808
ANALYTICAL CHEMISTRY
from the separatory funnel a t a rate sufficient to cause bubbling in the absorption tube' but substantially no escape of unabsorbed isobutylene into the inverted graduate. This requires 20 to 30 minutes and about 20 ml. of unabsorbed gas (chiefly air displaced from the system) are collected in the graduate. The reaction tube is then disconnected, the temperature of the bath is raised to 125", and heating is continued for an additional hour. The hot reaction mixture is then poured into 40 ml. of water and shaken vigorously by hand to remove the acid and any unreacted phenol. Crystallization occurs soon after shaking is begun. The colorless needles so obtained are filtered off by suction, washed with 5 to 10 ml. of p-ater, and dried to constant weight in a vacuum desiccator. Using this procedure, 2.45 grams (76% yield) of product melting a t 89.6" to 97.2" were obtained. An additional 0.1 gram of product was obtained by rinsing the reaction tube and extracting the aqueous filtrate with petroleum ether. Recrystallization by solution in aqueous alkali and acidification with hydrochloric acid raised the melting point to 98-100" and this recrystallized product did not depress the melting point of an authentic sample of 4tert-butylphenol. EXTENSION TO PENTENES
The reaction may be extended to the pentenes, 2-methyl-1butene and 2-methyl-%butene. Thus, 1.56 grams of %methyl-1-butene were distilled into an absorption tube containing 2.0 grams of phenol and 0.2 gram of 75% sulfuric acid. The mixture was heated for 1 hour. a t 125' C. and poured into 20 ml. of water. There were thus obtained 2.60 grams of nearly colorless needles of I-tert-pentylphenol, melting point
82" to 90" C., which after two cr stallisations from petroleum ether melted a t 94" to 96" C. a n i d i d not depress the melting point of an authentic sample of Ptert-pentylphenol. Correspondingly, 1.7 grams of 2-methyl-2-butene reacted with 2.3 grams of phenol containing 0.2 gram of 7570 sulfuric acid and gave 2.74 grams of crude Ptert-pentylphenol, melting a t 83' to 91 '. After two crystallizations from petroleum ether, the purified product melted a t 94" to 96" C. Although these two pentenes give the same ters-pentylphenol, they may be readily differentiated from one another by their physical constants. Thus, 2-methyl-1-butene boils a t 31.0 C. and has n y 1.3777 (6),whereas 2-methyl-2-butene boils a t 38.4' C. and has n y 1.3878 (5). ACKNOWLEDGMENT
The authors are indebted to Clora B. Heath for checking the method and for testing its applicability to the synthetic mixtures. LITERATURE CITED
(1) Davis, H. S., J . Am. Chem. SOC.,50,2778(1928). (2) Hurd, C.D.,and Spence, L. U., Ibid., 51,3568(1929). (3) Ipatieff, W.N., and Solonina, A. A., J . Russ. Phys. Chem. SOC., 33,496 (1901); Brit. Abstracts, 82,I, 1 (1902). (4) Natelson, S.,J.Am. Chem. Soc., 56,1585 (1934). (5) Sherrill, M. L.,and Walter, G. F., Ibid., 58, 744 (1936). (6) Sidorenko, K. W.,J. Russ. Phys. Chem. SOC.,38, 965 (1906); Brit. Abstracts, 92,I , 270 (1907). RECEIVED September 10, 1948.
Chemical Estimation of Tetraethyl Pyrophosphate A. R. WREATH
AND
E. J. ZICKEFOOSE', Victor Chemical Works, Chicago Heights, Ill.
The biologically active tetraethyl pyrophosphate content of the technical grade product and of the so-called "hexaethyl tetraphosphate" can be determined by a method which involves the preferential hydrolysis of the accompanying polyphosphoric acid esters in a 25% aqueous acetone solution at room temperature and the absorption of these products on an Amberlite IR-4B resin, followed by the alkaline hydrolysis of the column effluent containing the tetraethyl pyrophosphate by excess standard base and back-titration with standard acid.
T
HE chemical estimation of tetraethyl pyrophosphate pre-
sented here was developed during an investigation of socalled "hexaethyl tetraphosphate" made by either the Schrader (4) or Woodstock (6) process. Hall and Jacobsen (3) have reviewed the properties and preparation of hexaethyl tetraphosphate and of tetraethyl pyrophosphate and have concluded that the latter is the biologically active constituent. Toy has recently presented a new method for the preparation of several tetraalkyl pyrophosphates including tetraethyl pyrophosphate (6). The only method published to date (2) for the chemical estimation of tetraethyl pyrophosphate involves initial extraction of the active ingredient from an aqueous solution by chloroform. The tetraethyl pyrophosphate in the extract is subsequently hydrolyzed by heating to 50" C. overnight to form diethyl orthophosphate, which is then determined by titration with standard alkali. [hfter this manuscript was submitted for publication a paper on determination of tetraethyl pyrophosphate in mixtures of ethyl phosphate esters was published ( 1 ) . Tetraethyl pyrophosphate is removed from mixtures of ethyl phosphate esters by a, benzene extraction after selective h drolysis of the higher polyphosphate esters. It is subsequent6 determined by hydrolysis with alkali.] 1 Present
address, A. 9. Aloe Company, St. Louis, Mo.
