A du Pont Type Semimicronitrometer PHILIP J. ELVING AND WILRUR R. MCELROY, Purdue University, Lafayette, Ind.
determinations for which the usual Lunge or du Pont nitrometers are used. The advantages of the semimicronitrometer as compared to the compensating macronitrometers are use of a much smaller amount of mercury, compactness, and easier operation. In addition, the motor-operated model lessens the danger of explosions and decreases the man-time required by allowing the operator to weigh out samples or run other nitrometers while the apparatus is being shaken. The precieion is almost as good as that of the macroinstrument; the accuracy, slightly less.
The construction and operation of a semimicronitrometer are described. The nitrometer is of the compensating type, analogous to the du Pont nitrometer and the Lunge gasvolumeter, in which the quantity of gas is read a t the volume it would occupy at standard conditions of temperature and pressure. Two models of the nitrometer have been developed, one hand-shaken like the macronitrometers and the other motor-shaken. Various inorganic and organic nitrates, including nitrocelluloses, have been analyzed. The apparatus can also be applied to the other
T
HE nitrometer most widely used for the analysis of Method and Application mixed acids and organic and inorganic nitrates is the The nitrogen in inorganic or organic nitrates or nitrites is du Pont nitrometer (20) which is a development of Lunge’s readily evolved as nitric oxide by interaction of the substance gasvolumeter (13, 14). This instrument is used for one of with sulfuric acid and mercury. The volume of nitric oxide the more important and frequent determinations made on is measured and the percentage of nitrogen in the sample is explosives, the determination of nitrate and nitrite nitrogen. calculated. This so-called “nitrometer reaction” is given by The du Pont nitrometer, which has been standardized as compounds containing the NO, or NO group attached t o regards dimensions and operation (1, 16), is superior to the carbon through nitrogen (6) or attached to oxygen. This ordinary Lunge nitrometer in that a volume of gas in the method is extensively used for the determination of nitric acid measuring buret of the apparatus may be readily brought to in oleum and mixed acids (8). The nitrometer method the volume that it would occupy under standard conditions of temperature and pressure. This is accomplished by means of a compensating tube containing a quantity of gas whose volume A B C D at standard conditions is accurately known. The disadvantage of the du Pont nitrometer is its large size, which necessitates the use of 11 to 18 kg. (25 to 40 pounds) of mercury for its operation and makes the manual shaking of the reaction tube rather cumbersome. The possibility that the reaction tube may be broken by an explosion while being shaken introduces an element of danger. In view of this, a nitrometer modeled on the du Pont (1) and Lunge (14) compensating nitrometers was developed which has one tenth the capacity of the du Pont nitrometer. T o facilitate the operation of the apparatus, one model of the new nitrometer was mounted for mechanical shaking, thus eliminating the tedium of hand shaking and the danger to the operator of an explosion while shaking. An additional advantage of the mechanically shaken nitrometer is that several can be operated by the same operator, samples being weighed out while the apparatus is being shaken. Although no previous adaptation of the compensating type of nitrometer to smaller volumes or mechanical shaking could be found, Lunge semimicronitrometers have been described (4 I,%?) and a mechanically shaken Lunge nitrometer was recently mentioned (24). FIGURE1. HAND-SHAKEN MODEL O F SEMIMlCRONITROMETER 84
ANALYTICAL EDITION
January 15, 1942
85
Apparatus
MOTOR-SHAKEN MODEL SEMIMICRONITROMETER
FIGURE 2. OF
Two models of the semimicronitrometer were developed. The handshaken model, shown in Figure 1, has one tenth the capacity of the standard du Pont nitrometer (1) and was designed to give the required accuracy in reading the gaseous volume. The apparatus is made entirely of Pyrex glass, except for the leveling bulbs which are 60-ml. separatory funnels. The lower stopcock on the reaction tube may be omitted. Red rubber tubing of 4.8mm. internal diameter and 4.8mm. wail thickness is used. The capacity of each buret is about 25 ml.; the capacity of the reaction tube is 30ml. Graph paper scales are pasted securely on the two measuring burets; one unit on this scale equals 1 em. The scale on the nitrocellulose tube starts at the bottom of the bulb, on the universal tube at the bottom of the capillary from the stopcock. The two measuring tubes were calibrated by weighing the mercury delivered and calculating the volume. Weigbings were made every 5 om. of tube length. The universal tube is used for samples of unknown nitrogen content; the nitrocellulose tube for samples of approximately known nitrogen content, so that a sufficiently large sample can be taken to fill the large bulb with nitric oxide. The lesser diameter of the nitmcellulose tube permits a more accurate reading of thk volume. :;The motorshaken model, shown in Fieures 2. 3. and 4. is simolified hv hfiing the 'buret and reaition tub;
