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731. Rats 732 and 720, which received 2 and 3 mg. of dogfish liver oil daily, respectively, gained a t the same rate as Rat 738, which received 1 mg. per day. As will be noted, Rat 722 made an extremely rapid growth on 4 mg. of oil daily.
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It is apparent from the results obtained from this group of experiments as a whole that 1mg. of the dogfish liver oil under investigation contained sufficient vitamin A to meet the needs of albino rats for growth.
Determination of Moisture in Wheat and Flour’ Part I11 By Harry Snyder and Betty Sullivan RUSSELL-MILLER MILLINGCo.,MINNEAPOLIS, MI“.
The Brown-Duvel test for the determination of water in cereals gives results for wheat comparable with wateroven drying, against which i t is standardized. The double-walled flask distillation method proposed for the rapid determination of moisture in flour and meal is more difficult to operate t h a n the single-walled flask, and shows a tendency to yield slightly higher results t h a n water-oven drying, against which it is standardized. The method is consistent in its application in that i t yields results on a parity with the moisture test for wheat, as specified in the official grading of wheat provided under the Grain Standards Act of the United States. The operation of the double-walled flask test requires so much time, constant attention, and technical skill that i t is unsuitable for industrial purposes such as flour mill control.
There is need of a rapid, consistent, and reasonably accurate method for determining total water in wheat and flour. Since different methods give different results, in order to maintain a parity of moisture content between the raw material (wheat) and the finished product (flours and feeds), i t is necessary to make a correction when using different methods of testing. Such a provision is made in the official moisture test methods for grain and other substances. Nine moisture tests by the water-oven method, the basis of standardization of the official moisture test methods for wheat and flour (Brown-Duvel), gave a n average of 11.76 per cent moisture, and the same flours tested by the vacuum-oven method yielded a n average of 13.70 per cent moisture.
N PARTS I and I1 of this series are reported moisture results when flours are dried in water, air, and vacuum ovens, and over desiccating reagents without heat.2 The present article gives moisture results obtained by one of the distillation methods (Brown-Duvel), where the flour and the grain are heated in distillation flasks in the presence of hydrocarbon oils and the distillate (water) is collected and measured. The Brown-Duvel or official moisture method used in testing wheat and other grains in connection with the enforcement of the Grain Standards Act of the United States is standardized against water-oven drying described as follows: “The data here shown were secured by checking the results with moisture determinations, made by drying to constant weight in the common type of double-walled oven filled with water maintained a t the boiling point,” except in the case of flaxseed as noted.3 A special flask for the determination of water in flour and meal, also official and standardized against water-oven drying, is described by the Bureau of Plant Industry, U. S. Department of Agric~1tui-e.~ The Brown-Duvel Moisture Test Applied to Wheat The Brown-Duvel method was originally proposed as a quick method for the determination of water in corn, particularly wet corn, as it dispenses with grinding before testing, a process often resulting in a change of moisture content. The passage of the U. S. Grain Standards Act emphasized the need of a quick, reasonably accurate method for use by those unskilled in chemical analysis. Hence the Brown-Duvel method was extended to include other grains. Directions for its use are given in various publication~.~.586As the method is standardized against water-oven drying, it follows that any
observations concerning comparative differences of results between water and vacuum-oven drying would also apply with equal force to differences between the Brown-Duvel test and the vacuum method of drying. The Brown-Duvel test requires rigid adherence to directions. Uniformity as to time, rate of heating, exact temperature for extinguishing the flame, cooling, disconnecting the distillation flask, and other details are noted in the publications of the Research Laboratory, for Grain Investigations, U. S. Department of Agriculture.6 The use of other moisture methods giving results “equivalent to or are corrected to conform to those secured by the standard method specified” is also sanctioned in the enforcement of the Grain Standards Act. Hence it is important that any deviations arising from the use of moisture methods other than the standard be determined so that all necessary adjustments can be made. About a year after the Brown-Duvel method was published6 a special copper flask was devised and used in this company’s laboratory. It was provided with a block tin delivery tube attached to a threaded metallic stopper into which a standard thermometer was fitted, thus dispensing with rubber stoppers and connections so readily affected by oil and high temperature. The flask was patterned after the ordinary copper oxygen-generating flask supplied by dealers in chemical apparatus and was standardized against the official glass flask, also against water-oven drying. This copper flask was found more durable for testing wheat, particularly by laymen, than the glass flask. Since 1909 this company has had in daily use a number of glass flask, and copper flask, Brown-Duvel testers, heated in various ways-by gas, alcohol lamps, and electricity. Although discordant results by different operators in different localities not infrequently occur, reasonably concordant results are secured when all tests are made in a uniform conventional way, and these tests in turn have been found to check satisfactorily against water-oven drying.
