JULY 15, 1936
ANALYTICAL EDITION
lady for the design and construction of the vacuum-tube potentiometer.
Literature Cited Brown, A. S., J.Am. Chem. SOC.,53,645 (1931). Clarke. B. L.. Wooten. L. A.. and ComDton. _. - . K. C.. IND.ENG. CHEM.,Anal. Ed., 3,321 (1931). Dole, M., J . Am. Chem. Soc., 54, 3095 (1932). Evans, R. N., and Davenport, J. S., IND.ENG.C H m f . , Anal. Ed., 3, 82 (1931). Gen. Elec. Co., Pub. CET-249A, “Circuits for Amplification of Direct Currents Using the FP-54 Pictron,” February, 1932. MacInnes, D. A,, and Belcher, D., J . Am. Chem. SOC.,53, 3315 (1931).
Moles, F. J., Gen. Elec. Rev., 36, 156 (1933).
291
(8) O’Sullivan, J. B., Trans. Faraday SOC.,23, 52 (1927). (9) Perrakia, N., Compt. rend., 177, 879 (1923). (10) Ralston, R. R., Fellows, C. H., and Wyatt, K. S., IND.ENO. CHEM.,Anal. Ed., 4, 109 (1932). (11) Seltz, H., and McKinney, IND, ENG.CHEM.,20, 542 (1928). (12) Seltz, H., and Silverman, L., Ibid., Anal. Ed., 2, 1 (1930). (13) Timmermans, J., and Martin, F. S., J. chim. phys., 25, 411 (1928).
(14)
Wooten, L. A,, and Ruehle, A. E., IND.ENG.CHEM.,Anal. Ed.,
6, 449 (1934). (15) Yule, J. A. G., and (1931).
Wilson, C. P., IND.ENG.CHEM.,23,
1254
RI~~CDIIWD April 17, 1936. Presented in part at the Pittsfield meeting of the Committee on Electrical Insulation, Division of Engineering and Industrial Research, National Researoh Council, October, 1935.
Energy Equivalents of Vitamin D Units ROBERT W. HAMAN AND HARRY STEENBOCK, University of Wisconsin, Madison, Wis.
T
HE widespread acceptance of irradiated food products and
irradiated ergosterol, as well as the general use of cod liver oil and its concentrates] has demanded the formulation of unitary expressions of vitamin D activity. I n this country the Steenbock unit, prior to the acceptance of the International unit by the U. S. Pharmacopceia, had received widespread use. The Steenbock unit was originally defined in terms of the amount of calcium deposited in a standard rachitic rat under standard feeding conditions in 10 days. A reference preparation of irradiated ergosterol of known potency expressed in such units was made available for use in other laboratories. At the time of the adoption of the International unit by the Permanent Commission on Biological Standardization of the League of Nations in 1931, the authors immediately initiated experiments comparing the Steenbock unit with the International unit. Their initial experiments were of the therapeutic type. Preliminary experiments necessitating certain aomparisons revealed provisionally that 1 Steenbock unit was equivalent approximately t o 2.7 International units. This factor was sufficiently accurate for the purpose at that time (7). Unfortunately, it was accepted by others as the final conversion factor for purposes requiring a far greater degree of accuracy than demanded by the authors’ particular experiments. A limited number of quantitative studies relevant to the amount of energy required t o synthesize vitamin D from ergosterol have been reported (2, 4, 5 ) , and their results are generally concordant in showing that within certain limits there exists a definite relation between the amount of radiant energy absorbed and the amount of vitamin D synthesized. The authors have now used a similar technic to determine the amount of radiant energy required to synthesize one International unit of vitamin D in comparison with the Steenbock unit, using ergosterol as the substrate.
abled the authors to move either compartment into the path of the monochromatic radiations incident to the thermopile slit, thereby eliminating reflection and absorption by the quartz and the solvent as factors in the quantitative evaluations. As a source of light the authors used a capillary quartz mercury arc similar to one described by Daniels and Heidt (1). The degree of resolution of the spectrum from this arc as well as the energy values of different lines is shown in Figure 1. It is evident that the dis ersion was sufficient for the purpose. their first quantitative evaluations of the Steenbock unit the authors irradiated ergosterol in absolute alcohol solution with the 303 p mercury line. (The ergosterol was obtained from Chaa. Pfizer and Co. It had a melting point of 158” C., uncorrected, a rotation of CY)^ = -134.3‘ in chloroform, and an extinction coefficient of 11,000 at 282 mm.) Five cubic centimeters of a 0.1 per cent solution were placed in one compartment of the cell and 5 cc. of absolute alcohol in the other. The cell was sealed with a glass plate and placed on the rack in front of the thermopile slit. Readings of the galvanometer deflections for the determination of the energy transmitted by the absolute alcohol were made before and after the irradiation period for these short exposures. Since the radiant energy incident to the ergosterol solution waa all absorbed, this measurement represented the radiant energy absorbed. The am erage and voltage of the arc were always checked. Each soktion after proper exposure to measured amounts of radiant energy, ranging from 500 t o 5000 ergs, was fed in oil to rats using the 10-day line test technic with ration 2965 as the rachitogenic diet.
