ANALYTICAL CHEMISTRY
566 titration should be avoided-f%r example, contact of the hands with the upper portion of the microburet may introduce a significant error. Changes of room temperature during titration are usually so slight that no error is introduced. It is estimated that the accuracy and precision of the procedures desciibed are within: A, 0.3%; B, 0.5%; C, 1 to2%. The above procedures constituted a general method for titration of very small amounts of organic bases. They should be particularly useful for the determination of equivalent weights of pure organic bases where only a few milligrams or a few tenths of a milligram are available. As a large choice of solvents is possible, it should be feasible t o separate bases by microextractiqn procedures and titrate the extract directly, Where preliminary separation is necessary, the fact that carlion diouide does not interfeie is important.
ACKNOWLEDGMENT
The authors wish t o thank the Research Corp. for a grant in partial support of this work. LITERATURE CITED
(1) Blumrich and Bandel, Angew. Chenz., 54, 374 (1941). (2) Conant a n d Hall, J . Am. Cheni. SOC.,49, 3047, 3062 (1927). (3) Fritz, ANAL.CHEM.,22, 589 (1950). (4) Zbid., p. 1028. (5) K a h a n e , Bull. soc. chim., 18, 92 (1951). (6) Markunas and Riddick, ANAL.CHEM.,23,337 (1951). (7) Nadeau a n d Brauchen, J . A m . Chem. SOC.,57, 1363 (1935). (8) Niederl a n d Xiederl, “Micromethods of Quantitative Organic Analysis,” New York. John \Tiley & Sons, 1942. (9) Pifer and Tollish, A x ~ LCHmr., . 24. 519 (1952). R E C L I Y Efor D review J u n e 17, 1961.
Accepted October 17. 1951
Estimation of Boron in Plant Tissue Modification of Quinalizarin Method DANIEL MACDOUGALL AND D. .A. BIGGS Ontario Agricicltural College, Guelph, Ontario, Canada The organic reagent, quinalizarin, has been widely used for the colorimetric estimation of minute quantities of boron. The method has several objectionable features. The effective concentration range is small, the reagent in 98% sulfuric acid is cumbersome to prepare, store, and dispense, and small changes in moisture content affect the sensitivity of the reagent. By increasing the concentration of quinalizarin in 9 8 q ~sulfuric acid from 5 to 20 mg. per liter the effective range of the method was increased from 0-2 to 0-8 micrograms. With a concentration of 45 mg. of quinalizarin per liter, ordinary concentrated sulfuric acid was substituted for 98% acid without appreciably changing the range or sensitivity of the method. Small changes in moisture content have no effect on results with the latter reagent. Relationships of temperature, time of standing after mixing, and possible interfering ions to the amount of color developed have been investigated.
REAGENTS
Sulfuric acid, 0.36 S, 10 ml. of concentrated acid diluted to 1 liter (I). Calcium hydroxide, saturated solution. Sulfuric acid, 98%, prepared by mixing appropriate quantities of Nichols’ reagent grade fuming and concentrated acids. The mixture was standardized by the Berger and Tmog ( 2 ) method.
tion
T””
importance of boron in plant nutrition makes a simple method for its determination desirable. The nidely accurate ‘ used .4OAC quinalizarin method (1) has several objectionable features. First, the effective range of the determination (0 to 2 microgiams) is much smaller than the boron concentration range in many plant materials. Therefore, predetermination of the approximate boron level is necessary, so that sample weights may be selected t o give proper colorimeter readings. Secondly, the 98% sulfuric acid required is rather troublesome t o prepare, use, and store. As the percentage of moisture in the reagent is critical ( 2 ) , precautions must he taken to protert it from the atmosphere, It has been claimed ( 7 ) that for maximum sensitivity a final acid concentration of 93% is required. A new calibration curve must be prepared for each batch of reagent. It is obvious that the usefulness and efficiency of the method would be greatly enhanced if the effective concentration range were increased, the use of fuming sulfuric acid avoided, and the critical effect of moisture eliminated. This study was undertaken with these points in mind. q,
Rea
Figure 1. Apparatus for Dispensing Quinalizarin Reagent
V O L U M E 2 4 , NO. 3, M A R C H 1 9 5 2
561
Quinalizarin solutions. Eastman's quinalizarin ( S o . 2787) was used in all studies described. Standard boron solution, 2.857 grams of reagent grade boric acid per liter of water (500 micrograms of boron per ml.). Working standards were prepared by further dilution with water. APPARATUS
Transmittancy measurements were made with an Evelyn photoelectric colorimeter, using filter 620. Colorimeter tubes were calibrated with a solution of the boron-quinalizarin complex. Spectral absorpt,ion curves were determined on a Beckman Model DU spectrophotometer, using 1.000-em. cells. The reagent was dispensed from the apparatus shown in Figure 1. Reagent was allowed to drain freely until movement of the meniscus in the delivery tip stopped. T o further drainage time was allowed. The average deviation of the volume delivered from this apparatus, as determined gravimetrically, was 0.00076 ml. Boron-free glassware was used for storage of standard solutions. Soft glass was found to be satisfactory for all other glass\mre.
