods ma>-be adopted, but the :idopted time+ should he uniform in staiiilarti3 and uiikiio\~iis. Long standing in tlie smaller volume may result in cdor intensitie? which are initially highrr after dilution than the equilihri~uii ixlues for the 10-nil. T-olume, so t1i:tt a s l o ~diminution in intensit,! is observed. For precise x o r k n-itli tlic anthrariifin reagents, the adopted stantliiig time ~liouldbe adhered to vith reasoiiwhle care, although a wriation of 10 or 15 niinutes from the set time hetn-eeii mixing reagents aiid dilution 11-ill affect the results less than 37,. V-ith the dianiinochrysazin reagent. factor is the re~ic,tioii-tinie-and-~-olunie not important, if at least 15 minutes elapse Iietx~een mixing the reactants ;ili(l m:iliiiig the color measurement. C'hiefly for this reason the dianiinochrysazin compound is considered the superior reagent of the three clescrihed. \There rapid analyses are not required, it nin>- be more convenient to VOL. 2 9 , NO. 9, SEPTEMBER 1957
1253
dilute the solutions to final volume immediately and allow them t o stand (well stoppered) overnight or longer. This is permissible with any of the reagents. Once the colore have reached their equilibrium intensities, they are stable for several weeks, if the solutions are protected from. absorption of moisture. Temperature. T h e effect upon t h e color development of variations in temperature in t h e neighborhood of ordinary room temperatures vias determined b y measuring t h e color intensity produced by 5.05 y of boron when the recommended procedure was applied at 21', 2 i 0 , and 3 i o C. Table IV shows the relath e intensities of color formed a t the different temperatures, based upon that produced a t 27' as 100. It is estimated that a variation of 2' C. in room temperature affects the color developed with diaminochrysazin by approximately 1%; with tribromoanthrarufin, 2%; and with diaminoanthrarufin, 3%. The colored solutions with all three reagents fade 15 to 25% on heating for a n hour at 80' C., but the absorbances return to their original values when the solutions are brought back to room temperature. Restoration of the color with diaminochrysazin and tribronioanthrarufin is complete within a few minutes after cooling; with the more slowly reacting diaminoanthrarufin, several hours are required. Presence of Diverse Ions. Possible interference with t h e color development b y a number of common cations and anions was studied by applying t h e recommended procedure t o 2-ml.
0.18(
J
0.16(
0.w
0.12c
I-
:
0.10(
0
t
>OD8
c
0.06 0
a040
a02 o
0 0
01
0.2
0.3
04
05
06
0 7
0.8
09
IO
MOLE FRACTION R E A G E N T
Figure 2. Continuous variation curves for diaminochrysazin and borate
aliquots of solutions containing 5.05 y of boron plus a quantity of each foreign ion. I n the case of the cations, 1.0 mg. of ion was present, if the metallic sulfate was soluble t o that extent in 1 ml. of concentrated sulfuric acid; otherwise, the quantity of ion saturating 1 nil. of the acid was taken. With the anions, the quantities employed in the tests \$ere those shown in Table V. The actual amounts of the chloride and fluoride anions present in the solutions when the color was developed cannot he known, because the hydrogen halides volatilize rapidly from concentrated sulfuric acid. I n a n effort
t o minimize such loss, these ions were dissolved in the boron solution as solid alkali halides immediately before development of color. The extent of interference b y ions Table IV. Effect of Temperature on which caused either an increase or dimiColor Intensity nution of the color is shown in Table T; (Color intensity at 27" C. = 100) in terms of the percentage error which Relative Color Intensities M ould result in the determination of 5 Triy of boron in the presence of the indiDiamino- bromocated quantity of the foreign ion. An Temp., Diamino- anthraanthraobserved difference of 2% or less in ' C. chrysazin rufin rufin color intensity from that obtained with 21 102 6 90 1 95 1 similar htandards in the absence of 27 100 100 100 the foreign ion was considered a nil 37 95 2 108 G 101 6 interference. Titanium was the only metallic ion that interfered. Fluoride, which complexes readily with boron, must Table V. Interference by Diverse Ions be absent, as must oxidizing anions, (As yo eiioi- in drtermmation 01 5 y 131 such as nitrate and dichromate. The 1iiterfi.irnce latter decolorize the solutions with Qimitity Added, DiaminoIliaminoTiibromodianiinochrysazin and tribromoanthraIon Mg. chrysazin, ye anthrarufin,% anthrarufin, 7@ rufin, probably through oxidation of the Ti++++ Satd soh. Nil Nil +g organic substances, but form an in-10 5 $267 -S CrzO?-0 1 tensely blue-colored substance with 0 01 12 -60 +306 -34 SO30 1 diaminoan thrarufin. 0 01 22 Other ions which have been tested -44 - 49 - 76 F0 036 qualitatively with diaminochrysazin Ions showing no interference: i l l + + + B a + + Ca++ Co++, Cr++?-, Cu++, Fe+++, and found to give no observable interK+, Mg++, Mn++, Ka+, S i + + ,Pb++, Znk+?C1' ,PO,-'ference are: carbonate, acetate, for~~