-
and possible sources of error as applied to the determination of nitrogen, particularly in explosives, FIG3. MOTOR-SHAKEN MODEL have been fully discussed in the OF SEMIMICRONITROMETER, SIDE AND TOPVlnws literature and the interference of various substances which react with the nitric oxide has been pointed out (8, 3, 6, 8, 10, 11, 16-19, 81, 88, 83). The chief sources of error are the solubility of nitric oxide in sulfuric acid, the formation of ammonia on prolonged contact of the nitrate with sulfuric acid before shaking with mercury, the formation of carbon dioxide from cellulose nitrate under the same conditions, and the formation of nitrous oxide and nitrogen on prolonged shaking with mercury. The equation given (8) for the case of potassium nitrate is
+
~ K N O I 4H1S01
0
5
IO crn.
+ 3Hg = K,SO, + 3HgS0, + 4HzO + 2NO
The compensating type of nitrometer can be used to analyze other materials which liberate a gas on suitable treatment. It has been applied to the determination of available oxygen in peroxides, hypochlorites, and salts of peroxy-acids, and to the valuation of oxalic acid, formaldehyde, metals, and carbonates (8, 14, 86). In the case of the simple Lunge nitrometer, the volume of gas has t o be reduced t o standard conditions in order t o calculate the weight of gas evolved. This process of calculation is avoided by compensating the measuring buret with a volume of gas which has been calibrated t o standard conditions of temperature and pressure by bringing a gas in the measuring buret to the volume it would occupy at standard conditions. The manner of doing this is explained in the standardization procedure described later in this article.
3
I
1 I
FIQLTRE 4. MOTOR-SEAKEN MODELOF SEYMICRONITEOMETER, FRONT VIEW
86
INDUSTRIAL AND ENGINEERING CHEMISTRY
connected together as closely as ossible by a 2-mm. glass capillary tube, thus eliminating a stopcocf and a rubber tubing connection. The universal and nitrocellulose tubes, each with its reaction tube attached, are mounted separately on 9 X 73 cm. boards. In operation, a board is hinged at the bottom to the base and is moved in a reciprocal motion near the top by an eccentric mechanism actuated by a 0.05-horsepower motor, being shaken about 300 times per minute. The reaction tube moves throu h a horizontal distance of about 3 cm. The boards containing t%e two burets can be readily interchanged by removin the two screws a t the base and the one screw at the top. The%uret and reaction tube and the rubber tubin connecting the reaction tube with its leveling bulb are fastenej to the board with copper or lead stram. The apparatus stand should be screwed to or clamped on a firm surface to avoid excess vibration when in use. The compensating tube, which can be readily moved uf~and down as necessary, is held in place with two buret clamps, one holding the enlarged bulb and one located at the bottom. Mountin the compensating tube on a rack and pinion arrangement woud be advantageous. The three-branched connecting tube is held in a buret clamp with a moderately slack connection of 7.5 or 10 cm. (3 or 4 inches) of rubber tubing to the buret. About 45 cm. (18 inches) of rubber tubing should be allowed between the connecting tube and the compensating tube and 90 cm. (3 feet) of tubing between the connecting tube and its reservoir. The two measuring tubes could be readil mounted on the same large-scale installastand for shaking by the same motor. tions, a number of nitrometen could be operated by the same motor in a compact arrangement. The use of burets with etched volume gradations would be advantageous, increasing the accuracy of reading volumes. The air used in filling the compensating tube is desiccated by shaking with sulfuric acid in a separated reaction tube or in a similar tube having a simple two-way sto cock a t its u per end and a capillary tube a t its lower end. dercury is usezas confining liquid under the layer of sulfuric acid. Four or five pounds of mercury, which is used as confining liquid, are ample for operating the semimicronitrometer. Concentrated sulfuric acid, c. P. grade of no or low nitrogen content, should be used. The mercuric sulfate formed can be recovered and reclaimed. The use of a cellulose acetate face mask is advisable as a precaution in case of explosion (I).