81
Earlier results indicated that healing comparable t o that produced by one Steenbock unit was obtained when 3000
Experimental Known amounts of radiant energy of known wave lengths were obtained with the use of a Bausch and Lomb quartz monochromator equipped with a Coblena linear thermopile (12 copper-Constantan junctions) in conjunction with a Leeds & Northrup galvanometer (sensitivity, 10.4 mm. per mv.). The thermopile-galvanometer system was standardized with a carbon filament lamp obtained from the U. S. Bureau of Standards. The instrument was adapted for the purpose by the insertion of a quartz lens with a focal length of 5 cm. between the exit slit and the thermopile. This made it possible to place in front of the thermopile slit a cell, 2.5 cm. wide and 1.5 cm. thick, consisting of two compartments constructed of ground and polished plates of quartz. A rack provided for this cell en-
d
FIGURE 1
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INDUSTRIAL AND ENGINEERING CHEMISTRY
ergs of radiant energy of wave length 303 p were absorbed by the ergosterol solutions. These experiments have been repeated, using the following prominent lines of the mercury vapor arc: 249, 254, 265, 275, 280, 289,297,303, and 313 p. All of them with the exception of the 313 p line were found essentially equally effective per unit of erergy absorbed. Kon, Daniels, and Steenbock (4),using the line test, and Marshall and Knudson (6), using roentgenograms as criteria, have reported that within the synthesizing region the quantum efficiency was identical for the various wave lengths. The authors’ results represent an approximate 20 per cent difference in the number of quanta required to produce one Steenbock unit with the 248 p line as compared with the 303 pline. However, this difference may not be based on actual differences in potency, because of the limited accuracy of the line test with the animals used at that time. The authors have, however, repeatedly determined differences of 15 per cent in vitamin D activity, using the prophylactic method with 6 to 10 animals in each group of a series. The daily administration of as little as 0.025 Steenbock unit per day for 5 weeks produces a demonstrable response. One-tenth Steenbock unit per day, or a total of 3.5 Steenbock units for the experimental period of 5 weeks, has been found to be sufficient to protect a rat against rickets on rachitogenic diet 2965. To determine more accurately the relative effectiveness of the various wave lengths in the synthesizing region the authors repeated their irradiation with the various lines of the mercury vapor arc and fed the resultant preparations in oil to rats in prophylactic experiments with bone ash determinations as the criteria. The results of these experiments are presented in Table I, series 1 and 2. They confirm the results of previous therapeutic studies that the amount of vitamin D produced is proportional to the amount of radiant ehergy absorbed and independent of the wave length within a considerable range. It also confirms the fact that the upper limit of the ultraviolet zone capable of synthesizing vitamin D lies between 303 and 313 p . Hess and Anderson (8) reported in 1927 that the longest wave length producing antirachitic activity was 313 p. TABLEI. EFFECTIVENESS OF VARIOUSWAVE LENQTHS (Ash of femurs from rats fed daily the amount of vitamin D produced b y 150 ergs of various wave lengths) Averaee Number Gain-of in Wave Length Rats Weight Average Ash N Grams Gram % 40.55 43 0.0504 249 Series I 40.06 41 0.0481 265 0,0522 40.62 36 289 39.49 43 0.0499 302 42 0.0321 29.64 313 Negative 0.0253 26.11 36 controls Series 2 254 6 38 0.0617 47.04 265 6 31 0.0606 45.64 275 6 39 0,0523 43.74 280 6 38 0.0579 44.78 297 6 33 0.0501 43.81 Negative 29.46 33 0,0292 6 controls
The energy relations which the authors have expressed were obtained under rigidly controlled experimental conditions. It is to be expected that the energy relationship, 3000 ergs = 1 Steenbock unit, will not hold when the course of the reaction has proceeded to the point where the rate of destruction of the vitamin is greater than the rate of synthesis. However, the authors have found that the relationship still holds when lo4 ergs have been absorbed by 1 mg. of ergosterol in absolute alcohol. Their evaluations were made when approximately 103 ergs had been absorbed. They have also determined the amount of radiant energy required to synthesize one International unit of vitamin D
VOL. 8, NO. 4
from ergosterol in absolute alcohol solution. To make this determination they exposed an ergosterol solution to a measured amount of radiant energy of wave length 265 p . This preparation was diluted so that the daily dose of vitamin D was expressed in terms of radiant energy absorbed. Five dilutions were made and compared with the International standard preparation using rats in prophylactic experiments. From the data presented in Table I1 it is apparent that 0.165 International unit daily produced a somewhat greater deposition of ash than the daily intake of vitamin D produced by 140 ergs of radiant energy. The energy required to produce an International unit was, therefore, slightly greater than 850 ergs. It appears permissible to conclude that one International unit is equivalent to the vitamin D produced by 900 ergs of radiant energy of wave length 265 p . TABLE11. DETERMINATION OF RADIANT ENERQY REQUIRED TO PRODUCE ONE INTERNATIONAL UNIT
Preparation 119A. 119B. 119C. ll9D. 121A.
