V O L U M E 2 6 , NO. 1 2 , D E C E M B E R 1 9 5 4 of ethyl alcohol in the sample to be analyzed, in the silica gel, or in the chloroform used in fractions 0 t o 2 are likely to interfere with the effectiveness of the system in resolving steroids. If the stopcock remains open during the packing of the column or if the silica gel is allowed to become too warm during the process of chromatography, small vapor bubbles tend to form in the column and impede the flow of solvents. I n the chromatographic analysis of the above six steroids, cortisone and corticosterone have the greatest tendency to overlap. However, the quantitative analysis of corticosterone by fluoresence is not influenced by the presence of cortisone as the latter steroid has a.low index of fluoresence with the sulfuric acid technique. 17-Hydroxycorticosterone may also be measured in the presence of cortisone without appreciable error. Separation of desoxycorticosterone, 17-hydroxyl 1-desoxycorticosterone, and 4-pregnene1 1-p, 17-01, 20-8, 21-tetrol-3-one from each other and from cortisone, corticosterone, and 17-hydroxycorticosterone appear to present no particular problems. Morris and Williams ( 1 ) and Selson and Samuels ( 2 ) have reported the use of column chromatography in conjunction with their methods for evaluating corticosteroids in blood. These methods, however, require relatively large quantities of blood for analysis. The Xelson and Samuels method requires 20 to 30 ml. of blood. I t does not analyze for cortirosterone. The Morris and Williams method requires 50 ml. of blood and two separate chroniatographic columns €0 analyze for corticosterone and 17-hydroxycorticosterone. Resolution and analysis of the
1967 two steroids is accomplished by the present method with only 5 ml. of blood (2 ml. of plasma) on a single column. ACKNOWLEDGMENT
The author is indebted to George Sayers for advice and friendly criticism throughout the period of this investigation, to Gordon L. Farrell for the phenylhydrazine analyses, and to Ella Sandberg for her valuable technical assistance. The corticosterone, li-hydroxy-11-desoxycorticosterone and 4-pregnene-1 1-8,17-a, 20-8, 21-tetrol-3-one were supplied through the kindness of Leonard R. Axelrod, William J. Haines. Dan A. McGinty, and Tadelis Reichstein. LITERATURE CITED
(1) llorris, C. J. 0. R., and Williams, D. C., Biochem. J . , 54, 470 (1953). ~, (2) Nelson, D. H., and Samuels, L. T., J . Clin. Endocrinol. and Metabolzsm, 12, 519 (1952). (3) Porter, C . C., and Silber, R. H., J . Bzol. Chem., 185, 201 (1950). 26, 773 (1954). (4) Sweat, 31. L., ANAL.CHEM., (5j Sweat, AI. L., manuscript in preparation.
( 6 ) Sweat, AI. L., hbbott, W. E., Jeffries, W. Li., and Bliss, E. L.. Federation PTOC., 12, 141 (1953). (7) Sweat, M.L., and Farrell, G. L., J . Clin. Endocrinol. and Metabolism,12, 968 (1952). RECEIVED for review M a y 7, 1954. Accepted July 31, 1954. Investigation supported by a research grant (h-331) from t h e National l n s t i t u t e of Arthritis a n d Metabolic Diseases of the National Institutes of Health, Public Health Service.
Some Errors in the Determination of Calcium in Aged Blood Serum Eliminated by Flame Photometry P. S. CHEN, JR.,
and
T. Y . TORIBARA
School o f M e d i c i n e and Dentistry, University of Rochester, Rochester,
Errors in the measurement of the calcium in aged blood serum by precipitation as calcium oxalate, followed by titration of the oxalate, are manifold. The principal sources are the nonspecificity of the oxidizing agent, the incompleteness of calcium precipitation, and the contamination of the precipitate by oxalates other than calcium. The direct measurement of the calcium by flame photometry of acidified solutions from which protein has been removed eliminates most of the errors.