dr
Procedure The procedure for using the semimicronitrometer is based n large part on the instructions for operation of the du Pont nitrometer (1, 7, 8). The apparatus can be standardized with dry air or with nitric oxide from pure potassium nitrate. The latter procedure is the simpler and is recommended for
Vol. 14, No. 1
By this process the mercury comes to the same level in the two tubes and in their leveling bulb, while the desiccated air in the tubes comes to atmospheric pressure. During this process no undesiccated air can enter the tubes because the gas in the tubes was at higher than atmospheric pressure before being vented to the atmosphere via the reaction tubes. After noting the atmospheric pressure and temperature. the volume of gas in the measuring tube is calculated to standard conditions. With the s t o p cocks on the tubes closed, the leveling bulb is raised until the gas in the measuring tube occupies the calculated volume, the compensating tube being moved so that its mercury level is the same as that in the measuring tube. A strip of paper or some other mark is placed on the compensating tube at the mercury level, completing the standardization. To standardize the hand-shaken measuring burets, desiccated air is drawn into the compensating and measuring tubes aa before and the stopcocks are closed. The mercury columns in the two tubes are balanced. After a U-tube containing sulfuric acid has been attached to the outlet of the measuring tube, the stopcock on the latter is opened and the sulfuric acid levels are balanced, putting the gas in the buret at atmospheric pressure, The volume of gas in the buret is calculated to standard conditions. With the stopcock on the buret closed, the leveling bulb is regulated to bring the mercury level in both burets to the same point, which marks the calculated volume in the buret. The compensating tube is marked at the new mercury level, completing the standardization. STANDARDlZBTION USlNf3 POTASSIUM NITRATE. The compensating tube, buret, reaction tube, and all connections are completely filled with mercury. The separate reaction tube a-hich has been filled with air, desiccated by shaking with sulfuric acid in the tube, is attached to the compensating tube. With the compensating tube and the measuring buret in about the same position, desiccated air is drawn into the compensating tube to a point about opposite the 22-ml. mark on the buret. The stopcock on the compensatin tube is closed, and the attached reaction tube is removed. 100-mg. sample of c. P. potassium nitrate, which has been recrystallized twice from water, washed with alcohol, ground to pass a 100-mesh screen, and dried at 135" to 150' C., is accurately weighed and laced in the cup of the reaction tube with 1 ml. of sulfuric acii. The sample is drawn into the reaction tube and the cup is rinsed with 5 1-ml. ortions of sulfuric acid, care being taken t o admit no air. eh! ' instrument is shaken for two &minute periods aa described under Procedure for Analysis. The gas generated is transferred to the buret. The reservoir and compensating tube are then adjusted so that the mercury levels in both the buret and the compensating tube are the same and stand at the 22.15-ml. mark on the buret, which is the volume of nitric oxide, calculated to standard conditione of temperature and pressure, corresponding to 13.86 per cent of nitrogen in 100 mg. of potassium nitrate. This is the theoretical per cent of nitrogen in potassium nitrate. A strip of paper is aated on the Compensating tube a t the level of mercury, compkting the standardization. When using the hand-shaken apparatus the reaction tube is disconnected for shaking. The introduction of air from the capillaries must be avoided, since the oxygen in the air interacts with nitric oxide, forming nitrogen dioxide which results in a change in volume and in the attack of the mercury in the buret and the formation of a coating of mercury salts on
1
