ANALYTICAL CHEMISTRY
1166 hours’ elapsed time are the maximum required for a spectrophotometric analysis, the Bailey-rlndrew procedure requires 1 manhour per analysis and 7 hours’ elapsed time. Thus the spectrophotometric method reduces the time required to one third that previously necessary without sacrificing precision or accuracy. ACKNOWLEDGMENT
The authors wish to acknowledge the guidance and assistance received from L. W. Elder. Thanks are due to R. G. Moores for having provided samples of trigonelline and chlorogenic acid and for having given the authors the benefit of his experience with similar problems. I t is also desired to express appreciation for the great number of analytical determinations made for comparative purposes by J. J. Kelly, G. F. Lata, E. J. Sarna, and R. C. Sylvester. LITERATURE CITED (1) Assoc. O5cial Agr. Chem., J . Assoc. Official A g r . Chem., 30, 70-1 (1947).
( 2 ) hssoc. Official Agr. Chem., “Official and Tentative Methods of Analysis,” 6th ed., 18.14, p. 217, 1945. (3) Ibid., p. 220. (4) Castille, A., and Ruppol, E., Bull. soc. chim. biol., 10, 623 (1928), ( 5 ) Clifford, P. A,, J . Assoc. OflciaZ A g r . Chem., 14, 533 (1931). (6) Fendler, G., and Sttiber, W., 2. -Vahr. Genussm., 28, 9 (1941). (7) Gulland, J. M., Holiday, E. R., and Macrae, T. F., J . Chem. SOC.,1934, 1639-44. (8) Hartley, W.N., J . Chem. Soc., Trans., 87, 1796 (1905). (9) Hartley, W. N., Trans. R o y . Soe. (London), A, 176, 471 (1885). (10) Henri, V., ”Etudes de Photochimie,” Paris, Gauthier-Villars, 1919. (11) Holiday, E. R., Biochem. J., 24, 619 (1930). (12) Lendrich, K., and Nottbohm, F. E., Z . Nahr. Genussm., 17, 241 (1914). (13) Lepper, H. A,, J . Assoc. Official Agr. Chem., 4, 526 (1921). (14) Loofbourow, J. R., Stimson, M . M.,and Hart, M . J., J . A m . Chem. SOC.,65, 148 (1943). (15) .Moir, D. D., and Hinks, E., Analyst, 60, 439 (1935). (16) Snedecor, G. W., “Statistical Methods,” 4th ed., Ames. I o w a Collegiate Press, 1946. RECEIVED June 29, 1948. Presented before the Division of Analytical and Micro Chemistry a t the 113th Meeting of t h e AMERICAN CHEMICAL SOCIETY, Chicago, Ill.
Photometric Determination of Epinephrine in Pharmaceutical Products . I . HOY UOTY, Bureau of Chemistry, American Dental Association, Chicago. I l l . ired-blue color results from the addition of a solution of ferrous sulfate and a suitable buffer to a dilute solution of epinephrine. The transmittancy of this solution as determined at a wave length of 530 millimicrons is used to measure the amount of epinephrine. This method may be applied directly to many pharmaceutical products. The procedure is extremely simple and rapid and is capable of an accuracy of about 1% of the amount of epinephrine present. The optimum conditions for development of color are reported in some detail.
T
HE need for a reliable clieniical method for the t9stimatiori of epinephrine in solutions of local anesthetic agents led to the initiation of this investigation. -1 colorimetric procedure has been developed and tested over a period of approximately 3 years. The method is a modification of one described in 1923 by Mitchell (3) for the estimation of tannins and extended by Price (4)and Glasstone ( 2 ) to the analysis of related polyphenolic substances. The color varies with the pH of the solution. When an alkaline buffer is added to a slightly acid solution containing epinephrine and a ferrous salt, a blue color begins to develop at about pH 6.5 and gradually changes to the characteristic red-blue color which attains a maximum intensity at about pH 8 to pH 8.5. A similar procedure Fas mentioned unfavorably by Barker, Eastland, and Evers ( I ) , who did not find the reaction sufficiently sensitive for the estimation of epinephrine in adrenal glands. At least part of their difficulty may have been due to an attempt to use the blue color of lower intensity rather than the red-blue color that develops a t a higher pH. The present procedure may be employed to best advantage when the epinephrine content is a t least 10 p.p.m. Vogeler ( 5 ) reports briefly concerning a photometric study of the “ferrous iron-adrenaline complex.” Unfortunately his data are too meager to be of much value in formulating an analytical procedure. Yoe and Jones ( 6 ) , on the other hand, have suggested the use of disodium-1,2-dihydroxybenzene-3,5-disulfonate for the estimation of ferric iron. In the early part of this investigation a solution of a ferric salt was employed to develop the colored ironepinephrine complex. On the basis of further experience, however, it vias decided that the ferrous salt possessed some advantages and it was used throughout the remainder of this work.
