V O L U M E 20, NO, 3, M A R C H 1 9 4 8 Thit retention of the spherical form may be considered a dist inguisliing feature between the alumina and silica fumes. Fumes occurring above electric furnaces producing fused alumina from impure source materials containing silica may contain some crystalline forms but are predominantly spherical in form, i$7hen subjected to the vaporization treatment with hydrofluoric acid, many casts show only partial removal of the spherical particles (Figure 3). These particles may be silicates or eo-condensations of the two major components.
267 Further distinctions may be possible by submitting the siecimens to electron diffraction. LITERATURE CITED
(1) Schaefer, v. J., and Harker, D., J . A p p l i e d Phw., 1 3 , 4 2 7 (1942). (2) Schuster, M . C., and Fullam, E. F., IND. ENG.CHEM..ANAL.ED., 18, 653 (1946). RECEIVED May 26, 1947.
Colorimetric Microdetermination of Formic Acid Based on Reduction to Formaldehyde '
W. MORTON GRANT Howe Laboratory of Ophthalmology, Harvard University Medical School, Boston, Mass. A colorimetric method for determination of 0.25 t o 15 micrograms of formic acid in 0.5 ml. of solution is based on reduction to formaldehyde by means of magnesium with subsequent measurement of the formaldehyde by means of chromotropic acid. For determinations in blood, formic acid is first separated from interfering substances by quantitative vacuum microdistillation at a low temperature. In application to mixtures of formic acid and formaldehyde, the formaldehyde is first remoyed by reaction with phenvlhydrazirie.
ETHODS for detectionand estimation of formic acid have been based principally either on oxidation of formic acid by mercuric chloride or on reduction by magnesium. The sensitivity of measurement by means of mercuric chloride in a colorimetric procedure is sufficient for determination of quantities of formic acid as small as 5 to 30 micrograms, but greater sensitivity by this method does not appear readily attainable (6). The single quantitative procedure which has been described, based on reduction of formic acid by magnesium and measurement of the resultant formaldehyde, has hitherto been found satisfactory only in the range of 40 to 1000 micrograms (1). Honever, this reduction method has theoretical potentialities for application to sniallei quantities in view of the relativelj high vnsitivity of the procedui'ei available for colorimetiic deteiniination of foimaldehydc . T o increase the sensitivity of formic acid measurement, cspecially for toxicologic and microbiologic applications, factors influencing the reduction of formic acid to formaldehyde have been investigated further and it has been found possible t o utilize magnesium reduetion in conjunction n ith colorimetric determination of formaldehyde by means of chromotropic acid t o measure 0.25 t o 15 micrograms of formic acid in 0.5-nil. samples with an average error of 0.13 microgram for single analyses. For analysis of samples such as blood which contain protein and carbohydrate interfering substances, the formic acid may be separated by low-temperature vacuum distillation (6). For measurement of formic acid in samples containing formaldehyde, reaction n-ith phenylhydrazine may be utilized for preliminary removal of the formaldehyde. REAGENTS
Chrornotropic Acid .Reagent. To a solution of 0.6 gram of chromotropic acid (l,&dihydroxynaphthalene-3,6-disulfonic acid, Eastman Kodak Co., pract.) in 20 ml. of water are added 180 ml. of concentrated sulfuric acici. Magnesium Ribbon. Strips 10 em. long and 3 mm. wide, weighing approximately 80 mg., are rolled into coils 1 em. in diameter. The supply of ribbon should be protected from atmospheric attack in a desiccator containing sodium hydroxide.