+
+
~~
~
1254
ANALYTICAL CHEMlSTRY
mate, sulfite, and silicate; hydrogen peroxide bleaches the color of the reagent; formaldehyde produces a deep blue color. NATURE OF COLORED COMPLEX IN SOLUTION
The continuous variations technique (6, 1 1 ) was applied to determine the mole ratio with which the reagents combine with boron to produce the colored substance. The reactions of ciiaminochrysazin and diaminoanthrarufin were studied by this procedure. Solutions of equal molar concentrations of boron and reagents were eniployed, in a concentration of 0.111 pniole per ml. (1.11 x 10-4M). These m r e mixed in varying proportions to form a series of solutions having a total volume of 12 ml. throughout with iiiole ratios of reagent t o boron varying from 1 to 11 on one extreme to 10 to 2 on the other. The solutions were alloned to stand for 24 hours or longer to assure complete color development, and their absorbances were then rneasured at several selected m v e !rngths. Figures 2 and 3 are the continuous T ariations plots for diaminochrysazin xnd diaminonnthrarufin, where function I' as defined by T'osburgh and
Cooper (11) (difference between the measured absorbance and that calcuiated assuming no reaction) is plotted against the mole fraction of reagent in the reactants contained in each solution. For the diaminochrysazin in Figure 2, a 1 to 1 combining ratio of reagent to boron is clearly indicated at all n a v e lengths. For the diaminoanthrarufin, on the other hand, the curves in Figure 3, obtained at different wave lengths, are not uniform. The plot obtained a t 620 nip has its maximum very nearly a t the position indicating a 1 to 2 reagent-boron complex, on the curves taken at shorter wave lengths, the maximum is shifted in the direction of a 1 to 1 combining ratio. This is interpreted to indicate the coexistence of two complexes in the solutions with this reagent, one having two borate ions combined with one molecule of reagent and the other formed by a 1 to 1 combination of the reactants. It has been postulated by Feigl (4) that the color-forming reaction of borate with hydroxyanthraquinones takes place through reaction of metaboric acid, HB02, with the hydroxy-organic compound to form an inner-complex ester. On this assumption, and in light of the continuous variations data, the reactions of borate with thc two
isomeric hydroxyanthraquinone derivatives can be written: o'B\
0
d
0 Diamlnoc hrysazin
VC\t 0
NH2
\ OH
*
HI302
\c/ II
------+
'
HzSO,
"2
0
Diaminosnthrai u h
Additional qualitative support for the postulation of a t least two colored substances in the boron-diaminoanthrarufin solutions can be obtained by observing the color changes when an excess of boron is added to a solution uf diaminoanthrarufin. Almost immediately the solution becomes reddish brown; then the color changes to purple red and through magenta t o violet. This succession of color changeb requires I or 2 minutes; then, in about 10 minutes, the violet color becomes a clear dark blue. DETERMINATION
MOLE FRACTION R E A G E N T
Figure 3. Continuous variation curves for diaminoanthrarufin and borate
OF
BORON IN PLANT TISSUE
Separation of Boron by Wet Digestion and Distillation. For t h e determination of boron in plant tissue with these reagents, a procedure has been applied which employs wet digestion of t h e sample and separation of t h e boron by distillation as the methyl borate ester. It involves: (1) destroying the organic matter by diVOL. 2 9 , NO. 9 , SEPTEMBER 1957
1255
