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.
January 15, 1942
89
ANALYTICAL EDITION
Reagents Perchloric acid, 70 to 72 per cent; ammonium molybdate, 1.25 per cent; sulfite solution, containing 15 per cent of sodium bisulfite and 0.5 per cent of sodium sulfite. Aminonaphtholsulfonic acid stock solution, made by dissolving 16 mg. of recrystallized aminonaphtholsulfonic acid (9) in 25 cc. of the sulfite solution with heating on the steam bath. Dilute aminonaphtholsulfonic acid solution, made by diluting 1 cc. of the stock solution to.100 cc. Porcelain chips, heated with cleaning mixture and washed until they give no blank.
TABLE 11. RECOVERY OF PHOSPHORUS ADDEDTO LIPWB EXTRACTS (0.2 cc. of extract added to each flask) Extract 1 Extract 2 ----Phosphorus-PhosphorusSample Added Found Recovered Sample Added Found Recovered Microorams Macrograms 1 7.40 7 7.15 2 .. 7.70 .. 8 6.95 .. 3 7.55 9 6.95 4 5 12.50 4:95 10 5 11.85 4:83 5 5 12.50 4.95 11 5 12.10 6.08 6 5 12.55 5.00 12 5 12.10 5.08
7
.. ..
Method
Average percentage recovery 9 9 . 3
A scrupulously clean 50-cc. Erlenmeyer flask is clamped at an angle of about 45’ over a steam bath, and a portion of a lipide solution containin from 2 to 25 micrograms of phosphorus is
pipetted into the fowest [‘corner” of the flask, The solvent is removed; the position of the dry lipide is marked; 0.5 cc. of perchloric acid and 3 or 4 specially treated porcelain chips are added; the flask is placed on a digestion rack a t a 45’ angle with the lipide film down; a condenser (cold finger), previously cleaned by dipping in cleaning mixture and rinsing with distilled water, is inserted while still wet about halfway to the bottom of the flask (if the condenser is clean the small amount of water that adheres to it does not affect the result). and the sample ISheated until colorless (usually 5 to 10 minutes$. No white fumes escape past the cold finger except occasionally when bumping occurs. No loss of phosphorus has been observed even with the violent bumping which is frequently encountered when glass beads are used instead of porcelain chips. After cooling, the condenser is rinsed into the flask with 3 cc. of water from a pipet; the flask is removed from the rack and 2 cc. of the molybdate solution followed by 4 cc. of the dilute aminonaphtholsulfonic acid-sulfite solution are added from fast-flowing pipets with vigorous swirling of the flask during both additions. The color is read in an electrophotometer equipped with the filters described by McCune and Weech (6). (Identical results were obtained with and without the light shade Aklo, No. 396, filter. The filter pack has a low transmission and it may be desirable to omit this filter if the light source is not,sufficiently bri ht t o give a full deflection of the galvanometer with the blank S ~ U tion.) Blank solutions (usually 2 or 3) are prepared from the reagents in exactly the same way (without digestion), the average air setting of the instrument is determined for a blank setting of 100 per cent transmission, and the unknown sample is read against this setting 5 minutes or more after adding the aminonaphtholsulfonic acid solution. A cuvette 5 cm. long and 1 cm. in diameter is used. The volume of solution (9.5 cc.) is sufficient for two rinsings of the cuvette between readings. The method can be adapted to any electrophotometer equipped with the McCune and Weech filters, but the high sensitivity, down to 2 micrograms of phosphorus, depends on the development of color in a small volume and its reading in a long (5-cm.) cuvette. The readings are translated directly into uantities of phosphorus from a previously determined standar8 curve, plotted on semilogarithmic coordinate paper.
-
IN DETERMIKATION OF PHOSTABLE I. RANGEOF VARIATION PHORUS
(All data are actual readings of percentage transmission) 7 Phosphorus Present Sample 27‘ 5ya 7.57 lo70 1 2 . 5 7 1.57 207
25y
24.1 18.8 77.2 41.1 31.1 11.8 7.1 1 54.2 18.7 77.9 31.2 11.8 7 . 2 24.0 2 54.0 40.9 1 8 . 6 7 7 . 5 3 1 . 1 1 1.6 7.1 4 1 . 1 2 4 . 1 3 54.1 18.7 77.1 31.1 11.7 7.1 4 24.0 53.9 40.9 3 1 . 2 7.1 2 4 . 0 . . . . 53.9 77.6 5 31.4 24.0 77.7 .. 54.0 6 ... . . ... 54.0 77.6 7 . . 31.1 .. .. 77.5 54.0 .. 31.1 ... 8 .. . . 31.2 77.4 .. ... 54.1 9 .. 31.1 77.3 .. .. ... 64.1 10 77.8 53.9 11 .. 3 1 . 4 .. 31.2 77.3 .... 53.9 12 a Coefficients of variation (T’ = 100u/mean), calculated from the extinction coefficients, were 1.31, 0.35, and 0.30 per cent for the 2, 5, and 107 samplea, respectively.
