1414 -
ANALYTICAL CHEMISTRY ~
Table I. Found, Atom '% D 0.011 0.016
Sample4 14
Illustrative data of analyses with the mass spectrometer are shown in Table I.
Results .%tom % Excessb Found Calculated
0.017
2
0.080
0 Oil
0.063
3
0 ,>A 0 . %57
0 ,34 0 .ii
0 58 0 58
4
0.6.5 0 64 1 17 1.17
0 63 0 62
0 63 0 63
11.7
1 I6
5
113
1 Ih
6
2.83 2.74
;8:7 2 -.
2 90 2 90
7
2.90
2 8%
2 83
8
3.71 5.66 5.2:
3.70
3.61
5.64 5 23
a , 80
9
4 . 80
6.12 6 2.7 6.13 10 6.11 6.10 6.23 a Sample 1 is for Baltimore tap water. Samples 2 , 4, 7, and 8 were prepared b y dilution of deuterium oxide with t a p or once distilled water; samples 3, 5 , 6,9. and 10 were prepared b y dilution of deuterium oxide with whole blood. T h e actual values for calculated atom per cent excess may deviate by = t 2 % from the values given. 6 Atom % D minus average atom % D of tap water. C The average value, 0.015 atom % ' D is in agreement with the d a t a cited in ( 8 ) .
of the tube. As soon as the sample is frozen, the cock is turned to position I, and the sample tube is sealed a t the constriction. (The solid carbon dioxide need not be applied during the sealing.) Approximately 20 samples can be prepared per hour. (The vacuum pump should be protected from possible introduction of zinc dust by an appropriately placed plug of borosilicate glass Wool.)
The sample tubes are then placed in individual brass tubes (to prevent catastrophes in case one of the tubes explodes) and are heated in a suitable furnace a t 400" f 10" C. for 30 minutes. It is advisable to allow the furnace to cool to near room temperature before the door is opened.
DISCUSSION
The sample tubes after reduction of the mater are small bombs The samples of water used should be no greater than 0.01 ml. ; a pressure of approximately 24 atmospheres may be expected from 0.1 ml. of water in a 10-ml. sample tube. The sealed sample tubes should never be heated in an open flame. Data on samples containing greater enrichments of deuterium than 7 atom % excess are not included; in the experiments maximum sample enrichment is less than this value. Accurate analysis of highly enriched samples would have required conversion of the mass spectrometer to an instrument for deuterium gas. This was not considered justifiable in view of the purpose for which the mass spectrometer was intended: hydrogen isotope analysis. The "memory effect" is discussed in detail by Kirshenbaum (5). The procedure described here permits more numerous analyses to be made and reduces or eliminates certain of the sources of wror inherent in the conventional zinc reduction trains; it doe3 not affect in any n a v the memory effect arising from adsorbed water in the mass spectrometer itself. ACKNOWLEDGMENT
This work was supported in part by a grant from the Life Insurance Medical Research Fund and in part by contract No. \'-lo01 M-527 between the Veterans Administration and the Johns Hopkins University. LITERATURE CITED
(1) Friedman, L., and Irsa, -1. P., ANAL.CHEM.,24,876 (1952). (2) Graff, J.,and Rittenberg, D., Ibid., 24, 878 (1952). (3) Kirshenbaum, I., "Physical Properties and Analysis of Heavy Water," p. 130, New York, McGraw-Hill Book Co., 1951. (4)Orchin, M., mender, I., arid Friedel. R. .1.,.%s.\L. CHEY..21, 1072 (1919).
