341
V O L U M E 25, NO. 2, F E B R U A R Y 1 9 5 3 that utilizes a fixed volume rather thsn a fixed weight of sample. The data required to correct for such effects are not available. Nevertheless, in the analysis of synthetic benzene-cyclohexane mixtures, the sums of the concentrations found do not differ significantly from 100.0%. ACKNOWLEDGMENT
The assistance of E. H. Harclerode and N. G. Foster in establishing operating conditions for the required fractionation is gratefully acknowledged. LlTERATURE CITED
Am. Petroleum Inst., Research Project 44, “Catalog of Mass Spectral Data,” Pittsburgh, Pa., Carnegie Institute of Technology, 1947-. (2) Brown, R. A,, Taylor, R. C., Melpolder, F. W., and Young, W.S., ANAL.CHEM., 2 0 , 5 (1948). (3) Charlet, E. M . , Consolidated Engineering Corp., Pasadena, Calif., Muss Spectrometer Group Rept. 72 (19.50).
(1)
( 4 ) Ibid., 74 (1950).
(5) Lumpkin, H. E., and Thomas, B. W., ANAL.CHEM.,23, 1738 (1951). (6) Marschner, R. F., and Cropper, W. P., Ind. Eng. Chem., 38, 262 (1946). (7) Satl. Bur. Standards, “Selected Values of Properties of Hydrocarbons,” Washington, D. C., Government Printing O5ce, 1947. (8) Purdy, K. M., and Harris, R. J., ANAL.CHEM.,22, 1337 (1950). (9) Scatchard, G., Wood, S.E., and Mochel, J. M., J . Phys. Chem., 43, 119 (1939). (10) Starr, C. E., Jr., and Lane, T., ANAL.CHEM., 21, 572 (1949). (11 1’ Thomas, B. W.. Consolidated Engineering Corp., Pasadena, Calif., Mass Spectrometer Group E&. 88 (1951)(12) Union Oil Co. of California,Ibid., 18, (1945). (13) Washburn, H. W., Wiley, H. F., and Rock, S. M., IND. ENQ. CHEM.,ANAL.ED.,15, 541 (1943). (14) Washburn, H. W., Wiley, H. F., Rock, S. M., and Berry, C. E., Ibid., 17, 74 (1945). (15) Wiley, H. F., “Operation and Maintenance of the Consolidated Engineering Corp. Mass Spectrometer,” Vol. I, pp. 69-71, Pasadena, Calif., Consolidated Engineering Corp., 1946. RECEIVED for review March 14, 1952. Accepted October 4, 1952. Presented before the Pittsburgh conference on -4nalytical Chemistry and A p plied Spectroscopy, March 6, 1952.
Ultraviolet Spectrophotometric Determination of Nitrites With 4-Arninobenzenesulfonic Acid J. M. PAPPENHAGEN‘
WITH
M. G. M E L L O N , Purdue University, Lafayette, Ind.
HE Griess method ( 2 ) for the colorimetric determination of Tnitrites has been used for many years. An investigation of the color reaction was made by Rider ( 5 ) ,who determined the optimum conditions for diazotization of 4-aminobenzenesulfonic acid with nitrites and subsequent coupling of the diazo compound with 1-aminonaphthalene hydrochloride. The versatility of this method is shown by its acceptance as the standard method for the determination of nitrites in water and sewage ( 1 ). 1 Present address, Department of Chemistry, Kenyon College, Gambier. Ohio.