gestion with sulfuric acid and hydrogen peroxide; (2) separating the boron b y distillation with methanol, catching the distillate in an alkaline solution; (3) evaporating the distillate to dryness; (4) taking up the residue from evaporation in a measured volume of concentrated sulfuric acid; and ( 5 ) determining the boron spect'rophotometrically in an aliquot of the resulting solution. Wet Digestion of Plant Tissue. T h e organic matter i n leaf samples prior t o t h e determination of boron may be destroyed by dry ashing or wet digestion. T h e latter method lends itself well t o the subsequent eeparation of boron by methyl borate distillat,ion. The decomposition of plant niat,erial by wet digestion with concriitrateri sulfuric acid and hydrogen peroxide has been studied by Ellis, Zook, and Baudisch (3). who found no loss of boron in the process of digestion and boiling the resulting solutions to destroy excess peroxide. I n fact, Irith certain types of plant material significantly highcr recoveries of boron were obtained than with dry ashing. This is in line with the findings of Winsor (12). who shon-ed that appreciable quantities of boron may be expelled with the volatile matter during charring and ignition in a muffle furnace. I n the procedure adopted. the plant sample is decomposed initially by charring in concentrated sulfuric acid. The organic matter is then conipletely oxidized Iiy successive treatments with hydrogen peroxide (first with 307,, and finally. after vigorous reaction has ceased, with 90Yc peroxide). The bulk of the excess peroxide is then removed by cautious boiling, the last rmininder being destroyed by adding a little ferrous sulfate to reduce it. Separation of Borate as Methyl Ester. Yeparat'ion of boron as t h e trimethgl borate is more quantitative t h e more nearly anhydrous the solution, so t h a t absolute methanol should be employed. I n t h e present case, as it is necessary to minimize the amount of volatile acids n-hich pass into tlie distillate, the distilling flask sho1.ild not be heated to too high a temperature near the end of the distillation. The boiling point of sulfuric acid is sufficiently high to involve no tlangrr of volatilization; but much of this acid niay be converted to methyl sulfuric acid and dimethyl sulfate. nliich come over a t lower temperatures. and are hydrolyzed in the distillate. For this reason, heat is supplied to the distilling flask by a steam jacket. so that its teniperature never exceeds 100" C. Boron is separated by distilling off 80 ml. of methanol, in two 10-ml. portions. 50 gain in efficiency of separation is achieved by distilling thr meth1256
ANALYTICAL CHEMISTRY
0.31
.
I
I 0.r
t
0
5 I
8
7
6
9
10
It
I2
MICROGRAMS B IN SAMPLE
,
2
4
3
5
I 1
MICROGRAMS B IN 0.4 ML. ALIQUOT
Figure 4.
0 V
0
Calibration curves
Standards Standards carried through evaporation step Distillation standards
anol in three or four portions instead of two. Retention of Boron in Evaporation Step. T h e operation involving the greatest likelihood of loss of boron is evaporation of t h e distillate t o dryness. The distillate must, of course, be rendered alkaline before evaporation, to ensure complete hydrolysis of the methyl borate ester; but it must be definitely baqic. Boric acid, in addition to forming the volatile ester, is itself volatile from aqueous solutions; and as it is an extremely weak acid, its salts are extensively hydrolyzed even in \-ery dilute solutions a t p H 10 or higher. On the other hand, for the colorimetric measurement which is to follow, the amount of free alkali remaining in the residue from the evaporation must be held to a minimum. Excess alkali will produce water upon neutralization, when the residue is taken up in concentrated sulfuric acid, lowering the sensitivity of the color reaction. For a reduction of acid concentration of less than 0.1% under the conditions prevailing when the color is developed, the excess alkali should be less than about 0.8 meq. The evaporation step was tested by a series of experiments in which between 6 and 12 y of boron were added to methanol-water mixtures approximating the solution obtained at the end of a distillation. The solutions were made basic by addition of varying quantities of alkali and evaporated to dryness, after which the boron was determined colorimetrically in the residues. Boron losses were larger (50 to 85yc) when sodium hydroxide or calcium hydroxide, in quantities up to 0.5 meq., was used. V h e n calcium hydroxide is used with ammonium hy-