RECEIVED for review Sovember 3, 1932. Accepted April
1. 19S3
Colorimetric Determination of Calcium with Ammonium Purpurate MAX B. WILLIAMS
AND JAMES H. MOSER Oregon State College, Corcallis, Ore.
development of new colorimetric methods for the deterT mination of calcium has been curtailed by the limited availability of suitable reagents that change color in the presence of this HE
ion. The colorimetric method in the most general use a t this time consists of precipitating calcium oxalate with a known excess of oxalate reagent, then determining the residual oxalate concentration colorimetrically by measuring the color remaining after the addition of a known amount of standard permanganate or ceric solution. This method, while good and fairly free from interferences, is not applicable to very low concentrations of calcium (0.1 to 10 mg. per liter) for the possibi1it.v of precipitate loss is relatively great; also, it is often difficult to obtain complete precipitation of calcium ovalate when certain organic materials are present. Schwarzenbach and Gysling ( 4 ) investigated the formation of si colored complex of ammonium purpurate (murexide), ?;H*CsH,Os"., with calcium in aqueous solution. The structural forniula for the purpurate ion is
r
TH-C-0
/
O=C--SH
\
SH-CLO
They showed that the ratio of calcium ion to purpurate ion wm 1 to 1, with an equilibrium constant for the formation of the calcium complex of approximately 1380 a t a pH of 8.53. The value of the equilibrium constant varies from log IC = 2.6 a t pH 4.65 to 5.1 a t pH 12.50. The stability t,o decomposition of murexide decreases, however, with increasing pH of the solution. A possible structure of the calcium complex was suggest,ed, and! also, complexes of certain other cations wpre investigated. Ost,ertag and Rinck (2-3) described a method employing ammonium purpurate for the determination of macro amount,s of calcium. The aqueous solutions were compared at' pH 6 in a Lange photoelectric colorimeter equipped with a 500 to 550 mp filter. In their method, pH 6 was chosen, so that the dye would be more stable. I n this spectrophotometric study; it was found that the sen+ tivity of the colorimetric reaction is greatly increased by a considerahly higher pH, thus making possible its development into a micromethod. At the same time, errors caused by the decomposition of the dye in alkaline solut,ion v-ere minimized to the point where the method became reproducible and reliable.
1STABILITY O F A.MMONIUM PURPURATE SOLUTIONS
O=C--SH
The instability of ammonium purpurate in aqueous solution has been a drawback to the development of it3 w e in analytical
V O L U M E 25, NO. 9, S E P T E M B E R 1 9 5 3 procedures. Ostertag and Rinck (3) state that the pure dye 1more stable in neutral aqueous solution than are impure sampleof ammonium purpuiate. The authors studied the rate of decomposition of commercial lots of the dye in various organic m t e r miscible solvents, and found that the use of 70% by volume of e t h j l alcohol in water gave the slo\vest decomposition. Ammonium purpurate dissolved in this solvent was utilizable in the procedure described below, for at least 8 hours when stored at room temperature. Commercial lots of the dye were found to vary in purity, hut sniall diffeiences in concentration of aniiiionium purpurate in the reagent do not affect the accuracy 01 the method as developed.
01
ao WAVE LENGTH I N MILLIMICRONS
Figure 1. Absorption Spectra of Murexide Solutions with and without Calcium a t pH 11.3
Sinw the st:thility of the calcium purpurate complex ion is a function of pH, and the relative concentrat,ion of the complex ion to dye is great,est in an alkaline solution, it was necessary to investigate more thoroughly the effect of pH on the absorption spectra of animonium purpurate solutions and ammonium purpui,at,esolutions containing an excess of calcium. The dye decomposes too rapidly for practical use in solutions in which the pR is greater than 12. Boric acid-sodium borate buffei solutions were usrtl for pII 8, 9, and IO; for pH 11 t o 13 the adjustment wa': made x-it,h sodium hydroxidr. Th? largest difference hetweeri t l i e absorption spectra of the calcium-free ammonium purpurate solutions and the calcium-containing animoniuni purpurate solutions TWS found to exist at pH 11.3. Therefore, the pH of the solutions subsequently used was adjusted to pH 11.3 with sodium hydroxide. The desired accuracy \vas attainable without the use of a buffer system. The absorption spectra of ammonium purpurate with and without P X ~ P S Scalcium at pH 11.3 are shown in Figure 1.
