Karl Fischer titration of a sample placed in a water-free saturated ammonium perchlorate-in-methanol solution; internal water is determined after solution of oven-dried material by Karl Fischer in dry pyridine-methanol solvent, by Karl Fischer titration, and also evaluated from the difference between total and surface water contents. Examples of the type of results that are obtained for these determinations are shown in Table X. LITERATURE CITED
( 1 ) .Beckman Instruments, Inc., Instruc-
tion Manual Models KF-2 and KF-3
Aquameters, Fullerton, Calif., Decemb& 1958. ( 2 ) Burns, Eugene A., R . F. Muraca, Anal. Chim.Acta 23. 136 11960). (31, Burns, Eugene A’., R.‘ F. Muraca, Round Robin 13-C of the Joint ArmyKavy-.4ir Force Panel on the .4nalytical Chemistry of Solid Propellants: Evaluation of the Karl Fischer Method for the Determination of Water in Animonium Sitrate.” Progress Report No. 20-354, Jet Propulsion Laboratory, California Institute of Technology, April 1, 1958. (4) Dixon, W. J., Massev, F. J., “Introduction’ t o Statistical “Analysis,” X c Graw-Hill, Xew York, 1951. 1 5 ) JANAF-PACSP Handbook on Recommended Analytical Methods and Specifi-
cation Procedures, Method 300.1 and 701.0, July 1959. (6) McArthur, D. S.,Baldeschwieler, E. L.. White. W. H.. Anderson. J. S..ANAL. CHEM.26. 1012 11954). ’ ( 7 ) Muraca,’ R. F:, Burns, Eugene A., Chemist Analyst 5 0 , 121 (1961). (8) Rice, R. P., Summary of Round Robin conducted by American Potash and Chemical Corp., August 19, 1959. (9) Villars, D. S., “Statistical Design and Analysis of Experiments for Development Research,” FTilliam C. Brown Co., Dubuque, Ion-a, 1950. RECEIVEDfor review July 10, 1961. Accepted April 5, 1962. Division of Analytical Chemistry, 138th Meeting, ACS, New York, N. Y., September 1960.
N e w Method for Determination of Creatinine in Urine by Ion Exchange Separation and Ultraviolet Spectrophotometry WILLIAM S. ADAMS, FRANCES W. DAVIS, and LOUIS E. HANSEN Department of Medicine, School of Medicine, University of California at los Angeles, and Wadsworth Hospital, Veterans Administration, los Angeles, Calif.
A specific method for creatinine determination utilizes physical rather than chemical properties. The procedure is based on the separation of creatinine in urine by an ion exchange resin and subsequent ultraviolet spectrophotometric measurement of the compound a t pH 10.4. Triplicate analyses and standard recovery procedures show good reproducibility. To date, no interfering substances have been encountered. The reagents used in this procedure are stable over long periods of time. The number of variable factors which must b e closely controlled in colorimetric methods are minimized in this procedure.
A
entirely new method for the determination of creatinine in urine uses an ion exchange resin and the maximum ultraviolet absorption wavelength of creatinine at 234.5 mp in an alkaline solution. Because physical properties and not chemical characteristics are utilized, the method is specific, as well as simple, rapid, and accurate over a wide range of concentration. The creatinine value indicates the completeness of a 24hour urine collection in many laboratories. Several methods (2, 7, 8) may be used for the determination but most colorimetric procedures currently employed are not entirely specific or require close con854
N
ANALYTICAL CHEMISTRY
trol of many variables (3, 9). To eliminate the presence of possible chromogenic substances which interfere in the creatinine assay, two major modifications of the Jaffe reaction have been proposed in recent years. Dubos and Miller (4) used a microorganism, Clostridium ureafaciens,to destroy quantitatively the creatinine present, but this method is not without objections (9). Gaebler (5) employed Lloyd’s Reagent to separate creatinine from the chromogenic “pseudocreatinine,” and a modification has been described by Haugen and Blegen (6). The work in the authors’ laboratory involved the separation of various components of 24-hour urine collections b y ion exchange resins. T o standardize the difference in total output of patients, creatinine values were used as a criterion to determine the aliquot of urine t o be sorbed on to a resin column. Thus a rapid accurate procedure was necessary. The method of choice appeared to be the modification of Haugen and Blegen (6), but unsatisfactory reproducibility of results on some specimens was encountered, even though the HartmanLeddon Co. preparation of Lloyd’s Reagent was used. I n the technique employed for urinary fractionation with a Dowex 2 resin column, creatinine was found by ultraviolet absorption measurements to be in the first portion of the eluate ( I ) .