EXt’EKI\IEYTAL
The data for the absorption curve in Figure 1 were obtained with a Beckman Model DU spectrophotometer. Maximum absorption occur>at a wave length of 530 millimicrons. There is very little absorption due to the reagents alone a t this same wave length. A Coleman Model 10s spectrophotometer with a light slit designed to give an effective spectral band width of 30 milli100
I
Reapents no epinephrine 0.4 mg. epinejlhrine Beckman Spectrophotometer Bandwidth 2 millimicrons Cell Length = 1.000 cm. A.
8 . xeaeents
J
-
90 ~
80
70
60
Figure 1.
Spectral Absorption Curve for IronEpinephrine Complex
I
V O L U M E 20, NO. 12, D E C E M B E R 1 9 4 8 1.00
0.50
0.eo
1167 data in Table I, it is apparent that for best results each 10 ml. of epinephrine solution should contain 10 to 30 mg. of sodium bisulfite. Commercial epinephrine solutions often contain amounts of bisulfite that are adequate for this purpose. Excessive amounts of very strong reducing agents, such as sodium hydrosulfite, will reduce the intensity of the color. This bleaching effect is especially likely to occur if the colored solutions are allowed to stand in stoppered tubes so that atmospheric oxygen does not have ready access to the solution. Such difficulties have not been encountered in the analysis of commercial products.
0.70
Table I.
E
0.60
Sodium Bisulfite Added M g . / f O ml.
W z
a+.
0.4 0.4 0.5 0.5 0.5 0.5 10 20 20 20 30 60 59 100 100
0.M
r-
0
0.k
0.3c
a
Effect of Bisulfite upon Color Intensity Epinephrine Added M g . / l O ml. 0,200 0.400 0.200 0,200 0.400 0.400 0.200 0,200 0.200 0,400 0.200 0.200 0.400 0.200 0,400
Epinephrine Indicated Mg./lO ml. 0.192 0.384 0.197 0.198 0.380 0.390 0.201 0.199 0.202 0,400 0.200 0,198 0.385 0.12oa 0 . 240a
Error
% -4.0 -4.0 -1.5 -1.0 -5.0 -2.5 +0.5 -0.5 +1.0 0.0 0.0 -1.0 -3.8 -40 15
-
Color intensity increased upon standing with exposure to air.
0.2c
@.1C
O.@C
microns has been used in the greater part of the analytical work. Optical density measurements have been made a t a wave length of 530 millimicrons. An Evelyn photoelectric colorimeter with filter 540 has been employed in some instances. This instrument has been found t o be equally suitable for use with this procedure. The amount of ferrous salt which is necessary t o give maximum color development is shown in Figure 2. I n this experiment the amount of epinephrine was kept constant and the optical density TI as determined in each case after the addition of a known amount of ferrous sulfate. A straight-line relationship is observed until the point is reached a t which the molar ratio of iron to epinephrine is 0.4. The absorption increases further a t a molar ratio of 0.5 and slightly further a t a molar ratio of 0.6. There is no significant change in the density as the molar ratio increases from this point. This result is of Lome interest as indicating that approximately 0.5 mole of ferrous ion is the stoichiometric equivalent of 1 mole of epinephrine. I t further indicates that when equal molecular quantities of epinephrine and ferrous sulfate are present there will be an adequate excess of iron to give maximum color development. The results of numerous tests indicate that the color reaction is rather specific for compounds possessing a t least two phenolic groups attached to adjacent carbon atoms. S o color is produced with phenol, neosynephrine, resorcinol, hydroquinone, orcinol, or phloroglucinol. KO color is observed with phthalic acid but an atypical color response is observed with as little as 5 mg. of salicylic acid. Typical color reactions are produced by pyrocatechol (1,2-dihydroxybencene), pyrogallol, epinephrine, and cobefrin, in agreement with the earlier reports of Mitchell (S), Price (4, and Glasstone ( 8 ) . Of the inorganic ions ordinarily present in solutions for parenteral use, only bisulfite affects the color intensity. From the
The effects of other substances v,-hich have been suggested for use as epinephrine stabilizers are shown in Table 11. Slight interference results from the presence of ascorbic acid in the concentration indicated if the absorption is measured soon after the addition of the reagents. Table 11. Chromogenic Effect of Possible Epinephrine Stabilizers Epinephrine Added n l . Ng./lO rnl
Epinephrine Indicated K p . / l O mi.