1415
:hove) were pipetted into a 1-liter volumetric flask and diluted to volume. The acid in this solution was also titrated as ahove. One milliliter of this solution dilut,ed to 100 ml. producw a solution containing 0.100 mg. of calcium per liter. APPARATUS
A Beckman Model B spectrophotometer with a 3t.t of 1'0111. 1.00-cm. matched Cores cells was used for all measurenicsiit,s. PROCEDURE
Preparation of Working Curve.
the following manner. The series of solutions containing 0 to 1.2 mg. of calcium per liter were prepared by adding the calculated volume of standard calcium solution containing 10 mg. per liter to a 100-ml. volumetric flask hy means of a 10-ml. graduated pipet. The solution containing 100 mg. of calcium per liter was used similarly for the preparation of the solutions that contained more than 1.2 mg. of calcium per liter. The exact amount of standard base (as determined oreviouslv bv titration) was then ztdded to each flask to neutraliz'e the aci&i< the calcium-containing aliquot and to give a final hydroxyl ion concentration of 0.0020 N (pH 11.3). Distilled water was then added to each flask until it was a p proximately 0.8 full, in order to minimize the decomposition of the dye when i t x a s added. Exactly 10 ml. of the dye reagent were then DiDetted into each of the flasks. distilled water m-as added to t h e mark, and the contents were' shaken thoroughly. The time elapsing between the addition of the dye to a calcium solution and to the blank (0 mg. of calcium per liter) should not be excessive. It was found that a maximum time of 5 niinuteq did not affect the accuracy of the method. The absorbancies of the solutions thus prepared were deteimined with the spectrophotometer at 506 mp against the -elution containing no calcium. This procedure of using a blank containing all the reagents exrept calcium minimizes errors due to dye decomposition. The time elapsing between the final mixing of the solution? and the absorbancy determinations was not critical. Determination of Calcium. To an aliquot of the sample solution contained in a 100 ml. volumetric flask, the exact amount of hase was added so that on dilution to the mark a pH of 11.3 n a s attained. A blank was prepared at the same time
i
REAGENTS
Dye Reagent. Ammonium purpurate was obtained from the Hach Chemical Co., Ames, Iowa, and was used without further purification. The reagent is available elsewhere. Approximately 40 mg. of ammonium purpurate were dissolved in 75 ml. of distilled water, and the solution was filtered through a coarse sintered-glass filter funnel. Approximately 175 ml. of absolute ethyl alcohol were then added, and the solution was well mixed. Standard 0.1 N Sodium Hydroxide Solution, C.P. Standard Calcium Reagent, 100.0 Mg. per Liter. Anhydrous C.P. calcium carbonate (249.5 mg.) was placed in a 1-liter volumetric flask, dissolved in 10 ml. of 1 N hydrochloric acid, and distilled water was added to the mark. The excess acid in an aliquot of this solution was determined by titration with t,he standard sodium hydroxide. One milliliter of this calcium solution diluted to 100 ml. results i n a solution containing 1.00 nig. of calcium per liter. Standard Calcium Reagent, 10.0 Mg. per Liter. Exactly 100 nil. of the solution containing 100 mg. of calcium per liter (from
Solutions Containing 0, 0.2,
0.1, 0.6, 0.8, 1.0, 1.2, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 mg. of calcium per liter were prepared from the standard calcium solutions in
o.l
OD
Y
i
MILLIGRAMS P E R L I T E R CALCIUM
Figure 2. Calibration Curve for Calcium with Murexide at 506 mM and pH 11.3
The amount of standard base t o he added was deternrined i n t ~ v oways : (1) If no buffer species were present, the solution was titrated :is above, and the volume of base required was calculated. ( 2 ) Base and water m-ere added to a separate aliquot until the pH was 11.3 a t a total solution volume of 100 ml. The pH n-ae
ANALYTICAL CHEMISTRY
1416