.... ..
.. ..
.. ..
.
I
.. ..
...
Discussion Fiske and Subbarow (3) emphasized the great advantage of rapid development of color in determining phosphorus; with their procedure the maximum was reached in 5 minutes, as
..
.. .. ..
..
Average peroentage recovery 9 9 . 9
far as could be determined with a colorimeter (1, 2, 6). The proposed modification retains this advantage. Most of the color (in the spectral zone measured) develops within 1 minute; there is usually a slight intensification up t o 5 minutes, after which there is little or no change up to at least 1 hour. SENSITIVITY AND STABILITY OF REAQENTS. The concentration of none of the reagents is critical. Although there is little chance of losing perchloric acid with the condenser technique, it was found that the acid may be reduced by at least 20 per cent without materially affecting the result ( 4 4 ) . The same values have been obtained with 1.5 as with 1.25 per cent ammonium molybdate, and varying the amount of aminonaphtholsulfonic acid in the stock solution from 12 t o 20 mg. did not appreciably change the results. As the ammonium molybdate solution becomes older the blank increases and the color intensity decreases somewhat. The reagent should be checked occasionally against a standard phosphorus solution and renewed if the recovery tends to be low. The stock reagent gives some trouble due to the formation of crystals on standing, but if these are redissolved by heating the reagent appears t o be as good as before. It is not recommended, however, that this reagent be used more than 2 weeks (3) and it may be necessary to renew it oftener. It was anticipated that the dilute aminonaphtholsulfonic acid reagent would be unstable and throughout most of the work i t was diluted just before use. However, excellent results have been obtained with this reagent kept in a n ordinary flask in the laboratory for 5 days. PRECISION. The extinction coefficient is not constant over the entire range of measurement, but the statement of McCune and Weech, “When plotted on semilogarithmic coordinates the regression line of galvanometer reading on concentration is so nearly linear throughout its entire extent that the regions between the 5 points that are determined in its preparation [8 points were determined in the present work] can be considered to be strictly linear without introducing significant error”, applies to the findings with the proposed method. The actual data from which the calibration curve was plotted (Table I) show the range of variability of the method as applied to the determination of known quantitie i of phosphorus. (The calibration curve will probably vary somewhat with variations in light filters, etc.; it should be determined by each worker under his conditions.) Twenty-one 1-cc. portions of standard solutions were dried in 50-cc. flasks and carried through the digestion procedure. The samples covered the range from 2 to 20 micrograms of phosphorus; 11 contained 5 micrograms or less. The average recovery was 100.3 per cent and the standard deviation of the percentage error was 1.6 per cent. (This result shows that no pyrophosphate is formed during the digestion. I n a series of determinations the solution was boiled after addition of water following digestion with no effect on the recovery of phosphorus.)
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Vol. 14, No. 1
INDUSTRIAL AND ENGINEERING CHEMISTRY
RECOVERY FROM LIPIDEEXTRACTS. Samples containing 5 micrograms of phosphorus were dried in flasks, and 0.2-cc. portions of isopropyl ether extracts of brain were added and analyzed as described. The results (Table 11) show a quantitative recovery. SPECIFICITY. Fiske and Subbarow (Table 11, 3) tested a number of substances for their effect on the development of color with their method. Sodium chloride, potassium nitrate, or ammonium sulfate in the proportion of 10,000 times the quantity of phosphorus, sodium nitrite in the proportion of 100 times, and copper (added as copper sulfate) in the proportion of 10 times, do not interfere in the proposed modification. However, 50 micrograms of iron (added as ferric chloride) approximately double the intensity of color produced by 5 micrograms of phosphorus, and 1 mg. of silicon (added as sodium silicate) increases the color given by 5 micrograms of phosphorus by about one half. This result reduces the general applicability of the method, but does not seriously affect the use for which it was intended-i. e., the analysis of lipide extracts where appreciable quantities of iron or silicon would rarely, if ever, be present. The absence of significant solution of silicon during digestion with perchloric acid under the conditions described was shown by the quantitative recovery of phosphorus. EFFECT OF TEMPERATURE. Allen (1) found a considerable effect of variation in temperature on the development of color with the King (4, percliloric acid) modification of the Fiske and Subbarow method. With the proposed procedure identical readings were obtained with 5-microgram samples de-
veloped for 20 minutes a t 20", at room temperature (about 25'), and a t 38" C.