I n the hope of developing an alternate absorptiometric method, it seemed of interest to investigate the ultraviolet absorption spectra of 4aminobenzenesulfonic acid and its diazo compound. The spectral curves of these systems, obtained with a Cary recording spectrophotometer, &re shown in Figure 1. In addition, the spectrum of the diazo compound versus a 4-aminobenzenesulfonic acid blank is included. The nature of the increased absorption of the diazo compound, relative to that for the acid itself, indicated a possible method for the determination of nitrites. This paper presents the results of the investigation. APPARATUS AND REAGENTS
220
250
- 280 Wavelength, m p
310
3
Figure 1. Transmittance of 4-Aminobenzenesulfonic Acid and 4-Sulfobenzenediazonium Chloride pH 1.4
1-cm. cells
0.1 mp band width 0.4 p.p.m. nitrite nitrogen
A Beckman hlodel D U spectrophotometer, with matched I-cm. cells, was used for absorbance measurements. An investigation of the spectral curve of the diazo compound versus a reagent blank showed the wave length of maximum absorption to be 270 mp. Using this wave length for all quantitative measurements, the instrument was aperated a t a constant spectral band width of 1.8 mb. A stock nitrite solution was standardized according to directions given by Kolthoff and Sandell ( 3 ) . Approximately 1 gram of reagent grade sodium nitrite v a s placed in a 100-ml. volumetric flask and diluted to volume with distilled water. An excess of standard potassium permanganate solution was added to portions of this solution, and then potassium iodide. The iodine liberated by the excess permanganate solution was titrated with standard thiosulfate solution. Appropriate amounts of the standardized solution were diluted to 1 liter, and a few drops of chloroform were added as a preservative. The solution should be slightly basic, and a few milligrams of sodium hydroxide per liter may be added. The 4-aminobenzenesulfonic acid reagent was prepared by placing 0.60 gram of recrystallized material in about 50 ml. of distilled water. The solution was warmed to aid dissolution, cooled, and diluted to 100 ml. with water. An acid solution suitable for pH adjustment contained 20 ml. of reagent grade concentrated hydrochloric acid diluted to 100 ml. with water. All other chemicals were of reagent grade, and distilled water was used for all solutions and dilutions. Unless otherwise stated, all absorbance measurements were made versus a reagent blank. EFFECT O F SOLUTlON VARIABLES
In order to specify operating details for a recommended procedure, the effect of possible variable factors was studied.
ANALYTICAL CHEMISTRY
342
x
Table I.
Reproducibility Study
Each 25.0 ml. of sample contained 0.02 Kumber of tests, Range of absorbance values Average absorbance, X Standard deviation. u An estimate of true standard deviation, c’ Control limits f o r a sample size of 2 Range control limits for a sample size of 2, R
mg. of nitrite nitrogen
20
0.440 to 0.462 0.445 0.003795
-= 0.003945 U
= 0.9619 = X %
2 . 1 2 c’
= 0.453 to0.437
0 i o 0.0146
First of all, a tentative experimental solution was prepared for measurement. To a 50-ml. volumetric flask containing approximately 25 ml. of water, there were added, in succession, 3.8 ml. of nitrite solution (containing 0.02 mg. of nitrite nitrogen), 1.0 ml. of the 4-aminobenzenesulfonic acid solution, and 1 ml. of the hydrochloric acid solution. The contents n-ere mixed and diluted to volume. A time of 5 minutes was allowed for diazotization. Using this basic procedure, the effect of each factor rvas studied by varying it xhile holding the others constant. Acidity. The reaction involved during diazotization may be represented as follows: SHz
+K=N
As the equation indicates, a large excess of hydrogen ions should force the reaction to the right. An investigation was made to determine the optimum acidity by diazotizing a series of solutions at different p H values. The cgnclusions reached were identical with those given by Rider ( 4 ) ,and a p H of 1.4 is recommended.
limits on given in the table, it was assumed that there was no interference. The interfering diverse ions and the maximum permissible amounts of each are listed in Table 11. They are divided into two general classes, those that absorb radiant energy of wave length 270 mp and those that do not. This division is based on experiments in which the absorbances of solutions containing 200 p.p.m. of each diverse ion were measured versus a water blank. The following ions exhibited no interference: acetate, arsenite, borate, bromide, chloride, citrate, formate, phosphate, silicate, sulfate, tartrate, tetraborate, thiocyanate, aluminum, ammonium, barium, cadmium, calcium, cobalt(II), lithium, magnesium, manganese(II), nickel(II), potassium, sodium, strontium, thorium, and zinc. The reasons for the interference by the nonabsorbing diverse ions, as \Tell as for many of the ahsorbing ions which also interfere with the diazotization reaction, have been adequately explained by Rider ( 5 ) . Many of the diverse ions show interference common to each of the nitrite methods. However, the follon-ing interfering ions in the Griess method show less or no interference with the ultraviolet absorptiometric method: chromic, cobaltous, cupric, ferrous, mercuric, acetate, iodide, persulfate, and silicate. The reverse situation is true r i t h the following ions: chloroplatinate, uranyl, cyanide, dichromate, iodate, molybdate, nitrate, oualate, selenate, and tungstate.
