the colored zone was scraped off the illate, washed in acetone, and filtered through a cotton plug. The absorption sl)ectruni for each colored zone was obtained on a recording spectrophotometer. The absorption spectrum obtained for each spot matched the spectra rellorted in the literature. ACKNOWLEDGMENT
The assistance given to this project by G . P. Fitzgerald is greatly appreciated.
LITERATURE CITED
(1) Anwar, M. H., J . Chern. Educ. 40, 29 (1963). ( 2 ) Lind, E. F., Lane, H. C., Gleason, L. S., Plant Physiol. 28, 325 (1953). 13) Rollins. C.. J . Chert. Educ. 40. 32 ( 4 ) S,porer, A . H., Freed, S., Sancier, M.,
Sczence 119, 68-9 (1954). (5) Strain, H. H., J . Phys. Chem. 57, 638 (1953). 1~, 6 ) Strain. H. H.. Thirtv-Second Priestlev Lecture,' University Park, Pa., 1958. " ( 7 ) Strain, H. H., Murphy, G. W., ANAL. CHEM.24, 50 (1952).
18) . . Strain. H. H.. Sato. T. R.. b'. S . i l t . Energy Comrn. R e p t . No. TID7512, 1956.
HAKUMAT RAI G. FREDLEE
Water Chemistry Program Hydraulic and Sanitary Laboratory University of Wisconsin Madison, Wis. 53706
INVESTIGATION supported in art by a grant from the Wisconsin Afumni Research Foundation and P. H. S. Training Grant No. ITI-WP-22-01, Division of Water Supply and Pollution Control, Public Health Service.
Determination of Serum Creatinine by Ion Exchange Chromatography and Ultraviolet Spectrophotometry SIR: Serum creatinine methods now in use are based on the colorimetric measurement of a red chromogen formed by reaction of the compound with alkaline picrate [the Jaffe reaction ( 5 )J in a protein-free filtrate. Sumerous investigators (Z-i+] 6! 7 ) have demonstrated the Jaffe reaction to be nonspecific and unreliable without prior removal of interfering substances. Many biologically occurring compounds other than creatinine contribute to the Jaffe reaction, and attempts to remove these substances, as well as the serum proteins, by precipitation techniques are often accompanied by serious manipulative loss. h method recently reported by the authors for the determination of urinary creatinine involved preliminary separation of the compound by an anion exchange resin and subsequent ultraviolet spectrophotometric measurement of its concentration ( 1 ) . That procedure was not directly applicable to serum because of the high concentration of protein arid the relatively low creatinine concentration (about one twentieth that of urine). The quantitative separation of creatinine on a strong cation exchange resin from substances which interfere with the Jaffe reaction was recently descritjed by Teger-Kilsson ( 7 ) . His proredure, however, proved unsatisfactory for direct ultraviolet spectroIihotoinetric studies because of high intcrfering absorbance at 220 to 260 nip. In the present investigation, a two-column resin system was successfully employed for elimination of t,he substances responsible for the interfering absorbance, thus making feasible a ~iiectrophotometricdetermination of serum creatinine. Weakly acidified serum was ai)plietI to a cation exchange resin column which retained the creatinine, while wrum Iiroteins, uric acid, and some of the other interfering sub-
AIR
PRESSURE
4.
APPARATUS A Figure 1. Apparatus for Dowex 50 cation exchange resin column
stances were removed by washing. Creatinine was then eluted directly from that resin column through an anion exchange resin and the eluate quantitatively collected for ultraviolet spectrophotometric measurement. EXPERIMENTAL
Reagents. WATER. Deionized water was used throughout for all reagents, washing the resins, and final rinse of glassware.