droxide, retention of boron is quantitative (98 to 100%) if at least 0.4 meq. of calcium hydroxide is present with 15 meq. of ammonia. The corresponding combination of sodium hydroxide and ammonium hydroxide is not effective in retaining the boron, losses of 50 to 607, being observed when these alkalies are used. The relatively large quantity of ammonia evidently keeps the solution strongly alkaline in the early stages of the evaporation. until the borate has been entrained and fixed in the precipitate of calcium hydroxide and carbonate which separates as the evaporation proceeds. Being completely volatile, the ammonia does not add to the excess alkali in the residue. Calibration Curve. T h e separation of 6 to 12 y of boron b y distillation according to the procedure given below is 90 I=! 5% efficient. The results of analyses should therefore be calculated on the basis of procedure standards that have been carried through the distillation. For this purpose, a calibration curve may be constructed by distilling the boron from standard solutions containing between 5 and 15 y of the element (lower plot, Figure 4). For comparison, Figure 4 includes a curve (which is linear) of ordinary standards, as well as standards that have been carried through the evaporation step. PROCEDURE FOR ANALYSIS OF PLANT TISSUE
Weigh a sample of 0.15 to 0.25 gram, depending on the boron content (containing 6 to 12 y of boron), into a dry 200-ml. round-bottom flask (KO.7280 boron-free glass). Add 2 ml. of concentrated sulfuric acid, warm on a steam bath until the material is thoroughly charred, and cool under the tap. Add
0.5 ml. of 30yo hydrogen peroxide. dropwise and slowlj-, with swirling of the fluid mass. (The reaction is vigorous u-ith the first few drops of peroxide.) Warm on a steam bath for a few minutes! then cool and add 0.5 nil. of 30TGperoxide. Place on the st'earii bath for about 10 minutes. When the liquid is free of undecomposed black particles, remove the flask from tlie steam h t h and cautiously heat it with a very sniall flame until rapid evolution of gas ceases. Allow the flask t'o cool somewhat. then cool it under the tap. S o w :ttltl 2 drops of 90% hydrogen peroxide, r'over the flask with a small \vatch glasb, arid warm the liquid gently over tlie flame for a minute or two; then finally heat it' until evolution of gas ceases iiiitl the liquid has begun to boil. Cool anti repeat the addition of 2 drops of 90yc hydrogen peroxide, warming, and boiling. until the solution can be tirought t o the boiling point without turning (lark brown. (Usually three treatmeiits with 2 drops of 90% peroxide suffice.) Xow insert a small square of I)lat'inuni gauze, about 2 sq. cm., approximately 80 mesh, and keeping the flask covered with a watch glass to niininiize escape of steam, boil the solution gently for 2 ' o r 3 minutes. (Peroxide decomposes rapidly on the platinum surface. and the gauze aids in preventing buniping in the distillation to follow.) Cool tlic flask, remove the watch glas.. and allow it. to stand open for :I fen. niinutes for cnrbon dioxide to escape.
Add 2 drops of saturated ferrous sulfate solution, and attach the flask to the distillation apparatus. The receiver flask (a 250-ml. Erlenmeyer boron-free glass flask) should contain 10 ml. of saturated calcium hydroxide, and should be immersed in a beaker of ice water. Form a seal with a few drops of water in the guard-tube trap on the receiver flask. Fill the separatory funnel with absolute methanol, and from it add a 40-nil. portion of alcohol to the distilling flask. Surround the distilling flask with a covered beaker containing enough water to reach almost to the bottom of the flask, and heat the water to boiling. Adjust the heating to give a distillation rate of about 2 nil. per minute (approximately 6 drops in 5 seconds); the rate of heating may he increased toward the end of the distillation of a portion of bhe alcohol. Continue the distillation until it has become very slow (less than 2 drops per minute) or ceased entirely. Interrupt the heating of the steam bath, add a second 40i d . portion of met'hanol from t'he separatory funnel, and distill it over as before. At the end of the distillation of the second portion, disconnect t'he receiver flask and lower it : rinse the liquid in the guard t'ube over into t'he flask with Gietlianol. Sn-irl the liquid in the receiver fla*k to mix it thorounhlv. K i t h a stirring rod, remove 3 drop of tlie liquid and test it with red litmus paper. (It should give a distinctly alkaline reaction: if
Table VI.
