Vol. 1, No. 3
ANALYTICAL EDITION
136
Improvements in the Denighs Colorimetric Method for Phosphorus and Arsenic’ Emil Truog and A. H. Meyer DEPARTMENT OF SOILS, UNIVERSITY OF
A detailed study has been made of the Denigbs colorimetric method for phosphorus. The concentrations of reagents a t which phosphomolybdate gives a maximum blue color and interfering colors due to silicomolybdate and ammonium molybdate are eliminated have been definitely established. A greatly improved method of preparing and storing the stannous chloride solution used in the determination is described. I t has been found t h a t ferric iron when present in concentrations greater than 6 p. p. m. markedly depresses t h e formation of the blue color and also gives rise to greenish tints. Reduction to the ferrous condition eliminates this trouble. Titanium may be present up to 20 p. p. m. without interfering with the color.
WrSCONSIN, MADISON, WIS.
Salts of aluminum, manganese, calcium, and magnesium may be present in large amounts without interfering. Even nitrates may be present in considerable amounts without causing difficulty. An improved procedure has been developed which is rapid and more sensitive and accurate than the old. The method appears to be one of the most satisfactory in the field of colorimetric methods and should find wide application in biology, agriculture, and industry. The application of t h e same method to t h e determination of arsenic should find extensive use. Procedures for t h e single and joint determination of arsenic and phosphorus in a solution are given.
....... ...... N 1920 Denig&s( I ) ,* a French investigator, called attention to the extremely sensitive blue color reaction that occurs when the molybdate of phosphorus or arsenic is reduced with stannous chloride under suitable conditions. He then utilized this reaction as a basis for a colorimetric method for phosphorus and arsenic. At about this same time investigators in this country developed methods along similar lines, but employing different reducing agents. It appears that stannous chloride as utilized by DenigBs is far superior for this purpose than the other reducing agents suggested. The method of DenigBs has been successfully used by a number of investigators who have made slight modifications in the procedure. For a list of references and a review of the literature the reader is referred to the recent work of Parker and Fudge
I
(8 0
The Method I n the method as finally evolved by previous workers two reagents are employed, known as (A) and (B), which are compounded as follows: (A) consists of 100 cc. of 10 per cent ammonium molybdate, 150 cc. of concentrated sulfuric acid, and 150 cc. of distilled water. (B) consists of a stannous chloride solution prepared fresh each day by dissolving 0.1 gram of mossy tin in 2 cc. of concen-
trated hydrochloric acid to which one drop of 4 per cent copper sulfate has been added. Solution is hastened by gently heating after which a dilution to 10 cc. is made.
In the regular course of analysis an appropriate amount of solution (A) is added to the unknown solution to be tested for phosphorus and to the standard. Ammonium phosphomolybdate is thereby formed, but because of the high dilution a precipitate is not apparent. A few drops of solution (B) are then added, causing a reduction of the molybdenum which is in combination with the phosphate but not of that added in the excess of ammonium molybdate reagent. However, if the acidity is too low the ammonium molybdate will of itself give an intense blue color when stannous chloride is added. On the other hand, if the acidity is too high a blue color is not produced even if phosphates be present, probably because ammonium phosphomolybdate is not formed a t the ‘Received March 1, 1929. Pubfished with the permission of the director of the Wisconsin Agricultural Experiment Station. Italic numbers in parenthesis refer to literature cited at end of article.