determined with a Beckman p H meter using an alkaline glass electrode. The volume of base thus determined was then added to the 100-ml. volumetric flask containing the same size aliquot of sample as was used in this pH adjustment. Distilled water and dye reagent were then added in the same manner as described for preparation of the working curve. The absorbancy of the solution thus prepared was determined against a blank prepared a t the same time. Discussion. In Figure 2 are shown the results of absorbancy determinations by the above procedure. Beer’s law is followed for calcium concentrations up to 1.2 mg. per liter. This portion of the curve has been tested for 66 determinations by statistical methods and does not deviate significantly from linearity. For this reason, no points are included in the figure for this concentration range. The slope, 6, of the linear region is given in Table I. It was found that the working curve remained constant if the absorbancy of the blank versus distilled water at 506 mp was not less than 0.48. EFFECTS OF CERTAIN IONS ON THE WORKING CURVE
Schwareenbach and Gysling (4) studied the complex formation of murexide with bivalent magnesium, strontium, and barium and found that the equilibrium constants for complex formation were much smaller than the calcium complex. Bivalent zinc, cadmium, and copper were shown to react with murexide, and some color change was noted with bivalent nickel, cobalt, iron, manganese, and tin and trivalent iron. KO effect w&sobserved with bivalent lead, beryllium, mercury, and uranyl ion, with trivalent aluminum and chromium and with quadrivalent titanium. The following work was done in order to determine for the alkali metal ions, the alkaline earths, and certain common anions the maximum allowable concentrations which will not interfere with the colorimetric method developed for calcium. The procedure ueed was similar to that described above, with the followin.; additions: The constant given amount of the ion being studied was also added to the working solutions (0 to 1.2 mg. of calcium per liter), the blank included. The absorbancies were determined against this last solution for the preparation of the working curve. $nother blank (“pure blank”) was prepared a t the same time that contained neither calcium nor the metal ion being studied. The absorbancies of these two blanks were then determined against distilled water in order to be sure that the absorbancy of the pure blank was not less than 0.48 and to ascertain if there was a difference in absorbancies of the two blanks, which would be due to impurities in the salt concerned and/or the ion being considered. The results for the alkali metals, except lithium, and for the alkaline earths are presented in Table I. The deviation of the slope, b, from the established value of 3.55 for the calcium sohtions alone is a measure of the interference of the foreign ions. An analysis of covariance of this data showed that the slopes given in Table 1 do not differ significantly. The average slope was 3.55 mg. of calcium per liter per absorbancy unit with an estimated standard error of 0.03 mg. of calcium.
Table I. Maximum Concentrations of Certain Ions Causing No Change in Slope of Working Curve (506 LIP) Metal
Maximum Concn., Mg./Liter
Number of Detns.
66 12 18 18 6 30 I3
6
Slope b
3.55 3.49 3.62 3.52 3.49 3.56 3.57 3.66 -4.7.3.55
Higher concentrations of cesium were not studied due to the limited availability of the salt. Q
Table 11. Comparison of Analyses for Calcium in Solutions with and without Magnesium“ (505 hfp)
Calcium Known, Mg./Liter
Magnesium Known, Mg./Liter 0 5 Calcium found, Calcium found, mg./liter mg./liter
0.20 0.40 0.60 0.80 1.00 1.20
0.20 0.44 0.60 0.78 1.04 1.16
0.23 0.43 0.60 0.82 0.99 1.17
Results of single determinations.
Table 111. Effect of Certain Anions on the Working Curve (Sodium concentration, 400 mg. per liter; wave length, 506 mp) Anion Absorbancy Chloride Yitrate Sulfste Calcium, mg./liter
0,200 0.400 0.600 0.800 1.00 1.20
0.055 0.106 0.164 0.209 0.250 0.290 0.183
Means Absorbancy of Na blank5 0,495 Absorbancy of pure blsnka 0.505 a Against distilled water.