PROCEDURES
Solutions of formic acid which are free from interfering substances are analyzed by adding a 0.5-ml. sample containing not more than 15 micrograms of formic acid to an 80-mg. coil of magnesium ribbon in a test tube immersed in an ice bath. A total of 0.5 ml. of concentrated hydrochloric acid is then added in ten separate portions of 0.05 ml. each at intervals of not less than one minute. The tube is removed from t,he bath a minute after the last addition of hydrochloric acid and 1.5 ml. of chromotropic acid reagent are added. The misture is heated on a boiling water bath for 30 minutes with precautions against absorption of water vapor and uneven illumination, as described by MacFadyen ( 7 ) . The mixture is then centrifuged clear of whit,e precipitate and the color of the supernatant fluid is measured photoelectrically, using a filter with peak transmission a t 570 nip. The relationship betTyeen colorimeter reading and quantity of formic acid is established by measuring the color produced by a series of standards of 0 t o 15 micrograms of formic acid in 0.5 ml. of 0.01 A- hydrochloric acid solution submitted to the procedure already described. The relationship b e t w e n log galvanometer reading and amount of formic acid is linear in the range of 0 to 10 micrograms. Samples for analysis which may contain carbonates should be acidified to approximately pH 2 by addition of hydrochloric acid before submission to the magnesium reduction procedure. For measurement of formic acid in blood, protein is removed by mixing 0.5 to 1 ml. of blood with 2 volumes of a solution containing 5 % Sulfosalicylic acid in 0.25 M sulfuric acid. The mixture is centrifuged and the formic acid is separated from carbohydrates by low-temperature vacuum distillation of the supernatant fluid in a previously described apparatus ( 5 ) . This apparat,us employs a closed system; the distillate condenses a t the temperat'ure of dry ice and distillation of milliliter samples proceeds to dryness in approximately 2 hours without heating. The distillate is submitted to analysis by the procedure described for pure solutions. In calculation of the concentrat,ion in the original blood, correction should be made for the change in volume resulting from removal of nonvolatile substances. For average rabbit blood, the mixture of blood and sulfosalicylic acid solutions contain 9373 by volume volatile materials. When formaldehyde is present in the sample t.o be analyzed for formic acid, t'he formaldehyde may be removed preliminarily or the amount may be measured and correction made for the color produced by it in the analysis for formic acid. Formaldehyde is readily removed from blood samples by adding phenylhydrazine to the supernatant fluid from the mixture of blood with sul-
ANALYTICAL CHEMISTRY
268 fosalicylic and sulfuric acid before distillation in the proportions of 5 to 10 mg. of phenylhydrazine hydrochloride per milliliter of supernatant fluid. For concentrations up to 10 micrograms of formaldehyde per nil., an interval of 5 minutes is allowed for the reaction with phenylhydrazine to take place at room temperature before the mixture is frozen for distillation. EXPERIMENTAL
The use of magnesium and hydrochloric acid in the nianner described in the present procedure \vas found more favorable for the reduction of formic acid t o formaldehyde than the use of several other metals, acids, or conditions of reaction which were investigated. K i t h 5 to 50 mg. of sodium, lithium, calcium, aluminum, or zinc in conjunction with 4 t o 12 I\' hydrochloric acid a t room temperature no formaldehyde production was detectable from 20 micrograms of formic acid. Magnesium amalgam \vas considerably less effective than magnesium itself. Addition of platinum black did not increase formaldehyde production. Investigation of the effectiveness of various acids in combination with magnesium, when approximately 80 mg. of magnesium were employed x i t h 50 micrograms of formic acid in 1 ml. of acid a t room temperature, showed that n-it11 1 to 1 dilutions of concentrated sulfuric, metaphosphoric, or orthophosphoric, or saturated solutions of sulfurous or boric acids there was no appreciable formaldehyde production. With aminoacetic, lactic, oxalic, maleic, and sulfanilic acids there was much interfering discoloration. With acetic, monochloroacetic, trichloroacetic, citric, and