I
Received September 8 , 1924. 16, 741, 1163 (1924). a U.S. Depf. Agr., Bur. Plant Industry, Circ. 72, p. 11. 4 U.S. Depf. Agr., Bull. 66. ‘Bur. Planf Industry. Bull. 99 (1907). 6 U.S. G . S. A . , Mimeographed Circular, G . I.-15 (1922). 1
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Among the desirable features of the Brown-Duvel test are rapidity and its direct application to whole grains. The wheat is heated in oil for 20 minutes, until a temperature of 180" C. is reached for glass flasks, or 185' C. for copper flasks, when the flame is extinguished and a subsequent rise of temperature of several degrees occurs. The water collected as distillate is less than that liberated in the flask from the wheat, because some water condenses and remains in the condenser and on the delivery tube and walls of the flask. Additional heat will drive this water over, however. As the distillation proceeds particles of oil carried with the steam are deposited on the surfaces of the flask, delivery tube, and condenser. Inasmuch as water readily adheres to an oily surface, the water mechanically held in the apparatus by the oil film and that entrained in the oil are in excess of that delivered and measured in the cylinder. The test is regulated so as to yield a measurable distillate based on wateroven drying as noted, and this is not the total water of the wheat-namely, the ordinary forms of free or hygroscopic moisture plus other forms. That water remains in the distillation apparatus can be shown by making a test according to directions, then reconnecting, heating the flask again to 180" C. for glass flask, or 185' C. for copper, and cooling. On reheating the first time from 0.2 to 0.5 per cent more water is obtained. This process may be repeated four or five times, less additional water being obtained each time as the following typical example shows: first reheating, 0.5 per cent more water; second reheating, 0.3 per cent more water; third reheating, 0.2 per cent more water; fourth reheating, 0.2 per cent more water. In standardizing the Brown-Duvel method, the wheat is not ground when dried in the water oven. It is quite a problem to obtain an exact moisture determination on unground wheat by water-oven drying, even with a long drying period, because in drying whole kernels the heat does not penetrate the endosperm to the same extent that it does the outer covering. When the wheat is ground just before drying a change of water content is likely to occur. I n standardizing the Brown-Duvel method against water-oven drying, time is a very important factor. This varies considerably with the type of wheat dried, from about 70 to 140 hours. An example of the gradual loss of water when whole-wheat kernels are dried in a water oven, drying chamber a t 96' C., is given in Table I. Table I-Loss
of Moisture in Water-Oven Drying of Wheat Kernels Drying period Moisture Hours Per cent 11.84 19 11.39 12.65 43 12.01 12.63 48 11.98 12.75 67 12.17 .~
-4verage 67 hours 72 Average 72 hours
12.46 12.72 12.24 12.48
At the end of 72 hours one of the tests showed a slight loss and the other a gain in weight over the results a t 67 hours. Throughout the test the duplicates were consistently about 0.5 per cent apart, suggesting that the distribution of moisture was not even in all the wheat kernels. The Brown-Duvel test gave 12.50 per cent moisture against 12.48 per cent obtained by water-oven drying. This is a fair example of similarity of results by the Brown-Duvel test (glass flasks, made in conformity with the regulations) and by water-oven drying. I n previous articles of this series it has been shown that different methods for the determination of moisture in flour give different results. I n order to gain some idea of the extent to which further losses of moisture may occur in excess of water-oven drying, the lvheat yamples, having reached
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so-called constant weight in the water oven, were dried 19 hours longer in an air oven a t 133"C. At the end of this time an additional loss of 1.61 per cent moisture occurred, making a combined loss for the two methods of 14.09 per cent Then followed 5 hours' additional drying in a vacuum oven (100" C., vacuum 400 mm.) with a further loss of 0.22 per cent, showing an excess of 1.83 per cent of water for vacuum and air-oven drying over water-oven drying. This difference is similar to the difference observed when drying flour in water and in vacuum ovens. The action of the heated oil upon the wheat in the BrownDuvel test was also studied. The dark brown wheat residue left in the distillation flask was separated from the oil by filtration and soaked and washed in anhydrous ether until free from