iron is about 10 to 15 mg. or less, 400 ml. of 1.75N nitric acid can be wed for the elution of the trace elements. ACKNOWLEDGMENT
The author thanks c. J. c, 130thma of the National Chemical Research I~aboratorYfor her valuable assistance in doing most of the expcrimcnhl work.
LITERATURE CITED
(1) Gorbach, G., Pohl, F., Microchenie 38, 328 (1951). (2) Stetter, A,, Exler, H., Natuturissenschajlen 42, 45 (1955). (3) Strasheim, A., Eve, D. J., Fourie, R. M., J . South Ajrican Chem. Insl. 12, 75 (1959). (4) Strelow, F. W. E., ANAL. CHEW 31, 1201 (1959).
(5) Ibid., 32, 1185 (1960). (6) Strelow, F. W. E., National Chemical
Research Laborator South African Council for Scient& and Industrial Research Pretoria, South Africa, unpublished data. (7) StFasheim, A., Eve, D. J., J . South Afncan Chemical Instilute, to be publ. RECEIVED for review January 19, 1961 Accepted March 21, 1961.
Chromatographic Purity of Amino Acids Alanine, Aspartic Acid, Cystine, Histidine, Hydroxyproline, and Proline MAX S. DUNN and EDWARD A, MURPHY Chemical laboratory, University of California, Los Angeles, Calif.
b Paper chromatographic methods, analogous to those described previously for arginine, were employed to detect 0.1 to 0.2y0of any one of 26 amino acids present as impurity in alanine, aspartic acid, cystine, histidine, hydroxyproline, or proline.
I
been found possible to detect the prrsencc of a8 little as 0.1 to 0.2% of any one of 20 amino acids in any one of the six nanied amino acids, using modifications of thc procedure of Rockland and Underwood (7) described in our earlier paper (3). These modifications were necessary, since in some instances special solvent systems and/or color reagcnts were required to resolve and detect these amino acid impurities present in amounts as low aa 1 part in 500 parts of a tcst amino acid. Onedimensional chromatography and single, double, or triple development in a single solvent have been utilized when possible, T HAS
Procedures for Chromatographic Purity Tests
Designation A B C D E
Dimension One One One One One One Two Two
Development Triple Single Double Triple Triple F Triple G Single H Double Single I Triple Two Single J Two Double 4 Paper pretreated in buffer. ~
Solvent I I1 I1 ~IIIa IV. V I and I1 I I1 I I1 IandII
but more complicatcd procedures have been required in some cases. The authors' paper chromatographic procedures are designed to detect limit,ing amounts of amino acid impurities in a given test amino acid. Additional tests would be required to identify amino acid impurities. The R, values for amino acids listed in an earlier publication (3) may be referred to for this purpose. It was stated previously (3) that a Table 1.
trace of one optical form in a sample of the other antipode is not detectable by the authors' methods. Although allohydroxyproline may be an impurity in commercially available hydroxyproline, it has not been included in the impurities studied here. Some stereoisomers are separable on ion exchange columns (4, 6) or by lengthy paper chromatogr~phy(a, p. 141), but i t may not be possible to detect traces of one stereoisomeric amino acid pres-
Chromatographic Purity Tests for Six Amino Acids
Amino Acid Impurity
Test Amino Acid,n 500 pg. Spotted -Aspartic HydroxyAmino Acid Alanineb acidb Cyetinec Histidine* prolined Proline6 A A A B Alanine ... C A F A A Arginine A C A C A A Asparagine A C Aspartic acid A A A C A A C' A A A A Citrulline A A A A Cvsteine A I A A $stine A 1 A A A' C A utnmic acid C C A C A H A A Glycine A A A Hiptidine .. C C A A Hydroxyproline A A C A' A A A A A Isoleucine A A A A A A Leucine A A Lysine C A F A A A Norleucine A A A A A A A A A A Norvaline A A D A H H Ornithine A A P hcn yldanine A A A A A Proline A B B A A' C C B A Sarcosine A A C A Serine H A A A A Taurine A H A C A C Threonine J C A A A C Tryptophan G A A A A A Tyrosine A A C A A Valine A A A A C 4 ON HCI solution containing amino acid, mg. per ml. 1 pg./5oo
a.
loo.
20.
* 50. ~~
VOL. 33, NO. 8, JULY 1961
997
ent as impurity in the other stereoisomer.
V. Water-saturated collidine (2,4,6trimethylpyridine) (6).
G., “Paper Chromatography and Pa er ElectroDhoresis,” Academic Press, &w York, 1958. (3) Dum, M. 8. Murphy, E. A., ANAL. CHEM. 32,401 (1900). (4) Hamilton, P. B., Andereon, R. A,, J . B i d . C h q . 213,249 (1955). (5) McFarren, E. F., ANAL.CIiEM. 23, I
EXPERIMENTAL
Color Reagents. Isatin (9,p. 136) for proline impurity, isatin and pdimeth laminobenzaldehyde (S, p. 126) for hy&oxyproline impurit , 1-nitroso%naphthol (8, p. 139) &r tyrosine impurity in alanine and proline, and ninhydrin (7) for all other tests. Solvents. I. tert-Butyl alcoholformic acid-water, 70: 15: 15 (1). 11. Phenol-water, 78: 22 and then this mixture-concentrated ammonia, 94: 1 (7). 111. Phenol-buffer (pH 6.2), 78:22 (6). IV. Phenol-buffer (pH 9.0) 78:22.