CREATININE.A stock standard solution was made up in O.1N HC1 at a concentration of 1 mg. per ml. ALKALINE AMMONIUM CHLORIDEREAGENT. Ammonium chloride, 1.33 gram, was dissolved in about 800 ml. of water; 15 ml. of concentrated ammonium hydroxide were added and the volume was adjusted to 1 liter. Apparatus. U.V. SPECTROPHOTOMETER. Beckman Model DU or DK-2 with semimicro-absorption cells, 1-em. light path. T h e recording model was used for this investigation. MANIFOLD AND AIR PRESSURE REGULATOR. A glass manifold equipped with a stopcock a t each port opening was employed for independent control of the air pressure to each resin column. The regulator used for control of air pressure to the manifold was the Kullmatic .\ir Pressure Model 4015, Moore Products Co., Philadelphia. APPARATUSFOR RESIN COLUMNS. The design of the equipment for chromatography with Dowex 50 resin is shown in Figure 1. The narrow-bore section a t the bottom for sulqiort of the resin was provided by the glass tubing attached to the socket portion of a ball and socket joint. The reservoir a t the top for accommodation of the larger amounts of wash solutions could be attached by glass joint connections to the resin column below and to the manifold above. The column for the Dowex 2 resin is shown in Figure 2 . Procedure. P R E P l R A T I O N A K D REG E N E R A T I O N O F DOWEX 50 R E S I N COLUMN.The resin (Dowex 50 W-Xl2, H + form, 200 to 400 mesh, small porosity) was introduced as a slurry into the narrowbore column of ailparatus * I (Figure I ) with a filling pipet such as the 11-introbe. The ball and socket joint was then sealed with a Teflon-base sealant (Hoke Slic-Seal), secured with a pinch clamp, and the column w,s allowed to stand 24 hours to ensure drying of the sealant before the column was subjected to air pressure. .'\fter a column had been used for a serum creatinine analysis, the resin was regenerated by 1)assing VOL. 36, N O . 1 1 , OCTOBER 1964
2209
Table I.
Sample
Creatinine Recovery Experiments
APPARATUS A
Initial,
Added,
Found,
Recovery,
rg.
rg.
rg.
% I ML. SYRINGE BARREL IO CM. LONG
STANDARD 10.8 15.0 1R.O
0 0
FIG.1
11. 0 15.1
101.9 100 7
y GLASS WOOL
SERUM #1
#2 62
18.3 6.2
14.0 27.9
32.3 33.8
100.0 99.1
6.5 6.5
1.5 3 .O
7.9 9.3
98.7 97 9
#3 #3 #3 83
#4 #4
through in sequence 3 ml. of 0.5N NaOH, 5 ml. of water, 3 ml. of 3 N HC1, and finally water until the pH of the effluent was approximately 4 to 5 when checked with wide-range indicator paper. The column was stored with water in the reservoir between determinations.
and kept in a covered container to prevent the resin from drying. Shortly before a Dowes 2 column was to be used in conjunction with a cation exchange column in the analytical procedurc, about 3 ml. of water were passed through, followed by 3 nil. of the alkaline ainmoniuin chloride rePREPARATION AND REGENERATIONagent, and the column was drained OF D o w ~ x 2 RESINCOLUMN.A small completely without allo\ving the resin to become dry. The absorbance of pad of glass wool was gently tamped the alkaline aninioniuiii chloride soluinto the bottom of the syringe barrel (Figure 2), the resin (Dowex 2-X8, tion collected after it had passed through the column was measured at C1-, 200 to 400 mesh, medium po233 nip and should be less than 0.03. rosity) was introduced as a slurry with a The resin column was now ready filling pipet, and a sinal1 amount of for use in the analytical procedure. glass wool was placed on top of the To clean and regenerate the resin resin bed. The resin columns were for employment in a subsequent analysis washed with 3 nil. of water and were after it had been used with serum, the stored standing in a beaker containing column was washed with 3 ml. of water to a depth of a t least 1 inch water, with 3 ml. of 311; HC1, and finally with water until the pH of the effluent was apl)roxiniately 4 to 5 when checked with wide-range pH test Table II. Duplicate Analyses of Crepaper. 1 1.0-ml. ali~ N A L Y S I S OF S m o u . ; atinine in Normal and Pathological quot of serum was mixed in a 5-ml. Sera beaker with 1.0 ml. of 0.1A' HC1, and Serum creatinine, mg. 70 the mixture was t8hen transferred quantitatively by a Kintrobe pipet NORMAL FEMALES to the lower part of the reservoir of 0.76 0.77 apparatus A (Figure 1). This was followed by addition of 1 ml. of 0.05S HC1, which was first used to rinse the beaker. (If sufficient serum was available, a 2-ml. aliquot of seruni was mixed with an equal volume of 0.LV NORMAL MALES HCI and after evolution of carbon dioxide had ceased, a 2.0-ml. aliquot 0.89 0.90 of the mixt,ure was transferred by pipet 1.04 1.03 1.05 1.03 to the reservoir. A number of analyses 1.16 1.12 have also been carried out' succ,esjfully 1.18 1.13 with a quantitative transfer of a niix1.17 1.29 ture of 0.5 ml. of serum and 0.5 ml. 1.34 1.33 of acid.) The apparatus was attached to the RENALINSUFFICIENCY manifold and air pressure of 2 1). ' 4 67 4 78 was applied. The resin was allovied 5 38 5 40 to drain, air pressure was cut off a t 6 22 6 38 t,he manifold stopcock, and 3 ml. of a 11 0 11 0 0.9Yc aqueous ammonium chloride solu12 2 12 4 tion were introduced into t'he reservoir 13 6 13 9 and passed through t'he resin bed with 22 10