In the new and improved procedure for estimation of tetraethyl pyrophosphate in the technical grade product or in the hexaethyl tetraphosphate the sample is first dissolved in 25% aqueous acetone. The ethyl polyphosphates are hydrolyzed rapidly to acidic products which are removed by an acid-absorbing resin, Amberlite IR-4B. Under the same conditions tetraethyl pyrophosphate is not appreciably hydrolyzed and remains in solution. Alkaline hydrolysis of the latter to the diethyl orthophosphate is effected subsequently by an excess of standard base and this excess is determined by titration with standard acid. MATERIALS AND APPARATUS
Acetone, 25% solution in water. Five hundred milliliters of acetone, commercial grade, are mixed with 1500 ml. of water and cooled to 25' C. Sodium hydroxide, 0.1 iV. Hydrochloric acid, 0.1 N . Amberlite IR-4B resin, analytical grade, Resinous Products and Chemical Company, Philadelphia, Pa., obtained from Fisher Scientific Co., St. Louis, Mo. Beckman pH meter, Model G, or any other good glass electrode apparatus. Methyl red indicator, 0.1% aqueous solution. Preparation and Use of Resin Column. Thirt gram of hmberlite resin, screened to remove all particles under 30-mesh, are slurried with water and poured into a 100-ml. buret contain-
809
V O L U M E 21, NO. 7, J U L Y 1 9 4 9 or Net ml' Of
3'67 = % tetraethyl pyrophosphate
weight of sample
A single determination may be completed in 60 to 65 minutes. Samples can be so staggered that a series of ten determinations may be completed in 3.25 hours. DISCUSSION
I
e 7
0 8 5
IO
I5 20 T I M E IN MINUTES
25
30
F i g u r e 1. Hydrolysis of T e t r a e t h y l Pyrophosphate a n d Hexaethyl T e t r a p h o s p h a t e in Acetone-Water Solutions a t 23" C. Hexaethyl tetraphosphate in water (l),20% acetone (2). 25% acetone (3), 33% acetone (4). and 50% acetone ( 5 ) , tetraethyl pyrophosphate in water (6), 20% acetone (7), and 25% acetone (8).
ing a glass wool plug a t the bottom. The resin column is washed with 150 ml. of 3% aqueous sodium hydroxide solution a t a flow rate of approximately 5 ml. per minute and rinsed with mater at a flow rate of approximately 2.5 ml. per minute until the effluent is colorless to phenolphthalein. The water is displaced with 25% a ueous acetone solution and the column is ready for use. The c i u m n should not be allowed to run dry or channeling may result. The liquid level is maintained a t all times approximately 2.5 cm. (1 inch) above the resin bed. It is advisable to expand the resin bed after each determination before the introduction of a new sample, because the resin tends to pack in the column as it absorbs acidic material. This renovation is accomplished simply by back-washing with 25% aqueous acetone which is introduced a t the base of the column until the liquid level reaches the top of the buret. After the absorbent settles the solution is drained off to the customary height of 2.5 cm. above the resin bed. The column is now ready to receive the next sample. After eight to ten samples have been passed through the column it is necessary to remove the absorbed acidic material. Regeneration of the resin is accomplished by repeating the initial treatment with sodium hydroxide, water, and acetone as described above. PROCEDURE
Place the material to be analyzed in a glass-stoppered weighing bottle or weight buret and transfer a 2.5-gram sample (1.0 gram if the tetraethyl pyrophosphate content is over 50%), weighed to the nearest milligram by difference, to 50 ml. of 25% aqueous acetone contained ih a 125-ml. separatory funnel. Mix the sample with the acetone solution by swirling and allow it to stand 15 minutes a t 25" * 2 " C. The use of a glass weighing bottle having a ground-in dropper is recommended for the weighing operation in order to exclude atmospheric moisture. Permit the solution of the sample to run through the column by gravity a t a rate of approximately 25 ml. per minute. Wash the column and funnel with three 50-ml. portions of 25% aqueous acetone, added successively, and maintain the 2.5-em. liquid level above the resin at all times. Catch the combined effluent in a 250-ml. volumetric flask, dilute to volume with water, mix, and transfer a 100-ml. aliquot to a 250-ml. beaker. Add 50 ml. of 0.1 N sodium hydroxide, stir well, and allow to stand a t room temperature for 30 minutes; then back-titrate with 0.1 N hydrochloric acid to a p H of 6.0 using the glass electrode assembly. Methyl red may be used as a suitable indicator if a pH meter is not available. Calculate the percentage of tetraethyl pyrophosphate by use of the following formula : Net ml. of 0.1 N NaOH X 0.0145 X 1.016 X 2.5 X 100 weight of sample % tetraethyl pyrophosphate
The method presented here makes use of the fact that ethyl acid phosphates and ethyl acid polyphosphates are absorbed by a basic resin. Ethyl polyphosphates are first allowed to hydrolyze to acidic esters in a 25% aqueous acetone solution and the acidic compounds so formed are then removed by the acid-absorbing resin, Amberlite IR-4B. Tetraethyl pyrophosphate is not appreciably hydrolyzed under the recommended conditions. I t is, however, subsequently hydrolyzed in the presence of an excess of standard alkali to the diethyl orthophosphate; the excess alkali is determined with standard acid to a pH of 6.0.