Table 11. Interfering Diverse Ions (Each 50 ml. of solution contained 0.4 p.p.m. of nitrite nitrogen. p H 1.4 Measured a t 270 mp. 1.8-mp band width. 1-cm. cells) Diverse Added Maximum Permissible Ion as Amount, P . P . l I . Ions Absorbing a t 270 m p 0 100 20
Diazotee Concentration. The addition of 1.0 ml. of 4-aminobenzenesulfonic acid solution to the acidified nitrite sample as recommended by Rider was sufficient for sample solutions which contained from 0.003 to 0.05 mg. of nitrite nitrogen. Time of Diazotization. Using a p H of 1.4 for diazotization, a study showed that maximum absorption occurred after 3 minutes. The diazo compound showed irregular decomposition after 20 to 30 minutes. The time found to be satisfactory for all nitrite concentrations was from 3 to 15 minutes. Nitrite Concentration. Adherence to Beer’s law was observed and satisfactory determinations were made for samples containing from 0.003 to 0.05 mg. of nitrite nitrogen which gave an absorbance range of 0.06 to 1.13 using 1-em. cells. Precision. A study of precision was made in order to evaluate statistically the effect of the addition of diverse ions. The nitrite content of twenty solutions was determined, and the results of this reproducibility study are shown in Table I. Each final 50 ml. of solution contained 0.02 mg. of nitrite nitrogen, 1.0 ml. of 4-aminobenzenesulfonic acid, and 1.0 ml. of hydrochloric acid solution, and x a s diazotized for 5 minutes. Diverse Ions. The procedure used in the diverse ion study was to place 0.02 mg. of nitrite nitrogen and 20 mg. of the diverse ion in a 50-ml. volumetric flask containing approximately 25 ml. of distilled n-ater. The contents were mixed well. One milliliter of the 4aminobenzenesulfonic acid reagent and 1.0 ml. of hydrochloric acid solution were added, and the contents were diluted to volume and mixed. Absorbance measurements were made a t 270 mp after 5 minutes. If the range of absorbance values for each pair of solutions fell within the control limits for R given in Table I, the average absorbance was calculated. If the range exceeded these limits, the pair was rerun. If the average absorbance of each pair of given in Table I, solutions fell outside the control limits on progreesively smaller amounts of each diverse ion were added until this restriction was met. The procedure u s u a l l ~followed was to add successively 15, 10, 5, and 1 mg. of the diverse ion. If the average of the two absorbance values fell within the control
x
0 60 60
0 0 0 0 0
100 20
n n
PGt. 0 20 Ions S o t Absorbing at 270 nip 0 300 20 300 300 300 0 Ppt. 20 50 200 Ppt
.
The nitrate concentration is expressed as nitrate nitrogen. A reference solution containing 5 mg. of nitrate nitrogen per 50 ml. will compensate for nitrite samples containing from 0.3 t o 10 mg. of nitrate nitrogen. b An equivalent amount of nitrate ions was added t o reference solution. a
The interference of the cations does not present a great difficulty if the nature of the sample permits its passage through a cation exchange column containing a commercially available resin. The authors’ experience with a similar separation problem has shown that there is a complete recovery of nitrites and nitrates from samples passed over the Amberlite I R 120 cation exchange resin. The problem of nitrate ion interference can be overcome by adding a known amount of nitrates to the reference solution. Experiments have shown that the nitrate ion exhibits a general absorption in this region of the spectrum, and that the use of a reference solution containing 5 mg. of nitrate
343
V O L U M E 2 5 , NO. 2, F E B R U A R Y 1 9 5 3 nitrogen per 50 ml. permits the successful determination of nitrites in samples containing from 3 to 10 mg. of nitrate nitrogen. COYCLUSIONS
An ultraviolet absorptiometric method is described for the determination of nitrites by measuring diazotized 4-aminobenzenesulfonic acid versus a reagent blank. The method does not approach the sensitivity of the Griess method for nitrites since the molar absorptivity of the Griess method is 41,000 and that of the described method is 15,500. The new method does have the possible advantage of applicability a t higher nitrite doncentrations, where the uncertain precipitation of the relatively insoluble azo d!-e may present a problem. Another advantage is the rapidity with which sample determinations can be made. The time involved is considerably shorter than that required for the Griess method in that there is no coupling reaction. As is obvious from the absorbance maximum used, the sample should be relatively free from organic matter. RECOMMENDED PROCEDURE
Sample. Weigh or measure by volume an amount of the sample containing from 0.003 to 0.05 mg. of nitrite nitrogen. Any
interfering ions which may be present should be made to conform to the permissible concentrations given in Table 11. Desired Constituent. Place the sample in a 50-ml. volumetric flask and adjust the p H to 1.4; for previously neutralized unbuffered samples, 1 ml. of the hydrochloric acid solution is sufficient. To the acidified sample add 1.0 ml. of 4-aminobenzenesulfonic acid reagent, dilute to volume, and mix the contents. After 3 minutes, and less than 15 minutes, measure the absorbance of the diazo compound a t 270 mp in 1-cm. quartz cells versus a reagent blank. LITERATURE CITED
(1) American Public Health Association, Xew York, “Standard Methods for the Examination of X-ater and Sewage,’’pp. 71, 121, 1946. (2) Griess, P., Be?., 12, 427 (1879). (3) . . Kolthoff, I. M.. and Sandell. E. B “Textbook of Quantitative Inorganic iinalysis,” p. 603, S e w Tork, Macmillan Co., 1943. ( 4 ) Rider, B. F., thesis, Purdue University, 1944. (5) Rider, B. F., with Mellon, M. G., ISD. ENG.CHEM., ANAL.ED.,
.