*
high acidity. I n other words, stannous chloride easily reduces molybdenum in combination as a salt such as ammonium molybdate or ammonium phosphomolybdate, while under conditions in which it exists as the free acid reduction does not take place. Object of Study The writers were unable to find that a systematic study had ever been made regarding the influence of concentration of sulfuric acid, ammonium molybdate, and stannous chloride on the intensity of color produced. A study of this kind was therefore made with the hope that the method might thus be improved. Preliminary tests showed that the molybdenum in silicomolybdate, which is apparently formed a t low acidities, is also reduced by stannous chloride and gives the characteristic blue color. Because silica is often present in solutions to be tested for phosphate, a study was also made of the conditions under which silica might interfere. Preparation and Storage of Stannous Chloride Solution I n order to facilitate the work an effort was made a t t h e start to simplify the preparation and use of solution (B)the stannous chloride solution. It was found that ordinary c. P. SnCl2.2H20 dissolved in acidified water gives as good or better results than the solution prepared from metallic tin. The necessity of adding copper sulfate, which sometimes gives troublesome brownish tints t o the blue color, could thus be eliminated. It was also found that the stannous chloride solution can be preserved indefinitely if oxygen is excluded by covering with a layer of mineral oil. It was found advantageous to prepare the solution as follows: Twenty-five grams of SnC12.2Hz0 are dissolved in 1000 cc. of solution consisting of 900 cc. of water and exactly 100 cc. of concentrated hydrochloric acid. After complete solution, filtering is desirable if a turbidity exists. The solution is conveniently stored in a bottle fitted with a siphon or, better still, a s t o p cock outlet’ a t the bottom. The point of the outlet in either case is constricted and protected with a rubber cap when not in use. A layer about 5 mm. thick of white mineral oil sold under the trade name of “Stanolind” is floated over the surface. It was found desirable to use this solution in the proportion of six drops per 100 cc. of standard or unknown and this amount will hereafter be designated the regular amount.
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I N D U S T R I A L A N D ENGINEERING CHEMISTRY
July 15, 1929
Effect of Acidity on Development of Color from Ammonium Molybdate
A test was next made of the influence of acidity on the development of color with ammonium molybdate in the absence of phosphate. For this study there was made a 5 per cent water solution of ammonium molybdate, a normal solution of sulfuric acid and a standard solution containing 0.5 p. p. m. of phosphorus. The standard was made up to contain 40 cc. of normal sulfuric acid and 2 cc. of 5 per cent ammonium molybdate in 100 cc. Previous tests had shown that this amount of sulfuric acid entirely prevents the formation of blue color from ammonium molybdate itself when present in the concentration indicated. These concentrations of sulfuric acid and ammonium molybdate will hereafter be called the regular amounts. The results of this study are given in Table I. They show clearly that a t low acidities ammonium molybdate of itself gives an intense blue color and that it takes a t least 35 cc. of normal sulfuric acid in 100 cc. to prevent the formation of all but traces of blue color from this source. Table I-Influence of Acidity on Reduction of Molybdenum a n d Formation of Blue Color When Molybdenum Is Added a s (NH4)zMoOd in Absence of Phosphate STANDARD WITH STANDARD WITH COLOR DUE COLORDUE N H&OI TO 0.5 P. P. M. N His04 TO 0.5 P. P. M. ADDEDPER PHOSPHORUS ADDED PER PHOSPHORUS 100 cc. REQUIRED TO 100 cc. REQUIRED TO OF TEW MATCH 25 cc. OF TEST MATCH 25 CC. SOLN. TESTSOLN. SOL% TESTSOLN.
cc. e e
4 6
7.5 10.6 14.5 19 25 25 30
cc.
cc.
cc.
0 100
35 36 37
0 0 0
212 237 _. .
212 150 100 56 32 25 10
3R _.