Compound Added .\fg./lO Sodium thiosulfate 40 0,200 0.198 Acetone sodium bisulfite 40 0.200 0.200 Sodium metabisulfite 20 0,200 0.198 Sodium hydrosulfite (NazSlOr) 20 0.200 0.194 Thiourea 40 0,200 0.196 Sodium formaldehyde sulfoxalate 0,200 0.198 Sodium thioglycollate 4; 0.000 0.118O Sodium thioglycollate 5 0.200 0.3250 Ascorbic acid 20 0,000 0.007b Ascorbic acid 20 0.200 0.2076 a Intensity of color is variable. Color with thioglycollate appears much more susceptible to oxidation-reduction effects than iron-epinephrine color. b Variable.
REAGENTS
The following solutions are employed:
Iron Reagents. A. Dissolve 1.5 grams of ferrous sulfate heptahydrate in 200 ml. of distilled water to which have been added 1 ml. of 1 iV hydrochloric acid and 1.0 gram of sodium bisulfite. The amount of iron in 0.1 ml. of this solution is sufficient for the determination of quantities of epinephrine up to 0.5 mg. For larger amounts of epinephrine, it is necessary to employ a proportionately higher concentration of ferrous sulfate. B. Prepare ferrous sulfate-citrate solution by adding 0.5 gram of sodium citrate (Sa3C6H501.2H20) to 10 ml. of solution A. This solution should be prepared fresh each day. Buffer Reagent. Add 42 grams of sodium bicarbonate and 50 grains of potassium bicarbonate to about 180 ml. of distilled water. S o t all of the solids will be dissolved a t this stage. To another 180 ml. of water add 37.5 grams of aminoacetic acid and 17 ml. of strong ammonia solution (2gW0 "3). Now mix the two solutions and add sufficient water to adjust the volume to 500 ml. The remaining solids will be dissolved a t usual room temperatures after the final addition of water. hlannitol Solution. Dissolve 15 grams of mannitol i n diotilled water and adjust the volume of the solution to 100 ml.
.
1168
ANALYTICAL CHEMISTRY
60% Isopropyl Alcohol Solution. Dilute 60 ml. of reagent grade isopropyl alcohol to 100 ml. with distilled water. PROCEDURE
Pipet a 10-ml. sample of colorless anesthetic solution, which should contain about 20 mg. of sodium bisulfite and not more than 0.5 mg. of epinephrine, into a comparison tube, and add 0.1 ml. of the ferrous sulfate-citrate reagent, followed by 1.0 ml. of the buffer reagent. hlix the solution, allow it to stand 10 minutes, and examine it in the spectrophotometer a t a wave length of 530 millimicrons. The color quickly reaches its maximum intensity and remains essentially constant for some hours. The concentration of epinephrine is read directly from a calibration curve. The calibration curve is prepared in the usual fashion by plotting optical density against epinephrine. The density readings are obtained after the instrument has been set to read 0.0 when the comparison tube contains distilled water. Boric acid, which is sometimes present in tablet preparations, will interfere with the determination of epinephrine by the regular procedure. A modified procedure has been found suitable for such products. Dissolve a number of tablets in a sufficient volume of 0.2% sodium bisulfite solution or distilled water to provide a suitable concentration of epinephrine and bisulfite. To 5 ml. of this epinephrine solution add 4 ml. of the 15% mannitol solution, followed by 0.1 ml. of the ferrous sulfate-citrate reagent and 2 ml. of the b d e r reagent. The calibration curve for the regular procedure and for this modified procedure is identical if the 5 ml. of solution employed for the analysis contain not more than 60 mg. of boric acid. A straight line is obtained n-hen optical density is plotted against concentration of epinephrine,
These methods may be used on many local anesthetic products without performing any preliminary separations. I n the case of the usual procaine hydrochloride solution, no turbidity results from the addition of the buffer. Solutions of tetracaine hydrochloride, metycaine hydrochloride, and some other products become cloudy or milky \Then the buffer reagent is added. I n an instance of this nature, a 5-ml. sample of the anesthetic solution is added to 5 ml. of 60% isopropyl alcohol and the color is then developed 11-ith the usual reagents. ACKNOWLEDGMENT