concentration in 96% acid is small. In addition, thc vurvr.- obt'ained are similar in both range and sensitivity to that obtained with 20 mg. per liter of 98% acid. Olson and DeTurk (a) have reported a lower sensitivity when 96% acid is used. This effect is apparently offset,here l)y the increased quinalizarin ?oncentration. These results suggested a study of factors aflecting the amount of color developed under these conditions. As the reagent containing 45 mg. per liter of 96% sulfuric acid offeixd promise, the effects of small changes in acid concentratioii, time of standing, temperature of color development, and iuterferiiig ions were investigated n-ith this reagent. looh
1
EXPERIMENTAL
Effect of Quinalizarin Concentration. The effect of increasing quinalizarin concentration on the color developed was studied by preparing standard curves using 98YGsulfuric acid containing 5 and 20 micrograms of quinalizarin per liter. The results in Figure 2 indicate that a fourfold increase in the quinalizarin conwntration raises the maximum amount of boron determinable from 2 to 8 micrograms. The change in percentage transmittance per unit change in boron concentration is greater a t the higher quinalizarin level.
2080
-
3 60 W 0
z
e
-
r wl 2 4
a e
40
Table I. Effect of 1% Decrease in tcid Concentration on -4pparent Boron Content of Standard Solutions
20
Boron Found, ______ 96% acid
-
0 75 1 50 3 00 4 50 6 00
7 50 9 00
L
l
C
l
l
2
0
l
I
l
l
l
l
l
6
4
MICROGRAMS
Figure 2.
l
l
t
l
l
8
95% acid 0 70 1 48 3 00 4 50 5 94
7 60 9 00
Deviation
-0 05 -0 0 0 -0
+o
02 00 00 06
10 0 00
_ -
BORON
Effect of Increasing Quinalizarin Concentration in 9870 Sulfuric Acid A.
y_____
5 mg. of quinalizarin per liter
B. 20 m g . of quinalizarin per liter
Effect of Acid Concentration. The results of increasing quinalizarin concentration suggested a study of the reaction with still greater amounts of quinalizarin in weaker acid. Accordingly, solutions were prepared cdntaining 25, 3 5 and 45 mg. of quinalizarin per liter of reagent grade sulfuric acid (approximately 96%). The amount of color developed by each of these solutions with known amounts of boron was determined. The results in Figure 3 show t h a t in the range checked the effect of quinalizarin
Effect of 1% Change in Acid Concentration. Sufficient \\-atcr was added to reagent grade sulfuric acid to lower the acid concentration by 1%. After 45 mg. of quinalizarin had been dissolved in this acid, analyses were carried out on a series of solutions of known boron concentration. The values obtaintd i v ~ ~ r ( ~ calculated from a curve prepared with reagent grade acid. The results in Table I show t8hat decreasing the concentration of reagent grade sulfuric acid by 1% has no effect on the amount of color developed with boron. Small variations in the concentration of reagent grade sulfuric acid will not affect the results ohtained by this method. Spectral Characteristics. The spectral characteristics of the reagent and the boron complex ir-ere determined by transmittance
ANALYTICAL CHEMISTRY
568
measured a t IO-rnM wavelength intervals from 400 to 800 mp. solved in 15 ml. of acid and 1 ml. of this solution is used for the determination. Therefore 2.5 micrograms per ml. would correA blank determination and one containing 10 micrograms of spond to 37.5 p.p.m. in the original material. This is well above boron were prepared, and each was compared with sulfuric acid the concentration of fluoride found in moat plant-s (3). At higher of the same strength. The values shown in Figure 4 are based on fluoride concentrations the amounts of boron found are de100% transmittance for the sulfuric acid solution. This figure creased slightly. shows that the usable portion of the spectrum is from wavelength 600 to 700 mp. Determination in Plant Material. One-gram samples of dry In Figure 5 the absorption of the boron-quinaliearin complex is ground plant material were t,reated with 5 ml. of saturated calillustrated. The