sulfosalicylic acids, form-aldehyde production from formic acid was detectable, but there was also considerable color in blanks without formic acid. With propionic and sulfamic acids, formaldehyde was produced with little interference, but somewhat less efficiently than a ith hydrochloric acid. I n formic acid reduction by means of magnesium and hydrochloric acid, the manner of mixing and the temperature a t which the reduction is carried out influence the efficiency of the process. On the other hand, moderate variation in the total amounts of magnesium and hydrochloric acid employed is possible without markedly influencing the yield of formaldehyde. .4ddition of 0.5 ml. of hydrochloric acid in ten equal portions a t various intervals shorter than a minute resulted in definitely diminished yield of formaldehyde, while approximately constant yield resulted when intervals of from 1 to 3 minutes were employed. Similarly, if 0.5 nil. of hydrochloric acid was added in fewer portions than ten, even though with intervals greater than a minute, less color resulted, while addition in twenty portions gave the same yield as ten. When the reduction was carried out in an ice bath at 0" C. in the manner recommended in the present procedure, a better yield was obtained than in baths at -20°, -IOo, 2 j 0 , or 100" C. I t is noteworthy that 4ow addition of acid and employment of an ice bath in the reduction of formic acid by magnesium was previously recommended by Droller ( I ) , but his stated purpose was the prevention of separation of the fuchsin which he employed for formaldehyde measurement, rather than the improvement of formaldehyde yield. The quantity of magnesium recommended in the present procedure is based on a compromise between yield of formaldehyde and quantity of precipitate formed with the chromotropic acid reagent. I n the reduction of 5 micrograms of formic acid, formaldehyde production is increased as the amount of magnesium ribbon is increased a t the rate of approximately 10% per cm. from 7 to 11 cm. However, above 10 em. The amount of precipitate becomes excessive for direct application of the chromotropic acid. Attempts t o increase sensitivity by employing larger quantities of magnesium and hydrochloric acid with subsequent separation of the formaldehyde by distillation were unsuccessful because of difficulties in distilling the concentrated magnesium chloride solutions. Some limitation of the yield of formaldehyde from formic
acid is imposed by loss of formaldehyde through reduction by magnesium and hydrochloric acid. average loss of 15% occurred when solutions of 1 to 5 micrograms of formaldehyde per ml. were submitted to the procedure described for formic acid reduction. The reduetion of carbon dioxide to formaldehS;.de by magnesium, as noted by Fenton (S), n-as found to occur under the conditions of the present analytical procedure to a slight extent, indicated by production of a color equivalent to 5.2 micrograms of formic acid per nil. irhen 1 -1-sodium carbonate iyas submitted to this procedure. Interference from carbon dioxide v a s satisfactorily eliminated by preliminarv acidification of standards and experimental samples as pi-eviously described. Protection of the magnesium ribbon from atmospheric attack was also helpful in obtaining constant results. Separation of formic acid from interfering substances of blood by means of a procedure (1) employing successive treatments with metaphosphoric acid, copper sulfate, and calcium hydroxide mas investigated to determine its suitability for use in conjunction with the magnesium reduction and chromotropic acid reactions, but was found unsatisfactory. When normal rabbit blood and blood containing an additional 10 micrograms of formic acid per ml. were submitted to this procedure for removal of protein and carbohydrate and were then trebted with magnesium, hydrochloric acid, and chromotropic acid, turbid brown solutions were obtained which were indistinguishable and unsuitable for colorimetry. The separation of formic acid from blood by low-temperature vacuum distillation, which is recommended both in the present procedure and in a previously described procedure for determining formic acid by means of mercuric chloride (S), gave satisfactory recoveries of formic acid and eliminated nonvolatile interfering substances as well as carbon dioxide. Blank