18,96 (1946). RECEIVEDfor review August 4, 1952. Accepted October 22, 1952. hbstracted from a thesis presented by J. AI. Pappenhagen to the Graduate School of Purdue University in partial fulfillment of the requirements for t h e degree of doctor of philosophy.
Microgram and Submicrogram Determination of Phosphate -4pplications of Sealed Tube Digestion and Capillary Cell Spectrophotometry F R E D E R I C K L. SCHAFFER’, JEAN FONG, AND PAUL L. K I R K Dit-ision of Biochemistry, University of Calqornia Medical School, Berkeley, Calif.
CONSECTIOS it-ith the analysis of needle biopsy samples IXfrom the liver, in the study of infectious hepatitis and other
influences, it became necessary to determine microgram and submicrogram quantities of phosphorus. The tissue samples available, with a dry weight of about 1 mg., contain adequate phosphorus for microgram spectrophotometric analysis. The fractions in which the phosphorus exists, when analyzed separately require that the lower limit of analysis be reduced. Various phosphorus-containing fractions of tissue are of the greatest biochemical and possibly clinical interest, including nucleic acids, phospholipids, coenzymes, and metabolic intermediates. The method of Berenblum and Chain ( 1 ) involving extraction of the phosphomolybdic acid with isobutyl alcohol has several fundamental advantages over methods in which the molybdenum blue is developed in the aqueous phase. The medium is essentially constant in composition and the molybdenum blue appears to be in molecular solution. The method is also valuable for analysis of phosphate in colored solutions (9) and in the presence of labile phosphate compounds ( 3 , 9, 11, 12). When this procedure is employed with capillary absorption cells in the spectrophotometer, large blanks are obtained which reduce greatly the analytical range of concentration, and introduce additional uncertainties in the results. These difficulties have been overcome in the method described here by employing noctyl alcohol as the extracting agent. S o t only does octyl alcohol extract phosphomolybdic acid readily, but it apparently does not extract any appreciable quantity of molybdic acid, which is not the case with butyl alcohol. Martin and Doty (Q), who employed a mixture of isobutyl alcohol and benzene, indicated that various solvents may be applicable for the extraction of phosphomolybdic acid. The technic of extraction when the amount of sample is very 1 Lt. USNR. Office of Kava1 Research Unit S o . 1, University of California, Berkeley, Calif.
small has been approached with a number of extractor designs ( 7 ) . With amounts of phosphorus not less than 1 microgram, the extractor of Kirk and Danielson (8) is very satisfactory. In the submicrogram range, all liquid-liquid extractors for lighterthan-water solvents suffer from the necessity of unit operation and constant attention, combined with manipulative difficulties in some instances. An extraction technique for as many as eight simultaneous samples is presented here. hnother problem of importance is the method of digestion of biological samples prior to the determination of phosphorus. Digestion in a sealed tube, which has proved very satisfactory for the Kjeldahl determination of nitrogen on the microgram scale (6), may also be used for phosphate. In this paper are described procedures for analysis of phosphorus in quantities ranging from a few micrograms down to 2 my, making use of sealed tube digestion with sulfuric acid alone, extraction of phosphomolybdic acid with octyl alcohol, and final spectrophotometric measurement which in the lower ranges requires the use of capillary absorption cells. APPARATUS
Extractors. Shown in Figure 1, extractors (hIicrochemica1 Specialties Co., Berkeley, Calif.) were constructed of borosilicate glass tubing with over-all height of 90 mm., length of upper chamber of 47 mm., and outside diameter of 14 mm. The capillary bore of the large extractor, A , was 1 mm., and of the small extractor, B, 0.3 mm. The bulb capacities were 500 and 62.5 pl., respectively (to levels falling within the capillaries). The extractors were designed to fit into 15-ml. cups of the International clinical centrifuge. Centrifugal Pipets. Special pipets (Microchemical specialties Co., Berkeley, Calif.) of short length and 200 and 25 pl. capacities were constructed as shown in Figure 1. Each was provided with a plastic collar which was seated in the top of the extractor. Shaker. The edges of a hard rubber stopper were cut to form a pentagon. The stopper was then mounted on the shaft of a laboratory motor in a horizontal position.