ANALYTICAL CHEMISTRY
APPARATUS
B
Figure 2. Apparatus for Dowex 2 anion exchange resin column
2 1i.s.i. of air pressure. A final wash was made with 5 ml. of viater. (Once t'he water has been added, t'he procedure may be interrupted by turning off the pressure device and the analysis can be completed the following day without loss.) After t,he water had passed t,hrough the column, the air pressure lvas turned o f f , the effluent,s were discarded, and apparatus B (Figure 2) was placed directly under apparatus A . For elution of the weatminine,2 ml. of the alkaline ammonium chloride reagent were added to t8hereservoir of apparatus d and 0.5 p.s.i. of air pressure was applied. The eluate cont,aining the creatinine passed from the Dowex 50 column directly onto and through the Dowex 2 column into the 2-nil. volumet,ric flask. The volume was adjusted to 2 ml. with wat'er and the contents of the flask were thoroughly mixed before transfer to a cuvet'te. The absorbance of the sample was measured with water in the reference cell, but for greater precision it was corrected for t8he absorbance of t'he alkaline aninioniuni chloride control effluent. W t , h a nonrecording spectrophotometer the absorbance was first checked a t 233 mu; if the value was too high for the range of minimal instrumental error, measurements were t,hen made a t 250 mp, 255 nip, or 260 mu. 1 3 ~ selection of the appropriate viavelmpth, a wide range in concentration of creatinine could be determined without resorting to dilution. S o error was introduced if the eluates were allowed toppered flasks at, rooin temperature for 24 hours before spectrophotometry was performed.
STAXDARDIZATION. A 10-ml. aliquot of the stock creatinine standard (1 mg. per ml.) was diluted to 100 ml. with the alkaline ammonium chloride reagent. (Because the spectrum of creatinine exhibits a pronounced hypsochromic shift a t an acid pH, the spectrophotometric measurements were routinely made in the alkaline ammonium chloride reagent.) A graded series of aliquots of that solution was further diluted to 10 ml. with alkaline ammonium chloride to cover a range of 2.5 to 100 pg. per ml. The absorbances were determined at 233 mp, 250 mw, 255 mb, and 260 mp, and absorptivity a t each wavelength could be calculated or standard curves of absorbance us. concentration plotted. RESULTS AND DISCUSSION
Mean absorptivities, calculated individually with the data for the above wavelengths where the absorbance was between 0.2 and 0.9, were 61.8, 27.3, 13.5, and 4.9, respectively. Relative standard deviation using the same data was 0.6%, 1.7%, 1.5%, and 2.9%. Thus, in instances where relatively
high concentrations of creatinine are encountered, absorbance measurements a t wavelengths other than 233 mp may be used to circumvent further dilution of the sample or eluate. The reproducibility of the procedure was tested by performance of five replicate determinations on 0.5-rnl. aliquots of the same serum using different columns for each analysis. The mean of those determinations was 1.31 mg.% with a relative standard deviation of 1.9%. Recoveries of known amounts of creatinine, both alone and after addition to serum, are shown in Table I. Recoveries obtained were within ~ 2 . 1 7 , of theoretical. Duplicate analyses on a number of normal and pathological sera are shown in Table 11. The mean difference between duplicate analyses for the 18 sera was 2%. The spectra from different serum samples were identical to that of creatinine, and if interfering substances-e.g., from drugs-were to be encountered, a change in the characteristics of the spectral curves should be obvious. Xumerous sera from
various patients who were receiving a wide variety of drugs have been analyzed, and to date no interfering substances have been found. LITERATURE CITED
(1) Adams, W. S.,Davis, F. W., Hansen, L. E., ASAL. CHEM.34, 854 (1962). (2) .4rchibald, R. M., J . Biol. Chem. 237, 612 (1962). (3) Dubos, R., Miller, B. F., Ibid., 121, 427 (1937). ( 4 ) Haugen, H. X., Blegen, E. AI., Scand. J . Clin. Lab. Invest. 5,67 (1953). (5) Jaffe, 11. Z., Physiol. Chem. 10, 341 i 18861.