Test Conditione. The details of the procedures employed in the chromatographic purity testa of the six amino acids summarized in Table I are given on the preceding page. ACKNOWLEDGMENT
The authors are indebted to Moichi Itami for technical assistance. LITERATURE CITED
(1) Block, R. J., ANAL.CHEM.22, 1327 (1950). (2) Block, R. J., Durrum, E. L., Zweig,
(0)108 Michi, (lg51k ., Birnbaum 8. M., Winits,
“Abstracts of bapers,” 130th z i e t i n ACS, Atlmtic City, N. J., Se temker 1959, p. 19C. (7) &ockland, L. B., Underwood, J. C., ANAL.CHEM. 26,1557 (1954). RECEIVED for review January 18 1961. Accepted April 27, 1961. Work supported by grants from the American Cancer Society, U. 5. Public. Health Service, and University of California.
Spectro p hoto metric Extracti0 n Method Specific for PI uto nium WILLIAM J. MAECK, MAXINE ELLIOTT KUSSY, GLENN L. BOOMAN, and JAMES E. REIN Atomic Energy Division, Phillips Petroleum Co., Idaho Falls, Idaho
b A spectrophotometric extraction method for milligram levels of plutonium is described. Following a silver (11) oxidation, plutonium(V1) as the tetrapropylammonium trinitrate complex is extracted quantitatively into methyl isobutyl ketone from an aciddeficient aluminum nitrate salting solution. Though uranium and neptunium are extracted also, their spectra are sufficiently discrete so that a direct absorbance measurement of the separated organic phase can b e made for plutonium in the presence of the other two actinides. The method is fast, gives excellent decontamination from fission product nuclides, and is virtually free of diverse ion interference. It is applicable to the range of 2.0 to 15 mg. of plutonium with a 0.3% standard deviation at the 12.3mg. level, comparing favorably with redox titrimetric and coulometric methods. A method for the simultaneous determination of plutonium and uranium is described.
P
in aqueous media, exhibits sharp band spectra typically charactoristic of inner transition elements. Each oxidation state, 111, IV, V, and VI, has a discrete spectra with maximum molar absorptivities of 50 to 280. The main application of these data in the past has been the establishment of oxidation-reduction rates and kinetics of reaction rather than serving as bases for analytical methods. In a review article on the analytical chemistry of plutonium, Mets (7) states that the molar absorptivities are deLUTONIUM,
998
0
ANALYTICAL CHEMISTRY
pendent on temperature, pB, anion species, and anion concentrhtion. This behavior is due to the effect of these variables on the formation constants of the various anionic complexes. In addition, any method based on direct m e a urement in aqueous media would be sensitive to diverse , ion interference, especially for the elements in the transition and inner transition groups. Formation of the tetrapropylammonium uranium(V1) trinitrate complex and its extraction into methyl isobutyl ketone from an acid-deficient salting media followed by a direct absorptiometric measurement of the organic phase has been shown to be a rapid, reliable method for the determination of milligram quantities of uranium (6). The of tetrabutylammonium spectrum plutonyl trinitrate in methyl isobutyl ketone, reported by Berkman and Kaplan (S),indicated that a similar system would be applicable for plutonium and that the spectra of the two trinitrate complexes would be sufficiently different to permit a simultaneous determination of the two elements. The spectrum of the neptunium homolog is presented. EXPERIMENTAL
Apparatus and Reagents. All spectrophotometric measurements were made with a Gary Model 14 recording spectrophotometer and 1-cm. Corex cells. The extraction apparatus has been described (4). The source of chemicals and the preparation of the salting solution have been described (6). Silver(I1) oxide was obtained from
Handy and Harman, 82 Fulton St., New York 38, N. Y. Procedure. Samples of 0.5 ml. or leas containing u p to 6 meq. of acid and as much as 15 mg. of plutonium can be extracted from a salting solution which is 0.025M in tetrapropylammonium nitrate and 2N acid-deficient. With the 0.025M tetrapropylammonium nitrate reagent, the sum of plutonium, uranium, and neptunium in the sample aliquot is limited to 15 mg. Larger sums require higher concentrations of tetrapropylammonium nitrate. The preparation of a 0.25M tetrapropylammonium nitrate reagent - has been described (6). PiDet a chloride-free samde of 0.5 ml. i r less, containing from-0.5 to 15 mg. of plutonium, into a 125 X 15 mm. test tube. Add 0.05 ml. of 15.7M nitric acid and heat to boiling. Cool; then add 50 mg. of silver(I1) oxide and 5 ml. of 2.8M aluminum nitrate, 2N aciddeficient, 0.025M tetrapropylammonium nitrate salting solution. Add 4.0 ml. of methyl isobutyl ketone, stopper with a polyethylene stopper, and extract for 4 minutes. If possible, centrifuge to facilitate phase separation; otherwise let stand until the organic phase is clear. Transfer the organic phase to a 1-cm. Corex cell and scan over the wave length region of interest os. methyl isobutyl ketone. (A significant reagent blank has not been observed.) If chloride is present, place the sample in a test tube and either fume twice with 0.5-ml. volumes of 15.7M nitric acid and dilute to 0.5 ml. with 2M nitric acid before the silver(I1) oxide addition, or reduce the volume to 0.1 ml., add 0.4 ml. of 0.1M potassium permanganate, and heat 3 minutes b y holding the test tube 4 inches away from
