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 at room temperature. Citrazinic acid is formed by treatment of the aconitamide residue with 76% sulfuric acid at 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
V O L U M E 21, NO. 7, J U L Y 1 9 4 9 intensity of the ammonium citrazinate solution-that is, in the spectral region of 430 to 450 millimicrons. The specially constructed apparatus is illustrated in Figure 1. For the thionyl chloride reaction 8. 25-ml. Pyrex Erlenmeyer flask, A , is fitted to a Pyrex tube, B, 15 mm.-in diameter and 260 mm. in length, by means of a ground joint. The Pyrex tube serves as a reflux condenser and a drvine tube. A eonstriction in the tube 160 mm. from the groundjornt retains a plug of fine glass wool, 130 mm. of Dehydrite, and a covering plug of
joint will not'leak under vscuuk. For the'volatfiization and ;emoval of the excess thionyl chloride a three-way sto cock C, is connected to the upper end of the drying tube by 8-1, t; the vacuum DumD bv C-2. and to a mDillarv tube bv 0 3 . The trandfor&atibn of aeonityl ciiloride to acanitsmide is carried out in an ammonia chamber, D, which is BU Lshaped box of the following dimensions: horizontal section 5 om. wide, 8 om. high, &?d 30 cm. long; vertical section 5 cm. in both horizontal directions and 15 cm. high, The horizontal section accommodates six flasks at a time. Ammonia gas from the compressed gas cylinder enters through a tube on the right-hand end of D. After the flask with the reflux tube attached has been placed in the ohamber the tube is loosened with the aid of a wooden blook, E , and removed. The block is 150 mm. in length, fits loosely within the vertical section of the ammonia chamber, and has a hole 20 mm. in diameter bored through it lengthwise. It fits over the reflux tube and rests on the lip of the flask. The reflux tube, being considerably longer than the blook, is held by the top and the assembly is raised a few mm. from the floor of D while the block is tapped gently against the lip of the flask to make i t drop free of the tube. A cover, F , is placed over the opening on top of the ammonia, chamber in order to ret,ain a high ammonia concentration. FLUORESCENCE METEH SE'ITING AND CALIRRATION
The fluorometer is set to give a reading of 100 with standard sodium salicylrtte solution (2.000 grams per 1000 ml.) and a reading of 0 with distilled water. The fluorescence intensity of sodium salicylate a t this couceutration varies only slightly over the range of 20' to 30' C. and therefore temperature control is unnecessary. A calibration curve, which is very nearly linear, is prepared from instrument readings obtained from at least six well distributed points by carrying amounts of anhydrous citric acid ranging from 10 to 75 miorograms through the prooedure described below. Proper volumesaf the working standard (containing 64 micrograms of anhydrous citric acid per ml.) are measured out with accurately calibrated pipets. If more convenient, a series of
Figun 1.
811
dilutions of the citric acid may be prepared &reused in all cases.
EO
that 1-ml. smplea
nETERMlNATION O F CITRIC ACID IN SOLUTIONS FREE FROM INTERFERENCES
Analytical Procedure. A samole solution containina 10 to 75
anhydrous residue Approximately 15 mg. of anh drous sodium carbonate and 2 ml. of thionyl chloride are ad&d to the sample. The combination reflux condenser and drying tube, B, is connected to the reaction flask and the flask is heated in an oil bath maintained at
way stopcock. C. Durina this operation the reaction flask is
after the residue appeam dry and then air is allowed to flow into the flask by means of capillary C-3. The evacuation and flooding wit,h dry air are repeated three times, using 1-minute periods of evacuation. The flask with the condenser attached is introduced into the
from the chamber. The ;over is then replaced over the chamber opening. After 10 minutes the flask is removed from the ammonia chamber, approximately 2 ml. of 76% sulfuric acid are added, and the flask is tipped snd rotated to bring the sulfuric acid in contact with the entire residue. The h s k is then heated for 6 0.5 minutes a t 162" to 168' C. f
100-ml. glass-stoppered volumetric flask. usin'p 25 ml. of-wash water. The soluCion is made alkaliline'to l i h u s with dilute ammonium hydroxide ( 6 N ) , made up to 100 ml., mixed thoroughly, and brought to 24' 0.5' C., and the fluorescence iutensity is determined with the fluorometer. f
Discussion. The reaction between citric acid and thionyl chloride must take place in the complete absence of water in ordre to obtain reproducible results. The sample is dried effectively in a minimum of time without deoomoosition bv use of a vaouum oven a t 70" C. The direct action of thionvl chloride nu citric acid produces much tarry material aud the final oitrazinate solution is only weakly fluores cent. In the presence of anhydrous sodium carbonate darkening does not occur during this reaction and the find fluorescence intensity is increased approximately tenfold. From 10 to 50 mg. of sodium carbonate give maximum fluorescence. The thionyl chloride reaction reaches a maximum after 15 minutes' refluxing. Exposure to water during or after refluxing lowers the fluorescence intensity and therefore the reaction vessel is protected with a drying tube until it is placed in the atmosphere of gaseous ammonia. The only satisfactory drying agent found was Dehydrite. A water pump is satisfactory for the removal of excem thionyl chloride by evacuation. Ammonia gas rather than ammonium hydroxide is used to change the aconityl chloride to aeonitamide because i t facilitates the control of conditions in the reaction that follows. The time of exposure to ammonia is not critical. The reaction is apparently complete in less than 1 minute in some cases; long exposure does no Apparatus for Determining Citric Acid harm.