39 40 41 42 43 44 45
n 0
0 0 0
137
The influence of acidity on the development of a blue color due to the presence of silica was next studied. The standard stock solution of silica just described was employed to make up test solutions, all of which contained 700 p. p. m. of silica but varying amounts of acid as indicated in Table 111. The regular amounts of ammonium molybdate and stannous chloride were employed. The data show clearly that a t low acidities silica produces an additional blue color over that of ammonium molybdate alone, while at the higher acidities no color is produced. The addition of 40 cc. of normal sulfuric acid per 100 cc. of test solution is sufficient to prevent the formation of a blue color due to the presence of 700 p. p. m. of silica. This concentration of silica is very much above the maximum that is ordinarily encountered in solutions to be analyzed for phosphorus. Only in unusual cases is it therefore necessary to remove silica. Effect of Acidity on Formation of Blue Color by Phosphates
The influence of acidity on the development of the blue color due to the presence of phosphates was next studied. The results of this study (Table IV) show clearly that at too low an acidity the color is too intense due to the influence of ammonium molybdate itself, while at too high an acidity the color is too weak due probably to incomplete formation of ammonium phosphomolybdate. of Acidity on Intensity of Blue Color Produced by Phosphate Test solutions in all cases contained 0.5 p. p. m. of phosphorus and the regular amounts of (NH4)zMoOd and SnClz. Standard solution contained 0.5 p. p. m. of phosphorus and the regular amounts of acid and other reagents. N HzSOa STANDARD SOLN. N HzSO4 STANDARD SOLN. EMPLOYED IN REQUIRED TO EMPLOYED IN REQUIRED TO 100 CC. OF MATCH 25 CC. 100 CC. OF MATCH 25 CC. TESTSOLN. TESTSOLN. TESTSOLN. TESTSOLN. Table IV-Influence
0
cc.
cc.
cc.
cc.
0
30 35 36 37 38 39 40
29 27 27 27 26.5 26 25
41 42 43 45 68 80
24.5 24 23.5 23 7.5 0.5
0
Further investigation showed that if the concentration of ammonium molybdate is increased over that existing in the tests just described, additional sulfuric acid is necessary to prevent the reoccurrence of the blue color. It was also found that the use of 2 cc. of 5 per cent ammonium molybdate in 100 cc. of solution was a convenient and satisfactory amount to use as regards the development of a maximum blue color from phosphomolybdate. Formation of Blue Color from Silicomolybdate
General Discussion
I n Figure 1 the data of Tables I, 111, and IV are shown graphically. A study of this figure shows clearly that with an acidity of 40 cc. of normal sulfuric acid per 100 CC. of test solution the color developed is due entirely to phosphate pro-
The production of a blue color due to the presence of silica was next studied. A standard solution of silica was prepared by fusing silica of the highest purity with sodium carbonate and after solution diluting to definite volume. Appropriate volumes of this solution were then used to give the concentrations indicated in Table 11. The data in this table show that when the regular amounts of reagents are used silica can be present up to 700 p. p. m. without causing the develop ment of a blue color. Table 111-Influence of Aciditv nn Intensity of Blue Color-Pr~diicced Table 11-Production of Blue Color by 700 p. p . m. of SiOa in Presence by Silica in Presence of Regular of Regular Amounts of (NH4)zAmounts of Reagents Moo4 a n d SnClz STANDARD SOLN. N HlSO4 STANDARD SOLN. CONTG. 0.5 P. P. M. cc. OF NORMALHzSO, IN 100 cc. OF TEST~~OLUTION EMPLOYED IN CONTG. 0 5 P. P. M. PHOSPHORUS 100 cc. TEST PHOSPHORUS REQUIRED TO MATCH SOLN.CONTG. REQUIRED TO Figure 1-Influence of Acidity o n the Development of Blue Color in 25 cc. SILICA 700 P. P. x. MATCH 25 cc. t h e Solutions Indicated When Treated with Appropriate Reagents Si02 PRESENT TESTSOLN. SILICA SILICA TEST SOLN. cc. cc. CC. vided the silica present is not in excess of 700 p. p. m. and the 10 0 15 112 100 0 regular amounts of ammonium molybdate and stannous 20 84 500 0 25 62 chloride are used. This acidity is probably a little higher 700 0 30 40 750 0.5 35 20 than necessary, but owing to unavoidable errors in measuring SO0 1.0 40 0 45
0
reagents in routine analysis it is better to aim at using this
ANALYTICAL EDITION
138
