Rhodamine B Method for Microdetermination of Antimony LEON D. FREEDRIAN, Laboratory of Experimental Therapeutics, U . S . Public Health Service and The Johns Hopkins School of Hygiene, Baltimore 5, Md. phoric acid in the color development process. The author found that the iron interference could be eliminated by the addition a t this point of solid sodium pyrophosphate until the yellow color disappeared. The 0.02% rhodamine B solution was then added and the sample treated exactly as described by lfaren ( 3 ) . The color intensity of the antimony-rhodamine B complex was read in a Beckman quartz spectrophotometer a t a wave length of 562 millimicrons, the point of maximum extinction. Gellhorn, Krahl, and Fertig ( 1 ) have also used the Reckman for this purpose, but at a wave length of 565 millimicrons. The hydrogen alpha line a t 656.3 millimicrons was used in checking the calibration of the wave-length scale.
H E method recently described by Maren (3) for the microTdetermination of antimony has been used in this laboratory on a large number of specimens, including blood, urine, and tissues. Although the method was sensitive and rapid, the modifications explained here increased both the accuracy and specificity.
Table I.
Effect of Quantity of Sulfuric Acid on Color Intensity
(8 micrograms of antimony taken in each sample) Concentrated Sulfuric Acid Apparent Recovery of Antimony .If 1 \ficrograms, f r o m s t a n d a r d curve 2 5 9 5 5 0 8 0 6 0 10 0
With theze modifications evcellent recoverim uf antimony have been obtained from a variety of biological niateiials. Some typical rrcoveiies of antimony from 10 ml. of n-holrz hlood are shown in Table 11.
6 6
3 3
Table 11. Recovery of Antimony from Whole Blood
The quantity of sulfuric acid used in the digestion was found to affect the color intensity of the antimony-rhodamine B complex as shown in Table I. I n order to avoid this complication, it as necessary to keep the quantity of sulfuric acid used constant. Ten milliliters of 18 .Y sulfuric acid were found most satisfactory, and the digestion Tvas alwavs carried out in a manner to minimize loss of s u l h i c acid. Several hundred analyses showed that, if perchloric acid (0.5 ml. of the 60% solution) was used to comolete the digestion. both the sodium surfite reduction and the critic'al ceric sulfate oxidation could be omitted. The elimination of these two steps simplified the procedure considerably. Maren ( 2 ) independently suggested the elimination of thwe steps. As reported by AIaren ( 3 ) ,the presence of iron in excess of 1 mg. seriously interferes n ith the antimony determination, for iron forms xvith rhodamine B a color very similar to that of the antimony-dye complex. The presence of appreciable quantities of iron is indicated by the appearance of a distinct yellox color following the addi'tion of the 6 S hydrochloric acid and the 3 S'phos-
Antimony Taken Macrograms 0 1
.Intimony Found .llwrograms 0 1 2 2 3
2 2 9 7
8 4
10 6 16 1 20 1
LITERATURE CITED
(1) Gellhorn, .I.,Krahl, bl. E., and Fertig, J. W., J . PharmacoZ., 87, 169 (1946). (2) Maren, T., ASAL.CHEY.,19, 487 (1947). (3) Maren, T., Bull. Johns H o p k i n s Hosp., 77, 338 (1945).
Oxidation of Glycerinated Solutions in the Micro-Kjeldahl Determination of Nitrogen PAUL E. PORTNER, Biological Laboratory, Wyeth, Incorporated, Marietta, P a . ROTEIN solutions containing 0.1 to 0.3 mg. of nitrogen per 'ml. are readily oxidized by digestion with sulfuric acid and subsequent treatment \T-ith Superoxol (1). The usual procedure is to digest 1 to 2 ml of protein solution with 1 of 50% sulfuric acid until sulfur'trioxide is evolved, After 2 to 3 minutes' boiling, oxidationis completedby the addition of several drops of Superoxol and heating 1s continued several minutes. With aqueous solutions of pFotein little difficulty is encountered in oxidizing the carbon. The oxidized sample to 35 in a ~ ~digestion l i tube~ is diluted Tvithdistilled and then to 50 ml. with Nessler's reagent. A reading is made in a colorimeter and the nitrogen content is ascertained from a standard graph. Micro-Kjeldahl determinations are sometimes requested on solutions containing 50% or more of glycerol added as a preservative for biological preparations. Oxidation of protein solutions. containing glycerol in any quantity by the method described above is extremely difficult (3) and is attended by adverse reactions that require special precautions and technique. A glyceri-
nated solution will exhibit troublesonie foaming during digestion, and after all water has been evaporated and sulfur trioxide evolves, the solution becomes viscid. and caramelizes. Rapid carbonization follows with the formation of a large spongy mass of particulate carbon, Tvhich is not prevented by the use of potassium sulfate and copper sulfate. Further digestion of the carbonized sample for 3 to 5 hours yields an unsatisfactory solution for colorimetlic assay. The oxidation of glycerol with potassium bichromate ( 2 ) is time-consuming and involves considerable manipulation. Furthermore, potassium bichromate interferes Kith nesslerization, This method proved unsatisfactory because of time, manipulation, and interfering reagents. A simple, rapid, and useful method for oxidizing protein S d U tions containing glycerol utllizes pure bromine and Superoxol (30% hydrogel1 peroxide) as oxidizing agents in the presence of sulfuric acid. Care must be exercised to prevent overheating the digestion mixture, particularly during the initial stages of oxidation. When excessive heat is applied, the material carbonizes
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