routine use. STANDARDIZATION USINGDRYAIR. The compensating tube, buret, and all connections are completely iilled with mercury. The motor-shaken measuring burets are standardized as follows: The separate reaction tube filled with air, desiccated by shaking with sulfuric acid, is attached to the compensating tube. With the latter and the measuring tube in about the same position, desiccated air is drawn into the compensating tube to a Doint level with the 22-ml. Lark on the measuring tube and the stopcock on the compensating tube is closed. DesOF NITROGEN BT THE SEMIMICRONITROMETER TABLE I. DETERM~NAT~OX iccated air from the attached -Volume of Nitric Oxidereaction tube is then drawn Average into the measuring tube to the Average deviation ---NitrogenSample No. of Appa22-ml. mark and the stopcock Found Calculated Substance Weight Runs ratusa Mean deviation of mean is closed. The as in each Mg. M1. M1. Ml. % % tube is put unfer a slight 0.00 8 . 3 9 * 0.00 8.46 0.00 u-hl 22.13 3 165.0 Lead nitrate 13.86 0 . 0 3 1 3 . 8 4 * 0 . 0 2 positive pressure by having 0 . 1 6 22.13 N-H 21 100.0 Potassium nitrate 0.02 13.79 * 0 . 0 1 ... 0.07 22.04 N-M 15 the mercury level in the level13.79 * 0.01 0.02 0.07 22.05 ' 3 U-M ing bulb slightly above that in 16:48 16.46 * 0 . 0 2 0.03 0.Oi 22.36 U-41 85.0 Sodium nitrate 0.11 ... 10.33 * 0.06 the two tubes. The stopcock 0.36 20.65 N-H 11 125.0 Cellulose nitrate A 0.01 10.31 * 0 . 0 1 ... 0.03 20.59 U-M 5 on each reaction tube is then ... 0.03 11.31 h 0 . 0 1 0.02 22.59 4 N-H 125.0 Cellulose nitrate B opened to the air and their ... 11.30 * 0 . 0 1 0.02 0.05 22.58 U-M 5 0.01 11.31 * 0 . 0 1 0.03 mercury reservoirs are adjusted 22.60 N-M 5 13.26 =t0 . 0 1 13:is 0.03 0.01 21.20 7 U-M 100.0 Cellulose nitrate C so that the acid level in each is 12.66 0.01 12.56 0.01 0.02 20.08 U-M 100.0 3 Cellulose nitrate D 2.5 cm. (1 inch) from the top. 13.41 * 0 . 0 2 13.46 0.03 21.44 0.04 U-M 3 100.0 Yitroguanidine The stopcock on each reaction Pentaerythritol tetra. 17.73 17.51 * 0 . 0 1 0.02 0.01 U-M 20.99 3 75.0 nitrate tube is given several complete 0.06 4.65 * 0.01 4.88 0.10 U-M 18.95 3 255.0 Tetryl turns, thus venting the air in Apparatus used: N nitrocellulose tube, U universal tube, H hand-shaken apparatua. M motor-shaken appathe tube successively to the ratus. atmosphere and to the attached measuring tube. 5
ANALYTICAL E D I T I O N
January 15, 1942
the buret walls. Air can be excluded from the capillaries by filling them with mercury. It is advisable to run a sample of potassium nitrate occasionally-e. g., at the beginning of each day’s work-in order to check the calibration. The procedure using potassium nitrate given by the American Society for Testing Materials (1) calibrates the buret for 20’ C. and 760-mm. pressure, while the procedure given here is for 0’ C. and 760-mm. pressure. For the permanent standardization of the apparatus, which can be done if the lower opening of the compensating tube is kept continuously sealed with mercury, the stopcock on the compensating tube is replaced by a capillary tube which can be sealed after the introduction of dry air. PROCEDURE FOR ANALYSIS. The buret, reaction tube, and connections are completely filled with mercury. The size of the sample used should be sufficiently great to give from 20 to 24 ml. of nitric oxide-. g., 100 mg. for 14 nitrogen, 125 mg. for 11 per cent nitrogen. The weighecf%z$ is transferred to the cup of the reaction tube and is flushed into the tube with 1 ml. of sulfuric acid. I n some cases the sam le can be first dissolved in the acid. The cup is rinsed with zve 1-ml. portions of sulfuric acid. Care must be taken that no air bubbles enter the tube when the acid is admitted. With the lower stopcock on the reaction tube open and the mercury reservoir a little below the mid-point of the reaction tube, the apparatus is shaken for 5 minutes. The motor is stopped, and the reservoir on the buret and compensating tube is raised above the buret. The stopcock connecting the reaction tube and buret is opened, and any acid which is held in the upper capillary of the reaction tube is swept back into the tube by the flow of mercury. The reservoir of the buret is immediately lowered and all the gas is transferred t o the buret. The stopcock connecting the buret and the reaction bulb is closed. The reservoir on the reaction bulb is lowered to give a less than atmospheric pressure in the reaction tube. The apparatus is shaken for 5 minutes with the mercury in this position. The reduced pressure is especially helpful in removing dissolved gas from the sulfuric acid and the precipitate of mercuric sulfate. The gas formed during the second shaking operation is then transferred to the buret. Care must be taken to bring the acid layer in the reaction bulb flush with the upper face of the s t o p cock. If a considerable amount of gas was generated in the second shaking, the shaking process should be continued until nitric oxide is no longer evolved. With the stopcock on the buret closed, the compensating tube and reservoir are so