alight excess. The influence of this slight excess of acidity on the color due to phosphate is very slight and is of no consequence in a determination since the same excess is used in the standard. As is evident from the data presented, it is important to add the acid and ammonium molybdate and mix thoroughly before the stannous chloride is added. If the molybdate is added after the stannous chloride, some of it may become reduced and give rise to a color before it becomes mixed with the bulk of the acidified solution to be tested. When the reagents are used in the proportions indicated, a maximum blue color is developed immediately. Procedures previously described require 5 minutes for full color development. About 10 minutes after the color is developed it begins to fade. It may be regenerated, however, and kept practically constant for an hour by adding an additional drop of stannous chloride every 10 to 15 minutes. An attempt was made to find a substance which on addition would preserve the blue color. Gelatin was the only one tried that had a preservative effect. Its use is not permissible because it produces a cloudiness in the solution. A layer of lubricating oil placed over the surface prevents the entrance of oxygen and preserves the color somewhat, but its use is not practicable. Saturation of the solution with hydrogen from 8 tank helped to preserve the blue color, but not sufficiently t o make i t worth while. Effect of Ferric Iron on Color
The influence of various elements on the development of the blue color was next studied. Table V, showing the influence of ferric iron, shows conclusively that ferric iron affects the color markedly. I n most routine analytical work the presence of 4 to 6 p. p. m. is probably not serious. The ferric iron not only decreases the intensity of the color, but also produces troublesome greenish tints. It was found that ferrous iron does not have the undesirable effect of ferric iron. Therefore, it was thought that reduction of the ferric iron might solve the problem. Of the methods tried, reduction i n a Jones reductor using metallic cadmium gave the best results and may be used successfully to eliminate the undesirable effects of ferric iron. This reduction should be made just previous to the addition of the ammonium molybdate. 'Tahle V-Influence of Ferric Sulfate on Intensity of Blue Color in Colorimetric Determination of Phosphorus REGULARSTANDARD REOUlRED TO MATCH
Pea(S04)z ADDEDIN TERMS OF Fe P . 9. m. 2 4 6
a
10 50 100
23 c c . STANDARD TO WHICFIIRON BEENADDED
HAD
COLOR
CC.
24.5 23.5 23 22.5 21 16 8
Slight greenish tinge Distinct greenish tinge Distinct greenish tinge Distinct greenish tinge Distinct greenish tinge Distinct greenish tinge Distinct greenish tinge
Table VI-Influence of Titanium Sulfate on Intensity of Blue Color in Colorimetric Determination of Phosphorus
P . 9. m.
cc.
20 50
25 18 13 9
100 150 200 250
300
6 4
0
Blue Blue Blue Blue Blue Blue Yellow
Effect of Titanium on Color
Table VI gives the results of a similar study with titanium. 'The data show that 20 p. p. m. of titanium oxide may be present without harmful effect while higher amounts interfere
Vol. 1, No. 3
markedly. As is also the case with ferric iron, titanium retards the rate of development of the blue color and the effect may be partially overcome by allowing more time for the development of the color before the comparison is made. Effect of Aluminum, Manganese, Nitrate, Calcium, and Magnesium on Color Tests with aluminum and manganous salts showed that these may be present in considerable amounts without influence on the color. Nitrate added as potassium nitrate in an amount equivalent to 100 p. p. m. of nitrogen did not affect the color, while 200 p. p. m. reduced the intensity of the color about 10 per cent. The use of nitric acid in place of sulfuric for giving the desired acidity also reduced the intensity of the color about 10 per cent and caused it to fade rapidly. Tests with calcium and magnesium salts showed that these may be present in amounts up to a t least the equivalent of 1000 p. p. m. of CaO or MgO without influence on the color. Determination of Arsenic