0.053 0.109 0.165 0.210 0.260 0.302 0.179
0.046 0.097 0.I47 0.183 0.232 0.274 0.164
0.514
0.512
0.516
0.504
16 14
1.2 10
0.8 0.6 0.4
0.2 0.0
400
500
600
700
WAVE LENGTH IN MILLIMICRONS
Figure 3. Absorption Spectra of Murexide Solutions Containing Various Cations at pH 11.3
In the case of magnesium, slightly better results were obtained a t a wave length of 505 mp where an apparent isobestic point exists for ammonium purpurate solutions containing up to 5 mg. of magnesium per liter. Therefore, in the determination of calcium in solutions containing magnesium in this concentration range, 505 mp was the preferable m’ave length on our instrument. An example of a typical determination of calcium in solutions containing and not containing magnesium is given in Table 11. Lithium, as well as barium, strontium, and magnesium, reacts with ammonium purpurate. Absorption spectra of ammonium purpurate solutions containing an excess amount of each of these ions are given in Figure 3. This surprising complex ion formation of lithium with ammonium purpurate is being investigated by the authors w a possible basis for an analytical method applicable to that ion. Ostertag and Rinck ( 1 ) attempted to overcome the effects of interfering ions by deliberately adding salts of these ions until an ‘(indifferent” concentration was reached, after which a further increase in salt concentration was apparently without effect. Chloride or nitrate were the anions accompanying the metal ions in the above experiments. .4n experiment was conducted
V O L U M E 2 5 , NO. 9, S E P T E M B E R 1953.
1417
(Table 111) in which three working curves were obtained, the first of which was obtained in the presence of sodium chloride, the second in the presence of sodium sulfate, and the third in the presence of sodium nitrate, the salts being present in equimolar concentrations (400 mg. of sodium per liter). A statistical analysis of this data showed no significant difference between the curves obtained with the nitrate or chloride, but the slope of the sulfate curve differed significantly from the other two. In addition to the interferences already mentioned, it has been found that the approximate maximum concentration of ferric ion and of mercuric ion that may be tolerated is 1mg. per liter. This same concentration of hafnium or of zirconium strongly interferes. STATISTICAL ANALYSIS
The statistical method was used throughout this investigation, wherever applicable, with a significance level of 5%. The study of the effects of the certain cations was limited to the linear portion of the working curve, zero to 1.2 mg. per liter, in order to facilitate the analyses of covariance. ilnalysis of covariance is particularly useful in this instance, since it allows one to detect sig-
nificant differences among the slopes of working curves prepared under different conditions. I n the case of iron, mercury, hafnium, and zirconium, only a brief survey of possible interferences was desired, and the experimental design utilized was analysis of variance with multiple classification. The equation 2 = bA,, where 6 is the estimated calcium concentration corresponding to an absorbancy A,, and b is the estimate of the slope of the working curve, was fitted to the data h y the method of least squares. LITERATURE CITED
(1) Ostertag, H., and Rinck, E., Chim. anal., 34,108-9 (1952). (2) Ostertag, H., and Rinck, E., Compt. rend., 231, 1304-5 (1950). (3)Ibid., 232, 629-30 (1951). (4) Sohwarzenbach, G., and Gysling, H., Hela. Chim. Acta, 32, 131425 (1949). RECEIVED for review November 11, 1952. Accepted M a y 8, 1953. Preaented before the Section of Analytical Chemistry a t the Pacific Northweat CHEMICAL SOCIETY a t Corvallis, Ore., Regional Meeting of the AMERICAN J u n e 20-21, 1952. Approved for publication b y the Oregon S t a t e College Monograph Committee. Research Paper 229, Department of Chemistry, School of Science.