values of 1.4 to 8.4, averaging 5.2, micrograms of formic acid per ml., n-ere obtained for seven normal rabbit bloods. When 10 and 100 micrograms of formic acid per ml. were added to blood, 100.5 and 102.6%, respectively, were recovered. The treatment with phenylhydrazine which is recommended to eliminate small amounts of formaldehyde from samples to be analyzed for formic acid n-as found not t o interfere with recoveries of formic acid, and formaldehyde in concentrations up t o 10 micrograms per ml. was completely removed by this procedure. u p t,o 200 micrograms of formaldehyde per ml. could be removed if the phenylhydrazine mixture was heated for 5 minutes in a boiling water bath, but, under these conditions there was a moderate increase in the blank for formic acid, possibly due to some formation of formic acid. Colorimetric determination on the 1.5-ml. volumes of color solution yielded by the present analytical procedure was satisfactory when a Cenco Photelometer yas used, in Tvhich the lamp filament was turned horizontally and a piece of rubber stopper was placed in the cuvette compartment to raise the cuvette into appropriate position in the beam. Suitable spectral transmittance was obtained lvith a combinatfon filter consisting of Jena VG-3 (2 mm.), Corning 9780 (2.56 mm.), and Cenco KO. 2 (2 mm.) filters. DISCUSSION
The present procedure for measurement of formic acid provides advantages in increased convenience, sensitivity, and specificity over previous methods utilizing either mercuric chloride or magnesium. The use of chromotropic acid to measure formaldehyde in this procedure eliminates the uncertainty in color development inherent in the single previous quantitative magnesium method which employed fuchsin-sulfurous acid and required accurate titration of the magnesium by hydrochloric acid ( 1 ) . Use of chromotropic acid also eliminates the wait of 10 to 24 hours recommended for stabilization and clarification
V O L U M E 20, NO. 3, M A R C H 1 9 4 8
269
of solutions for colorimetry in the procedure employing fuchsinsulfurous acid. The sensitivity of determination of formic acid by the method described here, while superior to that of previous methods and probably adequate for many purposes, yet is somewhat less than the sensitivity which would be attained if the formic acid were completely converted to formaldehyde. At present a net yield of approximately 29Yc of theoretical is obtained, corresponding t o a total yield of 3-17 with a coincident loss of 15% of the formaldehyde formed. It \vould appear that if a larger proportion of magnesium could be conveniently utilized or conditions for more efficient reduction of formic acid x i t h sparing of the formaldehyde xere devised, the sensitivity might be increased slightly more than threefold. The specificity of thc chroniot ropic acid reagent for formaldehyde which has been demonstrated by Eegrixve (2j and MacFadyen ( 7 ) indicates a (mresporiding high degree of specificity for the determination of formic acid by the present procedure, since few substances other than formic acid yield formaldehyde on reduction by magnesium (4). Among possible interfering substances is carbonic acid, xvhich can be reduced to formaldehyde by magnesium, hut is readily eliminated by preliminary
acidification of solutions t o be tested. Interference by acetic acid or acetaldehyde would occur only a t high concentrations of these substances (4, 7 ) . Preformed formaldehyde would interfere a t low- concentrations, but is readily removed by means already described. Labile compounds or polymers of formaldehyde presumably would also interfere if not similarly removed. ACKNOWLEDGMENT
The author \\.ishe> to acknowledge the valuable technical as$i,tanw of 1Ivra Rolston. LITERATURE CITED
(11 Dioller, H , Z. physzol. Chem., 211, 57 (1932) (2) Eegriwe, E , Z. anal Chem., 110, 22 (1937). J . C'hem.Soc., 91, 687 (1907). (3'1 Fenton, H. J. H., (4) Fenton. H. J. H., and Sisson, H. A., Proc. Cambrzdge Phzlosoph. Soc., 1 4 , 3 8 5 (1908). (5) Giant, W. SI., ISD. ENG.CHEM, A s . 4 ~ED., . 18, 729 (194G). (6) Ibid., 19, 206 (1947). (7) MacFadjen. D A , J Biol. Chem., 158, 107 (1945) R E C E I V Z July D 9, 1 9 4 i
Quantitative Volumetric Analysis of Carbon-Bonded Halogen with Sodium Naphthalene F. L. BENTOY AhD W. H. HARlILL, Chemical Laboratories, L'niversity of S o t r e Dame, Notre Dame, Znd.