As would be expected, arsenates produce the blue color under exactly the same conditions as phosphates. Stringent precautions must therefore be observed to prevent errors and confusion from this source. Fortunately, arsenates give a maximum blue color a t about the same acidity as phosphates. A comparative test showed that if one solution contains a certain concentration of phosphorus as phosphate and another solution the same concentration of arsenic as arsenate the relation of the intensities of color which may be developed in the two is inversely as the atomic weights of phosphorus and arsenic. This is evidence, although not conclusive, that the concentration of reagents regularly employed causes all of both the arsenate and phosphate to take part in the reaction and allows a maximum blue color to be developed. Arsenates may thus be determined in exactly the same way as phosphates. If both are present, the two may first be determined together. I n another sample the arsenic may then be reduced with hydrogen sulfide or a little sodium sulfide in acid solution. After boiling to drive off the excess of hydrogen sulfide, free sulfur is removed by adding pure filter paper pulp, shaking vigorously, and filtering. The color due to phosphorus alone is then developed. By difference the amount of arsenic may be obtained. The arsenic and phosphorus may also be separated by distillation, after which each may be determined separately. The determination of arsenic, when present in sufficient amount, can probably be made more accurately by this colorimetric method than by the other methods now in use. A study of this matter deserves further attention. Preliminary tests indicated that ammonium arsenotungstate does not give a blue color on reduction under certain conditions, while the corresponding phosphorus compound does. The blue color in the latter case, however, appears to be too faint for the determination of small amounts. It is possible that this color would be more intense under certain conditions in which case it-might be utilized to determine phosphorus in the presence of arsenic. Reagents, Analytical Procedure, and Precautions AMMONIUM MOLYBDATE-SULFURIC ACID SoLuTroiv-Dissolve 25 grams of ammonium molybdate in 200 cc. of water heated to 60" C. and filter. Dilute 280 cc. of arsenic- and phosphorus-free concentrated sulfuric acid (approximately 36 N ) to 800 cc. After both solutions have cooled, add the ammonium molybdate solution, slowly with shaking, to the sulfuric acid solution. After the combined solution has cooled to room temperature, dilute with water to exactly 1000 cc. This is a 10 N sulfuric acid solution containing 2.5 grams of ammonium molybdate per 100 cc.
I N D UXTRIAL A N D ENGINEERING CHEMISTRY
July 15, 1929
STAXNOUS CHLORIDE SoLuTIox-Dissolve 25 grams of SnC12.2Hz0 in 1000 cc. of dilute (10 per cent by volume) hydrochloric acid solution. Filter if necessary. Store in a bottle with a siphon or side opening near the bottom, arranged with a glass stopcock for delivering the solution in drops. The solution should be protected from the air by floating a layer of white mineral oil about 5 mm. thick over the surface. STANDARD PHOSPHATE SoLuTIoN-Dissohe 0.2195 gram of (recrystallized) potassium-dihydrogen-phosphateand dilute to 1000 cc. This solution contains 50 p. p. m. of phosphorus and is too concentrated to use directly. A second stock solution is made by taking 50 cc. of the first stock solution and diluting to 500 cc. This second stock solution contains 5 p. p. m. and is used for making the standard solution for comparison. To make this standard solution take 5 cc. of the stock solution, dilute to 95 cc. with distilled water, add 4 cc. of the ammonium molybdate-sulfuric acid solution, and mix thoroughly by shaking in an Erlenmeyer flask. Add 6 drops of stannous chloride and shake. Dilute to exactly 100 cc., shake, and the solution is ready for use. It contains 0.25 p. p. m. of phosphorus. For very dilute solutions use 2 cc. of the stock solution, but the same amount of reagents, giving a standard which contains 0.1 p. p. m. of phosphorus. After standing 10 to 12 minutes, the standard starts to fade, and a drop more of stannous chloride should then be added to bring the full color back which will again be permanent for 10 to 12 minutes. ANALYTICAL PROCEDURE-In the analysis of water, water extracts of soils, minerals, fertilizers, etc., it is permissible to add the reagents directly to these unless they are colored, turbid, or decidedly acid or alkaline. Turbidity and color should be removed by appropriate means. A decided acid or alkaline reaction should be neutralized before adding the reagents. Organic materials may be ignited with magnesium nitrate. I n this case i t is best to keep the 10 N sulfuric acid and 2.5 per cent ammonium molybdate as separate solutions in order that the ignited residue may be dissolved with the sulfuric acid and the ammonium molybdate added after proper dilution, I n all cases the reagents should be present after final dilution in the proportion of 4 cc. of 10 N sulfuric acid, 4 cc. of 2.5 per cent ammonium molybdate, and 6 drops of