ANALYTICAL CHEMISTRY
812 Table I. Variation of Fluorescence Intensity with Sulfuric Acid Concentration for Con'stant Amounts of Citric Acid Concentration of Sulfuric Acid, %
Fluorometer Reading
68.9 72.7 74.8 76.9 78.5 80.0 81.5
67.9 83.4 85.5 85.3 85.7 76.5 68.2
Table IV.
Errors Incurred in Separation of Pure Citric Acid
Separation
Citric Acid Present
Citric Acid Found
Error
MU.
Mg.
70
Fluorometer Reading
Temperature of Oil Bath, a C.
Fluorometer Reading
160 165 170 175 180
82.1 83.9 83.8 80.8 80.0
61.9 60.9 62.9 61.C Av. 61.7
2.4
63.3
61.7 60.7 62.0 59.7 61.0
3.5
63.3
62.5 62.9 62.0 63.4 Av. 62.7
0.9
63.3
62.7 62.5 62.3 59.9 61.9
2.1
Av
Table 11. Variation of Fluorescence Intensity with Temperature of Oil Bath for Constant Amounts of Citric Acid Temperature of Oil Bath, C.
63.3
D
Av.
Table 111. Reproducibility of Fluorometric Method iiumber of samples, N Citric acid present in each sample Average fluorometric reading, 5 Average deviation of fluorometric readings Standard deviation of fluyo,metric fluorometric readings Standard deviation = [ 2 L $ ' ' ]
'/'
29 60 .O micrograms 77.0 units 1.2 units 1.6 units
L i v - 1
In the transformation of aconitamide to citrazinic acid the concentration of sulfuric acid and the temperature of the oil bath should be controlled rather closely. Table I illustrates the variations of fluorescence intensity as the concentration of sulfuric acid is varied and Table I1 illustrates the variations of fluorescence intensity as the temperature of the oil bath is varied. When 76 to 77% sulfuric acid and a temperature of 162" to 168" C. were used for 4 to 8 minutes, consistently good results were obtained. The final solution should be alkaline to litmus, but an excess of ammonium hydroxide does not change the fluorescence intensity. The temperature of the solution has a marked effect on the fluorescence intensity; an increase of 5' C. decreases the intensity approximately 10%. Table I11 demonstrates the reproducibility of fluorescence intensity readings for 29 of 31 samples taken. Each sample consisted of 60.0 micrograms of anhydrous citric acid. Compounds that decompose to discolor the solutions resulting from this series of reactions interfere. Tartaric and malic acids fa1l:into this classification. However, if tartaric and malic acids are present in amounts less than 100 micrograms per sample taken, the interference is slight; 500 micrograms per sample cause approximately 5% decrease in measured fluorescence intensity. Sulfate ion in very small amounts interferes, causing a darkening in the thionyl chloride reaction and a loss of fluorescence. Hygroscopic compounds interfere, owing to the presence of water of hydration after the drying period. The procedure requires 3' hours, of which 2 hours is the drying period. CITRUS JUICES
The method of the Association of Official Agricultural Chemists (1) for the separation of citric acid from citrus fruit juices has been modified in that hydrochloric acid is substituted for sulfuric acid and smaller volumes are used. In general, the protein matter is coagulated and separated by filtration along with solids from the juices. The citrate is precipitated as the lead salt, washed, and put back into solution as citric acid by precipitating the lead aa its sulfide. The elapsed time required for a complete determination by the fluorometric method is 4 hours compared to nearly 24 ,