Further analysis, both by spectro.photometry and paper chromatography revealed the additional presence of creatine and N-methyl-2-pyridone-5carboxamide. Creatine is normally found only in trace quantities in urine. The absorption curve of this compound exhibits no characteristic pattern except for an increased absorption in the region below 230 mp. However, to obtain this high end absorption, there must be a concentration far in excess of that found in urine. Consequently, with the possible exception of extreme cases of creatinuria there would be no distortion of the creatinine curve due t o creatine. I n a series of fractionations of normal and leukemic urine, an average of 34 mg. of N-methyl-2pyridone-5-carboxamide was found to be excreted in normal urine per 24 hours. The highest average value obtained for the different types of leukemia studied was 83 mg. per 24 hours although an isolated case of chronic granulocytic leukemia showed an excretion of 210 mg. However, since the pyridone a t p H 10.4 exhibits a minimum at 233 mp whereas creatinine has a maximum at 234.5 mp (10, 11), interference from the pyridone would be negligible. If creatinine had little affinity for the resin column and other substances eluting at the same time were either present in trace amounts or had an absorption maximum far removed from creatinine, a rapid
ground reading was made to be certain that the absorbance was less than 0.05. Standards. A standard containing 1.5 grams of creatinine in 1 liter of alkaline ammonium chloride u-as used as a stock solution. Dilutions varying from 1.5 to 15.0 bg. per ml. were used as working standards. Glassware. All glassware was washed in sulfuric acid-dichromate solution, rinsed with deionized water, and oven dried.
WAVELENGTH
Figure 1 . tinene
__ .. . .
mp
Absorption spectra of creaspectrum b e f o r e columnation a f t e r columnation
and accurate method of analysis could be developed. MATERIALS
Dowex 2-8X, 200- to 400- mesh, C1form. This resin must be well washed to remove background absorption at 234.5 mp. One hundred grams of resin were washed in a Buchner funnel alternately with 1 liter of 6N HCl and 1 liter of deionized water. When 4 liters of each had run through, the resin was washed with 1 liter of alkaline ammonium chloride. A background reading was made on the last few milliliters a t 234.5 mp. The entire washing procedure was repeated until the absorbance was less than 0.05. Once washed, the resin may be kept indefinitely slurried with the ammonium chloride solution. Alkaline ammonium chloride solution. Ammonium chloride, 1.2 grams, was diesolved in 1 liter of 0.25N NH40H. The p H of this solution should be 10.4. Column. The resin was packed as a slurry in a 7 mm. i.d. glass column to a height of 30 mm. The barrel of a 2-ml. long insulin syringe supported in Flexaframe clamps made a very satisfactory column. A small quantity of glass wool was placed in the bottom of the column to support the resin and at the top to prevent slurrying of the resin during sorption of the sample. Once packed, the column was washed with 15 ml. of alkaline ammonium chloride solution and another back-
Table I. Recovery of Creatinine Standards b y Ion Exchange Resin
Total Creatinine, Mg. yo Theoretical Calcd. 150.0 149.7 112.5 112.0 75.0 75.0 37.5 38.0 15.0 18.0
R
~
% 99.8 99.6 100.0 101.3 120.0
,oi ...
EXPERIMENTAL A N D RESULTS
200-
Procedures with Standards. One milliliter of each of several working standards was sorbed on t h e t o p of a resin column and allowed t o run dry. Four milliliters of alkaline ammonium chloride were then added without allowing t h e column t o run dry; however, when this inadvertently occurred, no discrepancies were noted in t h e results. T h e total eluate ( 5 ml.) was diluted to 10 ml. with the ammonium chloride solution and the absorbance spectrum measured with a Beckman DK-2A against a deionized water blank. The results of these tests are shown in Figure 1 and Table I. The spectral configuration of creatinine after columnation shows no alteration from that obtained initially. An inadequate washing of the resin manifests itself with a high end absorption, and the maximum a t 234.5 mp is distorted. The data in Table I show a quantitative recovery (99.6 to 101.3%) of creatinine over a range of 37.5 to 150 mg %. The 120% recovery obtained with the lowest standard is because the background was proportionately greater than in creatinine standards of higher concentration. Procedure with Urine. This method is basically t h e same as t h a t for t h e standards. One milliliter of a 24-hour urine is diluted t o 10 ml. with alkaline ammonium chloride. One milliliter of this dilution is then sorbed on a resin column a n d allowed t o r u n dry. Four milliliters of t h e alkaline ammonium chloride solution are added in 1-ml. increments and the total eluate (5 ml.) diluted to 10 ml. Readings are made a t a wavelength of 234.5 mp.