Summary A procedure for the electrophotometric determination of phosphorus (2 to 25 micrograms) in lipide extracts is described. To avoid a high blank, representing absorption in the ultraviolet, the concentration of aminonaphtholsulfonic acid was reduced to one fortieth, and of sulfite to one tenth of that employed in the procedure of Fiske and Subbarow (9). Full color development is obtained even though the quantity of aminonaphtholsulfonic acid may be less than that of phosphorus. Digestion is carried out with perchloric acid in a 50-cc. Erlenmeyer flask, using a condenser to facilitate rapid mixing which is essential in determining small quantitiee of phosphorus.
Acknowledgment
.
The author is indebted to A. Ashley Weech and to Donovan J. McCune for supplying unpublished details concerning their procedure.
Literature Cited Allen. R. J. L., Biochem. J.,34,858 (1840). Berenblum, J., and Chain. E., Ibid., 32, 286 (1938). Fiske. C. H., and Subbarow. Y.,J . B i d . Chm., 56,375 (1925). King. E. J , Biochem. J . , 26 292 (1932). McCune, D. J., and Weech, A. A., personal communication. (6) McCune, D. J., and Weech, A. A., Ptoc. SOC.E z p l l . B i d . Md., (1) (2) (3) (4) (5)
45, 559 (1940). (7) Weech, A. A., lbid., 45, 858 (1940).
A Micromethod for Determination of Arsenic SISTER EMILY CAHILL
A
AND
SISTER LOUISELLA WALTERS, Regis College, Weston, Mass.
CCURATE quantitative methods for the determination of 1 microgram and less of arsenic (4, 6, 9) are still problems for research, owing to interest in the arsenic content of normal tissue (9,8). Before it can be proved that arsenic is the causative agent in certain chronic conditions, a sensitive method for the analysis of normal tissue niust be established. To this end much work has been done with modifications of the original Gutzeit method (1) as used by the Association of Official Agricultural Chemists. In 1936, the author (R) used this method during a study of the accumulation of arsenic in tissues of the albino rat. For comparative purposes, it was necessary to analyze normal tissue simultaneously; therefore, as a possible extension to smaller quantities, pieces of ordinary cotton thread, No. 60, were impregnated with mercuric bromide and used as arsenic detectors instead of the Hanford Pratt strips. More sensitive results were obtained. It was thought, during the present investigation, that by using coarser thread in narrower capillary tubes, not only a more sensitive but also a more accurate and more definite stain could be produced, as How (6) has recently tried to prove with Morse and Kaley No. 8 knitting cotton. For this purpose two types of cotton threads were used in two types of capillaries: (1) No. 8 in a 1-mm. bore capillary, and (2) No. 24 in a 0.5-mm. bore capillary. The following investigation is a comparative study of these two types of impregnators and the Hanford Pratt strips, using in all analyses 1 microgram of arsenic.
Procedure and Experimental The regular Gutzeit method of the Association of Official Agricultural Chemists was used, with the following modification: In lace of dental rolls impregnated with lead acetate in the scrubler tubes, glass beads (7) which had been soaked in saturated lead acetate were used. These scrubber tubes were cleaned after every run, by taking out the beads, washing first with water, four times with concentrated hydrochloric acid, then four times with water. They were then soaked overnight in saturated lead acetate solution, owed out on filter paper, and put in the glass scrubber tubes, w h h were half filled with the beads. Instead of the regular detector tubes containing the Hanford Pratt strips of mercuric bromide paper (a series was run with these, however, for comparative purposes) ca illary tubes of 1-mm. bore, each containing a piece of 0. N. cotton thread No. 8 as a detector, were used in one series of determinations, and capillary tubes of 0.5-mm. bore, each containing a piece of Coat's cotton thread No. 24 as a detector, were used in a second series. . IMPREGNATION OF THREAD. It was found necessary to suspend the thread in the mercuric bromide solution in such a way that each bit of thread would be completely impregnated, without contamination of any kind from the time of initial impregnation t o the time of drying and placing in the capillary. To this end, a aiece of glass rod of 5-mm. bore was drawn out and shamd as shown in Figure 1. The device had two slight indentations at the top and one at the bottom. around which the thread was drawn loosely. The glass frame and thread were placed in a 50-ml. glass cylinder, and were held in place by means of the handle bent in a right angle at one end, which allowed the frame t o hang suspended in the center
8,