adjusted that the mercury level is the same in both tubes and comes to the standardization mark on the compensating tube. The volume found on the buret a t this point is the volume from which the per cent of nitrogen is calculated, The analytical procedure when using the hand-shaken apparatus is the same, suitably modified. I n emptying the buret after a determination, the nitric oxide is transferred to the reaction tube, from which it can then be vented to the air. The gas may be prevented from entering the atmosphere of the room by holding a suction line over the cup of the reaction bulb as the gas emerges. The sulfuric acid and mercuric sulfate are removed from the cup by suction and can be recovered by inserting a trap in the suction line.
CALCULATIONS OF RESULTS.
YON
=
vol. (ml.) of KO X weight (mg.) of N in 1 ml. of NO a t N. T. P. X 100 weight (mg.) of sample
The value given by the American Society for Testing Materials (1) for the volume of nitric oxide delivered at 20’ C . and 760-mm. pressure by 1 gram of sample containing 14.01 per cent of nitrogen is 240.36 ml. The weight in milligrams of nitrogen in 1.00 ml. of nitric oxide at normal temperature and pressure would, therefore, be 0.1401 X 1000 E o,6256 273 1 240.36 X 293.1
Practically the same value is obtained from Gray’s (9) value for t h e density of nitric oxide, 1.00 ml. of nitric oxide at N. T. P. weighing 1.3406 mg. The weight of nitrogen in 1.00 ml. of nitric oxide at N. T. P. is
81
14*01 X 30.01
1.3406 = 0.6258 mg.
Accordingly, the equation used t o calculate the results is
% ’
vol. (ml.) of NO X 62.56 weight (mg.) of sample
Experimental Results Table I shows the results obtained in analyzing various inorganic and organic compounds by the semimicronitrometer, using the “nitrometer reaction”. The inorganic substances used were potassium nitrate, Merck’s reagent grade; sodium nitrate, J. T. Baker’s C. P. grade; and lead nitrate, J. T. Baker’s c. P. grade; these were recrystallized twice from water and dried a t 150’ C. The organic compounds used were the ordinarily obtainable commercial grades and were not further purified; they were dried a t 100’ C. In general, compounds like these tend to run slightly low in their nitrogen content. The sample of pentaerythritol tetranitrate was analyzed by a government testing laboratory, using a regular du Pont nitrometer. Their reported value of 17.55 per cent nitrogen is in good agreement with the value, 17.51 per cent, found with the nitrometer described, The theoretical value is 17.73 per cent. The tetryl required 30 minutes shakin for complete evolution of the nitric oxide. The values given for nitrocelluloses C and D were presumably obtained by the regular du Pont nitrometer. As indicated in the table, both the universal and nitrocellulose tubes were used with the hand-shaken and motorshaken models. All determinations on otassium nitrate were made with the measuring buret calibrateiwith air. The results for the other materials are based on calibration of the buret with potassium nitrate. The terms used in referring t o the volumes of nitric oxide found have the following meaning: mean, the arithmetical average; average deviation, t h e sum of the deviations from the mean of the individual values, irrespective of sign, divided by the number of values; average deviation of the mean, the average deviation divided by the square root of the number of values. The value given for the per cent of nitrogen found is calculated from t h e mean value of the volume of nitric oxide, plus or minus the per cent equivalent t o the average deviation of the mean. The only experimental values omitted from Table I are those obtained in preliminary runs, testing the time of shaking necessary and other experimental factors, and those which were known t o be in error. The precision of the results is seen t o be excellent; in general the accuracy is not so good, the results tending t o be on the low side. The latter source of error may be due t o the solubility of nitric oxide in sulfuric acid. Kormally, 10 ml. of concentrated sulfuric acid dissolve 0.20 to 0.35 ml. of nitric oxide (16, 26) but by using the reduced pressure advocated in the Procedure for Analysis this figure is reduced. Further possible causes of low results are hygroscopicity of sample, loss .in transfer of the sample t o the cup of the reaction tube, and insufficient shaking. Several nitrometers of the motor-shaken type have been built at Purdue for use elsewhere and have been entirely satisfactory.