C
ALCIUM in blood serum has probably been determined most frequently by the Clark-Collip ( 4 ) modification of the Kramer-Tisdall ( 5 ) method, which is based on the precipitation of calcium oxalate by the addition of ammonium oxalate to serum. A thorough coverage of the variable results obtained by other investigators has been made by Sendroy (8) in the introduction of a paper in which he reports the results of a very detailed study of many of the sources of error. He shows that identical results are obtained whether precipitation of calcium by oxalate was carried out on dilute serum, ashed serum, or the trichloroacetic acid filtrate of serum (protein removed) after appropriate corrections have been made for the calcium content of the ashing reagents, filter paper, and trichloroacetic acid. His studies were carried out on human serum from blood freshly drawn from ambulant dispensary patients. A direct measurement of the calcium by the flame photometric procedure ( 3 ) has again shown that calcium may be precipitated completely as the oxalate from fresh serum. Analyses on aged serum samples, however, gave variable and erratic results. For
N. Y.
this reason a calcium balance was made on a number of aged serum samples, and a typical balance of a sample of calf serum which had been stored in a refrigerator for several weeks is shown in Table I. The trichloroacetic acid filtrate of serum contains all of the serum calcium, and all of this calcium can be precipitated by osalate by raising the p H to 5 . There was no detectable difference in the calcium content of the supernatant liquid when the protein was separated by centrifugation or by filtration through a Whatman No. 42 paper. S o error was introduced by the use of ashless filter paper in the preparation of protein-free filtrate-. The presence of trichloroacetic acid (Eastman white label) in the amounts used increased the emission a t 620 mp less than 1 yo. DISCUSSION
The inconsistencies in the results obtained with protein present may be attributed to the age and condition of the serum. In freshly drawn serum, the calcium may be completely precipitated
Table I.
Typical Calcium Balance Determined on Aged Calf Serum
Component .4nalyzed Serum, direct dilution, I t o 100 Serum, oxalate ppt. Serum after p p t n . by oxalate TrichlAroacetic acid filtrate, direct Trichloroacetic acid filtrate, oxalate p p t . Filtrate direct reading a f t e r pptn. Proteins, pptd. by T C A , washed twice with T C A
Calcium, hIg./100 hll 12.3 11.1 1.2 12.0
12.0 0.008
0.08
ANALYTICAL CHEMISTRY
1968 by the addition of oxalate but, in pa,rtially denatured or cloudy serum, the precipitation is incomplete. McLean and Hastings (6) have shown that a large fraction (up to 50%) of the calcium in serum is protein-bound. Protein which precipitates on standing a t physiological pH takes with it part of the calcium. When trichloroacetic acid is employed for protein removal, all the calcium is left in solution. Carr (2) has shown with bovine gerum albumin t’hat there is no protein-hound calcium when the pH is lowered to 4.5 and that the binding increases as the p H is raised above this figure. Trichloroacetic acid serves a dual purpose by releasing all the protein-hound calcium while removing the protein. While protein removal ensures complete precipitation of all the calcium in serum, the estimat’ion of the calcium through the oxalate content of the precipitate has several pitfalls. The theoretiral composition of the precipitate itself is in doubt (IO),for it is reported that the oxidizing agent must he standardized against a known calcium solution carried through the Pame precipitation steps as the unknown. A standardization against sodium osalate gives erroneous calcium values. The difficulties of separating calcium and mngnesiuni are discussed thoroughly in an elementary cpantitative analysis book (11). Smit,li et al. (9) used t,he direct spectrographic metliod of Boyle et a[.(1) for calcium and magnesium to make a thorough study of the precipitate obtained when ammonium oxalate is added to blood serum. They report that as much as 15% of the oxalate content may be due to magnesium. The uncertainties which may be encountered by determining d c i u i n , through the oxalate content of the precipitate, niay be c,liminated by a direct measurement of the calcium with a flame photometer. Nosher et al. ( 7 ) measured the calcium content l)y flame photometer after first wet-ashing the serum and separating the calcium as the phospha,te. On freshly drawn serunl the calcium may be determined directly after proper dilution, iising calcium chloride standard solutions which have been compensated for sodium anti pot ium (3). E’r)i. rloudy serum sam-
ples, complete protein removal vith trichloroacetic acid is brit to liberate the calcium from the suspended material. The cdcium content of these filtrates mag be determined by flame photometer with standard solutions containing phosphate or hy use of a suitable working curve (3). For the most accurate n.oi,k it is rrcommended that the calcium be precipitated as the oxalate from the trichloroacetic acid filtrate. The oxalate precipitate need not be washed carefully, as it has been shown ( 3 )that neithcr ammonium oxalate nor magnesium will interfere in the flame photometric determination of calcium. Since sodium, potassium, and phosphate have been removed, standards containing only calcium chloride may be used. LITERATURE CITED
(1) Boyle, A. J., Whit,ehead, T., Bird, E.
