spring-type thermobalances were not suitable. A t high pressures, the arrangement of a spring above a heating zone is very difficult to shield from conductive-convective heating. As no counterweight is possible, very large changes in apparent weight occur simply .because of the change in buoyancy on heating. Also, the weighkletecting system must be enclosed in the pressure system, thus preventing easy adjustments. The automatic torsion thermobalance, because of its compactness and special weightdetecting feature, is easily enclosed in a medium-sized pressure chamber. None of the elements are readily attacked by various gases. Befause of the counterweight feature, the buoyancy correction is small. For
applications to fast reactions, the present thermobalance is more suitable than the commercially available Chevenard thermobalance; otherwise, per€ormance is about the same, the Chevenard being slightly more sensitive. The thermobalance was not designed specifically for vacuum work, but has been used successfully to less than 1 mm. of mercury. For high vacuum work, large difhsion pumps and long p u m p a u t times are needed because of the difficulty of removing adsorbed water and gases from the insulating fibers and other parts of the chamber, although a furnace using radiation shields could avoid some degassing problems. The commercially available Aminco thermobalance performs well as
a high vacuum thermobalance, ‘becauseit is enclosed in a small glass chamber. LITERATURE CITED
(1) Bartlett, E. S., Williams, D. N., Rev. Sn’. Znstr. 28,919 (1957). (2) Guichard, M., Bull. SOC. him. 37, 62 (1925). (3) Holley, J. G., Can. J . Chem. 35, 374 (1957). ( 4 ) Honda, K., Sn’. Repts. Tohoku L’niv. Imp. 4, 97 (1915). (5) Pope, M. I., J. sei. Zmtr. 34, 229 (1957). (6) Powell, D. A., Ibid., 34, 225 (1955). (7) Rabatin, J. G., Gale, R. H., -ASAL. CHEM.28, 1314 (1956). ( 8 ) Wendlandt, W. W., Zbid., 30, 56 (1958). RECEIVED for review February 26, 1959. Accepted June 8, 1959.
Determination of Iron in Urine Using 4,7-DiphenylI ,I 0-phenanthroline PETER COLLINS and HARVEY DIEHL Department of Chemistry, Iowa State University, Ames, Iowa
b The determination of iron in urine can be made rapidly and accurately by wet ashing with nitric and perchloric acids and extracting the red color of iron(l1) with 4,7-diphenyl- 1 ,I O-phenanthroline (bathophenanthroline) into nitrobenzene.
T
demand for increased speed and improved accuracy brought about by the growing u8e being made in medical diagnosis of determinations of iron in blood s e d m and urine is attested by the growing body of literature on the subject. Not only has the clinical chemist been of help to the physician in applying the findings of biochemical rr-*arch, but he has been quick to utilize mort’ sensitive reagents as they have been offered and to increase his output by rmpioying modern instrumentation. The twnd is illustrated by two recent papers by Petcrson (2, 3) which make clw of the superlative iron reagent bathophenanthroline introduced by Case, Smith, %Curdy, and Diehl HE
(1, 4 ) .
A procedure which further increases the speed and accuracy of the determination of iron in urine with 4,745phtnyl - 1,10- phenanthroline (bathophenanthroline) takes advantage of the speed with which large samples of urine can be wet-ashed with nitric and perchloric acid. The d a c u l t y which 1692
0
A N A L Y l l W CHEMISTRY
plagued earlier applications of bathophenanthroline in the presence of perchlorates, the formation of a turbidity in the isoamyl alcohol extract of the ferrous-bathophenanthroline ion, is obviated completely by using nitrobenzene as the extracting liquid. The molar extinction coefficient of the ferrousbathophenanthroline perchlorate is somewhat larger in nitrobenzene than in water or isoamyl alcohol and repeated extractions can be made with ease if necessary, inasmuch as the nitrobenzene is the lower phase. Extraction Fvith nitrobenzene proceeds rapidly and the method preserves the advantages of the original isoamyl alcohol extraction procedure in affording a large concentration factor as well as a method of freeing the various reagents of iron and thus of reducing thc blank to essentially zero. For the concentrations of iron found in normal urine, tenths of a microgram per 50 ml., the method gives consistent results to -within 0.1 7 ; for the larger amounts of iron accompanying disease, the relative error is proportionally less. RECOMMENDED PROCEDURE
Reagents. Bathophenanthroline, 0.001M. Dissolve 33.2 mg. of 4,7diphenyl-l,l0-phenanthroline (bathophenanthroline) (available from the G. Frederick Smith Chemical Co., Columbus, Ohio) in 100 ml. of ethanol.