Acknowledgment One of t h e authors (W. R. M.) wishes t o acknowledge the financial assistance of the Purdue Research Foundation in the form of a research fellowship. The authors also wish t o thank W. E. Fish and J. V. Hession for constructing the motordriven shaking mechanism, and W. B. Ligett for running many of the analyses.
Literature Cited (1) Am. 600. Testing Materials, “Standard Specification8 and Testa
for Soluble Nitrocellulose”, Designation D301-33.
INDUSTRIAL AND ENGINEERING CHEMISTRY
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Beckett, E. G., J. Chem. Soc., 117, 220-35 (1920). Beckett, E.G., J. SOC.Chem. Ind., 33, 628-31 (1914). Bed, E., Hofmann, K., and Bremmann, R., Chem. Fabrilz, 1929, 359-60. Cope, W. C., and Barab, J., J . Am. Chem. SOC.,38, 2552-8 (1916). Cope, W. C., and Taylor, G. B., Bur. Mines, Tech. Paper 160, 15-18 (1917). Dennis, L. M., and Nichols, AI. L., “Gas Analysis”, pp. 432-6, New York, Macmillan Co., 1929. Furman, N. H., ed., “Scott’s Standard Methods of Chemical Analysis”, 5th ed., Vol. I, pp. 649-53, New York, D. Van Nostrand Go., 1939. Gray, R. W., J . Chem. Soc., 87, 1601-20 (1905). Hvde. A. L..J. Am. Chem. Soc.. 35. 1173-82 (1913). Joyce; C. G., and La Tourette, H., J. IND.’ENQ. CHEM.,5, 1017-18 (1913). Klemenc, A.,and Hayek, E., 2. anorg. allgem. Chem., 165, 15760 (1927). Lunge, G., J . Soc. Chem. Ind., 20, 100-2 (1901). Lunee, G..Z.anuew. Chem.. 3. 139-144 (1890): J . SOC.Chem.
Vol. 14, No. 1
(15) Manufacturing Chemists’ Assoc., “Standard Specifications for Laboratory Apparatus”, quoted in (1). (16) Marqueyrol, M.,M h .powlres, 21,326 (1924). (17) Marqueyrol, M., and Florentin, D., Bull. SOC. chim., 9,231-40 (1911). (18) Newfield, J., and Marx, J. S., J . Am. Chem. SOC.,28, 877-82 (1906). (19) Phelps, I. K.,J. Assoc. Oficial Agr. Chem., 5, 1065 (1921). (20) Pitman, J. R.,J. SOC.Chem. I&., 19, 982-6 (1900). (21) Snelling, W. O., and Storm, C. G., Bur. Mines, Bull. 51, 35-41 (1913). (22) Storm, C. G., Bur. Mines, Bull. 96,36-54 (1916). (23) Storm, C. G., 8th Intern. Cong. Appl. Chem., 4, 117-25 (1912). (24) Summers, R. E.,and Summers, TV. H., SOC.Chern. I d . Victoria Proc., 36, 1108-13 (1936). (25) Webb, W. H.,and Taylor, M.,J. SOC.Chem. Ind., 41, 362-4T (1922). (26) Wilson, J. A., Teztile Colorist, 44, 300-1 (1922). PREEENTED before the Division of Analytical and Micro Chemistry at the CHEMICAL SOCIETY, Atlantic City, S . J. 102nd Meeting of the AMERICAN
Electrophotometric Microdetermination of Phosphorus in Lipide Extracts WARREN M. SPERRY New York State Psychiatric Institute and Hospital and College of Physicians and Surgeons, Columbia University, New York, N. Y.