hours for the standard A.O.*4.C. pentabromoacetone gravimetric procedure. Procedure for Separation of Citric Acid from Citrus Juices. A weight of sample containing 40 to 75 mg. of citric acid is selected. The volume is brought to 15 ml. with water or by evaporation. ilfter 1.5 ml. of 1 N hydrochloric acid are added, the solution is heated to 50" C., and transferred quantitatively into a 100-ml. volumetric flask. The flask is cooled and brought to volume with 95% ethyl alcohol and the solution is well mixed. The solution is filtered through a folded filter paper covered with a watch glass. Fifty milliliters of filtrate are pipetted into a 100-ml. centrifuge tube, 2.5 to 3.0 ml. of lead acetate solution are added and the solution is thoroughly a t 1000 - - mixed, and centrifuged r.p.m. for 15 minutes. The supernatant liauid is tested with lead acetate solution for complete-precipitation. If additional precipitate appears, more lead acetate solution is added and the centrifuging is repeated. The liquid is carefully decanted and discarded, leaving the precipitate in the centrifuge tube. The precipitate is washed by adding 50 ml. of 80% ethyl alcohol in small portions, using a stirring rod to ensure a homogeneous mixture. The stirring rod is rinsed with the last portion of the 80% alcohol. The solution is again centrifuged and decanted as before. The precipitate is suspended by adding 50 ml. of water in small portions while using the stirring rod for thorough mixing. The solution is saturated with hydrogen sulfide and transferred quantitatively to a 100-ml. volumetric flask. I t is brought to volume with water; the contents are well mixed and filtered through a folded filter paper. The filtrate is diluted 1 part to 4 parts of water. One-milliliter portions are taken for fluorometric analysis. The citric acid in the original sample is found by multiplying the amount found in the 1-ml. portion by 1000.
Table V.
Citric Acid Determinations by Fluorometric and A.O.A.C. Methods for Citrus Juices Citric Acid by A.O.A.C. Method, % '
Citric Acid by Fluorometria Method, '%
Canned grapefruit juice
1.48 1.48 1.47 1.43
1.54 1.42 1.49 1.43
Canned orange juice
0.845 0.841 0.835
0.858 0.820 0.863 0.827
/
Discussion. The error incurred by passing known amounts of citric acid through this separation and the fluorometric determination is illustrated in Table N. Four samples, each containing 63.3 mg. of anhydrous citric acid, were used. In each of the four sepaqgtions four 1 4 . aliquot poTtions were analyzed. The er-
V O L U M E 21, NO. 7, J U L Y 1 9 4 9 Tors incurred in these separations and determinations are within the same range as those found in the determination alone. Values obtained by this method for some citrus juices are compared with values obtained by the use of the pentabromoacetone method of the Association of Official Agricultural Chemists ( I ) in given is an average Of four ali17. Each fluorometric quots.
813 LITERATURE CITED
(1) Assoc. Offic. AD. Chemists, “Official and Tentative Methods of Analysis,” 6th ed., pp. 391-4, 1945. (2) Feigl, F., Anger, v., and Frehden, O., ,Tfilzrochemie, 17, 35 (1935). RECErVED September 20, 1948. Abstracted f r o m a portion oE a thesis submitted by Sidney Kats in partial fulfillment of the requirements for the Ph.D. degree.