Hydroxylammonium Chloride, 10%. Remove iron from the solution by adding 10 ml. of 0.001M bathophenanthmline and extracting with 15 ml. of nitrobenzene. Sodium Acetate-Acetic Acid Buffer. Prepare a solution 2M in acetic acid and 2M in sodium acetate. Remove iron by adding 10 mi. of 10% hydroxylammonium chloride and 10 ml. of 0.001M bathophenanthroline and extracting -kith 15 ml. of nitrobenzene. Nitric Acid. Redistill reagent grade nitric acid from an all-glass still and store in a borosilicate glass container. Perchloric Acid, 70%. Use doubly distilled perchloric acid (available from the G. Frederick Smith Chemical Co., Columbus, Ohio). Ammonium Hydroxide. Use reagent grade ammonium hydroxide, or to reduce the blank tQ the ve.ry minimum, distill anhydrous ammonia into water. Deionized Water. Pass distilled water through a column of mixed cationanion exchange resins. Standard Iron Solution, .approximately 0.7 7 of iron per ml. Weigh accurately approximately 0.55 gram of electrolytic iron, dissolve in dilute hydrochloric acid, transfer to a 1-liter volumetric flask, and dilute to volume. Pipet 50.0 ml. of this solution into a 1liter volumetric flask, add 10 ml. of hydrochloric acid, and dilute to volume. From this solution pipet a 25.0-ml. aliquot into a 1-liter volumetric flask, add 10 ml. of hydrochloric acid, and dilute to volume. The concentration
of this final solution is: y Fe/ml. = g. Fe X 1.25. Procedure. Rinse all glassware with hydrochloric acid (1 t o 1) before using t o remove iron. Run a reagent blank along with the samples in exactly the same manner. Pipet 50 ml. of the urine into a 250-ml. conical flask. Add 25 ml. of nitric acid and 10 ml. of perchloric acid. Place a reflux head on the flask, heat to fumes of perchloric acid, and continue the digestion for 10 minutes. If a stone or Transite hood is not available for the wet ashing, use a glass fume eradicator (available from the G. Frederick Smith Chemical Co.). After the flask and contents have cooled to room temperature, rinse the reflux head with water and wash down the sides of the flask. Heat the solution to boiling to dissolve the precipitate of ammonium perchlorate and to remove chlorine. While still hot, transfer the solution to a 125ml. separatory fume1 and add 2.0 ml. of 10% hydroxylammonium chloride and 5.0 ml. of 0.001M bathophenanthroline. Place a small piece of Congo Red paper in the solution and dropwise add ammonium hydroxide until the paper turns red. Complete the adjustment of p H by adding 5.0 mi. of buffer. After the solution has cooled to room temperature add 4.0 ml. of nitrobenzene and shake vigorously for 1 .minute. Allow the phases to separate and gently swirl to dislodge any droplets of nitrobenzene clinging to the upper walls of the funnel. Collect the nitrobenzene layer in a 10ml. volumetric flask and repeat the extraction two more times, using 2.0-ml. portions of nitrobenzene. Dilute the combined extracts to exactly 10 ml. with ethanol and mix. Determine the absorbance of the solution a t 538 mp using l-cm. cells. Use a mixture of nitrobenzene and ethanol (4 to 1) as the reference solution. Correct the absorbance measured for the unknown solution by subtracting from it the absorbance of the reagent blank.
Table 1.