Ultraviolet Analysis of Isomeric Cresol Mixtures GERTRUDE E. CARNEY AND JANET K. SANFORD Research Laboratory, Barrett Division, Allied Chemical & Dye Corp., Glenolden, Pa.
techniques have been developed for analyzing mixtures Cryoscopic, colorimetric, infrared, and ultraviolet vapor (3) methods have beendescribed. Many of these methods are applicable only to binary mixtures. Ultraviolet spectrophotometry has been used in this work to analyze mixtures of any two or all three of the cresols simultaneously and directly in isooctane solution. The procedure is comparatively simple and rapid and can be used as a routine method. EVERAL
S of
0-,
m-, and pcresols.
Table I.
Analyses of Synthetic Mixtures of 0-, rn-,and p-Cresols
o-Cresol Known Found Error 90.1 9.0 0.0 10.2 1.9
89.1 9.0 -0.1 11.5 2.0
-1.0 0.0 -0.1 11.3 +0.1
W t . 7%) m-Cresol Known Found Error 9.9 11.3 91.0 90.5 89.8 89.1 69.2 67.6 17.5 17.2
+1.4 -0.5 -0.7 -1.6 -0.3
p-Cresol Known Found Error 0.0 0.0 10.2 20.6 80.6
-0.1 0.1 10.4 21.1 80.9
-0.1 +0.1 +0.2 +0.5
+0.3
Substances, other than cresols, which absorb between 2715 and 2860 A. will interfere with the analysis. Those most likely to be present and to cause interference are phenol and alkylphenols other than the cresols. The ultraviolet spectra of the simpler alkylphenols are so similar that it is impossible to prove by its ultraviolet spectrum alone that an unknown sample contains cresols only. Hence, care should be used to remove or demonstrate the absence of phenol, xylenols, and higher homologs. Since the three cresols, like most other phenols, have zero absorbances a t 3000 A., contaminants which absorb a t this wave length can easily be detected. To find the best set of wave lengths for the analysis, seven combinations of measurements made a t three wave lengths from the group 2719,2728,2774,2790,2798, and 2858 A. were used to calculate the compositions of five synthetic mixtures. At each of these wave lengths the absorptivity of a t least one isomer was strong, and the variation in absorptivity of every isomer with slight changes in wave length was small. Each set of three wave lengths was chosen so that the equations expressing the absorb-
ance-concentration relations had a high degree of independence and would yield relatively accurate analyses. Best results for the series of samples tested (Table I) were obtained a t 2728 (meta), 2774 (ortho), and 2858 A. (para). These are shown with the spectra of the individual cresols in Figure 1. From Beer's law, a t a given wave length the allsorbance, A , of a mixture of cresols is
A
=
(GC~
+
~
m
+
~ a p cnp ) b
(1)
where a is the absorptivity of a constituent in liters per gram centimeter; c, the concentration of a constituent in grams per liter of solution: and b, the optical path length in centimeters. The subscripts designate the individual isomers. For the general case where all three isomers may be present, measurements a t three wave lengths and three equations are required. In the special case where it is known that only two specific isomers are present, measurements need be made a t only the two wave lengths characteristic of these isomers, and only two equations need be solved. Deviations from Beer's law, resulting from association or the interaction of any of the cresols in isooctane solution, were not observed with concentrations having absorbances between 0.5 and 0.9 a t the characteristic wave length of the isomer. Spectrophotometric deviations, which reduce the precision of the method, were avoided by choosing slit widths to provide spectral band widths of 5 A. (2). EQUIPMENT
A Beckman quartz spectrophotometer, Model DUV, was used with a liquid-cooled lamp house thermostated a t 30" C. The wave length scale of the spectrophotometer was calibrated with a mercury arc lamp. To permit the use of narrow slits, a 10,000megohm phototube load resistor was substituted for the 2000megohm resistor. Solutions were measured in matched 1-em. quartz cells with covers. If the room temperature is variable, a thermostated cell compartment is desirable. ISOMERS
o-Cresol was purified to a constant melting point by four or five recrystallizations from a low boiling (b.p. 80" to 120' C.) petro-
