90
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 AND SISTER LOUISELLA WALTERS, Regis College, Weston, Mass.
A
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,
ANALYTICAL EDITION
January 15, 1942
of the mercuric bromide solution contained in the glass cylinder. The cylinder was filled t o a height of about 1 cm. from its opening
with 4 per cent mercuric bromide solution, and the thread was impregnated for 15 minutes, after which i t was removed by means of the glass handle, without contamination from any outside lifting device, and placed upon a iece of glass rod, bent in the form of a square, to dry. When d?y the thread was cut by scissorB at both ends of the frame, thus giving the required length for the ca illary tube. The individual threads were picked up by metal Forceps and fed through the required capillary by means of a vacuum line. By this technique the thread was untouched from the time it was impregnated until it was fed through the capillary tube and prepared for reaction with the arsine. ANALYSES.A standard solution of arsenious oxide was prepared according to Lachele (6). Dilutions of the original stock solution were made in such a way that the working standard contained 1 microgram in each 0.5 ml. One microgram was used in each of the following analyses (Table I). A series of analyses was run using the Hanford Pratt strips, the stains being measured in millimeters, and tabulated. A similar series was run, using the 1-mm. capillary and No. 8 thread. A similar series was run, using the 0.5-mm. capillary and No. 24 thread.
TABLEI. DETERMINATION FOR ONE MICROGRAM OF ARSENIC (All analyses were done under same conditions of time and temperature. Temperature 18' to 20' C. Time of each run 1.5 houra) Average Length of Stain Thread 24, Thread 8, Hanford Pratt No. of Run 0.5-mm. cap. 1-mm. cap. atripe, 3-mm. m p . Mm. Mm. Mm. 1 6.7 7 1.5 2 6.7 7.0
-
a
6.0 6.0 6.0 5.8 7.5 6.5 5.8 5 5 6.5
4 5 6 7
S
9 10
11 ~. 12 ._
6 8
13
6.0
6.3
14 15 16 17 18 19
5.0 6.0 6.0 6.3 6.0 6.5 6.0 6.0 6.0 6.3 6.0 6.0 5.0
2n -.
21 22 23
Discussion
24 25 26
In practically 50 per cent of the analyses, using the Hanford Pratt strips, the stains resulting from 1 microgram of arsenic were not measurable. Where it was possible to measure them, the result8 were far from accurate, because of the indefinite termination of the stain and the wide divergence in length of the stain on both sides of the filter-paper strips.
91
27
28 29
30
31
a
32 33 Not measurable.
6.0 6.5 6.0 6.3 6.0 6.0
7.0 6.4 5.0 7.0 5.2 7.7 6.5 5.5 5.0 4.0 4.0 4.0 5.0 5.0 5.5 5.0 6.0 6.0 5.0 4.0 5.0 4.8 4.5 4.5 5.0 4.8 4.5 4.5 6.2 6.5 6.5
1.6
1.5 15;
1.0 1.0 1.0 0.5 1.5 1.0 065
..
1.5 1.0 2.0 1.0 1.5 1.6 1.5 2.0 0
*
o
s
0.5 0
. s a
TABLE11. VARIATIONS IN LENGTHOF STAIN Length of Stain
Mm. 7 . 5 and over
L-
7,0-7.4 6.5-6.9 6.0-6.4 5.5-5.9 5.0-5.4 4.5-4.9 4.0-4.4
Thread 24 in Thread 8 in 0.5-Mm. Cap. I-Mm. Cap. Par cent of SS run8 3 3 3 9 12 18 61 12 9 6 6 28 0 18 0 12
variation with thread 24 in the 0.5-mm. bore capillary is not so great as with No. 8 in the I-mm. bore capillary, in the former 60 per cent of the results being between 6 and 6.5.
Conclusion and Summary
F I G U ~1.E DEVICE FOR IMPREGNATING ARSENIC THREADS A . Handle 8. Device for impregnating
C. Thread D. Glass rylinder cpntaininr
For the determination of 1 microgram of arsenic, thread as an arsenic detector in glass capillary tubes gives a more sensitive, more constant, and more definite stain than the usual Hanford Pratt strips. When No. 24 thread was used in a 0.5-mm. bore capillary, and No. 8 thread in a 1-mm. bore capillary, the former thread gave more constant stains. It would seem, therefore, that for the range of 1 microgram of arsenic, the closer the arsenic detector fits its glass container the more constant the stain.
m m m ~ A n hvnrnirla
The average length of the stain on the threads wm much greater than that on the Hanford Pratt strips, and for that reason could be measured more accurately. Including the thirty-three runs on each, the average lengths are aa follows: No. 24 thread in 0.5-mm. capillary, 6.1 mm. No. S thread in 1-mm. capillary, 5.4 mm. Hanford Pratt strips in 3-mm. capillary, 0.8 mm.
The variations in length of stain, using the two types of thread in different capillaries, are contained in Table 11. The
Literature Cited (1) Assoc. Official Agr. Chem., Official and Tentative Method8 of Analysis, 4th cd.. p. 370 (1933). (2) Cahill, Sister Emily, doctoral dimcrtation, Catholic Univereity,
1936. (3) Fairhall, L. T . , Am. J.Pub. Health, 28,825 (1938). (4) Griffon, H., and Thuret, J., Bull. SOC. cham., (6)5, 1129 (1938). (5) How, A. E., IND. ENQ.CHEM.,ANAL.ED.,10, 226 (1938). (6) Lachele, E. E.,Ibid.. 6,256(1934). (7) Longfield-Smith, L., U. 9. Dept. Agr. (Winter Haven Lab.), Rspt., p. 3 (1933). (8) Moneeau, P.,Griffon, E., and Nicolas, P., Ann. fala., 31, 262 (1938). (9) Stanley, J. P.,master's dissertation, Catholic University, 1938.