S.,Batchelor, T. AI.. 1sri.i. L. T., Jacobson, 6 . D., and LIyers, G. B., J . Lab. Clin. M e d . , 34, 625 (1949). (2) Carr, C. W., Arch. Biochetn. and Biophys., 43, 147 (1953). (3) Cheri, 12. S., Jr., and Torihara, T. Y., ANAL. CHEM.,25, 1Gt3 (1953). (4) Clark, E. P., and Collip, J. B., J . H i d . Chem., 63, 461 (1925). (5) Kramer, B., and Tisdall, F. F.. Ibid., 47, 475 (1921). (6) IIcLean, F. C., and Hastings, A. B., Ana. J . Med. Sci., 189, 601 (1935).
(7) llosher. 11. E., Itano, LI., Boyle, A. !J., Myers, G. B., and Iaeri, L. T., Am. J . Clin.Pathol., 21, 75 (1951). (8) Sendroy, J., Jr., J . Riol. Chem., 152, 539 (1944). (9) Smith, R. G., Craig, P., Bird, E. J., Boyle, A. J., Iseri, L. T., Jacobson, S.D., and Myers, G. B., Am 263 (1950). (IO) Willard, H. H., and Diehl, H., “Advanced Quantitative Analysis,” p. 296, New York, D. Van Sostrand Co., 1944. ( I 1 ) Willard, H. H., Furman, K. H., and Flagg, J. F., “A Short Course in Quantitative .inalysis,” pp. 211-15, Nen- York, D. Van Nostrand Co., 1944.
R E C ~ I V Efor D review November 30, 1933. -4ccepted J u n e 30, 19.54. Based on work performed under contract with the United States Atomic Energy Commission a t the Vniversirs of Rorhester Atolnir? Energy Project, Rorhester. X. Y.
CoIo rimet ric Dete rmination of CobaIt with 2,2 ’,2 ”-Te rpy ridine RONALD R. MILLER’ and WARREN W. BRANDT Department o f Chemistry, Purdue University, Lafayette,
An extraction adaptation of the colorimetric method for the determination of cobalt using 2,2’,2’’-terpyridine has doubled the sensitivitj and offset the disadvantage of the instability of the color. \-ariatiom in pH between 2 and 10 do not affect the colored species, and Beer’s law is valid for cohalt concentrations from 0.5 to 25 p.p.m. Interference hy most of the common metals except iron and copper above 100 p.p.rn. and nickel above 30 p.p.m. is not serious. Oxidizing agents and cyanide should he absent.
T
HE cobalt-2,2’,2”-terpylidiiie complex was first recognized by it,s interference with the iron terpyridine system (3). The composition of the orange salt \vas established hy Rforgari and Burstall to contain 2 molecules oi ligand per cobalt ion ( 2 ) . Oxidation of the bromide salt with chlorine gave a yellow cobalt( 111)chloride with the same polyaininn coordination. lloss and Mellon ( 4 ) proposed n c~~lorimetric method for cobalt using 2,2’,2”-terpyridine in the pH r:iiige 2 t o 10. Beer’s law is valid for cobalt concentrations from 0.5 to 50 p.p.m., and most common metals do not interfere. Copper, nickel, iron, cyanide, and dichromate interfere. This method suffers from a limited stability of the color which hiiidors its usefulness. I
Present address, Union Oil Co , R r m Calif.
Ind. This investigation proposes a method whereby the stability of the color is no longer a limiting factor, and in some instances allows a larger amount of interfering constituents to be prewiit. EXPERIMEVTAL WORK
Solutions and Apparatus. The csolor-forming reagent Mas a 0.1% aqueous solution of 2,2’,2”-terpyridine containing enough hydrochloric acid to dissolve the reagent. A standard solution of cobalt nitrate was prepared by dissolving the desired amount of reagent grade cobalt nitrate hexahydrate in iron-free distilled water. The nitrobenzene mas vacuum distilled a t 1- to 2-mm. pressure. Adjustments of p H were made with 6M sodium hydroxide and 6 M hydrochloric acid. The pH was measured with a line operated Leeds and Sorthrup p H indicator. Standard solutions of the anions studied were prepared from the alkali metal salts in most cases. Sitrate, chloride, and sulfate salts of the cations were used. Each solution contained 10 mg. per ml. of the ion in question. Spectrophotometric curves were obtained with a General Electric recording spectrophotometer with a band width of 10 mp. Individual absorbance measurements were made with a Beckman Model B epectrophotometer with a band width of 5 mp. Extraction Attempts. The success of Margerum and Banks in extracting the iron-phenanthroline complex ( I ) suggested the extraction of this complex into an immiscible organic solvent as a means of making the color more stable and of avoiding some