100-
Specificity a n d Recovery. T o determine t h a t the only compound measured was creatinine, absorption spectra were determined on resin eluates before a n d after removal of creatinine b y Lloyd’s Reagent. Several column eluates were pooled t o obtain a large volume. This solution was made acid with HC1 a n d a quant i t y of Lloyd’s Reagent equivalent t o 20 mg. per ml. was added. T h e resultant slurry was mixed a n d after 10 minutes i t was centrifuged a n d t h e ~ ~ ~ decanted. ~ ~The clear supernatant absorption spectrum of this material was measured, but because the creatinine spectrum at an acid p H shows a strong hypsochromic shift, i t was necessary t o adjust the p H to 10.4 with NH4OH. The resultant spectrum
WAVELENGTH
mp
Figure 2. Absorption spectra of urine eluates from ion exchange resin columns Numbers represent sample designation
showed a small amouiit of chroniogenic background and end absorption with no trace of creatinine, thus indicating that the ion exchange method was highly specific. The Lloyd’s Reagent was reslurried with alkaline ammonium chloride solution, mixed for 10 minutes, centrifuged, and again the clear supernatant was decanted. The absorption spectrum of the supernatant exhibited the typical creatinine curve, but because of the difficulty of obtaining a nonabsorbing background reading from Lloyd’s Reagent, due to chromogenic contamination eluted under the influence of the alkaline eluant quantitative determinations were not attempted. Glycosuric and alkaptonuric urines, and a variety of pharmaceuticals tested to date, including
Table It. Total Creatinine (Grams) in 24-Hour Urine Collections from 25 Adults
Triplicate Analyses
~
(1) 0.58 0.67 1.49 1.41 0.88 0.62 1.07 0.73 0.72 1.17 1.20 0.76 0.76 1.78 1.24 0.64 1.38 0.48 , 0.45 0.95 0.76 1.03
0.94
1.24 1.12
(2)
0.79 0.66 1.50 1.34 0.92 0.69 1.09 0.86 0.66 1 1 0 0 1 1 0 1 0 0 0 0 0
11 18
78 83 82 27 64 34 41 45 85 72 94 0.92 1.20 1.22
VOL. 34, NO. 7, JUNE 1962
(3) 0.80 0.66 1.46 1 37 ~-
0 95 0 61
1.02 0.81 0.68 1 15 1 18 0 73 0 74 1 82 1 30 0 60 1 49 0 44 0 42 0 92
0 73
1 00 0 91
1 11 1.12
855
barbituric acid derivatives and sulfonamides, do not affect the results of the creatinine assay because the potential interfering substances are not eluted from the ion exchange resin a t the specified pH. Additional analyses demonstrated recovery of known amounts of creatinine added to urine to be within 37,.
perienced frequently in obtaining duplicate checks, and analyses were repeated until satisfactory duplication was obtained. Results by the tlyo methods for 8 Of the 25 differed b y more than 10% (11 to 46%). It-appeared that the-modified Jaffe procedure was subject to rather large errors due to incomplete removal of interfering substances b y the Lloyd's
LITERATURE CITED
(1) Adams, W. S., Davis, F., Nakatsni, hf., Am. J . iwecz. 28, 726 (1960). ( 2 ) Barclay> J. Kenney~ '1. A , , Bzochem. J . 41, 5 (1947). (3) Carr, J. J., AKAL. CHEM. 25, 1859
(1953).