oil. The original wheat tested 2.58 per cent nitrogen (dry basis), and the wheat residue 2.36 per cent. The strawcolored wheat distillate was tested, 100 cc. of a composite of several distillates being used for the purpose. The unfiltered distillate yielded 0.218 per cent nitrogen, while the filtered distillate yielded 0.185 per cent, corrected for blanks. The loss of nitrogen from the wheat when distilled in the hydrocarbon oil and its presence in the distillate suggest that chemical changes affecting the composition of some of the wheat proteins have taken place. However, these changes incident to distillation, although of interest, do not seem to affect the final results, since the test is standardized to deliver a measured amount of distillate comparable with water-oven drying. Leavitt's work on drying wheat in the laboratory of the Bureau of Chemistry is of interest.' He used a waterjacketed vacuum oven and air-heated vacuum oven for drying ground wheat. I n the vacuum oven a t 100" C. and a vacuum of about 610 mm. (24 inches) of mercury, he obtained upwards of 1 per cent more moisture than when the temperature was maintained at 97" C., the temperature of the waterjacketed vacuum oven heated with boiling water. With the same vacuum he also reported higher results a t 102" C., than a t 100" C., his concluding statement being: No attempt is made in this article t o explain why it is that a long slow drying, a t a lower temperature than 100" C., did not give the same results as a t 100" C. Theoretically, we should expect the same results, but in the hands of the writer they hardly gave within 2 per cent of the same results, and further heating showed quite a rapid increase in weight of all samples examined, especially a t the lower temperatures.
The physical explanation offered by Nelson and Hulett8 also the loss of moisture at certain fixed points noted in previous articles of this series as being indicative of hydration changes, offer a feasible explanation of this phenomenon of failure of long heating at a lower temperature to be as effectual as a shorter heating period a t a higher temperature. So far as the determination of moisture in grain is concerned, it is unnecessary to entertain any theories as to why different methods of testing give different results, but only to recognize that such differences occur. A fair statement of the principle involved in the application of moisture tests to grain is made in the introduction of the "Report of Official Test Methods for Grain and Other Substances.'1B Owing to the numerous methods of making moisture determinations and the wide variations in the results obtained by the different methods, the Brown-Duvel moisture tester and its method of use described in Bureau of Plant Industry, Circular 72 and Department Bulletin 56, issued by the United States Department of Agriculture have been specified under the provisions of the United States Grain Standards Act as the standard on which the moisture factor in theofficial grain grades are based. This in no way
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precludes the use of other methods of making moisture determinations so long as the results of the other methods are equivalent to or are corrected to conform t o those secured by the Standard Method specified.
Hence the importance of determining any deviation in results arising from the use of other than the Brown-Duvel method, which in turn is standardized agahst water-oven drying in the manner designated. Use of Double-Walled Flask for Determining Moisture in Flour This method was published in 1914 as a rapid means for the determination of moisture in flour and meal,4 but it does not seem to have come into general use in flour-milling operations, doubtless because flour, owing to its fine state of subdivision, gives up its water readily enough by heating in an oven, unlike wheat kernels which cannot be dried in so short a time by ordinary methods. The double-walled flask is in reality a single flask provided with an oil-bath jacket into which 150 cc. of oil are placed. The original directions specify in detail the gradual rise in temperature necessary to be observed in making the test. The directions state : The proper temperature a t which to extinguish the flame in the apparatus was found by checking duplicate samples in the common type of double-walled water oven that was 11 inches high, 11 inches wide, and 10 inches deep, outside dimensions, and having a 1-inch space between the outer and the inner walls for the water. The water in the oven was kept boiling by two gas flames and was kept a t a uniform height. The different substances were allowed to remain in the oven until they came to constant weight.