Diphenylamine Test for Nitrates in Mixtures of Cellulose Esters A. G. ROBERTS, National Bureau of Standards, Washington, D . C . Diphenylamine in sulfuric acid is a sensitive reagent for indicating nitrates by producing a blue coloration. Earlier investigations were limited to nitrates in very dilute aqueous solution. Uncertainty exists concerning its use in the higher nitrate range and upon solid materials. An investigation was made of the speed and strength of color development when solutions containing diphenylamine and water were brought into contact with fiIms cast from mixtures of cellulose esters covering
D
IPHENYLAMINE in sulfuric acid has long been known ( 6 ) as a sensitive reagent for indicating the presence of nitrates by the production of a blue coloration. Because the blue color results from the oxidation of the diphenylamine, strong oxidizing agents such as nitrite, chromate, ferric salts, etc., interfere (6). The work of earlier investigators (3, 7, 10, 11) indicated that the sensitivity of the test depends upon the concentrations of diphenylamine and sulfuric acid. They employed reagents containing from 0.008 to 0.67 gram of diphenylamine and from 0 to 150 ml. of water per 100 ml. of concentrated sulfuric acid. These investigations were limited to the detection of very small amounts of nitrate in highly dilute aqueous solution, of the order of 1 mg. of nitrate nitrogen per liter (0.0001%). No investigation appears to have been made of the broad higher nitrate range (from 0.001 to 12.0% nitrogen) or of solid materials such as mixtures of cellulose esters, nor has the time for production of color been emphasized as an effective quantitative criterion. Standard reference works such as the “Modern Plastics Encyclopedia” (8), the “Handbook of Chemistry and Physics” (4), and others (2, IS) are a t variance with regard to the indicator reagent compositions recommended. It was the purpose of this investigation to determine the optimum concentrations of diphenylamine and sulfuric acid in the diphenylamine indicator solution for general use in the detection and estimation of nitrates in cellulosic films having a wide range of nitrate nitrogen compositions. EXPERIMENTAL
A series of diphenylamine indicator solutions was prepared covering the range of 0.01 to 1.0 gram of diphenylamine per 100 ml. of concentrated (96y0) sulfuric acid, and a range of 0 to 150 ml. of Kater per 100 ml. of concentrated sulfuric acid. These were evaluated with films of various nitrate contents, ranging from 12% nitrogen (as determined by a Kjeldahl titration) in.a film composed wholly of cellulose nitrate, to 0.001% nitrogen in a film consisting of a 1 to 12,000 mixture of cellulose nitrate and cellulose acetate butyrate. The cellulose nitrate employed was a dope grade complying with Army-Savy specifications (1). The mixed nitrate-butyrate compositions were obtained by casting films from homogeneous acetone solutions of cellulose
a wide range of nitrate compositions. The color development time is a minimum in the region of 2 to 6% nitrogen and increases at higher and lower concentrations. Water content is important; diphenylamine concentration exerts a minor influence. A suitable reagent for general use over a wide nitrate range contains 0.1 gram of diphenylamine, 100 ml. of concentrated sulfuric acid, and 30 ml. of water. Quantitative estimates are possible when films of known nitrate composition are used for comparison.
nitrate and cellulose acetate butyrate in the desired ratios. The latter was selected as the diluent because it is the standard material for doping fabric surfaces of naval aircraft (9) and is widely used in the aircraft industry. The diphenylamine solutions were prepared by suspending the diphenylamine in the water to be added, then adding the concentrated acid. The heat of mixing was thus utilized to effect rapid solution of the diphenylamine. (Maximum sensitivity is achieved only with freshly prepared reagents. The solution gradually deteriorates and should be discarded when it no longer produces an adequate color with a standard nitrate sample.) The compositions investigated are indicated in Table I. The time required for a particular color to develop after a single drop of the indicator solution was placed on a test film was found to depend in a consistent manner upon the nitrate content of the film. In order to standardize the time measurement, so as to permit visual resolution of nitrate composition differences among the samples, it was found convenient to use two arbitrarily selected reference colors-viz., a light blue (about 2.5PB 5 / 8 Munsell) and a deep blue (about 5.OPB 3/8 Munsell). I n the extremes of the nitrate compositions tested, where relatively little color is produced, the time required for the appearance of the first observable blue coloration was a useful criterion. Figure 1 shons the relation between nitrate nitrogen content and the color development time, using the several reference colors, with indicator reagents containing 30 and 50 ml., respectively, of water per 100 ml. of concentrated sulfuric acid. -4semilogarithmic plot has been employed solely for convenience in treating the data. Although the data presented are not inTable I. Compositions Investigated to Determine Sensitivity of Diphenylamine Test for Nitrate Radical Water added t o sulfuric acid, ml. per 100 ml. pf H+04 (96%) Sulfuric acid in reagent, Z ’ by weight Diphenylamine in reagent, grams per 100 ml. of &SO4 (96%)
Nitrate nitrogen in film, %
0,10,30,50,80, 150
96, 91, 82.5, 75.5, 67, 53 0.01,0.05, 0.10, 0.50, 1.00 0.000, 0.001, 0.005,0.015, 0.025, 0.050, 0.100, 0.185, 0.36, 0.71, 1.3, 2.4, 4.8, 7.2, 9.6, 10.6, 12.0
