418
ANALYTICAL CHEMISTRY
The lower limit of bubble frequency that: can be measured by this method is set by the least sweep frequency of the oscilloscope used, and by the image-retention time of the oscilloscope screen The DuMont 208-B oscilloscope has available sweep frequencies down to 2 cycles per second, but its screen does not have s u k cient persistence for frequency measurement below 4 cycles per second. A special cathode-ray tube can be obtained with longer persistence, but it is simpler to determine bubble frequencies below 4 cycles per second by actual bubble count. The upper limit of frequency measurement depends on the nature of the percussive trace, which is in turn a function of the components of the bubble noise. I n general, a high-pitched bubble sound permits the measurrment of higher bubble frequencies, because at the higher sweep frequencies required, the vertical displacement of the electron beam remains well-defined. Obviously, any sweep frequency above the lower audible limit of 20 cycles per second would obliterate the lower-toned components of the bubble shock. The pitch of the bubble sound is dependent on the size and shape of the reaction vessel, the properties of the liquid, and the size and thickness of the orifice device. Preliniinary consideration of the effect of these factors on hubblr sounds should yield a design suitable for the application of thii technique. The electronic technique has been applied in a study of bubble "twinning"-that is, the formation of a bubble pair per gas emission from the orifice. This phenomenon was earlier notcd bv Budge ( I ) , who attributed i t t o too large avolumeof gas between the orifice and the flow control valve. This particular bubble habit is easily followed on the oscilloscope. since the stntion:irv
pattern for pair formation consists of two regions of peak amplitude spaced closely together, followed by a comparatively long region of decreasing amplitude. This pattern is distinguishable from that of single-bubble formation at a bubble frequency twice the sweep frequency, because the latter gives two widely separated peak-amplitude regions. Studies of the twinning process with the oscilloscope indicates that a t low flows, bubbles of approximately equal size arc formed, while at higher gas rates, tlisproportionation takes placc. ACKNOWLEDGMENT
The interest and encouragement of the Monsanto Chemical Go., Texas City Division, are gratefully acknowledged. LITEH4TIJRE CITED
(1) Budge, E. A., J . Ant. Che7n. SOC.,53, 2451-3 (1931).
(2) Datta, R. L., Kapier, D. H., and Newitt, D. M., Trans. Inat. Chem. Engrs. ( L o n d o n ) , 28, 14-26 (1950). (3) Davidson, L., Ph.D. thesis i n chemical engineering, Columbia Universitv. New York. 1951. (4) Eversole, W.-G., Wagner, G. H., and Stackhouse, E., I n d Eng. Chem., 33, 1459-62 (1941). (5) Maier, C. G., U. S. Bur. Nines, Bull. 260 (1927). (6) Pattle, R. E., Trans. Inst. Chern. Engrs. (London),28, 32-i (1950). (7) Remy, H., and Seemann, W., Kolloid-Z., 72, 3-12, 279-91 (1935). (8) Rider, J. F., and Uslan, 8. D., "Encyclopedia on Cathode-Ray Oscilloscopes and Their Uses," pp. 427-75, New York. John F. Rider Publisher, Inc., 1950. RECEIIED for review July 31, 1053. Accepted October 30, 1953
Boron Determination in Soils and Plants Simplified Curcumin Procedure W. T. DIBLE', EMlL TRUOG, and K. C. BERGER University o f Wisconsin, Madison,
Wis.
F
OR the determination of boron in soils and plants, t v o colorimetric procedures are commonly used. One is based on n change in color from pink to blue when quinalizarin reacts with boric acid in concentrated sulfuric acid. This procedure, conimonly called the quinalizarin method, is widely used and has been described in several places (1, 2 ) . It gives reliable results, but has a disadvantage in that the color must be developed in very strong sulfuric acid, the strength of which must be carefully controlled. Recently hIacDougall and Biggs (9) have shown that by increasing the concentration of the quinalizarin, ordinary reagent grade sulfuric acid may be used, with a permissible variation of 1 % in strength. This partially overcomes the disadvantage mentioned. The other procedure is based on the rose-colored rosocyanine produced when an acid borate solution containing curcumin is evaporated to dryness. I t is commonly called the curcumin method, and was adapted to plant and soil analysis by Saftel (1f). Curcumin, 1,T-bis (.i-hydrosy-3-niethoxyphenyl)-1,6-heptadiene-3,5-dione, is a natural dyestuff obtained either from the rhizomes of Czcrc~c7mtznctora or by synthesis. Upon evitporation to dryness of an acidified solution containing boric acid and curcumin, a red reaction product soluble in alcohol is formed in amount proportional to the amount of boric acid present. This product has been identified by Schlumberger (13) as an isomeric form of curcumin and was named rosocyanine for the rose color of the acid form and the blue color of its metallic salt. The 1 Present
address, Potash Division, International hfineritls and Chemical Corp., Chicago, Ill.