WILLIAM S.ADAMS FRANCIS W. DAVIS LOUISE. HAKSEN Department of hledicine, School of Medicine Cniversity of California a t Los Angeles and Wadsworth Hospital, Veterans Administration Los Angeles, Calif. Work supported by U. S. Public Health Service Grant (CA 02433).
Analysis of Total Beryllium in Beryllium Metal Gamma Activation SIR: Methods now in use for the analysis of total beryllium in beryllium metal on an accurate, routine basis are not ideal. The accepted classical method is the complex and time-consuming oxide gravimetric method, which has a relative error of *0.4y0 a t a 957, confidence level (6). The object of this work was to develop a simple, rapid method of radioanalysis for high-purity beryllium metal with the accuracy comparable to or greater than the oxide gravimetric method using a 1-gram sample. Destructive and nondestructive methods of analysis were developed which employed the ( y , n ) reaction ( 2 ) in beryllium. A preliminary study of this reaction and its application to beryllium analysis was conducted by D. *J. Burkhart and coworkers using a 0.5-curie antimony124 gamma source and beryllium solutions (1). They concluded that ( y , n ) activation would be a feasible approach to the analysis of beryllium. Methods for the general analysis of beryllium by gamma activation with a relative error of +2.0% a t a 95% confidence level have been reported by -1.hI. Gaudin and J. H. Pannel ( 3 ) and by G. Goldstein ( 4 ) . F. IT.Postma and C. S. MeMurray ( 6 ) developed an analytical method for total beryllium content in beryllium
metal using a 10-curie antimony-124 gamma source, requiring 10- to 50-gram samples pressed in a 50-ton press. EXPERIMENTAL
Apparatus and Reagents. T h e gamma source was 9 grams of highpurity antimony metal, encapsulated in aluminum, and irradiated to a n activity of 3 curies of antimony-124. The neutron detector unit was a 1-inch diameter disk of 10-mil indium foil. The indium, which was activated in the neutron field produced from the (7,n) reaction and beryllium, was counted in a 2~ beta proportional counter. Beryllium samples, of greater than 97% total beryllium, from 0.4 to 2.0 grams, and in particle sizes ranging from 2 to 50, 50 to 200, and 100 to 500 microns were used nondestructively. Beryllium solutions for destructive analysis were prepared by dissolving the samples in 40 ml. of 4 N H2SO4 and diluting to 100 ml. The beryllium concentration ranged from 0.18 to 2.1 grams per 100 ml., the limit of solubility of beryllium sulfate in water. Procedure. Antimony-124, which had been irradiated in the Oak Ridge research reactor to a n activity of 3 curies, was used as the gamma source because of its long half life of 60 days, its 52% 1.69-m.e.v. gamma component, and its convenient production from natural antimony.
The y source was placed in a Lucite holder in the center of a 40-gallon drum of paraffin which is shielded with 8 inches of lead. I 2-inch diameter hole, with a movable lead cover, was left in the center of the top shielding to insert the samples. With this arrangement the average gamma radiation during operat'ion was 1.5 mr. per hour. The neutron detection unit was a disk of indium foil which was betacounted after irradiation in the neutron field from the (7,n) reaction in beryllium. This met'hod of detect,ion increases the accuracy by eliminating interferencr from the high-level gamma field of the antimony source encountered with B1@FFB (3). For nondestructive analysis a 1.0- by 0.5-inch diameter polyethylene vial containing beryllium powder was placed in the center of a Lucite irradiation chamber (Figure 1). The irradiation chamber was lowered over the y source with the indium neutron detector positioned 1 inch above the sample. The irradiation chamber held the geometry of the component parts constant. For the analysis, the samples were weighed and encapsulated in polyethylene vials. Each vial was positioned in the irradiation chamber, lowered over the gamma source, and irradiated for 1 hour. After irradiation the indium neutron detector was removed and counted for 10 consecutive 5-minute intervals. Each of the 5 VOL. 36, NO. 1 1 , OCTOBER 1964
221 1