Recovery of Iron Added to 50 MI. of Urine
Fe Originally
Fe Added. y
0.0 4.3 8.; 13.0
‘
-Prpwnt. - ----- -
Fe
Found. Absorbance
y
0.018 0.192 0.381
0.4 4.6 9.1 13.4
0.560
‘
in 50.0 M1. of Urine, y 0.4
0.3 0.4 0.4
Prepare a calibration curve by pipetting various volumes from 0 to 25 ml. of the standard iron solution into 125ml. separatory funnels. Add 10 ml. of 10% ammonium perchlorate, 2.0 ml. of 10% hydroxylammonium chloride, 5.0 ml. of 0.001M bathophenanthroline, and 8.0 mi. of buffer, and proceed with the extraction as directed in the preceding paragraph. U& the extract from the solution to which no iron was added as the reagent blank and subtract its absorbance from the absorbance of each of the other solutions. Prepare a plot of absorbance DS. concentration. DISCUSSION AND RESULTS
The absorption spectrum of ferrous bathophenanthroline perchlorate in nitrobenzene was obtained using a Cary Model 12 recording spectrophotometer. The wave length of maximum absorption was found to lie a t 538 mp. The system conforms to Beer’s law up to a concentration of iron a t least 4 X 10-6M. The molar extinction coefficient was found to be 23,300 using a Beckman Model DU spectrophotometer with a slit width of 0.03 mm. Although nitrobenzene has a slight yellow color. its absorbance is zero at 538 mp and no correction needs to be made for it. Experiments indicated that urine is
rapidly decomposed by digrstion with nitric and perchloric acids. The oxidation proceeds smoothiy and the decomposition requires only 30 to 45 rhinutes. The results on the determination of iron in urine deliberately spiked with known amounts of iron are given in Tab!e I. The urine used was very low in iron, 0.4 y per 50.0 ml., and the recovery of the d d e d iron is complete. The absolute error of the method is of the order of 0.1 y , about 25% of the iron present in the urine analyzed. In certain kidney disorders (6)the concentration of iron in urine increases markedly and on the larger amounts of iron the relative error, is; of course, proportionally less. SUMMARY
The turbidity difficulty often experienced in the use of bathophenanthroline for the colorimetric determination of iron following wet ashing with perchloric acid is obviated by extraction with nitrobenzene. The high sensitivity of bathophenanthroline pius the opportunity to extract the iron impuritics from the reagent solutions and thus of reducing the blank to zrro makes it possible to determine the iron in urinr with errors less than 0.2 y of iron per 50 ml. LITERATURE CITED (1) Case, F. H., J. Org. Chetn. 16, 1541 (1951). (2) Peterson, R. E., ANAL. CHEM.25, 1337 (1953). (3) Seven, M. J., Peterson, R. E., Zbid., 30, 2016 (1958).
(4) Smith, G. F., McCurdp, W. H., Diehl, H., Analyst 77, 418 (1952). (5) Vannotti, A., Delachaux, A., “Iron Metabolism and Its Clinical Significance,” p. 28, Grune & Stratton, New York, 1949.
RECEIVEDfor review April 22, 1959. Accepted July 6, 1959.
Phosphoryl Chloride Enhancement of Fluorescence and Absorbance of Estrogens in Sulfuric Acid JOSEPH C. TOUCHSTONE, JOHN W. GREENE, Jr., and WALTHER R. KUKOVETZ Department of Obstetrics and Gynecology, School of Medicine, University o f Pennsylvania, Philadelphia, Pa.
The addition of phosphoryl chloride to the sulfuric acid used for fluorometry of the estrogens causes a two- to threefold increase in the intensity of both the absorbance and fluorescence. The absorption and fluorescence properties of estrone, estradiol- 170, and estriol in phosphoryl chloridesulfuric acid mixtures were &died.
T
of submicrogram quantities of estrogens in biological fluids and tissues necessitates highly sensitive and reproducible methodology. The fluorescence properties of the estrogens have provided a basis for the development of methods which satisfy these criteria. Various investigators have used either phosphoric acid (3, 5) or HE INVESTIGATION
sulfuric acid (4, 6-81 for fluorometry of the estrogens. Veldhuis (IO) claimed that 700/, sulfuric acid gave maximal fluorescence with estrogens ir, contrast to the 88% reagent advocated b l Diczfalusy (4). The only thing these methods have in common is a strong acid treatment cambined with a short period of nesting. Bauld and GreenVOL. 31, NO. 10, OCTOBER 1959
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