mechanism of the reaction has not been clearly defined, hut Hafford ( 7 ) suggests that the rosocyanine is probably formed by a loose combination of the boratc with one of the hydrouyl groupfi of the curcumin molecule. Cassel and Gerrans (3') first outlined a colorimetric method in which a solution of oxalic acid, curcumin, and boric arid is evaporated to a dry residue, which is then taken up with ethyl alcohol. The steps involved w r c numerous and tedious, : i d Saftcl (11) and later Hafford ( 7 ) refined the method by improving the color development technique and adopting propos:tls by Gooch (6), whereby solutions containing boron are conc(mtrated by evaporation to dryness in the presence of excesh c-:rlcium hydroxide to prevent volatilization of boric acid. The curcumin method has an advantage over the quinalimrin method in that thp color reaction in the former is more sensitive to small amounts of boron and proceeds without the use of a strongly corrosive reagent like strong sulfuric acid; its disadvantage as commonly carried out lies in a multiplicity of timeconsuming evaporations and filtrations. Accordingly, the possibility of simplifying the curcumin method for plant and soil analysis was investigated. and a simplified procedure was evolved which requires 0111)- one evaporation and filtration after a water solution of the test sample is a t hand. Details of this procedure, comparative results obtained with i t and the quinalizarin procedure, and related matters are here presented. REAGEVTS
Standard Boron Stock Solutions. Dissolve 2.8578 grams of boric acid (reagent grade) in 1000 ml. of distilled water. This
419
V O L U M E 26, NO. 2, F E B R U A R Y 1 9 5 4 solution contains 0.5 mg. of horoii per ml. and serves as the primary base stock solution, A. Dilute 20 ml. of A to 1000 mi. with distilled water, giving standard solution B containing 0.01 mg. of boron per ml. Dilute 100 ml. of B to 1000 ml. with distilled water, giving standard solution C containing 0.001 nig. of boron per ml. Curcumin-Oxalic Acid Solution. Dissolve 0.04 gram of finely ground curcumin (Eastman Kodak No. 1179) and 5 grams of oxalic acid in 100 ml. of 95% ethvl alcohol. When stored in a cool dark place, the reagent-will "keep satisfactorily for several days; this may be lengthened to a week or more when storage is in an ice box without undue exposure to room temperature. Ethyl Alcohol. Use a good grade of 957, strength.
Table 11. Comparison of Curcumin and Quinalizarin Procedures in Determining Total Boron Content of Four Soils Boron Found. Curcumin Procedure A B Av. 47.5 47.6 47.4 3.5 3.6 3.4 51.2 52.4 50.0 34.8 35.2 34.0
~
COLOR DEVELOPMENT PROCEDURE
Having obtained from the sample under test a water solution of the boron to be determined, place a 1-ml. aliquot of this solution, containing from 0.0 to 2.07 of boron, in a 250-ml. beaker (boron-free glass). Add 4 ml. of the curcumin-oxalic acid solution and mix thoroughly by rotating the beaker. Evaporate on a water bath a t 55' rt 3" C., and then continue to bake the residue at this same temperature for a minimum of 15 minutes to ensure complete dryness. The importance of temperature control to within 5 3 ' C. mas indicated by Hafford ( 7 )and Naftel (11). Cool and then treat the reaction products n i t h 25 ml. of 95% ethyl alcohol. Filter directly into a comparison tube through a close-textured filter (Whatman S o . 2 is satisfactory), centrifuge, or allow to settle until clear. Record colorinieter transmittance readings using the appropriate filter (usually 540 nig) and determine the boron (soncentration of the unknown by reference to a standard curve prepared by using appropriate aniounts of standard stock solutions. DETERMINATION OF AVAILABLE SOIL BORON
Place 10 grams of soil, air-dried and ao-meshed, in a 125-ml. Florence flask (boron-free glass), add 20 ml. of distilled water, attach a reflux condenser, and boil for 5 minutes [see Berger and Truog ( 2 ) for basis of this extraction]. Disconnect the condenser and filter or centrifuge the suspension so as to obtain a clear extract. Clarification may be facilitated by the addition of not more than 0.02 gram of calcium chloride dihydrate. (Evaporation and ignition a t this point to destroy traces of organic matter are not generally necessary as under the quinaliznrin procedure.) Take a 1-ml. aliquot and proceed as outlined under color development procedure
Table I. Comparison of Curcumin and Quinalizarin Procedures in Determining Available Boron of Twelve Soils soli
Sample so.