+
(C,Hi),P,O7 H2O +2(C2Hs)2IIP04 2(C2135)2111'0, 2KaOH +2l;a(CaH&PO1 HzO. .: (C2Hj)4P207==2NaOH
+
+
-4previous investigation in this laboratory had demonstrated that acetone is one of a number of solvents with which tetraethyl pyrophosphate nxrp be conveniently diluted-Le., only slight reaction will occur over a considerable period of time as evidenced by the development of an acid reaction. I t is furthermore not necessary to remove the solvent later in the procedure. Solutions of so-called hesaethyl tetraphosphate were prepared using water arid aqueous solutions of acetone containing 20, 25, 33, and 50% of the latter. The acidity of each was determined after various time intervals. Data depicted graphically in Figure 1 indicate that hydrolysis in water solution increases markedly up t o 15 minutes, after which the change is much slower. Consequently, the 15-minute time period was chosen as the required time for hydrolysis of the polyphosphate esters. Solutions of tetraethyl pyrophosphate were also prepared using water and aqueous solutions of acet'one containing 20 and 25% of acetone. The increase in acidity of each of these as a function of time was likewise determined. On the basis of these data the 25% acetone in water solution was chosen for the analytical determination of tetraethyl pyrophosphate as permitting hydrolysis of the polyphosphates present, while minimizing that of the tetraethyl pyrophosphate. Curve 8 shows that pure tetraethyl pyrophosphate hydrolyzes to the extent of only 0.7% in 15 minutes. If a sample of pure tetraethyl pyrophosphate is treated according to the above method, a loss of approximately 1.6% is noted, presumably due to adsorption by the resin or hydrolysis, or both (Table I). This error is, however, fairly constant and is the basis for the factor 1.016 used in the calculation. Up to as many as eight determinations may be made using the same resin column before regeneration as indicated by successive Table I.
D e t e r m i n a t i o n of Tetraethyl Pyrophosphate Using Amberlite IR-4B Resin C o l u m n 7% Tetraethyl Pyrophosphate Pure T E P P
Determination 1
2 3 4 5 6 7 8
Average-Average deiiation Precision Not included in average.
(100%) 98.5 98.6 98.5 98.5 98.5 98.4 98.0 98.2
...
... 98.4 * 0.2 * 0.6
Commercial T E P P Series I Series I1 40.5 4C.6 40.3 40.9 40.9 40.6 40.9 40.5
35.2 35.3 35.6 35.5 35.4 35.F 35.3 35.4
... 40.7
36.1"
... d= t.
0.2 0.6
36.8" 35.4
* *
0.1
0.3
ANALYTICAL CHEMISTRY
810 Table 11. Determination of Tetraethyl Pyrophosphate Sample
Chemical Assay
Bioassay
%
%
40.0 40.0
39.0 37.0 11.0 35.0 41.0 37.0
9.0 35.0 40.0
38.0
methods. Using the bioassay the MLD/50 for pure tetraethyl pyrophosphate was found to be 0.80 mg. per kg., when male white mice were used. ACKNOWLEDGMENT
The authors wish to thank L. F. .4udrieth and W. H. Woodstock for their helpful suggestions, W. B. Coleman for conducting the bioassays of the samples employed in this study, and -4. D. F. Toy, who prepared and furnished samples of pure tetraethyl pyrophosphate.