An effort was made to compare the double-walled flask method for determining moisture in flour with water- and vacuum-oven drying. Flour, being finely divided, deports itself mechanically in a different way when heated in oil than do wheat kernels. It was found necessary to clean the distillation flask thoroughly after each test-a difficult operation because of the yellowish brown paste residue of flour and oil that sticks tenaciously to the walls of the flask. It was impossible to get rid of the flour residue by pouring out the oil and flour while still hot or by rinsing the flask with fresh oil as directed. The procedure adopted was to clean the flasks successively with petroleum ether, alcohol, and hot water, and then to dry thoroughly and cool to room temperature before using again. Unless this is done the variable portions of old flour residue left in the flask constitute a disturbing factor in standardization. In making the distillation there is always lag of moisture. When the test is made according to directions and the flask is reconnected and reheated, more water is secured, as noted in the case of the single-walled flask; the amount may be as high as 2 per cent, depending upon the technic followed. The original description of the double-walled flask specifies the use of either copper or glass. Preference seems to be given to copper, however. Although the temperature for extinguishing the flame is 190" C., the point of cooling before taking the reading is not specific. Later directions6 give this temperature as 160" C., but they do not specify interval rises in temperature as do the original directions.' If the latter directions supersede the former, then the interval rises in temperature are to be considered as fully offset by the conditions described in the later directions. A typical test made in accordance with the specifications' gave the following results: Per cent water Flame extinguished a t 190' C. 12.5 Subsequent rise to 1 9 5 O C. 12.7 Drop to 160' C., disconnected and read a t 150' C. 12.9
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Whereas, the double-walled flask yielded 12.9 per cent water, the water-oven gave 12.5, the vacuum-oven (100" C., 400 mm. Hg) 14.30, and one hour drying in an electric air oven a t 135" C.,14.40 per cent. I n making the double-walled flask tests, the cleaned flasks were rinsed with oil before using and the condenser was primed in accord with directions. Frequently, tests are lost by foaming and other causes, and it is often difficult to get satisfactory tests within the specified time limits. Tests were also made according to the original directions, observing as closely as possible the interval rises in temperature, the "cut-off" being made a t 190" C., since the "Method for Finding the Proper Temperature" was by water-oven drying in the manner previously described. Nine tests made in this way gave an average of 12.3 per cent moisture against 11.78 per cent by water-oven drying. The later directions (in Circular 15) gave results agreeing more closely with water-oven drying than the original directions. In the final analysis, differences in moisture tests between the official double-walled flask and vacuum drying must be the same or adjusted to the same basis as the differences between water- and vacuum-oven drying, inasmuch as the official double-walled moisture flask test is standardized against water-oven drying. Although comparative tests of water- and vacuum-oven methods of drying are reported in preceding*articles, a few additional tests are given in Table I1 as they also indicate the differences between the doublewalled official moisture test and vacuum drying moisture tests of flour. Table 11-Moisture
Sample
Average Difference
C o n t e n t of Flour by W a t e r - and Vacuum-Oven Methods Vacuum oven 5 hours, Dried in w a t a oven 100' C., 400 mm. Per cent Per cent 11.98 13.32 12.08 13.91 12.10 14.19 11.25 13.55 11.21 13.35 11.43 13.49 12.20 14.07 12.02 13.99 11.56 13.40 11 76 13 70 1 94 per cent
These results me quite similar to those reported in Part I of this series, where twenty-seven flour moisture tests, vacuum-oven drying, gave 1.87 per cent more moisture than water-oven drying. The Flour Residue and Distillate The heated oil, acting on the flour in the distillation flask a t a temperature of 190" C., and with a subsequent rise to 195" C. or more, completely changes the appearance and composition of the flour. The paste-like, sand-, or tan-colored flour residue separated from the oil by a cloth filter was treated with anhydrous ether until free from oil. The ether was removed by drying over sulfuric acid. This flour residue was largely soluble in water, yielding an abundant alcohol precipitate. Microscopic examination showed few unaltered wheat-starch granules having a characteristic starch structure, but numerous particles giving an iodine color reaction, suggesting amylodextrin. All the natural enzymes of the flour were destroyed by the distillation process. Flours containing originally 2.38 and 2.77 per cent nitrogen (dry basis) yielded residues after distillation containing 2.20 and 2.60 per cent, respectively, of nitrogen. The flour moisture distillate was practically colorless and more free from wheat odor than the straw-colored wheat distillate. The distillate from the flour contained an appreciable amount of nitrogen derived from the proteins of the flour.