T.7alrieqReported by Other
Lahoratoriesa
Sample Spinach
hletholi
P.p.m. of boron
Alfalfa 1
Chem.
56.5, 23.7, 58.0
Spect.
A V , 46.1 43.8, 44.0 48.0, 48.0
Analyses of Plant Tissue
.Inalyses in This Laboratory Kith Diaminochrysazin (r)istillatio~i With Quinalizarin ( D r y -4shing) Procedure) Sample Sample weight, g. y B found P.p.ni. of B weight, g. y B found P.p.m. of B 3 0, 50 0 1860 9 11 -lq 0 0 3051 2 4, 47 0 2082 10 97 52 7 0 2629 2 23 48 0 1808 9 61 53 1 0 2332 9 31 46 8 0 1989 0 2411 11 37 47 2 8 91 48 1 0 1850 -iv.48 .3 49 5 0 2411 1 501 62 0 1606 9 19 !?I7 1 0 2623
17 0,
.iv. oi all labs, 46.0 Alfalfa :3
34.0, 20.0, 16.0 hv. 33.3 Spec:. 30.5, 32 3 5 3 0, 30 :I Av. 36.5 .iv. of all labs. 35.2 Clieiii.
22.0 to 34.5 Alv.of 6 labs. 30.1 Spert. 20.0 to 32.0 Aiv.of 6 labs. 24.2 - i V . of A l l labs. 27.7 Chern. 28 0 to 83.0 Av. of 6 labs. 69.0 Spect. 54,8 to 85.0 -iv. of 4 labs. 58.2 .iv, of all labs. 64.7 p. 413); apple arid citrus leaves ( 7 ) . Chem.
a
Afdfa values-(9,
not, add calcium hydroxide solution in small increments until an alkaline reaction is obtained.) Add 5 ml. of saturated calcium hydroxide solution. Immediately before the evaporation, add 5 ml. of 3 X ammonium hydroside. Set the flask on a low temperature hot plate (temperature low enough to keep the solution just below boiling) with a 250-watt infrared heat lamp suspended about 3 inches above the mouth of tlie flask. Allow the solution to evaporate completely to dryness. K h e n conipletely dry, remove the flask from the hot plate, cap it with aluminum foil. and cool it to room temperature. K i t h a pipet, treat the residue with exact'ly 5 ml. of concentrated sulfuric acid. Immediately recap the flask n.ith aluminum foil. Dissolve the residue and mix the solution thoroughly by swirling the liquid and flowing it around the inner walls of the flask. Then withdraw a 2-nil. aliquot Ivith a pipet, transfer it to a 10-ml. volumetric flask. and develop the color with diaminochrysazin as directed in the Reconinienrled Procedure for Color Development. Determine bhe amount of boron present from a calibration curve obtained by running standards through the procedure. Distillation Standards and Blanks. T a k e a 2-nil. aliquot of a standard boron solution in concentrated sulfuric acid, containing between 5 and 15 y of boron. Transfer it to a 200-nil. distilling flask and add 1 nil. of water.
65
i v . 6.3 5
0 1483 0 1714
$1 46
8 25
55 7 55 3 56 0
0.3160
9.75
31
0.2152
7.40
:34 4
0 2793
9 50
34
0 2181 0 2417
7 08 7 92
32 5 32 9
0 2973 0 2775
9 30 8 50
0 1465 0.1592
12 60 12 90
.iv. 3 2 5
33 3
31 3 30 6
AV.