8 9 10 11 12
.4vailable Boron Found, P.P.M. Quinalizarin procedure, n AV. a\-. of two 1.205 1.20 1.20 0.725 0.70 0.80 0.745 0.80 0.77 0.415 0.45 0.41 0.755 0.75 0.78 0.58 0.55 0.68 0.885 0.85 0.89 1 .oo 1.02 1.01 1.05 1.04 1.05 1 .oo 1 .oo 0.98 3.94 3.90 3.78 6.04 5 995 :. 9 3
Curcumin Procedure
A 1.21 0.75 0.72 0.42 0.73 0.58 0.88 1.00 1.03 1 .oo 3.86 5.05,
-
In order that results obtained with the curcumin procedure might be compared to those obtained with the regular quinalizarin procedure, water extractions of twelve soils were made for available boron by the method just described. The amounts of boron in aliquots of these extracts were then determined by means of the two procedures. The quinalizarin procedure followed is the same as t h a t described by Berger and Truog ( 2 ) , except that the concentration of quinalizarin in the quinalixarin-sulfuric acid reagent used was increased from 5 to 25 mg. per liter so as to increase sensitivity. Results are given in Table I. The rcsulB obtained by the two methods are practically the same.
So11 Type
hliami silt loam Plainfield sand Superiorqlayloam Parr silt loam
P.P.M. Quinalizarin P r o c e d u r e A B Av. 46.0 16.2 46.4 3.S 3.5 3.5 46.4 47.2 48.0 33.2 33.2 33.2
Table 111. Comparison of Curcumin and Quinalizarin Procedures in Determining Boron in Alfalfa and Timothy Hay Follow-ing Ashing o f Tissue Specie* of Plant Alfalfa Tirnot ti). 1
2 3
Boron in Oven-Dry Plant Tissue, P.P.LI. Curcumin Proredure Quinalizarin Procedure A B Av. A B AV. 28.0 28.4 28.2 28.0 30.0 29.0 7.8 8.5 8.5
8.5 9.2 8.7
8,l5 8.85 8.6
98 . 5
9 .. 00
98 . 27 5
9.0
9.0
9.0
The small differences that exist are no greater than those tha occur between duplicate results by the same procedure. DETERMINATION OF TOTAL BORON I N SOILS
Fuse 0.5 gram of soil with 3 grams of anhydrous sodium carbonate in a platinum crucible. Cool and place the crucible in a 250-ml. beaker containing about 50 ml. of distilled water. Place a cover glass on the beaker and add approximately 4N sulfuric acid from time to time until the melt has disintegrated and the solution has a reaction in the range of pH 6.0 to 6.8. Transfer the resulting solution to a 500-ml. volumetric flask. Wash the beaker and crucible several times with distilled water and add the washings to the flask. The total volume of solution now should not exceed 150 ml. Add ethyl alcohol to the flask until a volume of nearly 500 inl. is reached, and mix the contents thoroughly. Then add just sufficient sodium carbonate to make the solution slightly alkaline, and bring up to full volume with alcohol. Filter the solution or centrifuge until the supernatant liquid is clear. Place a 400-ml. aliquot of the clear solution in a 500-ml. beaker (boron-free glass) and add about 100 ml. of distilled water to prevent subsequent precipitation. Evaporate to a small volume, and transfer to a platinum dish. Evaporate to dryness and ignite just enough to destroy organic matter. After cooling, add 5 ml. of 0.10S hydrochloric acid, and triturate thoroughly ivith a policeman. Take a 1-ml. aliquot of this solution and proceed as directed under Color Development Procedure. With the exception of several minor modifications, the method just described for obtaining a water solution of the total boron in n soil is the same as that previously described by Berger and Truog ( 1 ) . For purposes of comparison and checking, the total boron content of four soils was determined by application of the curcumin and quinalixarin procedures following procurement of water solutions of the boron as directed above. Results given in Table I1 confirm the reliability of the curcumin procedure for the purpose indicated. DETERMINATION OF TOT4L BORON IN PLANT TISSUE