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Need of a Rapid Method for Determining Flour Moisture
There is great need of a rapid, consistent method for determining flour moisture suitable for use by the layman. The time and skill required for operating the double-walled flask test do not permit its use for flour-mill control work. The test is correct in swfar as the basic commercial principles are involved in that a parity is maintained between the moisture in the wheat a miller uses and the resultant flours, since the tests for both wheat and flour are standardized in the same way. It would seem feasible to determine the moisture as total volatile products by drying flour in a strictly empirical way, heating to a higher temperature than the boiling point of water, as 125"C. to 135"C.,for a short period as one hour, and then applying an established factor for correcting the results to conform to the water-oven basis of the standard official methods as specified. Higher results are obtained by such a method than by the standard Brown-Duvel method for wheat or flour. As previously stated, such a procedure is in harmony with the principles governing moisture tests under the provision of the U. S. Grain Standards Act. I n this use of other methods giving higher results than the Brown-Duvel method, standardized against water-oven drying, no theories need be entertained as to the various forms in which water may exist in wheat and flour or why different moisture results are obtained at differenttemperatures and by different methods. As a chemical determination it would seem preferable to carry the drying to the point where rea-
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sonably constant results are obtained (loss of all volatile matter), rather than to stop a t a point where variable moisture results are obtained. Such additional losses a t higher temperatures may be due largely to physical causes or in part to water held in chemical combination with the carbohydrates and proteins as water of hydration or to other causes. Confusion arises when the Brown-Duvel method is used for testing wheat and the vacuum-oven for testing flour and no correction is made to reduce the results to a common basis. A miller's tempered wheat may test 14 per cent moisture (Brown-Duvel), his flour 14.5 per cent (vacuum basis), and a t the same time an appreciable loss of moisture may occur in milling. When the results are reduced to a common basis, however, the flour on the Brown-Duvel basis is 12.5 to 12.75 per cent moisture; or, placing the results on an exhaustive vacuum basis, a 14 per cent Brown-Duvel test is equivalent to about 15.75 per cent vacuum oven test. Without adjustments or a statement as to methods used, with the limitations of each, the chemist is discredited if he reports more moisture in the flour than is present in the wheat a t the rolls and the application of chemical tests to flour-milling are viewed with suspicion. Furthermore, dry matter comparisons of flour-milling operations between wheat used and flour and by-products recovered are not possible when the wheat is tested by one method and the flour by another, unless the results are corrected by the use of accurate factors for maintaining a parity between wheat and flour moisture tests.
Diphenylamine as Indicator in the Reduction of Vanadic Acid' By N. Howell Furman PRINCETON UNIVERSITY, PRINCETON, N. J.
K
NOP2 has s h o w n Diphenylamine has been shown to be a sensitive indicavanadyl salts in the concentrations encountered in that diphenylamine tor of the end point of the interaction between vanadic acid is a suitable indicaand ferrous iron. This fact, in conjunction with wellanalytical practice. tor of the end point of the established analytical methods, affords a simple method Nature of Color Reaction reaction between ferrous for the direct determination of vanadium in steels, ferroAccording to Kehrmann iron and bichromate, with allovs. I . and ores. and for the indirect determination of and others4 the deep blue application to the determichromium in steels. substance which is formed nation of either iron or by the action of oxidizing chromium. He has applied the method to the indirect determination of antimony.3 agents upon diphenylamine, is an imonium salt of N , N'-diI n the present investigation it was found that diphenyl- phenylbenzidine. Various reducing agents destroy the blue amine is a very sensitive indicator of the end point of the color with the formation of greenish quinhydrone salts. The oxidation of diphenylamine by vanadic acid has been reaction between ferrous iron and vanadic acid: previously reported by Hinrichs6 in a discussion of the inV O I - - - + Fe+ + 6H++VO+ + Fe+ + 3H20 terference of a number of oxidizing agents in the diphenylA very slight excess of ferrous sulfate causes the color of the amine method of detecting nitrates. Meaurio6 made use of solution to change from deep blue to the residual shade, the diphenylaminervanadic acid reaction in the colorimetric generally greenish, which is due to the salts present and to estimation of traces of vanadium in water. the reduction products of the indicator. The color change is It seems clear that the oxidation of diphenylamine yields not instantaneous. A convenient warning of the approach an oxidation-reduction indicator whose position on the normal of the end point is usually given by a slight fading of the color potential scale must lie between that of the vanadate-vanadyl within a drop or two of the equivalent point. Titrations are equilibrium and that of ferric-ferrous potential. Presumably, made at room temperature. Strong heating destroys the a number of reducing agents other than ferrous sulfate might blue color. No interference is caused by the blue color of be used. It was found qualitatively that diphenylamine +
'
+
+
+
1 Presented by title before the Division of Physical and Inorganic Chemistry at the 67th Meeting of the American Chemical Society, Washington, D.C . , April 21 t o 26, 1924. 2 J. A m . Chem. Soc., 46,263 (1924). 8 Z.anal. Chem., 69, 81 (1923).
4 Kehrmann and Miciewicz, B e y . , 46,2641 (1912); Heloetica Chim. Acta, 4,949 (1921); Kehrmann and Roy, Be?., SSB, 156 (1922). 5 Bull. SOL. chim., SS, 1002 (1905). 0 Anales SOC. guirn. Argentina, 5, 185 (1917),through C. A . , 12, 1221 (1918).