Q
UANTITATIJ*E organic analysis for halogen by the sodiuniin-liquid-ammonia method (2) is someJvhat limited because of the sparing solubility of many compounds in liquid ammonia at its boiling point. I t has, hoviever, suggested the possibility of using a solut'ion of sodium naphthalene in an appropriate oxygenated solvent (1j as a more corivenient analytical reagent. Choice of solvent is rather broad and includes ethylene glycol dimethj-1ether, ethylene glycol diethyl ether, diosane, and methyl isopropyl ether. Since man)- of these solvents are relatively high boiling, solubility limitation would be much less restrictive than for liquid ammonia. The volumetric procedure n-as chosen as the most rapid means of exploring the possibilities of this reagent. The appropriate gravimetric procedure is an obvious variation of the method described. EXPERI5IESTA L
Preparation of Sodium Naphthalene. The solutions of wdiuni naphthalene i n c s t liylenc~glycol dimethyl or diethylether n-ere pre~
.
~~~~
Table I. Compound E t h y l bromide Tetracliloroethylenr
Analyses.
3Iilliequiralents of Halogen Calculated Found 4.484 4.594 4.543 4.7Y0
5.Oi0 Carbon tetrachloride Brornoi)enzene g-Diclllorobenzent p-Chloroaniline p-Bromo-,~,N-dirnethylsni1ir.e ~-BronionaphthaIenc
4.841 5.210 4,319 5 , oe5 5.020 4 862 4.384 5 040 5.140 5,134
4.305 4.543 4.654 4.810 5.03; 4.81,
5.172 4.323 5,042 3,014 4,867 I-erJ-Ion 4.410
3.071 5,105 5 110
%
Error f0.4g -1 -~ 11 +1.11 70.41 -0.7; -0.49 -0.59 +0.09 -0,45 -0.12 fO.10 ~
+0.5"
-o.ei
-0.69 -0.85
p:tred acccrdiiig t o the directions of Scott (1) and were used at approsimately 0.5 molar. They were stored in and dispensed from an automatic buret and solutions of sodium naphthalene were transferred from flasks to buret under an atmosphere of dry nitrogen. Purification of Reagents and Samples. The ethylene glycol dimethyl and diethJ-1ethers were obtained from the Carbide and Carbon Chemical5 Corporation and purified by refluxing over sodium foy 3 houi.s, followed by fractional distillation. Samples of solid compounds for analysis were purified to maxiLiquids were fractionally distilled and that fraction of the distillate boiling within the range reported for the pure compound was employed for ahalysis. Analytical Procedure. Liquid samples ivere Tveiglletf in thinivalled, sealed glass bulbs and placed in a separatory funnel from \\.hich air was displaced by dry nitrogen, and 10 to 20 ml. (an escess) of the sodium naphthalene reagent were added. The separatory funnel was stoppered and shaken vigorously to break the bulb (as indicated by an abrupt temperature rise), and shaking was continued for 2 t o 3 minutes longer. The formation of sodium halide is probably instantaneous. Excess reagent was destroyed and the sodium halide was dissolved and separated by addingtwo to three consecutive portions of water up to a total of :tpprosimately 100 nil. The combined aqueous layers were acidulated n-ith nitric acid and titrated potentiometrically with 0.1 iV silver nitrate solution, using a silver-plated platinum n-ire and a saturated calomel electrode with an ammonium nitrate-agar gel bridge. Solid samples were introduced directly into the separatory funnel and 5 ml. of toluene were added to dissolve the samples to facilitate reaction. The procedure was not otherwise altered.
mum melting point by recrystallization.
DISCUSSION
It \vas not the purpose either t o establish the ultimate precision of this method, or to discover all limitations, but only t o demonstrate its applicability t o a considerable class of organic compounds with limits of precision which are usually acceptable. It is, of course, t o be expected that any compound conta.ining