The author wishes to acknowledge the technical aid of Genevieve Stein, Veronica Flood, and Janet Edwards who assisted with much of the experimental work in this study. LITERATURE CITED (1) Barker, J. H., Eastland, C. J., and Evers, N., Biochem. J . , 26,
2129-43 (1932). (2) Glasstone, S.,Analyst, 50,49-53(1925). (3) Mitchell, C.A,, Ibid., 48,2-15 (1923). Ibid., 49,361-6(1924). (4) Price, P.H., (5) Vogeler, G., Arch. ezptl. Path. Pharmakol., 194,281-3 (1940). (6) Yoe, J. H., and Jones, A. L., IND.ENG.CHEM.,ANAL. ED., 16, 111-15 (1944). RECEIVED May 13, 1948. Presented before the Division of Analytical and SOCIETY, Micro Chemistry a t the 113th Meeting of the .-1MERIcAx CHEMICAL Chicago, Ill.
AGRICULTURAL DUSTS Preparation of Dusts of Uniform Particle Size by Fractional Sedimentation H. P. BURCHFIELD, DELORA K. GULLSTROM, AND G. L. MCNEW' Naugatuck Chemical Dicision, United States Rubber Company, Naugatuck, Conn. A method based on fractional sedimentation is described for the isolation of particle size fractions from agricultural dusts. Mathematical procedures are developed by which it is possible to estimate the type and number of sedimentations required, as well as the mean radius, particle size distribution, and amount of each fraction that will be obtained. The isolation of fractions of definitely known distribution aids in the evaluation of those biological and physicochemical properties which are modified by changes in particle size.
T
HE protective value of a fungicidal dust depends on particle
size as d l as innate toxicity of the chemical to the organism. This Tyas clearly demonstrated by the J-vorlr of Kilcoxon and McCallan on sulfur (10) and Heuberger and Rorsfall on cuprous oxide ( 5 ) . In glass slide tests against Xacrosporzum sarcinaejorine Cae ., the latter authors showed that the percentage of spores not germinated increased from 53.0 to 98.3% when the mean particle size of the cuprous oxide was reduced from 2.57to 1.65-micron diameter at a constant deposition of 100 X 10-4 mg. of copper per square centimeter. I n addition to its effect on fungi toxicity, particle size may also be a factor in the stability of spray suspensions, the flowability and rate of settling of dusts, and the tenacity and chemical stability of spray or dust deposits on weathering. In the evaluation of new protectants it is frequently desirable to study these effects on a laboratory scale, using samples with narrow and clearly defined particle size ranges, in order to determine the optimum state of subdivision for the material. Samples ground in a laboratory hammer mill usually have a wide range o f 1
Present address, Department of Botany, Iowa State College, .4mes, Iowa.
particle size distribution; hence biological and physical tests carried out on them, reflect only the average properties of the materials without focusing attention on the size class that possesses the most desirable characteristics. Methods have been described for fractionating dusts by elutriation by water ( 1 ) and air ( 6 ) , but the apparatus is complicated and not readily available. The need for a rapid simple method for separating size fractions for biological assay led to the development of the sedimentation procedure described in this paper. EXPERI AI ENTA L
Separations were carried out on the organic fungicides Phygon (2,3-dichloro-l,4-naphthoquinone, 9) and Spergon (tetrachloro-pbenzoquinone, 8). Technical grade Phygon was purified by sublimation followed by recrystallization from benzene. The melting point was 190" C. and the specific gravity 1.645 a t 25'/ 25' C. The sample of Spergon was obtained by recrystallization of the crude product from acetic acid. I t had a melting point of 290 O C. and a specific gravity of 1.948 a t 25 O /25' C. The crystalline compounds were ground by two passes through a laboratory model Raymond pulverizer equipped with a 0.25mm. (0.01-inch) herringbone screen, The particle size distribu-