rum, the results for which are given in Table I. A second series of successive determinations, using a product of lower pyrophosphate content, revealed the presence of unabsorbed acid in the column effluent after it had been used for eight samples. The precision as calculated from three times the average deviation is *0.5%. A bioassay method and the above chemical method was compared on six samples of hexaethyl tetraphosphate and the technical grade product of tetraethyl pyrophosphate. The data presented in Table I1 show good agreement between the two
LITERATURE CITED
(1) Dvornikoff, M. N.,and Morrill, H. L., ANAL.CHEM.,20, 936 (1948). (2) Hall, S.A.,and Jacobsen, M., Agr. Chem., 3,30(1948). (3) Hall, S. A.,and Jacobsen, M., Ind. Eng. Chem., 40,694 (1948). (4) Schrader, G. (to I. G. Farbenindustrie), Ger. Patent 720,577 (1942) vested in Alien Property Custodian; U. 5. Patent 2,336,302(1943). ( 5 ) Toy, A. D. F., J. Am. Chem. Soc., 70, 3882 (1948). (6) Woodstock, W. H., U. S. Patent 7,402,703(1946). RECEIVED October 18, 1948
Fluorometric Method for Determination of Citric Acid ELMER LEININGEH
AND
SIDNEY KATZ, Michigan State College, East Lansing, Mich.
.4 quantitative fluorometric method is described for the determination of citric acid based upon its transformation into the highly fluorescent compound, ammonium citrazinate. The effect of changes in various operating conditions at each step in the procedure is discussed. The method is applicable to determinations of 10 to 75 micrograms of anhydrous citric acid. One hundred micrograms of tartaric and malic acids per sample do not interfere. Sulfate ion and hydroscopic compounds interfere. Application to determinations of citric acid in citrus juices is described.
A
FLUOROMETRIC method for the determination of citric acid and citrate ion is based upon the reactions employed by Feigl(2) in a qualitative test for citric acid. When anhydrous citric acid and anhydrous sodium carbonate are refluxed with thionyl chloride, aconityl chloride is formed. After the excess thionyl chloride is volatilized, aconitamide is formed by exposure of the aconityl chloride to ammonia gas a t room temperature. Citrazinic acid is formed by treatment of the aconitamide residue with 76% sulfuric acid a t 165' C. Upon neutralization with ammonium hydroxide the solution of ammonium citrazinate exhibits an intense blue fluorescence in ultravioLet light. The solution is diluted to a definite volume and the fluorescence intensity is determined.
0 ?OH
I
l
oc co I
I
0 0 H H Citric acid
I
l I
t:
c /\
HOd H;ZCH2
0 C S H,
0
CCI
+
/
HC
I
-
CH,
l
oc co I bl c1
Sconityl chloride
/\
HC,
CHZ
oc
A0
I
I
0
COH
-
NH2XHZ Aconitamide
,
i2
Sodium Carbonate, anhydrous, C.P. Ammonia, from a compressed gas cylinder. Sulfuric Acid, 76 to 77%. Concentrated sulfuric acid (73 ml.) is added to 35 ml. of water with cooling. The specific gravity (20"/4" C.) of the solution must be within the range of 1.681 to 1.692. Sodium Salicylate Solution. A standard solution is prepared by dissolving 2.000 grams of reagent grade sodium salicylate in water and diluting to 1000 ml. I t may be preserved from mold growth with a few drops of toluene. Citric Acid Solution. A standard stock solution is prepared by dissolving 7.000 grams of C.P. citric acid monohydrate in water and diluting to 1000 ml. The concentration is checked by titration with standard base. The standard stock solution may be preserved with a few drops of toluene. Two tenfold dilutions of this standard stock solution give a working standard containing 64 micrograms of anhydrous citric acid per ml. The working standard may be conveniently preserved by making it 0 3 A- with respect to hydrochloric acid during the dilution. Lead Acetate Solution. Normal lead acetate (75 grams) IS dissolved in water and 0.5 ml. of glacial acetic acid is added. The solution is diluted to 250 ml.
Hcf'CH
I I1 COH \/
HOC
K
Citrazinir acid
REAGENTS
Thionyl Chloride, Eastman No. 246. It must be protected from the moisture of the air a t all times. The practical grade causes lower fluorescence intensity and is not satisfactory.
APPARATUS
Fluorescence intensities are measured with a Lumetron fluorescence meter Model 402EF with 25-ml. cells. The primary filter permits maximum transmittance in the spectral region of 365 millimicrons. The secondary filters consist of a combination of a yellow filter, furnished by the Photovolt Corporation for use in vitamin B1 determinations, which does not transmit emission below 400 millimicrons and a Corning lantern blue filter KO. 5543. This combination of secondary filters permits maximum transmittance corresponding to the region of greatest fluorescence