absorption percentages shown are those for solutions containing 10 micrograms of boron when the absorption of the reagent is zero. Curves have been prepared in this way for the reagent containing 45 mg. of quinalizarin in 96% sulfuric acid, and 20 mg. of quinalizarin in 98% acid. The peak of both absorption curvw occurs about wave length 620 mp. Thus filter 620 was used for measurements with the Evelyn photoelectric colorimeter. Comparison of these two curves shows that the reagent prepared in 96% sulfuric acid is more sensitive than that prepared in 98% acid. Temperature of Color Development. This effect was studied by comparing results of determinations carried out a t 70°, 85", and 100' F. The values for 85' and 100' F. were read from the calibration curve prepared from 70' F. data. The results in Table I1 indicate that temperature has a marked effect on the amount of color developed during 24 hours of standing. Less color is developed a t the higher temperatures. Time of Standing after Addition of Reagent. The effects of time of standing and rate of cooling on the amount of color developed were studied. '0 Samples which were cooled to room temperature WAVE L E N G T H I N M I L L I M I C R O N S by being placed in cold water for 15 minutes Figure 4. Spectral Characteristics of Final Solution Using Proposed could be read immediately. There wae a further Reagent decreme in transmittance of about 1% during the 45 mg. of quinalizarin per liter of 96% sulfuric acid 4 hours following. When samples were allowed No boron present 10 y of boron present t o cool to room temperature in air, the color increased appreciably for the first 6 hours and then continued to darken very slowly. There w a a~ decrease in transmittance of approximately 1% during the 6- to 24hour period and a similar decrease from 24 to 72 hours. Satisfactory readings were obtained after standing for 6 hours when the mmples were air cooled and in 20 minutes after being water cooled. I n either case the time between addition of the reagent and reading should be standardized. In this study samples were read after cooling in air and standing for 24 hours. Effect of Interfering Ions. Analyses showed that results by the modified procedure check with those obtaiiicd when 9SUG sulfuric acid is used. Thus only the ions known to interfere with the quinalizerin determination @)-nitrate, dichromate, and fluoride-were investigated. Kitrate will be destroyed during ashing ( 2 ) and chromium occurs only in very small traces in plant material (4, 6). The effect of fluoride was investigated at two boron con580 600 620 640 660 680 centrations (Table 111). A concentration WAVE L E N G T H I N MILLIMICRON$ of 2.5 micrograms of fluoride per ml. of Figure 5. Comparison of Absorption of Boron-Quinalizarin Complex with solution does not interfere with the estimaDifferent Reagents tion of boron a t either of the levels 10 y of boron checked. I n the procedure outlined the , 0 45 mg. of quinalizarin per liter of 96% sulfuric acid ash from 1 gram of plant inaterial is disA 20 mg. of quinalizarin per liter of 98% sulfuric acid
-- - ---
V O L U M E 24, NO. 3, M A R C H 1 9 5 2
569
Table 11. Effect of Temperature of Color Development on Apparent Boron Content of Standard Solutions Boron Found, y/Ml.O
Boron Added, y/hfl.
85' F.
100' F.
1.50 3.00 4.50 6.00
7.50 3.00
the same manner as the sample solution but with the plant material omitted, the percentage trunsmittmce of the sample solutions waa determined. Recoveries were run with known amounts of boron added t o alsike clover. The results in Table IV show that the method is satisfactory for determination of microgram quantities in plant material. The method has been applied succesfifuIlyto the estimation of boron in soil extracts and water.
Values calculated from calibration curve prepared a t 70" F. DISCUSSION
Table 111. Effect of Fluoride on Apparent Boron Content of Standard Solutions Fluoride Added, 2 5 5.0 7.5 10.0
Table 1V.