Berger and Truog ( 1 ) have shown that vegetative tissues of plants contain sufficient bases to prevent the loss of boron during ashing. Results of tests by RIcHargue and Hodgkiss (10) and Hafford ( 7 ) substantiate this. I n the case of seeds, particularly those of an oily nature, i t may be advisable to add a base like calcium hydroxide prior to ashing. I n the procedure for vegetative plant material, no base is added. Place a 0.25- to 0.50-gram sample of plant material, ovendried and ground, in a porcelain or quartz crucible or dish and ash in a muffle furnace a t 550" C. or over an open flame to a gray-white ash. Dissolve the ash in 5 ml. of 0.10K hydrochloric acid and dilute nith distilled water to a definite volume, usually 10 to 20 ml., so as to come within the range of the stand-
ANALYTICAL CHEMISTRY
420 ard curve used. Take a 1-ml. aliquot of the clear solution and proceed as indicated under Color Development Procedure. I n Table 111, the total boron contents found on analysis of alfalfa and timothy hay tissue by means of the curcumin procedure are compared with those obtained with the quinalizarin procedure described by Berger and Truog ( 2 ) . Here, as in the case of soils, results by the two procedures appear to be of equal precision. SPECTRAL CHARACTEKISTICS OF CURCUMIh AND ROSOCYANINE SOLUTIONS
Curcumin dissolved in ethyl alcohol gives a solution t h a t is strongly yellow in color, while the product of the curcuminborate reaction, rosocyanine, is of a deep red color, giving the test solution a reddish hue. To show that curcumin and rosocyanine are spectrally separable, the absorption curve of each of the two pure substances dissolved in ethyl alcohol was determined by means of the Beckman spectrophotometer (Model DU). The rosocyanine used was synthesized according to the procedure of Clark and Jackson ( 4 ) .
1.c
i
permanganate, nitrite, and chlorate prevent or retard the reaction of curcumin with boric acid. Fortunately, none of the above interfering substances are ordinarily present in sufficient quantities in water extracts of soil or in plant ash to cause difficulty. Yoe and Sarver ( 1 4 ) state that beryllium, aluminum, iron, and magnesium form color lakes with curcumin. Kolthoff (8) has shown that these lake formations take place in alkaline solutions. The excess of oxalic acid present in the regular curcumin procedure for boron prevents by solution the formation of these interfering lakes. Schafer (12) has indicated that the presence of fluorides decreases the sensitivity of the reaction. but such presence in appreciable concentrations is rare in n a t e r extracts of soils because of the insolubility of calcium fluoride. K t r a t e s are known to interfere n i t h the curcumin-boron color reaction, and because nitrates are often present in a a t e r extracts of soils in significant amounts, their interference n-as investigated in solutions containing successive increments of nitrate nitrogen and known amounts of boron. The results obtained are presented in Table 1iT,and show that there is no detectahle interference when 20 p.p.m. or less of nitrate nitrogen is present, but a t 40 p.p.m. interference is appreciable. I n the regular procedure, this 20 p,p.m, mould correspond to 40 pounds of nitrate nitrogen per acre in the plow layer. Since soils generally do not contain this amount of nitrate, interference from this source will be rare, but should he guarded against. Sitrates may be eliminated from soil extracts by addition of 2 ml. of
100
80 70
I
I
1
I
I
I
i
-
60-
Ti= $ 2
WAVLLLNeTR , My
C
Y
The results obtained are given in Figure 1, where it will be noted that the absorption peak for rosocyanine falls a t approximately 540 mp, xhile that for curcumin is less than 480 mp. Curcumin does not absorb light over the range of maximum absorption of rosocyanine, thus eliminating any absorption effect due to excess of reagent in the determination of boron.