A
PROPOSED investigation of lipides in rat brain required
a method for determining phosphorus with reasonable accuracy in minimal quantities of lipide extracts. This communication describes such a procedure, together with some observations of general interest in the application of electrophotometry to the determination of phosphorus. McCune and Weech (6) noted that the blue color obtained in the method of Fiske and Subbarow (3) is not constant for a considerable time after development, as had been supposed, but continues to gain in intensity for a t least 72 hours. They solved the problem of measuring the Fiske and Subbarow blue color with the electrophotometer by their discovery that the amount of absorption of light in a zone in the far violet region of the spectrum is constant for between 30 and 60 minutes after mixing the reagents. By using a combination of filters which transmitted a band extending from about 350 millimicrons in the ultraviolet to about 430 in the visible spectrum, satisfactory determinations were obtained. In applying the observations of McCune and W7eech to the electrophotometric determination of phosphorus in lipide extracts an unexpectedly high blank was encountered. Solutions containing the reagents in the concentrations recommended by Fiske and Subbarow absorbed more than half of the light transmitted by the filters of McCune and Weech. The absorption was not in the visible portion of the spectrum, since the solutions were entirely colorless to the eye (this statement applies to all blanks referred to in this paper), and must have taken place in the ultraviolet. The high blank did not interfere with the procedure as employed by McCune and Weech (6) because in the instrument, of the test-tube type, which they used, the galvanometer was adjusted t o a reading of 100 per cent transmission with the tube containing the blank in place before each measurement; this was impossible in the instrument ( 7 ) employed by the author, as the “air setting” was considerably higher than the “blank setting”. The high blank absorption not only had this practical dis-
advantage, but was undesirable on theoretical grounds. A test of each reagent separately in the concentrations used in developing the blue color revealed that none absorbed an appreciable proportion of the light except the aminonaphtholsulfonic acid-sulfite reagent, which showed a strong fluorescence and absorbed even more than the combination of all reagents. Therefore the concentration of aminonaphtholsulfonic acid was reduced, eventually to one fortieth of that of Fiske and Subbarow in the final solution, without appreciable diminution in the intensity of color yielded by phosphorus, even though in some experiments less aminonaphtholsulfonic acid than phosphorus by weight was present. This, hon-ever, did not overcome the difficulty of the high blank. It was found that even though none of the reagents, including sulfite, gives a n appreciable blank alone, the combination without aminonaphtholsulfonic acid absorbs almost as much light as with it, but does not fluoresce. Apparently the absorption shown by aminonaphtholsulfonic acid and sulfite alone largely disappears when the other reagents are added, but instead there is another type of absorption more or less independent of the aminonaphtholsulfonic acid. This absorption, representing the high blank of the combined reagents, could be lowered to a point where the air setting was considerably less than the blank setting (7), by reducing the total sulfite concentration to one tenth of that used by Fieke and Subbarow. Highly erratic results were obtained when the dilute aminonaphtholsulfonic acid-sulfite reagent was applied to the determination of phosphorus. The extinction coefficient was much higher (100 per cent or more) with the smaller amounts of phosphorus (2 to 5 micrograms) than with the larger (10 to 20 microgram) quantities, and it was not reproducible from day to day. The difficulty was traced to the use of too concentrated reagents and too slow mixing (in cylinders or test tubes) ; it was avoided by carrying out the determination in Erlenmeyer flasks with rapid mixing as described herewith.