Irradiated ergosterol Irradiated ergosterol Irradiated ergosterol Irradiated ergosterol International standard preparation
Ergs per Day 115 140 165 190 0.165 International unit
Number of
Rats 10 10
10 10 13
Average Gain in Weight Grams 62 53 57 58 50
Average Gram 0.0407 0.0419 0.0451 0.0484 0.0412
Ash
% 34.80 35.77 37.94 39.78 35.97
From the authors’ data in the energy equivalents of one Steenbock unit it is obvious that it requires approximately 3.33 times as much radiant energy to produce one Steenbock unit as one International unit. TABLE111. COMPARISON OF STEENBOCK AND INTERNATIONAL UNITS (Percentage of ash roduoed by 0.025 Steenbock unit per day as compared with t h a t producea by different amounts of the International standard) Vitamin D Number Bverage Intake of Gain per Day Rats in Weight Average Ash Grams Gram % Series 1 Steenbock unit 0.025 12 35 0.0395 37.91 International unit 0.0475 12 36 0.0316 32.36 0.0575 12 33 0.0309 31.86 0.0675 11 30 0.0298 32.89 0.0775 12 30 0.0337 36.70 0.0875 12 30 0.0423 40.32 0 27.44 10 26 0.0246 Series 2 Steenbock unit 0.025 10 48 0.0376 35.60 International unit 0.0825 11 42 0.0356 36.03 0 12 29 0.0240 26.60
The ratio of the two units to each other which the authors established from their energy evaluations with independently executed biological tests has been checked by direct comparative biological tests. They prepared a standard preparation of known vitamin D activity expressed in Steenbock units with monochromatic light of wave length 265 p, using essentially the same technic as already described. This preparation was fed in oil from a calibrated dropper to rats a t a level of 0.025 Steenbock unit per day. Similarly, the International standard preparation as obtained from the Health Organization of the League of Nations was fed at daily levels of 0.0475, 0.0575, 0.0675, and 0.0875 International unit. From the resultant data, presented in Table 111, series 1, it is evident that the number of International units required to produce an ash equivalent to that produced by 0.025 Steenbock unit lies between 0.0775 and 0.0875 International unit. As this was too wide a range, another series of animals was fed 0.0825 International unit and 0.025
JULY 15, 1936
ANALYTICAL EDITION
Steenbock unit daily. The results of this series, presented in Table 111, series 2, show that the response obtained with these two preparations was identical. As the dosages of 0.025 to 0.0825are in the ratio of 1to 3.33, the results confirm the ratio obtained in the previous series. (Russel and Taylor, 6, have reported, while chis manuscript was being prepared; that one Steenbock unit is equivalent to 3.2 International units.)
Summary 1. One International unit of vitamin D was synthesized from ergosterol when 900 ergs of ultraviolet eneigy within the synthesizing region were absorbed. Similarly, one Steenbock unit was synthesized when 3000 ergs were absorbed. These values were found to be independent of the wave length within the synthesizing region. 2. A comparison of the energy equivalents of the two units, as obtained in independently executed series of assays, and potency Of the Interdirect Of the national Standard Preparation with a preparation produced
293
by a measured amount of monochromatic ultraviolet revealed that one Steenbock unit of vitamin D is equivalent to 3.33 International units.
Acknowledgment The authors acknowledge their indebtedness to Farrington Daniels for helpful counsel in the experimental work.