I n Figure 2 is presented a standard curcumin reference curve prepared from colorimeter transmittance readings obtained with the Evelyn colorimeter equipped with a Corning 540 mp filter when solutions of known boron content were used. The curve in the useful range is a straight line-that is, it follows Beer’s law. If less sensitivity is desired, as may be the case with plant tissue analysis, a similar curve can be constructed using a 580- or 600mp filter. I n the construction of the curves. care must be taken to develop the colors in the standard solutions in exactly the same manner as is done in the case of the unknown test solutions.
40-
I-
Figure 1. Spectral Characteristics of Curcumin and Rosocyanine Dissolved in Ethyl Alcohol
STANDARD REFERENCE CURVES
50-
20 00
02
0.6 Of
08
12
1.0
BORON
Figure 2. Standard Curcumin Reference Curve for Determination of Boron Micrograms of boron indicated give concentrations per ml. in original standard solutions which were diluted
25 times i n alcohol solutions on which photoelectric
readings were taken
Table IV.
Effect of Nitrates on Curcumin-Boric Acid Color Reaction
(As indicated by galvanometer deflections of photoelectric colorimeter) R n - n..r n ._
Present in Solution, P.P.M.
Kitrogen Present as Calcium Nitrate, P.P.M. 0.0 5.0 10.0 20.0 40.0 50.0
INTERFERING SUBSTANCES
Feigl ( 5 ) states t h a t in a n acid solution ferric iron, molybdenum, titanium, columbium, tantalum, and zirconium give a red-brown color with curcumin when present in sufficient amounts, and thus interfere with the color produced by boron. He also indicated that oxidizing agents such as peroxide, chromate,
0.4 MICROeRAMS
Galvanometer Deflection Readingsa 0.0
0.5 I .o
100.0 58.0 34.0
100.0 57.5 34.0
99.7 57.7 33.0
99.0
58.0 33.5
100.0 61.0 37.0
99.5 63.0 43.0
a One galvanometer scale division is equivalent t o a p roximately 0 . 0 1 ~ of boron in range of 34 to 63 galvanometer deflection reazngs.
V O L U M E 26, N O . 2, F E B R U A R Y 1954
421
saturated water solution of calcium hydroxide to a 10- to 20-ml. aliquot of test solution, followed by evaporation and gentle ignition. The residue is then dissolved in 10 ml. of 0.05N hydrochloric acid, after which the curcumin reagent is added, and the regular procedure followed. PRECAUTIONS
Grmt care must be exercised to prevent introduction into the test sample of appreciable amounts of baron via the chemicals, filter paper, and glassware used, or via dust, horon-containing fumes, and the operator's hands. Glassware made of Corning alkali-resistant glass No. 728 has proved to be m t i b factory. Storage of reagents in vessels of ordinary soft glass has not usually caused any difficulty. Ordinary C.P. and analytical grade chemicals h a w usually been satisfactory, b u t should be tested by means of a blank determination. The cureumin-oxalic acid reagent decomposes rapidly upon standing in direct light, b u t if stored as directed under "Reagents" will keep for a considerable period. Rosacyanine slowly hydrolyms to ourcumin; hence, all colorimetric readings should be made within 2 hours after solution of the color residue in alcohol.
LITERATURE CITED
Berger, X. C., and Truog, E.. IND. Exo. CHFM.,ANAL.E;..