31 0
86 0
81 0 Av. 83 5
VOL. 29, NO. 9, SEPTEMBER 1957
1257
Carry out the distillation and color measurement as directed above. The distillation standards do not include the blank on the ferrous sulfate and hydrogen peroxide. This correction could be included if the standards were treated with peroxide and the latter was decomposed as in the procedure; however, i t is less time-consuming to omit the peroxide from the standards and run this blank separately. Place in a distilling flask 2 ml. of concentrated sulfuric acid, 1 ml. of 30% hydrogen peroxide, and 6 drops of 90% hydrogen peroxide. Insert a square of platinum gauze, and heat the solution on a steam bath until evolution of gas on the platinum surface has nearly ceased. Then cover the flask with a watch glass and boil the solution with a small flame for several minutes, or until no further evolution of oxygen occurs. Add 2 drops of saturated ferrous sulfate, and proceed with the distillation and evaporation as described. I n the meantime, prepare a second blank by evaporating to dryness a solution containing 80 ml. of methanol, 15 ml. of calcium hydroxide, and 5 ml. of 337 ammonium hydroxide. Take up the residues from both blanks in 5 ml. of concentrated sulfuric acid, and carry a 2-ml. aliquot of each through the color development as in the procedure. The difference in absorbance, if any, b e h e e n the two blanks represents the blank correction for the peroxide and ferrous sulfate. It can be deducted from the absorbance readings of the unknowns, or its equiralence in micrograms of boron can be calculated and subtracted from the quantities of boron read from the calibration curve. ANALYTICAL RESULTS
Results of analyses of plant tissue samples by the procedure described are presented in Table VI. The analyses with quinalizarin which are given for comparison were made b y a procedure similar to that recommended by the Association of Official Agricultural
1258
ANALYTICAL CHEMISTRY
Chemists ( I ) , with improvements suggested by hfacDougal1 and Biggs (8), except that 98y0 sulfuric acid was used, instead of the 96y0 acid (with higher reagent concentration) preferred b y the latter authors. Samples were wet with calcium hydroxide solution and dried, and volatile matter was driven off by charring at a low temperature. They were then ashed in a muffle a t 600” C. The quinalizarin reagent solution was in 9870 sulfuric acid, containing 20 mg. of the reagent per liter. Absorbance measurements were made at 603 mp, which is the absorbance maximum for the quinalizarin-boron complex. For the data presented in Table VI, the precision of the distillation method, expressed as relative standard deviation from the average values, is within 4.0%.
with those of other reagents for boron described in the literature. The favorable spectral absorption characteristics of the reagents and their boron complexes, the high stability of reagent and complex solutions, and the excellent reproducibility of the color reactions make them attractive as substitutes for similar reagents of the anthraquinone type. The color development requires no bothersome heating or careful temperature control, which makes many existing procedures awkward for routine use. With the diaminochrysazin, the color produced reaches maximum intensity almost immediately, while nearly all other boron reagents described in the literature require prolonged color development. LITERATURE CITED
DISCUSSION
Separation of boron by wet digestion and methyl borate distillation is more time-consuming than direct determination after dry ashing, but where best accuracy is required it is to be preferred. Dry ashing is attended by danger of loss of boron, either from volatilization when the organic matter is destroyed or through its being incompletely leached out of the ignited ash when the latter is taken u p in acid. Moreover, in the separation by distillation, the boron is isolated from substances which might interfere in its determination. I n its direct determination in a n ash solution, on the other hand, the variable constituents of the ash are present, and these may include oxidizing substances or other materials which can introduce an error into the colorimetric measurement without making their presence known. The three reagents described can be adapted to the colorimetric measurement of boron following other already existing procedures for its separation of preliminary treatment of sample material. Their sensitivities compare well
(1) Assoc. Offic. Agr. Chemists, “Official Methods of Analysis,” 7th ed., p.
39, 1950.
( 2 ) Cogbill, E. C., Yoe, J. H., Anal.
Chi7n. Acta 12, 455-63 (1955). (3) Ellis, G. H., Zook, E. G., Baudisch, O., ANAL. CHEM. 21, 1345-8
(1949). (4) Feigl, ,F., “Specific, Selective and Sensitive Reactions” (tr. by R. E. Oesper), p. 355, -4cademic Press, New York. 1949. (5) Grob, R. L.,‘Yoe, J. H., Anal. Chim. Acta 14, 259 (1956). (6) Job, P., Ann. chim. (lo), 9, 113 (1928). ( 7 ) Kenivorthy, A. L., private communication. t o be aiiblished in Proc. Am. Sbc. Hort. ‘sei. (8) PrlscDougsll, D., Biggs, D. A., ANAL.CHEY.24, 566-9 (1952). (9) Mathis, IT‘. T., J . Assoc. O j k . Agr. Chemists 35, 406-18 (1952). (10) Sandel!, E,. B., “Colorimetric Determination of Traces of Metals.”
RECEIVEDfor review March 25, 1956, ilccepted March 12, 1957.