Literature Cited (1) Daniels, F., and Heidt, L., J . Am. Chem. SOC.,54, 2381 (1932). (2) Fosbinder, R. J., Daniels, F., and Steenbock, H., Ibid., 50, 923 (1928). (3) Hess, A. F., and Anderson, R. J., J . Am. Med. Assoc., 89, 1222 (1927). (4) Kon, S. K., Daniels, F., and Steenbock, H., J . Am. Chem. SOC.,50, 2573 (1928). (5) Marshall, A. L., and Knudson, A,, Ibid., 52, 2304 (1930). (6) Russel, W. U., and Taylor, M. W., J . Nutrition, 10, 613 (1935). (7) Steenbock, H.. Kletsein. S. W. F.. and Halpin. J. G., J . B i d . Chem., 97, 249 (1932). RECEIVED February 6, 1936. Published with the permission of the Director, RTisconsin Agricultural Experiment Station.
The Smoke Tendency of Refined Kerosene and Its Determination JOHN B. TERRY
AND
EDWARD FIELD, Standard Oil Company of California, San Francisco, Calif.
B
EFORE the present century, kerosene was not so well refined as it is now, various types of crude oils being used for its manufacture, often without segregation. The product sold varied widely in such characteristics as viscosity, capillarity, boiling range, and sulfur content, all of which are now closely controlled. The researches of Edeleanu were of inestimable value in showing that the aromatic hydrocarbons, which are removed from the crude kerosene by his well-known sulfur dioxide process, were responsible to a large extent for smoky flames, the removal of such compounds resulting in much larger flames having greater luminosity. Even though much better grades of kerosene have been marketed during the last 25 or 30 years, it is only within the last 15 years that attention has been paid to the quality of kerosene as judged by its tendency to smoke (1, 4, 7 , 8, 9). Burning tests have been devised and standardized with a view to determining oil consumption, wick incrustation, chimney fouling, and luminosity (candle power), all of which have produced very enlightening and indispensable information concerning quality. Oils are usually tested in lamps of varying design, depending on the particular purpose for which the oil is to be used. Lamps for such burning tests are not well adapted for smoke tendency tests because chimneys and flames differ in shape and size, so that grading of the oil is rendered difficult; large round wicks 2 or 3 inches in diameter, which are very susceptible to “pitting,” are often used; a considerable personal element is introduced; it is difficult, using such lamps, to grade oils to detect small differences in refinement; and test lamps of the same type are apt to vary when made by different manufacturers, so that an oil may burn with a different efficiency in two supposedly similar lamps.
Development of Smoke Tendency Tests In recent years, investigations of the chemical constitution of kerosenes of different degrees of refinement have shown that the constitution of any kerosene is closely related to its tendency to smoke in a given lamp. This is well illustrated
by the work of Minchin (6, 6) who states that the tendency to smoke is directly proportional to its aromatic or naphthene content. I n the case of homologous series, the tendency to smoke, with the exception of the paraffins, decreases with the increase in the number of carbon atoms or boiling point. The naphthene class has a flame height about three times that of the aromatic, and the paraffin about nine times that of the aromatic, Jackson (3) has further pointed out that a satisfactory test for smoke tendency gives all we need to know for test purposes regarding the percentages of aromatics, naphthenes, and paraffins present in the kerosene. Therefore, a reliable test for smoke tendency would be of great value in controlling the burning qualities of illuminating oils. On the basis of experimental work done in England, the Institution of Petroleum Technologists has standardized on a smoke point test for kerosene tZ), I. P. T. Serial Designation K.36.
Davis Factor Lamp I n the United States, similar experimental work has been proceeding for more than 10 years. One of the earliest lamps to be developed was the Davis factor lamp (Figure l), designed in 1923 by R. F. Davis of the Standard Oil Company of California. The basis of its design was that as increased refinement enables a given kerosene to burn with a higher flame in any one lamp without smoking, a lamp which produced a long narrow flame would be adaptable for control of refinement. The tall flame would be sensitive to oils differing by small increments of refining, and the heights of such a flame could be accurately read and would give a good index of quality. The original design of this lamp included a brass fount of approximately 4-ounce capacity, and regulating wick gears actuated by a larger milled wheel. A special cylindrical glass chimney 7 inches long, 1 inch in outside diameter, and graduated in tenths of an inch was mounted on a brass screen which allowed entrance of air t o the flame. In making a determination, the flame was turned up until a “tail” of smoke just appeared. The flame was then slowly turned down until this tail just disappeared,