( 4 ) Dubos, R., Miller, B. F., J . Biol. Chem. 121, 427 (1937). ( 5 ) Gaebler, 0. H., Zbid., 89, 451 (1930). (6) Haugen, H. N., ~ l E, ~nf., Scand. J . Clin. & Lab. Invest. 5, 67
REPRODUCIBILITY OF URINARY ANALYSES
Triplicate analyses, each on different days, were made by the ion exchange method on aliquots of 24-hour urine collections from 25 adults (Table 11). Absorption spectra of eluates from the resin columns for some of the urine specimens are shown in Figure 2. I n addition, each of the 25 urines were analyzed by the Haugen and Blegen modification of the Jaffe procedure (6). Considerable difficulty was ex-
variations Produced in the development and stability of the chromogen by external and internal influences. These factorsare not involved in the ion exchange method reported here, by which creatinine is not modified chemically, but is measured directly. ACKNOWLEDGMENT
The authors acknowledge the technical assistance of Ann Fein.
(laao).
(8) Langley, W. P., Evans, ill., J . Biol. Chem. 115, 333 (1936). (9) Owen, J. A., b o , B.,Scandrett, F. J.2 Stewart, C. p., Biochem. J . 58, 426
(1954). (10) Wollenberger, A., Acta Chem. Scand. 7 , 445 (1953). (11) Wollenberger, A., Yature 173, 205 (1954). RECEIVED for review February 1, 1962. Accepted April 16, 1962. Work supported by U. s. Public Health service Grant (CY-2433).
Spectrophotometric Determination of Titanium as Reduced Molybdotitanic Acid J. C. GUYON1 with M. G. MELLON Purdue University, lafayeite, Ind.
b The reduction product of a complex molybdotitanate is used as the basis of a spectrophotometric method for the determination of titanium(lV). An empirically devised method is described first. Factors affecting the color reactions are presented, and an application to the determination of titanium in glass is given. The useful range of the method for a 1-cm. cell is 6 to 30 mg. per liter. Also, using statistical methods, a regression equation is developed from which the concentration of titanium may be calculated from the absorbance at 755 rnp and the known concentrations of the reagents.
T
HE increased use of titanium especially in paint pigments and metallurgical products, indicates a need for accurate and sensitive methods for its determination. Although many chromogenic reagents have been proposed to determine the element spectrophotometrically, only one has been a heteropoly complex. Veitsman (9) measured titanium in the form of a complex, presumably the heteropoly anion molybdotitanophosphate. Present address, University of Missouri, Columbia, Mo.
856
ANALYTICAL CHEMISTRY
Work in this laboratory has shown the possibility of employing what has been assumed to be simple heteropoly species for the determination of vanadium and niobium. The postulated species, before reduction, were molybdovanadate (11) and tungstovanadate (10) for vanadium, and molybdoniobate (3) for niobium. This paper reports the resuIts of a spectrophotometric study to ascertain the possibility of determining titanium in a similar way. The assumed species, before reduction, is molybdotitanate. Various workers (4-8) have postulated the existence of heteropoly compounds containing titanium. However, these compounds have not been isolated, nor has the stoichiometry in solution been established. The possible structure and formulation of heteropoly compounds containing titanium have been the subject of some controversy. No suggested analytical applications of such complexes were found. EXPERIMENTAL W O R K
Apparatus. A Cary recording spectrophotometer, Model 10-11, having matched quartz absorption cells with a n optical p a t h of 1.000 0.002 cm., was used for the spectrophotometric
*
measurements. A Beckman Model G or Zeromatic meter was used for p H measurements. Reagents. A stock solution of sodium molybdate, approximately lo%, was prepared b y dissolving 100 grams of N a 2 M o O c . 2 H 2 0i n 500 ml. of distilled water and diluting t o 1 liter. A stock solution of sodium citrate, approximately 575, was prepared by dissolving 50 grams of NazCsHsO,.2H20 in water and diluting to a liter. A stock solution of chlorostannous acid, approximately lo%, was prepared by dissolving 110 grams of SnClz.2H20 in 170 ml. of concentrated hydrochloric acid and subsequently diluting to a liter. A few grams of mossy tin was added to the container. Dilutions of this solution were used as a reductant. A stock solution of titanium was prepared in two ways. Use of potassium titanyl oxalate: 4.00 grams of K2[TiO(C204)J.2H20 were dissolved in 25 ml. of hot concentrated sulfuric acid and then diluted to a liter. The solution contains 2.000 mg. T i per ml. Use of titanium dioxide: 1.000 gram of titanium dioxide, TiOz, was dissolved in 100 ml. of concentrated sulfuric acid containing 50 grams of ammonium sulfate and diluted to a liter. The resulting solution contained 0.5995 mg. Ti per ml. Either solution may be standardized by precipitation of the
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