139
stannous chloride per 100 cc. The reagents should be thoroughly mixed with the test solution before the stannous chloride is added, after which thorough mixing should again be accomplished. Comparison with the standard should be made within 10 minutes after adding the stannous chloride. PaECAuTIoNS-Reagents, filter paper, water, and glassware often contain appreciable quantities of phosphorus and arsenic. Blank tests should be made frequently in which all of the reagents and glassware come into play, and there should not be produced more than a very faint blue color if everything is satisfactory. New glassware should be thoroughly weathered by treatment with warm sulfuric acid dichromate solution for 24 hours. Filter paper may be tested by tearing up a sheet of it and throwing the shreds into a blank test and shaking. It is absolutely essential that every new lot of reagents be rigidly tested. Summary of Improvements
1-A stock solution of stannous chloride is prepared by dissolving the pure salt in acidified water and is preserved by covering with a layer of white mineral oil. This is far superior to the old method of preparing it each day as needed by dissolving mossy tin in hydrochloric acid. 2-In comparison to the old method the amount of ammonium molybdate is doubled and the acidity is increased. With these amounts and proportions of reagents the method is made considerably more sensitive, especially to small amounts, because the full effect of all the phosphate is probably brought into play. Evidence that this is the case is furnished by the fact that the full color development takes place immediately on adding the stannous chloride. In this respect there is a lag of 5 to 10 minutes in the old method due to the slowness of the reactions in coming to equilibrium on account of the less favorable conditions. 3-Effects from even high amounts of silica are entirely eliminated with the concentration of reagents recommended. Literature Cited (1) Denig&, Com9t. rend., 171, 802 (1920); Corn#;. rend. 875 (1921). (2) Parker and Fudge, SoiE Science, 34, 109 (1927).
SOC.
biol.. 84,
A Large Metal Soxhlet Extractor' 1,. R. Bryant ONTAEIO
I
AGRICULTURAL COLLEGE, GUELPA,
N T H E course of some work on poultry nutrition it was found necessary to prepare about 6.8 kg. per day of fat-
free material for feeding experiments. To meet this need a relatively inexpensive extractor was required. A large metal extractor of standard make2 was thought to be almost suitable and a drawing was made, modifying this extractor somewhat, and submitted to a local metal worker, who made it up in copper. The modified apparatus has been giving satisfactory service a t this institution for the past year and it is thought that the details of its construction would be of value to other workers in the field, as any good coppersmith should be able to make it up fairly cheaply. The apparatus is constructed in three sections. The lower section is a boiling vessel for the solvent and has a draining faucet a t the bottom. The center section comprises the extraction chamber with a tube for carrying up the solvent 1
Received February 14, 1929.
* Baird and Tatlock, Ltd., London, England.
ONT.
vapor, a siphon for returning the solvent and extract to t h e lower vessel, and a glass level gage to indicate the height of liquid in the extraction chamber. A faucet is provided for draining the extraction chamber. The cover of the lower chamber with the tubes attached forms a part of the center section. The top section is a reflux condenser consisting of a copper coil inside a copper tank. The center section fits into a groove in the top of the lower boiling chamber. An asbestos gasket has been placed in the bottom of t h e groove and the center section is provided with lugs so that the two sections may be fitted together and tightened by means of wing nuts, making an air-tight joint. The reflux condenser is fastened to the top of the extraction chamber in the same way. The material to be extracted is put into a linen bag, which is placed in a copper frame, shown a t right of diagram. An extra copper ring holds the bag firmly in place. The bag holder containing the material is then placed in the extrac-