11, 540 (1939). Berger, K. C., m d Truog, E.,Soil Sei., 57, 25 (1944). Cassel, C. E., and Gerrans, H., Chem. News, 87, 27 (1903). Clark, L.,and Jaokson, C. L.. Am. C h a . J . , 39, 696 (1908). Feigl, F., trans. by J. W. Matthew, "Spat Tests," p. 211, New York. Nordemann Publishine Co.. 1937. Gooch, F. A,, Am. Chem. J . , 9:23 (1887). Hafford, B. C..Ph.D. thesis, University of Wisoonsin, 1942. Kolthoff, I. M., J . Am. C h a . Soc., 50, 393 (1928). MatDougall, D.,and Biggs, D. A,, ANAL. CHEM.,24, 566 (1952). McIiargue, J. S.. and Hodgkiss, W. S., J . Assoc. OBc. A v . Chemists, 24, 518 (1941). Naftel, J. A,, IND. END.C n m . . ANAL.ED., 11, 407 (1939). Schiifer, H..Z.and. C h m . , 110, 11 (1937). Sehlumberger. M.E., Bull. SOC. ehim., 5, 11, 194 (1866). Yoe, . I . IT., and Sarver, L. A,, "Organic Analytical Reagents," p. 133, New York, John Wiley & Sona, 1941. Received for review November 7, 1951. Accepted October 2, 1953. Published with t h e permission of t h e director of t h e Wisconsin Aprioulturai Experiment Station. Work was supported in p a r t b y a fellomhip grant from t h e Paoific Coset Borar Co. a n d represents for t h e mort p m t a portion of a theais submitted b y t h e ~ e n i o rauthor in p & r t i d fulfillment oi t h e requirements for t h e degree of doctor of philosophy, University of Wisoonsin. The m t h o i r are indebted to L. C. W'ang for making t h e determinations of total boron in eoils reported in Table 11.
ptical and Crystallographic Properties of Socrose Memiheptakydrate FRANCIS T. JONES and FRANK E. YOUNG Westem Regional Research Laboratory, Albany, Calif.
A
N INCOMPLETE description of ~ u e r o ~hemihephhydrate e (C,,H,OII. 31/* RO),including x-ray powder data, has been
given by Young and Jones in their paper on the sucrose-water phase diagram ( 1 ) . Crystals suitable for the measurement of optical properties have since been ohtained by slow growth a t -7,50 c, in aqueous only slightly (Figure 1). Refractive indices of the oils used for immersion were measured on an Abbe refractometer in the same cooling circuit. All values are for sodium light. The composition of these crystals was determined by immersing and partially melting them under an oil having the refractive index of an squeons sugar solution whose composition %vas t h a t of sucro8e bemiheptahydrate (theoretical 84.4% sucrose). The refractive index of the resulting melt matched the index of the
d
Table I. Optioal and Crystallographic Properties
of Sucrose Herniheptahydrate Crystal Morphology crystal system. Orthorhombic. Form and habit. Spherulitio awegates of needles or blades when g r o m rapidly, tabulsr laths or prismatic rods (Figure 1) when g r o m slowly. The sides and ends are usually bevele' Optical Properties Refmotive indices. (5893 A.; 1 1 O C.) = 1.5135 0 1.5190 + 0.0005, y = 1.5205 + 0.0005. 2E = 849, zV = 52,50 caled, frvOptical axial 2E, 2V = 55O odcd. from e. 8 , and y. Dispersion. (v > ?) week. Optical (-1. Acute = a' Fusion Data M.P. 27.8". Crystals regrow slowly if fragments me left for seed. Sucrose hemipentahydrate or anhydrous sucrme crystals are likely to grow a t temperatures in the vicinity of 20'. I_
____
oil within the equivalent of 10.3% sucrose, leaving no doubt that this was the hemiheptahydrate. The common view of a tabular crystal lying on a pinacoid face (Figure 1) shows a centered optic normal interference figure with the slow ray (y) crosswise and the fast ray (a)lengthwise. End views showing acute bisectrix interference figures were obtained by cutting pieces off the large (approximately 2 mm.) crystals with a razor blade and propping the pieces in proper position in narrow slots cut in 1-em. squares of glass cut from thin spectrograph plates or microscope slides. Table I gives the optical and crystallographic properties. LITERATURE CITED
F i g u r e 1.
Sucrose Herniheptahydrate lOOX
(1) Young, F. E., and Jones, F. T., J . Phus. & Colloid C h m . , 53,1334 (1949).
R s c ~ r n oforreview
July 13. 1953. AooeDted September 28, 1053.