60
so
y
7.5 y boron added 7.50 7.35 7.25 7.00
Recovery of Boron Added to Alsike Clover
Boron .4dded,
0 20 '3 0
Boron Found, 2.5 y boron added 2.48 2.48 2.42 2 40
7
Recovery of Added Boron,
Boron Found, Y
%
13.2 33.4 52.5 72.9 33 7
101.0 98.5 99.5 100.5
The calibration curve for the proposed method is shown in Figure 3, C. The photometric accuracy, evaluated from the slope of the curve, varies between 90 and 30% transmittance from 2.4 t o 0.6% relative error for a 0.270 absolute photometric error. Maximum accuracy occurs at a boron level of about 7 micrograms. although the accuracy is e~sent~ially i ~ good s in the range from 2 t o 8 micrograms. In comparison wit'h the widely used method with 98% sulfuril, acid, the proposed method has t'he advantages of greater rangc: and greater sensitivity. I n addition the reagent is easier to prepare and dispense, and the color developed is less sensitive to small changes in acid concentration, LITERATURE CITED
(1) h m ~ c Offic. . Agr. Chcmists, "Official Methods of Analysis," 7th ed., p. 117, 1950. ( 2 ) Berger, K. C., and Truog, E., 1x0. EXQ.CHEM.,ANAL.ED., 11,
cium hydroxide solution and dried a t 105" C. After the volatile matter had been carefully driven off over a burner, samples were ashed for 1 hour a t 600' C. Experiments showed that 1 0 0 ~ o recovery of boron was obtained by this method. Exactly 15 ml. of 0.36 S sulfuric acid were added t o the ash and the resultant solution was filtered. One milliliter of the filtrate wm transferred to a comparator tube and an exact uantity (approximately 10 ml.) of quinalizarin reagent was a d d e l The tube was stoppered, and the contents were well mixed and allowed to stand for 24 hours a t room temperature. .kfter the colorimeter had been adjusted to 100% transmittance with a blank solution prepared in
540 (1939). (3) IIaohle, W., Scott, E. IT.,and Treon, J., Am. J . HYQ., 29A, 13S-45 (1939). 14) Mitchell, R. L., Proc. Nutrition Soc., 1, 183 (1944). (5) Olson, L. C., and DeTurk, E. E., Soil Sci., 50, 257 (1940). (6) Saint-Rat, L. de, Compt. rend., 227, 150-2 (1948). (7) Smith, G. G., Analyst, 60,735 (1035). RECEIVED for review July 11, 1351. Acaepted October 25, 1951. Presented to the Division of Agricultural Chemistry and Food Technology of the Chemical Institute of Canada a t the annual conference, Winnipeg, June 19.51.
Separation of Organic Insecticides from Plant and Animal Tissues LAWRENCE R. JONES
0""
AND
JOHN A. RIDDICK, Commercial Solvents Corp., Terre Haute, Znd.
of the most difficult analytical problems encountered in insecticide residue, penetration, and translocation studies on plants, and toxicity studies on animals, is the isolation of the insecticide from interfering biological substances. The methods for analyzing the insecticides per se generally are sufficiently sensitive and accurate for microgram quantities. There are specific methods for isolating microgram quantities of some insecticides from biological samples (&?'), (9, IO),but they usually are long and complicated. A general method that is simple and rapid for isolating insecticides from fats, waxes, and other biological substances should find wide application. The primary purpose of this study was the isolation of Dilan [an insecticide containing a mixture of 2-nitro-l,l-bis(p-chloropheny1)propane and 2-nitro-l,l-bis(p-chlorophenyl)butane ] from the several types of interfering biological materials. However, the method was extended to other insecticides to show its general applicability. The original method for Dilan (9),based on a simple n-hexane extraction, was modified to include an additional extraction step. It was found that acetonitrile Selectively extracted insecticides and many other organic compounds from n+
hexane. Other solvent pairs should also be satisfactory. However, they should meet the following requirements: The insecticide ahould be soluble in both solvents, but have a solubility preference for one of them. The extracted biological material should have a solubility preference for the other solvent. The two solvents should be mutually insoluble and low boiling. DISTRIBUTION OF INSECTICIDES BETWEEN n-HEXANE AND ACETONITRILE
The distribution between the solventa was determined for the followinginsecticides:
++%%,(9).1,2,3,4,5,6-hexrtchlorocyclohexane
Dilan, standard, 99 Lindane, standard 99 (19). \ - ' - I .
Chlordan, purifled technical grade, John Powell, Inc., and Velsicol Corp. Parathion standard, 99.8%, American Cyanamid Co. Methoxychlor, technical grade, 90.0%, E. I. du Pont de Nemours & Co., I n c DDT, technical grade, 90.4% p,p' isomer, Eimer and Amend,
